Protein kinase C-induced phosphorylation modulates the Na(+)-ATPase activity from proximal tubules.
This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from renal proximal tubule basolateral membranes (BLM) by protein kinase C (PKC). Two PKC isoforms were identified in BLM, one of 75 kDa and the other of 135 kDa. The former correlates with the PKC isoforms described in the literature but the latter seems to be a novel isoform, not yet identified. Both PKC isoforms of BLM are functional since a protein kinase C activator, TPA, increased the total hydroxylamine-resistant 32P(i) incorporation from [gamma-32P]ATP into the BLM. In parallel, TPA stimulated the Na(+)-ATPase activity from BLM in a dose-dependent manner, the effect being reversed by the PKC inhibitor sphingosine. The stimulatory effect of TPA on Na(+)-ATPase involved an increase in the V(max) (from 13.4+/-0.6 nmol P(i) mg(-1) min(-1) to 25.2+/-1.4 nmol P(i) mg(-1) min(-1), in the presence of TPA, P<0.05) but did not change the apparent affinity for Na(+) (K(0.5)=14.5+/-2.1 mM in control and 10.0+/-2.1 mM in the presence of TPA, P>0.07). PKC involvement was further confirmed by stimulation of the Na(+)-ATPase activity by the catalytic subunit of PKC (PKC-M). Finally, the phosphorylation of an approx. 100 kDa protein in the BLM (the suggested molecular mass of Na(+)-ATPase [1]) was induced by TPA. Taken together, these findings indicate that PKCs resident in BLM stimulate Na(+)-ATPase activity which could represent an important mechanism of regulation of proximal tubule Na(+) reabsorption.
Rangel
L B
LB
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS-Bloco G, 21949-900, Rio de Janeiro, RJ, Brazil.
Malaquias
A T
AT
Lara
L S
LS
Silva
I V
IV
De Souza
A M
AM
Lopes
A G
AG
Caruso-Neves
C
C
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D027682:Cation Transport Proteins; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000032:analysis; D007687:Kidney Tubules, Proximal / Q000201:enzymology / Q000378:metabolism*; D007700:Kinetics; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis / Q000378:metabolism*; D015398:Signal Transduction; D012964:Sodium / Q000378:metabolism; D013552:Swine
D027682:Cation Transport Proteins; D007527:Isoenzymes; D012964:Sodium; D011493:Protein Kinase C; D000251:Adenosine Triphosphatases; C021838:sodium-translocating ATPase