CALCIUM AND SODIUM TRANSPORT IN SMOOTH MUSCLE

平滑肌中钙和钠的转运

• 批准号: 2028550
• 负责人: MORDECAI P BLAUSTEIN
• 金额: $ 27.36万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2028550
• 关键词: antisense nucleic acid; artery; biological transport; caffeine; calcium; calcium binding protein; calcium channel blockers; calcium indicator; calcium metabolism; calcium transporting ATPase; digital imaging; fluorescent dye /probe; image processing; ion transport; laboratory rat; membrane transport proteins; mitochondria; muscle cells; sodium; stoichiometry; vascular smooth muscle

DESCRIPTION (adapted from the applicant's abstract): In vascular smooth muscle (VSM), a rise in the cytosolic free Ca2+ concentration, [Ca2+]cyt, is "necessary and sufficient" to trigger contraction. The overall goal of this project is to elucidate the factors that regulate intracellular Ca2+ in VSM, and to determine how they influence physiological and pathophysiological processes. The main hypothesis is that the plasmalemmal Na+ gradient helps regulate, via Na/Ca exchange (NCX), not only [Ca2+]cyt, but the intracellular stores of Ca2+ as well. Consequently, the Na+ gradient plays a critical role in the control of cell responsiveness. The emphasis will be on the NCX and its functional and spatial relationships to other, related transporters and to intracellular Ca2+ stores in VSM cells. The following specific aims are proposed: 1. To determine the contribution of NCX to regulation of intracellular Ca2+ storage in primary cultured rat arterial myocytes. Three spatially and functionally-distinct Ca2+ stores in VSM cells will be identified pharmacologically and their regulation by the Na+ gradient and NCX will be examined. Digital imaging methods with Ca2+- and Na+-sensitive dyes will be used to measure the Ca2+ content of the stores as well as cytoplasmic Na+ and Ca2+. These imaging studies will be correlated with contractile responses measured in arterial rings. 2. To determine the spatial distribution of key Na+ and Ca2+ transporters (NCX, Na+ and Ca2+ pumps) and relate them to sites of Ca2+ storage. These morphological data will be correlated with functional data from #1. Organelles (mitochondria and sarcoplasmic reticulum, SR) will be stained with fluorochromes [e.g., furaptra, DiOCb(3)]. Fluorescently labeled antibodies will be used to identify the NCX, Na+ pump, and plasmalemmal (PM) and SR Ca2+ pumps. Deconvolution and 3-D image restoration, as well as double-labeling [e.g., DiOCb(3) plus furaptra or a fluorescently labeled antibody], will be used to localize stores and transporters. Spatial correlation of the distribution of these organelles and transporters, to the PM NCX will be determined in freshly isolated as well as in cultured rat arterial myocytes. 3. To relate the physiological role of the NCX to other Ca2+ homeostasis in response to physiological stimuli and pharmacological perturbations will be characterized with digital imaging in fura-2 and furaptra-loaded cells. The NCX will be knocked down with antisense oligonucleotides, the PM Ca2+ pump will be inhibited with La3+, and intracellular stores will be emptied with selective agents. Standard molecular biological methods will be used for antisense studies.

描述（改编自申请人摘要）：血管光滑 肌肉（VSM），胞质游离钙浓度（[Ca 2 +]cyt）升高， “必要且充分”引发收缩。 总的目标是 本课题旨在阐明VSM细胞内Ca ~（2+）的调控因子， 并确定它们如何影响生理和病理生理 流程. 主要假设是质膜Na+梯度有助于 通过Na/Ca交换（NCX），不仅调节[Ca 2 +]cyt， 细胞内钙离子的储存。 因此，Na+梯度起着 在控制细胞反应性中起关键作用。 重点将 关于NCX及其与其他相关 运输和细胞内钙储存在VSM细胞。 以下 具体目标是：1。 为了确定NCX对 原代培养大鼠动脉细胞内Ca ~（2+）库的调节 肌细胞 VSM中三种不同空间和功能的钙库 细胞将被鉴定为β细胞，它们的调节由Na+ 将检查梯度和NCX。 数字成像方法与钙离子和 Na+敏感染料将用于测量钙离子的含量， 胞质Na+、Ca ~（2+）含量也有不同程度的降低。 这些影像学研究将与 在动脉环中测量收缩反应。 2. 确定 关键Na+和Ca 2+转运蛋白（NCX、Na+和Ca 2+）的空间分布 泵），并将它们与Ca 2+存储的网站。 这些形态学数据 将与来自#1的功能数据相关联。 细胞器（线粒体 和肌浆网，SR）将用荧光染料染色[例如， furaptra，DiOCb（3）]。 标记的抗体将用于 识别NCX、Na+泵、质膜（PM）和SR Ca 2+泵。 反卷积和3-D图像恢复，以及双标记[例如， DiOCb（3）加furaptra或荧光标记抗体]，将用于 本地化商店和运输。 分布的空间相关性 这些细胞器和转运蛋白，PM NCX将在 新鲜分离的以及在培养的大鼠动脉肌细胞中。 3. 到 将NCX的生理作用与其他Ca 2+稳态联系起来， 对生理刺激和药理学扰动的反应将是 其特征在于在Fura-2和Furaptra加载的细胞中的数字成像。 的 NCX将被反义寡核苷酸敲除，PM Ca 2+泵 将被La 3+抑制，细胞内储存将被排空， 选择剂。 标准分子生物学方法将用于 反义研究