The relationship between the endocannabinoid system in the renal medulla and the long-term regulation of blood pressure is not well understood. To investigate the possible role of the endocannabinoid system in renomedullary interstitial cells, mouse medullary interstitial cells (MMICs) were obtained, cultured and characterized for their responses to treatment with a selective inhibitor of fatty acid amide hydrolase (FAAH), PF-3845. Treatment of MMICs with PF-3845 increased cytoplasmic lipid granules detected by Sudan Black B staining and multilamellar bodies identified by transmission electron microscopy. HPLC analyses of lipid extracts of MMIC culture medium revealed a 205nm-absorbing peak that showed responsiveness to PF-3845 treatment. The biologic activities of the PF-3845-induced product (PIP) isolated by HPLC were investigated in anesthetized, normotensive surgically-instrumented mice. Intramedullary and intravenous infusion of PIP at low dose rates (0.5-1 AU/10 min) stimulated diuresis and natriuresis, whereas at higher doses, these parameters returned toward baseline but mean arterial pressure (MAP) was lowered. Whereas intravenous bolus doses of PIP stimulated diuresis, GFR and medullary blood flow (MBF) and reduced or had no effect on MAP, an intraperitoneal bolus injection of PIP reduced MAP, increased MBF, and had no effect on urinary parameters. Genetic or pharmacological ablation of the cannabinoid type 1 receptors in mice completely abolished the diuretic and vasodepressor properties of intramedullary infused PIP, suggesting that the PF-3845-induced product requires the presence of CB1 receptors in order to elicit its renal effects. In a radioactive competition binding assay, using Chinese hamster ovary cells expressing CB1 receptors, PIP successfully displaced the CB1 selective inverse agonist [3H] SR141716A, revealing that the lipid extract was able to compete for binding to CB1 receptors. Finally, we investigated the tubular location of diuretic activity that the PF-3845-induced lipid fraction exhibits. In a renal function in vivo experiment, we pre-treated anesthetized mice with an intramedullary infusion of one of four well-known diuretics. This procedure was followed by an intramedullary infusion of PIP (1AU). Only inhibition of the proximal tubule sodium reabsorption diminished the diuretic activity of the PF-3845-induced product, suggesting that the lipid fraction requires a physiologically intact proximal tubular reabsorption mechanism for it to produce diuresis. These data support a model whereby PF-3845 treatment of MMICs results in increased secretion of a neutral lipid which acts directly to promote diuresis and natriuresis and indirectly through metabolites to produce vasodepression. Efforts to identify the structure of the PF-3845-induced lipid and its relationship to the previously proposed renomedullary antihypertensive lipids are ongoing.

Considerable evidence supports anandamide (AEA) as an important mediator in the regulation of nausea and vomiting. The present study investigates the effect of inhibiting a protein reported to mediate AEA transport, FLAT (FAAH-1-like AEA transporter), on nausea and vomiting and the neural correlates of AEA regulated nausea in the visceral insular cortex (VIC). The systemic administration of the AEA transport inhibitor ARN272 was evaluated in LiCl-induced conditioned gaping in rats, and vomiting in shrews. The effect of intra-cranial administration of ARN272 into the VIC was also investigated using LiCl-induced conditioned gaping in rats. Systemic administration of ARN272 dose-dependently suppressed LiCl-induced conditioned gaping in rats, and was reversed by CB1 receptor antagonism with SR141716. Systemic administration of ARN272 also attenuated vomiting in shrews. Delivery of ARN272 into the VIC produced no effect on LiCl-induced conditioned gaping in rats. These results suggest that preventing the cellular reuptake of AEA through transport inhibition tonically activates CB1 receptors to regulate toxin-induced nausea, but that this is not AEA regulated within the VIC. / This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC-92057) to LAP.

Endocannabinoids are endogenous lipid mediators involved in various biological processes, and have immunomodulatory and anti-inflammatory activities. Anandamide (arachidonoyl ethanolamine, AEA) and 2-arachidonoyl glycerol (2-AG) are the main representatives of this group. The endocannabinoid receptors CB1 and CB2 with AEA have been found in human HaCaT keratinocytes and fibroblasts, but the metabolic pathway leading to endocannabinoid production in the skin has not been fully elucidated. This study aimed to investigate the profile of endocannabinoids and their main metabolizing enzymes in human skin cells and assess whether omega-3 polyunsaturated fatty acids (n-3 PUFA) altered these profiles. In addition, an investigation was carried out to check whether UV radiation could stimulate the production of endocannabinoids and N-acylethanolamines (NAE) in human skin cells. For this purpose HaCaT keratinocytes and 46RB.1N fibroblast cells were treated with 10 and 50µM of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or oleic acid (OA) in the presence or absence of UVR (15mJ/cm2). Data suggest that n-3 PUFA may both directly (by up-regulating NAPE-PLD levels) and indirectly (by decreasing FAAH levels) increased endocannabinoid and NAE levels in HaCaT keratinocytes and 46BR.IN fibroblasts. DHA treatment significantly decreased COX-2 expression in the absence of UVR and inhibited UVR-induced COX-2 overexpression in 46BR.IN fibroblasts. In contrast, DHA appeared to induce COX-2 up-regulation in the absence of UVR and did not prevent UVR induced COX-2 up-regulation in HaCaT keratinocytes. EPA appeared to induce COX-2 down-regulation in the absence of UVR and did not prevent UVR induced COX-2 up-regulation in both HaCaT keratinocytes and 46BR.IN fibroblasts. UVR did not have any significant effect on endocannabinoid and NAE biosynthesis. However, UVR induced endocannabinoid production in some experiments of this study. A clinical study was carried on 16 volunteers from two different ethnic groups and two different skin types. The purpose was to assess the effect of UVR on the serum endocannabinoids and NAE, therefore, the volunteers were subjected to multiple doses (1.3, SED/ 6 min) of UVR for 6 weeks. Data showed that UVR did not have major effect on human serum NAE in both skin phototypes II and V but increased 2-AG in human serum in both skin types but the more pronounced effect was evident in skin phototypes V rather than in skin phototypes II. Human serum docosahxaenoylethanolamide levels were found to be higher in White Caucasians group (skin phototypes II). Based on these it can be concluded that n-3 PUFA and UVR alter the endocannabinoids and NAE profile in HaCaT keratinocytes and 46BR.IN fibroblasts. In addition, results of the clinical study indicated that UVR has no major effects on serum endocannabinoids or NAE therefore, further studies are required to address this question in vivo.

No description available.

Recent evidence suggesting that the endogenous cannabinoid (ECB) system can be selectively manipulated to facilitate or impair the extinction of learned behaviours — specifically regarding drug-induced aversive memories — has important consequences for research on opiate withdrawal and abstinence. Data presented here support and expand previous findings that the ECB system has an important function in the extinction of aversively motivated behaviors and is mediated by i) an increase in available endogenous CB1 receptor agonists, primarily anandamide, and ii) the exogenous CB1 receptor agonist Δ9-THC, in a manner that is dependent upon both the dose and route of administration. Experiments demonstrated that the fatty acid amide hydrolase (FAAH) inhibitor, URB597, which blocks deactivation of endogenous CB1 ligands, such as anandamide, significantly facilitated extinction of naloxone-precipitated morphine withdrawal-induced conditioned cue aversion, whereas the CB1 receptor antagonist/inverse agonist SR141716 significantly impaired extinction. Several experiments demonstrated that neither the CB1 antagonist AM251 nor the FAAH inhibitor URB597 had any effect on extinction learning for morphine-induced conditioned cue preference. A method was developed for analysing cannabinoid levels in blood by liquid chromatography/mass spectrometry (LC/MS) to compare bioavailable levels of Δ9-THC and its primary psychoactive metabolite. Experiments were designed to meet three primary objectives: 1) to provide further support for the role of the ECB system in the extinction of aversively-motivated behaviours, 2) to compare bioavailable levels of Δ9-THC and its primary psychoactive metabolite, 11-OH-Δ9-THC, after pulmonary and parenteral administration, and 3) to demonstrate that the route of administration of Δ9-THC can have a significant impact on whether or not it facilitates or impairs extinction learning. Results showed that inhaled Δ9-THC dose- and time-dependently facilitated rates of extinction learning of the conditioned aversion whereas injected Δ9-THC significantly impaired extinction. These data suggest that the route of administration of Δ9-THC has important consequences for its resulting pharmacokinetic and behavioural effects, specifically, that pulmonary exposure facilitates, whereas parenteral exposure impairs, rates of extinction learning for conditioned cue aversion. Thus, pulmonary administration of Δ9-THC may prove more beneficial for pharmacological potentiation of extinction learning for aversive memories, such as those supporting drug-craving/seeking in opiate withdrawal-syndrome. / NSERC and OGS

Genetic factors underlying alcoholism, substance abuse, antisocial and violent behaviour, psychosis, schizophrenia and psychopathy are emerging to implicate dopaminergic and cannabinoid, but also monoaminergic and glutamatergic systems through the maze of promoter genes and polymorphisms. Candidate gene association studies suggest the involvement of a range of genes in different disorders of CNS structure and function. Indices of comorbidity both complicate the array of gene-involvement and provide a substrate of hazardous interactivity. The putative role of the serotonin transporter gene in affective-dissociative spectrum disorders presents both plausible genetic variation and complication of comorbidity. The position of genetic variation is further complicated through ethnic, contextual and social factors that provide geometric progressions in the comordity already underlying diagnostic obstacles. The concept of shared biological susceptibilty to two or more disorder conditions of comorbidity seems a recurring observation, e.g., bipolar disorder with alcoholism or schizophrenia with alcohol/substance abuse or diabetes with schizopsychotic disorder. Several lines of evidence seem to suggest that the factors influencing variation in one set of symptoms and those affecting one or more disorders are observed to a marked extent which ought to facilitate the search for susceptibility genes in comorbid brain disorders. Identification of regional genetic factors is awaited for a more compelling outline that ought eventually to lead to greater efficacy of symptom-disorder arrangements and an augmentation of current pharmacological treatment therapies.

Le système endocannaboïde (eCB) est constitué des ligands, des récepteurs – les plus étudiés étant les récepteurs CB1 et CB2 – et les enzymes de synthèse et de dégradation. Les ligands étant lipophiles, ils ne sont pas encapsulés dans des vésicules, ce qui place les enzymes de synthèse et de dégradation dans une position de régulateurs clés. Plusieurs études démontrent une participation du système eCB à des processus de développement dans le système nerveux central (SNC). La rétine est un modèle important pour l’étude de ces processus car elle contient plusieurs types cellulaires bien connus, dont le patron de développement est clairement établi. Pour l’instant très peu est connu sur l’expression du système eCB durant le développement rétinien. C’est dans ce cadre que les patrons d’expression du récepteur CB1 et de l’enzyme de dégradation FAAH ont été étudiés pendant le développement rétinien postnatal chez le rat. Pour identifier les types cellulaires exprimant ces protéines, des co-marquages ont été accomplis pour le récepteur CB1 ou FAAH et des marqueurs des types cellulaires rétiniens. À P1, les cellules ganglionnaires, amacrines, horizontales et mitotiques expriment le récepteur CB1. Les cellules ganglionnaires et amacrines cholinergiques sont FAAH-positives. Au cours du développement, certains types cellulaires démontrent une expression transitoire de ces deux protéines, suggérant une implication du système eCB dans les processus de développement. Nos données démontrent également une importante expression du système eCB dans la rétine adulte, ce qui soutient l’hypothèse de son implication dans la réponse rétinienne. En bref, des études fonctionnelles in vitro sur des rétines de non-mammifères ont révélées que le récepteur CB1 modulait la réponse des cônes et des cellules bipolaires. Malgré la récente démonstration de sa présence dans la rétine, il n’existe pas de d’étude sur le rôle du récepteur CB2 dans la rétine. Dans cette thèse, les conséquences fonctionnelles de l’élimination des récepteurs CB1 ou CB2 ont été évaluées chez des souris transgéniques. Les réponses rétiniennes ont été enregistrées par électrorétinographie chez des souris cnr1-/- (CB1R-KO) et cnr2-/- (CB2R-KO). Nos données suggèrent une implication différente pour chaque récepteur dans la formation de la réponse rétinienne / The endocannabinoid (eCBs) system is composed of the ligands, the receptors - the most studied are CB1R and CB2R – and the synthesizing and degradative enzymes. The lipophilic ligands are not stored in vesicles, thereby placing the synthesizing and degradative enzymes as key regulators of the receptor function. The eCB system is thought to participate to developmental processes in the central nervous system (CNS). The rodent retina is a valuable model to study CNS development, as it contains well identified cell types with established developmental timelines. Very little is known about the distribution of this neuromodulationsystem in the developing retina. In this thesis, the expression patterns of CB1R and eCB degradative enzyme FAAH were investigated in the rat retina during postnatal development. To identify the cells expressing these proteins, co-stainings were carried out for CB1R or FAAH and retinal cell type markers. At P1, CB1R was expressed in ganglion, amacrine, horizontal and mitotic cells, whereas FAAH was present in ganglion and cholinergic amacrine cells. In the course of development, both CB1R and FAAH were transiently expressed in some cell type, suggesting a role of the eCB system in developmental processes. Furthermore, our data demonstrated an important expression of both proteins in adult animals, supporting the hypothesis that the eCB system is involved in retinal functions. Briefly, functional in vitro studies on non-mammalian retinae have revealed an effect of CB1R on cone photoreceptors and bipolar cells response. Despite the recent demonstration of CB2R mRNA and protein presence in the retina, there are no data on CB2R functional role in retina have been published. In this thesis, the consequences of removing either CB1R or CB2R from the retina of transgenic mice were evaluated. Retinal response was recorded by electroretinogram in cnr1-/- (CB1R-KO) and cnr2-/- (CB2R-KO) mice. This data suggests that both receptors are involved in shaping the retinal response to light and they have different roles in this process.

Le système endocannaboïde (eCB) est constitué des ligands, des récepteurs – les plus étudiés étant les récepteurs CB1 et CB2 – et les enzymes de synthèse et de dégradation. Les ligands étant lipophiles, ils ne sont pas encapsulés dans des vésicules, ce qui place les enzymes de synthèse et de dégradation dans une position de régulateurs clés. Plusieurs études démontrent une participation du système eCB à des processus de développement dans le système nerveux central (SNC). La rétine est un modèle important pour l’étude de ces processus car elle contient plusieurs types cellulaires bien connus, dont le patron de développement est clairement établi. Pour l’instant très peu est connu sur l’expression du système eCB durant le développement rétinien. C’est dans ce cadre que les patrons d’expression du récepteur CB1 et de l’enzyme de dégradation FAAH ont été étudiés pendant le développement rétinien postnatal chez le rat. Pour identifier les types cellulaires exprimant ces protéines, des co-marquages ont été accomplis pour le récepteur CB1 ou FAAH et des marqueurs des types cellulaires rétiniens. À P1, les cellules ganglionnaires, amacrines, horizontales et mitotiques expriment le récepteur CB1. Les cellules ganglionnaires et amacrines cholinergiques sont FAAH-positives. Au cours du développement, certains types cellulaires démontrent une expression transitoire de ces deux protéines, suggérant une implication du système eCB dans les processus de développement. Nos données démontrent également une importante expression du système eCB dans la rétine adulte, ce qui soutient l’hypothèse de son implication dans la réponse rétinienne. En bref, des études fonctionnelles in vitro sur des rétines de non-mammifères ont révélées que le récepteur CB1 modulait la réponse des cônes et des cellules bipolaires. Malgré la récente démonstration de sa présence dans la rétine, il n’existe pas de d’étude sur le rôle du récepteur CB2 dans la rétine. Dans cette thèse, les conséquences fonctionnelles de l’élimination des récepteurs CB1 ou CB2 ont été évaluées chez des souris transgéniques. Les réponses rétiniennes ont été enregistrées par électrorétinographie chez des souris cnr1-/- (CB1R-KO) et cnr2-/- (CB2R-KO). Nos données suggèrent une implication différente pour chaque récepteur dans la formation de la réponse rétinienne / The endocannabinoid (eCBs) system is composed of the ligands, the receptors - the most studied are CB1R and CB2R – and the synthesizing and degradative enzymes. The lipophilic ligands are not stored in vesicles, thereby placing the synthesizing and degradative enzymes as key regulators of the receptor function. The eCB system is thought to participate to developmental processes in the central nervous system (CNS). The rodent retina is a valuable model to study CNS development, as it contains well identified cell types with established developmental timelines. Very little is known about the distribution of this neuromodulationsystem in the developing retina. In this thesis, the expression patterns of CB1R and eCB degradative enzyme FAAH were investigated in the rat retina during postnatal development. To identify the cells expressing these proteins, co-stainings were carried out for CB1R or FAAH and retinal cell type markers. At P1, CB1R was expressed in ganglion, amacrine, horizontal and mitotic cells, whereas FAAH was present in ganglion and cholinergic amacrine cells. In the course of development, both CB1R and FAAH were transiently expressed in some cell type, suggesting a role of the eCB system in developmental processes. Furthermore, our data demonstrated an important expression of both proteins in adult animals, supporting the hypothesis that the eCB system is involved in retinal functions. Briefly, functional in vitro studies on non-mammalian retinae have revealed an effect of CB1R on cone photoreceptors and bipolar cells response. Despite the recent demonstration of CB2R mRNA and protein presence in the retina, there are no data on CB2R functional role in retina have been published. In this thesis, the consequences of removing either CB1R or CB2R from the retina of transgenic mice were evaluated. Retinal response was recorded by electroretinogram in cnr1-/- (CB1R-KO) and cnr2-/- (CB2R-KO) mice. This data suggests that both receptors are involved in shaping the retinal response to light and they have different roles in this process.

Modifying the metabolism of the body’s own endocannabinoids is a novel approach for analgesia. Two key catabolic enzymes are fatty acid amide hydrolase (FAAH) and inflammation-inducible cyclooxygenase 2 (COX-2). The cellular uptake of the key endocannabinoid anandamide (AEA) has been found to be regulated by its FAAH-catalysed intracellular degradation, but COX-2 has not been investigated in this respect. We aimed to find out whether or not COX-2 in an in vitro inflammation setting would be able to gate AEA uptake. To achieve this, C6 cells and Raw 264.7 cells were stimulated with LPS/INF-γ and lysates then analyzed by immunoblot in order to verify COX-2 expression. AEA cellular uptake was quantified using a radioassay with [3H]-AEA. It was found that COX-2 was not inducible in C6 cells using the LPS/INF-γ conditions studied, while it was inducible in Raw 264.7 cells. AEA uptake in the COX-2-induced Raw 264.7 cells was not reduced by inhibitors of this enzyme. FAAH appeared to be down-regulated in the stimulated Raw 264.7 cells, and this was reflected in an overall lower AEA uptake. Our interpretation of the data points to FAAH as gating AEA uptake. Additional experiments are required to validate our findings by verifying significance.