動脈壁 Cyclic Nucleotides に関する研究 (IV)

Abstract

Cyclic GMP (c-GMP) 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 was difficult. We designed a microassay for cyclic GMP in 500μg d.w. 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. 500μg d.w. of intima or media of rabbit aorta was prepared under a streomicroscope. After deproteinization and lyophilization, the samples was dissolved with 60μl of H2O and 30μl of succinylation mixture (40mg succinic anhydride dissolved in 0.9ml dioxane was added: triethylamine=9:1). After succinylation, 40μl of each of the tissue extractions were 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 as compared to levels in the media and this difference was statistically significant (p<0.05).