Kithas P A, Artman M, Thompson W J, Strada S J
Department of Pharmacology, College of Medicine, University of South Alabama, Mobile 36688.
J Mol Cell Cardiol. 1989 May;21(5):507-17. doi: 10.1016/0022-2828(89)90790-6.
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.
从新生（NB；24至48小时）、未成熟（IM；14至16天）和成年（AD；6至8个月）兔的心室心肌中分离出胞质和颗粒型IV型（高亲和力）环磷酸腺苷磷酸二酯酶（PDE）活性。通过DEAE纤维素阴离子交换色谱法，将每个年龄组的胞质活性解析为三个不同的活性峰。当以0.25微摩尔/升环磷酸腺苷为底物进行测定时，在所有三个年龄组中，IV型PDE活性被确定为胞质峰III活性中的主要活性。每个年龄组中，颗粒型IV型PDE活性与肌浆网（SR）组分相关。颗粒型酶对环磷酸腺苷的亲和力（表观Km = 0.3至0.5微摩尔/升）没有明显的年龄相关变化，但这种与SR相关的活性的Vmax从新生兔的553±7皮摩尔/分钟/毫克增加到未成熟兔的725±9皮摩尔/分钟/毫克，以及成年兔的2450±33皮摩尔/分钟/毫克。在每个年龄组中，与胞质峰III活性相比，米力农、伊马唑旦、吡罗昔酮和吲哚利旦对与SR相关的活性是更强效的抑制剂。相比之下，RO 20 - 1724和咯利普兰是胞质峰III活性相对更具选择性的抑制剂。IV型PDE对抑制的敏感性的年龄相关差异取决于抑制剂的选择性和亚细胞酶分布。通过凝胶过滤色谱法，胞质峰III PDE活性进一步解析为两个峰。较高分子量峰对环磷酸腺苷的水解可被环磷酸鸟苷抑制（新生兔中IC50 = 0.25±0.07微摩尔/升，成年兔中为0.07±0.01微摩尔/升），而较低分子量峰的活性对环磷酸鸟苷的抑制相对不敏感（IC50大于100微摩尔/升）。与成年兔相比，较低分子量峰在新生兔的总峰III活性中占相对更大的比例。对环磷酸鸟苷抑制高亲和力环磷酸腺苷水解的动力学分析表明，与新生兔和成年兔的胞质峰III活性相比，与SR相关的活性中存在更多高亲和力（可能对药物敏感）的结合位点。这些结果支持这样的假设，即环磷酸鸟苷可抑制的IV型PDE活性可能是某些新型强心药物的主要作用位点。年轻心肌与成年心肌中药物作用的差异可能与强心药物对这种特定同工酶的选择性及其在成熟早期较低的比活性有关。
