INTERRUPTION OF ESCHERICHIA-COLI HEAT-STABLE ENTEROTOXIN-INDUCED GUANYLYL CYCLASE SIGNALING AND ASSOCIATED CHLORIDE CURRENT IN HUMAN INTESTINAL-CELLS BY 2-CHLOROADENOSINE

Diarrhea induced by Escherichia coli heat-stable enterotoxin (STa) is mediated by a receptor guanylyl cyclase cascade. The present study est ablishes that an intracellular nucleotide-dependent pathway disrupts t oxin-induced cyclic GMP (cGMP) production and the associated chloride (Cl-) flux that underlie intestinal secretion. Incubation of Caco 2 hu man intestinal epithelial cells with the nucleoside analog 2-chloroade nosine (2ClAdo) resulted in a concentration- and time-dependent inhibi tion of toxin-induced cGMP production. Inhibition of cGMP production c orrelated with the metabolic conversion of 2ClAdo to 2-chloroadenosine triphosphate. The effect of 2ClAdo did not reflect activation of aden osine receptors, inhibition of adenosine deaminase, or modification of the binding or distribution of STa receptors. Guanylyl cyclase activi ty in membranes prepared from 2ClAdo-treated cells was inhibited, in c ontrast to membranes from cells not exposed to 2ClAdo, demonstrating t hat inhibition of guanylyl cyclase C (GCC) was mediated by a noncompet itive mechanism, Treatment of Caco 2 cells with 2ClAdo also prevented STa induced Cl- current. Application of 8-bromo-cGMP, the cell-permean t analog of cGMP, to 2ClAdo-treated cells reconstituted the Cl- curren t, demonstrating that inhibition of Cl- flux reflected selective disru ption of ligand stimulation of GCC rather than the chloride channel it self. Thus, the components required for adenine nucleotide inhibition of GCC signaling are present in intact mammalian cells, establishing t he utility of this pathway to elucidate the mechanisms regulating ST-d ependent guanylyl cyclase signaling and intestinal fluid homeostasis. In addition, these data suggest that the adenine nucleotide inhibitory pathway may be a novel target to develop antisecretory therapy for en terotoxigenic diarrhea.