The aim of this work was to determine the molecular mechanism involved in the stimulation of the pig kidney proximal tubule Na+-ATPase by adenosine (Ado). To study the role of A2 Ado receptors, we added in all experiments 10(-6)M DPCPX, an A1 receptor-selective antagonist, since we have previously shown that Ado inhibits the enzyme activity through this receptor. Ado increased the Na+-ATPase activity with maximal effect observed at 10(-6)M. The presence of both A(2A) and A(2B) receptors were demonstrated by immunoblotting using specific polyclonal antibodies. The stimulatory effect of Ado was completely abolished by 5 x 10(-9)M DMPX, an antagonist of A2 receptor, and 10(-7)M SCH 58261, an A(2A) receptor-selective antagonist. DMPA (10(-7)M), a specific agonist of A(2A) receptor mimicked the stimulatory effect of Ado. Involvement of a Gs protein/adenylate cyclase/PKA pathway was evidenced by: (a) the reversion of Ado-induced effect by GDPbetaS; (b) stimulation of the Na+-ATPase activity in a similar and non-additive manner to Ado by 10(-8)M cholera toxin, 10(-7)M GTPgammaS, 10(-6)M forskolin, 10(-7)M cAMP or 1.25 U catalytic subunit of PKA; (c) the reversion of the stimulatory effect of Ado by 10(-8)M PKA inhibitor peptide; (d) Ado-produced two-fold increase of the PKA activity, which was completely reversed by 10(-6)M DMPX. These are the first evidences showing the modulation of a renal primary active sodium transporter by Ado through A(2A) receptor.