Different actions of protein kinase C isoforms α and ε on gastric acid secretion

The phorbol ester TPA, an activator of protein kinase C (PKC), inhibits cholinergic stimulation of gastric acid secretion but increases basal H+ secretion. Since these contradictory findings suggest the action of different PKC isozymes we analysed the role of calcium-dependent PKC-α, and calcium-independent PKC-e in gastric acid secretion. Inhibition of PKC-α by the indolocarbazole Go 6976 revealed that about 28% of carbachol-induced acid secretion was inhibited by PKC-α. In the presence of Go 6976 approximately 64% of the carbachol-induced signal transduction is mediated by Ca2+/calmodulin-dependent protein kinase II (CaMKII), and 14% is conveyed by PKC-e as deduced from the inhibition with the bisindolylmaleimide Ro 31-8220. Inhibition of carbachol-induced acid secretion by TPA was accompanied by a decrease in CaMKII activity. The stimulation of basal acid secretion by TPA was biphasic with a peak at a very low concentration (10 pM), resulting in an activation of the calcium-sensor CaMKII. The activation was determined with a phosphospecific polyclonal antibody against active CaMKII. The TPA-induced increase of H+ secretion was sensitive to the cell-permeable Ca2+-chelator BAPTA/AM, Ro 31-8220, and the CaMKII-inhibitor KN-62, but not to Go 6976. Since TPA induced the translocation of PKC-e but not of PKC-α in resting parietal cells, PKC-e seems to be at least responsible for an initial elevation of free intracellular calcium to initiate TPA-induced acid secretion. Our data indicate the different roles of two PKC isoforms: PKC-e activation appears to facilitate cholinergic stimulation of H+-secretion likely by increasing intracellular calcium. In contrast, PKC-α activation attenuates acid secretion accompanied by a down-regulation of CaMKII activity. Keywords: Bisindolylmaleimide, Ca2+/calmodulin-dependent protein kinase, calcium, carbachol, gastric acid secretion, muscarinic acetylcholine receptor, phorbol ester, protein kinase C, protein kinase inhibition, signal transduction Introduction Protein kinase C (PKC) comprises a family of multifunctional serine/threonine protein kinases which are ubiquitously distributed in a variety of eukaryotic cells. The PKC family is subdivided into three groups. The calcium- and diacylglycerol-dependent or ‘conventional' PKC-isoforms (cPKC) are activated by Ca2+ and diacylglycerol or the phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate), a structural analogue of diacylglycerol. The ‘novel' PKC's (nPKC) are activated by diacylglycerol or TPA but not by Ca2+. Finally, there are the ‘atypical' PKC's (aPKC) which are not regulated by Ca2+ or diacylglycerol or TPA (for reviews, see Nishizuka, 1992; Newton, 1997). In the case of rabbit gastric parietal cells, PKC-α is the only member of cPKC which could be detected by immunoblot analysis, and PKC-e the only member of nPKC, respectively (Nandi et al., 1996; Chew et al., 1997). There also appears to be at least two atypical PKC isoforms (ι, λ) expressed in parietal cells. The expression of PKC-ζ is uncertain, as reports are contradictory (Nandi et al., 1996; Chew et al., 1997). A related enzyme, PKC-μ or PKD, which is also expressed in rabbit parietal cells (Chew et al., 1997), displays multiple unique properties that makes it a distant relative of the PKC isozymes (Johannes et al., 1994). cPKC is capable of differentiating between different time-courses of [Ca2+]I (concentration of free intracellular Ca(II)-ions) and diacylglycerol. The rapid activation of cPKC by calcium is followed by the binding of diacylglycerol which potentiates the binding affinity of cPKC with the plasma membrane, and prolongs the kinase activity even after a decrease of [Ca2+]i (Oancea & Meyer, 1998). Upon stimulation of non-excitable secretory cells, PKC translocates from the cytosol to the cell membrane to be activated and targeted to the proximity of its substrate (Akita et al., 1994; Bastani et al., 1995; Hong et al., 1997; Yedovitzky et al., 1997). The involvement of PKC during H+ secretion has been extensively investigated especially by the use of the phorbol ester TPA to directly activate PKC (Anderson & Hanson, 1985; Muallem et al., 1986; Brown & Chew, 1987; Beil et al., 1987; Chiba et al., 1989; Nandi et al., 1996; Chew et al., 1997; Kopp & Pfeiffer, 2000). PKC inhibitors were also utilized to analyse the function of PKC in parietal cells (McKenna & Hanson, 1993; Chew et al., 1997). However, the data with regard to the role of PKC are conflicting, as either a stimulatory or inhibitory function of PKC in cholinergic stimulation of acid secretion was observed (reviewed in Fahrmann, 2000). Although some of those contradictory results may derive from the action of different PKC isoforms, the specific function of these isoforms in acid secretion has been rarely investigated. To show that different PKC-isoforms are effectively involved in gastric acid secretion we address the action of PKC-α and PKC-e in response to the muscarinic agent carbachol (2-hydroxyethyl)trimethylammonium chloride carbamate) in gastric parietal cells. The M3 acetylcholine muscarinic receptor is coupled to heterotrimeric Gq and phospholipase C. Cholinergic stimulation increases both intracellular calcium by action of inositol 1,4,5-trisphosphate, and diacylglycerol (Brown & Chew, 1989; Pfeiffer et al., 1989; 1990; Seidler & Pfeiffer, 1991; Kajimura et al., 1992) as well as acid secretion. As yet, the understanding of the molecular mechanism how [Ca2+]i and diacylglycerol mediate H+ secretion is incomplete.