ROLE OF A NOVEL GTP-SENSITIVE APICAL CHLORIDE CHANNEL IN CHOLERATOXIN- AND CYCLIC AMP-PROVOKED INTESTINAL CI' SECRETION AND ITS RELEVANCE IN CYSTIC FIBROSIS

Abstract

Chloride conductance measurements in apical membrane vesicles isolated from the HT-29 c.19A human colon carcinoma cell line and from rat enterocytes, and electrophysiological analysis of single Cl− channels in apical membrane patches excised from HT-29.cl 19A monolayers, revealed the existence of two major types of Cl− channels: (1) an outwardly rectifying 32 pS anion channel (A channel) activated by cyclic AMP and ATP in the presence of protein kinase A, and (2) an inwardly rectifying 20 pS channel (G channel) activated by GTPγS (10−5 M) and inhibited by GDPßS (10−4 M), indicative for the involvement of a GTP-binding protein in channel regulation. The GTP-activation of the G channel was inhibited by the pnospholipase A2 (PL-A2 inhibitor mepacrine (10−5 M) and mimicked by arachidonic acid (AA, 5.10−5 M), suggesting a role for PL-A2 and AA metabolites as coupling factors between the G-protein and the Cl− channel. Short circuit current (SCC) measurements on HT-29 monolayers and ileal mucosa from control and cystic fibrosis (CF) patients mounted in Ussing chambers showed that choleratoxin (CT)-and cyclic AMP-provoked active Cl− secretion was inhibited for 40-60% by mepacrine (2-5.10− M) and the lipoxygenase inhibitor NDGA (2.10−5 M) but was completely absent in CF patients carrying the major CF mutation (ΔF508). These data together suggest that (i) CT- and cyclic AMP-induced Cl− secretion is mediated in part by activation of PL-A2, resulting in AA release, formation of lipoxygenase products and activation of G channels; (ii) the residual Cl− secretion results from a cyclic AMP - and protein kinase A-induced phosphorylation and opening of apical A channels; (iii) since both types of Cl− channel apparently fail to respond to cAMP signals in CF, the CF gene-encoded protein (‘CFTR’) most likely functions as a common regulator of both channels rather than as a Cl− channel itself.