Mechanism of cholera toxin action on a polarized human intestinal epithelial cell line: role of vesicular traffic

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.

Proteolytic Activation of Cholera Toxin and Escherichia coli Labile Toxin by Entry into Host Epithelial Cells

“…If this is correct, it is also possible that the A 1 peptide of nicked CT or LT may not fully dissociate from the A 2 peptide and B subunit after entry into the cell. If so, this would fit nicely with our previous observations that in polarized cells signal transduction by CT is not diffusion-limited even after translocation of the A subunit (21) and that both the pentameric B subunit and translocated A subunit appear to travel together across the cell en route to the basolateral membrane (11). Alternatively, it remains possible that the A 1 peptide of both mutant and wt toxin may dissociate from the A 2 peptide/B subunit-G M1 complex after a small (but not measurable) amount of nicking by the same or another protease as discussed above or that proteolytic cleavage of the A subunit is not required for complete dissociation of A subunit from the B pentamer.…”

“…In contrast, when these same recombinant toxins were applied to apical membranes, there was little or no delay in the anticipated time course of the secretory response. In all past experiments (11,12,21,25,32), we consistently found that toxin preparations purified from V. cholerae supernatants elicited Cl Ϫ secretion with a shorter lag phase and more rapid rate of onset when applied to basolateral rather than apical surfaces of the same cells (Fig. 1, panel A).…”

“…Hanks' balanced salt solution (HBSS, containing in g/liter 0.185 CaCl 2 , 0.098 MgSO 4 , 0.4 KCl, 0.06 KH 2 PO 4 , 8 NaCl, 0.048 Na 2 HPO 4 , 1 glucose, to which was added 10 mM HEPES, pH 7.4) was used for all assays unless otherwise stated. T84 cells obtained from ATCC were cultured and passaged as described previously (20,21). Cells from passages 69 -88 were utilized for these experiments.…”

Role of platelet activating factor in the intestinal epithelial secretory and Chinese hamster ovary cell cytoskeletal responses to cholera toxin.

“…To determine whether cholera toxin could directly stimulate intestinal epithelial cells to secrete PAF, we assayed PAF in supernatants and cells ofthe human intestinal T-84 cell line (obtained from the laboratory of K. Dharmsathaphor at the University ofCalifornia in San Diego). This cell line has been shown to respond to cholera toxin and other secretagogues with electrogenic chloride secretion (26,27). Cell monolayers cultured in a 1:1 mixe of Dulbecco's modified Eagle's mem and Ham's F12 (Gibco, Grand Island, NY) with 5% fetal bovine serum were exposed to cholera toxin (1 ;g/ml) for 2 hr.…”

Structural Basis for the Differential Toxicity of Cholera Toxin and Escherichia coli Heat-labile Enterotoxin

“…The use of polarized human colonic epithelial T84 cells has greatly facilitated studies of Ctx and Etx, since toxicity can be readily monitored as the induction of electrogenic Cl Ϫ secretion (18). Toxin action is initiated by binding of the B-subunit moiety to cell surface receptors.…”