24,25(OH)2D3 attenuates the calcemic effect of 1,25(OH)2D3 in rats with reduced renal mass

The present study was undertaken to evaluate the effect of 24,25(OH)2D3 on serum calcium concentration in rats with reduced renal mass. Adult 5/6 nephrectomized male rats were divided into four groups: (i) control rats, (ii) rats treated with 1,25(OH)2D3, (iii) rats treated with 24,25(OH)2D3, and (iv) rats treated with 1,25(OH)2D3 and 24,25(OH)2D3. After 4 days, serum calcium in the 1,25(OH)2D3-treated group was 7.13 +/- 0.32 meq/liter (P less than 0.001 vs control). With the combination of 1,25(OH)2D3 and 24,25(OH)2D3 serum calcium was higher than that in control, 6.25 +/- 0.5 meq/liter (P less than 0.001 vs control), but lower than that in rats receiving 1,25(OH)2D3 alone (P less than 0.05). No change in serum calcium was seen in animals treated with 24,25(OH)2D3 alone. On the eighth day serum calcium in the 1,25(OH)2D3-treated group, 6.52 +/- 0.25, was higher than in the 1,25(OH)2D3 + 24,25(OH)2D3 group, 5.87 +/- 0.17 meq/liter, P less than 0.05, P less than 0.001 vs control. In both 1,25(OH)2D3- and 1,25(OH)2D3 + 24,25(OH)2D3-treated rats, hypercalciuria of similar magnitude occurred on the fourth and eighth day of treatment. No change in urinary calcium was seen in the control and 24,25(OH)2D3-treated rats. Thus, in 5/6 nephrectomized rats combined administration of 1,25(OH)2D3 and 24,25(OH)2D3 attenuates the calcemic response to 1,25(OH)2D3 without changes in urinary calcium excretion. These observations suggest that the effect of 24,25(OH)2D3 on serum calcium is different in 5/6 nephrectomized rats as compared to normal rats, in which an augmentation of serum calcium was observed following administration of both vitamin D metabolites. The effect of 24,25(OH)2D3 on serum calcium in rats with reduced renal mass may result from a direct effect of 24,25(OH)2D3 on the bone.     

24,25(OH)2D3 enhances the calcemic effect of 1,25(OH)2D3

The effect of 24,25(OH)2D3 on 1,25(OH)2D3-induced hypercalcemia was studied in normal rats. Serum (S) levels and urinary excretion of Ca2+ (UCaV) were measured in (a) control rats, (b) rats receiving a daily sc injection of 54 ng 1,25(OH)2D3, (c) rats receiving 24,25(OH)2D3 in the same dose and same manner, and (d) rats receiving 1,25(OH)2D3 + 24,25(OH)2D3. The animals were housed in metabolic cages and 24-hr urine specimens were collected. After 24 hr SCa2+ increased similarly with 1,25(OH)2D3 and with 1,25(OH)2D3 + 24,25(OH)2D3, while 24,25(OH)2D3 alone did not change SCa2+. UCaV after 24 hr increased significantly less (P less than 0.025) with 1,25(OH)2D3 + 24,25(OH)2D3 than with 1,25(OH)2D3 alone. After 5 days of 1,25(OH)2D3, SCa2+ rose from 5.1 +/- 0.15 to 6.29 +/- 0.08 whereas 1,25(OH)2D3 + 24,25(OH)2D3 effected a greater increase in SCa2+ up to 6.63 +/- 0.09 (P less than 0.01). 24,25(OH)2D3 alone did not change SCa2+. UCaV after 5 days of treatment rose similarly with 1,25(OH)2D3 and with 1,25(OH)2D3 + 24,25(OH)2D3. After 10 days of 1,25(OH)2D3 SCa2+ was 6.17 +/- 0.15 meq/liter while with the combination SCa2+ rose to 6.74 +/- 0.2 (P less than 0.025). 24,25(OH)2D3 alone did not change SCa2+. These results show that (a) 24,25(OH)2D3 alone does not alter SCa2+ in normal rats, (b) combined administration of 1,25(OH)2D3 + 24,25(OH)2D3 enhances the hypercalcemic response to 1,25(OH)2D3 without a parallel increase in UCaV, and (c) it is suggested that the effect of 24,25(OH)2D3 on serum Ca2+ level, at least partly, may result from its hypocalciuric effect.     

24,25(OH)2D3 enhances the calcemic effect of 1,25(OH)2D3 in PTX rats

The effect of 24,25(OH)2D3 on 1,25(OH)2D3-induced hypercalcemia was studied in parathyroidectomized (PTX) rats for 10 days. Serum (S) and urinary Ca excretion (UCaV) were measured in (a) control rats, (b) rats receiving a daily sc injection of 54 ng 1,25(OH)2D3, (c) rats receiving 24,25(OH)2D3 in the same dose and same manner, and (d) rats receiving 1,25(OH)2D3 + 24,25(OH)2D3. Our results show that (i) 24,25(OH)2D3 alone does not increase SCa2+ in PTX rats, (ii) combined administration of 1,25(OH)2D3 + 24,25(OH)2D3 enhances the hypercalcemic response to 1,25(OH)2D3 without a parallel increase in UCaV, (iii) combined administration of 1,25(OH)2D3 + 24,25(OH)2D3 reduces the rise in urinary excretion of Ca2+ compared with that of rats receiving 1,25(OH)2D3 alone for 10 days, and (iv) these alterations are independent of parathyroid hormone.     

Effects of 24,25(OH)2D3, 1,25(OH)2D3 and 25(OH)D3 on alkaline and tartrate-resistant acid phosphatase activities in fetal rat calvaria

The aim of this work was to evaluate the effects of 24,25-dihydroxyvitamin D3, 24,25(OH)2D3, on alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) activities in fetal rat calvaria cultures. These actions were compared with those of 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, and 25-hydroxyvitamin D3, 25(OH)D3, in similar experimental conditions. At 10 min, 30 min and at 24 h incubation time, 1,25(OH)2D3 (10(-10)M) and 25(OH)D3 (10(-7) M) produced a significant increase in AP and TRAP activities compared to control group (without vitamin D metabolites). However, 24,25(OH)2D3 (10(-7) M) only produced effects on phosphatase activities similar to those produced by 1,25(OH)2D3 and 25(OH)D3, after 24 h incubation time. These findings suggest that 1,25(OH)2D3 and 25(OH)2D3 could carry out actions in minutes (nongenomic mechanism), while 24,25(OH)2D3 needs longer periods of time to perform its biological actions (genomic mechanism).     

Phosphate uptake in chick kidney cells: effects of 1,25(OH)2D3 and 24,25(OH)2D3

Phosphate homeostasis is controlled in part by absorption from the intestine, and reabsorption in the kidney. While the effect of Vitamin D metabolites on enterocytes is well documented, in the current study we assess selected responses in primary cultures of kidney cells. Time course studies revealed a rapid stimulation of phosphate uptake in cells treated with 1,25(OH)(2)D(3), relative to controls. Dose-response studies indicated a biphasic curve with optimal stimulation at 300 pM 1,25(OH)(2)D(3) and inhibition at 600 pM seco-steroid. Antibody 099--against the 1,25D(3)-MARRS receptor - abolished stimulation by the steroid hormone. Moreover, phosphate uptake was mediated by the protein kinase C pathway. The metabolite 24,25(OH)(2)D(3), which was found to inhibit the rapid stimulation of phosphate uptake in intestinal cells, had a parallel effect in cultured kidney cells. Finally, the 24,25(OH)(2)D(3) binding protein, catalase, was assessed for longer term down regulation. In both intestinal epithelial cells and kidney cells incubated with 24,25(OH)(2)D(3) for 5-24h, both the specific activity of the enzyme and protein levels were decreased relative to controls, while 1,25(OH)(2)D(3) increased both parameters over the same time periods. We conclude that the Vitamin D metabolites have similar effects in both kidney and intestine, and that 24,25(OH)(2)D(3) may have effects at the level of gene expression.     

Mitochondrial alkaline phosphatase as an intracellular signal in the synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 in LLC-PK1 cells

In previous works we have found a mitochondrial alkaline phosphatase (AP) activity in LLC-PK1. The aim of this work has been to study the possible involvement of mitochondrial AP activity in the synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) from the substrate 25(OH)D3. Renal phenotype LLC-PK1 cells were incubated with 25(OH)D3 as substrate and treated with or without 1,25(OH)2D3, forskolin, 12-myristate-13-acetate (PMA) and 1,25(OH)2D3 in conjunction with PMA. Incubation of LLC-PK1 cells with forskolin (adenylate cyclase activator) not only stimulated the 1-hydroxylase and inhibited the 24-hydroxylase activities but also increased the mitochondrial AP activity. The addition of 1,25(OH)2D3, the main activator of 24-hydroxylase, produced a decrease of mitochondrial AP activity, a decrease of 1,25(OH)2D3 synthesis and an increase of the 24,25(OH)2D3 synthesis. Incubation with PMA, a potent activator of protein kinase C, did not produce any changes in mitochondrial AP activity, but an inhibition of 1,25(OH)2D3 and an activation of 24,25(OH)2D3 synthesis were found. Moreover, incubation of LLC-PK1 cells with PMA in conjunction with 1,25(OH)2D3 produced an additive effect in the decrease of 1,25(OH)2D3 and an increase of 24,25(OH)2D3 synthesis remaining mitochondrial AP activity as cells treated only with 1,25(OH)2D3. Our results suggest that mitochondrial AP activity could be involved as an intracellular signal in the regulation of 25(OH)D3 metabolism to the synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 in renal phenotype LLC-PK1 cells through cAMP protein kinase system.     

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.     

Effect of 1,25(OH)2D3 on the relative contributions of skeletal 45Ca and intestinal 40Ca to blood calcium in normal and thyroparathyroidectomized dogs

The effects of gradually increasing doses of 1,25(OH)2D3 on plasma calcium and 45Ca radioactivity were studied in young dogs that had been extensively prelabelled with 45Ca. The effects of orally and intravenously administered 1,25(OH)2D3 were evaluated in normal and thyroparathyroidectomized dogs fed a normal diet. In normal dogs when 1,25(OH)2D3 increased the plasma calcium within the normal range (2.9-3.1 mmol/L) there was no significant increase in plasma 45Ca. In thyroparathyroidectomized dogs, oral or intravenous 1,25(OH)2D3 increased the low blood calcium to a normal level (1.8-2.9 mmol/L) without significantly increasing plasma 45Ca. In normal and thyroparathyroidectomized dogs, any 1,25(OH)2D3-induced increase in plasma calcium above the normal range was associated with a significant increase in 45Ca, indicating mobilization of bone calcium. Intravenous administration of 1,25(OH)2D3 in the normal or thyroparathyroidectomized dogs had a much larger effect than oral doses in mobilizing bone 45Ca when inducing a similar level of hypercalcemia. The major physiological effect of 1,25(OH)2D3 in the low or normal range of plasma calcium is on intestinal absorption of calcium without a significant effect on mobilizing bone calcium. The pharmacological effect of 1,25(OH)2D3 in vivo is to mobilize bone calcium as well as dietary calcium into blood.     

Effect of 24,25-dihydroxyvitamin D3 in osteoclasts.

Previous results demonstrated that the administration of pharmacological doses of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) to animals reduces bone resorption and increases bone volume with a decrease in osteoclast number. In order to clarify whether 24,25(OH)2D3 has an effect to inhibit osteoclastic bone resorption, the effect of 24,25(OH)2D3 on the formation and function of osteoclastic cells was examined in vitro. Treatment of hemopoietic blast cells, which are progenitors of osteoclasts, with parathyroid hormone (PTH) or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) stimulated the formation of osteoclast-like multinucleated cells in a dose-dependent manner. Although 24,25(OH)2D3 in itself had little effect on osteoclast-like multinucleated cells formation, it inhibited the stimulatory effect of PTH on the formation of osteoclastic cells. In addition, 24,25(OH)2D3 also inhibited the stimulation of resorption pit formation by osteoclasts under stimulation with PTH. In contrast, 1,25(OH)2D3 stimulated the formation and function of osteoclastic cells even at low concentrations, and the effect was additive to PTH. These results could not be explained by either an agonistic or antagonistic effect of 24,25(OH)2D3 on 1,25(OH)2D3, and are consistent with the assumption that 24,25(OH)2D3 has a unique inhibitory effect on the formation and function of osteoclasts. Because 24,25(OH)2D3 is shown to stimulate the degradation of 1,25(OH)2D3 and because the formation of 24,25(OH)2D3 is stimulated by 1,25(OH)2D3 not only in the kidney but also in many of its target tissues, including bone, the inhibitory effect of 24,25(OH)2D3 on osteoclastic bone resorption may play a role in the local modulation of the actions of osteotropic hormones in bone.     

Biologic effect of 1,24(R)(OH)2D3 versus 1,25(OH)2D3 administration in chronic renal failure

1,24(R)(OH)2D3 is a synthetic analogue of 1,25(OH)2D3 which binds to the same receptors as the physiologic metabolite with a lower affinity. The aim of the present study was to compare the activity of 1,24(R)(OH)2D3 and 1,25(OH)2D3 on several target organs in patients with chronic renal failure. Treatment with 1,24(R)(OH)2D3 at doses of either 1 or 2 μg daily was carried out in two groups of 9 patients, with serum creatinine of 4.61 ± 1.59 and 4.66 ± 1.46 mg/dl, respectively. Doses of 1,25(OH)2D3 were 0.5 and 1 μg daily and were administered to 9 and 13 patients, serum creatinine of 4.52 ± 1.67 and 4.3 ± 1.16 mg/dl, respectively. Treatment periods were of 2 weeks. Administration of 1,25(OH)2D3, 1 μg, induced significant increments of intestinal calcium absorption (ICA), ionized calcium, osteocalcin, serum creatinine, urine Ca/GFR, and a decrease in iPTH. 1,25(OH)2D3, 0.5 μg, induced a significant increase in ICA and osteocalcin and a decrease in iPTH. Similarly 1,24(OH)2D3, 2 μg daily, significantly stimulated ICA and raised serum levels of osteocalcin and creatinine while lowering serum iPTH. In addition, 1,24(R)(OH)2D3 administration induced a significant fall of serum 1,25(OH)2D3. Following 1 μg, only osteocalcin increased. Therefore, the dose of 2 μg of 1,24(R)(OH)2D3 has biologic activity similar to 0.5 μg 1,25(OH)2D3 (4:1). However the activity ratio on osteocalcin production appears to be 2:1. In addition, 1,24(R)(OH)2D3 is able to inhibit renal tubular 1-hydroxylase. In conclusion 1,24(R)(OH)2D3 may prove to be useful in the treatment of metabolic bone disease.     

Responses of rachitic cartilage cells to metabolites of vitamin D3

Responses of cultured cartilage cells to metabolites of vitamin D3 were studied. Cells were obtained from the epiphyseal growth plate of rachitic chicks and were exposed to physiological and pharmacological concentrations of three metabolites of vitamin D3, 25 hydroxyvitamin D3 (25(OH)D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). 1,25(OH)2D3 was found to reduce L-[U-14C]leucine incorporation into proteins and Na2 35SO4 incorporation into proteoglycans. The synthesis of 24,25(OH)2D3 from 25(OH)D3 was stimulated upon addition of 1,25(OH)2D3 to the cultures. Physiological concentrations of 24,25(OH)2D3 stimulated protein and proteoglycan synthesis. These findings support the notion that vitamin D3, through its active dihydroxylated metabolites, is directly involved in cartilage cells metabolism and healing of rickets.     

Localization of vitamin D3-responsive alkaline phosphatase in cultured chondrocytes

Alkaline phosphatase activity appears to be altered when chondrocyte cultures are incubated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). This study examined whether the hormone-responsive enzyme activity is associated with alkaline phosphatase-enriched extracellular membrane organelles called matrix vesicles. Confluent, third passage cultures of rat costochondral growth cartilage (GC) or resting zone chondrocytes (RC) were incubated with 1,25-(OH)2D3 or 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) and enzyme specific activity was assayed in the cell layer or in isolated matrix vesicle and plasma membrane fractions. Alkaline phosphatase-specific activity in the matrix vesicles was enriched at least 2-fold over that of the plasma membrane and 10-fold over that of the cell layer. Matrix vesicle alkaline phosphatase was stimulated by 1,25-(OH)2D3 in GC cultures and by 24,25-(OH)2D3 in RC cultures. The cell layer failed to reveal these subtle differences. 1,25-(OH)2D3 increased GC enzyme activity but the effect was one-half that observed in the matrix vesicles alone. No effect of 1,25-(OH)2D3 on enzyme activity of the RC cell layer or of 24,25-(OH)2D3 on either GC or RC cell layers was detected. Thus, response to the metabolites is dependent on chondrocytic differentiation and is site specific: the matrix vesicle fraction is targeted and not the cells per se.     

Evidence for distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) in osteoblasts

1 alpha,25-(OH)(2)D(3) exerts its effects on chondrocytes and enterocytes via nuclear receptors (1,25-nVDR) and a separate membrane receptor (1,25-mVDR) that activates protein kinase C (PKC). 24R,25-(OH)(2)D(3) also stimulates PKC in chondrocytes, but through other membrane mechanisms. This study examined the hypothesis that osteoblasts possess distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) that are involved in the activation of PKC and that receptor expression varies as a function of cell maturation state. 1 alpha,25-(OH)(2)D(3) stimulated PKC in well differentiated (UMR-106, MC-3T3-E1) and moderately differentiated (ROS 17/2.8) osteoblast-like cells, and in cultures of fetal rat calvarial (FRC) cells and 2T3 cells treated with rhBMP-2 to promote differentiation. 24R,25-(OH)(2)D(3) stimulated PKC in FRC and 2T3 cultures that had not been treated to induce differentiation, and in ROS 17/2.8 cells. MG63 cells, a relatively undifferentiated osteoblast-like cell line, had no response to either metabolite. Ab99, a polyclonal antibody generated to the chick enterocyte 1,25-mVDR, but not a specific antibody to the 1,25-nVDR, inhibited response to 1 alpha,25-(OH)(2)D(3). 1 alpha,25-(OH)(2)D(3) exhibited specific binding to plasma membrane preparations from cells demonstrating a PKC response to this metabolite that is typical of positive cooperativity. Western blots of these membrane proteins reacted with Ab99, and the Ab99-positive protein had an Mr of 64 kDa. There was no cross-reaction with antibodies to the C- or N-terminus of annexin II. The effect of 24,25-(OH)(2)D(3) on PKC was stereospecific; 24S,25-(OH)(2)D(3) had no effect. These results demonstrate that response to 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) depends on osteoblast maturation state and suggest that specific and distinct membrane receptors are involved.     

Contributions of pro-oxidant and anti-oxidant conditions to the actions of 24,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on phosphate uptake in intestinal cells

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells, while the steroid 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by 1,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against the 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake. Incubation of cells in the presence of 50 nM catalase was also found to alleviate inhibition. In another series of experiments, isolated intestinal epithelial cells were incubated as controls or with 1,25(OH)2D3, each in the presence of the catalase inhibitor 3-amino-1,2,4-triazole, or with 1,25(OH)2D3 alone. Cells exposed to hormone alone again showed an increased accumulation of 32P, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for 32P uptake, occurred. Incubation of cells with phorbol-13-myristate (PMA) increased 32P uptake, while cells pretreated with 50 microM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity while cells exposed to H2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.     

Arachidonic acid is an autocoid mediator of the differential action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes

Prior studies have shown that 24,25-(OH)₂D₃ and 1,25-(OH)₂D₃ regulate protein kinase C (PKC) in costochondral chondrocytes in a cell maturation-dependent manner, with 1,25-(OH)₂D₃ affecting primarily growth zone (GC) cells and 24,25-(OH)₂D₃ affecting primarily resting zone (RC) cells. In addition, 1,25-(OH)₂D₃ has been shown to increase phospholipase A₂ activity in GC, while 24,25-(OH)₂D₃ has been shown to decrease phospholipase A₂ activity in RC. Stimulation of phospholipase A₂ in GC caused an increase in PKC, whereas inhibition of phospholipase A₂ activity in RC cultures increased both basal and 24,25-(OH)₂D₃-induced PKC activity, suggesting that phospholipase A₂ may play a central role in mediating the effects of the vitamin D metabolites on PKC. To test this hypothesis, RC and GC cells were cultured in the presence and absence of phospholipase A₂ inhibitors (quinacrine and oleyloxyethylphosphorylcholine [OEPC]), phospholipase A₂ activators (melittin and mastoparan), or arachidonic acid alone or in the presence of the target cell-specific vitamin D metabolite. PKC specific activity in the cell layer was determined as a function of time. Phospholipase A₂ inhibitors decreased both basal and 1,25-(OH)₂D₃-induced PKC activity in GC. When phospholipase A₂ activity was activated by inclusion of melittin or mastoparan in the cultures, basal PKC activity in RC was reduced, while that in GC was increased. Similarly, melittin and mastoparan decreased 24,25-(OH)₂D₃-induced PKC activity in RC and increased 1,25-(OH)₂D₃-induced PKC activity in GC. For both cell types, the addition of arachidonic acid to the culture media produced an effect on PKC activity that was similar to that observed when phospholipase A₂ activators were added to the cells. These results demonstrate that vitamin D metabolite-induced changes in phospholipase A₂ activity are directly related to changes in PKC activity. Similarly, exogenous arachidonic acid affects PKC in a manner consistent with activation of phospholipase A₂. These effects are cell maturation- and time-dependent and metabolite-specific. J. Cell. Physiol. 176:516–524, 1998. © 1998 Wiley-Liss, Inc.     

A-ring analogues of 1,25-(OH)2D3 with low affinity for the vitamin D receptor modulate chondrocytes via membrane effects that are dependent on cell maturation

1,25-(OH)₂D₃ (1,25) and 24,25-(OH)₂D₃ (24,25) mediate their effects on chondrocytes through the classic vitamin D receptor (VDR) as well as through rapid membrane-mediated mechanisms, which result in both nongenomic and genomic effects. In intact cells, it is difficult to distinguish between genomic responses via the VDR and genomic and nongenomic responses via membrane-mediated pathways. In this study, we used two analogues of 1,25 that have been modified on the A-ring (2a, 2b) and are only 0.1% as effective in binding to the VDR as 1,25, to examine the role of the VDR in the response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25 and 24,25. Chondrocyte proliferation ([³H]-thymidine incorporation), proteoglycan production ([³⁵S]-sulfate incorporation), and second messenger activation (activity of protein kinase C) were measured after treatment with 10^-8 M 1,25, 10^-7 M 24,25, or the analogues at 10^-9–10^-6 M. Both analogues inhibited proliferation of both cell types, as did 1,25 and 24,25. Neither 2a nor 2b had an effect on proteoglycan production by GCs or RCs. 2a caused a dose-dependent stimulation of protein kinase C (PKC) that was not inhibited by cycloheximide or actinomycin D in either GC or RC cells. 2b, on the other hand, had no effect on PKC activity in RCs and only a slight stimulatory effect in GCs. Both cells produce matrix vesicles, extracellular organelles associated with the initial stages of calcification, in culture that are regulated by vitamin D metabolites. Since these organelles contain no DNA or RNA, they provide an excellent model for studying the mechanisms used by vitamin D metabolites to mediate their nongenomic effects. When matrix vesicles were isolated from naive cultures of growth zone cells and treated with 2a, a dose-dependent inhibition of PKC activity was observed that was similar to that found with 1,25-(OH)₂D₃. Plasma membranes contained increased PKC activity after treatment with 2a, but the magnitude of the effect was less than that seen with 1,25-(OH)₂D₃. Analogue 2b had no affect on PKC activity in either membrane fraction. When matrix vesicles from resting zone chondrocyte cultures were treated with 24,25-(OH)₂D₃, a significant decrease in PKC activity was observed. No change in enzyme activity was found for either 1,25-(OH)₂D₃ or the analogues. PKC activity in the plasma membrane fraction, however, was increased by 24,25-(OH)₂D₃ as well as by analogue 2a. This study shows that these analogues, with little or no binding to the vitamin D receptor, can affect cell proliferation and PKC activity, but not proteoglycan production. The direct membrane effect is analogue specific and cell maturation dependent. Further, by eliminating the VDR-mediated component of the cellular response, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites. J. Cell. Physiol. 171:357–367, 1997. © 1997 Wiley-Liss, Inc.     

Enhanced effects of guggulsterone plus 1,25(OH)2D3 on 3T3-L1 adipocytes

Guggulsterone (GS) and 1,25-dihydroxyvitamin D3 [1,25(OH)₂D₃] have been shown to influence adipogenesis in 3T3-L1 cells. We investigated the ability of GS and 1,25(OH)₂D₃, alone and in combination to inhibit adipogenesis and induce apoptosis in 3T3-L1 adipocytes. Maturing preadipocytes were treated with 1,25(OH)₂D₃ in combination with GS for 6 days during differentiation. GS and 1,25(OH)₂D₃ each inhibited lipid accumulation, but the combination potentiated the inhibition of lipid accumulation. Apoptosis was increased by 1,25(OH)₂D₃ while GS had no effect, but GS + 1,25(OH)₂D₃ increased apoptosis more than either compound alone. Furthermore, GS + 1,25(OH)₂D₃ caused a potentiated decrease in the expression of aP2 and farnesoid X receptor expression more than either compound alone. In addition, 1,25(OH)₂D₃ increased vitamin D receptor expression after 6 days, while GS had no effect. GS + 1,25(OH)₂D₃, however, caused a potentiated increase in the expression of VDR. These findings show that GS potentiates 1,25(OH)₂D₃’s anti-adipogenic and pro-apoptotic effects in maturing 3T3-L1 preadipocytes.     

Induction of 25-OH-vitamin D3 24- and 23-hydroxylase activities in partially purified renal extracts from pigs given exogenous 1,25-(OH)2D3

A renal mitochondrial cytochrome P 450 preparation from pigs treated with exogenous 1,25-(OH)2D3 was reconstituted with an NADPH-generating system, adrenodoxin and adrenodoxin reductase. The reconstituted system catalyzed the conversion of the substrate, 25-OH-D3, to metabolites comigrating with authentic 23,25-(OH)2D3 and 24,25-(OH)2D3 in both straight- and reverse-phase high-performance liquid chromatography systems, which achieve separation of these metabolites from each other as well as from other vitamin D metabolites. The putative 23,25-(OH)2D3 product was resistant to periodate treatment, while the 24,25-(OH)2D3 product was sensitive, providing additional evidence for the identity of the products. Although induction of 24-hydroxylase activity has been studied using renal homogenates from several species, only recently have techniques become available to study the activity of the enzyme in a solubilized and reconstituted form. Using these techniques, the present study shows that production of 24,25-(OH)2D3 was increased more than 80-fold with 1,25-(OH)2D3 treatment compared with untreated controls, an effect much greater than that previously observed with homogenates. In addition, production of both 23,25-(OH)2D3 and 24,25-(OH)2D3 varied with substrate concentration and was consistent with a monooxygenase-linked enzyme reaction.     

1,25(OH)2D3 increases cytosolic Ca++ concentration of osteoblastic cells, clone MC3T3-E1

Effect of 1,25(OH)2D3 in vitro on cytosolic Ca++ concentration of osteoblastic cells (MC3T3-E1) was studied. Marked but transient increase of cytosolic Ca++ concentration of osteoblastic cells was observed following the addition of 10 pg/ml of 1,25(OH)2D3, but not with 10 pg/ml of 24,25(OH)2D3. The increase of cytosolic Ca++ concentration of osteoblastic cells by 1,25(OH)2D3 was not observed when the cells were incubated in Ca++ free medium. Therefore, it was concluded that 1,25(OH)2D3 increased cytosolic Ca++ concentration of osteoblastic cells through the increase of Ca++ influx into the cells.     

1,25(OH)2D3 inhibits in vitro and in vivo intracellular growth of apicomplexan parasite Toxoplasma gondii

The hormonal form of vitamin D, 1,25-dyhydroxyvitamin D3 (1,25(OH)2D3), is implicated in a wide range of functions other than its classical role in calcium and phosphorous homeostasis. When Toxoplasma gondii-infected BALB/c mice were treated with 1,25(OH)2D3, they succumb to death sooner than their counterparts. But they showed less parasite burden in tissues which was further supported by mild pathological lesions. As an effort to understand the physiological mechanism for the above observation an in vitro study was performed. Fewer parasites were observed when 1,25(OH)2D3 pre-treated murine intestinal epithelial cells were challenged with parasites. Moreover, the observed inhibition was dose-dependent and had a maximum effect with 10(-7)M of 1,25(OH)2D3. However, no observable difference was observed, when pre-incubated parasites were added to cells suggesting that the observed inhibition was a result of an effect from 1,25(OH)2D3 on Toxoplasma intracellular growth. Our data support the notion that 1,25(OH)2D3 may inhibit intra cellular T. gondii parasite proliferation in vivo and in vitro.