Na+/Ca2+ Exchanger type-1 in Arterial Contraction and Salt-induced Hypertension

描述（由申请人提供）：拟议研究的总体目标是确定血管NCX1 （Na+/Ca2+交换型-1）影响正常和盐依赖性高血压的动脉血压（BP）的分子和生理机制。两个关键的决定动脉直径，因此动脉血压在体内，交感神经活动（SNA）和肌张力（MT）。我的总体假设是，VSM NCX1增加sna介导的血管收缩和MT，通过净Ca2+内流增加VSM [Ca2+]，从而调节血压。将使用表达平滑肌NCX1水平改变的转基因小鼠（NCX1smTg/Tg，过表达者；NCX1sm-/-，敲除者）以及外源性（转基因）基于FRET的Ca2+/钙调素-肌球蛋白轻链激酶（MLCK）活性生物传感器分子。NCX1过表达将显示“功能获得”，敲除将显示NCX1功能的丧失。三个特定的目标将被测试：目标1：测试假设血管ncx1介导的Ca2+进入增加细胞质[Ca2+]和MLCK的激活在sna介导的血管收缩和MT在孤立的动脉；目的2：验证Ca2+通过VSM NCX1进入体内也有助于SNA和mt介导的动脉张力，从而调节血压的假设；目的3：验证盐依赖性高血压与NCX1活性进一步升高有关的假设。NCX1在sna介导的收缩中可能的细胞机制是Ca2+通过“反向模式”NCX1内流，这是G蛋白偶联受体（GPCR）诱导的Na+渗透瞬时受体电位规范通道TRPC6激活后局部Na+积累的结果。这将增加激活MLCK的肌浆网依赖性Ca2+波。NCX1在MT中的推测机制是Na+通道（TRPC6和/或TRPM4）拉伸激活后Ca2+内流。这将增加细胞质[Ca2+]。在sna介导的收缩过程中，共聚焦“4-D”成像将用于观察孤立的加压肠系膜小动脉的细胞质Ca2+波。使用的关键技术是麻醉活鼠体外小动脉的活体FRET成像，以观察[Ca2+]， MLCK激活，动脉直径和颈动脉血压。高盐摄入可诱导生物传感器/NCX1转基因小鼠盐依赖性高血压。血流（用于测定心输出量，CO）和遥测动脉血压将在有意识的，自由移动的小鼠中测量。总外围电阻（TPR）将计算自BP H CO x TPR。在体内实验中，药物GPCR阻滞剂和特定的NCX阻滞剂（SEA0400）将被全身或局部（被研究的动脉）施用。该研究将阐明BP与血管NCX1之间已建立的关联的分子机制。这些知识应该增加我们对盐依赖性高血压的理解，这是一个日益紧迫的健康问题。

DESCRIPTION (provided by applicant): The overall goal of the proposed research is to determine the molecular and physiological mechanisms by which vascular NCX1 (Na+/Ca2+ exchanger type-1) influences arterial blood pressure (BP) normally and in salt- dependent hypertension. Two key determinants of arterial diameter, and hence of arterial BP in vivo, are sympathetic nerve activity (SNA) and myogenic tone (MT). My overall hypothesis is that VSM NCX1 increases both SNA-mediated vasoconstriction and MT, via net Ca2+ influx that increases VSM [Ca2+], and thus regulates BP. Transgenic mice that express altered levels of smooth muscle NCX1 (NCX1smTg/Tg, overexpressors; NCX1sm-/-, knockouts) as well as an exogenous (transgenic) FRET based, Ca2+/calmodulin-myosin light chain kinase (MLCK) activity biosensor molecule will be used. The NCX1 overexpressors will reveal 'gain of function' and knockouts will reveal loss of NCX1 function. Three Specific Aims will be tested: Aim 1: test the hypothesis that vascular NCX1-mediated Ca2+ entry increases cytoplasmic [Ca2+] and activation of MLCK in both SNA-mediated vasoconstriction and in MT in isolated arteries; Aim 2: test the hypothesis that Ca2+ entry via VSM NCX1 also contributes to SNA- and MT-mediated arterial tone in vivo, hence regulates BP; Aim 3: test the hypothesis that salt-dependent hypertension involves further increases in NCX1 activity. The putative cellular mechanism for NCX1 in SNA-mediated contractions is Ca2+ influx via 'reverse mode' NCX1, as a result of local Na+ accumulation after G protein-coupled receptor (GPCR)-induced activation of Na+ permeant transient receptor potential canonical channels, TRPC6. This would increase sarcoplasmic reticulum-dependent Ca2+ waves that activate MLCK. The putative mechanism of NCX1 in MT is Ca2+ influx after stretch activation of Na+ channels (TRPC6 and/or TRPM4). This would increase cytoplasmic [Ca2+]. Confocal '4-D' imaging will be used to observe cytoplasmic Ca2+ waves in isolated pressurized mesenteric small arteries during SNA-mediated contraction. A key technique to be used is intra-vital FRET imaging of exteriorized small arteries of anesthetized living mice to observe [Ca2+], MLCK activation, artery diameter, and carotid artery BP. Salt-dependent hypertension will be induced in biosensor/NCX1 transgenic mice by high salt intake. Blood flow (to determine cardiac output, CO) and telemetric arterial BP will be measured in conscious, freely moving mice. Total peripheral resistance (TPR) will be calculated since BP H CO x TPR. Pharmacological GPCR blockers and a specific NCX blocker (SEA0400) will be administered systemically or locally (to the artery being studied) in the in vivo experiments. The research will elucidate the molecular mechanisms of the established association of BP with vascular NCX1. This knowledge should increase our understanding of salt-dependent hypertension, an increasingly urgent health problem.