RESUMEN
The discovery of hemoglobin allosteric properties is briefly summarized and contextualized in the frame of the main biochemical revelations that characterized the first half of the XX century. In particular, the historical background of DNA, RNA, and protein structure research is recalled and the new role that protein-protein interaction may have on allosteric regulation is discussed.
Asunto(s)
Hemoglobinas/química , Hemoglobinas/metabolismo , Regulación Alostérica , Sitio Alostérico , Animales , Humanos , Modelos Moleculares , Conformación Proteica , Mapas de Interacción de ProteínasRESUMEN
In this paper we report a procedure to analyze protein homodimer interfaces.We approached the problem by means of a topological methodology. In particular, we analyzed the subunits interface of about 50 homodimers and we have defined a few parameters that allow to organize these proteins in six different classes. The main characteristics of each class of homodimers have been discussed also taking into account their stabilization energy, as reported in the literature from the experimental measurements. A paradigmatic example for each class has been reported and a graphical representation proposed in order to better explain the meaning of the parameters chosen.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Proteínas/metabolismo , Sitios de Unión , Biología Computacional , Cristalografía por Rayos X , Bases de Datos de Proteínas , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de ProteínaAsunto(s)
ADN/química , Lípidos/química , Péptidos/química , Proteínas/química , ARN/química , Bioquímica , BiotecnologíaRESUMEN
Transactivation-proficient (TA) p73 is a transcription factor belonging to the p53 family, which regulates a variety of biological processes, including neurogenesis, differentiation, apoptosis, and DNA damage checkpoint response. In the present study, we adopted multiple Omics approaches, based upon the simultaneous application of metabolomics, lipidomics, and proteomics, in order to dissect the intracellular pathways activated by p73. As cellular model, we utilized a clone of the human osteosarcoma SAOS-2 cell line that allows the expression of TAp73α in an inducible manner. We found that TAp73α promoted mitochondrial activity (accumulation of metabolic intermediates and up-regulation of proteins related to the Krebs cycle), boosted glutathione homeostasis, increased arginine-citrulline-NO metabolism, altered purine synthesis, and promoted the pentose phosphate pathway toward NADPH accumulation for reducing and biosynthetic purposes. Indeed, lipid metabolism was driven toward the accumulation and oxidation of long-chain fatty acids with pro-apoptotic potential. In parallel, the expression of TAp73α was accompanied by the dephosphorylation of key proteins of the mitotic spindle assembly checkpoint. In conclusion, the obtained results confirm existing evidence from transcriptomics analyses and suggest a role for TAp73α in the regulation of cellular metabolism, cell survival, and cell growth.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Ciclo del Ácido Cítrico , Estrés del Retículo Endoplásmico , Regulación de la Expresión Génica , Glutatión/metabolismo , Glucólisis , Homeostasis , Humanos , Metabolismo de los Lípidos , Metaboloma , Datos de Secuencia Molecular , Vía de Pentosa Fosfato , Fosfatidilinositoles/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteolisis , Proteoma/química , Proteoma/metabolismo , Proteómica , Purinas/metabolismo , Biología de Sistemas , Transcripción Genética , Proteína Tumoral p73RESUMEN
In this study, we analyzed the components of the endocannabinoid system (ECS) in R6/2 mice, a widely used model of Huntington's disease (HD). We measured the endogenous content of N-arachidonoylethanolamine and 2-arachidonoylglycerol and the activity of their biosynthetic enzymes (N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D and diacylglycerol lipase, respectively) and hydrolytic enzymes [fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase, respectively] and of their target receptors (type 1 cannabinoid receptor, type 2 cannabinoid receptor, and transient receptor potential vanilloid-1) in the brains of wild-type and R6/2 mice of different ages, as well as in the striatum and cortex of 12-week-old animals. In addition, we measured FAAH activity in lymphocytes of R6/2 mice. In the whole brains of 12-week-old R6/2 mice, we found reductions in N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D activity, diacylglycerol lipase activity and cannabinoid receptor binding, mostly associated with changes in the striatum but not in the cortex, as well as an increase in 2-arachidonoylglycerol content as compared with wild-type littermates, without any other change in ECS elements. Then, our analysis was extended to HD43 cells, an inducible cellular model of HD derived from rat ST14A cells. In both induced and noninduced conditions, we demonstrated a fully functional ECS. Overall, our data suggest that the ECS is differently affected in mouse and human HD, and that HD43 cells are suitable for high-throughput screening of FAAH-oriented drugs affecting HD progression.
Asunto(s)
Endocannabinoides/fisiología , Enfermedad de Huntington/metabolismo , Amidohidrolasas/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Agonistas de Receptores de Cannabinoides/metabolismo , Línea Celular , Corteza Cerebral/metabolismo , Modelos Animales de Enfermedad , Endocannabinoides/metabolismo , Glicéridos/metabolismo , Humanos , Cinética , Linfocitos/enzimología , Ratones , Ratones Transgénicos , Neostriado/metabolismo , Alcamidas Poliinsaturadas , RatasRESUMEN
The discovery of the receptors for the most active compound of cannabis/marihuana opened the chase for the intrinsic, physiological ligands, which are collectively termed endocannabinoids. In just a few years, it has become difficult even for the followers of this field to keep up with the endocannabinoids literature, thus we thought it useful to offer the reader at least a compass to navigate such a mare magnum.
Asunto(s)
Endocannabinoides/fisiología , Transporte Biológico , Epigénesis Genética , Humanos , Receptores de Cannabinoides/genética , Receptores de Cannabinoides/metabolismoRESUMEN
The endocannabinoid system, composed of endogenous lipids, their target receptors and metabolic enzymes, has been implicated in multiple biological functions in health and disease, both in the central nervous system and in peripheral organs. Despite the exponential growth of experimental evidence on the key role of endocannabinoid signalling in basic cellular processes, and on its potential exploitation for therapeutic interventions, much remains to be clarified about the respective regulatory mechanisms. Epigenetics refers to a set of post-translational modifications that regulate gene expression without causing variation in DNA sequence, endowed with a major impact on signal transduction pathways. The epigenetic machinery includes DNA methylation, histone modifications, nucleosome positioning and non-coding RNAs. Due to the reversibility of epigenetic changes, an emerging field of interest is the possibility of an 'epigenetic therapy' that could possibly be applied also to endocannabinoids. Here, we review current knowledge of epigenetic regulation of endocannabinoid system components under both physiological and pathological conditions, as well as the epigenetic changes induced by endocannabinoid signalling.
Asunto(s)
Endocannabinoides/fisiología , Epigénesis Genética , Transducción de Señal , Animales , Metilación de ADN , Histonas/metabolismo , Humanos , MicroARNs/genética , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional , Receptores de Cannabinoides/genética , Receptores de Cannabinoides/metabolismoRESUMEN
Fatty acid amide hydrolase (FAAH) is a membrane protein that plays a relevant role in the metabolism of fatty acid amides and esters. It degrades important neurotransmitters such as oleamide and anandamide, and it has been involved in a number of human pathological conditions, representing therefore a valuable target for biochemical and pharmacological research. In this study, we have investigated in vitro the structure-function relationship of rat and human FAAHs. In particular circular dichroism, fluorescence spectroscopy and light scattering measurements have been performed, in order to characterize the structural features of the two proteins, both in the presence and absence of the irreversible inhibitor methoxyarachidonyl-fluorophosphonate. The results demonstrate that the structural dynamics of the two FAAHs are different, despite their high sequence homology and overall similarity in temperature-dependence. Additionally, membrane binding and kinetic assays of both FAAHs indicate that also the functional properties of the two enzymes are different in their interaction with lipid bilayers and with exogenous inhibitors. These findings suggest that pre-clinical studies of FAAH-dependent human diseases based only on animal models should be interpreted with caution, and that the efficacy of new drugs targeted to FAAH should be tested in vitro, on both rat and human enzymes.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/metabolismo , Ácidos Araquidónicos/farmacología , Inhibidores Enzimáticos/farmacología , Ácidos Grasos/metabolismo , Organofosfonatos/farmacología , Amidohidrolasas/química , Animales , Humanos , Cinética , Estabilidad Proteica , Estructura Secundaria de Proteína , Ratas , Especificidad por SustratoRESUMEN
We show that a fully functional endocannabinoid system is present in primary human melanocytes (normal human epidermal melanocyte cells), including anandamide (AEA), 2-arachidonoylglycerol, the respective target receptors (CB(1), CB(2), and TRPV1), and their metabolic enzymes. We also show that at higher concentrations AEA induces normal human epidermal melanocyte apoptosis (â¼3-fold over controls at 5 µM) through a TRPV1-mediated pathway that increases DNA fragmentation and p53 expression. However, at lower concentrations, AEA and other CB(1)-binding endocannabinoids dose-dependently stimulate melanin synthesis and enhance tyrosinase gene expression and activity (â¼3- and â¼2-fold over controls at 1 µM). This CB(1)-dependent activity was fully abolished by the selective CB(1) antagonist SR141716 or by RNA interference of the receptor. CB(1) signaling engaged p38 and p42/44 mitogen-activated protein kinases, which in turn activated the cyclic AMP response element-binding protein and the microphthalmia-associated transcription factor. Silencing of tyrosinase or microphthalmia-associated transcription factor further demonstrated the involvement of these proteins in AEA-induced melanogenesis. In addition, CB(1) activation did not engage the key regulator of skin pigmentation, cyclic AMP, showing a major difference compared with the regulation of melanogenesis by α-melanocyte-stimulating hormone through melanocortin 1 receptor.
Asunto(s)
Moduladores de Receptores de Cannabinoides/metabolismo , Endocannabinoides , Melaninas/metabolismo , Melanocitos/metabolismo , Monofenol Monooxigenasa/metabolismo , Receptor Cannabinoide CB1/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ácidos Araquidónicos/metabolismo , Ácidos Araquidónicos/farmacología , Western Blotting , Moduladores de Receptores de Cannabinoides/farmacología , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Glicéridos/metabolismo , Glicéridos/farmacología , Células HeLa , Humanos , Masculino , Melanocitos/citología , Melanocitos/efectos de los fármacos , Ratones , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Monofenol Monooxigenasa/genética , Piperidinas/farmacología , Alcamidas Poliinsaturadas/metabolismo , Alcamidas Poliinsaturadas/farmacología , Pirazoles/farmacología , Interferencia de ARN , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rimonabant , alfa-MSH/farmacologíaRESUMEN
Ascorbate oxidase (AAO) is a large, multidomain, dimeric protein whose folding/unfolding pathway is characterized by a complex, multistep process. Here we used fluorescence correlation spectroscopy to demonstrate the formation of partially folded monomers by pH-induced full dissociation into subunits. Hence, the structural features of monomeric AAO could be studied by fluorescence and CD spectroscopy. We found that the monomers keep their secondary structure, whereas subtle conformational changes in the tertiary structure become apparent. AAO dissociation has also been studied when unfolding the protein by high hydrostatic pressure at different pH values. A strong protein concentration dependence was observed at pH 8, whereas the enzyme was either monomeric or dimeric at pH 10 and 6, respectively. The calculated volume change associated with the unfolding of monomeric AAO, ΔV â¼ -55 mL·mol(-1), is in the range observed for most proteins of the same size. These findings demonstrate that partially folded monomeric species might populate the energy landscape of AAO and that the overall AAO stability is crucially controlled by a few quaternary interactions at the subunits' interface.
Asunto(s)
Ascorbato Oxidasa/metabolismo , Ascorbato Oxidasa/química , Dicroismo Circular , Concentración de Iones de Hidrógeno , Estructura Secundaria de Proteína , Espectrometría de FluorescenciaRESUMEN
Nonspecific binding of anandamide to plastic exhibits many features that could be mistaken as biological processes, thereby representing an important source of conflicting data on the uptake and release of this lipophilic substance. Herein, we propose an improved method to assay anandamide transport, by using glass slides (i.e., coverslips) as physical support to grow cells. Although the results obtained using plastic do not differ significantly from those obtained using glass, the new procedure has the advantage of being faster, simpler, and more accurate. In fact, the lack of aspecific adsorption of anandamide to the glass surface yields a lower background and a higher precision and accuracy in determining transport kinetics, especially for the export process. Remarkably, the kinetic parameters of anandamide uptake obtained with the old and the new procedures may be similar or different depending on the cell type, thus demonstrating the complexity of the interference of plastic on the transport process. In addition, the novel procedure is particularly suitable for visualization and measurement of anandamide transport in intact cells by using a biotinylated derivative in confocal fluorescence microscopy.
Asunto(s)
Ácidos Araquidónicos/química , Ácidos Araquidónicos/metabolismo , Artefactos , Alcamidas Poliinsaturadas/química , Alcamidas Poliinsaturadas/metabolismo , Adsorción , Transporte Biológico , Línea Celular Tumoral , Proliferación Celular , Endocannabinoides , Vidrio/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Plásticos/química , Silicatos/química , Espectrometría de Fluorescencia , Propiedades de SuperficieRESUMEN
The biological activity of endocannabinoids like anandamide (AEA) and 2-arachidonoylglycerol (2-AG) is subjected in vivo to a "metabolic control", exerted mainly by catabolic enzymes. AEA is inactivated by fatty acid amide hydrolase (FAAH), that is inhibited competitively by hydroxyanandamides (HAEAs) generated from AEA by lipoxygenase activity. Among these derivatives, 15-HAEA has been shown to be an effective (K(i) approximately 0.6 muM) FAAH inhibitor, that blocks also type-1 cannabinoid receptor (CB1R) but not other components of the "endocannabinoid system (ECS)", like the AEA transporter (AMT) or CB2R. Here, we extended the study of the effect of 15-HAEA on the AEA synthetase (NAPE-PLD) and the AEA-binding vanilloid receptor (TRPV1), showing that 15-HAEA activates the former (up to approximately 140% of controls) and inhibits the latter protein (down to approximately 70%). We also show that 15-HAEA halves the synthesis of 2-AG and almost doubles the transport of this compound across the membrane. In addition, we synthesized methyl and acetyl derivatives of 15-HAEA (15-MeOAEA and 15-AcOAEA, respectively), in order to check their ability to modulate FAAH and the other ECS elements. In fact, methylation and acetylation are common biochemical reactions in the cellular environment. We show that 15-MeOAEA, unlike 15-AcOAEA, is still a powerful competitive inhibitor of FAAH (K(i) approximately 0.7 muM), and that both derivatives have negligible interactions with the other proteins of ECS. Therefore, 15-MeOAEA is a FAAH inhibitor more selective than 15-HAEA. Further molecular dynamics analysis gave clues to the molecular requirements for the interaction of 15-HAEA and 15-MeOAEA with FAAH.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Moduladores de Receptores de Cannabinoides/metabolismo , Endocannabinoides , Alcamidas Poliinsaturadas/metabolismo , Acetilación , Amidohidrolasas/metabolismo , Animales , Ácidos Araquidónicos/farmacología , Cinética , Metilación , Ratones , Fosfolipasa D/efectos de los fármacos , Fosfolipasa D/metabolismo , Alcamidas Poliinsaturadas/farmacología , Receptor Cannabinoide CB1/efectos de los fármacos , Canales Catiónicos TRPV/metabolismoRESUMEN
Platelets are stored at 22 degrees C, since incubation at 37 degrees C results in loss of viability. Nonetheless, in our body (37 degrees C), platelets survive for 8-10 days. This discrepancy has been explained in terms of deprivation of viability factors or accumulation of apoptotic factors during storage. We report that the endocannabinoid anandamide (AEA) may be one of the agents allowing platelet survival. In fact, at 37 degrees C, human platelets enhance the expression of pro-apoptotic proteins (caspases, Bax, Bak) and decrease the expression of Bcl-xL, thus changing the Bcl-xL/Bak ratio, a key platelet biological clock. AEA or its non-hydrolyzable analogue, methanandamide, extend platelet life span, without reversing the changes in Bcl-xL/Bak ratio induced by heat stress. Instead, AEA binding to type-1 cannabinoid receptor activates Akt, which regulates, through phosphorylation of Bad, the interactions among different Bcl-2 family members. These findings could have implications for platelet collection and, potentially, for their clinical use.
Asunto(s)
Ácidos Araquidónicos/farmacología , Plaquetas/efectos de los fármacos , Conservación de la Sangre , Cannabinoides/farmacología , Supervivencia Celular/efectos de los fármacos , Alcamidas Poliinsaturadas/farmacología , Adulto , Ácidos Araquidónicos/metabolismo , Plaquetas/metabolismo , Plaquetas/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Endocannabinoides , Humanos , Alcamidas Poliinsaturadas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Cannabinoide CB1/metabolismo , Transducción de Señal/efectos de los fármacos , Manejo de EspecímenesRESUMEN
Anandamide (AEA) is an endogenous agonist of type 1 cannabinoid receptors (CB1R) that, along with metabolic enzymes of AEA and congeners, compose the "endocannabinoid system." Here we report the biochemical, morphological, and functional characterization of the endocannabinoid system in human neuroblastoma SH-SY5Y cells that are an experimental model for neuronal cell damage and death, as well as for major human neurodegenerative disorders. We also show that AEA dose-dependently induced apoptosis of SH-SY5Y cells. Through proteomic analysis, we further demonstrate that AEA-induced apoptosis was paralleled by an approximately 3 to approximately 5-fold up-regulation or down-regulation of five genes; IgG heavy chain-binding protein, stress-induced phosphoprotein-1, and triose-phosphate isomerase-1, which were up-regulated, are known to act as anti-apoptotic agents; actin-related protein 2/3 complex subunit 5 and peptidylprolyl isomerase-like protein 3 isoform PPIL3b were down-regulated, and the first is required for actin network formation whereas the second is still function-orphan. Interestingly, only the effect of AEA on BiP was reversed by the CB1R antagonist SR141716, in SH-SY5Y cells as well as in human neuroblastoma LAN-5 cells (that express a functional CB1R) but not in SK-NBE cells (which do not express CB1R). Silencing or overexpression of BiP increased or reduced, respectively, AEA-induced apoptosis of SH-SY5Y cells. In addition, the expression of BiP and of the BiP-related apoptotic markers p53 and PUMA was increased by AEA through a CB1R-dependent pathway that engages p38 and p42/44 mitogen-activated protein kinases. Consistently, this effect of AEA was minimized by SR141716. In conclusion, we identified BiP as a key protein in neuronal apoptosis induced by AEA.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácidos Araquidónicos/farmacología , Moduladores de Receptores de Cannabinoides/farmacología , Endocannabinoides , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas del Tejido Nervioso/biosíntesis , Neuronas/metabolismo , Alcamidas Poliinsaturadas/farmacología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Modelos Biológicos , Piperidinas/farmacología , Pirazoles/farmacología , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/metabolismo , RimonabantRESUMEN
The cellular uptake and the intracellular synthesis/degradation of anandamide are crucial steps for controlling its extracellular level and the duration of its activity. Although the biosynthesis and breakdown of anandamide are well understood, little is known about the mechanisms underlying its intracellular transport. Here, we investigated the presence of a potential carrier-mediated trafficking of anandamide within the cytosol, using a biotinylated analog as a tool to catch by affinity chromatography anandamide-interacting proteins. The identity of two of these anandamide-binding proteins, Hsp70 and serum albumin, was determined by mass spectrometry, confirmed by western blotting and confocal microscopy, and further validated through an anandamide-binding assay. These findings suggest that the trafficking of anandamide from the plasma membrane to the internal compartments of a cell occur via a nonvesicular mechanism mediated by cytosolic carriers.
Asunto(s)
Albúminas/metabolismo , Ácidos Araquidónicos/metabolismo , Proteínas Portadoras/metabolismo , Citosol/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Secuencia de Aminoácidos , Animales , Ácidos Araquidónicos/química , Transporte Biológico , Línea Celular Tumoral , Endocannabinoides , Humanos , Ratones , Alcamidas Poliinsaturadas/química , Unión ProteicaRESUMEN
Several G protein-associated receptors and synaptic proteins function within lipid rafts, which are subdomains of the plasma membranes that contain high concentrations of cholesterol. In this study we addressed the possible role of lipid rafts in the control of endocannabinoid system in striatal slices. Disruption of lipid rafts following cholesterol depletion with methyl-beta-cyclodestrin (MCD) failed to affect synthesis and degradation of anandamide, while it caused a marked increase in the synthesis and concentration of 2-arachidonoylglycerol (2-AG), as well as in the binding activity of cannabinoid CB1 receptors. Surprisingly, endogenous 2-AG-mediated control of GABA transmission was not potentiated by MCD treatment and, in contrast, neither basal nor 3,5-Dihydroxyphenylglycine-stimulated 2-AG altered GABA synapses in cholesterol-depleted slices. Synaptic response to the cannabinoid CB1 receptor agonist HU210 was however intact in MCD-treated slices, indicating that reduced sensitivity of cannabinoid CB1 receptors does not explain why endogenous 2-AG is ineffective in inhibiting striatal GABA transmission after cholesterol depletion. Confocal microscopy analysis suggested that disruption of raft integrity by MCD might uncouple metabotropic glutamate 5-CB1 receptor interaction by altering the correct localization of both receptors in striatal neuron elements. In conclusion, our data indicate that disruption of raft integrity causes a complex alteration of the endocannabinoid signalling in the striatum.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Cuerpo Estriado/fisiología , Glicéridos/metabolismo , Microdominios de Membrana/fisiología , Animales , Moduladores de Receptores de Cannabinoides/metabolismo , Colesterol/metabolismo , Cuerpo Estriado/metabolismo , Endocannabinoides , Potenciales Postsinápticos Inhibidores/fisiología , Ratones , Ratones Endogámicos C57BLRESUMEN
The role of the endocannabinoid system in haematopoietic cells is not completely understood. We investigated whether human erythroleukemia (HEL) cells were able to bind, metabolise and transport the main endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG). We also investigated whether AEA or 2-AG could modulate HEL differentiation. Although able to internalise both endocannabinoids, HEL cells had the machinery to metabolise 2-AG only, since they were devoid of the enzymes needed to synthesise and degrade AEA. Nonetheless, the intracellular transport of exogenous AEA might be required to activate the vanilloid receptors, with yet unknown implications for vascular biology. On the contrary, 2-AG appeared to play a role in lineage determination. Indeed, 2-AG itself drove HEL cells towards megakaryocytic differentiation, as it enhanced expression of beta3 integrin subunit, a megakaryocyte/platelet surface antigen, and glycoprotein VI, a late marker of megakaryocytes; in parallel, it reduced the amount of messenger RNA encoding for glycophorin A, a marker of erythroid phenotype. All these effects were mediated by activation of CB(2) cannabinoid receptors that triggered an extracellular signal-regulated kinase-dependent signalling cascade. In addition, classical inducers of megakaryocyte differentiation reduced 2-AG synthesis (although they did not affect the binding efficiency of CB(2) receptors), suggesting that levels of this endocannabinoid may be critical for committing HEL cells towards the megakaryocytic lineage.
Asunto(s)
Ácidos Araquidónicos/farmacología , Moduladores de Receptores de Cannabinoides/biosíntesis , Moduladores de Receptores de Cannabinoides/farmacología , Diferenciación Celular/efectos de los fármacos , Endocannabinoides , Regulación de la Expresión Génica/efectos de los fármacos , Glicéridos/farmacología , Megacariocitos/metabolismo , Antígenos de Diferenciación/biosíntesis , Ácidos Araquidónicos/metabolismo , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Humanos , Megacariocitos/citología , Alcamidas Poliinsaturadas/metabolismoRESUMEN
Anandamide (N-arachidonoylethanolamide; AEA) acts as an endogenous agonist of both cannabinoid and vanilloid receptors. During the last two decades, its metabolic pathways and biological activity have been investigated extensively and relatively well characterized. In contrast, at present, the effective nature and mechanism of AEA transport remain controversial and still unsolved issues. Here, we report the characterization of a biotinylated analog of AEA (b-AEA) that has the same lipophilicity of the parent compound. In addition, by means of biochemical assays and fluorescence microscopy, we show that b-AEA is accumulated inside the cells in a way superimposable on that of AEA. Conversely, b-AEA does not interact or interfere with the other components of the endocannabinoid system, such as type-1 and type-2 cannabinoid receptors, vanilloid receptor, AEA synthetase (N-acylphosphatidylethanolamine-hydrolyzing phospholipase D), or AEA hydrolase (fatty acid amide hydrolase). Together, our data suggest that b-AEA could be a very useful probe for visualizing the accumulation and intracellular distribution of this endocannabinoid.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Biotina/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Animales , Western Blotting , Línea Celular , Endocannabinoides , Técnica del Anticuerpo Fluorescente , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Microscopía Fluorescente , Receptores de Cannabinoides/metabolismoRESUMEN
Of the endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have received the most study. A functional interaction between these molecules has never been described. Using mouse brain slices, we found that stimulation of metabotropic glutamate 5 receptors by 3,5-dihydroxyphenylglycine (DHPG) depressed inhibitory transmission in the striatum through selective involvement of 2-AG metabolism and stimulation of presynaptic CB1 receptors. Elevation of AEA concentrations by pharmacological or genetic inhibition of AEA degradation reduced the levels, metabolism and physiological effects of 2-AG. Exogenous AEA and the stable AEA analog methanandamide inhibited basal and DHPG-stimulated 2-AG production, confirming that AEA is responsible for the downregulation of the other eCB. AEA is an endovanilloid substance, and the stimulation of transient receptor potential vanilloid 1 (TRPV1) channels mimicked the effects of endogenous AEA on 2-AG metabolism through a previously unknown glutathione-dependent pathway. Consistently, the interaction between AEA and 2-AG was lost after pharmacological and genetic inactivation of TRPV1 channels.
Asunto(s)
Ácidos Araquidónicos/farmacología , Ácidos Araquidónicos/fisiología , Moduladores de Receptores de Cannabinoides/farmacología , Cuerpo Estriado/efectos de los fármacos , Glicéridos/fisiología , Alcamidas Poliinsaturadas/farmacología , Amidohidrolasas/deficiencia , Animales , Cuerpo Estriado/citología , Regulación hacia Abajo/efectos de los fármacos , Interacciones Farmacológicas , Endocannabinoides , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Glutatión/metabolismo , Técnicas In Vitro , Metoxihidroxifenilglicol/análogos & derivados , Metoxihidroxifenilglicol/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Receptor Cannabinoide CB1/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genética , Canales Catiónicos TRPV/deficiencia , Factores de Tiempo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Alterations of the endocannabinoid system (ECS) have been recently implicated in a number of neuroinflammatory and neurodegenerative conditions so that the pharmacological modulation of cannabinoid (CB) receptors and/or of the enzymes controlling synthesis, transport, and degradation of these substances has emerged as a valuable option to treat neurological diseases. Here, we describe the current knowledge concerning the rearrangement of ECS in a primarily inflammatory disorder of the central nervous system such as multiple sclerosis (MS), and in a primarily degenerative condition such as amyotrophic lateral sclerosis (ALS). Furthermore, the data supporting a therapeutic role of agents modulating CB receptors or endocannabinoid tone in these disorders will also be reviewed. Complex changes of ECS take place in both diseases, influencing crucial aspects of their pathophysiology and clinical manifestations. Neuroinflammation, microglial activation, oxidative stress, and excitotoxicity are variably combined in MS and in ALS and can be modulated by endocannabinoids or by drugs targeting the ECS.