Pregnancy/NO Induced Changes in UAE Ca2+ Signaling

描述（由申请人提供）：我们实验室的重点是更全面地了解正常妊娠可以增强分布血管中的UA内皮功能的编程事件，以及血管是否可以将这些信号下游传播到阻力血管。为了更好地了解妊娠期正常UA内皮功能，我们可以开始了解异常/病变妊娠中与适应失败相关的血管功能障碍的分子起源。反过来，这将允许设计治疗策略来针对这些错误或通过补偿性干预最小化其影响。妊娠与UA内皮生成一氧化氮的增强有关，这主要是由于细胞内蛋白激酶和Ca2+信号通路的重新定位。Ca2+信号对激动剂（如ATP）的反应最明显的改变是导致持续和重复的Ca2+爆发，这反过来又与细胞持续NO输出的持续时间延长相平行。我们提供的初步数据表明，这些妊娠特异性爆发是由于细胞内TRPC与IP3-R更大的持续相互作用，并且这种持续的相互作用需要通过间隙连接的功能细胞-细胞偶联（CX43）。特别值得注意的是，这在P-UAEC中比在NP-UAEC中更常见，并且似乎是由CX43本身的磷酸化控制的。我们在本提案中的目标是测试特定目标A: TRPC的假设。IP3-R和CX43共同介导atp刺激的UAEC中fCa2+1i的爆发，这种方式在妊娠中进一步增强。具体而言，我们将研究以下建议：特定目的B：妊娠增强TRPC， IP3-R和CX43的相互作用，以控制[Ca2+]i，进而部分介导妊娠特异性ATP激活eNOS的增加。C：翻译到完整血管：由于CX43 GAP连接的增加，妊娠特异性增强了TRPC与IP3-R的相互作用，介导了UA Endo体内ATP对TCa2+1i爆发和eNOS活性的妊娠特异性增强升高。Aim C特别包括在内，因为我们的真正目标是为怀孕期间发生的生理适应建立基础。在整个研究过程中，这些平行的转化研究将在完整的子宫动脉上进行，以建立在体内运作的机制。将运行NO和[Ca2+]i的实时成像，以确定是否在怀孕期间持续的Ca2+爆发也与NO爆发有关。在初级分支（UAEC的来源）中启动的细胞间通信事件不仅招募更多的局部细胞参与响应，而且允许通过CX43进行下游通信/信号的可能性将通过对第二代、第三代和第四代血管的分析来考虑。

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.