Regulation and function of p38 protein kinase in isolated canine gastric parietal cells

We examined the regulation and functional role of p38 kinase in gastric aci d secretion. p38 kinase was immunoprecipitated from cell lysates of highly purified gastric parietal cells in primary culture, and its activity was qu antitated by in vitro kinase assay. Carbachol effects were dose- and time-d ependent, with a maximal 10-fold stimulatory effect detected after 30 min o f incubation. SB-203580, a highly selective inhibitor of p38 kinase, blocke d carbachol induction of p38 kinase activity, with maximal inhibition at 10 mu M. Stimulation by carbachol was unaffected by preincubation of parietal cells with the intracellular Ca2+ chelator BAPTA-AM, but incubation of cel ls in Ca2+-free medium led to a 50% inhibition of carbachol induction of p3 8 kinase activity. Because some of the effects of carbachol are mediated by the small GTP-binding protein Rho, we examined the role of Rho in carbacho l induction of p38 kinase activity. We tested the effect of exoenzyme C3 fr om Clostridium botulinum (C3), a toxin known to ADP-ribosylate and specific ally inactivate Rho. C3 led to complete ADP-ribosylation of Rho, and it inh ibited carbachol induction of p38 kinase by 50%. We then tested the effect of SE-203580 and C3 on carbachol-stimulated uptake of [C-14]aminopyrine (AP ). Inhibition of p38 kinase by SB-203580 led to a dose-dependent increase i n AP uptake induced by carbachol, with maximal (threefold) effect at 10 mu M SB-203580. Similarly, preincubation of parietal cells with C3 led to a tw ofold increase in AP uptake induced by carbachol. Thus carbachol induces a cascade of events in parietal cells that results in activation of p38 kinas e through signaling pathways that are at least in part dependent on Rho act ivation and on the presence of extracellular Ca2+. p38 kinase appears to in hibit gastric acid secretion.