Determination of hypoxic effect on neprilysin activity in human neuroblastoma SH-SY5Y cells using a novel HPLC method.
Alzheimer's disease (AD) is characterized by extracellular deposition of amyloid-beta-peptide (Abeta), which is closely associated with the metabolic balance between Abeta production and clearance activities. Neprilysin is one of the important enzymes to degrade Abeta in the brain and alternation of its activity would contribute to the AD neuropathology. However, measurement of neprilysin activity in neuronal cells, especially the extracellular activity, is very difficult because of its weak activities. In the present study, we established a sensitive method enough to estimate extracellular neprilysin activity of living cell cultivated in a 96-well plate using HPLC-fluorometric system, and investigated the effect of hypoxia, a closely associated event with neurodegenerative diseases, on neprilysin activity of human neuroblastoma SH-SY5Y cells. We demonstrated that chronic but not acute hypoxia significantly attenuated neprilysin activity without any alterations of neprilysin gene expression. The present study suggests that chronic hypoxia may down-regulate extracellular neprilysin activity of neuronal cells to impair Abeta degradation and associate with the development of amyloid pathology.
Oh-hashi
Kentaro
K
Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Nagai
Takeki
T
Tanaka
Tatsuhide
T
Yu
Hai
H
Hirata
Yoko
Y
Kiuchi
Kazutoshi
K
D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004789:Enzyme Activation; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D015260:Neprilysin / Q000032:analysis* / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
D015260:Neprilysin; D010100:Oxygen