Increase of particulate nitric oxide synthase activity and peroxynitrite synthesis in UVB-irradiated keratinocyte membranes
Increase of particulate nitric oxide synthase activity and peroxynitrite synthesis in UVB-irradiated keratinocyte membranes
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DOI：
10.1042/bj3200997
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发表时间：
1996-12-15
影响因子：
4.1
通讯作者：
Stavrides, JC
中科院分区：
文献类型：
--
作者：
Deliconstantinos, G;Villiotou, V;Stavrides, JC
在这里，我们证明，人角质形成细胞具有Ca 2 +/钙调蛋白依赖性颗粒NO合酶，可以被激活后，暴露于WE辐射释放NO。UVB照射（高达20 mJ/cm（2））人角质形成细胞质膜导致NO和L-[H-3]瓜氨酸产生的剂量依赖性增加，约100 mg/kg剂量可抑制该增加。在N-单甲基-L-精氨酸（L-NMMA）存在下，90%。在UVB照射质膜的时间过程实验中，NO产生的变化，其次是可溶性鸟苷酸环化酶（sGC）活性的类似变化。在重建实验中，当将颗粒状NO合酶加入到从角质形成细胞胞质溶胶中分离的纯化sGC中时，观察到cGMP增加了4倍;颗粒状NO合酶经UVB照射（高达20 mJ/cm（2））后合成的NO增加了cGMP。观察到UVB（20 mJ/cm（2））照射角质形成细胞微粒体后，超氧化物（O-2（-））增加5倍，NO形成增加7倍，随后过氧亚硝酸盐（ONOO-）产生增加8倍。UVB辐射（20 mJ/cm（2））降低质膜脂质流动性表明稳态荧光各向异性。L-NMMA可防止膜流动性的变化。颗粒状NO合酶的阿伦尼乌斯图的变化与UVB辐射诱导的变构特性的变化相结合，与该酶的脂质微环境流动性降低一致。目前的研究提供了重要的新线索的作用，一氧化氮和ONOO-释放的紫外线照射的人角质形成细胞在皮肤红斑和炎症。
Here we demonstrate that human keratinocytes possess a Ca2+/calmodulin-dependent particulate NO synthase that can be activated to release NO after exposure to WE radiation. UVB irradiation (up to 20 mJ/cm(2)) of human keratinocyte plasma membranes resulted in a dose-dependent increase in NO and L-[H-3]citrulline production that was inhibited by approx. 90% in the presence of N-monomethyl-L-arginine (L-NMMA). In time-course experiments with UVB-irradiated plasma membranes the changes in NO production were followed by analogous changes in soluble guanylate cyclase (sGC) activity. In reconstitution experiments, when particulate NO synthase was added to purified sGC isolated from keratinocyte cytosol, a 4-fold increase in cGMP was observed; the cGMP was increased by NO synthesized after UVB irradiation (up to 20 mJ/cm(2)) of particulate NO synthase. A 5-fold increase in superoxide (O-2(-)) and a 7-fold increase in NO formation followed by an 8-fold increase in peroxynitrite (ONOO-) production by UVB (20 mJ/cm(2))-irradiated keratinocyte microsomes was observed. UVB radiation (20 mJ/cm(2)) decreased plasma membrane lipid fluidity as indicated by steady-state fluorescence anisotropy. Membrane fluidity changes were prevented by L-NMMA. Changes in Arrhenius plots of particulate NO synthase in combination with changes in its allosteric properties induced by UVB radiation are consistent with a decreased fluidity of the lipid microenvironment of the enzyme. The present studies provide important new clues to the role of NO and ONOO- released by UVB-irradiated human keratinocytes in skin erythema and inflammation.