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CARDIOVASCULAR
Department of Cardiology, Marienhospital Herne, University of Bochum, Herne, Germany (B.B., R.B., D.D., M.V.B., M.M., M.P., I.W., H.J.T.); Centre for Experimental Surgery and Anaesthesiology, University of Leuven, Leuven, Belgium (R.M., K.M.); and Laboratory of Membrane Biophysics, Institute of Cardiology, Kaunas, Lithuania (R.M., V.G.).
Received August 5, 2003; accepted September 29, 2003.
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Abstract |
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0.6-0.8 µM) inhibited by KB. Receptor-independent guanosine 5'-O-(3-thio)triphosphate-induced and background IK(ACh), which contributes to the resting conductance of atrial myocytes, were equally sensitive to KB (IC50value of 0.9 µM). IK(ATP)induced in atrial myocytes during metabolic inhibition with 2,4-dinitrophenol (DNP) was also suppressed by KB, whereas IK1measured in ventricular myocytes was insensitive to the drug (KB 
50 µM). Although being effective when applied from the outside, intracellular application of KB via the patch pipette affected neither IK(ACh) nor IK(ATP). 3,3',5-triodo-L-thyronin, which shares structural groups with KB, did not have an effect on the K+currents. Consistent with the effects on single myocytes, KB did not depolarize the resting potential but antagonized the shortening of action potential duration by carbamylcholine-chloride or by DNP in multicellular preparations and antagonized the shortening of action potential duration by acetylcholine in single myocytes. It is concluded that KB inhibits IK(ACh)and IK(ATP)by direct drug-channel interaction at a site more easily accessible from extracellular side of the membrane.
).
The clinical benefits of amiodarone are complicated by many undesirable side effects. To develop drugs with an improved pharmacological profile, new molecules based on its structural modification have been synthesized. 2-Methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran; KB130015; KB) is one such novel compound that has been suggested to have antiarrhythmic properties and to inhibit the binding of T3 to nuclear receptors (Carlsson et al., 2002). Its relative effectiveness compared with amiodarone is not yet known. Given the structural similarity, some KB effects can be expected a priori. However, no prediction can be made about which of the amiodarone effects are conserved, or about their magnitude. As an example is the unexpected effect of KB on Na+ channel inactivation, which is nonexistent with amiodarone (Macianskiene et al., 2003b). Other structural analogs of amiodarone such as dronaderone (SR33589) have been shown to exert chronic and acute effects on ion channels similar to those of the parent agent amiodarone. The effects of KB on other K+ channels have not been investigated so far but could be important. Cardiac K+ currents that are acutely inhibited by amiodarone and its analog dronaderone include the background inward rectifying current (IK1), the G protein-coupled inwardly rectifying currents [IK(ACh) or IK(Ado)], and the ATP-sensitive current [IK(ATP); see Kodama et al., 1997; Altomare et al., 2000; Guillemare et al., 2000]. In addition, although initial reports suggested that the fast component of the delayed rectifier, IKr, is insensitive, a few recent studies indicate that it is inhibited by high concentrations of amiodarone (IC50 = 38 µM; Kamiya et al., 2001).
In the present study, we investigated the effects of KB on inward rectifying potassium currents in isolated guinea pig atrial and ventricular myocytes. IK(ACh) and IK(ATP) were measured in atrial cells. We found both currents to be markedly inhibited by KB, possibly via a direct drug-ion channel interaction. IK1 was studied in ventricular cells but was found to be insensitive to KB. In multicellular preparations, KB did not depolarize the resting membrane but antagonized action potential duration (APD) shortening by carbamylcholine-chloride (CCh) in atrial appendages, or by 2,4-dinitrophenol (DNP) in papillary muscles.
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Materials and Methods |
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Isolation and Culture of Myocytes. Single atrial myocytes were isolated via a Langendorff perfusion method described previously (Banach et al., 1993). Atrial myocytes were kept in culture for up to 48 h at 37°C. The culture medium was the bicarbonate-buffered M199 (Invitrogen, Dreiech, Germany) containing gentamycin and kanamycin (each at 25 pg ml-1; Sigma Chemie, Deisenhofen, Germany). The medium was not supplemented with fetal calf serum to prevent a time-dependent loss of sensitivity to ACh or Ado (Bunemann and Pott, 1995). Cells were plated at a density of several hundred per culture dish. About 50 to 70% of the cells attached within 16 to 24 h. No differences were found between freshly isolated and cultured myocytes in the effects studied here.
Multicellular Preparations. Left atrial appendages and left ventricular papillary muscles were dissected from guinea pig hearts. The preparations were pinned to the bottom of a tissue chamber and continuously superfused with the Tyrode's solution equilibrated with 100% O2. They were electrically stimulated with bipolar electrodes at 1 Hz with pulses of 2-ms duration and twice the diastolic threshold.
Solutions. For measurements in isolated myocytes the culture medium was replaced by a solution containing 120 mM NaCl, 20 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES-NaOH, pH 7.4. Pipettes were filled with a solution containing 110 mM K-aspartate, 20 mM KCl, 5 mM MgATP, 1 mM MgCl2, 2 mM EGTA, 0.1 mM GTP 0.1, and 10 mM HEPES-NaOH, pH 7.4. For activation of IK(ATP), MgCl2 was substituted for MgATP in an equimolar concentration. The chosen K+ concentrations yield an equilibrium potential for K+ of -48 mV. The Tyrode's solution used for multicellular preparations contained 144 mM NaCl, 4 mM KCl, 1.8 mM CaCl2,1mM MgCl2, 10 mM HEPES-NaOH, and 5 mM glucose, pH 7.4. Experiments on isolated cells were carried out at room temperature (21-24°C). For multicellular preparations, the bath temperature was kept at 36 ± 1°C, and pO2 was 580 to 600 mm Hg.
Materials. Standard salts were from Merck (Darmstadt, Germany). HEPES, EGTA, MgATP, GTP, ACh-chloride, CCh, adenosine (Ado), and DNP were from Sigma Chemie. KB (free acid) was from Karo-Bio AB (Huddinge, Sweden) and was dissolved as a 100 to 200 mM stock solution in dimethyl sulfoxide. The highest dimethyl sulfoxide concentration used [0.1% (v/v)] had no direct effect on the membrane currents and potentials under study nor did it affect the action of ACh, Ado, or DNP.
Ion Current Measurements and Action Potential Measurements in Single Atrial Myocytes. Membrane currents were measured under whole-cell patch-clamp (Hamill et al., 1981). Pipettes were fabricated from borosilicate glass with filament (Clark Electromedical, Pangbourne, UK) on a horizontal puller (DMZ, Munich, Germany) and were filled with the solution listed below. The d.c. resistance of the filled pipettes ranged from 3 to 6 M
. Currents were measured by means of a patch-clamp amplifier (List LM/EPC 7; List Electronics, Darmstadt, Germany). Signals were analog filtered (corner frequency 1-3 kHz) and were digitally stored on the hard disk of a computer equipped with a software package for voltage control, data acquisition, and data evaluation (ISO-2 by MFK, Frankfurt, Germany). Capacitance and series resistance were partially compensated. Unless stated otherwise the holding potential was -90 mV, chosen to cause large inward K+ currents because of the strong inward rectifying properties of G protein-coupled currents. Fast voltage ramps (-120 to +60 mV; 500 ms) were generated to provide current-voltage relations and to monitor the electrical access to the cell. Drug-induced changes in outward current were measured during the voltage ramps, and no qualitative difference was detected in the observed drug effects on inward and outward currents. Rapid exchange of superfusion solution was performed by means of solenoid-operated valves, which permitted switching between up to six different solutions. The half-time of solution exchange was about 0.5 s, as measured from the time course of IK(ACh) block upon switching from the standard (high-K+) to a K+-free, 5 mM Cs+-containing solution.
Current-clamp experiments were also performed in the whole-cell recording mode at 37 ± 1.0°C. External and pipette solutions were the same as those used in the whole-cell voltage-clamp experiments. However, a physiological external K+ concentration was chosen (5 mM). After the establishment of the whole-cell clamp mode, rectangular 2-ms currents were applied at a rate of 0.2 Hz. After a stabilization of action potential configuration, experiments were performed.
Action Potentials in Multicellular Preparations. For measurements in the multicellular preparation transmembrane potentials were recorded with glass microelectrodes filled with 3 M KCl (resistance 7-10 M). The microelectrodes were connected to the input stage of a high-impedance amplifier (MEZ-7101; Nihon Kohden America, Inc., Foothill Ranch, CA). The amplified signals were displayed on a dual-beam oscilloscope (C1-69) and sampled at 10 kHz using a 16-bit analog-to-digital converter (PCL816; Advantech France, Levallois Perret, France). After an equilibration period of 50 to 60 min with stable impalement, control recordings were first made. Then the atrial preparations were exposed to CCh (10 µM, for 10-15 min) followed by a washout of the agonist (15-25 min). Afterward, the same preparations were exposed to KB (50 or 200 µM; for 15-20 min), and CCh was reapplied and washed out in the presence of KB. In ventricular preparations, control measurements were followed by an application of 2,4-dinitrophenol (DNP; 50-100 µM) for 10 min, and of KB (200 µM; 25 min) added on top of DNP. Action potential durations were measured at 50% (APD50) and 90% (APD90) repolarization.
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Results |
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KB effectively inhibited both inward and outward components of IK(ACh). The KB-sensitive current was quantitatively larger than the ACh-induced current (see less inward current at holding potential in the presence of KB compared with predrug level in Fig. 1A). Because an agonist-independent opening of K(ACh) channels has been shown to contribute to the background current in atrial cells (Sakmann et al., 1983; Okabe et al., 1990; Kaibara et al., 1991), our results thus suggest that KB inhibited not only the ACh-induced IK(ACh) but also the basal G protein-activated current. The I-V relationship of the KB-inhibited background current (not illustrated) also displayed the same strong inward rectification as IK(ACh).
The effect of KB was concentration-dependent and reversible. Reversibility of the inhibition to the expected current level, taking into account the concurrent desensitization, was observed using low KB concentrations (Fig. 2A), but the effects of high concentrations (
10 µM) persisted for several minutes during drug washout. The concentration-effect curve obtained from experiments in 7 cells could be fitted by a Hill equation with IC50 of 0.82 µM and Hill coefficient of 1.12 (Fig. 2B, continuous line).
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To examine whether KB inhibition of IK(ACh) was due to an interaction at the muscarinic receptor level, or involved mechanisms downstream of the receptor, we tested its effect on G protein-coupled K+ current induced by other receptors. We therefore activated IK(ACh) by adenosine. Figure 3A illustrates an experiment that is representative for six different atrial myocytes, in which KB inhibited the adenosine-induced IK(Ado) (which is identical to IK(ACh)). Again the effect was concentration-dependent (IC50 = 0.57 µM; Fig. 2B, dashed line) and reversible within minutes. I-V relationships presented in Fig. 3B show that the KB-sensitive current displayed the same strong inward rectifying properties as IK(Ado).
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T3, which shares structural groups with KB and whose nuclear receptors can bind KB (Carlsson et al., 2002), failed to mimic the inhibitory action of this drug on the adenosine-induced potassium current (concentrations tested 3 µM). Figure 3A shows an experiment representative for four different myocytes. In this experiment, 30 nM T3, which is a physiological concentration of the hormone, had no effect on G protein-dependent potassium current.
KB Inhibits GTP
S-Induced IK(ACh). The above-mentioned experiments, in which KB inhibits IK(ACh) coupled to different receptors, make it unlikely that the drug acts at the receptor level. To further determine the level of KB action along the G protein-channel signal transduction cascade, we tested the effect of KB on IK(ACh) activated by GTPS (500 µM). The nonhydrolysable nucleotide was added to the patch pipette solution and yielded an irreversible activation of IK(ACh) upon repeated exposure to ACh. Figure 4 illustrates representative results of similar experiments in five atrial myocytes. While dialyzing with GTPS, the application of ACh resulted in an activation of IK(ACh) to its maximal level (Fig. 4A). The effect of ACh was only partially reversible after the first application and became irreversible after the third agonist application. Sustained IK(ACh) obtained after washout of ACh under these conditions was inhibited by KB (IC50 = 0.89 µM; Fig. 2B, dotted line). The KB-sensitive current displayed strong inward rectifying properties (Fig. 4B) and was larger than the current induced by GTPS and ACh (see less inward current at holding potential in the presence of KB compared with predrug level in Fig. 4A), suggesting that KB inhibited both GTPS-induced and background IK(ACh). These experiments indicate that KB inhibits IK(ACh) either by interfering with the G protein (
) subunit-channel interaction or by directly acting on K(ACh) ion channels.
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KB Inhibits the ATP-Sensitive K+ Channel. To investigate the sensitivity of IK(ATP) to KB, we performed experiments in atrial myocytes and superfused the cells with DNP (100 µM). Figure 5 shows a representative experiment for six cells superfused with DNP. After application of the metabolic uncoupler, IK(ATP) was slowly activated and resulted in an increase of inward current at the holding potential of -90 mV. Superfusion with KB in the continued presence of DNP resulted in a suppression of the inward current increase. I-V relationship of the DNP-induced current showed weak inward rectification, which is typical for IK(ATP), and was superimposable to the KB-inhibited current, indicating that IK(ATP) was the target of both agents.
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No Effect on the Background Inward Rectifier (IK1) Channel. If KB is an inhibitor of G protein-coupled and ATP-sensitive channels, other ion channels sharing structural similarities with these channels might be sensitive to the drug as well. For this reason, we studied the effect of KB on inward rectifying potassium current IK1. No effect of KB (10-50 µM) on that current was detected in five of five ventricular myocytes (not illustrated). These results are consistent with data from pig ventricular myocytes (Macianskiene et al., 2003a).
No Effect of KB Applied via the Patch Pipette. KB is a lipophilic compound and may be expected to easily cross the cell membrane upon extracellular superfusion. So far, the experiments did not differentiate between an intra- or an extracellular action site of the drug. To address this question, we added KB (50 µM) to the patch pipette-filling solution, and cell dialysis was allowed to take place during at least 20 min. A representative experiment for eight cells with this protocol is illustrated in Fig. 6. After rupturing the membrane patch, the whole cell was exposed to short periods of superfusion with ACh to monitor IK(ACh). The first response to ACh was elicited 10 s after getting access to the cell interior. At this time, diffusion of KB into the cell, if any, should be minimal because in experiments with intrapipette GTPS the first noticeable irreversible activation of IK(ACh) was observed only after a delay of about 40 s after patch rupture, with full activation being reached after a few minutes. Even after 30 min of cell dialysis with internal KB, no inhibition of IK(ACh) was detected. However, KB (50 µM) applied from the external side of the membrane completely inhibited IK(ACh). In line with the experiments described above, the background current was not affected by internal KB but was decreased by external KB. These results exclude that KB inhibits IK(ACh) by accessing its action site from the internal side of the membrane.
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Influence of KB on Action Potentials in Multicellular Preparations and in Single Atrial Myocytes. Given the above-mentioned KB effects on membrane currents, the drug may be expected to antagonize the shortening of action potential induced by muscarinic receptor activation in atrial cells. Figure 7A illustrates the effect of CCh before or after pretreatment with KB in multicellular preparations. Under control conditions (in the absence of KB), action potentials were markedly shortened in the presence of 10 µM CCh (Fig. 7A, left). After washout of the CCh effect (not illustrated; but see Table 1), treating the same preparation with 200 µM KB for 20 min caused no major change of the action potential. Reapplication of CCh after stabilization with KB caused less marked APD shortening compared with control conditions (Fig. 7A, right). Similar results were obtained in five preparations, as summarized in Fig. 7B. APD90 in the presence of both CCh and 200 µM KB was not significantly different from the value in the presence of 200 µM KB alone, but was significantly (P < 0.05) longer than the value in the presence of CCh before treatment with KB (Table 1). The inhibition of CCh effect by KB was concentration-dependent and was less with 50 µM KB. In single atrial myocytes KB 50 µM almost completely reversed an ACh (10 µM)-induced action potential shortening. An experiment being representative for six different cells is shown in Fig. 8A. The summary of the data of six different experiments (APD90) is shown in Fig. 8B.
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We also expected KB to antagonize the shortening of action potential induced upon KATP channel opening with DNP. This antagonism was tested in papillary muscles. It was rather difficult to stably record action potentials in the presence of DNP (100 µM), because of a frequent occurrence of contracture or of a complete loss of excitability in several experiments. Successful recordings in four preparations using 50 µM DNP (n = 2) or 100 µM DNP (n = 2) showed that KB could partly reverse the APD shortening caused by the metabolic poisoning. Figure 9A illustrates a typical result, and Fig. 9B and Table 2 summarize the data from all preparations. The APD prolongation caused by KB was significant (P < 0.01) but remained modest and did not restore the duration to its initial value.
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Discussion |
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S. Consistent with the suppression of IK(ACh) measured in single cells, KB decreased the APD shortening caused by the muscarinic agonist CCh in multicellular preparations. Similarly the drug suppressed IK(ATP) activated by metabolic inhibition with DNP in myocytes, and partially reversed the APD shortening by DNP in papillary muscles. In contrast, KB had no effect on IK1 recorded in myocytes and on the resting potential in multicellular preparations.
KB shares structural similarity with amiodarone, from which it was derived by chemical substitution (Carlsson et al., 2002), with the aim to produce a drug with less side effects and improved bioavailability. Preliminary animal toxicity studies of KB suggest that side effects after its chronic administration occur at doses similar to or higher than those of amiodarone, used as reference compound (Carlsson et al., 2002; supplementary materials at http://pubs.acs.org). Amiodarone, which is one of the most effective antiarrhythmic drugs, exerts many effects at the cellular level after either acute or chronic administration (Kodama et al., 1997). Although it is still unclear which of the complex effects of amiodarone are critical for its antiarrhythmic action, its effects on the function and expression of various ion channels are likely to play a predominant role. Amiodarone or its analogs, when acutely applied, decrease the availability or the open probability of many ion channels, including voltage-dependent Na+ channels, L-type Ca2+ channels, and various K+ channels (Kodama et al., 1997). Hence, KB could be expected to exert similar effects. However, besides the recently described and unexpected effects of KB on voltage-dependent Na+ and Ca2+ channels (Macianskiene et al., 2003b), its effects on other ion channels are largely unknown.
IK(ACh) represents the prototypical G protein-gated K+ channel and can be activated via the M2 muscarinic receptor (for reviews, see Kurachi, 1995; Dascal, 1997; Dascal, 2001) or via the A1 adenosine receptor (Kurachi et al., 1986). Our data show that IK(ACh) activated by agonists to either of these receptors (ACh and Ado, respectively) was suppressed by KB. The KB-sensitive current had the same reversal potential (-48 mV) and a similar (inward-rectifying) shape of the I-V relationship as the current induced by the agonists. Under our experimental conditions, IK(ACh) reached a peak shortly after the start of the agonist application and then decayed to a steady level due to desensitization (Carmeliet and Mubagwa, 1986). KB applied on top of the agonists suppressed IK(ACh) with a time course markedly faster than that of desensitization. In addition, upon washout of low KB concentrations, IK(ACh) rapidly returned to its steady-state level. The fast onset of and recovery from KB effect indicates that IK(ACh) suppression by KB involved a mechanism different from desensitization. The KB effect is likely to involve a mechanism downstream from the agonist-receptor interaction, at the G protein or K(ACh) channel level. This was suggested by the equal potency to suppress ACh and Ado effects (Fig. 2B) and was confirmed by the ability of KB to suppress IK(ACh) induced by GTPS. These results are similar to those obtained with amiodarone and dronaderone, which also inhibit IK(ACh) induced via various receptors or by internal GTPS (Watanabe et al., 1996; Altomare et al., 2000; Guillemare et al., 2000). The potency of KB to suppress IK(ACh) (IC50 = 0.8 µM) is similar to that of amiodarone (IC50 = 1-2.4 µM; Watanabe et al., 1996; Guillemare et al., 2000), but much lower compared with that reported for dronedarone (10-63 nM; Altomare et al., 2000; Guillemare et al., 2000). Our finding that KB suppresses activation of G protein-gated K+ channels by muscarinic and adenosine agonists does not exclude the possibility that it also acts at the receptor level. The parent compound amiodarone interferes with binding to the muscarinic receptor (Cohen-Armon et al., 1984; Colvin et al., 1989). As in the case of amiodarone, KB actions may be more potent at, but not restricted to, the channel.
During KB application the current at holding potential usually became smaller than the basal level. I-V relationships also showed quantitatively larger KB-sensitive currents compared with agonist- or GTPS-induced currents. This indicates that KB had another effect in addition to suppressing the effect of agonists and GTPS. The extra current suppressed by KB showed inward rectification and reversal potential similar to those of IK(ACh). Agonist-independent opening of K(ACh) channels has been shown to contribute to the background current in atrial cells (Sakmann et al., 1983; Okabe et al., 1990) and reflects basal activation of Gi(K) molecules by nonliganded receptors (Kaibara et al., 1991). Our results thus suggest that in the presence of KB not only the ACh-induced IK(ACh) but also basal G protein activated current was inhibited. Because agonist-independent opening of IK(ACh) may contribute to basal current in atrial cells, it has been proposed that APD prolongation by amiodarone and its analogs could contribute to antiarrhythmic action. Our experiments on atrial preparations, KB was applied alone after washout of CCh. At 50 µM there was no effect, but at 200 µM the drug caused a slight prolongation of the APD (Table 1). It is not possible to interpret this effect solely in terms of an effect on IK(ACh) because effects on other ion channels could contribute to the net change. KB has effects on Na+ and Ca2+ channels (Macianskiene et al., 2003a). Other ion currents (e.g., the Na+-K+ pump, the Na+-Ca2+ exchange, or the Na+-activated K+ currents that are sensitive to intracellular Na+ concentrations) might also be indirectly affected by the expected increase in Nai due to prolonged Na+ channel opening (Macianskiene et al., 2003b). In addition, the contribution of IK(ACh) might be overwhelmed by a larger contribution of IK1, especially at the resting potential.
In the present study, IK(ATP) in the whole-cell configuration was activated by uncoupling mitochondrial respiration from oxidative phosphorylation with DNP. Both the DNP-induced current and the current suppressed by KB in the presence of DNP showed similar inward-going rectification. Although outward rectifying currents have been induced by metabolic inhibition in other studies (Isenberg et al., 1983), inward rectification may be induced by intracellular Mg2+ and Na+ (Horie et al., 1987; Ciani and Ribalet, 1988). Inward rectifying K(ATP) currents have been obtained under experimental conditions similar to those used in the present study (Brandts et al., 2000). Hence, we can conclude that IK(ATP) induced by DNP was reversibly suppressed by KB. A similar effect (IC50 = 2.3 µM) has been previously noted using amiodarone on IK(ATP) induced by ATP depletion or by bimakalim in inside-out membrane patches of rat ventricular cells (Holmes et al., 2000). However, other studies showed a lower potency (IC50 = 19 µM) of amiodarone on the K+ conductance induced by ATP depletion in rat cardiocytes or a lack of effect on IK(ATP) induced by the K+ channel opener KRN2391 in Xenopus oocytes (Sakuta et al., 1992). The reason for this discrepancy remains unclear, but the difference in results might have been due to the cell type or the experimental conditions.
Of the K+ currents tested, IK1, which represents the strong inward rectifying background current responsible for setting the resting potential in atrial and ventricular myocytes, was the least sensitive to KB effect. Our results with guinea pig ventricular cells are similar to those obtained in pig cells, where KB was found to have no effect on IK1 or on the resting potential (Macianskiene et al., 2003a).
Our data also show that KB could antagonize the APD shortening induced by M2 muscarinic receptor activation in intact atrial muscle and isolated atrial myocytes. Similarly, the drug partially reversed the APD shortening induced by DNP in the multicellular preparation of ventricular muscle. In the multicellular preparation, KB concentrations of 50 µM were little or not effective and a concentration as high as 200 µM had to be given to obtain an effect. The reasons for the low potency of KB in multicellular preparations are unknown but could involve diffusion limitations imposed by superfusing instead of perfusing the preparations. This may result in reduced drug availability in the cell from which recordings are made. The same reason can be invoked to explain only partly the low efficacy of KB to oppose DNP-induced APD shortening. Additional factors (e.g., effects on other ion channels) implicated in the DNP effects or a change of sensitivity of K(ATP) channels by DNP are likely to be involved.
Although KB is highly lipophilic and its effect at high concentrations could not be easily washed out, the onset and washout of the effect of low concentrations was fast (Fig. 2). The latter finding makes it likely that KB was acting from a site easily accessible from the extracellular medium. This is supported by experiments in which intracellularly dialyzed KB failed to inhibit IK(ACh) (Fig. 6). A similar failure of intracellular KB (in contrast to extracellular KB) to modify Na+ channels has been observed (Macianskiene et al., 2003b). For amiodarone, based on 1) the high lipophilicity, 2) the slow time course of its action, and 3) the ability of bathapplied molecules to block IK(ACh) in cell-attached patches, it has been proposed that its effect on IK(ACh) is caused by drug diffused into the intracellular medium (Altomare et al., 2000). However, the same criteria would apply if the drug can diffuse within the membrane to reach its action site. Given the above-mentioned failure of intracellular KB to cause any effect, we propose that KB inhibits IK(ACh) and IK(ATP) by binding to the channels or their subunits from within the cell membrane, at a sites more easily accessible from the external side of the bilayer.
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Footnotes |
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ABBREVIATIONS:T3, 3,3',5-triodo-L-thyronine; Ado, adenosine; ACh, acetylcholine; CCh, carbamylcholine-chloride; APD, action potential duration; DNP, 2,4-dinitrophenol; I-V, current-voltage; GTPS, guanosine 5'-O-(3-thio)triphosphate.
Address correspondence to: Dr. Bodo Brandts, Medizinische Klinik II/Universitätsklinik Marienhospital Herne, Ruhr-Universität Bochum, Hölkeskampring 40, D-44625 Herne, Germany. E-mail: bodo.brandts{at}ruhr-uni-bochum.de
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