la,25-dihydroxyvitamin D3 in chick parathyroid glands (vitamin D/receptors/parathyroid hormone)

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 72, No. 12, pp , December 1975 Biochemistry Cytoplasmic and nuclear binding components for la,25-dihydroxyvitamin D3 in chick parathyroid glands (vitamin D/receptors/parathyroid hormone) PETER F. BRUMBAUGH, MARK R. HUGHES, AND MARK R. HAUSSLER Department of Biochemistry, College of Medicine, University of Arizona, Tucson, Ariz Communicated by John H. Northrop, September 29, 1975 ABSTRACT Specific binding of la,25-dihydroxyvitamin D3 [la,25(oh)2d3j to macromolecular components in the cytoplasm and nucleus is demonstrated in parathyroid glands of vitamin-d-deficient chicks. The interaction of la,25- (OH)WD3 with the cytoplasmic binding component is of high affinity (R4 = 3.2 X M) and high specificity [la,25- (OH)2D3 > 25-hydroxyvitamin D3 > la-hydroxyvitamin D3 > vitamin D3 in competing with radioactive la,25(0h)2d31. Both cytoplasmic and nuclear hormone-macromolecular complexes sediment at 3.1 S in 0.3 M KC-sucrose gradients, and agarose gel filtration of the components indicates an apparent molecular weight of 58,000. The 3.1S binding molecules are not observed in adrenal gland, testes, liver, or kidney, but similar receptors for la,25-(0h)2d3 have been found previously in intestine. Macromolecular species with a high affinity and preference for 25-hydroxyvitamin D3 [25(0H)D3] are also identified in parathyroid cytosol and differ from the parathyroid la,25(oh)wd3-binding component in that: (1) they sediment at 6 S in 0.3 M KC-sucrose gradients, (2) they are observed in all tissues examined, (3) they have a higher affinity for 25- (OH)D3 than la,25-(0h)2d3, and (4) they are not found in the nucleus of the parathyroid glands, in vitro. The discovery of unique la,25.oh)2d3-binding components in the parathyroid glands is consistent with the sterol hormone's action at this endocrine site and possible involvement in the regulation of parathyroid hormone synthesis and secretion. Vitamin D3 action to mobilize calcium and phosphate at intestine and bone is thought to be mediated by the metabolite la,25-dihydroxyvitamin D3 [la,25-(oh)2d3] (1-4). ts production from 25-hydroxyvitamin D3 [25-(OH)D3] by the renal la-hydroxylase appears to be regulated by calcium (5), phosphate (6, 7), parathyroid hormone (PTH) (7, 8), and the vitamin D status of the animal (9). Evidence suggests that hypocalcemia stimulates PTH secretion which in turn enhances the production of la,25-(oh)2d3 at the kidney (7, 8). Thus PTH, rather than calcium, may be the dominant modulator of the renal la-hydroxylase (10) and the finding of abnormal circulating 1a,25-(OH)2D3 in humans with parathyroid disease is consistent with this concept (11, 12). DeLuca has proposed that PTH and phosphate deficiency may be functioning through a common intracellular mechanism to enhance the la-hydroxylase by lowering the phosphate level in the renal cell (10). Moreover, Macntyre and associates (13) have suggested that la,25-(oh)2d3 might control its own biosynthesis directly at the kidney by a negative feedback mechanism involving the de novo synthesis of the la-hydroxylase enzyme. Clearly, the fashion in which la,25-(oh)2d3, PTH, and phosphate deficiency interact to control the formation of 1a,25-(OH)2D3 at its kidney endocrine site must be completely understood before the exact Abbreviations: 25-(OH)D3, 25-hydroxyvitamin D3; la-(oh)d3, la-hydroxyvitamin D3; la,25-(oh)2d3, la,25-dihydroxyvitamin D3; PTH, parathyroid hormone role of 1a,25-(OH)2D3 in the homeostatic regulation of calcium and phosphate can be elucidated. Henry and Norman (14) have recently reported that la,25-(oh)2[3h]da is localized in chick parathyroid glands following administration of the sterol, in vivo. n the present experiments, specific binding components for 1a,25- (OH)2D3 have been isolated from chick parathyroid glands and characterized in vitro. Previous reports (15, 16) have demonstrated that 1la,25-(OH)2D3 interacts with the intestine in a manner similar to the binding of steroid hormones to their respective target organs. 1a,25-(OH)2D3 enters the intestinal cell and binds to a 3.7S cytoplasmic receptor protein (17, 18). The hormone receptor complex then migrates into the nucleus in a temperature-dependent process, where it associates with the chromatin (15, 17-19). We report here that similar intracellular receptor proteins for la,25- (OH)2D3 exist in chick parathyroid glands. MATERALS AND METHODS- Materials. Animals used in experiments were White Leghorn cockerels (kindly donated by Demler Farms, Anaheim, Calif.) that were raised for 6 weeks on a vitamin-d-deficient diet (20). 25-Hydroxy[26(27)-methyl-3H]vitamin D3 (6.5 Ci/ mmol) was obtained from Amersham-Searle. Preparation of la,25-dihydroxy[3hjvitamin D3, n Vitro. la,25-dihydroxy[26(g7)-methyl-3h]vitamin D3 was prepared as previously described (19). Radiochemical purity of generated la,25-dihydroxy[3h]vitamin D3 was 98%. 25- Hydroxy[26(27)-methyl-3H]vitamin D3 substrate for the reaction was purified by Celite liquid-liquid partition chromatography (21). The radiochemical purity of the 25-hydroxy[3H]vitamin D3 was 95%, and its specific activity was determined by ultraviolet absorbance spectrophotometry at 265 nm. Exposure of Chick Tissue Subfractions to Radioactive Sterols, n Vitro. Homogenates [300 mg wet weight (20 parathyroid glands)/3 ml] were made in 0.25 M sucrose, 0.05 M Tris-HC, ph 7.4, M KC, and 5 mm MgC12 (0.25 M sucrose-buffer A) with a Potter-Elvehjem homogenizer equipped with a Teflon pestle at 00 by six passes, with 2 min cooling periods between passes. Homogenates were centrifuged at 1200 X g for 10 min. Nuclear pellets were removed, and the resulting supernatant was centrifuged at 100,000 X g for 1 hr at 0 to yield a final supernatant fraction (cytosol). The cytosol ( ml) was incubated with sterol (in 20,l ethanol) for 1 hr at 00 and then analyzed for sterol binding components. Purified nuclear extracts (chromatin) were prepared from nuclear pellets by a modification (7) of the method of Haussler et al. (1) and were resuspended in 0.01 M Tris-HCl, ph 7.5, and centrifuged for 20. min at 48,000 X g. The pellet from 300 mg of tissue was reconstituted with cytosol (2.5 ml)

2 4872 Biochemistry: Brumbaugh et al. and incubated for 1 hr at 25 with sterol (in 40 Al of ethanol). Chromatin was harvested and extracted with 0.3 M KC1, 0.01 M Tris-HCl, ph 7.5, 1.5 mm EDTA, 12 mm 1- thioglycerol (0.3 M KCl-Buffer B). Extracts were centrifuged at 48,000 X g for 20 min, and the resulting supernatants were analyzed for sterol-binding activity. Sucrose Gradient Centrifugation. Linear gradients (5.0 ml) of 5-20% sucrose in 0.3 M KC-Buffer B were prepared with a Buchler gradient mixer, Auto-Densi Flow, and Polystaltic pump. Aliquots (0.3 ml) of cytosol or nuclear extracts were layered on gradients and centrifuged at 234,000 X g (average force) for 24 hr at 00 with the use of a Beckman L3-50 ultracentrifuge and an SW 50.1 rotor. The fractions (6 drops each) were counted in 5 ml of liquid scintillation mixture A (3% Liquifluor in toluene-triton X-114, 3:1) in a Beckman LS-233 scintillation counter (35% efficiency). Sedimentation coefficients were estimated by comparison with protein markers (chymotrypsinogen, 2.5 S; ovalbumin, 3.67 S; and bovine serum albumin, 4.4 S). Agarose Gel Filtration. All chromatographic procedures were carried out at 1-3'. Agarose beads (Bio-Gel A-0.5m, 100 to 200 mesh from Bio-Rad) were equilibrated with 0.3 M KC-Buffer B and poured into a column (1.6 X 60 cm). Samples (1.0 ml) of nuclear extracts or cytosol incubations were applied to the column and 1-ml fractions were collected and counted (30% efficiency). The optical density of fractions was measured with a Gilford 240 spectrophotometer. Column flow rates were maintained at ml/hr with a Polystaltic pump. Filter Assay for Specific Macromolecular Binding. Separation of bound from free sterol was achieved by the filter assay method of Santi et al. (22). Aliquots of cytosol (0.2 ml) containing la,25(oh)2[3h]d3 and samples containing the same concentration of 1a,25-(OH)2[3H]Ds plus a 100-fold excess of unlabeled hormone were incubated at 00 for 2 hr. Cytosol (150 Al) was then applied to DEAE-cellulose filters (Whatman DE 81) and washed with three 1-ml portions of 1% Triton X-100 in 0.01 M Tris-HC, ph 7.5, with the use of a Millipore sampling manifold. The amount of la,25- (OH)2[3H]D3 specifically bound by the cytosol was determined as previously described (17). Efficiency of the filtration procedure, as determined by measurement of binding at low hormone concentrations, was 75%. RESULTS nitially, parathyroid gland was studied to determine if it contained a soluble binding component for the la,25- (OH)2D3 hormone. Parathyroid cytosol from vitamin-d-deficient chicks was incubated at 0 with ia,25-(oh)2[3h]da and binding of the sterol to macromolecules was analyzed by sucrose gradient centrifugation. As depicted in Fig. 1A, the radioactive hormone interacted with a macromolecular species sedimenting at 3.1 S. Parallel cytosol incubations containing excess unlabeled 1a,25-(OH)2D3 showed no radioactive hormone binding in the sedimentation position of the 3.1S-binding component (Fig. 1A), demonstrating that the interaction of hormone with this cytosol molecule is saturable and of high affinity. Sucrose gradients of the la,25- (OH)2D3-binding component in parathyroid cytosol reproducibly exhibited a faster-sedimenting shoulder on the primary peak. When a 2-fold excess of nonradioactive 25- (OH)D3 was included in the incubation (Fig. 1A), the shoulder was abolished without any detectable effect on la,25- (OH)2[3H]Dj binding to the 3.1S macromolecule. Thus, this macromolecule selectively binds the hormone and much higher concentrations of 25-(OH)D3 are required to effec- Proc. Nat. Acad. Sci. USA 72 (1975) 20 TOP 10 FRACTON NUMBER FG. 1. Sucrose gradient centrifugation of la,25-(oh)2d3 and 25-(OH)D3-binding components in parathyroid glands and nontarget organs. ncubations were carried out as follows: (A) Parathyroid cytosol (0.3 ml) at 00 for 1 hr with 3 nm la,25-(oh)2[3h]d3 (6.5 Ci/mmol) alone (0), or with 0.3 gm unlabeled la,25-(oh)2d3 (0), or with 6 nm nonradioactive 25-(OH)D3 (A). (B) Reconstituted cytosol-chromatin from parathyroid (@), adrenal (A), or testes (A) was incubated with 6 nm la,25-(oh)2[3h]d3 for 1 hr at 250. The chromatin was then extracted with 0.3 M KCl-Buffer B. Parallel incubation with parathyroid cytosol-chromatin, 6 nm lz,25-(oh)2[3h1d3, and 0.6 'M unlabeled la,25-(oh)2d3 was performed (0). (C) Parathyroid cytosol with 6 nm 25-(OH)[3H]D3 (6.5 Ci/mmol) alone (0), or with 60 nm nonradioactive 25-(OH)D3 (O). or with 60 nm unlabeled la,25-(oh)2d3 (A). (D) Testes cytosol with 6 nm 25-(OH)[3H]D3 (0); testes cytosol with 6 nm la,25- (OH)2[3H]D3 alone (A) or with 0.6 MM unlabeled la,25-(oh)2d3 (A) or with 60 nm nonradioactive 25-(OH)D3 (0). Arrows indicate sedimentation positions of protein standards: 1, chymotrypsinogen; 2, ovalbumin; 3, bovine serum albumin. tively compete with 1a,25-(OH)2D3 (see Table 1). Since the shoulder disappeared in the presence of 25-(OH)D3 and corresponded to the sedimentation position (6S) of the high-af- Table 1. nfluence of unlabeled sterols on the binding of labeled hormone to cytoplasmic parathyroid la,25-(oh)2d3-binding component Unlabeled sterol (concentration, nm) % Binding None 100 1a,25-(OH)2D3 (400) 0 1,25-(OH)2D3 (4) (OH)D3 (80) 55 la-(oh)d3 (2,000) 95 Vitamin D3 (10,000) 98 Aliquots of cytosol were incubated with 4 nm la,25-(oh)2- [3H]D3 and unlabeled sterol for 1 hr at 00. Binding of radioactive sterol to the 3.1S component was determined via sucrose gradient centrifugation.

3 Biochemistry: Brumbaugh et al. 0 -J 0() 0 z 0 U- LL N ) Proc. Nat. Acad. Sci. USA 72 (1975) J -j z ) 0~ H ) z C) v4 FRACTON NUMBER FG. 2. Agarose gel filtration of cytoplasmic and nuclear la,25-(oh)2d3-binding components of chick parathyroid glands. Reconstituted cytosol-chromatin (1 ml) was incubated with la,25-(oh)2[3h]d3 (4 nm) for 1 hr at 250. The chromatin was then extracted with 0.3 M KC1- Buffer B (0-*). One ml of parathyroid cytosol (prepared in 0.3 M KCl-Buffer B) was incubated with la,25-(oh)2[3hjd3 (6 nm) for 1 hr at 00 (0-0). (-), relative absorbance at 280 nm of cytosol eluate. Molecular weights of the components were estimated from a linear plot of Mr112 versus the distribution coefficient, KD1 3, with the use of myoglobin, chymotrypsinogen, pepsin, ovalbumin, and bovine serum albumin as standards. Vo and Vt are the void and total volumes, respectively. finity binding protein for 25-(OH)D3 discovered by Haddad and Birge in the rat (23), it was concluded that this shoulder represented association of the hormone with this 6S component. To further investigate the interrelationship of the 3.1S binding component and the 6S species which binds both 1a,25-(OH)2D3 and 25-(OH)D3, parathyroid cytosol was incubated with radioactive 25-(OH)D3. Sucrose gradient analysis (Fig. C) indicates that 25-(OH)[3H]D3 binds exclusively to a protein sedimenting at 6 S. Parallel incubations containing a 10-fold excess of either nonradioactive 25-(OH)D3 or la,25-(oh)2d3 show that this protein has a higher affinity for 25-(OH)D3 than for the hormone; binding to the 6S peak was reduced by 15% in the presence of 1a,25-(OH)2D3 and by about 90% in the presence of 25-(OH)D3 (Fig. C). The 25-(OH)D3-binding component in parathyroid cytosol is therefore similar to proteins found in a variety of rat tissues by Haddad and Birge (23) and to proteins reported in chick intestine, liver, and kidney by Brumbaugh and Haussler (24). n all cases, the binding component has a greater affinity for 25-(OH)D3 than 1a,25-(OH)2D3 and sediments at 6 S in sucrose gradients. Analysis of la,25-(oh)2[3h]da interactions with other tissue cytosols showed that the 3. S hormone-binding component exists only in parathyroid gland and is not present in testes (Fig. D), adrenal gland, liver, or kidney (data not shown). Detailed investigation of testes cytosol (Fig. D) revealed that the la,25-(oh)2d3 hormone associates predominantly with the 25-(OH)Ds-binding protein (sedimenting at 6 S). However, the preference of this protein for 25-(OH)D3 is demonstrated by the greater potency of unlabeled 25- (OH)D3 over la,25-(oh)2d3 in abolishing this 6S peak (Fig. id). A small amount of 1a,25-(OH)2[3H]D3 binds to molecules in testes cytosol sedimenting at 3-4 S, but this binding is low affinity and nonspecific because it is not reduced by excess unlabeled a,25-(oh)2d3. Therefore, with the exception of the target intestine, where a selective 3.7S hormone receptor is found (18), cytosol components specific for the la,25-(oh)2d3 hormone over its 25-(OH)D3 precursor are detected only in parathyroid glands. The association of la,25-(oh)2d3 with nuclear-binding components from parathyroid gland was also observed in vitro. la,25-(oh)2[3h]d3 was incubated with reconstituted cytosol-chromatin at 250 for 1 hr and the chromatin was then harvested and extracted with 0.3 M KC-Buffer B. Sucrose gradient analysis of this nuclear extract resulted in a 3.1S peak of bound 1a,25-(OH)2[3H]Da (Fig. 1B). Parallel incubations containing a 100-fold excess of unlabeled hormone showed a striking reduction in the radioactive sterol bound to this macromolecule. Also, the association of la,25- (OH)2[3H]D3 with a nuclear component was not observed in the adrenals or testes (Fig. 1B). Therefore, the parathyroid nuclear chromatin contains a tissue-specific, high-affinity binding component for 1a,25-(OH)2D3 which is indistinguishable from the cytosol macromolecular species by sucrose gradient centrifugation. The cytoplasmic and nuclear la,25-(oh)2d3-binding components were also isolated and compared by agarose gel filtration chromatography in 0.3 M KC-Buffer B (Fig. 2). The major peak represents [3H]sterol binding to a macromolecule of apparent molecular weight 58,000, which is resolved from the major protein peak (eluting in the void volume). No significant or reproducible difference could be demonstrated between the nuclear and cytoplasmic components when columns were run under identical, standardized conditions (Fig. 2) and both peaks of macromolecule- [3H]hormone complexes were abolished when incubations containing excess unlabeled la,25-(oh)2d3 were chromatographed (data not shown). Thus, the specific nuclear and cytoplasmic 1a,25-(OH)2D3-binding components could not be distinguished from each other by the ultracentrifugal and chromatographic techniques employed. These data are consistent with the concept that the nuclear component originates in the cytosol and suggest that 1a,25-(OH)2D3 may be functioning in the parathyroid gland at the level of the cell nucleus. Next the binding affinity and specificity of the cytoplasmic-binding components for 1a,25-(OH)2D3 in parathyroid glands were studied. ncubation of parathyroid cytosol with increasing concentrations of 1a,25-(OH)2[3H]D3 and determination of specific binding by the filter method of Santi et al. (22) showed the saturation of a limited number of binding sites (Fig. 3A). Saturation occurs at low concentrations of hormone (3 X 10-9 M). At this range of hormone concentration most of the binding detected by the filter assay is specific for la,25-(oh)2d3, since only a small

4 4874 Biochemistry: Brumbaugh et al. Proc. Nat. Acad. Sci. USA 72 (1975) FG. 3. Determination of dissociation constant for la,25-(oh)2d3-parathyroid cytosol macromolecule interaction. (A) Specific binding. of la,25-(oh)2[3h]d3 by parathyroid cytosol. Aliquots of cytosol (0.2 ml, 2.0 mg of protein per ml) were incubated with increasing amounts of la,25-(oh)2[3h]d3 in the presence (nonspecific) or absence (total) of a 100-fold excess of unlabeled la,25-(oh)2d3 for 2 hr at 00. (B) Scatchard analysis of specific binding in A. Three determinations indicate that the 1a,25-(OH)2[3H]D3 macromolecule interaction has a Kd = X M at 00, where the uncertainty is expressed as standard deviation. amount of labeled hormone is bound in the presence of a 100-fold excess of unlabeled hormone (nonspecific binding). Scatchard analysis of the specific binding (total minus nonspecific) is linear (Fig. 3B), suggesting a single class of binding sites. The dissociation constant for the hormone-macromolecule complex at 00 is 3.2 X M. Analogs of la,25-(oh)-d3 which lack a hydroxyl group at the la- and/or 25-position were tested for their ability to compete with the labeled hormone for binding to the 3.1S cytoplasmic component. As is shown in Table 1, the la,25- (OH)2D3-binding macromolecule is highly specific. The approximate relative affinity of these sterols for the 3.1S macromolecule is 1:1/20:<1/500:<1/2500 for la,25-(oh)2d3: 25-(OH)Ds:la-(OH)D3:vitamin D3. These values differ from the 1:1/500:1/800:<1/20,000 relative affinities for these sterols' association with the 3.7S receptor of intestine (25). The present results indicate that the parathyroid gland receptor for la,25-(oh)2d3 differs slightly from the wellcharacterized hormone receptor in the intestine in terms of sedimentation coefficient, affinity, and specificity. Yet it is clear that the parathyroid gland should be included with the intestine as a location of specific, high-affinity binding components for the la,25-(oh)2d3 hormone. DSCUSSON The existence of a specific la,25-(oh)2d3-binding macromolecule has been demonstrated in chick parathyroid glands. Although the function of this binding component is not known, it has been definitively distinguished from the 25-(OH)D3-binding protein which has been found in the rat and chick (23, 24). Table 2 summarizes data on the properties of chick vitamin D metabolite-binding proteins in terms of their sedimentation coefficients and subcellular location. A 6S 25-(OH)Ds-binding component has been identified in the cytosol of all organs examined in the chick, but this protein is not found in the nucleus. At present, a role for this macromolecule has not been found, but the fact that it does not transport 25-(OH)D3 into the nucleus suggests that it is not a classic steroid hormone receptor (27, 28). The tissue 25-(OH)Ds-binding protein can also be differentiated from the serum protein which binds either 25-(OH)D3 or la,25- (OH)2DW and sediments at 4.0 S in sucrose gradients (Table 2). We have also observed that although the ubiquitous 6S protein has a higher affinity for 25-(OH)D3 than the hormone, it will also bind la,25-(oh)2[3h]ds when incubated with the sterol at M in vitro (Fig. A and D, Table 2). However, in the target intestine and in the parathyroid gland, unique macromolecules sedimenting at 3.7 S and 3.1 S, respectively, are observed to selectively bind la,25- (OH)2D3 and transfer the hormone into the cell nucleus. These binding components are analogous to the classic steroid hormone receptors and, based upon the pattern of binding proteins established in Table 2, the parathyroid gland Table 2. Sedimentation coefficients of high-affinity binding proteins for 25-(OH)D3 and 1a,25-(OH)2D3 in the chick* Sedimentation coefficient, S (±SD) Sterol Site Cytosol Nuclear Refs. 25-(OH)D3 ntestine 6 t 24 Parathyroid 6 t Testes 6 t Liver 6 t 24 Kidney 6 t 24 Serum ± 0.1 lc,25-(oh)2d3 ntestine , 18 ± 0.1 ±0.1 Parathyroid ± Testes 6 t t Liver 6 t 26 Kidney 6 t 26 Serum ± 0.1 * Data from 0.3 M KCl-sucrose gradients. t Not observed. t Present study. Represents binding of la,25-(oh)2[3h]d3 to 25-(OH)D3 binding protein. Average of four sucrose gradient experiments; significantly different from 3.7S intestinal receptor (P < 0.005).

5 25-OH-D3 --- Biochemistry: LOW CALCUM PTH R ANDSE(-C PARATHYROD CELL NUCLEUS Xl2 0] ( )-1,25 ALCUM RETENTON \ PTH 1 HOSPHATE EXCRETON RENAL lc-ohase Brumbaugh et al.,25-(oh (CALC ( 3 APHOSF) JM/ PHATE - tmuu MDib Ll L ZATON HS - - LOW PHOSPHATE FG. 4. ntegration of possible 1a,25-(OH)2D3 action at parathyroid gland with the homeostatic regulation of serum calcium and phosphate by the concerted functioning of PTH and la,25- (OH)2D3. should perhaps be classified along with the intestine and bone as a target site for 1a,25-(OH)2D3. Little information was available until recently on the possible function of la,25-(oh)2d3 at the parathyroid gland. Oldham et al. (29) have detected and characterized a calcium-binding protein in parathyroid gland which is similar to the intestinal calcium-binding protein induced by la,25- (OH)2D3 (30, 31). t is possible that the sensing of serum calcium by the parathyroids is dependent upon 1a,25-(OH)2D3 and the synthesis of a calcium-binding protein in this gland. Also, Chertow et al. (32) have reported that la,25-(oh)2d3 suppresses PTH secretion in the rat, in vvo, and in isolated slices of bovine parathyroid gland. These observations suggest that 1a,25-(OH)2D3, like other steroid hormones (33), feedback inhibits its trophic counterpart, PTH. A model depicting the way in which this feedback loop may integrate into the complex scheme for the regulation of calcium and phosphate is illustrated in Fig. 4. The two primary signals in this endocrine system are postulated to be low circulating calcium and low phosphate (7, 10). Low calcium enhances the formation of 1a,25-(OH)2D3 via a stimulation of parathyroid hormone secretion (7, 8). The calcium mobilized from bone by the synergistic action of PTH and 1a,25-(OH)2D3 and that absorbed from intestine under the influence of enhanced sterol levels probably closes the hormone loop by abolishing further PTH secretion (34). Direct feedback of 1a,25-(OH)2D3 at the parathyroid gland may also be involved in the regulation of PTH secretion during the correction of low serum calcium. The significance of the operation of 1a,25-(OH)2D3 at the parathyroid gland may be more profound in terms of phosphate homeostasis. During phosphate depletion the kidney is stimulated to form more 1a,25-(OH)2D3 (Fig. 4; refs. 6 and 7). To maintain the phosphate mobilized by the sterol, PTH secretion must be curtailed (because PTH causes net phosphate excretion). Such curtailment can ultimately occur after hypercalcemia is established by the action of la,25- (OH)2D3, but a more plausible mechanism would be the direct inhibition of PTH secretion by 1a,25-(OH)2D3 (Fig. 4). The control of calcium and phosphate is apparently accomplished by a complex and delicate interrelationship between PTH and 1a,25-(OH)2D3 and a comprehension of this exact Proc. Nat. Acad. Scd. USA 72 (1975) 4875 relationship should further our understanding of both the normal physiology of these ions and of diseases of mineral metabolism such as primary hyperparathyroidism, idiopathic hypercalciuria, and vitamin D-resistant rickets. We gratefully acknowledge the expert technical assistance of Ms. Kristine Bursac and Ms. Patricia G. Jones. The authors wish to thank the National nstitutes of Health, Grant AM and Training Grant GM-01982, for support of this research. 1. Haussler, M. R., Myrtle, J. F. & Norman, A. W. (1968) J. Biol. Chem. 243, Fraser, D. R. & Kodicek, E. 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