Carbachol activates IkappaB kinase in isolated canine gastric parietal cells.
IkappaB kinase (IKK) is a recently discovered kinase complex composed of the kinases IKKalpha and beta, which plays a crucial role in the activation of NF-kappaB. In this study we examined the regulation of IKK by carbachol in isolated gastric parietal cells. IKKalpha and beta activities were measured by immune complex kinase assay. Carbachol induced both IKK alpha and beta in a time-dependent fashion, with a maximal stimulatory effect detected after 5 min of incubation. The action of carbachol was inhibited by the intracellular Ca(++) chelator BAPTA-AM, the PKC inhibitor GF109203X, and the NF-kappaB inhibitor PDTC. Carbachol also induced degradation of IkappaBalpha, which was reversed by addition of both GF109203X and PDTC and stimulated the activity of a NF-kappaB-luciferase reporter gene plasmid in COS-7 cells stably expressing the human M3 muscarinic receptor. In conclusion, carbachol induces IKK in the parietal cells via intracellular Ca(++)- and PKC-dependent signaling pathways. This observation represents a novel mechanism for the regulation of NF-kappaB through the activation of seven transmembrane G-protein-coupled receptors.
Todisco
A
A
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan, 48109-0682, USA.
Ramamoorthy
S
S
Pausawasdi
N
N
Tacey
K
K
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D004285:Dogs; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D007163:Immunosorbent Techniques; D007700:Kinetics; D018721:Muscarinic Agonists / Q000494:pharmacology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D010295:Parietal Cells, Gastric / Q000201:enzymology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000502:physiology
D002614:Chelating Agents; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D018721:Muscarinic Agonists; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D011976:Receptors, Muscarinic; D000072000:NF-KappaB Inhibitor alpha; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D002217:Carbachol; D017346:Protein Serine-Threonine Kinases; D051550:I-kappa B Kinase; D011493:Protein Kinase C; D002118:Calcium