Regulation of the Cardiac Na/Ca Exchanger

心脏钠/钙交换器的调节

• 批准号: 6537063
• 负责人: JOHN Paul REEVES
• 金额: $ 31.4万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-06-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6537063
• 关键词: CHO cells; actins; binding sites; calcium flux; calcium indicator; cardiac myocytes; caveolas; ceramides; conformation; inhibitor /antagonist; ion transport; laboratory rat; membrane activity; membrane structure; membrane transport proteins; phosphatase inhibitor; protein localization; protein structure function; sarcoplasmic reticulum; sphingosine; transfection; tumor necrosis factor alpha

The Na/Ca exchanger couples the movement of Ca ions across the plasma membrane to the movement of Na ions in the opposite direction. It is the principal Ca efflux mechanism in cardiac myocytes and plays an important role in controlling the Ca content of myocardial cells and in regulating cardiac contractility. Little is known about the cellular mechanisms that regulate exchange activity. In this project, the general hypothesis to be investigated is that sphingolipids are physiological regulators of Na/Ca exchange activity and block the conformational transition associated with regulatory Ca activation of the exchanger. This hypothesis is based on preliminary results demonstrating that short chain ceramide analogs and sphingosine inhibit exchange activity in a manner that is specifically targeted to the exchanger's regulatory properties. Exchange activity will be measured using fluorescent cytosolic Ca indicators in transfected CHO cells expressing the cardiac Na/Ca exchanger and in neonatal rat cardiac myocytes. The project has 6 specific aims: (1) Initial characterizations of the effects of exogenous and endogenous ceramide or sphingosine on exchange activity in the transfected cells will be completed. (2) A new kinetic formulation of exchanger regulation, which specifically links the process termed "Na-dependent inactivation" to changes in regulatory Ca-activation, will be developed. (3) The rate at which ceramide inhibition develops when cytosolic Ca is elevated or when internal Ca stores are filled will be measued in order to test the hypothesis that ceramide inhibition requires the formation of the Ca-depleted conformation of the exchanger. Other studies will test the prediction that ceramide does not inhibit the exchanger in the absence of Ca activation. (4) The hypothesis that ceramide inhibits exchange activity by perturbing lateral lipid subdomains such as lipid rafts and/or caveolae will be tested. (5) The studies in this aim will test the idea that pharmacological stabilization of the actin cytoskeleton locks the exchanger in an activated state, reducing its susceptibility to regulation by cytosolic Ca or inhibition by ceramide. (6) The physiological significance of ceramide inhibition of exchange activity will be assessed by measuring the influence of ceramide on (a) exchange activity, (b) the Ca-transient and (c) the amount of Ca in the sarcoplasmic reticulum in beating neonatal rat cardiac myocytes. Other studies will assess whether inhibition of exchange activity contributes to the cardiotoxic effects of TNFalpha.

钠/钙交换器将钙离子在质膜上的运动与钠离子在相反方向上的运动结合在一起。它是心肌细胞内钙外流的主要机制，在控制心肌细胞内钙含量和调节心肌收缩能力方面起着重要作用。人们对调节交换活动的细胞机制知之甚少。在这个项目中，要研究的一般假设是神经鞘脂是钠/钙交换活性的生理调节器，并阻止与调节性钙激活相关的构象转变。这一假说是基于初步结果表明，短链神经酰胺类似物和鞘氨醇以一种针对交易所调节特性的方式抑制交换活性。在表达心肌Na/Ca交换器的CHO细胞和新生大鼠心肌细胞中，将使用荧光胞浆钙指示剂来测量交换活性。该项目有6个具体目标：(1)初步表征外源性和内源性神经酰胺或鞘氨醇对转基因细胞交换活性的影响。(2)将开发一种新的交换器调节动力学公式，该公式将被称为“钠依赖失活”的过程与调节性钙激活的变化具体地联系起来。(3)当细胞内钙升高或内部钙库充满时，神经酰胺抑制的发展速度将被测量，以检验神经酰胺抑制需要形成交换的缺钙构象的假说。其他研究将检验神经酰胺在没有钙激活的情况下不会抑制交换器的预测。(4)神经酰胺通过干扰脂筏和/或小窝等侧向脂亚结构域而抑制交换活性的假说将得到验证。(5)这一目的的研究将检验这样一种观点，即肌动蛋白细胞骨架的药理稳定性将交换器锁定在激活状态，从而降低其对细胞内钙调节或神经酰胺抑制的敏感性。(6)通过测定神经酰胺对跳动乳鼠心肌细胞(A)交换活动、(B)钙瞬变和(C)肌浆网钙含量的影响，评价神经酰胺抑制交换活动的生理意义。其他研究将评估抑制交换活动是否有助于肿瘤坏死因子α的心脏毒性作用。