CALMODULIN-DEPENDENT ENDOTHELIUM-DERIVED RELAXING FACTOR NITRIC-OXIDE SYNTHASE ACTIVITY IS PRESENT IN THE PARTICULATE AND CYTOSOLIC FRACTIONS OF BOVINE AORTIC ENDOTHELIAL-CELLS
CALMODULIN-DEPENDENT ENDOTHELIUM-DERIVED RELAXING FACTOR NITRIC-OXIDE SYNTHASE ACTIVITY IS PRESENT IN THE PARTICULATE AND CYTOSOLIC FRACTIONS OF BOVINE AORTIC ENDOTHELIAL-CELLS
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DOI：
10.1073/pnas.88.5.1788
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发表时间：
1991-03-01
影响因子：
11.1
通讯作者：
MURAD, F
中科院分区：
文献类型：
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作者：
FORSTERMANN, U;POLLOCK, JS;MURAD, F
用牛主动脉内皮细胞及其亚细胞组分合成的内皮源性松弛因子/一氧化氮（EDRF/NO）对大鼠胎肺成纤维细胞（RFL-6细胞）可溶性鸟酰环化酶的刺激作用进行了研究。完整内皮细胞的EDRF/NO的释放可以被缓激肽、凝血酶或ADP刺激，并在无Ca2+的培养基中被消除。对亚细胞组分进行分析，发现细胞质组分中有一定的EDRF/ no合成活性，但大部分活性与颗粒组分有关。这两种酶合成EDRF/NO都需要l-精氨酸和NADPH，但均被N(G)-硝基- l-精氨酸和N(G)-甲基- l-精氨酸抑制，血红蛋白或亚甲基蓝消除了这两种酶产生的EDRF/NO的作用。两种酶均对Ca2+高度敏感；活性增加主要发生在100 ~ 500 nM游离Ca2+之间。当颗粒酶活性暴露于1 M KCl时，蛋白质活性降低39%，总活性降低46%，但添加外源钙调素（CaM）后活性恢复。进一步的KCl洗涤几乎没有引起蛋白质或EDRF/NO合成酶活性的进一步损失。氯化钾洗涤颗粒酶可与洗涤剂3-[（3-胆酰胺丙基）二甲酰胺]-1-丙磺酸溶解。CaM拮抗剂卡咪唑和三氟拉嗪以及CaM结合蛋白钙调神经磷酸酶抑制细胞质酶和颗粒酶合成EDRF/NO。这些效应部分被外源性CaM逆转。通过对腺苷2',5'-二磷酸- sepharose的亲和层析对胞质酶和溶解颗粒酶进行部分纯化，导致EDRF/NO合成酶活性依赖于外源CaM。我们得出结论，内皮细胞含有合成EDRF/NO的胞质酶和颗粒酶。这两种酶都受到游离Ca2+和CaM（至少部分地）的调节。
Endothelium-derived relaxing factor/nitric oxide (EDRF/NO) synthesized by bovine aortic endothelial cells and subcellular fractions thereof was assayed by its stimulating effect on soluble guanylyl cyclase of rat fetal lung fibroblasts (RFL-6 cells). The release of EDRF/NO by intact endothelial cells could be stimulated with bradykinin, thrombin, or ADP and was abolished in Ca2+-free medium. When subcellular fractions were analyzed, some EDRF/NO-synthesizing activity was found in the cytosolic fraction, but most of the activity was associated with the particulate fraction. Both enzyme activities required L-arginine and NADPH for EDRF/NO synthesis, both were inhibited by N(G)-nitro-L-arginine and N(G)-methyl-L-arginine, and hemoglobin or methylene blue abolished the effect of the EDRF/NO produced by both enzymes. Both enzymes were highly sensitive to Ca2+; the major increase in activity occurred between 100 and 500 nM free Ca2+. Exposure of the particulate enzyme activity to 1 M KCl removed 39% of the protein and reduced total activity by 46%, but the activity was restored when exogenous calmodulin (CaM) was added. Further KCl washes caused little further loss of protein or EDRF/NO synthase activity. The KCl-washed particulate enzyme could be solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The CaM antagonists calmidazolium and trifluoperazine as well as the CaM-binding protein calcineurin inhibited the EDRF/NO synthesis by both the cytosolic and the particulate enzyme. These effects were partially reversed with exogenous CaM. Partial purification of the cytosolic and solubilized particulate enzymes by affinity chromatography on adenosine 2',5'-bisphosphate-Sepharose resulted in EDRF/NO synthase activities dependent on exogenous CaM. We conclude that endothelial cells contain both cytosolic and particulate enzymes that synthesize EDRF/NO. Both enzymes are regulated by free Ca2+ and, at least in part, by CaM.