HETEROGENEITY OF PROTEIN KINASE-C-MEDIATED RAPID REGULATION OF NA K-ATPASE IN KIDNEY EPITHELIAL-CELLS/

Na/K-ATPase in renal epithelium is expressed at the basolateral surfac e and thus is critical for vectorial solute transport. One potential m ode of regulation of Na/K-ATPase involves the intracellular effector p rotein kinase C (PKC). In kidney cell lines, activation of PKC by the phorbol ester phorbol 12,13-dibutyrate (PDBu) (1 muM) inhibited Na/K-A TPase transport activity in OK cells (V(max) decreased 42%; p < 0.02), but not in LLC-PK1 cells. By immunoblot, both cell types expressed de tectable levels of PKCalpha and PKCzeta. In response to PDBu, PKCalpha translocated from the cytosol to the membrane fractions of both cell lines. Phorbol ester treatment increased incorporation of (PO4)-P-32 i n multiple substrates in both cell types, but a approximately 109-kDa substrate with neutral pI was detected only in the OK cell. Anti-LEAVE , directed against a highly conserved sequence in the H4-H5 loop of al l known a isoforms of Na/K-ATPase, recognized a approximately 109-kDa membrane protein from both cell lines. Anti-LEAVE also identified a pr otein that comigrated with the large phosphoprotein which was only pre sent in OK cells. Following (PO4)-P-32 loading and PDBu treatment, ant i-LEAVE immunoprecipitated a approximately 109-kDa phosphoprotein in O K but not LLC-PK, cells. These data support the notion that PKC is cap able of phosphorylating the a subunit and inhibiting Na/K-ATPase trans port activity in intact renal cells. Furthermore, they suggest that so me forms of Na/K-ATPase in the kidney are not susceptible to PKC phosp horylation and that this heterogeneity may contribute to response dive rsity.