RESUMEN

The emergence of new synthetic cathinones continues to be a matter of public health concern. In fact, they are quickly replaced by new structurally related alternatives. The main goal of the present study was to characterize the pharmacological profile, the psychostimulant and rewarding properties of novel cathinones (pentedrone, N-ethyl-pentedrone, α-PVP, N,N-diethyl-pentedrone and α-PpVP) which only differs in their amino terminal substitution. Rat synaptosomes were used for [3H]dopamine uptake experiments. HEK293 transfected cells (hDAT, hSERT, hOCT; human dopamine, serotonin and organic cation transporter) were also used for [3H]monoamine uptake and transporter binding assays. Molecular docking was used to investigate the effect of the amino substitutions on the biological activity. Hyperlocomotion and conditioned place preference paradigm were used in order to study the psychostimulant and rewarding effects in mice. All compounds tested are potent inhibitors of DAT with very low affinity for SERT, hOCT-2 and -3, and their potency for inhibiting DAT increased when the amino-substituent expanded from a methyl to either an ethyl-, a pyrrolidine- or a piperidine-ring. Regarding the in vivo results, all the compounds induced an increase in locomotor activity and possess rewarding properties. Results also showed a significant correlation between predicted binding affinities by molecular docking and affinity constants (Ki) for hDAT as well as the cLogP of their amino-substituent with their hDAT/hSERT ratios. Our study demonstrates the role of the amino-substituent in the pharmacological profile of novel synthetic cathinones as well as their potency inhibiting DA uptake and ability to induce psychostimulant and rewarding effects in mice.

Asunto(s)

Alcaloides/química , Alcaloides/farmacología , Simulación del Acoplamiento Molecular/métodos , Psicotrópicos/química , Psicotrópicos/farmacología , Recompensa , Animales , Estimulantes del Sistema Nervioso Central/química , Estimulantes del Sistema Nervioso Central/farmacología , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Locomoción/efectos de los fármacos , Locomoción/fisiología , Masculino , Ratones , Ratas

RESUMEN

3,4-Methylenedioxypyrovalerone (MDPV) acts as a dopamine transporter blocker and exerts powerful psychostimulant effects. In this study we aimed to investigate the bidirectional cross-sensitization between MDPV and cocaine, as well as to evaluate the role of the BDNF-TrkB signaling pathway in the development of locomotor sensitization to both drugs. Mice were treated with MDPV (1.5 mg/kg) or cocaine (10 or 15 mg/kg) once daily for 5 days. After withdrawal (10 days), animals were challenged with cocaine (8 mg/kg) or MDPV (1 mg/kg). For biochemical determinations, MDPV (1.5 mg/kg) or cocaine (15 mg/kg) were administered acutely or repeatedly, and BDNF, D3R and G9a transcription levels as well as pro- and mature BDNF protein levels were determined. Our results demonstrate that repeated administration of MDPV or cocaine sensitizes to cocaine and MDPV locomotor effects. After an acute or a repeated exposure to MDPV, cortical mRNA BDNF levels were increased, while a decrease in mBDNF protein levels in the nucleus accumbens 2 h after repeated exposure was evidenced. Interestingly, such decline was involved in the development of locomotor sensitization, thus the pretreatment with 7,8-dihydroxyflavone (10 mg/kg), a TrkB agonist, blocked the development of sensitization to MDPV but not to cocaine, for which no changes in the BDNF-TrkB signaling pathway were observed at early withdrawal. In conclusion, a bidirectional cross-sensitization between MDPV and cocaine was evidenced. Our findings suggest that decreased BDNF-TrkB signaling has an important role in the behavioral sensitization to MDPV, pointing TrkB modulation as a target to prevent MDPV sensitization.

Asunto(s)

Benzodioxoles/farmacología , Factor Neurotrófico Derivado del Encéfalo/fisiología , Cocaína/farmacología , Flavonas/farmacología , Glicoproteínas de Membrana/fisiología , Actividad Motora/efectos de los fármacos , Proteínas Tirosina Quinasas/fisiología , Pirrolidinas/farmacología , Animales , Inhibidores de Captación de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Actividad Motora/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Cathinona Sintética

RESUMEN

3,4-methylenedioxypyrovalerone (MDPV) is a synthetic cathinone with cocaine-like properties. In a previous work, we exposed adolescent mice to MDPV, finding sensitization to cocaine effects, and a higher vulnerability to cocaine abuse in adulthood. Here we sought to determine if such MDPV schedule induces additional behavioral-neuronal changes that could explain such results. After MDPV treatment (1.5 mg kg-1, twice daily, 7 days), mice were behaviorally tested. Also, we investigated protein changes in various brain regions. MDPV induced aggressiveness and anxiety, but also contributed to a faster habituation to the open field. This feature co-occurred with an induction of ΔFosB in the orbitofrontal cortex that was higher than its expression in the ventral striatum. Early after treatment, D2R:D1R ratio pointed to a preponderance of D1R but, upon withdrawal, the ratio recovered. Increased expression of Arc, CDK5 and TH, and decrease in DAT protein levels persisted longer after withdrawal, pointing to a neuroplastic lasting effect similar to that involved in cocaine addiction. The implication of the hyperdopaminergic condition in the MDPV-induced aggressiveness cannot be ruled out. We also found an initial oxidative effect of MDPV, without glial activation. Moreover, although initially the dopaminergic signal induced by MDPV resulted in increased ΔFosB, we did not observe any change in NFκB or GluA2 expression. Finally, the changes observed after MDPV treatment could not be explained according to the autoregulatory loop between ΔFosB and the epigenetic repressor G9a described for cocaine. This provides new knowledge about the neuroadaptive changes involved in the vulnerability to psychostimulant addiction.

Asunto(s)

Benzodioxoles/efectos adversos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Estimulantes del Sistema Nervioso Central/efectos adversos , Pirrolidinas/efectos adversos , Asunción de Riesgos , Trastornos Relacionados con Sustancias/metabolismo , Agresión/efectos de los fármacos , Agresión/fisiología , Animales , Ansiedad/inducido químicamente , Ansiedad/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Proteínas del Citoesqueleto/metabolismo , Dopamina/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Masculino , Ratones , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Distribución Aleatoria , Receptores Dopaminérgicos/metabolismo , Trastornos Relacionados con Sustancias/psicología , Factores de Tiempo , Cathinona Sintética

RESUMEN

BACKGROUND AND PURPOSE: 3,4-Methylenedioxypyrovalerone (MDPV) is a synthetic cathinone with powerful psychostimulant effects. It selectively inhibits the dopamine transporter (DAT) and is 10-50-fold more potent as a DAT blocker than cocaine, suggesting a high abuse liability. The main objective of the present study was to assess the consequences of an early (adolescence) MDPV exposure on the psychostimulant, rewarding and reinforcing effects induced by cocaine in adult mice. EXPERIMENTAL APPROACH: Twenty-one days after MDPV pretreatment (1.5 mg·kg-1 , s.c., twice daily for 7 days), adult mice were tested with cocaine, using locomotor activity, conditioned place preference and self-administration (SA) paradigms. In parallel, dopamine D2 receptor density and the expression of c-Fos and ΔFosB in the striatum were determined. KEY RESULTS: MDPV treatment enhanced the psychostimulant and conditioning effects of cocaine. Acquisition of cocaine SA was unchanged in mice pretreated with MDPV, whereas the breaking point achieved under a progressive ratio programme and reinstatement after extinction were higher in this group of mice. MDPV decreased D2 receptor density but increased ΔFosB expression three-fold. As expected, acute cocaine increased c-Fos expression, but MDPV pretreatment negatively influenced its expression. ΔFosB accumulation declined during MDPV withdrawal, although it remained elevated in adult mice when tested for cocaine effects. CONCLUSION AND IMPLICATIONS: MDPV exposure during adolescence induced long-lasting adaptive changes related to enhanced responsiveness to cocaine in the adult mice that seems to lead to a higher vulnerability to cocaine abuse. This particular behaviour correlated with increased expression of ΔFosB.

Asunto(s)

Benzodioxoles/farmacología , Cocaína/farmacología , Condicionamiento Psicológico/efectos de los fármacos , Locomoción/efectos de los fármacos , Pirrolidinas/farmacología , Refuerzo en Psicología , Animales , Benzodioxoles/administración & dosificación , Cocaína/administración & dosificación , Humanos , Inyecciones Subcutáneas , Masculino , Ratones , Pirrolidinas/administración & dosificación , Receptores de Dopamina D2/metabolismo , Recompensa , Autoadministración , Cathinona Sintética

RESUMEN

Methylenedioxypyrovalerone (MDPV) is a new psychostimulant cathinone acting as a selective dopamine transporter blocker. Due to the concomitant consumption of ethanol (EtOH) and new psychoactive substances, it is of interest to explore a possible pharmacological interaction between MDPV and EtOH. In locomotor activity assays, EtOH (1g/kg i.p.) elicited a reduction in the stimulant effect induced by low doses of MDPV (0.1-0.3mg/kg, s.c.) in rats, jointly with a decrease in blood and brain MDPV concentrations. Experiments in rat liver microsomes showed different effects depending on the [MDPV]/[EtOH] relationship, evidencing, at certain concentrations, the enhancing effect of EtOH on MDPV metabolism. These suggest that EtOH interacts with MDPV at microsomal level, increasing its metabolic rate. The interaction between both substances was also supported by results in plasma EtOH concentration, which were significantly increased by MDPV, in such a manner that EtOH elimination rate was significantly reduced. The possible toxicological impact of this phenomenon deserves further investigation. In contrast, the rewarding properties of MDPV were unaltered by EtOH. Microdialysis experiments verified that, in the NAcc, both substances could also act synergistically, in such a manner that extracellular dopamine concentrations are maintained. Finally, if the psychostimulant effect induced by MDPV decreased with EtOH, it could favor the boosting and re-dosing in search of the desired effects. However, as the rewarding effect of each dose of the substance would not decrease, the addictive liability could increase considerably. Moreover, we must warn about the increase in EtOH concentrations when consumed concomitantly with MDPV.

Asunto(s)

Alcaloides/metabolismo , Conducta Animal/efectos de los fármacos , Benzodioxoles/metabolismo , Benzodioxoles/farmacología , Estimulantes del Sistema Nervioso Central/metabolismo , Estimulantes del Sistema Nervioso Central/farmacología , Condicionamiento Clásico/efectos de los fármacos , Interacciones Farmacológicas , Etanol/metabolismo , Etanol/farmacología , Pirrolidinas/metabolismo , Pirrolidinas/farmacología , Animales , Benzodioxoles/administración & dosificación , Estimulantes del Sistema Nervioso Central/administración & dosificación , Etanol/administración & dosificación , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Microdiálisis , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Pirrolidinas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Cathinona Sintética

RESUMEN

(±)3,4-Methylenedioxymethamphetamine (MDMA) is a relatively selective dopaminergic neurotoxin in mice. This study was designed to evaluate whether MDMA exposure affects their recognition memory and hippocampal expression of plasticity markers. Mice were administered with increasing doses of MDMA once per week for 8 weeks (three times in 1 day, every 3 h) and killed 2 weeks (2w) or 3 months (3m) later. The treatment did not modify hippocampal tryptophan hydroxylase 2, a serotonergic indicator, but induced an initial reduction in dopaminergic markers in substantia nigra, which remained stable for at least 3 months. In parallel, MDMA produced a decrease in dopamine (DA) levels in the striatum at 2w, which were restored 3 months later, suggesting dopaminergic terminal regeneration (sprouting phenomenon). Moreover, recognition memory was assessed using the object recognition test. Young (2w) and mature (3m) adult mice exhibited impaired memory after 24-h but not after just 1-h retention interval. Two weeks after the treatment, animals showed constant levels of CREB but an increase in its phosphorylated form and in c-Fos expression. Brain-derived neurotrophic factor (BDNF) and especially Arc overexpression was sustained and long-lasting. We cannot rule out the absence of MDMA injury in the hippocampus being due to the generation of BDNF. The levels of NMDAR2B, PSD-95, and synaptophysin were unaffected. In conclusion, the young mice exposed to MDMA showed increased expression of early key markers of plasticity, which sometimes remained for 3 months, and suggests hippocampal maladaptive plasticity that could explain memory deficits evidenced here.

Asunto(s)

Envejecimiento/patología , Hipocampo/fisiopatología , Trastornos de la Memoria/fisiopatología , Plasticidad Neuronal , Animales , Biomarcadores/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Hipocampo/metabolismo , Masculino , Memoria , Ratones Endogámicos C57BL , N-Metil-3,4-metilenodioxianfetamina , Neuronas/metabolismo , Neuronas/patología , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tirosina 3-Monooxigenasa/metabolismo

RESUMEN

RATIONALE: Mephedrone (4-methylmethcathinone) is a still poorly known drug of abuse, alternative to ecstasy or cocaine. OBJECTIVE: The major aims were to investigate the pharmacokinetics and locomotor activity of mephedrone in rats and provide a pharmacokinetic/pharmacodynamic model. METHODS: Mephedrone was administered to male Sprague-Dawley rats intravenously (10 mg/kg) and orally (30 and 60 mg/kg). Plasma concentrations and metabolites were characterized using LC/MS and LC-MS/MS fragmentation patterns. Locomotor activity was monitored for 180-240 min. RESULTS: Mephedrone plasma concentrations after i.v. administration fit a two-compartment model (α = 10.23 h(-1), ß = 1.86 h(-1)). After oral administration, peak mephedrone concentrations were achieved between 0.5 and 1 h and declined to undetectable levels at 9 h. The absolute bioavailability of mephedrone was about 10% and the percentage of mephedrone protein binding was 21.59 ± 3.67%. We have identified five phase I metabolites in rat blood after oral administration. The relationship between brain levels and free plasma concentration was 1.85 ± 0.08. Mephedrone induced a dose-dependent increase in locomotor activity, which lasted up to 2 h. The pharmacokinetic-pharmacodynamic model successfully describes the relationship between mephedrone plasma concentrations and its psychostimulant effect. CONCLUSIONS: We suggest a very important first-pass effect for mephedrone after oral administration and an easy access to the central nervous system. The model described might be useful in the estimation and prediction of the onset, magnitude, and time course of mephedrone pharmacodynamics as well as to design new animal models of mephedrone addiction and toxicity.

Asunto(s)

Administración Intravenosa , Administración Oral , Estimulantes del Sistema Nervioso Central/administración & dosificación , Metanfetamina/análogos & derivados , Actividad Motora/efectos de los fármacos , Animales , Área Bajo la Curva , Disponibilidad Biológica , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Estimulantes del Sistema Nervioso Central/sangre , Estimulantes del Sistema Nervioso Central/metabolismo , Relación Dosis-Respuesta a Droga , Masculino , Redes y Vías Metabólicas , Metanfetamina/administración & dosificación , Metanfetamina/sangre , Metanfetamina/metabolismo , Unión Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem , Factores de Tiempo

RESUMEN

Aging is associated with an increased risk of depression in humans. To elucidate the underlying mechanisms of depression and its dependence on aging, here we study signs of depression in male SAMP8 mice. For this purpose, we used the forced swimming test (FST). The total floating time in the FST was greater in SAMP8 than in SAMR1 mice at 9 months of age; however, this difference was not observed in 12-month-old mice, when both strains are considered elderly. Of the two strains, only the SAMP8 animals responded to imipramine treatment. We also applied the dexamethasone suppression test (DST) and studied changes in the dopamine and serotonin (5-HT) uptake systems, the 5-HT2a/2c receptor density in the cortex, and levels of TPH2. The DST showed a significant difference between SAMR1 and SAMP8 mice at old age. SAMP8 exhibits an increase in 5-HT transporter density, with slight changes in 5-HT2a/2c receptor density. In conclusion, SAMP8 mice presented depression-like behavior that is dependent on senescence process, because it differs from SAMR1, senescence resistant strain.

Asunto(s)

Envejecimiento/genética , Envejecimiento/psicología , Conducta Animal , Depresión/epidemiología , Depresión/psicología , Ratones Endogámicos/genética , Ratones Endogámicos/psicología , Animales , Antidepresivos Tricíclicos/uso terapéutico , Corteza Cerebral/metabolismo , Depresión/tratamiento farmacológico , Modelos Animales de Enfermedad , Dopamina/metabolismo , Imipramina/uso terapéutico , Incidencia , Masculino , Ratones , Receptores de Serotonina/metabolismo , Natación/psicología , Resultado del Tratamiento , Triptófano Hidroxilasa/metabolismo

RESUMEN

The neurotoxicity of MDMA or "Ecstasy" in rats is selectively serotonergic, while in mice it is both dopaminergic and serotonergic. MDMA metabolism may play a key role in this neurotoxicity. The function of serotonin and dopamine transporter and the effect of MDMA and its metabolites on them are essential to understand MDMA neurotoxicity. The aim of the present study was to investigate and compare the effects of MDMA and its metabolite alpha-methyldopamine (MeDA) on several molecular targets, mainly the dopamine and serotonin transporter functionality, to provide evidence for the role of this metabolite in the neurotoxicity of MDMA in rodents. MeDA had no affinity for the serotonin transporter but competed with serotonin for its uptake. It had no persistent effects on the functionalism of the serotonin transporter, in contrast to the effect of MDMA. Moreover, MeDA inhibited the uptake of dopamine into the serotonergic terminal and also MAO(B) activity. MeDA inhibited dopamine uptake with a lower IC(50) value than MDMA. After drug washout, the inhibition by MeDA persisted while that of MDMA was significantly reduced. The effect of MDMA on the dopamine transporter is related with dopamine release from vesicular stores, as this inhibition disappeared in reserpine-treated animals. However, the effect of MeDA seems to be a persistent conformational change of this transporter. Moreover, in contrast with MDMA, MeDA did not show affinity for nicotinic receptors, so no effects of MeDA derived from these interactions can be expected. The metabolite reduced cell viability at lower concentrations than MDMA. Apoptosis plays a key role in MDMA induced cellular toxicity but necrosis is the major process involved in MeDA cytotoxicity. We conclude that MeDA could protect against the serotonergic lesion induced by MDMA but potentiate the dopaminergic lesion as a result of the persistent blockade of the dopamine transporter induced this metabolite.

Asunto(s)

Desoxiepinefrina/análogos & derivados , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Síndromes de Neurotoxicidad/metabolismo , Animales , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Cocaína/análogos & derivados , Cocaína/metabolismo , Desoxiepinefrina/toxicidad , Dopamina/metabolismo , Dopamina/fisiología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/química , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Inhibidores de Captación de Dopamina/metabolismo , Técnicas In Vitro , Masculino , Monoaminooxidasa/metabolismo , Inhibidores de la Monoaminooxidasa/farmacología , Células PC12 , Paroxetina/metabolismo , Conformación Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/efectos de los fármacos , Serotonina/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/química , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo

RESUMEN

During the last years, we have focused on the study of the neurotoxic effects of 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (METH) on the central nervous system (CNS) and their pharmacological prevention methods. In the process of this research, we have used a semipurified synaptosomal preparation from striatum of mice or rats as a reliable in vitro model to study reactive oxygen species (ROS) production by these amphetamine derivatives, which is well-correlated with their dopaminergic injury in in vivo models. Using this preparation, we have demonstrated that blockade of alpha7 nicotinic receptors with methyllycaconitine (MLA) prevents ROS production induced by MDMA and METH. Consequently, in vivo, MLA significantly prevents MDMA- and METH-induced neurotoxicity at dopaminergic level (mouse striatum), without affecting hyperthermia induced by these amphetamines. Additionally, when neuroprotection was assayed with memantine (MEM), a dual antagonist of NMDA and alpha7 receptors, an effective neuroprotection was obtained also ahead of serotonergic injury induced by MDMA in rats. MEM also prevents MDMA effect on serotonin transporter functionality and METH effect on dopamine transporter (DAT), suggesting that behavioral effects of these psychostimulants can also be modulated by MEM. Finally, we have demonstrated that MEM prevents the impaired memory function induced by MDMA, and also, using binding studies with radioligands, we have characterized the interaction of these substances with nicotinic receptors. Studies at molecular level showed that both MDMA and METH displaced competitively the binding of radioligands with homomeric alpha7 and heteromeric nicotinic acetylcholine receptors (nAChRs), indicating that they can directly interact with them. In all the cases, MDMA displayed higher affinity than METH and it was higher for heteromeric than for alpha7 subtype. Pre-incubation of differentiated PC12 cells with MDMA or METH induces nAChR upregulation in a concentration- and time-dependent manner, as many nicotinic ligands do, supporting their functional interaction with nAChRs. Such interaction expands the pharmacological profile of amphetamines and can account for some of their effects.

Asunto(s)

Estimulantes del Sistema Nervioso Central/toxicidad , Metanfetamina/toxicidad , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Síndromes de Neurotoxicidad/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Humanos , Síndromes de Neurotoxicidad/patología , Especies Reactivas de Oxígeno/metabolismo , Receptores Nicotínicos/efectos de los fármacos

RESUMEN

We hypothesize that 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (METH) interact with alpha-7 nicotinic receptors (nAChR). Here we examine whether memantine (MEM), an antagonist of NMDAR and alpha-7 nAChR, prevents MDMA and METH neurotoxicity. MEM prevented both serotonergic injury induced by MDMA in rat and dopaminergic lesion by METH in mice. MEM has a better protective effect in front of MDMA- and METH-induced neurotoxicity than methyllycaconitine (MLA), a specific alpha-7 nAChR antagonist. The double antagonism that MEM exerts on NMDA receptor and on alpha-7 nAChR, probably contributes to its effectiveness. MEM inhibited reactive oxygen species production induced by MDMA or METH in synaptosomes. This effect was not modified by NMDA receptor antagonists, but reversed by alpha-7 nAChR agonist (PNU 282987), demonstrating a preventive effect of MEM as a result of it blocking alpha-7 nAChR. In synaptosomes, MDMA decreased 5-HT uptake by about 40%. This decrease was prevented by MEM and by MLA but enhanced by PNU 282987. A similar pattern was observed when we measured the dopamine transport inhibited by METH. The inhibition of both transporters by amphetamine derivatives seems to be regulated by the calcium incorporation after activation of alpha-7 nAChR. MDMA competitively displaces [(3)H]MLA from rat brain membranes. MEM and METH also displace [(3)H]MLA with non-competitive displacement profiles that fit a two-site model. We conclude that MEM prevents MDMA and METH effects in rodents. MEM may offer neuroprotection against neurotoxicity induced by MDMA and METH by preventing the deleterious effects of these amphetamine derivatives on their respective transporters.

Asunto(s)

Antagonistas de Dopamina/uso terapéutico , Alucinógenos , Memantina/uso terapéutico , Metanfetamina , N-Metil-3,4-metilenodioxianfetamina , Fármacos Neuroprotectores , Síndromes de Neurotoxicidad/prevención & control , Animales , Benzamidas/farmacología , Temperatura Corporal/efectos de los fármacos , Compuestos Bicíclicos con Puentes/farmacología , Maleato de Dizocilpina/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Ratones , Neuroglía/efectos de los fármacos , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo

RESUMEN

d-Amphetamine (AMPH) and MDMA increased intracellular production of reactive oxygen species (ROS) in isolated mouse striatal synaptosomes. MDMA showed a maximal oxidative effect at 50-100 microM. However, for AMPH a double maximum was obtained, the first between 0.1 and 1 microM and the second at 1mM. No oxidative effect was present in synaptosomes from reserpinized mice. Cocaine and l-deprenyl inhibited MDMA and AMPH (0.1 microM) ROS production but not that of AMPH at a higher concentration (1mM). When this high concentration was used, its oxidative effect was abolished by a phospholipase A(2) inhibitor. Delta(9)-Tetrahydrocannabinol fully prevented the oxidative effect of AMPH and MDMA, by a CB(1) receptor-independent mechanism, as did it NPC 15437 and genistein. The pro-oxidative effect induced by AMPH and MDMA showed a strong dependence on calcium (extracellular and from internal stores) and also was inhibited by nicotinic receptor (nAChR) antagonists dihydro-beta-erythroidine, methyllycaconitine (MLA) and alpha-bungarotoxin. MDMA displaced [(3)H]epibatidine and [(3)H]MLA binding with higher affinity than AMPH. Both amphetamines competitively displaced [(3)H]epibatidine from heteromeric receptors but results obtained from [(3)H]MLA binding demonstrated a non-competitive profile. Preincubation of PC12 cells with AMPH or MDMA reduced [(3)H]dopamine uptake. For MDMA, this effect was prevented by MLA. To summarize, comparing AMPH and MDMA we have demonstrated that these drugs induce an oxidative effect dependent on drug concentration and also reduce dopamine uptake. Processes that are known to affect dopamine transporter functionality also seem to modulate amphetamine derivatives-induced ROS production. For MDMA, acute effects tested are blocked by nAChR antagonists, which points to the possibility that these antagonists could be used to treat some of the adverse effects described in MDMA abusers. Conversely, no implication of nicotinic receptors has been proved for AMPH-induced effects at concentrations achievable in CNS after its administration.

Asunto(s)

Anfetamina/farmacología , Cuerpo Estriado/efectos de los fármacos , N-Metil-3,4-metilenodioxianfetamina/farmacología , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Inhibidores de Captación Adrenérgica/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Inhibidores de Captación de Dopamina/farmacología , Sinergismo Farmacológico , Masculino , Ratones , Neuronas/metabolismo , Antagonistas Nicotínicos/farmacología , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/fisiología , Células PC12 , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Receptores Nicotínicos/metabolismo , Fracciones Subcelulares , Sinaptosomas

RESUMEN

MDMA (ecstasy) is an illicit drug causing long-term neurotoxicity. Previous studies demonstrated the interaction of MDMA with alpha-7 nicotinic acetylcholine receptor (nAChR) in mouse brain membranes and the involvement of alpha-7 nicotinic acetylcholine receptors (nAChR) in dopaminergic neurotoxicity induced by MDMA in mice. The aim of the present study was to investigate the utility of memantine (MEM), an alpha-7 nAChR antagonist used for treatment of Alzheimer's disease patients, to prevent neurotoxicity induced by MDMA in rats and the oxidative effect of this amphetamine derivative in mice striatal synaptosomes. In isolated mouse striatal synaptosomes (an in vitro model of MDMA neurotoxicity of dopaminergic origin), MDMA (50 microM)-induced reactive oxygen species (ROS) production that was fully inhibited by MEM (0.3 microM). This effect of MEM was fully prevented by PNU 282987 (0.5 microM), a specific agonist of alpha-7 nAChR. The preventive effect of MEM on this oxidative effect can be attributed to a direct antagonism between MDMA (acting probably as agonist) and MEM (acting as antagonist) at the alpha-7 nAChR. In Dark Agouti rats (an in vivo model of MDMA neurotoxicity of serotonergic origin), a single dose of MDMA (18 mg/kg) induced persistent hyperthermia, which was not affected by MEM pre-treatment. [(3)H]Paroxetine binding (a marker of serotonergic injury) was measured in the hippocampus of animals killed at 24h and 7 days after treatment. MDMA induced a significant reduction in [(3)H]paroxetine binding sites at both times of sacrifice that was fully prevented by pre-treatment with MEM. Since previous studies demonstrate that increased glutamate activity is not involved in the neurotoxic action of MDMA, it can be concluded that the effectiveness of MEM against MDMA-induced neurotoxicity would be the result of blockade of alpha-7 nAChR, although an indirect mechanism based on the interplay among the various neurotransmission systems leading to an increase in basal acetylcholine release should also be taken into account.

Asunto(s)

Inhibidores de Captación Adrenérgica/toxicidad , Antagonistas de Aminoácidos Excitadores/farmacología , Memantina/farmacología , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Sinaptosomas/efectos de los fármacos , Animales , Benzamidas/farmacología , Temperatura Corporal/efectos de los fármacos , Compuestos Bicíclicos con Puentes/farmacología , Cuerpo Estriado/ultraestructura , Interacciones Farmacológicas , Masculino , Ratones , Neuronas/ultraestructura , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo

RESUMEN

Methylenedioxymethamphetamine (MDMA) is a relatively selective dopaminergic neurotoxin in mice. Previous studies demonstrated the participation of alpha-7 nicotinic receptors (nAChR) in the neurotoxic effect of methamphetamine. The aim of this paper was to study the role of this receptor type in the acute effects and neurotoxicity of MDMA in mice. In vivo, methyllycaconitine (MLA), a specific alpha-7 nAChR antagonist, significantly prevented MDMA-induced neurotoxicity at dopaminergic but not at serotonergic level, without affecting MDMA-induced hyperthermia. Glial activation was also fully prevented by MLA. In vitro, MDMA induced intrasynaptosomal reactive oxygen species (ROS) generation, which was calcium-, nitric-oxide synthase-, and protein kinase C-dependent. Also, the increase in ROS was prevented by MLA and alpha-bungarotoxin. Experiments with reserpine point to endogenous dopamine (DA) as the main source of MDMA-induced ROS. MLA also brought the MDMA-induced inhibition of [3H]DA uptake down, from 73% to 11%. We demonstrate that a coordinated activation of alpha-7 nAChR, blockade of DA transporter function and displacement of DA from intracellular stores induced by MDMA produces a neurotoxic effect that can be prevented by MLA, suggesting that alpha-7 nAChR have a key role in the MDMA neurotoxicity in mice; however, the involvement of nicotinic receptors containing the beta2 subunit cannot be conclusively ruled out.

Asunto(s)

Aconitina/análogos & derivados , Dopamina/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Síndromes de Neurotoxicidad/tratamiento farmacológico , Receptores Nicotínicos/fisiología , Aconitina/uso terapéutico , Análisis de Varianza , Animales , Cocaína/análogos & derivados , Cocaína/farmacocinética , Modelos Animales de Enfermedad , Inhibidores de Captación de Dopamina/farmacocinética , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Citometría de Flujo/métodos , Masculino , Ratones , Actividad Motora/efectos de los fármacos , N-Metil-3,4-metilenodioxianfetamina , Síndromes de Neurotoxicidad/etiología , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Ensayo de Unión Radioligante/métodos , Especies Reactivas de Oxígeno/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Tritio/metabolismo

RESUMEN

The mechanisms underlying selective neuronal cell death in kainic acid-mediated neurodegeneration are not fully understood. We have recently demonstrated that in cerebellar granule neurons, kainic acid induces the expression of proteins associated with cell-cycle progression. In the present study we show that 3-amino thioacridone (3-ATA), a selective cyclin-dependent kinase 4 inhibitor, attenuates kainic acid-induced apoptosis in cerebellar granule neurons. When neurons were pre-treated with 3-ATA 10 microM for 24 h, they were less susceptible to damage induced by kainic acid 500 microM, since the number of dead cells decreased significantly. In flow cytometry studies using propidium iodide staining, 3-ATA also reduced the ratio of apoptotic cells induced by kainic acid. Moreover, 3-ATA decreased the proportion of cells with a condensed nucleus from 55% to 22%. Our data suggest that the cell cycle pathway is involved in the mechanism of apoptosis mediated by kainic acid and that cyclin-dependent kinase 4 plays a prominent role in this process. 3-ATA may to prevent the apoptosis associated with neurodegenerative disorders without the over-activation of excitatory amino acid receptors.

Asunto(s)

Aminoacridinas/farmacología , Apoptosis/efectos de los fármacos , Cerebelo/efectos de los fármacos , Quinasas Ciclina-Dependientes/metabolismo , Inhibidores Enzimáticos/farmacología , Ácido Kaínico , Neuronas/efectos de los fármacos , Proteínas Proto-Oncogénicas , Animales , Animales Recién Nacidos , Muerte Celular , Cerebelo/metabolismo , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Citometría de Flujo , Ácido Kaínico/toxicidad , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

We tested the potential cytoprotective role of C-phycocyanin in rat cerebellar granule cell cultures. Cell death was induced by potassium and serum (K/S) withdrawal. Cell viability was studied using the neutral red assay and laser scanning cytometry with propidium iodide as fluorochrome. C-phycocyanin (1-3 mg/ml) showed a neuroprotective effect against 24 h of K/S deprivation in cerebellar granule cells. After 4 h K/S deprivation this compound (3 mg/ml) inhibited formation of reactive oxygen species, measured as 2',7'-dichlorofluorescein fluorescence, showing its scavenger capability. Pre-treatment with C-phycocyanin reduced thymidine incorporation into DNA below control values and reduced dramatically apoptotic bodies as visualized by propidium iodide, indicating inhibition of apoptosis induced by K/S deprivation. Flow cytometry studies, using propidium iodide in TritonX100 permeabilized cells, indicated that 24 h K/S deprivation acts as a proliferative signal for cerebellar granule cells, which show an increase in S-phase percentage and cells progressed into the apoptotic pathway. C-phycocyanin protected cerebellar granule cells from the apoptosis induced by deprivation. These results suggest that C-phycocyanin prevents apoptosis in cerebellar granule cells probably through the antioxidant activity. It is proposed that K/S deprivation-induced apoptosis could be due, in part, to an alteration in the cell cycle mediated by an oxidative stress mechanism.

Asunto(s)

Apoptosis/efectos de los fármacos , Cerebelo/citología , Cerebelo/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Ficocianina/farmacología , Cloruro de Potasio/administración & dosificación , Animales , Animales Recién Nacidos , Apoptosis/fisiología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Cerebelo/metabolismo , Medio de Cultivo Libre de Suero/efectos adversos , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

The consequences of purinoceptor activation on calcium signalling, inositol phosphate metabolism, protein secretion and the actin cytoskeleton were demonstrated in the WRK-1 cell line. Extracellular ATP was used as a secretagogue to induce a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)), acting via P2x purinergic receptors, which causes actin skeleton disaggregation and protein secretion. ATP bound specifically to purinergic receptors, with Ki of 0.8 microM. The magnitude order for binding of different nucleotides was alpha beta-Met-ATP >or= dATPalphaS > ATP >or= ADP > UTP > AMP > suramin. No increase in inositol phosphates (IPs) was observed after ATP application suggesting that the purinergic sites in WRK-1 cells are not of a P2y type. ATP (1-100 microM) caused a concentration-dependent increase in [Ca(2+)](i)(EC(50)= 30 microM). The responses were reproducible without any desensitization over several applications. The response to ATP was abolished when extracellular calcium ([Ca(2+)](e)) was reduced to 100 nM. A non-specific purinergic antagonist, suramin, reversibly inhibited the ATP-response suggesting that ATP is able to bind to P2x purinergic sites to trigger Ca(2+) entry and increase of [Ca(2+)](i). ATP induced a concentration-dependent disaggregation of actin and exocytotic release of proteins both, which were dependent upon [Ca(2+)](e). Similarly, alpha,beta-Met-ATP, a potent P2x agonist also stimulated Ca(2+) mobilization, actin network destructuration, and protein release. In the isolated rat neurohypophysial nerve terminals, ATP was shown to act as a physiological stimulus for vasopressin release via Ca(2+) entry through a P2x receptor [6]. Here, we show that in these nerve terminals, ATP is also able to induce actin disaggregation by a Ca(2+) dependent mechanism. Thus, actin cytoskeleton alterations induced by ATP through activation of P2x receptors could be a prelude to exocytosis.

Asunto(s)

Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Señalización del Calcio/fisiología , Receptores Purinérgicos P2/metabolismo , Animales , Sitios de Unión , Calcio/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Nucleótidos de Desoxiadenina/metabolismo , Fosfatos de Inositol/metabolismo , Líquido Intracelular , Ratas , Tionucleótidos/metabolismo , Células Tumorales Cultivadas

RESUMEN

1. Previous studies indicate that 3-nitropropionic acid (3-NPA) neurotoxicity involves the excitotoxic activation of N-methyl-D-aspartate (NMDA) receptors. Thus, we examined the effect of orphenadrine (an anticholinergic drug with NMDA receptor antagonist properties) on 3-NPA neurotoxicity in both cultured rat cerebellar granule cells (CGCs) and in rats. 2. Orphenadrine protected CGCs from 3-NPA-induced mortality, as assessed by both the neutral red viability assay and laser scanning cytometry, using propidium iodide staining. 3. For rats, two indirect markers of neuronal damage were used: the binding of [(3)H]-PK 11195 to the peripheral-type benzodiazepine receptor (PBR), a microglial marker, and expression of the 27 kD heat-shock protein (HSP27), a marker of activated astroglia. Systemic administration of 3-NPA (30 mg kg(-1) per day for 3 days) induced a 170% increase in [(3)H]-PK 11195 binding, and expression of HSP27. 4. Both the increase in [(3)H]-PK 11195 and HSP 27 expression were prevented by previous administration of 30 mg kg(-1) per day of orphenadrine for 3 days. Lower doses (10 and 20 mg kg(-1)) had no protective effect. Orphenadrine also reduced 3-NPA-induced mortality in a dose-dependent manner. 5. We propose that orphenadrine or orphenadrine-like drugs could be used to treat neurodegenerative disorders mediated by overactivation of NMDA receptors.

Asunto(s)

Cerebelo/efectos de los fármacos , Orfenadrina/farmacología , Propionatos/toxicidad , Animales , Antihipertensivos/toxicidad , Western Blotting , Peso Corporal/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cerebelo/citología , Cerebelo/metabolismo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Isoquinolinas/farmacología , Masculino , Mortalidad , Antagonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/uso terapéutico , Síndromes de Neurotoxicidad/prevención & control , Nitrocompuestos , Orfenadrina/uso terapéutico , Propionatos/antagonistas & inhibidores , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Tritio

RESUMEN

Previous work from our laboratory has demonstrated the presence of high-affinity binding sites for [3H]nitrobenzylthioinosine ([3H]NBTI), a marker of adenosine uptake systems, in the mitochondrial fraction of rat testis. Here, we characterize this system functionally through [3H]adenosine uptake assays. This system (K(m)=2+/-1.3 microM; V(max)=86.2+/-15.5 pmol/mg protein/min) was found to be saturable, non sodium-dependent and sensitive to temperature, pH and osmolarity. [3H]Adenosine incorporation was potently inhibited by hydroxynitrobenzylthioguanosine (HNBTG, IC(50)=3 nM) although NBTI inhibited this uptake weakly (IC(50)=72. 7+/-37.1 microM). Dilazep>dipyridamole>/=hexobendine inhibited [3H]adenosine incorporation at low micromolar concentrations. The nucleosides inosine and uridine were weak inhibitors of this system. The adenosine receptor ligands N(6)-phenylisopropyladenosine (PIA) and 2-chloroadenosine inhibited the uptake only at micromolar concentrations. Neither 5'-(N-ethylcarboxamido)-adenosine (NECA) nor theophylline inhibited adenosine uptake by more than 60% but the mitochodrial benzodiazepine receptor ligands 4'-chloro-diazepam (Ro 5-4864) and 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl) isoquinoline carboxamide (PK 11195) were able to inhibit it. The lack of inhibition by the blockers of the mitochondrial adenine-nucleotide carrier, atractyloside and alpha, beta-methylene-ATP, indicates that [3H]adenosine uptake occurs via a transporter other than this carrier. All these results support the existence of an equilibrative adenosine transport system, which might mediate the passage of adenosine formed in the mitochondria to the cytoplasm.

Asunto(s)

Adenosina/metabolismo , Mitocondrias/metabolismo , Testículo/metabolismo , Adenosina/farmacología , Adenosina-5'-(N-etilcarboxamida)/farmacología , Animales , Benzodiazepinonas/farmacología , Transporte Biológico/efectos de los fármacos , Dilazep/farmacología , Dipiridamol/farmacología , Relación Dosis-Respuesta a Droga , Guanosina/análogos & derivados , Guanosina/farmacología , Hexobendina/farmacología , Concentración de Iones de Hidrógeno , Inosina/farmacología , Isoquinolinas/farmacología , Cinética , Masculino , Mitocondrias/efectos de los fármacos , Concentración Osmolar , Fenilisopropiladenosina/farmacología , Ratas , Ratas Sprague-Dawley , Sodio/farmacología , Fracciones Subcelulares , Temperatura , Testículo/efectos de los fármacos , Tioinosina/análogos & derivados , Tioinosina/farmacología , Tionucleósidos/farmacología , Factores de Tiempo , Tritio , Uridina/farmacología

RESUMEN

The peripheral adrenergic effects of orphenadrine, an antiparkinsonian drug, have been evaluated in the rat vas deferens to investigate whether these properties are the same as those of other phencyclidine ligands. In the low micromolar range, orphenadrine enhanced electrically-evoked and exogenous noradrenaline contractile responses in the epididymal portion of rat vas deferens. It also induced spontaneous activity that was inhibited by prazosin (1 microM) but not by atropine (20 nM). It inhibited accumulation of [3H]noradrenaline in rat vas deferens (IC50 = 14.2+/-2.3 microM). Orphenadrine competitively inhibited [3H]nisoxetine binding in rat vas deferens membranes (Ki = 1.05+/-0.20 microM). It can be concluded that orphenadrine, at low micromolar concentrations, interacts with the noradrenaline reuptake system inhibiting its functionality and thus potentiating the effect of noradrenaline.

Asunto(s)

Adrenérgicos/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Orfenadrina/farmacología , Fenciclidina/antagonistas & inhibidores , Animales , Atropina/farmacología , Unión Competitiva , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Estimulación Eléctrica , Epidídimo/efectos de los fármacos , Fluoxetina/análogos & derivados , Fluoxetina/farmacología , Técnicas In Vitro , Ligandos , Masculino , Antagonistas Muscarínicos/farmacología , Norepinefrina/metabolismo , Norepinefrina/farmacología , Prazosina/farmacología , Ratas , Ratas Sprague-Dawley , Conducto Deferente/efectos de los fármacos