Lugnier C, Schini V B
Laboratoire de Pharmacologie Cellulaire et Moléculaire, CNRS URA 600, Université Louis Pasteur de Strasbourg, Illkirch, France.
Biochem Pharmacol. 1990 Jan 1;39(1):75-84. doi: 10.1016/0006-2952(90)90650-a.
Experiments were carried out in order to isolate and characterize the cyclic nucleotide phosphodiesterase activities in primary and low passages of cultured bovine aortic endothelial cells. The subcellular characterization of the cyclic nucleotide hydrolytic activity showed that both cAMP and cGMP hydrolytic activities were predominant in the cytosolic rather than the particulate fraction of the endothelial cell homogenate. At a low substrate concentration (0.25 microM), the major hydrolytic activity was for cAMP while at a high concentration (20 microM) it was for both cAMP and cGMP. Both cAMP and cGMP hydrolytic activities were insensitive to calmodulin. Cytosolic cyclic nucleotide phosphodiesterase activity was resolved into two distinct phosphodiesterase forms using HPLC. The first eluted form was designated cGS-PDE: it hydrolysed both cAMP and cGMP and its cAMP hydrolytic activity was markedly enhanced by the presence of cGMP. The second form was designated cAMP-PDE: it selectively hydrolysed cAMP. The cytosolic cAMP-PDE was inhibited by micromolar concentrations of cAMP-PDE inhibitors such as trequinsin, rolipram, dipyridamole or papaverine. The cGS-PDE was inhibited by micromolar concentrations of trequinsin, dipyridamole and papaverine and was insensitive to rolipram, except for the hydrolysis of cAMP which was inhibited in the micromolar range. Both the cAMP-PDE and the cGS-PDE were relatively insensitive to the selective cGMP-PDE inhibitor, zaprinast which was about 750-fold less potent on endothelial PDEs than on smooth muscle cGMP-PDE. The identification of selective and specific PDE inhibitors of the different PDE forms may allow a better understanding of the regulation and the role of cyclic nucleotides in endothelial cells.
为了分离和鉴定培养的牛主动脉内皮细胞原代及低代培养物中的环核苷酸磷酸二酯酶活性，进行了相关实验。环核苷酸水解活性的亚细胞特征表明，在内皮细胞匀浆的胞质部分而非微粒部分，cAMP和cGMP水解活性均占主导。在低底物浓度（0.25微摩尔）时，主要的水解活性针对cAMP，而在高浓度（20微摩尔）时，针对cAMP和cGMP两者。cAMP和cGMP水解活性均对钙调蛋白不敏感。使用高效液相色谱法将胞质环核苷酸磷酸二酯酶活性解析为两种不同的磷酸二酯酶形式。第一种洗脱形式被命名为cGS-PDE：它能水解cAMP和cGMP，且cGMP的存在能显著增强其cAMP水解活性。第二种形式被命名为cAMP-PDE：它选择性地水解cAMP。胞质cAMP-PDE受到微摩尔浓度的cAMP-PDE抑制剂（如曲喹辛、咯利普兰、双嘧达莫或罂粟碱）的抑制。cGS-PDE受到微摩尔浓度的曲喹辛、双嘧达莫和罂粟碱的抑制，除了在微摩尔范围内cAMP水解受到抑制外，对咯利普兰不敏感。cAMP-PDE和cGS-PDE对选择性cGMP-PDE抑制剂扎普司特相对不敏感，扎普司特对内皮磷酸二酯酶的效力比对平滑肌cGMP-PDE低约750倍。鉴定不同磷酸二酯酶形式的选择性和特异性磷酸二酯酶抑制剂，可能有助于更好地理解环核苷酸在内皮细胞中的调节作用和功能。
