LOCALIZED CAMP SIGNALS IN ENDOTHELIAL CELLS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们实验室的长期目标是了解第二信使信号中信息编码的机制，包括钙离子和cAMP。已知这些信号通路调节多种过程，包括细胞兴奋性、增殖和基因表达。在过去的30年里，我们对钙信号的了解大大增加，这主要是由于单细胞测量细胞内钙的方法的发展。然而，我们对cAMP信号的理解一直滞后，这主要是由于缺乏高分辨率的单细胞测量技术。直到最近，才开发出可靠的方法来测量单细胞中的cAMP信号。这些方法提供了几种细胞类型质膜附近的环核苷酸信号的前所未有的视角。我们使用这些方法来提供证据，表明cAMP的有效扩散系数比之前认为的要慢得多(移动速度比单独使用自由扩散慢10,000倍)。此外，优雅的研究表明，区域化的cAMP信号对肺微血管内皮细胞(PMVECs)的屏障功能至关重要。基于这些研究，我们建议检验以下工作假设：磷酸二酯酶活性、缓冲和异常缓慢的cAMP扩散使cAMP信号定位于肺微血管内皮细胞。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term goal of our laboratory is to understand the mechanisms by which information is encoded within second messenger signals, including Ca2+ and cAMP. These signaling pathways are known to regulate diverse processes including cellular excitability, proliferation, and gene expression. Our understanding of Ca2+ signals has increased dramatically over the last 30 years, primarily due to the development of single-cell methods for measuring intracellular Ca2+. Our understanding of cAMP signals, however, has lagged behind, largely due to the lack of high-resolution, single-cell measurement techniques. Only recently have reliable approaches for measuring cAMP signals in single cells been developed. These approaches have provided an unprecedented view of cyclic nucleotide signals near the plasma membranes of several cell types. We have used these approaches to provide evidence suggesting that the effective diffusion coefficient of cAMP is considerably slower than had been previously thought (~10,000-fold slower movement than by free diffusion alone). In addition, elegant studies demonstrated that compartmentalized cAMP signals are critical for barrier function in pulmonary microvascular endothelial cells (PMVECs). Based upon these studies, we propose testing the following working hypothesis: phosphodiesterase activity, buffering, and anomalously slow cAMP diffusion localize cAMP signals in pulmonary microvascular endothelial cells.