Modulation of Expression of Endothelial Nitric Oxide Synthase by Nordihydroguaiaretic Acid, a Phenolic Antioxidant in Cultured Endothelial Cells SANTHINI RAMASAMY, GRANT R. DRUMMOND, JOON AHN, MICHAL STOREK, JAN POHL, SAMPATH PARTHASARATHY, and DAVID G. HARRISON Division of Cardiology (S.R., G.R.D., J.A., D.G.H.), Department of Gynecology and Obstetrics (S.P.), and Microchemical Facility (M.S., J.P.), Emory University, Atlanta, Georgia Received March 30, 1999; accepted April 5, 1999 This paper is available online at http://www.molpharm.org ABSTRACT Retrospective epidemiological studies have suggested that an- tioxidant therapy may decrease cardiovascular morbidity and mortality rates, although the mechanisms for this effect remain unclear. In the present study, we demonstrate that selective antioxidants can enhance expression of endothelial nitric oxide synthase (eNOS). We found that the antioxidants nordihy- droguaiaretic acid (NDGA), catechol, glutaryl probucol, and N-acetylcysteine increased eNOS expression in cultured bo- vine aortic endothelial cells (BAECs). NDGA seemed to be the most potent of the phenolic antioxidants, producing a 3-fold increase in eNOS mRNA. This effect of NDGA was enhanced by nonphenolic antioxidants such as N-acetylcysteine and ascor- bic acid. Nuclear run-on studies indicated that NDGA increased eNOS transcription. A similar increase in eNOS protein content was observed with Western blot analysis after treating BAECs or human aortic endothelial cells with NDGA. Exposure of BAECs to NDGA enhanced NO production, as measured by electron paramagnetic resonance spin trapping and eNOS ac- tivity, as measured by [ 14 C]arginine-to-[ 14 C]citrulline assay. Methylation of the phenolic hydroxyl groups completely inhib- ited the NDGA effect on eNOS mRNA levels. This effect of NDGA was not due to inhibition of lipoxygenase because cis- 5,8,11,14-eicosatetraynoic acid did not alter eNOS expression. We conclude that antioxidants may not only increase the bio- activity of nitric oxide but also enhance expression of the eNOS enzyme. Such an effect may prove useful in conditions such as hypertension and atherosclerosis, in which nitric oxide produc- tion and/or biological activity is impaired. Endothelium-derived nitric oxide (NO) plays an important role in the modulation of vascular tone and regulation of blood pressure. Moreover, NO inhibits platelet aggregation, smooth muscle proliferation, and leukocyte adhesion to the endothelium (Moncada and Higgs, 1993; Forstermann et al., 1994). In several pathological conditions, such as hypercho- lesterolemia, atherosclerosis, diabetes mellitus, and hyper- tension, endothelium-dependent relaxation is impaired. Re- active oxygen species appear to play an important role in the progression of most of these disease states (Freiman et al., 1986; Giugliano et al., 1995; Keaney and Vita, 1995; Rajago- palan et al., 1996). More specifically, it has been observed that there is increased production of vascular superoxide, which on reaction with NO results in loss of biological activ- ity of NO. In patients with non-insulin-dependent diabetes mellitus or hypertension or who are cigarette smokers, acute infu- sions of ascorbic acid have been shown to improve vasorelax- ation (Heitzer et al., 1996; Ting et al., 1996, 1997; Solzbach et al., 1997). In cholesterol-fed rabbits, the impairment in en- dothelium-dependent relaxation is, in part, reversed by treatment with antioxidants such as vitamin E (Keaney et al., 1993, 1994). Even oral administration of antioxidants has been shown to improve endothelium-dependent vasodilation (Simon et al., 1993; Levine et al., 1996). Although the mech- anism by which antioxidant treatment improves vasorelax- ation is not clear, it is generally assumed that antioxidants scavenge superoxide and thus increase the availability of functional NO. Kinetic constants, however, suggest that the rate of reactions between common antioxidants and superox- This work was supported by National Institutes of Health Grants HL390006 and HL48667m as well as American Heart Association Scientist Development Grant Award 9630119N (to S.R.). Portions of this work were presented at the American Heart Association 70th Meeting, November 1997, Orlando, FL; Society of Toxicology 37th Meeting, Seattle, WA, March 1998; and Vascular Biology, 1998, San Francisco, CA, April 1998. ABBREVIATIONS: NO, nitric oxide; NDGA, nordihydroguaiaretic acid; BHT, butylated hydroxy toluene; M199, Medium 199; EPR, electron paramagnetic resonance; MGD, N-methyl-D-glucamine dithiocarbamate; eNOS, endothelial nitric oxide synthase; HAEC, human aortic endothelial cell; NAC, N-acetylcysteine; L-NAME, N G -nitro-L-arginine methyl ester; BAEC, bovine aortic endothelial cell; SOD, superoxide dismutase; Sp1, simian virus 40 promoter factor 1; AP-1, activator protein 1; EGM, endothelial cell growth media; ARE, antioxidant response element; ETYA, cis-5,8,11,14-eicosatetraynoic acid. 0026-895X/99/010116-08$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 56:116 –123 (1999). 116 by guest on August 27, 2013 molpharm.aspetjournals.org Downloaded from