Norepinephrine induces endoplasmic reticulum stress and downregulation of norepinephrine transporter density in PC12 cells via oxidative stress.
Cardiac norepinephrine (NE) uptake is reduced in cardiomyopathy. This change is associated with a decrease of NE transporter (NET) receptor and can be reproduced in PC12 cells by extracellular NE. To study whether this effect of NE is mediated via impaired glycosylation and trafficking of NET in the endoplasmic reticulum (ER), we measured the distribution of glycosylated 80-kDa NET and unglycosylated 46-kDa NET in the membrane and cytosolic fractions of PC12 cells. We found that NE decreased glycosylated NET in both membrane and cytosolic fractions and increased cytosolic unglycosylated NET protein. Similar results were produced by tunicamycin and thapsigargin, two agents that induce ER stress by inhibiting N-glycosylation of membrane proteins and disrupting calcium homeostasis, respectively. Also, like the ER stressors, NE not only increased phosphorylation of both the alpha-subunit of eukaryotic initiation factor-2 and its upstream RNA-dependent protein kinase-like ER kinase over 12 h of treatment but also increased ER chaperone molecule glucose-regulated protein 78 and the nuclear transcription factor C/EBP homologous protein. Antioxidants superoxide dismutase and catalase prevented the downregulation of NET proteins and induction of ER stress signals produced by NE but not by tunicamycin or thapsigargin. The results indicate that the downregulation of membrane NET by NE is mediated by decreased N-glycosylation of NET proteins secondary to induction of ER stress pathways by NE-derived oxidative metabolites. Interventions involving the ER stress pathways may provide novel therapeutic strategies for the treatment of sympathetic dysfunction in heart failure.
Mao
Weike
W
Cardiology Unit, Department of Medicine, University of Rochester Medical Center, Rochester, New York 14642, USA.
Iwai
Chikao
C
Qin
Fuzhong
F
Liang
Chang-Seng
CS
D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics; D002374:Catalase / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D006031:Glycosylation; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D009638:Norepinephrine / Q000493:pharmacokinetics*; D050484:Norepinephrine Plasma Membrane Transport Proteins; D018384:Oxidative Stress / Q000187:drug effects*; D016716:PC12 Cells; D017510:Protein Folding; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013482:Superoxide Dismutase / Q000378:metabolism; D013566:Sympathomimetics / Q000493:pharmacokinetics*; D027981:Symporters / Q000378:metabolism*; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000235:genetics; D014316:Tritium; D015854:Up-Regulation / Q000187:drug effects; D000091342:Endoplasmic Reticulum Chaperone BiP
D022762:CCAAT-Enhancer-Binding Proteins; D002374:Catalase; D006360:Heat-Shock Proteins; D008024:Ligands; D018832:Molecular Chaperones; D009638:Norepinephrine; D050484:Norepinephrine Plasma Membrane Transport Proteins; D012333:RNA, Messenger; D013482:Superoxide Dismutase; D013566:Sympathomimetics; D027981:Symporters; D051742:Transcription Factor CHOP; D014157:Transcription Factors; D014316:Tritium; C497251:Ddit3 protein, rat; C051684:GRP78 protein, rat; D000091342:Endoplasmic Reticulum Chaperone BiP; C493226:Slc6a2 protein, rat