Guanine nucleotides regulate beta-adrenergic activation of Na-H exchange independently of receptor coupling to Gs.
We have previously shown that the beta-adrenergic receptor (beta-AR) stimulates activity of the ubiquitous Na-H exchanger (NHE-1) independently of changes in cAMP accumulation and independently of a cholera toxin-sensitive stimulatory GTP-binding protein (Gs). To further investigate the potential role of a GTP-binding protein in coupling the beta-AR to NHE-1, we have used a recently available nonhydrolyzable GTP analog, "caged" guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), to study time-dependent effects of GTP on NHE-1 in intact cells. By monitoring intracellular pH (pHi) in cells loaded with the fluorescent pH-sensitive dye, 2,7-biscarboxyethyl-5(6)-carboxyfluorescein, we determined NHE-1 activity in primary cultures of canine enteric endocrine cells, which express an endogenous beta-AR, and in mouse L cells stably transfected with either the wild type hamster beta 2-AR or a mutant construct of the hamster beta 2-AR containing a deletion in amino acid residues 222-229. This D(222-229)beta 2-AR is functionally uncoupled from Gs and adenylylcyclase. In all three cell types, NaF and GTP gamma S induced an increase in activity of the exchanger, determined by assessing the rate of pHi recovery from an acute intracellular acid load (dpHi/dt). This increase in pHi recovery was dependent on extracellular Na+ and sensitive to the amiloride analog ethylisopropylamiloride. GTP gamma S, but not NaF, also increased beta-adrenergic stimulation of resting NHE-1 activity. The alkalinization in response to isoproterenol was reversed by propranolol in the absence, but not the presence, of GTP gamma S and was completely blocked by GDP beta S. The ability of guanine nucleotides to regulate beta-adrenergic activation of NHE-1 in cells expressing the mutant D(222-229)beta 2-AR suggests that functional coupling of the beta-AR to NHE-1 may be mediated by a GTP-binding protein other than Gs.
Barber
D L
DL
Department of Stomatology, University of California, San Francisco 94143.
Ganz
M B
MB
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D004285:Dogs; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007082:Ileum; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D007739:L Cells; D008536:Meglumine / Q000494:pharmacology; D051379:Mice; D011433:Propranolol / Q000494:pharmacology*; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D012964:Sodium / Q000378:metabolism*; D012969:Sodium Fluoride / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection
D002352:Carrier Proteins; D006150:Guanine Nucleotides; D011943:Receptors, Adrenergic, beta; D017923:Sodium-Hydrogen Exchangers; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008536:Meglumine; C023427:guanosine 5'-O-(2-thiodiphosphate); D000584:Amiloride; D006160:Guanosine Triphosphate; D012969:Sodium Fluoride; D012964:Sodium; D011433:Propranolol; D019204:GTP-Binding Proteins; D007545:Isoproterenol; C039614:ethylisopropylamiloride