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Abstract
Fluorescence microscopy and the NO-sensitive indicator 4,5-diaminofluorescein were used to determine the effects of acetylcholine (ACh) on intracellular NO (NOi) 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 NOi. However, in field-stimulated cells, ACh exposure increased NOi, and ACh withdrawal elicited an additional, prominent increase in NOi production. During ACh exposure, addition of 1 μmol/L atropine increased NOi production similar to ACh withdrawal. ACh-induced increases in NOi 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 NOi. 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 NOi at local subsarcolemmal sites, and ACh withdrawal additionally increased NOi by recruiting additional subsarcolemmal release sites. Disruption of caveolae by 2 mmol/L methyl-β-cyclodextrin abolished ACh-induced NOi production. We conclude that in cat atrial myocytes, ACh stimulates NOi release from local subsarcolemmal sites. ACh-induced increases in NOi requires both muscarinic receptor–mediated Gi protein/phosphatidylinositol 3-kinase/Akt signaling and voltage-activated Ca2+ influx for stimulation of calmodulin-dependent endothelial NO synthase activity. Increases in NOi 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.
Several studies indicate that NO signaling mediates the inhibitory effects of acetylcholine (ACh) on cardiac function.1–3 These findings, however, have been disputed4–6 and remain controversial.7 In cat atrial myocytes, ACh-induced inhibition of basal L-type Ca2+ current (ICa,L) is not mediated by NO signaling.8 However, ACh withdrawal stimulates ICa,L above control levels, ie, rebound stimulation, and this response is mediated by NO signaling.8,9 The rebound stimulation of ICa,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 ICa,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 ICa,L in Purkinje fibers15 and ventricular myocytes prestimulated by β-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 signaling16,18 but rather is attributable to stimulation of adenylate cyclase by the βγ subunit of Gi 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 (NOi) production in cat atrial myocytes. We also sought to determine whether stimulation of muscarinic receptors increases NOi production via Gi proteins coupled to phosphatidylinositol 3-kinase (PI-3K)/protein kinase B (Akt) signaling, similar to NOi production elicited by β2-adrenergic receptor (AR) stimulation.19 The present results indicate that ACh exposure and withdrawal increase NOi production. ACh-induced increases in NOi require both muscarinic receptor–mediated Gi/PI-3K/Akt signaling and voltage-activated Ca2+ influx for stimulation of calmodulin (CaM)-dependent endothelial NO synthase (eNOS) activity. Moreover, ACh withdrawal increases NOi production above that induced during ACh exposure, consistent with the role of NO signaling in rebound stimulation of ICa,L.
Materials and Methods
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 NOi were obtained by incubating cells with the fluorescent NO-sensitive dye 4,5-diaminofluorescein (DAF-2),21,22 as previously described19,23 (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 NOi 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 NOi recorded by linescan were quantified by measuring the frequency and amplitude of NOi peaks. NOi peaks were defined as those changes in NOi 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-β-cyclodextrin, pertussis toxin (all from Sigma Chemical), and 4,5-diaminofluorescein diacetate (DAF-2 DA) (Calbiochem).
NOi 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
Figure 1A shows a typical recording of NOi 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 NOi, and withdrawal of ACh elicited an additional, prominent increase in NOi above that elicited during ACh exposure (n=16). In the quiescent cell (−FS), ACh exposure and withdrawal had no effect on NOi (n=6). However, in the same cell exposure to the NO donor, spermine/NO (Sper/NO; 100 μmol/L) prominently increased NOi (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 NOi production (data not shown; n=12). Previous work showed that termination of ACh action by atropine elicits rebound stimulation of ICa,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 NOi production similar to that elicited by ACh withdrawal (data not shown; n=3). The effects of ACh exposure and withdrawal on NOi production are summarized in Figure 1D and indicate that ACh exposure elicits a relatively modest increase in NOi production and that ACh withdrawal elicits additional, prominent increases in NOi above those elicited during ACh exposure. Moreover, the receptor-mediated effects of ACh to increase NOi 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 NOi (n=10). In the same cell, 100 μmol/L Sper/NO prominently increased NOi. In cardiac cells, eNOS is localized to caveolae.25 Methyl-β-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 NOi induced by ACh exposure and withdrawal (n=3). Together, these findings indicate that ACh increases NOi by stimulating eNOS activity localized to caveolae.
Figure 1. Effects of field stimulation, L-NIO, and methyl-β-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-β-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-β-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 NOi 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 NOi 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 NOi production elicited during ACh exposure and withdrawal (n=10), and 0.5 mmol/L [Ca2+]o essentially abolished the increase in NOi 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 NOi induced by ACh exposure and withdrawal (n=5). In the same cell, 100 μmol/L Sper/NO prominently increased NOi. To determine whether Ca2+ influx specifically contributes to the additional increase in NOi 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 NOi. However, ACh withdrawal in low [Ca2+]o failed to significantly increase NOi above that elicited by ACh exposure (n=4). In the four cells tested, NOi 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, NOi 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 NOi depends on Ca2+ influx, presumably to activate CaM-dependent eNOS.
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 ICa,L is mediated via muscarinic receptors coupled to Gi protein signaling.9 Moreover, β2-AR stimulation acts via Gi protein and PI-3K signaling to increase NOi.19 We therefore sought to determine whether muscarinic receptors act via a similar Gi protein/PI-3K signaling pathway to increase NOi. As shown in Figure 3A, compared with control responses, pretreatment with the muscarinic receptor antagonist (1 μmol/L) atropine blocked NOi 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 Gi protein signaling also abolished increases in NOi induced by ACh exposure and withdrawal (n=6). In the same PTX-treated cell (Figure 3B), 100 μmol/L Sper/NO prominently increased NOi. 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 β2-AR stimulation of NOi release.19 As shown in Figure 3C, inhibition of PI-3K signaling by LY294002 also inhibited NOi 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 NOi. The results are summarized in the graph in Figure 3D and indicate that ACh acts on muscarinic receptors coupled via Gi proteins and PI-3K signaling to stimulate NOi production. Apparently, muscarinic and β2-ARs act via the same Gi protein/PI-3K signaling pathway to stimulate NOi production.
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 NOi 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 NOi 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 NOi 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 NOi production, presumably to activate CaM-dependent eNOS, ACh-induced activation of Akt signaling is Ca2+-independent.
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 NOi 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 NOi at local sites along the cell periphery. ACh withdrawal elicited additional increases in NOi, 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-β-cyclodextrin, ACh failed to elicit any changes in NOi either during ACh exposure or withdrawal. Similar results were obtained in a total of four cells.
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-β-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 NOi production were examined in more detail by recording NOi 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 NOi average over time and expressed as F/F0 during ACh exposure (red trace) and ACh withdrawal (blue trace). ACh exposure increased NOi at local subsarcolemmal sites and elicited small increases in baseline NOi. ACh withdrawal elicited additional increases in NOi by enhancing NOi 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 NOi levels were increased additionally. Incubation of cells in 2 mmol/L cyclodextrin abolished all effects of ACh to increase NOi (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 NOi release events elicited by ACh exposure (red bars) and ACh withdrawal (blue bars) (n=5). ACh exposure increased both the amplitude and frequency of NOi release events, and these parameters were significantly greater during ACh withdrawal compared with ACh exposure.
Discussion
Previous studies from our laboratory have shown that in cat atrial myocytes, ACh exposure inhibits ICa,L and ACh withdrawal stimulates ICa,L above control levels, ie, rebound stimulation.9 Pharmacological analyses indicated that although NO signaling does not participate in ACh-induced inhibition of ICa,L, the rebound stimulation of ICa,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 NOi production and ACh withdrawal elicits an additional, prominent increase in NOi above that achieved during ACh exposure. The actions of ACh require both stimulation of muscarinic receptor–mediated Gi protein/PI-3K/Akt signaling and voltage-activated Ca2+ influx to elicit local, subsarcolemmal increases in NOi production.
The present results indicate that muscarinic receptor stimulation by ACh is unable to stimulate NOi production in quiescent atrial myocytes. β2-AR stimulation also requires electrical stimulation of atrial myocytes to increase NOi 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 NOi. Indeed, in electrically stimulated atrial myocytes, ACh-induced increases in NOi 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 NOi. 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 NOi production, it is not sufficient per se to elicit NOi 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 NOi production (unpublished observations). In electrically stimulated atrial myocytes, even marked increases in Ca2+ influx via ICa,L and presumably intracellular Ca2+ release induced by β1-AR stimulation fail to increase NOi.19 On the other hand, β2-AR stimulation elicits a similar increase in ICa,L and does increase NOi.19 Together, these findings indicate that, in atrial myocytes, voltage-activated Ca2+ influx and intracellular Ca2+ release per se are not sufficient to stimulate NOi production. However, Ca2+ influx is essential for NOi production stimulated by specific receptor-mediated signaling. The contribution of intracellular Ca2+ release to muscarinic receptor–mediated NOi production remains to be determined.
In the present study, ACh acts via muscarinic receptors coupled to Gi proteins and PI-3K/Akt signaling to activate CaM-dependent NOi production. Our previous studies have shown that in cat atrial myocytes, stimulation of ICa,L elicited by ACh withdrawal also is mediated via muscarinic receptors coupled to Gi 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 endothelial29,30 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 NOi 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 NOi 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 NOi 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 NOi production. Our previous experiments indicate that in cat atrial myocytes, β2-AR stimulation also requires both PI-3K signaling and voltage-activated Ca2+ influx to elicit NOi production.19 This dual signaling mechanism can account for the relatively small NOi production elicited during ACh exposure and the more prominent increase in NOi elicited by ACh withdrawal. Thus, ACh exposure stimulates PI-3K/Akt signaling at the same time that it decreases Ca2+ influx via ICa,L, thereby allowing only a modest increase in NOi 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 NOi production. Moreover, because ACh withdrawal results in the recovery of adenylate cyclase/cAMP signaling, increases in NOi stimulate cAMP-mediated increases in Ca2+ influx via ICa,L,8 which in turn contribute to additional Ca2+-dependent increases in NOi. Our interpretation that NOi production is modulated by Ca2+ influx is supported by experiments in which we recorded intracellular [Ca2+] and NOi 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 NOi 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 NOi production, NO signaling does not contribute to ACh-induced inhibition of ICa,L.8 In fact, in cat atrial myocytes, NO signaling stimulates ICa,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 ICa,L. In addition, the level of NOi 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 NOi primarily at the cell periphery and disruption of caveolae formation by cyclodextrin abolishes ACh-induced increases in NOi. 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 NOi release sites suggests that different release sites exhibit different thresholds for stimulation of NOi production. The preferential release of NOi from subsarcolemmal sites also is consistent with the local regulation of sarcolemmal channel function exerted by NOi signaling.19,43
Clearly, the prominent increase in NOi elicited by ACh withdrawal strongly supports our previous findings that rebound stimulation of ICa,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 NOi 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
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Original received May 30, 2003; revision received October 30, 2003; accepted October 30, 2003.
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