2017 Volume 68 Issue 1 Pages 52-61
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 et al. (Anal. Biochem. 1998). However, to make a new simultaneous comparison of two cyclic nucleotides, distinct measurement methods cause less reliability and longer measurement time. To overcome these problems, we developed a simultaneous measurement method for them. All adenosine nucleotides and GMP were enzymatically degraded using alkaline phosphatase and apyrase. The remaining GDP was converted to GTP by creatine kinase. Cyclic GMP and cAMP, absorbed into a Sep-Pak amino propyl cartridge, were eluted to separate from GTP, and then were simultaneously quantified using improved enzymatic fluorometric assay. The detection limits for cAMP and cGMP were 1 and 5 fmol, respectively. The total measurement time was about 10 h. Using this method, the basal cAMP and cGMP levels in rat aortic smooth muscle cells were confirmed to 4.1 and 0.042 pmol/mg protein, respectively. An adrenergic agonist, isoproterenol (1 μM) increased cAMP approximately 6 fold, while nitric oxide donor, S-nitroso-N-acetylpenicillamine (100 μM) increased cGMP approximately 230 fold. These results suggest that this simultaneous measurement of cGMP and cAMP can provide a more convenient assessment of them in biological samples.
