The Guanosine-Adenosine Mechanism

 描述(申请人提供)：1999年，邹丽红及其同事的基本发现是，在肾脏中，腺苷A2受体扩张髓质血管，导致利钠/利尿。在HL109002的前一次资助期间，我们发现胞外鸟苷通过抑制胞外腺苷的处置(我们称之为鸟苷-腺苷机制)显著增加胞外腺苷水平。我们还发现，8-氨基鸟苷[嘌呤核苷磷酸化酶(PNPase-代谢鸟苷的酶)的抑制剂]增加了细胞外的鸟苷，从而激活了鸟苷-腺苷机制，即增加了腺苷水平。结合邹某的结论和我们的研究结果，我们进行了初步实验，研究8-氨基鸟苷对肾功能的影响；这些初步实验的结果表明，8-氨基鸟苷确实诱导利钠/利尿，长期服用具有强大的降压作用。由于8-氨基鸟苷作为PNPase抑制剂的效力是8-氨基鸟苷的10倍，我们还假设8-氨基鸟苷的肾脏效应是通过转化为8-氨基鸟苷来实现的，这一假设已经经受住了初步测试。最后，由于有报道称组织中存在8-氨基鸟苷(由过氧亚硝酸根与鸟苷反应，然后将8-硝基鸟苷还原为8-氨基鸟苷)，我们建立了一种基于质谱学的8-氨基鸟苷和8-氨基鸟苷的分析方法，并证明了它们在肾脏和尿液中的存在。因此，我们的初步发现支持了一种令人兴奋的可能性，即我们已经发现了一种以前未被认识到的调节肾脏排泄功能和血压的系统。简而言之，我们的假设是8-氨基鸟嘌呤是一种内源性嘌呤，具有利钠、利尿和抗高血压活性，8-氨基鸟嘌呤通过阻断肾脏PNPase发挥作用，该酶参与肾脏的鸟苷-腺苷机制。我们建议在大鼠身上验证这一假设：1)表征静脉(IV)或直接进入肾动脉(IRA)给予8-氨基鸟苷(IV)和8-氨基鸟苷(IRA)的利尿/利钠、肾脏血流动力学效应和对尿嘌呤的影响；2)用LC-MS/MS测定IV和IRA对肾间质和肾组织8-氨基鸟苷、8-氨基鸟苷、鸟苷、腺苷和肌苷水平的影响；3)确定9-去氮鸟苷(PNPase特异性抑制剂)是否能模拟8-氨基鸟苷和8-氨基鸟苷对肾脏排泄功能的影响；4)使用基因敲除大鼠确定8-氨基鸟苷/8-氨基鸟苷的肾脏排泄作用是否需要A2A或A2B，而不是A1受体；5)测试过氧亚硝酸盐的产生是否决定内源性8-氨基鸟苷/8-氨基鸟苷的水平；以及6)使用放射遥测方法测定口服8-氨基鸟苷/8-氨基鸟嘌呤的长期降压活性。

 DESCRIPTION (provided by applicant): In 1999, Zou and colleagues made the fundamental discovery that in the kidney, adenosine A2 receptors vasodilate the medullary vasculature leading to natriuresis/diuresis. During the previous funding period for HL109002, we discovered that extracellular guanosine remarkably increases extracellular levels of adenosine by inhibiting the disposition of extracellular adenosine (we call this the guanosine-adenosine mechanism). We also discovered that 8-aminoguanosine [inhibitor of purine nucleoside phosphorylase (PNPase - the enzyme that metabolizes guanosine)] increases extracellular guanosine and thereby activates the guanosine- adenosine mechanism, i.e., increases adenosine levels. Combining Zou's conclusions with our findings prompted us to conduct preliminary experiments examining the effects of 8-aminoguanosine on renal function; and the results of these preliminary experiments suggest that 8-aminoguanosine does indeed induce natriuresis/diuresis and administered chronically is powerfully antihypertensive. Because 8-aminoguanine is 10-fold more potent as a PNPase inhibitor compared to 8-aminoguanosine, we also postulated that the renal effects of 8-aminoguanosine are mediated by conversion to 8-aminoguanine, a postulate that has withstood preliminary testing. Finally, because there are reports of 8-aminoguanosine in tissues (produced from peroxynitrite reacting with guanosine moieties followed by reduction of 8-nitroguanosine to 8- aminoguanosine), we developed a mass spectrometry-based assay for 8-aminoguanosine and 8- aminoguanine and demonstrated their existence both in kidneys and urine. Thus our preliminary findings support the exciting possibility that we have discovered a previously unrecognized system that regulates renal excretory function and blood pressure. In a nutshell, our hypothesis is that 8-aminoguanine is an endogenous purine that exerts natriuretic, diuretic, and antihypertensive activity and that 8- aminoguanine acts by blocking renal PNPase, which engages the guanosine-adenosine mechanism in the kidney. We propose to test this hypothesis in rats by: 1) Characterizing the diuretic/natriuretic, renal hemodynamic effects and effects on urinary purines of administering 8-aminoguanosine and 8-aminoguanine either intravenously (IV) or directly into the renal artery (IRA); 2) Determining using LC-MS/MS the effects of IV and IRA 8-aminoguanosine and 8-aminoguanine on renal interstitial levels and kidney tissue levels of 8- aminoguanosine, 8-aminoguanine, guanosine, adenosine, and inosine; 3) Determining whether 9- deazaguanine (specific PNPase inhibitor) can mimic the effects of 8-aminoguanosine and 8-aminoguanine on renal excretory function; 4) Determining, using knockout rats, whether the renal excretory effects of 8- aminoguanosine/8-aminoguanine require A2A or A2B, but not A1, receptors; 5) Testing whether peroxynitrite production determines the levels of endogenous 8-aminoguanosine/8-aminoguanine; and 6) Determining, using radiotelemetry, the long-term antihypertensive activity of oral 8-aminoguanosine/8-aminoguanine.