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Ouabain actions on the spontaneous activity and ionic currents in rabbit sino-atrial node cells
Pergamon
0306-3623(94)00140-5
Gen. Pharmac.Vol. 25, No. 8, pp. 1591-1598, 1994 Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0306-3623/94$7.00+ 0.00
Ouabain Actions on the Spontaneous Activity and Ionic Currents in Rabbit Sino-atrial Node Cells HIROYASU SATOH Department of Pharmacology, Nara Medical University, Kashihara, Nara 634, Japan ~Tel: (07442) 2-3051; Fax: (07442) 5-7657] (Received 7 April 1994)
Al~ract--l. The effects of ouabain on the action potentials and the membrane currents in spontaneously beating rabbit sino-atrial (SA) node cells were examined using the two-microelectrode technique. 2. Cumulative administrations of ouabain (10 -8 to 10-6 M) caused a negative chronotropic effect in a concentration-dependentmanner. The effect was not modified by atropine (10 -7 M). At 10-6 M, ouahain prolonged the duration of action potentials, but other parameters were unaffected to any significant extent. Ouabain elicited an arrhythmia, and increasing concentrations increased the incidence of arrhythmia (75% at 3 x 10-7 M). 3. Pretreatment with cionidine (10 -+ M), a selective agonist of presynaptic a2-adrenoceptors, completely blocked the development of arrhythmia induced by ouabain (3 x 10-7 M). Prazosin (10 -6 M), an a~ antagonist, had similar effects, and yohimbine (10 -6 to 10-5 M), an as antagonist, did not affect the arrhythmias. 4. Ouabain (10 -s to 10-6 M) inhibited the slow inward and the time-dependent outward currents, but enhanced the hyperpolarization-activated inward current, in a concentration-dependentmanner. The time course of inactivation phase for I~ was composed of two (fast and slow) components. Ouabain decreased the fast component and increased the slow component. The voltage of half-maximum activation for the outward current was not affected. Ouabain elicited a transient inward current on the repolarizing step, and also on the depolarizing step. 5. These results indicate that ouahain inhibits the slow inward and time-dependent outward currents, but elicits arrhythmias due to the induction of cellular calcium overload, which are modulated by ~t-adrenoceptors.
KeyWords: Ouabain, negative chronotropic effect, arrhythmia, cellular calcium overload, sino-atrial node
INTRODUCTION Cardiotonic steroids (cardiac glycosides) have been widely used to treat congestive failure for two centuries. The mechanism by which the glycosides increase the contractile force of heart muscle have demonstrated that an initial step is a remarkably specific N a + - K + ATPase inhibition, associated with an Na + pump (Lee and Fozzard, 1975; Akera and Brody, 1978; Eisner and Lederer, 1979). Secondly, it is concluded that the effect occurs because Na + increases close to the inner side of the plasma membrane, and this decreases Ca ~+ efltux through the N a - C a exchange mechanism (Langer et aL, 1979; Isenberg and Kl6ckner, 1980; Harding and Halliday, 1980). Digitalis actually increases the amplitude of the intracellular Ca 2+ ion transients that precede
tension development (Allen and Blinks, 1978; Wier and Hess, 1984). Other mechanisms of the cardiotonic action have also been proposed for the effects on the Ca 2+ current (/ca) through the voltage-gated Ca 2+ channels of the membrane and on release of Ca 2+ from this intracellular storage site (Lederer and Eisner, 1982; M a r b a n and Tsien, 1982). In electrophysiological studies, inconsistent findings have reported that cardiac glycosides produced a stimulatory or inhibitory action on the /ca (Josephson and Sperelakis, 1977; Weingart et al., 1978; M a r b a n and Tsien, 1982; Fischmeister et aL, 1986). The action potential duration (APD) is initially briefly prolonged and then shortened during exposure to glycosides (Miura and Biedert 1985). These results suggest that cardiac glycosides might possess direct and indirect actions on heart muscles,
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HIROYASUSATOH
resulting from the modulation by the glycosides of intracellular Ca 2÷ concentration ([Ca]i). In sino-atrial (SA) nodal cells, an electrogenic Na ÷ pump causes the K+-activated responses, and plays a role as a factor that modulates the heart rate under physiological conditions (Noma and Irisawa, 1974). The aim of the present experiments is to examine the effects of ouabain on spontaneous activity in rabbit sino-atrial (SA) node cells. Ouabain may also develop the Ca 2÷ overload in rabbit SA nodal cells, as in Purkinje fibres (Vassalle and Lin, 1979; Wier and Hess, 1984). Thus, how automaticity may be modulated by ouabain is examined, in addition examining the effects of ouabain on the underlying ionic currents of the SA node cells. Modulation by ctadrenoceptor drugs of spontaneous activity was also examined.
determined by taking the difference between the value of the current at the end of a long clamp pulse and the zero current level. The tail current was measured as the difference between its peak amplitude and the steady-state current value at the holding potentials. Values represent the m e a n _ SEM.
Solutions The bath solution had the following composition (in mM): NaCI 137, KCI 2.7, MgCI 2 0.5, CaCI 2 1.8, HEPES [N-(2-hydroxyethyl) piperazine-N'-2ethansulfonic acid] (Wako Pure Chemical Industries, Ltd., Osaka, Japan) 0.5, and glucose 5. The pH was adjusted to 7.4 with NaOH. The drugs used were ouabain (Sigma Chemical, St Louis, MO, U.S.A.), tetrodotoxin (TTX, Sankyo Co. Tokyo, Japan), clonidine hydrochloride (Sigma Chemical), prazosin hydrochloride (Wako Pure Chemical), and yohimbine hydrochloride (Wako Pure Chemical).
MATERIALS AND M E T H O D S
Preparations Rabbits weighing 1.5-2.0 kg were killed by a blow to the neck and exsanguinated. After removing the right atrium, strips of the SA node tissue were dissected from the tissue in a direction perpendicular to the crista terminalis. The specimens were made smaller by dissecting to a final dimension of approximately 0.2 × 0.2 mm. The specimens were superfused with a bath solution oxygenated by 100% 02 at 36°C, and left spontaneously beating.
Recordings of action potentials and ionic currents The two-microelectrode voltage-clamp technique used has already been described (Satoh and Hashimoto, 1988; Satoh, 1993a, c), and is similar to the method developed initially by Noma and Irisawa (1976). Test pulses were applied between - 7 0 and + 3 0 mV from a holding potential of - 4 0 mV, using a voltage-clamp amplifier (Dia Medical DP100, Tokyo, Japan). The membrane potential and ionic currents were displayed on an oscilloscope (Sony Tektronix, Tokyo, Japan). The responses progressed with time, and became significant 5--6 min after the initial application of ouabain. The data were obtained approximately 7-10min later. Tetrodotoxin (TTX, 3 x 10 -7 to 10 -6 M) was added to the bath solution to avoid the influences of the fast Na ÷ current. The amplitude of the slow inward current (lsi) was determined as the difference between the peak current and the current level measured at 100 ms after the onset of the step, following the method developed by McDonald and Trautwein (1978). The magnitudes of the outward current (IK) and the hyperpolarization-activated inward current (If) were
RESULTS The SA node preparations showed spontaneous action potentials. Ouabain (10 -8 to 10-6M) was cumulatively added to the bath solution (Fig. 1). Ouabain caused a negative chronotropic effect, and at 10 -6 M , the spontaneous activity was replaced by small oscillatory potentials. The negative chronotropic effect was not affected by atropine (10 -6 M). The percentage changes in action potential parameters are summarized in Table 1. The cycle length (CL) was prolonged with an increase in the concentrations of ouabain, and the action potential duration (APD) was significantly prolonged at 10 -6 M. These responses were concentration-dependent. The action potential amplitude (APA) and the maximum rate of depolarization (l?max) were decreased, and the maximum diastolic potential (MDP) was hyperpolarized, but these responses were not brought about to significant extent. After a washout, the responses were reversible. Figure 2 shows arrhythmias with oscillatory potentials during diastole in the presence of 3 x 10 -7 M ouabain. The incidence of arrhythmia is summarized in Table 1. Ouabain, even at low concentrations (10 -8 M), often elicited the arrhythmias. Furthermore, in 7 out of 16 preparations, a sinus arrest occurred at 10-6M ouabain, resulting in a resting potential (RP) of - 5 1 + 3 m V (Fig. 2A). After 5-10min washout, the arrhythmias (including the sinus arrest) disappeared, and a regular rhythm was resumed. During washout of 3 x 10 -7 M ouabain, the sinus arrest occurred in 3 of 20 preparations with arrhythmias (15% incidence). To elucidate the mechanisms for the effects of
Effects of ouabain on action potentials and membrane currents
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Fig. 1. Spontaneous action potentials of rabbit SA node cells in the presence of ouabain. (A) Spontaneously beating action potentials and maximum rate of depolarization (l;'m,). (B) Recordings at a fast time scale at a point above the recordings of A. The short line at the left of the action potential recordings represents 0 mV.
ouabain on the action potential configuration, voltage-clamp experiments were exerted using the two-microelectrode technique. Figure 3 shows the current traces on a depolarizing pulse of 0 mV and a hyperpolarizing pulse of - 7 0 mV. Ouabain (10 -8 to 1 0 - r M ) inhibited both the slow inward current (lsl) and the time-dependent outward current (Iz), whereas it enhanced the hyperpolarization-activated inward current (ln). The current-voltage curves for these ionic currents in the absence and presence of ouabain are shown in Fig. 4. The responses were concentration-dependent. Ouabain did not affect the peak of lsi (at + 10 mV) at 10 -8 and 10 -7 M, but inhibited it by 18.9+2.3% ( n = 6 , P < 0 . 0 1 ) at 10-rM. The voltages of half-maximum activation for I~ were almost identical in six preparations; - 2 0 . 1 + 2 . 1 m V in the control, - 2 0 . 2 + 1.8mV
at 10 - a M , , 2 0 . 4 + 1.2mV at 10 - 7 M , and - 2 1 . 9 + 1.7 mV at 10-SM of ouabain. The voltages of the half-maximum inactivation were also unaffected in five preparations; - 2 1 . 5 + 1.2mV at 10 - s M , -21.5_+ 1,8 mV at 10 -7 M, and - 2 0 . 7 + 2 . 3 m V at 10-6M of ouabain. The time course of the inactivation phase for I~ had two exponential components. The fast and slow time constants (el and zs) in six preparations were 7 . 2 + 0 . 3 m s and 22.3_+1.2ms in the control (Table 2). zr was decreased, whereas zs was increased by application of ouabain ( 1 0 - 7 t o 10 - 6 M). The I K current was also inhibited with an increase in ouabain concentrations (Figs 3 and 4). The percentage inhibitions at + 3 0 mV in six preparations was not significant at 10-SM (8.1_+1.5%), and 13.5 _ 1.8% (P < 0.05) at 10 -7 M, and 18.9 +_ 2.4%
Table 1. Effects of ouabain on spontaneous action potentials in rabbit sino-atrial node preparation Control n
APA APDs MDP CL
20 9 4 _ 3mV 68-+ 2ms -72_+2mV 12-+ I V/s 331 ± 2 4 m s
10 -8 M 15 +0.8 ± 1.2 - 0 . 1 _ 1.7 +3,0_+2.0 - 1 . 7 ± 3.5 +12.0_+2.6"
Incidence of arrhythmia
10 -7 M 16 -1.0.+1.6 -2.8_+3.1 +5.0-+2.2 -I.4.+5.6 +20.8-+3.6**
3 × 10-7 M 20 -4.1+0.9 -3.5-+2.5 +4.0-+ 1.0 -2.8±2.7 +15.0-+4.1"*
10 -6 M 16 -4.8_+2.8 - 1 2 . 1 _+ 3.3" +6.5_+2.1 -2.7.+4.7 +86.2+10.5"**
20
20
86
94
0
0
15
43
Incidence of sinus arrest
Values (%) represent mean -+ $EM.
total number of experiments. APA: amplitude of action potential. APDs0: duration of action potential. MDP: maximum diastolic potential. ~'m,x: maximum rate of depolarization. CL: Cycle length. *P < 0.05, **P < 0.01, ***P < 0.001, with respect to control value.
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HIROYASU SATOH
Ouabain 3x10-7 M
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2 rain Fig. 2. Arrhythmias induced by ouabain in the SA node preparations. Spontaneous action potentials and the maximum rate of depolarization represented at fast and slow time scales. Ouabain (3 x 10-7 M) induced oscillatory potentials during the pacemaker potential (arrows).
(P < 0.01)at 10-6M of ouabain. Simultaneously, the outward tail current was decreased in a concentration-dependent way (Fig. 5A). These activation curves were normalized by taking the amplitude at + 3 0 mV as 1.0 (Fig. 5B). The voltage of halfmaximum activation was unaffected, and the average value was - 2 . 3 + 0.3 mV (n = 6). On the other hand, the ]h current was enhanced by 23.3 __ 2.4% (n = 6, P<0.05) at l0 - s M , by 4 3 . 3 + 5 . 8 % ( n = 6 , P < 0 . 0 1 ) at 10-TM, and by 4 6 . 6 + 5 . 1 % ( n = 6 , P < 0.001) at l0 -6 M of ouabain. As shown in Fig. 3, the transient inward current (Its) occurred in the presence of ouabain (10 -s to 10 -6 M). Increasing concentrations of ouabain produced a more marked /ti current. The Iti at l0 -6 M ouabain was elicited on depolarizing steps as well as on repolarizing steps. Pretreatment with clonidine (10 -6 M) did not elicit arrhythmias, although ouabain (at 3 x l0 -7 M ) alone elicited these at 86% incidence (Fig. 6A and B).
Clonidine is a specific ct2-adrenoceptor agonist. Clonidine (10-6M) decreased the spontaneous activity (by 11.5 + 2.3%, n = 6, P < 0.05). Addition of ouabain (3 x 10 -7 M) caused a positive chronotropic effect (by 18.6 + 3.1%, n = 6, P < 0.01) and tended to increase APA and l?maX (P >0.05). Prazosin (10-6M), an ~]-adrenoceptor blocker, also caused similar effects in five preparations. In contrast, yohimbine (10 -6 to 10-SM), an ~2-adrenoceptor blocker, increased APA and Vmax significantly. But, yohimhine caused a negative chronotropic effect (by approximately 57 to 120%) and had no effect on arrhythmias (n = 3). DISCUSSION Cardiac glycosides are used extensively in the treatment of heart failure. Ouabain plays a role in numerous physiological and pathophysiological pro-
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io-6 ._~""--t---,~: Fig. 3. The effects of ouabain on ionic currents in rabbit SA node preparation. Test pulses were applied to + 10 mV and - 7 0 mV from a holding potential of - 4 0 mV. The current traces are the slow inward current (1,), the outward current (IK), and the hyperpolarization-activated inward current (lh) in the presence of ouabain (10 -s to 10-6 M). Note the appearance of a transient inward current during the steps repolarizing and depolarizing pulses. The short line at the left of the current recordings represents 0 current level.
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Fig. 4. Current-voltage relationships for the slow inward current, the time-dependent outward current, and the hyperpolarization-activated inward current in rabbit spontaneously boating SA node cells. The values were measured from the current records of Fig. 3. Symbols used are the control (open circles), at 10-SM (triangles), 10-7M (squares), and 10-6 M (filled circles) of ouabain.
Effects of ouabain on action potentials and membrane currents Table 2. Effects of ouabain on the time course of inactivation phase of the I~ in rabbit sino-atrial node preparations Control
6
7.2 + 0.3 ms
22.3 + 1.2ms
Ouabain lO-SM I0 7M 10-tM
6 6 7
7.1 __.0.6 6.6±0.2* 5.3:1:0.3"*
22.4___0.8 23.1±0.4 35.4+0.9***
Values represent mean_+SEM, n: number of experiments. Zr: fast component. ¢s: slow component. *P < 0.05, **P < 0.01, ***P < 0.001, with respect to control value.
cesses associated with altered fluid and electrolyte metabolism and deviations from the normal blood pressure-blood volume relationship (Blaustein, 1993). The results presented here in rabbit spontaneously beating SA node preparations show that; (1) ouabain inhibited lsi and IK but stimulated lh, (2) ouabain caused negative chronotropic effects, and at high concentrations (10 -6 M) prolonged the APD (by approximately 12%), (3) Iti (or sinus arrest) occurred, and (4) clonidine and prazosin protected the development of arrhythmia induced by ouabain. The changes in ionic currents appear to reflect the nA
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alterations of the action potential configuration. The APD prolongation would be due mainly to the time-dependent I~ inhibition. The depressions in APA and I'm,,, althOugh not significant, might be produced by the Isl inhibition. Ouabain actions MyoCardial sarcolemmal Na+/K÷-ATPase is the pharmacological receptor for the positive inotropic action of digitalis (Schwartz et aL, 1982). Binding studies have shown that digitalis binds specifically to Na+/K÷-ATPase and the amount of binding is directly related to the degree of the positive inotropic action, with a high affinity for dissociation constants (Kd) of the order of 1 0 - 9 M (Forbush, 1983; Sweadner, 1988; Blanco et al., 1990). The enzyme has and//catalytic components, and the g subunit of Na÷/K÷-ATPase contains the digitalis receptors. Ouabain (due to the effects on the Na + electrochemical gradient and indirectly on Na÷-Ca 2+ exchange) modulates the storage of Ca 2+ in the ER/SR of a large variety of cells, and thereby plays an important role in the regulation of cell responsiveness. An electroneutral active Na ÷ transport may indirectly affect the membrane potential of a cell by maintaining the Na + and K ÷ concentration gradients across the cell membrane (Akera and Brody, 1978). Also, an electrogenic Na + pump may directly contribute to the membrane potential by net charge transport. Inhibition of the Na ÷ pump by ouabain can be expected to influence the RP in two ways; (1) by altering the electrogenic pump current and (2) by altering the K ÷ equilibrium potential. The maximum steady-state voltage that can be generated by the electrogenic Na ÷ pump with a stoichiometry of 3Na+:2K + is only < 1 0 m V (Thomas, 1972). Ouabain (2 x 10-r M) decreased the RP (by approximately 5 mV) and the APDs0 (by approximately 8%), and increased the phase 4 depolarization in the Purkinje fibres (Miura and Biedert, 1985). In the present experiments, however, ouabain tended to hyperpolarize the MDP (by < 5 m V a t 1 0 - 6 M ) . Blaustein (1993) calculated the theoretical membrane potential, and the calculated potential at 145mM INa]o would expect a decline of only 0.2 mV during partial Na + pump inhibition. Therefore, these results indicate that ouabain may not affect the RP to a significant extent.
Fig. 5. A c t i v a t i o n curves for the o u t w a r d current in the
absence and presence of ouabain. The values were taken from the amplitude of the outward tail current by normalizing the amplitude at +30 mV. (A) Activation curves in the presence o f different concentrations of ouabain. (B) Normalized activation curves (p~) from part A. Symbols represent the control (open circles), 10-SM (triangles), 10-7 M (squares), and 10-6 M (fdled circles) concentrations of ouabain.
Induction of dysrhythmia Cardiac glycosides which have a very low therapeutic index produce cardiotoxicity as a side effect. In this study, ouabain elicited arrhythmias and produced/t~. The Iti current is elicited under cellular Ca z+ overload condition (Satoh, 1993c; Satoh and
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HIROYASUSATOH
CIoNdine + O u a b a i n 10-' M b 3 X 1 0 .7 M .
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L_LL L-L_L £.L-L L-LL L_L_L Fig. 6. Modulation by clonidine of ouabain-induced arrhythmia. (A) Spontaneous action potentials, maximum rate of depolarization, and sinus rate. (B) Recordings at a fast time scale. Clonidine (10-6 M) depressed the spontaneously beating action potentials. Additional application of ouabain (3 x 10 -7 M ) produced no arrhythmia, and brought about a positive chronotropic effect with a regular rhythm.
Vassalle, 1985; Satoh et al., 1989b). Thus, the arrhythmias would be due to cellular Ca 2+ overload. Ouabain actually increases the [Ca]i level (Allen and Blinks, 1978; Wier and Hess, 1984). It has already been demonstrated that the Ca 2÷ overload in cardiac muscles elicited arrhythmias by delayed afterdepolarization and Iti, which results from changes in the permeability of the cation channels to Na ÷ or K ÷, or to both ions (Lederer and Tsien, 1976; Kass et al., 1978; Eisner and Lederer, 1979). Satoh et al. (1989b; 1993a, b, c) have recently reported that arrhythmias in rabbit SA node cells are elicited by the lti and an inward tail current. During washout of ouabain in arrhythmiaoccurring preparations, the arrhythmia was replaced by a sinus arrest (about 38% incidence at 10 -6 M). This appears to be induced by a further elevation of [Ca]i. Because, in the single SA node cells overloaded with Ca 2+, as detected by Ca2+-sensitive fluorescent dye (fura-2), the [Ca]i level was transiently potentiated to increase during washout of drugs (by approximately 30%) (Satoh, 1993a). This rebound after a switch to lower [Ca]o is also induced in canine Purkinje fibres (Satoh and Vassalle, 1985, 1989). The [Ca]i elevation may stimulate the Ca 2÷activated I X of cardiac muscles (Clusin et aL, 1975; Colatsky and Hogan, 1975; Satoh et al., 1989a). However, this study showed that the voltagedependent IK current was inhibited in a concen-
tration-dependent manner. The inhibition of Ix produces the APD prolongation, which tends to elevate the [Ca]i level (Satoh and Hashimoto, 1986). Thus, the Ix inhibition might be one of the factors causing the positive inotropic effect and eliciting the arrhythmias. Negative chronotropic effect Ouabain caused a negative chronotropic effect in a concentration-dependent manner. The major effect of ouabain was the CL prolongation in the SA node cells. There are three hypotheses for the pacemaker current during diastole; the lsi hypothesis, the g r hypothesis and the Ih hypothesis, according to the review of Noble (1984). In the present experiment, ouabain inhibited Isi and I X and enhanced I h. The decreases in Isi and I X currents during the diastolic potential are considered to contribute to the formation of the pacemaker potential, resulting in a negative chronotropic effect. Since the negative chronotropic effect was not affected by atropine, it would be a direct action of ouabain. It might also be produced by an indirect action of ouabain. Satoh and colleagues (1989a; 1993a, b, c) have shown recently that several drugs cause the negative chronotropic effect because of development of a cellular Ca 2+ overload in the rabbit SA node cell. Therefore, the Ca 2+ overload induced by ouabain may somewhere relate to the negative ehronotropic effect.
Effects of ouabain on action potentials and membrane currents Actions on 1.,i current
Effects of cardiac glycosides on voltage-dependent Ca 2+ current (Ic,) of heart muscle cells are still controversial. The glycosides produce the stimulatory action on /ca (Weingart et al., 1978; Lederer and Eisner, 1982; Marban and Tsien, 1982; Fischmeister et al., 1986), whereas they produce the inhibitory action (Josephson and Sperelakis, 1977; Deslauriers et al., 1982). This discrepancy may result from a change in the [Call level (caused by inhibition of the Na + pump) or from differences of cell types, suggesting that ouabain may have direct and indirect actions o n / c a current. In this study, ouabain inhibited the l~ in a concentration-dependent way. The lc, inhibition may be explained by a mechanism including: (1) the decrease may be due to the depression in channel opening by cellular Ca 2+ overload induced by ouabain, as the current peak of 15 is depressed under Ca 2+ overload conditions (Satoh et al., 1989a; Satoh, 1993a, c). (2) The decrease may be due to a direct action of ouabain on the ionic channels. (3) Ouabain may induce damage to the cell membrane (or ionic channels). The time course of the inactivation phase for lsi is composed of two (fast and slow) components (xr and x~). Ouabain decreases xf and increased zs. The fast component is [Ca]~ dependent and the slow component is voltage-dependent (Eckert and Chad, 1984; Carbone and Swandulla, 1989). So, the zf decrease would be caused by an elevation in the [Ca]i level caused by ouabain application. The % increase would be a direct action of ouabain and may lead to an elevated [Ca]i level. Modulation o f cardiotoxicity by ~-adrenoceptor
It is well known that a-adrenoceptor blocking drugs prevent or suppress some experimental arrhythmias (Gould et al., 1969; Rothans and Powell, 1975). In in vivo experiments, cardiac glycosides increase neurotransmitter overflow from autonomic nerve endings in a variety of tissues including heart (Pace and GiUis, 1976; Powis, 1983). The changes in the sympathetic discharge to the heart may result in the non-uniformity of electrical properties of myocardial cells (Kim et al., 1984). Clonidine stimulates peripheral presynapthetic ~2-adrenoceptors, causing a diminished release of noradrenaline from the nerve endings towards the heart, and also reduces the peripheral sympathetic tone by stimulation of central • 2-adrenoceptors, decreasing the plasma catecholamines (Starke et al., 1974; Cavero and Roach, 1980). The reduction of sympathetic tone and the inhibition of the neurotransmitter release may be a contributing factor for the antihypertensive and antiarrhythmic
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activities of clonidine. Gillis and Quest (1979) reported that the concentration of cardiac glycosides required to produce ventricular arrhythmias is reduced by enhancing the sympathetic outflow to the heart. In the present experiments, small rabbit SA node preparations (0.2 x 0.2ram) were used, but nerve terminals would still be present. Pretreatment with clonidine (a2-adrenoceptor agonist) and prazosin (al-adrenoceptor antagonist) blocked the development of arrhythmias induced by ouabain (but unaffected by yohimbine), which is consistent with previous reports (Lechat and Schmitt, 1982; Thomas and Tripathi, 1986). They demonstrated that u:-receptor stimulation and a~-receptor blockage protect against the ventricular arrhythmias and cardiac arrest induced by ouabain in guinea-pig hearts. In addition, -adrenoceptor agonists inhibit the Isi in the SA node cells (Satoh and Hashimoto, 1988). Therefore, clonidine and prazosin could prevent the development of arrhythmias caused by the modulation of neurotransmitter release and Isl inhibition.
REFERENCES
Akera T. and Brody T. M. (1978) The role of Na +, K +ATPase in inotropic action of digitalis. Pharmac. Rev. 29, 187-220. Allen D. G. and Blinks J. R. (1978) Calcium transients in aequorin-injected frog cardiac muscle. Nature 273, 509-513. Blanco G., Berberian G. and Beaug6 L. (1990) Detection of a highly ouabain sensitive isoform of rat brainstem of Na, K-ATPase. Biochim. biophys. Acta 1027, 1-7. Blaustein M. P. (1993) Physiological Effects of endogenous ouabain: control of intracellular Ca2÷ stores and cell responsiveness. Am. J. Physiol. 264, C1367-C1387. Carbone E. and Swandulla D. (1989) Neuronal calcium channels: kinetics, blockade and modulation. Prog. biophys. Mol. Biol. 54, 31-58. Cavero I. and Roach A. G. (1980) Effects of clonidine on canine cardiac neuroeffector structures controlling heart rate. Br. J. Pharmac. 70, 269-276. Clusin W., Spray D. C. and Bennet M. V. L. (1975) Activation of voltage-insensitive conductance by inward calcium current. Nature 256, 425-427. Colatsky T. J. and Hogan P. M. (1975) Calcium modulation of action potential duration in cardiac Purkinje fibres. Fed. Proc. 34, 375. Deslauriers Y., Ruiz-Ceretti E., Schanne O. F. and Payet M. D. (1982) The toxic effects of ouabain. A voltageclamp study. Can. J. Physiol. Pharmac. 60, 1153-1159. Eckert R. and Chad J. E. (1984) Inactivation of Ca channels. Prog. biophys. Mol. Biol. 44, 215-267. Eisner D. A. and Lederer W. J. (1979) Does sodium pump inhibition produce the positive inotropic effects of strophanthidin in mammalian cardiac muscle? J. Physiol. (Lond.) 296, 75-76. Fischmeister R., Brocas-Randolph M., Lech6ne P., Argibay J. A. and Vassort G. (1986) A dual effect of cardiac glycisides in Ca current in single cells of frog heart. Pflfigers Arch. 406, 340-342. Forbush B. (1983) Cardiotonic steroid binding to Na, KATPase. Curt. Top. Membr. Transp. 19, 167-201.
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Gillis R. A. and Quest J. A. (1979) The role of the nervous system in the cardiovascular effects of digitalis. Pharmac. Rev. 31, 19-97. Gould L., Zahir M., Shariff M. and Guilliani M. G. (1969) Treatment of cardiac arrhythmias with phentolamine. Am. Heart J. 78, 189. Harding S. E. and Halliday J. (1980) Removal of sialic acid from cardiac sarcolemma does not affect contractile function in electrically stimulated guinea pig left atria. Nature 286, 819-821. Isenberg G. and Kl~ckner U. (1980) Glycocalyx is not required for slow inward calcium current in isolated rat heart myocytes. Nature 284, 358-360. Josephson I. and Sperelakis N. (1977) Ouabain blockade of inward slow current in cardiac muscle. J. Mol. Cell Cardiol. 9, 409-418. Kass R. S., Tsien R. W. and Weingart R. (1978) Ionic basis of transient inward current induced by strophanthidine in cardiac Purkinje fibres. J. Physiol. (Lond.) 281, 187-208. Kim D. H., Akera T., Kennedy R. H. and Stemmer P. M. (1984) Reduced tolerance to digitalis-induced arrhythmias caused by coronary flow alterations in isolated perfused heart of guinea pigs. Life Sci. 34, 105-112. Langer G. A., Frank J. S. and Nudd L. M. (1979) Correlation of calcium exchange, structure and function in myocardial tissue culture. Am. J. Physiol. 237, H239-246. Lechat P. and Schmitt H. (1982) Interactions between the autonomic nervous system and the cardiovascular effects of ouabain in guinea-pigs. Eur. J. Pharmac. 78, 21-32. Lederer W. J. and Eisner D. A. (1982) The effects of sodium pump activity on the slow inward current in sheep cardiac Purkinje fibres. Proc. R. Soc. B214, 249-262. Lederer W. J. and Tsien R. W. (1976) Transient inward current underlying arrhythmogenic effects of cardiotonic steroids in Purkinje fibers. J. Physiol. (Lond.)263, 73-100. Lee C. O. and Fozzard H. A. (1975) Activities of potassium and sodium ions in rabbit heart muscle. J. Physiol. (Lond.) 65, 695-708. Marban E. and Tsien R. W. (1982) Enhancement of calcium current during digitalis inotropy in mammalian heart: positive feed-back regulation by intracellular calcium? J. Physiol. (Lond.) 329, 589~514. McDonald T, F. and Trautwein W. (1978) Membrane current in cat myocardium: separation of inward and outward components. J. Physiol. (Lond.) 274, 193-216. Miura D. S. and Biedert S. (1985) Cellular mechanisms of digitalis action. J. Clin. Pharmacol. 25, 490-500. Noble D. (1984) The surprising heart: a review of recent progress in cardiac electrophysiology. J. Physiol. (Lond.) 353, 1-50. Noma A. and Irisawa H. (1974) Electrogenic sodium pump in rabbit sinoatrial node cell. Pfliiger Arch. 351, 177-182. Noma A. and Irisawa H. (1976) Membrane currents in the rabbit sinoatrial node cell as studied by the double microelectrode method. Pfliigers Arch. 364, 45-52. Pace D. R. and Gillis R. A. (1976) Neuroexcitatory effects of digoxin in the cat. J. Pharmac. exp. Ther. 199, 583~00. Powis D. A. (1983) Cardiac glycisides and autonomic neurotransmission. J. Auto. Pharmac. 3, 127-154. Rothans K. O. and Powell W. J. (1975) The role of alpha adrenergic receptors in digitoxin tachyarrhythmias. Fed. Proc. 34, 2976.
Satoh H. (1993a) Caffeine depression in spontaneous activity in rabbit sino-atrial node cells. Gen. Pharmac. 24, 555-563. Satoh H. (1993b) Positive and negative effects of caffeine in sponatneously beating sino-atrial node cells. Gen. Pharmac. 24, 1223--1230. Satoh H. (1993c) Class III antiarrhythmic drugs (amiodarone, bretylium and sotalol) on action potentials and membrane currents in rabbit sino-atrial node preparations. Naunyn-Schmiedeberg's Arch. Pharmac. 344, 674-681. Satoh H. and Hashimoto K. (1986) An electrophysiological study of amiloride on sino-atrial node cells and ventricular muscle of rabbit and dog action potentials and membrane. Naunyn-Schmiedeberg's Arch. Pharmac. 344, 674~i81. Satoh H. and Hashimoto K. (1988) Effect of ~q-adrenoceptor stimulations with methoxamine and phenylephrine on the spontaneously beating rabbit sino-atrial node cells. Naunyn-Schmiedeberg "s Arch. Pharmac. 337, 415-422. Satoh H. and Vassalle M. (1985) Reversal of caffeineinduced calcium overload in cardiac Purkinje fibers. J. Pharmac. exp. Ther. 234, 172-179. Satoh H. and Vassalle M. (1989) Role of calcium in caffeine-norepinephrine interactions in cardiac Purkinje fibers. Am. J. Physiol. 257, H226-H237. Satoh H., Tsuchida K. and Hashimoto K. (1989a) Electrophysiological actions of A23187 and X-537A in rabbit sino-atrial node cells. Naunyn-Schmiedeberg's Arch. Pharmac. 339, 320--326. Satoh H., Hasegawa J. and Vassalle M. (1989b) On the characteristics of the inward current induced by calcium overload. J. Mol. Cell Cardiol. 21, 5-20. Schwartz A., Whitmer K., Grupp G., Grupp I., Adams R. J. and Lee S. W. (1982) Mechanism of action of digitalis: is the Na, K-ATPase the pharmacological receptors? Ann. N.Y. Acad. Sci. 402, 253-271. Starke K., Montel H., Gayk W. and Merker R. (1974) Comparison of the effects of clonidine on pre- and post-synaptic adrenoceptors in the rabbit pulmonary artery. Naunyn-Schmiedeberg's Arch. Pharmac. 285, 133-147. Sweadner K. J. (1988) Isozymes of the Na+/K+-ATPase. Biochim. biophys. Acta. 988, 185-220. Thomas G. P. and Tripathi R. M. (1986) Effects of a-adrenoceptor agonists and antagonists on ouabaininduced arrhythmia and cardiac arrest in guinea-pig. Br. J. Pharmac. 89, 385-388. Thomas R. C. (1972) Electrogenic sodium pump in nerve and muscle cells. Physiol. Rev. 52, 563-594. Vassalle M. and Lin C. I. (1979) Effect of calcium on strophanthidin-induced electrical and mechanical toxicity in cardiac Purkinje fibers. Am. J. Physiol. 236, H689-H697. Weingart R., Kass R. S. and Tsien R. W. (1978) Is digitalis inotropy associated with enhanced slow inward Ca current? Nature 273, 389-392. Wier W. G. and Hess P. (1984) Excitation-contraction coupling in cardiac Purkinje fibers: effects of cardiotonic steroids on the intracellular [Ca 2+] transient, membrane potential, and contraction. J. Gen. Physiol. 83, 395-415.
0306-3623(94)00140-5
Gen. Pharmac.Vol. 25, No. 8, pp. 1591-1598, 1994 Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0306-3623/94$7.00+ 0.00
Ouabain Actions on the Spontaneous Activity and Ionic Currents in Rabbit Sino-atrial Node Cells HIROYASU SATOH Department of Pharmacology, Nara Medical University, Kashihara, Nara 634, Japan ~Tel: (07442) 2-3051; Fax: (07442) 5-7657] (Received 7 April 1994)
Al~ract--l. The effects of ouabain on the action potentials and the membrane currents in spontaneously beating rabbit sino-atrial (SA) node cells were examined using the two-microelectrode technique. 2. Cumulative administrations of ouabain (10 -8 to 10-6 M) caused a negative chronotropic effect in a concentration-dependentmanner. The effect was not modified by atropine (10 -7 M). At 10-6 M, ouahain prolonged the duration of action potentials, but other parameters were unaffected to any significant extent. Ouabain elicited an arrhythmia, and increasing concentrations increased the incidence of arrhythmia (75% at 3 x 10-7 M). 3. Pretreatment with cionidine (10 -+ M), a selective agonist of presynaptic a2-adrenoceptors, completely blocked the development of arrhythmia induced by ouabain (3 x 10-7 M). Prazosin (10 -6 M), an a~ antagonist, had similar effects, and yohimbine (10 -6 to 10-5 M), an as antagonist, did not affect the arrhythmias. 4. Ouabain (10 -s to 10-6 M) inhibited the slow inward and the time-dependent outward currents, but enhanced the hyperpolarization-activated inward current, in a concentration-dependentmanner. The time course of inactivation phase for I~ was composed of two (fast and slow) components. Ouabain decreased the fast component and increased the slow component. The voltage of half-maximum activation for the outward current was not affected. Ouabain elicited a transient inward current on the repolarizing step, and also on the depolarizing step. 5. These results indicate that ouahain inhibits the slow inward and time-dependent outward currents, but elicits arrhythmias due to the induction of cellular calcium overload, which are modulated by ~t-adrenoceptors.
KeyWords: Ouabain, negative chronotropic effect, arrhythmia, cellular calcium overload, sino-atrial node
INTRODUCTION Cardiotonic steroids (cardiac glycosides) have been widely used to treat congestive failure for two centuries. The mechanism by which the glycosides increase the contractile force of heart muscle have demonstrated that an initial step is a remarkably specific N a + - K + ATPase inhibition, associated with an Na + pump (Lee and Fozzard, 1975; Akera and Brody, 1978; Eisner and Lederer, 1979). Secondly, it is concluded that the effect occurs because Na + increases close to the inner side of the plasma membrane, and this decreases Ca ~+ efltux through the N a - C a exchange mechanism (Langer et aL, 1979; Isenberg and Kl6ckner, 1980; Harding and Halliday, 1980). Digitalis actually increases the amplitude of the intracellular Ca 2+ ion transients that precede
tension development (Allen and Blinks, 1978; Wier and Hess, 1984). Other mechanisms of the cardiotonic action have also been proposed for the effects on the Ca 2+ current (/ca) through the voltage-gated Ca 2+ channels of the membrane and on release of Ca 2+ from this intracellular storage site (Lederer and Eisner, 1982; M a r b a n and Tsien, 1982). In electrophysiological studies, inconsistent findings have reported that cardiac glycosides produced a stimulatory or inhibitory action on the /ca (Josephson and Sperelakis, 1977; Weingart et al., 1978; M a r b a n and Tsien, 1982; Fischmeister et aL, 1986). The action potential duration (APD) is initially briefly prolonged and then shortened during exposure to glycosides (Miura and Biedert 1985). These results suggest that cardiac glycosides might possess direct and indirect actions on heart muscles,
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HIROYASUSATOH
resulting from the modulation by the glycosides of intracellular Ca 2÷ concentration ([Ca]i). In sino-atrial (SA) nodal cells, an electrogenic Na ÷ pump causes the K+-activated responses, and plays a role as a factor that modulates the heart rate under physiological conditions (Noma and Irisawa, 1974). The aim of the present experiments is to examine the effects of ouabain on spontaneous activity in rabbit sino-atrial (SA) node cells. Ouabain may also develop the Ca 2÷ overload in rabbit SA nodal cells, as in Purkinje fibres (Vassalle and Lin, 1979; Wier and Hess, 1984). Thus, how automaticity may be modulated by ouabain is examined, in addition examining the effects of ouabain on the underlying ionic currents of the SA node cells. Modulation by ctadrenoceptor drugs of spontaneous activity was also examined.
determined by taking the difference between the value of the current at the end of a long clamp pulse and the zero current level. The tail current was measured as the difference between its peak amplitude and the steady-state current value at the holding potentials. Values represent the m e a n _ SEM.
Solutions The bath solution had the following composition (in mM): NaCI 137, KCI 2.7, MgCI 2 0.5, CaCI 2 1.8, HEPES [N-(2-hydroxyethyl) piperazine-N'-2ethansulfonic acid] (Wako Pure Chemical Industries, Ltd., Osaka, Japan) 0.5, and glucose 5. The pH was adjusted to 7.4 with NaOH. The drugs used were ouabain (Sigma Chemical, St Louis, MO, U.S.A.), tetrodotoxin (TTX, Sankyo Co. Tokyo, Japan), clonidine hydrochloride (Sigma Chemical), prazosin hydrochloride (Wako Pure Chemical), and yohimbine hydrochloride (Wako Pure Chemical).
MATERIALS AND M E T H O D S
Preparations Rabbits weighing 1.5-2.0 kg were killed by a blow to the neck and exsanguinated. After removing the right atrium, strips of the SA node tissue were dissected from the tissue in a direction perpendicular to the crista terminalis. The specimens were made smaller by dissecting to a final dimension of approximately 0.2 × 0.2 mm. The specimens were superfused with a bath solution oxygenated by 100% 02 at 36°C, and left spontaneously beating.
Recordings of action potentials and ionic currents The two-microelectrode voltage-clamp technique used has already been described (Satoh and Hashimoto, 1988; Satoh, 1993a, c), and is similar to the method developed initially by Noma and Irisawa (1976). Test pulses were applied between - 7 0 and + 3 0 mV from a holding potential of - 4 0 mV, using a voltage-clamp amplifier (Dia Medical DP100, Tokyo, Japan). The membrane potential and ionic currents were displayed on an oscilloscope (Sony Tektronix, Tokyo, Japan). The responses progressed with time, and became significant 5--6 min after the initial application of ouabain. The data were obtained approximately 7-10min later. Tetrodotoxin (TTX, 3 x 10 -7 to 10 -6 M) was added to the bath solution to avoid the influences of the fast Na ÷ current. The amplitude of the slow inward current (lsi) was determined as the difference between the peak current and the current level measured at 100 ms after the onset of the step, following the method developed by McDonald and Trautwein (1978). The magnitudes of the outward current (IK) and the hyperpolarization-activated inward current (If) were
RESULTS The SA node preparations showed spontaneous action potentials. Ouabain (10 -8 to 10-6M) was cumulatively added to the bath solution (Fig. 1). Ouabain caused a negative chronotropic effect, and at 10 -6 M , the spontaneous activity was replaced by small oscillatory potentials. The negative chronotropic effect was not affected by atropine (10 -6 M). The percentage changes in action potential parameters are summarized in Table 1. The cycle length (CL) was prolonged with an increase in the concentrations of ouabain, and the action potential duration (APD) was significantly prolonged at 10 -6 M. These responses were concentration-dependent. The action potential amplitude (APA) and the maximum rate of depolarization (l?max) were decreased, and the maximum diastolic potential (MDP) was hyperpolarized, but these responses were not brought about to significant extent. After a washout, the responses were reversible. Figure 2 shows arrhythmias with oscillatory potentials during diastole in the presence of 3 x 10 -7 M ouabain. The incidence of arrhythmia is summarized in Table 1. Ouabain, even at low concentrations (10 -8 M), often elicited the arrhythmias. Furthermore, in 7 out of 16 preparations, a sinus arrest occurred at 10-6M ouabain, resulting in a resting potential (RP) of - 5 1 + 3 m V (Fig. 2A). After 5-10min washout, the arrhythmias (including the sinus arrest) disappeared, and a regular rhythm was resumed. During washout of 3 x 10 -7 M ouabain, the sinus arrest occurred in 3 of 20 preparations with arrhythmias (15% incidence). To elucidate the mechanisms for the effects of
Effects of ouabain on action potentials and membrane currents
1593
Ouabain A
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Fig. 1. Spontaneous action potentials of rabbit SA node cells in the presence of ouabain. (A) Spontaneously beating action potentials and maximum rate of depolarization (l;'m,). (B) Recordings at a fast time scale at a point above the recordings of A. The short line at the left of the action potential recordings represents 0 mV.
ouabain on the action potential configuration, voltage-clamp experiments were exerted using the two-microelectrode technique. Figure 3 shows the current traces on a depolarizing pulse of 0 mV and a hyperpolarizing pulse of - 7 0 mV. Ouabain (10 -8 to 1 0 - r M ) inhibited both the slow inward current (lsl) and the time-dependent outward current (Iz), whereas it enhanced the hyperpolarization-activated inward current (ln). The current-voltage curves for these ionic currents in the absence and presence of ouabain are shown in Fig. 4. The responses were concentration-dependent. Ouabain did not affect the peak of lsi (at + 10 mV) at 10 -8 and 10 -7 M, but inhibited it by 18.9+2.3% ( n = 6 , P < 0 . 0 1 ) at 10-rM. The voltages of half-maximum activation for I~ were almost identical in six preparations; - 2 0 . 1 + 2 . 1 m V in the control, - 2 0 . 2 + 1.8mV
at 10 - a M , , 2 0 . 4 + 1.2mV at 10 - 7 M , and - 2 1 . 9 + 1.7 mV at 10-SM of ouabain. The voltages of the half-maximum inactivation were also unaffected in five preparations; - 2 1 . 5 + 1.2mV at 10 - s M , -21.5_+ 1,8 mV at 10 -7 M, and - 2 0 . 7 + 2 . 3 m V at 10-6M of ouabain. The time course of the inactivation phase for I~ had two exponential components. The fast and slow time constants (el and zs) in six preparations were 7 . 2 + 0 . 3 m s and 22.3_+1.2ms in the control (Table 2). zr was decreased, whereas zs was increased by application of ouabain ( 1 0 - 7 t o 10 - 6 M). The I K current was also inhibited with an increase in ouabain concentrations (Figs 3 and 4). The percentage inhibitions at + 3 0 mV in six preparations was not significant at 10-SM (8.1_+1.5%), and 13.5 _ 1.8% (P < 0.05) at 10 -7 M, and 18.9 +_ 2.4%
Table 1. Effects of ouabain on spontaneous action potentials in rabbit sino-atrial node preparation Control n
APA APDs MDP CL
20 9 4 _ 3mV 68-+ 2ms -72_+2mV 12-+ I V/s 331 ± 2 4 m s
10 -8 M 15 +0.8 ± 1.2 - 0 . 1 _ 1.7 +3,0_+2.0 - 1 . 7 ± 3.5 +12.0_+2.6"
Incidence of arrhythmia
10 -7 M 16 -1.0.+1.6 -2.8_+3.1 +5.0-+2.2 -I.4.+5.6 +20.8-+3.6**
3 × 10-7 M 20 -4.1+0.9 -3.5-+2.5 +4.0-+ 1.0 -2.8±2.7 +15.0-+4.1"*
10 -6 M 16 -4.8_+2.8 - 1 2 . 1 _+ 3.3" +6.5_+2.1 -2.7.+4.7 +86.2+10.5"**
20
20
86
94
0
0
15
43
Incidence of sinus arrest
Values (%) represent mean -+ $EM.
total number of experiments. APA: amplitude of action potential. APDs0: duration of action potential. MDP: maximum diastolic potential. ~'m,x: maximum rate of depolarization. CL: Cycle length. *P < 0.05, **P < 0.01, ***P < 0.001, with respect to control value.
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1594
HIROYASU SATOH
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2 rain Fig. 2. Arrhythmias induced by ouabain in the SA node preparations. Spontaneous action potentials and the maximum rate of depolarization represented at fast and slow time scales. Ouabain (3 x 10-7 M) induced oscillatory potentials during the pacemaker potential (arrows).
(P < 0.01)at 10-6M of ouabain. Simultaneously, the outward tail current was decreased in a concentration-dependent way (Fig. 5A). These activation curves were normalized by taking the amplitude at + 3 0 mV as 1.0 (Fig. 5B). The voltage of halfmaximum activation was unaffected, and the average value was - 2 . 3 + 0.3 mV (n = 6). On the other hand, the ]h current was enhanced by 23.3 __ 2.4% (n = 6, P<0.05) at l0 - s M , by 4 3 . 3 + 5 . 8 % ( n = 6 , P < 0 . 0 1 ) at 10-TM, and by 4 6 . 6 + 5 . 1 % ( n = 6 , P < 0.001) at l0 -6 M of ouabain. As shown in Fig. 3, the transient inward current (Its) occurred in the presence of ouabain (10 -s to 10 -6 M). Increasing concentrations of ouabain produced a more marked /ti current. The Iti at l0 -6 M ouabain was elicited on depolarizing steps as well as on repolarizing steps. Pretreatment with clonidine (10 -6 M) did not elicit arrhythmias, although ouabain (at 3 x l0 -7 M ) alone elicited these at 86% incidence (Fig. 6A and B).
Clonidine is a specific ct2-adrenoceptor agonist. Clonidine (10-6M) decreased the spontaneous activity (by 11.5 + 2.3%, n = 6, P < 0.05). Addition of ouabain (3 x 10 -7 M) caused a positive chronotropic effect (by 18.6 + 3.1%, n = 6, P < 0.01) and tended to increase APA and l?maX (P >0.05). Prazosin (10-6M), an ~]-adrenoceptor blocker, also caused similar effects in five preparations. In contrast, yohimbine (10 -6 to 10-SM), an ~2-adrenoceptor blocker, increased APA and Vmax significantly. But, yohimhine caused a negative chronotropic effect (by approximately 57 to 120%) and had no effect on arrhythmias (n = 3). DISCUSSION Cardiac glycosides are used extensively in the treatment of heart failure. Ouabain plays a role in numerous physiological and pathophysiological pro-
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io-6 ._~""--t---,~: Fig. 3. The effects of ouabain on ionic currents in rabbit SA node preparation. Test pulses were applied to + 10 mV and - 7 0 mV from a holding potential of - 4 0 mV. The current traces are the slow inward current (1,), the outward current (IK), and the hyperpolarization-activated inward current (lh) in the presence of ouabain (10 -s to 10-6 M). Note the appearance of a transient inward current during the steps repolarizing and depolarizing pulses. The short line at the left of the current recordings represents 0 current level.
\,
Fig. 4. Current-voltage relationships for the slow inward current, the time-dependent outward current, and the hyperpolarization-activated inward current in rabbit spontaneously boating SA node cells. The values were measured from the current records of Fig. 3. Symbols used are the control (open circles), at 10-SM (triangles), 10-7M (squares), and 10-6 M (filled circles) of ouabain.
Effects of ouabain on action potentials and membrane currents Table 2. Effects of ouabain on the time course of inactivation phase of the I~ in rabbit sino-atrial node preparations Control
6
7.2 + 0.3 ms
22.3 + 1.2ms
Ouabain lO-SM I0 7M 10-tM
6 6 7
7.1 __.0.6 6.6±0.2* 5.3:1:0.3"*
22.4___0.8 23.1±0.4 35.4+0.9***
Values represent mean_+SEM, n: number of experiments. Zr: fast component. ¢s: slow component. *P < 0.05, **P < 0.01, ***P < 0.001, with respect to control value.
cesses associated with altered fluid and electrolyte metabolism and deviations from the normal blood pressure-blood volume relationship (Blaustein, 1993). The results presented here in rabbit spontaneously beating SA node preparations show that; (1) ouabain inhibited lsi and IK but stimulated lh, (2) ouabain caused negative chronotropic effects, and at high concentrations (10 -6 M) prolonged the APD (by approximately 12%), (3) Iti (or sinus arrest) occurred, and (4) clonidine and prazosin protected the development of arrhythmia induced by ouabain. The changes in ionic currents appear to reflect the nA
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alterations of the action potential configuration. The APD prolongation would be due mainly to the time-dependent I~ inhibition. The depressions in APA and I'm,,, althOugh not significant, might be produced by the Isl inhibition. Ouabain actions MyoCardial sarcolemmal Na+/K÷-ATPase is the pharmacological receptor for the positive inotropic action of digitalis (Schwartz et aL, 1982). Binding studies have shown that digitalis binds specifically to Na+/K÷-ATPase and the amount of binding is directly related to the degree of the positive inotropic action, with a high affinity for dissociation constants (Kd) of the order of 1 0 - 9 M (Forbush, 1983; Sweadner, 1988; Blanco et al., 1990). The enzyme has and//catalytic components, and the g subunit of Na÷/K÷-ATPase contains the digitalis receptors. Ouabain (due to the effects on the Na + electrochemical gradient and indirectly on Na÷-Ca 2+ exchange) modulates the storage of Ca 2+ in the ER/SR of a large variety of cells, and thereby plays an important role in the regulation of cell responsiveness. An electroneutral active Na ÷ transport may indirectly affect the membrane potential of a cell by maintaining the Na + and K ÷ concentration gradients across the cell membrane (Akera and Brody, 1978). Also, an electrogenic Na + pump may directly contribute to the membrane potential by net charge transport. Inhibition of the Na ÷ pump by ouabain can be expected to influence the RP in two ways; (1) by altering the electrogenic pump current and (2) by altering the K ÷ equilibrium potential. The maximum steady-state voltage that can be generated by the electrogenic Na ÷ pump with a stoichiometry of 3Na+:2K + is only < 1 0 m V (Thomas, 1972). Ouabain (2 x 10-r M) decreased the RP (by approximately 5 mV) and the APDs0 (by approximately 8%), and increased the phase 4 depolarization in the Purkinje fibres (Miura and Biedert, 1985). In the present experiments, however, ouabain tended to hyperpolarize the MDP (by < 5 m V a t 1 0 - 6 M ) . Blaustein (1993) calculated the theoretical membrane potential, and the calculated potential at 145mM INa]o would expect a decline of only 0.2 mV during partial Na + pump inhibition. Therefore, these results indicate that ouabain may not affect the RP to a significant extent.
Fig. 5. A c t i v a t i o n curves for the o u t w a r d current in the
absence and presence of ouabain. The values were taken from the amplitude of the outward tail current by normalizing the amplitude at +30 mV. (A) Activation curves in the presence o f different concentrations of ouabain. (B) Normalized activation curves (p~) from part A. Symbols represent the control (open circles), 10-SM (triangles), 10-7 M (squares), and 10-6 M (fdled circles) concentrations of ouabain.
Induction of dysrhythmia Cardiac glycosides which have a very low therapeutic index produce cardiotoxicity as a side effect. In this study, ouabain elicited arrhythmias and produced/t~. The Iti current is elicited under cellular Ca z+ overload condition (Satoh, 1993c; Satoh and
1596
HIROYASUSATOH
CIoNdine + O u a b a i n 10-' M b 3 X 1 0 .7 M .
A
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L_LL L-L_L £.L-L L-LL L_L_L Fig. 6. Modulation by clonidine of ouabain-induced arrhythmia. (A) Spontaneous action potentials, maximum rate of depolarization, and sinus rate. (B) Recordings at a fast time scale. Clonidine (10-6 M) depressed the spontaneously beating action potentials. Additional application of ouabain (3 x 10 -7 M ) produced no arrhythmia, and brought about a positive chronotropic effect with a regular rhythm.
Vassalle, 1985; Satoh et al., 1989b). Thus, the arrhythmias would be due to cellular Ca 2+ overload. Ouabain actually increases the [Ca]i level (Allen and Blinks, 1978; Wier and Hess, 1984). It has already been demonstrated that the Ca 2÷ overload in cardiac muscles elicited arrhythmias by delayed afterdepolarization and Iti, which results from changes in the permeability of the cation channels to Na ÷ or K ÷, or to both ions (Lederer and Tsien, 1976; Kass et al., 1978; Eisner and Lederer, 1979). Satoh et al. (1989b; 1993a, b, c) have recently reported that arrhythmias in rabbit SA node cells are elicited by the lti and an inward tail current. During washout of ouabain in arrhythmiaoccurring preparations, the arrhythmia was replaced by a sinus arrest (about 38% incidence at 10 -6 M). This appears to be induced by a further elevation of [Ca]i. Because, in the single SA node cells overloaded with Ca 2+, as detected by Ca2+-sensitive fluorescent dye (fura-2), the [Ca]i level was transiently potentiated to increase during washout of drugs (by approximately 30%) (Satoh, 1993a). This rebound after a switch to lower [Ca]o is also induced in canine Purkinje fibres (Satoh and Vassalle, 1985, 1989). The [Ca]i elevation may stimulate the Ca 2÷activated I X of cardiac muscles (Clusin et aL, 1975; Colatsky and Hogan, 1975; Satoh et al., 1989a). However, this study showed that the voltagedependent IK current was inhibited in a concen-
tration-dependent manner. The inhibition of Ix produces the APD prolongation, which tends to elevate the [Ca]i level (Satoh and Hashimoto, 1986). Thus, the Ix inhibition might be one of the factors causing the positive inotropic effect and eliciting the arrhythmias. Negative chronotropic effect Ouabain caused a negative chronotropic effect in a concentration-dependent manner. The major effect of ouabain was the CL prolongation in the SA node cells. There are three hypotheses for the pacemaker current during diastole; the lsi hypothesis, the g r hypothesis and the Ih hypothesis, according to the review of Noble (1984). In the present experiment, ouabain inhibited Isi and I X and enhanced I h. The decreases in Isi and I X currents during the diastolic potential are considered to contribute to the formation of the pacemaker potential, resulting in a negative chronotropic effect. Since the negative chronotropic effect was not affected by atropine, it would be a direct action of ouabain. It might also be produced by an indirect action of ouabain. Satoh and colleagues (1989a; 1993a, b, c) have shown recently that several drugs cause the negative chronotropic effect because of development of a cellular Ca 2+ overload in the rabbit SA node cell. Therefore, the Ca 2+ overload induced by ouabain may somewhere relate to the negative ehronotropic effect.
Effects of ouabain on action potentials and membrane currents Actions on 1.,i current
Effects of cardiac glycosides on voltage-dependent Ca 2+ current (Ic,) of heart muscle cells are still controversial. The glycosides produce the stimulatory action on /ca (Weingart et al., 1978; Lederer and Eisner, 1982; Marban and Tsien, 1982; Fischmeister et al., 1986), whereas they produce the inhibitory action (Josephson and Sperelakis, 1977; Deslauriers et al., 1982). This discrepancy may result from a change in the [Call level (caused by inhibition of the Na + pump) or from differences of cell types, suggesting that ouabain may have direct and indirect actions o n / c a current. In this study, ouabain inhibited the l~ in a concentration-dependent way. The lc, inhibition may be explained by a mechanism including: (1) the decrease may be due to the depression in channel opening by cellular Ca 2+ overload induced by ouabain, as the current peak of 15 is depressed under Ca 2+ overload conditions (Satoh et al., 1989a; Satoh, 1993a, c). (2) The decrease may be due to a direct action of ouabain on the ionic channels. (3) Ouabain may induce damage to the cell membrane (or ionic channels). The time course of the inactivation phase for lsi is composed of two (fast and slow) components (xr and x~). Ouabain decreases xf and increased zs. The fast component is [Ca]~ dependent and the slow component is voltage-dependent (Eckert and Chad, 1984; Carbone and Swandulla, 1989). So, the zf decrease would be caused by an elevation in the [Ca]i level caused by ouabain application. The % increase would be a direct action of ouabain and may lead to an elevated [Ca]i level. Modulation o f cardiotoxicity by ~-adrenoceptor
It is well known that a-adrenoceptor blocking drugs prevent or suppress some experimental arrhythmias (Gould et al., 1969; Rothans and Powell, 1975). In in vivo experiments, cardiac glycosides increase neurotransmitter overflow from autonomic nerve endings in a variety of tissues including heart (Pace and GiUis, 1976; Powis, 1983). The changes in the sympathetic discharge to the heart may result in the non-uniformity of electrical properties of myocardial cells (Kim et al., 1984). Clonidine stimulates peripheral presynapthetic ~2-adrenoceptors, causing a diminished release of noradrenaline from the nerve endings towards the heart, and also reduces the peripheral sympathetic tone by stimulation of central • 2-adrenoceptors, decreasing the plasma catecholamines (Starke et al., 1974; Cavero and Roach, 1980). The reduction of sympathetic tone and the inhibition of the neurotransmitter release may be a contributing factor for the antihypertensive and antiarrhythmic
1597
activities of clonidine. Gillis and Quest (1979) reported that the concentration of cardiac glycosides required to produce ventricular arrhythmias is reduced by enhancing the sympathetic outflow to the heart. In the present experiments, small rabbit SA node preparations (0.2 x 0.2ram) were used, but nerve terminals would still be present. Pretreatment with clonidine (a2-adrenoceptor agonist) and prazosin (al-adrenoceptor antagonist) blocked the development of arrhythmias induced by ouabain (but unaffected by yohimbine), which is consistent with previous reports (Lechat and Schmitt, 1982; Thomas and Tripathi, 1986). They demonstrated that u:-receptor stimulation and a~-receptor blockage protect against the ventricular arrhythmias and cardiac arrest induced by ouabain in guinea-pig hearts. In addition, -adrenoceptor agonists inhibit the Isi in the SA node cells (Satoh and Hashimoto, 1988). Therefore, clonidine and prazosin could prevent the development of arrhythmias caused by the modulation of neurotransmitter release and Isl inhibition.
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