No abstract

Abstract. The massive secretion of salt and water in cholera-induced diarrhea involves binding of cholera toxin (CT) to ganglioside GM1 in the apical membrane of intestinal epithelial cells, translocation of the enzymatically active A~-peptide across the membrane, and subsequent activation of adenylate cyclase located on the cytoplasmic surface of the basolateral membrane. Studies on nonpolarized cells show that CT is internalized by receptor-mediated endocytosis, and that the A~-subunit may remain membrane associated. To test the hypothesis that toxin action in polarized cells may involve intracellular movement of toxin-containing membranes, monolayers of the polarized intestinal epithelial cell line 1'84 were mounted in modified Ussing chambers and the response to CT was examined. Apical CT at 37~ elicited a short circuit current (Isc: 48 • 2.1/zA/cm2; half-maximal effective dose, ED50 •0.5 nM) after a lag of 33 • 2 rain which bidirectional ~Na + and ~C1-flux studies showed to be due to electrogenic C1-secretion. The time course of the CT-induced Isc response paralleled the time course of cAMP generation. The dose response to basolateral toxin at 37~ was identical to that of apical CT but lag times (24 • 2 rain) and initial rates were significantly less. At 20~ the Isc response to apical CT was more strongly inhibited (30-50%) than the response to basolateral CT, even though translocation occurred in both cases as evidenced by the formation of A~-peptide. A functional rhodamine-labeled CT-analogue applied apically or hasolateraUy at 20~ was visualized only within endocytic vesicles close to apical or basolateral membranes, whereas movement into deeper apical structures was detected at 37~ At 15~ in contrast, reduction to the Arpeptide was completely inhibited and both apical and basolateral CT failed to stimulate Isc although Isc responses to 1 nM vasoactive intestinal peptide, 10 #M forskolin, and 3 raM 8Br-cAMP were intact. Re-warming above 32~ restored CT-induced Isc. Preincubating monolayers for 30 rain at 37~ before cooling to 15~ overcame the temperature block of basolateral CT but the response to apical toxin remained completely inhibited. These results identify a temperature-sensitive step essential to apical toxin action on polarized epithelial cells. We suggest that this event involves vesicular transport of toxin-containing membranes beyond the apical endosomal compartment.

Abstract. Migration of polymorphonuclear leukocytes across epithelia is a hallmark of many inflammatory disease states. Neutrophils traverse epithelia by migrating through the paracellular space and crossing intercellular tight junctions. We have previously shown (Nash, S., J. Stafford, and J. L. Madara. 1987. J. Clin. Invest. 80:1104-1113, that leukocyte migration across TM monolayers, a model human intestinal epithelium, results in enhanced tight junction permeability-an effect quantitated by the use of a simple, standard electrical assay of transepithelial resistance. Here we show that detailed time course studies of the transmigrationelicited decline in resistance has two components, one of which is unrelated to junctional permeability. The initial decrease in resistance, maximal 5-13 min after initiation of transmigration, occurs despite inhibition of transmigration by an antibody to the common beta subunit of neutrophil/32 integrins, and is paralleled by an increase in transepithelial short-circuit current. Chloride ion substitution and inhibitor studies indicate that the early-phase resistance decline is not attributable to an increase in tight junction permeability but is due to decreased resistance across epithelial cells resulting from chloride secretion. Since 1"84 cells are accepted models for studies of the regulation of C1-and water secretion, our results suggest that neutrophil transmigration across mucosal surfaces (for example, respiratory and intestinal tracts) may initially activate flushing of the surface by salt and water. Equally important, these studies, by providing a concrete example of sequential transcellular and paracellular effects on transepithelial resistance, highlight the fact that this widely used assay cannot simply be viewed as a direct functional probe of tight junction permeability.

Cholera toxin (CT) elicits a massive secretory response from intestinal epithelia by binding apical receptors (ganglioside Gm,) and ultimately activating basolateral effectors (adenylate cyclase). The mechanism of signal transduction from apical to basolateral membrane, however, remains undefined. We have previously shown that CT action on the polarized human intestinal epithelial cell line T84 requires endocytosis and processing in multiple intracellular compartments. Our aim in the present study was to test the hypothesis that CT may actually move to its site of action on the basolateral membrane by vesicular traffic. After binding apical receptors, CT entered basolaterally directed transcytotic vesicles. Both CT B subunits and to a lesser extent CT A subunits were delivered intact to the serosal surface of the basolateral membrane. The toxin did not traverse the monolayer by diffusion through intercellular junctions. Transcytosis of CT B subunits displayed nearly identical time course and temperature dependency with that of CT-induced Cl-secretion-suggesting the two may be related. These data identify a mechanism that may explain the link between the toxin's apical receptor and basolateral effector.