RESUMEN
2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid (EC), acting as a full agonist at both CB1 and CB2 cannabinoid receptors. It is synthesized on demand in postsynaptic membranes through the sequential action of phosphoinositide-specific phospholipase Cß1 (PLCß1) and diacylglycerol lipase α (DAGLα), contributing to retrograde signaling upon interaction with presynaptic CB1. However, 2-AG production might also involve various combinations of PLC and DAGL isoforms, as well as additional intracellular pathways implying other enzymes and substrates. Three other alternative pathways of 2-AG synthesis rest on the extracellular cleavage of 2-arachidonoyl-lysophospholipids by three different hydrolases: glycerophosphodiesterase 3 (GDE3), lipid phosphate phosphatases (LPPs), and two members of ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP6-7). We propose the names of AlterAG-1, -2, and -3 for three pathways sharing an ectocellular localization, allowing them to convert extracellular lysophospholipid mediators into 2-AG, thus inducing typical signaling switches between various G-protein-coupled receptors (GPCRs). This implies the critical importance of the regioisomerism of both lysophospholipid (LPLs) and 2-AG, which is the object of deep analysis within this review. The precise functional roles of AlterAGs are still poorly understood and will require gene invalidation approaches, knowing that both 2-AG and its related lysophospholipids are involved in numerous aspects of physiology and pathology, including cancer, inflammation, immune defenses, obesity, bone development, neurodegeneration, or psychiatric disorders.
Asunto(s)
Ácidos Araquidónicos , Endocannabinoides , Glicéridos , Lisofosfolípidos , Transducción de Señal , Endocannabinoides/metabolismo , Glicéridos/metabolismo , Lisofosfolípidos/metabolismo , Humanos , Ácidos Araquidónicos/metabolismo , Animales , Hidrolasas Diéster Fosfóricas/metabolismoRESUMEN
With impaired retinal ganglion cell (RGC) function and eventual RGC death, there is a heightened risk of experiencing glaucoma-induced blindness or other optic neuropathies. Poor RGC efficiency leads to limited transmission of visual signals between the retina and the brain by RGC axons. Increased focus on studying lipid messengers found in neurons such as endocannabinoids (eCBs) has importance due to their potential axonal pathway regenerative properties. 2-Arachidonoylglycerol (2-AG), a common eCB, is synthesized from an sn-1 hydrolysis reaction between diacylglycerol (DAG) and diacylglycerol lipase (DAGL). Examination of DAG production allows for future downstream analysis in relation to DAGL functionality. Here, we describe protocol guidelines for extracting RGCs from mouse retinas and subsequent mass spectrometry analysis of the DAG content present within the RGCs.
Asunto(s)
Diglicéridos , Células Ganglionares de la Retina , Transducción de Señal , Células Ganglionares de la Retina/metabolismo , Animales , Ratones , Diglicéridos/metabolismo , Endocannabinoides/metabolismo , Glicéridos/metabolismo , Lipoproteína Lipasa/metabolismo , Ácidos Araquidónicos/metabolismo , Espectrometría de Masas/métodos , Retina/metabolismoRESUMEN
Three decades ago, the first endocannabinoid, anandamide (AEA), was identified, and its analgesic effect was recognized in humans and preclinical models. However, clinical trial failures pointed out the complexity of the AEA-induced analgesia. The first synapses in the superficial laminae of the spinal cord dorsal horn represent an important modulatory site in nociceptive transmission and subsequent pain perception. The glutamatergic synaptic transmission at these synapses is strongly modulated by two primary AEA-activated receptors, cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid 1 (TRPV1), both highly expressed on the presynaptic side formed by the endings of primary nociceptive neurons. Activation of these receptors can have predominantly inhibitory (CB1) and excitatory (TRPV1) effects that are further modulated under pathological conditions. In addition, dual AEA-mediated signaling and action may occur in primary sensory neurons and dorsal horn synapses. AEA application causes balanced inhibition and excitation of primary afferent synaptic input on superficial dorsal horn neurons in normal conditions, whereas peripheral inflammation promotes AEA-mediated inhibition. This review focuses mainly on the modulation of synaptic transmission at the spinal cord level and signaling in primary nociceptive neurons by AEA via CB1 and TRPV1 receptors. Furthermore, the spinal analgesic effect in preclinical studies and clinical aspects of AEA-mediated analgesia are considered.
Asunto(s)
Ácidos Araquidónicos , Endocannabinoides , Alcamidas Poliinsaturadas , Médula Espinal , Transmisión Sináptica , Endocannabinoides/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Animales , Humanos , Ácidos Araquidónicos/metabolismo , Ácidos Araquidónicos/farmacología , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Transmisión Sináptica/fisiología , Transmisión Sináptica/efectos de los fármacos , Nocicepción/fisiología , Nocicepción/efectos de los fármacos , Nociceptores/metabolismo , Nociceptores/efectos de los fármacos , Nociceptores/fisiología , Receptor Cannabinoide CB1/metabolismo , Canales Catiónicos TRPV/metabolismoRESUMEN
Neuroinflammation is a hallmark of several neurodegenerative disorders that has been extensively studied in recent years. Microglia, the primary immune cells of the central nervous system (CNS), are key players in this physiological process, demonstrating a remarkable adaptability in responding to various stimuli in the eye and the brain. Within the complex network of neuroinflammatory signals, the fatty acid N-ethanolamines, in particular N-arachidonylethanolamine (anandamide, AEA), emerged as crucial regulators of microglial activity under both physiological and pathological states. In this study, we interrogated for the first time the impact of the signaling of these bioactive lipids on microglial cell responses to a sub-lethal acute UVB radiation, a physical stressor responsible of microglia reactivity in either the retina or the brain. To this end, we developed an in vitro model using mouse microglial BV-2 cells. Upon 24 h of UVB exposure, BV-2 cells showed elevated oxidative stress markers and, cyclooxygenase (COX-2) expression, enhanced phagocytic and chemotactic activities, along with an altered immune profiling. Notably, UVB exposure led to a selective increase in expression and activity of fatty acid amide hydrolase (FAAH), the main enzyme responsible for degradation of fatty acid ethanolamides. Pharmacological FAAH inhibition via URB597 counteracted the effects of UVB exposure, decreasing tumor necrosis factor α (TNF-α) and nitric oxide (NO) release and reverting reactive oxidative species (ROS), interleukin-1ß (IL-1ß), and interleukin-10 (IL-10) levels to the control levels. Our findings support the potential of enhanced fatty acid amide signaling in mitigating UVB-induced cellular damage, paving the way to further exploration of these lipids in light-induced immune responses.
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Amidohidrolasas , Microglía , Rayos Ultravioleta , Microglía/metabolismo , Microglía/efectos de los fármacos , Microglía/efectos de la radiación , Animales , Ratones , Amidohidrolasas/metabolismo , Amidohidrolasas/antagonistas & inhibidores , Rayos Ultravioleta/efectos adversos , Línea Celular , Carbamatos/farmacología , Benzamidas/farmacología , Estrés Oxidativo/efectos de los fármacos , Endocannabinoides/metabolismo , Ciclooxigenasa 2/metabolismo , Ciclooxigenasa 2/genética , Ácidos Araquidónicos/metabolismo , Ácidos Araquidónicos/farmacología , Interleucina-10/metabolismo , Alcamidas PoliinsaturadasRESUMEN
Understanding the endocannabinoid system in C. elegans may offer insights into basic biological processes and potential therapeutic targets for managing pain and inflammation in human. It is well established that anandamide modulates pain perception by binding to cannabinoid and vanilloid receptors, regulating neurotransmitter release and neuronal activity. One objective of this study was to demonstrate the suitability of C. elegans as a model organism for assessing the antinociceptive properties of bioactive compounds and learning about the role of endocannabinoid system in C. elegans. The evaluation of the compound anandamide (AEA) revealed antinociceptive activity by impeding C. elegans nocifensive response to noxious heat. Proteomic and bioinformatic investigations uncovered several pathways activated by AEA. Enrichment analysis unveiled significant involvement of ion homeostasis pathways, which are crucial for maintaining neuronal function and synaptic transmission, suggesting AEA's impact on neurotransmitter release and synaptic plasticity. Additionally, pathways related to translation, protein synthesis, and mTORC1 signaling were enriched, highlighting potential mechanisms underlying AEA's antinociceptive effects. Thermal proteome profiling identified NPR-32 and NPR-19 as primary targets of AEA, along with OCR-2, Cathepsin B, Progranulin, Transthyretin, and ribosomal proteins. These findings suggest a complex interplay between AEA and various cellular processes implicated in nociceptive pathways and inflammation modulation. Further investigation into these interactions could provide valuable insights into the therapeutic potential of AEA and its targets for the management of pain-related conditions.
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Ácidos Araquidónicos , Caenorhabditis elegans , Endocannabinoides , Alcamidas Poliinsaturadas , Canales Catiónicos TRPV , Animales , Caenorhabditis elegans/metabolismo , Endocannabinoides/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Alcamidas Poliinsaturadas/farmacología , Canales Catiónicos TRPV/metabolismo , Ácidos Araquidónicos/metabolismo , Ácidos Araquidónicos/farmacología , Receptores de Cannabinoides/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Reacción de Prevención/efectos de los fármacos , Reacción de Prevención/fisiología , Calor , Analgésicos/farmacologíaRESUMEN
BACKGROUND: Schizophrenia (SCZ) is a severe mental disorder, but its pathogenesis is still unknown, and its clinical treatment effect is very limited. Transient receptor potential vanilloid 1 (TRPV1) channel and the Endocannabinoid System (ECS)have been confirmed to be involved in the pathogenesis of SCZ, although their actions have not been fully clarified yet. The objective is to examine TRPV1 and ECS expression in the blood of schizophrenia patients and investigate their correlation with disease severity. METHODS: This is a cross-sectional investigation. Peripheral blood samples were gathered from normal controls (NC, n=37), as well as individuals with schizophrenia, including first episode (n=30) and recurrent (n=30) cases. We employed western blot and ELISA techniques to quantify TRPV1, cannabinoid receptors 1(CB1), anandamide (AEA), and 2-arachidonoylglycerol (2-AG), and assess the severity of the patient's symptoms by means of the PANSS scale. RESULTS: Compared to NC, TRPV1 levels showed a noticeable decrease in both first episode schizophrenia (f-SCZ group) and recurrent schizophrenia (r-SCZ group) subjects. Additionally, CB1 levels appeared increased in f-SCZ group. Furthermore, 2-AG levels were found to be elevated in both f-SCZ group and r-SCZ group compared to NC, whereas AEA levels were decreased in f-SCZ group but increased in r-SCZ group. Moreover, among schizophrenia patients, TRPV1 demonstrated a negative correlation with negative symptoms. Within r-SCZ subjects, CB1 displayed a negative correlation with relapse number, while 2-AG showed a correlation in the opposite direction. CONCLUSIONS: This study provides initial clinical evidence of changed TRPV1 expression in schizophrenia, potentially linked to negative symptoms. These results suggest a possible dysfunction of TRPV1 and the endocannabinoid system (ECS), which might offer new avenues for medical interventions.
Asunto(s)
Ácidos Araquidónicos , Endocannabinoides , Glicéridos , Alcamidas Poliinsaturadas , Esquizofrenia , Canales Catiónicos TRPV , Humanos , Canales Catiónicos TRPV/metabolismo , Esquizofrenia/metabolismo , Esquizofrenia/sangre , Endocannabinoides/metabolismo , Endocannabinoides/sangre , Masculino , Femenino , Adulto , Ácidos Araquidónicos/sangre , Ácidos Araquidónicos/metabolismo , Estudios Transversales , Glicéridos/sangre , Glicéridos/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Alcamidas Poliinsaturadas/sangre , Persona de Mediana Edad , Receptor Cannabinoide CB1/metabolismo , Adulto JovenRESUMEN
The endocannabinoid system (ECS) regulates neurotransmission linked to synaptic plasticity, cognition, and emotion. While it has been demonstrated that dysregulation of the ECS in adulthood is relevant not only to central nervous system (CNS) disorders such as autism spectrum disorder, cognitive dysfunction, and depression but also to brain function, there are few studies on how dysregulation of the ECS in the neonatal period affects the manifestation and pathophysiology of CNS disorders later in life. In this study, DO34, a diacylglycerol lipase alpha (DAGLα) inhibitor affecting endocannabinoid 2-AG production, was injected into C57BL/6N male mice from postnatal day (PND) 7 to PND 10, inducing dysregulation of the ECS in the neonatal period. Subsequently, we examined whether it affects neuronal function in adulthood through electrophysiological and behavioral evaluation. DO34-injected mice showed significantly decreased cognitive functions, attributed to impairment of hippocampal synaptic plasticity. The findings suggest that regulation of ECS activity in the neonatal period may induce enduring effects on adult brain function.
Asunto(s)
Animales Recién Nacidos , Ácidos Araquidónicos , Endocannabinoides , Glicéridos , Ratones Endogámicos C57BL , Animales , Endocannabinoides/metabolismo , Ácidos Araquidónicos/metabolismo , Glicéridos/metabolismo , Masculino , Ratones , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Lipoproteína Lipasa/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , CiclohexanonasRESUMEN
Resolution of inflammation is the cellular and molecular process that protects from widespread and uncontrolled inflammation and restores tissue function in the aftermath of acute immune events. This process is orchestrated by specialized pro-resolving mediators (SPM), a class of bioactive lipids able to reduce immune activation and promote removal of tissue debris and apoptotic cells by macrophages. Although SPMs are the lipid class that has been best studied for its role in facilitating the resolution of self-limited inflammation, a number of other lipid signals, including endocannabinoids, also exert protective immunomodulatory effects on immune cells, including macrophages. These observations suggest that endocannabinoids may also display pro-resolving actions. Interestingly, the endocannabinoid anandamide (AEA) is not only known to bind canonical type 1 and type 2 cannabinoid receptors (CB1 and CB2) but also to engage SPM-binding receptors such as GPR18. This suggests that AEA may also contribute to the governing of resolution processes. In order to interrogate this hypothesis, we investigated the ability of AEA to induce pro-resolving responses by classically-activated primary human monocyte-derived macrophages (MoDM). We found that AEA, at nanomolar concentration, enhances efferocytosis in MoDMs in a CB2- and GPR18-dependent manner. Using lipid mediator profiling, we also observed that AEA modulates SPM profiles in these cells, including levels of resolvin (Rv)D1, RvD6, maresin (MaR)2, and RvE1 in a CB2-dependent manner. AEA treatment also modulated the gene expression of SPM enzymes involved in both the formation and further metabolism of SPM such as 5-lipoxygenase and 15-Prostaglandin dehydrogenase. Our findings show, for the first time, a direct effect of AEA on the regulation of pro-resolving pathways in human macrophages. They also provide new insights into the complex interactions between different lipid pathways in activation of pro-resolving responses contributing to the reestablishment of homeostasis in the aftermath of acute inflammation.
Asunto(s)
Ácidos Araquidónicos , Endocannabinoides , Macrófagos , Alcamidas Poliinsaturadas , Receptor Cannabinoide CB2 , Receptores Acoplados a Proteínas G , Humanos , Endocannabinoides/metabolismo , Endocannabinoides/farmacología , Receptor Cannabinoide CB2/metabolismo , Receptor Cannabinoide CB2/genética , Alcamidas Poliinsaturadas/farmacología , Alcamidas Poliinsaturadas/metabolismo , Ácidos Araquidónicos/farmacología , Ácidos Araquidónicos/metabolismo , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Inflamación/metabolismo , Células Cultivadas , Transducción de Señal/efectos de los fármacos , Ácidos Docosahexaenoicos/farmacología , Ácidos Docosahexaenoicos/metabolismo , Araquidonato 5-Lipooxigenasa/metabolismoRESUMEN
Post-traumatic stress disorder (PTSD) is a debilitating mental health disease related to traumatic experience, and its treatment outcomes are unsatisfactory. Accumulating research has indicated that cannabidiol (CBD) exhibits anti-PTSD effects, however, the underlying mechanism of CBD remains inadequately investigated. Although many studies pertaining to PTSD have primarily focused on aberrations in neuronal functioning, the present study aimed to elucidate the involvement and functionality of microglia/macrophages in PTSD while also investigated the modulatory effects of CBD on neuroinflammation associated with this condition. We constructed a modified single-prolonged stress (SPS) mice PTSD model and verified the PTSD-related behaviors by various behavioral tests (contextual freezing test, elevated plus maze test, tail suspension test and novel object recognition test). We observed a significant upregulation of Iba-1 and alteration of microglial/macrophage morphology within the prefrontal cortex and hippocampus, but not the amygdala, two weeks after the PTSD-related stress, suggesting a persistent neuroinflammatory phenotype in the PTSD-modeled group. CBD (10 mg/kg, i.p.) inhibited all PTSD-related behaviors and reversed the alterations in both microglial/macrophage quantity and morphology when administered prior to behavioral assessments. We further found increased pro-inflammatory factors, decreased PSD95 expression, and impaired synaptic density in the hippocampus of the modeled group, all of which were also restored by CBD treatment. CBD dramatically increased the level of anandamide, one of the endocannabinoids, and cannabinoid type 2 receptors (CB2Rs) transcripts in the hippocampus compared with PTSD-modeled group. Importantly, we discovered the expression of CB2Rs mRNA in Arg-1-positive cells in vivo and found that the behavioral effects of CBD were diminished by CB2Rs antagonist AM630 (1 mg/kg, i.p.) and both the behavioral and molecular effects of CBD were abolished in CB2Rs knockout mice. These findings suggest that CBD would alleviate PTSD-like behaviors in mice by suppressing PTSD-related neuroinflammation and upregulation and activation of CB2Rs may serve as one of the underlying mechanisms for this therapeutic effect. The present study offers innovative experimental evidence supporting the utilization of CBD in PTSD treatment from the perspective of its regulation of neuroinflammation, and paves the way for leveraging the endocannabinoid system to regulate neuroinflammation as a potential therapeutic approach for psychiatric disorders.
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Encéfalo , Cannabidiol , Modelos Animales de Enfermedad , Microglía , Enfermedades Neuroinflamatorias , Receptor Cannabinoide CB2 , Trastornos por Estrés Postraumático , Animales , Cannabidiol/farmacología , Receptor Cannabinoide CB2/metabolismo , Masculino , Ratones , Trastornos por Estrés Postraumático/metabolismo , Trastornos por Estrés Postraumático/tratamiento farmacológico , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Ratones Endogámicos C57BL , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Corteza Prefrontal/efectos de los fármacos , Endocannabinoides/metabolismo , Inflamación/metabolismo , Inflamación/tratamiento farmacológico , Ácidos Araquidónicos/metabolismo , Ácidos Araquidónicos/farmacologíaRESUMEN
Cannabinoid receptors CB1R and CB2R are G-protein coupled receptors acted upon by endocannabinoids (eCBs), namely 2-arachidonoylglycerol (2-AG) and N-arachidonoyl ethanolamine (AEA), with unique pharmacology and modulate disparate physiological processes. A genetically encoded GPCR activation-based sensor that was developed recently-GRABeCB2.0-has been shown to be capable of monitoring real-time changes in eCB levels in cultured cells and preclinical models. However, its responsiveness to exogenous synthetic cannabinoid agents, particularly antagonists and allosteric modulators, has not been extensively characterized. This current study expands upon the pharmacological characteristics of GRABeCB2.0 to enhance the understanding of fluorescent signal alterations in response to various functionally indiscriminate cannabinoid ligands. The results from this study could enhance the utility of the GRABeCB2.0 sensor for in vitro as well as in vivo studies of cannabinoid action and may aid in the development of novel ligands.
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Endocannabinoides , Receptor Cannabinoide CB1 , Receptor Cannabinoide CB2 , Humanos , Receptor Cannabinoide CB2/metabolismo , Endocannabinoides/metabolismo , Receptor Cannabinoide CB1/metabolismo , Células HEK293 , Ligandos , Glicéridos/farmacología , Técnicas Biosensibles/métodos , Moduladores de Receptores de Cannabinoides/farmacología , Animales , Ácidos Araquidónicos/farmacología , Ácidos Araquidónicos/metabolismoRESUMEN
INTRODUCTION: Cyclic vomiting syndrome (CVS) is a disorder of gut-brain interaction often triggered by stress. Interventions such as meditation may improve psychological outcomes and health-related quality of life (HRQoL), but their efficacy and the underlying mechanism are unknown. METHODS: We conducted a 6-week single-arm pilot study to assess the effects of heartfulness meditation (HFM) in CVS using a custom-designed meditation app. Primary outcomes included state and trait anxiety and mood state changes pre vs post-meditation, and secondary outcomes were psychological distress, coping, sleep quality, and HRQoL at baseline and at weeks 3 and 6. Serum concentrations of endocannabinoids N -arachidonylethanolamine and 2-arachidonoylglycerol and related lipids were measured pre- and post-HFM at baseline and week 6. RESULTS: In 30 treatment completers, there was a significant improvement in state anxiety ( P < 0.001), total mood disturbance ( P < 0.001), and other mood states (all P values < 0.05) across the 3 time points. Trait anxiety was also improved at week 6. There was a significant improvement in psychological distress (Global Severity Index), sleep quality (daytime dysfunction), coping (using religion/spirituality), and HRQoL (mental and physical) across the 3 time points (all P < 0.05). Significant increases in N -arachidonylethanolamine and related lipids N -oleoylethanolamine and palmitoylethanolamide post vs pre-HFM were observed at week 6 ( P < 0.001, 0.002, 0.003, respectively). No adverse effects were noted. DISCUSSION: App-delivered HFM is feasible, safe, and effective and improves psychological outcomes and augments endocannabinoids. This provides insight into the mechanism underlying HFM and has potential for widespread use as a digital therapeutic in CVS and other disorder of gut-brain interaction.
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Ansiedad , Endocannabinoides , Meditación , Calidad de Vida , Vómitos , Humanos , Endocannabinoides/metabolismo , Endocannabinoides/sangre , Femenino , Masculino , Adulto , Vómitos/psicología , Vómitos/terapia , Proyectos Piloto , Meditación/métodos , Meditación/psicología , Ansiedad/psicología , Ansiedad/terapia , Ansiedad/sangre , Persona de Mediana Edad , Resultado del Tratamiento , Adaptación Psicológica , Afecto , Transducción de Señal , Glicéridos/sangre , Glicéridos/metabolismo , Ácidos Araquidónicos/sangre , Ácidos Araquidónicos/metabolismo , Adulto JovenRESUMEN
Muscle degeneration is a common feature in cancer cachexia that cannot be reversed. Recent advances show that the endocannabinoid system, and more particularly cannabinoid receptor 1 (CB1), regulates muscle processes, including metabolism, anabolism and regenerative capacity. However, it is unclear whether muscle endocannabinoids, their receptors and enzymes are responsive to cachexia and exercise. Therefore, this study investigated whether cachexia and exercise affected muscle endocannabinoid signaling, and whether CB1 expression correlated with markers of muscle anabolism, catabolism and metabolism. Male BALB/c mice were injected with PBS (CON) or C26 colon carcinoma cells (C26) and had access to wheel running (VWR) or remained sedentary (n = 5-6/group). Mice were sacrificed 18 days upon PBS/tumor cell injection. Cachexic mice exhibited a lower muscle CB1 expression (-43 %; p < 0.001) and lower levels of the endocannabinoid anandamide (AEA; -22 %; p = 0.044), as well as a lower expression of the AEA-synthesizing enzyme NAPE-PLD (-37 %; p < 0.001), whereas the expression of the AEA degrading enzyme FAAH was higher (+160 %; p < 0.001). The 2-AG-degrading enzyme MAGL, was lower in cachexic muscle (-34 %; p = 0.007), but 2-AG and its synthetizing enzyme DAGLß were not different between CON and C26. VWR increased muscle CB1 (+25 %; p = 0.005) and increased MAGL expression (+30 %; p = 0.035). CB1 expression correlated with muscle mass, markers of metabolism (e.g. p-AMPK, PGC1α) and of catabolism (e.g. p-FOXO, LC3b, Atg5). Our findings depict an emerging role of the endocannabinoid system in muscle physiology. Future studies should elaborate how this translates into potential therapies to combat cancer cachexia, and other degenerative conditions.
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Caquexia , Endocannabinoides , Ratones Endogámicos BALB C , Músculo Esquelético , Receptor Cannabinoide CB1 , Animales , Endocannabinoides/metabolismo , Masculino , Ratones , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Caquexia/metabolismo , Caquexia/patología , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB1/genética , Línea Celular Tumoral , Alcamidas Poliinsaturadas/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Condicionamiento Físico Animal , Ácidos Araquidónicos/metabolismoRESUMEN
The endocannabinoid system (ECS) is critically involved in the pathophysiology of Multiple Sclerosis (MS), a neuroinflammatory and neurodegenerative disease of the central nervous system (CNS). Over the past decade, researchers have extensively studied the neuroprotective and anti-inflammatory effects of the ECS. Inhibiting the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) has emerged as a promising strategy to mitigate brain damage in MS. In this study, we investigated the effects of a novel reversible MAGL inhibitor (MAGLi 432) on C57/BL6 female mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. We assessed its implications on motor disability, neuroinflammation, and synaptic dysfunction. Systemic in vivo treatment with MAGLi 432 resulted in a less severe EAE disease, accompanied by increased 2-AG levels and decreased levels of arachidonic acid (AA) and prostaglandins (PGs) in the brain. Additionally, MAGLi 432 reduced both astrogliosis and microgliosis, as evidenced by decreased microglia/macrophage density and a less reactive morphology. Flow cytometry analysis further revealed fewer infiltrating CD45+ and CD3+ cells in the brains of MAGLi 432-treated EAE mice. Finally, MAGLi treatment counteracted the striatal synaptic hyperexcitability promoted by EAE neuroinflammation. In conclusion, MAGL inhibition significantly ameliorated EAE clinical disability and striatal inflammatory synaptopathy through potent anti-inflammatory effects. These findings provide new mechanistic insights into the neuroprotective role of the ECS during neuroinflammation and highlight the therapeutic potential of MAGLi-based drugs in mitigating MS-related inflammatory and neurodegenerative brain damage.
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Ácidos Araquidónicos , Encefalomielitis Autoinmune Experimental , Endocannabinoides , Glicéridos , Ratones Endogámicos C57BL , Animales , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Glicéridos/metabolismo , Ratones , Endocannabinoides/metabolismo , Ácidos Araquidónicos/farmacología , Ácidos Araquidónicos/metabolismo , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/patología , Sinapsis/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Monoacilglicerol Lipasas/antagonistas & inhibidores , Monoacilglicerol Lipasas/metabolismoRESUMEN
The brain bidirectionally communicates with the gut to control food intake and energy balance, which becomes dysregulated in obesity. For example, endocannabinoid (eCB) signaling in the small-intestinal (SI) epithelium is upregulated in diet-induced obese (DIO) mice and promotes overeating by a mechanism that includes inhibiting gut-brain satiation signaling. Upstream neural and molecular mechanism(s) involved in overproduction of orexigenic gut eCBs in DIO, however, are unknown. We tested the hypothesis that overactive parasympathetic signaling at the muscarinic acetylcholine receptors (mAChRs) in the SI increases biosynthesis of the eCB, 2-arachidonoyl-sn-glycerol (2-AG), which drives hyperphagia via local CB1Rs in DIO. Male mice were maintained on a high-fat/high-sucrose Western-style diet for 60â d, then administered several mAChR antagonists 30â min prior to tissue harvest or a food intake test. Levels of 2-AG and the activity of its metabolic enzymes in the SI were quantitated. DIO mice, when compared to those fed a low-fat/no-sucrose diet, displayed increased expression of cFos protein in the dorsal motor nucleus of the vagus, which suggests an increased activity of efferent cholinergic neurotransmission. These mice exhibited elevated levels of 2-AG biosynthesis in the SI, that was reduced to control levels by mAChR antagonists. Moreover, the peripherally restricted mAChR antagonist, methylhomatropine bromide, and the peripherally restricted CB1R antagonist, AM6545, reduced food intake in DIO mice for up to 24â h but had no effect in mice conditionally deficient in SI CB1Rs. These results suggest that hyperactivity at mAChRs in the periphery increases formation of 2-AG in the SI and activates local CB1Rs, which drives hyperphagia in DIO.
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Dieta Alta en Grasa , Endocannabinoides , Glicéridos , Ratones Endogámicos C57BL , Obesidad , Transducción de Señal , Transmisión Sináptica , Animales , Endocannabinoides/metabolismo , Masculino , Obesidad/metabolismo , Ratones , Transmisión Sináptica/fisiología , Transmisión Sináptica/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Transducción de Señal/fisiología , Glicéridos/metabolismo , Ácidos Araquidónicos/metabolismo , Ingestión de Alimentos/fisiología , Ingestión de Alimentos/efectos de los fármacos , Antagonistas Muscarínicos/farmacología , Receptores Muscarínicos/metabolismo , Eje Cerebro-Intestino/fisiologíaRESUMEN
BACKGROUND AND PURPOSE: Neurotransmission and neuroinflammation are controlled by local increases in both extracellular ATP and the endocannabinoid 2-arachidonoyl glycerol (2-AG). While it is known that extracellular ATP stimulates 2-AG production in cells in culture, the dynamics and molecular mechanisms that underlie this response remain poorly understood. Detection of real-time changes in eCB levels with the genetically encoded sensor, GRABeCB2.0, can address this shortfall. EXPERIMENTAL APPROACH: 2-AG and arachidonoylethanolamide (AEA) levels in Neuro2a (N2a) cells were measured by LC-MS, and GRABeCB2.0 fluorescence changes were detected using live-cell confocal microscopy and a 96-well fluorescence plate reader. KEY RESULTS: 2-AG and AEA increased GRABeCB2.0 fluorescence in N2a cells with EC50 values of 81 and 58 nM, respectively; both responses were reduced by the cannabinoid receptor type 1 (CB1R) antagonist SR141617 and absent in cells expressing the mutant-GRABeCB2.0. ATP increased only 2-AG levels in N2a cells, as measured by LC-MS, and induced a transient increase in the GRABeCB2.0 signal within minutes primarily via activation of P2X7 receptors (P2X7R). This response was dependent on diacylglycerol lipase ß activity, partially dependent on extracellular calcium and phospholipase C activity, but not controlled by the 2-AG hydrolysing enzyme, α/ß-hydrolase domain containing 6 (ABHD6). CONCLUSIONS AND IMPLICATIONS: Considering that P2X7R activation increases 2-AG levels within minutes, our results show how these molecular components are mechanistically linked. The specific molecular components in these signalling systems represent potential therapeutic targets for the treatment of neurological diseases, such as chronic pain, that involve dysregulated neurotransmission and neuroinflammation.
Asunto(s)
Ácidos Araquidónicos , Endocannabinoides , Glicéridos , Neuronas , Receptores Purinérgicos P2X7 , Endocannabinoides/metabolismo , Glicéridos/metabolismo , Ácidos Araquidónicos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Animales , Ratones , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Monoacilglicerol Lipasas/metabolismo , Monoacilglicerol Lipasas/antagonistas & inhibidores , Receptor Cannabinoide CB1/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Línea Celular TumoralRESUMEN
Hypofunctioning of NMDA receptors, and the resulting shift in the balance between excitation and inhibition, is considered a key process in the pathophysiology of schizophrenia. One important manifestation of this phenomenon is changes in neural oscillations, those above 30 Hz (i.e., gamma-band oscillations), in particular. Although both preclinical and clinical studies observed increased gamma activity following acute administration of NMDA receptor antagonists, the relevance of this phenomenon has been recently questioned given the reduced gamma oscillations typically observed during sensory and cognitive tasks in schizophrenia. However, there is emerging, yet contradictory, evidence for increased spontaneous gamma-band activity (i.e., at rest or under baseline conditions). Here, we use the sub-chronic phencyclidine (PCP) rat model for schizophrenia, which has been argued to model the pathophysiology of schizophrenia more closely than acute NMDA antagonism, to investigate gamma oscillations (30-100 Hz) in the medial prefrontal cortex of anesthetized animals. While baseline gamma oscillations were not affected, oscillations induced by train stimulation of the posterior dorsal CA1 (pdCA1) field of the hippocampus were enhanced in PCP-treated animals (5 mg/kg, twice daily for 7 days, followed by a 7-day washout period). This effect was reversed by pharmacological enhancement of endocannabinoid levels via systemic administration of URB597 (0.3 mg/kg), an inhibitor of the catabolic enzyme of the endocannabinoid anandamide. Intriguingly, the pharmacological blockade of CB1 receptors by AM251 unmasked a reduced gamma oscillatory activity in PCP-treated animals. The findings are consistent with the observed effects of URB597 and AM251 on behavioral deficits reminiscent of the symptoms of schizophrenia and further validate the potential for cannabinoid-based drugs as a treatment for schizophrenia.
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Amidohidrolasas , Benzamidas , Carbamatos , Fenciclidina , Piperidinas , Esquizofrenia , Animales , Masculino , Ratas , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/metabolismo , Ácidos Araquidónicos/metabolismo , Ácidos Araquidónicos/farmacología , Benzamidas/farmacología , Carbamatos/farmacología , Modelos Animales de Enfermedad , Endocannabinoides/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Ritmo Gamma/fisiología , Ritmo Gamma/efectos de los fármacos , Fenciclidina/farmacología , Piperidinas/farmacología , Alcamidas Poliinsaturadas/metabolismo , Alcamidas Poliinsaturadas/farmacología , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Corteza Prefrontal/fisiopatología , Pirazoles/farmacología , Ratas Sprague-Dawley , Esquizofrenia/fisiopatología , Esquizofrenia/metabolismo , Esquizofrenia/tratamiento farmacológicoRESUMEN
Prefrontal cortex (PFC) maturation during adolescence is characterized by structural and functional changes, which involve the remodeling of GABA and glutamatergic synapses, as well as changes in the endocannabinoid system. Yet, the way PFC endocannabinoid signaling interacts with local GABA and glutamatergic function to impact its processing of afferent transmission during the adolescent transition to adulthood remains unknown. Here we combined PFC local field potential recordings with local manipulations of 2-AG and anandamide levels to assess how PFC endocannabinoid signaling is recruited to modulate ventral hippocampal and basolateral amygdalar inputs in vivo in adolescent and adult male rats. We found that the PFC endocannabinoid signaling does not fully emerge until late-adolescence/young adulthood. Once present, both 2-AG and anandamide can be recruited in the PFC to limit the impact of hippocampal drive through a CB1R-mediated mechanism whereas basolateral amygdalar inputs are only inhibited by 2-AG. Similarly, the behavioral effects of increasing 2-AG and anandamide in the PFC do not emerge until late-adolescence/young adulthood. Using a trace fear conditioning paradigm, we found that elevating PFC 2-AG levels preferentially reduced freezing behavior during acquisition without affecting its extinction. In contrast, increasing anandamide levels in the PFC selectively disrupted the extinction of trace fear memory without affecting its acquisition. Collectively, these results indicate a protracted recruitment of PFC endocannabinoid signaling, which becomes online in late adolescence/young adulthood as revealed by its impact on hippocampal and amygdalar-evoked local field potential responses and trace fear memory behavior.
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Amígdala del Cerebelo , Endocannabinoides , Miedo , Hipocampo , Corteza Prefrontal , Animales , Endocannabinoides/metabolismo , Endocannabinoides/fisiología , Miedo/fisiología , Miedo/efectos de los fármacos , Masculino , Corteza Prefrontal/metabolismo , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/fisiología , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Ratas , Amígdala del Cerebelo/metabolismo , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/fisiología , Ratas Sprague-Dawley , Alcamidas Poliinsaturadas/metabolismo , Condicionamiento Clásico/fisiología , Condicionamiento Clásico/efectos de los fármacos , Ácidos Araquidónicos/metabolismoRESUMEN
Feeding high-fat (HF) diets has been shown to cause hepatic and intestinal impairment in fish species, but the mode of action, especially the pathways involved in the intestine, has not been determined yet. In this study, the effects of resveratrol (RES) supplementation on the intestinal structure, microbial flora, and fat metabolism in red tilapia (Oreochromis niloticus) were determined. The results showed RES maintained the structural integrity of the intestine and significantly increased the number of goblet cells in the midgut. RES significantly induced interferon (IL)-1ß, IL-6, IL-10, and tumor necrosis factor (TNF)-α, serumal and fecal trimetlylamine oxide (TMAO) and lipopolysaccharides (LPS), intestinal acetic acid levels. However, the concentrations of bound bile acids increased in HF-fed red tilapia. Atp5fa1 and Pafah1b3 significantly increased, Pmt and Acss2 significantly decreased, respectively, with RES supplementation, which was alleviated and retained at the same level in the selisistat (EX527) group. While for transcriptome and proteomics results, RES was found to promote fatty acid ß-oxidation and arachidonic acid metabolism associated with the peroxisome proliferator-activated receptor (PPAR) signaling pathway. The next validation experiment showed some genes related to apoptosis and fatty acid metabolism pathways were altered by RES supplementation. Namely, sn6, loc100702698, new_14481, and prkaa1 were upregulated, while ffrs1, ap3s1, and loc100705861 were downregulated. RES significantly increased Planctomycetes and Verrucomicrobia while decreased Moonvirus, Citrobacter, and Pseudomonas. Akkermansia and Fusobacterium significantly increased and Aeromonas significantly decreased. Thus, unsaturated fatty acid biosynthesis significantly increased and carbohydrate/energy metabolism decreased. To conclude, RES enabled the body to complete fatty acid ß-oxidation and arachidonic acid metabolism, whereas the addition of inhibitors increased the expression of the phagosome transcriptome and reduced fatty acid ß-oxidative metabolism.
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Cíclidos , Tilapia , Animales , Tilapia/metabolismo , Cíclidos/metabolismo , Dieta Alta en Grasa , Resveratrol/metabolismo , Metabolismo de los Lípidos , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Receptores Activados del Proliferador del Peroxisoma/farmacología , Intestinos , Transducción de Señal , Ácidos Grasos/metabolismo , Ácidos Araquidónicos/metabolismo , Ácidos Araquidónicos/farmacología , Dieta , Suplementos Dietéticos , Alimentación Animal/análisisRESUMEN
We previously reported that 2-arachidonoylglycerol (2-AG) synthesis by diacylglycerol lipase (DAGL) and lysophosphatidate phosphohydrolase (LPAP) and hydrolysis by monoacylglycerol lipase (MAGL) in rod outer segments (ROS) from bovine retina were differently modified by light applied to the retina. Based on these findings, the aim of the present research was to evaluate whether 2-AG metabolism could be modulated by proteins involved in the visual process. To this end, ROS kept in darkness (DROS) or obtained in darkness and then subjected to light (BROS) were treated with GTPγS and GDPßS, or with low and moderate ionic strength buffers for detaching soluble and peripheral proteins, or soluble proteins, respectively. Only DAGL activity was stimulated by the application of light to the ROS. GTPγS-stimulated DAGL activity in DROS reached similar values to that observed in BROS. The studies using different ionic strength show that (1) the highest decrease in DROS DAGL activity was observed when both phosphodiesterase (PDE) and transducin α (Tα) are totally membrane-associated; (2) the decrease in BROS DAGL activity does not depend on PDE association to membrane, and that (3) MAGL activity decreases, both in DROS and BROS, when PDE is not associated to the membrane. Our results indicate that the bioavailability of 2-AG under light conditions is favored by G protein-stimulated increase in DAGL activity and hindered principally by Tα/PDE association with the ROS membrane, which decreases DAGL activity.
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Ácidos Araquidónicos , Endocannabinoides , Glicéridos , Segmento Externo de la Célula en Bastón , Animales , Endocannabinoides/metabolismo , Ácidos Araquidónicos/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Bovinos , Glicéridos/metabolismo , Fototransducción , Transducina/metabolismo , Luz , Lipoproteína Lipasa/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Visión Ocular/fisiología , Visión Ocular/efectos de los fármacosRESUMEN
Diacylglycerol lipase-beta (DAGLß) serves as a principal 2-arachidonoylglycerol (2-AG) biosynthetic enzyme regulating endocannabinoid and eicosanoid metabolism in immune cells including macrophages and dendritic cells. Genetic or pharmacological inactivation of DAGLß ameliorates inflammation and hyper-nociception in preclinical models of pathogenic pain. These beneficial effects have been assigned principally to reductions in downstream proinflammatory lipid signaling, leaving alternative mechanisms of regulation largely underexplored. Here, we apply quantitative chemical- and phospho-proteomics to find that disruption of DAGLß in primary macrophages leads to LKB1-AMPK signaling activation, resulting in reprogramming of the phosphoproteome and bioenergetics. Notably, AMPK inhibition reversed the antinociceptive effects of DAGLß blockade, thereby directly supporting DAGLß-AMPK crosstalk in vivo. Our findings uncover signaling between endocannabinoid biosynthetic enzymes and ancient energy-sensing kinases to mediate cell biological and pain responses.