The lack of relationships between vitamin D3 metabolites and calcium-binding protein in the eggshell gland of laying birds

The relationship of the metabolism of vitamin D3 and calcium-binding protein (CaBP) to calcium transport by the eggshell gland (ESG) was assessed in chickens. Plasma or ESG 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) and ESG CaBP were no different between periods of ESG inactivity and of shell calcification. A severe dietary calcium deficiency resulted in increased kidney 25-hydroxycholecalciferol-1-hydroxylase activity (542%), plasma and ESG 1,25(OH)2D3 concentrations (193 and 274%, respectively), but in decreased ESG CaBP (34%), associated with the production of poorly calcified eggs. Significant correlations were found between 25 hydroxycholecalciferol-1-hydroxylase, plasma 1,25(OH)2D3 and ESG 1,25(OH)2D3, but not between ESG 1,25(OH)2D3 and CaBP. Hens with a low shell density had a significantly lower (55%) ESG CaBP than those with high shell density, without any significant change in ESG 1,25(OH)2D3. Significant correlations were found between ESG CaBP and shell calcium. Total receptors for 1,25(OH)2D3 were lower in ESG than in the intestine. The results suggest that CaBP level and calcium transport in the ESG are not regulated by 1,25(OH)2D3.     

Independence of 1,25-dihydroxyvitamin D3-mediated calcium transport from de novo RNA and protein synthesis

The administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to rachitic chicks produces an increase in (a) RNA and protein synthesis, (b) calcium binding protein (CaBP) concentration, and (c) alkaline phosphatase activity in the duodenum. These events occur concomitantly with enhanced calcium transport. We inhibited RNA and protein synthesis in richitic chicks and measured the subsequent response to 1,25(OH)2D3. Actinomycin D, injected prior to and following 1,25(OH)2D3 administration, inhibited intestinal RNA polymerase activity, blocked the rise in serum calcium, reduced the amount of CaBP, and increased alkaline phosphatase activity. Cycloheximide injected in similar fashion, inhibited the 1,25(OH)2D3-mediated increase in intestinal protein synthesis, serum calcium, CaBP, and alkaline phosphatase activity. Neither inhibitor blocked the ability of 1,25(OH)2D3 to stimulate calcium transport as measured in isolated duodenal loops in vivo. The ability of either inhibitor to block 1,25(OH)2D3-mediated calcium transport despite inhibition of CaBP production and alkaline phosphatase activity (by cycloheximide) indicates that de novo RNA and protein synthesis, and in particular CaBP and alkaline phosphatase, are not required for the 1,25(OH)2D3 stimulation of calcium transport.     

Diabetes and renal calcium binding protein in the rat

Renal calcium binding protein (CaBP), a vitamin D-dependent protein of 28,000 Mr, may be involved in calcium transport by cells of the renal tubule. The streptozotocin-diabetic rat is hypercalciuric and shows markedly decreased concentration of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] in serum and of CaBP in small intestine. To examine the relationship of renal CaBP in diabetes to 1,25-(OH)2D3 and urinary calcium excretion, renal CaBP, serum 1,25-(OH)2D3, and urinary calcium were measured in control, diabetic, and insulin-treated diabetic rats. Treatment of the diabetic rat with insulin decreased urinary calcium excretion and elevated 1,25-(OH)2D3 toward normal. Renal CaBP was found to be the same in controls and diabetics despite a tenfold difference in concentration of 1,25-(OH)2D3 in serum, and to be unaffected by insulin treatment, which elevated 1,25-(OH)2D3 by a factor of 7 above untreated diabetics. It is concluded that in the diabetic rat either (1) the threshold concentration of 1,25-(OH)2D3 for inducing synthesis of renal CaBP is set at a much lower level than that for intestinal CaBP, or (2) since both 1,25-(OH)2D3 and renal CaBP are produced in the kidney, 1,25-(OH)2D3 exerts a paracrine effect on renal CaBP production because of its high local concentration. The increased urinary calcium excretion in the untreated streptozotocin-diabetic rat is not secondary to an alteration in renal CaBP.     

Intestinal vitamin D-induced calcium-binding protein: time-course of immunocytological localization following 1,25-dihydroxyvitamin D3

The vitamin D-induced calcium-binding protein (CaBP) was localized in histological sections of chick duodenum using the peroxidase-antiperoxidase immunocytochemical technique. The time-course of appearance of CaBP in rachitic chicks was investigated from 0 to 120 hr after stimulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). CaBP was not routinely detected at 0 hr after 1,25(OH)2D3 administration. CaBP was first noted in some, but not all, of the samples taken 2 hr following 1,25(OH)2D3 and was detected in all 2 1/2 hr samples. The number of CaBP-containing absorptive cells and the apparent CaBP concentration both increased to a maximum at about 16-24 hr. At later times, as CaBP free cells migrated up the villi, the CaBP-containing cells decreased in number, but even at 120 hr post 1,25(OH)2D3 dose there were significant numbers of CaBP-containing cells present. The relationships between time-course of CaBP location on intestinal villi, enterocyte migration rates, and the time-course of 1,25(OH)2D3 stimulated intestinal calcium transport are discussed.     

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.     

Effect of dietary calcium and phosphorus depletion on vitamin D metabolism and calcium binding protein in the growing pig

Twenty-four young pigs were divided into three groups and each fed a replete, low calcium (Ca) or low phosphorus (P) diet. It was found that the deficient diets induced rises in renal 25 hydroxy-vitamin D 1,hydroxylase (1-hydroxylase) activity, circulating 1,25 dihydroxy-vitamin D3 (1,25 (OH)2-D3) and Ca binding protein (CaBP) and intestinal 1,25(OH)2D3 and CaBP. All these rises were statistically significant in the low Ca group but only the rises in the 1-hydroxylase activity and intestinal 1,25(OH)2D3 were significant in the low P group. A high degree of correlation existed between the parameters. There was no enhancement of intestinal 1,25(OH)2D3 or CaBP concentration relative to the 1-hydroxylase activity in the low P pigs as occurs in the chick. The low-P-induced rise in 1-hydroxylase activity was independent of parathyroid hormone.     

Cholecalciferol and placental calcium transport

Transplacental movement of calcium from mother to fetus is essential for normal fetal development. In most species, fetal plasma calcium levels are higher than maternal levels at term. The role of cholecalciferol metabolites, with specific emphasis on 1,25-dihydroxycholecalciferol (1,25(OH)2D), in placental calcium transport and maintenance of the fetomaternal gradient has been extensively investigated. In rats, there is not an absolute demand for 1,25(OH)2D for maintenance of fetal calcium homeostasis in utero, even though it is essential for maintenance of maternal plasma calcium levels. However, in sheep, the absence of 1,25(OH)2D results in disruption of both maternal and fetal calcium homeostasis. It is known that rat and human placentas contain specific cytosolic binding proteins for 1,25(OH)2D that are similar to the well-characterized intestinal receptor. Two calcium-binding proteins (CaBP) have been detected in rat and human placentas: a protein immunologically identical to the vitamin D-dependent CaBP and a calcium-dependent ATPase. The levels of CaBP in rat placenta have been shown to increase in response to exogenously administered 1,25(OH)2D but cannot be obliterated with maternal vitamin D deficiency. No relationship has been shown between 1,25(OH)2D and placental Ca-ATPase in any species. Thus, the mechanism of action of 1,25(OH)2D in maintenance of the transplacental calcium gradient in sheep is unknown. In the pregnant rat (and perhaps human), 1,25(OH)2D is a critical factor in the maintenance of sufficient maternal calcium for transport to the fetus and may play a role in normal skeletal development of the neonate.     

Hydrocortisone and 1,25(OH)2D3: role in proliferative responses of the embryonic chick duodenum in organ culture

The present studies were conducted to determine if hydrocortisone (HC) and/or 1,25-dihydroxycholecalciferol (1,25(OH)2D3) alter proliferative responses of the cultured duodenum, and if such alterations could be related to the known augmentation of 1,25(OH)2D3-induction of a specific, intestinal calcium-binding protein (CaBP) by glucocorticoids. HC stimulated proliferation in the duodenal epithelium, as indicated by increased DNA synthesis (3H-thymidine uptake), increased cell number/villus, and increased mitotic index after colchicine treatment. Goblet cell numbers were not significantly increased with any hormone treatment. The presence of 1,25(OH)2D3 alone did not affect proliferative responses. CaBP concentration as a function of tissue weight was 50% greater in HC stimulated intestine, indicating that the proliferogenic action of HC on the duodenum alone could not account for the glucocorticoid-1,25(OH)2D3 interaction in CaBP synthesis.     

Interrelations of calcium-regulating hormones during normal pregnancy.

Profound changes in calcium metabolism occur during pregnancy. The mother has to make available extra calcium for fetal requirements while ensuring that her plasma and bone calcium concentrations are satisfactorily maintained. In a cross-sectional study plasma concentrations of the major calcium-regulating hormones--namely, calcitonin, parathyroid hormone, 25-hydroxyvitamin D (25-OHD), and 1,25-dihydroxyvitamin D (1,25-(OH)2D)--were measured to establish their interrelations during normal pregnancy. The major changes observed were increases in the circulating concentrations of 1,25-(OH)2D and calcitonin. Concentrations of parathyroid hormone and 25-OHD remained within the normal range. The increased concentrations of 1,25-(OH)2D enable the increased physiological need for calcium to be met by enhancing intestinal absorption of this element. The simultaneous rise in calcitonin opposes the bone-resorbing activities of 1,25-(OH)2D, thereby protecting the integrity of the maternal skeleton. Maternal calcium homeostasis is thus maintained yet the requirements of the fetus are fulfilled.     

Gene expression profiles in rat intestine identify pathways for 1,25-dihydroxyvitamin D(3) stimulated calcium absorption and clarify its immunomodulatory properties

Microarray technology has been used to discover 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) induced gene expression changes in rat small intestine in vivo. Here, we report gene expression changes related to intestinal absorption or transport, the immune system and angiogenesis in response to 1,25-(OH)(2)D(3). Vitamin D deficient rats were intrajugularly given vehicle or vehicle containing 730 ng of 1,25-(OH)(2)D(3)/kg of body weight. Intestinal mRNA was harvested from duodenal mucosa at 15 min, 1, 3, and 6 h post-injection and studied by Affymetrix microarrays. Genes significantly affected by 1,25-(OH)(2)D(3) were confirmed by quantitative RT-PCR with remarkable agreement. The most strongly affected gene in intestine was CYP24 with 97-fold increase at 6 h post-1,25-(OH)(2)D(3) treatment. Intestinal calcium absorption genes: TRPV5, TRPV6, calbindin D(9k), and Ca(2+) dependent ATPase all were up-regulated in response to 1,25-(OH)(2)D(3), supporting the currently accepted mechanism of 1,25-(OH)(2)D(3) induced transcellular calcium transport. However, a 1,25-(OH)(2)D(3) suppression of several intra-/intercellular matrix modeling proteins such as sodium/potassium ATPase, claudin 3, aquaporin 8, cadherin 17, and RhoA suggests a vitamin D regulation of tight junction permeability and paracellular calcium transport. Several other genes related to the immune system and angiogenesis whose expression was changed in response to 1,25-(OH)(2)D(3) provided evidence for an immunomodulatory and anti-angiogenic role of 1,25-(OH)(2)D(3).     

Fibroblast growth factor-23 abolishes 1,25-dihydroxyvitamin D₃-enhanced duodenal calcium transport in male mice

Despite being widely recognized as the important bone-derived phosphaturic hormone, whether fibroblast growth factor (FGF)-23 modulated intestinal calcium absorption remained elusive. Since FGF-23 could reduce the circulating level of 1,25-dihydroxyvitamin D? [1,25(OH)?D?], FGF-23 probably compromised the 1,25(OH)?D?-induced intestinal calcium absorption. FGF-23 may also exert an inhibitory action directly through FGF receptors (FGFR) in the intestinal cells. Herein, we demonstrated by Ussing chamber technique that male mice administered 1 μg/kg 1,25(OH)?D? sc daily for 3 days exhibited increased duodenal calcium absorption, which was abolished by concurrent intravenous injection of recombinant mouse FGF-23. This FGF-23 administration had no effect on the background epithelial electrical properties, i.e., short-circuit current, transepithelial potential difference, and resistance. Immunohistochemical evidence of protein expressions of FGFR isoforms 1-4 in mouse duodenal epithelial cells suggested a possible direct effect of FGF-23 on the intestine. This was supported by the findings that FGF-23 directly added to the serosal compartment of the Ussing chamber and completely abolished the 1,25(OH)?D?-induced calcium absorption in the duodenal tissues taken from the 1,25(OH)?D?-treated mice. However, direct FGF-23 exposure did not decrease the duodenal calcium absorption without 1,25(OH)?D? preinjection. The observed FGF-23 action was mediated by MAPK/ERK, p38 MAPK, and PKC. Quantitative real-time PCR further showed that FGF-23 diminished the 1,25(OH)?D?-induced upregulation of TRPV5, TRPV6, and calbindin-D(9k), but not PMCA(1b) expression in the duodenal epithelial cells. In conclusion, besides being a phosphatonin, FGF-23 was shown to be a novel calcium-regulating hormone that acted directly on the mouse intestine, thereby compromising the 1,25(OH)?D?-induced calcium absorption.     

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.     

Effects of suppression of eggshell calcification and of 1,25(OH)2D3 on Mg2+, Ca2+ and Mg2+HCO-3 ATPase, alkaline phosphatase, carbonic anhydrase and CaBP levels--II. The laying hen intestine

Activity of a HCO-3 stimulated Mg2+ dependent ATPase is demonstrated in mitochondrial fractions of the avian duodenum. Suppression of eggshell calcification resulted in a slight reduction in Mg2+, Ca2+ and Mg2+HCO-3 ATPase activities. Duodenal carbonic anhydrase activity was lower in birds laying soft-shelled eggs than in birds laying normal eggs. Alkaline phosphatase and calcium binding protein levels both decreased along the length of the small intestine, but the effect was more pronounced for alkaline phosphatase. Suppression of eggshell calcification and treatment of shell-less laying hens with 1,25(OH)2D3 influenced alkaline phosphatase activity only in the duodenal mucosa. Suppression of eggshell calcification reduced CaBP levels in all sections of the intestine. Treatment with 1,25(OH)2D3 restored CaBP levels. Regulation of intestinal CaBP levels by 1,25(OH)2D3 would therefore, seem to be controlled more directly by calcium requirements associated with eggshell calcification than by gonadal hormones.     

Influence of cycloheximide and 1,25-dihydroxyvitamin D3 on mitochondrial and vesicle mineralization in the intestine

1,25(OH)₂D₃ increases cell permeability to calcium. This increase is not mediated by proteins sensitive to cycloheximide or actinomycin D inhibition. We propose that CaBP may associate with intracellular membranes and organelles to prevent intracellular calcium accumulation and the potential cytotoxic effects of such accumulation. In support of this hypothesis, the amount of mitochondrial mineralization in chick intestinal cells was markedly increased by 1,25(OH)₂D₃ treatment when CaBP synthesis was simultaneously blocked by cycloheximide treatment. Mineral in membrane vesicles was increased by 1,25(OH)₂D₃ treatment, but was blocked by simultaneous treatment with cycloheximide.     

Duodenal calcium absorption in dexamethasone-treated mice: functional and molecular aspects

Reduced intestinal calcium absorption may be part of the pathogenesis of glucocorticoid-induced osteoporosis. 1,25(OH)2D3 is the major regulator of the expression of the active duodenal calcium absorption genes: TRPV6 (influx), calbindin-D9K (intracellular transfer) and PMCA1b (extrusion). We investigated the influence of dexamethasone (5 days: 2 mg/kg bw) on calcium absorption in vivo and on the expression of intestinal and renal calcium transporters in calcium-deprived mice. Total and free 1,25(OH)2D3-concentrations were halved, in line with decreased 25(OH)D3-1-alpha-hydroxylase and increased 24-hydroxylase expression. Nevertheless, no difference in duodenal or renal calcium transporter expression pattern could be detected between vehicle and dexamethasone-treated mice. Accordingly, dexamethasone did not affect in vivo calcium absorption. By contrast, increased calcemia and collagen C-terminal telopeptide levels reflected increased bone resorption. Decreased osteocalcin levels suggested impaired bone formation. Hence, short-term glucocorticoid excess in young animals affected bone metabolism without detectable changes in intestinal or renal calcium handling.     

1,25-Dihydroxyvitamin D3 enhances the growth of tumors in athymic mice inoculated with receptor rich osteosarcoma cells

We tested the influence of daily subcutaneous injections of 12.5 and 25 pmol of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the growth of tumors arising from intracutaneous inoculations of athymic nude mice with rat osteogenic sarcoma cells (ROS) and human melanoma cells. Both doses of 1,25(OH)2D3 increased plasma calcium levels after 3 weeks and produced a striking enhancement in tumor weight when the mice received 1,25(OH)2D3 receptor-rich ROS17/2.8 cells. In contrast, 1,25(OH)2D3 caused no consistent effect on tumor weight in mice given G-361 melanoma cells with low receptor copy number or receptor deficient ROS 24/1 cells. Thus, 1,25(OH)2D3 stimulated tumor growth in a receptor dependent fashion, in vivo, instead of inhibiting it as predicted from the reduction of proliferation of cultured cells in the presence of 1,25(OH)2D3.     

Synergistic effects of 1,25(OH)2D3 and 24,25(OH)2D3 on duodenal CaBP in rachitic chicks and on eggshell weight in Japanese quails

The role of 24,25(OH)2D3 in calcium homeostasis is still controversial. In the present study the administration of low doses of 1,25(OH)2D3 and of higher doses of 24,25(OH)2D3 either alone or in conjunction with each other, were studied in rachitic chicks and in Japanese quails. Whereas 24,25(OH)2D3 alone had no significant effect on duodenal CaBP and on alkaline phosphatase in chick serum, it increased the influence of 1,25(OH)2D3 on these two parameters strongly. Also, when 1,25(OH)2D3 and 24,25(OH)2D3 were given simultaneously to Japanese quails, calcium excretion via the egg shell was clearly higher than when either metabolite had been administered alone. These results indicate that 1,25(OH)2D3 and 24,25(OH)2D3 exert a strong synergistic effect in rachitic animals.     

Prednisolone-induced Ca2+ malabsorption is caused by diminished expression of the epithelial Ca2+ channel TRPV6

Glucocorticoids, such as prednisolone, are often used in clinic because of their anti-inflammatory and immunosuppressive properties. However, glucocorticoids reduce bone mineral density (BMD) as a side effect. Malabsorption of Ca2+ in the intestine is supposed to play an important role in the etiology of low BMD. To elucidate the mechanism of glucocorticoid-induced Ca2+ malabsorption, the present study investigated the effect of prednisolone on the expression and activity of proteins responsible for active intestinal Ca2+ absorption including the epithelial Ca2+ channel TRPV6, calbindin-D(9K), and the plasma membrane ATPase PMCA1b. Therefore, C57BL/6 mice received 10 mg/kg body wt prednisolone daily by oral gavage for 7 days and were compared with control mice receiving vehicle only. An in vivo 45Ca2+ absorption assay indicated that intestinal Ca2+ absorption was diminished after prednisolone treatment. We showed decreased duodenal TRPV6 and calbindin-D(9K) mRNA and protein abundance in prednisolone-treated compared with control mice, whereas PMCA1b mRNA levels were not altered. Importantly, detailed expression studies demonstrated that in mice these Ca2+ transport proteins are predominantly localized in the first 2 cm of the duodenum. Furthermore, serum Ca2+ and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] concentrations remained unchanged by prednisolone treatment. In conclusion, these data suggest that prednisolone reduces the intestinal Ca2+ absorption capacity through diminished duodenal expression of the active Ca2+ transporters TRPV6 and calbindin-D(9K) independent of systemic 1,25(OH)2D3.     

Is 24,25-dihydroxycholecalciferol a calcium-regulating hormone in man?

Small doses (1-10 microgram daily) of 24,25-dihydroxycholecalciferol (24,25-(OH)2D3), a renal metabolite of vitamin D of uncertain function, increased intestinal absorption of calcium in normal people and in patients with various disorders or mineral metabolism, including anephric subjects. In five of six patients studied, calcium balance increased, but, unlike 1,25-dihydroxycholecalciferol, 24,25-(OH)2D3 did not increase plasma or urinary calcium concentrations. These results suggest that 24,25-(OH)2D3 may be an important regulator of skeletal metabolism in man with potential value as a therapeutic agent.