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)     

Intestinal vitamin D-induced calcium-binding protein: time-course of immunocytological localization following 1,25-dihydroxyvitamin D3

The vitamin D-induced calcium-binding protein (CaBP) was localized in histological sections of chick duodenum using the peroxidase-antiperoxidase immunocytochemical technique. The time-course of appearance of CaBP in rachitic chicks was investigated from 0 to 120 hr after stimulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). CaBP was not routinely detected at 0 hr after 1,25(OH)2D3 administration. CaBP was first noted in some, but not all, of the samples taken 2 hr following 1,25(OH)2D3 and was detected in all 2 1/2 hr samples. The number of CaBP-containing absorptive cells and the apparent CaBP concentration both increased to a maximum at about 16-24 hr. At later times, as CaBP free cells migrated up the villi, the CaBP-containing cells decreased in number, but even at 120 hr post 1,25(OH)2D3 dose there were significant numbers of CaBP-containing cells present. The relationships between time-course of CaBP location on intestinal villi, enterocyte migration rates, and the time-course of 1,25(OH)2D3 stimulated intestinal calcium transport are discussed.     

CHANGES IN GROWTH KINETICS OF JEJUNAL EPITHELIUM IN MICE MAINTAINED ON AN ELEMENTAL DIET

Changes in the kinetics of the intestinal epithelium were observed in mice maintained on an elemental diet containing hydrolysed protein and medium chain triglycerides. An increase in the length of the villi seen shortly after commencement of the diet was followed by a reduction in the rate of proliferation in the crypt. After 7 days on the diet, an equilibrium state was reached with the cellularity of the villi being 120% that of control while the number of proliferative cells/crypt was reduced by 35%. The proliferative response of the crypt following irradiation occurrred 16 hr later in diet-fed mice than in controls. It was postulated that, because of the increased cellularity of the villus compartment in diet-fed mice, additional time was required to reduce the number of villus cells to a critical level at which a proliferative response is induced in the crypt.     

Changes in growth kinetics of jejunal epithelium in mice maintained on an elemental diet.

Changes in the kinetics of the intestinal epithelium were observed in mice maintained on an elemental diet containing hydrolysed protein and medium chain triglycerides. An increase in the length of the villi seen shortly after commencement of the diet was followed by a reduction in the rate of proliferation in the crypt. After 7 days on the diet, an equilibrium state was reached with the cellularity of the villi being 120% that of control while the number of proliferative cells/crypt was reduced by 35%. The proliferative response of the crypt following irradiation occurred 16 hr later in diet-fed mice than in controls. It was postulated that, because of the increased cellularity of the villus compartment in diet-fed mice, additional time was required to reduce the number of villus cells to a critical level at which a proliferative response is induced in the crypt.     

The influence of various cell kinetic conditions on functional differentiation in the small intestine of the rat. A study of enzymes bound to subcellular organelles

The process of cell maturation and cell ageing of absorptive epithelial cells was investigated in normal rat duodenum. The development of a number of enzymes bound to subcellular organelles was studied by using microchemical analyses on various cell compartments dissected from crypts and villi from freeze-dried cryostat sections. The development of the ultrastructural features of the absorptive epithelium was investigated by electron microscopy of various cell positions along the whole length of the crypt and the base of the villus. The data obtained were related to cell position along the crypt and villus and to cell age during migration from the bottom of the crypt to the tip of the villus.The influence of changes in the life-span of the cells and of increasing proliferative activity was studied by comparing normal rat duodenum with that from germfree rats and rats recovering from low radiation doses (72 hr after 400 R).Our data show that the specific activity of nonspecific esterases mainly localized in the endoplasmic reticulum increases when the cells migrate along the upper half of the crypt and the basal part of the villus. Activity of alkaline phosphatase, measured as a marker for the microvilli, is absent in the crypt, but increases linearly from the base of the villus to the tip. The longer life-span of villus cells in germfree animals does not result in a higher activity of these enzymes than in normal animals. An increased proliferative activity in the crypt, as present 72 hr after X-irradiation, is accompanied by a decreased activity of both enzymes but the pattern of activity during cell migration remains the same. The specific activity of enzymes bound to mitochondria or lysosomes (monoamineoxidase and β-N-acetylglucosaminidase) are not affected by changing crypt cell kinetics.Electrophoretic analyses of isolated cell compartments showed that the increase during normal differentiation or the decrease after X-irradiation of esterase activity is due to changes in overall activity, not to the appearance or disappearance of specific isoenzymes. Electron microscopy showed that in the normal intestine there is a gradual development of ultrastructural features during migration of the cell along the crypt while the most drastic changes in cell structure occur at the moment the cell enters the villus. Contrary to our expectation, the ultrastructural development was not influenced by increased proliferative activity in the crypt 72 hr after irradiation, and hence the decrease in enzyme activity found cannot be related to changes in ultrastructure.     

Effects of vitamin restriction and supplementation on rat intestinal epithelial cell apoptosis

Diet influences intestinal growth and function and vitamins modulate intestinal cell turnover. We have assessed the effects of chronic, moderate (50% of control) vitamin restriction and supplementation on intestinal epithelial cell (IEC) apoptosis and the relevance of this to alterations in tissue oxidative stress and antioxidant status. Feeding a vitamin-restricted diet to male, weanling WNIN rats for 20 weeks significantly increased IEC apoptosis, but only in the villi region, as evident from increased annexin V staining, M30 positivity, histological observations, DNA ladder formation, and reduced expression of Bcl-2. This was associated with elevated levels of lipid peroxides and protein carbonyls in the intestinal mucosa despite the increased activities of superoxide dismutase, catalase, and glutathione peroxidase. Consistent with the increased oxidative stress and apoptosis, structural and functional integrity of the villi were compromised as evident from the lowered ratio of villus height:crypt depth and the decreased activities of the membrane marker enzymes alkaline phosphatase and Lys-Ala dipeptidyl aminopeptidase. These changes were reversed by supplementation with a vitamin mixture or vitamin E alone, whereas riboflavin or folic acid supplementation reduced the apoptotic rates, but only partially. Further, oxidative stress was the least in vitamin E- or vitamin mixture-supplemented rats and correlated well with their IEC apoptotic rates. Increased tissue oxidative stress seems to mediate the vitamin-restriction-induced apoptosis of the IECs in rats.     

Development of the bovine ileal mucosa

The development of the bovine ileal mucosa was studied with particular reference to maturation during the fetal and neonatal period. In this region, by 4-5 months of fetal development, vacuolation of the epithelial cells had occurred on the villi, and the goblet and absorptive cells in the crypts were present. By 6-9 months, the villi were longer and more numerous than in the previous stages. At the same time, the vacuolated cells could be seen predominantly on the upper half of each villus. The absorptive cells and goblet cells were more distinct in the crypt and lower half of each villus. Moreover, the goblet cells showed differences in mucin, while in the submucosa the lymphoid follicles were seen to have enlarged to become a prominent feature of the Peyer's patches at this stage. At birth, in suckled animals, the ileal cells on the lower area of each villus and in the crypt appeared more like mature cells. In contrast, there were numerous inclusion bodies in epithelial cells on the upper half of each villus. They appeared in the apical portion of the cytoplasm as vacuoles with stainable or dense contents. By 1 week, however, epithelial cells no longer contained inclusion bodies, and absorptive and goblet cell populations had begun to emerge from the crypts. These histological results suggest that the bovine ileal mucosa has two distinct turning points during its development in the fetus and the neonate. Initially all the mucosal structures are present in fetuses at 6-7 months of gestation, and then the vacuolated cells covering the ileal villi are replaced by mature, nonpinocytosing epithelium which emerges from the crypts on or before the 7th day after birth (ileal closure).     

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.     

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.     

Diet, mucosal architecture and epithelial cell production in the small intestine of specified-pathogen-free and conventional rats.

Upper jejunum and terminal ileum were examined in specified-pathogen-free (SPF), conventional and conventional after SPF rearing (ex-SPF) rats. The effect of 2 differential diets on the last 2 groups was examined. Ex-SPF rats had taller villi and deeper crypts than SPF rats, but similar crypt to villus ratios and cell production rates. Ex-SPF rats had similar crypt depth and jejunal villus height to conventional rats on the same diet, but taller ileal villi and a lower cell production rate. Even after 6-8 weeks, in a conventional environment, ex-SPF rat intestine was still not identical with conventional rat intestine. Diet had a significant effect on mucosal architecture, and a smaller effect on cell production rate. It is concluded that diet, microbiological status of colony of origin, and environment after weaning, can all affect mucosal architecture and epithelial cell production, and should be properly controlled in experimental studies.     

Modulation of xanthine dehydrogenase and oxidase activities during the hormonal induction of vaginal epithelial differentiation in ovariectomized mice

In situ hybridization and immunocytochemical techniques have been used to examine the distribution of vitamin-D-induced calbindin mRNA and calbindin protein in enterocytes lining the crypts and villi of chicken small intestine. Basal villus enterocytes contained approximately twice as much calbindin but over three times as much calbindin mRNA compared to values found in basal crypt and upper villus enterocytes, all values being measured 2 days after vitamin D injection into D-deficient chickens. Virtually no calbindin mRNA was detected in tissues taken from control D-deficient birds. Direct proportionality found between calbindin mRNA and calbindin content in enterocytes of basal crypt, mid and upper villus suggests pre-translational control over calbindin synthesis. The implications of possible inefficient translation of calbindin mRNA in basal villus enterocytes are discussed. Present methods of analysis provide a novel way to study mechanisms controlling gene expression throughout the whole process of enterocyte differentiation.     

Intestinal calcium-binding protein in animals fed normal and rachitogenic diets: I. Rat studies.

Measurements were made of duodenal calcium-binding protein (CaBP) on rats during development of rickets and, subsequently, following vitamin-D2 treatment. Results showed a poor inverse correlation between duodenal CaBP and rickets. In rats fed a phosphate-deficient rachitogenic diet, duodenal CaBP concentration finally fell below detectable limits, but CaBP was still readily measurable 2 weeks after rickets was clearly established. Following a massive dose of vitamin D2 (50 000 I.U.) to rachitic animals, CaBP was formed. However, a small dose of vitamin D2 (500 I.U. daily for 4 days) was insufficient to demonstrate CaBP synthesis than vitamin-D treatment alone. The rachitogenic diet supplemented with phosphate, which caused osteoporosis but not rickets, inhibited CaBP synthesis. The results suggest that nutritional deficiencies from the rachitogenic diet, in addition to vitamin-D deficiency, inhibited CaBP synthesis.     

Intestinal villus structure contributes to even shedding of epithelial cells

In the intestinal epithelium, proliferated epithelial cells ascend the crypts and villi and shed at the villus tips into the gut lumen. In this study, we theoretically investigate the roles of the villi on cell turnover. We present a stochastic model that focuses on the duration over which cells migrate the shortest paths between the crypt orifices and the villus tips, where shedding cells are randomly chosen from among those older than the shortest-path cell migration times. By extending the length of the shortest path to delay cell shedding, the finger-like shape of the villus would tightly regulate shedding-cell ages compared with flat surfaces and shorter projections; the villus allows epithelial cells to shed at around the same age, which limits them from shedding early or staying in the epithelium for long periods. Computational simulations of cell dynamics agreed well with the predictions. We also examine various mechanical conditions of cells and confirm that coordinated collective cell migration supports the predictions. These results suggest the important roles of the villi in homeostatic maintenance of the small intestine, and we discuss the applicability of our approach to other tissues with collective cell movement.     

Effects of vitamin D supplementation on calcium balance and bone growth in young rats fed normal or low calcium diet

OBJECTIVE: We examined the effect of vitamin D supplementation on bone growth in young rats fed a normal or low calcium diet. METHODS: Fifty female Sprague-Dawley rats, 6 weeks of age, were randomized by stratified weight method into five groups with 10 rats in each group: baseline control, 0.5% (normal) or 0.1% (low) calcium diet, and 0.5 or 0.1% calcium diet + vitamin D (25 microg/100 g, food intake). Duration of the experiment was 10 weeks. RESULTS: Vitamin D supplementation stimulated intestinal calcium absorption and increased urinary calcium excretion in rats fed a low or normal calcium diet. Vitamin D supplementation prevented the reduction in periosteal bone gain but enhanced enlargement of the marrow cavity and reduced the maturation-related cancellous bone gain in rats fed a low calcium diet, and increased the maturation-related cancellous and cortical bone gains in rats fed a normal calcium diet. CONCLUSION: This study shows the differential effects of vitamin D supplementation on born growth in young rats fed a normal or low calcium diet.     

The kinetics of villus cell populations in the mouse small intestine

Abstract. The cell population kinetics of the villus epithelium of the mouse have been analysed with respect to the size, flux and time. Microdissection methods were employed to measure the villus cell population size and yielded reproducible, precise results. There was a proximodistal negative size gradient in villus cell population and, in those villi of normal morphology, there was a good correlation with the usual morphometric estimators such as height and row count, although correlation was improved by a product variable consisting of a height multiplied by a width parameter.
Flux onto the villus is the product of the crypt cell production rate, which was measured by a metaphase arrest method using vincristine and crypt microdissection, and the crypt:villus ratio; net villus influx was maximum proximally in the bowel, where the largest villi were found, and decreased distally. The distribution of transit times of labelled cells to the crypt: villus junction and to the villus tip was measured, allowing the measurement of the median villus transit time.
Comparison of the measured villus transit time with the theoretical transit time calculated from the villus influx and population size gave results consistent with a steady state hypothesis. It was found, at each level of the small intestine studied, that the number of epithelial cells on the villus was equivalent to the total number of crypt cells associated with the villus.     

Evidence that sodium deprivation influences vitamin D dependent rat renal calcium binding protein

In order to provide some insight concerning the role of renal calcium binding protein (CaBP) in the functioning of the mammalian kidney, the response of renal CaBP to dietary alterations was examined. Three week old rats were fed diets deficient in calcium, phosphorous or sodium supplemented with vitamin D for a four week period. The specific activity of renal CaBP (as measured by the chelex resin assay; Ca²⁺ bound protein/Ca²⁺ bound resin per mg protein) in the 28,000 M_r region was found to increase four fold in rats fed the low phosphorus diet and two fold rats fed the low calcium diet when compared to rats fed the control diet. Renal CaBP/mg protein from rats fed the low sodium diet decreased 50% from the control values. Changes in renal CaBP were confirmed by polyacrylamide gel analysis of the 28,000 M_r fraction by densitometric tracing using a purified CaBP marker. The greater response to dietary phosphorus restriction suggests that renal CaBP may be regulated by a mechanism different from that of intestinal CaBP. The decrease in renal CaBP in rats fed the low sodium diet suggests for the first time that sodium is required for vitamin D dependent distal tubular calcium transport processes.     

1,25-Dihydroxyvitamin D3-induced calcium-binding protein: localization in organ-cultured embryonic chick duodenum

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) induces de novo biosynthesis of a specific calcium-binding protein (CaBP) in embryonic chick duodenum in organ culture. Using a highly sensitive and specific, peroxidase-antiperoxidase immunocytochemical procedure, 1,25(OH)2D3-induced CaBP in the organ-cultured duodenum was found only in the cytoplasm of absorptive cells, corresponding to its localization in rachitic chick duodenal cells after a single injection of 1,25(OH)2D3 in vivo. This observation, along with evidence correlating CaBP with calcium transport, strongly supports the use of the embryonic chick duodenal organ culture system as a physiologically relevant model of the vitamin D-dependent calcium absorptive mechanism.     

In situ detection of vitamin D-induced calcium-binding protein (9-kDa CaBP) messenger RNA in rat duodenum

We have previously described the molecular cloning of a cDNA fragment synthesized from rat duodenal mRNA coding for a 9000-dalton vitamin D-induced calcium-binding protein (9-kDa CaBP) (3). We now report the use of this cloned cDNA to study the cytological distribution of 9-kDa CaBP mRNA in rat duodenum by in situ hybridization. Tissue sections, fixed in ethanol:acetic acid, were hybridized to the 3H-cDNA probe and processed for autoradiography. The specificity of the CaBP mRNA-DNA hybrid formation was checked using 3H-labeled plasmid pBR322 DNA as a control probe. 9k-Da CaBP mRNA, visualized by silver grains, was found only in the absorptive epithelial cells, and the concentration was greater in the cells at the villous tips than in those of the crypts. The 9k-Da CaBP mRNA was observed mainly in the cytoplasm of the columnar cells and less frequently in the nucleus. Labeling was not seen in the brush border and goblet cells. The submucosa, with Brunner's glands and muscularis, also showed no specific 9-kDa CaBP mRNA concentration. This demonstration of 9-kDa CaBP gene activity in the columnar cells of the rat duodenum illustrates the usefulness of in situ hybridization for characterization of specific cells involved in the expression of 1,25(OH)2 D3 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.