Presynaptic nicotinic acetylcholine receptors in the myenteric
plexus of guinea pig intestine
David A.
Schneider and
James J.
Galligan
Department of Pharmacology and Toxicology and Neuroscience Program,
Michigan State University, East Lansing, Michigan 48824-1317
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ABSTRACT |
Presynaptic nicotinic acetylcholine
receptors (nAChRs) were studied in myenteric plexus preparations from
guinea pig ileum using intracellular electrophysiological methods.
Microapplication of nicotine (1 mM) caused a biphasic depolarization in
all AH neurons (n = 30) and in 36 of 49 S neurons.
Cytisine (1 mM) caused fast depolarizations in S neurons and no
response in AH neurons. Mecamylamine (10 µM) blocked all responses
caused by nicotine and cytisine. TTX (0.3 µM) blocked slow excitatory
synaptic potentials in S and AH neurons but had no effect on fast
depolarizations caused by nicotine. Nicotine-induced slow
depolarizations were reduced by TTX in two of twelve AH neurons (79%
inhibition) and four of nine S neurons (90 ± 12% inhibition).
Slow nicotine-induced depolarizations in the remaining neurons were TTX
resistant. TTX-resistant slow depolarizations were inhibited
after neurokinin receptor 3 desensitization caused by senktide
(0.1 µM); senktide desensitization inhibited the slow
nicotine-induced depolarization by 81 ± 5% and 63 ± 15%
in AH and S neurons, respectively. A low-calcium and high-magnesium
solution blocked nicotine-induced slow depolarizations in AH neurons.
In conclusion, presynaptic nAChRs mediate the release of substance P
and/or neurokinin A to cause slow depolarizations of myenteric neurons.
presynaptic receptors; neurokinin receptor 3; myenteric
neurons
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INTRODUCTION |
PRESYNAPTIC
REGULATION OF neurotransmitter release is an important mechanism
governing synaptic transmission in the central and peripheral nervous
systems (28). In the enteric nervous system (ENS), there
are many receptor-mediated presynaptic mechanisms for inhibition of
transmitter release but only presynaptic 5-hydroxytryptamine (5-HT)4 receptor has been shown to facilitate enteric
neurotransmission (19, 32). However, in the
central nervous system and in other autonomic ganglia, presynaptic
nicotinic acetylcholine receptors (nAChRs) function to facilitate or
induce transmitter release (1, 12,
26, 27, 39). Although there is
evidence for presynaptic nAChRs on the nerve terminals of longitudinal
muscle motoneurons in guinea pig intestine (9), it is not
known if presynaptic nAChRs are present in enteric ganglia.
The fast sodium channel blocker TTX has been used to distinguish
the cellular location of presynaptic receptors. For example, the TTX
sensitivity of nAChR-mediated release of transmitter from synaptosomes
distinguishes two populations of presynaptic nAChRs (24,
39). One population of presynaptic receptors is located on
nerve terminals, and transmitter release mediated by these receptors is
resistant to blockade by TTX. Stimulation of presynaptic receptors
increases transmitter release either by causing direct depolarization
of the nerve terminal or by directly gating calcium entry into nerve
terminals, (39). Transmitter release mediated by
preterminal nAChRs is abolished by TTX as sodium-dependent action
potentials propagated along the axon are required for preterminal receptors to mediate depolarization and calcium entry into nerve terminals (39). Studies of the TTX sensitivity of a
variety of responses induced by nAChR agonists have provided mixed
results as to the existence of presynaptic nAChRs in the ENS. ACh
release induced by nAChR agonists is blocked by TTX in the ENS
(10, 37), but other nAChR-induced responses
in gastrointestinal tissues are TTX resistant (2,
3, 9, 10, 34,
38). Furthermore, recent immunohistochemical studies
(20, 31) of myenteric neurons of the guinea
pig ileum have demonstrated that nAChRs may be located on nerve
terminals. The experiments conducted here were designed to determine if
there are functional presynaptic nAChRs located on or near the nerve
terminals of myenteric neurons in the guinea pig ileum. The results
indicate that pharmacologically distinct populations of nAChRs are
present in the myenteric plexus, some of which induce TTX-resistant,
noncholinergic slow excitatory postsynaptic potentials (sEPSPs).
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MATERIALS AND METHODS |
Animal use and tissue preparation.
Male albino guinea pigs (250-450 g) obtained from the Michigan
Department of Public Health (Lansing, MI) were used. The care and use
of these animals were approved by the All-University Committee on
Animal Use and Care at Michigan State University. Animals were killed
by exsanguination after general anesthesia induced by inhalation of
halothane (Halocarbon Laboratories, River Edge, NJ). A segment of ileum
was removed and placed into a dissection bath lined with Sylgard 184 elastomer (Dow Corning, Midland, MI) and filled with oxygenated (95%
O2-5% CO2) Krebs solution of the following
composition (in mM): 117 NaCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 4.7 KCl, 25 NaHCO3, and 11 glucose. To minimize smooth muscle movement, nifedipine (1 µM) and scopolamine (1 µM) were added to the Krebs solution. The lumen of the bowel was opened along the longitudinal axis
and pinned mucosa-side up, and the bowel contents were rinsed away. The
mucosa, submucosa, and circular smooth muscle layers were sequentially
removed, leaving a preparation consisting of the longitudinal muscle
layer and attached myenteric plexus (LMMP). An LMMP preparation was
then removed and pinned, myenteric ganglia uppermost, in a 2-ml
elastomer-lined recording chamber filled with Krebs solution. The
recording chamber was secured to the stage of an inverted microscope
and continuously superfused (3-5 ml/min) with oxygenated Krebs
solution maintained at 37°C. Individual myenteric ganglia and
interconnecting fiber tracts were visualized at ×200 magnification
with Hoffman Modulation Contrast optics (Modulation Optics, Greenvale, NY).
Intracellular recording technique.
Membrane potential was recorded from individual myenteric neurons using
an Axoclamp-2A electrometer (Axon Instruments, Foster City, CA) and
current-clamp technique. Recordings were obtained using glass
microelectrodes filled with 2 M KCl and tip resistances of 80-120
M
. Data were sampled at 2 kHz, filtered at 5 kHz using a four-pole,
low-pass Bessel filter (Warner Instruments, Hamden, CT), and digitally
converted, monitored, and stored using acquisition and analysis
software (Axotape, version 2.0.2 and Axoscope, version 7.0; Axon
Instruments) and a desktop computer.
Experimental procedures.
Myenteric neurons were impaled, and the resting membrane potential was
measured 10-20 min after initial impalement to ensure stability of
the recording. In some experiments, a constant hyperpolarizing current
was passed through the recording microelectrode to facilitate a stable
impalement and to minimize the number of action potentials occurring
during drug- or nerve-stimulated depolarizations. Minimizing action
potential discharge allowed more accurate measurements of the peak
depolarization caused by drugs and nerve stimulation. Myenteric neurons
were categorized as AH neurons if a single somal action potential was
followed by an afterhyperpolarization
4 mV in amplitude and
4 s in
duration. S neurons were categorized as those cells in which single
electrical stimuli applied to interganglionic connectives elicited a
fast excitatory postsynaptic potential (fEPSP) (14).
Neurons for which these data were not complete were considered
unclassified. Synaptic responses were elicited using a Krebs
solution-filled glass stimulating microelectrode positioned over an
interganglionic fiber tract. Electrical stimuli were provided by a
Grass S44 stimulator and stimulus isolation unit (SIU 5, Grass
Instruments, Quincy, MA). Focal stimulation with single, 0.5-ms pulses
(10-150 V) was used to induce fEPSPs at a stimulus rate of 0.3 Hz.
Brief trains of stimuli (10 Hz for 300-800 ms) were used to evoke
sEPSPs. The presence of fEPSPs and sEPSPs was tested by stimulating
only one interganglionic fiber tract.
Local application of agonists was accomplished by microejection from
the tip of a micropipette (30- to 40-µm tip diameter) placed within
50 to 150 µm of the impaled neuron. Agonists were applied using short
pulses of nitrogen gas (3 to 35 ms, 10 psi) using a Picospritzer
II (General Valve, Fairfield, NJ). Antagonists and the neurokinin
receptor 3 (NK3) agonist senktide were superfused at
3-5 ml/min for a minimum of 5 min; there was a 30-s lag time for
drugs to reach the recording chamber.
Statistical analysis.
Responses were recorded as changes in membrane potential (mV) relative
to the resting membrane potential. Descriptive and analytical
statistics were computed using SAS, version 6.12 (SAS Institute, Cary,
NC). The assumptions of equal variance and normal distribution were
tested using
2 residual analysis and the Wilk-Shapiro
test, respectively. Maximum likelihood analysis was used to determine
appropriate transformations for statistical modeling. A
repeated-measures ANOVA was used to determine the effect of specific
antagonists on agonist-induced responses. When statistical differences
were detected, a Bonferroni t-test or specific linear
contrasts were used to separate the means. A paired t-test
was used to compare the responses induced by nicotine and cytisine
within the same neuron. Significant differences were declared when
P < 0.05. All data are presented as means ± SE.
Drugs.
The following drugs were purchased from Sigma Chemical (St. Louis, MO):
nicotine and cytisine (nAChR receptor agonists), senktide (NK3 receptor agonist), 5-HT, TTX (sodium channel
antagonist), scopolamine (muscarinic receptor antagonist), and
nifedipine (L-type calcium channel antagonist). The nAChR antagonist
mecamylamine hydrochloride was purchased from RBI (Natick, MA). All
reagents and drugs were diluted in distilled, deionized water except
for nifedipine, which was dissolved in 95% ethanol to make a 10 mM concentrated stock solution. The final working concentration of all
drugs was made daily by diluting concentrated stock solutions with
Krebs solution.
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RESULTS |
Responses of myenteric neurons to microejection of nicotine or
cytisine.
Membrane potential responses to microejection of nicotine (1 mM) or
cytisine (1 mM) were recorded from 124 myenteric neurons (30 AH, 67 S,
and 27 unclassified neurons). The initial resting membrane potentials
of AH, S, and unclassified neurons were:
70 ± 2,
54 ± 4, and
60 ± 6 mV, respectively. Nicotine caused a biphasic
response in 87 of 94 (93%) myenteric neurons. The first phase was a
fast depolarization, whereas the second phase was either slow
depolarization (65 neurons, 75%) or hyperpolarization (22 neurons,
25%) (Fig. 1, A-C). The
slow hyperpolarization in myenteric neurons that occurs after nAChR
activation has been described previously (37) and will not
be discussed further here. A monophasic fast depolarization occurred in
3 S (amplitude = 28 ± 9 mV) and 3 unclassified
(amplitude = 32 ± 7 mV) neurons, and one S neuron did not
respond to nicotine. Microejection of Krebs solution onto the ganglion
or microejection of nicotine onto the surrounding smooth muscle did not
affect the membrane potential of any neuron.

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Fig. 1.
Responses of myenteric neurons caused by microapplication
of nicotine (1 mM) or cytisine (1 mM). Arrowheads indicate drug
application. A-C: responses caused by nicotine were
biphasic, but the polarity of the slow phase varied between neurons. AH
neurons (n = 28) (A) and 18 of 25 S neurons
(B) responded to nicotine with biphasic depolarization.
C: 22 S neurons responded to nicotine with a fast
depolarization followed by hyperpolarization. D: cytisine
caused only fast depolarizations. In 14 of 20 S neurons, the fast
depolarization was followed by a hyperpolarization. Vertical scale
bars, 10 mV; horizontal scale bars, 30 s.
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The time course of the nicotine-induced fast depolarization was similar
for AH and S neurons with a rise time of 0.7 ± 0.5 s.
However, the amplitude of the fast depolarization in AH neurons (14.5 ± 2 mV, n = 28) was significantly smaller
than that recorded from S neurons (21 ± 2 mV, n = 25, P < 0.05). Although the amplitudes of the
nicotine-induced slow depolarization were similar in AH (15 ± 1 mV) and S (16 ± 1.5 mV, P > 0.05) neurons, the
time course of the slow depolarization was different (compare Fig. 1,
A and B). The time to reach the peak of the slow
depolarization in AH neurons was 25 ± 3 s but was only
5 ± 3 s in S neurons (P < 0.0001). In
addition, the half duration of the slow depolarization was 56 ± 11 s in AH vs. 15 ± 2 s in S neurons (P < 0.03).
Cytisine (1 mM) did not cause a slow depolarization in any neuron,
whereas in the same neurons nicotine caused both fast and slow
depolarizations (Fig. 2). In 10 of 12 AH
neurons, cytisine (1 mM, 5- to 15-ms pulse duration) did not cause a
fast depolarization, whereas in two AH neurons longer duration
applications of cytisine caused a only small-amplitude fast
depolarization (Fig. 2). In 14 of 20 S neurons (Fig. 1D),
cytisine induced a fast depolarization (26 ± 3 mV) that was
followed by hyperpolarization (
4.5 ± 1.5 mV), whereas the
remaining six S neurons did not respond at all to cytisine. All
responses caused by nicotine and cytisine were blocked by the nAChR
antagonist mecamylamine (10 µM) (Fig.
3).

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Fig. 2.
Responses of AH neurons to microapplication of nicotine
or cytisine (at the arrowheads, each at 1 mM). Nicotine (A)
caused a short latency, fast depolarization, and a slow depolarization
that took several seconds to develop and lasted >1 min. In contrast,
the same duration of cytisine application (B) did not cause
a response. Increasing the duration of application caused only a small
amplitude fast response (lower right trace). Vertical scale
bars, 10 mV; horizontal scale bars, 30 s.
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Fig. 3.
Depolarizations caused by nicotine and cytisine were
reversibly blocked by the nicotinic acetylcholine receptor (nAChR)
antagonist mecamylamine. A: data for inhibition by
mecamylamine (10 µM) of fast and slow depolarizations caused by
nicotine. Data are pooled responses from 15 neurons (11 AH and 4 S
neurons). B: mecamylamine inhibits fast depolarizations
caused by cytisine. Data are from 6 S neurons. Cytisine never caused a
slow depolarization. * P < 0.05, significantly
different from control responses.
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Responses to stimulation of interganglionic connectives.
Focal stimulation of a single interganglionic fiber tract induced a
fEPSP in 57 of 57 (100%) S neurons and a sEPSP in 45 of 57 (79%) S
neurons. In AH neurons (n = 30), focal stimulation never induced a fEPSP but always induced a sEPSP. Focal stimulation induced a sEPSP in 17 of 21 (81%) unclassified neurons. A slow depolarization in response to nicotine was observed in 63 of 73 (86%)
neurons in which focal stimulation induced a sEPSP. Microejection of
nicotine never induced slow depolarization in neurons in which focal
stimulation did not induce a sEPSP. Input resistance changes were
measured in four AH neurons during sEPSPs and nicotine-induced slow
depolarizations. In these neurons, input resistance increased from a
resting level of 212 ± 54 to 331 ± 74 M
at the peak of the sEPSP. In the same neurons, the nicotine-induced slow
depolarization was associated with an increase in input resistance from
a resting level of 234 ± 48 to 337 ± 87 M
at the peak of
the nicotine response.
Presynaptic nAChRs.
The effect of TTX (0.3 µM) on nicotine-induced slow depolarizations
was tested in 9 S neurons and 12 AH neurons. In four S neurons the
nicotine-induced slow depolarization was inhibited by 90 ± 12%
by TTX, whereas in two AH neurons, TTX inhibited this response by 79%.
TTX inhibited the nicotine-induced slow depolarization in the remaining
AH and S neurons by only 24 ± 7% and 18 ± 8%, respectively. These data indicate either that nicotine was causing the
slow depolarization by direct activation of nAChRs on the somatodendritic region of the neuron or that nicotine was causing TTX-resistant release of a mediator of the slow depolarization. To test
the possibility that a tachykinin peptide acting at NK3 receptors was a mediator of the nicotine-induced slow response, the
NK3 agonist senktide was used to desensitize
NK3 receptors. Senktide (0.3 µM) applied by superfusion
caused a slowly developing depolarization that returned to baseline
during continued application. Senktide-induced desensitization of
NK3 receptors inhibited the slow depolarization caused by
subsequent nicotine application in all AH neurons by 81 ± 5%
(n = 12) (Fig.
4A). In S neurons in which the
nicotine-induced slow depolarization was TTX resistant, NK3
receptor desensitization reduced the slow response by 63 ± 15%
(n = 5) (Fig. 4B). Data summarizing the
effects of sequential superfusion of TTX (0.3 µM) and the
NK3 receptor agonist senktide (0.1 µM) on fast and slow
depolarizations induced by nicotine are summarized in Fig.
5. These data show that in all AH neurons (n = 12) (Fig. 5A) and in a subset of S
neurons (n = 5) (Fig. 5B), in the presence
of TTX, NK3 receptor desensitization blocks the
nicotine-induced slow depolarization in a reversible manner. Although
the slow response was inhibited by NK3 receptor
desensitization, the fast nicotine-induced depolarization was not
inhibited during NK3 receptor desensitization. In fact, in
AH neurons, the nicotine-induced fast depolarization was increased in
amplitude during senktide superfusion (P < 0.05) (Fig.
5A). In a second subset of S neurons (n = 4), TTX inhibited the nicotine-induced slow depolarization by >90%.
After TTX was washed out and the nicotine slow depolarization returned
to the pre-TTX level, it was found that NK3 receptor desensitization reduced the slow response in these neurons by only
23 ± 8% (Fig. 5C).

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Fig. 4.
TTX-resistant slow depolarization (arrows) induced by
nicotine in an AH neuron (A) and an S neuron (B)
are inhibited after desensitization of neurokinin receptor 3 (NK3). TTX (0.3 µM) was present throughout the
recordings. Microapplication of nicotine (1 mM, arrowhead) caused a
slow depolarization in S and AH neurons (left traces).
Senktide (0.3 µM) was applied by superfusion, and after the
senktide-induced depolarization desensitized (middle
traces), the slow, but not fast, depolarizations induced by
nicotine were inhibited. Right traces were obtained after
senktide washout. Vertical scale bars, 10 mV; horizontal scale bars,
30 s.
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Fig. 5.
Summary of the effects of NK3 receptor
desensitization on biphasic depolarizations caused by nicotine (1 mM).
A: amplitudes of fast and slow nicotine-induced
depolarizations in AH neurons (n = 12) before, during,
and after NK3 receptor desensitization. B:
amplitudes of fast and slow nicotine-induced depolarizations in S
neurons in which the slow depolarization was TTX insensitive. In these
neurons (n = 5), the slow depolarization was inhibited
during NK3 receptor desensitization by senktide.
C: amplitudes of fast and slow nicotine-induced
depolarizations in S neurons (n = 4) in which the slow
depolarization was blocked by TTX (0.3 µM). The slow response was
allowed to recover after TTX washout before testing the effect of
NK3 receptor desensitization. In this group of neurons, the
nicotine-induced slow depolarization was only slightly inhibited during
NK3 receptor desensitization. * P < 0.05, significantly different from control.
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In a separate experiment, the effect of senktide-induced
desensitization on responses to microejection of senktide or 5-HT was
determined in the presence of TTX (0.3 µM). Microejection of senktide
(0.3 µM) induced a depolarization in AH neurons (n = 3) that lasted several minutes. Only AH neurons (n = 5)
in which microejection of 5-HT (1 mM) induced biphasic depolarization
were used for comparison with nicotine-induced responses.
Depolarization induced by microejection of senktide was abolished
during senktide-induced desensitization, whereas the amplitudes of the
biphasic depolarization induced by 5-HT were not changed (Fig.
6).

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Fig. 6.
Senktide-induced desensitization is selective for
NK3 receptors. A: microapplication of senktide
(3-ms pulse, 0.3 µM) induced a slow depolarization in AH neurons
(n = 3), which was blocked after senktide (0.1 µM)
superfusion. B: microapplication of 5-hydroxytryptamine
(5-HT) (5-ms pulse, 1 mM) induced a biphasic depolarization in AH
neurons (n = 5). Senktide superfusion did not affect
the fast or slow 5-HT-induced depolarizations. C: summary
data for experiments shown in A and B.
* P < 0.05, significantly different by paired
t-test. Vertical scale bars, 20 mV (A) and 10 mV
(B); horizontal scale bars, 20 s.
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Effects of low-calcium and high-magnesium solution.
The effect of a low-calcium (1.2 mmol/l) and high-magnesium (12 mmol/l)
Krebs solution on the nicotine-induced slow depolarizations recorded
from AH neurons (n = 6) was determined. Superfusion
with low-calcium and high-magnesium Krebs solution
depolarized AH neurons. Despite this complication, superfusion with
low-calcium and high-magnesium Krebs solution abolished sEPSPs and the
slow, but not the fast, depolarization induced by nicotine (Fig.
7). The control amplitudes for fast and
slow depolarizations were 15 ± 4 and 15 ± 3 mV,
respectively. After superfusion with the low-calcium and high-magnesium
solutions, the fast and slow depolarization amplitudes were 10 ± 3 (P > 0.05) and
1 ± 2 mV (P < 0.05), respectively.

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Fig. 7.
In AH neurons, low-calcium and high-magnesium Krebs
solution abolished the slow depolarization induced by nicotine. TTX
(0.3 µM) was present throughout these recordings. A:
control. B: superfusion with a low-calcium (0.25 mM) and
high-magnesium (12 mM) Krebs solution selectively blocked the slow
depolarization. Vertical scale bars, 10 mV; horizontal scale bars,
20 s.
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DISCUSSION |
TTX-resistant responses.
Because nAChRs are ligand-gated ion channels, responses mediated
directly at nAChRs have short latencies and durations (8). Consistent with this expectation, nicotine-induced fast depolarizations recorded from myenteric neurons were due to a direct action of nicotine
at somatodendritic nAChRs localized to impaled neurons. However, the
time course of nicotine-induced slow depolarizations suggests that this
response is mediated indirectly through the action of a slow excitatory
neurotransmitter. The slow responses could be due to activation of
somatodendritic nAChRs on presynaptic neurons with subsequent action
potential-dependent release of slow excitatory neurotransmitters
(17, 35, 37). Indeed, TTX
inhibited the nicotine-induced slow depolarization in some S neurons.
However, the slow depolarization induced by nicotine was resistant to
TTX in AH neurons and in a subset of S neurons. Therefore, release of
the slow transmitter was action potential independent and nAChRs
mediating release of the slow transmitter must be near the release site.
A low-calcium and high-magnesium Krebs solution was used to reduce
calcium entry into nerve terminals to test the hypothesis that nicotine
acts near nerve terminals to release a slow neurotransmitter. In AH
neurons, superfusion with low-calcium and high-magnesium Krebs solution
abolished both slow synaptic transmission induced by focal stimulation
of interganglionic connectives and the slow (but not the fast)
depolarization caused by nicotine. Superfusion with low-calcium and
high-magnesium Krebs solution produced a depolarization of AH neurons
that mimicked the sEPSP. Depolarization under these conditions is
mediated by inhibition of a resting calcium-activated potassium
conductance that contributes to the resting membrane potential of AH
neurons (11, 30). Despite this complication,
the nicotine-induced slow depolarization in AH neurons was blocked. We
conclude that the nicotine-induced slow depolarization is blocked by
low-calcium and high-magnesium solutions because nicotine-induced
transmitter release is blocked.
Mediator of slow depolarizations induced by nicotine.
The typical response to microejection of nicotine was biphasic; the
slow response to nicotine was always depolarizing in AH neurons but
either depolarizing or hyperpolarizing in S neurons. Hyperpolarization
after nicotinic depolarization has been studied previously and is
mediated by a calcium-sensitive potassium conductance (36). An ACh-induced biphasic depolarization has been
described in myenteric neurons previously and is similar to that
induced by nicotine (29). In contrast with our findings,
however, the ACh-induced slow depolarization was blocked by muscarinic
receptor antagonists. The slow depolarizations reported here are not
mediated by muscarinic receptors because they 1) persisted
in the presence of scopolamine, 2) are induced by nicotine,
and 3) are blocked by the nAChR antagonist mecamylamine.
Therefore, we designed experiments to test the hypothesis that the
nicotine-induced slow depolarization is mediated by nAChRs on neurons
releasing a noncholinergic neurotransmitter.
Several transmitters mediate slow excitatory neurotransmission in the
myenteric plexus, including ACh (29), tachykinin peptides (8), and 5-HT (25). Nicotinic ACh
receptor-mediated release of ACh has been demonstrated
(10, 35, 37) but is unlikely to
have participated in nicotine-induced slow depolarization as described
above. However, postsynaptic activation of NK3 receptors by
nAChR-mediated release of tachykinin peptides (15,
16) could contribute to the slow depolarization in
myenteric neurons. Activation of NK3 receptors is a
mechanism of slow excitatory neurotransmission in the myenteric plexus,
and we used continuous senktide application to induce selective
NK3 receptor desensitization. Selective NK3
receptor desensitization was confirmed by demonstrating that the
response to microejection of senktide was completely inhibited during
senktide superfusion, whereas slow depolarizations caused by 5-HT were
unaffected. In addition, fast depolarizations caused by nicotine and
5-HT were also unaffected during senktide superfusion. However,
nicotine-induced slow depolarizations were blocked during senktide
superfusion in all AH neurons and a subset of S neurons. Therefore, the
TTX-resistant slow depolarization induced by nicotine in AH neurons is
dependent on NK3 receptor activation. In some S neurons,
the nicotine-induced slow response was TTX sensitive and the slow
depolarization induced by nicotine was less affected by NK3
receptor desensitization. It is likely that in these neurons the slow
depolarization induced by nicotine is mediated by neurotransmitters in
addition to tachykinin peptides acting at NK3 receptors.
Pharmacology of nAChR-mediated responses.
The pharmacology of nAChRs of different subunit combinations has been
described previously (1, 5, 23,
27). Comparing responses induced by cytisine to those
induced by nicotine may discriminate receptors differing in
-subunit
composition (4, 5, 23,
33). Cytisine produces responses similar to nicotine when
nAChRs contain the
4-subunit, but cytisine is a weak agonist at
nAChRs containing the
2-subunit. Comparisons made in the present study reveal two notable findings: 1) AH neurons respond to
nicotine but not to cytisine, and 2) fast depolarizations
induced by nicotine and cytisine in S neurons were identical, whereas
cytisine never caused a slow depolarization. The equivalence of fast
depolarizations induced by cytisine and nicotine in S neurons suggests
that the nAChR(s) mediating this response contains the
4-subunit. In
contrast, only nicotine induced fast depolarizations in AH neurons,
suggesting that nAChRs in AH neurons do not contain
4-subunits.
Because cytisine never induced slow depolarizations, nAChRs mediating this response also do not contain
4-subunits. Therefore,
somatodendritic nAChRs on AH neurons and nAChRs on nerve terminals
releasing slow excitatory neurotransmitter(s) probably do not contain
4-subunits. This conclusion is consistent with the finding that the
pharmacological properties of TTX-resistant, nAChR-mediated
noncholinergic transmission to the longitudinal smooth muscle of guinea
pig ileum are characteristic of nAChRs containing
2-subunits
(9).
Postsynaptic nAChRs.
The most consistent response to nicotine was a fast depolarization that
was TTX resistant and occurred in the presence of scopolamine. The
receptors that mediate these fast depolarizations are nAChRs localized
to the somatodendritic region of postsynaptic neurons (20,
31). fEPSPs within the myenteric plexus are mediated partly by nAChRs (22). In the present study, fEPSPs were
recorded from S but not AH neurons. It is surprising, then, that a fast depolarization was induced by nicotine in most AH neurons. Using the
amplitude of single channel and whole cell currents activated by ACh,
it has been determined that myenteric AH neurons have fewer
somatodendritic nAChRs than S neurons (40), and this could account for the smaller nicotine-induced fast depolarizations in AH
neurons compared with responses in S neurons. The smaller response to
nicotine in AH neurons is consistent with immunofluorescence studies
demonstrating that nAChR immunoreactivity in S neurons is very dense
and is present in clusters on the somatodendritic region, contrasting
with nAChR immunoreactivity in AH neurons, which is less intense and
more diffuse (20).
Summary and conclusions.
TTX-resistant, nicotine-induced slow depolarizations of myenteric
neurons are mediated by stimulation of nAChRs localized to tachykinin
peptide-containing nerve terminals. Myenteric neurons contain and
release substance P (SP) and neurokinin A (NKA) (16), and
one or both of these peptides are the likely mediators of the
nicotine-induced slow response. The data presented here indicate that
SP and NKA act at postsynaptic NK3 receptors on S and AH neurons. AH neurons are intrinsic primary afferent neurons and provide
synaptic inputs to other AH neurons, interneurons, and motoneurons
(7). AH neurons connect with other AH neurons via slow
excitatory synapses (21). AH neurons express
NK3 receptors (18) and contain choline
acetyltransferase and tachykinin peptides (6), but ACh
released from the nerve terminals of AH neurons does not appear to play
a postsynaptic role at connections between AH neurons. The data
presented here suggest that ACh released at synaptic connections
between AH neurons could act presynaptically, at cytisine-insensitive
nAChRs, in a positive feedback mechanism to release SP and NKA (Fig.
8A). Somatodendritic
nAChRs are present on myenteric neurons. Because AH neurons do not
receive fast cholinergic synaptic input, somatodendritic nAChRs on AH
neurons must be extrasynaptic; these nAChRs are cytisine insensitive.
Because cytisine did not elicit slow responses in any neuron or
fast responses in AH neurons, nerve terminal nAChRs and those present
at the somatodendritic region of AH neurons may differ in subunit
composition from those on the somatodendritic region of S neurons. S
neurons in the myenteric plexus are interneurons and motoneurons, and
there are several classes of each of these functional groups
(6). The specific subset of S neurons receiving slow
excitatory input activated by nerve terminal nAChRs is unknown, but AH
neurons do make synaptic connections with both interneurons and
motoneurons (18). ACh released by AH neurons at
synaptic connections with S neurons acts postsynaptically to
mediate fEPSPs and sEPSPs (29) and may also act
presynaptically to facilitate SP and NKA release (Fig.
8B).

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|
Fig. 8.
nAChRs localized to nerve terminals and to the
somatodendritic region of AH and S neurons. A: nerve
terminal nAChRs mediate the release of substance P/neurokinin A
(SP/NKA), which act at NK3 receptors (arrow) to cause slow
depolarizations in AH neurons. As nerve terminal nAChRs are localized
near release sites, responses mediated by these receptors are TTX
resistant. Cytisine-insensitive nAChRs (shaded symbols) are localized
to nerve terminals and to extrasynaptic regions of AH neurons. These
receptors would be activated by nicotine (arrows). It is proposed that
ACh released from these nerve terminals stimulates the nerve terminal
nAChRs (arrow with ?). B: because S neurons (but not AH
neurons) receive fast synaptic input via nAChRs, these receptors must
be localized to synaptic regions (open symbol). Synaptically located
nAChRs on S neurons are nicotine and cytisine sensitive (indicated by
arrow). S neurons receive slow synaptic input mediated by SP/NKA acting
at NK3 receptors (arrows). Cytisine-insensitive nAChRs
(shaded symbol) are localized to nerve terminals providing slow
synaptic input to some S neurons. Nicotine activates these receptors
(arrow). It is also proposed that ACh released from these nerve
terminals activates the cytisine-insensitive, nerve terminal nAChRs
(arrow with ?).
|
|
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Neurological
Disorders and Stroke Grants T32-NS-07279 and NS-33289 and National Institute of Diabetes and Digestive and Kidney Diseases Grant 1 F32
DK-09935-01.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: J. J. Galligan, B440 Life Sciences Bldg., Dept. of Pharmacology and Toxicology, Michigan State Univ., East Lansing, MI 48824-1317 (E-mail:
galliga1{at}pilot.msu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 20 December 1999; accepted in final form 7 March 2000.
 |
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