MAINTENANCE OF MYOMETRIAL QUIESCENCE DURING HUMAN PREGNANCY

人类怀孕期间子宫肌层静止的维持

• 批准号: 5212470
• 负责人: RUTH A WORD
• 金额: --
• 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/5212470
• 关键词: biological signal transduction; birth; calbindin; calcium flux; calcium transporting ATPase; enzyme activity; gel electrophoresis; hormone regulation /control mechanism; human pregnant subject; muscle contraction; myometrium; myosin light chain kinase; northern blottings; nucleic acid probes; organ culture; phosphorylation; pregnancy; sarcoplasmic reticulum; scintillation spectrometry; sex hormones; steroid hormone; western blottings

The long-range goal of the research described in this project is to identify and characterize the pregnancy-induced biochemical modifications of myometrial cells that contribute to the maintenance of uterine quiescence during 95% of pregnancy (phase O of parturition). The intracellular concentration of free Ca2+ ({Ca2+]i) and the phosphorylation/dephosphorylation state of myosin light chain are the principal determinants of smooth muscle contraction. We will evaluate pregnancy-induced modifications of these fundamental processes, which govern uterine smooth muscle contraction/relaxation (viz., [Ca2+]i, Ca2+ - signal processing, and myosin light chain phosphorylation). The rate and extent of myosin light chain phosphorylation during contraction of myometrial tissue obtained from pregnant women (compared with that of myometrial tissue from nonpregnant women)is markedly attenuated; and, the [Ca2+]i in myometrial smooth muscle cells isolated from uterine tissues of pregnant women is low. We will investigate the possibility that there are alterations in the regulation of myometrial cell Ca2+ homeostasis during pregnancy that serve to decrease the availability of free cytoplasmic Ca2= and thereby attenuate the Ca2+/calmodulin-dependent phosphorylation of myosin light chain. We will quantify cytoplasmic free calcium in strips of myometrial tissue and in myometrial smooth muscle cells from nonpregnant women and from pregnant women at various stages of gestation before and after the onset of labor. We will ascertain whether myosin light chain kinase (MLCK), the enzyme that catalyzes the phosphorylation of myosin light chain, is phosphorylated and thereby desensitized to Ca2+ activation in the myometrium of pregnant women. Such a phenomenon could constitute yet another mechanism by which phosphorylation of myosin light chain is attenuated. We will investigate three cellular processes that may serve to limit the availability of free cytoplasmic Ca2+: (o) Ca2+ extrusion by the plasma membrane, (ii) Ca2+ sequestration by the sarcoplasmic reticulum, and (iii) Ca2+ buffering by cytoplasmic Ca2+ pumps (Ca2+ pumps (Ca2+-ATPases, i.e., number and function) facilitate extrusion and sequestration of cytoplasmic Ca2+ in myometrium of pregnant women. Calbindins may act to modify cellular responses to cytoplasmic Ca2+ by modifications in Ca2+ by modifications in Ca2+ signal transduction or by way of the Ca2+=buffering capacity of these proteins. We will assess the functional consequences of increased/decreased levels of calbindin-9K (induced in vitro by hormone treatment) in human myometrial cells in culture. We will investigate the role of estrogen and progesterone in effecting modifications in Ca2+ homeostasis and MLCK phosphorylation. Knowledge of the mechanisms by which myometrial cell refractoriness to contraction is effected is crucial to an understanding of phase O of parturition and thence the transitions of the uterus to phases 1 and 2.

该项目描述的研究的远程目标是 识别和表征妊娠诱导的生化修饰 肌层细胞有助于维持子宫 妊娠95％期间的静止（分娩期O期）。 这 自由Ca2+（{Ca2+] i）的细胞内浓度和 肌球蛋白轻链的磷酸化/去磷酸化状态是 平滑肌收缩的主要决定因素。 我们将评估 怀孕引起的这些基本过程的修改， 控制子宫平滑肌收缩/放松（即[Ca2+] I，Ca2+ - 信号处理和肌球蛋白轻链磷酸化）。 费率和 肌球蛋白轻链磷酸化的程度 从孕妇获得的肌层组织（与 来自非怀孕妇女的肌层组织显着减弱；而且， [Ca2+] i在从子宫组织中分离的肌层平滑肌细胞中 孕妇很低。 我们将调查有可能存在的可能性 在调节肌层细胞Ca2+稳态调节过程中的改变 怀孕可降低自由细胞质CA2的可用性 从而衰减Ca2+/钙调蛋白依赖性磷酸化的磷酸化 肌球蛋白轻链。 我们将量化细胞质的无钙 肌层组织和肌层平滑肌细胞中的非孕妇 妇女和孕妇在妊娠的各个阶段和 劳动发作后。 我们将确定肌球蛋白轻链是否 激酶（MLCK），催化肌球蛋白磷酸化的酶 轻链被磷酸化，从而脱敏Ca2+激活 在孕妇的子宫内。 这样的现象可能构成 肌球蛋白轻链磷酸化的另一种机制是 衰减。 我们将研究三个可能用于的蜂窝过程 限制自由细胞质Ca2+的可用性：（O）Ca2+挤出 质膜，（ii）Ca2+骨质网的隔离，并 （iii）CA2+细胞质Ca2+泵缓冲（Ca2+泵（Ca2+ -atpases，，， 即，数字和功能）有助于挤压和隔离 孕妇肌层中的细胞质Ca2+。 calbindins可能会采取行动 通过修饰Ca2+对细胞质Ca2+的细胞反应进行修饰 CA2+信号转导或通过Ca2+ =缓冲的修改 这些蛋白质的能力。 我们将评估功能后果 Calbindin-9K水平升高/降低（由激素在体外诱导 治疗）在培养的人肌层细胞中。 我们将调查 雌激素和孕酮在CA2+的作用修饰中的作用 稳态和MLCK磷酸化。 了解其机制 肌层细胞对收缩的耐磨性对 理解分娩的相位O及其之后的过渡 子宫至第1阶段。