Adenosine is an autacoid that regulates renal Na

Abstract. Regulation of renal apical Na ϩ /H ϩ exchanger 3 (NHE3) activity by adenosine has been suggested to contribute to acute control of mammalian Na ϩ homeostasis. The mechanism by which adenosine controls NHE3 activity in a renal cell line was examined. The adenosine analog, N 6 -cyclopentyladenosine (CPA) exerts a bimodal effect on NHE3: CPA concentrations Ͼ10 Ϫ8 M inactivate NHE3, whereas concentrations Ͻ10 Ϫ8 M stimulate NHE3 activity. Acute CPA-induced control of NHE3 was blocked by antagonists of A 1 adenosine receptors and inhibition of phospholipase C, pretreatment with BAPTA-AM (chelator of cellular calcium), and exposure to pertussis toxin. Stimulatory and to some extent also inhibitory CPA concentrations attenuated 8-bromo-cAMP and dopaminemediated inhibition of NHE3. BAPTA eliminated the ability of a stimulatory dose of CPA to attenuate 8-bromo-cAMP-induced suppression of NHE3 activity. Upon inhibition of protein kinase C, CPA at an inhibitory dose provoked activation of NHE3, which is partially reverted by 8-bromo-cAMP and suppressed by pre-incubation with BAPTA-AM. Cytochalasin B, an actin-modifying agent, selectively prevented downregulation but did not affect upregulation of NHE3 activity by CPA. In conclusion, these observations demonstrate that (1) CPA modulates NHE3 activity by elevation of cellular Ca 2ϩ exerting a negative control on adenylate cyclase activity, (2) protein kinase C is the determining factor leading to CPAinduced downregulation of NHE3 activity, and (3) alterations of surface NHE3 abundance may contribute to A 1 adenosine receptor-dependent inhibition of NHE3 activity.

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. 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 A2‐receptor activation and NHE3 inactivation by adenosine proceeds via two phases with distinct mechanisms.

The renal function of the A(3) adenosine receptor (A3AR) is poorly characterized. In this study, we report that the A3AR-selective agonist, 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purine-9-yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide (2-Cl-IBMECA) regulates the Na+/H+ exchanger-3 (NHE3) in a dose- and time-dependent fashion. In opossum kidney (OK) cells, 2-Cl-IBMECA at high (10(-6) M) and low (10(-8) M) dose inhibits NHE3 by a multiphasic time course with an acute phase of NHE3 inhibition from 15 min to 1 h, followed by a chronic phase of NHE3 inhibition from 24 to 48 h. Pre-incubation with either the selective A3AR-antagonist MRS1523 (10(-7) M) or the protein kinase C inhibitor, Calphostin C (10(-8) M) completely blocked 10(-6) M 2-Cl-IBMECA-induced acute (15 min) and chronic (24 h) phases of NHE3 inhibition. In contrast, the acute inhibitory phase (15 min) of 10(-8) M 2-Cl-IBMECA was completely prevented only when Calphostin C (10(-8) M) was added in conjunction with the protein kinase A inhibitor, H89 (10(-7) M). Acute (15 or 30 min depending on the A3AR-agonist concentration) A3AR-dependent inhibition of NHE3 activity was accompanied by decrease in cell surface NHE3 protein with no change in total NHE3 antigen. Chronic (24 h) A3AR-mediated down-regulation of NHE3 was associated with reduction of surface NHE3, decreased total NHE3 protein (70%) and a paradoxical rise of NHE3 RNA (40%). In summary, these results indicate that A3AR directly regulates NHE3 at multiple levels in a complex pattern. A3AR-dependent short- and long-term inhibition of NHE3 may be a fundamental mechanism of net sodium and fluid balance.