REBOUND STIMULATION OF THE CAMP-REGULATED CL- CURRENT BY ACETYLCHOLINE IN GUINEA-PIG VENTRICULAR MYOCYTES

1. Acetylcholine (ACh)-induced rebound stimulation of the cAMP-regulat ed Cl- current was studied in isolated guinea-pig ventricular myocytes using dialysing and dialysis-limiting configurations of the whole-cel l patch-clamp technique.2. Exposure to and subsequent washout of ACh p roduced a transient rebound stimulation of the Cl- current. However, t his rebound response was only observed in the presence of submaximally stimulating concentrations of the cAMP-producing agonists isoprenalin e (Iso) or histamine. ACh-induced rebound stimulation was not observed in the presence of maximally stimulating concentrations of Iso, nor w as it observed in the absence of Iso. 3. To prevent saturation of resp onses during rebound, the effects of ACh were studied in the presence of a subthreshold concentration of Iso (0.001 mu M). Varying the durat ion of exposure to ACh before washout demonstrated that the stimulator y effect of 1 mu M ACh approaches steady state with a time constant of 34. Exposing myocytes to varying concentrations of ACh for 90 s demon strated that the EC(50) for the stimulatory effect of ACh was 0.15 mu M with a maximum response equal to 67% of that obtained by a maximally stimulating concentration of Iso alone. 4. Rebound stimulation of the Cl- current could also be elicited by washing in 2 mu M atropine duri ng exposure to ACh, instead of washing out ACh. Furthermore, ACh-induc ed rebound was blocked by the M(2) muscarinic receptor antagonist meth octramine but not by the M(1), receptor antagonist pirenzepine. Reboun d was also blocked in pertussis toxin (PTX)-treated myocytes. 5. ACh-i nduced rebound stimulation was not blocked by: (a) L-NMMA, an inhibito r of nitric oxide synthase activity; (b) Methylene Blue, LY-83583, and ODQ, inhibitors of cGMP production; or (c) milrinone, an inhibitor of cGMP-dependent phosphodiesterase activity. 6. These results indicate that ACh can stimulate cAMP-regulated ion channel activity in cardiac ventricular myocytes by facilitating beta-adrenergic and histaminergic responses. This is opposite to the inhibitory actions more typically associated with muscarinic receptor stimulation in ventricular myocard ium. This stimulatory effect of ACh is mediated through M(2) muscarini c receptors and a PTX-sensitive G-protein, but it does not appear to i nvolve the production of nitric oxide or cGMP.