Nitric Oxide Modulates Cardiac Na+ Channel via Protein Kinase A and Protein Kinase G
We directly tested the effects of nitric oxide (NO) on Na+ channels in guinea pig and mouse ventricular myocytes using patch-clamp recordings. We have previously shown that NO donors have no observed effects on expressed Na+ channels. In contrast, NO (half-blocking concentration of 523 nmol/L) significantly reduces peak whole-cell Na+ current (INa) in isolated ventricular myocytes. The inhibitory effect of NO on INa was not associated with changes in activation, inactivation, or reactivation kinetics. At the single-channel level, the reduction in macroscopic current was mediated by a decrease in open probability and/or a decrease in the number of functional channels with no change in single-channel conductance. Application of cell permeable analogs of cGMP or cAMP mimics the inhibitory effects of NO. Furthermore, the effects of NO on INa can only be blocked by inhibition of both cGMP and cAMP pathways. Sulfhydryl-reducing agent does not reverse the effect of NO. In summary, although NO exerts its action via the known guanylyl cyclase (GC)/cGMP pathway, our findings provide evidence that NO can mediate its function via a GC/cGMP-independent mechanism involving the activation of adynylyl cyclase (AC) and cAMP-dependent protein kinase.
Nitric oxide (NO) is a uniquely diffusible and reactive molecular messenger, which is found in abundance and plays important regulatory roles in different systems throughout the body.1 In the cardiovascular system, NO is the major endothelium-derived relaxing factor (EDRF) and causes vasodilation and reduces blood pressure.2 In addition, NO functions as an important endogenous inhibitor of vascular lesion formation.3 The coronary endothelium is responsible for the bulk of the endogenous, physiological production of NO.4 However, NO can also be produced within the cardiac myocytes themselves by the constitutive NO synthase.5 NO modulates cardiac contractility both in vitro and in vivo.6 More recently, it was shown that NO can regulate both adenylyl cyclase (AC) and guanylyl cyclase (GC) in cardiac myocytes. High levels of NO induce large increases in cGMP and a negative inotropic effect, whereas low levels of NO increase cAMP and induce a positive contractile response.7
We have previously shown using heterologous expression systems that although Ca2+ channel can be directly modulated by NO, Na+ channels are unaffected by direct NO modulation.8 Here, we show that NO modulates Na+ channels in native cardiac myocytes. In addition, we provide evidence to demonstrate that NO modulates Na+ channels via second messenger pathways through activation of protein kinase G (PKG) and protein kinase A (PKA).
Materials and Methods
Single left ventricular myocytes were isolated from guinea pigs and mice (Charles River Laboratories, Wilmington, Mass) as previously described.9 See further details in the expanded Materials and Methods section that can be found in the online data supplement available at http://www.circresaha.org.
Whole-cell and cell-attached single-channel INa were recorded at room temperature using the patch-clamp techniques.10 Because of the variability in the current density among cells, we compared the control and treatment effects only within the same cells. Single-channel currents were recorded and analyzed as previously described.11,12
Saturated NO solution was prepared and diluted before used. NO concentration in the perfusing chamber was measured as described in the online expanded Materials and Methods section.
NO Inhibits INa in Guinea Pig and Mouse Ventricular Myocytes
Figure 1A shows whole-cell INa traces elicited from a holding potential of −100 mV using various depolarization steps in control, after application of NO (1:10 dilution from saturated NO stock) and after 1,4-dithiothreitol (DTT, 2 to 5 mmol/L), a sulfhydryl-reducing agent. Application of NO resulted in the inhibition of the currents. DTT was used to examine whether the inhibitory effects of NO were due to a direct modification of the channels. The time course of the current inhibition by NO is shown in Figure 1B. This effect was not reversible by DTT. Figure 1C summarizes the current-voltage relations from isolated guinea pig ventricular myocytes. The current density elicited at a test potential of −35 mV from a holding potential of −100 mV was decreased by 28±3% after application of NO (n=9, P=0.0001). Similar data were obtained in mouse ventricular myocytes (Figure 1G). The current density was decreased by 29±6% (P=0.015). Two different species were used in order to further assess that the inhibitory effects of NO on INa is not specific to certain species. The effects of NO and NO donor, S-nitroso-N-acetyl-d, l-penicillamine (SNAP), were tested in mouse ventricular myocytes.
Because the inhibitory effects of NO were only partially reversible after >20-minute of washout using external solution (data not shown), we directly tested for the possibility of “run down” of currents in our recording condition. No evidence of run down of the currents was observed during this recording time period (Figures 1D and 1E, n=3). The leftward shift in the steady-state inactivation curve of INa (h∞ curve) is well described and is more pronounced at the initial phase of establishment of gigaohm seal.13 Therefore, all our recordings were done 10 minutes after the whole-cell configuration was attained. In addition, in order to rule out the possibility that the inhibitory effects seen with NO resulted from the hyperpolarization shift in the steady-state channel availability, we reevaluated the effects of NO at a holding potential of −110 mV (Figure 1F). Similar percentages of block were observed at both holding potentials.
The effects of NO on INa were concentration-dependent, further suggesting the specificity of the inhibition. Figure 2A summarizes the dose-response relation illustrating the percentage of current inhibition against different NO concentrations. The solid line represents fit of the data to the logistic function yielding a half-blocking NO concentration of 523 nmol/L.
Figures 2B, 2C, and 2D examined 2 different classes of NO donors, SNAP and (Z)-1-(N-methyl-N-[6-(N-methyl-ammoniohexyl)amino])-diazen-1-ium-1,2-diolate] (MAHMA NONOate). Similar to the NO solution, both classes of NO donors led to inhibition of INa.
NO Did Not Alter the Kinetics of the Whole-Cell INa
The inhibitory effects of NO on INa were not associated with changes in steady-state activation or inactivation kinetics (online Figure 1). Similarly, there were no changes in the time-dependent inactivation or reactivation kinetics of INa after NO applications (online Figure 1).
Because of the known effects of F− on the activities of GS,14,15 we crosschecked the effects of NO on INa by replacing the F− in the internal solution by Cl−. The effects of SNAP were consistent with previous data when F− was used in the internal solution (online Figure 2).
Effects of NO on Single-Channel INa
The inhibitory changes observed at the macroscopic current level can result from a change in either permeation, gating, or the number of functional channels. To directly address this question, single-channel INa were recorded using cell-attached configuration. Application of NO resulted in a significant decrease in the open probabilities and/or channel number with no significant changes in single-channel conductance (online Figure 3). Putting together the macroscopic current findings, our data suggest that the inhibitory effects of NO are likely attributable to a change in the number of functional channels rather than changes in permeation or gating.
Inhibitory Effects of NO on INa Are Mediated in Part by cGMP Pathway
In order to test the involvement of second messenger pathways, we used inhibitors and agonists from both cGMP/PKG and cAMP/PKA pathways in different combinations (Figure 7D). Data will be presented in the next several sections.
First, we examined the effects of cell permeable analog of cGMP (8-bromo-cGMP, 100 μmol/L) on INa (Figure 3A). Similar to NO, application of 8-bromo-cGMP resulted in an inhibitory effect on INa; however, addition of NO to the perfusate containing 8-bromo-cGMP led to further reduction in INa. These inhibitory effects could not be reversed by DTT. Figure 3B shows the current density-voltage relationship obtained from a total of 7 cells. The magnitude of the current density obtained at a test potential of −35 mV is summarized in Figure 3C. Current density was significantly decreased by application of 8-bromo-cGMP (P=0.018) and 8-bromo-cGMP plus NO (P=0.01 compared with 8-bromo-cGMP alone). These data represent an 18±3% block of the total current density by cGMP alone, contrasting with an inhibition of ≈30% by NO (Figure 1). After application of cGMP plus NO, a current inhibition of 33±4% was produced. Taken together, these data suggest that the inhibitory effect of NO occurs at least, in part, via cGMP.
In order to rule out the possible time-dependent effects of cGMP, 2 groups of cells were exposed to 8-bromo-cGMP for 10 and 20 minutes (n=3 for each group). There were no significant differences in the degree of inhibition by 8-bromo-cGMP during a 10- versus a 20-minute exposure (Figure 3D). Finally, we further tested the effects of 8-bromo-cGMP on INa using a more hyperpolarized potential (−110 mV; Figure 3E). Similar percentages of block were observed at the 2 different holding potentials.
The effect of a specific inhibitor of phosphodiesterase (PDE) type 5, zaprinast, on INa was examined. PDE type 5 acts as a specific inhibitor of cGMP; therefore, the application of zaprinast would be expected to raise intracellular cGMP. Figure 3F shows the summary data obtained from application of zaprinast (20 μmol/L) and NO in the presence of zaprinast showing a 23±4% and 30±7% inhibition by zaprinast and zaprinast plus NO, respectively. The time course of the effects of zaprinast on the whole-cell INa is shown in Figure 3G. Similar to the direct application of cGMP, application of zaprinast led to an inhibitory effect on INa. However, further application of NO in the continued presence of zaprinast led to an additional block of INa, suggesting that another messenger pathway may be involved. The possible time-dependent effects of zaprinast were further tested by exposing 2 groups of cells to zaprinast for 10 and 20 minutes (n=3 for each group). There were no significant differences in the degree of inhibition by zaprinast during a 10- versus a 20-minute exposure (24.9±0.7% versus 23.8±1.4%, respectively).
Selective Inhibitor of NO-Sensitive Guanylyl Cyclase Only Partially Blocks the Inhibitory Effects of NO on INa
Figures 4A and 4B show the effects of ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one), an NO-sensitive guanylyl cyclase inhibitor, on INa. Application of ODQ (10 μmol/L) had no effects on INa; however, further application of NO in the presence of ODQ resulted in inhibitory effects on INa. This inhibitory effect represented a small percentage of block of the total current compared with NO alone. A decrease of INa by NO in the presence of ODQ was only 11±2% compared with ≈30% in the presence of NO alone, suggesting that the inhibitory effect of NO could be partially blocked by ODQ (Figure 4B). This data further suggest that the inhibitory effect of NO on INa may involve 2 separate second messenger pathways.
Involvement of cGMP-Dependent Protein Kinase
To test whether the signal transduction involves activation of cGMP-dependent protein kinase (PKG), the effects of an inhibitor of PKG (Rp-cGMP) was studied. Similar to ODQ, application of Rp-cGMP, a specific blocker of PKG, had no observable effects on INa. Further application of NO in the presence of Rp-cGMP resulted in reduction of the current but to a lesser degree than NO alone (Figure 4C). Figure 4D summarizes the data obtained from a total of 4 cells. In the presence of Rp-cGMP, the effect of NO on INa was much more moderate, representing only a 13±1% inhibition at a test potential of −35 mV compared with ≈30% by NO alone (Figure 1). In fact, the degree of block by NO in the presence of Rp-cGMP was similar to that of NO in the presence of ODQ (Figure 4B). The data are consistent with the idea that the action of NO on INa involves cGMP-dependent protein kinase and a separate second messenger pathway.
Application of Cell Permeable Analog of cAMP Leads to Similar Inhibitory Effects on INa
Next, we sought to investigate the alternative second messenger pathway involved in the action of NO on INa. Cell permeable analog of cAMP (dibutyryl cAMP, 100 μmol/L) was used. Dibutyryl cAMP led to an inhibition of INa; however, further application of NO in the presence of cAMP resulted in additional block of the current (Figures 5A and 5B). The data represent percentages inhibition by cAMP and cAMP plus NO of 20±2% and 28±3%, respectively. Previous data have suggested that cAMP/PKA activation may result in a shift in the steady-state inactivation curve of INa to more negative potentials;16 therefore, we tested the effects of cAMP at holding potentials of −100 and −140 mV. Application of cAMP resulted in similar inhibitory effects of the current at both holding potentials (Figure 5C).
Effect of Inhibition of PDE3, a cGMP-Inhibited cAMP-Specific PDE
We further investigated the possible crosstalk between cGMP and cAMP pathways. It has previously been shown that apart from interacting with the cGMP-specific PDE5, cGMP can also inhibit PDE3, thereby increasing intracellular cAMP levels. Therefore, we examined the effects of trequinsin, which is an inhibitor of PDE3, a cGMP-inhibited cAMP-specific PDE (Figure 5D). Application of trequinsin (2 μmol/L) resulted in inhibition of INa. After superfusion with trequinsin, further application of cGMP plus trequinsin resulted in additional block on INa. The degree of block by trequinsin and trequinsin plus cGMP was 13±4% and 29±7%, respectively. Taking together the results presented here and in Figures 4C and 4D, we conclude that cGMP exerts its effects on INa, at least partially, via cGMP-dependent protein kinase (see diagram in Figure 7D).
Presence of PKA Inhibitors Partially Blocks the Inhibitory Effects of NO on INa
To investigate the involvement of PKA in mediating the inhibitory effect of NO on INa, we conducted experiments using PKI (protein kinase A inhibitor 5-24 amide) in the pipette solution. Application of zaprinast resulted in INa inhibition (Figures 6A and 6B); however, in contrast to the result in Figures 3F and 3G, further application of NO in the presence of zaprinast did not lead to additional decrease in the current when PKI was included in the pipette solution. Figure 6B summarizes the current density obtained with PKI in the pipette solution, elicited at a test potential of −35 mV from a holding potential of −100 mV.
The involvement of the PKA was further tested using Rp-cAMP, a competitive inhibitor of PKA. Rp-cAMP (30 μmol/L) had no observable effects on INa. Further application of NO in the continued presence of Rp-cAMP resulted in an inhibitory effect on INa, however, to a much lesser degree compared with NO alone (Figure 6C and 6D), representing a ≈13% inhibition in the total current compared with ≈30% inhibition by NO alone (Figure 1C and 1G).
Inhibitory Effects of NO on INa Can Only Be Blocked by Inhibition of Both cGMP and cAMP Pathways
Figure 7 illustrates the combined effects of ODQ and PKI on the inhibitory action of NO. Experiments were performed using 10 nmol/L of PKI in the pipette solution. In the presence of PKI and ODQ (10 μmol/L), NO failed to induce inhibitory effects on INa. The corresponding current traces at each time point (a through f) are shown in Figure 7B. The current-voltage relations obtained from a group of 7 cells are shown in Figure 7C. The results provide direct evidence for the involvement of PKA and PKG in NO-mediated reduction of INa in ventricular myocytes (see summary diagram in Figure 7D).
In this study, we investigated the effects of NO on INa in isolated guinea pig and mouse ventricular myocytes. We demonstrated that NO modification of Na+ channels resulted in a reduction in whole-cell INa with no changes in the steady-state or time-dependent kinetics. At the single-channel level, the reduction in macroscopic current was mediated by a decrease in open probability and/or a decrease in the number of functional channels with no change in single-channel conductance. Taken together the macroscopic current findings, our data suggest that the inhibitory effects of NO most likely result from a change in the channel number rather than changes in permeation or gating. Finally, the inhibitory effects of NO on INa involve the activation of both cGMP- and cAMP-dependent protein kinases.
NO Inhibits INa via Indirect Pathway
The free radical NO can exert many of its effects through an indirect pathway involving activation of GC and increased levels of cGMP. In addition, the free radical NO can result in an array of interrelated redox forms with distinct properties and reactivities. These molecules encompass the actions of several naturally occurring nitrogen (N)-oxides, which display reactivity profiles that are different from NO itself.1 Regulation by redox state and NO/RSNOs has been described for numerous intracellular and extracellular proteins including ion channels.17,18 Direct effects of N-oxides appear to derive from reactions of vicinal thiols that serve as allosteric regulators of channel function.18
Our present study indicates that NO modulates INavia second messenger pathways. The inhibitory effects of NO on INa cannot be reversed by sulfhydryl-reducing agents as would be expected for a direct modulation. In contrast, it was previously shown in the native cardiac myocytes that the cardiac L-type Ca2+ channels can be modulated by both indirect (cGMP-dependent) and direct (S-nitrosylation/oxidation) pathways.19 Our previous report8 showed that NO did not exert any effects on heterologously expressed INa in Xenopus oocytes or mammalian cell line.8 However, only one NO donor (SNAP) was used on the expressed Na+ channel in our study.
Effects of NO on Other Ion Channels
NO modulates a wide variety of ion channels in different systems as diverse as neurons, vascular smooth muscles, carotid bodies, pancreatic cells, and hair cells in the inner ears.20,21 Indeed, the modulation of ion channels represents one of the important functional effects of NO. In the cardiac systems, NO can inactivate the cardiac ryanodine receptor Ca2+ release channel22 and plays an important role in cardiac pacemaking cells by mediating a muscarinic cholinergic attenuation of the L-type Ca2+ current in mammalian sinoatrial and atrioventricular node.23
Involvement of cGMP and cAMP
Previous studies have demonstrated that exogenously applied NO at high concentrations can produce a negative inotropic effect on cardiac contraction that is mediated by a cGMP-dependent PKG activation. In contrast, low concentrations of NO evoked a positive inotropic effect by a novel mechanism via a cGMP-independent activation of AC.7 An elevation of the intracellular levels of cAMP (and PKA activation) could occur because of a cGMP-dependent inhibition of the PDE 3.24 Low concentrations of cGMP (0.1 to 10 μmol/L) were found to have a stimulatory effect on L-type Ca2+ current likely due to the inhibition of cAMP degradation, mediated by the inactivation of the PDE 3 by low level of cGMP.25 An alternative mechanism involves the activation of AC by NO either directly or via G protein. Our results support a direct involvement of AC; even when basal cGMP increase was completely abolished by the presence of the selective inhibitor of soluble GC (ODQ), NO was still able to induce an inhibitory effect on INa (Figures 3A and 3B). In these settings, the additional block induced by NO must occur by a mechanism other than cGMP-mediated PDE inhibition, possibly by a cGMP-independent activation of AC. Indeed, our data suggest the direct involvement of both GC and AC as well as PKA and PKG (see Figure 7D).
Previous studies have provided evidence suggesting that NO can directly or indirectly activate AC in a cGMP-independent manner.26 The exact mechanism of NO activation of AC is uncertain; however, recent reports have demonstrated that NO can modulate G protein function.27 In addition, in endothelial cells, NO has been shown to selectively inhibit G proteins of the Gi and Gq family but not those of the Gs family, and that this modulation of G proteins could have a permissive action on the Gs-AC pathway.28 Therefore, it is possible that NO can activate AC via the potential modulation of G protein.
Effects of cAMP Modulation on INa
PKA and β-adrenergic receptor stimulation have been reported to cause diverse effects on cardiac Na+ channel function. Studies using rat ventricular myocytes have demonstrated an increase in INa with PKA stimulation.29,30 Using heterologous expression of cardiac Na+ channels in Xenopus oocytes and mammalian cell line, activation of PKA has been reported to increase INa derived from both the rat (rH1) and human (hH1) channels.31–33 In contrast, other studies have shown an inhibition of cardiac INa via PKA activation in guinea pig ventricular myocytes16,34 and neonatal rat cardiac myocytes.35 The effects of PKA activation may be species-specific. Consistent with our results in the present studies, PKA activation has been documented to inhibit the cardiac INa in guinea pig ventricular myocytes.16,34 In addition, the differences observed could result from data obtained in the native versus expression system.31–33 Previous evidence has suggested that β-adrenergic receptor–activated modulation of cardiac INa can occur via both a fast direct G-protein activation as well as by the slower indirect phosphorylation pathway.29,36 Activation of PKA results in phosphorylation of multiple sites (within the I-II linker) on cardiac Na+ channel that may cause different functional effects.37 Finally, PKA activation has been shown in some studies to result in a shift in the steady-state inactivation curve to more negative potentials.16 Therefore, variable results could result from differences in the holding potentials during the recordings. Nonetheless, we have performed our experiments using both holding potentials of −100 and −140 mV and did not find significant differences in the results.
In summary, we provide evidence to demonstrate that NO modulates cardiac Na+ channels via second messenger pathways through activation of protein kinase G and protein kinase A. This modulation occurs at a relatively high concentrations of NO. High concentration of NO has been well documented during pathological state, eg, sepsis.38 Therefore, it is reasonable to speculate that the modulation of NO on Na+ channels may play a significant functional role during these pathological states.
This study was supported by the AHA Scientist Development Grant (to N.C.) and the Veterans Administration Merit Review Grant (to N.C.).
Original received February 22, 2001; revision received October 15, 2001; accepted October 15, 2001.
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