Introduction

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The endothelium lining of blood vessels modulates vascular tone, inhibits platelet adhesion to the vessel wall, and maintains an anticoagulatory surface under normal conditions. Damage to the endothelium results in thrombus formation, platelet adhesion, and vascular smooth muscle cell proliferation. Injury to the endothelium plays an important role in the pathogenesis of atherosclerosis (Ross, 1993). It is well known that nitric oxide (NO) inhibits vascular smooth muscle cell proliferation both by interfering with processes common to all cells such as mitochondrial respiration and DNA synthesis (Garg et al., 1989; Kwon et al., 1991; Cornwell et al., 1994; Sarkar et al., 1995) and by apoptosis through a cGMP-independent mechanism (Nishio et al., 1996). On the other hand, atrial natriuretic peptide (ANP) belongs to a family of structurally related peptides with potent natriuretic, vasodilatory, and antimitogenic actions (Itoh et al., 1990; Furuya et al., 1991; Komatsu et al., 1996). ANP and brain natriuretic peptide are ligands for the natriuretic peptide-A receptor (Supaporn et al., 1996), highly expressed in vascular smooth muscle cells. These natriuretic peptides mediate their biological actions via binding to the guanylate cyclase-linked ANP receptor with the subsequent generation of second messenger cGMP (Kuno et al., 1986; Chinkers et al., 1989). The natriuretic peptide system may function in parallel with the NO system, which also participates in the control of cardiovascular function but via the action of soluble guanylate cyclase.

In a previous study carried out in our laboratory, we demonstrated that L-citrulline, the by-product of NO synthesis, relaxed rabbit vascular smooth muscle by increasing cGMP due to particulate guanylate cyclase (Ruiz and Tejerina, 1998). In the present study, we tested the possibility that L-citrulline could inhibit vascular smooth muscle proliferation and if this effect was related to an action on the guanylate cyclase-linked ANP receptor.

	Experimental Procedures

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Cell Culture and Cell Number Determination. Rat A10 vascular smooth muscle cells were obtained from the American Type Culture Collection (A10 CRL 1476). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) supplemented with glutamax I, 100 I.U./ml¹ penicillin G (sodium salt), 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (antibiotic-antimycotic solution, Life Technologies Inc., Gaithersburg, MD). To follow proliferation in the presence of L-citrulline (10⁸ M), cells were seeded at 2.0 × 10⁴ cells/well, detached with trypsin, and counted in a hemocytometer. Throughout the experiments, media were changed daily, and cultures were protected from the light.

Determination of DNA Synthesis. DNA synthesis, as assayed by the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into nuclei (Campana et al., 1988), was measured to study the effects of L-citrulline on cell proliferation. Cells were harvested by trypsinization and seeded onto glass coverslips in 24-well cluster culture plates at a density of 7000 cells per well in DMEM with 10% FCS. After 24 h of incubation, to allow for cell attachment, the cells were washed with DMEM with 0.4% FCS and incubated in the same medium for 48 h to obtain quiescent nondividing cells. The cultures were then incubated in DMEM with 10% FCS, containing vehicle alone, one of various concentrations of L-citrulline (10¹⁰ to 10⁷ M), L-citrulline plus zaprinast (10⁵ M), L-citrulline plus HS-142-1 (10⁵ M) (a particulate guanylate cyclase inhibitor), or L-citrulline plus oxadiazolo(4,3-a)quinoxalin-1-one (ODQ; 10⁶ M) (a soluble guanylate cyclase inhibitor) (to test the implication of cGMP) for 16 h. Next, the cultures were incubated for 2 h in the same medium containing BrdU (10 µM). BrdU incorporation was visualized by immunocytochemical staining. The cells on coverslips were fixed with acid-alcohol (90% ethanol, 5% acetic acid, 5% water) at room temperature for 30 min, rinsed in PBS, incubated for 1 h in a nuclease-containing anti-BrdU monoclonal antibody (Amersham Intl., Buckinghamshire, UK), and then washed again in PBS. Finally, the cells were incubated with a peroxidase-conjugated anti-mouse IgG antibody (Amersham) for an additional 30 min. Diaminobenzidine solution (0.5 mg/ml with 0.01% H₂O₂) was used as peroxidase substrate. Coverslips were counterstained and mounted in Eukit. The ratio of positively stained cell population to total cell population was calculated. Black staining at the BrdU-incorporated sites, indicating the cells initiating DNA replication, was detected by light microscopy. At least 500 nuclei were counted.

Cell Cycle Analysis. To estimate the proportions of cells in different phases of the cell cycle, cellular DNA contents were measured by flow cytometry. Cells were plated, allowed to attach overnight, and placed in DMEM plus 0.4% FCS for 48 h as described above. Ten percent FCS or 10% FCS plus L-citrulline (10⁸ M) was added for 18 h. The cells were then harvested by trypsinization, washed with PBS, pelleted, and resuspended in PBS containing 0.6% Nonidet P-40 and 100 µg/ml propidium iodide, to which RNase was added to a final concentration of 100 µg/ml. Flow cytometric analysis was carried out with a FACScan (Becton Dickinson, San Jose, CA) flow cytometer equipped with a 15-mW Argon laser emitting at 488 nm. Propidium fluoride fluorescence was recovered through a 575/24 BP filter; 10,000 cells were acquired per sample, and a double discriminator module was used only to detect single cells.

Measurements of cGMP Levels. Vascular smooth muscle cells were plated at 200,000 cells per well in 6-well plates, reaching confluence after 3 to 4 days. The cells were then incubated with L-citrulline (10¹⁰ to 10⁸ M) for 15 min in the presence of 5 × 10⁴ M of 3-isobutyl-1-methylxanthine to prevent cGMP degradation. The medium was removed, and the cells were lysed with a mixture of ethanol-distilled water (2:1) and scraped off the plates. The cell lysates were centrifuged in an Eppendorf centrifuge, and the supernatant was used for cGMP assay. Intracellular cGMP measurements were made using the cGMP radioimmunoassay kit from Amersham as described in the manufacturer's instructions. Data were expressed as picomole of cGMP per milligram of protein. Protein assay was performed following a method previously described (Lowry et al., 1951).

Materials. L-Citrulline (Sigma), zaprinast (Sigma), 3-isobutyl-1-methylxanthine (Sigma), and HS-142-1 (a generous gift from Kyowa Hakko Kogyo Co., Ltd.) were dissolved in PBS. ODQ (Tocris Cookson, Bristol, UK) was dissolved in dimethyl sulfoxide 50% (the final concentration of dimethyl sulfoxide was less than 0.1% in PBS). FCS, DMEM, and all other tissue culture reagents were obtained from GIBCO (Scotland). BrdU was obtained from Amersham.

Data Analysis. The results are expressed as the mean ± S.E.M. and accompanied by the number of observations. A statistical analysis of the data was carried out by a Student's t test or by a two-way ANOVA when necessary. Differences with a P value of less than .05 were considered statistically significant.

	Results

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L-Citrulline and Cell Proliferation. In the first series of experiments, cell counting was used to test L-citrulline ability to alter smooth muscle cell proliferation. Exposure to L-citrulline (10⁸ M) revealed a significant inhibition of proliferation (Fig. 1). 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% (F_(1,10) = 10.19, P < .01, n = 3 duplicated), respectively. To confirm that the above inhibitory effects were not due to toxicity or damage to the cells, trypan blue viability tests were carried out in cells treated in parallel to the proliferation studies. There was no loss in viability of cells treated with L-citrulline; less than 2% of the cells took up the dye. Furthermore, no floating cells were observed on any particular day of the entire proliferation curve. Thus, detachment and loss of cells did not account for the inhibition of cell proliferation.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effect of L-citrulline (108 M) on FCS (10%)-induced proliferation (cell number) of A10 rat vascular smooth muscle cells. L-Citrulline was added, and the cells were incubated for 7 days. Throughout the experiments, media were changed daily and cultures were protected from the light. Results are presented as the mean ± S.E.M. (vertical lines) of three separate experiments, each in duplicate, and expressed as fold increase in cell number, relative to that on day 1 of the experiment. [F(1,10) = 10.19, <0.01] (two-way ANOVA).

Effect of L-Citrulline on DNA Synthesis. The observed inhibitory effect of L-citrulline on cell proliferation could have resulted from an inhibition of DNA synthesis. To test this hypothesis, the drug was evaluated for concentration-related effects on DNA synthesis in smooth muscle cells that where synchronized by DMEM plus 0.4% FCS and then stimulated by the addition of DMEM plus 10% FCS or 10% FCS plus 10¹⁰ to 10⁸ M L-citrulline (Fig. 2). In quiescent cells, the incorporation of BrdU (as a measurement of DNA synthesis) was 5.7 ± 2.0%, whereas in stimulated cells it was 40.5 ± 1.3%. In L-citrulline-treated cells, these percentages were 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.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Effect of L-citrulline on serum-induced BrdU incorporation in A10 vascular smooth muscle cells. Data points represent a typical experiment; similar results were obtained in four determinations each in duplicate, and vertical lines represent the S.E.M. Results are expressed as percentage of control, defined as BrdU incorporation in the presence of 10% FCS and vehicle. The incorporation of BrdU in L-citrulline-treated cells was 36.0 ± 4.1%, 29.1 ± 2.0%, 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. *, P < .05; **, P < .01; ***, P < .001.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of 108 M L-citrulline, L-citrulline plus 105 M zaprinast, L-citrulline plus 105 M HS-142-1, or L-citrulline plus 106 M ODQ on serum-induced BrdU incorporation in A10 vascular smooth muscle cells. Data points represent a typical experiment; similar results were obtained in four determinations each in duplicate, and vertical lines represent the S.E.M. Results are expressed as percentage of control, defined as BrdU incorporation in the presence of 10% FCS and vehicle. *, P < .05 (with respect to 10% FCS-treated cells); ++, P < .01 (with respect to 108 M L-citrulline-treated cells).

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Effects of L-Citrulline on Cell Cycle in Synchronized A10 Vascular Smooth Muscle Cells. To ascertain whether L-citrulline indeed affected the transition from the G₀/G₁ to the S phase, the effects of the drug on cell-cycle progression were also analyzed. A10 vascular smooth muscle cells were initially characterized to confirm their synchrony and cell-cycle behavior (Table 1). After 48 h of exposure to DMEM plus 0.4% FCS, 12.1 ± 1.4% of cells were in S phase in the case of cells exposed to DMEM plus 10% FCS; this was 55.0 ± 3.2% and 40.0 ± 1.2% (P < .05, n = 3) in the case of cells exposed to 10% FCS plus 10⁸ M L-citrulline.

Effect of L-Citrulline on cGMP Levels in Aortic Smooth Muscle Cells in Culture. We also studied cGMP levels directly in aortic smooth muscle cells in cultures stimulated with 10¹⁰ to 10⁸ M L-citrulline for 15 min. As shown in Fig. 4, L-citrulline induced an increase in cGMP levels (with respect to basal levels). cGMP levels increased from 2.1 ± 0.2 pmol of cGMP/mg protein (basal level) to 2.6 ± 0.2 for 10¹⁰ M L-citrulline, 3.2 ± 0.1 for 10⁹ M L-citrulline (P < .01, n = 3, duplicate), and 4.1 ± 0.1 for 10⁸ M L-citrulline (P < .001, n = 3, duplicate).

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Effects of L-citrulline (1010 to 108 M) on cGMP levels (pmol/mg of protein) in rat aortic vascular smooth muscle cells (A10 cell line). Results are presented as the mean ± S.E.M. (vertical lines) of three separate experiments, each in duplicate.

	Discussion

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In the present work, we investigated the effect of L-citrulline, the by-product of NO synthesis, on serum-induced vascular smooth muscle proliferation. Our results show that L-citrulline was able to inhibit smooth muscle proliferation. This amino acid inhibited DNA synthesis measured by BrdU incorporation. In addition, flow cytometry analysis showed that L-citrulline also decreased serum-induced cell-cycle progression by blocking S-phase entrance. Although different authors have focused on the antiproliferative effect of NO, to our knowledge, this is the first report that shows an antiproliferative effect of L-citrulline, the by-product of NO synthesis. In a previous study carried out in our group (Ruiz and Tejerina, 1998), we reported a vasorelaxant effect of L-citrulline. This effect was due to the action of L-citrulline on guanylyl cyclase-linked ANP receptor, which increases intracellular cGMP levels in vascular smooth muscle cells causing relaxation. In this study, the inhibitory effect of L-citrulline on DNA synthesis was blocked by HS-142-1, a nonpeptidic ANP antagonist, but not by ODQ, suggesting an action of L-citrulline on the ANP receptor. The ANP receptor comprises a family of receptors that possess guanylyl cyclase activity per se. Guanylate cyclase, which catalyzes the formation of cGMP from GTP, exists in both soluble and particulate fractions in cells. The enzyme form found in the soluble fraction is a heterodimer that can be regulated by nitrovasodilators and by NO, whereas the membrane form exists as a single-chain polypeptide that can be regulated by various peptides, including ANP (Chinkers et al., 1989). At least two functionally and structurally distinct ANP receptors are present in a variety of tissues; guanylyl cyclase-linked and guanylyl cyclase-free receptors (Schenk et al., 1987). The guanylyl cyclase-linked receptors, which are presumed to be the functional receptors, contain at least four distinct domains: the ligand-binding domain (which binds ANP), the transmembrane domain, the protein kinase-like domain, and the catalytic domain (guanylate cyclase) (Schulz et al., 1989). HS-142-1 selectively recognizes the guanylyl cyclase-linked ANP receptor and decreases the affinity of [¹²⁵I]ANP to its receptor (Matsuda and Morishita, 1993). Since HS-142-1 also blocks L-citrulline inhibition of DNA synthesis, it may be hypothesized that L-citrulline needs to bind to the ANP-binding domain to initiate its effects. Experiments are being carried out in our laboratory to address this possibility.

The possible physiological role of L-citrulline in the control of vascular smooth muscle proliferation still remains unclear. However, the physiological plasma levels of L-citrulline in humans are around 10⁷ M (Mehta et al., 1996), and in our experiments 10⁷ M L-citrulline produced an important inhibition of smooth muscle proliferation (Figs. 1 and 2), suggesting that under normal conditions, L-citrulline may act, together with other factors, such as NO, in the control of the proliferation of vascular smooth muscle.