The Effect of Nitric Oxide Release Rates on the Oxidation of Human Low Density Lipoprotein

Steven P.A Goss,Neil Hogg,B Kalyanaraman

doi:10.1074/jbc.272.34.21647

Steven P.A Goss, Neil Hogg + Show 1 more

Open Access

https://doi.org/10.1074/jbc.272.34.21647

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Abstract

1-Substituted diazen-1-ium-1,2-diolates, a class of nitric oxide (. NO) donor compounds that spontaneously release .NO at different rates, were used to investigate the effect of .NO release rate upon the oxidation of low density lipoprotein (LDL). All donor compounds conferred an inhibitory effect upon the oxidation of LDL; however, the effect exhibited a biphasic dependence upon the rate of .NO release. The .NO release rate that maximally inhibited oxidation was dependent upon the rate of oxidation. When LDL was rapidly oxidized by copper(II) sulfate, a faster release rate was more effective. In contrast, when LDL was oxidized slowly by 2,2'-azobis-2-amidinopropane hydrochloride, a slower release rate was most effective. This biphasic relationship between .NO release rate and the duration of inhibition was also demonstrated when LDL oxidation was initiated with 5-amino-3-(4-morpholinyl)-1,2, 3-oxadiazolium, a peroxynitrite generator. We conclude that the antioxidant ability of .NO is dependent not only upon the rate of its release from .NO donors, but also upon the rate of oxidation. This conclusion is supported by a kinetic model of LDL oxidation in the presence of .NO.

Highlights

The mechanisms by which 1⁄7NO affects atherosclerotic lesion formation are unclear; there are many possibilities
We conclude that an optimal rate of 1⁄7NO release is required for maximal suppression of low density lipoprotein (LDL) oxidation, and that this optimal rate depends upon the rate and the mechanism of LDL oxidation
LDL oxidation for approximately 2 h, while DNN, with a t1⁄2 of 21.9 Ϯ 0.7 h, inhibited oxidation for 1 h. This indicates that there is an optimal rate of 1⁄7NO production for maximal suppression of Cu2ϩ-dependent LDL oxidation

Summary

EXPERIMENTAL PROCEDURES

Materials—Copper(II) sulfate was purchased from Fisher. 2,2Ј-Azobis-2-amidinopropane hydrochloride (ABAP) was obtained from Polysciences, Inc. (Z)-1-{N-Methyl-N-[6-(N-methylammoniohexyl)amino]} diazen-1-ium-1,2-diolate (MNN), (Z)-1-[N-(3-ammoniopropyl)-N-(npropyl)amino]}diazen-1-ium-1,2-diolate (PNN), (Z)-1-{N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino}diazen-1-ium-1,2-diolate (SNN), (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen1-ium-1,2-diolate (DNN), and 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium (SIN-1) were purchased from Cayman Chemical Co. LDL was isolated from human plasma as described previously [19]. Measurement of LDL Oxidation—LDL was incubated in phosphatebuffered saline (PBS) at 37 °C, either alone or in the presence of MNN, PNN, SNN, or DNN. Oxidation was initiated with copper(II) sulfate, ABAP, or SIN-1. Thiobarbituric Acid-reactive Substances (TBARS) Measurements— LDL (10 –20 ␮g) was incubated with thiobarbituric acid (0.5%, w/v, in H2SO4, 50 mM) for 30 min at 100 °C. TBARS concentration was calculated as malondialdehyde (MDA) equivalents using a MDA standard curve. Conjugated Diene Measurements—LDL (200 ␮g/ml), incubated in PBS at 37 °C, was continuously monitored at 234 nm to detect the formation of conjugated dienes as described previously [21]. ␣-Tocopherol Measurements—␣-Tocopherol was extracted into heptane and assayed by high performance liquid chromatography as described previously [23]. Integration of the equations shown under “Appendix,” using the rate constants defined in Table II, was performed using the Stiff Euler algorithm

RESULTS

DISCUSSION

CONCLUSIONS