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(Circulation Research. 1995;76:441-447.)
© 1995 American Heart Association, Inc.

Articles

Essential Role for Nitric Oxide in Neurogenic Inflammation in Rat Cutaneous Microcirculation

Evidence for an Endothelium-Independent Mechanism

Radhika Kajekar, Phillip K. Moore, Susan D. Brain

From the Pharmacology Group and Vascular Biology Research Centre, Division of Biomedical Sciences, King's College, London, UK.

Correspondence to Dr S.D. Brain, Pharmacology Group and Vascular Biology Research Centre, Division of Biomedical Sciences, King's College, Manresa Road, London SW3 6LX, UK.

	Abstract

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Abstract The possible modulatory role of nitric oxide (NO) in neurogenic edema formation in rat paw skin, induced by electrical stimulation of the saphenous nerve, was investigated by using two NO synthase inhibitors, N^G-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI). Both L-NAME (100 mg/kg IV, P<.05) and 7-NI (10 mg/kg IV, P<.05) caused an L-arginine (100 mg/kg IV, P<.01)–reversible inhibition of neurogenic edema as measured by ¹²⁵I-albumin accumulation, whereas D-NAME (inactive enantiomer of L-NAME) and 6-aminoindazole (structurally similar to 7-NI) were without inhibitory effect. L-NAME produced the predicted vasopressor effect (before, 115±18 mm Hg; 5 minutes after, 174±18 mm Hg; n=6; P<.05), whereas 7-NI showed no significant increase in blood pressure (before, 96±9 mm Hg; 5 minutes after, 102±10 mm Hg; n=6), and neither L-NAME nor 7-NI had any effect on basal or vasodilator calcitonin gene-related peptide (CGRP, 10 pmol per site)–stimulated local blood flow in rat skin, as measured by laser Doppler flowmetry. Furthermore, systemic and local 7-NI had no effect on edema formation induced by local administration of substance P (with or without CGRP) and histamine (with or without CGRP) in rat skin. Since 7-NI blocks edema produced by stimulation of the saphenous nerve, it is suggested that release of NO is involved in neurogenic edema formation, but the vasodilator action of NO is unimportant in this context. We suggest that NO is involved in the release of neuropeptides from sensory nerves.

Key Words: neurogenic inflammation • nitric oxide • substance P • calcitonin gene-related peptide • sensory nerves

	Introduction

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Neurogenic inflammation in the skin results from the proinflammatory actions in the microvasculature of vasoactive neuropeptides that include the well-described substance P and calcitonin gene-related peptide (CGRP).^{1 2} The neuropeptides are contained in, and released in response to the efferent stimulation of, sensory nerves that are mainly unmyelinated and of the C and A delta subtypes.^{3 4 5} Neurogenic inflammation is thought to be involved in vascular-associated diseases, which include migraine,⁶ asthma,⁷ and rheumatoid arthritis^{8 9} ; thus, there has recently been increasing interest in determining pharmacological mechanisms by which neurogenic inflammation can be inhibited.

The stimulation of the saphenous nerve in rats has become an established small animal model of neurogenic inflammation in which electrical stimulation leads to edema formation in addition to increased blood flow.¹⁰ Recent studies with selective antagonists have demonstrated that CGRP and substance P are responsible for the neurogenic inflammation. The increased blood flow is primarily due to CGRP,^{11 12} and the edema formation is mediated by substance P, which acts to increase microvascular permeability via NK₁ receptors,^{13 14} although CGRP, probably as a consequence of its vasodilator activity, has a potentiating effect on the edema formation.¹¹

One approach to developing drugs that can act to inhibit neurogenic inflammation is to develop selective neuropeptide antagonists that, if found to be effective in studies similar to those described above, have the potential to inhibit the neurogenic component of vascular and inflammatory diseases in humans. However, by virtue of their properties, selective receptor antagonists will only antagonize the effects mediated via the specific receptors that they antagonize; hence, an alternative approach is to inhibit neuropeptide release. There is evidence that presynaptic receptors for several endogenous agents are present on sensory nerves.¹⁵ We are interested in the possibility that nitric oxide (NO) may have an essential role in the release of neuropeptides from nerves.

The most common pharmacological tools used to investigate the function of endogenous NO have been NO synthase inhibitors, especially N^G-monomethyl-L-arginine¹⁶ and N^G-nitro-L-arginine methyl ester (L-NAME).¹⁷ However, these agents inhibit endothelial NO synthase, resulting in an inhibition in the release of vasodilator NO, which has a physiological role in maintaining vascular tone. A rise in blood pressure is observed after systemic administration of NO synthase inhibitors,¹⁸ and a decrease in skin blood flow is observed after intradermal administration.¹⁹ These actions complicate the interpretation of studies that are designed to investigate other possible functions of NO. For example, L-NAME inhibits cutaneous edema formation,^{20 21} but results suggest that this effect is secondary to a decrease in cutaneous blood flow.²⁰ The vasodilator effect of exogenous CGRP is NO independent in skin,^{22 23} but interestingly, L-NAME, but not a similar vasoconstrictor dose of noradrenaline, inhibits CGRP release induced by capsaicin in rabbit skin.^{23 24} Further, Lippe et al²⁵ have suggested that the neurogenic vasodilatation, but not edema formation, induced by mustard oil is inhibited by L-NAME; this finding led them to suggest that endothelium-derived NO is involved in neurogenic vasodilatation.

Recently, an alternative inhibitor of brain-derived NO synthase in vitro, which has no effect on blood pressure in vivo, has been identified.²⁶ This compound, 7-nitro indazole (7-NI), has been demonstrated to inhibit mouse cerebellar NO synthase activity in vitro.²⁶ In addition, 7-NI exhibits antinociceptive activity in the mouse, which has been observed after the formalin-induced hind paw licking and acetic acid–induced abdominal constriction assays.^{26 27} These results suggest a role of nerve-derived NO in the mediation of the afferent nociceptive activity of sensory nerves; thus, 7-NI has been suggested to be a useful pharmacological tool to enable the investigation of NO synthase in sensory nerves. In the present study, we have investigated the effect of 7-NI on the efferent activity of the rat saphenous nerve in stimulating neuropeptide-dependent edema formation. This has enabled us to investigate the influence of nerve-derived NO on neurogenic edema formation without concomitant vascular changes secondary to inhibition of endothelium-derived NO.

	Materials and Methods

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Saphenous Nerve–Induced Edema Formation in Rat Hind Paw
Edema formation in the rat hind paw after electrical stimulation of the saphenous nerve was measured according to a protocol previously described.¹¹ Male Wistar rats (250 to 350 g) were anesthetized with sodium pentobarbital (40 to 60 mg/kg IP, plus maintenance doses as necessary). Body temperature was maintained at 36°C to 38°C by automatic control of a heating pad. The hind paws were shaved, and the saphenous nerves in both paws (stimulated and sham [control] paw) were carefully dissected, ligated centrally, placed on bipolar platinum electrodes, and immersed in mineral oil. Thirty minutes after this procedure, ¹²⁵I-labeled human serum albumin (50 kBq) and Evans blue dye (0.5 mL of 2.5% [wt/vol] in saline) were administered intravenously through the tail vein 2 to 3 minutes before the administration of test agents. Test agents, respective vehicle controls, and other related compounds were injected intravenously 5 minutes before electrical stimulation (10 V, 1 millisecond, and 2 Hz for 5 minutes) of the saphenous nerve on one paw; the other paw acted as a control (sham paw). In one group of animals, L-NAME (10 mg/kg) was administered intravenously 15 minutes before electrical stimulation to study the effects of a longer pretreatment time. The parameters of electrical stimulation were selected from frequency-response studies to give a submaximal level of edema formation after stimulation of the saphenous nerve. Immediately after electrical stimulation was terminated, a cardiac blood sample (2 mL) was taken, and the animal was killed with an anesthetic overdose. The blood sample was centrifuged at 8000g for 4 minutes to obtain a plasma sample. The area of skin on the stimulated hind paw innervated by the saphenous nerve (demarked by Evans blue dye extravasation) was removed and weighed. A similar area of skin was removed from the unstimulated (sham) paw and weighed. Radioactivity was counted in the skin samples and in 100 µL of plasma. Edema formation was expressed as the volume of plasma accumulated in the skin samples, calculated by comparing the radioactivity present in each sample with that in 1 mL of plasma. The results are expressed as a ratio of plasma extravasation (microliters of edema per 100 mg tissue) in the stimulated and sham paws for each rat.

Effect of NO Synthase Inhibitors on Edema Formation Induced by Injected Neuropeptides in Rat Dorsal Skin
Effect of Intravenous 7-NI (10 mg/kg) on Edema Formation
Local edema formation in response to intradermally injected agents was measured by the extravascular accumulation of intravenously injected ¹²⁵I-labeled human serum albumin.²⁸ Male Wistar rats (250 to 350 g) were anesthetized with sodium pentobarbital (40 mg/kg IP, plus maintenance doses as necessary). The dorsal skin of the rat was shaved, and a randomized site plan was marked out. A mixture of ¹²⁵I-human serum albumin (50 kBq) and Evans blue dye was administered intravenously via the tail vein 2 minutes before intravenous test agents. 7-NI (10 mg/kg) and sodium carbonate (vehicle; 0.5% Na₂CO₃, 5 mL/kg) were given intravenously through the tail vein 5 minutes before intradermal agents. Edema-forming mediators were prepared in 0.1 mL Tyrode's balanced salt solution and injected intradermally in dorsal skin, in duplicate, according to a balanced site pattern. After a 30-minute accumulation period, a blood sample (2 mL) was taken by cardiac puncture, and a plasma sample was prepared as above. The animal was killed with anesthetic overdose. The dorsal skin was removed, and injection sites were punched out (diameter, 16 mm) and counted for radioactivity in a gamma counter. Edema formation at each site is expressed as the volume of plasma accumulated, calculated by comparing the radioactivity present at each site with that in 1 mL of plasma.

Effect of Intradermal L-NAME (100 nmol per Site) and 7-NI (100 nmol per Site) on Edema Formation
The above procedure was repeated, except L-NAME and 7-NI were coadministered intradermally with preformed mediators of inflammation in the rat dorsal skin.

Blood Pressure and Skin Blood Flow Measurement After Intravenous Administration of NO Synthase Inhibitors
Male Wistar rats were prepared as described above, and hind legs were shaved and depilated with a commercial cream. The carotid artery was exposed, ligated cranially, cannulated, and connected to a pressure transducer attached to a pen recorder (Lectromed, Multitrace 2). Blood pressure was monitored for 20 to 30 minutes before intravenous administration of test agents and for 30 minutes thereafter. Results are expressed as mean arterial pressure (MAP).

Concomitant skin blood flow measurements using laser Doppler flowmetry (dual-channel laser Doppler, MBF3D, Moor Instruments) was monitored on the hind paws. Laser probes were secured on each hind paw, under which intradermal CGRP (10 pmol per site, which represents a site of increased blood flow) or intradermal Tyrode's solution (100 µL per site, which represents a site of basal blood flow) was administered 10 minutes before intravenous test agents. Blood flow was monitored 10 minutes before intradermal injections and for 30 minutes after the administration of test agents. At the end of this period, the animal was killed with anesthetic overdose. Results are recorded as mean flux value (in arbitrary units, which represent the number of red blood cells moving in the volume measured by the path of the laser beam) and used as an index of skin blood flow (see Lawrence and Brain¹⁹ ).

Basal Blood Flow Measurements After Intradermal Administration of L-NAME (100 nmol per Site) and 7-NI (100 nmol per Site) in Rat Dorsal Skin
Male Wistar rats (250 to 300 g) were prepared as described above, and the dorsal skin was shaved and depilated. Two laser Doppler probes were secured onto the skin, and basal blood flow was measured. L-NAME (100 nmol per site) was intradermally injected at one site, while a concomitant control injection of Tyrode's solution was given at the other. Blood flow was measured for 30 minutes thereafter. The laser probes were then moved to alternative sites, where, after a basal blood flow measurement, an intradermal dose of 7-NI (100 mg/kg) and vehicle (0.0025% Na₂CO₃) was administered and continuously monitored for 30 minutes. The above procedure (subject to random-order injections) was repeated in each animal (ie, two replicates for each treatment).

Materials
The following drugs were used: L-NAME, the inactive enantiomer of L-NAME (D-NAME), L-arginine hydrochloride, substance P, histamine diphosphate salt, and Evans blue dye from Sigma Chemical Co; 7-NI from MTM Lancaster Ltd; 6-aminoindazole (6-AI) from Aldrich Ltd; human -CGRP, a gift from Dr U. Ney, Celltech; sodium pentobarbital (Sagatal) from May and Baker; ¹²⁵I-labeled human serum albumin from Amersham International; and Immac (used as a depilatory cream) from Reckitt & Colman. CGRP and substance P were stored in stock solutions at -20°C. All other drugs were weighed out on the day of use. Intradermally injected agents were diluted in Tyrode's solution containing (mmol/L) NaCl 136.89, KCl 2.68, NaH₂PO₄ 0.42, NaHCO₃ 11.9, MgCl₂ 1.05, and glucose 5.5. Intravenously administered drugs were dissolved in saline (0.9%) except for 7-NI and 6-AI, which were dissolved in Na₂CO₃ (0.5% [wt/vol]) by sonication and warming at a maximum concentration of 2 mg/mL, which represents the limit of solubility of 7-NI under these conditions.

Statistical Analysis
Results are expressed as mean±SEM. Skin blood flow and blood pressure differences, dorsal skin edema experiments, and the L-arginine study group were assessed for significance of treatments by Bonferroni's modified t test, which uses the SEM estimate for ANOVA to account for the fact that multiple tests were performed. For analysis of hind paw skin edema experiments after electrical stimulation (except the L-arginine group), unpaired Student's t test was used (P<.05 and P<.01 for both tests).

	Results

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Effect of NO Synthase Inhibitors on Neurogenic Edema Formation
Electrical stimulation of the saphenous nerve in control rats (treated with saline) induced edema formation in the skin of the stimulated hind paw (13.46±2.2 µL/100 mg tissue, n=6) compared with the sham paw, where basal values for albumin extravasation were observed (1.78±0.4 µL/100 mg tissue, n=6). Furthermore, there was no significant edema formation in the contralateral paw (sham) after intravenous administration of test agents or vehicles (microliters edema per 100 mg tissue in sham paw: L-NAME [10 mg/kg] 1.35±0.2, vehicle 1.67±0.11; L-NAME [100 mg/kg] 2.0±0.3, vehicle 1.78±0.4; 7-NI [3 mg/kg] 1.64±0.18, vehicle 1.46±0.1; and 7-NI [10 mg/kg] 1.47±0.13, vehicle 1.33±0.08 [n=5 to 7 rats]). Fig 1 shows the ratio of edema measured in the stimulated versus unstimulated paw where control rats received the drug solvents, saline (Fig 1a), or sodium carbonate (Fig 1b). The NO synthase inhibitor L-NAME was tested at two doses. A significant inhibition of edema formation was observed with the higher dose of L-NAME (100 mg/kg), with no effect observed with the same dose of D-NAME (Fig 1a). L-NAME (10 mg/kg) had no effect on edema formation when pretreated 5 minutes before electrical stimulation of the saphenous nerve or after a 15-minute pretreatment (as shown in Fig 1a). 7-NI inhibited paw skin edema formation induced by electrical stimulation of the saphenous nerve. Inhibition was dose dependent, with significance being observed at the 10 mg/kg dose of 7-NI (Fig 1b). 6-AI, which like 7-NI is an indazole derivative but exhibits minimal NO synthase inhibitory activity in vitro,²⁷ had no effect on saphenous nerve–induced edema formation (Fig 1b).

View larger version (21K): [in this window] [in a new window]   Figure 1. Bar graphs showing the effect of nitric oxide synthase inhibitors on edema formation after electrical stimulation (10 V, 1 millisecond, and 2 Hz for 5 minutes) of the rat saphenous nerve. a, Effect of intravenous NG-nitro-L-arginine methyl ester (L-NAME) and the inactive enantiomer of L-NAME (D-NAME) (solid bars) and, in each case, the response of a control group that received the vehicle (0.9% saline, 0.8 mL/kg) (open bars). b, Effect of intravenous 7-nitroindazole (7-NI) and 6-aminoindazole (6-AI) (solid bars) and, as above, the response of a control group that received the vehicle (0.5% Na2CO3, 5 mL/kg) (open bars). Results are expressed as the ratio of edema (microliters per 100 mg tissue, mean±SEM, n=4 to 7) in the stimulated and unstimulated hind paw. *Significant (P<.05) inhibitory response.

Effect of L-Arginine on the Ability of 7-NI and L-NAME to Inhibit Neurogenic Edema Formation
It is shown in Fig 2 that L-arginine (100 mg/kg IV) had no effect on neurogenic edema formation induced by electrical stimulation of the saphenous nerve. However, L-arginine significantly reversed the inhibition of edema induced by both L-NAME (100 mg/kg) and 7-NI (10 mg/kg). Further, intravenous L-arginine had no effect on basal blood flow measured in the rat contralateral (sham) hind paw (before, 29.82±3.46 flux; 5 minutes after, 32.33±5.65 flux; n=4; P>.05).

View larger version (32K): [in this window] [in a new window]   Figure 2. Bar graphs showing the effect of L-arginine on the ability of NG-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI) to inhibit saphenous nerve–induced edema. L-NAME, 7-NI, or the respective vehicle (saline/Na2CO3) was administered (intravenously) alone or together with L-arginine 5 minutes before electrical stimulation of the saphenous nerve (10 V, 1 millisecond, and 2 Hz for 5 minutes). a, Effect of L-arginine on the ability of L-NAME to affect saphenous nerve–induced edema formation. The edema response in groups of animals receiving L-NAME alone (100 mg/kg, solid bars), L-NAME (100 mg/kg) and L-arginine (100 mg/kg) together (crosshatched bars), L-arginine alone (100 mg/kg, stippled bars), and the vehicle (saline, open bars) is shown. b, Effect of L-arginine on the 7-NI response on edema formation. The edema response in groups of animals receiving 7-NI alone (10 mg/kg, solid bars), 7-NI (10 mg/kg) and L-arginine (100 mg/kg) together (crosshatched bars), L-arginine alone (100 mg/kg, stippled bars), and the vehicle (Na2CO3, open bars) is shown. Responses are expressed as the ratio of edema (microliters per 100 mg tissue, mean±SEM, n=4) measured in the stimulated and unstimulated hind paw. **Statistical differences (P<.01) between groups of animals receiving test agents alone and in the presence of L-arginine.

Effect of NO Synthase Inhibitors on Blood Pressure and Skin Blood Flow
The effect of intravenous L-NAME, 7-NI, and their related controls on MAP and cutaneous blood flow are shown in the Table. L-NAME (100 mg/kg) increased MAP by 51.3% (P<.05), which was recorded 5 minutes after intravenous administration, but had no significant effect on basal (Tyrode-injected site) or increased (CGRP-injected site) cutaneous blood flow measured on the rat hind paw. By contrast, 7-NI (10 mg/kg) and Na₂CO₃ (vehicle) had no effect on MAP^{26 27} or on basal or increased skin blood flow. Similarly, D-NAME (100 mg/kg IV) and saline (vehicle) were without effect on blood pressure or skin blood flow as shown in the Table. L-NAME (10 mg/kg) also increased MAP, with significant effects observed at 5 minutes (30.72% increase, P<.01), 15 minutes (46.29% increase, P<.001), and 30 minutes (45.60% increase, P<.001) after intravenous administration, with no significant increase in blood pressure observed in vehicle (saline)-treated animals.

View this table: [in this window] [in a new window]   Table 1. Blood Pressure and Local Skin Blood Flow Changes Before and After Addition of Systemic NG-Nitro-L-arginine Methyl Ester, 7-Nitroindazole, and Their Related Controls

The effect of intradermal L-NAME (100 nmol per site) and 7-NI (100 nmol per site) on basal blood flow in rat dorsal skin was investigated. L-NAME, as previously reported,^{19 20} significantly reduced basal blood flow (measured from 5 to 30 minutes after L-NAME administration, P<.05 for all time points, n=5 rats) compared with Tyrode's solution, whereas 7-NI had no significant effect on basal blood flow compared with Tyrode's solution.

Effect of NO Synthase Inhibitors on Edema Formation Induced by Injected Neuropeptides
The effects of intravenous 7-NI (10 mg/kg) and its vehicle, Na₂CO₃, on edema formation induced by intradermally injected mediators in the dorsal skin of the rat are shown in Fig 3a. The ability of substance P (10 and 30 pmol per site) and histamine (30 nmol per site) to induce edema formation in rat skin, in the presence and absence of an edema-potentiating dose of CGRP (10 pmol per site), is shown in both 7-NI (10 mg/kg)– and vehicle (Na₂CO₃)–treated animals. CGRP, as a consequence of its vasodilator activity, potentiates edema formation^{11 13 14 29 30} and is used to mimic neurogenic edema formation where both substance P and CGRP are involved. No significant inhibition of edema formation induced by the intradermal mediators was observed in the presence of intravenous 7-NI.

View larger version (23K): [in this window] [in a new window]   Figure 3. Bar graphs showing the effect of 7-nitroindazole (7-NI) on edema formation induced by intradermal mediators in the dorsal skin of the rat. a, Effect of intravenous 7-NI (10 mg/kg, solid bars) or 0.5% Na2CO3 (vehicle, 5 ml/kg; open bars). Mediators were injected intradermally 5 minutes after the intravenous agent, and edema formation was measured for 30 minutes. Responses to substance P (SP) and histamine (HA) with or without calcitonin gene-related peptide (CGRP) are shown. The dotted line represents the response at control sites injected with Tyrode's solution (100 µL) alone. Results are expressed as mean±SEM (n=4). No significant difference of treatments, assessed by Bonferroni's modified t test, was observed in 7-NI–treated and vehicle-treated animals. b, Effect of intradermal NG-nitro-L-arginine methyl ester (L-NAME) and 7-NI on edema induced by SP in rat skin. Edema induced by SP alone (open bars) with or without CGRP coinjected with L-NAME (100 nmol per site, hatched bars) or 7-NI (100 nmol per site, solid bars) is shown. The dotted line represents the response at control sites that received Tyrode's solution (100 µL) alone. The response of agents given alone is also shown. Results are expressed as mean±SEM (n=8 to 12). **Significant (P<.01) inhibitory effect on SP-induced edema.

The effects of locally injected (intradermal) L-NAME (100 nmol per site) and 7-NI (100 nmol per site) on edema formation are shown in Fig 3b. L-NAME, as previously reported,²⁰ significantly inhibited edema induced by substance P (100 pmol per site) but had no significant effect on edema induced by substance P in the presence of a vasodilatory dose of CGRP (10 pmol per site). 7-NI had no significant effect on either substance P–induced edema formation or substance P–induced edema observed in the presence of CGRP.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, two NO synthase inhibitors, L-NAME^{17 31} and 7-NI,^{26 27} selectively inhibited edema formation in rat skin induced by electrical stimulation of the saphenous nerve, thus suggesting a role for endogenous NO in the mediation of neurogenic edema formation. This inhibitory effect of both NO synthase inhibitors was reversed by the coadministration of intravenous L-arginine, whereas D-NAME (the inactive enantiomer of L-NAME) and 6-AI (which is structurally similar to 7-NI but devoid of NO synthase inhibitory activity) showed no inhibitory action on neurogenic edema formation. 7-NI is a selective inhibitor of NO synthase in vitro and in vivo,²⁷ and the present study confirms that the effect of intravenous 7-NI is not accompanied by cardiovascular effects or local vascular or edema effects when given intradermally. The inhibitory effect of 7-NI in the present study leads us to suggest that the observed modulation of neurogenic inflammation is mediated by the inhibition of NO synthase at the sensory nerve level.

Earlier investigations have suggested that neurogenic inflammation in the rat is mediated primarily by the release of endogenous neurokinins (eg, substance P) and CGRP. The neurokinins act to increase microvascular permeability to plasma proteins by the stimulation of neurokinin NK₁ receptors^{13 14} ; CGRP is involved in mediating the increased blood flow.¹¹ L-NAME, when injected intradermally, has been shown to inhibit basal blood flow in rat skin,¹⁹ thus providing evidence for a role of NO, produced locally in the cutaneous microvasculature, in the maintenance of vasodilator tone. However, it is unlikely that NO is involved in the CGRP-induced blood flow, because L-NAME has been shown to be ineffective in reducing the increased skin blood flow observed after local administration of CGRP in the rabbit²⁴ and rat.²² Furthermore, in the present study neither systemic L-NAME nor 7-NI, at doses that inhibited saphenous nerve–induced edema formation, had any effect on basal or CGRP-induced local blood flow in rat skin, where L-NAME produced the predicted vasopressor effect,¹⁸ whereas 7-NI showed no increase in blood pressure after intravenous administration.²⁷ The absence of any effect of L-NAME on basal skin blood flow is probably due to the fact that intravenous L-NAME is hypertensive and hence changes in skin blood flow are masked, as any changes are secondary to the effects in other vascular beds.

There is confusing evidence as to the involvement of NO in the substance P–induced edema response observed in inflammatory models. NO synthase inhibitors such as L-NAME have been used to suggest that NO generation is involved in the vasodilator²⁵ and exudative component of substance P–induced responses in rats.^{20 32} Evidence suggests that the inhibitory effect of L-NAME is secondary to inhibition of basal blood flow.²⁰ In a further study, substance P–induced plasma extravasation in rats has been demonstrated to be resistant to systemic NO synthase inhibitors, indicating that NO generation is not essential for the observation of neurogenic edema after the activation of NK₁ receptors.³³ In confirmation of the above, in the present study, intradermal L-NAME inhibited substance P–induced edema formation in rat skin but had no effect on edema induced by substance P in the presence of an edema-potentiating dose of the vasodilator CGRP. Hence, it is suggested that the inhibitory effect of L-NAME is secondary to NO-dependent basal blood flow. In the present study, intravenous and intradermal 7-NI had no effect on the actions of exogenous substance P (with or without CGRP), further supporting the concept that 7-NI does not act to inhibit endothelial NO synthase.

The evidence from the present study suggests a modulatory role for endogenous NO during neurogenic inflammation and is therefore in agreement with our previous study in rabbit skin, which suggests that NO plays a role in the release but not the action of sensory vasodilator CGRP.²⁴ This, together with studies showing that neurogenic vasodilation on application of mustard oil can be attenuated with NO synthase inhibitors²⁵ and others reporting that nitrovasodilators directly activate perivascular sensory fibers to release CGRP, which in turn leads to vasodilation,³⁴ adds further impetus to the suggestion that NO is acting prejunctionally or within peripheral neurons to mediate the release of neuropeptides during neurogenic inflammation in the cutaneous microvasculature.

It is yet unclear what the site of production and action of NO is, though there is evidence for the localization of NO synthase enzyme in both central and peripheral neurons.³⁵ This has led to the suggestion that NO may have a role as a neurotransmitter, because it has been shown to have a modulatory role in nonadrenergic noncholinergic neuronal function, including the relaxation of human³⁶ and guinea pig³⁷ airways. NO may act as a neurotransmitter in its own right, as has been reported in the enteric nervous system³⁸ ; however, evidence suggests that this is not the case in rat skin, because the increased blood flow is inhibited by a CGRP antagonist¹¹ and the edema formation is inhibited by an NK₁ receptor antagonist.^{13 14}

Thus, the exact mechanism of action of NO in modulating neurogenic inflammation remains unclear. However, several theories exist for mechanisms by which NO may act centrally, and these may be applicable to the peripheral role of neuronal NO. It is possible that NO has a cell-to-cell signaling function, where it diffuses out of the neuron in which it is generated to an adjacent neuron, where it interacts with its target to increase cGMP levels.³⁹ Evidence for the latter is provided by a study showing that NO synthase localized in dorsal root ganglion cells is associated with increases in intracellular cGMP levels in satellite cells.⁴⁰ This is unlikely to be the source of NO for the cutaneous sensory nerves. The most likely sources of NO in the skin, apart from nerves, are endothelial cells (see Lawrence and Brain¹⁹ ) and mast cells.⁴¹ However, this and previous studies provide evidence that 7-NI does not inhibit NO synthase in endothelial cells in vivo,^{26 27 42} and the total inhibition of edema formation by the NK₁ antagonists^{13 14} suggests that mast cells are unlikely to be involved in saphenous nerve–induced edema formation in the rat. Interestingly, capsaicin and bradykinin stimulate an increase in intracellular cGMP in dorsal root ganglion cells,^{43 44} the production of which is inhibited with NO synthase inhibitors in cultured primary afferent neurons. Thus, NO may stimulate an increase in cGMP within the neuron in which it is produced, which may in turn facilitate neuropeptide release. In this context, the colocalization of CGRP immunoreactivity and NADPH-diaphorase (an indicator of NO synthase) in afferent nerves of the rat penis⁴⁵ and the rat uterus⁴⁶ is of interest.

In conclusion, the present study suggests that NO is involved in mediating neurogenic edema formation by inhibiting the release of neuropeptides from sensory nerves. Furthermore, 7-NI, by virtue of its analgesic properties without cardiovascular side effects together with its ability to inhibit neurogenic edema, appears to be useful as a tool for the study of specific neuronal functions of NO. The role played by the L-arginine, NO, and cGMP system in inflammation and pain is not yet clear. However, these findings support the concept that NO can act independently of endothelial cells to influence neurogenic inflammation at the microvascular level. This indicates that NO is involved in influencing sensory nerve events at the peripheral nerve terminals in addition to the more established role of NO at central terminals. Thus, the possibility exists of a link between the activation of NO in sensory nerves in neurogenic vascular responses and pain, and this will be the subject of further study.

	Acknowledgments

 
R. Kajekar is the recipient of a Pfizer PhD studentship.

Received March 15, 1994; accepted December 2, 1994.

	References

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