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Blockade of the pacemaker current by intracellular application of UL-FS 49 and UL-AH 99 in sheep cardiac Purkinje fibers
European Journal of Pharmacology, 229 (1992) 55-62 © 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
55
EJP 52797
Blockade of the pacemaker current by intracellular application of UL-FS 49 and UL-AH 99 in sheep cardiac Purkinje fibers Pierre Paul Van Bogaert and Marnix Goethals Laboratory for Electrobiology, Department of Biochemistry, Physiology & Genetics, Uni~ersityof Antwerp (RUCA), B-2020Antwerpen, Belgium Received 27 August 1992, accepted 15 September 1992
UL-FS 49 (Zatebradine) and its quaternary derivative, UL-AH 99, were injected by iontophoresis in shortened sheep cardiac Purkinje fibres. The if pacemaker current changes were analyzed using the two-microelectrode voltage-clamp technique. Injection of either drug resulted in a decrease of the maximal diastolic depolarization rate as a consequence of a reduction in if amplitude, with no changes in the kinetics of this current or in voltage dependence. The if blockade was proportional to the total charge injected. After drug iontophoresis under conditions where no if current was activated, an exponential use-dependent decline in if tail current was observed during the application of a voltage-clamp pulse train activating if. A slow recovery from blockade, measured after prolonged hyperpolarizations, followed exponential kinetics. Recovery rate and extent of steady state recovery increased with more negative potentials. This suggests that bradycardiac agents interact with the if channel in cationic form from the inside of the cell. Bradycardiac agents; Sinus node inhibitors; Zatebradine; Pacemaker currents; Purkinje fibres; Iontophoresis; Use-dependent blockade; (Electrophysiology)
1. Introduction
When UL-FS 49 is added to the Tyrode bathing solution, the if p a c e m a k e r current of sheep cardiac Purkinje fibres is reversibly blocked in a use-dependent way (Van Bogaert et al., 1990). On the other hand, the blockade by extracellular cesium of the inward if current is fast, not use-dependent, and can rapidly be reversed by washout (Di Francesco, 1982). Presumably, cesium ions act by plugging the selectivity filter of the if channel from the outside. UL-FS 49's slow start of action and washout, both in vivo and in vitro (Kobinger and Lillie, 1988) suggest that this drug like some Na + and Ca 2+ channel blocking agents (Hescheler et al., 1982; Hille, 1992) has to pass a barrier constituted by the cell m e m b r a n e before it reaches its receptor. UL-FS 49 is a tertiary amine with a pKa of 8.73 and an octanol-buffer partition coefficient of 0.6 (fig. 1). At an extracellular p H of 7.4, 4.47% of the drug is present in uncharged form in the Tyrode solution. This form will equilibrate across the cell membrane. At an intracellu-
Correspondence to: P.P. Van Bogaert, Electrobiology, Department of Biochemistry, Physiology & Genetics, University of Antwerp (R.U.C.A.), Groenenborgerlaan 171, B-2020 Antwerpen, Belgium. Tel. 32/3/2180187, fax 32/3/2180217.
lar p H of 7.2 the steady state total intracellular drug concentration will be 1.43 times the total extracellular concentration with 96.8% in the cationic form (Roos and Boron, 1981). Based on the voltage dependence of the if current recovery rate constants after blockade by UL-FS 49, it had been assumed that this drug acts on the if channel in cationic form from the inside of the cell (Van Bogaert et al., 1990). This hypothesis was now tested by iontophoresis of UL-FS 49 and its permanently charged quaternary derivative U L - A H 99 (fig. 1) in shortened sheep cardiac Purkinje fibres. Preliminary notes on this study have already been published in abstract form (Van Bogaert and Goethals, 1988; Van Bogaert, 1992).
2. Materials and methods
2.1. Preparation and experimental protocol Short ( ~ 1 mm) Purkinje strands from sheep left ventricles were used with the two-microelectrode voltage-clamp technique as described earlier (Van Bogaert et al., 1990). Iontophoresis was done with a doublebarrelled microelectrode made of theta glass capillary with an o.d. of 2 m m (Hilgenberg, Malsfeld, Germany) used as a current-passing microelectrode. One barrel
56
was filled with 3 M KC1 and the other was filled with a saturated solution of UL-FS 49 (0.08 M) or U L - A H 99 (0.02 M) dissolved in distilled water. The microelectrode tip was bevelled to a resistance of _+4 MS2 with a Staehli FLM 80 beveller (A.W. Staehli, Pieterlen, Switzerland). Under control conditions the barrel containing 3 M KC1 was used to inject the transmembrane current. First, action potentials were elicited using short constant-current pulses. Under voltage-clamp conditions the fibre was clamped at a holding potential close to the normal resting potentials ( ~ - 8 0 mV). De- and hyperpolarizing voltage steps of various amplitude were imposed at regular intervals to analyze the if current kinetics under steady state conditions. After the control run, UL-FS 49 or U L - A H 99 was injected into the fibre by application of outward current pulses (50 to 100 nA amplitude, 400-ms duration and at a rate of 0.8 Hz) through the drug-containing barrel (Kass et al., 1982). The duration of the iontophoretic injection varied from a few minutes to more than 30 rain, depending on the amount of drug we wanted to inject. The drug-containing barrel was then disconnected from the constant current source and the KC1 barrel was used as a current-passing microelectrode. Action potentials were elicited to evaluate the effects of drug injection on the diastolic depolarization rate. Voltageclamp analysis of the if current was then performed under the same conditions as for the control. Membrane potential and transmembrane currents were recorded on a Brush 2400S pen recorder. With normally polarized Purkinje fibres constantcurrent injection of the drug resulted in the activation of action potentials with each current pulse. The if current blockade being use-dependent, this resulted in a certain amount of i t, current blockade at the end of the drug injection period. Control runs effected with the same amount of charge injected from the KCl-filled barrel did not show any change in if properties. In
order to accumulate the drug into the fibre without causing simultaneous blockade of the if current, the drug was injected at a m e m b r a n e potential at which the if channels are in closed configuration (positive to - 5 0 mV). No if blockade was previously observed under these conditions (Van Bogaert et al., 1990). A number of shortened Purkinje fibres are depolarized at plateau potential level as a consequence of damage caused either by the shortening procedure or by insertion of the microelectrodes. The drug was first injected by iontophoresis into these depolarized preparations. At the end of the injection, the fibre was voltage-clamped at a holding potential of - 8 0 mV and repetitively pulsed between - 8 0 mV and - 4 0 mV for 500 ms at a frequency between 0.8 and 0.04 Hz. At various time intervals the if tail current following a depolarizing pulse was allowed to decay completely by interruption of the train for _+ 10 s. The pulse train continued afterwards. Recovery from blockade was measured by imposing long-lasting hyperpolarizations between - 80 and - 100 mV after a steady state blockade had been obtained with a pulse train. At various time intervals the holding potential was reset at - 8 0 mV and a test pulse to - 3 5 m V was imposed to evaluate the amount of recovery of the amplitude of the if tail current.
2.2. Solutions and drugs The composition of the Tyrode solution was as follows (in mM): NaC1 139; KC1 4; CaC12 3.6; MgCI 2 1; glucose 5.5. The solution was buffered with 10 mM H E P E S (Fluka, A.G. Buchs, Switzerland) and neutralized to p H 7.4 with N a O H . The solution was gassed with 100% 0 2. The higher Ca 2+ content of the Tyrode solution helps to stabilize the fibres during the rather long duration of the experiments, especially when increasing amounts of charge have to be injected. The main effect of higher divalent ion concentrations is a few mV shift of the if current activation range to less
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Fig. 1. Chemical structures of UL-FS 49 and its quaternary derivative, U L - A H 99.
57 negative potentials. UL-FS 49 (1,3,4,5,-tetrahydro-7,8dimethoxy-3-[3-[[2-(3,4-dimethoxyphenyl)-ethyl]]methylimimo]-propyl]-2H-3-benzazepin-2-on hydrochloride) and U L - A H 99 (N-[2-(3,4-dimethoxyphenyl)-ethyl]-N(7,8-dimethoxy-3-benzazepin-2-on3-yl)-N,N-dimethylammoniumiodide) were kindly supplied by Dr. Karl Thomae Gmbh (Biberach, F.R.G). All solutions were prewarmed to 35°C. The temperature of the experimental chamber (volume 1.2 ml and flow rate 4 m l / m i n ) was 38°C.
2.3. Data analysis The if current steady state activation curve was obtained by plotting the peak values of the if tail currents, measured at the holding potential after deand hyperpolarizing voltage steps in the pacemaker voltage range, as a function of the step potential. The rate constants of if current activation and deactivation were calculated by fitting the current changes during and after the steps to single exponential functions. The rate constant of blockade induction during a pulse train was calculated by plotting the logarithm of the difference between the peak if tail current, following a voltage step of the train, and the steady state peak if tail current measured after a sufficient number of pulses as a function of the pulse number, N. This was fitted by a least square function. The recovery rate was calculated by plotting the logarithm of one minus the ratio of the peak if tail current, measured after a test pulse imposed after some time at the hyperpolarized membrane potential, and the peak if tail current measured after a very long time at the same potential, as a function of the time spent at the hyperpolarized membrane potential.
after iontophoresis of UL-FS 49. The holding current shifted in an outward direction, and the amplitude of the time- and voltage-dependent membrane currents in the pacemaker voltage range was reduced. This was the consequence of a decrease in total available if current, as illustrated by the reduced amplitude of the if steady state activation curve (fig. 3A). There was no change in the voltage dependence of if activation nor were the if current kinetics altered (fig. 3B). The fraction of fully activated if current blocked by UL-FS 49 iontophoresis increased sigmoidally with the logarithm of the total charge injected (fig. 4A).
3.1.2. Transient effects When particular precautions are taken to accumulate UL-FS 49 into a fibre without concomitant blockade of the if current, use-dependent blockade of this current can be clearly measured. In fig. 5A the first voltage-clamp pulse from - 8 0 mV to - 3 0 mV elicited
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3.1.1. Steady state effects Iontophoretic injections of UL-FS 49 resulted in a reversible reduction of the maximal diastolic depolarization rate (fig. 2A), proportional to the total charge injected up to complete elimination of the diastolic depolarization. Other changes in action potential configuration were a reduction of the amplitude of the 'notch' between phase 1 and phase 2 of the action potential, and a shift to more positive potentials of the action potential plateau. No change, some reduction or an increase in action potential duration at 90% of the repolarization was observed. The resting potential shifted to more negative potentials. Fig. 2B compares the properties of the if current measured under voltage-clamp conditions before and
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Fig. 2. (A) Action potentials recorded under control conditions and after iontophoresis of a total charge of 28.5/xC from the barrel filled with UL-FS 49. (B) Measurements of the if pacemaker current under voltage-clamp conditions. The holding potential was - 80 mV throughout the experiment. Membrane currents, measured during and after voltage-clamp steps to -35 and -100 mV, are illustrated before and after a total charge of 206.6 /~C was injected from the barrel filled with UL-FS 49. The holdingcurrent shifted from - 20 to - 10 nA.
58
an if peak tail current of 66 nA, close to the last control tail of 70 nA. With successive pulses of the train the peak tail current declined following an exponential time course with a rate constant, A, equal to 0.0127 N-~. Steady state blockade was obtained at a peak if tail current amplitude of 39 nA (59% of control). In fibres with comparable electrical properties (total available if current close to 100 nA) injected with 79.3 /xC of total charge the steady state if blockade averaged 50_+ 6% (n = 3) of the control if. The rate constant, A, was 0.019 + 0.006 N - 1 (n = 3). Subsequent hyperpolarizations, holding the membrane potential between - 8 0 mV and - 1 0 0 mV for several minutes, resulted in an exponential recovery from blockade with a rate constant of 0.006 s = at - 100 mV (fig. 5B). At - 80 mV the recovery rate was 0.0012 s 1. The more negative the holding potential, the faster and the greater was the extent of recovery from blockade.
3.2. Injections of UL-AH 99 3.2.1. Steady state effects After iontophoresis of 103.6 p~C total charge out of the barrel filled with UL-AH 99, the diastolic depolarization was completely suppressed (fig. 6A). The fast repolarization after the action potential spike (phase 1) was slowed down and the shoulder between the plateau and the phase 3 repolarization was greatly reduced. The resting potential shifted to more negative voltages. Under voltage-clamp conditions, the holding current needed to clamp the fibre at - 8 0 mV changed from 0 to +20 nA after UL-AH 99 iontophoresis. The timeand voltage-dependent currents, both during and after the voltage steps, were reduced in amplitude (fig. 6B). The amplitude of the steady state if activation curve was reduced with no change in voltage dependence or current kinetics (fig. 3C,D). In this particular experiment, the maximal available if current was 207 nA in
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Fig. 3. (A) Steady state activation curve of the if current as a function of the membrane potential. Control curve (open circles) and curve measured after iontophoresis of a total charge of 206.6 p~C (filled triangles) are described by the function: I t = [ l + e x p ( E m - E 0 . 5 ) / k ] I. E0. 5 = - 8 1 . 5 mV and k = 5.63 mV under control conditions and E0. 5 = - 8 2 . 5 mV and k = 4.88 mV after UL-FS 49 injection. The respective curve amplitudes are 123 nA and 81 nA. (B) Voltage-dependence of the rate constants of activation and deactivation for the i e current. Open circles are for control, and filled circles for after iontophoresis of 23/xC, open triangles for after 43.6 p~C and filled triangles for after application of the 140-p~C charge by iontopboresis from the barrel filled with UL-FS 49. (C) Steady state activation curve of the if current as a function of membrane potential. Control curve (open circles) and after the iontophoresis of 103.6 ~C total charge from the UL-AH 99-containing barrel (filled triangles). The continuous line is the solution of I t = [1 + exp(E m - E 0 . 5 ) / k ] - ~ with E0 s = - 87 mV and k = 5.85 mV in the control. After iontophoresis of UL-AH 99, E0. 5 = - 87 mV and k = 4.51 inV. The respective curve amplitudes were 206 and 40 hA. (D) Voltage-dependence of the rate constants of activation and deactivation for the if current. Open circles are for the control, filled circles for after 49.4/xC, open triangles for after 22.2 ,aC and filled triangles for after a 32-~C charge was applied by iontophoresis.
59 t h e c o n t r o l . I n j e c t i o n o f 49 /xC r e d u c e d if to 93 n A ( 4 5 % o f c o n t r o l ) a n d a s e c o n d i n j e c t i o n o f 22 / z C r e d u c e d it to 60 n A ( 2 9 % o f c o n t r o l ) . A f i n a l i n j e c t i o n o f 3 2 / z C r e d u c e d if to 40 n A ( 1 9 % o f c o n t r o l ) . T h e c u r v e r e l a t i n g t h e f r a c t i o n o f if b l o c k e d by i o n t o p h o r e s i s o f U L - A H 99 to t h e l o g a r i t h m o f t h e total amount of charge injected had a sigmoidal shape (fig. 4B).
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3.2.2. Transient effects U s e - d e p e n d e n t if b l o c k a d e w a s also o b s e r v e d a f t e r i o n t o p h o r e s i s o f U L - A H 99 (fig. 7B). T h e p e a k if tail c u r r e n t d e c l i n e d e x p o n e n t i a l l y w i t h a r a t e c o n s t a n t , A, e q u a l to 0.058 N t to a s t e a d y s t a t e v a l u e c l o s e to 4 5 % o f t h e initial p e a k tail c u r r e n t . Hyperpolarization of the preparation after a blocka d e - i n d u c i n g v o l t a g e - c l a m p p u l s e t r a i n l e d to r e c o v e r y o f t h e if c u r r e n t a m p l i t u d e . T h e s t e a d y s t a t e f r a c t i o n o f if c u r r e n t , r e c o v e r e d f r o m u s e - d e p e n d e n t b l o c k a d e ,
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Fig. 4. (A) Fraction of fully activated if current blocked, as a function of total charge of UL-FS 49 applied by iontophoresis. Different symbols represent different preparations receiving successive injections of UL-FS 49. In order to compare different preparations (n = 5) with amplitudes of if varying between 34 and 187 nA, all results were scaled to a representative preparation with a maximum available if of 100 nA. (B) Same curve calculated after injection of various amounts of UL-AH 99. All results were scaled to a preparation with a maximal available if of 100 nA in the control ((n = 5) and if amplitude varying between 62 and 259 nA).
Fig. 5. (A) Use-dependent blockade of the if current after iontophoresis of UL-FS 49. A total charge of 19 /xC was first injected under constant-current conditions from a depolarized potential of - 3 0 inV. The fibre was then clamped at - 8 0 mV and repetitively pulsed between - 80 mV and - 30 mV (duration 500 ms) at a rate of 0.4 Hz. The train was interrupted at regular intervals to allow complete decay of the tail current. The amplitude of the peak i t. tail current declined exponentially along the continuous line fitted to the data. The membrane currents measured during and after the first (n = 1) and last (n = 8) voltage-clamp pulse of the train are displayed. (B) Recovery from use-dependent blockade. After iontophoresis of UL-FS 49, the peak tail if current, measured at - 7 8 mV following a step to - 3 5 mV, declined from 54 nA in the control to 22 hA. The holding potential was set for increasing durations of time at -100 mV and the peak tail i t. current was measured at regular intervals, following a step identical to the control. The tail current recovered to 78% of its control amplitude with a rate constant of 0.006 s 1.
increased sigmoidally with more negative holding pot e n t i a l s in t h e v o l t a g e r a n g e b e t w e e n - 4 0 a n d - 1 2 0 m V (fig. 7A). R e c o v e r y was w i t h i n 9 5 . 7 % o f t h e c o n t r o l if a m p l i t u d e ( b e f o r e i o n t o p h o r e s i s ) w i t h an e s t i m a t e d r a t e c o n s t a n t o f r e c o v e r y o f 0.0065 s - 1 at - 1 2 0 m V . A t - 100 m V t h e if c u r r e n t r e c o v e r e d to 7 9 . 7 % o f t h e c o n t r o l w i t h a r a t e c o n s t a n t o f 0.004 s i a n d at - 8 0 m V if r e c o v e r e d to 3 7 % o f c o n t r o l at an e v e n s l o w e r rate. N o r e c o v e r y was o b s e r v e d at - 4 0 inV.
4. Discussion I n t r a c e l l u l a r i n j e c t i o n by i o n t o p h o r e s i s in m u l t i c e l l u lar c a r d i a c p r e p a r a t i o n s s u c h as P u r k i n j e f i b e r s has b e e n s u c c e s s f u l l y p e r f o r m e d w i t h cyclic A M P ( T s i e n , 1973), l i d o c a i n e a n d its q u a t e r n a r y d e r i v a t i v e , Q X - 3 1 4
60
(Glicklich and Hoffman, 1978), and with tetraethylammonium as well as with tetrabutylammonium ions (Kass et al., 1982). Based on previous experiments, the average intracellular UL-FS 49 needed to block 50% of the if current can be calculated as 1.43 times the extracellular concentration of 4.5 * 10 -7 M, i.e. 6.4 * 10 -7 M, when the pulse frequency is 0.04 Hz (Van Bogaert et al., 1990). This concentration is several orders of magnitude less than the intracellular tetrabutylammonium concentration that must be attained to block K + channels, i.e. 2mM. A molecular weight of = 500 in the case of UL-FS 49 and U L - A H 99 is not an obstacle for cell-to-cell diffusion through the intracellular hydrophillic channels connecting the multiple cells of the Purkinje fiber (De Mello, 1982). Using preparations with morphologic
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Fig. 7. (A) Steady stale fraction of if recovered from blockade after iontophoresis of a total charge of 103.6/xC UL-AH 99, as a function of membrane potential. Re-induction of blockade, after each recovery run at a different membrane potential, was obtained by a voltage clamp pulse train as described in fig. 7B. (B). Use-dependent blockade of the if current after iontophoresis of a total charge of 103,6 /zC UL-AH 99. First, recovery from blockade was obtained by prolonged hyperpolarization to - 1 0 0 mV. The i r blockade was re-induced by repetitively pulsing the fibre under voltage-clamp conditions, between - 8 0 and - 3 8 mV at a rate of 0.073 Hz (step duration: 500 ms). The peak tail i r current declined exponentially from a peak value of 29 nA to a steady state value of 45% of the initial value. The continuous line was fitted to the data and is I t = 13.1 + 16.4 exp(-0.0576 * N).
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fl. Fig. 6. (A) Action potentials recorded under control conditions and after iontophoresis of a total charge of 103.6 /xC from the barrel containing UL-AH 99. (B) Measurement of the if pacemaker current under voltage-clamp conditions. The holding potential was - 80 mV throughout the experiment. Membrane currents, measured during and after a voltage-clamp step to - 3 5 mV and - 1 0 0 mV, are illustrated under control conditions and after iontophoretic injection of a total charge of 103.6/.LC. The holding current changed from 0 to + 20 nA.
properties comparable to those described before (Kass et al. 1982), enough UL-FS 49 or U L - A H 99 could be applied to the cytoplasm of the Purkinje fibre to cause a reproducible blockade of the it. current, even if the transfer number of these drugs is presumably very low. Iontophoresis of UL-FS 49 and of U L - A H 99 had the same effects on resting and action potential as well as on the if pacemaker current as did the addition of UL-FS 49 to the Tyrode solution. Similarly, these effects can be explained as the consequence of a reduction in if current amplitude. Use-dependent blockade of the if current was also observed after iontophoresis of both substances. The kinetics of blockade induction as well as those of recovery are in reasonable agreement with those observed when the drug is added to the modified Tyrode solution. In the latter case the recovery process follows an exponential time course, with a very low rate constant of 0.0043 _+ 0.0003 s J (n = 7) at - 100 mV (Van
61 Bogaert et al., 1990). The faster recovery rate of 0.006 s - 1 measured at - 100 m V after iontophoresis of ULFS 49 can be explained by the higher Na ÷ content of the Tyrode solution compared to the modified Tyrode solution where only 35 mM Na ÷ was present. This clearly influences recovery kinetics (Van Bogaert, 1989). When the curves relating the fractional if reduction and the logarithm of the total charge injected are compared (cf. fig. 4A,B), clear differences can be noticed: 50% blockade is obtained after iontophoresis of 48.8 /xC U L - A H 99 versus 79.3 ~zC UL-FS 49, the maximal if reduction was 88% with U L - A H 99 versus 68% with UL-FS 49. The steepness of the curve was more pronounced in the case of U L - A H 99. The effects of tertiary amines are less pronounced and more rapidly reversible because a small fraction of the total drug (3% at p H i 7.2 in the case of UL-FS 49) exists in the uncharged form and will leak through the cell m e m b r a n e (Glicklich and Hoffman, 1978; Hescheler et al., 1982). This will cause a steep longitudinal gradient along the fibre and a lower average intracellular drug concentration. A clearly different voltage-dependence of the recovery process was seen when we compared the curve relating the steady state if fraction recovered as a function of m e m b r a n e potential (fig. 7A) and the same curve calculated from data obtained in modified Tyrode containing UL-FS 49 (fig. 9B in Van Bogaert et al., 1990). Both curves can be described as Boltzman functions with a half-maximal potential, E0. 5 = - 8 5 mV, and a steepness, k = 12 mV, in the case of U L - A H 99. With UL-FS 49 in modified Tyrode, the half-maximal potential E0. 5 was - 7 8 . 5 mV and k = 35.8 mV. The much steeper voltage dependence of if recovery after U L - A H 99 injection resulted in no recovery at all at m e m b r a n e potentials positive to - 4 0 m V while, with UL-FS 49, a substantial (25%) fraction of the if channels did recover at - 4 0 mV and beyond, albeit extremely slowly. This presumably results from the fact that the permanently charged U L - A H 99 molecules are trapped within the closed if channels. Only at voltages negative enough to open the if channels will unbinding of U L - A H 99 take place (Armstrong, 1971).
deduced from the use-dependent action of UL-FS 49 and its derivative, that the cationic drug will interact with the if channel once it is in the open configuration. The fact that p e r m e a n t ions like Na ÷ do influence the unbinding kinetics of UL-FS 49 can only be explained by a blocking site located in the channel itself (Van Bogaert, 1989). This is also the explanation for the voltage dependence of the recovery rate constants such as would be generated by the movement of a charged molecule down a substantial fraction of the m e m b r a n e electrical field. As for K + and Na + channels, the blocking site for UL-FS 49 is presumably located between the selectivity filter, controlling the entry of ions from the outside (Di Francesco, 1982), and the physical 'gate mechanism', controlling the channel configuration and the access to the cytoplasm (Hille, 1992). The use and voltage dependence of if current blockade by the bradycardiac agents shares many properties with the K + current blockade by T E A (Armstrong, 1971), the Na + channel blockade by local anaesthetics and the Ca 2+ channel blockade by organic Ca 2+ channel blockers ( H o n d e g h e m and Katzung, 1977; 1984; for a review see Hille, 1992). Provided the contribution of the if current to the diastolic depolarization process of SA node cells cannot simply be ignored and if this current is as sensitive to blockade by bradycardiac agents as the related if current of ventricular conductive tissue, bradycardiac agents like UL-FS 49 are then expected to selectively slow the firing rate of the SA node cell in a use- and frequency-dependent manner. This was demonstrated in conscious dogs: there is a strong correlation between the control sinus rate and the extent of the rate-lowering effect (cf. fig. 7 in Kobinger and Lillie, 1988). As these authors pointed out this correlation can be regarded as a 'self-limiting' mechanism: it might explain why sinoatrial standstill or extreme bradycardia has not been reported and has never been observed with any of the 'specific bradycardiac agents' except at toxic doses when presumably other currents like the Ca 2+ currents are blocked by these substances.
Acknowledgements
4.1. If blockade and topology of the if channel The mode of action of organic if blockers from inside the cell after having passed the cell m e m b r a n e in their uncharged form is similar to the action of local anesthetics on the sodium channel or organic calcium channel blockers ( H o n d e g h e m and Katzung, 1977; Hescheler et al., 1982; H o n d e g h e m and Katzung, 1984; Hille, 1992). The common mode of action of tertiary and quaternary derivatives of UL-FS 49 implies that the cationic form of the tertiary drug is active. It can be
This work was supported by a N.F.W.O. grant for equipment (S215-CD-E130) and by the Born-Bunge Stichting (Afd. Cardiovasculair Onderzoek).
References Armstrong, C.M., 1971, Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons, J. Gen. Physiol. 58, 413. Courtney, K.R., J.J. Kendig and E.N. Cohen, 1978, The rates of
62 interaction of local anesthetics with sodium channels in nerve, J. Pharmacol. Exp. Ther. 207, 594. De Mello, W.C., 1982, Cell-to-cell communication in heart and other tissues, Prog. Biophys. Mol. Biol. 39, 147. Di Francesco, D., 1982, Block and activation of the pace-maker channel in calf Purkinje fibers: effects of potassium, caesium and rubidium, J. Physiol. (London) 329, 485. Glicklich, J.I. and B.F. Hoffman, 1978, Sites of action and active forms of lidocaine and some derivatives on cardiac Purkinje fibres, Circ. Res. 43, 638. Hescheler, J., D. Pelzer, G. Trube and W. Trautwein, 1982, Does the organic calcium channel blocker D600 act from inside or outside on the cardiac cell membrane?, Pfluegers Arch. 393, 287. Hille, B., 1992, Ionic channels of excitable membranes, 2nd edn. (Sinauer Associates Inc, Sunderland, MA). Hondeghem, L. and B. Katzung, 1977, Time- and voltage-dependent interactions of antiarrhythmic drugs with cardiac sodium channels, Biochim. Biophys. Acta 472, 373. Hondeghem, L.M. and B.G. Katzung, 1984, Antiarrhythmic agents: the modulated receptor mechanism of action of sodium and calcium channel-blocking drugs, Ann. Rev. Pharmacol. Toxicol. 24, 387. Kass, R.S., T. Scheuer and K.J. Malloy, 1982, Block of outward current in cardiac Purkinje fibres by injection of quaternary ammonium ions, J. Gen. Physiol. 79, 1041. Kobinger, W. and C. Lillie, 1988, Falipamil (AQ-A39) and UL-FS 49, Cardiovasc. Drug Rev. 6, 35.
Roos, A. and W.F. Boron, 1981, lntraeellular pH, Physiol. Rev. 61, 296. Tsien, R.W., 1973, Adrenaline-like effects of intracellular iontophoresis of cyclic AMP in cardiac Purkinje fibres, Nature 245, 120. Van Bogaert, P.P., 1989, Extracellular caesium accelerates the onset of use-dependent if pacemaker current blockade, Biophys. J. 55, 291A. Van Bogaert, P.P., 1992, Kinetics of the blocking and unblocking processes of the if pacemaker current by bradycardic agents injected into isolated cardiac Purkinje fibres, J. Physiol. (London) 446, 342 P. Van Bogaert, P.P. and M. Goethals, 1987, Pharmacological influence of specific bradycardic agents on the pacemaker current of sheep cardiac Purkinje fibres. A comparison between three different molecules, European Heart J. 8 (Suppl. L), 35. Van Bogaert, P.P. and M. Goethals, 1988, Use-dependent block of the if pacemaker current by intracellular application of UL-FS 49 and its quaternary derivative UL-AH 99 in sheep cardiac Purkinje fibres, Pfluegers Arch. 412, $35. Van Bogaert, P.P., M. Goethals and C. Simoens, 1990, Use- and frequency-dependent blockade by UL-FS 49 of the if pacemaker current in sheep cardiac Purkinje fibres, European J. Pharmacol. 187, 241.
55
EJP 52797
Blockade of the pacemaker current by intracellular application of UL-FS 49 and UL-AH 99 in sheep cardiac Purkinje fibers Pierre Paul Van Bogaert and Marnix Goethals Laboratory for Electrobiology, Department of Biochemistry, Physiology & Genetics, Uni~ersityof Antwerp (RUCA), B-2020Antwerpen, Belgium Received 27 August 1992, accepted 15 September 1992
UL-FS 49 (Zatebradine) and its quaternary derivative, UL-AH 99, were injected by iontophoresis in shortened sheep cardiac Purkinje fibres. The if pacemaker current changes were analyzed using the two-microelectrode voltage-clamp technique. Injection of either drug resulted in a decrease of the maximal diastolic depolarization rate as a consequence of a reduction in if amplitude, with no changes in the kinetics of this current or in voltage dependence. The if blockade was proportional to the total charge injected. After drug iontophoresis under conditions where no if current was activated, an exponential use-dependent decline in if tail current was observed during the application of a voltage-clamp pulse train activating if. A slow recovery from blockade, measured after prolonged hyperpolarizations, followed exponential kinetics. Recovery rate and extent of steady state recovery increased with more negative potentials. This suggests that bradycardiac agents interact with the if channel in cationic form from the inside of the cell. Bradycardiac agents; Sinus node inhibitors; Zatebradine; Pacemaker currents; Purkinje fibres; Iontophoresis; Use-dependent blockade; (Electrophysiology)
1. Introduction
When UL-FS 49 is added to the Tyrode bathing solution, the if p a c e m a k e r current of sheep cardiac Purkinje fibres is reversibly blocked in a use-dependent way (Van Bogaert et al., 1990). On the other hand, the blockade by extracellular cesium of the inward if current is fast, not use-dependent, and can rapidly be reversed by washout (Di Francesco, 1982). Presumably, cesium ions act by plugging the selectivity filter of the if channel from the outside. UL-FS 49's slow start of action and washout, both in vivo and in vitro (Kobinger and Lillie, 1988) suggest that this drug like some Na + and Ca 2+ channel blocking agents (Hescheler et al., 1982; Hille, 1992) has to pass a barrier constituted by the cell m e m b r a n e before it reaches its receptor. UL-FS 49 is a tertiary amine with a pKa of 8.73 and an octanol-buffer partition coefficient of 0.6 (fig. 1). At an extracellular p H of 7.4, 4.47% of the drug is present in uncharged form in the Tyrode solution. This form will equilibrate across the cell membrane. At an intracellu-
Correspondence to: P.P. Van Bogaert, Electrobiology, Department of Biochemistry, Physiology & Genetics, University of Antwerp (R.U.C.A.), Groenenborgerlaan 171, B-2020 Antwerpen, Belgium. Tel. 32/3/2180187, fax 32/3/2180217.
lar p H of 7.2 the steady state total intracellular drug concentration will be 1.43 times the total extracellular concentration with 96.8% in the cationic form (Roos and Boron, 1981). Based on the voltage dependence of the if current recovery rate constants after blockade by UL-FS 49, it had been assumed that this drug acts on the if channel in cationic form from the inside of the cell (Van Bogaert et al., 1990). This hypothesis was now tested by iontophoresis of UL-FS 49 and its permanently charged quaternary derivative U L - A H 99 (fig. 1) in shortened sheep cardiac Purkinje fibres. Preliminary notes on this study have already been published in abstract form (Van Bogaert and Goethals, 1988; Van Bogaert, 1992).
2. Materials and methods
2.1. Preparation and experimental protocol Short ( ~ 1 mm) Purkinje strands from sheep left ventricles were used with the two-microelectrode voltage-clamp technique as described earlier (Van Bogaert et al., 1990). Iontophoresis was done with a doublebarrelled microelectrode made of theta glass capillary with an o.d. of 2 m m (Hilgenberg, Malsfeld, Germany) used as a current-passing microelectrode. One barrel
56
was filled with 3 M KC1 and the other was filled with a saturated solution of UL-FS 49 (0.08 M) or U L - A H 99 (0.02 M) dissolved in distilled water. The microelectrode tip was bevelled to a resistance of _+4 MS2 with a Staehli FLM 80 beveller (A.W. Staehli, Pieterlen, Switzerland). Under control conditions the barrel containing 3 M KC1 was used to inject the transmembrane current. First, action potentials were elicited using short constant-current pulses. Under voltage-clamp conditions the fibre was clamped at a holding potential close to the normal resting potentials ( ~ - 8 0 mV). De- and hyperpolarizing voltage steps of various amplitude were imposed at regular intervals to analyze the if current kinetics under steady state conditions. After the control run, UL-FS 49 or U L - A H 99 was injected into the fibre by application of outward current pulses (50 to 100 nA amplitude, 400-ms duration and at a rate of 0.8 Hz) through the drug-containing barrel (Kass et al., 1982). The duration of the iontophoretic injection varied from a few minutes to more than 30 rain, depending on the amount of drug we wanted to inject. The drug-containing barrel was then disconnected from the constant current source and the KC1 barrel was used as a current-passing microelectrode. Action potentials were elicited to evaluate the effects of drug injection on the diastolic depolarization rate. Voltageclamp analysis of the if current was then performed under the same conditions as for the control. Membrane potential and transmembrane currents were recorded on a Brush 2400S pen recorder. With normally polarized Purkinje fibres constantcurrent injection of the drug resulted in the activation of action potentials with each current pulse. The if current blockade being use-dependent, this resulted in a certain amount of i t, current blockade at the end of the drug injection period. Control runs effected with the same amount of charge injected from the KCl-filled barrel did not show any change in if properties. In
order to accumulate the drug into the fibre without causing simultaneous blockade of the if current, the drug was injected at a m e m b r a n e potential at which the if channels are in closed configuration (positive to - 5 0 mV). No if blockade was previously observed under these conditions (Van Bogaert et al., 1990). A number of shortened Purkinje fibres are depolarized at plateau potential level as a consequence of damage caused either by the shortening procedure or by insertion of the microelectrodes. The drug was first injected by iontophoresis into these depolarized preparations. At the end of the injection, the fibre was voltage-clamped at a holding potential of - 8 0 mV and repetitively pulsed between - 8 0 mV and - 4 0 mV for 500 ms at a frequency between 0.8 and 0.04 Hz. At various time intervals the if tail current following a depolarizing pulse was allowed to decay completely by interruption of the train for _+ 10 s. The pulse train continued afterwards. Recovery from blockade was measured by imposing long-lasting hyperpolarizations between - 80 and - 100 mV after a steady state blockade had been obtained with a pulse train. At various time intervals the holding potential was reset at - 8 0 mV and a test pulse to - 3 5 m V was imposed to evaluate the amount of recovery of the amplitude of the if tail current.
2.2. Solutions and drugs The composition of the Tyrode solution was as follows (in mM): NaC1 139; KC1 4; CaC12 3.6; MgCI 2 1; glucose 5.5. The solution was buffered with 10 mM H E P E S (Fluka, A.G. Buchs, Switzerland) and neutralized to p H 7.4 with N a O H . The solution was gassed with 100% 0 2. The higher Ca 2+ content of the Tyrode solution helps to stabilize the fibres during the rather long duration of the experiments, especially when increasing amounts of charge have to be injected. The main effect of higher divalent ion concentrations is a few mV shift of the if current activation range to less
OCH3 C H 3 0 ~
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MW = 5 9 8 . 5
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Fig. 1. Chemical structures of UL-FS 49 and its quaternary derivative, U L - A H 99.
57 negative potentials. UL-FS 49 (1,3,4,5,-tetrahydro-7,8dimethoxy-3-[3-[[2-(3,4-dimethoxyphenyl)-ethyl]]methylimimo]-propyl]-2H-3-benzazepin-2-on hydrochloride) and U L - A H 99 (N-[2-(3,4-dimethoxyphenyl)-ethyl]-N(7,8-dimethoxy-3-benzazepin-2-on3-yl)-N,N-dimethylammoniumiodide) were kindly supplied by Dr. Karl Thomae Gmbh (Biberach, F.R.G). All solutions were prewarmed to 35°C. The temperature of the experimental chamber (volume 1.2 ml and flow rate 4 m l / m i n ) was 38°C.
2.3. Data analysis The if current steady state activation curve was obtained by plotting the peak values of the if tail currents, measured at the holding potential after deand hyperpolarizing voltage steps in the pacemaker voltage range, as a function of the step potential. The rate constants of if current activation and deactivation were calculated by fitting the current changes during and after the steps to single exponential functions. The rate constant of blockade induction during a pulse train was calculated by plotting the logarithm of the difference between the peak if tail current, following a voltage step of the train, and the steady state peak if tail current measured after a sufficient number of pulses as a function of the pulse number, N. This was fitted by a least square function. The recovery rate was calculated by plotting the logarithm of one minus the ratio of the peak if tail current, measured after a test pulse imposed after some time at the hyperpolarized membrane potential, and the peak if tail current measured after a very long time at the same potential, as a function of the time spent at the hyperpolarized membrane potential.
after iontophoresis of UL-FS 49. The holding current shifted in an outward direction, and the amplitude of the time- and voltage-dependent membrane currents in the pacemaker voltage range was reduced. This was the consequence of a decrease in total available if current, as illustrated by the reduced amplitude of the if steady state activation curve (fig. 3A). There was no change in the voltage dependence of if activation nor were the if current kinetics altered (fig. 3B). The fraction of fully activated if current blocked by UL-FS 49 iontophoresis increased sigmoidally with the logarithm of the total charge injected (fig. 4A).
3.1.2. Transient effects When particular precautions are taken to accumulate UL-FS 49 into a fibre without concomitant blockade of the if current, use-dependent blockade of this current can be clearly measured. In fig. 5A the first voltage-clamp pulse from - 8 0 mV to - 3 0 mV elicited
00
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3.1.1. Steady state effects Iontophoretic injections of UL-FS 49 resulted in a reversible reduction of the maximal diastolic depolarization rate (fig. 2A), proportional to the total charge injected up to complete elimination of the diastolic depolarization. Other changes in action potential configuration were a reduction of the amplitude of the 'notch' between phase 1 and phase 2 of the action potential, and a shift to more positive potentials of the action potential plateau. No change, some reduction or an increase in action potential duration at 90% of the repolarization was observed. The resting potential shifted to more negative potentials. Fig. 2B compares the properties of the if current measured under voltage-clamp conditions before and
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Fig. 2. (A) Action potentials recorded under control conditions and after iontophoresis of a total charge of 28.5/xC from the barrel filled with UL-FS 49. (B) Measurements of the if pacemaker current under voltage-clamp conditions. The holding potential was - 80 mV throughout the experiment. Membrane currents, measured during and after voltage-clamp steps to -35 and -100 mV, are illustrated before and after a total charge of 206.6 /~C was injected from the barrel filled with UL-FS 49. The holdingcurrent shifted from - 20 to - 10 nA.
58
an if peak tail current of 66 nA, close to the last control tail of 70 nA. With successive pulses of the train the peak tail current declined following an exponential time course with a rate constant, A, equal to 0.0127 N-~. Steady state blockade was obtained at a peak if tail current amplitude of 39 nA (59% of control). In fibres with comparable electrical properties (total available if current close to 100 nA) injected with 79.3 /xC of total charge the steady state if blockade averaged 50_+ 6% (n = 3) of the control if. The rate constant, A, was 0.019 + 0.006 N - 1 (n = 3). Subsequent hyperpolarizations, holding the membrane potential between - 8 0 mV and - 1 0 0 mV for several minutes, resulted in an exponential recovery from blockade with a rate constant of 0.006 s = at - 100 mV (fig. 5B). At - 80 mV the recovery rate was 0.0012 s 1. The more negative the holding potential, the faster and the greater was the extent of recovery from blockade.
3.2. Injections of UL-AH 99 3.2.1. Steady state effects After iontophoresis of 103.6 p~C total charge out of the barrel filled with UL-AH 99, the diastolic depolarization was completely suppressed (fig. 6A). The fast repolarization after the action potential spike (phase 1) was slowed down and the shoulder between the plateau and the phase 3 repolarization was greatly reduced. The resting potential shifted to more negative voltages. Under voltage-clamp conditions, the holding current needed to clamp the fibre at - 8 0 mV changed from 0 to +20 nA after UL-AH 99 iontophoresis. The timeand voltage-dependent currents, both during and after the voltage steps, were reduced in amplitude (fig. 6B). The amplitude of the steady state if activation curve was reduced with no change in voltage dependence or current kinetics (fig. 3C,D). In this particular experiment, the maximal available if current was 207 nA in
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Fig. 3. (A) Steady state activation curve of the if current as a function of the membrane potential. Control curve (open circles) and curve measured after iontophoresis of a total charge of 206.6 p~C (filled triangles) are described by the function: I t = [ l + e x p ( E m - E 0 . 5 ) / k ] I. E0. 5 = - 8 1 . 5 mV and k = 5.63 mV under control conditions and E0. 5 = - 8 2 . 5 mV and k = 4.88 mV after UL-FS 49 injection. The respective curve amplitudes are 123 nA and 81 nA. (B) Voltage-dependence of the rate constants of activation and deactivation for the i e current. Open circles are for control, and filled circles for after iontophoresis of 23/xC, open triangles for after 43.6 p~C and filled triangles for after application of the 140-p~C charge by iontopboresis from the barrel filled with UL-FS 49. (C) Steady state activation curve of the if current as a function of membrane potential. Control curve (open circles) and after the iontophoresis of 103.6 ~C total charge from the UL-AH 99-containing barrel (filled triangles). The continuous line is the solution of I t = [1 + exp(E m - E 0 . 5 ) / k ] - ~ with E0 s = - 87 mV and k = 5.85 mV in the control. After iontophoresis of UL-AH 99, E0. 5 = - 87 mV and k = 4.51 inV. The respective curve amplitudes were 206 and 40 hA. (D) Voltage-dependence of the rate constants of activation and deactivation for the if current. Open circles are for the control, filled circles for after 49.4/xC, open triangles for after 22.2 ,aC and filled triangles for after a 32-~C charge was applied by iontophoresis.
59 t h e c o n t r o l . I n j e c t i o n o f 49 /xC r e d u c e d if to 93 n A ( 4 5 % o f c o n t r o l ) a n d a s e c o n d i n j e c t i o n o f 22 / z C r e d u c e d it to 60 n A ( 2 9 % o f c o n t r o l ) . A f i n a l i n j e c t i o n o f 3 2 / z C r e d u c e d if to 40 n A ( 1 9 % o f c o n t r o l ) . T h e c u r v e r e l a t i n g t h e f r a c t i o n o f if b l o c k e d by i o n t o p h o r e s i s o f U L - A H 99 to t h e l o g a r i t h m o f t h e total amount of charge injected had a sigmoidal shape (fig. 4B).
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3.2.2. Transient effects U s e - d e p e n d e n t if b l o c k a d e w a s also o b s e r v e d a f t e r i o n t o p h o r e s i s o f U L - A H 99 (fig. 7B). T h e p e a k if tail c u r r e n t d e c l i n e d e x p o n e n t i a l l y w i t h a r a t e c o n s t a n t , A, e q u a l to 0.058 N t to a s t e a d y s t a t e v a l u e c l o s e to 4 5 % o f t h e initial p e a k tail c u r r e n t . Hyperpolarization of the preparation after a blocka d e - i n d u c i n g v o l t a g e - c l a m p p u l s e t r a i n l e d to r e c o v e r y o f t h e if c u r r e n t a m p l i t u d e . T h e s t e a d y s t a t e f r a c t i o n o f if c u r r e n t , r e c o v e r e d f r o m u s e - d e p e n d e n t b l o c k a d e ,
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Fig. 4. (A) Fraction of fully activated if current blocked, as a function of total charge of UL-FS 49 applied by iontophoresis. Different symbols represent different preparations receiving successive injections of UL-FS 49. In order to compare different preparations (n = 5) with amplitudes of if varying between 34 and 187 nA, all results were scaled to a representative preparation with a maximum available if of 100 nA. (B) Same curve calculated after injection of various amounts of UL-AH 99. All results were scaled to a preparation with a maximal available if of 100 nA in the control ((n = 5) and if amplitude varying between 62 and 259 nA).
Fig. 5. (A) Use-dependent blockade of the if current after iontophoresis of UL-FS 49. A total charge of 19 /xC was first injected under constant-current conditions from a depolarized potential of - 3 0 inV. The fibre was then clamped at - 8 0 mV and repetitively pulsed between - 80 mV and - 30 mV (duration 500 ms) at a rate of 0.4 Hz. The train was interrupted at regular intervals to allow complete decay of the tail current. The amplitude of the peak i t. tail current declined exponentially along the continuous line fitted to the data. The membrane currents measured during and after the first (n = 1) and last (n = 8) voltage-clamp pulse of the train are displayed. (B) Recovery from use-dependent blockade. After iontophoresis of UL-FS 49, the peak tail if current, measured at - 7 8 mV following a step to - 3 5 mV, declined from 54 nA in the control to 22 hA. The holding potential was set for increasing durations of time at -100 mV and the peak tail i t. current was measured at regular intervals, following a step identical to the control. The tail current recovered to 78% of its control amplitude with a rate constant of 0.006 s 1.
increased sigmoidally with more negative holding pot e n t i a l s in t h e v o l t a g e r a n g e b e t w e e n - 4 0 a n d - 1 2 0 m V (fig. 7A). R e c o v e r y was w i t h i n 9 5 . 7 % o f t h e c o n t r o l if a m p l i t u d e ( b e f o r e i o n t o p h o r e s i s ) w i t h an e s t i m a t e d r a t e c o n s t a n t o f r e c o v e r y o f 0.0065 s - 1 at - 1 2 0 m V . A t - 100 m V t h e if c u r r e n t r e c o v e r e d to 7 9 . 7 % o f t h e c o n t r o l w i t h a r a t e c o n s t a n t o f 0.004 s i a n d at - 8 0 m V if r e c o v e r e d to 3 7 % o f c o n t r o l at an e v e n s l o w e r rate. N o r e c o v e r y was o b s e r v e d at - 4 0 inV.
4. Discussion I n t r a c e l l u l a r i n j e c t i o n by i o n t o p h o r e s i s in m u l t i c e l l u lar c a r d i a c p r e p a r a t i o n s s u c h as P u r k i n j e f i b e r s has b e e n s u c c e s s f u l l y p e r f o r m e d w i t h cyclic A M P ( T s i e n , 1973), l i d o c a i n e a n d its q u a t e r n a r y d e r i v a t i v e , Q X - 3 1 4
60
(Glicklich and Hoffman, 1978), and with tetraethylammonium as well as with tetrabutylammonium ions (Kass et al., 1982). Based on previous experiments, the average intracellular UL-FS 49 needed to block 50% of the if current can be calculated as 1.43 times the extracellular concentration of 4.5 * 10 -7 M, i.e. 6.4 * 10 -7 M, when the pulse frequency is 0.04 Hz (Van Bogaert et al., 1990). This concentration is several orders of magnitude less than the intracellular tetrabutylammonium concentration that must be attained to block K + channels, i.e. 2mM. A molecular weight of = 500 in the case of UL-FS 49 and U L - A H 99 is not an obstacle for cell-to-cell diffusion through the intracellular hydrophillic channels connecting the multiple cells of the Purkinje fiber (De Mello, 1982). Using preparations with morphologic
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Fig. 7. (A) Steady stale fraction of if recovered from blockade after iontophoresis of a total charge of 103.6/xC UL-AH 99, as a function of membrane potential. Re-induction of blockade, after each recovery run at a different membrane potential, was obtained by a voltage clamp pulse train as described in fig. 7B. (B). Use-dependent blockade of the if current after iontophoresis of a total charge of 103,6 /zC UL-AH 99. First, recovery from blockade was obtained by prolonged hyperpolarization to - 1 0 0 mV. The i r blockade was re-induced by repetitively pulsing the fibre under voltage-clamp conditions, between - 8 0 and - 3 8 mV at a rate of 0.073 Hz (step duration: 500 ms). The peak tail i r current declined exponentially from a peak value of 29 nA to a steady state value of 45% of the initial value. The continuous line was fitted to the data and is I t = 13.1 + 16.4 exp(-0.0576 * N).
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fl. Fig. 6. (A) Action potentials recorded under control conditions and after iontophoresis of a total charge of 103.6 /xC from the barrel containing UL-AH 99. (B) Measurement of the if pacemaker current under voltage-clamp conditions. The holding potential was - 80 mV throughout the experiment. Membrane currents, measured during and after a voltage-clamp step to - 3 5 mV and - 1 0 0 mV, are illustrated under control conditions and after iontophoretic injection of a total charge of 103.6/.LC. The holding current changed from 0 to + 20 nA.
properties comparable to those described before (Kass et al. 1982), enough UL-FS 49 or U L - A H 99 could be applied to the cytoplasm of the Purkinje fibre to cause a reproducible blockade of the it. current, even if the transfer number of these drugs is presumably very low. Iontophoresis of UL-FS 49 and of U L - A H 99 had the same effects on resting and action potential as well as on the if pacemaker current as did the addition of UL-FS 49 to the Tyrode solution. Similarly, these effects can be explained as the consequence of a reduction in if current amplitude. Use-dependent blockade of the if current was also observed after iontophoresis of both substances. The kinetics of blockade induction as well as those of recovery are in reasonable agreement with those observed when the drug is added to the modified Tyrode solution. In the latter case the recovery process follows an exponential time course, with a very low rate constant of 0.0043 _+ 0.0003 s J (n = 7) at - 100 mV (Van
61 Bogaert et al., 1990). The faster recovery rate of 0.006 s - 1 measured at - 100 m V after iontophoresis of ULFS 49 can be explained by the higher Na ÷ content of the Tyrode solution compared to the modified Tyrode solution where only 35 mM Na ÷ was present. This clearly influences recovery kinetics (Van Bogaert, 1989). When the curves relating the fractional if reduction and the logarithm of the total charge injected are compared (cf. fig. 4A,B), clear differences can be noticed: 50% blockade is obtained after iontophoresis of 48.8 /xC U L - A H 99 versus 79.3 ~zC UL-FS 49, the maximal if reduction was 88% with U L - A H 99 versus 68% with UL-FS 49. The steepness of the curve was more pronounced in the case of U L - A H 99. The effects of tertiary amines are less pronounced and more rapidly reversible because a small fraction of the total drug (3% at p H i 7.2 in the case of UL-FS 49) exists in the uncharged form and will leak through the cell m e m b r a n e (Glicklich and Hoffman, 1978; Hescheler et al., 1982). This will cause a steep longitudinal gradient along the fibre and a lower average intracellular drug concentration. A clearly different voltage-dependence of the recovery process was seen when we compared the curve relating the steady state if fraction recovered as a function of m e m b r a n e potential (fig. 7A) and the same curve calculated from data obtained in modified Tyrode containing UL-FS 49 (fig. 9B in Van Bogaert et al., 1990). Both curves can be described as Boltzman functions with a half-maximal potential, E0. 5 = - 8 5 mV, and a steepness, k = 12 mV, in the case of U L - A H 99. With UL-FS 49 in modified Tyrode, the half-maximal potential E0. 5 was - 7 8 . 5 mV and k = 35.8 mV. The much steeper voltage dependence of if recovery after U L - A H 99 injection resulted in no recovery at all at m e m b r a n e potentials positive to - 4 0 m V while, with UL-FS 49, a substantial (25%) fraction of the if channels did recover at - 4 0 mV and beyond, albeit extremely slowly. This presumably results from the fact that the permanently charged U L - A H 99 molecules are trapped within the closed if channels. Only at voltages negative enough to open the if channels will unbinding of U L - A H 99 take place (Armstrong, 1971).
deduced from the use-dependent action of UL-FS 49 and its derivative, that the cationic drug will interact with the if channel once it is in the open configuration. The fact that p e r m e a n t ions like Na ÷ do influence the unbinding kinetics of UL-FS 49 can only be explained by a blocking site located in the channel itself (Van Bogaert, 1989). This is also the explanation for the voltage dependence of the recovery rate constants such as would be generated by the movement of a charged molecule down a substantial fraction of the m e m b r a n e electrical field. As for K + and Na + channels, the blocking site for UL-FS 49 is presumably located between the selectivity filter, controlling the entry of ions from the outside (Di Francesco, 1982), and the physical 'gate mechanism', controlling the channel configuration and the access to the cytoplasm (Hille, 1992). The use and voltage dependence of if current blockade by the bradycardiac agents shares many properties with the K + current blockade by T E A (Armstrong, 1971), the Na + channel blockade by local anaesthetics and the Ca 2+ channel blockade by organic Ca 2+ channel blockers ( H o n d e g h e m and Katzung, 1977; 1984; for a review see Hille, 1992). Provided the contribution of the if current to the diastolic depolarization process of SA node cells cannot simply be ignored and if this current is as sensitive to blockade by bradycardiac agents as the related if current of ventricular conductive tissue, bradycardiac agents like UL-FS 49 are then expected to selectively slow the firing rate of the SA node cell in a use- and frequency-dependent manner. This was demonstrated in conscious dogs: there is a strong correlation between the control sinus rate and the extent of the rate-lowering effect (cf. fig. 7 in Kobinger and Lillie, 1988). As these authors pointed out this correlation can be regarded as a 'self-limiting' mechanism: it might explain why sinoatrial standstill or extreme bradycardia has not been reported and has never been observed with any of the 'specific bradycardiac agents' except at toxic doses when presumably other currents like the Ca 2+ currents are blocked by these substances.
Acknowledgements
4.1. If blockade and topology of the if channel The mode of action of organic if blockers from inside the cell after having passed the cell m e m b r a n e in their uncharged form is similar to the action of local anesthetics on the sodium channel or organic calcium channel blockers ( H o n d e g h e m and Katzung, 1977; Hescheler et al., 1982; H o n d e g h e m and Katzung, 1984; Hille, 1992). The common mode of action of tertiary and quaternary derivatives of UL-FS 49 implies that the cationic form of the tertiary drug is active. It can be
This work was supported by a N.F.W.O. grant for equipment (S215-CD-E130) and by the Born-Bunge Stichting (Afd. Cardiovasculair Onderzoek).
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