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增强分布血管中UA内皮功能的编程事件，以及血管是否可将此类信号向下游传播至阻力血管。在更好地了解妊娠期正常UA内皮功能的过程中，我们可以开始了解异常/患病妊娠中与适应失败相关的血管功能障碍的分子起源。这反过来又将允许设计治疗策略，以针对这些错误或通过补偿性干预尽量减少其影响。妊娠与UA内皮的NO生成增强有关，这主要是由于细胞内蛋白激酶和Ca 2+信号通路的重新映射。响应于激动剂如ATP的Ca 2+信号传导在引起持续和重复数量的Ca 2+爆发方面最显著地改变，并且这反过来又被细胞持续的NO输出的较长持续时间所抑制。我们目前的初步数据表明，这些妊娠特异性爆发是由于TRPC与IP 3-R在细胞内更大的持续相互作用，而且这种持续的相互作用需要通过缝隙连接（CX 43）进行功能性细胞-细胞偶联。特别值得注意的是，这在P-UAEC中比在NP-UAEC中更多地观察到，并且似乎受CX 43本身的磷酸化控制。我们在这个提案中的目标是检验特定目标A：TRPC的假设。IP 3-R和CX43共同介导UAEC中fCa 2 + 1 i的ATP刺激爆发，妊娠进一步增强了这种作用。具体而言，我们将研究以下建议：特定目的B：妊娠增强TRPC、IP 3-R和CX 43的相互作用以控制[Ca 2 +]i，进而部分介导由ATP引起的eNOS活化的妊娠特异性增加。具体目标C：完整船只的翻译：妊娠特异性增强TRPC与IP 3-R的相互作用，由于增加CX43 GAP连接介导妊娠特异性增强的TCa 2 + 1 i爆发和eNOS活性升高，以响应离体UA Endo中的ATP。目标C特别包括在内，因为我们的真正目标是建立怀孕期间发生的生理适应的基础。在整个研究期间，将在离体完整子宫动脉上进行这些平行平移研究，以确定体内运行机制。将对NO和[Ca 2 +]i进行真实的实时成像，以确定怀孕期间观察到的持续Ca 2+爆发是否也与NO爆发相关。通过分析血管的第二代、第三代和第四代，将考虑在初级分支（UAEC来源于初级分支）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CX 43向下游通信/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.