Calcium-binding Protein Content Research Articles

Effects of gonadal hormones on calcium-binding protein in chick duodenum

ARTICLESEffects of gonadal hormones on calcium-binding protein in chick duodenumRoberta J. Navickis, Ortrude K. Dial, Benita S. Katzenellenbogen, and Andrew V. NalbandovRoberta J. Navickis, Ortrude K. Dial, Benita S. Katzenellenbogen, and Andrew V. NalbandovPublished Online:01 Nov 1979https://doi.org/10.1152/ajpendo.1979.237.5.E409MoreSectionsPDF (2 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Download PDF FiguresReferencesRelatedInformation Cited ByDifferential Regulation of Calbindin in the Calcium-Transporting Organs of Birds with High Calcium RequirementsThe Journal of Poultry Science, Vol. 46, No. 4 More from this issue > Volume 237Issue 5November 1979Pages E409-E417 Copyright & PermissionsCopyright © 1979 by American Physiological Societyhttps://doi.org/10.1152/ajpendo.1979.237.5.E409PubMed227275History Published online 1 November 1979 Published in print 1 November 1979 Metrics

Vitamin D-dependent calcium-binding protein of rat intestine: changes during postnatal development and sensitivity to 1,25-dihydroxycholecalciferol.

To study the role of the vitamin D-endocrine system during the perinatal period, we monitored vitamin D-dependent calcium-binding protein (CaBP) in rat intestine by radial immunodiffusion and polyacrylamide disc gel electrophoresis. Small amounts of CaBp were present 2 days before birth; these levels increased 74-fold by day 38 after birth. Approximately 80% of the increase in CaBP concentration occurred in a 5-day period at the time of weaning (days 17--22 after birth). Before this period, the concentration of CaBP was comparable to that found in rachitic (adult) rats. Administration of 1,25-dihydroxycholecalciferol to suckling rats on days 15 and/or 16 was followed by a premature increase in the amount of intestinal CaBP. These data demonstrate that although vitamin D-dependent CaBP is low in preweaned rats, the rat intestine is responsive to exogeneous 1,25-dihydroxyvitamin D3 at least as early as day 15 after birth. The close temporal correspondence between the increases in CaBP and previously reported changes in calcium transport and vitamin D metabolism suggest that the vitamin D-endocrine system plays a role in postnatal intestinal maturation and adaptation during the weaning period.

Studies on the Mode of Action of Calciferol. XIII. Development of a Radioimmunoassay for Vitamin DDependent Chick Intestinal Calcium-Binding Protein and Tissue Distribution*

A RIA for chick intestinal calcium-binding protein (CaBP) has been developed with a sensitivity of 1 ng. The antiserum was generated in rabbits injected with highly purified vitamin D-dependent chick intestinal CaBP. The assay employs the double antibody technique, and 125-labeled CaBP was prepared using chloramine T. Low molecular weight peptide hormones and normal rabbit, rat, and human serum proteins show no cross-reactivity in the assay. Measurements of chick intestinal and kidney CaBP by RIA showed a good correlation with measurements of CaBP by the radial immunodiffusion method. The assay is reproducible (interassay variability, 16.3%) and precise (intraassay variability, 4.0%). The concentration of immunoreactive CaBP (iCaBP) in chick serum (2.7 ng/ml serum) can now be measured as early as 8 h after the administration of 6.5 nmol 1,25-dihydroxyvitamin D3; a maximum of 11 ng/ml is reached at 20 h. The level of CaBP in chick serum was found to be dependent on the dose of vitamin D3 or 1,25-dihydroxyv...

Intestinal Calcium-Binding Protein and Calcium Absorption in Cortisol-Treated Chicks: Effects of Vitamin D3and 1,25-Dihydroxyvitamin D3*

Vitamin D3 in rachitic chicks stimulates calcium absorption and induces the synthesis of two pools of intestinal calcium-binding protein (CaBP), one soluble and the other membrane bound. Cortisol acetate caused a decrease in calcium absorption which was accompanied by a decrease in soluble CaBP. Cortisol was similarly effective in 1,25-dihydroxyvitamin D3-dosed chicks, suggesting that the glucocorticoid effect was not entirely due to the defective synthesis of this metabolite. Ca absorption was directly correlated with soluble CaBP and alkaline phosphatase and inversely related to the ratio of bound to soluble CaBP. It was further observed that the slope of the Ca absorption vs. soluble CaBP regression line was greater in chicks given 1,25-dihyroxyvitamin D3 compared to those given vitamin D3, and this is interpreted to mean that another factor or condition, in addition to assayed concentrations of soluble CaBP, determines the degree of calcium absorption.

Embryonic chick intestine in organ culture: Hydrocortisone and vitamin D-mediated processes

The effects of the glucocorticoid, hydrocortisone (HC), on vitamin D-mediated responses were examined in the organ-cultured, embryonic chick duodenum. In this system tissue responses to vitamin D-steroids in the culture medium include increased cAMP concentration, de novo synthesis of a specific calcium-binding protein (CaBP), enhanced uptake and transmucosal transport of calcium, and increased alkaline phosphatase activity. HC at levels ≥ 27.5 n m increased vitamin D-induced CaBP concentration: This apparently represents the first report of an interaction of HC with another steroid, vitamin D, in the regulation of the concentration of a specific protein. High levels of HC (≥27.5 μ m) in the culture medium reduced duodenal calcium uptake and transmucosal transport regardless of the presence of vitamin D. However, at lower concentrations of HC (≤2.75 μ m), only vitamin D-independent calcium uptake (basal calcium uptake) was reduced. Actinomycin D had no effect on HC reduction of basal calcium uptake suggesting that new protein synthesis is not involved in this action. In other experiments either HC or vitamin D stimulated phosphate and glucose uptake, and this uptake was potentiated by the presence of both steroids. HC also stimulated alanine uptake. Either HC or vitamin D increased both alkaline phosphatase activity (APA) and cAMP concentration, but together their activities were only additive. The data accumulated thus far indicate that HC directly influences calcium (and other nutrient) uptake by the duodenum and increases the concentration of the vitamin D-induced CaBP. Other vitamin D-mediated responses (APA and cAMP) were influenced by HC but there was no readily discernible relationship to nutrient uptake.

Immunodiffusion using tissue sections

A simple radial immunodiffusion technique is described for determining relative antigen content of frozen tissue sections. Its use is illustrated with frozen sections of chick intestine containing the vitamin D-induced calcium-binding protein (CaBP). Diffusion of the intestinal CaBP from the sections placed on agar containing specific anti-CaBP anti-serum produced a precipitin ring, whose diameter was proportional to the CaBP content of the intestine. The technique allowed the determination of the relative antigen content in sections of tissue adjacent to others in which immunocytochemical localization or other histochemical studies were to be performed.

Calcium-Binding Protein and Kidney 25-hydroxycholecalciferol-1-hydroxylase Activity in Turkey Poults

A vitamin D-dependent calcium-binding protein (CaBP) has been found in the intestinal mucosa of turkeys (Meleagris gallopavo). This protein is similar, if not identical, to the corresponding one found in the intestine of the chicken (Gallus domesticus). The concentration of intestinal CaBP in the intestine of cholecalciferol-fed turkey poults was twice that found in the intestine of the domestic chicken of parallel age. Dietary calcium or phosphorus restriction resulted in a significant increase in intestinal CaBP. This increase was less pronounced than that obtained in chickens. The relationship between duodenal CaBP and dietary calcium appears to be linear. Cholecalciferol (vitamin D) deficiency or dietary calcium, but not phosphorus, restriction resulted in an increase of kidney 25 hydroxycholecalciferol-1-hydroxylase activity in vitro. The values of activity obtained in turkeys were higher than those obtained under the same experimental conditions in chicks. Bone ash was reduced by dietary calcium, phosphorus or cholecalciferol restriction. Plasma inorganic phosphorus was lower in either cholecalciferol- or phosphorus-restricted turkeys. Plasma calcium was significantly higher in the phosphorus-restricted turkeys than in the calcium-restricted or control turkeys.

Differential Response of Calcium Transport Systems in Laying Hens to Exogenous and Endogenous Changes in Vitamin D Status

Changes in intestinal calcium absorption, calcium deposition into egg shell, and intestinal, renal and uterine calcium-binding protein (CaBP) in laying hens were related to changes in 25 hydroxycholecalciferol-1-hydroxylase activity (1-hydroxylase), or to the supplementation of 1alpha-hydroxycholecalciferol (1alpha-OH-CC). The onset of egg production resulted in an increased kidney 1-hydroxylase activity and intestinal and uterine CaBP. Renal concentrations of CaBP remained unchanged. Intestinal calcium and phosphorus absorption, during the period of egg shell formation, and duodenal calcium-binding protein (CaBP), were higher in 1alpha-OH-CC-fed than in cholecalciferol-fed hens. Renal or uterine CaBP and calcium deposition into the egg shell did not fluctuate with the vitamin D source or concentration. 1alpha-OH-CC injection into non-laying Nicarbazinfed hens resulted in an increase in intestinal but not renal or uterine CaBP concentrations. It is suggested that (a) CaBP in various organs responded independently to the same stimuli; and (b) calcium deposition into egg shell and uterine CaBP level are not related to kidney 1-hydroxylase activity or concentration of 1,25 dihydroxycholecalciferol.

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

1. The rapid stimulation of intestinal Ca(2+) 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, Ca(2+) 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 Ca(2+) transport before calcium-binding-protein biosynthesis is induced. 2. The maximum increase in Ca(2+) transport observed after this dose of 1,25-dihydroxycholecalciferol (attained by 8h) is similar to that observed after 1.25-25mug 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 Ca(2+) 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 Ca(2+) transport. We propose that calcium-binding-protein synthesis is necessary for maintaining the stimulated rate of Ca(2+) transport, which is initiated by other factors.

Effect of lactose feeding on cell renewal, disaccharidase activity and calcium-binding protein content in the intestinal mucosa of rats

Effect of lactose feeding on cell renewal, disaccharidase activity and calcium-binding protein content in the intestinal mucosa of rats

Intestinal calcium-binding protein 3 months after massive small bowel resection in the piglet

Changes in intestinal calcium-binding protein and calcium binding activity were studied at resection and 3 months after 90% small bowel resection in piglets and one adult pig. A calcium-binding protein (MW congruent to 11.000) with calcium-dependent eletrophoretic mobility was partially purified from mucosal extract of proximal jejunum, mid-gut, and ileum. The concentration of calcium-binding protein and the calcium-binding activity of the intact animals were found highest in the proximal jejunal segment, lowest in the ileal segment. After resection in the four surviving animals out of nine, a significant increase in calcium-binding activity was observed in the proximal jejunum and in the distal ileal segment. The change in calcium-binding activity was much more marked in the ileum than the jejunum. These data demonstrate that pig intestinal mucosa possesses an adaptive capacity to increase the synthesis of calcium-binding protein after massive small bowel resection.

Intestinal Calcium Binding Protein Levels in the One Month Old Turkey Poult

Intestinal calcium binding protein (CaBP) was studied in one month old poults which had been fed a vitamin D3-deficient diet unsupplemented or with 300, 600, 900 or 1200 I.U. vitamin D3 added per kg. diet. CaBP levels increased with vitamin D3 supplementation up to 600 I.U. but showed no further increase with higher dietary vitamin D3. Poults supplemented with 600 to 1200 I.U. vitamin D3/kg. diet had an average of 1.03 mg. CaBP/g. of duodenal mucosa cells collected. The calcium binding activity of these samples was approximately 0.9% per mg. protein and was highly correlated with CaBP concentration (r = 0.94).

Chick Brain Calcium Binding Protein: Response to Cholecalciferol and Some Developmental Aspects

Following oral administration of 3H-cholecalciferol to rachitic chicks, the radioactive metabolites found in brain tissues were separated by Sephadex LH-20 chromatography. The parent vitamins and two biologically important metabolites, 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol were detected in the brain, although at relatively low levels. A comparison with other tissues suggested the brain was much less permeable to the vitamin D steroids than were other tissues. However, chronic cholecalciferol administration for 4 weeks to severely vitamin D deficient chicks elicited a significant increase in the cerebellar content of a calcium-binding protein (CaBP). The lowest level administered, 2 IU/day (130 pmole), increased the CaBP content by more than 50%, while 16 IU/day (1.04 nmole) doubled the CaBP content. Chick brain CaBP has the same physical characteristics and is immunologically identical to the vitamin D-induced CaBP present in chick intestine, it differs in that single acute doses of vitamin D do not increase the content of brain CaBP while intestinal CaBP synthesis is stimulated significantly. The time course of appearance and content of brain CaBP in embryonic chicks was monitored. It was first detectable in the brain at day 15 of incubation and increased to a near post-hatch level by day 20. It differed in its initial time of appearance from both kidney CaBP which first appears at day 10 and intestinal CaBP which is not detectable until hatch day.

The response of 25-hydroxycholecalciferol-1-hydroxylase activity, intestinal calcium absorption, and calcium-binding protein to phosphate deficiency in chicks

In two breeds of chicks. White Leghorn and White Rock, phosphate deficiency resulted in an increase in duodenal 47Ca absorption and calcium-binding protein (CaBP). Similar changes were observed in calcium-deficient chicks. No change was noted in the activity of 25-hydroxycholecalciferol-1-hydroxylase in the kidney in phosphate-deficient chicks, whereas a large increase in the activity of the enzyme occurred as a result of calcium restriction. During phosphate restriction, plasma inorganic phosphorus decreased continuously, plasma calcium increased, and body weight decreased. Slight changes were noted in the activity of 25-hydroxycholecalciferol-1-hydroxylase during 15 days of phosphate restriction. Concentration of CaBP in the intestinal mucosa reached a peak after ten days of phosphate restriction, followed by a decline. Switchback experiments indicated no inhibitory action on the activity of 25-HCC-1-hydroxylase by supernatants from phosphorus-deficient chicks. It is concluded that changes in calcium absorption due to phosphate deficiency are not associated with the modulation of the activity of kidney 25-hydroxycholecalciferol-1-hydroxylase, as measured in vitro.

Regulation of Intestinal Calcium Absorption in the Laying Quail: Independent of Kidney Vitamin D Hydroxylation

Two experiments were carried out with laying quail (Coturnix coturnix japonica) fed either cholecalciferol (40 mug/kg) or 1 alpha-hydroxycholecalciferol (8 mug/kg). Recovery following vitamin D depletion, as indicated by egg production and shell deposition, was faster in quail fed 1 alpha-hydroxycholecalciferol than in those fed cholecalciferol. Overall intestinal calcium absorption was higher in the 1 alpha-hydroxycholecalciferol-fed quail due to a higher absorption in the middle parts of the small intestine. This was associated with corresponding differences in the concentration of calcium binding protein. Intestinal calcium absorption was markedly higher during periods of shell formation than during periods of uterine inactivity in quail fed either cholecalciferol or 1 alpha-hydroxycholecalciferol. It is suggested, therefore, that the mechanism responsible for this difference is independent of vitamin D hydroxylation in the kidney.

Radioimmunoassay studies of intestinal calcium-binding protein in the pig. I. Identification of intestinal calcium-binding protein in blood and response to a low calcium diet.

We have developed a radioimmunoassay for porcine intestinal calcium-binding protein (CaBP) and have used it to detect CaBP in pig plasma. Plasma CaBP is identical to intestinal CaBP on the basis of immunological activity, molecular size, and molecular charge properties. The plasma CaBP concentration was greater in the portal blood than in mixed venous blood, suggesting that blood CaBP originates in the gut. Two of four 15-week-old littermate pigs were placed on a low calcium diet (0.15% calcium, 0.65% phosphorus) and two on a control diet (0.65% calcium, 0.65% phosphorus). After 2 weeks, the entire small intestine was removed and divided into nine 1.8-m segments. CaBP was assayed in both plasma and intestinal mucosa. When the two pigs on a low calcium diet were compared with two control pigs, there was a general increase in immunoreactive CaBP in both plasma and intestinal mucosa. However, there was no increment in immunoreactive CaBP in the first 1.8-m segment of small intestine. Seventy-one percent of the increment in CaBP occurred distal to the first two segments. The largest fractional low calcium diet effect occurred in the ileum. The mean CaBP concentration for the total small intestine increased by a factor of 1.9. The plasma CaBP concentration increased by a factor of 2.6. In these pigs, plasma CaBP was a more reliable indicator of change in CaBP status than was the measurement in the proximal gut segment which contained the duodenum. The assay of CaBP in blood is convenient and may obviate the sampling errors inherent in intestinal biopsy.

Radioimmunoassay studies of intestinal calcium-binding protein in the pig. II. The distribution of intestinal CaBP in pig tissues.

Using a specific radioimmunoassay for porcine intestinal calcium-binding protein (CaBP), we have measured the concentration of CaBP in the various tissues and organs of normal pigs. Intestinal CaBP was present in highest concentration in the upper small intestine, with lower concentrations in the distal small intestine. Intestinal CaBP was also found, in lower concentrations, in kidney, liver, thyroid, pancreas, and blood. In all other tissues, including parathyroid, bone, skeletal muscle, and brain, CaBP immunoreactivity was undetectable or less than in blood. The elution profile of calcium-binding activity and immunoreactivity from gel filtration analysis of kidney and parathyroid extracts suggest that the calcium-binding protein in the parathyroid gland, and the major calcium-binding protein(s) in the kidney, are chemically and immunochemically different from intestinal CaBP.

Calcium-binding protein in the duodenal mucosa of uremic patients and normal subjects

In the present study we measured the content and determined the localization of a calcium-binding protein (CaBP) in the intestinal biopsy specimen of ten patients with severe renal insufficiency and in eight healthy individuals. In each patient a biopsy specimen of the iliac crest was obtained for evaluation of the degree of osteodystrophy. The CaBP was isolated from human kidneys. Its identity with human intestinal CaBP was suggested by acrylamid gel electrophoresis. Specific antibodies against it were developed in rabbits. The content of CaBP in duodenal mucosa biopsy material was measured by quantitative radial immunodiffusion. Its mean value in the duodenum of patients with renal insufficiency was 3.65 +/- 1.14 mug; in that of normal persons, 10.80 +/- 3.20 mug/mg of protein in the supernatant. There was neither a correlation between the type and duration of renal insufficiency and the CaBP content of the specimens nor between the degree of renal osteodystrophy and CaBP content. In normal persons the specific immunohistological activity indicating the presence of CaBP was found uniformly along the brush border and basement membrane of duodenal epithelial and the goblet cells. In uremic patients the fluorescence was markedly reduced in the typical locations, especially in the brush border area.

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.

Fluorescent antibody localization of the vitamin D-dependent calcium-binding protein in the oviduct of the laying hen.

Fluorescein isothiocyanate-conjugated sheep antirabbit gamma-globulin was used in the indirect fluorescent antibody technique to localize the vitamin D-dependent calcium-binding protein (CaBP) in the shell gland of the laying hen. Intense specific fluorescence, indicative of the presence of CaBP, was found only in the tubular gland cells and not in the surface epithelium. CaBP was primarily associated with the luminal surface and other cell boundaries, and in lesser concentrations within the cells except for occasional intracellular regions of high concentration. The magnum, isthmus and vagina were essentially negative. Since the concentrations of intestinal CaBP is highly and directly correlated with the efficiency of calcium absorption, the present results suggest that the tubular gland cells might have a primary role in the transport of calcium associated with egg shell formation.