Deliconstantinos G, Villiotou V, Stavrides J C
Department of Physiology, University of Athens Medical School, Greece.
Biochem Pharmacol. 1996 Jun 28;51(12):1727-38. doi: 10.1016/0006-2952(96)00110-4.
In the present study, we demonstrated that NO synthase (cNOS) and xanthine oxidase (XO) of human keratinocytes can be activated to release NO, superoxide (O2-) and peroxynitrite (ONOO-) following exposure to ultraviolet B (UVB) radiation. We defined that this photo induced response may be involved in the pathogenesis of sunburn erythema and inflammation. Treatment of human keratinocytes with UVB (290-320 nm) radiation (up to 200 mJ/cm2) resulted in a dose-dependent increase in NO and ONOO- release that was inhibited by N-monomethyl-L-arginine (L-NMMA). NO and ONOO- release from keratinocytes was accompanied by an increase in intracellular cGMP levels. Treatment of human keratinocyte cytosol with various doses of UVB (up to 100 mJ/cm2) resulted in an increase in XO activity that was inhibited by oxypurinol. UVB radiation (up to 100 mJ/cm2) of keratinocytes resulted in a 15-fold increase in S-nitrosothiol formation, which directly increased purified soluble guanylate cyclase (sGC) activity by a mechanism characteristic of release of NO from a carrier molecule. In reconstitution experiments, when UVB-irradiated (20 mJ/cm2) purified cNOS isolated from keratinocyte cytosol was combined with UVB-irradiated (20 mJ/cm2) purified XO, a 4-fold increase in ONOO- production, as compared to nonirradiated enzymes, was observed. ONOO- synthesized by NO and O2- following UVB radiation of cNOS and XO was inhibited by oxypurinol (100 microM). UVB radiation of keratinocyte cytosol resulted in an increase in oxygen free radical production, consistent with the increased production of ONOO- by UVB-irradiated keratinocyte cytosol. In in vivo experiments, when experimental animals were subjected to UVB radiation, a protection factor (PF) of 6.5 +/- 1.8 was calculated when an emulsified cream formulation containing nitro-L-arginine (L-NA) (2%) and L-NMMA (2%) was applied to their skin. The present study indicates that UVB radiation acts as a potent stimulator of cNOS and XO activities in human keratinocytes. NO and ONOO- may exert cytotoxic effects in keratinocytes themselves, as well as in their neighboring endothelial and smooth muscle cells. This may be a major part of the integrated response leading to erythema production and the inflammation process.
在本研究中，我们证明，人角质形成细胞的一氧化氮合酶（cNOS）和黄嘌呤氧化酶（XO）在暴露于紫外线B（UVB）辐射后可被激活，释放一氧化氮（NO）、超氧阴离子（O2-）和过氧亚硝酸盐（ONOO-）。我们确定这种光诱导反应可能参与晒伤红斑和炎症的发病机制。用UVB（290 - 320 nm）辐射（高达200 mJ/cm2）处理人角质形成细胞，导致NO和ONOO-释放呈剂量依赖性增加，而N-单甲基-L-精氨酸（L-NMMA）可抑制这种增加。角质形成细胞释放NO和ONOO-的同时，细胞内cGMP水平升高。用不同剂量的UVB（高达100 mJ/cm2）处理人角质形成细胞胞质溶胶，导致XO活性增加，而氧嘌呤醇可抑制这种增加。角质形成细胞的UVB辐射（高达100 mJ/cm2）导致S-亚硝基硫醇形成增加15倍，这通过一种从载体分子释放NO的机制直接增加了纯化的可溶性鸟苷酸环化酶（sGC）的活性。在重组实验中，当将从角质形成细胞胞质溶胶中分离出的经UVB照射（20 mJ/cm2）的纯化cNOS与经UVB照射（20 mJ/cm2）的纯化XO结合时，与未照射的酶相比，观察到ONOO-生成增加了4倍。cNOS和XO经UVB辐射后由NO和O2-合成的ONOO-被氧嘌呤醇（100 microM）抑制。角质形成细胞胞质溶胶的UVB辐射导致氧自由基生成增加，这与经UVB照射的角质形成细胞胞质溶胶中ONOO-生成增加一致。在体内实验中，当对实验动物进行UVB辐射时，在其皮肤上涂抹含有硝基-L-精氨酸（L-NA）（2%）和L-NMMA（2%）的乳化乳膏制剂后，计算出保护因子（PF）为6.5±1.8。本研究表明，UVB辐射是人角质形成细胞中cNOS和XO活性的有效刺激物。NO和ONOO-可能对角质形成细胞本身及其邻近的内皮细胞和平滑肌细胞产生细胞毒性作用。这可能是导致红斑产生和炎症过程的综合反应的主要部分。
