Acrolein produces nitric oxide through the elevation of intracellular calcium levels to induce apoptosis in human umbilical vein endothelial cells: implications for smoke angiopathy.
Acrolein is a highly electrophilic alpha, beta-unsaturated aldehyde, the levels of which are increased in the blood of smokers. To determine if acrolein is involved in the pathology of smoke angiopathy, the effect of acrolein on human umbilical vein endothelial cells (HUVEC) was examined. Intracellular nitric oxide (NO) levels, determined using diaminofluorescein-2 diacetate (DAF-2 DA), an NO sensitive fluorescent dye, were found to be increased after treatment in HUVEC with 10 microM acrolein. The measurement of nitrite with 2,3-diaminonaphthalene and a Western blot analysis revealed that nitrite and S-nitroso-cysteine levels were increased in a dose-dependent manner, confirming that NO production is increased by acrolein. The increase was not reduced by treatment with 10mM N-acetyl-l-cysteine (NAC), an anti-oxidant, but was reduced with 10 microM of the intracellular calcium chelator, 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester. Acrolein-stimulated NO production was significantly reduced by pretreatment with 1mM N(G)-nitro-l-arginine-methyl ester (L-NAME), an NO synthase inhibitor. The cytotoxicity of acrolein was reduced by pretreatment with 10 microM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO), an intracellular NO scavenger, or 1mM L-NAME, whereas it was not reduced by 10mM NAC, 20 microM Curcumin, another peroxide scavenger, or 100 microM Mn(III)TMPyP, a superoxide dismutase mimic. Nuclear staining and a Western blot analysis using an anti-cleaved caspase 3 antibody revealed that the reduced viability of HUVEC by acrolein was due to apoptosis, which was reversed after pretreatment with 0.1mM carboxy-PTIO or 1mM L-NAME. Thus, acrolein increases intracellular calcium production to induce intracellular NO production by a calcium-dependent NO synthase, possibly eNOS, and the excess and rapid increase in NO might lead to the apoptosis of HUVEC. These data suggest that acrolein might be involved in the pathology of smoke angiopathy through the NO-induced apoptosis of endothelial cells.
Misonou
Yoshiko
Y
Department of Biochemistry, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Asahi
Michio
M
Yokoe
Shunichi
S
Miyoshi
Eiji
E
Taniguchi
Naoyuki
N
D000171:Acrolein / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D012907:Smoking / Q000009:adverse effects; D014471:Umbilical Veins / Q000166:cytology*
D003497:Cyclic N-Oxides; D007093:Imidazoles; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; C079391:2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; D009569:Nitric Oxide; D004533:Egtazic Acid; D000171:Acrolein; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester