Cell death induced by β-amyloid 1-40 in MES 23.5 hybrid clone: the role of nitric oxide and NMDA-gated channel activation leading to apoptosis

The molecular events associated with β-amyloid-induced neuronal injury remain incompletely characterized. Using a substantia nigra/neuroblastoma hybrid cell line (MES 23.5) synthetic β-amyloid 1-40 induced a time and dose-dependent apoptotic cell death which was characterized by cell shrinkage and fragmentation of DNA, and was inhibited by aurintricarboxylic acid (ATA), and cycloheximide (CHX). Following β-amyloid 1-40 treatment, cyclic GMP, an index of NO synthesis, was increased in MES 23.5 cells. The NO scavenger hemoglobin, as well as the NO synthase inhibitors N^G-monomethyl-l-arginine acetate (l-NMMA) andl-N⁵-(1-iminoethyl)ornithine hydrochloride (l-NIO) attenuated such increases. These same inhibitors and scavengers also significantly prevented cytotoxicity. β-Amyloid also induced an early and transient increase in intracellular calcium as monitored with laser scanning confocal microscopy and Fluo-3 imaging. These induced calcium transients could be significantly blocked by theN-methyl-d-aspartic acid (NMDA) receptor antagonist MK-801. Pretreatment with MK-801 or removal of extracellular Ca²⁺ also reduced β-amyloid-induced NO production and neurotoxicity. Furthermore, β-amyloid neurotoxicity was greatly enhanced in the absence of Mg²⁺ or in the presence of glutamate or NMDA. These data suggest that β-amyloid can lead to apoptotic cell death through a NO mediated process possibly triggered by Ca²⁺ entry through activated NMDA-gated channels.