Subcellular distribution of high-affinity type IV cyclic AMP phosphodiesterase activities in rabbit ventricular myocardium: Relations to post-natal maturation

Abstract

Cytosolic and particulate Type IV (high-affinity) cAMP phosphodiesterase (PDE) activities were isolated from the ventricular myocardium of newborn (NB; 24 to 48 h), immature (IM; 14 to 16 days) and adult (AD; 6 to 8 months) rabbits. Cytosolic activity from each age group was resolved into three distinct peaks of activity by DEAE cellulose anion exchange chromatography. Type IV PDE activity was identified as a predominant activity in the cytosolic peak III activity in all three age groups when measured with 0.25 μ m cAMP as substrate. A particulate Type IV PDE activity was associated with the sarcoplasmic reticulum (SR) fractions in each age group. No significant age-related changes in the affinity of the particulate enzyme for cAMP (apparent K m = 0.3 to 0.5 μ m) were evident, but the V max for this SR-associated activity increased from 553 ± 7 pmol/min/mg in the NB to 725 ± 9 pmol/min/mg in the IM and 2450 ± 33 pmol/min/mg in the AD. In each age group, milrinone, imazodan, piroximone and indolidan were more potent inhibitors of the SR-associated activity as compared with the cytosolic peak III activity. In contrast, RO 20-1724 and rolipram were relatively more selective inhibitors of the cytosolic peak III activity. Age-related differences in the sensitivity of type IV PDE to inhibition was dependent upon the selectivity of the inhibitor and the subcellular enzymic distribution. Cytostolic peak III PDE activity was further resolved by gel filtration chromatography into two peaks. Hydrolysis of cAMP by the higher molecular weight peak was inhibitable by cGMP (IC 50 = 0.25 ± 0.07 μ m in NB and 0.07 ± 0.01 μ m in AD) whereas the lower molecular weight peak activity was relatively insensitive to inhibition by cGMP (IC 50 > 100 μ m). The lower molecular weight peak constituted a relatively greater proportion of the total peak III activity in the NB as compared to the AD. Analysis of the kinetics of cGMP inhibition of high-affinity cAMP hydrolysis was consistent with the presence of a greater number of high-affinity (presumably drug-sensitive) binding sites in the SR-associated activity as compared to the cytosolic peak III activity in both NB and AD. These results support the hypothesis that the cGMP-inhibitable Type IV PDE activity may be the primary site of action for certain newer cardiotonic drugs. Differences in drug action in young versus adult myocardium may be related to the selectivity of the cardiotonic drugs for this specific isozyme and its lower specific activity during the early stages of maturation.