(Ca2+ + Mg2+)-ATPase activity in plasma membrane of circulating mononuclear cells. Lack of a direct effect of vitamin D

The in vivo effect of vitamin D on (Ca2+ + Mg2+)-ATPase activity was examined in a plasma membrane fraction of rat circulating mononuclear cells (MPM). Although there was no significant difference in the ATPase activities in red blood cell ghosts, (Ca2+ + Mg2+)-ATPase activity in MPM was significantly higher (p less than 0.05) in long-term vitamin D3-replete rats (100 IU/day for 6 months) than that in vitamin D-deplete rats (for 6 months). In rats maintained on vitamin D-deficient diets for 5-7 weeks, in vivo administration of either vitamin D3, 2,000 IU orally, 5 days prior to killing or 1,25-dihydroxyvitamin D3, 2.4 nmol, intraperitoneally, 24 h prior to killing failed to show any significant effect on (Ca2+ + Mg2+)-ATPase activity in MPM. (Ca2+ + Mg2+)-ATPase activity in MPM from rats maintained on vitamin D-deficient diet with high calcium content (1.8%) was significantly higher (p less than 0.05) than that from rats maintained on vitamin D-deficient diet with low calcium content (0.3%). Moreover, in vitro addition of vitamin D3 metabolites did not show any effect on (Ca2+ + Mg2+)-ATPase activity in MPM. These data suggest that decreased (Ca2+ + Mg2+)-ATPase activity in MPM from long-term vitamin D-deplete rats resulted from an adaptation to low extracellular calcium rather than vitamin D depletion.     

Oxygen-dependent 1,25-dihydroxycholecalciferol-induced calcium ion transport in rat intestine.

O2-dependent CA2+ uptake by rat duodenal discs has been characterized and used in a revised assay for 1,25-dihydroxycholecalciferol-induced intestinal Ca2+ transport. Although both muscle and mucosal surfaces are exposed in this free-floating-disc assay, the Ca2+ influx across the muscle surface is small, not O2- or vitamin D-dependent, and can be subtracted out. Depriving the animals of food for 9-14 h before assay increases the O2-dependent uptake by about 75%. Half-saturation values for O2-dependent Ca2+ uptake as determined with this assay are: 0.8mM-Ca2+ (fed) and 0.5mM-Ca2+ (food-deprived) for vitamin D-deficient rats, and 0.9mM-Ca2+ (fed) and 1.5mM-Ca2+ (food-deprived) for rats dosed with 1,25-dihydroxycholecalciferol. The maximum velocity of uptake varies from 6.7nmol of Ca2+ per cm2/min (fed) to 7.0nmol of Ca2+ per cm2/min (food-deprived) for vitamin D-deficient rats and 16.7nmol of Ca2+ per cm2/min (fed) to 29 nmol of Ca2+ per cm2/min (food-deprived) for 1,25-dihydroxycholecalciferol-treated rats. By using a 5 min preincubation and 15 min incubation with 1.0mM-Ca2+, duodenal tissue taken from vitamin D-treated rats shows about a 3-fold increase in O2-dependent Ca2+ uptake when compared with tissue taken from vitamin D-deficient animals. The calcium ionophore A23187, depending on concentration, either has no significant effect on or inhibits the O2-dependent uptake, rather than increasing it. Actinomycin D, at a dose of 2 micrograms/g, inhibits the O2-dependent uptake in intestinal discs from both vitamin D-deficient and vitamin D-treated rats by 58 and 80% respectively, when administered in vivo 3 1/2 h before assay.     

Age- and sex-dependent stimulation of calcium uptake in duodenal cells by prolactin in vitamin D-deficient rats

Administration of ovine-prolactin (O-PRL) stimulated Ca2+ uptake in isolated duodenal cells prepared from vitamin D-deficient rats. The time course of this effect was biphasic: uptake activity reached a peak in 2.5 hrs followed by a decrease at 5 hrs to original levels. This stimulatory effect of O-PRL was observed in vitamin D-deficient male, but not in female rats. This stimulatory effect was observed in 16- and 26-week old, but not 9 week old, animals. Increase in Ca2+ uptake in duodenal cells was not due to a decrease in intracellular Ca2+ efflux. We measured serum Ca concentration in vitamin D-deficient female rats and found that serum Ca increased in D-deficient female rats between 16 and 52 weeks whereas a minimal increase was observed in D-deficient male rats. Although prolactin was shown to stimulate duodenal Ca2+ uptake, it appears that the source of the increase in levels of serum Ca in D-deficient female rats was not derived from an increase in Ca2+ uptake by prolactin in duodenum. The increase in serum calcium with time may explain why female D-deficient rats survive longer then male.     

Calcium uptake in isolated brush-border vesicles from rat small intestine.

Ca2+ uptake in brush-border vesicles isolated from rat duodena was studied by a rapid-filtration technique. Ca2+ uptake showed saturation kinetics, was dependent on the pH and ionic strength of the medium and was independent of metabolic energy. Uptake activity was readily inhibited by Ruthenium Red, La3+, tetracaine, EGTA, choline chloride and Na+ or K+. The effect of variations in medium osmolarity on Ca2+ uptake and the ionophore A23187-induced efflux of the cation from preloaded vesicles indicated that the Ca2+-uptake process involved binding to membrane components, as well as transport into an osmotically active space. Scatchard-plot analyses of the binding data suggested at least two classes of Ca2+-binding sites. The high-affinity sites, Ka = (2.7 +/- 1.1) x 10(4) M-1 (mean +/- S.D.) bound 3.2 +/- 0.8 nmol of Ca2+/mg of protein, whereas the low-affinity sites (Ka = 60 +/- 6 M-1) bound 110 +/- 17 nmol of Ca2+/mg of protein. In the presence of 100 mM-NaCl, 1.7 and 53 nmol of Ca2+/mg of protein were bound to the high- and low-affinity sites respectively. Decreased Ca2+-uptake activity was observed in vesicles isolated from vitamin D-deficient as compared with vitamin D-replete animals and intraperitoneal administration of 1,25-dihydroxycholecalciferol to vitamin D-deficient rats 16 h before membrane isolation stimulated the initial rate of Ca2+ uptake significantly. The data indicated that Ca2+ entry and/or binding was passive and may involve a carrier-mediated Ca2+-uptake component that is associated with the brush-border membrane. Altering the electrochemical potential difference across the membrane by using anions of various permeability and selected ionophores appeared to increase primarily binding to the membrane rather than transport into the intravesicular space. Since there is considerable binding of Ca2+ to the vesicle interior, a comprehensive analysis of the transport properties of the brush-border membrane remains difficult at present.     

Relationship between parathyroid hormone and adenosine 3′,5′-monophosphate metabolism in the kidney of vitamin D-deficient rats

The effect of vitamin D administration on cyclic AMP metabolism in the kidney was examined in rats fed a vitamin D-deficient, low Ca diet. The renal cyclic AMP level in vitamin D-deficient rats was higher than that in normal rats fed a laboratory chow, and in significantly decreased after thyroparathyroidectomy. Parathyroid hormone administered in vitro and in vivo did not cause as great a cyclic AMP response in vitamin D-deficient rats as that seen in the normal rats. The response to calcitonin, however, was not blunted in vitamin D-deficient animals. The blunted cyclic AMP accumulation in the kidney seemed to be related to formation, rather than degradation, of the nucleotide. The rats fed the low Ca diet were still hypocalcemic even after supplementation of the diet with a daily dose of either 0.625 μg of vitamin D-3 for 3 weeks or 2.5 μg of vitamin D-3 for the last 3 days. Vitamin D supplementation did not influence either the basal level or parathyroid hormone-stimulated increase of cyclic AMP in the kidney. On the contrary, when animals maintained on the vitamin D-deficient, low Ca diet were switched to the vitamin D-deficient, high Ca diet containing lactose for several days, they recovered normocalcemia and a normal response. These results suggest that the blunted cyclic AMP response to parathyroid hormone in vitamin D deficiency is due to hypocalcemia or associated secondary hyperparathyroidism and not due to deficiency of vitamin D action.     

Studies on the role of calcium ions in the stimulation by adrenaline of amylase release from rat parotid

1. Mitochondrial and microsomal fractions were prepared from rat parotid glands. Both fractions were able to take up (45)Ca. The mitochondrial (45)Ca-uptake system could be driven by ATP (energy-coupled Ca(2+) uptake) or by ADP+succinate (respiration-coupled Ca(2+) uptake). Energy-coupled Ca(2+) uptake was blocked by oligomycin but not by carbonyl cyanide m-chlorophenylhydrazone; respiration-coupled Ca(2+) uptake was blocked by carbonyl cyanide m-chlorophenylhydrazone but not by oligomycin. Microsomal Ca(2+) uptake was dependent on the presence of ATP; the ATP-dependent Ca(2+) uptake was not affected by oligomycin or carbonyl cyanide m-chlorophenylhydrazone. Ca(2+) uptake by both fractions was inhibited by Ni(2+). 2. Incubation of parotid pieces with adrenaline increased the rate of release of amylase and the uptake of (45)Ca. The adrenaline-stimulated release of amylase was not dependent on the presence of extracellular Ca(2+). 3. The effect of adrenaline on the subcellular distribution of (45)Ca in parotid pieces incubated with (45)Ca was studied. In parotid tissue incubated with (45)Ca, both mitochondrial and microsomal fractions contained (45)Ca. Incubation with adrenaline increased the amount of (45)Ca incorporated into the mitochondrial fraction but not the microsomal fraction. In parotid tissue preloaded with (45)Ca subsequent incubation with adrenaline caused a decrease in the amount of (45)Ca found in both the mitochondrial and microsomal fractions. 4. From these data we conclude that the regulation of the cytosolic Ca(2+) concentration in the parotid may involve both mitochondrial and microsomal Ca(2+)-uptake systems. We suggest that the action of adrenaline on the parotid may be to increase the movement of Ca(2+) to the cytosol by increasing the flux of Ca(2+) across mitochondrial, microsomal and plasma membranes.     

Amylase release from rat parotid glands. II. Calcium kinetics.

The kinetics of 45Ca2+ uptake, efflux, and calcium potentiation of amylase release by slices of rat parotid glands were examined. Pretreatment of the tissue with 11.25 mM 45Ca2+ medium increased the total tissue 45calcium content. Lanthanum (1 mM) decreased tissue uptake, blocked the slow components of exchange and appeared to inhibit transcellular calcium movement. Neither dibutyryl cyclic AMP nor caffeine caused consistently significant effects on 45Ca2+ kinetics, or total 45calcium content. Carbamylcholine increased the initial rate of 45Ca2+ uptake, but had no effect on total uptake. Elevation of the extracellular Ca2+ concentration to 11.25 mM during stimulation of amylase release resulted in an initial decrease in the rate of amylase release followed by a potentiation of release which developed slowly, requiring 40--50 min to reach the maximal response. The inability to detect release-related changes in either calcium influx or mobilization, and the lengthy times and high Ca2+ concentrations required to achieve calcium potentiation suggests that calcium does not couple amylase release.     

Effects of vitamin D3 on the uptake and release of calcium by isolated intestinal mitochondria

Administration of vitamin D3 or 1,25 (OH)2D3 to rachitic chicks produces a decrease of 45Ca uptake by mitochondria from intestinal mucosa. This effect of vitamin D3 shows tissue specificity, since it was not observed in liver mitochondria from the same animals. The Km values were similar (about 10 microM) for intestinal mitochondria from rachitic and treated animals. The Ca2+ efflux in previously loaded mitochondria was not changed by treatment. The Ca content of recently isolated mitochondria was strikingly lower after vitamin D3 administration. It is concluded that vitamin D3 may participate in the mechanism which regulates the intramitochondrial Ca concentration.     

Regulation of vitamin D metabolism by calcium and phosphate ions in isolated renal tubules.

The acute and long-term effects of Ca2+ and Pi on vitamin D metabolism were studied in vitro with isolated renal tubules from vitamin D-deficient and vitamin D-supplemented chicks. Ca2+ depletion, achieved by isolating renal tubules in Ca2+-free buffers, led to suppression of 1 alpha-hydroxylase activity. Re-introduction of Ca2+ during incubation caused an acute stimulation of this enzyme. This stimulatory effect of Ca2+ was prevented by prior treatment of Ca2+-depleted renal tubules for 6 h with 1,25-dihydroxycholecalciferol. Ca2+ and Pi produced marked acute affects on 1 alpha-hydroxylase activity, which persisted for the whole 8 h experimental period, in Ca2+-depleted renal tubules from vitamin D-deficient chicks. The effects of either ion were influenced by the concentration of the other. However, the effects of these ions could not be reproduced in either Ca2+-depleted renal tubules from vitamin D-supplemented chicks or in renal tubules from vitamin D-deficient chicks, isolated in Ca2+-containing buffers. Isolation of renal tubules from vitamin D-supplemented chicks in Ca2+-containing buffers and subsequent incubation for 8 h in the presence of increased [Ca2+] led to a modest but statistically significant suppression of 1 alpha-hydroxylase and stimulation of 24-hydroxylase activity. It is concluded that the acute effects of Ca2+ and Pi on 1 alpha-hydroxylase activity of Ca2+-depleted renal tubules from vitamin D-deficient chicks are not regulatory but the results of the experimental conditions. In contrast the long-term effects of Ca2+ on both hydroxylases of renal tubules from vitamin D-supplemented chicks may be of physiological significance.     

A guanine nucleotide-binding protein mediates 1,25-dihydroxy-vitamin D-3-dependent rapid stimulation of Ca2+ uptake in skeletal muscle.

1,25-Dihydroxyvitamin D-3 (1,25(OH)2D3) has been shown to increase Ca2+ uptake readily in skeletal muscle through a dihydropyridine-sensitive pathway, cAMP levels and adenylate cyclase activity. In the present study, fluoride (F-), a potent guanine nucleotide binding protein (G protein) stimulator, rapidly increases vitamin D-deficient skeletal muscle Ca2+ uptake in a dose-dependent manner and with a similar time-course as 1,25(OH)2D3. The increment is detected within 1 min (15%) and steadily increases up to 15 min (60%). The effects of 1,25(OH)2D3 and F- are also observed in muscle from normal, vitamin D-replete chicks. AlCl3, which is required for G protein stimulation by F-, potentiates the effects of F-, Ca2+ uptake in 1,25(OH)2D3-dependent muscle is potentiated by F- and, analogous to the hormone, the effects of F- can be suppressed by Ca(2+)-channel antagonists. Direct exposure of microsomal membranes to 1,25(OH)2D3 reduces the specific binding of [gamma-35S]GTP to the membranes 40%. Pretreatment of muscle with Bordetella pertussis toxin (PTX), known to inhibit Gi, or with cholera toxin (CTX), known to stimulate Gs, produces an acute elevation of muscle Ca2+ uptake. 1,25(OH)2D3 potentiates CTX, but has no additional effect on PTX-dependent Ca2+ uptake. These results indicate that an interaction with an inhibitory G protein coupled to adenylate cyclase may be part of the mechanism by which 1,25(OH)2D3 increase Ca2+ uptake through regulation of Ca(2+)-channel gating by a cAMP-dependent pathway in skeletal muscle.     

Effect of vitamin D status on cyclic AMP-dependent protein kinase activity and its heat-stable inhibitor in chick kidney

Cyclic AMP-dependent protein kinase activity in supernatants of homogenates of kidneys from vitamin D-deficient chicks is decreased to 70% of the level measured in kidneys from normal chicks. Activity was restored to normal by oral administration of vitamin D or 1,25-dihydroxyvitamin D3 for 1 or 2 weeks. Both isozymes of cAMP-dependent protein kinase were reduced to the same extent by vitamin D deficiency. The decreased enzyme activity could not be accounted for by a shift to the particulate fraction nor by an increased requirement for cyclic AMP. A heat stable, trichloroacetic acid-precipitable, trypsin-labile inhibitor of protein kinase activity was identified and quantitated in kidneys from vitamin D-deficient chicks (16 to 26 units/mg of protein) and from those given vitamin D (2 to 6 units/mg of protein). The measured difference in inhibitor levels could not be attributed to differential stability in kidney homogenates from vitamin D-deficient or -repleted chicks. The observed increase in inhibitor level with vitamin D deficiency is not sufficient to account for the decrease in cyclic AMP-dependent protein kinase activity, suggesting that the total amount of this enzyme activity is reduced in vitamin D deficiency.     

1,25 (OH)2 vitamin D3-induced 45CA uptake in vascular myocytes cultured from spontaneously hypertensive and normotensive rats

The effect of 1,25 (OH)2 vitamin D3 on basal 45Ca uptake was examined in vascular smooth muscle cells cultured from mesenteric arteries of spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) normotensive rats. Basal uptake of 45Ca was significantly greater in myocytes of WKY than SHR at 5, 10, 30 and 60 min incubation with the isotope. Incubation with 1 ng/ml 1,25 (OH)2 vitamin D3 for 48 hr increased basal 45Ca uptake between 1-10 min in SHR and between 5-10 min in WKY. The dose-response relationship indicated that cells from both strains are equally sensitive to the calciotropic effects of 1,25 (OH)2 vitamin D3 with half-maximal stimulation occurring at approximately 0.3-0.4 ng/ml. In cells of both strains maximal stimulation of 45Ca uptake was achieved only after a 12-24 hr period of incubation with hormone and pretreatment with cycloheximide inhibited 1,25 (OH)2 vitamin D3-enhanced 45Ca uptake. Although 45Ca binding by extracellular matrix material was significantly greater in WKY than SHR, 1,25 (OH)2 vitamin D3 had no effect on the amount of matrix 45Ca binding in either strain. These results suggest that 1,25 (OH)2 vitamin D3 induces an increase in intracellular protein synthesis that results in enhanced 45Ca uptake. The similar responses of the two strains indicate that hypertensive smooth muscle is not more sensitive to 1,25 (OH)2 vitamin D3 and the Ca2+ response is a general property of vascular muscle.     

1,25-Dihydroxyvitamin D3-mediated intestinal calcium transport. Biochemical identification of lysosomes containing calcium and calcium-binding protein (calbindin-D28K)

A variety of intestinal cell organelles and proteins have been proposed to mediate 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated calcium absorption. In the present study biochemical analyses were undertaken to determine the subcellular localization of 45Ca after calcium transport in vivo in ligated duodenal loops of vitamin D-deficient chicks injected with 1.3 nmol of 1,25-(OH)2D3 or vehicle 15 h prior to experimentation. Separation of Golgi, mitochondria, basal lateral membrane, and lysosome fractions in the epithelial homogenates was achieved by differential sedimentation followed by centrifugation in Percoll gradients and evaluation of appropriate marker enzyme activities. Both vitamin D-deficient and 1,25-(OH)2D3-treated chicks had the highest levels of 45Ca-specific activity in lysosomal fractions. The lysosomes were also the only organelles to exhibit a 1,25-(OH)2D3-mediated difference in calcium content, increasing to 138% of controls. Lysosomes prepared from 1,25-(OH)2D3-treated chicks also contained the greatest levels of immunoreactive calbindin-D28k (calcium-binding protein). Chloroquine, a drug known to interfere with lysosomal function, was tested and found to inhibit 1,25-(OH)2D3-stimulated intestinal calcium absorption. Neither 1,25-(OH)2D3 nor chloroquine affected [3H]2O transport. In additional experiments, microsomal membranes (105,000 X g pellets) were subjected to gradient centrifugation. The highest levels of 45Ca-specific activity and calcium-binding protein in material from 1,25-(OH)2D3-treated chicks were found in fractions denser than endoplasmic reticulum and may represent endocytic vesicles. In studies on intestinal mucosa of 1,25-(OH)2D3-treated birds fractionated after 30 min of exposure to lumenal Ca2+ or Ca2+ plus chloroquine, 45Ca was found to accumulate in lysosomes and putative endocytic vesicles, relative to controls. A mechanism involving vesicular flow is proposed for 1,25-(OH)2D3-mediated intestinal calcium transport. Endocytic internalization of Ca2+, fusion of the vesicles with lysosomes, and exocytosis at the basal lateral membrane complete the transport process.     

Studies on the effect of vitamin D on calcium absorption and transport

1. Mucosal cells of the small intestine obtained from rats deprived of vitamin D or given excessive amounts of the vitamin accumulated significantly more calcium than did cells from control animals. 2. Mucosal cells from vitamin D-deficient rats released less calcium than did cells from normal or hypervitaminotic D animals. 3. Studies in vivo showed that the transfer of (45)Ca from the intestine to the blood was delayed in vitamin D deficiency, but was accelerated in hypervitaminosis D. 4. The findings support the thesis that vitamin D is involved in the release of calcium rather than in its uptake by mucosal cells. 5. Further evidence is presented suggesting that uptake of calcium by intestinal mucosal cells at 0 degrees is primarily passive, whereas at 38 degrees uptake and release are effected by an active process that depends on energy derived from glycolytic activity.     

Vitamin D3 and 1,25-dihydroxyvitamin D3 stimulate the skeletal muscle-calcium mobilization in rachitic chicks

The Ca content in skeletal muscle relative to vitamin D3 intake was studied in chicks. It was found that the Ca content in rachitic chick muscle was significantly higher than normal and it decreased with vitamin D3 treatment. In 4-week-old chicks fed a vitamin D-deficient diet, the Ca content in leg muscle reached 9.86 +/- 1.07 mg/100 g wet wt, although in chicks receiving vitamin D3 in doses of 100 and 500 IU/kg diet, it was 7.80 +/- 0.72 and 6.08 +/- 0.61 mg/100 g wet wt, respectively. A single i.m. dose of 0.50 micrograms of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or vitamin D3 caused a dramatic decrease in the muscle Ca content by 3 to 6 h after the injection. A simultaneous rise in the Ca level in blood serum was observed. However, at this time the Ca binding protein content in duodenal mucosa and the stimulation of Ca absorption were negligible. These findings allow the conclusion that the vitamin D deficiency in chicks leads to a surplus Ca accumulation in skeletal muscle. The administration of vitamin D3 or its metabolites causes rapid Ca release during the first 6 h. This may be the source of the Ca level increase in blood serum. In this respect 1,25(OH)2D3 was much more effective than vitamin D3.     

Characterization of the vitamin D-dependent Ca2+-binding sites in rat intestinal Golgi-enriched membrane fractions.

Rat intestinal Golgi-enriched membrane fractions take up Ca2+ by a vitamin D-dependent process that has been shown to recover within 15 min of repletion of vitamin D-deficient animals with intravenous 1,25-dihydroxycholecalciferol. The present paper reports studies characterizing the Ca2+-binding sites of these membrane fractions. Equilibrium binding of Ca2+ at concentrations between 5 and 400 microM showed significant decreases at all concentrations in membranes derived from vitamin D-deficient animals when compared with normal control-diet-fed animals. The predominant class of binding sites had a relatively high affinity for Ca2+ (KD approx. 3 microM). Vitamin D-deficiency did not change the affinity of this class of site, but decreased the number from 347 +/- 26 to 168 +/- 50 nmol of Ca2+ bound/mg of protein (means +/- S.D.). Mg2+ inhibited binding only at low Ca2+ concentrations, and the characteristics of this binding suggested positive co-operativity between two binding sites. Equimolar concentrations of Zn2+, La3+, Pb2+ and Mn2+ inhibited Ca2+ binding by over 50%. Increased ionic strength decreased Ca2+ binding by no more than half. Binding was maximal at pH 7.5 and half-maximal at pH 6.3. The large number of binding sites with relatively high affinity for Ca2+ suggests that it is unlikely that this binding is to any specific protein or to non-specific sites present on many proteins, and that the most likely sites are lipid molecules.     

Dietary calcium deficiency increases Ca2+ uptake and Ca2+ extrusion mechanisms in chick enterocytes

Ca2+ uptake and Ca2+ extrusion mechanisms were studied in enterocytes with different degree of differentiation from chicks adapted to a low Ca2+ diet as compared to animals fed a normal diet. Chicks adapted to a low Ca2+ diet presented hypocalcemia, hypophosphatemia and increased serum 1,25(OH)2D3 and Ca2+ absorption. Low Ca2+ diet increased the alkaline phosphatase (AP) activity, independently of the cellular maturation, but it did not alter gamma-glutamyl-transpeptidase activity. Ca2+ uptake, Ca2+-ATPase and Na(+)/Ca2+ exchanger activities and expressions were increased by the mineral-deficient diet either in mature or immature enterocytes. Western blots analysis shows that vitamin D receptor (VDR) expression was much higher in crypt cells than in mature cells. Low Ca2+ diet decreased the number of vitamin D receptor units in both kinds of cells. In conclusion, changes in Ca2+ uptake and Ca2+ extrusion mechanisms in the enterocytes by a low Ca2+ diet appear to be a result of enhanced serum levels of 1,25(OH)2D3, which would promote cellular differentiation producing cells more efficient to express vitamin D dependent genes required for Ca2+ absorption.     

Regulation of kidney calcium-binding protein in the bird (Gallus domesticus)

The possible involvement of plasma calcium and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in the regulation of the concentration of kidney calcium-binding protein (CaBP) was investigated. Chicks were fed diets varying in Ca2+ and P, with or without vitamin D. CaBP and 1,25(OH)2D3 were determined by competitive binding assays. A significant correlation between plasma and kidney 1,25(OH)2D3 was found, the linear regression equation of best-fit was plasma 1,25(OH)2D3 = 0.14 + 1.56 kidney 1,25(OH)2D3. In the vitamin D-fed chicks, kidney CaBP varied independently of the circulating or organ level of 1,25(OH)2D3 (P greater than 0.05), but was lower in the vitamin D-deficient than in the vitamin D-fed birds. A significant correlation was observed between kidney CaBP and plasma calcium (Cap). The regression equations were CaBP = Cap/(85.57-4.00 Cap) (R = 0.845) and CaBP = 0.0558 + 0.0404 Cap (R = 0.749), for vitamin D-treated and vitamin D-deficient chicks, respectively. The results suggest that the concentration of kidney CaBP is modulated by plasma calcium, but one or more of the vitamin D metabolites may be required for its synthesis.     

1,25-Dihydroxyvitamin D3 stimulates 45Ca2+ uptake by cultured adult rat ventricular cardiac muscle cells

This study tested the hypothesis that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and its previously described cardiac receptors play roles in regulating intracellular calcium homeostasis in cardiac muscle cells. This question was addressed by assessing whether 1,25-(OH)2D3 influences 45Ca2+ uptake by homogeneous cultures of adult rat ventricular cardiac muscle cells. Twenty-four h prior to the measurement of 45Ca2+ uptake, the cells were transferred to serum-free medium ([Ca2+], 1.0 mM) containing 1.0 nM 1,25(OH)2D3 or vehicle. The cells were then incubated with 45Ca2+ for periods up to 60 min at room temperature, followed by removal of excess external 45Ca2+ by washing repeatedly with La3+. Pretreating the cells with 1,25-(OH)2D3 caused 3-fold stimulation (p less than 0.005) of 45Ca2+ uptake. Stimulation of 45Ca2+ uptake required a prolonged (8-12 h) exposure to 1,25-(OH)2D3, suggesting a receptor-mediated phenomenon. Concentrations of 0.01-10 nM 1,25-(OH)2D3 yielded a dose-response curve which peaked at 1.0 nM and decreased at higher concentrations. Steroid specificity was established by the failure of 1.0 nM levels of 25-hydroxyvitamin D3, estradiol-17 beta, and progesterone to change 45Ca2+ uptake. Sucrose gradient analysis confirmed the presence of a specific 3-4 S 3H-1,25-(OH)2D3 binding component both in freshly isolated and in cultured ventricular cardiac muscle cells. The stimulatory effect of 1,25-(OH)2D3 on 45Ca2+ uptake was abolished by the concomitant incubation of the cells with cycloheximide or actinomycin D, demonstrating a requirement for protein and nucleic acid synthesis. In conclusion, these data demonstrate that 1,25-(OH)2D3 stimulates 45Ca2+ uptake in adult ventricular cardiac muscle cells by a mechanism resembling a receptor-mediated phenomenon.     

Effect of 1α,25-dihydroxyvitamin D3 in plasma membrane targets in immature rat testis: ionic channels and gamma-glutamyl transpeptidase activity

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is critical for the maintenance of normal reproduction since reduced fertility is observed in vitamin D-deficient male rats. The aim of this study was to investigate the effect of 1,25D(3) in 30-day-old rat testicular plasma membrane targets (calcium uptake and gamma-glutamyl transpeptidase (GGTP) activity), as well as to highlight the role of protein kinases in the mechanism of action of 1,25D(3). The results demonstrated that 1,25D(3) induced a fast increase in calcium uptake in rat testis through a nongenomic mechanism of action. This effect was dependent on PKA, PKC and MEK. Moreover, ionic channels, such as ATP- and Ca(2+)-dependent K(+) channels and Ca(2+)-dependent Cl(-) channels, are involved in the mechanism of action. The use of BAPTA-AM showed that [Ca(2+)](i) was also implicated, and the incubation with digoxin produced an increase in (45)Ca(2+) uptake indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, 1,25D(3) was able to increase the GGTP activity. Considered together, our results indicate a PKA/PKC/MEK-dependent 1,25D(3) pathway as well as ionic involvement leading to (45)Ca(2+) uptake in immature rat testis. These findings demonstrate that 1,25D(3) stimulates calcium uptake and increases GGTP activity which may be involved in male reproductive functions.