Cytoplasmic and nuclear binding components for la , 25-dihydroxyvitamin D 3 in chick parathyroid glands ( vitamin D / receptors / parathyroid hormone )

Content Provider	Semantic Scholar
Author	Brumbaugh, P. F. Hughes, Mark R. Haussler, Mark R.
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 10-10 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 KCI-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 KCI-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). Its 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, MacIntyre 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 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. In 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. MATERIALS AND METHODSMaterials. 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, In 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%. 25Hydroxy[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, In Vitro. Homogenates [300 mg wet weight (20 parathyroid glands)/3 ml] were made in 0.25 M sucrose, 0.05 M Tris-HCI, pH 7.4, 0.025 M KCI, 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 (0.2-1.0 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) Abbreviations: 25-(OH)D3, 25-hydroxyvitamin D3; la-(OH)D3, la-hydroxyvitamin D3; la,25-(OH)2D3, la,25-dihydroxyvitamin D3; PTH, parathyroid hormone.
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