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

Wang YG, Rechenmacher CE, Lipsius SL - J. Gen. Physiol. (1998)

Bottom Line: When ICa,L was prestimulated by 10 microM milrinone, an inhibitor of cGMP-inhibited phosphodiesterase (type III) activity, SP/NO failed to further increase ICa,L.In cells incubated in pertussis toxin (3.4 microg/ml for 6 h; 36 degrees C), ACh failed to affect ICa,L, but 100 microM SP/NO or 10 microM milrinone still increased basal ICa,L.Pertussis toxin-sensitive G-proteins couple M2 muscarinic receptors to NO signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, USA.

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 Ca2+ current (ICa,L) in cat atrial myocytes. Exposure to 1 microM ACh for 2 min inhibited basal ICa,L (-21 +/- 3%), and withdrawal of ACh elicited rebound stimulation of ICa,L above control (80 +/- 13%) (n = 23). Stimulation of ICa,L elicited by withdrawal of ACh (but not ACh-induced inhibition of ICa,L) was blocked by either 50 microM hemoglobin; 30 microM ODQ or 10 microM methylene blue, inhibitors of soluble guanylate cyclase; 10 microM W-7, a calmodulin inhibitor; or 10 microM L-NIO, an inhibitor of constitutive NO synthase (NOS). In cells incubated in 5 mM L-arginine, ACh-induced rebound stimulation of ICa,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 microM stimulated basal ICa,L. SP/NO-induced stimulation of ICa,L was inhibited by 50 microM hemoglobin, 30 microM ODQ, or 5 microM H-89, an inhibitor of PKA, and was unchanged by 50 microM MnTBAP, a peroxynitrite scavenger. When ICa,L was prestimulated by 10 microM milrinone, an inhibitor of cGMP-inhibited phosphodiesterase (type III) activity, SP/NO failed to further increase ICa,L. In cells incubated in pertussis toxin (3.4 microg/ml for 6 h; 36 degrees C), ACh failed to affect ICa,L, but 100 microM SP/NO or 10 microM milrinone still increased basal ICa,L. These results indicate that in cat atrial myocytes NO signaling mediates stimulation of ICa,L elicited by withdrawal of ACh but not ACh-induced inhibition of basal ICa,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 ICa,L. Pertussis toxin-sensitive G-proteins couple M2 muscarinic receptors to NO signaling. NO-mediated stimulation of ICa, 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.

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Effect of H-89, an inhibitor of PKA, on spermine/ NO-induced stimulation of ICa,L. The graph shows the percent  change in ICa,L amplitude elicited by either 100 μM spermine/NO  (A) or 30 μM spermine/NO (B) in the absence (open bar) and  presence (striped bar) of 5 μM H-89. Spermine/NO-induced stimulation of ICa,L was significantly blocked by inhibition of PKA activity.  In addition, H-89 exerted a more effective block at lower spermine/NO concentrations. *P < 0.05; **P < 0.001.
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Figure 11: Effect of H-89, an inhibitor of PKA, on spermine/ NO-induced stimulation of ICa,L. The graph shows the percent change in ICa,L amplitude elicited by either 100 μM spermine/NO (A) or 30 μM spermine/NO (B) in the absence (open bar) and presence (striped bar) of 5 μM H-89. Spermine/NO-induced stimulation of ICa,L was significantly blocked by inhibition of PKA activity. In addition, H-89 exerted a more effective block at lower spermine/NO concentrations. *P < 0.05; **P < 0.001.

Mentions: We previously reported that inhibition of cAMP-dependent PKA activity abolishes rebound stimulation of ICa,L elicited by ACh withdrawal (Wang and Lipsius, 1995). To determine whether exogenous NO stimulates ICa,L via cAMP, SP/NO was tested in the absence and presence of H-89, an inhibitor of cAMP-dependent PKA activity (Chijiwa et al., 1990). As shown in the bar graph in Fig. 11, 100 μM SP/NO elicited a mean increase in ICa,L of 108 ± 35%. In the presence of 5 μM H-89, the stimulatory effect of SP/NO on ICa,L (36 ± 7%) was significantly reduced compared with control (P < 0.05; n = 5). In three additional cells, the concentration of SP/NO was lowered (30 μM) to a level that elicited an increase in ICa,L comparable to that achieved by ACh withdrawal. In the absence and presence of H-89, 30 μM SP/NO elicited a mean increase in ICa,L of 61 ± 5% and 14 ± 5%, respectively (P < 0.001). These findings indicate that NO stimulates ICa,L primarily by increasing cAMP and are consistent with the role of NO signaling in stimulation of ICa,L elicited by ACh withdrawal.


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

Wang YG, Rechenmacher CE, Lipsius SL - J. Gen. Physiol. (1998)

Effect of H-89, an inhibitor of PKA, on spermine/ NO-induced stimulation of ICa,L. The graph shows the percent  change in ICa,L amplitude elicited by either 100 μM spermine/NO  (A) or 30 μM spermine/NO (B) in the absence (open bar) and  presence (striped bar) of 5 μM H-89. Spermine/NO-induced stimulation of ICa,L was significantly blocked by inhibition of PKA activity.  In addition, H-89 exerted a more effective block at lower spermine/NO concentrations. *P < 0.05; **P < 0.001.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1887767&req=5

Figure 11: Effect of H-89, an inhibitor of PKA, on spermine/ NO-induced stimulation of ICa,L. The graph shows the percent change in ICa,L amplitude elicited by either 100 μM spermine/NO (A) or 30 μM spermine/NO (B) in the absence (open bar) and presence (striped bar) of 5 μM H-89. Spermine/NO-induced stimulation of ICa,L was significantly blocked by inhibition of PKA activity. In addition, H-89 exerted a more effective block at lower spermine/NO concentrations. *P < 0.05; **P < 0.001.
Mentions: We previously reported that inhibition of cAMP-dependent PKA activity abolishes rebound stimulation of ICa,L elicited by ACh withdrawal (Wang and Lipsius, 1995). To determine whether exogenous NO stimulates ICa,L via cAMP, SP/NO was tested in the absence and presence of H-89, an inhibitor of cAMP-dependent PKA activity (Chijiwa et al., 1990). As shown in the bar graph in Fig. 11, 100 μM SP/NO elicited a mean increase in ICa,L of 108 ± 35%. In the presence of 5 μM H-89, the stimulatory effect of SP/NO on ICa,L (36 ± 7%) was significantly reduced compared with control (P < 0.05; n = 5). In three additional cells, the concentration of SP/NO was lowered (30 μM) to a level that elicited an increase in ICa,L comparable to that achieved by ACh withdrawal. In the absence and presence of H-89, 30 μM SP/NO elicited a mean increase in ICa,L of 61 ± 5% and 14 ± 5%, respectively (P < 0.001). These findings indicate that NO stimulates ICa,L primarily by increasing cAMP and are consistent with the role of NO signaling in stimulation of ICa,L elicited by ACh withdrawal.

Bottom Line: When ICa,L was prestimulated by 10 microM milrinone, an inhibitor of cGMP-inhibited phosphodiesterase (type III) activity, SP/NO failed to further increase ICa,L.In cells incubated in pertussis toxin (3.4 microg/ml for 6 h; 36 degrees C), ACh failed to affect ICa,L, but 100 microM SP/NO or 10 microM milrinone still increased basal ICa,L.Pertussis toxin-sensitive G-proteins couple M2 muscarinic receptors to NO signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, USA.

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 Ca2+ current (ICa,L) in cat atrial myocytes. Exposure to 1 microM ACh for 2 min inhibited basal ICa,L (-21 +/- 3%), and withdrawal of ACh elicited rebound stimulation of ICa,L above control (80 +/- 13%) (n = 23). Stimulation of ICa,L elicited by withdrawal of ACh (but not ACh-induced inhibition of ICa,L) was blocked by either 50 microM hemoglobin; 30 microM ODQ or 10 microM methylene blue, inhibitors of soluble guanylate cyclase; 10 microM W-7, a calmodulin inhibitor; or 10 microM L-NIO, an inhibitor of constitutive NO synthase (NOS). In cells incubated in 5 mM L-arginine, ACh-induced rebound stimulation of ICa,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 microM stimulated basal ICa,L. SP/NO-induced stimulation of ICa,L was inhibited by 50 microM hemoglobin, 30 microM ODQ, or 5 microM H-89, an inhibitor of PKA, and was unchanged by 50 microM MnTBAP, a peroxynitrite scavenger. When ICa,L was prestimulated by 10 microM milrinone, an inhibitor of cGMP-inhibited phosphodiesterase (type III) activity, SP/NO failed to further increase ICa,L. In cells incubated in pertussis toxin (3.4 microg/ml for 6 h; 36 degrees C), ACh failed to affect ICa,L, but 100 microM SP/NO or 10 microM milrinone still increased basal ICa,L. These results indicate that in cat atrial myocytes NO signaling mediates stimulation of ICa,L elicited by withdrawal of ACh but not ACh-induced inhibition of basal ICa,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 ICa,L. Pertussis toxin-sensitive G-proteins couple M2 muscarinic receptors to NO signaling. NO-mediated stimulation of ICa, 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.

Show MeSH
Related in: MedlinePlus