1‐Methyl‐4‐(2′‐Ethylphenyl)‐1,2,3,6‐Tetrahydropyridine‐ Induced Toxicity in PC12 Cells Is Enhanced by Preventing Glycolysis

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Abstract
The effects of l-methyl-4-(2′-ethylphenyl)-1,2,3,6-tetrahydropyridine (2′Et-MPTP), l-methyl-4-(2′-ethyl-phenyl)pyridinium (2′Et-MPP+), and the classic complex 1 inhibitor, rotenone, on toxicity as well as on rates of glucose use and lactate production were studied using the pheochro-mocytoma PC12 cell line. PC12 cells are neoplastic in nature and have a high rate of glycolysis accompanied by a large production of lactate and a low use of glucose carbon through the Krebs cycle. l-Methyl-4-phenylpyridinium (MPP+) and analogues such as 2′Et-MPP+ are actively accumulated by mitochondrial preparations in vitro and block NADH de-hydrogenase of complex 1. This blockade results in biochemical sequelae that are ultimately cytotoxic. In this study, untreated PC12 cells used glucose and concomitantly accumulated lactate in a time-dependent manner at all concentrations of glucose studied. Treatment with 50 pM 2′Et-MPP+ or 50 nAf rotenone increased both rates significantly, indicating a shift toward increased glycolysis. Cell death caused by the neurotoxins was also time and concentration dependent and markedly enhanced by glucose depletion in the medium. The increase in 2′Et-MPTP-induced toxicity in low glucose-supplemented cells was not due to an increase in pyridinium formation from the tetrahydro-pyridine, but rather to the lack of glucose for glycolysis. Moreover, inhibition of glycolysis with 2-deoxyglucose or io-doacetic acid also enhanced the lethality of the neurotoxins to the cells. The data in this study provide additional support to the hypothesis that 2′Et-MPP+ or related analogues act to kill cells by inhibiting mitochondrial respiration. Furthermore, these findings support the hypothesis that the ability of 2′Et-MPTP or its analogues to induce toxicity is dependent on the relative contributions of glycolysis and mitochondrial oxidation to the energy needs of a particular cell or model system.