Alpha1-adrenoceptor stimulation is known to produce electrophysiologic changes in cardiac tissues, which may involve modulations of the fast inward Na+ current (I(Na)). A direct prodysrhythmic alpha1-mediated interaction between catecholamines and halothane has been demonstrated, supporting the hypothesis that generation of halothane-epinephrine dysrhythmias may involve slowed conduction, leading to reentry. In this study, we examined the effects of a selective alpha1-adrenergic receptor agonist, methoxamine, on cardiac I(Na) in the absence and presence of equianesthetic concentrations of halothane and isoflurane in single ventricular myocytes from adult guinea pig hearts.
I(Na) was recorded using the standard whole-cell configuration of the patch-clamp technique. Voltage clamp protocols initiated from two different holding potentials (V(H)) were applied to examine state-dependent effects of methoxamine in the presence of anesthetics. Steady state activation and inactivation and recovery from inactivation were characterized using standard protocols.
Methoxamine decreased I(Na) in a concentration- and voltage-dependent manner, being more potent at the depolarized V(H). Halothane and isoflurane interacted synergistically with methoxamine to suppress I(Na) near the physiologic cardiac resting potential of -80 mV. The effect of methoxamine with anesthetics appeared to be additive when using a V(H) of -110 mV, a potential where no Na+ channels are in the inactivated state. Methoxamine in the absence and presence of anesthetics significantly shifted the half maximal inactivation voltage in the hyperpolarizing direction but had no effect on steady-state activation.
The present results show that methoxamine (alpha1-adrenergic stimulation) decreases cardiac Na+ current in a concentration- and voltage-dependent manner. Further, a form of synergistic interaction between methoxamine and inhalational anesthetics, halothane and isoflurane, was observed. This interaction appears to depend on the fraction of Na+ channels in the inactivated state. (Key words: Anesthetics, volatile: halothane; isoflurane; methoxamine. Patch clamp: whole-cell configuration; sodium current; ventricular guinea pig myocytes.)
Alpha1-Adrenoceptor stimulation produces electrophysiologic changes in several mammalian cardiac muscles, which may also involve alterations of the fast inward Na sup + current. [1,2] Particularly under ischemic conditions in vivo or in vitro, alpha1-adrenergic receptor stimulation has been found to elicit cardiac dysrhythmias. [3,4]
In the myocardium, volatile anesthetics differ substantially in their ability to potentiate the catecholamine-induced dysrhythmias in patients and laboratory animals, halothane being more “sensitizing” than either isoflurane or enflurane. [5,6] The cellular mechanisms of this catecholamine-sensitizing effect are not completely understood. Epinephrine or norepinephrine and halothane have synergistic negative dromotropic effects, slowing conduction of cardiac impulses to a greater extent than isoflurane. [7,8] Studies using specific adrenoceptor-blocking agents found that induction of ventricular dysrhythmias by epinephrine during anesthesia involves activation of alpha sub 1 - and beta1-adrenoceptors. [9,10] An alpha1-adrenoceptor-mediated potentiation of the slowing of cardiac conduction produced by halothane was first suggested by Reynolds and Chiz using epinephrine on Purkinje fibers.  This effect was blocked by phentolamine but not by propranolol. Another study demonstrated a direct prodysrhythmic alpha1-mediated interaction between catecholamines and halothane, indicating that the mechanism(s) underlying generation of ventricular dysrhythmias by epinephrine during halothane anesthesia may involve slowed conduction, leading to reentry. 
The present study investigated the interaction between inhalational anesthetics and catecholamines on cardiac Na sup + current. Using the whole-cell configuration of the patch-clamp technique, we systematically measured the effects of alpha1-agonist alone and combined with inhalational anesthetics on sarcolemmal Na sup + current, a major current flow responsible for propagation of the cardiac action potential.
Unless stated otherwise, the experiments in this study were conducted under conditions described in our preceding article.  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.  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.  A test was considered to be significant when P < 0.05. Data are presented as mean +/- SEM.
Concentration- and Holding Potential-dependent Inhibition of Cardiac Na sup + Current by alpha sub 1 -adrenergic Stimulation
To determine whether alpha1-adrenergic receptor stimulation modulates cardiac INa, 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.  Peak Na sup + currents were elicited by voltage steps to -20 mV from (1) a hyperpolarized VHof -110 mV, where all Na sup + channels are available for activation, and (2) a more depolarized VHof -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 INawhen using a VHof -110 mV. Figure 1(B) shows the dose-dependent block of peak INaby methoxamine from two different VHS. The plot shows a dependence on concentration and voltage with a Kdof 0.086 +/- 0.031 mM when using a VHof -80 mV and a Kdof 1.77 +/- 0.87 mM when using a VHof -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 VHof -80 mV, 10 and 100 micro Meter methoxamine reduced peak INaby 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 INa(Figure 2(A)). The effect of 100 micro Meter methoxamine in the presence of prazosin was significant, inhibiting INaby 9.24 +/- 0.88%, but less than in the absence of prazosin (Figure 2(B and C)).
Effects of Volatile Anesthetics and Methoxamine on Cardiac Na sup + Current
As shown by a recording from a representative cell in Figure 3(A and B), INawas initially depressed by 1.0 mM isoflurane and further inhibited by methoxamine. From a VHof -110 mV, equianesthetic concentrations of halothane (1.2 mM) and isoflurane (1.0 mM) suppressed the peak INaby 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. Figure 4shows the effects on the peak inward Na sup + current by halothane or isoflurane and methoxamine monitored over time using a VHof -110 mV. In the continued presence of anesthetics, methoxamine further decreased peak INa. The effect of anesthetic plus alpha1-adrenergic stimulation on peak INawas determined after the maximal anesthetic effects were observed. To compare the additional reduction of INaby 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 VHof -110 mV, when all Na sup + channels are in resting state, methoxamine alone (100 micro Meter) reduced the peak INaby 12.8 +/- 2.1%. Methoxamine in combination with halothane or isoflurane further decreased INaby 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 VHof -110 mV, are additive.
To test whether there is a voltage-dependent interaction between methoxamine and anesthetics, further experiments were conducted from a VHof -80 mV, which is near the physiologic resting potential in cardiac cells. From this VH, 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 INaby 17.0 +/- 1.3%(Figure 5(B)). However, methoxamine combined with halothane or isoflurane was more effective in depressing INa, 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 VHof -80 mV.
Shifts in Steady-state Inactivation and Activation Induced by Volatile Anesthetics and Methoxamine
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 INadepend 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.  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 INamay 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 VHof -80 mV is not caused by the shifts in steady-state activation or inactivation of the Na sup + channel.
Recovery from Inactivation
The effects of methoxamine and volatile anesthetics on the recovery from inactivation using a VHof -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.
Alpha sub 1 -adrenergic Inhibition of Cardiac Na sup + Current
The present study demonstrates that methoxamine, a specific alpha1-adrenergic receptor agonist, decreased INain guinea pig ventricular myocytes. Although most of the inhibitory response to 100 micro Meter methoxamine on INawas 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 INaby methoxamine is VH-dependent, being more pronounced at a depolarized VH. In this respect, the effect of methoxamine on cardiac INashows similarities to the effect of isoproterenol, a selective beta -adrenoceptor agonist, whose blocking effect is also enhanced at depolarized VHS. [16–18] However, at more hyperpolarizing VHS, the isoproterenol effects on INaare not clear. In experiments initiated from a VHof -90 mV, a current enhancement  or inhibition  was reported. Further, from a VHof -130 mV isoproterenol has been found to increase INa.  In contrast, in our experiments at a hyperpolarizing VHof -110 mV, methoxamine has been found to inhibit INa. However, an approximately 20-fold greater concentration was necessary to inhibit INaby 50% when using a hyperpolarized VHcompared with experiments initiated from a VHof -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.  In one respect, mechanisms of alpha1-adrenoceptor-mediated effects on INaappear 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 INa, 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 INa. The slope factor, k, was not changed for steady-state activation and inactivation, indicating that the channel's intrinsic voltage sensor was not affected.
Interaction between Volatile Anesthetics and alpha sub 1 -adrenergic Stimulation
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.  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 Vmax, 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 (VH=-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 INato 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.  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 INaby 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 INaby 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,  at a depolarized VH, 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 VHof -110 mV but irreversible when using a VHof -80 mV (data not shown). This further supports the stabilization of the inactivated state.
In experiments carried out with a VHof -80 mV, which is close to the physiologic diastolic membrane potential, methoxamine slowed INarecovery 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 VHof -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  where the slowing of the rate of recovery observed with a VHof -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 INacurrent 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 INaand 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.  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.  On the other hand, no effect on conduction velocity was demonstrated for methoxamine in the range of 1 nM to 10 micro Meter.  Our results obtained during conditions that are close to the physiologic cardiac diastolic membrane potential (-80 mV) demonstrate marked reductions in INaat relatively small methoxamine concentrations. This finding may suggest that decreased INacontributes 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 INamay 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.  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.  This is consistent with enhanced alpha-adrenergic responsiveness during myocardial ischemia, which has been reported to be a primary mediator for dysrhythmias.  The results from the present study show an increased inhibitory effect on INaby anesthetics and methoxamine in normally depolarized cells (VH=-80 mV) compared with hyperpolarized cells (VH=-110 mV). The inhibitory effect of halothane was significantly more potent at a depolarized membrane potential (VH=-65 mV).  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.