Effect of peroxynitrite scavenger on brain microvascular nitrotyrosination and S‐nitrosylation

Abstract

ObjectivePeroxynitrite, generated from nitric oxide and superoxide, plays a pathological role in both ischemic and neurodegenerative brain injury. Free radical mediated microvascular dysfunction is a key initiator of cerebrovascular and neurological pathologies. Thus, understanding the acute effects of peroxynitrite on brain microvessels will help identify potential therapeutic targets. The objective of the study was to investigate the impact of peroxynitrite scavenger on the nitrotyrosination and S‐nitrosylation of brain microvessels.MethodsBrain microvessels were isolated from the brains of 12‐week‐old female C57bl6 (WT, n= 4) mice by a combination of filtration (40μm – 300μm) and gradient centrifugation. Microvessels were treated with 2.5 μmol/L of peroxynitrite scavenger (FeTMPyP, Fe (III)tetrakis (1‐methyl‐4‐pyridyl) porphyrin pentachlorideporphyrin pentachloride) for 60 minutes at 37°C. Subsequently, western Blots were then performed to detect the nitrotyrosine residues in the protein lysates from microvessels. In addition, we determined the protein S‐nitrosylation in lysates by biotin‐switch method followed by immunoblotting. Nitrotyrosine residue is an indicator of peroxynitrite generation and protein‐S‐nitrosylation is an indirect measure for nitric oxide production.ResultsAcute treatment with FeTMPyP decreased nitrotyrosination of proteins from microvessels (3.16±0.63 vs 19.79±10.82, p=0.03, n=4 mice) in WT mice. In addition, 2.5 μM of FeTMPyP treatment decreased the protein S‐nitrosylation by 40% (p≤0.05).ConclusionsBrain microvessels form peroxynitrite at basal levels. Surprisingly, peroxynitrite inhibition decreases protein S‐nitrosylation in the brain microvessels.Support or Funding InformationSupport: Undergraduate Summer Research Fellowship by American Physiological Society (Albuck); and National Institute of Health: National Institute of General Medical Sciences and National Institute of Neurological Disorders and Stroke (Katakam: R01NS094834) and National Institute on Aging (Mostany: AG047296).