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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

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

Departments of Medicine (J.R., Z.M., N.A.S., E.K.J.) and Pharmacology and Chemical Biology (E.K.J.), Center for Clinical Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

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.

Address correspondence to: Dr. Edwin K. Jackson, Center for Clinical Pharmacology, Departments of Pharmacology and Chemical Biology and of Medicine, University of Pittsburgh School of Medicine, 100 Technology Drive, Suite 450, Pittsburgh, PA 15219-3130. E-mail: edj{at}pitt.edu