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7-nAChR-mediated cerebral nitrergic neurogenic vasodilation
Departments of 1Pharmacology and 4Neurology, School of Medicine, Southern Illinois University, Springfield, Illinois; 2Institute of Pharmacology and Toxicology, Tzu Chi University Center for Vascular Medicine, College of Life Sciences, and Neuro-Medical Scientific Center, Tzu Chi General Hospital, Hualien, Taiwan; and 3National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland
Submitted 11 November 2005 ; accepted in final form 7 February 2006
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ABSTRACT |
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7-nicotinic acetylcholine receptors (7-nAChRs) by nicotinic agonists releases norepinephrine, which then acts on parasympathetic nitrergic nerves, resulting in release of nitric oxide and vasodilation. Using patch-clamp electrophysiology, immunohistochemistry, and in vitro tissue bath myography, we tested this axo-axonal interaction hypothesis further by examining whether blocking norepinephrine reuptake enhanced 7-nAChR-mediated cerebral nitrergic neurogenic vasodilation. The results indicated that choline- and nicotine-induced 7-nAChR-mediated nitrergic neurogenic relaxation in endothelium-denuded isolated porcine basilar artery rings was enhanced by desipramine and imipramine at lower concentrations (0.03–0.1 µM) but inhibited at higher concentrations (0.3–10 µM). In cultured superior cervical ganglion (SCG) neurons of the pig and rat, choline (0.1–30 mM)-evoked inward currents were reversibly blocked by 1–30 µM mecamylamine, 1–30 µM methyllycaconitine, 10–300 nM -bungarotoxin, and 0.1–10 µM desipramine and imipramine, providing electrophysiological evidence for the presence of similar functional 7-nAChRs in cerebral perivascular sympathetic neurons of pigs and rats. In 7-nAChR-expressing Xenopus oocytes, choline-elicited inward currents were attenuated by -bungarotoxin, imipramine, and desipramine. These monoamine uptake inhibitors appeared to directly block the 7-nAChR, resulting in diminished nicotinic agonist-induced cerebral nitrergic vasodilation. The enhanced nitrergic vasodilation by lower concentrations of monoamine uptake inhibitors is likely due to a greater effect on monoamine uptake than on 7-nAChR blockade. These results further support the hypothesis of axo-axonal interaction in nitrergic regulation of cerebral vascular tone.
imipramine; desipramine; in vitro tissue bath; porcine basilar artery
7-nicotinic acetylcholine receptors (nAChRs) on the postganglionic sympathetic nerve terminals originating in the superior cervical ganglion (SCG), but not on the parasympathetic nitrergic neurons, and that these receptors play important roles in mediating cerebral nitrergic neurogenic vasodilation (25, 28). Activation of these sympathetic 7-nAChRs by nicotine and choline releases norepinephrine (NE), which acts on presynaptic 
2-adrenoceptors on the neighboring parasympathetic nitrergic nerve terminals, resulting in release of nitric oxide (NO) and vasodilation (14, 24–26). Accordingly, blockade of sympathetic neuronal uptake of NE would be expected to enhance nicotine- and choline-elicited 7-nAChR-mediated nitrergic vasodilation.
Several tricyclic antidepressants, such as imipramine and desipramine, are monoamine uptake inhibitors (19). These drugs have been shown to block NE reuptake into sympathetic neurons as well as antagonize nAChRs (11). The possible antagonistic effects of these monoamine uptake inhibitors on 7-nAChR-mediated cerebral neurogenic nitrergic vasodilation were also examined in the present study. Our results indicate that imipramine and desipramine at lower concentrations enhanced, but at higher concentrations attenuated, choline- and nicotine-induced cerebral nitrergic neurogenic vasodilation. The former effect of these monoamine uptake inhibitors is probably due to increased synaptic NE via blockade of its neuronal reuptake. The latter is most likely due to blockade of perivascular sympathetic 7-nAChRs by these inhibitors.
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MATERIALS AND METHODS |
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The care and use of animals reported on in this study were approved by the Southern Illinois University of Medicine Laboratory Animal Care and Use Committee and were in accordance with National Institutes of Health guidelines.
Rat and porcine SCG neurons were isolated and cultured as described in our previous reports (15, 25, 26). Briefly, SCGs from 6- to 10-wk-old male Sprague-Dawley rats or 60- to 100-kg adult pigs of either sex were carefully dissected bilaterally and placed in cold Hibernate A solution. The ganglia were minced into smaller pieces, transferred to Mg2+- and Ca2+-free Hanks' balanced salt solution containing 1 mg/ml collagenase D and 1 mg/ml trypsin (for rats) or 2 U/ml papain, 1.2 mg/ml collagenase D, and 4.8 mg/ml dispase (for pigs), and then incubated for 60 min at 37°C with shaking (150 rpm). Cells were released by gentle trituration at the end of the incubation. The cell suspension was centrifuged at 300 g for 5 min. The pellet was gently resuspended in neurobasal culture medium containing B27 (1:50 dilution), 0.5 mM L-glutamine, 100 U/ml antibiotic, and 100 ng/ml nerve growth factor. The cell suspension was plated into a four-well culture plate with a poly-D-lysine-coated (50 µg/ml; Sigma-Aldrich) glass coverslip (12-mm diameter; Carolina Biological Supply, Burlington, NC) in each well and maintained at 37°C in a humidified incubator with air containing 5% CO2.
Electrophysiology
Cultured SCG neurons of the pig and rat. Electrophysiological study by patch-clamp recording was carried out as described previously (15). Briefly, a glass coverslip containing cultured neurons was transferred from the growth medium to a recording chamber (model RC-26, Warner Instruments, Hamden, CT) containing the extracellular recording solution (see below) on a phase-contrast microscope (model IMT2, Olympus, Tokyo, Japan). The nicotinic currents were recorded in the whole cell configuration of the patch-clamp technique (10, 15) at room temperature.
Recording electrodes were manufactured from capillary glass (1.5-mm OD, 1.0-mm ID; model PG52151-4, World Precision Instruments, Sarasota, FL) using a glass microelectrode puller (model PP-830, Narishige, Tokyo, Japan), and the tips were fire polished using a microforge (model MF-830, Narishige). After the electrodes were filled with "intracellular solution" (see below), electrode impedance in the extracellular recording solution was 3–5 M
. This component of the series resistance was fully compensated using the bridge balance control of the Axoclamp 2B (Axon Instruments, Foster City, CA) used for recording. The electrode tip potential was also subtracted during the bridge mode. Tight seals >5 G were obtained by light suction. Voltage protocol generation and data acquisition were performed using pClamp software (Axon Instruments) and a digital data acquisition system (Digidata 1200, Axon Instruments). To evoke nicotinic currents, agonists were applied to cells that were kept at a holding potential of –60 mV. The current traces were low-pass filtered at 3 kHz and digitized at 10 kHz. Data were stored on a computer hard drive for later analysis.
Neurons were identified by their phase-bright appearance and spherical cell bodies using an inverted microscope with a x20 objective (model IMT2, Olympus). To limit the problems associated with uncontrolled voltage changes in long neurites, all patch-clamp experiments were carried out using SCG neurons during the first 4 days in culture. To minimize space-clamp problems, isolated cells without neurites or with short processes were selected for this study. Standard extracellular solution was constantly perfused at 2 ml/min.
Solutions and drug applications. Extracellular solution contained (in mM) 140 NaCl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES. Intracellular solution contained (in mM) 145 K-gluconate, 10 KCl, 1 EGTA, 10 HEPES, 5 K-ATP, and 0.25 GTP. ATP and GTP were used in the intracellular solution to prevent, to a large extent, the rundown of currents. The pH was adjusted to 7.35 using NaOH and osmolarity was adjusted to 320 mosM using sucrose to prevent activation of proton-sensitive or mechanoreceptive currents.
In experiments requiring precise timing of the onset and offset of drug application, a Master-8 (AMPI, Jerusalem, Israel)-controlled perfusion fast-step solution exchange system (model SF-77B, Warner Instrument) was used for rapid focal application of drugs onto SCG neurons (especially for agonists to minimize loss of response due to receptor desensitization). This system used a three-barreled glass perfusion head (<20-ms exchange at the cell). The distance from the perfusion head to the cell was 
200 µm, with flow controlled manually using a micromanipulator. External drug application was accomplished by gravity flow from a linear array of quartz tubes. The recording chamber was continually perfused, and the cells were exposed to a constant flow of bath solution between drug applications. To avoid possible degradation of nicotine because of its high sensitivity to light, the tube containing nicotine was covered with aluminum foil.
7-nAChR-Expressed Oocytes
Stage V and VI oocytes from Xenopus laevis were harvested and 7-nAChR RNA was injected using a nanoinjector (Drummond, Broomall, PA). The oocytes were maintained at 18°C. Membrane currents were recorded 2 days after the injection. The bath solution, ND96, contains (in mM) 96 NaCl, 2 KCl, 1.0 MgCl2, 1.8 CaCl2, and 5.0 HEPES (pH 7.5). During recording, the oocytes were continuously perfused with the bath solution at a rate of 10 ml/min. Stock solution of drugs was diluted in ND96 solution. Choline (0.3 mM) was applied directly onto the oocytes.
Two-electrode voltage clamp for the whole oocyte recording was performed at room temperature by using an amplifier (model OC-725C, Warner Instruments; unpublished observations). The borosilicate glass capillaries (1.5 mm OD; World Precision Instruments) were pulled using a microelectrode puller (model P-97, Sutter, Novato, CA). When filled with 3 M KCl, the resistance of the electrodes was 0.1–1 M. The membrane potential was held at –60 mV. Data acquisition and analysis were performed with pClamp 9.0 and Digidata 1322A (Axon Instruments). The traces were filtered at 1 kHz and sampled at 2 kHz. The maximum inward current was determined as the current amplitude. To compensate for the difference in the 7-nAChR expression level, the data were normalized and expressed as percentage of the choline-induced response.
For examination of effects of experimental drugs on choline-induced responses, the experimental drugs dissolved in the bath solution were perfused continuously into the bath chamber for 5 min. Choline was then applied for 1 s directly onto the oocytes to ensure its rapid interaction with 7-nAChRs. After responses of the experimental drugs on choline-induced inward currents had been established and the currents returned to the baseline, the experimental drugs and choline were washed out by switching to perfusate containing bath solution. After 10 min, the response to choline alone was repeated to obtain an additional control. Between drug applications, the oocytes were perfused continuously with the bath solution.
Immunohistochemistry.
SCG neurons cultured for 3–7 days were fixed in 4% paraformaldehyde for 20–60 min at room temperature or overnight at 4°C. After they were rinsed three times with PBS (pH 7.4), the cells were permeabilized, and nonspecific sites were blocked with 5% normal goat serum in 0.2% Triton X-100-PBS for 1 h at room temperature. The cells were rinsed again and then incubated simultaneously with neurofilament and anti-7-receptor antibody at room temperature overnight. The anti-7-receptor antibody was a monoclonal antibody raised from rat (1:100 dilution). The antibodies were diluted in 0.05% Triton X-100-PBS-1.5% normal goat serum. After incubation with the primary antibodies, the cells were rinsed with PBS three times before they were incubated with secondary antibodies. The secondary antibodies were fluorescein-conjugated anti-rabbit IgG (1:40 dilution) and rhodamine-conjugated anti-rat IgG (1:40 dilution). After 1 h of incubation with secondary antibodies at room temperature, the cells were rinsed three times with PBS (pH 8.2) and mounted in Vectashield mounting medium. The stained cells were observed and photographed under a fluorescence microscope (model BX50, Olympus) fitted with a fluorescein filter. With no change in field, the cells were photographed again using a rhodamine filter. Negative controls were obtained following the same incubation procedure with serum neutralized by corresponding antigen (15, 25).
In vitro tissue bath studies. In vitro tissue bath studies were carried out according to methods described in our previous reports (25, 26). Briefly, an artery ring segment was cannulated with a stainless steel rod and a short piece of platinum wire in a plastic tissue bath containing Krebs bicarbonate solution. The stainless steel rod was connected to a strain-gauge transducer for isometric recording of changes in force. The basilar artery ring segments were equilibrated in the Krebs solution for an initial 30 min and then mechanically stretched to a resting tension of 750 mg and contracted with 0.3–3 µM U-46619 to induce an active muscle tone of 0.5–0.75 g. Transmural nerve stimulation (TNS) at 8 Hz and nicotinic agonists at 0.1–1,000 µM were applied to induce relaxation. After relaxation induced by 8-Hz TNS and nicotinic agonists, the arteries were washed with prewarmed Krebs solution. A similar magnitude of active muscle tone was induced again with U-46619, and TNS was repeated (to serve as a control for comparison with the relaxation elicited by TNS before the wash). Experimental drugs were administered 30 min before TNS was repeated and before nicotinic agonists were applied at the same concentration used before the wash. The neurogenic origin of this TNS-induced response was verified by its complete blockade by 0.3 µM tetrodotoxin. The magnitude of a vasodilator response was expressed as a percentage of the maximum relaxation induced by 100 µM papaverine, which was added at the end of each experiment. The endothelial cells of all artery ring segments were mechanically removed. Complete removal of endothelial cells was verified by failure of nitro-L-arginine to increase basal tone (25).
Drugs.
Collagenase D was obtained from Roche Applied Science (Indianapolis, IN); Hibernate A, Hanks' balanced salt solution, dispase, and neurobasal culture medium from Life Technologies (Rockville, MD); nerve growth factor from Alomome Laboratories (Jerusalem, Israel); B27 from Invitrogen (Carlsbad, CA); anti-7-receptor antibody, fluorescein-conjugated anti-rabbit IgG, and Vectashield mounting medium from Vector Laboratories (Burlingame, CA); rhodamine-conjugated anti-rat IgG from Jackson Immunoresearch Laboratories (West Grove, PA); and acetylcholine chloride, -bungarotoxin (-BuTX), choline chloride, desipramine hydrochloride (DES), imipramine hydrochloride (IMI), mecamylamine (Mec), methyllycaconitine citrate (MLA), nicotine tartrate, nitro-L-arginine, papain, papaverine, poly-D-lysine, Triton X-100, trypsin, tetrodotoxin, U-46619, and all other reagents from Sigma-Aldrich (St. Louis, MO). Pharmacological agents were prepared as stock solutions (1 or 10 mM) and diluted to their required concentrations with extracellular solution just before the experiments. Choline was directly dissolved in extracellular solution.
Data Analysis and Statistics
For whole cell currents, Clampfit 6.0 (Axon Instruments) was used to determine current amplitude and rise/decay values. These values were exported to SigmaPlot 8 (SPSS, Chicago, IL) for statistical analysis and display. For individual agonist applications, peak amplitude values that were >10 times the baseline noise were characterized as "responsive." Values are means ± SE. Mean values for each parameter obtained from a number of neurons in different treatment groups were compared using Student's t-test.
Concentration-response curves were plotted on the logarithmic scale.
The following equation was used to evaluate the half-maximal effective concentration (EC50) of choline in the concentration-response relation: y = Emax(xn/EC
+ xn). The following equation was used to fit concentration curves to obtain half-maximal inhibition concentration (IC50): y = min + (max – min)/[1 + 10
].
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RESULTS |
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Consistent with our previous reports (14, 25, 28), porcine basilar arteries (without endothelial cells) in the presence of active muscle tone induced by 0.3 µM U-46619 relaxed exclusively on 8-Hz TNS and applications of 100 µM nicotine and 1 mM choline (Fig. 1). The relaxation induced by nicotine and choline was significantly reduced by 0.3 µM tetrodotoxin and was abolished by 30 µM N-nitro-L-arginine. These results were consistent with our previous reports that vasodilation induced by TNS, choline, and nicotine is due to release of neurogenic NO (24, 25, 28).
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In basilar arteries (without endothelial cells) in the presence of active muscle tone elicited by 0.3 µM U-46619, relaxation induced by 100 µM nicotine and 1 mM choline was enhanced by 0.03–0.1 µM IMI and DES but was blocked by 0.3–10 µM IMI and DES (Fig. 1). These two drugs at all concentrations examined, however, did not affect the relaxation elicited by TNS (Fig. 1), 1 nM–3 µM isoproterenol, or 10 nM–0.1 mM sodium nitroprusside (data not shown). Blockade of choline- and nicotine-induced relaxation by both drugs was completely reversible after washout (Fig. 1A).
Characteristics of Porcine and Rat SCG Neurons
The porcine and rat SCG cells were immunoreactive to neurofilament, 7-nAChR, and tyrosine hydroxylase (Fig. 2). 7-nAChR and tyrosine hydroxylase immunoreactivities were colocalized on the soma and axonal processes of rat and porcine SCG neurons. Electrophysiological recordings using the whole cell mode of the patch-clamp technique demonstrated the presence of voltage-activated Na+ and K+ currents as measured in response to depolarizing voltage steps (not shown). The mean membrane capacitance was 35.46 ± 1.25 pF (n = 99) for rat neurons and 32.87 ± 2.05 pF (n = 47) for porcine neurons (P > 0.05, by t-test), suggesting that the sizes of the SCG neurons from the rat and pig were similar.
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Choline has been reported to be a full and selective agonist at the 7-nAChR (3, 21), which has been found on the SCG neurons and their postganglionic sympathetic axon input to the cerebral circulation (25). Choline, therefore, was used to elucidate the characteristics of cerebral sympathetic 7-nAChRs in subsequent experiments. Choline (0.1–30 mM) in a concentration-dependent manner evoked inward currents in rat and porcine SCG neurons (Fig. 3A) as evidenced by similar concentration-response curves (Fig. 3B) and EC50 values of 1.6 (range 0.65–3.53) mM for the rat and 1.9 (range 0.73–3.71) mM for the pig (P > 0.05).
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We further characterized choline-induced responses by analyzing the current-voltage relation and the peak amplitude induced by 10 mM choline (300 ms) at various membrane potentials. On application of 10 mM choline, large amplitudes of the inward currents were recorded at negative membrane potentials, whereas no outward currents were evoked at a positive membrane potential. A plot of the peak amplitude vs. membrane potential for choline-evoked inward currents in the porcine SCG is shown in Fig. 3C.
Functional 7-nAChRs on SCG Neurons
Mec (a nonspecific nAChR antagonist) and MLA (a preferential 7-nAChR antagonist) were used to examine the properties of nAChR currents in cultured SCG neurons of rats and pigs. The choline-induced currents in these cells were blocked by 1–30 µM Mec (Fig. 4, A and C) and 1–30 µM MLA (Fig. 4, B and C) in a concentration-dependent manner. The blockade was completely reversible after washout of either antagonist (n = 4–6; Fig. 4, A and B). The IC50 values for Mec and MLA in the rat SCG neurons were 4.54 (range 0.61–9.48) µM and 5.78 (range 0.83–11.24) µM, respectively (P > 0.05).
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-BuTX (a preferential 7-nAChR antagonist) in porcine and rat SCG neurons. The blockade was almost completely reversible after washout of -BuTX (n = 5–7 for rat and n = 3–4 for pig; Fig. 5A). The IC50 values were 27.2 (range 10.65–82.35) nM for the rat and 64.46 (range 18.39–85.84) nM for the pig (P < 0.05).
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The peak currents elicited by 10 mM choline (300 ms) in rat and porcine SCG neurons were attenuated by IMI and DES (0.1–10 µM) in a concentration-dependent manner (Fig. 5, B and C). The blockade by both drugs was completely reversible after they were washed out. IMI or DES given alone had no direct gating action on neuronal nAChRs, because no membrane currents were detected on application of either of these agents (0.1–10 µM) for up to 10 or 15 min (not shown).
Effects of Monoamine Uptake Inhibitors on Choline-Evoked Inward Currents in 7-nAChR-Expressed Xenopus Oocytes
To investigate a possible direct effect of DES and IMI on 7-nAChR-mediated currents, we expressed 7-nAChRs as homopentamers in Xenopus oocytes, and the effects of choline (an 7-nAChR agonist) and other drugs on the inward currents mediated by these receptors were examined. The choline (0.3 mM)-induced, 7-nAChR-mediated currents desensitized rapidly and were sensitive to -BuTX and MLA. -BuTX and MLA at submicromolar concentrations completely inhibited choline- and nicotine-evoked currents in oocytes (data not shown). To achieve a stable choline-induced response, 0.3 mM choline was applied several times in a 10-min period, and its maximal negative deflection served as control. DES and IMI, in a concentration-dependent manner, profoundly inhibited choline-induced, 7-nAChR-mediated inward currents (Fig. 6; n = 3 for each drug). The IC50 values for DES and IMI were 8.50 (range 8.23–8.79) µM and 24.81 (range 18.31–33.61) µM, respectively (Fig. 6B). The DES-evoked blockade fully recovered after the drug was washed out (Fig. 6A). When the maximum concentration of IMI (300 µM) was administered, complete washout of this monoamine uptake inhibitor did not completely reverse blockade of the choline-induced currents (Fig. 6A).
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DISCUSSION |
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7-nAChR-mediated nitrergic neurogenic dilation of porcine basilar arteries. However, at all concentrations examined, these monoamine uptake inhibitors consistently blocked the choline-evoked, 7-nAChR-mediated inward currents in the SCG neurons of the pig and rat and 7-nAChR-expressing oocytes. These results support the hypothesis that there is an important axo-axonal interaction in cerebral nitrergic vasodilation (25, 28) and indicate that IMI and DES block the perivascular sympathetic 7-nAChRs.
We previously showed that cerebral perivascular 7-nAChRs on the postganglionic sympathetic nerve terminals originating in the SCG play important roles in regulating cerebral nitrergic neurogenic vasodilation (25, 28). Activation of these sympathetic 7-nAChRs by nicotine and choline releases NE, which then acts on presynaptic 2-adrenoceptors located on the neighboring parasympathetic nitrergic nerve terminals, resulting in release of NO and vasodilation (14, 24–26). IMI and DES are known to block neuronal monoamine reuptake (11, 27). The enhancement of choline- and nicotine-induced nitrergic vasodilation, therefore, is likely due to higher synaptic concentration of NE in the presence of these amine uptake inhibitors, which then bind presynaptic 2-adrenoceptors on the neighboring nitrergic neurons, causing an increased NO release (25).
IMI and DES at higher concentrations, however, blocked choline- and nicotine-induced neurogenic nitrergic vasodilation. DES and IMI, at the concentrations examined, did not affect TNS-elicited relaxation, suggesting that blockade of choline- and nicotine-induced neurogenic nitrergic vasodilation by these uptake inhibitors was not due to any possible local anesthetic effect. The possibility that IMI and DES attenuate neurogenic dilation by blocking 2-adrenoceptors on the nitrergic neurons and/or NO-cGMP coupling in the smooth muscle cells (25) is also unlikely, because these uptake inhibitors did not affect relaxation of the basilar arteries induced by isoproterenol (a nonpreferential -adrenoceptor agonist) or sodium nitroprusside (an NO releaser). Furthermore, attenuation of neurogenic vasodilation by these monoamine uptake inhibitors was not believed to be due to blockade of monoamine uptake transporters (11). Together, these findings support the possibility that monoamine uptake inhibitors may act by directly blocking sympathetic 7-nAChRs.
The presence of functional 7-nAChRs on the SCG neurons and the postganglionic sympathetic neurons mediating cerebral vasodilation (25) is further supported by the present finding that 7-nAChRs and tyrosine hydroxylase coexist in the same neurons of cultured SCGs from both species. The voltage-activated sodium and potassium currents observed in the SCG neurons from both species further indicate that these receptors are functional. Moreover, choline-induced inward currents in the SCGs were almost identical in both species and were blocked by Mec, MLA, and -BuTX, with -BuTX the most potent antagonist. This is consistent with previous findings from in vitro tissue bath studies that choline-induced nitrergic neurogenic vasodilation in porcine basilar arteries was blocked by these three antagonists in a similar order of potency (25), further suggesting the presence of similar functional 7-nAChRs on the SCGs of both species (25).
Choline has been well documented as an 7-nAChR agonist (1, 3, 21, 25). Inward whole cell currents were evoked by choline in a concentration-dependent manner in cultured SCG neurons of the rat and pig. The current-voltage relation exhibited a marked inward rectification, with no outward currents detected at positive membrane potentials. The strong voltage dependence of the choline-induced inward currents is possibly due to the positive charge of choline, suggesting that choline may interact with a specific site on the 7-nAChR protein. It may also imply that the ligand-gated ion channels open more readily at negative membrane potentials and allow the entry of cations. At positive membrane potentials, 7-nAChRs may not be functional because of their blockade by intracellular Mg2+ (2). Accordingly, 7-nAChRs may be fully functional only at negative membrane potentials (5).
The findings that IMI and DES, in a concentration-dependent manner, reversibly block choline-induced inward currents in the rat and porcine SCG neurons are consistent with observations in rat neonatal dorsal root ganglion neurons (8), bovine chromaffin cells (13), and rat hippocampal neurons (11). It has been shown that monoamine uptake inhibitors such as tricyclic antidepressants (7, 9, 11) and fluoxetine (11, 18) directly block nAChRs, including the 7-nAChR, in different preparations. Direct antagonism of nAChRs by monoamine uptake inhibitors in the rat neuronal 24-nAChRs and mouse muscle nAChRs expressed in X. laevis oocytes has also been demonstrated (17). This concept is further supported by the present findings that 7-nAChR-mediated inward current in X. laevis oocytes is blocked by -BuTX, IMI, and DES. Although EC50 values for blockade of 7-nAChR-mediated inward currents are higher for IMI, the potency of DES for blockade of 7-nAChR-mediated inward currents in oocytes is comparable to that in the SCG neurons. Together, these results suggest that the monoamine uptake inhibitors in the present study most likely directly block sympathetic 7-nAChRs, resulting in attenuation of choline-induced nitrergic neurogenic vasodilation.
Because perivascular sympathetic 7-nAChRs mediate cerebral nitrergic neurogenic vasodilation (25), blockade of these receptors by monoamine uptake inhibitors would be expected to cause a decrease in choline- and nicotine-induced cerebral blood flow. Indeed, several reports have indicated significant reductions in regional cerebral blood flow in the frontal and temporal regions in DES-treated patients (12, 20), although this finding is not universally accepted (22). The exact role of monoamine uptake inhibitors on influencing/attenuating the cerebral blood flow remains to be elucidated.
Although the present study was focused on cerebral arteries, our findings may provide important information for understanding the functional interaction between tricyclic antidepressants and the 7-nAChR in general. This information may be important in evaluating clinical effects of these monoamine uptake inhibitors and yield novel concepts toward the development of therapeutic compounds to treat diseases in which 7-nAChR activity is reduced. In particular, nAChR antagonists have been suggested to represent a novel class of therapeutic agents for treating mood disorders (7, 23). Therapeutic concentrations of antidepressants attainable in blood are on the order of 1 µM (4, 6). Antidepressants can reach even higher concentrations in the brain than in the plasma (4). These concentrations would be high enough to alter nAChR function or, more specifically, the 7-nAChR, as shown in the present study.
In summary, our hypothesis that activation of cerebral sympathetic 7-nAChRs leads to cerebral nitrergic neurogenic vasodilation via releases of sympathetic NE (25, 28) is further supported by the present results. This is based on the findings that cerebral nitrergic vasodilation is enhanced by lower concentrations of the monoamine uptake inhibitors IMI and DES. At higher concentrations, however, these inhibitors decrease neurogenic nitrergic vasodilation, most likely by blocking 7-nAChRs on the sympathetic nerves of SCG origin. These findings suggest that cerebral vascular hemodynamic effects of tricyclic antidepressants are likely a balance between blockade of neuronal reuptake of NE and the 7-nAChRs of the sympathetic neurons. The present findings also provide important information suggesting a possible role of the 7-nAChRs in the therapeutic and/or secondary effects of antidepressant drugs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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7-nicotinic acetylcholine receptors in the rat brain neurons. Eur J Neurosci 9: 2734–2742, 1997.[CrossRef][ISI][Medline]-bungarotoxin-sensitive receptor function and rectification induced by internal Mg2+. J Pharmacol Exp Ther 277: 432–444, 1996.2-adrenoceptors mediate nicotine-induced NOergic dilation in porcine basilar arteries. Am J Physiol Heart Circ Physiol 279: H808–H816, 2000.7-neuronal nicotinic acetylcholine receptor-mediated cerebral nitrergic vasodilation (Abstract). 33rd Annu Meeting Soc Neurosci New Orleans 2003.7 nicotinic receptors. Mol Psychiatry 3: 350–355, 1998.[CrossRef][Medline]7-subtype. Neurosci Lett 213: 201–204, 1996.[ISI][Medline]7-nicotinic acetylcholine receptor-mediated nitrergic neurogenic dilation in porcine basilar arteries. J Pharmacol Exp Ther 305: 1124–1131, 2003.7-Nicotinic acetylcholine receptors on cerebral perivascular sympathetic nerves mediate choline-induced nitrergic neurogenic vasodilation. Circ Res 91: 62–69, 2002.7-nicotinic acetylcholine receptors mediate nicotine-induced nitric oxidergic neurogenic vasodilation in porcine basilar arteries. J Pharmacol Exp Ther 298: 122–128, 2001.This article has been cited by other articles:
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  B. Lendvai and E. S. Vizi Nonsynaptic Chemical Transmission Through Nicotinic Acetylcholine Receptors Physiol Rev, April 1, 2008; 88(2): 333 - 349. [Abstract] [Full Text] [PDF]
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