A New Enzymatic Assay for Guanosine 3′:5′-Cyclic Monophosphate and Its Application to the Ductus Deferens of the Rat

摘要

A sensitive enzymatic procedure has been developed for the determination of guanosine 3′:5′-cyclic monophosphate (cyclic GMP). It is based on the conversion of cyclic GMP to GMP by cyclic nucleotide phosphodiesterase and on the transfer of ³²P from [γ-³²P]ATP to GMP by the action of a specific ATP:GMP phosphotransferase (EC 2.7.4.8). The [³²P]GDP is separated from the remaining [³²P]ATP by enzymatic degradation of ATP by myosin and by precipitation of the ³²Pi formed. The reaction blank, which is mostly caused by the nucleotide content of the enzymes, is doubled by about 0.1 pmol of cyclic GMP. The procedure has advantages in speed and/or accuracy over other methods in current use.Cyclic nucleotide concentrations were studied in the ductus deferens of the rat; two agents were used, carbachol and norepinephrine, which cause contraction. Incubation with 0.1 mM carbachol caused a 3-fold increase in cyclic GMP content, which was maximal about 2 min after carbachol addition. Cyclic AMP concentrations were not significantly changed. Addition of 0.01 mM norepinephrine increased cyclic GMP content by about 25% within 1 min and by 40% within 3 min; cyclic AMP concentrations were only slightly increased. A 3-min incubation with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (0.1 mM) doubled the cyclic GMP content and increased cyclic AMP concentration by 50%.