Pertussis Toxin-sensitive G Proteins Influence Nitric Oxide Synthase III Activity and Protein Levels in Rat Heart

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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, N (G) -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 microM) 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. (J. Clin. Invest. 1998. 101:1424-1431.)

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