Abstract WP245: Sex-dependent Regulation Of Mitochondrial Respiratory Function In Mouse Brain Microvessels By Peroxynitrite Decomposition Catalyst

Abstract

Background: Peroxynitrite (PN) is a strong oxidizing and nitrating molecule. PN is a potent inhibitor of mitochondrial respiration and mediates the ischemia-reperfusion injury following stroke. In isolated mouse brain mitochondria, we observed that PN donors inhibit mitochondrial respiratiory function whereas PN decomposition catalyst, Fe (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachlorideporphyrin pentachloride (FeTMPyP), enhanced mitochondrial state III and state IVo mitochondrial respiration. In addition, we demonstrated that mitochondrial respiration is the primary contributor of cellular energy in brain microvessels (BMVs). The present study tested the hypothesis that FeTMPyP negatively regulates the mitochondrial respiration in the mouse BMVs. Methods: BMVs were isolated from male and female mice (C57Bl/6, 2-4 months) using a combination of filters with pore sizes of 300μm and 40μm followed by gradient centrifugation. BMVs were treated with FeTMPyP at 37 o C for 60 minutes. Oxygen consumption rates (OCR) were measured using the Agilent Seahorse XFe24 analyzer and various respiratory parameters were determined following Mitostress test. Results: In the male BMVs, basal respiration, ATP production, and non-mitochondrial respiration were not altered by FeTMPyP treatment. Contrary to our hypothesis, in the male BMVs, PN scavenger decreased the mitochondrial maximal respiration by 24.6% (2.1 ± 0.4 vs 2.8 ± 0.3 picomoles of O 2 /min/μg protein;) whereas the spare respiratory capacity was reduced by 33.3% (1.2 ± 0.3 vs 1.8 ± 0.3 picomoles of O 2 /min/μg protein) (n=17 each, p<0.05). Proton leak was elevated by 70% (0.7 ± 0.1 vs 0.4 ± 0.1 picomoles of O 2 /min/μg protein ) by PN scavenger in male BMVs. In contrast, in the female BMVs, the PN scavenger failed to alter the mitochondrial respiratory parameters (n=15 each, p=NS). Interestingly, FeTMPyP increased non-mitochondrial respiration by 63.8% (0.95 ± 0.2 vs 0.58 ± 0.1 picomoles of O 2 /min/μg protein) in the female BMVs (n=17 each, p<0.05). Conclusion: BMVs display sex-dependent respenses to endogenous PN. Notably, in female mouse BMVs, PN appears to act as an antioxidant as PN inhibited the non-mitochondrial respiration which contributes to extramitochondrial superoxide generation.