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M₁ and M₂ Muscarinic Acetylcholine Receptor Subtypes Mediate Ca²⁺ Channel Current Inhibition in Rat Sympathetic Stellate Ganglion Neurons

¹Departments of Anesthesiology and ²Medicine, Penn State College of Medicine, Hershey, Pennsylvania; and ³Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland

Submitted 27 January 2006; accepted in final form 4 August 2006

	ABSTRACT

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Muscarinic acetylcholine receptors (mAChRs) are known to mediate the acetylcholine inhibition of Ca²⁺ channels in central and peripheral neurons. Stellate ganglion (SG) neurons provide the main sympathetic input to the heart and contribute to the regulation of heart rate and myocardial contractility. Little information is available regarding mAChR regulation of Ca²⁺ channels in SG neurons. The purpose of this study was to identify the mAChR subtypes that modulate Ca²⁺ channel currents in rat SG neurons innervating heart muscle. Accordingly, the modulation of Ca²⁺ channel currents by the muscarinic cholinergic agonist, oxotremorine-methiodide (Oxo-M), and mAChR blockers was examined. Oxo-M–mediated mAChR stimulation led to inhibition of Ca²⁺ currents through voltage-dependent (VD) and voltage-independent (VI) pathways. Pre-exposure of SG neurons to the M₁ receptor blocker, M₁-toxin, resulted in VD inhibition of Ca²⁺ currents after Oxo-M application. On the other hand, VI modulation of Ca²⁺ currents was observed after pretreatment of cells with methoctramine (M₂ mAChR blocker). The Oxo-M–mediated inhibition was nearly eliminated in the presence of both M₁ and M₂ mAChR blockers but was unaltered when SG neurons were exposed to the M₄ mAChR toxin, M₄-toxin. Finally, the results from single-cell RT-PCR and immunofluorescence assays indicated that M₁ and M₂ receptors are expressed and located on the surface of SG neurons. Overall, the results indicate that SG neurons that innervate cardiac muscle express M₁ and M₂ mAChR, and activation of these receptors leads to inhibition of Ca²⁺ channel currents through VI and VD pathways, respectively.

	INTRODUCTION

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Muscarinic acetylcholine receptors (mAChRs) play an important role in maintaining neuronal excitability. Cloning studies have revealed there are five mAChR subtypes (M₁–M₅; for review, see Wess 2004). These receptors belong to the G protein–coupled receptor superfamily. M₂ and M₄ mAChR subtypes couple to the pertussis (PTX)-sensitive G protein subunits, G_i/o, whereas M₁, M₃, and M₅ subtypes couple to PTX-insensitive G protein subunits, G_q/11.

In peripheral and central neurons, mAChR stimulation results in modulation of Ca²⁺ and K⁺ channels. The inhibition of Ca²⁺ channel currents is either voltage dependent (VD) and mediated by M₂ or M₄ mAChRs or voltage independent (VI) and occurs by odd-numbered mAChRs (Hille 1994). The VD inhibition of Ca²⁺ channels exhibits biophysical properties that consist of a biphasic Ca²⁺ current rising phase known as "kinetic slowing" and a relief of inhibition by a large depolarizing conditioning pulse termed "prepulse facilitation" (Elmslie et al. 1990; Ikeda and Dunlap 1999). On the other hand, the VI inhibition of Ca²⁺ currents is insensitive to membrane potential and involves a diffusible second messenger. The modulation of Ca²⁺ channels after mAChR stimulation has been well characterized in rat superior cervical ganglion (SCG) neurons and involves both VD and VI pathways. The former occurs through the M₄ and the latter through M₁ mAChR subtypes (Beech et al. 1992; Bernheim et al. 1991). Rat SCG neurons have also been reported to express M₂ mAChR, but Ca²⁺ channels do not couple to this receptor subtype (Fernandez-Fernandez et al. 1999).

Cardiac muscle is innervated by the sympathetic and parasympathetic branches of the autonomic nervous system. Previous electrophysiological studies in rat parasympathetic neurons have presented evidence that Ca²⁺ channels are modulated by M₂ mAChR in adults (Jeong and Wurster 1997) and through the M₄ receptor subtype in neonatal rats (Cuevas and Adams 1997). Stellate ganglion (SG) neurons represent the sympathetic branch of the autonomic nervous system that regulates cardiac function. Very few studies, however, have been carried out that have examined the signal transduction pathways in SG neurons. It is currently unknown which mAChR subtypes are expressed in SG neurons that modulate N-type Ca²⁺ currents. We have previously reported that the contribution of N-, P/Q-, and L-type Ca²⁺ channel subtypes to the total Ca²⁺ current in rat SG neurons are 54, 13, and 4%, respectively (Fuller et al. 2004). Similar findings have also been shown (Kukwa et al. 1998). Therefore the purpose of this study was to examine the mAChR modulation of N-type Ca²⁺ channels and to determine the expressed mAChR subtypes in acutely isolated SG ganglion neurons innervating cardiac muscle.

	METHODS

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Retrograde labeling and isolation of rat SG neurons

The experiments performed were approved by the Institutional Animal Care and Use Committee (IACUC). Ultrasound imaging was used to retrograde label rat SG neurons as previously described (Fuller et al. 2004). Adult male Wistar rats (225–450 g) were anesthetized with ketamine HCl (75 mg/kg) and xylazine (5 mg/kg), administered intraperitoneally. The heart was imaged on the ventral midline with a Sequoia C256 Echocardiography system (Siemens Medical Solutions, Mountain View, CA) equipped with a 14-MHz probe. Thereafter, DiIC₁₂(3) [1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, 1% in DMSO, Molecular Probes, Eugene, OR (DiI)] was injected into the ventricular muscle using a syringe with a 30-gauge needle. The volume of DiI injected ranged from 50 to 100 µl.

Four to 14 days after DiI injection, the rats were killed by CO₂ anesthesia and decapitated. The SG was removed and cleared of connective tissue in ice-cold Hanks' balanced salt solution. Thereafter, the SG was incubated for 60 min in a shaking water bath at 35°C in Earle's balanced salt solution with 0.6 mg/ml collagenase type D (Boehringer Mannheim, Indianapolis, IN), 0.4 mg/ml trypsin (Worthington Biochemicals, Freehold, NJ), and 0.1 mg/ml DNase type I (Sigma, St. Louis, MO). After the incubation period, the cells were dissociated in a culture flask by vigorous shaking, and the dispersed neurons were centrifuged twice for 6 min at 50g and resuspended in minimal essential medium (MEM; Gibco, Carlsbad, CA) supplemented with 10% fetal calf serum, 1% glutamine, and 1% penicillin-streptomycin solution (Gibco). Neurons were plated into 35-mm tissue culture plates coated with poly-L-lysine and stored in a humidified incubator containing 5% CO₂ in air at 37°C. Similar isolation protocol was performed in experiments where SCG neurons were isolated from adult rats.

Electrophysiology and data analysis

DiI-labeled SG neurons were identified with an inverted microscope (Diaphot 300, Nikon) equipped with an epifluorescence unit and G-2E/C filter cube set (Nikon) containing an excitation filter at 540 ± 12 nm, a dichroic beam splitter of 565 nm (long pass), and an emission filter at 620 ± 30 nm.

Ca²⁺ currents were recorded at room temperature (21–24°C) using the whole cell patch-clamp technique. The recording pipettes were pulled from borosilicate glass capillaries (Corning 7052, Garner Glass, Claremont, CA) on a Flaming-Brown (P-97) micropipette puller (Sutter Instrument Co., San Rafael, CA), coated with Sylgard (Dow Corning, Midland, MI) and fire polished. SG whole cell Ca²⁺ currents were acquired with a patch-clamp amplifier (Axopatch 1-C, Axon Instruments, Foster City, CA), analog filtered at 5–10 kHz (–3 dB; 4-pole Bessel), and digitized using custom designed software (S4) on a Power PC computer (Power Computing, Austin, TX) equipped with a 16-bit A/D converter board (ITC16, Instrutech, Elmont, NY). The cell's series resistance (80–85%) and membrane capacitance were electronically compensated. Data and statistical analyses were performed with the Igor Pro (Lake Oswego, OR) and Prism 4 (GraphPad Software, San Diego, CA) software packages, respectively, using one-way ANOVA with P < 0.05 considered statistically significant. Summary graphs and current traces were produced with the Igor Pro and Canvas 8.0 (Deneba Software, Miami, FL) software packages.

The pipette solution contained (in mM: 120 N-methyl-D-glucamine, 20 tetraethylammonium hydroxide (TEA-OH), 11 EGTA, 10 HEPES, 10 sucrose, 1 CaCl₂, 4 Mg-ATP, 0.3 Na₂GTP, and 14 Tris creatine phosphate. The pH was adjusted to 7.2 with methanesulfonic acid; the osmolality was 296–302 mosmol/kg. The external recording solution consisted of (in mM) 155 Tris hydroxymethylaminomethane, 20 HEPES, 10 glucose, 10 CaCl₂, and 0.0003 TTX. The pH was adjusted to 7.4 with TEA-OH, and the osmolality was 317–323 mosmol/kg. With these solutions, the free Ca²⁺ concentration was 10.5 nM.

The oxotremorine-methiodide (Oxo-M)–mediated concentration–response curve was determined by the sequential application of concentrations of the receptor agonist. No more than three different concentrations were used with each cell to avoid desensitization. The results were pooled, and each point represents mean ± SE. The concentration–response curve was fit to the Hill equation: I = I_max/(1 + IC₅₀/[Oxo-M])^nH), where I is the Ca²⁺ current inhibition, I_max is maximum inhibition, IC₅₀ is the half-inhibition concentration, [Oxo-M] is Oxo-M concentration, and nH is Hill coefficient.

Solution and drugs

Stock solutions of Oxo-M (Research Biochemicals International, Natick, MA), methoctramine (Meth), M₄-toxin, -conotoxin GVIA (all from Sigma), PTX toxin (List Biological Laboratories, Campbell, CA), M₁-toxin (Peptides International, Louisville, KY), and -agatoxin IVA (Alomone Labs) were prepared in H₂O. All drugs were diluted in the external solution to their final concentrations before use. Cells were pretreated for 5 min with M₁- and M₄-toxins before seal formation and maintained for the duration of the experiment. Neurons were pretreated with Meth (5 µM) for 2 min before Oxo-M application. The solutions containing M₁- and M₄-toxins also contained 0.1 mg/ml cytochrome c (Sigma) to prevent binding of the peptides to the perfusion lines. PTX was added to the culture medium (12–20 h) at a final concentration of 500 ng/ml. Percentage inhibition of muscarinic response was calculated as decrease in current of the third sweep in Oxo-M (30 s, to allow steady state to be reached) compared with the last sweep before application of drug. All drugs were applied to the neuron under study with a custom, gravity-fed perfusion system positioned 50 µm from the cell.

Immunofluorescence and deconvolution microscopy

SG and SCG neurons were plated into 35-mm glass bottom culture dishes coated with poly-D-lysine (MatTek, Ashland, MD) and incubated overnight in MEM prepared as described above. Cells were initially rinsed five times with 1x PBS, fixed in 2% formaldehyde/2% sucrose for 20 min, and rinsed again five times with 1x PBS. The cells were permeabilized with 0.05% TWEEN in 1x PBS + 5% goat serum for 10 min at 37°C and followed by a 15-min incubation in 1x PBS + 5% goat serum. The cells were incubated for 60 min with the primary antibody diluted in 1x PBS + 5% goat serum at room temperature. The primary antibodies used were rabbit anti-M₁ (1:200; Chemicon International, Temecula, CA or Research and Diagnostics Antibodies, Benicia, CA) and mouse anti-M₂ (1:200; Abcam, Cambridge, MA). Thereafter, the cells were rinsed five times with 1x PBS and incubated for 60 min at room temperature in 1x PBS + 5% goat serum. This was followed by incubating the cells with the secondary antibody in 1x PBS + 5% goat serum for 45 min at 4°C. The secondary antibodies used were Alexa Fluor 488 goat anti-rabbit Ig and Alexa Fluor 488 goat anti-mouse IgG1 (both from Molecular Probes) at a final concentration of 3–5 µg/ml. In a separate set of experiments, the primary or secondary antibodies were omitted from the solutions. Under these conditions, minimal or no fluorescence background was observed.

Fluorescence images were obtained with a Nikon TE2000U (Nikon) microscope using a x60 oil objective (plus x1.5 magnification), the X-Cite 120 (EXFO Life Sciences Group, Ontario, Canada) for illumination, and acquired with an Orca-ER 1394 digital CCD camera (Hamamatsu Photonics) and IPLab software (Scanalytics, Fairfax, VA). Fluorescence images of DiI-labeled neurons were obtained with a filter set (G-2E/C, Nikon) containing an excitation filter at 540 ± 15 nm, a dichroic beam splitter of 585 nm [long pass (LP)], and an emission filter at 620 ± 30 nm. Optical sections of Alexa Fluor 488–labeled neurons were obtained with a filter set (B-2E/C, Nikon) containing an excitation filter at 480 ± 15 nm, a dichroic beam splitter of 505 nm (LP), and an emission filter at 535 ± 20 nm. Section planes were collected in 0.3-µm steps covering the z-axis field using the Pro Scan II (Prior Scientific, Cambridge, UK) motorized stage. The acquired images were processed with the Huygens Essential software package (Scientific Volume Imaging, Hilbersum, The Netherlands) and pseudocolored with IPLab software.

Single-cell RT-PCR analysis

Single-cell RT-PCR was carried out using the OneStep RT-PCR Kit (Qiagen, Valencia, CA) and the following primer sequences (expected product sizes given in parenthesis)—GAPDH forward: CCA AAA GGG TCA TCA TCT CCG, GAPDH backward: AGA CAA CCT GGT CCT CAG TGT AGC (501 bp); M1 forward: AGC AGC TCA GAG AGG TCA CAG CCA, M1 backward: GGG CCT CTT GAC TGT ATT TGG GGA (273 bp); M2 forward: CAA GAC CCA GTA TCT CCA AGT CTG, M2 backward: CGA CGA CCC AAC TAG TTC TAC AGT (369 bp); M3 forward: ACA GAA GCG GAG GCA GAA AAC TTT, M3 backward: CTT GAA GGA CAG AGG TAG AGT AGC (561 bp); M4a forward: GTT CCG CCG TCT GTC CGG CAC C, M4a backward: CAC AAC AGT CAC CAG GCT CAG GGA G (219 bp); and M4b forward: GTT CCG CCG TCT GTC CGG CAC C, M4b backward: CCA GCC ACA AGT CAC AGA CCA CG (403 bp).

Acutely dissociated SG neurons were rinsed twice in a solution containing (in mM) 130 NaCl, 5.4 KCl, 10 HEPES, 0.8 MgCl₂, 10 CaCl₂, 15 glucose, and 15 sucrose. Thereafter, neurons were collected into SigmaCote-coated (Sigma) glass capillaries (Corning 7052) with a final volume of 10–15 µl. The number of neurons collected per capillary ranged from three to six. The cells were placed in PCR tubes containing reagents supplied by the manufacturer (Qiagen). Marathon Ready whole rat brain cDNA (Clontech) was used as a positive control. The tubes were placed in a PCR thermocycler (Eppendorf) and subjected to the following protocol: 94°C for 1 min, 58°C for 1 min, and 74°C for 1.5 min, for a total of 44 cycles. The PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining and UV illumination. Gel images were captured using a digital gel imaging system (Eastman Kodak, Rochester, NY).

	RESULTS

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The muscarinic modulation of N-type Ca²⁺ channels in SG neurons innervating rat myocardium was examined in this study. Previous reports have shown N-type Ca²⁺ channels contribute most of the total Ca²⁺ current in these neurons (Fuller et al. 2004; Kukwa et al. 1998). The first set of experiments was carried out to determine the concentration–response relationship of the Oxo-M–mediated Ca²⁺ channel current inhibition. Ca²⁺ currents were evoked every 10 s with a 50-ms depolarizing pulse to +10 mV from a holding potential of –80 mV (Fig. 1A, top). The Ca²⁺ current amplitude was measured isochronally 10 ms into the test pulse in the presence and absence of agonist. Figure 1B is a plot of the time-course of Ca²⁺ current amplitude obtained before and after the sequential application of 0.1 and 1 µM Oxo-M. Beforeo Oxo-M application, the current amplitude was nearly 2.5 nA. After application of 0.1 µM Oxo-M, the Ca²⁺ current was blocked by 25% to 1.9 nA (Fig. 1, A and B). After the recovery of the Ca²⁺ current, exposure of the cell to 1 µM Oxo-M resulted in a 55% inhibition of the current amplitude to 1.1 nA. The results of the Oxo-M concentration–response curve are plotted in Fig. 1C. The Ca²⁺ current inhibition was normalized to 10 µM Oxo-M (highest concentration used) and plotted against the log[Oxo-M]. The data were fitted to the Hill equation, and the estimated EC₅₀ was 49 nM and a Hill coefficient of 0.75 (n = 3–9 neurons). A separate group of neurons was pretreated with PTX to determine the contribution of the non–PTX-mediated pathway. The Oxo-M–induced Ca²⁺ current inhibition was attenuated after PTX treatment, and the EC₅₀ and Hill coefficient were 816 nM and 1.5 (n = 2–11 neurons), respectively. The results, plotted in Fig. 1C, show the rightward shift of Ca²⁺ current inhibition by PTX treatment. Figure 1D shows the Ca²⁺ current I-V curves recorded from an SG neuron before and during Oxo-M (10 µM) exposure. The Ca²⁺ currents were evoked from a holding potential of –80 mV to various depolarizing steps every 3 s. The maximal Oxo-M–mediated inhibition occurred over the range of –10 to +35 mV. The difference current shows that no significant voltage shift occurred in the presence of Oxo-M; similar results were obtained in six neurons.

View larger version (21K): [in this window] [in a new window]   FIG. 1. Oxotremorine-methiodide (Oxo-M) concentration–response relationship of DiIC12(3) (1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled stellate ganglion (SG) neurons. A: superimposed Ca2+ current traces from an SG neuron were evoked every 10 s by a single 50-ms test pulse to +10 mV from a holding potential of –80 mV before and during agonist application. B: time-course of Ca2+ current amplitude inhibition acquired from the sequential application of 0.1 and 1 µM Oxo-M to neuron in A. C: summary of the Oxo-M–mediated Ca2+ current inhibition in control () and pertussis (PTX)-treated (500 ng/ml; ) SG neurons. Peak Ca2+ current inhibition was normalized to peak current evoked by 10 µM Oxo-M. Data point represents mean ± SE, except for 0.01 µM Oxo-M in PTX-treated cells, where n = 2. Numbers in parentheses indicate number of experiments. D: current-voltage (I-V) curve of Ca2+ currents of a DiI-labeled SG neuron elicited by 70-ms depolarizing pulses from a holding potential of –80 mV to different test potentials from –80 to +80 mV before () and during () exposure to 10 µM Oxo-M. , difference in current.

The subtype of mAChRs that couples to Ca²⁺ channels was examined in the next set of experiments. The double-pulse voltage paradigm (Fig. 2A, top) was used to determine the presence of either VD (e.g., M₂ or M₄) or VI (e.g., M₁ or M₃) components of the Oxo-M–mediated Ca²⁺ current modulation. The voltage protocol consists of a test pulse to +10 mV (prepulse), followed by a conditioning pulse to +80 mV, a return to –80 mV, and a second test pulse to +10 mV (postpulse) before returning to –80 mV. Figure 2A shows Ca²⁺ current traces that were evoked with this paradigm. Before agonist application, the current trace (bottom trace) shows that the prepulse amplitude is slightly less than the postpulse amplitude, a result of low-level tonic G protein activation (Ikeda 1991). Application of 10 µM Oxo-M resulted in a greater inhibition of the Ca²⁺ current during the prepulse than the postpulse (top trace), indicating that the modulation is both VD and VI. The VD inhibition of the currents can be observed by the kinetic slowing of the prepulse current and facilitation of the postpulse current. The summary plot in Fig. 2E shows the Oxo-M–mediated Ca²⁺ current inhibition for prepulse (filled bars) and postpulse (empty bars). Figure 2B shows the effect of pretreatment with the M₁ mAChR subtype blocker, M₁-toxin (0.1 µM) on Ca²⁺ currents. The M₁-toxin has been shown to have a greater selectivity for M₁ over M₂–M₅ mAChR with an estimated IC₅₀ of 90 nM (Carsi and Potter 2000). The mean Ca²⁺ current inhibition of the prepulse (54.8 ± 6.1%) was significantly less (P < 0.01) compared with control neurons (68.5 ± 1.6%; Fig. 2E). The facilitation ratios (postpulse current/prepulse current amplitude) of control and M₁-toxin–treated cells increased from 1.38 ± 0.03 to 2.12 ± 0.04 and from 1.20 ± 0.05 to 2.22 ± 0.12 after Oxo-M application, respectively. Next, a separate group of cells were pretreated overnight with PTX to remove the VD component (e.g., G_i/o-coupled M₂ and M₄ mAChR). The current trace in Fig. 2C shows that PTX pretreatment resulted in significantly less (P < 0.01) inhibition of the prepulse current (36.0 ± 4.0%) compared with control neurons. In addition, the facilitation ratio was 1.16 ± 0.02 before exposure to Oxo-M and 1.30 ± 0.02 after Oxo-M application, indicating the inhibition of the currents was mostly VI. The current trace in Fig. 2D and the summary in Fig. 2E show that the Oxo-M–mediated Ca²⁺ current modulation was completely abolished (2.7 ± 1%) when SG neurons were pretreated with both PTX and M₁-toxin. These results suggest that the VI modulation of Ca²⁺ currents in SG neurons is mediated by the G_q/11-coupled M₁ mAChR and not through M₃ or M₅ receptors.

View larger version (29K): [in this window] [in a new window]   FIG. 2. Oxo-M–mediated Ca2+ current inhibition is voltage dependent (VD) in the presence of the M1 mAChR blocker (M1-toxin) and voltage independent (VI) after PTX pretreatment. Superimposed Ca2+ current traces from DiI-labeled neurons evoked with the double-pulse protocol (A, top) in the absence (bottom traces) and presence (top traces) of 10 µM Oxo-M in control (A) and in cells exposed to 0.1 µM M1-toxin (B), pretreated overnight with PTX (500 ng/ml, C), and treated with both M1-toxin and PTX (D). Ca2+ current amplitude was measured isochronally 10 ms after initiation of both prepulse and postpulse. E: summary graph showing mean ± SE prepulse (solid bars) and postpulse (empty bars) Ca2+ current inhibition mediated by Oxo-M in control and in neurons treated with M1-toxin, PTX, and M1-toxin + PTX. *P < 0.01 compared with control. Numbers in parentheses indicate number of experiments.

View larger version (30K): [in this window] [in a new window]   FIG. 3. Application of the M2 muscarinic acetylcholine receptor (mAChR) blocker methoctramine results in VI modulation, whereas the M4 mAChR toxin is without effect. Superimposed Ca2+ current traces from DiI-labeled neurons evoked with the double-pulse protocol (Fig. 2A, bottom) in the absence (bottom traces) and presence (top traces) of 10 µM Oxo-M in control (A) and in cells exposed to 5 µM methoctramine (B), 5 µM methoctramine +0.1 µM M1-toxin (C), and 0.2 µM M4-toxin (D). E: summary graph showing mean ± SE prepulse (solid bars) and postpulse (empty bars) Ca2+ current inhibition mediated by Oxo-M in control (Con) and in neurons treated with 10 methoctramine (Meth) alone or in the presence of 0.1 µM M1-toxin (Meth and M1-toxin) and in the presence of M4-toxin. *P < 0.01 compared with control. Numbers in parentheses indicate number of experiments.

View larger version (43K): [in this window] [in a new window]   FIG. 4. SG neurons express M1 and M2 mAChR as revealed by immunofluorescence. Deconvolution fluorescence (A and C) and fluorescence (B and D) images of SG neurons isolated from an adult rat after DiI injection to cardiac muscle. Rabbit antibody to M1 (A) and mouse antibody to M2 (C) were the primary antibodies used and were followed by appropriate Alexa Fluor 488-labeled secondary antibodies. Neurons were imaged at x60 (plux x1.5 aux. magnification), and sections were collected in 300-nm steps covering the entire thickness of cells with a filter set containing an excitation filter at 480 nm and an emission filter at 535 nm. Images were deconvolved and pseudocolored. DiI fluorescence images (B and D) were obtained with a filter set specific for DiI (540-nm excitation and 620-nm emission) and pseudocolored; scale bar represents 20 µm.

View larger version (19K): [in this window] [in a new window]   FIG. 5. mAChR subtype mRNA expression in rat SG neurons and whole brain. Single-cell RT-PCR analyses of RNA isolated from DiI-labeled SG neurons (A) and rat brain (B) using primer pairs for M1–M4 mAChR. PCR products were separated by 1.3% agarose gel electrophoresis. Expected molecular size for each PCR product is indicated on the top of each gel. Number of SG neurons tested for each mAChR subtype ranged from 3 to 6 cells. Approximately 40 ng of whole brain mRNA was used per reaction. SG neurons were sampled from 3 separate rat dissections.

	DISCUSSION

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Normally, the modulation of N-type Ca²⁺ channel currents mediated by mAChR activation is analyzed by examining the biophysical characteristics of the currents when the double-pulse voltage protocol is used (Elmslie et al. 1990; Ikeda and Dunlap 1999). The VD inhibition of the currents occurs through the PTX-sensitive, G_i/o-coupled M₂ or M₄ mAChRs. On the other hand, the VI inhibition occurs through the PTX-resistant, G_q/11-coupled M₁, M₃, or M₅ mAChR subtypes (for review, see Hille 1994). In this study, application of the mAChR agonist Oxo-M resulted in both VD inhibition, as observed by the kinetic slowing and facilitation of the post-pulse current and VI inhibition of Ca²⁺ currents. The estimated EC₅₀ (49 nM) observed was comparable with that recorded in rat cardiac parasympathetic neurons (41 nM) (Jeong and Wurster 1997) and 10-fold lower than that observed in rat SCG neurons (Bernheim et al. 1992), magnocellular cholinergic basal forebrain neurons (Allen and Brown 1993), and neostriatal cholinergic interneurons (Yan and Surmeier 1996). However, the EC₅₀ of the PTX-treated SG neurons was similar to that reported in rat SCG neurons also pretreated with PTX (Bernheim et al. 1992) and both presumably activated M₁ mAChRs. The observed differences can be reconciled when considering that different mAChR are expressed in these neuron types and/or the same receptor subtypes expressed may not couple to Ca²⁺ channels. That is, in rats, Ca²⁺ channels are modulated by M₁ and M₂ in SG neurons (this study), M₂ in magnocellular cholinergic basal forebrain and intracardiac neurons (Allen and Brown 1993; Jeong and Wurster 1997), M₁ and M₄ in SCG (Bernheim et al. 1992) and striatal neurons (Howe and Surmeier 1995), and M₂ and M₄ in neostriatal cholinergic interneurons (Yan and Surmeier 1996). Finally, a separate study has shown in rat pyramidal neurons that N- and P-type Ca²⁺ channels are modulated by PTX-sensitive G-coupled mAChRs in a VD manner (Stewart et al. 1999). However, the specific mAChR subtype (i.e., M₂ or M₄) was not identified.

The electrophysiological results in this study showed that the VI inhibition of Ca²⁺ currents in SG neurons is mediated by the M₁ mAChR subtype. Application of the specific M₁ receptor blocker M₁-toxin resulted in VD inhibition of the Ca²⁺ currents. This toxin has been successfully used previously to eliminate the VI component observed in rat SCG neurons (Kammermeier et al. 2000). These findings are also consistent with those previously described in rat (Beech et al. 1992; Bernheim et al. 1991; Delmas et al. 1998) and mice SCG (Shapiro et al. 1999) and rat sympathetic major pelvic ganglion (MPG) neurons (Zhu and Yakel 1997). In rat MPG neurons, however, the mAChR subtype (M₁, M₃, or M₅) responsible for the VI modulation of the Ca²⁺ channels was not identified (Zhu and Yakel 1997). In this study, the M₃ or M₅ mAChR subtypes did not seem to contribute to the modulation of the currents because both M₁-toxin and PTX abolished the Oxo-M–mediated Ca²⁺ current inhibition. This conclusion is further supported by the single-cell RT-PCR assays that showed the M₃ mAChR mRNA was not present in DiI-labeled neurons. Finally, using the M₁-specific antibodies confirmed that M₁ receptors are present on the cell membrane and are responsible for the VI modulation of the Ca²⁺ channels.

As previously mentioned, mAChR are capable of modulating K⁺ channel currents. One of these K⁺ currents is known as the M-current, originally described in bullfrog sympathetic neurons (Brown and Adams 1980). The inhibition of M-currents through mAChR activation results in increased cell excitability and the ability of neurons to maintain a high-frequency discharge (Brown et al. 1997). In rat SCG neurons, activation of M₁ receptors not only results in Ca²⁺ current inhibition, but also inhibits M-currents through G_q/11 proteins (Brown et al. 1997; Marrion et al. 1989). M-currents were recorded in five SG neurons, and the application of Oxo-M (10 µM) resulted in inhibition of M currents by 94 ± 1% (Ruiz-Velasco, unpublished observations). Thus M₁ mAChR expressed in SG neurons are capable of modulating both N-type Ca²⁺ and M-currents, in agreement with previous observations (Beech et al. 1992; Bernheim et al. 1991; Delmas et al. 1998).

Application of Meth revealed that the VD inhibition of Ca²⁺ currents was mediated through M₂ mAChRs. The immunofluorescence and single-cell RT-PCR results presented in this study showed that the M₂ receptor subtype is expressed and located on the cell surface of SG neurons. These observations are similar to those found in adult rat parasympathetic neurons innervating cardiac muscle (Jeong and Wurster 1997). However, in neonatal rats, electrophysiological and pharmacological studies have shown that only M₄ mAChR modulate N-type Ca²⁺ channels (Cuevas and Adams 1997). It is interesting to note that both studies showed evidence that odd-numbered mAChRs do not couple to Ca²⁺ channels. The mAChR subtype expressed in neonatal rat SG neurons innervating cardiac muscle remains to be determined. Our data and the aforementioned studies suggest that expression of mAChRs in SG and parasympathetic neurons innervating heart muscle may be related to age (i.e., M₂ and M₄ mAChR are expressed in adults and neonates, respectively). Because both M₂ and M₄ receptors modulate Ca²⁺ currents in a VD manner and use PTX-sensitive G protein subunits, the reason for the exclusive expression of one receptor subtype over the other is uncertain.

Rat SCG neurons express M₂ mAChRs, but this receptor subtype does not seem to couple to N-type Ca²⁺ channels (Fernandez-Fernandez et al. 1999). This coupling selectivity was shown by heterologously expressing G protein–gated inwardly rectifying K⁺ (GIRK) channels in SCG neurons. M₂ and M₄ mAChR activation modulated GIRK and Ca²⁺ currents through G_o and G_i G proteins, respectively. Unlike rat SCG neurons, our data suggest that M₄ mAChR are not present in SG neurons. First, exposure of SG neurons to the selective M₄ mAChR M₄-toxin had no effect on the Oxo-M–mediated inhibition of Ca²⁺ currents. The amount used (0.2 µM), however, did block the M₄-mediated Ca²⁺ current inhibition by Oxo-M in SCG neurons. This concentration is well over the EC₅₀ (11 nM) reported in rat parasympathetic neurons (Cuevas and Adams 1997). Second, single cell RT-PCR of SG neurons failed to detect M₄ mAChR mRNA. Brain tissue, a positive control, did show expression of the M₄ mAChR message. Interestingly, a recent study using mAChR knockout mice found that the muscarinic inhibition of N-type Ca²⁺ channels occurred through both M₁ and M₂ mAChRs and not M₄ receptor subtype (Shapiro et al. 1999).

In sympathetic postganglionic processes, the inhibition of NE release occurs primarily through the VD Ca²⁺ current inhibition after stimulation of PTX-sensitive mAChRs (Dunlap et al. 1995; Hille 1994). Previous experiments have shown that exogenously applied Oxo-M decreased the stimulation-evoked overflow of radiolabeled NE from sympathetic nerves innervating mice cardiac muscle (Schelb et al. 2001). A separate study using mAChR knockout mice also found that electrically evoked sympathetic tritriated NE release in atrial muscle was significantly decreased, but not abolished, after carbachol application in M₂ receptor-knockouts and unaffected in M₄ receptor-knockouts (Trendelenburg et al. 2003). In a subsequent report, the authors presented evidence showing that the postganglionic sympathetic neurons innervating the atrium use both M₂ and M₃ mAChR subtypes to inhibit NE release (Trendelenburg et al. 2005). ACh release by preganglionic sympathetic fibers, on the other hand, involves the modulation of ion channels in the cell soma of postganglionic neurons, particularly through stimulation of mAChR. It is generally accepted that the response of sympathetic neuron soma to ACh is mediated through M₁ receptor stimulation with subsequent M current inhibition (Brown et al. 1997; Delmas et al. 1998). In addition, N-type Ca²⁺ channel inhibition would decrease Ca²⁺ entry and attenuate the hyperpolarization by inhibiting Ca²⁺-activated K⁺ channels. Thus it seems that the ACh-mediated inhibition of Ca²⁺ channels in rat SG neurons is associated with 1) a negative feedback regulation of NE secretion through M₂ mAChRs at the nerve terminals and 2) a reduced Ca²⁺ influx at the soma to facilitate and maintain a high-frequency discharge through M₁ mAChRs (Delmas et al. 1998).

In conclusion, the results of this study show that adult rat cardiac sympathetic SG neurons use both M₁ and M₂ mAChRs to modulate Ca²⁺ currents. Activation of the M_I receptor uses the PTX-insensitive G protein subunits and produces VI regulation of the Ca²⁺ channel currents. The M₂ mAChRs couple to Ca²⁺ channels through PTX-sensitive G protein (G_i/o) subunits and modulate Ca²⁺ currents in a VD manner. Furthermore, both M₁ and M₂ receptors are expressed on the cell surface as indicated by immunofluorescence.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study was supported by National Heart, Lung, and Blood Institute Grant HL-074311 to V. Ruiz-Velasco.

	FOOTNOTES

 
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.

Address for reprint requests and other correspondence: V. Ruiz-Velasco, Dept. of Anesthesiology, H187, Penn State College of Medicine, 500 University Dr., Hershey, PA 17033-0850 (E-mail: vruizvelasco{at}psu.edu)

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