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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Department of Chemistry and Biochemistry, Edison Biotechnology Institute, Ohio University, Athens, OH 45701, United States.
Lei
L
Wang
Wei
W
Liu
Suzanne H
SH
Parker
Shiyong
S
Wu
D017209:Apoptosis / Q000502:physiology* / Q000528:radiation effects*; D015153:Blotting, Western; D002614:Chelating Agents / Q000494:pharmacology; D004789:Enzyme Activation / Q000502:physiology*; D005029:Ethylenediamines / Q000494:pharmacology; D005434:Flow Cytometry; D006706:Homeostasis / Q000502:physiology*; D015603:Keratinocytes / Q000502:physiology / Q000528:radiation effects; D009569:Nitric Oxide / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014466:Ultraviolet Rays; D015032:Zinc / Q000378:metabolism*
D002614:Chelating Agents; D005029:Ethylenediamines; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D011065:Poly(ADP-ribose) Polymerases; D015032:Zinc; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine