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(The FASEB Journal. 1999;13:1810-1816.)
© 1999 FASEB

Constitutive expressions of type I NOS in human airway smooth muscle cells: evidence for an antiproliferative role

HEMA J. PATEL*, MARIA G. BELVISI*,{dagger}, LOUISE E. DONNELLY*, MAGDI H. YACOUB{ddagger}, K. FAN CHUNG* and JANE A. MITCHELL§1

* Department of Thoracic Medicine and
{ddagger} Cardiothoracic Surgery, Imperial College School of Medicine at the National Heart and Lung Institute, London, SW3 6LY, U.K;
{dagger} Pharmacology Department, Rhône-Poulenc Rorer, Dagenham Research Centre R&D, Dagenham, Essex RM10 7XS, U.K.; and
§ Unit of Critical Care Medicine, Imperial College School of Medicine at the Royal Brompton Hospital, London, SW3 6NP, U.K.

1Correspondence: Unit of Critical Care Medicine, Royal Brompton Hospital, Sydney Street, London SW3 6NP, U.K. E-mail: j.a.mitchell{at}ic.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In airway diseases, smooth muscle cells can proliferate at exaggerated rates; thus, the identification of endogenous pathways that limit proliferative responses is important. Here we show that human airway smooth muscle express type I nitric oxide synthase (NOS), which results in inhibition of DNA synthesis and cell proliferation. In addition, superoxide dismutase (SOD), a cell-permeable mimetic that increases the biological half-life and therefore enhances the biological activity of endogenously released nitric oxide (NO), or NO-releasing drugs also greatly reduce DNA synthesis and cell proliferation. Observations in this study have important clinical implications: 1) NOS inhibition may exacerbate airway disease and 2) inhaled SOD/mimetics or NO/nitrovasodilators may be therapies for the treatment of asthma or chronic obliterative pulmonary disease.—Patel, H. J., Belvisi, M. G., Donnelly, L. E., Yacoub, M. H., Chung, K. F., Mitchell, J. A. Constitutive expressions of type I NOS in human airway smooth muscle cells: evidence for an antiproliferative role.


Key Words: hyperplasia • human respiratory tract • nitric oxide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NITRIC OXIDE (NO)2 IS NOW recognized to be an important mediator in many physiological and pathophysiological processes, including smooth muscle relaxation (1 , 2) and inhibition of proliferative responses (3) . NO is produced from a wide variety of cell types after the conversion of the amino acid L-arginine by a family of NO synthase enzymes (NOS). Several isoforms of NOS—type I (nNOS), type II (iNOS), and type III (eNOS)—-were first identified in brain, macrophages, and vascular endothelial cells, respectively (4 5 6 7) . Type I and type III NOS have an absolute requirement for calcium and thereby tend to form discrete quanta of NO. NO formed by these constitutive forms of NOS are involved in regulation of physiological processes such as maintenance of blood pressure, gastrointestinal integrity, and airway caliber (8) . By contrast, type II NOS, which usually requires the presence of some inducing agent (e.g., cytokines) for expression, does not require free calcium for activity and consequently forms large amounts of NO. Thus, type II NOS activity is generally thought to mediate pathophysiological events associated with some inflammatory diseases (8) .

NOS isoenzymes have been identified in different cell types that comprise human airways including nerves (9) , epithelial (10 , 11) and endothelial cells (12) , as well as resident leukocytes (13 14 15) . In the case of nerves and epithelium, the release of NO regulates airway tone by activating guanylyl cyclase in airway smooth muscle cells (16) . However, the possibility that airway smooth muscle cells express NOS has not been addressed.

In this study, we have therefore investigated the possibility that human airway smooth muscle (HASM) cells express NOS. In other smooth muscle cells (e.g., vascular), NO is a powerful inhibitor of cell growth (3) , an aspect of airway smooth muscle cell physiology that has not been studied. Since hyperplasia and hypertrophy of airway smooth muscle are thought to contribute to airway diseases such as asthma and chronic obliterative pulmonary disease (COPD), we have also investigated any effects of endogenously released and exogenously applied NO on DNA synthesis and cell proliferation of these cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of HASM cells
As described previously (17 , 18) , tracheal rings from either heart/heart and lung transplantation donors (three female, three male, aged 12–55 years) were dissected under sterile conditions in Hanks' balanced salt solution (HBSS; in mM: NaCl 136.8, KCl 5.4, MgSO4 0.8, Na2HPO4. 7H2O 0.4, CaCl2. 2 H2O 1.3, NaHCO3 4.2, and glucose 5.6) supplemented with the antibiotics penicillin (100 U/ml), streptomycin (100 µg/ml), and the antifungal amphotericin B (2.5 µg/ml). The smooth muscle layer was dissected free of adherent connective tissue and cartilage; the epithelial layer was removed using a rounded scalpel blade. The smooth muscle section was then incubated for 30 min at 37°C in 5% CO 2/95% air in HBSS containing 10 mg/ml bovine serum albumin (BSA) and the enzymes collagenase (type XI, 1 mg/ml) and elastase (type I, 3.3 U/ml). After the removal of any remaining connective tissue, the smooth muscle was chopped finely and incubated for an additional 150 min in the enzyme solution outlined above, with the elastase content increased to 15 U/ml. Cells were centrifuged (100 x g, 5 min) at 4°C and resuspended in Dulbecco's modified Eagles medium (DMEM) containing heat-inactivated fetal calf serum (FCS; 10%v/v), sodium pyruvate (1 mM), L-glutamate (2 mM), nonessential amino acids (1x), and antimicrobial agents as described previously.

Primary culture of HASM cells
The HASM cellular suspension was placed in a tissue culture flask (75 cm2) with 6 ml of supplemented DMEM and incubated at 37°C in 5% CO2/95% air. The cells adhere after ~12 h; the culture medium was replaced after 4–5 days (12 ml) and then every 3–4 days. After ~10–14 days, the cells reach confluence and are identified by their typical `hill and valley' appearance and positive immunostaining for {alpha}-actin. Cells were plated onto either 6-, 24-, 48-, or 96-well plates (Costar UK Ltd., High Wycombe, U.K.). At subconfluence the cells were growth arrested by being placed in DMEM containing apotransferin (5 µg/ml), insulin (1 µM), ascorbate (100 µM), and bovine serum albumin (0.1%) for 24 h. The medium was then replaced with DMEM containing 3% FCS and the treatment drugs, as described below.

Western blot analysis
Western blot analysis was performed as outlined previously (18) . Cells were grown to subconfluence on 6-well plates with an initial seeding density of 30,000 cells/well. After the growth arrest period, cells were restimulated by the addition of DMEM with or without FCS (3%) for 24 h; the medium was then removed and the cells washed with HBSS. Proteins were extracted in buffer (Tris, 50 mM; EDTA, 10 mM; triton X-100, 1% v/v; phenylmethylsulfonyl fluoride, 1 mM; pepstatin A, 50 µM, and leupeptin, 0.2 mM). The resulting cell extract was boiled (3 min) in a ratio of 1:1 with gel loading buffer [Tris, 50 mM; sodium dodecyl sulfate (SDS), 10% w/v; glycerol, 10% v/v; 2-mercaptoethanol, 10% v/v, and bromphenol blue 2 mg/ml]. Samples were centrifuged at 10,000 x g for 2 min before being resolved on a 7.5% SDS separating gel (~15 µg of protein was loaded/lane, as determined by a Bradford protein assay). The separated proteins were then transferred to nitrocellulose. The nitrocellulose filters were blocked overnight at 4°C using 10% w/v dried minimal-fat milk in phosphate buffered saline (PBS). The blots were washed in PBS containing 0.05% v/v Tween 20 and then incubated for 1 h with a rabbit polyclonal antibody raised against either NOS I (1:1000), II (1:10,000), or III (1:1000) diluted in PBS containing 1% BSA. The antibodies have no cross-reactivity for the other NOS isoforms. The blots were washed excessively and then incubated with a secondary antibody raised against rabbit immunoglobulin G conjugated to horseradish peroxidase 1:4000 in PBS containing 1% dried milk. Antibody bound protein was visualized by enhanced chemiluminescence (ECL: Amersham plc, U.K.).

Nitrite assay
For these experiments cells were grown to subconfluence on 24-well plates. After the growth arrest period, 400 µl of medium (phenol red-free DMEM) containing the nonselective NOS inhibitor L-NAME (N{omega}-nitro-L-arginine methyl ester; 1–100 µM), the selective NOS II inhibitor, 1400W [N-(3-(aminomethyl)benzyl)acetamidine; 1–100 µM], or vehicle (3% FCS) was added to the wells for 24 h. The amount of nitrite in the cell culture medium was measured using a modification of the method of Misko et al. (19) . Briefly, 200 µl of conditioned media or nitrite standard was mixed with 100 µl of a suspension of 2% (w/v) charcoal in 0.2% (w/v) dextran. The suspension was centrifuged at 10,000 x g for 10 min and 100 µl of the cleared supernatant was placed into a well of a 96-well plate. This was mixed with 10 µl of 0.05 mg/ml 2, 3-diaminonapthalene (DAN) in 0.625 M HCl and the plate was incubated in the dark at room temperature for 10 min; the reaction was stopped by the addition of 10 µl of 1.4M NaOH. The fluorescence was measured using a Biolite F1 plate fluorimeter (Labtech, Uckfield, U.K.), with the excitation wavelength set at 360 nm and the emission wavelength set at 460 nm. The sensitivity of the fluorimeter was set between 40 and 50%. The amount of nitrite in each sample was calculated using a standard curve of known nitrite concentrations. The limit of detection for this assay under these conditions was 10 pmol/100 µl.

Measurement of DNA synthesis
HASM cells were cultured onto 48-well plates with an initial seeding density of 4000 cells/well. At subconfluence (~80%), their growth cycle was synchronized by withdrawal of serum for 24 h. Cells were then treated with a range of drugs including the NO donors SNP (sodium nitroprusside; 1 µM–1 mM) or NOC 18 [2,2'-(hydroxynitrosohydrazino)bis-ethanamine; 1 µM–1 mM], L-NAME (1 µM–100 µM) and its inactive enantiomer, D-NAME (N{omega}-nitro-D-arginine methyl ester; 1 µM–100 µM), superoxide dismutase (SOD; 10–300 U/ml), or the SOD mimetic MnTMPyP [Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride; 1 µM–100 µM] or vehicle (culture medium). Proliferation was initiated by the addition of FCS at 1, 3, 10, or 20%. We have previously established that serum stimulation results in a `typical' S-phase of proliferation in these cells, which is linear between 24 and 32 h (20) . Thus, at 26 h after serum/drug treatment, [3H]-methyl thymidine (1 µCi/well; specific activity 25 Ci/mmol) was added to the cells for 2 h. Cells were washed twice with 0.5 ml HBSS to remove any loosely attached radioactive tracer before the addition of 0.5 ml of 5% trichloroacetic acid for 30 min. After two 0.5 ml washes with 95% ethanol, the remaining material on the plate, which represents the DNA fraction, was solubilized by a 30 min incubation with 0.5 ml of 2% Na2CO3 in 0.1 M NaOH. Samples were mixed with scintillation fluid (1:9 v/v) and radioactivity determined with a beta counter. We have shown that changes in thymidine uptake are representative of changes in differentiation of HASM cells (20) . In preliminary studies, we found that 3% FCS produced a submaximal (50% max) proliferative response. Thus, this concentration of serum was used for further experiments to stimulate cells.

Quantification of cell number
Cell proliferation was assayed using a CyQUANT cell proliferation assay kit. Cells were grown to subconfluence on 96-well plates with an initial seeding density of 2000 cells/well. After a 24 h growth arrest period, the cells were treated with L-NAME (100 µM), SNP (1 mM), NOC 18 (1 mM), and MnTMPyP (100 µM) diluted in DMEM containing 3% FCS or serum-free medium alone for 4 days. The medium was discarded and the plates were stored at -70°C until needed. The CyQUANT GR dye used in this assay exhibits strong fluorescence enhancement when bound to DNA or RNA. Hence, the cells were thawed and incubated with 50 µl DNA-free RNase (1.35 Kunitz units/ml) for 1 h at room temperature to eliminate any binding of the dye to RNA. One hundred microliters of 2x CyQUANT GR reagent was added to all wells and incubated for 4 min in the dark. Fluorescence was then measured using a Biolite F1 plate fluorimeter (Labtech, Uckfield, U.K.) with the excitation wavelength set at 480 nm and the emission wavelength set at 520 nm. The assay was linear over a range of 50 to 50,000 cells under these conditions. The sensitivity of the fluorometer was set between 40 and 50%. The number of cells in the treated wells was calculated using a standard curve, which was constructed by serial dilutions from a top concentration of known cell number counted with a hemacytometer.

Cell viability
In a separate series of experiments, cells were cultured onto 96-well plates to assess the effects of different drug treatments on viability, measured by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan, as described previously (21) .

Materials
Radiochemicals and ECL reagent were obtained from Amersham International (Amersham, Bucks, U.K.). 1400W and antibodies raised against NOS I and III were purchased from Alexis Corporation (Alexis Corporation Ltd., Nottingham, U.K.). The antibody against NOS II was purchased from Transduction Laboratories (Lexington, Ky.). Amphotericin B, nonessential amino acids, and sodium pyruvate were purchased from Life Technologies (Life Technologies Ltd., Paisley, U.K.). MnTMPyP and NOC-18 were from Calbiochem (Calbiochem-Novachem Ltd., Nottingham, U.K.). Immunoglobulin G conjugated to horseradish peroxidase was purchased from DAKO (DAKO Ltd., Ely, Cambridge, U.K.). The CyQUANT cell proliferation kit was purchased from Molecular Probes (Eugene, Oreg.). All other materials were from Sigma Chemical Company (Poole, U.K.). All reagents used were either of tissue culture grade or filtered under sterile conditions.

Statistical analysis
Results are shown as the mean ± SE of n determinations from HASM cells obtained from at least three patients. Normalized data were analyzed by a one-sample t test. All treatments were compared to control values and P < 0.05 was considered to be significant. EC50 values were calculated by iterative curve fitting using Graphpad Inplot (Graphpad Inc., San Diego, Calif.)


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of nitrite release by HASM cells
HASM cells released detectable levels of nitrite when cultured for 24 h in the presence (411.2±81.1 pmol/well; n=6) or absence (1815.2±203.2 pmol/well: n=6) of FCS. In separate experiments, the NOS inhibitor (types I, II, and III) L-NAME (1–100 µM) inhibited nitrite release from HASM cells stimulated with serum by ~50% (Fig. 1 ). A similar effect was seen in unstimulated HASM cells (data not shown). By contrast, the selective NOS II inhibitor, 1400W (1–100 µM), had no effect on nitrite release by HASM cells (Fig. 1) . To assess the possibility that human airway smooth muscle cells can express type II NOS, cells were treated with mixture of cytokines (interleukin 1ß, tumor necrosis factor {alpha}, and interferon {gamma}, 10 ng/ml for each) with or without serum for 24 h. In these experiments, stimulation with cytokines did not increase nitrite release by HASM cells (data not shown; n=6).



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  		Figure 1. Effect of L-NAME and 1400W on nitrite release from HASM cells. The nonselective NOS inhibitor L-NAME (1–100 µM) significantly inhibits the release of nitrite from HASM cells stimulated with 3% FCS ({blacksquare}). In contrast, the selective NOS II inhibitor 1400W (1–100 µM) had no effect on nitrite release ({blacktriangleup}) from these cells under the same conditions. Results are shown as the mean ± SEM of six determinations from three patients. 100% is defined as the amount of nitrite released from control cells (pmol/well) Treatment groups were compared by a one-sample t test to control values (*P<0.05).

Identification of type I, but not types II or III, in HASM cells
A single band of ~150 kDa was recognized in protein extracts from HASM cells by antibodies specific for type I NOS (Fig. 2 ). No difference in the level of this protein was observed when cells were cultured for 24 h without serum (Fig. 2) . In separate experiments using antibodies specific for type II or type III NOS, no protein bands were detectable in extracts of HASM cells treated with or without cytokines in the presence or absence of serum (data not shown).



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  		Figure 2. Expression of NOSI protein in HASM cells. A representative Western blot with a specific antibody to NOSI that recognizes a single band of ~150 kDa in protein extracts from HASM cells. No difference in the level of NOSI protein was observed when cells were cultured for 24 h with or without serum.

Effect of endogenous NO on DNA synthesis in HASM cells
The level of 3H-thymidine incorporation into the cells was increased when cells were stimulated with culture medium containing 3% FCS (0.38±0.02 pmol/well, n=6) compared to cells treated with culture medium without FCS (0.05±0.003 pmol/well, n=6).

L-NAME at 1, 10, and 100 µM significantly increased DNA synthesis in a concentration-dependent manner (Fig. 3 ). In contrast, D-NAME at the same concentrations had no effect on DNA synthesis in HASM cells (Fig. 3) . In a separate series of experiments, the superoxide dismutase mimetic MnTMPyP (1, 10, and 100 µM) and authentic SOD (150 U/ml), both of which increase the biological activity of NO by prolonging its half-life, reduced DNA synthesis in human airway smooth muscle cells (Fig. 4 ).



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  		Figure 3. Endogenous NO inhibits DNA synthesis in HASM cells. L-NAME ({blacksquare}; 1–100 µM) significantly increases DNA synthesis in HASM cells stimulated with 3% FCS in a concentration-dependent manner. In contrast, D-NAME (•; 1–100 µM) under identical conditions has no effect on DNA synthesis in HASM cells. Results are shown as the mean ± SEM of at least six determinations from three patients. 100% is defined as the amount of 3H-thymidine incorporated into control cells. Treatment groups were compared by a one-sample t test to control values (*P<0.05).



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  		Figure 4. The effect MnTMPyP and authentic SOD on DNA synthesis in HASM cells. A) The SOD mimetic, MnTMPyP (1–100 µM), significantly inhibits DNA synthesis in HASM cells stimulated with 3% FCS in a concentration-dependent manner. Under identical conditions, authentic SOD (B); 150 U/ml) also inhibited proliferation of these cells to a similar extent as 100 µM MnTMPyP. Results are shown as the mean ± SEM of at least six determinations from three patients. 100% is defined as the amount of 3H-thymidine incorporated into control cells. Treatment groups were compared by a one-sample t test to control values (*P<0.05).

Effect of exogenous NO on DNA synthesis in HASM cells
The nitrodilators SNP and NOC 18 (1 µM–1 mM), which release NO, inhibited DNA synthesis in HASM cells in a concentration-dependent manner. SNP and NOC 18 both produced maximum inhibition at 1 mM, with EC50 values of 0.21 mM and 0.17 mM, respectively (Fig. 5 ).



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  		Figure 5. Exogenous NO inhibits proliferation of HASM cells. SNP ({square}; 1 µM–1 mM) and NOC 18 ({diamondsuit}; 1 µM–1 mM) both inhibited proliferation of HASM cells stimulated by 3% FCS in a concentration-dependent manner. Results are shown as the mean ± SEM of at least nine determinations from three patients. 100% is defined as the amount of 3H-thymidine incorporated into control cells. Treatment groups were compared by a one-sample t test to control values (*P<0.05).

Effect of L-NAME, SNP, NOC 18, and MnTMPyP on HASM cell number
Cells stimulated for 24 h with culture medium containing 3% FCS increased in number compared with cells treated without serum (from 4780±388 cells to 8228±354 cells; n=6,). Furthermore, in the presence of L-NAME (100 µM), cell number was further enhanced by 70.7 ± 8.9% (n=6). In contrast, incubation of the stimulated cells with SNP (1 mM), NOC 18 (1 mM), or MnTMPyP (100 µM) reduced cell number to the level seen with unstimulated cells (Fig. 6 ).



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  		Figure 6. Effect of L-NAME, NO donors, and MnTMPyP on cell proliferation. L-NAME (100 µM) significantly increased cell number compared to cells treated with serum alone (control). In contrast, the NO donors SNP (1 mM) and NOC 18 (1 mM) and the SOD mimetic, MnTMPyP (100 µM), completely abolished proliferation induced by 3% FCS. Results are shown as the mean ± SEM of at least six determinations from three patients. 100% is defined as the number of cells in the control wells (cell treated with 3% FCS alone). Treatment groups were compared by a one-sample t test to control values (**P<0.005).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The lung represents an important target organ for the generation of NO. Type I NOS has been identified in different cells of the airways, including nerves and epithelium (1) . Type III NOS is present in pulmonary endothelium as well as in the blood vessels that supply the airways (22) . Furthermore, type II NOS is expressed in bronchial epithelium and lung macrophages (23) . Here we show for the first time that human airway smooth muscle cells also express NOS. In these cells, we found a constitutive expression of type I NOS protein, accompanied by the release of nitrite. In addition, we found no evidence for the expression of the other constitutive isoform, type III NOS. When cells were stimulated with a mixture of cytokines shown to induce type II NOS in other cell types (12) , no expression was detected in human airway smooth muscle cells. Thus, these cells appear to express type I NOS only, even under inflammatory conditions.

We found that the release of nitrite by human airway smooth muscle cells was inhibited by L-NAME, which inhibits all types of NOS (24 , 25) , but not by 1400W, which is highly selective for type II NOS (26) . These observations are consistent with a constitutive isoform being responsible for NO release by these cells. The findings that cytokines did not increase nitrite release or the expression of type II NOS are in contrast to observations in other cells. Indeed, rat airway smooth muscle cells readily express type II NOS under inflammatory conditions (27) . The reason for this inconsistency may be related to species differences between human and rat in the case of type II NOS expression. Alternatively, type II NOS may not be expressed in human airway smooth muscle because they contain type I NOS constitutively. This is supported by numerous findings that nerve cells expressing type I NOS do not readily express type II (28 , 29) .

In some other cell types, NO can inhibit cellular proliferation. However, the role of NO in the growth of airway smooth muscle has not previously been addressed. Since we had identified type I NOS as an active enzyme in these cells, we extended our study to address the effects of NO on DNA synthesis in human airway smooth muscle cells. We found that exogenous NO, supplied in the form of two structurally unrelated NO-releasing drugs (SNP and NOC-18) produced clear and effective reductions in DNA synthesis. These observations are in keeping with others using vascular smooth muscle cells in culture (3) . We then specifically ascertained whether endogenously released NO could influence cell growth. In fact, we found that when human airway smooth muscle cells were stimulated to proliferate, L-NAME increased DNA synthesis in a concentration-dependent manner. These observations suggest that endogenously released NO indeed acts in an autocrine fashion to suppress DNA synthesis in human airway smooth muscle cells.

In many tissues, the effects of NO are limited by its very short half-life (30) . However, when superoxide anions are removed by SOD, the biological activity of NO is increased dramatically. We found that when human airway smooth muscle cells were stimulated to divide in the presence of SOD, DNA synthesis was reduced. Moreover, when experiments were repeated in the presence of a cell-permeable SOD mimetic, DNA synthesis was virtually abolished. To demonstrate that the changes induced in DNA synthesis by these compounds lead to changes in cell proliferation, cell number experiments were performed. Indeed, L-NAME increased proliferation by 70% above that induced by 3% FCS. Furthermore, the NO donors and the SOD mimetic completely inhibited proliferation to that of unstimulated levels. These observations support the notion that endogenously released NO inhibits proliferation in these cells.

We found that the release of nitrite by human airway smooth muscle cells was greatly reduced when cells were stimulated to proliferate with serum. However, this was not associated with a reduction in the level of NOS protein, suggesting that cell division is associated with posttranslational modifications of NOS activity. Little or no data exist as to how the activity of type I NOS changes in proliferating cells. However, a number of studies, have shown that type III NOS is altered when cells are stimulated to divide. One study has shown that type III NOS mRNA and protein are increased in growing vs. resting cells (31) . By contrast, another group found that type III NOS mRNA was actually less stable, resulting in lower levels of enzyme in proliferating vs. resting cells (32) . These conflicting observations may reflect the complexity of responses produced by NO in different cells and also the variability in responses of cultures at different passages in different laboratories.

Hyperplasia and hypertrophy of airway smooth muscle are thought to contribute to airway diseases. Indeed, enhanced proliferation of airway cells is associated with the chronic stages of asthma and COPD in some patients. The findings in this study have important therapeutic relevance for two reasons. First, the finding that airways smooth muscle cells express type I NOS, which inhibits proliferative responses, suggests that inhibitors of NOS may exacerbate diseases some airway disease. Second, the finding that cell-permeable mimetics strongly suppress DNA synthesis and cell proliferation of these cells suggests that this class of drug may be useful in the treatment of diseases such as asthma or COPD.


   ACKNOWLEDGMENTS
 
This work was supported by a grant form the Wellcome Trust. J.A.M. is a Wellcome Trust Career Development Fellow. L.D. is supported by a grant from the National Asthma Campaign.


   FOOTNOTES
 
2 Abbreviations: BSA, bovine serum albumin; COPD, chronic obliterative pulmonary disease; DMEM, Dulbecco's modified Eagle's medium; D-NAME, N{omega}-nitro-D-arginine methyl ester; FCS, fetal calf serum; HASM, human airway smooth muscle; HBSS, Hanks' balanced salt solution; L-NAME, N{omega}-nitro-L-arginine methyl ester; MnTMPyP, Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride; NO, nitric oxide; NOC 18, 2,2'-(hydroxynitrosohydrazino)bis-ethanlamine; eNOS, endothelial NO synthase; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; SOD, superoxide dismutase.

Received for publication February 19, 1999. Accepted for publication May 12, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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