The Guanosine-Adenosine Mechanism

描述(申请人提供)：腺苷和鸟苷是天然存在的，结构相似的嘌呤。此外，自然产生的腺苷和鸟苷的环状类似物也参与了类似的生理过程(例如，cAMP和cGMP都介导血管扩张)。奇怪的是，乍一看，进化似乎打破了腺苷和鸟苷之间的对称性，赋予细胞腺苷受体，而不是鸟苷受体。因此，标准的观点是，腺苷通过激活自己的细胞表面受体来作为细胞功能的生理调节器，而鸟苷只是一个无所事事的旁观者。这项申请的目的是挑战这一公认的观点，提出鸟苷在细胞生理学中也发挥重要作用，不是通过它自己的受体，而是通过参与涉及腺苷的强大合作机制。在这一点上，我们假设胞外鸟苷是腺苷胞外水平的主要生理决定因素，因为鸟苷阻止了腺苷从胞外隔室的处置，从而由于胞外腺苷水平的增加而允许更大的腺苷细胞表面受体的激活。我们将这种鸟苷-腺苷相互作用称为鸟苷-腺苷机制。我们的初步实验有力地证明了我们的假设是正确的。在肾小球前血管平滑肌细胞(微血管平滑肌细胞的模型系统)中，我们观察到，对于任何给定量的腺苷添加到细胞中，当鸟苷也添加到细胞中时，细胞外的腺苷浓度[通过高效液相色谱-质谱仪(LC-MS/MS)测量]增加了10倍以上。我们还发现，细胞应激/损伤会增加细胞外鸟苷和腺苷的水平。此外，我们发现鸟苷在体内可以显著增强腺苷的生物效应！这些发现表明，胞外鸟苷通过阻断胞外腺苷的处置，是迄今发现的影响胞外腺苷水平的最重要的生理决定因素。我们建议测试鸟苷-腺苷机制的存在、机制和重要性，具体有以下六个目的：目的1-确定在血管和肾细胞类型中是否存在鸟苷-腺苷机制；目的2-确定鸟苷-腺苷机制是由鸟苷直接介导的，还是通过鸟苷向鸟苷的转化间接介导的；目标3-确定鸟苷-腺苷机制是否涉及鸟苷对腺苷代谢相关酶的抑制；目标4-确定鸟苷-腺苷机制是否涉及鸟苷抑制参与腺苷摄取的转运体；目的5-确定鸟苷-腺苷机制是否对细胞应激和能量耗竭后细胞外腺苷水平的升高起重要作用；以及目标6-确定鸟苷-腺苷机制是否在体内放大腺苷的作用。

DESCRIPTION (provided by applicant): Adenosine and guanosine are naturally-occurring and structurally similar purines. Moreover, naturally- occurring cyclic analogues of adenosine and guanosine are involved in similar physiological processes (e.g., both cAMP and cGMP mediate vasodilation). Strangely, at first blush it would seem that evolution broke the symmetry between adenosine and guanosine by bestowing cells with adenosine, but not guanosine, receptors. Thus the standard view is that adenosine serves as a physiological regulator of cellular function via activation of its own cell-surface receptors, while guanosine is merely an idle bystander. The purpose of this application is to challenge this received view by proposing that guanosine also plays an important role in cellular physiology, not via its own receptors but rather by engaging a powerful collaborative mechanism involving adenosine. In this regard, we hypothesize that extracellular guanosine is THE major physiological determinant of extracellular levels of adenosine because guanosine blocks the disposition of adenosine from the extracellular compartment allowing greater activation of adenosine cell-surface receptors due to increased extracellular adenosine levels. We refer to this guanosine-adenosine interaction as the GUANOSINE- ADENOSINE MECHANISM. Our pilot experiments strongly suggest that our hypothesis is correct. In preglomerular vascular smooth muscle cells (a model system for microvascular smooth muscle cells) we observe that for any given amount of adenosine added to cells, the extracellular concentrations of adenosine [as measured by high performance liquid chromatography-mass spectrometry (LC-MS/MS)] are more than 10- fold higher when guanosine is also added to the cells. We also find that cellular stress/injury increases extracellular levels of BOTH guanosine and adenosine. Moreover, we find that guanosine PROFOUNDLY augments the biological effects of adenosine in vivo! These findings suggest that extracellular guanosine, by blocking the disposition of extracellular adenosine, is the most important physiological determinant of extracellular levels of adenosine yet discovered. We propose to test the existence of, the mechanism of and the importance of the Guanosine-Adenosine Mechanism with the following six Specific Aims: Aim 1 - To determine the existence of the Guanosine-Adenosine Mechanism in vascular and renal cell types; Aim 2 - To determine whether the Guanosine-Adenosine Mechanism is mediated directly by guanosine or indirectly by conversion of guanosine to guanine; Aim 3 - To determine whether the Guanosine-Adenosine Mechanism involves inhibition by guanosine of enzymes involved in adenosine metabolism; Aim 4 - To determine whether the Guanosine-Adenosine Mechanism involves inhibition by guanosine of transporters involved in adenosine uptake; Aim 5 - To determine whether the Guanosine-Adenosine Mechanism importantly contributes to elevated extracellular levels of adenosine following cellular stress with energy depletion; and Aim 6 - To determine whether the Guanosine-Adenosine Mechanism amplifies the effects of adenosine in vivo.