Parietal cell MAP kinases: multiple activation pathways.
Epidermal growth factor (EGF) is a potent mitogen for many cell types; however, the best known effect of EGF on gastric parietal cell HCl secretion is inhibition of this response. Using rabbit parietal cells in primary culture, we recently showed that the effect of EGF is biphasic with acute inhibition followed by sustained enhancement of acid secretory-related responses. We hypothesized that EGF might activate a mitogen-activated protein (MAP) kinase signaling pathway in parietal cells, and this pathway might play a role in mediating sustained and/or acute effects of EGF on parietal cell acid secretory-related functions [C. S. Chew, K. Nakamura, and A. C. Petropolous. Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G818-G826, 1994]. We used several methodological approaches to demonstrate the presence of MAP kinase (MAPK) isoforms, extracellular signal-regulated kinases (ERKs) 1 and 2, in parietal cells and to begin to characterize their mechanisms of activation in this highly differentiated cell type. In acutely isolated, 90-98% enriched parietal cells, EGF biphasically activated ERK-1 and ERK-2, with peak response occurring at approximately 5 min followed by a sustained lower level of activation for at least 2 h. The EC50 for EGF (1.2 +/- 0.4 nM) was similar to the previously determined EC50 for the stimulatory effect of EGF on acid secretory responses. In contrast to EGF, the phorbol ester protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) induced a sustained activation of ERK-1 and ERK-2 for at least 2 h. Carbachol also activated ERK-1 and ERK-2; however, this response was weaker and monophasic. Neither the Ca2+ ionophore ionomycin nor the adenylyl cyclase activator forskolin altered basal or stimulated ERK activity. Carbachol, but not EGF or TPA, also activated an unidentified 70-kDa protein kinase as detected with in-gel myelin basic protein (MBP) kinase renaturation assays. Parietal cell MAPK activation was not correlated to a shift in apparent relative molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, suggesting that basal phosphorylation of ERK isoforms may be higher in parietal cells compared with actively proliferating cell lines. Also, in contrast to observations in neutrophils, the phosphatidylinositol 3-kinase (PtdIns 3-kinase) inhibitor, wortmannin (0.3-3 microM), failed to inhibit ERK activation in response to EGF, carbachol, or TPA. The combined data indicate that 1) EGF, TPA, and carbachol activate overlapping as well as distinct intracellular signaling pathways in gastric parietal cells, 2) EGF activates ERKs and enhances parietal cell acid secretory related functions via receptors with similar affinities, and 3) in contrast to some cell types, the parietal cell ERK-signaling cascade does not appear to be directly modulated by the PtdIns 3-kinase pathway or by elevated intracellular free Ca2+ or adenosine 3',5'-cyclic monophosphate concentrations.
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta 30912-3175, USA.
K
K
Nakamura
C J
CJ
Zhou
J
J
Parente
C S
CS
Chew
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005753:Gastric Mucosa / Q000201:enzymology*; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010277:Parasympathomimetics / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011817:Rabbits; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D007527:Isoenzymes; D010277:Parasympathomimetics; D010750:Phosphoproteins; D019000:Phosphotyrosine; D004815:Epidermal Growth Factor; D002217:Carbachol; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D000077191:Wortmannin