Alpha1-adrenergic receptor stimulation has been shown to inhibit cardiac Na+ current (INa). Furthermore, some form of synergistic interaction of alpha1-adrenergic effects on INa in combination with volatile anesthetics has been reported. In this study, the authors investigated the possible role of G proteins and protein kinase C in the effects of halothane and isoflurane in the absence and presence of alpha1-adrenergic stimulation on the cardiac INa.
The standard whole-cell configuration of the patch-clamp technique was used. INa was elicited by depolarizing test pulses from a holding potential of -80 mV in reduced Na+ solution (10 mM). The experiments were conducted on ventricular myocytes enzymatically isolated from adult guinea pig hearts.
The inhibitory effect of halothane (1.2 mM) and isoflurane (1 mM) on peak INa was significantly diminished in the presence of guanosine 5'-O-[2-thiodiphosphate (GDPbetaS). In myocytes pretreated with pertussis toxin (PTX), the potency of halothane was significantly enhanced, but the isoflurane effect was unchanged. In the presence of the protein kinase C (PKC) inhibitor bisindolylmaleimide (BIS), the effect of halothane was unchanged. In contrast, the effect of isoflurane on INa in the presence of BIS was significantly enhanced. The positive interaction between methoxamine and halothane was evident in the presence of G protein and PKC inhibitors. In contrast, the effect of methoxamine with isoflurane was additive in the presence of GDPbetaS or BIS.
Different second messenger systems are involved in the regulation of cardiac Na+ current by volatile anesthetics. The effect of halothane involves a complex interaction with G proteins but is independent of regulation by PKC. In contrast, PKC is involved in the modulation of cardiac INa by isoflurane. In addition, non-PTX-sensitive G proteins may contribute to the effects of isoflurane. The positive interaction between methoxamine and anesthetics are independent of G proteins and PKC for halothane. In the case of isoflurane, the positive interaction with methoxamine is coupled to PTX-insensitive G proteins and PKC.
At the cellular level, stimulation of alpha1-adrenergic receptors has been shown to modify the activity of several different cardiac ion channels, including Na sup + channels. In guinea pig ventricular myocytes, alpha1-adrenergic receptor activation by methoxamine inhibits Na sup + current amplitude in a concentration- and voltage-dependent manner. Anesthetic potentiation of alpha1-adrenergic effects in the heart has been suggested to contribute to the genesis of halothane-epinephrine dysrhythmias by markedly slowing cardiac conduction. This is thought to be a key component in facilitating dysrhythmias by reentry mechanisms. The mechanisms by which anesthetics and alpha1-adrenergic stimulation depress conduction may involve a reduction of the fast cardiac inward Na sup + current (INa). A recent study showed a potentiation of alpha1-adrenergic depressant effects on cardiac Na sup + current in ventricular myocytes in the presence of the volatile anesthetics halothane and isoflurane. This positive interaction of alpha1-effects in combination with anesthetics may contribute to the generation of dysrhythmias, especially in the ischemic heart.
The regulation of the cardiac Na sup + current by volatile anesthetics may involve G-protein-dependent pathways. Furthermore, inactivation of the inhibitory G protein (Gi) seems to be involved in the facilitation of catecholamine-induced dysrhythmias in the heart. However, not all alpha1-adrenergic responses in cardiac tissue are mediated by a Giprotein. Furthermore, many responses to alpha1-adrenergic receptor stimulation are linked to protein kinase C (PKC). [1,7]A potential role of PKC in modulating anesthetic effects was shown recently, in which halothane and enflurane inhibited PKC activity. However, for volatile anesthetics and alpha1-adrenergic stimulation, the role of G proteins and PKC within the signal transduction pathway regulating the cardiac Na sup + channel is unclear.
The objective of the present study was to investigate the subcellular mechanisms underlying the depression of peak INaby (1) the volatile anesthetics halothane and isoflurane and by (2) methoxamine (an alpha1-adrenergic agonist) in combination with anesthetics. The possible role of G proteins linking alpha1-adrenoceptor activation and anesthetic action to the modulation of cardiac INawas also evaluated. Further, we examined the possible role of PKC in mediating the methoxamine and anesthetic effects on the cardiac Na sup + current. The whole-cell patch-clamp technique was used to measure the effects of anesthetics and methoxamine on the fast inward Na sup + current in single ventricular myocytes obtained from guinea pig hearts.
Unless stated otherwise, the experiments in this study were conducted under conditions described in an earlier 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 method. 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) and 1 mM propranolol (Sigma Chemical) were freshly prepared each day and diluted in the external bath solution. Pertussis toxin (List Biological Laboratories, Campbell, CA) was first prepared in distilled water. The final concentration of pertussis toxin (PTX) in Tyrode solution was 2 micro gram/ml. Bisindolylmaleimide (BIS; Calbiochem, La Jolla, CA) was initially prepared in dimethyl sulfoxide (Sigma Chemical) and further diluted in external solution. The desired final BIS concentration of 200 nM was achieved by 1:1000 dilution with the appropriate external solution. Dimethyl sulfoxide (0.1%) alone had no significant effect on INa(n = 4 cells).
Statistical analysis within one experimental group was computed using one-way repeated measures analysis of variance. Differences between treatment means were evaluated with the Bonferroni test. However, in some cases the tests for normality and equality of variance within groups were not satisfied. For those cases a one-way repeated measure analysis of variance on ranks (post hoc Student-Newmann-Keuls test) was used. When different groups of anesthetics were compared, data were expressed as percentage change and a two-way repeated measures analysis of variance was performed. Differences between group means were evaluated using the Bonferroni test. Statistical analysis was determined using commercially available software (SigmaStat, Jandel Scientific, Corte Madera, CA, and SuperANOVA, Abacus Concepts, Berkeley, CA). For experiments comparing shifts in steady-state inactivation, the predicted background shift was subtracted from the obtained shifts, as has been previously described, before performing statistical analysis. A test was considered to be significant when P < 0.05. Data are presented as means +/- SEM.
(Figure 1(A)) shows halothane (1.2 mM) inhibition of peak I sub Na by 38.5 +/- 2.9%. Halothane inhibition of cardiac INavia a G-protein-dependent pathway is demonstrated in experiments using guanosine 5'-O-(2-thiodiphosphate)(GDP beta S) in the pipette solution and in cells pretreated for 2–5 h with PTX. A nonhydrolyzable GDP analog, GDP beta S competitively inhibits G protein activation by GTP and GTP analogs. Petussin toxin inhibits activity of Giand Goproteins. As shown in Figure 1(A), the effect of halothane was significantly diminished with GDP beta S, decreasing current amplitude by 23.4 +/- 2.5%. This corresponds with the results of our previous study. In contrast to experiments with GDP beta S, for myocytes pretreated with PTX, the potency of halothane is significantly enhanced, inhibiting INaby 54.1 +/- 2.7%. Experiments including GDP beta S in PTX-pretreated cells resulted in an inhibition of INaby 23.6 +/- 3.1%, which is virtually unchanged compared with halothane effect with GDP beta S alone. The result from the GDP beta S and PTX combination indicated no further inhibition of PTX-sensitive G protein activity. To investigate the role of PKC in the halothane effect on INa, 200 nM BIS, a highly specific PKC inhibitor, was added to the extracellular solution. The BIS concentration used in our experiments is approximately 65 times greater than the Kifor inhibition of PKC activity. In the presence of BIS, the depressant effect of halothane on Na sup + current amplitude remained unchanged (36.7 +/- 4.2%) compared with the halothane effect alone (Figure 1(A)).
(Figure 1(B)) shows the average effects of isoflurane on I sub Na in untreated myocytes and in combination with inhibitors. Isoflurane (1 mM) alone inhibited INaby 21.2 +/- 2.0%. In the presence of GDP beta S, the effect of isoflurane is significantly diminished, decreasing INaby 13.7 +/- 2.4%. Unlike the effect of halothane, the effect of isoflurane on INawas not significantly different in PTX-pretreated cells compared with untreated cells, decreasing INaby 24.2 +/- 3.1%. The combination of GDP beta S and PTX with isoflurane depressed INaby 10.8 +/- 0.9%, which was similar to the result obtained with GDP beta S alone. Further contrasting the effect of halothane, the depressant action of isoflurane in the presence of BIS was significantly enhanced (35 +/- 2.9%).
(Figure 2(A) and Figure 3(A)) illustrate the effect of halothane and isoflurane, respectively, in combination with methoxamine on peak inward Na sup + current. For both anesthetics, the maximal suppressing effects were observed within 3 min after drug application. Methoxamine further decreased INain the presence of either halothane or isoflurane. Washout of anesthetic and methoxamine, however, did not consistently result in complete recovery of the Na sup + current amplitude. As cited previously, this partial reversal can be attributed to the stabilization of the inactivated state of the channel by the anesthetics. [2,15]The effects of methoxamine in the presence of halothane or isofiurane were further investigated under conditions in which G protein and PKC activities were inhibited. The time courses of peak INaunder the various conditions are depicted in Figure 2(B) and Figure 3(B) and the results are summarized in Figure 4. To compare the additional reduction of INaby methoxamine in combination with anesthetics, data were analyzed from the steady state obtained during anesthetic exposure. Thus the current obtained after the maximal effect of anesthetic served as the new “control,” as shown by the dotted lines in Figure 2and Figure 3. The effect of methoxamine on INain the presence of anesthetics was significantly enhanced compared with that of methoxamine alone (Figure 4(A)). The effect of methoxamine on INain combination with halothane was also significantly enhanced under conditions of GDP beta S, PTX, GDP beta S plus PTX, and BIS (Figure 4(B)). In contrast to halothane, the effect of methoxamine with isoflurane appears to be additive in the presence of GDP beta S, GDP beta S plus PTX, or BIS (Figure 4(B)). However, a greater decrease of INaby methoxamine in combination with isoflurane was found in PTX-pretreated cells.
The effects of anesthetics and alpha1-adrenergic stimulation by methoxamine on the steady-state inactivation parameters of the Na channel were also investigated. To distinguish drug-induced shifts from the spontaneous background shifts inherent for INa, [16,17]steady-state inactivation curves were evaluated over time under control conditions. We have previously reported a rate of shift in steady-state inactivation of INaof -0.27 +/- 0.01 mV/min under control (drug-free) conditions. In the presence of either BIS or in PTX-pretreated cells, the rate of shift remained unchanged (n = 6, data not shown). However, GDP beta S prevented the spontaneous shift in steady-state inactivation (n = 6 cells, data not shown). Steady-state inactivation was monitored after allowing for the diffusional exchange of GDP beta S into the cell (approximately 25 min). Thus spontaneous shifts were corrected for, except in the presence of GDP beta S. In the example shown in Figure 5, which is corrected for the spontaneous shift, halothane alone decreased current amplitude at all potentials and shifted the potential for half-maximal inactivation (V1/2) in the hyperpolarizing direction. Methoxamine in the continued presence of halothane further reduced Na sup + currents, and the steady-state inactivation curve was further shifted leftward. On washout of halothane and methoxamine, the current amplitude at hyperpolarized potentials returned to control values. The halothane and methoxamine effects were readily reversible when using a holding potential of -110 mV. In all cases, no significant changes in the slope factor k were observed.
A summary of the effects of anesthetics and methoxamine on V sub 1/2 is shown in Table 1. For halothane, the shift in steady-state inactivation was significantly enhanced in PTX-treated cells. For the isoflurane and methoxamine effects, there were no significant differences in shifts in V1/2 in the presence of various inhibitors. No significant differences were found between methoxamine alone and methoxamine in combination with anesthetics in the presence or absence of the specific inhibitors.
The regulation of the cardiac N channel by volatile anesthetics involves complex interactions of several distinct mechanisms. Studies have shown that volatile anesthetic action on the cardiac Na channel may, in part, be a result of direct interaction between the anesthetic and the channel protein. [18,19]In addition, anesthetic action on ion channel function may be induced by effects on the boundary lipids surrounding the channel proteins. [18,20]In the present study, the PKC inhibitor, BIS, did not affect halothane's effect on INa, suggesting that PKC is not involved. With GDP beta S, the depressant effect of halothane on INais significantly diminished. However, surprisingly, in PTX-pretreated cells, the halothane effect on INais significantly enhanced. Because GDP beta S should have blocked G-protein activities, including those of Gi, it is not clear why inhibiting activities of PTX-sensitive G proteins (Giand Go) would lead to an enhanced effect of halothane. Halothane has been reported to stimulate adenylyl cyclase activity by inhibiting the function of Gi(PTX sensitive) proteins. Thus if Giwere involved, we would have expected that in PTX-pretreated cells halothane would be less effective in inhibiting INa. This result suggests that in the presence of PTX, the subcellular mechanisms of halothane action on the Na sup + channel appear to be fully activated. Pertussis toxin has been reported to catalyze the adenosine diphosphate-ribosylation of the alpha-subunits of both Giand Go, which have been identified in cardiac tissue. Halothane's effect on Gois not known. It is unlikely that halothane enhances G sub o activity in PTX-pretreated cells. However, it cannot be excluded because the halothane effect was greater in those cells.
Alpha1-adrenergic-mediated interactions between catecholamines and halothane results in marked slowing of conduction. [3,22]In the presence of halothane, methoxamine produces a disproportionately large decrease of peak inward current, suggesting a type of synergistic interaction between halothane and methoxamine. This indicates a strong contribution to the observed conduction changes. Studies have shown that several responses mediated by alpha1-adrenergic receptor stimulation in cardiac myocytes can be blocked by PTX. [23,24]However, not all alpha1-adrenergic responses in cardiac muscle are PTX sensitive. The present results show that the mechanisms of interaction between methoxamine and halothane do not include G proteins and PKC.
The mechanisms of action of isoflurane on INaappears to be distinct from those of halothane. The preceding study showed that isoflurane, like halothane, acts through a G-protein-dependent pathway but, unlike halothane, not via a cyclic adenosine monophosphate-dependent pathway. Our result shows that the G-protein pathway involved in the isoflurane effect on INadoes not involve PTX-sensitive G proteins. Thus this excludes the Giand G sub o proteins. This is supported by a study showing that isoflurane has no effect on either basal or stimulated adenylyl cyclase activity, which is regulated by the inhibitory G protein Gi. Although we did not tested it in this study, one possible mechanism of action may be that isoflurane acts on cardiac Na channels through the direct membrane-delimited pathway via Gsbecause a regulation of cardiac I sub Na by isoproterenol has been shown to include this pathway. Another possibility includes a pathway via PTX-insensitive G proteins and PKC because stimulation of PKC has been shown to inhibit cardiac Na sup + current. Our findings show a significant enhancement of the suppressing effect of isoflurane in the presence of the PKC inhibitor, BIS, indicative of an involvement of PKC. However, inhibition of PKC should have diminished the suppressing effect of isoflurane. Many studies have been done of the anesthetic effects on PKC. Enhancement of PKC-mediated smooth muscle vasoconstriction by isoflurane and stimulation of brain PKC by halothane and propofol have been reported. Inhibition of PKC by anesthetics in neuronal tissues have also been reported. Attenuation of PKC by isoflurane in cardiac cells is unlikely because this would not lead to suppression of INa. Yet enhancement of PKC in cardiac ventricular myocytes would not explain the greater effect of isoflurane in the presence of BIS. If isoflurane suppressed INavia PKC stimulation, in the presence of BIS, the suppression would be less. Thus the mechanism for the greater isoflurane effect in the presence of BIS is unresolved.
Similar to halothane, the effect of methoxamine was significantly enhanced in combination with isoflurane compared with the methoxamine effect alone, indicating a type of synergistic interaction between these two agents. However, unlike the effect of halothane, the effect of isoflurane and methoxamine was additive in the presence of GDP beta S, GDP beta S plus PTX, and BIS. The isoflurane and methoxamine effect was unaffected in cells pretreated with PTX. Our findings clearly show that the interaction between isoflurane and methoxamine includes PTX-insensitive and PKC pathways.
The hyperpolarizing background shift, inherent in INarecordings under whole-cell configuration, was unaffected by inhibition of PKC and in PTX-pretreated cells. However, the background shift was not evident in the presence of GDP beta S. These results provide evidence that PTX-insensitive G proteins are involved in the time-dependent shift of steady-state inactivation. The negative shifts in V1/2 for the steady-state inactivation induced by halothane and isoflurane in the present study are in the range reported previously. The shift in steady-state inactivation was significantly enhanced by halothane in PTX-pretreated cells, indicating that a larger decrease of peak current amplitude is due in part to the shift in inactivation. However, the shifts in steady-state inactivation for either halothane or isoflurane in the absence or presence of GDP beta S, GDP beta S plus PTX, and BIS and for isoflurane also in combination with PTX showed no significant differences. Consequently, the shifts in steady-state inactivation in combination with inhibitors cannot account for the differential effects of each anesthetic on INa. Furthermore, inactivation shifts induced by methoxamine in the absence and presence of anesthetics and the different inhibitors were not significantly different. Thus the effects on INaby methoxamine and in combination with anesthetics cannot be explained by effects on steady-state inactivation.
Our results show a similar positive interaction between both anesthetics, halothane and isoflurane, with methoxamine. Thus alpha1-stimulation alone cannot explain the observation that epinephrine or norepinephrine with halothane, more so than isoflurane, have synergistic negative dromotropic effects. [31,32]Our preceding study showed some form of synergistic interaction in suppressing INaonly for halothane, and not for isoflurane, during beta-adrenoceptor stimulation by isoproterenol. Consequently, the less potent isoflurane effect in decreasing conduction velocity in combination with epinephrine appears to be related more to differential beta-adrenergic-mediated effects rather than alpha1-adrenergic effects. However, further experiments are necessary to examine the combined effects of volatile anesthetics and simultaneous alpha- and beta-adrenoceptor stimulation, for example by epinephrine, on cardiac INa. In addition to the effect of alpha1- and beta-stimulation on INabeing differentially affected by volatile anesthetics, the mechanisms of interaction are also different. As reported earlier, the enhanced effect of beta-stimulation by halothane involves a G-protein-dependent, PKA-independent pathway. In contrast, the interaction between alpha1-stimulation and halothane is a G-protein-independent pathway and is also regulated by PKC. A type of synergistic interaction between alpha1-stimulation and isoflurane includes a PTX-insensitive G-protein pathway and also PKC.
In summary, the present study provides strong evidence that intracellular signal transduction pathways are involved in the regulation of cardiac Na sup + current by volatile anesthetics. The mechanism of the halothane effect includes a PTX-sensitive, G-protein-dependent pathway and is independent of regulation by PKC. For isoflurane, the mechanism is distinct from that of halothane and includes a PTX-insensitive G-protein pathway. The isoflurane effect on I sub Na is also regulated by PKC. Halothane and isoflurane enhance the effect of alpha1-stimulation, although the mechanisms involved differ.