Regulation of Renal Cortical Adenosine Levels

描述(申请人提供)：我们在前一个资助期间的工作建立了一个产生细胞外腺苷的机制，即细胞外3‘，5’-cAMP-腺苷途径，它包括四个连续步骤：1)腺酰环化酶在细胞内产生3‘，5’-cAMP；2)多药耐药蛋白(MRPs)介导细胞内3‘，5’-cAMP外流到细胞表面；3)3‘，5’-cAMP通过ecto-3‘，5’-cAMP细胞外代谢为5‘-AMP；4)CD73将5‘-AMP胞外转化为腺苷。在包括肾脏和心血管系统在内的许多器官系统中，这一途径被认为是腺苷生物合成的重要机制。最近，我们设想，除了细胞外3‘，5’-cAMP-腺苷途径外，可能还存在细胞外2‘，3’-cAMP-腺苷途径：mRNA转换-细胞内2‘，3’-cAMP-细胞外2‘，3’-cAMP-细胞外2‘-AMP/3’-AMP-腺苷。这一假说的基本原理是：1)信使核糖核酸酶参与切割形成2‘，3’-cAMP的核糖核酸酶；2)一些核糖核酸酶将环核苷酸快速转运到胞外空间；3)存在胞外-2‘，3’-cAMP-磷酸二酯酶和胞外-2‘/3’-核苷酸酶，分别将胞外2‘，3’-cAMP和胞外2‘-AMP/3’-AMP水解为腺苷。每当细胞受到应激刺激时，这一途径在产生细胞外腺苷方面可能非常重要，这些刺激会增加mRNA的周转，从而提供“报复性”代谢物--腺苷，以减轻细胞损伤。为了验证这一假设，我们最近在实验室建立了一项创新技术，使我们能够以极高的准确性、特异性和灵敏度测量嘌呤代谢物，即Thermo Electron TSQ量子-超液相色谱质谱计。利用这台仪器，我们在完整的肾脏和培养的肾小球前血管平滑肌细胞和系膜细胞中进行了有趣的初步观察，支持肾脏产生更多的细胞外2‘，3’-cAMP的概念，并且这个2‘，3-cAMP被代谢成腺苷。这些确实是潜在的变革性的初步观察结果！因此，这一竞争性更新应用的总体目标是确定肾细胞外2‘，3’-cAMP-腺苷途径是否存在(特定目标1和2)，它是否由与3‘，5’-cAMP-腺苷途径不同的胞外酶介导(特定目标3)，以及2‘，3’-cAMP-腺苷途径是否有可能引起生物效应(特定目标4)。 与公共健康相关：该项目的目标是更好地了解人体如何产生一种名为腺苷的化学物质。腺苷是一种自然产生的化学物质，会影响身体的每个器官系统。因此，了解人体如何制造腺苷对于阐明许多疾病的机制非常重要，包括肾脏、心脏、血管和大脑的疾病，以及炎症性疾病和癌症。腺苷也是一种重要的药物，具有多种用途，例如用于治疗心律失常、诊断冠状动脉疾病和治疗心肌缺血/再灌注损伤。这个项目将研究另一种自然产生的化学物质2‘，3’-cAMP到腺苷的新陈代谢。2‘，3’-cAMP很可能是一种“腺苷前体药物”，作为一种更安全的腺苷形式，它在临床医学上可能具有多种用途。雷帕霉素(西罗莫斯)是一种用于药物洗脱冠状动脉支架以防止再狭窄的药物。有趣的是，雷帕霉素的药理学的一个主要方面是它可以激活腺苷生产系统，这是这个项目的主题。可以想象，该项目将提供导致更安全和更有效的药物洗脱支架的信息。

DESCRIPTION (provided by applicant): Our work during the previous funding period established the existence of a mechanism that produces extracellular adenosine, i.e., the extracellular 3',5'-cAMP-adenosine pathway that involves four sequential steps: 1) intracellular production of 3',5'-cAMP by adenylyl cyclases; 2) efflux of intracellular 3',5'-cAMP to the cell surface mediated by multidrug resistance proteins (MRPs); 3) extracellular metabolism of 3',5'-cAMP to 5'- AMP by ecto-3',5'-cAMP-phosphodiesterases; and 4) extracellular conversion of 5'-AMP to adenosine by CD73. This pathway is gaining recognition as an important mechanism for adenosine biosynthesis in many organ systems, including the kidney and the cardiovascular system. Recently, we conceived the hypothesis that there may exist, in addition to the extracellular 3',5'-cAMP- adenosine pathway, an extracellular 2',3'-cAMP-adenosine pathway: mRNA turnover-intracellular 2',3'- cAMP-extracellular 2',3'-cAMP-extracellular 2'-AMP/3'-AMP-extracellular adenosine. The rationale for this hypothesis is: 1) mRNA turnover involves ribonucleases that cleave the phosphodiester bonds in the polyadenine tail of mRNA forming 2',3'-cAMP; 2) Some MRPs rapidly transport cyclic nucleotides into the extracellular space; and 3) Enzymes exist that could serve as ecto-2',3'-cAMP-phosphodiesterases and ecto- 2'/3'-nucleotidases to hydrolyze extracellular 2',3'-cAMP to 2'-AMP/3'-AMP and extracellular 2'-AMP/3'-AMP to adenosine, respectively. This pathway could be extremely important in producing extracellular adenosine whenever cells are exposed to stressful stimuli that enhance mRNA turnover, thus providing the "retaliatory" metabolite, adenosine, to mitigate cellular damage. To test this hypothesis, we recently established an innovative technology in our lab that allows us to measure purine metabolites with extreme accuracy, specificity and sensitivity, i.e., the Thermo Electron TSQ Quantum-Ultra liquid chromatograph-mass spectrometer. Using this instrument, we made fascinating preliminary observations in intact kidneys and in cultured preglomerular vascular smooth muscle cells and mesangial cells supporting the concept that the kidney produces more extracellular 2',3'-cAMP than 3',5'- cAMP and that this 2',3-cAMP is metabolized to adenosine. These are indeed potentially transformative preliminary observations! Accordingly, the overall goal of this competing renewal application is to determine whether the renal extracellular 2',3'-cAMP-adenosine pathway exists (Specific Aims 1 and 2), whether it is mediated by ecto-enzymes that are different from those mediating the 3',5'-cAMP- adenosine pathway (Specific Aim 3) and whether the 2',3'-cAMP-adenosine pathway has the potential to cause biological effects (Specific Aim 4). PUBLIC HEALTH RELEVANCE: The goal of this project is to better understand how the body makes a chemical called adenosine. Adenosine is a naturally occurring chemical that affects every organ system in the body. Therefore, knowledge regarding how the body makes adenosine is important for elucidating mechanisms of many diseases including diseases of the kidney, heart, blood vessels and brain, as well as inflammatory diseases and cancer. Adenosine is also an important pharmaceutical (drug) that has multiple uses, for example to treat cardiac arrhythmias, to diagnosis coronary artery disease and to treat myocardial ischemia/reperfusion injury. This project will examine the metabolism of another naturally occurring chemical called 2',3'-cAMP to adenosine. It is likely that 2',3'-cAMP is an "adenosine prodrug" that could have multiple uses in clinical medicine as a safer form of adenosine. Rapamycin (sirolimus) is a drug used in drug-eluting coronary stents to prevent a process called restenosis. Interestingly, a major aspect of the pharmacology of rapamycin is that it may activate the adenosine-producing system that is the subject of this project. It is conceivable that this project will provide information leading to safer and more effective drug-eluting stents.