Abstract Effects of Two General Anesthetics, Halothane

Japanese Journal of Physiology, 36, 107-121, 1986

Reduction in the Myocardial Sodium Current by Halothane and Thiamylal

Yoshimi IKEMOTO,* Atsuko YATANI,* °* * Yutaka IMOTO,* and Hlroyukl ARIMURA***

*Department of Physiology, and ***Department of Anesthesiology, Faculty of Medicine, Kyushu University, Fukuoka, 812 Japan

Abstract Effects of two general anesthetics, halothane and thiamylal, on the fast sodium inward current (INa) of enzymatically isolated single rat ventricular cells were studied under current clamp and voltage clamp conditions. A suction pipette technique was used for potential measurement, current injection and internal perfusion of isolated cells. In current clamp experiments, sodium action potential was elicited in a Ca-free Co Krebs solution and the action potential was reduced by 0.5°c halothane and 5 x 10-5 M thiamylal. In voltage clamp experiments, the calcium current was suppressed by replacing Ca with Co and the potassium current was eliminated by replacing K with Cs and adding 4-aminopyridine and tetraethylammonium. Both anesthetics decreased INa, in a dose dependent manner, without changing the shape of the current-voltage curve. Halothane (l°) shifted the steady state inactivation curve in a negative direction along the potential axis by 8.5±2 mV (mean + S.D., n=4). Thiamylal, 5 x 10-5 and 10-4M, shifted the curve in a negative direction by 4.4 ± 0.8 mV (n =5) and 8.6 + 3.2 mV (n = 5), respectively. Both agents slightly reduced the maximum sodium conductance (gNa). Halothane (1 %) increased half recovery time from inactivation measured at - 80 mV from 30±15 to 80 + 25 ms (n=4). Thiamylal (104M)-prolonged it at - 75 mV from 50± 20 to 110± 15 ms (n=5). With a test pulse duration of 50 ms, neither drug produced a use-dependent inhibition of INa. Halothane and thiamylal depress the INa of cardiac muscles mainly by shifting the steady state inactivation curve in a negative direction along the potential axis. Relatively small prolongation of half recovery time from inactivation and no sign of use-dependent inhibition suggest a molecular mechanism which differs in some respects from the local anesthetics.

Key words : halothane, isolated single cells, rat myocardium, sodium current, thiamylal.

Received for publication September 24, 1985 ** Present address: Department of Physiology and Biophysics, University of Texas Medical Branch, Galvestone, Texas 77550, U.S.A.

107 108 Y. IKEMOTO, A. YATANI, Y. IMOTO, and H. ARIMURA

Effects of general anesthetics on the action potential of various cardiac tissues have been studied. However, little is known of their effects on membrane currents of cardiac muscle cells. Depressant action of general anesthetics on the calcium inward current was suggested from the finding of a reduction in the slow action potential by these agents (IKEMOTO,1977; LYNCHet al., 1981, 1982). The fast sodium inward current (INa)plays an important role in conduction of impulse within the heart and in generation and cessation of some types of arrythmia. Since halothane is one of the most popular general anesthetics of current clinical use, there have been some reports about the halothane effects on INa. It however, technically difficult to study INa of multicellular cardiac muscle preparations under a voltage clamp due to limitations caused by the complex cellular geometry (JOHNSONand LIEBERMAN, 1971; ATTWELLand CoHEN, 1977). Therefore some investigators studied the effects of anesthetics on the INa using the maximum rate of rise of action potential generated at normal resting membrane potential. HAUSWIRTH(1969) reported that 2 % halothane depressed the maximum rate of rise of action potential of sheep cardiac Purkinje fiber, along with hyperpolarization of resting membrane potential. On the other hand, LYNCH et al. (1981) showed no significant change in the maximum rate of rise with 0.5 to 3% halothane, using a guinea pig papillary muscle. They sometimes observed an increase in the upstroke velocity. Single ventricular cells can now be isolated enzymatically and these cells seem to be little damaged by collagenase treatments (PowELL et al., 1980; BROWNet al., 1981a, b). The electrical properties of INa of the isolated ventricular cells are essentially similar to those of intact cardiac Purkinje fibers and embryonic heart cells, which have less complex geometry and were studied using the two- microelectrode voltage-clamp technique (CoLATSKYand TSIEN,1979; EBIHARAet al., 1980). In the present experiments, we studied the effects of two popular general anesthetics, halothane and thiamylal (an ultrashort-acting barbiturate for induction of anesthesia) on the INa of single isolated rat ventricular cells, using internal perfusion with a suction pipette technique. This approach eliminated problems when using the voltage clamp to measure the fast ionic current, INa. Halothane and thiamylal reduced INa in a dose dependent manner.

MATERIALSAND METHODS

1) Cell isolation. Ventricular myocardial cells were isolated from rats using an enzymatic dissociation procedure much the same as that of PowELL et al. (1980) and YATANIand GoTo (1983). Adult rats weighing 200-250g were killed by a blow on the head, the chest of each rat was opened and the heart extracted quickly and washed with a Krebs solution. Retrograde perfusion through the aorta was performed at 37°C using a Langendorff apparatus. Perfusion was as follows: 4 min with a nominally Ca-free Krebs solution containing 1 mg/ml bovine serum albumin which was essentially fatty acid free (Sigma); 20 min with an enzyme medium containing 0.4 mg/ml collagenase type I (Sigma). Following perfusion, the ventricle

JapaneseJournal of Physiology MYOCARDIAL INa AND GENERAL ANESTHETICS 109 was cut into 1 mm slices which were incubated at 37°C for 12-15 min in an enzyme solution containing an additional 10 mg/ml bovine serum albumin. The composition of the Ca-free Krebs solution for cell isolation was in mM: NaCI 118.41; KC1 2.55; MgSO4 1.16; KH2P04 1.32; NaHC03 14.52; glucose 11.1. The solution was saturated with 95% 02 and 5 % C02. Dispersed cells were filtered and stored at 4°C in "Kraft-bruhe," or "power soup," identical to that described by IsENBERGand KLOCKNER(1980, 1982). The composition of the "Kraftbruhe" was in mM: KC1 80; KH2SO4 30; Na2ATP 5; MgSO4 5; pyruvic acid 5; succinic acid 5; creatine 5; Nat ethyleneglycol-bis-($- aminoethylether)-N,N'-tetraacetic acid (EGTA) 0.1. The pH was adjusted to 7.4 with KOH. Cells were generally used after 2-6 h. 2) Solutions. At first the cells were superfused with a Krebs solution of the following composition (mM): NaC1120; KC12.5; CaC12 3.6; MgC12 1.2; glucose 5.5 (pH 7.4). In this Ca-containing solution 30-60% of the cells remained viable and were rod-shaped and quiescent. In current clamp experiments, the solution was changed to a Ca-free Co Krebs solution of the following composition (mM): NaCI 120; KC1 5; MgCl2 2.5; CoC12 5; glucose 5.5 (pH 7.4). The composition of the internal solution which perfused the cell interior through the pipette was (mM): K- aspartate 130; glucose 5.5; EGTA 0.1 (pH 7.3). In voltage clamp experiments, INa was separated by suppression of calcium and potassium currents by perfusion with external and internal solutions of the following composition (mM). External solution: NaCI 60; CsCI 5; tetraethylammonium (TEA) chloride 20; MgC12 2.5; CoCl2 5; 4-aminopyridine 5; glucose 5.5; sucrose 80 (pH 7.4). Internal solution: Cs- aspartate 130; NaOH 15; glucose 5.5; EGTA 0.1 (pH 7.3). pH of all solutions was adjusted with N-2-hydroxyethylpiperazine-N'-2-ethansulfonic acid (HEPES) and tris(hydroxymethyl)aminomethane hydroxychloride (Tris). 3) Electrical recording. All experiments were performed using the suction pipette method, which allows for internal perfusion of the isolated cell and current or voltage clamp as described previously (YATANIand AKAIKE,1984). Potential measurement and current passing were carried out with a high frequency sample- and-hold preamplifier. This apparatus makes it possible to voltage-clamp a cell with a single electrode, switching rapidly (0.5-20 kHz) from voltage recording to current passing through the suction pipette electrode. The voltage is measured only when the discontinuous or chopped current is zero, yielding an accurate measurement of the membrane potential. Switching frequencies ranging between 10 and 20 kHz were used. With the same preparation as that used in the present experiments, AKAIKEet al. (1984) checked the voltage control during the clamp pulse using a separate microelectrode. They obtained good voltage control with an INa of about 50 nA. Upstroke velocity of the action potential was measured with a differentiator which responded linearly to the rate of voltage change up to 500 V/s. Action potential and differentiated upstrokes were displayed on a storage oscilloscope, photographed and stored on magnetic tapes for subsequent analysis. INa was also monitored using a storage oscilloscope and magnetic tapes. INa was measured by subtracting the

Vol. 36, No. 1, 1986 110 Y. IKEMOTO, A. YATANI, Y. IMOTO, and H. ARIMURA maximum inward current from the steady background current at the end of the test clamp pulse, because in the present experimental conditions, the leakage current was linear in the voltage range explored (PowELL et al., 1980). 4) Drugs. Halothane was vaporized in compressed air flowing through a Fluotec III vaporizer. The halothane-containing air was then bubbled into the test solution for at least 45 min before and during application. When equilibrated at 20°C, 1% halothane results in a 0.63 mM concentration in solution, according to the solubility coefficient, while 0.5% halothane results 0.31 mM (HALSEY,1980). The potential loss of halothane due to vaporization during perfusion was minimized by locating the perfusate outlet directly in the small chamber which has a volume of 0.5 ml and by perfusing the preparation rapidly. Thiamylal was dissolved in the test solution just before use and perfused to the test chamber. S) Data analysis. All experiments were carried out at room temperatures between 18 and 21 °C, with the range maintained within 0.5°C. In current clamp experiments the action potential, elicited every 5s, was maintained for about 30 min without any change in the absence of the drugs. Drug effects were apparent within 2 min and reached a steady state within 5 min. The reversion was not complete (to about 90% of initial control value) after a 20 min washout following a 5 min application of 0.5% halothane or 5 x 10-5 M thiamylal. Therefore, the effects of drugs were expressed as the per cent change from the initial control value (mean ± S.D., n = number of experiments). In voltage clamp experiments it was possible to maintain INa unchanged for 15-20 min in the absence of the anesthetics, elicited every 10 s by 50 ms clamp pulses to -10 mV from a holding potential of - 80 mV. The steady state inactivation curve and the recovery time course from inactivation did not change during the first 15 min of the experiment without the drugs (n = 3 for each phenomenon). Drug effects appeared within 2 min and reached a steady state in 4-5 min. Following 5-6 min application of higher concentrations of the drugs, however, the reversion was not complete after a washout of about 10 min (to 60-90% of initial control value, depending on the concentration used). Therefore, the effects were expressed as changes from the initial control value (mean ± S.D., n = number of experiments). Tests of significance were performed using Student's t-test. The differences were considered to be significant when the p value was less than 0.05.

RESULTS

1) Current clamp experiments The resting membrane potential of isolated single rat ventricular cells with a rod shape in a Ca-free Co Krebs solution ranged from - 50 to - 70 mV. An inward DC current of 0.5 to 1.0 nA was injected to hyperpolarize the membrane to - 80 mV. Action potential was elicited with a 3 ms depolarizing pulse at a frequency of 0.2 Hz. Amplitude and maximum rate of rise of the action potential were 120 + 8 mV and 320±36 V/s (mean + S.D., n = 24), respectively. Under these con-

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Fig. 1. Effects of O.5° halothane on the action potential (a and b) and its first derivative (c and d) of an isolated single rat ventricular cell. The cell was perfused with a Ca-free Co Krebs solution to generate Na-action potentials. Control action potential (a) and its upstroke velocity (c) were reduced by 4 min application of 0.5 halothane (b and d). Action potentials were elicited from - 80 mV by 3 ms of depolarizing stimulation every 5 s. ditions, the preparation had a high input resistance between 50 and 100 mQ. The duration of action potential was short and ranged between 10 and 40 ms. 0.5 halothane decreased the overshoot and maximum rate of rise of action potential without affecting the resting membrane potential (Fig. 1). In the presence of 0.5 halothane, the overshoot and maximum rate of rise decreased significantly to 81 + 2 and 82± 3 % of the initial control value (n= 11), respectively. Similar results were obtained with thiamylal 5 x 10 - 5 M.In 11 cells, the overshoot significantly decreased to 80± 4% and the maximum rate of rise to 81 + 3 % of the initial control.

2) Voltage clamp experiments 1. Current-voltage (I-V) relationship. Figure 2A shows records of INa in the absence (a) and the presence (b, c) of 100 halothane in response to depolarizing steps of -25, -15, -5, and + 5 mV from a holding potential of -85 mV (a, b) and - 95 mV (c). No correction was made for leak or capacitive current. One halothane depressed INa. The depression was reversed to some extent by making the holding potential more negative (-95 mV, Fig. 2Ac), suggesting a negative shift of the steady state inactivation curve on the voltage axis (see also Fig. 5). The current- voltage relationship of INais illustrated in Fig. 2B, where the peak of INais plotted as a function of the clamp pulse potential. One % halothane decreased INa without affecting the threshold potential. The reversal potential of +32± 3 mV in 8 cells was unchanged by 1% halothane. Clamp pulses were applied every 10 s. Figure 3A shows current records of INa in the absence and the presence (indicated by white dots) of 10_4 M thiamylal. Currents were elicited by clamp pulses of - 20, -10, and 0 mV from a holding potential of - 80 mV. Thiamylal clearly reduced the INa.This reduction is illustrated on an I-V curve in Fig. 3B. The

Vol. 36, No. 1, 1986 112 Y. IKEMOTO, A. YATANI, Y. IMOTO, and H. ARIMURA

Fig. 2. Effects of l % halothane on the INa of an isolated single rat ventricular cell. Panel A shows original current records before (a) and 3 min after adding 1 halothane (b), elicited by depolarizing voltage steps of 10 mV increments from - 25 to + 5 mV from a holding potential (VH) of -85 mV. In c, the holding potential was hyperpolarized to - 95 mV and clamp pulses were applied to the same potentials as in a and b in the presence of 100 halothane. Panel B shows the effects of the anesthetic on the current-voltage relationship for the sodium current. Clamp pulses were applied every l0 s from a holding potential of -85 mV. drug did not change the threshold potential. The reversal potential of +30±3 mV in 9 cells was not affected by 10_4 M thiamylal. Voltage clamp pulses were applied every 10 s. Effects of various concentrations of halothane and thiamylal on the maximum INa on the I-V curve were studied using 7 cells for each concentration (Fig. 4). Clamp pulses were applied every 10 s from a holding potential of - 80 mV. Both agents decreased INa in a dose dependent manner. 2. Inactivation of the sodium system. The steady state inactivation of the Na system was determined by a pulse program in which a constant test pulse was preceded by a conditioning pre-pulse of different amplitude and direction (Fig. 5 inset). A sequence of pre- and test clamp pulses was applied every 10 s. The amplitude of INa during the test pulse is a measure of availability of the sodium system at the membrane potential before the test pulse. As the conditioning pre-

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Fig. 3. Effects of 10-4 Mthiamylal on INa of an isolated single rat ventricular cell. A: superimposed current traces before and after (indicated by white dots) addition of thiamylal (10 _ 4 M), elicited by depolarizing voltage steps of - 20 (a), -10 (b), and 0 mV (c) from a holding potential of - 80 mV. B: effects of 10-4 M thiamylal on the 1 V curve. Clamp pulses were applied every 10 s from a holding potential of - 80 mV. pulse was hyperpolarized to a greater extent, the degree of inactivation was reduced and the INa evoked by the test pulse was enlarged until saturation occurred, under conditions of extreme hyperpolarization (- 140 mV). The saturated INawas then used to normalize the test INathat resulted from other conditioning test pulse potentials. Figure 5A shows the effects of 100 halothane on the steady state inactivation curve. This anesthetic significantly shifted the midpotential of the curve in a negative direction along the voltage axis by 8.5 + 2.0 mV (n =4) and reduced the sodium current measured with a pre-pulse of -140 mV to 91% (significant), thereby suggesting a slight reduction in the maximum available sodium conductance (gNa). Under the effects of 0.5% halothane, 2 out of 3 cells produced a slight negative shift but the other cell did not. gNawas reduced to 97% (not significant) of the control in an average of the three cells. Figure SB illustrates the effects of thiamylal on the steady state inactivation curve. Thiamylal 5 x 10-s and 10_4 M significantly shifted the midpotential in a negative direction by 4.4 + 0.8 (n = 4) and 8.6 + 3.2 mV (n = 5), respectively. gNa was decreased to 96 (not significant) and 92% (significant) of the

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Fig. 4. Dose-dependent inhibitory action of halothane and thiamylal on INa. Per cent inhibition of peak INa on the I-V curve corresponding to control value is plotted against drug concentration. Clamp pulses were applied every 10 s from a holding potential of - 80 mV. Data were obtained 3 to 5 min after adding each concentration of the drugs. Concentration of halothane was calculated using a water- gas partition coefficient at 20°C. The symbols respresent mean + S.D. (n = 7). The continuous curve was drawn by hand.

Fig. 5. Effects of halothane (A) and thiamylal (B) on the steady state inactivation curve of INa. Clamp pulses shown in the inset were applied every 10 s. Abscissa is the pre-pulse potential and ordinate is the relative amplitude of INa elicited by the test pulse with various amplitudes of pre-pulse to that elicited with a pre-pulse of -140 mV. Both anesthetics shifted the curve in a negative direction along the voltage axis.

control for each concentration of thiamylal, respectively. 3. Recovery from inactivation of sodium current. In the heart muscle, development of re-entrant arrythmias is in part dependent on the length of the refractory period which is attributed to recovery of the sodium conductance from inactivation (GETTESand REUTER,1974).Hence, effects of the drugs on the recovery of INa from inactivation were studied by use of the classical two pulse produre (HODGKINand HUXLEY,1952). In Fig. 6A, the recovery at -80 mV was assessed by

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Fig. 6. Effects of halothane (A) and thiamylal (B) on the recovery of INa from inactivation. Insets show the double pulse program, where the holding potential was - 80 mV in (A) and - 75 mV in (B). Each two pulse sequence was followed by a 10 s rest period. Abscissa is the interval between conditioning pulse (50 ms duration) and test pulse. Ordinate is the ratio of INaelicited by the test pulse (] J to that elicited by the conditioning pulse (I~). measuring INa in response to paired 50 ms depolarizing pulses of - 30 mV, separated by intervals varying from 5 to 400 ms. We chose a rather short conditioning pulse of 50 ms because the action potential of this preparation is short. Each two-pulse sequence was followed by a 10 s rest period. The recovery curve was plotted as a relation of INa (test)/INa(conditioning), abbreviated as IJI~, to the interval between the coupled pulses. One % halothane significantly prolonged the half recovery time from 30 ± 15 to 80±25 ms (n = 4). The half time was chosen for the recovery index because the INa recovery of these ventricular cells does not follow a simple exponential curve (BROWNet al., 1981b). The effects of thiamylal on INarecovery are shown in Fig. 6B. In this case the holding potential was - 75 mV instead of - 80 mV, which lengthened the control half recovery time. Thaamylal 10_4 M significantly prolonged the half recovery time from 50±20 to 110 + 15 ms (n = 5). With clamp pulses of 50 ms duration, we observed no use-dependent inhibition

Vol. 36, No. 1, 1986 116 Y. IKEMOTO, A. YATANI, Y. IMOTO, and H. ARIMURA of INa in the presence of either anesthetic at frequencies up to 5 Hz (n = 4 with 1 halothane and n = 5 with 10 _4 M thiamylal).

DISCUSSION

1) Depression of the sodium action potential. The action potential duration of single rat ventricular cells in the present study was short due to blockade of the calcium current and to the early outward current described by JOSEPHSONet al. (1984). Halothane and thiamylal depressed the overshoot and the maximum rate of the rise of action potential of isolated rat ventricular cells in a Ca-free Co Krebs solution, suggesting a depression of INa by these agents. HAUSw1RTH(1969) reported no significant change with 1 % halothane and a depression with 2 % of the anesthetic in the maximum rate of rise of the action potential of sheep Purkinje fiber. In the present experiments, the action potential was already depressed by O.5° halothane. The discrepancy may arise from differences in the preparation, the temperature at which the experiments were carried out, and the following two factors: 1) HAUSWIRTH(1969) bubbled the halothane-containing gas into a Tyrode solution kept at 37°C. According to the partition coefficient, 1 % halothane should have resulted at this temperature in a 0.31 mM concentration, which is calculated to be almost equal to that equilibrated with O.5° of the anesthetic at 20°C in the present experiments (HALSEY,1980). 2) In his report the resting membrane potential was hyperpolarized from - 82.4 to - 87 mV by 1% halothane, with a significant decrease in the overshoot of the action potential. The hyperpolarization may to some extent have cancelled the depressant effect on the maximum rate of increase, as suggested by the present result that the anesthetic shifted the steady state inactivation curve in a negative direction (Fig. SA). No increase in the maximum rate of increase was seen with either anesthetic, as was observed with halothane by LYNCHet al. (1981). There seems to be no literature available on the plasma concentration of thiamylal during clinical anesthesia. MORGANet al. (1981) reported that the venous plasma concentration of thiopental was 20-30 µg/ml 5-7 min after intravenous administration of 275-450 mg (mean 355 mg) of the drug to nonpregnant patients (n =5, 49-73 kg weight, mean 60 kg), and expected higher arterial concentrations in the early period. Assuming a higher concentration of 100,ig/ml in very early circulation due to less initial apparent volume of distribution and taking into account the plasma protein binding ratio of 75°c given in their report, the free plasma concentration of thiopental would be 25 µg/ml. Resemblance of thiamylal and thiopental in molecular structure and potency might mean a similar pharmaco- kinetic profile of thiamylal following intravenous administration. A 10_4 M concentration of thiamylal is calculated to be 25.4,ug/ml, which can be attained in clinical anesthesia. 2) Decrease in the sodium current and antiarrythmic characteristics. In voltage clamp experiments, potassium currents were blocked by replacing KCl with

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CsCI and adding 4-aminopyridine and tetraethylammonium to the external solution, and by replacing K-aspartate with Cs-aspartate in the internal solution. The calcium current was blocked by omitting CaCl2 and adding CoCl2 to the external solution. Blockade of potassium and calcium currents may not have influenced the drugs' effects on INa, since INa is determined by gNa and by time and voltage dependent kinetics, both of which are not affected by these currents. Halothane and thiamylal reduced INa in a dose dependent manner without affecting the shape of the current-voltage curve, namely, the threshold and reversal potentials for the current were not changed. SCHRIVASTAVet at. (1976) reported that trichloroethylane, a general anesthetic, greatly shifted the reversal potential for the INa of squid giant axon's in a negative direction. They attributed the shift to a decrease in selectivity of the channel to cations and to an internal accumulation of sodium ions. Our findings suggest no change in ion selectivity or in sodium concentration inside the cardiac muscle cells. Both anesthetics in the present experiments shifted the steady state inactivation curve in a negative direction along the voltage axis and there was a slight reduction in gNa.These findings suggest that depression of INa by the two drugs is mainly due to the negative shift of the steady state inactivation curve. gNa of axons was not reported to be significantly affected by high concentrations of volatile anesthetics (4-5 mM halothane, 100-200 mM diethyl ether etc.), and no decrease in the number of the functioning sodium channels was suggested (BEAN et at., 1981; HAYDONand URBAN,1983c). Shift of the curve in a negative direction was noted in various preparations, with a variety of drugs including general anesthetics (SCHRIVASTAVet at., 1976; BEANet at., 1981; HAYDONand URBAN, 1983c), local anesthetics (HILLS, 1977a, b; BEANet at., 1983), hydrocarbones (HAYDONand URBAN, 1983a), alcohols (HAYDONand URBAN, 1983b; HARPERet at., 1983), and barbiturates (SCHWARZ,1979).Reduction in INawill decrease velocity of the impulse conduction in cardiac muscles, as noted with an increased concentration of halothane in the canine His-Purkinje system in vivo (ATLEEand ALEXANDER,1977; TURNERet at., 1980). The decrease in the conduction velocity could create a block in both limbs of a re-entrant circuit, making it incapable of sustaining a re-entrant arrythmia. The antiarrythmic property of the agents is also suggested by a prolongation of the recovery time from inactivation (Fig. 6), which may induce a lengthening of the refractory period in some circumstances. But the prolongation was much smaller than that produced by local anesthetics (BEANet at., 1983). The refractory period depends also on the duration of the action potential, which was reportedly decreased by halothane and barbiturates, probably through reduction in the slow inward current (HAUSWIRTH,1969; IKEMOTO,1977; LYNCHet at., 1981). Actually, TURNERet at. (1980) did an in vivo study and found that an increased concentration of halothane (1.5 to 2.4%) slightly shortened the functional refractory period. They found no significant shortening by l.5° halothane during basal thiopental anesthesia. 3) How do anesthetics affect the sodium current? It was reported that short

Vol. 36, No. 1, 1986 118 Y. IKEMOTO, A. YATANI, Y. IMOTO, and H. ARIMURA clamp pulses induced little use-dependent depression of INa in this preparation (LEE et al., 1979; SANCHEZ-CHAPULAet al., 1983). Lidocaine was shown to produce a small use-dependent depression with short clamp pulses (50 ms), whereas a new antiarrythmic drug produced a prominent use-dependent block (YATANI and AKAIKE,1984). Halothane and thiamylal did not produce a use-dependent depression with a 50 ms clamp pulse up to 5 Hz. The effects of various local anesthetics on INa of single myelinated nerve fibers were studied and it was reported that benzocaine, a neutral molecule with a physiological pH, did not show a use- dependent inhibition of the current and did shift the inactivation curve in a negative direction (HILLE, 1977b). The use-dependent inhibition by local anesthetics was related to the charged form (CoUTNEY,1975; HILLE, 1977a; GINTANTet al., 1983), which was indicated to have a separate site of action from an uncharged form such as benzocaine (HUANG and EHRENSTEIN,1981). HILLE (1977a, b) proposed a "Modulated Receptor Hypothesis" to account for the effect of local anesthetics on nerves and which was also related to the effects of these agents on cardiac muscles (HONDEGHEMand KATZUNG,1977; BEANet al., 1983). For development of the use- dependent inhibition, drug access to open channels through the hydrophilic phase and a high affinity binding between the inactivated state of the channel and drugs have been suggested. Barbiturates were reported to block the INa of axonal membranes in the uncharged molecular form which has high lipid solubility (NARAHASHIet al., 1971; HARPER et al., 1983). Halothane is a hydrophobic substance which may reach the blocking site of sodium channels from the lipid region of the membrane. FRANKSand LIEB (1978, 1982) reported that a high concentration of halothane (12 times the surgical concentration) had no effect on lipid bilayer structures and that octanol, possessing both polar and apolar characteristics, gave an excellent correlation for all anesthetic compounds in a potency vs. solvent/water partition coefficient plot. They suggested involvement of protein at the primary site of action of general anesthetics. These arguments indicate that halothane and thiamylal reach the sodium channel protein via the lipid phase of the membrane to shift the inactivation curve without producing a use- dependent inhibition of INa. In conclusion, halothane and thiamylal depress the INa of isolated rat ventricular cells mainly by shifting the steady state inactivation curve in a negative direction along the voltage axis. Recovery time from inactivation was prolonged by both drugs but a use-dependent depression was not observed up to 5 Hz. These effects might be explained in terms of adsorption of the anesthetics into the lipid region of cell membrane and consequent pertubation of the channel proteins.

We thank Ms. M. Ohara for reading the manuscript.

REFERENCES

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