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)，它们可以从多肽或蛋白质中裂解C端精氨酸(Arg)。我们还发现，酶促释放Arg可以调节一氧化氮(NO)的合成。鉴于损伤部位精氨酸的酶促释放和NO的产生可能在肺血管内皮细胞屏障功能的调节中起关键作用，在我们初步研究结果的基础上，我们将检验以下假设：细胞羧肽酶增加Arg对NO合成酶的利用率，并放大NO合成酶和放大NO的产生，作为自分泌和旁分泌信号改变肺血管内皮细胞的通透性。在特定的目标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产生NO方面的作用，方法是：(I)测定特定的羧肽酶底物增加NO产生的能力和羧肽酶抑制剂阻止NO产生的能力；(Ii)研究羧肽酶对内皮细胞和单核/巨噬细胞摄取Arg的调节。在特定的目标2中，我们将通过(I)在有或没有炎症介质或单核/巨噬细胞共同培养的情况下，通过测定羧肽酶底物和抑制剂对体外培养的肺血管内皮细胞通透性的影响来研究羧肽酶诱导的NO产生对内皮屏障功能的影响；(Ii)在大鼠肺灌流模型中测定羧肽酶底物和抑制剂对NO产生和肺水肿形成的影响。在特定的目标3中，我们将确定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，并确定负责这一调节的基因启动子区域。这些研究将为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.