A novel molecular mechanism for stimulating uterine contractility by oxytocin

项目摘要 在怀孕期间，子宫逐渐从静止状态过渡，其特征是虚弱，异步， 局部收缩到激活状态，其中收缩的力度、频率和同步性增加， 在足月时排出胎儿这种转变的主要驱动力是子宫肌层平滑肌的逐渐去极化 细胞（MSMC）膜电位。随着膜内部的负电荷减少， 子宫肌层变得更加兴奋。然而，控制这种转变的分子途径是未知的， 阻碍了我们制定策略来调节子宫收缩力以防止早产或足月分娩的能力。 在这里，我们建议测试中心假设，钠（Na+）信号复合物形成的Na+- 激活的钾（K+）通道SLO 2.1和Na+泄漏通道NALCN调节该转换。这 假设是建立在我们从以前的R 01获得的资助的已发表和初步数据上的。在 在足月时分离的原代人MSMCs中，我们发现Na+通过NALCN进入激活K+通过 SLO 2.1和超极化膜。接下来，我们证明了这种复合物的激活降低了细胞内的张力。 子宫条最后，我们报道了抑制这种复合物诱导MSMC去极化，触发钙离子通道， (Ca2+）通过电压依赖性Ca 2+通道进入并促进收缩性。这些数据表明， NALCN/SLO 2.1复合物是控制MSMC膜电位的强有力的候选者。然而，在这方面， 因为我们使用的是人体组织，我们不能确定这种复合物在逐渐去极化中的作用。 MSMC膜电位的变化。为了解决这一局限性并充分验证我们的假设，我们 目的探讨NALCN/SLO 2.1复合物在小鼠妊娠过程中的功能和调控 MSMCs。该项目的目标是：1）定义整个妊娠期间NALCN/SLO 2.1复合物的活性，2）评估 NALCN/SLO 2.1复合物活性对细胞内Ca 2+和子宫收缩力的影响; NALCN/SLO2.1复合物的其他成员，并确定其对MSMCs功能的影响。 复杂.在完成这些目标，我们将确定的主要调节MSMC膜电位和如何 它们会随着怀孕的进展而改变这项工作将促进未来旨在开发治疗方法的努力， 抑制NALCN/SLO 2.1复合物以促进分娩或激活复合物以促进静止， 预防早产。

Project Summary During pregnancy, the uterus gradually transitions from a quiescent state characterized by weak, asynchronous, regional contractions to an activated state in which contractions increase in force, frequency, and synchrony to expel the fetus at term. A major driver of this transition is gradual depolarization of the myometrial smooth muscle cell (MSMC) membrane potential. As the inside of the membrane becomes less negatively charged, the myometrium becomes more excitable. However, the molecular pathways controlling this transition are unknown, hampering our ability to develop strategies to regulate uterine contractility to prevent pre- or post-term labor. Here, we propose to test the central hypothesis that a sodium (Na+) signaling complex formed by the Na+- activated potassium (K+) channel SLO2.1 and the Na+ leak channel NALCN regulates this transition. This hypothesis is founded on published and preliminary data we obtained with funding from our previous R01. In primary human MSMCs isolated at term, we showed that Na+ entry through NALCN activated K+ efflux through SLO2.1 and hyperpolarized the membrane. Next, we showed that activation of this complex reduced tension in uterine strips. Finally, we reported that inhibiting this complex induced MSMC depolarization, triggering calcium (Ca2+) entry through voltage-dependent Ca2+ channels and promoting contractility. Together, these data indicate that the NALCN/SLO2.1 complex is a strong candidate to control the MSMC membrane potential. However, because we used human tissues, we could not determine the role of this complex in the gradual depolarization of the MSMC membrane potential over pregnancy. To address this limitation and fully test our hypothesis, our objective is to define the function and regulation of the NALCN/SLO2.1 complex across pregnancy in mouse MSMCs. The goals of this project are to: 1) Define NALCN/SLO2.1 complex activity across pregnancy, 2) Assess the effects of NALCN/SLO2.1 complex activity on intracellular Ca2+ and uterine contractility and 3) Identify additional members of the NALCN/SLO2.1 complex in MSMCs and determine their effects on functionality of the complex. In completing these aims, we will define the main regulators of MSMC membrane potential and how they change as pregnancy progresses. This work will facilitate future efforts aimed at developing therapeutics to inhibit the NALCN/SLO2.1 complex to promote labor or to activate the complex to promote quiescence and prevent preterm labor.