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The putative endothelin-converting enzyme (ECE) has been the focus of intense research, both within academia and the pharmaceutical industry. Interest in ECE stems mainly from the hypothesis that development of inhibitors of ECE will provide an effective means of preventing production of endothelin in circumstances where it may play a pathogenic role. Both an aspartic and a metalloprotease have been identified that have characteristics of this putative enzyme. Evidence suggests that the metalloprotease, which is inhibited by phosphoramidon, may be the physiologically relevant converting enzyme. However, it remains to be demonstrated conclusively that any inhibitor of an ECE activity directly alters endogenous endothelin production and/or the pathogenesis of a disease condition in which endothelin is thought to play a primary role.

Recent evidence indicates that tumor necrosis factor-␣ (TNF-␣) is up-regulated following brain injury andin neurodegenerative disorders such as stroke, multiple sclerosis, Parkinson's disease, and Alzheimer's disease. TNF-␣ elicits its biological effects through two distinct TNF receptor (TNFR) subtypes: p55 TNFR (TNFR1) and p75 TNFR (TNFR2). Studies have demonstrated that the p55 TNFR contributes to cell death, whereas the role of the p75 TNFR in neuronal viability is unclear. To better understand the role of p75 TNFR, we treated human neuronal SH-SY5Y cells with phosphorothioate-modified antisense oligonucleotides (ASO) for p75 TNFR and established that ASO inhibited p75 TNFR expression. Treatment of SH-SY5Y cells with ASO alone did not affect cell viability, whereas treatment with both ASO and human TNF-␣ significantly increased cell death relative to treatment with TNF-␣ alone. Moreover, addition of ASO significantly increased the level of cell injury observed following hypoxic conditions or exposure of ␤-amyloid peptide. These results indicate that inhibition of p75 TNFR using ASO increases the vulnerability of neurotypic cells to insults and suggest that the p75 TNFR may not be required for normal neuronal cell viability but rather plays a protective role following injury.Tumor necrosis factor-␣ (TNF-␣), 1 an inflammatory mediator, is recently reported to be up-regulated following brain trauma (1, 2) and ischemic injury (3, 4), and in multiple sclerosis (5, 6), Parkinson's disease (7), and Alzheimer's disease (8). Studies have shown that TNF-␣ can elicit either a trophic or toxic effect, which is dependent on the target cell type. For example, TNF-␣ is toxic to human oligodendrocytes (9) and neuronal cells (10, 11), but it is trophic to rat hippocampal neurons (12). Since TNF-␣ elicits its biological effects through activation of two distinct receptors, p55 TNFR and p75 TNFR, both of which have been cloned (13-15), we hypothesized that the distinct TNF receptor subtypes might contribute to the multiple biological functions of TNF-␣. Reports indicate that the p55 TNFR may contribute to cell death (16), whereas the role of the p75 TNFR in neuronal cell function is not clear. Here we show that inhibition of p75 TNFR by antisense oligonucleotides (ASO) increased the magnitude of neuronal cell death induced by TNF-␣, hypoxic injury or ␤-amyloid peptide (A␤) as measured by lactate dehydrogenase (LDH) release. This novel finding provides evidence that the p75 TNFR might play a protective role following neuronal insult. EXPERIMENTAL PROCEDURESCell Cultures-Human neuronal-like cells, SH-SY5Y, were cultured in a 1:1 ratio of minimum Eagle's medium/F-12 medium, 15% fetal calf serum (Life Technologies, Inc.), and 10 M retinoic acid (RA, Sigma). The cells were seeded at 25,000 cells/well in a 24-well plate for cytotoxicity assays or 3 ϫ 10 6 cells in Petri dishes for radioimmunoprecipitation study. The medium was replaced every 3 days. Treatments were given to the cells in serum-free medium with 1:100 N2-supplement (Li...

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