Involvement of protein kinase C in the mechanism of action of Escherichia coli heat-stable enterotoxin (STa) in a human colonic carcinoma cell line, COLO-205.
The present study was undertaken to determine the involvement of calcium-protein kinase C pathway in the mechanism of action of Escherichia coli heat stable enterotoxin (STa) apart from STa-induced activation of guanylate cyclase in human colonic carcinoma cell line COLO-205, which was used as a model cultured cell line to study the mechanism of action of E. coli STa. In response to E. coli STa, protein kinase C (PKC) activity was increased in a time-dependent manner with its physical translocation from cytosol to membrane. Inhibition of the PKC activity in membrane fraction and inhibition of its physical translocation in response to IP3-mediated calcium release inhibitor dantrolene suggested the involvement of intracellular store depletion in the regulation of PKC activity. Among different PKC isoforms, predominant involvement of calcium-dependent protein kinase C (PKC(alpha)) was specified using isotype-specific pseudosubstrate, which showed pronounce enzyme activity. Inhibition of enzyme activity by PKC(alpha)-specific inhibitor Gö6976 and immunoblott study employing isotype-specific antibody further demonstrated the involvement of calcium-dependent isoform of PKC in the mechanism of action of E. coli STa. Moreover, inhibition of guanylate cyclase activity by PKC(alpha)-specific inhibitor Gö6976 suggested the involvement of PKC(alpha) in the regulation of guanylate cyclase activity.
Gupta
Dyuti Datta
DD
Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, P-33, C. I. T. Road, Scheme-XM, Beliaghata, Kolkata-700 010, India.
Saha
Subhrajit
S
Chakrabarti
Manoj K
MK
D001427:Bacterial Toxins / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology*; D002227:Carbazoles / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004768:Enterotoxins / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D029968:Escherichia coli Proteins; D006162:Guanylate Cyclase / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*
D001427:Bacterial Toxins; D002227:Carbazoles; D004768:Enterotoxins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D007211:Indoles; D007527:Isoenzymes; C034151:heat stable toxin (E coli); C081021:Go 6976; D011493:Protein Kinase C; D006162:Guanylate Cyclase; D002118:Calcium