Molecular aspects of acute inhibition of Na⁺-H⁺ exchanger NHE3 by A₂-adenosine receptor agonists

Adenosine regulates Na⁺ homeostasis by its acute effects on renal Na⁺ transport. We have shown in heterologously transfected A6/C1 cells (renal cell line from Xenopus laevis) that adenosine-induced natriuresis may be effected partly via A₂ adenosine receptor-mediated inactivation of the renal brush border membrane Na⁺-H^- exchanger NHE3. In this study we utilized A6/C1 cells stably expressing wild-type as well as mutated forms of NHE3 to assess the molecular mechanism underlying A₂-dependent control of NHE3 function. Cell surface biotinylation combined with immunoprecipitation revealed that NHE3 is targeted exclusively to the apical domain and that the endogenous Xenopus NHE is located entirely on the basolateral side of A6/C1 transfectants. Stimulation of A₂-adenosine receptors located on the basolateral side for 15 min with CPA (N6-cyclopentyladenosine) acutely decreased NHE3 activity (microspectrofluorimety). This effect was mimicked by 8-bromo-cAMP and entirely blocked by pharmacological inhibition of PKA (with H89) or singular substitution of two PKA target sites (serine 552 and serine 605) on NHE3. Down-regulation of NHE3 activity by CPA was attributable to a reduction of NHE3 intrinsic transport activity without change in surface NHE3 protein at 15 min. At 30 min, the decrease in transport activity was associated with a decrease in apical membrane NHE3 antigen. In conclusion, two highly conserved target serine sites on NHE3 determine NHE3 modulation upon A₂-receptor activation and NHE3 inactivation by adenosine proceeds via two phases with distinct mechanisms.