Effects of okadaic acid, calyculin A, and PDBu on state of phosphorylation of rat renal Na+-K+-ATPase.
Several indirect lines of evidence suggest that protein kinases and phosphatases modulate the activity of renal Na+-K+-ATPase. The aim of this study was to examine whether such regulation may occur via modulation of the state of phosphorylation of Na+-K+-ATPase. Slices from rat renal cortex were prelabeled with [32P]orthophosphate and incubated with the inhibitors of protein phosphatase (PP)-1 and PP-2A, okadaic acid (OA) and calyculin A (CL-A), respectively, the protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), or the PP-2B inhibitor, FK-506. Phosphorylation of Na+-K+-ATPase alpha-subunit was evaluated by measuring the amount of [32P]phosphate incorporation into the immunoprecipitated protein. Incubation with either OA, CL-A, or PDBu caused four- to fivefold increases in the amount of [32P]phosphate incorporation into immunoprecipitated Na+-K+-ATPase alpha-subunit. OA 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 phosphorylation, 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 proximal 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-2A, it is concluded that the tubular effect is mainly due to inhibition of PP-1. The PP-1 activity in rat renal cortex was approximately 1.5 nmol Pi. mg protein-1. min-1. Using a monoclonal anti-alpha antibody that fails to recognize the subunit when Ser23 is phosphorylated by PKC, we demonstrated that the dose response of PDBu inhibition of Na+-K+-ATPase correlated with the 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 and phosphatases.
Li
D
D
Departments of Woman and Child Health, Karolinska Institute, S-171 76 Stockholm, Sweden.
Cheng
S X
SX
Fisone
G
G
Caplan
M J
MJ
Ohtomo
Y
Y
Aperia
A
A
D000818:Animals; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology*; D008297:Male; D008387:Marine Toxins; D009419:Nerve Tissue Proteins / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology*; D010080:Oxazoles / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010750:Phosphoproteins; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D009419:Nerve Tissue Proteins; D010080:Oxazoles; D010750:Phosphoproteins; D019319:Okadaic Acid; D015240:Phorbol 12,13-Dibutyrate; C059041:calyculin A; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D000254:Sodium-Potassium-Exchanging ATPase