Protein kinase C expression and translocation in dispersed chief cells from guinea-pig stomach.
The protein kinase C (PKC) family of enzymes is comprised of at least nine isoforms that vary with respect to co-factor dependence, cellular distribution and substrate specificity. Using specific antibodies for alpha, beta, gamma, delta, epsilon, zeta and eta PKC isoforms, and Western blot analysis, we found that alpha and zeta PKC are expressed in gastric chief cells. We then used these methods to examine the effects of carbamylcholine, a cholinergic agonist that increases cellular calcium and diacylglycerol concentrations, and PMA, a phorbol ester that activates PKC, on the subcellular distribution of these isoforms. Carbamylcholine and PMA caused an increase in membrane-associated alpha PKC, but did not alter the subcellular distribution of zeta PKC. Comparison of the dose-response curves for carbamylcholine-induced pepsinogen secretion and alpha PKC membrane-association indicates that PKC translocation is not required for carbamylcholine-induced secretion. Nevertheless, maximal carbachol-induced secretion occurs at concentrations that also cause translocation of the alpha isoform. Whereas treatment of chief cells with PMA (300 nM) for 4 h down-regulated levels of alpha PKC by 61%, there was no change in the levels of zeta PKC. Separation of the two PKC isoforms in chief cell lysates by DEAE-column chromatography revealed that kinase activity in fractions containing the alpha isoform was increased more than 3-fold by calcium and lipids. In contrast, kinase activity in fractions containing the zeta isoform was not altered. In gastric chief cells, translocation and activation of alpha PKC occurs in response to agonist-induced increases in calcium and diacylglycerol. Zeta PKC may be involved in the regulation of basal pepsinogen secretion.
Raffaniello
R D
RD
Department of Medicine, State University of New York-Health Science Center at Brooklyn 11203-2098.
Raufman
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D015536:Down-Regulation; D005753:Gastric Mucosa / Q000187:drug effects / Q000201:enzymology*; D006168:Guinea Pigs; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000819:agonists / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007527:Isoenzymes; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium