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(Circulation Research. 2003;93:1233.)
© 2003 American Heart Association, Inc.

Cellular Biology

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

Elena N. Dedkova, Xiang Ji, Yong Gao Wang, Lothar A. Blatter, Stephen L. Lipsius

From the Department of Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Ill.

Correspondence to Stephen L. Lipsius, PhD, Department of Physiology, Loyola University Medical Center, 2160 S First Ave, Maywood, IL 60153. E-mail slipsiu{at}lumc.edu

	Abstract

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Fluorescence microscopy and the NO-sensitive indicator 4,5-diaminofluorescein were used to determine the effects of acetylcholine (ACh) on intracellular NO (NO_i) in cat atrial myocytes. Field stimulation (1 Hz) of cells or exposure of quiescent cells to ACh (1 to 10 µmol/L) had no effect on NO_i. However, in field-stimulated cells, ACh exposure increased NO_i, and ACh withdrawal elicited an additional, prominent increase in NO_i production. During ACh exposure, addition of 1 µmol/L atropine increased NO_i production similar to ACh withdrawal. ACh-induced increases in NO_i were reduced by prior exposure to 1 mmol/L extracellular Ca²⁺ ([Ca²⁺]_o) and prevented by 0.5 mmol/L [Ca²⁺]_o, 1 µmol/L verapamil, 1 µmol/L atropine, 10 µmol/L L-N⁵-(1-iminoethyl)ornithine, 10 µmol/L W-7, or incubating cells in pertussis toxin or 10 µmol/L LY294002 (inhibits phosphatidylinositol 3-kinase). Switching to 0.5 mmol/L [Ca²⁺]_o during ACh withdrawal prevented the additional increase in NO_i. ACh exposure increased phosphorylation (Ser⁴⁷³) of protein kinase B (Akt), and this effect was blocked by LY294002 and unaffected in low (0.5 mmol/L) [Ca²⁺]_o. Confocal microscopy revealed that ACh exposure increased NO_i at local subsarcolemmal sites, and ACh withdrawal additionally increased NO_i by recruiting additional subsarcolemmal release sites. Disruption of caveolae by 2 mmol/L methyl-ß-cyclodextrin abolished ACh-induced NO_i production. We conclude that in cat atrial myocytes, ACh stimulates NO_i release from local subsarcolemmal sites. ACh-induced increases in NO_i requires both muscarinic receptor–mediated G_i protein/phosphatidylinositol 3-kinase/Akt signaling and voltage-activated Ca²⁺ influx for stimulation of calmodulin-dependent endothelial NO synthase activity. Increases in NO_i elicited by ACh withdrawal result from the recovery of Ca²⁺ influx after ACh inhibition. NO signaling elicited by ACh withdrawal stimulates rapid recovery from cholinergic atrial inhibition.

Key Words: phosphatidylinositol 3-kinase • protein kinase B/Akt • calmodulin • calcium

	Introduction

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Several studies indicate that NO signaling mediates the inhibitory effects of acetylcholine (ACh) on cardiac function.^1–3 These findings, however, have been disputed^4–6 and remain controversial.⁷ In cat atrial myocytes, ACh-induced inhibition of basal L-type Ca²⁺ 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 Ca²⁺-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 Ca²⁺ 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 cat⁸ and human¹³ 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.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 fibers¹⁵ and ventricular myocytes prestimulated by ß-adrenergic agonists.^16,17 However, in contrast to atrial myocytes, the stimulatory effect of ACh withdrawal in ventricular myocytes is not mediated via NO or cGMP signaling^16,18 but rather is attributable to stimulation of adenylate cyclase by the ß subunit of G_i protein.¹⁶

In the present study, we used fluorescence microscopy and the NO-sensitive indicator DAF-2 to directly determine the effects of ACh on intracellular NO (NO_i) production in cat atrial myocytes. We also sought to determine whether stimulation of muscarinic receptors increases NO_i production via G_i proteins coupled to phosphatidylinositol 3-kinase (PI-3K)/protein kinase B (Akt) signaling, similar to NO_i production elicited by ß₂-adrenergic receptor (AR) stimulation.¹⁹ The present results indicate that ACh exposure and withdrawal increase NO_i production. ACh-induced increases in NO_i require both muscarinic receptor–mediated G_i/PI-3K/Akt signaling and voltage-activated Ca²⁺ influx for stimulation of calmodulin (CaM)-dependent endothelial NO synthase (eNOS) activity. Moreover, ACh withdrawal increases NO_i production above that induced during ACh exposure, consistent with the role of NO signaling in rebound stimulation of I_Ca,L.

	Materials and Methods

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Adult cats of either sex were anesthetized with sodium pentobarbital (50 mg/kg IP). Once anesthetized, the heart was rapidly excised and mounted on a Langendorff perfusion apparatus. Atrial myocytes were dispersed by enzymatic (collagenase; type II, Worthington Biochemical) digestion, as previously reported.²⁰ Measurements of NO_i were obtained by incubating cells with the fluorescent NO-sensitive dye 4,5-diaminofluorescein (DAF-2),^21,22 as previously described^19,23 (for additional details, see the online data supplement, available at http://www.circresaha.org). Experiments were performed at room temperature. Changes in cellular DAF-2 fluorescence intensities (F) were normalized to the level of fluorescence recorded before stimulation (F₀), and changes in [NO]_i are expressed as F/F₀. Activation of DAF-2 by NO is irreversible, and therefore fluorescence intensity remains constant even if NO_i levels decrease. In a few experiments, perforated patch recording methods were used to measure electrical activity of atrial myocytes, as previously described.¹¹

Two-dimensional (2D) and fast one-dimensional (linescan) imaging was performed using a confocal scanning unit (LSM 410, Zeiss) attached to an inverted microscope (Axiovert 100, Zeiss) fitted with a 40 oil-immersion objective lens (Plan-Neofluar, numerical aperture=1.3, Zeiss). Atrial myocytes were loaded with the NO-sensitive indicator DAF-2, as described above. DAF-2 fluorescence was excited with a 488-nm line of an argon ion laser, and emitted fluorescence was collected at wavelength >515 nm. For linescan imaging, the specimen was scanned repetitively at 5-ms intervals. All linescan images were recorded at a central focal plane and oriented along the longitudinal axis of the cell within the subsarcolemmal region. Increases in NO_i recorded by linescan were quantified by measuring the frequency and amplitude of NO_i peaks. NO_i peaks were defined as those changes in NO_i that reached 50% above baseline (F/F₀).

Immunoblots were used to analyze ACh-induced phosphorylation of Akt (protein kinase B). Isolated atrial cells were treated with control media (M199), 10 µmol/L ACh, ACh plus 10 µmol/L LY294002, low (0.5 mmol/L) [Ca²⁺]_o, or ACh plus 0.5 mmol/L [Ca²⁺]_o before harvesting. Cells were incubated with LY294002 for 10 minutes, followed by a 2-minute exposure to ACh (for additional details, see the online data supplement).

Drugs in this study included acetylcholine chloride, atropine, LY294002, L-N⁵-(1-iminoethyl)ornithine (L-NIO), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), verapamil, methyl-ß-cyclodextrin, pertussis toxin (all from Sigma Chemical), and 4,5-diaminofluorescein diacetate (DAF-2 DA) (Calbiochem).

NO_i measurements obtained from two groups of cells were analyzed using Student’s unpaired t test for significance at P<0.05. Multiple groups were analyzed using ANOVA followed by Student Newman-Keuls test for significance at P<0.05.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

	Results

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Figure 1A shows a typical recording of NO_i production obtained from a field-stimulated (+FS, 1 Hz) atrial myocyte and a second quiescent (-FS) atrial myocyte exposed to 1 µmol/L ACh. In the field-stimulated cell, ACh exposure elicited an increase in NO_i, and withdrawal of ACh elicited an additional, prominent increase in NO_i above that elicited during ACh exposure (n=16). In the quiescent cell (-FS), ACh exposure and withdrawal had no effect on NO_i (n=6). However, in the same cell exposure to the NO donor, spermine/NO (Sper/NO; 100 µmol/L) prominently increased NO_i (Figure 1A). The response to Sper/NO indicates that the DAF-2 indicator was functioning normally and thereby serves as a positive control. Field stimulation alone also had no effect on NO_i production (data not shown; n=12). Previous work showed that termination of ACh action by atropine elicits rebound stimulation of I_Ca,L typically obtained by ACh withdrawal.⁹ In the present study, after 3 minutes of ACh exposure, addition of 1 µmol/L atropine elicited an increase in NO_i production similar to that elicited by ACh withdrawal (data not shown; n=3). The effects of ACh exposure and withdrawal on NO_i production are summarized in Figure 1D and indicate that ACh exposure elicits a relatively modest increase in NO_i production and that ACh withdrawal elicits additional, prominent increases in NO_i above those elicited during ACh exposure. Moreover, the receptor-mediated effects of ACh to increase NO_i require electrical stimulation of the cell, presumably to increase Ca²⁺ influx (see below). Therefore, all additional experiments were performed on myocytes field-stimulated at 1 Hz. Figure 1B shows the effects of ACh recorded from another atrial myocyte in the presence of 10 µmol/L L-NIO, a specific inhibitor of eNOS activity.²⁴ As summarized in Figure 1D, compared with control responses, inhibition of eNOS essentially abolished the effects of ACh exposure and withdrawal to increase NO_i (n=10). In the same cell, 100 µmol/L Sper/NO prominently increased NO_i. In cardiac cells, eNOS is localized to caveolae.²⁵ Methyl-ß-cyclodextrin (cyclodextrin) solubilizes cholesterol and thereby disrupts caveolae formation.²⁶ ACh was tested on atrial myocytes incubated (1 hour at 37°C) in 2 mmol/L cyclodextrin. As shown in Figure 1C and summarized in Figure 1D, cyclodextrin abolished the increases in NO_i induced by ACh exposure and withdrawal (n=3). Together, these findings indicate that ACh increases NO_i by stimulating eNOS activity localized to caveolae.

View larger version (25K): [in this window] [in a new window]   Figure 1. Effects of field stimulation, L-NIO, and methyl-ß-cyclodextrin on NOi production induced by 1 µmol/L ACh in atrial myocytes. A, In a field-stimulated cell (+FS, 1 Hz), ACh exposure increased NOi and ACh withdrawal elicited an additional, prominent increase in NOi. In a quiescent atrial myocyte (-FS), ACh had no effect on NOi. In the same cell, 100 µmol/L spermine/NO (Sper/NO) prominently increased NOi. B, Pretreatment with 10 µmol/L L-NIO inhibited ACh-induced increases in NOi. In the same cell, 100 µmol/L Sper/NO increased NOi. C, Incubation in 2 mmol/L methyl-ß-cyclodextrin abolished ACh-induced increases in NOi. D, Graph shows mean±SE, summarizing the effects of ACh exposure (ACh) and withdrawal (ACh/w) on NOi production in the presence of L-NIO (gray bar; n=10) and methyl-ß-cyclodextrin (dextrin; open bars; n=3) compared with control (black bars; n=16). *P<0.05 compared with control.

The fact that ACh-induced increases in NO_i require that cells be electrically stimulated suggests that voltage-activated Ca²⁺ influx is an essential signaling element, consistent with the Ca²⁺-CaM dependence of eNOS activity. We therefore tested the effects of ACh to increase NO_i when the extracellular Ca²⁺ concentration ([Ca²⁺]_o) was reduced to either 1 or 0.5 mmol/L before ACh exposure. As shown in Figure 2A and summarized in Figure 2D, compared with control (2 mmol/L), reducing [Ca²⁺]_o to 1 mmol/L decreased NO_i production elicited during ACh exposure and withdrawal (n=10), and 0.5 mmol/L [Ca²⁺]_o essentially abolished the increase in NO_i elicited during ACh exposure and withdrawal (n=5). Similar results were obtained by exposure to 1 µmol/L verapamil, an L-type Ca²⁺ channel antagonist (data not shown; n=3). In separate experiments, bathing cells in 0.5 mmol/L [Ca²⁺]_o or verapamil did not prevent electrical excitation elicited by field stimulation (n=3). To determine the role of CaM, ACh was tested on atrial myocytes pretreated with 10 µmol/L W-7, a potent CaM inhibitor.²⁷ As shown in Figure 2B and summarized in Figure 2D, compared with control, W-7 abolished increases in NO_i induced by ACh exposure and withdrawal (n=5). In the same cell, 100 µmol/L Sper/NO prominently increased NO_i. To determine whether Ca²⁺ influx specifically contributes to the additional increase in NO_i elicited by ACh withdrawal, we switched to 0.5 mmol/L [Ca²⁺]_o specifically during ACh withdrawal. As shown in Figure 2C and summarized in Figure 2D, ACh exposure in normal (2 mmol/L) [Ca²⁺]_o elicited a typical increase in NO_i. However, ACh withdrawal in low [Ca²⁺]_o failed to significantly increase NO_i above that elicited by ACh exposure (n=4). In the four cells tested, NO_i levels during ACh exposure in normal [Ca²⁺]_o (1.041±0.009 F/F₀) and during ACh withdrawal in 0.5 mmol/L [Ca²⁺]_o (1.053±0.011 F/F₀) were not different. As summarized in Figure 2D, NO_i during ACh withdrawal was significantly smaller in 0.5 mmol/L [Ca²⁺]_o (hatched bar) compared with control ACh withdrawal (black bar). These findings indicate that the ability of ACh exposure and withdrawal to increase NO_i depends on Ca²⁺ influx, presumably to activate CaM-dependent eNOS.

View larger version (30K): [in this window] [in a new window]   Figure 2. Effects of ACh on NOi are dependent on [Ca2+]o and CaM. A, In control (2 mmol/L Ca2+), 1 µmol/L ACh elicited typical increases in NOi. In 2 different cells, lowering [Ca2+]o to 1 mmol/L and 0.5 mmol/L decreased and prevented, respectively, ACh-induced increases in NOi. B, Pretreatment with 10 µmol/L W-7, a potent CaM inhibitor, abolished ACh-induced increases in NOi. In the same cell, 100 µmol/L Sper/NO prominently increased NOi. C, ACh exposure in normal [Ca2+]o elicited a typical increase in NOi. However, superfusing 0.5 mmol/L [Ca2+]o specifically during ACh withdrawal prevented the increase in NOi. D, Graph shows mean±SE, summarizing the effects of ACh exposure (ACh) and withdrawal (ACh/w) in 1 mmol/L [Ca2+]o (shaded bars; n=10), 0.5 mmol/L [Ca2+]o (open bars; n=5), W-7 (hatched bars; n=5), and ACh withdrawal in 0.5 mmol/L [Ca2+]o (cross-hatched bar; n=4) compared with control (black bars; n=16). *P<0.05 compared with control.

Previous findings in cat atrial myocytes indicate that the effect of ACh withdrawal to stimulate I_Ca,L is mediated via muscarinic receptors coupled to G_i protein signaling.⁹ Moreover, ß₂-AR stimulation acts via G_i protein and PI-3K signaling to increase NO_i.¹⁹ We therefore sought to determine whether muscarinic receptors act via a similar G_i protein/PI-3K signaling pathway to increase NO_i. As shown in Figure 3A, compared with control responses, pretreatment with the muscarinic receptor antagonist (1 µmol/L) atropine blocked NO_i production induced by ACh exposure and withdrawal (n=6). In Figure 3B, incubating cells in pertussis toxin (PTX) (3.5 µg/mL; 3 hours, 36°C) to inhibit G_i protein signaling also abolished increases in NO_i induced by ACh exposure and withdrawal (n=6). In the same PTX-treated cell (Figure 3B), 100 µmol/L Sper/NO prominently increased NO_i. To examine the role of PI-3K signaling, cells were incubated in 10 µmol/L LY294002, an inhibitor of PI-3K signaling,²⁸ for 30 minutes before being tested with ACh. Previous work has shown that either LY294002 or wortmannin, another inhibitor of PI-3K signaling, inhibits ß₂-AR stimulation of NO_i release.¹⁹ As shown in Figure 3C, inhibition of PI-3K signaling by LY294002 also inhibited NO_i production elicited by ACh exposure and withdrawal (n=7). Once again, in the same cell treated with LY294002, 100 µmol/L Sper/NO prominently increased NO_i. The results are summarized in the graph in Figure 3D and indicate that ACh acts on muscarinic receptors coupled via G_i proteins and PI-3K signaling to stimulate NO_i production. Apparently, muscarinic and ß₂-ARs act via the same G_i protein/PI-3K signaling pathway to stimulate NO_i production.

View larger version (27K): [in this window] [in a new window]   Figure 3. Effects of ACh on NOi are mediated by muscarinic receptors coupled via PTX-sensitive Gi protein and PI-3K signaling. A, In control, 1 µmol/L ACh elicited typical increases in NOi. Pretreatment with 1 µmol/L atropine abolished ACh-induced increases in NOi. B, Incubation of cells in PTX abolished ACh-induced increases in NOi. In the same cell, 100 µmol/L Sper/NO increased NOi. C, Incubation of cells in 10 µmol/L LY294002, a specific PI-3K inhibitor, prevented ACh-induced increases in NOi. In the same cell, 100 µmol/L Sper/NO increased NOi. D, Graph shows mean±SE, summarizing the effects of ACh exposure (ACh) and withdrawal (ACh/w) in the presence of atropine (gray bars; n=6), PTX (open bars; n=6), and LY294002 (hatched bars; n=7) compared with control (black bars; n=5). *P<0.05 compared with control.

In endothelial cells, PI-3K signaling phosphorylates protein kinase B (Akt), which in turn activates eNOS.^29,30 We therefore used immunoblots to determine the effects of ACh on Akt phosphorylation. As shown in Figure 4, compared with control, 10 µmol/L ACh significantly increased phosphorylation of Akt. Pretreatment with 10 µmol/L LY294002 prevented ACh-induced phosphorylation of Akt (n=5). These results are consistent with the present findings that inhibition of PI-3K signaling (LY294002) inhibits ACh-induced NO_i production (Figure 3C). It is also important to note that in the experiments designed to measure Akt phosphorylation, cells were quiescent. That is, under these conditions, voltage-activated Ca²⁺ influx is not operating and therefore the cells are not capable of ACh-induced NO_i production (see Figure 1A). This indicates that ACh-induced stimulation of PI-3K/Akt signaling occurs independently of voltage-activated Ca²⁺ influx and is not capable per se of stimulating NO_i production. To gain additional insight into the Ca²⁺ dependence of ACh-induced phosphorylation of Akt, we tested the effects of ACh in control (2 mmol/L) and low (0.5 mmol/L) [Ca²⁺]_o (see online Figure 2, available at http://www.circresaha.org). The results of these experiments show that in low [Ca²⁺]_o, ACh still increased Akt phosphorylation. We therefore conclude that although Ca²⁺ is necessary for ACh-induced NO_i production, presumably to activate CaM-dependent eNOS, ACh-induced activation of Akt signaling is Ca²⁺-independent.

View larger version (47K): [in this window] [in a new window]   Figure 4. Western blots showing the effects of ACh exposure on Akt phosphorylation. A, Top, typical bands obtained by phospho-Akt (p-Akt)-specific antibodies. Bottom, Akt-loading controls. ACh (10 µmol/L) significantly increased Akt phosphorylation, and pretreatment with 10 µmol/L LY294002 prevented ACh-induced Akt phosphorylation. Graph shows mean±SE normalized against control (1.0) (n=5). *P<0.05 compared with control.

We next used high-resolution confocal imaging to examine spatial patterns of NO_i release induced by ACh exposure and withdrawal. Figure 5A shows typical 2D surface plots of atrial cells during control, ACh exposure, and ACh withdrawal. Compared with control, ACh exposure increased NO_i at local sites along the cell periphery. ACh withdrawal elicited additional increases in NO_i, primarily by recruiting additional release sites along the cell periphery. Figure 5B shows that in another atrial myocyte pretreated (1 hour) with 2 mmol/L methyl-ß-cyclodextrin, ACh failed to elicit any changes in NO_i either during ACh exposure or withdrawal. Similar results were obtained in a total of four cells.

View larger version (43K): [in this window] [in a new window]   Figure 5. 2D surface plots from 2 atrial myocytes showing spatial patterns of NOi production induced by ACh exposure and withdrawal. A, Compared with control, ACh (1 µmol/L) exposure increased NOi primarily along the cell periphery. ACh withdrawal (ACh/w) elicited additional increases in NOi primarily by recruiting additional release sites along the cell periphery. B, In another atrial myocyte, incubation in 2 mmol/L methyl-ß-cyclodextrin (cyclodextrin) abolished increases in NOi typically induced by ACh exposure and withdrawal. C, Spatial profiles obtained from linescan images of local changes in NOi average over time (250 ms), recorded between contractile events to avoid mechanical artifacts. A repetitively scanned line was positioned parallel with the longitudinal axis of the cell within the subsarcolemmal region. ACh exposure (red trace) increased NOi at local subsarcolemmal sites and slightly raised baseline NOi. ACh withdrawal (blue trace) elicited additional increases in NOi by enhancing NOi release at sites previously stimulated during ACh exposure (site 1) and by recruiting additional subsarcolemmal release sites (sites 2, 3, and 4). Baseline NOi levels also were increased additionally. D, Graph summarizes the increases in NOi elicited by ACh exposure (red bars) and ACh withdrawal (blue bars) by quantifying the amplitude and frequency of NOi release events recorded by linescan (n=5). NOi peaks were defined as those changes in NOi that reached 50% above baseline (dashed line). Frequency of NOi peaks were normalized to scanned cell length (peaks/µm). Cells were electrically field–stimulated at 1 Hz. *P<0.05.

Spatial changes in ACh-induced NO_i production were examined in more detail by recording NO_i using a repetitively scanned line positioned parallel with the longitudinal axis of the cell within the subsarcolemmal region. The graph in Figure 5C shows spatial profiles of local changes in NO_i average over time and expressed as F/F₀ during ACh exposure (red trace) and ACh withdrawal (blue trace). ACh exposure increased NO_i at local subsarcolemmal sites and elicited small increases in baseline NO_i. ACh withdrawal elicited additional increases in NO_i by enhancing NO_i release at some sites previously stimulated during ACh exposure (for example, site 1) and by recruiting additional subsarcolemmal release sites (for example, sites 2, 3, and 4). Also, baseline NO_i levels were increased additionally. Incubation of cells in 2 mmol/L cyclodextrin abolished all effects of ACh to increase NO_i (data not shown). Similar results were obtained in a total of five cells. The graph in Figure 5D summarizes the increases in amplitude (in relation to baseline) and frequency of NO_i release events elicited by ACh exposure (red bars) and ACh withdrawal (blue bars) (n=5). ACh exposure increased both the amplitude and frequency of NO_i release events, and these parameters were significantly greater during ACh withdrawal compared with ACh exposure.

	Discussion

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Previous studies from our laboratory have shown that in cat atrial myocytes, ACh exposure inhibits I_Ca,L and ACh withdrawal stimulates I_Ca,L above control levels, ie, rebound stimulation.⁹ Pharmacological analyses indicated that although NO signaling does not participate in ACh-induced inhibition of I_Ca,L, the rebound stimulation of I_Ca,L elicited by ACh withdrawal is mediated by NO signaling.⁸ The present study directly demonstrates for the first time that in atrial myocytes, ACh exposure increases NO_i production and ACh withdrawal elicits an additional, prominent increase in NO_i above that achieved during ACh exposure. The actions of ACh require both stimulation of muscarinic receptor–mediated G_i protein/PI-3K/Akt signaling and voltage-activated Ca²⁺ influx to elicit local, subsarcolemmal increases in NO_i production.

The present results indicate that muscarinic receptor stimulation by ACh is unable to stimulate NO_i production in quiescent atrial myocytes. ß₂-AR stimulation also requires electrical stimulation of atrial myocytes to increase NO_i production.¹⁹ Given the Ca²⁺-CaM dependence of eNOS activity, these findings suggest that voltage-activated Ca²⁺ influx is essential for receptor-mediated stimulation of NO_i. Indeed, in electrically stimulated atrial myocytes, ACh-induced increases in NO_i are decreased (1 mmol/L) or abolished (0.5 mmol/L) by reducing [Ca²⁺]_o, inhibition of L-type Ca²⁺ channels (verapamil), or inhibition of CaM activity (W-7). In addition, lowering [Ca²⁺]_o specifically during ACh withdrawal prevented the additional, prominent increase in NO_i. In endothelial cells, removal of extracellular Ca²⁺³¹ or exposure to CaM antagonists³² also abolishes agonist-induced NO formation. Although electrical stimulation is required for receptor-mediated NO_i production, it is not sufficient per se to elicit NO_i production. In other words, in atrial myocytes, basal voltage-activated Ca²⁺ influx and presumably intracellular Ca²⁺ release induced by Ca²⁺ influx are not capable of stimulating eNOS activity. This is in contrast to findings in rat ventricular myocytes in which basal Ca²⁺ influx elicited by electrical stimulation is sufficient to increase nitrite levels.³³ We have obtained similar results in cat ventricular myocytes, where electrical stimulation alone is sufficient to increase NO_i production (unpublished observations). In electrically stimulated atrial myocytes, even marked increases in Ca²⁺ influx via I_Ca,L and presumably intracellular Ca²⁺ release induced by ß₁-AR stimulation fail to increase NO_i.¹⁹ On the other hand, ß₂-AR stimulation elicits a similar increase in I_Ca,L and does increase NO_i.¹⁹ Together, these findings indicate that, in atrial myocytes, voltage-activated Ca²⁺ influx and intracellular Ca²⁺ release per se are not sufficient to stimulate NO_i production. However, Ca²⁺ influx is essential for NO_i production stimulated by specific receptor-mediated signaling. The contribution of intracellular Ca²⁺ release to muscarinic receptor–mediated NO_i production remains to be determined.

In the present study, ACh acts via muscarinic receptors coupled to G_i proteins and PI-3K/Akt signaling to activate CaM-dependent NO_i production. Our previous studies have shown that in cat atrial myocytes, stimulation of I_Ca,L elicited by ACh withdrawal also is mediated via muscarinic receptors coupled to G_i proteins and activation of CaM-dependent NO signaling.⁸ In a variety of cell systems, PI-3K signaling leads to phosphorylation and activation of Akt signaling.³⁴ In both endothelial^29,30 and cardiac³⁵ cells, PI-3K/Akt signaling phosphorylates and activates eNOS to produce NO. The present experiments indicate that ACh is not capable of eliciting NO_i production in quiescent atrial cells because of the requirement for voltage-activated Ca²⁺ influx. However, the immunoblot experiments show that ACh is able to stimulate PI-3K/Akt signaling in quiescent cells (Figure 4A). These findings suggest that muscarinic receptor–mediated stimulation of PI-3K/Akt signaling is not capable per se of stimulating NO_i production and that stimulation of this signaling pathway occurs independently of voltage-activated Ca²⁺ influx. The latter finding is supported by the fact that lowering [Ca²⁺]_o to a level (0.5 mmol/L) that prevents ACh-induced NO_i production failed to prevent ACh-induced Akt phosphorylation (see online Figure 2). This is consistent with reports that Akt activation is Ca²⁺-independent.³⁶ We therefore conclude that receptor-mediated PI-3K/Akt signaling plus voltage-activated Ca²⁺ influx are both required for stimulation of NO_i production. Our previous experiments indicate that in cat atrial myocytes, ß₂-AR stimulation also requires both PI-3K signaling and voltage-activated Ca²⁺ influx to elicit NO_i production.¹⁹ This dual signaling mechanism can account for the relatively small NO_i production elicited during ACh exposure and the more prominent increase in NO_i elicited by ACh withdrawal. Thus, ACh exposure stimulates PI-3K/Akt signaling at the same time that it decreases Ca²⁺ influx via I_Ca,L, thereby allowing only a modest increase in NO_i production. However, once PI-3K/Akt signaling is stimulated by ACh exposure, rapid removal of ACh from its receptor allows rapid recovery of Ca²⁺ influx, resulting in the additional, prominent stimulation of NO_i production. Moreover, because ACh withdrawal results in the recovery of adenylate cyclase/cAMP signaling, increases in NO_i stimulate cAMP-mediated increases in Ca²⁺ influx via I_Ca,L,⁸ which in turn contribute to additional Ca²⁺-dependent increases in NO_i. Our interpretation that NO_i production is modulated by Ca²⁺ influx is supported by experiments in which we recorded intracellular [Ca²⁺] and NO_i simultaneously (see online Figure 1). In endothelial cells, receptor-mediated signaling by bradykinin can act independently of PI-3K/Akt signaling to enhance the binding of CaM to eNOS and thereby enhance the Ca²⁺ sensitivity of eNOS activity.³⁷ This mechanism results in high-output Ca²⁺-dependent NO production. It seems unlikely, however, that a similar mechanism plays a primary role in atrial myocytes given the present finding that ACh-induced increases in NO_i are entirely dependent on PI-3K/Akt signaling.

The contribution of NO signaling to muscarinic receptor–mediated inhibition of cardiac function differs among different species, tissues, and reports from different laboratories.⁷ Although the present results show that ACh exposure modestly increases NO_i production, NO signaling does not contribute to ACh-induced inhibition of I_Ca,L.⁸ In fact, in cat atrial myocytes, NO signaling stimulates I_Ca,L via cAMP-dependent protein kinase A signaling⁸ and therefore would not be expected to contribute to the inhibitory effects of ACh. Moreover, the fact that ACh inhibits basal adenylate cyclase/cAMP activity would preclude any significant effects of NO signaling on cAMP-mediated regulation of I_Ca,L. In addition, the level of NO_i production during ACh exposure may be below the threshold required for activation of cGMP-mediated signaling and modulation of channel function.

The present results indicate that ACh increases NO_i primarily at the cell periphery and disruption of caveolae formation by cyclodextrin abolishes ACh-induced increases in NO_i. These findings are consistent with reports that in cardiac cells eNOS is localized to caveolae through binding to the scaffolding protein caveolin-3.^25,38 The binding of caveolin holds eNOS in an inactive conformation.^38,39 Increases in Ca²⁺ concentration activate CaM binding to eNOS, thereby disrupting the inhibitory eNOS-caveolin complex and activating eNOS activity.³⁹ M₂ muscarinic receptors are thought to translocate to caveolae once stimulated by agonist.⁴⁰ Moreover, stimulation of M₂ muscarinic receptors causes a reversible translocation of eNOS from caveolae and may partition the enzyme into both noncaveolar plasma membrane and more hydrophilic regions of the cell.⁴¹ Because cat atrial myocytes lack T-tubules,⁴² the peripheral sarcolemmal membrane is the only site of voltage-activated Ca²⁺ influx and the region most abundant in caveolae. The finding that ACh withdrawal stimulates additional NO_i release sites suggests that different release sites exhibit different thresholds for stimulation of NO_i production. The preferential release of NO_i from subsarcolemmal sites also is consistent with the local regulation of sarcolemmal channel function exerted by NO_i signaling.^19,43

Clearly, the prominent increase in NO_i elicited by ACh withdrawal strongly supports our previous findings that rebound stimulation of I_Ca,L elicited by ACh withdrawal is mediated by NO signaling.⁸ In vivo, the actions of ACh at the muscarinic receptor are terminated almost instantaneously by cholinesterase activity. We therefore propose that the NO signaling mechanisms reported here play an important role in ensuring rapid recovery of both chronotropic¹⁰ and inotropic⁹ activities after cholinergic inhibition of atrial function. In fact, we have reported that NO_i signaling elicited by ACh withdrawal may contribute to the nonadrenergic component of postvagal tachycardia¹⁰ and the potential development of Ca²⁺-mediated atrial dysrhythmias¹¹ induced by withdrawal of parasympathetic nerve activity.⁴⁴

	Acknowledgments

 
Support was provided by NIH grants HL63753 (to S.L.L.) and HL62231 (to L.A.B.). We thank Anne Pezalla for her technical assistance with these experiments.

	Footnotes

 
Original received May 30, 2003; revision received October 30, 2003; accepted October 30, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Han X, Shimoni Y, Giles WR. An obligatory role for nitric oxide in autonomic control of mammalian heart rate. J Physiol. 1994; 476.2: 309–314.

2. Han X, Kubota I, Feron O, Opel DJ, Arstall MA, Zhao YY, Huang P, Fishman MC, Michel T, Kelly RA. Muscarinic cholinergic regulation of cardiac myocytes I_Ca-L is absent in mice with targeted disruption of endothelial nitric oxide synthase. Proc Natl Acad Sci U S A. 1998; 95: 6510–6515.[Abstract/Free Full Text]

3. Han X, Kobzik L, Balligand J-L, Kelly RA, Smith TW. Nitric oxide synthase (NOS3)-mediated cholinergic modulation of Ca²⁺ current in adult rabbit atrioventricular nodal cells. Circ Res. 1996; 78: 998–1008.[Abstract/Free Full Text]

4. Vandecasteele G, Eschenhagen T, Scholz H, Stein B, Verde I, Fischmeister R. Muscarinic and ß-adrenergic regulation of heart rate, force of contraction and calcium current is preserved in mice lacking endothelial nitric oxide synthase. Nat Med. 1999; 5: 331–334.[CrossRef][Medline] [Order article via Infotrieve]

5. Belevych AE, Harvey RD. Muscarinic inhibitory and stimulatory regulation of the L-type Ca²⁺ current is not altered in cardiac ventricular myocytes from mice lacking endothelial nitric oxide synthase. J Physiol. 2000; 528: 279–289.[Abstract/Free Full Text]

6. Godecke A, Heinicke T, Kamkin A, Kiseleva I, Strasser RH, Decking UKM, Stumpe T, Isenberg G, Schrader J. Inotropic response to ß-adrenergic receptor stimulation and anti-adrenergic effect of ACh in endothelial NO synthase-deficient mouse hearts. J Physiol. 2001; 532.1: 195–204.

7. Massion PB, Balligand J-L. Modulation of cardiac contraction, relaxation and rate by the endothelial nitric oxide synthase (eNOS): lessons from genetically modified mice. J Physiol. 2003; 546: 63–75.[Abstract/Free Full Text]

8. Wang YG, Rechenmacher CE, Lipsius SL. Nitric oxide signaling mediates stimulation of L-type Ca²⁺ current elicited by withdrawal of acetylcholine in cat atrial myocytes. J Gen Physiol. 1998; 111: 113–125.[Abstract/Free Full Text]

9. Wang YG, Lipsius SL. Acetylcholine elicits a rebound stimulation of Ca²⁺ current mediated by pertussis toxin-sensitive G protein and cAMP-dependent protein kinase A in atrial myocytes. Circ Res. 1995; 76: 634–644.[Abstract/Free Full Text]

10. Wang YG, Lipsius SL. A cellular mechanism contributing to post-vagal tachycardia studied in isolated pacemaker cells from cat right atrium. Circ Res. 1996; 79: 109–114.[Abstract/Free Full Text]

11. Wang YG, Hüser J, Blatter LA, Lipsius SL. Withdrawal of acetylcholine elicits Ca²⁺-induced delayed afterdepolarizations in cat atrial myocytes. Circulation. 1997; 96: 1275–1281.[Abstract/Free Full Text]

12. Endoh M, Blinks JR. Effects of endogenous neurotransmitters on calcium transients in mammalian atrial muscle. In: Fleming WW, Graefe K-H, Langer SZ, Weiner N, eds. Neuronal and Extraneuronal Events in Autonomic Pharmacology. New York, NY: Raven Press; 1984: 221–230.

13. Kirstein M, Rivet-Bastide M, Hatem S, Benardeau A, Mercadier J-J, Fischmeister R. Nitric oxide regulates the calcium current in isolated human atrial myocytes. J Clin Invest. 1995; 95: 794–802.[Medline] [Order article via Infotrieve]

14. Linden J. Enhanced cAMP accumulation after termination of cholinergic action in the heart. FASEB J. 1987; 1: 119–124.[Abstract]

15. Ehara T, Mitsuiye T. Transient increase in the slow inward current following acetylcholine removal in catecholamine-treated guinea-pig Purkinje fibers. Jpn J Physiol. 1984; 34: 775–779.[Medline] [Order article via Infotrieve]

16. Belevych AE, Sims C, Harvey RD. ACh-induced rebound stimulation of L-type Ca²⁺ current in guinea-pig ventricular myocytes, mediated by ß-dependent activation of adenylyl cyclase. J Physiol. 2001; 536: 677–692.[Abstract/Free Full Text]

17. Song Y, Shryock JC, Belardinelli L. Potentiating effect of acetylcholine on stimulation by isoproterenol of L-type Ca²⁺ current and arrhythmogenic triggered activity in guinea pig ventricular myocytes. J Cardiovasc Electrophysiol. 1998; 9: 718–726.[Medline] [Order article via Infotrieve]

18. Bett GCL, Dai S, Campbell DL. Cholinergic modulation of the basal L-type calcium current in ferret right ventricular myocytes. J Physiol. 2002; 542: 107–117.[Abstract/Free Full Text]

19. Wang YG, Dedkova EN, Steinberg SF, Blatter LA, Lipsius SL. ß₂-Adrenergic receptor signaling acts via NO release to mediate ACh-induced activation of ATP-sensitive K⁺ current in cat atrial myocytes. J Gen Physiol. 2002; 119: 69–82.[Abstract/Free Full Text]

20. Wu J, Vereecke J, Carmeliet E, Lipsius SL. Ionic currents activated during hyperpolarization of single right atrial myocytes from cat heart. Circ Res. 1991; 68: 1059–1069.[Abstract/Free Full Text]

21. Kojima H, Nakatsubo N, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Hirata Y, Nagano T. Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem. 1998; 70: 2446–2453.[Medline] [Order article via Infotrieve]

22. Nakatsubo N, Kojima H, Kikuchi K, Nagoshi H, Hirata Y, Maeda D, Imai Y, Irimura T, Nagano T. Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins. FEBS Lett. 1998; 427: 263–266.[CrossRef][Medline] [Order article via Infotrieve]

23. Dedkova EN, Wang YG, Blatter L, Lipsius SL. Nitric oxide signalling by selective ß₂-adrenoceptor stimulation prevents ACh-induced inhibition of ß₂-stimulated Ca²⁺ current in cat atrial myocytes. J Physiol. 2002; 542: 711–723.[Abstract/Free Full Text]

24. Rees DD, Palmer RMJ, Schulz R, Hodson H, Moncada S. Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br J Pharmacol. 1990; 101: 746–752.[Medline] [Order article via Infotrieve]

25. Feron O, Belhassen L, Kobzik L, Smith TW, Kelly RA, Michel T. Endothelial nitric oxide synthase targeting to caveolae. J Biol Chem. 1996; 271: 22810–22814.[Abstract/Free Full Text]

26. Smart EJ, Anderson RGW. Alterations in membrane cholesterol that affect structure and function of caveolae. Methods Enzymol. 2002; 353: 131–139.[Medline] [Order article via Infotrieve]

27. Hidaka H, Sasaki Y, Tanaka T, Endo T, Ohno S, Fujii Y, Nagata T. N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin antagonist, inhibits cell proliferation. Proc Natl Acad Sci U S A. 1981; 78: 4354–4357.[Abstract/Free Full Text]

28. Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem. 1994; 269: 5241–5248.[Abstract/Free Full Text]

29. Fulton D, Gratton J-P, McCabe TJ, Fontana J, Fuijo Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 1999; 399: 597–601.[CrossRef][Medline] [Order article via Infotrieve]

30. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999; 399: 601–605.[CrossRef][Medline] [Order article via Infotrieve]

31. Luckhoff A, Pohl U, Mulsch A, Busse R. Differential role of extra- and intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells. Br J Pharmacol. 1988; 95: 189–196.[Medline] [Order article via Infotrieve]

32. Busse R, Mulsch A. Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. FEBS Lett. 1990; 265: 133–136.[CrossRef][Medline] [Order article via Infotrieve]

33. Kaye DM, Wiviott SD, Balligand J-L, Simmons WW, Smith TW, Kelly RA. Frequency-dependent activation of a constitutive nitric oxide synthase and regulation of contractile function in adult rat ventricular myocytes. Circ Res. 1996; 78: 217–224.[Abstract/Free Full Text]

34. Kandel ES, Hay N. The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB. Exp Cell Res. 1999; 253: 210–229.[CrossRef][Medline] [Order article via Infotrieve]

35. Vila Petroff MG, Kim SH, Pepe S, Dessy C, Marbán E, Balligand J-L, Sollott SJ. Endogenous nitric oxide mechanisms mediate the stretch dependence of Ca²⁺ release in cardiomyocytes. Nat Cell Biol. 2001; 3: 867–873.[CrossRef][Medline] [Order article via Infotrieve]

36. Conus NM, Hemmings BA, Pearson RB. Differential regulation by calcium reveals distinct signaling requirements for the activation of Akt and p70S6k. J Biol Chem. 1998; 273: 4776–4782.[Abstract/Free Full Text]

37. Fleming I, Fisslthaler B, Dimmeler S, Kemp BE, Busse R. Phosphorylation of Thr⁴⁹⁵ regulates Ca²⁺/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res. 2001; 88: 68–75.[CrossRef]

38. Okamoto T, Schlegel A, Scherer PE, Lisanti MP. Caveolins, a family of scaffolding proteins for organizing "preassembled signaling complexes" at the plasma membrane. J Biol Chem. 1998; 273: 5419–5422.[Free Full Text]

39. Michel JB, Feron O, Sacks D, Michel T. Reciprocal regulation of endothelial nitric-oxide synthase by Ca²⁺-calmodulin and caveolin. J Biol Chem. 1997; 272: 15583–15586.[Abstract/Free Full Text]

40. Feron O, Smith TW, Michel T, Kelly R. Dynamic targeting of the agonist-stimulated m2 muscarinic acetylcholine receptor to caveolae in cardiac myocytes. J Biol Chem. 1997; 272: 17744–17748.[Abstract/Free Full Text]

41. Feron O, Saldana F, Michel JB, Michel T. The endothelial nitric-oxide synthase-caveolin regulatory cycle. J Biol Chem. 1998; 273: 3125–3128.[Abstract/Free Full Text]

42. Hüser J, Lipsius SL, Blatter LA. Calcium gradients during excitation-contraction coupling in cat atrial myocytes. J Physiol. 1996; 494: 641–651.[Abstract/Free Full Text]

43. Dittrich M, Jurevicius J, Georget M, Rochais F, Fleischmann BK, Hescheler J, Fischmeister R. Local response of L-type Ca²⁺ current to nitric oxide in frog ventricular myocytes. J Physiol. 2001; 534: 109–121.[Abstract/Free Full Text]

44. Verrier RL, Dickerson LW. Central nervous system and behavioral factors in vagal control of arrhythmogenesis. In: Levy MN, Schwartz PJ, eds. Vagal Control of the Heart: Experimental Basis and Clinical Implications. Armonk, NY: Futura Publishing Co; 1994: 557–577.

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