Department of Physiology, Medical College of Georgia, Augusta, Georgia, USA.
Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, USA.
Redox Biol. 2019 Oct;27:101191. doi: 10.1016/j.redox.2019.101191. Epub 2019 Apr 16.
Hv1 is a voltage-gated proton channel highly expressed in immune cells where, it acts to maintain NAD(P)H oxidase activity during the respiratory burst. We have recently reported that Hv1 is expressed in cells of the medullary thick ascending limb (mTAL) of the kidney and is critical to augment reactive oxygen species (ROS) production by this segment. While Hv1 is associated with NOX2 mediated ROS production in immune cells, the source of the Hv1 dependent ROS in mTAL remains unknown. In the current study, the rate of ROS formation was quantified in freshly isolated mTAL using dihydroethidium and ethidium fluorescence. Hv1 dependent ROS production was stimulated by increasing bath osmolality and ammonium chloride (NHCl) loading. Loss of either p67phox or NOX4 did not abolish the formation of ROS in mTAL. Hv1 was localized to mitochondria within mTAL, and the mitochondrial superoxide scavenger mitoTEMPOL reduced ROS formation. Rotenone significantly increased ROS formation and decreased mitochondrial membrane potential in mTAL from wild-type rats, while treatment with this inhibitor decreased ROS formation and increased mitochondrial membrane potential in mTAL from Hv1 mutant rats. These data indicate that NADPH oxidase is not the primary source of Hv1 dependent ROS production in mTAL. Rather Hv1 localizes to the mitochondria in mTAL and modulates the formation of ROS by complex I. These data provide a potential explanation for the effects of Hv1 on ROS production in cells independent of its contribution to maintenance of cell membrane potential and intracellular pH.
Hv1 是一种电压门控质子通道，在免疫细胞中高度表达，在呼吸爆发期间，它作用于维持 NAD(P)H 氧化酶的活性。我们最近报道 Hv1 在肾脏髓质升支粗段（mTAL）的细胞中表达，并且对于增强该节段的活性氧物种（ROS）的产生至关重要。虽然 Hv1 与免疫细胞中 NOX2 介导的 ROS 产生有关，但 mTAL 中依赖 Hv1 的 ROS 的来源仍不清楚。在当前的研究中，使用二氢乙啶和 ethidium 荧光法在新鲜分离的 mTAL 中定量 ROS 的形成速率。通过增加浴渗透压和氯化铵（NHCl）加载来刺激 Hv1 依赖性 ROS 的产生。p67phox 或 NOX4 的缺失并没有消除 mTAL 中 ROS 的形成。Hv1 位于 mTAL 内的线粒体中，线粒体超氧化物清除剂 mitoTEMPOL 减少了 ROS 的形成。鱼藤酮显著增加了野生型大鼠 mTAL 中的 ROS 形成并降低了线粒体膜电位，而用该抑制剂处理降低了 Hv1 突变型大鼠 mTAL 中的 ROS 形成并增加了线粒体膜电位。这些数据表明，NADPH 氧化酶不是 mTAL 中 Hv1 依赖性 ROS 产生的主要来源。相反，Hv1 在 mTAL 中定位于线粒体，并通过复合物 I 调节 ROS 的形成。这些数据为 Hv1 对细胞中 ROS 产生的影响提供了一个潜在的解释，而与其对维持细胞膜电位和细胞内 pH 的贡献无关。
