Assessment of Nerve Stimulation-Induced Release of Purines from Mouse Kidneys by Tandem Mass Spectrometry

Abstract

Adenosine, formed from AMP and metabolized to inosine, modulates renal sympathetic neurotransmission. The present study had two goals: 1) to develop ultrasensitive and specific mass spectrometry-based assays for cAMP, AMP, adenosine, inosine, and, for comparison, guanosine using state-of-the-art tandem liquid chromatography-mass spectrometry (LC-MS-MS); and 2) to quantify the effects of renal sympathetic nerve stimulation on the release of cAMP, AMP, adenosine, inosine, and guanosine from the isolated, perfused mouse kidney. Using LC-MS-MS, we developed highly sensitive (detection limit of 0.02-0.05 pg/microl) and accurate (r(2) > 0.99) assays for all the aforementioned compounds. In the perfused mouse kidney (n = 9), periarterial (renal sympathetic) nerve stimulation elicited a frequency-dependent (0, 3, 5, 7, and 9 Hz) and significant (p = 0.0148, repeated measures analysis of variance) increase in the concentration of inosine in the renal venous perfusate (29 +/- 8, 51 +/- 8, 54 +/- 11, 65 +/- 15, and 80 +/- 20 pg/microl, respectively), yet concomitantly decreased (p = 0.0239, repeated measures analysis of variance) the concentration of AMP in the renal venous perfusate (3.8 +/- 1.3, 3.2 +/- 1.7, 2.4 +/- 1.5, 2.0 +/- 1.1, and 1.1 +/- 0.4 pg/microl, respectively). No significant changes were observed in the levels of adenosine, cAMP, or guanosine in the renal venous perfusate. These results indicate that using state-of-the-art mass spectrometric methods, it is possible to investigate trace amounts of purines released from mouse organs in perfusion and that renal sympathetic nerve stimulation is associated with a robust increase in the main metabolite of adenosine (inosine), while concomitantly decreasing AMP. This suggests that renal sympathetic nerve stimulation influences the efficiency of AMP conversion to adenosine and hence to inosine.