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 because of 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 min, compared with 6.3 min for authentic 3′,5′-cAMP. We hypothesized that the unknown substance was an isomer of 3′,5′-cAMP. 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/g kidney weight, respectively; n = 18). Isoproterenol (1 μM; β-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 μM; activator of mRNA turnover) and iodoacetate + 2,4-dinitrophenol (50 μM; 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.