CHANGES IN MYOMETRIAL ANNEXIN ACTIVITIES AT PARTURITION

分娩时子宫肌层膜联蛋白活性的变化

• 批准号: 3757288
• 负责人: WILLIAM V EVERSON
• 金额: --
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3757288
• 关键词: SDS polyacrylamide gel electrophoresis; antisense nucleic acid; birth; calcium channel; cell cell interaction; electron microscopy; electrophysiology; gap junctions; hormone regulation /control mechanism; laboratory rat; light microscopy; membrane proteins; muscle contraction; myometrium; posttranslational modifications; premature labor; protein biosynthesis; protein structure function; protein tyrosine kinase; receptor coupling; receptor expression; southern blotting; steroid hormone; western blottings

The annexins are a group of calcium and phospholipid binding proteins which may function as regulators of intracellular free Ca2+ or as mediators of Ca2+ action in the cell. Currently, the role of annexins in myometrium is unknown. We propose that annexins, under the control of steroid hormones, may regulate the contractile activity of the myometrium at parturition. Annexin II may regulate the formation of gap junctions which are essential for cell-cell coupling underlying the coordinate contraction of adjacent cells. Annexin VI may regulate the IP3-sensitive calcium release channel of the sarcoplasmic reticulum, leading to regulation of intracellular free Ca2+ and modulation of myosin light chain kinase activity. Our studies will include the following objectives: 1. Expression of annexins II and VI will be determined during normal gestation, in hormonally manipulated rats and in control and preterm human myometrial tissue. Intracellular localization of annexins II and VI will be performed, with selective co-localization by light and EM microscopy of annexin II with connexin 43 and of annexin VI with the IP3-sensitive calcium release channel. 2. The regulation of annexin II and VI expression by steroid hormones, using both hormonally manipulated rats and cultured myometrial cells will be determined. Annexins will be separated by subcellular fractionation. The nature and extent of covalent modifications in each subcellular fraction will be quantitated and evaluated to determine their role in regulation of annexin function. 3. The effect of suppression of connexin 43 or of annexin II using S- oligonucleotides antisense to their respective mRNAs will be examined. Concurrent measurements of gap junction formation and cell-cell coupling by electrophysiological methods will be made. The effect of agents which alter connexin and annexin activity (in particular, pp60arc tyrosine kinase) will be measured in conjunction with measurement of cell-cell coupling and subcellular localization of connexin 43. 4. The effect of annexin VI on the IP3-sensitive calcium release channel from the sarcoplasmic reticulum will be examined in lipid bilayer. These findings will be supported in vivo through the use of antisense against annexin VI to suppress intracellular concentration of annexin VI. Contractile properties and free calcium dynamics and ion channel activity will be examined following suppression of annexin VI in cultured myometrial cells. These studies will establish the role of annexins in two aspects of myometrial function which are essential to coordinated contractile activity. A more detailed understanding of the mediation and action of intracellular calcium will allow the development of novel approaches directed toward prevention of preterm labor.

膜联蛋白是一组钙和磷脂结合蛋白， 可能作为细胞内游离钙离子的调节器或作为 钙离子在细胞内的作用。目前，膜联蛋白在子宫肌层中的作用是 未知。我们认为，膜联蛋白在类固醇激素的控制下， 可调节分娩时子宫肌层的收缩活动。 膜联蛋白II可能调节缝隙连接的形成，缝隙连接是必不可少的 对于在相邻的坐标收缩基础上的细胞-细胞耦合 细胞。膜联蛋白VI可能调节IP3敏感的钙释放通道 肌浆网，导致细胞内游离调节 钙离子与肌球蛋白轻链激酶活性的调节。我们的研究 将包括以下目标： 1.在正常情况下，将测定膜联蛋白II和VI的表达 在荷尔蒙操纵的大鼠、对照组和早产儿中的妊娠 子宫肌层组织。膜联蛋白II和VI的细胞内定位 通过光学显微镜和EM显微镜进行选择性共定位 膜联蛋白II与连接蛋白43和膜联蛋白VI与IP3敏感 钙释放通道。 2.类固醇激素对膜联蛋白II和膜联蛋白VI表达的调节 同时使用激素处理的大鼠和培养的子宫肌层细胞将 要下定决心。膜联蛋白将通过亚细胞分级分离。 每个亚细胞中共价修饰的性质和程度 将对分数进行量化和评估，以确定它们在 对膜联蛋白功能的调控。 3.S抑制缝隙连接蛋白43或膜联蛋白II的作用 将检测与其各自的mRNAs反义的寡核苷酸。 缝隙结形成和细胞-细胞耦合的同时测量 将制定电生理学方法。药剂的作用 改变连接蛋白和膜联蛋白的活性(特别是pp60Arc酪氨酸 将结合细胞-细胞的测量进行测量 连接蛋白43的偶联和亚细胞定位。 4.Annexin VI对IP3敏感性钙释放通道的影响 从肌浆网中会检测到脂质双层。这些 这些发现将通过使用反义核酸在体内得到支持。 Annexin VI抑制细胞内Annexin VI的浓度。 收缩特性、游离钙动力学和离子通道活性 将在抑制培养的子宫肌层膜联蛋白VI后进行检查 细胞。 这些研究将确定膜联蛋白在以下两个方面的作用 协调收缩所必需的子宫肌层功能 活动。更详细地了解调解和行动 细胞内钙离子将使新方法的发展成为可能 旨在预防早产。