Premium
Autophagic flux alters hepatic iNOS protein accumulation, functional form, and localization (151.8)
Author(s) -
Loughran Patricia,
Sun Qian,
Scott Melanie,
Stolz Donna,
Mastroberardino Pier,
Friedman Peter,
Watkins Simon,
Billiar Timothy
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.151.8
Subject(s) - chemistry , nitric oxide synthase , nitric oxide , microbiology and biotechnology , autophagy , peroxisome , biochemistry , inflammation , biology , immunology , receptor , apoptosis , organic chemistry
The hepatic iNOS response to different stressors varies with the amount, duration, cell type and source of nitric oxide. Autophagy is known to be regulated by inducible nitric oxide synthase (iNOS) and nitric oxide (NO), but little is known about how autophagic flux impacts iNOS protein levels. Regulation of iNOS accumulation is important to understand as many metabolic processes are influenced by oxygenation/nitrosylation mediated by iNOS/NO during inflammation. We have also previously shown that iNOS binding to ezrin radizin moesin binding phosphoprotein 50 (EBP50) allows transport of iNOS to peroxisomes and then degradation via pexophagy. The aim of this study was to identify the role of alterations of autophagic flux on iNOS in hepatocytes after stimulation with cytokine mix (CM: TNF[500U/ml]; IL1[200U/ml]; IFN [100U/ml]). Rat hepatocytes were stimulated with CM +/‐ autophagic inhibitors for 8h. Cells were analyzed by Griess, immunoblot, and confocal microscopy to determine the localization, functional form and accumulationof iNOS. iNOS peroxisomal localization is maximal with the inclusion of 3MA and to a lesser degree the NO inhibitor, L‐NIO. This form of iNOS was determined by immunoblot to be monomeric. Accumulation of iNOS and its reactive products at the various stages of autophagic sequestration is of interest due to the shift of metabolic processes that rely on heme proteins susceptible to modification by oxygen and nitrogen radicals characteristic in surgical models generated in inflammation. Grant Funding Source : R37‐GM044100 awarded to TRB