Cyclic nucleotide-dependent protein kinase inhibition by H-8: effects on ion transport.

We explored the potential role of cyclic nucleotide-dependent protein phosphorylation in regulating ion transport across flounder intestinal mucosa by studying the effects of N-[2(methylamino)-ethyl]-s-isoquinolinesulfonamide (H-8), a selective inhibitor of cyclic nucleotide-dependent protein kinase in vitro. Addition of H-8 reversed the inhibitory effects of 8-bromoguanosine 3',5'-cyclic-monophosphate (8-BrcGMP), 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), atriopeptin III (AP III), and vasoactive intestinal peptide (VIP) on the short-circuit current (Isc) and transepithelial potential difference (PD). Flux measurements established that these changes in Isc and PD directly reflected changes in Na and Cl absorption by the intestine. H-8 was unable, however, to reverse the inhibitory effects on Isc and PD of the Ca ionophore ionomycin and of substance P at dosages exceeding those needed to reverse the effects of AP III, VIP, and the cyclic nucleotides. We conclude that 1) H-8 (100 microM or less) does not exert toxic effects, 2) exogenously added cyclic nucleotide analogues inhibit ion transport through activation of cyclic nucleotide-dependent kinases resulting in protein phosphorylation, 3) activation of these kinases is an essential intermediate step in the inhibitory action of AP III and VIP on ion transport, and 4) the Ca ionophore ionomycin and substance P appear to inhibit ion transport by a mechanism that is independent of cyclic nucleotide-dependent protein phosphorylation.