O sistema endocanabinóide (eCB) tem sido reconhecido como um importante modulador da homeostase energética e recentemente estudos o apontam como um possível integrador da homeostase hidroeletrolítica. Estudos recentes do nosso laboratório demonstraram a participação do receptor canabinóide do tipo 1 (CB1R) no controle da secreção neurohipofisária em resposta a expansão hipertônica do volume extracelular. Dessa forma, o presente trabalho visou esclarecer a participação do sistema eCB, particularmente do CB1R, nas respostas neuronais, neuroendócrinas e comportamentais induzidas pela sobrecarga salina de 4 dias (SS). Uma vez que os animais em SS apresentam hipofagia induzida pela hiperosmolalidade, buscou-se avaliar as vias de integração do controle da homeostase energética e do balanço hidroeletrolítico por meio da introdução de um grupo em dieta pareada (pair fed, PF). De forma a investigar a hipótese acima, utilizou-se como ferramenta farmacológica o agonista seletivo CB1R, araquidonil-2-cloroetilamida (ACEA - 0,1g/5L), administrado por via intracerebroventricular (icv). Inicialmente, nosso trabalho mostrou que a SS promoveu aumento da expressão de CB1R tanto nos núcleos supra-óptico (NSO) e paraventricular (NPV) do hipotálamo quanto em estruturas da lâmina terminal [órgão subfornicial (SFO), o órgão vasculoso da lâmina terminal (OVLT) e o núcleo pré-óptico mediano (MnPO)]. Tais observações foram reforçadas pela análise microscópica destas regiões cerebrais por imunofluorescência, que evidenciou aumento da imunomarcação para CB1R no NPV, NSO e SFO em animais submetidos a SS. Estes resultados também mostraram que a maioria dos terminais pré-sinápticos CB1R-positivos estão localizados predominantemente na porção ventral do NSO, na qual predominam neurônios vasopressinérgicos. Os dados mostram ainda que todas as respostas induzidas pela SS foram revertidas após a reintrodução dos líquidos (RL, água e NaCl 0,3M). Já no grupo PF, não foram observadas alterações na expressão de CB1R em nenhuma das áreas avaliadas. No entanto, após a RL, os animais PF apresentaram hipoosmolalidade plasmática e aumento da expressão de CB1R na LT, sendo este último efeito aparentemente mediado por um aumento da expressão deste receptor no SFO. Em animais normoidratados, a administração central de ACEA produziu um aumento significativo na ingestão alimentar, sendo esta resposta ausente nos animais submetidos a SS, apesar do aumento da expressão de CB1R no hipotálamo verificada neste grupo. Entretanto, o pré-tratamento com ACEA foi capaz de potencializar a ingestão de água induzida pela SS, não produzindo efeitos significativos sobre este parâmetro no grupo PF. Este estudo demonstrou ainda que a SS não alterou as concentrações plasmáticas de angiotensina II (ANGII), porém promoveu aumento signficativo nas concentrações plasmáticas de corticosterona, vasopressina (AVP) e ocitocina (OT), além de diminuir a secreção de peptídeo natriurético atrial (ANP). Em animais submetidos a SS, o prétratamento com ACEA potencializou a secreção de corticosterona e preveniu o aumento da secreção de AVP e OT. Por outro lado, não foram observados efeitos da administração de ACEA sobre a secreção de ANP e ANGII induzida pela SS. Após a RL, o grupo SS apresentou normalização das concentrações plasmáticas hormonais, não sendo observados quaisquer efeitos da administração de ACEA nestas condições experimentais. No grupo PF, por sua vez, após a RL foi observada diminuição na secreção de OT e aumento nas concentrações plasmáticas de ANGII, efeitos estes não alterados pelo pré-tratamento com ACEA. Em conjunto, nossos dados sugerem que o CB1R participa ativamente das respostas homeostáticas comportamentais e neuroendócrinas desencadeadas pela SS, sendo estas respostas especificamente relacionadas ao controle da homeostase hidrossalina e não secundárias à hipofagia induzida pela hiperosmolalidade. Desta forma, conclui-se que a participação do CB1R na homeostase hidroeletrolítica ocorre em paralelo e independentemente da modulação exercida por este receptor sobre a homeostase energética. / The endocannabinoid system (eCB) has been recognized as an important modulator of energy homeostasis and recent studies suggest that this system may play a possible integrator role on hydromineral homeostasis. Recent studies from our laboratory demonstrated the involvement of the type 1 cannabinoid receptor (CB1R) in the control of neurohypophyseal secretion in response to hypertonic extracellular volume expansion. Therefore, the present study aimed to clarify the involvement of the ECB system, particularly of CB1Rs, in neuronal, neuroendocrine and behavioral responses induced by 4 days of salt load (SS). Since the animals submitted to SS exhibit a well known state of hyperosmolality-induced hypophagia, we attempted to evaluate the integrated control of energy homeostasis and hydroelectrolytic balance through the introduction of a paired diet group (pair fed, PF). In order to achieve these goals, this study employed as a pharmacological tool the CB1R selective agonist, arachidonoyl-2\'-chloroethylamide (ACEA-0.1g/5L), administered intracerebroventricularly (icv). Initially, our work showed that SS increased the expression of CB1R in both supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus, as well as in the structures of the lamina terminalis [subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT) and median preoptic nucleus (MnPO)]. These observations were reinforced by the microscopic analysis of these brain regions by immunofluorescence, which showed increased immunostaining for CB1R in the PVN, SON and SFO in animals submitted to SS. These results also showed that most of the presynaptic CB1R-positive terminals are located predominantly in the ventral part of the SON, which is characterized by the presence of vasopressinergic neurons. The data also showed that all SS-induced responses were reversed after reintroduction of fluids (RF, water and 0,3M NaCl). On the other hand, no changes in the expression of CB1R in any of the evaluated areas were observed in the PF group. However, after RF, PF animals showed hypoosmolality and increased expression of CB1R in the LT, being the latter effect apparently mediated by increased expression of this receptor in SFO. In euhydrated animals, the central administration of ACEA produced a significant increase in food intake, being this response absent in animals submitted to SS, despite the increased expression of CB1R in the hypothalamus observed in this group. However, pretreatment with ACEA was able to potentiate SS-induced water intake, producing no significant effect on this parameter in the PF group. This study also demonstrated that SS did not alter plasma concentrations of angiotensin II (ANG II), but significantly increased plasma concentrations of corticosterone, vasopressin (AVP) and oxytocin (OT), and decreased the secretion of atrial natriuretic peptide (ANP). In animals submitted to SS, pretreatment with ACEA enhanced the secretion of corticosterone and prevented the increased secretion of AVP and OT. Moreover, no effect of ACEA was observed on the SS-induced ANG II and ANP secretion. After RF, the SS group showed normalization of hormonal plasma concentrations, and no effects of ACEA administration were verified under these experimental conditions. After RF, the PF group exhibited a decrease in OT secretion and increased plasma concentrations of ANG II, effects that were not altered by pretreatment with ACEA. Taken together, our data suggest that CB1Rs actively participate in behavioral and neuroendocrine homeostatic responses triggered by SS, and that these responses were specifically related to the control of hydromineral homeostasis and not secondary to the hyperosmolality-induced hypophagia. Therefore, we conclude that the involvement of CB1R in electrolyte homeostasis occurs in parallel and independently of the modulation exerted by this receptor on energy homeostasis.

O sistema endocanabinóide (eCB) tem sido reconhecido como um importante modulador da homeostase energética e recentemente estudos o apontam como um possível integrador da homeostase hidroeletrolítica. Estudos recentes do nosso laboratório demonstraram a participação do receptor canabinóide do tipo 1 (CB1R) no controle da secreção neurohipofisária em resposta a expansão hipertônica do volume extracelular. Dessa forma, o presente trabalho visou esclarecer a participação do sistema eCB, particularmente do CB1R, nas respostas neuronais, neuroendócrinas e comportamentais induzidas pela sobrecarga salina de 4 dias (SS). Uma vez que os animais em SS apresentam hipofagia induzida pela hiperosmolalidade, buscou-se avaliar as vias de integração do controle da homeostase energética e do balanço hidroeletrolítico por meio da introdução de um grupo em dieta pareada (pair fed, PF). De forma a investigar a hipótese acima, utilizou-se como ferramenta farmacológica o agonista seletivo CB1R, araquidonil-2-cloroetilamida (ACEA - 0,1g/5L), administrado por via intracerebroventricular (icv). Inicialmente, nosso trabalho mostrou que a SS promoveu aumento da expressão de CB1R tanto nos núcleos supra-óptico (NSO) e paraventricular (NPV) do hipotálamo quanto em estruturas da lâmina terminal [órgão subfornicial (SFO), o órgão vasculoso da lâmina terminal (OVLT) e o núcleo pré-óptico mediano (MnPO)]. Tais observações foram reforçadas pela análise microscópica destas regiões cerebrais por imunofluorescência, que evidenciou aumento da imunomarcação para CB1R no NPV, NSO e SFO em animais submetidos a SS. Estes resultados também mostraram que a maioria dos terminais pré-sinápticos CB1R-positivos estão localizados predominantemente na porção ventral do NSO, na qual predominam neurônios vasopressinérgicos. Os dados mostram ainda que todas as respostas induzidas pela SS foram revertidas após a reintrodução dos líquidos (RL, água e NaCl 0,3M). Já no grupo PF, não foram observadas alterações na expressão de CB1R em nenhuma das áreas avaliadas. No entanto, após a RL, os animais PF apresentaram hipoosmolalidade plasmática e aumento da expressão de CB1R na LT, sendo este último efeito aparentemente mediado por um aumento da expressão deste receptor no SFO. Em animais normoidratados, a administração central de ACEA produziu um aumento significativo na ingestão alimentar, sendo esta resposta ausente nos animais submetidos a SS, apesar do aumento da expressão de CB1R no hipotálamo verificada neste grupo. Entretanto, o pré-tratamento com ACEA foi capaz de potencializar a ingestão de água induzida pela SS, não produzindo efeitos significativos sobre este parâmetro no grupo PF. Este estudo demonstrou ainda que a SS não alterou as concentrações plasmáticas de angiotensina II (ANGII), porém promoveu aumento signficativo nas concentrações plasmáticas de corticosterona, vasopressina (AVP) e ocitocina (OT), além de diminuir a secreção de peptídeo natriurético atrial (ANP). Em animais submetidos a SS, o prétratamento com ACEA potencializou a secreção de corticosterona e preveniu o aumento da secreção de AVP e OT. Por outro lado, não foram observados efeitos da administração de ACEA sobre a secreção de ANP e ANGII induzida pela SS. Após a RL, o grupo SS apresentou normalização das concentrações plasmáticas hormonais, não sendo observados quaisquer efeitos da administração de ACEA nestas condições experimentais. No grupo PF, por sua vez, após a RL foi observada diminuição na secreção de OT e aumento nas concentrações plasmáticas de ANGII, efeitos estes não alterados pelo pré-tratamento com ACEA. Em conjunto, nossos dados sugerem que o CB1R participa ativamente das respostas homeostáticas comportamentais e neuroendócrinas desencadeadas pela SS, sendo estas respostas especificamente relacionadas ao controle da homeostase hidrossalina e não secundárias à hipofagia induzida pela hiperosmolalidade. Desta forma, conclui-se que a participação do CB1R na homeostase hidroeletrolítica ocorre em paralelo e independentemente da modulação exercida por este receptor sobre a homeostase energética. / The endocannabinoid system (eCB) has been recognized as an important modulator of energy homeostasis and recent studies suggest that this system may play a possible integrator role on hydromineral homeostasis. Recent studies from our laboratory demonstrated the involvement of the type 1 cannabinoid receptor (CB1R) in the control of neurohypophyseal secretion in response to hypertonic extracellular volume expansion. Therefore, the present study aimed to clarify the involvement of the ECB system, particularly of CB1Rs, in neuronal, neuroendocrine and behavioral responses induced by 4 days of salt load (SS). Since the animals submitted to SS exhibit a well known state of hyperosmolality-induced hypophagia, we attempted to evaluate the integrated control of energy homeostasis and hydroelectrolytic balance through the introduction of a paired diet group (pair fed, PF). In order to achieve these goals, this study employed as a pharmacological tool the CB1R selective agonist, arachidonoyl-2\'-chloroethylamide (ACEA-0.1g/5L), administered intracerebroventricularly (icv). Initially, our work showed that SS increased the expression of CB1R in both supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus, as well as in the structures of the lamina terminalis [subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT) and median preoptic nucleus (MnPO)]. These observations were reinforced by the microscopic analysis of these brain regions by immunofluorescence, which showed increased immunostaining for CB1R in the PVN, SON and SFO in animals submitted to SS. These results also showed that most of the presynaptic CB1R-positive terminals are located predominantly in the ventral part of the SON, which is characterized by the presence of vasopressinergic neurons. The data also showed that all SS-induced responses were reversed after reintroduction of fluids (RF, water and 0,3M NaCl). On the other hand, no changes in the expression of CB1R in any of the evaluated areas were observed in the PF group. However, after RF, PF animals showed hypoosmolality and increased expression of CB1R in the LT, being the latter effect apparently mediated by increased expression of this receptor in SFO. In euhydrated animals, the central administration of ACEA produced a significant increase in food intake, being this response absent in animals submitted to SS, despite the increased expression of CB1R in the hypothalamus observed in this group. However, pretreatment with ACEA was able to potentiate SS-induced water intake, producing no significant effect on this parameter in the PF group. This study also demonstrated that SS did not alter plasma concentrations of angiotensin II (ANG II), but significantly increased plasma concentrations of corticosterone, vasopressin (AVP) and oxytocin (OT), and decreased the secretion of atrial natriuretic peptide (ANP). In animals submitted to SS, pretreatment with ACEA enhanced the secretion of corticosterone and prevented the increased secretion of AVP and OT. Moreover, no effect of ACEA was observed on the SS-induced ANG II and ANP secretion. After RF, the SS group showed normalization of hormonal plasma concentrations, and no effects of ACEA administration were verified under these experimental conditions. After RF, the PF group exhibited a decrease in OT secretion and increased plasma concentrations of ANG II, effects that were not altered by pretreatment with ACEA. Taken together, our data suggest that CB1Rs actively participate in behavioral and neuroendocrine homeostatic responses triggered by SS, and that these responses were specifically related to the control of hydromineral homeostasis and not secondary to the hyperosmolality-induced hypophagia. Therefore, we conclude that the involvement of CB1R in electrolyte homeostasis occurs in parallel and independently of the modulation exerted by this receptor on energy homeostasis.

Acyl coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the intracellular synthesis of cholesteryl esters (CE). Both ACAT isoforms, ACAT1 and ACAT2, play key roles in the pathophysiology of atherosclerosis and ACAT inhibition retards atherosclerosis in animal models. Rimonabant, a type 1 cannabinoid receptor (CB1) antagonist, produces anti-atherosclerotic effects in humans and animals by mechanisms which are not completely understood. Rimonabant is structurally similar to two other cannabinoid receptor antagonists, AM251 and SR144528, recently identified as potent inhibitors of ACAT. Therefore, we examined the effects of Rimonabant on ACAT using both in vivo cell-based assays and in vitro cell-free assays. Rimonabant dose-dependently reduced ACAT activity in Raw 264.7 macrophages (IC50=2.9±0.38μM) and isolated peritoneal macrophages. Rimonabant inhibited ACAT activity in intact CHO-ACAT1 and CHO-ACAT2 cells and in cell-free assays with approximately equal efficiency (IC50=1.5±1.2μM and 2.2±1.1μM for CHO-ACAT1 and CHO-ACAT2, respectively). Consistent with ACAT inhibition, Rimonabant treatment blocked ACAT-dependent processes in macrophages, oxysterol-induced apoptosis and acetylated-LDL induced foam cell formation. From these results we conclude that Rimonabant is an ACAT1/2 dual inhibitor and suggest that some of the atherosclerotic beneficial effects of Rimonabant are, at least partly, due to inhibition of ACAT.

L’architecture polarisée des neurones est mise en place est maintenue grâce à un adressage hautement contrôlé de protéines vers l’axone ou vers le compartiment somatodendritique. Parmi ces protéines, les récepteurs aux protéines G (RCPG) neuronaux sont des cibles pharmacologiques clés. Cependant, leur pharmacologie est généralement étudiée dans des lignées cellulaires non polarisées et les résultats obtenus dans ces systèmes ne caractérisent pas correctement les effets physiologiques de l’activation des RCPG présents dans le cerveau. Par conséquent, un des principaux sujets de recherche de notre équipe est de comprendre comment la polarité neuronale influe sur la pharmacologie des RCPG, en étudiant l’un des RCPG les plus abondants dans le cerveau : le récepteur cannabinoïque de type-1 (CB1R). Les études précédentes de notre groupe ont suggéré que CB1R acquiert une polarisation axonale grâce à un adressage transcytotique : après leur synthèse, ces récepteurs apparaissent sur la membrane plasmique somatodendritique d’où ils sont rapidement enlevés par endocytose constitutive puis adressés à la membrane plasmique axonale où ils s’accumulent du fait d’une endocytose réduite. Au début de ma thèse, nous avons directement mesuré cette endocytose différentielle et le transport transcytotique de CB1R en utilisant des neurones de rats mis en culture dans des dispositifs microfluidiques. De plus, nous avons montré que des traitements pharmacologiques prolongés peuvent fortement changer la distribution de RCPG à la surface neuronale. Ces résultats démontrent que l’équilibre endocytotique dépendant du compartiment neuronal, qui est contrôlable pharmacologiquement, est important pour la distribution des RCPG neuronaux. Dans une seconde partie, nous avons étudié si le trafic différentiel de CB1R entre axones et dendrites est corrélé avec une pharmacologie différentielle. CB1R est majoritairement couplé à des protéines de type Gi/o et est connu pour inhiber la production d’AMPc. Nous avons donc développé l’imagerie par Föster Resonance Energy Transfer (FRET) appliqué aux cultures de neurones d’hippocampe de rats afin de mesurer la modulation de la voie de signalisation AMPc/PKA en aval de CB1R endogènes dans l’ensemble des compartiments neuronaux : somata, dendrites, mais aussi dans les axones matures très fins. Nos résultats montrent que CB1R possède une pharmacologie différente entre les dendrites et les axones. Notamment, son activation conduit à une diminution plus forte de l’activité basale de la PKA dans les axones comparé aux dendrites, lié au plus grand nombre de récepteurs présents sur la membrane de ce compartiment. De plus, nous démontrons que, contrairement aux récepteurs axonaux, les CB1R somatodendritiques inhibent constitutivement la voie AMPc/PKA. Cette différence est due à la distribution polarisée de la DAGLipase, l’enzyme synthétisant l’endocannabinoïde principal, le 2-arachidonoyglycerol (2-AG). De plus, l’inhibition pharmacologique de la DAGL modifie l’efficacité de plusieurs agonistes de CB1R dans le compartiment somatodendritique mais pas dans l’axone. Cet effet pourrait être dû à une modulation allostérique. Dans une troisième partie, nous avons étudié si les résultats ci-dessus peuvent être généralisés à d’autres RCPG. Etant donné que l’adressage axonal et la pharmacologie in vitro des récepteurs sérotoninergiques 5-HT1B montrent de fortes similitudes avec ceux de CB1R, nous avons étudié la pharmacologie de ces récepteurs en utilisant la technique de FRET développée précédemment. De façon similaire, nous avons trouvé une pharmacologie différentielle entre l’axone et les dendrites. / Polarized neuronal architecture is achieved and maintained mainly through highly controlled targeting of proteins to axons versus to the somatodendritic compartment. Among these proteins, neuronal G protein coupled receptors (GPCRs) are key therapeutic targets. However, their pharmacology is generally studied in non-polarized cell lines, and results obtained in such systems likely do not fully characterize the physiological effects of brain GPCR activation. Therefore, a main research subject of our group is to understand how neuronal polarity influences GPCR pharmacology, by studying one of the most abundant GPCR in the brain: the type-1 cannabinoid receptor (CB1R). Previous studies of the group suggested that CB1Rs achieve axonal polarization through transcytotic targeting: after their synthesis, these receptors appear on the somatodendritic plasma membrane from where they are removed rapidly by constitutive endocytosis and then targeted to the axonal plasma membrane where they accumulate due to relatively reduced endocytosis rate. At the beginning of my PhD project we directly demonstrated this differential endocytosis and transcytotic transport of CB1Rs by using cultured neurons in microfluidic devices. Moreover, we showed that chronic pharmacological treatments may strongly change neuronal GPCR distribution on the neuronal surface. These results demonstrate that subdomain-dependent steady-state endocytosis, which is pharmacologically controllable, is important for GPCR distribution in neurons. In a second part, we asked if differential traffic of CB1Rs between axons and dendrites is correlated with differential pharmacology. CB1R is predominantly coupled to Gi/o proteins and is known to inhibit cAMP production. Thus, we developed live Föster Resonance Energy Transfer (FRET) imaging in cultured hippocampal neurons in order to measure basal cAMP/PKA pathway modulation downstream of endogenous CB1Rs in all neuronal compartments: in somata, in dendrites but also in the very thin mature axons. Our results show that CB1R displays differential pharmacology between axon and dendrites. Notably, its activation leads to a stronger decrease of PKA activity in axons compared to dendrites, due to increased number of membrane receptors in this compartment. Moreover, we demonstrate that somatodendritic CB1Rs constitutively inhibit cAMP/PKA pathway, while axonal receptors do not. This difference is due to polarized distribution of DAGLipase, the enzyme that synthesizes the major endocannabinoid 2-arachidonoylglycerol (2-AG). Moreover, blocking DAGL by pharmacological treatment modifies somatodendritic, but not axonal effects of several CB1R agonists, possibly through allosteric action. In a third part, we asked if the above results may be generalized to other GPCRs. Because the axonal targeting and in vitro pharmacology of 5-HT1B serotonin receptors demonstrate strong similarities with CB1Rs, we studied their neuronal pharmacology by using the previously developed FRET technique. We found similar differential responses to pharmacological treatments between axon and dendrites. In a fourth part, we investigated the role of the threonine 210 (T210) residue in the constitutive activity of neuronal CB1R. We showed that the hypoactive mutant T210A-CB1R do not constitutively recruit signaling pathways even in somatodendritic compartment, where 2-AG is present. This result demonstrates that T210 is necessary for constitutive CB1R activation by 2-AG.Finally, previous results of our group demonstrated the involvement of CB1R in neuronal development. Notably, CB1R activation was shown to have an overall inhibitory effect on the development of polarized neuronal morphology. We established a bibliographic review on this subject. The published literature data suggest that not only neuronal polarization influences both CB1R traffic and pharmacology but CB1Rs also contribute to the achievement of neuronal polarization. (...)