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

描述（由申请人提供）：拟定研究的总体目标是确定血管NCX 1（Na+/Ca 2+交换器1型）正常和盐依赖性高血压影响动脉血压（BP）的分子和生理机制。动脉直径以及体内动脉BP的两个关键决定因素是交感神经活性（SNA）和肌源性张力（MT）。我的总体假设是，VSM NCX 1通过增加VSM [Ca 2 +]的净Ca 2+内流增加SNA介导的血管收缩和MT，从而调节BP。将使用表达改变水平的平滑肌NCX 1（NCX 1 smTg/Tg，过表达; NCX 1 sm-/-，敲除）以及基于外源（转基因）FRET的Ca 2 +/钙调蛋白-肌球蛋白轻链激酶（MLCK）活性生物传感器分子的转基因小鼠。NCX 1过表达子将揭示“功能的获得”，敲除将揭示NCX 1功能的丧失。将测试三个特定目的：目的1：测试血管NCX 1介导的Ca 2+内流在SNA介导的血管收缩和MT中增加细胞质[Ca 2 +]和MLCK激活的假设;目的2：测试通过VSM NCX 1的Ca 2+内流也有助于SNA和MT介导的体内动脉张力，从而调节BP的假设;目的3：测试盐依赖性高血压涉及进一步增加NCX 1活性的假设。在SNA介导的收缩中，NCX 1的假定细胞机制是通过“反向模式”NCX 1的Ca 2+内流，这是G蛋白偶联受体（GPCR）诱导的Na+渗透性瞬时受体电位经典通道TRPC 6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Ca 2+波。NCX 1在MT中的假定机制是Na+通道（TRPC 6和/或TRPM 4）牵张激活后的Ca 2+内流。这将增加细胞质[Ca 2 +]。共聚焦“四维”成像将用于观察SNA介导收缩期间离体加压肠系膜小动脉中的细胞质Ca 2+波。使用的关键技术是活体FRET成像的外部小动脉的麻醉活小鼠观察[Ca 2 +]，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/NCX 1转基因小鼠产生盐依赖性高血压。将在清醒、自由活动的小鼠中测量血流量（以测定心输出量，CO）和遥测动脉BP。将计算总外周阻力（TPR），因为BP H CO x TPR。在体内实验中，药理学GPCR阻断剂和特异性NCX阻断剂（SEA 0400）将全身或局部（研究动脉）给药。这项研究将阐明BP与血管NCX 1建立联系的分子机制。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.