Ouabain-insensitive Na+-ATPase activity is an effector protein for cAMP regulation in basolateral membranes of the proximal tubule

Abstract 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 5×10 −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 5×10 −6 M d-cAMP, respectively. Similarly, the addition of cholera toxin (CTX), forskolin (FSK) or guanosine 5′- O -(3-thiotriphosphate) (GTPγS) 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 GTPγS, and is completely abolished by 200 μM 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 5×10 −6 M d-cAMP or 10 −4 M FSK on the Na + -ATPase activity. Incubation of the basolateral membranes with [γ- 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.