Department of Physiology and Pharmacology, University of Queensland, Brisbane, QLD 4072, Australia
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ABSTRACT |
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Cuevas, Javier and David J. Adams. Substance P Preferentially Inhibits Large Conductance Nicotinic ACh Receptor Channels in Rat Intracardiac Ganglion Neurons. J. Neurophysiol. 84: 1961-1970, 2000. The effects of substance P (SP) on nicotinic acetylcholine (ACh)-evoked currents were investigated in parasympathetic neurons dissociated from neonatal rat intracardiac ganglia using standard whole cell, perforated patch, and outside-out recording configurations of the patch-clamp technique. Focal application of SP onto the soma reversibly decreased the peak amplitude of the ACh-evoked current with half-maximal inhibition occurring at 45 µM and complete block at 300 µM SP. Whole cell current-voltage (I-V) relationships obtained in the absence and presence of SP indicate that the block of ACh-evoked currents by SP is voltage independent. The rate of decay of ACh-evoked currents was increased sixfold in the presence of SP (100 µM), suggesting that SP may increase the rate of receptor desensitization. SP-induced inhibition of ACh-evoked currents was observed following cell dialysis and in the presence of either 1 mM 8-Br-cAMP, a membrane-permeant cAMP analogue, 5 µM H-7, a protein kinase C inhibitor, or 2 mM intracellular AMP-PNP, a nonhydrolyzable ATP analogue. These data suggest that a diffusible cytosolic second messenger is unlikely to mediate SP inhibition of neuronal nicotinic ACh receptor (nAChR) channels. Activation of nAChR channels in outside-out membrane patches by either ACh (3 µM) or cytisine (3 µM) indicates the presence of at least three distinct conductances (20, 35, and 47 pS) in rat intracardiac neurons. In the presence of 3 µM SP, the large conductance nAChR channels are preferentially inhibited. The open probabilities of the large conductance classes activated by either ACh or cytisine were reversibly decreased by 10- to 30-fold in the presence of SP. The single-channel conductances were unchanged, and mean apparent channel open times for the large conductance nAChR channels only were slightly decreased by SP. Given that individual parasympathetic neurons of rat intracardiac ganglia express a heterogeneous population of nAChR subunits represented by the different conductance levels, SP appears to preferentially inhibit those combinations of nAChR subunits that form the large conductance nAChR channels. Since ACh is the principal neurotransmitter of extrinsic (vagal) innervation of the mammalian heart, SP may play an important role in modulating autonomic control of the heart.
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INTRODUCTION |
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While the vagus nerve is the
predominant source of neural input into the mammalian cardiac ganglia,
efferent and afferent sympathetic and sensory neurons also project onto
the cardiac nerve plexus (see Ardell 1994;
Moravec and Moravec 1987
). Whereas the efferent nerves
primarily release norepinephrine to the heart from nerve terminals
found in close proximity to parasympathetic nerve terminals
(Jacobowitz et al. 1967
), afferent neurons of spinal and
vagal origin also form a moderately dense network of substance P
(SP)-immunoreactive fibers in the myocardium and around blood vessels
in the atria (Dalsgaard et al. 1986
; Forsgren et al. 1990
). These SP-containing nerve fibers have been found
closely associated with the parasympathetic intracardiac ganglia, often surrounding ganglion cells (Dalsgaard et al. 1986
;
Hardwick et al. 1995
; Hoover and Hancock
1988
; Steele et al. 1994
). Further evidence
suggesting that SP may play a major role in the cardiac ganglia is the
fact that this neuropeptide is also found in neurons of vagal sensory
origin. Treatment of the vagus nerve with capsaicin, which depletes
nerves of SP content, significantly reduced SP immunoreactivity in the
right atrium of guinea pigs (Dalsgaard et al. 1986
;
Papka et al. 1981
). SP may be acting as a
neurotransmitter or neuromodulator in the intracardiac ganglia, thus
influencing neuronal transmission and ultimately cardiac function.
In canine hearts, SP modulates autonomic nerve activity, increasing
cardiac contractility during vagal stimulation and decreasing contractility during stellate ganglion stimulation (Smith et al. 1992). In guinea pig hearts, SP has been shown to activate a
nonselective cation conductance via an NK3
receptor in intracardiac neurons (Hardwick et al. 1997
)
and to induce bradycardia (Tompkins et al. 1999
).
However, less is known about the effects of SP on rat intracardiac
neurons, and species differences in responses to neuropeptides have
been reported in autonomic neurons (see Liu et al.
2000
).
The physiological effects of SP have been attributed to alterations in
membrane properties, which are tissue specific. In sympathetic neurons
of the rat prevertebral ganglia, SP and other tachykinins activate two
distinct depolarizing responses; a fast response attributed to an
increase in a Ca2+-dependent cationic conductance
and a slow response produced by a decrease in K+
conductance (Konishi et al. 1992). SP has also been
shown to attenuate nicotinic ACh-evoked currents in chick sympathetic
and ciliary ganglion neurons (Margiotta and Berg 1986
;
Role 1984
). The inhibition of neuronal nicotinic
ACh-evoked current amplitude by SP has been attributed to either an
acceleration of the rate of desensitization or open channel blockade
(Clapham and Neher 1984
).
The effects of SP are mediated by neurokinin (NK) tachykinin
cell-surface receptors
(NK1-NK3) (Otsuka
and Yoshioka 1993), which couple with both pertussis
toxin-sensitive and -insensitive G proteins (Nakajima et al.
1988
; Shapiro and Hille 1993
), and activation
may increase intracellular cAMP and/or phospholipase A2 (Catalan
et al. 1995
). However, at the nicotinic ACh receptor (nAChR)
channel, SP also exerts its effects by acting directly on membrane ion
channels (Clapham and Neher 1984
). This investigation studied the effects of SP on membrane currents in isolated
parasympathetic intracardiac neurons and characterized the signal
transduction pathway(s) involved. SP was found to attenuate nicotinic
ACh-evoked current amplitude. This effect was not mediated by cAMP,
protein kinase C, or protein phosphorylation, suggesting that SP may be acting directly on the neuronal nAChR channel. A preliminary report of
some of these results has been presented (Cuevas and Adams 1997
).
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METHODS |
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Preparation
Membrane currents were studied in isolated parasympathetic
neurons of neonatal rat intracardiac ganglia. The procedures for isolation of the neurons have been described in detail previously (Cuevas and Adams 1996). Briefly, neonatal rats (3-8
days old) were killed by decapitation, and the heart was excised and
placed in a saline solution containing (in mM) 140 NaCl, 3 KCl, 2.5 CaCl2, 0.6 MgCl2, 7.7 glucose, and 10 histidine (pH to 7.2 with NaOH). Atria were removed and
incubated for 1 h at 37°C in saline solution containing
collagenase (1 mg/ml, Type 2, Worthington Biochemical, Freehold, NJ).
Following enzymatic treatment, clusters of ganglia were dissected from
the epicardial ganglion plexus, transferred to a sterile culture dish
containing high glucose culture media (Dulbecco's modified Eagle's
media), 10% (vol/vol) fetal calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin, and triturated using a fine-bore Pasteur pipette.
The dissociated neurons were then plated onto 18-mm glass cover slips
coated with laminin, and incubated at 37°C for 24-72 h under a 95%
air, 5% CO2 atmosphere.
Electrical recordings and data analysis
Neurons plated on glass cover slips were transferred to a 0.5-ml
recording chamber mounted on an inverted phase contrast microscope (×400 magnification), allowing electrophysiological experiments to be
performed on isolated neurons. Membrane currents in intracardiac neurons were studied under voltage-clamp mode using the whole cell and
excised outside-out membrane patch recording configurations of the
patch-clamp technique (Hamill et al. 1981). In whole
cell experiments, electrical access was achieved either by rupturing the membrane patch and dialyzing the cell, or through the use of the
perforated-patch method (Horn and Marty 1988
).
Whereas the intracellular integrity of the neurons is maintained using the perforated patch method, cytosolic components are lost with cell
dialysis, altering the functional response of these neurons (Cuevas and Adams 1994
).
For perforated patch experiments, a stock solution of 60 mg
ml1 of amphotericin B in
dimethylsulfoxide (DMSO) was prepared the day of the experiment and
kept on ice, in the dark. Immediately prior to use, the amphotericin B
stock solution was diluted in pipette solution to yield a final
concentration of 360 µg/ml amphotericin B in 0.6% DMSO. Following
gigaseal formation, the neurons were held at
70 mV, and voltage
pulses (20 ms) to
80 mV were applied at 1 Hz. Amphotericin B
incorporation into the membrane patch resulted in an increase in a fast
capacitive transient, the appearance of a slow capacitive transient,
and a decrease in the series resistance (Rs). Experiments were continued only
if Rs decreased to
10 M
within 10 min of seal formation. Final series resistance was usually
4 M
following approximately 50% Rs
compensation. Rupture of the membrane under the pipette tip was
indicated by a rapid change in the holding current, at which time the
experiment was terminated.
Electrodes for whole cell and excised membrane patch experiments were
pulled from thin- and thick-walled borosilicate glass (GC150F; Clark
Electromedical Instruments, Reading, UK), respectively. For
single-channel experiments, pipettes were coated with dental wax to
within 100 µm of the tip. Final pipette resistance was 1-3 M for
conventional and perforated patch whole cell recordings and 4-6 M
for excised membrane patch recordings. Membrane currents evoked by
agonist application were amplified and low-pass filtered (10 kHz) using
an Axopatch 200A patch-clamp amplifier (Axon Instruments, Foster City,
CA) and recorded on digital audio tape (DAT) using a digital tape
recorder (DTR-1204, BioLogic Science Instruments, Claix, France).
Membrane currents were continuously monitored on a digital oscilloscope
and a chart recorder. In some experiments, the effects of SP on the
membrane current-voltage relationship were investigated using slow
voltage ramps, whereby the membrane voltage was increased linearly from
150 to +50 mV at a rate of 50 mV/s.
For computer analysis, ACh-evoked current records were played back from
digital tape, filtered, and digitized (Digidata 1200A interface, Axon
Instruments). Whole cell currents were filtered at either 1 or 3 kHz
(3 dB; 4-pole Bessel filter), sampled at 3 and 10 kHz, respectively,
and analyzed using the Axotape 2.0 program (Axon Instruments). Unitary
current records were transferred from DAT cassette to a PC Pentium
100-MHz computer using the pClamp 6.0 acquisition program Fetchex (Axon
Instruments). Analysis of unitary currents was performed using the
pClamp 6.0 programs Fetchan and pSTAT. To minimize attenuation, mean
unitary current amplitude was determined from data filtered at 10 kHz,
whereas data obtained for analysis of nAChR channel kinetics was
filtered at 5 kHz (
3 dB, 4-pole Bessel filter). The sampling rate for
both conditions was set at 50 kHz, and the threshold for detection of
events was 100 µs for amplitude and kinetic data. Unitary current
amplitude distributions were obtained from outside-out patch recordings by measuring the difference between the amplitude of each event and the
baseline current. Each individual event contributed one point to the
amplitude distribution for the patch. Frequency distribution histograms
were constructed from idealized unitary current records using a
detection threshold for channel openings set at 50% of the amplitude
of a single-channel opening, and a minimum resolvable time of 100 µs
(see Colquhoun and Sigworth 1995
). Exponential and
Gaussian curves to the data were fit using a nonlinear, minimum
2 method.
Dose-response curves were obtained by measuring the peak current
amplitude at each agonist concentration and the experimental data
points were fit using the equation
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(1) |
Data are expressed as means ± SE. For statistical analysis, data in the absence and presence of SP were compared using a paired t-test and were considered significant if P < 0.05. For comparison of results obtained from neurons under different conditions unpaired t-tests were used.
Solutions and reagents
The control external solution for dialyzed and perforated patch
whole cell recordings was physiological saline solution (PSS) containing (in mM) 140 NaCl, 3 KCl, 2.5 CaCl2,
1.2 MgCl2, 7.7 glucose, and 10 HEPES-NaOH, pH
7.2. The pipette solution for conventional whole cell recordings
consisted of (in mM) 140 CsCl, 2 MgATP, 0.1 GTP sodium salt (GTP), 2 Cs4 BAPTA, and 10 HEPES-CsOH, pH 7.2. The
perforated patch pipette solution contained (in mM) 75 K2 SO4, 55 KCl, 5 MgSO4, and 10 HEPES (pH to 7.2 with
N-methyl-D-glucamine). The recording chamber was
continuously perfused (2 ml/min) with the indicated solutions at room
temperature (22-23°C). ACh-evoked responses were elicited during
whole cell recordings by focal application of 100 µM ACh or 100 µM
ACh + SP in PSS via pressure ejection (15 psi; Picospritzer II, General
Valve, Fairfield, NJ) from an extracellular pipette (3-5 µm diam).
The pressure ejection pipette was positioned 50 µm from the cell
soma to evoke maximal responses to agonist. The concentration of bath
applied drugs (e.g., atropine, mecamylamine, and spantide) at the cell
surface during pressure application of ACh or ACh + SP was equal to or lower than that of the bulk solution. The external solution used for
recording single-channel currents in outside-out membrane patches
contained (in mM) 140 NaCl, 2.5 CaCl2, 1.2 MgCl2, 7.7 glucose, and 10 HEPES-NaOH, pH 7.2, and the intracellular pipette solution was composed of (in mM) 140 CsCl, 2 Cs4 BAPTA, 2 MgATP, 0.1 Na-GTP, and 10 HEPES-CsOH, pH 7.2. The osmotic activities of the solutions (285-295
mmol/kg) were monitored with a vapor pressure osmometer (Wescor 5500, Logan City, UT). In outside-out patch experiments, 3 µM ACh or
cytisine were bath applied in external solution in the absence or
presence of SP. In some experiments, the neurons were dialyzed with a
pipette solution containing the nonhydrolyzable ATP analogue, AMP-PNP.
Stock solutions of forskolin and phorbol 12,13-dibutyrate were made in DMSO and were later diluted to the final concentration in extracellular solution. 8-Bromoadenosine 3':5'-cyclic monophosphate and 1-(5-isoquinoline sulfonyl)-2-methyl-piperazine, dihydrochloride (H-7) were added to the extracellular solution at the concentration stated. Experiments involving these compounds required a 10-min pretreatment of the neurons.
All chemical reagents used were of analytical grade. Acetylcholine chloride, ATP magnesium salt, 5'-adenylyl-imidodiphosphate tetralithium salt (AMP-PNP), adenosine 3':5'-cyclic monophosphate (cAMP), 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP), atropine sulfate, cytisine, mecamylamine hydrochloride, phorbol 12,13-dibutyrate (PDBu), and [D-Arg1, D-Trp7,9, Leu11]-substance P (spantide) were obtained from Sigma Chemical (St. Louis, MO), substance P from Research Biochemicals (Natick, MA), and forskolin and 1-(5-isoquinoline sulfonyl)-2-methyl-piperazine, dihydrochloride (H-7) were obtained from Calbiochem (San Diego, CA).
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RESULTS |
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Bath application of SP (10 nM to 300 µM) alone evoked no direct membrane response (change in holding current), and had no effect on the whole cell current-voltage (I-V) relationship of rat intracardiac neurons (n = 6). SP (100 µM) also had no effect on depolarization-activated Na+, K+, or Ca2+ currents in these neurons (data not shown).
SP inhibition of nicotinic ACh-evoked currents
Given that SP has been shown to modulate muscle and neuronal nAChR
channels (Clapham and Neher 1984; Role
1984
; Simasko et al. 1985
), the effect of SP on
ACh-evoked currents was investigated in rat intracardiac neurons. The
onset and recovery from SP inhibition was examined during prolonged
application of ACh to the cell soma. During a maintained inward current
evoked by bath application of 5 µM ACh onto a neuron held at
70 mV,
brief application (10 ms) of 5 µM ACh onto the cell soma had no
effect on the time course of decay of the inward current (Fig.
1A1). However, brief focal applications of 100 µM SP together with ACh transiently and
reversibly reduced the amplitude of maintained ACh-evoked current
evoked by bath application of 5 µM ACh (Fig. 1A2).
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The nicotinic ACh-evoked current at negative potentials has been shown
in perforated patch experiments to be biphasic, composed of a transient
and a sustained inward current components (see Cuevas and Adams
1994). To determine whether SP affects both components of the
ACh-evoked current, currents were examined during prolonged exposure to
ACh in the absence and presence of SP. Membrane currents evoked by
focal application (30 s) of 100 µM ACh and 100 µM ACh + 100 µM SP
to the soma membrane held at
70 mV, both in the presence of 1 µM
atropine to inhibit muscarinic ACh-mediated responses, are shown in
Fig. 1B1. In all cells examined, SP attenuated both the
transient and the sustained current evoked by ACh by approximately 70%
(n = 4). Figure 12 shows the superimposed
currents on an expanded time scale. The SP-induced inhibition of
ACh-evoked current amplitude was accompanied by a decrease in the
half-time of current decay. In four experiments, the half-time of
ACh-evoked current decay decreased from 1.60 ± 0.19 s
(mean ± SE, control, absence of SP) to 0.24 ± 0.04 s
in presence of 100 µM SP. The approximately sixfold increase in the
rate of decay of the ACh-evoked current in the presence of SP was
statistically significant (P < 0.03).
Figure 2A shows representative
whole cell membrane currents evoked by focal application of 100 µM
ACh to the cell soma held at different membrane potentials.
Co-application of 100 µM ACh and 50 µM SP inhibited the ACh-evoked
current amplitude at all membrane potentials in the absence (Fig.
2A2) and presence (Fig. 2A3) of the nonselective
tachykinin receptor antagonist, spantide. Whole cell I-V
relations obtained for peak ACh-evoked currents obtained in the absence
and presence of 50 µM SP and 50 µM SP + 3 µM spantide are shown
in Fig. 2B. SP reversibly depressed ACh-evoked current
amplitude at all membrane potentials examined. This inhibition was
voltage insensitive, with the ACh-evoked current amplitude reduced by
70% at all membrane potentials in the presence of 50 µM SP
(n = 3).
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The concentration dependence of SP-mediated inhibition of ACh-evoked
currents was examined by co-application of 100 µM ACh and SP at
various concentrations. Representative ACh-evoked currents obtained
from a neuron held at 100 mV in the absence and presence of SP at the
concentrations indicated are shown in Fig.
3A. The dose-response
relationship obtained for mean peak ACh-evoked current amplitude
normalized to control (absence of SP) for four cells held at
100 mV
plotted as a function of SP concentration is shown in Fig.
3B. The solid line is a fit of the data using Eq. 1 yielded half-maximal inhibition of ACh-evoked current at 45 µM
SP, and near complete block was obtained with 300 µM SP. The Hill
coefficient calculated for the data was 1.02.
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ATP-activated and muscarinic ACh receptor-mediated whole cell currents
were unaffected by 100 µM SP (data not shown). Co-application of SP
with ATP had no effect on either the ATP-evoked current amplitude or
the time course of current decay. ATP-evoked current density at 100
mV was 15.1 ± 1.0 pA/pF (n = 4) under control conditions and 15.5 ± 1.2 pA/pF (n = 4) in the
presence of SP. Half-time of decay of ATP-evoked currents at
100 mV
was 5.3 ± 0.7 s and 5.5 ± 0.6 s in the absence
and presence of SP, respectively.
Lack of evidence for a role of second messengers in SP inhibition of ACh-evoked currents
Dialysis of the cytoplasm with the pipette solution, which occurs
during conventional whole cell recording, has been shown to inhibit
cellular processes, which depend on diffusible cytosolic second
messengers (Cuevas and Adams 1994; Horn and Marty
1988
). Given that SP inhibition of ACh-evoked currents is
observed in dialyzed whole cell recording conditions argues against the
possible involvement of a diffusible cytosolic second messenger(s) in
mediating SP inhibition. To determine whether cAMP or protein kinase C
(PKC) are constituents in the signal transduction pathway mediating SP
inhibition of nAChR channels, SP modulation of ACh-evoked currents was
examined in the presence of compounds that either inhibit, activate, or
mimic the effects of these second messengers, respectively. The percent
inhibition of ACh-evoked current at
100 mV by SP, phorbol
12,13-dibutyrate, forskolin, and 8-Br-cAMP is summarized in Fig.
4. Bath application of the synthetic PKC
activator, PDBu (10 µM), decreased ACh-evoked current amplitude by
79 ± 5% (n = 3; P < 0.05),
similar to that produced by 100 µM SP (81 ± 6%, n = 4; P < 0.03). Forskolin (100 µM), an activator of adenylate cyclase, also significantly decreased
ACh-evoked current amplitude by 70 ± 7% (n = 3, P < 0.05). However, bath application of the membrane-permeant analogue of cAMP, 8-Br-cAMP (1 mM) had no effect on
the ACh-evoked current amplitude, nor did intracellular application of
0.1 mM cAMP (n = 4). The increase in the rate of decay
of the ACh-evoked current observed in the presence of SP was not
mimicked by any of the above compounds.
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Membrane currents evoked by focal application (300 ms) of 100 µM ACh and 100 µM ACh + 100 µM SP in the absence and presence of bath-applied 5 µM H-7, an inhibitor of PKC, are shown in Fig. 4B1. H-7 alone reduces the ACh-evoked current by 35 ± 6% (n = 3), but does not affect SP-mediated inhibition of ACh-evoked currents.
Phosphorylation of nAChR channels has been shown to increase the rate
of desensitization of the channel, and increase the rate of current
decay (Eusebi et al. 1985; Hopfield et al.
1988
; Huganir et al. 1986
). To examine whether
phosphorylation is involved in SP-mediated modulation of ACh-evoked
currents, cells were dialyzed for
20 min with a pipette solution
containing 2 mM AMP-PNP, which has been shown to inhibit processes that
are phosphorylation dependent. Figure 42 shows currents
evoked at
70 mV by 10-s pulse application of 100 µM ACh or 100 µM
ACh + 100 µM SP. AMP-PNP did not affect SP-induced attenuation of
ACh-evoked currents or alter the half-time of current decay in all
neurons examined (n = 3).
Effects of SP on neuronal nAChR channels
To examine the mechanism(s) underlying the inhibition of
ACh-evoked whole cell currents by SP in rat intracardiac neurons, single-channel activity was studied in excised outside-out membrane patches (n = 8). Bath application of 3 µM ACh
elicited unitary currents occurring as clusters of bursts of openings
separated by long closed times, while bursts within a cluster are
separated by closings of intermediate length. Figure
5, A and B, shows
representative records obtained from a single outside-out patch in the
presence of 3 µM ACh (A) and 3 µM ACh + 3 µM SP
(B) at 60 mV. Single-channel current amplitude
distributions shown in Fig. 5, C and D, were constructed from the experiment shown in Fig. 5, A and
B. Histograms were best fit by Gaussian distributions with
mean values of
1.25 ± 0.28 pA,
2.09 ± 0.11 pA, and
2.83 ± 0.22 pA (Fig. 5C, ACh alone) and
1.21 ± 0.23 pA,
2.14 ± 0.09 pA, and
2.80 ± 0.33 pA (Fig.
5D, ACh + SP). These unitary current amplitudes correspond with mean conductances of 21 ± 5 pS, 35 ± 2 pS, and 47 ± 4 pS in the absence of SP and 20 ± 4 pS, 36 ± 1 pS, and
47 ± 6 pS in the presence of SP. The mean values obtained for
four similar experiments are shown in Table
1. There was no SP-mediated change in the
mean unitary current amplitude for any of the three conductance classes
observed. Three of the four patches examined with ACh exhibited all
three conductance classes, and one patch contained only the 20- to
21-pS and the 35- to 36-pS conductance classes.
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Previous experiments demonstrated differences in nicotinic agonist
efficacy among rat intracardiac neurons, and this variability reflected
the heterogeneity in nAChR subunits expressed in these neurons
(Poth et al. 1997). Given that SP has shown selectivity for
4-containing nAChRs and cytisine preferentially activates such
receptors (Stafford et al. 1994
), we examined the
effects of SP on nAChRs using cytisine as the agonist
(n = 4). Figure 6 shows
representative records of AChR activity from a single outside-out patch
in the presence of 3 µM cytisine (A) and 3 µM cytisine + 3 µM SP (B) at
60 mV. Single-channel current amplitude distributions shown in Fig. 6, C and D, were
constructed from the experiment shown in Fig. 6, A and
B. Histograms were best fit by Gaussian distributions with
mean values of
0.96 ± 0.19 pA,
1.76 ± 0.12 pA, and
2.59 ± 0.26 pA with 3 µM cytisine (Fig. 6C) and
0.96 ± 0.18 pA,
1.76 ± 0.13 pA, and
2.56 ± 0.28 pA with 3 µM cytisine + 3 µM SP (Fig. 6D).
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The macroscopic ACh-evoked current amplitude is a function of unitary currents and is defined by the equation: I = i NPo, where I is the whole cell current amplitude, i is the unitary current amplitude, N is the number of channels, and Po is the single-channel open probability. Given that SP does not alter i, the effects of SP on Po were studied, as were the effects of SP on the apparent mean open time of single-channel events. Table 1 summarizes the results obtained in eight experiments using ACh or cytisine as the agonist. Whereas SP had no effect on the mean amplitude of the three conductance classes (types A-C) when either ACh (Fig. 7, A1, B1, and C1) or cytisine (Fig. 7, A2, B2, and C2) was used as the agonist, SP reduced the mean open time of the large conductance class (type C) when these channels were activated by either ACh (58 ± 7% decrease) or cytisine (44 ± 11% decrease; Fig. 7B). However, SP did not affect the mean open time of the smaller conductance classes (types A and B).
|
The open channel probability (Po) observed for conductance classes B and C was significantly greater (P < 0.05) with cytisine than with ACh (Fig. 7C). The mean Po of conductance class A was more than double that of conductance class C with ACh, but these were nearly identical with cytisine as the agonist. SP decreased the open channel probability for all conductance classes with either ACh or cytisine as the agonist. However, the Po of conductance classes B and C showed greater sensitivity to neuropeptide inhibition, decreasing 88 ± 4% and 96 ± 2%, respectively, in the presence of SP. In contrast, conductance class A only decreased 51 ± 11% following application of the neuropeptide.
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DISCUSSION |
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In perforated patch recordings from rat intracradiac
neurons, ACh evoked a biphasic inward current in the presence of
atropine, which was inhibited by mecamylamine indicating that it is
mediated by nAChR activation (Cuevas and Adams 1994).
Application of SP (10 nM to 300 µM) reversibly decreased the
amplitude of both the transient and sustained components of the
ACh-evoked current. SP-mediated inhibition of the transient component
was not voltage sensitive, suggesting that SP does not inhibit the
nAChR channel by acting as an open-channel blocker, as had been
previously suggested for SP inhibition of ACh-evoked currents in bovine
chromaffin cells (Clapham and Neher 1984
). A similar
voltage-independent reduction of ACh-evoked current amplitude by SP has
been reported in bullfrog sympathetic neurons (Akasu et al.
1983
).
Application of SP together with ACh also increased the rate of
ACh-evoked current decay in rat intracardiac neurons. In chick sympathetic neurons, SP produced a comparable increase in the rate of
ACh-evoked current decay but did not attenuate ACh-evoked current
amplitudes (Role 1984; Valenta et al.
1993
). The SP-mediated increase in the rate of current decay
has been attributed to an augmentation of nAChR channel desensitization
(Clapham and Neher 1984
; Role 1984
;
Stallcup and Patrick 1980
; Valenta et al.
1993
).
In rat sympathetic neurons, ATP-induced attenuation of nicotinic
ACh-evoked currents has been suggested to occur via a cAMP-dependent pathway (Nakazawa 1994). Increased cAMP levels have also
been shown to increase the rate of nAChR channel desensitization in rat
skeletal muscle (Albuquerque et al. 1986
;
Middleton et al. 1986
). To determine whether cAMP was
involved in the signal transduction pathway mediating SP attenuation of
ACh-evoked currents in rat parasympathetic neurons, the effects of
second-messenger activators and inhibitors were examined. An activator
of adenylate cyclase, forskolin, reduced ACh-evoked current
amplitude by ~70%; however, the membrane-permeant analogue of cAMP,
8-Br-cAMP, failed to alter ACh-evoked current amplitude or time course.
The lack of effect by 8-Br-cAMP suggests that the effect of forskolin
on ACh-evoked currents may be independent of increased cAMP levels.
Forskolin has been shown to alter nAChR gating by an open-channel block mechanism similar to that observed for local anesthetics
(Wagoner and Pallotta 1988
; White 1988
).
Intracellular dialysis with normal pipette solution under conventional
whole cell recording conditions did not alleviate the SP-mediated
decrease in ACh-evoked current amplitude, indicating that a diffusable
cytosolic second messenger is unlikely to be involved in the SP signal
transduction pathway.
An alternative pathway for SP modulation of ACh receptor-channels may
involve PKC. Substance P has been shown to stimulate inositol phospholipid metabolism in the rat superior cervical ganglion
in vitro (Horwitz et al. 1986), and diacylglycerol
produced from inositol phospholipid hydrolysis may activate PKC. It has previously been proposed that this signal transduction pathway mediates
SP inhibition of ACh-evoked currents in embryonic chick sympathetic
ganglion neurons (Downing and Role 1987
). This idea is
supported by the finding that the Torpedo nAChR channel
contains a putative PKC phosphorylation site on the
subunit, which
may regulate nAChR channel desensitization (Huganir et al.
1986
). In the present study, bath application of the synthetic
diacylglycerol activator, phorbol dibutyrate, attenuated ACh-evoked
current amplitudes, but in contrast to SP, phorbol dibutyrate did not
alter the kinetics of ACh-evoked currents. Further evidence against a
PKC signal transduction pathway mediating SP inhibition comes from a
series of experiments involving the PKC inhibitor, H-7. H-7 alone
depressed ACh-evoked currents, as previously reported in
Xenopus oocytes expressing BC3H-1 nACh receptors
(Reuhl et al. 1992
); however, H-7 failed to inhibit the
actions of SP on ACh-evoked currents in rat intracardiac neurons. This
result differs from that found in chick sympathetic ganglion neurons
where the PKC inhibitor, staurosporine, blocked SP modulation of nAChR
channel currents (Simmons et al. 1990
). Also, cell
dialysis with the nonhydrolyzable ATP analogue, AMP-PNP, did not
inhibit the actions of SP in rat intracardiac neurons, suggesting that
protein phosphorylation is not a constituent of the signaling mechanism
mediating the effects of SP.
Previous studies have speculated that SP might modulate ACh-evoked
currents by a direct allosteric interaction with the nAChR channel
(Akasu et al. 1983; Clapham and Neher
1984
; Stallcup and Patrick 1980
). This idea is
supported by the finding that SP inhibits ACh-evoked currents resulting
from the activation of different combinations of rat neuronal nAChR
subunits expressed in Xenopus oocytes (Stafford et
al. 1994
). This inhibition is believed to reflect direct action
of SP on nAChRs because tachykinin receptors are not endogenously
expressed in Xenopus oocytes (Fong et al. 1992
). Half-maximal inhibition of macroscopic ACh-evoked
currents by SP (45 µM) in rat intracardiac neurons is within the
range observed for the modulation by SP of different subunit
combinations (2.8-74 µM) expressed in Xenopus oocytes. A
Hill coefficient of 1.02 was calculated for SP-mediated inhibition of
nAChR channels in rat intracardiac neurons, which is similar to that
determined in Xenopus oocytes (0.71-2.4) (Stafford
et al. 1994
), suggesting that one SP molecule interacts with
each nAChR-channel.
In the present study, no evidence was found for a second-messenger
molecule being involved in SP inhibition of nAChR channels in rat
parasympathetic neurons. While the actions of SP appear to be due to a
direct interaction of the neuropeptide with the nAChR channel, a second
putative mechanism for SP modulation of nAChRs also exists whereby
changes in the lipid environment induced by SP (Keire and
Fletcher 1996) may affect nAChR function. However, given that
SP selectively inhibits nAChR channels and not other ligand- or
voltage-gated ion channels in these neurons suggests that it is
unlikely that SP interaction with the lipid membrane can explain the
inhibition of nAChR channel activity by SP.
Although SP has been reported to directly modify other membrane
conductances in mammalian autonomic neurons, such as inhibiting N-type
Ca2+ channel currents in rat sympathetic neurons
(Shapiro and Hille 1993) and decreasing
K+ permeability in neonatal rat sympathetic
neurons (Dun and Mo 1988
) and adult guinea pig celiac
(Vanner et al. 1993
) and stellate ganglion neurons
(Gilbert et al. 1998
), such effects of SP were not
observed in rat parasympathetic intracardiac neurons. SP also failed to
alter muscarinic ACh-sensitive K+ currents, in
contrast to findings in frog sympathetic neurons where SP inhibits the
M-current (see Brown 1988
) and did not activate a
nonspecific cation conductance as has been reported in guinea pig
intracardiac ganglion neurons (Hardwick et al. 1997
).
Furthermore, ATP-evoked currents in rat intracardiac neurons were also
not affected by SP, suggesting that SP acts exclusively as a
neuromodulator of nicotinic ACh transmission in rat intracardiac ganglia.
SP inhibition of ACh-activated unitary currents was observed in
excised, outside-out membrane patches. Co-application of SP and ACh or
cytisine did not alter the single-channel amplitude, consistent with
findings previously reported in chick sympathetic neurons
(Simmons et al. 1990). However, SP (3 µM)
differentially modulated the frequency of occurrence and apparent mean
open time of the three distinct nAChR channel conductances observed in
rat intracardiac neurons. The mean apparent channel open times for the
large conductance nAChR channels were only slightly decreased, but an
increase in the closed times was observed that resulted in an
approximately 10- to 30-fold decrease in
NPo for the large conductance (classes
B and C) channels. Two possible mechanisms may explain the observed
effects of SP on ACh- and cytisine-activated unitary currents:
1) a decrease in the affinity of the nAChR channel for ACh
and cytisine and 2) a decrease in the number of functional channels. However, it has been shown that SP does not affect the binding of [3H]acetylcholine to neuronal nAChRs
(Lukas and Eisenhour 1996
; Weiland et al.
1987
), suggesting that the effects are likely due to a decrease
in the number of functional channels.
The preferential inhibition of the large conductance (class B and C)
nAChR channels by SP is most likely to account for SP inhibition of
whole cell nicotinic ACh-evoked currents. A decrease in the number of
functional channels due to enhanced receptor desensitization was
proposed to explain SP inhibition of nicotinic ACh-evoked currents in
chick sympathetic ganglion neurons (Simmons et al.
1990). Although only single-channel openings were used for
kinetic analysis, it is impossible to rule out that multiple channels
with low Po and not a single channel
are present in the membrane patches studied. A decrease in the number
of functional channels in the membrane patch could account for the
observed increase in the closed times and may be due to an increase in the rate of desensitization of the nAChR (see Sakmann et al.
1980
). An increase in the long closed time observed in the
presence of SP suggests that SP may increase the time a channel remains
in the desensitized state, effectively reducing the number of
functional channels available for ACh activation at any given time.
Neuronal nAChRs formed from different combinations of and
subunits exhibited a differential sensitivity to SP with those containing
4 subunits having a 25-fold higher affinity than those having
2 subunits (Stafford et al. 1994
). Chimeric
and site-directed mutagenesis studies of
subunits coexpressed with
3 in Xenopus oocytes indicate that amino acids of the
first extracellular domain and within the putative channel lining
transmembrane domain M2 contribute to the apparent affinity of SP
(Stafford et al. 1998
). AChRs containing the
4
subunit also show greater affinity for cytisine (Luetje and
Patrick 1991
). Interestingly, the large conductance class of
nAChRs is both preferentially activated by cytisine and more sensitive
to SP, consistent with these nAChRs containing the
4 subunit.
Vagal innervation of the heart is mediated by the intrinsic
cardiac ganglia and is predominantly cholinergic. The presence of
SP-containing neurons and nerve fibers associated with mammalian intracardiac ganglia (Dalsgaard et al. 1986;
Hardwick et al. 1995
; Steele et al. 1994
)
and the release of SP from intrinsic and extrinsic sources may modulate
vagal transmission within the ganglia and reflect an integrative
function of the mammalian cardiac ganglia (Hardwick et al.
1995
). SP inhibits the inotropic effects produced by vagal
stimulation in mammals (Hoover 1990
; Smith et al.
1992
) and evokes bradycardia in isolated guinea pig hearts
(Tompkins et al. 1999
). Direct application of SP to
spontaneously active canine intrinsic cardiac neurons in situ can both
increase and decrease neuronal activity, resulting in concomitant
changes in heart rate (Armour et al. 1993
). Some of
these effects of SP may be attributed to modulation of membrane
conductances and increased excitability (Hardwick et al.
1997
; Tompkins et al. 1999
). However, the
present study suggests that the effects of SP on the mammalian heart
may also be mediated by modulation of nicotinic ACh ganglionic transmission. Given that ACh is the primary neurotransmitter mediating vagal innervation of the mammalian heart, SP inhibition of neuronal nicotinic ACh-evoked responses is likely to alter neuronal activity within intrinsic cardiac ganglia and ultimately affect cardiac performance.
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ACKNOWLEDGMENTS |
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This work was supported by National Health and Medical Research Council of Australia Grant 961138 to D. J. Adams and by American Heart Association Grant 9930259N to J. Cuevas.
Present address of J. Cuevas: Dept. of Pharmacology and Therapeutics, University of South Florida College of Medicine, Tampa, FL 33612.
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FOOTNOTES |
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Address for reprint requests: D. J. Adams (E-mail: dadams{at}plpk.uq.edu.au).
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.
Received 13 March 2000; accepted in final form 9 June 2000.
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REFERENCES |
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