G-protein stimulation inhibits amiloride-sensitive Na/H exchange independently of cyclic AMP.
G-proteins are heterotrimeric proteins involved in many transmembrane signaling events. Both the renal basolateral membrane and the renal brush border membrane contain large quantities of these proteins. G-proteins appear related to hormonal signaling in the basolateral membrane and presumably affect ion gating in the brush border. We investigated the influence of G-proteins on the amiloride-sensitive Na/H exchanger, the activity of which is regulated at least in part by cAMP-dependent protein kinase, by measuring the amiloride-sensitive component of [22Na+] uptake in rat renal brush border membrane vesicles (BBMV) in the presence of a pH gradient. Incubation of vesicles with AlF4- (10 microM Al3+, 10 mM F-) resulted in significant inhibition of amiloride-sensitive [22Na+] uptake at both 20 seconds and 5 minutes of incubation. Incorporation of GTP gamma S into BBMV by transient hypotonic lysis also resulted in significantly reduced amiloride-sensitive [22Na+] uptake compared to controls at both time points. This inhibition could be reversed by GDP beta S. Similar lysis in the presence of 10 microM GDP beta S alone had no significant effect. When Na(+)-dependent [14C]-D-glucose uptake into BBMV was studied no significant effect of these G-protein modulating agents was observed. Adenylate cyclase activity could not be stimulated in these BBMV preparations using standard techniques. Furthermore, cAMP-dependent protein kinase activity, strongly stimulated in these BBMV by exogenously added cAMP, was not stimulated by 10 microM GTP gamma S alone. These findings suggest that the amiloride-sensitive Na/H exchanger can be regulated by G-proteins independently of adenylate cyclase and cAMP-dependent protein kinase.
Brunskill
N J
NJ
Renal Division, Washington University School of Medicine, St. Louis, Missouri.
Morrissey
J J
JJ
Klahr
S
S
D000262:Adenylyl Cyclases / Q000378:metabolism; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology; D066298:In Vitro Techniques; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D015398:Signal Transduction / Q000502:physiology; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D006150:Guanine Nucleotides; D017923:Sodium-Hydrogen Exchangers; D005576:Colforsin; D000584:Amiloride; D012964:Sodium; D000242:Cyclic AMP; D011494:Protein Kinases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D005947:Glucose