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Published ahead of print on August 11, 2004, doi:10.1164/rccm.200306-775OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 941-946, (2004)
© 2004 American Thoracic Society
doi: 10.1164/rccm.200306-775OC

Original Article

Cannabinoid Receptor Agonists Inhibit Sensory Nerve Activation in Guinea Pig Airways

Shigemi Yoshihara, Hiroshi Morimoto, Yumi Yamada, Toshio Abe and Osamu Arisaka

Department of Pediatrics, Dokkyo University School of Medicine, Tochigi; and Department of Pharmacology, Medical Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan

Correspondence and requests for reprints should be addressed to Shigemi Yoshihara, M.D., Department of Pediatric, Dokkyo University School of Medicine, 880 Kitakobayashi, Mibu-machi, Shimotsuga-gun, Tochigi 321-0293, Japan. E-mail: shigemi{at}dokkyomed.ac.jp


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We examined the effects of cannabinoid receptor agonists on various respiratory reactions induced by the activation of capsaicin-sensitive afferent sensory nerves (C-fibers). (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-merpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone (WIN 55212–2) dose-dependently inhibited electrical field stimulation– and capsaicin-induced guinea pig bronchial smooth muscle contraction, but not the neurokinin A–induced contraction. A cannabinoid CB2 receptor antagonist, {N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide} (SR 144528), reduced the inhibitory effect of WIN 55212–2, but not a cannabinoid CB1 antagonist, [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride] (SR 141716A). A cannabinoid CB2 agonist, JWH 133, also inhibited electrical field stimulation–induced guinea pig bronchial smooth muscle contraction and its inhibitory effect was blocked by SR 144528. The inhibitory effect of WIN 55212–2 on electrical field stimulation-induced bronchial contraction was reduced by the pretreatment of large conductance Ca2+-activated K+ channel (Maxi-K+ channel) blockers, iberiotoxin and charybdotoxin, but not other K+ channel blockers, dendrotoxin or glibenclamide. A Maxi-K+ channel opener, 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)benzimidazolone (NS1619), inhibited bronchial contraction induced by electrical field stimulation. WIN 55212–2 and JWH 133 blocked the capsaicin-induced release of substance P-like immunoreactivity from guinea pig airway tissues. These findings suggest that WIN 55212–2 inhibit the activation of C-fibers via cannabinoid CB2 receptors and Maxi-K+ channels in guinea pig airways.

Key Words: airway • cannabinoid • C-fibers • guinea pig • Maxi-K+ channels

Several previous studies showed that neurogenic inflammation in the airway must have an important role in the pathogenesis of asthma (1, 2). By using tachykinin antagonists, Murai and coworkers (3, 4) directly demonstrated that airway inflammation is generated by tachykinin released from capsaicin-sensitive afferent sensory nerves (C-fibers), which are stimulated by various types of irritants like cigarette smoke (5), cold air (6), and hypertonic saline (7). These irritants activate C-fibers by opening nonselective cation channels with a high Ca2+ permeability (8). Morimoto and colleagues (9) have showed that {omega}–conotoxin GVIA blocked the activation of C-fibers and the release of substance P from their endings, and suggested that the opening of N-type voltage-dependent Ca2+ channels might activate C-fibers in guinea pig airway tissues.

Cannabinoids are a direct class of psychoactive compounds that produce a wide array of effects including hypothermia, depressed motor activity, hypotension, inhibition of intestinal motility, and antinociception (10). The biological effects of cannabinoids are mediated by specific cell surface receptors, CB1 and CB2, in the central nervous system and in peripheral tissues (11–13). Over the last 10 years much evidence has accumulated favoring the concept that cannabinoids are endogenous modulators of neuronal activity in several tissues (14, 15). Cannabinoid receptor agonists have been shown to inhibit the function of Ca2+ channels (16, 17).

In this report, we now discovered a cannabinoid receptor agonist, WIN 55212–2 inhibited the electrical field stimulation–induced guinea pig bronchial smooth muscle contraction. We examined to clarify this inhibitory effect of WIN 55212–2 and showed that cannabinoid receptors negatively regulate the release of tachykinins from the endings of C-fibers in guinea pig airways by the inhibition of Ca2+ influx to C-fibers via the opening of Maxi-K+ channels. Our results suggested that cannabinoid receptor agonists may be a valuable tool for the treatment of inflammatory diseases induced by the activation of C-fibers in the airways.

In these experiments, we have used the nonselective agonist WIN 55212–2, the CB2-selective agonist JWH 133, the CB1 receptor antagonist SR 141716A, and the CB2-receptor antagonist SR 144528 as pharmacologic tools with which to characterize the cannabinoid receptor subtype involved in this response. WIN 55212–2 has essentially the same affinity for CB1 and CB2 receptors. The affinities for both receptors are in the nanomolar range, and this agonist exhibits relatively high efficacy at both these receptor type (18). JWH 133 is the most selective CB2 receptor agonist. Its binding affinities (Ki) for CB2 and CB1 receptors are 3.4 ± 1.0 and 677 ± 132 nM, respectively (19). SR 141716A was developed by Sanofi-Synthelabo Recherche (Montpellier, France), and is a highly potent and selective CB1 receptor antagonist (Ki = 5.9 nM for CB1 and > 1 µM for CB2) (20). SR 144528 is also developed by Sanofi that binds with higher affinity to CB2 than CB1 receptors (Ki = 0.6 nM for CB2 and 437 nM for CB1) (21).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Twelve-week-old male Hartley guinea pigs (260–455 g) were purchased from SLC (Hamamatsu, Japan) at least 1 week before the experiments. Animals were housed in a temperature- and humidity-controlled environment under a 12:12 hour light/dark cycle with light on at 7:00 A.M. Animals were allowed free access to food and water ad libitum.

Contractile Response of Isolated Guinea Pig Bronchi
The procedure of Fox and coworkers (22) and Patel and colleagues (23) was used with certain modifications. Male Hartley guinea pigs were killed and their bronchi were removed rapidly. A ring preparation with 4 to 5 mm length of the main bronchi (only one bronchi/animal) was mounted in 5-ml organ baths filled with warmed (37°C) and oxygenated (95% O2, 5% CO2) standard Tyrode's solution (pH 7.5) containing 137.0 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1.05 mM MgCl2, 0.4 mM NaHPO4, 5.6 mM dextrose, 11.9 mM NaHCO3 under a resting tension of 0.5 g. After a 60-min equilibration period, bronchi were stimulated by pre-established (data not shown) submaximal concentrations or stimuli of 1 nM neurokinin A, 1 µM capsaicin, or electrical field stimulation in the presence of atropine, propranolol, and phosphoramidon, respectively, 1 µM. Electrical field stimulation was delivered using two parallel platinum wires connected to a stimulator, and square-wave pulses of supramaximal voltage (50 V) and 1 ms pulse duration were applied for 30 s every 30 min at a frequency of 10 Hz. After two reproducible responses were obtained (control response) and the tension of the preparation returned to basal levels by washing, the nonselective cannabinoid agonst, WIN 55212–2, or the CB2 receptor agonist, JWH 133, was added and 10 min later contraction was induced with the same stimuli of neurokinin A, capsaicin, or electrical field stimulation. The contractile response obtained in the presence of drug was compared with the control response. In separate experiments, K+ channel blockers, the CB1 receptor antagonist SR 141716A or the CB2 receptor antagonist SR 144528 were added 10 min before application of the agonist. Only one concentration of one agonist and/or antagonist was tested per bronchi preparation. All of the drugs employed in this study did not affect the baseline tone.

Substance P Release from Guinea Pig Airway Tissues
The procedure of Ray and coworkers (24) was used with certain modifications. Male Hartley guinea pigs were killed and lungs were perfused (6 ml/min, 37°C) with oxygenated (95% O2, 5% CO2) Krebs-Ringer HEPES buffer (pH 7.5) containing 138.0 mM NaCl, 5.6 mM KCl, 1.0 mM CaCl2, 1.0 mM MgCl2, 1.0 mM NaH2PO4, 11 mM NaHCO3, 10 mM dextrose, 20 mM HEPES, 30 µM bacitracin, 1 µM phosphoramidon via a cannula that was inserted into the pulmonary artery through the right ventricle. The left atrium was opened to collect the outflow. Fifteen minutes after the start, perfusates from one period (15 min; i.e., 90 ml) were collected on ice in beakers containing hydrochloric acid to give a final concentration of 0.1 M. Each fraction was desalted on Sep-Pak C18 cartridges (Waters, Milford, MA) as described for somatostatin (25), and the peptides were concentrated to a final volume of 1 ml. The recovery from the Sep-Pak cartridge was more than 90% for radiolabeled substance P. Chemical irritation of tissues was achieved by perfusion with buffer containing 1 µM capsaicin for 5 min during the second collection period. Substance P–like immunoreactivity was measured by radioimmunoassay. The amount of substance P released by capsaicin was calculated by subtracting the level detected in the first period perfusate from that in the second period perfusate. Drugs were added in Krebs-Ringer HEPES buffer throughout the experiment. After the experiments finished, the lungs, including trachea and bronchi, were dissected out and weighed. The increase of substance P–like immunoreactivity release was calculated in fmol per gram of tissues.

Materials
Substance P, neurokinin A, iberiotoxin, charybdotoxin, and phosphoramidon were purchased from Peptide Institute Inc. (Osaka, Japan). Bacitracin was obtained from Sigma Chemical Co. (St. Louis, MO). Capsaicin, JWH 133, dendrotoxin, and glibenclamide were from Nakalai Tesque Chemical Co. (Kyoto, Japan). 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)benzimidazolone (NS1619) were purchased from Funakoshi Chemical Co. (Tokyo, Japan). (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone (WIN 55212–2) was synthesized in Fujisawa Pharmaceutical Co. Ltd. (Osaka, Japan). [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride] (SR141716A) and {N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide} (SR 144528) were kindly supplied by Sanofi-Synthelabo Recherche. [125I]-Substance P (74 TBq/mmol) and anti–substance P antiserum for the radioimmunoassay were purchased from Amersham Int. Ltd. (Buckinghamshire, UK). All drugs were dissolved in dimethylsulfoxide. In in vitro experiments, concentrated solutions of drugs prepared in dimethylsulfoxide were diluted in Tyrode's solution or Krebs-Ringer HEPES buffer. The final bath concentration of dimethylsulfoxide was 0.1%. The solvent alone or 0.1% dimethylsulfoxide, had no effect on the responses in the study.

Statistical Analysis
Results are each given as the means ± SEM of five experiments. Statistical analyses were performed by either ANOVA followed by Dunnett's multicomparison test (contractile response of isolated guinea pig bronchi) or by means of the unpaired Student's t test (others). p Values less than or equal to 0.05 were considered indicative of significance.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Cannabinoid Receptor Agonists on Isolated Guinea Pig Bronchial Smooth Muscle Contraction
The effects of cannabinoid receptor agonists on isolated guinea pig bronchial smooth muscle contraction were examined. After electrical field stimulation (26) and capsaicin (27) in the presence of atropine and propranolol, isolated guinea pig bronchial smooth muscles evoke tachykinin-dependent prolonged contraction. Electrical field stimulation, 1 µM capsaicin, and 1 nM neurokinin A elicited the guinea pig isolated bronchial smooth muscle contraction of 0.25 ± 0.03, 0.31 ± 0.02, and 0.21 ± 0.03 g, respectively. A cannabinoid receptor agonist, WIN 55212–2 (0.0191–19.1 µM), inhibited electrical field stimulation– and capsaicin-induced tachykinin-dependent contraction in a dose-dependent manner (Table 1). On the other hand, neurokinin A–induced guinea pig bronchial smooth muscle contraction was not affected by WIN 55212–2 at dose of 19.1 µM. To identify the cannabinoid receptor subtype that was involved in these effects of WIN 55212–2, we examined the influences of selective cannabinoid CB1 (SR 141716A) and CB2 (SR 144528) receptor antagonists. SR 144528 (10 nM) reduced the inhibitory effect of WIN 55212–2, but not SR 141716A (10 nM) on the guinea pig bronchial smooth muscle contraction induced by electrical field stimulation (Figure 1) and by capsaicin (Figure 2). A selective cannabinoid CB2 receptor agonist, JWH 133 (0.1–100 µM), also dose-dependently reduced electrical field stimulation–induced guinea pig bronchial smooth muscle contraction (Table 2). The inhibitory effect of JWH 133 was also reduced by SR 144528 (10 nM), but not by SR 141716A (10 nM) (Figure 3).


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  		TABLE 1. Effect of win 55212-2 on guinea pig isolated bronchial smooth muscle contraction induced by electrical field stimulation, capsaicin, and neurokinin a

 


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  		Figure 1. Effects of WIN 55212–2 on isolated guinea pig bronchial smooth muscle contraction induced by electrical field stimulation with cannabinoid receptor antagonists, 10 nM SR 141716A (open circles), 10 nM SR 144528 (squares) or vehicle (filled circles). **Significantly different from vehicle, p < 0.01. Values are given as % inhibition of control contraction and are the mean ± SEM of five experiments.

 


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  		Figure 2. Effects of WIN 55212–2 on isolated guinea pig bronchial smooth muscle contraction induced by capsaicin with cannabinoid receptor antagonists, 10 nM SR 141716A (open circles), 10 nM SR 144528 (squares) or vehicle (filled circles). *Significantly different from vehicle, p < 0.05. **Significantly different from vehicle, p < 0.01. Values are given as % inhibition of control contraction and are the mean ± SEM of five experiments.

 

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  		TABLE 2. Effect of a cannabinoid CB2 receptor agonist, jwh 133, on guinea pig isolated bronchial smooth muscle contraction induced by electrical field stimulation and neurokinin a

 


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  		Figure 3. Effects of a cannabinoid receptor agonist, JWH 133 on isolated guinea pig bronchial smooth muscle contraction induced by electrical field stimulation with SR 141716A (open circles), SR 144528 (squares) or vehicle (filled circles). **Significantly different from vehicle, p < 0.01. Values are given as % inhibition of control contraction and are the mean ± SEM of five experiments.

 
We examined the influence of K+ channel blockers on inhibitory effects of WIN 55212–2 on C-fibers. Maxi-K+ channel antagonists, iberiotoxin (0.1 µM) and charybdotoxin (0.01 µM), greatly reduced the inhibitory effect of WIN 55212–2 on electrical field stimulation–induced guinea pig bronchial smooth muscle contraction (Figure 4). However, a voltage-sensitive A-type K+ channel blocker, dendrotoxin (1 µM), or an ATP-sensitive K+ channel blocker, glibenclamide (1 µM), had no effect. Then, a Maxi-K+ channel opener, NS 1619 (0.0276–27.6 µM) inhibited electrical field stimulation–induced contraction in a dose-dependent manner, but not the neurokinin A–induced contraction at dose of 27.6 µM (Table 3).



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  		Figure 4. Effects of WIN 55212–2 on isolated guinea pig bronchial smooth muscle contraction induced by electrical field stimulation with K+ channel blockers, 1 µM dendrotoxin (open squares), 1 µM glibenclamide (triangles), 0.01 µM charybdotoxin (filled circles), 0.1 µM iberiotoxin (filled squares) or vehicle (open circles). *Significantly different from vehicle, p < 0.05. **Significantly different from vehicle, p < 0.01. Values are given as % inhibition of control contraction and are the mean ± SEM of five experiments.

 

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  		TABLE 3. Effect of ns 1619 on guinea pig isolated bronchial smooth muscle contraction induced by electrical field stimulation and neurokinin a

 
Effects of Cannabinoid Receptor Agonists on Substance P–like Immunoreactivity Release from Guinea Pig Airway Tissues
To clarify the mechanism involved in cannabinoid receptor agonist inhibition of electrical field stimulation– and capsaicin-induced guinea pig bronchial smooth muscle contraction, their effects on capsaicin-induced substance P–like immunoreactivity release from guinea pig airway tissues were examined (Table 4). WIN 55212–2 (0.00287–2.87 µM) dose-dependently inhibited the capsaicin-induced release of substance P–like immunoreactivity from guinea pig airway tissues. JWH 133 (0.1–10 µM) also significantly reduced substance P–like immunoreactivity release. In the presence of SR 144528 (10 nM) and WIN 55212–2 (2.87 µM) or JWH 133 (10 µM), the capsaicin-induced release of substance P–like immunoreactivity from guinea pig airway tissues were 48.3 ± 1.1 (control; 49.6 ± 1.6) or 44.2 ± 2.5 (control; 43.3 ± 2.1) fmol/ g tissue, respectively (not significantly different from control). SR 141716A (10 nM), however, did not reduce the inhibitory effects of WIN 55212–2 and JWH 133 (data not shown).


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  		TABLE 4. Effect of cannabinoid agonists on substance p–like immunoreactivity release from guinea pig airway tissues induced by capsaicin

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has been suggested that various stimuli act on irritant receptors in respiratory mucosa and produce various respiratory reactions (e.g., bronchoconstriction, tracheal plasma extravasation, and mucus hypersecretion) via the release of tachykinins from the endings of C-fibers (28). The influx of Ca2+ into presynaptic nerve endings through voltage-dependent Ca2+ channels is essential for the release of neurotransmitters within the nervous system. Ca2+ influx is a key step in excitation-release coupling in C-fibers (8, 29). For asthma research, it is important to examine how Ca2+ influx into C-fibers is modulated. Morimoto and colleagues (9) suggested that N-type voltage-dependent Ca2+ channels are involved in the activation of C-fibers.

WIN 55212–2 has been known to show the cannabismimetic activity and prevent voltage-dependent Ca2+ channels (16, 17). In this study, we found that WIN 55212–2 inhibited electrical field stimulation– and capsaicin-induced isolated guinea pig bronchial smooth muscle contraction. This reaction is dependent on tachykinins because it was inhibited by tachykinin receptor antagonists (4, 5, 27). In contrast, WIN 55212–2 did not influence neurokinin A–induced guinea pig bronchial smooth muscle contraction. These results suggested that WIN 55212–2 blocks the release of tachykinins from C-fibers, but does not antagonize the interaction of tachykinins on their receptors. Indeed, WIN 55212–2 did significantly reduce the capsaicin-induced release of substance P–like immunoreactivity from guinea pig airway tissues. Ralevic (30) has reported that cannabinoid-induced inhibition of neurotransmission is mediated by the effector systems such as calcium or potassium channels. Mackie and Hille (17) suggested that inhibitory effect of WIN 55212–2 on voltage-dependent Ca2+ channels might be mediated by its interaction with other ion channels coupled either directly or indirectly to G proteins, e.g., potassium channels. Deadwyler and coworkers (31) has shown that WIN 55212–2 increases voltage-dependent potassium-A current in cultured hippocampal cells. It has been reported that ATP-sensitive K+ channels (32) and Maxi-K+ channels (28, 33) show inhibitory modulations on the activation of C-fibers. Fox and colleagues (22) reported that NS 1619 inhibited a single C-fiber firing induced by bradykinin and excitatory nonadrenergic noncholinergic contraction induced by electrical field stimulation in guinea pig bronchi at the same dose of 30 µM as shown in our study. Our results well support these evidences and suggest that inhibitory effect of WIN 55212–2 on guinea pig bronchial contraction induced by the activation of C-fibers, may be caused by the inhibition of voltage-dependent Ca2+ influx and release of substance P from sensory nerves via the opening of Maxi-K+ channels.

In the second part of this study, we examined what kind of cannabinoid receptors are involved in the inhibitory effect of cannabinoid receptor agonists on the activation of C-fibers. The biological effects of cannabinoids are mediated by specific receptors, CB1 and CB2, in the central nervous system and in peripheral tissues (11–13). WIN 55212–2 binds to and activates both CB1 and CB2 receptors (18). In this study, inhibitory effects of WIN 55212–2 on electrical field stimulation– and capsaicin-induced isolated guinea pig bronchial smooth muscle contraction, and capsaicin-induced release of substance P from guinea pig airway tissues, were reduced by a specific CB2 cannabinoid receptor antagonist, SR 144528, but not by a CB1 antagonist, SR 141716A. A specific CB2 cannabinoid receptor agonist, JWH 133 (19) also dose-dependently inhibited electrical field stimulation–induced bronchial contraction and capsaicin-induced release of substance P, and its effect was reversed by SR 144528 but not by SR 141716A. According to these facts, it is possible that CB2 receptors exist on the C-fibers in guinea pig airway and that CB2 receptors downregulate the activation of C-fibers and tachykinin release as a physiologic role. It has been reported that the activation of cannabinoid CB1 receptors caused the inhibition of ACh, glutamate, dopamine, noradrenaline, and GABA (34) release from brain slices, and noradrenaline release in the guinea pig lung (35). Recently, it has been reported that an endogenous cannabinoid agonist, anandamide, interacts and activates a nonselective cation channel, TRPV1 receptor, with a high calcium permeability, which is activated by a broad spectrum of stimuli, including capsaicin, heat, and low pH (36, 37). In guinea pig airways, anandamide stimulates sensory nerves by inducing depolarization (38, 39). Our results might support the inactivation of calcium influx in the opposite direction against their reports by the interaction on CB2 receptors, but not CB1 or maybe TRPV1. Indeed, Patel and coworkers (23) reported that activation of the CB2 receptors inhibited sensory nerve activation of guinea pig and human vagus nerve, and the cough reflex in guinea pigs. They showed the dose-dependent inhibition of a nonselective cannabinoid agonist, CP 55940, and JWH 133 on depolarization of guinea pig and human vagus nerve by capsaicin, prostaglandin E2, and hypertonic saline. Tucker and colleagues (38), however, indicated CP 55,940 failed to attenuate the excitatory nonadrenergic noncholinergic response in guinea pig airways. Although the same dose of CP 55940 (1 and 10 µM) was used in both studies, their results were discrepant. The reasons for this discrepancy remain to be established, but in our study, a nonselective cannabinoid agonist, WIN 55212–2 and a selective CB2 agonist, JWH 133, significantly inhibited the excitatory nonadrenergic noncholinergic response in guinea pig isolated bronchi and airway tissues in the same micromolar range of doses as the report of Patel and coworkers (23). Some investigators have reported that activation of CB2 receptors was neuroprotective for rat cerebellar granule cells (40) and neuronal expression of CB2 receptor could be regulated by culture conditions (41). These evidences and our results suggest that cannabinoid CB2 receptors exist in nervous system to downregulate Ca2+ influx to neuronal cells and the transmitter releases. Especially, in airway tissues, they may act to inhibit the activation of afferent sensory nerves, C-fibers. Calignano and colleagues (42) suggested that the parallel activation of peripheral CB1- and CB2-like receptors inhibited the peripheral pain initiation. They examined the effects of endogenous cannabinoid agonists, anandamide for CB1 and palmitylethanolamide for CB2, and concluded that endogenous cannabinoids might participate in buffering emerging pain signals at sites of tissue injury by the activation of local CB1- and CB2-like receptors. Endogenous cannabinoids may play a physical role to regulate several neuronal responses, including airway tissues.

When excitatory C-fibers are stimulated, not only substance P but other neuropeptides also, e.g., neurokinin A and calcitonin gene-related peptide, are released from their nerve endings and exert various respiratory reactions (43). The inflammatory effects of these neuropeptides on airway tissues may be of pathologic relevance in human bronchial hyperreactivity, and we showed that antiasthmatic compounds, sodium cromoglycate and nedocromil sodium, inhibited hypertonic saline–induced plasma extravasation in guinea pig airways by the activation of C-fibers (7). We conclude that a cannabinoid receptor agonist, WIN 55212–2 which inhibits the activation of C-fibers and the release of these neuropeptides from their endings via the opening of Maxi-K+ channels by the activation of CB2 receptors, will be a valuable tool for the therapy on airway inflammatory diseases such as asthma.


   Acknowledgments
 
The authors thank Sanofi-Synthelabo Recherche for the sample supply. They thank Dr. Makoto Ohori, Department of Molecular Science, Exploratory Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., for his help in preparing this manuscript.


   FOOTNOTES
 
Conflict of Interest Statement: S.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; H.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; Y.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; T.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; O.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form June 12, 2003; accepted in final form August 6, 2004


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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