The release of endothelium-derived relaxing factor via alpha 2-adrenergic receptor activation is specifically mediated by Gi alpha 2.
The purpose of this study is to determine which pertussis toxin-sensitive guanine nucleotide-binding protein (Gi) mediates alpha 2-adrenergic receptor stimulation of endothelium-derived relaxing factor (EDRF) release. Bovine aortic endothelial cells were treated with pertussis toxin (0-100 ng/ml) for 16 h and stimulated with an alpha 2-adrenergic receptor agonist, UK14304, to release EDRF in a bioassay system. Pertussis toxin produced a concentration-dependent decrease in EDRF release with maximal inhibition (80%) occurring at 5 ng/ml. This correlated with a decrease in receptor-G protein coupling as measured by 87% loss of high affinity agonist binding sites and 94% decrease in agonist-stimulated GTPase activity. Immunoprecipitation of [32P]NAD-ribosylated membranes using specific Gi protein antisera demonstrated that complete ADP-ribosylation of Gi alpha 2 occurred at 5 ng/ml compared to 30 ng/ml for Gi alpha 3. When bovine aortic endothelial cell membranes were treated with carboxyl terminus-directed antisera to Gi alpha 2 (P4) and Gi alpha 3 (EC), the P4 antisera abolished 86% of the high affinity agonist binding sites and 93% decrease in agonist-stimulated GTPase activity, while the EC antisera had minimal effect (12%). These results indicate that Gi alpha 2 mediates most of the EDRF released via the alpha 2-adrenergic receptor.
Liao
J K
JK
Cardiovascular Division, Brigham and Women's Hospital, Boston, Massachusetts.
Homey
C J
CJ
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000502:physiology; D001667:Binding, Competitive; D000068438:Brimonidine Tartrate; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D004837:Epinephrine / Q000494:pharmacology*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007106:Immune Sera; D007700:Kinetics; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D009243:NAD / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis* / Q000378:metabolism; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D037342:Pertussis Toxin; D011810:Quinoxalines / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects / Q000502:physiology*; D010566:Virulence Factors, Bordetella / Q000378:metabolism / Q000494:pharmacology; D015016:Yohimbine / Q000378:metabolism
D000316:Adrenergic alpha-Agonists; D007106:Immune Sera; D009842:Oligopeptides; D011810:Quinoxalines; D011942:Receptors, Adrenergic, alpha; D010566:Virulence Factors, Bordetella; D009243:NAD; D015016:Yohimbine; D009569:Nitric Oxide; D006165:Guanylyl Imidodiphosphate; D000068438:Brimonidine Tartrate; D037342:Pertussis Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D004837:Epinephrine