A novel mechanism of NO synthase uncoupling involving isolevuglandin adduction

Abstract

We have previously shown that hypertension leads to modification of self‐proteins in antigen presenting cells by isolevuglandin (isoLG) lipid oxidation products. These modified proteins seem to be immunogenic. IsoLG‐modified peptides are presented in major histocompatibility complex (MHC) class 1 and drive CD8+ T cell proliferation. To understand alterations in the peptidome presented by MHC1 in hypertension, we created transgenic mice expressing a truncated form of MHC1 Kb lacking the transmembrane domain, driven by the CD11c promoter. The lack of a transmembrane domain causes MHC1 to be shed into the media when DCs are cultured. We treated these mice with either 2 weeks sham or angiotensin (ang) II infusion, isolated splenic DCs and placed these in culture for 72 hours. The shed soluble MHC1 Kb was harvested and associated peptides were analyzed by mass spectroscopy. One peptide identified in DCs from hypertensive mice was an identical match for a portion of the neuronal nitric oxide synthase (nNOS) containing an isoLG adduct at lysine 225. Because this lysine is positioned between the heme and arginine binding sites, we hypothesized that its modification would disrupt normal NO production and instead lead to superoxide production, i.e. NOS uncoupling. In subsequent studies, we found that nNOS expression is increased 5‐fold in DCs of ang II‐treated compared to sham‐treated mice (n = 6). Importantly, ang II infusion increased superoxide production of DCs from 37±3 to 111±5 pmol/mg protein (mean ± SE, p = 0.0001), and this was completely blocked by treatment of the cells with the selective nNOS inhibitor L‐vinyl NIO, suggesting that nNOS is a source of superoxide in DCs in hypertension. Next we expressed wild type nNOS and a mutant nNOS in which lysine 225 was mutated to an alanine (nNOS K225A) in human epithelial kidney (HEK) cells. Untransfected HEK cells failed to produce NO, as measured by electron spin resonance and the spin trap Fe[DETC]2. HEK cells transfected with either the WT or K225A nNOS produced 152 ± 5 vs 156 ± 6 arbitrary units of NO/mg protein (p = NS). However, exposure of the transfected HEK cells to tertbutyl hydroperoxide, which promotes isoLG formation, reduced NO production by cells expressing the wild‐type enzyme to 86 ± 6 units/mg protein, while having no effect on cells transfected with the mutant K225A (p = 0.003, n = 5 – 7), further implicating lysine 225 as a site of oxidative modification in nNOS. In summary, hypertension increases nNOS expression in DCs and this enzyme is altered by isoLG adduction at lysine 225. This leads to an increase in superoxide and reduced NO production in these immune cells. We conclude that isoLG adduction represents a novel mechanism of NOS uncoupling and a source of reactive oxygen species in dendritic cells.Support or Funding InformationAmerican Heart Association grants POST290900 and SFRN204200, and National Institutes of Health grants K01HL130497, R01HL125865, R01HL039006 and P01HL129941This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.