Simultaneous Measurement of Adenosine 3',5'-Cyclic Monophosphate and Guanosine 3',5'-Cyclic Monophosphate in Biological Samples

Two sensitive enzymatic fluorometric assays have been developed for adenosine 3’,5’-cyclic monophosphate（cAMP） by Sugiyama et al. （Anal. Biochem. 1990） and for guanosine 3’,5’-cyclic monophosphate （cGMP） by Seya al. （Anal. Biochem. 1998）. However, to make a new simultaneous comparison of two cyclic nucleotides, distinct methods cause less reliability and longer measurement time. To overcome these problems, we a simultaneous measurement method for them. All adenosine nucleotides and GMP were enzymatically using alkaline phosphatase and apyrase. The remaining GDP was converted to GTP by creatine kinase. GMP and cAMP, absorbed into a Sep-Pak amino propyl cartridge, were eluted to separate from GTP, and were simultaneously quantified using improved enzymatic fluorometric assay. The detection limits for cAMP cGMP were 1 and 5 fmol, respectively. The total measurement time was about 10 h. Using this method, the cAMP and cGMP levels in rat aortic smooth muscle cells were confirmed to 4.1 and 0.042 pmol/mg protein,. An adrenergic agonist, isoproterenol （1 μM） increased cAMP approximately 6 fold, while nitric oxide, S-nitroso-N-acetylpenicillamine （100 μM） increased cGMP approximately 230 fold. These results suggest that simultaneous measurement of cGMP and cAMP can provide a more convenient assessment of them in biological samples.