The effect of age and 1,25-dihydroxyvitamin D3 treatment on the intestinal calcium-binding protein of suckling rats.

The intestinal level of the vitamin D-dependent duodenal calcium-binding protein was assayed by an equilibrated column technique in rat embryos, neonates, and pups. Calcium-binding protein was undetectable in unborn, newborn, and 1- to 2-day-old rats i.e., the level was lower than in severely vitamin D-deficient animals. Calcium-binding protein was detected after the animals were 5-days old and thereafter rose monotonically as a function of body weight. Treatment with 1,25-dihydroxyvitamin D₃ failed to raise the calcium-binding protein levels of newborn or 1-day-old rats, but doubled the level in 11- or 12-day-old pups. Plasma calcium was raised in all treated animals. The failure to detect calcium-binding protein in vitamin D-replete suckling animals provides evidence for a dissociation between calcium absorption and calcium binding protein.     

Calcium uptake by isolated rat intestinal cells

Intestinal cells were isolated by a combination of mechanical and enzymatic means, and their calcium uptake was assayed by a rapid filtration procedure. Calcium uptake was a time- and concentration-dependent process that was markedly elevated at 25 and 37°C, as compared to 0°C. Cells isolated from rat duodenum exhibited higher uptakes than cells from jejunum, which in turn took up more calcium than cells from the ileurn. Duodenal cells from vitamin D-deficient animals took up less calcium than cells from vitamin D-replete cells. In vivo vitamin D repletion with 1,25-dihydroxyvitamin D₃ raised calcium uptake by duodenal cells from treated animals toward that of cells from replete rats. Furthermore, calcium uptake by duodenal cells from vitamin D-deficient animals approximated that of ileal cells from replete rats. These findings with isolated cells parallel prior findings of tissue calcium transport and suggest that cellular calcium uptake may be related to the saturable component of intestinal calcium absorption. Isolated intestinal cells may therefore constitute one experimental model for the study of transcellular calcium transport.     

Characterization of 1,25-dihydroxyvitamin D3-dependent calcium uptake in isolated chick duodenal cells

Summary Thein vivo andin vitro effects of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) on calcium uptake by isolated chick duodenal cells were studied.In vivo, 1,25-(OH)₂D₃ given orally to vitamin D-deficient chicks increased the initial rate of calcium uptake by cells prepared 1 hr after administration of the hormone. The rate was stimulated approximately 100%, 17 to 24 hr after repletion.In vitro, pre-incubation of 1,25-(OH)₂D₃ with cells from D-deficient chicks increased the cellular rate of calcium uptake in a concentration-dependent relationship. Enhancement was found with 10^–15 m, was maximal at 10^–13 m, and was diminished at higher (10^–11 m) concentrations. Stimulation was observed after a pre-incubation period as brief as 1 hr. The potency order for vitamin D₃ analogs was 1,25-(OH)₂D₃=1-(OH)D₃>25-(OH)D₃>1,24,25-(OH)₃D₃>24,25-(OH)₂D₃>D₃. The maximal enhancement in calcium uptake induced by the analogs was the same, only the concentration at which the cell responded was different. The effectiveness of 1,25-(OH)₂D₃ was five orders of magnitude greater than D₃. Kinetically, 1,25-(OH)₂D₃ increased theV _max of calcium uptake; the affinity for calcium (K _m=0.54mm) was unchanged. The enhanced uptake found after the cells were pre-incubated for 2 hr with the hormone was completely blocked by inhibitors of protein synthesis. 1,25-(OH)₂D₃,in vitro, also increased calcium uptake in cells isolated from D-replete chicks. The maximal rates of uptake were the same in cells from D-deficient and D-replete animals. The hormone had no effect of calcium efflux from cells. Calcium uptake in microvillar brush-border membrane vesicles was increased by 1,25-(OH)₂D₃. These findings suggest that thein vitro cell system described in this paper represents an appropriate model to examine the temporal relationships between 1,25-(OH)₂D₃ induction of calcium transport and specific biochemical correlates.     

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.     

Modulation of rat calbindin-D28 gene expression by 1,25-dihydroxyvitamin D3 and dietary alteration

We have used a specific cDNA to the mammalian 28,000 Mr vitamin D-dependent calcium binding protein (calbindin-D28k) to study the regulation of the expression of this mRNA in rat kidney and brain. The effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and dietary alteration on genomic expression were characterized by both Northern and slot blot analysis. Administration of 1,25-(OH)2D3 for 7 days (25 ng/day) to vitamin D-deficient rats resulted in a marked increase in renal calbindin-DmRNA, renal calbindin, and serum calcium. When vitamin D-deficient rats were supplemented for 10 days with calcium (3% calcium gluconate in the water, 2% calcium in the diet) serum calcium levels were similar to the levels observed in the 1,25-(OH)2D3-treated rats. However, in the calcium-supplemented rats the levels of renal calbindin and renal calbindin mRNA were similar to the levels observed in the vitamin D-deficient rats, suggesting that calcium alone without vitamin D does not regulate renal calbindin gene expression in vivo. In dietary alteration studies in vitamin D-replete rats, renal calbindin protein and mRNA increased 2.5-fold in rats fed diets low in phosphate providing evidence that in the rat the nutritional induction of calbindin is accompanied by a corresponding alteration in the concentration of its specific mRNA. Under low dietary calcium conditions, the levels of renal calbindin protein and mRNA were similar to the levels observed in control rats, although 1,25-(OH)2D3 serum levels were markedly elevated, suggesting that factors in addition to 1,25-(OH)2D3 can modulate renal calbindin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Skin calcium-binding protein: effect of vitamin D deficiency and vitamin D treatment

The amount of skin calcium-binding protein, evaluated using a sensitive radioimmunoassay and indirect immunofluorescence, was decreased in vitamin-D deficient rats and increased after one week vitamin D3 or 1 alpha-hydroxyvitamin D3 treatment. In vitamin D replete and in vitamin D-deficient animals, skin calcium-binding protein was not sensitive to changes in dietary and/or serum calcium concentrations. These results indicate that this protein is different from other calcium-binding proteins such as parvalbumin and calmodulin which are not vitamin D-dependent, and also different from intestinal calcium-binding protein which, in D replete animals, is sensitive to changes in dietary and serum calcium concentrations. Skin calcium-binding protein may, therefore, represent a new class of vitamin D-dependent protein.     

24R,25-dihydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 are both indispensable for calcium and phosphorus homeostasis

The essential role of vitamin D throughout the life of most mammals and birds as a mediator of calcium homeostasis is well established. In view of the complex endocrine system existent for the regulated metabolism of vitamin D₃ to both 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 24R,25-dihydroxyvitamin D₃ [24R,25-(OH)₂D₃] (both produced by the kidney), an intriguing problem is to elucidate whether only one or both of these dihydroxyvitamin D₃ metabolites is required for the generation of all the biological responses mediated by the parent vitamin D₃. In contrast to the accumulated knowledge concerning the short term actions of 1,25(OH)₂-D₃ on stimulating intestinal calcium absorption and bone calcium reabsorption, relatively little is known of the biological function of 24,25(OH)₂D₃. We report now the results of a nine month study in which chicks were raised on a vitamin D-deficient diet from hatching to sexual maturity and received as their sole source of “vitamin D” either 24,25(OH)₂D₃ or 1,25(OH)₂D₃ singly or in combination. Specifically we are describing the integrated operation of the vitamin D endocrine system as quantitated by the individual measurement in all birds of 22 variables related to “vitamin D status” and as evaluated by the statistical procedure of multivariate discriminant analysis. Twelve of these variables involved detailed analysis of the bone including quantitative histology and the other 10 variables reflect various manifestations of vitamin D action, e.g. serum Ca²⁺ and Pi levels, vitamin D-dependent calcium binding protein (CaBP) in the intestine and kidney, egg productivity etc. As evaluated by the multivariate analysis, it is clear that 24,25(OH)₂D₃ and 1,25(OH)₂D₃ are simultaneously required for normalization of calcium homeostasis.     

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.     

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.     

Intestinal calcium-binding protein (CaBP) and bone calcium mobilization in response to 25R,26 AND 25S,26-dihydroxycholecalciferol in intact and nephrectomized rats

Since intestinal calcium-binding protein (CaBP) can he regarded as an expression of the hormone-like action of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) on the duodenal enterocyte we have investigated the potential biological activity of 25R and 25S,26-(OH)₂D₃ (two recently synthesized epimers of vitamin D₃ metabolite) to promote intestinal CaBP production as compared to bone calcium mobilization in vitamin D and calcium-deficient rats. In our assay steroids exhibited a 72 hour calcemic response. Our results show a linear relationship between CaBP synthesis and the logarithm of the dose (130–2080 pmol dose range) of either 25R or 25S epimer. The CaBP response was comparable for both epimers. Similarly bone calcium mobilization response was dose related as a linear function of the logarithm of the administered dose. Again, calcemic response was comparable for both epimers. In our model these two epimers were about as active on intestine to increase CaBP amount as on bone to elevate serum calcium level. Bilateral nephrectomy abolished CaBP response to a large dose (1040 pmol) of either 25R or 25S epimer but did not abolish it to a 130 pmol dose of 1α, 25-(OH)₂D₃.     

The relationship between vitamin D-stimulated calcium transport and intestinal calcium-binding protein in the chicken

1. The rapid stimulation of intestinal Ca²⁺ transport observed in vitamin D-deficient chicks after receiving 1,25-dihydroxycholecalciferol has necessitated a re-evaluation of the correlation hitherto observed between this stimulation and the induction of calcium-binding protein synthesis. By 1h after a dose of 125ng of 1,25-dihydroxycholecalciferol, Ca²⁺ transport is increased. This is at least 2h before calcium-binding protein can be detected immunologically and 1h before synthesis of the protein begins on polyribosomes, and thus the hormone stimulates Ca²⁺ transport before calcium-binding-protein biosynthesis is induced. 2. The maximum increase in Ca²⁺ transport observed after this dose of 1,25-dihydroxycholecalciferol (attained by 8h) is similar to that observed after 1.25–25μg of cholecalciferol, but the stimulation is only short-lived, in contrast with the effect observed after the vitamin. At later times after the hormone, however, when Ca²⁺ transport has declined to its basal rate, the cellular content of calcium-binding protein remains elevated. 3. Calcium-binding protein is synthesized on free rather than membrane-bound polyribosomes, which implies that it is an intracellular protein. 4. Rachitic chicks require the presence of dietary calcium for maximum stimulation of calcium-binding protein production by cholecalciferol. 5. These results suggest that calcium-binding protein is an intracellular protein, and that its synthesis may be a consequence of the raised intracellular calcium content of the intestinal epithelial cells resulting from 1,25-dihydroxycholecalciferol-stimulated Ca²⁺ transport. We propose that calcium-binding-protein synthesis is necessary for maintaining the stimulated rate of Ca²⁺ transport, which is initiated by other factors.     

Homologous up-regulation of the 1,25 (OH)2 vitamin D3 receptor in rats

This study investigates the ability of vitamin D-metabolites to regulate 1,25(OH)2D3 receptors in vivo. Rats made vitamin D-deficient were treated with 1,25(OH)2D3 or vehicle for 1-5 days. In treated animals, receptors for 1,25(OH)2D3 in kidney increased dramatically compared with control levels. An increase in specific binding to 220% of control was seen after 2 doses of hormone, which reached to 336% after 5 days of treatment. Intestinal receptors increased to only 130% of control levels after 5 days of treatment. In vitamin D-replete animals, the difference between control and treated groups was slightly greater when endogenously occupied sites were measured by exchange (TPCK). However, significant changes were observed only after 4 days of hormone treatment. The data indicate that homologous up-regulation of the 1,25(OH)2D3 receptor occurs in vivo. The difference in response in kidney and in intestine suggests differential importance of up-regulation in various organs.     

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.     

Metabolism of 25-hydroxyvitamin D3 in kidney homogenates of chicks supplemented with vitamin D3.

Kidney homogenates from chicks fed a vitamin D-deficient diet for 10 days and supplemented with 6.5 nmol of vitamin D₃ 48 hr prior to sacrifice metabolized $\text{in}\text{vitro}\text{[}{}^3\text{H}\text{]}$-25-hydroxyvitamin D₃ (25-OH-D₃) to 24,25-dihydroxyvitamin D₃ [24,25-(OH)₂-D₃] and 3 other metabolites (peaks A, C and E). When the homogenates were incubated with purified [³H]-24,25-(OH)₂-D₃, 3 similar metabolites (peaks A′, C′ and E′) were produced. On high pressure liquid chromatography, peaks A, C and E migrated to exactly the same respective positions as peaks A′, C′ and E′. Kidney homogenates from D-deficient chicks failed to produce these metabolites from [³H]-25-OH-D₃ or [³H]-24,25-(OH)₂-D₃. These results strongly suggest that the new metabolites reported here are synthesized via 24,25-(OH)₂-D₃ in the kidney of chicks supplemented with vitamin D₃.     

Serum concentrations of 1,25-dihydroxyvitamin D3 in Platyrrhini and Catarrhini: A phylogenetic appraisal

We measured the serum concentration of 25-hydroxyvitamin D₃ (25-OH-D₃) and 1,25-dihydroxyvitamin D₃ (1,25-[OH]₂-D₃) in 23 different Platyrrhines from four different genera and in 21 Catarrhines from six different genera in residence at the Los Angeles Zoo. The mean (±S.E.) serum concentration of 1,25-(OH)₂-D₃ was significantly greater in Platyrrhines (810 ± 119 pg/ml) than in Catarrhines (61 ± 5 pg/ml), suggesting that high circulating concentrations of the active vitamin D hormone were a characteristic of New World primates in both the Cebidae and Callitrichidae family. This increase in the serum concentration of 1,25-(OH)₂-D₃ is probably an adaptational response on the part of Platyrrhini to offset a relative decrease in the concentration of specific receptor for 1,25-(OH)₂-D₃ in target tissues for the hormone.     

Effect of dietary calcium, phosphate and vitamin D deprivation on the pharmacokinetics of 1,25-dihydroxyvitamin D3 in the rat

The in vivo regulation of circulating 1,25(OH)2D3 concentrations by vitamin D status and by dietary calcium and phosphate deficiency was studied. Adult rats were cannulated in the jugular vein and the clearance of physiological doses of 1,25(OH)2D3 monitored. In vitamin D-replete rats we investigated the effects of dietary calcium and phosphate deficiency on the elimination half life of 1,25(OH)2D3 The results showed no effect of dietary phosphate deficiency on the elimination half life of 1,25(OH)2D3. Dietary calcium deficiency resulted in a small increase of the 1,25(OH)2D3 elimination half life (P = 0.04) (normal diet: 16.3 +/- 1.8 hrs, n = 6; -Ca diet: 18.6 +/- 1.1 hrs, n = 5; -P diet: 16.0 +/- 1.4 hrs, n = 6; mean +/- SD). The experiments with the vitamin D deficient rats showed a marked increase in the elimination half life of 1,25(OH)2D3 (36.4 +/- 6.8 hrs, n = 7), when compared to the rats on the normal diet (P = 0.001). From the experiments in the vitamin D replete rats one can infer that regulation of circulating 1,25(OH)2D3 concentrations by dietary calcium or phosphate takes place at the production site and not by changes in elimination rate. However, vitamin D status appears to regulate circulating 1,25(OH)2D3 concentrations also through an effect on the elimination rate.     

Effect of 1,25-dihydroxyvitamin D3 on human keratinocytes grown under different culture conditions

Summary 1,25-Dihydroxyvitamin D₃ (1,25-(OH)₂-D₃) is known to decrease the proliferation and increase the differentiation of different cell types including human keratinocytes. The growth and differentiation of keratinocytes in the presence of 1,25-(OH)₂-D₃ using serum-free media formulations has been described previously. This investigation extends these studies to describe various culture conditions with human foreskin keratinocytes to determine the optimal antiproliferative activity of 1,25-(OH)₂-D₃. Keratinocytes were plated onto tissue culture dishes using one of three basic serum-free media protocols; a) with no feeder layer in keratinocyte growth medium (KGM); b) onto mitomycin C-treated 3T3 mouse embryo fibroblasts; or c) onto mitomycin C-treated dermal human fibroblasts. The last two protocols utilized Dulbecco's modified Eagle's Medium (DMEM) supplemented with growth factors. Keratinocyte cell growth was greatest in the KGM medium. Although the growth of keratinocytes on either feeder layer was similar, there were differences in the ability of the cells to form envelopes in the presence of 1,25-(OH)₂-D₃. The addition of hydrocortisone and cholera toxin to the medium also affected the response of the keratinocytes to 1,25-(OH)₂-D₃. The antiproliferative effect of 1,25-(OH)₂-D₃ was not altered by varying the extracellular calcium levels from 0.25 to 3 mM. The antiproliferative activity of 1,25-(OH)₂-D₃ is attenuated in cells at low density. Our results suggest that an optimal condition to investigate the ability of 1,25-(OH)₂-D₃ to inhibit keratinocyte proliferation is at preconfluent cell density in the presence of KGM supplemented with 1.5 mM calcium without a feeder layer. These conditions are not appropriate for investigating the enhancement of differentiation by 1,25-(OH)₂-D₃, but can be used to assay other agents that modulate keratinocyte proliferation. Portions of this work were presented and abstracted at the April 1988 meeting of the Society of Investigative Dermatology (J. Inv. Derm. 90(4): 586; 1988) and the February 1988 meeting of New York Academy of Sciences (Ann NY Acad. Sci. 548: 341–342; 1988).     

Studies on the mechanism of action of calciferol VII. Localization of 1,25-dihydroxy-vitamin D3 in chick parathyroid glands.

When 1,25(OH)₂-vitamin D₃ was administered to vitamin D-deficient chicks, within two hours the parathyroid glands were observed to accumulate this steroid to a concentration four times that present in the blood and equivalent to levels observed in the target intestine. Similarly, when 25-(OH)-vitamin D₃ was administered, the parathyroid glands had 2.4 times the concentration of the metabolite, 1,25-(OH)₂-vitamin D₃ as that seen in the blood and 60% of that found in the intestine. These results are consistent with the concept that the hormonally active form of vitamin D, 1,25-(OH)₂-vitamin D₃, may interact with the parathyroid glands to effect changes in parathyroid hormone secretion.