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

METH induces LC3 recruitment and vesicular compartmentalization of LAMP1. To further determine if acute METH treatment induces an autophagic pro-survival response, we monitored different autophagy markers: LC3 vesicular recruitment and LAMP1. To analyze autophagosome formation, we used anti-LC3 immunofluorescence in a cell-based detection system with HUVECs. In 1 mM METH-treated cells, LC3 protein increased in total abundance and was aggregated in vesicular structures ( Figure 1d ). LC3 (ATG 8) is first cleaved by ATG4 to generate LC3-I and then lipidated to produce LC3-II. LC3-II is incorporated into the inner and outer surfaces of autophagosomes, and as such, remains associated with the autophagosome throughout the pathway. The lipidated (LC3-II) form can be detected as a faster-migrating band in western blots. 11, 12 The amount of LC3-II in the presence and absence of saturating levels of inhibitors, which can be used to examine the transit of LC3-II through the autophagic pathway; if autophagy flux is occurring, the levels of LC3-II will be higher in the presence of the inhibitors. Lysosomal degradation can be prevented through the use of protease inhibitors (e.g., pepstatin A and E-64d). 13 Our data demonstrate that the LC3-I/LC3-II conversion occurred rapidly, within 6 h, after METH treatment and was still detectable at 24 h post treatment in the presence of the protease inhibitors pepstatin A and E-64d (Figures 1e and f) .