Regulation of Renal Cortical Adenosine Levels

描述（由申请人提供）：我们在上一个资助期内的工作建立了一种产生细胞外腺苷的机制，即细胞外3',5'-cAMP-腺苷途径，包括四个连续步骤：1)通过腺苷酸环化酶在细胞内产生3',5'-cAMP；2)多药耐药蛋白介导的细胞内3′，5′-cAMP向细胞表面外排；3)胞外3′，5′- camp磷酸二酯酶将3′，5′- camp代谢为5′- AMP；4) CD73将5′-AMP细胞外转化为腺苷。这一途径被认为是许多器官系统（包括肾脏和心血管系统）中腺苷生物合成的重要机制。最近，我们提出了一个假设，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翻转-细胞内2’，3’- camp -细胞外2’，3’- camp -细胞外2’- amp /3’- amp -细胞外腺苷。这一假设的基本原理是：1)mRNA的转换涉及核糖核酸酶，它在mRNA的多腺嘌呤尾部切割磷酸二酯键，形成2',3'-cAMP；2)一些MRPs快速将环核苷酸转运到细胞外空间；3)细胞外2’、3’- camp -磷酸二酯酶和2’/3’-核苷酸酶可分别将细胞外2’、3’- camp水解为2’- amp /3’- amp和细胞外2’- amp /3’- amp水解为腺苷。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，从而增加mRNA的转换，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，我们最近在我们的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，即热电子TSQ量子超液相色谱-质谱联用仪。使用该仪器，我们在完整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令人兴奋的初步观察，支持肾脏产生比3',5'- cAMP更多的细胞外2',3'-cAMP的概念，并且这种2'，3-cAMP被代谢为腺苷。这些确实是潜在的革命性的初步观察！因此，这项竞争性更新申请的总体目标是确定肾脏细胞外2’，3’- camp -腺苷途径是否存在（Specific Aims 1和2），是否由不同于介导3’，5’- camp -腺苷途径的外泌酶介导（Specific Aim 3），以及2’，3’- camp -腺苷途径是否具有引起生物学效应的潜力（Specific Aim 4）。

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.