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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 l-arginine on ACh-induced  regulation of ICa,L. In control cells (not incubated  in l-arginine), ACh induced a typical inhibition  followed by rebound stimulation of ICa,L elicited  by ACh withdrawal (A). In another cell incubated  in 5 mM l-arginine for 3 h, withdrawal of ACh  elicited a significantly larger rebound stimulation  of ICa,L (B).
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Figure 5: Effect of l-arginine on ACh-induced regulation of ICa,L. In control cells (not incubated in l-arginine), ACh induced a typical inhibition followed by rebound stimulation of ICa,L elicited by ACh withdrawal (A). In another cell incubated in 5 mM l-arginine for 3 h, withdrawal of ACh elicited a significantly larger rebound stimulation of ICa,L (B).

Mentions: As noted earlier, l-arginine is the substrate used by NOS to synthesize NO (Mayer, 1995). If NO signaling participates in ACh-induced regulation of ICa,L, then exposure to a relatively high concentration of l-arginine should accentuate the stimulatory response elicited by ACh withdrawal. In one approach, we tested 1 μM ACh in the absence and presence of 5 mM l-arginine in the same cell. The cell was acutely exposed to l-arginine for 8 min between the first and second exposures to ACh. In the absence of l-arginine, ACh decreased ICa,L by 21 ± 5% and withdrawal of ACh stimulated ICa,L by 49 ± 6% (not shown). Exposure to l-arginine elicited a small decrease in ICa,L (−13 ± 6%). After 8 min in l-arginine, a second exposure to ACh inhibited ICa,L by 23 ± 4% and withdrawal of ACh stimulated ICa,L by 73 ± 13% (n = 8). Although the stimulation of ICa,L elicited by ACh withdrawal was enhanced, it did not reach statistical significance (P = 0.09). It seemed possible that an 8-min exposure to l-arginine was not long enough to significantly raise intracellular l-arginine concentrations. Therefore, in a second approach, we incubated atrial myocytes in 5 mM l-arginine for at least 3 h before testing their response to ACh. Cells incubated in l-arginine for 3 h and those cells exposed to l-arginine for 8 min were obtained from the same two hearts. Cells incubated in l-arginine (3 h) exhibited basal ICa,L amplitudes not significantly different from control cells. Fig. 5, A and B shows original ICa,L traces from a typical experiment. In a cell not incubated in l-arginine (Fig. 5 A), exposure to ACh induced inhibition and withdrawal of ACh induced stimulation of ICa,L (49%). In another cell incubated in l-arginine (Fig. 5 B), ACh-induced inhibition of ICa,L was similar to control, but stimulation of ICa,L elicited by withdrawal of ACh was enhanced (98%). In a total of six cells incubated in l-arginine, exposure to ACh decreased ICa,L by 11 ± 5% and withdrawal of ACh stimulated ICa,L by 109 ± 18%. Although ACh-induced inhibition of ICa,L was smaller in cells incubated in l-arginine (−11 ± 5%) compared with control cells (−21 ± 5%), the differences in these unpaired data did not reach statistical significance. Stimulation of ICa,L elicited by withdrawal of ACh, however, was significantly larger in l-arginine-treated cells (109 ± 18%) compared with control (49 ± 6%) (P < 0.05). To examine the stereo specificity of arginine, an additional experiment was performed where cells were incubated in 5 mM d-arginine. Cells incubated in d-arginine showed a 25% smaller rebound stimulation of ICa,L induced by ACh withdrawal than control cells from the same heart (n = 2). The results are consistent with the view that additional substrate (l-arginine) for NO signaling augmented the stimulation of ICa,L elicited by withdrawal of ACh. In our previous study, we noted that the increase in ICa,L amplitude elicited by ACh withdrawal ranged between 5 and 232% (SD ± 49%; n = 53) in any given cell (Wang and Lipsius, 1995). Based on the present results, this variability in response could be due to the dependence of the rebound response on l-arginine concentration, which may vary in individual myocytes after isolation.


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 l-arginine on ACh-induced  regulation of ICa,L. In control cells (not incubated  in l-arginine), ACh induced a typical inhibition  followed by rebound stimulation of ICa,L elicited  by ACh withdrawal (A). In another cell incubated  in 5 mM l-arginine for 3 h, withdrawal of ACh  elicited a significantly larger rebound stimulation  of ICa,L (B).
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Related In: Results  -  Collection

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Figure 5: Effect of l-arginine on ACh-induced regulation of ICa,L. In control cells (not incubated in l-arginine), ACh induced a typical inhibition followed by rebound stimulation of ICa,L elicited by ACh withdrawal (A). In another cell incubated in 5 mM l-arginine for 3 h, withdrawal of ACh elicited a significantly larger rebound stimulation of ICa,L (B).
Mentions: As noted earlier, l-arginine is the substrate used by NOS to synthesize NO (Mayer, 1995). If NO signaling participates in ACh-induced regulation of ICa,L, then exposure to a relatively high concentration of l-arginine should accentuate the stimulatory response elicited by ACh withdrawal. In one approach, we tested 1 μM ACh in the absence and presence of 5 mM l-arginine in the same cell. The cell was acutely exposed to l-arginine for 8 min between the first and second exposures to ACh. In the absence of l-arginine, ACh decreased ICa,L by 21 ± 5% and withdrawal of ACh stimulated ICa,L by 49 ± 6% (not shown). Exposure to l-arginine elicited a small decrease in ICa,L (−13 ± 6%). After 8 min in l-arginine, a second exposure to ACh inhibited ICa,L by 23 ± 4% and withdrawal of ACh stimulated ICa,L by 73 ± 13% (n = 8). Although the stimulation of ICa,L elicited by ACh withdrawal was enhanced, it did not reach statistical significance (P = 0.09). It seemed possible that an 8-min exposure to l-arginine was not long enough to significantly raise intracellular l-arginine concentrations. Therefore, in a second approach, we incubated atrial myocytes in 5 mM l-arginine for at least 3 h before testing their response to ACh. Cells incubated in l-arginine for 3 h and those cells exposed to l-arginine for 8 min were obtained from the same two hearts. Cells incubated in l-arginine (3 h) exhibited basal ICa,L amplitudes not significantly different from control cells. Fig. 5, A and B shows original ICa,L traces from a typical experiment. In a cell not incubated in l-arginine (Fig. 5 A), exposure to ACh induced inhibition and withdrawal of ACh induced stimulation of ICa,L (49%). In another cell incubated in l-arginine (Fig. 5 B), ACh-induced inhibition of ICa,L was similar to control, but stimulation of ICa,L elicited by withdrawal of ACh was enhanced (98%). In a total of six cells incubated in l-arginine, exposure to ACh decreased ICa,L by 11 ± 5% and withdrawal of ACh stimulated ICa,L by 109 ± 18%. Although ACh-induced inhibition of ICa,L was smaller in cells incubated in l-arginine (−11 ± 5%) compared with control cells (−21 ± 5%), the differences in these unpaired data did not reach statistical significance. Stimulation of ICa,L elicited by withdrawal of ACh, however, was significantly larger in l-arginine-treated cells (109 ± 18%) compared with control (49 ± 6%) (P < 0.05). To examine the stereo specificity of arginine, an additional experiment was performed where cells were incubated in 5 mM d-arginine. Cells incubated in d-arginine showed a 25% smaller rebound stimulation of ICa,L induced by ACh withdrawal than control cells from the same heart (n = 2). The results are consistent with the view that additional substrate (l-arginine) for NO signaling augmented the stimulation of ICa,L elicited by withdrawal of ACh. In our previous study, we noted that the increase in ICa,L amplitude elicited by ACh withdrawal ranged between 5 and 232% (SD ± 49%; n = 53) in any given cell (Wang and Lipsius, 1995). Based on the present results, this variability in response could be due to the dependence of the rebound response on l-arginine concentration, which may vary in individual myocytes after isolation.

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