Nitric oxide (NO) is produced by endothelial NO synthase (eNOS) and plays a key role in maintaining vascular health and renal function. Diabetic levels of glucose promote oxidation of tetrahydrobiopterin (BH4), an essential eNOS cofactor, resulting in accumulation of dihydrobiopterin (BH2). BH4 insufficiency triggers a switch in the eNOS product from NO to superoxide, resulting in endothelial dysfunction (ED), a major diabetic complication that leads to blindness, amputations, kidney failure and death. We discovered that BH4 and BH2 exhibit equal binding affinity for eNOS and infer that the balance of these species is a major determinant of vascular health. Mitochondria (Mt) are hypothesized to provide the source of superoxide that initiates BH4 oxidation in diabetes, whereas BH2-bound (uncoupled) eNOS derived superoxide may sustain BH4 oxidation and cause ED. Notably, we showed that eNOS directly associates with Mt via a pentabasic peptide in the autoinhibitory domain of eNOS (residues 629-633 in the bovine isoform) and a proteinase K-cleavable site on the outer Mt membrane. We hypothesize that this protein- protein interaction is dynamic and contributes to the NO-mediated regulation of Mt activities. Localization at the outer membrane strategically places eNOS in proximity to the major source of cellular superoxide, emanating from the Mt inner membrane due to inefficiencies in electron transport. Owing to the diffusion- limited reaction of eNOS-derived NO with electron transport-derived superoxide, a gradient of peroxynitrite would arise at the interface of these two fluxes, at the intermembrane space in Mt. Notably, the rate of electron transport-generated superoxide is accelerated by hyperglycemia - accordingly, we hypothesize that in diabetic blood vessels peroxynitrite production by Mt would accelerate, increasing the oxidation of BH4, leading to superoxide-producing, BH2-bound, eNOS on Mt. Redistribution of this uncoupled eNOS from Mt to other subcellular loci would promote BH4 oxidation at non-Mt sites, disseminating the NO insufficiency.
Aim 1 of this research is to define the molecular basis for eNOS association with Mt, the consequences for NO production by eNOS and targets of eNOS-derived NO in Mt. Studies will rely on our development of strategies for the selective placement and displacement of Mt eNOS. We will employ engineered cell lines and a novel proteomic approach for unbiased identification of proteins and their specific Cys residues that undergo reversible S-nitrosylation. Preliminary experiments have already identified endogenous SNO- modified proteins in mitochondria from NOS-rich tissues - the functional consequences of these modifications remain to be established.
Aim 2 will test the hypothesis that mitochondria are the primary site of glucose and oxLDL-induced BH4 oxidation, resulting in suppressed NO signaling.
Aim 3 will evaluate N?- hydroxyarginine as a superoxide-dependent NO donor, for its ability to protect against BH4 oxidation, vascular lesion development and endothelial dysfunction in a murine genetic model of diabetes.
This aim i s a direct translation of our basic research and may provide for the selective delivery of NO to vascular sites where superoxide overproduction is greatest and hence. NO bioactivity is most compromised.
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