Effects of L‐arginine/citrulline–simvastatin (SIM) association on nitric oxide production in endothelial cells

Abstract

Hypercholesterolemia is a major risk factor for atherosclerosis. It is usually treated by statins, which also increase endothelial NO synthase (eNOS) activity producing NO. NO, which has vasodilatory and antiatherosclerotic properties, is synthesized from arginine (ARG) or citrulline (CIT) by regenerating ARG with argininosuccinate synthetase and lyase (ASS and ASL). We hypothesized that a therapeutic strategy associating a statin to activate eNOS, with ARG or CIT to increase eNOS substrate availability, would be more efficient than using a statin alone. Methods Bovine aortic endothelial cells were used. NO production was measured after various treatments (SIM: 0 to 10 mM with ARG or CIT: 0 to 5 mM, 24 h of incubation; n=4/treatment group). Protein quantity (eNOS) was assessed by Western blot analysis and mRNA levels (eNOS, ASS and ASL) were assessed by RT-PCR. Results NO production was increased by combining ARG or CIT with SIM. ARG was 1.5-fold more efficient in producing NO than equimolar CIT. SIM induced a 2-fold increase in eNOS mRNA levels, and an optimal concentration of ARG or CIT in association with SIM was required to induce a significant increase (x3) in eNOS protein. ASS and ASL mRNA levels were doubled with SIM, whereas high concentrations of substrate (ARG or CIT at 1mM) decreased (/3) ASL mRNA levels. Conclusion The association of ARG or CIT with SIM promotes NO production in endothelial cells by increasing the quantity of eNOS proteins. These results show the potential clinical benefit of this association in the prevention and treatment of atherosclerosis.