Microassay of cyclic nucleotides in vessel wall: V. Simultaneous assay of cyclic AMP and cyclic GMP

Abstract

Cyclic GMP (cGMP) is an important key substance in cell function; however, the related mechanisms have not been entirely elucidated, as the content of this nucleotide within the cell is so small that an accurate estimation is difficult. We designed a microassay for cyclic GMP in 500 μg dry weight of tissue samples, using succinylation, and were able to assay, separately, the content of cyclic GMP in the intima or media of the arterial wall. In addition, we attempted to develop a method for a simultaneous determination of both cyclic AMP and cyclic GMP in a small amount of the same assay mixture. Five hundred micrograms dry weight of intima or media of rabbit aorta was prepared under a stereomicroscope. After deproteinization and lyophilization, the samples were dissolved with 60 μl of H 2O and 30 μl of succinylation mixture. After succinylation, 40 μl of each of the tissue extractions was sampled to determine the levels of cyclic AMP and cyclic GMP. The radioimmunoassay was performed, using Yamasa's kit. The recovery rates of cyclic AMP and cyclic GMP in our method were 86.2 ± 1.2 and 87.6 ± 1.3%, respectively. The levels of cyclic GMP in the intima and media of aorta of cow, pig, and rabbits were estimated to be, intima: 0.18 ± 0.02, 0.06 ± 0.03, and 0.20 ± 0.01 pmole/mg protein, media: 0.08 ± 0.01, 0.04 ± 0.01, and 0.09 ± 0.01. The cyclic GMP levels in the intima of aorta of cow and rabbits were high compared to levels in the media and this difference was statistically significant ( P < 0.05).