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Updated: Jul 12, 2026
Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry
Published on: November 21, 2012
Different oxidative profile and nicotinic receptor interaction of amphetamine and 3,4-methylenedioxy-methamphetamine
C Chipana1, S García-Ratés, J Camarasa
1Unitat de Farmacologia i Farmacognòsia, Facultat de Farmàcia, Nucli Universitari de Pedralbes, Universitat de Barcelona, 08028 Barcelona, Spain.
Abstract:
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.
Insights
d-Amphetamine (AMPH) and MDMA increase reactive oxygen species (ROS) production in mouse brain synaptosomes, with effects modulated by drug concentration and calcium. Nicotinic receptor antagonists block MDMA
Area of Science:
- Neuroscience
- Pharmacology
- Biochemistry
Background:
- d-Amphetamine (AMPH) and MDMA are psychoactive substances with known neurological effects.
- Reactive oxygen species (ROS) play a role in cellular signaling and damage.
- Understanding the mechanisms of amphetamine-induced neurotoxicity is crucial for public health.
Purpose of the Study:
- To investigate the comparative effects of d-Amphetamine (AMPH) and MDMA on intracellular ROS production in mouse striatal synaptosomes.
- To explore the mechanisms underlying AMPH and MDMA-induced oxidative stress, including the roles of calcium, receptor interactions, and dopamine uptake.
- To evaluate the potential of nicotinic acetylcholine receptor (nAChR) antagonists in mitigating MDMA's acute effects.
Main Methods:
- Measurement of intracellular ROS production in isolated mouse striatal synaptosomes following exposure to AMPH and MDMA.
- Assessment of drug concentration-dependent effects, including double-maximum responses for AMPH.
- Investigation of the involvement of calcium, phospholipase A(2), CB(1) receptors, and nAChRs using specific inhibitors and antagonists.
- Analysis of [(3)H]epibatidine and [(3)H]MLA binding to study receptor interactions.
- Evaluation of [(3)H]dopamine uptake in PC12 cells after preincubation with AMPH or MDMA.
Main Results:
- Both AMPH and MDMA significantly increased ROS production in a concentration-dependent manner, with AMPH exhibiting a double-maximum effect.
- The pro-oxidative effects were dependent on extracellular and intracellular calcium stores and were inhibited by nAChR antagonists (dihydro-beta-erythroidine, MLA, alpha-bungarotoxin).
- MDMA, but not AMPH at relevant CNS concentrations, showed significant interaction with nAChRs, and MLA blocked MDMA-induced reduction in dopamine uptake.
- Delta(9)-Tetrahydrocannabinol, NPC 15437, and genistein also prevented the oxidative effects via a CB(1) receptor-independent pathway.
- AMPH and MDMA reduced [(3)H]dopamine uptake in PC12 cells.
Conclusions:
- AMPH and MDMA induce concentration-dependent oxidative stress and reduce dopamine uptake, with mechanisms involving calcium and, for MDMA, nAChRs.
- nAChR antagonists effectively blocked acute MDMA effects, suggesting potential therapeutic applications for managing adverse effects in MDMA users.
- The study highlights distinct mechanistic pathways for AMPH and MDMA, particularly concerning nAChR involvement in their neurochemical effects.
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