Nitric oxide signaling mediates stimulation of L-type Ca2+ current elicited by withdrawal of acetylcholine in cat atrial myocytes

A perforated-patch whole-cell recording method was used to determine whether nitric oxide signaling participates in acetylcholine (ACh)-induced regulation of basal L-type Ca2+ current (I-Ca,I-L) in cat atrial myocytes. Exposure to 1 mu M ACh for 2 min inhibited basal I-Ca,I-L (-21 +/- 3%), and withdrawal of ACh elicited rebound stimulation of I-Ca,I-L above control (80 +/- 13%) (n = 23). Stimulation of I-Ca,I-L elicited by withdrawal of ACh (but not ACh-induced inhibition of I-Ca,I-L) was blocked by either 50 mu M hemoglobin; 30 mu M ODQ or 10 mu M methylene blue, inhibitors of soluble guanylate cyclase; 10 mu M W-7, a calmodulin inhibitor; or 10 mu M L-NIO, an inhibitor of constitutive NO synthase (NOS). In cells incubated in 5 mM L-arginine, ACh-induced rebound stimulation of I-Ca,I-L was enhanced compared with control responses. Histochemical assay (NADPH diaphorase) indicated that atrial myocytes express constitutive NOS. NO-donor, spermine/NO (SP/NO), >1 mu M stimulated basal I-Ca,I-L. SP/NO-induced stimulation of I-Ca,I-L was inhibited by 50 mu M hemoglobin, 30 mu M ODQ, or 5 mu M H-89, an inhibitor of PKA, and was unchanged by 50 mu M MnTBAP, a peroxynitrite scavenger. When I-Ca,I-L was prestimulated by 10 mu M milrinone, an inhibitor of cGMP-inhibited phosphodiesterase (type III) activity, SP/NO failed to further increase I-Ca,I-L. In cells incubated in pertussis toxin (3.4 mu g/ml for 6 h; 36 degrees C), ACh failed to affect I-Ca,I-L but 100 mu M SP/NO or 10 mu M milrinone still increased basal I-Ca,I-L. These results indicate that in cat atrial myocytes NO signaling mediates stimulation of I-Ca,I-L elicited by withdrawal of ACh but not ACh-induced inhibition of basal I-Ca,I-L. NO activates cGMP-induced inhibition of phosphodiesterase (type III) activity. Upon withdrawal of ACh, this mechanism allows cAMP to recover to levels above control, thereby stimulating I-Ca,I-L. Pertussis toxin-sensitive G-proteins couple M-2 muscarinic receptors to NO signaling. NO-mediated stimulation of I-Ca,I-L elicited by withdrawal of ACh may be an important mechanism that rapidly restores cardiac pacemaker and contractile functions after cholinergic suppression of atrial activity.