MECHANISM OF CHOLERA-TOXIN ACTION ON A POLARIZED HUMAN INTESTINAL EPITHELIAL-CELL LINE - ROLE OF VESICULAR TRAFFIC

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 A1-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 A1-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 T84 were mounted in modified Ussing chambers and the response to CT was examined. Apical CT at 37-degrees-C elicited a short circuit current (Isc: 48 +/- 2.1-mu-A/cm2; half-maximal effective dose, ED50 approximately 0.5 nM) after a lag of 33 +/- 2 min which bidirectional Na-22+ and Cl-36- flux studies showed to be due to electrogenic Cl- 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-degrees-C was identical to that of apical CT but lag times (24 +/- 2 min) and initial rates were significantly less. At 20-degrees-C, 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 A1-peptide. A functional rhodamine-labeled CT-analogue applied apically or basolaterally at 20-degrees-C was visualized only within endocytic vesicles close to apical or basolateral membranes, whereas movement into deeper apical structures was detected at 37-degrees-C. At 15-degrees-C, in contrast, reduction to the A1-peptide was completely inhibited and both apical and basolateral CT failed to stimulate Isc although Isc responses to 1 nM vasoactive intestinal peptide, 10-mu-M forskolin, and 3 mM 8Br-cAMP were intact. Re-warming above 32-degrees-C restored CT-induced Isc. Preincubating monolayers for 30 min at 37-degrees-C before cooling to 15-degrees-C 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.