Modulation of Cardiac Sodium Current by α¹-stimulation and Volatile Anesthetics 

Unless stated otherwise, the experiments in this study were conducted under conditions described in our preceding article. [13] Briefly, single cardiac myocytes were obtained by retrograde perfusion of guinea pig hearts with an enzyme. Na sup + current was measured using the whole-cell configuration of the patch-clamp methodology. In most cases, linear leak current was digitally subtracted using the P/N method. [14] To exclude possible beta-adrenergic activation, 100 nM propranolol was added to the external solution. Stock solutions of 10 mM methoxamine (Sigma Chemical Co., St. Louis, MO), 1 mM propranolol (Sigma), and 0.1 mM prazosin-hydrochloride (Pfizer, New York, NY) were freshly prepared each day and diluted in the external bath solution.

Statistical analyses were computed using paired t tests when comparing two sample means when a single cell served as its own control. A one-way analysis of variance (ANOVA) was used when different groups were compared. Differences between group means were evaluated with the Bonferroni test. In experiments in which shifts in steady-state inactivation and activation were compared, the predicted background shift was first subtracted, and a paired t test was performed as previously reported. [15] A test was considered to be significant when P < 0.05. Data are presented as mean +/- SEM.

To determine whether alpha¹-adrenergic receptor stimulation modulates cardiac I^Na, guinea pig ventricular myocytes were exposed to various concentrations of methoxamine, a specific alpha sub 1 -adrenergic receptor agonist. Propranolol (100 nM) was present throughout all phases of the experiment to block possible beta-adrenergic influences. [16] Peak Na sup + currents were elicited by voltage steps to -20 mV from (1) a hyperpolarized V^Hof -110 mV, where all Na sup + channels are available for activation, and (2) a more depolarized V^Hof -80 mV, which is within the physiologic resting potential where a fraction of channels are in the inactivated state. As shown in *figure 1*(A), 1 mM methoxamine reversibly inhibits I^Nawhen using a V^Hof -110 mV. Figure 1(B) shows the dose-dependent block of peak I^Naby methoxamine from two different V^HS. The plot shows a dependence on concentration and voltage with a K^dof 0.086 +/- 0.031 mM when using a V^Hof -80 mV and a K^dof 1.77 +/- 0.87 mM when using a V^Hof -110 mV. The Hill coefficients were significantly different with 0.72 and 1.02 mM, respectively.

To determine whether the inhibitory effect of methoxamine on I sub Na was the result of an activation of alpha¹-adrenergic receptors or of a direct effect on the channel protein, the effect in 1 micro Meter prazosin, an antagonist of the alpha¹-adrenergic receptor, was examined. In the absence of prazosin, from a V^Hof -80 mV, 10 and 100 micro Meter methoxamine reduced peak I^Naby 17.03 +/- 1.33 (n = 16) and 50.03 +/- 7.1%(n = 6), respectively. However, in the presence of 1 micro Meter prazosin, there was no significant effect of 10 micro Meter methoxamine on I^Na(Figure 2(A)). The effect of 100 micro Meter methoxamine in the presence of prazosin was significant, inhibiting I^Naby 9.24 +/- 0.88%, but less than in the absence of prazosin (Figure 2(B and C)).

As shown by a recording from a representative cell in Figure 3(A and B), I^Nawas initially depressed by 1.0 mM isoflurane and further inhibited by methoxamine. From a V^Hof -110 mV, equianesthetic concentrations of halothane (1.2 mM) and isoflurane (1.0 mM) suppressed the peak I^Naby 18.2 +/- 2.0%(n = 7) and 9.8 +/- 2.1%(n = 6), respectively. These values are significantly different and in the range previously observed. [15]Figure 4shows the effects on the peak inward Na sup + current by halothane or isoflurane and methoxamine monitored over time using a V^Hof -110 mV. In the continued presence of anesthetics, methoxamine further decreased peak I^Na. The effect of anesthetic plus alpha¹-adrenergic stimulation on peak I^Nawas determined after the maximal anesthetic effects were observed. To compare the additional reduction of I^Naby methoxamine, data were analyzed from the steady-state obtained during anesthetic exposure. The current obtained after the maximal effect of anesthetic served as new “control” as shown by the dotted lines in Figure 4(A and B).

The effects of methoxamine alone and combined with anesthetics on Na sup + current are summarized in Figure 5. When using a V^Hof -110 mV, when all Na sup + channels are in resting state, methoxamine alone (100 micro Meter) reduced the peak I^Naby 12.8 +/- 2.1%. Methoxamine in combination with halothane or isoflurane further decreased I^Naby 17.5 +/- 3.0% and 11.8 +/- 2.4%, respectively (Figure 5(A)). Hence, the effects of methoxamine in the absence or presence of halothane and isoflurane, respectively, were not significantly different. These results indicate that the combined effects of anesthetics with methoxamine, when using a V^Hof -110 mV, are additive.

To test whether there is a voltage-dependent interaction between methoxamine and anesthetics, further experiments were conducted from a V^Hof -80 mV, which is near the physiologic resting potential in cardiac cells. From this V^H, halothane and isoflurane depressed I sub Na by 33.5 +/- 2.9%(n = 6) and 19.7 +/- 2.1%(n = 7), respectively. Methoxamine alone at 10 micro Meter blocked I^Naby 17.0 +/- 1.3%(Figure 5(B)). However, methoxamine combined with halothane or isoflurane was more effective in depressing I^Na, further reducing the current amplitude by 39.1 +/- 3.1% and 36.5 +/- 2.3%, respectively (Figure 5(B)). The results suggest some form of synergistic interaction between anesthetics (halothane and isoflurane) and methoxamine when using a V^Hof -80 mV.

Changes in inactivation or activation properties of the cardiac Na sup + channel can alter the cardiac action potential characteristics and, therefore, set the stage for development of arrhythmias. The previously described results show that the effects of anesthetics and methoxamine on I^Nadepend on the holding potential. Thus, we investigated the influence of anesthetics and methoxamine on the steady-state inactivation and activation characteristics of the Na sup + channel. Shifts for steady-state inactivation and activation are corrected for the spontaneous background shifts. [15] As shown in Figure 6, halothane produced shifts in the inactivation and activation curves in the hyperpolarizing direction. An additional hyperpolarizing shift for steady-state inactivation was observed after application of methoxamine (10 micro Meter) in the continued presence of halothane (1.2 mM). In contrast, the steady-state activation curve remained unchanged (Figure 6).

A summary of the shifts in the inactivation and activation parameters, V¹/2 and k are shown in Table 1. The values for the shifts in V¹/2 shown in the table are corrected for the spontaneous background shifts. After correction for the predicted background shifts, methoxamine, at 10 and 100 micro Meter, produced a significant hyperpolarizing shift in steady-state inactivation (Table 1(A)). However, no statistically significant differences were found within the two groups of concentrations of methoxamine. In contrast, methoxamine did not induce shifts in steady-state activation. These results reveal that the concentration-dependent effect of methoxamine on peak I^Namay be related to mechanisms other than shifts in activation or inactivation parameters. The slope factor, k, for either the steady-state inactivation or activation was not significantly affected by methoxamine.

Halothane (1.2 mM) and isoflurane (1.0 mM), produced significant hyperpolarizing shifts in V¹/2 for inactivation and activation without affecting the slope factors (Table 1(B)). At approximately equianesthetic concentrations, no significant differences were found within the different groups of anesthetics for shifts in inactivation and activation. Methoxamine combined with halothane and isoflurane produced a significant shift in V¹/2 in steady-state inactivation and activation compared with control, drug-free conditions (Table 1). However, when accounting for anesthetic-induced shifts, the methoxamine effects in the presence of anesthetics are similar to the effects of methoxamine in the absence of anesthetics (Table 1). After subtracting the anesthetic effects, methoxamine combined with halothane or isoflurane shifted the V¹/2 of steady-state inactivation by -1.5 +/- 0.4 and -1.4 +/- 0.4 mV, respectively, which is similar to the shift produced by methoxamine alone (-1.6 +/- 0.4 mV). No significant shifts in V¹/2 of steady-state activation were observed. These results suggest that the enhanced effect of methoxamine in the presence of anesthetics from a V^Hof -80 mV is not caused by the shifts in steady-state activation or inactivation of the Na sup + channel.

The effects of methoxamine and volatile anesthetics on the recovery from inactivation using a V^Hof -80 mV were monitored. Channel recovery in the absence and presence of drugs was well fit by a single exponential function. In the presence of methoxamine (10 micro Meter) and in the absence of anesthetics, the rate of recovery from inactivation was not significantly affected as shown in *figure 7*(A). The mean recovery time constant was 58.3 +/- 2.8 ms for control and 64.8 +/- 4.0 ms for methoxamine (n = 6). As demonstrated in representative cells in Figure 7(B and C), the rate of recovery was significantly slower after application of halothane (86.1 +/- 4.5 ms) versus control (63.3 +/- 2.0 ms) and isoflurane (76.9 +/- 6.2 ms) versus control (58.2 +/- 2.5 ms); n = 6 cells in each group. The rate of recovery for methoxamine combined with halothane was 105.0 +/- 3.1 ms and combined with isoflurane 106.0 +/- 8.9 ms. Hence, only in combination with anesthetic did methoxamine significantly slow recovery of Na sup + channel from inactivation.

The present study demonstrates that methoxamine, a specific alpha¹-adrenergic receptor agonist, decreased I^Nain guinea pig ventricular myocytes. Although most of the inhibitory response to 100 micro Meter methoxamine on I^Nawas prevented with the alpha¹-adrenergic receptor antagonist prazosin (1 micro Meter), the block was not completely abolished. Propranolol (100 nM) was present throughout all phases of the experimental protocols to exclude possible beta-adrenergic influence. Because methoxamine and prazosin act competitively at the alpha sub 1 -adrenergic receptor site, a possible explanation could be that the high methoxamine concentration partially overcame the antagonistic effect of 1 micro Meter prazosin.

The inhibition of I^Naby methoxamine is V^H-dependent, being more pronounced at a depolarized V^H. In this respect, the effect of methoxamine on cardiac I^Nashows similarities to the effect of isoproterenol, a selective beta -adrenoceptor agonist, whose blocking effect is also enhanced at depolarized V^HS. [16–18] However, at more hyperpolarizing V^HS, the isoproterenol effects on I^Naare not clear. In experiments initiated from a V^Hof -90 mV, a current enhancement [16] or inhibition [17] was reported. Further, from a V^Hof -130 mV isoproterenol has been found to increase I^Na. [18] In contrast, in our experiments at a hyperpolarizing V^Hof -110 mV, methoxamine has been found to inhibit I^Na. However, an approximately 20-fold greater concentration was necessary to inhibit I^Naby 50% when using a hyperpolarized V^Hcompared with experiments initiated from a V^Hof -80 mV where approximately 15% of the available channels are in the inactivated state (see Figure 6).

In guinea pig ventricular myocytes, isoproterenol produced significant hyperpolarizing shifts in V¹/2 of steady-state activation and inactivation, which has been shown to be related to cAMP-mediated channel phosphorylation. [16] In one respect, mechanisms of alpha¹-adrenoceptor-mediated effects on I^Naappear to be different from those of beta-adrenoceptor stimulation. Our study shows that methoxamine shifted the steady-state inactivation curve in the negative direction, but steady-state activation was not affected. Interestingly, these shifts in V¹/2 showed no concentration-dependence between the two concentrations examined (10 and 100 micro Meter). Thus, in contrast to the concentration-dependent effects on peak I^Na, the shift in inactivation appears to saturate at concentrations of 10 micro Meter or lower. Hence, shifts in steady-state inactivation may contribute but cannot, by themselves, account for the depressant effects of methoxamine on I^Na. The slope factor, k, was not changed for steady-state activation and inactivation, indicating that the channel's intrinsic voltage sensor was not affected.

The mechanisms by which anesthetics and adrenergic agents depress conduction and, thereby, set the stage for development of dysrhythmias, may include a combination of alpha¹- and beta-adrenoceptor-mediated effects on the cardiac Na sup + current. [10] In the present study, we focused on largely unknown mechanisms underlying alpha¹-adrenoceptor mediated depression of conduction combined with anesthetics. Alpha¹-mediated interactions between catecholamines and halothane resulting in marked slowing of conduction have been reported. [2,11] However, these changes in conduction velocity did not involve reduction of V^max, an indicator of Na sup + current influx, and thus, it was proposed to be a result of alterations in cell-to-cell coupling. Our findings during conditions near the physiologic cardiac diastolic membrane potential (V^H=-80 mV) that methoxamine in the presence of anesthetics (halothane and isoflurane) produced a significant decrease in peak Na sup + current amplitude indicates a strong contribution of I^Nato the observed conduction changes. Hence, the mechanisms by which anesthetics and methoxamine depress cardiac conduction velocity may involve a combination of actions on Na sup + current block and cellular uncoupling.

A direct prodysrhythmic alpha¹-mediated interaction between catecholamines and halothane has been reported. [2] Further, epinephrine, or norepinephrine, and halothane have synergistic negative dromotropic effects, slowing conduction of cardiac impulses in dog Purkinje fibers to a greater extent than isoflurane. [7,8] One possible explanation might be that the magnitude of overall depression of I^Naby methoxamine combined with halothane is greater than that with isoflurane. On the other hand, our results show an enhanced effect of methoxamine with halothane and isoflurane. Thus, alpha¹-stimulation alone does not seem to explain the distinct effects found in cardiac impulse propagation.

No significant differences in the shifts in inactivation or activation were found between methoxamine alone or methoxamine combined with anesthetics after subtraction of the anesthetic effect. Hence, the enhanced suppression of peak I^Naby alpha¹-adrenergic stimulation in the presence of halothane or isoflurane cannot be explained by the shifts in steady-state activation or inactivation. This effect of methoxamine combined with anesthetics is only apparent when a fraction of the available channel is in the inactivated state. As discussed in the preceding study, [13] at a depolarized V^H, halothane appears to stabilize the Na sup + channel in an inactivated state. The interaction between anesthetics and methoxamine that is evident from a depolarized holding potential suggests that methoxamine may enhance this stabilization. That this effect is not evident from -110 mV implies interaction of methoxamine and anesthetics with inactivated channels. The depressant effects of methoxamine in the presence of halothane or isoflurane were reversible in experiments using a V^Hof -110 mV but irreversible when using a V^Hof -80 mV (data not shown). This further supports the stabilization of the inactivated state.

In experiments carried out with a V^Hof -80 mV, which is close to the physiologic diastolic membrane potential, methoxamine slowed I^Narecovery from inactivation after exposure to equianesthetic concentrations of halothane and isoflurane. The increased channel recovery time induced by methoxamine only in the presence of anesthetics further demonstrates a positive interaction between these agents. Further, in experiments from a V^Hof -110 mV, methoxamine in the presence of halothane still slowed the rate of recovery from inactivation compared with halothane alone, with the rate constant increasing from 8.83 +/- 0.98 ms to 12.65 +/- 1.62 ms (n = 6, data not shown). This is in contrast to the results reported for halothane alone [13] where the slowing of the rate of recovery observed with a V^Hof -80 mV was abolished with a V sub H of -110 mV. Thus, this result provides yet another evidence that methoxamine in the presence of anesthetic further stabilizes an inactivated state of the channel.

The fast cardiac I^Nacurrent is responsible for the generation of the rapid upstroke of the cardiac action potential and contributes to the rate of impulse propagation throughout the heart. Catecholamines and volatile anesthetics may decrease cardiac I^Naand consequently conduction velocity, which is, in physiologic and in pathophysiologic conditions, likely to be of great clinical relevance. The dysrhythmogenic effects of epinephrine in the heart in the absence and presence of halothane has been shown to involve synergistic alpha¹- and beta-adrenoceptor-mediated actions. [10] However, reports of isolated alpha¹-adrenergic effects on impulse propagation in dog Purkinje fibers are conflicting. Turner et al. reported a significant decrease of conduction velocity by phenylephrine. [2] On the other hand, no effect on conduction velocity was demonstrated for methoxamine in the range of 1 nM to 10 micro Meter. [19] Our results obtained during conditions that are close to the physiologic cardiac diastolic membrane potential (-80 mV) demonstrate marked reductions in I^Naat relatively small methoxamine concentrations. This finding may suggest that decreased I^Nacontributes to the reported decrease in conduction velocity by alpha¹-adrenergic stimulation in the heart and, thus, to dysrhythmogenesis. However, because our results are based on experiments conducted at room temperature, studies at 37 [degree sign] Celsius will be needed to confirm this finding.

Interactions of volatile anesthetics and catecholamines in facilitating cardiac dysrhythmias have been reported in in vivo and in vitro studies. [5,6] Our results suggest a prodysrhythmic interaction between alpha¹-adrenoceptor stimulation and both tested volatile anesthetics, halothane and isoflurane. At the channel level, the positive interaction in block of cardiac I^Namay be one of the mechanisms generating dysrhythmias. Further, recovery from inactivation of Na sup + channels is essential to the normal conduction and refractoriness properties of the myocardium. [20] At higher stimulus frequencies, a prolonged recovery may decrease conduction velocity and lengthen the refractory period. Under these circumstances, volatile anesthetics, especially combined with alpha¹-adrenoceptor stimulation, may promote dysrhythmias.

In pathophysiologic conditions, such as during myocardial ischemia, alpha¹-adrenoceptor sites are significantly increased in the heart. [21] This is consistent with enhanced alpha-adrenergic responsiveness during myocardial ischemia, which has been reported to be a primary mediator for dysrhythmias. [22] The results from the present study show an increased inhibitory effect on I^Naby anesthetics and methoxamine in normally depolarized cells (V^H=-80 mV) compared with hyperpolarized cells (V^H=-110 mV). The inhibitory effect of halothane was significantly more potent at a depolarized membrane potential (V^H=-65 mV). [13] One might speculate that further cell depolarization (positive to -80 mV) may lead to even more enhanced inhibitory effects of anesthetics and alpha-adrenergic stimulation, causing a further decrease in cardiac conduction in the ischemic cells. Inhomogeneities in cardiac tissue ischemic and nonischemic zones can produce electrophysiologic alterations that facilitate or block reentrant dysrhythmias, depending on the conduction properties and refractoriness of the tissue. [23]