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Recent studies have sparked renewed interest in the therapeutic potential of psychedelics for treating depression and other mental health conditions. Simultaneously, the novel psychoactive substances (NPS) phenomenon, with a huge number of NPS emerging constantly, has changed remarkably the illicit drug market, being their scientific evaluation an urgent need. Thus, this study aims to elucidate the impact of amino-terminal modifications to the 5-MeO-DMT molecule on its interactions with serotonin receptors and transporters, as well as its psychoactive and thermoregulatory properties. Our findings demonstrated, using radioligand binding methodologies, that all examined 5-MeO-tryptamines exhibited selectivity for 5-HT1AR over 5-HT2AR. In fact, computational docking analyses predicted a better interaction in the 5-HT1AR binding pocket compared to 5-HT2AR. Our investigation also proved the interaction of these compounds with SERT, revealing that the molecular size of the amino group significantly influenced their affinity. Subsequent experiments involving serotonin uptake, electrophysiology, and superfusion release assays confirmed 5-MeO-pyr-T as the most potent partial 5-HT releaser tested. All tested tryptamines elicited, to some degree, the head twitch response (HTR) in mice, indicative of a potential hallucinogenic effect and mainly mediated by 5-HT2AR activation. However, 5-HT1AR was also shown to be implicated in the hallucinogenic effect, and its activation attenuated the HTR. In fact, tryptamines that produced a higher hypothermic response, mediated by 5-HT1AR, tended to exhibit a lower hallucinogenic effect, highlighting the opposite role of both 5-HT receptors. Moreover, although some 5-MeO-tryptamines elicited very low HTR, they still act as potent 5-HT2AR agonists. In summary, this research offers a comprehensive understanding of the psychopharmacological profile of various amino-substituted 5-MeO-tryptamines, keeping structural aspects in focus and accumulating valuable data in the frame of NPS. Moreover, the unique characteristics of some 5-MeO-tryptamines render them intriguing molecules as mixed-action drugs and provide insight within the search of non-hallucinogenic but 5-HT2AR ligands as therapeutical agents.
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The utility of classical drugs used to treat psychiatric disorders (e.g., antidepressants, anxiolytics) is often limited by issues of lack of efficacy, delayed onset of action or side effects. Psychoactive substances have a long history of being used as tools to alter consciousness and as a gateway to approach the unknown and the divinities. These substances were initially obtained from plants and animals and more recently by chemical synthesis, and its consumption evolved toward a more recreational use, leading to drug abuse-related disorders, trafficking, and subsequent banning by the authorities. However, these substances, by modulation of certain neurochemical pathways, have been proven to have a beneficial effect on some psychiatric disorders. This evidence obtained under medically controlled conditions and often associated with psychotherapy, makes these substances an alternative to conventional medicines, to which in many cases the patient does not respond properly. Such disorders include post-traumatic stress disease and treatment-resistant depression, for which classical drugs such as MDMA, ketamine, psilocybin and LSD, among others, have already been clinically tested, reporting successful outcomes. The irruption of new psychoactive substances (NPS), especially during the last decade and despite their recreational and illicit uses, has enlarged the library of substances with potential utility on these disorders. In fact, many of them were synthetized with therapeutic purposes and were withdrawn for concrete reasons (e.g., adverse effects, improper pharmacological profile). In this review we focus on the basis, existing evidence and possible use of synthetic cathinones and psychedelics (specially tryptamines) for the treatment of mental illnesses and the properties that should be found in NPS to obtain new therapeutic compounds.
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N-ethyl-pentedrone (NEPD, 2-(ethylamino)-1-phenyl-1-pentanone) is one of the latest synthetic cathinone derivatives that emerged into the illicit drug market. This drug has psychostimulant properties and has been related with several intoxications and even fatalities. However, information about the consequences of its acute and repeated consumption is lacking. Thus, the aim of our study was to investigate the behavioral effects after both acute and repeated NEPD exposure as well as the neurochemical changes. Male OF1 mice were treated with an acute dose (1, 3 or 10 mg/kg, i.p.) or received repeated injections of these doses (twice/day, 5 days) of NEPD. Shortly after drug-exposure or during drug-withdrawal, anxiety-like behavior, aggressiveness, social interaction, depressive-like symptoms, body weight and temperature were assessed. Also, monoamine synthesis enzymes, levels of neurotransmitters and their precursors and main metabolites, as well as ΔFosB, were determined in striatum and prefrontal cortex from post-mortem tissue. Acute administration of NEPD induced anxiolytic effects and reduced social exploration whereas during withdrawal after repeated administration the anxiolytic effect had vanished, and the reduced social exploration was still present and accompanied with increased aggressive behavior. Moreover, NEPD (10 mg/kg) induced slight hyperthermia and reduced weight gain during the repeated administration, whereas increased locomotor activity and lack of depressive symptoms were found during withdrawal. This was accompanied by increased plasma corticosterone and decrease in striatal dopamine. Finally, the long-lasting and robust increase in ΔFosB levels found in striatum after NEPD chronic exposure suggests a high risk of dependence. The increased aggressivity and locomotor activity, together with this potential of inducing dependence justify a warning about the risks of consumption of NEPD if translated to humans.
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Estimulantes del Sistema Nervioso Central , Pentanonas , Agresión , Animales , Masculino , Metilaminas , RatonesRESUMEN
Changes in the molecular structure of synthetic cathinones has led to an increase in the number of novel emerging drugs in the illicit drug market at an unprecedented rate. Unfortunately, little is known about the neuropsychopharmacology of recently emerged halogen-substituted α-PVP derivatives. Thus, the aim of this study was to investigate the role of para- and meta-halogen (F-, Cl-, and Br-) substitutions on the in vitro, in silico, and in vivo effects of α-pyrrolidinopentiophenone (α-PVP) derivatives. HEK293 cells expressing the human dopamine or serotonin transporter (hDAT and hSERT) were used for the uptake inhibition and transporter affinity assays. Molecular docking was used to model the interaction mechanism against DAT. Swiss CD-1 mice were used for the horizontal locomotor activity, open field test, and conditioned place preference paradigm. All compounds demonstrated potent DA uptake inhibition and higher DAT selectivity than cocaine. Meta-substituted cathinones showed higher DAT/SERT ratios than their para- analogs, which correlates with an increased psychostimulant effect in vivo and with different meta- and para-in silico interactions at DAT. Moreover, all compounds induced rewarding and acute anxiogenic effects in mice. In conclusion, the present study demonstrates the role of meta- and para-halogen substitutions in the mechanism of action and provides the first evidence of the rewarding and anxiety-like properties of halogenated α-PVP derivatives.
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Estimulantes del Sistema Nervioso Central/efectos adversos , Halógenos/efectos adversos , Drogas Ilícitas/efectos adversos , Pentanonas/efectos adversos , Pirrolidinas/efectos adversos , Animales , Ansiedad/inducido químicamente , Ansiedad/metabolismo , Línea Celular , Cocaína/efectos adversos , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Células HEK293 , Humanos , Locomoción/efectos de los fármacos , Masculino , Ratones , Simulación del Acoplamiento Molecular/métodos , Recompensa , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismoRESUMEN
N-ethyl-pentylone (NEP), also known as 'ephylone' and N-ethylnorpentylone, has been identified as one of the most recent novel psychostimulants to emerge into the illicit drug market and it has been associated with some intoxications and even fatalities. However, little is known about the consequences of its repeated consumption as well as the role of the monoaminergic system in such consequences. Thus, the aim of our study was to investigate the neurochemical profile and the behavioural effects after both acute and repeated NEP exposure. Male OF1 mice were acutely (1, 3, 10 mg/kg, i.p.) or repeatedly (1, 3, 10 mg/kg, i.p., 5 days, twice/day) exposed to NEP, and anxiety-like behaviour, aggressiveness, social interaction, depressive-like symptoms, body temperature, changes in monoaminergic enzymes and neurotransmitters levels as well as ΔFosB in striatum and prefrontal cortex (PFC) from post-mortem tissue were analysed short after drug-exposure or during drug-withdrawal. Acute administration of NEP induced anxiolytic effects but also an aggressive behaviour and social exploration deficits in mice, which persist during NEP-withdrawal. Moreover, NEP induced hyperthermia as well as depressive-like symptoms after repeated administrations that may be related to the decrease in serotonin and noradrenaline levels observed in striatum and PFC. Finally, the long-term increase in ΔFosB levels in striatum after NEP chronic exposure points to a high risk of dependence. Altogether indicates that NEP consumption induces different neurological and neuropsychiatric disorders accompanied by changes in the monoaminergic system, posing a threat to public health.
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Conducta Animal/efectos de los fármacos , Benzodioxoles/toxicidad , Butilaminas/toxicidad , Estimulantes del Sistema Nervioso Central/toxicidad , Animales , Masculino , RatonesRESUMEN
Several new synthetic cathinones, which mimic the effect of classical psychostimulants such as cocaine or MDMA, have appeared in the global illicit drug market in the last decades. In fact, the illicit drug market is continually evolving by constantly adding small modifications to the common chemical structure of synthetic cathinones. Thus, the aim of this study was to investigate the in vitro and in vivo structure-activity relationship (SAR) of six novel synthetic cathinones currently popular as recreational drugs, pentedrone, pentylone, N-ethyl-pentedrone (NEPD), N-ethyl-pentylone (NEP), 4-methyl-pentedrone (4-MPD), and 4-methyl-ethylaminopentedrone (4-MeAP), which structurally differ in the absence or presence of different aromatic substituents and in their amino terminal group. Human embryonic kidney (HEK293) cells expressing the human isoforms of SERT and DAT were used for the uptake inhibition and release assays. Moreover, Swiss CD-1 mice were used to investigate the psychostimulant effect, rewarding properties (3, 10, and 30 mg/kg, i.p.), and the induction of immediate-early genes (IEGs), such as Arc and c-fos in the dorsal striatum (DS) and ventral striatum (VS) as well as bdnf in the medial prefrontal cortex (mPFC), of the test compounds. Our results demonstrated that all tested synthetic cathinones are potent dopamine (DA) uptake inhibitors, especially the N-ethyl analogs, while the ring-substituted cathinones tested showed higher potency as SERT inhibitors than their no ring-substituted analogs. Moreover, unlike NEP, the remaining test compounds showed clear "hybrid" properties, acting as DAT blockers but SERT substrates. Regarding the locomotion, NEP and NEPD were more efficacious (10 mg/kg) than their N-methyl analogs, which correlates with their higher potency inhibiting the DAT and an overexpression of Arc levels in the DS and VS. Furthermore, all compounds tested induced an increase in c-fos expression in the DS, except for 4-MPD, the least effective compound in inducing hyperlocomotion. Moreover, NEP induced an up-regulation of bdnf in the mPFC that correlates with its 5-HTergic properties. Finally, the present study demonstrated for the first time that NEP, 4-MPD, and 4-MeAP induce reward in mice. Altogether, this study provides valuable information about the mechanism of action and psychostimulant and rewarding properties as well as changes in the expression of IEGs related to addiction induced by novel second-generation synthetic cathinones.
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3,4-Methylenedioxypyrovalerone (MDPV) is a new psychoactive substance (NPS) and the most widespread and life-threatening synthetic cathinone of the "bath salts". Preclinical research has proven the cocaine-like psychostimulant effects of MDPV and its potential for abuse. Cannabidiol (CBD) is a non-psychotropic phytocannabinoid that has emerged as a new potential treatment for drug addiction. Here, we tested the effects of CBD (20 mg/kg) on MDPV (2 mg/kg)-induced conditioned place preference and MDPV (0.05 and 0.075 mg/kg/infusion) self-administration paradigms. In addition, we assessed the effects of the co-administration of CBD and MDPV (3 and 4 mg/kg) on anxiety-like behaviour using the elevated plus maze (EPM). CBD mitigated the MDPV-induced conditioned place preference. On the contrary, CBD administration throughout the MDPV (0.075 mg/kg/infusion) self-administration increased drug-seeking and taking behaviours, but only in the high-responders group of mice. Furthermore, CBD exerted anxiolytic-like effects, exclusively in MDPV-treated mice. Taken together, our results indicate that CBD modulation of MDPV-induced motivational responses in mice varies depending on the requirements of the learning task, resulting in a complex response. Therefore, further research attempting to decipher the behavioural and molecular interactions between CBD and MDPV is needed.
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Ansiedad/tratamiento farmacológico , Conducta Animal/efectos de los fármacos , Benzodioxoles/toxicidad , Cannabidiol/farmacología , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Pirrolidinas/toxicidad , Inhibidores de Captación Adrenérgica/toxicidad , Animales , Anticonvulsivantes/farmacología , Ansiedad/inducido químicamente , Ansiedad/patología , Condicionamiento Clásico/efectos de los fármacos , Masculino , Ratones , Cathinona SintéticaRESUMEN
BACKGROUND: 3,4-Methylenedioxymethamphetamine (MDMA) is still one of the most consumed drugs by adolescents. Its abuse is related with cognitive impairment, which seems due to maladaptive plasticity and neural stress. In turn, new hypotheses suggest that Alzheimer's disease (AD) may be promoted by neural stressors. AIMS AND METHODS: To test if there is an increase in vulnerability to AD after chronic MDMA consumption, we investigated the effects of this drug on recognition memory and its neurotoxic and neuroplastic effects in a transgenic mouse model of presymptomatic familiar AD (APP/PS1 dE9, Tg). RESULTS: MDMA-treated animals showed recognition memory deficits, regardless of genotype, which were accompanied by changes in plasticity markers. Tg mice showed an impaired expression of Arc compared with wild-type animals, but exposure to MDMA induced an increase in the expression of this factor of the same percentage in both genotypes. However, the expression of c-fos, BDNF and p-CREB was not significantly altered by MDMA treatment in Tg mice. Although Tg mice had higher free choline levels than wild-type mice (about 123%), MDMA did not modify these levels in any case, ruling out any specific effect of this drug on the acetylcholine pathway. MDMA treatment significantly increased the presence of cortical amyloid plaques, as well as Aß40, Aß42 and secreted APPß levels in Tg mice. These plaques were accompanied by increased tau phosphorylation (S199), which does not seem to occur via the canonic pathway involving AKT, CDK5 or GSK3ß. CONCLUSIONS: The present results support previous evidences that MDMA can contribute to the amyloid cascade.
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Péptidos beta-Amiloides/metabolismo , N-Metil-3,4-metilenodioxianfetamina/farmacología , Plasticidad Neuronal/efectos de los fármacos , Fosforilación/efectos de los fármacos , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/metabolismo , Modelos Animales de Enfermedad , Humanos , Masculino , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Nicotine (NIC), the main psychostimulant compound of smoked tobacco, exerts its effects through activation of central nicotinic acetylcholine receptors (nAChR), which become up-regulated after chronic administration. Recent work has demonstrated that the recreational drug 3,4-methylenedioxy-methamphetamine (MDMA) has affinity for nAChR and also induces up-regulation of nAChR in PC 12 cells. Tobacco and MDMA are often consumed together. In the present work we studied the in vivo effect of a classic chronic dosing schedule of MDMA in rats, alone or combined with a chronic schedule of NIC, on the density of nAChR and on serotonin reuptake transporters. MDMA induced significant decreases in [(3)H]paroxetine binding in the cortex and hippocampus measured 24h after the last dose and these decreases were not modified by the association with NIC. In the prefrontal cortex, NIC and MDMA each induced significant increases in [(3)H]epibatidine binding (29.5 and 34.6%, respectively) with respect to saline-treated rats, and these increases were significantly potentiated (up to 72.1%) when the two drugs were associated. Also in this area, [(3)H]methyllycaconitine binding was increased a 42.1% with NIC+MDMA but not when they were given alone. In the hippocampus, MDMA potentiated the α7 regulatory effects of NIC (raising a 25.5% increase to 52.5%) but alone was devoid of effect. MDMA had no effect on heteromeric nAChR in striatum and a coronal section of the midbrain containing superior colliculi, geniculate nuclei, substantia nigra and ventral tegmental area. Specific immunoprecipitation of solubilised receptors suggests that the up-regulated heteromeric nAChRs contain α4 and ß2 subunits. Western blots with specific α4 and α7 antibodies showed no significant differences between the groups, indicating that, as reported for nicotine, up-regulation caused by MDMA is due to post-translational events rather than increased receptor synthesis.
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Encéfalo/efectos de los fármacos , N-Metil-3,4-metilenodioxianfetamina/farmacología , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/efectos de los fármacos , Aconitina/análogos & derivados , Aconitina/metabolismo , Animales , Western Blotting , Encéfalo/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Esquema de Medicación , Sinergismo Farmacológico , Inmunoprecipitación , Inyecciones Subcutáneas , Masculino , N-Metil-3,4-metilenodioxianfetamina/administración & dosificación , Nicotina/administración & dosificación , Agonistas Nicotínicos/administración & dosificación , Paroxetina/metabolismo , Piridinas/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Abstract The 20th century brought with it the so-called club drugs (the most notorious being amphetamine derivatives), which are used by young adults at all-night dance parties. Methamphet-amine and 3,4-methylenedioxymethamphetamine (MDMA or ecstasy) are synthetic drugs with stimulant and psychoactive properties that belong to the amphetamine family. Here, we have reviewed the literature about the cognitive impairment induced by these two amphetamine derivatives and the preclinical and clinical outcomes. Although there is controversial evidence about the effect of methamphetamine and MDMA on learning and memory in laboratory animals, results from published papers demonstrate that amphetamines cause long-term impairment of cognitive functions. A large number of pharmacological receptors have been studied and screened as targets of amphetamine-induced cognitive dysfunction, and extensive research efforts have been invested to provide evidence about the molecular mechanisms behind these cognitive deficits. In humans, there is a considerable body of evidence indicating that methamphetamine and MDMA seriously disrupt memory and learning processes. Although an association between the impairments of memory performance and a history of recreational amphetamine ingestion has also been corroborated, a number of methodological difficulties continue to hamper research in this field, the most important being the concomitant use of other illicit drugs.
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Previous work by our group demonstrated that homomeric alpha7 nicotinic acetylcholine receptors (nAChR) play a role in the neurotoxicity induced by 3,4-methylenedioxymethamphetamine (MDMA), as well as the binding affinity of this drug to these receptors. Here we studied the effect of MDMA on the activation of nAChR subtypes, the consequent calcium mobilization, and calpain/caspase 3 activation because prolonged Ca(2+) increase could contribute to cytotoxicity. As techniques, we used fluorimetry in Fluo-4-loaded PC12 cells and electrophysiology in Xenopus oocytes. MDMA produced a rapid and sustained increase in calcium without reaching the maximum effect induced by ACh. It also concentration-dependently inhibited the response induced by ACh, nicotine, and the specific alpha7 agonist PNU 282987 with IC(50) values in the low micromolar range. Similarly, MDMA induced inward currents in Xenopus oocytes transfected with human alpha7 but not with alpha4beta2 nAChR and inhibited ACh-induced currents in both receptors in a concentration-dependent manner. The calcium response was inhibited by methyllycaconitine (MLA) and alpha-bungarotoxin but not by dihydro-beta-erythroidine. These results therefore indicate that MDMA acts as a partial agonist on alpha7 nAChRs and as an antagonist on the heteromeric subtypes. Subsequently, calcium-induced Ca(2+) release from the endoplasmic reticulum and entry through voltage-operated calcium channels are also implicated as proved using specific antagonists. In addition, treatment with MDMA for 24 h significantly increased basal Ca(2+) levels and induced an increase in alpha-spectrin breakdown products, which indicates that calpain and caspase 3 were activated. These effects were inhibited by pretreatment with MLA. Moreover, pretreatment with MDMA induced functional upregulation of calcium responses to specific agonists of both heteromeric and alpha7 nAChR. Sustained calcium entry and calpain activation could favor the activation of Ca(2+)-dependent enzymes such as protein kinase C and nitric oxide synthase, which are involved in the generation of ROS and the blockade of the dopamine transporter. This, together with caspase 3 activation, must play a role in MDMA-induced cytotoxicity.
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Calcio/metabolismo , Calpaína/metabolismo , Caspasa 3/metabolismo , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Receptores Nicotínicos/efectos de los fármacos , Acetilcolina/metabolismo , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Activación Enzimática , Humanos , Nicotina/metabolismo , Agonistas Nicotínicos/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Células PC12 , Ratas , Receptores Nicotínicos/metabolismo , Espectrina/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Xenopus/metabolismoRESUMEN
Previous work from our group indicated that alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) potentially play a role in methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA) neurotoxicity. The aims of the present study were two-fold: (1) to demonstrate the interaction of METH and MDMA with homomeric alpha7 nAChR ([(3)H]methyllycaconitine binding) and other heteromeric subtypes ([(3)H]epibatidine binding); and (2) to show the effects of amphetamine derivative pretreatment on the density of binding sites. METH and MDMA displaced [(3)H]methyllycaconitine and [(3)H]epibatidine binding in membranes from NGF-differentiated PC 12 cells and mouse brain, with K(i) values in the micromolar range, MDMA revealing a greater affinity than METH. In addition, METH and MDMA induced a time- and concentration-dependent increase in [(3)H]methyllycaconitine and [(3)H]epibatidine binding; which had already been apparent after 6 h of pretreatment, and which peaked in differentiated PC 12 cells after 48 h. The highest increases were found in [(3)H]epibatidine binding, with MDMA inducing higher increases than METH. Treatment with METH and MDMA increased B(max) of high-affinity sites for both radioligands without affecting K(d). The heightened binding was inhibited by pretreatment with cycloheximide, suggesting the participation of newly synthesised proteins while inhibition of protein trafficking to plasma membrane did not block up-regulation. The effects of protein kinase and cyclophilin inhibitors on such up-regulation were explored, revealing a rapid, differential and complex regulation, similar to that described for nicotinic ligands. All of these results demonstrate that METH and MDMA have affinity for, and can interact with, nAChR, inducing their up-regulation, specially when higher doses are used. Such effects may have a role in METH- and MDMA-induced neurotoxicity, cholinergic neurotransmission, and in processes related to addiction and dependence.
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Encéfalo , Membrana Celular/efectos de los fármacos , Metanfetamina/farmacología , N-Metil-3,4-metilenodioxianfetamina/farmacología , Receptores Nicotínicos/biosíntesis , Animales , Unión Competitiva , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Células PC12 , Ensayo de Unión Radioligante , Ratas , Factores de Tiempo , Regulación hacia Arriba , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Ecstasy (MDMA) street tablets often contain several other compounds in addition to MDMA, particularly caffeine. Then, it becomes necessary to study the consequences of caffeine plus MDMA combination. MDMA (1 mg/kg) elicited an analgesic response both at the spinal and supraspinal levels. However, when associated, MDMA and caffeine did not show any synergistic interaction. When caffeine was administered prior to MDMA, a potentiation of locomotor activity was observed, which consisted in an increase in maximal values and in a prolonged time of activity. In the neurotoxicity studies, a hyperthermic effect of MDMA was observed. Although caffeine alone failed to alter body temperature, it potentiated MDMA-induced hyperthermia. This association also significantly increased MDMA lethality (from 22% to 34%). Following administration of MDMA to rats, there was a persistent decrease in the number of serotonin transporter sites in the cortex, striatum and hippocampus, which was potentiated by caffeine co-treatment. This MDMA toxicity in rats was accompanied by a transient dopaminergic impairment in the striatum, measured as decreased [(3)H]WIN35428 binding sites, by 31% 3 days after treatment, which was not modified by caffeine. A transient down-regulation of 5-HT(2) receptors occurred in the cortex of MDMA-treated rats, whose recovery was slowed by co-treatment with caffeine. In conclusion, the association of MDMA with caffeine does not generate any beneficial effects at the antinociceptive level. The acute effects stemming from this association, in tandem with the final potentiation of serotonergic terminals injury, provide evidence of the potentially greater long-term adverse effects of this particular recreational drug combination.
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Analgésicos/efectos adversos , Encéfalo/efectos de los fármacos , Cafeína/efectos adversos , N-Metil-3,4-metilenodioxianfetamina/efectos adversos , Dolor/tratamiento farmacológico , Analgésicos/toxicidad , Animales , Temperatura Corporal/efectos de los fármacos , Temperatura Corporal/fisiología , Encéfalo/metabolismo , Encéfalo/fisiopatología , Cafeína/toxicidad , Estimulantes del Sistema Nervioso Central/efectos adversos , Estimulantes del Sistema Nervioso Central/toxicidad , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Interacciones Farmacológicas/fisiología , Fiebre/inducido químicamente , Fiebre/fisiopatología , Alucinógenos/efectos adversos , Alucinógenos/toxicidad , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Neurotoxinas/efectos adversos , Neurotoxinas/toxicidad , Dolor/inducido químicamente , Dolor/fisiopatología , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Dopamina D2/efectos de los fármacos , Receptores de Dopamina D2/metabolismo , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina/metabolismo , Serotonina/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismoRESUMEN
In a previous study, we demonstrated that in rat striatal synaptosomes, methamphetamine (METH)-induced reactive oxygen species (ROS) production was prevented by methyllycaconitine (MLA), a specific antagonist of alpha7 neuronal nicotinic acetylcholine receptors (alpha7 nAChR). The aim of this study was to test the influence of MLA on acute METH effects and neurotoxicity in mice, using both in vivo and in vitro models. MLA inhibited METH-induced climbing behavior by 50%. Acute effects after 30-min preincubation with 1 microM METH also included a decrease in striatal synaptosome dopamine (DA) uptake, which was prevented by MLA. METH-induced neurotoxicity was assessed in vivo in terms of loss of striatal dopaminergic terminals (73%) and of tyrosine hydroxylase levels (by 90%) at 72 h post-treatment, which was significantly attenuated by MLA. Microglial activation [measured as 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide binding] was also present at 24 h post-treatment and was fully prevented by MLA, tending to confirm its neuroprotective activity. MLA had no effect on METH-induced hyperthermia. Additionally, flow cytometry assays showed that METH-induced ROS generation occurs inside synaptosomes from mouse striatum. This effect implied release of vesicular DA and was calcium-, neuronal nitric-oxide synthase-, and protein kinase C-dependent. MLA and alpha-bungarotoxin, but not dihydro-beta-erythroidine (an antagonist that blocks nAChR-containing beta2 subunits), fully prevented METH-induced ROS production without affecting vesicular DA uptake. The importance of this study lies not only in the neuroprotective effect elicited by the blockade of the alpha7 nicotinic receptors by MLA but also in that it proposes a new mechanism with which to study METH-induced acute and long-term effects.
Asunto(s)
Aconitina/análogos & derivados , Estimulantes del Sistema Nervioso Central/antagonistas & inhibidores , Metanfetamina/antagonistas & inhibidores , Neostriado/metabolismo , Antagonistas Nicotínicos/farmacología , Receptores Nicotínicos/efectos de los fármacos , Aconitina/farmacología , Animales , Antioxidantes/farmacología , Western Blotting , Temperatura Corporal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Calcio/fisiología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Estimulantes del Sistema Nervioso Central/toxicidad , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Inhibidores Enzimáticos/farmacología , Inmunohistoquímica , Isoquinolinas , Masculino , Metanfetamina/toxicidad , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Actividad Motora/efectos de los fármacos , Neostriado/efectos de los fármacos , Síndromes de Neurotoxicidad/metabolismo , Síndromes de Neurotoxicidad/psicología , Ensayo de Unión Radioligante , Especies Reactivas de Oxígeno/metabolismo , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
The pro-oxidative effect of methamphetamine (METH) in dopamine terminals was studied in rat striatal synaptosomes. Flow cytometry analysis showed increased production of reactive oxygen species (ROS) in METH-treated synaptosomes, without reduction in the density of dopamine transporters. In synaptosomes from dopamine (DA)-depleted animals, METH did not induce ROS production. Reserpine, in vitro, completely inhibited METH-induced ROS production. These results point to endogenous DA as the main source of ROS induced by METH. Antioxidants and inhibitors of neuronal nitric oxide synthase and protein kinase C (PKC) prevented the METH-induced oxidative effect. EGTA and the specific antagonist methyllycaconitine (MLA, 50 microM) prevented METH-induced ROS production, thus implicating calcium and alpha7 nicotinic receptors in such effect. Higher concentrations of MLA (>100 microM) showed nonspecific antioxidant effect. Preincubation of synaptosomes with METH (1 microM) for 30 min reduced [(3)H]DA uptake by 0%. The METH effect was attenuated by MLA and EGTA and potentiated by nicotine, indicating that activation of alpha(7) nicotinic receptors and Ca(2+) entry are necessary and take place before DAT inhibition. From these findings, it can be postulated that, in our model, METH induces DA release from synaptic vesicles to the cytosol. Simultaneously, METH activates alpha(7) nicotinic receptors, probably inducing depolarization and an increase in intrasynaptosomal Ca(2+). This would lead to DAT inhibition and NOS and PKC activation, initiating oxidation of cytosolic DA.
Asunto(s)
Estimulantes del Sistema Nervioso Central/toxicidad , Radicales Libres/metabolismo , Metanfetamina/toxicidad , Neostriado/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Sinaptosomas/efectos de los fármacos , Animales , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Técnicas In Vitro , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Neostriado/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Sinaptosomas/metabolismo , Pruebas de Toxicidad , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
The neurotoxic action of the abuse drugs methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA) on cerebellar granule neurones (CGNs) culture was examined. Treatment for 48 h with METH or MDMA (1-5 mM) induced a higher decrease in viability than 24 h treatment. z.VAD.fmk (100 microM) but not MK-801 nor NBQX recovered control viability values. In both cases, cell death was characterised as apoptotic rather than necrotic by morphology cell observation. Apoptosis measured by flow cytometry indicated an increase in the hypodiploid population after 48 h treatment with METH and MDMA. Apoptosis was reverted by the presence of z.VAD.fmk (100 microM) but not by 10 microM MK-801 or NBQX. Similar results were obtained by analysing nuclear chromatine condensation. These results ruled out excitotoxic participation in amphetamine derivative-induced neurotoxicity in CGNs. Participation of radical oxygen species (ROS) was evaluated using alpha-tocopherol (1-15 microM) and cytometric studies. The co-treatment with 4 mM METH or MDMA for 48 h partially reverted neurotoxic action and apoptotic features, indicating ROS implication in CGNs death by amphetamine derivatives. Alteration of mitochondrial function induced cytochrome C (Cyt C) release after 48-h treatment with METH and MDMA (4 mM). There was also indication of caspase-3-like activation, measured by immunoanalysis and biochemically. Finally, neurodegenerative action caused by amphetamine derivatives may be prevented by using caspase inhibitors.
Asunto(s)
Clorometilcetonas de Aminoácidos/farmacología , Caspasas/metabolismo , Cerebelo/efectos de los fármacos , Cerebelo/enzimología , Metanfetamina/antagonistas & inhibidores , Metanfetamina/toxicidad , N-Metil-3,4-metilenodioxianfetamina/antagonistas & inhibidores , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Fármacos Neuroprotectores/farmacología , Animales , Apoptosis/efectos de los fármacos , Caspasa 3 , Cerebelo/citología , Cerebelo/metabolismo , Citocromos c/metabolismo , Maleato de Dizocilpina/farmacología , Interacciones Farmacológicas , Activación Enzimática , Citometría de Flujo , Microscopía Fluorescente , Microscopía de Contraste de Fase , Mitocondrias/enzimología , Mitocondrias/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/metabolismo , Quinoxalinas/farmacología , Ratas , Especies Reactivas de Oxígeno/metabolismo , alfa-Tocoferol/farmacologíaRESUMEN
Neuronal apoptosis may be partly due to inappropriate control of the cell cycle. We used serum deprivation as stimulus and reduced potassium from 25 to 5mM (S/K deprivation), which induces apoptosis in cerebellar granule neurons (CGNs), to evaluate the direct correlation between re-entry in the cell cycle and apoptosis. Roscovitine (10 microM), an antitumoral drug that inhibits cyclin-dependent kinase 1 (cdk1), cdk2 and cdk5, showed a significant neuroprotective effect on CGNs deprived of S/K. S/K deprivation induced the expression of cell cycle proteins such as cyclin E, cyclin A, cdk2, cdk4 and E2F-1. It also caused CGNs to enter the S phase of the cell cycle, measured by a significant incorporation of BrdU (30% increase over control cells), which was reduced in the presence of roscovitine (10 microM). On the other hand, roscovitine modified the expression of cytochrome c (Cyt c), Bcl-2 and Bax, which are involved in the apoptotic intrinsic pathway induced by S/K deprivation. We suggest that the antiapoptotic effects of roscovitine on CGNs are due to its anti-proliferative efficacy and to an action on the mitochondrial apoptotic mechanism.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular , Cerebelo/efectos de los fármacos , Potasio/administración & dosificación , Purinas/farmacología , Animales , Bromodesoxiuridina , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Cerebelo/citología , Medio de Cultivo Libre de Suero , Activación Enzimática , Inmunohistoquímica , Ratas , Ratas Sprague-Dawley , RoscovitinaRESUMEN
Flavopiridol was developed as a drug for cancer therapy due to its ability to inhibit cell cycle progression by targeting cyclin-dependent kinases (CDKs). In this study, we show that flavopiridol may also have a neuroprotective action. We show that at therapeutic dosage (or at micromolar range), flavopiridol almost completely prevents colchicine-induced apoptosis in cerebellar granule neurones. In agreement with this, flavopiridol inhibits both the release of cyt c and the activation of caspase-3 induced in response to colchicine treatment. We demonstrate that in this cellular model for neurotoxicity, neither re-entry in the cell cycle nor activation of stress-activated protein kinases, such as c-Jun N-terminal kinase (JNK) or p38 MAP kinase, is involved. In contrast, we show that colchicine-induced apoptosis correlates with a substantial increase in the expression of cdk5 and Par-4, which is efficiently prevented by flavopiridol. Accordingly, a cdk5 inhibitor such as roscovitine, but not a cdk4 inhibitor such as 3-ATA, was also able to protect neurons from apoptosis as well as prevent accumulation of cdk5 and Par-4 in response to colchicine. Our data suggest a potential therapeutic use of flavopiridol in disorders of the central nervous system in which cytoskeleton alteration mediated by cdk5 activation and Par-4 expression has been demonstrated, such as Alzheimer's disease.
Asunto(s)
Apoptosis , Colchicina/farmacología , Flavonoides/farmacología , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas JNK Activadas por Mitógenos , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Piperidinas/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Animales Recién Nacidos , Antracenos/farmacología , Antibacterianos/farmacología , Proteínas Reguladoras de la Apoptosis , Western Blotting , Bromodesoxiuridina/metabolismo , Quinasas CDC2-CDC28/metabolismo , Proteínas Portadoras/metabolismo , Caspasa 3 , Caspasas/metabolismo , Recuento de Células , Supervivencia Celular , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/fisiología , Cromatina/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Quinasa 5 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Citometría de Flujo , Inmunohistoquímica , Ácido Kaínico/farmacología , MAP Quinasa Quinasa 4 , Microtúbulos/metabolismo , Minociclina/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neuronas/fisiología , Purinas/farmacología , Ratas , Ratas Sprague-Dawley , Roscovitina , Factores de Tiempo , Tubulina (Proteína)/metabolismoRESUMEN
The neuroprotective effect of carnosine, an endogenous antioxidant, was examined against methamphetamine-induced neurotoxicity in rats. Carnosine pretreatment had no effect on dopamine terminal loss induced by methamphetamine (assessed by [3H]1-(2-[diphenylmethoxy]ethyl)-4-[3-phenylpropyl]piperazine([3H]GBR 12935) binding) but prevented microgliosis (increase in [3H]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide ([3H]PK 11195) binding) in striatum. The 27-kDa heat-shock protein (HSP27) expression was used as indicator of astroglial stress. Methamphetamine treatment induced the expression of HSP27 in striatum and hippocampus, which was inhibited by carnosine, indicating a protective effect. Carnosine had no effect on methamphetamine-induced hyperthermia. Thus, carnosine prevents the microgliosis in striatum (where we did not detect loss of serotonergic terminals by [3H]paroxetine binding) and the expression of HSP27 in all the areas, but fails to prevent methamphetamine-induced loss of dopamine reuptake sites. Therefore, carnosine inhibits only some of the consequences of methamphetamine neurotoxicity, where reactive oxygen species play an important role.
Asunto(s)
Carnosina/farmacología , Gliosis/inducido químicamente , Gliosis/prevención & control , Metanfetamina/toxicidad , Terminales Presinápticos/efectos de los fármacos , Receptores Dopaminérgicos/metabolismo , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Piperazinas/metabolismo , Terminales Presinápticos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
1. We examined the role of non-NMDA receptors in kainic acid (KA)-induced apoptosis in cultures of rat cerebellar granule cells (CGCs). KA (1 - 500 microM) induced cell death in a concentration-dependent manner, which was prevented by NBQX and GYKI 52466, non-NMDA receptor antagonists. Moreover, AMPA blocked KA-induced excitotoxicity, through desensitization of AMPA receptors. 2. Similarly, KA raised the intracellular calcium concentration of CGCs, which was inhibited by NBQX and GYKI 52466. Again, AMPA (100 microM) abolished the KA (100 microM)-induced increase in intracellular calcium concentration. 3. KA-induced cell death in CGCs had apoptotic features, which were determined morphologically, by DNA fragmentation, and by expression of the prostate apoptosis response-4 protein (Par-4). 5. KA (500 microM) slightly (18%) increased caspase-3 activity, which was strongly enhanced by colchicine (1 microM), an apoptotic stimulus. However, neither Z-VAD.fmk, a pan-caspase inhibitor, nor the more specific caspase-3 inhibitor, Ac-DEVD-CHO, prevented KA-induced cell death or apoptosis. In contrast, both drugs inhibited colchicine-induced apoptosis. 5. The calpain inhibitor ALLN had no effect on KA or colchicine-induced neurotoxicity. 6. Our findings indicate that colchicine-induced apoptosis in CGCs is mediated by caspase-3 activation, unlike KA-induced apoptosis.