Ang II and Norepinephrine in Cardiac Sympathetic Nerves

超出提供的空间。心肌缺血时心脏交感神经末梢(CSNE)病理性释放去甲肾上腺素(NE)可导致心律失常和死亡。负责的机制仍然不清楚。这笔赠款的总体目标是解决重要的生物学问题，即局部心脏肾素-血管紧张素系统(RAS)是否在心肌缺血时cSNE释放NE方面发挥重要作用。我们的初步结果表明，心肌肥大细胞是肾外肾素样蛋白的来源，能够将血管紧张素原分解为Ang I。我们推测，心肌肥大细胞在缺血时脱颗粒是心脏SNE附近局部RAS激活和Ang II形成的关键。在特定的目标I中，我们将鉴定从大鼠、人、野生型和肥大细胞缺陷的小鼠心脏以及人肥大细胞系HMC-1细胞中分离的肥大细胞来源肾素的分子同一性。我们还将在豚鼠和人类的缺血心脏模型上研究局部合成的Ang II是否以旁分泌模式作用于AT1受体(AT1R)以诱导NE释放。为了证实肥大细胞在心脏RAS中的作用，将在肥大细胞缺陷小鼠中进行平行实验。由于Ang II是一种已知的Na/H交换器(NHE)刺激剂，我们假设在缺血时，Ang II刺激NHE会导致细胞内Na_2增加，从而通过NE转运体触发NE的过度释放。我们将在分离的表达天然Ang II受体的cSNE和转染克隆的大鼠AT1A受体(SH-SY5Y-AT_A)的人神经母细胞瘤细胞中验证这一假说。血管紧张素转换酶II通过血管紧张素Ⅱ受体对去甲肾上腺素释放的影响的信号转导途径将在特定的目的II进行研究。初步实验表明，血管紧张素转换酶II刺激cSNE中的PLC-PKC通路，调节NHE活性和去甲肾上腺素释放。因此，我们将研究Ang II受体介导的信号通路中的关键元件在NHE激活和相关NE释放中的作用。实验将集中在PLC、CAI、PKC和钙调蛋白。进行这些实验的总体前提是，如果我们了解Ang II在心脏中形成的生理基础，以及Ang II是如何刺激NE释放的，那么我们也将确定潜在的可用于治疗缺血性心律失常和相关心功能障碍的靶点。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. Pathologic release of norepinephrine (NE) from cardiac sympathetic nerve endings (cSNE) during myocardial ischemia leads to arrhythmias and death. The mechanisms responsible remain obscure. The overall objective of this grant is to address the important biological issue of whether a local cardiac renin-angiotensin system (RAS) plays a significant role in NE release from cSNE in myocardial ischemia. Our preliminary results demonstrate that cardiac mast cells are a source of extra-renal renin-like protein capable of cleaving angiotensinogen to ANG I. We hypothesize that degranulation of cardiac mast cells in ischemia is pivotal for local RAS activation and ANG II formation, proximal to cardiac SNE. In Specific Aim I, we will characterize the molecular identity of mast-cell derived renin in SNE isolated from rat, human, wild-type and mast cell deficient mouse hearts, and from the human mast cell line, HMC-1 cells. We will also investigate in ischemic cardiac models from guinea-pigs and humans, whether locally synthesized ANG II acts in a paracrine mode on AT1 receptors (AT1R) to elicit NE release. To confirm the role of mast cells in this cardiac RAS, parallel experiments will be conducted in mast-cell deficient mice. Because ANG II is a known stimulant of the Na+/H + exchanger (NHE),Iwe hypothesize that in ischemia, NHE stimulation by ANG II will lead to an increase in intracellular Na _, thereby triggering excessive NE release via the NE transporter. We will test this hypothesis in isolated cSNE expressing native ANG II receptors, and in human neuroblastoma cells transfected with cloned rat AT1A receptor (SH-SY5Y-AT_A). The signal transduction pathways mediating the effects of ANG II via AT1R on NE release will be studied in Specific Aim II. Pilot experiments demonstrate that ANG II stimulates the PLC-PKC pathway in cSNE, regulating NHE activity and NE release. Hence, we will study the roles of key elements of ANG II receptor-mediated signaling pathways involved in NHE activation and associated NE release. The experiments will focus on PLC, Cai, PKC, and calmodulin. The overall premise for conducting these experiments is that if we understand the physiological basis for the formation of ANG II in the heart, and how ANG II stimulates NE release, then we will also have identified targets that potentially can be exploited for therapies for ischemic arrhythmias and associated cardiac dysfunctions. PERFORMANCE SITE ========================================Section End===========================================