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Am J Physiol Gastrointest Liver Physiol 278: G24-G31, 2000;
0193-1857/00 $5.00
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Vol. 278, Issue 1, G24-G31, January 2000

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

N. Pausawasdi, S. Ramamoorthy, V. Stepan, J. del Valle, and A. Todisco

Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0682

We examined the regulation and functional role of p38 kinase in gastric acid secretion. p38 kinase was immunoprecipitated from cell lysates of highly purified gastric parietal cells in primary culture, and its activity was quantitated by in vitro kinase assay. Carbachol effects were dose- and time-dependent, with a maximal 10-fold stimulatory effect detected after 30 min of incubation. SB-203580, a highly selective inhibitor of p38 kinase, blocked carbachol induction of p38 kinase activity, with maximal inhibition at 10 µM. Stimulation by carbachol was unaffected by preincubation of parietal cells with the intracellular Ca2+ chelator BAPTA-AM, but incubation of cells in Ca2+-free medium led to a 50% inhibition of carbachol induction of p38 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 carbachol induction of p38 kinase activity. We tested the effect of exoenzyme C3 from Clostridium botulinum (C3), a toxin known to ADP-ribosylate and specifically inactivate Rho. C3 led to complete ADP-ribosylation of Rho, and it inhibited carbachol induction of p38 kinase by 50%. We then tested the effect of SB-203580 and C3 on carbachol-stimulated uptake of [14C]aminopyrine (AP). Inhibition of p38 kinase by SB-203580 led to a dose-dependent increase in AP uptake induced by carbachol, with maximal (threefold) effect at 10 µM SB-203580. Similarly, preincubation of parietal cells with C3 led to a twofold increase in AP uptake induced by carbachol. Thus carbachol induces a cascade of events in parietal cells that results in activation of p38 kinase through signaling pathways that are at least in part dependent on Rho activation and on the presence of extracellular Ca2+. p38 kinase appears to inhibit gastric acid secretion.

gastric acid secretion; protein kinases; mitogen-activated protein kinase/extracellular signal-regulated protein kinase; GTP-binding proteins; Rho proteins; cellular cytoskeleton.


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