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Bunazosin, an alpha-adrenoceptor antagonist, blocks calcium current in guinea-pig ventricular myocytes
Camp. Eiochem. Physiol.Vol. IOOC,No. 3, pp. 40404, FVinteAin Great Britain
0306-4492/91 $3.00 + 0.00 0 1991 Pergamon Press plc
1991
BUNAZOSIN, AN ALPHA-ADRENOCEPTOR ANTAGONIST, BLOCKS CALCIUM CURRENT IN GUINEA-PIG VENTRICULAR MYOCYTES HIROSHI KOTAKE, NORIYASUNOGUCHI, SATOSHIMATSUOKA, ICHIRO HISATOME, JUNICHI HASEGAWA and HIROTO MASHIBA First Department of Internal Medicine, Tottori University, 36-1, Nishimachi 36-1, Yonago, 683, Japan (Telephone 0859-34-8101, Fax: 0859-34-8099) (Received 19 November 1990)
Abstract-l. Effect of bunazosin, an alpha,-adrenoceptor antagonist, upon the slow inward Ca*+ current (Z,) was studied in single ventricular myocytes of the guinea-pig using a whole-cell patch-clamp technique. 2. Bunazosin (10-100 PM) decreased Zc, in a concentration-dependent manner with an Q, of 60 PM during depolarization to + 10 mV from the holding potential of - 40 mV. 3. As for the inactivation process of ZQ, the steady-state inactivation (fco) curve was shifted toward more negative potentials from - 12 mV to - 17 mV and -21 mV at fan = 0.5, by 30 PM and 70 PM bunazosin. 4. Inhibition of Zti by the compound (10 PM) was also dependent on stimulation frequency, with greater block induced at 2 Hz (50%) than at 0.33 Hz (13%). 5. It is concluded that bunazosin possesses a direct CaZ+ channel-blocking (class 4) action in a rate-dependent fashion.
INTRODUCTION
METHODS
Alpha-adrenoceptor antagonists are found to possess antiarrhythmic properties besides their vasodilating
actions (Gould et al., 1975; Igarashi et al., 1977; Williams et al., 1978; Kawasaki et al., 1981). Although several investigators demonstrated the role of enhanced alpha-adrenergic responsiveness in arrhythmias during myocardial ischemia and reperfusion (Sheridan et al., 1980; Thandroyen ef al., 1983), direct membrane actions of alphaadrenoceptor antagonists are also being watched with interest. In vitro studies showed that an antiarrhythmic effect of these compounds is attributed to Na+ channel blocking action (Ledda and Marchetti, 1971; Rosen et al., 1971), namely a class I type antiarrhythmic property (Singh and Hauswirth, 1974; Vaughan Williams, 1984). Moreover, Dukes and Vaughan Williams (1984) and Kotake and colleagues (1988a,b) showed that alphaadrenoceptor antagonists produce a negative chronotropic effect in rabbit sino-atria1 node preparations. Considering these electrophysiological findings, alphaadrenoceptor antagonists are expected to exert inhibitory actions not only on Na+ channels but also on Ca*+ channels of the heart. However, the effect of these agents on myocardial Ca*+ channels has not been fully elucidated. In the present study, we examined the action of an alpha,- adrenoceptor antagonist, bunazosin (4amino-2-(4_butyrylhexahydro- 1H- 1,4-diazepin- 1-yl)6,7-dimethoxyquinazoline hydrochloride), upon the Ca’+ inward current (I& of single guinea-pig ventricular cells by means of a whole-cell patch-clamp method.
Single ventricular cells of guinea-pig heart were obtained with enzymatic dissociation technique similar to that previously described (Powell et al., 1980; Isenberg and Kliickner, 1982; Ehara er nl., 1989). In brief, guinea-pigs (200-350 g) were anaesthetized with sodium pentobarbital (20-50mg/kg). Under artificial respiration, the chest was opened and the aorta was cannulated in situ. The heart was excised maintaining the LangendorlT perfusion with control Tyrode solution and then perfused with Ca2+-free Tyrode solution until the heart beat stopped. The perfusate was switched to a Ca2+ Tyrode so lution containing collagenase (Sigma type I, 0.4mg/ml) and trypsin inhibitor (Sigma, 0.4 mg/ml) for about 30 min at 37°C. After this period, the heart was perfused with “KB medium” (Isenberg and Klackner, 1982). The ventricle was then further dissected in a dish filled with KB medium. The dispersed ventricular cells were kept in KB medium before use. Membrane currents of single ventricular cells were recorded using the whole-cell clamp technique, which was essentially the same as that described by Hamill et al. (1988). The membrane current and voltage-were recorded with video recorder (Mitsubishi. HV-F73) through a PCM data recorder @ho& EM, PCM-DP16) for latercomputer analysis (NEC, PC98XL). The control Tyrode solution contained (in mM): NaCl 140; KC1 5.4; MgCl, 0.5; CaCl, 1.8; NaH,PO, 0.33; glucose 5.5; HEPES 5.0 (pH = 7.4 with NaOH). The composition of internal solutiins is; KOH 110; asp&tate 110; KC1 20; EGTA 5.0: K,ATP 5.0: Na,creatineuhosnhate 5.0: M&l, 5.0; CaCl; 3.;; HEPEs 5.5 (pH = j.4 \;ith KOI$. -?hi free Ca2+ (146 nM) and Mg2+ (0.5 mM) concentrations were calculated by using Fabiato and Fabiato’s equations (1979) with the correction by Tsien and Rink (1980). All experiments were performed at 36-37°C. Drugs used were bunazosin (Eisai Co.) and tetrodotoxin (Sankyo Co.).
401
402
H.
KOTAKE et al.
RESULTS
Time course of changes in I,
by bunazosin
To investigate the effect of bunazosin on the calcium current (I,,), voltage-clamp experiments were performed using the whole-cell clamp method. The cell membrane potential was clamped at -40 mV and transiently depolarized to + 10 mV (300msec in duration) at 0.33 Hz. At potentials positive to -40mV the fast inward Na+ current can be inactivated and the inward current induced by depolarization is considered to be Zc, (Pappano, 1970). Figure 1 shows the time course of the changes in Zca amplitude with and without bunazosin (100 PM). The compound rapidly decreased Zc,, and a 3-min administration of the drug induced an obvious reduction of Zc, from 1.00 nA to 0.23 nA. Subsequent washout of the drug was followed by slow recovery but not to the control level. After returning to the control Tyrode solution, the amplitude of Zc, recovered to 78% of the control value (N = 3).
0
I) 0
,
,
wa&ut , , ,
,
2
4
6
,
, 8
,
,
,
10 min
Fig. 1. Effect of bunazosin on the time course of changes in The amplitude of Z, was decided as the difference between the peak inward current and the current at 150 msec after the onset of the clamp. I, was plotted every 15 set in the absence (open circle) and the presence (closed
I,,.
circle) of 1OOpM bunazosin. (a)-(c) Show the practical membrane current recordings. Buna: bunazosin. Frequency-dependent
block of I,
by bunazosin
Previous voltage clamp experiments (McDonald Tsien, 1983) showed that the inhibitory effect of Ca2+ antagonistic agents like verapamil and diltiazem upon Zc, is accentuated by repetitive membrane depolarization (frequencydependent block of I,,). We examined whether a similar effect accounts for some of the I,, depression observed in the present study. In Fig. 5, the depolarizing train pulses were applied at 0.33, 1, and 2 Hz in the absence and the presence of 10 PM bunazosin. Zc, inhibition by the compound was obviously dependent on stimulation frequency. In three experiments of this kind, ZG inhibition was 13 and 50% at
et al., 1980; Lee and Voltage- and concentration-dependent bunazosin on I,
effects
of
Figure 2 shows the voltage-dependent decrease in I,-, by bunazosin. The depolarizing test pulses of 300 msec in duration were applied from - 40 mV with 10 mV steps. Zc, was obviously activated at - 20 mV, reaching peak amplitude at + 10 mV, and then reduced at more positive membrane potentials. When the I,, amplitude was measured as the difference between the peak inward current and the current amplitude measured at 150 msec after the onset of the test clamp, the current-voltage relationship for Zc, was obtained. This graph indicates that bunazosin clearly decreased Zc, concentration-dependently without a significant voltage shift. Figure 3 shows a representative dose-response curve of bunazosin on Zc, measured at + 10 mV. On this voltage, an lcsO of bunazosin for Zc, is 60 PM. Effect of bunazosin on the steady-state inactivation curve of I,
The effect of bunazosin on the inactivation curve of Zc, was examined. The protocol involved conditioning clamp from the holding potential of -40 mV to various values ( - 60 to 0 mV), and then the clamp pulse was applied from the various levels of the conditioning clamp to +lOmV in 50pM tetrodotoxin containing Tyrode solution. The entire pulse protocol was repeated every 10 sec. Figure 4A shows the amplitudes of Zc, plotted against the prepulse potentials. Bunazosin (30-70 FM) clearly decreased Zc, at every membrane potential. In Fig. 4B, the current values elicited at each prepulse potential were normalized to the maximal current value and plotted as a function of prepulse potentials. Thus, the sigmoidal curve obtained represented a steady-state inactivation curve of I,-, (f co curve). After superfusion with the compound, this curve was shifted towards more negative potentials from -12mV to -17mV and to -2lmV atfoo =0.5 by 30 p M and 70 PM bunazosin.
Fig. 2. Effect of bunazosin on the current-voltage relationship for 6. The upper panel shows the membrane current traces at + 10, + 20, + 30, and + 40 mV. The lower shows the Z-V relationship before (0) and after exposure to 30pM (A) and 100pM (0) bunazosin.
403
Ca channel block of bunazosin
OJ , 1
10 1OO)lM Concantration Fig. 3. Effect of increasing concentrations of bunazosin on Za. Preparations were clamped to + 10 mV from the holding potential of - 40 mV at 0.33 Hz.
0.33 and 2 Hz, respectively, after exposure to 10 PM bunazosin. DISCUSSION
Alpha-adrenoceptor antagonistic agents are found to exert an antiarrhythmic property by Na+ channel blocking action which results in reduced upstroke velocity of the action potential (Ledda and Marchetti, 1971; Rosen et al., 1971; Williams et al., 1978; Gluch et al., 1986). Moreover, these compounds produced a negative chronotropic effect in rabbit sino-atria1 node preparations (Dukes and Vaughan Williams, 1984; Kotake el al., 1988a,b). Since the depolarization of dominant pacemaker cells in the sinus node is almost entirely dependent on the Ca*+ conductance system (Irisawa, 1978), these electrophysiological findings may suggest that alpha-adrenoceptor antagonists inhibit not only Na+ channels but also Ca*+ channels of the heart. In the present study, we have clearly shown that alpha,-adrenoceptor antagonist bunazosin decreased the slow inward Ca*+ current (I,,) of single guineapig ventricular myocytes in a concentration-dependent manner (rcso = 60 PM) by means of whole-cell patch-clamp technique. As for the bunazosin-induced inhibition of I,,, the compound altered its inactivation process. Bunazosin shifted the steady-state inactivation (foe) curve to more negative potentials dose-dependently. Another interesting observation of the present study was the strong influence exerted by the stimuA
B 1.0.
\>,\ -7 A
Z0.5.
5lmam*b
I’
0 olu * 30pY u 7oJn
\ i,
OLC -6omv
,
* -40
,
\ ;$
,
-20
0
Fig. 4. Effect of bunaxosin on the steady-state inactivation of I,,. (A) Z, amplitudes during second pulse were plotted as a function of membrane potential in the absence (0) and the presence of 30 PM (A) and 70 PM (0) bunaxosin. An inset shows the double-pulse protocol. (B) Steady-state inactivation curve for Zti with and without bunazosin.
01, , , , 1
(
,
*
5 Pulse
,
,
( 10th
number
Fig. 5. Frequency-dependent block of Z, by bunazosin. Depolarizing test pulses to + 10 mV from -40 mV were applied at 0.33 (O), 1 (A), and 2 Hz (a) in the presence of 10 PM bunaxosin. Before each protocol, the test pulses were discontinued for 2 min. An inset shows membrane current recordings during 1, 4, 7, and 10th test pulse at 1 Hz after exposure to bunazosin.
lation frequency on the Zc, block after exposure to bunazosin. Ca*+ antagonistic (class 4 antiarrhythmic) agents also show similar findings, namely, the frequency-dependent inhibition of Zca(McDonald et al., 1980; Trautwein et al., 1981; Lee and Tsien, 1983; Tung and Morad, 1983). Since antiarrhythmic agents are used for the treatment of tachyarrhythmias, such a property seems to be quite important for the antiarrhythmic effect of bunazosin. As for the genesis of cardiac arrhythmias during myocardial ischemia and reperfusion, enhanced alpha-adrenergic involvement has been demonstrated (Sheridan et al., 1980; Thandroyen et al., 1983). Alpha-adrenergic involvement in arrhythmia has also been found even in non-ischemic conditions (Lechat and Schmitt, 1982; Kimura et al., 1984; Thomas and Tripathi, 1986). Alpha,-adrenergic antagonists AR-C 239 and prazosin eliminated the arrhythmogenic effect of ouabain, although an alphaadrenoceptor agonist phenylephrine potentiated arrhythmogenic actions. Anyhow, these observations suggest that alpha-adrenergic stimulation potentiates a certain cardiac arrhythmias and, therefore, alphaadrenoceptor antagonists are expected to exert an antiarrhythmic property. However, recent studies showed that alpha-adrenoceptor antagonists have additional direct membrane actions, namely, class I and probably Class IV properties (Ledda and Marchetti, 1971; Rosen et al., 1971; Dukes and Vaughan Williams, 1984; Kotake et al., 1988a,b). Our experiments have clearly shown that an alphaadrenoceptor antagonist bunazosin inhibits the calcium current (a class IV property) concentrationand frequency-dependently with a shift of the steadystate inactivation curve of Zca to be more negative potentials. These electrophysiological observations suggest that bunazosin has antiarrhythmic properties not only through an alpha-adrenoceptor antagonistic action but also its direct membrane action, at least, independently of blockade of cardiac alphaadrenoceptors. Acknowledgements-The present study was supported by Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science and Culture, Japan.
404
H.
KOTAM E
REFERENCES
et al, effects of ouabain in guinea-pigs. Eur. J. Pharmac. 78, 21-32.
Dukes I. D. and Vaughan Williams E. M. (1984) Electrophysiological effects of alpha-adrenoceptor antagonists in rabbit sine-atrial node, cardiac Purkinje cells and papillary muscles. 31. J. Pharmac. 83, 419-426. Ehara T., Matsuoka S. and Noma A. (1989) Measurement of reversal potential of Na+Ca’+ exchange current in single guinea-pig ventricular cells. J. Physiol., Land. 410, 221-249.
Fabiato A. and Fabiato F. (1979) Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skin&d muscle cells. J.PhysioZ., Z.ond. 75,~4633505. Gluch R. W.. Mohrmann J. and Schultheiss S. 11986) Influence of the alpha-adrenoceptor antagonist urapidil on transmembrane action potentials of mammalian myocardium. Armeimittel Forsch. 36, 14%1480. Gould L., Reddy C. V. R., Weinsterin T. and Gomprecht R. F. (1975) Antiarrhythmic prophylaxis with phentolamine in acute myocardial infarction. J. &in. Pharmae. 15, 191-197. Hamill 0. P., Marty A., Neber E., Sakmann B. and Sigworth F. J. (1988) Improved patch-clamp techniques for high-resolution current recording from cells and cellfree membrane patches. Pptgers Arch. ges. Physiol. 391, 85-100. Igarashi T., Nakajima Y. and Ohtake S. (1977) Antihypertensive effect of combined treatment with alpha- and beta-adrenergic blockers in the spontaneously hypertensive rat. Jap. Circ. J. 41, 903-911. Irisawa H. (1978) Comparative physiology of the cardiac pacemaker mechanism. Physiol. Rev. 58, 461480. Isenberg G. and Klijckner U. (1982) Calcium tolerant ventricular myocytes prepared by preincubation in a “KB medium”. ppiigers Arch. ges. Physiol. 395, 6-18. Kawasaki T., Uexono K., Abe I., Nakamura S., Ueno M., Kawazoe N. and Qrnae T. (1981) Antihypertensive effect of E-643, a new alpha-adrenergic blocking agent. Eur. J. Pharmac. 20, 399-405.
Kimura S., Cameron J. S., Kozlovski P. L., Bassett A. L. and Myerburg R. J. (1984) Delayed after depolarizations and triggered activity induced in feline Purkinje fibers by alpha-adrenergic stimulation in the presence of elevated calcium levels. Circulation 70, 1074-1082. Kotake H., Hirai S., Watanabe M., Miyamoto J., Takami T., Hasegawa J. and Mashiba H. (1988a) Effect of the alpha-adrenoceptor antagonist urapidil on the pacemaker activity of rabbit sino-atria1 node preparations. Arch. Znt. Phurmacodyn. Z’her. 294, 149-158.
Kotake H., Saitoh M., Ogino K., Kurata Y., Ohtahara A., Hasegawa J. and Mashiba H. (1988b) Inhibition of Ca2+ inward current in rabbit sinoatrial node by bunazosin, and alpha-adrenoceptor antagonist. Eur. J. Pharmac. 136, 113-117. Lechat P. and Schmitt H. (1982) Interactions between the autonomic nervous system and the cardiovascular
Ledda F. and Marchetti P. (1971) Effects of phentolamine on transmembrane action potentials of sinale Purkinie fibers of sheep heart. Pharmac. Res. Commun~3,155_16b. Lee K. S. and Tsien R. W. (19831 Mechanism of calcium channel blockade by verapamil, D600, diltiaxem and nitrendipine in single dialyzed heart cells. Nature 303, 790-194. McDonald T. F., Pelzer D. and Trautwein W. (1980) On the mechanism of the slow calcium channel block in heart. PjIcgers Arch. ges. Physiof. 385, 171-1’79. Pappano A. J. (1970) Calci~~~ndent action potentials produced by catecholamines in guinea-pig atria1 muscle fibers depolarized by potassium. Circulation Z&s. 27, 379-390.
Powell T., Terrar D. A. and Twist V. W. (1980) Electrical properties of individual cells isolated from adult rat ventricular myocardium. J. Physiol., Land. 302, 131-153. Rosen M. R., Gelband H. and Hoffman B. F. (1971) Effects of phentolamine on el~trophysiolo~~ properties of isolated canine Purkinje fibers. .I. Pharmac. exp. Ther. 179, 586-593.
Sheridan D. J., Penkoske P. A., Sobel B. E. and Corr P. B. (1980) Alpha-adrenergic contributions to dysrhythmia during myocardial ischemia and reperfusion in cats. J. c&n. Znuest. 65, 161-171. Singh B. N. and Hauswirth 0. (1974) Com~rative mechanism of action of antia~hyt~c drugs. Am. Heart J. 87, 367-382.
Thandroyen F. T., Worthington M. G., Higgiuson L. M. and Opie L. H. (1983) The effect of alpha- and betaadrenoceptor antagonistic agents on reperfusion ventricular fibrillation and metabolic status in the isolated perfused rat heart. .I. Am. Call. Card&I. 1, 1056-1066. Thomas G. P. and Trioathi R. M. 11986) Effects of alnhaadrenoceptor agonists and antagonists on ouabait&duced arrhythmias and cardiac arrest in guinea-pig. Br. J. Pharmac. 98, 385-388.
Trautwein W., Pelzer D., McDonald T. F. and Osterrieder W. (1981) AQA 39, a new bradycardiac agent which blocks myocardial calcium (Ca) channels in a frequencyand voltagede~ndent manner. Noun-~e~iedeberg~s Arch. Pharmac. 317,228-232.
_
Tsien R. Y. and Rink T. J. (1980) Neutral carrier ionselective microelectrodes for measurement of intracellular free calcium. Biochim. biophys. Acta 599, 623-638. Tung L. and Morad M. (1983) Voltage- and frequencydependent block of diltiazem on the slow inward current and generation of tension in frog ventricular muscle. P’zZgers Arch. ges. Physiol. 398, 189-198, Vaughan Williams E. M. (1984) El~trophysiolo~~al basis for a rational approach to antidysrhythmic drug therapy. Adv. Drug Res. 9, 69-101. Williams B. J., Griffith W. H. and Albrecht C. M. (1978) The projective effect of phentolamine against cardiac arrrhythmias in the rat. Eur. J. Pharmac. 49, 7-15.
0306-4492/91 $3.00 + 0.00 0 1991 Pergamon Press plc
1991
BUNAZOSIN, AN ALPHA-ADRENOCEPTOR ANTAGONIST, BLOCKS CALCIUM CURRENT IN GUINEA-PIG VENTRICULAR MYOCYTES HIROSHI KOTAKE, NORIYASUNOGUCHI, SATOSHIMATSUOKA, ICHIRO HISATOME, JUNICHI HASEGAWA and HIROTO MASHIBA First Department of Internal Medicine, Tottori University, 36-1, Nishimachi 36-1, Yonago, 683, Japan (Telephone 0859-34-8101, Fax: 0859-34-8099) (Received 19 November 1990)
Abstract-l. Effect of bunazosin, an alpha,-adrenoceptor antagonist, upon the slow inward Ca*+ current (Z,) was studied in single ventricular myocytes of the guinea-pig using a whole-cell patch-clamp technique. 2. Bunazosin (10-100 PM) decreased Zc, in a concentration-dependent manner with an Q, of 60 PM during depolarization to + 10 mV from the holding potential of - 40 mV. 3. As for the inactivation process of ZQ, the steady-state inactivation (fco) curve was shifted toward more negative potentials from - 12 mV to - 17 mV and -21 mV at fan = 0.5, by 30 PM and 70 PM bunazosin. 4. Inhibition of Zti by the compound (10 PM) was also dependent on stimulation frequency, with greater block induced at 2 Hz (50%) than at 0.33 Hz (13%). 5. It is concluded that bunazosin possesses a direct CaZ+ channel-blocking (class 4) action in a rate-dependent fashion.
INTRODUCTION
METHODS
Alpha-adrenoceptor antagonists are found to possess antiarrhythmic properties besides their vasodilating
actions (Gould et al., 1975; Igarashi et al., 1977; Williams et al., 1978; Kawasaki et al., 1981). Although several investigators demonstrated the role of enhanced alpha-adrenergic responsiveness in arrhythmias during myocardial ischemia and reperfusion (Sheridan et al., 1980; Thandroyen ef al., 1983), direct membrane actions of alphaadrenoceptor antagonists are also being watched with interest. In vitro studies showed that an antiarrhythmic effect of these compounds is attributed to Na+ channel blocking action (Ledda and Marchetti, 1971; Rosen et al., 1971), namely a class I type antiarrhythmic property (Singh and Hauswirth, 1974; Vaughan Williams, 1984). Moreover, Dukes and Vaughan Williams (1984) and Kotake and colleagues (1988a,b) showed that alphaadrenoceptor antagonists produce a negative chronotropic effect in rabbit sino-atria1 node preparations. Considering these electrophysiological findings, alphaadrenoceptor antagonists are expected to exert inhibitory actions not only on Na+ channels but also on Ca*+ channels of the heart. However, the effect of these agents on myocardial Ca*+ channels has not been fully elucidated. In the present study, we examined the action of an alpha,- adrenoceptor antagonist, bunazosin (4amino-2-(4_butyrylhexahydro- 1H- 1,4-diazepin- 1-yl)6,7-dimethoxyquinazoline hydrochloride), upon the Ca’+ inward current (I& of single guinea-pig ventricular cells by means of a whole-cell patch-clamp method.
Single ventricular cells of guinea-pig heart were obtained with enzymatic dissociation technique similar to that previously described (Powell et al., 1980; Isenberg and Kliickner, 1982; Ehara er nl., 1989). In brief, guinea-pigs (200-350 g) were anaesthetized with sodium pentobarbital (20-50mg/kg). Under artificial respiration, the chest was opened and the aorta was cannulated in situ. The heart was excised maintaining the LangendorlT perfusion with control Tyrode solution and then perfused with Ca2+-free Tyrode solution until the heart beat stopped. The perfusate was switched to a Ca2+ Tyrode so lution containing collagenase (Sigma type I, 0.4mg/ml) and trypsin inhibitor (Sigma, 0.4 mg/ml) for about 30 min at 37°C. After this period, the heart was perfused with “KB medium” (Isenberg and Klackner, 1982). The ventricle was then further dissected in a dish filled with KB medium. The dispersed ventricular cells were kept in KB medium before use. Membrane currents of single ventricular cells were recorded using the whole-cell clamp technique, which was essentially the same as that described by Hamill et al. (1988). The membrane current and voltage-were recorded with video recorder (Mitsubishi. HV-F73) through a PCM data recorder @ho& EM, PCM-DP16) for latercomputer analysis (NEC, PC98XL). The control Tyrode solution contained (in mM): NaCl 140; KC1 5.4; MgCl, 0.5; CaCl, 1.8; NaH,PO, 0.33; glucose 5.5; HEPES 5.0 (pH = 7.4 with NaOH). The composition of internal solutiins is; KOH 110; asp&tate 110; KC1 20; EGTA 5.0: K,ATP 5.0: Na,creatineuhosnhate 5.0: M&l, 5.0; CaCl; 3.;; HEPEs 5.5 (pH = j.4 \;ith KOI$. -?hi free Ca2+ (146 nM) and Mg2+ (0.5 mM) concentrations were calculated by using Fabiato and Fabiato’s equations (1979) with the correction by Tsien and Rink (1980). All experiments were performed at 36-37°C. Drugs used were bunazosin (Eisai Co.) and tetrodotoxin (Sankyo Co.).
401
402
H.
KOTAKE et al.
RESULTS
Time course of changes in I,
by bunazosin
To investigate the effect of bunazosin on the calcium current (I,,), voltage-clamp experiments were performed using the whole-cell clamp method. The cell membrane potential was clamped at -40 mV and transiently depolarized to + 10 mV (300msec in duration) at 0.33 Hz. At potentials positive to -40mV the fast inward Na+ current can be inactivated and the inward current induced by depolarization is considered to be Zc, (Pappano, 1970). Figure 1 shows the time course of the changes in Zca amplitude with and without bunazosin (100 PM). The compound rapidly decreased Zc,, and a 3-min administration of the drug induced an obvious reduction of Zc, from 1.00 nA to 0.23 nA. Subsequent washout of the drug was followed by slow recovery but not to the control level. After returning to the control Tyrode solution, the amplitude of Zc, recovered to 78% of the control value (N = 3).
0
I) 0
,
,
wa&ut , , ,
,
2
4
6
,
, 8
,
,
,
10 min
Fig. 1. Effect of bunazosin on the time course of changes in The amplitude of Z, was decided as the difference between the peak inward current and the current at 150 msec after the onset of the clamp. I, was plotted every 15 set in the absence (open circle) and the presence (closed
I,,.
circle) of 1OOpM bunazosin. (a)-(c) Show the practical membrane current recordings. Buna: bunazosin. Frequency-dependent
block of I,
by bunazosin
Previous voltage clamp experiments (McDonald Tsien, 1983) showed that the inhibitory effect of Ca2+ antagonistic agents like verapamil and diltiazem upon Zc, is accentuated by repetitive membrane depolarization (frequencydependent block of I,,). We examined whether a similar effect accounts for some of the I,, depression observed in the present study. In Fig. 5, the depolarizing train pulses were applied at 0.33, 1, and 2 Hz in the absence and the presence of 10 PM bunazosin. Zc, inhibition by the compound was obviously dependent on stimulation frequency. In three experiments of this kind, ZG inhibition was 13 and 50% at
et al., 1980; Lee and Voltage- and concentration-dependent bunazosin on I,
effects
of
Figure 2 shows the voltage-dependent decrease in I,-, by bunazosin. The depolarizing test pulses of 300 msec in duration were applied from - 40 mV with 10 mV steps. Zc, was obviously activated at - 20 mV, reaching peak amplitude at + 10 mV, and then reduced at more positive membrane potentials. When the I,, amplitude was measured as the difference between the peak inward current and the current amplitude measured at 150 msec after the onset of the test clamp, the current-voltage relationship for Zc, was obtained. This graph indicates that bunazosin clearly decreased Zc, concentration-dependently without a significant voltage shift. Figure 3 shows a representative dose-response curve of bunazosin on Zc, measured at + 10 mV. On this voltage, an lcsO of bunazosin for Zc, is 60 PM. Effect of bunazosin on the steady-state inactivation curve of I,
The effect of bunazosin on the inactivation curve of Zc, was examined. The protocol involved conditioning clamp from the holding potential of -40 mV to various values ( - 60 to 0 mV), and then the clamp pulse was applied from the various levels of the conditioning clamp to +lOmV in 50pM tetrodotoxin containing Tyrode solution. The entire pulse protocol was repeated every 10 sec. Figure 4A shows the amplitudes of Zc, plotted against the prepulse potentials. Bunazosin (30-70 FM) clearly decreased Zc, at every membrane potential. In Fig. 4B, the current values elicited at each prepulse potential were normalized to the maximal current value and plotted as a function of prepulse potentials. Thus, the sigmoidal curve obtained represented a steady-state inactivation curve of I,-, (f co curve). After superfusion with the compound, this curve was shifted towards more negative potentials from -12mV to -17mV and to -2lmV atfoo =0.5 by 30 p M and 70 PM bunazosin.
Fig. 2. Effect of bunazosin on the current-voltage relationship for 6. The upper panel shows the membrane current traces at + 10, + 20, + 30, and + 40 mV. The lower shows the Z-V relationship before (0) and after exposure to 30pM (A) and 100pM (0) bunazosin.
403
Ca channel block of bunazosin
OJ , 1
10 1OO)lM Concantration Fig. 3. Effect of increasing concentrations of bunazosin on Za. Preparations were clamped to + 10 mV from the holding potential of - 40 mV at 0.33 Hz.
0.33 and 2 Hz, respectively, after exposure to 10 PM bunazosin. DISCUSSION
Alpha-adrenoceptor antagonistic agents are found to exert an antiarrhythmic property by Na+ channel blocking action which results in reduced upstroke velocity of the action potential (Ledda and Marchetti, 1971; Rosen et al., 1971; Williams et al., 1978; Gluch et al., 1986). Moreover, these compounds produced a negative chronotropic effect in rabbit sino-atria1 node preparations (Dukes and Vaughan Williams, 1984; Kotake el al., 1988a,b). Since the depolarization of dominant pacemaker cells in the sinus node is almost entirely dependent on the Ca*+ conductance system (Irisawa, 1978), these electrophysiological findings may suggest that alpha-adrenoceptor antagonists inhibit not only Na+ channels but also Ca*+ channels of the heart. In the present study, we have clearly shown that alpha,-adrenoceptor antagonist bunazosin decreased the slow inward Ca*+ current (I,,) of single guineapig ventricular myocytes in a concentration-dependent manner (rcso = 60 PM) by means of whole-cell patch-clamp technique. As for the bunazosin-induced inhibition of I,,, the compound altered its inactivation process. Bunazosin shifted the steady-state inactivation (foe) curve to more negative potentials dose-dependently. Another interesting observation of the present study was the strong influence exerted by the stimuA
B 1.0.
\>,\ -7 A
Z0.5.
5lmam*b
I’
0 olu * 30pY u 7oJn
\ i,
OLC -6omv
,
* -40
,
\ ;$
,
-20
0
Fig. 4. Effect of bunaxosin on the steady-state inactivation of I,,. (A) Z, amplitudes during second pulse were plotted as a function of membrane potential in the absence (0) and the presence of 30 PM (A) and 70 PM (0) bunaxosin. An inset shows the double-pulse protocol. (B) Steady-state inactivation curve for Zti with and without bunazosin.
01, , , , 1
(
,
*
5 Pulse
,
,
( 10th
number
Fig. 5. Frequency-dependent block of Z, by bunazosin. Depolarizing test pulses to + 10 mV from -40 mV were applied at 0.33 (O), 1 (A), and 2 Hz (a) in the presence of 10 PM bunaxosin. Before each protocol, the test pulses were discontinued for 2 min. An inset shows membrane current recordings during 1, 4, 7, and 10th test pulse at 1 Hz after exposure to bunazosin.
lation frequency on the Zc, block after exposure to bunazosin. Ca*+ antagonistic (class 4 antiarrhythmic) agents also show similar findings, namely, the frequency-dependent inhibition of Zca(McDonald et al., 1980; Trautwein et al., 1981; Lee and Tsien, 1983; Tung and Morad, 1983). Since antiarrhythmic agents are used for the treatment of tachyarrhythmias, such a property seems to be quite important for the antiarrhythmic effect of bunazosin. As for the genesis of cardiac arrhythmias during myocardial ischemia and reperfusion, enhanced alpha-adrenergic involvement has been demonstrated (Sheridan et al., 1980; Thandroyen et al., 1983). Alpha-adrenergic involvement in arrhythmia has also been found even in non-ischemic conditions (Lechat and Schmitt, 1982; Kimura et al., 1984; Thomas and Tripathi, 1986). Alpha,-adrenergic antagonists AR-C 239 and prazosin eliminated the arrhythmogenic effect of ouabain, although an alphaadrenoceptor agonist phenylephrine potentiated arrhythmogenic actions. Anyhow, these observations suggest that alpha-adrenergic stimulation potentiates a certain cardiac arrhythmias and, therefore, alphaadrenoceptor antagonists are expected to exert an antiarrhythmic property. However, recent studies showed that alpha-adrenoceptor antagonists have additional direct membrane actions, namely, class I and probably Class IV properties (Ledda and Marchetti, 1971; Rosen et al., 1971; Dukes and Vaughan Williams, 1984; Kotake et al., 1988a,b). Our experiments have clearly shown that an alphaadrenoceptor antagonist bunazosin inhibits the calcium current (a class IV property) concentrationand frequency-dependently with a shift of the steadystate inactivation curve of Zca to be more negative potentials. These electrophysiological observations suggest that bunazosin has antiarrhythmic properties not only through an alpha-adrenoceptor antagonistic action but also its direct membrane action, at least, independently of blockade of cardiac alphaadrenoceptors. Acknowledgements-The present study was supported by Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science and Culture, Japan.
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