Energetics of Na+-dependent sugar transport by isolated intestinal cells: evidence for a major role for membrane potentials

Intestinal epithelial cells isolated from 6-wk-old chickens maintain the capability for Na+-dependent concentrative accumulation of 3-O-methylglucose (3-OMG). Cells depleted of ATP exhibit a transient accumulation of 3-OMG in response to imposed Na+ gradients ([Na+]o greater than [Na+]i) or when transmembrane ion diffusion potentials (cell interior negative) are established. Phlorizin or lack of extracellular Na+ prevents formation of sugar gradients in every case. A nonconcentrative, non-Na+-dependent sugar transport system is also operative in these cells. The latter system is inhibited to various degrees by phloretin, theophylline, cytochalasin B, and a variety of flavonones and flavones, including apigenin. These agents also act to inhibit efflux of sugar from the cell via this pathway. The concentrative system normally operates against a "leak" of sugar through the nonconcentrative carrier. If the passive system is made inoperative by any of the agents named above, a significant enhancement of steady-state sugar gradients maintained by the cells is observed. With cytochalasin B, gradients as large as 30-fold are established. The energy inherent in cellular Na+ gradients cannot account for sugar gradients of this magnitude unless both chemical electrical driving forces are considered. When the passive leak is maximmally inhibited, more than half of the total energy required must be derived from the membrane potential.     

Calcium transport by permeabilised rabbit small intestinal epithelial cells

Intestinal epithelial cells isolated from rabbit small intestine and whose plasma membrane had been rendered highly permeable were used to study the role of intracellular structures in Ca²⁺ buffering. Monitoring free Ca²⁺ with a selective electrode revealed that the cells could reduce Ca²⁺ concentration in the medium to a level of 3.6×10^–7 M independently, within a certain range, of the initial Ca²⁺ concentration or amount of cells used. Ca²⁺ buffering by permeabilised enterocytes was Mg²⁺-and ATP-dependent and was abolished in the presence of the Ca²⁺ ionophore A23187.The rapidity of Ca²⁺ buffering, but not the final Ca²⁺ level attained, was reduced in the combined presence of the mitochondrial inhibitors azide and oligomycin or in the presence of ruthenium red. Buffering of Ca²⁺ was abolished in the presence of vanadate, although some uptake was still observed. Complete blocking occurred in the presence of hoth vanadate and mitochondrial inhibitors.Measurement of initial rates of uptake with radioactive calcium, revealed that mitochondrial uptake plays a role at relatively high Ca concentrations but that at the presumably physiological levels most of the uptake is into a nonmitochondrial compartment. Non-differentiated crypt cells scemed to handle intracellular Ca²⁺ in a similar way as mafure villus cells, although they appeared to buffer at a level about 2×10^–7 M lower.     

Glutamine transport in isolated epithelial intestinal cells. Identification of a Na+-dependent transport mechanism,highly specific for glutamine

L-glutamine transport was evaluated in isolated cells from the guinea-pig small intestine by measuring [(3)H]- L-glutamine uptake. Villous and crypt cells expressed Na(+)-dependent and Na(+)-independent transport mechanisms. Glutamine transport systems were identified using various amino acids and analogues as inhibitors. In both villous and crypt cells, 2-(methylamino)-isobutyrate (MeAIB), a system A inhibitor, did not inhibit Na(+)-dependent glutamine influx. 2-Aminobicyclo(2,2,1)heptane-2-carboxylate (BCH), a system B(0) and B(0,+) substrate, had no effect on Na(+)-dependent influx. Serine, cysteine and threonine, system ASC inhibitors, reduced Na(+)-dependent influx by 50%. Asparagine, but not histidine, system N inhibitors, reduced Na(+)-dependent glutamine influx by 50%, however the effect of asparagine was not additive to that of threonine. The remaining Na(+)-dependent glutamine influx (50%) was only inhibited by glutamine itself, by Na(+) substitution ( N-methyl-glucamine, K(+), Li(+)) or by external pH reduction. Phenyl-acetyl-glutamine (PAG), a synthetic amino acid analogue, also inhibited this Na(+)-dependent, threonine-insensitive glutamine influx (IC(50) 2.45 mM). The Na(+)-independent uptake was partially inhibited by BCH, a system L inhibitor, and other neutral amino acids, but was not affect by PAG. Our results suggest that glutamine is transported in both villous and crypt cells by the Na(+)-independent system L, by the Na(+)-dependent system ASC and by an as yet undescribed Na(+)-dependent transport mechanism, highly specific for glutamine.     

Effects of phloretin and theophylline on 3-O-methylglucose transport by intestinal epithelial cells

Phloretin and theophylline each exert an immediate inhibitory effect on the Na+-independent, facilitated-diffusion transport system for sugar associated with intestinal epithelial cells. Phloretin inhibits approximately 50% more of the total Na+-independent sugar flux than theophylline. Neither agent has an immediate effect on the Na+-dependent, concentrative sugar transport system, although preincubation of the cells with phloretin causes a significant inhibition. The slowly developing effect is correlated with a decrease in cellular adenosine triphosphate (ATP) and an elevation of intracellular Na+. Other agents which elevate cell Na+ also inhibit Na+-dependent sugar influx, even if ATP levels are not depleted. On the other hand, if ATP is depleted by phloretin under conditions in which the cells do not gain Na+, the inhibitory effect on Na+-dependent sugar flux tends to disappear. The slow-onset phloretin effects are due to transinhibition of the Na+-dependent sugar carrier by cellular Na+. When the passive sugar carrier is inhibited by phloretin or theophylline, the concentrative system can establish an enhanced sugar gradient. Because of the secondary metabolic effects of phloretin, theophylline induces a greater gradient enhancement despite its more limited effect on the passive sugar-transport system. Sugar gradients as large as 20-fold are induced by theophylline, in contrast to 12-fold gradients observed in the presence of phloretin and approximately 7- to 8-fold for untreated cells. These results are discussed in terms of conceptual questions regarding the energetics of Na+-dependent transport systems.     

Sensitization of HeLa cells for viral RNA infection by cytochalasin B.

Incubation of HeLa cells in medium containing 5 μg cytochalasin B/ml for 5–10 min at 37° results in: (a) 30- to 80-fold increase in the sensitivity of cells for infection by isolated poliovirus RNA, (b) reduced viral RNA adsorption and penetration, and (c) immediate inhibition of incorporation of labeled amino acids into proteins. Protein synthesis in poliovirus-infected cells, however, continues unabated for 15 min in the presence of a ten-fold higher concentration of cytochalasin B (50 μg/ml). Apparently, viral RNA can efficiently direct protein synthesis under conditions where host RNA translation is inhibited.     

Human T-lymphocyte rosette formation: inhibition by cytochalasin B.

The effects of cytochalasin B, colchicine and vinblastine on the rosette formation of human T lymphocytes with neuraminidase-treated human erythrocytes (nHRBC) and with sheep red blood cells (SRBC) have been studied. Pretreatment of the lymphocytes with cytochalasin B which affects microfilament action reversibly inhibits both nHRBC and SRBC rosette formation. Colchicine and vinblastine known to interact with microtubules causes no major reduction in rosette-forming cells. The results suggest that normally functioning microfilaments are necessary for nHRBC and SRBC rosetting, whereas microtubules are not essential for the blinding of the erythrocytes to the lymphocytes.     

Effects of cytochalasin B and dimethylsulphoxide on isosmotic fluid transport by rabbit gall-bladder in vitro.

1. Net fluid transport rate, transepithelial ohmic resistance and potential difference (p.d.), and unidirectional fluxes of Na+ were measured in rabbit gall-bladder preparations in vitro exposed on both sides to Ringer solutions of identical electrolyte composition. 2. Bilateral application of 1% dimethylsulphoxide (DMSO), the solvent for cytochalasin B, rapidly and reversibly depressed net fluid transport rate by 15% and increased the lumen positive p.d. by 1-5-2-0 mV. Resistance did not change significantly. These effects of DMSO were shown to be non-specific osmotic effects. 3. Cytochalasin B (10(-5)M) applied bilaterally caused: (a) a progressive inhibition of net fluid transfer rate to 40-50% of its control value within 60 min; the effect was partly reversible within 60 min and independent of the substrates glucose, glutamate and pyruvate; (b) a progressive depression of the mucosal-to-serosal Na+ flux within the first 30 min with no further change in the flux during the following 30 min of exposure to cytochalasin B; the effect was partly reversible within 70 min; (c) a rapid but moderate increase in the passive serosal-to-mucosal Na+ flux, which continued to increase gradually during the entire 60 min period of exposure to cytochalasin B; the effect was completely reversible within 70 min; (d) a prompt drop in ohmic resistance (30%) and p.d. (40%) with no further changes in these parameters during the following 60 min of exposure to cytochalasin B. The effect on resistance was partly reversible within 90 min; the effect on p.d. was completely reversible within 30 min. 4. The results are interpreted to indicate an early inhibitory action of cytochalasin B on the active transcellular pump mechanism and to suggest a cytochalasin B-mediated progressive increase in cell membrane permeability to sodium resulting ultimately in a highly leaky epithelium. The results are compatible with the concept that a mechanochemical process is involved in isosmotic transcellular transport of fluid across low-resistance epithelia.     

Capsule reduces adherence of enterotoxigenic Escherichia coli to isolated intestinal epithelial cells of pigs.

Previous reports have demonstrated that heat-stable (A-type) capsule on piliated enterotoxigenic Escherichia coli enhances colonization of enterotoxigenic E. coli in the small intestine and enhances virulence of enterotoxigenic E. coli. In this report, four encapsulated enterotoxigenic E. coli strains and one encapsulated nonenterotoxigenic strain of E. coli and their nonencapsulated mutants were tested for adhesion to isolated intestinal epithelial cells or brush borders from neonatal pigs. The enterotoxigenic E. coli also expressed the K99 pilus antigen. The nonencapsulated mutants of the four enterotoxigenic E. coli adhered in higher numbers than did the encapsulated parental strains. Both the encapsulated and nonencapsulated forms of enterotoxigenic E. coli 431 grown at 18 degrees C (K99 production suppressed) adhered poorly to the isolated cells. The nonenterotoxigenic E. coli 1793 which does not express K99 antigen also adhered poorly in both encapsulated and nonencapsulated forms. Fab fragments of anticapsular immunoglobulin G failed to block the effect of capsule on adherence of strain 431. The results indicated that K99 was the principal mediator of in vitro adhesion of the enterotoxigenic E. coli strains and that capsule impedes the in vitro adhesion. They also suggested that the capsular enhancement of colonization by such strains in vivo probably is by some mechanism other than enhanced adhesion to epithelium.     

Interaction of Escherichia coli heat-stable enterotoxin B with cultured human intestinal epithelial cells.

Binding of Escherichia coli heat-stable enterotoxin B (STb) to the human intestinal epithelial cell lines T84 and HT29 and to polarized T84 cells was studied to define the initial interaction of this peptide toxin with target cells. Equilibrium and competitive binding isotherms showed that 125I-STb bound specifically to T84 and HT29 cells; however, the toxin-epithelial cell interactions could be characterized by low-affinity binding (< or = 10(5) M(-1)) to a high number of binding sites (> or = 10(6) per cell). STb binding to T84 and HT29 cells as a function of 125I-STb concentration did not approach saturation at levels well above the effective biological concentration of STb for fluid secretion. Treatment of the 125I-STb-bound T84 and HT29 cells with an acidic saline solution to remove surface-bound toxin revealed that only approximately 55% +/- 10% of 125I-STb could be removed by this treatment at 4 degrees C, suggesting that approximately half of the bound STb was stably associated with the plasma membrane and/or internalized into the cytoplasm. Similar results were obtained when binding and internalization experiments were conducted at 22 and 37 degrees C. Immunofluorescence studies demonstrated that the strongest signal for STb appeared in the plasma membrane even after acid treatment. Toxin-treated cells also displayed diffuse cytoplasmic staining, indicating that once cell bound, STb did not appear to preferentially associate with membrane vesicles or cellular organelles. Binding and subsequent internalization of 125I-STb were not affected by treatment of the cells with trypsin, endoglycosidase F/peptide N-glycosidase F, Vibrio cholerae neuraminidase, tunicamycin, or 5 mM sodium chlorate, which blocks sulfation of surface proteoglycans. In addition, the internalization process was not altered by preincubation of the cells with the cytoskeleton inhibitors cytochalasin D and colchicine or cellular perturbants (i.e., 0.45 M sucrose and 5 mM sodium azide), indicating that cell surface proteins or carbohydrates did not function as STb receptors. The binding of 125I-STb to polarized T84 cells was also examined, and the total and nonspecific binding isotherms were found to overlap, indicating that the apical surface of polarized T84 cells did not contain a specific receptor for STb. In comparison to undifferentiated cells, twice the amount of bound STb (approximately 80% +/- 10%) was removable from polarized T84 cells after treatment with acidic solution. The percentage of surface-bound STb to polarized T84 cells did not vary significantly with the transepithelial electrical resistance of the cells or when STb was applied basolaterally. Together, our results indicate that STb binds with relatively low affinity to the plasma membrane of cultured intestinal epithelial cells and polarized T84 cells, probably to membrane lipids, and becomes stably associated with the lipid bilayer. The fact that a significant portion of the bound STb becomes free in the cytoplasm, even at a low temperature, suggests that the bound toxin may directly traverse the membrane bilayer.     

Potassium transport in epithelial cells isolated from small intestine of the chicken

The transport of potassium has been studied in epithelial cells isolated from chicken small intestine using⁸⁶Rb as a tracer for K⁺. (i) The uptake studies revealed that about 60% of the total K⁺ net flux is inhibited by ouabain and therefore mediated by the Na⁺–K⁺-ATPase. About 20% of the ouabain-insensitive K⁺ net influx was inhibited by furosemide, bumetanide and by either Na⁺ or Cl^– removal from the incubation solution, suggesting that a Na⁺/Cl^–/K⁺ cotransport system might be present in chicken enterocytes. (ii) The efflux of K⁺ was measured from cells preloaded with⁸⁶Rb. K⁺ efflux was inhibited by Ba²⁺, quinine and verapamil; it was stimulated by A23187, and it was unaffected by 3,4-diaminopyridine. Apamin, that has no effect on basal rates of K⁺ efflux, abolished the effect of A23187. These findings suggest that K⁺ efflux appears to occur through Ca²⁺-activated K⁺ channels.     

Role of intestinal epithelial cells in immune effects mediated by gram-positive probiotic bacteria: involvement of toll-like receptors

The mechanisms by which probiotic bacteria exert their effects on the immune system are not completely understood, but the epithelium may be a crucial player in the orchestration of the effects induced. In a previous work, we observed that some orally administered strains of lactic acid bacteria (LAB) increased the number of immunoglobulin A (IgA)-producing cells in the small intestine without a concomitant increase in the CD4(+) T-cell population, indicating that some LAB strains induce clonal expansion only of B cells triggered to produce IgA. The present work aimed to study the cytokines induced by the interaction of probiotic LAB with murine intestinal epithelial cells (IEC) in healthy animals. We focused our investigation mainly on the secretion of interleukin 6 (IL-6) necessary for the clonal expansion of B cells previously observed with probiotic bacteria. The role of Toll-like receptors (TLRs) in such interaction was also addressed. The cytokines released by primary cultures of IEC in animals fed with Lactobacillus casei CRL 431 or Lactobacillus helveticus R389 were determined. Cytokines were also determined in the supernatants of primary cultures of IEC of unfed animals challenged with different concentrations of viable or nonviable lactobacilli and Escherichia coli, previously blocked or not with anti-TLR2 and anti-TLR4. We concluded that the small intestine is the place where a major distinction would occur between probiotic LAB and pathogens. This distinction comprises the type of cytokines released and the magnitude of the response, cutting across the line that separates IL-6 necessary for B-cell differentiation, which was the case with probiotic lactobacilli, from inflammatory levels of IL-6 for pathogens.     

Decay of murine intestinal epithelial cells under extrusion.

Electron microscopic findings of this study revealed 2 types of extrusion of brush border cells from the summits of intestinal villi: a) By expansion of initial vacuolation the degenerating cell enlarges and elevates itself above the level of the extrusion zone. Cells about to lift become sphered and are shed into the lumen for degradation to continue. b) Another group of cells vacuolating to a similar extend already may start apical decay in situ. In this case mechanical elevation, apparently, is inefficient or retarded such as to offer a cell the opportunity to decompose at its proper place in the mucosal lining. Contrary to type A extrusion, i.e. the desquamation of the entire degenerating cell, type B produces cellular debris to be shed into the lumen. Either type of exfoliation may be associated with the formation of gaps the closure of which proceeds slowly. The light microscopic test of cell viability by means of horseradish peroxidase demonstrated many brush border cells leaky such as to become loaded with the tracer irrespective of its vascular or enteric approach to the extrusion zone. Damage of the cell membrane due to a brief pretreatment with digitonin resulted in a dramatic rise of horseradish peroxidase positive cells. With respect to these findings, one cannot escape the conclusion that both types of extrusion confer intermittent leakage on the summits of intestinal villi which at least could account for part of the diffusion of macromolecules in either direction.