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Cryosection-Elemental Analysis Studies on the Regulatory Mechanism of Contraction by Calcium bound to Inner Surface of Smooth Muscle Plasma Membrane

平滑肌质膜内表面钙结合的收缩调节机制的冰冻切片元素分析研究

基本信息

批准号：
07670063
负责人：
SUZUKI Suechika
金额：
$ 1.47万
依托单位：
Teikyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

To make clear the regulatory mechanism of contraction byCa ions which are nound to the inner surface of smooth muscle plasma membrane in the resting state, intracellular Ca localization and its translocation during the contraction-relaxation cycle in the anterior byssal retractor muscle (ABRM) of Mytilus edulis were studied by the electron probe X-ray microanalysis of cryosections. By the spot analyzes of inner surface of plasma membrane in the resting fibers, significant amounts of Ca (-8mmol/kg dry wt) were detected frequently, although in some cases the Ca concentration was negligible. The averaged Ca concentration at the inner surface of plasma membrane was approximately 3.0mmol/kg dry wt (n=25). On the other hand, the Ca concentration detected by the spot analyzes of myoplasm was low (-1.5mmol/kg dry wt). This result indicates that the Ca accumulation occurs actually at the inner surface of plasma membrane. In the spot analysis along the inner surface of plasma membrane, a siginificant amount of Ca is not always detected. This fact may be related to the distribution of Ca-binding protein (MCBP-450) isolated recently from the plasma membrane fraction of ABRM.Then, we further investigated the ultrastructural features of this Ca-binding protein. The low-angle rotary shadowing replicas of purified MCBP-450 showed that the protein is roughly ellipsoidal in shape. The dimensions measured by digital image processor were approximately 22 nm and 18 nm in long and short diameters respectively. Electron microscope observations of freeze-fracture and freeze-etching replicas showed that a part of some intramembranous particles, possibly the Ca-binding prtein, protrudes into the myoplasm at the inner surface of plasma membrane. Such a protein of molecules may play a role as Ca-binding sites, and release Ca during the membrane excitation. Further experiments to reveal the Ca translocation during contraction is now in progress.

为明确静息状态下平滑肌质膜内表面的钙离子对收缩的调控机制，采用冷冻切片的电子探针x射线微量分析研究了贻贝前背牵肌（ABRM）在收缩-松弛循环过程中的细胞内钙定位及其移位。通过对静息纤维质膜内表面的现场分析，经常检测到大量的Ca (-8mmol/kg dry wt)，尽管在某些情况下Ca浓度可以忽略不计。质膜内表面Ca的平均浓度约为3.0mmol/kg dry wt （n=25）。另一方面，肌浆斑点分析检测到的Ca浓度较低（-1.5mmol/kg dry wt）。这一结果表明钙的积累实际上发生在质膜的内表面。在沿着质膜内表面的斑点分析中，并不总是检测到大量的Ca。这可能与最近从ABRM的质膜部分分离到的ca结合蛋白（MCBP-450）的分布有关。然后，我们进一步研究了该钙结合蛋白的超微结构特征。纯化的MCBP-450的低角度旋转阴影复制品显示该蛋白的形状大致为椭球状。数字图像处理器测量的尺寸长、短直径分别约为22 nm和18 nm。冻裂和冻蚀复制品的电镜观察显示，部分膜内颗粒，可能是钙结合蛋白，在质膜内表面突出到肌浆中。这种蛋白分子可能作为钙结合位点，在膜激发过程中释放钙。进一步的实验揭示收缩过程中的钙转运目前正在进行中。