Förstermann U, Gorsky L D, Pollock J S, Ishii K, Schmidt H H, Heller M, Murad F
Abbott Laboratories, Abbott Park, Illinois 60064.
Mol Pharmacol. 1990 Jul;38(1):7-13.
Stimulation of soluble guanylyl cyclase in rat fetal lung fibroblasts (RFL-6 cells) was used as a sensitive assay for endothelium-derived relaxing factor/nitric oxide (EDRF/NO) formation. Intact N1E-115 cells released an EDRF/NO-like material that enhanced cyclic GMP levels in RFL-6 cells. The synthesis of this substance could be stimulated with the receptor agonist neurotensin (10 microM) or by addition of the EDRF/NO substrate L-arginine (100 microM). In Ca2(+)-free Locke's solution, stimulation of EDRF/NO production by both neurotensin and L-arginine was abolished. The EDRF/NO-synthesizing activity was localized in the cytosol of N1E-115 cells. The activity was lost after boiling and it was highly sensitive to Ca2+ with the major increase in activity occurring between 100 and 500 nM Ca2+. L-Arginine and NADPH were required for maximal synthesis of EDRF/NO by the enzyme(s). The synthesis of EDRF/NO was inhibited by the following antagonists of calmodulin-regulated functions (with the approximate IC50 values given in parentheses): calmidazolium (7 microM), trifluoperazine (10 microM), fendiline (80 microM), W-7 (N-[6-aminohexyl]-5-chloro-1-naphthalenesulfonamide) (120 microM), and compound 48/80 (3 micrograms/ml). The EDRF/NO-synthesizing activity was partially purified from N1E-115 cytosol by DE 52 anion exchange chromatography. The activity was eluted with 0.1 M KCl. The enzyme(s) showed very little activity in the presence of L-arginine (100 microM) and NADPH (100 microM), but the activity could be fully restored by addition of exogenous calmodulin (EC50, approximately 2 units/ml). At 0.3 M KCl, a fraction eluted from the DE 52 column that was also able to fully restore the EDRF/NO-synthesizing activity. Thus, this fraction is likely to contain the endogenous Ca2(+)-binding protein. It is concluded that the activity of the EDRF/NO-synthesizing enzyme(s) in N1E-115 neuroblastoma cells is regulated by Ca2+ and calmodulin.
在大鼠胎儿肺成纤维细胞（RFL - 6细胞）中刺激可溶性鸟苷酸环化酶，被用作一种检测内皮衍生舒张因子/一氧化氮（EDRF/NO）生成的灵敏方法。完整的N1E - 115细胞释放出一种类似EDRF/NO的物质，该物质可提高RFL - 6细胞中的环磷酸鸟苷水平。这种物质的合成可被受体激动剂神经降压素（10微摩尔）刺激，或者通过添加EDRF/NO底物L - 精氨酸（100微摩尔）来刺激。在无钙的洛克溶液中，神经降压素和L - 精氨酸对EDRF/NO生成的刺激作用均被消除。EDRF/NO合成活性定位于N1E - 115细胞的胞质溶胶中。煮沸后该活性丧失，并且它对钙离子高度敏感，在100至500纳摩尔钙离子之间活性主要增加。L - 精氨酸和NADPH是该酶最大程度合成EDRF/NO所必需的。EDRF/NO的合成被以下钙调蛋白调节功能的拮抗剂抑制（括号内给出近似的IC50值）：氯米帕明（7微摩尔）、三氟拉嗪（10微摩尔）、芬地林（80微摩尔）、W - 7（N - [6 - 氨基己基] - 5 - 氯 - 1 - 萘磺酰胺）（120微摩尔）和48/80化合物（3微克/毫升）。通过DE 52阴离子交换色谱法从N1E - 115胞质溶胶中部分纯化了EDRF/NO合成活性。该活性用0.1M氯化钾洗脱。在存在L - 精氨酸（100微摩尔）和NADPH（100微摩尔）的情况下，该酶显示出极低的活性，但通过添加外源性钙调蛋白（EC50，约2单位/毫升）可使活性完全恢复。在0.3M氯化钾时，从DE 52柱上洗脱的一个组分也能够完全恢复EDRF/NO合成活性。因此，该组分可能含有内源性钙离子结合蛋白。结论是，N1E - 115神经母细胞瘤细胞中EDRF/NO合成酶的活性受钙离子和钙调蛋白调节。
