Kauffman R F, Crowe V G, Utterback B G, Robertson D W
Mol Pharmacol. 1986 Dec;30(6):609-16.
LY195115 selectively inhibited the peak III isozyme of cardiac cyclic nucleotide phosphodiesterase (PDE) eluted from DEAE-cellulose columns. Inhibition curves were biphasic, suggesting heterogeneity within this preparation. Since peak III PDE is reported to be derived from membranes, effects of LY195115 upon PDE associated with cardiac membranes were examined. LY195115-sensitive PDE measured in the various membrane fractions correlated well with the sarcoplasmic reticulum marker Ca2+-ATPase (r = 0.94; p less than 0.001), but not with Na+,K+-ATPase or azide-sensitive ATPase. Membrane disruption failed to reveal latent LY195115-sensitive PDE in sarcolemmal vesicles known to be primarily right side out. The results suggest that LY195115-sensitive PDE is located within sarcoplasmic reticulum membranes with a distribution similar or identical to that of Ca2+-ATPase. Accordingly, LY195115-sensitive PDE was referred to as SR-PDE. A subfraction of sarcoplasmic reticulum vesicles (free SR vesicles) was sufficiently homogeneous with respect to SR-PDE activity to carry out steady state kinetic studies. Double reciprocal plots of cAMP hydrolysis were linear, yielding Km and Vmax values of 0.46 +/- 0.03 microM and 700 +/- 90 pmol/min/mg of vesicle protein, respectively. LY195115 was a linear competitive inhibitor of SR-PDE with a Ki of 80 +/- 10 nM. -LogIC50 values for inhibition of SR-PDE by a series of structural analogues of LY195115 correlated highly with published -logED50 values for stimulation of cardiac contractility in vivo (r = 0.91, p less than 0.001). Consequently, in vivo effects of LY195115 upon the heart appear to result primarily from competitive inhibition of SR-PDE, or from binding to a site with a topography similar or identical to that of the catalytic site of SR-PDE.
LY195115能选择性抑制从DEAE - 纤维素柱洗脱的心脏环核苷酸磷酸二酯酶（PDE）的Ⅲ峰同工酶。抑制曲线呈双相性，表明该制剂存在异质性。由于据报道Ⅲ峰PDE来源于细胞膜，因此研究了LY195115对与心脏细胞膜相关的PDE的影响。在各种膜组分中测得的对LY195115敏感的PDE与肌浆网标志物Ca2 + - ATP酶相关性良好（r = 0.94；p < 0.001），但与Na + ，K + - ATP酶或叠氮化物敏感的ATP酶无关。膜破裂未能在已知主要为外翻的肌膜小泡中揭示潜在的对LY195115敏感的PDE。结果表明，对LY195115敏感的PDE位于肌浆网膜内，其分布与Ca2 + - ATP酶相似或相同。因此，对LY195115敏感的PDE被称为SR - PDE。肌浆网小泡的一个亚组分（游离SR小泡）在SR - PDE活性方面足够均匀，可进行稳态动力学研究。cAMP水解的双倒数图呈线性，分别产生的Km和Vmax值为0.46±0.03 microM和700±90 pmol / min / mg小泡蛋白。LY195115是SR - PDE的线性竞争性抑制剂，Ki为80±10 nM。一系列LY195115结构类似物对SR - PDE抑制的 - LogIC50值与已发表的体内刺激心脏收缩性的 - LogED50值高度相关（r = 0.91，p < 0.001）。因此，LY195115对心脏的体内作用似乎主要源于对SR - PDE的竞争性抑制，或源于与SR - PDE催化位点拓扑结构相似或相同的位点结合。
