Expression and characterization of protein kinase C in isolated rabbit parietal cells

We investigated the expression, characterization and distribution of protei n kinase C (PKC) isozymes in isolated rabbit parietal cells (PC). Cellular extracts of PC were analyzed by Western blot using isozyme-specific antibod ies. The Ca2+-independent PKC-epsilon was detected in cytosolic, membrane a nd cytoskeletal fractions of basal and histamine-stimulated PC whereas the Ca2+-dependent PKC-alpha was confined to the cytosolic and membrane fractio ns. Cytosolic and membrane fractions were partially purified by DEAE cellul ose column chromatography with elution of increasing NaCl concentration. El uates of 0.15 M and 0.3 M NaCl PC fractions were identified as PKC-alpha an d -epsilon isoforms, respectively. Phorbol 12-myristate 13-acetate (TPA) tr eatment of PC for 15, 30 and 60 sec decreased significantly cytosolic PKC-a lpha and increased membranc-associated PKC-alpha. In contrast to the distri bution of PKC-alpha, TPA did not alter membrane or cytosolic level of PKC-e psilon. Comparison of the dose-response curves between TPA-induced hydrogen (H+) secretion, as measured by aminopyrine (AP) uptake, and the membrane-a ssociated PKC-zeta suggests that translocation of PKC-alpha is not involved in the H+ secretory process in PC. Furthermore, a PKC inhibitor, staurospo rine, produced a concentration-dependent enhancement of histamine-stimulate d H+ secretion. These findings suggest that PKC-alpha plays a negative modu latory role, rather than an obligatory role, in H+ secretion. The localizat ion and distribution of PKC-epsilon into the cytoskeletal fraction of PC al so suggests that this isozyme may be involved in the cellular regulation of reversible morphological transformation during stimulation.