The effects of Met-enkephalin on Ca2+-dependent K+ channel activity were investigated using the cell-attached patch recording technique on isolated parasympathetic neurones of rat intracardiac ganglia. Large-conductance, Ca2+-dependent K+ channels (BKCa) were examined as an assay of agonist-induced changes in the intracellular free calcium ion concentration ([Ca2+](i)). These BKCa channels had a conductance of similar to 200 pS and were charybdotoxin- and voltage-sensitive. Caffeine (5 mM), used as a control, evoked a large increase in BKCa channel activity, which was inhibited by 10 mu M ryanodine. Met-enkephalin (10 mu M) evoked a similar increase in BKCa channel activity, which was dependent on the presence of extracellular Ca2+ and inhibited by either ryanodine (10 mu M) or naloxone (1 mu M) In Fura-2-loaded intracardiac neurones, Met-enkephalin evoked a transient increase in [Ca2+](i). Mel-enkephalin-induced mobilization of intracellular Ca2+ may play a role in neuronal excitability and firing behaviour in mammalian intracardiac ganglia. (C) 1999 Published by Elsevier Science Ireland Ltd. All rights reserved

Adams

Connor

Davies

Eriksson

Grynkiewicz

Hamill

Hughes

Kostyuk

Musha

Muñoz

North

Smart

Sutko

Tang

Twitchell

Weitzell

Smith, A. B.

Adams, D. J.

University of Queensland eSpace

Neuroscience Letters (1999) 264 (1-3): 105-108.  doi: 10.1016/S0304-3940(99)00180-9 
 
Met-enkephalin-induced mobilization of intracellular Ca2+ in 
rat intracardiac ganglion neurones  
 
Amanda B. Smith and D. J. Adams 
  
Department of Physiology and Pharmacology, University of Queensland, Brisbane, QLD 4072, Australia  
 
Abstract 
 
The effects of Met-enkephalin on Ca2+-dependent K+ channel activity were investigated using 
the cell-attached patch recording technique on isolated parasympathetic neurones of rat 
intracardiac ganglia. Large-conductance, Ca2+-dependent K+ channels (BKCa) were examined 
as an assay of agonist-induced changes in the intracellular free calcium ion concentration 
([Ca2+]i). These BKCa channels had a conductance of ˜200 pS and were charybdotoxin- and 
voltage-sensitive. Caffeine (5 mM), used as a control, evoked a large increase in BKCa 
channel activity, which was inhibited by 10 μM ryanodine. Met-enkephalin (10 μM) evoked a 
similar increase in BKCa channel activity, which was dependent on the presence of 
extracellular Ca2+ and inhibited by either ryanodine (10 μM) or naloxone (1 μM). In Fura-2-
loaded intracardiac neurones, Met-enkephalin evoked a transient increase in [Ca2+]i. Met-
enkephalin-induced mobilization of intracellular Ca2+ may play a role in neuronal excitability 
and firing behaviour in mammalian intracardiac ganglia.  
 
Keywords: Parasympathetic ganglia; Intracellular Ca2+; Caffeine; Ryanodine; Opioid receptor; Ca2+-
dependent K+ channel  
 
Index Terms: heart innervation  
 
 
Endogenous opioid peptides have been found in the extrinsic and intrinsic innervation of the rat heart [7] and 
vagal transmission to the heart has been shown to be inhibited by exogenous opioid substances [11, 18]. 
Opioid receptor activation has been shown to evoke numerous cellular responses via Gi/Go proteins, for 
example, inhibition of N-type Ca2+ channels, inhibition of adenylyl cyclase, activation of K+ currents (for 
review, see [12]) and intracellular Ca2+ mobilization [2, 8]. Mu-opioid receptor activation in cultured bovine 
adrenal medullary chromaffin cells has also been shown to potentiate Ca2+-dependent K+ channel activation 
[17]. Opioid receptor-mediated increases in intracellular Ca2+ has been reported to arise from two mechanisms, 
namely, Ca2+ influx across the plasmalemma [4, 16] and Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-
sensitive Ca2+ stores [2, 8]. 
In rat intracardiac neurones, activation of μ-opioid receptors has been shown to inhibit N-type Ca2+ 
channels via a Pertussis toxin-sensitive G-protein [1]. However, the action potential after-hyperpolarization due 
to a Ca2+-dependent K+ conductance(s) in these neurones was unaltered in the presence of Met-enkephalin. In 
the present study, the actions of Met-enkephalin and caffeine, a known intracellular Ca2+ mobilizing agent, on 
intracellular Ca2+ mobilization and activation of the large conductance Ca2+-dependent K+ channels (BKCa) were 
investigated. 
Parasympathetic neurones from neonatal rat intracardiac ganglia were isolated as previously described 
[19]. The dissociated neurones were plated onto glass coverslips coated with laminin, incubated at 37°C in 95% 
O2 – 5% CO2 atmosphere and used for experiments within 36–72 h. At the time of experiments, the neurone-
plated glass coverslips were transferred to a recording chamber (0.5 ml volume) and mounted on the stage of an 
inverted phase contrast microscope. Unitary current recordings were obtained using the cell-attached patch 
recording configuration of the patch clamp technique [6]. In the cell-attached configuration, K+ currents in the 
membrane patch follow the relationship iK=γ(Vm−Vh−EK). When the cells were bathed in a depolarizing external 
solution (Vm ˜0 mV) and Vh was held at 0 mV, the membrane potential in the patch was close to 0 mV. Unitary 
currents were recorded using an Axopatch 200B patch clamp amplifier (Axon Instruments, Foster City, CA), 
filtered at 5 kHz, and stored on digital tape using a DAT digital recorder (DTR-1204, Biologic Science 
Instruments, Claix, France). Records were transferred to a Pentium computer using an analogue-to-digital 
converter (TL-1 DMA interface) and pClamp and Axotape software (Axon Instruments). Membrane currents 
were filtered at either 500 Hz or 2.5 kHz and sampled at 2 or 10 kHz, respectively. 
Neuroscience Letters (1999) 264 (1-3): 105-108.  doi: 10.1016/S0304-3940(99)00180-9 
 
The activity of BKCa channels was monitored as an assay of agonist-induced changes in intracellular 
free Ca2+ concentration ([Ca2+]i) at the cytoplasmic face of the plasma membrane [10]. The neurones were 
superfused with physiological salt solution (PSS) composed of (mM): 140 NaCl, 3 KCl, 2.5 CaCl2, 0.6 MgCl2, 
7.7 glucose and 10 HEPES-NaOH, pH 7.2. In a series of experiments, external NaCl was isotonically replaced 
by KCl. The patch pipette contained a high K+ solution of the following composition (mM): 140 KCl, 2.5 CaCl2, 
0.6 MgCl2, 7.7 glucose and 10 HEPES-KOH, pH 7.2. The osmolality of the solutions was in the range 285–295 
mmol/kg. The following drugs were used at the final concentrations stated: methionine enkephalin acetate 
(Met5-enkephalin) and caffeine (Sigma, St. Louis, MO), naloxone hydrochloride (RBI, Natick, MA), ryanodine 
(Calbiochem, San Diego, CA) and charybdotoxin (Auspep, Parkville, Victoria, Australia). Experiments were 
carried out at room temperature (22°C). 
Microfluorometric measurements were carried out on Fura-2 loaded neurones using standard 
ratiometric techniques. Isolated neurones, incubated for 1 h in PSS containing 5 μM Fura-2/AM, were 
illuminated with 340 and 380 nm light, and a Hatamatsu R928 photomultiplier tube collected the emission 
fluorescence (510 nm band pass filter) through a variable aperture which was set around the cell image. The 
output signal was digitized using a PTI interface (Photon Technology International, South Brunswick, NJ) and 
sampled at 5 Hz using Felix 1.1 software and a Pentium computer. Changes in [Ca2+]i were calculated as 
previously described [5]. Data are expressed as the mean±SEM of n, the number of observations. 
Ca2+-dependent K+ channels were studied in dissociated neurones from rat intracardiac ganglia in the 
cell-attached membrane patch recording configuration. In the presence of PSS, depolarization of the membrane 
by>20 mV evoked unitary outward currents, whose amplitude and open probability increased with further 
depolarization. The single channel conductance determined from the slope conductance of unitary current-
voltage (I–V) relations was ˜200 pS (n=3). Changing the external K+ concentration shifted the reversal potential 
of the I–V relationship as predicted by the Nernst equation for a K+-selective electrode (unpublished data). In 
four out of four patches, charybdotoxin (100 nM), added to the patch pipette solution, abolished the activity of 
the large conductance channels (data not shown). The large K+-selective single channel conductance and 
charybdotoxin- and voltage-sensitivity suggest that these channels are BKCa channels. 
The effects of caffeine were investigated to determine the presence of a caffeine-induced Ca2+ release 
from intracellular stores in rat intracardiac neurones. In all patches (n=11), 5 mM caffeine, applied to the 
superfusing solution, induced a large increase in BKCa channel activity in neurones held at 0 mV (Fig. 1A). The 
caffeine-induced increase in outward current was reversed after prolonged washout (>10 min) (not shown). 
Ryanodine (10 μM), a plant alkaloid that binds to the ryanodine receptor on the endoplasmic reticulum (ER) and 
reduces ER Ca2+ permeability at micromolar concentrations [15], inhibited the caffeine-induced activation of 
BKCa channels in all (seven/ seven) patches studied (Fig. 1B). In all membrane patches studied (n=7), bath 
application of ryanodine alone for (15 min had no effect on BKCa channel activity in these neurones.  
 
Neuroscience Letters (1999) 264 (1-3): 105-108.  doi: 10.1016/S0304-3940(99)00180-9 
 
 
 
Fig. 1. Activation of BKCa channels by caffeine and Met-enkephalin in rat intracardiac neurones. 
Membrane currents recorded from cell-attached patches in the presence of high K+ pipette solution. Membrane 
patch was depolarized by applying a pipette potential of −50 mV. (A) Bath application of caffeine (5 mM) 
evoked an increase in BKCa channel activity. Inset. Unitary currents recorded on an expanded time scale. (B) 
Effect of caffeine (5 mM) in the presence of ryanodine (10 μM) applied throughout the trace. (C) Bath 
application of Met-enkephalin (10 μM) produced an increase in BKCa channel activity. Inset. Unitary currents 
recorded on an expanded time scale. (D) Effect of Met-enkephalin (10 μM) in the presence of ryanodine (10 
μM) applied continuously throughout the recording.  
To investigate whether opioid receptor stimulation could mobilize intracellular Ca2+ and activate BKCa 
channels in rat intracardiac neurones, Met-enkephalin was added to the superfusing solution. In 21 of 25 
patches, 10 μM Met-enkephalin evoked BKCa channel activity in cell-attached patches held at 0 mV (Fig. 1C). 
Prolonged washout (>60 min) was required to reverse the increase in BKCa channel activity. In all neurones 
examined, desensitization of the response to Met-enkephalin occurred upon subsequent application of Met-
enkephalin to the same cell or to other neurones in the dish (Fig. 3). In four out of four patches, 10 μM 
ryanodine inhibited the increase in BKCa channel activity induced by Met-enkephalin (Fig. 1D) suggesting that 
ryanodine-sensitive Ca2+ stores may be the source of Ca2+ for the activation of BKCa channels by Met-
enkephalin. 
To determine if the Met-enkephalin-induced increase in [Ca2+]i was dependent on Ca2+ influx, the 
effects of Ca2+-free external solutions and the inorganic Ca2+ channel blocker, cadmium (Cd2+) were studied on 
BKCa channel activity. In the presence of Ca2+-free solution (1 mM EGTA), 10 μM Met-enkephalin failed to 
evoke a significant increase in BKCa channel activity (n=4; Fig. 2A). However, the activation of BKCa channels 
by Met-enkephalin was observed when extracellular Ca2+ (2.5 mM) was restored (Fig. 2B). In the presence of an 
isotonic K+ external solution which depolarized the neurone to ˜0 mV, Met-enkephalin failed to evoke an 
increase in BKCa channel activity (n=5; not shown). Bath application of 100 μM Cd2+, to block Ca2+ influx 
through voltage-gated Ca2+ channels, also inhibited the Met-enkephalin-induced increase in BKCa channel 
activity (n=3; not shown). Taken together, these results suggest that Met-enkephalin-induced mobilization of 
intracellular Ca2+ is dependent on Ca2+ influx through plasmalemmal Ca2+ channels.  
Neuroscience Letters (1999) 264 (1-3): 105-108.  doi: 10.1016/S0304-3940(99)00180-9 
 
The activation of BKCa channels by Met-enkephalin was inhibited by the opioid receptor antagonist, 
naloxone. In the presence of 1 μM naloxone, Met-enkephalin activation of BKCa channels was reversibly 
inhibited (n=5) (Fig. 2C), suggesting that Met-enkephalin mobilizes intracellular Ca2+ by activating cell-surface 
opioid receptors and not from a non-specific effect of Met-enkephalin. 
 
 
 
 
 
 
 
 
Microfluorometric measurements were carried out to directly measure the Met-enkephalin-induced 
increase in [Ca2+]i in Fura-2 loaded neurones. In isolated rat intracardiac neurones, resting [Ca2+]i, was 55±8 nM 
(n=9). Bath application of 10 μM Met-enkephalin induced a biphasic increase in [Ca2+]i with an initial rapid, 
Neuroscience Letters (1999) 264 (1-3): 105-108.  doi: 10.1016/S0304-3940(99)00180-9 
 
transient rise followed by a sustained, slowly decaying phase (Fig. 3). The mean peak increase in [Ca2+]i 
(Δ[Ca2+]i), evoked by Met-enkephalin was 284±113 nM (n=7). In the presence of Ca2+-free external solution 
containing 1 mM EGTA, Met-enkephalin failed to evoke an increase in [Ca2+]i, (n=3; not shown). Subsequent 
application of Met-enkephalin evoked an attenuated Ca2+ response even after >10 min washout (Fig. 3). 
Mobilization of intracellular Ca2+ by caffeine and Met-enkephalin was demonstrated in rat intracardiac 
neurones using the activation of large conductance, charybdotoxin-sensitive BKCa channels as an assay of 
changes in submembrane [Ca2+]i. Caffeine (5 mM) evoked a large increase in BKCa channel activity, which was 
inhibited by ryanodine (10 μM) suggesting that caffeine released Ca2+ from intracellular ryanodine-sensitive 
Ca2+ stores. Similarly, Met-enkephalin (10 μM) produced an increase in BKCa channel activity, which was 
dependent on extracellular Ca2+ and inhibited by ryanodine (10 μM), Cd2+ (100 μM) and K+-induced membrane 
depolarization. Naloxone (1 μM) inhibited the Met-enkephalin-induced increase in BKCa activity consistent with 
the activation of μ-opioid receptors in these neurones [1]. Fura-2 fluorescence measurements confirm that Met-
enkephalin (10 μM) and caffeine evoke a mean increase in [Ca2+]i of ˜300 nM sufficient to activate BKCa 
channels in excised, inside-out membrane patches (unpublished data). 
The inhibition of caffeine- and Met-enkephalin-induced increases in [Ca2+]i by ryanodine suggests that 
ryanodine-sensitive Ca2+ stores are present in rat intracardiac neurones and that Ca2+-induced Ca2+ release 
(CICR) mechanisms may contribute to the action potential after-hyperpolarization [3]. Recent 
immunocytochemical and autoradiographic studies have detected a widespread and heterogeneous distribution 
of ryanodine receptors (RyR) throughout the nervous system (for review, see [9]) suggesting that intraneuronal 
Ca2+ stores and CICR may play an important role in neuronal Ca2+ dynamics. Although all three isoforms of the 
ryanodine receptor (RyR1–3) have been found in the CNS (RyR2 is the most prevalent), the function(s) of RyRs 
in neurones remains relatively unknown. 
The cellular effects of opioids, including Met-enkephalin, have been shown to be mediated via the IP3 
signal transduction pathway [13]. Mu-opioid activation of SH-SY5Y cells has also been reported to stimulate 
Ca2+ influx via L-type Ca2+ channels [14]. Furthermore, opioids have been shown to mobilize Ca2+ from 
intracellular stores in SH-SY5Y cells but require ongoing muscarinic receptor activation [2]. 
In conclusion, opioid receptor activation produces opposing actions on Ca2+ mobilization in rat 
intracardiac neurones; inhibition of Ca2+ entry through ω-conotoxin GVIA-sensitive Ca2+ channels [1] and 
stimulation of Ca2+ release from ryanodine-sensitive intracellular Ca2+ stores.  
 
Acknowledgements 
 
This work was supported by the National Health and Medical Research Council of Australia, grant No. 
96/NHMRCO088G.  
 
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Met-enkephalin-induced mobilization of intracellular Ca2+ in rat intracardiac ganglion neurones

Met-enkephalin-induced mobilization of intracellular Ca2+ in rat intracardiac ganglion neurones

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