Cyclic 3′, 5′-AMP phosphodiesterase of rabbit aorta

Abstract

Listen

Translate article

Cyclic AMP and cyclic GMP phosphodiesterase activities (3′ : 5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17) were demonstrated in the isolated intima, media, and adventitia of rabbit aorta. The activity for cyclic AMP hydrolysis in the intima was 2.7-fold higher than that for cyclic GMP hydrolysis. The activity for cyclic AMP hydrolysis in the media was approximately equal to that for cyclic GMP hydrolysis, but in the adventitia, cyclic GMP hydrolytic activity was 2.1-fold higher than cyclic AMP hydrolytic activity. Distribution of the activator of the phosphodiesterase was studied in the three layers. Each layer contained the activator. The activator was predominantly localized in the smooth muscle layer (the media). The effect of the activator and Ca 2+ on the media cyclic AMP and cyclic GMP phosphodiesterase was also briefly studied. The activity of the cyclic GMP phosphodiesterase was stimulated by micromolar concentration of Ca 2+ in the presence of the activator. However, the activity of the cyclic AMP Phosphodiesterase was not significantly stimulated by Ca 2+ up to 100 μM in the presence of the activator. Above 90% of cyclic nucleotide phosphodiesterase activity in the whole aorta was found to be derived from the media. A major portion (60–70%) of the media enzyme was found in 105 000 × g supernatant. Cyclic AMP phosphodiesterase in the supernatant was partially purified through Sepharose 6B column chromatography and partially separated from cyclic GMP phosphodiesterase. Using a partially purified preparation from the 105 000 × g supernatant the main kinetic parameters were specified as follows: 1. 1)|The pH optimum was found to be about 9.0 using Tris-maleate buffer. The maximum stimulation of the enzyme by Mg 2+ was achieved at 4 mM of MgCl 2. 2. 2)|High concentration of cyclic GMP (0.1 mM) inhibited noncompetitively the enzyme activity, and the activity was not stimulated at any tested concentration of cyclic GMP. 3. 3)|Activity-substrate concentration relationship revealed a high affinity ( K m = 1.0 μM ) and low affinity ( K m = 45 μM ) for cyclic AMP. The homogenate and 105 000 × g supernatant of the media also showed non-linear kinetics similar to the Sepharose 6B preparation and their apparent K m values for cyclic AMP hydrolysis were 1.2 μM and 36–40 μM and an enzyme extracted by sonication from 105 000 × g precipitate also exhibited non-linear kinetics ( K m = 5.1 μM and 70 μM . 4. 4)|Papaverine exhibited much stronger inhibition on the aorta cyclic AMP phosphodiesterase (50% inhibition of the intima enzyme, I 5 0 at 0.62 μM, I 5 0 of the media at 0.62 μM and 5 0 of the adventitia at 1.0 μM) than on the brain ( I 5 0 at 8.5 μM) and serum ( I 5 0 at 20 μM) cyclic AMP phosphodiesterase, while theophylline inhibited these enzymes similarly. However, cyclic GMP phosphodiesterases in all tissues examined were inhibited similarly, not only by theophylline but also by papaverine.