Souness J E, Diocee B K, Martin W, Moodie S A
Dagenham Research Centre, Rhône-Poulene Ltd., Health Care Division, Essex, U.K.
Biochem J. 1990 Feb 15;266(1):127-32. doi: 10.1042/bj2660127.
Two cyclic nucleotide phosphodiesterase (PDE) activities were identified in pig aortic endothelial cells, a cyclic GMP-stimulated PDE and a cyclic AMP PDE. Cyclic GMP-stimulated PDE had Km values of 367 microM for cyclic AMP and 24 microM for cyclic GMP, and low concentrations (1 microM) of cyclic GMP increased the affinity of the enzyme for cyclic AMP (Km = 13 microM) without changing the Vmax. This isoenzyme was inhibited by trequinsin [IC50 (concn. giving 50% inhibition of substrate hydrolysis) = 0.6 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 0.6 microM for cyclic GMP hydrolysis] and dipyridamole (IC50 = 5 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 3 microM for cyclic GMP hydrolysis). Cyclic AMP PDE exhibited a Km of 2 microM for cyclic AMP and did not hydrolyse cyclic GMP. This activity was inhibited by trequinsin (IC50 = 0.2 microM), dipyridamole (IC50 = 6 microM) and, selectively, by rolipram (IC50 = 3 microM). Inhibitors of cyclic GMP PDE (M&B 22948) and of low Km (Type III) cyclic AMP PDE (SK&F 94120) only weakly inhibited the two endothelial PDEs. Incubation of intact cells with trequinsin and dipyridamole induced large increases in cyclic GMP, which were completely blocked by LY-83583. Rolipram, SK&F 94120 and M&B 22948 did not significantly influence cyclic GMP accumulation. Dipyridamole enhanced the increase in cyclic GMP induced by sodium nitroprusside. Cyclic AMP accumulation was stimulated by dipyridamole and trequinsin with and without forskolin. Rolipram, although without effect alone, increased cyclic AMP in the presence of forskolin, whereas M&B 22948 and SK&F 94120 had no effects on resting or forskolin-stimulated levels. These results suggest that cyclic GMP-stimulated PDE regulates cyclic GMP levels and that both endothelial PDE isoenzymes contribute to the control of cyclic AMP.
在猪主动脉内皮细胞中鉴定出两种环核苷酸磷酸二酯酶（PDE）活性，一种是环鸟苷酸刺激的PDE，另一种是环腺苷酸PDE。环鸟苷酸刺激的PDE对环腺苷酸的Km值为367微摩尔，对环鸟苷酸的Km值为24微摩尔，低浓度（1微摩尔）的环鸟苷酸增加了该酶对环腺苷酸的亲和力（Km = 13微摩尔），而不改变Vmax。这种同工酶被曲喹辛抑制[IC50（导致底物水解50%抑制的浓度）= 0.6微摩尔，用于在环鸟苷酸存在下的环腺苷酸水解；IC50 = 0.6微摩尔，用于环鸟苷酸水解]和双嘧达莫（IC50 = 5微摩尔，用于在环鸟苷酸存在下的环腺苷酸水解；IC50 = 3微摩尔，用于环鸟苷酸水解）。环腺苷酸PDE对环腺苷酸的Km值为2微摩尔，不水解环鸟苷酸。该活性被曲喹辛（IC50 = 0.2微摩尔）、双嘧达莫（IC50 = 6微摩尔）选择性地被咯利普兰（IC50 = 3微摩尔）抑制。环鸟苷酸PDE（M&B 22948）和低Km（III型）环腺苷酸PDE（SK&F 94120）的抑制剂仅微弱地抑制这两种内皮PDE。用曲喹辛和双嘧达莫孵育完整细胞会导致环鸟苷酸大幅增加，这被LY-83583完全阻断。咯利普兰、SK&F 94120和M&B 22948对环鸟苷酸积累没有显著影响。双嘧达莫增强了硝普钠诱导的环鸟苷酸增加。双嘧达莫和曲喹辛在有或没有福斯可林的情况下均刺激环腺苷酸积累。咯利普兰虽然单独无作用，但在有福斯可林存在时增加环腺苷酸，而M&B 22948和SK&F 94120对静息或福斯可林刺激的水平无影响。这些结果表明，环鸟苷酸刺激的PDE调节环鸟苷酸水平，并且两种内皮PDE同工酶都有助于控制环腺苷酸。
