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1.
Methods Mol Biol ; 2576: 317-327, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152199

RESUMO

Cytochrome P450 enzymes are a large family of heme-containing proteins that have important functions in the biotransformation of xenobiotics, including pharmacologic and environmental agents, as well as endogenously produced chemicals with broad structural and functional diversity. Anandamide and 2-arachidonoylglycerol (2-AG) are substrates for P450s expressed in multiple tissues, leading to the production of a diverse set of mono- and di-oxygenated metabolites. This chapter describes tools and methods that have been used to identify major endocannabinoid metabolizing P450s and their corresponding products using subcellular tissue fractions, cultured cells, and purified recombinant enzymes in a reconstituted system.


Assuntos
Sistema Enzimático do Citocromo P-450 , Endocanabinoides , Sistema Enzimático do Citocromo P-450/metabolismo , Endocanabinoides/metabolismo , Heme/metabolismo , Microssomos Hepáticos , Oxirredução , Proteínas Recombinantes/metabolismo , Xenobióticos/metabolismo
2.
Methods Mol Biol ; 2576: 1-8, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152173

RESUMO

Endocannabinoids (eCBs) are endogenous lipids able to bind to cannabinoid receptors, the primary molecular targets of the cannabis (Cannabis sativa) active principle Δ9-tetrahydrocannabinol. During the last 20 years, several N-acylethanolamines and acylesters have been shown to act as eCBs, and a complex array of receptors, metabolic enzymes, and transporters (that altogether form the so-called "eCB system") has been shown to finely tune their manifold biological activities. It appears now urgent to develop methods and protocols that allow to assay in a specific and quantitative manner the distinct components of the eCB system and that can properly localize them within the cell. A brief overview of eCBs and of the proteins that bind, transport, and metabolize these lipids is presented here, in orderto put in a better perspective, the relevance of methodologies that help to disclose molecular details of eCB signaling in health and disease. Proper methodological approaches form also the basis for a more rationale and effective drug design and therapeutic strategy to combat human disorders.


Assuntos
Dronabinol , Endocanabinoides , Agonistas de Receptores de Canabinoides , Endocanabinoides/metabolismo , Humanos , Receptores de Canabinoides/metabolismo , Transdução de Sinais
3.
Methods Mol Biol ; 2576: 49-66, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152177

RESUMO

The adequate quantification of endocannabinoids and related N-acylethanolamines can be complex due to their low endogenous levels, structural diversity, and metabolism. Therefore, advanced analytical approaches, involving LC-MS, are required to quantify these molecules in plasma, tissues, and other matrices. It has been shown that endocannabinoid congeners synthesized from n-3 poly-unsaturated fatty acids (n-3 PUFAs), such as docosahexaenoylethanolamide (DHEA) and eicosapentaenoylethanolamide (EPEA), have interesting immunomodulatory and tumor-inhibiting properties. Recent work has shown that DHEA and EPEA can be further enzymatically metabolized by cyclo-oxygenase 2 (COX-2), forming oxygenated metabolites. Here, an LC-MS-based method for the quantification of the n-3 PUFA-derived endocannabinoid congeners DHEA and EPEA is described, which is also suited to measure a wider spectrum of endocannabinoids. The chapter contains a step-by-step protocol for the analysis of (n-3) endocannabinoids in plasma, including sample collection and solid phase extraction, LC-MS analysis, and data processing. In addition, protocol modifications are provided to allow quantification of n-3 PUFA-derived endocannabinoids and their COX-2 metabolites in tissues and cell culture media. Finally, conditions that alter endocannabinoid concentrations are briefly discussed.


Assuntos
Endocanabinoides , Ácidos Graxos Ômega-3 , Ciclo-Oxigenase 2 , Desidroepiandrosterona , Endocanabinoides/metabolismo , Etanolaminas , Ácidos Graxos Ômega-3/metabolismo
4.
Methods Mol Biol ; 2576: 201-211, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152188

RESUMO

The wide distribution of the endocannabinoid system (ECS) throughout the body and its pivotal pathophysiological role offer promising opportunities for the development of novel therapeutic drugs for treating several diseases. However, the need for strategies to circumvent the unwanted psychotropic and immunosuppressive effects associated with cannabinoid receptor agonism/antagonism has led to considerable research in the field of molecular alternatives, other than type-1 and type-2 (CB1/2) receptors, as therapeutic targets to indirectly manipulate this pro-homeostatic system. In this context, the use of selective inhibitors of proteins involved in endocannabinoid (eCB) transport and metabolism allows for an increase or decrease of the levels of N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) in the sites where these major eCBs are indeed needed. This chapter will briefly review some preclinical and clinical evidence for the therapeutic potential of ECS pharmacological manipulation.


Assuntos
Endocanabinoides , Endocanabinoides/metabolismo , Receptores de Canabinoides/metabolismo
5.
Methods Mol Biol ; 2576: 275-283, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152195

RESUMO

The endocannabinoid 2-arachidonoylglycerol (2-AG) exerts its physiological action by binding to and functionally activating type-1 (CB1) and type-2 (CB2) cannabinoid receptors. It is thought to be produced through the action of sn-1 selective diacylglycerol lipase (DAGL) that catalyzes 2-AG biosynthesis from sn-2-arachidonate-containing diacylglycerols. Different methodological approaches for measuring DAGL activity in biological samples are now available. Here, a highly sensitive radiometric assay to assess DAGL activity, by using 1-oleoyl[1-14C]-2-arachidonoylglycerol as the substrate, is reported. All the steps required to perform lipid extraction, fractionation by thin-layer chromatography (TLC), and quantification of radiolabeled [14C]-oleic acid via scintillation counting are described in detail.


Assuntos
Endocanabinoides , Lipase Lipoproteica , Diglicerídeos/metabolismo , Endocanabinoides/metabolismo , Lipase Lipoproteica/metabolismo , Ácido Oleico , Receptores de Canabinoides
6.
Methods Mol Biol ; 2576: 307-316, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152198

RESUMO

The endocannabinoids anandamide and 2-arachidonoylglycerol are not only metabolized by serine hydrolases, such as fatty acid amide hydrolase, monoacylglycerol lipase, and α,ß-hydrolases 6 and 12, but they also serve as substrates for cyclooxygenases, cytochrome P450s, and lipoxygenases. These enzymes oxygenate the 1Z,4Z-pentadiene system of the arachidonic acid backbone of endocannabinoids, thereby giving rise to an entirely new array of bioactive lipids. Hereby, a protocol is provided for the enzymatic synthesis, purification, and characterization of various oxygenated metabolites of anandamide generated by lipoxygenases, which enables the biological study and detection of these metabolites.


Assuntos
Alcadienos , Endocanabinoides , Ácido Araquidônico , Ácidos Araquidônicos , Citocromos , Endocanabinoides/metabolismo , Lipoxigenases , Monoacilglicerol Lipases , Alcamidas Poli-Insaturadas , Prostaglandina-Endoperóxido Sintases/metabolismo , Serina
7.
Methods Mol Biol ; 2576: 385-393, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152204

RESUMO

Measuring protein levels of receptors and enzymes involved in endocannabinoid metabolism is an important step for understanding the distribution, function, and regulation of these components of the endocannabinoid system. A common approach for detecting proteins from complex biological systems is western blotting. In this chapter, we describe a general approach to western blotting protein components of the endocannabinoid system using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose membranes with a focus on detecting type 1 cannabinoid (CB1) receptors. When this technique is carefully used, with due attention paid to the validation of the primary antibodies used, it can provide quantitative information on protein expression levels. Additional information can also be inferred from western blotting such as potential pre- and post-translational modifications (e.g., alternative splicing, phosphorylation, or glycosylation) that can be further evaluated by specific analytical techniques.


Assuntos
Canabinoides , Endocanabinoides , Western Blotting , Colódio , Endocanabinoides/metabolismo , Dodecilsulfato de Sódio
8.
Methods Mol Biol ; 2576: 437-451, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152208

RESUMO

Single-molecule localization microscopy (SMLM) opened new possibilities to study the spatial arrangement of molecular distribution and disease-associated redistribution at a previously unprecedented resolution that was not achievable with optical microscopy approaches. Recent discoveries based on SMLM techniques uncovered specific nanoscale organizational principles of signaling proteins in several biological systems including the chemical synapses in the brain. Emerging data suggest that the spatial arrangement of the molecular players of the endocannabinoid system is also precisely regulated at the nanoscale level in synapses and in other neuronal and glial subcellular compartments. The precise nanoscale distribution pattern is likely to be important to subserve several specific signaling functions of this important messenger system in a cell-type- and subcellular domain-specific manner.STochastic Optical Reconstruction Microscopy (STORM) is an especially suitable SMLM modality for cell-type-specific nanoscale molecular imaging due to its compatibility with traditional diffraction-limited microscopy approaches and classical staining methods. Here, we describe a detailed protocol for STORM imaging in mouse brain tissue samples with a focus on the CB1 cannabinoid receptor, one of the most abundant synaptic receptors in the brain. We also summarize important conceptual and methodical details that are essential for the valid interpretation of single-molecule localization microscopy data.


Assuntos
Endocanabinoides , Microscopia , Animais , Endocanabinoides/metabolismo , Camundongos , Microscopia/métodos , Receptores de Canabinoides , Receptores de Neurotransmissores , Sinapses/metabolismo
9.
Methods Mol Biol ; 2576: 453-459, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152209

RESUMO

A still unsolved, although critical, issue in endocannabinoid research is the mechanism by which the lipophilic anandamide (AEA) moves from its site of synthesis, crosses the aqueous milieu, and reaches the different intracellular membrane compartments, where its metabolic and signaling pathways take place. The difficulty of studying intracellular AEA transport and distribution results from the lack of specific probes and techniques to track and visualize this bioactive lipid within the cells. Herein, we describe the use of a biotinylated, non-hydrolyzable derivative of AEA (biotin-AEA, b-AEA) for visualizing the subcellular distribution of this endocannabinoid by means of confocal fluorescence microscopy.


Assuntos
Biotina , Endocanabinoides , Transporte Biológico , Biotina/metabolismo , Endocanabinoides/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Alcamidas Poli-Insaturadas/metabolismo
10.
Glia ; 71(1): 5-35, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36308424

RESUMO

It is hard to overestimate the influence of the endocannabinoid signaling (ECS) system on central nervous system (CNS) function. In the 40 years since cannabinoids were found to trigger specific cell signaling cascades, studies of the ECS system continue to cause amazement, surprise, and confusion! CB1 cannabinoid receptors are expressed widely in the CNS and regulate cell-cell communication via effects on the release of both neurotransmitters and gliotransmitters. CB2 cannabinoid receptors are difficult to detect in the CNS but seem to "punch above their weight" as compounds targeting these receptors have significant effects on inflammatory state and behavior. Positive and negative allosteric modulators for both receptors have been identified and examined in preclinical studies. Concentrations of the endocannabinoid ligands, N-arachidonoylethanolamine and 2-arachidonoylglycerol (2-AG), are regulated by a combination of enzymatic synthesis and degradation and inhibitors of these processes are available and making their way into clinical trials. Importantly, ECS regulates many essential brain functions, including regulation of reward, anxiety, inflammation, motor control, and cellular development. While the field is on the cusp of preclinical discoveries providing impactful clinical and therapeutic insights into many CNS disorders, there is still much to be learned about this remarkable and versatile modulatory system.


Assuntos
Canabinoides , Endocanabinoides , Endocanabinoides/metabolismo , Receptores de Canabinoides/metabolismo , Transdução de Sinais , Sistema Nervoso Central/metabolismo , Receptor CB1 de Canabinoide
11.
Glia ; 71(1): 71-90, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36222019

RESUMO

Microglia, the innate immune cells of the central nervous system (CNS), execute their sentinel, housekeeping and defense functions through a panoply of genes, receptors and released cytokines, chemokines and neurotrophic factors. Moreover, microglia functions are closely linked to the constant communication with other cell types, among them neurons. Depending on the signaling pathway and type of stimuli involved, the outcome of microglia operation can be neuroprotective or neurodegenerative. Accordingly, microglia are increasingly becoming considered cellular targets for therapeutic intervention. Among signals controlling microglia activity, the endocannabinoid (EC) system has been shown to exert a neuroprotective role in many neurological diseases. Like neurons, microglia express functional EC receptors and can produce and degrade ECs. Interestingly, boosting EC signaling leads to an anti-inflammatory and neuroprotective microglia phenotype. Nonetheless, little evidence is available on the microglia-mediated therapeutic effects of EC compounds. This review focuses on the EC signals acting on the CNS microglia in physiological and pathological conditions, namely on the CB1R, CB2R and TRPV1-mediated regulation of microglia properties. It also provides new evidence, which strengthens the understanding of mechanisms underlying the control of microglia functions by ECs. Given the broad expression of the EC system in glial and neuronal cells, the resulting picture is the need for in vivo studies in transgenic mouse models to dissect the contribution of EC microglia signaling in the neuroprotective effects of EC-derived compounds.


Assuntos
Microglia , Fármacos Neuroprotetores , Animais , Camundongos , Microglia/metabolismo , Endocanabinoides/farmacologia , Endocanabinoides/metabolismo , Transdução de Sinais , Camundongos Transgênicos , Fármacos Neuroprotetores/farmacologia
12.
Glia ; 71(1): 91-102, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35411970

RESUMO

In the central nervous system, oligodendrocytes synthesize the myelin, a specialized membrane to wrap axons in a discontinuous way allowing a rapid saltatory nerve impulse conduction. Oligodendrocytes express a number of growth factors and neurotransmitters receptors that allow them to sense the environment and interact with neurons and other glial cells. Depending on the cell cycle stage, oligodendrocytes may respond to these signals by regulating their survival, proliferation, migration, and differentiation. Among these signals are the endocannabinoids, lipidic molecules synthesized from phospholipids in the plasma membrane in response to cell activation. Here, we discuss the evidence showing that oligodendrocytes express a full endocannabinoid signaling machinery involved in physiological oligodendrocyte functions that can be therapeutically exploited to promote remyelination in central nervous system pathologies.


Assuntos
Endocanabinoides , Oligodendroglia , Endocanabinoides/metabolismo , Oligodendroglia/metabolismo , Bainha de Mielina/metabolismo , Axônios/metabolismo , Sistema Nervoso Central/metabolismo , Diferenciação Celular/fisiologia
13.
Glia ; 71(1): 44-59, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35822691

RESUMO

The study of the astrocytic contribution to brain functions has been growing in popularity in the neuroscience field. In the last years, and especially since the demonstration of the involvement of astrocytes in synaptic functions, the astrocyte field has revealed multiple functions of these cells that seemed inconceivable not long ago. In parallel, cannabinoid investigation has also identified different ways by which cannabinoids are able to interact with these cells, modify their functions, alter their communication with neurons and impact behavior. In this review, we will describe the expression of different endocannabinoid system members in astrocytes. Moreover, we will relate the latest findings regarding cannabinoid modulation of some of the most relevant astroglial functions, namely calcium (Ca2+ ) dynamics, gliotransmission, metabolism, and inflammation.


Assuntos
Astrócitos , Canabinoides , Astrócitos/metabolismo , Endocanabinoides/metabolismo , Neurônios/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia
14.
J Pharm Biomed Anal ; 222: 115090, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36252552

RESUMO

Endocannabinoids are endogenous lipids with the main function recognized to act as neuromodulators through their cannabinoid receptors. Dysregulation of the endocannabinoid system is implicated in various pathologies, such as inflammatory and neurodegenerative diseases. In this study we describe a sensitive UHPLC-MS/MS method for the analysis of trace levels of 7 endocannabinoids in cerebrospinal fluid samples. The analytes covered comprised 1- and 2-arachidonoylglycerol 1- and 2-AG (which were analysed as sum due to their interconversion), 2-arachidonylglycerol ether 2-AGE, anandamide AEA, N-linoleoyl ethanolamide LEA, N-palmitoyl ethanolamide PEA and N-oleoyl ethanolamide OEA. Analytes were extracted from the biofluid by a simple monophasic procedure involving protein precipitation with acetonitrile (MeCN). The analytical method is based on chromatographic separation of the analytes with solid-core (core-shell, superficially porous) particle column Cortecs C18+ . Gradient elution with changing proportion of water and acetonitrile and constant concentration of formic acid provided reasonable separation of analytes, close elution of analytes and their internal standards and minimized matrix effects in biological samples. For specific detection of the endocannabinoids a triple-quadrupole tandem mass spectrometer with electrospray ionisation (ESI) and selected reaction monitoring (SRM) mode was used, and it provided good assay selectivity. The developed method required a minute volume of the biological samples (50 µL) and achieved excellent sensitivity (the lower limit of detection was between 4.15 and 30.18 pM of the biological sample). Linear calibration was achieved in the range from 25 to 10,545 pM for AEA, 90-3802 pM for 1-AG, 90-724 pM for 2-AG, 12-5226 pM for LEA, 33-13,942 for OEA, 34-23,850 pM for 2-AGE, 72-30,190 for PEA and 10-4218 for AEA-d4 in CSF. The method was validated and revealed relative errors in the range of - 14.7 to + 12.3% at LLOQ and - 14.1 to + 14.2% for the remaining validation range. Precisions were in the acceptable range (< 20% RSD at LLOQ, and <15% for the remaining levels) as well. It was finally used to quantify endocannabinoids in human cerebrospinal fluid obtained from 118 donors. Accurate quantification of endogenous compounds in biological samples was achieved by using two different principal approaches (surrogate matrix for AEA, 2-AG, OEA, 2-AGE, LEA and PEA, and surrogate calibrant for AEA only) and they were evaluated by use of the Passing-Bablok regression. Concentrations (median) of CSF samples of patients suffering from CNS infection and controls were found to be around 160 pM for 1- and 2-AG, 86 pM for AEA, 62 for 2-AGE, 58 for LEA, 93 pM for PEA, and 83 pM for OEA.


Assuntos
Endocanabinoides , Espectrometria de Massas em Tandem , Humanos , Idoso , Pessoa de Meia-Idade , Endocanabinoides/metabolismo , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos , Acetonitrilas
15.
Methods Mol Biol ; 2576: 21-40, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152175

RESUMO

Different mass spectrometric techniques have been used over the past decade to quantify endocannabinoids (eCBs) and related lipids. Even with the level of molecular fingerprinting accuracy of an instrument like the most advanced triple quadrupole mass spectrometer, if one is not getting the most optimized sample to the detector in a way that this improved technology can be of use, then advancements can be stymied. Here, our focus is on review and discussion of sample preparation methodologies used to isolate the eCB anandamide and its close congeners N-acyl ethanolamines and structural congeners (i.e., lipo amino acids, lipoamines, N-acyl amides) in biological fluids. Most of our focus will be on the analysis of these lipids in plasma/serum, but we will also discuss how the same techniques can be used for the analysis of saliva and breast milk.


Assuntos
Endocanabinoides , Etanolaminas , Aminoácidos , Animais , Endocanabinoides/metabolismo , Feminino , Humanos , Espectrometria de Massas , Leite/química
16.
Nat Commun ; 13(1): 6941, 2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36396957

RESUMO

Chronic obstructive airway diseases are a global medical burden that is expected to increase in the near future. However, the underlying mechanistic processes are poorly understood so far. Herein, we show that the endocannabinoid anandamide (AEA) induces prominent airway relaxation in vitro and in vivo. In contrast to 2-arachidonlyglycerol-induced airway relaxation, this is mediated by fatty acid amide hydrolase (FAAH)-dependent metabolites. In particular, we identify mouse and also human epithelial and airway smooth muscle cells as source of AEA-induced prostaglandin E2 production and cAMP as direct mediator of AEA-dependent airway relaxation. Mass spectrometry experiments demonstrate reduced levels of endocannabinoid-like compounds in lungs of ovalbumin-sensitized mice indicating a pathophysiological relevance of endocannabinoid signalling in obstructive airway disease. Importantly, AEA inhalation protects against airway hyper-reactivity after ovalbumin sensitization. Thus, this work highlights the AEA/FAAH axis as a critical regulator of airway tone that could provide therapeutic targets for airway relaxation.


Assuntos
Ácidos Araquidônicos , Endocanabinoides , Animais , Camundongos , Humanos , Endocanabinoides/metabolismo , Ovalbumina , Ácidos Araquidônicos/metabolismo , Alcamidas Poli-Insaturadas/metabolismo
17.
Int J Mol Sci ; 23(21)2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36361803

RESUMO

The endocannabinoid system is involved in physiological and pathological processes, including pain generation, modulation, and sensation. Its role in certain types of chronic orofacial pain (OFP) has not been thoroughly examined. By exploring the profiles of specific salivary endocannabinoids (eCBs) in individuals with different types of OFP, we evaluated their use as biomarkers and the influence of clinical parameters and pain characteristics on eCB levels. The salivary levels of anandamide (AEA), 2-arachidonoyl glycerol (2-AG), and their endogenous breakdown product arachidonic acid (AA), as well as the eCB-like molecules N-palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), were assessed in 83 OFP patients and 43 pain-free controls using liquid chromatography/tandem mass spectrometry. Patients were grouped by diagnosis: post-traumatic neuropathy (PTN), trigeminal neuralgia (TN), temporomandibular disorder (TMD), migraine, tension-type headache (TTH), and burning mouth syndrome (BMS). Correlation analyses between a specific diagnosis, pain characteristics, and eCB levels were conducted. Significantly lower levels of 2-AG were found in the TN and TTH groups, while significantly lower PEA levels were found in the migraine group. BMS was the only group with elevated eCBs (AEA) versus the control. Significant correlations were found between levels of specific eCBs and gender, health-related quality of life (HRQoL), BMI, pain duration, and sleep awakenings. In conclusion, salivary samples exhibited signature eCBs profiles for major OFP disorders, especially migraine, TTH, TN, and BMS. This finding may pave the way for using salivary eCBs biomarkers for more accurate diagnoses and management of chronic OFP patients.


Assuntos
Transtornos da Cefaleia , Transtornos de Enxaqueca , Humanos , Endocanabinoides/metabolismo , Qualidade de Vida , Biomarcadores , Dor Facial/diagnóstico
18.
Int J Mol Sci ; 23(21)2022 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-36362014

RESUMO

Opportunities for developing innovative and intelligent drug delivery technologies by targeting the endocannabinoid system are becoming more apparent. This review provides an overview of strategies to develop targeted drug delivery using the endocannabinoid system (ECS). Recent advances in endocannabinoid system targeting showcase enhanced pharmaceutical therapy specificity while minimizing undesirable side effects and overcoming formulation challenges associated with cannabinoids. This review identifies advances in targeted drug delivery technologies that may permit access to the full pharmacotherapeutic potential of the ECS. The design of optimized nanocarriers that target specific tissues can be improved by understanding the nature of the signaling pathways, distribution in the mammalian body, receptor structure, and enzymatic degradation of the ECS. A closer look at ligand-receptor complexes, endocannabinoid tone, tissue distribution, and G-protein activity leads to a better understanding of the potential of the ECS toolkit for therapeutics. The signal transduction pathways examine the modulation of downstream effector proteins, desensitization, signaling cascades, and biased signaling. An in-depth and overall view of the targeted system is achieved through homology modeling where mutagenesis and ligand binding examine the binding site and allow sequence analysis and the formation of libraries for molecular docking and molecular dynamic simulations. Internalization routes exploring receptor-mediated endocytosis and lipid rafts are also considered for explicit signaling. Furthermore, the review highlights nanotechnology and surface modification aspects as a possible future approach for specific targeting.


Assuntos
Canabinoides , Endocanabinoides , Animais , Endocanabinoides/metabolismo , Simulação de Acoplamento Molecular , Ligantes , Canabinoides/farmacologia , Transdução de Sinais , Mamíferos/metabolismo
19.
Horm Behav ; 146: 105277, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36356457

RESUMO

The mechanisms involved in hedonic regulation of food intake, including endocannabinoid system (ECs) are scarcely known in fish. We recently demonstrate in rainbow trout the presence of a rewarding response mediated by ECs in hypothalamus and telencephalon when fish fed a lipid-enriched diet, and that central administration of main agonists of ECs namely AEA or 2-AG exert a bimodal effect on feed intake in fish with low doses inducing an increase that disappears with the high dose of both endocannabinoids (EC). To assess the precise involvement of the different receptors of the ECs (CNR1, TRPV1, and GPR55) in this response we injected intracerebroventricularly AEA or 2-AG in the absence/presence of specific receptor antagonists (AM251, capsazepine, and ML193; respectively). The presence of antagonists clearly counteracts the effect of EC supporting the specificity of EC action inducing changes not only in ECs but also in GABA and glutamate metabolism ultimately leading to the increase observed in food intake response.


Assuntos
Endocanabinoides , Oncorhynchus mykiss , Animais , Endocanabinoides/farmacologia , Endocanabinoides/metabolismo , Oncorhynchus mykiss/fisiologia , Hipotálamo/metabolismo , Ingestão de Alimentos , Telencéfalo
20.
Artigo em Inglês | MEDLINE | ID: mdl-36231760

RESUMO

The endocannabinoid (eCB) system is critically involved in the modulation of synaptic transmission in the central nervous system, playing an important role in the control of emotional responses, neurodevelopment and synaptic plasticity among other functions. The eCB system is also present in the retina, with studies indicating changes in function after application of cannabinoid receptor agonists, antagonists and in knockout models. Whether eCBs are tonically released in the retina and their physiological functions is, however, still unknown. We investigated the role of the eCB system in the modulation of response strength of retinal ganglion cells (RGCs) to light stimulation, their receptive field organization, contrast sensitivity and excitability properties by performing whole-cell patch-clamp recordings in mouse RGCs before and after bath application of URB597, an inhibitor of the enzyme that degrades the eCB anandamide. Our results show that URB597 application leads to a reduction in the strength of synaptic inputs onto RGCs but paradoxically increases RGC excitability. In addition, URB597 was shown to modulate receptive field organization and contrast sensitivity of RGCs. We conclude that tonically released eCBs modulate retinal signaling by acting on traditional cannabinoid receptors (CB1R/CB2R) as well as on non-cannabinoid receptor targets. Thus, a thorough understanding of the effects of drugs that alter the endogenous cannabinoid levels and of exogenous cannabinoids is necessary to fully comprehend the impact of their medical as well as recreational use on vision.


Assuntos
Agonistas de Receptores de Canabinoides , Endocanabinoides , Animais , Benzamidas , Carbamatos/farmacologia , Endocanabinoides/metabolismo , Endocanabinoides/farmacologia , Camundongos , Retina
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