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Cannabinoid CB2 receptor activation prevents bronchoconstriction and airway oedema in a model of gastro-oesophageal reflux
Abstract
Cannabinoids have been shown to inhibit sensory nerve activation in guinea-pigs and humans. Their effects are mediated by specific activation of two types of receptors, named CB(1) and CB(2). The purpose of this study was to investigate the effects of WIN 55,212-2, (R)-(+)-[2,3-dihydro-5methyl-3-[(4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone, a non selective agonist of cannabinoid receptors, and JWH 133, (6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran a selective cannabinoid CB(2) receptor agonist, on the sensory nerve component of intraoesophageal (i.oe.) HCl-induced airway microvascular leakage and bronchoconstriction in guinea-pigs. We also tested the effect of WIN 55,212-2 on substance P-induced plasma extravasation and bronchoconstriction. Airway microvascular leakage and bronchoconstriction induced by i.oe. HCl was inhibited by the cannabinoid CB(1)/CB(2) agonist WIN 55,212-2 (0.3-3 mg/kg i.p.) in a dose-dependent manner (maximal inhibition at the dose of 3 mg kg(-1), P<0.01). The effect of WIN 55,212-2 was inhibited by a cannabinoid CB(2) receptor antagonist SR 144528, [N-[(1S)-endo-1,3,3-trimethylbicyclo[2,2,1] heptan-2yl]-5-(-4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide], but not by a CB(1) receptor antagonist, SR 141716, [N-(piperidin-1yl)-5-(-4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride]. The cannabinoid CB(2) agonist JWH 133 (0.3-3 mg/kg i.p.) mimicked the inhibitory effect of WIN 55,212-2 on HCl-induced microvascular leakage. Under similar conditions, WIN 55,212-2 (1 mg kg (-1) i.p.) was unable to counteract the airway microvascular leakage and bronchoconstriction induced by substance P. These results suggest that inhibition by WIN 55,212-2 of airway plasma extravasation and bronchoconstriction induced by i.oe. HCl instillation in guinea-pigs is mediated through cannabinoid CB(2) receptor activation.
... Due to the immunomodulatory effect of cannabinoids, a significant variety of studies were focused on their possible therapeutic potential on inflammatory diseases like asthma [43,67,68,92,93,104,[109][110][111][112][113][114]. The molecular mechanisms mediating the effects of cannabinoids in allergic airway responses mainly depend on their effects on immune cells and the related release of cytokines [115]. ...
... This was due to an inhibition of C-fiber nerve activity and mediated by the activation of CB 2 receptors. WIN-55,212-2 was also shown to inhibit airway plasma extravasation and bronchoconstriction induced by intra-esophageal HCl instillation in guinea-pigs, an effect which was also mediated through CB 2 activation [113]. CB 2 receptor modulation of airway sensory nerve activity and related cough reflex have been highlighted in several different studies [110,117]. ...
Cannabinoids and the mammalian endocannabinoid system is an important research area of interest and attracted many researchers because of their widespread biological effects. The significant immune-modulatory role of cannabinoids has suggested their therapeutic use in several inflammatory conditions. Airways are prone to environmental irritants and stimulants, and increased inflammation is an important process in most of the respiratory diseases. Therefore, the main strategies for treating airway diseases are suppression of inflammation and producing bronchodilation. The ability of cannabinoids to induce bronchodilation and modify inflammation indicates their importance for airway physiology and pathologies. In this review, the contribution of cannabinoids and the endocannabinoid system in the airways are discussed, and the existing data for their therapeutic use in airway diseases are presented.
... Gastroesophageal reflux is a prevalent clinical disease linked with several respiratory symptoms, such as bronchoconstriction and chronic cough, and it is more common in patients with asthma (Leggett et al., 2005). Contrary to the nonprotective effect of JWH133 in airway inflammation, a study by Cui et al. (2007) found that JWH133 suppressed microvascular airway leakage and bronchoconstriction induced by intraoesophageal HCl in guinea pigs. However, the protective effect of JWH133 was reversed by SR144528, indicating that the effect was mediated by CB2R stimulation. ...
The endocannabinoid system has attracted attention as a pharmacological target for several pathological conditions. Cannabinoid (CB2)-selective agonists have been the focus of pharmacological studies because modulation of the CB2 receptor (CB2R) can be useful in the treatment of pain, inflammation, arthritis, addiction, and cancer among other possible therapeutic applications while circumventing CNS-related adverse effects. Increasing number of evidences from different independent preclinical studies have suggested new perspectives on the involvement of CB2R signaling in inflammation, infection and immunity, thus play important role in cancer, cardiovascular, renal, hepatic and metabolic diseases. JWH133 is a synthetic agonist with high CB2R selectivity and showed to exert CB2R mediated antioxidant, anti-inflammatory, anticancer, cardioprotective, hepatoprotective, gastroprotective, nephroprotective, and immunomodulatory activities. Cumulative evidences suggest that JWH133 protects against hepatic injury, renal injury, cardiotoxicity, fibrosis, rheumatoid arthritis, and cancer as well as against oxidative damage and inflammation, inhibits fibrosis and apoptosis, and acts as an immunosuppressant. This review provides a comprehensive overview of the polypharmacological properties and therapeutic potential of JWH133. This review also presents molecular mechanism and signaling pathways of JWH133 under various pathological conditions except neurological diseases. Based on the available data, this review proposes the possibilities of developing JWH133 as a promising therapeutic agent; however, further safety and toxicity studies in preclinical studies and clinical trials in humans are warranted.
... Experiments on guinea pigs showed that when CB2 receptors are activated, both bronchoconstriction and microvascular leakage of gastric acid decrease. 18 This property of CB2 activation could prove useful in preventing gastroesophageal reflux disease. 19 ...
... However, there are at least 100 chemically related compounds currently known (122,(143)(144)(145)(146). While some of them have been legally controlled on individual level, recent legislation in Germany now considers the lead structures and attempts to control whole drug classes. It is expected that this will make it more difficult to simply replace single banned compounds by their substituted analogs in the synthetic cannabinoid preparations (122,(147)(148)(149)(150)(151)(152)(153). ...
A feature of human culture is that we can learn to consume chemical compounds, derived from natural plants or synthetic fabrication, for their psychoactive effects. These drugs change the mental state and/or the behavioral performance of an individual and can be instrumentalized for various purposes. After the emergence of a novel psychoactive substance (NPS) and a period of experimental consumption, personal and medical benefits and harm potential of the NPS can be estimated on evidence base. This may lead to a legal classification of the NPS, which may range from limited medical use, controlled availability up to a complete ban of the drug form publically accepted use. With these measures, however, a drug does not disappear, but frequently continues to be used, which eventually allows an even better estimate of the drug’s properties. Thus, only in rare cases, there is a final verdict that is no more questioned. Instead, the view on a drug can change from tolerable to harmful but may also involve the new establishment of a desired medical application to a previously harmful drug. Here, we provide a summary review on a number of NPS for which the neuropharmacological evaluation has made important progress in recent years. They include mitragynine (“Kratom”), synthetic cannabinoids (e.g., “Spice”), dimethyltryptamine and novel serotonergic hallucinogens, the cathinones mephedrone and methylone, ketamine and novel dissociative drugs, γ-hydroxybutyrate, γ-butyrolactone, and 1,4-butanediol. This review shows not only emerging harm potentials but also some potential medical applications.
... CB 2 receptor activation prevents bronchoconstriction and airway edema in a guinea-pig model of gastro-esophageal reflux. 160 Coughing is a very common clinical symptom, and current therapies are largely ineffective, indicating a major pharmacological need. CB 2 agonists appear to be protective in the cough reflex, and this effect is blocked by the presence of CB 2 antagonists, suggesting the direct involvement of this receptor. ...
The purpose of this review is to analyze the molecular characterization, distribution, and signal transduction pathways CB2 receptors together with their pharmacological role in some of the most relevant biological systems. The structure and activity relationships of several interesting CB2 agonists, including synthetic schemes to clarify the chemistry involved in this field, are reported in this review. The corresponding pharmacological and beneficial potentials of selected CB2 compounds have been focused upon in the review, along with the patent literature and the identification of promising therapeutic candidates.
... CB-R1 receptors are primarily found in the nervous system, whereas CB-2R receptors are mainly found in the spleen, tonsils, and cells of the immune system. Although CB-1R receptors can be found in non-neuronal cells and tissues, including reproductive organs, the pituitary gland, and immune cells, they are predominantly found on central and peripheral nerve terminals, where they mediate the inhibition of transmitter release (Sachs et al., 2015;Ortega et al., 2015;Latorre and Schmidt, 2015;Soderstrom and Tian, 2008;Cui et al., 2007;Cox et al., 2007;Darmani, 2001;Acquas et al., 2000;Buckley et al., 2000). ...
... The 1 mg kg À1 dose of CBD significantly reduced OVA-induced airway obstruction as detected by an inhibition of the reduction in C dyn . This bronchoprotective effect of CBD against allergen-induced bronchoconstriction supports other work developing a bronchoprotective effect of the synthetic cannabinoid receptor agonists, WIN55212-2 and JWH133 against neurogenic airway inflammation in the guinea-pig [34,35] and is consistent with our in vitro observations that CBD can influence OVA-induced contraction of isolated guinea-pig bronchi. ...
(-)-Δ9-Tetrahydrocannabinol has been demonstrated to have beneficial effects in the airways, but its psychoactive effects preclude its therapeutic use for the treatment of airways diseases. In the present study we have investigated the effects of (-)-cannabidiol, a non-psychoactive component of cannabis for its actions on bronchial smooth muscle in vitro and in vivo. Guinea-pig bronchial smooth muscle contractions induced by exogenously applied spasmogens were measured isometrically. In addition, contractile responses of bronchial smooth muscle from ovalbumin-sensitized guinea-pigs were investigated in the absence or presence of (-)-cannabidiol. Furthermore, the effect of (-)-cannabidiol against ovalbumin-induced airway obstruction was investigated in vivo in ovalbumin-sensitized guinea-pigs. (-)-Cannabidiol did not influence the bronchial smooth muscle contraction induced by carbachol, histamine or neurokinin A. In contrast, (-)-cannabidiol inhibited anandamide- and virodhamine-induced responses of isolated bronchi. A fatty acid amide hydrolase inhibitor, phenylmethanesulfonyl fluoride reversed the inhibitory effect of (-)-cannabidiol on anandamide-induced contractions. In addition, (-)-cannabidiol inhibited the contractile response of bronchi obtained from allergic guinea-pigs induced by ovalbumin. In vivo, (-)-cannabidiol reduced ovalbumin-induced airway obstruction. In conclusion, our results suggest that cannabidiol can influence antigen-induced airway smooth muscle tone suggesting that this molecule may have beneficial effects in the treatment of obstructive airway disorders.
... As recently shown, both CB1 and CB2 receptors are abundantly expressed in humans along the vagovagal neural pathway involved in the triggering of transient lower oesophageal sphincter relaxations [34] . Interestingly, activation of CB2 receptors reduced microvascular leakage and bronchoconstriction when acid was instilled in a guinea pig model of gastro-oesophageal reflux [35] . Therefore, an increased CB tone could be useful for the treatment of gastro-oesophageal reflux as an adjunct therapy with acid inhibition [3] . ...
The marijuana plant Cannabis sp. and its derivatives and analogues, known as cannabinoids (CBs), induce many effects throughout the whole body. Herein we briefly review the gastrointestinal (GI) pharmacology of CBs, with special focus on motor function. Some drugs are available to treat nausea and emesis, and evidences in humans and animal models suggest that other GI motility alterations (gastro-oesophageal reflux, inflammatory bowel conditions or paralytic ileus) might benefit from modifications of the CB tone throughout the gut. However, central and peripheral (including GI) side effects may occur upon acute and chronic CB administration. Hopefully, the ongoing worldwide intense research on CBs will soon provide new, safer CB-based medicines.
... Central and peripheral vagal mechanisms are involved in these functional changes (Beaumont et al., 2009). An interesting study by Cui et al. (2007) extends the potential therapeutic benefits of CBs in gastroesophageal reflux disease. They demonstrated in a guinea pig model of gastroesophageal reflux that CB 2 , but not CB 1 , receptor activation was capable of reducing the microvascular leakage and bronchoconstriction observed by instillation of hydrochloric acid into the oesophagus. ...
Cannabis has been used to treat gastrointestinal (GI) conditions that range from enteric infections and inflammatory conditions to disorders of motility, emesis and abdominal pain. The mechanistic basis of these treatments emerged after the discovery of Delta(9)-tetrahydrocannabinol as the major constituent of Cannabis. Further progress was made when the receptors for Delta(9)-tetrahydrocannabinol were identified as part of an endocannabinoid system, that consists of specific cannabinoid receptors, endogenous ligands and their biosynthetic and degradative enzymes. Anatomical, physiological and pharmacological studies have shown that the endocannabinoid system is widely distributed throughout the gut, with regional variation and organ-specific actions. It is involved in the regulation of food intake, nausea and emesis, gastric secretion and gastroprotection, GI motility, ion transport, visceral sensation, intestinal inflammation and cell proliferation in the gut. Cellular targets have been defined that include the enteric nervous system, epithelial and immune cells. Molecular targets of the endocannabinoid system include, in addition to the cannabinoid receptors, transient receptor potential vanilloid 1 receptors, peroxisome proliferator-activated receptor alpha receptors and the orphan G-protein coupled receptors, GPR55 and GPR119. Pharmacological agents that act on these targets have been shown in preclinical models to have therapeutic potential. Here, we discuss cannabinoid receptors and their localization in the gut, the proteins involved in endocannabinoid synthesis and degradation and the presence of endocannabinoids in the gut in health and disease. We focus on the pharmacological actions of cannabinoids in relation to GI disorders, highlighting recent data on genetic mutations in the endocannabinoid system in GI disease.
... Moreover, PEA has been demonstrated to down-modulate mast cell degranulation induced either by neurogenic (substance P) or immune-mediated stimuli, both in vitro and in vivo (88,89). Interestingly, it was also observed that substance P induced bronchoconstriction and airway oedema could be alleviated by CB 2 R activation (90). In addition, activation of peripheral CB 2 R decreased the spinal cord inflammation in animal model of multiple sclerosis (91,92). ...
Cannabinoids and their derivatives are group of more than 60 biologically active chemical agents, which have been used in natural medicine for centuries. The major agent of exogenous cannabinoids is Delta(9)-tetrahydrocannabinol (Delta(9)-THC), natural psychoactive ingredient of marijuana. However, psychoactive properties of these substances limited their use as approved medicines. Recent discoveries of endogenous cannabinoids (e.g. arachidonoylethanolamide, 2-arachidonoylglycerol or palmithyloethanolamide) and their receptors initiated discussion on the role of cannabinoid system in physiological conditions as well as in various diseases. Based on the current knowledge, it could be stated that cannabinoids are important mediators in the skin, however their role have not been well elucidated yet. In our review, we summarized the current knowledge about the significant role of the cannabinoid system in the cutaneous physiology and pathology, pointing out possible future therapeutic targets.
Significant growth of interest in cannabis (Cannabis sativa L.), especially its natural anti-inflammatory and antioxidative properties, has been observed recently. This narrative review aimed to present the state of the art of research concerning the anti-inflammatory activity of all classes of cannabinoids published in the last five years. Multimodal properties of cannabinoids include their involvement in immunological processes, anti-inflammatory, and antioxidative effects. Cannabinoids and non-cannabinoid compounds of cannabis proved their anti-inflammatory effects in numerous animal models. The research in humans is missing, and the results are unconvincing. Although preclinical evidence suggests cannabinoids are of value in treating chronic inflammatory diseases, the clinical evidence is scarce, and further well-designed clinical trials are essential to determine the prospects for using cannabinoids in inflammatory conditions.
Introduction: Overdose fatalities associated with the opioid epidemic are predictably attributable to drug-induced respiratory depression. In terms of illicit opioid abuse, fentanyl is the synthetic opioid responsible for the largest number of overdose deaths. There is, therefore, an urgent need to identify safe and effective therapeutics that can attenuate fentanyl-induced respiratory depression. Identification of effective alternate analgesic strategies that lessen the respiratory depression associated with narcotics would also help improve current strategies for pain management. Our laboratory recently reported that the G protein-biased CB2 cannabinoid receptor agonist LY2828360 suppressed chemotherapy-induced neuropathic nociception and attenuated both morphine tolerance and physical dependence in paclitaxel-treated mice. However, the impact of LY2828360 on other undesirable side effects of opioids, such as opioid-induced respiratory depression, remains unknown.
Materials and Methods: We used whole-body plethysmography to assess the impact of the CB2 cannabinoid agonist LY2828360 on fentanyl-induced respiratory depression using wild-type (WT) and CB2 knockout (CB2KO) mice.
Results: Fentanyl reduced minute ventilation and respiratory frequency without altering tidal volume in both WT and CB2KO mice. In WT mice, the high dose of fentanyl (0.2 mg/kg intraperitoneal [i.p.]) produced a greater suppression of respiratory parameters compared with the low dose of fentanyl (0.1 mg/kg i.p.). Coadministration of a behaviorally active dose of LY2828360 (3 mg/kg i.p.) with fentanyl (0.2 mg/kg i.p.) attenuated fentanyl-induced respiratory depression in WT mice. Notably, LY2828360 (3 mg/kg i.p.) did not attenuate fentanyl-induced respiratory depression in CB2KO mice, consistent with mediation by CB2 receptors. Moreover, LY2828360 (3 mg/kg i.p.) alone lacked intrinsic effects on respiratory parameters in either WT or CB2KO mice.
Conclusion: The combination of a CB2 agonist with fentanyl may represent a safer adjunctive therapeutic strategy compared with a narcotic analgesic alone by attenuating the development of opioid-induced respiratory depression. Moreover, the CB2 agonist, administered alone, did not alter respiration. Our findings suggest that the CB2 cannabinoid agonist LY2828360 may provide CB2-mediated protection against fentanyl-induced respiratory depression, a detrimental and unwanted side effect of opioid use and abuse.
Background
Irritable bowel syndrome (IBS) is a common disease with intestinal dysmotility, whose mechanism remains elusive. The endocannabinoid system is emerging as an important modulator of gastrointestinal (GI) motility in multiple diseases, but its involvement in IBS is unknown. We aimed to determine whether cannabinoid 2 (CB2) receptor modulates intestinal motility associated with stress‐induced IBS.
Methods
A rat IBS model was established by chronic water avoidance stress (WAS). Colonic pathological alterations were detected histologically and intestinal motility was assessed by intestinal transit time (ITT) and fecal water content (FWC). Visceral sensitivity was determined by visceromotor response (VMR) to colorectal distension (CRD). Real‐time PCR, western blot, and immunostaining were performed to identify colonic CB2 receptor expression. Colonic muscle strip contractility was studied by isometric transducers and nitric oxide (NO) was detected by the Griess test. The effects of AM1241, a selective agonist of CB2 receptors, on colonic motility were examined.
Key Results
After 10 days of WAS exposure, ITT was decreased and FWC elevated while VMR magnitude in response to CRD was significantly enhanced. Colon CB2 protein and mRNA levels increased and density of CB2‐positive macrophages in the mucosa and enteric neurons in the myenteric plexus was higher than in controls. Pharmacological enhancement of CB2 activity by AM1241 relieved colonic hypermotility in WAS rats in a concentration‐dependent manner via inhibition of p38 phosphorylation and elevation of NO production.
Conclusion
CB2 receptor may exert an important inhibitory effect in stress‐induced colonic hypermotility by modulating NO synthesis through p38 mitogen‐activated protein kinase signaling. AM1241 could be used as a potential drug to treat disorders with colonic hypermotility.
The endocannabinoid system (ECS) primarily consists of cannabinoid receptors (CBRs), endogenous ligands, and enzymes for endocannabinoid biosynthesis and inactivation. Although the presence of CBRs, both CB1 and CB2, as well as a third receptor (G-protein receptor 55 [GPR55]), has been established in the gastrointestinal (GI) tract, few studies have focused on the role of cannabinoids on esophageal function. To date, studies have shown their effect on GI motility, inflammation and immunity, intestinal and gastric acid secretion, nociception and emesis pathways, and appetite control. Given the varying and sometimes limited efficacy of current medical therapies for diseases of the esophagus, further understanding and investigation into the interplay of the ECS on esophageal health and disease may present new therapeutic modalities that may help advance current treatment options. In this brief review, the current understanding of the ECS role in various esophageal functions and disorders is presented.
The endogenous cannabinoid system and its role in the physiology and pathophysiology of the digestive system is presented. The potential use of agonists and antagonists of cannabinoid receptors in such disorders as nausea and vomiting, peptic ulcer disease, gastroesophageal reflux disease, irritable bowel syndrome, inflammation, and paralytic ileus are discussed. Differences in the pharmacological action of these substances in animals and humans as well as the observed side effects are also considered. Pharmacological modulation of the endogenous cannabinoid system could provide a new therapeutic target for the treatment of a number of gastrointestinal diseases.
The objective of this thesis was to study the time-course of the respiratory effects of THC/ethanol and GHB/ethanol associations. Respiration was studied using whole body plethysmography in non-anesthetized rats during the four hours following intraperitoneal injection. In a first step, both GHB and ethanol affected the respiratory pattern. Ethanol caused bradypnea at doses greater than 3 g.kg-1 and GHB induced apneusis above 600 mg.kg-1. Arterial blood gases modifications were metabolic: acidemia with ethanol and alkalosis with GHB. At those doses, both substances altered consciousness and body temperature of animals. Ethanol induced hypothermia and GHB a temperature triphasic change: hypothermia/ hyperthermia/hypothermia. Blood determinations and kinetic studies reinforced the likelihood, and clinical and forensic relevancies of our animal model. Association studies showed that the respiratory effects of THC and GHB were unchanged in presence of ethanol. Association with 3 g.kg-1 ethanol dose only provoked a reduction in minute volume and in mean inspiratory flow, but mechanisms were different. Ethanol and THC induced a decrease in tidal volume; ethanol and GHB an increase in expiratory apnea duration. The observed kinetic interaction between ethanol and GHB did not explain phenomenon intensity; a potentialization could occur.
Marijuana has been used for thousands of years to affect human health. Dissecting the peripheral effects from the central psychotropic effects has revealed a complex interplay between cannabinoids, endocannabinoids and their receptors. This review examines recent advances in understanding the expression, regulation and utilization of the CB2 receptor. Here we highlight the molecular aspects of the CB2 receptor, CB2 receptor signaling and new ligands for this receptor. We focus in the rest of the review on recent findings in the immune system, the gastrointestinal tract and liver, the brain and the cardiovascular system and airways as examples of areas where new developments in our understanding of the CB2 receptor have occurred. Early studies focused on expression of this receptor under baseline physiologic conditions; however, perturbations such as those that occur during inflammation, ischemia/reperfusion injury and cancer are revealing a critical role for the CB2 receptor in regulating these disease processes amongst others. As a result, the CB2 receptor is an appealing therapeutic target as well as a useful tool for shedding new light on physiological regulatory processes throughout the body.
Although neurogenic inflammation of the airways via activation of C-fibers is thought to be important in the pathogenesis of asthma, the mechanisms regulating C-fiber activity remain uncertain.
The influence of a cannabinoid receptor agonist, WIN 55,212-2, on C-fiber activation in guinea pig airways was investigated, as was the mechanism by which cannabinoids regulate antigen-induced airway inflammation.
The inhibitory effect of WIN 55,212-2 on antigen-induced plasma extravasation was assessed in guinea pig tracheal tissues by photometric measurement of extravasated Evans blue dye after extraction with formamide.
Pretreatment with WIN 55,212-2 (0.001, 0.01 or 0.1 mg/kg) significantly and dose-dependently reduced tracheal plasma extravasation induced by inhaling a 5% ovalbumin solution for 2 min after pretreatment with a neutral endopeptidedase inhibitor (phosphoramidon at 2.5 mg/kg i.v.). A cannabinoid CB2 receptor antagonist (SR144528) blunted the inhibitory effect of WIN 55,212-2, while a cannabinoid CB1 antagonist (SR141716A) did not. Pretreatment with a neurokinin-1 receptor antagonist (FK888) significantly reduced ovalbumin-induced extravasation of Evans blue dye. Pretreatment with the combination of WIN 55,212-2 and FK888 reduced antigen-induced plasma extravasation more markedly than FK888 alone.
These findings suggest that WIN 55,212-2 inhibits C-fiber activation via the cannabinoid CB2 receptor and thus suppresses antigen-induced inflammation in guinea pig airways.
The identification of endocannabinoids and cannabinoid CB1 receptors in key areas of the intestinal wall, such as cholinergic neurons, supports a role for cannabinoids in the control of gastrointestinal motility. Activation of CB1 receptors inhibits the peristaltic reflex and slows down gastrointestinal and colonic transit. Endocannabinoids play an important inhibitory role in the control of the occurrence of transient lower esophageal sphincter relaxations. Cannabinoid receptor agonists inhibit gastric emptying and intestinal motility in humans. There is strong anatomical support for a role of CB1 receptors in the control of gastrointestinal perception, since these receptors have been identified in key sites of the neuronal circuitry involved in the transmission of visceral pain. Experimental data indicate a visceral antinociceptive action of cannabinoid receptor agonists, which remains to be confirmed in humans.
Two cannabinoid receptors have been identified: CB1, present in the central nervous system (CNS) and to a lesser extent in other tissues, and CB2, present outside the CNS, in peripheral organs. There is evidence for the presence of CB2-like receptors in peripheral nerve terminals. We report now that we have synthesized a CB2-specific agonist, code-named HU-308. This cannabinoid does not bind to CB1\ (Ki>10\ mu M), but does so efficiently to CB2\ (Ki=22.7± 3.9\ nM); it inhibits forskolin-stimulated cyclic AMP production in CB2-transfected cells, but does so much less in CB1-transfected cells. HU-308 shows no activity in mice in a tetrad of behavioral tests, which together have been shown to be specific for tetrahydrocannabinol (THC)-type activity in the CNS mediated by CB1. However, HU-308 reduces blood pressure, blocks defecation, and elicits anti-inflammatory and peripheral analgesic activity. The hypotension, the inhibition of defecation, the anti-inflammatory and peripheral analgesic effects produced by HU-308 are blocked (or partially blocked) by the CB2 antagonist SR-144528, but not by the CB1 antagonist SR-141716A. These results demonstrate the feasibility of discovering novel nonpsychotropic cannabinoids that may lead to new therapies for hypertension, inflammation, and pain.
The major active ingredient of marijuana, delta 9-tetrahydrocannabinol (delta 9-THC), has been used as a psychoactive agent for thousands of years. Marijuana, and delta 9-THC, also exert a wide range of other effects including analgesia, anti-inflammation, immunosuppression, anticonvulsion, alleviation of intraocular pressure in glaucoma, and attenuation of vomiting. The clinical application of cannabinoids has, however, been limited by their psychoactive effects, and this has led to interest in the biochemical bases of their action. Progress stemmed initially from the synthesis of potent derivatives of delta 9-THC, and more recently from the cloning of a gene encoding a G-protein-coupled receptor for cannabinoids. This receptor is expressed in the brain but not in the periphery, except for a low level in testes. It has been proposed that the nonpsychoactive effects of cannabinoids are either mediated centrally or through direct interaction with other, non-receptor proteins. Here we report the cloning of a receptor for cannabinoids that is not expressed in the brain but rather in macrophages in the marginal zone of spleen.
Based on both binding and functional data, this study introduces SR 144528 as the first, highly potent, selective and orally active antagonist for the CB2 receptor. This compound which displays subnanomolar affinity (Ki = 0.6 nM) for both the rat spleen and cloned human CB2 receptors has a 700-fold lower affinity (Ki = 400 nM) for both the rat brain and cloned human CB1 receptors. Furthermore it shows no affinity for any of the more than 70 receptors, ion channels or enzymes investigated (IC50 > 10 microM). In vitro, SR 144528 antagonizes the inhibitory effects of the cannabinoid receptor agonist CP 55,940 on forskolin-stimulated adenylyl cyclase activity in cell lines permanently expressing the h CB2 receptor (EC50 = 10 nM) but not in cells expressing the h CB1 (no effect at 10 microM). Furthermore, SR 144528 is able to selectively block the mitogen-activated protein kinase activity induced by CP 55,940 in cell lines expressing h CB2 (IC50 = 39 nM) whereas in cells expressing h CB1 an IC50 value of more than 1 microM is found. In addition, SR 144528 is shown to antagonize the stimulating effects of CP 55,940 on human tonsillar B-cell activation evoked by cross-linking of surface Igs (IC50 = 20 nM). In vivo, after oral administration SR 144528 totally displaced the ex vivo [3H]-CP 55,940 binding to mouse spleen membranes (ED50 = 0.35 mg/kg) with a long duration of action. In contrast, after the oral route it does not interact with the cannabinoid receptor expressed in the mouse brain (CB1). It is expected that SR 144528 will provide a powerful tool to investigate the in vivo functions of the cannabinoid system in the immune response.
Two cannabinoid receptors have been identified: CB(1), present in the central nervous system (CNS) and to a lesser extent in other tissues, and CB(2), present outside the CNS, in peripheral organs. There is evidence for the presence of CB(2)-like receptors in peripheral nerve terminals. We report now that we have synthesized a CB(2)-specific agonist, code-named HU-308. This cannabinoid does not bind to CB(1) (K(i) > 10 microM), but does so efficiently to CB(2) (K(i) = 22.7 +/- 3.9 nM); it inhibits forskolin-stimulated cyclic AMP production in CB(2)-transfected cells, but does so much less in CB(1)-transfected cells. HU-308 shows no activity in mice in a tetrad of behavioral tests, which together have been shown to be specific for tetrahydrocannabinol (THC)-type activity in the CNS mediated by CB(1). However, HU-308 reduces blood pressure, blocks defecation, and elicits anti-inflammatory and peripheral analgesic activity. The hypotension, the inhibition of defecation, the anti-inflammatory and peripheral analgesic effects produced by HU-308 are blocked (or partially blocked) by the CB(2) antagonist SR-144528, but not by the CB(1) antagonist SR-141716A. These results demonstrate the feasibility of discovering novel nonpsychotropic cannabinoids that may lead to new therapies for hypertension, inflammation, and pain.
Two types of cannabinoid receptor have been discovered so far, CB(1) (2.1: CBD:1:CB1:), cloned in 1990, and CB(2) (2.1:CBD:2:CB2:), cloned in 1993. Distinction between these receptors is based on differences in their predicted amino acid sequence, signaling mechanisms, tissue distribution, and sensitivity to certain potent agonists and antagonists that show marked selectivity for one or the other receptor type. Cannabinoid receptors CB(1) and CB(2) exhibit 48% amino acid sequence identity. Both receptor types are coupled through G proteins to adenylyl cyclase and mitogen-activated protein kinase. CB(1) receptors are also coupled through G proteins to several types of calcium and potassium channels. These receptors exist primarily on central and peripheral neurons, one of their functions being to inhibit neurotransmitter release. Indeed, endogenous CB(1) agonists probably serve as retrograde synaptic messengers. CB(2) receptors are present mainly on immune cells. Such cells also express CB(1) receptors, albeit to a lesser extent, with both receptor types exerting a broad spectrum of immune effects that includes modulation of cytokine release. Of several endogenous agonists for cannabinoid receptors identified thus far, the most notable are arachidonoylethanolamide, 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether. It is unclear whether these eicosanoid molecules are the only, or primary, endogenous agonists. Hence, we consider it premature to rename cannabinoid receptors after an endogenous agonist as is recommended by the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. Although pharmacological evidence for the existence of additional types of cannabinoid receptor is emerging, other kinds of supporting evidence are still lacking.
Gastro-oesophageal reflux is a common clinical disorder associated with a variety of respiratory symptoms, including chronic cough and exacerbation of asthma. In this study, the potential role of acid-induced tachykinin release was examined in guinea pigs and rabbits, by examining the effects of the tachykinin NK1 and NK3 receptors antagonists (SR 140333 and SR 142801, respectively) (1-10 mg x kg(-1)) on plasma protein extravasation induced in airways by hydrochloric acid (HCl) infusion in the oesophagus. Guinea pigs were anaesthetised with urethane, while rabbits were subject to neuroleptoanalgesia with hypnorm. Airway vascular leakage was evaluated by measuring extravasation of Evans blue dye. All animals were pretreated with atropine (1 mg x kg(-1) i.p.), propranolol (1 mg x kg(-1) i.p.), phosphoramidon (2.5 mg x kg(-1) i.v.) and saline or tachykinin receptor antagonists (1-10 mg x kg(-1) i.p.). Infusion of 1 N HCl into the oesophagus led to a three- and five-fold increase in plasma extravasation in the main bronchi and trachea, respectively. This increase was largely prevented by the tachykinin NK1 and NK3 receptor antagonists SR 140333 and SR 142801 (1-10 mg x kg(-1)). These results suggest that protein extravasation in the airways, as induced by intraoesophageal HCl infusion, is mainly dependent on the release of tachykinins, and that both NK1 and NK3 tachykinin receptors are involved. The results suggest that HCl-induced sensory nerve stimulation may act in the periphery on intermediate neurons and/or ganglia where NK3 receptors have been shown to play an important role.
Cannabinoid receptor agonists inhibit inflammatory hyperalgesia in animal models. Nonselective cannabinoid receptor agonists also produce central nervous system (CNS) side effects. Agonists selective for CB2 cannabinoid receptors, which are not found in the CNS, do not produce the CNS effects typical of nonselective cannabinoid receptor agonists but do inhibit acute nociception. The authors used the CB2 receptor-selective agonist AM1241 to test the hypothesis that selective activation of peripheral CB2 receptors inhibits inflammatory hyperalgesia.
Rats were injected in the hind paw with carrageenan or capsaicin. Paw withdrawal latencies were measured using a focused thermal stimulus. The effects of peripheral CB2 receptor activation were determined by using local injection of AM1241. CB2 receptor mediation of the actions of AM1241 was shown by using the CB2 receptor-selective antagonist AM630 and the CB1 receptor-selective antagonist AM251.
AM1241 fully reversed carrageenan-induced inflammatory thermal hyperalgesia when injected into the inflamed paw. In contrast, AM1241 injected into the contralateral paw had no effect, showing that its effects were local. AM1241 also reversed the local edema produced by hind paw carrageenan injection. The effects of AM1241 were reversed by the CB2 receptor-selective antagonist AM630, but not by the CB1 receptor-selective antagonist AM251. AM1241 also inhibited flinching and thermal hyperalgesia produced by hind paw capsaicin injection.
Local, peripheral CB2 receptor activation inhibits inflammation and inflammatory hyperalgesia. These results suggest that peripheral CB2 receptors may be an appropriate target for eliciting relief of inflammatory pain without the CNS effects of nonselective cannabinoid receptor agonists.
To determine the prevalence of gastroesophageal reflux disease (GERD)-both symptoms and objective evidence-using 24-h dual-probe pH monitoring in difficult asthma, and the relationship between the presence and treatment of GERD to clinical outcome.
As part of a systematic evaluation protocol, 68 subjects with difficult-to-control asthma attending a difficult asthma clinic were referred for dual-probe ambulatory pH esophageal monitoring.
Esophageal probe data were available in 52 patients (76%) with difficult asthma. The prevalence of GERD/GERD-associated asthma symptoms was 75% (39 of 52 patients; 95% confidence interval [CI], 63 to 84.7%). The prevalence of GERD as evidenced by an abnormal pH profile at the distal esophageal probe was 55% (29 of 52 patients; 95% CI, 40 to 69%). The prevalence of GERD at the proximal probe was 34.6% (18 of 52 patients; 95% CI, 23.6 to 51%). The prevalence of GERD was similar in asthmatic subjects who responded to intervention and those who remained difficult to control (therapy resistant). Asymptomatic GERD was present in 9.6% (5 of 52 patients); 16% of cough episodes correlated with acid reflux.
In difficult-to-control asthma, GERD is common, but identification and treatment of GERD do not appear to relate to improvement in asthma control in this population.
We examined the effects of a cannabinoid receptor agonist, (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), on various respiratory reactions induced by the activation of capsaicin-sensitive afferent sensory nerves (C-fibers). WIN 55212-2 significantly inhibited capsaicin-induced guinea pig bronchoconstriction, but not the neurokinin A-induced reaction. Intravenous injection of WIN 55212-2 also blocked cigarette smoke-induced rat tracheal plasma extravasation. However, substance P-induced rat tracheal plasma extravasation was not affected by the administration of WIN 55212-2. A cannabinoid CB(2) 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 effects of WIN 55212-2, but not a cannabinoid CB(1) antagonist, [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride] (SR 141716A). A Maxi-K(+) channel opener, 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)benzimidazolone (NS 1619), specifically inhibited capsaicin-induced guinea pig bronchoconstriction and cigarette smoke-induced rat tracheal plasma extravasation. These findings suggest that WIN 55212-2 inhibits the activation of C-fibers via cannabinoid CB(2) receptors and Maxi-K(+) channels and reduces airway neurogenic inflammatory reactions in vivo.
In a recent study so far published in abstract form, it was reported that the CB(2) receptor selective agonist AM1241 diminishes oedema produced as a result of mast cell degranulation in vivo. It is, however, not known whether other structurally different CB(2) agonists share this effect, and whether this is due to a direct effect on mast cell function. In the present study, we have investigated the effects of JWH133, a CB(2) receptor selective agonist, together with the anti-inflammatory agent palmitoylethanolamide and its analogue palmitoylisopropylamide, on compound 48/80-induced oedema and degranulation in vivo and in vitro. JWH133 (20 and 200 microg/mouse i.p.) significantly reduced the ability of compound 48/80 to induce oedema in vivo in the anaesthetised mouse following its injection into the ear pinna. Palmitoylethanolamide (200 microg/mouse i.p) also reduced the response to compound 48/80, whereas no firm conclusions could be drawn for palmitoylisopropylamide (20 and 200 microg/mouse i.p.). The CB(2) selective antagonist/inverse agonist SR144528 (60 microg/mouse i.p.) appeared to produce anti-inflammatory effects per se in this model, making it hard to interpret the effects of JWH133 in terms of CB(2) receptor mediated activation. In contrast to the situation in vivo, neither JWH133 (0.3 and 3 microM) nor palmitoylethanolamide (10 microM) affected mast cell degranulation, measured by following the release of the granular protein beta-hexosaminidase, produced by compound 48/80 in vitro in mouse skin slices. The two compounds were also ineffective in inhibiting the binding of [(3)H]pyrilamine to histamine H(1) receptors in vitro. It is concluded that the ability of JWH133 to affect mast cell dependent inflammation in vivo may be mediated by an indirect action upon the mast cells.
The active principle in marijuana, Δ9-tetrahydrocannabinol (THC), has been shown to have wide therapeutic application for a number of important medical conditions, including pain, anxiety, glaucoma, nausea, emesis, muscle spasms, and wasting diseases. Δ9-THC binds to and activates two known cannabinoid receptors found in mammalian tissue, CB1 and CB2. The development of cannabinoid-based therapeutics has focused predominantly on the CB1 receptor, based on its predominant and abundant localization in the CNS. Like most of the known cannabinoid agonists, Δ9-THC is lipophilic and relatively nonselective for both receptor subtypes. Clinical studies show that nonselective cannabinoid agonists are relatively safe and provide therapeutic efficacy, but that they also induce psychotropic side effects. Recent studies of the biosynthesis, release, transport, and disposition of anandamide are beginning to provide an understanding of the role of lipid transmitters in the CNS. This review attempts to link current understanding of the basic biology of the endocannabinoid nervous system to novel opportunities for therapeutic intervention. This new knowledge may facilitate the development of cannabinoid receptor-targeted therapeutics with improved safety and efficacy profiles.
1. We have studied the effect of the sensory neuropeptides substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and calcitonin gene-related peptide (CGRP) on microvascular permeability in guinea-pig airways in vivo and investigated whether CGRP would potentiate the effect of SP. We used the extravasation of intravenously-injected Evans blue dye as an index of permeability. 2. The tachykinins SP, NKA and NKB (0.025-5.0 nmol kg-1, i.v.) significantly (P less than 0.05) increased extravasation of dye in a dose-related manner and with a similar pattern of distribution; they were most potent in the trachea and main bronchi, less potent in the larynx and intrapulmonary airways, and had little significant effect in the bladder. 3. SP was significantly more potent in causing extravasation of dye than NKA or NKB with ED50 values (nmol kg-1) in the range 0.04-0.1, depending on the airway level, compared with values in the range 0.3-0.7 for the neurokinins. 4. CGRP (0.0025-2.5 nmol kg-1, i.v.) had no significant effect on microvascular permeability and did not potentiate SP-induced extravasation of dye. 5. Each neuropeptide decreased mean arterial blood pressure, indicating vasodilatation, in a dose-related manner. Co-injection of CGRP and SP produced additive decreases in arterial pressure. 6. We conclude that, in guinea-pig airways, tachykinins increase microvascular permeability via tachykinin receptors of the NK-1 sub-type (indicated by an order of potency of SP greater than NKA = NKB) on endothelial cells. The response appears to be related to mechanisms in addition to vasodilatation. The relevance of the responses to the tachykinins in asthma is discussed.
The determination and characterization of a cannabinoid receptor from brain are reported. A biologically active bicyclic cannabinoid analgetic CP-55,940 was tritium-labeled to high specific activity. Conditions for binding to rat brain P2 membranes and synaptosomes were established. The pH optimum was between 7 and 8, and specific binding could be eliminated by heating the membranes to 60 degrees. Binding to the P2 membranes was linear within the range of 10 to 50 micrograms of protein/ml. Specific binding (defined as total binding displaced by 1 microM delta 9-tetrahydrocannabinol (delta 9-THC) or 100 nM desacetyllevonantradol) was saturable. The Kd determined from Scatchard analysis was 133 pM, and the Bmax for rat cortical P2 membranes was 1.85 pmol/mg of protein. The Hill coefficient for [3H]CP-55,940 approximated 1, indicating that, under the conditions of assay, a single class of binding sites was determined that did not exhibit cooperativity. The binding was rapid (kon approximately 2.6 x 10(-4) pM-1 min-1) and reversible (Koff approximately 0.016 min-1) and (koff' greater than 0.06 min-1). The two Kd values estimated from the kinetic constants approximately 55 pM and exceeded 200 pM, respectively. The binding of the agonist ligand [3H]CP-55,940 was decreased by the nonhydrolyzable GTP analog guanylylimidodiphosphate. The guanine nucleotide induced a more rapid dissociation of the ligand from the binding site, consistent with an allosteric regulation of the putative receptor by a G protein. The binding was also sensitive to MgCl2 and CaCl2. Binding of [3H]CP-55,940 was displaced by cannabinoid drugs in the following order of potency: CP-55,940 greater than or equal to desacetyllevonantradol greater than 11-OH-delta 9-THC = delta 9-THC greater than cannabinol. Cannabidiol and cannabigerol displaced [3H]CP-55,940 by less than 50% at 1 microM concentrations. The (-)-isomer of CP-55,940 displaced with 50-fold greater potency than the (+)-isomer. This pharmacology is comparable to both the inhibition of adenylate cyclase in vitro and the analgetic activity of these compounds in vivo. The criteria for a high affinity, stereoselective, pharmacologically distinct cannabinoid receptor in brain tissue have been fulfilled.
Gastroesophageal reflux (GER) has been shown to be more frequent in people with asthma, but the mechanism by which it might aggravate asthmatic symptoms remains unclear. We compared the effects on maximal expiratory flow at 50% of VC (MEF50) of esophageal perfusion of hydrochloric acid (HCl) and of normal saline (NaCl) in 12 asthmatic subjects chosen at random. In all subjects, HCl perfusion did not change MEF50 but potentiated the bronchoconstriction induced by isocapnic hyperventilation of dry air (maximal decrease in MEF50 = 44 +/- 7% with HCl versus 22 +/- 5% with NaCl; p less than 0.001) or methacholine (provocative dose producing a 20% decrease in FEV1 = 349 +/- 99 micrograms with HCl versus 496 +/- 119 micrograms with NaCl; p less than 0.01). Seven of the asthmatic subjects were found to have GER on esophageal pH monitoring. In these subjects, HCl alone decreased MEF50 slightly but significantly (-17.5 +/- 5.5%; p less than 0.05), possibly reflecting the higher degree of basal bronchial hyperreactivity observed in this group. Thus, perfusion of acid into the distal esophagus caused slight but significant bronchoconstriction in asthmatic subjects with GER and increased the bronchoconstriction produced by isocapnic hyperventilation and by methacholine in asthmatic subjects without regard for the presence of GER.
To determine a possible mechanism for the association between gastroesophageal reflux and obstructive pulmonary disease, we quantitatively compared the short latent airway response after acid infusion into the trachea or esophagus in 13 anesthetized adult cats. Total lung resistance was calculated from synchronous measurements of air flow and intrapleural pressure differences from those at end expiratory level. Tracheal infusion of as little as 0.05 ml of 0.2 N HCl evoked an average 4.65-fold increase in total lung resistance from baseline in all animals tested (p less than 0.005). Intratracheal saline had no effect. The response to intratracheal acid infusion was rapidly adapting, pH dependent, and vagally mediated. Infusion of a much larger volume of 10 ml of 0.2 N HCl into the esophagus produced an average 1.47-fold increase in total lung resistance from baseline (p less than 0.05). No change was seen with intraesophageal saline. In contrast to intratracheal acid infusion, a clearly significant increase in resistance was seen in only 8 of 13 animals tested after intraesophageal acidification. When it occurred, the response was sustained for at least 60 s after acid infusion. The magnitude of the response was not augmented by the presence of severe esophagitis. These studies strengthen the concept that reflex pathways in the trachea and esophagus may explain a causal relationship between gastroesophageal reflux and obstructive pulmonary diseases. The results support the view that microaspiration into the trachea is a much more likely mechanism for bronchospasm associated with gastroesophageal reflux than simple acid reflux into the esophagus.
0.1 N hydrochloric acid instilled into the esophagi of dogs with esophagitis caused a significant fall in respiratory conductance (P<.01). When the esophagi were distended with an intraesophageal balloon, a fall of similar magnitude occurred. These responses disappeared with bilateral interruption of the vagal nerves. These findings support previous results in human subjects which suggested reflexes from the esophagus could cause bronchoconstriction.
The capacity of substance P (SP) and endogenously released tachykinins to liberate histamine was examined in isolated tracheally perfused guinea pig lungs. Increasing doses of tracheally injected SP were associated with the recovery of increasing amounts of histamine from lung effluent. The mechanism of SP-induced histamine liberation was explored in studies with neurokinin-(NK) receptor agonists and antagonists. Tracheal injection of either the NK1 agonist [Sar9,Met(O2)11]SP or the NK2 agonist [beta-Ala8]-neurokinin A-(4-10) was associated with a significant increase in histamine recovery from lung effluent. In addition, both the NK1 antagonist CP-99994 and the NK2 antagonist SR-48968 significantly inhibited SP-induced histamine release. These findings support the hypothesis that SP can liberate histamine from guinea pigs lungs by a mechanism that depends predominantly on NK1- and NK2-receptor activation. The liberation of endogenous tachykinins by acute tracheal injection of capsaicin was also associated with augmented histamine recovery, which was inhibited by combined NK1- and NK2-receptor blockade. Tracheal injection of SP was associated with an increase in the percentage of airway mast cells exhibiting histological evidence of degranulation. This study demonstrates that exogenous SP, as well as endogenous tachykinins released from capsaicin-sensitive neurons, can liberate histamine, most likely from airway mast cells, by a mechanism that depends predominantly on the activation of NK1 and NK2 receptors.
SR141716A is the first selective and orally active antagonist of the brain cannabinoid receptor. This compound displays nanomolar affinity for the central cannabinoid receptor but is not active on the peripheral cannabinoid receptor. In vitro, SR141716A antagonises the inhibitory effects of cannabinoid receptor agonists on both mouse vas deferens contractions and adenylyl cyclase activity in rat brain membranes. After intraperitoneal or oral administration SR141716A antagonises classical pharmacological and behavioural effects of cannabinoid receptor agonists. This compound should prove to be a powerful tool for investigating the in vivo functions of the anandamide/cannabinoid system.
Cannabinoid receptor mRNA was localized in adult rat brain by 35S-tailed oligonucleotide probes and in situ hybridization histochemistry. Labelling is described as uniform or non-uniform depending on the relative intensities of individual cells expressing cannabinoid receptor mRNA within a given region or nucleus. Uniform labelling was found in the hypothalamus, thalamus, basal ganglia, cerebellum and brainstem. Non-uniform labelling that resulted from the presence of cells displaying two easily distinguishable intensities of hybridization signals was observed in several regions and nuclei in the forebrain (cerebral cortex, hippocampus, amygdala, certain olfactory structures). Olfactory-associated structures, basal ganglia, hippocampus, and cerebellar cortex displayed the heaviest amounts of labelling. Many regions that displayed cannabinoid receptor mRNA could reasonably be identified as sources for cannabinoid receptors on the basis of well documented hodologic data. Other sites that were also clearly labelled could not be assigned as logical sources of cannabinoid receptors. The localization of cannabinoid receptor mRNA indicates that sensory, motor, cognitive, limbic, and autonomic systems should all be influenced by the activation of this receptor by either exogenous cannabimimetics, including marijuana, or the yet unknown endogenous "cannabinoid" ligand.
Mast cells play a key role in inflammatory reactions triggered by tissue injury or immune perturbations. Little is known about endogenous molecules and mechanisms capable of modulating inappropriate mast cell activity. N-(2-Hydroxyethyl)hexadecanamide (palmitoylethanolamide), found in peripheral tissues, has been proposed to act as a local autacoid capable of negatively regulating mast cell activation and inflammation-hence the acronym Autacoid Local Inflammation Antagonism (ALIA). Recently, N-(2-hydroxyethyl)hexadecanamide (LG 2110/1) has been reported to down-modulate mast cell activation in vitro by behaving as an agonist at the peripheral cannabinoid CB2 receptor. Here, we have characterized and functionally correlated the anti-inflammatory actions of LG 2110/1 with its ability to control mast cell activation, when given orally in a battery of rodent models of inflammation. LG 2110/1 diminished, in a dose-dependent and correllated manner, the number of degranulated mast cells and plasma extravasation induced by substance P injection in the mouse ear pinna. In addition, LG 2110/1 reduced dose dependently plasma extravasation induced by passive cutaneous anaphylaxis reaction. In adult rats LG 2110/1 decreased, in a dose-dependent manner, carrageenan-induced hindpaw edema and hyperalgesia, but not phospholipase A2-induced hindpaw edema. Further, anti-edema effects were observed when utilizing dextran and formalin, known to also cause mast cell activation. Locally administered LG 2110/1 was likewise effective in minimizing dextran-induced hind paw edema. In contrast, equivalent amounts of palmitic acid plus ethanolamine were ineffective against plasma extravasation provoked by substance P. LG 2110/1 did not decrease plasma extravasation induced by the substance P fragment, substance P-(6-11), known to be inactive on mast cells. These results indicate that orally administered N-(2-hydroxyethyl)hexadecanamide is effective in: (a) directly down-modulating mast cell activation in vivo; (b) suppressing pathological consequences initiated by mast cell activation independently of the activating stimuli; (c) exerting an anti-inflammatory action distinguishable from that of classical steroidal and non-steroidal anti-inflammatory agents. These findings raise the possibility that N-(2-hydroxyethyl)hexadecanamide and related saturated N-acylamides ('ALIAmides') represent novel therapeutic agents useful in the management of inflammatory disease conditions.
To investigate whether tachykinins are released in the airways in response to stimulation of the esophagus, we studied the airway plasma extravasation induced by intraesophageal HCl in the presence or absence of neutral endopeptidase inhibitor phosphoramidon and NK1-receptor antagonist FK-888 in anesthetized guinea pigs. The airway plasma leakage was evaluated by measuring extravasated Evans blue dye in the animals pretreated with propranolol and atropine. Infusion of 1 N HCl into the esophagus significantly increased plasma extravasation in the trachea. Phosphoramidon significantly potentiated plasma extravasation in the trachea and main bronchi, whereas FK-888 significantly inhibited that extravasation in a dose-related manner. In the capsaicin-treated animals, airway plasma extravasation was completely inhibited even in the presence of phosphoramidon. Tracheal plasma extravasation potentiated by phosphoramidon was significantly inhibited in the bilateral vagotomized animals. These results suggest that 1) tachykinin-like substances are released to cause plasma extravasation in the airways as a result of intraesophageal HCl stimulation and 2) there are neural pathways communicating between the esophagus and airways, including the vagus nerve.
The effects of arachidonic acid ethanolamide (anandamide), palmitoylethanolamide and delta9-tetrahydrocannabinol on the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-4, interleukin-6, interleukin-8, interleukin-10, interferon-gamma, p55 and p75 TNF-alpha soluble receptors by stimulated human peripheral blood mononuclear cells as well as [3H]arachidonic acid release by non-stimulated and N-formyl-Met-Leu-Phe (fMLP)-stimulated human monocytes were investigated. Anandamide was shown to diminish interleukin-6 and interleukin-8 production at low nanomolar concentrations (3-30 nM) but inhibited the production of TNF-alpha, interferon-gamma, interleukin-4 and p75 TNF-alpha soluble receptors at higher concentrations (0.3-3 microM). Palmitoylethanolamide inhibited interleukin-4, interleukin-6, interleukin-8 synthesis and the production of p75 TNF-alpha soluble receptors at concentrations similar to those of anandamide but failed to influence TNF-alpha and interferon-gamma production. The effect of both compounds on interleukin-6 and interleukin-8 production disappeared with an increase in the concentration used. Neither anandamide nor palmitoylethanolamide influenced interleukin-10 synthesis. delta9-Tetrahydrocannabinol exerted a biphasic action on pro-inflammatory cytokine production. TNF-alpha, interleukin-6 and interleukin-8 synthesis was maximally inhibited by 3 nM delta9-tetrahydrocannabinol but stimulated by 3 microM delta9-tetrahydrocannabinol, as was interleukin-8 and interferon-gamma synthesis. The level of interleukin-4, interleukin-10 and p75 TNF-alpha soluble receptors was diminished by 3 microM delta9-tetrahydrocannabinol. [3H]Arachidonate release was stimulated only by high delta9-tetrahydrocannabinol and anandamide concentrations (30 microM). These results suggest that the inhibitory properties of anandamide, palmitoylethanolamide and delta9-tetrahydrocannabinol are determined by the activation of the peripheral-type cannabinoid receptors, and that various endogenous fatty acid ethanolamides may participate in the regulation of the immune response.
Dexanabinol, HU-211, a synthetic cannabinoid devoid of psychotropic effects, improves neurological outcome in models of brain trauma, ischemia and meningitis. Recently, HU-211 was found to inhibit brain tumor necrosis factor (TNFalpha) production after head injury. In the present study, we demonstrate the ability of HU-211 to suppress TNFalpha production and to rescue mice and rats from endotoxic shock after LPS (Escherichia coli 055:B5) inoculation. In BALB/c mice, a dose of 10 mg/kg LPS, injected i.p., caused 57% and 100% mortality, at 24 and 48 hr, respectively. HU-211, administered i.p. 30 min before lipopolysaccharide (LPS), reduced lethality to 9 and 67% at these time points (P < .05). When coinjected with D-galactoseamine (i.p.), LPS was 100% lethal within 24 hr, whereas eight hourly injections of HU-211 caused mortality of C57BL/6 mice to drop to 10% (P < .001). Administration of LPS to Sprague-Dawley rats resulted in a 30% reduction in the mean arterial blood pressure within 30 min, which persisted for 3 hr. HU-211, given 2 to 3 min before LPS, completely abolished the typical hypotensive response. Furthermore, the drug also markedly suppressed in vitro TNFalpha production and nitric oxide generation (by >90%) by both murine peritoneal macrophages and rat alveolar macrophage cell line exposed to LPS. HU-211 may, therefore, have therapeutic implications in the treatment of TNFalpha-mediated pathologies.
We investigated the effect of the cannabinoid agonist (+)WIN-55212-2 on human ileum longitudinal smooth muscle preparations, either electrically stimulated or contracted by carbachol. Electrical field stimulation mostly activated cholinergic neurons, since atropine and tetrodotoxin (TTX), alone or co-incubated, reduced twitch responses to a similar degree (85%). (+)WIN-55212-2 concentration-dependently inhibited twitch responses (IC50 73 nm), but had no additive effect with atropine or TTX. The cannabinoid CB1 receptor antagonist SR 141716 (pA2 8.2), but not the CB2 receptor antagonist, SR 144528, competitively antagonized twitch inhibition by (+)WIN-55212-2. Atropine but not (+)WIN-55212-2 or TTX prevented carbachol-induced tonic contraction. These results provide functional evidence of the existence of prejunctional cannabinoid CB1-receptors in the human ileum longitudinal smooth muscle. Agonist activation of these receptors prevents responses to electrical field stimulation, presumably by inhibiting acetylcholine release. SR 141716 is a potent and competitive antagonist of cannabinoid CB1 receptors naturally expressed in the human gut.
British Journal of Pharmacology (1998) 125, 1393–1395; doi:10.1038/sj.bjp.0702190
The synthesis and pharmacology of 15 1-deoxy-delta8-THC analogues, several of which have high affinity for the CB2 receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-delta8-THC (5), 1-deoxy-delta8-THC (6), 1-deoxy-3-butyl-delta8-THC (7), 1-deoxy-3-hexyl-delta8-THC (8) and a series of 3-(1',1'-dimethylalkyl)-1-deoxy-delta8-THC analogues (2, n = 0-4, 6, 7, where n = the number of carbon atoms in the side chain-2). Three derivatives (17-19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB1 and CB2 receptors were determined employing previously described procedures. Five of the 3-(1',1'-dimethylalkyl)-1-deoxy-delta8-THC analogues (2, n = 1-5) have high affinity (Ki = < 20 nM) for the CB2 receptor. Four of them (2, n = 1-4) also have little affinity for the CB1 receptor (Ki = > 295 nM). 3-(1',1'-Dimethylbutyl)-1-deoxy-delta8-THC (2, n = 2) has very high affinity for the CB2 receptor (Ki = 3.4 +/- 1.0 nM) and little affinity for the CB1 receptor (Ki = 677 +/- 132 nM).
Smoking marijuana or administration of its main active constituent, delta9-tetrahydrocannabinol (delta9-THC), may exert potent dilating effects on human airways. But the physiological significance of this observation and its potential therapeutic value are obscured by the fact that some asthmatic patients respond to these compounds with a paradoxical bronchospasm. The mechanisms underlying these contrasting responses remain unresolved. Here we show that the endogenous cannabinoid anandamide exerts dual effects on bronchial responsiveness in rodents: it strongly inhibits bronchospasm and cough evoked by the chemical irritant, capsaicin, but causes bronchospasm when the constricting tone exerted by the vagus nerve is removed. Both effects are mediated through peripheral CB1 cannabinoid receptors found on axon terminals of airway nerves. Biochemical analyses indicate that anandamide is synthesized in lung tissue on calcium-ion stimulation, suggesting that locally generated anandamide participates in the intrinsic control of airway responsiveness. In support of this conclusion, the CB1 antagonist SR141716A enhances capsaicin-evoked bronchospasm and cough. Our results may account for the contrasting bronchial actions of cannabis-like drugs in humans, and provide a framework for the development of more selective cannabinoid-based agents for the treatment of respiratory pathologies.
Cannabinoid research underwent a tremendous increase during the last 10 years. This progress was made possible by the discovery of cannabinoid receptors and the endogenous ligands for these receptors. Cannabinoid research is developing in two major directions: neurobehavioral properties of cannabinoids and the impact of cannabinoids on the immune system. Recent studies characterized the cannabinoid-induced response as a very complex process because of the involvement of multiple signalling pathways linked to cannabinoid receptors or effects elicited by cannabinoids without receptor participation. The objective of this review is to present this complexity as it applies to immune response. The functional properties of cannabinoid receptors, signalling pathways linked to cannabinoid receptors and the modulation of immune response by cannabinoid receptor ligands are discussed. Special attention is given to 'endocannabinoids' as immunomodulatory molecules.
The endogenous cannabinoid agonist, anandamide produced a modest contractile response in guinea-pig isolated bronchus compared with the vanilloid receptor agonist capsaicin. The contractile response to both anandamide and capsaicin was inhibited by the vanilloid receptor antagonist, capsazepine. Furthermore, the NK2-selective antagonist, SR48968 but not the NK1-selective antagonist, SR140333 inhibited contractile responses to anandamide.
The contractile response to anandamide was abolished in tissues desensitized by capsaicin. However, anandamide failed to cross-desensitize the contractile response to capsaicin.
The contractile response to anandamide was not significantly altered in the presence of the CB1 receptor antagonist, SR141716A, nor the amidase inhibitor, phenylmethylsulphonyl fluoride (PMSF) but was significantly increased in the presence of the neutral endopeptidase inhibitor, thiorphan.
The cannabinoid agonist, CP55,940 failed to significantly attenuate the excitatory non-adrenergic non-cholinergic (eNANC) response in guinea-pig airways. In contrast, the ORL1 receptor agonist, nociceptin, significantly inhibited this response.
The results demonstrate that anandamide induces a modest contractile response in guinea-pig isolated bronchus that is dependent upon the activation of vanilloid receptors on airway sensory nerves. However, cannabinoid receptors do not appear to play a role in this regard, nor in regulating the release of neuropeptides from airway sensory nerves under physiological conditions.
British Journal of Pharmacology (2001) 132, 1127–1135; doi:10.1038/sj.bjp.0703906
Nerve growth factor induces an airway hyperresponsiveness in vivo in guinea-pigs, as we have shown previously. Since antagonizing the neurokinin-1 (NK1) receptor can prevent this NGF-induced airway hyperresponsiveness and since sensory nerves release tachykinins, we investigated the role of sensory nerves in the NGF-induced airway hyperresponsiveness.
We used isolated tracheal rings from guinea-pigs to measure tracheal contractility. In these rings sensory nerve endings are present, but these endings lack any contact with their cell bodies.
In this in vitro system, NGF dose-dependently induced a tracheal hyperresponsiveness to histamine. The NK1 receptor antagonist SR140333 could block the induction of tracheal hyperresponsiveness.
To further investigate the involvement of sensory nerve endings we used the cannabinoid receptor 1 (CB1) agonist R-methanandamide to inhibit excitatory events at the nerve terminal. The CB1 receptor agonist was capable of blocking the tracheal hyperresponsiveness to NGF in the isolated system, as well as the airway hyperresponsiveness to NGF in vivo.
This indicates that NGF can induce an increase in airway responsiveness in the absence of sensory nerve cell bodies. NGF may act by increasing substance P release from sensory nerve endings, without upregulation of substance P in the neurons. Substance P in its turn is responsible for the induction of the NGF-induced airway hyperresponsiveness.
British Journal of Pharmacology (2001) 134, 771–776; doi:10.1038/sj.bjp.0704310
In asthma patients, microaspiration of acid into the lower airways (ie, airway acidification) causes such respiratory responses as cough and bronchoconstriction. The mechanism of bronchoconstriction induced by airway acidification is unknown, although evidence is emerging that increasing proton concentrations in airway tissues can activate a subpopulation of primary sensory neurons, so-called capsaicin-sensitive primary sensory neurons, that contain such neuropeptides as the tachykinins substance P (SP) and neurokinin A (NKA). Protons activate a capsaicin-operated channel/receptor, located in the afferents of capsaicin-sensitive neurons, with the subsequent opening of ion channels that are permeable to sodium, potassium, and calcium ions. This event initiates a propagated action potential that antidromically depolarizes collateral fibers and triggers neuropeptide release from nerve fiber varicosities. The tachykinins SP and NKA, released from terminals of primary sensory neurons in peripheral tissues, cause all the major signs of inflammation (neurogenic inflammation) by means of activation of NK(1) and NK(2) receptors. Exposure of the airways to acidic solutions stimulates sensory nerve endings of capsaicin-sensitive sensory neurons and causes different airway responses, including bronchoconstriction. Recently, the NK(2), and to a lesser extent the NK(1), receptors have been shown to be involved with citric acid-induced bronchoconstriction in the guinea pig, which is in part mediated by endogenously released bradykinin. Tachykinins and bradykinin, released by airway acidification, could also modulate citric acid-induced bronchoconstriction by their ability to subsequently release the epithelially derived bronchoprotective nitric oxide (NO). Further study with selective tachykinin NK(1) and NK(2) agonists demonstrated that only the septide-insensitive tachykinin NK(1) receptor releases NO. Thus, bronchoconstriction induced by citric acid inhalation in the guinea pig, mainly caused by the tachykinin NK(2) receptor, is counteracted by bronchoprotective NO after activation of bradykinin B(2) and tachykinin NK(1) receptors in airway epithelium. If a similar mechanism is involved in the pathogenesis of bronchial asthma associated with gastroesophageal reflux in the respiratory tract, new therapeutic strategies should be investigated.
The antiinflammatory activity of synthetic cannabinoid nabilone in the rat model of carrageenan-induced acute hindpaw inflammation was compared with that of the endocannabinoid palmitoylethanolamide and the nonsteroidal antiinflammatory drug indomethacin.
Preliminary experiments in rats used a tetrad of behavioural tests, specific for tetrahydrocannabinol-type activity in the CNS. These showed that the oral dose of nabilone 2.5 mg kg−1 had no cannabinoid psychoactivity.
Intraplantar injection of carrageenan (1% w v−1) elicited a time-dependent increase in paw volume and thermal hyperalgesia.
Nabilone (0.75, 1.5, 2.5 mg kg−1, p.o.), given 1 h before carrageenan, reduced the development of oedema and the associated hyperalgesia in a dose-related manner. Nabilone 2.5 mg kg−1, palmitoylethanolamide 10 mg kg−1 and indomethacin 5 mg kg−1, given p.o. 1 h before carrageenan, also reduced the inflammatory parameters in a time-dependent manner.
The selective CB2 cannabinoid receptor antagonist {N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3 carboxamide} (SR 144528), 3 mg kg−1 p.o. 1 h before nabilone and palmitoylethanolamide, prevented the anti-oedema and antihyperalgesic effects of the two cannabinoid agonists 3 h after carrageenan.
Our findings show the antiinflammatory effect of nabilone and confirm that of palmitoylethanolamide indicating that these actions are mediated by an uncharacterized CB2-like cannabinoid receptor.
British Journal of Pharmacology (2002) 135, 181–187; doi:10.1038/sj.bjp.0704466
Cannabinoids are known to downregulate immune response but the role for cannabinoid receptors in cannabinoid-induced immunosuppression is still unclear. To address this question, the interference of CB1 and CB2 receptor antagonists with the inhibition of TNF-alpha production by synthetic cannabinoid WIN 55,212-2 was studied using human peripheral blood mononuclear cells (PBMC) in vitro. CB2 (SR 144528) but not CB1 (SR 141716A) receptor antagonist dose dependently interfered with WIN 55,212-2-induced inhibition of TNF-alpha synthesis. Also, WIN 55,212-2 decreased fMLP-induced reactive oxygen species generation in lipopolysaccharide (LPS)-primed PBMC. However, the high concentrations of cannabinoid receptor ligands needed to achieve significant effects suggest that the observed effects may be in part cannabinoid receptor independent.
There is considerable interest in elucidating potential endogenously derived agonists of the vanilloid receptor and the role of anandamide in this regard has received considerable attention. In the present study, we have used an electrophysiological technique to investigate the mechanism of activation of vanilloid receptors in an isolated vagal preparation.
Both capsaicin and anandamide depolarized de-sheathed whole vagal nerve preparations that was antagonized by the VR1 antagonist, capsazepine (P<0.05) whilst this response was unaltered by the cannabinoid (CB1) selective antagonist SR141716A or the CB2 selective antagonist, SR144528, thereby ruling out a role for cannabinoid receptors in this response.
The PKC activator, phorbol-12-myristate-13-acetate (PMA) augmented depolarization to both anandamide and capsaicin and this response was significantly inhibited with the PKC inhibitor, bisindolylmaleimide (BIM) (P<0.05).
The role of lipoxygenase products in the depolarization to anandamide was investigated in the presence of the lipoxygenase inhibitor, 5,8,11-Eicosatriynoic acid (ETI). Depolarization to anandamide and arachidonic acid was significantly inhibited in the presence of ET1 (P<0.05). However, in the absence of calcium depolarization to anandamide was not inhibited by ETI.
Using confocal microscopy we have demonstrated the presence of vanilloid receptors on both neuropeptide containing nerves and nerves that did not stain for sensory neuropeptides.
These results demonstrate that anandamide evokes depolarization of guinea-pig vagus nerve, following activation of vanilloid receptors, a component of which involves the generation of lipoxygenase products. Furthermore, these receptors are distributed in both neuropeptide and non-neuropeptide containing nerves.
British Journal of Pharmacology (2002) 137, 39–48. doi:10.1038/sj.bjp.0704840
Because the CB1 receptor antagonist SR141716 was previously reported to modulate food intake in rodents, we studied its efficacy in reducing obesity in a diet-induced obesity (DIO) model widely used for research on the human obesity syndrome. During a 5-wk treatment, SR141716 (10 mg. kg(-1). day(-1) orally) induced a transient reduction of food intake (-48% on week 1) and a marked but sustained reduction of body weight (-20%) and adiposity (-50%) of DIO mice. Furthermore, SR141716 corrected the insulin resistance and lowered plasma leptin, insulin, and free fatty acid levels. Most of these effects were present, but less pronounced at 3 mg. kg(-1). day(-1). In addition to its hypophagic action, SR141716 may influence metabolic processes as the body weight loss of SR141716-treated mice was significantly higher during 24-h fasting compared with vehicle-treated animals, and when a 3-day treatment was compared with a pair feeding. SR141716 had no effect in CB1 receptor knockout mice, which confirmed the implication of CB1 receptors in the activity of the compound. These findings suggest that SR141716 has a potential as a novel anti-obesity treatment.
Gastroesophageal acid reflux (GER) is a common disorder associated with the exacerbation of asthma. In this study we investigated the effects on the airways of intraoesophageal HCl instillation in the rabbit and the role of tachykinins in these effects. In anaesthetized New Zealand rabbits bronchopulmonary functions [total lung resistance (R(L)) and dynamic compliance (C(dyn))] were calculated before and after HCl intraoesophageal instillation. Infusion of HCl induced a significant bronchoconstriction (P < 0.05) in the terms of R(L) and C(dyn) changes, that were increased by phosphoramidon pre-treatment and reduced by capsaicin pre-treatment. Moreover, a pre-treatment with SR 48968, a tachykinin NK2 receptor antagonist, or SR 140333, a NK1 receptor antagonist, significantly inhibited the bronchoconstriction induced by intraoesophageal HCl infusion in terms of R(L) and C(dyn)changes. Finally, the HCl induced bronchoconstriction was unaffected by SR 142801, a tachykinin NK3 receptor antagonist. In conclusion these results suggest that bronchoconstriction induced by intraoesophageal HCl infusion is mainly dependent on the release of tachykinins and that both NK1 and NK2 tachykinin receptors are involved.
The effects of cannabinoid drugs on the cholinergic response evoked by electrical field stimulation (0.2 ms pulse width, 20 V amplitude, 10 Hz, 7.5 s train duration) in guinea-pig tracheal preparations were investigated. The stable analogue of the endocannabinoid anandamide, R(+)-methanandamide (10(-7)-10(-4) M), produced a dose-dependent inhibition (up to 27+/-5% of control) of electrical field stimulation-mediated atropine-sensitive response. This effect was not blocked by the selective cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3 carboxamide hydrochloride (SR 141716A; 10(-6) M), and was not reproduced with the cannabinoid CB(1)/CB(2) receptor agonist R(+)-[2,3-dihydro-5-methyl-[(morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl)methanone mesylate) (WIN 55,212-2; 10(-8)-10(-5) M) or the cannabinoid CB(2) receptor selective agonist 1-propyl-2-methyl-3-(1-naphthoyl)indole (JWH-015; 10(-8)-10(-5) M); it was, on the contrary, antagonized by the vanilloid antagonist 2-[2-(4-chlorophenyl)ethyl-amino-thiocarbonyl]-7,8-dihydroxy-2,3,4,5-tetrahydro-1H-2 benzazepine (capsazepine; 10(-6) M). At the postjunctional level, neither R(+)-methanandamide nor WIN 55,212-2 nor JWH-015 did affect tracheal contractions induced by exogenous acetylcholine (10(-6) M). An inhibitory vanilloid receptor-mediated effect on the cholinergic response evoked by electrical stimulation was confirmed with the vanilloid agonist capsaicin, at doses (3-6 x 10(-8) M) which poorly influenced the basal smooth muscle tone of trachea. In conclusion, our data indicate that in guinea-pig trachea (a) neither CB(1) nor CB(2) cannabinoid receptor-mediated modulation of acetylcholine release occurs; (b) vanilloid VR1-like receptors appear involved in R(+)-methanandamide inhibitory activity on the cholinergic response to electrical field stimulation.
Cannabinoids are cell membrane-derived signalling molecules that are released from nerves, blood cells and endothelial cells, and have diverse biological effects. They act at two distinct types of G-protein-coupled receptors, cannabinoid CB(1) and CB(2) receptors. Cannabinoid CB(1) receptors are highly localised in the central nervous system and are also found in some peripheral tissues, and cannabinoid CB(2) receptors are found outside the central nervous system, in particular in association with immune tissues. Novel actions of cannabinoids at non-CB(1) non-CB(2) cannabinoid-like receptors and vanilloid VR1 receptors have also recently been described. There is growing evidence that, among other roles, cannabinoids can act at prejunctional sites to modulate peripheral autonomic and sensory neurotransmission, and the present article is aimed at providing an overview of this. Inhibitory cannabinoid CB(1) receptors are expressed on the peripheral terminals of autonomic and sensory nerves. The role of cannabinoid receptor ligands in modulation of sensory neurotransmission is complex, as certain of these (anandamide, an "endocannabinoid", and N-arachidonoyl-dopamine, an "endovanilloid") also activate vanilloid VR1 receptors (coexpressed with cannabinoid CB(1) receptors), which excites sensory nerves and causes a release of sensory neurotransmitter. The fact that the activities of anandamide and N-arachidonoyl-dopamine span two distinct receptor families raises important questions about cannabinoid/vanilloid nomenclature, and as both compounds are structurally related to the archetypal vanilloid capsaicin, all three are arguably members of the same family of signalling molecules. Anandamide is released from nerves, but unlike classical neurotransmitters, it is not stored in and released from nerve vesicles, but is released on demand from the nerve cell membrane. In the central nervous system, cannabinoids function as retrograde signalling molecules, inhibiting via presynaptic cannabinoid CB(1) receptors the release of classical transmitter following release from the postsynaptic cell. At the neuroeffector junction, it is more likely that cannabinoids are released from prejunctional sites, as the neuroeffector junction is wide in some peripheral tissues and cannabinoids are rapidly taken up and inactivated. Understanding the actions of cannabinoids as modulators of peripheral neurotransmission is relevant to a variety of biological systems and possibly their disorders.
There is considerable interest in novel therapies for cough, since currently used agents such as codeine have limited beneficial value due to the associated side effects. Sensory nerves in the airways mediate the cough reflex via activation of C-fibres and RARs. Evidence suggests that cannabinoids may inhibit sensory nerve-mediated responses.
We have investigated the inhibitory actions of cannabinoids on sensory nerve depolarisation mediated by capsaicin, hypertonic saline and PGE2 on isolated guinea-pig and human vagus nerve preparations, and the cough reflex in conscious guinea-pigs.
The non-selective cannabinoid (CB) receptor agonist, CP 55940, and the selective CB2 agonist, JWH 133 inhibited sensory nerve depolarisations of the guinea-pig vagus nerve induced by hypertonic saline, capsaicin and PGE2. These responses were abolished by the CB2 receptor antagonist SR144528, and unaffected by the CB1 antagonist SR141716A. Similarly, JWH 133 inhibited capsaicin-evoked nerve depolarisations in the human vagus nerve, and was prevented by SR144528.
Using a guinea-pig in vivo model of cough, JWH 133 (10 mg kg−1, i.p., 20 min) significantly reduced citric acid-induced cough in conscious guinea pigs compared to those treated with the vehicle control.
These data show that activation of the CB2 receptor subtype inhibits sensory nerve activation of guinea-pig and human vagus nerve, and the cough reflex in guinea-pigs, suggesting that the development of CB2 agonists, devoid of CB1-mediated central effects, will provide a new and safe antitussive treatment for chronic cough.
British Journal of Pharmacology (2003) 140, 261–268. doi:10.1038/sj.bjp.0705435
Growing evidence suggests that a major physiological function of the cannabinoid signaling system is to modulate neuroinflammation. This review discusses the anti-inflammatory properties of cannabinoid compounds at molecular, cellular and whole animal levels, first by examining the evidence for anti-inflammatory effects of cannabinoids obtained using in vivo animal models of clinical neuroinflammatory conditions, specifically rodent models of multiple sclerosis, and second by describing the endogenous cannabinoid (endocannabinoid) system components in immune cells. Our aim is to identify immune functions modulated by cannabinoids that could account for their anti-inflammatory effects in these animal models.
British Journal of Pharmacology (2004) 141, 775–785. doi:10.1038/sj.bjp.0705667
The antigen-induced release of histamine from sensitized guinea pig mast cells was dose-dependently reduced by endogenous (2-arachidonylglycerol; 2AG) and exogenous [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP55,940)] cannabinoids. The inhibitory action afforded by 2AG and CP55,940 was reversed by N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide (SR144528), a selective cannabinoid 2 (CB(2)) receptor antagonist, and left unchanged by the selective CB(1) antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251). The inhibitory action of 2AG and CP55,940 was reduced by the unselective nitric-oxide synthase (NOS) inhibitor N-monomethyl-L-arginine methylester (l-NAME) and reinstated by L-arginine, the physiological substrate. The inhibitory action of 2AG and CP55,940 was also reduced by the unselective cyclooxygenase (COX) inhibitor indomethacin and the selective COX-2 blocker rofecoxib. Both 2AG and CP55,940 significantly increased the production of nitrite from mast cells, which was abrogated by L-NAME and N-(3-(aminomethyl)benzyl)acetamidine (1400W), a selective inducible NOS (iNOS) inhibitor. Nitrite production consistently paralleled a CP55,940-induced increase in the expression of iNOS protein in mast cells. Both 2AG and CP55,940 increased the generation of prostaglandin E(2) from mast cells, which was abrogated by indomethacin and rofecoxib and parallel to the CP55,940-induced expression of COX-2 protein. Mast cell challenge with antigen was accompanied by a net increase in intracellular calcium levels. Both cannabinoid receptor ligands decreased the intracellular calcium levels, which were reversed by SR144528 and l-NAME. In unstimulated mast cells, both ligands increased cGMP levels. The increase was abrogated by SR144528, l-NAME, indomethacin, and rofecoxib. Our results suggest that 2AG and CP55,940 decreased mast cell activation in a manner that is susceptible to a CB(2) receptor antagonist and to inhibition of nitric oxide and prostanoid pathways.
The biological effects of cannabinoids (CB) are mediated by CB1 and CB2 receptors. The role of CB2 receptors in the gastrointestinal tract is uncertain. In this study, we examined whether CB2 receptor activation is involved in the regulation of gastrointestinal transit in rats.
Basal and lipopolysaccharide (LPS)-stimulated gastrointestinal transit was measured after instillation of an Evans blue-gum Arabic suspension into the stomach, in the presence of specific CB1 and CB2 agonists and antagonists, or after treatment with inhibitors of mediators implicated in the transit process.
In control rats a CB1 (ACEA; 1 mg kg−1), but not a CB2 (JWH-133; 1 mg kg−1), receptor agonist inhibited basal gastrointestinal transit. The effects of the CB1 agonist were reversed by the CB1 antagonist AM-251, which alone increased basal transit. LPS treatment increased gastrointestinal transit. This increased transit was reduced to control values by the CB2, but not the CB1, agonist. This inhibition by the CB2 agonist was dose dependent and prevented by a selective CB2 antagonist (AM-630; 1 mg kg−1).
By evaluating the inhibition of LPS-enhanced gastrointestinal transit by different antagonists, the effects of the CB2 agonist (JWH-133; 1 mg kg−1) were found to act via cyclooxygenase, and to act independently of inducible nitric oxide synthase (NOS) and platelet-activating factor. Interleukin-1β and constitutive NOS isoforms may be involved in the accelerated LPS transit.
The activation of CB2 receptors in response to LPS is a mechanism for the re-establishment of normal gastrointestinal transit after an inflammatory stimulus.
British Journal of Pharmacology (2004) 142, 1247–1254. doi:10.1038/sj.bjp.0705889
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 Ca(2+)-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.
Effects of the CB2-selective cannabinoid agonist AM1241 on activity evoked in spinal wide dynamic range (WDR) neurons by transcutaneous electrical stimulation were evaluated in urethane-anesthetized rats. Recordings were obtained in both the absence and the presence of carrageenan inflammation. AM1241, administered intravenously or locally in the paw, suppressed activity evoked by transcutaneous electrical stimulation during the development of inflammation. Decreases in WDR responses resulted from a suppression of C-fiber-mediated activity and windup. Abeta- and Adelta-fiber-mediated responses were not reliably altered. The AM1241-induced suppression of electrically evoked responses was blocked by the CB2 antagonist SR144528 but not by the CB1 antagonist SR141716A. AM1241 (33 microg/kg intraplantar [i.p.l.]), administered to the carrageenan-injected paw, suppressed activity evoked in WDR neurons relative to groups receiving vehicle in the same paw or AM1241 in the opposite (noninflamed) paw. The electrophysiological effects of AM1241 (330 microg/kg intravenous [i.v.]) were greater in rats receiving i.p.l. carrageenan compared with noninflamed rats receiving an i.p.l. injection of vehicle. AM1241 failed to alter the activity of purely nonnociceptive neurons recorded in the lumbar dorsal horn. Additionally, AM1241 (330 microg/kg i.v. and i.p.l.; 33 microg/kg i.p.l.) reduced the diameter of the carrageenan-injected paw. The AM1241-induced decrease in peripheral edema was blocked by the CB2 but not by the CB1 antagonist. These data demonstrate that activation of cannabinoid CB2 receptors is sufficient to suppress neuronal activity at central levels of processing in the spinal dorsal horn. Our findings are consistent with the ability of AM1241 to normalize nociceptive thresholds and produce antinociception in inflammatory pain states.
Peripheral cannabinoid 2 receptors (CB2 receptors) modulate immune responses and attenuate nociceptive behaviour in models of acute and persistent pain. The aim of the present study was to investigate whether peripheral CB2 receptors modulate spinal processing of innocuous and noxious responses and to determine whether there are altered roles of CB2 receptors in models of persistent pain. Effects of local administration of the CB2 receptor agonist JWH-133 (5 and 15 microg/50 microL) on mechanically evoked responses of spinal wide dynamic range (WDR) neurons in noninflamed rats, rats with carrageenan-induced hindpaw inflammation, sham operated rats and spinal nerve-ligated (SNL) rats were determined in anaesthetized rats in vivo. Mechanical stimulation (von Frey filaments, 6-80 g) of the peripheral receptive field evoked firing of WDR neurons. Mechanically evoked responses of WDR neurons were similar in noninflamed, carrageenan-inflamed, sham-operated and SNL rats. Intraplantar injection of JWH-133 (15 microg), but not vehicle, significantly (P < 0.05) inhibited innocuous and noxious mechanically evoked responses of WDR neurons in all four groups of rats. In many cases the selective CB2 receptor antagonist, SR144528 (10 microg/50 microL), attenuated the inhibitory effects of JWH-133 (15 microg) on mechanically evoked WDR neuronal responses. The CB1 receptor antagonist, SR141716A, did not attenuate the inhibitory effects of JWH-133 on these responses. Intraplantar preadministration of JWH-133 also inhibited (P < 0.05) carrageenan-induced expansion of peripheral receptive fields of WDR dorsal horn neurons. This study demonstrates that activation of peripheral CB2 receptors attenuates both innocuous- and noxious-evoked responses of WDR neurons in models of acute, inflammatory and neuropathic pain.
1. Nociceptin/orphanin FQ (N/OFQ) is the endogenous peptide ligand for a specific G-protein coupled receptor, the N/OFQ peptide receptor (NOP). The N/OFQ-NOP receptor system has been reported to play an important role in pain, anxiety and appetite regulation. In airways, N/OFQ was found to inhibit the release of tachykinins and the bronchoconstriction and cough provoked by capsaicin. 2. Here we evaluated the effects of NOP receptor activation in bronchoconstriction and airway microvascular leakage induced by intraesophageal (i.oe.) hydrochloric acid (HCl) instillation in rabbits. We also tested the effects of NOP receptor activation in SP-induced plasma extravasation and bronchoconstriction. 3. In anesthetized New Zealand rabbits bronchopulmonary function (total lung resistance (R(L)) and dynamic compliance (C(dyn))) and airway microvascular leakage (extravasation of Evans blue dye) were evaluated. 4. Infusion of i.oe. HCl (1 N) led to a significant increase in bronchoconstriction and plasma extravasation in the main bronchi and trachea of rabbits pretreated with propranolol, atropine and phosphoramidon. 5. Bronchoconstriction and airway microvascular leakage were inhibited by N/OFQ (3-30 microg kg(-1) i.v.) in a dose-dependent manner. The NOP receptor agonist [Arg14,Lys15]N/OFQ mimicked the inhibitory effect of N/OFQ, being 10-fold more potent, UFP-101, a peptide selective NOP receptor antagonist, blocked the inhibitory effects of both agonists. 6. Under the same experimental conditions, N/OFQ and [Arg14,Lys15]N/OFQ did not counteract the bronchoconstriction and airway microvascular leakage induced by substance P. 7. These results suggest that bronchoconstriction and airway plasma extravasation induced by i.oe. HCl instillation are inhibited by activation of prejunctional NOP receptors.
To date, two cannabinoid receptors have been identified, CB1 and CB2. Activation of these receptors with non-selective cannabinoid receptor agonists reduces pain sensitivity in animals and humans. However, activation of CB1 receptors is also associated with central side effects, including ataxia and catalepsy. More recently, a role for selective CB2 agonists in pain modification has been demonstrated. GW405833, a selective CB2 agonist, was recently reported to partially reverse the inflammation and hyperalgesia in a rat model of acute inflammation. In the current report, we extend the characterization and therapeutic potential of this compound. For the first time, we show that GW405833 selectively binds both rat and human CB2 receptors with high affinity, where it acts as a partial agonist (approximately 50% reduction of forskolin-mediated cAMP production compared to the full cannabinoid agonist, CP55,940). We also report for the first time that intraperitoneal administration of GW405833 (0.3-100 mg/kg) to rats shows linear, dose-dependent increases in plasma levels and substantial penetration into the central nervous system. In addition, GW405833 (up to 30 mg/kg) elicits potent and efficacious antihyperalgesic effects in rodent models of neuropathic, incisional and chronic inflammatory pain, the first description of this compound in these models. In contrast, analgesia, sedation and catalepsy were not observed in this dose range, but were apparent at 100 mg/kg. Additionally, GW405833 was not antihyperalgesic against chronic inflammatory pain in CB2 knockout mice. These data support the tenet that selective CB2 receptor agonists have the potential to treat pain without eliciting the centrally-mediated side effects associated with non-selective cannabinoid agonists, and highlight the utility of GW405833 for the investigation of CB2 physiology.
Migraine is a common and disabling neurological disorder that involves activation or the perception of activation of the trigeminovascular system. Cannabinoid (CB) receptors are present in brain and have been suggested to be antinociceptive. Here we determined the effect of cannabinoid receptor activation on neurons with trigeminovascular nociceptive input in the rat. Neurons in the trigeminocervical complex (TCC) were studied using extracellular electrophysiological techniques. Responses to both dural electrical stimulation and cutaneous facial receptive field activation of the ophthalmic division of the trigeminal nerve and the effect of cannabinoid agonists and antagonists were studied. Nonselective CB receptor activation with R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2, 3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl) (WIN55,212; 1 mg kg(-1)) inhibited neuronal responses to A-(by 52%) and C-fiber (by 44%) afferents, an effect blocked by the CB(1) receptor antagonist SR141716 [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; 3 mg kg(-1)] but not the CB2 receptor antagonist AM630 (6-iodopravadoline; 3 mg kg(-1)). Anandamide (10 mg kg(-1)) was able to inhibit both A- and C-fiber-elicited TCC firing, only after transient receptor potential vanilloid 1 receptor inhibition. Activation of cannabinoid receptors had no effect on cutaneous receptive fields when recorded from TCC neurons. The data show that manipulation of CB1 receptors can affect the responses of trigeminal neurons with A- and C-fiber inputs from the dura mater. This may be a direct effect on neurons in the TCC itself or an effect in discrete areas of the brain that innervate these neurons. The data suggest that CB receptors may have therapeutic potential in migraine, cluster headache, or other primary headaches, although the potential hazards of psychoactive side effects that accompany cannabinoid treatments may be complex to overcome.
Gastro-oesophageal acid reflux may cause airway responses such as cough, bronchoconstriction and inflammation in asthmatic patients. Our previous results suggest that microvascular leakage induced, in the guinea-pig airways, by intra-oesophageal hydrochloric acid (HCl) infusion was mainly dependent on the release of tachykinins. Nociceptin, an endogenous ligand of the opioid receptor NOP, has been shown to inhibit bronchoconstriction and cough in guinea-pig or cat by inhibiting tachykinin release.