We hypothesized that NO^• is generated in isolated cardiac mitochondria as the source for ONOO⁻ production during oxidative stress. We monitored generation of ONOO⁻ from guinea pig isolated cardiac mitochondria subjected to excess Ca²⁺ uptake before adding succinate and determined if ONOO⁻ production was dependent on a nitric oxide synthase (NOS) located in cardiac mitochondria (mtNOS). Mitochondria were suspended in experimental buffer at pH 7.15, and treated with CaCl₂ and then the complex II substrate Na-succinate, followed by menadione, a quinone redox cycler, to generate O₂^•−. L-tyrosine was added to the mitochondrial suspension where it is oxidized by ONOO⁻ to form dityrosine (diTyr) in proportion to the ONOO⁻ present. We found that exposing mitochondria to excess CaCl₂ before succinate resulted in an increase in diTyr and amplex red fluorescence (H₂O₂) signals, indicating that mitochondrial oxidant stress, induced by elevated mtCa²⁺ and succinate, increased mitochondrial ONOO⁻ production via NO^• and O₂^•−. Changes in mitochondrial ONOO⁻ production dependent on NOS were evidenced by using NOS inhibitors L-NAME/L-NNA, TEMPOL, a superoxide dismutase (SOD) mimetic, and PTIO, a potent global NO^• scavenger. L-NAME and L-NNA decreased succinate and menadione-mediated ONOO⁻ production, PTIO decreased production of ONOO⁻, and TEMPOL decreased ONOO⁻ levels by converting more O₂^•− to H₂O₂. Electron microscopy showed immuno-gold labeled iNOS and nNOS in mitochondria isolated from cardiomyocytes and heart tissue. Western blots demonstrated iNOS and nNOS bands in total heart tissue, bands for both iNOS and nNOS in β-tubulin-free non-purified (crude) mitochondrial preparations, and a prominent iNOS band, but no nNOS band, in purified (Golgi and ER-free) mitochondria. Prior treatment of guinea pigs with lipopolysacharride (LPS) enhanced expression of iNOS in liver mitochondria but not in heart mitochondria. Our results indicate that release of ONOO⁻ into the buffer is dependent both on O₂^•− released from mitochondria and NO^• derived from a mtCa²⁺-inducible nNOS isoform, possibly attached to mitochondria, and a mtNOS isoform like iNOS that is non-inducible.

我们假设 NO ^•是在分离的心脏线粒体中产生的，作为氧化应激期间ONOO ^-产生的来源。我们在添加琥珀酸之前监测了来自接受过量 Ca ²⁺摄取的豚鼠分离的心脏线粒体的 ONOO ^{- 的}生成，并确定 ONOO ^{- 的}产生是否依赖于位于心脏线粒体 (mtNOS) 中的一氧化氮合酶 (NOS)。将线粒体悬浮在 pH 7.15 的实验缓冲液中，用 CaCl ₂处理，然后用复合物 II 底物 Na-succinate，接着用甲萘醌（一种醌氧化还原循环仪）处理以生成 O ₂ ^•-. L-酪氨酸被添加到线粒体悬浮液中，在那里它被 ONOO ^-氧化以形成与 ONOO ^-成比例的二酪氨酸（diTyr）。我们发现，在琥珀酸之前将线粒体暴露于过量的 CaCl ₂导致 diTyr 和复合红色荧光 (H ₂ O ₂ ) 信号增加，表明由升高的 mtCa ²⁺和琥珀酸诱导的线粒体氧化应激通过以下方式增加了线粒体 ONOO ^{- 的}产生NO ^•和O ₂ ^•-。线粒体 ONOO 的变化^-通过使用 NOS 抑制剂 L-NAME/L-NNA、TEMPOL（一种超氧化物歧化酶 (SOD) 模拟物）和 PTIO（一种有效的全球 NO ^•清除剂）证明了依赖于 NOS 的生产。L-NAME和L-NNA琥珀酸盐和甲萘醌-介导的降低ONOO ^-生产，PTIO产量下降的ONOO ^-和TEMPOL ONOO降低^-通过将多个O级别₂ ^{• -}至H ₂ ö ₂. 电子显微镜显示从心肌细胞和心脏组织分离的线粒体中的免疫金标记的 iNOS 和 nNOS。蛋白质印迹证明了总心脏组织中的 iNOS 和 nNOS 条带，不含 β-微管蛋白的未纯化（粗制）线粒体制剂中的 iNOS 和 nNOS 条带，以及纯化的（高尔基体和 ER -免费）线粒体。预先用脂多糖 (LPS) 处理豚鼠后，iNOS 在肝线粒体中的表达增强，但在心脏线粒体中没有。我们的结果表明，ONOO ⁻释放到缓冲液中取决于线粒体释放的O ₂ ^•−和来自 mtCa ^{2+ 的}NO ^•- 诱导型 nNOS 异构体，可能附着在线粒体上，以及非诱导型 iNOS 等 mtNOS 异构体。