Ultraviolet B (UVB) Radiation Induces Peroxinitrite (ONOO−) Formation by Human Endothelial Cells
Ultraviolet B radiation (UVB 290–320 nm) up to 100 mJ/cm2 of human endothelial cells increased nitric oxide (NO), cGMP, superoxide O2− and hydroperoxide (H2O2) production. UVB irradiated endothelial cells elicited luminol-dependent chemiluminescence (CL) that increased linearly by the addition of L-Arg. N-mono-methyl-L-arginine (L-NMMA) (1 mM) inhibited CL by up to 70%. SOD inhibited CL response indicating that O2− was a key intermediate for chemiexcitation. Neither NO nor O2− alone were capable in directly inducing luminol CL suggesting that ONOO¯ was responsible for the CL of luminol. Cysteine (3 mM), and desferoxamine (200 µM) reduced the peak CL by 90% while mannitol (100 mM) and DMSO (100 mM) was ineffective. A similar CL response was also obtained using authentic ONOO¯. UV spectra were recorded 1 hour after UVB radiation (100 mJ/cm2) of endothelial cells in a Hank’s Balanced Salt Solution (HBSS) made basic (pH: 8.0) by Na2CO3. The concentration of ONOO¯ formed by irradiated endothelial cells based on an ε382 nm value of 1670 <¯1 cm¯1 was estimated to be 50 nmol ONOO per 106 cells. Spectroscopy of human erythrocyte lysates showed absorbance at 540 and 576 nm. When intact erythrocytes (106 cells) were cultured with UVB (100 mJ/cm2) irradiated endothelial cells (l08 cells) the absorption spectrum of this lysate demonstrated a decrease (80%) of these peaks, findings consistent with those obtained using authentic ONOO¯. ONOO¯ (up to 100 µM) increased four folds the soluble guanylate cyclase activity of endothelial cells. This effect was reduced by approximately 90% in the presence of desferioxamine and by approximately 50% by bicarbonate. UVB radiation causes lipid peroxidation as evidenced by the formation of thiobarbituric acid reactive substances (TBARS) and the stimulation of endothelial cells oxygen consumption. ONOO¯ initiated lipid peroxidation of endothelial cells in an extent dependent on ONOO¯ concentration, with the greatest yield occurring at 200 µM ONOO¯. Oxygen consumption was conducted using a Clark electrode, in a 1.8 ml mater-jacked chamber maintained at 37°C. In UVB irradiated endothelial cells a slow phase reaction of oxygen consumption lasting for 15 min was observed. ONOO¯ decomposition in HBSS resulted in oxygen evolution in the medium. Oxygen evolution from the decomposition of ONOO¯ was complete within 5–10 sec. In ONOO¯ endothelial cells reaction an initial rapid phase of oxygen consumption lasting for 10 min was observed. Our results showed that ultraviolet radiation of human endothelial cells results in the elaboration of NO and O2− leading to the production of ONOO¯. Radiation of the human skin may lead to the production of ONOO¯ by endothelial cells resulting in inflammation that accompanies human sunburn reactions.