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 a ctivity from proximal tubule basolateral membranes by cAMP. An increase in dibutyryl-cAMP (d-cAMP) concentration from 10(-8) to 5 X 10(-5) M stimulate s the ouabain-insensitive Na+-ATPase activity. The ATPase activity increase s from 6.0 +/- 0.4 to 10.1 +/- 0.7 nmol Pi mg(-1) min(-1), in the absence a nd presence of 5 X 10(-6) M d-cAMP, respectively. Similarly, the addition o f cholera toxin (CTX), forskolin (FSK) or guanosine 5'-O-(3-thiotriphosphat e) (GTP gamma S) 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, respective ly. The effect of 10(-8) M CTX is not additive to the effect of GTP gamma S , and is completely abolished by 200 mu M guanosine 5'-O-(2-thiodiphosphate ). The stimulatory effects of CTX and FSK on the Na+-ATPase activity are ac companied by an increase in cAMP formation by the basolateral membranes of the proximal tubule cells. Furthermore, 10-8 M protein kinase A peptide inh ibitor (PKAi) completely abolishes the stimulatory effect of 5 X 10(-6) M d -cAMP or 10(-4) M FSK on the Na+-ATPase activity. Incubation of the basolat eral membranes with [gamma-P-32]ATP in the presence of d-cAMP or FSK increa ses the global hydroxylamine-resistant phosphorylation and especially promo tes an increase in phosphorylation of protein bands of approximately 100 an d 200 kDa. This stimulation is not seen when 10(-8) M PKAi is added simulta neously. Taken together these data suggest that activation of a cAMP/PKA pa thway modulates the Na+-ATPase activity in isolated basolateral membranes o f the proximal tubule. (C) 2000 Elsevier Science B.V. All rights reserved.