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The phorbolester 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C (PKC), inhibits cholinergic stimulation of gastric acid secretion. We observed that this effect strongly correlated with the inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity in rabbit parietal cells. The aim of this study was to specify the function of PKC-α in cholinergically stimulated H+ secretion. PKC-α represents the only calcium-dependent PKC isoenzyme that has been detected in rabbit parietal cells. Go 6976, an inhibitor of calcium-dependent PKC, concentration-dependently antagonized the inhibitory effect of TPA, and, therefore, revealed the action of PKC-α on carbachol-induced acid secretion in rabbit parietal cells. TPA exerted no additive inhibition of carbachol-stimulated acid secretion if acid secretion was partially inhibited by the potent CaMKII inhibitor 1-[N,O-bis(5-isoquinolinsulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine (KN-62). Since both kinase modulators, TPA and KN-62, affected no divergent signal transduction pathways in the parietal cell, an in vitro model has been used to study if PKC directly targets CaMKII. CaMKII purified from parietal cell-containing gastric mucosa of pig, was transphosphorylated by purified cPKC containing PKC-α up to 1.8 mol Pi per mol CaMKII in vitro. The autonomy site of CaMKII was not transphosphorylated by PKC. The phosphotransferase activity of the purified CaMKII was in vitro inhibited after transphosphorylation by PKC if calmodulin was absent during transphosphorylation. Attenuation of CaMKII activity by PKC showed strong similarity to the downregulation of CaMKII by basal autophosphorylation. Our results suggest that PKC-α and CaMKII are closely functionally linked in a cholinergically induced signalling pathway in rabbit parietal cells. We assume that in cholinergically stimulated parietal cells PKC-α transinhibits CaMKII activity, resulting in an attenuation of acid secretion. British Journal of Pharmacology (2003) 139, 545–554. doi:10.1038/sj.bjp.0705211 |
