Marian R. Walters

1,25-Dihydroxyvitamin D3 receptors identified in the rat heart

Specific receptors for 1,25-dihydroxyvitamin D3, the active hormonal form of vitamin D3, were demonstrated in low salt chromatin preparations from normal rat hearts. Sucrose gradient analysis of KCl-extracted chromatin yielded a significant (P less than 0.005) peak of specific [3H]1,25-dihydroxyvitamin D3 binding in the 3.6S region. The peak of [3H]1,25-dihydroxyvitamin D3 binding was abolished by excess 1,25-dihydroxyvitamin D3, but not by 50 nM 25-hydroxyvitamin D3 nor by 1.0 microM levels of estradiol-17B, cortisol, or promegestone, demonstrating steroid specificity characteristic for such receptors. Upon Scatchard analysis this putative cardiac 1,25-dihydroxyvitamin D3 receptor yielded a single binding component with high affinity (KD = 0.36 nM) and low capacity (Nmax = 33 fmol/g tissue). Coupled with evidence for the presence of calcium binding proteins in this tissue, these observations suggest functional roles for 1,25-dihydroxyvitamin D3 and its receptors in cardiac muscle, possibly in regulating intracellular effects of calcium.

An estrogen-stimulated 1,25-dihydroxyvitamin D3 receptor in rat uterus

Abstract Sucrose density gradient analysis was utilized to determine whether 1,25-dihydroxyvitamin D3 receptors are present in the rat uterus. A distinct 3.6S [3H]1,25-dihydroxyvitamin D3 binding component was observed in chromatin extracts of estrogen-primed, ovariectomized rat uteri. Binding to this putative 1,25-dihydroxyvitamin D3 receptor was inhibited by excess 1,25-dihydroxyvitamin D3, but not by 25-hydroxyvitamin D3, estradiol-17β, promegestone, or cortisol. Low levels of the receptor seemed to be present in the unprimed uterus. Estrogen injection significantly increased the number of 1,25-dihydroxyvitamin D3 receptors and progesterone co-administration reduced, but did not abolish, this effect.

Possible significance of new target tissues for 1,25-dihydroxyvitamin D3

A sensitive sucrose gradient procedure provided evidence for specific 3.6S 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] receptor-like binding components with low affinity for 25(OH)D3 in several reproductive target tissues in the rat, including testis, uterus and probably epididymis. Thus, the presence of 1,25(OH)2D3 receptors in non-vitamin D endocrine tissues is not restricted to the hormone secreting glands. Estrogen-stimulated uterine growth paralleled increased levels of the putative 1,25(OH)2D3 receptors, providing in vivo evidence for the concept of a relationship between these phenomena. However, a similar correlation was not observed in the growing testis (prepubertal vs mature rats). Whether this result stems from receptor localization in a cell type prevalent in the adult or a true dissociation between testis growth and receptor levels is unclear. Finally, significant levels (10.3 +/- 1.0% vs intestinal mucosa) of the putative 1,25(OH)2D3 receptors were found in the heart, an organ in which calcium mediates many specialized functions. Taken collectively, these observations on new target tissues of quite different overall function suggest that 1,25(OH)2D3 and its receptors may play a role in intracellular calcium homeostasis and possibly in regulating specialized intracellular functions of calcium.

Multiple sites of action of the vitamin D endocrine system: FSH stimulation of testis 1,25-dihydroxyvitamin D3 receptors.

1,25-Dihydroxyvitamin D [1,25(OH)2D] receptors exist in numerous unexpected tissues. These include, for example, rat lung, heart, testis, and uterus, but not prostate and bladder. The issues of 1,25(OH)2D effects on and receptor location in the testis were addressed by (a) physiological and pharmacological manipulations of tubule cell types and (b) histological examination of testes of vitamin D-deficient rats. FSH treatment in hypophysectomized adult rats increased 1,25(OH)2D receptor levels by 135% (P less than 0.01). Busulfan treatment reduced testis receptor levels by 35% (P less than 0.05) after 35 days (maximum effect), and the effect was reversed after recovery (85 d). Cryptorchidism for 5 or 50 days resulted in modest (33%, P less than 0.05) or substantial (79%, P less than 0.001) reductions in receptor levels. Only the FSH treatment and 50 days cryptorchidism reduced receptor levels in the residual tissue. The testes of vit. D-deficient rats showed incomplete spermatogenesis and degenerative changes. Although interpretation is complicated by the intricate communication among testis cell types, these data suggest that the Sertoli cell is a primary site of action of 1,25(OH)2D in the testis. Moreover, these data indicate that 1,25(OH)2D receptor function in the testis relates to germ cell division/maturation, although this may be an indirect effect via the Sertoli cells.

Glucocorticoid Receptors: Documentation in the Rat Uterus

The question of the presence of specific glucocorticoid receptors in the rat uterus was reassessed. A high speed supernatant obtained from uteri of ovariectomized or intact 45-day old rats was incubated 18 h at 4 c in the presence of [3H]triamcinolone acetonide ([3H]TA) with or without excess dexamethasone, followed by a 10 min exposure to dextran-coated charcoal. A specific uterine glucocorticoid receptor was detected with a Kd=5.1 nM and the number of binding sites equal to 1.1 pmol/uterus. [3H]dexamethasone was observed to underestimate the number of specific receptor sites, probably due to the instability of the dexamethasone-receptor complex during treatment with charcoal. In addition, unlabeled TA was unsatisfactory for determining nonspecific binding because of the cross-reactivity with the progesterone receptor also present in uterine cytosol. The uterine glucocorticoid receptor exhibited the appropriate steroid specificity. Sucrose density gradient analysis of uterine cytosol revealed peaks of dexamethasone-competable [3H]TA binding at 7.2S and 5.75 in low salt and at 4S in 0.4M KCL. Similar components were observed in cytosol and nuclear fractions, respectively, 20 min after in vivo injection of 25 mu Cl [3H]TA. The physical characteristics of the specific glucocorticoid receptor were consistent with those determined for other receptor proteins. The documentation of the presence of glucocorticoid receptors in the rat uterus is important physiologically because of numerous reports of glucocorticoid effects on this reproductive tissue. If receptors could not be demonstrated, extensive studies of the mechanism of these effects would be necessary for a full understanding of uterine endocrinology.

What is vitamin D deficiency

Vitamin D derived from the diet or produced in the skin is converted to its active form by two sequential hydroxylation reactions (1, 2). The first occurs in the liver, forming 25-hydroxyvitamin D3 (25-(OH)D3), and the second occurs in the kidney, forming the active hormonal form 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). The activity of the renal 25-(OH)D3-1 α-hydroxylase enzyme (1-hydroxylase), which is tightly regulated according to the calcium and phosphorus requirements of the body, is stimulated predominantly by parathyroid hormone and low plasma calcium and phosphate concentrations. 1,25-Dihydroxyvitamin D3 acts to increase intestinal active calcium transport and, in concert with parathyroid hormone, to stimulate renal tubular calcium reabsorption and bone mineral mobilization (1, 2). Vitamin D restriction by dietary deficiency and/or by reduced exposure to sunlight can lead to the development of vitamin D-deficiency rickets (2, 3). The best recognized definition of vitamin D-deficiency, i.e., bony rickets, has its origin in the evolving understanding of this bone disease and the fact that vitamin D deficiency is the most frequent cause (Table I). In more recent times, many investigators have considered animals to be vitamin D deficient if they exhibit rickets, reduced intestinal calcium transport, hypocalcemia, or “undetectable” levels of 25-(OH)D3 (Table II; 7–27). However, in a series of studies (28–30) directed toward establishing a normocalcemic rat model exhibiting vitamin D-deficiency, we realized that understanding or defining vitamin D deficiency is extremely complex. For example, most workers would probably agree that vitamin D deficiency should entail 1,25-(OH)2D3 deficiency. However, as described below, absence of 1,25-(OH)2D3 may not always be associated with absence of vitamin D or 25-(OH)D3. Conversely, physiological levels of 1,25-(OH)2D3 can exist in the presence of extremely low levels of vitamin D or 25-(OH)D3.

1,25-Dihydroxyvitamin D3 receptors: intermediates between triiodothyronine and steroid hormone receptors

Abstract Comparison of the biochemical properties of the 1,25-dihydroxyvitamin D 3 receptor with receptors for classical steroid hormones and for triiodothyronine suggests that these diverse biological regulators are more closely linked than previously thought. A general model is presented to describe the subcellular location of unoccupied and occupied receptors for both the steroid and triiodothyronine hormone.

Homologous Up‐Regulation of Vitamin D Receptors Is Tissue Specific in the Rat

1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) receptors (VDR) are expressed in multiple tissues within the body. VDR levels are increased by 1,25(OH)2D3 in intestine and kidney and in numerous cell models. The ability of 1,25(OH)2D3 to affect VDR levels in other target tissues in vivo was studied by assessing VDR levels by the3H‐1,25(OH)2D3 binding assay under varied physiological conditions in the rat. When compared with vitamin D–deficient (−D) controls, rats raised on a normal vitamin D–sufficient (+D) diet showed elevated VDR levels in kidney (391 ± 53 vs. 913 ± 76 fmol/g of tissue; p < 0.05), but not in testis, heart, or lung. Up‐regulation of the VDR also occurred in kidney of +D rats 1 day after a single 100‐ng dose of 1,25(OH)2D3 (454 ± 43 vs. 746 ± 113 fmol/mg of DNA; p < 0.05), but no changes were seen in intestine, testis, or lung. Because 1,25(OH)2D3‐induced hypercalcemia may independently affect VDR regulation, 1,25(OH)2D3 was infused into −D rats, and normocalcemia was maintained by reduced dietary calcium intake. In this model, the renal VDR was again up‐regulated (446 ± 115 vs. 778 ± 58 fmol/mg of DNA; p < 0.05), but VDR levels in testis and lung were unaffected. Scatchard analysis and tests of 1,25(OH)2D3 dose (1–100 ng/day for 7 days) and temporal (100 ng/day for 1–7 days) responsiveness further supported the tissue‐specific nature of the homologous VDR regulation. Assay of VDR levels by l‐1‐tosylamido‐2‐phenylethyl chloromethyl ketone–3H‐1,25(OH)2D3 exchange assay ruled out differences in endogenous 1,25(OH)2D3 occupancy as the basis for the observed differences in VDR regulation. Finally, coidentity of the VDR‐like sites in kidney versus testis was confirmed by competitive binding analysis comparing their relative affinities for 25(OH)D3 versus 1,25(OH)2D3 (30.5 ± 6.4 vs. 35.6 ± 3.6 in kidney and testis, respectively) and by immunoblot analysis using a highly specific monoclonal anti‐rat VDR antibody. Thus, under a wide variety of experimental conditions, homologous up‐regulation of the VDR occurs in the rat kidney in vivo, but not in several other target tissues which do not regulate plasma calcium homeostasis. Moreover, this differential VDR regulation did not result from secondary changes in plasma calcium, from differential 1,25(OH)2D3 responsiveness in the various tissues, nor from differences in endogenous 1,25(OH)2D3 occupancy of the VDR. These studies thus establish that, in contrast to observations in vitro, the widely described phenomenon of homologous VDR up‐regulation in kidney and intestine is not a universal property of 1,25(OH)2D3 target tissues in vivo in the rat.

Specific 1,25-dihydroxyvitamin D3 binding sites in choroid plexus

Quantitative autoradiographic analysis of [3H] 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) binding in vitamin D deficient mice provided evidence for high levels of specific binding in choroid plexus and, to a lesser extent, ventral hippocampus. Sucrose gradient analysis yielded a 3-4S peak of specific [3H]1,25(OH)2D3 binding in bovine choroid plexus, but not amygdala or hippocampus. Scatchard analysis of [3H]1,25(OH)2D3 binding in bovine choroid plexus yielded KD = 0.23 +/- 0.06 nM and Nmax = 43.5 +/- 0 fmol/g tissue (n = 5). This result indicates the presence of significant receptor-like [3H]1,25(OH)2D3 binding sites in the choroid plexus and, thus, suggests roles for this hormone in regulating the entry of calcium into the brain and/or in the central regulation of calcium homeostasis.

Evidence for two classes of 1,25-dihydroxyvitamin D3 binding sites in classical vs. nonclassical target tissues

Possible differences in 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] binding sites in classical and nonclassical target tissues were tested by Scatchard analysis of [3H]1,25(OH)2D3 binding in parallel chromatin preparations of rat kidney vs. testis. Two distinct binding components were resolved in kidney (p less than 0.005). Moreover, the single binding site in testis exhibited a 10-fold lower Kd (p less than 0.05) than did the principal binding site in kidney (50 +/- 4 vs. 405 +/- 142 pM). Secondly, regulation of [3H]1,25(OH)2D3 binding sites also differed. 1,25(OH)2D3 injection resulted in increased 1,25(OH)2D3 binding (p less than 0.05) in kidney (92%) and intestine (415%), but not in testis, lung or heart. These results suggest that the principal 1,25(OH)2D3 binding sites in classical targets kidney and intestine may be intrinsically different from those in at least some nonclassical targets.