Related Experiment Videos
Subcellular distribution of high-affinity type IV cyclic AMP phosphodiesterase activities in rabbit ventricular
P A Kithas1, M Artman, W J Thompson
1Department of Pharmacology, College of Medicine, University of South Alabama, Mobile 36688.
Insights
Type IV phosphodiesterase (PDE) activity in rabbit myocardium changes with age. The sarcoplasmic reticulum-associated activity increases, suggesting a key role for this enzyme in cardiotonic drug action during maturation.
Area of Science:
- Biochemistry
- Cardiovascular Physiology
- Pharmacology
Background:
- Type IV phosphodiesterase (PDE) enzymes play crucial roles in regulating intracellular cyclic adenosine monophosphate (cAMP) levels.
- Understanding the developmental changes in PDE activity is essential for comprehending cardiac function and drug response in different age groups.
Purpose of the Study:
- To investigate the age-related changes in cytosolic and particulate Type IV cAMP PDE activities in rabbit ventricular myocardium.
- To characterize the kinetic properties and inhibitor sensitivity of these PDE activities.
- To explore the potential role of specific PDE isozymes in the mechanism of action of cardiotonic drugs.
Main Methods:
- Isolation and characterization of cytosolic and particulate PDE activities from rabbit ventricular myocardium of different age groups (newborn, immature, adult).
- DEAE cellulose anion exchange and gel filtration chromatography for enzyme resolution.
- Kinetic analysis (Km, Vmax) and inhibitor studies using selective PDE inhibitors (milrinone, imazodan, piroximone, indolidan, RO 20-1724, rolipram) and cGMP.
Main Results:
- Cytosolic Type IV PDE activity resolved into three peaks, with peak III being predominant and showing age-related changes in its proportion of cGMP-inhibitable activity.
- Particulate Type IV PDE activity associated with sarcoplasmic reticulum (SR) exhibited a significant increase in Vmax with age, while affinity remained constant.
- SR-associated activity was more potently inhibited by cardiotonic drugs (milrinone, etc.) compared to cytosolic peak III activity, which was more sensitive to RO 20-1724 and rolipram.
Conclusions:
- Age-dependent alterations in the distribution and activity of Type IV PDE isozymes occur in rabbit myocardium.
- The cGMP-inhibitable Type IV PDE activity, particularly the SR-associated fraction, is a likely target for cardiotonic drugs.
- Differences in drug efficacy between young and adult hearts may be attributed to variations in PDE isozyme selectivity and activity during 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 microM 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 Km = 0.3 to 0.5 microM) were evident, but the Vmax 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. Cytosolic 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 (IC50 = 0.25 +/- 0.07 microM in NB and 0.07 +/- 0.01 microM in AD) whereas the lower molecular weight peak activity was relatively insensitive to inhibition by cGMP (IC50 greater than 100 microM). 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.