Tnf– Production in Glutamate–Induced Apoptosis in a Differentiated Neuronal Cell Line

Abstract

Purpose: : Glutamate, a major excitatory neurotransmitter, is implicated in certain ocular diseases, including proliferative diabetic retinopathy, optic neuropathy and glaucoma. We previously reported that glutamate enhanced TNF–α production in differentiated PC12h cells. The objective of the present study is to investigate whether TNF–α production by glutamate mediates NMDA receptor and whether induction of caspase–8 by glutamate is involved in TNF–α production.

Methods: : PC12h cells were cultured in F12/DMEM supplemented with 10% heat–activated fetal calf serum (FCS). For differentiation, the cells were cultured in the presence of 100ng/ml NGF for three days in F12/DMEM medium contained 1% FCS. Cells were incubated with or without glutamate (0.1mM, 1mM, 5mM) for several days (1day, 2days, 3days). Cell viability was assessed using MTS assay. Apoptotic cells were detected by TUNEL assay. The density of TNF–α in the supernatant fluid of medium after exposure of glutamate with or without APV (specific NMDA receptor antagonist) was measured by using TNF–α ELISA kit. Cells were pretreated with soluble TNF–R1 prior to glutamate exposure and collected for Western blotting analysis.

Results: : Treatment of differentiated PC12h cells with glutamate resulted in dose–dependent and time–dependent cell death. In addition, glutamate induced apoptosis in differentiated PC12h cells in a dose dependent manner. Glutamate increased TNF–α production and the expression of caspase–8 and caspase–3. APV prevented the glutamate–induced TNF–α production. Moreover soluble TNF–R1 inhibited the increase of caspase–8, but not caspase–3 in differentiated neuronal cells.

Conclusions: : These results suggest that glutamate induces TNF–α production via NMDA receptor in neuronal cells and that increase of caspase–8 induced by glutamate may be related to TNF–α induction with the involvement of TNF–R1.

Keywords: excitatory amino acid receptors • retinal degenerations: cell biology • cell survival