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CARDIOVASCULAR
2-Receptor Ligand-Mediated Inhibition of Inwardly Rectifying K+ Channels in the HeartLaboratoire de Neurobiologie et de Pharmacologie Cardiovasculaire, Facultéde Médecine, Inserm, U-715, Facultéde Médecine, Strasbourg, France (L.M., H.G., D.Z., J.-D.E., P.B.); Laboratoire de Pharmacologie, Facultéde Médecine St Antoine, Paris, France (J.W., P.J.); and Inserm U-533 and Institut du Thorax, Université de Nantes, Facultéde Médecine, Nantes, France (B.M., C.B., I.B.)
Received March 1, 2007; accepted April 24, 2007.
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2-receptor agonist, ifenprodil, was suggested as an inhibitor of G protein-coupled inwardly rectifying potassium channels. Nevertheless, an analysis of the role of 2 receptors in cardiac electrophysiology has never been done. This work aims i) to identify the roles of cardiac 2 receptors in the regulation of cardiac K+ channel conductances and ii) to check whether 2-receptor agonists exhibit class III antiarrhythmic properties. The 2-receptor agonists ifenprodil, threo-ifenprodil, LNP250A [threo-8-[1-(4-hydroxyphenyl)-1-hydroxy-propan-2-yl]-1-phenyl-1,3,8-triazaspiro[4,5]decane-4-one] (a derivative of ifenprodil devoid of 
1-adrenergic and N-methyl-D-aspartate glutamate receptor-blocking properties), and 1,3-di(2-tolyl)guanidine were used to discriminate the effects linked to 2 receptors from those of the 1 subtype, induced by (±)-N-allylnormetazocine (SKF-10,047). The 2-receptor antagonist 3--tropanyl-2(pCl-phenoxy)butyrate (SM-21) was employed to characterize 2-mediated effects in patch-clamp experiments. In rabbits, all 2-receptor agonists reduced phenylephrine-induced cardiac arrhythmias. They prolonged action potential duration in rabbit Purkinje fibers and reduced human ether-a-go-go-related gene (HERG) K+ currents. (+)-SKF-10,047 was completely inactive in the last two tests. The effects of threo-ifenprodil were not antagonized by SM-21. In HERG-transfected COS-7 cells, SM-21 potentiated the ifenprodil-induced blockade of the HERG current. These data suggest that 2-receptor ligands block IKr and that this effect could explain part of the antiarrhythmic properties of this ligands family. Nevertheless, an interaction with HERG channels not involving 2 receptors seems to share this pharmacological property. This work shows for the first time that particular caution has to be taken toward ligands with affinity for 2 receptors. The repolarization prolongation and the early-afterdepolarization can be responsible for "torsades de pointe" and sudden cardiac death.
Receptors were initially proposed as a subtype of opiate receptors. The idea for this classification came from the induction of hallucinations by a synthetic opioid, (±)-N-allylnormetazocine (SKF-10,047), which could not be attributed to its actions on µ, 
,or 
receptors (Martin et al., 1976
). However, naloxone and naltrexone could not block these actions (Vaupel, 1983). Thus, currently, it is clear that receptors do not constitute a subtype of opiate receptors. The receptors are found in the central nervous system as well as in other organs (heart, spleen, and liver) (Monassier and Bousquet, 2002). Based on different binding profiles, two subtypes have been proposed. The one that binds (+)-6,7-benzomorphanes, such as (+)-pentazocine, with high affinity is termed 1, whereas the one with low affinity for these ligands is termed 2 (Quirion et al., 1992). The two subtypes were identified in the heart, in saturation studies, using (+)-[3H]PPP [(+)3-hydroxyphenyl-N-(1-propyl)-piperidine] for 1 receptors and [3H]DTG [1,3-di(2-tolyl)guanidine] for 2 receptors (Dumont and Lemaire, 1991; Ela et al., 1994). In the rat heart, the activation of the 1 subtype seems to mainly induce an increase of inotropism linked to an intracellular elevation of inositol 1,4,5-trisphosphate followed by a cellular Ca2+ overload (Novakova et al., 1998). In this tissue, the functions of the 2 receptors remain unknown. It was suggested by Jeanjean et al. (1993) that a link between 2 receptors and the K+ channels targeted by class III antiarrhythmic drugs could exist (Jeanjean et al., 1993). Moreover, the classic 2-receptor agonist, ifenprodil, was recently described as a blocker of G protein-activated inwardly rectifying potassium channels (Kobayashi et al., 2006). Taken together, these data suggest a link between 2 receptors and K+ channels; however, the mechanisms and the consequences of this link have never been investigated. The aim of the present study was to further investigate the role of the 2 receptor on cardiac repolarization, HERG K+ channels, and on cardiac arrhythmias by using 2-receptor ligands with various pharmacological profiles. The following compounds were used: i) racemic ifenprodil (mixture of erythro- and threo-isomers), because it has been proposed as a reference ligand for 2 receptors (Hashimoto et al., 1994), but it is also an antagonist of both the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors and 1-adrenergic receptors (Hashimoto and London, 1995); ii) 1,3-di(2-tolyl)guanidine as a reference 2-receptor ligand, with a chemical structure completely different from that of ifenprodil (DeHaven-Hudkins and Hudkins, 1991); iii) threo-ifenprodil, which is more selective for 2 receptors than the racemic ifenprodil (it has no effect on 1-adrenergic receptors) (Hashimoto and London, 1995); and iv) threo-8-[1-(4-hydroxyphenyl)-1-hydroxy-propan-2-yl]-1-phenyl-1,3,8-triazaspiro[4,5]decane-4-one (LNP250A), synthesized in our laboratory as a derivative of threo-ifenprodil devoid of action on NMDA and 1-adrenergic receptors. For the in vitro experiments, these 2-receptor ligands were compared with the reference 1-receptor agonist SKF-10,047 (Vaupel, 1983), and 3--tropanyl-2(pCl-phenoxy)butyrate (SM-21) was used as a 2-receptor antagonist (Ghelardini et al., 2000). Here we show that all 2-receptor agonists exhibit antiarrhythmic properties in vivo that are attributed to a class III-mediated effect, because they increase repolarization duration in rabbit Purkinje fibers. In this last model, high concentrations were also able to provoke early-afterdepolarizations. We confirm this property by demonstrating the ability of these compounds to block HERG K+ channels, an effect not antagonized by the usual 2-receptor antagonist SM-21. Therefore, this work suggests that 2-receptor ligands constitute a family of molecules that deeply modify cardiac repolarization, contributing to interesting class III antiarrhythmic properties but which could, as a consequence, also promote "torsades de pointe" and cardiac sudden death (Redfern et al., 2003). |
Materials and Methods |
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) and with the French law concerning experiences on vertebrate laboratory animals (Décret 2001-464, 2001).
Radioligand Binding Assays. The binding profile of the putative ligand LNP250A was evaluated according to the method employed by Ganapathy et al. (1999) for 1 sites and by Hashimoto and London (1995) for 2 sites. In brief, 1-binding experiments were performed on cultured Human Jurkat cell membrane preparations. Membranes (250-µg protein) were incubated with [3H]haloperidol for 3 h at room temperature. Nonspecific binding was determined in the presence of 10 µM unlabeled haloperidol. With regard to 2-binding experiments, aliquots of crude rat brain membrane preparations (100 µg of protein) were incubated with [3H]ifenprodil for1hat 37°C. Nonspecific binding was measured in the presence of 10 µM unlabeled ifenprodil. The binding profile of LNP250A on a few other receptors was also investigated and is presented under Results.
In Vivo Evaluation of the Cardiovascular Effects of 2-Receptor Ligands in Rabbits. Normotensive male rabbits (Zika strain) weighting between 2.5 and 3.5 kg were anesthetized with sodium pentobarbitone (40 mg/kg) injected through the marginal vein of the ear; anesthesia was maintained with another i.v. injection of pentobarbitone (5 mg/kg) just before the control period of the experiment. Rectal temperature was maintained at 38 ± 0.5°C, with a warming blanket as soon as anesthesia was induced (Harvard Apparatus Mills, MA). The animals were tracheotomized, immobilized with pancuronium bromide (0.3 mg/kg i.v.), and artificially ventilated as described previously (Monassier et al., 1994). During the surgical procedure, the depth of anesthesia was regularly assessed by pinching the ear and checking whether there was any blood pressure response or any modification of the heart rate. In case of such a response, an additional dose of pentobarbitone (5 mg/kg) would have been injected. The right femoral vein was catheterized to permit i.v. injections, and instantaneous arterial pressure was continuously monitored through an arterial femoral catheter placed in the abdominal aorta via the right femoral artery as described previously (Monassier et al., 1994).
We measured systolic arterial pressure (SAP) and diastolic arterial pressures, respectively, and calculated the mean arterial pressure as the diastolic arterial pressure plus one-third the differential pulse pressure. Heart rate was derived from the continuous pressure and electrocardiographic recordings. The electrocardiogram was continuously recorded from the standard leads (Hellige EK 512E; Hellige, Freiburg, Germany) by mean of four stainless steel electrodes inserted subcutaneously at the origin of the four limbs.
Cardiac arrhythmias were induced by the intravenous injection of 100 µg/kg phenylephrine (PE), a selective agonist of the 1-adrenergic receptors. Three injections of PE were performed every 20 min. The first PE administration (control injection) was performed after 10 min of hemodynamic stability (less than 10% variation among different recordings). Ten minutes later, the tested drug was intravenously injected in a single slow bolus of 30 s duration (with the exception of di-tolylguanidine, which was perfused over 5 min), and the second PE injection was performed 10 min after the beginning of each treatment. The last PE injection was performed 30 min after the administration of the tested compound. The cardiovascular effects of drugs were analyzed just before and during each PE injection. Arrhythmias were evaluated according to the Lambeth convention (Walker et al., 1988). The incidence, occurrence (number of episodes), and duration (s) of the following arrhythmias were recorded: ventricular ectopic beats (VEB) and ventricular tachycardia (VT). We never observed ventricular fibrillation. The number of ventricular ectopic beats included single ectopic beats as well as those being part of VT. VT was regarded as a run of five or more ectopic beats. All of the arrhythmic events occurred during a maximum of 5 min after the phenylephrine injection and stopped spontaneously. They were all taken into account for pro- and antiarrhythmic evaluations.
Evaluation of the Effects of 2-Receptor Ligands on Isolated Purkinje Fibers. Electrophysiological experiments were performed using a method described previously by Adamantidis et al. (1995). In brief, New Zealand white rabbits of either sex (1.5-2.5 kg) were sacrificed. The heart was quickly excised and placed in a modified Tyrode's solution (high KCl, 27 mM; high glucose, 55 mM) bubbled continuously with 95% O2,5%CO2 at room temperature and warmed at 36.5 ± 0.5°C. Running Purkinje fibers were perfused at 10 ml/min with this solution and changed for normal Tyrode's solution after 30 min. Electrical stimulation was initiated at 120 bpm and, 15 min later, reduced to the baseline-driven rate of 60 bpm (1-ms duration, 1.5 times the diastolic threshold current, DTU 200; Bloom Associates, Reading, PA). Intracellular action potentials (APs) were recorded with borosilicate glass microelectrodes filled with 3 mM KCl (15-25 M
) using high-impedance electrometer (VF180; Bio-logic, Claix, France), displayed on a digital oscilloscope (Gould 1600; Gould, Ballainvilliers, France), and analyzed by means of a high resolution (50 KHz, 12 bits) data acquisition system (Notocord HEM, Croissy, France). The following parameters of the APs were analyzed: resting membrane potential; APA (amplitude); and Vmax, APD50 and APD90 (AP duration at 50 and 90% repolarization, respectively). After at least2hof stabilization, drugs were added to the bathing solution at increasing cumulative concentrations of 0.1 and 1 mM, each perfused for 45 min. At drug-free concentration and during the last 15min of each drug superfusion, the stimulation frequency was changed from 60 to 15 bpm for 3 min and from 60 to 120 bpm for 3 other min before return to the baseline driven rate (60 bpm). Each drug was dissolved in 500 µl of dimethyl sulfoxide (DMSO) and then in Tyrode's solution (maximal DMSO concentration: 0.05%). A control set of experiments was created to check the effect of 0.05% DMSO solution using the same timing and driven rates (Control group).
Evaluation of the Effects of 2-Receptor Ligands on Human Recombinant HERG Potassium Channels in COS-7 Cells. COS-7 cells (ATCC, Manassas, VA) were cultured and transfected as described previously, with HERG cDNA (a kind gift from S. Kupershmidt, D. Snyders, and D. Roden, Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, TN) subcloned into the mammalian expression vector pSI (Promega, Madison, WI) (Potet et al., 2001). In brief, 0.4 µg of pSI-HERG and 1.6 µg of pTR-GFP plasmids per milliliter of culture medium were complexed with polyethyleneimine using a polyethyleneimine/cDNA ratio of 5 Eq. Ionic currents from HERG-transfected cells were recorded using the whole-cell configuration of the patch-clamp technique in Cl--free solutions at 35°C, as described previously (Mohammad-Panah et al., 1999). Stimulation, data recording, and analysis were performed through an A/D converter (Tecmar TM100 Labmaster; Scientific Solutions, Solon, OH) using Acquis1 software (Bio-Logic) as described previously (Potet et al., 2001).
Solutions and Drugs. The following drugs were used: sodium pentobarbitone (Sanofi Santé Animale, Libourne, France); pancuronium bromide (Organon Teknica, Fresnes, France); 2-(4-benzylpiperidino)-1-(4-hydroxyphenyl)-1-propanol (ifenprodil; Tocris, Bioblock, Illkirch, France); and DTG, SM-21, and SKF-10,047 (Sigma/RBI, Natick, MA). LNP250A and threo-ifenprodil were synthesized in the Laboratoire de Neurobiologie et de Pharmacologie Cardiovasculaire (INSERM U-715, Strasbourg, France). For binding experiments, [3H]DTG and [3H]ifenprodil were purchased from PerkinElmer Life and Analytical Sciences (Paris, France). Tyrode's solution for Purkinje fiber experiments contained 110 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 1.8 mM NaH2PO4, 2 mM NaHCO3, and 11 mM glucose, pH 7.45 ± 0.05. Patch-clamp study. The standard extracellular medium contained 145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, and 5 mM glucose, with pH adjusted to 7.4 with NaOH. The intracellular medium contained 145 mM potassium gluconate, 5 mM HEPES, 2 mM EGTA, 2 mM hemi-magnesium gluconate (free-Mg2+, 0.1), and 2 mM K2ATP, pH 7.2 with KOH, and the extracellular medium used to record K+ currents contained 145 mM sodium gluconate, 4 mM potassium gluconate, 7 mM hemi-calcium gluconate (free-Ca2+, 1), hemi-4 mM magnesium gluconate (free-Mg2+, 1), 5 mM HEPES, and 5 mM glucose, pH 7.2 with NaOH (all powders from Sigma-Aldrich Chimie SARL, St Quentin Fallavier, France). Free activities were calculated using a software designed by G. L. Smith (University of Glasgow, Glasgow, UK). Threo-ifenprodil, SKF-10,047, and LNP250A were dissolved in DMSO to obtain 10-2 M stock solutions and then diluted in DMSO to reach concentrations of 10-3,10-4, and 10-5 M. The stock solutions were then further diluted with the Cl--free extracellular medium to the final concentration. DMSO was added in experimental solutions when needed to reach the same final concentration (1%). The 10-2 M DTG stock solution was prepared from the chlorohydrate DTG salt in distilled water, and the pH of the experimental solutions was readjusted.
Synthesis of LNP250A. After protection of the phenol group of para-hydroxy-propiophenone by benzoylation, the resulting compound was treated with bromine in carbon tetrachloride to give 4-benzyloxy--bromo-propiophenone. This compound was then reacted with 1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one to give 8-[1-(4-benzyloxyphenyl)-1-oxo-propan-2-yl]-1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one-, which in turn was reduced with sodium borohydride to give the corresponding threo-alcohol. Deprotection of this benzyloxy derivative by hydrogenation gave rise to the target compound threo-8-[1-(4-hydroxyphenyl)-1-hydroxy-propan-2-yl]-1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one (Fig. 1).
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1, 
1, 2); dopamine (D1,D2); GABAA and GABAB; histamine (H1, H2), muscarinic (M1), and glutamatergic (NMDA). With regard to receptors, LNP250A had no affinity for the 1 subtype (Ki > 10-5 M) but exhibited a Ki of 1.6 µM toward 2. This affinity was lower than that of ifenprodil, but compared with this drug, the selectivity of this compound was markedly improved.
In Vivo Evaluation of the Cardiovascular Effects of 2-Receptor Ligands in Rabbits
Cardiovascular Effects of Bolus Intravenous Injections of Phenylephrine. In pentobarbitone-anesthetized rabbits, the bolus intravenous injection of the 1-adrenergic agonist phenylephrine (100 µg/kg) markedly increased blood pressure (SAP to approximately 185 mm Hg) and induced, just after the peak of the blood pressure, numerous ventricular ectopic beats (approximately 450 VEBs), part of them included in ventricular tachycardia (mean duration 77 s) (Fig. 2; Tables 1 and 2). These arrhythmias stopped spontaneously. In most cases, the blood pressure returned to initial values, but a slight secondary reduction (-10 mm Hg) was observed in a few animals. In control rabbits, saline affected neither the pressive nor the proarrhythmic responses to phenylephrine (Table 2). When the mean blood pressure decreased by more than 15 mm Hg under the basal value after the first phenylephrine injection (before any administration of the tested compounds), the experiments were excluded from our analysis. As a matter of fact, an important decrease in the blood pressure was probably attributed to a cardiotoxic effect of phenylephrine and associated with the reduction of afterload; these phenomena could interfere with the evaluation of the pro- or antiarrhythmic effects of the compounds.
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In Vitro Evaluation of the Effects of 2-Receptor Ligands in Isolated Purkinje Fibres and Recombinant HERG Potassium Channels
Evaluation of the Cardiovascular and Antiarrhythmic Properties of the 2-Receptor Ligands. Among the different 2-receptor ligands tested in the present work, only the 1 mg/kg dose of ifenprodil significantly reduced the resting blood pressure (-14% for the SAP; p < 0.05) (Table 1). The peak systolic arterial pressure during the second phenylephrine injection was also diminished by this treatment in a similar extent (-15%; p < 0.05). As a consequence, the amplitude of the pressive response to phenylephrine was not modified. These actions were obtained, in our experimental conditions, without change of the heart rate. The other drugs, di-tolylguanidine (250 µg/kg), threo-ifenprodil (2 mg/kg), and LNP250A (1 mg/kg), did not affect blood pressure, heart rate, and the pressive response to phenylephrine. The 250 µg/kg dose of ifenprodil was selected because preliminary data showed that it does not reduce the blood pressure. DTG was administered in an infusion regimen (50 µg/kg/min during 5 min) because the injection of the same dose as a bolus provoked QT interval enlargement and in some cases cardiac arrhythmias. With this procedure, the QT interval was not significantly modified by DTG: 86 ± 5to87 ± 6ms(p > 0.05). None of the other drugs provoked such a phenomenon after systemic bolus injection at the doses tested here (data not shown).
With regard to the proarrhythmic response to phenylephrine, all of the 2-receptor ligands reduced the number of VEBs 30 min after the drug delivery (Table 2). The order of potencies was as follows: ifenprodil (1 mg/kg) > LNP250A (1 mg/kg) > ifenprodil (250 µg/kg) > DTG (250 µg/kg) > threo-ifenprodil (2 mg/kg), each dose selected as the most active one that did not affect blood pressure (at the exception of the highest dose of ifenprodil).
Evaluation of the Effects of 1- and 2-Receptor Ligands on Isolated Purkinje Fibers. Purkinje fibers were unaffected by application of the vehicle only (DM) or by application of (+)-SKF-10,047 (0.1 to 1 mM), the 1-selective ligand, at any stimulation rate (Table 3, Fig. 3).
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Micromolar concentrations of LNP250A increased APD50 and APD90 slightly when stimulated at 60 bpm, respectively, from 201 ± 14sto240 ± 17 ms for APD50 and from 270 ± 18 to 320 ± 20 ms for APD90 (n = 7; p < 0.01). No early-afterdepolarization (EAD) was observed at a 15-bpm stimulation rate (Fig. 3). These concentrations slightly decreased Vmax at 60- and 120-bpm stimulation rates (Table 3).
Micromolar concentration of ifenprodil increased APD50 and APD90 at 60 bpm, respectively, from 173 ± 12 to 215 ± 16 ms for APD50 and from 249 ± 13s to 339 ± 30 ms for APD90 (n = 7; p < 0.01). These effects were more pronounced at 15-bpm driven rates, allowing EADs to occur in two experiments (Fig. 3). Such a concentration affected neither APA nor Vmax, even at 120 bpm (Table 3).
The main effect of threo-ifenprodil was to increase APD50 and APD90 at all stimulation rates at both of the tested concentrations (Fig. 3). At 15 bpm, EADs were observed at 10-7 (n = 2) and 10-6 M(n = 3) concentrations (Fig. 4). Furthermore, at 120 bpm, threo-ifenprodil slightly decreased APA (p < 0.01) at micromolar concentration (Table 3). At high doses, DTG decreased mainly Vmax and APD50 (-38 ± 6.3%: p < 0.001, -37 ± 8.3% at fast rates; data not shown) but also APD90 and APA (-17 ± 4.6%, p < 0.01; -8,6 ± 2.1%, p < 0.01 at fast rates).
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Evaluation of the Effects of 2-Receptor Ligands on Recombinant HERG Potassium Channels in COS-7 Cells. Upon transfection with HERG cDNA, COS-7 cells expressed a large K+ current with biophysical properties that were reminiscent of those of the rapid component of the cardiac delayed rectifier K+ current recorded in human cardiac cells (Fig. 5A) (Wang et al., 1994). When repolarized to -60 mV, expressing COS-7 cells exhibited a large deactivating current (I tail). As illustrated in Fig. 5B, the HERG-related K+ current activated at positive potentials up to -50 mV and exhibited strong inward rectification. Deactivating K+ currents reached a maximal value after a prepulse to approximately 10 mV and did not further increase when depolarizing pulses to more positive potentials were applied (Fig. 5C).
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Threo-ifenprodil also decreased activating (Ipp) and deactivating currents (Fig. 6, A, B, and C). At a concentration of 10-7 M, the drug induced a slow decay of the activating current recorded at +10 mV with less effects at the initial phase of activation. In the presence of threo-ifenprodil, the deactivating current density decreased in a dose-dependent manner from 10.14 ± 1.82 pA/pF in control to 0.45 ± 0.29 pA/pF, with 10-6 M threo-ifenprodil (n = 6; Fig. 6D). The IC50 value for threo-ifenprodil to block recombinant HERG current (8.8.10-8 M, Hill coefficient = 1.0; n = 6-7) was approximately a half-log smaller than that for LNP250A. Furthermore, the threo-ifenprodil-induced block of the tail current was significantly more effective at depolarized potentials (p < 0.001, n = 4; Fig. 6C, inset). The drug effect was highly reversible; 69 ± 8% of the control tail current amplitude was restored after washout of the drug (n = 6).
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To evaluate 2-receptor involvement in HERG response to these specific agonists, we tested the effects of SM-21, a 2-receptor antagonist, on threo-ifenprodil effects. It has been shown that SM-21 has a high affinity for the receptor in the same concentration range as threo-ifenprodil [
70 nM (Mach et al., 1999) and 77 nM, respectively]. Therefore, we first investigated SM-21 effects on HERG current. Surprisingly, we observed a concentration-dependent inhibition of the K+ current and calculated an IC50 of 9.8 x10-7 M and a Hill coefficient of 1.2 (n = 5; Fig. 7A).
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2-receptor agonists could have direct effects on HERG channels and indirect effects via the receptor, we tested the effects of 10-7 M threo-ifenprodil in the presence of 10-7 M SM-21, assuming that, at this concentration, SM-21 has a nonsignificant direct effect on HERG channel where it can antagonize threo-ifenprodil effects on 2 receptor. At this concentration, threo-ifenprodil should inhibit about half of the HERG-mediated current, and if part of the effects is via 2-receptor activation, the presence of SM-21 should impair these effects. The results are summarized in Fig. 7B. In a new set of experiments, the addition of 10-7 M SM-21 reduced the K+ tail current from 14.64 ± 3.65 to 12.87 ± 3.42 pA/pF (n = 13). Compared with the spontaneous rundown with time of HERG current (11.69 ± 2.11 to 10.43 ± 1.74 pA/pF, when measured after the same delay as SM-21 application; n = 17), these effects were not significant. The addition of 10-7 M threo-ifenprodil significantly reduced the remaining tail K+ current to 8.86 ± 2.13 pA/pF (p < 0.01). On another set of cells, when threo-ifenprodil was first applied, the tail current was significantly decreased from 8.05 ± 1.67 to 5.26 ± 1.10 pA/pF (n = 13; p < 0.001), and when SM-21 was added, it reached 4.15 ± 0.97 pA/pF (n = 13). When the SM-21 effects in the presence of threo-ifenprodil were compared with those of time (from 5.35 ± 0.72 to 4.62 ± 0.64 pA/pF; n = 7), the current change due to SM-21 was considered not significant. SM-21 did not reverse the current decrease induced by threo-ifenprodil. The effects of threo-ifenprodil plus SM-21 on HERG activity were similar to whatever drug was first applied, indicating that SM-21 pretreatment did not antagonize threo-ifenprodil. Altogether these data show that SM-21 did not alter HERG block by threo-ifenprodil.
Effects of the 1-Selective Ligand SKF-10,047 on HERG K+ Currents Expressed in COS-7 Cells. Unlike 2 agonists, the classic 1-selective ligand (+)-SKF-10,047 did not significantly alter the deactivating current amplitude (6.86 ± 1.58 pA/pF at 10-7 M versus 7.73 ± 1.87 pA/pF in control; n = 8) until higher concentrations (10-5 M) were reached (4.46 ± 1.15 versus 6.72 ± 1.89 pA/pF in control, n = 7).
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2-receptor agonists exhibit cardiac antiarrhythmic effects in a rabbit model of premature ventricular beats and left ventricular tachycardia. This property is due to a prolongation of myocardial repolarization demonstrated in rabbit Purkinje fibers, arguing in favor of class III antiarrhythmic actions. These compounds block the fast component of the delayed rectifier K+ current generated by the human K+ channel HERG in transfected COS-7 cells. This effect is not prevented by the 2-receptor antagonist SM-21. All of these results strongly suggest that the cardiac current IKr is deeply affected by 2-receptor agonists, a property that can drive to antiarrhythmic activities but also to QT prolongation and consequently to torsades de pointe and sudden cardiac death.
In humans, IKr, the fast component of the delayed rectifier K+ current is linked with HERG, and mutations of this gene are associated with electrophysiologic troubles observed in the congenital long-QT syndrome. Numerous drugs are known to block HERG channels and provoke QT interval prolongations and cardiac arrhythmias, such as torsades de pointe. Among them, several psychoactive drugs, such as haloperidol or chlorpromazine, prolong the QT interval, an effect that is not related to their antagonism on dopamine D2 receptors (Kriwisky et al., 1990; O'Brien et al., 1999). In fact, this blockade could be due to a direct antagonism of the HERG channel, with haloperidol able to displace [3H]dofetilide from its binding site in the HERG channel pore (Finlayson et al., 2001), or due to an interaction with receptors known to regulate K+ channels. Haloperidol has been described to have a high affinity for receptors (Tam and Cook, 1984). The regulation of K+ channels by receptors is well established. It has been shown in neurohypophysial nerve terminals (Wilke et al., 1999) that K+ currents are modulated by different 1-receptor ligands. Furthermore, the activation of 1 receptors has recently been shown to depress the excitability of rat intracardiac neurons (Zhang and Cuevas, 2005). Finally, the direct protein-protein interaction of the 1 receptor and a K+ channel was demonstrated recently by Aydar et al. (2002). They showed that the K+ current generated Kv1.5, a K+ channel present in pituitary cells, that is regulated by coexpressed 1 receptors in Xenopus oocytes and coimmunoprecipitation of both proteins in the native tissue. The second member of this family, the 2 receptor, is expressed in the heart (Dumont and Lemaire, 1991) but until now has never been cloned and has yet to be considered a binding site. The class III antiarrhythmic properties of some compounds, including amiodarone, have been attributed to their action on 2 receptors (Jeanjean et al., 1993); however, the mechanisms of these 2-receptor-mediated effects have never been investigated. In this work, we studied the effects of the ligands (+)-SKF-10,047, DTG, and ifenprodil (and some of its derivatives) on cardiac repolarization in isolated rabbit Purkinje fibers. In a RNase protection assay, it was shown that the cardiac tissue of the rabbit contains HERG mRNA at similar levels than in humans (Wymore et al., 1997) and that Purkinje fibers isolated from this species are currently used as a model to test class III antiarrhythmics (Valenzuela et al., 1996). For these reasons, we decided to investigate the effects of 2-receptor ligands on Purkinje fibers. Racemic ifenprodil exhibits a high affinity for the 2 receptors (Hashimoto and London, 1995), but it is also an 1-adrenergic and NMDA receptor antagonist (Honda et al., 1988; Carter et al., 1989). Its stereoisomer, threo-ifenprodil, has no detectable affinity for 1-adrenergic receptors but retains NMDA receptor-blocking activity (Chenard et al., 1991). For this last reason, we synthesized an analogous of threo-ifenprodil called LNP250A, which binds neither to NMDA nor to 1-adrenergic receptors up to concentrations of 10 µM. This improvement of pharmacological selectivity was associated to a reduction of its affinity toward the 2 receptors compared with the reference substances. Nevertheless, we used it as a selective 2-receptor pharmacological tool. At a functional point of view, the absence of blockade of 1-adrenergic receptors by threo-ifenprodil and LNP250A was confirmed by the in vivo experiments, because these two drugs affected neither the resting blood pressure nor the pressive response to intravenous phenylephrine. In fact, all of the 2-receptor ligands dose-dependently prolonged action potential duration on Purkinje fibers, with threo-ifenprodil exhibiting the greatest efficacy. This prolongation promoted early-afterdepolarizations at the higher concentrations, suggesting that such drugs may have proarrhythmic effects. The fact that racemic ifenprodil was less potent than the threo-isomer alone could be due, at least in part, to the 1-adrenergic antagonist properties of the erythro-isomer. In rabbits, the stimulation of 1-adrenergic receptors by methoxamine prolongs cardiac repolarization (Carlsson et al., 1990). In our preparation, the simultaneous blockade of 1-adrenergic receptors that shortens action potential and stimulation of 2 receptors could result in a functional antagonism. Such prolongation was also obtained with LNP250A; its lower efficacy was probably due to its lower affinity at 2 receptors. In these conditions, neither threo-ifenprodil nor LNP250A affected the maximal slope of upstroke (Vmax) and the amplitude (APA) of the action potential ruling out some actions on channels involved in the depolarization phase. Moreover, the resting potential of the membrane also was not modified. Surprisingly, the usual 2 ligand, 1,3-di(2-tolyl)guanidine, did not exhibit any effect up to a concentration of 10-5 M. This phenomenon has been described by others, the binding site of this very hydrophilic compound on the 2 receptor potentially located intracellularly (Ela et al., 1994). This problem could not occur with ifenprodil and its derivatives, which probably are lipophilic enough at physiological pH to cross the cellular membrane. In contrast to 2-receptor ligands, the selective 1-receptor agonist (+)-SKF-10,047, known as a blocker of neurohypophysial K+ channels (Lupardus et al., 2000), had no effect on action potential duration in the rabbit Purkinje fibers, even at very high concentrations. Taken together, these data argue in favor of a preferential modulation of K+ currents by 2-receptor ligands.
To further test this hypothesis, we analyzed the effects of threo-ifenprodil, LNP250A, DTG, and SKF-10,047 on HERG K+ channels in whole-cell patch-clamp experiments. All of the 2-receptor ligands inhibited the K+ currents in a voltage- and dose-dependent manner. The order of potencies and the range of concentrations of drugs were nearly superimposable to those observed in Purkinje fibers, suggesting that this action underlies the APD prolongation. Nevertheless, the 2-receptor antagonist SM-21 (Ghelardini et al., 2000) was unable to block the effects of the 2-receptor agonists, arguing in favor of a direct interaction of these compounds on the HERG channels. A similar direct interaction of 2-receptor ligands with potassium channels was described previously in the brainstem (Nguyen et al., 1998). Taken together, these data argue in favor of a class III antiarrhythmic profile of 2-receptor ligands. This was confirmed in vivo, as both compounds prevented the cardiac arrhythmias induced by the intravenous injection of phenylephrine irrespective of their affinity for the 1-adrenergic receptors. In the present study, we used the intravenous administration of phenylephrine in rabbits as a model to trigger cardiac arrhythmias and to study the antiarrhythmic properties of -receptor ligands. This is an adaptation of the classic methoxamine-sensitized rabbit heart model developed in the early 1990s (Carlsson et al., 1990), in which the intraduodenal administration of this 1-adrenergic agonist predisposes the myocardium to arrhythmias because of improper calcium handling. This model is extensively used to screen drugs that could induce torsades de pointe but also to extend our knowledge on the mechanisms of class III antiarrhythmics (Lawrence et al., 2005). By using bolus injections of phenylephrine, we obtained a rapid and transient myocardium destabilization that mimicked the methoxamine model. This short-lasting effect allowed the employment of rabbits as their own controls and prevented the risk of torsades de pointe that usually occur minutes after the QT interval prolongation induced by methoxamine. Therefore, this model appears as a simple way to test class III antiarrhythmics, and we show that 2-receptor ligands are antiarrhythmics. Nevertheless, this work shows for the first time that particular caution has to be taken with ligands with affinity for these receptors, the repolarization prolongation, and the early-afterdepolarization potentially responsible of torsades de pointe and sudden cardiac death.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: SKF-10,047, (±)-N-allylnormetazocine; (+)-[3H]PPP, (+)3-hydroxyphenyl-N-(1-propyl)-piperidine; DTG, 1,3-di(2-tolyl)guanidine; NMDA, N-methyl-D-aspartate; LNP250A, threo-8-[1-(4-hydroxyphenyl)-1-hydroxy-propan-2-yl]-1-phenyl-1,3,8-triazaspiro[4,5]decane-4-one; AP, action potential; APA, action potential amplitude; Vmax, maximal slope of AP upstroke; APD, action potential duration; bpm, beats per min; SM-21, 3--tropanyl-2(pCl-phenoxy)butyrate; CL, cycle length; IKr, rapidly activating delayed rectifier K+ current; SAP, systolic arterial pressure; IKs, slowly activating delayed rectifier K+ current; PE, phenylephrine; DTG, 1,3-di(2-tolyl)guanidine; DMSO, dimethyl sulfoxide; VT, ventricular tachycardia; VEB, ventricular ectopic beat; HERG, human ether-a-go-go-related gene; EAD, early-afterdepolarization.
Address correspondence to: Laurent Monassier, Laboratoire de Neurobiologie et de Pharmacologie Cardiovasculaire, Facultéde Médecine, INSERM U-715, 11 rue Humann, 67085 Strasbourg, France. E-mail: laurent.monassier{at}medecine.u-strasbg.fr
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) and in the presence of 10-6 M LNP250A (
; n = 5). Inset, voltage protocol consisted of 500-ms depolarizing and hyperpolarizing prepulses to various potentials between -100 and +60 mV followed by a test pulse to -60 mV for 500 ms. Holding potential, -80 mV; frequency, 0.33 Hz. C, current-voltage relation of the deactivating tail current density in control conditions (
) and in the presence of 10-6 M LNP250A (
; n = 5) measured after 500-ms depolarization to +10 mV. Statistical significance versus I tail/Cm in control conditions (*, p < 0.05; **, for prepulse potentials of 0 mV and above, p < 0.01). Inset, inhibition ratio ([I tail in control conditions - I tail in the presence of 10-6 M LNP250A]/I tail control) versus prepulse potential (n = 4-5). D, dose-response curve for HERG K+ tail current (
