Abstract

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 thatl-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 (10⁻⁸ M) significantly decreased rat aortic (A10 cell line) vascular smooth muscle proliferation. The percentage of inhibition exerted byl-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 inl-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 10⁻¹⁰, 10⁻⁹, 10⁻⁸, and 10⁻⁷ 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 (10⁻⁵ 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 (10⁻⁸ M) (P < .001,n = 3). These findings suggest thatl-citrulline decreases vascular smooth muscle cell proliferation in the A10 cell line by acting on DNA synthesis by mechanisms that involve the ANP receptor.