Role of insulin in the stimulation of renal 25-hydroxyvitamin D3-1 alpha-hydroxylase by phosphorus deprivation in rats

The increase in serum 1,25(OH)2D concentration in response to dietary phosphorus (P) depreviation is dependent on the presence of insulin in rats. The present study was undertaken to clarify whether insulin exerts its effects by affecting the renal production of 1,25(OH)2D. The 25(OH)D-1 alpha-hydroxylase activity in kidney homogenates was markedly stimulated by P deprivation in control rats (0.20 +/- 0.06 pmol/g tissue/min in the rats on a normal P diet v 1.3 +/- 0.15 pmol/g/min in the rats on a low P diet; 6.5-fold increase). In contrast, in streptozotocin-diabetic rats, the increase in the renal 1 alpha-hydroxylase activity in response to P deprivation (0.25 +/- 0.01 pmol/g/min; 3.6-fold increase) as well as the enzyme activity in the rats on a normal P diet (0.07 +/- 0.01 pmol/g/min) was markedly suppressed. Furthermore, all the changes in the renal 1 alpha-hydroxylase activity in insulin-deficient rats disappeared by insulin replacement (0.16 +/- 0.01 pmol/g/min in the rats on a normal P diet v 1.3 +/- 0.01 pmol/g/min in the rats on a low P diet; eightfold increase). These results demonstrate that the stimulation of 1 alpha-hydroxylase in response to dietary P deprivation is blunted by insulin deficiency and is fully restored by insulin replacement. It is suggested that insulin, in addition to its direct stimulatory effect on 1 alpha-hydroxylase, alters the responsiveness of renal 1 alpha-hydroxylase to P deprivation. These effects of insulin on 1 alpha-hydroxylase may be responsible for the change in serum 1,25(OH)2D concentration in response to dietary P deprivation, although the possibility cannot be ruled out that insulin also affects the metabolic clearance of 1,25(OH)2D.     

Dietary phosphorus deprivation induces 25-hydroxyvitamin D(3) 1alpha-hydroxylase gene expression

Dietary phosphorus deprivation causes hypophosphatemia and an increase in serum 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] concentrations. To determine the molecular mechanisms of this regulation, the effects of dietary phosphorus deprivation and hypophysectomy on 25-hydroxyvitamin D(3) 1alpha-hydroxylase (1alpha-hydroxylase) protein and messenger RNA (mRNA) expression were examined in rats. A low phosphorus diet (LPD) for 4 days resulted in hypophosphatemia and an increase in serum 1,25-(OH)(2)D(3) levels. This increase was caused by the induction of 1alpha-hydroxylase protein and mRNA expression (4- and 10-fold increases, respectively). Administration of the LPD or normal phosphorus diet to hypophysectomized (HPX) rats resulted in hypophosphatemia and suppression of 1alpha-hydroxylase gene expression, indicating that hypophosphatemia itself is not sufficient to induce 1alpha-hydroxylase mRNA expression. Administration of GH to HPX rats fed LPD could partially restore 1alpha-hydroxylase mRNA expression, whereas supplementation with insulin-like growth factor I, T(3), estrogen, or corticosterone had no effect. We also examined Phex gene expression in the bone, because the clinical features of X-linked hypophosphatemia resemble those of HPX rats. Phex mRNA expression, however, was not altered in HPX rats. In conclusion, we demonstrated that the increase in serum 1,25-(OH)(2)D(3) levels caused by dietary phosphorus deprivation is due to the induction of 1alpha-hydroxylase mRNA expression, and this increase is mediated in part by a GH-dependent mechanism.     

The role of insulin in the stimulation of renal 1,25-dihydroxyvitamin D synthesis by parathyroid hormone in rats

To evaluate the role of insulin in 1,25-dihydroxyvitamin D [1,25(OH)2D] production in response to PTH, 25-hydroxyvitamin D-1 alpha-hydroxylase activity in kidney homogenates as well as serum 1,25(OH)2D concentration was measured both after dietary calcium (Ca) deprivation and after PTH infusion in control and streptozotocin-diabetic rats. Although serum Ca and phosphate (Pi) levels did not change significantly after dietary Ca deprivation for 1 week, urinary cAMP excretion increased significantly, indicating that dietary Ca deprivation caused secondary hyperparathyroidism without a significant change in serum Ca level. In control rats, renal 1 alpha-hydroxylase activity increased markedly from 0.11 +/- 0.05 to 1.70 +/- 0.46 ng/300 mg tissue/20 min in parallel with the change in serum 1,25(OH)2D level from 121 +/- 8 to 360 +/- 54 pg/ml in response to Ca deprivation. In contrast, serum 1,25(OH)2D level (82 +/- 3 pg/ml) and 1 alpha-hydroxylase activity (0.07 +/- 0.02 ng/300 mg tissue.20 min) were lower in the diabetic rats on a normal Ca diet than those in control rats, and the increase in both 1,25(OH)2D level and 1 alpha-hydroxylase activity in response to Ca deprivation was suppressed in diabetic rats (136 +/- 24 pg/ml and 0.38 +/- 0.12 ng/300 mg tissue.20 min, respectively, after Ca deprivation). Insulin treatment of the diabetic rats restored the baseline levels of serum 1,25(OH)2D (125 +/- 14 pg/ml) and renal 1 alpha-hydroxylase activity (0.21 +/- 0.02 ng/300 mg tissue.20 min) as well as those after Ca deprivation (340 +/- 52 pg/ml and 2.05 +/- 0.30 ng/300 mg tissue.20 min, respectively). Furthermore, when control and diabetic rats were thyroparathyroidectomized and infused with a maximal stimulatory dose of PTH, the increase in serum 1,25(OH)2D and renal 1 alpha-hydroxylase activity in response to PTH was markedly inhibited in diabetic rats. In addition, the baseline levels of serum 1,25(OH)2D and renal 1 alpha-hydroxylase activity in thyroparathyroidectomized diabetic rats were not different from those in control rats. These results are consistent with the conclusion that insulin plays an important role in the regulation of renal 1 alpha-hydroxylase activity and serum 1,25(OH)2D levels in response to PTH.     

Dietary phosphorus and 1,25-dihydroxyvitamin D metabolism: influence of insulin-like growth factor I

Hypophysectomy abolishes the increase in serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] induced by restriction of dietary phosphorus. Administration of GH increases circulating insulin-like growth factor I levels (IGF-I) and restores, in part, the responsiveness of serum 1,25-(OH)2D to restriction of dietary phosphorus. To determine whether the GH-dependent increase in serum 1,25-(OH)2D induced by restriction of dietary phosphorus is mediated by IGF-I, we measured the serum concentration of 1,25-(OH)2D in hypophysectomized rats treated with either GH (100 micrograms/day) or recombinant human IGF-I (150 micrograms/day) and fed either a normal or low phosphorus diet for 6 days. Restriction of dietary phosphorus in sham-hypophysectomized rats increased serum 1,25-(OH)2D from 97 +/- 13 to 251 +/- 36 pg/ml, or 159%, but had no effect on serum 1,25-(OH)2D in hypophysectomized rats. Restriction of dietary phosphorus in rats receiving GH increased, (P less than 0.001) serum 1,25-(OH)2D from 52 +/- 8 to 133 +/- 18 pg/ml, or 156%. Restriction of dietary phosphorus in rats receiving IGF-I increased (P less than 0.001) serum 1,25-(OH)2D from 33 +/- 5 to 94 +/- 11 pg/ml, or 185%, an increase equivalent to that observed in animals receiving GH. For a given diet, no significant differences were seen between the serum concentrations of 1,25-(OH)2D in animals receiving GH or IGF-I. These data indicate that IGF-I can restore the increase in serum 1,25-(OH)2D induced by restriction of dietary phosphorus to the same degree as GH. This strongly suggests that the GH-dependent increase in serum 1,25-(OH)2D induced by restriction of dietary phosphorus is mediated by IGF-I.     

Evidence that somatomedins mediate the effect of hypophosphatemia to increase serum 1,25-dihydroxyvitamin D3 levels in rats

The present studies were undertaken in an effort to determine whether somatomedins (SMs) play a role in the elevation of serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] levels during dietary phosphate deprivation. Serum 1,25-(OH)2D3,SM-C, and phosphate levels were measured in rats fed diets containing adequate or very low levels of dietary phosphorus under circumstances known to affect SM levels, including hypophysectomy with and without GH replacement, normal protein vs. low protein diets, and streptozotocin-induced diabetes with and without insulin replacement. In all circumstances, serum 1,25-(OH)2D3 concentrations were directly related to serum SM-C levels. However, the slope for the relationship was increased 2- to 10-fold in animals fed the low phosphorus diets. As observed previously, serum 1,25-(OH)2D3 levels were inversely related to serum phosphate levels, but the slope for this relationship was deceased in the presence of low SM levels and absent in animals with very low SM levels. These results suggest that SM are required for elevation of serum 1,25-(OH)2D3 levels in response to phosphate deprivation.     

Evidence for a difference in vitamin D metabolism between spontaneously hypertensive and Wistar-Kyoto rats

It has been contended that the metabolism of vitamin D in spontaneously hypertensive rats (SHR) is different from that in Wistar-Kyoto rats (WKY). To investigate this possibility, the plasma concentration of 1,25-dihydroxycholecalciferol (1,25[OH]2D) and several known determinants of its production rate were measured in SHR and WKY given normal and restricted amounts of dietary phosphorus. In 12-week-old male SHR given a normal amount of dietary phosphorus, the mean plasma concentration of 1,25(OH)2D (72 +/- 5 pg/ml) was significantly lower than that in age-matched WKY (129 +/- 6 pg/ml; p less than 0.001). The lower plasma concentration of 1,25(OH)2D in the SHR could not be attributed to higher circulating levels of inorganic phosphorus or ionized calcium, lower plasma concentrations of 25-hydroxycholecalciferol, or acidosis. However, in the SHR, urinary excretion of cyclic adenosine 3',5'-monophosphate (12.5 +/- 0.4 nmol/mg creatinine) was significantly lower than that in WKY (15.2 +/- 0.3 nmol/mg creatinine; p less than 0.001). In both SHR and WKY, restriction of dietary phosphorus for 1 week induced an increase in the plasma concentration of 1,25(OH)2D without affecting blood pressure. The current findings indicate that in 12-week-old male SHR, 1,25(OH)2D metabolism is different from that in age-matched WKY. The activity of 25-hydroxyvitamin D-1 alpha-hydroxylase, however, appears to be at least partially responsive to short-term restriction of dietary phosphorus. In SHR, the activity of 25-hydroxyvitamin D-1 alpha-hydroxylase may be lower than that in WKY, perhaps due in part to some impairment in the renal metabolism of, or responsiveness to, cyclic adenosine 3',5'-monophosphate.     

Effect of dietary calcium or phosphorus restriction and 1,25-dihydroxyvitamin D administration on rat intestinal 24-hydroxylase.

1,25-Dihydroxyvitamin D-24-hydroxylase (24-hydroxylase) modulates the biological effects of 1,25-dihydroxyvitamin D [1,25-(OH)2D] in tissues. The presence of 24-hydroxylase in intestinal mucosa and the mass of the intestine suggest that the intestine is a major site of catabolism of 1,25-(OH)2D. How intestinal levels of 24-hydroxylase are regulated under various dietary conditions, such as calcium (Ca) or phosphorus (P) restriction, is poorly understood. In a series of trials on weanling and mature rats, the effects of dietary Ca or P restriction were compared with the effects of exogenous 1,25-(OH)2D3 administration on intestinal 24-hydroxylase activity. Exogenous administration of 1,25-(OH)2D3, by single bolus injection or constant infusion, increased intestinal 24-hydroxylase activity significantly. Dietary Ca and P restriction both resulted in increased plasma 1,25-(OH)2D3 concentrations several-fold above control rat values (P less than 0.001) and to levels higher than those achieved by constant infusion of 1.3 ng 1,25-(OH)2D3/h. Dietary Ca restriction increased intestinal 24-hydroxylase 6- to 20-fold above that of rats fed a Ca-replete diet (P less than 0.001). Dietary P restriction had no significant effect on intestinal 24-hydroxylase activity. These data suggest that dietary Ca restriction results in increased plasma levels of 1,25-(OH)2D3, which, in turn, leads to up-regulation of intestinal 24-hydroxylase. Conversely, dietary P restriction prevents 1,25-(OH)2D3-mediated up-regulation of 24-hydroxylase.     

Contrasting effects of exogenous 1,25-dihydroxyvitamin D [1,25-(OH)2D] versus endogenous 1,25-(OH)2D, induced by dietary calcium restriction, on vitamin D receptors

The biological actions of 1,25-dihydroxyvitamin D [1,25-(OH)2D] are mediated by specific binding of the hormone with an intracellular vitamin D receptor, which ultimately regulates expression of genes within the target tissues. The quantity of vitamin D receptors varies between target tissues and within target tissues, depending on the physiological state of the animal. One factor that can modulate tissue vitamin D receptor content is 1,25-(OH)2D. In the present study performed in male rats, exogenous administration of 36 ng 1,25-(OH)2D3/day for 7 days increased plasma 1,25-(OH)2D concentrations 5-fold above those in control rats (to 261 +/- 17 pg/ml). Compared with those in control rats, 1,25-(OH)2D3 treatment resulted in a 1.5-fold increase in duodenal vitamin D receptor content (351 +/- 16 vs. 520 +/- 21 fmol/mg protein) and a 3-fold increase in renal vitamin D receptor content (60.3 +/- 5.2 vs. 193.8 +/- 22.7 fmol/mg protein). The effects of endogenously produced 1,25-(OH)2D on tissue vitamin D receptor content were studied by feeding rats either a 0.02% or 1% calcium diet for 2, 7, 14, or 21 days. Rats fed the low calcium diet exhibited plasma 1,25-(OH)2D concentrations similar to (day 7) or exceeding (days 14 and 21) those achieved by exogenous administration of 1,25-(OH)2D3, yet duodenal vitamin D receptor content was not up-regulated by dietary calcium restriction at any time point. The renal vitamin D receptor content of calcium restricted rats was 20-38% lower (P less than 0.05) than that in rats fed a calcium-replete diet 7, 14, and 21 days after initiation of the dietary treatments. These data suggest that under physiological conditions, increased plasma concentrations of 1,25-(OH)2D do not result in up-regulation of tissue vitamin D receptor concentrations, and that dietary calcium restriction must induce some factor(s) that results in down-regulation of vitamin D receptors in the kidney.     

Role of insulin in the increase in serum 1,25-dihydroxyvitamin D concentrations in response to phosphorus deprivation in streptozotocin-induced diabetic rats

To evaluate the role of insulin in the regulation of circulating 1,25-dihydroxyvitamin D [1,25(OH)2D] levels, serum 1,25(OH)2D concentrations in response to phosphorus (P) deprivation were examined in control, streptozotocin-diabetic and insulin-treated diabetic rats. Dietary P deprivation for 1 week caused a marked increase in serum 1,25(OH)2D level from 75 +/- 4 pg/ml to 274 +/- 16 pg/ml in control rats. In contrast, serum 1,25(OH)2D level was significantly lower in diabetic rats on a normal P diet (20 +/- 2 pg/ml) compared to that in control rats and increased only slightly by P deprivation (33 +/- 4 pg/ml). Treatment of the diabetic rats on normal P diet with insulin caused an increase in serum 1,25(OH)2D concentration to a level (82 +/- 10 pg/ml) similar to that in control rats and restored the increase in serum 1,25(OH)2D concentration in response to P deprivation (315 +/- 38 pg/ml). Although there was a marked decrease in serum phosphate level by P deprivation in all groups of animals, the rise in serum calcium level by P deprivation seen in control rats was abolished in diabetic rats. In addition, while bone mineral contents decreased significantly in response to P deprivation in control rats, no significant changes in either bone calcium or P contents were observed after P deprivation in diabetic rats. Insulin treatment of the diabetic rats recovered the responsiveness to P deprivation in both serum calcium level and bone mineral contents. P deprivation did not affect plasma glucose or serum creatinine level in any group of rats. These results suggest that insulin, either directly or indirectly, is required for the increase in circulating 1,25(OH)2D concentrations in response to P deprivation, and that the rise in serum 1,25(OH)2D level may play a role in the hypercalcemic response to P deprivation.     

Osteocalcin during the reproductive cycle in normal and diabetic rats

Concentrations of osteocalcin were measured in plasma and bone of normal and diabetic rats during the reproductive cycle and compared with plasma 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) concentrations. The age-dependence of osteocalcin was also examined. Plasma concentrations of osteocalcin levels were low but detectable in 21-day-old fetuses (3.7 +/- 0.3 nmol/l); osteocalcin concentrations were highest in weaning rats (104 +/- 9 nmol/l) and decreased thereafter. In adult rats, plasma concentrations of both osteocalcin and 1,25-(OH)2D3 increased during the last days of normal pregnancy, and even more so in rats fed a diet low in calcium and phosphate. After an early post-partum decline, osteocalcin concentrations in plasma remained at non-pregnant levels in lactating rats fed a high calcium/phosphate diet while their 1,25-(OH)2D3 concentrations were higher than in non-pregnant rats; however, lactating rats fed a low calcium/phosphate diet showed increasing osteocalcin concentrations. In spontaneously diabetic BB rats, plasma osteocalcin concentrations were severely decreased compared with those in non-diabetic rats, more than would have been expected from their decreased 1,25-(OH)2D3 concentrations. Moreover, plasma osteocalcin did not increase during pregnancy or lactation in diabetic rats, even when fed a low calcium/phosphate diet. Fetuses of diabetic rats also had lower plasma osteocalcin levels than fetuses from non-diabetic rats or than weight-matched fetuses from semistarved rats. In contrast to plasma osteocalcin concentrations, bone osteocalcin concentrations and content were not altered by pregnancy, lactation, low calcium/phosphate diet or diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of hypophysectomy and growth hormone treatment on renal hydroxylation of 25-hydroxycholecalciferol in rats

Growth hormone stimulates intestinal calcium absorption. This action has been linked to vitamin D metabolism. We have investigated the effects of hypophysectomy and GH treatment on renal metabolism of 25-hydroxycholecalciferol (25-OH-D3). Renal hydroxylation of 25-OH-D3 was measured in vitro using the renal slice technique. Experiments were performed in young F344 rats fed a vitamin D-replete, low calcium diet for 4 weeks. In hypophysectomized rats, renal conversion of 25-OH-D3 to 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was markedly reduced compared with sham-operated rats. Renal conversion of 25-OH-D3 to 24,25-(OH)2D3 was markedly increased in hypophysectomized rats compared with sham-operated rats. Treatment of hypophysectomized rats with rat GH (rGH) for 10 days resulted in a significant increase in renal conversion of 25-OH-D3 to 1,25-(OH)2D3 and a significant decrease in conversion to 24,25-(OH)2D3. Rat GH treatment caused no significant changes in serum levels of immunoreactive parathyroid hormone. Serum calcium concentrations were similar in all groups, and serum phosphorus was low in hypophysectomized rats. Treatment of hypophysectomized rats with ovine GH for 6 days caused changes which were much less pronounced than those induced by rGH. Renal conversion of 25-OH-D3 to 1,25-(OH)2D3 and 24,25-(OH)2D3 correlated well with growth rate (weight gain). These results suggest that GH, either directly or indirectly, modulates renal metabolism of 25-OH-D3.     

1,25-Dihydroxycholecalciferol effect on serum phosphorus homeostasis in rats.

It has recently been shown that 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) increases the serum phosphorus concentration of rats on a low-phosphorus diet. While studying the biological activity of 1,25(OH)2D3, we observed that under certain circumstances 1,25-(OH)2D3 would decrease the serum phosphorus concentration. The analysis of all data obtained in rat experiments during the past 3 years revealed highly significant linear correlations (P less than 0.001) between changes of serum phosphorus concentrations after the administration of 1,25-(OH)2-D3 (130 pmol/d for 1 or 5 days) and serum phosphorus or calcium levels in the animals before injection. Similar correlations could only be found with the higher dose of 25-hydroxycholecalciferol (130 pmol/d for 5 days). Another vitamin D3 metabolite, 24,25-dihydroxycholecalciferol, had no effect on serum phosphorus concentrations under our experimental conditions. The 1,25-(OH)2D3 effect on serum phosphorus concentration does not require the presence of circulating parathormone and/or calcitonin. We suggest that 1,25-(OH)2D3 might be an important factor in serum phosphorus homeostasis.     

Regulation of vitamin D-1alpha-hydroxylase and -24-hydroxylase expression by dexamethasone in mouse kidney

We investigated the effects of dexamethasone on vitamin D-1alpha-hydroxylase and -24-hydroxylase expression and on vitamin D receptor (VDR) content in the kidneys of mice fed either a normal (NCD) diet or a calcium- and vitamin D-deficient (LCD) diet for 2 weeks. For the last 5 days mice received either vehicle or dexamethasone (2 mg/kg per day s.c.). Dexamethasone significantly increased plasma calcium concentrations without changing plasma concentrations of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) in both NCD and LCD groups. Northern blot and enzyme activity analyses in NCD mice revealed that dexamethasone increased renal VDR mRNA expression modestly and greatly increased 24-hydroxylase mRNA abundance and enzyme activity, but did not affect 1alpha-hydroxylase mRNA abundance and enzyme activity. In mice fed an LCD diet, dexamethasone increased renal VDR mRNA expression 1.5-fold, decreased 1alpha-hydroxylase mRNA abundance (52%) and activity (34%), and markedly increased 24-hydroxylase mRNA abundance (16-fold) and enzyme activity (9-fold). Dexamethasone treatment did not alter functional VDR number (B(max) 125-141 fmol/mg protein) or ligand affinity (K(d) 0.13-0.10 nM) in LCD mice. Subcutaneous injections of 1,25(OH)(2)D(3) (0.24 nmol/kg per day for 5 days) into NCD mice strongly increased renal 24-hydroxylase mRNA abundance and enzyme activity, while there was no effect of dexamethasone on renal 24-hydroxylase expression in these mice. This may be due to overwhelming induction of 24-hydroxylase by 1,25(OH)(2)D(3). These findings suggest that glucocorticoid-induced osteoporosis is caused by direct action of the steroids on bone, and the regulatory effect of glucocorticoids on renal 25-hydroxyvitamin D(3) metabolism may be less implicated in the initiation and progression of the disease.     

The effect of 22-oxacalcitriol on serum calcitriol.

1,25-Dihydroxyvitamin D3 (1,25D) regulates its own levels in circulation by affecting its rates of synthesis and degradation, 22-Oxacalcitriol (OCT), a vitamin D analog with low calcemic activity, decreases circulating PTH levels, one of the regulators of renal 1 alpha-hydroxylase, and stimulates vitamin D degradation in vitro. The purpose of this study was to examine the effects of OCT administration on serum levels of 1,25D. In normal rats, OCT administration (4-200 ng, ip, daily for 5 days) caused a dose-dependent reduction in serum calcitriol levels. At a dose of 200 ng, OCT reduced serum 1,25D from 34.5 +/- 2.7 to 10.9 +/- 0.7 pg/ml (P less than or equal to 0.01) without significant changes in ionized Ca or phosphorus levels. The contribution of the suppression of PTH by OCT to the reduction of serum 1,25D was examined by administering OCT to parathyroidectomized (PTX) rats. Two hundred nanograms of OCT, ip, daily for 5 days significantly reduced serum calcitriol from 29.7 +/- 7.6 to 9.1 +/- 0.5 pg/ml (P less than or equal to 0.01) in rats fed a normal calcium diet. Because OCT increased total calcium (TCa) in this group from 7.4 +/- 0.1 to 9.5 +/- 0.3 mg/dl, similar doses of OCT were given to PTX rats fed a calcium-deficient diet. OCT decreased 1,25D from 58.9 +/- 8.9 to 10.3 +/- 0.4 pg/ml and increased TCa from 4.8 +/- 0.2 to 7.4 +/- 0.1 mg/dl. Comparison of serum 1,25D for identical TCa levels in PTX rats (normal calcium diet controls vs. calcium-deficient diet, OCT-treated) clearly indicates that OCT per se reduced serum 1,25D. Further support for a direct effect of OCT was provided by studies in PTX rats fed a low phosphorus diet. OCT decreased serum 1,25D from 125.8 +/- 15.6 to 10.9 +/- 0.6 pg/ml without significant changes in TCa. To further characterize the mechanisms involved in this effect, similar studies were performed in six normal dogs. Intravenous administration of 0.75 micrograms OCT every other day for 1 week decreased serum calcitriol from 25.4 +/- 3.2 to 12.2 +/- 1.3 pg/ml (P less than or equal to 0.002). Ionized Ca and phosphorus remained unchanged. Despite the short half-life of OCT in the circulation, 1,25D levels returned to basal concentrations 96 h after the last dose of OCT.(ABSTRACT TRUNCATED AT 400 WORDS)     

The relationship between bone apposition rate and vitamin D activity in phosphate-deficient rats

In rats, phosphorus deficiency (P-) has been shown previously to stimulate the linear bone apposition rate (BAR) and this P- effect is dependent on adequate intake of vitamin D. To investigate further the relative importance of the vitamin D3 metabolites, 1,25(OH)2D3, 24,25(OH)2D3, and 25(OH)D3, in BAR stimulation, we studied, in P- rats, the relationships between BAR and plasma levels of these three vitamin D3 metabolites following vitamin D3 deprivation. Three groups of rats were placed on diets differing only in phosphorus (P) and vitamin D3(D3) content, with one group diet deficient in both P and D3, one diet, P-, D3 replete, and one diet both P and D3 replete. Plasma levels of the three vitamin D3 metabolites, plasma Ca and P, isotopic Ca absorption and BAR measurements were carried out at 1, 3, and 5 weeks after onset of the test diets. In P-, D3 replete rats, both plasma levels of 1,25(OH)2D3 and BAR were increased throughout the 1 to 5 week study period, while 25(OH)D3 and 24,25(OH)2D3 levels were not significantly different from P and D3 replete controls. In P-, D3 restricted rats, BAR was decreased by one week, prior to any reduction in plasma levels of 25(OH)D3 and 24,25(OH)2D3 and while plasma 1,25(OH)2D3 levels were still well above control values. In this P- rat model, the vitamin D dependent BAR stimulation does not appear to be directly related to alterations in the plasma levels of 1,25(OH)2D3, 24,25(OH)2D3, or 25(OH)D3.     

Extrarenal production of calcitriol in normal and uremic humans

We have previously reported low serum levels of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and increased 1,25-(OH)2D3 production after the administration of 25-hydryoxyvitamin D (25OHD) to anephric humans. Since normal alveolar macrophages are known to synthesize 1,25-(OH)2D3 when stimulated with gamma-interferon or lipopolysaccharide, we determined whether macrophages derived from peripheral blood monocytes could be an extrarenal source of 1,25-(OH)2D3. Our results demonstrated that macrophages from normal individuals synthesize 1,25-(OH)2D3. The apparent Km for 25OHD3 was 6.6 +/- 0.5 nM and the maximum velocity was 47.4 +/- 13.7 fmol 1,25-(OH)2D3/h.microgram DNA. The activity of this enzyme was reduced 37.2 +/- 3.1% by physiological concentrations (96 pmol/L) of 1,25-(OH)2D3 in the incubation medium. Normal macrophages further hydroxylated 1,25-(OH)2D3 to more polar metabolites, and this catabolic activity was significantly enhanced by physiological concentrations of 1,25-(OH)2D3. In chronic renal failure, peripheral macrophages exhibited an enhanced 1 alpha-hydroxylase activity (8.2 +/- 0.8 vs. 4.2 +/- 0.5 fmol 1,25-(OH)2D3/microgram DNA.h in controls) and a decreased capacity to degrade 1,25-(OH)2D3. Exogenous 1,25-(OH)2D3, in physiological concentrations, reduced 1,25-(OH)2D3 synthesis to a degree (23.6 +/- 8.5%) comparable to that observed in normal cells. 1,25-(OH)2D3 production by macrophages did not correlate with the severity of hyperparathyroidism. Moreover, human PTH-(1-34) in supraphysiological concentrations (20,000 and 100,000 ng/L) did not stimulate the 1 alpha-hydroxylase activity of macrophages from either normal or uremic subjects. These results demonstrate that 1) normal peripheral macrophages metabolize 25OHD3 and 1,25-(OH)2D3; 2) macrophages in uremia display higher rates of 1,25-(OH)2D3 synthesis and lower rates of catabolism than normal macrophages; and 3) 1,25-(OH)2D3 deficiency, but not hyperparathyroidism, may play a role in the stimulation of 1,25-(OH)2D3 production by macrophages in chronic renal failure.     

Cyclosporin-A increases synthesis of 1,25-dihydroxyvitamin D3 in the rat and mouse

We have previously observed elevated serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D] levels in male rats treated with oral cyclosporin-A (CsA). This elevation was independent of changes in PTH, ionized calcium, or phosphate. This paper investigates the potential sources and mechanisms for this increase in both rats and mice. Kidney homogenates from rats treated for 14 days with (15 mg/kg) had a significant increase in 25-hydroxyvitamin D (25OHD)-24-hydroxylase (24-hydroxylase) activity (149 +/- 20 vs. 89 +/- 16 fmol/mg.min; P less than 0.05), but nonsignificant increases in 25OHD-1 alpha-hydroxylase (1 alpha-hydroxylase) activity compared to controls. Kidney homogenates from C57b16J mice after the administration of 30-50 mg/kg CsA for 3 days revealed a linear dose-related increase in renal 1 alpha-hydroxylase (r = 0.96; P less than 0.05), which became significant with doses of 30 mg/kg CsA or more (P less than 0.05). To investigate the source of this 1,25-(OH)2D production, serum 1,25-(OH)2D was measured before and 48 h after bilateral nephrectomy in rats receiving CsA for 16 days. The percent decrease in serum 1,25-(OH)2D values was not significantly different in CsA-treated and untreated rats (33.9 +/- 4.9% vs. 47.5 +/- 4.9%), indicating little or no contribution from nonrenal sources. Studies of MCRs and production rates (PRs) revealed that the elevated 1,25-(OH)2D values were due to enhanced production and not altered clearance (PR, 12.4 +/- 1.2 vs. 19.1 +/- 1.9 fmol/mg.min; P less than 0.01). CsA increases 1 alpha-hydroxylase activity and produces significant elevations in serum 1,25-(OH)2D levels in both rats and mice. This increase may have an impact on bone mineral metabolism and immune modulation in postorgan transplantation patients.     

Inhibition by prostaglandin E2 of renal effects of calcitonin in rats

To investigate the possible role of prostaglandin E2 (PGE2) in modulating the actions of PTH and calcitonin (CT) in the kidney, the effects of PGE2 were examined on the in vivo conversion of [3H]25-hydroxyvitamin D3 to [3H]1,25-dihydroxyvitamin D3 ([3H]1,25-(OH)2D3) in vitamin D-deficient thyroparathyroidectomized (T-PTX) rats and on the urinary excretion of phosphate (Pi) in vitamin D-replete T-PTX rats in the presence of either PTH or CT. Plasma accumulation of [3H] 1,25-(OH)2D3 increased from 12.2 +/- 0.6 pmol/100 ml in controls to 19.5 +/- 1.1 (P less than 0.01) by 20 micrograms/h PGE2 to 29.8 +/- 1.8 (P less than 0.001) by 7.5 U/h PTH, and to 23.3 +/- 0.7 (P less than 0.01) by 500 mU/h CT. Administration of PGE2 inhibited CT-stimulated accumulation of 1,25-(OH)2D3 to levels not different from those by PGE2 alone (17.8 +/- 1.0 pmol/100 ml). In contrast, PGE2 had no effect on PTH-stimulated 1,25-(OH)2D3 accumulation. PTH and CT caused an increase in urinary Pi excretion and a decrease in plasma Pi levels. PGE2 abolished the effects of CT, but not of PTH, on both urinary Pi excretion and plasma Pi levels. Administration of PGE2 alone caused no significant changes in plasma Pi levels and only minimal increase in urinary Pi excretion. PGE2 did not suppress urinary cAMP excretion stimulated by CT. These results demonstrate that PGE2 specifically suppresses the effects of CT to stimulate synthesis of [3H]1,25-(OH)2D3 from [3H]25-hydroxyvitamin D3 and to inhibit tubular reabsorption of Pi without affecting urinary cAMP excretion. Since CT appears to stimulate 1 alpha-hydroxylase and inhibit Pi reabsorption in proximal tubules, nephron segments devoid of CT-sensitive adenylate cyclase, these data suggest that PGE2 modulates the actions of CT, but not of PTH, on proximal tubular functions.