Nitric Oxide Signaling Mediates Stimulation of L-Type Ca2+ Current Elicited by Withdrawal of Acetylcholine in Cat Atrial Myocytes

Abstract

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 Ca 2� current (I Ca,L ) in cat atrial myocytes. Exposure to 1 �M ACh for 2 min inhibited basal I Ca,L (�21 � 3%), and withdrawal of ACh elicited rebound stimulation of I Ca,L above control (80 � 13%) (n � 23). Stimulation of I Ca,L elicited by withdrawal of ACh (but not ACh-induced inhibition of I Ca,L ) was blocked by either 50 �M hemoglobin; 30 �M ODQ or 10 �M methylene blue, inhibitors of soluble guanylate cyclase; 10 �M W-7, a calmodulin inhibitor; or 10 �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,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 �M stimulated basal I Ca,L . SP/NO-induced stimulation of I Ca,L was inhibited by 50 �M hemoglobin, 30 �M ODQ, or 5 �M H-89, an inhibitor of PKA, and was unchanged by 50 �M MnTBAP, a peroxynitrite scavenger. When I Ca,L was prestimulated by 10 �M milrinone, an inhibitor of cGMP-inhibited phosphodiesterase (type III) activity, SP/NO failed to further increase I Ca,L . In cells incubated in pertussis toxin (3.4 �g/ml for 6 h; 36�C), ACh failed to affect I Ca,L , but 100 �M SP/NO or 10 �M milrinone still increased basal I Ca,L . These results indicate that in cat atrial myocytes NO signaling mediates stimulation of I Ca,L elicited by withdrawal of ACh but not ACh-induced inhibition of basal I Ca,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,L . Pertussis toxin-sensitive G-proteins couple M 2 muscarinic receptors to NO signaling. NO-mediated stimulation of I Ca,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.

Signaling Mechanisms That Mediate Nitric Oxide Production Induced by Acetylcholine Exposure and Withdrawal in Cat Atrial Myocytes

“…By this mechanism, NO signaling mediates the stimulation of I Ca,L elicited by ACh withdrawal. 8 These findings are consistent with studies in chick heart cells, in which ACh withdrawal stimulates cAMP above control levels. 14 Others laboratories have reported that ACh withdrawal stimulates I Ca,L in Purkinje fibers 15 and ventricular myocytes prestimulated by ␤-adrenergic agonists.…”

“…7 In cat atrial myocytes, AChinduced inhibition of basal L-type Ca 2ϩ current (I Ca,L ) is not mediated by NO signaling. 8 However, ACh withdrawal stimulates I Ca,L above control levels, ie, rebound stimulation, and this response is mediated by NO signaling. 8,9 The rebound stimulation of I Ca,L elicited by ACh withdrawal results in stimulation of atrial contraction, 9 atrial pacemaker activity, 10 and the potential development of Ca 2ϩ -mediated delayed afterdepolarizations and arrhythmic atrial activity.…”

“…8 However, ACh withdrawal stimulates I Ca,L above control levels, ie, rebound stimulation, and this response is mediated by NO signaling. 8,9 The rebound stimulation of I Ca,L elicited by ACh withdrawal results in stimulation of atrial contraction, 9 atrial pacemaker activity, 10 and the potential development of Ca 2ϩ -mediated delayed afterdepolarizations and arrhythmic atrial activity. 11 These findings are consistent with reports in multicellular atrial preparations that ACh withdrawal elicits rebound stimulation of intracellular Ca 2ϩ transients and contraction.…”

“…12 The fact that rebound stimulation is exhibited by multicellular tissue indicates that the stimulatory response to ACh withdrawal is not unique to isolated myocytes but rather is a physiological mechanism responsible for rapid recovery from cholinergic inhibition of atrial function. In both cat 8 and human 13 atrial myocytes, NO acts via cyclic GMP (cGMP)-induced inhibition of phosphodiesterase type III activity to increase endogenous cAMP levels. By this mechanism, NO signaling mediates the stimulation of I Ca,L elicited by ACh withdrawal.…”

β-Eudesmol Induces Neurite Outgrowth in Rat Pheochromocytoma Cells Accompanied by an Activation of Mitogen-Activated Protein Kinase

“…W7 (10 M) inhibited MAPK (ERK 1, 2) and CREB phosphorylation by 80.9, 69.5, and 71.0%, respectively, whereas H89 (10 M) reduced the degree of phosphorylation by 67.2, 67.7, and 55.0%, respectively. For determinations of involvements of these molecules, the concentrations of inhibitors used in this study were referred to these articles (Wang et al, 1998;Grewal 2000a,b). On the other hand, the protein kinase C (PKC) inhibitor GF109203X (1 M) did not affect ␤-eudesmol-induced MAPK activation (data not shown).…”