Ductus Arteriosus Regulation by Anion Channels

项目总结 该建议代表了两个互补实验室之间的多PI合作，该实验室具有独特的 在动脉导管(Reese博士)和血管氯(Cl-)通道方面的专业知识(Lamb博士)。这样做的目的是 建议确定阴离子调节动脉导管(DA)的机制。血管 出生时的过渡需要迅速收缩DA。DA(PDA)的持续性通畅是一个严重的问题 影响超过30%的早产儿和1500克，但目前的治疗方法有许多缺点。这个 调节胎儿DA开放和出生后关闭的机制尚未完全解决。我们之前 确定了一些离子通道，这些离子通道是DA特异性治疗的有希望的靶点。其中，氯离子 渠道是一个耐人寻味的前景。尽管阳离子通道在DA中已经确立了作用，但实际上没有 关于氯电流和DA收缩的信息已经存在。一般而言，氯离子在血管中的运输是光滑的 肌细胞(VSMC)是动态的，由特定的通道和辅助转运体精确调节。中的更改 血管氯电流与高血压和其他血管异常有关。此外，众所周知， 利尿剂和氯离子转运抑制剂速尿的临床应用与早产儿动脉导管未闭相关。 这些数据表明，氯-平衡在调节DA音调中起作用。探讨氯离子助熔剂在土壤中的重要性 对于DA，我们首先确定了在DA中显著丰富的氯离子相关基因的子集。我们 结果表明，在DA中，特定的氯输入基因的表达增加，而氯输出基因的表达减弱 在发育过程中，在胎儿时期有利于氯-的输出，但在出生时转向氯-的积累。 分离的DA SMC的膜片钳记录证实了特定氯电流的存在。肌造影术 研究证实了这些电流的功能意义。抑制特定的氯离子通道可阻断O2-和 激动剂对小鼠离体多巴胺的收缩作用。速尿对体外小鼠DA的松弛作用， 不受一氧化氮或前列腺素作用的影响。此外，使用非选择性氯离子通道抑制剂进行治疗 在新生小鼠中产生了一种PDA表型。这些数据强烈地暗示了氯离子转运和氯离子电流 重要的，未被发现的DA收缩调节因子。 在这个提议中，我们将检验这一假设，即氯电流是DA音调的关键介体，并且 出生时阴离子转运的变化DA VSMC跨膜Cl-梯度由抑制转变为 令人兴奋。目标1将确定氯离子电流对子宫DA松弛和 出生后DA关闭。目标2将确定氯离子转运中的发育开关是否调节DA 收缩能力和驱使出生后DA关闭。分子、遗传学、药理学和电生理学 这些方法将确定在体外和体内调节DA通畅性的氯依赖机制。 这些目标的完成将提供有关DA中氯离子稳态的临床相关信息，并可能 为调节DA音寻找新的治疗靶点。

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.