Muscarinic receptors couple to modulation of nicotinic ACh receptor desensitization in myenteric neurons
Erika N. Brown and
James J. Galligan
Department of Pharmacology and Toxicology and The Neuroscience Program,
Michigan State University, East Lansing, Michigan 48824
Submitted 31 January 2003
; accepted in final form 26 February 2003
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ABSTRACT
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Signaling mechanisms coupled to activation of different neurotransmitter
receptors interact in the enteric nervous system. ACh excites myenteric
neurons by activating nicotinic ACh receptors (nAChRs) and muscarinic
receptors expressed by the same neurons. These studies tested the hypothesis
that muscarinic receptor activation alters the functional properties of nAChRs
in guinea pig small intestinal myenteric neurons maintained in primary
culture. Whole cell patch-clamp techniques were used to measure inward
currents caused by ACh (1 mM) or nicotine (1 mM). Currents caused by ACh and
nicotine were blocked by hexamethonium (100 µM) and showed complete cross
desensitization. The rate and extent of nAChR desensitization was greater when
recordings were obtained with ATP/GTP-containing compared with ATP/GTP-free
pipette solutions. These data suggest that ATP/GTP-dependent mechanisms
increase nAChR desensitization. The muscarinic receptor antagonist scopolamine
(1 µM) decreased desensitization caused by ACh but not by nicotine, which
does not activate muscarinic receptors. Phorbol 12,13-dibutyrate (10100
nM), an activator of protein kinase C (PKC), but not 4-
-phorbol
12-myristate 13-acetate (a PKC inactive phorbol ester), increased nAChR
desensitization caused by ACh and nicotine. Forskolin (1 µM), an activator
of adenylate cyclase, increased nAChR desensitization, but this effect was
mimicked by dideoxyforskolin, an adenylate cyclase inactive forskolin analog.
These data indicate that simultaneous activation of nAChRs and muscarinic
receptors increases nAChR desensitization. This effect may involve activation
of a PKC-dependent pathway. These data also suggest that nAChRs and muscarinic
receptors are coupled functionally through an intracellular signaling pathway
in myenteric neurons.
enteric nervous system; electrophysiology; intracellular signaling
IT HAS BEEN KNOWN for some time that "cross-talk"
between intracellular signaling pathways activated by different classes of
cell surface receptors is a mechanism for integration of information coming
into individual cells, including neurons
(12,
24). Typically, these
interactions involve G protein-linked receptors and two or more intracellular
signaling pathways composed of multiple enzymatic processes and diffusible
signaling molecules (12,
23). Recently, it has been
shown that there are interactions between different ligand-gated ion channels
that result in inhibition of signals mediated by each receptor type. This
interaction does not involve diffusible second messenger molecules but instead
may involve direct protein-protein interaction. For example, in myenteric
neurons, nicotinic ACh receptors (nAChRs) and P2X receptors exhibit
cross inhibition when these receptors are activated simultaneously
(9,
26). In the submucosal plexus,
a similar direct inhibitory interaction occurs between P2X
receptors and 5-HT3 receptors
(1).
ACh, acting at nAChRs and at M1 muscarinic receptors, is an
important excitatory neurotransmitter in the enteric nervous system (ENS; see
Refs. 6,
7,
15,
16). M1 muscarinic
receptors are G protein-coupled receptors that link to activation of
phospholipase C (PLC) and generation of inositol trisphosphate and
diacylglycerol (DAG; see Ref.
19). DAG activates protein
kinase C (PKC), and PKC can phosphorylate a variety of intracellular targets,
including ion channels (21).
M1 muscarinic receptors mediate some slow excitatory postsynaptic
potentials (sEPSPs) in the ENS
(17,
18). Furthermore, drugs that
inhibit the PLC-PKC signaling pathways can inhibit sEPSPs, whereas drugs that
activate this system can mimic sEPSPs
(2). The sEPSPs are associated
with a long-lasting increase in excitability. ACh acting at nAChRs mediates
most fast excitatory postsynaptic potentials (fEPSPs) in the ENS. There are
neurons in the ENS that receive dual excitatory synaptic input mediated by ACh
acting at nAChRs and muscarinic receptors
(17,
18). The long-lasting increase
in excitability subsequent to muscarinic receptor activation can potentiate
fEPSPs mediated by nAChRs on the same neuron. This is a mechanism by which two
different receptors can interact in the ENS. However, it is also known that
nAChRs are targets for phosphorylation, which alters desensitization and other
functional properties of the nAChR
(4,
5,
8,
20). It is not known if the
functional properties of nAChRs in the ENS can be modified after activation of
intracellular signaling pathways. In the present study, we tested the
hypothesis that simultaneous activation of nAChRs and muscarinic receptors is
associated with an alteration in desensitization of nAChRs expressed by the
same myenteric neurons. Data from these studies would provide evidence that
different types of receptor for the same transmitter can interact functionally
and therefore modify interneuronal signaling.
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MATERIALS AND METHODS
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Primary culture of myenteric neurons. The procedures used in these
studies are similar to previously published methods using similar preparations
(9,
26,
27). Newborn (1 to 2 days old)
guinea pigs were anesthetized by halothane inhalation, stunned, and
exsanguinated by severing major neck blood vessels. The entire small intestine
was placed in 4°C Krebs bicarbonate buffer of the following composition
(in mM): 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2
NaH2PO4, 25 NaHCO3, and 11 glucose. The
longitudinal muscle myenteric plexus was stripped free using a moist cotton
swab and then was cut into 5-mm pieces. Dissected tissues were divided into
two equal aliquots, and each aliquot was transferred to 1 ml of
sterile-filtered Krebs solution containing 1,600 units of trypsin (Sigma
Chemical, St. Louis, MO) for 30 min at 37°C. After trypsin incubation,
tissues were triturated 30 times through a fire-polished Pasteur pipette and
centrifuged at 900 g for 10 min using a bench-top centrifuge. The
supernatant was discarded, and the pellet was resuspended in 1 ml Krebs
solution containing 4,000 units crab hepatopancreas collagenase
(Calbiochem-Novabiochem, La Jolla, CA). The suspension was triturated using a
fire-polished Pasteur pipette and then centrifuged for 10 min. The pellet was
resuspended in Eagle's MEM containing 10% FBS, 10 µM gentamycin, 100 U/ml
penicillin, and 50 mg/ml streptomycin (all from Sigma). Aliquots (200 µl)
were plated on sterile, poly-L-lysine (mol wt 30,00070,000;
Sigma)-coated glass cover slips placed in 35-mm plastic culture dishes
containing 3 ml MEM. After 2 days of incubation, cytosine arabanoside (10
µM) was added to the MEM to limit smooth muscle and fibroblast
proliferation. Cultures were maintained at 37°C in a tissue culture
incubator containing a 5% CO2 atmosphere. Cultures were maintained
up to 2 wk after plating with medium replacement every 2 days.
Whole cell patch-clamp recordings. The glass cover slips
containing neurons was removed from the culture dishes and placed in a
Plexiglas recording chamber (3 ml volume) with a glass bottom. The recording
chamber was placed on the stage of an inverted microscope, and neurons were
viewed using Hoffman modulation contrast optics. The extracellular solution
was the standard Krebs solution described above. The Krebs solution was
superfused through the recording chamber at a flow rate of 4 ml/min.
Fire-polished patch-clamp pipettes were fabricated from borosilicate glass
capillary tubes (World Precision Instruments, Sarasota, FL). The fire-polished
pipettes had a tip resistance of 37 M
. Electrode liquid junction
potentials and series resistance were compensated. The standard intracellular
(pipette) solution for patch-clamp recording was as follows (in mM): 160 CsCl,
2.9 MgCl2, 10 EGTA, 10 HEPES, 0.5 ATP, and 0.25 GTP; pH was
adjusted to 7.4 using CsOH. The CaCl2-to-EGTA ratio yielded a
resting level of free Ca2+ concentration of <100 nM.
Experiments were performed using an Axopatch 200B patch-clamp amplifier, a
Digidata 1200 analog-to-digital converter, and pCLAMP 6.01 programs for
acquisition, storage, and analysis of data (Axon Instruments, Burlingame, CA).
Data were filtered at 2 kHz using a four-pole Bessel filter (Warner
Instruments, New Haven, CT) and were digitized at a rate of 5 kHz and stored
on the computer hard drive. Unless otherwise indicated, the holding potential
was 60 mV.
Drug application. Drugs were applied via flow tubes gated by
computer-controlled solenoid valves. Four of these flow-through tubes were
glued together at the tip, and each was connected via polyethylene tubing to a
reservoir syringe (10 ml) containing a known concentration of drug. The
reservoir platform was positioned above the recording chamber so that drugs
were gravity fed through the flow tubes. Platform height was adjusted to
provide a flow rate of 0.1 ml/min. The flow tube array was mounted on a
micromanipulator, and drugs were applied by positioning the flow tubes over
the neuron. This method permitted rapid adjustments between control and
drug-containing solution (equilibrium <100 ms).
Drugs. All drugs were obtained from Sigma Chemical. ACh and
nicotine were prepared as 1 M stock solutions in deionized water. Phorbol
12,13-dibutyrate (PDBu), 4-
-PMA, forskolin, and dideoxyforskolin (DDF)
were prepared as stock solutions in 50% (vol/vol) ethanol-deionized water and
were then diluted to working concentrations in Krebs buffer. The final ethanol
concentration in the buffer did not exceed 0.05%.
Statistics. All data were expressed as means ± SE, and
n values refer to the number of neurons from which data were
obtained. Differences between treatment groups were established using
Student's t-test for paired and unpaired data, and one-way ANOVA and
the Student-Newman-Keuls test for multiple comparisons. A P value
<0.05 was taken as the level of statistical significance.
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RESULTS
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ACh and nicotine activate nAChRs in myenteric neurons. ACh (1 mM) caused
rapidly developing and desensitizing inward currents in >90% of neurons
tested (Fig. 1A). ACh
caused inward currents that did not decline in amplitude over 20 min, the time
course of most experiments done here. In eight neurons, the initial ACh
response was 1.3 ± 0.3 nA, whereas at 20 min the response amplitude was
1.5 ± 0.3 nA (P > 0.05). These data are similar to those
published previously (27).
ACh-induced currents were the result of activation of nAChRs because they were
mimicked by nicotine (1 mM; Fig.
1A). In addition, ACh- and nicotine-induced currents were
blocked completely and reversibly by the nAChR antagonist hexamethonium
(Fig. 1B). ACh and
nicotine also activated the same receptors as responses caused by nicotine,
and ACh exhibited cross desensitization. In these experiments, ACh (1 mM) was
applied for 2 s, and, after a 5-min recovery, nicotine was applied for 2 s.
After an additional 5 min of recovery, ACh was applied again for 7 s, and the
inward current decayed to a steady-state level during this time (see below).
Nicotine was applied again at this point, and the nicotine-induced current was
reduced significantly (Fig.
1C). After a 5-min recovery period, the ACh response
returned to near its initial amplitude
(Fig. 1C).
Modulation of nAChR desensitization during muscarinic receptor
activation. ACh-induced currents decayed in the continued presence of
agonist, and the ACh-induced current reached a steady-state level within 7 s
(Fig. 2, A and
B). The time course of desensitization was quantified as
the time to half decay (T1/2) of the ACh current in each
neuron. The average time course for the decay of the ACh response under
different recording conditions is presented in
Fig. 2B. The ACh
current amplitude was measured at the peak response
(Ipeak), and this was set as time 0. The
amplitude of the ACh current was then measured at 1-s intervals
(Itime) until the current reached a steady-state level
(Isteady state; Fig.
2A). The amount of nAChR desensitization was quantified
by calculating the ratio of Isteady state versus
Ipeak in the same neuron
(Fig. 2A). ACh-induced
currents declined by one-half in <3 s and reached a steady-state level of
77% of the initial peak current (Fig.
2B; Table
1).

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Fig. 2. A: representative recording of an inward current caused by ACh.
ACh-induced responses desensitized to a steady-state level
(Isteady state) within 7 s. ACh was applied during the
period indicated by the bar on top. B: time course of desensitization
of ACh responses under control recording conditions (drug-free extracellular
solution, with 1 mM ATP and 0.25 mM GTP in the whole cell pipette solution)
when ATP and GTP were omitted from the pipette solution (without ATP/GTP) and
when scopolamine (1 µM) was added to the extracellular solution (with ATP
and GTP in the pipette solution). The rate and extent of desensitization were
reduced either by omitting ATP and GTP from the pipette solution or by adding
scopolamine to the extracellular solution (see
Table 1 for statistical
analysis).
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Desensitization of nAChRs can be modulated by a variety of intracellular
signaling pathways and receptor phosphorylation
(20). Therefore, we tested the
effects of removing ATP and GTP from the pipette solution on the rate and
extent of nAChR desensitization in myenteric neurons. The peak amplitude of
the ACh-induced current recorded using an ATP/GTP-free pipette solution was
not different from the peak amplitude obtained using ATP/GTP-containing
pipette solutions (Table 1).
Furthermore, the ACh-induced current recorded without ATP/GTP in the recording
pipette was stable in amplitude over a 20-min recording period. When an
ATP/GTP-free pipette solution was used, the initial ACh-induced current was
1.5 ± 0.5 nA, whereas 20 min after establishing the whole cell
recording the ACh response amplitude was 1.4. ± 0.5 nA (n = 6,
P > 0.05). When recordings were obtained using an ATP/GTP-free
pipette solution, the T1/2 for decay of the ACh-induced
current increased to 4 s, whereas the steady-state current was
60% of the
initial peak current (Fig.
2B and Table
1).
Myenteric neurons express muscarinic cholinergic receptors that can couple
to activation of one or more intracellular signaling pathways, leading to
excitation of myenteric neurons. It is possible that simultaneous activation
of muscarinic receptors by ACh could modulate nAChR function. Therefore, we
tested the effects of scopolamine (1 µM), a muscarinic receptor antagonist,
on the rate and extent of nAChR desensitization recorded using an
ATP/GTP-containing pipette solution. The peak amplitude of the ACh-induced
current recorded in the presence of scopolamine was not different from that
recorded under control conditions (Table
1). Furthermore, it was found that scopolamine decreased both the
rate and extent of nAChR desensitization
(Fig. 2B and
Table 1). Scopolamine also
increased the rate of recovery from desensitization of nAChRs caused by ACh.
This conclusion is based on studies in which ACh (1 mM) was applied for 7 s to
cause nAChR desensitization, and ACh was reapplied at several time points
after the initial response to determine the time course of recovery from
desensitization (Fig.
3A). Under control recording conditions, the ACh response
had a time to one-half recovery of 0.1 ± 0.04 min (n = 7),
whereas in the presence of scopolamine (1 µM) the time to one-half recovery
was reduced to 0.03 ± 0.01 min (n = 6, P < 0.05;
Fig. 3B).

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Fig. 3. Blockade of muscarinic receptors increases the rate of recovery from
desensitization caused by ACh. A: representative recordings, under
control conditions, of inward currents caused by a 7-s application of ACh at
time 0 (Iinitial) and at several time points
after the initial application (Itime). B: time
course of recovery of nAChRs from desensitization caused by a 7-s application
of ACh in the absence (Control) and presence of scopolamine (1 µM). Data
points are means ± SE of measurements made on 7 neurons for each
group.
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Scopolamine does not alter nAChR desensitization caused by
nicotine. It is possible that scopolamine could alter the rate and extent
of nAChR desensitization through mechanisms other than blockade of muscarinic
receptors. To rule out this possibility, we tested the effects of scopolamine
on currents caused by nicotine, which will not activate muscarinic receptors.
It was found that both the time course and extent of nAChR desensitization
caused by nicotine were unaffected by scopolamine
(Fig. 4A and
Table 1). Scopolamine also did
not alter the time course of recovery from desensitization caused by nicotine
(Fig. 4B). The time to
one-half recovery from nicotine desensitization under control conditions was
0.67 ± 0.1 min, whereas in the presence of scopolamine this value was
0.51 ± 0.1 min (P > 0.05).

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Fig. 4. Scopolamine does not alter desensitization or recovery from desensitization
of nAChRs caused by nicotine. A: time course or extent of nAChR
desensitization caused by a 7-s application of nicotine is not changed by
scopolamine (1 µM). Data are means ± SE of measurements made in 7
neurons in the control group and 9 neurons in the scopolamine group.
B: recovery from desensitization caused by nicotine is not altered by
scopolamine. Data are means ± SE of measurements made in 8 neurons in
the control group and 10 neurons in the scopolamine group.
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Bethanechol increases nAChR desensitization. The data presented
above indicate that muscarinic receptor activation can increase nAChR
desensitization. This issue was investigated further by using nicotine to
selectively activate and desensitize nAChRs in the absence and presence of the
muscarinic receptor agonist bethanechol (BeCh; 100 µM). It was found that
under control conditions, the amount of desensitization in the same neuron was
stable on successive nicotine applications
(Fig. 5, left).
However, when neurons were treated with BeCh, the amount of nAChR
desensitization caused by nicotine increased significantly
(Fig. 5, right). BeCh
did not change the amplitude of the peak nicotine-induced current. The control
peak nicotine current was 1.7 ± 0.5 nA, whereas in the presence of BeCh
this value was 1.8 ± 0.6 nA (P > 0.05, n = 5).

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Fig. 5. Muscarinic receptor agonist bethanechol (BeCh) increases nAChR
desensitization caused by nicotine. Nicotine caused similar amounts of nAChR
desensitization during successive applications in the absence or presence of
the BeCh vehicle (H2O; n = 10). In a separate group of
neurons (n = 5), addition of BeCh to the extracellular medium
increased desensitization caused by a second application of nicotine.
*Significantly different from control (P < 0.05).
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Phorbol ester-induced modulation of nAChR desensitization.
Myenteric neurons express M1-type muscarinic receptors that couple
via Gq to the phosphatidylinositol (PI)-dependent signaling pathway
and activation of PKC (19).
Therefore, PDBu was used to activate PKC to investigate a role for this enzyme
in modulating nAChR desensitization. ACh (1 mM; in the presence of 1 µM
scopolamine) or nicotine (1 mM) was applied after pretreating neurons with
vehicle (0.05% ethanol) or PDBu (10300 nM). It was found that PDBu
increased the maximum nAChR desensitization occurring during a 7-s application
of ACh (Fig. 6A) or
nicotine (Fig. 6B).
However, PDBu did not change the amplitude of the peak ACh- or
nicotine-induced current (Table
2). To test that PDBu was not acting via a PKC-independent
mechanism to alter nAChR desensitization, a PKC-inactive phorbol ester analog,
4-
-PMA, was tested. 4-
-PMA (10300 nM) did not alter the
amount of nAChR desensitization caused by either ACh
(Fig. 6A) or nicotine
(Fig. 6B).
Furthermore, 4-
-PMA did not change the peak ACh- or nicotine-induced
currents (Table 2).
Effects of forskolin on nAChR desensitization. The next set of
experiments tested the possibility that adenylate cyclase-dependent mechanisms
could also modulate nAChR desensitization. These studies used forskolin (1
µM) to stimulate adenylate cyclase. Forskolin did not alter the peak
current amplitude caused by either ACh or nicotine
(Table 2). Forskolin increased
the maximum nAChR desensitization caused by both ACh (scopolamine present) and
nicotine (Fig. 7, A and
B). However, it was found that DDF, a forskolin analog
that does not activate adenylate cyclase, also caused an apparent increase in
nAChR desensitization caused by ACh (Fig.
7A) and nicotine (Fig.
7B). These data suggest that forskolin decreased the
steady-state nAChR current at least partly through an adenylate
cyclase-independent mechanism.

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Fig. 7. Forskolin and dideoxyforskolin (DDF) increase nAChR desensitization.
A: amount of nAChR desensitization caused by ACh is increased after
treatment with forskolin and the adenylate cyclase inactive forskolin analog
DDF. Scopolamine (1 µM) was present in the extracellular solution
throughout these studies. B: nicotine-induced nAChR desensitization
is also increased after treatment with forskolin and DDF. In A and
B, data are expressed as the means ± SE percent change in the
amount of desensitization caused by a 7-s application of ACh or nicotine
before and after treatment with vehicle (0.05% ethanol), forskolin, or DDF.
*Significantly different from the amount of desensitization occurring before
forskolin or DDF treatment.
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DISCUSSION
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ACh is the principal excitatory neurotransmitter in the ENS, and ACh acts
at nAChRs and M1 muscarinic receptors expressed by the same neurons
to cause synaptic excitation
(6,
7,
17,
18). The different time course
and signaling pathways coupled to nAChRs and M1 muscarinic
receptors provide a basis for integration of synaptic input and output of the
neuron receiving the mixed cholinergic input. In addition, the data from the
present study indicate that activation of muscarinic receptors can alter the
time course of responses mediated by nAChRs on the same neurons.
nAChR-mediated inward currents in myenteric neurons. Previous
studies have shown that ACh acts at nAChRs to cause an inward current in
myenteric neurons maintained in primary culture
(24). These results were
confirmed in the present study where it was shown that ACh caused a rapidly
developing and desensitizing inward current that was blocked completely by the
nAChR antagonist hexamethonium. The ACh-induced current was mimicked by the
selective nAChR agonist, nicotine, and the nicotine-induced current was also
blocked by hexamethonium. In addition, the ACh- and nicotine-induced currents
cross-desensitized, indicating that these responses were mediated at the same
population of nAChRs. The amplitude of the ACh-induced current is stable over
the period of at least 20 min (see also Ref.
27), and omitting ATP and GTP
from the recording pipette solution did not alter the peak ACh current over
this time period. However, it was found that the time course and extent of
nAChR desensitization were altered by omitting ATP and GTP from the pipette
solution. This result suggests that ATP- and or GTP-dependent processes modify
nAChR desensitization. Because ATP and GTP are substrates for kinase-dependent
reactions, phosphorylation of the nAChR is a mechanism by which
desensitization can be modulated
(8,
16).
Muscarinic receptor activation increases nAChR desensitization.
Myenteric neurons express both nAChRs and M1 muscarinic receptors
(6,
7,
17,
18). M1 muscarinic
receptors are G protein-coupled receptors that are linked to activation of PLC
and PI hydrolysis (19). PI
hydrolysis can lead to release of DAG and PKC activation
(21). The data from the
present study indicate that simultaneous activation of nAChRs and muscarinic
receptors increases the amount of nAChR desensitization occurring during a 7-s
application of ACh. This conclusion is based on the result showing that the
addition of scopolamine, a muscarinic receptor antagonist, to the
extracellular solution decreased the rate and extent of nAChR desensitization
caused by ACh. Therefore, simultaneous activation of muscarinic receptors
modulates the function of nAChRs expressed by the same neuron. M1
muscarinic receptors mediate sEPSPs in the myenteric plexus
(17,
18). The
M1-mediated sEPSP is the result of an inhibition of potassium
channels that are open near the resting membrane potential
(18). The pipette solution
used in the present study contained cesium ions to block all potassium
channels. Therefore, changes in resting conductance of the neurons would not
contribute to the muscarinic receptor-mediated change in nAChR
desensitization.
Scopolamine could have effects on myenteric neurons that are independent of
muscarinic receptor antagonism. For example, scopolamine could interact
directly with the nAChR ion channel to alter its functional properties
(14). To test the possibility
that scopolamine altered nAChR desensitization by a muscarinic
receptor-independent mechanism, nicotine was used as an nAChR agonist. Because
nicotine does not activate muscarinic receptors, scopolamine should not alter
nAChR desensitization if it acts only at muscarinic receptors. It was found
that the rate and extent of nicotine-induced nAChR desensitization was
unaffected by scopolamine; therefore, scopolamine did not interact with the
nAChR via a muscarinic receptor-independent mechanism.
The data discussed above suggest that simultaneous activation of muscarinic
receptors alters nAChR desensitization. To test this hypothesis more directly,
nicotine and the muscarinic receptor agonist BeCh were applied simultaneously
to individual neurons. When BeCh was coapplied with nicotine, the amount of
nAChR desensitization increased compared with the level occurring during the
control recording. Taken together, these data indicate that activation of
muscarinic receptors increases nAChR desensitization during simultaneous
activation of the two cholinergic receptors.
PKC activation modulates nAChR desensitization. Because
M1 muscarinic receptors couple to generation of DAG
(19) and DAG activates PKC
(21), we tested the effects of
the PKC activator PDBu on nAChR desensitization. PDBu caused a
concentration-dependent increase in the amount of nAChR desensitization caused
by ACh (in the presence of scopolamine to block muscarinic receptors) and by
nicotine. However, PDBu can alter nAChR desensitization by an action that is
independent of PKC activation
(15). To test this
possibility, we used 4-
-PMA, a phorbol ester that does not activate
PKC, in our desensitization protocol. 4-
-PMA, in a concentration range
identical to that used for PDBu, did not alter nAChR desensitization caused by
either ACh or nicotine. These data suggest that PDBu-induced changes in nAChR
desensitization were mediated by PKC activation. Although PDBu increased nAChR
desensitization, the phorbol ester did not alter the peak currents caused by
either ACh or nicotine. This result indicates that PKC activation does not
alter the affinity of the nAChR for agonist. In rat cardiac parasympathetic
neurons, vasoactive intestinal peptide and pituitary adenylate
cyclase-activating peptide (PACAP) both cause an increase in peak
nAChR-mediated currents, and this increase in response has been attributed to
an increase in affinity of the nAChR for ACh
(11). Vasoactive intestinal
peptide and PACAP receptors modify nAChR function through a mechanism that is
independent of a diffusible second messenger but may use a membrane-delimited
but G protein-dependent pathway
(11). Also, in rat cardiac
parasympathetic neurons, receptors for substance P couple to an inhibition of
a subset of nAChRs. This mechanism also does not involve a diffusible,
cytosolic second messenger, and it may involve a change in desensitization or
the conducting properties of the nAChR ion channel
(3). Both of these mechanisms
are different from the pathway activated by muscarinic receptors in myenteric
neurons where the increase in PKC activity may alter the transition of the
nAChR from the open and activated state to one or more desensitized states
(20). Because peak currents
are not altered by muscarinic receptor activation or treatments that mimic
down-stream signaling, the change in function of the nAChR is likely to be
independent of changes in ACh binding.
Regulation of nAChR desensitization by PKC is not unique to myenteric
neurons. Earlier studies done on desensitization of nAChRs at the
neuromuscular junction and in sympathetic ganglia demonstrated that PKC
activation increases nAChR desensitization
(4,
5,
10,
16). Therefore, regulation of
desensitization by PKC is a general mechanism by which the function of nAChRs
can be modulated. Protein kinase A (PKA) has also been shown to phosphorylate
nAChRs and alter desensitization of these receptors
(16,
20). On the basis of these
previous data, we tested the effects of forskolin, an activator of adenylate
cyclase that can lead to stimulation of PKA. Forskolin increased nAChR
desensitization to a similar degree as PDBu; however, forskolin can act as an
nAChR ion channel blocker
(25). Therefore, we tested the
adenylate cyclase-inactive forskolin analog DDF for its effects on nAChR
desensitization. It was found that forskolin and DDF caused similar increases
in nAChR desensitization, leading to the conclusion that the effects of
forskolin on desensitization are likely to be independent of adenylate cyclase
and PKA activation.
Functional significance. There are many examples of cotransmitters
released simultaneously by autonomic nerves producing responses in synaptic
targets that differ from responses produced by the individual transmitters
acting alone (13). These
effects can be mediated presynaptically, resulting in either a facilitation or
inhibition of transmitter release, or transmitter interactions can be
postsynaptic where one transmitter may sensitize the target cell to the
actions of the second transmitter
(13). In addition, it has been
shown previously that nicotinic and P2X receptors and
P2X and 5-HT3 receptors exhibit cross inhibition when
these receptors are activated simultaneously
(1,
9,
27). The data from the present
paper indicate that there is a potential for postsynaptic modulation of
responses to a single transmitter (ACh) mediated by different classes
(nicotinic and muscarinic) of receptor for that transmitter. Muscarinic
receptor-mediated acceleration of the desensitization rate of nAChRs could
function to limit postsynaptic excitation during periods of high cholinergic
nerve activity. The delay in recovery from desensitization of the nAChR would
serve a similar function.
 |
FOOTNOTES
|
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Address for reprint requests and other correspondence: J. J. Galligan, Dept.
of Pharmacology and Toxicology, Life Science B440, Michigan State Univ., East
Lansing, MI 48824 (E-mail:
galliga1{at}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. Section 1734
solely to indicate this fact.
 |
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