Regulation of NHE3 activity by G protein subunits in renal brush-border membranes

NHE3 activity is regulated by phosphorylation/ dephosphorylation processes and membrane recycling in intact cells. However, the Na+/H+ exchanger (NHE) can also be regulated by G proteins independent of cytoplasmic second mess engers, but the G protein subunits involved in this regulation are not know n. Therefore, we studied G protein subunit regulation of NHE3 activity in r enal brush-border membrane vesicles (BBMV) in a system devoid of cytoplasmi c components and second messengers. Basal NHE3 activity was not regulated b y G(s)alpha or G(i)alpha, because antibodies to these G proteins by themsel ves were without effect. The inhibitory effect of D-1-like agonists on NHE3 activity was mediated, in part, by G(s)alpha, because it was partially rev ersed by anti-G(s)alpha antibodies. Moreover, the amount of G(s)alpha that coimmunoprecipitated with NHE3 was increased by fenoldopam in both brush-bo rder membranes and renal proximal tubule cells. Furthermore, guanosine 5'-O -(3-thiotriphosphate) but not guanosine 5'-O-(2-thiodiphosphate), the inact ive analog of GDP, increased the amount of G(s)alpha that coimmunoprecipita ted with NHE3. The alpha(2)-adrenergic agonist, UK-14304 or pertussis toxin (PTX) alone had no effect on NHE3 activity, but UK-14304 and PTX treatment attenuated the D-1-like receptor-mediated NHE3 inhibition. The ability of UK-14304 to attenuate the D-1-like agonist effect was not due to G(i)alpha, because the attenuation was not blocked by anti-G(i)alpha antibodies or by PTX. Anti-G beta(common) antibodies, by themselves, slightly inhibited NHE 3 activity but had little effect on D-1-like receptor-mediated NHES inhibit ion. However, anti-G beta(common) antibodies reversed the effects of UK-143 04 and PTX on D-1-like agonist-mediated NHE3 inhibition. These studies prov ide concrete evidence of a direct regulatory role for G(s)alpha, independen t of second messengers, in the D-1-like-mediated inhibition of NHES activit y in rat renal BBMV. In addition, beta/gamma dimers of heterotrimeric G pro teins appear to have a stimulatory effect on NHE3 activity in BBMV.