Sex‐dependent Regulation of Mitochondrial Respiratory Function in Mouse Brain Microvessels by Peroxynitrite Decomposition Catalyst

Abstract

BackgroundPeroxynitrite (PN) is a strong oxidizing and nitrating molecule. PN is a potent inhibitor of mitochondrial respiration and promotes ischemia‐reperfusion injury following stroke. In isolated mouse brain mitochondria, we observed that PN donors inhibit mitochondrial respiratiory function whereas the PN decomposition catalyst, Fe (III) tetrakis (1‐methyl‐4‐pyridyl) porphyrin pentachlorideporphyrin pentachloride (FeTMPyP), enhances 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 mitochondrial respiration in the mouse BMVs.MethodsBMVs 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 37oC for 60 minutes. Oxygen consumption rates (OCR) were measured using the Agilent Seahorse XFe24 analyzer and various respiratory parameters were determined following Mitostress test.ResultsIn male BMVs, basal respiration, ATP production, and non‐mitochondrial respiration were not altered by FeTMPyP treatment. Contrary to our hypothesis, in male BMVs, PN decomposition catalyst decreased the mitochondrial maximal respiration by 24.6% (2.1 ± 0.4 vs 2.8 ± 0.3 picomoles of O2/min/µg protein;) whereas the spare respiratory capacity was reduced by 33.3% (1.2 ± 0.3 vs 1.8 ± 0.3 picomoles of O2/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 O2/min/µg protein) by PN decomposition catalyst in male BMVs. In contrast, in the female BMVs, the PN decomposition catalyst failed to alter 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 O2/min/µg protein) in the female BMVs (n=17 each, p<0.05).ConclusionBMVs 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 mostly contributes to extramitochondrial superoxide generation.