ACh-induced rebound stimulation of L-type Ca(2+) current in guinea-pig ventricular myocytes, mediated by Gbetagamma-dependent activation of adenylyl cyclase.

1. The effects that muscarinic receptor stimulation have on the cAMP-dependent regulation of L-type Ca(2+) 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 Ca(2+) 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 amplitude of the Ca(2+) current. The ACh-induced stimulatory effect was not observed 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 stimulation was not observed in the presence of forskolin, which enhances AC activity in a G(s)-independent manner. 4. Pertussis toxin (PTX) treatment blocked both the stimulatory and inhibitory effects of ACh. Intracellular dialysis with QEHA, a peptide that binds free G protein betagamma 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 stimulated by Gbetagamma but only in the presence of Galpha(s), was obtained by Western blot analysis of guinea-pig ventricular myocyte membrane preparations. 6. These results suggest that muscarinic receptor stimulation facilitates as well as inhibits cAMP-dependent regulation of the Ca(2+) current and that the 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 betagamma subunits of a PTX-sensitive G protein.