Extracellular norepinephrine reduces neuronal uptake of norepinephrine by oxidative stress in PC12 cells

Cardiology Unit, Department of Medicine, and Department of Radiation Oncology, University of Rochester Medical Center, Rochester, New York 14642 Submitted 8 December 2003 ; accepted in final form 9 February 2004 Cardiac norepinephrine (NE) uptake activity is reduced in congestive heart failure. Our studies in intact animals suggest that this effect on the cardiac sympathetic nerve endings is caused by oxidative stress and/or NE toxic metabolites derived from NE. In this study, we investigated the direct effects of NE on neuronal NE uptake activity and NE transporter (NET), using undifferentiated PC12 cells. Cells were incubated with NE (1–500 µM) either alone or in combination of Cu 2+ sulfate (1 µM), which promotes free radical formation by Fenton reaction for 24 h. NE uptake activity was measured using [ 3 H]NE. Cell viability was determined with the use of Trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay, and cellular oxidative stress by dichlorodihydrofluorescein fluorescence and the GSH/GSSG ratio. Cell viability was reduced by NE >100 µM. At lower doses, NE produced oxidative stress and a dose-dependent reduction of NE uptake activity without affecting cell viability significantly. Cu 2+ , which has no direct effect on NE uptake activity, potentiated oxidative stress and reduction of NE uptake activity produced by NE. This decrease of NE uptake activity was associated with reductions of NE uptake binding sites and NET protein expression by using the radioligand assay and Western blot analysis, but no changes in NET gene expression. In addition, the free-radical scavenger mannitol, and antioxidant enzymes superoxide dismutase and catalase, reduced oxidative stress and attenuated the reductions of NE uptake activity and NET protein produced by NE/Cu. Thus our results support a functional role of oxidative stress in mediating the neuronal NE uptake reducing effect of NE and that this effect of NE on NET is a posttranscriptional event. Cu 2+ ; reactive oxygen species; antioxidants Address for reprint requests and other correspondence: C.-s. Liang, Univ. of Rochester Medical Center, Cardiology Unit, Box 679, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: chang-seng_liang{at}urmc.rochester.edu ).