Rapid action of 1,25-dihydroxyvitamin D3 on hepatocyte phospholipids

Recent studies have reported cellular effects of 1,25-dihydroxyvitamin D3 within 15 minutes, a time period too rapid to be mediated by nuclear activation. The vitamin increases hepatocyte cytosolic calcium levels in the absence of extracellular calcium within 5 minutes. Since metabolites of phosphatidylinositol have been implicated as second messengers in the regulation of cytosolic calcium, we examined the effect of 1,25-dihydroxyvitamin D3 on hepatocyte phosphatidylinositol turnover and compared these effects to those produced by vasopressin. In isolated hepatocytes labeled with [3H]inositol, 1,25-dihydroxyvitamin D3 (4 nM) increased [3H]glycerophosphorylinositol by 16% (p less than 0.01) within 2.5 minutes, by 18% (p less than 0.01) after 5 minutes, and by 11% (p less than 0.05) after 10 minutes. At a concentration of 20 nM, 1,25-dihydroxyvitamin D3 increased [3H]glycerophosphorylinositol by 27% (p less than 0.01) after 5 minutes. Vitamin D did not affect [3H]inositol polyphosphates. Conversely, vasopressin had no effect on [3H]glycerophosphorylinositol but significantly increased [3H]inositol phosphate, [3H]inositol bisphosphate, and [3H]inositol triphosphate. 1,25-Dihydroxyvitamin D3 (4 nM) decreased [3H]phosphatidylinositol by 10% (p less than 0.05) after 5 minutes and by 16% (p less than 0.01) after 10 minutes. At a concentration of 20 nM, 1,25-dihydroxyvitamin D decreased [3H]phosphatidylinositol by 18% (p less than 0.01) after 5 minutes. The vitamin did not affect [3H]phosphatidylinositol bisphosphate or [3H]phosphatidylinositol trisphosphate. 24,25-Dihydroxyvitamin D had no effect on inositol phospholipids. The effects of 1,25-dihydroxyvitamin D3 on inositol phospholipids were blocked by quinacrine. Bromophenacylbromide inhibited the effects of 1,25-dihydroxyvitamin D3 on inositol phospholipids and also blocked the vitamin-induced increments in cytosolic calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

1 Alpha,25-dihydroxyvitamin D3 rapidly increases cytosolic calcium in clonal rat osteosarcoma cells lacking the vitamin D receptor.

1 alpha,25-Dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] rapidly increases cytosolic calcium in a variety of cell types. Although these rapid effects do not appear to directly involve genome activation, the requirement for the classic vitamin D receptor is unclear. Clonal rat osteosarcoma cells, ROS 17/2.8, respond to 1 alpha,25-(OH)2D3 with an increase in osteocalcin message but ROS 24/1 cells do not. The lack of the receptor for vitamin D in the ROS 24/1 cells has been confirmed by the absence of any detectable vitamin D-receptor complex binding to the vitamin D-responsive element (VDRE) of the osteocalcin gene and the absence of vitamin D receptor mRNA in the cells. Quin-2-loaded ROS 17/2.8 and ROS 24/1 cells were treated with 1 alpha,25-(OH)2D3 in the presence and absence of extracellular calcium and with the inactive epimer, 1 beta,25-dihydroxyvitamin D3 [1 beta,25-(OH)2D3]. The 1 alpha,25-(OH)2D3 increased cytosolic calcium in the ROS 17/2.8 and 24/1 cells after 5 minutes in a dose-responsive manner and in the presence and absence of extracellular calcium. Pretreatment of both cell lines with 1 beta,25-(OH)2D3 for 30 s blocked the hormone-induced rise in cytosolic calcium. The rapid effects of 1 alpha,25-(OH)2D3 on ROS cells with and without the vitamin D receptor and the ability of the inactive epimer to inhibit these effects indicate that the signaling system mediating the hormone's rapid actions is not the classic vitamin D receptor.     

1Alpha,25-dihydroxyvitamin D(3) - not just a calciotropic hormone

1Alpha,25-dihydroxyvitamin D(3), the hormonal form of vitamin D(3), is widely appreciated to play a central role in calcium and phosphorous homeostasis. It is becoming increasingly clear, however, that the sterol also plays an important role in the regulation of cellular growth, central nervous system function, and immune responsiveness. In this review, I will highlight some of the mechanisms by which 1alpha,25-dihydroxyvitamin D(3) regulates cellular growth, alters central nervous system function, and immune function.     

Evidence for abnormal regulation of circulating 1 alpha, 25-dihydroxyvitamin D in patients with pulmonary tuberculosis and normal calcium metabolism

Available evidence indicates that hypercalcemia in pulmonary tuberculosis results from increases in circulating 1 alpha, 25-dihydroxyvitamin D [1 alpha, 25(OH)2D]. To further characterize vitamin D metabolism in this disorder, the effects of vitamin D, 100,000 units a day for 4 days, were compared in 25 normal subjects and 11 patients with active pulmonary tuberculosis who were normocalcemic and had not had hypercalcemia. Serum calcium, phosphorus, 25-hydroxyvitamin D (25-OHD) and 1 alpha, 25(OH)2D were measured. Whereas vitamin D increased mean serum 25-OHD from 20 +/- 2 (+/- SE) to 40 +/- 5 ng/ml (P less than 0.001) and did not change mean serum 1 alpha, 25(OH)2D in the normals (33 +/- 2 vs. 31 +/- 2 pg/ml), it increased mean serum 25-OHD from 21 +/- 4 to 55 +/- 13 ng/ml (P less than 0.05) and mean serum 1 alpha, 25(OH)2D from 28 +/- 2 to 35 +/- 3 pg/ml (P less than 0.05) in the patients. Serum calcium was normal and remained within the normal range in all subjects and patients. The findings indicate that there is a modest but significant abnormality in the regulation of circulating 1 alpha, 25(OH)2D in normocalcemic patients with pulmonary tuberculosis. The results are similar to those previously reported by us in normocalcemic patients with sarcoidosis.     

Inhibitory effect of 1 alpha,25-dihydroxyvitamin D3 on the growth of the renal carcinoma cell line

We studied the effect of vitamin D compounds on the growth of the human renal carcinoma cell line (KU-2) and discovered a receptor protein specific for the active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3. The KU-2 cell line was established from a pulmonary metastasis of renal cell carcinoma in a patient with hyperhemoglobinemia. The cells were tumorigenic in nude mice and clonogenic in a soft agar culture. Vitamin D3 derivatives suppressed proliferation of KU-2 cells in a monolayer culture and also clonogenicity in a soft agar culture dose-dependently. Of the vitamin D3 derivatives tested, 1 alpha,25-dihydroxyvitamin D3 was the most potent in inhibiting cell growth, followed successively by 1 alpha,24R,25-trihydroxyvitamin D3, 25-hydroxyvitamin D3, 1 alpha-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 in that order. Analysis of the cell cycle phase of treated and non-treated KU-2 cells revealed that the action of 1 alpha,25-dihydroxyvitamin D3 was not phase-specific but simply extended the doubling time of the cells. Radioreceptor assay and sucrose density gradient analysis of the cytosol showed that KU-2 cells contained a 3.2S receptor protein to which 1 alpha,25-dihydroxyvitamin D3 was specifically bound (Kd = 20.8 +/- 4.8 pM, Nmax = 87 +/- 24 fmole/mg protein, 4000 molecules/cell). On the other hand, the equilibrium dissociation constant of internalization of 1 alpha,25-dihydroxyvitamin D3 (Kint) by intact KU-2 cells was 1.2 nM and the internalizing capacity was 33 fmole/8 X 10(6) cells (2500 molecules/cell) in the 10% serum medium, which was the same as that used in the growth study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetics of active vitamins D3, 1 alpha-hydroxyvitamin D3 and 1 alpha, 25-dihydroxyvitamin D3 in patients on chronic hemodialysis

Two active forms of vitamin D3, 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25 [OH] 2D3) and 1 alpha-hydroxyvitamin D3 (1 alpha, [OH] D3) are widely used for treating osteodystrophy in patients with chronic renal failure. Since the pharmacokinetics of these agents during long-term oral administration are unclear, we measured the circulating concentrations of 1 alpha, 25 (OH) 2D before and after their long-term oral administration in patients receiving maintenance hemodialysis. After 12 weeks of treatment with a daily dose of 1 alpha, (OH) D3 (0.5 micrograms), the administration of a single dose (2 micrograms) of 1 alpha, (OH) D3 showed that the area under the curve over 24 h (AUC) was increased significantly compared with day 1 of therapy. In contrast, after the treatment with a daily dose of 1 alpha, 25 (OH) 2D3 (0.25 micrograms), a single dose of 1 alpha, 25 (OH) 2D3 (1 microgram) did not exhibit this effect on the AUC. A single dose of each agent produced no significant change in either the peak increment above basal values or the elimination half-time (T 1/2) of circulating plasma 1 alpha, 25 (OH) 2D. The overall basal concentration of 1 alpha, 25 (OH) 2D achieved by 1 alpha, (OH) D3 after 12 weeks of administration was cumulative, but this effect was not observed in patients on 1 alpha, 25 (OH) 2D3. These data indicate that the pharmacokinetics of the two forms of active vitamin D3 did not differ as to peak increment and T1/2 except for the AUC, even after long-term dosage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of retinoic acid on the activation and fusion of mouse alveolar macrophages induced by 1 alpha,25-dihydroxyvitamin D3

1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] directly induces both fusion and cytotoxicity in murine alveolar macrophages. Unlike 1 alpha,25(OH)2D3, retinoic acid per se did not induce fusion of alveolar macrophages, but it greatly enhanced the 1 alpha,25(OH)2D3-induced fusion every time the macrophages were treated simultaneously with the two vitamins. The giant cells induced by the two vitamins were much larger than those induced by 1 alpha,25(OH)2D3 alone. The macrophages treated with 1 alpha,25(OH)2D3 started to fuse 36 h after the addition of the vitamin, whereas the macrophages pretreated with retinoic acid for 24 h began to fuse immediately after 1 alpha,25(OH)2D3 was added. 1 alpha,25(OH)2D3 and retinoic acid activated alveolar macrophages similarly, measured by the enhancement of glucose consumption and the induction of cytotoxicity against tumor cells, though 1 alpha,25(OH)2D3 was 100 times more potent than retinoic acid on a molar basis. Simultaneous treatment with physiological concentrations of 1 alpha,25(OH)2D3 (0.12 nM) and retinoic acid (10 nM) induced cytotoxicity additively. Morphological examinations revealed that the treated cells were enlarged and flattened with numerous filopodia. These results clearly indicate that both 1 alpha,25(OH)2D3 and retinoic acid similarly activate alveolar macrophages, and the activated state is prerequisite to the fusion of macrophages induced by 1 alpha,25(OH)2D3.     

Differential effects of 1,25-dihydroxyvitamin D3 on cytosolic calcium in two human cell lines (HL-60 and U-937)

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to induce maturational changes in the human promyelocytic leukemia cell line HL-60 and in the human monocytic cell line U-937. Changes in cytosolic calcium have been reported to regulate cellular processes. We used the fluorescent dye Quin 2 to examine the effects of vitamin D metabolites on cytosolic calcium levels in HL-60 and U-937 cells. 1,25-(OH)2D3 (20 nM) increases cytosolic calcium by 24% over a 5-min period in HL-60 but not in U-937 cells. 1,25-(OH)2D3 (0.2 nM and 4 nM) has no effect on cytosolic calcium levels in either cell type. 24,25-(OH)2D3 (20 nM) has no effect on cytosolic calcium in HL-60 cells. Nifedipine (1 mM) has no effect on cytosolic calcium levels over 30 min and likewise does not block the 1,25-(OH)2D3-induced increase in cytosolic calcium in HL-60 cells. However, chelation of extracellular calcium with EGTA (10 mM) blocks the 1,25-(OH)2D3-induced increment in cytosolic calcium, but does not block the 1,25-(OH)2D3-induced maturational changes in HL-60 cells. The data suggest that 1,25-(OH)2D3 but not 24,25-(OH)2D3 increases cytosolic calcium in HL-60 cells within 5 min and the increment is due to increased influx of calcium. 1,25-(OH)2D3 modifies membrane permeability to calcium independent of calcium channels sensitive to nifedipine. Finally, 1,25-(OH)2D3-induced maturational changes in HL-60 cells can take place without an increase in cytosolic calcium.     

5alpha-dihydrotestosterone inhibits 1alpha,25-dihydroxyvitamin D3-induced expression of CYP24 in human prostate cancer cells

BACKGROUND: A cross-talk between 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)] and 5alpha-dihydrotestosterone (DHT) in the growth inhibition has been demonstrated, but the mechanism is unknown. METHODS: The expression of 25-hydroxyvitamin D(3) 24-hydroxylase (24-hydroxylase) was measured using a real-time quantitative RT-PCR assay and the catabolism of 1alpha,25-(OH)(2)D(3) was measured using a radioreceptor assay. RESULTS: Real-time RT-PCR showed that DHT at 1-100 nM significantly inhibited 1alpha,25-(OH)(2)D(3)-induced expression of 24-hydroxylase in LNCaP cells. Furthermore, the catabolism of 1alpha,25-(OH)(2)D(3) was decreased by 10 nM DHT. An androgen receptor (AR) antagonist, Casodex antagonized the DHT effect, whereas an AR agonist (due to the mutant AR in LNCaP cells) hydroxyflutamide did not. CONCLUSIONS: We demonstrated, for the first time, that DHT reduces the ability of 1alpha,25-(OH)(2)D(3) to induce 24-hydroxylase expression. Our results not only support the earlier finding of a cross-talk between androgen and vitamin D in human prostate cancer cells but also provide a possible mechanism how androgen and vitamin D signaling pathways may interact.     

Newly established assay method for 25-hydroxyvitamin D3 24-hydroxylase revealed much lower Km for 25-hydroxyvitamin D3 than for 1alpha,25-dihydroxyvitamin D3.

An accurate assay method of 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) was established. Kidney mitochondria prepared from vitamin D-replete rats were treated with polyoxyethylenesorbitan monolaurate. The solubilized suspension was ultracentrifuged at 100,000g for 60 minutes and an aliquot of the supernatant was incubated under the saturating concentrations of substrate NADPH and the mitochondrial-type electron transferring proteins, adrenodoxin and NADPH-adrenodoxin reductase. Products were analyzed by high-performance liquid chromatography (HPLC) monitoring effluents at a wavelength of 265 nm. The maximal velocity of the enzyme in vitamin D-replete rats was 400 pmol/minute per mg of protein, which was considerably higher than those reported by previous authors who used intact kidney mitochondria as the enzyme source. In applying the new assay method, an interesting property was found; Michaelis constant of 24-hydroxylase for 25-hydroxyvitamin D3 [25(OH)D3] was 0.6 microM, which was 35-fold lower than that for 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] which was 20.9 microM. This fact indicates that affinity of the enzyme to 25(OH)D3 is 35-fold higher than that to 1alpha,25(OH)2D3. These data suggest that 25(OH)D3 is the preferred substrate to 1alpha,25(OH)2D3.     

24,25-dihydroxyvitamin D3 suppresses the rapid actions of 1, 25-dihydroxyvitamin D3 and parathyroid hormone on calcium transport in chick intestine.

Studies were undertaken to determine whether 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) modulates the rapid effects of 1, 25-dihydroxyvitamin D3 (1,25(OH)2D3) and parathyroid hormone (PTH) on calcium transport in the perfused chick intestine. Perfusion with control media resulted in a transport ratio (treated/average basal) of 1.07 +/- 0.06 at t = 40 minutes, while perfusion with 65, 130, 300, or 650 pM 1,25(OH)2D3 yielded ratios of 1.92 +/- 0.23, 2.6 +/- 0.4, 2.8 +/- 0.08, and 3.34 +/- 0.37, respectively. Simultaneous perfusion with each of these doses and 6.5 nM 24,25(OH)2D3 reduced treated/average basal ratios to approximately 1.4 after 40 minutes of perfusion. Vascular perfusion with 65 pM bovine PTH [bPTH(1-34)] stimulated intestinal calcium transport ratios to 3.0 +/- 0.5 after 40 minutes, while the inclusion of 6.5 nM 24,25(OH)2D3 reduced ratios at this time point to 0.56 +/- 0.19. To investigate the effect of these agents on signal transduction, isolated intestinal cells were monitored for intracellular calcium changes using the indicator dye fura-2. After establishing a stable baseline, addition of 130 pM 1,25(OH)2D3 induced rapid calcium oscillations. Intestinal cells exposed to 6.5 nM 24,25(OH)2D3 also exhibited rapid oscillations in fluorescence, which were not further altered by subsequent addition of 1,25(OH)2D3. Incubation of isolated cells with 130 pM 1,25(OH)2D3 was found to increase protein kinase C (PKC) activity within 5 minutes, and protein kinase A (PKA) activity within 7 minutes. Exposure of cells to 65 pM bPTH(1-34) had minimal effect on PKC activity, but resulted in pronounced increases in PKA activity. Stimulation of protein kinases by either secosteroid or peptide hormone was inhibited in the presence of 6.5 nM 24,25(OH)2D3. It is concluded that 24,25(OH)2D3 may exert endocrine actions on intestine.     

Effects of 1 alpha-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 on bone remodeling

The present study was undertaken to evaluate the effects of 1 alpha-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 on bone remodeling in dogs with osteomalacia induced by vitamin D depletion. To assess the rates of skeletal remodeling, intravital tetracycline labeling and morphometry of surface pattern were employed. Either vitamin D3 derivative accelerated the appositional growth rate, increased the percentage of osteoid seams labeled, and decreased the number, width, and perimeter of new osteoid seams. But the derivatives differed in bone resorbing activity: 1 alpha-hydroxyvitamin D3 increased the number and perimeter of resorption sites whereas 24R,25-dihydroxyvitamin D3 decreased them. Thus the results show that the former is a better bone remodeler while the latter may be useful in treating osteoporosis.     

Effect of 1,25-dihydroxyvitamin D3 on cytosolic calcium in dispersed parathyroid cells

We examined the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on cytosolic calcium ([Ca]i) of dispersed bovine parathyroid cells, using the fluorescent dye indo-1. The addition of 10(-8) M 1,25-(OH)2D3 caused an increase in [Ca]i by 23.4 +/- 2.7% over a 10 minute period. There was a significant increase in [Ca]i within two minutes of the addition of 1,25-(OH)2D3. 1,25-(OH)2D3 increased [Ca]i in a dose-dependent manner and this occurred with as little as 10(-10) M. Neither 10(-7) M 25-(OH)D3 nor 10(-7) M 24, 25-(OH)2D3 caused a significant increase in [Ca]i. Chelation of extracellular calcium with EGTA blocked the 1,25-(OH)2D3-induced increase in [Ca]i, suggesting that the increase was mainly from extracellular calcium. Neither 10(-5) M verapamil nor 10(-4) M diltiazem blocked the 1,25-(OH)2D3-induced increase in [Ca]i. The present data suggest that 1,25-(OH)2D3 might modify membrane permeability to calcium independent of voltage-dependent calcium channels sensitive to verapamil or diltiazem. The rapid effect of 1,25-(OH)2D3 raises the possibility that its mechanism is independent of genome activation, perhaps attributable to direct interaction with components of the parathyroid cell plasma membrane.     

大鼠睾丸中1，25－双羟维生素D＿3受体的免疫组织化学定位

用抗１，２５－（ＯＨ）２Ｄ３受体单克隆抗体（９Ａ７），以免疫组化法对正常大鼠睾丸行１，２５－（ＯＨ）２Ｄ３受体定位研究。成年雄性大鼠在苯巴比妥腹腔注射麻醉下，用生理盐水对心脏灌注放血致死，取睾丸置Ｂｏｕｎ＇ｓ液中固定２４小时，用振荡切片机制备５０μｍ厚切片，以ＡＢＣ法进行免疫反应，ＤＡＢ显色。在普通光镜下观察，发现睾丸曲精管内，特别是基底部的细胞有明显免疫反应。这表明雄性动物睾丸组织可能是１，２５－（ＯＨ）２Ｄ３的靶器官。     

Effect of a short course of 1,25-dihydroxyvitamin D3 on biochemical markers of bone remodeling in adult male volunteers.

To investigate the stimulatory effect of vitamin D on biochemical markers of bone remodeling, 15 normal men (aged 26-45 years, mean 33.2) were treated orally with 1,25-dihydroxyvitamin D3, 2 micrograms daily for 7 days, and followed for a total of 16 weeks. Serum concentrations of 1,25-dihydroxyvitamin D3 rose 43% during the first week (p less than 0.01), with no significant alteration in the level of 25-hydroxyvitamin D3. Serum level of immunoreactive parathyroid hormone (1-84) (iPTH) decreased markedly (p less than 0.02), and the maximal renal reabsorption capacity of phosphate (TmP/GFR) increased (p less than 0.05), both indicating the impact of the raised vitamin D level on target tissues. Serum phosphate and serum calcium increased during the treatment week (p less than 0.05), as did the fasting renal excretion of phosphate and calcium (p less than 0.01). However, a gradual fall in the excretion of hydroxyproline was seen in the observation period. The serum activity of acid phosphatase increased in the first weeks after vitamin D treatment, reaching significance at the end of week 2 (p less than 0.05). Acid phosphatase activity was still increased at the end of the observation period (p less than 0.02). These observations suggest a synchronization and recruitment of new bone resorptive cells. The immediate response to 1,25-dihydroxyvitamin D administration on the biochemical markers of formative bone cells was a marked increase in the serum level of osteocalcin (BGP), (p less than 0.002) with a gradually fall during the next weeks. A secondary increase, however, was observed in the last two months of the follow-up period.(ABSTRACT TRUNCATED AT 250 WORDS)     

An ultrastructural study on the multinucleation process of mouse alveolar macrophages induced by 1 alpha,25-dihydroxyvitamin D3

The multinucleation process of isolated alveolar macrophages induced by 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] was examined using a scanning electron microscope (SEM) and a transmission electron microscope (TEM). At the beginning of culture, most of the macrophages were spherical in shape. During incubation with 1.2 X 10(-8) M 1 alpha,25(OH)2D3, spreading macrophages appeared among the spherical macrophages, and they increased in number. Spreading macrophages extended many cytoplasmic processes toward adjacent macrophages, and interdigitations of these processes between those of neighboring cells were often seen. Two types of cell contact have been observed in the 1 alpha,25(OH)2D3-treated cells. In some, cytoplasmic processes were put into the cytoplasm of the adjacent cells, where clathrinlike structures were observed at the inner membrane of the concave portion. In others, spreading macrophages occasionally came in contact with adjacent cells by a peripheral rim of their cytoplasm with gap junctions. Cytoplasmic continuity was rarely observed at the boundaries between the closely associated cells. The two types of cell contact were also found, though not frequently, in the untreated cells. These results indicate that 1 alpha,25(OH)2D3 promotes multinucleation of alveolar macrophages through spreading forms with the formation of gap junctions and the coated membrane invagination.