LOCALIZED CAMP SIGNALS IN ENDOTHELIAL CELLS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者的研究机构。 我们实验室的长期目标是了解信息在第二信使信号（包括Ca 2+和cAMP）中编码的机制。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Ca 2+信号的理解急剧增加，主要是由于测量细胞内Ca 2+的单细胞方法的发展。 然而，我们对cAMP信号的理解已经落后，主要是由于缺乏高分辨率的单细胞测量技术。直到最近才开发出测量单细胞中cAMP信号的可靠方法。这些方法提供了一个前所未有的观点，环核苷酸信号附近的几种细胞类型的质膜。我们使用这些方法提供的证据表明，cAMP的有效扩散系数比以前认为的要慢得多（比单独的自由扩散慢约10，000倍）。此外，优雅的研究表明，区室化的cAMP信号对肺微血管内皮细胞（PMVEC）的屏障功能至关重要。基于这些研究，我们提出测试以下工作假设：磷酸二酯酶活性，缓冲，和非常缓慢的cAMP扩散本地化肺微血管内皮细胞中的cAMP信号。

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.