Gail Hecht

Enteropathogenic Escherichia coli dephosphorylates and dissociates occludin from intestinal epithelial tight junctions

Enteropathogenic Escherichia coli (EPEC) increases tight junction permeability in part by phosphorylating the 20 kDa myosin light chain (MLC20) that induces cytoskeletal contraction. The impact of this enteric pathogen on specific tight junction (TJ) proteins has not been investigated. We examined the effect of EPEC infection on occludin localization and phosphorylation in intestinal epithelial cells. After infection by EPEC, a progressive shift of occludin from a primarily TJ‐associated domain to an intracellular compartment occurred, as demonstrated by immunofluorescent staining. A reverse in the ratio of phosphorylated to dephosphorylated occludin accompanied this morphological change. Eradication of EPEC with gentamicin resulted in the normalization of occludin localization and phosphorylation. The serine/threonine phosphatase inhibitor, calyculin A, prevented these events. The EPEC‐associated decrease in transepithelial electrical resistance, a measure of TJ barrier function, returned to baseline after gentamicin treatment. Non‐pathogenic E. coli, K‐12, did not induce these changes. Transformation of K‐12 with the pathogenicity island of EPEC, however, conferred the phenotype of wild‐type EPEC. Deletion of specific EPEC genes encoding proteins involved in EPEC type III secretion markedly attenuated these effects. These findings suggest that EPEC‐induced alterations in occludin contribute to the pathophysiology associated with this infection.

Innate mechanisms of epithelial host defense: spotlight on intestine

The single layer of epithelial cells lining the intestinal tract is charged with a most difficult task: protecting the underlying biological compartments from both the normal commensal flora that reside within the intestinal lumen as well as the uninvited pathogens. To such an end, the intestinal epithelial cells are equipped with a panoply of defense mechanisms, both constitutive and inducible. This review focuses only on those defense mechanisms that are initiated and executed by the intestinal epithelial cell. Fitting these strict criteria are three major categories of epithelial host defense: enhanced salt and water secretion, expression of antimicrobial proteins and peptides, and production of intestinal mucins. Each of these areas is discussed in this review.

Enteropathogenic E. coli attenuates secretagogue-induced net intestinal ion transport but not Cl− secretion

Enteric bacterial pathogens often increase intestinal Cl- secretion. Enteropathogenic Escherichia coli(EPEC) does not stimulate active ion secretion. In fact, EPEC infection decreases net ion transport in response to classic secretagogues. This has been presumed to reflect diminished Cl- secretion. The aim of this study was to investigate the influence of EPEC infection on specific intestinal epithelial ion transport processes. T84 cell monolayers infected with EPEC were used for these studies. EPEC infection significantly decreased short-circuit current ( I sc) in response to carbachol and forskolin, yet125I efflux studies revealed no difference in Cl- channel activity. There was also no alteration in basolateral K+ channel or Na+-K+-2Cl-cotransport activity. Furthermore, net36Cl-flux was not decreased by EPEC. No alterations in either K+ or Na+ transport could be demonstrated. Instead, removal of basolateral bicarbonate from uninfected monolayers yielded an I sc response approximating that observed with EPEC infection, whereas bicarbonate removal from EPEC-infected monolayers further diminished I sc. These studies suggest that the reduction in stimulated I sc is not secondary to diminished Cl-secretion. Alternatively, bicarbonate-dependent transport processes appear to be perturbed.

Human colonic epithelial cells express galanin-1 receptors, which when activated cause Cl− secretion

Galanin is a peptide hormone widely expressed in the central nervous system and gastrointestinal (GI) tract. Within the GI tract galanin is present in enteric nerve terminals where it is known to modulate intestinal motility by altering smooth muscle contraction. Recent studies also show that galanin can alter intestinal short-circuit current ( I sc) but with differing results observed in rats, rabbits, guinea pigs, and pigs. In contrast, nothing is known about the ability of galanin to alter ion transport in human intestinal epithelial tissues. By RT-PCR, we determined that these tissues express only the galanin-1 receptor (Gal1-R) subtype. To evaluate Gal1-R pharmacology and physiology, we studied T84 cells. Gal1-R expressed by these cells bound galanin rapidly (half time 1-2 min) and with high affinity (inhibitor constant 0.7 ± 0.2 nM). T84 cells were then studied in a modified Ussing chamber and alterations in I sc, a measure of all ion movement across the tissue, were determined. Maximal increases in I sc were observed in a concentration-dependent manner around 2 min after stimulation with peptide, with 1 μM galanin causing I sc to rise more than eightfold and return to baseline occurring within 10 min. The increase in galanin-induced I sc was shown by125I efflux studies to be due to Cl- secretion, which occurred independently of alterations in cAMP and phospholipase C. Rather, Cl- secretion is mediated via a Ca2+-dependent, pertussis toxin-sensitive mechanism. These data suggest that galanin released by enteric nerves may act as a secretagogue in the human colon by activating Gal1-R.

Myosin regulation of NKCC1: effects on cAMP-mediated Cl− secretion in intestinal epithelia

The basally located actin cytoskeleton has been demonstrated previously to regulate Cl- secretion from intestinal epithelia via its effects on the Na+-K+-2Cl- cotransporter (NKCC1). In nontransporting epithelia, inhibition of myosin light chain kinase (MLCK) prevents cell-shrinkage-induced activation of NKCC1. The aim of this study was to investigate the role of myosin in the regulation of secretagogue-stimulated Cl- secretion in intestinal epithelia. The human intestinal epithelial cell line T84 was used for these studies. Prevention of myosin light chain phosphorylation with the MLCK inhibitor ML-9 or ML-7 and inhibition of myosin ATPase with butanedione monoxime (BDM) attenuated cAMP but not Ca2+-mediated Cl- secretion. Both ML-9 and BDM diminished cAMP activation of NKCC1. Neither apical Cl- channel activity, basolateral K+ channel activity, nor Na+-K+-ATPase were affected by these agents. Cytochalasin D prevented such attenuation. cAMP-induced rearrangement of basal actin microfilaments was prevented by both ML-9 and BDM. The phosphorylation of mosin light chain and subsequent contraction of basal actin-myosin bundles are crucial to the cAMP-driven activation of NKCC1 and subsequent apical Cl- efflux.The basally located actin cytoskeleton has been demonstrated previously to regulate Cl-secretion from intestinal epithelia via its effects on the Na+-K+-2Cl-cotransporter (NKCC1). In nontransporting epithelia, inhibition of myosin light chain kinase (MLCK) prevents cell-shrinkage-induced activation of NKCC1. The aim of this study was to investigate the role of myosin in the regulation of secretagogue-stimulated Cl- secretion in intestinal epithelia. The human intestinal epithelial cell line T84 was used for these studies. Prevention of myosin light chain phosphorylation with the MLCK inhibitor ML-9 or ML-7 and inhibition of myosin ATPase with butanedione monoxime (BDM) attenuated cAMP but not Ca2+-mediated Cl- secretion. Both ML-9 and BDM diminished cAMP activation of NKCC1. Neither apical Cl- channel activity, basolateral K+ channel activity, nor Na+-K+-ATPase were affected by these agents. Cytochalasin D prevented such attenuation. cAMP-induced rearrangement of basal actin microfilaments was prevented by both ML-9 and BDM. The phosphorylation of mosin light chain and subsequent contraction of basal actin-myosin bundles are crucial to the cAMP-driven activation of NKCC1 and subsequent apical Cl- efflux.