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PubMed1999

Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein.

A line of mice deficient in vitamin D binding protein (DBP) was generated by targeted mutagenesis to establish a model for analysis of DBP's biological functions in vitamin D metabolism and action. On vitamin D-replete diets, DBP-/- mice had low levels of total serum vitamin D metabolites but were otherwise normal. When maintained on vitamin D-deficient diets for a brief period, the DBP-/-, but not DBP+/+, mice developed secondary hyperparathyroidism and the accompanying bone changes associated with vitamin D deficiency. DBP markedly prolonged the serum half-life of 25(OH)D and less dramatically prolonged the half-life of vitamin D by slowing its hepatic uptake and increasing the efficiency of its conversion to 25(OH)D in the liver. After an overload of vitamin D, DBP-/- mice were unexpectedly less susceptible to hypercalcemia and its toxic effects. Peak steady-state mRNA levels of the vitamin D-dependent calbindin-D9K gene were induced by 1,25(OH)2D more rapidly in the DBP-/- mice. Thus, the role of DBP is to maintain stable serum stores of vitamin D metabolites and modulate the rates of its bioavailability, activation, and end-organ responsiveness. These properties may have evolved to stabilize and maintain serum levels of vitamin D in environments with variable vitamin D availability.

Animals↗
PubMed1996

Role of calbindin-D9k in buffering cytosolic free Ca2+ ions in pig duodenal enterocytes.

1. The aim of the present study was to test whether the vitamin D-dependent Ca(2+)-binding protein calbindin-D9k could function as an important cytosolic Ca2+ buffer in duodenal enterocytes while facilitating transepithelial active transport of Ca2+ ions. For the investigations we used dual-wavelength, fluorescence ratio imaging, with fura-2 as the Ca(2+)-sensitive dye, to measure changes in cytosolic concentrations of free Ca2+ ions ([Ca2+]i) in isolated pig duodenal enterocytes affected by different cytosolic calbindin-D9k concentrations. 2. Epithelial cells were obtained from weaned piglets with normal calbindin-D9k concentrations (con-piglets), from piglets with low calbindin-D9k levels due to inherited calcitriol deficiency caused by defective renal 25-hydroxycholecalciferol D3-1 alpha-hydroxylase activity (def-piglets), and from piglets with reconstituted calbindin-D9k concentrations, i.e. def-animals treated with high doses of vitamin D3 which elevated plasma calcitriol levels by extrarenal production (def-D3-piglets). Basal levels of [Ca2+]i ranged between 170 and 205 nM and did not differ significantly between the groups. 3. After addition of 5 mM theophylline, the [Ca2+]i in enterocytes from con-piglets doubled during the 10 min incubation. This effect, however, was three times higher in enterocytes from def-piglets compared with those from con-piglets. Similar results were obtained after 4 min incubation of enterocytes from con- and def-piglets in the presence of 1 microM ionomycin. In preparations from def-D3-piglets, ionomycin-induced increases in [Ca2+]i were significantly lower compared with enterocytes from def-piglets and were not different from the control values. 4. From the results, substantial support is given for the hypothesis that one of the major functions of mucosal calbindin-D9k is the effective buffering of Ca2+ ions.

Animals↗
PubMed1998

Effect of 26,26,26,27,27,27-Hexafluoro-1,25-dihydroxyvitamin D3 on the expression of vitamin-D-responsive genes in vitamin-D-deficient mice.

26,26,26,27,27,27-Hexafluoro-1,25-dihydroxyvitamin D3 (ST-630) is a newly developed agent to maintain the levels of calcium and phosphorus in blood. Herein, we investigated the effect of this compound on the expression of vitamin-D-responsive genes in vitamin-D-deficient mice. ST-630 was more effective than 1, 25-dihydroxyvitamin D3 [1,25(OH)2D3] with respect to the induction of Cyp24 and calbindin-D9k mRNAs in the kidney and in the small intestine. Moreover, the increase in mRNA levels of vitamin-D-responsive genes induced by ST-630 lasted longer than that induced by 1,25(OH)2D3. These results indicate that ST-630 was more effective in inducing Cyp24 and calbindin-D9k gene expression than 1, 25(OH)2D3 when both compounds were injected into vitamin-D-deficient mice.

Animals↗
PubMed1998

Thyroid hormone receptor does not heterodimerize with the vitamin D receptor but represses vitamin D receptor-mediated transactivation.

The 9,000 Mr calcium-binding protein calbindin-D9k (CaBP9k) is markedly induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in mammalian intestine. However, although a vitamin D response element (VDRE) has been reported in the promoter of the rat CaBP9k gene (at -490/-472), the CaBP9k promoter is weakly transactivated by 1,25-(OH)2D3. Previous studies indicated that when MCF-7 cells are transfected with the rat CaBP9k VDRE ligated to the thymidine kinase promoter and treated with both 1,25-(OH)2D3 and T3 there is an enhancement of the response observed with 1,25-(OH)2D3 alone, suggesting direct cross-talk between thyroid hormone and the vitamin D endocrine system and activation via the formation of vitamin D receptor (VDR)-thyroid hormone receptor (TR) heterodimers. To determine whether the weak response of the rat CaBP9k natural promoter to 1,25-(OH)2D3 could be enhanced by T3, CaBP9k promoter/reporter chloramphenicol acetyltransferase constructs were transfected in MCF-7 cells, and the cells were treated with the two hormones alone or in combination. No induction with T3 alone and no enhancement of reporter activity in the presence of both hormones was observed. To determine whether a lack of effect by T3 was specific for the CaBP9k promoter and to further examine the possibility of cross-talk between the TR- and VDR-signaling pathways, the 1,25-(OH)2D3-responsive rat 24 hydroxylase [24(OH)ase] promoter and the rat osteocalcin VDRE (-457/-430), both fused to reporter genes were similarly examined in MCF-7 cells. Again, no enhancement of the response to 1,25-(OH)2D3 was observed in the presence of T3. In addition, a similar lack of response to T3 but responsiveness to 1,25-(OH)2D3 was observed when UMR106-01 osteosarcoma cells [which, like MCF-7 cells, express VDR, TR, and the retinoid X receptor (RXR) endogenously] were transfected with a 1,25-(OH)2D3 responsive mouse osteopontin promoter reporter. In vitro DNA binding assays were carried out using purified human VDR, human RXRalpha, and chick T3Ralpha and 24(OH)ase, osteocalcin, osteopontin, and CaBP9k VDRE oligonucleotide probes. No VDR-TR heterodimer binding on any of these VDREs was observed, although, as expected, there was binding by the VDR-RXR complex and strong TR-RXR binding to a consensus thyroid hormone response element. Simultaneous gel retardation assays using similar and lower concentrations of TR with RXR showed strong binding of TR-RXR on a 32P-labeled thyroid response element. Studies using the yeast two-hybrid system also did not provide evidence for the formation of a VDR-TR protein-protein interaction. In addition, in vivo data showed that transfection of TR, in fact, repressed VDR-mediated transcription and that the repression could be reversed by the addition of RXR. Thus, in vitro and in vivo experiments do not support ligand-sensitive transactivation mediated by VDR-TR heterodimer formation but rather suggest that TR expression can repress 1,25-(OH)2D3-induced transcription predominantly by sequestering RXR.

Animals↗
PubMed1998

Regulation of osteopontin gene expression during egg shell formation in the laying hen by mechanical strain.

The aim of this study is to evaluate the regulation of the osteopontin (OPN) gene expression by non-hormonal stimuli, such as calcium flux and mechanical strain during the daily egg cycle in the oviduct of the laying hen. After the egg enters the eggshell gland (ESG), the OPN gene is expressed by the epithelium cells in two waves: first by the basal cells and only then by the apical cells of the epithelium. A reduction in OPN gene expression was observed 1 h prior to laying. The calbindin gene, which marks the onset of calcification, was found to be expressed in the glandular epithelium starting 2 h after OPN gene expression. In addition, the formation of soft shells was accompanied by a reduction in calbindin, but not in OPN, gene expression. The application of a mechanical strain comparable to that induced by an egg led to induction of OPN gene expression at a normally quiescent phase in the cyclical expression of this gene. The induction of the gene was time- and strain-dependent and temporally similar to that induced by the entry of the egg into the ESG. In contrast, the calbindin gene was not affected by mechanical strain. The ESG of the laying hen provides a system to study the effect of a mechanical strain on matrix protein production in vivo, in a relevant physiological setting. The finding suggests that, in contrast to calbindin, OPN gene expression is not regulated by calcium flux but rather by the mechanical strain imposed by the resident egg.

Animals↗
PubMed2006

Exogenous 1,25-dihydroxyvitamin D3 exerts a skeletal anabolic effect and improves mineral ion homeostasis in mice that are homozygous for both the 1alpha-hydroxylase and parathyroid hormone null alleles.

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and PTH each modulate calcium and skeletal homeostasis. To identify 1,25(OH)(2)D(3)-mediated skeletal and mineral ion actions independent of PTH, double-knockout mice, which are homozygous for both the 1alpha-hydroxylase and PTH null alleles, were treated with 1,25(OH)(2)D(3), sc, from d 4 to 14 and compared with vehicle-treated animals. Serum calcium rose in 1,25(OH)(2)D(3)-treated double-knockout mice, and messenger RNA and protein levels of the renal calcium transporters TRPV5, calbindin-D(28K), calbindin-D(9K), and Na(+)/Ca(2+) exchanger 1 were up-regulated. Parameters of endochondral bone formation, including long bone length, epiphyseal volume, chondrocyte proliferation and differentiation, and cartilage matrix mineralization, were all increased by 1,25(OH)(2)D(3), Exogenous 1,25(OH)(2)D(3) also increased both trabecular and cortical bone; augmented both osteoblast number and type I collagen deposition in bone matrix; and up-regulated expression levels of the osteoblastic genes alkaline phosphatase, type I collagen, and osteocalcin. Furthermore, in 1,25(OH)(2)D(3)-treated double mutants, osteoclastic bone resorption appeared to decline. The results indicate that administered 1,25(OH)(2)D(3) used intestinal and renal but not skeletal mechanisms to elevate serum calcium and that this sterol can promote endochondral and appositional bone increases independent of endogenous PTH.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMedDate not supplied

Kindling induces transient changes in neuronal expression of somatostatin, neuropeptide Y, and calbindin in adult rat hippocampus and fascia dentata.

Fully hippocampus-kindled rats were examined 1 day and 1 month after the last stimulation for changes in somatostatin (SS)-, neuropeptide Y (NPY)-, and calbindin (CaBP)-immunoreactivity (ir) and SS- and NPY-mRNA in situ hybridization (ISH). One day after the last stimulation, there was marked, bilateral increase in SS- and NPY-ir in the outer part of the dentate molecular layer. The cell bodies of dentate hilar SS- and NPY-containing neurons, known to project to this area, also appeared to display increased immunoreactivity as well as an increased ISH signal for SS and NPY mRNA. Bilateral de novo expression of NPY-ir in dentate mossy fiber projection to dentate hilus and CA3 was also evident, but we noted no corresponding NPY-mRNA signal in the parent cell bodies, the dentate granule cells. After 1 month, the levels of NPY-ir and ISH signal appeared essentially normal. In contrast, the levels of SS apparently were decreased, although not yet normal. CaBP-ir was markedly and selectively reduced in dentate granule cell bodies, dendrites, and mossy fibers 1 day after the last stimulation, but after 1 month CaBP-ir appeared essentially normal. Because kindling, once established, is a permanent phenomenon, the observed transient changes in SS, NPY, and CaBP in specific hippocampal terminal fields and neuronal populations cannot be associated specifically with kindling. Rather, they relate to the repeated high-frequency stimulations and may serve as protective measures against deleterious effects of such stimulations.

Animals↗
PubMed2002

Co-expression of VRL-1 and calbindin D-28k in the rat sensory ganglia.

The co-expression of vanilloid receptor 1-like receptor (VRL-1), a newly cloned capsaicin-receptor homologue, with calbindin D-28k was examined in the rat sensory ganglia. The co-expression was rare in the dorsal root, trigeminal and jugular ganglia and abundant in the petrosal and nodose ganglia. In the dorsal root ganglion, none of VRL-1-immunoreactive (ir) neuron co-expressed calbindin D-28k-immunoreactivity (ir). Of the VRL-1-ir neurons, 9 and 5% showed calbindin D-28k ir in the trigeminal and jugular ganglia, respectively. On the other hand, 35 and 63% of VRL-1-ir neurons in the petrosal and nodose ganglia, respectively, co-expressed these substances. The retrograde tracing method indicated that petrosal neurons which co-expressed VRL-1-and calbindin D-28k-ir innervated taste buds in the circumvallate papilla. The present findings may suggest that VRL-1 is associated with chemosensory functions in visceral sensory neurons.

Animals↗
PubMed2001

Expression of calbindin-D(28k) in a pancreatic islet beta-cell line protects against cytokine-induced apoptosis and necrosis.

Cytokines produced by immune system cells that infiltrate pancreatic islets are candidate mediators of islet beta-cell destruction in autoimmune (type 1) diabetes mellitus. Because the calcium binding protein, calbindin-D(28k), can prevent apoptotic cell death in different cell types, we investigated the possibility that calbindin-D(28k) may prevent cytokine-mediated islet beta-cell destruction. Using the expression vector BSRalpha, rat calbindin-D(28k) was stably expressed in the pancreatic islet beta-cell line, betaTC-3. Calbindin-D(28k) expression resulted in increased cell survival in the presence of the cytotoxic combination of the cytokines IL-1beta (30 U/ml), TNFalpha (10(3) U/ml), and interferon gamma (10(3) U/ml). The greatest protection was observed in the betaTC-3 cell clone expressing the highest concentration of calbindin-D(28k). Apoptotic cell death was detected by annexin V staining and by the TdT-mediated dUTP-X nick end labeling assay in vector-transfected betaTC-3 cells incubated with cytokines (14-15% apoptotic cells). The number of apoptotic cells was significantly decreased in calbindin-D(28k)-overexpressing betaTC-3 cells incubated with cytokines (5-6% apoptotic cells). To address the mechanism of the antiapoptotic effects of calbindin, studies were done to examine whether calbindin inhibits free radical formation. The stimulatory effects of the cytokines on lipid hydroperoxide, nitric oxide, and peroxynitrite production were significantly decreased in the calbindin-D(28k)-expressing betaTC-3 cells. Our findings indicate that calbindin-D(28k), by inhibiting free radical formation, can protect against cytokine-mediated apoptosis and destruction of beta-cells. These findings suggest that calbindin-D(28k) may be an important regulator of cell death that can protect pancreatic islet beta-cells from autoimmune destruction in type 1 diabetes.

Animals↗
PubMed1999

Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines.

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).

1-Methyl-3-isobutylxanthine↗
PubMed1991

Immunoreactive calbindin-D9K in bone matrix vesicle.

This electron microscope study describes the subcellular occurrence and distribution of immunoreactive calbindin-D9K in the trabecular metaphyseal and compact cortical bone of normal rats, rachitic vitamin-D-deficient rats, and rachitic rats given 1,25-(OH)2D3. Undecalcified bones were embedded in Lowicryl K4M and calbindin-D9K antigenicity was detected by the protein A-gold method. Immunoreactive calbindin-D9K was localized in the cytoplasm and cell processes of osteoblasts and osteocytes. Immunoreactive calbindin-D9K was also found within matrix vesicles and calcifying matrix vesicles, where it lay over the needle-shaped crystallites, at the apparent site of initial crystal formation, but not along the whole crystallites. In fully mineralized bone it occurred at the same site, over the crystallites. Calibindin-D9K was vitamin-D-dependent in the osteoblasts and matrix vesicles, where its presence was correlated with the reappearance of crystallites in 1,25-(OH)2D3-treated vitamin-D-deficient rats. This suggests that immunoreactive calbindin-D9K is involved in mineral deposition in bone matrix vesicles. Abnormal intracellular calcification associated with calbindin-D9K antigenicity in the osteoblasts of 1,25-(OH)2D3-treated vitamin-D-deficient rats indicates that immunoreactive calbindin-D9K may also play a part in abnormal intracellular mineral deposition.

Animals↗
PubMed1993

The mechanism for the disparate actions of calcitriol and 22-oxacalcitriol in the intestine.

22-Oxacalcitriol (OCT) is one of several new analogs of vitamin D that retain many of the therapeutically useful properties of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], but have much less calcemic activity. In the present study we examined the actions of OCT on intestinal calcium absorption and calbindin D9k mRNA in vitamin D-deficient rats. After ip injection of OCT (1 microgram/kg), calcium absorption increased significantly by 2 h and was maximal at 4 h (2.5-fold above control), but returned to pretreatment levels by 16 h. In contrast, the same dose of 1,25-(OH)2D3 caused a 3-fold increase in calcium absorption, which lasted more than 48 h. The transient effect of OCT on calcium absorption was also observed when the analog was infused at a dose of 1 micrograms/kg.day for 3 days. At the end of the infusion period, calcium absorption was 3-fold higher than that in vehicle-infused controls, but fell to pretreatment levels by 24 h after removing the minipumps. The time courses for induction of calbindin D9k mRNA were similar for OCT and 1,25-(OH)2D3, with no change observed until more than 4 h after injection. However, calbindin mRNA levels returned to pretreatment values more rapidly in the OCT-treated rats. Consistent with these findings, we observed that a 1 microgram/kg dose of [3H] OCT was completely cleared by 4-6 h after injection. This was paralleled by a loss of [3H]OCT associated with the intestinal vitamin D receptor. The rapid clearance of OCT is probably due to its low affinity for the serum vitamin D-binding protein. This low affinity would also be expected to allow greater accessibility to target cells. In support of this, we found that higher amounts of OCT than 1,25-(OH)2D3 were associated with the intestinal vitamin D receptor after the injection of several doses of these tritiated ligands. In summary, our results indicate that the pharmacokinetic properties of OCT are responsible at least in part for its low calcemic activity. Furthermore, comparison of the transient elevation of calcium absorption by OCT with its more prolonged effects on PTH and calbindin D9k indicates that each action of vitamin D compounds has a distinct biological half-life. The short circulating half-life of OCT can exploit these differences to provide a therapeutic advantage in the treatment of vitamin D-responsive diseases.

Animals↗
PubMed1988

Expression of calbindin immunoreactivity by subpopulations of primary sensory neurons in chick embryo dorsal root ganglion cells grown in coculture or conditioned medium.

Primary sensory neurons which innervate neuromuscular spindles in the chicken are calbindin-immunoreactive. The influence exerted by developing skeletal muscle on the expression of calbindin immunoreactivity by subpopulations of dorsal root ganglion (DRG) cells in the chick embryo was tested in vitro in coculture with myoblasts, in conditioned medium (CM) prepared from myoblasts and in control cultures of DRG cells alone. Control cultures of DRG cells grown at the 6th embryonic day (E6) did not show any calbindin-immunostained ganglion cell. In coculture of myoblasts previously grown for 14 days, about 3% of calbindin-immunoreactive ganglion cells were detected while about 1% were observed in some cultures grown in CM. Fibroblasts from various sources were devoid of effect. Skin or kidney cells were more active than myoblasts to initiate calbindin expression by subpopulations of DRG cells in coculture or, to a lesser degree, in CM. The results suggest that cellular factors would rather induce calbindin expression in certain sensory neurons than ensure a selective neuronal survival.

Animals↗
PubMed1987

Vitamin D metabolism and calbindin (calcium-binding protein) in aged laying hens.

Calbindin (calcium-binding protein) concentration, vitamin D metabolism and shell quality were investigated in young (7- to 9-mo-old) and aged (19- to 21-mo-old) laying hens consuming normal or low levels of calcium (Ca). Although egg weight and percent of cracked eggs were higher and egg production and shell density (mg/cm2) were lower (significantly, P less than 0.01) in aged hens, shell weight, plasma Ca and duodenal and egg shell gland calbindin were similar to those of young hens. Dietary Ca restriction reduced shell weight, shell density and structural bone and plasma Ca in both the young and aged birds. The production of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and its concentration in the plasma were higher in hens fed low dietary levels of Ca than in hens fed normal Ca levels only in the younger hens. However, a slightly higher production of 1,25(OH)2D3 and concentration of duodenal calbindin were also observed in severely Ca-restricted (1.4% Ca for 19 d) aged hens than in the younger hens. The results suggest that the aged hen loses its ability to adapt to changes in Ca intake or needs through mechanisms involving modulation of vitamin D metabolism.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1996

Urinary 28-kD calbindin-D as a new marker for damage to distal renal tubules caused by cisplatin-based chemotherapy.

Calbindin-D, a vitamin D-dependent calcium-binding protein of 28 kD, is found predominantly in the distal tubules of the kidney and central nervous system tissues in humans. To evaluate damage to the renal tubules caused by cisplatin-based chemotherapy, levels of urinary and serum calbindin-D were determined in patients treated with cisplatin- or carboplatin-based chemotherapies using a highly sensitive enzyme immunoassay system developed in our laboratory. Levels of urinary 28-kD calbindin-D were also determined in patients with benign and malignant urological diseases. The mean urinary calbindin-D level was 2.44 + or - 0.31 (mean + or - SE) ng/mg creatinine in 40 healthy subjects. Urinary calbindin-D levels were elevated (>10 ng/mg creatinine) in 2 of 33 patients (6%) with benign and 1 of 50 (2%) with malignant urological diseases. Urinary calbindin-D levels were significantly increased after cisplatin-based chemotherapy in 14 patients, with peaks (71.8 + or - 13.5 ng/mg creatinine) being found 8 days after administration of cisplatin, and then a gradual return to the baseline. On the other hand, 7 patients receiving carboplatin-based chemotherapy demonstrated no significant elevation (highest level 7.7 + or - 2.5 ng/mg creatinine). In 7 patients treated with cisplatin-based chemotherapy the serum calbindin-D level was also raised after treatment, with a good correlation to urinary values. These findings suggest that urinary and serum calbindin-D may be kidney-derived and that 28-kDa calbindin-D is a useful marker for damage to the distal renal tubules associated with cisplatin-based chemotherapy.

Acetylglucosaminidase↗
PubMed1993

Demonstration that 1 beta,25-dihydroxyvitamin D3 is an antagonist of the nongenomic but not genomic biological responses and biological profile of the three A-ring diastereomers of 1 alpha,25-dihydroxyvitamin D3.

The steroid hormone 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) generates biological responses via both genomic and nongenomic mechanisms. This article reports the biological profile of four A-ring diastereomers of this secosteroid (results are expressed as percentage of the response of 1 alpha,25-(OH)2D3. The activity of the compounds, 1 alpha,25-(OH)2D3, 1 alpha,25-(OH)2-3-epivitamin D3, 1 beta,25-(OH)2D3, 1 beta,25-(OH)2-3-epivitamin D3, for in vivo intestinal Ca2+ absorption and bone Ca2+ mobilization and in vitro binding to the nuclear receptor (genomic responses) was, respectively, 100, 2.8, < 0.1, and < 0.1% (intestinal Ca2+ absorption); 100, 1.5, < 0.1, and < 0.1% (bone Ca2+ mobilization); 100, 24, 0.2, and 0.8% (receptor binding). In the in vivo nongenomic transcaltachia assay the results were 100, 80, 0, and 20-30%, and in ROS 17/2.8 cells (45Ca2+ uptake through voltage-gated Ca2+ channels) 1 alpha,25-(OH)2D3 had 100% activity and 1 beta,25-(OH)2D3 (the only diastereomer evaluated) had no agonist activity. Keratinocyte proliferation was inhibited in the order 1 alpha,25-(OH)2D3 > 1 alpha,25-(OH)2-3-epivitamin D3 > 1 beta,25-(OH)2D3 > 1 beta,25-(OH)2-3-epivitamin D3. 1 beta,25-(OH)2D3 was a potent antagonist of 1 alpha,25-(OH)2D3-mediated transcaltachia and 45Ca2+ uptake in ROS 17/2.8 cells but was unable to block the genomic 1 alpha,25-(OH)2D3 induction of chick calbindin-D28k (in vivo), induction of MG-63 cell osteocalcin, and HL-60 cell differentiation. These results suggest that analogs of 1 alpha,25-(OH)2D3 may be synthesized which are selective agonists or antagonists of genomic or nongenomic responses in the vitamin D endocrine system.

Animals↗
PubMed1993

Evidence for calcium mediated conformational changes in calbindin-D28K (the vitamin D-induced calcium binding protein) interactions with chick intestinal brush border membrane alkaline phosphatase as studied via photoaffinity labeling techniques.

The role of the vitamin D-induced calcium binding protein termed calbindin-D (CaBP) in the biological response to 1,25-dihydroxyvitamin D3 was assessed by photoaffinity labeling techniques. The heterobifunctional cross-linking reagent methyl-4-azidobenzoimidate was employed for studies with the 28 KD chick intestinal calbindin-D28K. Calcium-dependent interactions were evident with purified chick intestinal CaBP-immunoglobulins and bovine intestinal alkaline phosphatase; in the absence of Ca2+ there was a greatly diminished crosslinking process. There were also at least two membrane components of chick intestinal brush border membranes, with M(R) = 60,000 and 130,000, which were photoaffinity cross-linked with CaBP in a calcium-dependent manner. Similar interactions were demonstrated following incubations of CaBP with phosphatidylinositol-specific phospholipase C (PI-PLC)-treated supernatant fractions from chick intestinal brush borders. PI-PLC was shown to release 14% of the alkaline phosphatase from chick intestinal brush borders compared to greater than 80% for rabbit and chick kidney BBM preparations. Specific interactions between CaBP and brush border membrane proteins could also be demonstrated in the absence of photoaffinity labeling by Sephadex G-150 chromatography of Triton X-100 solubilized incubations between calbindin-D28K and chick intestinal BBMS, with 17% of the radiolabelled CaBP comigrating with alkaline phosphatase activity. These studies collectively demonstrate that calbindin-D28K undergoes calcium-dependent conformational changes which alter its subsequent interactions with cellular proteins in a way consistent with other calcium-binding proteins such as calmodulin or troponin C.

Affinity Labels↗
PubMed2003

Thiazide-induced hypocalciuria is accompanied by a decreased expression of Ca2+ transport proteins in kidney.

INTRODUCTION: Thiazide diuretics have the unique characteristic of increasing renal Na+ excretion, while decreasing Ca2+ excretion. However, the molecular mechanism responsible for this thiazide-induced hypocalciuria remains unclear. The present study investigates the effect of thiazides on the expression of the proteins involved in active Ca2+ transport as well as the role of extracellular volume (ECV) status. METHODS: Hydrochlorothiazide (HCTZ), 12 mg/24 hours, was administered during 7 days to Wistar rats by osmotic minipumps. In addition, ECV contraction was either prevented by Na+ repletion or induced by a low-salt diet. Expression levels of the proteins involved in active Ca2+ transport [i.e., epithelial Ca2+ channel (TRPV5/ECaC1), calbindin-D28K, Na+/Ca2+ exchanger (NCX1)], as well as the thiazide-sensitive Na+ Cl- cotransporter (NCC) were determined by real-time quantitative polymerase chain reaction (PCR) and semiquantitative immunohistochemistry. RESULTS: HCTZ significantly reduced urinary Ca2+ excretion (22%+/- 5% relative to controls). Hematocrit was significantly increased, confirming ECV contraction. In addition, Na+ depletion virtually abolished Ca2+ excretion (8%+/- 1%), while Na+ repletion during HCTZ treatment prevented both ECV contraction and hypocalciuria. HCTZ significantly decreased mRNA expression of TRPV5 (71%+/- 6%), calbindin-D28K (53%+/- 6%), NCX1 (51%+/- 8%) and NCC (50%+/- 11%), regardless of ECV status or calciuresis. Immunohistochemistry revealed reduced TRPV5 (43%+/- 2%), calbindin-D28K (59%+/- 1%) and NCC (56%+/- 4%) abundance. Furthermore, during HCTZ treatment, the subset of tubules coexpressing NCC and calbindin-D28K was significantly reduced (43%+/- 5%) and a disturbed cellular localization of NCC was observed. CONCLUSION: These data suggest that ECV contraction is a critical determinant of the thiazide-induced hypocalciuria, which is accompanied by a decreased expression of Ca2+ transport proteins.

Animals↗