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 receptors 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 proteins), 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 identified by molecular cloning. To examine their intrinsic responsiveness to Gr protein and second messenger stimulation, three of these isoforms, NHE-1, -2, and -3, were stably expressed in mutant Chinese hamster ovary cells devoid of endogenous NHE activity (AP-1 cells). Incubation of cells with either AlF4-, a general agonist of G proteins, or cholera toxin, a selective activator of G alpha(s) that stimulates adenylate 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 with agents (i.e. forskolin, 8-(4-chlorophenylthio)-cAMP, and isobutylmethylxanthine) that lead to activation of cAMP-dependent protein kinase (PKA) also stimulated transport by NHE-1 and NHE-2 but depressed that by NHE-3. The effects of phorbol 12-myristate 13-acetate were blocked by depleting cells of PKC or by inhibiting PKC using chelerythrine chloride, confirming a role for PHC in modulating NHE isoform activities. Likewise, the PKA antagonist, H-89, attenuated the effects of elevated cAMP(i) on NHE-1, -2, and -3, further demonstrating the regulation by PKA. Unlike cAMP(i), elevation of cGMP(i) by treatment with dibutyryl-cGMP or 8-bromo-cGMP had no influence on NHE isoform activities, thereby excluding the possibility of a role for cGMP-dependent protein kinase in these cells. These data support the concept that the NHE isoforms are differentially responsive to agonists of the PKA and PKC pathways.