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INHIBITORY EFFECTS OF CANNABINOID RECEPTOR LIGANDS ON ELECTRICALLY-EVOKED RESPONSES IN RAT ISOLATED TRACHEAL RING SEGMENTS
Pharmacological Research, Vol. 40, No. 5, 1999 Article No. phrs.1999.0532, available online at http:rrwww.idealibrary.com on
INHIBITORY EFFECTS OF CANNABINOID RECEPTOR LIGANDS ON ELECTRICALLY-EVOKED RESPONSES IN RAT ISOLATED TRACHEAL RING SEGMENTS MARIAM H.M. YOUSIFU and MABAYOJE A. ORIOWO Department of Pharmacology and Toxicology, Faculty of Medicine, Kuwait Uni¨ ersity, P.O. Box 24923, Safat 13110, Kuwait Accepted 5 May 1999
We have examined the possible existence of cannabinoid receptors in the isolated rat tracheal ring segments by studying the effects of some cannabinoid receptor ligands on electrically-induced contractions. Anandamide Ž10y8 ]3 = 10y5 M., an endogenous ligand for cannabinoid receptors, and WIN 55,212-2 Ž10y9 ]3 = 10y5 M., a moderately selective CB 2 agonist, inhibited electrically evoked contractions of the rat tracheal ring segments in a concentration-related manner. Addition of phentolamine Ž10y6 M. to Krebs Henseleit solution to block a 2-adrenoceptors did not affect anandamide-induced inhibition of the electrically evoked contractions. The EC 25 Žylog M. values were 5.25" 0.2 and 5.8" 0.4 for anandamide and WIN 55,212-2, respectively. The maximal inhibition produced by the highest concentration of the agonists used was 51.4" 5.8% for anandamide and 35.1" 19.5% for WIN 55,212-2. WIN 55,212-3 also produced a concentration-dependent inhibition of the electrically evoked contractions. The maximal inhibition produced by WIN 55,212-3 was 15.8" 2.4. The inhibitory effects of anandamide and WIN 55,212-2 were not attenuated by SR141716A Ž10y6 M., a selective CB1 receptor antagonist. Anandamide Ž10y8 ]3 = 10y5 M. did not relax rat tracheal ring segments pre-contracted with carbachol Ž10y6 M.. These results suggest that anandamide and WIN 55,212-2 produce pre-junctional inhibitory effects in the rat trachea and that these effects were likely mediated through cannabinoid CB 2 receptors. These effects were probably non-cannabinoid receptor-mediated considering the high concentrations of the agents required to produce inhibitory responses and the effectiveness of WIN 55,212-3. Q 1999 Academic Press KEY
WORDS:
CB receptors, anandamide, WIN 55,212-2, SR141716A.
INTRODUCTION Marijuana has, for centuries, been used as a recreational drug. It has also been used therapeutically. It has been used in Chinese medicine in the treatment of rheumatic pains, constipation and female disorders w1x. The active constituent of marijuana was later identified as D9-tetrahydro cannabinol Ž D9THC.. It is now known that D9-THC produces its actions via specific cannabinoid receptors. Two subtypes of the cannabinoid receptors, CB1 and CB 2 receptors, have been identified. Both of these receptors have been cloned and are coupled to G proteins w2, 3x. CB1 receptors were originally reported to be U
Corresponding author.
1043]6618r99r110415]07r$30.00r0
located mainly in the CNS, but it is now known that they are also present in some peripheral tissues w4x. CB 2 receptors are located mainly in peripheral tissues w5x. Endogenous ligands for these receptors have been identified, the prototype of which is anandamide Žarachidonylethanolamide.. Recently, more potent cannabinoid receptor agonists have been synthesised. These include HU210, CP-55,940 and WIN 55,212-2 w6]8x. WIN 55,212-2 shows a modest degree of selectivity Ž20-fold. for cannabinoid CB 2 receptors w6, 9x. Using these agonists, it has been shown that cannabinoid receptors are located prejunctionally in the mouse vas deferens and guinea pig small intestine, where they mediate inhibition of neurotransmission w10, 11x. They have also been shown to produce endothelium-independent vascular smooth muscle relaxation w12]14x. It was even Q 1999 Academic Press
416
proposed that anandamide could be the endothelium-derived hyperpolarising factor, even though this has not been proven. The effect of cannabinoids on neurotransmission in airway smooth muscles has not been investigated. Since most people who use marijuana for recreational purposes prefer to smoke it, we decided to study the effect of anandamide, an endogenous cannabinoid receptor ligand and WIN 55,212,2, a potent agonist with modest selectivity for CB 2 receptors, on electrically-induced contractions of the rat tracheal ring segment. The interaction between these ligands and SR141716A, a selective CB1 receptor antagonist, was also examined in an attempt to determine which subtypeŽs. of cannabinoid receptors is Žare. present in this preparation.
MATERIALS AND METHODS Adult male Sprague]Dawley rats weighing 250]300 g were used in this study. The animals were killed by decapitation under light ether anaesthesia. The tracheas were isolated and excess connective tissue was removed. Tracheas were cut into rings of approximately 4 mm in length. The preparations were mounted in organ-baths containing 25 ml of Krebs]Henseleit ŽKH. solution ŽpH 7.4.. The composition of the Krebs]Henseleit solution was as follows ŽmM.: NaCl Ž118.3., KCl Ž4.7., CaCl 2 Ž2.5., MgSO4 Ž1.2., NaHCO3 Ž25., KHPO4 Ž1.2. and glucose Ž11.2.. The tissue bath solution was maintained at 378C and was aerated with a 95% O 2 and 5% CO 2 mixture. Each ring was allowed to equilibrate for 60 min under a resting tension of 1.0 g. During this period of equilibration, the tissues were rinsed at 15-min intervals with KH solution. Isometric contractions of the tracheal ring segments were recorded through UFI dynamometers, on a Grass polygraph Žmodel 7D..
Electrical stimulation
Pharmacological Research, Vol. 40, No. 5, 1999
contractions was observed. This was done to confirm the neurogenic nature of the contractions.
Effect of anandamide on electricallyinduced contractions When the contractions to field stimulation had stabilised Žfour or five consecutive contractions of the same amplitude., anandamide Ž10y8 ]3 = 10y5 M. was added cumulatively to the baths and its effects on the contractions were recorded. The response to any given concentration was allowed to peak Žfour consecutive contractions of the same amplitude. before adding the next concentration. In another series of experiments, the effect of anandamide was tested in the presence of phentolamine Ž10y6 M. to determine whether prejunctional a 2-adrenoceptors were involved in the actions of anandamide. Concentration]response curves for anandamide Ž10y8 ]3 = 10y5 M. were established in the absence and also in the presence of phentolamine Ž10y6 M. which was added to the bath 30 min before adding anandamide.
Effect of SR141716A on anandamideinduced inhibition of electrically e¨ oked contractions In order to determine the subtype of cannabinoid receptor mediating the inhibitory effects of anandamide, inhibitory responses to a submaximal concentration of anandamide were obtained in the absence and also in the presence of SR141716A Ž10y6 M.. Since recovery from the effect of anandamide was not complete, antagonist studies were carried out on paired tissues. Experiments on control and SR141716A-treated tissues were run simultaneously. In all studies involving SR141716A, the antagonist was added to the tissue and allowed to equilibrate with the preparation for 30 min before the tissues were stimulated.
Effect of anandamide on carbacholinduced contraction in the tracheal rings
For electrical stimulation, the tracheal rings were suspended between platinum electrodes and were stimulated with square wave pulses delivered by a Grass S 88 stimulator. Trains of pulses were applied at 20 Hz for 10 s every 90 s Ž60-V and 0.5-ms duration.. The response to electrical stimulation was considered to have stabilised when three or four consecutive contractions were of the same amplitude.
After establishing a steady basal tone, carbachol Ž10y6 M. was added to the organ baths. This concentration of carbachol produced a submaximal Žless than 80% of the maximum. contraction. Once the contraction has reached a plateau, increasing concentrations of anandamide Ž10y8 ]3 = 10y5 M. were added cumulatively, to the baths to generate data for a concentration]response curve. The contact time for each concentration of anandamide was 10 min.
Effect of tetrodotoxin (TTX ) on electricallyinduced contractions
Effect of WIN55,212-2 and WIN55,212-3 on electrically-induced contractions
In some preliminary experiments, after establishing a stable response to electrical stimulation, a single concentration of TTX Ž10y6 M. was added to the bath and its effect on the electrically-induced
In this series of experiments, the inhibitory effects of WIN55,212-2 and WIN55,212-3 Ž10y9 ]3 = 10y5 M. were also examined. After obtaining stable contractions to electrical stimulation, increasing
Pharmacological Research, Vol. 40, No. 5, 1999
concentrations of WIN55,212-2 or WIN55,212-3 were added to the baths in a cumulative manner. The effect of any given concentration of the agonists was allowed to peak before the next concentration was added. The effect of SR141716A Ž10y6 M. on the inhibitory effect of a submaximal concentration of WIN55,212-2 Ž10y5 M. was also examined using the same protocol that was used for anandamide.
Statistical analysis Agonist effect was expressed as a percentage reduction in the amplitude of electrically-induced contractions. The immediate contraction before the drugs were added was considered as the reference contraction. Results are presented as means " SE. Concentration]response curves were analysed using Graph-Pad Prism W software. The potency Žexpressed as ylog EC 25 value. for the agonists was obtained from the cumulative concentration response curves. The EC 25 value is the concentration of the agonist that reduced electrically-induced contractions by 25%. EC 25 value was determined since the inhibitory responses, at the maximum concentration ranged between 30 and 60%. Higher concentrations of the drugs were not used to avoid any possible effect of the solvents. Standard statistical evaluation of the data was done using Student’s t-test Žunpaired.. The difference was considered significant ŽU . when the P value was less than 0.05.
417
Drugs and chemicals Carbachol and phentolamine were obtained from Sigma chemicals ŽSt Louis, MO, USA.. Anandamide, SR141716A w N-Žpiperidin-1-yl.-5-Ž4-chlorophenyl.-1Ž2,4-dichlorophenyl.-4-methyl-1H-pyrazole-3-carboxamide hydrochloridex, WIN55,212-2 mesylate Ž RŽq.w2,3-dihydro-5-methyl-3-wŽmorpholinyl.methylxpyrrolo w1,2,3-dex-1,4-benzoxazinylx-Ž1-naphthalenyl methanone mesylate., WIN55,212-3 and tetrodotoxin were obtained from Research Biochemicals International. SR141716A, WIN55,212-2 and WIN55,212-3 were dissolved in DMSO, while anandamide and tetrodotoxin were dissolved in ethanol. Carbachol and phentolamine were dissolved in distilled water. Drug concentrations quoted in the text represent final baths concentrations in all cases. The final concentration of the solvents in the organ-baths in all experiments did not exceed 0.1%. The concentration of ethanol or DMSO did not affect tissue responsiveness to electrical stimulation.
RESULTS
Effect of anandamide on electricallyinduced contractions Electrical field stimulation produced reproducible contractions of the rat tracheal ring segment. Tetrodotoxin Ž10y6 M. abolished the electrically-
Fig. 1. ŽA. Effect of TTX Ž. Ž10y6 M. on electrically-induced contractions in the isolated rat tracheal ring segments. ŽB. A typical trace of anandamide-induced inhibition Žv. Ž10y8 ]3 = 10y5 M. of electrically-evoked contractions in the isolated rat tracheal ring segments.
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induced contractions, indicating neurogenic origin of the contractions wFig. 1ŽA.x. Anandamide Ž10y8 ]3 = 10y5 M. produced a concentration-dependent inhibition of the contractions evoked by electrical stimulation wFig. 1ŽB.x. The highest concentration of anandamide used in this study Ž3 = 10y5 M. produced approximately 51.4" 5.8% Ž n s 8. inhibition of electrically-induced contractions ŽFig. 2.. A higher concentration of anandamide was not tested because the final ethanol concentration in the baths would be increased to 1%. At this concentration, ethanol alone reduced electrically-induced contractions. The ylog EC 25 value for anandamide was 5.25" 0.2. In order to determine the possible involvement of a 2 adrenergic receptors in the effects of anandamide, the effects of anandamide were tested in the presence of phentolamine 10y6 M. Results showed that there was no significant difference in the inhibitory effect of anandamide in the presence or absence of phentolamine Ž10y6 M.. The ylog EC 25 value for anandamide in the presence of phentolamine Ž10y6 M. was 5.6" 0.2 Ž n s 5.. This observation would exclude any contribution of a 2-adrenergic receptors to the inhibitory effect of anandamide on electrically-evoked contractions.
Effect of SR141716A on anandamideinduced inhibition of electrically-e¨ oked contractions
SR141716A Ž10y6 M. on its own did not affect the electrically-induced contractions. As shown in Fig. 3, SR141716A Ž10y6 M. also had no effect on anandamide Ž10y5 M.-induced inhibitory responses. There was a tendency towards enhanced inhibition of electrically-induced contractions by anandamide in the presence of SR141716A Ž10y6 M.. This increase, however, did not reach the level of statistical significance. The maximal inhibition in electrically-
evoked contractions was 55.3" 5.3% Ž n s 8. at the concentration of 3 = 10y5 M Žcompared to 51.4" 5.8% in the absence of SR141716A..
Effect of anandamide on carbacholinduced contraction of the rat tracheal ring segments In rat tracheal ring segments pre-contracted with a submaximal concentration of carbachol Ž10y6 M., a cumulative concentration]response curve for anandamide Ž10y8 ]3 = 10y5 M. did not produce any inhibitory effect ŽFig. 4.. In other experiments, anandamide Ž10y5 M. had no effect on the concentration]response curve to carbachol.
Effect of WIN55,212-2 and WIN55,212-3 on electrically-induced contractions
WIN55,212-2 Ž10y9 ]3 = 10y5 M. had no effect on electrically-induced contractions at low concentrations Ž- 10y9 M.. However, higher concentrations of WIN55,212-2 produced a concentration-dependent inhibition of the electrically evoked contractions ŽFig. 2.. The ylog EC 25 value was 5.8" 0.4. The reduction in contractions induced by the highest concentration of WIN55,212-2 in this study was 35.1%" 19.5 Ž n s 4.. WIN55,212-3 also produced a concentration-dependent inhibition in the electrically evoked contractions. There was no significant difference between the effectiveness of WIN55,212-2 and WIN55,212-3 in this study. The maximal inhibition produced by WIN55,212-3 was 15.8" 2.4% Ž n s 6..
Effect of SR141716A on WIN55,212-2induced inhibition of electrically-e¨ oked contractions
The effect of SR141716A Ž10 y 6 M . on WIN55,212-2-induced inhibition of the electrically induced contractions was also studied using the same procedure as for anandamide. As shown in Fig. 3, WIN55,212-2 Ž10y5 M. induced inhibition of electrically evoked contractions was not reduced in the presence of SR141716A Ž10y6 M.. Instead, there was a tendency towards an increased inhibition Žthough not significantly. of the electrically-evoked contractile response. WIN55,212-2 Ž10y5 M. reduced electrically-induced contractions by 22.4 and 32.9% in the absence and presence of SR141716A Ž10y6 M., respectively.
DISCUSSION
Fig. 2. Effect of ŽI. anandamide Ž10y8 ]3 = 10y5 M., ŽB . WIN55,212-2 Ž10 y9 ] 3 = 10 y 5 M . and Ž$ . WIN55,212-3 on electrically-evoked contractions in isolated rat tracheal preparations. Mean " SE Ž n s 7]8..
As stated in the Introduction, the main objective of this study was to determine Žfractionally . whether cannabinoid receptors are present in the airway smooth muscle and the type of function Žexcitatory or inhibitory. they mediate. We also attempted to
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Fig. 3. ŽA. Effect of ŽB. SR141716A Ž10y6 M. on ŽI. anandamide Ž10y5 M.-induced inhibition of electricallyevoked contraction in isolated rat tracheal preparations. Mean " SE. Ž n s 5.. ŽB. Effect of ŽB. SR141716A Ž10y6 y5 M. on ŽI. WIN 55,212-2 Ž10 M.-induced inhibition of electrically-evoked contraction in isolated rat tracheal preparations. Mean " SE Ž n s 4..
determine the receptor subtype involved in this response. Electrical field stimulation of rat trachea produced contractions that were abolished by TTX indicating that they were nerve-mediated. We have therefore used this test system to study the effect of some cannabinoid agonists on neurotransmission in the rat trachea ring segments. Anandamide inhibited electrically-induced neurogenic contractions of the rat trachea at concentrations that had no effect on carbachol-induced contractions. Thus anandamide produced its actions via a prejunctional mechanism, probably inhibiting neurotransmitter release. Similar prejunctional inhibitory effects of anandamide have been reported in other preparations including mouse vas deferens w15, 16x and rat vas deferens and atria w17x. The potency
Fig. 4. Effect of anandamide Žv. Ž10y8 ]3 = 10y5 tracheal preparation.
M.
of anandamide observed in our study was however, slightly less than that reported by Ishac et al. w17x in the rat vas deferens and atria. Thus, at 10 m M, anandamide inhibited noradrenaline release by approximately 50% in the vas deferens and 40% in the atria w17x, while we have observed a 35% reduction in electrically-induced contractions at this concentration and 51.4% reduction at 30 m M. The effect of anandamide was not affected by pre-treatment of the tissues with phentolamine to block prejunctional a 2-adrenoceptors, indicating that prejunctional a 2adrenoceptors were not involved in this response. Cannabinoids activate two types of receptors, CB1 and CB 2 . These receptors can be differentiated using SR141716A which binds with a high Žnanomolar. affinity for CB1-receptors w18, 19x, but displays low Žmicromolar. affinity for CB 2-receptors w16, 20x. Using this antagonist, it has been shown that CB1receptors mediated prejunctional inhibitory effects of the cannabinoids in the mouse vas deferens w15, 16x and rat atria w17x. We therefore tested the effect of SR141716A on inhibition of electrically-induced contractions induced by anandamide. The results showed that SR141716A Ž1 m M. on its own did not affect the responses to electrical stimulation, indicating that endogenously released cannabinoids, acting on CB1 receptors, did not modulate these responses. The results also showed that SR141716A Ž1 m M. did not attenuate the inhibitory effect of anandamide in the rat trachea. This would confirm that the inhibitory responses were not mediated via CB1 receptors. WIN55,212-2 is a potent cannabinoid receptor ligand, which displays a modest Ž20-fold. selectivity for CB 2-receptors w6, 9x. In the present study, WIN55,212-2 produced a concentration-dependent inhibition of electrically-induced contractions. In this study, WIN55,212-2 showed a lower potency compared to anandamide. However, Izzo et al. w21x have reported a higher potency for WIN55,212-2 compared to anandamide in circular smooth muscle of
on Ž. carbachol Ž10y6
M.
induced pre-contraction of isolated rat
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the guinea-pig ileum. The inhibitory effect produced by WIN55,212-2 was not reduced by SR141716A indicating that CB1-receptors were not involved in this response. A higher concentration of SR141716A was not used in this study since the concentration used Ž1 m M. was already approximately 100-fold higher than the pA 2 value of 8.17 reported by Rinaldi-Carmona et al. w16x and tenfold higher than the p K i of 10y7 M reported by Ishac et al. w17x in the rat atria. Secondly, White and Hiley w14x have shown that higher concentrations Ž) 3 m M. of SR141716A can behave as inverse agonists and are therefore not suitable for studies of cannabinoid receptor mechanisms. In our study, SR141716A Ž1 m M., as stated above, did not reduce the response of the tissue to electrical stimulation but it increased the inhibitory effect Žnot significantly. of both anandamide and WIN55,212-2. These results therefore, tend to suggest that the prejunctional inhibitory effect of anandamide and WIN55,212-2 in the rat trachea were possibly mediated via CB 2-receptors. Alternatively, considering Ža. the weak agonistic effect of anandamide and WIN55,212-2; Žb. the fact that WIN55,212-2 was only marginally Žthough not significant. more effective than anandamide in the rat trachea; and Žc. that anandamide had a higher potency than WIN55,212-2, it is possible that these compounds interacted with sites other than Ži.e. a non-specific effect. cannabinoid receptors. The observation in this study that WIN55,212-3, a less active stereoisomer of WIN55,212-2 was almost as potent as WIN55,212-2 in producing neuroinhibitory effects. This would tend to support a non-specific non-receptor-mediated effect of these compounds in the rat trachea.
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ACKNOWLEDGEMENTS
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SR141716A was provided by Research Biochemicals International as part of the chemical synthesis program of the National Institute of Mental Health Žcontract N01 MH 30003..
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1. Felder CC, Glass M. Cannabinoid receptors and their endogenous agonists. Annu Re¨ Pharmacol Toxicol 1998; 38: 179]200. 2. Caulfield M, Brown D. Cannabinoid receptor agonists inhibit Ca current in NG108-15 neuroblastoma cells via a pertussis toxin-sensitive mechanism. Br J Pharmacol 1992; 106: 231]2. 3. Pan X, Ikeda S, Lewis D. Rat brain cannabinoid receptor modulates N-type Ca2q channels in a
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Characterization and distribution of binding sites for w 3 HxSR141716A, a selective brain ŽCB1 . cannabinoid receptor antagonist, in rodent brain. Life Sci 1996; 58: 1239]47. 20. Showalter VM, Compton D, Martin B, Abood M. Evaluation of binding in a transfected cell line expressing a peripheral cannabinoid receptor ŽCB 2 .:
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identification of cannabinoid receptor subtype selective ligands. J Pharmacol Exp Ther 1996; 278: 989]99. 21. Izzo AA, Mascola N, Borrelli F, Capasso F. Excitatory transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of cannabinoid receptors. Br J Pharmacol 1998; 124: 1363]8.
INHIBITORY EFFECTS OF CANNABINOID RECEPTOR LIGANDS ON ELECTRICALLY-EVOKED RESPONSES IN RAT ISOLATED TRACHEAL RING SEGMENTS MARIAM H.M. YOUSIFU and MABAYOJE A. ORIOWO Department of Pharmacology and Toxicology, Faculty of Medicine, Kuwait Uni¨ ersity, P.O. Box 24923, Safat 13110, Kuwait Accepted 5 May 1999
We have examined the possible existence of cannabinoid receptors in the isolated rat tracheal ring segments by studying the effects of some cannabinoid receptor ligands on electrically-induced contractions. Anandamide Ž10y8 ]3 = 10y5 M., an endogenous ligand for cannabinoid receptors, and WIN 55,212-2 Ž10y9 ]3 = 10y5 M., a moderately selective CB 2 agonist, inhibited electrically evoked contractions of the rat tracheal ring segments in a concentration-related manner. Addition of phentolamine Ž10y6 M. to Krebs Henseleit solution to block a 2-adrenoceptors did not affect anandamide-induced inhibition of the electrically evoked contractions. The EC 25 Žylog M. values were 5.25" 0.2 and 5.8" 0.4 for anandamide and WIN 55,212-2, respectively. The maximal inhibition produced by the highest concentration of the agonists used was 51.4" 5.8% for anandamide and 35.1" 19.5% for WIN 55,212-2. WIN 55,212-3 also produced a concentration-dependent inhibition of the electrically evoked contractions. The maximal inhibition produced by WIN 55,212-3 was 15.8" 2.4. The inhibitory effects of anandamide and WIN 55,212-2 were not attenuated by SR141716A Ž10y6 M., a selective CB1 receptor antagonist. Anandamide Ž10y8 ]3 = 10y5 M. did not relax rat tracheal ring segments pre-contracted with carbachol Ž10y6 M.. These results suggest that anandamide and WIN 55,212-2 produce pre-junctional inhibitory effects in the rat trachea and that these effects were likely mediated through cannabinoid CB 2 receptors. These effects were probably non-cannabinoid receptor-mediated considering the high concentrations of the agents required to produce inhibitory responses and the effectiveness of WIN 55,212-3. Q 1999 Academic Press KEY
WORDS:
CB receptors, anandamide, WIN 55,212-2, SR141716A.
INTRODUCTION Marijuana has, for centuries, been used as a recreational drug. It has also been used therapeutically. It has been used in Chinese medicine in the treatment of rheumatic pains, constipation and female disorders w1x. The active constituent of marijuana was later identified as D9-tetrahydro cannabinol Ž D9THC.. It is now known that D9-THC produces its actions via specific cannabinoid receptors. Two subtypes of the cannabinoid receptors, CB1 and CB 2 receptors, have been identified. Both of these receptors have been cloned and are coupled to G proteins w2, 3x. CB1 receptors were originally reported to be U
Corresponding author.
1043]6618r99r110415]07r$30.00r0
located mainly in the CNS, but it is now known that they are also present in some peripheral tissues w4x. CB 2 receptors are located mainly in peripheral tissues w5x. Endogenous ligands for these receptors have been identified, the prototype of which is anandamide Žarachidonylethanolamide.. Recently, more potent cannabinoid receptor agonists have been synthesised. These include HU210, CP-55,940 and WIN 55,212-2 w6]8x. WIN 55,212-2 shows a modest degree of selectivity Ž20-fold. for cannabinoid CB 2 receptors w6, 9x. Using these agonists, it has been shown that cannabinoid receptors are located prejunctionally in the mouse vas deferens and guinea pig small intestine, where they mediate inhibition of neurotransmission w10, 11x. They have also been shown to produce endothelium-independent vascular smooth muscle relaxation w12]14x. It was even Q 1999 Academic Press
416
proposed that anandamide could be the endothelium-derived hyperpolarising factor, even though this has not been proven. The effect of cannabinoids on neurotransmission in airway smooth muscles has not been investigated. Since most people who use marijuana for recreational purposes prefer to smoke it, we decided to study the effect of anandamide, an endogenous cannabinoid receptor ligand and WIN 55,212,2, a potent agonist with modest selectivity for CB 2 receptors, on electrically-induced contractions of the rat tracheal ring segment. The interaction between these ligands and SR141716A, a selective CB1 receptor antagonist, was also examined in an attempt to determine which subtypeŽs. of cannabinoid receptors is Žare. present in this preparation.
MATERIALS AND METHODS Adult male Sprague]Dawley rats weighing 250]300 g were used in this study. The animals were killed by decapitation under light ether anaesthesia. The tracheas were isolated and excess connective tissue was removed. Tracheas were cut into rings of approximately 4 mm in length. The preparations were mounted in organ-baths containing 25 ml of Krebs]Henseleit ŽKH. solution ŽpH 7.4.. The composition of the Krebs]Henseleit solution was as follows ŽmM.: NaCl Ž118.3., KCl Ž4.7., CaCl 2 Ž2.5., MgSO4 Ž1.2., NaHCO3 Ž25., KHPO4 Ž1.2. and glucose Ž11.2.. The tissue bath solution was maintained at 378C and was aerated with a 95% O 2 and 5% CO 2 mixture. Each ring was allowed to equilibrate for 60 min under a resting tension of 1.0 g. During this period of equilibration, the tissues were rinsed at 15-min intervals with KH solution. Isometric contractions of the tracheal ring segments were recorded through UFI dynamometers, on a Grass polygraph Žmodel 7D..
Electrical stimulation
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contractions was observed. This was done to confirm the neurogenic nature of the contractions.
Effect of anandamide on electricallyinduced contractions When the contractions to field stimulation had stabilised Žfour or five consecutive contractions of the same amplitude., anandamide Ž10y8 ]3 = 10y5 M. was added cumulatively to the baths and its effects on the contractions were recorded. The response to any given concentration was allowed to peak Žfour consecutive contractions of the same amplitude. before adding the next concentration. In another series of experiments, the effect of anandamide was tested in the presence of phentolamine Ž10y6 M. to determine whether prejunctional a 2-adrenoceptors were involved in the actions of anandamide. Concentration]response curves for anandamide Ž10y8 ]3 = 10y5 M. were established in the absence and also in the presence of phentolamine Ž10y6 M. which was added to the bath 30 min before adding anandamide.
Effect of SR141716A on anandamideinduced inhibition of electrically e¨ oked contractions In order to determine the subtype of cannabinoid receptor mediating the inhibitory effects of anandamide, inhibitory responses to a submaximal concentration of anandamide were obtained in the absence and also in the presence of SR141716A Ž10y6 M.. Since recovery from the effect of anandamide was not complete, antagonist studies were carried out on paired tissues. Experiments on control and SR141716A-treated tissues were run simultaneously. In all studies involving SR141716A, the antagonist was added to the tissue and allowed to equilibrate with the preparation for 30 min before the tissues were stimulated.
Effect of anandamide on carbacholinduced contraction in the tracheal rings
For electrical stimulation, the tracheal rings were suspended between platinum electrodes and were stimulated with square wave pulses delivered by a Grass S 88 stimulator. Trains of pulses were applied at 20 Hz for 10 s every 90 s Ž60-V and 0.5-ms duration.. The response to electrical stimulation was considered to have stabilised when three or four consecutive contractions were of the same amplitude.
After establishing a steady basal tone, carbachol Ž10y6 M. was added to the organ baths. This concentration of carbachol produced a submaximal Žless than 80% of the maximum. contraction. Once the contraction has reached a plateau, increasing concentrations of anandamide Ž10y8 ]3 = 10y5 M. were added cumulatively, to the baths to generate data for a concentration]response curve. The contact time for each concentration of anandamide was 10 min.
Effect of tetrodotoxin (TTX ) on electricallyinduced contractions
Effect of WIN55,212-2 and WIN55,212-3 on electrically-induced contractions
In some preliminary experiments, after establishing a stable response to electrical stimulation, a single concentration of TTX Ž10y6 M. was added to the bath and its effect on the electrically-induced
In this series of experiments, the inhibitory effects of WIN55,212-2 and WIN55,212-3 Ž10y9 ]3 = 10y5 M. were also examined. After obtaining stable contractions to electrical stimulation, increasing
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concentrations of WIN55,212-2 or WIN55,212-3 were added to the baths in a cumulative manner. The effect of any given concentration of the agonists was allowed to peak before the next concentration was added. The effect of SR141716A Ž10y6 M. on the inhibitory effect of a submaximal concentration of WIN55,212-2 Ž10y5 M. was also examined using the same protocol that was used for anandamide.
Statistical analysis Agonist effect was expressed as a percentage reduction in the amplitude of electrically-induced contractions. The immediate contraction before the drugs were added was considered as the reference contraction. Results are presented as means " SE. Concentration]response curves were analysed using Graph-Pad Prism W software. The potency Žexpressed as ylog EC 25 value. for the agonists was obtained from the cumulative concentration response curves. The EC 25 value is the concentration of the agonist that reduced electrically-induced contractions by 25%. EC 25 value was determined since the inhibitory responses, at the maximum concentration ranged between 30 and 60%. Higher concentrations of the drugs were not used to avoid any possible effect of the solvents. Standard statistical evaluation of the data was done using Student’s t-test Žunpaired.. The difference was considered significant ŽU . when the P value was less than 0.05.
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Drugs and chemicals Carbachol and phentolamine were obtained from Sigma chemicals ŽSt Louis, MO, USA.. Anandamide, SR141716A w N-Žpiperidin-1-yl.-5-Ž4-chlorophenyl.-1Ž2,4-dichlorophenyl.-4-methyl-1H-pyrazole-3-carboxamide hydrochloridex, WIN55,212-2 mesylate Ž RŽq.w2,3-dihydro-5-methyl-3-wŽmorpholinyl.methylxpyrrolo w1,2,3-dex-1,4-benzoxazinylx-Ž1-naphthalenyl methanone mesylate., WIN55,212-3 and tetrodotoxin were obtained from Research Biochemicals International. SR141716A, WIN55,212-2 and WIN55,212-3 were dissolved in DMSO, while anandamide and tetrodotoxin were dissolved in ethanol. Carbachol and phentolamine were dissolved in distilled water. Drug concentrations quoted in the text represent final baths concentrations in all cases. The final concentration of the solvents in the organ-baths in all experiments did not exceed 0.1%. The concentration of ethanol or DMSO did not affect tissue responsiveness to electrical stimulation.
RESULTS
Effect of anandamide on electricallyinduced contractions Electrical field stimulation produced reproducible contractions of the rat tracheal ring segment. Tetrodotoxin Ž10y6 M. abolished the electrically-
Fig. 1. ŽA. Effect of TTX Ž. Ž10y6 M. on electrically-induced contractions in the isolated rat tracheal ring segments. ŽB. A typical trace of anandamide-induced inhibition Žv. Ž10y8 ]3 = 10y5 M. of electrically-evoked contractions in the isolated rat tracheal ring segments.
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induced contractions, indicating neurogenic origin of the contractions wFig. 1ŽA.x. Anandamide Ž10y8 ]3 = 10y5 M. produced a concentration-dependent inhibition of the contractions evoked by electrical stimulation wFig. 1ŽB.x. The highest concentration of anandamide used in this study Ž3 = 10y5 M. produced approximately 51.4" 5.8% Ž n s 8. inhibition of electrically-induced contractions ŽFig. 2.. A higher concentration of anandamide was not tested because the final ethanol concentration in the baths would be increased to 1%. At this concentration, ethanol alone reduced electrically-induced contractions. The ylog EC 25 value for anandamide was 5.25" 0.2. In order to determine the possible involvement of a 2 adrenergic receptors in the effects of anandamide, the effects of anandamide were tested in the presence of phentolamine 10y6 M. Results showed that there was no significant difference in the inhibitory effect of anandamide in the presence or absence of phentolamine Ž10y6 M.. The ylog EC 25 value for anandamide in the presence of phentolamine Ž10y6 M. was 5.6" 0.2 Ž n s 5.. This observation would exclude any contribution of a 2-adrenergic receptors to the inhibitory effect of anandamide on electrically-evoked contractions.
Effect of SR141716A on anandamideinduced inhibition of electrically-e¨ oked contractions
SR141716A Ž10y6 M. on its own did not affect the electrically-induced contractions. As shown in Fig. 3, SR141716A Ž10y6 M. also had no effect on anandamide Ž10y5 M.-induced inhibitory responses. There was a tendency towards enhanced inhibition of electrically-induced contractions by anandamide in the presence of SR141716A Ž10y6 M.. This increase, however, did not reach the level of statistical significance. The maximal inhibition in electrically-
evoked contractions was 55.3" 5.3% Ž n s 8. at the concentration of 3 = 10y5 M Žcompared to 51.4" 5.8% in the absence of SR141716A..
Effect of anandamide on carbacholinduced contraction of the rat tracheal ring segments In rat tracheal ring segments pre-contracted with a submaximal concentration of carbachol Ž10y6 M., a cumulative concentration]response curve for anandamide Ž10y8 ]3 = 10y5 M. did not produce any inhibitory effect ŽFig. 4.. In other experiments, anandamide Ž10y5 M. had no effect on the concentration]response curve to carbachol.
Effect of WIN55,212-2 and WIN55,212-3 on electrically-induced contractions
WIN55,212-2 Ž10y9 ]3 = 10y5 M. had no effect on electrically-induced contractions at low concentrations Ž- 10y9 M.. However, higher concentrations of WIN55,212-2 produced a concentration-dependent inhibition of the electrically evoked contractions ŽFig. 2.. The ylog EC 25 value was 5.8" 0.4. The reduction in contractions induced by the highest concentration of WIN55,212-2 in this study was 35.1%" 19.5 Ž n s 4.. WIN55,212-3 also produced a concentration-dependent inhibition in the electrically evoked contractions. There was no significant difference between the effectiveness of WIN55,212-2 and WIN55,212-3 in this study. The maximal inhibition produced by WIN55,212-3 was 15.8" 2.4% Ž n s 6..
Effect of SR141716A on WIN55,212-2induced inhibition of electrically-e¨ oked contractions
The effect of SR141716A Ž10 y 6 M . on WIN55,212-2-induced inhibition of the electrically induced contractions was also studied using the same procedure as for anandamide. As shown in Fig. 3, WIN55,212-2 Ž10y5 M. induced inhibition of electrically evoked contractions was not reduced in the presence of SR141716A Ž10y6 M.. Instead, there was a tendency towards an increased inhibition Žthough not significantly. of the electrically-evoked contractile response. WIN55,212-2 Ž10y5 M. reduced electrically-induced contractions by 22.4 and 32.9% in the absence and presence of SR141716A Ž10y6 M., respectively.
DISCUSSION
Fig. 2. Effect of ŽI. anandamide Ž10y8 ]3 = 10y5 M., ŽB . WIN55,212-2 Ž10 y9 ] 3 = 10 y 5 M . and Ž$ . WIN55,212-3 on electrically-evoked contractions in isolated rat tracheal preparations. Mean " SE Ž n s 7]8..
As stated in the Introduction, the main objective of this study was to determine Žfractionally . whether cannabinoid receptors are present in the airway smooth muscle and the type of function Žexcitatory or inhibitory. they mediate. We also attempted to
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Fig. 3. ŽA. Effect of ŽB. SR141716A Ž10y6 M. on ŽI. anandamide Ž10y5 M.-induced inhibition of electricallyevoked contraction in isolated rat tracheal preparations. Mean " SE. Ž n s 5.. ŽB. Effect of ŽB. SR141716A Ž10y6 y5 M. on ŽI. WIN 55,212-2 Ž10 M.-induced inhibition of electrically-evoked contraction in isolated rat tracheal preparations. Mean " SE Ž n s 4..
determine the receptor subtype involved in this response. Electrical field stimulation of rat trachea produced contractions that were abolished by TTX indicating that they were nerve-mediated. We have therefore used this test system to study the effect of some cannabinoid agonists on neurotransmission in the rat trachea ring segments. Anandamide inhibited electrically-induced neurogenic contractions of the rat trachea at concentrations that had no effect on carbachol-induced contractions. Thus anandamide produced its actions via a prejunctional mechanism, probably inhibiting neurotransmitter release. Similar prejunctional inhibitory effects of anandamide have been reported in other preparations including mouse vas deferens w15, 16x and rat vas deferens and atria w17x. The potency
Fig. 4. Effect of anandamide Žv. Ž10y8 ]3 = 10y5 tracheal preparation.
M.
of anandamide observed in our study was however, slightly less than that reported by Ishac et al. w17x in the rat vas deferens and atria. Thus, at 10 m M, anandamide inhibited noradrenaline release by approximately 50% in the vas deferens and 40% in the atria w17x, while we have observed a 35% reduction in electrically-induced contractions at this concentration and 51.4% reduction at 30 m M. The effect of anandamide was not affected by pre-treatment of the tissues with phentolamine to block prejunctional a 2-adrenoceptors, indicating that prejunctional a 2adrenoceptors were not involved in this response. Cannabinoids activate two types of receptors, CB1 and CB 2 . These receptors can be differentiated using SR141716A which binds with a high Žnanomolar. affinity for CB1-receptors w18, 19x, but displays low Žmicromolar. affinity for CB 2-receptors w16, 20x. Using this antagonist, it has been shown that CB1receptors mediated prejunctional inhibitory effects of the cannabinoids in the mouse vas deferens w15, 16x and rat atria w17x. We therefore tested the effect of SR141716A on inhibition of electrically-induced contractions induced by anandamide. The results showed that SR141716A Ž1 m M. on its own did not affect the responses to electrical stimulation, indicating that endogenously released cannabinoids, acting on CB1 receptors, did not modulate these responses. The results also showed that SR141716A Ž1 m M. did not attenuate the inhibitory effect of anandamide in the rat trachea. This would confirm that the inhibitory responses were not mediated via CB1 receptors. WIN55,212-2 is a potent cannabinoid receptor ligand, which displays a modest Ž20-fold. selectivity for CB 2-receptors w6, 9x. In the present study, WIN55,212-2 produced a concentration-dependent inhibition of electrically-induced contractions. In this study, WIN55,212-2 showed a lower potency compared to anandamide. However, Izzo et al. w21x have reported a higher potency for WIN55,212-2 compared to anandamide in circular smooth muscle of
on Ž. carbachol Ž10y6
M.
induced pre-contraction of isolated rat
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the guinea-pig ileum. The inhibitory effect produced by WIN55,212-2 was not reduced by SR141716A indicating that CB1-receptors were not involved in this response. A higher concentration of SR141716A was not used in this study since the concentration used Ž1 m M. was already approximately 100-fold higher than the pA 2 value of 8.17 reported by Rinaldi-Carmona et al. w16x and tenfold higher than the p K i of 10y7 M reported by Ishac et al. w17x in the rat atria. Secondly, White and Hiley w14x have shown that higher concentrations Ž) 3 m M. of SR141716A can behave as inverse agonists and are therefore not suitable for studies of cannabinoid receptor mechanisms. In our study, SR141716A Ž1 m M., as stated above, did not reduce the response of the tissue to electrical stimulation but it increased the inhibitory effect Žnot significantly. of both anandamide and WIN55,212-2. These results therefore, tend to suggest that the prejunctional inhibitory effect of anandamide and WIN55,212-2 in the rat trachea were possibly mediated via CB 2-receptors. Alternatively, considering Ža. the weak agonistic effect of anandamide and WIN55,212-2; Žb. the fact that WIN55,212-2 was only marginally Žthough not significant. more effective than anandamide in the rat trachea; and Žc. that anandamide had a higher potency than WIN55,212-2, it is possible that these compounds interacted with sites other than Ži.e. a non-specific effect. cannabinoid receptors. The observation in this study that WIN55,212-3, a less active stereoisomer of WIN55,212-2 was almost as potent as WIN55,212-2 in producing neuroinhibitory effects. This would tend to support a non-specific non-receptor-mediated effect of these compounds in the rat trachea.
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ACKNOWLEDGEMENTS
15.
SR141716A was provided by Research Biochemicals International as part of the chemical synthesis program of the National Institute of Mental Health Žcontract N01 MH 30003..
16.
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