A Maillard reaction product enhances eNOS enzymatic activity in human endothelial cells

Nitric oxide (NO) produced by the endothelial nitric oxide synthase (eNOS) is an essential signaling molecule in the cardiovascular system. Reduced eNOS activity is associated with the development of atherosclerosis [1]. Maillard reaction products (MRP), formed by the ubiquitious reaction between sugars and amins, possess antioxidant activity and other pharmacological effects [2]. We investigated a MRP, which is formed by the reaction between starch and a primary amine, and examined its effects on eNOS in the human endothelial cell line EA.hy926 [3]. We used EA.hy926 cells stably transfected with a plasmid containing 3600bp of the human eNOS promotor driving a luciferase reporter gene for measuring human eNOS promotor activity and western blot to quantify protein levels. ENOS enyzyme activity was investigated by an [¹⁴C]L-arginine/L-citrulline conversion assay. NO was quantified by the reaction with the fluorescent probe DAF-2 [4]. After 18 hours of incubation (30µM –300µM) we observed a significant and concentration-dependent increase of eNOS activity. NO production peaked at a concentration of 100µM. Surprisingly we found a tendency towards a slight decrease of human eNOS promotor activity and protein levels. A time course with incubation times ranging from 30 minutes to 24 hours showed that eNOS enzyme activity was slightly attenuated during the first eight hours, but increased significantly afterwards. We therefore hypothesize that the de novo synthesis of another protein is needed to mediate this effect. This is the first time that positive effects of MRPs on eNOS activity and NO production are demonstrated in-vitro. Given the regular nutritional uptake of MRPs due to their great abundance in food, these results could be of physiologic importance.

Acknowledgments: The authors would like to thank Dr. C.-J.S. Edgell (University of North Carolina) for EA.hy926 cells and Daniel Schachner for excellent technical assistance.

References: 1. Naseem, K.M. (2005), Mol. Aspects Med. 26:33–65. 2. Somoza, V. (2005), Mol. Nutr. Food. Res. 49: 663–672. 3. Edgell, C.-J.S. et al. (1983), Proc. Natl. Acad. Sci. USA 80: 3734–3737. 4. Leikert, J.F. et al. (2001), FEBS Lett. 506: 131–134.