Calcium-binding Protein Content Research Articles

Measurement of the 10,000-molecular weight calcium-binding protein in small-intestinal biopsy specimens from patients with malabsorption syndromes.

Calcium-binding protein (CaBP) (molecular weight, 10,000) was measured in small-intestinal biopsy specimens from 36 patients with malabsorption syndromes: short-bowel syndrome (n = 13), untreated coeliac disease (n = 4), coeliac disease in remission (n = 7), patients with intestinal bypass owing to morbid obesity (n = 5), and in patients with chronic diarrhoea of unknown cause (n = 7). Twelve patients with no signs of malabsorption who had the irritable bowel syndrome were used as controls. Patients with small-bowel resections showed reduced concentrations of CaBP (p less than 0.01) and low intestinal calcium absorption (p less than 0.05). Small amounts of CaBP were found in intestinal specimens from patients with coeliac disease in remission (p less than 0.01), and CaBP was almost undetectable in patients with a newly diagnosed coeliac disease and avillous jejunal biopsy findings (p less than 0.001). Patients with chronic diarrhoea and patients with an intestinal bypass had CaBP concentrations comparable to those of the control group. A direct correlation was found between CaBP and the fractional calcium absorption in all patients (p less than 0.05). CaBP may therefore be considered an indicator of the efficiency of the small intestine to absorb calcium.

Endocrine control of plasma concentrations of calcium-binding protein in the pig.

The aetiology of the rise in plasma calbindin-D9K (vitamin D-induced calcium-binding protein; CaBP), following insulin-induced hypoglycaemia, was studied in the pig. ACTH led to a rise in plasma concentrations of both CaBP and cortisol. Metyrapone, which blocks cortisol synthesis, abolished the increases in plasma concentrations of CaBP and cortisol normally observed in response to insulin-induced hypoglycaemia. However, there was no significant rise in plasma concentrations of CaBP in response to pharmacological or physiological doses of cortisol. Injection of clonidine, an alpha 2-adrenergic agonist, led to a rise in plasma concentrations of CaBP, whereas phenylephrine, an alpha 1-adrenergic agonist, tended to exert an inhibitory effect. Also, administration of phentolamine (an alpha-adrenergic blocker) before injection of insulin abolished the usual increase in plasma concentrations of CaBP, whereas propranolol (a beta-adrenergic blocker) enhanced the normal increase in plasma concentrations of CaBP in response to insulin-induced hypoglycaemia. Isoproterenol, a beta-adrenergic agonist, was without effect on plasma CaBP. Neither GH nor glucagon appear to be involved in the rise in plasma CaBP following insulin-induced hypoglycaemia. Although atropine abolished the effect of acute hypoglycaemia on plasma CaBP, carbamylcholine was without effect on plasma CaBP concentration. It is concluded that the increases in plasma CaBP induced by either ACTH or alpha 2-adrenergic stimulation may be interrelated since the administration of ACTH can lead to raised plasma concentrations of catecholamines.

Effect of 1,25-dihydroxyvitamin D3 on plasma concentrations of calcium-binding protein in normal and rachitic (vitamin D-dependent rickets type I) pigs.

An i.v. injection of calcitriol (1,25-(OH)2D3) had no effect within 2.5 h on plasma concentrations of calbindin-D9K (vitamin D-induced calcium-binding protein; CaBP) in hypocalcaemic pigs with inherited vitamin D-dependent rickets type I or in their normocalcaemic siblings or half-siblings. Three days later the plasma concentration of CaBP had doubled in the hypocalcaemic pigs, but was unaltered in the normocalcaemic siblings and half-siblings. Following daily i.v. injections of 1,25-(OH)2D3 for a further 5 days (days 4-8) plasma concentrations of CaBP increased in both the hypocalcaemic (days 4-8) and normocalcaemic (day 8) pigs, the effect being more rapid and greater in the hypocalcaemic 1,25-(OH)2D3-deficient animals. An i.v. injection of 1,25-(OH)2D3 to pure Yucatan pigs also had no effect on plasma concentrations of CaBP within 1.5 h, but in the following 1 h there was some indication of an increase in plasma CaBP levels. In contrast to the normal pigs, insulin-induced hypoglycaemia did not lead to a peak in plasma CaBP concentrations in the hypocalcaemic pigs. There was also no change in the plasma concentrations of 1,25-(OH)2D3 associated with the peak in plasma CaBP following insulin-induced hypoglycaemia in normocalcaemic pigs. These results suggest that changes in plasma concentrations of 1,25-(OH)2D3 are not directly involved in mediating the increase in plasma CaBP which follows hypoglycaemia induced by insulin in normal pigs, although 1,25-(OH)2D3 probably plays a permissive role.

1,25-Dihydroxyvitamin D3 induces the synthesis of vitamin D-dependent calcium-binding protein in cultured chick kidney cells.

A 28,000 mol wt vitamin D-dependent calcium-binding protein (CaBP), first isolated from avian intestine, has recently been shown to exist in kidney and other tissues. To study the mechanism regulating the production of renal CaBP, we used primary cultures of chick kidney cells as an in vitro model. Renal cortical tubules isolated from vitamin D-deficient chicks were grown in serum-free, hormone-supplemented medium. Confluent cells were epithelioid and expressed CaBP, as demonstrated by immunocytochemistry and a specific RIA. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] increased cellular CaBP content in a dose-dependent manner (10(-10)-10(-7) M) from basal concentrations of 50-240 ng/mg protein to maximal concentrations of 600-1200 ng/mg protein 48 h after dosing. Cycloheximide (2 microM) inhibited 1,25-(OH)2D3 induction of CaBP, indicating that the mechanism requires new protein synthesis. Western blotting of cell extracts confirmed the identity of the inducible protein as the 28,000 mol wt CaBP. 25-Hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were effective but somewhat less potent inducers of CaBP, whereas vitamin D3 was without significant effect. The activities of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 are attributed to their rapid conversion by kidney cells to 1 alpha-hydroxylated metabolites. The dose-response profiles for two additional bioresponses in these cells, namely induction of 24-hydroxylase and inhibition of 1 alpha-hydroxylase activity, were comparable to those for CaBP induction. To our knowledge, this is the first description of a cell culture system that exhibits 1,25-(OH)2D3-inducible CaBP in vitro. This model should permit the study of certain aspects of the physiology of 1,25-(OH)2D3 and CaBP in kidney that are not possible in vivo.

Analysis and in situ detection of cholecalcin messenger RNA (9000 Mr CaBP) in the uterus of the pregnant rat

The molecular cloning of a cDNA fragment synthesised from rat duodenal mRNA coding for cholecalcin (calbindin), a 9000 Mr vitamin D-induced calcium-binding protein (CaBP), has been previously described. DNA/RNA hybridisation assays have been used to examine CaBP mRNA production in the uterine horns and duodena of pregnant (21 day) rats using the cloned CaBP cDNA. Northern hybridisation studies showed that the 32P cDNA sequence hybridised to a single 500-600 nucleotide species in both the uterus and the duodenum, thus demonstrating identical CaBP mRNA processing in both tissues. Dot blot hybridisation studies showed that the CaBP mRNA concentration was greatest in the duodenum while that of the uterine horns was about 10% of the duodenal level. The observed differences in CaBP mRNA levels correlate well with the in vivo CaBP concentrations. In situ hybridisation histochemistry using 3H cDNA revealed that CaBP mRNA visualised by silver grains was found in all the parts of the endometrium and the myometrium. However, CaBP mRNA was more concentrated in the outer and inner muscular fibres and in the luminal cells of the endometrium than in the stroma cells. These results demonstrate that the CaBP gene is expressed in specific cells of the rat uterus.

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.

Decreased content of integral membrane calcium-binding protein (IMCAL) in tissues of the spontaneously hypertensive rat.

Prior studies report that plasma membranes of the spontaneously hypertensive rat (Okamoto-Aoki strain) bind less calcium than do the corresponding preparations from Wistar Kyoto controls. The possibility that the differences result from a decrease in the content of integral membrane calcium-binding protein (IMCAL) was explored by the application of immunoassays with polyclonal antisera and a mouse monoclonal antibody. IMCAL binds calcium with relatively high affinity, and its content in many rat tissues is regulated by vitamin D and the level of dietary calcium. Immunoassays of tissue IMCAL demonstrate significant reductions in content in the erythrocyte ghost, intestinal mucosa, kidney, heart, testis, and liver of the spontaneously hypertensive rat as compared to the control strains. The decreases are observed both at 4-5 weeks of age, before the onset of severe hypertension and at 8-9 weeks in the presence of severe hypertension. Moreover, the magnitude of the decrease in erythrocyte IMCAL can account for much of the decrease in the calcium-binding capacity of erythrocyte membranes reported by others. The results are especially significant because an abnormality in the membrane binding of calcium and in the regulation of cytosolic calcium ion concentration could underlie the pathogenesis of the hypertension.

Regulation of kidney calcium-binding protein in the bird ( Callus domesticus)

1. 1. The possible involvement of plasma calcium and 1,25-dihydroxyvitamin D, [1,25(OH) 2D 3] in the regulation of the concentration of kidney calcium-binding protein (CaBP) was investigated. 2. 2. Chicks were fed diets varying in Ca 2+ and P, with or without vitamin D. CaBP and 1,25(OH) 2D 3 were determined by competitive binding assays. 3. 3. A significant correlation between plasma and kidney 1,25(OH) 2D 3 was found, the linear regression equation of best-fit was plasma 1,25(mOH) 2D 3 = 0.14 + 1.56 kidney 1,25(OH) 2D 3. 4. 4. In the vitamin D-fed chicks, kidney CaBP varied independently of the circulating or organ level of 1,25(OH) 2D 3 ( P>0.05), but was lower in the vitamin D-deficient than in the vitamin D-fed birds. 5. 5. A significant correlation was observed between kidney CaBP and plasma calcium (Ca p). The regression equations were CaBP = Ca p /(85.57-4.00 Ca p ) (R = 0.845) and CaBP = 0.0558 + 0.0404 Ca p ( R = 0.749), for vitamin D-treated and vitamin D-defieient chicks, respectively. 6. 6. 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.

The effects of chronic prednisone administration on intestinal receptors for 1,25-dihydroxyvitamin D3 in the dog.

Glucocorticoid inhibits intestinal calcium absorption. To further explore the mechanism of this inhibition, we studied dogs during the administration of oral prednisone (1.2-1.5 mg/kg X day) for 20 to 28 weeks in comparison to untreated dogs. Prednisone administration had no effect on serum 25-hydroxyvitamin D concentrations, but was accompanied by a fall in serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations from 87 +/- 20 pM (control) to 62 +/- 28 pM (prednisone-treated; P less than 0.01). Cytosol prepared from the duodenal, jejunal, and ileal mucosa of control dogs was found to contain a specific 3.2S [3H]1,25-(OH)2D3 binder analogous to the binder that has been observed in the intestine of other species and in other tissues. The apparent concentration of this binder decreased progressively from duodenum to ileum. Prednisone administration increased the apparent duodenal concentration of the binder from 170 +/- 91 (control) to 363 +/- 124 fmol/mg protein (prednisone-treated; P less than 0.025). The intestinal content of calcium-binding protein also declined progressively from the duodenum to the ileum, but was not affected by prednisone administration. These data suggest that events other than alterations in intestinal 1,25-(OH)2D3 receptors must mediate the inhibition of intestinal calcium absorption during chronic glucocorticoid administration.

Distribution of Ca 2+-ATPase, ATP-dependent Ca 2+-transport, calmodulin and vitamin D-dependent Ca 2+-binding protein along the villus-crypt axis in rat duodenum

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Ca 2+-ATPase activity and ATP-dependent Ca 2+-transport rates in basolateral membranes from rat duodenum were measured during migration along the crypt-villus axis. In addition, vitamin D-dependent calcium-binding protein and calmodulin content were measured in homogenates of six cell populations which were sequentially derived from villus tip to crypt base. Alkaline phosphatase activity was highest at the tip of the villus (fraction I) and decreased more than 20-fold towards the crypt base (fraction VI). ( Na + + K + )-ATPase activity also decreased along the villus-crypt axis but in a less pronounced manner than alkaline phosphatase. ATP-dependent Ca 2+-transport in fraction II ( 8.2 ± 0.3 nmol Ca 2+/min per mg protein ) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca 2+-transport activity ( 0.9 ± 0.1 nmol Ca 2+/min per mg protein ). The distribution of high-affinity Ca 2+-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca 2+-transport. The activity of Na +/Ca 2+ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP-dependent Ca 2+-transport system, the Na +/Ca 2+ exchanger is of minor importance in villus cells but may play a more significant role in crypt cells. Calcium-binding protein decreased from mid-villus towards the villus base and was undetectable in crypt cells. Calmodulin levels were equal along the villus-crypt axis. It is concluded that vitamin D-dependent calcium absorption takes primarily place in villus cells of rat duodenum.

Cell-Free Translational Analysis of Messenger Ribonucleic Acid Coding for Vitamin D-Dependent Rat Renal Calcium-Binding Protein*

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] has been found to exert its effects in a manner entirely analogous to that of other steroid hormones and is known to induce the synthesis of a calcium-binding protein (CaBP). The effects of 1,25-(OH)2D3 and dietary alteration on genomic expression in rat kidney were studied by in vitro translation of poly(A+)-containing RNA and by immunoprecipitation. Poly(A+)RNA from rat kidneys was translated in a rabbit reticulocyte lysate system in the presence of [35S]methionine, and the renal CaBP mRNA translation product was identified and quantitated by specific immunoprecipitation. Total translation products and specific immunoprecipitable products were visualized on sodium dodecyl sulfate-gels, followed by fluorography. After the addition of affinity purified rat renal CaBP antiserum to the 35S-labeled translation products, only one protein band, electrophoretically indistinguishable from that of purified renal CaBP (mol wt, 28,000), was observed. When 10 micrograms purified renal CaBP were added to the translation product before addition of the antiserum, immunoprecipitation of the 35S-labeled 28,000 mol wt protein was not observed. A comparison of the peptides produced after limited digestion with trypsin of 125I-labeled CaBP and [3H]tyrosine-labeled translation product indicated a good coincidence of peaks from purified 125I-labeled CaBP and the immunoprecipitated translation product, suggesting that the immunoprecipitated translation product is indeed vitamin D-dependent renal CaBP. When 100 ng 1,25-(OH)2D3 were injected for 7 days to 8-week-old vitamin D-deficient rats, there was a 4-fold increase in CaBP mRNA levels in the kidney (quantitated by densitometry of immunoprecipitates analyzed on sodium dodecyl sulfate-polyacrylamide gels). This increase in mRNA was accompanied by a corresponding increase in the concentration of renal CaBP, as measured by RIA, thus establishing a bridge between CaBP and the putative transcriptional effect of 1,25-(OH)2D3 in rat kidney. Similarly, both the concentration of renal CaBP and renal CaBP mRNA levels increased 4-fold in rats fed low phosphorus diets, increased 2-fold in rats fed low calcium diets, and decreased 67% in rats fed low sodium diets, providing evidence that the nutritional induction or decrease in renal CaBP is accompanied by a corresponding alteration in the concentration of its specific translatable mRNA. These results are consistent with a transcriptional control mechanism for the induction of renal CaBP.

Reduction of rat hippocampal calcium-binding protein following commissural, amygdala, septal, perforant path, and olfactory bulb kindling.

The calcium-binding protein (CaBP) content of the hippocampal formation was determined by radioimmunoassay in control and kindled rats. Kindling of a number of different sites resulted in a reduction in the CaBP content of the hippocampal formation, which was shown immunohistochemically to be restricted to the dentate granule cells and their processes. The maximum decline in CaBP varied with the different kindling sites: perforant path, 33%; commissural path, 32%; septum, 30%; amygdala, 18%; and olfactory bulbs, 15%. There were no changes in the CaBP content of the stimulated areas themselves. In cases where the kindling stimulus was delivered unilaterally (perforant path and amygdala), the maximum decrease in hippocampal CaBP was observed ipsilateral to the site of stimulation when the criterion for full kindling was established (six consecutive stage 5 motor seizures). Further kindling trials were required to produce a similar magnitude decrease in the CaBP content of the contralateral hippocampus. These observations are discussed both in relation to the possible role of CaBP in the establishment of a seizure response to kindling and also as a potential compensatory mechanism that may serve to overcome the epileptogenic effects of kindling.

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.

Vitamin D-dependent rat renal calcium-binding protein: development of a radioimmunoassay, tissue distribution, and immunologic identification.

A sensitive double antibody RIA has been developed for the 28,000 mol wt rat renal vitamin D-dependent calcium-binding protein. Using this assay, concentrations of calcium-binding protein (CaBP) as low as 30 ng can be measured. The assay is precise (intraassay variability, 5.0%) and reproductible (interassay variability, 8.2%). Measurements of renal CaBP by RIA showed a good correlation with measurements of CaBP by the chelex resin assay and by polyacrylamide gel analysis by densitometric tracing using a purified CaBP marker. The concentration of CaBP in the vitamin D-replete rat kidney is 7.3 +/- 1.0 (mean +/- SEM) micrograms/mg protein. In vitamin D-deficient rats the level of renal CaBP is 2.6 +/- 0.3 micrograms/mg protein. Tissue distribution of immunoreactive rat renal CaBP showed the highest concentration of CaBP in the rat cerebellum (38.3 +/- 5.1 micrograms/mg protein). Lower concentrations of immunoreactive CaBP were detected in several other rat tissues. No immunoreactive CaBP was detected in rat or human serum. In necropsy human kidney and cerebellum, high levels of immunoreactive CaBP were also detected (1.5 +/- 0.1 and 27.3 +/- 2.1 micrograms/mg protein, respectively). When extracts of rat kidney and brain and human cerebellum and kidney were assayed at several dilutions, immunodisplacement curves parallel to that of pure renal CaBP were observed, indicating immunochemical similarity. Fractionation of extracts of rat cerebellum, human kidney, and human cerebellum on Sephadex G-100 revealed immunoreactivity and calcium-binding activity in the 28,000 mol wt region similar to rat kidney.

Pancreatic vitamin D-dependent calcium binding protein: Biochemical properties and response to vitamin D

The biochemical properties of a chick pancreatic calcium binding protein (CaBP) and its response to vitamin D status and dietary calcium and phosphorus levels were studied and compared with the known vitamin D-dependent CaBPs present in the chick intestine and kidney. Pancreatic CaBP is homologous to the intestinal CaBP on the basis of immunological cross-reactivity, molecular size (28,200 Da), and charge properties (chromatographic mobility on DEAE-Sephadex in the presence of either EDTA or Ca 2+). Pancreatic levels of CaBP respond to changes in vitamin D status and dietary Ca and P level in a fashion similar to the intestinal CaBP. Thus, in the absence of dietary vitamin D, both pancreatic and intestinal CaBPs were essentially undetectable, while in the presence of dietary vitamin D, a low dietary P (0.05%) elevated the pancreatic and intestinal CaBP 1.5× and 1.6×, respectively, compared to the CaBP levels present with normal dietary Ca and P (1.0%, 1.0%). The tissue levels of pancreatic CaBP (6–10 ng/mg protein) are about 0.2% of the intestine (5000 ng/mg protein) and 1% of the kidney CaBP (700 ng/mg protein). However, when corrections are made for the CaBP distribution in the tissues and expressed as CaBP concentration per CaBP-containing cells, the pancreatic CaBP level was 30% of the intestine and 10% of the kidney. Collectively, these results suggest that the chick pancreatic vitamin D-dependent CaBP is a homologous protein to the intestinal CaBP, both with regards to its relative cellular concentration as well as in its response to changing dietary levels of Ca and P.

Vitamin D-dependent calcium-binding protein in rat kidney. Purification and physiocochemical and immunological characterization.

A vitamin D-dependent Mr = 28,000 calcium-binding protein (CaBP) has been isolated from rat kidney. Rat renal CaBP was purified from heat-treated post-mitochondrial supernatants by gel filtration on Sephadex G-100 followed by preparative gel electrophoresis. The specific properties and characteristics of the protein were examined. Rat renal CaBP was found to have a pI of 4.8 and showed increased electrophoretic mobility during polyacrylamide gel electrophoresis in the presence of 1 mM EDTA. Amino acid analysis of renal CaBP revealed a high content of glutamic and aspartic acids and a low level of methionine, histidine, cysteine, and tyrosine, similar but not identical to chick intestinal CaBP. Circular dichroism studies indicated that the alpha-helical content of renal CaBP was of the order of 31% and was changed to a minor degree by the addition of calcium. A study of the thermal stability indicated that renal CaBP is heat-stable up to 75 degrees C. Binding studies utilizing the technique of equilibrium dialysis established a dissociation constant of 2.1 X 10(-6) M and binding sites with a capacity of approximately 4 mol Ca2+/mol of CaBP. Immunologically, using Ouchterlony immunodiffusion, a precipitin line which joined with total coalescence with that due to purified renal CaBP was observed with extracts of rat brain and kidney but not with extracts of rat parathyroid, skeletal muscle, myocardium, bone, pancreas, intestine, and liver. In summary, these studies present the first purification and characterization of vitamin D-dependent rat renal calcium-binding protein. The antibody as well as the protein will be useful for the development of a radioimmunoassay and for the determination of the molecular mechanisms of induction of renal calcium-binding protein.

Effects of long-term vitamin D deficiency and response to vitamin D repletion in the mature and aging male and female rat

This study investigated the sex- and age-related alterations in calcium homeostasis in 39- to 82-week-old rats raised from weaning on a vitamin D deficient (−D) diet. It was found that vitamin D deprivation decreased the life span of male, but not female, rats. Female −D animals exhibited a study increase in serum calcium with age of 39 to 82 weeks, although circulating calcium of −D animals never reached normocalcemic levels. There was no attenuation of the secondary hyperparathyroidism. Serum calcium of −D males was significantly lower than that of age-matched females at all ages when sufficient males were alive to make the comparison. Serum parathyroid hormone levels were decreased in −D females when serum clacium was elevated to hypercalcemic levels by calcium injection. Similarly, administration of vitamin D 3 or 1,25-dihydroxyvitamin D 3 elevated serum calcium and depressed parathyroid hormone in 14- and 22-month-old −D females. These animals also exhibited increased intestinal calcium binding protein content. Administration of vitamin D 3 or dihydroxyvitamin D 3 repaired renal adenylate cyclase refractoriness to parathyroid hormone. The sex- and diet-related alterations in serum phosphorus that were found at earlier ages disappeared by 67 weeks of age. Serum calcitonin was elevated in mature and aging +D males and females and −D females relative to younger animals. In −D males, calcitonin levels were less markedly elevated. The results of this study indicate that there are several important sex differences related to the regulation of calcium homeostasis in mature and aging rats. In addition, it was found that mature (14 month) and old (22 month) chronically −D female rats were able to respond to repletion with dihydroxyvitamin D 3 or vitamin D within 10 days.

Biochemical and immunohistochemical correlates of kindling-induced epilepsy: role of calcium binding protein

The levels and distribution of calcium binding protein (CaBP) were examined in the central nervous system of rats exhibiting kindling-induced epilepsy. Following commissural kindling, the concentration of CaBP in the hippocampal formation was significantly reduced but no change was observed in levels of total soluble protein or calmodulin, another calcium-related protein. Histochemical examination of kindled animals revealed a localized depletion of CaBP-like immunoreactivity restricted to the dentate granule cell-mossy fiber system of the hippocampal formation. These data demonstrate a correlation between the loss of CaBP from dentate granule cells and kindling-induced epilepsy, the underlying mechanism of which may involve an impairment of granule cells to regulate their intracellular calcium environment when challenged with high frequency electrical stimulation.

Hydrocortisone and 1,25(OH)2D3: role in proliferative responses of the embryonic chick duodenum in organ culture.

The present studies were conducted to determine if hydrocortisone (HC) and/or 1,25-dihydroxycholecalciferol (1,25(OH)2D3) alter proliferative responses of the cultured duodenum, and if such alterations could be related to the known augmentation of 1,25(OH)2D3-induction of a specific, intestinal calcium-binding protein (CaBP) by glucocorticoids. HC stimulated proliferation in the duodenal epithelium, as indicated by increased DNA synthesis (3H-thymidine uptake), increased cell number/villus, and increased mitotic index after colchicine treatment. Goblet cell numbers were not significantly increased with any hormone treatment. The presence of 1,25(OH)2D3 alone did not affect proliferative responses. CaBP concentration as a function of tissue weight was 50% greater in HC stimulated intestine, indicating that the proliferogenic action of HC on the duodenum alone could not account for the glucocorticoid-1,25(OH)2D3 interaction in CaBP synthesis.

Studies of calcium-binding protein in cattle.

We have developed and validated a double-antibody radioimmunoassay for quantifying bovine calcium-binding protein (CaBP). Cross-reactivity between the antiserum and microgram quantities of thyrocalcitonin, calmodulin, gastrin, cholecystokinin, vasoactive intestinal polypeptide, serum albumin and concentrated extract of bovine pituitary gland was insignificant. Slight cross-reactivity (6%) of the antiserum with parathyroid hormone was demonstrated. Assay sensitivity was .25 ng/ml and intraassay and interassay coefficients of variation ranged from 4 to 11% and 10 to 24%, respectively. The CaBP immunoreactivity was not affected by endogenous Ca concentrations. Plasma and serum concentrations of immunoreactive CaBP were similar. The CaBP concentrations were unaffected when coagulated and anticoagulated blood samples were stored at 4 or 22 C for up to 72 h and when serum was stored at -20, 4 or 22 C for 8 d. Serum CaBP concentrations in cattle were not affected by gonadal steroids, but may have been influenced by age. Treatment with 500 mg of vitamin D3, but not 50 mg of dihydrotachysterol, significantly increased serum Ca and CaBP concentrations in Holstein heifers after a lag period of 7 to 10 d. Serum Ca and CaBP concentrations began to increase in serum at approximately the same time and both exhibited parallel responses to treatment with vitamin D3. Serum Ca concentrations were positively correlated (r = .81) with CaBP concentrations and this relationship was described by the equation, Y = 6.85 + 1.01X - .03X2. Serum Ca and CaBP concentrations were still elevated in heifers 75 d after initial treatment with vitamin D3. The radioimmunoassay we describe provides an opportunity to investigate the role of CaBP in Ca homeostasis during growth, pregnancy, lactation, parturient paresis and other physiological and pathological states in cattle.