Oxidized Lipoproteins Induce Long-Lasting Inhibition of Nitric Oxide Synthase from a Murine Endothelioma Cell Line (bEnd.4)

Abstract

Background: The vascular endothelium produces nitric oxide, which has vasodilatory properties. It has been postulated that some lipoproteins may increase arterial vascular tone by decreasing the availability of endothelium-derived nitric oxide. The mechanism underlying this effect, however, is still poorly understood.

Methods: We investigated the effect of native and oxidized human low- and high-density lipoproteins on the nitric oxide synthetic activity of an endothelioma cell line (bEnd.4). Oxidized lipoproteins were obtained by incubation with CuSO₄. The production of nitric oxide by the cells was monitored by quantifying the nitrite concentration in the medium using the Greiss reagent.

Results: The synthesis of nitric oxide by the bEnd.4 cell line was calcium-dependent and was abolished by a selective inhibitor of the constitutive nitric oxide synthase. Incubation with oxidized lipoproteins caused a time- and dose-dependent inhibition of nitric oxide synthetic activity. At a concentration of 100 μg/ml cholesterol, oxidized low- and high-density lipoproteins inhibited the production of nitric oxide by 27 and 51%, respectively, within 6 h. Normal nitric oxide synthetic activity was regained by the cells only after 24 h. The lipid fraction obtained from the native or the oxidized lipoproteins mimicked the effect of the intact lipoproteins.

Conclusion: These results support the involvement of oxidized lipoproteins in the modulation of endothelial functions relevant to the pathogenesis of cardiovascular diseases.