RESUMEN
Acute stress reduces pain sensitivity by engaging an endocannabinoid signaling circuit in the midbrain. The neural mechanisms governing this process and molecular identity of the endocannabinoid substance(s) involved are unknown. We combined behavior, pharmacology, immunohistochemistry, RNA interference, quantitative RT-PCR, enzyme assays, and lipidomic analyses of endocannabinoid content to uncover the role of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in controlling pain sensitivity in vivo. Here, we show that footshock stress produces antinociception in rats by activating type 5 metabotropic glutamate receptors (mGlu(5)) in the dorsolateral periaqueductal gray (dlPAG) and mobilizing 2-AG. Stimulation of mGlu(5) in the dlPAG with DHPG [(S)-3,5-dihydroxyphenylglycine] triggered 2-AG formation and enhanced stress-dependent antinociception through a mechanism dependent upon both postsynaptic diacylglycerol lipase (DGL) activity, which releases 2-AG, and presynaptic CB(1) cannabinoid receptors. Pharmacological blockade of DGL activity in the dlPAG with RHC80267 [1,6-bis(cyclohexyloximinocarbonylamino)hexane] and (-)-tetrahydrolipstatin (THL), which inhibit activity of DGL-α and DGL-ß isoforms, suppressed stress-induced antinociception. Inhibition of DGL activity in the dlPAG with THL selectively decreased accumulation of 2-AG without altering levels of anandamide. The putative 2-AG-synthesizing enzyme DGL-α colocalized with mGlu(5) at postsynaptic sites of the dlPAG, whereas CB(1) was confined to presynaptic terminals, consistent with a role for 2-AG as a retrograde signaling messenger. Finally, virally mediated silencing of DGL-α, but not DGL-ß, transcription in the dlPAG mimicked effects of DGL inhibition in suppressing both endocannabinoid-mediated stress antinociception and 2-AG formation. The results indicate that activation of the postsynaptic mGlu(5)-DGL-α cascade triggers retrograde 2-AG signaling in vivo. This pathway is required for endocannabinoid-mediated stress-induced analgesia.
Asunto(s)
Analgesia/métodos , Ácidos Araquidónicos/metabolismo , Moduladores de Receptores de Cannabinoides/farmacología , Endocannabinoides , Glicéridos/metabolismo , Lipoproteína Lipasa/metabolismo , Dolor/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Análisis de Varianza , Animales , Moduladores de Receptores de Cannabinoides/agonistas , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Ciclohexanonas/farmacología , Relación Dosis-Respuesta a Droga , Terapia Electroconvulsiva/métodos , Antagonistas de Aminoácidos Excitadores/farmacología , Masculino , Metoxihidroxifenilglicol/administración & dosificación , Metoxihidroxifenilglicol/análogos & derivados , Ratones , Microscopía Inmunoelectrónica , Dolor/tratamiento farmacológico , Dolor/patología , Sustancia Gris Periacueductal/efectos de los fármacos , Sustancia Gris Periacueductal/metabolismo , Piperidinas/farmacología , Inhibidores de Proteasas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , ARN Mensajero/metabolismo , ARN Interferente Pequeño/uso terapéutico , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/metabolismo , Receptor del Glutamato Metabotropico 5 , Rimonabant , Sinapsis/metabolismo , Sinapsis/ultraestructura , Espectrometría de Masas en TándemRESUMEN
The steroid 17ß-estradiol (E2) is well known to influence hippocampal functions such as memory, affective behaviors, and epilepsy. There is growing awareness that in addition to responding to ovarian E2, the hippocampus of both males and females synthesizes E2 as a neurosteroid that could acutely modulate synaptic function. Previous work on acute E2 actions in the hippocampus has focused on excitatory synapses. Here, we show that E2 rapidly suppresses inhibitory synaptic transmission in hippocampal CA1. E2 acts through the α form of the estrogen receptor to stimulate postsynaptic mGluR1-dependent mobilization of the endocannabinoid anandamide, which retrogradely suppresses GABA release from CB1 receptor-containing inhibitory presynaptic boutons. Remarkably, this effect of E2 is sex specific, occurring in females but not in males. Acute E2 modulation of endocannabinoid tone and consequent suppression of inhibition provide a mechanism by which neurosteroid E2 could modulate hippocampus-dependent behaviors in a sex-specific manner.
Asunto(s)
Región CA1 Hipocampal/citología , Moduladores de Receptores de Cannabinoides/metabolismo , Endocannabinoides , Estradiol/farmacología , Estrógenos/farmacología , Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Benzodioxoles/farmacología , Benzoxazinas/farmacología , Biofisica , Moduladores de Receptores de Cannabinoides/agonistas , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Castración , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Femenino , Antagonistas del GABA/farmacología , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Lactonas/farmacología , Masculino , Morfolinas/farmacología , NAD/farmacología , Naftalenos/farmacología , Neuronas/fisiología , Orlistat , Técnicas de Placa-Clamp , Fenoles , Piperidinas/farmacología , Pirazoles/farmacología , Piridazinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Chronic pain remains unsatisfactorily treated, and few novel painkillers have reached the market in the past century. Increasing the levels of the main endogenous opioid peptides - enkephalins - by inhibiting their two inactivating ectopeptidases, neprilysin and aminopeptidase N, has analgesic effects in various models of inflammatory and neuropathic pain. Stemming from the same pharmacological concept, fatty acid amide hydrolase (FAAH) inhibitors have also been found to have analgesic effects in pain models by preventing the breakdown of endogenous cannabinoids. Dual enkephalinase inhibitors and FAAH inhibitors are now in early-stage clinical trials. In this Review, we compare the effects of these two potential classes of novel analgesics and describe the progress in their rational design. We also consider the challenges in their clinical development and opportunities for combination therapies.
Asunto(s)
Analgésicos/farmacología , Analgésicos/farmacocinética , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Moduladores de Receptores de Cannabinoides/farmacocinética , Dolor Crónico/tratamiento farmacológico , Encefalinas/antagonistas & inhibidores , Amidohidrolasas/antagonistas & inhibidores , Animales , Moduladores de Receptores de Cannabinoides/farmacología , Dolor Crónico/metabolismo , HumanosRESUMEN
Disruptions of cell death signalling occur in pathological processes, such as cancer and degenerative disease. Increased knowledge of cell death signalling has opened new areas of therapeutic research, and identifying key mediators of cell death has become increasingly important. Early triggering events in cell death may provide potential therapeutic targets, whereas agents affecting later signals may be more palliative in nature. A group of primary mediators are derivatives of the highly unsaturated fatty acids (HUFAs), particularly oxygenated metabolites such as prostaglandins. HUFAs, esterified in cell membranes, act as critical signalling molecules in many pathological processes. Currently, agents affecting HUFA metabolism are widely prescribed in diseases involving disordered cell death signalling. However, partly due to rapid metabolism, their role in cell death signalling pathways is poorly characterized. Recently, HUFA-derived mediators, the resolvins/protectins and endocannabinoids, have added opportunities to target selective signals and pathways. This review will focus on the control of cell death by HUFA, eicosanoid (C20 fatty acid metabolites) and docosanoid (C22 metabolites), HUFA-derived lipid mediators, signalling elements in the micro-environment and their potential therapeutic applications. Further therapeutic approaches will involve cell and molecular biology, the multiple hit theory of disease progression and analysis of system plasticity. Advances in the cell biology of eicosanoid and docosanoid metabolism, together with structure/function analysis of HUFA-derived mediators, will be useful in developing therapeutic agents in pathologies characterized by alterations in cell death signalling.
Asunto(s)
Muerte Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Eicosanoides/metabolismo , Ácidos Grasos/metabolismo , Microdominios de Membrana/efectos de los fármacos , Terapia Molecular Dirigida , Transducción de Señal/efectos de los fármacos , Animales , Agonistas de Receptores de Cannabinoides , Antagonistas de Receptores de Cannabinoides , Moduladores de Receptores de Cannabinoides/agonistas , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Moduladores de Receptores de Cannabinoides/metabolismo , Membrana Celular/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Eicosanoides/agonistas , Eicosanoides/antagonistas & inhibidores , Ácidos Grasos/agonistas , Ácidos Grasos/antagonistas & inhibidores , Humanos , Microdominios de Membrana/metabolismo , Metabolómica/métodosRESUMEN
Synaptic activity in magnocellular neurosecretory neurones is influenced by the retrograde (i.e. somatodendritic) release of vasopressin, oxytocin and cannabinoids (CBs). For oxytocin neurones, oxytocin exerts constitutive effects on pre-synaptic activity through its ability to release CBs post-synaptically. In the present study, we examined evoked inhibitory post-synaptic currents (eIPSCs) and spontaneous inhibitory post-synaptic currents (sIPSCs) in identified vasopressin (VP) neurones in coronal slices from virgin rats to determine: (i) the extent to which CBs may also tonically modulate VP synaptic activity; and (ii) to determine whether depolarisation-induced suppression of inhibition was present in VP neurones, and if so, whether it was mediated by VP or CBs. The CB1 antagonists AM251 (1 µm) and SR14171 (1 µm) consistently increased the frequency of sIPSCs in VP neurones without affecting their amplitude, suggesting a tonic CB presence. This effect on frequency was independent of action potential activity, and blocked by chelating intracellular calcium with 10 mm ethylene glycol tetraacetic acid (EGTA). AM251 also increased the amplitude of eIPSCs and decreased the paired-pulse ratio (PPR) in VP neurones-effects that were completely blocked with even low (1 mm EGTA) internal calcium chelation. Bouts of evoked firing of VP neurones consistently suppressed sIPSCs but had no effect on eIPSCs or the PPR. This depolarisation-induced suppression of IPSCs was reduced by AM251, and was totally blocked by 10 µm of the mixed vasopressin/oxytocin antagonist, Manning compound. We then tested the effect of vasopressin on IPSCs at the same time as blocking CB1 receptors. Vasopressin (10-100 nm) inhibited sIPSC frequency but had no effect on sIPSC or eIPSC amplitudes, or on the PPR, in the presence of AM251. Taken together, these results suggest a tonic, pre-synaptic inhibitory modulation of IPSCs in VP neurones by CBs that is largely dependent on post-synaptic calcium, and an inhibitory effect of VP on IPSCs that is independent of CB release.
Asunto(s)
Moduladores de Receptores de Cannabinoides/fisiología , Neuronas GABAérgicas/fisiología , Potenciales Postsinápticos Inhibidores/fisiología , Vasopresinas/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Arginina Vasopresina/análogos & derivados , Arginina Vasopresina/farmacología , Calcio/fisiología , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Ácido Egtácico/farmacología , Femenino , Neuronas GABAérgicas/efectos de los fármacos , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/fisiología , Piperidinas/farmacología , Pirazoles/farmacología , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/fisiología , Rimonabant , Vasopresinas/antagonistas & inhibidores , Vasopresinas/farmacologíaRESUMEN
The endocannabinoid (eCB) system and the cannabinoid CB1 receptor (CB1R) play key roles in the modulation of brain functions. Although actions of eCBs and CB1Rs are well described at the synaptic level, little is known of their modulation of neural activity at the network level. Using microelectrode arrays, we have examined the role of CB1R activation in the modulation of the electrical activity of rat and mice cortical neural networks in vitro. We find that exogenous activation of CB1Rs expressed on glutamatergic neurons decreases the spontaneous activity of cortical neural networks. Moreover, we observe that the net effect of the CB1R antagonist AM251 inversely correlates with the initial level of activity in the network: blocking CB1Rs increases network activity when basal network activity is low, whereas it depresses spontaneous activity when its initial level is high. Our results reveal a complex role of CB1Rs in shaping spontaneous network activity, and suggest that the outcome of endogenous neuromodulation on network function might be state dependent.
Asunto(s)
Encéfalo/citología , Moduladores de Receptores de Cannabinoides/metabolismo , Endocannabinoides , Red Nerviosa/fisiología , Neuronas/fisiología , 4-Aminopiridina/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Animales , Animales Recién Nacidos , Benzotiadiazinas/farmacología , Moduladores de Receptores de Cannabinoides/agonistas , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Células Cultivadas , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas del GABA , Técnicas In Vitro , Ratones , Ratones Transgénicos , Red Nerviosa/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Inhibición Neural/genética , Neuronas/clasificación , Neuronas/efectos de los fármacos , Compuestos Organofosforados/farmacología , Picrotoxina/farmacología , Piperidinas/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Pirazoles/farmacología , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/deficiencia , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacología , Valina/análogos & derivados , Valina/farmacologíaRESUMEN
Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for central control of fertility. Regulation of GnRH neurons by long-loop gonadal steroid feedback through steroid receptor-expressing afferents such as GABAergic neurons is well studied. Recently, local central feedback circuits regulating GnRH neurons were identified. GnRH neuronal depolarization induces short-term inhibition of their GABAergic afferents via a mechanism dependent on metabotropic glutamate receptor (mGluR) activation. GnRH neurons are enveloped in astrocytes, which express mGluRs. GnRH neurons also produce endocannabinoids, which can be induced by mGluR activation. We hypothesized the local GnRH-GABA circuit utilizes glia-derived and/or cannabinoid mechanisms and is altered by steroid milieu. Whole cell voltage-clamp was used to record GABAergic postsynaptic currents (PSCs) from GnRH neurons before and after action potential-like depolarizations were mimicked. In GnRH neurons from ovariectomized (OVX) mice, this depolarization reduced PSC frequency. This suppression was blocked by inhibition of prostaglandin synthesis with indomethacin, by a prostaglandin receptor antagonist, or by a specific glial metabolic poison, together suggesting the postulate that prostaglandins, potentially glia-derived, play a role in this circuit. This circuit was also inhibited by a CB1 receptor antagonist or by blockade of endocannabinoid synthesis in GnRH neurons, suggesting an endocannabinoid element, as well. In females, local circuit inhibition persisted in androgen-treated mice but not in estradiol-treated mice or young ovary-intact mice. In contrast, local circuit inhibition was present in gonad-intact males. These data suggest GnRH neurons interact with their afferent neurons using multiple mechanisms and that these local circuits can be modified by both sex and steroid feedback.
Asunto(s)
Encéfalo/citología , Moduladores de Receptores de Cannabinoides/metabolismo , Endocannabinoides , Hormona Liberadora de Gonadotropina/metabolismo , Neuronas/fisiología , Prostaglandinas/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Potenciales de Acción/efectos de los fármacos , Vías Aferentes/efectos de los fármacos , Vías Aferentes/fisiología , Análisis de Varianza , Andrógenos/administración & dosificación , Animales , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Dihidrotestosterona/administración & dosificación , Interacciones Farmacológicas , Estimulación Eléctrica , Estradiol/administración & dosificación , Estrógenos/administración & dosificación , Femenino , Hormona Liberadora de Gonadotropina/genética , Proteínas Fluorescentes Verdes/genética , Técnicas In Vitro , Masculino , Ratones , Ratones Transgénicos , Red Nerviosa/efectos de los fármacos , Red Nerviosa/fisiología , Neuronas/efectos de los fármacos , Ovariectomía , Técnicas de Placa-Clamp , Piperidinas/farmacología , Pirazoles/farmacología , Rimonabant , Transmisión Sináptica/efectos de los fármacosRESUMEN
The lack of satisfactory results of alcohol dependence treatment force us to search for new directions of research. Recent studies concentrate on endocannabinoid transmission. The results show an interplay between the endocannabinoid and dopaminergic signaling in activation of the limbic reward system. The mechanisms leading to development of dependence are very complex and poorly recognized. Endogenous cannabinoids seem to have an important role in the functioning of this system, both directly and indirectly affecting the level of different neurotransmitters. The effect of alcohol on the endocannabinoid system is also complex and involves changes at the molecular level. Experimental studies have demonstrated an important role of the CB1 receptors in the neurochemical mechanism of alcohol consumption and its regulation. SR141716 (rimonabant), a CB1 receptor antagonist, significantly lowers voluntary alcohol intake and motivation for its consumption in various experimental studies. Very encouraging results of preclinical studies were not completely confirmed in the clinical studies. However, further clinical studies are still necessary.
Asunto(s)
Alcoholismo/etiología , Moduladores de Receptores de Cannabinoides/fisiología , Piperidinas/uso terapéutico , Pirazoles/uso terapéutico , Receptores de Cannabinoides/fisiología , Consumo de Bebidas Alcohólicas , Alcoholismo/tratamiento farmacológico , Antagonistas de Receptores de Cannabinoides , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Ensayos Clínicos como Asunto , Humanos , RimonabantRESUMEN
The frequency and duration of glutamatergic inputs to the striatum are strong determinants of the net effect of retrograde endocannabinoid (eCB) signaling, and key factors in determining if long-term depression (LTD) has a net disinhibitory or inhibitory action in striatum. Low to moderate frequency stimulation in the dorsolateral striatum elevates eCB levels to an extent that primarily depresses transmitter release at inhibitory synapses, leading to a long-lasting disinhibition (DLL) of synaptic output. The aim of this study was to further characterize the basic features of endocannabinoid-mediated DLL of striatal output induced by moderate frequency stimulation (5 Hz, 60 s). DLL was inhibited in slices treated with the group 1 metabotropic glutamate receptor (mGluR) antagonists MPEP (40 µM) and CPCCOEt (40 µM), the dopamine D2 receptor antagonist sulpiride (5 µM), the L-type calcium channel blocker nifedipine (20 µM), the nicotinic receptor antagonist mecamylamine (10 µM), the muscarinic agonist oxotremorine sesquifumarate (10 µM), and strychnine (0.1 µM). Strychnine did not block DLL induced by WIN55,212-2 (250 nM), showing that glycine receptor-mediated modulation of eCB signaling occurs upstream from CB(1)R activation. Scopolamine (10 µM) restored DLL in strychnine-treated slices, suggesting that inhibition of glycine receptors on cholinergic interneurons could modulate eCB signaling by enhancing muscarinic receptor activation and reducing the opening of L-type calcium channels in response to depolarization. These data suggests that similar activation points are required for stimulation-induced DLL as for LTD at excitatory striatal synapses, and that cholinergic interneurons are key modulators of stimulation-induced eCB signaling in the striatum.
Asunto(s)
Moduladores de Receptores de Cannabinoides/metabolismo , Cuerpo Estriado/citología , Endocannabinoides , Interneuronas/metabolismo , Inhibición Neural/fisiología , Transducción de Señal/fisiología , Animales , Animales Recién Nacidos , Benzazepinas/farmacología , Bicuculina/farmacología , Biofisica , Moduladores de Receptores de Cannabinoides/agonistas , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Colinérgicos/farmacología , Cromonas/farmacología , Antagonistas de Dopamina/farmacología , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Técnicas In Vitro , Interneuronas/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Modelos Neurológicos , Inhibición Neural/efectos de los fármacos , Piperidinas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Sulpirida/farmacología , Valina/análogos & derivados , Valina/farmacologíaRESUMEN
Alterations in the endogenous cannabinoid system have been described in almost every category of disease. These changes can alternatively be protective or maladaptive, such as producing antinociception in neuropathic pain or fibrogenesis in liver disease, making the system an attractive therapeutic target. However, the challenge remains to selectively target the site of disease while sparing other areas, particularly mood and cognitive centers of the brain. Identifying regional changes in cannabinoid receptor-1 and -2 (CB(1)R and CB(2)R) expression is particularly important when considering endocannabinoid system-based therapies, because regional increases in cannabinoid receptor expression have been shown to increase potency and efficacy of exogenous agonists at sites of disease. Although there have been extensive descriptive studies of cannabinoid receptor expression changes in disease, the underlying mechanisms are only just beginning to unfold. Understanding these mechanisms is important and potentially relevant to therapeutics. In diseases for which cannabinoid receptors are protective, knowledge of the mechanisms of receptor up-regulation could be used to design therapies to regionally increase receptor expression and thus increase efficacy of an agonist. Alternatively, inhibition of harmful cannabinoid up-regulation could be an attractive alternative to global antagonism of the system. Here we review current findings on the mechanisms of cannabinoid receptor regulation in disease and discuss their therapeutic implications.
Asunto(s)
Regulación de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptores de Cannabinoides/metabolismo , Animales , Agonistas de Receptores de Cannabinoides , Antagonistas de Receptores de Cannabinoides , Moduladores de Receptores de Cannabinoides/agonistas , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Moduladores de Receptores de Cannabinoides/metabolismo , Cannabinoides/agonistas , Cannabinoides/antagonistas & inhibidores , Cannabinoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Terapia Molecular Dirigida , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Neuronas/efectos de los fármacos , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/antagonistas & inhibidores , Receptor Cannabinoide CB2/genética , Receptor Cannabinoide CB2/metabolismo , Receptores de Cannabinoides/genéticaRESUMEN
OBJECTIVE: To study the influences of endocannabinoid-anandamide (AEA) on the proliferation and apoptosis of the colorectal cancer cell line (CaCo-2) and to elucidate the effects of CB1 and lipid rafts, and to further elucidate the molecular mechanism and the effect of AEA on the generation and development of colorectal cancer. METHODS: Human colorectal cancer cell line CaCo-2 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in 5% CO(2) atmosphere at 37°C. CaCo-2 cells were divided into different groups and treated with different concentrations of AEA, AEA + SR141716A, AEA + AM630 and AEA + methyl-ß-cyclodextrin (MCD). MTT assay was used to determine the effects of AEA, its putative CB1, CB2 receptor antagonists (SR141716A and AM630) and MCD on the proliferation of CaCo-2 cells. Annexin V-PE/7AAD binding assay was used to detect apoptosis in the CaCo-2 cells. Western-blot was applied to check the expressions of CB1, CB2, p-AKT and caspase-3 proteins in different groups of CaCo-2 cells. RESULTS: AEA inhibited the proliferation of CaCo-2 cells in a concentration-dependent manner and the effect could be antagonized by SR141716A and MCD. The inhibiting rates were (21.52 ± 0.45)%, (42.16 ± 0.21)%, (73.64 ± 0.73)% and (83.28 ± 0.71)%, respectively, at different concentrations of AEA (5, 10, 20 and 40 µmol/L). The three groups (20 µmol/L AEA, 20 µmol/L AEA + 10 µmol/L SR141716A and 20 µmol/L AEA + 1 mmol/L MCD) showed different inhibiting rates [(73.64 ± 0.73)%, (16.15 ± 0.75)% and (12.58 ± 0.63)%], respectively. Annexin V-PE/7AAD binding assay showed that AEA induced apoptosis in the CaCo-2 cells and MCD could antagonize this effect. The apoptosis rates of the three groups (control, 20 µmol/L AEA and 20 µmol/L AEA + 1 mmol/L MCD) were (2.95 ± 0.73)%, (39.61 ± 0.73)% and (14.10 ± 0.64)%, respectively. The expressions of CB1, CB2, p-AKT and Caspase-3 proteins were all observed in the CaCo-2 cells. AEA inhibited p-AKT protein expression and induced caspase-3 protein expression. The two actions were also antagonized by MCD. CONCLUSIONS: AEA can strongly suppress the proliferation of colorectal cancer CaCo-2 cells via the CB1 receptor and membrane cholesterol-LRs and induce apoptosis via lipid rafts. Anandamide plays a very important role in the carcinogenesis and development of colorectal cancer. MCD is a critical member in this system.
Asunto(s)
Ácidos Araquidónicos/farmacología , Moduladores de Receptores de Cannabinoides/farmacología , Proliferación Celular/efectos de los fármacos , Microdominios de Membrana/metabolismo , Alcamidas Poliinsaturadas/farmacología , Receptor Cannabinoide CB1/antagonistas & inhibidores , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ácidos Araquidónicos/antagonistas & inhibidores , Células CACO-2 , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Caspasa 3/metabolismo , Relación Dosis-Respuesta a Droga , Endocannabinoides , Humanos , Indoles/farmacología , Piperidinas/farmacología , Alcamidas Poliinsaturadas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirazoles/farmacología , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/antagonistas & inhibidores , Receptor Cannabinoide CB2/metabolismo , Rimonabant , beta-Ciclodextrinas/metabolismoAsunto(s)
Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Colesterol/metabolismo , Hígado Graso/tratamiento farmacológico , Resistencia a la Insulina/fisiología , Obesidad Mórbida/tratamiento farmacológico , Piperidinas/uso terapéutico , Pirazoles/uso terapéutico , Adulto , Biomarcadores/metabolismo , Femenino , Humanos , Hipercolesterolemia/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico , Obesidad Mórbida/metabolismo , Estudios Retrospectivos , Rimonabant , Retirada de Medicamento por Seguridad , Triglicéridos/metabolismo , Pérdida de Peso/efectos de los fármacosAsunto(s)
Depresores del Apetito/uso terapéutico , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Dexfenfluramina/uso terapéutico , Diseño de Fármacos , Agonismo Inverso de Drogas , Piperidinas/uso terapéutico , Pirazoles/uso terapéutico , Receptor Cannabinoide CB1/antagonistas & inhibidores , Animales , Depresores del Apetito/efectos adversos , Dexfenfluramina/efectos adversos , Humanos , Obesidad/tratamiento farmacológico , Piperidinas/efectos adversos , Pirazoles/efectos adversos , Rimonabant , Agonistas de Receptores de Serotonina/efectos adversos , Agonistas de Receptores de Serotonina/uso terapéuticoRESUMEN
BACKGROUND AND PURPOSE: Presynaptic CB1 cannabinoid receptors can be activated by endogenous cannabinoids (endocannabinoids) synthesized by postsynaptic neurones. The hypothesis of the present work was that activation of calcium-permeable transmitter-gated ion channels in postsynaptic neurones, specifically of P2X purine receptors, can lead to endocannabinoid production and retrograde synaptic signalling. EXPERIMENTAL APPROACH: GABAergic inhibitory postsynaptic currents (IPSCs) were recorded with patch-clamp techniques in Purkinje cells in mouse cerebellar slices. Purine receptors on Purkinje cells were activated by pressure ejection of ATP from a pipette. KEY RESULTS: ATP evoked an inward current in Purkinje cells, most likely due to P2X receptor activation. The ATP-evoked currents were accompanied by currents via voltage-gated calcium channels. ATP suppressed electrical stimulation-evoked IPSCs and miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin, and these effects were prevented by the CB1 antagonist rimonabant and the calcium chelator BAPTA (applied into the Purkinje cell). ATP also suppressed mIPSCs when voltage-gated calcium channels were blocked by cadmium, and intracellular calcium stores were depleted by thapsigargin. However, ATP failed to suppress mIPSCs when the extracellular calcium concentration was zero. CONCLUSIONS AND IMPLICATIONS: ATP elicits CB1 receptor-dependent retrograde synaptic suppression, which is probably mediated by an endocannabinod released by the postsynaptic neurone. An increase in intracellular calcium concentration in the postsynaptic neurone is necessary for this retrograde signalling. We propose that ATP increases the calcium concentration by two mechanisms: calcium enters into the neurone via the P2X receptor ion channel and the ATP-evoked depolarization triggers voltage-gated calcium channels.
Asunto(s)
Moduladores de Receptores de Cannabinoides/metabolismo , Corteza Cerebelosa/metabolismo , Endocannabinoides , Receptores Purinérgicos/metabolismo , Transmisión Sináptica , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/efectos de los fármacos , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Corteza Cerebelosa/efectos de los fármacos , Técnicas In Vitro , Cinética , Ratones , Técnicas de Placa-Clamp , Agonistas Purinérgicos/farmacología , Células de Purkinje/efectos de los fármacos , Células de Purkinje/metabolismo , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptores Purinérgicos P2X/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Potenciales Sinápticos/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacosRESUMEN
The cannabinoid receptor type 1 (CB1) and the central nucleus of the amygdala (CeA) are both known to have crucial roles in the processing of fear and anxiety, whereby they appear to be especially involved in the control of fear states. However, in contrast to many other brain regions including the cortical subregions of the amygdala, the existence of CB1 in the CeA remains enigmatic. In this study we show that CB1 is expressed in the CeA of mice and that CB1 in the CeA mediates short-term synaptic plasticity, namely depolarization-induced suppression of excitation (DSE) and inhibition (DSI). Moreover, the CB1 antagonist AM251 increased both excitatory and inhibitory postsynaptic responses in CeA neurons. Local application of AM251 in the CeA in vivo resulted in an acutely increased fear response in an auditory fear conditioning paradigm. Upon application of AM251 in the basolateral nucleus of the amygdala (BLA) in an otherwise identical protocol, no such acute behavioral effects were detected, but CB1 blockade resulted in increased fear responses during tone exposures on the subsequent days. Moreover, we observed that the efficacy of DSE and DSI in the CeA was increased on the day following fear conditioning, indicating that a single tone-shock pairing resulted in changes in endocannabinoid signaling in the CeA. Taken together, our data show the existence of CB1 proteins in the CeA, and their critical role for ensuring short-term adaptation of responses to fearful events, thereby suggesting a potential therapeutic target to accompany habituation-based therapies of post-traumatic symptoms.
Asunto(s)
Adaptación Psicológica/fisiología , Amígdala del Cerebelo/fisiología , Moduladores de Receptores de Cannabinoides/metabolismo , Condicionamiento Psicológico/fisiología , Endocannabinoides , Miedo/fisiología , Transducción de Señal/fisiología , Potenciales de Acción/efectos de los fármacos , Amígdala del Cerebelo/citología , Amígdala del Cerebelo/efectos de los fármacos , Animales , Conducta Animal , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Condicionamiento Psicológico/efectos de los fármacos , Estimulación Eléctrica/métodos , Antagonistas de Aminoácidos Excitadores/farmacología , Extinción Psicológica/efectos de los fármacos , Extinción Psicológica/fisiología , Miedo/efectos de los fármacos , Antagonistas del GABA/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Técnicas In Vitro , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ácidos Fosfínicos/farmacología , Piperidinas/farmacología , Propanolaminas/farmacología , Pirazoles/farmacología , Piridazinas/farmacología , Quinoxalinas/farmacología , Receptor Cannabinoide CB1/deficiencia , Células Receptoras Sensoriales/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Tiempo , Valina/análogos & derivados , Valina/farmacologíaRESUMEN
Endocannabinoids and their receptor CB1 play key roles in brain function. Astrocytes express CB1Rs that are activated by endocannabinoids released by neurons. However, the consequences of the endocannabinoid-mediated neuron-astrocyte signaling on synaptic transmission are unknown. We show that endocannabinoids released by hippocampal pyramidal neurons increase the probability of transmitter release at CA3-CA1 synapses. This synaptic potentiation is due to CB1R-induced Ca(2+) elevations in astrocytes, which stimulate the release of glutamate that activates presynaptic metabotropic glutamate receptors. While endocannabinoids induce synaptic depression in the stimulated neuron by direct activation of presynaptic CB1Rs, they indirectly lead to synaptic potentiation in relatively more distant neurons by activation of CB1Rs in astrocytes. Hence, astrocyte calcium signal evoked by endogenous stimuli (neuron-released endocannabinoids) modulates synaptic transmission. Therefore, astrocytes respond to endocannabinoids that then potentiate synaptic transmission, indicating that astrocytes are actively involved in brain physiology.
Asunto(s)
Astrocitos/fisiología , Moduladores de Receptores de Cannabinoides/metabolismo , Endocannabinoides , Células Piramidales/fisiología , Transmisión Sináptica/fisiología , Animales , Animales Recién Nacidos , Benzoatos/farmacología , Biofisica , Calcio/metabolismo , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Moduladores de Receptores de Cannabinoides/farmacología , Quelantes/farmacología , Interacciones Farmacológicas , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Estimulación Eléctrica/métodos , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/genética , Glicina/análogos & derivados , Glicina/farmacología , Hipocampo/citología , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Placa-Clamp/métodos , Fotólisis , Piperidinas/farmacología , Células Piramidales/efectos de los fármacos , Pirazoles/farmacología , Piridinas/farmacología , Receptor Cannabinoide CB1/deficiencia , Resorcinoles/farmacología , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genética , Tapsigargina/farmacologíaRESUMEN
Peripheral cannabinoid receptors exert a powerful inhibitory control over pain initiation, but the endocannabinoid signal that normally engages this intrinsic analgesic mechanism is unknown. To address this question, we developed a peripherally restricted inhibitor (URB937) of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the endocannabinoid anandamide. URB937 suppressed FAAH activity and increased anandamide levels outside the rodent CNS. Despite its inability to access brain and spinal cord, URB937 attenuated behavioral responses indicative of persistent pain in rodent models of peripheral nerve injury and inflammation and prevented noxious stimulus-evoked neuronal activation in spinal cord regions implicated in nociceptive processing. CB1 cannabinoid receptor blockade prevented these effects. These results suggest that anandamide-mediated signaling at peripheral CB1 receptors controls the access of pain-related inputs to the CNS. Brain-impenetrant FAAH inhibitors, which strengthen this gating mechanism, might offer a new approach to pain therapy.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Ácidos Araquidónicos/uso terapéutico , Moduladores de Receptores de Cannabinoides/metabolismo , Moduladores de Receptores de Cannabinoides/uso terapéutico , Endocannabinoides , Dolor/prevención & control , Alcamidas Poliinsaturadas/metabolismo , Alcamidas Poliinsaturadas/uso terapéutico , Amidohidrolasas/deficiencia , Amidohidrolasas/metabolismo , Animales , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Cannabinoides/farmacología , Cannabinoides/uso terapéutico , Carragenina , Cromatografía Liquida/métodos , Modelos Animales de Enfermedad , Vías de Administración de Medicamentos , Esquema de Medicación , Inhibidores Enzimáticos , Reacción de Fuga/efectos de los fármacos , Glicoles de Etileno/metabolismo , Conducta Alimentaria/efectos de los fármacos , Formaldehído , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/genética , Indoles/uso terapéutico , Masculino , Espectrometría de Masas/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monoacilglicerol Lipasas/metabolismo , Actividad Motora/efectos de los fármacos , Proteínas Oncogénicas v-fos/metabolismo , PPAR alfa/deficiencia , Dolor/inducido químicamente , Dolor/genética , Dolor/patología , Dimensión del Dolor/efectos de los fármacos , Umbral del Dolor/efectos de los fármacos , Enfermedades del Sistema Nervioso Periférico , Piperidinas/uso terapéutico , Pirazoles/uso terapéutico , Ratas , Ratas Sprague-Dawley , Rimonabant , Ciática/tratamiento farmacológico , Médula Espinal/metabolismo , Estadísticas no Paramétricas , Factores de Tiempo , Distribución Tisular/efectos de los fármacos , TritioRESUMEN
Prolonged exposure to drugs of abuse, such as cannabinoids and opioids, leads to pharmacological tolerance and receptor desensitization in the nervous system. We found that a similar form of functional antagonism was produced by sustained inactivation of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol. After repeated administration, the MAGL inhibitor JZL184 lost its analgesic activity and produced cross-tolerance to cannabinoid receptor (CB1) agonists in mice, effects that were phenocopied by genetic disruption of Mgll (encoding MAGL). Chronic MAGL blockade also caused physical dependence, impaired endocannabinoid-dependent synaptic plasticity and desensitized brain CB1 receptors. These data contrast with blockade of fatty acid amide hydrolase, an enzyme that degrades the other major endocannabinoid anandamide, which produced sustained analgesia without impairing CB1 receptors. Thus, individual endocannabinoids generate distinct analgesic profiles that are either sustained or transitory and associated with agonism and functional antagonism of the brain cannabinoid system, respectively.
Asunto(s)
Moduladores de Receptores de Cannabinoides/metabolismo , Endocannabinoides , Monoacilglicerol Lipasas/metabolismo , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/metabolismo , Analgésicos/administración & dosificación , Analgésicos/farmacología , Animales , Benzodioxoles/administración & dosificación , Benzodioxoles/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Animales , Monoacilglicerol Lipasas/antagonistas & inhibidores , Monoacilglicerol Lipasas/genética , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Dolor/tratamiento farmacológico , Dolor/metabolismo , Piperidinas/administración & dosificación , Piperidinas/farmacología , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/fisiologíaRESUMEN
A growing body of evidence supports an important role for the endocannabinoid system as a regulator of appetite, body weight, and systemic metabolism, which is overactive in obesity and type 2 diabetes. While initial attempts to target this system using the cannabinoid receptor inverse agonist rimonabant were successful in producing modest weight loss and improving obesity-related metabolic complications in humans, adverse central nervous system side effects precluded introduction of this drug into clinical practice. However, new data, presented by Tam and colleagues in this issue of the JCI, demonstrate that selective blockade of peripheral cannabinoid receptors may be a novel successful therapeutic approach.
Asunto(s)
Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Endocannabinoides , Obesidad/tratamiento farmacológico , Receptor Cannabinoide CB1/antagonistas & inhibidores , Animales , Regulación del Apetito , Peso Corporal , Moduladores de Receptores de Cannabinoides/fisiología , Humanos , Ratones , Piperidinas/uso terapéutico , Pirazoles/uso terapéutico , RimonabantRESUMEN
IMPORTANCE OF THE FIELD: Addiction to opiates is one of the most severe forms of substance dependence, and despite a variety of pharmacological approaches to treat it, relapse is observed in a high percentage of subjects. New pharmacological compounds are necessary to improve the outcome of treatments and reduce adverse side effects. Moreover, drugs that act on the opioid system can also be of benefit in the treatment of alcohol or cocaine addiction. AREA COVERED BY THIS REVIEW: Recent preclinical studies of pharmacological agents for the treatment of opiate addiction (2008 to the present date). WHAT THE READER WILL GAIN: The reader will be informed of the latest drugs shown in animal models to modify dependence on opiates and the reinforcing effects of these drugs. In addition, reports of the latest studies to test these compounds in models of other drug addictions are reviewed. TAKE HOME MESSAGE: The classic clinical pharmacotherapy for opiate dependence, involving mu-opioid receptor agonists or antagonists, has not yielded a high success rate in humans. In pharmacotherapy for opioid dependence, new options are emerging and different pharmacological strategies are now being tested.