The phytocannabinoid cannabidiol (CBD) has a complex pharmacology which is thought to include, but is not limited to, an ability to act as an inverse agonist at the CB1 and CB2 receptors and an antagonist of GPR55. Moreover, is has been shown to reduce infarct size and ameliorate reductions in left ventricular function in vivo. These improvements in the pathogenesis of experimental MI are accompanied by a reduction in inflammatory cell migration to the area at risk. More recently it has been shown that CBD is anti-arrhythmic in acute experimental MI. Thus, it was suggested that the cardioprotective effects of CBD might be due to an anti-inflammatory action. In addition, GPR55 receptor activation is acknowledged to mediate mobilisation of intracellular Ca2+ (Ca2+i) which could potentially be pro-arrhythmic and so CBD, as an antagonist may confer cardioprotection via GPR55. However, the receptors and/or mechanisms responsible for mediating the cardioprotective effects of CBD are get to be determined. The present studies were therefore performed to; (1) better understand the pharmacology of CBD by assessing haemodynamic responses to CBD and other cannabinoids ligands in anaesthetised rats, (2) investigate the receptors involved in the anti-arrhythmic effect of CBD in a rat model of coronary artery occlusion (CAO), and (3) investigate if CBD can alter [Ca2+]i in isolated rat cardiomyocytes. The characterisation of the pharmacology of CBD in vivo showed that; firstly, CB1 receptor activation causes a hypotensive response which can be dose-dependently inhibited by AM251; secondly, both CBD and AM251 alone (a CB1 receptor antagonist and GPR55 agonist) can induce vasodepressor responses and finally, CBD can potentiate the AM251-mediated hypotension when co-administered, suggesting possible cross-talk between the CB1 and GPR55. Results from CAO studies showed that CBD and AM251 each have the capacity to reduce arrhythmias. Moreover, when CBD and AM251 were co-administered the anti-arrhythmic capacity of either alone was potentiated. However, the degree of potentiation was dependent on the order of administration, suggesting that more than one receptor is involved in the summative anti-arrhythmic effects. The investigation of cardiomyocyte [Ca2+]i suggested that AM251 can modulate [Ca2+]i at the level of the cardiomyocyte, while CBD cannot. These data give novel insight into the anti-arrhythmic effects of CBD and, moreover, for the first time demonstrate that AM251 is anti-arrhythmic. In addition, these data suggest a role for GPR55 in increasing [Ca2+]i via AM251.

A ativação do receptor CB1, leva a modulação de processos intracelulares que muda a resposta celular de acordo com o estímulo, além de estar envolvida em mecanismos de proliferação, diferenciação, movimentação e morte celular. O objetivo desse trabalho foi avaliar a participação desse sistema em processos oxidativo e inflamatório relacionados à neurodegeneração in vitro. Foi utilizado a linhagem de neuroblastoma Neuro2a diferenciada em células dopaminérgicas que foram expostas a três condições: com 6OHDA, H2O2 e LPS e co-tratadas com o agonista do receptor CB1 ACEA e o antagonista/agonista inverso AM251 por 24 horas. Utilizamos parâmetros funcionais de viabilidade celular, produção de espécies reativas de oxigênio e técnica de western blot. O tratamento com ACEA ou ACEA/AM251 produziram um aumento da viabilidade celular nos três modelos de exposição propostos; redução da produção de espécies reativas de oxigênio e ativação da via da proteína ERK1/2, além da inibição da morte celular pela diminuição da expressão da caspase 3. Concluímos que os canabinoides escolhidos foram capazes de proteger as células dopaminérgicas do dano oxidativo e inflamatório através do aumento da sobrevida celular por diminuição da produção de ROS. / The CB1 receptor activation leads to modulation of intracellular processes that change the cellular response according to the stimulus, as well as being involved in mechanisms of proliferation, differentiation, cell movement and death. The present study evaluated the participation of this system in oxidative and inflammatory processes related to neurodegeneration in vitro. We have used the Neuro2A neuroblastoma lineage, which those were differentiated into dopaminergic cells, and exposed to 6OHDA, H2O2 and LPS. They were co-treated with ACEA, CB1 receptor agonist, and AM251, the CB1 receptor antagonist/inverse agonist, for 24 hours. We used functional parameters of cell viability, production of reactive oxygen species and protein analyses by western blot. Treatment with ACEA or ACEA/AM251 produced an increase in cell viability; reduced production of reactive oxygen species and activation of the ERK1/2 protein, in addition to inhibition of cell death by decreasing the expression of caspase 3 in all three models proposed. We concluded that chosen cannabinoids were able to protect dopaminergic cells from oxidative damage and inflammation through the increased cell survival by decreasing the production of ROS.

A neurodegeneração é o resultado da destruição progressiva e irreversível dos neurônios no sistema nervoso central, apresentando causas desconhecidas e mecanismos patológicos não totalmente elucidados. Fatores como a idade, o aumento da formação de radicais livres e/ou estresse oxidativo, defeito no metabolismo energético, a inflamação e acúmulo de elementos neurotóxicos e de proteínas malformadas no lúmen do retículo endoplasmático (RE) contribuem para o desenvolvimento dos processos neurodegenerativos. O sistema canabinoide tem sido proposto como neuroprotetor em diversos modelos de neurodegeneração como hipóxia aguda e epilepsia, isquemia cerebral, lesão cerebral e modelos de estresse oxidativo. Assim, este trabalho teve como objetivo investigar o papel do sistema canabinoide em uma linhagem de neuroblastoma (Neuro 2a) submetida a condições de estresse oxidativo (H2O2), inflamação (LPS) e estresse do RE (tunicamicina), avaliando parâmetros de viabilidade celular e vias de sinalização envolvidas. Nossos resultados mostram que o agonista canabinoide ACEA foi capaz de proteger as células da morte celular causada pela inflamação e pelo estresse de retículo endoplasmático, mas não pelo estresse oxidativo. Esse efeito neuroprotetor exercido pelo ACEA parece pelo menos em parte ocorrer via receptor CB1 no modelo de inflamação e ser independente deste receptor no modelo de estresse de RE. Os efeitos neuroprotetores observados envolveram a modulação dos níveis de proteínas pré-apoptóticas, CHOP e Caspase 12, e da proteína relacionada à sobrevivência celular ERK 1/2. Nossos dados sugerem um papel neuroprotetor do sistema canabinoide em mecanismos relacionados aos processos neurodegenerativos e propõem a manipulação desse sistema como possível alvo terapêutico. / Neurodegeneration is the result of progressive and irreversible destruction of neurons in the central nervous system, with unknown causes and pathological mechanisms not fully elucidated. Factors such as age, increased formation of free radicals and/or oxidative stress, defects in energetic metabolism, inflammation and accumulation of neurotoxic factors and misfolded proteins in the lumen of the endoplasmic reticulum (ER) contribute to the development of neurodegenerative processes. The cannabinoid system has been proposed as neuroprotector in several models of neurodegeneration such as acute hypoxia and epilepsy, cerebral ischaemia, brain injury and oxidative stress models. This work aimed to investigate the role of the cannabinoid system in a neuroblastoma line (Neuro 2a) submitted to oxidative stress (H2O2), inflammation (LPS) and ER stress (tunicamycin) conditions, assessing cell viability parameters and signaling pathways involved. Our results show that the ACEA cannabinoid agonist was able to protect cells from cell death caused by inflammation and ER stress, but not from oxidative stress. This neuroprotective effect exerted by ACEA appears to occur at least in part via the CB1 receptor in inflammation model and it seems to be independent of this receptor in the ER stress model. The neuroprotective effects observed involved the modulation of the levels of pre-apoptotic proteins CHOP and Caspase 12 and the cell survival related protein ERK 1/2. Our data suggest a neuroprotective role of the cannabinoid system in mechanisms related to neurodegenerative processes and propose it as possible therapeutic target.

The endocannabinoid system is a novel pharmacological target in the treatment of metabolic syndrome. Antagonism of the endocannabinoid-1 receptor (CB1R) leads to a transient reduction in food intake, a sustained decrease in body weight and an improvement in metabolic parameters in animal models of obesity. Skeletal muscle is the primary tissue involved in glucose uptake in response to insulin, and insulin sensitivity of skeletal muscle is vital to the maintenance of whole-body euglycemia. Little is known regarding the effects of CB1R antagonism on skeletal muscle glucose transport activity. The purpose of this dissertation was to test the hypothesis that antagonism of the CB1R activates signaling molecules of the insulin signaling pathway to increase glucose transport activity in normal and insulin-resistant skeletal muscle, thereby improving whole-body glucose tolerance. CB1R antagonism with SR141716 directly enhanced basal and insulin-stimulated glucose transport activity in skeletal muscle from lean and obese Zucker while activation of the CB1R with ACEA, decreased glucose transport activity. Key proteins associated with regulation of glucose transport activity were not altered by either CB1R agonism (ACEA) or antagonism (SR141716). Chronic CB1R antagonist treatment (10 mg/kg SR141716 i.p. / 14 days) also enhanced insulin-stimulated glucose transport activity in skeletal muscle of both lean and obese animals, again with no alteration in relevant signaling factors. Plasma free fatty acids (FFAs) were decreased in chronically-treated lean and obese animals and whole-body insulin sensitivity was improved in obese Zucker rats. The enhanced insulin sensitivity seen in chronically-treated obese animals was associated with a dramatic reduction in insulin secretion following a glucose challenge. Acute CB1R antagonism in obese animals also elicited a reduction in insulin secretion following a glucose challenge; however, with no improvement of whole-body insulin sensitivity. Acute CB1R antagonist treatment did not alter skeletal muscle glucose transport activity or circulating FFAs for any animals. These data suggest that although CB1R antagonism directly enhances basal and insulin stimulated glucose transport in skeletal muscle of lean and obese rats, direct action on the skeletal muscle is not responsible for the improvement in insulin-stimulated glucose transport activity and whole-body insulin sensitivity seen in chronically-treated obese animals.

Introduction: Tobacco dependence is a chronic disorder that carries the risk of relapse at any time point during abstinence. It is a major health issue in the world and current pharmacotherapies have had limited efficacy. Therefore, development and validation of novel treatments are required. Objective: Investigate the novel neutral cannabinoid-1 receptor antagonist AM4113 on nicotine (main psychoactive ingredient in tobacco)-taking behaviour in animals. Methods: Using the nicotine intravenous- and food control- self administration paradigms, we tested the acute and chronic (10-days) effects of AM4113 on nicotine- and food-taking behaviour. Results: Acute AM4113 treatments (1-, 3-, 10-mg/kg) reduced nicotine self administration. Chronic AM4113 administration (10mg/kg) produced a sustained reduction of nicotine-taking behaviour during the course of the treatment. In the similar food control self administration experiments, AM4113 overall produced no effect. Conclusion: AM4113 can attenuate nicotine-taking behaviour and its effect is sustained under chronic treatment.

Introduction: Tobacco dependence is a chronic disorder that carries the risk of relapse at any time point during abstinence. It is a major health issue in the world and current pharmacotherapies have had limited efficacy. Therefore, development and validation of novel treatments are required. Objective: Investigate the novel neutral cannabinoid-1 receptor antagonist AM4113 on nicotine (main psychoactive ingredient in tobacco)-taking behaviour in animals. Methods: Using the nicotine intravenous- and food control- self administration paradigms, we tested the acute and chronic (10-days) effects of AM4113 on nicotine- and food-taking behaviour. Results: Acute AM4113 treatments (1-, 3-, 10-mg/kg) reduced nicotine self administration. Chronic AM4113 administration (10mg/kg) produced a sustained reduction of nicotine-taking behaviour during the course of the treatment. In the similar food control self administration experiments, AM4113 overall produced no effect. Conclusion: AM4113 can attenuate nicotine-taking behaviour and its effect is sustained under chronic treatment.

The role of 2-arachidonoylglycerol (2-AG) in nausea and vomiting was evaluated using a shrew (Suncus murinus) model of emesis and nausea-like behavior in rats, conditioned gaping. Shrews received JZL184, a selective MAGL inhibitor, prior to treatment with emetogenic lithium chloride (LiCl). The potential of exogenously administered 2-AG and arachidonic acid (AA) to regulate conditioned gaping was assessed in rats. The role of cannabinoid receptors and cyclooxygenase (COX) inhibition in suppression of vomiting and conditioned gaping was also evaluated. JZL184 dose-dependently suppressed vomiting in shrews, and was shown to inhibit MAGL in shrew brain tissue. The anti-emetic effects of JZL184 were prevented by the CB1 antagonist, AM251. Exogenous 2-AG suppressed LiCl-induced conditioned gaping, but was not prevented by AM251 or the CB2 antagonist, AM630. Instead, the COX inhibitor, indomethacin, prevented the suppressive effects of 2-AG, as well as AA. These results suggest that manipulations that elevate 2-AG may have anti-emetic/anti-nausea potential. / This research was supported by research grants from the Natural Sciences and Engineering Research Council of Canada (NSERC 92057) to Linda Parker, the Israel Science Foundation (DA009789) to Raphael Mechoulam, and the National Institutes of Health (DA009789, DA017259) to Benjamin Cravatt.

During marijuana and alcohol consumption as well as during inflammation the reproductive axis is inhibited, mainly through the inhibition of luteinizing hormone-releasing hormone release. In male rats, this inhibitory effect is mediated, at least in part, by the activation of hypothalamic cannabinoid type 1 receptors (CB1). During inflammation, this activation of the endocannabinoid system seems to be mediated by an increase in TNF-α production followed by anandamide augmentations, similarly the effect of intragastric administration of ethanol (3 g/kg) seems to be due to an increase in anandamide. On the other hand, a number of different actions mediated by the endocannabinoid system in various organs and tissues have been described. Both cannabinoid receptors, CB1 and CB2, are localized in the submandibular gland where they mediate the inhibitory effect of intrasubmandibular injections of the endocannabinoid anandamide (6 × 10–5M) on salivary secretion. Lipopolysaccharide (5 mg/kg/3 h) injected intraperitoneally and ethanol (3 g/kg/1 h) injected intragastrically inhibited the salivary secretion induced by the sialogogue metacholine; this inhibitory effect was blocked by CB1 and/or CB2 receptor antagonists. Similar to the hypothalamus, these effects seem to be mediated by increased anandamide. In summary, similar mechanisms mediate the inhibitory actions of endocannabinoids and cannabinoids in both hypothalamus and submandibular gland during drug consumption and inflammation. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

A ativação do receptor CB1, leva a modulação de processos intracelulares que muda a resposta celular de acordo com o estímulo, além de estar envolvida em mecanismos de proliferação, diferenciação, movimentação e morte celular. O objetivo desse trabalho foi avaliar a participação desse sistema em processos oxidativo e inflamatório relacionados à neurodegeneração in vitro. Foi utilizado a linhagem de neuroblastoma Neuro2a diferenciada em células dopaminérgicas que foram expostas a três condições: com 6OHDA, H2O2 e LPS e co-tratadas com o agonista do receptor CB1 ACEA e o antagonista/agonista inverso AM251 por 24 horas. Utilizamos parâmetros funcionais de viabilidade celular, produção de espécies reativas de oxigênio e técnica de western blot. O tratamento com ACEA ou ACEA/AM251 produziram um aumento da viabilidade celular nos três modelos de exposição propostos; redução da produção de espécies reativas de oxigênio e ativação da via da proteína ERK1/2, além da inibição da morte celular pela diminuição da expressão da caspase 3. Concluímos que os canabinoides escolhidos foram capazes de proteger as células dopaminérgicas do dano oxidativo e inflamatório através do aumento da sobrevida celular por diminuição da produção de ROS. / The CB1 receptor activation leads to modulation of intracellular processes that change the cellular response according to the stimulus, as well as being involved in mechanisms of proliferation, differentiation, cell movement and death. The present study evaluated the participation of this system in oxidative and inflammatory processes related to neurodegeneration in vitro. We have used the Neuro2A neuroblastoma lineage, which those were differentiated into dopaminergic cells, and exposed to 6OHDA, H2O2 and LPS. They were co-treated with ACEA, CB1 receptor agonist, and AM251, the CB1 receptor antagonist/inverse agonist, for 24 hours. We used functional parameters of cell viability, production of reactive oxygen species and protein analyses by western blot. Treatment with ACEA or ACEA/AM251 produced an increase in cell viability; reduced production of reactive oxygen species and activation of the ERK1/2 protein, in addition to inhibition of cell death by decreasing the expression of caspase 3 in all three models proposed. We concluded that chosen cannabinoids were able to protect dopaminergic cells from oxidative damage and inflammation through the increased cell survival by decreasing the production of ROS.

A neurodegeneração é o resultado da destruição progressiva e irreversível dos neurônios no sistema nervoso central, apresentando causas desconhecidas e mecanismos patológicos não totalmente elucidados. Fatores como a idade, o aumento da formação de radicais livres e/ou estresse oxidativo, defeito no metabolismo energético, a inflamação e acúmulo de elementos neurotóxicos e de proteínas malformadas no lúmen do retículo endoplasmático (RE) contribuem para o desenvolvimento dos processos neurodegenerativos. O sistema canabinoide tem sido proposto como neuroprotetor em diversos modelos de neurodegeneração como hipóxia aguda e epilepsia, isquemia cerebral, lesão cerebral e modelos de estresse oxidativo. Assim, este trabalho teve como objetivo investigar o papel do sistema canabinoide em uma linhagem de neuroblastoma (Neuro 2a) submetida a condições de estresse oxidativo (H2O2), inflamação (LPS) e estresse do RE (tunicamicina), avaliando parâmetros de viabilidade celular e vias de sinalização envolvidas. Nossos resultados mostram que o agonista canabinoide ACEA foi capaz de proteger as células da morte celular causada pela inflamação e pelo estresse de retículo endoplasmático, mas não pelo estresse oxidativo. Esse efeito neuroprotetor exercido pelo ACEA parece pelo menos em parte ocorrer via receptor CB1 no modelo de inflamação e ser independente deste receptor no modelo de estresse de RE. Os efeitos neuroprotetores observados envolveram a modulação dos níveis de proteínas pré-apoptóticas, CHOP e Caspase 12, e da proteína relacionada à sobrevivência celular ERK 1/2. Nossos dados sugerem um papel neuroprotetor do sistema canabinoide em mecanismos relacionados aos processos neurodegenerativos e propõem a manipulação desse sistema como possível alvo terapêutico. / Neurodegeneration is the result of progressive and irreversible destruction of neurons in the central nervous system, with unknown causes and pathological mechanisms not fully elucidated. Factors such as age, increased formation of free radicals and/or oxidative stress, defects in energetic metabolism, inflammation and accumulation of neurotoxic factors and misfolded proteins in the lumen of the endoplasmic reticulum (ER) contribute to the development of neurodegenerative processes. The cannabinoid system has been proposed as neuroprotector in several models of neurodegeneration such as acute hypoxia and epilepsy, cerebral ischaemia, brain injury and oxidative stress models. This work aimed to investigate the role of the cannabinoid system in a neuroblastoma line (Neuro 2a) submitted to oxidative stress (H2O2), inflammation (LPS) and ER stress (tunicamycin) conditions, assessing cell viability parameters and signaling pathways involved. Our results show that the ACEA cannabinoid agonist was able to protect cells from cell death caused by inflammation and ER stress, but not from oxidative stress. This neuroprotective effect exerted by ACEA appears to occur at least in part via the CB1 receptor in inflammation model and it seems to be independent of this receptor in the ER stress model. The neuroprotective effects observed involved the modulation of the levels of pre-apoptotic proteins CHOP and Caspase 12 and the cell survival related protein ERK 1/2. Our data suggest a neuroprotective role of the cannabinoid system in mechanisms related to neurodegenerative processes and propose it as possible therapeutic target.