The Guanosine-Adenosine Mechanism

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 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.