| Lipid peroxidation is thought to contribute to the development of atherosclerosis and its associated impairment of endothelium-derived nitric oxide (NO ·) bioactivity. Investigation into the role of lipid peroxidation and endothelium-derived NO· bioactivity has focused primarily on the oxidation of low-density lipoprotein and its impact on the endothelium. However, endothelial cells contain considerable oxidizable lipid in the plasma membrane and the consequences of endothelial cell lipid peroxidation on NO· action and metabolism is unknown. Therefore, a model of endothelial cell lipid peroxidation was established using cultured porcine aortic endothelial cells (PAECs) exposed to a flux of aqueous peroxyl radicals from 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) or lipid-soluble peroxyl radicals from 2,2′-azobis(2,4-dimethyl-valeronitrile) (AMVN).; PAECs exposed to AAPH or AMVN received a steady-state flux of peroxyl radicals for 0 to 5 hours and demonstrated significant accumulation of membrane lipid peroxidation markers without evidence of cytotoxicity. Intact PAECs exposed to AAPH in this manner demonstrated impaired NO· bioactivity, measured as cGMP accumulation, when stimulated with a calcium ionophore but not with exogenous NO·-donors, suggesting a defect in NO· production. This defect was not accompanied by a reduction in endothelial nitric oxide synthase (eNOS) protein. To elucidate the possible mechanism(s) for the decrease in NO· bioactivity, membrane preparations from treated cells were isolated, the required cofactors were added back, and eNOS activity was assessed by measuring the extent of conversion of L-arginine to L-citrulline. Membrane preparations from cells exposed to AAPH or AMVN exhibited decreased eNOS catalytic activity. Several potential mechanisms were investigated to explain the decrease in eNOS activity. These included eNOS cofactor status, thiol status, phosphorylation status, and eNOS dimer interaction. We found none of these mechanisms was of major importance in the effect of peroxyl radicals to inhibit eNOS activity in endothelial cells. However, direct exposure of membrane preparations to peroxyl radicals decreased eNOS enzymatic activity. These data imply that lipid peroxyl radicals may mediate impaired NO· bioactivity through inhibition of eNOS catalytic activity by a mechanism(s) yet to be elucidated. These studies demonstrate one potential means by which oxidative stress can reduce NO · bioactivity during atherosclerosis. |
