GLUTAMATE-INDUCED CYTOTOXICITY IN PC12 PHEOCHROMOCYTOMA CELLS - ROLE OF OXIDATION OF PHOSPHOLIPIDS, GLUTATHIONE AND PROTEIN SULFHYDRYLS REVEALED BY BCL-2 TRANSFECTION

Incubation of mock-transfected PC12 rat pheochromocytoma cells (PC12) for 2 h with increasing concentrations of glutamate caused progressive loss of viability (e.g., 67% with 15 mM glutamate). In contrast, the viability of bcl-2-transfected cells (PC12/bcl-2) was unaffected by gl utamate, Neither PC12 nor PC12/bcl-2 cells showed a significant incide nce of apoptosis in response to glutamate. Conventional phospholipid a nalysis by high-performance TLC and phosphorous determination showed n o significant changes in the phospholipid composition of either cell l ine incubated with I 15 mM glutamate, Phospholipid peroxidation was qu antified in the cells using our newly developed method based on fluore scence-HPLC analysis of metabolically incorporated oxidation-sensitive and fluorescent fatty acid, cis-parinaric acid. Unlike previous studi es that measured total phospholipid oxidation, this novel technology p ermitted quantitation of oxidative stress in different classes of labe led phospholipids (the amount of labeled phospholipids in the cells di d not exceed 1% of total phospholipids). Significant peroxidation of p hosphatidylcholine and phosphatidylethanolamine occurred in PC12 cells treated with > 5 mM glutamate. The peroxyl radical initiator 2,2'-azo bis(2,3-dimethylvaleronitrile) caused a pronounced loss of all major p hospholipid classes in PC12 cells, but no loss of cell viability. No p hospholipid peroxidation was detected in PC12/bcl-2 cells incubated wi th less than or equal to 15 mM glutamate or with 2,2'-azobis(2,4-dimet hylvaleronitrile). These results directly demonstrate that peroxidatio n of membrane phospholipids is not responsible for the cytotoxicity of glutamate in PC12 cells. Total cellular thiol, protein thiol and GSH reserves were quantified by a previously described electron paramagnet ic resonance spectrometric method. Total thiols were ca. 1.5-fold grea ter hi PC12/bcl-2 than in PC12 cells. Glutamate (I 5 mM) caused a prog ressive and equally significant decrease in total thiols and GSH in bo th PC12 and PC12/bcl-2 cells. High glutamate concentrations caused oxi dation of protein sulfhydryls in PC12 cells, but not in PC12/bcl-2 cel ls. The results suggest that the changes in cellular milieu caused by bcl-2 gene transfection protect PC12 cells from the toxic effects of g lutamate in a manner consistent with prevention of protein sulfhydryl oxidation, (C) 1998 Elsevier Science B.V. All rights reserved.