Carboxypeptidase regulated NO production and endothelial barrier

羧肽酶调节 NO 产生和内皮屏障

• 批准号: 6584673
• 负责人: Randal A Skidgel
• 金额: $ 26.65万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6584673
• 关键词: aminoacid transport; arginine; autocrine; biological signal transduction; carboxypeptidase; enzyme activity; enzyme inhibitors; genetic promoter element; inflammation; laboratory mouse; laboratory rat; lung; macrophage; mixed tissue /cell culture; monocyte; nitric oxide; nitric oxide synthase; paracrine; vascular endothelium permeability

We have characterized and cloned two carboxypeptidases present in monocytes/macrophages and endothelial cells, carboxypeptidases M (CPM) and D (CPD), that cleave C-terminal arginine (Arg) from peptides or proteins. We have also found that enzymatic release of Arg can regulate the synthesis of nitric oxide (NO). As the enzymatic release of Arg and the production of NO at sites of injury may be critical in the regulation of pulmonary vascular endothelial barrier function, and based on our preliminary findings, we will test the following hypothesis: cellular carboxypeptidases increase the availability of Arg to NO synthase and amplify NO synthase and amplify NO production, which acts as an autocrine and paracrine signal to alter pulmonary vascular endothelial permeability. In Specific Aim 1, we will elucidate the role of predominantly extracellular CPM and of intracellular CPD in regulating NO production in monocytes/macrophages and pulmonary vascular endothelial cells by: (i) determining the ability of specific carboxypeptidase substrates to increase NO production and of carboxypeptidase inhibitors to prevent NO production and (ii) investigating the regulation by carboxypeptidases of uptake of Arg into endothelial cells and monocytes/macrophages. In Specific Aim 2, we will investigate the functional consequences of carboxypeptidase-induced NO production on endothelial barrier function by: (i) measuring the effects of carboxypeptidase substrates and inhibitors on permeability of pulmonary vascular endothelial cells in vitro in the presence or absence of inflammatory mediators or co-cultured monocytes/macrophages; (ii) determining the effects of carboxypeptidase substrates and inhibitors on NO production and pulmonary edema formation in a perfused rat lung model. In Specific Aim 3, we will define the regulation of carboxypeptidase protein in pulmonary vascular endothelial cell and monocytes/macrophages in response to inflammatory mediators and identify the gene promoter regions responsible for this regulation. These studies will provide novel information on the role of carboxypeptidases in regulating endothelial- and macrophage-induced NO production and the role of NO in signaling changes in endothelial barrier function.

我们表征并克隆了单核细胞/巨噬细胞和内皮细胞中存在的两种羧肽酶，即羧肽酶 M (CPM) 和 D (CPD)，它们可将 C 端精氨酸 (Arg) 从肽或蛋白质中裂解出来。我们还发现精氨酸的酶促释放可以调节一氧化氮（NO）的合成。由于损伤部位精氨酸的酶促释放和 NO 的产生可能对肺血管内皮屏障功能的调节至关重要，根据我们的初步研究结果，我们将检验以下假设：细胞羧肽酶增加 Arg 对 NO 合酶的可用性，并放大 NO 合酶和 NO 的产生，从而作为自分泌和旁分泌信号来改变肺血管 内皮通透性。在具体目标 1 中，我们将通过以下方式阐明主要细胞外 CPM 和细胞内 CPD 在调节单核细胞/巨噬细胞和肺血管内皮细胞中 NO 产生中的作用：（i）确定特定羧肽酶底物增加 NO 产生的能力和羧肽酶抑制剂阻止 NO 产生的能力，以及（ii）研究羧肽酶的调节作用 内皮细胞和单核细胞/巨噬细胞对 Arg 的摄取。在具体目标 2 中，我们将通过以下方式研究羧肽酶诱导的 NO 产生对内皮屏障功能的功能影响：（i）在存在或不存在炎症介质或共培养的单核细胞/巨噬细胞的情况下，在体外测量羧肽酶底物和抑制剂对肺血管内皮细胞通透性的影响； (ii)确定羧肽酶底物和抑制剂对灌注大鼠肺模型中NO产生和肺水肿形成的影响。在具体目标 3 中，我们将定义肺血管内皮细胞和单核细胞/巨噬细胞中羧肽酶蛋白响应炎症介质的调节，并确定负责这种调节的基因启动子区域。这些研究将提供关于羧肽酶在调节内皮细胞和巨噬细胞诱导的 NO 产生中的作用以及 NO 在内皮屏障功能信号变化中的作用的新信息。