Abstract: OBJECTIVE: To study the neuroprotective effects of amiloride (Ami), non-selective blocker of acid-sensing ion channels，in the cell death and apoptosis induced by extracellular acid in PC12 cells, and investigate the effects of Ami on chaperone-mediated autophagic pathway. METHODS：The cell viability following acid exposure (pH6.0) was analyzed with 3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay. The degree of cell injury was assessed by a quantitative measurement of lactate dehydrogenase (LDH) released from cytosol to culture medium. Double staining of treated cells with Annexin V and PI was assayed with flow cytometry to determine the apoptotic rate. Western blot was used to examine the expression levels of receptor lysosome-associated membrane protein 2a (Lamp2a). RESULTS: Acid exposure significantly decreased the cell viability, increased the activity of LDH which released to culture medium and raised apoptosis rate. Western bolt analysis shown that acidic exposure led to compensatory induction of Lamp2a protein signaficantly. Co-incubation with Ami (100μM) significantly increased the cell viability, inhibited the release of LDH, reduced the apoptosis rate, and inhibited the overexpression of Lamp2a. CONCLUSION: Ami protects PC12 cells against acid-induced injury via chaperone-mediated autophagic pathway.

Key words: amiloride, chaperone-mediated autophagy, acid sensing ion channels, PC12 cells, Parkinson’s disease