title: Pertussis toxin-sensitive G proteins influence nitric oxide synthase III activity and protein levels in rat heart.
abstract: Inhibitory G protein activity (Gi) and nitric oxide (NO) modulate muscarinic-cholinergic (MC) inhibition of cardiac beta-adrenergic inotropic responses. We hypothesized that Gi mediates MC-NO synthase (NOS) signal transduction. Isoproterenol (0.2-0.8 microg/min) and acetylcholine (1 microM) were administered to isolated perfused rat hearts pretreated with saline (controls; n = 8) or pertussis toxin (PT; 30 microg/kg intraperitoneally 3 d before study; n = 20). PT abrogated in vitro ADP-ribosylation of Gi protein alpha subunit(s) indicating near-total decrease in Gi protein function. Isoproterenol increased peak +dP/dt in both control (peak isoproterenol effect: +2, 589+/-293 mmHg/s, P < 0.0001) and PT hearts (+3,879+/-474 mmHg/s, P < 0.0001). Acetylcholine reversed isoproterenol inotropy in controls (108+/-21% reduction of +dP/dt response, P = 0.001), but had no effect in PT hearts. In controls, NG-monomethyl-L-arginine (100 microM) reduced basal +dP/dt, augmented isoproterenol +dP/dt (peak effect: +4,634+/-690 mmHg/s, P < 0.0001), and reduced the MC inhibitory effect to 69+/-8% (P < 0.03 vs. baseline). L-arginine (100 M) had no effect in controls but in PT hearts decreased basal +dP/dt by 1, 426+/-456 mmHg/s (P < 0.005), downward-shifted the isoproterenol concentration-effect curve, and produced a small MC inhibitory effect (27+/-4% reduction, P < 0.05). This enhanced response to NO substrate was associated with increased NOS III protein abundance, and a three- to fivefold increase in in vitro calcium-dependent NOS activity. Neomycin (1 microM) inhibition of phospholipase C did not reverse L-arginine enhancement of MC inhibitory effects. These data support a primary role for Gi in MC receptor signal transduction with NOS in rat heart, and demonstrate regulatory linkage between Gi and NOS III protein levels.
authors.affiliation: Department of Medicine, Cardiology Division, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287-6568, USA. jhare@welchlink.welch.jhu.edu
authors.forename: J M
authors.initials: JM
authors.lastname: Hare
authors.forename: B
authors.initials: B
authors.lastname: Kim
authors.forename: N A
authors.initials: NA
authors.lastname: Flavahan
authors.forename: K M
authors.initials: KM
authors.lastname: Ricker
authors.forename: X
authors.initials: X
authors.lastname: Peng
authors.forename: L
authors.initials: L
authors.lastname: Colman
authors.forename: R G
authors.initials: RG
authors.lastname: Weiss
authors.forename: D A
authors.initials: DA
authors.lastname: Kass
mesh_terms: D000109:Acetylcholine / Q000378:metabolism / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001120:Arginine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D006321:Heart / Q000187:drug effects*; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009355:Neomycin / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D037342:Pertussis Toxin; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
chemical_list: D000318:Adrenergic beta-Agonists; D004791:Enzyme Inhibitors; D011500:Protein Synthesis Inhibitors; D011506:Proteins; D011943:Receptors, Adrenergic, beta; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D009569:Nitric Oxide; D000244:Adenosine Diphosphate; D001120:Arginine; D019001:Nitric Oxide Synthase; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D009355:Neomycin; D007545:Isoproterenol; D000109:Acetylcholine; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester