Ductus Arteriosus Regulation by Anion Channels

项目摘要 该提案代表了两个互补实验室之间的多PI合作， 在动脉导管（Reese博士）和血管氯（Cl-）通道（Lamb博士）方面的专业知识。这个目标 建议是确定阴离子调节动脉导管（DA）的机制。血管 出生时的过渡期需要DA的快速收缩。DA的持续通畅（PDA）是一个严重的问题 影响超过30%的<1500 g的早产儿，但目前的治疗方法有许多缺点。的 调节胎儿DA开放和产后关闭的机制尚未完全解决。我们之前 鉴定了许多离子通道，这些离子通道是DA特异性治疗的有希望的靶点。其中，C1- 渠道是一个有趣的前景。虽然阳离子通道在DA中已经确立了作用，但实际上没有 存在关于Cl-电流和DA收缩性的信息。总的来说，血管平滑肌的Cl-转运 肌肉细胞（VSMC）是动态的，并通过特定的通道和协同转运蛋白进行精确调节。的改变 血管Cl-电流与高血压和其它血管异常有关。此外，据了解， 临床上使用利尿剂和氯离子转运抑制剂呋塞米与早产儿动脉导管未闭有关。 这些数据表明Cl-平衡在调节DA张力中的作用。探讨氯离子通量在植物生长发育中的重要性， 在DA中，我们首先确定了一个在DA中显著富集的Cl-相关基因的子集。 确定了特定的Cl-输入基因的表达增加，而Cl-输出基因在DA中减弱 在发育过程中，有利于在胎儿期输出Cl-，但在出生时转换为Cl-积累。 膜片钳记录隔离DA SMC确定存在特定的Cl-电流。肌造影 研究证实了这些电流的功能意义。特异性Cl-通道的抑制阻断了O2-和 激动剂诱导的分离的小鼠DA的收缩。呋塞米松弛离体小鼠DA， 不依赖于NO或前列腺素的作用。此外，用非选择性Cl-通道抑制剂治疗 在新生小鼠中产生PDA表型。这些数据强烈暗示Cl-运输和Cl-电流， 重要的，未开发的DA收缩介质。 在这个建议中，我们将测试的假设，Cl-电流的DA张力的关键介质， 出生时阴离子转运的变化将DA VSMC跨膜Cl-梯度从抑制性转换为 兴奋目的1将确定氯电流有助于子宫内DA松弛的机制， 产后DA关闭。目的2将确定氯离子转运的发育开关是否调节DA 收缩力和驱动产后DA关闭。分子、遗传、药理学和电生理学 方法将定义在体外和体内调节DA开放性的氯依赖性机制。 这些目标的完成将提供DA中Cl-稳态的临床相关信息， 鉴定用于调节DA张力新的治疗靶点。

PROJECT SUMMARY This proposal represents a multi-PI collaboration between two complementary laboratories with unique expertise in the ductus arteriosus (Dr. Reese) and vascular chloride (Cl-) channels (Dr. Lamb). The goal of this proposal is to determine the mechanisms by which anions regulate the ductus arteriosus (DA). Vascular transition at birth requires rapid constriction of the DA. Persistent patency of the DA (PDA) is a serious problem affecting over 30% of preterm infants <1500g, yet current therapies have many shortcomings. The mechanisms that regulate fetal DA patency and postnatal closure are not fully resolved. We previously identified a number of ion channels that are promising targets for DA-specific therapies. Among them, Cl- channels are an intriguing prospect. Although cation channels have established roles in the DA, virtually no information exists regarding Cl- currents and DA contractility. In general, Cl- transport in vascular smooth muscle cells (VSMC) is dynamic and precisely tuned by specific channels and co-transporters. Alterations in vascular Cl- currents have been linked to hypertension and other vascular abnormalities. Moreover, it is known that clinical use of furosemide, a diuretic and Cl- transport inhibitor, is associated with PDA in preterm infants. These data suggest a role for Cl- balance in regulating DA tone. To probe the importance of Cl- flux in the DA, we first identified a subset of Cl--associated genes that were significantly enriched in the DA. We determined that the expression of specific Cl- import genes increased while Cl- export genes waned in the DA over the course of development, favoring Cl- export during fetal life, but switching to Cl- accumulation at birth. Patch-clamp recordings of isolated DA SMCs identified the presence of specific Cl- currents. Myography studies verified the functional significance of these currents. Inhibition of specific Cl- channels blocked O2- and agonist-induced constriction of the isolated mouse DA. Furosemide relaxed the ex vivo mouse DA, independent of NO or prostaglandin actions. Furthermore, treatment with a non-selective Cl- channel inhibitor produced a PDA phenotype in newborn mice. These data strongly implicate Cl- transport and Cl- currents as important, unexplored mediators of DA contractility. In this proposal, we will test the hypothesis that Cl- currents are critical mediators of DA tone, and that changes in anion transport at birth switch the DA VSMC transmembrane Cl- gradient from inhibitory to excitatory. Aim 1 will identify the mechanisms by which Cl- currents contribute to in utero DA relaxation and postnatal DA closure. Aim 2 will determine whether a developmental switch in Cl- transport modulates DA contractility and drives postnatal DA closure. Molecular, genetic, pharmacological, and electrophysiological approaches will define chloride-dependent mechanisms that regulate DA patency in vitro and in vivo. Completion of these aims will provide clinically relevant information on Cl- homeostasis in the DA and may identify new therapeutic targets for modulation of DA tone.