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Identification and quantitative analysis of the cell membrane MgィイD1[2+]ィエD1 transporter in cardiac and smooth muscles.

心肌和平滑肌细胞膜 MgD1[2+]D1 转运蛋白的鉴定和定量分析。

基本信息

批准号：
09670055
负责人：
KONISHI Masato
金额：
$ 2.05万
依托单位：
Tokyo Medical University (1999)Jikei University School of Medicine (1997-1998)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

A fluorescent MgィイD12+ィエD1 indicator, furaptra (mag-fura-2), was loaded into smooth muscle cells of guinea-pig tenia cecum, and cytoplasmic MgィイD12+ィエD1 concentration ([MgィイD12+ィエD1]ィイD2iィエD2) was measured. Lowering extracellular NaィイD1+ィエD1 concentration ([NaィイD1+ィエD1]ィイD2oィエD2)in the absence of CaィイD12+ィエD1 caused gradual elevation of [MgィイD12+ィエD1]ィイD2iィエD2,the rate was strongly dependent on [NaィイD1+ィエD1]ィイD2oィエD2 with the maximal rate of rise of 0.16μM./s.We also measured [MgィイD12+ィエD1]ィイD2iィエD2 in rat ventricular myocytes. [MgィイD12+ィエD1]ィイD2iィエD2 was elevated when cells were treated with ionomycin in 10 mM extracellular MgィイD12+ィエD1([MgィイD12+ィエD1]ィイD2oィエD2). The rat of rise of [MgィイD12+ィエD1]ィイD2iィエD2 was reduced in the higher [NaィイD1+ィエD1]ィイD2oィエD2. The net efflux of MgィイD12+ィエD1 from the loaded cells was also markedly inhibited by the presence of 140 mM NaィイD1+ィエD1.These results suggest the exixtence of the NaィイD1+ィエD1-dependent MgィイD12+ィエD1 transport (NaィイD1+ィエD1-MgィイD12+ィエD1 exchange) in smooth muscle and cardiac muscle cells. The rate of MgィイD12+ィエD1 transport was estimated to be 0.026 pmol/cmィイD12ィエD1・s in tenia and 0.085 pmol/cmィイD12ィエD1・ s in cardiac myocytes at 25℃. It is unlikely that the NaィイD1+ィエD1-CaィイD12+ィエD1 exchanger is mainly responsible for this MgィイD12+ィエD1 transport, because only the slight NaィイD1+ィエD1-MgィイD12+ィエD1 exchange activity was observed in the CCL39 cells overexpressing the NaィイD1+ィエD1-CaィイD12+ィエD1 exchanger.

将荧光Mg ~（2+）D_1荧光指示剂furaptra（mag-fura-2）加载到豚鼠盲肠平滑肌细胞中，测定细胞质Mg ~（2+）D_1浓度（[Mg ~（2+）D_1]~（2+）D_1）。在缺乏Ca ~（2+）D_1的情况下，降低细胞外Na ~（2+）D_1 + Mg ~（2+）D_1浓度（[Na ~（2+）D_1] Mg ~（2+）D_1）~（2+）D_2逐渐升高，其速率强烈依赖于[Na ~（2+）D_1 + Mg ~（2+）D_1] Mg ~（2+）D_2 ~（2+）D_2，最大升高速率为0.16μM。测定了大鼠心室肌细胞[Mg ~（2+）D_1] Mg ~（2+）D_2和[Mg ~（2+）D_1] Mg ~（2+）D_2。[Mg当细胞在10 mM细胞外Mg（OH）2 D12+ Mg（OH）2 D1（[Mg（OH）2 D12+ Mg（OH）2 D1] Mg（OH）2 D2）中用离子霉素处理时，[Mg（OH）2 D12 + Mg（OH）2 D1] Mg（OH）2 D2升高。[Na + D1]+ Na + D2 + Na + D1+ Na + D2 + Na + 140 mM Na + D1+ Mg 2 + D1也能明显抑制Mg + D1的净流出。提示平滑肌和心肌细胞中存在Na + D1+ Mg 2 + D1依赖的Mg + D1转运（Na + D1 + Mg 2 + D1-Mg + D1交换）。在25℃时，心肌细胞Mg ~（2+）转运速率为0.085pmol/cm ~（2+）D_（12）~（1+）D_（12）~（1+）D_（12）· s。Na + D1+ Ca 2 + D1-Ca + D12+ Ca 2 + D1交换体不太可能主要负责这种Mg + D12+ Ca 2 + D1转运，因为在过度表达Na + D1+ Ca 2 + D12+ Ca 2 + D1交换体的CCL 39细胞中仅观察到轻微的Na + D1 + Ca 2 + D1-Mg + D12 + Ca 2 + D1交换活性。