NO synthase and xanthine oxidase activities of rabbit brain synaptosomes: Peroxynitrite formation as a causative factor of neurotoxicity |
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Authors: | George Deliconstantinos Vassiliki Villiotou |
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Institution: | (1) Department of Experimental Physiology, University of Athens Medical School, GR-115 27 Athens, Greece |
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Abstract: | In the present study we demonstrated that synaptosomes isolated from rabbit brain cortex contain NO synthase and xanthine
oxidase that can be activated by ultraviolet B radiation and Ca2+ accumulation to produce nitric oxide and superoxide which react together to form peroxynitrite. Irradiation of synaptosomes
with ultraviolet B (up to 100 mJ/cm2), or increase the intrasynaptosomal calcium concentration using various doses (up to 100 μM) of the calcium ionophore A 23187,
a gradual increase in both nitric oxide and peroxynitrite release that was inhibited by N-monomethyl-L-arginine (100 μM) was
observed. The rate of nitric oxide release and cyclic GMP production by NO synthase and soluble guanylate cyclase, both located
in the soluble fraction of synaptosomes (synaptosol), were increased approximately eight fold after treatment of synaptosomes
with Ultraviolet B radiation (100 mJ/cm2). In reconstitution experiments, when purified NO synthase isolated from synaptosol was added to xanthine oxidase, in the
presence of the appropriate cofactors and substrates, a ten fold increase in peroxynitrite production at various doses (up
to 20 mJ/cm2) of UVB radiation was observed. Ultraviolet B irradiated synaptosomes promptly increased malondialdehyde production with
subsequent decrease of synaptosomal plasma membrane fluidity estimated by fluorescence anisotropy of 1-4-(trimethyl-amino-phenyl)-6-phenyl-hexa-1,3,5-triene.
Desferrioxamine (100 μM) tested in Ultraviolet B-irradiated synaptosomes showed a decrease (approximately 80%) in malondialdehyde
production with subsequent restoration of the membrane fluidity to that of non-irradiated (control) synaptosomes. Ca2+-stimulated ATPase activity was decreased after Ultraviolet B (100 mJ/cm2) radiation of synaptosomes indicating that the subsequent increase of intrasynaptosomal calcium promoted peroxynitrite production
by a calmodulin-dependent increase of NO synthase and xanthine oxidase activities. Furthermore, it was shown that UVB-irradiated
synaptosomes were subjected to higher oxidative stress by exogenous peroxynitrite (100 μM) compared to non-irradiated (control)
synaptosomes. In summary, the present results indicate that activation of NO synthase and xanthine oxidase of brain cells
lead to the formation of peroxynitrite providing important clues in the role of peroxynitrite as a causative factor in neurotoxicity. |
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Keywords: | Nitric oxide (NO) peroxynitrite (ONOO− ) NO-synthase xanthine oxidase soluble guanylate cyclase ultraviolet radiation membrane fluidity synaptosomes neurotoxicity |
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