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 microM ZVAD-fmk), an antioxidant (25 mM N-acetyl-l-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 microM, 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 microM 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-l-cysteine or EGF treatment.
Yoshinaga
N
N
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Murayama
T
T
Nomura
Y
Y
D015655:1-Methyl-4-phenylpyridinium / Q000494:pharmacology*; D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004347:Drug Interactions; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism; D016166:Free Radical Scavengers / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D020011:Protective Agents / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats
D003574:Cytochrome c Group; D016166:Free Radical Scavengers; D020011:Protective Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011493:Protein Kinase C; D020169:Caspases; D015655:1-Methyl-4-phenylpyridinium; D000111:Acetylcysteine