Immunocytochemical localization of actin in epithelial cells of rat small intestine by light and electron microscopy

We used post-embedding immunocytochemical techniques and affinity-purified anti-actin antibody to evaluate localization of actin in epithelial cells of small intestine by fluorescence and electron microscopy. Small intestine was fixed with 2% formaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M. One-micron or thin sections were stained with antibody followed by rhodamine- or colloidal gold-labeled goat anti-rabbit IgG, respectively. Label was present overlying microvilli, the apical terminal web, and the cytoplasm directly adjacent to occluding and intermediate junctions. Label was associated with outer mitochondrial membranes of all cells and the supranuclear Golgi region of goblet cells. Lateral cytoplasmic interdigitations between mature cells and subplasmalemmal filaments next to intrusive cells were densely labeled. The cytoplasm adjacent to unplicated domains of lateral membrane was focally labeled. Label was prominent over organized filament bundles within the subplasmalemmal web at the base of mature cells, whereas there was focal labeling of the cytoplasm adjacent to the basal membrane of undifferentiated cells. Basolateral epithelial cell processes were labeled. Label was focally present overlying the cellular ground substance. Our results demonstrate that actin is distributed in a distinctive fashion within intestinal epithelial cells. This distribution suggests that in addition to its function as a structural protein, actin may participate in regulation of epithelial tight junction permeability, in motile processes including migration of cells from the crypt to the villus tip, in accommodation of intrusive intraepithelial cells and in adhesion of cells to one another and to their substratum.     

Immunocytochemical localization of alkaline phosphatase in absorptive cells of rat small intestine after colchicine treatment

Summary The purpose of this study was to investigate the effect of colchicine and vinblastine on the localization of alkaline phosphatase (AlPase) in rat duodenum in relation to structural changes. AlPase was localized on the membranes of rough endoplasmic reticulum, Golgi stacks, cytoplasmic vesicles, microvilli, on lateral plasma membranes, and in some lysosomes of the duodenal epithelial cells of rats treated with either lumicolchicine or 0.9% NaCl alone. Microvilli were most intensely stained, and AlPase-positive Golgi stacks were regularly distributed in the supranuclear regions. After colchicine treatment, microvilli were shortened and the staining intensity became weaker, whereas basal as well as lateral plasma membranes showed stronger staining. The AlPase-positive microvilli appeared not only on the luminal surfaces, but also on the baso-lateral plasma membranes and even on the surfaces of characteristic intracytoplasmic cysts. Golgi stacks became smaller and their distribution became less localized, and the staining intensity of the Golgi stacks became weaker. AlPase localization in rats treated with vinblastine was almost identical with that of rats treated with colchicine. Thus, colchicine and vinblastine appeared to have elicited a disorientation of intracellular transport of intestinal AlPase by inhibiting microtubule organization.     

Immunocytochemical identification and localization of immunoglobulin A within Paneth cells of the rat small intestine.

Light microscopic immunocytochemistry was used to identify Paneth cells by their lysozyme content and to detect immunoglobulin antigens within a subpopulation of these cells. Antisera specific for the heavy chains of rat or human immunoglobulin A and for immunoglobulin light chain antigens produced specific staining of rat Paneth cells. The distribution of immunoglobulin staining varied between adjacent Paneth cells in the same crypt and between Paneth cells in adjacent crypts, as well as between Paneth cell populations of different animals. No staining of rat Paneth cells was detected using antisera specific for the heavy chain of immunoglobulins G or M. The specific staining of Paneth cells for immunoglobulin A and light chain antigens was blocked by absorption of each antiserum with its respective purified antigen. Absorption of these antisera with purified rat lysozyme did not affect staining and thereby eliminated the possibility of immunologic cross-reactivity between lysozyme and immunoglobulin antigens. It is suggested, in light of current concepts of Paneth cell function, that the immunoglobulin staining of Paneth cells may reflect their ability to phagocytize immunoglobulin A-coated microorganisms or immune complexes containing immunoglobulin A.     

Paracellular absorption of D-glucose by rat small intestine in vivo

D-glucose diffusion in both jejunum and ileum using a perfusion system in vivo was determined. 2,4,6-triaminopyrimidine (20 mM) induced an inhibition on D-glucose diffusion of 32% in the two segments of the small intestine studied. Glucose net efflux from the jejunum into the lumen was higher than that from the ileum. Phlorizin increased the sugar efflux in both areas.     

Immunofluorescent localization of transglutaminase in rat small intestine

The distribution of intestinal transglutaminase was investigated by immunofluorescence microscopy using rabbit anti-guinea pig transglutaminase immunoglobulin. Transglutaminase-related antigen was demonstrated principally in the cytoplasm of villous core interstitial cells with some activity in the brush border region of the villous epithelial cells. Implications for the pathogenesis of coeliac disease are discussed.     

Electrical charge on protein regulates its absorption from the rat small intestine

The effect of the electrical charge on the intestinal absorption of a protein was studied in normal adult rats. Chicken egg lysozyme (Lyz), a basic protein with a molecular weight of 14,300, was selected and several techniques for chemical modification were applied. Then the intestinal absorption of Lyz derivatives was evaluated by measuring the radioactivity in plasma and tissues, after the administration of an (111)In-labeled derivative to an in situ closed loop of the jejunum. After the administration of (111)In-Lyz, the level of radioactivity in plasma was comparable with the lytic activity of Lyz, supporting the fact that the radioactivity represents intact Lyz. (111)In-cationized Lyz showed a 2-3 times higher level of radioactivity in plasma, whereas the radioactivity of (111)In-anionized Lyz was much lower. The absorption rate of (111)In-Lyz derivatives calculated by a deconvolution method was correlated for the strength of their positive net charge. A similar relationship was observed using superoxide dismutase. These findings indicate that the intestinal absorption of a protein is, at least partially, determined by its electrical charge.     

Sterol absorption by the small intestine

PURPOSE OF REVIEW: Cholesterol absorption is a selective process in that plant sterols and other non-cholesterol sterols are absorbed poorly or not at all. Recent research on the sterol efflux pumps adenosine triphosphate-binding cassette transporter G5 and adenosine triphosphate-binding cassette transporter G8 has not only provided an explanation for this selectivity, but also, together with the discovery of a new class of cholesterol absorption inhibitor, has yielded new insights into the mechanisms that potentially regulate the flux of cholesterol across the enterocyte. This review discusses these recent developments and their importance to the regulation of whole body cholesterol homeostasis. RECENT FINDINGS: Adenosine triphosphate-binding cassette transporters G5/8 regulate plant sterol absorption and also the secretion into bile of cholesterol and non-cholesterol sterols. Loss of adenosine triphosphate-binding cassette transporter G5/8 function results in sitosterolemia. Ezetimibe, a novel, potent and selective inhibitor of cholesterol absorption which is effective in milligram doses, lowers plasma plant sterol concentrations in sitosterolemic subjects, thus suggesting that this drug might be inhibiting the activity of a putative sterol permease in the brush border membrane of the enterocyte that actively facilitates the uptake of cholesterol as well as other non-cholesterol sterols. SUMMARY: Intestinal cholesterol absorption represents a major route for the entry of cholesterol into the body's miscible pools and therefore can potentially impact the plasma LDL-cholesterol concentration. The combined use of agents that inhibit the absorption and synthesis of cholesterol provides a powerful new approach to the prevention and treatment of atherosclerosis.     

In situ absorption of aflatoxins in rat small intestine

To evaluate the rate at which the four main aflatoxins (aflatoxins B₁, B₂, G₁ and G₂) are able to cross the luminal membrane of the rat small intestine, a study about intestinal absorption kinetics of these mycotoxins has been made. In situ results obtained showed that the absorption of aflatoxins in rat small intestine is a very fast process that follows first-order kinetics, with an absorption rate constant (k _a ) of 5.84±0.05 (aflatoxin B₁), 4.06±0.09 (aflatoxin B₂), 2.09±0.03 (aflatoxin G₁) and 1.58±0.04 (aflatoxin G₂) h^–1, respectively.     

Structural-functional analysis of diffusion in glucose absorption by rat small intestine enterocytes

To elucidate mechanisms providing transport of sugars across intestinal epithelium, on taking into account the current hypotheses (active transport, participation of paracellular transport and passive component of transcellular transport), it was important to reveal structural changes of tight junctions and distribution of the carriers of facilitated diffusion of GLUT2 and protein kinase C during absorption of glucose. On using confocal and electron microscopy, ultrastructural and immunocytochemical studies of enterocytes after perfusion of isolated rat small intestine fragment with 75 mM glucose (chronic experiment) have shown: 1) fluorescent labels of transporter GLUT2 and PKCbetaII are located in the apical area of enterocytes situated at the upper half of the villus. Antibodies against GLUT2, conjugated with gold, are revealed at the microvilli or apical membrane and in the area of terminal network; 2) no ultrastructural changes of the tight junction are detected on ultrathin sections and freeze--fracture replics. At the same time, fluorescent and gold labels against actin are concentrated in the vicinity of the lateral membrane in the tight junction area. The results obtained can serve a confirmation of a hypothesis that at high glucose concentrations GLUT2 participates in its transfer across the apical membrane.     

Iron absorption by small intestine of chickens

Iron (Fe) absorption by three segments (duodenum, jejunum, and ileum) of the small intestine of chickens was studied by a perfusion technique in vivo in closed circuit using⁵⁹Fe Cl₃ and was related to the histological characteristics of each segment. The serosal transfers of Fe for the duodenum and jejunum were the same (14%/cm), but significantly different (p<0.05) from those of the ileum (9%/cm), which may be explained by the morphological and histological properties of the gut of chickens. However, the presence of Fe in blood and in liver was significantly lower after perfusion of the jejunum and ileum than after perfusion of the duodenum. It is concluded that chickens show an early adaptation of small intestine to Fe absorption in response to the considerable loss of Fe suffered during the laying process.     

Immunocytochemical localization of substance P in mammalian intestine

Summary In mammalian intestine immunoreactive Substance P is localized not only in the plexuses of Auerbach and Meissner, as could be anticipated, but also in a number of basally situated, often basigranular, endocrine cells which have been identified tentatively as enterochromaffin.The presence of a neurohormone in cells of this type confirms their close association with the nervous system, noted by Masson (1924), and suggests that their postulated origin from the nervous system (Danisch, 1924) may well be correct.     

The absorption of phenformin and its effects on glucose and water absorption in isolated perfused rat small intestine

The effects of phenformin on glucose and water absorption from isolated perfused rat small intestine were studied. Luminal phenformin inhibited glocose and water absorption progressively as its concentration was increased from 0-1-1-0 mg.ml-1. At 0-5 mg phenformin ml-1, inhibition increased with time of exposure to phenformin up to 15 min and thereafter remained constant. Arterial infusion of phenformin (1-0 mg-ml-1) produced less inhibition of glucose and water absorption. The site of phenformin's action appeared to be intracellular. Phenformin absorption from a luminal perfusate (0-5 mg-ml-1) was measured. Although it was rapidly absorbed (22 microgram.cm intestine-1.h-1) from the lumen, less than 2 microgram.cm-1.h-1 appeared at the serosal surface of the intestine. In subsequent phenformin-free perfusion, only 25% of the absorbed phenformin was recovered in the luminal and serosal effluents.     

Cytoskeletal control of calcium absorption across the rat small intestine

1. The effect of colchicine, cytochalasin-B and procaine on calcium transport across the rat small intestine was investigated. The results obtained show the following: 2. Colchicine and cytochalasin-B at different concentrations inhibited significantly (P less than 0.001) calcium accumulation in rat intestinal cells, whereas procaine at different concentrations increased significantly (P less than 0.001) calcium accumulation in the rat small intestine. 3. Unidirectional influx of calcium across the rat small intestine was significantly inhibited (P less than 0.01) in the presence of colchicine and cytochalasin-B in the preincubation medium. Procaine, on the other hand, caused a significant increase (P less than 0.01) in the unidirectional influx of calcium across the rat intestinal cells. 4. The cell water content was not altered in the presence of the different drugs indicating that the changes in calcium transport across the rat intestinal cells are not due to alterations in the structure of the cell membrane.     

Nickel absorption and distribution from rat small intestine In situ

The aim of this work was to study the absorption of nickel chloride in rats by means of the intestinal perfusion in situ technique at nickel concentrations of 1, 5, 10, 25, and 100 mg/L. Active transport and facilitated diffusion seem to play an important role in the intestinal absorption of nickel at concentrations≤10 mg/L. At higher concentrations, the absorption rate would be limited by saturation of the carriers. The distribution of the absorbed nickel was studied by intestinal perfusion of a 10-mg Ni/L solution for 30 or 60 min. Both in concentration and amount, the jejunum showed the higher values of absorbed nickel, followed by the kidneys and liver. When all of the collected organs (brain, heart, liver, lungs, spleen, kidneys, and testicles) and blood, but not the small intestine, are analyzed following a 60-min perfusion, it was found that 1% of the initial concentration had passed through the intestinal barrier.     

Immunocytochemical demonstration of vasopressin binding in rat kidney

We investigated the immunoperoxidase demonstration of vasopressin (VSP) bound to paraffin-embedded sections of rat kidney and the effects of various fixatives. Slices of rat kidney from normal and 4-day water-deprived rats were incubated with 10(-7) M VSP, fixed, and embedded in paraffin. Hydrated sections of these tissues were again incubated with 10(-7) M VSP or 10(-7) M VSP and 10(-5) M oxytocin (OXY). VSP bound to the sections was demonstrated using rabbit anti-Arg8 VSP antiserum and peroxidase-labeled second antibody. In sections of kidney from both normal and water-deprived rats, immunoperoxidase labeling was most intense in the renal papilla and was restricted to the cells of the ducts of Bellini and loops of Henle. In the medulla, the collecting ducts and medullary thick ascending limbs of Henle were moderately stained. In the normal kidney sections there was no staining of the proximal tubules, distal convoluted tubules (DCT), and only slight staining of the cortical collecting ducts (CCD). However, in the water-deprived rats there was a considerable increase in the staining of the DCT and CCD. Simultaneous incubation in OXY and VSP resulted in reduced immunoperoxidase labeling of the tubules. Omission of VSP incubation led to a similar decrease in stain intensity, indicating a specificity for the sites of VSP binding. This technique allows the identification of cells responsible for the binding of VSP in the kidney.