Effect of 1,25-dihydroxyvitamin D3 on intestinal calcium absorption in strontium-fed rats.

These studies investigated the initial stimulation of intestinal calcium absorption in the rat by 1,25-dihydroxyvitamin D₃. To produce a functional vitamin D₃-deficiency, rats were fed a diet containing 2.4% strontium. After 10 days on the diet, intestinal calcium uptake, as measured by everted gut sacs, was significantly depressed. Strontium-fed rats were dosed orally with 1,25-dihydroxyvitamin D₃, and changes in intestinal calcium uptake, intestinal alkaline phosphatase activity, and intestinal calcium-binding protein were measured as a function of time after dose. Calcium uptake was significantly increased in the proximal 2.5 cm of the duodenum at 4 h and along the whole duodenum by 7 h. Intestinal alkaline phosphatase activity, measured in a Triton extract of the mucosal homogenate and in isolated brush border complexes, was also increased by 7 h. Using both gel electrophoresis and immunodiffusion against a specific antiserum, an increase in intestinal calcium-binding protein was detected in intestinal supernate at 4 h after dosing. Almost no calcium-binding protein was detectable in strontium-fed rats dosed with propylene glycol only. These time studies are consistent with a role for both alkaline phosphatase and calcium-binding protein in the 1,25-dihydroxyvitamin D₃-stimulated uptake of calcium by the intestine. In addition, the usefulness of strontium feeding for producing a functional vitamin D₃ deficiency in rats is demonstrated.     

Evidence for calcium mediated conformational changes in calbindin-D28K (the vitamin D-induced calcium binding protein) interactions with chick intestinal brush border membrane alkaline phosphatase as studied via photoaffinity labeling techniques

The role of the vitamin D-induced calcium binding protein termed calbindin-D (CaBP) in the biological response to 1,25-dihydroxyvitamin D₃ was assessed by photoaffinity labeling techniques. The heterobifunctional cross-linking reagent methyl-4-azidobenzoimidate was employed for studies with the 28 KD chick intestinal calbindin-D_28K. Calcium-dependent interactions were evident with purified chick intestinal CaBP-immunoglobulins and bovine intestinal alkaline phosphatase; in the absence of Ca²⁺ there was a greatly diminished crosslinking process. There were also at least two membrane components of chick intestinal brush border membranes, with M_R = 60,000 and 130,000, which were photoaffinity cross-linked with CaBP in a calcium-dependent manner. Similar interactions were demonstrated following incubations of CaBP with phosphatidylinositol-specific phospholipase C (PI-PLC)-treated supernatant fractions from chick intestinal brush borders. PI-PLC was shown to release 14% of the alkaline phosphatase from chick intestinal brush borders compared to greater than 80% for rabbit and chick kidney BBM preparations. Specific interactions between CaBP and brush border membrane proteins could also be demonstrated in the absence of photoaffinity labeling by Sephadex G-150 chromatography of Triton X-100 solubilized incubations between calbindin-D_28K and chick intestinal BBMS, with 17% of the radiolabelled CaBP comigrating with alkaline phosphatase activity. These studies collectively demonstrate that calbindin-D_28K undergoes calcium-dependent conformational changes which alter its subsequent interactions with cellular proteins in a way consistent with other calcium-binding proteins such as calmodulin or troponin C.     

Effect of alcohol on renal vitamin D metabolism in chickens.

Intraperitoneal administration of ethanol to young chickens (both vitamin D-replete and vitamin D-deficient) produced a significant impairment of renal 25 hydroxyvitamin D₃ 1α-hydroxylase (EC 1.14.13.13) activity with no significant change in serum calcium or phosphorus. In ethanol treated D-replete chicks the renal 25 hydroxyvitamin D₃ 24-hydroxylase activity was enhanced, and serum 25 hydroxyvitamin D₃ was significantly increased. The alkaline phosphatase levels in the D-deficient ethanol treated chicks were significantly less than the controls. Our data suggest that the impairment of the metabolic effects of vitamin D due to ethanol occurs chiefly via a renal, rather than a hepatic mechanism. Furthermore, 1α -hydroxylated metabolites of vitamin D would appear to be the logical treatment of choice for the bone disease of alcoholism.     

Purification and characterization of chick intestine brush border membrane Effects of 1α(OH) vitamin D3 treatment

A new technique has been developed for the isolation of membrane vesicles from the vitamin D-deficient and vitamin D-treated chick intestinal brush border membrane. The technique involves removal of nuclei from a low speed pellet by discontinuous sucrose gradient centrifugation. The resulting intact brush borders are then homogenized in 0.5 M Tris and the membrane fragments purified on a glycerol gradient. This preparation represents a 20-fold purification of the brush border marker sucrase. After 1α-hydroxyvitamin D₃ treatment there is a significant increase in membrane phospholipid phosphorous, an alteration in the fatty acid composition of the phosphatidylcholine fraction of membrane phospholipid, and a decrease in sucrase specific activity.     

Intestinal calcium transport: evidence for two distinct mechanisms of action of 1,25-dihydroxyvitamin D3

The response of the small intestine in the vitamin D-deficient rat to a single intrajugular injection of 1,25-dihydroxyvitamin D₃ has been studied. The time course of 1,25-dihydroxyvitamin D₃-induced transport suggests that two separate responses occur. The first or initial response reaches a maximum at 6 h after 1,25-dihydroxy vitamin D₃ administration, decays, and is effectively gone by 12 h postinjection. This response does not appear to be associated with alkaline phosphatase activity. The second or late response first appears roughly 12 h after dosing, reaches a maximum at 24 h, and remains elevated for up to 72 h. This response is accompanied by an elevation of alkaline phosphatase activity and appears to be mediated through the action of 1,25-dihydroxyvitamin D₃ on the absorptive cell during its normal differentiation and migration up the villus.     

Ion microscopic imaging of calcium during 1,25-dihydroxyvitamin D-mediated intestinal absorption

A combination of ion microscopic and conventional radionuclide techniques was employed to investigate the temporal-spatial dynamics of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]-stimulated intestinal calcium (Ca) absorption. At varying times following the administration of a single intravenous dose of 1,25(OH)₂D₃, to vitamin D-deficient chicks, transepithelial transport and tissue retention of Ca were quantitated in vivo, using the ligated duodenal loop technique and⁴⁷Ca as the tracer. The localization of Ca in the intestinal tissue during absorption was monitored by ion microscopy, using the stable Ca isotope,⁴⁴Ca, as the absorbed species. There was little transepithelial absorption of Ca in the vitamin D-deficient animals despite a substantial tissue accumulation of luminally derived Ca, the latter localizing predominantly in the brush border region of the enterocyte, as shown by the⁴⁴Ca-ion microscopic images. The early (30 min-1 h) response to 1,25(OH)₂D₃ was an increased tissue uptake of luminal⁴⁷Ca, which also primarily associated with the brush border region, again as shown by ion microscopy. At 2–4 h after the 1,25(OH)₂)D₃ dose, there was a progressive redistribution of Ca from the brush border region throughout the cytoplasm and into the lamina propria. At 8–16 h,⁴⁷Ca absorption was maximal and⁴⁴Ca was sparsely distributed in the intestinal tissue.⁴⁷Ca absorption gradually declined and reached pre-dose levels by 72 h. At this time, tissue⁴⁴Ca was again largely limited to the brush border region. These results provide support for the multiple actions of 1,25(OH)₂D₃ on the intestinal Ca absorption     

Binding Proteins from Animals with Possible Transport Function

Several proteins from various animal tissues with possible transport function have been briefly described, with emphasis given to a vitamin D-induced calcium-binding protein (CaBP) implicated in calcium translocation across epithelial membranes. The latter protein was shown to be present in the small intestine, colon, kidney, and the uterus (shell gland) of the chicken. CaBP was also found in the small intestine of the rat, dog, bovine, and monkey. This protein has been isolated in high purity from chick intestinal mucosa and some of its properties determined. Its molecular weight is about 28,000, its formation constant, about 2.6 x 10⁵ M^-1, and its binding capacity, 1 calcium atom per protein molecule. Correlative studies have shown that CaBP concentration in intestinal mucosa varies with the calcium absorptive capacity of the gut, thereby suggesting that CaBP is intimately involved in the process of calcium absorption. CaBP has been localized in the brush border region of the intestinal absorptive cell and within goblet cells. Among other proteins mentioned were the intrinsic factor required for vitamin B₁₂ absorption and the protein(s) associated with iron translocation.     

Intestinal brush border hydrolase topography. Effects of vitamin D-3 and filipin

Intestinal brush borders were isolated from vitamin D-3-treated and vitamin D-deficient chicks, and protein topography in the paired preparations assessed by the enzymatic release of four marker hydrolases. Exposure of the brush borders to the protease bromelain resulted in soluble levels of alkaline phosphatase, leucine aminopeptidase, maltase, and sucrase activities from preparations of vitamin D-3-treated birds that were 42%, 75%, 64%, and 56%, respectively, of corresponding activities released in preparations from rachitic chicks. Analyses for recovery of enzyme activity revealed that bromelain treatment selectively inactivated 43% of the alkaline phosphatase activity of brush borders obtained from vitamin D-3-replete birds, and preferentially diminished recovered sucrase activity in preparations from vitamin D-deficient chicks. In additional experiments, brush borders isolated from rachitic birds were treated in vitro with the polyene antibiotic filipin or an equivalent volume of vehicle. Subsequent exposure of such preparations to bromelain resulted in little or no differences in levels of marker hydrolase specific activities released from filipin- or vehicle-treated brush borders. However, analyses of membrane-bound specific activities after treatment of brush border preparations with a range of filipin concentrations, revealed a biphasic inhibition of approx. 30% for both maltase and sucrase, relative to vehicle controls, and a smaller effect on alkaline phosphatase and leucine aminopeptidase.     

Metabolism of vitamin D3 in the chick embryo

In agreement with previous reports, chick intestinal calcium-binding protein does not appear in the chick embryo until 1 day after hatching while intestinal alkaline phosphatase begins to appear at 19–20 days of embryonic life. The ability of chick embryo to metabolize vitamin D₃ to 25-hydroxyvitamin D₃, 1,25-dihydroxyvitamin D₃, and 24,25-dihydroxyvitamin D₃ is present at least by day 18 of embryonic life as demonstrated by in vivo and in vitro techniques. It also illustrates that metabolism of vitamin D₃ was not the limiting factor in the appearance of calcium-binding protein and alkaline phosphatase in intestine. Instead, the uptake of 1,25-dihydroxyvitamin D₃ by the duodenum was very low prior to hatching, even though significant amounts were present in the yolk sac. Injection of a physiological dose of 1,25-dihydroxyvitamin D₃ to chick embryo at 9 days failed to stimulate appearance of calcium binding protein by 18 days of embryonic life. Thus, it appears that either the normal mechanism for transport of 1,25-dihydroxyvitamin D₃ to intestine or its receptors in intestine may not be present prior to day 18–19.A large fraction of radioactive vitamin D₃ injected into the yolk sac was found esterified especially in the embryonic liver. The significance of this is not yet understood.Injection of 1,25-dihydroxyvitamin D₃ at 325 pmoles/per egg at 9 days resulted in 70% mortality of embryos while a 32-pmole dose resulted in no significant increase in mortality. The basis for this toxicity is not yet understood.     

Subcellular localization of enterokinase (Enteropeptidase EC 3.4.21.9) in rat small intestine

The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3 in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (M_r, 2·10^?6) and two larger peaks of free enzyme (M_r, 3·10⁵ and 9·10⁵). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.     

Biological activity of 1 alpha-hydroxyvitamin D2 in the rat.

The biological activity of 1α-hydroxyvitamin D₂ has been determined in vitamin D-deficient rats. In the calcification of the rachitic epiphyseal plate, 1α-hydroxyvitamin D₂ is more active than 25-hydroxyvitamin D₃, while it is equally active in stimulating intestinal calcium absorption. On the other hand, it is much less active (one-third to one-fifth) than 25-hydroxyvitamin D₃ in the mobilization of calcium from bone. In both the intestinal and bone responses, 1α-hydroxyvitamin D₂ (312 pmol) is active in nephrectomized rats while 25-hydroxyvitamin D₃ is not.     

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.     

Embryonic chick intestine in organ culture. A unique system for the study of the intestinal calcium absorptive mechanism

Duodena from 20-day-old chick embryos can be maintained in large scale organ culture on specially designed stainless-steel grids in contact with serum-free medium for 48 h with excellent preservation of mucosal structure at both the light and electron microscope levels. Although mitotic rate was subnormal, several other factors attest to the essential viability of the cultured intestine: L-leucine incorporation into protein, as well as the synthesis of a specific vitamin D₃-induced calcium-binding protein (CaBP), increased over a 48-h culture period, and the electropotential gradient across the intestine was maintained throughout the culture period as was a concentration gradient for calcium. The tissue responded to vitamin D₃ in the medium by synthesizing the calcium-binding protein within 6 h and by exhibiting enhanced ⁴⁵Ca uptake within 12–24 h. Concentrations of vitamin D₃, or its 25-hydroxylated derivative, higher than necessary for CaBP induction, also increased the activity of alkaline phosphatase. The 1,25-dihydroxylated derivative of vitamin D₃, at a level extremely potent in CaBP induction, did not stimulate alkaline phosphatase. Mucosal to serosal transport of ⁴⁵Ca could also be measured in everted duodenal sacs, subsequent to culture under similar conditions, and was also increased by vitamin D₃ in the medium. Other embryonic organs, esophagus, stomach, liver, pancreas, lung, skin, and muscle, did not produce CaBP in response to vitamin D₃ in the culture medium. However, CaBP-synthesizing capacity was present in the entire intestinal tract, exclusive of the rectum. ⁵⁹Fe and ³²P uptake by cultured duodenum were also stimulated by vitamin D₃. The system has proven quite useful in the study of the vitamin D-mediated calcium absorptive mechanism but should be applicable to the study of the absorption of other nutrients, drugs, hormones, etc., as well as other studies of intestinal function.     

Solubilization of brush borders of hamster small intestine and fractionation of some of the components

About 90% of the protein of hamster intestinal brush borders was solubilised in 0.25% (w/v) sodium dodecyl sulphate without total loss of biological activity. Detergent-polyacrylamide gel electrophoresis of the solubilised protein separated 10–15 bands and partially resolved maltase, lactase, sucrase-maltase, trehalase and alkaline phosphatase activities. The disaccharidases, which were associated with the higher molecular weight proteins, were preferentially solubilised with 0.1%. (w/v) Triton X-100, butanol or papain, whereas Tris and NaI extracted only the lower molecular weight proteins, possible derived from the core filaments.Electrophoresis of brush border proteins metabolically labelled with [¹⁴C] glucosamine suggested that many of the membrane-bound enzymes are glycoproteins. However, chromatography of a papain digest on Sephadex G-200 showed that the sucrase-maltase complex can be separated nearly free of carbohydrate without total loss of activity.The importance of characterizing membrane proteins solubilised by a number of techniques is discussed.     

24,24-Difluoro-1,25-dihydroxyvitamin D3: In vitro production,isolation, and biological activity

24,24-Difluoro-1,25-dihydroxyvitamin D₃ has been synthesized by in vitro incubation of vitamin D-deficient chick kidney homogenates with 24,24-difluoro-25-dihydroxyvitamin D₃. The compound produced was isolated and purified by successive high-performance liquid chromatographic steps and then identified by means of ultraviolet absorption spectrophotometry and mass spectrometry. The difluoro analog of 1,25-dihydroxyvitamin D₃ is found to be highly active in stimulating intestinal calcium transport and bone calcium mobilization in vitamin D₃-deficient rats.     

Biological activity of 1alpha-hydroxyvitamin D3 in the rat.

The biological activity of 1α-hydroxyvitamin D₃ has been determined in vitamin D-deficient rats. In the accumulation of mineral in bone and cartilage, maintenance of serum calcium, and in efficiency of calcium absorption the 1α-hydroxyvitamin D₃ was approximately two to five times more active than vitamin D₃ or 80–200 units of activity per microgram.     

Effect of dietary calcium and phosphorus on intestinal calcium absorption and vitamin D metabolism.

To understand better dietary regulation of intestinal calcium absorption, a quantitative assessment of the metabolites in plasma and duodenum of rats given daily doses of radioactive vitamin D₃ and diets differing in calcium and phosphorus content was made. All known vitamin D metabolites were ultimately identified by high-pressure liquid chromatography. In addition to the known metabolites (25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, 1,25-dihydroxyvitamin D₃, 25,26-dihydroxyvitamin D₃, and 1,24,25-trihydroxyvitamin D₃), several new and unidentified metabolites were found. In addition to 1,25-dihydroxyvitamin D₃ and 1,24,25-trihydroxyvitamin D₃, the levels of some of the unknown metabolites could be correlated with intestinal calcium transport. However, whether or not any of these metabolites plays a role in the stimulation of intestinal calcium absorption by low dietary calcium or low dietary phosphorus remains unknown.     

Anti-clotting activity of endothelial cell cultures and heparan sulfate proteoglycans

A photoaffinity probe for the vitamin D-dependent chick intestinal calcium binding protein (CaBP) has been prepared by conjugation of methyl-4-azidobenzoimidate (MABI) to lactoperoxidase-¹²⁵I-iodinated CaBP to yield ¹²⁵I-CaBP-MABI: [3 moles MABI per mole CaBP]. After incubation $\text{in}\text{vitro}$ of ¹²⁵I-CaBP-MABI (28,000 daltons) in model systems with bovine intestinal alkaline phosphatase (AP) (67,000 daltons), a UV light-dependent crosslinking occurred to yield a conjugate with a molecular weight of 95,000 (by SDS-gel electrophoresis); no crosslinking occurred with $\text{E.}\text{coli}$ alkaline phosphatase. The formation of the ¹²⁵I-CaBP-MABI-AP was found to occur only in the presence of calcium.     

Characterization of dietary phosphorus-dependent duodenal calcium uptake in vitamin D-deficient chicks

The effect of dietary phosphorus on intestinal calcium uptake was examined in duodenal cells isolated from vitamin D-deficient chicks. Cells from chicks on a high phosphorus diet accumulated calcium at a rate 38% higher than cells from animals on a normal phosphorus diet. Diet high in calcium did not affect calcium absorption in duodenal cells. The dietary phosphorus effect on calcium absorption was specific. Uptake of -methyl glucoside was not altered. Increase in calcium absorption by a high phosphorus diet was not due to a change in cellular energy metabolism nor to the content of phosphorus in cells. Kinetically, a high phosphorus diet increased the V _max of calcium uptake; the affinity for calcium was unaffected. The effectiveness of dietary phosphorus to enhance the intestinal calcium uptake could also be demonstrated in brush border membrane vesicles. The increase in calcium uptake was not due to an alteration in membrane binding capacity nor to calcium efflux from vesicles. To test the hypothesis that a high phosphorus diet may affect membrane transport by altering phospholipid metabolism in duodenal cells, we examined the phospholipid content in isolated brush border membranes. The content of phosphatidylcholine, phosphatidylserine, phosphatidyinositol and phosphatidylethanolamine was not altered by the high phosphorus diet. These findings suggest that the vitamin D-independent and dietary phosphorus-dependent effect on intestinal calcium absorption was primarily due to a change in the calcium flux at the luminal side of the cells. However, the precise mechanism is still not clear.