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Some applications to man of specific markers of the molecular action of vitamin D (1.25(OH)2D3 receptors and antibodies to hormone-dependent proteins (CaBP and cDNA] are reported in this study. On case of type II vitamin-dependent rickets was characterized by 1.25(OH)2D3 plasma level greater than 250 pg/ml and a ten-fold decrease of the number of binding sites of the hormone in cultured skin fibroblasts. We propose that CaBP 28K and/or 9K-containing cells, such as Purkinje's cells and chondroblasts may be targets for vitamin D action. Detection in fetuses, from the 20th week of gestation, of CaBP 9K messenger RNA in the duodenum and sternum and presence of CaBP 28K and 9K in the chondroblasts of the upper extremity of tibia, suggest that vitamin D acts on the nucleus of its target-cells during fetal development. Finally, discovery of the gene of CaBP 9K in man opens the prospect of studies which will improve the understanding of the mechanism of action of vitamin D.

Chronic metabolic acidosis entails hyperparathyroidism and osteopathy. In order to elucidate the role of the thyroparathyroids in this bone lesion production the effects of acidic diet for 7 weeks were studied in parathyroidectomized (PTX), thyroparathyroidectomized (TPTX) and shamoperated (Sh-O) growing rats. In all animals urinary excretion of calcium, phosphate, ammonium and titrable acidity was similarly increased. The rise in hydroxyproline excretion and urinary 85-sr (that was injected previous to acidic feeding) was more marked in PTX and TPTX rats. Moreover, in these animals the serum calcium level was increased, the blood pH was decreased. According to these data, an acidic diet intake that is not sufficient to elicit a fall in blood pH of normal young rats can induce severe acidosis in chronically parathyroidectomized or thyroparathyroidectomized animals; moreover the bone resorption appears more marked. It is concluded that parathyroids are involved in the extra-cellular fluid defense mechanism against acidosis by a no bone resorptive mechanism. We hypothesize that the parathyroids permit the necessary and adequate supply of bicarbonates by the bone to maintain blood pH homeostasis.

The mechanism by which inaktin, a thiobarbiturate, promotes a moderate drop in serum calcium has been investigated. The effect was cancelled in hypocalcemic parathyroidectomized rats. On the other hand inaktin antagonized the serum calcium raising effects of parathyroid extract in these animals. In rats wit intact parathyroid glands inaktin caused a two-fold increase in urinary calcium excretion and a marked decrease in body and bone calcium turnover (measured with 45 Ca). These results support the view that inaktin impairs the parathyroid hormone effects on calcium metabolism.

Lung mechanics was studied at 50 days of age in 7 rachitic rats born from mothers deprived of vitamin D. They were compared with 7 control rats raised in the same conditions but fed a diet supplemented with vitamin D. The animals were anesthetized, tracheotomized, and paralyzed. Quasi-static pressure-volume curves of the respiratory system and of the lungs were obtained. Body weight of the rachitic rats was within the range of the control rats, but dry lung weight (LW) was significantly lower (p less than 0.01). Lung volumes in absolute terms and when normalized for LW were significantly lower than in the control rats. Chest wall compliance (Ccw) of the rachitic rats was within the range of values of the control rats, except for 2 animals with an infinite Ccw. Analysis of the pressure-volume curves of the lungs of the rachitic rats compared with those in the control animals showed a significant decrease in lung compliance (CL) and in CL/LW (p less than 0.01), indicating a decrease in lung distensibility. The more severe the rickets (according to microradiographic criteria of the tibia), the lower the CL/LW. It is speculated that decrease in lung distensibility may be related to abnormal lung growth caused by disturbed alveolar formation and lung connective tissue development. These abnormalities could be due to vitamin D deficiency acting on the growing lung, as on the growing bones, by a mechanism involving proteoglycans.

The tissue distribution, developmental changes, and the vitamin D dependence in the rat of two calcium-binding proteins [CaBPs, 28,000 and 10,000 Mr (28 and 10 K)] were examined. The radioimmunoassays used employed specific antibodies to either the human cerebellar CaBP (28 K protein) or to the smaller rat intestinal CaBP (10 K protein). The assay for the 28 K CaBP may be used to detect this protein in a number of mammalian species and tissues, whereas the 10 K CaBP assay appears to be specific only for the rat intestinal CaBP. This report demonstrates that the tissue distribution of the two CaBPs is different in the rat. High levels of the 28 H protein were found in the cerebellum and kidney, whereas the smaller CaBP was concentrated in the duodenum, jejunum, and cecum. Many other organs and tissues contained small quantities of both CaBPs. Developmental studies indicated some variability in the concentration of the CaBPs. Duodenum, kidney, and cerebellum all contained small amounts of one of the CaBPs prior to birth. Adult levels in all three tissues were already reached at 30 postpartum days. Levels of both CaBPs began to decline in rats older than 2 mo. The vitamin D dependence appeared to reflect cell turnover in that the duodenal and kidney CaBPs showed a vitamin D dependence not observed for the cerebellar protein.

Clinical observation of patients with disordered phosphocalcium metabolism has demonstrated that dyschromia and/or dental dysplasias systematically accompany such disorders. A certain action of this steroid on dental buds has been demonstrated after analysis of the effects of experimental vitamin D deficiency in the rat: vitamin D would seem to control the behaviour of cells undergoing differentiation and also after this process is complete. Dentinogenesis and amelogenesis would appear principally to be affected. Two proteins, calbindins D-9K and -28K, may constitute the molecular mediators of this ameloblastic regulation.

Vitamin D-dependent CaBP isolated from Rat renal cortex (rCaBP) was measured in phosphorus-depleted (OP) and control (C) Rats, either vitamin D-deficient (OD) or vitamin D-supplemented (1 or 10 i. u.). A low molecular weight fraction was isolated from renal cortex by "Sephadex G-100" chromatography and rCaBP activity quantitated by saturation analysis using a 45 Ca chelex assay. The results indicated that phosphorus deprivation resulted in the increase in the vitamin D-dependent rCaBP as well as in the intestinal CaBP. As a marked hypercalciuria was noted in all OP Rats and as the rCaBP activity was high in vitamin D-supplemented Rats and hardly detectable in vitamin D-deficient Rats, the implication of the rCaBP in the large hypercalciuria can be definitely ruled out. Furthermore when vitamin D-supplementation ranged from 1 to 10 i. u. vitamin D, while the serum calcium level was increasing a decrease could be noticed in the large hypercalciuria. This deserves to be related to the increase in rCaBP activity. The high CaBP activity probably resulting from the renal synthesis of 1,25-dihydroxycholecalciferol stimulated by phosphorus-deprivation could represent the molecular basis of the calcium tubular reabsorption increased by vitamin D. Thus a vitamin D-dependent protein implicated in an ion-selective transport could be involved in the tubular calcium reabsorption as well as in the intestinal calcium absorption.

It has been suggested that vitamin D is involved in the process of cell differentiation and extracellular mineralization during tooth development. One of the best-defined molecular markers of the action of vitamin D is a calcium-binding protein of Mr 28,000 called calbindin D-28 K (CaBP 28 K). Since this protein is present in growing teeth, we have examined its synthesis in teeth from vitamin D-replete and -deplete rats by Western blotting and immunocytochemistry with an antiserum to CaBP 28 K purified from rat kidney. The CaBP 28 K present in the enamel organ is a single molecular species migrating near 30 k Da, similarly to the kidney protein. The differentiation and maturation of odontogenic cells were followed during early postnatal development (2-12 days) in rat molars. At the light-microscope level, CaBP 28 K was only found in a single cell-type, the ameloblasts. The expression of this protein appeared to be developmentally controlled, since its distribution varied with the cell stage and the functional steps of amelogenesis. The protein was localized in the basal compartment of ameloblasts from the presecretory stage. During the early secretory stage, the concentration of cytoplasmic CaBP 28 K formed a gradient from the apical to the basal pole of the ameloblasts. Staining appeared homogeneous in the cytoplasm of later secretory ameloblasts. CaBP 28 K was discontinuously distributed during the maturation stage. This discontinuity might be related to cyclical changes in mature ameloblasts. In all stages, ameloblasts from vitamin-D-deficient rats appeared depleted of CaBP 28 K.