Renal 25-hydroxyvitamin D-3 1α-hydroxylase in guinea-pig: Activity variations during development and pregnancy

The renal 25-hydroxyvitamin D-3-1α-hydroxylase (1α-hydroxylase) activity and circulating levels of 1,25-dihydroxyvitamin D (1,25(OH)₂D) were measured in pregnant guinea-pigs and their offspring. Serum levels of 1,25(OH)₂D were significantly elevated in pregnant guinea-pigs but the renal enzyme activity was not different from non-pregnant animals. The fetal renal 1α-hydroxylase activity was about 6-fold higher than the maternal level, whereas circulating 1,25(OH)₂D was low. Treatment with pharmacological doses of 1,25(OH)₂D3 increased circulating 1,25(OH)₂D and depressed the renal 1α-hydroxylases both in the mother and the fetus. In newborn guinea-pigs the enzyme activity was up to 10-times that seen in adults. It declined over the first 3 weeks, showing no difference between the sexes. In sexually mature animals the males had a significantly higher 1α-hydroxylase activity than the female. However, this higher enzyme activity was not correlated to serum testosterone. Around the time the animals reached sexual maturity serum 1,25(OH)₂D increased in both sexes. In the males this rise was correlated to an increase in serum testosterone. It is concluded that the maternal renal 1α-hydroxylase activity is unchanged in late pregnancy, compared to non-pregnant females. The data indicate that the fetus produces 1,25(OH)₂D, and may contribute to the maternal circulating 1,25(OH)₂D. The sex difference in 1α-hydroxylase activity previously demonstrated is manifest at about the time of puberty.     

Role of pituitary hormones in regulating renal vitamin D metabolism in man.

Studies in animals and tissue culture have shown the importance of prolactin and growth hormone in regulating renal 1 alpha-hydroxylase activity and plasma concentrations of 1,25-dihydroxycholecalciferol (1,25(OH)2D3). Evidence for a similar role for these hormones in man was sought by using a radioreceptor assay to measure plasma 1,25(OH)2D3 concentrations in 20 normal subjects, 12 patients receiving dialysis, 11 patients with primary hyperparathyroidism, 10 pregnant women, seven women with prolactinoma, and 14 patients with acromegaly. Circulating 1,25(OH)2D3 concentrations were appreciably raised in the patients with primary hyperparathyroidism and the pregnant women (P less than 0.001), slightly but significantly increased in the patients with prolactinoma (P less than 0.05), and greatly raised in those with acromegaly (P less than 0.001). These results suggest that prolactin and growth hormone are important regulators of renal vitamin D metabolism in the physiological conditions of pregnancy, lactation, and growth in man.     

Effects of 1,25-dihydroxycholecalciferol administration on the rat renal vitamin K-dependent carboxylating system

We have shown previously that the in vitro activity of the renal vitamin K-dependent gamma-glutamyl carboxylase toward synthetic oligopeptide substrates is stimulated by administration of either parathyroid hormone (PTH) or 1,25-dihydroxycholecalciferol [1,25(OH)2D3] to rats [(1983) J. Biol. Chem. 258, 12783-12786]. Here we report that administration of 1,25(OH)2D3 to rats increases their levels of endogenous carboxylase substrate as well. Rats fed a vitamin D-deficient diet had highly elevated serum PTH levels while vitamin D-replete animals had undetectable levels. Furthermore, since PTH increases 1,25(OH)2D3 levels by stimulating renal 25-hydroxyvitamin D-1 alpha-hydroxylase, it is very likely that the stimulatory effects of PTH on the renal vitamin K-dependent carboxylating system are mediated by 1,25(OH)2D3.     

Plasma vitamin D metabolite levels in pregnant and nonpregnant ewes

1. Maternal calcium homeostasis during pregnancy is strained due to fetal mineral requirements for bone formation. 2. In most species, the mother adjusts to the mineral requirements of the fetus with alterations in her metabolism of vitamin D that include a decrease in plasma 25-(OH)D levels and an increase in circulating levels of the hormone, 1,25-(OH)2D. 3. Plasma 25-(OH)D and 1,25-(OH)2D levels in adult male, adult female and pregnant sheep were measured by specific radioreceptor binding assays. 4. Pregnancy did not alter circulating levels of 25-(OH)D or 1,25-(OH)2D in the sheep. 5. The pregnant ewe differs from all species studied to date in that maternal plasma 1,25-(OH)2D levels do not rise as a result of pregnancy.     

Hypercalcemia produced by parathyroid hormone suppresses experimental autoimmune encephalomyelitis in female but not male mice

Besides its role in regulating serum levels of calcium and phosphorus, 1alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) has potent effects on the immune system and suppresses disease in several animal models of autoimmune disorders including experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. While the amount of 1,25-(OH)2D3 needed to prevent EAE is dependent on the gender of the mouse and amount of calcium available in the diet, the minimum levels of 1,25-(OH)2D3 sufficient to prevent disease cause hypercalcemia. To test if hypercalcemia independent of high levels of 1,25-(OH)2D3 can suppress EAE, we used a 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-hydroxylase) knockout mouse strain. Because these 1alpha-hydroxylase knockout mice lack the parathyroid hormone (PTH)-regulated enzyme that synthesizes 1,25-(OH)2D3, hypercalcemia from increased bone turnover was created by continuous administration of PTH without changing the circulating levels of 1,25-(OH)2D3. This PTH-mediated hypercalcemia generated after EAE induction prevented disease in female mice but not male mice. When hypercalcemia was prevented by diet manipulation, PTH administration no longer prevented EAE. We conclude that hypercalcemia is able to prevent EAE after disease induction in female mice.     

The mechanism of 1,25-dihydroxyvitamin D(3) autoregulation in keratinocytes

The synthesis of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) from its precursor, 25-dihydroxyvitamin D(3) (25(OH)D(3)), is catalyzed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase). It has been generally assumed that 1,25(OH)(2)D(3) inhibits the activity of this enzyme by regulating its expression at the genomic level. We confirmed that 1,25(OH)(2)D(3) reduced the apparent conversion of 25(OH)D(3) to 1,25(OH)(2)D(3) while stimulating the conversion of 1,25(OH)(2)D(3) and 25(OH)D(3) to 1,24,25(OH)(3)D(3) and 24,25(OH)(2)D(3), respectively. However, 1,25(OH)(2)D(3) failed to reduce the abundance of its mRNA or its encoded protein in human keratinocytes. Instead, when catabolism of 1,25(OH)(2)D(3) was blocked with a specific inhibitor of the 25-hydroxyvitamin D(3)-24-hydroxylase (24-hydroxylase) all apparent inhibition of 1alpha-hydroxylase activity by 1,25(OH)(2)D(3) was reversed. Thus, the apparent reduction in 1alpha-hydroxylase activity induced by 1,25(OH)(2)D(3) is due to increased catabolism of both substrate and product by the 24-hydroxylase. We believe this to be a unique mechanism for autoregulation of steroid hormone synthesis.     

A role for the cytoskeleton in renal vitamin D metabolism

Conversion of circulating 25-hydroxyvitamin D3 (25(OH)D3) to its active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) occurs in the renal tubule mitochondrion. Recent reports have implicated the cytoskeleton in certain other steroid metabolizing cells as a mediator of a rate-limiting mitochondrial transport step. Whilst the activity of the renal converting enzyme, a typical steroid hydroxylase, is known to be regulated closely by a number of well studied factors, no information is available to indicate whether an analogous transport step is relevant to the regulation of vitamin D metabolism. Cytochalasin B and vinblastine were used as chemical antagonists of the microfilamentous and microtubular elements of the cytoskeleton. Both agents inhibited the conversion of 25(OH)D3 to 1,25(OH)2D3 by isolated vitamin D-deficient chick renal tubules in a dose-dependent manner. At the concentrations required to inhibit 25(OH)D3-1 alpha-hydroxylase activity in whole cells, these agents inhibited neither isolated mitochondrial 1,25(OH)2D3 production, nor 24,25(OH)2D3 synthesis by vitamin D-replete tubules. The cytoskeletal antagonists were found to increase the content of labelled 1,25(OH)2D3 and 25(OH)D3 in a mitochondrial fraction prepared by Percoll fractionation of tubule cells pre-exposed to the antagonists and labelled 25(OH)D3 substrate. The data suggest that disruption of the cytoskeleton may result in inhibition of transport of newly synthesised 1,25(OH)2D3 out of the mitochondrion and through the cell, and accumulating 1,25(OH)2D3 may oppose its further synthesis. This is consistent with a transport process mediated by the cytoskeleton being involved in the regulation of renal vitamin D metabolism.     

Altered vitamin D metabolism in the kidney of the spontaneously hypertensive rat.

A decrease in plasma Ca2+ and increases in plasma immunoreactive parathyroid hormone (PTH) have been reported in spontaneously hypertensive (SH) rats as compared with normotensive Wistar-Kyoto (WKy) rats. These changes should lead to a higher plasma 1,25(OH)2D (1,25-dihydroxycholecalciferol/1,25-dihydroxyergocalciferol) concentration in SH rat if the kidney responds appropriately. Plasma 1,25(OH)2D, however, has been reported to be normal in SH rats, suggesting possible impairments of vitamin D metabolism in this animal model of hypertension. To test this possibility, we studied the effect of PTH on renal production of 1,25(OH)2D in SH rats before (4 weeks of age) and after (12 weeks of age) the onset of hypertension. Basal serum levels of 1,25(OH)2D were normal in SH rats at both ages. At 4 weeks of age, the rise in serum 1,25(OH)2D after PTH injection (50 units subcutaneously every 2 h; four times) was also normal in SH rats. By contrast, at 12 weeks of age, the rise in serum 1,25(OH)2D was approximately one-half of that in WKy rats, despite the similar rises in serum Ca2+ levels in both groups by PTH injection. The attenuated rise in serum 1,25(OH)2D in SH rats was consistent with the impaired response of renal 1-hydroxylase (25-hydroxycholecalciferol 1 alpha-hydroxylase) activity to PTH. Basal 1,25(OH)2D production by the kidney in SH rat was higher than that in WKy rats both at 4 and 12 weeks of age. These data suggest that, in SH rats: serum 1,25(OH)2D is inappropriately low in relation to the elevated PTH and this may be due, at least in part, to the impaired responsiveness to PTH of renal 1-hydroxylase and to the enhanced metabolism of 1,25(OH)2D, and elevated PTH or other agents may stimulate the 1-hydroxylase in the kidney even before the onset of hypertension.     

Effect of 24,25-dihydroxyvitamin D3 on 1,25-dihydroxyvitamin D3 metabolism in calcium-deficient rats.

The effect of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] metabolism was examined in rats fed on a low-calcium diet. These rats exhibit hypocalcaemia, high urinary cyclic AMP excretion, a markedly elevated serum 1,25(OH)2D concentration and low serum concentrations of both 24,25(OH)2D and 25(OH)D. When the rats are treated orally with 1, 5 or 10 micrograms of 24,25(OH)2D3/100 g every day, there is a dramatic decrease in serum 1,25(OH)2D concentration in a dose-dependent manner concomitant with an increase in serum 24,25(OH)2D concentration. Serum calcium concentration and urinary cyclic AMP excretion are not significantly affected by the 24,25(OH)2D3 treatment, which suggests that parathyroid function is not affected by the 24,25(OH)2D3 treatment. The 25(OH)D3 1 alpha-hydroxylase activity measured in kidney homogenates is markedly elevated in rats on a low-calcium diet but is not affected by any doses of 24,25(OH)2D3. In contrast, recovery of intravenously injected [3H]1,25(OH)2D3 in the serum is decreased in 24,25(OH)2D3-treated rats. Furthermore, when [3H]1,25(OH)2D3 is incubated in vitro with kidney or intestinal homogenates of 24,25(OH)2D3-treated rats there is a decrease in the recovery of radioactivity in the total lipid extract as well as in the 1,25(OH)2D3 fraction along with an increase in the recovery of radioactivity in the water-soluble phase. These results are consistent with the possibility that 24,25(OH)2D3 has an effect on 1,25(OH)2D3 metabolism, namely that of enhancing the degradation of 1,25(OH)2D3. However, because a considerable proportion of the injected 24,25(OH)2D3 is expected to be converted into 1,24,25(OH)3D3 by renal 1 alpha-hydroxylase in 24,25(OH)2D3-treated rats, at least a part of the decrease in serum 1,25(OH)2D concentration may be due to a competitive inhibition by 24,25(OH)2D3 of the synthesis of 1,25(OH)2D3 from 25(OH)D3. Thus the physiological importance of the role of 24,25(OH)2D3 in regulating the serum 1,25(OH)2D concentration as well as the mechanism and metabolic pathway of degradation of 1,25(OH)2D3 remain to be clarified.     

25(OH)D3 and 1,25(OH)2D3 serum concentration and breast tissue expression of 1alpha-hydroxylase, 24-hydroxylase and Vitamin D receptor in women with and without breast cancer

1,25(OH)2D3 is an antiproliferative agent that may inhibit proliferation of breast cancer (BC) cells in vitro and BC development in animals. Epidemiological studies have shown a high incidence of BC in people less exposed to solar rays. To unravel the role of Vitamin D3 in BC patients, we have investigated serum levels of 25(OH)D3 and its active form 1,25(OH)2D3 as well as tissue expression of 1alpha-hydroxylase, 24-hydroxylase, and Vitamin D-receptor (VDR), determined by semiquantitative RT-PCR, in 88 Brazilian BC patients and 35 women without cancer (submitted to mammoplasties or resection of benign lesions). Median age of women with and without cancer was 51 and 46 years, respectively, and the majority of BC patients were classified as clinical stage II (67%). Although no differences in 25(OH)D3 serum concentration were found, 1,25(OH)2D3 (40+/-21 pg/ml) levels in BC patients were lower than in women without cancer (53+/-23). Our results indicate that 24-hydroxylase, VDR and 1alpha-hydroxylase mRNA tissue expression is similar in both groups and no correlation between 24-hydroxylase, 1alpha-hydroxylase, and VDR expression in breast tumors was found. A low 1,25(OH)2D3 serum concentration seems to be associated to breast cancer, however, the mechanism involved in this regulation is still unclear.     

Stimulation of 1,25-dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3 in the hypocalcaemic rat.

Serum 1,25-dihydroxyvitamin D3 concentration and renal 25-hydroxyvitamin D 1 alpha-hydroxylase activity were measured in rats fed various levels of calcium, phosphorus and vitamin D3. Both calcium deprivation and phosphorus deprivation greatly increased circulating levels of 1,25-dihydroxyvitamin D3. The circulating level of 1,25-dihydroxyvitamin D3 in rats on a low-calcium diet increased with increasing doses of vitamin D3, whereas it did not change in rats on a low-phosphorus diet given increasing doses of vitamin D3. In concert with these results, the 25-hydroxyvitamin D 1 alpha-hydroxylase activity was markedly increased by vitamin D3 administration to rats on a low-calcium diet, whereas the same treatment of rats on a low-phosphorus diet had no effect and actually suppressed the 1 alpha-hydroxylase in rats fed an adequate-calcium/adequate-phosphorus diet. The administration of 1,25-dihydroxyvitamin D3 to vitamin D-deficient rats on a low-calcium diet also increased the renal 25-hydroxy-vitamin D 1 alpha-hydroxylase activity. These results demonstrate that the regulatory action of 1,25-dihydroxyvitamin D3 on the renal 25-hydroxyvitamin D3 1 alpha-hydroxylase is complex and not simply a suppressant of this system.     

Activation of protein kinase C modulates dihydroxycholecalciferol synthesis in rat renal tubules.

1,25(OH)2D3, the biologically active metabolite of vitamin D, is produced from 25(OH)D3 by the renal mitochondrial 25(OH)D3 1 alpha-hydroxylase. Several studies have implicated reversible phosphorylation and a possible role for protein kinase C (PKC) in acute regulation of 1,25(OH)2D3 production. In the experiments described here, we studied 1,25(OH)2D3 production in freshly isolated rat renal tubules treated with activators and inhibitors of PKC. In this mammalian system, TPA, but not its inactive analogue 4 alpha PDD, inhibited 1,25(OH)2D3 production in a dose-dependent fashion within 20 min. The acute inhibition of 1,25(OH)2D3 production by TPA exposure was preceded by an increase in membrane associated PKC activity, which was paralleled by a decrease in cytosolic PKC activity. Pre-incubation of tubules with staurosporine, a PKC inhibitor, abolished the inhibitory effect of TPA on 1,25(OH)2D3 production. Chronic (18 h) exposure of tubules to high dose TPA resulted in down regulation of both membrane and cytosolic PKC activity and re-exposure to TPA did not affect PKC translocation or 1,25(OH)2D3 production in down regulated tubules. Our data strongly suggest that modulation of renal PKC activity may be an important mechanism for acute regulation of 1,25(OH)2D3 production.     

Cholecalciferol and placental calcium transport

Transplacental movement of calcium from mother to fetus is essential for normal fetal development. In most species, fetal plasma calcium levels are higher than maternal levels at term. The role of cholecalciferol metabolites, with specific emphasis on 1,25-dihydroxycholecalciferol (1,25(OH)2D), in placental calcium transport and maintenance of the fetomaternal gradient has been extensively investigated. In rats, there is not an absolute demand for 1,25(OH)2D for maintenance of fetal calcium homeostasis in utero, even though it is essential for maintenance of maternal plasma calcium levels. However, in sheep, the absence of 1,25(OH)2D results in disruption of both maternal and fetal calcium homeostasis. It is known that rat and human placentas contain specific cytosolic binding proteins for 1,25(OH)2D that are similar to the well-characterized intestinal receptor. Two calcium-binding proteins (CaBP) have been detected in rat and human placentas: a protein immunologically identical to the vitamin D-dependent CaBP and a calcium-dependent ATPase. The levels of CaBP in rat placenta have been shown to increase in response to exogenously administered 1,25(OH)2D but cannot be obliterated with maternal vitamin D deficiency. No relationship has been shown between 1,25(OH)2D and placental Ca-ATPase in any species. Thus, the mechanism of action of 1,25(OH)2D in maintenance of the transplacental calcium gradient in sheep is unknown. In the pregnant rat (and perhaps human), 1,25(OH)2D is a critical factor in the maintenance of sufficient maternal calcium for transport to the fetus and may play a role in normal skeletal development of the neonate.     

Parathyroid hormone stimulation of the renal 25-hydroxyvitamin D-1alpha-hydroxylase--effect of age and free radicals

The capacity of parathyroid hormone (PTH) to increase serum 1,25(OH)(2)D levels declines with age in both rats and humans. In young rats, PTH stimulates renal 1,25(OH)(2)D production and increases mRNA levels for the terminal mitochondrial P450 of the 1alpha-hydroxylase complex (CYP27B1 or CYP1alpha). However, in older rats PTH increases mRNA levels but not 1,25(OH)(2)D production. This suggests that in old animals there is either decreased CYP1alpha protein levels in response to PTH or that the protein produced lacks functionality. The CYP1alpha protein is located on the inner mitochondrial membrane, the site of increased free radical production with age. To study these possibilities, we examined the effect of PTH and free radicals on CYP1alpha expression in a model system-AOK-B50 renal tubular cells. PTH increased CYP1alpha mRNA and protein in a similar time-dependent manner, suggesting that CYP1alpha protein levels were largely regulated by mRNA levels. The effect of free radicals was determined by preincubation with hydrogen peroxide (H(2)O(2)), a standard model for studying free radical damage. H(2)O(2) inhibited PTH-stimulated CYP1alpha protein levels and 1,25(OH)(2)D production in a dose dependent manner. However, 1,25(OH)(2)D production was more sensitive to H(2)O(2) than was CYP1alpha protein levels. This suggests that the catalytic activity of the CYP1alpha protein may be reduced by free radical damage in these cells. Future studies will focus on detecting oxidative damage in this model system and in vivo.     

Measurement of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in mammalian kidney

Until recently measurement of 25-OH-D3-1 alpha-hydroxylase activity in mammalian kidney has not been possible due to the presence of a protein which inhibits the enzyme by reducing available substrate. However, utilization of sufficient unlabeled 25-OH-D3 (80 nmol/ml renal homogenate) to overcome the effect of the inhibitor while maintaining optimal concentration for 1-hydroxylation has made quantitation of enzyme activity possible. We have modified this existing technique in order to increase the sensitivity and to permit detailed study of 1 alpha-hydroxylate regulation in mouse kidney. The modifications that we have incorporated include (i) simplifying the purification scheme for obtaining measurable 1,25-(OH)2D3 by reducing to one the necessary number of high-performance liquid chromatography steps and (ii) quantifying 1,25-(OH)2D3 by radioligand assay. The sensitivity of the assay is 10 pg, which, corrected for fractionation and recovery (50-60%), allows the measurement of 0.5 fmol 1,25-(OH)2D3 produced per milligram kidney per minute. Moreover, reliability and precision of the assay have been confirmed by demonstrating that samples from carefully matched, identically treated mice have reproducible enzyme activity (interassay coefficient of variation = 9.1%, n = 5) and show appropriate dilution characteristics. We have also demonstrated appropriate modulation of enzyme activity by known effectors of 1-hydroxylation. Kidneys from D-deficient mice exhibit significantly higher enzyme activity (15.28 +/- 1.17, n = 21) than do normal mouse kidneys (5.14 +/- 0.26, n = 33). In contrast, enzyme activity is suppressed significantly in kidneys obtained from calcium-loaded (1.20 +/- 0.04, n = 5) and parathyroidectomized animals (2.94 +/- 0.29, n = 5). Our assay now permits the indepth study of 1 alpha-hydroxylase regulation in mammalian (mouse) kidneys.     

Longitudinal changes in maternal serum 1,25-dihydroxyvitamin D and insulin like growth factor I levels in pregnant women who developed preeclampsia: comparison with normotensive pregnant women

This study was undertaken to determine the longitudinal changes of serum 1,25-dihydroxyvitamin D (1,25-(OH)(2)D) and insulin like growth factor I (IGF-I) levels at 20.7, 27.6, and 35.5 week periods of gestation in 40 pregnant women who remained normotensive (NT) and in 10 women who developed preeclampsia (PE). As compared with the first period, significant increases (P < 0.01) in maternal serum 1,25-(OH)(2)D and IGF-I were observed in the NT group. In the PE group, a similar increase in serum 1,25-(OH)(2)D was observed. In contrast, significant (P < 0.05) lower IGF-I levels were observed in the PE group at the moment of diagnosis. In addition a high incidence of subjects with low increase in IGF-I levels (相似文献   

The effects of aluminum loading on the renal response to parathyroid hormone in the vitamin D-replete rat

Aluminum (Al) may cause vitamin D-resistant osteomalacia and depress the serum levels of immunoreactive parathyroid hormone (iPTH) in patients treated with maintenance dialysis and those on total parental nutrition (TPN). Both conditions have been associated with low serum levels of 1,25(OH)2-vitamin D (1,25(OH)2D). Al may inhibit PTH secretion in vitro; however, induction of hypocalcemia can enhance endogenous PTH secretion in Al-loaded dogs and TPN patients. Despite hypocalcemia and/or increased endogenous iPTH levels, Al-loaded TPN patients fail to show the expected rise in serum 1,25(OH)2D levels. Such observations suggest that Al may impair the renal response to PTH. We studied vitamin D-replete rats given Al or saline vehicle IP for 5 days. Al and control rats then received a saline infusion with an IV bolus of PTH 1-34. Urinary cyclic AMP and P excretion rose in Al and control rats by 1 hr post-PTH, without differences between the groups. Serum P and ionized Ca levels were not different between Al and control rats. In other Al and control rats, serum 1,25(OH)2D levels were measured after saline without PTH. Serum 1,25(OH)2D levels were higher in controls given PTH than in those without, but 1,25(OH)2D levels were not different between Al rats given PTH and those with none. Thus, aluminum does not affect cyclic AMP or P excretion but may impair 25(OH)D-1 alpha-hydroxylase activity in response to PTH.     

Regulation of renal vitamin D receptor is an important determinant of 1alpha,25-dihydroxyvitamin D(3) levels in vivo

The synthesis of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) is most strongly regulated by dietary calcium and the action of parathyroid hormone to increase 1alpha-hydroxylase (1alpha-OHase) and decrease 24-hydroxylase (24-OHase) in kidney proximal tubules. This study examines the hypothesis that 1,25-(OH)(2)D(3) synthesis, induced by dietary calcium restriction, is also the result of negative feedback regulation blockade. Rats fed a low calcium (0.02%, -Ca) diet and given daily oral doses of vitamin D (0, 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 microg) remained hypocalcemic despite increasing levels of serum calcium in relation to the vitamin D dose. Plasma levels of 1,25-(OH)(2)D(3) rose to high levels (1200 pg/ml) at the high vitamin D dose levels. As expected, thyroparathyroidectomy caused a rapid fall in serum 1,25-(OH)(2)D(3). In rats fed a 0.47% calcium diet (+Ca) supplemented with vitamin D (4 microg/day), exogenous 1,25-(OH)(2)D(3) suppressed renal 1alpha-OHase and stimulated the 24-OHase. In rats fed the -Ca diet, vitamin D was unable to suppress the renal 1alpha-OHase or stimulate the renal 24-OHase. In contrast, vitamin D was fully able to stimulate intestinal 24-OHase. Intestinal vitamin D receptor (VDR) was present under all circumstances, while kidney VDR was absent under hypocalcemic conditions and present under normocalcemic conditions. It appears that tissue-specific down-regulation of VDR by hypocalcemia blocks the 1,25-(OH)(2)D(3) suppression of the 1alpha-OHase and upregulation of the 24-OHase in the kidney, causing a marked accumulation of 1,25-(OH)(2)D(3) in the plasma.