Naunyn-Schmiedeberg's Arch. Pharmacol. 307, 9-19 (1979) Naunyn-Schmiedeberg's Archives of Pharmacology (cid:14)9 by Springer-Verlag 1979 The Relation Between the Current Underlying Pacemaker Activity and Beta-Adrenoceptors in Cardiac Purkinje Fibres: A Study Using Adrenaline, Procaine, Atenolol and Penbutolol Keitaro Hashimoto*, Otto Hauswirth, Heinz D. Wehner, and Rolf Ziskoven Physiologisehes Institut II, Universit/it Bonn, Wilhelmstrasse 31, D-5300 Bonn, Federal Republic of Germany Summary. The pacemaker current - iK2 -- in cardiac Purkinje fibres was analysed using the voltage clamp technique described by Deck et al. (1964). (-)- Adrenaline (5.5 (cid:12)9 10 -6 M) causes the wellknown shift of the Hodkin-Huxley kinetics in the depolarizing direction. Procaine (7.3.10 -4 M) does not cause any further shift of s~ in the presence of adrenaline. Atenolol (3.8-10- s M) causes a backshift of the kinetics in the negative direction in the presence of adrenaline and procaine. The instantaneous current-voltage re- lationship (iK2) is altered neither with adrenaline, nor with procaine or atenolol. The results exclude the possibility that the local anaesthetic side effect of many beta-adrenoceptor blocking agents may be involved in the backshift of the s-kinetics. The voltage de- pendence of the reciprocals of the time constants is shifted in a similar way as s~ by the sympathomimetic or blocking drugs. Following the application of (-)- adrenaline (5.5-10 -6 M) the (-)-isomere ofpenbutolol (1.7 and 3.5.10-6M) is about equally effective in shifting the kinetics back as the (+)-isomere (3.5 (cid:12)9 10 -5 M). In the presence of (-)-adrenaline, the (+)- and (-)-forms of penubutolol cause virtually no change Of the instantaneous current-voltage relation- ship, iK2. Thus, (-)-adrenaline and (+)- and (-)- penbutolol are aiming for the s-kinetics whose voltage dependence is controlled by the electric field near the iK2-channel of the membrane and do not influence the number of the iK2-channels. These findings suggest that the sympathomimetic or blocking agents influence the s-kinetics of the pacemaker current iK2 by altering the electric field; the fully activated current-voltage re- lationship which is proportional to the number of the open iKz-channels is not subject to any appreciable modification. The results conclusively show that the Send offprint requests to O. Hauswirth at the above address * Present address: Department of Pharmacology, University of Niigata, Niigata, Japan kinetics of the pacemaker current can be controlled by beta-adrenoceptors. Key words: Pacemaker current - Beta-adrenoceptors - Voltage clamp analysis. Introduction The pacemaker potential in cardiac Purkinje fibres is brought about by decline of slow outward current (Deck et al., 1964; Vassalle, 1966; McAllister and Noble, 1966, 1967; Noble and Tsien, 1968), which allows the non time dependent steady state background current (Peper and Trautwein, 1969) to depolarize the membrane. Noble and Tsien (1968) gave a detailed description of the kinetics and the rectifier properties of this pacemaker current which they designated iK2. This current component is described by two factors: iK2 = zK2 "s (1) where iK2 is the instantaneous current-voltage re- lationship depending on voltage only and showing inward going rectification with a marked negative slope in the region positive to about - 70 mV (Noble and Tsien, 1968). s is a dimensionless variable analogous to n (Hodgkin and Huxley, 1952) which controls the degree of activation and which follows from a chemical reaction [1 - s] [s]. [s] is described under the assumption of a first order kinetic: ds dt e~ (l-s)- fis s. (2) 0028-1298/79/0307/0009/$02.20
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`10 Naunyn-Schmiedeberg's Arch. Pharmacol. 307 (1979) ~s and/~ are rate constants which are voltage dependent only and which empirically have been found to be exponential functions (see Hodgkin and Huxley, 1952; Noble, 1962; McAllister et al., 1975; Tsien, 1974a). In the specialized conducting tissue (Purkinje fibre) the accelerating effect of adrenaline on the pacemaker potential has been analysed by Hauswirth et al. (1968) and Tsien (1974a, b). It was shown that adrenaline and isoprenaline shift the voltage dependence of the kinetics "of iK2 in the depolarizing direction (see also Hauswirth et al. 1976b). Likewise it was shown that beta- adrenoceptor blocking agents like pronethalol (10-6M) (Hauswirth et al., 1968) and propranolol (10-6 M) (Tsien, 1974 a) shift the kinetics back towards their original position. Tsien (1974a) suggested that i~2 may be controlled via beta-adrenoceptors since only beta-sympathomimetic compounds like adrenaline and isoprenaline cause a shift of the s-kinetics, and since, in striking contrast to this finding, the application of alpha-stimulating drugs did not cause a measurable shift of the kinetics of iKz. Cranefield et al. (1971) and Giotti et al. (1968, 1973) provided evidence that in Purkinje fibres alpha- receptors are also present. In contrast to the findings of Giotti et al. (1968, 1973), Quadbeck and Reiter (1975) in guinea-pig papillary muscle did not find any partici- pation of alpha-adrenoceptors in the prolonging effect of the action potential caused by noradrenaline (10-7 to 10 -6 M) or isoprenaline (10 -s M). From the quali- tative resemblence of the effects of noradrenaline (10- v to 10 -6 M) and isoprenaline (i0 -s M) both of which were inhibited by propranolol (5.10 -6 M), these au- thors concluded that the prolongation of the action potential induced by noradrenaline (10-7 to 10-6 M) or isoprenaline (10 -8 M) is mediated solely by beta- adrenoceptors. Tsien (1974a) did not find any detect- able shift of the steady-state degree of activation of iK2 with phenylephi'ine (10 -s M) in the presence of pro- pranolol (10 .6 M). this suggestion was confirmed by Hauswirth et al. (1976a) in a somewhat extended study using methoxamine (1.6.10-4M) a very selective alpha-stimulating agent with beta-adrenoceptor block- ing activity (hnai et al., 1961). However, some of the beta-adrenoceptor blocking agents such as pronethalol and propranolol exert a strong local anaesthetic side effect (Gill and Vaughan- Williams, 1964; Morales Aguilerfi and Vaughan- Williams, 1965; Papp and Vaughan-Williams, 1969). Taking all this information into account the follow- ing questions should be answered in voltage clamp experiments : 1. Does the local anaesthetic side effect of beta- blockers play any role in the backshift of the s-kinetics ? 2. Do beta-blockers without prior administration of adrenaline alter the s-kinetics? 3. Do the drugs mentioned above alter the voltage dependence of the kinetics or the instantaneous current-voltage relationship? 4. Does the well known and widely studied stereo- specificity of beta-adrenoceptor stimulating and block- ing agents hold true also in case of a particular current component, i.e. of a single membrane channel? In order to answer these questions a full voltage clamp analysis of iKz consisting of the measurements of the fully activated current-voltage relationship, the steady state activation curve and the time and rate constants is required. Methods The present experiments were performed in Purkinje fibres of sheep, the approach being similar to the previous work of Noble and Tsien (1968), Hauswirth et al. (1968), Tsien (1974a, b) and Hauswirth et al. (1976a, b). The pacemaker potassium current, iK2, was analysed in preparations which were shorter than one space constant (1 -2 mm, Weidmann, 1952) using two microelectrode voltage clamp technique (Deck et al. 1964). Preparations. Sheep hearts were obtained from the slaughter house immediately after sacrifice. After the ventricles were opened and rinsed, the hearts were placed in a cooled (about 8 ~ C) bathing solution. Purkinje fibres were cut out of both ventricles not later than 60 min after slaughter and kept in oxygenated bathing solution at about 35 ~ C. The fibres were given at least 1 h for healing over before they were impaled. The used perspex chamber contained about 0.8 ml solution and was surrounded by aqueous heating fluid which maintained the temperature of the bathing fluid near 35 - 36 ~ C. The bathing solution aerated with 95 % 02 and 5 % CO2 flowed from one of several reservoirs continously through the chamber; the exchange of the solution in the bath was quickly performed through a short (30 cm) common tube between the chamber and a tap where the flow could be switched from one reservoir to another. At a flow rate of 1 ml/min the exchange of the bathing solution was 95 ~o complete after about 180s. The composition of the bath solution was the following (mM) : Na+: 148,3; K+:4.0; Ca2+:1.8; Mg2+:0.5; C1-:144.6; HCO~: 12.0; HzPO2: 0.35; glucose: 15. (-)-Adrenaline hydrochloride (5.5.10- 6 M) Merck, Darmstadt, Germany) was used as a catecholamine to stimulate beta- adrenoceptors. In order to exclude the possibility that the local anaesthetic side effect of many beta-adrenoceptor blocking agents (Gill and Vaughan-Williams, 1964; Papp and Vaughan-Williams, 1969) might be involved in the backshift of the kinetics of iKz, procaine (novocaine hydrochloride, Hoechst, Frankfurt/Main, Germany) was administered in rather large concentration (7.3-10-4M) following the application of adrenaline. Finally, a racemate of atenolol (38 (cid:12)9 10 -6 M) (Imperial Chemical Industries, Macclesfield0 Cheshire, England) was added as a beta-adrenoceptor blocking agent exerting practically no local anaesthetic activity in the concentration used (Hashimoto and Hauswirth, unpublished). Since only very small amounts of (+)- and (-)-atenolol could be made available to us, we have decided to use an additional beta- blocker, penbutolol (Hoechst, Frankfurt/Main, Germany), whose optical isomeres were available in larger quantities. In all experiments, the fibres were given about 30min for equilibration to every new solution before any measurements were obtained. This holds also true in experiments where successively several solutions were administered to the same fibre.
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`K. Hashimoto et al. : Pacemaker Current and Beta-Adrenoceptors l] Current and Voltage Recording Intracellular microelectrodes filled with 3 M KC1 which had a d.c. resistence between 10 and 20 mr2 were used for voltage recording and injecting current. Short Purkinje fibres (1 - 2 ram) were impailed with two microelectrodes. The current passing microelectrode was in- serted midway between the two cut ends of the preparation. The second microelectrode was impailed about 300 ~tm apart. The input stages connected to the intracellular voltage measuring microelectrode and to the reference electrode consisted of integrated circuits with field effect inputs (Ri, = 1012 ~2) which are arranged as voltage followers. The transmembrane current was measured by an operational amplifier connected to the bath by an Ag-AgC1 half cell electrode. This signal was recorded together with the membrane potential signal on a number of various devices: A R 5103 N storage oscilloscope (Tektronix), a Tektronix 565 oscilloscope where the beam was often photographed with a camera (Grass, Quincy, Mass., U.S.A.), a Brush 240 four channel pen recorder whose frequence response was diminished by 3dB at 150Hz and a TR3200 tape recorder (Bell & Howell, Basingstoke, Hampshire, England) which, in a second run, allowed a signal to be displayed on the oscilloscope or pen recorder with different amplification and time scale than was used in the original experiment. In some experiments, a Schwarzer PEE/4B pen recorder was used whose frequency response was reduced by 3 dB at 300 Hz. In most experiments, the current signal was additionally filtered by a RC-circuit and displayed on one chart recorder channel at high amplification. The filtering reduced noise but did not affect the measurement of iK2 since this current component has a time constant of the order of two seconds in the range of the normal pacemaker potential. Experimental Procedure The analysis of the kinetics and rectifier properties of iK2 has been extensively described by Noble and Tsien (1968) and Tsien (1974a). Imposing rectangular voltage pulses on the membrane, the steady state degree of activation of iK2 (Soy) was measured as the amplitude of current tails on the return to the holding potential. The time constant of iKa at potentials positive to the threshold of the excitatory sodium current was measured by progressively shor- tening the clamp pulses and measuring the amplitudes of current tails following depolarizing clamp pulses (envelope test). The instan- taneous current-voltage relationship was obtained by calculating the quotient of the amplitudes of iKz during and following depolari- zations to various clamp levels (iA and iB; see Fig. 2A inset). This rectifier ratio (ig/iB) is then multiplied by the amplitude of s 2 obtained at the same holding potential resulting in the values of the "rectifier function', t'K2 (Noble and Tsien, 1968). Possible Errors a) Voltage Drift. The most obvious source of error is the drift of voltage in this kind of experiments (see Tsien, 1974a). The electronic device caused little drift over several hours - as tested on a model circuit the most serious source is the tip of the voltage measuring microelectrode (see also Tsien, 1974a). In general, the experiments were performed with continuous impailements of the microelec- trodes. In the present experiments the voltage drift somewhat less than 1 mV/h. b) Voltage Non-Uniformity. The possibility of voltage non-uniform- ity has been discussed by Tsien (1974a) on the grounds of experi- mental findings of Deck et al. (1964), Sommer and Johnson (1968), MoNey and Page (1972), McGuigan (1974) and Weidmann (1952) and taking into account theoretical considerations of Jack et al. (1975) and of Johnson and Lieberman (1971). The essence of Tsien's discussion shows that distortions of voltage distribution are consider- able in the attempts to clamp iNA but much less pronounced in the case of a small, slowly changing outward current like iKa where the degree of non-uniformity should be small (Tsien, 1974a; Jack et al. 1975). Results Local Anaesthetics do not Cause any Shift of s~ in the Presence of Adrenaline Weld and Bigger (1976) showed that lidocaine itself (1 rag/l) had no influences on the iK2 current system. After the administration of adrenaline and shifting the s-kinetics in the depolarizing direction (5 preparations), procaine (7.3-10-4M) was not able to restore the kinetics towards their original position (Fig. 1, 1 prep- aration). It is shown in Figs. 2 and 3 that the ad- ditional application of a beta-adrenoceptor blocker (atenolol 38.10-6 M) is needed to cause a backshift of the s-kinetics (3 preparations). In Fig. 1, panel A shows the tracing of the clamp current under control con- ditions : The most important and characteristic feature is that the current tails on the return to the holding potential (iB, see also inset in Fig. 2A) of -80 mV fol- lowing depolarizing and hyperpolarizing clamp pulses are almost equal in their amplitudes. Panel B shows the wellknown alteration measured 30 min after the appli- cation of adrenaline (5.5.10 -6 M) (Hauswirth et al., 1968): The current tail on the return to the holding potential following depolarization to -50 mV is in- creased in its amplitude and somewhat accelerated; following the hyperpolarizing clamp to -90mV the current record shows virtually no remaining time dependence. In addition, the current level at the holding potential is shifted in the inward direction as described previously by Hauswirth et al. (1968) and indicated in Fig. 2 by arrows. PanelC demonstrates that, in the presence of adrenaline, there is essentially no change 30min after the addition of procaine (7.3.10-*M) (1 experiment). Although the time constants of the cur- rents during and following depolarization to -50 mV are further accelerated, this has to be regarded as the full development of the adrenaline effect and was seen already prior to the administration of procaine. Finally, in the last panel it is shown that the beta- adrenoceptor blocking agent atenolol (38-10-6M) cause a backshift of the voltage dependence of the s- kinetics in the presence of adrenaline and procaine (3 experiments): the time courses of the time dependent currents during and following the depolarizing clamp are slower again and the amplitude of the current tail of the return to the holding potential is smaller. Moreover, the occurrence of time dependent currents in response to hyperpolarization can be observed. Figure 2A shows
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`12 Naunyn-Schmiedeberg's Arch. Pharmacol. 307 (1979) mV -50 -80 -90 / I 165 -1 -2 o[ 107A -1 -2 ,...-..-,.-.-- I 0 f I ! I 20 40 60 seconds A B D Fig. 1 One example of original records of mem- brane currents of a Purkinje fibre as responses to long lasting depolarizations and hyperpolarizations under various conditions. Holding potential: - 80 inV. PanelA: Control; PanelB: Influence of adrenaline (5.5-10 6M); PanelC: Effect of procaine (730 - 10 -6 M) plus adrenaline. Note that this tracing is practically identical with that in PanelB; PanelD: Effect of the beta- adrenoceptor blocker atenolol (38.10-6 M) in the presence of adrenaline (5.5.10 -6 M) and procaine (730.10 -6 M). The beta blocker diminishes the amplitude of the tail current following depolarization and restores the time dependent of the current following hyperpolarization. Ordinate: top: Membrane voltage; below (PanelAD): membrane current. Abscissa: time; Starting the experiment, the pulses as well as the intervalls lasted for 15 s. With adrenaline, the time constant at the depolarized voltage level was slowed and the amplitude of the current on the return to the holding potentials was increased. Therefore, PanelB, C and D show durations of pulses as well as of the intervalls of 20 s. Horizontal arrows ~ indicate control holding current; --+ indicate holding current shifted in inward direction in the presence of adrenaline and procaine the curve relating the steady state degree of activation of iK2 and the membrane potential measured as the current tails (iB) on the return to the holding potential under the conditions mentioned above. Adrenaline shifts s~ (the normalized curve of Fig. 2A) in the depolarizing direction (Hauswirth et al., 1968; Tsien, 1974a) (Fig. 2B). The current tails ob- tained with plocaine in the presence of adrenaline do not show any additional shift of the curve. The beta- adrenoceptor blocking agent, however, produces a substantial backshift although not entirely to the original position. The average shift of the s-kinetics caused by adrenaline (5.5.10- 6 M) amounted to 20 mV (+ 2.2 mV); the backshift ranged close to 2/3 of the adrenaline shift with atenolol (38.10 6M) or (-)- penbutolol (17.10 -6 M) (Fig. 6) independently of the degree of the shift caused by a particular concentration of adrenaline (5.5.10 -6 M). With atenolol (racemate) as well as with (+)- or (-)-penbutolol (Fig. 6) the back- shift of the s-kinetics was not complete. On the other hand, Hauswirth et al. (1968) obtained a complete remission (see also Noble 1975) with a comparable concentration of a beta-adrenoceptor blocker (pro- nethalol 4-10 -6 M). However, the results of Hauswirth et al. (1968) were presumably due to the lower adre- naline concentration (2.5-10 _6 M). Nevertheless, it is shown in Fig. 6 that the effect of the beta-adrenoceptor blocker is dependent upon the concentration adminis- tered and whether the (+)- or (-)-isomere is applied. This is shown in Fig. 2 B more clearly: The normalized s~ curve is shifted by adrenaline by 18.5 mV in the positive direction; with the additional application of procaine no further shift occurs. The betablocker causes a shift by 12 mV in the negative direction which
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`K. Hashimoto et al. : Pacemaker Current and Beta-Adrenoceptors 13 @ 14 12 10 I B 8 10 -SA 6 Fig. 2 (A) Steady state degree of activation of iK2 measured as the amplitudes of current tails on return to the holding potential under various conditions (]B - see inset). (O) control; (zX) adrenaline (5.5.10 -6 M); (e) procaine (730.10 -6 M) plus adrenaline; ([3) atenolol (38 (cid:12)9 10 -6 M) plus adrenaline and procaine. Note ~) 1.0 that procaine does not cause any further shift 0.9 of the activation curve in the presence of adrenaline. The voltage dependence of the o.8 steady state degree of activation is again shifted o.7 in the negative direction by the additional application of the beta-blocker. Ordinate: 0.6 current in 10 -8 Ampere; Abscissa: membrane voltage in mVolt. (B) Normalized curves (s~) s = 0.5 Here the voltage shift of the curves can be 0.4 observed as 18.5 mV in the depolarizing direction by adrenaline and 12 mV in the o.3 backward direction by the beta-blocker. 0.2 Ordinate: arbitrary units for the activation degree between 0 and 1 for sod. Abscissa: o.1 membrane voltage in reVolt 0 CONTROL AADRENALINE 5,5xl()6 MOLI L ePROCAINE 730 xl(~6MOL/L +~ e~! OATENOLOL 38 xlO"6MOL]L +~+e o z~ o~ (cid:12)9 VOLTAGE , ....... ..... .... , .... -~ o ~ %__- 400 ' -~o -13o '7o 4,0 -~o -~.o mV 2x o ~ a 185mV ~o ' -~o ' -do ' -s'o ' Ao mV means that the catecholamine effect is compensated by two thirds using this particular concentration (38" 10-6 M) of the beta-adrenoceptor blocking agent. According to the Hodgkin-Huxley theory the time constants showed a behaviour similar to that of the activation curves. Figure3A shows the amplitudes of current tails on the return to the holding potential following voltage clamp pulses of different duration to -40mV and -50inV. At both potentials, this was done under control conditions, adrenaline and the beta- blocker. It is shown that at both voltage levels the time constant is slowed under the influence of adrenaline and again accelerated in the presence of adrenaline and the betablocker. The effects of adrenaline and the beta-adrenoceptor blocking agent on the rate of change of iK2 is sum- marized in Fig. 3 B showing the reciprocals of the time constant in relation to the membrane potential. As already known from Figs. 2B and 3A, the s-kinetics are shifted in the depolarizing direction by adrenaline and restored to a large extent by the betablocker. The Shift of the Rate Constants According to the Hodgkin-Huxley theory, when ds/dt = O, the steady state value of s follows as" O~ s s~ - as+/?s (3) and ~-1 = c~s + /~. (4) ~ and/~ are the rate constants of the foreward and backward reactions (see page9) which are voltage
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`14 Naunyn-Schmiedeberg's Arch. Pharmacol. 307 (1979) | | '~ l 15 18 1 0 0,1 0,2 0,3 0,4 0,5 1 ,[ 5 S@C 1 2- 3- 4- i .8 61 8~ 10: 3 o ENVELOPE- TEST at -50mV T- 0,1 2 T- 1,4 sec O" 0 0,1 0,2 0,3 0,4 0,5 1 $er 0 CON TOOL ~AORENALINE 5,Sxl(~6 MOL/L DATENOLOL 38 xlO'6MOLIL +~ -;oo___ ' -~o ' -8o ' -~o ' -~o_ ' mV / J H Y -go ' -~4o Fig. 3. (A) Pacemaker current tails on the return to the holding potential following depolarizing clamp pulses of various durations plotted on semilogarithmic scale. This procedure was called "envelope test" by Noble and Tsien (1968). Holding potential: -80 inV. The symbols are the same as in Fig. 2. With procaine, no envelope test was performed since the tracings did not show any change in comparison to the ones obtained with adrenaline. Note that in the range of relatively low membrane potentials the time constant of iK2 is slowed with adrenaline and accelerated again with the beta-blocker in the presence of adrenaline. Ordinate : Logarithmicplot of the amplitudes of current tails as (ico - /) where i~ is the amplitude in the steady state following a relatively long pulse and i is the amplitude of current tails in response to shorter pulses. Abscissa: Time (duration of the voltage clamp pulse). (B) Rate of change of iK2 plotted as the reciprocals of the time constants (l/z) in relation to the membrane potential. The symbols are the same as in Fig. 2. Holding potential: - 80 mV. Note that positive to - 75 mV the time course of i~2 is slowed with adrenaline and speeded up again with the beta-blocker. Ordinate: 1/time in s-1. Abscissa: membrane potential in mV dependent only (Hodgkin and Huxley, 1952) and which together determine the rate of current change at a given potential [cf. Eq. (4)]. From Eqs. (3) and (4) we obtain So~ ~= "C s and / ~s -- -- l" s (5) (6) (More detailed explanation of the theoretical back- ground is given by Hauswirth and Singh, 1979.) In Fig 4A and 4B, e~ and/~ are plotted against the membrane potential. It appears that, with adrenaline, es is shifted more than/~ in the depolarizing direction. A conductance change underlying a time dependent current may be treated as a chemical reaction running in the foreward and backward direction (Tsien and Noble, 1969). Does Adrenaline Influence the Instantaneous Current-Voltage Relationship ? The instantaneous of fully activated current-voltage relationship shows essentially no modification, neither with adrenaline, nor with procaine or under the in- fluence of the betablocker (Fig. 5). This holds true for the absolute magnitude of this curve (also called i~:2, Noble and Tsien, 1968) as well as for the rectifying properties, especially for the marked negative slope in the potential range positive to -70 mV (Noble and Tsien, 1968). Although the current levels at the holding potential of -80mV are slightly different due to the different amplitudes of the s~ curves under various conditions (see Fig. 2) the scatter within the voltage range tested is remarkably small. This means that iK2 (first factor in Eq. 1) which is proportional to the number of iKz-channels is not subject to any considerable change under the influence of the drugs used.
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`K. Hashimoto et al.: Pacemaker Current and Beta-Adrenoceptors 0 gONTROL 1 1 rnATENOLOL 38 xlO'6mOL/L +~ 1,0 ser \ \ \\ 1,0 I I I / / / i iiI/'li IO I / I / sec4 - I 0,5 \ 0.5 O "" \ "-. / / / ~ / [] o J 0 i z~ -100 -90 -80 -70 -60 -90 -8'0 "7~0 -60 -5'0 4'0 mV mV 15 Fig. 4. (A) Rate constants/?~ obtained from (1-s ~ )/z~ under control conditions, adrenaline (5.5.10-6 M) and atenolol (38.10-6 M) in the presence of adrenaline (5.5.10- 6 M). With the catecholamine,/?s is shifted by about 8 mV in the depolarizing direction and restored by atenolol towards its original position. Note that in comparison to the shift of the rate constant fi~ the shift of c~ shown in B is larger. The symbols are the same as in Fig. 2. The dashed line is predicted by the equation 3~ = 0.00005 exp[- (Em -}- 52)/14.93] given by McAllister, Noble and Tsien (1975). (B) Rate constants c~ calculated from s~/z s under the same conditions as in A. With the agent stimulating the beta-adrenoceptor, % is shifted by about 17 mV in the depolarizing direction and restored by the beta-adrenoceptor blocking agent toward its original positio{a. The dashed line is again given by a theoretical model of McAllister, Noble and Tsien (1975): es = 0.00I "(Era + 52)/(1 -exp[-(E m fi- 52)/5]. Ordinates: arbitrary units for e~ and 3s. Abscissas: membrane potentials in mV However, it is difficult to decide whether the drugs mentioned above affect the absolute magnitude of iK2 or whether they may shift the reversal potential of iK2 (Cohen et al., 1976), thereby pretending an increase or decrease of iK2, respectively. Moreover, Weld and Bigger (1976) did not show any essential change of the amplitude of so~ with lidocaine although, with a high concentration of lidocaine, a depression of the acti- vation curve was found. Effect of Optical Isomeres of a Beta-Adrenoceptor Blocking Substance after Prior Administration of Adrenaline In this section it will be shown that as it is well known from other pharmacological studies, optical isomers are differently effective in antagonizing the action of adrenaline on the pacemaker current iK2. Figure 6A shows the effect of (+)- and (-)-penbutolol following the administration of adrenaline. After a stepwise REGTIFIED FUNCTION OF LN 2 15 ~ (cid:12)9 o Lx L~ o GONTROL 0 ts ADRENALINE 55,1()6 MOLIL p iPROCAINE 730 xl(~6MOL]L +~ OATENOLOL 38 xlO'6MOL]L +z~+O -16o -9'o -eo -:,o e'o -go - ~o mV Fig. 5. Instantaneous current-voltage relationship of i~2 under vari- ous conditions. This relation was obtained by building the quotient of the amplitudes of iK2 during and following polarization to various clamp levels. The so obtained "rectifier ratio" is then multipled by the amplitude ofs:o measured at the same holding potential resulting in the value of the "rectifier function" iK2. The vertical bar indicates the holding potential. The symbols are the same as in Fig. 2. Ordi- nate : membrane current in 10- 8 A. Abscissa: membrane potential in mV 10 16e A 5
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`16 Naunyn-Schmiedeberg's Arch. Pharmacol, 307 (1979) | -41 mlt ~ .I -66m1" CONTROL A AORs S,5 xlO "6 MOL/L T II .8 | (*)Ps _ 35110"6MOL L ~ A 15 20 (-)PEnUroLoL 1,7.106 MOL L § A 2"- O,55se/ f i 2 3 4 5 Sec ENVELOPE-TEST -65mu to -4Stay | (cid:14)9 CONTROL O, 10, 20, ~-.-1 ~, ADRENALINE 5,5x10-6 MOL//- seconds (cid:12)9 (-)PENBIJTOLOL 17x10"6MOL/L | 2 soo o CONTROL ADRENALINE 5,5 x 10 -6 MOLIL 1Oq eO,)PERBUrOLOL 35 x IO'6 BOLIL § z~ O i(-)PfDRUTOLOL 3,5 ~ 10 "6 EOL~L + z~ / /~'/ / :t I (-)'EDRUTRLOL 17 xlO "6 mOi/L +/~/ sea-11 . (cid:12)9 O0,4J -51 5 4.0,5 O 1,2t 0,2 e i [] -65 -60 -55 -50 -45 -75 -70 -65 -60 -55 -50 -45 -40 -35 mr/ Fig. 6. (A) Tails of membrane current (iB) under the influence of adrenaline and the optical isomeres of a beta-blocker (penbutolol). Top : Time course of the membrane potential during identical voltage clamp pulses from - 66 mV (holding potential) to - 41 mV (clamp potential). Below: Membrane currents under control conditions, adrenaline (5.5"10-6M), (§ (35.10 -6 M) and its (-)- isomere (1.7.10-6M). (B) Steady state degree of activation under control conditions (O), adrenaline (5.5.10- 6 M) (L}~), (+)-penbutolol (35.10 - 6 M) (e) and (-)-penbutolol in concentrations : (1.7.10- 6 M) (IZ), (3.5-10 -6 M) (I-~) and (I7.10 -6 M) (I). Adrenaline shifts s~c by 16mV in the depolarizing direction, the (+)-isomere of the beta- blocker and the 10 and 20 times smaller concentration of the (-)- isomere cause a backshift by about 1/3 (5.5 mV). 17.10 6M of the (-)-isomere restore the voltage dependence to z/3 (9 mV). In addition. adrenaline increases the steepness of sc~ - By application of the beta blocker following the adrenergic stimulating the original slope of s z is restored again. Ordinate: arbitrary units from 0 to 1 for s~. Abscissa: membrane potential in mV repolarization from a more positive potential to the holding potential iK2 declines. This deactivation is accelerated and continued to a lower level with adre- naline (5.5.10- 6 M). With a high concentration of (+)-penbutolol the action of adrenaline can be partly compensated. A concentration of 35.10-6M of (+)-penbutolol is ap- proximately equal to 1.7.10 -6 M of (-)-penbutolol in its adrenolytic effect (6runs in 1 experiment). This -30 mlt Fig. 7. (A) Envelope test (as described in Fig.3) determining the time constants at -45mV. Under the influence of adrenaline v~ is prolonged from 0.55s to 3.95s; under the influence of the beta- adrenoceptor blockade z~ is again accelerated to 1.2s. The symbols are the same as in Fig. 6. (B) Rate of change of current plotted as the reciprocals of the time constants (vs -1) versus the membrane potential. Like xjj, adrenaline shifts the U-shaped rate curve in the depolarizing direction, the beta-blocker causes a backshift to a large extent. The symbols are the same as in Fig. 6 corresponds well to other pharmacological studies. Figure6B shows the voltage dependence of the acti- vation curves of iK2 under the influence of adrenaline and with the (+)- and (-)-isomeres of the beta- adrenoceptor blocker. Adrenaline shifts this curve in the depolarizing direction; a high concentration of the (+)-isomere and a concentration of its (-)-isomere which is 10 and 20 times smaller have roughly the same effect, namely to shift s~ back by about one third; a larger concentration of the (-)-isomere of penbutolol (17.10 .6 M) causes a backshift by almost two third. Figure7A shows the time constants at -45mV measured by using the envelope test.