Apoptosis induction by a dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP+), and inhibition by epidermal growth factor in GH3 cells

A dopaminergic neurotoxin, 1-methyl-4 phenyl-1,2,3,6-tetrahydropyridine (MPTP), can induce dopaminergic denervation and Parkinsonism in humans. The active metabolite of MPTP is the 1-methyl-4-phenylpyridinium ion (MPP+). Previously we reported that MPP+ is incorporated via the dopamine transport system and causes delayed cell death in GH3 cells, a clonal strain from the rat anterior pituitary. In this study, we investigated whether MPP+ induces apoptosis. GH3 cells cultured with MPP+ exhibited DNA laddering and fragmentation in a time and concentration-dependent manner. The effect of MPP+ was inhibited in GH3 cells treated with a pan-caspase inhibitor (100 mu M ZVAD-fmk), an antioxidant (25 mM N-acetyl-1-cysteine), or epidermal growth factor (EGF; 50 ng/mL). Because EGF stimulated tyrosine phosphorylation of the EGF receptor and tyrphostin AG1478 [4-(3-chloroanilino)-6,7-dimethoxyquinazoline; 5 mu M, a specific inhibitor of EGF receptor kinase] abolished EGF inhibition, involvement of EGF receptor kinase is assumed. Protein kinase C-dependent processes and Bcl-2 protein expression were shown not to be involved in EGF inhibition. MPP+ increased cytochrome c immunoreactivity in cytosolic fractions in GH3 cells. The addition of 200 mu M MPP+ to isolated mitochondrial fractions from GH3 cells stimulated the release of a 13-kDa protein that cross-reacted with anti cytochrome c antibody. The release was inhibited in EGF-treated GH3 cells. Our findings demonstrated that (i) MPP+ induces apoptosis of GH3 cells via cytochrome c release and caspase activation and (ii) apoptosis by MPP+ can be blocked by N-acetyl-1-cysteine or EGF treatment. BIOCHEM PHARMACOL 60;1: 111-120, 2000. (C) 2000 Elsevier Science Inc.