Regulation of Renal Cortical Adenosine Levels

肾皮质腺苷水平的调节

• 批准号: 7687240
• 负责人: EDWIN Kerry JACKSON
• 金额: $ 36.54万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7687240
• 关键词: 2' ,3' -Cyclic-Nucleotide Phosphodiesterases; 2' -adenylic acid; 5' -Nucleotidase; Adenine Nucleotides; Adenosine; Adenylate Cyclase; Affect; Alkaline Phosphatase; Anabolism; Arrhythmia; Arts; Attenuated; Binding; Biochemical; Biological; Biological Assay; Blood Vessels; Brain; Cardiovascular system; Cell membrane; Cell surface; Cells; Chemicals; Cleaved cell; Clinical Medicine; Collecting Cell; Coronary; Coronary Arteriosclerosis; Cyclic AMP; Cyclic Nucleotides; Data; Diagnosis; Diphosphates; Disease; Drug usage; Duct (organ) structure; Electrons; Enzymes; Extracellular Space; Family; Funding; Glomerular Mesangial Cell; Goals; Growth; Head; Heart; Human; Hydrolysis; Inflammatory; Inosine; Kidney; Kidney Diseases; Knockout Mice; Knowledge; Laboratories; Left; Link; Lipids; Liquid Chromatography; Liquid substance; Malignant Neoplasms; Measurement; Measures; Mediating; Membrane; Messenger RNA; Metabolism; Microcirculation; Microdialysis; Molecular Weight; Mus; Myocardial Ischemia; Nucleosides; Nucleotidases; Nucleotides; P-Glycoproteins; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Pilot Projects; Play; Poly(A) Tail; Process; Prodrugs; Production; Progress Reports; Property; Publishing; Pump; Purinergic P1 Receptors; Purines; Rattus; Regulation; Reperfusion Injury; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleases; Role; Sensitivity and Specificity; Side; Sirolimus; Site; Small Interfering RNA; Smooth Muscle Myocytes; Solid; Sorting - Cell Movement; Stents; Stimulus; Structure; Structure of renal vein; Surface; System; Tail; Testing; Time; Tissues; Vascular Endothelial Cell; Western Blotting; Work; adenylyl cyclase 2; body system; ecto-nucleotidase; extracellular; fascinate; gastrointestinal; in vivo; innovation; innovative technologies; instrument; kidney cell; mass spectrometer; meetings; mesangial cell; methylxanthine; multidrug resistance protein 3; novel; nucleotidase; phosphodiester; phosphodiesterase IV; phosphoric diester hydrolase; prevent; public health relevance; purine; quantum; research study; restenosis; skeletal; tandem mass spectrometry

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.

描述（由申请人提供）：我们在上一个资助期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）腺苷酸环化酶在细胞内产生3 ′，5 ′-cAMP; 2）多药耐药蛋白（MRP）介导的细胞内3 ′，5 ′-cAMP外排至细胞表面; 3）外-3 ′，5 ′-cAMP-磷酸二酯酶在细胞外将3 ′，5 ′-cAMP代谢为5 ′-AMP;和4）通过CD 73将5 ′-AMP细胞外转化为腺苷。该途径作为许多器官系统（包括肾脏和心血管系统）中腺苷生物合成的重要机制得到了认可。最近，我们设想了这样的假设，即除了细胞外的3 '，5'-cAMP-腺苷途径之外，可能存在细胞外的2 '，3'-cAMP-腺苷途径：mRNA周转-细胞内的2 '，3'-cAMP-细胞外的2 '，3'-cAMP-细胞外的2 '-AMP/3'-AMP-细胞外的腺苷。这一假说的基本原理是：1）mRNA周转涉及核糖核酸酶，其切割mRNA的聚腺嘌呤尾中的磷酸二酯键形成2 '，3'-cAMP; 2）一些MRP将环核苷酸快速转运到细胞外空间;和3）存在可作为胞外-2 ′，3 ′-cAMP-磷酸二酯酶和胞外-2 ′/3 ′-核苷酸酶水解胞外2 ′，3 ′-cAMP转化为2 ′-AMP/3 ′-AMP，细胞外2 ′-AMP/3 ′-AMP转化为腺苷。当细胞暴露于增强mRNA周转的应激刺激时，这种途径在产生细胞外腺苷方面可能是极其重要的，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种创新技术，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 Quantum-Ultra液相色谱-质谱仪。使用这种仪器，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，支持肾脏产生比3 '，5'-cAMP更多的细胞外2 '，3'-cAMP并且这种2 '，3-cAMP代谢为腺苷的概念。这些确实是潜在的变革性初步观察！因此，这一竞争性更新申请的总体目标是确定肾细胞外2 ′，3 ′-cAMP-腺苷途径是否存在（具体目的1和2），是否由与介导3 '，5'-cAMP-腺苷途径的酶不同的胞外酶介导（具体目标3）以及2 '，3'-cAMP-腺苷途径是否有可能引起生物学效应（具体目标4）。 公共卫生相关性：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，影响身体的每个器官系统。因此，关于身体如何制造腺苷的知识对于阐明许多疾病的机制是重要的，所述疾病包括肾脏、心脏、血管和大脑的疾病，以及炎性疾病和癌症。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。该项目将研究另一种天然存在的化学物质2 '，3'-cAMP到腺苷的代谢。2 '，3'-cAMP很可能是一种“腺苷前药”，作为一种更安全的腺苷形式，在临床医学中可能有多种用途。雷帕霉素（西罗莫司）是一种用于药物洗脱冠状动脉支架的药物，用于预防称为再狭窄的过程。有趣的是，雷帕霉素药理学的一个主要方面是它可以激活腺苷产生系统，这是这个项目的主题。可以想象，该项目将提供更安全和更有效的药物洗脱支架的信息。