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NEUROPHARMACOLOGY

Receptor Activation Blocks Potassium Channels and Depresses Neuroexcitability in Rat Intracardiac Neurons

Department of Pharmacology and Therapeutics, University of South Florida College of Medicine, Tampa, Florida

Received January 26, 2005; accepted March 8, 2005.

	Abstract

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The receptors have been implicated in the regulation of the cardiovascular system, and -1 receptor transcripts have been found in parasympathetic intracardiac neurons. However, the cellular function of -1 receptors in these cells remains to be determined. Effects of receptor activation on voltage-activated K⁺ channels and action potential firing were studied in isolated intracardiac neurons using whole-cell patch-clamp recording techniques. Activation of receptors reversibly blocked delayed outwardly rectifying potassium channels, large conductance Ca²⁺-sensitive K⁺ channels, and the M-current with maximal inhibition >80%. The inhibition of K⁺ channels by ligands was dose-dependent, and the rank order potency of (+)-pentazocine > ibogaine > 1,3-di-O-tolyguanidin (DTG) suggests that the effect is mediated by -1 receptor activation. Preincubation of neurons with the irreversible receptor antagonist metaphit blocked DTG-induced inhibition of K⁺ channels, confirming that the effect is mediated by receptor activation. Although bath application of ligands depolarized intracardiac neurons, the number of action potentials fired by the cells in response to depolarizing current pulses was decreased in the presence of these drugs. Neither dialysis of the neurons nor application of intracellular 5'-O-(2-thiodiphosphate) trilithium salt inhibited the effect of receptors on K⁺ channels, which suggests that the signal transduction pathway does not involve a diffusible cytosolic second messenger or a G protein. Together, these data suggest that -1 receptors are directly coupled to K⁺ channels in intracardiac neurons. Furthermore, activation of -1 receptors depresses the excitability of intracardiac neurons and is thus likely to block parasympathetic input to the heart.

The receptors also seem to play a role in the regulation of the cardiovascular system, as suggested by pharmacological studies using receptor ligands. For example, it has been shown that the receptor ligand DuP 754 raises the threshold of ventricular fibrillation in rats (Lishmanov et al., 1999). Conversely, the receptor agonists (+)-SKF-10,047, 1,3-di-O-tolyguanidin (DTG), and (+)-3-[3-hydroxyphenyl]-N-(1-propyl)piperidine have been shown to produce tachycardia by activating central and peripheral receptors (Wu and Martin, 1989; Lishmanov et al., 2000). Some of the effects of receptor ligands on the cardiovascular system are likely due to activation of receptors on cardiac muscle. The receptors have been detected in cardiac myocytes from neonatal and adult rats, and stimulation of these receptors was shown to elicit direct inotropic and chronotropic effects on cardiac muscle (Ela et al., 1994; Novakova et al., 1995). However, receptors have also been found in autonomic neurons that regulate the cardiovascular system (Zhang and Cuevas, 2002), and modulation of the electrical activity of these neurons by endogenous or exogenous ligands is likely to have profound effects on cardiovascular function.

The receptor ligands, including haloperidol, (+)-pentazocine, DTG, and ibogaine, were previously shown to inhibit high-voltage-activated calcium channels in intrinsic cardiac neurons (Zhang and Cuevas, 2002). The pharmacological profile of the receptor mediating these effects was consistent with a -2 receptor. Although -1 receptor transcripts were detected in intracardiac neurons, no cellular effects have been attributed to this receptor. One possible role for -1 receptors in these cells is the modulation of voltage-activated K⁺ channels. Recent experiments have shown that currents mediated by Kv1.4 and Kv1.5 are depressed by the ligand (+)-SKF-10,047 in oocytes injected with Kv subunits and the -1 receptor but not in oocytes injected with the Kv subunits alone (Aydar et al., 2002). Furthermore, -1 receptors were shown to alter the kinetic properties of Kv1.4 channels in the absence of ligand, and Kv1.4 and Kv1.5 coimmunoprecipitated with the -1 receptor (Aydar et al., 2002), which led those investigators to propose that the -1 receptor functions as a ligand-regulated auxiliary K⁺ channel subunit. The inhibition of delayed outwardly rectifying K⁺ channels, such as Kv1.5, by verapamil has been shown to depress action potential firing in intrinsic cardiac neurons, and this phenomenon has been implicated in the tachycardia associated with this type IV antiarrhythmic drug (Hogg et al., 1999). However, it remains to be determined whether -1 receptors couple to voltage-activated K⁺ channels in intracardiac neurons, and whether receptor activation can alter the active membrane proprieties of these cells. By regulating the function of K⁺ channels in parasympathetic intracardiac neurons, -1 receptors, and therefore drugs that act on these receptors, may have significant influence on the heart.

Experiments were undertaken to determine the effects of receptors on currents mediated by voltage-activated K⁺ channels (I_K) and the active membrane properties of intracardiac neurons from neonatal rats. The receptors were shown to decrease the peak amplitude of currents mediated by delayed outwardly rectifying K⁺ channels [I_K(DR)], large conductance Ca²⁺-sensitive K⁺ channels (I_BK), and the M-current (I_M). The rank-order potency of K⁺ channel inhibition by different ligands suggest that the effect is mediated by -1 receptors. Neither cell dialysis nor intracellular application of guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDP--S) blocked the effects of -1 receptors on I_K, consistent with a direct interaction between -1 receptors and the K⁺ channels. Activation of -1 receptors also decreased action potential firing and changed the action potential configuration in intracardiac neurons. Therefore, -1 receptor activation depresses the excitability of intracardiac neurons and is likely to attenuate parasympathetic input to the heart.

	Materials and Methods

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Preparation. The isolation and culture of neurons from neonatal rat intracardiac ganglia has been described previously (Cuevas and Adams, 1994). Briefly, neonatal rats (2–7 days old) were euthanized by decapitation, and the hearts were excised and placed in a physiological saline solution containing 140 mM NaCl, 3 mM KCl, 2.5 mM CaCl₂, 0.6 mM MgCl₂, 7.7 mM glucose, and 10 mM histidine, pH to 7.2 with NaOH. Atria were removed and incubated for 1 h at 37°C in physiological saline solution containing 1 mg/ml collagenase (type 2, Worthington Biochemicals, Freehold, NJ). After enzymatic treatment, clusters of ganglia were dissected from the epicardial ganglion plexus and dispersed by trituration in a high-glucose culture medium (Dulbecco's modified Eagle's medium), 10% fetal calf serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. The dissociated neurons were then plated onto glass coverslips coated with poly-L-lysine and incubated at 37°C under a 95% air/5% CO₂ atmosphere for 48 to 72 h.

Electrical Recordings. Electrophysiological recording methods used were similar to those described previously (Cuevas et al., 1997). Active membrane properties and voltage-activated K⁺ channel currents in intracardiac neurons were studied under current-clamp and voltage-clamp mode, respectively, using the whole cell patch-clamp technique. Electrical access was achieved through the use of the amphotericin B perforated-patch method to preserve the intracellular integrity and prevent the loss of cytoplasmic components and subsequent alteration of the functional responses of these neurons (Cuevas and Adams, 1994). For perforated patch experiments, a stock solution of 60 mg/ml amphotericin B in dimethyl sulfoxide was prepared and diluted in pipette solution immediately before use to yield a final concentration of 198 µg/ml amphotericin B in 0.33% dimethyl sulfoxide. Final patch pipette resistance was 1.0 to 1.3 M to permit maximal electrical access under the present recording configuration.

Action potentials were elicited by depolarizing current pulses (+100 pA) in absence and presence of ligands. Currents through depolarization-activated K⁺ channels were elicited by step depolarizations from –90 mV to more positive potentials (-50 to +70 mV). For experiments studying the effects of receptor activation on I_M, cells were held at -30 mV to activate the channel and repolarized to -60 mV to deactivate the channels. Membrane voltages and currents were amplified using an Axopatch 200B patch-clamp amplifier (Axon Instruments, Inc., Union City, CA), filtered at 5 kHz (-3 dB; four-pole Bessel filter), and digitized at 20 kHz (Digidata 1200B).

Data Analysis. Analyses of these data were conducted using the SigmaPlot 2000 program (SPSS Inc., Chicago, IL). Data points represent means ± S.E.M. Statistical difference was determined using paired t test for within-group experiments and unpaired t test for between groups experiments and was considered significant if p < 0.05.

Solutions and Reagents. The bath solution for action potential experiments was a physiological saline solution (PSS) containing 140 mM NaCl, 1.2 mM MgCl₂, 3 mM KCl, 2.5 mM CaCl₂, 7.7 mM glucose, and 10 mM HEPES, pH to 7.2 with NaOH. Potassium channel currents were isolated by adding tetrodotoxin (TTX; 400 nM) and cadmium chloride (CdCl₂; 100 µM) to the PSS to inhibit voltage-activated Na⁺ channel and Ca²⁺ channel currents, respectively. Pipette solution used for perforated patch experiments contained 75 mM K₂SO₄, 55 mM KCl, 5 mM MgSO₄, and 10 mM HEPES (adjust to pH 7.2 with N-methyl-D-glucamine). For studies using conventional (dialyzing) whole cell recording configuration, the pipette solution contained140 mM KCl, 2 mM MgCl₂, 2 mM EGTA, 2 mM Mg₂ATP, 0.1 mM GTP lithium salt (GTP), and 10 mM HEPES-KOH, pH to 7.2. In some experiments, GTP was replaced with 100 µM GDP--S to inhibit G protein activation. All chemical reagents used were of analytical grade. Haloperidol, ibogaine hydrochloride, (+)-pentazocine, DTG, (+)-SKF-10,047, TTX; tetraethylammonium chloride (TEA), and linopirdine were purchased from Sigma-Aldrich (St. Louis, MO).

	Results

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Receptor Activation Inhibits Delayed Outward Rectifying K⁺ Currents. Mammalian intracardiac neurons exhibit currents mediated by delayed outwardly rectifying K⁺ channels (Xi-Moy and Dun, 1995; Hogg et al., 1999) and express various subunits that contribute to these channels, including KCNA5 and KCNA6 (J. Cuevas, unpublished observation). Given that -1 receptors have been shown to depress heterologously expressed KCNA5 channels (Aydar et al., 2002) and that inhibition of these channels may have significant effects on the function of intracardiac neurons, it seemed prudent to test the effects of receptor activation on currents mediated by voltage-activated K⁺ channels in these cells. Figure 1A shows a family of depolarization-activated K⁺ currents recorded from a single intracardiac neuron in the absence and presence of 100 µM DTG. Bath application of 100 µM DTG depressed peak voltage-activated K⁺ current amplitude at potentials positive to +20 mV. The voltage dependence and kinetics of the currents observed are consistent with the delayed outwardly rectifying K⁺ current [I_K(DR)] previously reported in intracardiac neurons (Xi-Moy and Dun, 1995). Figure 1B shows a plot of the mean peak I_K(DR) amplitude in the absence and presence of DTG as a function of voltage. The DTG-induced block of I_K(DR) was voltage-dependent, with the current being blocked by 26.5 ± 6.2% at +70 mV but only by 1.5 ± 0.1% at +10 mV (n = 7). This difference was statistically significant (p < 0.01).

View larger version (18K): [in this window] [in a new window]   Fig. 1. The receptor ligands inhibit IK(DR) in rat intracardiac neurons. A, depolarization-activated (-50 to +70 mV; 300 ms) K+ currents recorded from a single neuron held at -90 mV in the absence (control) and presence of 100 µM DTG (DTG) or after washout of drug (wash). B, peak IK(DR) current density as a function of voltage recorded in a similar manner from seven neurons in the absence () and presence () of 100 µM DTG.

Concentration-Dependent Inhibition of I_K(DR) by Ligands.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Dose-dependent inhibition of IK(DR) by receptor ligands in rat intracardiac neurons. A, whole cell K+ currents evoked from three intracardiac neuron by step depolarizations to +50 mV from a holding potential of -90 mV in the absence (control) and presence of (+)-pentazocine, ibogaine, and DTG at the indicated concentrations. B, peak whole cell IK(DR) current amplitude, evoked by depolarizing to +50 mV from -90 mV, normalized to control, and plotted as a function of ligands concentration. Data points represent mean ± S.E.M. for four or more observations. The curves represent best fit to the data using the Hill equation. Half-maximal inhibition was 9.7 ± 0.5 µM for haloperidol, 76.4 ± 7.7 µM for (+)-pentazocine, 218.1 ± 7.0 µM for ibogaine, 295.4 ± 45.4 µM for (+)-SKF-10,047, and 341.3 ± 26.0 µM for DTG. The corresponding Hill coefficients were 1.01 ± 0.05, 0.69 ± 0.05, 1.14 ± 0.04, 0.84 ± 0.11, and 0.97 ± 0.07, respectively.

The Receptor Antagonist Metaphit Depresses the Effect of DTG on K⁺ Channels.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Attenuation of DTG-mediated inhibition of IK(DR) by the receptor antagonist metaphit. A, depolarization-activated (-90 to 50 mV) IK(DR) recorded from two intracardiac neurons in the absence (control) and presence of 100 µM DTG (DTG). Bottom traces are from a neuron preincubated in metaphit (50 µM in PSS, 10 min). B, bar graph of the percent inhibition of mean peak IK(DR) (±S.E.) produced by 100 µM DTG in control cells that were not exposed to metaphit (DTG) or cells preincubated in metaphit (50 µM; 10 min; metaphit + DTG). IK(DR) was evoked by step depolarizations (-90 to +50 mV). Data were collected from seven neurons for each condition, and the asterisk denotes significant difference between the groups (p < 0.05).

Effect of Intracellular Dialysis with GTP and GDP--S on Receptor Inhibition of I_K(DR). Controversy exists in the literature as to whether receptors couple to G proteins and whether these second messengers are involved in the regulation of ion channels by receptors. To determine whether receptor-mediated inhibition of K⁺ channels in intrinsic cardiac neurons is dependent on G protein activation, neurons were dialyzed with pipette solution containing either 100 µM GTP or the G protein inhibitor GDP--S (100 µM). Cell dialysis with 100 µM GDP--S has previously been shown to successfully block G protein-mediated signal transduction in intrinsic cardiac neurons (Cuevas and Adams, 1994, 1997; Zhang and Cuevas, 2002). Figure 4A shows representative currents in response to step depolarizations from -90 to +50 mV recorded from two neurons dialyzed with either GTP (top traces) or GDP--S (bottom traces) in the absence and presence of 100 µM DTG. The inhibition of I_K(DR) evoked by this concentration of DTG under dialyzing conditions, with GTP in the pipette, was similar to that observed when the intracellular milieu was preserved (30%). The DTG-induced attenuation of I_K(DR) was present when the cells were dialyzed with GDP--S (Fig. 4A, bottom traces). A summary of the peak I_K(DR) amplitudes elicited upon depolarization to +50 mV under the different experimental conditions and normalized to their respective control values is presented in Fig. 4B. The difference observed between the two groups was not statistical significant. The current-voltage relationships for six neurons dialyzed with either GTP or GDP--S are shown in Fig. 4, C and D, respectively. Whereas dialyzing cells with GDP--S decreased the peak I_K(DR) amplitude, compared with the cells dialyzed with GTP, DTG attenuated peak amplitudes under both conditions to a similar extent. These data suggest that neither cell dialysis nor inhibition of G protein activation blocks -1 receptor-mediated inhibition of I_K(DR). Thus, neither a diffusible cytosolic second messenger nor a G protein couples -1 receptors to I_K(DR).

View larger version (19K): [in this window] [in a new window]   Fig. 4. The receptor inhibition of IK(DR) is not blocked by intracellular GDP--S. A, depolarization activate (-90 to +50 mV) K+ currents recorded from neurons dialyzed with pipette solutions containing either 100 µM GTP or GDP--S in the absence (control) or presence of 100 µM DTG (DTG). B, mean (±S.E.M.) peak IK(DR) recorded from neurons dialyzed with either GTP (n = 6) or GDP--S (n = 6) in the presence of 100 µM DTG. Currents are normalized to their respective controls (absence of drug). Mean (±S.E.) peak IK(DR) current density as a function of depolarization voltage (-90-mV holding potential) recorded from neurons dialyzed with pipette solution containing either 100 µM GTP (C; n = 6) or 100 µM GDP--S (D; n = 6) in the absence () and presence () of 100 µM DTG.

Effects of Receptor Ligands on Action Potential Firing. Studies have shown that inhibition of I_K(DR) by verapamil in intracardiac neurons can depress action potential firing (Hogg et al., 1999). Experiments were thus conducted to determine whether receptor modulation I_K(DR) has a similar effect on neuroexcitability in these cells. The effects of ligands on the active membrane properties of isolated intracardiac neurons were studied using the amphotericin B perforated patch method under current-clamp mode. Figure 5A shows a family of action potentials elicited from a single intracardiac neuron in response to depolarizing current pulses (100 pA; 400 ms) in the absence (control) and presence of 100 µM DTG (DTG), and after washout of drug (wash). DTG depolarized the neuron and decreased the number of action potentials evoked by the current injection in a rapidly reversible manner. In similar experiments, 100 µM DTG depolarized neurons from a control value of -50.8 ± 2.1 to -49.2 ± 2.4 mV (n = 9). DTG also decreased the number of action potentials evoked by depolarizing membrane pulses by 85% (Fig. 5B). Both of these changes were statistically significant (p < 0.01 and p < 0.001, respectively). DTG also altered the action potential configuration by decreasing both the action potential overshoot and afterhyperpolarization (AHP) and by slowing both the rate of depolarization and rate of repolarization (Fig. 5A, inset; Table 1). The effects of DTG were mimicked by 10 µM haloperidol, 200 µM ibogaine, and 50 µM (+)-pentazocine, which depressed action potential firing by 65, 85, and 65%, respectively and changed the configurations of the waveform in a reversible manner. The effects of the ligands on the action potential firing and configuration are summarized in Table 1.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Inhibition of action potential firing by receptor ligand DTG in rat intracardiac neurons. A, action potentials fired from an isolated intracardiac neuron in response to 100-pA depolarizing current pulses in the absence (control) and presence of 50 µM DTG (DTG) and following washout of drug (wash). Inset is superimposition of first action potential from control and DTG traces. B, bar graph of the number of action potentials fired in the absence (control) or presence of 100 µM DTG (DTG) and after washout of the drug (wash). Asterisk denotes significant difference from control and DTG (p < 0.01) (n = 7).

View this table: [in this window] [in a new window]   TABLE 1 Effects of ligands on the resting membrane potential and action potential waveform parameters Action potentials were evoked by 100-pA current injections. All drugs were bath applied, and the data are shown as mean ± S.E. for the number of cells (n) shown in the table.

Role of M-Currents in the Effects of Receptor Activation. One possible mechanism by which -1 receptor activation depolarizes intracardiac neurons is via a block of the M-current. Previous studies have shown that inhibition of I_M by muscarinic receptor activation depolarizes intracardiac neurons (Cuevas et al., 1997). The M-channels mediating I_M are noninactivating and close slowly in response to membrane repolarization from depolarizing holding potentials. The closing of these channels in intracardiac neurons upon repolarization results in a characteristic inward current relaxation (Cuevas et al., 1997). Figure 6A shows whole cell currents recorded from a single cell in response to repolarizing steps to -60 mV from a holding potential of -30 mV in the absence (control) and presence of 1 mM DTG. The inward relaxation observed under control conditions is abolished by application of DTG. Similarly, 20 µM linopirdine, a specific blocker of I_M (Wallace et al., 2002), eliminates the inward relaxation (Fig. 6B). Figure 6, C and D, shows bar graphs of mean peak I_M recorded under control conditions (absence of drug) and when either DTG (n = 4) or linopirdine (n = 6) was added, respectively. I_M was completely blocked by application of either drug, and this effect was reversible after washout in both cases (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 6. The receptor ligand DTG inhibits the M-current in rat intracardiac neurons. A, slow inward current relaxation (M-current) induced by repolarizing step (-30 to -60 mV; 250 ms) recorded from a single intracardiac neuron before (control) and after application of 1 mM DTG (DTG). B, M-current recorded in the absence (control) and in the presence of 20 µM linopirdine (linopirdine). Bar graph of mean current amplitudes in the absence (control) or presence of 1 mM DTG (C; n = 4) and 20 µM linopirdine (D; n = 6). The amplitude of the M-current was defined as the difference between the peak current observed at the start of the relaxation (A, left trace, black arrow) and the holding current at the end of the 250-ms repolarizing step (A, left trace, gray arrow). Asterisks denote significant difference from control (p < 0.001).

Effects of Receptor Activation on BK Currents.

View larger version (22K): [in this window] [in a new window]   Fig. 7. The receptor activation blocks BK in rat intracardiac neurons. Traces of BK-mediated currents recorded from two neurons, expressing fast inactivating (A) and slowly inactivating BK channels (B), in the absence (control) and presence of 1 mM DTG (DTG). BK-mediated currents were evoked by step depolarizations to +50 mV from a holding potential of –90 mV and defined as the net TEA-sensitive current. The net TEA-sensitive current was determined by subtracting the outward current recorded in the presence of 500 µM TEA (with or without DTG) from the outward current obtained in the absence of TEA (with or without DTG). Bar graph of the peak and sustained BK current amplitudes recorded in the absence (control) and presence of DTG (DTG; 1 mM) in cells expressing fast-inactivating (C; n = 6) or slowly inactivating (D; n = 4) BK channels. Asterisks denote significant difference from control (p < 0.05).

	Discussion

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The results presented here show that in neonatal rat intracardiac neurons activation of -1 receptors reversibly inhibited I_K(DR),I_K(Ca), and I_M through a pathway that involved neither a diffusible cytosolic second messenger nor a G protein. Furthermore, activation of receptors resulted in the depolarization of the neurons and depression of neuroexcitability.

Studies in our laboratory have demonstrated that -1 receptors are expressed in parasympathetic intracardiac neurons, but the cellular function of these receptors remained unknown. Whereas -2 receptors in these cells couple to Ca²⁺ channels, -1 receptors modulate the function of various K⁺ channels in these neurons. Although the modulation of Ca²⁺ channels in intracardiac neurons by neurotransmitters is well documented, less is known about the regulation of K⁺ channels in these cells (Adams and Cuevas, 2004). Activation of M₁ muscarinic receptors in neonatal rat intracardiac neurons has been shown to block I_M in these cells (Cuevas et al., 1997), and activation of a prostaglandin E₂ receptor inhibits a small-conductance I_K(Ca) in adult guinea pig intracardiac neurons (Jelson et al., 2003). The -1 receptor activation, however, inhibits multiple K⁺ channels subtypes in intracardiac neurons. The modulation of K⁺ channels by receptors has been reported in mouse sympathetic neurons, frog melanotrophs, and mouse neurohypophysial nerve terminals (Kennedy and Henderson, 1990; Soriani et al., 1998, 1999a,b; Wilke et al., 1999). The K⁺ channel types that have been shown to be modulated by receptor activation include A-type K⁺ channels (I_A), I_K(Ca), I_K(DR), and I_M, and multiple K⁺ channels types are frequently affected in individual cells (Soriani et al.,1999a,b; Wilke et al.,1999). All of these K⁺ channel subtypes, except A-type K⁺ channels, which are not expressed in intracardiac neurons, were shown to be affected by receptor activation in our study.

Although receptors have been shown to block K⁺ channels in native cells, the receptor subtype mediating these effects has not been definitively identified. The rank-order potency for I_K inhibition by ligands reported here, haloperidol > (+)-pentazocine > ibogaine > DTG, suggests that the effect is mediated by -1 receptor activation. In contrast, the rank-order potency for ligand inhibition of Ca²⁺ channels in intracardiac neurons is haloperidol > ibogaine > (+)-pentazocine > DTG, which is consistent with a -2 receptor-mediated effect. The IC₅₀ values for the various ligands tested here are in agreement with those reported in the literature for modulations of voltage-gated K⁺ channels. For example, in frog pituitary melanotrophs, (+)-pentazocine inhibited delayed outwardly rectifying K⁺ channels with an IC₅₀ of 37 µM, compared with the IC₅₀ of 42 µM reported here. Similarly, 100 µM (+)-SKF-10,047 blocked approximately 50% of the voltage-activated K⁺ currents recorded in neurohypophysial nerve terminals (Wilke et al., 1999). Thus, -1 receptors are likely to be responsible for inhibition of K⁺ channels in intracardiac neurons and in other native cells studied. The conclusion that receptors mediate the inhibition of I_K by ligands in intracardiac neurons is strengthened by the observation that metaphit, an irreversible, antagonist of receptors, blocks the effects of DTG on I_K in these cells.

Considerable controversy exists as to the mechanisms by which receptors modulate K⁺ channels. In frog pituitary melanotrophs, cell dialysis with GTP--S and preincubation in cholera toxin were shown to inhibit receptor effects on I_A and I_K(DR) (Soriani et al., 1999a,b), suggesting that receptors couple to the K⁺ channels via a G protein. G proteins have also been implicated in both receptor-mediated activation of phospholipase C and regulation of Ca²⁺ release from intracellular stores (Morin-Surun et al., 1999; Hayashi et al., 2000). However, neither cell dialysis nor application of intracellular GTP--S blocked the inhibition of I_K by -1 receptors in intracardiac neurons. Thus, receptors couple to K⁺ channels in these cells via a membrane-delimited signal transduction cascade that does not involve a G protein. Our observation is in agreement with the direct protein-protein interaction between -1 receptors and K⁺ channels shown by Jackson and colleagues (Lupardus et al., 2000; Aydar et al., 2002).

The net effect of receptor modulation of ion channels in intracardiac neurons at rest is a depolarization of the membrane potential. We have previously shown that the I_M contributes to the resting membrane potential of intracardiac neurons and that inhibition of this channel depolarizes the cells (Cuevas et al., 1997). Given that application of DTG blocks I_M, receptor modulation of these channels likely accounts for the depolarizations reported here. Further support for this conclusion comes from the fact that neither inhibition I_K(DR) nor I_K(Ca) has been linked to changes in the resting membrane potential of these neurons (Xu and Adams, 1992; Hogg et al., 1999; Jelson et al., 2003). Inhibition of voltage gated Ca²⁺ channels by -2 receptor activation is also unlikely to account for this depolarization since removal of extracellular Ca²⁺ does not alter the resting membrane potential of these cells (DeHaven and Cuevas, 2004).

The block of I_K by receptors in intracardiac neuron is associated with decreased action potential firing, whereas previous studies have shown that receptor block of I_K enhances neuroexcitability (Soriani et al., 1998). Our laboratory has shown that inhibition of I_M is associated with increased excitability of intracardiac neurons (Cuevas et al., 1997), and thus receptor modulation of non-I_M K⁺ channels must be responsible for the decreased excitability observed. The depressed neuroexcitability reported here likely results from modulation of I_K(DR) by receptors. Verapamil has been shown to convert tonic and adapting intracardiac neurons into phasic neurons via a direct block of I_K(DR) (Hogg et al., 1999). However, high concentrations of verapamil failed to abolish action potential firing in intracardiac neurons, whereas high concentrations of ligands completely blocked the genesis of action potentials in these cells. The concentrations of (+)-pentazocine (50 µM) and ibogaine (200 µM) required to appreciably decrease action potential firing and alter the action potential configuration in our study suggest that the effects of ligands on the active membrane properties of these cells are primarily mediated by -1 receptors. However, inhibition of voltage-activated K⁺ channels alone cannot explain the complete block of action potential firing at high concentrations of ligands, and thus receptors are likely to affect other channel types that regulate action potential firing (e.g., voltage-gated sodium channels).

The inhibition of I_K(Ca) by receptors is also likely to contribute to changes in the active membrane properties of intracardiac neurons. Two types of receptor-regulated BK currents were found in rat intracardiac neurons, one which exhibited rapid time-dependent inactivation, BK_i, and a second that showed little or no inactivation, BK_s. The presence of distinct subpopulations of BK channels with dissimilar inactivation kinetics has been reported in rat adrenal chromaffin cells (Solaro et al., 1995). The inhibition of BK currents by receptors would account, at least in part, for the increase in action potential duration and decrease in AHPs reported here, since inhibition of BK by 200 µM TEA produces similar effects on the action potentials of these cells (Franciolini et al., 2001). Although inhibition of voltage-gated Ca²⁺ channels may contribute to the decrease in I_K(Ca) amplitude observed in our studies, it is unlikely to exclusively account for the block of BK channels observed here. Evidence for this conclusion comes from the fact that concentrations of Cd²⁺ (100 µM) that inhibit all Ca²⁺ channels in these neurons (Cuevas and Adams, 1997) fail to eliminate the AHPs in these cells (Franciolini et al., 2001). Furthermore, in our study, 100 µMCd²⁺ blocked <5% of I_K, whereas 500 µM TEA or Ca²⁺-free extracellular solution blocked 25% of I_K (data not shown). Together, these data suggest that -1 receptors are likely having a direct effect on BK channels in intracardiac neurons. However, receptor modulation of other mechanisms of Ca²⁺ entry or homeostasis must also be examined to confirm a direct effect on I_K(Ca).

There has been considerable speculation about the role of receptors in the cardiovascular system. Although the endogenous ligand responsible for activating receptors under physiological and pathophysiological conditions remains to be determined, there are data suggesting that some putative receptor ligands may affect the heart and coronary vasculature. For example, pregnenolone, a putative ligand (Maurice et al., 2001), increases heart rate and cardiac output in anesthetized dogs (Hogskilde et al., 1991). Such effects may in part be due to activation of receptors on intracardiac neurons. Similarly, various drugs that act on receptors, such as haloperidol, have significant effects on the heart (Monassier and Bousquet, 2002). Our observations raise the possibility that the cardiovascular effects of these compounds may be mediated by their modulation of the electrical activity of intrinsic cardiac neurons. In conclusion, stimulation of -1 receptors inhibits multiple voltage-gated K⁺ channel subtypes and depresses excitability in intracardiac neurons. Thus, activation of -1 receptors in these cells is likely to attenuate parasympathetic input to the heart, and consequently, to affect cardiovascular function.

	Acknowledgements

 
We thank Dr. Christopher Katnik, Wayne I. DeHaven, and Nivia Cuevas for comments on a draft of this manuscript.

	Footnotes

 
This study was supported by National Institutes of Health Grant R01 HL63247 (to J.C.) and American Heart Association Florida/Puerto Rico Affiliate Awards (grant-in-aid to J.C. and Predoctoral Fellowship to H.Z.). A preliminary report of some of these results has been presented in abstract form (Zhang and Cuevas, 2003).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.084152.

ABBREVIATIONS: (+)-SKF-10,047, 2S-(2a,6a,11R^*]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol hydrochloride; DTG, 1,3-di-O-tolyguanidin; Kv, voltage-activated K⁺ channel; I_K, voltage-activated K⁺ channel current; K_(DR), delayed outwardly rectifying K⁺ channel; I_M, M-current; GDP--S, guanosine 5'-O-(2-thiodiphosphate) trilithium salt; PSS, physiological saline solution; TTX, tetrodotoxin; GTP, GTP lithium salt; TEA, tetraethylammonium chloride; BK, large conductance K_(Ca) channel; AHP, afterhyperpolarization; K_(Ca), Ca²⁺-sensitive K⁺ channel; SK, small conductance K_(Ca) channel; IK, intermediate conductance K_(Ca) channel; BK_i, fast inactivating BK current; BK_s, noninactivating BK current; SM-21, (±)-tropanyl 2-(4-chlorophenoxy)butanoate maleate; BD-1047, N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide; BD-1063, 1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride.

Address correspondence to: Dr. Javier Cuevas, Department of Pharmacology and Therapeutics, University of South Florida College of Medicine, 12901 Bruce B. Downs Blvd., MDC 9, Tampa, FL 33612-4799. E-mail: jcuevas{at}hsc.usf.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

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