Pregnancy/NO Induced Changes in UAE Ca2+ Signaling

描述(由申请人提供)：我们的实验室重点是更全面地了解正常妊娠通过哪些编程事件可以增强UA血管分配血管的内皮功能，以及血管是否可以将这种信号向下游传播到阻力血管。随着对正常妊娠尿酸内皮细胞功能的进一步了解，我们可以开始了解异常/疾病妊娠中与适应失败相关的血管功能障碍的分子起源。这反过来将允许设计针对此类错误的治疗策略，或通过补偿性干预将其影响降至最低。妊娠与尿酸内皮细胞产生的NO增加有关，这主要是由于蛋白激酶和钙信号通路在细胞内的重新映射。钙信号对激动剂如三磷酸腺苷的反应发生了最显著的改变，导致了持续和重复的钙爆发，而这反过来又与细胞持续产生NO的较长时间相平行。我们提供的初步数据表明，这些妊娠特异性猝发是由于TRPC与IP3-R在细胞内更大的持续相互作用，而且这种持续相互作用需要通过缝隙连接(Cx43)的方式实现细胞与细胞之间的功能耦合。特别值得注意的是，这在P-UAEC中比在NP-UAEC中更常见，并且似乎受Cx43自身的磷酸化控制。我们在这个提案中的目标是测试特定目标A：TRPC的假设。IP3-R和Cx43共同作用于介导ATP刺激的UAEC内fCa~(2+)1i的爆发，这种作用因怀孕而进一步增强。具体地说，我们将研究以下建议：特定的目标B：妊娠增强的TRPC、IP3-R和Cx43的相互作用，以控制[Ca+]i，进而部分地介导由ATP引起的妊娠特异性eNOS激活的增加。特定目标C：转化为完整的血管：由于Cx43缝隙连接增加而导致TRPC与IP3-R相互作用的妊娠特异性增强，介导了UA Endo体外反应中TCa+1i猝发和eNOS活性的妊娠特异性增强。尤其包括目标C，因为我们的真正目标是建立发生在怀孕期间的生理适应的基础。这些平行的翻译研究将在整个研究过程中对完整的子宫动脉进行，以建立在体内运行的机制。将对NO和[Ca~(2+)]i进行实时成像，以确定妊娠期间持续的Ca~(2+)猝发是否也与NO~(2+)猝发有关。通过对第二代、第三代和第四代血管的分析，将考虑在初级分支(UAEC的来源)发起的细胞-细胞通信事件不仅招募更多本地细胞参与反应，而且允许通过Cx43向下游传递/信号的可能性。

DESCRIPTION (provided by applicant): Our laboratories focus is to understand more fully the programming events through which normal pregnancy can enhance UA endothelial function in distributing vessels and if vessels can propagate such signals downstream to resistance vessels. In achieving a greater understanding of normal UA endothelial function in pregnancy we can begin to understand the molecular origins of vascular dysfunction associated with failed adaptation in abnormal/diseased pregnancies. This in turn will allow the design of therapeutic strategies to target such errors or minimize their impact through compensatory intervention. Pregnancy is associated with enhanced NO production by UA endothelium, largely due to a remapping of both protein kinase and Ca2+ signaling pathways within the cell. Ca2+ signaling in response to agonists such as ATP is most notably altered in bringing about a sustained and repeated number of Ca2+ bursts and this in turn is paralleled by the longer duration of sustained NO output by the cells. We present preliminary data that these pregnancy specific bursts are due to greater continued interaction of TRPC with IP3-R within the cells and further that such sustained interaction requires functional cell-cell coupling by way of Gap junctions (CX43). Of particular note this is seen more in P-UAEC than in NP-UAEC, and appears to be controlled by phsophorylation of CX43 itself. Our goal in this proposal is to test the hypothesis that Specific Aims A: TRPC. IP3-R and CX43 work together to mediate ATP-stimulated bursts in fCa2+1i in UAEC, in a manner further enhanced by pregnancy. Specifically we will investigate the proposal that: Specific Aims B: Pregnancy enhanced interactions of TRPC, IP3-R and CX43 to control [Ca2+]i in turn mediate, in part, pregnancy specific increases in eNOS activation by ATP . Specific Aims C: Translation to Intact Vessels: Pregnancy specific enhancement of interactions of TRPC with IP3-R due to increased CX43 GAP junctions mediates the pregnancy specific enhanced elevation of TCa2+1i bursts and eNOS activity in response to ATP in UA Endo ex vivo. Aim C in particular is included since our true goal is to establish the basis for the physiologic adaptation occurring in pregnancy. These parallel translational studies will be run on intact Uterine Artery ex vivo throughout the study, in order to establish the mechanisms operating in vivo. Real time imaging of NO and [Ca2+]i will be run to establish in particular if sustained Ca2+ bursts seen in pregnancy are also associated with NO bursts. The possibility that cell-cell communication events initiated in the primary branches (from which UAEC are derived) not only recruit more local cells to the response but allow communicate/signal downstream via CX43 will be considered by analysis of secondary, tertiary and quaternary generations of vessels.