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 surface and thus is critical for vectorial solute transport. One potential mode of regulation of Na/K-ATPase involves the intracellular effector protein kinase C (PKC). In kidney cell lines, activation of PKC by the phorbol ester phorbol 12,13-dibutyrate (PDBu) (1 μM) inhibited Na/K-ATPase transport activity in OK cells (V_max decreased 42%; p < 0.02), but not in LLC-PK₁ cells. By immunoblot, both cell types expressed detectable levels of PKCa and PKCζ. In response to PDBu, PKCα translocated from the cytosol to the membrane fractions of both cell lines. Phorbol ester treatment increased incorporation of ³²PO₄ in multiple substrates in both cell types, but a ∼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 all known α isoforms of Na/K-ATPase, recognized a ∼109-kDa membrane protein from both cell lines. Anti-LEAVE also identified a protein that comigrated with the large phosphoprotein which was only present in OK cells. Following ³²PO₄ loading and PDBu treatment, anti-LEAVE immunoprecipitated a ∼109-kDa phosphoprotein in OK but not LLC-PK₁ cells. These data support the notion that PKC is capable of phosphorylating the α subunit and inhibiting Na/K-ATPase transport activity in intact renal cells. Furthermore, they suggest that some forms of Na/K-ATPase in the kidney are not susceptible to PKC phosphorylation and that this heterogeneity may contribute to response diversity.