Heat-stable enterotoxin (STa) stimulates intestinal Cl(-) secretion by activating guanylate cyclase C (GCC) to increase intracellular cyclic GMP (cGMP). In the colon, cGMP action could involve protein kinase (PK) G-II or PKA pathways, depending on the segment and species. In the human colon, both PKG and PKA pathways have been implicated, and, therefore, the present study examined the mechanism of cGMP-mediated Cl(-) transport in primary cultures of human distal colonocytes and in T84, the colonic cell line. Both cell preparations express mRNA for CFTR, Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), GCC and PKG-II as detected by RT-PCR. The effects of STa and the PKG-specific cGMP analogues, 8Br-cGMP and 8pCPT-cGMP, on Cl(-) transport were measured using a halide-sensitive probe. In primary human colonocytes and T84 cells, STa, the cGMP analogues and the cAMP-dependent secretagogue, prostaglandin E(1) (PGE(1)), enhanced Cl(-) transport. The effects of 8Br-cGMP and 8pCPT-cGMP suggested the involvement of PKG, and this was explored further in T84 cells. The effects of 8pCPT-cGMP were dose-dependent and sensitive to the PKG inhibitor, H8 (70 microM), but H8 had no effect on PGE(1)-induced Cl(-) secretion. In contrast, a PKA inhibitor, H7 (50 microM), blocked PGE(1)-mediated but not 8pCPT-cGMP-induced Cl(-) transport. 8pCPT-cGMP enhanced phosphorylation of the PKG-specific substrate, 2A3, by T84 membranes in vitro. This phosphorylation was inhibited by H8. These results strongly suggest that cGMP activates Cl(-) transport through a PKG-II pathway in primary cells and in the T84 cell line of the human colon.