Disruption of guanylyl cyclase signalling pathway mediating intestinal secretion

The present studies establish that an intracellular nucleotide dependent pathway disrupts cyclic GMP (cGMP) production by guanylyl cyclase C (GCC) and guanylyl cyclase A (GCA). Inhibition requires the kinase-homology domain since deletion of this region yields a guanylyl cyclase insensitive to the nucleotide inhibitor, 2-chloro-ATP (2ClATP). Immunopurificd GCA is insensitive to 2ClATP suggesting additional factors are required for inhibition. Diarrhea induced by E. coli heat-stable enterotoxin (STa) is mediated by guanylyl cyclase C (GCC). Incubation of Caco 2 human intestinal epithelial cells with 2-chloroadenosine (2ClAdo) prevented STa dependent cGMP production and associated chloride (Cl-) flux which underlies intestinal secretion. Inhibition was concentration and time dependent correlating with the metabolic conversion of 2ClAdo to 2ClATP. Guanylyl cyclase activity in membranes prepared from 2ClAdo treated cells was inhibited, compared to membranes from control cells, demonstrating a noncompetitive mechanism. Treatment of Caco 2 cells with 2ClAdo also prevented STa-induced Cl- flux. Incubation of 2ClAdo treated cells with the cell permeant analog 8Br-cGMP reconstituted the Cl- current, demonstrating that inhibition of Cl- flux reflected selective disruption of ligand stim-ulation of GCC rather than the chloride channel. Thus, the components required for adenine nucleotide inhibition of receptor guanylyl cyclase signaling are present in intact mammalian cells, establishing the utility of this pathway for elucidating the mechanisms regulating GCC and other receptor guanylyl cyclases.