Vol. 290, Issue 1, 310-313, July 1999

L-Citrulline, the By-Product of Nitric Oxide Synthesis, Decreases Vascular Smooth Muscle Cell Proliferation

Emilio Ruiz, Marcela Del Rio, Beatriz Somoza, Patricia Ganado, Mercedes Sanz and Teresa Tejerina

Department of Pharmacology, School of Medicine, Complutense University, Madrid, Spain

Endothelium injury plays an important role in atherosclerosis. Damage to the endothelium results in vascular smooth muscle cell proliferation. Natriuretic peptides present a potent antimitogenic action, mediating their biological effects via the binding of guanylate cyclase-linked atrial natriuretic peptide (ANP) receptor and the production of cyclic GMP. In a previous study, we demonstrated that L-citrulline, the by-product of nitric oxide synthesis, could relax rabbit aortic rings by stimulating the guanylate cyclase-linked ANP receptor. In this work, we investigated the effect of L-citrulline on vascular smooth muscle cell proliferation. L-Citrulline (10⁸ M) significantly decreased rat aortic (A10 cell line) vascular smooth muscle proliferation. The percentage of inhibition exerted by L-citrulline on days 3, 5, and 7 of the proliferation curve was 20.0 ± 0.5%, 37.5 ± 8.3%, and 28.5 ± 7.2%, respectively. In addition, L-citrulline also inhibited serum-induced DNA synthesis, measured as 5-bromo-2'-deoxyuridine incorporation. 5-Bromo-2'-deoxyuridine incorporation into nuclei of vehicle-treated cells was 40.5 ± 2.4%, whereas in L-citrulline-treated cells the percentage decreased to 36.0 ± 4.1%, 29.1 ± 2.0% (P < .01, n = 4), 30.5 ± 2.4% (P < .05, n = 4), and 23.1 ± 0.5% (P < .001, n = 4) for 10¹⁰, 10⁹, 10⁸, and 10⁷ M, respectively. Zaprinast, a phosphodiesterase type V inhibitor, enhanced 5-bromo-2'-deoxyuridine incorporation in serum-stimulated cells. Moreover, L-citrulline inhibition of serum-stimulated DNA synthesis was abolished by HS-142-1 (10⁵ M), an ANP receptor antagonist. In another group of experiments, L-citrulline was shown to increase intracellular cyclic GMP levels from 2.1 ± 0.2 pmol of cGMP/mg protein to 4.1 ± 0.1 for L-citrulline (10⁸ M) (P < .001, n = 3). These findings suggest that L-citrulline decreases vascular smooth muscle cell proliferation in the A10 cell line by acting on DNA synthesis by mechanisms that involve the ANP receptor.