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. 2015 Dec 2:5:17555.
doi: 10.1038/srep17555.

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

Raphael Caio Tamborelli Garcia  1   2   3   4 , Livia Mendonça Munhoz Dati  1 , Larissa Helena Torres  1 , Mariana Aguilera Alencar da Silva  1 , Mariana Sayuri Berto Udo  1 , Fernando Maurício Francis Abdalla  5 , José Luiz da Costa  6 , Renata Gorjão  7 , Solange Castro Afeche  5 , Mauricio Yonamine  1 , Colleen M Niswender  3   4 , P Jeffrey Conn  3   4 , Rosana Camarini  8 , Maria Regina Lopes Sandoval  5 , Tania Marcourakis  1
Affiliations

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

Raphael Caio Tamborelli Garcia et al. Sci Rep. .

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 [(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.

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Figures

Figure 1
Figure 1. Effect of AEME on total [3H]inositol phosphates accumulation and on intracellular calcium release in hippocampal cell after culturing.
(A) Concentration-effect curves of carbachol and AEME on total [3H]inositol phosphate accumulation in hippocampal cells. Maximum inositol phosphate accumulation was obtained with 10−5 M (10 μM) AEME and carbachol. Each point and vertical line represent the mean ± SEM of three to five experiments performed in duplicate. The basal level of the total [3H]inositol phosphate was 30626 ± 4250 dpm/106 cells. (B) Intracellular calcium release after exposure to 10−5, 10−4 and 10−3 M AEME. w/o: without AEME. *p < 0.05 and ***p < 0.001, compared with control group (w/o), ##p < 0.01 and ###p < 0.001 intergroup comparison (ANOVA and Newman-Keuls multiple comparison). Each bar and vertical line represent the mean ± SEM of ten independent experiments, each one performed in duplicate.
Figure 2
Figure 2. Percentage of hippocampal cells with fragmented DNA after exposure to AEME for: (A) 12 and (B) 24 hours.
Each bar and vertical line represent the mean ± SEM of three to four independent experiments. **p < 0.01, compared with control group, #p < 0.05 intergroup comparison (ANOVA and Newman-Keuls multiple comparison).
Figure 3
Figure 3. AEME competition binding curves obtained in CHO cells expressing individual subtypes of muscarinic receptors (data represent the mean of three independent experiments performed in triplicate).
The Ki values for AEME (μM) were plotted alongside with the competition binding curves. The affinity for rat M2 was greater than rat M4 (p < 0.05), rat M4 was greater than rat M3 (p < 0.05), and rat M1 greater than rat M3 (p < 0.05). There was no difference among rat M4, rat M1 and rat M5. The averages of absolute values of controls are: 2274 ± 187, 888 ± 25, 3698 ± 303, 2831 ± 231, and 4059 ± 415 dpm/25 μg of protein for rat M1, rat M2, rat M3, rat M4 and rat M5, respectively. Data presented as mean ± SEM.
Figure 4
Figure 4. AEME concentration-response curve normalized to the maximum acetylcholine response. CHO cells expressing rat M1, rat M3 and rat M5 were studied (data represent the mean of three independent experiments, each one performed in triplicate).
The AEME concentration effect was greater than 100 μM for both rat M1 and rat M3. The averages of absolute values of control are: 21950 ± 133, 23107 ± 77, and 20117 ± 1323 arbitrary units/4 × 10 cells for rat M1, rat M3 and rat M5, respectively. Data presented as mean ± SEM.
Figure 5
Figure 5. Calcium mobilization assay in CHO-K1 cells stably expressing all five subtypes of muscarinic receptors (data represent the mean of three independent experiments, each one performed in duplicate): rat M1 (A), rat M2 (B), rat M3 (C), rat M4 (D) and rat M5 (E).
The averages of absolute values of control are: 20307 ± 149, 18233 ± 86, and 23438 ± 205 arbitrary units/4 × 104 cells for rat M1, rat M3 and rat M5, respectively; 21937 ± 178 and 20218 ± 165 arbitrary units/6 × 104 cells for rat M2 and rat M4, respectively. Data presented as mean ± SEM.
Figure 6
Figure 6. Acetylcholine concentration-response curves in the absence or presence of different concentrations of AEME (10−6 to 10−3.5 M) in a calcium mobilization assay using CHO-K1 cells stably expressing rat M5.
Schild regression of the dose ratios (DR) derived from the AEME antagonism of acetylcholine was presented and the slope was 0.95 ± 0.07. The average of absolute values of control is 21255 ± 124 arbitrary units/4 × 104 cells. Data presented as mean ± SEM. ACh, acetylcholine.
Figure 7
Figure 7. Effect of M1 selective antagonist pirenzepine (10 nM), M3 selective antagonist p-fluoro-hexahydrosila-difenidol (p-F-HHSiD, 10 nM) and PLC inhibitor (U73122, 10 nM) after 24 hours of exposure to 10−3 M AEME (n = 4).
Potassium chloride (250 mM) was used as a positive control of neuronal death. ***p < 0.001, compared with neurobasal medium (control group), #p < 0.05, ##p < 0.01, ###p < 0.001compared with 10−3 M AEME (ANOVA and Newman-Keuls multiple comparison).
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References

    1. UNODC (United Nations Office on Drugs and Crime), World Drug Report (2014). United Nations Publication, Sales No., E.14.XI.7. Available at: http://www.unodc.org/documents/wdr2014/World_Drug_Report_2014_web.pdf (Date of access: 04/09/2014).
    1. Schwartz B. G., Rezkalla S. & Kloner R. A. Cardiovascular effects of cocaine. Circulation 122, 2558–2569 (2010). - PubMed
    1. Mao J. T. et al. Cocaine inhibits human endothelial cell IL-8 production: the role of transforming growth factor-β. Cell Immunol 181(1), 38–43 (1997). - PubMed
    1. Heesch C. M. et al. Cocaine activates platelets and increases the formation of circulating platelet containing microaggregates in humans. Heart 83(6), 688–695 (2000). - PMC - PubMed
    1. Valente M. J., Carvalho F., Bastos Md., de Pinho P. G. & Carvalho M. Contribution of oxidative metabolism to cocaine-induced liver and kidney damage. Curr Med Chem 19(33), 5601–5606 (2012). - PubMed

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