Evidence for the presence of cGMP-dependent protein kinase-II in human distal colon and in T84, the colonic cell line.
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.
Selvaraj
N G
NG
Department of Physiology and Biophysics, University of Illinois, Chicago, 60612-7342, USA.
Prasad
R
R
Goldstein
J L
JL
Rao
M C
MC
D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D003106:Colon / Q000201:enzymology*; D006152:Cyclic GMP / Q000378:metabolism; D062967:Cyclic GMP-Dependent Protein Kinase Type II; D017869:Cyclic GMP-Dependent Protein Kinases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004768:Enterotoxins / Q000494:pharmacology; D005456:Fluorescent Dyes; D006162:Guanylate Cyclase / Q000235:genetics; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D028021:Sodium-Potassium-Chloride Symporters
C505032:CFTR protein, human; D002352:Carrier Proteins; D002712:Chlorides; D004768:Enterotoxins; D005456:Fluorescent Dyes; D028021:Sodium-Potassium-Chloride Symporters; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D062967:Cyclic GMP-Dependent Protein Kinase Type II; D017869:Cyclic GMP-Dependent Protein Kinases; C569729:PRKG2 protein, human; D006162:Guanylate Cyclase; D006152:Cyclic GMP