Action of 1,25-dihydroxyvitamin D3 and parathyroid hormone on 45calcium uptake by the yolk sac membrane of chick embryos

During development, the chick embryo mobilizes the calcium it needs from two extraembryonic sources, initially from the yolk and later from the eggshell. Calcium may be hormonally regulated during avian embryogenesis, but details of this regulation are lacking. We investigated the effects of 1,25-dihydroxycholecalciferol [1,25(OH)2D3], bovine parathyroid hormone [bPTH], and vehicle [ethanol or saline] on blood calcium values and incorporation of 45Ca into the yolk sac membrane of 9, 12, and 15 day chick embryos. Control data were also collected from uninjected 6 day embryos. Solutions were injected directly into the yolk sac compartment 48 and 24 hours prior to the experiment. Exogenous 1,25(OH)2D3 induced hypercalcemia in all age groups examined, while exogenous PTH induced hypercalcemia in day 12 and 15 embryos. Small disks of yolk sac membrane were incubated in medium to which 45Ca was added and assayed for 45Ca content at various intervals after start of incubation. In control yolk sac tissue, the uptake of 45Ca was greatest in younger embryos with decreasing uptake at developmentally more advanced ages; 1,25(OH)2D3 treatment significantly enhanced the uptake of 45Ca into yolk sac tissue in all groups (9, 12, and 15 day embryos). PTH treatment caused a significant elevation in 45Ca uptake in the day 12 and 15 embryos.     

Vitamin D and chick embryonic yolk calcium mobilization: identification and regulation of expression of vitamin D-dependent Ca2(+)-binding protein, calbindin-D28K, in the yolk sac

The developing chick embryo acquires calcium from two sources. Until about Day 10 of incubation, the yolk is the only source; thereafter, calcium is also mobilized from the eggshell. We have previously shown that during normal chick embryonic development, vitamin D is involved in regulating yolk calcium mobilization, whereas vitamin K is required for eggshell calcium translocation by the chorioallantoic membrane. We have studied here the biochemical action of 1,25-dihydroxy vitamin D3 in the yolk sac by examining the expression and regulation of the cytosolic vitamin D-dependent calcium-binding protein, calbindin-D28K. Two types of embryos are used for this study, normal embryos developing in ovo and embryos maintained in long-term shell-less culture ex ovo, the latter being dependent solely on the yolk as their calcium source. Our findings are (1) calbindin-D28K is expressed in the embryonic yolk sac, detectable at incubation Days 9 and 14; (2) the embryonic yolk sac calbindin-D28K resembles that of the adult duodenum in both molecular weight (Mr 28,000) and isoelectric point, as well as the presence of E-F hand Ca2(+)-binding structural domains; (3) systemic calcium deficiency caused by shell-less culture of chick embryos results in enhanced expression of calbindin-D28K in the yolk sac during late development; (4) yolk sac calbindin-D28K expression is inducible by 1,25-dihydroxy vitamin D3 treatment in vivo and in vitro; and (5) immunohistochemistry revealed that yolk sac calbindin-D28K is localized exclusively to the cytoplasm of the yolk sac endoderm. These findings indicate that the chick embryonic yolk sac is a genuine target tissue of 1,25-dihydroxy vitamin D3.     

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.     

Ca2(+)-channel agonist BAY K8644 mimics 1,25(OH)2-vitamin D3 rapid enhancement of Ca2+ transport in chick perfused duodenum.

To further understand the molecular mechanism by which 1,25(OH)2-vitamin D3 [1,25(OH2D3] rapidly stimulates intestinal calcium transport (termed "transcaltachia"), the effect of the calcium channel agonist BAY K8644 was studied in vascularly perfused duodenal loops from normal, vitamin D-replete chicks. BAY K8644, 2 mu M, was found to stimulate 45Ca2+ transport from the lumen to the vascular effluent to the same extent as physiological levels of 1,25(OH)2D3. The sterol and the Ca2+ channel agonist both increased 45Ca2+ transport 70% above control values within 2 min and 200% after 30 min of vascular perfusion. The effect of the Ca2+ channel agonist was dose dependent. Also, 1,25(OH)2D3-enhanced transcaltachia was abolished by the calcium channel blocker nifedipine. Collectively, these results suggest the involvement of 1,25(OH)2D3 in the activation of basal lateral membrane Ca2+ channels as an early effect in the transcaltachic response.     

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

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

Regulatory activities of 2 beta-(3-hydroxypropoxy)-1 alpha, 25-dihydroxyvitamin D3, a novel synthetic vitamin D3 derivative, on calcium metabolism

Regulatory activities of 2 beta-(3-hydroxypropoxy)-1 alpha, 25-dihydroxyvitamin D3 [ED-71], a novel synthetic vitamin D3 derivative, on calcium metabolism were investigated. The compound behaved similar to 1 alpha, 25-dihydroxyvitamin D3 [1,25(OH)2D3] in the ex vivo intestinal calcium transport using rat everted gut sac and the in vivo bone mobilization using vitamin D-deficient rats. By means of Raisz's assay method, 45Ca releasing activity of ED-71 was not greater than that of 1,25(OH)2D3. The time course curve of ED-71 in plasma made a mild round shape compared with that of 1,25(OH)2D3 and the former's plasma concentration remained increased longer than the latter's. The therapeutic effect of ED-71 for the animal models with osteoporosis seemed to be better than that of 1,25(OH)2D3. The results suggest that ED-71 may be a promising drug for therapy of osteoporosis.     

The effect of essential fatty acid deficiency on the stimulation of intestinal calcium transport by 1,25-dihydroxyvitamin D3

The effect of altering the lipid composition of the brush-border membrane on the ability of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) to stimulate calcium transport across the intestinal mucosa was examined by raising chicks on a vitamin D, essential fatty acid-deficient diet (-DEFAD) and measuring calcium absorption from duodenal sacs in situ and calcium uptake into brush-border membrane vesicles in vitro. Administration of 1,25-(OH)2D3 to -DEFAD and to -D control chicks led to the same increase in calcium transport in situ, whereas calcium transport in isolated brush-border membrane vesicles was not stimulated in the EFAD group, but responded normally in the control group. When the incubation temperature was increased to 34 degrees C, brush-border membrane vesicles from 1,25-(OH)2D3-treated essential fatty acid-deficient (+DE-FAD) chicks accumulated calcium at a faster rate than did vesicles from -DEFAD chicks. There was a marked decrease in the linoleic acid content and an increase in the oleic acid content of both the total lipid extract of the brush-border membrane as well as the phosphatidylcholine and phosphatidylethanolamine fractions, which could explain the temperature sensitivity of the in vitro system. When the diet of the EFAD chicks was supplemented with linoleic acid, the rate of calcium uptake into subsequently isolated vesicles from +DE-FAD chicks correlated with the amount of linoleic acid in the brush-border membranes. These results support the concept that the action of 1,25-(OH)2D3 on membrane lipid turnover and structure plays a critically important role in the 1,25-(OH)2D3-mediated cellular transport responses.     

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.     

Calcium-transport in quiescent and 1,25-dihydroxycholecalciferol-stimulated human osteosarcoma cells. Role in 24 hydroxylase enhancement

The present study evaluates in osteosarcoma cells, the effects of a calcium channel inhibitor nicardipine in 24-hydroxylase activity and 45Ca desaturation curve in presence of 1,25-dihydroxycholecalciferol (1,25(OH)2D3). This sterol induced an increase in 24-OHase activity and 45Ca fluxes. Nicardipine reversed the effect of 1,25(OH)2D3 on 45Ca fluxes but reinforced the enhancement of the 24-OHase activity. The fact that the effects of 1,25(OH)2D3 were reduced by cycloheximide support the hypothesis of a de novo protein synthesis. Our study has allowed us to dissociate the effects of 1,25(OH)2D3 on 24-OHase enhancement from those on Ca2+ transport.     

Regulation of rat yolk sac 25-hydroxy- and 1,25-dihydroxyvitamin D3 24-hydroxylase activities

The yolk sac of the pregnant rat which functions as a true placenta is a target organ for vitamin D. This tissue can hydroxylate in position 24 both 25-hydroxy- and 1,25-dihydroxyvitamin D3 (25-OHD3 and 1,25-(OH)2D3). The present report describes an in vitro model for the study of 1,25-(OH)2D3 action on the further metabolism of 25-OH[3H]D3 and 1,25-(OH)2[3H]D3 by yolk sac. The tissue explants were preincubated with 1,25-(OH)2D3 for 18 h in a serum-free culture medium. Physiological concentrations of 1,25-(OH)2D3 were the most effective in stimulating (7.5-fold) the 1,25-(OH)2D3 24-hydroxylase, while the 25-OHD3 24-hydroxylase stimulation (4-fold) required a 1,25-(OH)2D3 concentration of 10(-7) M. The stimulating effect of 1,25-(OH)2D3 on the 1,25-(OH)2D3 24-hydroxylase was temperature-dependent, and, since its was inhibited by actinomycin D and cycloheximide, required de novo protein synthesis. 1,24,25-(OH)3D3, 25-OHD3, and 24,25-(OH)2D3 were 10- to 1000-fold less potent than 1,25-(OH)2D3 in inducing the 1,25-(OH)2D3 hydroxylase. Our results strongly suggest that 1,25-(OH)2D3 regulated the 1,25-(OH)2D3 24-hydroxylase by a receptor-mediated process. Furthermore, 1,25-(OH)2D3 at 10(-9) M induced within 4 h an increase of its own degradation and the formation of an as yet unidentified major 1,25-(OH)2[3H]D3 metabolite. We conclude that the yolk sac can participate in the regulation of 1,25-(OH)2D3 concentration in the fetoplacental unit.     

Biological activity of vitamin D metabolites and analogs: dose-response study of 45Ca transport in an isolated chick duodenum perfusion system

We have previously reported that vascular perfusion of the normal vitamin D3-replete chick duodenum with physiological amounts of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] increases the unidirectional movement of 45Ca from the lumen to the venous effluent under conditions of normal (0.9 mM) Ca2+ concentrations in both the lumen and vascular perfusate [Endocrinology 115: 1476 1984)]. The purpose of the present study was to determine the dose responsivity of this perfused intestinal calcium transport system for 1,25(OH)2D3 and some structurally related congeners. The dose-response curve was biphasic for all compounds studied; for 1,25(OH)2D3 initial stimulation of transport was detected at only 30 pM [the plasma concentration of 1,25(OH)2D3 is normally 125 pM] while maximal stimulation was 154% above control at a concentration of 650 pM. Above 650 pM 1,25(OH)2D3 the stimulation fell off sharply and transport had returned to basal levels by 1.3 nM. The relative potency of the D homologs tested was respectively 1,25(OH)2D3: 10,000; 1-alpha-hydroxyvitamin D3: 400; 25-hydroxyvitamin D3: 200; 24R,25-dihydroxy-vitamin D3: 137; vitamin D3: 34; 5,6-trans-25-hydroxyvitamin D3: 3. These results establish the usefulness of the perfused intestinal calcium transport system to study the nongenomic actions of 1,25(OH)2D3 on intestinal calcium transport.     

Single cell analysis of changes in cytosolic calcium induced by vitamin D3 metabolites in cultured rat mesangial cells.

The acute effects of 1,25-Dihydroxy-vitamin D3 [1,25(OH)2D3] on the concentration of cytoplasmic ionized calcium [Ca2+] of cultured rat mesangial cells were studied at the single cell level by microspectrofluorometry of fura-2-loaded cells. Addition of 1,25(OH)2D3 produced an immediate increase of [Ca2]+. This rise in [Ca2+] was sustained and similar to that caused by the Ca2+ channel agonist BAY K 8644. Comparable changes were also observed in cultured human mesangial cells. The effects of the hormone (10 (-10)-10(-7) M) were dose-dependent (62% and 285%). Only 30-40% of the cells responded to stimulation with 1,25(OH)2D3. 25OHD3 also increased Ca2+ whereas 24,25(OH)2D3 and 1aOHD3 were inactive. Addition of 1 mM CoCl2 or 2-5 microM nifedipine largely blocked the effects of 1,25(OH)2D3 suggesting the involvement of Ca2+ channel activation in the rapid 1,25(OH)2D3-induced increase in mesangial cell [Ca2+]. 45Ca uptake studies are consistent with This interpretation.     

Ca2+ priming during vitamin D-induced monocytic differentiation of a human leukemia cell line

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) induces monocytic differentiation of the human promyelocytic leukemia line, HL-60, and enhances Ca2+ transport in target cells of the mineral metabolism system. Hence, we determined whether the steroid's maturational effect on HL-60 involves alterations of intracellular calcium [( Ca2+]i). We found that, as detected by indo-1 fluorescence, [Ca2+]i increases in a slow tonic manner from 99 +/- 11 nM in virgin HL-60 to 182 +/- 19 nM (p less than 0.001) in those treated with 1,25-(OH)2D3 for 24 h. The first apparent rise in [Ca2+]i occurs at between 6 and 12 h and parallels expression of alpha-thrombin and N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptors. This increase in [Ca2+]i is derived from extracellular calcium as its reduction abolishes the effect. The increase in [Ca2+]i is associated with an increase in inositol trisphosphate-stimulated Ca2+ flux from intracellular stores. Interestingly, 1,25-(OH)2D3-mediated HL-60 differentiation as manifest by expression of the macrophage-specific antigen, 63D3, is not blocked by low extracellular calcium. In contrast, the fMLP-induced superoxide ion generation is diminished if the increase in [Ca2+]i is prevented. Furthermore, fMLP-stimulated signal transduction is also reduced by limiting the stimulation of [Ca2+]i during 1,25-(OH)2D3 treatment. Thus, although differentiation of HL-60 to the monocytic phenotype by 1,25-(OH)2D3 is Ca2+-independent, expression of response to regulatory stimuli requires priming of cellular Ca2+ stores. The latter appears to be induced by 1,25-(OH)2D3 via stimulated Ca2+ entry through the plasma membrane.     

Vitamin D3 metabolites modulate dihydropyridine-sensitive calcium currents in clonal rat osteosarcoma cells

A slowly inactivating inward calcium current was identified in the rat osteosarcoma cell line ROS 17/2.8 using a combination of ion flux and electrophysiological techniques. Voltage dependence, dihydropyridine sensitivity, divalent cation selectivity, and single channel properties identified this current as a high threshold, "L-type" calcium current. Ion flux experiments using 45Ca2+ confirmed that calcium uptake through these channel represents a major pathway for calcium entry into osteosarcoma cells. In resting cells, i.e. at negative membrane potentials, stimulation of both calcium current and rapid 45Ca2+ influx could be elicited by concentrations of 1,25-(OH)2-vitamin D3 between 0.1 and 3 nM. At these concentrations, 1,25-(OH)2-vitamin D3 shifted the threshold for activation of inward calcium current to more negative potentials. At higher concentrations (5-10 nM), inhibitory effects became predominant. These opposing effects are functionally similar to those of the dihydropyridine BAY K 8644. Other vitamin D3 metabolites (25-(OH)-D3 and 24,25-(OH)2-D3) exhibited less potent stimulatory effects and greater inhibition of calcium current than 1,25-(OH)2-D3. These results suggest that (i) vitamin D3 acts as a potent modulator of calcium channel function in osteosarcoma cells, and (ii) intracellular Ca2+-dependent signaling processes may be affected acutely by physiological concentrations of vitamin D3 metabolites.     

Sources and timing of calcium mobilization during embryonic development of the corn snake, Pantherophis guttatus

Embryos of oviparous Reptilia (=turtles, lepidosaurs, crocodilians and birds) extract calcium for growth and development from reserves in the yolk and eggshell. Yolk provides most of the calcium to embryos of lizards and snakes. In contrast, the eggshell supplies most of the calcium for embryonic development of turtles, crocodilians and birds. The yolk sac and chorioallantoic membrane of birds recover and transport calcium from the yolk and eggshell and homologous membranes of squamates (lizards and snakes) probably transport calcium from these two sources as well. We studied calcium mobilization by embryos of the snake Pantherophis guttatus during the interval of greatest embryonic growth and found that the pattern of calcium transfer was similar to other snakes. Calcium recovery from the yolk is relatively low until the penultimate embryonic stage. Calcium removal from the eggshell begins during the same embryonic stage and total eggshell calcium drops in each of the final 2 weeks prior to hatching. The eggshell supplies 28% of the calcium of hatchlings. The timing of calcium transport from the yolk and eggshell is coincident with the timing of growth of the yolk sac and chorioallantoic membrane and expression of the calcium binding protein, calbindin-D28K, in these tissues as reported in previous studies. In the context of earlier work, our findings suggest that the timing and mechanism of calcium transport from the yolk sac of P. guttatus is similar to birds, but that both the timing and mechanism of calcium transport by the chorioallantoic membrane differs. Based on the coincident timing of eggshell calcium loss and embryonic calcium accumulation, we also conclude that recovery of eggshell calcium in P. guttatus is regulated by the embryo.     

1,25-Dihydroxyvitamin D₃ contributes to regulating mammary calcium transport and modulates neonatal skeletal growth and turnover cooperatively with calcium

To assess the interaction of 1,25(OH)(2)D(3) and dietary calcium on mammary calcium transport in lactating dams and skeletal growth and turnover in the neonate, female lactating 1α(OH)ase(+/-) or 1α(OH)ase(-/-) mice were fed either a high-calcium diet containing 1.5% calcium in the drinking water or a "rescue diet." Dietary effects on the expression of molecules mediating mammary calcium transport were determined in the dams, and the effects of milk calcium content were assessed on skeletal growth and turnover in 2-wk-old 1,25(OH)(2)D(3)-deficient pups. Results showed that the reduction of milk calcium levels in the 1α(OH)ase(-/-) dams and the elevation of milk calcium levels in dams fed the rescue diet were associated with the down- or upregulation of calbindin D(9k) and plasma membrane Ca(2+) ATPase isoform 2b expression, respectively, in mammary epithelial cells. The action of ambient calcium in stimulating skeletal growth in the neonates appeared to supercede the direct action of 1,25(OH)(2)D(3), and the response of chondrocytes in the neonates to elevated calcium was more sensitive in hypocalcemic animals. Osteopenia was more apparent in pups nursed by dams with lower milk calcium than in 1,25(OH)(2)D(3)-deficient pups nursed by dams with higher milk calcium. Bone formation parameters were increased significantly in all pups fed by dams on the rescue diet but were still lower in 1α(OH)ase(-/-) pups than in 1α(OH)ase(+/-) pups. Consequently, there is an important contributory role of calcium in conjunction with 1,25(OH)(2)D(3) to mammary calcium transport in lactating dams and skeletal growth and turnover in the neonate.     

A specific binding protein for 1 alpha,25-dihydroxyvitamin D in the chick embryo chorioallantoic membrane

A 3.7 S binding protein for the steroid hormone and vitamin D metabolite 1 alpha-25-dihydroxyvitamin D (1,25-(OH)2-D) was observed in high salt cytosol extracts of chick embryo chorioallantoic membrane. The binding protein was characterized after partial purification of cytosol extracts by ammonium sulfate fractionation. The binding of 1,25-(OH)2-D was saturable, had a high affinity (Kd = 0.16 nM), and was specific for hormonally active vitamin D metabolites. Analysis of the displacement of [3H]1,25-(OH)2-D by unlabeled analogues showed the affinities of vitamin D metabolites to be in the order of 1,25-(OH)2-D = 1,24R,25-(OH)3-D much greater than 25-OH-D = 1-OH-D greater than 24R,25-(OH)2-D. Hormone binding was sensitive to pretreatment with sulfhydryl-blocking reagents. The chorioallantoic membrane 1,25-(OH)2-D-binding protein associated with the chromatin fraction after homogenization of membranes in low salt buffer, and bound to DNA-cellulose columns, eluting as a single peak at 0.215 M KCl. These findings support identification of this 1,25-(OH)2-D-binding protein as a steroid hormone receptor, with properties indistinguishable from 1,25-(OH)2-D receptors in other chick tissues. The chorioallantoic membrane functions in the last third of embryonic development to reabsorb calcium from the eff shell for deposition in embryonic bone. 1,25-(OH)2-D binding activity in the chorioallantoic membrane increased 4- to 5-fold from day 12 to day 16 of incubation, immediately preceding the onset of shell reabsorption. This finding suggests that 1,25-(OH)2-D may act to regulate shell mobilization and transepithelial calcium transport by the chorioallantoic membrane. Finally, the similarity of shell mobilization to bone resorption, which is also stimulated by 1,25-(OH)2-D, suggests that the chorioallantoic membrane is a useful alternate model for the study of 1,25-(OH)2-D action on bone mineral metabolism.     

1,25 dihydroxyvitamin D3 enhances the calcium response of keratinocytes

The steroid hormone 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) regulates cell proliferation and differentiation. Intracellular calcium (Cai) concentrations play a crucial role in these events. From our previous studies, we have demonstrated a calcium receptor (CaR) in keratinocytes which appears to regulate the initial release of Cai from intracellular stores in response to extracellular calcium (Cao) and so is likely to participate in the differentiation process. In this study, we determined whether the ability of 1,25(OH)2D3 to enhance Ca++ -induced differentiation was mediated at least in part through changes in the CaR. Keratinocytes were grown in keratinocyte growth medium (KGM) with 0.03 mM, 0.1 mM, or 1.2 mM Ca and treated with 10(-8) M 1,25(OH)2D3 till harvest after 5, 7, 14, and 21 days. CaR mRNA levels were quantitated by polymerase chain reaction. The results were compared to the ability of 1,25(OH)2D3 to enhance calcium-stimulated increases in Cai. In cells grown in 0.03 mM Ca, the CaR mRNA levels decreased with time. 1,25(OH)2D3 stimulated the levels at 5 days and prevented the falloff over the subsequent 16 days. On the other hand, in cells grown in 0.1 or 1.2 mM Ca, the message levels remained high, and 1,25(OH)2D3 had no further effect. To study the functional relationship, we harvested cells after 5 and 7 days in culture following a 24 h treatment with 1,25(OH)2D3 or vehicle to measure the Cai response to 2 mM Cao. The preconfluent cells grown in 0.03 mM Ca showed a nearly twofold increase in the Cai response to Cao when pretreated with 1,25(OH)2D3, whereas the confluent cells and those grown in 1.2 mM Ca showed no enhancement by 1,25(OH)2D3. Studies with 45Ca influx into keratinocytes revealed that 1,25(OH)2D3 enhanced the influx in preconfluent and confluent cells when grown in KGM containing 0.03 mM Ca but not in cells grown in 1.2 mM calcium. We conclude that 1,25(OH)2D3 maintains the CaR mRNA levels in cells grown in 0.03 mM Ca, thus maintaining their responsiveness to Cao and so ensuring their ability to differentiate in response to the calcium signal.     

The effect of calcium-regulating hormones on transport of calcium across the chorioallantoic membrane of the chicken embryo

The hormonal form of vitamin D3 (1,25(OH)2D3), parathyroid hormone (PTH), or appropriate vehicle were injected into the yolk sac of eggs of domestic fowl on days 16 and 17 of incubation. The chorioallantoic membrane (CAM) and overlying inner shell membrane were removed from eggs on day 18 and mounted in a Ussing-type apparatus. Transport of calcium was assessed by monitoring movements of radiolabeled calcium. Transport of calcium from the chorionic aspect of the CAM to the allantoic aspect increased considerably with time for all treatment groups except the one receiving PTH. "Back-flux" of calcium (movement of calcium from the allantoic aspect to the chorionic) was negligible for all treatment groups at all sampling periods. PTH treatment did not affect flux of calcium from allantois to chorion but reduced flux from chorion to allantois considerably. The underlying cause of this effect has not been identified. The hormonal form of vitamin D3 did not affect flux of calcium in either direction. These data raise the possibility that control of calcium transport by the CAM may not be the primary function of the vitamin D hormone.     

Regulation of duodenal insulin-like growth factor I and active calcium transport by ovariectomy in female rats.

There is a significant body of data that supports the concept that reproductive hormones in females have effects on duodenal calcium transport that are not mediated via altered circulating concentrations of 1,25-dihydroxyvitamin D (1,25(OH)2D). Previously, we have shown parallel alterations in duodenal Ca transport and longitudinal bone growth rate in sexually maturing female rats in response to ovariectomy and estradiol (E) treatment of ovariectomized (OVX) rats (OVX+E) without any change in circulating levels of 1,25(OH)2D or parathyroid hormone. Results are presented here from experiments designed to: (i) further explore the relationship between 1,25(OH)2D and ovarian status in the regulation of duodenal calcium transport, and (ii) determine whether OVX and E replacement alter circulating and duodenal levels of insulin-like growth factor I (IGF-I) that might be related to effects on Ca transport. Growth hormone, which has been shown to affect intestinal Ca absorption and vitamin D metabolism, is thought to act indirectly by stimulating IGF-I. Six-week-old female rats were OVX, given estradiol implants (OVX+E), and fed a diet containing either 0.5% or 0.1% Ca for 3 weeks. In both diet groups, the OVX animals exhibited a higher level of Ca transport, as measured by the everted gut sac method, than either the intact controls or the OVX+E group; there was no difference in calcium transport between the different diet groups. Although there was no difference in circulating levels of 1,25(OH)2D among the intact, OVX, and OVX+E groups fed either diet, animals fed the 0.1% Ca diet had higher circulating levels of 1,25(OH)2D than those fed the 0.5% Ca diet. There was no difference in duodenal levels of calbindin9K among intact, OVX, and OVX+E animals in either diet group, although the animals fed the 0.1% Ca diet had higher levels of calbindin9K than the animals fed the 0.5% Ca diet. In animals fed the 0.5% Ca diet, OVX resulted in elevated serum and duodenal levels of IGF-1, as compared with intact and OVX+E animals on the same diet. In animals fed the 0.1% Ca diet, there was no elevation of IGF-I in the OVX group relative to intact and OVX+E animals. These results lend additional support to the concept that alterations in duodenal active calcium transport that occur with alterations in ovarian hormones are not mediated by changes in serum levels of 1,25(OH)2D, but may be related to some factor related to growth, possibly IGF-I.