2',3'-cGMP exists in vivo and comprises a 2',3'-cGMP-guanosine pathway

The discovery in 2009 that 2',3'-cAMP exists in biological systems was rapidly followed by identification of 2',3'-cGMP in cell and tissue extracts. To determine whether 2',3'-cGMP exists in mammals under physiological conditions, we used ultraperformance LC-MS/MS to measure 2',3'-cAMP and 2',3'-cGMP in timed urine collections (via direct bladder cannulation) from 25 anesthetized mice. Urinary excretion rates (means ± SE) of 2',3'-cAMP (15.5 ± 1.8 ng/30 min) and 2',3'-cGMP (17.9 ± 1.9 ng/30 min) were similar. Mice also excreted 2'-AMP (3.6 ± 1.1 ng/20 min) and 3'-AMP (9.5 ± 1.2 ng/min), hydrolysis products of 2',3'-cAMP, and 2'-GMP (4.7 ± 1.7 ng/30 min) and 3'-GMP (12.5 ± 1.8 ng/30 min), hydrolysis products of 2',3'-cGMP. To validate that the chromatographic signals were from these endogenous noncanonical nucleotides, we repeated these experiments in mice (n = 18) lacking 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), an enzyme known to convert 2',3'-cyclic nucleotides to their corresponding 2'-nucleotides. In CNPase-knockout mice, urinary excretions of 2',3'-cAMP, 3'-AMP, 2',3'-cGMP, and 3'-GMP were increased, while urinary excretions of 2'-AMP and 2'-GMP were decreased. Infusions of exogenous 2',3'-cAMP increased urinary excretion of 2',3'-cAMP, 2'-AMP, 3'-AMP, and adenosine, whereas infusions of exogenous 2',3'-cGMP increased excretion of 2',3'-cGMP, 2'-GMP, 3'-GMP, and guanosine. Together, these data suggest the endogenous existence of not only a 2',3'-cAMP-adenosine pathway (2',3'-cAMP → 2'-AMP/3'-AMP → adenosine), which was previously identified, but also a 2',3'-cGMP-guanosine pathway (2',3'-cGMP → 2'-GMP/3'-GMP → guanosine), observed here for the first time. Because it is well known that adenosine and guanosine protect tissues from injury, our data support the concept that both pathways may work together to protect tissues from injury.