Substance P Preferentially Inhibits Large Conductance Nicotinic ACh Receptor Channels in Rat Intracardiac Ganglion Neurons

Javier Cuevas and David J. Adams

Department of Physiology and Pharmacology, University of Queensland, Brisbane, QLD 4072, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 ml-1 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 MOmega within 10 min of seal formation. Final series resistance was usually <= 4 MOmega 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 MOmega for conventional and perforated patch whole cell recordings and 4-6 MOmega 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 chi 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
<IT>I</IT><IT>/</IT><IT>I</IT><SUB><IT>max</IT></SUB><IT>=1/</IT>[<IT>1+</IT>([<IT>A</IT>]<IT>/EC<SUB>50</SUB></IT>)<SUP><IT>n</IT></SUP>] (1)
where I/Imax represents the relative current, [A] is the agonist concentration, EC50 is the concentration giving half-maximal activation, n is the Hill coefficient.

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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1. A: rapid and reversible inhibition of ACh-evoked currents by substance P (SP) in dialyzed intracardiac neurons. Whole cell currents evoked in a neuron held at -70 mV by bath application of 5 µM ACh for the duration indicated (solid line). Brief pulses (10 ms) of either 5 µM ACh (1) or 5 µM ACh + 100 µM SP (2) were applied (indicated by arrowheads) during the decay phase of the ACh-evoked current. B: superimposed currents evoked from an isolated neuron by a 30-s pulse of either 100 µM ACh alone (Control) or 100 µM ACh + 30 µM SP (+SP). B2: ACh-evoked currents shown in B1 scaled to peak amplitude and displayed on an expanded time scale.

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).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. Inhibition of ACh-evoked current in rat intracardiac neurons by SP. A: whole cell ACh-evoked currents elicited from an isolated neuron by focal application (10-ms pulse) at the membrane potentials, -100, -60, -20, and +20 mV. ACh-evoked currents obtained from the same neuron by focal application of 100 µM ACh + 50 µM SP in the absence (B) and presence (C) of bath applied tachykinin receptor antagonist, spantide (3 µM). D: current-voltage relation obtained in response to ACh alone (open circle , control), ACh + SP (), and ACh + SP in the presence of spantide (down-triangle). Data points represent means ± SE for 4 neurons.

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. 



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3. Concentration-dependent inhibition of ACh-evoked whole cell currents by SP. A: ACh-evoked currents obtained from a neuron held at -100 mV in the absence and presence of SP at the concentrations indicated. B: ACh-evoked current amplitude normalized to control (100 µM ACh) plotted as a function of SP concentration (n = 6). Curve is a best fit to the data using Eq. 1. Half-maximal inhibition occurred at 45 µM SP, with a Hill coefficient of 1.02.

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.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4. Modulation of ACh-evoked currents by SP and activators of second-messenger pathways. A: bar graph of peak ACh-evoked whole cell current amplitude at -100 mV normalized to control (ACh alone) in the presence of bath applied 100 µM SP (n = 4), 10 µM phorbol 12,13-dibutyrate (PDBu; n = 3), 100 µM forskolin (n = 3), or 1 mM 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP; n = 4). Asterisks indicate significant difference relative to control (P < 0.05). B: lack of effect of 1-(5-isoquinoline sulfonyl)-2-methyl-piperazine, dihydrochloride (H-7), and intracellular dialysis with 5'-adenylyl-imidodiphosphate tetralithium salt (AMP-PNP) on SP-mediated modulation of ACh-evoked whole cell currents. B1: currents evoked from a single neuron by focal application (300 ms) of either 100 µM ACh alone or 100 µM ACh + 100 µM SP (+SP) in the absence and presence of bath applied 5 µM H-7. B2: Whole cell currents evoked by 10-s pulse of either 100 µM ACh or 100 µM ACh + 100 µM SP from a single neuron dialyzed with pipette solution containing the nonhydrolyzable ATP analogue, AMP-PNP (2 mM).

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.



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 5. ACh-evoked single-channel currents obtained in the absence and presence of SP. A and B: unitary currents recorded from an excised outside-out membrane patch held at -60 mV and perfused with physiological saline solution (PSS) containing either 3 µM ACh (A) or 3 µM ACh + 3 µM SP (B). Continuous line represents closed state (C), and dashed lines represent open states (O). C and D: amplitude histograms of unitary currents obtained from the same patch. Histograms are fitted with Gaussian distributions with amplitudes of -1.25 ± 0.28 pA (51% total fit area), -2.09 ± 0.11 pA (22%), and -2.83 ± 0.22 pA (27%) for ACh (C); and -1.21 ± 0.23 pA (99%), -2.14 ± 0.09 pA (<1%), and -2.80 ± 0.33 pA (<1%) for ACh + SP (D).


                              
View this table:
[in this window]
[in a new window]
 
Table 1. Effects of SP on ACh- and cytisine-evoked unitary currents in rat intracardiac neurons

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 beta 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).



View larger version (45K):
[in this window]
[in a new window]
 
Fig. 6. Cytisine-evoked single-channel currents obtained in the absence and presence of SP. A and B: unitary currents recorded from an excised outside-out membrane patch held at -60 mV and perfused with PSS containing either 3 µM cytisine (A) or 3 µM cytisine + 3 µM SP (B). Continuous line represents closed state (C), and dashed lines represent open states (O1, O2, O3). C and D: amplitude histograms of unitary currents obtained from the same patch. Histograms are fitted with Gaussian distributions with mean amplitudes of -0.96 ± 0.19 pA (48% of total fit area), -1.76 ± 0.12 pA (25%) and -2.59 ± 0.26 pA (27%) for cytisine (C) and -0.96 ± 0.18 pA (74%), -1.76 ± 0.13 pA (14%) and -2.56 ± 0.28 pA (12%) for cytisine + SP (D).

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).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 7. Effects of SP on unitary currents evoked by ACh or cytisine. A: mean current amplitude for the 3 conductance classes observed in the absence () and presence () of 3 µM SP when either 3 µM ACh (A1; n = 4) or 3 µM cytisine (A2; n = 4) was used as the agonist. B: mean apparent open time for events within a burst when either 3 µM ACh (B1; n = 4) or 3 µM cytisine (B2; n = 4) was used as the agonist in the absence () and presence () of 3 µM SP. C: bar graph of the mean open-channel probability for the 3 conductance classes in the absence () and presence () of 3 µM SP when either 3 µM ACh (C1; n = 4) or 3 µM cytisine (C2; n = 4) was used as the agonist.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha  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 alpha  and beta  subunits exhibited a differential sensitivity to SP with those containing beta 4 subunits having a 25-fold higher affinity than those having beta 2 subunits (Stafford et al. 1994). Chimeric and site-directed mutagenesis studies of beta  subunits coexpressed with alpha 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 beta 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 beta 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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society