Methamphetamine induces autophagy as a pro-survival response against apoptotic endothelial cell death through the Kappa opioid receptor

Ma, J; Wan, J; Meng, J; Banerjee, S; Ramakrishnan, S; Roy, S

Inhibiting METH-induced autophagy triggers apoptotic cell death. Under pathological conditions, autophagy is considered to have a pro-survival role. 24 Therefore, we asked whether suppression of METH-induced autophagy may accelerate cell death. Therefore, two well-characterized inhibitors of autophagy, 3-methyladenine (3-MA) and chloroquine, were used in parallel in this study. 3-MA and chloroquine have different structures, different mechanisms of action and act at early and late stages of autophagy progression, respectively. 3-MA is a class III phosphatidylinositol 3-kinase (PI3K) inhibitor that prevents autophagy at the earliest stage of autophagosome formation. Chloroquine is a weak base that concentrates in acidic vesicles such as lysosomes and disrupts vesicular acidification compromising their degradative and recycling capacity. 26 First, we used 3-MA to examine whether inhibition of autophagy can enhance METH-induced apoptosis. HUVECs were either pretreated with or without 3-MA before the addition of 1 mM METH for 72 h. Cells were stained with Annexin V (green) and analyzed by fluorescence microscopy. 3-MA markedly increased apoptosis induction by METH at 72 h, as measured by Annexin V labeling. 3-MA alone caused minimal apoptosis at 72 h (Figures 5a and b) . These findings suggest that inhibition of autophagy can accelerate apoptosis. Second, 100 mM chloroquine also significantly increased METHinduced caspase-3 activity (Figure 5c ). These findings suggest that although 3-MA and chloroquine have different mechanisms of action, they both can accelerate METHinduced apoptosis by inhibiting autophagy.