3-nitrotyrosine and Cadaver

Recently we have shown that diabetes-induced retinal neurodegeneration positively correlates with oxidative stress and peroxynitrite. Studies also show that peroxynitrite impairs nerve growth factor (NGF) survival signaling in sensory neurons. However, the causal role of peroxynitrite and the impact of tyrosine nitration on diabetes-induced retinal neurodegeneration and NGF survival signaling have not been elucidated.. Expression of NGF and its receptors was examined in retinas from human and streptozotocin-induced diabetic rats and retinal ganglion cells (RGCs). Diabetic animals were treated with FeTPPS (15 mg x kg(-1) x day(-1) ip), which catalytically decomposes peroxynitrite to nitrate. After 4 weeks of diabetes, retinal cell death was determined by TUNEL assay. Lipid peroxidation and nitrotyrosine were determined using MDA assay, immunofluorescence, and Slot-Blot analysis. Expression of NGF and its receptors was determined by enzyme-linked immunosorbent assay (ELISA), real-time PCR, immunoprecipitation, and Western blot analyses.. Analyses of retinal neuronal death and NGF showed ninefold and twofold increases, respectively, in diabetic retinas compared with controls. Diabetes also induced increases in lipid peroxidation, nitrotyrosine, and the pro-apoptotic p75(NTR) receptor in human and rat retinas. These effects were associated with tyrosine nitration of the pro-survival TrkA receptor, resulting in diminished phosphorylation of TrkA and its downstream target, Akt. Furthermore, peroxynitrite induced neuronal death, TrkA nitration, and activation of p38 mitogen-activated protein kinase (MAPK) in RGCs, even in the presence of exogenous NGF. FeTPPS prevented tyrosine nitration, restored NGF survival signal, and prevented neuronal death in vitro and in vivo.. Together, these data suggest that diabetes-induced peroxynitrite impairs NGF neuronal survival by nitrating TrkA receptor and enhancing p75(NTR) expression.

Topics: Animals; Cadaver; Cell Death; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Humans; Lipid Peroxidation; Middle Aged; Nerve Degeneration; Nerve Growth Factor; Optic Nerve; Peroxynitrous Acid; Rats; Reference Values; Retinal Ganglion Cells; RNA, Messenger; Streptozocin; Tyrosine

Altered nitric oxide (NO) metabolism has been shown to contribute to ischemia-reperfusion (IR) injury in animal models. However, similar studies have not been performed in human liver transplantation (LT). In this study, we examined nitrate, nitrite, and nitrosothiols (NOx), NO synthases (endothelial [constitutive] nitric oxide synthase [eNOS] and inducible nitric oxide synthase [iNOS]), and nitrotyrosine in early IR injury after human LT.. Paired biopsies were obtained from nine donor livers before cold ischemia (retrieval biopsy) and after reimplantation (reperfusion biopsy). Sections were graded for reperfusion injury using the Suzuki score. NO was detected by chemiluminescence after reduction of NOx. Expression of eNOS and iNOS was by Western blot and reverse transcriptase polymerase chain reaction and peroxynitrite by immunodetection of 3-nitrotyrosine.. Reperfusion biopsies showed histologic evidence of injury (median Suzuki score: retrieval 2, reperfusion 6, P=0.008) and neutrophil infiltration. NOx was reduced after reperfusion from 5.41 microM/100 mg (median, range 2.17-13.39 microM) to 3.51 microM (1.45-5.66 microM, P=0.05). eNOS protein was reduced after reperfusion from 0.6 units (median, range 0.45-1 unit) in retrieval biopsies to 0.39 units in reperfusion biopsies (range 0.2-0.79 units, P=0.007). There was no change in eNOS or iNOS mRNA expression or iNOS protein. Western blotting showed increased nitrotyrosine formation after reperfusion, median 0.42 (range 0.16-0.87) units in retrieval biopsies and 0.68 (0.29-1.06) units in reperfusion samples (P=0.007) and localized to periportal regions.. iNOS protein may not contribute to early reperfusion injury during human LT. However, reduced NO bioavailability caused by reduced eNOS may contribute significantly to damage at this time point.

Topics: Adolescent; Adult; Aged; Cadaver; Female; Humans; Immunohistochemistry; Liver Transplantation; Male; Middle Aged; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Reoperation; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Donors; Tyrosine

An HPLC method was used for quantification of 3-nitrotyrosine (3-NT) in human postmortem brain tissue. A peak with similar retention time to 3-NT was detected in brain tissue from patients with Parkinson's disease, Huntington's chorea, multiple system atrophy, and Alzheimer's disease but not in control tissue. The peak was lost on reduction with dithionite, a criterion often used to identify 3-NT. Tissue from the same neurodegenerative diseases was analysed by HPLC using a photodiode array detector in series with an amperometric electrochemical detector, but the peak was found not to be 3-NT. The absorbance spectrum, fragmentation pattern on mass spectroscopy, and electrochemical profile of this peak do not match authentic 3-NT. A search of the mass spectroscopy databases failed to reveal its identity. The presence of this closely eluting, dithionite-reducible peak could confound analysis of human tissues for 3-NT. In vitro experiments showed that high concentrations of peroxynitrite were needed to achieve detectable levels of 3-NT in human brain tissue.

Topics: Artifacts; Brain; Cadaver; Chromatography, High Pressure Liquid; Humans; Nerve Degeneration; Nervous System Diseases; Reference Values; Tyrosine