Ontogeny of vitamin D action on the morphology and calcium transport properties of the chick embryonic yolk sac

The ontogeny of the calcium transport properties and hormonal modulation of the yolk sac membrane in amniote embryos is presently poorly understood. We investigated the role of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on plasma calcium values, yolk sac morphology and the ability of the yolk sac membrane to transport 45Ca from yolk to embryo. 1,25-(OH)2D3 treatment caused significant hypercalcaemia in 9-, 12- and 15-day embryos. Additionally, this hormone caused a hypertrophy of the endodermal cell layer that comprises the bulk of the yolk sac membrane. Both of these effects were the most dramatic in the 15-day embryo, the oldest age tested. 45Ca added to the yolk was transported into the blood rapidly across the yolk sac membrane. 1,25-(OH)2D3 significantly enhanced this transport in all age groups. [14C]Inulin was also taken across the yolk sac membrane, but at a slower rate than 45Ca; this transport was unaffected by 1,25-(OH)2D3. Thus, the yolk sac responds to 1,25-(OH)2D3 treatment both morphologically and functionally. The mechanism for transport appears to be a specific one, rather than a simple enhancement of non-specific endocytosis.     

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.     

Membrane receptor-initiated signaling in 1,25(OH)2D3-stimulated calcium uptake in intestinal epithelial cells

Demonstrating 1,25(OH)2D3-stimulated calcium uptake in isolated chick intestinal epithelial cells has been complicated by simultaneous enhancement of both uptake and efflux. We now report that in intestinal cells of adult birds, or those of young birds cultured for 72 h, 1,25(OH)2D3-stimulates 45Ca uptake to greater than 140% of corresponding controls within 3 min of addition. Such cells have lost hormone-stimulated protein kinase C (PKC) activity, believed to mediate calcium efflux. To further test this hypothesis, freshly isolated cells were preincubated with calphostin C, and calcium uptake monitored in the presence or absence of steroid. Only cells treated with the PKC inhibitor demonstrated a significant increase in 45Ca uptake in response to 1,25(OH)2D3, relative to corresponding controls. In addition, phorbol ester was shown to stimulate efflux, while forskolin stimulated uptake. To further investigate the mechanisms involved in calcium uptake, we assessed the role of TRPV6 and its activation by beta-glucuronidase. beta-Glucuronidase secretion from isolated intestinal epithelial cells was significantly increased by treatment with 1,25(OH)2D3, PTH, or forskolin, but not by phorbol ester. Treatment of cells with beta-glucuronidase, in turn, stimulated 45Ca uptake. Finally, transfection of cells with siRNA to either beta-glucuronidase or TRPV6 abolished 1,25(OH)2D3-enhanced calcium uptake relative to controls transfected with scrambled siRNA. Confocal microscopy further indicated rapid redistribution of enzyme and calcium channel after steroid. 1,25(OH)2D3 and PTH increase calcium uptake by stimulating the PKA pathway to release beta-glucuronidase, which in turn activates TRPV6. 1,25(OH)2D3-enhanced calcium efflux is mediated by the PKC pathway.     

Hypercalcemia in glucocorticoid withdrawal

We found severe hypercalcemia in the course of hydrocortisone withdrawal in a patient who had undergone unilateral adrenalectomy to resect a cortisol-hypersecreting adenoma. Serum calcium gradually but progressively increased after unilateral adrenalectomy. Severe hypercalcemia developed on the 77th postoperative day (the 15th day after discontinuing hydrocortisone replacement). The serum concentration of calcium, PTH, 25(OH)D, and 1,25(OH)2D were 8.0 mEq/l, less than 100 pg/ml, 10.1 ng/ml and 29.6 pg/ml, respectively. This hypercalcemia was accompanied by marked urinary hydroxyproline excretion and less calcium excretion in the urine than the prevailing level of serum calcium. Serum concentrations of 25(OH)D, 1,25(OH)2D and PTH were not elevated during the severe hypercalcemia. We concluded that the hypercalcemia in this patient was due in part to enhanced bone resorption and increased renal tubular reabsorption of calcium as a result of glucocorticoid withdrawal, but not to the elevation of serum PTH or serum 25(OH)D and serum 1,25(OH)2D.     

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.     

Hypercalcemia produced by parathyroid hormone suppresses experimental autoimmune encephalomyelitis in female but not male mice

Besides its role in regulating serum levels of calcium and phosphorus, 1alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) has potent effects on the immune system and suppresses disease in several animal models of autoimmune disorders including experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. While the amount of 1,25-(OH)2D3 needed to prevent EAE is dependent on the gender of the mouse and amount of calcium available in the diet, the minimum levels of 1,25-(OH)2D3 sufficient to prevent disease cause hypercalcemia. To test if hypercalcemia independent of high levels of 1,25-(OH)2D3 can suppress EAE, we used a 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-hydroxylase) knockout mouse strain. Because these 1alpha-hydroxylase knockout mice lack the parathyroid hormone (PTH)-regulated enzyme that synthesizes 1,25-(OH)2D3, hypercalcemia from increased bone turnover was created by continuous administration of PTH without changing the circulating levels of 1,25-(OH)2D3. This PTH-mediated hypercalcemia generated after EAE induction prevented disease in female mice but not male mice. When hypercalcemia was prevented by diet manipulation, PTH administration no longer prevented EAE. We conclude that hypercalcemia is able to prevent EAE after disease induction in female mice.     

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.     

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.     

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.     

Production of hypercalcemia in the chick embryo by an extract of Solanum malacoxylon.

White Leghorn eggs were injected on the 15th day of incubation with various doses of an acqueous extract of Solanum malacoxylon (SME). Most of the embryos died after the injection of 0.2 ml but the dose of 0.1 ml was well tolerated. The concentration of calcium in the sera from 15-day embryos injected with 0.1 ml SME was determined. Three hr after the injection the concentration of calcium had increased significantly; this increase lasted for at least 3 hr more but had disappeared 12 hr after the injection. It is suggested that this hypercalcemia may be produced by a water-soluble analog of 1,25-(OH)2D3 the presence of which has been demonstrated in the SME by other authors. It is also assumed that the mortality produced by the higher doses may be related to the hypercalcemia.     

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.     

Hypercalcemia and Acromegaly-Clarifying the Connections: A Case Report and Review of the Literature

ObjectiveHypercalcemia in patients with acromegaly is rare and usually due to co-existent primary hyperparathyroidism. The etiology of hypercalcemia directly related to acromegaly is debated.MethodsWe present a case report of 1,25(OH)2D3-mediated hypercalcemia in a patient with acromegaly and discuss potential pathophysiological mechanisms contributing to the development of hypercalcemia late in the course of the disease.ResultsA 67-year-old female presented with classical features of acromegaly. A review of her previous photographs suggested a disease duration of approximately 10 years, and her serum calcium (Ca) was normal during this period. A biochemical work up confirmed a combined growth hormone (GH-) and prolactin (PRL-) cosecreting tumor with a GH level of 92.03 ng/mL (normal 0-3.61), an insulin-like growth factor-1 (IGF-1) level of 1,498 ng/ mL (59-225), and a PRL level of 223.3 ng/mL (2-17.4). Magnetic resonance imaging (MRI) of the pituitary showed a 1.9-cm macroadenoma. Her preoperative work up revealed new onset hypercalcemia with a corrected serum Ca level of 10.7 mg/dL (8.5-10.5), an ionized Ca level of 1.37 mmol/L (1.08-1.30), a parathyroid hormone (PTH) level of 13.0 pg/mL (10-60), and a high 1,25(OH)2D3 level of 72.6 pg/mL (15-60). She underwent resection of the pituitary adenoma with normalization of GH and PRL levels, and her IGF-1 level decreased to 304 ng/mL. Her serum Ca (9.3 mg/dL), ionized Ca(1.22) and 1,25(OH)2D3 levels (38.6 pg/mL) normalized after surgery.ConclusionWhile overt hypercalcemia in acromegaly is rare, it tends to occur late in the disease course. The hypercalcemia is mediated by elevated 1,25(OH)2D3 levels rather than PTH. (Endocr Pract. 2014;20:e86-e90)     

Lead inhibits 1,25-dihydroxyvitamin D-3 regulation of calcium metabolism in osteoblastic osteosarcoma cells (ROS 17/2.8).

We have determined the dose-response of 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) on the intracellular free calcium-ion concentration ([Ca2+]i) in the osteoblastic osteosarcoma cells, ROS 17/2.8, using 19F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA). The dose-response demonstrated an inverted U-shaped relationship with maximal elevation of [Ca2+]i at doses of 1 to 10 nM 1,25-(OH)2D3. At 10 nM, 1,25-(OH)2D3 elevated the [Ca2+]i from a control level of 118 +/- 4 nM to a peak value of 237 +/- 8 nM within 40 min. 1,25-(OH)2D3 also increased the initial rate of Ca2+ influx into ROS 17/2.8 cells, measured by 45Ca uptake, with a dose-response relationship which paralleled its effect on [Ca2+]i. Treatment of ROS 17/2.8 cells with Pb2+ at 1 and 5 microM significantly increased [Ca2+]i but significantly reduced the 1,25-(OH)2D3-induced elevation of [Ca2+]i. Simultaneous treatment of naive cells with 1,25-(OH)2D3 and Pb2+ produce little reduction of 1,25-(OH)2D3-induced 45Ca uptake while 40 min treatment with Pb2+ before addition of 1,25-(OH)2D3 significantly reduced the 1,25-(OH)2D3-induced increase in 45Ca influx. These findings suggest that Pb2+ acts by inhibiting 1,25-(OH)2D3-activation of Ca2+ channels and interferes with 1,25-(OH)2D3 regulation of Ca2+ metabolism in osteoblastic bone cells.     

Effects of vitamin D3 metabolites on cytosolic free calcium in confluent mouse osteoblasts

A fluorescent Ca2+ indicator, acetoxymethyl Quin2, was used to quantify changes in the cytosolic free calcium concentration ([Ca2+]i) of confluent mouse osteoblasts. 1,25 - Dihydroxycholecalciferol (1,25 - (OH)2D3, 10-100 pM), 25-hydroxycholecalciferol (25-(OH)D3, 10-100 nM), parathyroid hormone (PTH(1-84), 0.1-10 nM), and prostaglandin E2 (PGE2, 10-1000 nM) all induced immediate (t less than 15 s) transient increases in [Ca2+]i, from a basal level of 135 +/- 8 nM to levels of 179-224 nM. These increases rapidly returned to a plateau approximately 10% higher than the basal level. 24,25-Dihydroxycholecalciferol (24,25-(OH)2D2, 0.1-10 nM) induced a rapid increase in [Ca2+]i which remained elevated for 5 min before decreasing. The 1,25-(OH)2D3- and PTH-induced spikes were abolished by the prior addition of EGTA and Ca2+ entry blockers (verapamil, nifedipine, 1 microM) while the responses to 25-(OH)D3, 24,25-(OH)2D3, and PGE2 were unaffected. Addition of 1,25-(OH)2D3 + EGTA or PTH + EGTA caused enhanced Ca efflux. Addition of drugs which interfere with calcium sequestration by the endoplasmic reticulum (ER) (caffeine, 4 mM; 8-(diethyl-amino)-octyl 3,4,5-trimethoxybenzoate HCl, 0.5 mM) or mitochondria (antimycin, 10 microM; oligomycin, 5 microM) showed that 25-(OH)D3 and PGE2 mainly mobilized Ca2+ from ER. 1,25-(OH)2D3 and bovine PTH caused a transient increase in [Ca2+]i, 70% of which resulted from Ca2+ influx from outside the cells and 30% by release from the ER. The [Ca2+]i increase induced by 24,25-(OH)2D3 included a 30% contribution from the ER and 70% from the mitochondria.     

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.     

1α,25-Dihydroxyvitamin D(3) signaling pathways on calcium uptake in 30-day-old rat Sertoli cells

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is the active metabolite of vitamin D(3) and the major calcium regulatory hormone in tissues. The aim of this work was to investigate the mechanism of action of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells from 30-day-old rats. Results showed that 10(-9) and 10(-12) M 1,25D(3) increased the rate of (45)Ca(2+) uptake 5 and 15 min after hormone exposure and that 1α,25(OH)(2) lumisterol(3) (JN) produced a similar effect suggesting that 1,25D(3) action occurs via a putative membrane receptor. The involvement of voltage-dependent calcium channels (VDCC) in 1,25D(3) action was evidenced by using nifedipine, while the use of Bapta-AM demonstrated that intracellular calcium was not implicated. Moreover, the incubation with ouabain and digoxin increased the rate of (45)Ca(2+) uptake, indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, we demonstrated that the mechanism underlying the hormone action involved extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) activation in a phospholipase C-independent way. Furthermore, a local elevation of the level of cAMP, as demonstrated by incubating cells with dibutyryl cAMP or a phosphodiesterase inhibitor, produced an effect similar to that of 1,25D(3), and the inhibition of protein kinase A (PKA) nullified the hormone action. In conclusion, the stimulatory effect of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells occurs via VDCC, as well as PKA, PKC, and ERK activation. These protein kinases seem to act by inhibiting Na(+)/K(+)-ATPase or directly phosphorylating calcium channels. The Na(+)/K(+)-ATPase inhibition may result in Na(+)/Ca(2+) exchanger activation in reverse mode and consequently induce the uptake of calcium into the cells.     

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.     

Rapid effects of calciotropic hormones on female rat enterocytes: combined actions of 1,25(OH)2-vitamin D3, PTH and 17beta-estradiol on intracellular Ca2+ regulation.

1,25(OH)(2)-Vitamin D(3) [1,25(OH)(2)D(3)], PTH and 17beta-estradiol increase intracellular Ca(2+) levels ([Ca(2+)](i)) in rat enterocytes by stimulating inner Ca(2+) store mobilization and voltage-dependent Ca(2+) channels through non-genomic activation of second-messenger cascades. The participation of store-operated Ca(2+) (SOC) channels in 17beta-estradiol regulation of enterocyte [Ca(2+)](i) has also been suggested. The aim of this work was to investigate whether PTH and/or 17beta-estradiol exert additive or synergistic effects acting in concert with the classic intestinal calciotropic hormone 1,25(OH)(2)D(3). Fura-2-loaded rat duodenal cells were stimulated using rPTH (10 nM), 17beta-estradiol (0.1 nM) or 1,25(OH)(2)D(3) (0.1 nM). The resulting Ca(2+) signal was characterized by an almost immediate rise in [Ca(2+)](i) (within 30 s) rapidly reaching peak levels, followed by a plateau phase that remained sustained as long as the cells were exposed to the stimulus. The addition of PTH at the sustained phase induced by 1,25(OH)(2)D(3) or, conversely, the addition of the secosteroid after the PTH-induced effect, did not induce additional increases in [Ca(2+)](i). Simultaneous treatment with both hormones resulted in an elevation of [Ca(2+)](i) equivalent to the maximal level caused by either agonist alone, suggesting common components for [Ca(2+)]i stimulation by PTH and 1,25(OH)(2)D(3). Treatment with 17beta-estradiol at the sustained phase induced by 1,25(OH)(2)D(3) or, conversely, treatment with the secosteroid after the 17beta-estradiol effect, induced additional increments in [Ca(2+)](i) (58 % and 63 %, respectively). Simultaneous treatment of enterocytes with both steroids potentiated their individual effects to the same extent as when added sequentially, also indicative of additive actions mediated by different sources of calcium signaling cascades. Moreover, 17beta-estradiol failed to further increase the 1,25(OH)(2)D(3)-induced initial Ca(2+) elevation in Ca(2+)-free medium, thus suggesting that extracellular influx mechanisms rather than intracellular Ca(2+) mobilization account for estrogen potentiation of 1,25(OH)(2)D(3) modulation of [Ca(2+)](i) in duodenal cells.     

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.     

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.