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Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois 62794-9629
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Nitric oxide (NO)
is a major transmitter in mediating cerebral neurogenic vasodilation in
several species. Recent findings have suggested that acetylcholine,
which is costored with NO in cerebral perivascular nerves, plays a role
in modulating NO release, presumably by acting on muscarinic (M)
receptors on nitric oxidergic nerve terminals. The present study was
designed using an in vitro tissue bath technique to pharmacologically
characterize the presynaptic muscarinic-receptor subtype(s) that
mediate modulation of NO release and therefore neurogenic vasodilation
and to investigate further the possible mechanisms involved in this
presynaptic modulation in porcine basilar arteries. Transmural nerve
stimulation (TNS) elicited a frequency-dependent,
tetrodotoxin-sensitive relaxation. The relaxation was abolished by
nitro-L-arginine (30 µM) and
was completely reversed by
L-arginine and
L-citrulline, but not by their D-enantiomers. Atropine
(0.01-1 µM), pirenzepine (an
M1-receptor antagonist,
0.01-1 µM), and methoctramine (an
M2-receptor antagonist, 0.01-1 µM), but not 4-DAMP (an
M3-receptor antagonist) or
tropicamide (an M4-receptor
antagonist) at concentrations as high as 10 mM, significantly increased
the TNS-elicited relaxation. This relaxation, on the other hand, was
significantly attenuated by arecaidine but-2-ynyl ester tosylate (an
M2-receptor agonist, 0.1 µM) but was not affected by McN-A-343 (an
M1-receptor agonist, 1 µM). Double-labeling immunohistochemical study demonstrated that
perivascular M2
receptor-immunoreactive fibers were completely coincident with NADPH
diaphorase fibers. Furthermore, the muscarinic receptor-mediated modulation of TNS-elicited relaxation was completely prevented by
-conotoxin GVIA (0.1 µM), a specific N-type
Ca2+ channel inhibitor, but was
still observed in the presence of tetraethylammonium (1 mM),
8-bromo-cAMP (0.5 mM), and pertussis toxin. It is concluded that the
presynaptic M2 receptors on
porcine cerebral perivascular nitric oxidergic nerves mediate
inhibition of NO release. The inhibition is due primarily to a
decreased Ca2+ influx through
N-type Ca2+ channels.
nitric oxide; presynaptic muscarinic receptor; transmural nerve stimulation; M2 receptor-immunoreactive fibers; N-type calcium channel; cerebral blood vessels; porcine
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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NITRIC OXIDE (NO) has been shown to play a predominant role in mediating cerebral neurogenic vasodilation in several species (13, 34, 48). Release of NO from cerebral perivascular nerves has been demonstrated (13, 14). Because of its chemical properties, NO is not likely to be stored in the vesicles or released from neurons by the exocytotic mechanism, like classic neurotransmitters such as acetylcholine (ACh; see Ref. 37). The exact mechanism of NO release from perivascular nerves remains unclear.
NO synthase (NOS) and choline acetyltransferase (ChAT) have been found to coexist in the parasympathetic ganglion and perivascular nerves in cerebral blood vessels of several species (27, 46). Results from pharmacological studies, however, have demonstrated that NO mediates the major component of the neurogenic vasodilator response (33), whereas endogenous ACh exhibits a negligible direct effect on vascular smooth muscle tone, possibly due to its low synaptic concentration and wide synaptic distance (31). In fact, the direct effect of exogenous ACh on cerebral vascular smooth muscle is a constriction rather than a dilation (30, 31). Furthermore, the possibility that endogenous ACh, which is released from adventitial nerves, may induce an endothelium-dependent neurogenic vasodilation is very unlikely, at least in the large cerebral arteries (31). Accordingly, ACh has been proposed to act presynaptically to be more like a modulator (32). This hypothesis has recently been supported by the findings that endogenous ACh may act on presynaptic muscarinic (M) receptors on nitric oxidergic nerves to inhibit release of cotransmitter NO and therefore diminished vasodilation (4, 47). The exact mechanism of this presynaptic cholinergic modulation of NO-mediated vasodilation, however, is not clarified.
Four subtypes of muscarinic receptors (M1-M4) have been pharmacologically characterized, and all subtypes have been reported to be involved in the presynaptic modulation of neurotransmitter release in both central and peripheral neurons (12). In perivascular nerves, although the presynaptic M2-receptor subtype is most frequently reported to mediate muscarinic modulation of neurotransmitter release (2, 45, 47, 53), other subtypes are also involved. For example, the M3-receptor subtype has been shown to mediate inhibition of ACh and norepinephrine (NE) release in bovine cerebral arteries (19), whereas the M1-receptor subtype mediates inhibition of NE release in guinea pig carotid arteries (10). The prejunctional muscarinic receptors mediating inhibition of NE release in the rabbit ear arteries do not appear to be the M1, M2, or M3 subtype (16). Most of these studies were examining effects of presynaptic muscarinic receptors on release of classic neurotransmitters. The present study was designed to investigate, by a pharmacological approach in porcine basilar arteries, the presynaptic muscarinic-receptor subtype and its related mechanism(s) that are involved in inhibition of NO-mediated neurogenic vasodilation.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In Vitro Tissue Bath
Fresh heads of adult pigs of either sex were collected from a local slaughterhouse. The entire brain was removed and placed in Krebs-bicarbonate solution equilibrated with 95% O2-5% CO2 at room temperature. The composition of the Krebs-bicarbonate solution was as follows (in mM): 122.0 NaCl, 5.2 KCl, 1.33 CaCl, 1.2 MgSO4, 25.0 NaCO3, 0.03 disodium EDTA, 0.01 L-ascorbic acid, and 11.0 glucose (pH 7.4).Basilar arteries were dissected and cleaned of surrounding tissue under a dissecting microscope. The endothelium was mechanically denuded according to our previous reports (31, 34). A complete removal of endothelial cells was verified by the failure of nitro-L-arginine (L-NNA) in raising U-46619-induced basal tone further (34, 35). The ring segment (4 mm long) was cannulated with a stainless steel rod (30-gauge hemispherical section) and a short piece of platinum wire and was mounted in tissue bath containing 6 ml of Krebs-bicarbonate solution equilibrated with 95% O2-5% CO2 at 37°C. Tissues were equilibrated in the Krebs-bicarbonate solution for the initial 30 min and then were mechanically stretched to a resting tension of 0.75 g (34, 35). U-46619 (0.1-1 µM) was applied to induce an active muscle tone of ~0.75 g. Tissues were electrically, transmurally stimulated with a pair of platinum electrodes through which 100 biphasic square-wave pulses of 0.6 ms in duration and constant 200 mA in intensity were applied at various frequencies (31). Stimulation parameters were continually monitored on a Tektronix oscilloscope (31). The neurogenic origin of this transmural nerve stimulation (TNS)-induced response was verified by its complete blockade by tetrodotoxin (TTX; 1 µM). Papaverine (300 µM) was applied to each tissue to induce maximum relaxation at the end of the experiment. The magnitude of a vasodilator response during the experiments was expressed as a percentage of the maximum response induced by papaverine (31).
All drugs, unless otherwise stated, were dissolved in deionized water and were added directly to the tissue baths after control relaxation induced by TNS was established (55). The concentrations of drugs reported were the final concentrations in the bath. TNS was elicited 15 min after each experimental drug was added. Each tissue preparation served as its own control.
Immunohistochemistry
The basilar and middle cerebral arteries were dissected and immediately fixed in 4% paraformaldehyde in 0.1 phosphate-buffered saline (PBS; pH 7.4) at 4°C overnight. The fixed arterial preparations were washed in 0.01 M PBS and blocked with 1% normal goat serum (NGS) diluted in 0.05% Triton X-100-PBS for 30 min at room temperature. The specimens then were incubated with rat monoclonal anti-m2/M2 muscarinic receptor antibody (1:100 diluted in 0.05% Triton X-100-PBS-1.5% NGS; Chemicon, Temcula, CA) at 4°C overnight. The m2 receptor antibody was raised against the third intracellular loop of m2-receptor fusion proteins. After a wash in PBS, the specimens were incubated with biotinylated goat anti-rat IgG antibody (1:200 diluted in PBS; Vector Laboratories) for 1 h at room temperature. After another wash in PBS, the specimens were incubated with FITC-labeled avidin D (1:80 diluted in PBS; Vector Laboratories) in the dark for 1 h at room temperature. The specimens were then rinsed and covered with coverslips and Vectashield mounting medium (Vector Laboratories) for photography under a fluorescence microscope fitted with proper filters (Olympus BX50 microscope). After the immunofluorescence fibers were photographed, the specimens were washed in PBS and processed for NADPH diaphorase (NADPHd) histochemistry (27, 54) as described below. For negative control, no m2 receptor-immunoreactive fibers were observed when tissues were incubated with nonimmunized serum or without m2-receptor antiserum (54).NADPHd Histochemical Staining
After immunofluorescence labeling of m2 receptor and photographing as described above, the specimens were washed in PBS and then incubated in 0.1 M phosphate buffer containing 0.5 mg/ml NADPH (reduced form), 0.1 mg/ml nitro blue tetrazolium, and 0.3% Triton X-100 at 37°C for 1 h (54). The specimens were rinsed with PBS, mounted with Gel Mount (Biomeda, Foster City, CA), and examined under a microscope. The same field previously photographed for m2-receptor immunoreactivity was rephotographed for NADPHd fibers. For negative control, no NADPHd fibers were found in tissues incubated in the absence of NADPH.Statistical Methods and Drugs Used
The data were computed as means ± SE and were evaluated by Student's t-test for paired samples and ANOVA for multigroup comparisons. The following drugs were used: atropine, L-NNA, L-arginine, L-citrulline, TTX, NADPH, U-44619 (9,11-dideoxy-9
,11-epoxymethanoprostaglandin F2
), arecaidine but-2-ynyl
ester tosylate (ABET), tetraethylammonium (TEA), 8-bromo-cAMP, nitro
blue tetrazolium, sodium nitroprusside (SNP), all from Sigma Chemical
(St. Louis, MO); and tropicamide, 4-diphenylacetoxy-N-(2-chloroethyl)-piperidene (4-DAMP) mustard hydrochloride, methoctramine hydrochloride, McN-A-343, pirenzepine, -conotoxin GVIA (CTX), and pertussis toxin (PTX), all from RBI (Natick, MA).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Atropine Enhances TNS-Elicited Neurogenic Vasodilation
In the presence of active muscle tone induced by U-46619, porcine cerebral arterial rings without endothelial cells relaxed exclusively upon TNS (Fig. 1A). The relaxation was abolished by TTX (0.1 µM) and L-NNA (30 µM), a result similar to that reported previously in the same arterial preparations (34). The relaxation induced by TNS at various frequencies (2, 4, and 8 Hz) was significantly enhanced in the presence of atropine (0.1 µM; Fig. 1, A and B, and Fig. 2). The enhanced relaxation was completely inhibited by L-NNA (30 µM; Fig. 1, A and B) and nitro-L-arginine methyl ester (30 µM, n = 6, data not shown). The inhibition was fully reversed by L-arginine (1 mM) and L-citrulline (1 mM) but not by D-arginine or D-citrulline (n
8, Fig. 1,
A and
B). TNS-induced relaxation, on the
other hand, was significantly inhibited by exogenously applied
physostigmine (0.3 µM) and ACh (1 µM;
n = 6-8, Fig.
3).
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Effects of Selective Muscarinic-Receptor Antagonists and Agonists on TNS-Elicited Vasodilation
Pirenzepine (0.1-10 µM), a selective M1-receptor antagonist, and methoctramine (0.01-10 µM), a selective M2-receptor antagonist, in a concentration-dependent manner significantly increased TNS-induced relaxation in basilar arteries without endothelial cells (Fig. 4, A and B). The threshold concentration, however, was 10-fold higher for pirenzepine than methoctramine. 4-DAMP (0.01-10 µM), a selective M3-receptor antagonist, did not significantly affect TNS-induced relaxation when compared with the effect produced by its solvent, DMSO (Fig. 4C). Tropicamide (0.01-10 µM), a selective M4-receptor antagonist, did not affect TNS-induced relaxation either (Fig. 4D).
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Furthermore, ABET (0.01-10 µM), a selective M2-receptor agonist that slightly increased the basal tone in arteries without endothelial cells, significantly and concentration dependently inhibited relaxation elicited by TNS at various frequencies (Fig. 5B, EC50 = 0.05, 0.17, and 0.44 µM for 2, 4, and 8 Hz, respectively). On the other hand, McN-A-343 (0.01-10 µM), a selective M1-receptor agonist that did not change the basal vascular tone, did not affect TNS-induced relaxation until its concentration reached 10 µM (Fig. 5A, EC50 values were at least 8 µM for all 3 stimulating frequencies).
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Coincidence of m2 Receptor-Immunoreactive Fibers and NADPHd Fibers in Porcine Cerebral Arteries
In whole mount porcine basilar and middle cerebral arteries, m2 receptor-immunoreactive fibers were found to consist of bundles of various sizes and fine fibers (Fig. 6A). Most m2 receptor-immunoreactive fibers were coincident with NADPHd fibers (Fig. 6B). In negative control in which the tissue was incubated with nonimmunized serum or denervated by cold storage for a week, no m2 receptor-immunoreactive or NADPHd fibers were found (data not shown).
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Effect of PTX on Muscarinic Receptor-Mediated Inhibition of TNS-Induced Relaxation
After the endothelium-denuded arterial preparations were incubated with PTX (500 ng/ml-25 µg/ml) at 37°C for 4-7 h according to our previous report (26), ABET still significantly inhibited TNS-induced relaxation in the presence of active muscle tone induced by U-46619, and the inhibition was completely reversed by atropine (0.1 µM, n = 7). There was no significant difference between the control and PTX-treated preparations on responses to ABET and atropine (Fig. 7).
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Effects of Ca2+ Channel Blocker, cAMP Analog, and Potassium Channel Blocker on Muscarinic Receptor-Mediated Inhibition of Cerebral Neurogenic Vasodilation
CTX. CTX, a selective N-type Ca2+ channel blocker that did not affect the U-46619-induced active muscle tone of basilar arteries without endothelial cells, significantly inhibited the relaxation elicited by TNS at 2 and 4 Hz (Fig. 8). In the presence of CTX (0.1 µM), the residual relaxation induced by TNS was not affected by ABET or atropine (n = 6 for each group, Fig. 8).
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8-Bromo-cAMP. 8-Bromo-cAMP (0.5 mM), a membrane-permeable cAMP analog that slightly relaxed the basal vascular tone in arteries without endothelial cells, did not significantly affect TNS-induced relaxation (Fig. 9). In the presence of 8-bromo-cAMP, ABET still inhibited TNS-induced relaxation (Fig. 9), and the inhibition was reversed by atropine (0.1 µM; data not shown). In parallel studies, ABET inhibition of vasodilation elicited by TNS at 2 and 4 Hz was partially reversed by 21.0 ± 9.2 and 30.9 ± 17.0%, respectively, after addition of 8-bromo-cAMP (0.5 mM; n = 8 for each group, Fig. 9B).
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TEA. TEA (1 mM), a nonselective
potassium channel blocker that significantly increased (20-50%)
the basal tone of basilar arteries without endothelial cells,
significantly enhanced the neurogenic vasodilation elicited by TNS at 2 and 4 Hz (Fig.
10A). TEA (1 mM), however, slightly but significantly inhibited the relaxation induced by SNP (an NO-donor; 0.1-100 µM). The
EC50 values for SNP in inducing
relaxation in the control and TEA-treated tissues were 2.94 (1.59-5.43) × 10
7 M and 8.47 (6.0-12.0) × 107
M (n = 4, P < 0.05), respectively. In the
presence of TEA, ABET (1 µM) significantly inhibited the relaxation
induced by TNS at all frequencies examined
(n = 8 for each group, Fig.
10B), and the inhibition was
reversed by atropine (0.1 µM; data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The present study demonstrated that TNS-induced, NO-mediated relaxation in porcine cerebral arteries without endothelial cells was significantly enhanced by atropine and M1- and M2-receptor antagonists. The relaxation however was significantly inhibited by an M2 (but not M1-)-receptor agonist, ACh, and physostigmine. These muscarinic receptor-mediated responses were completely prevented by CTX. Together with the presence of coincident m2 receptor-immunoreactive fibers and NADPHd fibers in cerebral arteries, it is suggested that activation of presynaptic M2 receptors on cholinergic-nitric oxidergic nerve terminals (27) by endogenous ACh results in inhibition of NO-mediated neurogenic vasodilation. The inhibition appears to result from an M2-receptor-mediated decrease in Ca2+ influx via N-type Ca2+ channels and therefore a diminished synthesis and release of NO.
Results from morphological studies have indicated that NOS and ChAT are colocalized in the same nerve fibers in cerebral blood vessels of several species, including the pig (27, 46, 54). It has also been shown in porcine cerebral arteries that the TNS-induced relaxation is abolished by NOS inhibitors and NO scavengers and that L-citrulline, the by-product of NO synthesis, can be recycled to form L-arginine for synthesizing NO in cerebral perivascular nerves (13, 14, 35, 54). These results provide evidence indicating that NO can be released from the perivascular nerves to induce neurogenic vasodilation. Release of ACh from perivascular nerves has also been demonstrated in isolated cerebral arteries from several species, such as the rabbit (18) and pig (our pilot studies). Therefore, it is reasonable to suggest that both NO and ACh are coreleased from the perivascular nerves upon TNS.
The relaxation of cerebral arteries from several species elicited by TNS, however, is predominantly mediated by NO (33, 34). The endogenous ACh does not exhibit any direct effect on the vascular smooth muscle. This is based on the findings that, although a direct effect of exogenous ACh on cerebral vascular smooth muscle is a constriction (30, 33), in the presence of L-NNA (34) or oxyhemoglobin (an NO scavenger; see Ref. 36) to abolish NO-mediated relaxation, TNS has never elicited a cholinergic receptor-mediated constriction in cerebral arteries without endothelial cells. These findings indicate that neuronally released ACh upon TNS does not directly affect the postsynaptic smooth muscle, possibly due to a combination of long synaptic distance and a low synaptic concentration of ACh (31). Accordingly, it has been hypothesized that ACh acts more like a presynaptic transmitter (31, 32).
In the present study, TNS-elicited neurogenic vasodilation in porcine basilar arteries was enhanced by atropine. This enhancement was most likely due to increased NO release from perivascular nerves, since it was abolished by NOS inhibitors and was fully reversed by L-arginine (the precursor of NO synthesis) and L-citrulline, which has been shown to be recycled to form L-arginine for synthesizing NO in porcine cerebral perivascular nerves (14). In addition, the exogenously applied ACh and physostigmine, a cholinesterase inhibitor, inhibited the TNS-elicited relaxation, suggesting that the endogenous ACh coreleased with NO from cholinergic-nitric oxidergic nerves (27) acts on presynaptic muscarinic receptors to inhibit further release of NO and NO-mediated relaxation. This finding is consistent with those found in the bovine and monkey cerebral arteries (4, 47).
Four muscarinic-receptor subtypes have been defined pharmacologically as M1, M2, M3, and M4 subtypes, corresponding to their genes of m1, m2, m3, and m4, respectively (12, 24). M1 receptors are defined by high affinity for pirenzepine and low affinity for methoctramine and 4-DAMP. M2 receptors are defined by high affinity for methoctramine and low affinity for pirenzepine and 4-DAMP. M3 receptors have high affinity for 4-DAMP but not for pirenzepine (12), and M4 receptors have high affinity for tropicamide (28, 29), although they have high affinity for pirenzepine, methoctramine, and 4-DAMP as well (12). All four muscarinic-receptor subtypes have been reported to be involved in presynaptic modulation of transmitter release in various tissue preparations of both peripheral and central nervous systems (12). In the present study, neither 4-DAMP nor tropicamide affected TNS-induced relaxation in porcine basilar arteries, suggesting that M3 and M4 receptors are unlikely to mediate the inhibition of NO release by endogenous ACh. On the other hand, pirenzepine and methoctramine, like atropine, significantly enhanced TNS-induced relaxation, suggesting that M1 and M2 receptors may be involved in mediating the modulation of NO release. TNS-induced relaxation, however, was significantly inhibited by ABET, a selective M2-receptor agonist (42), with EC50 values ranging from 0.05 to 0.4 µM, depending on stimulating frequency, but was not inhibited by McN-A-343, a selective M1-receptor agonist (39), until its concentration was >10 µM. These results suggest that the M2 receptor is the most likely muscarinic subtype that is involved in the presynaptic inhibition by endogenous ACh of NO release. This is further supported by results from immunohistochemical studies that m2 receptor-immunoreactive fibers were coincident with NADPHd fibers in porcine cerebral arteries. Because NADPHd is a reliable marker for NOS in the porcine cerebral arteries (54), the present results suggest that m2 receptors are present on the nitric oxidergic nerve fibers. Although pirenzepine enhanced TNS-induced relaxation, the high concentration of McN-A-343 needed to affect NO-mediated relaxation may indicate a possible nonspecific effect of the M1-receptor antagonist and/or agonist. The possible involvement of presynaptic M1 receptors, however, can not be ruled out completely, since M1-receptor immunoreactivities were not performed due to inaccessibility of M1-receptor antibodies.
It is well established that neurotransmitter release in many tissues is dependent on Ca2+ influx via voltage-sensitive N-type Ca2+ channels, which are found in neurons but not in the smooth muscle (6, 38, 40). Release of NO evoked by electrical stimulation has also been shown to be Ca2+ dependent (7, 8). In the present study, the relaxation of porcine basilar arteries induced by TNS at different frequencies was significantly decreased by CTX by at least 40%, suggesting that Ca2+ influx via N-type Ca2+ channels plays a significant role in activating NOS and therefore NO release in perivascular nerves. The residual relaxation may be due to activation of NOS by other sources of Ca2+, such as Ca2+ influx from L-type channels or release of intracellular Ca2+ (8). In the presence of CTX, the residual relaxation in porcine basilar arteries elicited by TNS was no longer affected by either atropine or ABET. These findings suggest that M2 receptor-mediated inhibition of TNS-elicited nitric oxidergic vasodilation was due exclusively to a negative coupling of M2 receptors to Ca2+ influx via N-type Ca2+ channels. This finding is consistent with those reported by others that muscarinic receptors mediate inhibition of Ca2+ current in many neuronal preparations (1, 11, 20, 41, 49, 52).
The exact mechanism by which activation of the
M2 receptor is coupled to
inhibition of N-type Ca2+ channels
is not established. Activation of
M2 receptors has been shown to be
negatively coupled to adenylate cyclase (51). Evidence however has also
been presented to indicate that cAMP is not essential in modifying
voltage-dependent Ca2+ channels
after activation of G protein-coupled receptors, including the
muscarinic receptor in neuronal preparations (3, 5, 15, 17, 21). This
is in line with the findings of the present study that PTX failed to
prevent the inhibitory effect of muscarinic (including
M2)-receptor agonists or the
potentiating effect of muscarinic-receptor antagonists on TNS-induced
relaxation. The exact reason for the failure of PTX in modifying the
muscarinic-receptor response in the present study remains to be
determined. An almost identical PTX incubation condition used in our
previous study in porcine pial veins indicated that PTX significantly
blocked 2-adrenoceptor-mediated
NE-induced vasoconstriction in these veins (26). Several reports by
others also have indicated that presynaptic
M2 receptor- and
2-adrenoceptor-mediated
inhibition of NE release from sympathetic nerve terminals are
PTX-insensitive (43, 53). Consistent with these findings is the recent
suggestion of a direct interaction between voltage-dependent
Ca2+ channels and G
protein-coupled receptors via a membrane mechanism (23) and the
observation that the 
-subunit of G protein may play an important
role in this direct modification (22, 23, 25). Whether this mechanism
is involved in the presynaptic inhibition of NO release from cerebral
perivascular nerves remains to be examined.
The insignificant role of cAMP in negative coupling of M2 receptor to N-type Ca2+ channels is further supported by the present findings that the M2-receptor agonist-produced inhibition of TNS-induced relaxation was still observed in the presence of a high concentration (0.5 mM) of 8-bromo-cAMP, a membrane-permeable cAMP analog. When it was administered after ABET inhibition of neurogenic vasodilation, however, 8-bromo-cAMP slightly but significantly reversed the neurogenic effect of ABET. Although the exact mechanism of this partial reversal by 8-bromo-cAMP remains unknown, this finding suggests that, should it be involved in M2 receptor-mediated inhibition of neurogenic vasodilation, the intracellular cAMP is not a major factor in causing M2-receptor-mediated downregulation of N-type Ca2+ channels in cerebral perivascular nerves.
Activation of muscarinic receptors has also been shown to increase potassium conductance, resulting in membrane hyperpolarization and a decrease in voltage-dependent Ca2+ channel openings in neurons (6, 9). In the present study, however, TEA (1 mM), a nonspecific potassium channel blocker, significantly enhanced TNS-elicited neurogenic vasodilation. This enhancement was most likely due to increased release of NO from perivascular nerves, since TEA did not increase but rather decreased relaxation induced by SNP (an NO-donor; 0.1-100 µM). Furthermore, in the presence of TEA, the ABET inhibition and atropine potentiation of relaxation elicited by TNS at different frequencies were not different from the control. These results suggest that presynaptic potassium channels are not involved significantly in M2 receptor-mediated inhibition of neurogenic vasodilation.
In conclusion, the present study demonstrated that endogenous ACh, by acting on presynaptic M2 receptors on perivascular nitric oxidergic-cholinergic nerves in porcine basilar arteries, inhibited NO release and therefore diminished NO-mediated neurogenic vasodilation. This M2 receptor-mediated inhibition of NO release and NO-mediated vasodilation appear to be mainly due to a negative coupling of M2 receptors to N-type Ca2+ channels. This results in diminished Ca2+ influx, leading to a decreased NOS activity, NO synthesis, and neurogenic vasodilation. The exact mechanism underlying this negative coupling remains undetermined. However, it is not likely mediated by a decrease in cAMP synthesis and/or potassium channel function. The present finding on the role of muscarinic cholinergic receptors in mediating inhibition of neurogenic vasodilation in large cerebral arteries at the base of the brain is different from that found in the cortical microvascular circulation. Electrical stimulation of a central cholinergic system originating in the nucleus basalis of Meynert and substantia innominata has been shown to contribute to the cortical vasodilator response via activation of muscarinic cholinergic receptors (44), although nicotinic cholinergic receptors have been shown to mediate the vasodilator response in both cortical circulation and large arteries at the base of the brain (44, 50, 55). This finding on the regional difference in cholinergic receptor mechanisms can be important for a complete elucidation of the physiological role of cholinergic innervation in regulating cerebral vascular function.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. G. Yu for technical advice on immunohistochemistry.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-27763 and HL-47574 and Southern Illinois University Central Research Committee Grant 6-23069.
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. §1734 solely to indicate this fact.
Address for reprint requests: T. J. F. Lee, Dept. of Pharmacology, Southern Illinois Univ., School of Medicine, PO Box 19230, Springfield, IL 62794-9629.
Received 13 July 1998; accepted in final form 9 September 1998.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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P < 0.01, significant difference from atropine + L-NNA group.
D-Arginine
(D-Arg) and
D-citrulline
(D-Cit) did not reverse
L-NNA-induced inhibition
(P > 0.05). Atropine-enhanced
relaxation was abolished by TTX. Data represent means ± SE;
n indicates at least 8 experiments in
each group.
