3,4-Methylenedioxy-methamphetamine induces in vivo regional up-regulation of central nicotinic receptors in rats and potentiates the regulatory effects of nicotine on these receptors

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-methylenedioxymethamphetamine (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 [3H]paroxetine binding in the cortex and hippocampus measured 24 h 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 [3H]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, [3H]methyllycaconitine binding was increased a 42.1% with NIC + MDMA but not when they were given alone. In the hippocampus, MDMA potentiated the a7 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 a4 and b2 subunits. Western blots with specific a4 and a7 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

Interacció dels derivats amfetamínics amb els receptors nicotínics: Aspectes moleculars i funcionals

[spa] En treballs anteriors del nostre grup de recerca es va demostrar que l’antagonista específic del receptor nicotínic α7, metillicaconitina (MLA), inhibia in vitro la producció d’espècies reactives d’oxigen (EROS) i protegia de la neurotoxicitat in vivo induïda per metamfetamina (METH) i per la 3,4-metilendioxi-N-metamfetamina (MDMA). En aquesta tesi, es descriu un nou mecanisme d’acció dels derivats amfetamínics. Mitjançant assajos de fixació de radiolligands, es va comprovar que ambdós derivats amfetamínics competien amb els radiolligands específics dels receptors nicotínics α7 ([3H]Metillicaconitina), i dels heteromèrics ([3H]Epibatidina), el que indicava que en les cèl ·lules PC12, el nostre model experimental, i també en una preparació de cervell total de ratolí, aquestes substàncies interaccionaven directament amb els receptors nicotínics. L’MDMA mostrava més afinitat per ambdós subtipus de receptors. Està descrit que el tractament crònic amb nicotina provoca un augment en la densitat de receptors nicotínics tan in vivo com in vitro en cèl ·lules PC12. Ambdós derivats amfetamínics van provocar una regulació a l’alça dels dos subtipus de receptors ja a les 6 hores de pretractament. A la vegada, in vivo, l’MDMA i la Nicotina van provocar la regulació a l’alça que va ser potenciada per la seva associació en determinades zones cerebrals on s’expressen cada subtipus de nAChR. De l’estudi dels mecanismes implicats en aquesta regulació a l’alça, mitjançant inhibidors a diferents nivells, es va concluir que, igual com passa amb la nicotina, es produeixen a nivell postraduccional. A nivell funcional, vam determinar que aquests derivats amfetamínics eren capaços d’activar els receptors nicotínics i, d’acord amb les hipòtesis de treballs anteriors, induir una entrada de calci i de sodi que podria estar implicada en els esdeveniments que comportarien la seva neurotoxicitat. Per una banda, l’MDMA i la METH es van comportar com agonistes parcials dels nAChR α7 induïnt un increment de Ca2+ citosòlic. Per altra banda, l’MDMA es va comportar com antagonista dels nAChR heteromèrics i la METH com agonista parcial induint l’entrada de Na+ de la mateixa manera, el qual explicaria diferències a nivell de dependència, ja que els nAChR α4β2 estan implicats en la via mesolímbica o de recompensa. Paral ·lelament a l’increment de fixació de radiolligands, es va determinar que la preincubació amb MDMA indueix un increment en la resposta per activació de receptors nicotínics, demostrant que l’ MDMA també indueix regulació a l’alça funcional. Alhora, es va observar que la preincubació de les cèl ·lules durant 24 hores amb MDMA dona lloc a un increment perllongat dels nivells basals de Ca2+, el qual indica que l’MDMA inhibeix la desensibilització dels receptors i fa que entri calci durant un temps més llarg. Aquesta entrada persistent podria estar implicada en fenòmens de neurotoxicitat ja que va seguida de l’activació de vies dependents de calci com la calpaïna i la caspasa-3.[eng] During the last years, our emphasis has focused in the study of the neurotoxic effects of MDMA and methamphetamine (METH) on central nervous system and their pharmacological prevention. It has been demonstrated that these amphetamine derivatives produce oxygen species (ROS) in an in vitro model of synaptosomes. In previous works, we demonstrated that blockade of alpha7 nicotinic receptors with methyllycaconitine (MLA) prevented ROS production induced by MDMA and METH, consequently the alpha7 receptor would be involved in the neurotoxicity induced by these drugs. Studies at molecular level, using radioligand binding assays, showed the interaction of METH and MDMA with homomeric alpha7 nAChR and heteromeric subtypes of nicotinic receptors, such as aplha4 beta2. In addition, we investigated the effects of pretreatment with METH and MDMA on nAChR densities. We used PC 12 cells as an experimental model due to the fact other authors have similarly utilised them to evaluate the neurotoxicity of amphetamines. Moreover, they not only express nAChR, including the alpha7 subtype, but also provide an in vitro model for the up-regulation of nAChR, which occurs in vivo following chronic exposure to nicotine. In recent works, we demonstrated in vitro that Ca2+ chelation with EGTA prevented the production of reactive oxygen species (ROS) to a similar extent as nAChR blockade. This indicates that calcium influx, probably through alpha7 nAChR, is a key step in this process. Consequently, one of the objectives of this work was to use a fluorimetric method to investigate the effect of MDMA on Ca2+ and Na+ levels in cultured PC12 cells and the involvement of different nAChR subtypes and other cell pathways related to Ca2+ mobilization. In addition, we used electrophysiology in transfected Xenopus oocytes to corroborate the effects on alpha7 and alpha4 beta2 nAChR. Moreover, pretreatment with MDMA induced functional upregulation by potentiating the effects of specific nAChR agonists or whether it provoked a persistent Ca2+ increase, leading to calpain, caspase 3, NFκB, GSK-3 and Cyt C activation, which was involved in toxicity

Involvement of nicotinic receptors in methamphetamine and MDMA induced neurotoxicity: Pharmacological studies

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/32392During the last years, our emphasis has focused in the study of the neurotoxic effects of 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (METH) on central nervous system and their pharmacological prevention. 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) and memantine (MEM) prevents ROS production induced by MDMA and METH. Studies at molecular level showed that both, MDMA and METH, displaced competitively the binding of radioligands for homomeric alpha7 and heteromeric 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. Preincubation of differentiated PC12 cells with MDMA or METH induces nicotinic acetylcholine receptors (nAChR) up-regulation 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

Additive toxicity of β-amyloid by a novel bioactive peptide in vitro: possible implications for Alzheimer's disease.

BACKGROUND: β-amyloid is regarded as a significant factor in Alzheimer's disease: but inefficient therapies based on this rationale suggests that additional signalling molecules or intermediary mechanisms must be involved in the actual initiation of the characteristic degeneration of neurons. One clue could be that acetylcholinesterase, also present in amyloid plaques, is aberrant in peripheral tissues such as blood and adrenal medulla that can be implicated in Alzheimer's disease. The aim of this study was to assess the bioactivity of a fragment of acetylcholinesterase responsible for its non-enzymatic functions, a thirty amino acid peptide ("T30") which has homologies with β-amyloid. METHODS: Cell viability was measured by sulforhodamine B assay and also lactate dehydrogenase assay: meanwhile, changes in the status of living cells was monitored by measuring release of acetylcholinesterase in cell perfusates using the Ellman reagent. FINDINGS: T30 peptide and β-amyloid each have toxic effects on PC12 cells, comparable to hydrogen peroxide(.) However only the two peptides selectively then evoke a subsequent, enhanced release in acetylcholinesterase that could only be derived from the extant cells. Moreover, unlike hydrogen peroxide, the T30 peptide selectively shifted a sub-threshold dose of β-amyloid to a toxic effect, which also resulted in a comparable enhanced release of acetylcholinesterase. INTERPRETATION: This is the first study comparing directly the bioactivity of β-amyloid with a peptide derived from acetylcholinesterase: the similarity in action suggests that the sequence homology between the two compounds might have a functional and/or pathological relevance. The subsequent enhanced release of acetylcholinesterase from the extant cells could reflect a primary 'compensatory' response of cells prone to degeneration, paradoxically providing further availability of the toxic C-terminal peptide to modulate the potency of β-amyloid. Such a cycle of events may provide new insights into the mechanism of continuing selective cell loss in Alzheimer's disease and related degenerative disorders

Dose-response curves representing cell viability (% of control) after 1 and 6 h of treatment with AChE C-terminus peptides of 30 amino acids (T30) with concentrations.

<p>Data are presented as the percentage (of control) of the mean ± S.E.M. from five independent experiments carried out on triplicates (*<i>P<</i>0.05, N = 5).</p

Involvement of nicotinic receptors in methamphetamine and MDMA induced neurotoxicity: Pharmacological studies

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/32392During the last years, our emphasis has focused in the study of the neurotoxic effects of 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (METH) on central nervous system and their pharmacological prevention. 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) and memantine (MEM) prevents ROS production induced by MDMA and METH. Studies at molecular level showed that both, MDMA and METH, displaced competitively the binding of radioligands for homomeric alpha7 and heteromeric 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. Preincubation of differentiated PC12 cells with MDMA or METH induces nicotinic acetylcholine receptors (nAChR) up-regulation 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

Neuronal nicotinic receptors as new targets foramphetamine-induced oxidative damage and neurotoxicity

Amphetamine derivatives such as methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) are drugs widely abused in a recreational context. This has led to concern because of the evidence that they are neurotoxic in animal models and cognitive impairments have been described in heavy abusers. The main targets of these drugs are plasmalemmal and vesicular monoamine transporters, leading to reverse transport and increased monoamine efflux to the synapse. As far as neurotoxicity is concerned, increased reactive oxygen species (ROS) production seems to be one of the main causes. Recent research has demonstrated that blockade of 7 nicotinic acetylcholine receptors (nAChR) inhibits METH- and MDMA-induced ROS production in striatal synaptosomes which is dependent on calcium and on NO-synthase activation. Moreover, 7 nAChR antagonists (methyllycaconitine and memantine) attenuated in vivo the neurotoxicity induced by METH and MDMA, and memantine prevented the cognitive impairment induced by these drugs. Radioligand binding experiments demonstrated that both drugs have affinity to 7 and heteromeric nAChR, with MDMA showing lower Ki values, while fluorescence calcium experiments indicated that MDMA behaves as a partial agonist on 7 and as an antagonist on heteromeric nAChR. Sustained Ca increase led to calpain and caspase-3 activation. In addition, modulatory effects of MDMA on 7 and heteromeric nAChR populations have been found

Representative table of cell viability (% of Control) after 15 and 60 minutes treatments with Hydrogen peroxide (H₂O₂) 100 µM, β-Amyloid (Aβ) 0.7 µM, AChE C-terminus peptides of 30 amino acids (T30) 0.7 αM and the residual 15 amino acid sequence (T15) 0.7 αM. Data are presented as the percentage of the mean ± S.E.M. from five independent experiments carried out on triplicates (N = 5).

*<p><i>P</i><0.05 and **<i>P</i><0.01 vs Control.</p

Bar graph showing the additive effect of β-Amyloid (Aβ) and AChE C-terminus peptides of 30 amino acids (T30) on cell viability.

<p>Cells were treated for 1 hour. Data is presented as % cell viability ± SEM (N = 3). * vs Control: **<i>P</i><0.01, **<i>P<</i>0.001; ^# vs T30: ^##<i>P</i><0.01, ^###<i>P</i><0.001;^& vs Aβ: ^&<i>P</i><0.05, ^&&<i>P</i><0.01.</p

3,4-Methylenedioxy-methamphetamine induces in vivo regional up-regulation of central nicotinic receptors in rats and potentiates the regulatory effects of nicotine on these receptors

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-methylenedioxymethamphetamine (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 [3H]paroxetine binding in the cortex and hippocampus measured 24 h 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 [3H]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, [3H]methyllycaconitine binding was increased a 42.1% with NIC + MDMA but not when they were given alone. In the hippocampus, MDMA potentiated the a7 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 a4 and b2 subunits. Western blots with specific a4 and a7 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