Dikalov Sergey I, Dikalova Anna E, Kirilyuk Igor A
Vanderbilt University Medical Center, Nashville, TN, United States.
N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Novosibirsk, Russia.
Front Cardiovasc Med. 2022 Jul 28;9:942736. doi: 10.3389/fcvm.2022.942736. eCollection 2022.
Superoxide radical plays an important role in redox cell signaling and physiological processes; however, overproduction of superoxide or insufficient activity of antioxidants leads to oxidative stress and contributes to the development of pathological conditions such as endothelial dysfunction and hypertension. Meanwhile, the studies of superoxide in biological systems represent unique challenges associated with short lifetime of superoxide, insufficient reactivity of the superoxide probes, and lack of site-specific detection of superoxide. In this work we have developed N-and deuterium-enriched spin probe N-CAT1H for high sensitivity and site-specific detection of extracellular superoxide. We have tested simultaneous tracking of extracellular superoxide by N-CAT1H and intramitochondrial superoxide by conventional N-containing spin probe mitoTEMPO-H in immune cells isolated from spleen, splenocytes, under basal conditions or stimulated with inflammatory cytokines IL-17A and TNFα, NADPH oxidase activator PMA, or treated with inhibitors of mitochondrial complex I rotenone or complex III antimycin A. N-CAT1H provides two-fold increase in sensitivity and improves detection since EPR spectrum of N-CAT1 nitroxide does not overlap with biological radicals. Furthermore, concurrent use of cell impermeable N-CAT1H and mitochondria-targeted N-mitoTEMPO-H allows simultaneous detection of extracellular and mitochondrial superoxide. Analysis of IL-17A- and TNFα-induced superoxide showed parallel increase in N-CAT1 and N-mitoTEMPO signals suggesting coupling between phagocytic NADPH oxidase and mitochondria. The interplay between mitochondrial superoxide production and activity of phagocytic NADPH oxidase was further investigated in splenocytes isolated from Sham and angiotensin II infused C57Bl/6J and Nox2KO mice. Angiotensin II infusion in wild-type mice increased the extracellular basal splenocyte superoxide which was further enhanced by complex III inhibitor antimycin A, mitochondrial uncoupling agent CCCP and NADPH oxidase activator PMA. Nox2 depletion attenuated angiotensin II mediated stimulation and inhibited both extracellular and mitochondrial PMA-induced superoxide production. These data indicate that splenocytes isolated from hypertensive angiotensin II-infused mice are "primed" for enhanced superoxide production from both phagocytic NADPH oxidase and mitochondria. Our data demonstrate that novel N-CAT1H provides high sensitivity superoxide measurements and combination with mitoTEMPO-H allows independent and simultaneous detection of extracellular and mitochondrial superoxide. We suggest that this new approach can be used to study the site-specific superoxide production and analysis of important sources of oxidative stress in cardiovascular conditions.
超氧阴离子自由基在氧化还原细胞信号传导和生理过程中起着重要作用；然而，超氧阴离子的过量产生或抗氧化剂活性不足会导致氧化应激，并促使诸如内皮功能障碍和高血压等病理状况的发展。同时，在生物系统中对超氧阴离子的研究面临着独特的挑战，这些挑战与超氧阴离子的短寿命、超氧阴离子探针的反应活性不足以及缺乏超氧阴离子的位点特异性检测有关。在这项工作中，我们开发了富含氮和氘的自旋探针N-CAT1H，用于细胞外超氧阴离子的高灵敏度和位点特异性检测。我们测试了在基础条件下或用炎性细胞因子IL-17A和TNFα、NADPH氧化酶激活剂PMA刺激后，或用线粒体复合物I鱼藤酮或复合物III抗霉素A抑制剂处理后，从脾脏分离的免疫细胞（脾细胞）中，用N-CAT1H同时追踪细胞外超氧阴离子和用传统的含氮自旋探针mitoTEMPO-H追踪线粒体内超氧阴离子的情况。N-CAT1H的灵敏度提高了两倍，并改善了检测效果，因为N-CAT1氮氧化物的电子顺磁共振谱不与生物自由基重叠。此外，同时使用细胞不可渗透的N-CAT1H和线粒体靶向的N-mitoTEMPO-H可以同时检测细胞外和线粒体内的超氧阴离子。对IL-17A和TNFα诱导的超氧阴离子的分析表明，N-CAT1和N-mitoTEMPO信号平行增加，这表明吞噬性NADPH氧化酶与线粒体之间存在偶联。在从假手术组和输注血管紧张素II的C57Bl/6J和Nox基因敲除小鼠分离的脾细胞中，进一步研究了线粒体内超氧阴离子产生与吞噬性NADPH氧化酶活性之间的相互作用。在野生型小鼠中输注血管紧张素II会增加脾细胞外的基础超氧阴离子水平，复合物III抑制剂抗霉素A、线粒体解偶联剂CCCP和NADPH氧化酶激活剂PMA会进一步增强这种超氧阴离子水平。Nox2基因缺失减弱了血管紧张素II介导的刺激，并抑制了细胞外和线粒体内PMA诱导的超氧阴离子产生。这些数据表明，从输注血管紧张素II的高血压小鼠中分离的脾细胞“准备好”增强吞噬性NADPH氧化酶和线粒体产生超氧阴离子的能力。我们的数据表明，新型N-CAT1H提供了高灵敏度的超氧阴离子测量，并且与mitoTEMPO-H结合可以独立且同时检测细胞外和线粒体内的超氧阴离子。我们建议这种新方法可用于研究位点特异性超氧阴离子的产生以及分析心血管疾病中氧化应激的重要来源。
