Small Intestinal Crypt Cells Research Articles

Alterations in the physical state and composition of brush border membrane lipids of rat enterocytes during differentiation

The physical state of the membrane lipid of brush border membranes, prepared from rat small intestinal villus and crypt cells, was examined by steady-state fluorescence polarization using three lipid-soluble fluorophors. Membranes prepared from crypt cells were found to possess a higher lipid fluidity than those of villus cells with each probe. Analysis of the composition of these membranes revealed that those from crypt cells had lower ratios of cholesterol/phospholipid (mol/mol), protein/lipid ( w w ), and saturated fatty acyl chains/unsaturated chains ( w w ). Alterations in the levels of stearic (18:0) and oleic (18:1) acids were responsible for differences in the latter ratio. The results, therefore, demonstrate that alterations in the lipid composition and fluidity of brush border membranes of enterocytes occur during the process of differentiation.

Glucocorticoid receptors in isolated intestinal epithelial cells in rats.

There is evidence that glucocorticoid hormones influence a variety of epithelial cell functions in the small intestine. To exert their effect, glucocorticoids bind to cytoplasmic glucocorticoid receptors (GR), which, following translocation to the nucleus, stimulate mRNA transcription and then protein synthesis and ultimately trigger hormonal actions. To determine the distribution of GR in small intestinal villus and crypt cells, we measured GR activity in enriched villus and crypt cell fractions by use of [3H]dexamethasone. In normal rats GR activity was present in all cell fractions but was lowest in fully mature villus cells of the upper villus and greatest in immature crypt cells. In contrast, 1 wk after adrenalectomy and ovariectomy or sham operation, both of which reduced plasma corticosterone levels, GR activities were comparable in enriched villus cell and enriched crypt cell fractions. Kinetic studies indicated a single class of binding sites with comparable Kd values in villus and crypt cells of normal and steroid-depleted animals, suggesting that there are more glucocorticoid-binding sites in crypt than in villus cells of normal animals. Immature crypt cells may be more susceptible to glucocorticoid modulation than fully mature villus cells in normal rats in view of their higher GR activity.

Expression of YBB 3/10 Antigen in Human Tumor Colon Cell Lines and Its Induction by <italic>N,N</italic>-Dimethylformamide<xref ref-type="fn" rid="FN1">2</xref>

A monoclonal antibody (YBB 3/10) raised against rat small intestinal brush border membranes defined a high-molecular-weight antigen present on the surface membrane of the epithelial cells in fetal and newborn rat small intestine but appeared specific for the undifferentiated crypt cells in adult rat small intestine. In rat colon and rectum, expression of the YBB 3/10 antigen was limited to a specific period of development, from day 17 to 18 of gestation in the fetus to the first few days after birth. This antigen was detected in 11 of 12 human tumor intestinal cell lines. By immunofluorescent staining, it was found present only in subsets of the cultured tumor cells appearing as colonies of variable size. In contrast, it could not be detected in a large panel of normal rat and human cell lines. Expression of the YBB 3/10 antigen could be induced in the only negative colon tumor cell line (DLD-1 cells) by treatment with N,N-dimethylformamide (CAS: 68-12-2). These results suggested that the YBB 3/10 antigen represented a marker for the differentiated state of human colon tumor cells and that it would be useful to investigate the relationship between the state of cell differentiation and the pattern of development of small and large intestine during fetal and postnatal life.

The kinetics of villus cell populations in the mouse small intestine

Cytosine arabinoside was used to kill large numbers of proliferating crypt cells in the mouse small intestine, and the recovery sequence was analysed in terms of the villus population size, the crypt population size, the crypt: villus and the net villus cell influx: because the crypt: villus ratio changed considerably, it was found appropriate to express the crypt population in terms of the crypt cell mass per villus in order to relate it to the villus cell population. All parameters measured showed a series of damped oscillations over the experimental period, and analysis of the relationship between the several population changes strongly suggest that negative feedback control mechanisms are responsible for the response pattern. The importance of measuring the correct kinetic parameters in growth control studies on renewing epithelia is stressed.

Glycosphingolipids and the differentiation of intestinal epithelium

Changes in lipid-bound cell surface carbohydrates during cell differentiation were studied in epithelial cells of rat small intestine (crypt and villus tip cells). The identity of the cells was established by morphological and enzymatic criteria. Acid and non-acid glycosphingolipids were analysed by thin-layer chromatography, with a separate study by mass spectrometry for identification purposes. The amount of glucosylceramide in the epithelial cell increased 2–3-fold and N-glycoloylneuraminosyllactosylceramide (hematoside) 6–7-fold from crypt to villus tip, but globotriaosyl- and tetrahexosylceramide decreased. Fucolipids with blood group A activity (black-white strain) and H activity (white and black-white strain) showed no difference in this respect. No lengthening of the carbohydrate chains was noted, in contradiction to earlier suggestions concerning cell surface carbohydrate and differentiation. The increased amount of sialic acid of the villus tip cells may facilitate the cell-cell dissociation needed for cell extrusion. There was an increase in 2-hydroxy fatty acids vs non-hydroxy fatty acids of major glycolipids from crypt to villus tip.

Amino acid and hexose transport by cultured crypt cells from rat small intestine.

The characteristics of amino acid and sugar transport in intestinal crypt epithelial cells have been examined by measuring substrate uptake in an established epithelial cell line. These cells (IEC-6 cells) have been characterized as derived from rat small intestinal crypt cells on the basis of morphological criteria (J. Cell. Biol. 80: 248-265, 1979). Amino acid transport appeared to be mediated by both Na+-dependent and Na+-independent systems. Hexose uptake was stereospecific and Na+ independent, and was markedly inhibited by phloretin and cytochalasin B. Since glucocorticoids are known to have profound effects on maturation of the intestinal epithelium in vivo, their effects on transport properties of the cultured crypt cells were studied. Hydrocortisone, while completely inhibiting cell growth, increased the initial uptake rates of various hexoses, while having little or nor effect on the initial rate of amino acid uptake. The increased hexose uptake appeared to be due to a change in Vmax rather than Km. Appearance of the Na+-dependent hexose transport system, which is present in differentiated enterocytes, was not elicited by in vitro treatment with glucocortcoids.

Modeling to determine dose dependence of drug and cell kinetic parameters

In this work, we report on the use of a computer-based mathematical model which allows us to quantitate the in vivo effects of various chemotherapeutic agents on both malignant and normal cells. The usefulness of the model is shown by obtaining the dose response of the small intestinal crypt cells to Ara-C. The use of such an approach in determining optimal chemotherapeutic protocols for cancer treatment is discussed.

A vitamin D-related inhibition of growth of an epithelioid cell line derived from rat small intestine

A recently established epithelioid cell line derived from rat small intestinal crypt cells was tested for the effects on growth of rat serum obtained from normal, vitamin D-deficient and 25-(OH)D 3 repleted animals as compared to the growth curves established with fetal calf serum. The results suggest that there is a heat-labile growth-inhibiting factor(s) present in normal and 25-(OH)D 3 repleted animals which is not present in vitamin D deficient animals.

Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria.

Rat small intestinal epithelial cell lines have been established in vitro and subcultured serially for periods up to 6 mo. These cells have an epithelioid morphology, grow as monolayers of closely opposed polygonal cells, and during the logarithmic phase of growth have a population doubling time of 19--22 h. Ultrastructural studies revealed the presence of microvilli, tight junctions, an extensive Golgi complex, and the presence of extracellular amorphous material similar in appearance to isolated basement membrane. These cells exhibit a number of features characteristic of normal cells in culture; namely, a normal rat diploid karyotype, strong density inhibition of growth, lack of growth in soft agar, and a low plating efficiency when seeded at low density. They did not produce tumors when injected in syngeneic animals. Immunochemical studies were performed to determine their origin using antisera prepared against rat small intestinal crypt cell plasma membrane, brush border membrane of villus cells and isolated sucrase-isomaltase complex. Antigenic determinants specific for small intestinal epithelial (crypt and villus) cells were demonstrated on the surface of the epithelioid cells, but they lacked immunological determinants specific for differentiated villus cells. An antiserum specifically staining extracellular material surrounding the cells cultured in vitro demonstrated cross-reactivity to basement membrane in rat intestinal frozen sections. It is concluded that the cultured epithelioid cells have features of undifferentiated small intestinal crypt cells.

Effect of a feeding schedule on crypt cell proliferation in the small intestine of the rat.

Using a stathmokinetic technique, the mitotic rate in the small intestinal crypts of two groups of male Sprague-Dawley rats was studied. The first group was fed solids for 3 h daily only. The second group was fed solids ad libitum. In the first group it was found that the mean daily weight of food consumed rose quickly to normal 24-hour levels but the mean daily weight of the rats fell rapidly and did not approach the original level throughout the experiment. There was no significant difference in the mitotic rate of the small intestinal crypt cells between the two groups.

Alterations in cell surface membrane components of adapting rat small intestinal epithelium: Studies with lectins after massive proximal jejunoileal resection and jejunoileal transposition

After proximal resection or transposition of intestinal segments, the small intestinal mucosa of rats was examined with fluorescein-conjugated lectins derived from Ricinus communis and Triticum vulgare (wheat germ). These agglutinins are thought to be specific for the nonreducing end-terminal carbohydrate residues, β-d-galactose and β-N-acetyl-d-glucosamine, respectively. After 70% proximal jejunoileal resection as well as transposition of ileal segments to jejunum, ileal villus cell surface labeling by both lectins was changed to a pattern characteristic of normal rat proximal intestine. Alterations were not detected proximal to the surgical anastomosis after massive resection or in the jejunal segments transposed to ileum. Although the labeling pattern of goblet cell mucin differed in proximal duodenum from the remainder of control small intestine in normal animals, no differences were detected in goblet cell mucin labeling between normal and resected or transposed animals. This study confirms the previously reported differential lectin-labeling patterns of the small intestinal crypt and villus cells and further emphasizes the differences between cell surface carbohydrate and secretory mucins of goblet cells. Furthermore, the changes induced in the cell surfacelabeling pattern by massive proximal jejunoileal resection and transposition suggest that cell surface and secretory components may be altered differently during adaptation of the small intestine.

Fibronectin synthesis by epithelial crypt cells of rat small intestine.

Synthesis of fibronectin in an epithelial cell line (IEC-6) established from rat small intestine was demonstrated by using immunofluorescence, radioimmunoassay, and collagen-binding. Internally labeled radioactive fibronectin isolated from the IEC-6 cells gave a single main band in sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing conditions. Fibronectin isolated from rat plasma gave two closely spaced bands. The slower one had the same mobility as the epithelial cell fibronectin. The distribution of fibronectin in IEC-6 cells as detected by immunofluorescence was different from that described for fibroblasts and other cell types; fibronectin was present exclusively in regions of cell-to-cell contact. No fluorescence was detected on the surface membrane facing the culture medium or underneath the cells. This suggests that fibronectin may not be involved in the adhesion of the epithelial cells to the growth surface but could mediate cell-to-cell contacts. In microscopic sections of the small intestine, immunofluorescent staining with antifibronectin serum was strong in the basement membrane underlying the epithelial cells in the crypts. The in vitro synthesis of fibronectin by the crypt cells and its abundant presence in the basement membrane underlying the same cells in vivo suggests that fibronectin is a structural component of the basement membrane, and that it may be, at least in part, synthesized and deposited by the intestinal epithelium.

Properties of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in villous and crypt cells of the rat small intestine.

Some properties of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase in microsomes of villous and crypt cells from the jejunal and ileal epithelia of rats fed commercial pellet were studied. The optimum pH of the microsomal reductase from villi and crypts was 7.0_??_7.2 and the Km for HMG-CoA was 41.7μM. The reductase specifically required dithiothreitol for its activity. The activity was higher in ileal populations than in the jejunum. Responses of the reductase in the villous fraction to feeding cholesterol and taurocholate in combination or cholestyramine resembled those observed in crypt cells. Thus, the properties of microsomal HMG-CoA reductase in villous and crypt cells from the small intestine are similar each other, and they are possibly the same enzyme.

Ultrastructure of cell loss in intestinal mucosa

Small intestinal crypt cell production is balanced by an equal cell loss from the villus. In mouse small intestine this cell loss appears to be largely achieved by the extrusion of individual cells by a process of intercellular vacuolation resulting in detachment of the entire cell. This process is similar to that involved in the extrusion of effete goblet cells, apoptotic cells, and the migration of individual epidermal basal cells (reported elsewhere). Scanning electron microscopy reveals a sequence of surface changes concomitant with the stages of cell loss. Though not clearly defined, there was some suggestion of circadian variations in the level of cell loss.

The Effect of60Co Gamma-radiation and Hydroxyurea on thein VivoChain Growth of DNA in Crypt Cells of the Small Intestine of the Mouse

DNA chain growth has been studied in small intestinal crypt cells of the mouse in vivo using a sensitive method. The method is designed primarily to study radiation-induced DNA-breaks and their repair; but since there are breaks in DNA at the replicating fork, it is also possible to study DNA chain growth after a 3H-thymidine pulse. We found that DNA chain growth is not depressed by 200 rad of 60Co gamma radiation. This finding supports the hypothesis that irradiation interfers mainly with the initiation of new replicons in mammalian cells affecting DNA chain growth only at higher doses. Hydroxyurea at sufficient dosage, however, depresses or even stops DNA chain growth in mouse crypt cells in vivo.

Intestinal Villus and Crypt Cell Responses to Cholera Toxin

Adenylate cyclase activity was measured in rat small intestinal villus and crypt cells after in vivo and in vitro exposure to cholera toxin. The increase in intestinal adenylate cyclase induced by cholera toxin in vivo appeared to be largely confined to the villus cell with the largest increase observed for upper villus cells. Crypt cell adenylate cyclase was not responsive to cholera toxin. No response could be demonstrated for isolated villus or crypt cells incubated with cholera toxin in vitro. In vivo incubation with 125I-cholera toxin demonstrated binding to only villus cells. These results suggest that the major effect of cholera toxin was on villus cells rather than crypt cells and this was due to the greater accessibility or binding capacity of the villus cell to luminal cholera toxin.

Adenylate and Guanylate Cyclase Activities and Cellular Differentiation in Rat Small Intestine

Adenylate and guanylate cyclase activities were measured in rat small intestinal villus and crypt cells to determine possible correlations with cellular differentiation. Isolated intestinal cells were prepared by a method which effectively separates differentiated villus cells from undifferentiated crypt cells (J Biol Chem 248:2542, 1973). Crypt cells were found to have a significantly lower guanylate cyclase activity than villus cells. Adenylate cyclase activity was higher in crypt cells than villus cells, although the difference was less striking than the reverse gradient observed for guanylate cyclase. There was no gradient of activity for cyclic guanosine 3':5'-monophosphate phosphodiesterase. However, cyclic adenosine 3':5'-monophosphate phosphodiesterase activity was lower in villus cells. No villus to crypt gradient of cyclic adenosine 3':5'-monophosphate concentration was detected in mucosa frozen rapidly in liquid nitrogen. The properties and subcellular localization of the cyclases were also evaluated, and of particular interest was the localization of guanylate cyclase to the microvillus membrane and the confirmation of adenylate cyclase activity in the lateral-basal membrane. The villus to crypt gradient of guanylate cyclase suggests that this enzyme has a specialized role in the differentiated villus cell. The contrasting subcellular localization of the cyclases suggests that the cyclases may be interrelated, possibly reflecting the epithelial cell polarity for absorption and secretion.

Properties of guanylate cyclase and levels of cyclic GMP in rat small intestinal villous and crypt cells

A number of studies on the mechanism of action of bacterial enterotoxins [1,2], prostaglandins [2] and the recently discovered gastrointestinal hormone vasoactive intestinal polypeptide (VIP)* [3] on small intestinal epithelium stress the role of cyclic AMP as a regulator of water and electrolyte trans- port in this tissue. The function of cyclic GMP in the small intestine is however entirely unknown. Its level as well as the activity of the particulate form of the enzyme guanylate cyclase in rat small intestinal epithelium are relatively high as compared to most other mammalian tissues [4-7]. The soluble form of guanylate cyclase which, at least in rat heart [6] and lung [8], clearly differs from the particulate enzyme in kinetic and physical properties, is nearly absent in small intestinal mucosa [4,6,7]. Our previous work [7] showed that the major part of the intestinal enzyme activity is associated with the microvillous membrane of the brushborder, the remaining part being localized in the basal-lateral plasma membranes. Accordingly, proliferative crypt cells displayed a much lower guanylate cyclase activity than the mature villous cells [7]. Since intestinal brushborders: which are easily obtained in a relatively pure state, provide one of the richest sources of particulate guanylate cyclase in the rat,

Possible precursors of aminopeptidase and alkaline phosphatase in the proximal tubules of kidney and the crypts of small intestine of mice

Two hydrolytically inactive proteins, one having common antigenic determinants with aminopeptidase and the other with alkaline phosphatase, have been localised in the apical cytoplasm of crypt cells of small intestine and in the cytoplasm of proximal convoluted tubules. In addition, the two proteins are also differently heat labil. Although they could not be detected with mere histochemical stain methods, they were detected by the immunofluorescence sandwich technique using specific antibody directed against either of the solubilised enzymes. The findings were confirmed using the previously described immunohistochemical method (Wachsmuth, 1973). The cellular and subcellular localisation implies that these two proteins are precursors of the hydrolytically active brush border membrane enzymes.

Cytoplasmic Changes in the Crypt Cells of the Mouse Small Intestine

Cytoplasmic Changes in the Crypt Cells of the Mouse Small Intestine