 |
INTRODUCTION |
Vagal transmission to the mammalian heart was shown to be inhibited by exogenous opioid substances. Kosterlitz and Taylor (1959)
found that intravenous doses of morphine reduced the bradycardia produced by stimulation of the vagus nerve in anesthetized rats and rabbits. Similarly, met-enkephalin and [D-Ala2, D-Leu5] enkephalin reduced bradycardia produced by vagal stimulation and naloxone antagonized the effects of these substances in rabbit (Weitzell et al. 1984
) and canine heart (Musha et al. 1989
).
Endogenous opioid peptides were found in rat and guinea pig heart associated with cardiac nerves (Hughes et al. 1977
; Weihe et al. 1985
). Immunohistochemical studies had localised enkephalin-immunoreactive axons that were predominantly perivascular within the guinea pig heart (Reinecke and Forssmann 1984
; Weihe et al. 1985
). The dramatic reduction in the levels of enkephalin and prodynorphinderived peptides after chemical sympathectomy suggested that enkephalin was present in sympathetic nerves (Lang et al. 1983
). Immunoreactivity for prodynorphin- and proenkephalin-derived peptides was also recently observed in populations of parasympathetic postganglionic neurons in guinea pig cardiac ganglia and their axons (Steele et al. 1994
, 1996
). Proenkephalin mRNA was also found in rat atria and ventricles and the enkephalin peptides were shown to be synthesized and secreted from cardiac myocytes (Springhorn and Claycomb 1992
).
Although the release of opioid peptides from cardiac nerves and myocytes and the presence of high affinity saturable opioid binding were shown in rat atria, the mechanisms of action of enkephalin peptides in the mammalian heart remain largely unknown. Leu-enkephalin was reported to decrease the intracellular Ca2+ transient and contraction amplitudes in individual cardiac ventricular cells, in part, by reducing L-type Ca2+ channel currents (Xiao et al. 1993
). Local administration of enkephalin to the right atrial ganglion plexus was also shown to increase the neuronal activity generated by in situ canine intracardiac neurons (Armour et al. 1993
).
Opioids were previously shown to inhibit neuronal, voltage-dependent Ca2+ currents and activate inwardly rectifying K+ currents (see review by North 1993
). In the present study, the effects of enkephalin peptides on depolarization-activated ionic currents were investigated in isolated parasympathetic neurons of rat intracardiac ganglia by using the amphotericin B, perforated-patch configuration of the whole cell patch clamp technique. A preliminary report of some of these results was presented (Adams and Trequattrini 1995
).
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METHODS |
Preparation
Isolated parasympathetic neurons, dissociated from neonatal rat intracardiac ganglia, were obtained as described previously (Xu and Adams 1992a
). Briefly, atria were dissected from neonatal rats (3-10 days old) and incubated in Krebs solution containing 1 mg/ml collagenase (Type 2, Worthington Biochemical, Freehold, NJ) for 1 h at 37°C. Individual ganglia were then dissected from the epicardial ganglion plexus, transferred to a culture dish containing high glucose culture medium (Dulbecco's Modified Eagle Media, 10% (vol/vol) fetal calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin), triturated with a fine bore pasteur pipette and plated onto 18-mm glass coverslips coated with laminin. The dissociated cells were incubated at 37°C in 95% O2-5% CO2 atmosphere and used for the experiments after 24-72 h. At the time of experiments, the glass coverslip was transferred to a low volume (0.5 ml) recording chamber and viewed at ×400 magnification with an inverted phase contrast microscope. Experiments were conducted at room temperature (21-23°C).
Electrical recording and analysis
Membrane current and voltage were recorded from voltage- and current-clamped neurons, respectively, with the perforated-patch method (Korn et al. 1991
). Amphotericin B was used to obtain electrical access to the cell and gave a mean access resistance of <10 M
within 10 min of establishing the cell-attached configuration. To minimize voltage error, series resistance (RS) was typically compensated by 60% to
4 M
. In a series of experiments, the effects of cell dialysis were examined with the standard whole cell recording configuration (Hamill et al. 1981
).
Patch electrodes were pulled from thin-walled borosilicate glass (GC150TF; Clark Electromedical Instruments, Reading, UK) to a final resistance of 1-3 M
. Membrane current and voltage were recorded by using a patch-clamp amplifier (Dagan 3900A, Dagan Corporation, Minneapolis, MN), filtered at 10-20 kHz (
3 dB), with a 4-pole Bessel filter and digitized at 50 kHz with an A/D converter (TL-1 DMA interface, Axon Instruments, Foster City, CA) and stored on a PC 486/50 MHz computer. Membrane current and voltage were continuously monitored on a digital oscilloscope and on a chart recorder.
Depolarization-activated Ca2+ channel currents were elicited with voltage steps from -90 mV to more positive potentials. Capacitive transients were minimized by using analog circuitry of the amplifier and linear leak currents were subtracted with the
P/4 protocol. Data acquisition and analysis was carried out with the pClamp 5.5 program (Axon Instruments) and Boltzmann distributions for determining the voltage dependence of Ca2+ channel activation and inactivation were fit with SigmaPlot 3.0 (Jandel Scientific, San Rafael, CA). Dose-response curves were obtained by measuring the peak current amplitude at each agonist/antagonist concentration and the experimental data points were fit using the equation:
|
(1)
|
where I/Imax is the fractional current, Io is the noninhibitable current, [A] is the agonist/antagonist concentration, IC50 is the half-maximal concentration and n is the Hill coefficient. Results are presented as mean ± SE, with the number of experiments in parentheses. Statistical significance was determined by Student's t-test and was considered significant if P < 0.05.
Solutions and drugs
The bath solution (physiological salt solution, PSS) used in these experiments contained (in mM) 140 NaCl, 3 KCl, 2.5 CaCl2, 0.6 MgCl2, 7.7 D-Glucose, 10 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid (HEPES) and adjusted to pH 7.4 with NaOH. Ca2+ channel currents were obtained in the presence of 1 µM tetrodotoxin (TTX) and 70 mM NaCl replaced by tetraethylammonium (TEA)Cl. Unless specified, BaCl2 (5 mM) was used as the charge carrier to maximize Ca2+ channel current amplitude and to minimize any Ca2+-activated currents and Ca2+-dependent current rundown (Xu and Adams 1992b
). The pipette solution used for perforated patch experiments contained (in mM) 75 Cs2SO4, 55 CsCl, 5 MgSO4, 10 HEPES, pH 7.2 and 180 µg/ml amphotericin B in 0.6% dimethyl sulfoxide (DMSO). Amphotericin-containing solutions were prepared daily, kept on ice and light protected. Cs2SO4 and CsCl were replaced by equimolar concentrations of K2SO4 and KCl, respectively, for K+ current recordings. In experiments carried out with the perforated-patch method, Ca2+ channel current run-down was not observed. For standard (dialyzed) whole cell recordings, the internal solution contained (in mM) 130 CsCl, 10 ethylene glycol bis(
-aminoethyl ether)-N,N,N
,N
-tetraacetic acid (EGTA), 2 Mg2ATP, 0.1 guanosine 5
-triphosphate (GTP) sodium salt, and 10 HEPES-CsOH, pH 7.2. The osmolality of the extra- and intracellular solutions (285-290 mM/kg) was monitored with a vapor pressure osmometer (Wescor 5500, Logan, UT). The opioid agonists or antagonists were bath applied, diluted to the final concentration stated. In experiments in which Met-enkephalin and norepinephrine were studied, the drugs were briefly (<2 s) applied during continuous bath perfusion of the control solution by means of a pressure ejection system (Picospritzer II, General Valve, Fairfield, NJ). Complete replacement of the bath solution at a perfusion rate of ~2 ml/min required <1 min.
In a series of experiments, neurons were preincubated in 200 ng/ml Bordetella pertussis toxin (PTX) for 24 h before the experiments. Neurons from the same cardiac ganglia explant, cultured in different dishes, were used as controls.
All the solutions were made from analytic grade reagents. The following drugs were used: amphotericin B, dynorphin A, GTP, guanosine 5
-(3-O-thio)-triphosphate (GTP-
-S), Leucine enkephalin acetate (Leu5-enkephalin), Methionine enkephalin acetate (Met5-enkephalin), and B. pertussis toxin were obtained from Sigma Chemical Co. (St Louis, MO). TTX,
-conotoxin GVIA (
-CgTX) and nifedipine were obtained from Calbiochem (San Diego, CA). (±)-bremazocine hydrochloride, DADLE ([D-Ala2, D-Leu5]-enkephalin), DAMGO ([D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin), DPDPE ([D-Pen2, D-Pen5]-enkephalin), naloxone hydrochloride, and naltrindole hydrochloride were obtained from Research Biochemicals International (Natick, MA).
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RESULTS |
The effects of enkephalins on the membrane potential and depolarization-activated ionic currents in neonatal rat intracardiac neurons were investigated in 54 isolated neurons. In normal PSS, neurons had a resting membrane potential ranging from
50 to
58 mV, an input resistance of
750 M
at 22°C and action potentials of amplitude
90 mV could be elicited by brief current pulses (100 pA, 10 ms) (see Xu and Adams 1992a
).
Effects of Met-enkephalin on the passive and active electrical properties
The effects of enkephalins on the resting membrane and action potentials were investigated in current-clamped neurons by using the perforated-patch method. Bath application of 1-10 µM Met-enkephalin altered the action potential waveform, reducing the maximum amplitude and slowing the rate of rise and the repolarization (Fig. 1A). The mean amplitude was 101.5 ± 2.0 mV in control conditions and 77.0 ± 3.5 mV (n = 13) after application of 10 µM Met-enkephalin; the maximum rate of rise of the action potential was reduced from 123.0 ± 8.3 V/s to 69.8 ± 4.8 V/s and the duration measured at
55 mV was increased by 40% from 6.9 ± 0.5 ms to 11.6 ± 0.2 ms. The amplitude and duration of the afterhyperpolarization were not appreciably altered in the presence of 10 µM Met-enkephalin. Metenkephalin did not alter either the resting membrane potential or the input resistance of the neurons.

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| FIG. 1.
Effects of Met-enkephalin on action potential and depolarization-activated ionic currents in rat intracardiac neurons. A: superimposed action potentials elicited in response to depolarizing current pulses (100 pA, 2 ms) obtained in absence (Control, Recovery) and presence of 10 µM Met-enkephalin bath applied. Resting membrane potential, 55 mV. B: superimposed Ca2+ currents evoked by depolarizing voltage steps from 90 to 0 mV in absence (Control, Recovery), and in presence of 10 µM Met-enkephalin. External physiological salt solution (PSS) solution contained 5 mM Ca2+ and 1 µM tetrodotoxin (TTX). C: depolarization-activated K+ currents evoked at 20 mV from a holding potential of 90 mV in absence (Control) and presence of 10 µM Met-enkephalin. External PSS solution contained 5 mM Ca2+, 100 µM Cd2+ and 1 µM TTX.
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Met-enkephalin was shown to inhibit voltage-dependent Ca2+ currents in guinea-pig submucous neurons (Surprenant et al. 1990
) and rat dorsal root ganglion (DRG) neurons (Moises et al. 1994b
; Schroeder et al. 1991
) and potentiate inwardly rectifying K+ currents in guinea-pig submucous neurons (Mihara and North 1986
; North et al. 1987
). To determine the mechanism by which enkephalin modified the action potential, the effects of enkephalin were examined on both depolarization-activated Ca2+ and K+ currents. In isolated voltage-clamped neurons, 10 µM Met-enkephalin reversibly inhibited depolarization-activated Ca2+ currents but not delayed rectifier K+ currents. Figure 1B shows superimposed Ca2+ currents evoked by depolarizing pulses to 0 mV before, during, and after washout of PSS containing 10 µM enkephalin. The inhibition of Ca2+ currents by Met-enkephalin was more pronounced for the peak current(52.3 ± 2.3%, n = 10), than at the end of a 120-ms pulse (31.4 ± 1.8%). Furthermore, the rate of Ca2+ channel activation was slowed by Met-enkephalin and the time to peak was increased from 12.3 ± 1.3 ms to 46.0 ± 1.8 ms (n = 10). Figure 1C shows an example from four similar experiments that Met-enkephalin had no effect on either the amplitude or kinetics of the delayed rectifier outward K+ currents. Ca2+-activated K+ currents were completely inhibited by blocking Ca2+ channels with 100 µM Cd2+ applied externally.
Enkephalin inhibition of high-voltage-activated Ca2+ channel currents
To examine enkephalin inhibition of Ca2+ channel currents in rat intracardiac neurons, inward currents obtained with Ba2+ as the charge carrier were elicited from a holding potential of
90 mV with depolarizing voltage pulses ranging from
30 to 40 mV. Figure 2 shows families of Ba2+ currents (IBa) obtained in the absence (control) and presence of 10 µM Met-enkephalin (Fig. 2, A and B) and the corresponding current-voltage (I-V) relationships (Fig. 2C). The inhibition of IBa by Met-enkephalin is voltage dependent, being less effective at more positive membrane potentials. The percent inhibition of IBa was 57% at
10 mV and only 41% at 30 mV. Superimposed traces of IBa evoked on depolarization to 0 mV in the absence and presence of 10 µM Met-enkephalin are shown in Fig. 2B. Under control conditions, the time course of inactivation of IBa was biphasic and best fit by the sum of two exponential functions with mean time constants of 90 ms and 1.6 s (see Xu and Adams 1992b
, 1993
). In the presence of Met-enkephalin, the inward current inactivation was best fit by a single exponential function with a mean time constant of 2.1 ± 0.5 s (n = 4).

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| FIG. 2.
Met-enkephalin inhibition of voltage-dependent Ca2+ channels in rat intracardiac neurons. A: depolarization-activated Ba2+ currents evoked by depolarizing steps (10 mV increments) from 30 to 40 mV from a holding potential of 90 mV. Family of Ba2+ currents recorded in absence (Control, top) and in presence of 10 µM Met-enkephalin (Met-Enkephalin, bottom). B: superimposed Ba2+ current traces evoked on depolarization to 0 mV from 90 mV obtained in absence (Control, Recovery) and in presence of 10 µM Met-enkephalin. C: Whole cell peak I-V relations obtained in absence ( ) and presence ( ) of Met-enkephalin.
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Effects of enkephalin on the voltage dependence of activation and inactivation
The voltage dependence of activation of Ca2+ channel currents was examined by measuring tail current amplitude in the absence and presence of 10 µM Met-enkephalin, by using a double pulse protocol. Neurons were held at
90 mV and 30 ms steps to various test potentials were applied before a hyperpolarizing voltage step to
100 mV. Figure 3A shows Ba2+ currents obtained in the absence and presence of Met-enkephalin in response to voltage steps to 0, 50, and 100 mV and the ensuing tail currents on repolarization to
100 mV. The corresponding I-V relationships obtained for the tail currents are shown in Fig. 3B. Ca2+ channels exhibit sigmoidal activation at potentials positive to
40 mV in both the absence and presence of 10 µM Met-enkephalin, which could be best fit by two component Boltzmann distributions. A saturating concentration of Met-enkephalin slightly reduced the maximum tail current amplitude (<5% at 100 mV). Half-maximal activation of the first component (Vh1) was shifted slightly from
12 mV in the absence (control) to
6.8 mV in the presence of Met-enkephalin, whereas the second component (Vh2) shifted from 8 mV (control) to 34 mV (Met-enkephalin). The relative contribution of the two components of IBa was changed by Met-enkephalin whereby i1/i2 was 1.8 in the absence (control) and 0.6 in the presence of Met-enkephalin. Similarly, the slope parameters changed from 3.6 and 9.2 mV/e-fold change in open probability in control to 5.7 and 14.9 mV/e-fold change in the presence of Met-enkephalin. The effect of Met-enkephalin on the voltage dependence of Ca2+ channel activation was completely reversible on washout.

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| FIG. 3.
Voltage-dependence of Metenkephalin inhibition of Ba2+ currents through open Ca2+ channels. A: superimposed traces of dialyzed whole cell Ba2+ currents evoked by step depolarization to 0, 50, and 100 mV from 100 mV in absence (Control) and presence of 10 µM Met-enkephalin (Met-Enk). Tail currents were recorded at higher sampling rate (100 kHz, filtered at 20 kHz) and are displayed on an expanded time scale. B: current-voltage relations for Ba2+ tail currents measured in absence ( ) and presence of Met-enkephalin ( ). Peak tail-current amplitudes were obtained by fitting tail currents with sum of 2 exponential functions. Smooth curves were drawn according to a 2 component Boltzmann distribution:
where A is maximum amplitude ( 0.89 nA, Control and 0.81 nA, enkephalin), i1 and i2 are fraction contributed by each component (see text for details), Vh1 and Vh2 are half-maximal activation voltages and k1 and k2 are slope factors. Values for i1, i2, Vh1, Vh2, k1, and k2 were 0.63, 0.36, 12, 8, 3.6, and 9.2 for control and 0.36, 0.63, 6.8, 34.1, 5.7, and 14.9 in presence of Met-enkephalin, respectively.
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The effect of Met-enkephalin on steady-state inactivation of Ca2+ channels in rat intracardiac neurons was examined by using a double pulse protocol. Neurons were initially held at
90 mV and 4-s prepulses from
120 to 10 mV were applied in 10 mV increments before a voltage step to 20 mV to activate (open) the available Ca2+ channels. The steady-state inactivation of IBa exhibited a sigmoidal dependence on voltage and was best fit with a single Boltzmann function according to the equation: IBa/IBa(max) = 1/{1 + exp[(V
Vh)/k]}. A fit of the mean relative current(IBa/IBa(max))-voltage relationship for three neurons exhibited half-maximal steady-state inactivation (Vh) at
57 mV under control conditions and
54 mV in the presence of Met-enkephalin (n = 3). The slope parameter (k) was
13 mV both in the absence and presence of Met-enkephalin (data not shown).
Concentration-dependent inhibition of Ca2+ channel currents by Met-enkephalin
The concentration dependence of Met-enkephalin inhibition of Ca2+ channel currents was examined by measuring the peak amplitude of IBa elicited at 0 mV before and after application of various concentrations of Met-enkephalin. The peak inward current amplitude was measured before and after exposure to Met-enkephalin and the mean percent inhibition obtained for 3-5 cells was plotted as a function of Met-enkephalin concentration. Data fitted according to Eq. 1 gave a half-maximal inhibitory concentration (IC50) of 270 nM and a maximal percent inhibition of IBa of 52%. At least three different concentrations were tested on each cell, which were bracketed by the amplitude of IBa obtained in control solution. Figure 4A shows superimposed traces of IBa obtained at 0 mV from the same cell exposed to three different concentrations of Met-enkephalin and Fig. 4B shows the corresponding dose-response relation.

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| FIG. 4.
Dose-response relation for Met-enkephalin inhibition of Ca2+ channel currents. A: superimposed Ba2+ currents elicited on depolarization to 0 mV from 90 mV recorded from same neuron before (Control) and after application of 100 nM, 1 µM, and 30 µM Met-enkephalin. B: mean relative peak Ba2+ current amplitude(±SE) determined in 3-5 neurons plotted as a function of Met-enkephalin concentration. Data were fitted according to Eq. 1 where half-maximal inhibitory concentration, IC50 = 270 nM and Hill coefficient,n = 1.3. At least three different concentrations of Met-enkephalin were examined on each neuron.
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Effects of opioid agonists and antagonists on Ca2+ channel currents
Opioid receptors are commonly divided into three different classes, µ-,
-, and
-opioid receptors, on the basis of their affinity to specific opioid receptor agonists and antagonists and molecular cloning studies (Loh and Smith 1990
; Reisine 1995
). The actions of relatively specific opioid agonists and antagonists were examined in at least 15 different neurons to determine which opioid receptor type mediates the modulation of Ca2+ channel currents in rat intracardiac neurons. Bath application of Leu-enkephalin was equipotent to Met-enkephalin in the inhibition of Ca2+ channel currents. Given that Met- and Leu-enkephalin are relatively nonselective for µ- and
-opioid receptors (Reisine 1995
), the efficacy of these two agonists in the micromolar range cannot be considered a definitive test for the characterization of the receptor type. The enkephalin derivative, DPDPE, a selective agonist for the
-opioid receptor, did not affect IBa amplitude or kinetics at concentrations up to 10 µM (Fig. 5A), whereas DADLE, which barely distinguishes between
from µ receptors (Goldstein and Naidu 1989
), reduced IBa amplitude at a maximally effective concentration of 5 µM (Fig. 5B). The presence of a µ-opioid receptor was further demonstrated with the selective µ-receptor agonist, DAMGO, which reversibly inhibited IBa amplitude by ~50% at a concentration of 30 nM and was maximally effective at 1 µM (n = 4; Fig. 5C). Bath application of either dynorphin A(1 µM) or bremazocine (10 µM), selective
-receptor agonists, failed to affect Ca2+ channel currents (Fig. 5D), suggesting the absence of this receptor type in neonatal rat intracardiac neurons.

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| FIG. 5.
Effects of opioid agonists on Ca2+ channel currents. A-D: superimposed Ba2+ currents evoked by depolarizing pulses to 0 mV from a holding potential of 90 mV. Ba2+ currents were obtained before (Control) and during bath application of 10 µM DPDPE (A), 10 µM DADLE (B), 1 µM DAMGO (C), and 10 µM bremazocine (D). DAMGO and DADLE were applied at maximally effective concentrations and inhibition of Ba2+ currents by these agonists was reversible on washout (Recovery).
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The presence of µ- and
-opioid receptors was also examined by using the opioid receptor antagonists, naloxone and naltrindole, which is selective by more than three orders of magnitude for
-opioid receptors (Reisine 1995
). Bath application of either naloxone or naltrindole antagonized the inhibition of IBa produced by 10 µM Met-enkephalin (Fig. 6, A and B). The dose-response relations obtained for the antagonism of Met-enkephalin inhibition of IBa amplitude by naloxone and naltrindole were best fit by Eq. 1 with IC50s of 84 nM and 1 µM for naloxone and naltrindole, respectively (Fig. 6C).

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| FIG. 6.
Effects of opioid antagonists on Ca2+ channel inhibition by Met-enkephalin. A and B: superimposed Ba2+ currents evoked by step depolarizations to 0 mV from 90 mV in absence (Recovery) and presence of 10 µM Met-enkephalin and after addition of opioid anatagonists, naloxone (A) and naltrindole (B) to external solutions at concentrations stated. C: dose-response relations obtained for antagonism of Met-enkephalin inhibition of Ba2+ currents by naloxone (n = 2, ) and naltrindole (n = 2, ). Averaged data were fitted according to Eq. 1 where values obtained for IC50 and n were 84 nM and 1.2 for naloxone and 0.97 µM and 1.0 for naltrindole, respectively.
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Enkephalin modulation of
-conotoxin GVIA-sensitive Ca2+ channels
Rat intracardiac parasympathetic neurons express at least three pharmacologically distinct, high-voltage-activated Ca2+ channels:
-CgTX GVIA-sensitive, dihydropyridine (DHP)-sensitive, and
-CgTX- and DHP-insensitive (Jeong and Wurster 1997
; Xu and Adams 1992b
). To determine which Ca2+ channel types were affected by enkephalins, experiments were carried in which the effects of Metenkephalin (10 µM) were examined after selective block of populations of Ca2+ channels with either
-CgTX (300 nM) or nifedipine (10 µM), a DHP antagonist, or both. Figure 7A shows superimposed traces of IBa evoked by step depolarizations to 0 mV from
90 mV obtained in the absence (control, recovery) and presence of 10 µM Met-enkephalin, in the presence of
-CgTX alone and
-CgTX plus Met-enkephalin. Met-enkephalin had no effect on the
-CgTX-insensitive current, which constitutes about 25% of the total Ca2+ channel current. Addition of Met-enkephalin after exposure to 300 nM
-CgTX, which blocked 74.0 ± 6.3%(n = 5) of the total current failed to cause a further decrease of the residual current. A summary of the results obtained from five cells is presented as a bar graph shown in Figure 7B. In the presence of Met-enkephalin, IBa peak amplitude could be further reduced by bath application of 10 µM nifedipine whereas prior application of nifedipine did not affect Met-enkephalin inhibition of IBa (data not shown). These data are consistent with the insensitivity of the DHP-sensitive, L-type neuronal Ca2+ channels in rat intracardiac neurons to enkephalins.

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| FIG. 7.
Enkephalin modulation of -conotoxin GVIA-sensitive Ca2+ channels. A: superimposed Ba2+ currents evoked by step depolarization to 0 mV from 90 mV in absence (Control and Recovery) and presence of 10 µM Met-enkephalin, 300 nM -CgTX GVIA and 10 µM Met-enkephalin plus 300 nM -CgTX. Met-enkephalin failed to produce any further inhibition of Ba2+ current amplitude after bath application of 300 nM -CgTX. B: percent inhibition of peak Ba2+ current amplitude (mean ± SE, n = 5) produced by bath application of 10 µM Met-enkephalin, 300 nM -CgTX, and Met-enkephalin and -CgTX applied together.
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| FIG. 8.
Met-enkephalin inhibition of -conotoxin GVIA-sensitive Ca2+ channels is mediated by a pertussis toxin-sensitive G-protein. A: superimposed Ba2+ currents evoked by step depolarization to 0 mV from a holding potential of 90 mV in a dialyzed neuron before (Control) and after bath application of 10 µM Met-enkephalin. Ba2+ currents were recorded 10 min after establishing of whole cell recording configuration to permit dialysis of cell. B: Ba2+ currents recorded from a neuron dialyzed with a pipette "intracellular" solution containing 0.1 mM GTP in absence (Control,Recovery) and presence of 10 µM Met-enkephalin (Met-Enkephalin). C: perforated patch whole cell recording of Ba2+ currents evoked at 0 mV from a holding potential of 90 mV in absence (Control) and presence of Met-enkephalin in a neuron pretreated with 200 ng/ml pertussis toxin (PTX) for 24 h.
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Enkephalin inhibition of Ca2+ channels is mediated by a PTX-sensitive G-protein
Modulation of neuronal Ca2+ channels by neurotransmitters was shown to be mediated by specific guanine nucleotide binding protein (G-protein) coupled receptors (see reviews by Dolphin 1995
; Hille 1994
). To determine whether or not enkephalins modulate Ca2+ channels in rat intracardiac neurons via a G-protein coupled pathway, a series of experiments were carried out by using the conventional whole cell recording configuration in which the cell interior is dialyzed by a GTP-free pipette solution. Under such conditions, 10 µM Met-enkephalin failed to inhibit Ca2+ channel currents (Fig. 8A). Met-enkephalin inhibition was observed, however, in all dialyzed cells with 0.1 mM GTP added to the pipette intracellular solution (n = 6; Fig. 8B), suggesting that a G-protein was involved in Ca2+ channel inhibition in these neurons. In contrast, dialysis of cells with 0.1 mM GTP-
-S, a nonhydrolyzable GTP analog, produced a rapid decrease in IBa amplitude until it stabilized to a new level and any further inhibition by Met-enkephalin was occluded (data not shown). The involvement of a B. pertussis toxin (PTX)-sensitive G-protein was examined in neurons pretreated with PTX (200 ng/ml for 24 h). In neurons pretreated with PTX (n = 4), 10 µM Met-enkephalin failed to inhibit IBa (Fig. 8B), indicating that enkephalin inhibition of Ca2+ channel currents is mediated by a PTX-sensitive G-protein.
 |
DISCUSSION |
This study demonstrates that enkephalins inhibit a high-voltage-activated Ca2+ conductance in neurons of neonatal rat intracardiac ganglia. Met- and Leu-enkephalin selectively and reversibly inhibited the peak amplitude of high-threshold Ca2+ channel currents by activating µ-opioid receptors in the cell soma. The activation of µ-opioid receptors was previously shown to inhibit high-threshold Ca2+ channel currents in rat DRG neurons (Moises et al. 1994b
; Nomura et al. 1994
; Schroeder et al. 1991
), human neuroblastoma cell line SH-SY5Y (Seward et al. 1991
), and neurons isolated from the nucleus tractus solitarius of the rat (Rhim and Miller 1994
). Given that Met-enkephalin failed to further inhibit Ba2+ currents after exposure to
-CgTX (see Fig. 7) suggests that Met-enkephalin inhibits predominantly the
-CgTX-sensitive (N-type) Ca2+ channels in neonatal ratintracardiac neurons. Met-enkephalin maximally inhibited the Ca2+ channel current at 0 mV by ~52%, whereas the
-CgTX-sensitive component constitutes 65-75% of the total whole cell Ca2+ current (Jeong and Wurster 1997
; Xu and Adams 1992b
).
In the presence of Met-enkephalin, the activation kinetics of the remaining Ca2+ channel current was slowed significantly: enkephalin caused a three- to fourfold increase in the time to peak of IBa. A similar decrease in the rate of activation of Ca2+ current in these neurons was reported for activation of
-adrenoceptors by norepinephrine (Xu and Adams 1993
) and muscarinic receptors by acetylcholine (ACh) (Cuevas and Adams 1997
). The voltage-dependence of Ca2+ channel activation obtained in the absence and presence of Met-enkephalin was best fit by the sum of two Boltzmann distributions. In the presence of 10 µM Met-enkephalin, the activation curve was reversibly shifted to more positive potentials and the maximum tail amplitude slightly reduced (<5% at 100 mV). Half-maximal activation of the two components, Vh1 and Vh2, was shifted by 5.2 mV and 26 mV, respectively, in the presence of Met-enkephalin. The effects of Metenkephalin on the relative contribution and voltage-sensitivity of the two components of the Boltzmann distribution of the Ca2+ channel activation curve are consistent with Met-enkephalin converting a fraction of Ca2+ channels from a "willing" to a "reluctant" state, which requires stronger depolarizations to open in the presence of the neuromodulator (Bean 1989
). Met-enkephalin reversibly decreased the willing component from 63 to 36%, which is similar to that reported for norepinephrine-induced inhibition of Ca2+ channels in rat intracardiac neurons (Xu and Adams 1993
). A shift of Ca2+ channel gating is sufficient to account for Met-enkephalin inhibition of IBa and, in contrast to norepinephrine and ACh (Cuevas andAdams 1997; Xu and Adams 1993
), the inhibition of Ca2+ channel currents appears to be predominantly through a voltage-dependent mechanism. Met-enkephalin did not alter the voltage-dependence of steady-state inactivation of the Ca2+ channels in rat intracardiac neurons.
Met- and Leu-enkephalin were found to be equipotent, with half-maximal inhibition of IBa obtained at ~300 nM and maximal inhibition at concentrations
10 µM. The enkephalin derivatives, DADLE and DAMGO, selective µ-opioid receptor agonists, inhibited IBa whereas DPDPE, a selective
-opioid receptor agonist and the
-opioid receptor agonists, dynorphin A and bremazocine, did not affect Ca2+ channel current amplitude or kinetics (see Fig. 5). Given that DAMGO and DPDPE are selective by more than three orders of magnitude for µ- and
-opioid receptors, respectively (Goldstein and Naidu 1989
; Reisine 1995
), these data suggest that enkephalin-induced inhibition of Ca2+ channel currents in rat intracardiac neurons is mediated primarily by the µ-opioid receptor type. The dose-response relationships obtained for antagonism of Met-enkephalin-induced inhibition of IBa by naloxone and naltrindole, with IC50s of 84 nM and 1 µM, respectively, are consistent with the involvement of a µ-opioid receptor (see Reisine 1995
).
The involvement of a PTX-sensitive G-protein in the signal transduction pathway is suggested by the absence of enkephalin inhibition of Ca2+ channel currents after either dialysis of the cell with a GTP-free solution or a solution containing the nonhydrolysable GTP analog, GTP-
-S, or by preincubation of the neurons in PTX. Norepinephrine and ACh were also shown to modulate Ca2+ channel currents in these neurons via a PTX-sensitive G-protein (Cuevas and Adams 1997
; Xu and Adams 1993
). Opioid receptor-mediated inhibition of Ca2+ currents in rat DRG neurons was shown to result from the coupling of µ-opioid receptors to
-CgTX-sensitive Ca2+ channels via the PTX-sensitive Go subclass of G-proteins (Moises et al. 1994a
). Furthermore, the rapid kinetics and tight localization of the µ-opioid receptor-mediated inhibition of high-threshold Ca2+ currents is consistent with a membrane delimited G-protein coupling of the receptor and channel (Wilding et al. 1995
).
Inhibition of Ca2+ channel currents by a saturating concentration (30 µM) of Met-enkephalin alone is enhanced by coapplication of maximally effective doses of Met-enkephalin and norepinephrine (data not shown) suggesting that Met-enkephalin may not activate all the available PTX-sensitive G-proteins coupled to
-CgTX-sensitive Ca2+ channels. Norepinephrine-induced inhibition of Ca2+ channel currents in these neurons was also shown to result from the inhibition
-CgTX-sensitive Ca2+ channels (Xu and Adams 1993
). The saturation of the response to Met-enkephalin may reflect the saturation of the available µ-opioid receptors and not of the common G-protein coupled pathway.
Functional significance
The presence of enkephalin-immunoreactive neurons and nerve fibers in the mammalian heart (Steele et al. 1994
, 1996
) and the synthesis and release of enkephalin peptides from cardiac myocytes (Springhorn and Claycomb 1992
), suggests that enkephalins may play a physiological role in modulating neurotransmission in mammalian intracardiac ganglia. Enkephalins were shown to participate in circulatory regulation in vivo in dogs through the modification of vagal control (Caffrey et al. 1995
). Furthermore local application of DADLE to epicardial ganglia increases the neuronal activity generated by in situ canine intracardiac neurons resulting in concomitant increases in heart rate (Armour et al. 1993
). Enkephalin inhibition of Ca2+ channel currents may regulate neuronal excitability and transmitter release from both intrinsic and extrinsic nerve fibres within mammalian intracardiac ganglia. Enkephalins were shown to produce presynaptic inhibition in rat sympathetic ganglia (Konishi et al. 1979
) and cat parasympathetic ciliary ganglia (Katayama and Nishi 1984
) and reduce the stimulated-induced release of ACh from cholinergic nerve fibers in rat and rabbit atria prelabeled with tritiated choline (Wong-Dusting and Rand 1985
). Neuropeptides were shown to modulate Ca2+ influx into nerve terminals and attenuate depolarization-evoked transmitter release by inhibiting N-type Ca2+ channels in autonomic ganglia (e.g., Toth et al. 1993
). If the terminals of postganglionic intracardiac neurons have µ-opioid receptors and N-type Ca2+ channels similar to those found in the cell soma, the release of enkephalins from autonomic and sensory nerve terminals during transmission could act to diminish subsequent release of ACh onto the heart. Thus the activation of µ-opioid receptors in postganglionic intracardiac neurons is likely to regulate vagal transmission to the mammalian heart and influence heart rate and contractility.