Ouabain-insensitive Na(+)-ATPase activity is an effector protein for cAMP regulation in basolateral membranes of the proximal tubule.
This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from proximal tubule basolateral membranes by cAMP. An increase in dibutyryl-cAMP (d-cAMP) concentration from 10(-8) to 5x10(-5) M stimulates the ouabain-insensitive Na(+)-ATPase activity. The ATPase activity increases from 6.0+/-0.4 to 10.1+/-0.7 nmol Pi mg(-1) min(-1), in the absence and presence of 5x10(-6) M d-cAMP, respectively. Similarly, the addition of cholera toxin (CTX), forskolin (FSK) or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) also increases the Na(+)-ATPase activity in a dose-dependent manner, with maximal effect at 10(-8) M, 10(-6) M and 10(-7) M, respectively. The effect of 10(-8) M CTX is not additive to the effect of GTPgammaS, and is completely abolished by 200 microM guanosine 5'-O-(2-thiodiphosphate). The stimulatory effects of CTX and FSK on the Na(+)-ATPase activity are accompanied by an increase in cAMP formation by the basolateral membranes of the proximal tubule cells. Furthermore, 10(-8) M protein kinase A peptide inhibitor (PKAi) completely abolishes the stimulatory effect of 5x10(-6) M d-cAMP or 10(-4) M FSK on the Na(+)-ATPase activity. Incubation of the basolateral membranes with [gamma-(32)P]ATP in the presence of d-cAMP or FSK increases the global hydroxylamine-resistant phosphorylation and especially promotes an increase in phosphorylation of protein bands of approximately 100 and 200 kDa. This stimulation is not seen when 10(-8) M PKAi is added simultaneously. Taken together these data suggest that activation of a cAMP/PKA pathway modulates the Na(+)-ATPase activity in isolated basolateral membranes of the proximal tubule.
Caruso-Neves
C
C
Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, 21949-900 Rio de Janeiro, RJ, Brazil.
Rangel
L B
LB
Vives
D
D
Vieyra
A
A
Coka-Guevara
S
S
Lopes
A G
AG
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D027682:Cation Transport Proteins; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis* / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology*; D013552:Swine
D027682:Cation Transport Proteins; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010042:Ouabain; D002772:Cholera Toxin; D000242:Cyclic AMP; D000251:Adenosine Triphosphatases; C021838:sodium-translocating ATPase