Elsevier

Biochemical Pharmacology

Volume 51, Issue 12, 28 June 1996, Pages 1727-1738
Biochemical Pharmacology

Research paper
Alterations of nitric oxide synthase and xanthine oxidase activities of human keratinocytes by ultraviolet B radiation: Potential role for peroxynitrite in skin inflammation

https://doi.org/10.1016/0006-2952(96)00110-4Get rights and content

Abstract

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 μM). 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.

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    This work was supported by grants from the Ministry of Research and Technology (92 SYN 17) and from the University of Athens.

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