PLASMA-MEMBRANE NA+ H+ EXCHANGER ISOFORMS (NHE-1, NHE-2, AND NHE-3) ARE DIFFERENTIALLY RESPONSIVE TO 2ND MESSENGER AGONISTS OF THE PROTEIN-KINASE-A AND PROTEIN-KINASE-C PATHWAYS/

Na+/H+ exchanger (NHE) activity is regulated by several types of recep tors directly coupled to distinct classes (i.e. G(s), G(i), G(q), and G(12)) of heterotrimeric (alpha beta gamma) GTP-binding proteins (G pr oteins), which, upon activation, modulate production of various second messengers (e.g. cAMP, cGMP, diacylglycerol, inositol trisphosphate, and Ca2+). Recently, four isoforms of the rat Na+/H+ exchanger were id entified by molecular cloning. To examine their intrinsic responsivene ss to Gr protein and second messenger stimulation, three of these isof orms, NHE-1, -2, and -3, were stably expressed in mutant Chinese hamst er ovary cells devoid of endogenous NHE activity (AP-1 cells). Incubat ion of cells with either AlF4-, a general agonist of G proteins, or ch olera toxin, a selective activator of G alpha(s) that stimulates adeny late cyclase, accelerated the rates of amiloride-inhibitable Na-22(+) influx mediated by NHE-1 and 2, whereas they inhibited that by NHE-3. Similarly, short term treatment with phorbol 12-myristate 13-acetate, which mimics diacylglycerol activation of protein kinase C (PKC), or w ith agents (i.e. forskolin, 8-(4-chlorophenylthio)-cAMP, and isobutylm ethylxanthine) that lead to activation of cAMP-dependent protein kinas e (PKA) also stimulated transport by NHE-1 and NHE-2 but depressed tha t by NHE-3. The effects of phorbol 12-myristate 13-acetate were blocke d by depleting cells of PKC or by inhibiting PKC using chelerythrine c hloride, confirming a role for PHC in modulating NHE isoform activitie s. Likewise, the PKA antagonist, H-89, attenuated the effects of eleva ted cAMP(i) on NHE-1, -2, and -3, further demonstrating the regulation by PKA. Unlike cAMP(i), elevation of cGMP(i) by treatment with dibuty ryl-cGMP or 8-bromo-cGMP had no influence on NHE isoform activities, t hereby excluding the possibility of a role for cGMP-dependent protein kinase in these cells. These data support the concept that the NHE iso forms are differentially responsive to agonists of the PKA and PKC pat hways.