Acetylcholine Elicits a Rebound Stimulation of Ca 2+ Current Mediated by Pertussis Toxin–Sensitive G Protein and cAMP-Dependent Protein Kinase A in Atrial Myocytes

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Abstract
Cholinergic inhibition of atrial contraction is typically followed by a rebound positive inotropic response. In the present study, we used a nystatin–perforated patch whole-cell recording method to determine whether acetylcholine (ACh) elicits a rebound stimulation of L-type Ca 2+ current (I Ca,L ) in cat atrial myocytes. ACh (1 μmol/L) decreased basal I Ca,L (−19±2%). Within ≈30 s of returning to ACh-free solution, basal I Ca,L exhibited a rebound increase above the control level (+61±7%) that returned to the control level within 4 to 5 minutes. ACh elicited concomitant changes in cell shortening, ie, a decrease followed by a rebound increase. The EC 50 and maximal response of ACh-induced inhibition and rebound stimulation of I Ca,L were 1.9×10 −9 mol/L and −30%, respectively, and 2.9×10 −8 mol/L and +64%, respectively. All effects of ACh on I Ca,L were blocked by prior exposure to 1 μmol/L atropine or 100 μmol/L AFDX116 and unaffected by 0.2 μmol/L pirenzepine or 1 μmol/L propranolol. In the presence of ACh, exposure to atropine elicited stimulation of I Ca,L . ACh-induced inhibition and rebound stimulation of current were independent of external Ca 2+ . Rebound stimulation of I Ca,L was associated with a negative shift in the voltage dependence of I Ca,L activation. Inhibition of protein kinase A by 50 μmol/L Rp-cAMPs decreased basal I Ca,L by 36±1% and abolished the rebound stimulation of I Ca,L . Forskolin (0.01 μmol/L) or isoproterenol (0.01 μmol/L) had no effect on basal I Ca,L , but each accentuated the rebound increase in I Ca,L . When adenylate cyclase was maximally stimulated with 1 μmol/L isoproterenol plus 2 μmol/L forskolin, ACh decreased I Ca,L but failed to elicit rebound stimulation of I Ca,L . Milrinone (10 μmol/L) increased basal I Ca,L by 70±7% and significantly attenuated the rebound stimulation of I Ca,L . Exposure to 1 mmol/L 8-bromo-cGMP elicited a small decrease in basal I Ca,L , attenuated ACh-induced inhibition, and enhanced the rebound stimulation of I Ca,L . Incubation in pertussis toxin prevented all ACh-induced changes in I Ca,L . Inhibition of nitric oxide synthase by 100 μmol/L N G -monomethyl- l -arginine (L-NMMA) decreased basal I Ca,L by −20±5%, prevented ACh-induced inhibition, and markedly attenuated the rebound stimulation of I Ca,L . We conclude that in cat atrial myocytes ACh acts via M 2 muscarinic receptors and pertussis toxin–sensitive G protein to inhibit basal I Ca,L and that on withdrawal ACh elicits a rebound stimulation of I Ca,L . Rebound stimulation of I Ca,L is mediated via cAMP-dependent protein kinase A enhanced by ACh-induced inhibition of phosphodiesterase. This mechanism contributes directly to the positive inotropic response that follows cholinergic inhibition of atrial contraction.