Pregnancy/NO Induced Changes in UAE Ca2+Signaling

描述(由申请人提供)：在美国，先兆子痫(PE)对多达20%的怀孕有不利影响。最早的事件之一是母体血管内皮血管扩张功能无法适应，与正常妊娠相关的NO增加被钝化或丢失。在我们之前的资助期间，我们在新鲜分离的子宫动脉内皮细胞上进行了[Ca~(2+)]i和NO的双重成像，表明在正常妊娠中，Ca~(2+)信号的增强是NO产生增加的主要原因，这种增强依赖于Ca~(2+)信号介质功能的特定变化。持续的钙离子通过TRPC通道进入UA内皮细胞，但我们的初步数据也表明，妊娠促进的TRPC通道的开放反过来依赖于Cx43缝隙连接通讯，这种细胞间的耦合可能发生在电水平。我们的数据进一步表明，KCA型通道的膜超极化连接了增加的缝隙连接功能，从而增强了妊娠期间的TRPC功能。与此一致的是，UAEC对KCA功能拮抗剂(Apamin和Tram34)表现出敏感性，导致钙信号转导减少。如果这个模型是正确的，那么未来对于PE妊娠中钙信号适应失败的治疗可以通过使用SK或IK类型的KCA通道激动剂来实现。在这次更新中，我们建议测试这一模型，从培养中的分离细胞转移到体外完整的血管，再到整个动物研究。我们将检测1)在低密度和高密度条件下，NP-和P-UAEC细胞膜电位和KCA电流活性对ATP的响应变化，以及这些变化与缝隙连接功能的依赖关系，并确定拮抗剂Apamin和TRAM-34或选择性激动剂NS309对钙通道活性的影响；2)检测这些拮抗剂和激动剂对完整UA血管钙离子、膜电位和NO产生的影响；3)确定KCA激动剂NS309在调节妊娠期间在体子宫动脉血流量的NO依赖性增加方面是否与ATP/UTP的作用相当，以及SK和/或IK类型的KCA通道在多大程度上介导了这一作用。通过这种方式，我们利用我们在妊娠血流适应机制知识方面的最新进展，现在确立了在怀孕期间使用KCA通道激动剂在体内最大化这种反应的有效性，特别是那些合并PE的患者。成功可能会对改善人类PE妊娠治疗的战略产生重大影响，在这种情况下，这种正常的适应性反应已经失败，并且对当前的治疗方法具有抵抗力。 与公共卫生相关：子痫前期(PE)是一种潜在的破坏性妊娠疾病，可威胁母亲和未出生婴儿的生命。它影响了多达20%的人口。即使是轻微的PE也是危险的，可能会导致早产，这会增加婴儿成年后患上多种疾病的风险，如血压、糖尿病和肥胖症。人们认为，在PE中，子宫动脉血管衬里的细胞未能增加其功能，否则在正常妊娠中会发生这种情况。我们有证据表明，正常的适应是由于电变化，它们的失败可能是用一种特定的药物NS309“修复”的。这个项目的目的是通过在动物模型上进行测试来找出我们的怀疑是否正确。如果我们是正确的，我们可能会提出一种治疗PE妊娠的方案。对怀孕期间细胞功能如何调控有了更全面的了解，肯定会让我们离克服这种潜在的破坏性疾病又近了一步，这种疾病影响着国内外的许多人。

DESCRIPTION (provided by applicant): Preeclampsia (PE) adversely affects as many as 20% of pregnancies in the USA. One of the earliest events is the failure of maternal vascular endothelium vasodilatory function to adapt, with the normal pregnancy-associated increase in NO being blunted or lost. In our previous funding period we used dual imaging of [Ca2+]i and NO in freshly isolated uterine artery endothelium to show that in normal pregnancy, enhancement of Ca2+ signaling is the primary cause of enhanced NO production, and such enhancement depends on specific changes in function of Ca2+ signaling mediators. Sustained Ca2+ entry into UA endothelium occurs through TRPC channels but our preliminary data also suggests the pregnancy enhanced opening of TRPC channels in turn depends on CX43 Gap junction communication, and that such coupling of cells may occur at an electrical level. Our data further suggests membrane hyperpolarization by KCa type channels connects increased Gap junction function to enhance TRPC function in pregnancy. Consistent with this, UAEC show sensitivity to antagonists of KCa function (Apamin and Tram34) with a resulting reduction in Ca2+ signaling. If this model is correct then future therapy for failed adaptation of Ca2+ signaling in PE pregnancy could be achieved by the use of SK or IK type KCa channel agonists. In this renewal we propose to test this model, moving from isolated cells in culture to whole vessels ex vivo to whole animal studies. We will examine 1) Changes in membrane potential and KCa current activity in response to ATP in NP- and P-UAEC in cells at low vs high density, dependence of these changes on gap junction function, and determine the effect of antagonists Apamin and Tram-34 or the selective agonist NS309 on Ca2+ burst activity to implicate KCa channels in this response; 2) Examine the effects of these same antagonists and agonists on changes in Ca2+, membrane potential and NO production in intact UA vessels ex vivo; 3) determine if the KCa agonist NS309 is comparable to the effects of ATP/UTP in mediating NO dependent increases in blood flow in uterine artery in vivo during pregnancy, and also the extent to which SK and/or IK types of KCa channel mediate this effect. In this way we take our recent advances in mechanistic knowledge of pregnancy adaptation of blood flow to now establish the validity of using KCa channel agonists to maximize this response in vivo in pregnancy and particularly those complicated by PE. Success could have major impact on strategies to improve treatment of human PE pregnancy where this otherwise normal adaptive response has failed and is resistant to current therapies. PUBLIC HEALTH RELEVANCE: Preeclampsia (PE) is a potentially devastating disease of pregnancy that can threaten the life of both mother and the unborn child. It affects as many as 20% of the population. Even mild PE is dangerous and can cause preterm (early) birth and this then gives the baby an increased risk to develop several diseases such as blood pressure, diabetes and obesity as an adult. It is believed that in PE the cells lining the uterine artery blood vessels fail to increase their function as would otherwise happen in normal pregnancy. We have evidence to suggest normal adaptation is due to electrical changes and their failure may be 'fixed' with a specific drug NS309. The aim of this project is to find out if our suspicions are correct by testing in an animal model. If we are right we may be able to come up with a treatment plan fro PE pregnancy. Having more complete knowledge of how cell function is regulated in pregnancy will certainly take us one step closer to overcoming this potentially devastating disease that effects so many in this country and beyond.