Effects of okadaic acid, calyculin A, and PDBu on state of phosphorylationof rat renal Na+-K+-ATPase

Several indirect lines of evidence suggest that protein kinases and phospha tases modulate the activity of renal Na+-K+-ATPase. The aim of this study w as to examine whether such regulation may occur via modulation of the state of phosphorylation of Na+-K+-ATPase. Slices from rat renal cortex were pre labeled with [P-32]orthophosphate and incubated with the inhibitors of prot ein phosphatase (PP)-1 and PP-2A, okadaic acid (OA) and calyculin A (CL-A), respectively, the protein kinase C (PKC) activator, phorbol 12,13-dibutyra te (PDBu), or the PP-2B inhibitor, FK-506. Phosphorylation of Na+-K+-ATPase alpha-subunit was evaluated by measuring the amount of [P-32]phosphate inc orporation into the immunoprecipitated protein. Incubation with either OA, CL-A, or PDBu caused four- to fivefold increases in the amount of [P-32]pho sphate incorporation into immunoprecipitated Na+-K+-ATPase alpha-subunit. O A and PDBu had a synergistic effect on the state of phosphorylation of Na+- K+-ATPase alpha-subunit. FK-506 did not affect Na+-K+-ATPase phosphorylatio n, neither alone nor in the presence of PDBu. Each of the drugs, OA, CL-A, and PDBu, inhibited the activity of Na+-K+-ATPase in microdissected proxima l tubules. PDBu potentiated OA-induced inhibition of Na+-K+-ATPase activity . Inhibition of Na+-K(+)ATPase required a lower dose of CL-A than of OA. On the basis of the inhibitory constant values of CL-A and OA for PP-1 and PP -BA, it is concluded that the tubular effect is mainly due to inhibition of PP-1. The PP-1 activity in rat renal cortex was similar to 1.5 nmol P-i.mg protein(-l) min(-1). Using a monoclonal anti-a antibody that fails to reco gnize the subunit when Ser(23) is phosphorylated by PKC, we demonstrated th at the dose response of PDBu inhibition of Na+-K+-ATPase correlated with th e dose response of phosphorylation of the enzyme. The results suggest that the state of phosphorylation and activity of proximal tubular Na+-K+-ATPase are determined by the balance between the activities of protein kinases an d phosphatases.