Received for publication October 3, 2008.
Revised November 24, 2008.
Accepted for publication November 24, 2008.

Identification and Quantification of 2',3'-cAMP Release by the Kidney

Abstract

We recently developed a sensitive assay for 3',5'-cAMP using high performance liquid chromatography-tandem mass spectrometry. Using this assay, we investigated the release of 3',5'-cAMP from isolated, perfused rat kidneys. To our surprise, we observed a dominant chromatographic peak that was due to an endogenous substance that had the same parent ion as 3',5'-cAMP and that fragmented to the same daughter ion (adenine) as 3',5'-cAMP. However, the retention time of this unknown was approximately 2.9 minutes compared to 6.3 minutes for authentic 3',5'-cAMP. We hypothesized that the unknown substance was an isomer of 3',5'-cAMP. Indeed, the unknown substance had the same retention time and mass spectral properties as authentic 2',3'-cAMP. Renal venous secretion of 2',3'-cAMP was greater in kidneys from 20-week-old genetically-hypertensive rats compared with age-matched normotensive rats (12.49 ± 2.14 versus 5.32 ± 1.97 ng/min per gram kidney weight, respectively; n=18). Isoproterenol (1 µmol/L; -adrenoceptor agonist) increased renal venous 3',5'-cAMP secretion (approximately 690% of control), but had no effect on 2',3'-cAMP production. In contrast, rapamycin (0.2 µmol/L; activator of mRNA turnover) and iodoacetate + 2,4-dinitrophenol (50 µmol/L; metabolic inhibitors) increased the renal venous secretion of 2',3'-cAMP (approximately 1000% and 4100% of control, respectively) while simultaneously decreasing the renal venous secretion of 3',5'-cAMP. In conclusion, 2',3'-cAMP is a naturally-occurring isomer of 3',5'-cAMP that is: 1) not made by adenylyl cyclase; 2) released from kidneys into the extracellular compartment; 3) released more by kidneys from rats with long-standing hypertension; 4) derived from mRNA turnover; and 5) increased by energy depletion.

Key words: adenosine 2',3'-cyclic monophosphate, adenosine 3',5'-cyclic monophosphate, isolated, perfused kidney, kidney, spontaneously hypertensive rat, tandem liquid chromatography-mass spectrometry