INTRACELLULAR STORAGE OF CALCIUM IN ARTERIAL MYOCYTES

动脉肌细胞内钙的储存

• 批准号: 2226959
• 负责人: MIKHAIL L BORIN
• 金额: $ 9.52万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-13 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2226959
• 关键词: calcium indicator; cations; digital imaging; fluorescent dye /probe; growth factor; image processing; intracellular; muscle cells; muscle contraction; phosphorylation; protein kinase C; sodium channel; stimulant /agonist; tissue /cell culture; vascular smooth muscle; vasoactive agent; western blottings

Na+, the predominant cation in the extracellular fluid, is involved in numerous physiological and pathophysiological processes. Arterial myocytes, like most other types of cells, normally maintain a low intracellular Na+ concentration. The Na+ gradient is involved in the regulation of intracellular Ca2+ concentration [Ca2+]i and pH, contractility, cell volume and membrane potential, in the distribution of numerous solutes, and many other functions. For vascular smooth muscle cells there is little quantitative information available regarding the intracellular Na+ concentration ([Na+]i), and the temporal and spatial changes in [Na+]i, such as those that are evoked by vasoconstrictors and vasodilators. We have obtained evidence that various agonists evoke a common Na+ response, namely a sustained elevation of [Na+]i. The goals of this project are to investigate: whether such an increase in [Na+]i is a general phenomenon for arterial myocytes; what the mechanisms underlying these effects are, and what implications arise in [Na+]i, evoked by different stimuli, has on [Ca2+]i, the main determinant of muscle contraction. In this project I will: 1) Determine changes in [Na+]i evoked by selected vasoconstrictors, growth factors and vasodilators. Digital imaging analysis of cells loaded with the Na+-sensitive fluorescent dye, SBFI, will be used to determine temporal and spatial changes in [Na+]i. 2) Determine whether agonist-induced changes in [Na+]i are due to net agonist-induced changes in Na+ influx and/or Na+ efflux. 3) Elucidate the mechanisms of acute agonist-induced regulation of the Na+ pump, the main determinant of [Na+]i. The main emphasis of this part of the project is placed on testing the hypothesis that the Na+ pump activity is regulated, in part, by phosphorylation/dephosphorylation processes. Effects of intracellular kinases (e.g., protein kinase C) and phosphatases (e.g., phosphatase 1) on the Na+ pump activity and phosphorylation status will be determined and compared with the effects of the physiological agonists. The Na+ pump activity will be evaluated in single cultured cells (measurements of SBFI fluorescence) and in native tissue (i.e. arterial rings; measurements of 86Rbuptake). To determine the phosphorylation status of the Na+ pump, the enzyme will be immunoprecipitated, using specific antibodies, from 32P04 labelled cells. 4) Compare the regulation of [Na+]i in activated myocytes from conduit and muscular arteries. 5) Demonstrate the critical role of the rise in {Na+]i in augmentation of the amount of stored Ca2+. The accumulation of Ca2+ in stores and its release will be evaluated using digital imaging analysis of cells loaded with Ca2+-sensitive fluorescent dyes, chlortetracycline and fura-2, respectively. The results of these studies should provide new information about the regulation of Na+ in arterial myocytes and its relation to the regulation of Ca2+. Moreover, these findings may be useful in elucidating pathophysiological processes, such as those associated with (Na+-sensitive) hypertension or ischemia.

Na+是细胞外液中的主要阳离子， 许多生理和病理生理过程。 动脉 像大多数其他类型的细胞一样，肌细胞通常保持低水平的 细胞内Na+浓度。 Na+梯度参与了 调节细胞内Ca 2+浓度[Ca 2 +] i和pH， 收缩性、细胞体积和膜电位，在分布中 多种溶质以及许多其他功能。 对于血管平滑 肌肉细胞，几乎没有定量信息， 细胞内Na+浓度（[Na +] i），以及时间和 [Na +] i的空间变化，例如由 血管收缩剂和血管扩张剂。 我们有证据表明 不同的激动剂引起共同的Na+反应，即持续的 [Na +] i升高。 该项目的目标是调查： [Na +] i的这种增加是否是动脉的普遍现象， 肌细胞;这些影响的机制是什么，以及 不同刺激引起的[Na +] i的影响， [Ca2+] i，肌肉收缩的主要决定因素。 在这个项目中，我 将：1）确定[Na +] i的变化诱发的选择 血管收缩剂、生长因子和血管扩张剂。 数字成像 用Na +-敏感荧光染料，SBFI， 将用于确定[Na +] i的时间和空间变化。 （二） 确定激动剂诱导的[Na +] i变化是否是由于净 激动剂诱导的Na+内流和/或Na+外流的变化。 3)阐明 急性激动剂诱导的Na+泵调节机制， [Na +] i的主要决定因素。 本部分的主要重点是 该项目被放置在测试的假设，Na+泵活动 在某种程度上是由磷酸化/去磷酸化过程调节的。 细胞内激酶（例如，蛋白激酶C）和 磷酸酶（例如，磷酸酶1）对Na+泵活性的影响， 将确定磷酸化状态并与效果进行比较 生理激动剂的作用。 将评价Na+泵活性 在单个培养的细胞中（SBFI荧光的测量）和 天然组织（即动脉环;测量86Rbuptake）。 到 确定Na+泵的磷酸化状态，酶将被 使用特异性抗体从32P04标记的细胞免疫沉淀。 4)导管激活心肌细胞[Na +] i调节的比较 和肌肉动脉。 5)显示了在全球化进程中， {Na+] i增加了钙的贮存量。 积累 将使用数字成像技术评估钙离子在储存物中的含量及其释放情况 分析装载有Ca2+敏感性荧光染料的细胞， 金霉素和Fura-2。 这些研究的结果 为研究动脉血Na+的调节提供了新的信息 心肌细胞及其与钙调节的关系。 而且这些 这些发现可能有助于阐明病理生理过程， 如与（Na+敏感性）高血压或局部缺血相关的那些。