␣7-Nicotinic Acetylcholine Receptors on Cerebral
Perivascular Sympathetic Nerves Mediate Choline-Induced
Nitrergic Neurogenic Vasodilation
Min-Liang Si, Tony J.F. Lee
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Abstract—It has been suggested in isolated porcine cerebral arteries that stimulation by nicotine of ␣7-nicotinic
acetylcholine receptors (␣7-nAChRs) on sympathetic nerves, but not direct stimulation of parasympathetic nitrergic
nerves, caused nitrergic neurogenic dilation. Direct evidence supporting this hypothesis has not been presented. The
present study, which used in vitro tissue bath and confocal microscopy techniques, was designed to determine whether
choline, a selective agonist for ␣7-nAChRs, induced sympathetic-dependent nitrergic dilation of porcine basilar arterial
rings. Choline and several nAChR agonists induced exclusive relaxation of basilar arterial rings without endothelium.
The relaxation was blocked by tetrodotoxin, nitro-L-arginine, guanethidine, and ␤2-adrenoceptor antagonists. Furthermore, the relaxation was blocked by methyllycaconitine and ␣-bungarotoxin (preferential ␣7-nAChR antagonists) and
mecamylamine but was not affected by dihydro-␤-erythroidine (a preferential ␣4-nAChR antagonist). Confocal
microscopic study demonstrated that choline and nicotine induced significant calcium influx in cultured porcine superior
cervical ganglionic cells but failed to affect calcium influx in cultured sphenopalatine ganglionic cells, providing direct
evidence that choline and nicotine did not act directly on the parasympathetic nitrergic neurons. The increased calcium
influx in superior cervical ganglionic cells was attenuated by ␣-bungarotoxin and methyllycaconitine but not by
dihydro-␤-erythroidine. These results support our hypothesis that activation of ␣7-nAChRs on cerebral perivascular
sympathetic nerves causes calcium influx and the release of norepinephrine, which then act on presynaptic
␤2-adrenoceptors located on the neighboring nitrergic nerve terminals, resulting in NO release and vasodilation.
Endogenous choline may play an important role in regulating cerebral sympathetic activity and vascular tone. (Circ Res.
2002;91:62-69.)
Key Words: choline 䡲 ␣7-nicotinic acetylcholine receptors 䡲 cerebral neurogenic vasodilation 䡲 nitric oxide
䡲 sympathetic-parasympathetic interaction
he ␣-bungarotoxin (␣-BGTX)–sensitive nicotinic acetylcholine receptor (nAChR), which is one of the predominant nAChR subtypes in the brain, consists of the ␣7-subunit,
possesses calcium permeability, and exhibits rapid desensitization.1 Choline, which is a precursor of acetylcholine (ACh)
synthesis and a product of ACh hydrolysis by acetylcholinesterase, has been shown to act as a relatively selective agonist
for ␣7-nAChRs in the central nervous system.2,3 By activating
presynaptic ␣7-nAChRs, choline has been shown to elicit the
release of neurotransmitters, including norepinephrine
(NE).2,4 The presence of nAChRs on sympathetic adrenergic
nerve terminals is also well established.5,6 However, the
functional significance of choline and ␣7-nAChRs in the
perivascular neurons has not been clarified.
We reported recently that nicotine-induced NO-mediated
neurogenic vasodilation in porcine basilar arteries and feline
middle cerebral arteries was dependent on intact perivascular
sympathetic adrenergic innervation.7–9 Results from pharma-
T
cological studies using nicotinic receptor antagonists further
suggest that nicotine acts on ␣7-nAChRs located on presynaptic sympathetic nerve terminals to release NE, which then
acts on presynaptic ␤2-adrenoceptors located on the neighboring nitrergic nerve terminals, resulting in the release of
NO and dilation of porcine basilar arteries. These results do
not support the idea that nicotine acts directly on nitrergic
nerves to induce nitrergic vasodilation.7–9 However, direct
evidence supporting this “indirect” nitrergic neurogenic vasodilation mediated by ␣7-nAChRs located on presynaptic
sympathetic neurons has not been presented.
Nicotine is a nonspecific nAChR agonist.1,2 To further confirm
that nicotine-induced cerebral neurogenic vasodilation is mainly
mediated initially through sympathetic presynaptic ␣7-nAChRs, we
used choline (a selective ␣7-nAChR agonist)2 and, for comparison,
several nonselective nAChR agonists, namely, epibatidine, 1,1dimethyl-4-phenylpiperazinium iodide (DMPP), and cytisine,10,11 to
determine whether these nicotinic agonists induced an NO-
Original received March 12, 2002; revision received May 8, 2002; accepted May 15, 2002.
From the Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Ill.
Correspondence to Dr Tony J.F. Lee, Department of Pharmacology, Southern Illinois University, School of Medicine, PO Box 19629, Springfield, IL
62794-9629. E-mail [email protected]
© 2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000024417.79275.23
62
Si and Lee
mediated relaxation in porcine basilar arteries and whether the
relaxation was blocked by guanethidine (a sympathetic neuronal
blocker) and antagonists of ␤2-adrenoceptors and ␣7-nAChRs. In
addition, cultured porcine superior cervical ganglionic (SCG) cells
(the origin of cerebral perivascular sympathetic nerves) and sphenopalatine ganglionic (SPG) cells (one of the major origins of
cerebral perivascular parasympathetic nitrergic nerves)12 were used
to determine whether nicotine and choline induced ␣7-nAChR–
mediated calcium influx in the former but not the latter cells.
Materials and Methods
General Procedure
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Fresh heads of adult pigs (60 to 100 kg) of either sex were collected
at local packing companies (Excel and Y-T). The entire brain, with
dura matter attached, was removed and placed in Krebs’ bicarbonate
solution equilibrated with 95% O2/5% CO2 at room temperature. The
composition of the Krebs’ solution was as follows (mmol/L): NaCl
122.0, KCl 5.16, CaCl2 1.2, MgSO4 1.22, NaHCO3 25.6, EDTA 0.03,
L-ascorbic acid 0.1, and glucose 11.0 (pH 7.4). Basilar arteries were
dissected under a dissecting microscope.7
In Vitro Tissue Bath Studies
An arterial ring segment (4 mm long) was cannulated with a
stainless-steel rod and a short piece of platinum wire in a plastic
tissue bath containing 6 mL Krebs’ bicarbonate solution. The
stainless-steel rod was connected to a strain-gauge transducer for
isometric recording of changes in force, as described in our previous
report.7 The temperature of the Krebs’ solution was maintained at
37°C. Tissues were equilibrated in the Krebs’ solution for an initial
30 minutes and then mechanically stretched to a resting tension of
750 mg.7–9
The basilar arterial ring segments were then contracted with
U-46619 (0.3 to 3 ␮mol/L) to induce an active muscle tone of 0.5to 0.75-g transmural nerve stimulation (TNS) at 8 Hz, and concentrations of nicotinic agonists (0.1 to 1000 ␮mol/L) were applied to
induce a relaxation. The concentrations that induce the maximum
relaxation by most nicotinic agonists were used in subsequent
experiments. After relaxation induced by TNS at 8 Hz and nicotinic
agonists, the arteries were washed with prewarmed Krebs’ solution.
A similar magnitude of active muscle tone was induced with
U-46619 again, and TNS was repeated (to serve as a control for
comparison with the relaxation elicited by TNS before the wash).
Experimental drugs were then administered 30 minutes before
repeating TNS and application of nicotinic agonists at the same
concentration before the wash. To avoid the possible development of
tachyphylaxis on repeated applications of nAChR agonists, at least
90 minutes with 6 washes (every 15 minutes) was allowed before the
next application of nicotinic agonists.7–9
For TNS, tissues were electrically and transmurally stimulated
with a pair of electrodes through which 100 biphasic square-wave
pulses of 0.6 ms in duration and 200 mA in intensity (continuously
monitored on a Tektronix oscilloscope) were applied at various
frequencies.7–9 The neurogenic origin of this TNS-induced response
was verified by its complete blockade by tetrodotoxin (TTX, 0.3
␮mol/L). The magnitude of a vasodilator response was expressed as
a percentage of the maximum relaxation induced by 100 ␮mol/L
papaverine, which was added at the end of each experiment.7–9 EC50
values (the concentration that produces 50% of the maximum
relaxation) and IC50 values (the concentration that produces 50%
inhibition of agonist-induced relaxation) were determined for each
arterial ring. From these values, the geometric means EC50 and IC50,
with 95% CIs,13 were calculated.
The endothelial cells of all arterial ring segments were mechanically removed.7 A complete removal of endothelial cells was verified
by lack of effect of nitro-L-arginine (L-NNA) in increasing basal
tone.7
Choline and Cerebral Neurogenic Vasodilation
63
SCG and SPG Cell Cultures
Freshly dissected porcine SCG and SPG cells were placed in cold
Hibernate A (Life Technologies) solution.14 After being cut into
smaller pieces, the ganglia were transferred to Mg2⫹/Ca2⫹-free
Hanks’ balanced salt solution containing papain (2 U/mL, Sigma),
collagenase D (1.2 mg/mL, Boehringer-Mannheim), and dispase (4.8
mg/mL, GIBCO) and were incubated for 50 minutes at 37°C. Cells
were released by gentle triturating at the end of the incubation. The
cell suspension was centrifuged at 300g for 5 minutes. The pellet was
gently resuspended in Neurobasal culture medium (Life Technology)
containing B27 (1:50 dilution, Life Technology), L-glutamine
(0.5 mmol/L), L-glutamate (25 ␮mol/L), and nerve growth factor (50
ng/mL, Alomome Laboratory Ltd).14 All media and Hanks’ balanced
salt solution contained 100 U/mL penicillin G, 100 ␮g/mL streptomycin, and 0.25 ␮g/mL amphotericin B (Fisher Scientific Inc). The
cell suspension was plated into a 4-well culture plate with a
poly-D-lysine– coated (50 ␮g/mL, Sigma) glass coverslip (12-mm
diameter, Fisher) in each well and incubated with air containing 5%
CO2 at 37°C. The growth medium was changed next day.
Intracellular Calcium Imaging
Between 3 and 7 days in culture, the SCG and SPG cells were used
to examine the effects of nicotine and choline on calcium influx in
these cells by confocal microscopy. The cells were washed with
physiological buffer (mmol/L: NaCl 130, KCl 5, HEPES 10, glucose
5, CaCl2 2, and MgCl2 2, pH 7.3) and were loaded with 3 ␮mol/L
fluo 4-AM in physiological buffer and incubated at room temperature for 30 minutes. The cells were washed with calcium indicator–
free buffer to remove any dye that was nonspecifically associated
with the cell surface and then incubated for a further 30 minutes to
allow complete deesterification of intracellular AM esters. Nicotine
(100 ␮mol/L) or choline (1 mmol/L) was then applied, and the
calcium influx was measured. nAChR antagonists were added 15
minutes before the application of nicotine, choline, or KCl (50 mmol/
L). Calcium fluorescence images were examined with a Fluoview
Olympus confocal microscope. Fluo 4 was excited at 488 nm, and
emitted fluorescence was filtered with a 535⫾25-nm bandpass filter,
read into a computer running Fluoview software, and quantified by
using this software.15
Drugs and Statistical Analysis
The following drugs were used: (⫺)-nicotine, ACh, choline chloride,
cytisine, DMPP, hemicholinium, atropine, methyllycaconitine
(MLA), ␣-BGTX, mecamylamine, dihydro-␤-erythroidine (DH␤E),
atenolol, guanethidine, propranolol, L-NNA, tetrodotoxin, and papaverine (all from Sigma), ICI 118,551 hydrochloride (Imperial
Chemical), U46619 (Upjohn), and fluo 4-AM (Molecular Probes
Inc). All drugs, otherwise stated, were dissolved in deionized water
and added directly to the tissue baths. The drug concentrations
reported were the final concentrations in the bath.
Results were expressed as mean⫾SEM. Statistical analysis was
evaluated by ANOVA and Student’s t test for paired or unpaired
samples as appropriate. A value of P⬍0.05 was accepted as
significant.
Results
Choline- and TNS-Induced Neurogenic
Vasodilation in Porcine Basilar Arteries
The porcine basilar arteries without endothelial cells in the presence
of active muscle tone induced by U-46619 (0.3 ␮mol/L) relaxed
exclusively with TNS at 8 Hz and applications of choline (10 to
1000 ␮mol/L) in a concentration-dependent manner (Figures 1 and
2). Similar to choline and nicotine,9 several nAChR agonists, such
as epibatidine, DMPP, and cytisine, induced dilation of porcine
basilar arteries without endothelial cells (Figure 2 and online Table,
which appears in the online data supplement available at http://
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July 12, 2002
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Figure 1. Effects of TTX, atropine, L-NNA, and MLA on relaxation, estimated as papaverine (PPV)-induced maximum relaxation, of porcine basilar arteries (denuded of endothelium) induced by choline and TNS. A, Representative tracing showing effect of TTX on relaxation elicited by choline (1 mmol/L) and TNS at 8 Hz in a basilar artery in the presence of active muscle tone induced by U-46619 (0.3
␮mol/L). ACh at 1 mmol/L induced a contraction in the same artery. B, Representative tracing showing effect of atropine and MLA on
relaxation elicited by choline (1 mmol/L) and TNS at 8 Hz in a basilar artery in the presence of active muscle tone induced by U-46619
(0.3 ␮mol/L). Atropine at 10 ␮mol/L potentiated the relaxation induced by TNS but did not affect that induced by choline (lower tracing
of panel B). Contraction induced by ACh (1 mmol/L) was reversed to relaxation after atropine treatment (upper tracing of panel B). This
residual relaxation induced by ACh (1 mmol/L) and that induced by choline were abolished by preferential ␣7-nAChR antagonist MLA
(10 ␮mol/L), which, however, did not affect the relaxation elicited by TNS at 8 Hz. C, Summary of effect of TTX on choline- and TNSinduced relaxation (n⫽4). D, Summary of effect of L-NNA on choline- and TNS-induced relaxation (n⫽4). Arrowheads in panels A and B
indicate repeated washings. Values are mean⫾SEM; n indicates number of experiments. *P⬍0.05 vs respective controls.
www.circresaha.org). The rank order of potency of these agonists is
epibatidine⬎DMPPⱖcytisineⱖnicotine⬎choline.
The relaxation of porcine basilar arteries induced by TNS,
choline, and nAChR agonists was almost completely blocked
by TTX (0.3 ␮mol/L, Figures 1A and 1C, n⫽4) and L-NNA
(30 ␮mol/L, Figure 1D, n⫽4), was abolished by cold-storage
denervation7 (n⫽5, data not shown), and was endothelium
independent (n⫽6, data not shown). These results suggest
that the relaxation induced by TNS, choline, and other
nAChR agonists was due to the release of neurogenic NO. In
parallel studies, ACh (up to 1 mmol/L) usually induced a
constriction of arteries without endothelial cells (Figures 1A
and 1B). Only in the presence of atropine (10 ␮mol/L) did
ACh induce a small relaxation, which was blocked by MLA.
On the other hand, choline-induced relaxation was not affected by atropine, which potentiated TNS-induced relaxation
(Figure 1B).
pranolol, and ICI 118,551 (a preferential ␤2-adrenoceptor
antagonist) in a concentration-dependent manner. The IC50
values for guanethidine and ICI 118,551 were 6.98 (4.22 to
11.54)⫻10⫺7 mol/L and 10.9 (4.9 to 24.2)⫻10⫺7 mol/L,
respectively (Figures 3A and 3B, n⫽5). However, the
choline-induced relaxation was not affected by atenolol (a
preferential ␤1-adrenoceptor antagonist) even at concentrations as high as 10 ␮mol/L (Figure 3A, n⫽6). Similarly,
relaxation induced by cytisine, DMPP, and epibatidine was
blocked by guanethidine, ICI 118,551, and propranolol but
not atenolol (data not shown). The blockade was readily
recovered after washing off these antagonists in the bath
(Figure 3A, n⫽5 for each antagonist). However, these
antagonists had no effect on TNS-induced vasodilation
(Figure 3A).
Guanethidine, ICI 118,551, and Propranolol
Blockade of Relaxation Induced by Choline and
Other Nicotinic Agonists
Choline is known to be taken up via choline transporters on
the synaptic membrane into nerve terminals for the synthesis
of ACh.16 Preincubation with hemicholinium (10 ␮mol/L), a
neuronal choline uptake inhibitor, potentiated relaxation induced by choline (1 mmol/L, n⫽8). However, hemicholinium
Choline-induced relaxation in isolated porcine basilar arteries
without endothelial cells was blocked by guanethidine, pro-
Effects of Choline Uptake Inhibition on
Choline-Induced Relaxation
Si and Lee
Choline and Cerebral Neurogenic Vasodilation
65
Blockade by ␣7-nAChR Antagonists of Neurogenic
Vasodilation Induced by Choline and Other
nAChR Agonists
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had no significant effect on relaxation induced by TNS at 8
Hz or other nAChR agonists, such as nicotine and cytisine at
the maximum concentrations examined (n⫽5; see online
Figure, available at http://www.circresaha.org).
Because TNS at 8 Hz and most nicotinic agonists at 100
␮mol/L (except choline at 1 mmol/L and epibatidine at 10
␮mol/L) induced maximum relaxation, these parameters were
used in the subsequent studies. In the presence of active
muscle tone induced by U-46619 in basilar arteries without
endothelial cells, relaxation induced by choline was
concentration-dependently blocked by selective ␣7-nAChR
antagonists MLA and ␣-BGTX17 (Figure 4A), with the IC50
values of 5.16 (1.39 to 19.21)⫻10⫺7 mol/L and 5.15 (1.10 to
24.23)⫻10⫺8 mol/L, respectively (Figure 4B). Cholineinduced relaxation was also blocked by mecamylamine (10
␮mol/L, a nonspecific nAChR antagonist; Figure 4A). Blockade of choline-induced relaxation by these specific and
nonspecific ␣7-nAChR antagonists was fully recovered after
washing off these antagonists (Figure 4A). However, cholineinduced relaxation was not appreciably affected by ␣4nAChR antagonist DH␤E (10 ␮mol/L, Figure 4A; n⫽6).
Similar results were found; ie, these nAChR antagonists
(MLA, ␣-BGTX, and mecamylamine but not DH␤E) blocked
relaxation induced by epibatidine, DMPP, and cytisine (Figure 5). MLA, ␣-BGTX, mecamylamine, and DH␤E at the
Figure 3. Effects of guanethidine, ICI 118,551, propranolol, and
atenolol on relaxation, estimated as percentage of PPV-induced
maximum relaxation, of porcine basilar arteries (denuded of endothelium) induced by choline and TNS. A, Summary of the
effects of guanethidine (10 ␮mol/L), ICI 118,551 (10 ␮mol/L),
propranolol (10 ␮mol/L), and atenolol (10 ␮mol/L) on relaxation
elicited by choline (1 mmol/L) and TNS at 8 Hz in a basilar
artery in the presence of active muscle tone induced by
U-46619 (0.3 ␮mol/L). B, Blockade of choline-induced relaxation
by guanethidine and ICI 118,551 was concentration dependent.
Values are mean⫾SEM; n indicates number of experiments.
*P⬍0.05 vs respective controls.
Figure 4. Effects of different nAChR antagonists on relaxation,
estimated as percentage of PPV-induced maximum relaxation,
of porcine basilar arteries (denuded of endothelium) induced by
choline (1 mmol/L) and TNS (8 Hz). A, Summary of the effects of
preferential ␣7-nAChR antagonists MLA (10 ␮mol/L) and
␣-BGTX (1 ␮mol/L), a nonspecific nAChR agonist
mecamylamine (10 ␮mol/L), and a preferential ␣4-nAChR antagonist DH␤E (10 ␮mol/L) on relaxation induced by choline and
TNS and recovery from blockade of choline-induced relaxation
after washing off the nAChR antagonists (n⫽6). B, Blockade of
choline-induced relaxation by MLA and ␣-BGTX was concentration dependent (n⫽5). Values are mean⫾SEM; n indicates number of experiments. *P⬍0.05 vs respective controls.
Figure 2. Concentration-dependent relaxation, estimated as
percentage of PPV-induced relaxation, of porcine basilar arteries
(denuded of endothelium) induced by choline, cytisine, DMPP,
nicotine, and epibatidine. All nicotinic agonists, in a
concentration-dependent manner, induce relaxation of porcine
basilar arteries with different EC50 values (as shown in the online
Table, available at http://www.circresaha.org).
66
Circulation Research
July 12, 2002
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mals) and 3.32⫾0.45-fold (n⫽8 cells from 8 different animals), respectively. The addition of KCl (50 mmol/L) did not
further increase intracellular calcium. The nicotine- and
choline-induced calcium influx was drastically attenuated in
cells pretreated with ␣-BGTX (1 ␮mol/L, Figure 6 [panel C3]
and Figure 7B) and MLA (10 ␮mol/L, Figure 7C), both of
which alone did not affect calcium influx (Figure 6 [panel
C2] and Figure 7B and 7C). In the presence of blockade of
calcium influx by ␣-BGTX and MLA, KCl (50 mmol/L) still
induced significant calcium influx (Figure 6 [panel C4] and
Figures 7B and 7C), which was comparable to that seen in
preparations in the absence of antagonists (Figure 7A),
suggesting the specificity of blockade by ␣-BGTX and MLA.
In parallel studies, ␣4-nAChR antagonist DH␤E (10 ␮mol/L)
had no significant effect on nicotine-, choline-, or KClinduced calcium influx in cultured SCG cells (Figure 7D).
In contrast to the calcium influx found in SCG cells,
choline and nicotine in the same concentrations did not
increase calcium influx in cultured SPG cells (1343 cells in 8
plates from at least 8 animals, Figure 7E). However, significant calcium influx was induced by the addition of KCl
(50 mmol/L) in the same cells, which was comparable to that
seen in the SCG cells.
Discussion
Figure 5. Effects of different nAChR antagonists (MLA, ␣-BGTX,
mecamylamine, and DH␤E) on relaxation, estimated as percentage of PPV-induced maximum relaxation, of porcine basilar
arteries (denuded of endothelium) induced by TNS and different
nAChR agonists (epibatidine, DMPP, and cytosine) and recovery
from blockade of agonist-induced relaxation after washing off
the nAChR antagonists. Values are mean⫾SEM; n indicates
number of experiments. *P⬍0.05 vs respective controls.
concentrations used did not affect the TNS-elicited relaxation
(Figures 4A and 5).
Choline- and Nicotine-Induced Calcium Influx in
Cultured SCG and SPG Cells
We previously reported that cultured SCG cells, like cerebral
perivascular sympathetic neurons, contain dense ␣ 7 nAChRs.9 Because ␣7-nAChRs form membrane cation channels that possess high calcium permeability,1 we used the
intracellular calcium imaging indicator fluo 4-AM to determine whether the activation of ␣7-nAChRs by choline and
nicotine would induce calcium influx. As shown in Figure 6,
both nicotine (100 ␮mol/L) and choline (1 mmol/L) induced
a significant increase in calcium image in the SCG cells
(1818 of 2108 cells in 10 plates from at least 10 animals and
1507 of 1706 cells in 8 plates from at least 8 animals,
respectively). Quantitative analysis on single cells (Figure
7A) indicated that both nicotine (100 ␮mol/L) and choline
(1 mmol/L) significantly increased calcium image in the SCG
cells by 2.94⫾0.18-fold (n⫽10 cells from 10 different ani-
Our previous studies demonstrated that nicotine-induced
NO-mediated neurogenic relaxation in porcine basilar arteries
was dependent exclusively on intact sympathetic innervation.7 This conclusion was based on the findings indicating
that after a complete blockade of sympathetic transmission
with guanethidine or chemical denervation of sympathetic
nerves with 6-hydroxydopamine, nicotine never induced a
relaxation, whereas TNS-elicited NO-mediated relaxation in
the same preparations remained unchanged.7 These results
suggest that in porcine basilar arteries, nicotine does not act
directly on nitrergic nerves to release transmitter NO. Rather,
nicotine acts on the nicotinic receptors located on sympathetic nerves to release NE, which then diffuses to act on
adrenoceptors located on the neighboring nitrergic nerves,
causing release of NO from these nerves and relaxation of the
smooth muscle7–9 (Figure 8). The role of NE as the mediator
was supported by the findings that ␤2-adrenoceptors on the
nitrergic nerves but not adrenoceptors on the smooth muscle
mediated nicotine-induced relaxation.8 This functional axoaxonal or sympathetic-parasympathetic interaction is also
supported by morphological evidence indicating that close
apposition (25 nm) between the adrenergic nerve terminals
and the nonadrenergic nerve terminals is a characteristic of
innervation of cerebral arteries at the base of the brain in
several species.18 –21
Our findings, based on studies using nicotinic receptor
antagonists, further suggested that ␣7-nAChRs on sympathetic nerves were mediating nicotine-induced nitrergic neurogenic vasodilation in porcine basilar arteries.9 This was
supported by the presence of ␣7-nAChR immunoreactivities
on both cerebral perivascular sympathetic nerves and SCG
cells.9 This is further supported by results of the present
study, which indicated that choline acts as a selective agonist
for ␣7-nAChR on sympathetic nerves in porcine basilar
Si and Lee
Choline and Cerebral Neurogenic Vasodilation
67
further supported by the fact that choline and nicotine did not
elicit any calcium influx, whereas KCl did, in the parasympathetic SPG cells. This result provides convincing evidence
supporting our hypothesis that choline and other nicotinic
agonists do not act directly on the SPG cells to release NO.
Rather, these agonists act initially on ␣7-nAChRs on sympathetic nerves, resulting in NE release and the aforementioned
nitrergic vasodilation7 (Figure 8).
␣7-nAChRs have been shown to belong to the evolutionarily oldest group of nAChRs.22 It is possible that choline
rather than ACh was originally the natural transmitter for
cholinergic receptors. It appears feasible that in the developed
cerebral vascular system, choline rather than ACh remains to
be the endogenous ligand for ␣7-nAChRs. In the present study
in isolated porcine basilar arteries without endothelial cells,
choline and other nAChR agonists consistently induced
NO-mediated neurogenic vasodilation. In contrast, ACh
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Figure 6. Effect of nicotine and choline on calcium influx in cultured porcine SCG cells. Cultured SCG cells were loaded with
fluo 4-AM (3 ␮mol/L) in physiological buffer and incubated at
room temperature for 30 minutes (panels A1, B1, and C1). Nicotine (100 ␮mol/L) or choline (1 mmol/L) followed by KCl
(50 mmol/L) was applied to the medium, and the calcium image
in the neuronal cells was examined. Both choline (panel A2) and
nicotine (panel B2) induced a significant calcium influx. ␣-BGTX
(1 ␮mol/L, panel C2), which did not change the basal intracellular calcium concentrations, blocked choline-elicited calcium
influx (panel C3). In the presence of ␣-BGTX blockade of the
choline effect, KCl (50 mmol/L) induced significant calcium influx
in these cells (panel C4).
arteries to induce NO-mediated neurogenic vasodilatation
with a mechanism of action similar to that of nicotine.7–9 This
conclusion was based on the findings that choline-induced
relaxation was neurogenic, nitrergic, and sensitive to
guanethidine, ICI 118,551, and propranolol (10 ␮mol/L) but
not atenolol. Furthermore, the relaxation induced by choline
was blocked specifically by preferential ␣7-nAChR antagonists. Similar results of the blockade of neurogenic vasodilation induced by various nAChR agonists in the presence of
guanethidine, preferential ␤2-adrenoceptor antagonists, and
preferential ␣7-AChR antagonists were obtained. Finally,
both nicotine and choline induced significant calcium influx
into sympathetic ganglionic neurons, and the influx was
specifically blocked by preferential ␣7-nAChR antagonists.
The sympathetic-dependent nitrergic neurogenic vasodilation induced by choline and other nicotinic agonists was
Figure 7. Summary of the effects of choline (1 mmol/L), nicotine
(100 ␮mol/L), and KCl (50 mmol/L) on intracellular calcium concentration ([Ca2⫹]i) in cultured porcine SCG cells (A through D),
as described in Figure 6, and SPG cells (E). A, Changes in intracellular calcium were compared with the basal control calcium
concentration, which served as control (100%). *P⬍0.05 vs
respective control. B and C, Effects of 1 ␮mol/L BGTX (B) and
10 ␮mol/L MLA (C) on choline-, nicotine-, and KCl-induced calcium influx. $P⬍0.05 vs respective agonist. D, Failure of DH␤E
in blocking calcium influx induced by choline, nicotine, or KCl.
&P⬍0.05 vs blocker. DH␤E alone did not increase calcium
influx. E, Summary showing that neither choline nor nicotine significantly increased [Ca2⫹]i in the cultured porcine SPG cells.
However, the addition of KCl significantly increased calcium
influx in these cells. Values are mean⫾SEM; n indicates number
of experiments.
68
Circulation Research
July 12, 2002
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Figure 8. Summary diagram showing close apposition of an
adrenergic and a cholinergic-nitrergic nerve terminal33 in large
cerebral arteries at the base of the porcine brain. The axoaxonal distance between these 2 different nerve terminals is
closer than that between the nerves and the smooth muscle.
Choline, nicotinic agonists, and ACh act on presynaptic
␣7-nicotinic receptors located on the adrenergic nerve terminal,
causing release of NE (⫹), which then acts on presynaptic
␤2-adrenoceptors located on the adjacent cholinergic-nitrergic
nerve terminal. This effect of NE results in stimulating NO
release (⫹), which activates guanylate cyclase (GC), increases
cGMP synthesis from GTP, and relaxes the smooth muscle.
However, NE released from sympathetic adrenergic nerves is a
weak postsynaptic transmitter (as indicated by a question mark).
NO is not stored in vesicles and is synthesized from L-arginine
(L-Arg) in the presence of NO synthase (NOS). L-Citrulline
(L-Cit), the byproduct of NO synthesis, is actively converted to
L-Arg.34 This L-Cit–L-Arg cycle provides evidence for the neuronal source of NO. Furthermore, NO is colocalized and coreleased with ACh, which is stored in neuronal vesicles. ACh
released from cholinergic–NO-ergic nerves acts on presynaptic
muscarinic M2 receptors (M2), resulting in G-protein–mediated
negative coupling (⫺) to the N-type calcium channels, which
leads to suppression of calcium influx through this type of calcium channel,14 a decreased NOS activity accompanied by a
decrease in NO formation and release, and diminished relaxation of the smooth muscle cell. ACh, like NE, is a very weak or
negligible postsynaptic transmitter, inasmuch as these 2 classic
transmitter substances released on TNS do not directly affect
the smooth muscle cell tone on the basis of animal studies.8,12
never induced a relaxation but did induce a small contraction
in these arterial preparations. This lack of effect of ACh in
inducing NO-mediated neurogenic vasodilation is consistent
with previous findings that ACh attenuates TNS-induced
nitrergic relaxation of porcine cerebral arteries by acting on
presynaptic muscarinic M2 receptors on nitrergic nerves,
which are negatively coupled to the neuronal calcium influx
via N-type calcium channels, resulting in decreased release of
its cotransmitter NO12,23 (Figure 8).
The contraction induced by ACh is most likely because of
its direct effect on muscarinic receptors on the smooth muscle
cells, resulting in calcium influx via L-type calcium channels.24 However, in the presence of atropine, ACh-induced
contraction was converted to a relatively small relaxation
compared with that induced by choline in the same preparations (Figure 1B). The residual relaxation was blocked by
MLA, suggesting that ACh also acted on ␣7-nAChRs. In
addition, desensitization to choline-induced relaxation oc-
curred after pretreatment of ACh, suggesting that ACh acts on
nicotinic receptors with significantly less efficacy than does
choline. In contrast, choline does not seem to bind the
muscarinic receptors, inasmuch as choline-induced nitrergic
neurogenic vasodilation was not significantly affected by
atropine, which nevertheless potentiated TNS-induced relaxation (a result similar to our previous findings23). These
results suggest that choline is more effective than ACh as an
endogenous ligand for ␣7-nAChRs on the sympathetic nerves
of SCG origin.
The present study demonstrates that ␣7-nAChR selective
and nonselective agonists induce relaxation in porcine basilar
arteries with different potency and efficacy. Epibatidine, a
well-described nAChR agonist,25 is the most potent agonist,
with the highest efficacy among the agonists examined.
Although choline is a full ␣7-nAChR agonist, a relatively high
concentration (high EC50) is needed to induce a relaxation.
However, this concentration is comparable to the concentration of choline needed to induce neurotransmitter release in
the central nervous system.26
The possible physiological significance of choline is further supported by the finding of the present study, ie, that
NO-mediated neurogenic vasodilation induced by choline but
not nicotine or cytisine is potentiated by hemicholinium (a
choline uptake blocker). Furthermore, choline is present in
significant concentration in the cerebral spinal fluid27 and
blood serum.28 Accordingly, activation of ␣7-nAChRs by
endogenous choline may play an important role in regulating
cerebral sympathetic activity and vascular tone.
The sympathetic-dependent neurogenic vasodilation induced by nAChR agonists has also been demonstrated in the
mesenteric vascular beds,29 although the specific subtype of
nAChRs in this vascular bed is not determined. Furthermore,
acute cigarette smoking has been shown to cause pulmonary
vasodilation in pigs30 and to cause increased cerebral blood
flow in humans.31,32 Because nicotine is a major constituent
in the cigarette and because NE is a vasoconstrictor, it is very
likely that these vasodilatory effects of cigarette smoking are
due to the initial release of NE, which then diffuses to the
neighboring nerves to release potent dilator transmitters from
these nerves. Together, it is very likely that activation of
sympathetic nerves will lead to activation of parasympathetic
nerves. Surprisingly, the subtype(s) of nicotinic receptors
found on the sympathetic nerves in peripheral vasculatures is
practically unavailable; however, ␣3␤4-nicotinic receptors on
sympathetic nerves in the rat stomach25 have been shown to
be involved in regulating nicotine-induced NE release. It is
possible that variations in the functional subunits of nAChRs
on adrenergic neurons in regulating NE release in different
vascular beds may exist. Because choline is an endogenous
ligand for ␣7-nAChRs, identification of the nAChR subtype
becomes critical for a complete understanding of the sympathetic control of systemic circulation.
In conclusion, the present study demonstrates for the first
time that choline and all nAChR agonists examined do not act
directly on the nitrergic nerves but bind ␣7-nAChRs on
sympathetic nerve terminals, causing subsequent nitrergic
vasodilation in porcine basilar arteries. This is consistent with
our hypothesis that activation of ␣7-nAChRs on cerebral
Si and Lee
perivascular sympathetic nerves results in calcium influx and
release of NE. The released NE then diffuses to act on
␤2-adrenoceptors located on the neighboring nitrergic nerves,
causing release of NO and, therefore, vasodilation (Figure 8).
The endogenous choline may play an important role in
regulating cerebral perivascular sympathetic activity and,
therefore, the cerebral circulation.
Acknowledgments
This study was supported by the NIH (grants HL-27763 and
HL-47574), the American Heart Association/Illinois Heart Affiliate
(grant 9807871), and Southern Illinois University–Central Research
Committee/Excellence in Academic Medicine. We thank Susan
Sarwinski and Dr Raj Mishra for technical advice on cell culture,
Carol Morgan for preparing the manuscript, and Drs L. Premkumar
and D. Caspary for reading the manuscript.
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α7-Nicotinic Acetylcholine Receptors on Cerebral Perivascular Sympathetic Nerves
Mediate Choline-Induced Nitrergic Neurogenic Vasodilation
Min-Liang Si and Tony J.F. Lee
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Circ Res. 2002;91:62-69; originally published online June 6, 2002;
doi: 10.1161/01.RES.0000024417.79275.23
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