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: 3843009
• 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/3843009
• 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Ca 2+的调节剂或作为 Ca 2+在细胞中的作用。 目前，膜联蛋白在子宫肌层中的作用是 未知 我们认为，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可以调节间隙连接的形成， 对于细胞-细胞耦合，其基础是相邻细胞的协调收缩， 细胞 Annexin VI可能调节IP 3敏感性钙释放通道 肌浆网的调节，导致细胞内自由基的调节 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. 膜联蛋白II和VI的表达将在正常生长期间测定。 妊娠，在经口操作的大鼠和对照组及早产人中 子宫肌层组织 膜联蛋白II和VI的细胞内定位将 通过光学和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IP 3敏感性 钙释放通道 2. 类固醇激素对膜联蛋白II和VI表达的调节， 使用激素处理的大鼠和培养的子宫肌层细胞， 被确定。 将通过亚细胞分级分离来分离膜联蛋白。 每个亚细胞中共价修饰的性质和程度 部分将被定量和评估，以确定其作用， 膜联蛋白功能的调节。 3. 使用S-半乳糖苷酶抑制连接蛋白43或膜联蛋白II的作用 将检查与其各自mRNA反义的寡核苷酸。 缝隙连接形成和细胞-细胞偶联的同时测量 电生理方法。 药剂的作用， 改变连接蛋白和膜联蛋白活性（特别是pp 60酪氨酸 激酶）将与细胞-细胞 连接蛋白43的偶联和亚细胞定位。 4. 膜联蛋白VI对IP 3敏感性钙释放通道的影响 将在脂质双层中检查来自肌浆网的蛋白质。 这些 这些发现将在体内通过使用反义抗 膜联蛋白VI以抑制膜联蛋白VI的细胞内浓度。 收缩特性和游离钙动力学和离子通道活性 将在培养的子宫肌层中抑制膜联蛋白VI后进行检查 细胞 这些研究将确定膜联蛋白在两个方面的作用， 子宫肌层的功能，这是必要的协调收缩 活动 更详细地了解调解和行动， 细胞内钙离子将允许开发新的方法 旨在预防早产。