Expression and characterization of protein kinase C in isolated rabbit parietal cells.
We investigated the expression, characterization and distribution of protein kinase C (PKC) isozymes in isolated rabbit parietal cells (PC). Cellular extracts of PC were analyzed by Western blot using isozyme-specific antibodies. The Ca2+-independent PKC-epsilon was detected in cytosolic, membrane and cytoskeletal fractions of basal and histamine-stimulated PC, whereas the Ca2+-dependent PKC-alpha was confined to the cytosolic and membrane fractions. Cytosolic and membrane fractions were partially purified by DEAE cellulose column chromatography with elution of increasing NaCl concentration. Eluates of 0.15 M and 0.3 M NaCl PC fractions were identified as PKC-alpha and -epsilon isoforms, respectively. Phorbol 12-myristate 13-acetate (TPA) treatment of PC for 15, 30 and 60 sec decreased significantly cytosolic PKC-alpha and increased membrane-associated PKC-alpha. In contrast to the distribution of PKC-alpha, TPA did not alter membrane or cytosolic level of PKC-epsilon. Comparison of the dose-response curves between TPA-induced hydrogen (H+) secretion, as measured by aminopyrine (AP) uptake, and the membrane-associated PKC-alpha suggests that translocation of PKC-alpha is not involved in the H+ secretory process in PC. Furthermore, a PKC inhibitor, staurosporine, produced a concentration-dependent enhancement of histamine-stimulated H+ secretion. These findings suggest that PKC-alpha plays a negative modulatory role, rather than an obligatory role, in H+ secretion. The localization and distribution of PKC-epsilon into the cytoskeletal fraction of PC also suggests that this isozyme may be involved in the cellular regulation of reversible morphological transformation during stimulation.
Nandi
J
J
Department of Medicine, Division of Gastroenterology, State University of New York Health Science Center, Syracuse, NY 13210, USA.
Loo
A
A
Kim
S W
SW
Levine
R A
RA
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000632:Aminopyrine / Q000493:pharmacokinetics; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002927:Cimetidine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D010295:Parietal Cells, Gastric / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D011522:Protons; D011817:Rabbits; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011522:Protons; D000632:Aminopyrine; D002927:Cimetidine; D006632:Histamine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium