To clarify the mechanism(s) of anesthetic depression of myocardial Ca2+ currents, the effects of isoflurane and sevoflurane on the inactivation kinetics of Ca2+ current in single bullfrog atrial myocytes were studied.
Freshly isolated bullfrog atrial myocytes were obtained with an enzymatic dispersion procedure. Ca2+ currents were recorded with a whole-cell voltage-clamp technique.
Both isoflurane (1.25, 2.5, 5.0 vol%) and sevoflurane (2.5, 5.0 vol%) decreased the peak amplitude of Ca2+ current ICa with a minimal change in the time to peak and the reversal potential. The inactivation kinetics studies revealed that (1) isoflurane (2.5 vol%) and sevoflurane (5.0 vol%) markedly reduced the time constant of inactivation in ICa to 55% and 75% of control, respectively; (2) isoflurane (2.5 vol%) shifted the midpoint (V1/2) of steady-state inactivation curve of ICa toward negative by 2.3 mV; and (3) isoflurane (2.5 vol%) delayed the reactivation time constant of ICa to 119% of control. The further computer-simulation study demonstrated that the observed decrease of time constant by isoflurane (1.25, 2.5 vol%) and sevoflurane (2.5 vol%) can explain the reduction in amplitude of ICa.
The depression of ICa by lower concentration of isoflurane (1.25, 2.5 vol%) and sevoflurane (2.5 vol%) mainly is due to the decrease of time constant and, at higher concentration, isoflurane and sevoflurane may affect the other membrane components.
Key words: Anesthetics, volatile: isoflurane; sevoflurane. Animal: bullfrog. Heart: calcium current; inactivation.
VOLATILE anesthetics have been reported to depress cardiac Calcium2+ current (ICa) in atrial [1,2]and ventricular myocytes. [3-6]According to the theory of the Hodgkin and Huxley model, the membrane conductance to Calcium2+ ions (GCa) is initially activated then inactivated during a depolarization. The activation is a rapid process that opens Calcium2+ channels and the inactivation is a slower process that closes Calcium2+ channels. Inactivated channels cannot be activated until their inactivation is removed. The inactivation process overrides the tendency of the activation process to open channels. Thus, the modulations of the inactivation process might be responsible for the anesthetic depression of ICain myocardium. Terrar and Victory reported that isoflurane speeded the decay of ICain guinea pig ventricular myocytes. Conversely, Bosnjak et al. showed that enflurane, but neither isoflurane nor halothane, enhanced ICadecay in canine ventricular myocytes. The controversy prompted us to investigate the effects of inhalational agents on the inactivation process of ICa.
In the current study, we studied the effects of isoflurane and sevoflurane on the inactivation kinetics of Calcium2+ current. Bullfrog atrial myocytes were used because they offer certain advantages to study the kinetics of ICadecay. Because the decay of ICais well fitted by a single exponential time course, it is easier to simulate ICawith the Hodgkin and Huxley model. The gradual decline of ICa(run down) is expected minimal in amphibian myocytes compared with mammalian preparations, as well.
Materials and Methods
All solutions were made with glass-distilled water and were kept saturated with 100% oxygen. The following solutions were used:
1. Standard Ringer's solution (mmol *symbol* l sup -1): NaCl 110.0, KCl 2.5, CaCl22.5, MgCl25.0, HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) 5.0, glucose 10.0 (pH was adjusted to 7.4 with 1N NaOH).
2. Potassium sup + -free Caesium sup + solution: NaCl 110.0, CsCl 20.0, CaCl22.5, MgCl25.0, HEPES 5.0, glucose 10.0 (pH was adjusted to 7.4 with 1N NaOH).
3. Caesium sup + internal solution: Caesium aspartate 120.0, EGTA (ethyleneglycol-bis-[beta-aminoethyl ether]-N,N,N',N'-tetraacetic acid) 0.5, HEPES 5.0 (pH was adjusted to 7.2 with 1N CsOH).
The calcium-free Ringer's solution was identical to the standard Ringer's solution except that CaCl2was omitted.
The current technique for isolation of atrial myocytes from bullfrog hearts is a modification of the method described previously by Hirota et al. After obtaining approval from the Animal Care Committee of Toyama Medical and Pharmaceutical University, adult bullfrogs (Rana Catesbeiana) were killed by decapitation under ether anesthesia and their hearts removed. The atrium was dissected and then transferred into a 20-ml solution of calcium-free Ringer's solution containing 0.15% crude bacterial collagenase (Type I; Sigma, St Louis, MO) and 0.1% bovine pancreatic trypsin (Type III; Sigma). The enzyme solution was passed through 0.22-micro meter micropore filter before use. The atrial myocytes were dispersed by gently triturating the tissue segment with a Pasteur pipette. The isolated atrial myocytes were stored in a calcium-free Ringer's solution containing 0.1% bovine albumin (Sigma).
Small aliquots of the cell suspension were placed in the recording chamber (volume 0.5 ml) mounted on the stage of an inverted microscope. After cells had settled at the bottom of the chamber, a Potassium sup + -free Caesium sup + solution was superfused at a rate of 1 ml *symbol* min sup -1.
A single suction micropipette technique was used for the whole-cell voltage clamp recordings. The voltage clamp experiments controls the membrane potential by holding it at chosen values and keeping it uniform within the cell so that ionic currents can be recorded. Electrodes were made from 1.0-mm ID, 1.5-mm OD borosilicate capillary tubing (World Precision Instruments, Sarasota, FL) and the electrode resistance was 1-5 M omega when filled with the Caesium sup + internal solution. A List-Medical Electronics (Darmstadt, Germany) EPC7 amplifier was used for voltage-clamp recordings and corrections for liquid junction potentials between the external and internal solutions. .
Membrane currents were monitored on a storage oscilloscope and were digitized simultaneously (1024 samples per record; 12-bit resolution) using a type DT2801A (Data Translation, Marlborough, MA) A/D converter board, and then stored on a microcomputer (IBM AT). Scientific graphing software (SigmaPlot; Jandel Scientific, San Rafael, CA) was used for analysis, curve fits and plots of the digitized data and computer simulations of ICa.
The methods for recording of the Calcium2+ currents and the analysis of the inactivation kinetics in the Calcium2+ currents basically are the same as those described by Hume and Giles and by Isenberg and Klockner. Calcium2+ currents were recorded in response to 200-ms depolarizing pulses from a holding potential of -40 mV with a Potassium sup + free Caesium sup + external solution and a Caesium sup + internal solution, a condition that induced a complete blockage of the Sodium sup + and Potassium sup + currents. The steady-state inactivation curve for ICawas obtained from double-pulse protocols. Every 10 s a 200-ms prepulse from -40 mV to different potentials was followed by a 200-ms test pulse from -40 mV to +20 mV. The amplitude of I sub Ca elicited by the test-pulse was plotted as percentage of the ICaevoked in the absence of a prepulse. Reactivation (recovery from inactivation) was analyzed by depolarizing the cell every 10 s from -40 mV to +20 mV, returning to -40 mV for 11 intervals from 0 ms to 200 ms.
All experiments were performed at room temperature (20-22 degrees C).
Administration of Isoflurane and Sevoflurane
Isoflurane and sevoflurane were vaporized with isoflurane (Muraco, Tokyo, Japan) and sevoflurane vaporizers (Ohmeda, West Yorkshire, UK), respectively, in 100% oxygen (1 liter *symbol* min sup -1) and then bubbled into the Potassium sup + -free Caesium sup + solution in a reservoir for at least 5 min before application to cells. Anesthetic concentrations are presented as volume percentage (vol%) in 100% oxygen. The concentration of isoflurane and sevoflurane in the gas phase were verified by an anesthetic gas analyzer (Capnomac; Datex, Finland) and the concentration in solution was analyzed by a gas chromatograph (Shimazu, Kyoto, Japan). The concentrations of isoflurane and sevoflurane in solution were found to be linear (0.64 and 0.55 mmol *symbol* 1 sup -1 per 1% respectively) with the percentage in gas phase, up to 5.0%.
When breaking the patch after a gigaseal was made with an electrode, there was a gradual decline or increase in ICathat could be related to "run down" or "run up" of current during the stabilizing period. After ICahad reached its steady state, control data were measured. Subsequently, cells were exposed to the anesthetic-equilibrated solution for at least 3 min, which was sufficient to produce stable effects, and the effects of anesthetics were examined. Recovery responses were recorded at least 15 min after washout of the anesthetic-containing solution from the chamber. Data were analyzed only if complete recovery (95-105% of control) occurred on washout of anesthetics.
Forty-six atrial myocytes from 18 bullfrogs were used for the current experiments. Values were expressed as mean+/-SEM, and comparisons were made by paired t test. A probability of chance occurrence of less than 5% (P < 0.05) was considered significant.
Effects of Isoflurane on the Current-Voltage Relationship of the Peak Amplitude of Calcium sup 2+ Current
(Figure 1) shows the representative recordings of ICaand the current-voltage curve in the presence and the absence of isoflurane (5.0 vol%) and sevoflurane (5.0 vol%), respectively. The currents elicited at different potentials for each cell under both conditions were normalized to the peak ICaat the maximum response in the absence of anesthetic (control). Calcium2+ currents were strongly depressed by isoflurane and sevoflurane. However, they had no significant effect on either the activation threshold or the apparent reversal potential for ICa. Effects of isoflurane (1.25-5.0 vol%) and sevoflurane (2.5-5.0 vol%) on characteristics of ICaare summarized in Table 1. Isoflurane and sevoflurane decreased the amplitude of Calcium2+ currents in a dose-dependent manner.
Effects of Isoflurane on Inactivation of Peak Amplitude of Calcium sup 2+ Current
(Figure 2) shows kinetics of the inactivation of ICain the presence and absence of isoflurane (2.5 vol%) and sevoflurane (2.5 vol%). Figure 2(A) shows the representative recordings of the effects of isoflurane on ICa. In Figure 2(B and C), time courses of inactivation of ICa(indicated by dashed squares in Figure 2(A)) were plotted in semilogarithmic charts. The time decays of currents were satisfactorily fitted by a single exponential equation of the form: Equation 1where t is the time after a peak of ICa, ICa,max is the maximum ICaamplitude, and taufis the time constant for inactivation time course of ICa. The calculated taufvalues from ICarecordings in Figure 2(B) were 52.3 ms in the absence of isoflurane. Isoflurane (2.5 vol%) markedly reduced the taufof ICato 25.2 ms. Sevoflurane (2.5 vol%) decreased the taufof ICafrom 41.8 ms to 34.2 ms (Figure 2(C)).
Effects of isoflurane (1.25-2.5 vol%) and sevoflurane (2.5-5.0 vol%) on taufin ICaare summarized in Table 1. Every concentration of isoflurane and sevoflurane significantly reduced tauf.
Effects of Isoflurane on the Steady-state Inactivation of the Peak Amplitude of Calcium sup 2+ Current
(Figure 3)(A) shows the effects of isoflurane (2.5 vol%) on the steady-state inactivation of ICa(n = 5). After a 200-ms conditioning prepulse between -40 and +20 mV (to inactivate ICa), remaining ICawere measured by a 200 ms test-pulse at +20 mV. The test pulse to +20 mV elicits ICawhose peak depends on the prepulse potential. I/Imaxis (peak ICaelicited by test-pulse)/(maximum ICa). When the prepulse was +20 mV (inactivation was maximal), peak ICaof the test pulse was minimal. When the prepulse went to -40 mV, inactivation was least pronounced and ICawas largest. The observed curves were fitted to a Boltzmann function of the form: Equation 2where Vmis prepulse potential, V1/2 is the mid-point potential, and k is the slope factor. The value of V1/2 in ICawas -0.3+/-0.9 mV in control state. Isoflurane (2.5 vol%) significantly shifted the V1/2 toward the negative to -2.5+/-0.1 mV without a change in the slope factor (k). Thus, the data indicate that isoflurane increases the probability of closing the inactivation gate of the Calcium2+ channel.
Effects of Isoflurane on Reactivation Curve of I sub Ca
(Figure 3)(B) shows a double-pulse experiment measuring the time course of reactivation (recovery from inactivation) of ICa. The prepulse to +20 mV was used to inactivate ICa. The test pulse separated by several intervals (0, 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 ms) determines what fraction has recovered in that time.
Peak amplitude of Calcium2+ current recovers with an approximately exponential time course of the form: Equation 3where t is the interval between prepulse and test-pulse, and tauris the time constant for the reactivation of ICa.
The taurof ICa(57.8+/-2.7 ms [n = 5]) was significantly prolonged by isoflurane (2.5 vol%) to 68.9+/-3.7 ms.
According to the modification of the Hodgkin and Huxley model by Bassingthwaighte and Reuter, ICais represented by: Equation 4where GCais the membrane conductance to Calcium2+, and d(t) and f(t), the parameters of activation and inactivation of ICa, respectively, are defined by: Equation 5, Equation 6where taudand taufare the time constant of activation and inactivation of I sub Ca, respectively.
(Equation 4) attributed ICainactivation to a purely voltage-dependent process, analogous to inactivation of the Sodium sup + current. Although the model is widely used, the idea of intracellular Calcium2+ -dependent inactivation of ICahas been suggested. Because the inactivation of ICadepends on the intracellular Calcium2+ concentration as well as the membrane potential, the Calcium2+ -dependent inactivation might be involved in the observed ICainactivation. In the current study, however, we used EGTA as the Calcium2+ chelator to prevent the Calcium2+ -dependent inactivation. Additionally, the Calcium sup 2+ transient due to the release of Calcium2+ from intracellular Calcium2+ stores could be excluded because of the lack of sarcoplasmic reticulum in bullfrog myocytes. This would be helpful to minimize the interference due to the Calcium2+ -dependent inactivation.
The results showed that isoflurane and sevoflurane reduced not only the amplitude of ICabut also tauf. However, the apparent reversal potential remained unchanged in the presence of isoflurane and sevoflurane. The changes of taudby both anesthetics presumably are negligible because isoflurane (1.25-2.5 vol%) and sevoflurane (2.5-5.0 vol%) had no significant effect on the time to peak.
Here, we hypothesized that isoflurane and sevoflurane (up to 2.5 vol%) have no effect on GCaand that the decrease of taufis mainly responsible for the ICadepression, and we then simulated I sub Ca using Equation 4. Figure 4shows the computer-simulated ICaconstructed with taufvalues from Figure 2(B). The configurations of ICain Figure 2were well simulated in Figure 4. The peak amplitudes of the simulated ICaafter isoflurane (2.5 vol%) and sevoflurane (2.5 vol%) calculated with taufvalues were 75.5 +/-8.6% (n = 5) and 91.7+/-4.3% (n = 8) of control, respectively. The results were not significantly different from the observed reduction by isoflurane (2.5 vol%) and sevoflurane (2.5 vol%) in amplitudes (71.1+/-5.2% and 89.9+/-3.9% of control; Table 1). These are consistent with our hypothesis that the observed decreases in ICaby isoflurane and sevoflurane are mainly caused by the depression of tauf. The model that the local anesthetic receptor of the Sodium sup + channel has additional states differing in their binding affinities and rate constants is called the modulated receptor model for local anesthetic action. There is the possibility of an additional inactivated state of the Calcium2+ channel with the application of isoflurane or sevoflurane.
The reduction in tauf, however, was not found to be linear with the reduction in amplitude at 5.0 vol% of isoflurane and sevoflurane (Figure 5). The observation suggests that a higher concentration of isoflurane and sevoflurane may affect the other components of the membrane currents, because a high concentration of isoflurane (5.0 vol%) significantly prolonged the time to peak (Table 1). It has been reported that a relatively high concentration of volatile anesthetics (4.0 vol% of sevoflurane and 2.0 vol% of halothane ) reduces the intracellular concentration of myocardial cyclic adenosine 3',5'-monophosphate. The myocardial cyclic adenosine 3',5'-monophosphate depression [23,24]may explain the reduction of ICacaused by the decrease of GCaafter the high concentration of volatile anesthetics application.
In summary, isoflurane and sevoflurane modified the inactivation process of ICain frog atrial myocytes. These results suggest that the volatile anesthetics enhance the probability of closing the inactivation gate of the Calcium2+ channel, and the volatile anesthetic depression of myocardial Calcium2+ current is mainly due to the acceleration of the inactivation in ICa.
The authors thank Dr. K. Toriizuka, for performing assays of isoflurane and sevoflurane bath concentration; Dinabot Co. Ltd., Osaka, Japan, for supplying isoflurane and a isoflurane vaporizer; and Maruishi Pharmaceutical Co., Osaka, Japan, for supplying sevoflurane and a sevoflurane vaporizer.