ACh-induced rebound stimulation of L-type Ca2+ current in guinea-pig ventricular myocytes, mediated by G beta gamma-dependent activation of adenylyl cyclase

1. The effects that muscarinic receptor stimulation have on the cAMP-depend ent regulation of L-type Ca2+ currents were studied in isolated guinea-pig ventricular myocytes using the whole-cell configuration of the patch-clamp technique. 2. The muscarinic agonist ACh inhibited the Ca2+ current stimulated by the beta -adrenergic agonist isoprenaline (Iso), and washout of ACh revealed a stimulatory response that appeared as a transient rebound increase in the a mplitude of the Ca2+ current. The ACh-induced stimulatory effect was not ob served in the absence of Iso. 3. ACh-induced rebound stimulation was also observed in the presence of H-2 histamine receptor activation and cholera toxin treatment, which like beta -adrenergic receptor activation enhance adenylyl cyclase (AC) activity in a stimulatory G protein (G(s))-dependent manner. ACh-induced rebound stimul ation was not observed in the presence of forskolin, which enhances AC acti vity in a G(s)-independent manner. 4. Pertussis toxin (PTX) treatment blocked both the stimulatory and inhibit ory effects of ACh. Intracellular dialysis with QEHA, a peptide that binds free G protein,beta gamma subunits, selectively antagonized the stimulatory effect, leaving an enhanced inhibitory effect. 5. Evidence for the expression of AC4, an isoform of AC that can be stimula ted by G beta gamma but only in the presence of G alpha (s), was obtained b y Western blot analysis of guinea-pig ventricular myocyte membrane preparat ions. 6. These results suggest that muscarinic receptor stimulation facilitates a s wen as inhibits cAMP-dependent regulation of the Ca2+ current and that th e net response is a balance between these two actions. We suggest that the stimulatory effect is due to a direct activation of AC4 by the beta gamma s ubunits of a PTX-sensitive G protein.