Molecular aspects of acute inhibition of Na(+)-H(+) exchanger NHE3 by A(2)-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 A(2) 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 A(2)-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 A(2)-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. Downregulation 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 A(2)-receptor activation and NHE3 inactivation by adenosine proceeds via two phases with distinct mechanisms.
Di Sole
Francesca
F
Department of Physiology and Pathophysiology, Division of Vegetative Physiology and Pathophysiology, Georg-August-University of Göttingen, D-37073 Göttingen, Germany.
Cerull
Robert
R
Casavola
Valeria
V
Moe
Orson W
OW
Burckhardt
Gerhard
G
Helmle-Kolb
Corinna
C
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active / Q000502:physiology; D015153:Blotting, Western; D002460:Cell Line; D003470:Culture Media; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D051379:Mice; D058906:Purinergic P1 Receptor Agonists; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014162:Transfection
D003470:Culture Media; D004791:Enzyme Inhibitors; D058906:Purinergic P1 Receptor Agonists; D012333:RNA, Messenger; C000613386:Slc9a3 protein, mouse; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D015124:8-Bromo Cyclic Adenosine Monophosphate; C048599:N(6)-cyclopentyladenosine; D017868:Cyclic AMP-Dependent Protein Kinases; D000241:Adenosine