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

描述(申请人提供)：拟议研究的总体目标是确定血管NCX1(钠/钙交换因子-1)在正常和盐依赖型高血压中影响动脉血压(BP)的分子和生理机制。动脉直径的两个关键决定因素是交感神经活动(SNA)和肌源性张力(MT)，从而决定了体内动脉血压。我的总体假设是，VSM NCX1通过净钙内流增加VSM[Ca~(2+)]，从而增加SNA介导的血管收缩和MT，从而调节血压。将使用表达改变水平的平滑肌NCX1(NCX1smTg/TG，过表达；NCX1sm-/-，敲除)以及基于外源(转基因)FRET的、具有钙/钙调蛋白-肌球蛋白轻链激酶(MLCK)活性的生物传感器分子的转基因小鼠。NCX1的过表达将揭示功能的获得，而敲除将揭示NCX1功能的丧失。目的1：检验血管NCX1介导的钙离子内流增加SNA介导的血管收缩和MT中的胞浆[Ca2+]和MLCK激活的假说；目的2：检验通过VSM NCX1介导的钙内流也有助于体内SNA和MT介导的动脉张力，从而调节血压的假说；目标3：检验盐依赖型高血压涉及NCX1活性进一步增加的假说。NCX1在SNA介导的收缩中可能的细胞机制是，在G蛋白偶联受体(GPCRa)激活Na+预指的瞬时受体潜在规范通道TRPC6后，由于局部Na+积累，NCX1通过反向模式内流。这将增加激活MLCK的肌浆网依赖的钙波。NCX1在MT中的作用机制可能是牵张激活Na+通道(TRPC6和/或TRPM4)后的钙内流。这会增加细胞质内的钙离子浓度。共聚焦‘4-D’成像将被用来观察在SNA介导的收缩过程中，分离的加压肠系膜小动脉中的胞浆钙波。要使用的一项关键技术是对麻醉活着的小鼠体外小动脉进行活体FRET成像，以观察[Ca~(2+)]、MLCK激活、动脉直径和颈动脉血压。高盐摄入可诱导BiosensorNCX1转基因小鼠发生盐依赖型高血压。将在清醒、自由活动的小鼠身上测量血流量(以确定心输出量，CO)和遥测动脉血压。总外周阻力(TPR)将从BP H CO x TPR开始计算。在活体实验中，系统地或局部地(对被研究的动脉)给予药理上的GPCR阻滞剂和特定的NCX阻滞剂(SEA0400)。这项研究将阐明BP与血管NCX1建立联系的分子机制。这些知识应该会增加我们对盐依赖型高血压的理解，盐依赖型高血压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.