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Nitric oxide synthase activation and oxidative stress, but not intracellular zinc dyshomeostasis, regulate ultraviolet B light-induced apoptosis.

ABSTRACT: To investigate the role of nitric oxide synthase (NOS) and intracellular free zinc ion (Zn(2+)) in regulation of ultraviolet B light (UVB)-induced cell damage and apoptosis.Real-time confocal microscopy measurement was used to determine the changes of intracellular free zinc concentration under different conditions. Cell apoptotic death was determined using fluorescein isothiocyanate (FITC) conjugated-annexin V (ANX5)/PI labeling followed by flow cytometry. Western analysis was used to determine cell apoptosis and eNOS uncoupling.UVB induced an elevation of Zn(2+) within 2 min of exposure. The UVB-induced intracellular Zn(2+) elevation was dependent on the increase of constitutive nitric oxide synthase (cNOS) activity and production of superoxide. Removal of Zn(2+) with a lower concentration (<25 microM) of N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a Zn(2+)-specific chelator, did not induce cell death or prevent cells from UVB-induced apoptosis. However, a higher [TPEN] (>50 microM) was cytotoxic to cells, but prevented cells from further UVB-induced apoptosis. The higher [TPEN] also induced cNOS uncoupling. Furthermore, treating the cells with a membrane permeable superoxide dismutase (PEG-SOD) inhibited Zn(2+) release and reduced apoptotic cell death after UVB treatment. The results demonstrated a complex and dynamic regulation of UVB-induced cell damage.Our findings not only advance our understanding of the correlations between cNOS activation and Zn elevation, but also elucidated the role of cNOS in regulation of oxidative stress and apoptosis upon UVB-irradiation.

SUBMITTER: Wang L

PROVIDER: S-EPMC2834872 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Nitric oxide synthase activation and oxidative stress, but not intracellular zinc dyshomeostasis, regulate ultraviolet B light-induced apoptosis.

Wang Lei L Liu Wei W Parker Suzanne H SH Wu Shiyong S

Life sciences 20100208 11-12

<h4>Aims</h4>To investigate the role of nitric oxide synthase (NOS) and intracellular free zinc ion (Zn(2+)) in regulation of ultraviolet B light (UVB)-induced cell damage and apoptosis.<h4>Main methods</h4>Real-time confocal microscopy measurement was used to determine the changes of intracellular free zinc concentration under different conditions. Cell apoptotic death was determined using fluorescein isothiocyanate (FITC) conjugated-annexin V (ANX5)/PI labeling followed by flow cytometry. West ...[more]

PMID: 20149802

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Project description:BACKGROUND:Nutrition with ammonium (NH4+) can enhance the drought tolerance of rice seedlings in comparison to nutrition with nitrate (NO3-). However, there are still no detailed studies investigating the response of nitric oxide (NO) to the different nitrogen nutrition and water regimes. To study the intrinsic mechanism underpinning this relationship, the time-dependent production of NO and its protective role in the antioxidant defense system of NH4+- or NO3--supplied rice seedlings were studied under water stress. RESULTS:An early NO burst was induced by 3?h of water stress in the roots of seedlings subjected to NH4+ treatment, but this phenomenon was not observed under NO3- treatment. Root oxidative damage induced by water stress was significantly higher for treatment with NO3- than with NH4+ due to reactive oxygen species (ROS) accumulation in the former. Inducing NO production by applying the NO donor 3?h after NO3- treatment alleviated the oxidative damage, while inhibiting the early NO burst by applying the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) increased root oxidative damage in NH4+ treatment. Application of the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester(L-NAME) completely suppressed NO synthesis in roots 3?h after NH4+ treatment and aggravated water stress-induced oxidative damage. Therefore, the aggravation of oxidative damage by L-NAME might have resulted from changes in the NOS-mediated early NO burst. Water stress also increased the activity of root antioxidant enzymes (catalase, superoxide dismutase, and ascorbate peroxidase). These were further induced by the NO donor but repressed by the NO scavenger and NOS inhibitor in NH4+-treated roots. CONCLUSION:These findings demonstrate that the NOS-mediated early NO burst plays an important role in alleviating oxidative damage induced by water stress by enhancing the antioxidant defenses in roots supplemented with NH4+.

Project description:In previous studies [Delker, S. L., et al. (2010), J. Am. Chem. Soc. 132, 5437-5442], we determined the crystal structures of neuronal nitric oxide synthase (nNOS) in complex with nNOS-selective chiral pyrrolidine inhibitors, designed to have an aminopyridine group bound over the heme where it can electrostatically interact with the conserved active site Glu residue. However, in addition to the expected binding mode with the (S,S)-cis inhibitors, an unexpected "flipped" orientation was observed for the (R,R)-cis enantiomers. In the flipped mode, the aminopyridine extends out of the active site where it interacts with one heme propionate. This prompted us to design and synthesize symmetric "double-headed" inhibitors with an aminopyridine at each end of a bridging ring structure [Xue, F., Delker, S. L., Li, H., Fang, J., Jamal, J., Martásek, P., Roman, L. J., Poulos, T. L., and Silverman, R. B. Symmetric double-headed aminopyridines, a novel strategy for potent and membrane-permeable inhibitors of neuronal nitric oxide synthase. J. Med. Chem. (submitted for publication)]. One aminopyridine should interact with the active site Glu and the other with the heme propionate. Crystal structures of these double-headed aminopyridine inhibitors in complexes with nNOS show unexpected and significant protein and heme conformational changes induced by inhibitor binding that result in removal of the tetrahydrobiopterin (H(4)B) cofactor and creation of a new Zn(2+) site. These changes are due to binding of a second inhibitor molecule that results in the displacement of H(4)B and the placement of the inhibitor pyridine group in position to serve as a Zn(2+) ligand together with Asp, His, and a chloride ion. Binding of the second inhibitor molecule and generation of the Zn(2+) site do not occur in eNOS. Structural requirements for creation of the new Zn(2+) site in nNOS were analyzed in detail. These observations open the way for the potential design of novel inhibitors selective for nNOS.

Dataset Information

Nitric oxide synthase activation and oxidative stress, but not intracellular zinc dyshomeostasis, regulate ultraviolet B light-induced apoptosis.

Publications

Nitric oxide synthase activation and oxidative stress, but not intracellular zinc dyshomeostasis, regulate ultraviolet B light-induced apoptosis.

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