Carboxypeptidase regulated NO production and endothelial barrier

我们已经鉴定并克隆了存在于单核/巨噬细胞和内皮细胞中的两种羧肽酶，即羧基肽酶M （CPM）和D (CPD)，它们从肽或蛋白质中切割c端精氨酸（Arg）。我们还发现，酶促释放精氨酸可以调节一氧化氮（NO）的合成。由于损伤部位Arg的酶促释放和NO的产生在肺血管内皮屏障功能的调节中可能是至关重要的，基于我们的初步研究结果，我们将验证以下假设：细胞羧肽酶增加了Arg对NO合成酶的可用性，放大了NO合成酶和放大了NO的产生，这是一种自分泌和旁分泌信号，可以改变肺血管内皮的通透性。在Specific Aim 1中，我们将阐明细胞外CPM和细胞内CPD在调节单核/巨噬细胞和肺血管内皮细胞NO生成中的主要作用：(i)确定特定羧肽酶底物增加NO生成和羧肽酶抑制剂阻止NO生成的能力；（ii）研究羧肽酶对内皮细胞和单核/巨噬细胞摄取精氨酸的调节作用。在专项目标2中，我们将通过以下方式研究羧肽酶诱导NO生成对内皮屏障功能的功能影响：(i)测量在体外存在或不存在炎症介质或共培养单核细胞/巨噬细胞的情况下，羧肽酶底物和抑制剂对肺血管内皮细胞通透性的影响；（ii）测定羧肽酶底物和抑制剂对灌注大鼠肺模型NO生成和肺水肿形成的影响。在Specific Aim 3中，我们将定义肺血管内皮细胞和单核/巨噬细胞对炎症介质的反应中羧肽酶蛋白的调节，并确定负责这种调节的基因启动子区域。这些研究将为羧基肽酶在调节内皮和巨噬细胞诱导的NO生成中的作用以及NO在内皮屏障功能信号变化中的作用提供新的信息。

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.