In vitro production of 1,25-dihydroxyvitamin D3 by rat placental tissue.

Weanling female rats were fed a vitamin D-deficient diet for 4 months until they reached maturity. They were mated with normal, vitamin D-replete male rats and, at 20 days of pregnancy, the female rats were killed and their placentae were removed, homogenzied, and incubated with 25-hydroxyvitamin D3. The incubation mixtures were extracted and the extracts were subjected to Sephadex LH-20 column chromatography followed by high-pressure liquid chromatography. The 1,25-dihydroxyvitamin D3 region of the high-pressure liquid chromatogram was recycled to purity and the structure of the product was identified as 1,25-dihydroxyvitamin D3 by ultraviolet absorption spectrophotometry and by mass spectrometry. Thus it is now evident that placenta, in addition to renal tissue, is capable of converting 25-hydroxyvitamin D3 to the hormonal form of vitamin D, 1,25-dihydroxyvitamin D3.     

Prolactin but not growth hormone stimulates 1,25-dihydroxyvitamin D3 production by chick renal preparations in vitro

We studied the effect of PRL from two species (bovine and turkey) and GH from two species (bovine and turkey) on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] production by two whole cell preparations from vitamin D-deficient chick kidneys (slices and tubules). We observed that 8 ng/ml turkey PRL stimulated 1,25(OH)2D3 production by renal tubules and slices. Ovine PRL had a similar effect on 1,25(OH)2D3 production but at higher concentrations. In contrast, neither bovine GH nor turkey GH stimulated 1,25(OH)2D3 production appreciably at doses up to 1000 ng/ml. The effect of PRL on 1,25(OH)2D3 production by renal tubules required a 3-h preincubation, although its effect on 1,25(OH)2D3 production by renal slices was immediate. We conclude that PRL, but not GH, directly stimulates 1,25(OH)2D3 production by the chick kidney.     

A role for calmodulin in renal production of 1,25-dihydroxyvitamin D3

The conversion of circulating 25-hydroxyvitamin D3 (25OHD3) to its active metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in the renal tubule mitochondrion by the enzyme 25OHD3-1 alpha-hydroxylase is closely regulated in vivo according to the physiological need for calcium and phosphorus. The mechanism by which this regulation is achieved at the cellular level has not been clarified, although a number of lines of evidence suggest that calcium ions (Ca2+) are involved. This study was designed to determine whether calmodulin, the ubiquitous cell protein that binds and mediates many of the regulatory functions of Ca2+, plays a role in the regulation of renal vitamin D metabolism. The calmodulin antagonists trifluoperazine (TFP), Janssen R24571, and the naphthalene sulfonamides W5 and W7 inhibited conversion of 25OHD3 to 1,25-(OH)2D3 by isolated renal tubules from vitamin D-deficient chicks in a dose-dependent manner (ED50: TFP, 12 mumol/liter; R24571, 10 mumol/liter; W7, 30 mumol/liter; W5, 75 mumol/liter). TFP did not inhibit production of the alternative metabolite 24,25-(OH)2D3 by chick renal tubules. In a similar manner, TFP, W7, and W5 inhibited conversion of 25OHD3 to 1,25-(OH)2D3 by isolated energized chick renal mitochondria, with no detrimental effect on mitochondrial respiratory indices. Bovine brain calmodulin in a concentration of 1 X 10(-7) mol/liter enhanced 1,25-(OH)2D3 production by isolated chick renal mitochondria in Ca2+ -containing medium, but not in the absence of Ca2+. Preincubating mitochondria with anticalmodulin antiserum resulted in decreased conversion of 25OHD3 to 1,25-(OH)2D3, an effect that was prevented by exogenous calmodulin. These data support the notion of a role for calmodulin in the Ca2+ -mediated control of renal 1 alpha-hydroxylase activity.     

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.     

Insulin modulates the stimulation of renal 1,25-dihydroxyvitamin D3 production by parathyroid hormone

Previous studies have shown that there is an impairment in renal production of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the major biologically active metabolite of vitamin D3, in diabetes. This impairment is not due to a deficiency in the parathyroid hormone (PTH), a major stimulator of renal 1,25(OH)2D3 production. Therefore, we have investigated the capacity of PTH to stimulate 1,25(OH)2D3 production in insulin deficiency and with insulin replacement. Experiments were performed in rats fed a 0.6% calcium, vitamin D sufficient diet for 2 weeks. Thyroparathyroidectomy was performed on all rats. Rats to be rendered diabetic were injected with streptozotocin immediately after surgery. In non-diabetic rats, PTH administration significantly increased renal 1,25(OH)2D3 production (11 +/- 2 vs 46 +/- 5 pg/min/g; P less than 0.05). In diabetic rats, however, PTH caused only a modest increase in 1,25(OH)2D3 production (11 +/- 1 vs 19 +/- 4 pg/min/g; P less than 0.05). With insulin replacement, PTH stimulation of 1,25(OH)2D3 production was markedly increased over that seen in diabetic rats (48 +/- 12 vs 19 +/- 4 pg/min/g; P less than 0.05). PTH was equally effective in raising serum calcium, depressing serum phosphorus and tubular reabsorption of phosphate in non-diabetic as well as in diabetic rats. These results demonstrate that insulin is necessary for the maximal stimulation of renal 1,25(OH)2D3 production by PTH. However, insulin is not necessary for PTH action in terms of renal handling of phosphate and inducing hypercalcaemia. These results suggest multiple pathways for the action of PTH, only some of which are insulin requiring.     

Inhibition of interleukin-1 production by 1,25-dihydroxyvitamin D3

The hormonal form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], inhibits the proliferation of T lymphocytes and production of growth-promoting factors (including interleukin-2) (IL2) in CTLL2 murine cells. In this study, we investigated the role of monocytes in this hormone-mediated inhibitory effect, by testing the effects of 1,25-(OH)2D3 on the ability of the mitogenic lectin phytohemagglutinin (PHA) to induce T cell activation in either a monocyte-dependent or phorbol myristate acetate (PMA)-driven (monocyte-independent) system. The results indicate that proliferation of T cells and production of growth-promoting factors are inhibited by 1,25-(OH)2D3 only in the monocyte-dependent system. Preincubation of monocytes with 1,25-(OH)2D3 for various periods of time and subsequent removal of the hormone resulted in inhibition of the PHA-driven proliferation of T cells. Preincubation for 2 h resulted in 20% inhibition, while preincubation for 36 h reduced proliferation to 50% of the control value [no 1,25-(OH)2D3 exposure]. These data suggested that monocytes are important participants in 1,25-(OH)2D3-mediated events. Therefore, we tested the effects of the hormone on the production of IL1, a monocyte-derived product thought to be involved in the induction of IL2 release and the subsequent development of the T cell proliferative response. 1,25-(OH)2D3 inhibited the production of both extracellular and cell-associated immunoreactive IL1 alpha and IL1 beta. Indomethacin, a prostaglandin synthetase inhibitor, did not alter the inhibitory properties of 1,25-(OH)2D3, suggesting that prostaglandins are not responsible for the inhibitory phenomenon. We conclude that part of the ability of 1,25-(OH)2D3 to inhibit T cell proliferation may be due to direct effects on monocytes by down-regulating IL-1 production. However, it is unlikely that the immunoregulatory properties of 1,25-(OH)2D3 on T cells are mediated solely through monocytes, and it is possible that the hormone also exerts its influence directly on T cells.     

Calcitonin stimulates 1,25-dihydroxyvitamin D production in diabetic rat kidney

In diabetic animals, there is a decrease in serum 1,25-dihydroxyvitamin D [1,25(OH)2D] and in renal production of 1,25(OH)2D. In nondiabetic animals, renal 1,25(OH)2D production is markedly stimulated by parathyroid hormone (PTH) and calcitonin (CT). There is evidence that diabetes impairs the responsiveness of the kidney to PTH. The effect of diabetes on responsiveness to CT is unknown. The studies reported here determined the effect of streptozotocin-induced diabetes on renal responsiveness to PTH and CT. Experiments were performed in 7- to 8-week-old rats that were fed a diet sufficient in calcium and vitamin D and were thyroparathyroidectomized (TPTX) 5 days before hormone treatment. PTH (0.33 U/g body weight at 24, 12, and 2 hours before death) significantly increased renal 1,25(OH)2D production by threefold in nondiabetic rats. This effect was markedly attenuated by diabetes. On the other hand, CT (20 U/100 g body weight at 12 and 2 hours before death) produced a maximal response in both groups of animals. In diabetic rats, CT stimulated renal 1,25(OH)2D production fivefold, whereas PTH stimulated production only 1.5-fold. Diabetes did not affect the capacity of PTH to increase serum calcium or decrease renal tubular reabsorption of phosphorus (TRP). These findings suggest that the decrease in renal 1,25(OH)2D production seen in experimental diabetes may be due to decreased renal responsiveness to PTH, but not to decreased responsiveness to CT.     

Suppression of serum 1,25-dihydroxyvitamin D in humoral hypercalcemia of malignancy is caused by elaboration of a factor that inhibits renal 1,25-dihydroxyvitamin D3 production

Although many patients with humoral hypercalcemia of malignancy exhibit reduced serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] levels, N-terminal fragments of recently identified PTH-related protein as well as PTH itself elevate serum 1,25-(OH)2D concentrations. In the present study, the effect of tumor extracts from human tumor-implanted hypercalcemic nude rat models with high and low serum 1,25-(OH)2D on renal 1,25-(OH)2D3 production was examined using rat kidney cells in culture. Whereas tumors from rats with high serum 1,25-(OH)2D levels (OCC rats) contained only a single peak of cAMP production-stimulating activity (CPSA) in osteogenic sarcoma cells on reverse phase HPLC, tumor extracts from rats with low serum 1,25-(OH)2D levels (UCC rats) contained at least two peaks of CPSA. The main peak (peak A) was estimated to be approximately 17K by gel permeation chromatography, which was the same as the molecular size of the hitherto identified PTH-related protein, and a minor peak of CPSA (peak B) was estimated to be about 25K. When peak A or crude extracts of OCC tumors as well as human PTH-(1-34) were added to primary cultures of rat kidney cells, the production of 1,25-(OH)2D3 was significantly stimulated. In contrast, although peak B or crude UCC tumor extracts had no effect on 1,25-(OH)2D3 production in themselves, when they were added together with peak A or human PTH-(1-34) the stimulation of 1,25-(OH)2D3 production was almost completely inhibited. Both peak A and peak B enhanced cAMP production in cultured kidney cells, and the cAMP production by peak A was not affected by peak B. These results are consistent with the possibility that elaboration of an additional factor from tumor cells may be the mechanism by which serum 1,25-(OH)2D levels are suppressed in patients with humoral hypercalcemia of malignancy. The nature as well as the mechanism of action of this factor remain to be elucidated.     

Dexamethasone increases 1,25-dihydroxyvitamin D3 receptor levels and augments bioresponses in rat osteoblast-like cells

Glucocorticoids increase the level of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors in primary cultures of rat calvarial osteoblast-like (OB) cells. The present study investigated how this dexamethasone (DEX) up-regulation of 1,25-(OH)2D3 receptors modulates three 1,25-(OH)2D3 bioresponses: inhibition of collagen synthesis, stimulation of osteocalcin synthesis, and induction of 25-hydroxyvitamin D3-24-hydroxylase activity. Pretreatment of OB cells with 13 nM DEX for 24 h doubled the 1,25-(OH)2D3 receptor level without changing receptor affinity for 1,25-(OH)2D3 to study bioresponses. After DEX treatment to increase the 1,25-(OH)2D3 receptor level, the magnitude and sensitivity of all three 1,25-(OH)2D3 bioresponses were enhanced. The maximal 1,25-(OH)2D3 inhibition of collagen synthesis was increased by DEX pretreatment compound to control values: 30 to 50% (1 day treatment) and 50 to 70% (2 day treatment). The sensitivity to 1,25-(OH)2D3, as measured by reduction of the half-maximal inhibitory dose (ED50), was increased 50%. This potentiation of 1,25-(OH)2D3 inhibitory action on collagen synthesis was still evident after correction for the inhibitory effect on collagen synthesis by DEX alone. The maximal stimulation of osteocalcin by 1,25-(OH)2D3 was also enhanced from 2- to 3-fold in controls to over 4- to 5-fold by DEX pretreatment. Similarly, the ED50 of the response was reduced 50%. For the induction of 25-hydroxyvitamin D3-24-hydroxylase activity, DEX doubled the enzyme activity over that seen with 1,25-(OH)2D3 alone, but only slightly affected the sensitivity of the enzyme induction. In conclusion, after DEX up-regulation of 1,25-(OH)2D3 receptor levels, there was a general potentiation of 1,25-(OH)2D3 bioresponses in rat OB cells. However, the detailed patterns of the augmented responses were different for each of the three biological functions we studied.     

Evidence for 1,25-dihydroxyvitamin D3 production by cultured porcine alveolar macrophages

Previous studies have demonstrated that human alveolar and bone marrow macrophages when activated in vitro can metabolize 25-hydroxyvitamin[3H]D3 to 1 alpha,25-dihydroxyvitamin[3H]D3; however, to date no animal models to study this system have been available. In the present study, cultured porcine pulmonary alveolar macrophages from two animals were assayed for their capability for metabolism of 25-hydroxyvitamin[3H]D3. The porcine alveolar macrophages constitutively produced a metabolite of 25-hydroxyvitamin[3H]D3 which was identified as 1 alpha,25-dihydroxyvitamin[3H]D3 by high performance liquid chromatography. The apparent KM was in the range of 300 nM. Unlike human macrophages, treatment of porcine alveolar macrophages with lipopolysaccharide did not stimulate 1 alpha,25-dihydroxyvitamin[3H]D3 production. Addition of 1 alpha,25-dihydroxyvitamin-D3 to macrophages cultures led to a sensitive proportional inhibition of 1 alpha,25-dihydroxyvitamin[3H]D3 synthesis.     

Regulation of the murine renal vitamin D receptor by 1,25-dihydroxyvitamin D3 and calcium

Renal vitamin D receptor (VDR) is required for 1,25-dihydroxyvitamin D3-[1,25(OH)2D3]-induced renal reabsorption of calcium and for 1,25(OH)2D3-induced 1,25(OH)2D3 24-hydroxylase. The long-term effect of vitamin D and dietary calcium on the expression of renal VDR was examined in the nonobese diabetic mouse. Vitamin D-deficient and vitamin D-replete mice were maintained on diets containing 0.02%, 0.25%, 0.47%, and 1.20% calcium with or without 50 ng of 1,25(OH)2D3 per day. Vitamin D-replete mice on a 1.20% calcium diet had renal VDR levels of 165 fmol/mg protein. Calcium restriction caused renal VDR levels to decrease to <30 fmol/mg protein in vitamin D-deficient mice and to approximately 80 fmol/mg protein in vitamin D-replete mice. When dietary calcium was present, 50 ng of 1,25(OH)2D3 elevated the VDR levels 2- to 10-fold, depending on vitamin D status and the level of calcium. In the absence of either vitamin D or calcium, the VDR mRNA was expressed at a basal level. 1,25(OH)2D3 supplementation caused relative VDR mRNA to increase 8- to 10-fold in the vitamin D-deficient mouse when dietary calcium was available. This increase was completely absent in the calcium-restricted mice. This in vivo study demonstrates that 1,25(OH)2D3 and calcium are both required for renal VDR mRNA expression above a basal level, furthering our understanding of the complex regulation of renal VDR by 1,25(OH)2D3 and calcium.     

Induction of human monocyte to macrophage maturation in vitro by 1,25-dihydroxyvitamin D3

Cells of the mononuclear phagocyte system arise from circulating blood monocytes (MO) that undergo further maturation on leaving the vasculature and migration into the various tissues and body cavities. This terminal differentiation step is also observed in vitro when blood MO are cultured in the presence of serum. Yet, the inducing signals present in serum are not defined. We have established primary cultures from elutriation-purified blood MO and found that the active metabolite of vitamin D3 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) could induce maturation of MO to macrophages (MAC) in the absence of any serum proteins. Cells were cultured for 7 days with AB-group serum or 1,25(OH)2D3, respectively, and MO maturation analyzed by morphology, functional activity, and the expression of lineage-restricted maturation-associated antigens (MAX.1, MAX.3). At an optimal concentration of 10(-8) mol/L, 1,25(OH)2D3 promoted the development of fully differentiated MAC whose phenotype and functional competence in terms of cytokine release (tumor necrosis factor alpha, interleukin-6, fibronectin, and lysozyme) was comparable with MAC grown in serum. In conclusion, our data may add to the immunoregulatory potential of 1,25(OH)2D3, which may play an essential role in the ontogeny of the mononuclear phagocyte system.     

Enhanced production rate of 1,25-dihydroxyvitamin D in sarcoidosis

We determined the metabolic clearance and production rates of 1,25-dihydroxyvitamin D [1,25-(OH)2D] in 5 patients with sarcoidosis who had either hypercalciuria or hypercalcemia to examine whether abnormalities in the metabolism of this hormone existed. The mean MCR in the 5 patients with sarcoidosis [40 +/- 9 (+/- SD) mL/min] was similar to that in 13 normal subjects (37 +/- 6 mL/min) and that in 9 patients with absorptive hypercalciuria and renal stones (35 +/- 4 mL/min). However, the mean serum 1,25-(OH)2D concentration was significantly higher in the patients with sarcoidosis (211 +/- 60 pmol/L) than in either of the other 2 groups. The mean 1,25-(OH)2D production rate was markedly elevated in the patients with sarcoidosis (12.4 +/- 5.3 mumol/day), being more than 2-fold greater than the normal mean value (5.4 +/- 1.2 mumol/day). The highest production rates were found in patients with hypercalcemia, whereas subjects with hypercalciuria had production rates comparable to those in the patients with absorptive hypercalciuria. These data indicate that there is no impairment in the clearance of 1,25-(OH)2D in patients with sarcoidosis and that the elevated serum 1,25-(OH)2D levels are due to an increase in its production rate.     

1,25-Dihydroxyvitamin D3 up-regulates the 1,25-dihydroxyvitamin D3 receptor in vivo.

The level of mRNA encoding the 1,25-dihydroxyvitamin D3 receptor in the intestine of vitamin D-deficient rats given 1,25-dihydroxyvitamin D3 was determined by Northern blot analysis using a 32P-labeled cDNA probe to the 1,25-dihydroxyvitamin D3 receptor. mRNA levels increased 10-fold above deficiency levels at 6 and 12 hr after an intravenous dose of 1,25-dihydroxyvitamin D3, returning to predosing levels at 24 hr. Total receptor protein level determined by an immunoradiometric assay was increased 2-fold at 12 hr. No change in unoccupied receptor levels determined by ligand-binding assay was observed during this period. These results suggest that 1,25-dihydroxyvitamin D3 increases receptor mRNA and total receptor level to maintain constant levels of unoccupied receptor.     

Effect of 1,25-dihydroxyvitamin D3 on rat pituitary prolactin release

The effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on PRL secretion from rat pituitary in vivo and in vitro was investigated. Treating the rats for three days with 0.05 microgram/kg per day had no effect on basal PRL secretion, whereas the TRH-induced PRL secretion was increased (P less than 0.001). The enhancing effect of 1,25(OH)2D3 was blunted by verapamil. Incubating dispersed anterior pituitary cells with 10(-8) mol/l 1,25(OH)2D3 induced a significant increase in PRL secretion after 96 h (364 +/- 30 ng/well vs 481 +/- 33 ng/well, P less than 0.001; mean +/- SEM) compared with control cells. The TRH-induced PRL secretion was increased in cells incubated with 1,25(OH)2D3 for 144 h (0.766 +/- 0.061 vs 1.024 +/- 0.076 microgram/well, P less than 0.05; mean +/- SEM) compared with control cells. Neither 25-hydroxyvitamin D3 (25OH-D3) nor 24,25-dihydroxyvitamin D3 had any effects on the PRL secretion. However, when the cells were incubated with both 10(-8) mol/l 1,25(OH)2D3 and 10(-6) mol/l 25OHD3, the enhancing effect of 1,25(OH)2D3 on the basal PRL secretion was blunted. The results suggest that 1,25(OH)2D3 possibly affects the regulation of PRL release from the rat pituitary and that this effect is specific for 1,25(OH)2D3.