Ca2+-independent protein kinase C isoforms may modulate parietal cell HCl secretion.
Although activation of adenosine 3',5'-cyclic monophosphate by histamine and of Ca2+-dependent signaling pathways by cholinergic agonists is a generally recognized mechanism for increasing parietal cell HCl secretion, the role of protein kinase C (PKC) in this process is controversial. In this study, acid-secretory responses of gastric glands from rabbits [measured as accumulation of aminopyrine (AP)] were found to be relatively resistant to the PKC inhibitors calphostin C, chelerythrine chloride, staurosporine, and the bisindolylmaleimide-like inhibitors Ro 31-8220, Gö 6976, and bisindolylmaleimide I hydrochloride. Western analyses of the PKC isozyme profile in highly enriched parietal cells (98% purity) indicated that this cell type expresses abundant levels of the novel isoforms PKC-epsilon and PKC-mu and abundant levels of the atypical isoforms PKC-iota, PKC-lambda, and PKC-zeta. In contrast, there appeared to be low to undetectable expression of the classical isoforms PKC-alpha and PKC-beta1/beta2, respectively. Relatively high concentrations of Ro 31-8220 potentiated both carbachol- and histamine-stimulated AP accumulation (IC50 857 +/- 100 and 910 +/- 98 nM, respectively). There was a similar dose dependence for Ro 31-8220 inhibition of in situ phosphorylation of a parietal cell phosphoprotein, pp66 (IC50 750 +/- 120 nM). Similar concentrations of Ro 31-8220 also inhibited phosphorylation of the cytoskeletal, actin membrane cross-linking phosphoprotein ezrin, but not other phosphoproteins. Ezrin phosphorylation was increased by carbachol and 12-O-tetradecanoylphorbol 13-acetate (TPA). Because carbachol and TPA stimulate pp66 phosphorylation in a Ca2+-independent manner, our results suggest that one or more novel PKC isoforms may be involved in negative regulation of HCl secretion. In related experiments, PKC-epsilon, but not PKC-mu, was immunolocalized by confocal microscopy to a parietal cell compartment that bore a striking resemblance to that containing filamentous actin. Moreover, pp66 was enriched in a Triton X-100-insoluble parietal cell fraction, suggesting a potential cytoskeletal localization for this unknown protein. Given their location and sensitivity to Ro 31-8220, it is possible that pp66 and ezrin interact in a PKC-dependent manner to regulate the well-known morphological changes that occur in concert with agonist-dependent activation of parietal cell HCl secretion.
Chew
C S
CS
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta 30912-3175, USA.
Zhou
C J
CJ
Parente
J A
JA
D000199:Actins / Q000378:metabolism; D000632:Aminopyrine / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000502:physiology*; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006851:Hydrochloric Acid / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000502:physiology*; D008297:Male; D010295:Parietal Cells, Gastric / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D011817:Rabbits; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D000099149:Ezrin
D000199:Actins; D000632:Aminopyrine; D002118:Calcium; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D006851:Hydrochloric Acid; D007211:Indoles; D007527:Isoenzymes; D010750:Phosphoproteins; D011493:Protein Kinase C; D000099149:Ezrin; C064758:Ro 31-8220