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

Raphael Caio Tamborelli Garcia,Mariana Aguilera Alencar Da Silva,Mariana Sayuri Berto Udo,Livia Mendonça Munhoz Dati,P Jeffrey Conn,José Luiz Da Costa,Renata Gorjão,Solange Castro Afeche,Colleen M Niswender,Tania Marcourakis,Rosana Camarini,Mauricio Yonamine,Maria Regina Lopes Sandoval,Fernando Maurício Francis Abdalla,Larissa Helena Torres

doi:10.1038/srep17555

Abstract

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 [3H]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-3H]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.

Highlights

South America (1.8 and 1.2% annual prevalence rates, respectively), Oceania (1.5%), and Western and Central Europe (1%)[1]
To the best of our knowledge, the data available about this substance far includes only studies in the peripheral system, i.e., reductions in blood pressure and heart rate in rabbits with an increase in the respiratory rate[13]; a negative inotropic effect in vitro possibly mediated by muscarinic cholinergic receptors, since it was reversed by atropine, a nonspecific muscarinic receptor antagonist[14]; cardiovascular effects, e.g., hypotension and tachycardia in sheep, which are antagonized by intravenous administration of atropine, again consistent with a muscarinic cholinergic effect[15]
Maximum inositol phosphate accumulation was obtained with 10−5 M (10 μ M) anhydroecgonine methyl ester (AEME) and carbachol (Fig. 1A)

Summary

Introduction

South America (1.8 and 1.2% annual prevalence rates, respectively), Oceania (1.5%), and Western and Central Europe (1%)[1]. It blocks the uptake of serotonin, norepinephrine and dopamine in presynaptic nerve terminals, as well as voltage-specific sodium channels, responsible for the local anesthetic effect[2]. Crack cocaine is the smoked form of cocaine and has greater addictive potential than other routes of cocaine administration[10]. Caspase-3 activity in the hippocampal neurons was increased after 6 hours of exposure and seems to be one of the main mechanisms of AEME-induced neurotoxicity. Atropine prevented AEME-induced neurotoxicity, reinforcing that mAChRs are involved in AEME’s effects. It is important to emphasize that all five mAChRs are expressed in the hippocampus[19,20] and they modulate hippocampal function through the inhibition of synaptic activity and/or the increase of neuronal excitability[21]

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