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.     

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.     

1,25-二羟维生素D3的生物学效应

1,25-二羟维生素D3是维生素D3的活性形式,其生物学效应是由基因组与非基因组两种机制介导的。维生素D3除了具有钙磷代谢调节作用外,还具有其他更为广泛的生物学效应。1,25-二羟维生素D3能够抑制多种类型细胞的增殖,诱导细胞的凋亡和分化,调节机体免疫系统功能,保护中枢神经系统,以及保护基因等。     

The cellular and molecular regulation of 1,25(OH)2D3 production.

The synthesis of 1,25(OH)₂D₃ is a critical control point in the regulation of calcium metabolism, and possibly in the growth and differentiation of a number of cell types. This paper reviews our current understanding of the regulation of this process at the cellular and molecular levels, with the emphasis on the mechanisms of feedback control 1,25(OH)₂D₃ itself, control of parathyroid hormone, the roles of cyclic AMP dependent protein kinase and protein kinase C, and the interaction between the various intracellular regulators of 1,25(OH)₂D₃ production.     

Regulation of 1,25(OH)2D3 (calcitriol) receptor by vitamin B6

In vitamin B6-deficient rats the concentration of in vivo occupied nuclear and total cellular receptors of 1.25(OH)2D3 increases 1.3-1.7 times, whereas the binding of in vitro occupied receptors to DNA-cellulose increases by 40%. Pyridoxal-5'-phosphate (PLP) added in vitro to solubilized receptors of 1.25(OH)2D3 lowers the ligand binding by 15-25% but causes no dissociation of hormone-receptor complexes formed in vivo. The association of in vitro occupied receptors of 1.25(OH)2D3 with DNA-cellulose is suppressed by PLP (3.5-4.5-fold). It has been shown for the first time that vitamin B6 is a physiological regulator of 1.25(OH)2D3 receptor binding by chromatin and DNA which diminish the concentration of occupied receptors and thus suppress the hormonal response.     

The effect of 1,25-dihydroxyvitamin D3 on in vitro immunoglobulin production in human B cells

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently suppressed immunoglobulin (Ig) production of human B cells, as evaluated by IgG-plaque-forming cells (IgG-PFC) in the culture of pokeweed mitogen (PWM)-activated B cells. Similar suppressive effect of 1,25(OH)2D3 on Ig production of B cells was observed in the Staphylococcus aureus Cowan I(SAC)-induced Ig-producing system. The mean percentage of inhibitions at a concentration of 10(-9) M were 60.0 +/- 8.2% (mean +/- SE, n = 6) and 65.1 +/- 4.7% (n = 10) in PWM- and SAC-stimulated cultures, respectively. The suppression was strongly exhibited only when 1,25(OH)2D3 was added at the start of the 6-day culture, accompanied by a decrease in DNA synthesis of B cells in both culture systems. On the other hand, the addition of 1,25(OH)2D3 on day 4, when DNA synthesis reached at plateau and IgG-PFC began to be detectable, had no noticeable affect on either the number of PFC or DNA synthesis of B cells. Furthermore, 1,25(OH)2D3 suppressed Ig production even when B cells were exposed to the agent for 4 hr after the activation with PWM or SAC, but not before the activation. These results indicate that 1,25(OH)2D3 inhibits B cell proliferation before differentiation to Ig-secreting cells, consequently reducing Ig production; and that its action appears to be mediated by the cytosol receptors expressed on activated B cells. Thus, the agent may serve as an immunoregulating hormone in vivo, as well as in vitro.     

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.     

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.     

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.     

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.     

Role of the 1,25D3-MARRS receptor in the 1,25(OH)2D3-stimulated uptake of calcium and phosphate in intestinal cells

We have used mice with a targeted knockout (KO) of the 1,25D(3)-MARRS receptor (ERp57/PDIA3) in intestine to study rapid responses to 1,25-dihydroxyvitamin D(3) [1,25D(3)] with regards to calcium or phosphate uptake. Western analyses indicated the presence of the 1,25D(3)-MARRS receptor in littermate (LM) mice, but not KO mice. Saturation analyses for [(3)H]1,25D(3) binding revealed comparable affinities for the hormone in lysates from female and male LM, but a reduced B(max) in females. Binding in lysates from KO mice was absent or severely reduced. Enterocytes from KO mice failed to respond to hormone with regard to either ion uptake, while cells from LM mice exhibited an increase in uptake. For calcium uptake, the protein kinase (PK) A pathway mediated the response to 1,25D(3). Enterocytes from LM mice responded to 1,25D(3) with enhanced PKA activity, while cells from KO mice did not, although both cell types responded to forskolin. Calcium transport in LM mice in vivo was greater than in KO mice. Cells from LM and KO mice had cell surface VDR; however, anti-VDR antibodies had no effect on ion uptake. Unlike chicks, the PKC pathway was not involved in phosphate uptake. As in chicks and rats, intestinal cells from adult male mice lost the ability to respond to 1,25D(3) with enhanced phosphate uptake, whereas in female mice, uptake in cells from adults was greater than that observed in young mice. Finally, when we tested phosphate uptake in vivo, we found that young female mice had a much greater rate of transport than young male mice.     

1,25(OH)2D3 increases membrane associated protein kinase C in MDBK cells.

To determine whether 1,25-dihydroxycholecalciferol [1,25(OH)2D3] affects protein kinase C (PKC) activity in kidney, as has been demonstrated in HL-60 cells we measured 1,25(OH)2D3 binding, PKC activity and PKC immunoreactivity in Madin Darby bovine kidney (MDBK) cells, a normal renal epithelial cell line derived from bovine kidney. Our data demonstrate that MDBK cells exhibit specific high affinity binding for 1,25(OH)2D3, indicating the presence of the vitamin D receptor (VDR). Treatment of MDBK cells with 1,25(OH)2D3 for 24 h increased membrane PKC activity and immunoreactivity. The effect of 1,25(OH)2D3 was dose-dependent, with a peak effect observed at 10(-7)M 1,25(OH)2D3. The 1,25(OH)2D3 induced increase in membrane PKC was paralleled by a comparable decrease in cytosolic PKC activity and amount. Although time course studies were consistent with a VDR mediated effect of 1,25(OH)2D3 on PKC protein synthesis, total PKC activity was not increased by 1,25(OH)2D3, suggesting an effect on PKC translocation or localization. These results suggest that 1,25(OH)2D3 modulates PKC mediated events in kidney, a classic target for this steroid hormone.     

1,25(OH)2 vitamin D3 sites of action in the brain

Summary After injection of ³H 1,25(OH)₂ vitamin D₃ to adult rats and mice, under normal or vitamin D deficient diet, the hormone was found to be accumulated in nuclei of neurons in certain brain regions. Nuclear concentration was prevented or diminished, when excess unlabeled 1,25 (OH)₂ vitamin D₃ was injected before ³H 1,25(OH)₂ vitamin D₃, while excess 25 (OH) vitamin D₃ did not prevent nuclear labeling.Highest nuclear concentration of ³H 1,25 (OH)₂ vitamin D₃ is observed in certain neurons in the nucleus interstitialis striae terminalis, involving its septo-preoptic pars dorsolateralis and its anterior hypothalamic-thalamic portion, and in the nucleus centralis of the amygdala, all constituting a system of target neurons linked by a component of the stria terminalis. Nuclear concentration of ³H 1,25 (OH)₂ vitamin D₃ is also found in neurons in the periventricular nucleus of the preoptic-hypothalamic region, including its extensions, the parvocellular paraventricular and arcuate nucleus, in the ventromedial nucleus, supramammillary nucleus, reticular nucleus of the thalamus, ventral hippocampus, caudate nucleus, pallium, in the midbrain-pontine central gray, dorsal raphe nucleus, parabrachial nuclei, cranial motor nuclei, substantia gelatinosa of the sensory nucleus of the trigeminus, Golgi type II cells of the cerebellum, and others.The extensive distribution of target neurons suggests that 1,25(OH)₂ vitamin D₃ regulates the production of several aminergic and peptidergic messengers, and influences the activity of certain endocrine-autonomic, sensory and motor systems.     

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).