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.     

Intestinal calcium-binding protein

Summary The vitamin D-dependent calcium-binding protein (CaBP) was studied in relation to the age of the cell, in isolated epithelial cell populations removed from rat duodenum. Alkaline phosphatase and thymidine kinase activities were used as markers to characterize differentiated villus cells and undifferentiated (mitotically active) crypt cells, respectively. CaBP distribution along the length of the villus, as established by radioimmunoassay, appears as a gradient increasing from the crypt to the tip of the villus. CaBP concentration in cells is shown to be (i) negatively correlated with the thymidine kinase activity of cells, and (ii) positively correlated with the alkaline phosphatase activity of cells. This indicates that CaBP is absent in crypt cells and appears in differentiated cells with the development of the brush border. Thus CaBP, like alkaline phosphatase, can be considered as an indicator of enterocyte maturation. These data were also confirmed by studying the cellular localization of the protein. In addition both indirect immunofluorescence and immunoperoxidase staining methods reveal that antibody against CaBP decorates the terminal web, but not the microvilli of the brush border of mature absorptive cells. The results suggest that CaBP may act as a modulator of some Ca²⁺-mediated biochemical processes at the level of the enterocyte brush border.Portions of this work were presented at the Fourth International Workshop on Calcified Tissues, Israel (March 1980)     

Distribution of Ca2+-ATPase, ATP-dependent Ca2+-transport, calmodulin and vitamin D-dependent Ca2+-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 Ca2+-ATPase activity and ATP-dependent Ca2+-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 Ca2+-transport in basolateral membranes was highest in fraction II (8.2 +/- 0.3 nmol Ca2+/min per mg protein) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca2+-transport activity (0.9 +/- 0.1 nmol Ca2+/min per mg protein). The distribution of high-affinity Ca2+-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca2+-transport. The activity of Na+/Ca2+ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP-dependent Ca2+-transport system, the Na+/Ca2+ 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.     

Distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase and alkaline phosphatase activities in isolated ileal epithelial cells of fed, fasted, cholestyramine-fed, and 4-aminopyrazolo[3,4-d]pyrimidine-treated rats.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, E.C. 1.1.1.34), the major rate-limiting enzyme of the sterol biosynthetic pathway, was studied in ileal epithelial cells isolated in a villus-to-crypt gradient according to Weiser (Weiser, M. M. 1973. J. Biol. Chem, 248:2536-2541). Alkaline phosphatase (E.C. 3.1.3.1) served as a marker for the mature villus cells. Protease effects on activity determinations were negligible. The intracellular location of HMG-CoA reductase could not be precisely determined. The activity of ileal reductase was predominantly associated with the less differentiated lower villus and crypt cells, while the reverse gradient occurred with alkaline phosphatase. This distribution of enzymes persisted in both fed and fasted rats injected with control saline-phosphate, although fasting decreased total reductase units in the ileum by 86% in 72 hr. Treatment with cholestyramine and with 4-aminopyrazolo[3,4-d]pyrimidine (APP) enhanced reductase activity in ileal cells. The percent stimulation in both cases was higher in the upper villus cells than in the crypt cells, leading to abolition of the gradient in enzyme activity. However, APP treatment caused a 98% loss in total alkaline phosphatase units and a 55% loss in total epithelial cell protein in 72 hr. Thus, there was no increase in total reductase units. These data show that APP affects ileal cell metabolism directly. Furthermore, it appears that the regulation of sterol synthesis in the intestinal mucosa, via HMG-CoA reductase, involves a complex interplay of the effects exerted by the level of alimentation, the enterohepatic circulation of bile, and the levels of plasma lipoproteins.     

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.     

Independence of 1,25-dihydroxyvitamin D3-mediated calcium transport from de novo RNA and protein synthesis

The administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to rachitic chicks produces an increase in (a) RNA and protein synthesis, (b) calcium binding protein (CaBP) concentration, and (c) alkaline phosphatase activity in the duodenum. These events occur concomitantly with enhanced calcium transport. We inhibited RNA and protein synthesis in richitic chicks and measured the subsequent response to 1,25(OH)2D3. Actinomycin D, injected prior to and following 1,25(OH)2D3 administration, inhibited intestinal RNA polymerase activity, blocked the rise in serum calcium, reduced the amount of CaBP, and increased alkaline phosphatase activity. Cycloheximide injected in similar fashion, inhibited the 1,25(OH)2D3-mediated increase in intestinal protein synthesis, serum calcium, CaBP, and alkaline phosphatase activity. Neither inhibitor blocked the ability of 1,25(OH)2D3 to stimulate calcium transport as measured in isolated duodenal loops in vivo. The ability of either inhibitor to block 1,25(OH)2D3-mediated calcium transport despite inhibition of CaBP production and alkaline phosphatase activity (by cycloheximide) indicates that de novo RNA and protein synthesis, and in particular CaBP and alkaline phosphatase, are not required for the 1,25(OH)2D3 stimulation of calcium transport.     

The effect of vitamin D3 metabolites on membrane proteins of chick duodenal brush borders.

Chick intestinal brush border proteins were examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate. Following injection of 25-hydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₃, a large molecular weight protein present in the vitamin D-deficient brush borders diminishes and a larger protein appears. This change occurs before calcium binding protein can be detected by Chelex assay and prior to the increase in total alkaline phosphatase but correlates closely with increased intestinal calcium absorption in response to the metabolites. The two brush border proteins have been solubilized with n-butanol and partially characterized. The vitamin D-deficient protein has a molecular weight of about 200,000 and has alkaline phosphatase activity but no detectable calcium binding activity. The protein which appears in response to metabolites has a molecular weight of 230,000, binds calcium, and also has alkaline phosphatase activity.     

Distribution of Ca-ATPase, ATP-dependent Ca-transport, calmodulin and vitamin D-dependent Ca-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 Ca²⁺-ATPase activity and ATP-dependent Ca²⁺-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 Ca²⁺-transport in fraction II (8.2 ± 0.3 nmol Ca²⁺/min per mg protein) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca²⁺-transport activity (0.9 ± 0.1 nmol Ca²⁺/min per mg protein). The distribution of high-affinity Ca²⁺-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca²⁺-transport. The activity of Na⁺/Ca²⁺ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP-dependent Ca²⁺-transport system, the Na⁺/Ca²⁺ 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.     

RNA synthesis by villus and crypt cell nuclei of rat intestinal mucosa

Rat intestinal mucosa was separated by eversion and vibration to provide a sequence of fractions from predominantly villus cells to predominantly crypt cells. The proportions of these cell types in each fraction were computed from the concentrations of alkaline phosphatase (villus cells) and thymidine kinase (crypt cells) in each population. The isolated mucosal fractions varied from about 90% villus cells to 90% crypt cells. Following injection of the rats with [³H]thymidine, the nuclei were isolated from each mucosal cell fraction and the amount of radioactivity incorporated into DNA was measured as an index of crypt cell abundance. The isolated nuclei were also incubated with ribonucleoside triphosphates and the amount of RNA synthesized was measured. Nuclei labeled with [³H]thymidine were found only in fractions rich in crypt cells, whereas capacity for RNA synthesis remained very active in mucosal fractions consisting predominantly of villus cells. It is concluded that non-dividing villus cells continue to make RNA.     

Time dependency of 1,25(OH)2D3 induction of calbindin mRNA and calbindin expression in chick enterocytes during their differentiation along the crypt-villus axis

Quantitative methods of in situ hybridization and immunocytochemistry have been used to measure 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) induction of calbindin mRNA and calbindin protein expressed in jejunal enterocytes at all points along the crypt-villus axis over a 24 h period. Small amounts of calbindin mRNA detected in vitamin D3 deficient (D-deficient) chick intestine increased rapidly to maximal values 8 h after hormone injection. The magnitude of this response was inversely related to age of enterocyte measured separately by injecting tritiated thymidine into D-deficient and 1,25(OH)2D3-injected birds. Enterocytes of all ages expressed small amounts of calbindin 3 h after hormone injection. This amount of calbindin then increased up to 24 h after hormone injection. Maximal calbindin expression took place in basal villus enterocytes. Later decrease in the ability of upper villus enterocytes to express calbindin was associated with a similar fall in calbindin mRNA expression. Previously it was suggested that inefficient translation to calbindin mRNA might take place in basal villus enterocytes 48 h after vitamin D injection. Present work using 1,25 (OH)2D3 shows that calbindin expression takes place at a constant rate during this early stage of enterocyte development. Secondary events limiting higher rates of calbindin synthesis in upper crypt and basal villus enterocytes remain to be identified.     

Distribution of 3-hydroxy-3-methylglutaryl-CoA reductase in isolated villus and crypt cells of chick duodenum, jejunum and ileum

3-Hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34), the major rate-limiting enzyme of cholesterogenesis, was studied in epithelial cells isolated in a villus to crypt gradient from chick duodenum, jejunum and ileum, in order to resolve the apparent controversy that exists on the anatomical localization of sterol synthesis in the intestine. Consistent separation was demonstrated by using the marker enzymes alkaline phosphatase, specific to the villus cells, and thymidine kinase, specific to the crypt cells. No relative difference in stability was observed, as shown by the equal distribution of acid phosphatase. Cells were 90-95 per cent viable. The highest specific activity of reductase was located in the microsomal fraction (41 per cent of the total). The mitochondria had lower specific activity (8 per cent of the total). The distribution of reductase activity in epithelial cells of the villus-crypt axis was also studied. The specific activity in each cell fraction from chick duodenum was clearly lower than that in jejunum and ileum. The jejunal and ileal crypt regions showed lower specific activity than the villus cells. About 70 per cent of total reductase activity was found in cells from the upper and the mid villus fraction in each intestinal segment.     

Molecular mechanisms involved in the enhancement of mitochondrial malate dehydrogenase activity by calcitriol in chick intestine

Mitochondrial malate dehydrogenase (mMDH) from the intestine is the NAD-linked oxidoreductase of the tricarboxylic acid cycle with the highest activity and response to vitamin D treatment in vitamin D-deficient chicks (?D). The aim of this study was to elucidate potential molecular mechanisms by which cholecalciferol or calcitriol enhances the activity of this enzyme. One group of animals used was composed of ?D and ?D treated with cholecalciferol or with calcitriol. A second group consisted of ?D and ?D supplemented with high Ca²⁺ diet. A third group included chicks receiving either a normal or a low Ca²⁺ diet. In some experiments, animals were injected with cycloheximide. Data showed that either vitamin D (cholecalciferol or calcitriol) or a low Ca²⁺ diet increases mMDH activity. High Ca²⁺ diet did not modify the intestinal mMDH activity from ?D. The mMDH activity from ?D remained unaltered when duodenal cells were exposed to 10^?8 mol/L calcitriol for 15 min. The enhancement of mMDH activity by calcitriol was completely abolished by simultaneous cycloheximide injection to ?D. mMDH mRNA levels, detected by RT-PCR, indicate that calcitriol did not affect gene expression. In contrast, Western blots show that calcitriol enhanced the protein expression. In conclusion, calcitriol stimulates intestinal mMDH activity by increasing protein synthesis. No response of mMDH activity by rapid effects of calcitriol or activation through increment of serum Ca²⁺ was demonstrated. Consequently, ATP production would be increased, facilitating the Ca²⁺ exit from the enterocytes via the Ca²⁺-ATPase and Na⁺/Ca²⁺ exchanger, which participate in the intestinal Ca²⁺ absorption.     

Intestinal basolateral membrane Ca-ATPase activity with properties distinct from those of the Ca-pump

The Ca-pump in rat intestinal basolateral membranes had been studied previously as vesicular ATP-dependent Ca-uptake. In the present studies, Ca-stimulated ATP hydrolysis (Ca-ATPase activity) was measured and found to differ from the Ca-pump in having higher activity and being insensitive to vanadate. Whereas the pump was specific for ATP, hydrolytic activity was found with ATP, GTP or ADP but not with AMP or p-nitro-phenyl-phosphate. In contrast to Ca-pump activity, Ca-ATPase activities were similar for different intestinal segments, for duodenal villus/crypt cell-fractions and for vitamin D-deficient animals. Thus, as usually measured, intestinal basolateral membrane Ca-ATPase activity is not equivalent to the Ca-pump.     

Hydrocortisone and vitamin D3 stimulation of 32Pi-phosphate accumulation by organ-cultured chick embryo duodenum.

Either vitamin D3 (or 1 alpha,25--(OH)2-D3) or hydrocortisone (HC) stimulated phosphate accumulation by organ-cultured embryonic chick duodenum. In combination, these two steroids stimulated phosphate uptake synergistically. Phosphate accumulation appeared to be independent of other vitamin D3-stimulated processes: CaBP concentration, cAMP concentration, or alkaline phosphatase activity. L-phenylalanine, a reported alkaline phosphatase inhibitor, when added to the culture medium progressively inhibited either D3- or HC-stimulated phosphate uptake subsequent to culture, but did not inhibit the synergistic action. Under these conditions L-phenylalanine had no consistent effect on alkaline phosphatase activity but unexpectedly, greatly inhibited vitamin D3-stimulated CaBP concentration, but only in the absence of HC. Some limited suggestion of an intestinal phosphoprotein sensitive to either vitamin D3 or HC was observed.     

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.     

Vitamin D-inducible calcium transport and gene expression in three Caco-2 cell lines

The parental cell line (P) of Caco-2 cells and two clones, BBe and TC7, were studied at 11 days postconfluence to test the facilitated diffusion model of vitamin D-mediated intestinal calcium absorption (CaTx). Nuclear vitamin D receptor (nVDR) and calbindin D(9k) (CaBP) were measured by Western blot; 1,25-hydroxyvitamin D(3) 24-hydroxylase (CYP24), CaBP, plasma membrane Ca-ATPase (PMCA), and Ca transport channel (CaT1) mRNA levels were examined by RT-PCR; and net apical-to-basolateral CaTx was examined after treating cells with vehicle or 10 nM calcitriol for 8 (mRNA levels) or 48 h (protein, CaBP mRNA, CaTx). nVDR level was lowest in BBe (38% P value) and directly related to CYP24 induction (TC7 = P, which were 1.56 times greater than BBe). nVDR was inversely related to the vitamin D-induced levels of CaT1 mRNA, CaBP mRNA, PMCA mRNA, and net CaTx, with the highest induction seen in BBe. Basal CaBP mRNA (86 times greater than P) and protein levels were highest in TC7 cells and were not associated with higher net CaTx, suggesting CaBP may not be rate limiting for CaTx in these cells.     

Intestinal enzymes activities in isolated villus and crypt cells during postnatal development of the rat

Summary A modification of Weiser's (1973) cell isolation method was used in order to study the developmental pattern of various intestinal enzyme activities in villus and crypt cells of normal rats from 5 days after birth until 8 weeks. Alkaline phosphatase and enterokinase activities were always located in the upper villus zone during postnatal development. Enterokinase activity was higher in the upper villus cells during the third week of life than after this period. Aminopeptidase activity was located in the crypt cells during the first week, its maximum activity remained in this area until the third week. At this time, sucrase activity appeared in the crypt cells, then aminopeptidase and sucrase activities rose to the villus zone during the fourth week. Amylase activity was detected along the entire crypt-villus axis 5 days after birth, reaching maximum activity in crypt cells at the end of the first week and in the upper villus cells after the fourth week. In contrast with the other enzymes studied almost all amylase activity was soluble in the youngest animals whereas at weaning most of the activity appeared in a particulate form in the villus cells. But in the crypt cells the ratio between particulate and soluble form remained unchanged until the adult stage. Various hypotheses are advanced to explain the patterns of evolution of the different enzymes.     

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.