Pertussis toxin-sensitive G proteins influence nitric oxide synthase III activity and protein levels in rat heart

Inhibitory G protein activity (G(i)) and nitric oxide (NO) modulate muscarinic-cholinergic (MC) inhibition of cardiac β-adrenergic inotropic responses. We hypothesized that G(i) mediates MC-NO synthase (NOS) signal transduction. Isoproterenol (0.2-0.8 μg/min) and acetylcholine (1 μM) were administered to isolated perfused rat hearts pretreated with saline (controls; n = 8) or pertussis toxin (PT; 30 μg/kg intraperitoneally 3 d before study; n = 20). PT abrogated in vitro ADP-ribosylation of G(i) protein α subunit(s) indicating near-total decrease in G(i) 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 μM) 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 μM) 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 μM) inhibition of phospholipase C did not reverse L-arginine enhancement of MC inhibitory effects. These data support a primary role for G(i) in MC receptor signal transduction with NOS in rat heart, and demonstrate regulatory linkage between G(i) and NOS III protein levels.