Carboxypeptidase regulated NO production and endothelial barrier

我们已经表征并克隆了存在于单核细胞/巨噬细胞和内皮细胞中的两种羧肽酶，羧肽酶M（CPM）和D（CPD），其从肽或蛋白质切割C-末端精氨酸（Arg）。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Arg的酶促释放和NO的产生可能在肺血管内皮屏障功能的调节中至关重要，基于我们的初步发现，我们将检验以下假设：细胞羧肽酶增加Arg对NO合酶的可用性并扩增NO合酶和扩增NO产生，其作为自分泌和旁分泌信号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防止NO产生的能力，和（ii）研究羧肽酶对内皮细胞和单核细胞/巨噬细胞摄取Arg的调节。在具体目标2中，我们将通过以下方法研究羧肽酶诱导的NO产生对内皮屏障功能的功能后果：（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，测量羧肽酶底物和抑制剂对体外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/巨噬细胞中的羧肽酶蛋白对炎症介质的调节，并确定负责这种调节的基因启动子区域。这些研究将提供新的信息羧肽酶在调节内皮细胞和巨噬细胞诱导的NO产生的作用和NO在内皮屏障功能的信号变化的作用。

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.