氨基甲酰磷酸合成酶1(CPS1)是最丰富的肝细胞线粒体基质蛋白。低渗应激增加了分离的小鼠肝细胞中CPS1的释放,而没有细胞死亡。我们假设低渗透过程中CPS1释放的增加是选择性的,并且与线粒体形态的改变有关。评估离体和体内模型。用等渗或低渗(35mosM)缓冲液攻击小鼠肝细胞和肝脏。小鼠腹膜内注射水(10%体重),有或没有抗利尿剂。使用差速离心分离线粒体和胞质部分,然后通过免疫印迹分析以评估四种线粒体蛋白的亚细胞再分布:CPS1,鸟氨酸转碳淀粉酶(OTC),吡咯啉-5-羧酸还原酶1(PYCR1),和细胞色素c。使用电子显微镜检查线粒体形态改变。全肝或肝细胞的低渗处理导致优先或增加的线粒体释放,分别,CPS1与两种线粒体基质蛋白(OTC/PYCR1)和膜间隙蛋白相比,细胞色素c。肝细胞中使用星形孢菌素的线粒体凋亡诱导的通道开放导致优先的CPS1和细胞色素c释放。CPS1选择性变化伴随着超微结构线粒体形态的戏剧性改变。在老鼠身上,低渗透/低钠血症导致肝脏血管充血增加,胆汁中CPS1增加,但血液中CPS1不增加,再加上线粒体结构改变。相比之下,与低渗条件相比,血管内体积的等渗增加导致线粒体大小减小,胆汁CPS1的变化有限,并且没有低渗透相关的组织学改变.一起来看,肝细胞CPS1响应低渗透/低钠血症而选择性释放,并提供线粒体损伤的独特生物标志物。新的和注意的暴露孤立的小鼠肝脏,原代培养肝细胞,或处于低渗透/低钠血症状态的小鼠会引起明显的线粒体形状改变,并伴随着线粒体基质蛋白CPS1的优先释放,CPS1是一种尿素循环酶。相比之下,膜间隙蛋白,细胞色素c,和另外两种基质蛋白,包括尿素循环酶鸟氨酸转碳淀粉酶,保留优先保留在线粒体中。因此,肝细胞CPS1表现出独特的线粒体应激反应区室化,是线粒体低渗/低钠损伤的敏感传感器。
Carbamoyl phosphate synthetase 1 (CPS1) is the most abundant hepatocyte mitochondrial matrix protein. Hypoosmotic stress increases CPS1 release in isolated mouse hepatocytes without cell death. We hypothesized that increased CPS1 release during hypoosmosis is selective and associates with altered mitochondrial morphology. Both ex vivo and in vivo models were assessed. Mouse hepatocytes and livers were challenged with isotonic or hypoosmotic (35 mosM) buffer. Mice were injected intraperitoneally with water (10% body weight) with or without an antidiuretic. Mitochondrial and cytosolic fractions were isolated using differential centrifugation, then analyzed by immunoblotting to assess subcellular redistribution of four mitochondrial proteins: CPS1, ornithine transcarbamylase (OTC), pyrroline-5-carboxylate reductase 1 (PYCR1), and cytochrome c. Mitochondrial morphology alterations were examined using electron microscopy. Hypoosmotic treatment of whole livers or hepatocytes led to preferential or increased mitochondrial release, respectively, of CPS1 as compared with two mitochondrial matrix proteins (OTC/PYCR1) and with the intermembrane space protein, cytochrome c. Mitochondrial apoptosis-induced channel opening using staurosporine in hepatocytes led to preferential CPS1 and cytochrome c release. The CPS1-selective changes were accompanied by dramatic alterations in ultrastructural mitochondrial morphology. In mice, hypoosmosis/hyponatremia led to increased liver vascular congestion and increased CPS1 in bile but not blood, coupled with mitochondrial structural alterations. In contrast, isotonic increase of intravascular volume led to a decrease in mitochondrial size with limited change in bile CPS1 compared with hypoosmotic conditions and absence of the hypoosmosis-associated histological alterations. Taken together, hepatocyte CPS1 is selectively released in response to hypoosmosis/hyponatremia and provides a unique biomarker of mitochondrial injury.NEW & NOTEWORTHY Exposure of isolated mouse livers, primary cultured hepatocytes, or mice to hypoosmosis/hyponatremia conditions induces significant mitochondrial shape alterations accompanied by preferential release of the mitochondrial matrix protein CPS1, a urea cycle enzyme. In contrast, the intermembrane space protein, cytochrome c, and two other matrix proteins, including the urea cycle enzyme ornithine transcarbamylase, remain preferentially retained in mitochondria. Therefore, hepatocyte CPS1 manifests unique mitochondrial stress response compartmentalization and is a sensitive sensor of mitochondrial hypoosmotic/hyponatremic injury.