The presence of dopamine receptors in mammalian heart has been demonstrated using radioligand binding, autoradiography, and Western blot techniques.1–5 In the guinea pig heart, the D3 and D4 receptors have been found mainly distributed in the right atria, mediating negative chronotropic and inotropic effects.5 Gomez et al5 have shown, using the Langendorff system, that the D3-dopamine receptor agonist 7-hydroxy-dipropylaminotetralin (7-OH-DPAT) produces a concentration-dependent decrease of the cardiac rate. The negative chronotropic effect of 7-OH-DPAT at nanomolar concentrations was antagonized by D2/D3 antagonists. The effect of 7-OH-DPAT at micromolar concentrations was not antagonized even at high concentrations of antagonists. They suggest a dual mechanism of 7-OH-DPAT, a direct mechanism at nanomolar concentrations mediated by activation of D3 receptors, and a secondary effect observed at micromolar concentrations, probably not mediated by dopamine receptors.5
Effects of different dopamine receptor agonists have been found on the action potential duration (APD) of canine Purkinje fibers. Apomorphine and ropinirole produced an increase in APD, sumanirole did not modify APD, and pergolide significantly shortened APD. Apomorphine, ropinirole, and pergolide did block hERG currents, and sumanirole only partially blocked human Ether-a-go-go-related Gene (hERG) currents at high concentrations.6
To explain the electrophysiologic cardiac effects of the D3-dopamine receptor agonist 7-OH-DPAT, we studied the effects of the drug on action potentials of rabbit sinoatrial node and cat Purkinje fibers. We also studied the effects of the drug on different potassium currents of cat ventricular myocytes under voltage-clamp conditions. In addition, we studied the effects of 7-OH-DPAT on hERG channels expressed in HEK293 cells and Xenopus laevis oocytes.
MATERIALS AND METHODS
Standard Microelectrode Technique
Purkinje Fiber Preparations
Adult cats (2 to 4 kg) were anesthetized with sodium pentobarbital (35 mg/kg and treated with heparin, 1000 U/kg). Free-running Purkinje strands were obtained from the left ventricle of the cat hearts. The Purkinje strands were fixed to the Sylgard (Dow Corning Co, Midland, MI)-coated bottom of a Plexiglass chamber (2-mL volume) with micropins.
Sinoatrial Node Preparations
Rabbits (1.5 to 2.5 kg) were anesthetized with sodium pentobarbital (35 mg/Kg and treated with heparin, 1000 U/Kg). By dissection perpendicular to the crista terminalis, small strips of sinoatrial node were obtained, which were then ligated with fine thread to obtain small (balllike) preparations of approximately 0.5 mm×0.5 mm.7
The preparations were fixed to the Sylgard-coated bottom of a Plexiglass chamber (2-mL volume) with micropins. We used only preparations that exhibited spontaneous firing. The electrical activity exhibited was clearly that of the central region of the node. The preparations were superfused with a solution containing 125 mM NaCl, 24 mM NaHCO3, 0.43 mM NaH2PO4, 4 mM KCl, 1.8 CaCl2, MgCl2, and 11 mM glucose. The solution was equilibrated with 95% O2+5% CO2 (pH 7.4). Temperature was kept constant at 36°C. The preparations were allowed to equilibrate for 60 minutes before experimental protocols were performed. During this time, the Purkinje fibers were stimulated at a frequency of 1 Hz with rectangular stimuli (3-ms duration, 1.5 times diastolic threshold intensity). Sinoatrial node preparations were not externally stimulated. Action potentials were recorded using glass microelectrodes filled with 3 M KCl (resistance 10 to15 mΩ for Purkinje fibers, 20 to 30 MΩ for sinoatrial node cells) and coupled to the input of a high-impedance preamplifier (World Precision Instruments, New Haven, CT). Action potential signals were digitized at a sampling rate of 10 kHz by use of an analog-to-digital converter (Digidata 1200 interface; Molecular Devices Corporation) and stored on a hard disk, Axotape data-acquisition software (Molecular Devices Corp), and a personal computer. Data analysis was performed using pClamp software (version 8.0; Molecular Devices Corp). All drug effects on action potentials recording from multicellular tissue were from continuous impalements.
Voltage Clamp of Oocytes
Isolation and maintenance of Xenopus oocytes and cRNA injection were performed as described.8 A GeneClamp 500 amplifier (Molecular Devices Corp) and standard 2-microelectrode voltage-clamp techniques9 were used to record currents. Currents were recorded at room temperature (22 to 24°C), 2 to 4 days after cRNA injection. Glass microelectrodes were filled with 3 M KCl and their tips broken to obtain resistances of 0.8 to 1.2 MΩ. The external low Cl− solution contained 96 mM NaMES (2-[morpholino]ethanesulfonic acid), 2 mM KMES, 2 mM CaMES2, 5 mM HEPES, and 1 mM MgCl2, and adjusted to pH 7.6 with methanesulfonic acid. Voltage commands were generated using pCLAMP software (version 8; Molecular Devices Corp). Specific voltage-clamp protocols are described in the Results section. Currents were not corrected for leak or endogenous currents and capacitance transients were not nulled.
The experiments with animals were approved by the Ethics Committee of the University of Colima.
Voltage Clamp in Cell Line
hERG was stably transfected into HEK-293H cell line with the use of Lipofectamine-Plus and selected with 750 mg/mL G418 (Life Technologies). Current recordings in HEK-293H cells were carried out at room temperature and were obtained using the whole-cell “perforated patch” clamp method10 with an Axopatch 200B amplifier (Molecular Devices Corp). Data acquisition and generation of voltage-clamp pulse protocols were performed with a Digidata 1322A interface (Molecular Devices Corp) controlled by pCLAMP8.0 software (Molecular Devices Corp). Currents were low-pass filtered at 1 kHz and sampled at 2 kHz. Micropipettes were pulled from borosilicate glass capillary tubes (WPI, World Precision Instruments, Sarasota, FL) on a programmable puller (Sutter Instruments, Novato, CA). When micropipettes were filled with the pipette solution, tip resistance ranged from 1 to 3 MΩ. After obtaining whole-cell access, series resistance was compensated to minimize the duration of the capacitive transient. The external solution contained (mmol/L) 130 NaCl, 4 KCl, 1 MgCl2, 10 HEPES, 1.8 CaCl2, and 10 Glucose, with pH adjusted to 7.35 with NaOH. Patch pipettes were filled with (mmol/L) 5 K4BAPTA, 100 KCl, 10 HEPES, 5 MgCl2, 5 ATP-K2, and 200 μg/mL amphotericin B, with the pH adjusted to 7.2 with KOH. All junction potentials were zeroed before formation of the seals. Specific voltage-clamp protocols are also described in the Results section.
7-OH-DPAT (Sigma Chemicals, St Louis, MO) was dissolved directly in the external solution at the desired concentrations. Oocytes and HEK-293 cells were exposed to 7-OH-DPAT solutions until steady-state effects were achieved, usually in about 15 minutes. Multicellular preparations were exposed to 7-OH-DPAT solutions until steady-state effects were achieved, usually in about 30 minutes.
Data are presented as mean±SEM (n=number of cells). Clampfit (pClamp 8) software (Axon Instruments) was used to do nonlinear least-square kinetic analyses of time-dependent currents. The fractional block of current (f) was plotted as a function of drug concentration ([D]) and the data fit with a Hill equation,
Equation (Uncited)Image Tools
to determine the concentration (IC50) required for 50% block of current magnitude and the 0 Hill coefficient, nH. The voltage dependence of hERG activation was determined from tail currents measured at −70 mV after 4-second test depolarizations. Normalized tail current amplitude (In) was plotted versus test potential (Vt) and fitted to a Boltzmann function, In=1/(1+exp[(V1/2−Vt)/k]), using Origin software (Northampton, MA). V1/2 is the voltage at which the current is half-activated, and k is the slope factor of the relationship.
Statistical comparisons between experimental groups were performed using 1-way ANOVA and Dunnet method. Differences were considered significant at P<0.05.
Effect of (±) 7-OH-DPAT on Electrical Activity in Central Balls of Sinoatrial Node Tissue
Figure 1 shows the effect of the D3-dopamine agonist 7-OH-DPAT at different concentrations on action potentials recorded from small balllike preparations of central sinoatrial node tissue. 7-OH-DPAT (1 and 3 μM) significantly prolonged cycle length and APD, depolarized maximum diastolic potential, and reduced the upstroke velocity of the action potential. These effects were concentration-dependent (see Table 1). At higher concentrations (10 μM), spontaneous activity ceased in all cells tested (n=4; data not shown). These experiments with 7-OH-DPAT on preparations of the central sinoatrial node tissue were also performed in the presence of the D3-dopamine receptor antagonist U99194 at a concentration of 1 μM. The effects of 7-OH-DPAT in the presence of the antagonist on sinoatrial node cells were similar to those found in its absence (Table 2), suggesting that effects of 7-OH-DPAT on sinoatrial node cells are not related to activation of D3-dopamine receptors. In addition, another series of experiments were performed using preparations of the central sinoatrial node tissue from rabbits pretreated with reserpine (data not shown). The effects were similar to those found in preparations obtained from rabbits not so treated, suggesting that an effect of 7-OH-DPAT inhibiting noradrenaline release from presynaptic neurons is not involved.
Effect of 7-OH-DPAT on Electrical Activity in Cat Purkinje Fibers
The effect of 7-OH-DPAT was evaluated for its effects on the duration of the action potential (APD) in cat Purkinje fibers. Action potentials were evoked at different frequencies before and during exposure of 7-OH-DPAT at different concentrations. Figure 2A shows recorded action potentials evoked at 1 Hz, before and during exposure of different concentrations of the drug (0.01, 0.03, 0.1, 0.3, 1, and 3 μM). 7-OH-DPAT produced an increase in APD. When studied at different frequencies, it showed “reversal frequency-dependency”, that is more effect at lower stimulation frequencies (Fig. 2B).
Effect of 7-OH-DPAT on Different Potassium Currents in Cat Ventricular Myocytes
The experiments studying the effect of 7-OH-DPAT on different potassium currents were conducted in voltage-clamped cat ventricular myocytes. The inward rectifying potassium current IK1 was elicited by applying hyperpolarizing and depolarizing pulses to different membrane potentials (from −120 to −20 mV) from a holding potential (HP) of −40 mV (Fig. 3A). 7-OH-DPAT did not affect the IK1 I–V relationship of the current measured at the end of the 500-ms pulses under control conditions and during superfusion of 3 μM 7-OH-DPAT with the results shown in Figure 3B.
Figure 4 shows the effect of 3 μM 7-OH-DPAT on the transient outward potassium current Ito. This current, Ito, was caused by applying depolarizing pulses to membrane potentials ranging from −50 to +50 mV from an HP of −70 mV. 7-OH-DPAT did not significantly affect the peak current amplitude or time course of Ito. Peak current–voltage relationships are shown in Figure 4C.
In cat ventricular myocytes, as in other mammalian species, the delayed, rectifying outward potassium current IK is composed of a rapid component IKr and slow component IKs.11,12 IK was activated with 3-second depolarizing pulses to membrane potentials between −30 and +50 mV applied from an HP of −40 mV. Tail currents were recorded upon repolarization to −40 mV. Figure 5A shows current records obtained under control conditions and during superfusion with 3 μM 7-OH-DPAT (Fig. 5B). 7-OH-DPAT at different concentrations ranging from 300 nM to 10 μM decreased the amplitude of the currents elicited during the depolarizing pulses and tail current amplitude in a concentration-dependent manner (Figs. 5C, D).
In an attempt to dissect out the effects of 7-OH-DPAT on the 2 components of IK, we applied, from an HP of −40 mV, a 3-second depolarizing pulse to +50 mV followed by a repolarizing pulse to 0 mV for 3 seconds and then a repolarization to −40 mV for 3 seconds. The depolarizing pulse to +50 mV activates both the IK components, IKr and IKs. The repolarization pulse to 0 mV causes deactivation of IKs so the decaying tail current recorded at this potential mainly represents IKs. The tail current recorded upon repolarization to −40 mV mainly represents IKr deactivation.13–15 Figure 6 shows superimposed current traces obtained under control conditions and during superfusion with 3 μM 7-OH-DPAT. The drug slightly decreased the current during the depolarizing pulse and tail current upon repolarization to 0 mV. However, during repolarization to −40 mV, the tail current was abolished. The tail current measured at 0 mV was not significantly changed (−7%±4%) from control by 10 μM 7-OH-DPAT (n=5). The tail current measured upon repolarization to −40 mV was significantly decreased to 6%±4% by the drug (n=5). These experiments suggest that 7-OH-DPAT mainly inhibited the rapid component IKr of the delayed rectifier potassium current.
Effect of 7-OH-DPAT on hERG Channels Expressed in the HEK293 Cell Line
hERG subunits coassemble to form channels that conduct IKr.16 Experiments on hERG currents expressed in HEK293 cells were conducted using the perforated patch clamp technique. herg currents were inhibited by 7-OH-DPAT in a concentration-dependent manner (Fig. 7A). hERG currents were elicited by 2-second depolarizing steps to a membrane potential of +20 mV, followed by repolarization to −40 mV to cause slowly decaying outward tail currents. The pulses were applied every 20 seconds. Figure 7A shows current traces obtained under control conditions and in the presence of 7-OH-DPAT at concentrations ranging from 0.3 to 10 μM. The concentration–response curve for the tail current measured after a depolarizing pulse to +20 mV is shown in Figure 7B. The IC50 was 3.1±0.3 μM and the Hill coefficient nH=0.79±0.05. The effect of 7-OH-DPAT on hERG was completely reversible after 7.5 minutes of washout (Fig. 7C).
Block of WT hERG Channels Expressed in Oocytes by 7-OH-DPAT
The effect of 7-OH-DPAT on the hERG current–voltage (I–V) relationship was investigated under isochronal-recording conditions. Oocytes were clamped at an HP of −80 mV. Depolarizing pulses were applied for 4 seconds to voltages between −70 and +50 mV, in 10 mV increments, and tail currents were recorded upon repolarization to −70 mV for 4 seconds. Families of current traces from 1 oocyte are shown for control conditions (Fig. 8A) and during superfusion with 1 μM 7-OH-DPAT (Fig. 8B). Current activated during depolarizations positive to −70 mV reached a maximum at −10 mV and declined at more positive potentials, producing the bell-shaped I–V curve (Fig. 8C). Figure 8D displays peak tail current amplitudes as a function of the preceding test hERG-pulse potential, resulting in activation curves. The peak tail current increased with voltage steps from −70 to +10 mV, and then plateaued for test pulse potentials positive to +10 mV. 7-OH-DPAT reduced the current during the depolarizing pulse and also the tail currents. These effects were concentration-dependent (Figs. 8C, D). 7-OH-DPAT significantly affected the half-maximum activation voltage (V1/2), which was –27.6±2.1 mV under control conditions, −33.2±1.9 mV in the presence of 1 μM, and−37.6±1.8 mV in the presence of 5 μM 7-OH-DPAT (n=5 oocytes).
7-OH-DPAT is a D3-dopaminergic receptor agonist, which has been found to produce cardiac negative chronotropic and inotropic effects.5 In the present work, we found in rabbit sinoatrial tissue that 7-OH-DPAT decreased spontaneous activity and increased APD. In cat Purkinje fibers, 7-OH-DPAT increased APD. In cat ventricular myocytes, 7-OH-DPAT inhibited IKr without effect on IK1 and Ito. The effect on IKr was corroborated by the blocking effects of the drug on hERG currents expressed in the HEK293 cell line and Xenopus oocytes.
The effects of 7-OH-DPAT on rabbit sinoatrial node cells were not antagonized by the D3-dopamine receptor antagonist U99194. In addition, the effects of 7-OH-DPAT were not prevented by pretreatment of the animals with reserpine. These results suggest that the effects of 7-OH-DPAT were not mediated by D3-dopamine receptors localized presynaptically or postsynaptically. The presence of a type or subtype of receptor in a tissue is frequently supported by the use of supposedly specific agonist or antagonist. However, care should be taken when studying possible D3-dopamine-mediated effects in cardiac tissues by the use of 7-OH-DPAT at micromolar concentrations because of its direct blocking effect on IKr and hERG. On the other hand, erg-mediated currents have also been recorded in various other types of cells, for example, sympathetic ganglia neurons, glomus cells of the carotid body, and smooth muscle myocytes.17
To obtain information about the possible electrophysiologic mechanism of action to explain the effects of 7-OH-DPAT on sinoatrial node cells and Purkinje fibers, we studied the effects of the drug on different cardiac potassium currents. In voltage-clamped cat ventricular myocytes, we found that 7-OH-DPAT inhibited the fast component of the delayed rectifier potassium current IKr, without affecting Ito and IK1. These results can at least partially explain the electrophysiologic effects of the drug on sinoatrial node cells and Purkinje fibers. It is known that the blocking of IKr by E-4031 produces effects similar to those observed in our work with 7-OH-DPAT.18,19 E-4031 (0.1 μM) significantly prolonged cycle length and APD, depolarized the maximum diastolic potential, and reduced both the upstroke velocity of the action potential and the diastolic depolarization rate. In the presence of higher concentrations of E-4031 (1 to 10 μM), spontaneous activity ceased in all cells (n=4). In Purkinje fibers of different species, E-4031 increases APD without affecting other electrophysiologic variables of the action potential.20
The inhibitory effects of 7-OH-DPAT on hERG channel current measured in the cell line and oocytes corroborated the effects of the drug on IKr. Different dopamine receptor agonists developed for the treatment of Parkinson disease have been found to inhibit hERG currents. Pergolide, ropinirole, and apomorphine produced inhibition of hERG currents in CHO-K1 cells, with IC50 of 0.12, 1.2, and 2.4 μM.6 In our work, we found that 7-OH-DPAT inhibited hERG current in HEK293 cells with an IC50 of 3.1 μM. The effect on hERG currents was corroborated in Xenopus oocytes in this heterologous system.
Drugs that prolong ventricular repolarization are associated with the acquired long QT syndrome, which is associated with malignant ventricular arrhythmias (especially the distinctive polymorphic ventricular-tachycardia called torsade de pointes). Prolongation of ventricular repolarization by drugs is caused by the blockade of cardiac potassium channels responsible for mediating ventricular repolarization. Different types of drugs, including some antiarrhythmics, antihistamines, antibiotics, gastrointestinal prokinetics, and antipsychotics, have been found to block hERG and IKr channels.21,22 In our work, we found that 7-OH-DPAT prolongs cardiac repolarization and decreases the frequency of spontaneous sinoatrial node cells caused by the inhibition of hERG and IKr currents. However, there is no clinical or experimental (electrocardiographic) information about long QT prolongation with 7-OH-DPAT.
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