Effects of okadaic acid, calyculin a and pdbu on the state of
phosphorylation of rat renal na+-k+-atpase.
Li, Dailin ), Sam Xian Jun Cheng(), Gilberto Fisone(), Michael J.
Caplan(), Yoshiyuki Ohtomo(), and Anita Aperia(),.
Departments of Woman and Child Health, Pediatric Unit(1),, and
Neuroscience(2), Karolinska Institute, Stockholm, Sweden, and
Department of Cellular and Molecular Physiology, Yale University
School of Medicine, New Haven, Connecticut 06510(3).
APStracts 5:0144F, 1998.
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), the protein kinase C
(PKC) activator, phorbol 12, 13-dibutyrate (PDBu), or the PP-2B
inhibitor, FK506. Phosphorylation of Na+-K+-ATPase a subunit was
evaluated by measuring the amount of 32P-phosphate incorporation into
the immunoprecipitated protein. Incubation with either OA, CL-A or
PDBu caused 4- to 5-fold increase in the amount of 32P-phosphate
incorporation into immunoprecipitated Na+-K+-ATPase a subunit. OA and
PDBu had a synergistic effect on the state of phosphorylation of Na+
-K+-ATPase a subunit. FK506 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 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/min. Using a monoclonal anti-a 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.
Received 4 June 1996; accepted in final form 17 August 1998.
APS Manuscript Number F165-6.
Article publication pending Am. J. Physiol. (Renal Physiology).
ISSN 1080-4757 Copyright 1998 The American Physiological Society.
Published in APStracts on 21 September 1998