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Electrophysiological and electron microprobe studies of ion transport in cold acclimated brown adipocyte.

冷适应棕色脂肪细胞中离子传输的电生理学和电子微探针研究。

基本信息

批准号：
08670092
负责人：
SASAKI Sadao
金额：
$ 1.09万
依托单位：
Hyogo College of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670092/
关键词：
brown adipose tissue cold acclimation heat production Ca^<2+>CLSM ion uncoupling protein

项目摘要

Brown adipose tissue cell has much quantity of Lipid droplets and motochondria which contain heat productive protein, thermogenin (uncoupling protein) and electron transfer system in the inner membrane. These are involved in the process of heat production during cold acclimation in animals.The transport of ions in warm (WA) and cold (CA) acclimated rat brown adipocytes and the patch clamp method of reconstituted purified mitochondria of adipocytes were investigated.The electrophysiological recordings of Xenopus oocytes injected with total RNA extracted from CA and WA were also performed.Morphometric analyses of brown adipocytes showed that lipid droplets occupied 75% of the WA cells and 37% of the CA cells. The area of a single mitochondrion in CA cells was three times that of one in the WA cells. X-ray microanalysis showed that Na was higber, and K and Cl were lower in CA than WA, in both the nucleus and the mitochondria. Moreover, Ca (total) was higher in the mitochondria, whereas was lower in the nucleus of CA cells than in WA cells. Confocal laser scanning microscopy was performed also. Acridine orange (AO), a fluorescent dye which can indicate a H^+ gradient, was localized exclusively in the mitochondria rich cytoplasm of CA cells. The fluorescence intensity of fluo-3 (Ca^<2+>indicating dye), which was loaded for 30 ruin, increased within 1 min of noradrenaline (NA) exposure in the cytoplasm of WA and CA cells. In contrast, in the nucleus with NA application fluorescence intensity rose after an initial decrease in WA, and in CA decreased gradually. The patch clamp record shows achannel activity when the reconstituted mitochondrial membrane has some voltage and pH gradient. The whole cell clamp record from Oocyte injected with total RNA of CA shows the voltage dependent outward currents.

棕色脂肪组织细胞内含有大量的脂滴和运动蛋白，其内膜含有产热蛋白、解偶联蛋白和电子传递系统。在动物冷适应过程中，这些离子参与了产热过程。本文研究了大鼠棕色脂肪细胞的培养方法和重组纯化脂肪细胞线粒体的膜片钳技术，以及用CA和WA提取的总RNA注射非洲爪蟾卵母细胞的电生理记录。棕色脂肪细胞的形态计量学分析表明，脂肪细胞内的脂滴在细胞膜上形成，并在细胞膜上形成一个完整的细胞膜占WA细胞的75%和CA细胞的37%。CA细胞中单个胞体的面积是WA细胞中单个胞体的3倍。X射线微区分析表明，无论是细胞核还是线粒体，CA的Na含量均高于WA，而K和Cl含量低于WA。CA细胞线粒体Ca（total）高于WA细胞，而细胞核Ca（total）低于WA细胞。还进行了共聚焦激光扫描显微镜。吖啶橙子（AO）是一种能够指示H^+梯度的荧光染料，它只位于CA细胞富含线粒体的细胞质中。去甲肾上腺素（NA）作用于WA和CA细胞的胞质后1分钟内，荧光素3（Ca^2+指示染料）的荧光强度增加，荧光素3的浓度为30 μ mol/L。与此相反，在核NA应用荧光强度上升后，在WA的初始下降，并在CA逐渐下降。当重构的线粒体膜具有一定的电压和pH梯度时，膜片钳记录显示出achannel活性。注射CA总RNA的卵母细胞的全细胞钳夹记录显示电压依赖性外向电流。