1,25-dihydroxycholecalciferol: the metabolite of vitamin D responsible for increased intestinal calcium transport

1,25-Dihydroxycholecalciferol was prepared from [26,27-³H]-25-hydroxycholecalciferol and from [1,2-³H]-25-hydroxycholecalciferol enzymatically and purified chromatographically. Injection of 62.5 pmoles of 1,25-dihydroxycholecalciferol intravenously into vitamin D-deficient chicks resulted in the accumulation of a maximum of 5.9% of the dose in the intestine. During the 12 hr period following injection, this radioactivity was found almost entirely as 1,25-dihydroxycholecalciferol. It has previously been shown that intestinal calcium absorption is initiated by 1,25-dihydroxycholecalciferol during this period. These results provide strong evidence that the 1,25-dihydroxycholecalciferol is not metabolized further before it initiates intestinal calcium absorption.     

Effects of 1,25-dihydroxycholecalciferol administration on the rat renal vitamin K-dependent carboxylating system

We have shown previously that the in vitro activity of the renal vitamin K-dependent gamma-glutamyl carboxylase toward synthetic oligopeptide substrates is stimulated by administration of either parathyroid hormone (PTH) or 1,25-dihydroxycholecalciferol [1,25(OH)2D3] to rats [(1983) J. Biol. Chem. 258, 12783-12786]. Here we report that administration of 1,25(OH)2D3 to rats increases their levels of endogenous carboxylase substrate as well. Rats fed a vitamin D-deficient diet had highly elevated serum PTH levels while vitamin D-replete animals had undetectable levels. Furthermore, since PTH increases 1,25(OH)2D3 levels by stimulating renal 25-hydroxyvitamin D-1 alpha-hydroxylase, it is very likely that the stimulatory effects of PTH on the renal vitamin K-dependent carboxylating system are mediated by 1,25(OH)2D3.     

Intestinal lead and calcium absorption: effect of 1,25-dihydroxycholecalciferol and lead status

This study was designed to investigate, in some detail, the relative effects of the hormonal form of vitamin D (1,25-dihydroxycholecalciferol) on duodenal Pb and Ca absorption as a function of dietary Pb level. When cholecalciferol-deficient chicks were chronically repleted with physiologic levels of 1,25-dihydroxycholecalciferol (1,25(OH)2D3), as the sole source of the vitamin, 203Pb and 47Ca absorption were enhanced over 4- and 8-fold, respectively. Ingestion of Pb during the repletion period had no significant effect on the intestinal Ca absorption response to 1,25-(OH)2D3 even at a very high dietary Pb level. The efficiency of intestinal 203Pb absorption was, however, significantly diminished by dietary Pb, in an apparent dose-dependent fashion. The results indicate that the extent to which systemic Ca homeostatic mechanisms influence intestinal Pb absorption is dependent, in large part, on Pb status.     

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.     

1,25-Dihydroxyvitamin D3 increases the expression of the CaT1 epithelial calcium channel in the Caco-2 human intestinal cell line

Background  

The active hormonal form of vitamin D (1,25-dihydroxyvitamin D) is the primary regulator of intestinal calcium absorption efficiency. In vitamin D deficiency, intestinal calcium absorption is low leading to an increased risk of developing negative calcium balance and bone loss. 1,25-dihydroxyvitamin D has been shown to stimulate calcium absorption in experimental animals and in human subjects. However, the molecular details of calcium transport across the enterocyte are not fully defined. Recently, two novel epithelial calcium channels (CaT1/ECaC2 and ECaC1/CaT2) have been cloned and suggested to be important in regulating intestinal calcium absorption. However, to date neither gene has been shown to be regulated by vitamin D status. We have previously shown that 1,25-dihydroxyvitamin stimulates transcellular calcium transport in Caco-2 cells, a human intestinal cell line.     

Lysosomal proliferation in rachitic avian intestinal absorptive cells following 1,25-Dihydroxycholecalciferol

Lysosomes in chick intestinal absorptive cells from rachitic (vitamin D-deficient) and vitamin D-replete animals were studied utilizing transmission electron microscopic histochemistry and ultrastructural morphometry. Absorptive cells from rachitic animals, serum calcium = 7.3±0.3 mg%, contained an average of 4.0±0.3 supranuclear lysosomes. In rachitic chicks sacrificed 9 hr post-injection of 1,25-dihydroxycholecalciferol, the active metabolite of vitamin D, the values for both serum calcium, 9.8 ± 0.2 mg%, and the number of apical absorptive cell lysosomes, 12.9±0.6, were increased over non-injected or vehicle-only injected animals. Lysosomes in vitamin D-replete absorptive cells were characterized by their intense staining with pyroantimonate, indicative of their high calcium content. The same organelles also produced a positive reaction for acid phosphatase. Rachitic lysosomes, also acid phosphatase positive, were only lightly stained with pyroantimonate. The lysosomal proliferation apparently induced by 1,25-dihydroxycholecalciferol may be a further indication that these organelles play a role in intestinal calcium transport and/or intracellular calcium homeostasis within the absorptive cell.     

Duodenal expression of the epithelial calcium transporter gene TRPV6: is there evidence for Vitamin D-dependence in humans?

Intestinal absorption of dietary calcium is regulated by 1,25-dihydroxycholecalciferol (1,25(OH)(2)D(3)) in humans and in experimental animals but interspecies differences in responsiveness to 1,25(OH)(2)D(3) are found, possibly due to differences in the promoters of genes for intestinal calcium transport proteins or of the Vitamin D receptor (VDR). The epithelial calcium transporter, known as ECAC2 or CAT1, the product of the TRPV6 gene expressed in proximal intestinal enterocytes, is the first step in calcium absorption and studies in mice have shown that its expression is Vitamin D-dependent. In contrast in man, we showed that duodenal TRPV6 mRNA expression was independent of blood 1,25(OH)(2)D(3), although in Caco-2 cells, 1,25(OH)(2)D(3)-dependent changes have been demonstrated. We sought to explain these findings. A consensus Vitamin D response element in the mouse gene is absent in the human gene. We re-analysed our duodenal expression data according to a CDX2-site polymorphism in the VDR promoter. Mean TRPV6 expression was the same, but there was evidence of different responsiveness to 1,25(OH)(2)D(3). In the GG genotype group, but not the AG, duodenal TRPV6 expression increased with 1,25(OH)(2)D(3). We postulate that lower levels of expression of VDR in the GG group produce greater sensitivity to 1,25(OH)(2)D(3).     

Hypertrophy and calcification of rabbit permanent chondrocytes in pelleted cultures: synthesis of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor

Cartilage calcification at specific sites is a key event that leads to skeletal development and growth. To obtain insights into the control of cartilage calcification, we examined whether cells distributed in permanent cartilage regions might have the ability to express the calcification-related phenotype in a permissive environment. Chondrocytes were isolated from the permanent and growth plate cartilages of 4-week-old rabbit ribs. They were seeded as a pelleted mass in a centrifuge tube and cultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum. These cells proliferated for several generations, and then synthesized large amounts of proteoglycans, yielding a cartilage-like tissue in 16 days. Cultures from the permanent and growth plate cartilages showed similar time courses for increases in DNA synthesis and proteoglycan production that reached similar maximal levels. Thereafter, they initiated the syntheses of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor and induced matrix calcification without additional phosphate. The increases in alkaline phosphatase, 1,25-dihydroxycholecalciferol receptor, and calcium contents in cultures from the permanent cartilage were consistently delayed for 4-7 days relative to the growth plate-derived cells, but caught up by Day 28. The maximal levels of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor in the cultures from the permanent cartilage were 40- to 100-fold higher than that of the in vivo permanent cartilage. These results provide evidence that permanent cartilage cells in postnatal young rabbit ribs have the capacity to express alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor and induce calcification in a permissive environment, although they never express these calcification-related phenotypes in vivo.     

Differentiation of the changes in alkaline phosphatase from calcium ion-activated adenosine triphosphatase activities associated with increased calcium absorption in chick intestine.

The pattern of response of the intestinal enzymes Ca2+-activated adenosine triphosphatase and alkaline phosphatase in the chick to 1,25-dihydroxycholecalciferol is consistent with a role for the former but not the latter enzyme in the vitamin D-dependent absorption of calcium.     

Early rise in cyclic GMP after 1,25-dihydroxycholecalciferol administration in the chick intestinal mucosa

cGMP and cAMP levels were measured in the duodenal mucosa of 12-day-old chicks that had been raised from hatching in vitamin D-depleting conditions and at the time of use were moderately hypocalcemic. After administration of a dose (250 ng) of 1,25-dihydroxycholecalciferol, the cGMP levels increased about twofold in 2–3 hr and returned to control levels between 4 and 6 hr. Our data suggest that 1,25-dihydroxycholecalciferol behaves like other steroid hormones which induce an early rise in cGMP in their respective target tissues.     

Vitamin D-dependent calcium-binding protein. Changes during gestation, prenatal and postnatal development in rats

During the perinatal period, calcium metabolism is stressed. As intestinal Ca-binding protein is considered as a molecular expression of the hormonal effect of 1,25-dihydroxycholecalciferol (1,25(OH)2D3), Ca-binding protin measurements may document the vitamin D roles during this period. We describe the variations of Ca-binding protein concentrations in the rat during the last 5 days of gestation, in the maternal duodenum, placentas, fetal membranes and fetal intestines. We also report intestinal Ca-binding protein changes from birth until weaning. The evolution of the maternal intestinal Ca-binding protein, which increases on day 19.5 of gestation, is consistent with that of calcium intestinal absorption and may be explained by increased 1,25(OH)2D3 production. Placental Ca-binding protein rises from day 17.5 until the end of gestation, and may be related to the profile of calcium transfer from mother to fetuses. It is noteworthy that the placental Ca-binding protein is predominantly found in the fetal part of the organ where materno-fetal exchanges occur. The yolk sac synthesizes substantial amounts of Ca-binding protein. In the fetal membranes, Ca-binding protein plateaus from day 17.5 until day 20.5 and decreases on day 21.5. The Ca-binding protein presence in the fetal placenta and in the yolk sac may suggest that these tissues are also targets for vitamin D. In the fetus the intestinal Ca-binding protein s is detected as early as day 17.5 of gestation and increases markedly during the last day of gestation. From birth and during the first 3 weeks of postnatal life, the intestinal Ca-binding protein concentration does not change. It undergoes a sharp rise just at the time of weaning. We have also shown that the specific distribution of Ca-binding protein along the intestine is acquired during intrauterine life and does not change with sucking or weaning. The two main changes of intestinal Ca-binding protein, observed just before birth and at weaning, may reflect the intestinal maturation and/or variations in vitamin D metabolism.     

Studies on calciferol metabolism. VII. The effects of actinomycin D and cycloheximide on the metabolism, tissue and subcellular localization, and action of vitamin D3

It was originally postulated, primarily on the basis of experiments employing actinomycin D, that calciferol (vitamin D) mediated its characteristic physiological responses in the intestine via the activation of information stored in the intestinal genome. A more recent alternative hypothesis suggested that actinomycin D blocked the biological response to calciferol by inhibiting the mandatory metabolism of cholecalciferol to 1,25-dihydroxycholecalciferol. Presented in this paper are the results of recent experiments studying the effects of both actinomycin D and cycloheximide on the metabolism, subcellular localization, and action of cholecalciferol or its metabolites, 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol. Actinomycin D was found to inhibit calcium transport stimulated by cholecalciferol or its metabolites without inhibiting their metabolism or localization in the target tissue, the intestinal mucosa. However, actinomycin D had to be administered in four doses at 2-hr intervals to block the stimulation of calcium transport by 1,25-dihydroxycholecalciferol. Actinomycin D was also found not to lower the renal levels of 25-hydroxycholecalciferol-1-hydroxylase, which were measured in vitro. In contrast, cycloheximide was found to inhibit the localization of the sterols in the intestine. Also cycloheximide lowered the renal enzyme levels which were measured in vitro following administration of the antibiotic in vivo. From these data it can be calculated that the 25-hydroxycholecalciferol-1-hydroxylase appears to have a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of approximately 3 hr. Thus, the inhibition of intestinal calcium transport by these two antibiotics may in fact occur at two different target organs; cycloheximide by a lowering of the kidney levels of 25-hydroxycholecalciferol-1-hydroxylase and actinomycin D by blocking the action of 1,25-dihydroxycholecalciferol in the intestine.     

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.     

Vitamin D: its role in cancer prevention and treatment

Vitamin D, the sunshine vitamin, has been recognized for almost 100 years as being essential for bone health. Vitamin D provides an adequate amount of calcium and phosphorus for the normal development and mineralization of a healthy skeleton. Vitamin D made in the skin or ingested in the diet, however, is biologically inactive and requires obligate hydroxylations first in the liver to 25-hydroxyvitamin D, and then in the kidney to 1,25-dihydroxyvitamin D. 25-Hydroxyvitamin D is the major circulating form of vitamin D that is the best indicator of vitamin D status. 1,25-dihydroxyvitamin D is the biologically active form of vitamin D. This lipid-soluble hormone interacts with its specific nuclear receptor in the intestine and bone to regulate calcium metabolism. It is now recognized that the vitamin D receptor is also present in most tissues and cells in the body. 1,25-dihydroxyvitamin D, by interacting with its receptor in non-calcemic tissues, is able to elicit a wide variety of biologic responses. 1,25-dihydroxyvitamin D regulates cellular growth and influences the modulation of the immune system. There is compelling epidemiologic observations that suggest that living at higher latitudes is associated with increased risk of many common deadly cancers. Both prospective and retrospective studies help support the concept that it is vitamin D deficiency that is the driving force for increased risk of common cancers in people living at higher latitudes. Most tissues and cells not only have a vitamin D receptor, but also have the ability to make 1,25-dihydroxyvitamin D. It has been suggested that increasing vitamin D intake or sun exposure increases circulating concentrations of 25-hydroxyvitamin D, which in turn, is metabolized to 1,25-dihydroxyvitamin D(3) in prostate, colon, breast, etc. The local cellular production of 1,25-dihydroxyvitamin D acts in an autocrine fashion to regulate cell growth and decrease the risk of the cells becoming malignant. Therefore, measurement of 25-hydroxyvitamin D is important not only to monitor vitamin D status for bone health, but also for cancer prevention.     

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.     

Regulation of Metabolism of 25-Hydroxycholecalciferol by Kidney Tissue <Emphasis Type="Italic">in vitro</Emphasis> by Dietary Calcium

IT is now recognized that hydroxylated metabolites of vitamin D (that is, cholecalciferol) function as effectors of the physiological actions originally attributed to the unaltered vitamin¹. The activation of vitamin D by specific hydroxylation reactions and sequestration of the resultant metabolites by target tissues represents a hormonal control loop which is feed-back sensitive. 25-Hydroxycholecalciferol (25-HCC) and 1,25-dihydroxycholecalciferol (1,25-DHCC) have been shown to be participants in the control loop, vitamin D being first metabolized in the liver to 25-HCC² which in turn is hydroxylated in the C-1 position to 1,25-DHCC in the kidney^3,4. The metabolically active form in the intestine appears to be 1,25-DHCC^5,6.     

Calcitonin secretion during postnatal development in the rat: beta-adrenergic agents and vitamin D3 metabolites

The possible contribution of catecholamines and vitamin D3 metabolites to the high plasma calcitonin (CT) levels in suckling baby rats is unknown. So, in vivo and in vitro (using a perifusion system) effects of beta-adrenergic agents and vitamin D3 metabolites on CT release were studied in the rat during the postnatal development. In 13-day-old rats, the increase in plasma CT levels induced by isoproterenol injection (0.1 micrograms/kg b.w.) was inhibited by a previous administration of propranolol. A significant decrease in plasma CT levels was observed after propranolol injection in baby rats (0.68 +/- 0.05 ng/ml vs. 0.93 +/- 0.01 ng/ml). A daily injection of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3; 25 pmoles/rat/day during 4 days) induced a marked rise in plasma calcium (16.1 +/- 0.2 mg/dl), and a great decrease in thyroidal CT contents (approximately 70% of control values) in 13-day-old rats while no change was noted with 24,25-dihydroxycholecalciferol (24,25-(OH)2D3). A negative correlation between plasma calcium and thyroidal CT stores was found in suckling and in weaning rats treated with different doses of 1,25-(OH)2D3, suggesting an indirect effect of 1,25-(OH)2D3 on CT secretion. The mobilization of the thyroidal CT content was greater in weaning than in suckling rats in response to a given hypercalcemia. In vitro, 5 X 10(-5) M isoproterenol induced a rapid increase in CT secretion rate while 1,25-(OH)2D3 inhibited the rise in CT release induced by 3.0 mM calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of pituitary hormones in regulating renal vitamin D metabolism in man.

Studies in animals and tissue culture have shown the importance of prolactin and growth hormone in regulating renal 1 alpha-hydroxylase activity and plasma concentrations of 1,25-dihydroxycholecalciferol (1,25(OH)2D3). Evidence for a similar role for these hormones in man was sought by using a radioreceptor assay to measure plasma 1,25(OH)2D3 concentrations in 20 normal subjects, 12 patients receiving dialysis, 11 patients with primary hyperparathyroidism, 10 pregnant women, seven women with prolactinoma, and 14 patients with acromegaly. Circulating 1,25(OH)2D3 concentrations were appreciably raised in the patients with primary hyperparathyroidism and the pregnant women (P less than 0.001), slightly but significantly increased in the patients with prolactinoma (P less than 0.05), and greatly raised in those with acromegaly (P less than 0.001). These results suggest that prolactin and growth hormone are important regulators of renal vitamin D metabolism in the physiological conditions of pregnancy, lactation, and growth in man.     

1,25-Dihydroxyvitamin D3 has opposite effects on the expression of parathyroid secretory protein and parathyroid hormone genes

Previous studies have shown that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] decreases levels of mRNA for prepro-PTH as well as PTH secretion after chronic exposure (24-48 h) of parathyroid cells in tissue culture. We have now extended these studies to determine the effects of the vitamin D3 metabolite on parathyroid secretory protein (PSP) gene expression. Primary cultures of bovine parathyroid cells were incubated with 10(-8) M 1,25-(OH)2D3 for periods of time ranging from 24-72 h. As observed in earlier experiments, prepro-PTH mRNA decreased to less than 50% of the control value after 72 h. In marked contrast, PSP mRNA showed a 2.5-fold increase by 24 h and greater than 7-fold stimulation by 72 h. In the same studies, PTH secretion was suppressed (to 60% of control), while PSP secretion was increased by 40% over control values. Exposure to high (2.5 mM) or low (0.5 mM) calcium had no effect on PSP mRNA, even though low calcium stimulated the secretion of PSP while high calcium suppressed secretion. These studies showed that 1,25-(OH)2D3 has opposite effects on the gene expression of PSP and PTH in bovine parathyroid cells in tissue culture.