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 messengers, but the G protein subunits involved in this regulation are not known. Therefore, we studied G protein subunit regulation of NHE3 activity in renal brush-border membrane vesicles (BBMV) in a system devoid of cytoplasmic components and second messengers. Basal NHE3 activity was not regulated by G(s)alpha or G(i)alpha, because antibodies to these G proteins by themselves 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 reversed by anti-G(s)alpha antibodies. Moreover, the amount of G(s)alpha that coimmunoprecipitated with NHE3 was increased by fenoldopam in both brush-border membranes and renal proximal tubule cells. Furthermore, guanosine 5'-O-(3-thiotriphosphate) but not guanosine 5'-O-(2-thiodiphosphate), the inactive analog of GDP, increased the amount of G(s)alpha that coimmunoprecipitated 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-Gbeta(common) antibodies, by themselves, slightly inhibited NHE3 activity but had little effect on D(1)-like receptor-mediated NHE3 inhibition. However, anti-Gbeta(common) antibodies reversed the effects of UK-14304 and PTX on D(1)-like agonist-mediated NHE3 inhibition. These studies provide concrete evidence of a direct regulatory role for G(s)alpha, independent of second messengers, in the D(1)-like-mediated inhibition of NHE3 activity in rat renal BBMV. In addition, beta/gamma dimers of heterotrimeric G proteins appear to have a stimulatory effect on NHE3 activity in BBMV.
Albrecht
F E
FE
Departments of Pediatrics, Georgetown University Medical Center, Washington, District of Columbia 20007, USA.
Xu
J
J
Moe
O W
OW
Hopfer
U
U
Simonds
W F
WF
Orlowski
J
J
Jose
P A
PA
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001552:Benzazepines / Q000494:pharmacology; D000068438:Brimonidine Tartrate; D002461:Cell Line, Transformed; D018491:Dopamine Agonists / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D018818:Fenoldopam / Q000494:pharmacology; D044385:GTP-Binding Protein alpha Subunits; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000737:chemistry / Q000166:cytology / Q000201:enzymology*; D008297:Male; D008871:Microvilli / Q000737:chemistry / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology; D037342:Pertussis Toxin; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D011921:Rats, Inbred WKY; D017447:Receptors, Dopamine D1 / Q000378:metabolism; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology; D012979:Sodium Radioisotopes / Q000493:pharmacokinetics; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000316:Adrenergic alpha-Agonists; D001552:Benzazepines; D018491:Dopamine Agonists; C118981:G-protein Beta gamma; D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D018696:Neuroprotective Agents; D011810:Quinoxalines; D017447:Receptors, Dopamine D1; C000613387:Slc9a3 protein, rat; D012979:Sodium Radioisotopes; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D010566:Virulence Factors, Bordetella; C084618:olfactory G protein subunit alpha olf; D000068438:Brimonidine Tartrate; C067113:SK&F 81297; D000584:Amiloride; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins; D018818:Fenoldopam; C039614:ethylisopropylamiloride