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/
Rk. Kandasamy et al., 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/, The Journal of biological chemistry, 270(49), 1995, pp. 29209-29216
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.