Introduction

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NO acts as a neurotransmitter in autonomic efferent nerves innervating blood vessels (Toda and Okamura, 1996), gastrointestinal tracts (Rand and Li, 1995), corpora cavernosa (Anderson, 1993), anococcygeus muscles (Martin and Gillespie, 1991), and so on. We have demonstrated that not only canine (Toda and Okamura, 1990a, 1990b) but also primate cerebral arteries (Toda and Okamura, 1990c; Toda, 1993) are innervated by NO-mediated vasodilator nerves. However, questions have arisen as to whether the substance liberated from the nerve is free NO or its stable analog, like R-SNO (Myers et al., 1990), since the response to NO derived from the nerve or endothelium is resistant to antioxidants, superoxide anion-generating substances (Toda and Okamura, 1990b, 1990c; Gillespie and Shen, 1990), which are recognized to effectively scavenge NO (Gryglewski et al., 1986).

Vasodilatation induced by nitroxidergic nerve stimulation of monkey cerebral arteries are attenuated by endogenous and exogenous acetylcholine which possibly impair the release of NO by acting on prejunctional muscarinic M₂ receptors (Toda et al., 1997). Adrenergic, cholinergic and nitroxidergic nerves innervate cerebral arteries and various mediators, such as neuropeptide Y, ATP and opiates (Morris et al., 1995), are liberated, together with norepinephrine, from the adrenergic nerve. However, pre- or postjunctional modulations by these substances and neurotransmitters of nitroxidergic nerve functions have not been elucidated.

Aims of the present study were to determine whether NO or R-SNO participates in the neurogenic relaxation of monkey cerebral arteries and to clarify effects of neuropeptide Y, ATP, clonidine, morphine, PACAP and sodium nitroprusside on the response to NO derived from perivascular nerves.

	Methods

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Fifteen Japanese monkeys (Macaca fuscata) of either sex, weighing 6 to 10 kg, were used for these experiments. The Animal Care and Use Committee at our university approved the use of monkey blood vessels in this study.

Each monkey was anesthetized with intramuscular injections of ketamine (40 mg/kg) and sodium pentobarbital (30 mg/kg) and was killed by bleeding from carotid arteries. Pieces of middle and posterior cerebral and basilar arteries (0.2-0.3 mm outside diameter) were rapidly removed from the brain. The arteries were helically cut into strips of approx. 20 mm long. Two to four strips were obtained from each monkey, but any given series of experiments was carried out on strips from different monkeys. The endothelium was removed by gently rubbing the intimal surface with a cotton ball. Endothelial denudation was verified by abolishment of the relaxation induced by Ca²⁺ ionophore A23187 (10⁷ M). The specimen was vertically fixed between hooks in a muscle bath containing the modified Ringer-Locke solution, which was maintained at 37 ± 0.3°C and aerated with a mixture of 95% O₂ and 5% CO₂. The hook anchoring the upper end of the strips was connected to the lever of a force-displacement transducer. The resting tension was adjusted to 1.0 g which is optimal for inducing the maximal contraction. The composition of the solution was as follows (mM): NaCl 120, KCl 5.4, CaCl₂ 2.2, MgCl₂ 1.0, NaHCO₃ 25.0, and dextrose 5.6. The pH of the solution was 7.38 to 7.43. Before the start of experiments, all of the strips were allowed to equilibrate for 90 to 120 min in the bathing media, during which time the fluid was replace every 10 to 15 min.

Isometric mechanical responses were displayed on an ink-writing oscillograph. The contractile response to 30 mM K⁺ was first obtained, and the preparations were repeatedly rinsed and equilibrated. The arterial strips were placed between stimulating electrodes. A train of .2 msec square pulses of supramaximal intensity were applied transmurally at a frequency of 5 Hz for 40 sec, which produced submaximal and reproducible responses (Toda et al., 1997). The stimulus pulses were delivered by an electronic stimulator. In order to obtain the relaxant response to transmural electrical stimulation or agonists, the arterial strips were partially contracted with PGF₂, the contraction being in a range between 28% and 42% of K⁺ (30 mM)-induced contraction. Papaverine (10⁴ M) was added at the end of each series of experiments to obtain the maximal relaxation. Relaxations induced by transmural electrical stimulation or agonists were expressed as values relative to those caused by 10⁴ M papaverine. The strips were treated for 20 min or longer with blocking agents, after the responses to electrical stimulation or agonists were determined to be reproducible.

The results shown in the text and figures are expressed as mean values ± S.E. All reported n values refer to the number of strips from separate monkeys used. Statistical analyses were made using the Student's paired and unpaired t test for two groups and the Tukey's method after one-way analysis of variance for more than three groups. Drugs used were 2,3,5,6-tetramethyl-1,4-benzoquinone (DQ), yohimbine, L-arginine (Nacalai Tesque, Kyoto, Japan), DETCA, SOD, clonidine hydrochloride (Sigma Chemical, St. Louis, MO), pertussis toxin (Kaken Pharm, Tokyo, Japan), acetylcholine chloride (Daiichi, Tokyo), atropine sulfate (Tanabe Seiyaku, Osaka, Japan), tetrodotoxin, morphine hydrochloride (Sankyo, Tokyo), N^G-nitro-L-arginine, PACAP (Peptide Institute, Minoh, Japan), adenosine triphosphate (ATP), Ca²⁺ ionophore A23187 (Boehringer Mannheim GmbH, Mannheim, Germany), neuropeptide Y (Peninsula Lab., Belmont), sodium nitroprusside (SNP; Merck, Japan, Tokyo), PGF₂ (Pharmacia-Upjohn Co., Tokyo), and papaverine hydrochloride (Dainippon Co., Osaka). Responses to NO were obtained by adding the NaNO₂ solution adjusted at pH 2 (Furchgott, 1988), and concentrations of NO applied were expressed as those of NaNO₂ solution.

	Results

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In PGF₂-contracted monkey cerebral arterial strips denuded of the endothelium, transmural electrical stimulation at 5 Hz for 40 sec produced moderate relaxations which were abolished by tetrodotoxin (3 × 10⁷ M). Treatment with L-NA (10⁶ to 10⁵ M) abolished the response which was restored by L-arginine, as demonstrated in our previous reports (Toda and Okamura, 1990c; Toda et al., 1997).

Modifications by duroquinone of the response to nitroxidergic nerve stimulation. Treatment with duroquinone (10⁵ M) slightly attenuated the response to nerve stimulation. SOD (200 U/ml) was without significant effect in the duroquinone-treated strips (fig. 1). Typical recordings are illustrated in figure 2. Modifications by treatment with DETCA of the duroquinone action were evaluated in a pair of strips obtained from the same monkeys. Data on the paired analysis are summarized in figure 1. In the strips treated with DETCA (10³ M) for 45 min and rinsed, duroquinone inhibited the neurogenic response to a greater extent, as compared with that without the treatment (29.6 ± 5.5% vs. 68.0 ± 10.1% inhibition, P < .01, unpaired t test). In 3 out of 7 strips treated with DETCA, duroquinone abolished the response, as demonstrated in the lower tracing of figure 2. SOD (200 u/ml) partially restored the response depressed by 10⁵ M duroquinone in DECTA-treated strips; the value was identical with that seen in the presence of duroquinone plus SOD in nontreated strips (fig. 1). The stimulation-induced relaxation was not significantly influenced by DETCA-treatment; mean values before and after the treatment were 35.3 ± 4.9 and 30.1 ± 4.7% (93.3 ± 6.9% of control, n = 7). In all of 3 additional strips from separate monkeys treated with DETCA, relaxations induced by electrical stimulation were abolished by raising the concentration of duroquinone to 3 × 10⁵ M. 

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Modifications by duroquinone (DQ, 105 M) and SOD (200 u/ml) of the relaxant response to transmural electrical stimulation (5 Hz for 40 sec) in monkey cerebral arterial strips under nontreated and DETCA (103 M for 45 min)-treated conditions. Two strips from the same monkeys were used for control and treated series of experiments; numbers in parentheses indicate the number of strips from separate monkeys. The ordinate denotes the stimulation-induced response relative to that elicited by 104 M papaverine which produced the maximal relaxation. The number in each column represents the value relative to that in control media (C). Significantly different from control (C), aP < .01 (Tukey's method); bP < .001; cP < .01; dP < .02 (paired t test). Significantly different from the value with duroquinone, eP < .01 (paired t test). Vertical bars represent mean values ± S.E.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Tracings of the response to transmural electrical stimulation (5 Hz) of the strips obtained from the same monkey, as affected by duroquinone (DQ, 105 M), SOD (200 u/ml) and tetrodotoxin (TTX, 3 × 107 M) under control (upper tracing) and DETCA (103 M for 45 min)-treated condition (lower). The strips were partially contracted with PGF2. Dots denote the application of electrical stimulation. The upward arrow indicates the addition of supplemental dose of PGF2 to raise the tone. PA represents 104 M papaverine that produced the maximal relaxation.

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View larger version (21K): [in this window] [in a new window]   Fig. 3.   Modifications by duroquinone (DQ) and SOD (200 U/ml) of the relaxant response to exogenous NO in nontreated (left) and DETCA-treated (right) strips of monkey cerebral arteries. The ordinate denotes the NO-induced relaxation relative to that induced by 104 M papaverine. Significantly different from control, aP < .01; significantly different from the value with 105 M duroquinone, bP < .01, cP < .05; significantly different from the value with 3 × 105 M duroquinone, dP < .01, eP < .05 (Tukey's method). Numbers of strips used were 7 for nontreated and DETCA-treated series obtained from separate monkeys. Vertical bars represent mean vlaues ± S.E.

Modifications by agonists of the response to nitroxidergic nerve stimulation. Relaxations induced by transmural nerve stimulation were not influenced by treatment with NPY in concentrations from 3 × 10⁹ to 3 × 10⁸ M (fig. 4), which contracted the arterial strips by 23 ± 10 mg (n = 4), 113 ± 36 mg (n = 7) and 121 ± 29 mg (n = 5), respectively. Clonidine (10⁷ and 10⁶ M), yohimbine (10⁷ M) and morphine (10⁶ M) did not alter the tone of arterial strips contracted with PGF₂ nor the response to nerve stimulation (table 1). SNP (3 × 10⁸ and 10⁷ M) produced relaxations averaging 13.6 ± 2.9% and 34.2 ± 7.2% of papaverine (10⁴ M) (n = 5), respectively, and the higher concentration of SNP attenuated the response to nerve stimulation (table 1). ATP (10⁷ M) and PACAP (10⁷ M) relaxed the strips by 11.0 ± 1.5% (n = 4) and 47.4 ± 5.3% (n = 5), respectively, and also unaffected the stimulation-induced relaxation (table 1). On the other hand, acetylcholine inhibited the relaxations induced by transmural electrical stimulation in a dose-dependent manner (table 1). In the arterial strips treated with SNP, ATP, PACAP, or ACh the tone was adjusted by PGF₂ to a level similar to that prior to the addition of the vasodilators.

View larger version (48K): [in this window] [in a new window]   Fig. 4.   Effects of neuropeptide Y (NPY) on the response to transmural electrical stimulation (5 Hz) of monkey cerebral arterial strips. The ordinate denotes the relaxant response relative to that elicited by 104 M papaverine which produced the maximal relaxation. The number in each column represents the value relative to that obtained in control media (C). Numbers in parentheses indicate the number of strips used. Vertical bars denote mean values ± S.E.

	Discussion

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Monkey cerebral arterial strips responded to electrical nerve stimulation with relaxations which were abolished by treatment with NO synthase inhibitors and restored by L-, but not D-, arginine (Toda and Okamura, 1990c, 1996). Release of NO, measured as NO_x, during nerve stimulation has been detected. NO synthase-immunoreactive nerve fibers innervate the cerebral arterial wall (Yoshida et al., 1994). Therefore, NO is hypothesized to be a neurotransmitter responsible for vasodilatation in monkey cerebral arteries. However, antioxidants generating superoxide anion do not inhibit the vascular response mediated by NO derived from the endothelium and perivascular nerve (Gillespie and Sheng, 1990; Toda and Okamura, 1990c), allowing us to consider that NO-containing mediators resistant to superoxide anion, like R-SNO, are involved in the response (Meyers et al., 1990). The inability of antioxidants to depress the NO-mediated response are postulated to be due to a difficult access of the agents to the site of NO generation or due to a protective mechanism against pathogenic products, such as superoxide anions. In the present study, monkey arterial relaxation in response to nerve stimulation was slightly inhibited by duroquinone in a concentration (10⁵ M) sufficient to abolish the relaxation induced by a concentration (10⁷ M) of exogenous NO equipotent to electrical nerve stimulation (5 Hz). However, this inhibition was not sensitive to SOD. Since duroquinone generates superoxide anion and presumably increases its concentrations intra- and extracellularly (Lilley and Gibson, 1995) and SOD applied exogenously scavenges only extracellular superoxide, intracellular superoxide may be sufficient to inhibit the neurogenic relaxation even in the presence of endogenous SOD. However, the possibility that the inhibition is due to the action of duroquinone other than superoxide generation cannot be excluded. When the arteries were treated with DETCA, an inhibitor of Cu-Zn SOD (Cocco et al., 1981; Kelner et al., 1989), the neurogenic relaxation was markedly attenuated in 4 out of 7 strips or abolished in the remaining 3 by the same concentration of duroquinone. The reduced response was partially reversed by SOD. Increasing the concentration of duroquinone to 3 × 10⁵ M abolished the response in all of the strips used. These findings strongly suggest that endogenous SOD in the vicinity of vasodilator nerve terminal and smooth muscle protects nitroxidergic nerve function by degrading superoxide generated intra- and extracellularly. This suggests that NO per se, not R-SNO, is the transmitter in nitroxidergic nerves innervating monkey cerebral arteries. Incomplete reversal by SOD of the inhibitory action of duroquinone may be due to barriers to the intra- and extracellular sites of superoxide anion generation. Similar results with DETCA were also obtained in extravascular tissues innervated by nonadrenergic, noncholinergic nerves (Martin et al., 1994; Lilley and Gibson, 1995; Paisley and Martin, 1996).

Neurotransmitters and modulators in peripheral nerves are expected to interact in their release from nerve terminals or synthesis in nerve terminals. NPY is known to modulate adrenergic and cholinergic nerve functions and to inhibit the vasodilator response to nerve stimulation, mediated by CGRP in rat mesenteric arteries (Kawasaki et al., 1991). The authors suggest that the release of CGRP is impaired by NPY, since the relaxation induced by exogenous CGRP is not influenced. However, this is not the case for nitroxidergic nerve, since NPY at the same concentration that inhibits CGRP nerve function did not alter the relaxant response to nitroxidergic nerve stimulation. Clonidine, an agonist of adrenergic alpha-2 receptors that mediates the inhibition of transmitter release from adrenergic and cholinergic nerves (Starke, 1981; Langer, 1981), and yohimbine, an alpha-2 receptor antagonist, did not change the neurogenic relaxation. ATP, a substance liberated from stimulated adrenergic nerves, did not affect the response either. Endogenous opiates have also been proposed to modulate adrenergic and cholinergic nerve functions via kappa opioid receptors (Gibbins, 1992; Morris et al., 1995). However, exogenously applied morphine, a kappa and mu receptor agonist, did not affect the response to vasodilator nerve stimulation.

PACAP, an activator of adenylate cyclase, was recently found in the ovine hypothalamus (Miyata et al., 1989) and was shown to elicit cerebral vasodilatation (Uddman et al., 1993; Tong et al., 1993; Seki et al., 1995). Because this peptide coexists with VIP in cat pial arteries (Uddman et al., 1993) and VIP is present in parasympathetic ganglia and nerve fibers together with NO synthase and acetylcholinesterase (Hara et al., 1985; Minami et al., 1994), modulation of nitroxidergic neurological responses by PACAP was evaluated. Modulation by VIP of the response to nitroxidergic nerve stimulation has not been observed, but effects of PACAP were not previously determined (Toda et al., 1997). PACAP dilated monkey cerebral arteries but did not change the nitroxidergic nerve function in the current study. However, sodium nitroprusside, an NO donor that increases the production of cyclic GMP in vascular smooth muscle, relaxed monkey cerebral arteries dose-dependently and inhibited the response to nerve stimulation at high dose. It is possible that exogenous NO inhibits the activity of neuronal NO synthase as a negative feedback mechanism (Ignarro et al., 1994) or that increased production of cyclic GMP in smooth muscle interferes with the relaxation mediated by cyclic GMP (Toda and Okamura, 1991; Matsumoto et al., 1993). Unfortunately, the inhibitory action could not be observed in low concentrations insufficient to elicit smooth muscle relaxation. On the other hand, acetylcholine inhibition of the nitroxidergic nerve function was previously reported (Toda et al., 1997), suggesting that endogenous NO production can inhibit neural nitroxidergic dilatation.

In summary, it is concluded that vasodilatation induced by perivascular nerves in monkey cerebral arteries is mediated mainly if not entirely by free NO. Although neurogenic acetylcholine appears to be important in the control of nitroxidergic and adrenergic nerves (Ayajiki et al., 1993; Toda et al., 1997; Zhang et al., 1997), other neurotransmitters and modulators such as neuropeptide Y, ATP, norepinephrine, opiates and PACAP liberated from neighboring or coexisting efferent nerves do not appear to participate in regulating NO-mediated, vasodilator nerve function.