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(Circulation Research. 1995;76:305-309.)
© 1995 American Heart Association, Inc.

Articles

Nitric Oxide–Generating Compounds Inhibit Total Protein and Collagen Synthesis in Cultured Vascular Smooth Muscle Cells

Valeri Kolpakov, David Gordon, Thomas J. Kulik

From the Department of Pediatrics (Division of Pediatric Cardiology) and the Department of Pathology (D.G.), University of Michigan Medical School, Ann Arbor.

Correspondence to Dr Thomas J. Kulik, Division of Pediatric Cardiology, University of Michigan Hospitals, MCHC F 1310, Box 0204, Ann Arbor, MI 48109-0204.

	Abstract

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Abstract Nitric oxide (NO) participates in the regulation of vascular tone and smooth muscle cell proliferation, but little is known of its effect on total protein and matrix synthesis in smooth muscle. We studied the effects of the NO-generating compounds S-nitroso-N-acetylpenicillamine (SNAP, 0.4 to 1.2 mmol/L) and sodium nitroprusside (SNP, 0.1 to 0.5 mmol/L) on total protein (using [³H]leucine) and collagen (using [³H]proline) synthesis in cultured rabbit aortic smooth muscle cells. Both agents caused dose-dependent inhibition of the relative rate of protein (maximum reduction of 87% [SNAP] and 80% [SNP]) and collagen synthesis, as measured by trichloroacetic acid–precipitated label. The magnitudes of percent inhibition of total protein and collagen synthesis were approximately equal. Inhibition of protein synthesis by SNAP and SNP was prevented by hemoglobin (10 µmol/L), suggesting that the protein synthesis inhibition was due to NO release. Inhibition of protein synthesis was reversible after removal of SNAP and SNP and was not caused by damage to the cells. These results suggest that NO may function as a modulator of vascular smooth muscle cell protein synthesis and production of extracellular matrix components.

Key Words: nitric oxide • protein synthesis inhibitors • collagen • vascular smooth muscle

	Introduction

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Nitric oxide (NO) is a potent endogenous mediator, and its ability to cause relaxation of vascular smooth muscle has been extensively characterized.^{1 2 3 4} Recently, NO-generating compounds have also been shown to inhibit smooth muscle cell proliferation^{5 6} (one of the key events in atherogenesis⁷ ), restenosis after angioplasty,⁸ and stenosis of arteriovenous bypass grafts.⁹ The effect of NO on smooth muscle cell growth (as judged by the rate of total protein synthesis) or the rate of synthesis of extracellular proteins has been incompletely characterized (see "Discussion"). The NO-generating agents S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) spontaneously release NO in aqueous medium and have been used as surrogates for authentic NO in experiments designed to assess the effect of NO on smooth muscle cell physiology.^{10 11 12} The present study was undertaken to determine whether these agents affect the relative rate of total protein synthesis in cultured aortic smooth muscle cells. We also determined whether any NO-induced inhibition of total protein synthesis is also reflected in the rate of synthesis of collagen, a protein exported from vascular smooth muscle cells and important in the genesis of atherosclerotic lesions.¹³

	Materials and Methods

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Reagents
SNAP was purchased from BIOMOL Research Laboratories, Inc; collagenase form III, from Advance Biofactures Corp; and L-[2,3,4,5-³H]proline and L-[4,5-³H]leucine, from NEN Research Products. All other reagents were from Sigma Chemical Co.

Cell Culture
Smooth muscle cells were obtained from the thoracic aortas of adult New Zealand White rabbits by using enzymatic digestion (collagenase and elastase).¹⁴ Medium 199 containing 10% fetal calf serum (FCS), L-glutamine (2 mmol/L), penicillin (100 U/mL), streptomycin (100 µg/mL), and amphotericin B (0.25 µg/mL) was the culture medium used. The cells were kept at 37°C in a humidified atmosphere with 5% CO₂/95% air. Cells between the 10th and 14th subpassages were used for the experiments.

Experimental Protocol
The smooth muscle cells were seeded at a density of 2x10⁴ cells per square centimeter in 35-mm plastic culture dishes and allowed to reach confluence in the medium described above supplemented with sodium ascorbate (50 µg/mL). To achieve quiescence, the cells were washed three times with serum-free medium and cultured for 2 days in medium lacking FCS but supplemented with 0.5% bovine serum albumin.^{12 15} The cells were then cultured for 24 hours in medium 199 supplemented with 10% FCS and containing (except for controls) SNAP (0.4, 0.8, or 1.2 mmol/L) or SNP (0.1, 0.3, or 0.5 mmol/L). In some experiments, SNAP or SNP was added to medium containing 0.5% bovine serum albumin and no FCS. SNAP was dissolved in 0.1 mol/L ethanol (10 µL/mL of medium), and an identical volume of ethanol was added to control dishes. SNP was dissolved in medium. L-[2,3,4,5-³H]Proline (127 Ci/mmol, 10 µCi/mL of medium) or L-[4,5-³H]leucine (60 Ci/mmol, 10 µCi/mL of medium) was added to culture medium for the last 2 hours of incubation.

Hemoglobin is a well-established antagonist of NO^{16 17} and has been used to determine whether the effects of SNAP and SNP on smooth muscle cells may be due to NO generation.^{12 15} Therefore, we determined the effect of SNAP and SNP on protein synthesis in the presence or absence of this agent. Hemoglobin (10 µmol/L), prepared as described below, was added to the culture medium with SNAP (1.2 mmol/L) or SNP (0.5 mmol/L).

After incubation with the experimental agents, the relative rate of total protein synthesis in the smooth muscle cells was measured as leucine incorporation into trichloroacetic acid (TCA)-precipitated material. The medium was collected, and the cells were rinsed three times with 4°C phosphate-buffered saline, rinsed once with cold 5% TCA, and scraped into 5% TCA. The cellular material was pelleted, washed once with cold 5% TCA and twice with cold 95% ethanol, dried, and dissolved in 300 µL of 0.1N NaOH. An aliquot was dissolved in EcoLite LSC counting fluid, and the activity was measured with a Beckman LS 7500 liquid scintillation counter.

To determine the relative rate of collagen synthesis, a technique previously described was used.¹⁸ Culture medium was removed and added (vol/vol) to cold buffer containing 0.65 mol/L NaCl, 0.1 mol/L Tris (pH 7.4), 4.7 mmol/L CaCl₂, and 2.5 mg/mL N-ethylmaleimide. Bovine serum albumin (100 µg/mL) was added as a carrier. An aliquot was removed, 10% TCA was added, and the material was allowed to flocculate for 30 minutes at 4°C. The TCA-precipitated material was pelleted, washed twice with 5% TCA, washed twice with cold 95% ethanol, dried, dissolved in 0.1N NaOH, and counted as above. A second aliquot was digested with a highly specific collagenase (collagenase form III, 10 U/mL medium) for 90 minutes at 37°C and then treated identically as the nondigested aliquot. The relative rate of collagen synthesis was determined, assuming that the ratio of proline residues in collagen relative to noncollagen protein is 5.4.¹⁹

Lactate dehydrogenase (LDH) in cell culture medium was measured in the chemical pathology laboratory of the University of Michigan Hospitals.

Preparation of Hemoglobin
Pure hemoglobin was prepared as previously described²⁰ by adding to a 1-mmol/L solution of rabbit hemoglobin in distilled water a 10-fold molar excess of the reducing agent sodium dithionite. The sodium dithionite was then removed by dialysis against 100 vol distilled water for 2 hours at 4°C.

Statistical Analysis
The data are reported as mean±SEM. Multiple comparisons were made by two-way ANOVA, with post hoc comparisons by the Tukey test.²¹ A value of P<.05 was considered significant.

	Results

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Effect of NO Donors on Total Protein Synthesis
Both SNAP and SNP inhibited the relative rate of total protein synthesis in the rabbit aortic smooth muscle cells in a concentration-dependent fashion (n=3 independent experiments in triplicate, Fig 1). At the highest concentration of SNAP tested (1.2 mmol/L), it induced an 87±2% decrease in the rate of total protein synthesis in the cell layer, whereas SNP reduced the rate of protein synthesis by 80±2% at the highest dose (0.5 mmol/L). Treating cells with experimental agents in serum-free medium (hence, the cells were serum-deprived throughout the experiment) also caused a significant reduction in protein synthesis: 75±8% with SNAP (1.2 mmol/L) and 64±9% with SNP (0.5 mmol/L) (n=3 independent experiments in triplicate). Normalizing the number of disintegrations per minute to the total amount of protein per dish did not significantly alter the percent change in protein synthesis caused by the NO donors (data not shown).

View larger version (21K): [in this window] [in a new window]   Figure 1. Bar graph showing the relative rates of total protein synthesis by cultured aortic smooth muscle cells in the presence of S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) compared with untreated cells. Cells were treated with the agents noted for 24 hours and labeled for 2 hours with [3H]leucine, and trichloroacetic acid–precipitated counts were determined from the cell layer (n=3 independent experiments [each performed in triplicate]). *P<.05 vs control; #P<.05 vs control and previous dose.

The inhibition of protein synthesis caused by SNAP and SNP was reversible. After removal of the NO donors and incubation of cells in serum-supplemented medium for 24 hours, total protein synthesis returned to control levels (mean, 12 473±126 cpm per dish for SNAP; mean, 12 169±174 cpm per dish for SNP; and mean, 12 736 ±153 cpm per dish for controls; n=2 experiments in triplicate).

The inhibition of the protein synthesis by NO-generating agents conceivably could be caused by degradation (by a free radical–mediated mechanism) of the compounds in the culture medium that activate protein synthesis. To rule out this possibility, we preincubated the culture medium (with 10% FCS) with 1.2 mmol/L SNAP (the highest concentration used) for 3 days, a period of time sufficient for degradation of SNAP to N-acetylpenicillamine, nitrite, and nitrate.¹² Preincubated SNAP decreased protein synthesis by 16±4% (P=NS, n=2 experiments in triplicate) relative to serum-containing medium preincubated without SNAP for 3 days; medium containing the same concentration of fresh SNAP inhibited protein synthesis by 87±2%.

Effect of NO Donors on Collagen Synthesis
SNAP and SNP also inhibited the production of collagen in a concentration-dependent fashion (n=2 experiments in triplicate, Fig 2). The percent inhibition by either SNAP or SNP was similar for both total protein and collagen (both measured in the media). Measurement of the rate of collagen synthesis using the cell/extracellular matrix layer also showed NO donor inhibition of collagen synthesis: SNAP (1.2 mmol/L) inhibited collagen production by 62%; SNP (0.5 mmol/L), by 42% (P<.05, n=2 experiments in triplicate).

View larger version (38K): [in this window] [in a new window]   Figure 2. Bar graph showing the relative rates of total protein and collagen synthesis by cultured aortic smooth muscle cells in the presence of S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) compared with untreated cells. Cells were treated as in Fig 1 and labeled with [3H]proline, and trichloroacetic acid–precipitated counts were determined in the medium. Collagenase was used to differentiate total protein from collagen synthesis (n=2 independent experiments [each performed in triplicate]). *P<.05 vs control; #P<.05 vs control and previous dose.

Effect of Hemoglobin on SNAP- and SNP-Mediated Decreases in Protein Synthesis
Hemoglobin alone had no significant effect on either total protein or collagen synthesis. However, hemoglobin abolished both the SNAP- and SNP-mediated reduction in the total protein and collagen synthesis in the smooth muscle cells (n=3 experiments in triplicate) (Fig 3).

View larger version (52K): [in this window] [in a new window]   Figure 3. Bar graph showing that hemoglobin (Hgb) abolishes nitric oxide donor–mediated reduction in total protein and collagen synthesis. Cultured aortic smooth muscle cells were treated as in Figs 1 and 2, but Hgb (10 µmol/L) was added to the medium with S-nitroso-N-acetylpenicillamine (SNAP, 1.2 mmol/L) or sodium nitroprusside (SNP, 0.5 mmol/L) and to untreated cells. Hgb alone had no effect on protein synthesis but abolished the SNAP- and SNP-mediated reduction in total protein and collagen synthesis. For all values, P=NS compared with controls (n=3 independent experiments [each performed in triplicate]).

SNAP and SNP Do Not Cause Cell Toxicity
It is possible that the inhibition of protein synthesis by SNAP and SNP was due to cell toxicity. Cells were treated with SNAP (1.2 mmol/L) or SNP (0.5 mmol/L) for 24 hours and examined at x200. Cell morphology and density appeared identical in treated and control cells (n=3 experiments in triplicate) (Fig 4). The cell number after adding the experimental agents for 24 hours (2.91±0.28x10⁵ cells per well for SNAP [1.2 mmol/L] and 3.03±0.44x10⁵ cells per well for SNP [0.5 mmol/L]) did not differ from control (3.24±0.31x10⁵ cells per well). The number of the detached cells also did not differ (80±44 cells per well for SNAP, 60±38 cells per well for SNP, and 60±23 cells per well for control). There was no significant release of LDH after treatment with the experimental agents for 24 hours (202±15 IU for SNAP, 195±8 IU for SNP, and 191±9 IU for controls; n=1 experiment in triplicate).

View larger version (79K): [in this window] [in a new window]   Figure 4. Photomicrographs of control aortic smooth muscle cells (A) and cells treated with 1.2 mmol/L S-nitroso-N-acetylpenicillamine (SNAP, B) and 0.5 mmol/L sodium nitroprusside (SNP, C) for 24 hours. The cells appear normal after SNAP or SNP treatment (Harris hematoxylin staining, original magnification x400).

	Discussion

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Although it is well established that NO inhibits proliferation in cultured vascular smooth muscle cells,^{5 6 12 15} there are few data regarding the effect of NO on smooth muscle protein synthesis and none regarding the effect of NO on collagen production in these cells. Garg and Hassid²² reported that SNAP inhibited total protein synthesis in cultured rat aortic smooth muscle cells but only when protein synthesis was measured 12 hours after addition of 5% FCS to serum-deprived cells; cells exposed to FCS for 4 or 20 hours showed no inhibition of protein synthesis with SNAP. The effect of other NO donors, the effect of these agents on collagen production, and whether hemoglobin altered the effect of SNAP were not reported. Little else has been published regarding the effect of NO on protein synthesis, although one report established that SNAP and SNP inhibit protein synthesis in cultured hepatocytes.²³

We found that the structurally dissimilar NO donors SNAP and SNP reduced the relative rates of total protein and collagen synthesis in cultured aortic smooth muscle cells, although to a lesser extent than previously reported in hepatocytes.²³ The reduction in the rate of protein synthesis was dose dependent, and the percent reduction in synthetic rate was roughly the same in total protein and collagen synthesis. We did not measure aminoacyl-tRNA; hence, it is conceivable that changes in incorporation of label reflect NO-related changes in precursor levels. That hemoglobin prevents the SNAP- and SNP-mediated reduction in protein synthesis suggests that the inhibition was due to NO formation rather than a nonspecific effect of these agents.

The inhibition of protein synthesis was unlikely to be due to NO-induced cell damage, on the basis of our findings that even at the highest concentration used, neither SNAP nor SNP altered cell morphology, caused cell detachment, or caused release of the cytosolic enzyme LDH. These data are consistent with those of other investigators who found that incubation of cultured vascular smooth muscle cells with SNAP (1 mmol/L for 22 hours) did not cause release of LDH or significant cell loss.¹²

The molecular mechanism(s) of the NO-mediated reduction in protein synthesis is unknown. Considerable data indicate that NO causes smooth muscle relaxation via activation of guanylate cyclase and generation of cGMP.²⁴ However, recent work suggests that NO can directly activate calcium-dependent potassium channels⁴ or perhaps cause smooth muscle relaxation through ADP-ribosylation of proteins,²⁵ indicating that NO may have important non–cGMP-related effects. Experiments are inconclusive regarding the role of cGMP in NO-mediated reduction in smooth muscle proliferation. Some (but not all²⁶ ) studies have found 8-bromo-cGMP to inhibit cultured smooth muscle proliferation.^{12 27} However, proliferation of BALB/c 3T3 fibroblasts lacking soluble guanylate cyclase activity is inhibited by NO donors,¹⁵ suggesting that at least in these cells, cGMP is not required for NO-related suppression of proliferation. Furthermore, the addition of exogenous 8-bromo-cGMP did not cause inhibition of total protein synthesis in cultured hepatocytes, suggesting that cGMP alone cannot account for the NO-induced suppression of protein synthesis.²³ Hence, one or more of several biochemical intermediates may be involved in the NO-mediated reduction of protein synthesis.

Vascular smooth muscle cell proliferation and production of extracellular matrix components (mainly collagen) are key events in a variety of types of vascular pathology.^{7 8 9 28} In vitro studies showing NO to inhibit cell replication^{12 15} are complemented by those demonstrating that endothelial cells tonically generate NO²⁹ and that deendothelialization is associated with proliferation of underlying smooth muscle cells and production of extracellular matrix.^{30 31} These observations, and the data presented here, suggest that the endothelium may play an important role in modulating abnormal proliferation, hypertrophy, and connective tissue synthesis of vascular smooth muscle cells.

	Acknowledgments

 
This study was supported by National Heart, Lung, and Blood Institute grants HL-42908 and HL-42119. The authors are grateful to Carolyn Work for technical help and Dr Mark D. Rekhter for his helpful comments.

Received June 8, 1994; accepted October 27, 1994.

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