Adenosine stimulation of Na⁺ transport is mediated by an A₁ receptor and a [Ca²⁺]_i-dependent mechanism

Studies were performed to determine the primary signal transduction mechanism that mediates adenosine stimulation of electrogenic sodium transport in renal epithelial cells. Experiments were performed on cultured amphibian A₆ cells with an adenosine analogue that preferentially binds to the A₁ receptor, cyclohexyladenosine (CHA). Sodium transport was assessed by the equivalent short circuit current (Ieq). CHA was found to stimulate Ieq via activation of an A₁ receptor because (1) the threshold concentration was 1 nM compared to that of 10 μM for the specific A₂ agonist CGS21680, (2) CHA inhibited vasopressin (AVP)-stimulated cAMP production by a pertussis toxin-sensitive mechanism, and (3) the action of CHA was inhibited by the A₁ antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). CHA increased intracellular Ca²⁺ ([Ca²⁺]_i) and stimulated phosphoinositide turnover at concentrations that increased Ieq and in a time course that paralleled the increase in Ieq. Ion transport was stimulated by a Ca²⁺-dependent mechanism because the CHA induced increase in Ieq was inhibited by chelating [Ca²⁺]_i with 5,5′dimethyl BAPTA in a dose-dependent manner, with a K_i of approximately 10 μM. The increase in Ieq was also dose-dependently inhibited by the specific PKC inhibitors dihydroxychlorpromazine and chelerythrine, and by trifluoperazine which inhibits PKC and calmodulin. Further studies indicated that CHA-stimulated Ieq was independent of cAMP generation because CHA did not induce an increase in cAMP accummulation parallel to the increase in Ieq in a dose-response analysis, and the adenylate cyclase inhibitor 2′,5′ dideoxy-adenosine (DDA) did not affect the CHA-induced increase in Ieq. These studies indicate, therefore, that adenosine stimulation of Ieq occurs, at least in part, through calcium-dependent signal transduction events and not through regulation of adenylate cyclase.