Vitamin D-dependent Intestinal Calcium-binding Protein Research Articles

Influence of exogenous porcine growth hormone on vitamin D metabolism and calcium and phosphorus absorption in intact pigs.

The influence of growth hormone (GH) on vitamin D metabolism and calcium and phosphorus absorption in vivo is not clear. We, therefore, measured calcium and phosphorus balance, plasma 1,25-dihydroxyvitamin D (1,25(OH)2D), and intestinal vitamin D-dependent calcium-binding protein (CaBP 9k) in intact growing pigs given exogenous GH. Six 10-week-old pigs were given two daily subcutaneous injections of 50 micrograms porcine GH/kg body weight for 2 months; six control pigs were given vehicle. They were all fed a diet containing 1.1% Ca, 0.6% P, and 1000 IU vitamin D3/kg. Apparent Ca and P absorption and retention were measured in a 10-day balance trial at the end of the 2 months. The plasma levels of Ca, P, 1,25(OH)2D, IGF-I, and GH were determined, and the duodenal and jejunal mucosal CaBP 9k content was measured at slaughter. The plasma Ca and P of GH-treated pigs were unchanged, but all aspects of mineral metabolism, including the plasma 1,25(OH)2D concentration (40%), Ca absorption and retention (70%), P absorption (33%) and retention (45%), and jejunal CaBP 9k (40%), were stimulated, in addition to an increase in the circulating IGF-I concentration.

Developmental changes of Ca2+, PO4, and calcitriol metabolism in spontaneously hypertensive rats

We have measured Ca and P balance, serum calcitriol, and vitamin D-dependent intestinal calcium-binding protein (CaBP9k) in the spontaneously hypertensive rat (SHR) and its normotensive control, the Wistar-Kyoto rat (WKY) at 4-5 and again at 13-14 wk of age. In rats on a 1% Ca diet, P balance was significantly more positive in the 5-wk-old SHR than WKY (P less than 0.01); Ca balance tended to be greater in the 5-wk-old SHR. In contrast, in the 14-wk-old SHR, P and Ca balance were less positive than in the WKY (P was 5.8 +/- 1.3 vs. 13 +/- 1.7 mg/day, P less than 0.01, and Ca was 53 +/- 4.6 vs. 67 +/- 2.7 mg/day, P less than 0.05). On the 1% Ca diet, plasma calcitriol levels of the 5-wk-old SHR were higher than those of the WKY (58 +/- 3.2 vs. 40 +/- 2.1 pg/ml, P less than 0.002), whereas at 12 wk there was no difference. On Ca-deficient (0.1%) diets, plasma calcitriol was increased in 5-wk-old and 12-wk-old SHR and WKY, compared with the 1% Ca diet (P less than 0.001). Calcitriol metabolic clearance rate was the same in the 13-wk-old SHR and WKY on either Ca diet. Intestinal CaBP9k content of the 5-wk-old (38 +/- 2.6 vs. 40 +/- 3.9 micrograms/mg) and the 12-wk-old (11 +/- 1.6 vs. 14 +/- 1.4 micrograms/mg) SHR and WKY were similar on the 1% Ca diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal Growth Factor Increases Intestinal Calbindin-D9kand 1,25-Dihydroxyvitamin D Receptors in Neonatal Rats*

Epidermal growth factor (EGF) has been reported to increase intestinal calcium absorption in suckling rats. The mechanism of this effect is unknown, as are the roles of vitamin D-dependent and independent pathways. The present studies were undertaken to investigate the ability of EGF to accelerate the postnatal induction of the vitamin D-dependent intestinal calcium-binding protein, calbindin-D9k. Subcutaneous administration of EGF increased duodenal calbindin-D9k in suckling rats by more than 100% (P less than 0.001). The effect of EGF was not seen in older weaned animals or when EGF was given to suckling rats by gavage. Administration of EGF simulated the changes of normal development. 1) It increased calbindin-D9k, and the effect was greater in proximal than distal duodenum. 2) EGF increased alkaline phosphatase activity to the same extent in proximal and distal duodenum. 3) EGF increased sucrase more markedly in distal than in proximal epithelium. Maximal and half-maximal effects of EGF on each of these proteins were observed at twice daily doses of 0.1 and 0.04 microgram/g BW, respectively. 4) EGF at the maximally effective dose produced a small (30%) but statistically significant (P less than 0.005) increase in serum 1,25-dihydroxyvitamin D. 5) Most importantly, EGF treatment resulted in a 2-fold increase in intestinal 1,25-dihydroxyvitamin D receptors (VDR) in the proximal segments of the small intestine (P less than 0.001). EGF effects on calbindin-D9k and VDR were specific for the intestine, as EGF did not change kidney calbindin-D9k or kidney VDR. Thus, EGF was able to prematurely initiate a complex series of molecular changes that occur during normal development. The mechanism of EGF's action to stimulate calcium absorption appears to involve a maturation effect on the vitamin D-dependent pathway.

Calmodulin binding to the intestinal brush-border membrane: Comparison to other calcium-binding proteins

The intestinal brush-border membrane contains a high concentration of calmodulin bound to a 105,000 dalton (105 kDa) protein. Binding of radioiodinated calmodulin to this protein does not require calcium but is inhibited by trifluoperazine and excess unlabelled calmodulin. Recent evidence suggests that the 105 kDa protein in conjunction with calmodulin may be involved in the regulation of calcium transport across the brush-border membrane. In this report, we evaluated the binding of the 105 kDa protein to other radioiodinated calcium-binding proteins including the vitamin D-dependent intestinal calcium-binding protein. We observed that troponin C and S100 beta protein both bound strongly to the 105 kDa protein. The binding of S100 beta was inhibited by EGTA, but was little affected by trifluoperazine and excess unlabelled S100 beta, whereas that of troponin C was inhibited by trifluoperazine and excess unlabelled troponin C, but was little affected by EGTA. Both troponin C and S100 beta bound to a large number of proteins to which calmodulin did not bind. The vitamin D-dependent calcium-binding protein (calbindin) from chick intestine and rat kidney also bound to the 105 kDa protein, albeit more weakly than troponin C, S100 beta and calmodulin. The binding of the calbindins was increased by EGTA and was little affected by trifluoperazine and excess unlabelled calbindin. Parvalbumin, rat osteocalcin, and alpha-lactalbumin showed little binding to any brush-border membrane protein. Our results indicate that the 105 kDa calmodulin-binding protein of the intestinal brush border can bind to a variety of calcium-binding proteins all of which contain homologous regions thought to be the calcium-binding sites. Only the binding of troponin C resembles the binding of calmodulin, however, in being inhibited by trifluoperazine and excess unlabelled ligand. The functional significance of these observations in terms of regulating calcium transport across the brush-border membrane remains to be established.

The identification of a normal rat gene located close to the gene for the potential myoepithelial cell calcium-binding protein, p9Ka.

The potential calcium-binding protein p9Ka is related to S-100 protein and the vitamin D-dependent intestinal calcium-binding protein. p9Ka accumulates abundantly in cultured rat mammary myoepithelial-like cells but is very much less abundant in the parental cuboidal epithelial cells. p9Ka mRNA is found in normal rat mammary gland, and preliminary experiments suggest that it is found in the mammary myoepithelial cells. A 17-kilobase pair fragment of cloned normal rat DNA contains the gene for p9Ka, but it also contains the gene for two additional polypeptides of molecular mass 6 kDa that are resolved as two isoelectric focusing variants by two-dimensional gel electrophoresis. These two isoelectric focusing variants correspond to two abundant polypeptides present in the cultured myoepithelial cells and probably arise from postsynthetic modification of the product of a single gene. The mRNA for the product of this gene and the p9Ka mRNA are both found in the normal rat mammary gland, but these two mRNAs are differentially expressed in certain tumor-derived rat cell lines.

Localization of Ca 2+-binding proteins of rat cortex on two-dimensional gels—II. analysis of Ca 2+ binding proteins in ammonium sulfate fractions of rat brain

A total of seven high-affinity calcium-binding proteins have been detected in rat brain. This was accomplished using a combination of ammonium sulfate fractionation, two-dimensional gel electrophoresis, western blotting and 45Ca 2+-autoradiography. Of these seven proteins, three are detectable in a crude tissue punch of rat cortex while four are seen only after protein enrichment with ammonium sulfate. Four of the seven proteins detected in this study have been identified: calmodulin, the B subunit of calcineurin, the intestinal vitamin D-dependent calcium-binding protein and parvalbumin. The identities of the other three proteins visualized by 45Ca 2+-autoradiography in this study are unknown.

Observations on the binding of lanthanides and calcium to vitamin D-dependent chick intestinal calcium-binding protein. Implications regarding calcium-binding protein function.

The binding of calcium and terbium to purified chick vitamin D-dependent intestinal calcium-binding protein was studied by terbium fluorescence, circular dichroism, and intrinsic protein fluorescence techniques. Calcium-binding protein bound, with high affinity, at least 3 mol of terbium/mol of protein; numerous low affinity terbium-binding sites were also noted. The three highest affinity sites were resolved into one very high affinity site (site A) and two other sites (sites B and C) with slightly lower affinity. Resonance energy transfer from tryptophan residues to terbium occurred only with site A. This site was filled before sites B and C. Competition experiments in which calcium was used to displace terbium bound to the protein showed that larger amounts of calcium were needed to displace terbium from site A than from sites B and C. Energy transfer from terbium to holmium indicated that the terbium-binding sites (B and C) were located close to each other (about 7-12 A) but were distant (greater than 12 A) from site A. The addition of EDTA to calcium-binding protein resulted in a 25% decrease in intrinsic protein fluorescence, suggesting a conformational change in the protein. The titration of EDTA-treated calcium-binding protein with calcium resulted in recovery of intrinsic protein fluorescence. A reversible calcium-dependent change in the ellipticity of calcium-binding protein in circular dichroism experiments was also seen. These observed properties suggest that vitamin D-dependent chick intestinal calcium-binding protein behaves in a manner similar to other well-known calcium-binding regulatory proteins.

Molecular cloning of the cDNA for a growth factor-inducible gene with strong homology to S-100, a calcium-binding protein.

We have identified a cDNA whose sequence is preferentially expressed when quiescent fibroblasts are stimulated to proliferate. The steady-state levels of the mRNA corresponding to this clone, called 2A9, are increased by serum, platelet-derived growth factor, and epidermal growth factor, but not by insulin or platelet-poor plasma. mRNA levels of 2A9 are also increased in human acute myeloid leukemia. The 2A9 cDNA has been molecularly cloned from an Okayama-Berg library, and its complete nucleotide sequence has been determined. It has an open reading frame of 270 nucleotides, which has a 55% homology with the coding sequence of the beta-subunit of the S-100 protein, a calcium-binding protein that belongs (like calmodulin and the vitamin D-dependent intestinal calcium-binding protein) to the family of calcium-modulated proteins and is found in abundance in several human tumors, including melanoma. The S-100 protein and the deduced aminoacid sequence of 2A9 are also partially homologous to the small subunit of a protein complex that serves as a cellular substrate to tyrosine kinase. The partial homology of 2A9 (whose RNA is inducible by growth factors and is overexpressed in human acute myeloid leukemias) to the S-100 protein, other calcium-modulated proteins, and the subunit of a substrate for tyrosine kinase, is particularly interesting in view of the role attributed to calcium and tyrosine kinases in the regulation of cell proliferation.

Vitamin D-dependent calcium-binding protein of mouse yolk sac. Biochemical and immunochemical properties and responses to 1,25-dihydroxycholecalciferol.

The present studies were performed to further characterize a mouse yolk sac protein which is similar or identical to the vitamin D-dependent intestinal calcium-binding protein (CaBP). Yolk sac protein and purified rat intestinal CaBP displayed full identity upon immunodiffusion (Ouchterlony) using antiserum to the rat intestinal CaBP. Immunoreactive CaBP in yolk sac homogenates eluted from gel permeation columns with the low molecular weight peak of 45Ca2+ binding (Chelex assay), and the electrophoretic mobility of the protein was markedly increased by EDTA. On days 11-13 of gestation, the concentrations of immunoreactive CaBP in yolk sac were 4-5-fold higher than in placenta; by days 16-17, the concentrations in yolk sac and placenta were similar. Incubation of yolk sac with [3H]leucine demonstrated synthesis of immunoprecipitable [3H]CaBP. A single band of 3H-labeled protein was seen on sodium dodecyl sulfate gel electrophoresis of the immunoprecipitate. This protein co-migrated with radioactive placental CaBP with an apparent Mr of 10,050. Addition of 1,25-dihydroxycholecalciferol (calcitriol) to organ culture media with or without serum increased the amount and concentration of CaBP in yolk sac (p less than 0.001) at 48 h. CaBP synthesis in yolk sac appeared to be independent of calcitriol concentrations in the maternal circulation since injection of the hormone into the maternal compartment produced no change in yolk sac CaBP despite increases of maternal intestinal and renal CaBP. These studies demonstrate that yolk sac immunoreactive CaBP is synthesized in yolk sac and has an apparent molecular size and calcium-binding properties characteristic of mammalian vitamin D-dependent calcium-binding proteins. The in vitro response of yolk sac CaBP to calcitriol is the first evidence of a vitamin D effect on the fetal membranes and suggests one function for calcitriol receptors in these tissues.

Rat brain synthesizes two ‘vitamin D-dependent’ calcium-binding proteins

Two proteins from rat brain reacting against anti-chick intestinal vitamin D-dependent calcium-binding protein were characterized in terms of their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their molecular size. The proteins were present in the isolated cytoplasm and were produced following translation of brain mRNA in the rabbit reticulocyte lysate system. Their apparent molecular weight was 29,000 and 27,000 daltons whereas rat kidney contained only one protein cross-reacting with this antiserum and with a molecular weight of 27,000 daltons.

Low levels of intestinal vitamin D-dependent calcium-binding protein in juvenile X-linked hypophosphatemic mice.

Hyp mice are a model for human X-linked hypophosphatemia, the most common form of vitamin D-resistant rickets. The developmental appearance of intestinal vitamin D-dependent calcium-binding protein (CaBP) and alkaline phosphatase was studied in Hyp mice and normal mice from the perinatal period to adulthood. Both intestinal proteins were increased in the duodenum during weeks 2 and 3 of age, with values rising 10-fold or more above values measured in intestines of 1-week-old mice. During this developmental period and at most other ages, Hyp mice had levels of alkaline phosphatase and total intestinal protein comparable to those in control mice. On the other hand, the concentration of intestinal CaBP was decreased in juvenile Hyp mice during the weaning period at 2-3 weeks of age (35-65% of normal) and further depressed in the rapid growth phase at 4-6 weeks of age (15-45% of normal). During adulthood (7-35 weeks of age) Hyp mice maintained a CaBP concentration that averaged 71% of the level of control mice. These maturational defects in the Hyp intestine may play a contributory role in the bone disease in young Hyp mice.

Immunocytochemical localization of rat intestinal vitamin D-dependent calcium-binding protein.

Vitamin D-dependent calcium-binding protein (CaBP) was localized in intestinal tissue sections obtained from rats raised under three different nutritional conditions: a normal vitamin D-replete diet, a vitamin D-free diet followed by supplementation with vitamin D3, or a vitamin D-free diet without additional supplementation. An indirect immunoperoxidase technique, with immunocontrols, was used to visualize the specific sites of CaBP. CaBP was visualized only in the cytoplasm of absorptive cells. In the duodenum of animals raised on a normal diet, CaBP was present in absorptive cells from the upper crypt region to the villus tips. In the jejunum, many fewer absorptive cells contained CaBP, while in the ileum only random absorptive cells near the villus tips contained CaBP. In rats raised on a vitamin D-deficient diet then supplemented with vitamin D3, CaBP was present in cells at the full depth of the crypts and in absorptive cells along the total villus length in the duodenum. Rats raised on the same deficient diet but without supplementation with additional vitamin D exhibited no CaBP in crypt cells nor in absorptive cells more than half way up the villi. Absorptive cells higher on the villi contained immunoreactive CaBP but the intensity of immunostaining and number of CaBP-containing cells was markedly reduced compared to the vitamin D-supplemented group.

Sequence of rat intestinal vitamin D-dependent calcium-binding protein derived from a cDNA clone. Evolutionary implications.

We have recently reported molecular cloning of the cDNA synthesized from rat duodenal mRNA-encoding intestinal calcium-binding protein (ICaBP), a vitamin D3-induced protein (Desplan, C., Thomasset, M., and Moukhtar, M. S. (1983) J. Biol. Chem. 258, 2762-2765). Nucleotide sequence analysis of the longest cDNA insert (375 base pairs) permitted the assignment of 207 nucleotides of the coding region and 104 nucleotides of the entire 3'-noncoding region of the mRNA. Although the derived amino acid sequence for rat ICaBP differed from the bovine and porcine sequences by 16 and 14 residues, respectively, all the residues of each calcium-binding site met the proposed requirements of the "EF hand" theory. In contrast, several differences found in the linker regions might explain the absence of cross-immunoreactivity between rat and porcine ICaBPs. Analysis of nucleotide sequence homologies between the coding and noncoding regions showed that the region coding for the two calcium-binding sites (I and II) was immediately followed in the noncoding region by a sequence very similar to the sequence coding for site I. This suggests that rat ICaBP mRNA contains the remains of an untranslated calcium-binding site III-like structure and that low Mr ICaBP could result in early termination of the translation of a larger molecule containing four sites.

Human growth hormone increases intestinal vitamin D-dependent calcium-binding protein in hypophysectomized rats.

We examined the effects of hypophysectomy and pituitary hormone replacement on vitamin D-dependent calcium-binding protein (CaBP) in rat small intestine. The concentration of immunoreactive CaBP per mg intestinal protein was decreased by at least 56% in hypophysectomized rats compared to that in intact pair-fed controls. Alkaline phosphatase and total protein also were reduced by hypophysectomy, but pair-feeding produced comparable decreases. Daily injections of 2, 10, or 50 micrograms human GH (hGH) for 9 days produced a dose-dependent increase in CaBP. At the highest hGH dose (50 micrograms), the content of CaBP was increased 2- to 4-fold to intact levels. By comparison, the increases in total protein and alkaline phosphatase were small (25% to 40% and 80% to 90%, respectively). The induction of CaBP preceded the other protein responses; half-maximal increases in CaBP occurred after 2 days of hGH (50 micrograms/day) treatment before statistically significant changes in total protein or alkaline phosphatase activity. hGH was the most potent pituitary hormone tested; ovine TSH (25 mU/day) had no effect on CaBP, and ovine PRL (10 or 50 micrograms/day) increased CaBP by only 25-27% (P = 0.014). These studies indicate that the vitamin D-dependent intestinal CaBP in hypophysectomized rats is regulated by GH and provide further evidence that the pituitary may be involved in regulating vitamin D-dependent intestinal adaptations.

Facilitated calcium diffusion by intestinal calcium-binding protein.

The calcium flux through an aqueous compartment was determined using a flow-through dialysis cell in which two dialysis membranes isolated the aqueous compartment. Addition of the intestinal vitamin D-dependent calcium-binding protein (CaBP) significantly enhanced the calcium flux at near physiological calcium concentrations (1 X 10(-6) M). Bovine serum albumin had no effect on the calcium flux. CaBP appears to be one of a class of low molecular weight, soluble binding proteins that enhance ligand diffusion.

Immunocytochemical localization of the vitamin D-dependent calcium binding protein in chick duodenum.

The vitamin D-dependent calcium binding protein (CaBP) of chick duodenum has been localized by immunocytochemistry and by radioimmunoassay. Light microscopically, CaBP was seen to be present in the absorptive cells of the villi while in other cell types of the villi and the crypts, including goblet cells and endocrine cells, no CaBP was seen. At the electron microscopic level, CaBP was shown to be localized in the cytosol and the euchromatin of the nucleus but not in membrane-bounded cytoplasmic compartments. Quantitative evaluation of the immunocytochemical protein A-gold label showed that the terminal web and the cytosol of basal cellular regions were most highly labeled while the brush border was weakly labeled. The radioimmunoassay evaluation of intestinal subcellular fractions indicated that 96% of the homogenate CaBP is in the cytosol high-speed supernatant fraction. Collectively, these results support the hypothesis that the vitamin D-dependent intestinal CaBP may play a role in either regulation of intracellular calcium concentration or movement of calcium across the brush border membrane from the gut lumen.

Specific neurons in chick central nervous system stain with an antibody against chick intestinal vitamin D-dependent calcium-binding protein

A specific antiserum against intestinal vitamin D-dependent calcium-binding protein (CaBP) was used for a systematic immunohistochemical evaluation of immunoreactive sites in the central nervous system of chick. CaBP was observed in the perikarya, dendrites and axons of a specific population of neurons. It is concluded that CaBP represents a marker for selected neurons in the central nervous system of chick.

Relation of vitamin D-dependent intestinal calcium-binding protein to calcium absorption during the ovulatory cycle in Japanese quail.

SummaryDuodenal CaBP levels and the efficiency of 47Ca absorption by the duodenal segment of ovulating Japanese quail were determined as a function of eggshell formation. No differences in these parameters were noted in quail in which eggs were being calcified and in quail with no egg in the calcification stage. Thus, there is a correlation between the efficiency of Ca absorption and the level of vitamin D-dependent intestinal calcium-binding protein in this physiological state in Japanese quail.

Immunological Interaction of Vitamin D-dependent Intestinal Calcium-binding Protein of Chick and Rat

Immunological Interaction of Vitamin D-dependent Intestinal Calcium-binding Protein of Chick and Rat

Regulation of intestinal calcium-binding protein calcium intake in the rat.

Analytical gel electrophoresis of the vitamin D-dependent intestinal calcium-binding protein (CaBP) has demonstrated two protein bands (1 and 2) of similar molecular weight and similar specific binding activity. The mucosal concentration of CaBP, measured by a quantitative competitive binding assay, has been shown to vary reproducibly and inversely with calcium intake and the mucosal calcium concentration. These same factors also influence the relationship of bands 1 and 2. When animals on a high-calcium diet were placed on a low-calcium diet, their CaBP increased by 35% in 24 h and by 48% in 48 h and reached a level typical of animals on a low-calcium diet. Measurement of the diurnal variation of CaBP and mucosal calcium in animals allowed access to feed only at night revealed significant, but inverse, oscillations. These observations are interpreted as reflecting a regulation of CaBP by the mucosal calcium concentration, which appears to reflect absorbed calcium in transit.