Department of Internal Medicine, The University of Michigan Medical
Center, Ann Arbor, Michigan
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Introduction |
cAMP and Ca++ are
ubiquitous second messengers in a majority of cell types (Katz and
Miledi, 1965
; Sutherland, 1972; Berridge and Irvine, 1984; Tsien and
Tsien, 1990; Tsunoda, 1993). Previous studies demonstrated that VIP and
CCK stimulate cholinergic transmission via the cyclic AMP pathway
(Kusunoki et al., 1986; Wiley and Owyang, 1987).
Furthermore, it has been reported that activation of the cyclic AMP
pathway in the myenteric plexus results in ACh release (Wiley and
Owyang, 1987; Yau et al., 1987). It is widely accepted that
the release of ACh from cholinergic neurons requires an influx of
extracellular calcium (Katz and Miledi, 1965).However, the mechanism by
which cAMP modifies Ca++ channel activities in the ganglia
is not clear. In bovine adrenal chromaffin cells cAMP has been shown to
modify the gating properties of L-type calcium channels (Doupnik and
Pun, 1992). In cardiac muscle cells, cAMP-dependent protein kinase A
phosphorylates the alpha-1 subunit of the L-type calcium
channels (Yatani and Brown, 1989). Furthermore cAMP in pancreatic 
cells increases [Ca++]i via the opening of
the L-type Ca++ channels (Grapengiesser et al.,
1991; Hsu et al., 1991). In addition to these cAMP coupled
L-type Ca++ channels, in olfactory cillia, cAMP increases
membrane conductance resulting in membrane depolarization
via activation of the Golf and superfamily II
Na+/Ca++ channels (Nakamura and Gold, 1987).
Although these studies suggest that the cAMP pathways are linked to
either the L-type Ca++ channels or the
Golf/Ca++ channels, the type of
Ca++ channels associated with cAMP-mediated ACh release in
neural tissue has not been clearly identified.
In the majority of cells, G proteins are coupled to the membrane
receptors to mediate the intracellular signal transduction systems
(Gilman, 1987). Among these, the inhibitory G proteins (Gi/Go) are capable of regulating the
receptor-mediated Ca++ flux across the plasma membrane in
several cell types including the neurons. For instance, the
somatostatin receptor is coupled to the Ca++ channels
via the PTX-sensitive G proteins in chick ciliary ganglion (Dryer et al., 1991). Also in the SV40 transformed hamster
[beta] cell line, somatostatin inhibits insulin secretion
by inhibiting Ca++ influx via the PTX-sensitive G proteins
(Hsu et al., 1991). The G protein Go regulates
neuronal Ca++ channels in neuroblastoma X glioma hybrid
cells (Hescheler et al., 1987). In rat myenteric neuron the
neuropeptide Y receptor, which is coupled to the PTX-sensitive G
protein, regulates the voltage-sensitive N-type calcium channels
(Hirning et al., 1990). Similarly in dorsal root ganglion
cells, sympathetic neurons and pituitary cells, dopamine2-,
GABAB-, alpha-2-,
A1-adenosine-receptors and
M2-muscarinic-receptors may also inhibit the N-type
Ca++ channels through the PTX-sensitive
Gi/Go proteins (Gross and MacDonald, 1987;
Schultz et al., 1990). Furthermore, opioid peptides are
known to inhibit intestinal motility by reducing cholinergic transmission via mediation of inhibitory G proteins (Cherubini and
North, 1985; Nakayama et al., 1990; Gross et al.,
1990). Recently, we reported that inhibition of cholinergic
transmission by opioids in ileal myenteric plexus is mediated by the
kappa opioid receptor (Kojima et al., 1994).
Thus, kappa receptors modulate ACh release by the inhibition
of N-type voltage-sensitive Ca++ channels via a
PTX-sensitive G-protein. The objectives of this study were to
characterize the Ca++ channels associated with
cAMP-mediated ACh release and to examine if this process can be
inhibited by agents like opioids which inhibit N-type Ca++
channels via a PTX-sensitive G-protein.
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Materials and Methods |
Preparations.
A portion of the ileum (10 cm proximal to the
ileocecal junction) from adult male Hartley guinea pigs (weighing
300-350 g) was excised after laparotomy. The longitudinal
muscle with the myenteric plexus was prepared according to the method
of Yau et al. (1989). Briefly a piece of 10-cm ileum was
stretched on a glass rod (4 mm in diameter) and the mesentery was
removed. The strips were divided into 2-cm pieces and were
enzymatically digested at 37°C for 20 min in a 95%
O2/5% CO2-gassed Dubnoff metabolic incubator
in a mixture of the Krebs bicarbonate buffer and enzymes consisting of
the following composition: NaCl, 118; KCl, 4.8; CaCl2 2.5;
MgSO4 1.19; NaHCO3, 25.0; KH2PO4,
1.18; glucose 11 (in millimolar), collagenase type II 2.5 mg/ml,
protease 2 mg/ml and 0.1% bovine serum albumin. At the end of
digestion the partly digested tissue was mechanically disrupted by
repeated suctions into a 5-ml pipette to free the ganglia. A suspension
was centrifuged for 2 min at 200 × g and the resultant
supernate containing dispersed muscle cells was discarded. The pellet
was resuspended with Krebs buffer and this procedure was repeated three
times. After the third centrifuge, resuspended ganglia were filtered
through a 500-µm Nitex. 200 ganglia per test tube were harvested with
a 20-ml capillary pipette under a dissecting microscope.
3H-ACh release and
45Ca++ efflux
studies.
The 3H-ACh release studies were carried out
according to the method as described previously (Takahashi et
al. 1992). The collected ganglia were incubated in a test tube
with either 3H-choline (10 µCi/ml) or
45Ca++ (10 µCi/ml) in the Krebs buffer
containing 50 µM physostigmine for 45 min in an incubator at 37°C
which was gassed with 95% O2 and 5% CO2. The
radiolabeled ganglia were subsequently placed in a water-jacketed
column over a filter paper (5.0-µm pore size) on a bed of Bio-Gel P2
(Bio-Rad, Richmond, CA) at the bottom, which was perfused at a rate of
1 ml/min with the 95% O2/5% CO2-gassed Krebs
buffer containing 50 µM physostigmine and 10 µM hemicholinium-3. Experiments were started 30 min after perfusion at 37°C, a time when
the spontaneous release of 3H-ACh or
45Ca++ had approached a plateau. The perfusates
were collected using a fraction collector. In studies to evaluate the
effects of various Ca++ channel blockers, nifedipine or
nickel were added 10 min before and during the stimulation with
8Br-cAMP or forskolin. In other experiments, ganglia were treated with
-CgTx for 20 min before and during the stimulation. When the effects
of U50488H, a kappa receptor agonist, were tested, the drug
was applied 5 min before and during the stimulation. These chemicals
were further coincubated with secretagogues at each time interval. In
figures 2, 4 and 6, the cells were stimulated with secretagogues for 1 min. In studies using PTX, ganglia were pretreated with PTX (300 ng/ml) at 37°C for 3 hr and further coincubated with 3H-choline
for 45 min. PTX was not included in the superfusion medium when ganglia
were stimulated by either 8Br-cAMP or forskolin.
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Fig. 2.
Effects of -CgTx on 8Br-cAMP-induced
3H-ACh release. Ganglia were treated with -CgTx at
37°C 20 min prior to and during 8Br-cAMP stimulation for 1 min.
-CgTx significantly inhibited the release of 3H-ACh
evoked by 8Br-cAMP at 10 to 100 nM. Concentration of reagent used:
8Br-cAMP, 1.0 × 10 3 M. Data are
expressed as mean ± S.E.M. from six separate experiments. *P < .05 compared with control.
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Fig. 4.
Effects of U50488H on (A) 8Br-cAMP- and (B)
forskolin-evoked 3H-ACh release. Ganglia were treated with
U50488H at 37°C 5 min before to and during 8Br-cAMP or forskolin
stimulation for 1 min. 8Br-cAMP- or forskolin-evoked 3H-ACh
release was inhibited by U50488H, a kappa receptor
agonist, in a dose-dependent manner. Concentrations of reagents used:
8Br-cAMP, 1.0 × 103 M; forskolin,
1.0 × 104 M. Data are expressed as
mean ± S.E.M. from six separate experiments. *P < .05 compared with control.
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Fig. 6.
Effects of pertussis toxin on (A) 8Br-cAMP and (B)
forskolin-induced 3H-ACh release in the presence or absence
of U50488H. Ganglia were pretreated with 300 ng/ml PTX or vehicle for 3 hr at 37°C before cell stimulation. U50488H was applied 5 min before
and during 8Br-cAMP or forskolin stimulation for 1 min. Concentrations
of reagents used: 8Br-cAMP, 1.0 × 103
M; U50488H 1.0 × 109 M (A); forskolin,
1.0 × 104 M; U50488H, 3.0 × 1010 M (B). Data are expressed as mean ± S.E.M. from seven separate experiments. Abbreviations used: 8-Br,
8Br-cAMP; U50, U50488H; FK, forskolin.
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Fractional release was calculated by expressing the radioactivity in
each vial as a percentage of the total radioactivity in the tissue at
the time of collection. The amount of radioactivity in the tissue at
the time of collection was almost equal to the sum of the c.p.m. of
released radioactivity in all vials plus the c.p.m. contained in the
tissue at the end of the experiments which were determined by removing
tissues from a water-jacketed column. Quantitative values of the c.p.m.
effluxed from the superfused ganglia showed significant variations
depending on the amounts of tissue used and loading conditions of the
radioactive choline or Ca++. Therefore, results were
presented as a percent of increase over basal release. Assessment of
the percentage of labeled metabolites in the form of 3H-ACh
was performed on an ion exchange columns as described previously (Wu
et al., 1982). We observed that more than 85% of the
labeled metabolites of choline were in the form of 3H-ACh
during stimulated conditions.
Statistical analysis of the data was carried out with an unpaired
Student's t test or a one-or two-way analysis of variance. P < 0.05 was considered to be significant. The means ± S.E.M. were used throughout this report.
Materials.
Chemicals were purchased from the following
sources: physostigmine, hemicholinium-3, 8Br-cAMP, -CgTx GIVA,
nifedipine, forskolin, collagenase type II, protease from Sigma
Chemical (St. Louis, MO); nickel chloride from Mallinckrodt (Paris,
KY); 3H-choline chloride and 45Ca++
from Amersham (Arlington Heights, IL); PTX from List Biological Laboratories (Campbell, CA). U50488H was a gift from Dr. James Woods,
the University of Michigan. All chemicals except nifedipine and
forskolin were dissolved in distilled water, stocked -20°C and
diluted by the Krebs bicarbonate buffer before experiments. Nifedipine
was dissolved in dimethyl sulfoxide and forskolin was dissolved in
ethanol, both of which the final concentrations did not exceed 0.1%
(v/v). This concentration (0.1%) of dimethyl sulfoxide or ethanol did
not change the release of, 3H-ACh and
45Ca++ efflux.
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Results |
Effects of Ca++ channel blockers on cyclic
AMP-stimulated 3H-Ach release and
45Ca++ efflux.
Application of the cell permeant cAMP analogue, 8Br-cAMP (1 mM), to the
superfused ganglia resulted in a 50% increase of 3H-ACh
release over basal after 1 min cell stimulation. This increase gradually declined over the next 7 min. 3H-ACh release was,
however, sustained above the basal level during the entire study (fig.
1A). Because our quantitative superfusion systems were
incapable of directly measuring energy independent 45Ca++ influx into the ganglia, we measured
energy dependent 45Ca++ efflux, as an indirect
index of the Ca++ transport system because we have
previously shown that Ca++ influx and efflux are highly
synchronized (Tsunoda, 1993). A 60% increase in
45Ca++ efflux from superfused ganglia was
observed after 8Br-cAMP stimulation. The peak increase coincided with
the peak increase of 3H-ACh release (fig. 1A). However,
release of 45Ca++ was more transient and
returned to the basal level 4 min after the beginning of stimulation
(fig. 1A). A similar pattern of 3H-ACh release as well as
45Ca++ efflux was observed when the ganglia
were stimulated with forskolin, an agent that directly activates
adenylyl cyclase (Seamon and Daly, 1981). Forskolin, 0.1 mM, caused
both a 70% increase over basal in 3H-ACh release and
45Ca++ efflux from the dispersed ganglia within
2 min of cell stimulation (fig. 1B). Similar to the observation when
8Br-cAMP was used as a stimulant, the forskolin-stimulated
3H-ACh release was sustained over 7 min during cell
stimulation, whereas the 45Ca++ efflux was more
transient (fig. 1B). These results indicate that cyclic AMP mobilizes
Ca++ and elicits ACh release in the myenteric ganglia.
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Fig. 1.
Effects of (A) 8Br-cAMP and (B) forskolin on
3H-ACh release and 45Ca++ efflux.
Application of 8Br-cAMP and forskolin resulted in a stimulation of
3H-ACh release and 45Ca++ efflux.
Basal c.p.m. values were around 200 to 500 in both 3H-ACh
and 45Ca++ radioactivities in figures 1 through
6. Cells were stimulated with secretagogues as indicated by arrow lines
in figures 1, 3 and 5. Concentrations of reagents used: 8Br-cAMP,
1.0 × 103 M; forskolin, 1.0 × 104 M. Data are expressed as mean ± S.E.M. from seven separate experiments.
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To characterize the Ca++ channel(s) involved in
45Ca++ efflux, we investigated the effects of
-CgTx (GIVA) (preferential N-channel blocker), nifedipine (specific
L-channel blocker) and nickel (relative T-channel blocker) (Tsien and
Tsien, 1990, Spedding and Paoletti, 1992). As shown in figure
2, pretreatment of the myenteric ganglia with -CgTx
at 10 and 100 nM resulted in a significant inhibition of
8Br-cAMP-evoked 3H-ACh release for 1 min. However, the
KD and IC50 values of -CgTx (GIVA) acting on
other tissues and myenteric ganglia stimulated with veratridine were
approximately 1 nM (Dolphin, 1995; Kojima et al., 1994;
McEnery et al., 1991; Reynolds et al., 1986;
Witcher et al., 1993). -CgTx at 10 nM completely
abolished 8Br-cAMP-stimulated 45Ca++ efflux
(fig. 3A). In contrast, nifedipine (1 µM) and nickel
(100 µM) had no significant effects on 8Br-cAMP-induced
45Ca++ efflux (fig. 3B). The concentrations of
nifedipine and nickel were sufficient to block the opening of the
L-type and T-type Ca++ channels, respectively (Dolphin,
1995; Godfraind, 1983; Spedding and Paoletti, 1992). These data suggest
that the N-type voltage-dependent Ca++ channel is the
predominant Ca++ channel mediating cAMP-dependent ACh
release from the myenteric ganglia.
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Fig. 3.
Effects of (A) -CgTx, (B) nifedipine and nickel
on 8Br-cAMP-induced 45Ca++ efflux. Ganglia were
treated with -CgTx and nifedipine or nickel at 37°C for 20 and 10 min, respectively, before and during 8Br-cAMP application. -CgTx
completely blocked 8Br-cAMP-induced 45Ca++
efflux, whereas nifedipine or nickel had no effect. Concentrations of
reagents used: -CgTx, 1.0 × 108 M;
nifedipine, 1.0 × 106 M, nickel
1.0 × 104 M. Data are expressed as
mean ± S.E.M. from six separate experiments.
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Effects of kappa receptor agonist (U50488H) on cAMP-mediated
3H-Ach release and
45Ca++ efflux.
We
next investigated the effects of U50488H, a kappa receptor
agonist, on 3H-Ach release and
45Ca++ release from the myenteric ganglia. As
shown in figure 4A, 8Br-cAMP- (1 mM) evoked
3H-ACh release for 1 min was significantly inhibited by
pretreatment of ganglia U50488H at 1 to 100 nM (fig. 4A). Similarly,
the forskolin- (0.1 mM) stimulated 3H-ACh release for 1 min
was also significantly inhibited by U50488H at 1 to 100 nM (fig. 4B).
This value was close to that observed in other tissues (Cherubini and
North, 1985; Knapp et al., 1995; Reisine and Bell, 1993;
Takemori and Portoghese, 1992; Watson and Girdlestone, 1996).
Furthermore, 8Br-cAMP-elicited 45Ca++ efflux
from myenteric ganglia was completely inhibited by 10 nM U50488H during
the entire period of cell stimulation (fig. 5). The
inhibitory effects of U50488H on 8Br-cAMP- or forskolin-evoked 3H-ACh release for 1 min were significantly reversed by 3 hr pretreatment of the myenteric ganglia with PTX (300 ng/ml). The
concentration of PTX used was sufficient to inactivate the
Gi/Go proteins (Birnbaumer et al.,
1990) (fig. 6A, B). These data suggest that cAMP-induced Ca++ mobilization and ACh release can be regulated by the
kappa opioid receptor through PTX-sensitive G proteins.
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Fig. 5.
Effects of U50488H on 8Br-cAMP-induced
45Ca++ efflux. Ganglia were treated with
U50488H at 37°C 5 min before and during 8Br-cAMP stimulation.
8Br-cAMP-elicited 45Ca++ efflux was inhibited
by U50488H. Concentrations of reagents used: 8Br-cAMP, 1.0 × 103 M; U50488H, 108
M. Data are expressed as mean ± S.E.M. from six separate
experiments.
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Discussion |
Electrophysiological studies on myenteric plexus neurons suggest
that many neuropeptides may stimulate cholinergic transmission via a
cAMP pathway (Palmer et al., 1987). Neuropeptides such as CCK, VIP, gastrin releasing peptide and substance P generated slow
exitatory post synaptic potentials, a characteristic pattern observed
with muscarinic cholinergic depolarization. With the exception of
substance P, the generation of slow exitatory post synaptic potentials
by these peptides was abolished by prior treatment with adenosine, an
inhibitor of adenylyl cyclase, whereas the same neurons continued to
respond to substance P (Palmer et al., 1987). This implies
that the receptors for all these peptides except substance P are
coupled to adenylyl cyclase. This possibility is further supported by
the direct demonstration that VIP and CCK stimulated acetylcholine
release from the myenteric plexus which was inhibited by 2
,
5,-dideoxyadenosine, an inhibitor of adenylyl cyclase (Kusunoki
et al., 1986; Wiley and Owyang, 1987).
Direct evidence that cAMP may serve as an intracellular mediator for
cholinergic transmission in the myenteric neurons has been provided by
several laboratories. In guinea pig myeneric plexus, activation of cAMP
pathway resulted in ACh release during stimulation with forskolin,
dibutyryl-cAMP and 8Br-cAMP (Reese and Cooper, 1984; Yau et
al., 1987). Palmer et al. (1986) and Nemeth et
al. (1986) reported that forskolin and intracellular injection of
cAMP evoked slow synaptic excitation in AH/type 2 myenteric neurons of
guinea pig. Yau et al. (1987) demonstrated that
8Br-cAMP-induced ACh release was sustained even after removal of the
agent. Our study indicated that 45Ca++ efflux
occurred in a transient manner despite continuous application of
forskolin or 8Br-cAMP, whereas ACh release was sustained for at least 7 min above the prestimulation level. Kurahashi (1990) also observed
similar phenomenon in ciliary cells that the cAMP-induced Ca++ current was transient despite continuous
administration of cAMP from the patch pipette. It is conceivable that
the transient Ca++ influx from the extracellular space may
trigger the initial ACh release and that some intracellular relay
signals may be responsible for the subsequent sustained ACh release.
Alternatively, the signal initiated by the initial [Ca++
]i increase may be "locked in," so that subsequent ACh
release may be less Ca++ dependent (Tsunoda, 1993). Further
studies are needed to examine these possibilities.
Recent studies indicate that activation of the cAMP pathway resulted in
the opening of L-type voltage-sensitive Ca++ channels in
several cell types. In cardiac muscle cells, the L-type calcium
currents may be mediated by at least two different mechanisms. In
addition to the cAMP/protein kinase A system (Yatani and Brown, 1989),
GS may directory stimulate the L-type Ca++
channels by a cAMP independent mechanism (Brown and Birnbaumer, 1988;
Yatani et al., 1987). GS is responsible for the
small but early phosphorylation that accounts for 20% of the L-type
Ca++ currents, whereas the longer and slower
phosphorylation is regulated by the cyclic AMP/protein kinase A system
which accounts for 80% of the activation of the L-type
Ca++ channels. A similar observation have been made
regarding the L-type Ca++ channels in pancreatic beta cells
(Grapengiesser et al., 1991, Hsu et al., 1991)
and the adrenal chromaffin cells (Doupnik and Pun, 1992). Furthermore,
in olfactory receptor cilia, the operation of the Ca++
channels in the ciliary plasma membrane is directly gated by cAMP
(Nakamura and Gold, 1987, Kurahashi, 1990). However, our studies showed
that in the myenteric plexus, cAMP-stimulated Ca++ efflux
and ACh release were both inhibited by -CgTx, but not by nifedipine
and nickel. This suggests that the type of Ca++ channel(s)
associated with the cAMP system may be tissue specitific. In the
peripheral neurons, cAMP-stimulated Ca++ efflux and ACh
release appears to be mediated mainly by N-type Ca++
channels. This finding is in agreement with previous observations that
N-type Ca++ channels are the preferential Ca++
channels in the myenteric neurons (Hirning et al., 1988,
Takahashi, et al., 1992).
The ability of an agent to inhibit cholinergic transmission evoked by
different peptides may be determined by the intracellular mechanisms
used by a specific neuropeptide (Kowal et al., 1989). For
example, somatostatin demonstrated differential effectiveness to alter
ACh release evoked by CCK and substance P (Teitelbaum et
al., 1984, Yau et al., 1986). This appears to be
related to the fact that CCK but not substance P stimulated ACh release
by a cAMP-dependent pathway. Somatostatin, which regulates adenylyl cyclase activity via Gi, inhibited the cAMP-dependent
component of CCK-mediated cholinergic transmission via activation of a
pertussis toxin-sensitive G protein (Kowal et al., 1989).
However, somatostatin was ineffective against cholinergic transmission
evoked by substance P, whose receptors appear to be coupled directly to
Ca++ channels independent of any second messengers within
the cell (Wood, 1987). We reported that the kappa receptors
mediated the inhibitory action of opiates on cholinergic transmission
in the myenteric plexus via a PTX-sensitive inhibitory G protein that is coupled to voltage-sensitive N-type Ca++ channels
(Kojima et al., 1994). Because cAMP evoked Ca++
efflux and ACh release also involves N-type Ca++ channels,
it is not surprising that the kappa receptor agonist, U50488H very potently inhibited the Ca++ efflux and ACh
release stimulated by 8Br-cAMP.
In our study we demonstated that PTX reversed the inhibitory action of
the kappa receptor agonist (U50488H) on Ach release evoked
by 8Br-cAMP. It seems unlikely that the effects of the kappa
opioid receptor agonists are due to inactivation of the adenylyl
cyclase system through PTX-sensitive G proteins because 8Br-cAMP acts
at a step distal to the formation of cAMP. It has been proposed that
the cellular stimulation of the kappa opioid receptor may
activate a PTX-sensitive G protein, which may result in
hyperpolarization and subsequent inhibition of Ca++ influx
from the extracellular space. In fact it has been demonstrated that
opioids hyperpolarize a subpopulation of neurons in the myenteric plexus of the guinea pig ileum (North and Tonini, 1977) and this may be
due to K+ efflux from the cell because it is well known
that the PTX-sensitive Gi/Go family of G
proteins activates K+ channels and K+ efflux,
which result in cellular hyperpolarization (Birnbaumer et
al., 1990).
Our myenteric ganglia preparation contains a mixed population of
"S" and "AH" neurons (Hirst et al., 1974). Because
it has been shown that the action of forskolin in the myenteric neurons is limited to AH neurons although the mu opioid agonists act
primarily on S neurons and does not involve changes in cellular levels
of cAMP (Johnson and Pillai, 1990), we believe that the inhibitory action of the kappa agonists on cAMP-stimulated ACh release
is probably on the AH neurons. The S and AH neurons may differentially contribute to the regulation of smooth muscle contraction. In general,
the S neurons are the population which innervate the longitudinal
smooth muscle while the AH neurons may be sensory neurons without any
contact with the longitudinal smooth muscle. However, these AH neurons
may serve as interneurons and synaptically contact the S neurons to
promote release of neurotransmitter(s). Cherubini and North (1985)
reported that opioids can reduce ACh release by activation of the
mu receptors which result in increased K+
conductance in the S neuron soma or stimulation of kappa
receptors that suppress the release of ACh by directly reducing the
Ca++ conductance at the level of the nerve terminal. It is
therefore conceivable that the kappa receptor agonist
U50488H may reduce ACh release at the nerve terminal of the AH neurons
by reducing Ca++ entry evoked by the cAMP pathway.
Additional studies are needed to clarify this issue.
In conclusion, using dissociated ideal myenteric ganglia, we have
demonstrated that cyclic AMP-stimulated Ca++ efflux and Ach
release were mediated by N-type Ca++ channels. This process
can be inhibited by activation of the kappa opioid receptor
through pertussis toxin-sensitive G protein(s).
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Abstract
Introduction
