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1.
Nature ; 583(7817): 603-608, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32641832

RESUMO

Astrocytes take up glucose from the bloodstream to provide energy to the brain, thereby allowing neuronal activity and behavioural responses1-5. By contrast, astrocytes are under neuronal control through specific neurotransmitter receptors5-7. However, whether the activation of astroglial receptors can directly regulate cellular glucose metabolism to eventually modulate behavioural responses is unclear. Here we show that activation of mouse astroglial type-1 cannabinoid receptors associated with mitochondrial membranes (mtCB1) hampers the metabolism of glucose and the production of lactate in the brain, resulting in altered neuronal functions and, in turn, impaired behavioural responses in social interaction assays. Specifically, activation of astroglial mtCB1 receptors reduces the phosphorylation of the mitochondrial complex I subunit NDUFS4, which decreases the stability and activity of complex I. This leads to a reduction in the generation of reactive oxygen species by astrocytes and affects the glycolytic production of lactate through the hypoxia-inducible factor 1 pathway, eventually resulting in neuronal redox stress and impairment of behavioural responses in social interaction assays. Genetic and pharmacological correction of each of these effects abolishes the effect of cannabinoid treatment on the observed behaviour. These findings suggest that mtCB1 receptor signalling can directly regulate astroglial glucose metabolism to fine-tune neuronal activity and behaviour in mice.


Assuntos
Astrócitos/metabolismo , Metabolismo Energético , Glucose/metabolismo , Mitocôndrias/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Agonistas de Receptores de Canabinoides/farmacologia , Células Cultivadas , Dronabinol/farmacologia , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Ácido Láctico/metabolismo , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Oxirredução , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Receptor CB1 de Canabinoide/agonistas , Comportamento Social
2.
Neuron ; 107(3): 538-551.e7, 2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32502461

RESUMO

Pain is a source of substantial discomfort. Abnormal activity in both the zona incerta (ZI) and posterior complex of the thalamus (Po) are implicated in neuropathic pain, but their exact roles remain unclear. In particular, the precise cell types and molecular mechanisms of the ZI-Po circuit that regulate nociception are largely uncharacterized. Here, we found that parvalbumin (PV)-positive neuronal projections from the ventral ZI (ZIv) to the Po (ZIv-Po) are critical for promoting nocifensive behaviors, whereas selectively inhibiting ZIv-Po activity reduces nocifensive withdrawal responses. Furthermore, cannabinoid type 1 receptors (CB1Rs) are expressed specifically at ZIv-Po axon terminals in this circuit, and cannabinoids attenuate nocifensive responses through presynaptic inhibition. Selective inhibition of the ZIv-Po circuit or administration of cannabinoids into the Po are sufficient to ameliorate pathological pain. These findings identify the critical role of the ZIv-Po circuit and its modulation by endocannabinoids in controlling nocifensive behaviors.


Assuntos
Neurônios/fisiologia , Nociceptividade/fisiologia , Dor/fisiopatologia , Núcleos Posteriores do Tálamo/fisiologia , Receptor CB1 de Canabinoide/metabolismo , Zona Incerta/fisiologia , Animais , Comportamento Animal , Endocanabinoides , Camundongos , Inibição Neural , Vias Neurais , Neurônios/metabolismo , Dor/metabolismo , Parvalbuminas , Núcleos Posteriores do Tálamo/citologia , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Zona Incerta/citologia
3.
J Vasc Res ; 57(3): 152-163, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32248195

RESUMO

Palmitoylethanolamide is an endogenous lipid that exerts complex vascular effects, enhances the effects of endocannabinoids and induces a direct hypotension, but the mechanisms involved have been poorly explored. Hence, this study investigated in Wistar pithed rats the role of CB1, CB2, TRPV1 and GPR55 receptors in the inhibition by palmitoylethanolamide of the vasopressor responses produced by sympathetic stimulation or exogenous noradrenaline. Frequency- and dose-dependent vasopressor responses were analysed before and during intravenous (i.v.) continuous infusions of palmitoylethanolamide in animals receiving i.v. bolus of the antagonists NIDA41020 (CB1), AM630 (CB2), capsazepine (TRPV1), and/or cannabidiol (GPR55). Palmitoyletha-nolamide (0.1-3.1 µg/kg/min) dose-dependently inhibited the sympathetically induced and noradrenaline-induced vasopressor responses. Both inhibitions were: (i) partially blocked by 100 µg/kg NIDA41020, 100 µg/kg capsazepine, or 31 µg/kg cannabidiol; (ii) unaffected by 310 µg/kg AM630; and (iii) abolished by the combination NIDA41020 + capsazepine + cannabidiol (100, 100, and 31 µg/kg, respectively). The resting blood pressure was decreased by palmitoylethanolamide (effect prevented by NIDA41020, capsazepine or cannabidiol, but not by AM630). These results suggest that: (i) palmitoylethanolamide inhibits the vasopressor responses to sympathetic stimulation and exogenous noradrenaline and that it induces hypotension; and (ii) all these effects are mediated by prejunctional and vascular CB1, TRPV1 and probably GPR55, but not by CB2, receptors.


Assuntos
Artérias/efeitos dos fármacos , Etanolaminas/farmacologia , Ácidos Palmíticos/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Artérias/inervação , Artérias/metabolismo , Estado de Descerebração , Estimulação Elétrica , Masculino , Norepinefrina/farmacologia , Ratos Wistar , Receptor CB1 de Canabinoide/metabolismo , Receptores de Canabinoides/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Sistema Nervoso Simpático/fisiologia , Simpatomiméticos/farmacologia , Canais de Cátion TRPV/metabolismo
4.
Cell ; 180(4): 655-665.e18, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32004463

RESUMO

Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.


Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Receptor CB1 de Canabinoide/química , Receptor CB2 de Canabinoide/química , Transdução de Sinais , Regulação Alostérica , Sítio Alostérico , Animais , Células CHO , Agonistas de Receptores de Canabinoides/química , Canabinoides/química , Canabinoides/farmacologia , Linhagem Celular Tumoral , Colesterol/química , Colesterol/farmacologia , Cricetinae , Cricetulus , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Simulação de Dinâmica Molecular , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Células Sf9 , Spodoptera
5.
PLoS One ; 15(2): e0228729, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32053633

RESUMO

BACKGROUND: There is a correlation between the endocannabinoid system and hepatic fibrosis based on the activation of CB1 and CB2 receptors; where CB1 has profibrogenic effects. Gene therapy with a plasmid carrying a shRNA for CB1 delivered by hydrodynamic injection has the advantage of hepatic tropism, avoiding possible undesirable effects of CB1 pharmacological inhibition. OBJECTIVE: To evaluate hydrodynamics-based liver transfection in an experimental model of liver cirrhosis of a plasmid with the sequence of a shRNA for CB1 and its antifibrogenic effects. METHODS: Three shRNA (21pb) were designed for blocking CB1 mRNA at positions 877, 1232 and 1501 (pshCB1-A, B, C). Sequences were cloned in the pENTR™/U6. Safety was evaluated monitoring CB1 expression in brain tissue. The silencing effect was determined in rat HSC primary culture and CCl4 cirrhosis model. Hydrodynamic injection in cirrhotic liver was through iliac vein and with a dose of 3mg/kg plasmid. Serum levels of liver enzymes, mRNA levels of TGF-ß1, Col IA1 and α-SMA and the percentage of fibrotic tissue were analyzed. RESULTS: Hydrodynamic injection allows efficient CB1 silencing in cirrhotic livers and pshCB1-B (position 1232) demonstrated the main CB1-silencing. Using this plasmid, mRNA level of fibrogenic molecules and fibrotic tissue considerably decrease in cirrhotic animals. Brain expression of CB1 remained unaltered. CONCLUSION: Hydrodynamics allows a hepatotropic and secure transfection in cirrhotic animals. The sequence of the shCB1-B carried in a plasmid or any other vector has the potential to be used as therapeutic strategy for liver fibrosis.


Assuntos
Inativação Gênica , Hidrodinâmica , Cirrose Hepática/patologia , RNA Interferente Pequeno/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Actinas/genética , Actinas/metabolismo , Alanina Transaminase/sangue , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/sangue , Aspartato Aminotransferases/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Fígado/metabolismo , Masculino , Plasmídeos/metabolismo , RNA Interferente Pequeno/administração & dosagem , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/genética , Transfecção , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
6.
PLoS One ; 15(2): e0228909, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32049991

RESUMO

BACKGROUND/OBJECTIVE: Patients with non-small cell lung cancer (NSCLC) develop resistance to antitumor agents by mechanisms that involve the epithelial-to-mesenchymal transition (EMT). This necessitates the development of new complementary drugs, e.g., cannabinoid receptors (CB1 and CB2) agonists including tetrahydrocannabinol (THC) and cannabidiol (CBD). The combined use of THC and CBD confers greater benefits, as CBD enhances the effects of THC and reduces its psychotropic activity. We assessed the relationship between the expression levels of CB1 and CB2 to the clinical features of a cohort of patients with NSCLC, and the effect of THC and CBD (individually and in combination) on proliferation, EMT and migration in vitro in A549, H460 and H1792 lung cancer cell lines. METHODS: Expression levels of CB1, CB2, EGFR, CDH1, CDH2 and VIM were evaluated by quantitative reverse transcription-polymerase chain reaction. THC and CBD (10-100 µM), individually or in combination (1:1 ratio), were used for in vitro assays. Cell proliferation was determined by BrdU incorporation assay. Morphological changes in the cells were visualized by phase-contrast and fluorescence microscopy. Migration was studied by scratch recolonization induced by 20 ng/ml epidermal growth factor (EGF). RESULTS: The tumor samples were classified according to the level of expression of CB1, CB2, or both. Patients with high expression levels of CB1, CB2, and CB1/CB2 showed increased survival reaching significance for CB1 and CB1/CB2 (p = 0.035 and 0.025, respectively). Both cannabinoid agonists inhibited the proliferation and expression of EGFR in lung cancer cells, and CBD potentiated the effect of THC. THC and CBD alone or in combination restored the epithelial phenotype, as evidenced by increased expression of CDH1 and reduced expression of CDH2 and VIM, as well as by fluorescence analysis of cellular cytoskeleton. Finally, both cannabinoids reduced the in vitro migration of the three lung cancer cells lines used. CONCLUSIONS: The expression levels of CB1 and CB2 have a potential use as markers of survival in patients with NSCLC. THC and CBD inhibited the proliferation and expression of EGFR in the lung cancer cells studied. Finally, the THC/CBD combination restored the epithelial phenotype in vitro.


Assuntos
Canabidiol/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células/fisiologia , Dronabinol/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Neoplasias Pulmonares/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Células A549 , Adulto , Idoso , Idoso de 80 Anos ou mais , Agonistas de Receptores de Canabinoides/farmacologia , Canabinoides/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Psicotrópicos/farmacologia
7.
J Sports Med Phys Fitness ; 60(2): 320-328, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31974335

RESUMO

BACKGROUND: Exercise has been reported to enhance cognitive functions via mechanism(s) yet to be fully understood. The endogenous cannabinoid system (ECS) is involved in regulating cognitive function, including learning and memory. This system may also be involved in enhancing learning and memory after exercise. The objective of this study is to explore whether and how ECS participates in the enhancement of learning and memory after exercise. METHODS: In this study, a treadmill exercise training model was established. Wild-type C57BL/6J mice and those deficient in the cannabinoid receptor 1 (CB1R) coding gene, Cnr1, specifically in the glutamatergic neurons, γ-aminobutyric acid (GABA) neurons or glial cells were randomly grouped for 4 weeks' moderate treadmill exercise. The Morris water maze was used to evaluate the spatial learning and memory abilities of mice in each group. The expression of brain-derived neurotrophic factor (BDNF) and CB1R in hippocampus was detected by western blot. The dendritic spine density of pyramidal cells in the hippocampal CA1 region was analyzed by quantitative Golgi staining. This study consisted of eight single-factor inter-subject designs, and each batch of experiments was divided into two groups. Corresponding experimental items and data analysis were carried out according to the experimental objectives. RESULTS: CB1R antagonist administration or CB1R knockout in glutamatergic neurons eliminated the effect of exercise on learning and memory, and counteracted exercise-elicited upregulation of BDNF in the hippocampus; CB1R-specific knockout on GABAergic neurons and glial cells did not affect the moderate exercise-induced enhancement of learning and memory. In addition, the results of Golgi staining showed that exercise increased dendritic spine density in hippocampal neurons, which was abolished by specific CB1R depletion in glutamatergic neurons. CONCLUSIONS: The ECS, particularly CB1R signaling in glutamatergic neurons, mediates the enhancement of learning and memory by exercise, which involves increased BDNF production and dendritic spine density.


Assuntos
Endocanabinoides/metabolismo , Aprendizagem , Memória , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Condicionamento Físico Animal , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo
8.
Psychopharmacology (Berl) ; 237(4): 1063-1079, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31919563

RESUMO

RATIONALE: The behavioural effects elicited by chemical constituents of Cannabis sativa, such as cannabidiol (CBD), on the ventromedial hypothalamus (VMH) are not well understood. There is evidence that VMH neurons play a relevant role in the modulation of unconditioned fear-related defensive behavioural reactions displayed by laboratory animals. OBJECTIVES: This study was designed to explore the specific pattern of distribution of the CB1 receptors in the VMH and to investigate the role played by this cannabinoid receptor in the effect of CBD on the control of defensive behaviours and unconditioned fear-induced antinociception. METHODS: A panic attack-like state was triggered in Wistar rats by intra-VMH microinjections of N-methyl-D-aspartate (NMDA). One of three different doses of CBD was microinjected into the VMH prior to local administration of NMDA. In addition, the most effective dose of CBD was used after pre-treatment with the CB1 receptor selective antagonist AM251, followed by NMDA microinjections in the VMH. RESULTS: The morphological procedures demonstrated distribution of labelled CB1 receptors on neuronal perikarya situated in dorsomedial, central and ventrolateral divisions of the VMH. The neuropharmacological approaches showed that both panic attack-like behaviours and unconditioned fear-induced antinociception decreased after intra-hypothalamic microinjections of CBD at the highest dose (100 nmol). These effects, however, were blocked by the administration of the CB1 receptor antagonist AM251 (100 pmol) in the VMH. CONCLUSION: These findings suggest that CBD causes panicolytic-like effects and reduces unconditioned fear-induced antinociception when administered in the VMH, and these effects are mediated by the CB1 receptor-endocannabinoid signalling mechanism in VMH.


Assuntos
Canabidiol/toxicidade , Medo/fisiologia , Medição da Dor/métodos , Transtorno de Pânico/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Núcleo Hipotalâmico Ventromedial/metabolismo , Animais , Canabidiol/administração & dosagem , Medo/efeitos dos fármacos , Medo/psicologia , Injeções Intraventriculares , Masculino , N-Metilaspartato/administração & dosagem , Medição da Dor/efeitos dos fármacos , Medição da Dor/psicologia , Transtorno de Pânico/induzido quimicamente , Piperidinas/administração & dosagem , Pirazóis/administração & dosagem , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/antagonistas & inibidores , Núcleo Hipotalâmico Ventromedial/efeitos dos fármacos
9.
Molecules ; 25(2)2020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31968549

RESUMO

Cannabinoid receptor 1 (CB1) activation has been reported to reduce transient receptor potential cation channel subfamily V member 1 (TRPV1)-induced inflammatory responses and is anti-nociceptive and anti-inflammatory in corneal injury. We examined whether allosteric ligands, can modulate CB1 signaling to reduce pain and inflammation in corneal hyperalgesia. Corneal hyperalgesia was generated by chemical cauterization of cornea in wildtype and CB2 knockout (CB2-/-) mice. The novel racemic CB1 allosteric ligand GAT211 and its enantiomers GAT228 and GAT229 were examined alone or in combination with the orthosteric CB1 agonist Δ8-tetrahydrocannabinol (Δ8-THC). Pain responses were assessed following capsaicin (1 µM) stimulation of injured corneas at 6 h post-cauterization. Corneal neutrophil infiltration was also analyzed. GAT228, but not GAT229 or GAT211, reduced pain scores in response to capsaicin stimulation. Combination treatments of 0.5% GAT229 or 1% GAT211 with subthreshold Δ8-THC (0.4%) significantly reduced pain scores following capsaicin stimulation. The anti-nociceptive effects of both GAT229 and GAT228 were blocked with CB1 antagonist AM251, but remained unaffected in CB2-/- mice. Two percent GAT228, or the combination of 0.2% Δ8-THC with 0.5% GAT229 also significantly reduced corneal inflammation. CB1 allosteric ligands could offer a novel approach for treating corneal pain and inflammation.


Assuntos
Lesões da Córnea/tratamento farmacológico , Dronabinol/análogos & derivados , Hiperalgesia/tratamento farmacológico , Indóis/administração & dosagem , Inflamação/tratamento farmacológico , Receptor CB1 de Canabinoide/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Cauterização , Lesões da Córnea/complicações , Lesões da Córnea/etiologia , Modelos Animais de Doenças , Dronabinol/administração & dosagem , Dronabinol/farmacologia , Sinergismo Farmacológico , Técnicas de Inativação de Genes , Hiperalgesia/metabolismo , Indóis/farmacologia , Inflamação/etiologia , Inflamação/metabolismo , Ligantes , Camundongos , Receptor CB1 de Canabinoide/química , Receptor CB2 de Canabinoide/genética , Transdução de Sinais
10.
Chemistry ; 26(6): 1380-1387, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31961047

RESUMO

The endocannabinoid (eCB) system is implied in various human diseases ranging from central nervous system to autoimmune disorders. Cannabinoid receptor 2 (CB2 R) is an integral component of the eCB system. Yet, the downstream effects elicited by this G protein-coupled receptor upon binding of endogenous or synthetic ligands are insufficiently understood-likely due to the limited arsenal of reliable biological and chemical tools. Herein, we report the design and synthesis of CB2 R-selective cannabinoids along with their in vitro pharmacological characterization (binding and functional studies). They combine structural features of HU-308 and AM841 to give chimeric ligands that emerge as potent CB2 R agonists with high selectivity over the closely related cannabinoid receptor 1 (CB1 R). The synthesis work includes convenient preparation of substituted resorcinols often found in cannabinoids. The utility of the synthetic cannabinoids in this study is showcased by preparation of the most selective high-affinity fluorescent probe for CB2 R to date.


Assuntos
Aminas/química , Canabinoides/química , Dronabinol/análogos & derivados , Receptor CB2 de Canabinoide/metabolismo , Sítios de Ligação , Canabinoides/metabolismo , Dronabinol/química , Dronabinol/metabolismo , Humanos , Cinética , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/química
11.
Int J Mol Sci ; 21(2)2020 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-31940843

RESUMO

Contact hypersensitivity (CHS) is an established animal model for allergic contact dermatitis. Dendritic cells (DCs) play an important role in the sensitization phase of CHS by initiating T cell responses to topically applied haptens. The cannabinoid receptors 1 (CB1) and 2 (CB2) modulate DC functions and inflammatory skin responses, but their influence on the capacity of haptenized DCs to induce CHS is still unknown. We found lower CHS responses to 2,4-dinitro-1-fluorobenzene (DNFB) in wild type (WT) mice after adoptive transfer of haptenized Cnr2-/- and Cnr1-/-/Cnr2-/- bone marrow (BM) DCs as compared to transfer of WT DCs. In contrast, induction of CHS was not affected in WT recipients after transfer of Cnr1-/- DCs. In vitro stimulated Cnr2-/- DCs showed lower CCR7 and CXCR4 expression when compared to WT cells, while in vitro migration towards the chemokine ligands was not affected by CB2. Upregulation of MHC class II and co-stimulatory molecules was also reduced in Cnr2-/- DCs. This study demonstrates that CB2 modulates the maturation phenotype of DCs but not their chemotactic capacities in vitro. These findings and the fact that CHS responses mediated by Cnr2-/- DCs are reduced suggest that CB2 is a promising target for the treatment of inflammatory skin conditions.


Assuntos
Células Dendríticas/imunologia , Dermatite Alérgica de Contato/imunologia , Receptor CB2 de Canabinoide/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Quimiotaxia , Células Dendríticas/citologia , Dermatite Alérgica de Contato/genética , Dinitrofluorbenzeno/toxicidade , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/genética , Receptores CCR4/genética , Receptores CCR4/metabolismo , Receptores CCR7/genética , Receptores CCR7/metabolismo
12.
J Biomed Sci ; 27(1): 7, 2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31915019

RESUMO

BACKGROUND: Stress-induced analgesia (SIA) is an evolutionarily conserved phenomenon during stress. Neuropeptide S (NPS), orexins, substance P, glutamate and endocannabinoids are known to be involved in stress and/or SIA, however their causal links remain unclear. Here, we reveal an unprecedented sequential cascade involving these mediators in the lateral hypothalamus (LH) and ventrolateral periaqueductal gray (vlPAG) using a restraint stress-induced SIA model. METHODS: Male C57BL/6 mice of 8-12 week-old were subjected to intra-cerebroventricular (i.c.v.) and/or intra-vlPAG (i.pag.) microinjection of NPS, orexin-A or substance P alone or in combination with selective antagonists of NPS receptors (NPSRs), OX1 receptors (OX1Rs), NK1 receptors (NK1Rs), mGlu5 receptors (mGlu5Rs) and CB1 receptors (CB1Rs), respectively. Antinociceptive effects of these mediators were evaluated via the hot-plate test. SIA in mice was induced by a 30-min restraint stress. NPS levels in the LH and substance P levels in vlPAG homogenates were compared in restrained and unrestrained mice. RESULTS: NPS (i.c.v., but not i.pag.) induced antinociception. This effect was prevented by i.c.v. blockade of NPSRs. Substance P (i.pag.) and orexin-A (i.pag.) also induced antinociception. Substance P (i.pag.)-induced antinociception was prevented by i.pag. Blockade of NK1Rs, mGlu5Rs or CB1Rs. Orexin-A (i.pag.)-induced antinociception has been shown previously to be prevented by i.pag. blockade of OX1Rs or CB1Rs, and here was prevented by NK1R or mGlu5R antagonist (i.pag.). NPS (i.c.v.)-induced antinociception was prevented by i.pag. blockade of OX1Rs, NK1Rs, mGlu5Rs or CB1Rs. SIA has been previously shown to be prevented by i.pag. blockade of OX1Rs or CB1Rs. Here, we found that SIA was also prevented by i.c.v. blockade of NPSRs or i.pag. blockade of NK1Rs or mGlu5Rs. Restrained mice had higher levels of NPS in the LH and substance P in the vlPAG than unrestrained mice. CONCLUSIONS: These results suggest that, during stress, NPS is released and activates LH orexin neurons via NPSRs, releasing orexins in the vlPAG. Orexins then activate OX1Rs on substance P-containing neurons in the vlPAG to release substance P that subsequently. Activates NK1Rs on glutamatergic neurons to release glutamate. Glutamate then activates perisynaptic mGlu5Rs to initiate the endocannabinoid retrograde inhibition of GABAergic transmission in the vlPAG, leading to analgesia.


Assuntos
Analgesia , Neuropeptídeos/metabolismo , Receptores de Orexina/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Estresse Psicológico/metabolismo , Núcleos Ventrais do Tálamo/metabolismo , Animais , Masculino , Camundongos , Estresse Psicológico/patologia , Estresse Psicológico/fisiopatologia , Núcleos Ventrais do Tálamo/patologia , Núcleos Ventrais do Tálamo/fisiopatologia
13.
Nat Rev Neurol ; 16(1): 9-29, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31831863

RESUMO

Anecdotal evidence that cannabis preparations have medical benefits together with the discovery of the psychotropic plant cannabinoid Δ9-tetrahydrocannabinol (THC) initiated efforts to develop cannabinoid-based therapeutics. These efforts have been marked by disappointment, especially in relation to the unwanted central effects that result from activation of cannabinoid receptor 1 (CB1), which have limited the therapeutic use of drugs that activate or inactivate this receptor. The discovery of CB2 and of endogenous cannabinoid receptor ligands (endocannabinoids) raised new possibilities for safe targeting of this endocannabinoid system. However, clinical success has been limited, complicated by the discovery of an expanded endocannabinoid system - known as the endocannabinoidome - that includes several mediators that are biochemically related to the endocannabinoids, and their receptors and metabolic enzymes. The approvals of nabiximols, a mixture of THC and the non-psychotropic cannabinoid cannabidiol, for the treatment of spasticity and neuropathic pain in multiple sclerosis, and of purified botanical cannabidiol for the treatment of otherwise untreatable forms of paediatric epilepsy, have brought the therapeutic use of cannabinoids and endocannabinoids in neurological diseases into the limelight. In this Review, we provide an overview of the endocannabinoid system and the endocannabinoidome before discussing their involvement in and clinical relevance to a variety of neurological disorders, including Parkinson disease, Alzheimer disease, Huntington disease, multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain injury, stroke, epilepsy and glioblastoma.


Assuntos
Canabinoides/metabolismo , Endocanabinoides/metabolismo , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/metabolismo , Analgésicos/metabolismo , Analgésicos/uso terapêutico , Animais , Canabidiol/metabolismo , Canabidiol/uso terapêutico , Canabinoides/uso terapêutico , Dronabinol/metabolismo , Dronabinol/uso terapêutico , Combinação de Medicamentos , Endocanabinoides/uso terapêutico , Humanos , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo
14.
J Ethnopharmacol ; 246: 112218, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31494202

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Located throughout the body, cannabinoid receptors (CB1 and CB2) are therapeutic targets for obesity/metabolic diseases, neurological/mental disorders, and immune modulation. Phytocannabinoids are greatly important for the development of new medicines with high efficacy and/or minor side effects. Plants and fungi are used in traditional medicine for beneficial effects to mental and immune system. The current research studied five fungi from the genus Ganoderma and five plants: Ganoderma hainanense J.D. Zhao, L.W. Hsu & X.Q. Zhang; Ganoderma capense (Lloyd) Teng, Zhong Guo De Zhen Jun; Ganoderma cochlear (Blume & T. Nees) Bres., Hedwigia; Ganoderma resinaceum Boud.; Ganoderma applanatum (Pers.) Pat.; Carthamus tinctorius L. (Compositae); Cynanchum otophyllum C. K. Schneid. (Asclepiadaceae); Coffea arabica L. (Rubiaceae); Prinsepia utilis Royle (Rosaceae); Lepidium meyenii Walp. (Brassicaceae). They show immunoregulation, promotion of longevity and maintenance of vitality, stimulant effects on the central nervous system, hormone balance and other beneficial effects. However, it remains unclear whether cannabinoid receptors are involved in these effects. AIM OF THE STUDY: This work aimed to identify components working on CB1 and CB2 from the above plants and fungi, as novel phytocannabinoids, and to investigate mechanisms of how these compounds affected the cells. By analyzing the structure-activity relationship, we could identify the core structure for future development. MATERIALS AND METHODS: Eighty-two natural compounds were screened on stably transfected Chinese hamster ovary (CHO) cell lines, CHO-CB1 and CHO-CB2, with application of a label-free dynamic mass redistribution (DMR) technology that measured cellular responses to compounds. CP55,940 and WIN55,212-2 were agonist probe molecules, and SR141716A and SR144528 were antagonist probes. Pertussis toxin, cholera toxin, LY294002 and U73122 were signaling pathway inhibitors. The DMR data were acquired by Epic Imager software (Corning, NY), processed by Imager Beta 3.7 (Corning), and analyzed by GraphPad Prism 6 (GraphPad Software, San Diego, CA). RESULTS: Transfected CHO-CB1 and CHO-CB2 cell lines were established and characterized. Seven compounds induced responses/activities in the cells. Among the seven compounds, four were purified from two Ganoderma species with potencies between 20 and 35 µM. Three antagonists: Kfb68 antagonized both receptors with a better desensitizing effect on CB2 to WIN55,212-2 over CP55,940. Kga1 and Kfb28 were antagonists selective to CB1 and CB2, respectively. Kfb77 was a special agonist and it stimulated CB1 in a mechanism different from that of CP55,940. Another three active compounds, derived from the Lepidium meyenii Walp. (Brassicaceae), were also identified but their effects were mediated through mechanisms much related to the signaling transduction pathways, especially through the stimulatory Gs protein. CONCLUSIONS: We identified four natural cannabinoids that exhibited structural and functional diversities. Our work confirms the presence of active ingredients in the Ganoderma species to CB1 and CB2, and this finding establishes connections between the fungi and the cannabinoid receptors, which will serve as a starting point to connect their beneficial effects to the endocannabinoid system. This research will also enrich the inventory of cannabinoids and phytocannabinoids from fungi. Yet due to some limitations, further structure-activity relationship studies and mechanism investigation are warranted in future.


Assuntos
Canabinoides/química , Canabinoides/farmacologia , Ganoderma/química , Magnoliopsida/química , Animais , Células CHO , Canabinoides/metabolismo , Cricetinae , Cricetulus , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Relação Estrutura-Atividade
15.
Proc Natl Acad Sci U S A ; 117(1): 650-655, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31843894

RESUMO

Even a brief exposure to severe stress strengthens synaptic connectivity days later in the amygdala, a brain area implicated in the affective symptoms of stress-related psychiatric disorders. However, little is known about the synaptic signaling mechanisms during stress that eventually culminate in its delayed impact on the amygdala. Hence, we investigated early stress-induced changes in amygdalar synaptic signaling in order to prevent its delayed effects. Whole-cell recordings in basolateral amygdala (BLA) slices from rats revealed higher frequency of miniature excitatory postsynaptic currents (mEPSCs) immediately after 2-h immobilization stress. This was replicated by inhibition of cannabinoid receptors (CB1R), suggesting a role for endocannabinoid (eCB) signaling. Stress also reduced N-arachidonoylethanolamine (AEA), an endogenous ligand of CB1R. Since stress-induced activation of fatty acid amide hydrolase (FAAH) reduces AEA, we confirmed that oral administration of an FAAH inhibitor during stress prevents the increase in synaptic excitation in the BLA soon after stress. Although stress also caused an immediate reduction in synaptic inhibition, this was not prevented by FAAH inhibition. Strikingly, FAAH inhibition during the traumatic stressor was also effective 10 d later on the delayed manifestation of synaptic strengthening in BLA neurons, preventing both enhanced mEPSC frequency and increased dendritic spine-density. Thus, oral administration of an FAAH inhibitor during a brief stress prevents the early synaptic changes that eventually build up to hyperexcitability in the amygdala. This framework is of therapeutic relevance because of growing interest in targeting eCB signaling to prevent the gradual development of emotional symptoms and underlying amygdalar dysfunction triggered by traumatic stress.


Assuntos
Complexo Nuclear Basolateral da Amígdala/fisiologia , Emoções/efeitos dos fármacos , Endocanabinoides/metabolismo , Transdução de Sinais/fisiologia , Estresse Psicológico/fisiopatologia , Administração Oral , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Animais , Antagonistas de Receptores de Canabinoides/administração & dosagem , Modelos Animais de Doenças , Emoções/fisiologia , Inibidores Enzimáticos/administração & dosagem , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Humanos , Masculino , Ratos , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/psicologia
16.
J Pharm Pharmacol ; 72(1): 84-91, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31722122

RESUMO

OBJECTIVES: To determine if diminished orthosteric agonist binding due to mutations in extracellular loops 1 or 2 of the cannabinoid receptor 1 (CB1 ) can be overcome by an allosteric modulator and restore agonist binding. METHODS: Binding assays were performed using a range of concentrations of orthosteric compound, in the presence or absence of a set concentration of the allosteric modulator PSNCBAM-1 to determine the EC50 in its absence or presence. KEY FINDINGS: Single mutations in extracellular loop 1 or 2 of CB1 showed weak or no binding of agonist CP55940 to the receptor. Interestingly, upon addition of the allosteric modulator PSNCBAM-1, this binding was restored typically to wild-type CB1 levels. In a few cases, the allosteric modulator ORG27569 was compared with PSNCBAM-1 for CP55940 binding and it also restored binding. Further, wild-type levels of inverse agonist bound the CB1 mutants in the absence of modulator, suggesting the mutants were originally folded like the wild type. CONCLUSIONS: Based on our findings, we provide evidence of a therapeutic application for allosteric modulators in situations where a mutation in the receptor may hinder its function. By utilizing allosteric modulators, restoration of orthosteric binding may be possible.


Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Cicloexanóis/farmacologia , Indóis/farmacologia , Compostos de Fenilureia/farmacologia , Piperidinas/farmacologia , Piridinas/farmacologia , Receptor CB1 de Canabinoide/agonistas , Rimonabanto/farmacologia , Sítios de Ligação , Agonistas de Receptores de Canabinoides/metabolismo , Cicloexanóis/metabolismo , Células HEK293 , Humanos , Indóis/metabolismo , Ligantes , Mutação , Compostos de Fenilureia/metabolismo , Piperidinas/metabolismo , Ligação Proteica , Conformação Proteica , Piridinas/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Rimonabanto/metabolismo , Relação Estrutura-Atividade
17.
Cell Rep ; 29(13): 4295-4307.e6, 2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31875541

RESUMO

A large number of experiments have indicated that precise spike times, firing rates, and synapse locations crucially determine the dynamics of long-term plasticity induction in excitatory synapses. However, it remains unknown how plasticity mechanisms of synapses distributed along dendritic trees cooperate to produce the wide spectrum of outcomes for various plasticity protocols. Here, we propose a four-pathway plasticity framework that is well grounded in experimental evidence and apply it to a biophysically realistic cortical pyramidal neuron model. We show in computer simulations that several seemingly contradictory experimental landmark studies are consistent with one unifying set of mechanisms when considering the effects of signal propagation in dendritic trees with respect to synapse location. Our model identifies specific spatiotemporal contributions of dendritic and axo-somatic spikes as well as of subthreshold activation of synaptic clusters, providing a unified parsimonious explanation not only for rate and timing dependence but also for location dependence of synaptic changes.


Assuntos
Córtex Cerebral/fisiologia , Dendritos/metabolismo , Potenciação de Longa Duração/fisiologia , Modelos Neurológicos , Células Piramidais/metabolismo , Sinapses/fisiologia , Potenciais de Ação/fisiologia , Animais , Córtex Cerebral/citologia , Simulação por Computador , Dendritos/ultraestrutura , Expressão Gênica , Hipocampo/citologia , Hipocampo/fisiologia , Técnicas de Patch-Clamp , Células Piramidais/ultraestrutura , Ratos , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/ultraestrutura , Transmissão Sináptica/fisiologia
18.
Int J Mol Sci ; 21(1)2019 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-31881740

RESUMO

The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron's axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search identified stathmin-2, a protein controlling microtubule dynamics, previously demonstrated to be coexpressed with CB1R and now shown to be downregulated upon interference with CB1R in zebrafish. Together, these results raise the likely possibility that embryonic exposure to low doses of CB1R-interfering compounds could impact on the development of the neuroendocrine systems controlling sexual maturation, reproduction and food intake.


Assuntos
Proteína Relacionada com Agouti/metabolismo , Axônios/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Ácido Pirrolidonocarboxílico/análogos & derivados , Receptor CB1 de Canabinoide/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Benzoxazinas/farmacologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Morfolinas/farmacologia , Morfolinos/metabolismo , Naftalenos/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ácido Pirrolidonocarboxílico/metabolismo , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética
19.
Int J Mol Sci ; 20(23)2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31771126

RESUMO

The cannabinoid receptor type 1 (CB1R), a G protein-coupled receptor (GPCR), plays an essential role in the control of many physiological processes such as hunger, memory loss, gastrointestinal activity, catalepsy, fear, depression, and chronic pain. Therefore, it is an attractive target for drug discovery to manage pain, neurodegenerative disorders, obesity, and substance abuse. However, the psychoactive adverse effects, generated by CB1R activation in the brain, limit the use of the orthosteric CB1R ligands as drugs. The discovery of CB1R allosteric modulators during the last decade provided new tools to target the CB1R. Moreover, application of the site-directed mutagenesis in combination with advanced physical methods, especially X-ray crystallography and computational modeling, has opened new horizons for understanding the complexity of the structure, function, and activity of cannabinoid receptors. In this paper, we present the latest advances in research on the CB1R, its allosteric modulation and allosteric ligands, and their translational potential. We focused on structural essentials of the cannabinoid 1 receptor- ligand (drug) interactions, as well as modes of CB1R signaling regulation.


Assuntos
Agonistas de Receptores de Canabinoides/química , Receptor CB1 de Canabinoide , Regulação Alostérica , Animais , Agonistas de Receptores de Canabinoides/uso terapêutico , Cristalografia por Raios X , Humanos , Mutagênese Sítio-Dirigida , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Dor/tratamento farmacológico , Dor/genética , Dor/metabolismo , Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Relação Estrutura-Atividade , Transtornos Relacionados ao Uso de Substâncias/tratamento farmacológico , Transtornos Relacionados ao Uso de Substâncias/genética , Transtornos Relacionados ao Uso de Substâncias/metabolismo
20.
PLoS One ; 14(11): e0220542, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31738771

RESUMO

The impact of performing exercise on the immune system presents contrasting effects on health when performed at different intensities. In addition, the consequences of performing chronic exercise have not been sufficiently studied in contrast to the effects of acute bouts of exercise. The porpoise of this work was to determine the effect that a popular exercise regimen (chronic/moderate/aerobic exercise) has on the proportion of different immune cell subsets, their function and if it affects the cannabinoid system with potentially functional implications on the immune system. A marked increase in several immune cell subsets and their expression of cannabinoid receptors was expected, as well as an enhanced proliferative and cytotoxic activity by total splenocytes in exercised animals. For this study male Wistar rats performed treadmill running 5 times a week for a period of 10 weeks, at moderate intensity. Our results showed a significant decrease in lymphocyte subpopulations (CD4+, Tγδ, and CD45 RA+ cells) and an increase in the cannabinoid receptors expression in those same cell. Although functional assays did not reveal any variation in total immunoglobulin production or NK cells cytotoxic activity, proliferative capability of total splenocytes increased in trained rats. Our results further support the notion that exercise affects the immunological system and extends the description of underlying mechanisms mediating such effects. Altogether, our results contribute to the understanding of the benefits of exercise on the practitioner´s general health.


Assuntos
Imunidade Celular , Condicionamento Físico Animal/fisiologia , Receptores de Canabinoides/metabolismo , Animais , Composição Corporal , Proliferação de Células , Corticosterona/sangue , Citotoxicidade Imunológica , Imunoglobulina G/metabolismo , Ativação Linfocitária , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Masculino , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Corrida/fisiologia
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