INTRODUCTION

Lyso-PC¹ is a major component of atherogenic lipoproteins and a product of phospholipase A₂ which accumulates in atherosclerotic and inflammatory lesions of blood vessels (1-3). This polar phospholipid has multiple atherogenic effects. It stimulates macrophage proliferation and plays a critical role in foam cell formation (4). Lyso-PC also induces chemotaxis of leukocytes and activates endothelial expression of leukocyte adhesion molecules, resulting in the recruitment of leukocytes into the arterial intima (5, 6). In addition, lyso-PC promotes intimal thickening of blood vessels by stimulating SMC proliferation. Lyso-PC induces gene expression of potent SMC growth factors such as PDGF and heparin-binding epidermal growth factor-like protein in vascular cells and directly stimulates SMC proliferation (7-10). However, the mechanism by which lyso-PC induces SMC growth is not known. Recent studies indicate that the synthesis of polyamines plays an integral role in the mitogenic response of vascular SMC. SMC proliferation is preceded by increases in cellular polyamine content and inhibition of polyamine formation prevents cell growth (11-13). Furthermore, the exogenous addition of polyamines to SMC stimulates DNA synthesis (11).

The polyamines putrescine, spermidine, and spermine are naturally occurring polycations found in all mammalian cells. Putrescine is generated from the basic amino acid L-ornithine via a decarboxylation reaction catalyzed by the enzyme ODC, whereas spermidine and spermine are synthesized from putrescine through the sequential addition of a propylamine moiety from S-adenosylmethionine (14). Induction of ODC activity and polyamine synthesis is one of the earliest biochemical events associated with cell growth (14, 15). Both arterial injury-induced and PDGF-mediated SMC proliferation are associated with a striking increase in ODC activity (11, 13, 16). Moreover, the inhibition of ODC activity inhibits both arterial injury- and PDGF-mediated SMC proliferation, indicating that ODC activity is essential for SMC growth (11, 13, 17).

Although ODC activity is often believed to be rate-limiting in the polyamine biosynthetic pathway, the availability of L-ornithine also plays a crucial role in regulating polyamine synthesis. Previous studies demonstrate that the steady-state level of L-ornithine markedly influences polyamine formation and DNA synthesis in neoplastic cells (18, 19). More recently, we have shown that PDGF-induced polyamine synthesis and vascular SMC mitogenesis are dependent on the transcellular transport of L-ornithine (13). The transport of cationic (basic) amino acids, such as L-ornithine and L-arginine, by vascular SMC is mediated by the system y⁺ carrier (20, 21). This particular transport system is characterized by its recognition of basic amino acids with high affinity, its Na⁺-independence, and the ability of substrate on the opposite (trans) side of the membrane to increase transport activity (22). Recently, the genes encoding the proteins responsible for the activity of the system y⁺ carrier have been cloned and designated as CAT-1, CAT-2, and CAT-2A (23-26). Both CAT-1 and CAT-2 are low capacity transporters that have a high affinity (K_m  100 µM) for cationic amino acids. In contrast, CAT-2A is an alternate splice variant of CAT-2 which possesses low affinity but high transport capacity (26). In a previous study we found that vascular SMC express mRNA for both of the high affinity transporters, CAT-1 and CAT-2, but do not express message for CAT-2A (13).

In addition to transcellular transport, L-ornithine can be obtained from intracellular sources by endogenous synthesis. In this respect, the basic amino acid L-arginine, which enters cells via the same CATs as L-ornithine, is metabolized to L-ornithine and urea by arginase (27). This enzyme is found in a high concentration in the liver and has recently been identified in vascular cells (28, 29). Presently, little is known about the regulation of arginase activity in vascular SMC.

Because lyso-PC stimulates SMC proliferation and because polyamines play a critical role in mediating cell growth, the present study was designed to determine whether lyso-PC regulates polyamine synthesis. We now report that lyso-PC coordinately induces the gene expression of CAT proteins and ODC, resulting in markedly increased production of polyamines by vascular SMC. In addition, lyso-PC promotes the intracellular metabolism of L-arginine to polyamines by stimulating arginase activity. These lyso-PC-induced actions that promote SMC proliferation are amplified by lyso-PC inhibition of the synthesis of antiproliferative NO by inhibition of iNOS protein expression.

EXPERIMENTAL PROCEDURES

Fetal calf serum, sodium dodecyl sulfate, EDTA, L-arginine, L-ornithine, trichloroacetic acid, penicillin, streptomycin, elastase, collagenase, aprotonin, trypan blue, pyridoxal 5-phosphate, ammonium hydroxide, chloroform, formamide, methanol, Triton X-100, Tris, Tes, HEPES, ninhydrin spray, and TLC plates (Silica Gel 25) were purchased from Sigma. Lyso-PC (palmitoyl, C16:0) and PC (dipalmitoyl) were obtained from Avanti Polar Lipids (Alabaster, AL). L-NOHA was from Alexis Corporation (San Diego). Minimum essential medium was from ICN (Costa Mesa, CA). Leupeptin and MTT were from Boehringer Mannheim. Guanidine isothiocyanate and CsCl were from Life Technologies, Inc. Dithiothreitol and the Bradford protein assay were from Bio-Rad. Ribonuclease A and T1 and GAPDH cDNA were from Ambion Inc. (Austin, TX). Bicinchoninic acid protein assay was from Pierce. GeneScreen Plus membranes were from NEN Life Science Products. Murine monoclonal antibody to iNOS was from Transduction Laboratories (Lexington, KY). PDGF antibody was from Upstate Biotechnology (Lake Placid, NY). DFMO was generously provided by Dr. Ekkhardt H. W. Bohme (Marion Merrill Dow Inc., Cincinnati, OH). L-[³H]Ornithine (55 Ci/mmol) and L-[³H]arginine (58 Ci/mmol) were from American Radiolabeled Chemicals (St. Louis). L-[1-¹⁴C]Ornithine (55 mCi/mmol), L-[guanido-¹⁴C]arginine (52 mCi/mmol), [-³²P]UTP (400 Ci/mmol), and chemoluminescence reagents were from Amersham Corp.

Vascular SMC were isolated by elastase and collagenase digestion of rat thoracic aorta and characterized by morphological and immunological criteria (30). Cells were cultured serially in minimum essential medium containing Earle's salts, 5.6 mM glucose, 2 mM L-glutamine, 20 mM Tes-NaOH, 20 mM HEPES-NaOH, 100 units/ml penicillin, and 100 units/ml streptomycin. Subcultured strains were used between passages 6 and 26. When cells reached confluence, the culture media were replaced with serum-free media containing bovine serum albumin (0.1%) for 24 h. Treatment of vascular SMC with lyso-PC was restricted to those concentrations that did not affect cell viability, as determined by trypan blue exclusion (30) and by the mitochondria-dependent reduction of MTT to formazan (31).

Cationic amino acid transport was determined by measuring the influx of radiolabeled L-ornithine or L-arginine into SMC (21). Similar results were obtained with either basic amino acid. Cells were washed with HEPES buffer (140 mM choline chloride, 5.0 mM KCl, 1.0 mM MgCl₂, 0.9 mM CaCl₂, 5.6 mM D-glucose, and 25 mM HEPES, pH 7.4) and then incubated for 45 s in HEPES buffer containing L-[³H]ornithine or L-[³H]arginine (50 µM; 1 µCi). Transport activity was terminated by aspirating the media and rapidly washing the cells with ice-cold HEPES buffer. Cells were then solubilized by the addition of 0.2% sodium dodecyl sulfate in 0.2 N NaOH, and a portion of the extract was collected for liquid scintillation counting. The remaining extract was used for the determination of protein concentration utilizing the bicinchoninic acid method with serum albumin as the standard.

SMC were harvested in ice-cold Tris buffer (20 mM Tris, 0.1 mM EDTA, 2 mM dithiothreitol, and 0.1 mM pyridoxal 5-phosphate, pH 7.4), sonicated, and centrifuged at 14,000 × g for 20 min at 4 °C. The supernatant (soluble fraction) was collected, and ODC activity was determined by measuring the release of ¹⁴CO₂ from L-[1-¹⁴C]ornithine, as described previously (32). Protein concentration was determined by the Bradford assay using -globulin as the standard, and enzymatic activity was expressed in pmol of CO₂/mg of protein/h.

Polyamine formation was determined by incubating SMC with L-[³H]ornithine or L-[³H]arginine and monitoring the intracellular formation of radiolabeled putrescine, as described previously (13). SMC were incubated with L-[³H]ornithine or L-[³H]arginine (20 µCi/mmol) for 24 h and the reactions stopped by removing the radiolabel-containing media and washing the cells with ice-cold phosphate-buffered saline, pH 7.4. Cells were solubilized in Tris buffer (20 mM, pH 7.4) containing Triton X-100 (0.01%), and aliquots of the supernatant were spotted onto TLC plates and developed in the solvent system chloroform/methanol/ammonium hydroxide/water (1:4:2:1, v/v/v). After drying, putrescine was detected by ninhydrin spray, and [³H]putrescine was identified by co-chromatography with unlabeled putrescine, scraped, and quantified by liquid scintillation counting.

Arginase activity was determined by monitoring the formation of [¹⁴C]urea from L-[guanido-¹⁴C]Larginine (33). SMC were lysed for 30 min in Tris buffer (10 mM, pH 7.4) containing Triton X-100 (0.4%), leupeptin (10 mg/ml), and aprotonin (10 mg/ml). Samples were centrifuged at 14,000 × g for 10 min at 4 °C, and an aliquot of the supernatant was added to an equal volume of Tris buffer (10 mM, pH 7.4) containing MnCl₂ (10 mM). Arginase was then activated by heating for 10 min at 56 °C and the arginase reaction initiated by adding Tris buffer (10 mM, pH 9.6) containing L-arginine (10 mM) and L-[ guanido-¹⁴C]Larginine (0.25 Ci). Samples were incubated at 37 °C for 20 min and reactions terminated by adding ice-cold sodium acetate (250 mM, pH 4.5) containing urea (100 mM). [¹⁴C]Urea was separated from basic amino acids by adding a continuously stirred suspension of Dowex resin (50W-X8 100-200 mesh; 1 g/ml water) to the samples. Samples were shaken, centrifuged at 1,000 × g for 5 min, and an aliquot was collected for scintillation counting. Arginase activity was expressed in µmol of urea/mg of protein/h.

Total cellular RNA was obtained by the guanidine isothiocyanate/CsCl procedure and RNA concentration determined by absorbance spectrophotometry at 260 nm (34). CAT mRNA levels were determined by solution hybridization/ribonuclease protection analysis; ODC mRNA levels were determined by Northern blotting (13, 35, 36). For both ribonuclease protection assays and Northern blotting, relative mRNA levels were quantified by scanning densitometry (LKB 2222-020 Ultrascan XL laser densitometer, Bromma, Sweden) and normalized with respect to GAPDH mRNA.

The expression of iNOS by SMC was determined by measuring NO release and by monitoring iNOS protein levels. NO synthesis was assessed by measuring the extracellular release of nitrite, the stable oxidation product of NO (37), with the Griess reagent (38). iNOS protein levels were determined by Western blotting using anti-mouse iNOS IgG, as described previously (39, 40).

Results are expressed as the means ± S.E. Statistical analysis was performed with the use of Student's two-tailed t test and an analysis of variance when more than two treatments were compared; p values < 0.05 were considered to be statistically significant.

RESULTS

Treatment of vascular SMC with lyso-PC stimulated the transport of L-ornithine and L-arginine in a concentration-dependent manner (Fig. 1). In contrast, PC had no effect on cationic amino acid transport (Fig. 1). Time course studies demonstrated that lyso-PC (100 µM) had a biphasic effect on cationic amino acid transport (Fig. 2). Initially, lyso-PC inhibited the transport of basic amino acids, but by 6 h of lyso-PC treatment a significant rise in transport was observed; this was increased further after 24 h of treatment. Incubation of SMC with a neutralizing antibody directed against PDGF had no effect on lyso-PC-stimulated L-ornithine transport but completely prevented the PDGF-mediated increase in uptake (data not shown).

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In subsequent kinetic studies, saturable uptake of radiolabeled L-ornithine (5-500 µM) was measured. As evident from a representative Eadie-Hofstee plot (Fig. 3), high affinity uptake of L-ornithine by vascular SMC was mediated by a single carrier. Data from several experiments (n = 5) indicated that this transporter had a Michaelis constant (K_m) of 128.3 ± 13.1 µM and a maximum transport velocity (V_max) of 770 ± 77 pmol/mg of protein/45 s. Pretreatment of vascular SMC with lyso-PC (100 µM) for 24 h significantly increased both the K_m (216.6 ± 14.5 µM; p < 0.05) and V_max (3,206 ± 195 pmol/mg of protein/45 s; p < 0.05) of the L-ornithine transporter. The increase in V_max of cationic amino transport by lyso-PC was completely abolished with actinomycin D (2 µg/ml) (data not shown).

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Treatment of vascular SMC with lyso-PC (100 µM) rapidly induced the expression of the mRNA of both cationic amino acid transporters, CAT-1 and CAT-2 (Fig. 4). Both CAT transcripts peaked 2 h after exposure to lyso-PC; however, the increase in CAT-2 message (20-fold) was much greater than that of CAT-1 (3-fold) (Fig. 4). The elevated levels of CAT mRNA decayed rapidly, but messages for CATs reappeared again at 24 h at levels comparable to those observed at 2 h (Fig. 4).

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Incubation of vascular SMC with lyso-PC also induced ODC activity. Significant increases in enzyme activity were evident 4 h after lyso-PC (100 µM) addition, reached maximum activity at 8 h (20-fold), and then declined to near basal levels by 24 h (Fig. 5A). Increases in ODC activity were dependent on the concentration of lyso-PC (Fig. 5B) and were blocked by actinomycin D (data not shown). In contrast, PC failed to stimulate ODC activity (data not shown). SMC expressed two faint ODC transcripts of approximately 2.6 and 2.2 kb (Fig. 6). Treatment of SMC with lyso-PC resulted in a concentration-dependent increase in ODC message with proportional increases (12-fold) in the two mRNA species (Fig. 6).

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Treatment of vascular SMC with lyso-PC increased the capacity of SMC to generate the polyamine putrescine from extracellular L-ornithine in a concentration-dependent manner (Fig. 7A). The stimulatory effect of lyso-PC on putrescine synthesis was inhibited by the cationic amino acid transport inhibitor N^G-methyl-L-arginine (21) and by the selective ODC inhibitor DFMO (15) (Fig. 7B).

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Vascular SMC also expressed constitutive arginase activity (1.6 ± 0.2 µmol of urea/mg of protein/h, n = 4) which was increased by lyso-PC in a concentration-dependent manner (Fig. 8A). In addition, lyso-PC increased the capacity of SMC to generate putrescine from extracellular L-arginine (Fig. 8B). The latter experiments were based on the rationale that L-arginine must be converted to L-ornithine by arginase to be metabolized to putrescine by the action of ODC. Therefore, stimulation of arginase by lyso-PC would result in increased synthesis of [³H]putrescine from L-[³H]arginine. The stimulatory effect of lyso-PC on putrescine synthesis was inhibited by the arginase inhibitor L-NOHA (41, 42) and by the ODC inhibitor DFMO (Fig. 8B).

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Finally, incubating SMC with interleukin-1 (5 ng/ml) for 24 h stimulated the accumulation of nitrite in the incubation medium and induced the expression of iNOS protein (Fig. 9). The addition of lyso-PC (100 µM) to SMC inhibited the interleukin-1-stimulated production of the NO oxidation product, nitrite, in a concentration-dependent manner and reduced markedly the cytokine-induced rise in iNOS protein (Fig. 9). In the absence of interleukin-1, lyso-PC had no effect on SMC NO production or iNOS protein levels (Fig. 9).

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DISCUSSION

The present study demonstrates that lyso-PC stimulates polyamine synthesis in vascular SMC by modulating the expression of the genes that regulate the transport and metabolism of cationic amino acids (see Fig. 10). In particular, lyso-PC increases the transcellular transport of basic amino acids and the intracellular metabolism of L-ornithine to polyamines by stimulating the expression of the genes for both CAT and ODC. In addition, lyso-PC directs the intracellular metabolism of L-arginine to polyamines by stimulating arginase activity and by blocking iNOS. These effects are specific for the lysophospholipid because PC has no effect on cationic amino acid transport or metabolism. The capacity of lyso-PC to augment the transport of basic amino acids and direct their metabolism to growth-stimulatory polyamines (and away from growth-inhibitory NO) may contribute to the proliferative actions of lyso-PC.

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Treatment of vascular SMC with lyso-PC stimulates the transport of cationic amino acids in both a time- and concentration-dependent manner. Kinetic experiments indicate that high affinity (K_m  130 µM) basic amino acid transport is mediated by a single carrier system and that lyso-PC increases both the V_max and K_m of this transport system. These kinetic data suggest that the lyso-PC-induced decrease in cationic amino acid uptake observed at early time points likely arises from a lyso-PC-mediated decrease in affinity of the transporter. In contrast, the increase in basic amino acid transport observed at later time points may result from the de novo induction of transport protein. Studies in our laboratory and others have demonstrated that high affinity transport of cationic amino acids by vascular SMC is mediated by CAT-1 and CAT-2 (13, 43). Consistent with this, our current study demonstrates that lyso-PC stimulates CAT-1 and CAT-2 mRNA expression. Although the relative contribution of these CAT proteins to the overall activity of the system y⁺ carrier is difficult to determine because of their similar kinetic properties, the much greater increase in CAT-2 mRNA compared with CAT-1 suggests that CAT-2B mediates most of the lyso-PC-mediated increase in L-ornithine transport. Interestingly, whereas CAT mRNA expression is elevated during the initial 2 h after lyso-PC treatment, transport activity is increased several hours later when CAT transcripts have decayed to near basal levels. This dissociation between CAT message and transport activity may reflect the time required for the translation, post-translational modification, and eventual insertion of the CAT protein into the plasma membrane to yield a functional transporter.

The co-expression of both CAT-1 and CAT-2 mRNA in vascular SMC after lyso-PC treatment is also observed after the administration of PDGF or angiotensin, but it contrasts with the selective expression of CAT-2 by inflammatory cytokines (13, 43, 44). Interestingly, lyso-PC induces PDGF synthesis from vascular cells (7), raising the possibility that the lyso-PC effect is secondary to PDGF release. However, neutralizing antibodies to PDGF fail to modulate lyso-PC-mediated increases in basic amino acid uptake. It is possible that SMC-derived PDGF exerts its effect in an intracrine manner, which would be unaffected by neutralizing antibodies. The ability of lyso-PC to stimulate CAT gene expression directly and to evoke the release of secondary autocrine mediators may account for the biphasic temporal expression of CAT transcripts.

In addition to stimulating L-ornithine transport, lyso-PC stimulates the metabolism of intracellular L-ornithine to polyamines in vascular SMC. The lyso-PC-mediated increase in ODC activity is paralleled by an increase in ODC mRNA, suggesting that lyso-PC stimulates ODC gene transcription. The inhibition of lyso-PC-stimulated ODC activity by the transcriptional inhibitor actinomycin D is consistent with this notion. The presence of two ODC mRNA species in vascular SMC is also in agreement with previous studies utilizing other cell types and inducing stimuli (36, 45). The two ODC mRNA species arise from the alternative use of two different polyadenylation signals (46, 47).

The transport and metabolism of L-ornithine appear to be closely coupled after lyso-PC administration. This is reflected by the delayed, parallel concentration-dependent increase in CAT and ODC activities. The co-induction of these two proteins by lyso-PC may provide a mechanism by which increased levels of substrate (L-ornithine) are provided to SMC during activation of the ODC enzyme. In this respect, treatment of vascular SMC with lyso-PC is associated with a prominent increase in the capacity of SMC to generate putrescine. This lyso-PC-mediated effect is blocked by the cationic amino acid transport inhibitor N^G-methyl-L-arginine and by the selective ODC inhibitor DFMO, indicating that both the transcellular transport of L-ornithine and ODC activity are limiting factors that regulate the capacity of lyso-PC to generate polyamines in vascular SMC.

Interestingly, whereas lyso-PC results in a transient elevation in ODC activity which returns to basal levels after 24 h of lyso-PC exposure, cationic amino acid transport activity increases progressively during this time. These findings indicate that L-ornithine uptake is dissociated from polyamine synthesis at later time points. The elevated rate of transport at this time may function to provide the necessary amino acids required for the synthesis of new proteins during cell growth.

Because lyso-PC stimulates the transcellular transport of L-arginine as well as L-ornithine into SMC, the effect of lyso-PC on intracellular L-arginine metabolism was also investigated. We found that vascular SMC possess substantial arginase activity and that lyso-PC increases this activity. This finding complements recent studies demonstrating constitutive arginase activity in endothelial cells (28, 29). Although the physiological role of arginase activity in vascular cells is unknown, we suggest that arginase may function to shunt L-arginine to L-ornithine metabolism to generate biologically relevant polyamines. In support of this proposal, we found that lyso-PC increases the capacity of SMC to generate putrescine from L-arginine and that this lyso-PC effect is blocked by the arginase inhibitor L-NOHA. The ability of lyso-PC to induce coordinately the transcellular transport and the intracellular synthesis of L-ornithine may function to maximize the cellular capacity for polyamine biosynthesis.

In vascular SMC L-arginine is also metabolized to NO and L-citrulline by iNOS (48). NO is an important antiatherogenic molecule. In addition to being a potent vasodilator, it inhibits platelet aggregation, vascular SMC proliferation, and monocyte adherence, all of which are key contributors in the development of atherosclerosis (49-52). In the present study we found that lyso-PC attenuates cytokine-stimulated SMC NO synthesis by inhibiting iNOS protein expression. In contrast, an earlier study demonstrated that lyso-PC stimulates the transcriptional activation of the constitutive endothelial NOS gene (53). These results indicate that lyso-PC has divergent isoform-specific regulatory effects on NOS gene expression. The ability of lyso-PC to shunt the metabolism of L-arginine from the iNOS to the arginase pathway in vascular SMC may serve to promote further the mitogenic and atherogenic effects of lyso-PC.

The concentration of lyso-PC required to regulate cationic amino acid transport and metabolism in vascular SMC is similar to the level needed to induce gene expression of other proteins in vascular cells (6, 7, 53). Although this concentration is higher than the level of lyso-PC present in oxidized low density lipoprotein (10 µM) (8, 54, 55), the response of SMC to lyso-PC may be physiologically relevant because cells can generate additional lyso-PC by the action of phospholipase A₂ on membrane PC (56). Moreover, the actual concentration of free lyso-PC in our study is much lower than the reported concentration because the experiments were performed in the presence of albumin, which has a high affinity for lysophospholipids (57).

In conclusion, these studies demonstrate that lyso-PC stimulates the transport of cationic amino acids by inducing the expression of the genes for CAT-1 and CAT-2 and increases L-ornithine metabolism by inducing ODC activity. In addition, lyso-PC redirects the metabolism of L-arginine from the formation of antiproliferative NO to the synthesis of growth-stimulatory polyamines. As shown in the model in Fig. 10, these multiple sites of action of lyso-PC found in this study appear to be coordinately directed toward promoting the proliferative capacity of vascular SMC. The actions of lyso-PC to up-regulate basic amino acid transport and direct their metabolism to polyamines may contribute to SMC proliferation and atherosclerotic lesion formation.