Vitamin D: Molecular, Cellular and Clinical Endocrinology. Proceedings of the Seventh Workshop on Vitamin D, Rancho Mirage, California, USA, April 1988 9783110846713, 9783110114775

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Vitamin D Molecular, Cellular and Clinical Endocrinology

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2D) Receptor Concentration is Increased During Lactation in the Rat J. P. Goff, R. L. Horst, Τ. Α. Reinhardt

246

The 1,25-Dihydroxycholecalciferol Level in Plasma does not Influence the Properties of its Intestinal Receptor in Piglets B. Schröder, R. Kaune, J. Harmeyer 248 The Number of 1,25-Dihydroxyvitamin D Receptors in Rat Intestinal Mucosa have been Markedly Underestimated Steve D. Antrobus, Marian R. Walters

250

DNA Binding Property of Vitamin D3 Receptors Associated with 26,26,26,27,27,27-Hexafluoro-l,25(OH)2D3 S. Okuno, M. Inaba, K. Yukioka, Y. Nishizawa, A. Inoue, H. Morii

256

XVIII

Contents

Vitamin D3 Binding Protein of Phaseolus Vulgaris Roots. Biochemical and Functional Characterization M. A. Vega, M. Pintos, R. L. Boland

258

Cell Differentiation / Hematopoiesis / Immunology Interaction between the Hematopoietic System and the Vitamin D Endocrine System H. Phillip Koeffler, Anthony W. Norman

263

I,25-(OH)2D3 and Gene Expression During HL-60 Differentiation Y. Cayre, D. H. Solomon, D. Bories, M. C. Raynal

267

Vitamin D: A Steroid Hormone of the Immune System, by the Immune System, for the Immune System W.F.C.Rigby

276

Immunoregulatory Properties of l,25(OH)2D3: Cellular Requirements and Mechanisms S. C. Manolagas

282

1,25-Dihydroxyvitamin D3 and the Regulation of Cancer Cell Replication J. A. Eisman, R. L. Sutherland, D. H. Barkla, P. J. M. Tutton

291

MC 903 - A Novel Vitamin D Analogue with Potent Effects on Cell Proliferation and Cell Differentiation Lise Binderup

300

la, 25-Dihydroxy-22-Oxavitamin D3: A New Synthetic Analogue of Vitamin D3 Having Potent Differentiation-Inducing Activity without Inducing Hypercalcemia in Vivo and in Vitro J. Abe, M. Morikawa, Y. Takita, K. Miyamoto, S. Kaiho, M. Fukushima, C. Miyaura, E. Abe, T. Suda, Y. Nishii

310

Mechanisms of Cell Fusion Induced by la,25-Dihydroxyvitamin D3 T. Suda, H. Tanaka, C. H. Jin, C. Miyaura, T. Shinki, N. Takahashi, T. Akatsu, A. Segawa, E. Abe

320

l,25(OH)2D3 Promotes Differentiation of Resting Chondrocytes to Hypertrophic Cells in Vitro: A Model System for Evaluating the Potency of Vitamin D Analogs L. Gerstenfeld | , M . von Deck, C. Kelly, M.Uskokovic, J. Liant

330

la,25-Dihydroxyvitamin D 3 Synthesis by Synovial Fluid Macrophages in Arthritic Disease Μ. E. Hayes, J. Denton, A. J. Freemont, Ε. B. Mawer

332

Contents

XIX

l,25-Dihydroxyvitamin-D3 Prolongs Rat Cardiac Allograft Survival S. C. Jordan, M. Nigata, Y. Mullen

334

1,25-Dihydroxyvitamin D3 as a Synergistic Agent of in Vitro Cyclosporin Α-Induced Suppressive Activity in Rheumatoid Arthritis P. Gepner, B. Amor, C. Fournier

336

Changes in Phosphoinositide Metabolism During l,25(OH)2D3 Induced Cell Differentiation Μ. B. Hill, B. L. Brown, R. G. G. Russell, D. F. Guilland-Cumming

338

Increase in [Ca2+]i in Cultured Human Keratinocytes by l,25(OH)2D3 is Calcium Dependent and Coincides with an Increase in Phosphatidylinositol Metabolism J. MacLaughlin, L. Cantley, M. Holick 340 The Effects of la,25(OH)2D3, Diltiazem (D-Cis, L-Cis) on Induction of Differentiation of HL-60 Cells T. Taoka, T. Itano, M. Tokuda, Y. Kubota, T. Tanaka, O. Hatase, S.Irino

342

Ια-Hydroxylated Vitamin D Metabolites Promote Differentiation and 24,25-Dihydroxy Vitamin D Synthesis in Human Leukaemia (HL60) Cells Μ. E. Hayes, Ε. B. Mawer 344 1,25 Dihydroxyvitamin-D3 Prolongs Skin Graft Survival in Mice S. C. Jordan, R. Shibuka, Y. Mullen

346

Effects of l,25(OH)2D3 on Myelopoiesis and Β Lymphopoiesis in Long-term Marrow Cultures K. Dorshkind, H. Reichel, A. W. Norman 348 Vitamin D-Deficiency Disturbs the Differentiation Process in the Dental Papilla: An Ultrastructural Study A. Berdal, N. Balmain, P. Cuisinier-Gleizes, H. Mathieu

350

1,25-Dihydroxyvitamin D3 Inhibits Delayed-Type Hypersensitivity Mediated by T-Cell Clones Inducing Experimental Autoimmune Encephalomyelitis Jaques M. Lemire, Richard A. Miller

352

1,25-Dihydroxyvitamin D3 Acts Directly on Human Lymphocytes and Interferes with the Cellular Response to Interleukin-2 A. Ravid, R. Koren, L. Maron, A. Novogrodsky, U. A. Liberman

354

la,25-Dihydroxyvitamin D3 Synthesis by Normal and Myelofibrotic Human Spleen Cells Μ. E. Hayes, M. Davies, J. A. Liu-Yin, Ε. B. Mawer 356

XX

Contents

Pretreatment of Τ Cell or Monocytic Cell Lines with l,25(OH)2D3 Markedly Increases IL2 or IL1 mRNA Levels Following Cell Activation J. L. Prehn, S. C. Jordan 358 Phorbol Ester Stimulation of 250H-Vitamin D 1-Hydroxylase Activity in the Monoblastic Cell Line U937 M. Hewison, S. Barker, A. Brennan, D. R. Katz, J. L. H. O'Riordan

360

An Evaluation of Analogs of 1,25-Dihydroxyvitamin D3 on the Inhibition of Proliferation and Induction of Terminal Differentiation in Cultured Human Normal Keratinocytes M. F. Holick, M. Uskokovic, K. Persons, R. Horst, Ε. G. Baggiolini, G. A. Truitt

362

1,25-Dihydroxyvitamin D3 Potentiates the Immunomodulatory Action of Prostaglandin E2 and Histamine by Interacting with cAMP-Dependent Pathways U. A. Liberman, A. Ravid, R. Narinsky, C. Rotem, A. Novogrodsky, R.Koren

364

Effects of 1,25-Dihydroxyvitamin D3 and other Steroid Hormones on Protein Syntheses in Primary Cultures of Adult Rat Hepatocytes K. Taniguchi, K. Itoh, S. Morimoto, T. Shiraishi, T. Onishi

366

The Separation of Keratinocyte Populations Using a Gravity Sedimentation Chamber: A Potential Tool for the Study of l,25(OH)2D3 Action on Cells at Different Stages of Differentiation J. H. Pavlovitch, M. Rizk-Rabin, M. Garabedian

368

Evidence that Osteoinduction by Implants of Demineralized Allogenic Bone Matrix is Diminished in Vitamin D-Deficient Normocalcemic Rats R. T. Turner, J. J. Vandersteenhoven, Ν. H. Bell

370

l,25(OH)2D3 Binds Specifically to Rat Vascular Smooth Muscle Cells and Stimulates their Proliferation in Vitro E. Koh, S. Morimoto, K. Fukuo, R. Morita, S. Kim, T. Onishi, Y. Kumahara

372

Involvement of Intracellular Free Calcium and pH in the Effect of l,25(OH)2D3 on Leukemic Cells S. Shany, E. Barnea, P. Hazav, R. Levy

374

Gene Regulation by l,25(OH) 2 D 3

Modifications in the Interactions of Regulatory Proteins with the Promoter of a Cell Growth Regulated Gene Induced to Differentiate by l,25(OH)2D3 G. S. Stein, J. B. Lian 379

Contents

XXI

Inhibition of 1,25-Dihydroxyvitamin D3 Stimulated BGP mRNA Synthesis in ROS 17/2 Cells after Prolonged Treatment with 1,25-Dihydroxyvitamin D3 Georgia Theofan, Paul A. Price 381 l,25(OH)2Ö Regulates Oncogene Expression in U937 Cells R. Karmali, A. K. Bhalla, S. M. Farrow, Μ. M. Williams, S. Lai, P. M. Lydyard, J. L. H. O'Riordan

383

Regulation of Calbindin-D9K (CaBP9K) Gene Expression by 1,25-Dihydroxycholecalciferol and Calcium in Organ Cultures of Fetal Rat Duodenum A. Brehier, C. Perret, Μ. Thomasset

385

Enhancement of the Genomic Action of 1,25-Dihydroxycholecalciferol by Triiodothyronine is a Regulatory Factor of Intestinal Calcium and Phosphate Transport H. S. Cross, M. Peterlik

387

Influence of Vitamin D3 upon the Expression of PEP-19, a Putative Neuron-Specific Calcium-Binding Protein A. K. Hall, L. Sangameswaran, J. I. Morgan

389

Induction of Osteocalcin Synthesis in Human Osteosarcoma Cells by I,25-Dihydroxyvitamin D and 24,24-Difluoro-l,25-Dihydroxyvitamin D A. Mahonen, A. Pirskanen, M. Haukilahti, P. H. Mäenpää

391

Biological Actions of Vitamin D Metabolites The Role of 24,25 Dihydroxy Vitamin D3 During Development of Skeletal and Non-Skeletal Tissues S. Sömjen, Y. Weisman, S. Harell, Y. Earon, A. Harell, E. Berger, Z. Shimshoni, A. Waisman, A. M. Kaye, I. Binderman

395

The Regulation of Intracellular Ionized Calcium by Calcitriol S. Edelstein, S. Bar, A. Harell, C. Lidor

405

Rapid Action of l,25(OH)2D3 in Liver Cells D. Baran, A. Sorensen, T. Honeyman

416

l,25(OH)2D3 directly Modulates Human Melanocyte Function M. Ranson, S. Posen, R. S. Mason

419

Stimulation of Synthesis of Myoblast Membrane Proteins by 25-HydroxyVitamin D3 T. Bellido, R. Boland

421

XXII

Contents

Stimulation of Rat Duodenal Brush Border Alkaline Phosphatase Activity within 10 Minutes of in Vivo Calcitriol Administration P. A. Lucas, L. Ben Nasr, J.-D. Monet, T. Driieke

423

1,25-Dihydroxy-Vitamin D3 Increases 3H-Thymidine-Uptake and Potentiates the Effect of Epidermal Growth Factor in FRTL-5 Cells C. Lamberg-Allardt, T. Nyman

425

1,25-Dihydroxyvitamin D3 Stimulates 45Ca2+ Uptake by Cultured Vascular Smooth Muscle Cells Derived from Rat Aorta Tsutomu Inoue, Hiroyuki Kawashima

427

l,25(OH)2D3 Activates Protein Kinase C in Rat Colonic Crypts R. Wali, C. Baum, Μ. Sitrin, Τ. Brasitus

429

Free 1,25-Dihydroxyvitamin D3 Levels in Normal and Rachitic Piglets R. Kaune, B. Schröder, J. Harmeyer

431

l,25(OH)2 Vitamin D3 Analogs: Comparison of 1,25(0FO2D3 Receptor Ligand Specificity and Biological Activity in the Chick and Human Promyelocyte (HL-60) Cells E. D. Collins, G. I. Jones, M. Uskokovic, H. P. Koeffler, A. W. Norman

433

24,25-Dihydroxy-Vitamin D3 Binding in Bovine Thyroid C. Lamberg-Allardt, M. Forss

435

Effects of 1,25-Dihydroxy-Vitamin D3 on Phospholipid Metabolism in Cultured Myoblasts L. N. Drittanti, A. R. de Boland

437

Vitamin D Status and Intestinal Absorption of Aluminum in Rats with Normal or Reduced Renal Function Τ. H. Ittel, Η. G. Sieberth

439

Direct Effect of Vitamin D Metabolites on Matrix Vesicle Alkaline Phosphatase Z. Schwartz, B. D. Boyan, D. L. Schlader, V. Ramirez

441

Proliferative Effects of 1,25-Dihydrocholecalciferol (1,25-DHCC), 24,25Dihydroxycholecalciferol (24,25-DHCC), 25-Hydroxycholecalciferol (25-HCC) and Ια-Hydroxycholecalciferol (la-HCC) D. B. Evans, M. Thavarajah, J. A. Kanis

443

The Effect of Pertussis Toxin on PTH- and la,25(OH)2D3-Induced Desensitization in Rat Osteoblasts of the cAMP Response to PTH Μ. P. M. Herrmann-Erlee, J. P. Τ. M. van Leeuwen, M. P. Bos, J. M. van der Meer

445

Synergistic Effects of Vitamin D3 Metabolites W. A. Rambeck, Η. Weiser, W. Meier, Η. Zucker

448

Contents

XXIII

25(OH)D3, but not l,25(OH) 2 D 3 Cures Osteomalacia in Marmoset Monkeys H. Zucker, C. I. Flurer, U. Hennes, W. A. Rambeck

450

Acute Release of Bioactive PTH by Intravenous 1,25 Dihydrocholecalciferol in Chronic Renal Failure Caje Moniz, Mahesh Dixit, Victor Parsons, David Taube, Anthony Care

452

I,25-Dihydroxy-Vitamin D3 Increases Calmodulin Binding to Skeletal Muscle Membrane Proteins L. M. Fernandez, V. Massheimer, A. R. de Boland

454

Hypoparathyroidism in Hereditary Resistance to l,25(OH)2D During Long-Term Treatment with Excessive Doses of Vitamin D3 K. Kruse, Ε. Feldmann, Η. Bartels

456

Effects of l-alpha-OHD3 in Intestinal Radiocalcium Absorption and Serum Bone GLA Protein in Normal Subjects and Osteoporotic Women R. Nuti, V. Turchetti, G. Martini, G. Righi, F. Lore, A. Caniggia

458

Rapid Effects of l,25(OH)2-Vitamin D3 on Calcium Uptake by Cardiac Muscle J. Seiles, R.L. Boland

460

Ια, 25-Dihydroxyvitamin D3 in Rat Skin: Identification, Quantification and Biological Function T. Okano, N. Tsugawa, T. Kobayashi

462

1,25-Dihydroxyvitamin D3 Stimulates Ca-ATPase and Regulates Cellular Calcium in Vascular Smoosth Muscle Cells Hiroyuki Kawashima

464

Calbindins (Biochemistry, Molecular Biology, Biological Actions) Calbindin-DgK (CaBP9K) Gene: Expression, Regulation, Structure and Evolution M. Thomasset, A. Brehier, N. Lomri, J. M. Dupret, N. Gouhier, M. Eb, M. Warembourg, C. Perret

469

1,25-Dihydroxyvitamin D3, its Receptor and the Eukaryotic Genome Phillip P. Minghetti, Leonor Cancela, Hitoshi Ishida, Anthony W. Norman

479

Structure and Evolution of the Chick Calbindin Gene D. Ε. M. Lawson, M. Harding, E. Muir, P. W. Wilson

489

XXIV

Contents

Regulation of Rat Calbindin-D28k Gene Expression S. Christakos, S. Varghese, Y. C. Huang

499

Molecular Cloning and Sequencing of Calbindin-Dcuc cDNA from Mouse Placenta Τ. E. Mifflin, W. R. Pearson, J. Reinhart, D. E. Bruns, Μ. Ε. Bruns

507

High Conservation of Calbindin D28 in Evolution: Implications for its Function Igor Bendik, Alfred W. A. Hahn, Willi Hunziker

509

Functional Analysis of the Promoter Region of the Gene Encoding Chicken Calbindin-D28K S. Ferrari, R. Battini, E. Drusiani, M. Fregni

512

Cellular Localization of Brain Calbindin-Ö28k A. Heick, N. Aronin, M. Di Figlia, S. Christakos

514

Vitamin D Dependence of Calbindin D 9K and Calbindin D 28K Synthesis in Various Rat Tissues M. Thomasset, A. Tenenhouse

516

Immunohistochemical Demonstration of Vitamin D-Dependent Calcium-Binding Protein, Calbindin-D 28K (CaBP28K) in the Spinal Cord Motoneurons of Teleost Fish J. P. Denizot, A. Brehier, B. O. Bratton, M. Thomasset 518 In Situ Hybridization of the Rat Calbindin D-28 J. M. Sequier, W. Hunziker, H. Weiser, J. G. Richards

520

Spectroscopic Studies on Chick Intestinal Calbindin-D28K Suggest the Existence of 4-6 Functional Ca2+ Binding Sites V. Leathers, T. Drakenberg, C. Johansson, S. Linse, S. Forsen, A. W. Norman

523

Evolution of the "EF-Hand" CaBP Family: Exon Shuffling and Intron Insertion C. Perret, Ν. Lomri, Μ. Thomasset

525

Effects of Altered Thyroid States and Undernutrition on Calbindin-D28K (Calcium-Binding Protein) in the Hippocampal Formation of the Developing Rat A. Rami, N. Lomri, A. Brehier, M. Thomasset, A. Rabie

527

Expression of Calbindin D Decreases with Age in Intestine and Kidney H. J. Armbrecht, Μ. Boltz, R. Strong, Μ. Ε. H. Bruns, S. Christakos

529

Evidence for Tissue-Specific Regulation of Calbindin 28K Gene Expression in the Chick by 1,25-Dihydroxyvitamin D3 T. L. Clemens, S. A. McGlade, K. P. Garrett, N. Horiuchi, G. N. Hendy

531

Contents

XXV

Evidence for the Presence of two Vitamin D-Dependent Calcium Binding Proteins in Brain C. Gabrielides, A. Heick, S. Christakos

533

The Human Calbindin D28 Gene Igor Bendik, Alfred W. A. Hahn, Willi Hunziker

535

Effect of Vitamin D Deficiency and 1,25-Dihydroxycholecalciferol Treatment on Epidermal Calcium Binding Protein (ECaBP) RNA Activity M. Rizk-Rabin, J. Pavlovitch

537

Complete Amino Acid Sequences of Mouse Calbindin-D9K Isoforms Determined by Tandem Mass Spectrometry: Protein Modification by Internal Insertion of a Single Amino Acid D. E. Bruns, Μ. Ε. Bruns, J. R. Yates, D. F. Hunt

539

Developmental Pattern and Vitamin D-Dependency of the Calbindins CaBP 9KDa and CaBP 28KDa in the Ameloblasts of Rodent Teeth: An Immunocytochemical Study A. Berdal, Ν. Baimain, A. Brehier, Μ. Thomasset, D. Hotton, P. Cuisinier-Gleizes, H. Mathieu

541

Calbindin (CaBP28kDa) Localization in the Peripheral Vestibular System of Various Vertebrates and Comparison with its Distribution During Development of the Mouse and Human C. J. Dechesne, M. Thomasset, A. Sans

543

Nuclear Magnetic Resonance and Terbium Fluorescence Studies on the Vitamin D-Dependent 28-Kilodalton Chick Intestinal Calcium-Binding Protein : Evidence for Calcium-Dependent Conformational Change M. D. Gross, B. Sykes, R. Kumar

545

Intestinal and Renal Ca and Ρ Transport

Transcaltachia, Vesicular Calcium Transport, and Microtubule-Associated Calbindin-D28K Ilka Nemere, Anthony W. Norman

549

Vitamin D: Effects on Intestinal Calcium Absorption and Peripheral Nerve Function R. H. Wasserman, C. S. Fullmer, C. Hu, Q. Cai, D. N. Tapper 558 ATP-Dependent Ca 2+ Pumps in Endoplasmic Reticulum and Plasma Membranes are not Affected by 9kDa Calbindin-D and Vit. D-Deficiency J. A. H. Timmermans, E. J. J. M. van Corven, C. H. van Os

565

XXVI

Contents

Effects of Vitamin D-Deficiency on Different Activities of the Ca 2+ -Pump in Rat Intestinal Basolateral Membranes JulianR.F.Walters

567

ATP-Driven Ca2+ Pumps in Duodenal Plasma Membranes and Endoplasmic Reticulum from Piglets with Inherited Rickets R. Kaune, C. H. van Os

569

Effect of l,25(OH)2D3 on Phosphorus Absorption in Sheep Κ. M. Schneider, D. D. Leaver

571

Reduced Calcium Absorption in X-Linked Hypophosphatemic Rickets 0 . Mehls, Ε. Wilhelm, Ε. Werner, Τ. Floren, F. Manz, Β. Tönshoff.

573

Phosphate Transport in the Intestine of the Rat During Early Development: Role of Vitamin D J. K. Yeh, J. F. Aloia

575

The 105 kD Calmodulin (CAM) Binding Protein from Intestinal Brush Border Membrane (BBM) is a Calcium Transport Protein D. D. Bikle, S. Munson

577

Bone and Action of Vitamin D Metabolites The Role of 1,25-Dihydroxyvitamin D3 in the Generation and Regulation of Osteoclasts T. J. Chambers

581

Vitamin D and Osteoclastogenesis D. R. Clohisy, Z. Bar-Shavit, H. C. Blair, J. D. Konsek, A. J. Kahn, S. L. Teitelbaum

591

Dentin Formation and Plasma Ca Levels in PTX Rats given l,25(OH)2D3 S. Matsumoto, M. Yamaguchi

596

Elevated 1,25-Dihydroxyvitamin D3 and Intestinal Calbindin-D9K Levels in the Osteopetrotic Toothless Rat M. F. Seifert, R. W. Gray, Μ. E. Bruns

598

Influence of 24-Fluorinated Analogues of l,25(OH)2D3 on Bone Matrix Formation and Bone Resorption in Vitro 1. R. Dickson

600

Modulation of the Action of l,25(OH)2Ü3 on the Osteocalcin Production of Human Osteoblast-Like Cells by Agents Affecting Adenylate Cyclase Activity D. B. Evans, R. G. G. Russell, B. L. Brown, P. R. M. Dobson

602

Contents

XXVII

Increased Bone Minerals in Vitamin D-Replete Rabbit by the Massive Administration of 24R,25(OH) 2 D 3 T. Nakamura, T. Kurokawa, H. Orimo

604

Studies on the Interactions between Retinoic Acid and l,25(OH)2Ö3 on Human Bone-Derived Osteoblast-Like Cells D. B. Evans, R. A. D. Bunning, R. G. G. Russell

606

Immunosuppressive Agent Cyclosporin A and Various Cell Growth Factors Affect Cloned Osteoblastic Cell Line MC3T3-E1 Cells K. Okano, M. Yajima, Y. Yamada, S. Fujibayashi, S. Kou, K. Sasagawa, S. Suzuki, S. Naito, K. Ohira, C. Nawa, N. Sekita, K. Someya 608 23(S)25(R)-l,25(OH)2D3-26,23-Lactone Stimulates Bone Formation in Vivo H. Tanaka, Y. Seino, M. Shima, K. Yamaoka, H. Yoshikawa, K. Takaoka, S. Ishizuka, A. W. Norman, H. Yabuuchi 610 Side Chain Modifications Increase the Potency of 1,25-Dihydroxycholecalciferol (Calcitriol) on Human Bone-Derived Osteoblast-Like Cells D. B. Evans, M. Thavarajah, R. A. D. Bunning, M. R. Uskokovic, J. A. Kanis

612

Activity of Ornithine Decarboxylase in Hard Tissue of Vitamin D-Deficient and -Replete Rats: Effect of l,25(OH)2D3, Uremia and Aluminum Intoxication Τ. H. Ittel, L. Walter, H. G. Sieberth

614

Production of Bone-Resorbing and Osteoclast-Inducing Factors by Osteoblasts in Organ Culture and Modulation by l,25(OH)2D3 and other Agonists I. R. Dickson, B. A. A. Scheven

616

Circulating Osteocalcin in Magnesium Deficiency: Response to l,25(OH) 2 D T. Carpenter, M. Mitnick, P. Johnson, C. Gundberg

618

l,25(OH)2D3 Mediates the Effect of Parathyroid Hormone on New Bone Formation in the Rat U. G. Lempert, H. W. Minne, S. H. Scharia, Η. Schmidt-Gayk, R. Ziegler

620

Circulating Osteocalcin and 1,25-Dihydroxyvitamin D Concentrations and Urinary y-Carboxyglutamic Acid in Healthy Subjects and in Patients with Metabolic Bone Disease A. Pirskanen, Μ. T. Parviainen, M. Haukilahti, P. H. Mäenpää, E.M.Alhava

622

XXVIII

Contents

Regulation of Fibronectin and Collagen Synthesis by 1,25-Dihydroxyvitamin D3 R. T. Franceschi, P. R. Romano, K.-Y. Park, J. Young

624

1,25-Dihydroxyvitamin D3 Stimulates in Vitro Growth of Avian and Mammalian Cartilage Warner M. Burch

626

Nutritional Aspects of Vitamin D Minimal Intravenous Vitamin D Requirement During Parenteral Nutrition in Infancy W. Koo, S. Krug-Wispe, P. Succop, R. Tsang

631

Serum 25(OH)D 3 and l,25(OH) 2 D 3 Levels in Wild and Laboratory-Bred Wood Mice and Bank Voles R. F. Shore, Μ. E. Hayes, R. J. Balment, Ε. B. Mawer

633

Effect of Acute Ethanol Administration on the Circulating Parameters of Bone Mineral Metabolism C. Movsowitz, S. Thomas, S. Epstein, P. Jowell, F. Ismail

635

Plasma 25-OHvitamin D and PTH in the Detection of Osteomalacia in Asian Outpatients J. B. Eastwood, J. A. Nisbet, K. W. Colston, L. Ang, A. M. Flanagan, T. J. Chambers, J. D. Maxwell

637

The Vitamin D Content of Finnish Hospital Diets Μ. T. Parviainen, J. Kumpulainen, M. Sinisalo, K. Nyyssönen

640

Zinc, Vitamin D and Bone Mass in the Elderly G. Toss, L. Larsson, L. Wahl

642

The Importance of Vitamin C for Hydroxylation of Vitamin D3 to la,25(OH) 2 D 3 and of 24R,25(OH)2D3 to a More Active Metabolite H. Weiser, Μ. Schlachter, Η. Bachmann

644

Plasma Magnesium in Pigs with Pseudo Vitamin D Deficient Rickets; Type 1. J. Harmeyer, U. Duchatz, Κ. M. Schneider, D. D. Leaver

654

Sunlight Degradation of Vitamin D 3 A. R. Webb, B. Decosta, M. F. Holick

656

Fatty Acid and Calcium Absorption in Rats. Role of Vitamin D D. A. McCredie, V. Groves, S. W. Cosby

658

Contents

XXIX

Clinical Studies on the Mechanism of Acquired Vitamin D Deficiency M. R. Clements, M. Davies, G. A. Lumb, Μ. E. Hayes, C. Hickey, Ε. B. Mawer, P. H. Adams

660

How is Plasma l,25(OH)2Ö3 Concentration Regulated by Dietary Calcium? D. D. Leaver, U. Trechsel, H. Fleisch

662

Studies of Rickets and its Prevention in Beijing, China X. C. Chen, R. W. Li, H. C. Yen, Q. M. Xu, D. S. Liu

664

Vitamin D Binding Proteins DBP and AIDS: Fact or Fiction? H. van Baelen, I. Surmont, R. Bouillon

669

Plasma Vitamin D Binding Protein: Plasma Scavenger Function and Origin of Cell-Surface DBP J. G. Haddad, K. D. Harper, J. McLeod, J. E. Nestler, M. Guoth

674

Partial Structure of the Gene for Human Vitamin D-Binding Protein W. F. Witke, F. Yang, Β. H. Bowman, A. Dugaiczyk

680

A New Interaction between Gc (Vitamin D-Binding Protein) and Unsaturated Fatty Acids R. M. Galbraith, G. M. P. Galbraith, Μ. H. Williams

685

Two 25-Hydroxycholecalciferol-Binding Proteins in Carp Serum K. Allewaert, H. van Baelen, R. Bouillon

692

HPLC Analysis of the Cyanogen Bromide Digest of Human Serum Vitamin D Binding Protein Photoaffinity Labelled with a 25-Hydroxyvitamin D3 Analog R. Ray, M. F. Holick

694

Effect of Calcium Ions upon Interactions between Gc (Vitamin D-Binding) Protein and Cibacron Blue C. Suck, Μ. H. Williams, D. L. Emerson, G. R. Lattanze, P. Arnaud, R. M. Galbraith 696 Partial Purification and Properties of Bat Vitamin D Binding Protein Meropi Cavaleros, John M. Pettifor, F. Patrick Ross

698

XXX

Contents

Assay Methodology: Vitamin D and Metabolites Measurement of Free Vitamin D Metabolite Levels: Biological and Clinical Significance D. D. Bikle, E. Gee

703

The Assay of Circulating l,25(OH)2D using Non-End-Capped Cis Silica (C18-OH): Performance and Validation B. W. Hollis, T. Kilbo

710

Simplified Assays for the Determination of 25-OHD, 24,25(OH)2D and l,25(OH)2D T. A. Reinhardt, R. L. Horst

720

Stable Isotope Dilution Mass Fragmentography for the Measurement of Metabolites of Vitamins D2 and D3 in Human Plasma Ruth D. Coldwell, D. J. H. Trafford, Η. L. J. Makin, M. J. Varley, D.N. Kirk

727

Well Established Physiological Functions are a Prequisite for Introducing Biochemical Parameters M. Schlachter, Η. Weiser, Η. Bachmann

728

The Quantification of Serum 25-Hydroxyvitamin D Using a Competitive Protein-Binding Assay after Preliminary Purification Μ. T. Parviainen, M. Haukilahti, K. E. Savolainen, E. Kumpusalo

730

Changes in Plasma Levels of Vitamin D2, D3 and Their Metabolites after Oral Administration of the Vitamins to Vitamin D-Deficient Rats S. Masuda, T. Okano, T. Kobayashi

732

Age-Related Changes in Immunoradiometric PTH and 25-Hydroxyvitamin D C. W. Lo, M. Alpert, E. Flynn, P. Burdette-Miller, D. Schoenfeld, R. Neer

734

Relevance of Long-Term Variation in Vitamin D Metabolite Measurements. Implications for Longitudinal Study Design. O. R. Leeuwenkamp, R. Barto, W. J. F. van der Vijgh, P. J. M. Elders, F. C. van Ginkel, P. Lips, J. C. Netelenbos

735

Other Basic Science Topics Prospects for the Modelling of Vitamin D Activity in Man J. A. Kanis, Μ. K. Drezner, D. B. Evans, R. L. Horst, Η. Η. Malluche, A. W. Norman, Μ. Thavarajah, Μ. R. Uskokovic

739

Contents

XXXI

Isolated Rat Proximal Tubule Cell (PTC) Cultures are cAMP Responsive to Parathyroid Hormone (PTH), Calcitonin (CT), and Prostaglandins (PGE) but not to Vasopressin (VP) L. G. Rao, F. Baksh

749

Lipid Composition of Basolateral Membranes from Rat Enterocytes: Effects of Vitamin D Deficiency E. J. J. M. van Corven, D. Α. Η. M. van Groningen, W. J. de Grip, C.H.vanO s

751

Possible Origin of Extremely Large Amounts of Vitamin D3 in Some Kinds of Fish Liver T. Kobayashi, A. Takeuchi, T. Okano

753

Hepatotoxic Effects of Aluminium: Implications for Vitamin D Metabolism G. L. Klein, T. C. Lee, M. B. Heyman, W. R. Bidlack, R. C. Brown, D. K. Rassin, A. C. Alfrey 755 Studies on Kinetics of Previtamin D Formation Reaction, Effects of Irradiation Intensity at 296nm on Previtamin D Formation Zhiren Lu, Yafang Pang, Chengfa Zheng

757

Vitamin D Deficiency and Cardiac Function in Isolated Chick Hearts E. Hochhauser, J. Barak, T. Kushnir, G. Navon, M. S. Meyer, S. Edelstein, Β. A. Vidne

760

Vitamin D Effects on Ca-Stimulated ATPase Activity and Protein Composition of the Rat's Skeletal Muscle Miofibrills V. M. Kodentsova, V. V. Risnic, A. A. Sokolnikov, I. N. Sergeev, V. B. Spirichev

762

The Spontaneously Hypertensive Rat (SHR): A Model of Abnormal Vitamin D Metabolism P. A. Lucas, J. Merke, G. Cournot, M. Thomasset, R. C. Brown, D. A. McCarron, E. Ritz, Τ. Drüeke

763

Vitamin D3- and Stigmasterol-Induced Calmodulin Synthesis in Phaseolus Vulgaris Roots is Mediated by Ca +2 and Plant Growth Hormone-Like Mitogenic Stimulation M. A. Vega, E. C. Santamaria, R. L. Boland

765

AVery Rapid Receptor Mediated Action of 1,25-Dihydroxyvitamin D3: Increase of Intracellular Cyclic GMP in Human Skin Fibroblasts J. Barsony, S. J. Marx

767

Effects of Vitamin D3 Metabolites on Cytosolic Free Calcium in Confluent Mouse Osteoblasts: A Possible Involvement of Protein Kinase C Michele Lieberherr 769

XXXII

Contents

Renal Osteodystrophy

Plasma Kinetics of Intravenous Calcitriol in Normal and Dialysed Subjects and Acute Effect on Serum nPTH Levels. I. B. Salusky, W. G. Goodman, R. Horst, G. V. Segre, K. C. Norris, J. W. Coburn

781

Bioavailability of Calcitriol after Oral, Intravenous and Intraperitioneal Doses in Dialysis Patients I. B. Salusky, W. G. Goodman, K. C. Norris, R. Horst, R. N. Fine, J. W. Coburn

783

Intravenous Administration of Ια-Hydroxycholecalciferol in Haemodialysed Patients: Dose-Dependent Increase in Circulating 1,25-Dihydroxycholecalciferol S. E. Papapoulos, H. v. d. Berg, M. Frölich, R. M. Valentijn

785

Ten Years Experience in the Prevention of Renal Osteodystrophy by Vitamin D-Prophylaxis K. Lindenau, F. Kokot, Κ. Abendroth, U. Futh, I. Grossmann, C. Schmidt, C. Rehse, P. T. Fröhling

787

Successful Treatment of Renal Osteodystrophy with Keto Acids (KA) and Vitamin D P. T. Fröhling, Κ. Abendroth, F. Kokot, Κ. Vetter, C. Rehse, K. Lindenau

789

Effect of l,25(OH)2D3 and 24R,25(OH)2D3 on the Accumulation of Aluminum in Bone in Rats with Renal Failure Τ. H. Ittel, Η. G. Plückelmann, F. Hofstädter, Η. G. Sieberth

791

The Influence of Vitamins D2 and D3 on Intestinal Calcium Absorption in Patients with Renal Insufficiency and in Dialysis Patients R. Fünfstück, G. Stein, K. Abendroth, F. Kokot, K. Günther, U. Tietz, G.Wessel

793

la(OH)D3 i.v. Suppression of PTH Secretion in Hemodialysis Patients L. Brandl, Ε. Tvedegaard, Η. Daugaard, Τ. Storm, Κ. Olgaard

795

Clinical and Biochemical Effects of 1,25-Dihydroxyvitamin D3 Administration in Normal and Renal Impaired Dogs D. A. Dzanis, F. A. Kallfelz

797

Hyper- and Normo- Calcemic Dogs with Chronic Renal Failure: Relations of Serum PTH and Calcitriol to PTG Ca++ Set-Point L. A. Nagode, C. L. Steinmeyer, D. J. Chew, B. D. Hansen

799

Contents

XXXIII

Dietary Intake and Status of Vitamin D and Calcium in Patients with Chronic Renal Failure (CRF) Treated by Haemodialysis (HD) at home F. T. Pender, R. J. Winney, N. R. Belton

801

Osteocalcin (OC) in Adolescents with Advanced Renal Failure J. Merke, A. Zlotkowski, G. Müller-Wiefel, Ε. Ritz, Ο. Mehls

803

Osteoporosis Long-Term Calcitriol Treatment in Post-Menopausal Osteoporosis: Follow-Up of two Hundred Patients A. Caniggia, R. Nuti, F. Lore, G. Martini, G. Righi, V. Turchetti

807

Osteoporosis and Vitamin D Metabolism: The State of our Knowledge as of 1988 B. L. Riggs

817

The Rationale for Calcitriol Therapy in Osteoporosis Β. E. C. Nordin, A. G. Need, H. A. Morris, M. Horowitz

826

Calcitriol Therapy in the Management of Osteoporosis J. C. Gallagher, D. Goldgar, J. O'Neill

836

Alfacalcidol in Prednisone Treatment: Effect on Bone Mineral Content in Lumbar Spine and Femur Ole P. Schaadt, Hans H. Bohr

838

Decrease of Plasma 1,25-Dihydroxyvitamin D3 (l,25(OH)2D3) and Loss of Spinal Bone Mass in Women During Treatment with GnRH Agonists S. H. Scharia, Η. W. Minne, A. Schaible, S. Waibel, U. G. Lempert, H. Schmidt-Gayk, R. Ziegler

840

Effect of 26,27-Hexafluoro-l,25-Dihydroxyvitamin D 3 (F6-l,25(OH)2D3) on Osteoporosis Induced by Immobilization Combined with Ovariectomy in the Rats S. Higuchi, M. Harada, T. Takamura, S. Otomo, H. Aihara, H. Okumura, T. Yamamuro, N. Ikekawa, T. Kobayashi

842

Some Biochemical Markers in Distal Fore-Arm and Hip Fractures R. Nilsson, O. Löfman, G. Toss, J. Gillquist, L. Larsson

844

The Short Term Effects of Alfacalcidol in Elderly Osteoporotic Women R. M. Francis, C. J. Robinson, C. E. Davison, A. Rodgers

846

Calcitonin Secretion in Osteoporosis of Turner Syndrome G. Saggese, S. Bertelloni, G. I. Baroncelli, B. Buggiani, A. Papini

848

XXXIV

Contents

Determinants of Calcium Malabsorption in Postmenopausal Osteoporosis H. A. Morris, P. D. O'Loughlin, A. G. Need, M. Horowitz, Β. E. C. Nordin 850 Sarcoidosis Regulated Expression of the Sarcoid Macrophage 25-Hydroxyvitamin D-l-Hydroxylation Reaction J. S. Adams, Μ. M. Diz, M. A. Gacad

855

Accentuated Transpleural Gradient of "Total" and "Free" 1,25-Dihydroxyvitamin D in Patients with Tuberculous Pleuritis P. Ε Barnes, R. L. Modlin, D. B. Endres, D. D. Bikle, J. S. Adams

857

The Effects of Ketoconazole and Metyrapone on Activated Alveolar Macrophages A. M. Pryke, R. S. Mason, C. Duggan

859

I,25(OH)2D3 Production by Lung Τ Lymphocytes from Tuberculosis Patients J. Cadranel, M. Garabedian, H. Guillozo

861

Cancer and Vitamin D Treatment of Myelodysplastic Syndrome and AML with la Hydroxyvitamin D3 T. Taoka, Y. Kubota, T. Tanaka, S. Irino

865

Nude Mouse Model (CAC-8) of Humoral Hypercalcemia of Malignancy (HHM) with Increased Serum Levels of 1,25-Dihydroxycholecalciferol (l,25(OH)2D): In Vivo and in Vitro Studies T. J. Rosol, C. C. Capen, L. J. Deftos, R. L. Horst

867

Effects of Treatment with the Bisphosphonate APD on 1,25-Dihydroxyvitamin D in Patients with Malignant Hypercalcaemia Ε. B. Mawer, P. E. Still, A. R. Morton, D. C. Anderson

869

Effect of Ια-Hydroxyvitamin D3 on Growth of Nitrosomethylurea-Induced Rat Mammary Tumors K. W. Colston, P. Shah, R. C. Coombes 871 Diabetes and Vitamin D Interaction between Vitamin D, Insulin and Diabetes Mellitus R. Bouillon, J. Verhaeghe, B. L. Nyomba, W. J. Visser, H. van Baelen, M. Thomasset

875

Contents

XXXV

Endogenous Diabetes Decreases the Number of 1,25-Dihydroxyvitamin D3 Receptors in both Intestine and Kidney H. Ishida, N. S. Cunningham, H. L. Henry, A. W. Norman

885

Plasma Concentrations of Vitamin D Metabolites in Newly Diagnosed Young Insulin-Dependent Diabetics before and after Insulin Treatment L. Aksnes, O. Rodland, S. Sjöblad, Η. Tornquvist, D. Aarskog

887

The Effect of Hypocalcemia and Vitamin D-Deficiency on Glucose Utilization in Piglets C. Schlumbohm, J. Harmeyer, A. Dwenger

889

Vitamin D-Induced Increase in Calcium Content in Secretory Granules of Β Cells Plays a Role in Recovery of Insulin Secretion in Vitamin D-Deficient Rats K. Ohzono, Y. Seino, Y. Tanaka, H. Yabuuchi, H. Fujita

891

Increase in Insulin Response to Glucagon Following UV-B Irradiation in Healthy Adults C. Colas, M. Garabedian, A. Fontbonne, H. Guillozo, N. Desplanque, G. Slama, G. Tchobroutsky

893

Gerontology and Vitamin D

Serum l,25(OH)2D3 in the Elderly and Osteoporotic Subjects from Southern Italy. A Preliminary Report F. P. Cantatore, M. Carrozzo, D. M. Magli, M. D'Amore, V. Pipitone

897

Effect of Age on the Rat Intestinal l,25(OH)2D3 Receptor S. Takamoto, C. T. Liang, Y. Seino, B. Sacktor

899

Effect of Age and l,25(OH)2D3 on Calcium Uptake in Rat Duodenum Cells C. T. Liang, J. Barnes, S. Takamoto, B. Sacktor

901

Low Serum Concentrations of 25-Hydroxyvitamin D, Selenium and Osteocalcin in Finnish Elderly Men P. H. Mäenpää, A. Pirhonen, A. Pirskanen, A. Nissinen, S.-L. Kivelä

903

Response of the Serum Level of l,25(OH)2D to Restriction of Dietary Phosphorus: Effect of Advanced Age A. A. Portale, Β. P. Halloran, Ε. T. Lonergan, R. C. Morris, Jr.

905

Intestinal End-Organ Resistance to 1,25-Dihydroxyvitamin D Stimulation of Calcium Absorption in the Senescent Rat R. J. Wood, C. L. Theall, J. H. Contois, I. H. Rosenberg

907

XXXVI

Contents

Pregnancy / Neonatology Serum Vitamin D, 25-OHD, and l,25(OH)2D, Bone Mineral Content (BMC) and Mineral Homeostasis in Premature Infants Fed High Mineral Premature Formula L. Hillman

911

Normal Plasma l,25(OH)2Ö3 in a Breast Fed Premature Infant with Early Hypophosphatemia Rickets B. R. Thomas, S. Groh Wargo, B. W. Hollis, G. S. Reddy

913

Changes in 1,25-Dihydroxyvitamin D3 Receptor (1,25 DR) Content of Rat .Mammary Gland During Pregnancy and Lactation K. W. Colston, U. Berger, P. Wilson, Η. M. Earl, R. C. Coombes

915

Concentration of Vitamin D Metabolites in Amniotic Fluid and Serum During Pregnancy S. Issa, R. Mallmann, W. Burmeister

917

l,25(OH)2D3 and Ostoecalcin Concentrations in Fetuses and Neonates from Rats Fed two Different Calcium-Phosphate Diets J. Verhaeghe, R. Bouillon

919

Intact PTH and Vitamin D Metabolites in Early Neonatal Hypocalcemia G. Saggese, G. I. Baroncelli, S. Bertelloni

921

Other Clinical Topics Vitamin D and the Skin: Site of Synthesis, Target Tissue, and New Therapeutic Approach for Psoriasis M.F.Holick

925

Inborn Errors in Vitamin D Metabolism - Their Contribution to the Understanding of Vitamin D· Metabolism U. A. Liberman

935

Vitamin D and Human Antituberculosis Immunity A. J. Crowle, E. J. Ross, Μ. H. May

948

Successful Treatment of Psoriasis with Topical Application of the Active Vitamin D3 Analog, la,24-Dihydroxycholecalciferol (TV-02) H. Tagami, T. Kato, T. Terui, T. Tadaki

958

Evidence that Alteration of the Vitamin D-Endocrine System in Obesity Results from Vitamin D Deficiency Ν. H. Bell, Y. Liel, B. W. Hollis, S. Epstein

968

Abnormal l,25(OH)2D3 Receptor Relation of Parathyroid Gland in Lead Intoxication A. Szabo, J. Merke, U. Hügel, G. Mall, E. Ritz

976

Contents

XXXVII

Hypocalcemia and Decreased l,25(OH)2Ö Blood Levels in Septic Patients M. S. Meyer, K. Heller, S. Shibolet, S. Edelstein 977 Serum Vitamin D and Calcium Metabolism in Tuberculosis Patients Resident in two Tropical Environments P. D. O. Davis, H. A. Church, R. C. Brown, A. Charumilind, S. Bejrachandra, S. Bovornkitti

979

Abnormal Vitamin D Metabolism in Primary Aldosteronism and Experimental Mineralocorticoid Excess L. Resnick, J. Gertner, J. Laragh

981

Vitamin D Metabolites and Intact PTH in Spasmophilia G. Saggese, S. Bertelloni, G. I. Baroncelli, B. Buggiani, M. Gualtieri

983

Vitamin D Status in Patients with Cystic Fibrosis: Seasonal Variations and the Importance of Vitamin D Supplementation: A. Zittermann, S. Issa, J. A. Falch, W. Burmeister

985

An Epidemiological Association between Vitamin D Deficiency and Tuberculosis P. D. O. Davies

987

Conversion of la-OHDß into l,25(OH)2D3 in Normal Subjects, Cirrhotic Patients and Postmenopausal Osteoporotic Women F. Lore', G. di Cairano, M. Nobili, F. D'Ubaldo, G. Manasse, G. Miracapillo, M. Romei, R. Nuti, A. Caniggia

989

The Role of 1,25-Dihydroxyvitamin D in Salt Sensitive Essential Hypertension: Effects of Calcium Channel Blockade L. Resnick, J. Nicholson, J. Laragh

991

Vitamin D3 Induced Glycolic Aciduria in an Experimental Stone Model A. Halabe, N. L. M. Wong, L. Hägen, Η. Hughes, R. A. L. Sutton

993

Vitamin D Metabolism in Benign Transient Hyperphosphatasaemia P. M. Crofton, N. R. Belton

995

Serum Calcium can Modulate the Production of 1,25-Dihydroxyvitamin D Independently of PTH in Man S. E. Papapoulos, O. L. M. Bijvoet, M. Frölich, Η. v. d. Berg 997 Age-Dependent Changes of Vitamin D Status and l,25(OH)2D3 Receptor Expression in Spontaneously Hypertensive (SH) Rats J. Merke, P. Lucas, A. Szabo, T. Drüeke, R. Bouillon, E. Ritz

999

Behavioral Changes in Chronically D-Hypervitaminotic Animals P. A. de Viragh, D. Wolfer, H. P. Lipp, Μ. R. Celio

1001

XXXVIII

Contents

1,25-Dihydroxyvitamin D3 is an Effective Therapeutic Agent for the Treatment of Psoriasis Vulgaris and Erythroderma Psoriasis M. F. Holick, E. L. Smith, S. Pincus

1007

Vitamin D, Calcium and Hypertension R. C. Morris, jr., T. W. Kurtz

1009

Dietary Vitamin D Level and Mineral (P, Ca) Absorption in Pigs Fed Phytic Ρ Diets A. Pointiiiart, N. Fontaine, M. Eb, M. Thomasset

1013

Effect of Active Vitamin D3 on Enterocytes and Fatty Acid Binding Substance in Microsomal Fraction of Intestinal Mucosa of Rats M. Inada, S. Watanabe, K. Wakatsuki, H. Kudo, M. Murakami

1015

Vitamin D Therapy in Renal Failure - Challenges and Problems for the Future E. Ritz, J. Merke, A. Szabo

1017

Synthesis of 24R,25-Dihydroxyvitamin D3 from Vitamin D2 and Study on Inclusion Complexes of Vitamin D Derivatives with /?-Cyclodextrin N. A. Bogoslovsky, Β. I. Kurganov, N. G. Samochvalova, T. A. Isaeva, N. P. Sugrobova, V. M. Gurevich, I. E. Valashek, G. I. Samochvalov 1021

Author Index

1025

Key Word Index

1033

Cell Line Index

1067

Vitamin D Workshop Zoo

1069

Summary of the Dates and Locations of the Seven Vitamin D Workshops

1071

Vitamin D Workshop Logo

1072

TRIBUTE TO Ο. NEAL MILLER Neal Miller was indeed a friend of vitamin D and a friend and stalwart supporter of the Vitamin D Workshop. He was a warm and friendly person with a subtle ana wiy sense of humor. Publicly, he tended to be rather quiet and generally worked behind the scenes, particularly as it had to do with the science and clinical application of vitamin D and the role of the Pharmaceutical giant, Hoffmann-La Roche. He tended to take a fatherly view of the scurrying of the various "leaders" in the vitamin D fields. He would often subtly give advice, particularly about the best way to present a point, and he would do this in a clever congenial way. If he were to be here to listen to this now, I suspect that he would say, "Jack, what have you done that you are on the program to give an obituary?" Who was Neal Miller and what was his background? He was a native of Missouri, where he was born in 1918. After obtaining his bachelors degree from the University of Missouri, he obtained his Ph.D. in biochemistiy from Harvard. After short periods in Michigan at Wayne State and in Texas, he moved to Tulane University, where he was Professor of Biochemistry from 1953 to 1968. He then joined Hoffmann-La Roche in Nutley, Ν J., where he became Director of Nutritional Biochemistiy in 1976. His status changed to consultant in 1983. Neal had a long list of publications dealing with nutrition, enzymology, and vitamin research. He was member of many societies, including American Society of Biological Chemists, the American Institute of Nutrition, the American Society for Clinical Nutrition, the American Society of Pharmacology and Experimental Therapeutics, and many more. When did Neal become interested in vitamin D and an active supporter of the Vitamin D Workshop? Record of such events are not kept very well; however, the Vitamin D Workshop probablv joined the "big time" in Asilomar in January, 1977 when the attendance grew from the 220 present in Wiesbaden (2nd Workshop in Oct. 1974) to 335 in Pacific Grove, California. It was at this time that Neal Miller and Roche-Nutley put their support behind the Workshop. It should be noted that Neal was a major force behind the strong financial support provided by Hoffmann-La Roche, Nutley. As a skilled pharmacologist, it was Neal who helped engineer the rather simple but straight-forward multicenter clinical trials with "1,25," or calcitnol as we should call it, that resulted in its FDA approval for clinical use within a "record" time from the very first administration to a patient with renal failure in 1971. Neal's death from cancer was untimely. He is survived by his wife, Gloria, and two children. He was an able biochemist, a skilled clinical pharmacologist, and an excellent administrator in the pharmaceutical industry. He was also a fine person and truly a "nice" man. We hold his memory highly, the Vitamin D Workshop misses Neal Miller; we all miss Neal Miller. In his memory, I would ask that we have a moment of silence.

Jack W. Coburn, M.D. April 26,1988

Chemistry of Vitamin D Seco-Steroids

DEVELOPMENT OF SYNTHETIC METHODS FOR FLUORINATED VITAMIN D ANALOGS Yoshiro Kobayashi* and Takeo Taguchi, Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan.

Introduction As a general consideration of fluorine substitution in biologically active compounds, the following characteristic properties of fluorine and fluorinated compounds are important with respect to their biological responses (1,2). 1) The fluorine atom is the second smallest substituent as measured by the covalent radius and the bond length of a carbon-fluorine bond is most similar to that of a carbon-hydrogen bond (Table 1). The fluorine atom mimics a hydrogen atom in biologically active compounds with respect to steric requirements at the enzyme receptor site. 2) Since the bond strength of a carbon-fluorine bond exceeds that of a carbon-hydrogen bond (Table 2), introduction of fluorine into an organic molecule causes increased oxidative and thermal stability. In this respect a carbon-fluorine bond is usually biologically more stable. 3) Owing to the high electronegativity (Table 1), the fluorine atom alters electronic effects, which resuls in changing the properties of the neighboring functional group or the conformation of the molecule through the formation of a hydrogen bond or dipole interaction. 4) Fluorine s presence increases the lipophilicity of the molecule which results in enhancing the rate of absorption and transportation of fluorinated biologically active compound in vivo. 5) In spite of the great strength of the carbon-fluorine bond, under certain circumstances fluorine acts as a leaving group as is consistent with its native character as a halogen atom. These are the basic features of fluorinated compounds which should be considered in fluorine substitution

Table 1. Element X

Electronegativity and Steric Size of Some Elements Electronegativity (Pauling)

Bond Length(r ) (CH^X, A)

Van der Waal s Radii(r 2 , A)

r +r (A)

Η

2.1

1.09

1.20

2.29

F

4.0

1.39

1.35

2.74

CI

3.0

1.77

1.80

3.57

0(0H)

3.5

1.43

1.40

2.83

Table 2. Bond C-H C-F

Comparative Bond Energies kcal mol 98 108-116

1

(kJ mol _ 1 ) (410) (451-485)

C-Cl

81

(339)

C-Br

68

(284)

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · New York - Printed in Germany

4 in biologically active compounds. On the basis of the characteristic properties of fluorinated compounds and the metabolism and mechanism of functions of vitamin D fluorinated analogs were designed, synthesized and their biological activities were studied in collaboration with Prof. DeLuca (Wisconsin Univ.) and Prof. Ikekawa (Tokyo Institute of Technology). In this report our development of synthetic methods for the preparations of the fluorine-modified vitamin D analogs is described. Preparation of Fluorinated Vitamin D Analogs Needless to say, investigations on the metabolism and function of vitamin D^ revealed that it must undergo metabolic conversion before acting. Among the metabolites discovered so far, 1,25-dihydroxyvitamin D^ (1,25(OH) D ) the most biologically active form, is now regarded as a hormon which plays the central role in calcium and inorganic phosphorous homeostatis (3-6). Substitution of hydrogen atom(s) at the posiotion(s) where metabolic hydroxylation occurs with fluorine atom(s) would be expected to block the hydroxylation, thereby such compound becoming a useful tool for the study to elucidate the physiological significance of the metabolic hydroxylation of D^. Moreover, such analogs would be expected to give important information on the structure-activity relationships. The fluorinated analogs we have synthesized are shown below.

HO'^R RsHorOH, R=CF3 r=h,r=ch 3 R=H, R'sH

HO

>

F

R=H or OH

Γ

^—S>

R=H or OH

ΗΟΠΓ^ΟΗ F R=H or OH

24,24-F^- and 23,23-F2-25-OH-D and The lg-Hydroxylated Analogs In our first synthesis of 24,24-F2~25-OH-D3 1, the ring opening reaction of acetoxydifluorocyclopropane derivative was used (7). Reaction of the enol acetate with difluorocarbene gave the cyclopropane in 72% conversion yield. Alkaline hydrolysis of the cyclopropane gave the desired difluoroketone along with the formation of the fluoroenone. The ratio of the products

5 s t r o n g l y d e p e n d s o n t h e s t r u c t u r e of t h e c y c l o p r o p a n e a s c o m p a r e d w i t h t h e r e s u l t of t h e b i c y c l i c c o m p o u n d , p r e s u m a b l y d u e t o t h e e a s e o f t h e a n t i p e r i p l a n a r a r r a n g e m e n t of t h e a n i o n a n d t h e f l u o r i n e a t o m i n t h e f o r m e r c a s e w h e n t h e o x i d o - p r o m o t e d r i n g - c l e a v a g e o c c u r r e d (8). The difluorok e t o n e w a s c o n v e r t e d t o t h e c h o l e s t e r o l f o r m .2 b y t h e G r i g n a r d r e a c t i o n .

OAc

OAc ^ r t ) Ac ο Λ Λ Τ

CICiyOONa^

riL·)-'

r

LIOH

dlglym· 72

*'·

to·/.

63·/.

ΌΗ AcO'

ξ ^

AeO up

Cr+£f 0

F

58 V.

0

18·/.

A n a l t e r n a t i v e r o u t e f o r t h e i n t r o d u c t i o n of t h e g e m i n a l f l u o r i n e s w a s a c h i e v e d t h r o u g h t h e r e a c t i o n of t h e k e t o e s t e r ,3 w i t h d i e t h y l a m i n o s u l f u r trifluoride (DAST). The keto ester was effectively p r e p a r e d from the keto sulfoxide through the Pummerer rearrangement followed by the acetyl migration. R e a c t i o n of t h e a c t i v a t e d c a r b o n y l g r o u p w i t h D A S T p r o v i d e d t h e difluoride, which was converted to the cholesterol form 2 by the Grignard r e a c t i o n (9). A t the nearly same time T e i k y o g r o u p w a s also p r e p a r e d the same c o m p o u n d by D A S T w a y (10).

-γ—V0CH3 THPO*^

o

V--^Y' o c h 3

v-JCsPh THPO^-^ 89*/.

τ η ρ 0

4) CH 2 N 2

Λ>Ο 51·/.

6 T r a n s f o r m a t i o n of the difluorocholesterol to the corresponding D^ form 1 was a c h i e v e d by the conventional m e t h o d . Isolation and identification of the ΐ α - h y d r o x y l a t e d c o m p o u n d was successfully carried o u t by incubation of 1, w i t h D - d e f i c i e n t chick kidney h o m o g e n a t e (11). The 24-difluoro analog 4, has a potency of a p p r o x i m a t e l y 5-10 times that of Ι ^ δ ί Ο Η ) ^ in the known in vivo vitamin D responsive system (12,13). Induction of calcium-binding p r o t e i n in o r g a n - c u l t u r e d chick intestine by _4 w a s about 4 times more p o t e n t than that of l , 2 5 ( O H ) 2 D 3 (14). ~

A c ο ί ώ ^

Dh» Δ

it)

0

^

0

lsJ—I

Js

Owns irrt» D)jgi /xylene 1) 5*/*KOH/CH0H

HCrkJ^t/

Chick Kidney Homogenate

For the synthesis of the geminal d i f l u o r i d e a t 23 p o s i t i o n w a s also u s e d the reaction of the keto ester 5, w i t h DAST. Further e l a b o r a t i o n for the conversion of the difluoroester 6 to the D form 2 w a s i n v o l v e d the carbon chain extention b y the reaction of the triflate w i t h p o t t a s i u m salt of m a l o n a t e (15). 23,23-?^!,25(OH) D^ Q w a s synthesized enzymatically (16). Unlike fluorine s u b s t i t u t i o n on Ehe C-24, C - 2 6 and C-27, the 23-fluorinated analogs were less active than the c o r r e s s p o n d i n g n o n f l u o r i n a t e d compounds in general v i t a m i n D activity (16).

CH3

A c 0

78*/.

Vy^OTt i) LIAIH A H)Tf20-Py

80*/.

95·/.

YvyCOOEt KCHtOOOEtfc

'

THPO

74·/.

F

00061

THF-HMPA ' ' 81 ·/. 1 8

R-H R>0H

7 Analogs with Fluorine on the Terminal carbon For the synthesis of analogs with fluorine at C-26 and C-27, the sulfone intermediates were used. Lithiation of the sulfone £ and subsequent reaction with hexafluoroacetone gave the adduct 10, which was converted to the carbinol 11 by reductive desulfonylation with Na-Hg (17). However, similar reaction of the trifluoro compound 12 with Na-Hg afforded the olefinic compound 14 as the major product, as in the case of nonfluorinated compound. In the latter case, desulfonylation of the keto sulfone 15 with Al-Hg gave a satisfactory result (18). LDA

Na(Hg)

CFJCOCFJ

10

61·/.

11

84*/.

—*Si02Ph

a^·

Ph

LDA CF3COCH3

~:F3

No(Hg)

OH

THPO·^^

12

quant.

14

13

J

53·/.

MeMgl

LDA

>

CFjCOOEt 15

— Ύ

ä02Ph JyCFa AI(Hfl) β

78·/.

(6A·/.;

16

Introduction of la-hydroxyl group into 17 was achieved through the Birch reduction of the epoxydienone 18 without affecting the hexafluorocarbinol, This was converted to the D^ form 19 by the standard method (19). The hexafluoro analog is about 5-10 times more active than the native active form in general vitamin D activity and of importance is that the action of this fluoro analog is long lasting campared with that of l,25(OH)2D3 (20). •CF3 OH

CP?" 30*ftH,0, 5·/.Να0Η' 18

97 V.

8 Analogs with Fluorine on Ring A Introduction of fluorine into ring A of D is of considerable interest, since the 1 -hydroxyl group is essential for eliciting biological activity. 23-F-lct-OH-D^ 20 (21), la-F-D3 21 and its 25-hydroxylated compound (22) were synthesized in stereoslective manner using trans diaxial ring opening of epoxides with fluoride anion.

khf2 RC1—Ό

(CH^HJj, 170"C, I5h

R-H 48·/. R'Ac

') khf 2

Ηα
9 i) LiC=CH (Y:98%) 12-KL (ref. 7,8) ii) PhSOCl, Py (Y:86%) 13-^2 (ref. 9) la,25-(0H)2-D3[1]

References 1) Takayama, Η., Ohmori, Μ., and Yamada, S. (1980) Tetrahedron Lett. 21, 5027-5028 2) Yamada, S., Nakayama, Κ., and Takayama, H. (1981) Tetrahedron Lett. Z2, 2591-2594 3) Yamada, S., Shiraishi, Μ., Ohmori, Μ., and Takayama, H. (1984) Tetrahedron Lett. 25, 3347-3350 4) Ohmori, Μ., Yamada, S., Takayama, Η., and Ochi, K. (1982) Tetrahedron Lett. 23, 4709-4712 5) Ohmori, Μ., Takano, Υ., Yamada, S., and Takayama, H. (1986) Tetrahedron Lett. 27, 71-74; Idem., J. Org. Chem. in press 6) Dodson, R.M., Goldkamp, Α.Η., and Muir, R.D. (1960) J. Am. Chem. Soc. 82, 40264033 7) Barton, D.H.R., Hesse, R.H., Pechet, M.M. and Rizzardo, E. (1974) J. Chem. Soc., Chem. Commun. 203-204 8) Narwid, T.A., Blount, J.F., Iacobelli, J.A., and Uskokovic, M.R. (1974) Helv. Chim. Acta 57, 781-789 9) Ikekawa, Ν., Morisaki, Μ., Koizumi, N., Kato, Y., and Takeshita, T. (1975) Chem. Pharm. Bull. 23, 695-697

ON OXIDATION OF THE VITAMIN D TRIENE SYSTEM- - A SIMPLE AND CONVENIENT ENTRY INTO THE 19-NOR-10-0X0-DERIVATIVES OF VITAMIN D WOLFGANG REISCHL Institut für Organische Chemie der Universität Wien, Austria Introduction Since the discovery of 5E and 5Z 19-nor-10-oxo-derivatives of vitamin D as metabolites, their biological production and their biological profile have attracted considerable attention (1), but no convenient preparation of these compounds appeared in the literature. Since there is no oxidation reagent known which attacks the exomethylene group of the triene leaving the diene bridge untouched a different strategy has been chosen. It was anticipated to convert the exomethylene group into a heterocycle which could be directly fragmentated to the desired dienone system. 1,3 Dipolar Addition Nitriloxides generated in situ according to Mukaiyama's procedure (2) add selectively to the exomethylene group of 5Z vitamin D acetate J_ to give a seperable diastereomeric mixture of 2 and _4 in almost quantitative yield. 5E vitamin D acetate behaves in an identical manner to yield the Cs-2isoxazolineacetates 1_ and 9^. The acetates could be saponified by treatment with sodiummethoxide in dry methanol to 5_, £ and 10. Fragmentation to the Dienones 11 and 13 Treatment of 2/4 with Mo(CO)5 in refluxing acetonitrile afforded JM_ in 70 to 75% yield along with traces of 13. Analogous treatment of 7/9 results in the production of only 13 in good yield. It is not clear whether J_3 is produced during the reaction of 2/4 or during work up and purification. Oxidation studies on 11 and 13 7-8 Oxiranes of J_2 and 1_3 appeared in the literature (3) . The stereochemistry of these compounds has not been given. V\_ is oxidised with MCPBA cleanly to the oxirane J_5· It's 7R stereochemistry was established by converting it to the known oxirane J_7 (4) . In contrast 1_4 or V3 always produces upon treatment with MCPBA mixtures of 1_9 (_1_8) and the overoxidation product 2J (20). References (1) Brown A.J. and DeLuca H.F. (1985) J. Biol. Chem. 1413214136 and references cited (2) Mukaiyama T. and Hoshino T. (1960) J. Org. Chem. 53395342 (3) DeLuca H.F., Schnoes H.K., Tanaka Y. and Alper L.B. (1981) CA 94: 20981 η (4) Kratky C., Reischl W. and Zbiral E. (1984) Monatsh. Chem. 1453-1458

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · New York - Printed in Germany

67

1 Κ a ΟΑβ 3 Κ a OA· —ι 4ft-OA·—ι 3Ha OH -J 3fta OH -J

ft

• ft-OA·

3/4ft- OA·

7rtft- OA·

7 Ä-OA« —. Ift-OH—J

•ft-OA·—ι 10 Ks OH —J

11 Κ - OA« —1 12 2 - OH -J

13ft- OA· —i 14ft-OH—-1

13ft— OA· —, 14ft— OH —1

ft

11ft3 OA· 12ft- OH

13ft- OA· -ι 16ft— OH

13ft3 OA· 14fta OH

IIfta OA· 19ft= OH

17

20ft=. OA· 21 ft-OH

SYNTHESIS O F 25-HYDROXYDIHYDROTACHYSTEROL2

R. Boer Rookhuizen, R. Bosch, L. Castedo, J.G. Cota, J. Granja, M.A. Maestro, E. Martinez de Marigorta, and A. Mourifio. Departamento de Quimica Orgänica. Facultad de Quimica y Secciön de Alcaloides del C.S.I.C. Santiago de Compostela (Spain) and Department of Internal Medicine, Universitary Hospital, 3500CG Utrech (The Netherlands). Dihydrovitamins D are a class of compounds derived by reduction of the natural vitamin D3, the unnatural vitamin D2 and their 5E-isomers (5,6-frans-derivatives). Among them, dihydrotachysterol2 (DHT2, l a ) , first isolated by Von Werder (1), is considered an interesting analog of la,25-dihydroxyvitamin D3, the hormonal form of vitamin D3 , because the former's 3ß-OH group has a similar topological orientation to that of the letter's 1a-OH (2). In spite of the availability of 1a,25-dihydroxyvitamin D3, DHT2 is still widely used in the treatment of hypoparathyroidism and to prevent bone disorders attending renal failure (3). DHT2 was recently labeled with 3h by R. Bosch et al., and after biological transformation in rats they isolated a more polar metabolite that was identified by mass spectrometry as 25-hydroxydihydrotachysterol2 (250H-DHT2, l b ) (4). We have now synthesized 25-OH-DHT2 (Hi) in order to corroborate its structure and to obtain material for the evaluation its biological properties. ι

R'

R'

16 s t e p s

Vitamin D 2 30% TBSO*' La R = H•·, D H'T' ,2 ' —' L b , R = O H , 2 5 - O H - D H T ,2

£(58%)

5.(12%)

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · New York - Printed in Germany

4

69 Following

published procedures (5,6) the protected 25-hydroxyvitamin D2 2. was

synthesized in 16 steps from vitamin D2 in 30% overall yield. Deprotection of the tert-butyldimethylsilyl group with n-BuNF afforded the protected 25-hydroxyvitamin D2 3. in a 75% yield. Equilibration with iodine (7) led to the desired 5,6-frans-isomer £ in 55% yield after HPLC separation (Partisil M9 10/50 / 60% EtOAc/Hexanes, flow rate 2 mL/min). Reduction of ± with the system Cp2TiCl2-LiAIH4 (2:2.4 ratio with respect to the substrate) afforded the desired 25-protected dihydrotachysterol2 £ after flash chromatography in 58% yield together with the epimer 5. (12%) (8). Deprotection with AG 50W-X4 cation-exchange resin afforded 25-OH-DHT2 (Hi) quantitatively. The synthesis of 25-OH-DHT2 (111) was thus completed in 20 steps from vitamin D2 in 7% overall yield (4 steps from 2,23% yield) (9). Acknowledgements We gratefully acknowledge the financial support of the CAICYT (Spain) and the Academish Ziekenhuis Utrech and the generous gift of vitamin D2 by Hoffmann-La Roche (Basel). M.A.M thanks the Spanish Ministry of Education and Science for an F.P.I, fellowship. References and notes (1) Von Werder, F., (1939) Ζ. Physiol., 260,119. (2) (a) Holick, M.F., Garabedian, M., DeLuca, H.F., (1972) Science, 176, 1247; (b) Okamura, W.H., Norman, A.W., Wing, R.M., (1974) Proc. Natl. Acad. Sei. USA, 71, 4194. (3) (a) Holick, M.F., DeLuca, H.F., "Advances in Steroid Biochemistry"; Briggs, Μ.Η.; Christie, C.A., Eds.; Academic Press: New York (1974), 4; (b) Norman, A.W. "Vitamin D: the Calcium Homeostatic Steroid Hormone"; Academic Press: New York (1979); (c) DeLuca, H.F., Anast, C.S., "Pediatric Diseases Related to Calcium"; Blackwell Scientific Pub.: New York (1980) (4) Bosch, R., Versluis,C., Terlouw, J.K., Thijssen, J.H.H.,Duursma, S.A.,"Vitamin D. A Chemical, Biochemical and Clinical Update", Walter de Gruyter & Co., Berin-New York, 37 (1985). (5) Sardina, F.J., Mourino, Α., Castedo, L„ (1986) J. Org. Chem., 51,1264. (6) Castedo, L., Granja.J., Maestro,M.A., Mourino, Α., (1987) Tetrahedron Lett, 28, 4589. (7) Mourino, Α., Okamura, W.H., (1978) J. Org. Chem., 43,1653. (8) Attemps to carry out regioselective 10,19-double bond reduction with Wilkinson's catalyst were unsuccessful. (9) This work was carried out at the Departamento de Quimica Orgänica, Santiago de Compostela (Spain).

STEREOCHEMISTRY AT C(23) OF 23.25-DIHYDROXY-24-OXOVITAMIN D3 S. YAMADA,11* K. YAMAMOTO,' K. SAKAIDA,11 Η. ΤΑΚ AY ΑΜΑ J1 Τ. SHINKI,§ Τ. SUDA§, Υ. ΠΤΑΚΑ,Ί' A. ITAI * A F a c u l t y of P h a r m a c e u t i c a l S c i e n c e s , T e i k y o U n i v e r s i t y , S a g a m i k o , Kanagawa 1 9 9 - 0 1 ; § D e p a r t m e n t of B i o c h e m i s t r y , S c h o o l of D e n t i s t r y , Showa U n i v e r s i t y , H a t a n o d a i , S h i n a g a w a - k u , Tokyo 1 4 2 ; φ F a c u l t y of P h a r m a c e u t i c a l S c i e n c e s , Tokyo U n i v e r s i t y , Hongo, Bunkyo-ku, Tokyo 113, J a p a n ( * p r e s e n t a d d r e s s : I n s t i t u t e f o r M e d i c a l and D e n t a l E n g i n e e r i n g , Tokyo M e d i c a l and D e n t a l U n i v e r s i t y , K a n d a - S u r u g a d a i , C h i y o d a k u , Tokyo 101) 2 3 , 2 5 - ( 0 H ) 2 - 2 4 - o x o - D ^ ( I ) i s a m a j o r m e t a b o l i t e of 24R,25-(0H) 2 Do p r o d u c e d iji^_vit£0 by k i d n e y s f r o m v i t a m i n D - s u p p l e m e n t e d c h i c k s ( 1 ) and r a t s (2,3). We r e p o r t e d p r e v i o u s l y t h e s y n t h e s i s of two C(23) e p i m e r s of t h e m e t a b o l i t e and s h o w e d t h a t one of t h e s y n t h e t i c e p i m e r s w a s i d e n t i c a l w i t h t h e i s o l a t e d m e t a b o l i t e i n i t s s p e c t r a l p r o p e r t i e s and HPLC b e h a v i o r ( 4 ) . However t h e s t e r e o c h e m i s t r y a t C(23) of t h e m e t a b o l i t e h a s n o t been d e t e r m i n e d . By m o d i f y i n g t h e p r e v i o u s l y r e p o r t e d m e t h o d , we s y n t h e s i z e d t h e t w o e p i m e r s i n q u a n t i t y and d e t e r m i n e d t h e s i d e - c h a i n s t e r e o c h e m i s t r y by t h e s i n g l e c r y s t a l X-ray analysis. We h e r e r e p o r t s y n t h e s i s d e t a i l s and t h e s t e r e o c h e m i c a l s t r u c t u r e of t h e m e t a b o l i t e . In t h e s y n t h e s i s (Scheme 1), we used t h e 2 5 - h y d r o x y - 2 4 - o x o - 7 - d e h y d r o c h o l e s t e r o l d e r i v a t i v e ( I I ) a s t h e s t a r t i n g m a t e r i a l , which was o b t a i n e d r e a d i l y and i n h i g h o v e r a l l y i e l d (73%) f r o m C ( 2 2 ) - s t e r o i d s u l f o n e ( I I I ) and e p o x y a l c o h o l ( I V ) , a s r e p o r t e d ( 5 ) . The h y d r o x y l group a t C(23) was i n t r o d u c e d v i a o x i d a t i o n of t h e s i l y l e n o l e t h e r of t h e k e t o n e I I . The k e t o n e p r o d u c e d a s i n g l e t - b u t y l s i l y l e n o l e t h e r (V) r e g a r d l e s s of t h e c o n d i t i o n s u s e d , w i t h o r w i t h o u t HMPA a s t h e cosolvent. This i n d i c a t e s t h a t t h e bulky t - c a r b i n o l group c a n n o t be s i t u a t e d i n t h e p o s i t i o n c i s t o t h e bulky s t e r o i d r e s i d u e and t h e r e f o r e t h e s t e r e o c h e m i s t r y of t h e s i l y l e n o l e t h e r w a s a s s i g n e d t o be Z. A f t e r t h e e n d o c y c l i c d i e n e f u n c t i o n of t h e Β r i n g was p r o t e c t e d , t h e e n o l e t h e r was o x i d i z e d w i t h MCPBA t o g i v e two e p i m e r i c 2 3 - h y d r o x y l a t e d c o m p o u n d s V i a and V I b i n a 7 : 2 r a t i o . The t w o e p i m e r s w e r e r e a d i l y s e p a r a t e d by s i m p l e c o l u m n c h r o m a t o g r a p h y . The d i e n e f u n c t i o n was d e p r o t e c t e d and s u b s e q u e n t l y t h e p r o t e c t i n g g r o u p s of t h e h y d r o x y l f u n c t i o n s w a s r e m o v e d t o a f f o r d t h e d e s i r e d p r o v i t a m i n D ( V i l a and Vila). The s t e r e o c h e m i s t r y a t C(23) of t h e two i s o m e r i c p r o v i t a m i n Ds was d e t e r m i n e d by t h e s i n g l e c r y s t a l X-ray a n a l y s i s of t h e m a j o r i s o m e r (Vila). The c r y s t a l b e l o n g s t o a m o n o c l i n i c s p a c e group P2i w i t h u n i t c e l l p a r a m e t e r s of a = 1 2 . 2 5 8 ( 4 ) , b = 3 1 . 8 6 8 ( 8 ) , c = 6 . 3 6 0 ( 2 ) Ä and β = 92.06(2) i n c l u d i n g two m o l e c u l e s i n an a s y m m e t r i c u n i t . The f i n a l Rf a c t o r was 5.6%. F i g u r e 1 shows t h e m o l e c u l a r s t r u c t u r e of V i l a drawn by ORTEP. The s t e r e o c h e m i s t r y a t C(23) of V i l a was c l e a r l y d e t e r m i n e d t o be £ and t h a t of t h e minor i s o m e r ( V l l b ) t o be S. Each i s o m e r ( V i l a and V l l b ) w a s c o n v e r t e d t o t h e c o r r e s p o n d i n g v i t a m i n D ( l a and l b ) by UV i r r a d i a t i o n f o l l o w e d by t h e r m a l i s o m e r i z a t i o n . (23R)and ( 2 3 S J 2 3 , 2 5 - ( 0 H ) 2 - 2 4 - o x o - D 3 ( l a and l b ) t h u s s y n t h e s i z e d w e r e c o m p a r e d by HPLC w i t h t h e m e t a b o l i t e p r e p a r e d f r o m 25-(0H)-24-oxo-Ü3 by i n c u b a t i n g w i t h k i d n e y homogenates from r a t s or c h i c k s . The m e t a b o l i t e o b t a i n e d f r o m e i t h e r r a t s or c h i c k s c o m i g r a t e d w i t h t h e 23S i s o m e r ( l b ) . Thus

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988Walterde Gruyter&Co., Berlin · New York-Printed in Germany

71

the stereochemistry at C(23) of the natural 23,25-(OH)2-24-oxo-Do was determined to be S^ irrespective of the animals producing it. It is interesting to note that the stereochemistry at C(23) is the same as that of other 23-hydroxylated metabolites of vitamin Do, 23S^25-(0H)2Do (6), 23S, 25ji,26-(0H)ßDo (7), 25R-(0H)Do 26,23S.-lactone (8), 25R-(0HjD 3 26,23S-lactol (7) and their la-hydroxylated analogs (9).

b (235) R - TBDMS

VII Q(23R) b(23S)

α(23R) b (23SJ

S c h e m e 1. 1) 3 , 4 - e p o x y - 2 - m e t h y l - 2 - b u t a n o l (IV), LDA, -2.0'C, 921; 2) Na-Hg, 961; 3) D M S O , Py-S0 3 , E t 3 N , 90%; 4) PPTS, EtOH-CH 2 Cl 2 , 97%j 5) t-BDMSCl, Imidazole, 947.; 6) LDA, THF, - 2 0 ' C then t - B D M S C l , THF-HMPA, 83%; or LDA, THP-HMPA then t-BDMSCl, 847.; 7) phenyltrlazolinedione, 831; 8) M C P B A , 82%; 9) K 2 C 0 3 , DMSO, 60* C, 75%; 10) n - B u 4 N F , 72%; 11) hv, 39%; 12) room temp. 78%.

Fig. 1. Molecular structure of Vila by ORTEP drawing. 1. 2.

3.

4.

5. 6. 7. 8. 9.

Yamada, S., Ohmori, M., Takayama, H., Takasaki, Y., Suda, T. (1983) J. Biol. Chem. 258, 457-463 Mayer, E., Reddy, G. S., Chandraratna, R. A. S., Okamura, W. H., Kruse, J. R., Popjak, G., Bishop, J. E., Norman, A. W. (1983) Biochemistry 22, 1798-1805 Yamada, S., Ino, E., Takayama, H., Horiuchi, N., Shinki, T., Suda, T., Jones, G., DeLuca, H. F. (1985) Proc. Natl. Acad. Sei. USA 8 2 , 7485-7489 Yamada, S., Ino, E., Takayama, H., Suda, T. (1985) in "Vitamin D Chemical. Biochemical and Clinical Update" Norman, A. W. et al. (ed.) Walter de Gruyter, Berlin, 13-22 Yamada, S., Ohmori, M., Takayama, H., Suda, T., Takasaki, Y. (1981) Chem. Pharm. Bull. 29, 1187-1188 Tanaka, Y., DeLuca, H. F., Schnoes, Η. K., Ikekawa, N., Eguchi, T. (1981) Proc. Natl. Acad. Sei. USA 7 8 , 4805-4808 Yamada, S., Nakayama, K., Takayama, H., Shinki, T., Takasaki, Y., Suda, T. (1984) J. Biol. Chem. 259, 884-889 Yamada, S., Nakayama, K., Takayama, H. (1981) Chem. Pharm. Bull. 29, 2393-2396 Ishizuka, S., Oshida, J., Tsuruta, H., Norman, A. W. (1985) Arch. Biochem. Biophys. 242, 82-89

SYNTHESIS AND BIOLOGICAL ACTIVITY OF HIGHLY ACTIVE VITAMIN D ANALOGS: GENERAL APPROACHES TO THE SYNTHESIS OF BIOLOGICALLY POTENT VITAMIN D STEROLS H.S. GILL, J.M. LONDOWSKI, R.A. CORRADINO, and R. KUMAR Mayo Clinic and Foundation, Rochester, MN; Cornell University, Ithaca, NY The influence of alterations in the structure of the vitamin D3 molecule such as alterations in the side chain, the A ring, the triene structure, and the C ring has yielded information about the features of the sterol needed to display biological activity (1-4 and references therein). Modification of the side chain of 1,25-(0H)2D3 occurs during its further metabolism to biologically inert metabolites (1). We reasoned that we would be able to synthesize more potent or long lived vitamin D sterols if we could block side chain metabolism by the addition of less biodegradable moieties. To this end, we synthesized 25-hydroxy-26,27dimethylvitamin D3, 9 and l,25-dihydroxy-26,27-dimethylvitamin D3, 14 from chol-5-enic acid-3ß-ol. We tested the biological activities of these compounds in vivo and in vitro. 25-Hydroxy-26,27-dimethylvltamin D3, 9 was found to be a highly potent vitamin D analog in vivo. It was equally potent with 25-(0H)D3 in mobilizing bone calcium and in increasing intestinal calcium transport in everted duodenal sacs. 9 bound to rat vitamin D binding protein with approximately one-third the affinity of 25-(0H)D3· In a duodenal organ culture system and in a competitive binding assay with chick intestinal I,25-(0H)2d receptor, it was found to be more potent than 25-(0H)D3·

1 II

V

AcaO-Py

SOCI2

VI

ElMger

NBS

III

CHjN2

VII

Coadin·

IV

C,H,COOAfl

VIII

4-PiwnyV-1,2,4-trlazo«ne-3.S-{#one

Fig. 1.

κ umh4 X

hf

XI

Ethanol. Reflux

Synthesis of 25-hydroxy-26,27-dimethylvitamin D3

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

73

1

τ«α. py

> MeOH. N a H C O , HI

SoO,. B u ' O O H

IV

AcOH ηβυπτΒβυπ

V

Fig. 2.

Synthesis of l,25-dihydroxy-26,27-dimethylvitamin D3

l,25-Dihydroxy-26,27-dimethyl D3, 14, was also highly active iri vivo. When tested in an intestinal calcium transport and bone calcium mobilization assay at doses of 1000-5000 pmoles/rat, its action was more sustained than that of 1,25-(0H)2D3· 14, bound to vitamin D binding protein about 18 times less effectively than 1,25-(0H)2D3· bound to the chick intestinal cytosol receptor with an affinity approximately 0.5 times that of 1,25-(OH)2^3· In a duodenal organ culture system, 14, was about one-half as active as 1,25-(0H)2D3· We conclude that extension of the sterol side chain, at C-26 and C-27, by methylene groups, prolongs the bioactivity of a vitamin D sterol hydroxylated at both C-l and C-25. The corresponding sterol, hydroxylated only at C-25, does not show any alteration in its bioactivity in vivo. These newly synthesized analogs of vitamin D3 may potentially be oT~use in various mineral disorders. References 1. 2. 3. 4.

Kumar, R. (1984) Physiol. Rev. Gill, H. S., Londowski, J. M., Steroid Biochem. 28, 147-153. Gill, H. S., Londowski, J. M., Steroids 48, 93-108. Gill, H. S., Londowski, J. M., Steroid Biochem. in press.

64, 478-504. Corradino, R. and Kumar, R. (1987) J. Corradino, R. and Kumar, R. (1986) Corradino, R. and Kumar, R. (1988) J.

SYNTHESES OF LABELED SIDE CHAINS OF VITAMIN D METABOLITES AND ANALOGS. L. Castedo, M. C. Domarco, J. Granja, F. J. Maestro, M. A. Maestro, J. L. Mascarefias, J. M. Mendez, A. Mourifio, C. Postigo, J. J. Santiago, L. Sarandeses and R. Tojo. Departamentos de Quimica Orgänica y Pediatria. Universidad de Santiago de Compostela. Spain The determination of the principal vitamin D metabolites is of crucial importance in the diagnosis and treatment of patients with a wide range of mineral and skeletal disease states. Several methods of carrying out these measurements make use of labeled vitamin D analogs (1). We describe here efficient methods that allow the side chains of the principal metabolites of vitamin D3 and vitamin D2 to be labeled with 3H, 2H, or

14

C.

The synthesis of vitamin D3 side chain synthons 2 and 4 starts with the Inhoffen-Lythgoe diol (D, which is easily obtainable in 85% yield by degradation of vitamin D2 (O3, MeOH-py; NaBH4) (see FIG.1) (2). Compound 1 was transformed into the iodide Ζ by the sequence (p-TsCI, py; Nal, acetone). Construction of the desired side chain synthon 2. was accomplished in 78% yield by sonication of an oxygen-free mixture of Cul (1 equiv), Zn (4 equiv), iodide Ζ and methyl vinyl ketone (1.3 equiv) in Et0H:H20 (7:3) for 20 min and further sonication for 30 min after the addition of more Cul (0.5 equiv) and Zn (2 equiv) at RT (4 steps from vitamin D2,58%) (3). FIG.1

Synthons 2 and 4 can at this stage be reacted the appropriate labeled reagent or transformed into the principal 25-functionalized metabolites of vitamin D3p which should be susceptible to labeling in the last steps of the syntheses. Side chain-labeled 25-OH-D2 or la,25-(OH)2-D2 can be obtained in the last steps of their syntheses as depicted in FIG.2. Carbamate £ was recently obtained in 77% yield from the aldehyde £ (4). Rather than being reacted with Li2Cu3Mes for the construction of 25-functionalized vitamin D2 side chain as previously reported (2), it was debenzoylated (NaOH, EtOH, 87%), and the resulting alcohol oxidized (PDC, PPTS, CH2CI2, 97%) to the ketone Z- Wittig-Horner coupling of 2 with the anion of the phosphine oxide corresponding to the bottom part of vitamin D afforded the carbamate a (96%). Finally, treatment of a with Li2Cu3Me5 followed by deprotection (AG50W-X4)

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · New York - Printed in Germany

75 furnished 25-OH-D2 (ä, X=CH3) in 70% yield. Alternatively, compound fi. or its 1a-OH-protected derivative should react in a similar fashion with labeled high order cuprates to afford, after deprotection, the desired labeled analog. FIG. 2

vCH0

ςβ ;

7 steps 77%

BzO

H

S

HO'" Acknowledgements

TBSO

This study was supported by a grant from the Comisiön Asesora de Investigaciön Cientifica y T6cnica (CAICVT). We thank Hoffmann-La Roche for the generous gift of vitamin D2. The valuable suggestions of Professor Okamura are also acknowledged. References (1)

(a) Norman, A.W., "Vitamin D, the Calcium Homeostatic Steroid Hormone"; Academic Press: New York, 1979; (b) "Vitamin D. A Chemical, Biochemical and Clinical Update", Walter de Gruyter & Co., Berlin-New YorK 1985.

(2)

Sardina, F.J., Mourifio, Α., Castedo, L„ (1986) J. Org. Chem., 5L. 1264.

(3)

Castedo, L., Mascarenas, J.L., Mourifto, Α., Sarandeses, L.A., (1988) Tetrahedron Lett., 22,

(4)

Castedo, L., Granja, J., Maestro, M.A., Mourifto, Α., (1987) Tetrahedron Lett., 2fi, 4587.

1203.

SYNTHESIS OF 22-OXA-, AND 20-OXA-ANALOGUES OF VITAMIN D 3 K.Miyamoto, E.Murayama, N.Kubodera and T.Mori. Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co.Ltd., 135, Komakado 1 chome, Gotemba-shi, Shizuoka 412, Japan There has been an increasing interest in the synthesis of vitamin D3 analogues since la,25-dihydroxyvitamin D3 was shown to have a differentiationinducing activity on myeloid leukemia cells in addition to its regulatory activity on calcium and phosphorus metabolism. But the severe hypercalcemia caused by la,25-dihydroxyvitamin D3 prevents us from using it as an antileukemic. Our efforts to separate these activities led to the vitamin D3 analogues having an oxygen atom in the side chain. Attempting to synthesize these analogues, we attached great importance to the following two points: i) selection of a suitable hydroxyl-protecting group, ii) introduction of the diene system in an early stage. These were accomplished by synthesizing the 5,7-diene (2) as a key intermediate. 5.7-diene (2)

The ketodiol (1) , prepared by microbiological Ια-hydroxylation of dehydroepiandrosterone, was converted to the 5,7-diene (2) by following three steps: i) tert-butyldimethylsilylation , ii) bromination, iii) dehydrobromination. The tert-butyldimethylsilyl ether in the 5,7-diene (2) was stable enough under all conditions used in our synthesis, the protecting group was, however, easily removed after irradiation and thermal isomerization . The 5,7-diene (2) is a common intermediate for the title compounds.

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · N e w York - Printed in Germany

77

OH

22-oxa-analogue (3)2) ''"„.•OH

2

-

1)EtPPh3Br *-Β"οκ 2)9-BBN

3)Η2θ2 NaOH

02 PdCI 1) CuCI2

TBDMSO *

TBDMSO 1Bum:5u

TBDMSO

2)CH3MgBr

TBDMSO

On the differentiation inducing activity, 3 was about 10-times as effective as la,25-dihydroxyvitamin Ü3f and 4 about 1/5.3) Whereas, concerning the boneresorbing activity, both 3 and 4 were remarkably less active than la,25dihydroxyvitamin D3 .3) These results suggest that the two biological activities are structurally separable. REFERENCES 1. Kubodera, N., Miyamoto, K., Ochi, K. and Matsunaga, I. (1986) Chem. Pharm.Bull. 34 (5), 2286-2289 · 2. Murayama, E., Miyamoto, K., Kubodera, N., Mori, T. and Matsunaga, I. (1986 ) Chem.Pharm. Bull. 34(10), 4410-4413 3. Abe, J., Morikawa, M., Miyamoto, K., Kaiho, S., Fukushima, M., Miyaura,C., Abe, E., Suda, T. and Nishii, Y. (1987) FEBS Lett. 226(1), 58-62

COMPUTER CALCULATIONS OF THE ACTIUE CONFORMATION OF 1,25-DIHYOROHY U I T A M I N 03 S. R. WILSON, R. UNWRLLR RND Ii). CUI Department of Chemistry, Neui Vork Uniueristy, UJashington Square, New Vork, NV 10003 The a c t i u e c o n f o r m a t i o n of fleHible m o l e c u l e s is an i m p o r t a n t issue, since it has been s h o w n that rigid a n a l o g s of bioactiue molecules, held in the correct "actiue c o n f o r m a t i o n " are significantly more actiue. The problem houjeuer is to define that c o n f o r m a t i o n , since flexible m o l e c u l e s haue n u m e r o u s possible c o n f o r m a t i o n s . UJe haue deueloped a new tool for the c o m p u t e r modeling of fleHible m o l e c u l e s b a s e d on s i m u l a t e d annealing.' This technique s i m u l a t e s an a n n e a l i n g p r o c e s s ujhereby a molecule is heated to a high t e m p e r a t u r e and then s l o w l y c o o l e d until it s l o w l y f r e e z e s into its m o s t s t a b l e c o n f o r m a t i o n . This p r o g r a m , called A n n e a l - C o n f o r m e r has been reported r e c e n t l y 2 for peptides and is applied here to the problem of 1,25-D3 1.

1

OH

2

3

In the c a s e of uitamin D3, the 6 s i n g l e b o n d s (dihedral a n g l e s ) w h i c h haue free r o t a t i o n leads to a l a r g e number of p o s s i b l e low e n e r g y local m i n i m a . The " g l o b a l m i n i m u m problem" is the p r o b l e m of f i n d i n g the l o w e s t e n e r g y c o n f o r m a t i o n ouerall and not a local minimum. Up to now the only technique auailable has been to produce a large number of p o s s i b l e s t a r t i n g g e o m e t r i e s , minimize then and discard any duplicates. IDe h a u e c a r r i e d out this p r o c e s s for 1 - h y d r o H y - u i t a m i n D3 u^ing the m u l t i c o n f o r m e r option of the program Macromodel3. R o t a t i o n a r o u n d the 6 r o t a t a b l e d i h e d r a l s at a 6 0 ° r e s o l u t i o n , we produced 30879 c o n f o r m a t i o n s w h i c h m u s t be m i n i m i z e d ! UJhile this is f a r b e y o n d the c a p a c i t y of our UaH c o m p u t e r s , w e w e r e able to break the problem into t w o parts g e n e r a t i n g 22 c o n f o r m e r s of ring R f r a g m e n t 2 and 585 c o n f o r m a t i o n s of C/D f r a g m e n t 3. M i n i m i z a t i o n of all of these s t r u c t u r e s , f o l l o w e d by fusion of the l o w e s t energy c o n f o r m a t i o n of the top half with the si» l o w e s t e n e r g y c o n f o r m a t i o n s of the b o t t o m half, leads to 6 c o n f o r m e r s of e n e r g y (197.7, 198.9, 199.9, 204.0, 204.6 and 208.6 K j o u l / m o l ) . Surprizingly, the l o w e s t energy conformation has ring-R folded ouer the top of the C-ring. The second l o w e s t conformation a p p e a r s more like the tradition representation of

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · New York - Printed in Germany

79

l o w e s t energy conformer 197.7 k j o u l / m o l

second l o w e s t energy 198.8 k j o u l / m o l

Figure 1: Superposition of 1,25-03 and Cortisol u i t a m i n D. Using our flnneal-Conformer program, 1 - h y d r o x y - u i t a m i n D3 m a s s l o w l y c o o l e d f r o m a h i g h t e m p e r a t u r e . Our p r o g r a m u s e s a M o n t e Carlo a l g o r i t h m to r o t a t e a r a n d o m b o n d a r a n d o m n u m b e r of d e g r e e s p r o d u c i n g a r a n d o m w a l k a r o u n d c o n f o r m a t i o n s p a c e . The e n e r g y o f e a c h c o n f o r m a t i o n is c a l c u l a t e d and a B o l t z m a n n d i s t r i b u t i o n at e a c h t e m p e r a t u r e is p r o d u c e d . The e n e r g y of t h e m o l e c u l e is a l l o w e d to go b o t h d o w n h i l l and uphill at h i g h e r t e m e r a t u r e s , but a s the t e m e r a t u r e is l o w e r e d the uphill m o u e s ( w h i c h are c a l c u l a t e d p r o b a b i l i s t i c a l l y ) are d e c r e a s e d until the m o l e c u l e is t r a p p e d in an e n e r g y well. The 1 , 2 5 - D 3 is l o c k e d into the s a m e R - r i n g f o l d e d c o n f o r m a t i o n a s w e f o u n d u s i n g the " b r u t e f o r c e " approach described aboue. The f o l d e d c o n f o r m a t i o n of u i t a m i n D s h o w s t h e critical I - h y d r o n y g r o u p is b r o u g h t into p r o x i m i t y w i t h t h e C - 1 1 p o s i t i o n of the s t e r o i d nucleus. This l e a d s us το the h y p o t h e s i s t h a t the 1 - a l p h a h y d r o x y l g r o u p of u i t a m i n D3 is r e l a t e d to the I I - b e t a s u b s t i t u e n t k n o w n to be of c r i t i c a l i m p o r t a n c e to g l u c o c o r t i c o i d s . The recently r e p o r t e d 4 c-DNR s e q u e n c e f o r the auian uitamin D receptor also s u g g e s t h o m o l o g y of uitamin D receptor with other steroid receptors. F i g u r e 1 s h o w s the s u p e r p o s i t i o n o f the l o w e s t e n e r g y c o n f o r m a t i o n o f 1 - 0 H - D 3 w i t h an 1 l - b e t a - O H s t e r o i d Cortisol. References: 1. 2.

3. 4.

K i r k p a t r i c k , S.,. Gelatt, Jr. C. D, a n d Uecchi, M . P., ( 1 9 8 7 ) , Science. 220, 6 7 1 - 6 7 5 . a. M o s k o w i t z , J. 111., S c h m i d t , Κ. E., W i l s o n , S. R. a n d Cui, LU.,( 1 988), Int J. Q u a n t u m Chem. in p r e s s . b. UJilson, S. R., Cui, UJ., M o s k o w i t z , J. ID., S c h m i d t , Κ. E., submitted. M a c r o m o d e l , c o p y r i g h t 1986, UJ. C. Still, C o l u m b i a U n i u e r s i t y . M c D o n n e l l , D. P., M a n g e l s d o r f , D. J., Pike, J. UJ., H a o s s l e r , M . R., O ' M a l l e y , B. UJ., (1987), Science, 235, 1 2 1 4 - 1 2 1 7 .

Vitamin D Metabolism and Catabolism

PRECHOLECALCIFEROL FORMATION BY A N INVERTEBRATE, PSAMMECHINUS MILIARIS P.J. YATES, R.N. HOBBS AND J.F. PENNOCK. Biochemistry Department, University of Liverpool L69 3BX, U.K. Introduction Cholecalciferol (vitamin D ^ ) is formed in skin and in the laboratory b y the same m e t h o d ie. irradiation of 7-dehydrocholesterol w i t h UV light of around 295nm produces precholecalciferol and this is converted to cholecalciferol by heat (see Figure 1).

7-Dehydrocholesterol

Precholecalciferol HO

Cholecalciferol Figure 1:

Formation of cholecalciferol from 7-dehydrocholesterol

Holick and coworkers (1) showed that application of tritiated 7-dehydrocholesterol to the skin of rats followed by irradiation (250-350nm) produced precholecalciferol. T h e precholecalciferol w a s shown to undergo a temperature-dependent thermal isomerization to cholecalciferol in skin which took 3 days to complete (2). The equilibrium between precholecalciferol a n d cholecalciferol w a s shown to be

40 % Precholecalciferol

20

0 0

40

80

120

Temperature (°C) Figure 2:

Relationship between temperature and proportion of precholecalciferol present in equilibrium mixture w i t h cholecalciferol

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · N e w York - Printed in Germany

84 temperature-dependent by Velluz et al. (3) and the kinetics of the reaction were determined by Hanewald et al. (4). Using these data it can be shown (Figure 2) that precholecalciferol is present in an equilibrium mixture at 37 at approximately 10% and below this temperature the percentage falls. The higher the temperature, the higher the proportion of precholecalciferol. It can also be shown that it takes three days to reach equilibrium at this temperature starting with precholecalciferol. The importance of the photobiogenesis of cholecalciferol in skin is emphasised by the scarcity of the vitamin in dietary components (5). Cholecalciferol in marine animals Fish liver oils contain the highest levels of cholecalciferol to be found naturally (6) but the origin of the vitamin in these cases is unclear. There is little or no evidence for cholecalciferol formation either in fish or phytoplankton (7,8,9) and two main problems are faced by any organism forming the vitamin. a) Sunlight penetrates sea water to a very limited extent so reducing the photoisomerization of 7-dehydrocholesterol to precholecalciferol (10). b) Animals which breath by gills have a body temperature equivalent to that of the environment, which in the sea is near 10 . This is because thermal equilibration in the gills takes place many times more rapidly than does gaseous equilibration. Although there would be about 95% cholecalciferol in an equilibrium mixture at 10 this equilibrium would take in excess of 100 days to achieve. Figure 3 shows the rate of

Days Figure 3:

Formation of cholecalciferol from precholecalciferol at 10°

formation of cholecalciferol from precholecalciferol. As can be seen it would take about 17 days for 50% of the precholecaliferol to be converted

85 to cholecalciferol. It was decided to study the formation of the vitamin in a marine animal to understand how these problems may be overcome. Cholecalciferol metabolism in Psammechinus miliaris P. miliaris, the small green sea urchin, is widespread in seas around the British Isles and appeared ideally suited as an experimental animal since its shell (or test) is composed of calcareous plates containing principally calcium carbonate with small amounts of magnesium carbonate, calcium sulphate and calcium phosphate. P. miliaris feeds on seaweed and animal debris and has a relatively simple structure and physiology. Tubular feet protrude through the shell and propel the animal using a hydraulic system. The central cavity, the coelem, contains a fluid similar to sea water together with a selection of coelomocytes which have various functions including transport of essential substances, excretion, regeneration, some are phagocytic and others may be involved in clotting. Five large gonads are arranged radially and there is a gut with associated digestive organs. Gills are small and found near the mouthparts. In ^he initial experiments (11) [2- Cjmevalon ic acid and [7- H]-7-dehydrocholesterol were used as possible precursors. Label from mevalonic acid was found in squalene, lanosterol, cholesterol and 7-dehydrocholesterol but not in cholecalciferol or precholecalciferol. For many invertebrates eg. coelenterates, crustaceans, some protozoans, molluscs and annelids, cholesterol or other sterols are a dietary requirement and sterol synthesis in these animals is blocked at the level of squalene formation from farnesyl diphosphate (12). P. miliaris in common with all other echinoderms studied synthesizes cholesterol and 7-dehydrocholesterol is thought to be the immediate precursor of cholesterol. This was proven by the experiment with tritiated 7-dehydrocholesterol, 75% of the recovered radioactivity being located in cholesterol. When the animals were injected with [7- H]-7-dehydrocholesterol and the aquarium irradiated with a UV lamp a very small incorporation into cholecalciferol was detected and cholecalciferol was also detected chemically with the mass spectrometer

(11). Incubation of P. miliaris specimens for 24-48 hours with [1,2- H]-cholecalciferol (11) gave somewhat surprising results. About 1% of the recovered radioactivity appeared in cholecalciferol esters, 4-5% in 25-hydroxycholecalciferol and 30-45% in precholecalciferol. No 1,25-dihydroxychol^calciferol was detected even following incubation with 25-hydroxy-[26,27- H]-cholecalciferol. The presence of over 40% precholecalciferol in a mixture with cholecalciferol was unexpected since at 10° the equilibrium between these two compounds should contain only 5.5% precholecalciferol. Furthermore the time taken to produce 5.5% precholecalciferol from calciferol would be extremely long; it would take 54 days to produce 5% precholecalciferol. The thermal equilibrium between the vitamin and the previtamin requires a temperature of around 130° to allow for 40% previtamin! Two factors are immediately obvious. The equilibrium between the two compounds has been disturbed heavily towards precholecalciferol and also

86 the speed at which isomerization has taken place is very rapid. The equilibrium between vitamin and previtamin Clearly a catalyst may be capable of influencing the speed of isomerization but how can the theoretical equilibrium between the cholecalciferol and precholecalciferol be disturbed? It must be remembered that the kinetics of the reaction as determined by Hanewald (4) were for equilibrium in ethanol. In another solvent or perhaps adsorbed on a protein or inorganic material the equilibrium might not be the same. Holick et al. (2) found the conversion of previtamin to vitamin to be about 20% faster in vivo (rat skin) than in vitro but ruled out a catalytic role because incubations with skin homogenates agreed with theoretical values. The authors suggested that removal of the product, cholecalciferol, by translocation on a binding protein would promote cholecalciferol formation. This is clearly possible but there is also the possibility that adsorption onto a cutaneous surface of lipid or protein may have enhanced the reaction rate. Yamamoto and Borch (13) found up to 15 fold enhancement for the thermal isomerization of precholecalciferol if it was incorporated into either egg phosphatidylcholine or dipalmitoylphosphatidylcholine liposomes. Havinga (14) suggested that the vitamin occurred predominantly in a mixture with the previtamin because the exocyclic double bond reduces the strain imposed by the trans attached D ring. He showed that removal of the D ring gave a model compound in which the previtamin predominated and another model compound with the 18-methyl in the α-configuration (ie.

Precholecalciferol (18-P-CH3, 14-a-H)

Hypothetical compound (18-a-CH3, 14-a-H)

18-a-methyl, 14-a-hydrogen) also predominated in the previtamin form. It seemed possible that the disturbance of the equilibrium between the vitamin and previtamin as found in P. miliaris might be explainable if in fact the 'previtamin' had a structure modified so as to reduce the strain of the D ring. Precholecalciferol has an 18-ß-methyl and 14-a-hydrogen and isomerization of the methyl to an 18-a-position is not possible without removing the methyl. However it would be possible to modify the 14-a-hydrogen to 14-ß using perhaps the intermediacy of isotachysterol.

CH

Hypothetical compound (18-ß-CH3, 14-3-H) Isotachysterol

3

87 This would produce a connection between the C and D ring similar to that where there is an 18-a-methyl and 14-a-hydrogen and this too may be stable in the previtamin form. Jeganathan et al. have made a very similar compound (ie. with 18-ß-methyl and 14-3-hydrogen) but with a 1-hydroxyl in place of the 3-hydroxyl and this compound is stable in the previtamin form (15). If the 'precholecalciferol' produced in P. miliaris had this sort of structural modification which brought about the disturbance in position of equilibrium with cholecalciferol then it would not conform with the kinetic data of Hanewald (4). Following incubation of [1,2- H]-cholecalciferol with P. miliaris, the tritiated precholecalciferol was isolated and was mixed with authentic unlabelled precholecalciferol. The mixture was dissolved in hexane and heated to 40 and the rates of conversion to cholecalciferol examined. The labelled and unlabelled material isomerized to cholecalciferol at a similar rate in agreement with the theoretical value. This indicates that the P. miliaris isomer when isolated is precholecalciferol but perhaps when present in the animal, adsorbed onto protein or inorganic material, modification to a more stable isomer may occur. Distribution of cholecalciferol and precholecalciferol in tissues of P. miliaris In the initial experiments the whole anima^ was treated to lipid extraction following incubation with [1,2- H]-cholecalciferol and a knowledge of where the precholecalciferol was formed was deemed essential. To investigate the uptake of cholecalciferol and formation of precholecalciferol, a number of specimens of P. miliaris were injected w i t h [1,2- H]-cholecalciferol (lpCi/animal) and pairs of animals were sacrificed at 3,6,12,24 and 48 hours. Tissues were obtained by dissection. Five 'tissues' were removed; the shell together with the bony mouthparts (also known as Aristotle's lantern); the gonads; the gut; the coelomic fluid and the coelomocytes (obtained by centrifugation of the coelomic fluid at lOOOxg. for 10 minutes). P. miliaris is quite small and it was impossible to isolate every tissue and the various fractions isolated are unlikely to be homogeneous. Coelomocytes are found in the coelomic fluid but also they occur in most tissues of this sea urchin. The shell had parts of the hydraulic system attached and the small gills may well have been with the shell. Nevertheless it was felt that this relatively crude separation would still give some idea of the distribution of the vitamin and previtamin. Another problem concerned the site of injection. The radiolabelled cholecalciferol was taken up in 0.2% Tween 80 in sterile sea water (the cholecalciferol was taken up into Tween 80 which was then suspended in the sea water) and 1μ0ί/0.2πι1 of 0.2% Tween 80/sea water was injected per animal. The injection was through the peristomial membrane (which holds the mouthparts) and into the coelomic cavity. However there was always the chance that one of the tissues suspended in the coelomic cavity was penetrated on injection. Because of the size of the injection (0.2ml), the majority of the material would finish up in the coelomic fluid but small amounts may have entered individual tissues. This may explain some of the variation in uptake by tissues. The animals were maintained in sea water aquaria at 10° for the period of the incubation. Although the

88 radiolabelled cholecalciferol is excreted in part (possibly via the gut and anal opening) marine animals are extremely adept at concentrating material from the surrounding sea water, perhaps by uptake by the gills, and so, much of the excreted cholecalciferol may be taken up once more during the course of the incubation. Figure 4 shows the uptake of radioactivity into the various tissues analysed. As can be seen, over a period of 48 hours most of the recovered radioactivity appeared in the 'shell' fraction. The radiolabelled cholecalciferol was injected into the coelomic fluid and disappears either by uptake by the tissues or excretion. The coelomocytes appear to take up the radiolabel over the first 12 hours and as some components of the coelomocytes are known to be involved in transport it seems likely that they are involved here in uptake and transport of cholecalciferol.

AOO -

300

-

•H > •H

200

-

PS

100

Shell —α Coelomocytes -· Coelomic fluid —ο Gonads Gut

•μ •P υ cd ο •Η Ό Λ

20 Figure 4:

Hours

Incorporation of cholecalciferol into tissues of P.miliaris

Figure 5 shows the proportion of cholecalciferol and precholecalciferol in each tissue. Precholecalciferol appears in quite high concentration in gut, gonads and shell with the shell being quantitatively the largest. The coelomic fluid contained only very small proportions of precholecalciferol, agreeing with the amounts expected by theoretical considerations. The coelomocytes contained relatively large amounts of radioactivity over the first 12 hours and the proportion of precholecalciferol was never very large, (11% of the total was precholecalciferol at 12 hours). It had been hoped that these experiments would give some idea of the origin of the

89 precholecalciferol but it was not the case. The proportion of precholecalciferol built up in the gut, gonads and shell over the period of 48 hours and either all these tissues are capable of catalysing formation of the previtamin or transport between tissues must be extensive.

400-1

ε

Ο. X» •P •P υ Π) ο •Η Ό (β Ρί

λ Β C D Ε

3hr A Β C D Ε Figure 5:

-

ABCDE 6hr

coelomic fluid coelomocytes gut gonads shell

ABCDE 12hr

24hr

48hr

Cholecalciferol Precholecalciferol

Precholecalciferol and cholecalciferol in tissues of P. miliaris

Metabolism of precholecalciferol The process illustrated in the experiments described so far may be something of an artefact. If P. miliaris does form vitamin D then it is likely that the photoconversion of 7-dehydrocholesterol to precholecalciferol takes place in the shell. The shell contains about half the total 7-dehydrocholesterol to be found in P. miliaris (about 0.5mg 7-dehydrocholesterol/10g animal) (11) and the required light could not penetrate the shell to internal tissues. Therefore the precholecalciferol formed in the shell would either be converted to cholecalciferol in situ or transported to another tissue. As stated earlier some 25-hydroxycholecalciferol was formed by P. miliaris and Weiner et al. (16) also found this compound to be produced in the land snails Levantina hiersolyma and Theba pisana. In neither case was any 1,25-hydroxycholecalciferol produced. The formation of 25-hydroxycholecalciferol in P. miliaris appeared not to be connected with

90 calcium levels since maintenance of animals in either low calcium or high calcium sea water did not effect production. Thus the 25-hydroxycholecalciferol may not be a hormone or related to a hormone in this animal and it perhaps significant that some invertebrate species introduce a hydroxyl in the 25-position of sterols in ecdysone synthesis (17).2 To investigate the metabolism of precholecalciferol, a sample of [1,2-^H]-precholecalciferol was prepared by refluxing [1,2- H]-cholecalciferol in ethanol and separating the previtamin by thin layer chromatography. This was injected into P. miliaris and after 24 hours the resulting mixture of previtamin and vitamin isolated from the animal contained 35% previtamin and 65% vitamin. This is very similar to the mixture obtained starting with cholecalciferol. In vitro studies Four tissues dissected from P. miliaris ie. gut, gonad, slpll and coelomic fluid (containing coelomocytes) were incubated with [1,2- H]cholecalciferol in sea water containing glucose and antibiotics to prevent bacterial contamination (11). Only the shell produced appreciable amounts of precholecalciferol (21% of the recovered radioactivity). The incubations were carried out at 15 for 18 hours. Further studies using shell fragments or ground shell suggested that the catalyst was not an enzyme since treatment with 8M urea or trypsin or boiling had little effect. Extraction of shell with methanol severely reduced the catalytic activity but the lipid extract was not an effective agent. At this point it appeared that the inorganic material of the shell, perhaps in the presence of lipid might be the catalytic and binding agent which disturbed the equilibrium. 5,6-Trans cholecalciferol formation With the increasing evidence that shell material was involved in the formation of precholecalciferol from cholecalciferol a series of inorganic salts were tested for catalytic activity. Small columns of absorbent were prepared and l-2mg of cholecalciferol was applied in hexane and the column was eluted with either hexane or ether. The eluate contained an isomer of vitamin D which could be separated from precholecalciferol and was shown to be 5,6-trans cholecalciferol (11). The 5,6-trans cholecalciferol migrated with authentic material, prepared by the method of Lawson and Bell (18), on h.p.l.c. (Ultrasphere 5μ Silica column, 10% methyl-t-butyl ether solvent). Elution volumes were 5,6-trans cholecalciferol, 10.4ml, precholecalciferol, 11.4ml and cholecalciferol, 16.4ml. Conversions of up to 75% could be achieved by reducing flow rate and increasing the size of the inorganic salt column (Table 1). The trans cholecalciferol was formed using CaCO^ and CaCl^ but not CaO, Ca(0H)2, CaSO^ or CaitK^^· KHC03 could carry out the reaction but not NaHCO^. KCl and NaCl were unable to isomerize the cholecalciferol whereas MgCl^ and KF had the ability. The relationship between the catalytic property and the nature of the salt was not obvious and requires clarification. As the shell of P. miliaris is composed of several inorganic salts it was decided to investigate whether it too could carry out the formation of

91 5,6-trans cholecalciferol. Accordingly columns of dried, ground shell material were prepared and cholecalciferol was applied as above. However no 5,6-trans cholecalciferol was formed. Table 1 Formation of 5,6-trans cholecalciferol on inorganic salts Salt CaCOCa(OH)» Ca(NO j CaSO CaCllJ KCl NaCl CdCl„ MgCli FeCl^ CsCl"

% 5,6-Trans cholecalciferol 72 0 0 0 75 0 0 21 67 10 34

Salt

% 5,6-Trans cholecalciferol 0 72 12 75 42 74 0 19

NaHCO. KHCOK S0 2 A KHOKF 3 KBr KI

lmg. Vitamin D^ in hexane was applied to a column of 3g. of inorganic salt and the material was eluted with hexane/ether. Conclusions P. miliaris is capable of converting cholecalciferol to precholecalciferol and vice versa at very much enhanced rates and the resulting mixture of about 40% precholecalciferol and 60% cholecalciferol is not in keeping with theoretical values. The disturbance in the position of equilibrium which should at this temperature be 5.5% precholecalciferol and 94.5% cholecalciferol could come about by removal of precholecalciferol from the reaction by some form of preferential binding. This could occur in many ways and two possibilities are shown. UV 1)

7-DHC

2)

7-DHC-
2-95% air, and its production increased time-dependently up to 48 h (2-6%).

Since it was assumed that some

components of the medium caused the conversion of 25-(OH)Dg to 10-oxo19-nor-25-(0H)Dß, we investigated conditions to produce the compound chemically.

In preliminary experiments, we found that a combination of

iron(II) and molecular oxygen effects the conversion of 25-(0H)Dß to 10-oxo-19-nor-25-(0H)Dß

(21).

This

indicates

that

iron and

some

components in the medium that can act as electron transporting agents cause the autooxidative production of 10-oxo-19-nor-25-(0H)Dß. The fact that the addition of the adrenal reconstitution system, where adrenodoxin and adrenodoxin reductase are included,

improved the production

of 10-oxo-19-nor-25-(0H)Dß apparently supports this supposition.

So

we suggested the following mechanism for the formation of (5E)-10-oxo19-norvitamin D from vitamin D by the action of Fe(II) and molecular oxygen (Scheme 2). Iron(II) and dioxygen f.orm a complex of Fe(III) and superoxide anion radical (Fe(III)-02V).

Iron(III) abstracts one π -

105 e l e c t r o n from the t r i e n e part of v i t a m i n D to y i e l d c a t i o n r a d i c a l ( V I I ) which r e a c t s at C(19) with the superoxide anion r a d i c a l . The resulting z w i t t e r ionic peroxide c y c l i z e s to form dioxetane (IX) which undergoes retro-cycloaddition to a f f o r d ( 5 E ) - 1 0 - o x o - 1 9 - n o r v i t a m i n D. This mechanism explains why 5E-isomer i s produced from the (5Z)-vitamin D. Namely, since the bond between C(5) and C(6) can rotate in either the cation radical ( V I I ) or the zwitter ionic species ( V I I I ) , s e l e c t i v e formation of the thermodynamically more stable (5E)-dioxetane (IX) i s quite reasonable. Similar mechanisms have been suggested in autooxidation of l i p i d s (22) and in some enzymatic o x i d a t i o n s c a t a l y z e d by dioxygenases (23). T h e r e f o r e , i f 10-oxo-19-norvitamin D i s produced by an enzymatic process, a similar mechanism, supposedly, i s operating. Scheme 2

Production of new metabolite: When 25-(0H)Dß was incubated in mouse a l v e o l a r macrophages f o r a longer t i m e , a new and prominant peak appeared on HPLC ( F i g . 1) (20,21). The new peak did not appear when 25-(0H)Dß was incubated with heatd e a c t i v a t e d c e l l s or i t was i n c u b a t e d w i t h the i n c u b a t i o n medium alone without the c e l l s . The new peak was eluted just a f t e r 24R,25-(0H)2D3 on a s t r a i g h t phase HPLC with 2-propanol-hexane as the eluent. The new m e t a b o l i t e was a l s o produced by other phagocytic c e l l s , Ml, HL-60, and U937 ( F i g . 2). The new m e t a b o l i t e produced by the Ml c e l l s was i s o l a t e d f o r structural determination. Thus, Ml c e l l s (8 χ 10 8 c e l l s ) were

14.1XOHM). ZJIOHID,

10-oio-H-nor1XOMID,

2B,2C(OH^O) III (E)

m.IVOHI^J,

[B]

10 I«« ?· 'o a X i· 0

5

10

15

20

25

30

35

Retention Time (mln)

F i g . 1. HPLC p r o f i l e of the metabolites produced by mouse alveolar macrophages incubated w i t h 2 5 - ( 0 H ) D 3 f o r 24 h. (Finepak SIL, 10% 2-Propanol in hexane, 1 ml/min)

40

106

1 h

24 h

new metabolite

j f J Μφϊ

U937

HI

Ü HL-60

F i g . 2. Conversion of 25-(0H)Ü2 t o the new metabolite. The c e l l s were incubated with 25-(0H)[ 3 H]D 3 f o r 1 hour (empty columns) or 24 hours (hatched columns).

incubated w i t h 25-(0H)D 3 (640 Ug) f o r 18 h at 37 'C, under 5% C0 2 , 95% a i r in 400 mL of serum-free medium. A f t e r the incubation, the c e l l s and the medium together were extracted with dichloromethane-methanol and the o r g a n i c e x t r a c t s were b r i e f l y p u r i f i e d by SEP-PACK. The dihydroxyvitamin D f r a c t i o n of the SEP-PACK column was p u r i f i e d three t i m e s on HPLC to g i v e a homogeneous m e t a b o l i t e (10.5 y g ) . The new metabolite showed an absorption maximum at 295 nm (95% ethanol) and a minimum at 245 nm (Fig. 3), showing that the conjugated triene part of vitamin D had been changed. The wavelength of the maximum suggests the presence of a conjugated dienone function as the chromophore. The IR spectrum of the metabolite (Fig. 4) showed absorption due to a conjugated carbonyl group at 1660 cm-^. The mass spectrum (Fig. 5) showed a parent ion at m/e 414. I t s elemental composition was determined to be C27H42O3 by high r e s o l u t i o n mass s p e c t r o m e t r y . The formula corresponds to an incorporation of one oxygen atom and the loss of two hydrogen atoms from the starting 25-(0H)Dß. The ^H NMR spectrum (Fig. 6) showed f i v e o l e f i n i c protons which appear in a r e l a t i v e l y l o w e r f i e l d . This i s in accord with the presence of double bonds conjugated with a carbonyl group: two protons on a trans-oriented double bond as AB-type d o u b l e t s at . Chem. 45, 3253-3258 20. H a y a s h i , T., Yamada, S . , M i y a u r a , C., T a n a k a , H., Yamamoto, K., Abe, Ε . , Takayama, Η., Suda, Τ. (1987) FEBS L e t t e r s 218, 200-204 21. Yamada, S. Yamamoto, K., Takayama, H., H a y a s h i , T., M i y a u r a , C., T a n a k a , H., Abe, Ε., Suda, Τ. ( 1 9 8 7 ) J . B i o l . Chem. 2 6 2 , 1 2 9 3 9 12944 22. K a p p u s , H. ( 1 9 8 5 ) in O x i d a t i v e S t r e s s S i e s , H.(ed), Academic P r e s s , London, Chap. 12 23. Matuura, T. (1977) Tetrahedron 33, 2869-2905

EXTRARENAL PRODUCTION OF CALCITRIOL IN CHRONIC RENAL FAILURE A. Dusso, S. Lopez-Hilker, N. Rapp, and E. Slatopolsky. Renal Division, Department of Medicine Washington University School of Medicine St. Louis, Missouri, U.S.A. Introduction Circulating l,25(OH)2D concentrations are not greatly affected by increased vitamin D intake in normal adults (1,2) or intact chicks and rats (3). However, an increase in serum calcitriol after 25(OH)D administration was found in hypoparathyroidism (4), in normal children (5,6) and there are also conflicting reports regarding the effects of 25(OH)D therapy in uremia (7-12). Our experiments were conducted to provide further insight into the mechanisms affecting l,25(OH)2D levels in chronic renal failure. Methods 4 normal volunteers, 4 anephric patients undergoing hemodyalisis, 3 normal dogs and 7 5/6 nephrectomized dogs were studied. 200 ug of 25(OH)D were administered daily to normal and anephric humans. Dogs received 25(OH)D, at a dose of 100 ug, every other day for two weeks, followed by 50 ug for two more weeks. Studies were continued 2 weeks after cessation of 25(OH)D administration. Fasting blood samples were analyzed for ionized calcium (ICa), phosphorus (P) and creatinine following standard procedures. Nterminal PTH was quantitated in dogs (13) and the mid region C-terminal fragment was measured in humans (14). 25(OH)D was measured after C18 Sep Pak extraction with a radioreceptor assay using sheep Vitamin D binding protein (15). l,25(OH)2D was measured according to Reinhardt (16). No significant amounts of 19-nor-10-keto-25(OH)D interfering with l,25(OH)2D measurements were detected. In anephrics, the amount of l,25(OH)2D measured as described, was confirmed after 3 different HPLC purifications of the l,25(OH)2D fraction. Results and Discussion By the end of the second week of receiving 25(OH)D, normal dogs increased serum 25(OH)D levels from 33.4 + 1.5 to 137.8 + 8.6 ng/ml. No significant changes in l,25(OH)2D concentrations were found. On the contrary, uremic dogs significantly increased calcitriol levels from 16.4 +. 0.9 to 28.0 +. 1.9 pg/ml (p< 0.001). A significant correlation coefficient between serum levels of l,25(OH)2D and 25(OH)D was found for each uremic dog (pcO.OOl). No significant changes in serum ICa, P, or N-terminal PTH were found, suggesting that 25(OH)D, per se, enhanced l,25(OH)2D levels in uremic dogs. To test the contribution of extrarenal sources to the response to 25(OH)D administration, we studied 4 anephric patients undergoing hemodyalisis. Normal volunteers increased 25(OH)D levels from 29.0 + 1.7 to 152.2 ± 8.2 ng/ml after two weeks of oral 25(OH)D intake. Again, l,25(OH)?D levels remained unchanged. In anephric patients, the increase in 25(OH)D levels

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

113

(Basal:23.2 ± 1.8 ng/ml; 2nd week: 203.9 ± 23.4 ng/ml; ρ < 0.001) was accompanied by a concomitant enhancement in l,25(OH)2D concentrations (Basal 5.5 ± 1.2 pg/ml; 2nd week: 19.6 ± 4.9; ρ < 0.02). A correlation coefficient of 0.72 (p < 0.001; n=18) was found for the relationship between serum levels of substrate (25(OH)D) and product (l,25(OH)2D) of the extra renal enzyme. No significant changes in ICa, Ρ or PTH were detected either for normal or for anephric humans. This strongly suggests that the increase in l,25(OH)2D levels following 25(OH)D administration is not exclusively a consequence of enhanced substrate availability to renal 1-alpha-hydroxylase. 25(OH)D can stimulate extrarenal production in the absence of renal mass. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. -

Chesney R.W., Rosen J.F., Hamstra A.J., Smith C., Mahaffey K., De Luca H.F. (1981) J. Clin. Endocrinol. Metab. 53:139-142. Hughes M.R., Baylink D.J., Jones P.E., Haussler M.R., (1976) J. Clin. Invest. 58:61-70. Hughes M.R., Baylink D.J., Gonnerman W.A., Toverud K.U., Ramp W.K., Haussler M.R. (1977) Endocrinology 100:799-806. Lund B.J., Sorensen O.H., Lund B.I., Bishop J.E., Norman A.W. (1980) -J. Clin. Endocrinol. Metab. 51:606-610. Taylor A.T., Norman M.E., (1984) Pediatric Res. 18:886-890. Stem P.H., Taylor A.B., Bell N., Epstein S. (1981) J. Clin. Invest. 68:1374-1377. Taylor Α., Norman M.E. (1982). Met. Bone Dis. Rel. Res. 4:255-261 Langman C.B., Mazur A.T., Baron R., Norman M.E. (1982) J. Pediatr. 100:815-820. Zerwekh J.E., McPhaul J.J., Parker T.F., Pak C.Y. (1983) Kidney Int. 23:401-406. Lucas P.A., Lucas R.C., Brown R.C., Woodhead J.S., Coles G.A. (1986) Nephrology: 25:7-10. Halloran B.P., Schaefer P., Lifschitz M., Levens Μ., Goldmsted R.S. (1984) J. Clin. Endocrin. ' Metab. 59:1063-1069. Eastwood J.B., Stamp T.B.C., De Wardener H.E., Bordier P.J., Arnaud (1977). Clin. Science Mol. Med. 52: 499-508. Lopez Hilker S., Galceran T., Chan Y.L., Rapp N., Martin K., Slatopolsky E. (1986) J. Clin. Invest. 78:1097-1102. Hruska K., Kopelman R., Rutherford E., Klahr S., Slatopolsky E. (1975) J. Clin. Invest. 56:39-48. Horst R. (1983) In Assay of calcium regulating Hormones. D. Bikle (ed) Chapter 2. pg. 41-47. Springer Verlag. Reinhardt T.A., Horst R.L., Orf W., Hollis B.W. (1984) J. Clin. Endocrinol. Metab. 58:91-98.

METABOLISM OF DUODENA FROM NORMAL CHICKS * l,25(OH) *D- IN PERFUSED * Y. * YOSHIMOTO , Κ. OHNO , T. FUJITA AND S. ISHIZUKA 3rd Division, Department of Medicine, Kobe University, Kobe 650 and **Teijin Institute for Bio-Medical Research, Tokyo 191, Japan. Introduction The most active form of vitamin D3, 1,25-dihydroxyvitamin Dß(1,25(0H)2Ü3), mediates the primary biological actions classically attributed to vitamin D in the intestine, bone and other tissues, although the pathways of 1,25(0H)2D3 metabolism are not totally understood. The present study t le was designed to explore the metabolism of 1,25(0H)2D3 ' intestine using vascularly perfused duodena of normal chicks. Materials and Methods 8 to 10 weeks-vit D-replete chicks were anesthetized with pentobarbital and duodenal loop surgically exposed and kept moist with physiological saline. The celiac artery and vein were canulated with PE-50 tubing and perfused with modified Gey's Balanced Salt Solution (GBSS) at a flow rate of 2 ml/min. The perfusion apparatus was equipped with two peristaltic pumps. [1-3H]1,25(0H)2D3 ( ΙΟ - 8 M, specific activity 18.2 Ci/ mmol ) was introduced into the perfusion medium through a second auxiliary pump in GBSS lacking bicarbonate and the collected perfusate was recirculated. Perfusate was collected every 30 min and the used duodena were obtained at the end of experiments (2 hours after perfusion) for metabolites analysis. The metabolites of 1,25(0H)2D3 were separated and identified by high performance liquid chromatography (HPLC). Results and Conclusion With time the amounts of radioactivity in the perfusate present as 1 , 2 5 ( 0 H ) 2 D 3 diminished and 2 known metabolites, 1 , 2 4 ( R ) 2 5 ( 0 H ) 3 D 3 and 1,25(0H)2-24-oxo-D3 and one unknown metabolite appeared after 60 min in perfusate, solution into duodena and duodena (Fig. 1). About 70 % of labelled l,25(OH)2D3 was distributed to duodena. In another experiments we investigated 1,25(0H)2Ü3 metabolites concentration in chick serum and duodena after intravenous administration of 160 ng of [ 1 - 3 H ] 1 , 2 5 ( 0 H ) 2 D 3 to vitamin D-replete chick. As shown in Fig. 2A, in serum 1,24(R)25(0H)QDO» 1 , 2 5 ( 0 H ) 2 - 2 4 - O X O - D 3 and 2 3 ( S ) 2 5 ( R ) - l , 2 5 ( 0 H ) 2 D 3 - 2 6 , 2 3 - l a c t o n e appeared within 2 hours and 40 % of 1 , 2 5 ( 0 H ) 2 D 3 was metabolized to 2 3 ( S ) 2 5 ( R ) - 1 , 2 5 (0H)2Do-26,23-lactone 24 hours after administrations. On the other hand, in intestine 1 , 2 4 ( R ) 2 5 ( 0 H ) 3 D 3 and l , 2 5 ( O H ) 2 - 2 4 - o x o - D o appeared within 1 0 min and their concentrations reached the maximum at 2 hours and then decreased rapidly. But 23(S)25(R)-1,25(0H) 2 D 3 -26,23-lactone did not appear until 24 hours after administrations (Fig. 2B). We also investigated the production of 1,25(0H)2D3 metabolites in chick intestinal mucosa homogenates, which showed the major metabolite was 23(S)25(R)-1,25 (0H) 2 D 3 -26,23-lactone ( 7th Workshop on Vitamin D; Abstract No. 294 ) . In summary, although the major metabolite of l,25(OH)2Ü3 was 1,25(0H)2Ü326,23-lactone in chick in vivo study and in vitro study, we could not find its existence in chick intestine itself in the perfusion system and in vivo system. The precise mechanism of this discrepancy should be crarified, but the concentrations of 1,25(0H)2Ü3 in tissues may be the key role of further metabolism of l,25(OH)2D3·

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · New York - Printed in Germany

115 159ίΙΡΑ in n-hexane

RETENTION TIME (min)

Fig. 1. HPCL profile of extracts of perfusate 2 hours after perfusion of duodenum. The column was eluted with 15% isopropanol in n-hexane. 1or,2S-{OH)iD* Metabolites Concentration in Chick Serum 1 a, 25-(OH)iDs Metabolites Concentration in Chick Intestine After i.v. Administration of 1«r, 25-3 has a direct effect on osteoblasts (1,2). It has been proposed that all target tissues of 1,25- (OH) ^Dg possess the enzymatic capability for the 24-oxidation of this steroid hormone, a process which results in its inactivation (3). In order to understand how the action of 1,25-(CH) 2D3 on osteoblasts may be regulated, we examined the metabolism of 1,25(OH) ^D^ by a human osteoblastic osteosarcoma cell line, U-20S. In this preliminary study we identify by «migration on 3 HFLC systems, four metabolites produced from l,25-(OH)2D3 by the U-20S cells. Methods and Materials U-20S cells were maintained in McCoy's 5A medium containing 10% FCS. When nearly confluent the FCS was removed and 24 h later 1,25-(OH) 2D3 (3.5 χ ΙΟ - 7 Μ, 8 Ci/mole) was added in absolute ethanol (0.1% v/v). Incubation with substrate occurred at 37 °C, 5% C0 2 with continuous shaking for 18 h. Hie lipid soluble vitamin D metabolites were then extracted frcm the cells and medium using methanol-chloroform. Separation of the vitamin D3 metabolites was achieved by HPLC using a Zorbax-SIL 3 μ (6.2 mm χ 15 cm) column with a solvent system of hexane, iscpropanol, methanol (iyi/Μ) (88/10/2) at a flow rate of 2.0 ml/min., followed by HPLC on Zorbax-CN using H/I/M (91/7/2) at a flow rate of 1.3 ml/min. A final purification step WEIS achieved Jay HPK: on Zorbax-SIL using H/I/M (88/10/2). Results Putative vitamin D metabolites were located by virtue of their vitamin D chromophore using a diode-array spectrophotometric detector. Identification of four metabolites of 1,25- (CH) 2D3 was achieved based on their ocsnigration on each HPLC system with authentic standards (Figure 1) and by mass spectrometry. These metabolites were identified as 1,24,25(ΟΗ)3Ε>3; 24-oxo-l,25-(OH)2C>3; 24-οχο-1,23,25-(0Η)3Ε>3; and 24,25,26,27tetranor-l,23-(0H)2D3· Metabolism of 1,25-(0Η)2Ι>) was not detected when substrate was incubated in the absence of cells. Discussion We have demonstrated for the first time that a human bone cell line can metabolize 1,25-(0H)2E>3 by side chain oxidation. The metabolites produced form the 24-oxidation pathway for 1,25- (CH) -^Th, which has been demonstrated in other 1,25-(ΟΗ)2Ε>3 target tissues of kidney and intestine (4,5). While the role of this side chain hydroxylation of 1,25-(OH)2D3 in a target tissue is unknown, available evidence seems to suggest that this pathway for l,25-(OH)2E>3 has a catabolic function (6). Hence this may be one way that the action of this hormone in osteoblasts is regulated.

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · N e w York - Printed in Germany

123

5 'Μ ιη

•Ν C4 ι^ι

f.

4

1AJV

j

1Θ Time

Figure 1:

Θ

1:

HPLC of lipid extract of U-20S cells incubated with 1,25(OH) 2D3. Peaks numbered 1 through 6 possessed the vitamin D chromophore and were identified as: 1. Unidentified 2. 1,25-(OH)2D3 3. 24-oxo-l,25-(CH)2D3 4. 24,25,26,27-tetranor-l,23-(OH)2D3 5. 24-0X0-1,23,25-(CH)3D3 6. 1,24,25-(CH)3D3

References 1. Price, P.A., Baukol, S.A. (1980) J.Biol.Chem. 255:11660-11663. 2. Mulkins, M.A., Manolagas, S.C., Deftos, L.J., Sussmani, H.H. (1983) J.Biol.Chem. 258:6219-6225. 3. Lahnes, D., Jones, G. (1987) J.Biol.Chem. 262:14393-14401. 4. Jones, G., Vriezen, D., Ißhnes, D., Palda, V., Edwards, N.S. (1987) Steroids 49:29-55. 5. Napoli, J.L., Horst, R.L. (1983) Biochemistry 22:5848-5835. 6. Haussler, M.R. (1986) Ann.Rev.Nutr. 6:527-562.

THE VITAMIN D DEPLETION OF CHRONIC CHOLESTASIS IS INDEPENDENT OF THE HEPATIC METABOLISM OF VITAMIN D3. M. GASCON-BARRß, V. PLOURDE, P.M. HUET and B. WILLEMS. Centre de recherche clinique Andr6-Viallet, Höpital Saint-Luc, and Facult6 de M6decine, Universit6 de Montr6al, Montrdal, Qu6bec, CANADA. INTRODUCTION In biliary cirrhosis, administration of large amounts of vitamin D (D) has been reported to be followed by increases in the plasma 25(OH)D suggesting that the D-25 hydroxylase is responsive if sufficient D is brought to the liver (1-3). However, is vitro data have shown pronounced inhibition of the enzyme in cholestatic rats leaving unanswered the question of the capacity of the cholestatic liver to metabolize D3 (4). Moreover, the influence of cholestatic liver disease on the uptake and C-25 hydroxylation of D3 has not yet been studied in experimental models in vivo. The purpose of our studies was to evaluate, in an in vivo model of biliary cirrhosis, the hepatic uptake and C-25 hydroxylation of D3 in order to determine the role of the liver as a contributory factor in the D deficiency associated with chronic cholestatic liver disease. MATERIALS AND METHODS Chronic bile duct ligation (CBDL) was performed in dogs, as described by Bosch al (5), while controls underwent diversion of bile flow through the urinary bladder using a choledococystostomy anastomosis in order to achieve a malabsorption of fat soluble substances similar to that observed in the presence of cholestasis. Ten to 12 weeks after surgery, D3 uptake and metabolism were evaluated in vivo by direct intraportal injection of the vitamin, and the subsequent hepatic vein collection of blood samples over the following 150 min. Evaluation of D3 and 25(OH)D3 was done by HPLC RESULTS AND DISCUSSION Histologic evaluation of the hepatic lesion revealed that CBDL dogs had severe cholestasis and macronoaular cirrhosis. Controls had normal livers and their handling of D3 was similar to that of D-depleted dogs with intact biliary function. Data obtained on the plasma 25(OH)D at the time of surgery, and at different time point after surgery show that the absence of bile flow in the intestine led to a progressive depletion of D reserve in CBDL as well as in controls (plasma 25(OH)D from 34.5 to 5.4 ng/mL within 25 days). However, as shown in Table 1, neither the fractional hepatic D3 uptake the hepatic systemic clearance, nor the hepatic intrinsic clearance were significantly affected by chronic cholestasis. Moreover, the transformation of D3 into 25(OH)D3 was found to increase linearly with time in both groups of animals to reach values of 44.8 ± 4.4 and 38.3 ± 6.1% of the dose of D* injected, 150 min after D3 administration in controls and CBDL animals respectively (N.S.).

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · N e w York - Printed in Germany

125

Table 1 $ρπιτη fripchemistiy, and hepatiff Mfftakf Λ η Λ clearance nf pH]D3

Parameters

1.

2.

Control dogs (n=5)

Serum .Albumin, g/dL .Total bilirubin, mg/dL .ALT, IU/L .Alk. phosphatase, IU/L .25(OH)D .l,25(OH) 2 D

2.2 ± 0.1 ± 38.6 ± 81.0 ± 8.0 ± 52.3 ±

Hepatic parameters of [2H]D3 .Fractional uptake, % .Systemic clearance, mL/min/kg .Intrinsic clearance, mL/min/kg

31.8 ± 7.4 19.9 ± 3.7 33.4 ± 10.5

0.2 0.0 7.6 19.1 1.5 2.3

CBDLdogs (n=5)

Ρ

1.2 ± 0.1 6.2 ± 0.9 539.6 ± 141.2 1886 ± 558.8 5.2 ± 0.8 27.8 ± 3.3

95

>95

•

•

>95

94

>95

10 >95 65

>95 >95 >95

>95

>95 >95 >95

>95 >95 89

>95 >95 >95

83

94

•

•

•

•

*

52

•

•

•

•

51

58

>95

31

51

73

35

•

•

83 67

1,25 (OH)2D3

>95 >95

>95

*

•

53

>95

•

30

•

15

60 85

Oig) 0 4 16

•

72 • •

•

*

*

62

52

0 4 16 0 4 16 0 4 16 0 4 16

Insufficient counts (or analysis

Table 1. Percentage of counts from Figure 1 migrating with authentic metabolite in isocratic HPLC.

In serum, 25 hydroxyvitamin D remained the major metabolite in all situations. However, in the storage organs, the higher dosage of vitamin D resulted in a larger percentage of polar metabolites, of which only a fraction showed identity with authentic material on HPLC (Table 1). M o s t notably, fat stored large amounts of polar material, which contained only a small percentage of the two major dihydrox.y compounds. We suggest that these polar compounds may contribute to the soft tissue calcification seen in vitamin D excess. We are currently investigating their identity, using gradient H P L C in several solvent systems. REFERENCES 1.

2.

Ratzkowski, C., Fine N. and Edelstein, S. (1982). Tsr. J. Med. .Sei., 18, 695-700. Mawer, B.E., Backhouse, J., Holman, C., Lumb., G.A. and Stanbury, S.W. (1972). Clin. Sei., 43, 413-431.

This research was supported by the South African MRC.

IDENTIFICATION OF A SUBSET OF DAIRY COWS THAT FAIL TO PRODUCE 1,25-DIHYDROXYVITAMIN D (1,25-(OH)2D) AT THE ONSET OF THE HYPOCALCEMIA ASSOCIATED WITH PARTURIENT PARESIS. J.P. GOFF, T.A. REINHARDT, and R.L. HORST National Animal Disease Center, USDA, ARS, Ames, IA 50010 USA. Introduction Parturient paresis (milk fever) is a hypocalcemic disorder of dairy cows which occurs when the Ca homeostatic mechanisms fail to meet the Ca demands imposed by the onset of lactation. Generally, the hypocalcemia of parturient paresis (PP) is accompanied by a great increase in plasma concentrations of PTH and 1,25-(OH)2D. Typically, cows suffering from PP recover following a single intravenous Ca treatment. However, about 20% of cows treated for parturient paresis suffer a second episode of severe hypocalcemia necessitating further treatment. In this study, the plasma concentration of 1,25-(OH)2D of relapsing PP cows is compared to that of non-relapsing PP cows. Materials and Methods Jersey cows with previous histories of PP were placed on a high calcium diet prior to parturition to induce PP. Daily blood samples were taken beginning 10 d before parturition and for at least 10 d after parturition. Additional blood samples were obtained as paresis developed and prior to treatment with intravenous Ca. Plasma was analyzed for its l,25-(OH)2D, PTH, and Ca content. Results and Discussion Plasma 1,25-(OH)2D and Ca concentration profiles from five relapsing PP and five non-relapsing PP cows are presented in the Figure. Plasma 1,25-(OH)2D concentration of non-relapsing PP cows increased 4- to 5-fold as hypocalcemia developed. However, in relapsing PP cows, plasma 1,25-(OH)2D did not exhibit the same increase as was seen in non-relapsing PP cows, despite the fact that the cows suffered similar degrees of hypocalcemia. Relapsing PP cows did eventually exhibit a 5- to 6-fold increase in plasma 1,25-(OH)2D concentration, but this increase was delayed by 24-48 h when compared to non-relapsing PP cows. The profile of radioimmunoassayable PTH in the plasma of relapsing PP and non-relapsing PP cows was similar to that seen for 1,25-(OH)2D (data not shown), except that delayed secretion of PTH by relapsing PP cows in response to the first hypocalcemic episode was not as clearly defined as it was with 1,25-(OH)2D. Non-relapsing PP cows returned to normocalcemia about 2.5 d after being treated with intravenous calcium after first becoming paretic. Relapsing PP cows required two or more intravenous calcium treatments to effect a complete recovery and did not become normocalcemic until about 5 d

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter&Co., Berlin · N e w York- Printed in Germany

138 after the f i r s t h y p o c a l c e m i a episode. R e c o v e r y f r o m the h y p o c a l c e m i a a s s o c i a t e d w i t h PP seems to occur only after there is a s i g n i f i c a n t increase i n p l a s m a c o n c e n t r a t i o n o f 1,25-(OH)2D.

220-1

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NON-RELAPSING COWS RELAPSING COWS

I I I I I I I—I - 1—I

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-6

-5

-4

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- 1 0

DAYS A R O U N D

1

2

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4

5

6

7

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PARTURITION

Figure. P l a s m a C a a n d l,25-(OH)2D c o n c e n t r a t i o n s b e f o r e a n d after p a r t u r i t i o n i n relapsing a n d n o n - r e l a p s i n g p a r t u r i e n t p a r e t i c cows.

W e h a v e i d e n t i f i e d a subset of dairy cows in w h i c h 1 , 2 5 - ( O H ) 2 D (and p o s s i b l y PTH) p r o d u c t i o n is d e f i c i e n t in r e s p o n s e to the h y p o c a l c e m i a a s s o c i a t e d w i t h PP. M o s t r e l a p s i n g PP cows e x h i b i t e d a d e l a y e d p r o d u c t i o n of 1,25-(OH)2D (2 of 7 r e l a p s i n g PP cows e x a m i n e d e x h i b i t e d no d e l a y e d increase in p l a s m a 1,25-(OH)2D) implying that lack of p r o d u c t i o n of 1 , 2 5 - ( 0 H ) 2 D is a n i m p o r t a n t factor in p r e d i s p o s i n g cows to relapses of p a r t u r i e n t paresis. These d a t a s u g g e s t that therapeutic i n t e r v e n t i o n w i t h 1,25-(OH)2D m a y b e of some a i d in p r e v e n t i n g relapses of p a r t u r i e n t paresis. I n t e r e s t i n g l y , p r e p a r t a l p l a s m a l , 2 5 - ( O H ) 2 D a n d PTH c o n c e n t r a t i o n s of r e l a p s i n g PP cows w e r e also lower t h a n that of n o n - r e l a p s i n g PP cows suggesting that the two c l i n i c a l types o f PP m a y be d i s t i n g u i s h a b l e p r i o r to p a r t u r i t i o n .

THE INTACT HEPATOCYTE HYPOTHESIS APPLIES TO THE C-25 HYDROXYLATION OF VITAMIN D3 IN ALCOHOUC CIRRHOSIS. M. GASCON-BARRE, V. PLOURDE, C. DUBE, N. BENBRAHIM, P. COULOMBE, S. VAT ,T.TERES, C. TREMBLAY and P.M. HUET. Centre de recherche clinique Andri-Viallet, Höpital Saint-Luc, and Faculty de M£decine, Universit6 ae Montr6al, Montr6al, Qu6bec, CANADA INTRODUCTION Studies on vitamin D (D) metabolism in liver disease indicate that the factors responsible for the disturbed D homeostasis are different in cholestatic and hepatocellular diseases (1,2). In alcoholic liver disease, a wide spectrum of prevailing D nutritional status as well as responses to D challenge have been reported (3-5). As yet, no unifying concept on the capacity of the liver to metabolize D at C-25 is yet clearly emerging from the available studies. The purpose of our studies was to evaluate the hepatic C-25 hydroxylation of D3 in an experimental model of liver cirrhosis and to try to relate the metabolic response to the severity of the hepatocellular injury. MATERIAL AND METHODS Micronodular cirrhosis was induced in the rat by chronic CCI4 administration (6). The uptake, and C-25 hydroxylation was D3 were studied in isolated-perfused liver preparations. Furthermore, the transformation of D3 into 25(OH)D3 was also studied in hepatocytes isolated from controls or cirrhotic animals. Separation of Dß metabolites was achieved by HPLC. Evaluation of the hepatic lesions was achieved by histomorphometry (7). RESULTS AND DISCUSSION Cirrhotic rats had a significantly lower fractional hepatic D3 uptake than controls (20.6 ± 2.6% vs 30.7 ± 4.6%, ρ C3 Ο ζ 5Γ Ω 0 SC in CM ζ i Ο C-3 CO Ζ αCM Ο CO χ cc ο LLJ ΙΟ > CSJ Ζ ', ο ο χ CO

SS

10 -

0

2 4 6 8 PERFUSION TIME (HOURS) FIG. CONVERSION RATE OF 3H-(OH)D3 TO 3H-1,25(OH)2D3 IN ISOLATED PERFUSED KIDNEY

TABLE.

%(3H)-25(OH)D3 converted Perfusate Perfusate to (3H) l,25(OH)2D/g kidcalcium (mM) phosphate (mM) Ν ney (mean + SE) Group I 2.5 2.0 5 10.51+2.15 Group II 1.25 1.0 4 9.38 ± 2.05 Group III 1.25 2.0 6 12.00 ± 1.67 Group IV 2J> 1^0 3 8.27 + 1.64 Data in the Table show no significant differences in 3H-1.25(OH)2D3 production between groups suggesting that acute changes in calcium and phosphate do not have a direct effect on renal Ι-α-hydroxylase activity in the isolated perfused rat kidney. Studies are in progress to evaluate the in vitro effect of parathyroid hormone and other known Jji vivo modulators on renal Ι-α-hydroxylase activity in the isolated perfused rat kidney. Reference 1. 2. 3. 4. 5. 6.

Armbrecht, H.J., Wongsurawat, N., Zenser, T.V. and Davis, B. (1983) Archieves of Biochemistry Biophysics, 220 (No.l), 52-29. Hughes, M.R., Brumbaugh, P.F. and Haussler, M.R. (1975) Science, 190, 578-580. Tanaka, Y. and Deluca, H.F. (1984) Am. J. Physiol. 246 (Endo. Metab. 9E168-E173. Trechel, V., Eisman, J.Α., Fischer, J.Α., Bonjour, J.P. and Fleisch, Η. (1980) Am. J. Physiol. 239 (Endo. Metab. 2) E119-E124. Armbrecht, Η.J., Zenser, T.V. and Davis, B.B. (1982) Endocrinology 110, 1983-1988. Reddy, G.S., Jones, G., Kooh, S.W. and Fräser, D. (1982) J. Physiol. 243 (Endocrinol. Metab. 6), E265-E271.

DOES THE ADMINISTRATION O F PHOSPHATE INCREASE THE SERUM 1 , 2 5 ( 0 H ) 2 D CONCENTRATION IN X-LINKED HYPOPHOSPHATEMIC RICKETS? Η. TANAKA, Y. SEINO, Κ. YAMAOKA, S. NAKAJIMA, Κ. OHZONO, M . SHIMA·, H. KUROSE and H. YABUUCHI. Department of Pediatrics, Osaka University School of Medicine, Osaka, Japan· Introduction X-linked hypophosphatemic rickets (XLH) is a prototype of vitamin D resistant rickets. Previous reports indicate a blunted renal 250HD-1hydroxylase response to a potent stimulator, phosphate restriction (1-4·). However, the effect of an elevated serum Ρ level induced by a h i g h phosphate diet on 1-hydroxylase has never been reported except for our report on hypophosphatemic mice (5). Recently, it had been reported t h a t in normal healthy men w i t h dietary phosphate supplementation, the 1,25(0H) 2 D production rate was decreased, followed by a decrement in the serum concentration of 1 , 2 5 ( 0 H ) 2 D (6,7). We examined here an effect of single oral phosphate loading on the serum 1 , 2 5 ( 0 H ) 2 D level in XLH patients compared with normal healthy subjects. Materials and Methods Seven normal adult volunteers and 9 XLH patients were studied. Seven patients who were being treated with vitamin D metabolites and/or phosphate were taken off all medication for one month prior to this study. Two had never been treated for the X L H disorders. After a d m i n i s t ration of phosphate tablets at 4-0-50 mg/kg (max 2,000 mg) of elemental phosphorus w i t h 200-4-00 ml of tap water, blood and urine samples were collected sequentially over a period of 6 h in the XLH patients or 24 h in the normal men. Results In the normal men, the serum Ρ level was increased at 2 h after a d m i n i s t ration of phosphate, and it was gradually decreased until 24 h. In contrast, serum Ca level decreased significantly at 4 h after ingestion. Serum 250HD, 2 4 , 2 5 ( 0 H ) 2 D and 1 , 2 5 ( 0 H ) 2 D levels did not change significantly, although serum intact PTH and mid-region PTH levels were s i g n i f i c a n tly increased from 2 h to 10 h after ingestion. Urinary cAMP were also significantly increased, while the serum CT levels did not change. In the XLH patients, although serum 250HD and 2 4 , 2 5 ( 0 H ) 2 D levels did not change, serum concentration of 1 , 2 5 ( 0 H ) 2 D w a s significantly increased 6 h after loading as shown in Figure. Especially in the untreated patients 1 , 2 5 ( 0 H ) 2 D level increased more than in the previously treated patients. Discussion In normal subjects, our single oral phosphate loading induced the h y p e r parathyroidism and mild reduction of serum Ca level w h i c h are w e l l k n o w n

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter&Co., Berlin · New York - Printed in Germany

190 to stimulate 1-hydroxylation. On the contrary, phosphate supplementation is reported to inhibit the 1-hydroxylation (6,7). In this circumstance, the serum 1,25(0H) 2 D level did not change significantly throughout the study period in the normal subjects. However, in XLH patients, serum 1 ,25( O H ^ D level was increased significantly at 6 h after phosphate loading. Although hyperparathyroidism and mild reduction of serum Ca had the stimulator action, it had been reported that, in patients with XLH (1) and Hjrg mice (8), the 1-hydroxylase activity shows a blunted response to PTH as a stimulator, moreover in Hyp mice the in vivo signal for hypocalcemic stimulation of renal 1-hydroxylase activity may be set at a different threshold (9). Therefore, our study demonstrated that this paradoxical increment in 1 ,25( O H ^ D seems to be induced by elevation of the serum Ρ concentration.

40' a j

30

oj C 20 10

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1 6

Hours after phosphate loading * : p 9 5 % a N A E p o s i t i v e ) were purified by adherence. AM n u m b e r s w e r e 1 2 0 x l 0 e a n d 7 0 x l 0 e per animal, respectively. The AM ( 1 0 6 c e l l s / p o i n t ) w e r e p l a t e d in 2 4 - w e l l p l a t e s in s e r u m - f r e e a M E M w i t h o u t a n t i b i o t i c s (1 m l / p o i n t ) . Reagents [la,25(0H)2Da, 1 i p o p o l y s a c c h a r i d e ] or v e h i c l e w e r e a d d e d at t h i s t i m e . A f t e r 24 hr, t h e m e d i a w e r e c h a n g e d to 0 . 5 m l s e r u m - f r e e m e d i a / p o i n t , a n d 2 5 ( 0 H ) [ 3 H ] D a (40 n M ) w a s a d d e d as substrate. A f t e r f o u r h o u r s , the r e a c t i o n w a s s t o p p e d b y a d d i t i o n of 0 . 5 m l m e t h a n o 1 / p o i n t . Porcine circulating lymphocytes were isolated, c u l t u r e d and a s s a y e d for l a , 2 5 ( 0 H ) 2 D a p r o d u c t i o n e x a c t l y as d e s c r i b e d (6). M e t a b o l i s m of 2 5 ( 0 H ) [ 3 H ] D a a n d r e c h r o m a t o g r a p h y of m e t a b o l i t e p e a k s w e r e d e t e r m i n e d by H P L C e x a c t l y as d e s c r i b e d p r e v i o u s l y (2, 3). Results and D i s c u s s i o n P o r c i n e AM f r o m b o t h a n i m a l s c o n s t i t u t i v e l y s y n t h e s i z e d a 25(0H) [ 3 H ] D 3 - m e t a b o l i t e w h i c h was identified as l a , 2 5 ( 0 H ) 2 [ 3 H ] D a by c o - e l u t i o n w i t h a u t h e n t i c l a , 2 5 ( 0 H ) 2 D a on t h r e e H P L C s y s t e m s ( F i g s . 1, 2). The conversion e f f i c i e n c i e s w e r e b e t w e e n 1 8 - 3 0 % of 4 0 n M 2 5 ( 0 H ) [ 3 H ] D a . P o s s i b l y , l a , 2 5 ( 0 H ) 2 D a f r o m AM h a s c o n t r i b u t e d to h o r m o n e s y n t h e s i s in a n e p h r i c p i g s a f t e r a d m i n i s t r a t i o n of l a r g e v i t a m i n D d o s e s (5). Activated circulating lymphocytes did n o t p r o d u c e Ι α , 2 5 ( O H )2Üa.

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter&Co., Berlin · New York- Printed in Germany

195

Characteristics of Ια,25(0H)2Ds-synthesis by porcine AM were: (i) Bacterial lipopolysaccharide had no effects on hormone production. (ii) Exogenous la,25(0H)2Ö3 inhibited hormone production (EDso ca. 5 x l 0 _ l o M ) but did not induce 25(0H)Ö3-24-hydroxyläse activity. (iii) The Km of the 1ahydroxylation reaction was in the range of 300 nM. In summary, we have obtained evidence in. vitro for constitutive la,25(0H)2Da synthesis by porcine AM. Since murine macrophages from several tissues had under comparable conditions no evidence for hormone production in vitro. we conclude that species differences are operative with respect to extrarenal la,25(0H)2D3 synthesis. τ

1

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-

1 -

1 0

1 4

1

1 β

1 12

16

1 20

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RETENTION TIME (min)

Fig. 1 25(0H)C 3 H]D 3 metabolism by porcine AM. HPLC: RadialPak^Porasil cartridge, isopropanol in n-hexane; 4-60% gradient; arrows show elution positions of standards.

Fig. 2: Rechromatography of putative 1,25(0H )z[ 3 H]Ds produced by porcine AM; A) Zorbax-Sil column, dichloromethane/isopropanol 92/8 vol/vol; B) Zorbax ODS reverse phase column, methanol/ water 85/15 vol/vol.

References 1. Adams, J.S., Gacad, M.S. (1985) J.Exp.Med. 161:755-765. 2. Reichel, Η., Koeffler, Η.P., Barbers, R.,Norman, A.W. (1987) J.Clin.Endocrinol.Metab. £5.: 1201-1209 . 3. Reichel, Η., Koeffler, Η.P., Norman, A.W. (1987) J.Biol. Chem . 232.·· 10931-10937 . 4. Hayes, M.E., O'Donoghue, D.J.O., Ballardie, F.W., Mawer, E.B. (1987) FEBS Lett. ΖΖΩ.· 307-310. 5. Littledike, E.T, Horst R.L. (1982) Endocrinology 111:2008-2013. 6. Reichel, Η., Koeffler, Η.P., Norman, A.W. (1987) J. CI in. Endocrinol .Metab. £5.: 519-526.

EVIDENCE THAT RAT KIDNEY 25-HYDROXYVIΤΑΜIN D-24-HYDROXYLASE CAN EXIST IN AN INACTIVE STATE. HEINHOLD VIETH Department of Clinical Biochem., University of Toronto, and Research Inst. Queen Elizabeth Hosp., Toronto, Canada M5G 2A2 In vitro studies consistently show that 1,25-(0H)2D is the primary stimulus to increase the amount of 24-OHase. Paradoxically, 24,25(0H)2D and 1,25(0H)2D synthesis are inversely related in vivo. It has been proposed that when 24-OHase and 1-OHase are present simultaneously, a lower Km of 1-OHase would give it a competitive advantage over 24-OHase when 25(OH)D is low (1). An alternative possibility is that 1,25(OH)2D directly inhibits 24-OHase (2). In a previous report, hypocalcemic rats did not generate 24,25(0H)2D in vivo, yet near-normal 24-OHase levels were measured (3). The following experiments were done to test whether 24-OHase can be present but inactive in vivo. METHODS. Hats were fed low-Ca, vit D-deficient diet for 4 wk. In vivo metabolism of intracadrial [26,27-3H]-25(OH)D3, 0.5 uCi, (20 Ci/mmol, Amersham) was expressed as % serum [3H] comigrating with authentic metabolites (3). Mitochondrial assays were as described (3). Treatment with l,25(OH)2D3 (Hoffman-LaLoche) was lug orally in ethanol and lug with the intracardial [3H]-25(OH)D3. Results are mean +SE (n=3). HPLC was on Zorbax CN (Dupont) eluted with a gradient of hexane/isopropanol/methanol, 99:1:0 (A) to 90:9:1 (B). RESULTS. In rats deficient in both calcium and vitamin D, 1-OHase was 320+78 fmol/mg/min (U) and 24-OHase was undetectable ( M, «1» | 1 » « r , 1 . M, t n I n t n Μ» » . 1 t h r

CM O0T AM A*C C M ATC ATA CTC A^'ACA AAA CM GAA C M CCC m K C U t CCT HC CK TCC^CM GM C M ATC « Μ « X «T^ATA 4 4 1 « 1 . . r , l y - « r , , 1 « M t t u l . u l y . r , l y . , 1 « , 1 « , 1 « . 1 . V«1 M r l y . 1 4 « l y 1 « . , 1 . «1. 3 iE JE u i i\. 1 , 1 >01 Ml 741 441 T^® ^ ^ OAC AOT CTC AOC CCC AM CT A TCT GAA CAA CAA C M CAC AM TTC C M CTG OOC CT6 AM AM CTC AAC TTA CAT OAC CAA CAC AOOC » y j ^ 1 « · l y . M p Η Γ 1 « ! . r , pro l y . 1 « M i , 1 » , 1 . f l n « I n h l . ly« , 4 » « I n v . l « l y 1 « , l y . l y , lau U . I mk l . f l u ^ ^

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S41 Ϊ*1 941 AM CTC ATC OCC ΤΤΓ CCA AM ATC ATC CCA GGA TTC ACC CAT CTC k e r a l y « v . l I I a « l y pha . 1 . l y . M t 1 1 . p r o « l y p h . . r q a a p l . t 1 1201 1221 CAOCCCA GCAOTCCCTC CTCCCCTTTC TOCACTTCM ItXUCCTCT CCCATCCC

Fig. 4. Nucleotide and deduced amino acid sequence of the partial rat V D R cDNA. U n d e r l i n e d sequence = second DNA b i n d i n g "finger", •denotes conserved cysteine residues. T R A N S C R I P T I O N A L R E G U L A T I O N O F T H E OSTEOCALCIN G E N E BY VITAMIN D R E C E P T O R Considerable evidence suggests that the mechanism of action of the v i t a m i n D hormone involves m o d i f i c a t i o n of gene expression (21). This hypothesis is strongly supported directly by transcriptional i n h i b i t o r studies (22) and nuclear r u n - o f f experiments (23) with a variety of v i t a m i n D responsive genes, and by the observed s t r u c t u r a l organization of the VDR protein itself. However, a direct interaction between the VDR and cloned hormone-responsive gene f r a g m e n t s has not been i d e n t i f i e d to date, nor has the sequence of a v i t a m i n D responsive element ( V D R E ) been d e f i n e d . So that such studies can be i n i t i a t e d , we have obtained the h u m a n osteocalcin gene (24), cloned the 5' f l a n k i n g sequence, which contains the T A T A box and approximately 1300 nucleotides upstream of that site, into a reporter chloramphenicol acetyltransferase (CAT) expression vector

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Human/Rat VDR

K Kd = \ d= 5.8 χ 1 0 _ 1 1 Μ \ 4 . 6 χ 1 0 " 1 1 Μ

Ο 0.1 0.2 0.3 0.4 Bound 1 , 2 5 ( 0 H ) 2 D 3 (ηΜ) Fig. 5. Expression and 1,25 (OHJ^D^ saturation analysis of the rat VDR hormone binding domain and h u m a n / r a t VDR chimera. VDR cDNA encoding the rat hormone-binding domain or encoding the human DNA binding domain spliced together with the rat hormone-binding domain at a common Stu I site were expressed in COS-1 cells and the cytosols subjected to saturation analysis. (phOsteo-CAT), and assessed its activity in cultured cells in response to 1,25(OH)2Do. Transfection of this hybrid gene into rat osteosarcoma cells (ROS 17/2.8) resulted in significant basal CAT enzyme activity as seen in Fig. 6. As anticipated, the addition of 10 Μ l ^ C O H ^ D j simultaneously led to a substantial increase in the activity of the reporter gene. In contrast, transfection of this Plasmid into the ROS 24/1 cell line , a line which is VDR-negative and in which the endogenous osteocalcin gene cannot be stimulated by hormone, resulted in neither basal nor stimulatable CAT activity. These experiments suggest that the native osteocalcin promoter is capable of significant basal activity only in the osteoblast-like cell line (22), and also provides the first direct evidence that the gene is inducible by l,25(OH)2Dß. In order to prove a direct involvement of the receptor in phOsteo-CAT expression by vitamin D, we carried out related experiments utilizing the receptornegative monkey cell line CV-1 (14). Transfection of phOsteo-CAT into this cell line resulted in low and uninducible reporter gene activity. In contrast, when VDR was added in this cell line by cotransfection of the VDR expression plasmid together with the target gene phOsteo-CAT, the activity of the latter acquired inducibility by l ^ i O H U D ^ . Similar results are observed in Fig. 7 when the ROS 24/1 cell line was complemented by the addition of VDR through transfection. Inducibility by the hormone displayed both receptor orientation and concentration dependence. Of primary interest was the observation that cotransfection of the expression plasmid directing synthesis of only the N-terminal peptide (which contains the intact DNA binding domain) along with phOsteo-CAT provided neither an enhancement of basal activity nor hormone inducibility. These results suggest that the absence of the VDR is a major determinant for the lack of reponsiveness of the osteocalcin gene to 1,25(0^1)^03 induction in ROS 24/1 cells. In addition, they suggest that the N-terminal portion of the VDR, which is

221

D—»kadfll

" Π 1,25(OH)2D3 ( 1 0 _ β Μ) Cell Line

_

+

-

ROS 17/2.8

+

ROS 24/1

F i g . 6. R e g u l a t i o n o f chloramphenicol acetyltransferase ( C A T ) expression by 1 , 2 5 ( O H ) 2 D j v i a p r o m o t e r / e n h a n c e r e l e m e n t s o f the h u m a n o s t e o c a l c i n gene. T h e p r o m o t e r a n d 5' r e g u l a t o r y r e g i o n o f the human o s t e o c a l c i n g e n e c o n t a i n i n g a p p r o x i m a t e l y 1300 n u c l e o t i d e s was c l o n e d i n t o a C A T e x p r e s s i o n v e c t o r d e s i g n a t e d p h O s t e o - C A T . T h e construct was t r a n s f e c t e d i n t o R O S 17/2.8 or R O S 24/1 cells and its a c t i v i t y assessed in response to e t h a n o l ( - ) or l , 2 5 ( O H ) 2 D 3 (10 M ) ( + ) . C A T a c t i v i t y was d e t e r m i n e d using C - c h l o r a m p h e n i c o l . S = substrate, Μ = monoacetylated product, D = diacetylated product.

Receptor

C

WTIx

WT+ 1x

WT2x

WT+A114 2χ

F i g . 7. R e s t o r a t i o n o f o s t e o c a l c i n g e n e response to 1 , 2 5 ( O H ) 2 D i in R O S 24/1 cells by t r a n s f e c t i o n o f human V D R expression v e c t o r . p h O s t e o - C A T w a s t r a n s f e c t e d i n t o 24/1 cells w i t h c o n t r o l D N A ( C ) , w i t h V D R e x p r e s s i o n v e c t o r c o n t a i n i n g e i t h e r the f u l l - l e n g t h human V D R in p o s i t i v e ( W T + ) or n e g a t i v e ( W T - ) o r i e n t a t i o n , or w i t h a p a r t i a l V D R s e q u e n c e e n c o d i n g the N - t e r m i n a l D N A b i n d i n g d o m a i n ( d e l t a 114). C e l l s w e r e then e x p o s e d to e i t h e r e t h a n o l ( - ) or l , 2 5 ( O H ) 2 D ß ( + ) as a b o v e . T r a n s f e c t i o n o f f u l l - l e n g t h V D R c D N A at t w i c e the c o n c e n t r a t i o n is i n d i c a t e d as 2x. E x t r e m e l e f t lane is a pure C A T e n z y m e c o n t r o l .

222 s u f f i c i e n t to c o n f e r heterologous DNA binding activity upon this macromolecule, is incapable of i n d e p e n d e n t l y directing e f f i c i e n t transcription f r o m the osteocalcin promoter. While the latter results d i f f e r in general with t h a t observed f o r several other steroid receptors (25-28), the VDR is u n i q u e in that its N-terminus exhibits considerable t r u n c a t i o n when compared to other members of this gene f a m i l y (6). Thus, while this large domain in other steroid receptors may c o n t r i b u t e strongly to their t r a n s c r i p t i o n a l capacity, our conclusion is that either transcriptional regulation by the V D R is less complex or analogous domains have evolved in this protein C-terminal to the domain which c o n f e r s DNA binding capacity. F u t u r e experiments involving VDR cDNA mutagenesis will be necessary to d e f i n e and evaluate receptor sequences which are important to transcriptional regulation of the osteocalcin gene as well as the host of other genes which are regulated by vitamin D.

T H E S T R U C T U R A L O R G A N I Z A T I O N OF T H E H U M A N V I T A M I N D R E C E P T O R CHROMOSOMAL G E N E Insight into the evolution of the vitamin D receptor and its interrelationship with other members of the steroid receptor gene f a m i l y , requires characterization of the chromosomal gene. To this end, we have recovered large overlapping genomic clones f r o m a human pCV-109 liver DNA library through hybridization screening utilizing the h u m a n VDR cDNA as probe. These clones were mapped through the use of restriction endonucleases, and probe-hybridizable DNA f r a g m e n t s i d e n t i f i e d , subcloned, and then sequenced by the dideoxy chain t e r m i n a t i o n method using d e n a t u r e d double-stranded plasmid D N A as template. As illustrated in Fig. 8, the chromosomal gene f o r the h u m a n vitamin D receptor encompasses approximately 44 kilobases of DNA, and is comprised of nine exons. T h e introns which intersperse these small exons range in size f r o m 183 nucleotides between exons 7 and 8 to over 13 kb between exons 2 and 3. The leader sequence, which represents the f i r s t exon, is interrupted by an intron such that exon 2 contains the r e m a i n d e r of the leader sequence, the translation initiation codon, and the f i r s t of the two dissimilar "finger" structures. Exon 3 contains the second "finger", thus completing the DNA binding domain of the VDR. Exons 4-6 comprising the "hinge" portion of the molecule are located in a cluster downstream, as are the f i n a l three exons which comprise the bulk of the l , 2 5 ( O H ) 2 D j - b i n d i n g domain. Exon 9 is the largest of the exons w i t h i n the V D R gene, a n d contains the f i n a l approximately 250 nucleotides of coding sequence as well as the entire 3' non-coding tail. Despite the complexity of this gene, its overall organization provides important clues as to the evolution of its f u n c t i o n a l domains. A comparison of this gene with that f o r the steroid receptors and p a r t i c u l a r l y those f o r the thyroid and vitamin A hormones will be of major interest in d e t e r m i n i n g the evolutionary relationship between these related e f f e c t o r proteins. Clearly, "steroid" hormone action requires reclassification, not on the basis of the chemical n a t u r e of the ligands, but on the evolution a n d common molecular a r c h i t e c t u r e of the proteins which mediate these ligands' physiologic activities in e u k a r y o t i c organisms.

223

10 I

Exon

10

20 ι

30

_ι

456

40 ι

50 K i l o b a s e s

78 9

Fig. 8. Structural organization of the human V D R chromosomal gene. The overall organization of the human V D R gene is indicated. The locations of the nine exons are indicated as vertical bars. Their position with respect to the c D N A and protein is discussed in the text.

SUMMARY In this chapter, we have documented recent experiments which demonstrate that vitamin D action on transcription is mediated by the receptor protein f o r 1,25(OH)2Dj. The overall structure of this molecule exhibits the essential features of the steroid, thyroid, and vitamin A hormone receptor gene family, leaving no doubt that the mechanism by which the vitamin D hormone operates involves transcriptional regulation. Studies using the human osteocalcin gene promoter as a target gene and receptor complementation in cell lines which do not normally express the V D R , provide the first unequivocal evidence that the vitamin D receptor is involved in the action of l,25(OH)2D3. Characterization of the gene f o r the human V D R and the identification of its promoter will provide essential insight into both the evolution and regulation of this macromolecule. Most importantly, the sequence of the V D R gene will now permit definition of genetic lesions which are proposed to form the basis f o r dysfunctional receptors found in humans with.vitamin D-dependency rickets, type 2. The availability of a V D R expression system will be essential in evaluating these disease related mutations as well as those introduced experimentally by mutagenesis.

REFERENCES 1. 2.

Green, S., Walter, P., Kumar, V., Krust, Α., Bornert, J-M., Argos, P., Chambon, P. (1984) Nature (London) 320:134-139. Conneely, O.M., Sullivan, W.P., T o f t , D.O., Birnbaumer, Μ, Cook, R.G., Maxwell, B.L., Zarucki-Schulz, T „ Greene, G.L., Schräder, W.T., O'Malley, B.W. (1986) Science 211:767-770.

224 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

Hollenberg, S.M., Weinberger, C., Ong, E.S., Cerelli, G., Oro, Α., Lebo, R., Thompson, E.B., Rosenfeld, M.G., Evans, R.M. (1985) Nature (London) 318:635-641. Weinberger, C., Thompson, C.C., Ong, E.S., Lebo, R., Gruol, DJ., Evans, R.M. (1986) Nature (London) 324:641-646. McDonnell, D.P., Mangelsdorf, DJ., Pike, J.W., Haussler, M.R., O'Malley, B.W. (1987) Science 235:1214-1217. Baker, A.R., McDonnell, D.P., Hughes, M , Crisp, T.M., Mangelsdorf, DJ., Haussler, M.R., Pike, J.W., Shine, J., O'Malley, B.W. (1988) Proc. Natl. Acad. Sei.USA, § 1 (in press). Giguere, V., Ong, E.S., Segui, P., Evans,R.M (1987) Nature (London) 330:624629. Pike, J.W., Kerner, S., Scott, R.A., (unpublished results). Miller, I., McLachlan, A.D., Klug,A. (1985) EMBO Journal 4:1609-1614. Berg, J.M. (1986) Science 232:485-487. Nagai, K., Nakaseko, Y., Nasmyth, K., Rhodes, D. (1988) Nature (London) 332:184-186. Redemann, N., Gaul, U., Jackie, Η. (1988) Nature (London) 332:90-92. Yamaoka, Κ., Mangelsdorf, D.J., Pike, J.W., Haussler, M.R. (manuscript submitted). McDonnell, D.P., Scott, R.A., O'Malley, B.W., Pike, J.W. (manuscript in preparation). Pike, J.W. (1984) J. Biol. Chem. 2^9:1167-1173. Allegretto, E.A., Pike, J.W., Haussler, M R . (1987) J.Biol.Chem. 262:1312-1319. Pike, J.W., Sleator, N.M., Haussler, M.R. (1987) J. Biol. Chem. 262:1305-1311. Pike, J.W., Mangelsdorf, D.J., Allegretto, E.A., Haussler, M.R. (1987) in Sterol and Steroid Hormone Action, Eds. T.C. Spelsberg and R. Kumar, pp. 339-354, Martinus N i j h o f f , Boston. Conneely, O.M, Maxwell, B., Toft, D.O., Schräder, W„ O'Malley, B.W. (1987) Biochem. Biophys. Res. Commun. 149:493-501. Pike, J.W., Haussier, M R . (1983) J. Biol. Chem. 2I&8554-8560. Pike, J.W. (1987) Steroids 49:3-27. Kream, B.E., Rowe, D., Smith, MD., Mäher, V., Majeska, R. (1986) Endocrinology 119:1922-1928. Dupret,J-M., Brun, P., Perret, C., Lomri, N., Thomasset, Μ , Cuisinier-Gleizes, P. (1987) J. Biol. Chem. 262:16553-16557. Celeste, A.J., Rosen, V., Buecker, J.L., Kriz, R., Wang, E.A., Wozney, J.M. (1986) EMBO Journal 1:1885-1890. Waterman, ML., Adler, S., Nelson, C., Greene, G.L., Evans, R.M, Rosenfeld, MG. (1988) Molecular Endocrinology 2:14-21. Hollenberg, S.M., Giguere, V., Segui, P., Evans, R.M. (1987) Cell 42:39-46. Miesfeld, R„ Godowski, Ρ J., Maler, Β. Α., Yamamoto, K.R. (1987) Science 2M:423-427. Kumar, V., Green, S., Stack, G., Berry, Μ , Jin, J-R., Chambon, P. (1987) Cell 51:941-951.

DEFINITION OF THE FUNCTIONAL DOMAINS OF THE VITAMIN D 3 RECEPTOR D.P. MCDONNELL, R. SCOTT, B.W. O'MALLEY and J.W. PIKE Departments of Cell Biology and Pediatrics, Baylor College of Medicine, Houston, Texas 77030. Introduction A comparison of the deduced amino acid sequence of the cloned human vitamin D^ receptor (VDR) with that of other cloned steroid and thyroid hormone receptors, reveals three distinct regions of primary sequence conservation (C^, C^ and C^) (1). The 70 amino acid, cysteine rich C^ region is most highly conserved. This equivalent region in the glucocorticoid receptor has been demonstrated to be involved in DNA binding (2). The C„ region is a 60 amino acid hydrophobic domain located towards the center of the molecule. A third homologous region (C3) close to the carboxyl terminus of the protein is found only in comparisons of the primary sequence of VDR and the receptor for thyroid hormone (TR). The receptors for glucocorticoid and progesterone, which are biochemically similar, contain an analogous sequence. Thus, the C^ domain may be conserved only among closely related receptors and may suggest a similar evolutionary origin for VDR and TR (3). Results A series of deletion mutants was constructed in order to define the nature of these conserved sequences and the roles they pley In the functional properties of the receptor (Fig. 1). C1 5'—I M ^ ·

C2

C3 I· I —1

,,

3'

Δ1 14-427 Δ1 90-427 Δ282-427 Δ363-427 Δ373-427

ι • ι Figure 1.

1 1 1 1

Δ1-104 Δ1-114 Δ1-166 Δ1-282

Deletion mutants of hVDR. (A) Linear organization of the receptor protein showing the location of the three regions found to be conserved among the steroid receptors. C, = Solid, C 2 = Hatched and C = Stippled. (B) A series of 3' and 5' deletions was constructed by linearizing the cDNA and introducing a stop codon to produce a 3' deletion or a start codon to produce a 5' deletion.

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · N e w York - Printed in Germany

226 The mutants were inserted into the eukaryotic expression vector P91023b and the recombinant protein was isolated following transfection into COS Ml cells. Initially the proteins were examined for their ability to bind hormone. Receptor mutants with as few as 50 amino acids deleted from the carboxyl terminus failed to bind hormone, whereas 100 amino acids could be deleted from the amino terminus and still retain wild type binding characteristics. The DNA binding properties of these proteins was examined using DNA cellulose chromatography. All mutants that contained the entire C^ region bound with high affinity to DNA. The smallest protein that demonstrated wild type binding was 12.5 kDA, corresponding to the amino terminal 114 amino acids. Using these constructs we sought also to define the sequences responsible for regulation of transcription. For this purpose a construct containing 1300 bp of the 5' flanking region of the vitamin D responsive human osteocalcin gene, fused to the coding region of chloramphenicol acetyl transferase (CAT) was made. When this was co-transfected into CV-1 cells with a plasmid encoding either wild type or mutant receptor, it was observed that only the cells containing wild type receptor exhibited hormone dependent regulation of the reporter gene. We were unable to show a constitutive activity with mutant receptors containing only the DNA binding domain, as has been observed with other receptors (2). Conclusions These data suggest that the DNA and hormone binding domains of the vitamin D receptor are separate and distinct. Those sequences responsible for DNA binding reside within a 114 amino acid stretch at the amino terminus. The hormone binding domain is more complex and extends from residue 104-427, encompassing the entire carboxyl half of the molecule. We have not yet been able to specifically localize the sequences involved in regulation of transcription. These results agree with the proteolytic mapping studies of Allegretto, et al. (4), which defined distinct hormone and DNA binding regions within the receptor. References 1. 2. 3. 4.

Baker, A.R., McDonnell, D.P., Hughes, Μ., Crisp, T.M., Mangelsdorf, D.J., Haussler, M.R., Pike, J.W., Shine, J. and O'Malley, B.W. (1988) Proc. Natl. Acad. Sei. USA (in press). Hollenberg, S.M., Gigure, U., Segui, P. and Evans, R.M. (1987) Cell 49:39-46. McDonnell, D.P., Pike, J.W. and O'Malley, B.W. (1988) J. Steroid Biochem. (in press). Allegretto, E.A., Pike, J.W. and Haussler, M.R. (1987) J. Biol. Chem. 262:1312-1319.

HYPERTHYROIDISM INCREASES FEMALE RAT LIVER.

NUCLEAR

1,25-DIHYDROXYVITAMIN

D3

W.E. DUNCAN, A.R. GLASS, D. WHITEHEAD, and H.L. WRAY Walter Reed Army Medical Center, Washington, DC and the Services University of the Health Sciences, Bethesda, MD.

BINDING

IN

Uniformed

Introduction 1,25-Dihydroxyvitamin D 3 (1,25(0H)2D 3 ), like other steroid hormones, exerts its actions through receptors in target tissues. Changes in l,25(OH)2Dß receptor concentration correlate with hormone responsiveness (1). We have recently identified a 1,25(0H)2D 3 receptor in rat liver nuclei (2) whose ligand specificity, sedimentation coefficient, binding affinity, trypsin sensitivity and nuclear location are identical to the well characterized l,25(OH)2D 3 receptor proteins identified in other tissues (3). This hepatic nuclear l,25(OH)2D 3 binding protein is also found in mice, rabbits, and chicks. We have previously demonstrated that the activity of this liver nuclear receptor is increased by estrogen (4). This study examines the effects of another hormone, triiodothyronine, on l ^ S C O H ^ D ^ binding in extracts of liver nuclei. Materials and Methods Female Sprague-Dawley rats (25 days old) were rendered hyperthyroid by infusion of triiodothyronine (T^) administered by miniosmopump (3 mcg/d). Prior to T 3 infusion, some animals were ovarectomized, and some of the ovarectomized rats were concurrently given estrogen replacement by estradiol-containing silastic implants. At sacrifice (12-14 days later), blood was obtained and livers were dissected, frozen in liquid nitrogen, and stored at -70°C until the tissue was processed. Liver nuclei were isolated in a low ionic strength buffer and the Ι ^ δ ί Ο Η ^ ϋ β receptor was then extracted with a 26 mM Tris (pH 7.4) buffer containing 0.3 Μ KCl, 5 nM dithiothreitol, 1 mM EDTA, and 10 mM sodium molybdate. Specific l ^ S i O H ^ D ß binding was quantitated by a hydroxylapatite method after incubation of the the extract with ^Η-labeled ± unlabeled sterol for 18 hours at 4 C. Results and Discussion Table 1. Effect of Triiodothyronine on Specific 1,25(0H) 2 D 3 Binding in Extracts of Hepatic Nuclei from Intact Female Rats. Treatment Control T

3

Ν 6 6

Thyroxine (meg/dl) 3.86 ± 0.30* < 1.8

T 3 (ng/dl) 67.4 ± 4.52* 391.7 ± 43.0

TSH (ng/ml) 1.28 ± 0.20*

Χ to

I A

20

40

£ 22a

60

80

elution volume (ml) Fig-2: Interaction of root sterol binder with DNA-cellulose. Left: DNA-cellulose binding of -vitamin D 3 incubated with root cytosol. Right: sucrose density gradient analysis of ^H-vitamin D3 binding to root cytosol after the incubation with DNA-cellulose. Cytosol preincubated with 3 H -vitamin D3 at 0 °C for 4 h followed by 30 min at 0 °C (·,ο) or 25 °C (o) was incubated with DNA-cellulose (n) or buffer (o,·). REFERENCES 1.-Vega,M.A.,Boland,R.L. (1988) Biochim J., in press 2.-Vega,M.A.,Santamaria,E.C..Morales,A.,Boland,R.L. (1985) Physiol. Plant. 65:423-426 3.-Radparvar,S.,Mellon,W.S. (1982) Arch.Biochem.Biophys. 654:181-186 4.-Hamilton,R.H.,Künsch,U.,Temperli,A. (1972) Anal.Biochem. 49:48-57 5.-Boland,R.L.,Vega,M.A. (1987) in Calcium Regulation and Bone Metabolism. Basic and Clinical Aspects. Cohn D.V., Martin,Τ.J., eds., p.370, Excerpta Medica, Amsterdan.

Cell Differentiation Hematopoiesis Immunology

INTERACTION BETWEEN THE HEMATOPOIETIC SYSTEM AND THE VITAMIN D ENDOCRINE SYSTEM H. PHILLIP KOEFFLER and ANTHONY W. NORMAN Department of Medicine, Division of Hematology-Oncology, The University of California, Los Angeles and Department of Biochemistry, The University of California, Riverside, California. Synthesis of 1,25 Dihydroxyvitamln D^ by Macrophages The 1,25 dihydroxyvitamin D 3 [1,25(0H)2D3] is predominately synthesized by the kidneys and causes calcium absorption from the intestine and calcium r e a b s o r p t i o n from bone. A d a m s e t al. (1) has shown that m a c r o p h a g e s from the lungs of patients with sarcoidosis can produce I,25(0H)2D3· We have found that normal human macrophages can synthesize 1,25(OH>303 after their activation (2-4). This activation occurred w i t h exposure to interferon (IFN) - T or - a or to lipopolysaccharide derived f r o m t h e c e l l w a l l s of g r a m n e g a t i v e b a c t e r i a . This putative 1,25(0H)2D3 was purified to homogeneity and was shown: 1) to co-migrate with chemically synthesized 1,25(0H>2D3 on four different systems of high pressure liquid chromatography; 2) to have a UV-spectra typical of a vitamin D sterol with a maximum absorption of 265 nM and a minimum absorption at 228 nM; 3) to have a mass spectra typical of 1,25(0H) 2 D3; 4) to have the exact mass and elemental composition (C27,H44,03) as authentic 1,25(0H) 2 D3; 5) to compete in an equal molar fashion w i t h chemically synthesized 1,25(0H) 2 D3 for binding to l,25(OH)2D3 receptors; 6) to c a u s e I n t e s t i n a l c a l c i u m a b s o r p t i o n (ICA) and bone calcium mobilization (BCM) in an equal molar amount to that produced by chemically synthesized 1,25(0H)2D 3 ; 7) to induce differentiation of HL-60 promyelocytes to macrophages at an equal potency as chemically synthesized l,25(OH)2D 3 . W e f o u n d t h a t a c t i v a t e d m a c r o p h a g e s and monocytes were capable of synthesis of 1,25(0H)2D3, but neutrophils, myeloid leukemia cell lines, and Β and T-lymphocytes were incapable of this synthesis. The pulmonary alveolar macrophages from rabbits and miniature pigs were capable of production of the l,25(OH)2D3· In contrast, the macrophages from mice and rats did not produce appreciable amounts of l,25(OH)2D3· Patients with sarcoidosis can have elevated blood levels of 1,25(0H)2D3 r e s u l t i n g in hypercalcemia, even in those patients who are anephric (5). In v i t r o , t h e m a c r o p h a g e s from these patients constitutively produced l,25(OH)2D3· Hypercalcemia associated with elevated serum 1,25(0H)2D3 levels have been reported in several patients with other g r a n u l o m a t o u s d i s e a s e s i n c l u d i n g t u b e r c u l o s i s (6,7), leprosy (8), disseminated candidiasis (9) and silicone-induced granuloma (10). In vivo significance of synthesis of 1,25(0H)2D3 by normal human m a c r o phages is unknown. Levels of l,25(OH)2D3 are very low or undetectable in anephric individuals (11-13). Therefore, any synthesis of 1,25(0H)2D3 by macrophages would probably have its effect predominately in a paracrine fashion.

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter&Co., Berlin · New York-Printed in Germany

264 Inhibition of Lymphoklne Synthesis by 1,25(0H) ? D^ The synthesis of interferon gamma (IFN-Y) by peripheral blood T-lymphocytes in vitro is inhibited in a dose-dependent fashion by 1,25(0H)2D3 (14). A 50 percent inhibition occurs at about 1 0 - 9 M 1,25(0H) 2 D3. The inhibition of INF-Y occurs independent of inhibition of IL-2 synthesis and is dependent on the T—lymphocyte expressing 1,25(0H) 2 D3 receptors. The 1,25(0H)2D3 is also a potent inhibitor of synthesis of granulocytemacrophage colony stimulating factor (GM-CSF) (15,16). The GM-CSF is the growth factor that stimulates both the production of myeloid progenitor cells and their differentiation into granulocytes and macrophages. The 1,25(0H) 2 D3 reduces GM-CSF mRNA accumulation in a dose-dependent manner with a 50 percent effective dose (ED50) of "5 χ 10 _ 1 1 M. A fifty percent decrease of GM-CSF mRNA occurs in 2D3

Time course of inhibitory effects of high concentrations of l,25-(OH)2D3 on cell cycle parameters. Cells were grown in medium supplemented with normal FCS and treated with ethanol vehicle or l,25-(OH)2D3 at the concentrations as indicated at days 0,2 and 4

was seen with all concentrations of l,25-(OH)2Ö3 tested. At the lower concentrations of l,25-(OH)2D3 this was accompanied with a small decrease in G0/G1 phase cells. However at the highest concentration of l,25-(OH)2Ü3 the increase in G2 + Μ phase was accompanied by a marked depletion of S phase

295

cells, indicating that l,25-(OH)2Ö3 treatment had little effect on progression through S phase. The anti-estrogen Tamoxifen had quite different effects, i.e., a reduction of cells in both G2 + Μ and S phases with cells accumulating in G0/G1 phase. The concentration of 10"8 Μ l,25-(OH)2D3 , which reduced cell numbers to about half of that in control cultures, approximately doubled the proportion of cells in G2 + Μ phase from 9.7% to 19.6%, significant by paired analysis ( ρ < 0.002). This was accompanied by a small decrease in the number of cells in G()/GI from 71% to 63%. Tamoxifen, which caused a similar degree of inhibition, resulted in cells accumulating in G0/G1 without any increase but in fact a decrease in G2 + Μ phase cells. The increase in G2 + Μ phase cells after exposure to l,25-(OH)2D3 was dose-dependent, first seen after 3 days of exposure and persisted throughout the 6 day exposure period. GQ/GI exit kinetics were not affected by moderately high concentrations of l,25-(OH)2D3 but were delayed after treatment with 10"7 Μ l,25-(OH)2D3; the ti/2 of exit being prolonged in the l,25-(OH)2D3-treated cells from 13 hours (control) to 90 hours.

Treated >ο

3 >

u

Figure 2.

Effect of l,25-(OH)2D3 on the growth of the human colonic cancer cell line (COLO 206F) xenografts in immunosuppressed mice. Groups of animals received i.p. injections three days per week of either 0.2 ml com oil vehicle or 0.1 μg l,25-(OH)2D3 in vehicle over the first 20 days.

296

For the colon carcinoma COLO 206F xenografts, volume doubling time in control mice was 7 days. The growth of these xenografts was markedly inhibited by treatment with 1,25-(ΟΗ)203 over the entire treatment period as we have shown previously (11). However when the l,25-(OH)2D3 treatment was ceased the xenografts recommenced growth with a doubling time similar to that of the untreated control xenografts (Figure 2). A factor in optimising the effects of l,25-(OH)2D3 on cellular replication is the time of exposure to the hormone. As we have shown previously the stimulatory effects are first seen in this cell line after 6 days of exposure to l,25-(OH)2D3 (8), i.e, about three population doubling times. Moreover the stimulation is seen only in the presence of charcoal treated FCS (8), as has been shown for estrogen stimulation of breast cancer cell replication (9). In another l,25-(OH)2D3-responsive human cancer cell line (HL-60), l,25-(OH)2D3 at high concentration (10~9 "Μ -10~7 M) has been shown to inhibit the production of c-myc mRNA (21). The depletion of the oncogene product would be expected to lag behind the inhibition of its mRNA synthesis. Hence it is possible that l,25-(OH)2Ö3 may exert some of its effects in these cells by the regulation of the transcription and/or translation of c-myc or other oncogene products. It is of considerable interest that the inhibitory concentrations of l,25-(OH)2D3 cause an accumulation of cells in the G2 + Μ phase of the cell cycle, suggesting a primary effect of l,25-(OH)2Ö3 on breast cancer cell replication of transition delay through G2 or mitosis. At higher concentrations of l,25-(OH)2D3, where the inhibitory effects on replication are maximal, increased G2 + Μ and G0/G1 transit times increase the overall cell cycle time. Indeed at the highest concentrations tested virtually no cells appear to remain in S phase, indicating that progression out of G j phase is blocked, although progression through S phase can proceeed. Explanation of these data necessitate differential doseresponsiveness for 1,25-(ΟΗ)203 effects in Go/Gj and G2 + Μ phases. Recent studies of the effects of l,25-(OH)2D3 in cells of the hemopoietic lineage have shown accumulation of cells in Go/Gj and a block of progression of cells from the early, low RNA to the late, higher RNA compartment of Gi (22, 23). These observations are consistent with the effects we have reported here with the highest dose of l,25-(OH)2D3 in this human breast cancer cell line. They suggest a distinct mechanism of regulation of cell cycle progression and substantial differences in the dose-responses of the two major l,25-(OH)2Ö3 inhibitory effects, i.e., on progression through Gl and through G2 + M. These effects, distinct from the effects seen with tamoxifen where the predominant

297

effect is to block the progression of cells through G j phase (12-15), are being studied further. A small number of other studies from our own and other groups have also shown an accumulation of cells in G2 + Μ phase using continuous exposure to tamoxifen (24), high concentrations of oestrogen (25), treatment with phorbol ester (26) or very low (0.25%) concentrations of FCS (27). In the latter study autocrine growth factor production appeared to be involved since proliferation rates and cell cycle kinetic parameters were partially restored by addition of growth factors. Also we have recently shown that treatment with l,25-(OH)2D3 or phorbol ester down-regulates the number of epidermal growth factor receptors on human breast cancer cells (28). The inhibition of the growth of the colonic cancer cell (COLO 206F) xenografts is unequivocal with the l,25-(OH)2D3-treated xenografts being less than 1 % of the relative volume of the control xenografts, consistent with the presence of receptor in these cell line-derived solid tumor xenografts (Figure 2). The rapid recovery of growth of the l,25-(OH)2D3-treated xenografts, when the hormone is withdrawn indicates that treatment must be continuous for effective control of tumor growth. However this transient effect is also seen with antiestrogens, which do have an important role in clinical therapy of breast cancer. The transient effect, of apparent inhibition of cell cycle progression without cell death, would be consistent with suppression of production of autocrine growth factor(s) by the cells or of inhibition of cellular responsiveness to such factors. Several normal and malignant cells of haemopoietic lineages possess l,25-(OH)2D3 receptor and respond to the hormone with inhibition of replication and stimulation of differentiation (21, 29-33) and in the HL-60 cell line inhibition the c-myc cellular oncogene expression (21). If similar mechanisms apply as in leukaemic cells, i.e., differentiation of the cells by the hormone, then this effect must be transient as has been suggested in some recent studies in leukaemic cells (34). The marked inhibition of growth of l,25-(OH)2Ö3 receptor-positive human xenografts and our data on cell cycle kinetic changes establish the value of and provide a background for further studies, particularly towards development of less hypercalcaemic analogues of l,25-(OH)2D3 with enhanced and perhaps longer-lasting anti-proliferative activity. References:

1. Eisman, J.A., Martin, TJ., Maclntyre, I., and Moseley, J.M. Lancet, ii: 1335-1336, 1979.

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2. Eisman, J.A., Martin, TJ., Maclntyre, I., Frampton, R.J., Moseley, J.M., Whitehead, R. Biochem. Biophys. Res. Commun., 93: 9-15,1980. 3. Findlay, D.M., Michelangeli, V.P., Eisman, J.A., Frampton, R.J., Moseley, J.M., Maclntyre, I., Whitehead, R., and Martin, TJ. Cancer Res., 40:4764-4767, 1980. 4. Frampton, R.J., Suva, L.J., Eisman, J.A., Findlay, D.M., Moore, G.E., Moseley, J.M., and Martin, T.J. Cancer Res., 42: 1116-1119,1982. 5. Freake, H.C., Marcocci, C., Iwasaki, J., and Maclntyre, I. Biochem. Biophys. Res. Commun., 101:1131-1138,1981. 6. Eisman, J.A. In: Kumar, R. (ed.), Vitamin D Metabolism: Basic and Clinical Aspects, pp. 365-382. The Hague: Martinus Nijhoff, 1983. 7. Colston, K., Colston, M.J., and Feldman, D. Endocrinology, 108:10831086, 1981. 8. Frampton, R.J., Omond, S.A., and Eisman, J.A. Cancer Res., 43:44434447, 1983. 9. Lipmann, M.E., and Bolan, G. Nature 256: 592-593,1975. 10. Honma, Y., Hozumi, M., Abe, Ε., Konno, K., Fukushima, M., Hata, S., Nishil, Y., DeLuca, H.F., and Suda, T. Proc. Natl. Acad. Sei., U.S.A., 80:201-204, 1983. 11. Eisman, J.A., Barkla, D.H. and Tutton, PJ.M. Cancer Res., 47:21-25, 1987. 12. Sutherland, R.L., Hall, R.E., and Taylor, I.W. Cancer Res., 43:39984006, 1983. 13. Taylor, I.W., Hodson, P.J., Green, M.D., and Sutherland, R.L. Cancer Res., 43:4007-4011,1983. 14. Reddel, R.R., Murphy, L.C., and Sutherland, R.L. Cancer Res., 44:23982403, 1984. 15. Reddel, R.R., Murphy, L.C., Hall, R.E., and Sutherland, R.L. Cancer Res., 45:1525-1531, 1985. 16. Taylor, I.W. J. Histochem. Cytochem., 28:1021-1024, 1980. 17. Milthorpe, B.K. Comput. Biomed. Res., 13: 417-429,1980. 18. Tutton P.J.M., Barkla D.H. Br. J. Cancer, 41:47-51, 1980. 19. Tutton P.J.M., Barkla D.H. Virchows Arch. B, 38:351-355,1981. 20. Barkla D.H., Tutton PJ.M. J. Natl. Cancer Inst., 67: 1207-1212,1981. 21. Reitsma, P.H., Rothberg, P.G., Astrin, S.M., Trial, J., Bar-Shavit, Z., Hall, Α., Teitelbaum, S.L., and Kahn, A.J. Nature, 306:492-494,1983. 22. Rigby, W.F., Noelle, R J., Krause, Κ. and Fanger, M.W. J Immunol., 135:2279-2286, 1985. 23. Studzinski, G.P., Bhandal, A.K. and Brelvi, Z.S. Cancer Res., 45:38983905, 1985.

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24. Lykkesfeldt, A.E., Larsen, J.K., Christensen, I.J., and Briand, P. Brit. J. Cancer, 49:717-722,1984. 25. Reddel, R.R., and Sutherland, R.L. Cancer Res., 44:5323-5525,1987. 26. Valette, Α., Gus, Ν., Jozan, S., Roubinet, F. Dupont, M.A., Bayard, F. Cancer Res., 47:1615-1620, 1987. 27. Reddel, R.R., Hedley, D.W., Sutherland, R.L. Expl. Cell Res., 161:277284, 1985. 28. Koga, M., Eisman, J.A., Sutherland, R.L. Cancer Res., 48,1988 (in press) 29. Bhalla, A.K., Amento, A.P., Clemens, T.L., Hollick, M.F., and Krane, S.M. J. Clin. Endocrinol. Metab., 57:1308-1310,1983. 30. Ravid, Α., Koren, R., Novorodsky, Α., and Liberman, U.A. Biochem. Biophys. Res. Commun., 123:163-169,1984. 31. Lemire, J.M., Adams, J.S., Sakai, R., and Jordon, S.C. J. Clin. Invest. 74:657-661, 1984. 32. Tsoukas, C.D., Prowedini, D.M., and Manolagas, S.C. Science 224:1438-1440, 1984. 33. Bar-Shavit, Z., Teitelbaum, S.L., Reitsma, P., Hall, Α., Pegg, L.E., Trial, J., Kahn, A.J. Proc. Natl. Acad. Sei., U.S.A., 80:5907-5911,1983. 34. Bar-Shavit, Z., Kahn, A.J., Stone, K.R., Trial, J., Hilliard, T., Reitsma, P.H., Teitelbaum, S.L. Endocrinology 118:679-686,1986. Acknowledgements

We thank Jean-Charles Fragonas, Lynne McMenemy, Grace Pang and Narelle Harley for technical assistance and Elizabeth Musgrove for valuable discussion. This study was supported by grants from the National Health and Medical Research Council of Australia, the New South Wales State Cancer Council, the Anti-Cancer Council of Victoria and Hoffmann-La Roche, U.S.A.

MC 903 - A NOVEL VITAMIN D ANALOGUE WITH POTENT EFFECTS ON CELL PROLIFERATION AND CELL DIFFERENTIATION.

LISE BINDERUP Department of Biology, Leo Pharmaceutical Products, DK-2750, Ballerup, Denmark. INTRODUCTION Recent investigations have shown that la,25-dihydroxycholecalciferol (1,25(OH)2D_), the hormonally active form of vitamin D„, is able to mediate a number of effects in tissues other tnan the classical targets for calcium and phosphate metabolism (1). Specific receptors for l,25(OH) 2 Dg have been found in skin, muscle, and pancreas (2,3,4), in mononuclear cells (5) and in a variety of tumour cells (6). After interaction with the receptor 1,25(0H) 2 D„ may promote cell differentiation and inhibit cell proliferation (7,8). 1,25(0H)2D„ may thus be able to exert a regulatory function in diseases characterized by excessive cell proliferation and low or defective cell differentiation.

1,25(OH)_Dg and its analogues may therefore find application in the therapy of proliferative disorders. Recent evidence indicates that psoriasis vulgaris, a skin disease characterized by hyperproliferation and incomplete differentiation of epidermis, improves after oral or topical administration of l,25(OH) 2 D 3 or its analogues 1 , 2 4 ( O H K D - and la-hydroxycholecalciferol (9,10,11). Unfortunately, the use of these highly active vitamin D derivatives is limited by their potent effects on calcium metabolism. Hypercalcemia is induced by systemic doses higher than a few pg per day, and topical application may be complicated by the risk of transdermal absorption of the active compound in areas of psoriatic skin lesions.

Therefore, new vitamin D analogues with potent cell regulating properties, but with lower risk of inducing calciumrelated side effects, are needed. This study describes the effects of MC 903, a novel vitamin D analogue with a promising pharmacological and therapeutic profile (12,13). MC 903 is presently undergoing clinical evaluation in patients with psoriasis vulgaris.

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · New York - Printed in Germany

301

MATERIALS AND METHODS Cellular effects of MC 903 were studied using the human histiocytic lymphoma U 937 cell line, the human promyelocytic cell line HL-60 and freshly established epidermal cells from newborn Balb/c mice. MC 903 was added at concentrations from 10 1 0 to 10~7 Μ and cell proliferation was assessed at the end of culture by counting of cells or by measurement of the cellular DNA-content. Induction of cell differentiation was followed by the appearance of nonspecific esterase activity in the U 937 cells (14) and by the reduction of nitroblue tetrazolium in the HL-60 cells (15). Differentiation of epidermal keratinocytes was assessed as described by Hosomi et al (16). All experiments were performed in triplicate, and control cultures with vehicle alone or with Ι ^ δ ζ Ο Η ^ Ο ^ were run in parallel. In vivo effects of MC 903 on calcium metabolism were studied in rats. Female LEW/MOL rats (140-160 g) were used to assess the effects of MC 903 (1 to 100 yg/kg/day p.o. for 7 days) on calcium levels in urine and serum (12). Infantile Wistar rats fed a calcium and vitamin D-deficient diet were used to assess the effects of MC 903 (1 to 100 pg/kg/day i.m. for 3 days) on intestinal calcium absorption and bone calcium mobilization (17). Control rats received l,25(OH)2D_ (0.5 or 1.0 pg/kg/day) or vehicle alone. Receptor binding studies with the U 937 cellular receptor were performed according to Manolagas and Deftos (18). Binding to the receptor from rachitic chick intestine was assessed using Amersham's assay reagents system TRK.870. Five hundred μΐ of receptor proteins were incubated with approximately 10,000 dpm 3 H-l,25(0H) 2 D 3 (Amersham TRK. 656) and increasing concentrations of unlabelled MC 903 or 1,25(0H) 2 D 3 · After incubation for 60 minutes at 22°C the bound ana tree 3 H-l, 25(ΟΗ) 2 ϋ 3 were separated on dextrancoated charcoal. Binding of MC 903 to serum proteins was studied using rat serum, undiluted or diluted with 0.05 Μ phosphate buffer pH 7.4, containing 0.05 Μ KCl, 1 mM dithiothreitol and 1 mg/ml gammaglobulin. Aliquots of 500 μΐ were incubated with 3 H-MC 903 (175 mCi/mmol) for 60 minutes at 0°C. Separation of bound and free radioactivity was performed with dextran-coated charcoal at 0°C. Samples with 3 H-l,25(OH) 2 D 3 (170 Ci/mmol) were run in parallel.

302

RESULTS Figure 1 shows the chemical structure of MC 903 and its relationship to the naturally occurring metabolite of vitamin D_, 0 l,25(OH) 2 D 3 .

1.25 (OH) D

MC 903 OH

Figure 1. Structures of MC 903 and l,25(OH)_D The synthesis of MC 903 from vitamin D, has previously been z described (19 ). Cellular effects of MC 903. The ability of MC 903 to affect cell differentiation and proliferation was studied in two different human tumour cell lines and in freshly established cultures of mouse keratinocytes. The effects of MC 903 were compared to those obtained with 1,25(OH)„Do· Table 1 shows that MC 903 induced cell differentiation of human histiocytic lymphoma U 937 cells and of human promyelocytic HL-60 cells in concentrations comparable to those of l,25(OH) 2 D 3 . Both compounds induced cell differentiation via the monocyte-macrophage pathway. The proliferation of U 937 cells was inhibited slightly more effectively by MC 903 than by l,25(OH)_D„, whereas none of the compounds inhibited_the growth of rhe HL-60 cells in concentrations up to 10 7 M . Cell viability was not affected by the treatments.

303

Table 1.

Effects of MC 903 on human tumour cells in vitro.

Compound

Cell

tested

line

MC 903 l,25(OH) 2 D 3

Induction of cell differentiation (M)

Inhibition of cell proliferation IC (M) 50

U 937 HL-60

-9 1.0x10 -9 5.0x10

1.4x10

U 937 HL-60

-9 1.0x10 -9 5.0x10

2.8x10

>10

-8

-7 - 8

>10'

Keratinocytes from newborn mice possess high affinity receptors for 1,25(0H) 2 D 3 . Table 2 shows that incubation with MC 903 or 1,25(0H) 2 D 3 Induced cell differentiation, as visualized by epidermal cell stratification, appearance of squamous cells and of cornified envelopes. Induction of differentiation started at lxlO - 9 Μ with l,25(OH) 2 D 3 , whereas MC 903 was fully active at this concentration (tne lower limit remains to be determined). MC 903 was more potent than l,25(OH)_D.as an inhibitor of cell proliferation, as measured by tne total DNA-content of treated keratinocyte cultures.

Table 2. Effect of MC 903 on cultured mouse keratinocytes. Compound tested MC 903 l,25(OH) 2 D 3

Induction of cell differentiation (M)

Cellular DNA-content % change compared to control

Cl.OxlO" 9

-22 (at 10~ 8 M)

l.OxlO - 9

- 3 (at 1 0 _ 8 M )

Effects of MC 903 on calcium metabolism in vivo. The ability of MC 903 to cause hypercalciuria and hypercalcemia after oral administration was studied in adult female LEW/MOL rats. MC 903 was administered at 1, 10 and 100 μg/ kg/day for 7 days, control rats received vehicle alone (propylene glycol) or l,25(OH) 2 D 3 (0.5 pg/kg/day, 7x). Figure 2 shows the excretion of calcium in a 24-hours sample of urine, collected on day 7 of treatment.

304

Calcium in Urine μπιοΙ/24 hrs 160 . Control

MC 903

1.25 (OHL D,

120

80 .

40

.

τ-Π" 0

1 10 100 Dosage in μ ς ^ / ό β γ p.ο. 7χ

0.5

Figure 2. Effects of MC 903 and 1,25(0H) 2 D 3 on calcium excretion in the urine. Mean ± S.D., n=10-14, *=p), dexamethasone (Dex) or 1,25(CH)^ (from Chugßi Pharmaceutical Go. Tokyo, Japan) and further incubated far 24 hrs. Next the mediun was replaced by serun-free phosphate buffered saline (FBS) containing the same steroid hormones. After incubation for 24 hrs, the mediun was collected and the protein, precipitated with 60i? saturation of annoniun .sulfate, was collected by centrifug^tion at 30,OCD χ g for 30 min, dispersed in PK and dialyzad against the same buffer overnight at 4°C. Proteins were examined by SD5polyacrylanri.de gel electrophoresis (SDS-PAGE) (6) followed by silver staining (7). Western blotting (8) and semi-quantitive dot blot (8) analyses were carried out with anti-albunin and anti-transferrin antibodies (Abs) (fron Cooper RLanedical Inc. Malvern, PA). Results and Discussion Mare than 95% of the cells excluded trypan blue at the time of mediun collection. As drawn in PÜg. 1, 1,25-(CH)2&3 affected the electrophoretic profiles of the proteins secreted by the cultured nepatocyte, causing decrease in albunin synthesis and increases in the syntheses of several other proteins, one with a slightly higher molecular weight than albunin. Next we investigated the proteins by inmmological methods. As shown in Pig. 2 A, Dex (1CT8 M) and (1CT8 M) increased the synthesis and secretion of albunin. These results were consistent with those reported by others (4). Ch the contrary, 1,25-(CH)2E^ (10"^ M) significantly decreased albunin synthesis. Pig. 2B shews that one protein, whose synthesis was stimilated by 1,25((IO2D3 (10 M) was iumunologically crossreactive with anti-tranrferrin Ab. Dot blot semi-quantitive analyses confirmed that both 1CT^ Μ Dex and 10~~ Μ E^ increased albunin synthesis to about 1.5-fold the control level, and that 1CT8 Μ 1,25-(®)2&} decreased the albumin synthesis to about two-third t h i s level and increased transferrin synthesis to about 1.5-fold the control level (data not shown). These

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter&Co., Berlin · New York- Printed in Germany

367

findings that l . Z H C H ) ^ decreased the synthesis of albumin and enhanced that of transferrin are noteworthy because both proteins >ere major products of hepatocytes and they are thought to be markers of differentiation of the cells (3). Moreover the serun concentration of transferrin in analfaimnenic rats i s repeated to be higher than that in normal rats (9). Fran these results we suggest that hepatocytes are new target cells of l . Z H C H ) ^ , and that this sterol may affect the differentiation of these cells. Further investigations are in progress on the presence of a specific receptor for 1,23-(CH)2&j in hepatocytes and i t s intracellular mediator. Fig. 1: Profile on SDS polyacrylanri.de gel electrophoresis (12.5 of proteins secreted by cultured rat hepatocytes. Lane a: l . Z H C H ) ^ (1CT7 M), lane b: control, lane c: globulin heavy chain, lane d: albunin. Fig. 2: Identifications of proteins by Western blotting using anti-elbunin (A) and anti-transferrin (B) antibodies. lane ra: marker protön (A:albwrin, B:transferrin), Jane a: control, lane b: Ej (1CT8 M), lane c: Dex (1CT8 Μ), lane d: l , ^ ® ) ^ (1(T

Fig.l a

b

top

C

d

Fig.2 A

Β

a n t i - a l b u m i n Ab

a n t i - t r a n s f e r r i n Ab

mabed

mabed

Ü

74K 50K

•

25K

•

12K

•

...

Dye fron'c

BaCuraices 1. Abe,E., Miyaura.Y., Sakaganii,H., Takeda,M., Κοπηο,Τ., Yamazaki,T., Yoshiki,S., Suda.T. (1981) Proc.Natl.Acad.Sci.USA. 78:4990-4994. 2. Koh,E., Morimoto,S., Fukuo.K., I t o h , K . , H i r o n a k a . T . , S h i r a i s h i . T . , Otiishi.T., Kunahara.Y. (1988) Life Sei. 42:215-223. 3. Nawa.K., Nakamira.T., Kunatani.A., Noda.C., Ichihara,A. (1986) J.Biol.Chem. 261:16883-16888. 4. Moshage,H., Huard.H., Princen.H., Yap.S. (1985) Biochim.BLophys.Acta. 824:27-33. 5. Tanaka.K., Sato,M., Tanita,Y., Ichihara.A. (1978) J.Biochem. 84:937-946. 6. Laeranli.U.K. (1970) Nature 227:680-685. 7. Ohsawa.K., Ebuta.N. (1983) Anal.Biochan. 135:409-415. 8. Towbin.H., StaehelinJ., Gordon,J. (1979) Proc.Natl.Acad.Sci.USA. 76:435CW354. 9. Sugiyama.K., Izimi.S., Tonino.S., Nagase.S (1987) J.Biochem. 102: 967-970.

THE SEPARATION OF KERATINOCYTE POPULATIONS USING A GRAVITY SEDIMENTATION CHAMBER : A POTENTIAL TOOL FOR THE STUDY OF 1.25(OH) 2 D 3 ACTION ON CELLS AT DIFFERENT STAGES OF DIFFERENTIATION. J.H. PAVLOVITCH, Μ. RIZK-RABIN and M. GARABEDIAN. CNRS UA.583, Laboratolre des Tissus calcifies, Hop!tal Necker-Enfants-Malades, Paris, France. INTRODUCTION 1,25-dihydroxycholecalciferol (l,25(OH)2Ü3), the active form of vitamin D3, has been shown to be one of the important factors for maintaining normal epidermal structure and metabolism through an effect on cell differentiation (1,2,3). Furthermore, the epidermal keratinocytes itself can synthesize 1,25(0H)2D3 (4). The keratinocytes are genetically programmed to divide at a constant rate in the basal layer and to enter the differentiation pathway. The epidermis, therefore, represents an heterogeneous system of the same type of cells at different stage of differentiation. As part of our study on the vitamin D action in epidermis, we attempted to separate the keratinocyte populations of different stage of differentiation pathway. We investigated, then, the ability of these cell populations to synthesize l,25(OH)2D3 and to respond to this hormone. MATERIALS AND METHODS New-born rat keratinocytes were separated on the basis of their size in a unit gravity velocity sedimentation chamber Into different fractions. Cell size distribution of the keratinocytes was determined using a Coulter Counter with channel analyzer. Flow cytometric study In living and alcohol (70%) fixed cells was performed after staining respectively with Hoescht 33528, Acrldin Orange and Propidium Iodide. DNA, RNA and cell size distributions were analyzed by univariate and bivariate histograms. Growth parameters of cells from different fractions were studied in culture on a feeder layer of mitomycin C treated 3T3 cells (seeding density 10 χ 10 4 per 1 cnr). In order to test the 3 H25(0H)D 3 -24 hydroxylase response to l,25(OH)2D3 and the l,25(OH)2D3 synthesis, cells were Incubated in a protein-free medium (2 χ 10 cells/ml) for 18 hours at 37°C under 5% C0 2 in the presence of ΙΟ - 9 Μ l,25(OH) 2 D 3 or of its ethanol solvent (0.5%). 3H-25(OH)D3 (2 nM) was then added to the medium and cells were Incubated for two additional hours. Media were collected and extracted with 4 volumes of Methanol-Chloroform (50:50). Chloroform-soluble extracts were cochromatographed with synthetic 24,25(OH)2D3 and 1,25(0H)2D3 on a straight-phase HPLC system (92.8 η Hexane-Isopropanol, 1.5 ml/min). The absorbance of the elution fluid was recorded at 254 nm and radioactivity measured in allquots of the 1.5 ml collected fracions. The l,25(OH)2D3 regions were rechromatographed on another HPLC system (95.5 methylene chloride-Isopropanol, 1 ml/min) and the radioactivity measured In the elution fluid. RESULTS

The use of the unit gravit gravity velocity sedimentation chamber enabled us to separate rat keratinocytes Into different cell fractions. The first fractions(3-5) consisted of pure population of uniformly small (about 7.5 u) cells with large nuclei and invisible cytoplasms. The majority of them (90%) were in G 0 /G^ cell cycle phase with low RNA content. These cells growed in forming small colonies of cells with basal morphology, and no signs of differentiation.

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · N e w York - Printed in Germany

369 The cells from the next 2-3 fractions were larger (about 8.5 u) due to an increased volume of cytoplasm. They were heterogeneous with respect to their cell cycle position and RNA content. Their morphology, Increased amount of cytoplasm and higher RNA content, indirectly suggests that they are more differentiated, rapidly dividing basal keratinocytes. They gave colonies of mixed composition with signs of differentiation and stratification in culture. Several cell populations were present in the last fractions (11-15) : spherical cells of slightly larger size, spinous and early granular cells in G 0 /G\, S or G2M phases. These cells rose to terminally differentiated daughter cells in culture. Preliminary results concerning both the vitamin D metabolism and l,25(OH)2D3 sensitivity of keratinocyte subpopulatlons suggest that the less differentiated stem cells from the first fractions have the highest capacity to produce. 1,25(011)203, whereas the more differentiated cells from the last fractions possess a 25(OH)D3-24 hydroxylase response to l,25(OH) 2 D 3 . Tn conclnslon : The method described in this work allows keratinocyte separation into satisfactorily homogeneous subpopulatlons : (1) basal stem cells, (2) more differentiated basal cells and (3) mature early spinal and granular cells. These populations are different regarding their capacity to metabolize 25(0H) 2 D 3 into 1 ,25(0H)2D3-like metabolite and their 25(0H)D3~24 hydroxylase sensitivity to l,25(OH) 2 D 3 . In the case the produced metabolite is 1,25-(0H)2D3, our results suggest the existence of a paracrine system within the mammalian skin,with certain keratinocyte populations being sensitive to the 1,25-(0H)2D3 locally produced by neighbouring keratinocytes at a different stage of differentiation. KF TERENCES 1. Hosomi, J., Kosoi, J., Abe, E., Suda, T., Kuroki, T.C. (1983) Endocrinology 113:1950-1957. 2. Pavlovitch, J.H., Galoppin, L., Rizk, M., Didierjean, L., Balsan, S. (1984) Am. J. Physiol. K):E4228-E4233. 3. Smith, E.L., Walworth, N.C., Holick, M.F. (1986) J. Invest. Dermatol. 86: 709-714. 4. Bikle, D.D., Nemanic, M.K., Gee, E., Ellas, P. (1986) J. Clin. Invest. 78: 556-557.

EVIDENCE THAT OSTEOINDUCTION BY IMPLANTS OF DEMINERALIZED ALLOGENIC BONE MATRIX IS DIMINISHED IN VITAMIN D-DEFICIENT NORMOCALCEMIC RATS. R.T. Turner, J.J. Vandersteenhoven and N.H. Bell Orthopedics, Mayo Clinic, Rochester, Minnesota, and V.A. Medical Center and Medical University of South Carolina, Charleston, South Carolina. Introduction The initiation of bone induction by demineralized allogenic bone matrix (DABM) implants at heterotopic sites is believed to be mediated by the release of factors from the matrix (1). W h e n DABM is implanted into vitamin D deficient (-D) rats, resorption of implant matrix, bone formation, and implant mineralization are each greatly reduced (2). In addition, the extractable bone cell mitogenic activity and osteoinductive potential of bone matrix prepared from - D rats are reduced (3,4). It is not clear, however, whether the requirement for vitamin D for osteoinduction is a direct effect, secondary to the action of vitamin D in maintaining mineral homeostasis, or both. To determine the role of serum calcium (Ca) in osteoinduction and the subsequent cellular processes, we implanted DABM into rats maintained on - D diets, on Ca-deficient (-Ca) diets (0.1% Ca) to induce hypocalcemia in animals receiving adequate vitamin D (+D) on - D diets supplemented with 60 pmol/day 1,25-dihydroxyvitamin D3, ( 1 , 2 5 ( 0 ^ 2 0 3 ) to induce hypercalcemia and on - D diets containing high lactose (20%) to normalize serum Ca in - D rats by increasing vitamin D independent Ca transport. Methods DABM was prepared from the long bones of normal rats, and the powder was implanted into the three - D groups and two groups of +D rats as previously described (2). Implants were recovered at three and six weeks postimplantation. Serum Ca, 25-hydroxyvitamin D (25-OHD), and l,25(OH)2D3 and implant histology were measured as described previously (2). Serum immunoreactive PTH (iPTH) was measured by radioimmunoassay. Results and Discussion - D rats had severe hypocalcemia which was corrected by a high lactose diet and over corrected by l,25(OH)2D3 treatment. - C a rats were also hypocalcemic but to a lesser extent than - D animals. Bone induction was decreased in - D rats. These animals had more of the original matrix than did the controls. This finding suggests that - D resulted in a defect in bone resorption as well as bone formation. Implant Ca was reduced in - D rats to a much greater extent than was induced bone indicating that the bone matrix that formed was under mineralized. Implants from - C a rats were qualitatively similar to those from - D animals except that the deficiency in mineralization of the newly formed matrix appeared to be less severe in the former group. Thus, hypocalcemia per se, resulted in many of the changes associated with - D . Administration of l,25(OH)2D3 to - D rats reversed the direction of

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · N e w York - Printed in Germany

371 all of the changes: the rats became hypercalcemic, had increased induced bone, decreased original implant matrix and increased implant Ca. When - D rats were fed a diet containing 20% lactose, serum Ca and phosphorous (data not shown) as well as original implant matrix were normal. In contrast, implant Ca and induced bone were reduced to an extent comparable to the values in -D, hypocalcemic rats. Serum 25-OHD and l,25(OH)2D3 were reduced as expected in - D rats, and serum iPTH was elevated in the - C a rats. The findings of these studies suggest that osteoinduction is vitamin D dependent. This is in agreement with our previous results demonstrating that bone matrix from - D rats has greatly reduced osteoinductive activity when implanted into normal rats and contains reduced bone cell mitogenic activity (3). In contrast, resorption of the implant does not appear to require vitamin D but may be calcium dependent. In other studies, we demonstrated that bone turnover in DABM implants is altered by systemic factors that regulate bone turnover in the skeleton including ovarian hormones (4) and fluoride (unpublished). Further, vitamin D metabolites, ovarian hormones, and fluoride each regulate osteoinduction in a manner consistent with the skeletal action of these agents. Therefore, it is interesting that - C a results in a decrease in implant resorption in the presence of elevated serum PTH. Table 1: The Effects of Vitamin D and Calcium on Osteoinduction by DABM Powder Measurement -D +D -D -D Low Ca +l,25(OH)2D3 High Lactose Serum Ca Original Implant Matrix Induced Bone Implant Ca

51 + 2** 151 + 2**

68 + 9* 9 + 2**

117 + 2*

103 + 2

227 + 6**

75 + 7*

105 + 11

23 + 8**

105 + 18

61 + 11*

23 + 7**

113 + 15

62 + 12*

91 +

Values are expressed as % of control _+ SEM; η = 6-9. * ρ < .05, ** ρ < .01 compared to +D control by non-paired t test. References 1. 2. 3. 4.

Reddi, A.H. (1985) In Bone and Mineral Research, Peck, W.A., Ed. Elsevier, New York, pp 27-47. Vandersteenhoven, J.J., DeLustro, F.A., Bell, N.H., and Turner, R.T. (1984) Calcif. Tiss. Int. 42,39-45. Turner, R.T., Farley, J., Vandersteenhoven, J.J., Bell, N.H., and Baylink, D.J. (1988) J. Clin. Invest, (in press). Turner, R.T., Vandersteenhoven, J.J. and Bell, N.H. (1987) J. Bone Min. Res. 2,115-122.

Supported in part by Grant AR 35651 from the NIH.

l f 25-(0H) 2 D 3 BINDS SPECIFICALLY TO RAT VASCULAR SMOOTH MUSCLE CELLS AND STIMULATES THEIR PROLIFERATION IN VITRO. Ε. KOH, S. MORIMOTO, K. FUKUO, R. MORITA, S. KIM, T. ONISHI and Y. KUMAHARA Dep. Med. & Geriat., Osaka Univ. Med. Sch., Osaka, Japan. Introduction l,25-(OH)2D3 is known to affect proliferation of certain cells that have a specific receptor for it (1,2). However, there have been no reports on the receptor of 1,25-(0H)2D3 and its effect on the proliferation of vascular smooth muscle cells (VSMC) in vitro. In this study, we found that VSMC have a specific receptor for 1,25-(0H)2D3 and that 1,25-(0H)2D3 induced increase in their proliferation concomitant with change in their morphology. Materials and Methods VSMC were prepared from the aorta of Wistar rats and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (3). The cytosol fraction of VSMC was prepared for examinations of equilibrium binding studies, sucrose density gradient analysis and DNAcellulose chromatography by the methods described elsewhere (4). Inocula of 2 χ 10* VSMC were introduced into 35 mm culture dishes. After attachment of the cells (24 h), the medium was replaced by fresh medium containing vehicle with or without vitamin D3 metabolites, PTH, CT or additional CaCl2 (day 0). These media were renewed on day 3. On day 6, cells were harvested from triplicate dishes by trypsinization and counted in a Coulter counter. Results and Discussion Cultured VSMC were found to contain a specific receptor for 1,25(0H) 2 D 3 . Its Kd (5.0 χ 10" 1 1 M) and capacity (22.9 fmol/mg) for 1,25-(0H)2D3, its sedimentation coefficient on a sucrose density gradient (3.2 S), its relative affinities for various vitamin D metabolites [l t 25-(0H) 2 D 3 » 25-0HD 3 > 24,25-(0H) 2 D 3 » D 3 ] and its affinity for DNA-cellulose were similar to those reported for the 1,25-(0H) 2 D 3 receptor in other tissues (2). 1,25-(0H) 2 D 3 at concentrations of more than 10 - 10 Μ caused significant and dosedependent stimulation of cell proliferation. Other vitamin D3 metabolites also enhanced cell proliferation, but to lesser extents, [1,25-(0H) 2 D 3 » 25-OHD3 > 24,25-(0H) 2 D 3 > D 3 ] (Table 1). Control VSMC grew in multi-layers as polygonal cells with obscure outlines, but in the presence of 1,25-(0H)2D3 at 10 - ^ Μ or more they became smaller and grew as multi-layers of spindle-shaped cells with clear outlines. Similar morphological changes were seen with 25-OHD3 and 24,25-(0H) 2 D 3 at concentrations of 1 0 - 6 M. PTH, CT or increase in extracellular Ca concentration had no effect on the proliferation or the morphology of VSMC. Recent studies revealed that proliferation of VSMC is important in the pathogenesis of arteriosclerosis (3). Moreover, Ca and Ca-regulating hormones were reportedly associated with development of arteriosclerosis in vivo (5-7). However, little is known about the effects of these hormones on the proliferation of VSMC in vitro. In the present study, we found that PTH, CT and increase in the extracellular Ca

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter&Co., Berlin · N e w York-Printed in Germany

373 concentration had no significant effect on the proliferation or morphology of VSMC, but that vitamin D3 metabolites, especially 1,25-(0Η)203, stimulated proliferation of VSMC with change in their morphology in vitro. Moreover a specific receptor for 1,25-(0H)2D3 was found in the cytosol of VSMC. At present, it is not known how the proliferation of VSMC is regulated by 1,25-(0H) 2 D3 and 25-0HD 3 in vivo. However, excess ingestion of vitamin D induces marked elevation of 25-OHD3 and slight elevation of 1,25-(0H)2D3 in the blood (8). The present studies iji vitro suggest that vitamin D metabolites may stimulate proliferation of VSMC in vivo and that arteriosclerosis induced by vitamin D intoxication may depend at least in part on the stimulation of proliferation of VSMC by vitamin D metabolites. Table 1. Effects of Ca and Ca-regulating hormones on the growth of vascular smooth muscle cells. Mean + SD. p3

ΡΤΗ CT CaCl 2

ΙΟ" 8 Μ 10~ 7 Μ 10~ 6 Μ

101+4 104+2 129+5**

10-10 Μ ΙΟ" 8 Μ

108+2 110+6

ΙΟ" 5 U 10~ 3 U

96+3 93+2

2.3 mM 3.3 mM 4.3 mM

98+5 91+5 89+5

References 1. Abe,E., Miyaura,C., Sakagami,H., Takeda.M., Κοηηο,Κ., Yamazaki,T., Yoshiki,S., Suda,T. (1981) Proc.Natl.Acad.Sei.USA. 78:4990-4994. 2. Dokoh.S., Donaldson,C.A., Haussler,M.R. (1984) Cancer Res. 44:2103-09. 3. Ross.R., Glomset,J.A. (1976) N.Engl.J.Med. 295:369-377 and 420-425. 4. Koh,E., Morimoto,S., Fukuo,K., Itoh,K., Hironaka.T., Shiraishi,T., 0nishi,T., Kumahara,Y. (1988) Life Sei. 42:215-223. 5. Peng,S.Κ., Taylor,C.B. (1980) Arterial Wall. 6:63-68. 6. Herbert,F.K., Miller,H.G., Richardson,G.0. (1941) J.Pathol.Bacteriol. 53:161-182. 7. Robert,L., Brechemier,D., Godeau,G., Labat,M.L., Milhaud,G. (1977) Biochem.Pharmacol. 26:2129-2135. 8. Mawer,Ε.Β., Hann,J.Τ., Davies,M. (1985) Clin.Sei. 68:135-141.

INVOLVEMENT O F INTRACELLULAR F R E E CALCIUM AND pH IN THE E F F E C T O F 1.25(OH)2D3 ON LEUKEMIC CELLS. S. SHANY, E. BARNEA, P. HAZAV and R. LEVY Clinical Biochemistry Unit, Faculty of Health Sciences, University of the Negev, Beer Sheva, Israel.

Ben-Gurion

Introduction Attention has recently been focused on the potential action of 1.25dihydroxyvitamin D3 [1.25(OH)2D3] in controlling cell growth and differentiation. 1.25(OH)2D3 was found to suppress proliferation of human leukemic cells (HL-60) and to induce their differentiation to monocyte-like cells. In a previous study (1) we showed that limitation of available calcium ions by means of EGTA or verapamil enhanced the inhibitory effect of 1.25(OH)2D3 on proliferation and caused a decrease in the number of differentiated cells obtained by 1.25(OH)2D3 alone. The purpose of the present study was to examine the role of intracellular free calcium ions [Ca 2+ i] and intracellular pH (pHi) alterations in the effect of 1.25(OH)2D3 on inhibition of proliferation and induction of differentiation of HL-60 cells to monocyte-like cells. Materials and Methods HL-60 cells were grown in stationary suspension culture in RPMI-1640 medium containing 10% fetal calf serum, 2mM L-glutamine, 100U/ml penicillin, 100μg/ml streptomycin and 12.5U/ml nystatin (Biological Industries, Beth Haemek, Israel), at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Cell number and viability were determined by trypan blue exclusion, using a hematocytometer, and by (methyl 3 H)-thymidine incorporation. Differentiated cells (monocytes) were determined by using the monoclonal antibody Mac-1 (2). [Ca 2+ i] concentrations were measured by using the fluorescent indicator quin-2AM (3). Fluorescence intensity was recorded with a spectrofluorometer (Perkin-Elmer LS-5). pHi was measured using B C E C F [2'7'-bis(carboxyethyl)-5-(6)-carboxyfluorescein] (4). Changes in pHi altered the fluorescence. Calibration was carried out in every sample by solubilizing the cells with 0.05% Triton X-100, followed by the addition of small amounts of HCl or NaOH and measuring the fluorescence. Results and Discussion The treatment of HL-60 cells with 1.25(OH)2D3 caused a significant increase in [Ca 2+ i] concentration (from 101 ±4 nM to 134±5 mN, pF-Q-£

S-LFLTE

PEP-19

P-Ö-I-Ü-M-IM-P-S-T-E-S-A

Fig.2. E-F Hands of various CaBPs compared to that of PEP-19.

(1) McBurney.R.M. and Neering.I.R.(1987), TINS,10(4),164-169. (2) Ziai,R.,Pan,Y-C.E.,Hulmes,J.D., Sangameswaran,L and Morgan,J.I.(1987) Proc.Natl.Acad.Sei.80,8420-8423 (3) Auffray,C and Rougeon,F.(1980), Eur.J.Biochem.107.3 03-314. (4) Taylor,A.N.(1974),Arch.Biochem.Bioph ys.161,100-108. (5) Huang,Y.C.,et al.,(1988),ASBMR(Abstract)(In Press).

INDUCTION OF OSTEOCALCIN SYNTHESIS IN HUMAN OSTEOSARCOMA CELLS BY 1,25-DIHYDROXYVITAMIN D AND 24,24-DIFLUORO-l,25-DIHYDROXYVITAMIN D A. MAHONEN, A. PIRSKANEN, M. HAUKILAHTI and P.H. MÄENPÄÄ Department of Biochemistry, University of Kuopio, Kuopio, Finland Introduction l,25(OH)2D3, the most active metabolite of vitamin D, exerts diverse effects on normal growth and development through the action of specific intracellular receptor molecules (1,2). These receptors are thought to function as trans-acting regulatory proteins by interacting with chromatin and modulating the transcription of specific genes in target cells. The l,25(OH)2D3 receptor, which is present in very low abundance, has been shown to vary in size from 52-54 kd to 58-60 kd and there is an inverse relationship between the receptor mass and the phylogenetic status (3). Vitamin D is known to control calcium homeostasis via effects on intestine, bone and kidney along with other defined biological functions (4). The most studied protein, that appears to be produced specifically by osteoblastic bone cells, is osteocalcin, which may regulate mineral deposition in bone (5). In this study, we describe uptake of l,25(OH)2D3 into cultured human osteosarcoma cells and induction of osteocalcin synthesis in these cells. Materials and Methods Human osteosarcoma cells, MG-63 ( ATCC, Rockville MD), were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 100 U/ml penicillin and 100 Hg/ml streptomycin in a humidified 95% air /5% CO2 incubator. The production of osteocalcin by human osteosarcoma cells in vitro was stimulated by l,25(OH)2D3, 24,25(OH)2D3 and 24,24-F2l,25(OH)2D3 (a gift from Hoffman-La Roche, Inc.) as a function of time and concentration. Intracellular and secreted osteocalcin was measured by radioimmunoassay (Immuno Nuclear Co.). Specific binding sites for l,25(OH)2D3 were quantified by incubating intact MG-63 cells for various times up to 24 h with 3 H-l,25(OH)2D 3 alone or with 200-fold excess of l,25(OH) 2 D3· For a saturation analysis, the cells were incubated at different hormone concentrations. The effect of hormone concentration on the cellular content of receptors was further evaluated by incubating the cells with various concentrations of ,25(OH>2D3 and then filling unoccupied sites by incubating with a saturating dose of l,25(OH)2D3Results and Discussion Human osteosarcoma cell line (MG-63) contains receptors for l,25(OH)2D3 and exhibits a hormone-dependent accumulation of specific binding sites for this metabolite. Saturation analysis of hormone binding indicated that human osteosarcoma cells possess classical receptors for 3H-1^5(OH)2D3 corresponding to about 4400 receptor molecules per cell. Receptors in MG-63 cells appeared to be saturated with 3 H-l,25(OH)2D3 in 3 hours after hormone addition and further exposure of the cells to the hormone resulted in an up-regulation of receptors (Fig.l). The level of hormone binding was elevated by as little as 50 pM of l,25(OH)2D3 and maximum levels of liganded receptors were achieved with nanomolar hormone concentrations. Human osteosarcoma cells also respond to l,25(OH)2D3 with two- and fourfold increases, respectively, of intracellular and secreted levels of osteocalcin (Fig. 2). Time course experiments demonstrated that osteocalcin first appear in the medium 10 hours after the addition of l,25(OH) 2 D 3 . The stimulatory response is specific for l,25(OH) 2 D 3 and 24,24-F2-l,25(OH) 2 D 3 . 24,25(OH)2D3 has no effect on the basal production of osteocalcin. The regulation of vitamin D receptor levels demonstrates that the magnitude and dose dependence of the response is also influenced by the duration of the hormone exposure. The cellular response to l,25(OH)2Ö3 was consistent with its predicted effects in modulating receptor levels and stimulating osteocalcin

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · New York - Printed in Germany

392 synthesis. The results show for the first time that human osteosarcoma cells (MG-63) contain receptors for l,25(OH)2D3 and exhibit hormone-dependent stimulation of osteocalcin synthesis and secretion.

0

50

200

350

700

WSiOHijDg concentration (pM) Figure 1. l,25(OH>2D3-dependent accumulation of specific binding sites in MG-63 cells.

Time (h) Figure 2. Synthesis and secretion of osteocalcin by MG-63 cells after addition of l,25(OH>2D3. References 1. Wecksler, W.R., Norman, A.W. (1980) J. Steroid Biochem. 13: 977-989. 2. Haussler, M.R., Pike, J.W., Chandler, J.S., Manolagas, S.C., Deftos, L.J. (1981) Ann. N.Y. Acad. Sei. 372:502-517. 3. Mangelsdorf, D.J., Pike, J.W., Haussier, M.R. (1987) Biochemistry 84: 354-358. 4. Haussler, M.R. (1986) Annu. Rev. Nutr. j>: 527-562. 5. Price, P.A. (1983), in Bone and Mineral Research Annual, Peck, W.A., Ed., Excerpta Medica, New York, Vol 1., pp. 157-190.

Biological Actions of Vitamin D Metabolites

TOE BOLE OF 24,25 DIHYDROXY VITAMIN D3 DURING DEVELOPMENT OF SKELETAL AND NON-SKELETAL TISSUES D. Samjen, Y. Weisman*, S. Harell**, Y. Earon**, A. Harell, E. Berger, Z. Shimshcni, A. Waisman***, A.M. Kaye*** and I. Binderman Hard Tissues Laboratory, *Vitamin Research Laboratory, and **Department of Pediatrics, Ichilov Hospital, Tel Aviv 64239, and ***Department of Ησπηοηβ Research, The Weizmann Institute of Science, Rehovot 76100, Israel. INIBCOUCTICN While the role of the vitamin D metabolite l,25(CH)^Do (calcitriol) has been well established in regulation of calcium metabolism, the role of the mare abundant metabolite 24,25(CH)2D3 is less clear. This paper reviews experimental data which suggest a role for 24,25(CH)2D3 in the embryonic stage of skeletal and non-skeletal tissues and during their development in the perinatal and postnatal period. We propose that, among other functions, 24,25(CH)oD3 is involved in the development of tissue responsiveness to 1,25(0H)2Ö3, in post-natal and maturation stages of several tissues. Like other steroids, 1,25(0H)OD3 interacts with specific intracellular binding proteins (1-3) which participate in regulating mRNA for the specific proteins which mediate its biological activity (1,2). Receptors for 1,25(GH)2D3 have been demonstrated in a variety of organs (1,3), leading to the realization that the vitamin D endocrine system extends far beyond its original classical sites of action (1), the intestine (4,5) and bone (7), where its main role is in calcium absorption (6) and bone mineral mobilization (8). l^SiOHJoDo has also been shown to be associated with cell proliferation and differentiation (1,2). In rat diaphyseal osteoblasts which contain specific receptors for its action (9) 1,25(0H)2D3 stimulates DNA synthesis and ornithine decarboxylase (10) and creatine kinase (CK) specific activities (11). Mature rat kidney also contains receptors for 1,25(0H)2D3 (2,12-15) which stimulate CK activity both in normally-fed and vitamin D-depleted rats (16). We have also recently demonstrated that 1,25(OH^3 stimulates the specific activity of CX in mature rat and rabbit cerebellum and in mandibular condyle after the first weeks of life (17). The vitamin D metabolite 24,25(CH)^3,which exists in high concentration in the circulation, is not implicated in calcium transport but was shewn to have a role in the development of endochondral bene (18-20) and hatching of chick embryos (21,22). 24,25(0Η)203 binds specifically to receptors in parathyroid gland (23), chondrocytes (24), epiphyseal growth plates (9), limb bud mesenchymal cells (25) and immature rat kidney (16). In seme of these tissues, 24,25(0H)2Ü3 also stimulated CK specific activity and DNA synthesis (17). It was, therefore, intriguing to test for 1 T the role of 24,25{r"^ n ~ it acting in immature organs in the same way as Does it have an independent mechanism of actio ___.. t paring skeletal and ncn-skeletal tissues far responsiveness to 1,25 (OH ^ 3 in later life?

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter&Co., Berlin · New York- Printed in Germany

396 DEVELOPMENTAL RESPONSIVENESS TO VITAMIN D METABOLITES Differential responsiveness of skeletal tissues to vitamin D metabolites. 1,25(0H)2D3 binds to specific receptors (9)rinduces DNA synthesis (10) and stimulates ODC activity (10) and CK activity (11) in diaphyseal osteoblasts of vitamin D-depleted rats, at all ages. In chrondroblasts from epiphyseal cartilage it is 24,25(0H)2D3 which stimulates the same activities at all ages tested (17). The rabbit, which is a perinatal developer like humans (unlike the rat, in which the development of seme organs is shifted postnatally), shews the same distinction. Epiphyses of normally-fed rabbits, 6 days before birth to 15 days after birth, respond by increased CK activity to 24,25(0Η)2Ο3, while 1,25(0H)2D3 has no effect cn enzyme activity (17). In contrast, condylar chcndroblasts from rat show a developmental change in responsiveness, responding to 24,25(CH)2D3 in early development, from 6-20 days after birth, and subsequently developing responsiveness to 1,25(0H)2D3 beginning at 21 days after birth (17). Sequential responsiveness of kidney to vitamin D metabolites. The kidney shews an autocrine response to vitamin D metabolites, since it is the principal site of synthesis of the dihydroxylated metabolites of vitamin D (28-30) to which it also responds. Receptors for l,25(0H)2Do (2,12-15) are detected in adult kidneys. There are also two vitamin D-dependent Ca binding proteins induced by 1,25(0H)2D3 (31). One of these is similar to that found in the intestine (32-35); its concentration in kidney (31) and intestine (36) varies with age, with a marked increase starting at day 18 after birth. We have shown (16) that there is a change in binding activity in the kidney with age. 24,25(0H)2D3 binds predcminantly in 3-15 day old rats, showing low level after 21 days. By contrast, 1,25(0*1)203 is bound in increasing concentration with age till a plateau is reached at day 18; this day is also a cross-crver point at which there is equal binding for the two metabolites (16). Fran 21 days an, there is predcminantly binding of l,25(0H)2Do. These changes in binding are reflected in changes in responsiveness using either CK activity (16) or DNA synthesis (17) as criteria. The same changes are seen in normally-fed rats and vitamin D-depleted rats (16). In the rabbit, the same pattern of response is found, although shifted to the perinatal rather than the postnatal period. At 6 days before birth up to 3 days after birth, 24,25(0H)2D3 but not 1,25(0H)2D3 increases CK specific activity in kidney of normally fed rabbits. Ar 15 days after birth, only 1,25(0H)2D3 increases CK specific activity while at 8-9 days after birth both metabolites stimulate activity (17). Sequential responsiveness of brain to vitamin D metabolites. The brain also contains vitamin D-dependent calcium binding proteins (37,38) similar to those induced by 1,25(0H)2D3 in the intestine. Calcium binding protein as well was found by immunbhistociiemistry in cerebellum following vitamin D administration (39). Specific rat brain nuclei, which contain 1,25(0H)2D3 receptors and/or calcium binding protein, shewed an increase in choline acetyl transferase activity after treatment with 1,25(0H)2D3 (40). Therefore, we tested whether the brain of either

397 rat or rabbit is responsive to vitamin D metabolites. The normal developmental increase of CK specific activity in the cerebellum during the postnatal period is prevented in vitamin D-depleted rats (Fig. 1). The developmental increase in cerebellar CK specific activity in normal rats is paralleled by changes in the steady state 8

Normally - fed rats

Α|c4 ο a> υο α >CT °· εξ3.

in

kidney

cell

cultures

by

Cell cultures from kidneys of 1 week old rats respond by both increased CK specific activity and increased DNA synthesis to 24,25(0Η)ΟΓ>3 but not 1,25(CH)2D3 (17). On the other hand, cell cultures prepared from kidney of 5 week old rats show an exclusive responsiveness to l,25(CH)2Do. AS in vivo (16), at an intermediate stage, 3 week old rat cell cultures from kidney respond to both metabolites (17). Cells treated daily with either vehicle or 1,25(01)203, shew no change in their pattern of responsiveness to either metabolite at any age, in terms of either increased CK activity (Table 3) or augmented DNA synthesis (Table 3). However, one week of treatment with 24,25(0H)oD3 causes a precocious development of responsiveness to 1,25(0H)2D3 in cell cultures made from kidneys of 1 week old rats. Similarly, in cultures from kidneys of 3 week old rats, 24,25(0H)2D3 treatment increases the responsiveness to 1,25(0H)2D3 and abolishes the responsiveness to 24,25(0H)2D3. In cell cultures from kidneys of 5 week old rats, 24,25 (OH ^03 no longer has any effect on hormonal responsiveness (Tables 3,4). Daily treatment with

400

Table 3.

The effect of daily treatment for 1 week with either vehicle (control, 10 jul/ml of 20% ethanol in saline) 1,25(0H)2D3 (1.2 nM) or 24,25(CH)2D3 (12 nM) on the responsiveness of renal cell cultures to vitamin D metabolites by increased CK activity. Stimulation of CK activity (E/C) by 1,25(0H)2D3 by 24,25(CH)2D3

Age

Chronic treatment

1 week

Control 1,25(0H)2D3 24,25(0H)2D3

0.89±0.16 0.89±0.13 2.26*0.17**

1.51*0.16* 1.46*0.20* 2.26*0.14**

3 weeks

Control 1,25(0H)2D3 24,25(^)^53

1.60*0.21* 1.40*0.13* 1.86±0.19**

1.49*0.19* 1.44*0.17* 1.06*0.27

5 weeks

Gcntrol 1,25(CH)2D3 24,25(0Η)^3

2.00*0.11** 1.91*0.06 2.06±0.16**

1.06*0.17 0.89*0.17 1.00*0.17

Two days after the last addition, the responsiveness to either horn m e was measured. Results are expressed as experimental divided by control means ± SE for η = 6-9. Table 4.

The effect of daily treatment for 1 week with either vehicle (control) 1,25(0H)2D3 or 24,25(CH)2Üo on the responsiveness of renal cell cultures to vitamin D metabolites by increased DNA synthesis.

Age

Chronic treatment

1 week

Control

Stimulation of DNA synthesis (E/C) by 1,25(0H)2D3 by 24,25(0H)2D3

24,25(OH)2^3

1.00*0.11 1.03*0.09 1.63*0.09**

1.40*0.06* 1.60*0.05** 1.69*0.09**

3 weeks

Control 1,25(CH)2D3 24,25(CH)2D3

1.66*0.17* 1.46*0.17* 1.80*0.18**

1.86*0.17** 1.49*0.17* 1.26*0.19

5 weeks

Control 1,25(0H)2D3 24,25(CH)2D3

1.80*0.14*** 1.53*0.14** 1.51*0.17*

0.69*0.14 0.97*0.19 0.83*0.17

Treatment was described in the legend to Table 3. Results are expressed as experimental divided by control means * SE for η = 6-9.

401 either PTH (1 U/wl) or 25(0H)E>3 (12 r*I) has no effect. Combined treatment with the previously given doses of 1,25(0H)2D3 and 24,25(OH)2D3 caused the same effect as 24,25(0H)2Do alone (17). Cell cultures frctn rat epiphyseal cartilage (27) respond acutely to 24,25(0H)2D3 but not 1,25(01)203 in terms of increased CK specific activity and increased DNA synthesis. When these cartilage cells were treated chronically with either vitamin D metabolite, no change in their pattern of responsiveness to vitamin D metabolites could be observed (Table 5). Table 5. The effect of daily treatment for 1 week (as described for Tables 3,4) on the responsiveness of epiphyseal cell cultures to vitamin D metabolites by increased DNA. synthesis.

Chronic treatment Control 1,25(CH)2D3 24,25(CH)^3

Stimulation of DNA synthesis (E/C) by 1,25(0H)2D3 by 24,25(CH)2D3 1.07±0.10 1.07±0.14 1.20±0.12

1.73*0.08** 1.78±0.04** 1.89*0.23**

Results are expressed as experimental divided by control means ± SE for η = 8. CONCLUSIONS AND OCWENTS The predominant responsiveness during early development to 24,25(GH)oDo and not to 1,25(0H)2D3 in non-skeletal tissues such as kidney, cerebellum and pituitary, suggests that this vitamin D metabolite should be tested far a role in differentiation in other organs and in species other than rat and rabbit. We have demonstrated in rat kidney (16) a correlation between the type and concentration of renal binding proteins for vitamin D metabolites and response to these metabolites by increased CK activity. This suggests a specific role during development for each of the two metabolites. A developmental change in responsiveness to vitamin D has also been reported in the intestine (31), in which increase in calcium binding protein in response to l,25(OH)2D3 starts only from day 18 (31). Our previous results (17) are the first to demonstrate an acute effect of vitamin D metabolites on any portion of the brain. Moreover, the lack of increase in CK activity as a function of age in vitamin D-deficient rats in both cerebellum and cerebrum is strikingly different from the normal growth curve for CK activity in brain areas and in whole brain (43) in normally-fed rats. The changes in mRNA for α tubulin which differ from those for CKB confirm that the pattern of changes in mRNA for CKB is specific. The deficiency in CK activity upon vitamin D-depletion suggests that the changes in enzyme activity may have a deleterious effect on brain development. The acceleration of maturation parameters of kidney cell cultures by chronic treatment with 24,25(0H)2D3,but not l,25(OH)2D3 provides support

402

for the general hypothesis that 24,25(0H)2Ü3 acts in kidney (and in other tissues) as a maturation factor, possibly by induction of receptors to 1,25(CH)2D3 accomplished by down regulation of its cwn receptors. This concept nas been previously investigated in (44) different cell lines in which 24,25(0H)2DQ, like other steroids, caused an increase in the number of receptors to l,25(0H)2D3 without altering their affinity. Since in cells containing receptors for 1,25(011)^3 there is induction of 24 hydroxylase activity (3), they might contain sufficient 24,25(0H)2D3 to dcwn regulate its own receptors, leaving the cells with essentially only 1,25(CH)2D3 receptors. On the other hand, in epiphyseal cartilage cells which have exclusively receptors for and respond to 24,25(0H)2D3 (26), there is no induction of receptors to 1,25(0H)2D3 by the other metabolite. Moreover, in chick intestine, 24,25(0H)2Ü3 allosterically modulates the binding of 1,25(0H)2D3 to its chromatin receptor (45), separately frcm its own specific binding domain (45). We, therefore, suggest that 24,25(0H)2D3 may act as a maturation factor during early development, possibly by induction of receptors for 1,25(0H)2D3 parallel to the induction of progesterone receptors by estrogen. ACKNOWLEDGMENTS We thank Mrs. Rena Levin far her skilled processing of this manuscript, Dr. Pamela Benfield for CK cDNA. clone and Prof. Irit Ginzburg for α tubulin cDNA. AMK is the Joseph Moss Professor of Molecular Endocrinology in the Weizmann Institute of Science. REFERENCES 1.

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T H E R E G U L A T I O N OF I N T R A C E L L U L A R IONIZED CALCIUM B Y CALCITRIOL S. E D E L S T E I N , S. BAR, A. H A R E L L and C. LIDOR Biochemistry Department, The Weizmann Institute of Science, Rehovot Israel.

76100,

Introduction The mechanism of action of vitamin D is still not known, though the presence or absence of a receptor for 1,25(OH)2D.j (calcitriol) in selected cells is used by many investigators to identify new target organs for the vitamin. These include skin (1), pituitary (2), pancreas (3), lymphocytes and monocytes (4), placenta (5) and others.

In classical target organs for vitamin D action such as

intestine or kidney, the action of calcitriol is known to involve calcium absorption and translocation (6), a process which depends on the calcitriol protein, calbindin (7, 8).

However, in many of the non-classical

induced

target

cells

listed above, calbindin was not detected, thus, raising the question what is the function of calcitriol in these receptor-containing cells. Cytosolic free calcium ( [ C a * ^ ] ^

is the key regulator for the activity

of

most cells and the action of a very large number of hormones involve changes in [ C a ^ j j (9, 10). Utilizing isolated cartilage cells from rachitic chicks we were able to show that calcitriol decreases the concentration of intracellular free calcium.

This action is receptor-mediated and involves modulations of activity of

the C a ^ - p u m p i n g ATPase, Materials and Methods Isolation of cartilage cells. Rachitic chicks aged 3-4 weeks were sacrificed and the legs (tibia) were chilled on ice. Slices from epiphyseal growth plate were digested at 3 7 ° C in a bath shaker using proteolytic enzymes dissolved in synthetic cartilage fluid buffer (10 ml per 1 g, tissue) (11). After one hour of incubation with 0.25% hyaluronidase (Sigma, Type 1-S 300 N F units/mg solid) and with 0.25% trypsin (Sigma Type III-S, 13,000 B A E E units/mg protein) the supernatant was decanted and the slices were further digested in 0.2% collagenase (Sigma, Type II 300 units/mg protein) for another 2 hours. At the end of the second incubation the slices were washed with cold buffer, minced with glass rod and the cells filtrated through nylon mesh (45 μ m). The cells were washed twice with cold buffer.

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · New York - Printed in Germany

406 Incubation of cartilage cells with vitamin D metabolites.

Isolated washed

chondrocytes were finally suspended in BGJ medium (Fitton-Jackson Modificar tion) containing 1.5 mM calcium, supplemented with 2.5% serum obtained from rachitic

chicks.

Cells

4-6x10® cells/ml

were

medium.

incubated Vitamin

at

37°C,

5%

CO2

D metabolites dissolved

in plastic

tubes,

in ethanol

added at final centration 0.25% ethanol for various periods of time.

were

The con-

trol probes were treated with ethanol. Manipulating intracellular calcium.

Cartilage cells after overnight incubation

in BGJ medium were treated with 4 μ Μ Chemicals

Ltd.,

Jerusalem,

Israel).

of

Reduction

the ionophore A2gjg7 of

intracellular

(Makor

calciuai

was

achieved by adding 35 μ Μ quin 2 acetoxymethylester (Sigma Chemical CO.) to cells after overnight

incubation in BGJ medium containing very low calcium

(0.04 mM). Measurement

of

intracellular

free calcium.

the fluorescent indicator quin2 (12).

[Ca^ + ]j was determined using

Calibration of fluorescence signals and the

final estimation of [ C a 2 + ] were done essential according to Powell et al. (13). [Ca2~^]j was calculated using the formula, [Ca2+] =

115 (F* - Fmin)/(Fmax-F)

where 115 nM is the apparent Kd for the quin2-Ca2"^ complex at 37°C, F is the recorded fluorescence of the loaded washed cells, Fmax is the maximal signal of indicator at 1 mM calcium obtained by adding digitonin (35 μ Μ ) and diethylenetriaminepentaacetic acid (10 μ Μ ) , and Fmin is the minimal fluorescence signal still remaining after displacing Ca2"^ from the Ca-saturated indicator with Mn2+

(MnCl2 0.5 mM).

F* is the net fluorescence within the loaded cells

determined separately by adding M n C ^ (0.5 mM) which quenches the leaked extracellular dye. Alkaline

phosphatase

(AP)

activity.

At the end of the incubation periods

the cell suspensions were centrifuged at 800 g (Sorvall, R T 6000).

The pellets

were washed twice to remove all medium fluids and homogenized for 10 sec in maximum speed, using Polytron homogenizer in 2 ml ice-cold 0.15 Μ NaCl containing 3 mM NaHCOß (pH=7.4).

The homogenizations were assayed for alka-

line phosphatase activity with p-nitrophenyl phosphate (purchased from Sigma) as a substrate in a 0.1 Μ sodium barbital buffer pH 9.3 (14).

Protein was

determined (15), and the results were expressed as units per mg protein. unit of phosphatase is defined as the enzyme activity that liberated

One

1 μπιοί

p-nitrophenol/0.5 h at 37°C per mg protein. C a 2 + -ATPase

activity.

The isolated cartilage cells were incubated for 3 h

and 40 h with 5 ng/ml calcitriol.

At the end of incubation periods the cells

were washed twice with 150 mM NaCl, 3 mM NaCl (pH 7.4), and redissolved in 10 mM HEPES (pH 7.4) containing protease inhibitor

[aprotinin 0.1 mM

PMSF (phenylmethane sulfonylfluoride), Sigma], on ice for 1 h (20 ml/1 ml cell

407 pellet).

T h e suspensions were passed through a syringe with a 27G needle, and

were centrifuged at 2000xg (Sorvall, RC-5B) for 10 min at 4 ° C .

The

pellets

were redissolved in 1 ml of 250 mM sucrose, 10 mM H E P E S , 1 % Triton X-100 pH 7.4 (containing the proease inhibitors).

After 1 h of extraction and solubili-

zation supernatants were prepared by centrifugation for 3 min in an Eppendorf 3200 centrifuge at 4 ° C .

The Ca2+-ATPase

after obtaining the supernatants.

activity was assessed

immediately

Microassays were carried out in a final vol-

ume of 60 μ I, for 60 min at 37°C.

Each assay tube contained 12-14 μ g pro-

tein, 5 mM A T P , 200 cpm Α Τ Ρ - [ 7 - 3 2 Ρ ] per 1 μ Μ A T P (adenosine

5'-[τ-32Ρ]

triphosphate purchased from Amersham International pic, Buckinghamshire, England), 50 mM Tris-HCl, 200 μ Μ E G T A (pH 7.4) and various concentrations of calcium (10"® Μ - 10" 2 M).

After 1 h of incubation the reaction was stopped

by adding 300 μ\ 5% P C A and 1 mM inorganic phosphate.

In order to form

complexes of molybdophosphoric acid, 100 μ ΐ of 5% Mb(NH^) in 4 Ν I ^ S O ^ were added.

700 μ 1 of isobutanol was added for extraction

these

complexes.

Following rapid and short centrifugation, 500 μ ΐ of the extracted isobutanol was taken for radioactivity determination, as above. The activity was expressed as μπιοΐ/mg protein/min, after subtracting the activity in the absence of calcium from that achieved in the presence of calcium. A T P a s e s activities.

These assayes were performed without solubilization of the

enzyme in Triton x-100.

After 2 days of incubation with 5 ng/ml of calcitriol

a 1000xg pellet was prepared, as described earlier.

The pellet was resuspended

in buffer containing 250 mM sucrose, 50 mM Tris-HCl (pH 8.0) and assayed (16). N u c l e a r binding of ^ - H - l ^ S f O H l o D g

by cartilage cells.

After overnight

resting in suspension cartilage cells (7-8x10® cells/3 ml medium in each

tube)

were incubated with 0.13 nM l,25-dihydroxy(26,27-methyl- H) cholecalciferol (180 3

Ci/mmol,

Amersham

International

pic, Buckinghamshire,

England) for 3 h at

37°C in the presence or absence of 100-fold excess of radioinert metabolites of cholecalciferol.

All operations following 3 h incubation were carried out at 4 ° C .

The cells were washed twice with phosphate buffer saline consisting of NaCl, 0 . 1 9 % N a 2 H P 0 4

0.8%

0 . 0 2 % Κ Η 2 Ρ Ο φ 0 . 0 2 % KCl, (PBS), suspended in cold

sucrose buffer (13) and homogenized for 10 seconds with Polytron (Kinematica, Luzern, Switzerland).

homogenizer

The nuclei were collected by centrifugation

at 1500 g for 10 minutes. The nuclear pellet was washed with P B S , dissolved in 300 μ ΐ 0.5 Ν NaOH and its radioactivity and DNA content (17) were measured. P r e p a r a t i o n of crude cytosol.

Cartilage cells after overnight incubation were

washed twice

in cold P B S

about 40x10

cells/ml and homogenized for 10 seconds with Polytron homogen-

suspended

in cold

hypertonic

buffer (18)

to give

izer.

The cytosolic fraction was obtained by centrifugation at 100,000 g 1 h at

4°^.

Portions of crude cytosol were kept at - 7 0 ° C until used.

408 Competitive

binding

experiments on

crude

cytosol.

3

H-l,25(OH) 2 D 3

was

incubated at final concentration of 80 pM with 0.8 ml of crude cytosol containing 0.16 mg protein.

The incubation was performed in the presence or absence

of 100 fold excess of radioinert analogues of cholecalciferol.

After 1 h incubar

tion at 2 0 ° C the unbound sterol was removed with the aid of charcoal coated with dextran. Sedimentation 3

analysis.

Aliquots of cytosolic cartilage were incubated with

H - l , 2 5 ( O H ) 2 D 3 for 1 h at 20°C..

dextran-coated

charcoal,

300 μ\

After separation of the free metabolite by of labelled

cytosol were layered on 4.7

ml

5-20% linear sucrose gradient prepared in hypertonic buffer and centrifuged for 18 h at 260,000 g at 4 ° C .

Cytosolic fraction prepared from the intestinal

mucosa was manipulated in the same way for comparison.

Sedimentation coef-

ficients were estimated by parallel runs of known proteins (chymotrypsin, 2.5 S; ovalbumin, 3.6 S; bovine serum albumin, 4.4 S; aldolase, 7.8 S). R e s u l t s a n d Discussion Calcitriol decreases [Ca^^Jj in cartilage cells isolated from the epiphyseal growth plate of rachitic chicks. The effect was noted after an overnight incubation with the hormone (Table 1). AP activity is lowered as well by calcitriol. These effects on [Ca 2 +]i and on AP activity in rachitic cartilage cells in apparently specific for calcitriol as the two other metabolites, 25(OH)D 3 and 24,25(OH) 2 D 3 are without any effects on these parameters. Table 1. Metabolite

Response to cartilage cells to vitamin D metabolites Concen. ng/ml)

[Ca2+] i

Concen. (ng/ml)

(nM)

Control

_

264±17.1la

l,25(OH) 2 D 3

5

168±24.9

25(OH)D 3

50

24,25(OH) 2 D 3

50

_

AP (UP) 7.52±0.282a

0.05

4.89±0.14b

235±20.0a

5

6.94±0.28a

264±19.9

50

6.64±0.42a

b

a

Means bearing a common superscript within a column are not significantly different.

1.

p2D3 administration or alteration of dietary phosphorus or calcium, suggesting differential regulation of calbindin gene expression.

DEVELOPMENTAL CHANGES IN KIDNEY

«* if"

18d fetus Birth 1w

2w

3w

6w

8w

CaBP-

CALMODULIN

Fig. 6. Developmental changes in renal calbindin-D mRNA assessed by Northern blot hybridization. Poly(A + )RNA (8 ug) was electrophoresed on a denaturing agarose gel, transferred to a nylon membrane and hybridized with calbindin and calmodulin probes. Poly(A + )RNA was prepared from tissue pooled from 3-9 rats. Calbindin-D mRNA was observed in the 18 day old fetus after longer autoradiographic exposure. Similar results were obtained in a duplicate Northern blot analysis using poly(A + )RNA from a separate group of rats. NOTE: Unlike calbindin mRNA, the calmodulin mRNA levels in developing kidney were rather constant.

505

NORTHERN BLOT ANALYSIS OF RENAL CaBP mRNA ^,3.2 kb 2.8 kb 1.9 kb

C

-P

Fig. 7. N o r t h e r n analysis of calindin-D28k rnRNA in kidneys f r o m v i t a m i n D r e p l e t e rats f e d e i t h e r a c o n t r o l diet (C), a low phosphorus diet (-P) or a low c a l c i u m diet ( - C a + + ) . P o l y ( A + ) R N A was isolated f r o m r a t kidney and f r a c t i o n a t e d (8 ug/lane) on a 1.7% formaldehyde agarose gel. The R N A was t r a n s f e r r e d t o a nylon membrane and hybridized w i t h calbindin c D N A . Similar results were observed in t w o additional experiments. In additional studies, we have assigned the calbindin-D28k gene t o human c h r o m osome 8 by h y b r i d i z a t i o n of the calbindin c D N A probe t o spots of individual human chromosomes f l o w sorted o n t o nitrocellulose f i l t e r s (studies done in c o l l a b o r a t i o n w i t h Dr. L. Deaven, r e f . 18). Southern analysis of human genomic D N A indicates t h a t the calbindin gene is probably a single copy. In summary, these studies represent the f i r s t analysis o f r a t calbindin-D28k gene* expression. Our results demonstrate d i f f e r e n t i a l r e g u l a t i o n of calbindin-D28k gene expression. R e c e n t l y , a r a t Hae III genomic library has been screened using the 1.2 kb calbindin c D N A and t w o overlapping clones have been isolated and are c u r r e n t l y being analyzed. With the a v a i l a b i l i t y of t h e probes for calbindin we can now i n i t i a t e new biochemical studies which should lead t o an increased understanding of the physiological significance of calbindin. In a d d i t i o n , f u t u r e studies concerning the c h a r a c t e r i z a t i o n of the p r o m o t e r and the 5' f l a n k i n g region of the r a t calbindin-D28k gene should result in new advances which w i l l allow us t o define more precisely the basic molecular mechanisms of 1,25(OH)2D3 a c t i o n .

ACKNOWLEDGEMENTS Supported by grants t o S.C. f r o m N I H (NS20270 and DK-38961).

506

REFERENCES 1. Spencer, R., Charman, M., Wilson, P. and Lawson, D.E.M. (1978) Biochem. J. 170:93-101. 2. Christakos, S. and N o r m a n , A.W. (1980) A r c h . Biochem. Biophys. 203: 809-815. 3. Thomasset, Μ., Desplan, C., Parkes, Ο. (1983) Eur. J. Biochem. 129: 519-524. 4. Pansini, A . R . and Christakos, S. (1985) Endocrinology 117: 1652-1660. 5. H u n z i k e r , W., Siebert, P.D., King, M.W., Stucki, P., D u g a i c z y k , A . and Norman, A.W. (1983) Proc. N a t l . Acad. Sei. USA 80: 4228-4232. 6. Wilson, P.W., Harding, M. and Lawson, D.E.M. (1985) N u c l e i c Acids Res. 13: 8867-8881. 7. Desplan, C., Thomasset, Μ. and Moukhatar, M. (1983) J. Biol. Chem. 258: 2762-2765. 8. Wood, T . L . , Kobayashi, Y . , Franz, G., Varghese, S., Christakos, S. and Tobin, A.J. (1988) D N A , in press. 9. Pansini, A . R . and Christakos, S. (1984) J. Biol. Chem. 259: 9735-9741. 10. Varghese, S. and Christakos, S. (1987) Anal. Biochem. 72: 248-254. 11. Thomas, P. (1980) Proc. N a t l . Acad. Sei. USA 77:5201-5205. 12. Blum, M., McEwen, B.S. and Roberts, J.L. (1987) J. Biol. Chem. 262: 817-821. 13. Wuenschell, C.W., Fisher, R.S. K a u f m a n , D . L . and Tobin, A . J . (1986) Proc. N a t l . Acad. Sei. USA 83: 6193-6197. 14. Y a m a k u n i , T., Kuwano, R., Odani, S., M i k i , N., Yamaguchi, K . and Takahashi, Y. (1987) J. Neurochem. 48: 1590-1596. 15. Friedlander, Ε.J., Henry, H . L . , and Norman, A.W. (1977) J. Biol. Chem. 252: 8677-8683. 16. Hughes, M.R., Brumbaugh, P.F., Haussler, M . R . , Wergedel, J.Ε. and Baylink, D. (1975) Science 190: 578-580. 17. Gray, R.W. and Napoli, L. (1983) J. Biol. Chem. 258: 1152-1155. 18. Deaven, L . L . , V a n D i l l a , M . A . , B a r t h o l d i , M . F . , Carrano, A . V . , C r a m , L.S., Fuscoe, J.C., Gray, J.W., Hildebrand, C.E., Moyzis, R . K . and Perlman, J. (1986) C o l d Spring Harbor Symp. Quant. Biol. 51; 159-167.

MOLECULAR CLONING AND SEQUENCING OF CALBINDIN-Dg^ CDNA FROM MOUSE PLACENTA. T.E. MIFFLIN, W.R. PEARSON, J. REINHART, D.E. BRUNS AND Μ.Ε. BRUNS Departments of Pathology and Biochemistry, University of Virginia, Charlottesville, VA 22908, USA. Introduction Previous work on calbindin-D^ cDNA has been performed with rat intestinal RNA (1,2), but no similar work has been performed in mice. We are interested in the mouse because the mouse is a well-characterized model of developmental expression of calbindin (3,4), and multiple genetic models of rickets (Hyp, oc, Gy) have been defined in mice (5,6). In mice, calbindin-Dg^ is expressed in kidney and is vitamin D-stimulated (4). Recently calbindin has also been found in murine cell lines (7). Materials and Methods: RNA extraction (8), Northern blot analysis and cloning procedures were done as outlined (9). The lambda gtll mouse placental library was a gift from D. Linzer (Northwestern Univ.). Results and Discussion: We constructed a 42-base oligonucleotide based upon the sequence of rat calbindin-Dg^ cDNA (1), and used it to examine tissue-specific expression of murine calbindin mRNA and to identify a murine calbindin cDNA clone. The probe sequence corresponded to a highly conserved amino acid sequence within the first calcium binding domain of mammalian calbindins. The probe hybridized to a single 500600 nt RNA species on northern blots of RNA from duodenum and did not hybridize with liver RNA. In a separate experiment,»' RNA extracts from mouse duodenum, kidney, spleen, brain, and lung were examined by northern blots. A strong hybridization was observed at 600 bases in kidney and intestine and a similar sized but weak signal was seen in lung; no hybridization was observed with liver, brain or spleen RNA. This tissue distribution of calbindin mRNA was identical to the protein distribution determined immunochemically. Approximately 200,000 plaques of a murine placental cDNA library were screened using the olignucleotide and six hybridizing plaques were identified. DNA sequencing of one insert revealed a nucleotide sequence similar to that found between nucleotides 10 and 180 (EcoRl site) of the published

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · New York - Printed in Germany

508

rat calbindin cDNA sequence. The murine cDNA shared 92% sequence identity (157/171) with the reported rat calbindin cDNA sequence. The predicted amino acid sequences for rat and mouse calbindins were identical at 56 of 57 positions (98%). The predicted sequence for the mouse protein shared 91% and 82% sequence identity with the bovine and porcine proteins, respectively. The predicted mouse sequence was in complete agreement with that found by direct protein sequencing. Importantly, Asn-24 of rat calbindin was replaced by Asp. This non-neutral change from the rat protein helps to explain the marked electrophoretic differences between rat and mouse calbindins. References 1. 2. 3. 4. 5. 6. 7.

8. 9.

Desplan,C., Heidmann,0., Lillie,J.W., Auffray,C., Thomasset,Μ. (1983) J.Biol.Chem. 258:13502-13505. Perret,C., Desplan,C., Brehier,A., Thomasset,Μ. (1985) Eur.J.Biochem. 148 : 61-66 . Delorme,A.C., Danan,J.L., Ripoche,Μ.Α., Mathieu,H. (1982) Biochem.J. 205:49-57. Bruns,Μ.Ε., Kleeman.E., Bruns,D.E., (1986) J.Biol.Chem. 261:7485-7490. Bruns,Μ.Ε., Meyer,R.Α., Meyer,Μ.Η. (1984) Endocrinology 15:1459-1463. Seifert,Μ.F., Gray,R.W., Bruns,Μ.Ε. (1988) Endocrinology 122 :1067-1073. Bruns,Μ.Ε., Burnet,S.H., Damjanov.A., Damjanov,E., Bruns,D.E. (1988) Abstract in Proceedings of the 10th Meeting American Society for Bone and Mineral Research (In Press) . Chirgwin,J.Μ., Przybyla,A.E., MacDonald,R.J., Rutter,W.J. (1979) Biochemistry 18:5294-5299. Maniatis,Τ., Fritsch,E.F., Sambrook,J. (1982) in Molecular Cloning (Maniatis,Τ., Fritsch,Ε.F., Sambrook, J.,Eds.) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

HIGH CONSERVATION O F CALBINDIN D28 IN EVOLUTION; IMPLICATIONS FOR ITS FUNCTION. IGOR BENDIK, ALFRED W.A. HAHN and WILLI HUNZIKER, Central Research Units, F. Hoffmann-La Roche & Co, Ltd., CH-4002 Basel, Switzerland Calbindin D28 was originally discovered in the chicken intestine as a protein induced by vitamin D (1) and is thought to be involved in the transcellular transport of calcium in response to vitamin D. Later it was found that Calbindin D28 is also present in a variety of other tissues that do not appear to be directly involved in a vitamin D dependent transcellular calcium transport (2,3). The protein binds 3-4 moles of calcium or terbium per mol of protein (4,5). The calcium is bound by the EF-hand principle where two amphipatic helices flank a loop of 12 amino acids. At certain positions in this loop there are amino acids containing oxygen in their side chains that coordinates the calcium (6) as shown in Fig.l. Numerous other calcium binding proteins (e.g. calmodulin, parvalbumin, troponin C etc.) bind calcium by the same principle. Therefore all these proteins are thought to have evolved from a common ancestor by gene duplications and comprise the troponin C superfamily. Little is known about the function of Calbindin D28. A hypothesis put forward by Kretsinger et al. (7) that is supported by Bronner et al. (8), and also by Feher in a cell free system (9), is that Calbindin D28 facilitates the diffusion of calcium through the cell and/or serves as an intracellular calcium buffer. If that were the sole function of the protein, then the only requirement for its function would be to preserve the calcium binding sites. Since the amino acid requirements for a calcium binding site are rather relaxed, as shown in the test sequence in Fig.l, one would expect a high rate of divergence of such a protein in evolution. This is further supported by the fact that there is only a low amino acid sequence homology among the members of the troponin C superfamily. Particularly Calbindin D28 only shows a limited homology to other calcium binding proteins in the loop region (10). Therefore maintenance of a calcium binding site is a weak conservation pressure in evolution. Potential other or additional physiological functions however could pose a much stronger conservation pressure, as evidenced for example in calmodulin, which needs to interact with a variety of other proteins in order to regulate their function in a calcium dependent manner. In order to address the question of such additional functions we investigated the conservation of Calbindin in evolution. We have cloned and sequenced Calbindin D28 cDNA from chicken (11) and rat (12) as well as the human Calbindin D28 gene (13) and derived the encoded amino acid sequence. We found that Calbindin D contains six EF-hand like domains two of which (domains II and VI in Fig.l) have most likely lost their calcium binding ability due to mutations in critical positions of the calcium binding loop (11). Fig.l shows the amino aicd sequence of the chicken (11,14), rat (12,15), bovine (16) and human (13,17) Calbindin D28. There is a 79% homology between chick and rat and a 98% homology between rat and human Calbindin D28.

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

510 From this data we put forward the concept that a mere calcium binding function of Calbindin, which is sufficent for the facilitated diffusion/ buffer hypothesis, cannot explain the high degree of conservation of the protein in evolution. Therefore we postulated calcium dependent interactions of Calbindin D28 with other molecules within the cell (18-20,13) instead of, or in addition to, the facilitated diffusion hypothesis. LinAtA Ta^t

Helix

sequence

Loop

HeUlx.

ΙΛΛΛλλ

Ε

bowie human λ at chick

Domain

M A E S H L Q S S L I T A S Q F F I I U H F

I HT

Τ

O

V

I

S

*

Η

bovine. human AOt

Domain

II

G L Ε L S Ρ Ε M K T F V D Q Y 0

O W M

III

3

Λ Tti Domain IV

chick

human *at chick bovine human *at c/u.ck

S

Ν

Q 0 A R Κ Κ A L

r

MD

Domain. V

VI

Τ

V Q Q I K M C O K E V

. Domain

D

L A H V L P T E

DM

F N K A F E L Y

A Ρ 3 L D I Ν Ν I Τ Τ Υ Κ Κ Ν S A S

Ε Ν F L L L F F

Q

D T D H 3 Q F I B T E E S

0

EI

C Q R D D Q K I G I V E

Κ A Τ

V D D T K L A E Y T O L H L K L F I K S

bovine.

A

I

R C Q Q L K S C E E F H K T W R K Y

chick bovine human tat

Ϋ

L Q N L I Q E L

Ε

chick bovine human

D A D G S G Y L E G K E

L K N F L K O L

D S

L Ε Κ A Ν Κ Τ

3

D S N N D G K L E L T E A

M A R L L P V Q

Ε Ν F L 1 Κ F

L

D D Q D G N G Y I D E N E

Q

9

I

L D A L L K D L

C Ε Κ Ν Κ Q D Ε Κ Ε

Μ

IM A L S D O G K L Y R T O

3 L A L I L C A G

D Ν

3 A Ε

Κ Ε

Fig. 1. Amino acid sequence comparison between the human, bovine, rat chicken Calbindin D28. The human Calbindin D28 with its 6 potential EFhand structures (I-Vl) is shewn and is used as a reference. Amino acids that differ in the other species are given in their respective positions.

There is preliminary evidence that such interactions of Calbindin D28 with other proteins indeed could exist. Freund and Christakos (21) describe a calcium dependent binding of Calbindin D28 to kidney microsomal membranes. Shimura and Wasserman (22) show an association of Calbindin D28 with purified chick intestinal membranes and identify two Calbindin D28 binding protein bands in a gel overlay assay, neither of the two bands is alkaline phosphatase. Norman and Leathers (23) on the other hand find a calcium dependent association of Calbindin D28 with chick intestinal phosphatase using a photoaffinity labeled Calbindin D28. A Calbindin D28 dependent effect on the calmodulin regulated Ca/Mg ATPase was observed by Morgan et al. (24) and Freund and Christakos (21), however, 300 fold higher concentrations of Calbindin D28 than calmodulin were required for a similar response. Taken collectively the conservation in evolution strongly suggests an interaction of Calbindin D28 with other cellular molecules and a regulation of their function in a calcium dependent manner. The identification of such molecules will yield clues to the function of Calbindin D28 and could open up a whole new area of research.

511 References 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

22. 23. 24.

Wasserraan, R.H., and Taylor, A.M. (1966) Science 152, 791-793 Christakos, S., Friedlander, E.J., Frandsen, B.R., and Norman, A.W. (1979) Endocrinology 104, 1495-1503 Norman, A.W., Roth, J.. and Orci, L. (1982) Endocr. Rev. 3, 331-336 Brederman, P.J., and Uasserman, R.H. (1974) Biochemistry 13. 1687-1697 Gross, M.D., Nelsestuen, G.L., and Kumar, R. (1987) J. Biol. Chem. 262, 6539-6545 Kretsinger, R.H. (1976) Ann. Rev. Biochem. 45, 239-266 Kretsinger, R.H., Mann, J.E., and Simmonds, J.T. (1982) In: Vitamin D: Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism (Norman, A.W., Schaefer, Κ., Herrotti, D.U., Grigoleit, H.-G., eds) pp 233-248, de Gruyter, Berlin Bronner, F., Pansu, D., and Stein, W.D. (1986) Am. J. Physiol. 250, G561-G569 Feher, J.J. (1983) Am. J. Physiol. 244, C303-C307 Hunziker, W., and Schrickel, S. (1987) In: Calcium binding proteins in Health and Disease (Norman, A.W., Vanaman, T.C., Means, A.R., eds) pp 458-468, Academic Press, NY Hunziker, W. (1986) Proc. Natl. Acad. Sei. USA 83, 7578-5782 Hunziker, W., and Schrickel, S. (1988) Molec. Endocrinology, in press Bendik, I., Hahn, A.W.Α., and Hunziker, W. (in preparation) Wilson, P.W., Harding, Μ., and Lawson, D.E.M. (1985) Nucleic Acids Res. 13, 8867-8881 Yamakuni, T., Kuwanao, R., Odani, S., Miki, N., Yamaguchi, K., and Takahashi, Y. 81987) J. Neurochem. 48, 1590-1596 Takagi, Τ., Nojiri. Μ., Konishi, Κ., Maruyama, κ., and Nonomura, Y. (1987) FEBS Lett. 201, 41-45 Parmentier, Μ., Lawson, D.E.M., and Vassart, G. (1987) Eur. J. Biochem. 170, 207-215 Hunziker, W.. and Schrickel, S. (1986) Program of the Eigth Annual Meeting of the American Soc. for Bone and Mineral Res., Anaheim, CA, #22 Hunziker, W., and Schrickel, S. (1986) Program of the IXth International Conference on Calcium Regulating Hormones and Bone Metabolism, Nice, France, #756 Hunziker, W., and Schrickel, S. (1986) Program of the Fifth International Symposium on Calcium Binding Proteins in Health and Disease, Asilomar, CA, #110 Freund, T.S., and Christakos, S. (1985) In: Vitamin D: Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism (Norman, A.W., Schaefer, Κ., Herrotti, D.U., Grigoleit, H.-G., eds) pp 369-370, de Gruyter, Berlin Shimura, F., and Wasserman, R.H. (1984) Endocrinology 115, 1964-1972 Norman, A.W., and Leathers, U. (1982) Biochem. Biophys. Res. Commun. 108' 220-226 Morgan, D.W., Welton, A.F., Heick, A.E., and Christakos, S. (1986) Biochem. Biophys. Res. Commun. 138, 547-553

FUNCTIONAL ANALYSIS OF THE PROMOTER REGION OF THE GENE ENCODING CHICKEN CALBINDIN-D 00 toK S. F e r r a r i , R. B a t t i n i , E. Drusiani and M. Fregni I s t i t u t o di Chimica B i o l o g i c a , Universitä di Modena, 41100 Modena, I t a l y .

Via Campi 287,

Introduction Vitamin D-induced C a l b i n d i n - D ^ i s an i n t r a c e l l u l a r calciufii binding protein (1). I t i s found in many chicken t i s s u e s , but i t s dependency on 1,25(OH)^D^ varies among the different s i t e s where i t i s expressed (2). I t i s conceivable that the functional a n a l y s i s of cloned DNA fragments representing portions of the Calbindin-D gene might provide information on the regulation of t h i s gene product, as i t occurred for other hormone regulated proteins. Materials and Methods The plasmid pCBCAT-2200 was constructed by i n s e r t i n g the DNA r e s t r i c t i o n fragment representing the 5'-terminal region of the Calbindin D gene from about nt-2200 to nt +50 into the polylinker of BlueCAT. Tne other members of the pCBCAT series (pCBCAT-2200 to pCBCAt-151) were obtained by 5' deletions of pCBCAT-2200. Transfection of cultured c e l l s was performed by either the calcium-phosphate (3) or the DEAE-dextrane (4) methods. Cell l i n e s permanently transformed with pCB-451 were obtained by cotransfection with pRSVneo DNA, followed by selection with Geneticin (Sigma) (5). The CAT a c t i v i t y of cell homogenates was assayed as described (3). Results and Discussion The two recombinant chicken genomic clones CBA1 and CBC1 contain a portion of the gene encoding Calbindin-D ? _ and sequences which flank the 5'-end by 0.45 kb and 4.4 kb respectively. We have recently shown that the Bam ΗI/Sac 11 fragment of CBA1 (coordinates-451+50), when inserted in a CAT vector, promotes CAT a c t i v i t y in transiently transfected f i b r o b l a s t s 3T6 and MDCK-1 c e l l s (6). However the expression of CAT i s t ie independent on the presence of 1,25(OH)2^3 ' culture medium. Constitutive expression i s observed over a broad concentration range of 1,25(0H) D and in different cell l i n e s (HL-60, U-937). To exclude the p o s s i b i l i t y that 1 ^ ( O H J ^ D , present in the FCS or that the procedures involved in transfecting c e l l s could interfere with the induction of the Calbindin-D^ promoter, we established cell lines permanently transfected with pCB-451. Several cell clones were expanded in completely synthetic medium and analyzed for the induction of CAT by 1,25(0H) D . CAT a c t i v i t y appeared to be c o n s t i t u t i v e in all cases examined,

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter&Co., Berlin · New York-Printed in Germany

513

independently on the copy number of plasmid DNA molecules inserted in the genome. Since the p o s s i b i l i t y s t i l l existed that the vitamin D responsive elements could reside further 5' of the analyzed promoter fragments, we repeated t r a n s i e n t transfection experiments with a recombinant CAT plasmid containing up to about 2.2 kb of the 5 ' - f l a n k i n g region. Again no s i g n i f i c a n t l y inducible expression was detected. We further i n v e s t i g a t e d , by deletion mutant a n a l y s i s , whether control elements, other than the vitamin D responsive ones, were present in the Calbindin-D^g^. promoter. As the f i g u r e shows, MDCK-1 c e l l s t r a n s i e n t l y transfected with pCB-182 and pCB-151 display s i g n i f i c a n t l y higher CAT a c t i v i t y than cells transfected with longer 5'-f1anking segments (pCB-370 to pCB-2200). I t i s suggested that a control element with s i l e n c e r a c t i v i t y might be located between -370 and -182. 70

Figure. Deletion mutant a n a l y s i s of the 5 ' - e n d of the chicken Calbindi n ~^28K 9 e n e ' Pro" moter a c t i v i t y i s measuij-ijd as percent of C-Chloramphenicol converted to acetyl d e r i v a t i v e s after incubation with homogenates of MDCK-1 c e l l s t r a n s i e n t l y transfected with CAT Plasmids of the pCB series.

4Q 3Q t< BO

Ο

β

I 126

. ι 054

1

1

ι 0.72

ι 040

1

1

ι ΙΟβ

I 1.26

C o * * CONCENTRATIONS mM

Fig. 2.

Effect of calcium concentrations ranging from 0.54-

1.26 m M in the vascular perfusate on basal l,25(OH)2D3-mediated duodenum,

filled

containing

0.9

45

with mM

perfused for the

Ca

2+

transport

tracer

CaCl 2

(right panels).

quantities

(control

(left panels) of

medium),

Each

in was

and DPBS

vascularly

first 20 min w i t h control m e d i u m and

then

w i t h DPBS containing different concentrations of C a C l 2 ,

1.25

mg/ml

BS A,

and

either vehicle

or

650 pM

l,25(OH)2D3.

The

m e a n + SEM for the indicated number of experiments are shown 20 and 40 m i n after changing the calcium concentration in the buffer for b o t h groups. *, Ρ < 0.05; **, Ρ < 0.01; ***, Ρ < 0.001

[vs. duodena perfused with 650 pM l , 2 5 ( O H ) 2 D 3

in DPBS

containing 0.9 m M C a C l 2 ] . Table inhibitors.

II

summarizes

the results

of

studies with

other

553 Table II. Summary of the effect of inhibitory compounds on calcium transport in vascularly perfused duodena. Inhibition of 1,25(OH) 2 D 3 mediated Ca transport

Compound

Target

Actinomycin D Leupeptin Pepstatin Honensin Colchicine Cytochalasin Β

Nuclear Transcription Cathepsin Β Cathepsin D, Pepsin Golgi Microtubules Microfilaments

No Yes No No Yes No

These findings suggested that lysosomes among

other

hydrolases

microtubules vesicles) also

the

(intracellular

are

indicated

involved that

x

protease tracks'

in calcium

the

next

(which contain

cathepsin

for

the

of

and

transport

transport.

line

B)

The

inquiry

of

results

should

be

addressed to the subcellular localization of calcium during the absorptive process. Subcellular fractionation studies Fractionation

of

duodenal

mucosa

after

in

vivo

45

Ca

absorption, using a combination of differential- and Percollgradient

centrifugation,

contained

the

activity (8).

highest

revealed levels

that of

lysosomal

fractions

radionuclide

specific

Comparison of gradients prepared from vitamin

D-deficient

chicks

l,25(OH) 2 D 3

15

h

treated prior

to

with

vehicle

or

experimentation

1.3

nmols

indicated

of

that

lysosomal fractions exhibited hormone-enhanced levels of the radionuclide, as well as the highest levels of calbindin-D 28K specific immunoreactivity. microsomal

membranes

endocytic vesicles, specific transport,

revealed

the

also enriched

activities.

lysomotrophic

Similar fractionation studies on

agent

completely

inhibited

of

putative

in radionuclide and

Moreover, chloroquine

existence

introduction into

the

of

lumen

1,25(OH)2D3-augmented

CaBP the

during 45

Ca

554

absorption,

but

intestinal

had

no

effect

epithelium.

3

on

Transport

of

H20

movement

water

by

across

fluid

phase

pinocytosis largely bypasses lysosomal organelles for 60 m i n after initiation of uptake.

More recently, these

vesicular

fractions h a v e b e e n subjected to time course studies (9). revealed

in

Table

III,

parallels intestinal radioactivity, substantial

45

lysosomal

Ca

calcium absorption,

whereas

amounts

the

of

endocytic

radionuclide

content

closely

as judged by vesicles

prior

to

As

serum

accumulate

the

times

of

maximum transport. 45 Ca

Table III. Time course of lysosomal and vesicle accumulation Time A f t e r Serum 4 5 C a Lysosomal 4 5 C a l , 2 5 ( O H ) 2 D 3 (+D/-D) (+D/-D; Fr 1-3) 2.5 5 10 15 43

1.08+0.12 1.64+0.29 3.00+0.35 3.4 0+0.3 9 1.69+0.08

1.02 1.47 3.10 1.88 1.29

22+17 1054+788 971+121 820+200 840+325

Vesicle 4 5 C a (+D/-D; Fr 3) 2.5 5 10 15 43

Lysosomel CaBP (ng/mg protein; Fr 3)

Vesicle CaBP (ng/mg protein; Fr 3)

1.32 1.87 2.05 1.72 1.36

11+1 255+31 13+9 523+295 1320+319

The origin of the vesicle-associated b e e n postulated internalized

to

with

arise the

from brush

endocytic

transferred to lysosomes

(8) .

calbindin-D28K

border

calbindin-D28£,

vesicles,

and

ultimately

It should be stated

however,

that only a small percent of the total c a l b i n d i n - D 2 8 K is ever found

in

vesicles; "soluble". the

use

association most

of

the

with

either

lysosomes

1,25(OH)2D3-induced

or

protein

endocytic remains

Although this observation could be attributed to of

calcium

redistribution

inhibitors

in

the

555

gradients, it was also deemed possible that

calbindin-D 28K ,

in analogy to calmodulin, might have a role as a microtubule associated protein—perhaps mediating attachment of calciumbearing vesicles to the cytoskeletal transport elements. Biochemical analyses of microtubules Microtubules different

were

biochemical

subsequently approaches:

isolated (1)

by

two

three

cycles

of

polymerization and depolymerization; (2) taxol stabilization; (3)

affinity

Sepharose-4B calbindin-D 28 K

chromatography columns. was

In

on

anti-tubulin

each

procedure,

conjugated

immunoreactive

found in association with purified tubulin

(Table IV). Table IV.

Polym.: Depolym. Taxol Affinity Chromatog. nq iCaBP/mq protein + range MT a Sup MT a MT a Sup

Vit D Status -D 2.5h c 5h c 10h c 15h c 24h c 43h c +D

Microtubule associated CaBP

160+ 30

190+110

960+270 2370

1290+690 4080

7950+600

10500+690

36± 9 38+18 100+16 130±14 2 2 0+2 8

45±19

11

85+21 99+21 108± 4

120 310 840

170±79

2460

a

MT=Microtubules; ^Sup=High-speed supernatants; c Time after l,25(OH) 2 D 3 Immunocytochemical

studies

are

determine whether the association calbindin-D 28 K exists in situ.

now

in

progress

to

between microtubules

and

Preliminary results suggest

that both tubulin and CaBP colocalize in brush borders and along basal lateral membranes (data not shown). Thus,

the combined data support

the model

transport schematically presented in Fig. 3.

of

calcium

556 INTESTINAL EPITHELIAL CELL

BRUSH BORDER

KEY .

= C.

2

+

• B= C a B P ± C « 2 + OA - Q O L Q I

APPARATUS

Ν * NUCLEUS Ρ

= PINOCYTIC

L I = PRIMARY

VESICLE

LYSOSOME

L2 = S E C O N D A R Y

LYSOSOME

MVB= MULTIVESICULAR μΛ

BODY

= MICROTUBULE

(Äl -

MEMBRANE R E C O G N I T I O N SITE

[ n l - NUCLEAR

RECEPTOR

Λ » 1.25(OH)2D3

Fig.

3.

Intracellular

elements

of

Intestinal

calcium

transport At the brush border, calcium is recognized by a specific moiety, perhaps calbindin-D 28K , and internalized by endocytosis.

The endocytic vesicles are conveyed along microtubules

to lysosomes where fusion of the membrane-bounded organelles occurs.

The lysosomes, in turn, move along microtubules to

the basal lateral membrane where exocytosis of calcium and CaBP completes the transport process. The observations that vitamin D 3 repletion tremendously increases brush border calbindin-D2gK, fluorescence

microscopy,

and

as

influences

judged by immunothe

tubulin, the protein subunits of microtubules

expression

of

(10), suggest

that the presence of these proteins are the underlying basis for the to

'biochemical competence' that allows transcaltachia

proceed

in

the

normal

chick

duodenum.

The

question

557

remains, however, as to how l,25(OH)2D3 in association with a nongenomic receptor, initiates transcaltachia. References 1.

Nemere,

I.,

Szego,

C.M.

(1981)

Endocrinology

128:1450-1462. 2.

Nemere, I., Putkey, J.Ά., Norman, A.W. Biochem. Biophys.

3.

Nemere,

I.,

(1983)

Arch.

222:610-620.

Norman,

A.W.

(1986)

Endocrinology

119:1406-1408. 4.

Nemere, I., Norman, A.W.

(1987)

J. Bone. Min. Res.

2.: 167-169. 5.

Yoshimoto, Y., Norman, A.W.

(1986)

J. Steroid Biochem.

1^:905-909. 6.

Nemere,

I.,

Yoshimoto,

Endocrinology 7.

Yoshimoto,

Y.,

Endocrinology 8.

Norman,

A.W.

(1984)

Norman,

A.W.

(1986)

115:1476-1483. Nemere,

I.,

118:2300-2304.

Nemere, I., Leathers, V., Norman, A.W. Chem.

9.

Υ.,

(1986)

J. Biol.

261:16943-16947.

Nemere, I., Norman, A.W.

(1988)

Endocrinology

(in

press). 10.

Nemere, I., Theofan, G., Norman, A.W. Biophys. Res. Commun. 148:1270-1276.

(1987)

Biochem.

VITAMIN

D:

EFFECTS

ON

INTESTINAL

CALCIUM

ABSORPTION

AND

PERIPHERAL NERVE FUNCTION. R.H. WASSERMAN, C.S. FULLMER, C. HU, Q. CAI AND D.N. TAPPER. Department and Section of Physiology, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 1Ψ853. Vitamin D and Calcium Absorption The physiology, biochemistry and molecular biology of vitamin D action on the intestinal transport of calcium has been subjected to a number of reviews in recent years (1-17). From the information cited in some of these reviews, the following concepts are generally agreed upon: (a) the absorption of calcium occurs by an active transport process and by a non-saturable process. The predominating process is dependent upon the intraluminal concentration of Ca2+· (b) the e f f i c i e n c y of calcium absorption increases in response to various stresses, such as low calcium intake, growth, lactation and pregnancy. These increases in calcium absorption coincide with increases in the formation of l,25(OH)2D3 by the kidney hydroxylase system; (c) the enterocyte possesses a specific receptor for l,25(OH)2D3 that, when complexed with l,25(OH)?D3, interacts with the genomic apparatus to induce the synthesis of an mRNA for mRNAs) coding for proteins potentially involved in calcium transport. Among these are the vitamin D-induced calcium-binding protein (CaBP), now termed calbindin-D28K t^e avian species and calbindin-D9K in mammalian species; (d) l,25(OH)2D3 also stimulates the activity of a number of enzymes and the formation of other proteins, including adenylate cyclase, guanylate cyclase, ornithine decarboxylase, phospholipase A2 and other enzymes involved in phospholipid metabolism; (e) in addition to increasing Ca^+ absorption, l,25(OH>2D3 also increases sodium-dependent phosphate transport. Existing data have been used to formulate models of Ca^ + transport by the intestine (which probably apply to other epithelial tissues) and there is some agreement (and disagreement) as to the physiological and biochemical mechanisms. Brought to bear is information derived from a variety of experimental approaches, including the use of ligated loops in situ, everted gut sacs in vitro, embryonic chick intestine in organ culture, in vitro perfusion systems, in vivo perfusion systems, intestinal slices, Ussing chambers, isolated brush border and basolateral membrane vesicles, e t c . With these preparations, a game with many players has been (is) the attempt to identify the earliest event modified by l,25(OH)2D3. This is the Vitamin D Marathon, with the supposition that this early event is a primary or the primary e f f e c t of l,25(OH)2D3 on C a 2 + absorption. A very early event wouI3 suggest a non-genomic response; a later event, a genomic-protein synthetic response. As it turns out, l,25(OH)2D3 seems to influence many aspects of the intestinal cell that might bear on increasing the absorption of Ca^+. From our perspective, a requirement for l,25(OH)2D3-stimulated Ca 2 + absorption is the de novo synthesis of a protein, and the best characterized (and only) gene product is CaBP. The absorption of Ca2+ under a variety of situations is positively correlated with CaBP concentrations (with correlation c o e f f i c i e n t s of 0.9 or better) and, in a number of experiments by different investigators, the appearance of CaBP in the intestinal mucosa of rachitic animals after exposure to l,25(OH)2D3 coincides with an

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter&Co., Berlin · New York-Printed in Germany

559

increase in calcium absorption. However, there are circumstances in which this relationship does not seem to hold (18). Further, the very rapid e f f e c t of l,25(OH>2D3 and PTH (19,20) on transepithelial C a 2 + movement (in minutes) indicates a non-genomic e f f e c t but these responses occur only in guts from vitamin D-replete animals. This is reminiscent of the prior observations of Harrison and Harrison (21) showing that dibutyryl cAMP increases Ca 2 + transport by everted gut sacs, but only in those sacs derived from vitamin D-replete rats. Returning to the Vitamin D Marathon, what are some of the early l,25(OH)2D3stimulated events that might not require de novo protein synthesis? Attention is drawn to the stimulation of cAMP synthesis, which occurs within 30-60 min. or less (since earlier time points were not assessed) (22,23). Stimulation of cGMP synthesis also occurs and, with a human skin fibroblast system, a presumed receptor-mediated synthesis of cGMP occurred at 1 min. {2k). With the assumption that cyclic nucleotide synthesis is the non-genomic event, the early e f f e c t s of l,25(OH)2D3 and PTH on C a 2 + transport by vitamin D-replete guts (19,20) could be explained since both hormones stimulate cAMP synthesis. Together, the non-genomic and genomic responses to l,25(OH)2D3 appear to yield a system that is competent to facilitate transport of C a 2 + from lumen to cell to extracellular fluid. The delayed retention of CaBP by the intestine a f t e r C a 2 + absorption has returned to basal, pre-l,25(OH)2D3 levels could be due to the absence of l,25(OH)2D3 and the ability of this hormone to e f f e c t a non-genomic response. Another proposed non-genomic e f f e c t is an increase in the fluidity state of the phospholipid membrane of the brush border (1^,25). Various studies have not revealed any changes in membrane fluidity by biophysical procedures (cf. ref. Ψ for discussion and references). However, the use of a probe to monitor the "dynamic" rather than the "static" state (as above) of the membrane showed an increase in "dynamic" fluidity at 1 hr after l,25(OH)2D3 adminsitration (26). But an increase in calcium absorption did not occur until 5 hrs. after l,25(OH)2D3 administration, sufficient time for CaBP to be synthesized. Another contender for a role in C a 2 + uptake is calmodulin, found to associate with a 102-105 kD brush border protein; the complex was considered to influence C a 2 + uptake by brush border membrane vesicles (27). Consistent with an involvement of cyclic nucleotides, recent studies in our laboratory (C. Hu and R.H. Wasserman, unpublished data) revealed that E. coli heat-stable enterotoxin (Sta) increased the in vitro C a 2 + uptake by intestinal preparations from either vitamin D-deficient or vitamin D-replete chicks (Fig. 1). However, Sta effected an increase in C a 2 + absorption only in the vitamin D-replete animals using the ligated loop procedure (Fig. 2). This enterotoxin is known to activate a brush border membrane-associated guanylate cyclase, and Vesely and Juan (28) previously showed that l,25(OH)2D3 increases cGMP synthesis in rat renal cortical tissue. Thus, cyclic nucleotide metabolism could indeed be a significant factor in the early responses to l,25(OH)2D3. If this is the case, a direct e f f e c t of l,25(OH)2D3 on cyclic nucleotide synthesis (i.e., in contrast to an e f f e c t that is secondary to elevated intracellular Ca^+ levels) implies the presence of a receptor (surface ?, cytosolic ?) that activates the cyclases upon binding l,25(OH)2D3· Barsony and Marx (2Ψ), as mentioned, recently reported the stimulation of cGMP synthesis by human skin fibroblasts within 1 min a f t e r

560 In vitro effect of St 0 on Co 2 + (0.25mM) uptake by jejunum from vitamin D deficient and replete chicks

c

1=3 - S t 0 +St 0 (100ng/ml)

Ε > 3 » §. s ε

Pre—incubation:1 Omin. Uptake Period : 3min.

η

+

Ο CM Ο Ο -s ι

CM

D—deficient

Figure 1:

D—replete

E f f e c t of E. coli heat (Sta) stable enterotoxin. Presumably the toxin activated guanylate cyclase and the resultant cGMP is responsible for the increased in vitro uptake of Ca-^5 by both -D and +D everted jejunal tissue. Calcium concentration = 0.25 mM. Values are the mean + SE of 3 determinations. E.coli heat-stable enterotoxin and C a 2 + absorption

60

ι

I Absorption

Tissue Accumulation ν m -g * 40 a

Ο ι-* 20

D

-

-

Sta

-

+

+

Sta (0.5ug) per os 2.5hrs. before experiment

Figure 2:

E f f e c t of E. coli heat stable enterotoxin (Sta) on the absorption of Ca*f7 by chick duodenum. Absorption was determined, using the in situ ligated loop technique (M). Residual tissue Ca-^7 was determined by counting the intestinal tissue after rinsing with saline. Tissue Ca-47 in the -D tissue was significantly increased by Sta and the absorption of Ca-47 by the +D tissue were significantly increased at ρ < .05. Values are mean + SEM of 5-6 chicks per group. The Sta was administered orally 2.5 hrs. before experiment.

561

exposure to l,25(OH)2D3; other data by the authors suggested mediation of this effect through a specific l,25(OH)2D3 receptor. Models of Caj+ Transport Three processes of Ca^+ permeation across the intestine have been proposed. These are, as follows: (a) the endocytotic-exocytotic-vesicular flow model, (b) the paracellular diffusional model and (c) diffusional-active transport model. The endocytotic-exocytotic-vesicular flow model The endocytotic-exocytotic-vesicular flow model has early support from histological studies and electron probe analysis of intestinal tissue (see ref. k for references). The more recent studies of Nemere et al (29) provide biochemical evidence for Ca2+ containing vesicular components in the intestinal cell that are vitamin D-responsive. Also Rubinoff and Nellans (30) isolated an endoplasmic reticular-enriched microsomal fraction from rat intestine which accumulated Ca2+ by an ATP-dependent mechanism and might be involved in transepithelial Ca 2 + transport. Consistent with the idea of an endocytotic-exocytotic mechanism was our observation on the transfer of CaBP from intestinal mucosa to the vasculature draining the intestine (31). It was shown that intraluminal Ca^ + , presumably as a consequence of its transcellular movement, increased the amount of a CaBP appearing in the blood of the duodeno-pancreatic vein. The CaBP extruded from the intestinal cell could very well be that previously incorporated in the intracellular vesicles identified by Nemere et al (29) and others. However, the amount of CaBP appearing in blood was small compared to the residual concentration of CaBP in the intestinal mucosa. If vesicular flow does occur, its quantitative relation to overall Ca2+ absorption needs to be assessed. The paracellular diffusional model The non-saturable component of Ca 2 + absorption, which becomes increasingly evident as intraluminal increases, is thought to be diffusion via the paracellular path. Evidence for this path is based primarily on theoretical grounds and the biphasic nature of the intraluminal [Ca 2 + ]vs. Ca2+ absorption curve (1), and the response of Ca2+ translocation across the intestine to applied transcellular voltages, using in vitro preparations (32). The latter approach provided evidence for the electrophoretic transfer of Ca 2 + across the epithelial cell layer, presumably via the paracellular route. The diffusion-active transport model This model of Ca^+ absorption is considered today to be the most probable manner by which Ca2 + is absorbed when intraluminal Ca2+ is in the low mM range. Transfer of Ca2+ across the brush border membrane appears to have a vitamin Dsensitive component. The extremely early increase in cGMP synthesis in response to l,25(OH)2D3 in one cell type (24) and the fast absorption response to l,25(OH)2Ö3 and PTH (19,20) speaks for a cyclic nucleotide-sensitive event that leads to the proposal of phosphorylation of a latent Ca2+ channel. Closure of the channel would result from the action of a phosphatase (alkaline phosphatase ?). The cAMP- and cGMP-dependent phosphorylation of the intestinal brush border membrane proteins was previously demonstrated (39).

562 The transfer of Ca^+ through the cytosol to the basal lateral membrane is considered to be a function of vitamin D-induced calbindin. How could calbindin function in facilitating the cytosolic transport of calcium? The affinity constant of calbindin and the high concentration of this protein within the intestinal cell indicate that it is quite suitable to act as a calcium buffer, potentially preventing Ca2 + from reaching toxic levels during the course of calcium absorption. In addition to this buffer function of calbindin, it was proposed on theoretical grounds that calbindin could facilitate the transfer of Ca^+ from the brush border region to the basal lateral membrane (33). The primary consideration for this mechanism is that the diffusion of Ca2+ to the region of the calcium pump occurs as the f r e e Ca2+ ion and Ca^+ bound to calbindin. Since the affinity of the Ca pump for Ca2+ exceeds that of calbindin, Ca^+ available for active extrusion across the basal lateral membrane can be derived from both the f r e e diffusible and that Ca?+ released from calbindin in the region of the Ca pump. Support for calbindin serving as a diffusional facilitator comes from the studies by Feher (3*0, using an in vitro three compartment model system. The responsiveness of the calcium pump to vitamin D is another area of uncertainty at the present time. Ghijsen and Van Os (35) reported that the Ca pump of rat intestine is vitamin D-responsive, as did others (36,37). However, vitamin D was considered by Van Corven et al (38) to protect the Ca pump of the basal lateral membrane from proteolytic and lipolytic activity and not activate the pump directly. The validity of this notion requires further investigation. In actuality, it is possible that acceleration of overall calcium transport by vitamin D might not require stimulated pump activity. In the view of some (1,8), the Ca pump is normally "starved" for Ca2+ and additional Ca2+ presented to the pump during the course of vitamin D-stimulated calcium absorption can be readily accommodated. In the view of others (37), an increase in pump activity might be required. Vitamin D and Non-epithelial Tissues The e f f e c t of the vitamin D hormone on tissues other than those classically associated with systemic calcium homeostasis is receiving increasing attention. Disease states attributed to vitamin D deficiency, other than abnormal bone formation, are hypertension, immune deficiency, and muscle dysfunction. Our attention was drawn to a specific nerve in chicks, the intestinalis nerve. The calbindin content of this nerve is a f f e c t e d by vitamin D status and dietary calcium levels (40). The CaBP content of this nerve decreases in vitamin D deficiency, and increases in chicks fed a vitamin D-replete, calcium deficient diet. Subsequent studies have now shown that the conduction velocity of this nerve is altered by vitamin D-deficiency. Intestinalis nerves from vitamin D-deficient birds have a greater conduction velocity than those from normal birds, and vitamin D repletion of the deficient birds decreases the conduction velocity of the nerve towards that seen with normal intestinalis nerves. The CaBP in this nerve is present in the cell body and in varicosities within the nerve axon and these latter structures presumably represent sites of neurotransmitter release. The influx of Ca2+ is apparently a requisite for the release of neurotransmitters by nerves in general. Assuming a Ca2+ buffer function for CaBP in non-epithelial tissues, the presence of CaBP in normal or vitamin D-replete nerves would dampen the rise of intracellular Ca^+ following depolarization and thereby possibly decrease the number and rate of neurotransmitter quanta released. In the absence of CaBP, the Ca^+ rise would be greater, with possibly a faster release of more neurotransmitter

563 molecules with each depolarizing event. It is also possible that vitamin D directly influences the properties of the nerve membrane. These alternatives need to be sorted out. References 1. Bronner, F., Pansu, D., Stein, W.D. (1986) Am.J.Physiol. 250:G561-G569. 2. Norman, A.W. (1987) J.Nutr. 117:797-807. 3. Toverud, S.U., Dostal, L.A. (1986) J.Pediatric.Gastroenterol.Nutr. 5:688-695. 4. Wasserman, R.H., Fullmer, C.S., Shimura, F. 1984 in Vitamin D. Basic and Clinical Aspects, Kumar, R., Ed. Martinus Nijhoff Publishing, Boston, pp. 233-257. 5. DeLuca, H.F. (1988) FASEB 3. 2:224-236. 6. Haussler, M.R. (1986) Annu.Rev.Nutr. 6:527-562. 7. Henry, H.L., Norman, A.W. (1984) Annu.Rev.Nutr. 4:493-520. 8. Van Os, C.H. (1987) Biochim.Biophys.Acta 906:195-222. 9. Nemere, I., Norman, A.W. (1982) Biochim.Biophys.Acta 694:307-327. 10. Wasserman, R.H., Fullmer, C.S. (1983) Annu.Rev.Physiol. 45:375-390. 11. DeLuca, H.F. Schnoes, H.K. (1983) Annu.Rev.Biochem. 52:411-439. 12. Weiser, Μ.Μ. 1984 in Absorption and Malabsorption of Mineral Nutrients, Solomons, N.W., Rosenberg, I.H., Eds. Alan R. Liss, New York, pp. 15-68. 13. Levine, B.S., Walling, M.W., Coburn, J.W. 1982 in Disorders of Mineral Metabolism, Academic Press, New York, Vol. 11:103-188. 14. Rasmussen, H., Fontaine, O., Goodman, D.B.P. 1980 in Pediatric Diseases Related to Calcium, DeLuca, H.F., Anast, C.S., Eds., Elsevier, New York, pp. 133-142. 15. Schachter, D., Kowarski, S. 1980 in Pediatric Diseases Related to Calcium, DeLuca, H.F., Anast, C.S., Eds., Elsevier, New York, pp. 143-152. 16. Bikle, D.D., Morrissey, R.L., Zolock, D.T. (1979) Am.J.Clin.Nutr. 32:23222338. 17. Favus, M.J. (1985) Am.J.Physiol. 248:G147-G157. 18. Lawson, D.E.M. 1984 in Vitamin D. Basic and Clinical Aspects. Kumar, R., Ed. Martinus Nijhoff Publishing, Boston, pp. 303-324. 19. Nemere, I., Yoshimoto, Y., Norman, A.W. (1984) Endocrinology 115:14761483. 20. Nemere, I., Norman, A.W. (1986) Endocrinology 119:1406-1408. 21. Harrison, H.C., Harrison, H.E. (1970) Endocrinology 86:756-760. 22. Corradino, R.A. 1977 in Vitamin D: Biochemical Chemical and Clinical Aspects Related to Calcium Metabolism, Norman, A.W. et al, Eds., de Gruyter, Berlin, pp. 231-240. 23. Wasserman, R.H., Corradino, R.A., Feher, J., Armbrecht, H.J. 1977 in Vitamin D: Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism, Norman, A.W. et al, Eds., de Gruyter, Berlin, pp. 331-340. 24. Barsony, J., Marx, S.J. (1988) Proc.Natl.Acad.Sci.USA 85:1223-1226. 25. Rasmussen, H., Matsumoto, T., Fontaine, O., Goodman, D.B.P. (1982) Fed.Proc. 41:72-77. 26. Brasitus, T.A., Dudeja, P.K., Eby, B., Lau, Κ. (1986) J.BioI.Chem. 261:1640416409. 27. Bikle, D.D., Munson, S. (1985) J.Clin.Invest. 76:2312-2316. 28. Vesely, D.L., Juan, D. (1984) Am.J.Physiol. 246:E115-E120. 29. Nemere, I., Leathers, V., Norman, A.W. (1986Tj.Biol.Chem. 261:16106-16114. 30. Rubinoff, M.J., Nellans, H.N. (1985) J.BioI.Chem. 260:7824-7828. 31. Lee, Y.S., Reimers, T.J., Cowan, R.G., Fullmer, C.S., Wasserman, R.H. (1988) Arch.Biochem.Biophys. 261:27-34.

564 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.

Nellans, H.N., Kimberg, D.V. (1978) Am.3.Physiol.236:E726-E737. Kretsinger, R.H., Mann, J.E., Simmonds, J.G., 1982 in Vitamin D: Chemical, Biochemical and Clinical Endocrinology, Norman, A.W., et al., Eds., De Gruyter, Berlin, pp. 233-248. Feher, J . J . (1983) Am.J.Physiol. 2M:C303-C307. Ghijsen, W.E.J.M., Van Os, C.H. (1982) Biochim.Biophys.Acta 689:170-172. Meyer, S.A., Wasserman, R.H. (1983) Fed.Proc. ^2:1367. Walters, J.R.F., Weiser, Μ.Μ. (1987) Am.J.Physiol. 252:G170-G177. Van Corven, E.J.J.M., De Jong, M.D., Van Os, C.H.TT986) Cell Calcium 7:8999. Donowitz, Μ., Cohen, Μ.Ε., Gudewich, R., Taylor, L., Sharp, G.W.G. (1984) Biochem.J. 219:573-581. Lee, Y.S., Taylor, A.N., Reimers, T.J., Edelstein, S., Fullmer, C.S., Wasserman, R.H. (1987) Proc.Natl.Acad.Sci. USA S4i7344-734Z. Morrissey, R.L., Wasserman, R.H. (1971) Am.J.Physiol. 220:1509-1515.

ATP-DEPENDENT C a 2 + PUMPS IN ENDOPLASMIC RETICULUM AND PLASMA MEMBRANES A R E NOT AFFECTED BY 9kDa CALBINDIN-D AND VIT.D-DEFICIENCY. J.A.H. TIMMERMANS, E.J.J.M. v a n CORVEN and C.H. v a n OS Department of Physiology, Unive-rsity of Nijmegen, Nijmegen. The Netherlands. Introduction 2+ Free Ca concentration in cells is maintained by a concerted action of C a 2 + transport systems in intracellular organelles and in the plasma m e m brane. In duodenal cells intracellular Ca^ homeostasis is challenged by variable rates of transcellular C a 2 + transport regulated by l,25(OH)2D3 (1). Active C a ^ + sequestration b y intracellular organelles and buffering by Calbindin-D may be involved in handling in transit through the cytosol. W e have studied the effects of vit.D-deficiency and of 9kDa Calbindin-D on ATP-dependent Ca^-1" transport in endoplasmic reticulum, m i t o c h o n d r i a and plasma membranes. Methods Duodenal cells were isolated and permeabilized w i t h saponin as recently described (2). Pig intestinal and rat intestinal 9kDa Calbindin-D was isolated according to Gleason and Lankford (3). Microsomes and m i t o c h o n d r i a were isolated by differential centrifugation. ATP-dependent Ca^"1" uptake by permeabilized cells, microsomes, mitochondria and basolateral membrane vesicles, BLMV, was studied by means of millipore filtration as previously (2). Vit.D-deficient rats w e r e raised as before (4). Results and Discussion 45 Permeabilized cells from rat duodenum accumulate C a w h e n provided w i t h ATP. The use of oxalate, vanadate and ruthenium red indicate that n o n mitochondrial as well as mitochondrial pools are involved. 0.5 m M vanadate inhibits completely non-mitοchondrial C a 2 + uptake, w h i l e 10 μΜ ruthenium red inhibits mitochondrial Ca2+ accumulation (2). K i n e t i c analysis of n o n mitochondrial C a 2 + uptake reveals a % ^ 0.1 μΜ C a ^ + and a ^ 1*5 nmol Ca^ + /min.mg protein. A microsomal preparation w i t h low ( N a + - K ) - A T P a s e activity and 2.4-fold enrichment in NADPH-cyt.-C-reductase accumulates Ca^"1" w i t h similar Kju and 2-fold higher V m a x as in permeabilized cells. In Fig.l it is shown that the affinity and the V m a x of ATP-dependent Ca^"1" uptake by the non-mitochondrial pool and by microsomes are unchanged in vit.D-deficiency. The mitochondrial pool and isolated mitochondria started to accumulate C a ^ + at 0.3 μΜ free C a ^ + . The rate of uptake increases sigmoidally up to 5 μΜ C a ^ + (2). In Fig.2 we show that vit.D-deficiency does not influence mitochondrial C a 2 + handling. The effect of 9kDa Calbindin-D from pig intestine on the non-mitochondrial C a ^ + store is shown in Fig.3 and the effect on the BLM C a ^ + pump in Fig.4. Despite the h i g h concentrations applied no effects are observed. References 1. v a n Os,C.H. (1987) Biochim.Biophys.Acta 906: 195-222. 2. v a n Corven,Ε.J.J.Μ., Verbost,P.Μ., de Jong,Μ.D., v a n Os,C.H. (1987) Cell Calcium 8^: 197-206. 3. Gleason,W.A., Lankford,G.L. (1981) Anal.Biochem. _1_Ηκ 256-263. 4. Ghijsen,W.E.J.M. , v a n Os.C.lI., Heizmann,C.W., Murer,H. (1986) Am.J. Physiol. 251: G223-G229.

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · N e w York - Printed in Germany

566

ISOLATED 45

ATP -dependent •

^Ca-uptake

ATP - dependent

- D

Ca-uptake

n m o l / m i n . m g . prot. \ Z Z

nmol/min.mg. p r o t .

+

D

MICROSOMES

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4 η

•

- D

Ea 3-

SAPONIN TREATED C E L L S + RUTHENIUM

150·

100-

1 -

50-

0.1

0.1

1.0 f i M free

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Fig. 2 .

ATP-dependent

Ca

p+

.

1

0.6

10

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•

Figures 1 and 2;

+ d

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2 -

.

ij Μ free Ca

.

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Effect of vit.D-deficiency on active Ca* uptake by non-mitοchondrial and mitochondrial C a ^ + stores.

uptake p e r m e a b i l i z e d cells 0.1 μ Μ t r e e C a z

150 τ

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30

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Effect of pig intestinal 9kDa Calbindin-D on ATPdependent C a 2 + transport by non-mitochondrial C a 2 + stores and basolateral membranes.

-t-

300

EFFECTS OF VITAMIN D-DEFICIENCY ON DIFFERENT ACTIVITIES OF THE Ca 2+ -PUMP IN RAT INTESTINAL BASOLATERAL MEMBRANES. JULIAN RF WALTERS.

SUNY Division of Gastroenterology, Buffalo General Hospital, Buffalo, NY 14203, USA. Calcium absorption in the intestine is well-established as being regulated by 1,25-dihydroxycholecalciferol. Extrusion of calcium from the enterocyte by the ATF-dependent Ca2+-pump in the basolateral membrane is the energy-requiring process in Ca2+ absorption and is a potential site for control. In other cells, the partial reactions of the Ca2+-pump (the "Ca2+-ATPase") and several means of regulation have been described. Various activities of this Ca2+-pump were compared in normal and vitamin D-deficient rats. METHODS

Isolated duodenal cells from male rats were used to prepare basolateralenriched membranes and Ca2+ uptake in EGTA/Ca2+ buffers at 0.5μΜ free-Ca2+ was performed as previously described [1]. Ca2+-ATPase activity was measured at ΙμΜ free-Mg2+ and 0.5mM EGTA in the presence and absence of ΙμΜ free-Ca2+ [2] . To detect the phosphorylated-intermediate of the pump, membrane proteins were incubated with [7-32P]-ATP on ice for 1 min in the presence of Ca2+ and with La3* to stabilize the pump phosphoprotein. Proteins were separated on acidic gels and detected by autoradiography [3] . CaBP stimulation of the Ca2+-pump was shown with CaBP dialysed extensively against an EGTA-free buffer in order to avoid changes in *5Ca2+ specific activity (measured free-Ca2+ activity of 7μΜ). Ca2+ uptake was then determined in this EGTA-free system with 10-60μΜ CaBP. RESULTS AND DISCUSSION

Vesicular Ca2* uptake. As shown in our previous publication [1] , Ca2+ transport was reduced in vesicles prepared from vitamin D-deficient animals (control 6.55 ± 0.37, D-def. 3.56 ± 0.69 nmol/min/mg protein, ρ < 0.01). Similar findings were reported by Ghijsen and Van Os [4]. Van Corven et al. [5] repeated these results with an isolated cell method but found that modifications to this method, or an alternative method with vibration, removed the vitamin D-dependent effect. They postulated that in the isolated cell method, vitamin D-deficient membranes were more sensitive to inactivation by pancreatic enzymes; they could overcome this difference by: (1) "lipase" inhibition with diethyl p-nitrophenol phosphate, (2) proteolytic inhibition with PMSF/aprotinin, and (3) fasting the animals for 16 hours. However, in this laboratory, fasted rats were always used. The difference in vitamin D-deficients was found in villus-tip cell which had the shortest exposure to possible enzyme action [1]. did not increase uptake and membrane preparation by a vibration gave lower values. It may be relevant to note that proteolytic also can activate the Ca2+-pump [3] .

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · New York - Printed in Germany

greatest membranes Inhibitors method treatment

568 Ca2*-dependent AIP-hydrolysis. "Ca2+-ATPase" activity, i.e. ATP hydrolysis in the presence and absence of 1 μΜ Ca2+, was not different in vitamin D-deficient animals (3.97 ± 1.64 vs. 4.66 ± 1.76 μιηοΐ/h/mg protein). However, in Intestinal basolateral membranes, as in liver, kidney and some other cells, Ca2+-ATPase activity was shown to not be equivalent to Ca2+-transport activity by the Ca2+-pump. In the intestine, differing properties of Ca2+-ATPase activity were: (1) 20-fold higher activity than transport rates; (2) a Kc, of Ο.βμΗ (transport 0.3/iM); (3) insensitive to vanadate or calmodulin; (4) wider substrate specificity including ADP and GTP; and (5) different villus/crypt and proximal/distal distributions [2]. Phosphorylated intermediate of the pump. The formation of a 130 kDa phosphoprotein was enhanced by La3+; it had similar acyl-phosphate properties to the red-cell Ca2+-pump [3], No difference in the intensity of this band was detected in vitamin D-deficiency. The 130 kDa phosphoprotein was purified from intestine by calmodulin affinity chromatography. Calbindin-D9k stimulation of transport activity. Stimulation of Ca2+ uptake was seen in an EGTA-free system using physiological concentrations of CafiP. EGTA mimicked the stimulation by CaBP (the EGTA-effect). Stimulation of Ca2+ transport by CaBP appears to be one means of regulation of Ca2+-pump activity by vitamin D. CONCLUSION It is still unclear how the duodenal basolateral membrane Ca2+-pump is affected in vitamin D-deficiency. Similar levels of the phosphorylated intermediate at 130 kDa suggest that the amount of protein is not changed. Effects of regulatory enzymatic actions and direct stimulation by calbindin-D9k are possible modes of vitamin D action. REFERENCES 1. 2. 3. 4. 5.

Walters, J.R.F. and Weiser, Μ.Μ. (1987) Am. J. Physiol. 252, G170-G177. Moy, T.C., Walters, J.R.F. and Weiser, Μ.Μ. (1986) Biochem. Biophys. Res. Commun. 141, 979-985. Sarkadi, B., Enyedi, Α., Foldes-Papp, Z. and Gardos, G. (1986) J. Biol. Chem. 261, 9552-9557. Ghijsen, W.E.J.M., and van Os, C.H. (1982) Biochim. Biophys. Acta 689, 170-172. Van Corven, E.J.J.M., de Jong, M.D. and van Os, C.H. (1987) Endocrinology 120, 868-873.

ATP-DRIVEN C A 2 + PUMPS IN DUODENAL PLASMA MEMBRANES AND ENDOPLASMIC RETICULUM FROM PIGLETS WITH INHERITED RICKETS. R. KAUNE and C.H. v a n Os*. Institute of Physiology, School of Veterinary Medicine, D-3000 Hannover 1, Bischofsholer Damm 15, ERG, *Department of Physiology, University of Nijmegen, Nijmegen, The Netherlands. Introduction Active intestinal absorption of calcium mainly occurs in the duodenum and depends on circulating levels of l,25(OH>2D3, which cause a rise in intestinal vitamin D-dependent calcium binding protein (1). The exact mechanism by which l,25(OH)2D3 stimulates the active calcium transport is still unknown. A n effect on the basolateral ATP-dependent C a 2 + - p u m p has been reported (2,3), but recently it has been shown, that this effect might have been caused by the procedure applied for cell isolation (4). It was the aim of this study, to measure basolateral and microsomal C a 2 + pumps in duodenum of piglets with an inherited form of rickets ("Hannover Pig Strain") and low l,25(OH) 2 D3 levels in comparison with healthy controls . Materials and Methods Piglets with pseudo vitamin .D deficiency rickets, type I were used. Rickets is caused in the animals by a lack of renal 1-hydroxylation of 25OHD3. Pathogenesis and etiology of the disease have b e e n described (5,6). Immediately after killing the animals by stunning, the duodenum was removed, frozen in liquid nitrogen and stored at -70°C. Basolateral membranes (BLM) »were prepared from scrapings as described by Ghijsen et al. (7). For preparing microsomes (MSM), the lOOOOxg supernatant of a mitochondrial preparation (8) was centrifuged at lOOOOOxg for lh and the pellet resuspended in transport medium. C a ^ - u p t a k e s were measured at 3 7 e C by the rapid filtration technique (7) with 0.1 mM IACI3 added to the stop buffer. The free Ca 2 + -concentrations of the media were adjusted to 1 μΜ and the free Mg2"1"-concentrations to 1.45-1.5 mM as described in detail by v a n Heeswijk et al. (9). ATP was added as Tris-ATP (BLM) or M g 2 + - A T P (MSM) to give a final concentration of 3 mM (BLM) or 10 m M (MSM). Results and Discussion Basolateral membrane vesicles were purified 5 to 6 fold in (Na + -K+)ATPase activity and the microsomal fraction was prepared with a 3 to 4 fold purification in NADPH-cyt.-C-reductase activity, containing only minor amounts of (Na + -K + )ATPase and mitochondrial markers. ATP-dependent C a 2 + uptake at 1 μΜ free calcium and 37°C in BLM-preparations from control and rachitic animals are shown in the table. No significant differences were found between the two groups, especially when the values were related to the individual enrichment factors for (Na'^-IC4")ATPase. ATP-dependent C a 2 + uptake in BLM was inhibited by A23187 and vanadate but mitochondrial inhibitors as ruthenium red or NaN3 had no effect.

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin • New York - Printed in Germany

570 Table: ATP-dependent Ca^ + -uptake into basolateral membrane vesicles from control and rachitic piglets at 1 μΜ free Ca^ + : Ca^ + -uptake [nmol/mg protein] Incubation time [min]

Control

Rachitic

Ca^ + -uptake / enrichment factor of (Na + -K + )ATPase Control

Rachitic

1

2.59 ± 0.37

3.22 ± 0.39

0.55 ± 0.07

0.58 ± 0.06

5

5.60 ± 0.52

6.05 ± 0.56

1.28 ± 0.15

1.08 ± 0.12

10

7.20 ± 0.58

7.75 ± 0.80

1.66 ± 0.21

1.36 ± 0.11

Mean values are given ± SEM from 5-6 different pairs of control and rachitic litter mates. There are no significant differences between the two groups. ATP-dependent Ca^ + -uptake at 1 μΜ free and 37*C into microsomal membrane vesicles were estimated in the presence of 10 μΜ ruthenium red to Inhibit mitochondrial uptake. Uptake amounted to 1.2 ± 0.02 and 1.79 ± 0.36 nmol/(min χ mg protein) in control and rachitic animals, respectively (n-3,SEM) and was inhibited by A23187 and vanadate. ATP-dependent C a 2 + uptake in the microsomal fraction was markedly stimulated by addition of 20 mM oxalate. Stimulation 10 min after start of incubation ranged from 6.76 ± 0.47 (without oxalate) to 12.0 ± 1 . 8 nmol/mg protein (with oxalate) in controls (n-5) and from 9.10 ± 1.72 to 14.24 ± 2.95 (n-4) in rachitic piglets, (mean ± SEM). We conclude, that ATJP-dependent pumps in duodenum of piglets with pseudo vitamin D'deficiency rickets, type I are not reduced. References 1. Van Os.C.H. (1987) Biochim.Biophys.Acta 906:195-222. 2. Ghijsen.W.E.J.M., van Os.C.H. (1982) Biochim.Biophys.Acta 68£:170-172. 3. Chandler,J.S., Meyer,S.A., Wasserman.R.H. (1985) in Vitamin D, Chemical, Biochemical and Clinical Update, Norman,A.W., Schaefer.K., Grigoleit.H.G., Herrath,v.D. Ed., Walter de Gruyter, Berlin:408-409. 4. Van CorVen.E.J.J.M.. de Jong,Μ.D., van Os.C.H. (1987) Endocrinology 120:868-873. 5. Harmeyer,J., Grabe,v.C., Winkler,I. (1982) Exp. Biol. Med. 2:117-125. 6. Kaune,R., Harmeyer,J. (1987) Acta Endocrin. (Copenh.) 115:345-352. 7. Ghijsen.W.E.J.M., de Jong.M.D., van Os.C.H. (1982) Biochim.Biophys. Acta 689:327-336. 8. Van Corven.E.J.J.M. (1987) Thesis Univ. of Nijmegen,ISBN:90-9001754-2 Nijmegen, The Netherlands. 9. Van Heeswijk.M.P.E., Geertsen,J.A.M., van Os.C.H. (1984) J. Membrane Biol. 22:19-32.

EFFECT OF l,25(OH)2D3 ON PHOSPHORUS ABSORPTION IN SHEEP. K.M. SCHNEIDER and D.D. LEAVER Department of Pharmacology, University of Melbourne, Vic. 3052 Australia. Introduction 32 Following administration of Ρ into the abomasum or fourth stomach of the sheep, the blood tracer appearance and disappearance curve has two separate peaks, which have been quantified as two separate absorptive compartments by fitting a compartmental model to the tracer data (Figure 1)(1). Since these peaks reflect the time tracer takes to pass down the intestine, the restriction of the sodium-dependent phosphate transport system to the ileum could provide a physiological basis for the two compartments (2). If this was the case, then it should be possible to manipulate the rates of uptake from the two compartments by the administration of l,25(OH)2D3, as in the rabbit l,25(OH)2D3 increases phosphate absorption by enhancing the activity of the sodium-dependent absorption process. The purpose of this study was to examine the effect of 1,25(OH)jD^ on the amount of Ρ absorbed from the two absorptive compartments. Methods Five sheep were treated with 1,25(0Η)203 (25 ng/kg s.c.) daily for 4 days. Absorption of Ρ was measured by compartmental analysis by giving simultaneous injections of into the abomasum and 3 3 P into the jugular vein, and assaying sequential blood and abomasal samples for P, 3 2 P and 33 P (1). Results and Discussion Administration of l,25(OH)2D^ increased plasma Ρ concentration from 1.34 ± 0.10 to 2.39 ± 0.12 mM and abomasal Ρ from 0.15 ± 0.05 to 0.26 ± 0.06 g. Absorption of phosphorus was also increased, but in 3 sheep the major site was absorptive compartment 1 (AC1) rather than 2 (AC2), Figure 1A. In 2 sheep, l,25(OH)2D3 increased absorption in AC2, see Figure IB. Since sodium-independent phosphate transport is distributed throughout the intestine, whereas sodium-dependent uptake is confined to the ileum, absorption from AC1 is believed to reflect the former process, and absorption from AC2 reflects both processes. If this is the case, 1,25(OH)2DJ should increase absorption from AC2 only, but this hypothesis 32 Table 1. The absorption of Ρ from AC1 and AC2 in the presence and absence of l,25(OH)2D3, mean ± SE (n = 4 unless marked) Absorption % Absorption, g/d AC1 AC2 AC1 AC2 Control 54 ± 8* 31 ± 7* 2.24 ± 0.90 0.97 ± 0.14 l,25(OH)2D3 85 ± 7(3) 13 ± 7(3) 9.73 ± 4.78 1.85 ± 0.69 «Significantly different from treated sheep, P3 per kg feed), this fact might be of importance, since feedback regulation and thus physiological functions are not impaired (25). Vit.C participates in many functions of bone metabolism, enhancing collagen synthesis, stimulating hydroxylation of lysine and proline and contributing to the cross-linking of fibrils and their stabilization. The mineralization step depends on v i t . D 3 , whose transformation into the active D 3 metabolites is promoted by vit.C. W i t h the additional vit.C supplementation, above all increased 1.25(OH) 2 D and slightly higher Ca plasma levels were observed. The binding capacity of duodenal CaBP and the proportion of bone AP related to total AP were enhanced. Bone weights were also higher without an essential change in the ratio of organic and inorganic components. In the lower dosis ranges of vit.D3 and 2 4 R , 2 5 ( O H ) 2 D 3 , vit.C supplementation resulted in a significant promotion of the body weight in all cases.

653

References 1. Murad, S. , Grove. D., Lindberg, Κ.A. and Reynolds,G. (1981) Proc. Natl. Acad. Sei. USA 78, 2879-2882. 2. Levine, M. (1986) N. Engl. J. Med. 314. 892-902. 3. Pinnell, S.R.. Murad. S. and Darr, D. (1987) Arch. Dermatol. 123, 684-1686. 4. Barnes, M.J. (1975) Ann. N.Y. Acad. Sei. 258, 264-277. 5. Pinnell, S.R. and Martin. G.R. (1968) Proc. Natl. Acad. Sei. USA 61. 708-714. 6. Bird. T.A. and Levene. C.I. (1982) Biochem. Biophys. Res. Commun. 108. 1172-1180. 7. Bird, T.A. and Levene. C.I. (1983) Biochem. J. 210, 633-638. 8. Eyre, D.R., Paz, M.A. and Gallop, P.M. (1984) Am. Rev. Biochem. 53. 714-748. 9. Benke, P.J., Fleshood, H.L. and Pitot, H.C. (1972) Biochem. Med. 6, 526-535. 10. Trechsel, U.. Eisman. J.Α., Fisher. J.Α., Bonjour. J.P. and Fleisch. Η. (1980) Am. J. Physiol. 239. E119. 11. Gray, R.W. (1981) Calcif. Tissue Int. 33, 485-488. 12. Littledike. E.T. and Goff. J. (1987) J. Anim. Sei. 65. 1727-1743. 13. Hornig. D. and Frigg, M. (1979) Arch. Geflügel*. 43, 108-112.

14. Mallon. J.P., Hamilton, J.G., Nauss-Karol. C.. Karol, R.J., Ashley, C.J., Matuszewski, D.S., Tratneyk, C.A. and Miller, N.O. (1980) Arch. Biochem. Biophys. Res. Commun. 83, 441-448. 15. Borgers, Μ. and Thone. F. (1975) Histochemistry 44, 277-280. 16. Wasserman. R.H., Corradino, R.A. and Taylor, A.N. (1968) J. Biol. Chem. 243, 3978-3986. 17. Wolinsky. I., Simkin, A. and Guggenheim, K. (1972) Am. J. Physiol. 233. 46-50. 18. Bell. N.H., Shaw, S. and Turner. R.T. (1984) J. Clin. Invest. 74, 1540-1544. 19. Gates, S.. Shavy. J.. Turner. R.T.. Wallach. S. and Bell. N.H. (1986) J. Bone Mineral Res. 1. 221-226. 20. Milue, M.L. and Bavau. D.T. (1985) Arch. Biochem. Biophys. 242, 488-492. 21. Vieth, R.. Fräser, D. and Kooh. S.W. (1987) J. Nutr. 117. 914-918. 22. Ishida. M.. Bulos. B.. Takamotos. S. and Sacktor, B. (1987) Endocrinology 121, 443-448. 23. Francis, R.M. and Beaumont, D.M. (1987) N. Engl. J. Med. 316. 215-216. 24. Riggs. B.L. and Melton. L.J. (1987) N. Engl. J. Med. 326. 216. 25. Weiser. Η. and Schlachter, Μ. (1987) in Generalized Bone Diseases, ed. by Kuhlencordt, F., Dietsch, P.. Keck, E., Kruse, Η.-P.. Springer-Verlag Berlin, Heidelberg, New York, London. Paris, Tokio, p. 71-76.

PLASMA MAGNESIUM IN PIGS WITH PSEUDO VITAMIN D DEFICIENT RICKETS; TYPE 1. J. HARMEYER, U. DUCHATZ, K.M. SCHNEIDER and D.D. LEAVER Institute of Physiology, School of Vet. Med., 3000 Hannover, FRG. Introduction Pigs genetically deficient in renal 1-hydroxylase have low circulating concentrations of 1,25(OH) 2 D 3 and require Supplemente of vitamin D 3 to prevent rickets and death. Since there is evidence that vitamin D influences magnesium (Mg) (1,2) as well as calcium (Ca) and phosphorus (P) metabolism, the effect of vitamin D supplements on the concentration of Mg in plasma and urine hae been assessed. Methods At birth, piglets were divided into rachitic and healthy groups, and the rachitic piglets were further subdivided into treated and untreated groups. Pigs were weaned between 40 and 50 days, and were then fed ad libitum pig starter diet, containing 1% Ca, 0.8% Ρ and 18% crude protein, followed at 10 weeks with a twice daily ration of pelleted pig diet with 0.9% Ca, 0.8% Ρ and 16% crude protein. Treated pigs were given vitamin D 3 supplements (5 mg) at weaning, then the supplements were withheld until the pigs developed signs of lameness, when they were given a second treatment with l,25(OH) 2 D 3 (1-5 μg/d), 1ocOHD3 (5 Mg/d), 24,25(OH) 2 D 3 (1020 μg/d), all i.V., and the plants Solanum malacoxvlon l g/d and Trlsetum flavescens 180 g/d, orally. Body weight changes and plasma and urine concentrations of Mg, Ca, Ρ and parathyroid hormone (ΡΤΗ) were measured. Results and Discussion All the piglets were normal at birth, but between 30 and 40 days, control and treated piglets showed a greater weight gain than untreated rachitic animals. Thereafter the feed intake of treated pigs also decreased as the rachitic condition progressed, and at three months of age their weight gains were less than the controls. Untreated animals died between 80-90 days after birth. Following weaning, plasma Mg of the treated pigs gradually increased to a mean of 0.94 ± 0.03 mM for the two weeks prior to the second treatment. During the second treatment (with l,25(OH) 2 D 3 , laOHD^, or Solanum malacoxvlon) plasma Mg declined steadily, the lowest concentration (0.56 mM) being attained after 22 d supplementation with laOHD^. Urine Mg increased during treatment from 18 ± 3 to 36 ± 8 mmoles Mg/d. Plasma ΡΤΗ declined during the second treatment, but plasma Ca and Ρ both increased, see Figure 1A and B. Neither 24,25(OH) 2 D 3 nor Trisetum flavescens produced any effect on the parameters measured. The rapid decline in plasma Mg following treatment with 1,25(OH) 2 D 3 or its analogues was associated with an increase in the renal excretion of Mg. Such an increase may arise indirectly because of competition between Ca and Mg for reabsorption in the nephron, as the rise in plasma Ca would

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter& Co., Berlin · New York - Printed in Germany

655

Age

(Days)

Age

(Day·)

Figure 1. A. Plasma Mg and PTH before, during and after treatment of rachitic piglets with 5 \xg/d laOHD^, and B. Plasma Ca and Ρ during the same experiment. lead to an increase in the filtered load of Ca in the kidney. In addition the decline in plasma concentration of PTH that followed treatment would also decrease Mg absorption in the nephron (3). Moreover, the reversal of these processes could have led to the increase in plasma Mg that preceded the second treatment. Such an explanation implies that l ^ S f O H ^ D ^ has little direct effect on renal Mg excretion, but in the sheep treated with l,25(OH) 2 D 3 net retention of Mg doubled even though plasma Mg declined (4). Since the plasma Mg of these pigs is dependent on their l,25(OH) 2 D 3 status, they should be a suitable model to examine whether 1,25(OH) 2 D 3 h a s a direct action on the cellular distribution of Mg. References 1. Schneider, K.M., Parkinson, G.B., Houston, J.C. and Leaver, D.D. (1985) Aust.Vet.J. 62:82-5. 2. Miller, E.R., Ullrey, D.E., Zutaut, C.L. Baltzer, B.V., Schmidt, D.A., Vincent, B.H., Hoefer, J.A. and Luecke, R.W. (1964) J.Nutr. 83:140-8. 3. Massrey, S.G., Coburn, J.W. and Kleeman, C.R. (1969) Am.J.Physiol. 216:1460-7. 4. Schneider, K.M. and Leaver, D.D. (1985) in Vitamin D, Chemical, Biochemical and Clinical Update, Norman, A.W., Schaefer, Κ., Grigoleit, H.-G. and Herrath, D.v., Eds. de Gruyter, Berlin, ρ 571-2.

SUNLIGHT DEGRADATION OF VITAMIN D 3 A.R.WEBB, B.DECOSTA AND M.F.HOLICK. Boston University School of Medicine, Boston, MA 02118. Introduction Casual exposure to sunlight is responsible for providing the vitamin D requirement of much of the world's population. During exposure to sunlight, high energy ultraviolet radiation of wavelengths between 290 and 315 nm penetrate into the epidermis and cause the photochemical conversion of 7-dehydrocholesterol (7-DHC) to previtamin D 3 (pre-D-j) (1) . Once formed, pre-D 3 undergoes a thermally induced isomerisation to vitamin D 3 (D 3 )(2). It was shown in the 1940's that vitamin D could be photodegraded by exposure to very high intensity, high energy UV radiation that resulted in a variety of photoproducts (3). We now report that cutaneous Do is exquisitely sensitive to sunlight, exposure resulting in the formation of 3 major photoproducts. Materials and Methods The photolability of D 3 in sunlight was investigated in human skin samples and an in vitro model previously developed to predict the ability of sunlight to produce preD 3 in skin (4). Quartz tubes containing H-D 3 in MeOH were exposed to sunlight for 3 hours from 11:30 to 14:30 EST on clear days throughout the year in Boston (42.2°N). Aliquots were taken hourly and analyzed by hplc. Products were further purified by reverse phase hplc and identification was made by cochromatography, mass and NMR spectroscopy. Human skin samples, previously irradiated and incubated at 37°C to generate cutaneous D 3 , were also exposed to sunlight for 3-hour periods, followed by extraction and hplc analysis of the epidermis with stratum basale (1,4). Action spectrum studies of the photodegradation of D 3 used the in vitro model. Results and Discussion The D 3 content of skin exposed to sunlight was significantly reduced compared to unexposed control samples. Quantitation and photoproduct identification was made possible by the in vitro model. Using an in-line radioactivity detector the H-D-j showed photodegradation to 3 photoproducts, only one of which absorbs UV light at 254 nm. Degradation was observed year round (Fig.l) with a dramatic 95% reduction in the original D-j after 3 hours exposure in June. A corresponding depletion of 30% was observed in December, resulting in the formation of the photoproducts 5,6-transvitamin D 3 and the suprasterols I and II (3) (Fig.2). This is in contrast to similar studies with H-7DHC which have shown that in Boston between the months of November and February solar irradiation does not convert cutaneous 7-DHC to pre-D 3 (4).

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter&Co., Berlin · N e w York - Printed in Germany

657

< 29 «> 2 ω

α:

il

10 ο

tM

SΟΝ ο

MONTH

Figur· 1

Figure 2

Figure Legends 1. Photodegradation of H-D 3 after 3 hours irradiation by Boston sunlight for each month of the year. Columns represent mean values, points represent the years 1985 (•) , 1986 (·), 1987 (Ο) , 1988 (*) . 2. The structures of the photoproducts of D 3 after exposure to sunlight. In explanation, our action spectrum studies showed radiation of 2906 ratio of 0.08 to 0.5 in replete rats, and 0.5 to 2.6 in deficient rats. % * 7 Ca absorption + SD ± SEM EHBF alone EHBF + Calcitriol

EFA replete rats (η - 12) EFA deficient rats (η - 11) Table II:

30.5 ± 9.6 + 2.8%

45.4 + 11.5 + 3.3%

ρ Ϊ- ΙΟ Ο Ο

η Η 0 ο

CO 0) •Η 0 α 0) 3

•

ο ο

Ο

α •Η

rH to 00 CM CM rH rH rH rH

η CO rH 90% of actin eventually became complexed with Gc (19,20). In addition, plasma clearance of complexed Gc was more rapid (tl/2 c. 30 min) than that of rabbit Gc (tl/2 c. 20-24 hr) (19,20). Thus, although another similar study in rats did not show preferential uptake of Gc:G-actin (21), these studies collectively confirm an emerging function for Gc in actin homeostasis. Interaction of Gc with the Lipid Bilaver of Cells Initial reports of Gc in the cytosol of most nucleated cells (22,23) were greeted with some scepticism, since it was possible that the small amounts observed merely represented plasma contamination of cell homogenates. However, these experiments were confirmed by the finding of Gc in relation to the membrane of several cells, in particular Β lymphocytes and monocytes (2327). Furthermore, this interaction was found to be extremely stable; essentially Gc behaved as an integral membrane protein, requiring chaotrope, denaturant or urea for removal (24-26). Furthermore, Gc underwent co-distribution with at least one integral membrane protein, the membrane immunoglobulin (mlg) receptor of Β cells, upon crosslinking of the latter with anti-Ig and warming to 37°C (25,26), and was phosphorylated in signal-dependent fashion by Ca2+/phospholipid-dependent C-kinase (27).. Physicochemical studies have shown that the membrane homologue exhibits MW and PI indistinguishable from those of the serum form (26), and no evidence of synthesis by lymphocytes or monocytes has been obtained either by hybridization analysis of mRNA with cDNA for Gc, or in biosynthetic labeling experiments. In addition, some evidence of binding of Gc, albeit non-saturable, has recently been obtained in Τ lymphocytes activated by monoclonal antisera to Τ cell antigen receptor-associated proteins, and also in liposomes (unpublished observations).

687

Interaction of Gc with Unsaturated Fatty Acids Taken together, the above observations suggested the possibility that Gc was interacting with component lipids of the bilayer. In addition, sequencing of human Gc from cDNA (28,29) showed 40-45% nucleotide homology with albumin, which is known to act as a carrier protein for fatty acids (30,31). The indistinguishable physicochemical properties of membrane and serum Gc, and the observation that 5iQBlpD, prnHiiflftd Γιοι/10 c e l l s / h J 0.0 319±43 2.5 297±15 0.5 251+12· 1.0 186+32*· REFERENCES. 1) Adams J . S . , S i n g e r F.R., Gacad M.A., Sharma O.P., Hayes J . , Vouros P. and Holick H.F. (1985) JCEM Vol 60(5):960 2) Mason R.S., F r a n k e l T . I . , Chan I . T . , L i s s n e r D. and Posen S. (1984) Ann. I n t e r n . Med. Vol 100:59 3) Henry H.L. (1985) J . S t e r o i d Blochen. Vol 23:991 4) R e i c h e l Η., K o e f f l e r H.P., B a r b e r s R. and Norman A.W. (1987) JCEM Vol 65(6): 1202 5) Mason R.S. (1985) Vitamin D: Chemical- Biochemical and C l i n i c a l dpdate. Ed. Norman A.W. Walter de Gruyter, Berlin.

1,25(0H)2D3 PRODUCTION BY LUNG Τ LYMPHOCYTES FROM TUBERCULOSIS PATIENTS. J. CADRANEL, M. GARABEDIAN, H. GUILLOZO, A. HANCE. CNRS UA. 563. HApltOl des Enfants Mel odes, end INSERM U. B2, Hftpltol Xovler Blchat, Paris, France. Introduction Recent evidence demonstrates that extra renal production of 1 ^SCOHj^occurs In ο variety of granulomatous diseases (l)ond that this vitamin D metabolite can modify the activity of both macrophages and Τ lymphocytes (2), the predominant cell types present ingranulometous lesions. Although 1,25(0H)2D3 production by granulomatous tissue and cultured macrophages hasbeen demonstrated (3,4), It Is unclear whetherT lymphocytes participate In 1,25(0H)2t>3 production (5) or ore only targets for 1,ΣδίΟΗ)^ produced by macrophages (2). To study this question vre evaluated production of 1,25(0H)2C>3by lung τ lymphocytes from patients with pulmonary tubeixuloslsandnormalcolclum metabolism. Material sand Methods Fresh lung cells were recovered, by bronchoalveolar lavage, from 7 patients with pulmonary tuberculosis and In 3 control subjects. LungT lymphocytes (more than 95% purity) were then obtained by passing fresh lung cells thru nylon wool columns (6). LungT lymphocytes, were incubated at lo5cells/mllnDulbecco'smod1f1ed Eagle s medium for 150 m1n (37eC; 5% C0 2 ) In the presence of 2.5 nM ^H 25(0H)C>3 (SA: 20 C1/mmole) and/or 2.5 uM 25(0H)D3 (SA: 0.02 C1/mmole).

Vitamin D. Molecular, Cellular and Clinical Endocrinology © 1988 Walter de Gruyter&Co., Berlin · New York-Printed in Germany

862 Resul tsendDI scussl on Lung Τ1 ymphocytes from 6 of 7 patl ents with tubercul osl s, but not those from controls,produceda polar metabolite of 25(0H)D3Wh1chwas1dent1f1edas ι ,25(0Η)2θ3 by the roll owing criteri 6: Dcoeiutl on with synthetic i,25(0H)2D3 using two successive HPLC systems (92/8 nhexane/lsopropanol then 95/5 methylene chlorlde/lsopropanol); 2) specific activity (cpm/A254)ldentlcel to that of the 25(0H)D; substrate (0.016 ± 0.004 vs 0.020 ± 0.001 Cl/mmole); 3) ability to compete wlthsynthetlc^H 1,25(OH)2D3forb1ndlngtoch1ck intestinal cytosol Identical tothot of synthetic I ^ S C O H ) ^ (Kd: 0.1 I 0 " 9 M). These results suggest that extra renal production of 1 ^ S t O H J ^ b y granulomatous tissue 1s, at least In part, due to the production of this metabolite byT lymphocytes.

References 1. Lemenn J., Gray R.W. (1984) N. Engl. J. Med. i l l : 1115-1116. 2. Rlgby W.F.C. (1988) Immunology Today £ 54-58. 3. Mason R.S., Frankel Τ., Chan Y., Llssner D., Posen S. (1984) Ann. Intern. Med. 100: 59-61.

4. Adams J.5., Sharma O.P., Gocad M.A., Singer F.R. (1983) J. Clin. Invest. 22: 1856-1860. 5. Fetchlck D.A., Bertollnl D.R., Sarin P.S., Welntreub S.T., Mundy G.R., Dunn J.F., (1986) J. Clin. Invest. 2fl: 592-596. 6. Julius M.H., Simpson E., Herzenberg L.A., (1973) Eur. J. Immunol. £ 645-649.

Cancer and Vitamin D

TREATMENT OF MYELODYSPLASTIC SYNDROME AND AML WITH HYDROXYVITAMIN

la

D3

Τ. TAOKA, Y. KUBOTA, Τ. TANAKA and S. IRINO. 1st Department of Internal Medicine, Kagawa Medical School, Kagawa,

Japan.

Introduction 1 « , 25(OH)2D 3 is known as one of the inducers of non-lymphoid leukemia cell differentiation to monocyte-macrophages in vitro (1).

Although a few clinical studies have

been reported (2, 3), the usefulness of vitamin D3 therapy for myelodysplastic syndrome (MDS) and acute myelocytic leukemia (AML) is still unclear.

In this present study,

we administered high doses of 1 a ( O H ) D 3 , which is converted in vivo to l i H Z

C3 ΙΛ Ν

fe Ρ ρ

)

α* W 0) (β Λ U Φ > ΙΛ -a·

73 4) η Οβ 00 o> φ h rH ο «β Ο) θ Τ) β) (Η II II ρ Ρ* Λ 03 β 1) h Ο (β C θ II II Ζ £

880 studied to evaluate the possible effects of maternal diabetes and/or calc ium restriction on the fetal vitamin D and bone metabolism (44,45). Results 1. Adult male rats with untreated diabetes for 3 weeks maintain a normal protein-corrected serum calcium despite severe hypercalciuria and disappearance of active calcium absorption, by increasing their food and calcium intake and by increased passive intestinal calcium diffusion (46,47). The serum concentration of 25-OHD remains normal but serum l,25-(OH)2D decreases significantly in diabetic male rats. This decrease can be partly explained by their increased food and calcium intake but as the serum vitamin D-binding protein decreases even more than total l,25-(OH)aD, the free 1,25(OH)aD index increases in diabetic animals whereas in calcium-intake matched non-diabetic rats the expected decrease in free l,25-(OH)2D is observed (Table 1). The end organs show a significant disappearance or reduction TABLE 2.

Synoptic overview of abnormalities in calcium/vitamin D/bone metabolism in diabetic male rate. Values are compared with non-diabetic littermates fed at libitum. Diabetic Rats

Non-diabetic Rats (wt-matched)

X weight loss during - 29 X 3 week period protein-corrected Ν serum calcium serum phosphate Ν D total serum 1,25-(OH)2D D DBP I free l,25-(OH)»D I urinary calciuflk excretion D active calcium absorption D duodenal CaBP Net calcium balance Ν D Bone mineralization rate D Osteoblast surface D Serum osteocalcin

- 31 X

(Ca-matched) unchanged

D

Ν

D Ν D I Ν

Ν D Ν D I

Ν

D

D D Ν Ν Ν

D Ν =

=

Ν

wt-matched: non-diabetic rats on semistarvation to obtain a weight-matched control group Ca-matched: non-diabetic rats on a calcium supplemented diet to obtain a calcium-intake matched control group.

881 in vitamin D-dependent proteins : the duodenal CaBP concentration is low and the active duodenal calcium absorption (evaluated by the everted gut sac technique) disappears. The osteoblast-derived vitamin D and vitamin Itdependent protein, osteocalcin or bone Gla protein, also decreases markedly in untreated diabetic male rats (values about 20% of normal non-diabetic rats). This low osteocalcin level corresponds to a low osteoblast function as assessed by histomorphometry and calcein labeling of bone (Table 2). 2. Female rats with mi Idly-treated diabetes for about 8 weeks also have a profound disturbance of vitamin D metabolites and vitamin D-dependent tissues. Although the protein-corrected serum calcium remains normal, serum l,25-(OH)iD levels are lower in diabetic rats than in non-diabetic littermates, whether on a high or low Ca/P diet (Fig. 3). Similar to the observations in untreated diabetic male rats, very low osteocalcin levels were observed in diabetic female rats. These low serum osteocalcin concentrations could not be stimulated by situations known to be a "calcium stress". Indeed, a low calcium/phosphate diet in diabetic rats could increase total and free l,25-(OH)2D concentrations but not osteocalcin levels. Moreover, pregnancy was not associated with increased 1,25-(OH)2D or osteocalcin concentrations as in non-diabetic rats. The trabecular bone volume, measured in the tibial metaphysis, showed a marked reduction in non-pregnant diabetic rats on a high and even more on a low calcium/phosphate diet. Pregnant diabetic rats, however, did not suffer from a diabetesinduced bone loss, although their osteoblast and osteoclast surface percentage and osteocalcin levels were similar to the diabetic non-pregnant rats (43), indicating that pregnancy could protect bone in diabetic rats by yet unknown mechanisms. Fig. 3. Total serum 1, 25-(OH)a D concentration in female diabetic rats, fed a high (0.85/0.7 X) or low (0.2/0.2 X) calcium/phosphate diet. The rats were about 5 months old with a diabetes duration of about 8 weeks. Pregnant rats were studied at day 21 of pregnancy. Serum 1, 25-(OH)a D and osteocalcin were measured by radioimmunoassay (modified from ref. 44).

882

3. The fetuses of diabetic rats show vitamin D and bone abnormalities similar to the disturbances found in adult diabetic rats. Their l,25-(OH)2D and DBP levels are lower than in fetuses from non-diabetic controls and their plasma osteocalcin levels are similarly reduced. Their total body calcium, mainly representing bone mineralization, is also severely depressed even when corrected for a lower total body weight (44). Conclusions Diabetes mellitus, either untreated or undertreated with insulin results in several abnormalities of the vitamin D metabolism and action. (1) At the serum level, protein-corrected calcium remains normal. Total serum l,25-(OH)2D usually decreases but this is largely due to decreased serum DBP levels so that the unbound 1,25-(OH)2D levels are normal or increased. (2) The duodenal response to vitamin D is impaired as the active duodenal calcium absorption is abolished and duodenal CaBP levels are low. The net calcium balance can however be greatly maintained by increased calcium intake and passive intestinal calcium diffusion. (3) The bone turnover is severely impaired. Especially the osteoblast number and function is suppressed (in untreated male rats to about 20 % of normal values). The osteoclast number is also decreased. The total bone mass and trabecular volume decreases after 3 to 8 weeks of diabetes both in bones with predominantly cortical bone (tibia) as in bones with a higher trabecular content (vertebrae). The decrease in bone mass is accelerated in diabetic rats fed a low calcium/low phosphorus diet but can be prevented by pregnancy. The low serum osteocalcin levels are unresponsive to stimulation by endogenous or exogenous l,25-(OH)2D indicating vitamin D resistance of the osteoblasts. (4) Fetuses from diabetic rats have a marked growth retardation but their bone mineralization is even more severely impaired. (5) It is presently unclear whether the effects of insulin deficiency on vitamin D metabolism and action are due to a direct effect or mediated by other hormones, especially since insulin-like growth factors, androgens and thyroxine are also markedly suppressed in diabetic animals. Acknowledgments We thank Prof. D. Pipeleers (Brussels) for his cooperation in providing isolated pancreatic B-cells for immunocytochemietry. The work was supported by an FGWO grant n* 3.0029.85.

883 References Ϊ. Clark,S.A., Stumpf.W.E., Sar,M., DeLuca,Η.F., Tanaka,Y. (1980) Cell. T i s s u e Res. 2 0 9 : 5 1 5 - 5 5 2 . 2. C l a r k , S . A . , S t u m p f , W . E . , Sar,M., D e L u c a , Η . F . (1987) Am. J. P h y s i o l . 2 5 3 : E 9 9 - E 1 0 5 . 3. C h r i s t a k o s , S . , N o r m a n , A . W . (1979) B i o c h e m . B i o p h y s . R e s . Commun. 89:56-63. 4. P i k e , J . W . ( 1 9 8 1 ) J. S t e r o i d B i o c h e m . 1 6 : 3 8 5 - 3 9 5 . 5. Roth,J., Bonner-Weir,S., Norman,A.W., Orci,L. (1982) Endocrinol. 110:2216. 6. Christakos,S., Friedlander,Ε.J., Frandsen,B.R., Norman, A.W. (1979) E n d o c r i n o l . 104:1495. 7. Morrisey.R.L., Bucci,T.J., Empson,R.Ν., Lufkin,E.G. (1975) P r o c . Soc. Exp. B i o l . M e d . 149:56. 8. Arnold,B.B., Kuttner,M., Willis,D.M., Hitchman,J.W., Harrison,J.Ε., Murray,T.M. (1975) Can. J. Physiol. P h a r m a c o l . 5 3 : 113 5. 9. Pipeleers D.G., in't Veld,P.Α., Van de Winkel,Μ., Maes,Μ., Schuit,F.C., Gepts,W. (1985) Endocrinol. 117:806-816. 10. Kaidowak i, S . , N o r m a n A . W . ( 1 9 8 4 ) A r c h . B i o c h e m . B i o p h y s . 233:228-236. 11. Sonnenberg,J., Pansini,A.R., Christakos,S. (1984) E n d o c r i n o l . 115 : 6 4 0 - 6 4 8. 12. Norman,A.W., Frankel,Β.J., Heidt,Α.Μ., Grodsky,G.M. (1980) S c i e n c e 2 0 9 : 8 2 3 - 8 2 5 . 13. Kadowaki,S., Norman,A.W. (1984). J. Clin. Invest. 73:759. 14. K a d o w a k i , S . , N o r m a n , A . W . (1985) D i a b e t e s 3 4 : 3 1 5 - 3 2 0 . 15. Nyomba,B.L., Bouillon,R., Lissens,W., Van Baelen,H., De Moor,P. (1985) E n d o c r i n o l . 1 1 6 : 2 4 8 3 - 2 4 8 8 . 16. Kadowaki,S., Norman,A.W. (1985) Endocrinol. 117:17651771. 17. C a d e , C . , N o r m a n , A . W . (1986) E n d o c r i n o l . 1 1 9 : 8 4 - 9 0 . 18. Cade,C., N o r m a n , A . W . (1987) E n d o c r i n o l . 1 2 0 : 1 4 9 0 - 1 4 9 7 . 19. Nyomba,B.L., Auwerx,J., Bornans,V., Peeters,Τ.L., Pelemans,W., Reynaert,J., Bouillon,R., Vantrappen,G., De M o o r , P . (1986) D i a b e t o l o g i a 2 1 9 : 3 4 - 3 8 . 20. G e d i k , Ο . , A k a l i n . S . (1986) D i a b e t o l o g i a 2 9 : 1 4 2 - 1 4 5 . 21. Chertow,Β.S., Sivitz,W.I., Baranetsky,Ν.G., Clark, S., W a i t e . A . , D e L u c a , H . F . (1983) E n d o c r i n o l . 1 1 3 : 1 5 1 1 . 22 Frankel,B.J., Sehlin,J., Taljedal,I.Β. (1985) Acta Physiol. Scand. 123:61-66. 23. Harmeyer,J.f Knorz,S., Dwenger,A., Winkler,I. (1985) Zbl. Vet. Med. A. 32:606-615. 24. Auwerx,J., Dequeker,J., Bouillon,R., Geusens,P., Nijs,P. (1988) D i a b e t e s 37:8-12. 25. Nyomba,B.L. (1986) Thesis, Katholieke Universiteit Leuven. 26. Schneider,L.Ε., Schedl,H.P. (1972) Am. J. Physiol. 223:1319-1323. 27. Schneider,L.E., Wilson,H.D., Schedl,H.P. (1974) A m . J. Physiol. 227:832-838.

884

28. 29. 30. 31. 32. 33.

34. 35. 36. 37. 38.

39.

40. 41.

42. 43. 44. 45. 46. 47.

Schneider,L.Ε., Omdahl,J., Schedl,H.P. (1976) Endocrinol. 99:793-799. Schneider,L.Ε., Schedl,H.P., McCain,T., Haussier,M.R. (1977) Science 196 : 1452-1454. Schedl,Η.Ρ., Heath,Η., Wenger,J. (1978) Endocrinol. 103:1368-1373. Spencer,Ε.Μ., Khalil,M., Tobiassen,0. (1980) Endocrinol. 107:300-305. Charles,M.A., Tirunaguru,Ρ., Zolock,D.T., Morissey,R.L. (1981) Miner Electrolyte Metab. 5:15-22. Shires f R., Teitelbaum,S.L., Bergfeld,M.A., Fallon,M.D., Slatopolsky,Ε., Avioli.L.V. (1981) J. Lab. Clin. Med. 97:231-240. Hough,S., Russell,J.Ε. , Teitelbaum,S.L. , Avioli,L.V. (1982). Am. J. Physiol. 242:E451-E456. Wilson,H.D., Horst,R.L., Schedl,Η.P. (1982) Diabetes 31:401-405. Hough,S., Slatopolsky,Ε., Avioli.L.V. (1983) Calcif. Tissue Int. 35:615-619. Seino,Y., Sierra,R.I., Sonn,Y.M., Jafari,A., Birge, S.J., Avioli.L.V. (1983) Endocrinol. 113:1721-1725. Wongsurawat,Ν·, Armbrecht,Η·J·, Zenser,Τ·V·, Davis, B.B. , Thomas,Μ.L., Forte,L.R. (1983) Diabetes 32:302306. Schedl,Η.P., Christakos,S., Wilson,Η.D., Malkowitz,L., Horst,R.L. (1984) Proc. Soc. Exp. Biol. Med. 177:176179. Wood,R.J., Allen,L.H., Bronner,F. (1984) Am. J. Physiol. 247:R120-R123. Ishida,H., Seino.Y., Nishi,S., Kitano.N., Seno,M., Taminato.T., Matsukura,S., Ishizuka,S., Imura.H. (1985) Acta Endocrinol. 108:231-236. Nyomba,B.L., Bouillon,R., Lissens,W., Van Baelen,H., De Moor,Ρ. (1985) Endocrinol. 116:2483-2488. Matsumoto,Τ., Kawanobe,Y., Ezawa,I., Shibuya,N., Hata,K., Ogata,E. (1986) Endocrinol. 118:1440-1444. Verhaeghe,J., Bouillon,R., Lissens,W., Visser,W.J., Van Assche,F.A. (1988) Am. J. Physiol. 254 (in press). Verhaeghe,J, Thomasset,Μ., Brehier,Ä., Van Assche,F.A., Bouillon,R. (1988) Am. J. Physiol. 254 (in press). Nyomba,B.L., Verhaeghe,J., Thomasset,Μ., Lissens,W., Bouillon, R. (submitted). Verhaeghe,J., Suiker,A.M.H., Nyomba,B.L., Visser.W.J., Einhorn,Τ.Α., Dequeker,J., Bouillon,R. (submitted).

E N D O G E N O U S DIABETES D E C R E A S E S T H E R E C E P T O R S IN B O T H I N T E S T I N E A N D KIDNEY.

NUMBER

OF

1,25-DIHYDROXYVITAMIN

DO

H. Ishida*, N.S. Cunningham*, K L Henry # , and A.W. Norman**, Division of Biomedical Sciences* and Department of Biochemistry*, University of California, Riverside, CA 92521. Introduction Alterations in calcium and vitamin D metabolism and some suggestive evidence for the decrease in the biological actions of 1,25-dlhydroxyvltamin D 3 (1,25(OH)2DQ) have been observed in human diabetic patients (1,2). The strain of genetically diabetic mice, C57BL/KsJ db/db, Is a model of endogenous diabetes, which bears a resemblance to human type 2 diabetes. The present study was designed to evaluate 1,25(OH) 2 D 3 receptors in intestine and kidney of these endogenous diabetic mice and to investigate the alterations of calcium and vitamin D metabolism. Materials and Methods Male weanling diabetic db/db mice and their controls were raised for 9-11 weeks on a vitamin Ddeficient diet (1.0% Ca, 1.0%P). A group of vitamin D-replete animals were administered orally 0.5IU/g body weight of vitamin D 3 three times a week, and a second group was given the solvent alone. Plasma levels of glucose, insulin, calcium and 25(OH)D were determined in the fed state. The 1,25(OH) 2 D 3 receptors in Intestine and kidney were investigated via sucrose density gradient analysis and Scatchard analysis in vitamin D-deflcient animals. The renal conversion of [ H]25(OH)D 3 to [ 3 H]-24,25(OH) 2 D 3 and [ 3 H]-1,25(OH) 2 D 3 was also studied in diabetic and control mice. Results a)

In vivo experiment: The body weight and plasma glucose In diabetic mice were significantly elevated (P