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Prolongation of the action potential and reduction of the delayed outward K+ current by halothane in single frog atrial cells
European Journal of Pharmacology, 126 (1986) 293-295
293
Elsevier
Short communication
P R O L O N G A T I O N OF T H E ACTION P O T E N T I A L AND R E D U C T I O N O F T H E DELAYED O U T W A R D K + CURRENT BY H A L O T H A N E IN S I N G L E F R O G ATRIAL CELLS KOHKI HIROTA *, YASUNORI MOMOSE *, RYUJI TAKEDA *, SUEHIRO NAKANISHI * and YUSUKE ITO
Department of Anesthesiology, and * Department of Pharmacology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan Received 28 April 1986, accepted 20 May 1986
K. HIROTA, Y. MOMOSE, R. TAKEDA, S. NAKANISHI and Y. ITO, Prolongation of the action potential and reduction of the delayed outward K + current by halothane in single frog atrial cells, European J. Pharmacol. 126 (1986) 293-295. Halothane has been shown to produce significant changes in heart rate and strength of contraction. We have studied the mechanism(s) of these effects by recording action potentials and transmembrane ionic currents in single cardiac cells from bullfrog atrium. Our results show (i) that the action potential was prolonged and its plateau was depressed; and (ii) that there was a significant inhibition of a potassium current, It<, and a Ca2÷ current, Isi, following halothane (2%) application. Halothane
Single atrial cell
K + current
I. Introduction
2. Materials and methods
Halothane has previously been shown to depress the plateau phase of the action potential in myocardial tissues (Hauswirth, 1969; Lynch et al., 1981). This effect is likely to arise from inhibition of a Ca 2+ current or a K ÷ current since these generate the action potential plateau and initiate repolarization, respectively. Recently, Ikemoto et al. (1985) reported that halothane specifically inhibited the Ca 2+ current in rat ventricular cells. In the present study, using single atrial ceils from bullfrog, after halothane treatment we observed a marked prolongation of the action potential associated with depression of the plateau. Significant effects of halothane on transmembrane K ÷ currents of the cardiac cells have not been described in detail. Accordingly, we attempted to define the electrophysiological mechanism(s) of action of halothane on cardiac myocytes using a whole cell voltage clamp technique to measure the ionic currents underlying the action potential.
The right atrium of the bullfrog (Rana catesbeiana) was used. The enzymatic dispersion procedure for isolating single cells and the experimental setup for recording membrane potentials and transmembrane ionic currents were essentially the same as those described by Hume and Giles (1983). Frog Ringer solution, which was kept saturated with 95% 0 2 and 5% C O 2 (pH 7.4) and contained (mM): NaC1 90.6, KC1 2.5, CaC12 2.5, N a H C O 3 20.0 and glucose 10.0 was used at 2024°C. Halothane was vaporized in 95% O 2 and 5% CO 2 gas mixture and then bubbled into the Ringer solution. Halothane concentrations are presented as volume % in the gas mixture. Ca 2+ and K + currents were recorded in the presence of 3 /~M tetrodotoxin (TTX) so that the Na + current, INa , was blocked. Ca 2÷ currents were elicited by 200 ms depolarization voltage steps from a holding potential of - 9 0 mV. The time- and voltage-dependent K ÷ currents were obtained using 5 s clamp pulses to between - 2 0 and + 60 mV from the holding potential.
* To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
294 ---------
-90 . mV
. ~
I sec
Control
.
.
.
i
Fig. 1. Effect of halothane (2%) on the resting potential and the action potential recorded in single cells from bullfrog atrium. Stimuli (0.5 ms, 10 nA) were applied every 5 s. Note the depression of the plateau height and the lengthening of the action potential but absence of change in resting potential.
3. Results Exposure of single atrial cells to halothane at concentrations ranging from 0.5 to 4.0% significantly depressed the initial plateau height and lengthened the action potential (fig. 1). However, halothane had no effect on the resting membrane
2.l~t, Halolhane
A
r5 ~
Conlrol
B
-
Ii
2.0%Halothane
-
200ms
]100pA
Fig. 2. (A) Effect of halothane (2%) on the time- and voltagedependent K + current (IK) in bullfrog atrium; 5 s voltage clamp pulses from - 9 0 to + 10 and + 30 mV were used to activate I K. Halothane produced a significant decrease in I K at both of these potential levels. (B) Inhibition of the Ca 2÷ current, Isi, by halothane (2%). The control record (left panel) was elicited by a 200 ms depolarization from - 90 to 0 mV. An identical voltage clamp pulse after halothane treatment (right panel) elicited a substantial reduction in Isi.
potential. The effects on the height and duration of the action potential appeared to be dose-dependent, and reversible. The action potential duration measured at 90% repolarization increased to 160 + 7% (mean + S.D., n = 5) of that of the control after 2% halothane. In corresponding voltage clamp experiments, halothane significantly depressed the time- and voltage-dependent K + current, I K. After 2% halothane I K was reduced to 34 + 9% (mean + S.D., n = 5) of its control value as measured by 5 s 'isochronal' pulses to +10 and +30 mV from - 9 0 mV (fig. 2A). IK was completely but reversibly suppressed with 4% halothane. As shown in fig. 2B, halothane (2%) also decreased the peak Ca 2÷ current. However, it had no significant effect on either the activation threshold or the apparent reversal potential for the Ca 2÷ current (not shown).
4. Discussion In summary, halothane was found to significantly increase action potential duration in single cells from frog atrium. Voltage clamp experiments revealed that halothane decreased the time- and voltage-dependent K ÷ current which triggers repolarization of the action potential, and also suppressed the Ca 2÷ current, Isi , which generates the plateau. However, since the resting potential did not change, halothane at this level seems to have no effect on IKt, the background K + current. Thus, halothane at the 2% level seems to selectively affect one K ÷ current (IK) but not the other (IK1). Since the action potential upstroke was not changed significantly it also appears that halothane (2%) can inhibit Isi without blocking INa. It has been reported that halothane decreased the action potential duration in guinea-pig papillary muscle (Lynch et al., 1981), and that it slightly increased action potential duration in rabbit atrial cells (Hauswirth, 1969). Changes in the duration of action potential recorded at a fixed stimulation frequency suggest that halothane changes the K ÷ current(s) responsible for repolarization. Ikemoto et al. (1985) did not find any effect of halothane on K + current in rat ventricular cells. However, in the rat ventricle the most
295 p r o m i n e n t time- a n d v o l t a g e - d e p e n d e n t K + current ( J o s e p h s o n et al., 1984) is a transient c u r r e n t similar to the I A c u r r e n t first d e s c r i b e d in neurons. I n contrast, frog a t r i u m exhibits a n o n - i n a c t i v a t ing b u t time- a n d v o l t a g e - d e p e n d e n t K + c u r r e n t ( H u m e a n d Giles, 1983). T h u s these a p p a r e n t differences i n e x p e r i m e n t a l r e s u l t s ' following h a l o t h a n e t r e a t m e n t m a y arise f r o m the different n a t u r e of the ionic currents, especially K + currents, which c o n t r i b u t e to r e p o l a r i z a t i o n of a c t i o n p o t e n t i a l s in these two c a r d i a c tissues (Noble, 1979). O u r o b s e r v a t i o n that h a l o t h a n e d e p r e s s e d the C a 2÷ c u r r e n t s a n d decreased the a c t i o n p o t e n tial p l a t e a u is consistent with the results d e s c r i b e d b y L y n c h et al. (1981) a n d I k e m o t o et al. (1985). T h e p r e s e n t results, i.e. m a r k e d lengthening of the a c t i o n p o t e n t i a l d u e to i n h i b i t i o n of t r a n s m e m b r a n e K ÷ a n d C a 2÷ currents, m a y e x p l a i n the d e p r e s s e d c o n t r a c t i o n o r decreased h e a r t rate o b served in m a n a n d e x p e r i m e n t a l a n i m a l s following h a l o t h a n e inhalation.
Acknowledgement The authors are grateful to Dr. Wayne R. Giles for many helpful discussions and suggestions touching this manuscript.
References Hauswirth, O., 1969, Effects of halothane on single atrial, ventricular, and Purkinje fibers, Circ. Res. 24, 745. Hume, J.R. and W. Giles, 1983, Ionic currents in single isolated bullfrog atrial cells, J. Gen. Physiol. 81, 153. Ikemoto, Y., A. Yatani, H. Arimura and J. Yoshitake, 1985, Reduction of the slow inward current of isolated rat ventricular cells by thiamylal and halothane, Acta Anesthesiol. Scand. 29, 583. Josephson, I.R., J. Sanchez-Chapula and A.M. Brown, 1984, Early outward current in rat single ventricular cells, Circ. Res. 54, 157. Lynch, C., S. Vogel and N. Sperelakis, 1981, Halothane depression of myocardial slow action potentials, Anesthesiology 55, 360. Noble, D., 1979, The Initiation of the Heartbeat, 2nd edition (Clarendon Press, Oxford).
293
Elsevier
Short communication
P R O L O N G A T I O N OF T H E ACTION P O T E N T I A L AND R E D U C T I O N O F T H E DELAYED O U T W A R D K + CURRENT BY H A L O T H A N E IN S I N G L E F R O G ATRIAL CELLS KOHKI HIROTA *, YASUNORI MOMOSE *, RYUJI TAKEDA *, SUEHIRO NAKANISHI * and YUSUKE ITO
Department of Anesthesiology, and * Department of Pharmacology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan Received 28 April 1986, accepted 20 May 1986
K. HIROTA, Y. MOMOSE, R. TAKEDA, S. NAKANISHI and Y. ITO, Prolongation of the action potential and reduction of the delayed outward K + current by halothane in single frog atrial cells, European J. Pharmacol. 126 (1986) 293-295. Halothane has been shown to produce significant changes in heart rate and strength of contraction. We have studied the mechanism(s) of these effects by recording action potentials and transmembrane ionic currents in single cardiac cells from bullfrog atrium. Our results show (i) that the action potential was prolonged and its plateau was depressed; and (ii) that there was a significant inhibition of a potassium current, It<, and a Ca2÷ current, Isi, following halothane (2%) application. Halothane
Single atrial cell
K + current
I. Introduction
2. Materials and methods
Halothane has previously been shown to depress the plateau phase of the action potential in myocardial tissues (Hauswirth, 1969; Lynch et al., 1981). This effect is likely to arise from inhibition of a Ca 2+ current or a K ÷ current since these generate the action potential plateau and initiate repolarization, respectively. Recently, Ikemoto et al. (1985) reported that halothane specifically inhibited the Ca 2+ current in rat ventricular cells. In the present study, using single atrial ceils from bullfrog, after halothane treatment we observed a marked prolongation of the action potential associated with depression of the plateau. Significant effects of halothane on transmembrane K ÷ currents of the cardiac cells have not been described in detail. Accordingly, we attempted to define the electrophysiological mechanism(s) of action of halothane on cardiac myocytes using a whole cell voltage clamp technique to measure the ionic currents underlying the action potential.
The right atrium of the bullfrog (Rana catesbeiana) was used. The enzymatic dispersion procedure for isolating single cells and the experimental setup for recording membrane potentials and transmembrane ionic currents were essentially the same as those described by Hume and Giles (1983). Frog Ringer solution, which was kept saturated with 95% 0 2 and 5% C O 2 (pH 7.4) and contained (mM): NaC1 90.6, KC1 2.5, CaC12 2.5, N a H C O 3 20.0 and glucose 10.0 was used at 2024°C. Halothane was vaporized in 95% O 2 and 5% CO 2 gas mixture and then bubbled into the Ringer solution. Halothane concentrations are presented as volume % in the gas mixture. Ca 2+ and K + currents were recorded in the presence of 3 /~M tetrodotoxin (TTX) so that the Na + current, INa , was blocked. Ca 2÷ currents were elicited by 200 ms depolarization voltage steps from a holding potential of - 9 0 mV. The time- and voltage-dependent K ÷ currents were obtained using 5 s clamp pulses to between - 2 0 and + 60 mV from the holding potential.
* To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
294 ---------
-90 . mV
. ~
I sec
Control
.
.
.
i
Fig. 1. Effect of halothane (2%) on the resting potential and the action potential recorded in single cells from bullfrog atrium. Stimuli (0.5 ms, 10 nA) were applied every 5 s. Note the depression of the plateau height and the lengthening of the action potential but absence of change in resting potential.
3. Results Exposure of single atrial cells to halothane at concentrations ranging from 0.5 to 4.0% significantly depressed the initial plateau height and lengthened the action potential (fig. 1). However, halothane had no effect on the resting membrane
2.l~t, Halolhane
A
r5 ~
Conlrol
B
-
Ii
2.0%Halothane
-
200ms
]100pA
Fig. 2. (A) Effect of halothane (2%) on the time- and voltagedependent K + current (IK) in bullfrog atrium; 5 s voltage clamp pulses from - 9 0 to + 10 and + 30 mV were used to activate I K. Halothane produced a significant decrease in I K at both of these potential levels. (B) Inhibition of the Ca 2÷ current, Isi, by halothane (2%). The control record (left panel) was elicited by a 200 ms depolarization from - 90 to 0 mV. An identical voltage clamp pulse after halothane treatment (right panel) elicited a substantial reduction in Isi.
potential. The effects on the height and duration of the action potential appeared to be dose-dependent, and reversible. The action potential duration measured at 90% repolarization increased to 160 + 7% (mean + S.D., n = 5) of that of the control after 2% halothane. In corresponding voltage clamp experiments, halothane significantly depressed the time- and voltage-dependent K + current, I K. After 2% halothane I K was reduced to 34 + 9% (mean + S.D., n = 5) of its control value as measured by 5 s 'isochronal' pulses to +10 and +30 mV from - 9 0 mV (fig. 2A). IK was completely but reversibly suppressed with 4% halothane. As shown in fig. 2B, halothane (2%) also decreased the peak Ca 2÷ current. However, it had no significant effect on either the activation threshold or the apparent reversal potential for the Ca 2÷ current (not shown).
4. Discussion In summary, halothane was found to significantly increase action potential duration in single cells from frog atrium. Voltage clamp experiments revealed that halothane decreased the time- and voltage-dependent K ÷ current which triggers repolarization of the action potential, and also suppressed the Ca 2÷ current, Isi , which generates the plateau. However, since the resting potential did not change, halothane at this level seems to have no effect on IKt, the background K + current. Thus, halothane at the 2% level seems to selectively affect one K ÷ current (IK) but not the other (IK1). Since the action potential upstroke was not changed significantly it also appears that halothane (2%) can inhibit Isi without blocking INa. It has been reported that halothane decreased the action potential duration in guinea-pig papillary muscle (Lynch et al., 1981), and that it slightly increased action potential duration in rabbit atrial cells (Hauswirth, 1969). Changes in the duration of action potential recorded at a fixed stimulation frequency suggest that halothane changes the K ÷ current(s) responsible for repolarization. Ikemoto et al. (1985) did not find any effect of halothane on K + current in rat ventricular cells. However, in the rat ventricle the most
295 p r o m i n e n t time- a n d v o l t a g e - d e p e n d e n t K + current ( J o s e p h s o n et al., 1984) is a transient c u r r e n t similar to the I A c u r r e n t first d e s c r i b e d in neurons. I n contrast, frog a t r i u m exhibits a n o n - i n a c t i v a t ing b u t time- a n d v o l t a g e - d e p e n d e n t K + c u r r e n t ( H u m e a n d Giles, 1983). T h u s these a p p a r e n t differences i n e x p e r i m e n t a l r e s u l t s ' following h a l o t h a n e t r e a t m e n t m a y arise f r o m the different n a t u r e of the ionic currents, especially K + currents, which c o n t r i b u t e to r e p o l a r i z a t i o n of a c t i o n p o t e n t i a l s in these two c a r d i a c tissues (Noble, 1979). O u r o b s e r v a t i o n that h a l o t h a n e d e p r e s s e d the C a 2÷ c u r r e n t s a n d decreased the a c t i o n p o t e n tial p l a t e a u is consistent with the results d e s c r i b e d b y L y n c h et al. (1981) a n d I k e m o t o et al. (1985). T h e p r e s e n t results, i.e. m a r k e d lengthening of the a c t i o n p o t e n t i a l d u e to i n h i b i t i o n of t r a n s m e m b r a n e K ÷ a n d C a 2÷ currents, m a y e x p l a i n the d e p r e s s e d c o n t r a c t i o n o r decreased h e a r t rate o b served in m a n a n d e x p e r i m e n t a l a n i m a l s following h a l o t h a n e inhalation.
Acknowledgement The authors are grateful to Dr. Wayne R. Giles for many helpful discussions and suggestions touching this manuscript.
References Hauswirth, O., 1969, Effects of halothane on single atrial, ventricular, and Purkinje fibers, Circ. Res. 24, 745. Hume, J.R. and W. Giles, 1983, Ionic currents in single isolated bullfrog atrial cells, J. Gen. Physiol. 81, 153. Ikemoto, Y., A. Yatani, H. Arimura and J. Yoshitake, 1985, Reduction of the slow inward current of isolated rat ventricular cells by thiamylal and halothane, Acta Anesthesiol. Scand. 29, 583. Josephson, I.R., J. Sanchez-Chapula and A.M. Brown, 1984, Early outward current in rat single ventricular cells, Circ. Res. 54, 157. Lynch, C., S. Vogel and N. Sperelakis, 1981, Halothane depression of myocardial slow action potentials, Anesthesiology 55, 360. Noble, D., 1979, The Initiation of the Heartbeat, 2nd edition (Clarendon Press, Oxford).