Anhydroecgonine methyl ester, a cocaine pyrolysis product, contributes to cocaine-induced rat primary hippocampal neuronal death in a synergistic and time-dependent manner.

Crack cocaine users are simultaneously exposed to volatilized cocaine and to its main pyrolysis product, anhydroecgonine methyl ester (AEME). Although the neurotoxic effects of cocaine have been extensively studied, little is known about AEME or its combination. We investigated cell death processes using rat primary hippocampal cells exposed to cocaine (2 mM), AEME (1 mM) and their combination (C + A), after 1, 3, 6 and 12 h. Cocaine increased LC3 I after 6 h and LC3 II after 12 h, but reduced the percentage of cells with acid vesicles, suggesting failure in the autophagic flux, which activated the extrinsic apoptotic pathway after 12 h. AEME neurotoxicity did not involve the autophagic process; rather, it activated caspase-9 after 6 h and caspase-8 after 12 h leading to a high percentage of cells in early apoptosis. C + A progressively reduced the percentage of undamaged cells, starting after 3 h; it activated both apoptotic pathways after 6 h, and was more neurotoxic than cocaine and AEME alone. Also, C + A increased the phosphorylation of p62 after 12 h, but there was little difference in LC3 I or II, and a small percentage of cells with acid vesicles at all time points investigated. In summary, the present study provides new evidence for the neurotoxic mechanism and timing response of each substance alone and in combination, indicating that AEME is more than just a biological marker for crack cocaine consumption, as it may intensify and hasten cocaine neurotoxicity.

M1 and M3 muscarinic receptors may play a role in the neurotoxicity of anhydroecgonine methyl ester, a cocaine pyrolysis product.

The smoke of crack cocaine contains cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). AEME possesses greater neurotoxic potential than cocaine and an additive effect when they are combined. Since atropine prevented AEME-induced neurotoxicity, it has been suggested that its toxic effects may involve the muscarinic cholinergic receptors (mAChRs). Our aim is to understand the interaction between AEME and mAChRs and how it can lead to neuronal death. Using a rat primary hippocampal cell culture, AEME was shown to cause a concentration-dependent increase on both total [(3)H]inositol phosphate and intracellular calcium, and to induce DNA fragmentation after 24 hours of exposure, in line with the activation of caspase-3 previously shown. Additionally, we assessed AEME activity at rat mAChR subtypes 1-5 heterologously expressed in Chinese Hamster Ovary cells. l-[N-methyl-(3)H]scopolamine competition binding showed a preference of AEME for the M2 subtype; calcium mobilization tests revealed partial agonist effects at M1 and M3 and antagonist activity at the remaining subtypes. The selective M1 and M3 antagonists and the phospholipase C inhibitor, were able to prevent AEME-induced neurotoxicity, suggesting that the toxicity is due to the partial agonist effect at M1 and M3 mAChRs, leading to DNA fragmentation and neuronal death by apoptosis.