NOVEL ANGIOTENSIN II-FORMING PATHWAY IN THE HUMAN HEART

我们已经证明了血管紧张素H受体的存在， 心肌、交感神经和人体心脏的传导系统。 血管紧张素II在人心脏中具有正性肌力作用。在 人心室的匀浆，主要（>75%）酶 血管紧张素I向血管紧张素II的转化是由丝氨酸蛋白酶产生的， 不受血管紧张素转化酶（ACE）抑制剂的抑制。我们有 纯化并克隆了该蛋白酶。从结构上讲，这种酶属于 胰凝乳蛋白酶样蛋白酶的糜酶组， 称为人心脏糜酶（hHC）。与胰凝乳蛋白酶不同， 事实上，其他糜酶，hHC是最特异和有效的血管紧张素II- 形成酶描述。 使用心脏小梁的初步研究显示， 人心脏中血管紧张素II形成的功能途径 ACE通路的一部分因为高水平的免疫反应性hHC 与肌细胞周围的细胞外基质相关，我们 推测心脏血管紧张素II形成的ACE非依赖性途径 确实依赖于hHC。 我们建议开发特异性底物和抗体为基础的抑制剂 的hHC，以明确确定hHC是主要的功能性血管紧张素II- 在人体心脏中形成酶。这些抑制剂将用于显示 人心脏小梁中血管紧张素I向血管紧张素II的主要转化， 以及由Ang I引起的反应是由于hHC。 在平行研究中，我们计划将hHC以天然形式结晶， 以抑制剂复合形式研究其独特的结构 hHC的决定因素负责其高底物特异性。研究 也计划在功能上定位延伸的底物结合位点 的hHC使用血管紧张素I类似物。在人类心脏中，免疫反应性hHC是 储存在心脏肥大细胞颗粒中。为了研究hHC的加工过程，我们 提出开发表达hHC的人心脏肥大细胞系。 该细胞系还将用于研究顺式作用元件， 反式作用因子，只允许肥大细胞的一个子集表达 hHC。 人类心脏中hHC的存在表明心脏血管紧张素II 在治疗高血压期间， 血管紧张素转换酶抑制剂导致充血性心力衰竭的实现 这些研究的目标最终将帮助我们了解 在正常和衰竭心脏中心脏血管紧张素II形成的结果， 并将提高我们对心力衰竭及其治疗的理解。

We have shown the presence of angiotensin (Ang) H receptors in the myocardium, sympathetic nerves and conduction system of the human heart. Ang II elicits a positive inotropic effect in the human heart. In homogenates of the human cardiac ventricle, the major (>75%) enzymatic conversion of Ang I to Ang II is produced by a serine proteinase that is not inhibited by Ang I-converting enzyme (ACE) inhibitors. We have purified and cloned this proteinase. Structurally, this enzyme belongs to the chymase group of chymotrypsin-like proteinases and will be referred to as human heart chymase (hHC). Unlike chymotrypsin and, indeed, other chymases, hHC is the most specific and efficient Ang II- forming enzyme described. Preliminary studies using cardiac trabeculae show the presence of a functional pathway for Ang II formation in the human heart independent of the ACE pathway. Because high levels of immunoreactive-hHC are associated with the extracellular matrix surrounding myocytes, we speculate that the ACE-independent pathway of cardiac Ang II formation is indeed hHC-dependent. We propose to develop specific substrate-and antibody-based inhibitors of hHC to definitively establish that hHC is the major functional Ang II- forming enzyme in the human heart. These inhibitors will be used to show that the major conversion of Ang I to Ang II in human cardiac trabeculae, and that the response elicited by Ang I, are due to hHC. In parallel studies, we plan to crystallize hHC in the native form and in the inhibitor complexed form to study the unique structural determinants of hHC responsible for its high substrate specifity. Studies are also planned to functionally map the extended substrate binding site of hHC using Ang I analogs. In the human heart, immunoreactive-hHC is stored in cardiac mast cell granules. To study the processing of hHC, we propose to develop a human cardiac mast cell line that expresses hHC. This cell line will also be used to study the cis-acting elements and trans-acting factors that allow only a subset of mast cells to express hHC. The presence of hHC in the human heart suggests that cardiac Ang II formation may not cease during the treatment of hypertension and congestive heart failure with ACE inhibitors. The achievement of the goals of the proposed studies will ultimately help us understand the consequence of cardiac Ang II formation in the normal and failing heart, and will improve our understanding of heart failure and its treatment.