Specific receptor-guanine nucleotide binding protein interaction mediates the release of endothelium-derived relaxing factor.
High affinity agonist-binding (HAB) sites are formed from specific receptor interaction with guanine nucleotide-binding (Gi) proteins. To determine whether the release of endothelium-derived relaxing factor (EDRF) is regulated by specific receptor-Gi protein coupling, we treated bovine aortic endothelial cells with 100 ng/ml pertussis toxin (PTX) for 16 hours to effect receptor-Gi protein uncoupling. The degree of receptor uncoupling as measured by the loss of HAB sites for the alpha 2-adrenergic receptor and bradykinin receptor was assessed by radioligand binding studies using partially purified bovine aortic endothelial cell membranes. The release of EDRF in response to UK14304 (an alpha 2-adrenergic receptor agonist) and bradykinin stimulation was measured with a bioassay apparatus. The Gi protein isoforms were characterized by Western blotting, and complete ADP-ribosylation of these proteins was confirmed by PTX-catalyzed [32P]NAD ribosylation. PTX produced a greater inhibition of EDRF release via the alpha 2-adrenergic receptor pathway compared with the bradykinin receptor pathway (80% versus 46%, p less than 0.01). This corresponded to the loss of HAB sites from the alpha 2-adrenergic receptor and bradykinin receptor pathway (72% versus 46%, p less than 0.01) as compared with complete loss of both HAB sites in the presence of GppNHp (0.1 mM). Since loss of HAB sites from PTX-mediated receptor uncoupling parallels the inhibition of EDRF release, these data suggest that Gi proteins contribute to a greater proportion of HAB sites derived from alpha 2-adrenergic receptor rather than bradykinin receptor interaction and that the inhibition of EDRF release by PTX is mainly due to the loss of these HAB sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Liao
J K
JK
Cardiovascular Division, Brigham & Women's Hospital, Boston.
Homcy
C J
CJ
D000818:Animals; D001665:Binding Sites; D001681:Biological Assay; D015153:Blotting, Western; D001920:Bradykinin / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism*; D037342:Pertussis Toxin; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D011942:Receptors, Adrenergic, alpha; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter; D010566:Virulence Factors, Bordetella; D009569:Nitric Oxide; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D001920:Bradykinin