Deliconstantinos G, Villiotou V, Stavrides J C
Department of Experimental Physiology, University of Athens Medical School, Greece.
Biochem J. 1996 Dec 15;320 ( Pt 3)(Pt 3):997-1003. doi: 10.1042/bj3200997.
Here we demonstrate that human keratinocytes possess a Ca2+/calmodulin-dependent particulate NO synthase that can be activated to release NO after exposure to UVB radiation. UVB irradiation (up to 20 mJ/cm2) of human keratinocyte plasma membranes resulted in a dose-dependent increase in NO and L-[3H]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/cm2) of particulate NO synthase. A 5-fold increase in superoxide (O2-) and a 7-fold increase in NO formation followed by an 8-fold increase in peroxynitrite (ONOO-) production by UVB (20 mJ/cm2)-irradiated keratinocyte microsomes was observed. UVB radiation (20 mJ/cm2) 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.
在此我们证明，人类角质形成细胞拥有一种Ca2+/钙调蛋白依赖性颗粒型一氧化氮合酶，暴露于中波紫外线（UVB）辐射后，该酶可被激活以释放一氧化氮。对人类角质形成细胞质膜进行UVB照射（高达20 mJ/cm2）会导致一氧化氮和L-[3H]瓜氨酸生成呈剂量依赖性增加，在N-单甲基-L-精氨酸（L-NMMA）存在的情况下，这种增加被抑制了约90%。在用UVB照射的质膜进行的时间进程实验中，一氧化氮生成的变化之后伴随着可溶性鸟苷酸环化酶（sGC）活性的类似变化。在重组实验中，当将颗粒型一氧化氮合酶添加到从角质形成细胞胞质溶胶中分离出的纯化sGC中时，观察到环磷酸鸟苷（cGMP）增加了4倍；颗粒型一氧化氮合酶经UVB照射（高达20 mJ/cm2）后合成的一氧化氮使cGMP增加。观察到经UVB（20 mJ/cm2）照射的角质形成细胞微粒体中超氧化物（O2-）增加了5倍，一氧化氮生成增加了7倍，随后过氧亚硝酸盐（ONOO-）生成增加了8倍。如稳态荧光各向异性所示，UVB辐射（20 mJ/cm2）降低了质膜脂质流动性。L-NMMA可防止膜流动性变化。颗粒型一氧化氮合酶的阿累尼乌斯曲线变化及其由UVB辐射诱导的别构性质变化与该酶脂质微环境流动性降低一致。本研究为UVB照射的人类角质形成细胞释放的一氧化氮和过氧亚硝酸盐在皮肤红斑和炎症中的作用提供了重要的新线索。
