Division of Respiratory Medicine, City Hospital, Nottingham NG5 1PB, United Kingdom
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ABSTRACT |
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Airway smooth muscle (ASM) hypertrophy and
hyperplasia are important determinants of bronchial responsiveness in
asthma, and agents that interfere with these processes may prevent
airway remodeling. We tested the hypothesis that activators of soluble and particulate guanylyl cyclases would inhibit human ASM cell (HASMC)
proliferation. We report that the nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine
(SNAP; 106 to
10
4 M) and sodium
nitroprusside (10
5 to
10
3 M) and human atrial
natriuretic peptide [ANP-(1
28);
10
8 to
10
6 M], which
activate soluble and particulate guanylyl cyclases, respectively,
inhibited serum- and thrombin-induced proliferation of cultured HASMCs.
The antimitogenic effect of SNAP was reversed by hemoglobin
(10
5 M), an NO scavenger,
suggesting that NO donation was involved. The antiproliferative effects
of SNAP and ANP-(1
28) were potentiated by the cGMP-specific
phosphodiesterase zaprinast and mimicked by 8-bromo-cGMP
(10
6 to
10
3 M), suggesting that
cGMP-dependent mechanisms were involved. However, first, ANP-(1
28)
produced a smaller antiproliferative effect than SNAP in contrast to
their abilities to elevate cGMP, and second, rat ANP-(104
126), which
binds selectively to ANP clearance receptors without elevating cGMP,
had a small antiproliferative effect, suggesting that cGMP-independent
mechanisms were also involved. These results provide
evidence for a novel antiproliferative effect of NO and ANP in HASMCs
mediated through cGMP-dependent and cGMP-independent mechanisms.
nitric oxide; atrial natriuretic peptide; asthma; guanosine 3',5'-cyclic monophosphate; hypertrophy; hyperplasia
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INTRODUCTION |
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HISTOPATHOLOGICAL STUDIES of airways removed at
postmortem from patients with severe asthma show that the area of
airway smooth muscle (ASM) is substantially increased in both large
(10, 24, 27, 58) and small (50) airways. This ASM
thickening is believed to be one of the principal contributors to
airway wall thickening and the resulting poorly reversible airflow
obstruction, which characterizes chronic asthma (29). It is not clear
whether the increase in ASM mass occurs as a result of overproduction
of growth factors, a loss of inhibitory influence, or both. Recent
studies (25, 32) have identified several growth factors for ASM in a
number of species, but little is known about the mechanisms and
pathways inhibiting ASM proliferation. Ligands binding to adenylyl
cyclase-linked receptors
(2-adrenoceptor
agonists, PGE2, and vasoactive
intestinal peptide) (12, 36, 59), heparin (30), and interleukin-4 (23)
all have some antiproliferative properties in ASM.
The synthesis of cGMP is catalyzed by guanylyl cyclases (GCs), which exist in two forms: a soluble (cytosolic) form activated by nitric oxide (NO) donors (38) and a particulate (membrane-bound) form activated by natriuretic peptides (33). Hamad et al. (21) recently performed a pharmacological characterization of GCs in cultured human ASM cells (HASMCs). This study showed that both GC forms were abundant, suggesting that they are functionally important. This is consistent with in vivo studies (1, 51) showing that both NO and ANP can regulate human bronchial tone. NO is produced by the airway epithelium where it has a paracrine bronchoprotective role (20, 48) and by nonadrenergic noncholinergic nerves where it acts as an inhibitory neurotransmitter causing cGMP-mediated ASM relaxation (4, 61). Consistent with this relaxant effect, NO and NO donors cause bronchodilatation when administered exogenously to both normal and asthmatic subjects (31, 51). The physiological role of atrial natriuretic peptide (ANP) is less clear. However, ANP is produced locally in the lung (47), and it acts as a relaxant in ASM in vitro (2) and a bronchodilator when administered exogenously to asthmatic subjects (28). Collectively, these studies suggested that ANP may have a physiological role in the regulation of ASM tone. Thus there is a large body of evidence to suggest that NO donors and natriuretic peptides act to regulate airway caliber. Their effects on other aspects of ASM function, particularly proliferation, are unknown.
We hypothesized that pathways linked to cGMP accumulation might have an
inhibitory role on ASM proliferation. We therefore studied the effect
of activators of soluble and particulate GCs on HASMC proliferation and
determined whether their effects were cGMP dependent. Because these
enzymes generate cGMP in different intracellular compartments that may
have differential access to antiproliferative signal transduction
pathways or cGMP phosphodiesterases (PDEs), we postulated that they may
have different effects on proliferation. Both SNAP and ANP have
cGMP-independent effects on proliferation in other biological systems
(7, 15, 22, 52), and we postulated that cGMP-independent effects may
also occur in ASM. To test the hypothesis that cGMP is involved in the
effects of NO donors or ANP on proliferation, we determined whether
their effects could be potentiated by a cGMP-specific PDE inhibitor,
zaprinast, and compared those effects with those of 8-bromo-cGMP, a
cell-permeable cGMP analog. In the case of ANP, we compared the effect
of human ANP-(128), which is known to elevate cGMP with rat
ANP-(104
126), which activates ANP clearance receptors without
elevating cGMP, to determine whether ANP could have cGMP-independent effects.
We found that activators of soluble GC
[S-nitroso-N-acetylpenicillamine
(SNAP) and sodium nitroprusside (SNP)] and particulate GC
[ANP-(128)] had potent inhibitory effects on serum- and
thrombin-induced proliferation of HASMCs and that these effects
occurred through cGMP-dependent and cGMP-independent pathways. Our
studies suggest that activation of GCs may provide a novel target for
the prevention of airway remodeling in asthma.
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MATERIALS AND METHODS |
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Cell Culture
Primary cultures of HASMCs were prepared from explants of ASM according to methods previously described (21, 43, 44). We (21) have shown, using morphological and immunohistochemical staining, that this method produces a relatively pure (>95%) population of ASM cells. Frozen aliquots of cells were thawed before use and plated at a density of 2 × 104 cells/ml in culture plates containing DMEM plus 10% fetal bovine serum (FBS), 50 U/ml of penicillin G, 50 µg/ml of streptomycin, 2.5 µg/ml of amphotericin B, and 4 × 10Proliferation Assays
[3H]thymidine incorporation. Cells were grown in 96-well plates in DMEM plus 10% FBS until 90-100% confluent. The cells were then growth arrested in serum-free DMEM for 24 h. Growth-arrested cells were used because they can be synchronized in the G0/G1 phase of the cell cycle and at this baseline minimally incorporate [3H]thymidine (42). The cells were stimulated with either 10% FBS or 1 U/ml of human thrombin for 30 h. During the last 6 h, HASMCs were labeled with 4 µCi/ml of [3H]thymidine. DNA was isolated with a cell harvester (Automash 2000, Dynatec) adsorbed onto fiberglass filters, treated with 200 µl of 0.01 M potassium hydroxide, and immersed in 4 ml of scintillation fluid (Ready Protein, Beckman). Radioactivity was quantified with a scintillation counter (Minaxi 4000, Packard) with a counting efficiency of 40%.
Cell counts. Cells were grown in DMEM plus 10% FBS in 24-well plates for 2-3 days (until 30-40% confluent) and then growth arrested in serum-free DMEM for 24 h. They were then treated with either DMEM alone or DMEM plus 10% FBS with and without the appropriate test substance (day 0). Fresh medium containing the same concentration of the test substance was added every 48 h over 8 days. The cells were counted on days 2, 4, 6 and 8 after being detached with trypsin (0.25%) and EDTA (0.02%) in PBS, centrifuged, pelleted, and then resuspended in 20 µl of trypan blue. Viable cells were counted with a hemacytometer. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used as a measure of the proliferation response (26). MTT cleavage into a blue-colored formazan product by the mitochondrial enzyme succinyl dehydrogenase occurs exclusively in living cells. Cells were grown in DMEM plus 10% FBS in 96-well plates for 2-3 days (until 30-40% confluent) and then growth arrested in serum-free DMEM for 24 h. They were then treated with either DMEM alone or DMEM plus 10% FBS with and without the appropriate test substance (day 0). Fresh medium containing the same concentration of the test substance was added every 48 h over 8 days. An MTT assay was performed every second day as previously described (45). Briefly, 20 µl of a 5 mg/ml MTT solution were added to each well, and the cells were incubated at 37°C for 1 h. After removal of the medium, 200 µl of DMSO were added to solublize the blue formazan product, and the plates were shaken for 5 min. The optical density at 570 nm was compared with that of control wells with a plate reader (MR 5000, Dynatec).cGMP Measurement
cGMP was measured as previously described (21). Briefly, the cells were trypsinized and cGMP was extracted with 0.1 M hydrochloric acid. The resulting suspension was freeze-dried (SB9, Lab Plant, Huddersfield, UK) before the measurement of cGMP content. cGMP was measured with a commercially available enzyme-linked immunosorbant assay kit (Amersham, Little Chalfont, UK). The samples were first acetylated with a mixture of acetic anhydride and triethylamine to increase the sensitivity of the assay to 4 fmol/100 µl. The coefficient of variation of the assay was 11%. All samples were assayed in duplicate. The cells from six wells not used for cGMP assay were used to give a representative estimate of protein content in each experiment with the method of Bradford (5).Chemicals
[3H]thymidine was obtained from Amersham. FBS was purchased from JRH Biosciences (Sera-Lab). Human ANP-(1Analyses
Results are shown as means ± SE of the indicated number (n) of individual observations from representative experiments. Each experiment was repeated two to three times with similar results. [3H]thymidine incorporation data are expressed as disintegrations per minute per well (or as percent inhibition of stimulated control cells to allow comparison between experimental protocols). MTT assay data are expressed as absorbance at 570 nm per well. Cell-counting data are expressed as cell number per well. Significance of the drug effect was assessed by one-way ANOVA followed by Student's t-test with the SPSS software program (SPSS, Chicago, IL). A P value ![]() |
RESULTS |
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Effect of Serum and Thrombin on HASMC Proliferation
Preliminary experiments showed that a 30-h time point was optimal for measuring thymidine incorporation in cells stimulated with FBS or thrombin (data not shown). Both FBS and thrombin caused a concentration-dependent increase in thymidine incorporation, with a maximal increase of 56 ± 4.2- and 8 ± 0.4-fold in cells treated with 10% FBS and 1 U/ml of thrombin, respectively (P < 0.001 for both; n = 6; Fig. 1).
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Effect of SNAP on HASMC Proliferation
SNAP concentration dependently inhibited thymidine incorporation induced by 10% FBS and 1 U/ml of thrombin. FBS-induced thymidine incorporation was inhibited by 31 ± 7.0% at 10
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Because thymidine incorporation measures DNA synthesis that may occur
in some systems without cell division (8), we compared data from
thymidine incorporation experiments with data from MTT assays (26) and
cell-counting experiments (with 10% FBS as the mitogenic stimulus).
SNAP at 104 M
abolished the FBS-induced increase in MTT conversion on
day 4 and inhibited the FBS-induced
increase in MTT by 78 ± 1.9 and 61 ± 4.6% on
days 6 and
8, respectively
(P < 0.001 for all;
n = 12; Fig.
4A). In
cell-counting experiments,
10
4 M SNAP inhibited the
FBS-induced increase in cell number by 82 ± 12.7, 87 ± 2.7, 88 ± 1.6, and 91 ± 0.6% on days
2, 4,
6, and 8, respectively
(P < 0.01, 0.001, 0.001, and 0.001, respectively; n = 4; Fig.
4B).
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Effect of Hemoglobin on the Antimitogenic Effect of SNAP
To determine whether the inhibitory effect of SNAP was related to NO donation, we studied the effect of hemoglobin (Hb), an NO scavenger, on the antimitogenic effect of SNAP. Cells were pretreated with 10
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Effect of Human ANP-(128) on HASMC Proliferation
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Effect of Rat ANP-(104126) on HASMC Proliferation
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Effects of Zaprinast on the Action of SNAP and ANP-(128)
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Effect of 8-Bromo-cGMP on HASMC Proliferation
To further elucidate the role of cGMP in the antimitogenic effect of SNAP and ANP-(1
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MTT Conversion by HASMCs Treated With 8-Bromo-cGMP,
ANP-(128), and Rat ANP-(104
126)
Assessment of Cell Viability
None of the chemicals or vehicles used caused inhibition below the mean values for unstimulated cells when proliferation was assessed by thymidine incorporation, MTT assay, or cell counting nor was there evidence of cytotoxicity when measured by trypan blue exclusion. Moreover, the effects of SNAP, SNP, and ANP were reversible. In experiments where they were removed from the culture medium, the cells regained their ability to proliferate in response to mitogens (data not shown). Experiments were attempted with the GC inhibitors methylene blue and 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one, but both compounds produced toxic effects with prolonged incubation and could not therefore be used. ![]() |
DISCUSSION |
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We have shown for the first time that the NO donors SNAP and SNP, which
activate soluble GC, and human ANP-(128), which activates particulate
GC, inhibited the proliferation of HASMCs in response to serum and
thrombin. The fact that the antimitogenic effects of these agents could
be potentiated by a cGMP-specific PDE inhibitor, zaprinast, and were
mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP suggests
that the effects were mediated, at least in part, by cGMP.
We used serum as a mitogenic stimulus because the increase in ASM growth seen with increasing concentrations of serum in culture may provide an in vitro correlate of the situation in vivo created by increased vascular permeability with plasma leakage in asthmatic airways (55). Thrombin was used as a more specific mitogen because it is generated at the sites of inflammation in the airways and has been shown to potently stimulate DNA synthesis in HASMCs (41). The change in thymidine incorporation produced by 10% FBS was significantly greater than that produced by 1 U/ml of thrombin, which is consistent with the results of previous studies in HASMCs (23, 57). The magnitude of the changes in the proliferative indexes seen in response to serum and thrombin in our study are broadly similar to those previously reported in HASMCs (41).
We used thymidine uptake as our main proliferation index. In selected experiments, we confirmed that the changes in thymidine incorporation were paralleled by the changes in MTT conversion and cell number, suggesting that thymidine uptake was a measure of cell proliferation under our experimental conditions.
We used SNAP as the main NO donor because it is a direct NO donor and produced the largest change in cGMP of NO donors in HASMCs in the previous study by Hamad et al. (21). Both FBS and thrombin induced significant increases in thymidine incorporation, which were prevented by SNAP and SNP. Similarly, the FBS-induced increase in cell number was inhibited by SNAP. SNAP and SNP inhibited thrombin-induced proliferation to a much greater extent than serum-induced proliferation, which may be due to the lesser degree of proliferation induced by thrombin (57). Alternatively, it may reflect differential inhibition of the intracellular pathways mediating the thrombin response. The inhibitory effects of NO donors may therefore relate to the type of mitogenic stimulus. The inhibition by SNAP of cell counts was greater than that by thymidine incorporation, consistent with experiments in vascular smooth muscle cells (17). This difference is likely to reflect the fact that these indexes measure different aspects of proliferation at different times related to cell cycling. Thymidine incorporation is a measure of thymidine uptake across the cell membrane as well as thymidine incorporation into DNA, whereas cell counts reflect the balance between cell proliferation and apoptosis. The antiproliferative effect of SNAP in our study was likely to be NO mediated because Hb, an NO scavenger that acts in a competitive manner (18), reduced the antimitogenic effect of SNAP. This is consistent with previous studies in other cell systems (9, 13, 15). Hb had an antiproliferative effect on its own, which was reported in other cell systems (14, 15). This effect of Hb was not due to toxicity because we saw no effect of Hb on MTT assay or trypan blue exclusion (data not shown). Because Hb was dissolved in DMEM, there was no possibility of a vehicle effect. The mechanism, therefore, is not clear, but possibilities include enhanced release of antiproliferative cytokines in the presence of Hb or an oxygen-scavenging effect altering mitochondrial respiration. Having demonstrated an antimitogenic effect of SNAP, we performed experiments to probe the role of cGMP. Zaprinast, a cGMP-specific PDE inhibitor, given at a concentration selective for cGMP-specific PDE (3) potentiated the antimitogenic effect of SNAP parallel to an increase in cGMP accumulation. Moreover, the cell-permeable cGMP analog 8-bromo-cGMP could mimic the antimitogenic effect of SNAP, although it was less potent. Taken together, these results support the view that at least part of the effect of SNAP was mediated by cGMP. However, the fact that 8-bromo-cGMP was less effective than SNAP and SNP at inhibiting proliferation suggests that additional cGMP-independent mechanisms may also be involved. The suggestion that NO donors may have both cGMP-dependent and -independent effects has been made by workers in a variety of cell lines, including vascular smooth muscle cells (16, 37, 54). Several NO donors and the cGMP analog 8-bromo-cGMP have been shown to inhibit serum-induced mitogenesis and proliferation in cultured rat aortic and renal arteriolar smooth muscle cells via a cGMP-dependent mechanism (9, 15, 39). In contrast, NO inhibition of rat aortic smooth muscle cells is independent of cGMP (52, 53). Several cGMP-independent mechanisms have been suggested for the antiproliferative effect of NO, including inhibition of thymidine kinase (17) and ribonucleotide reductase, the rate-limiting enzyme in DNA synthesis (34). Future studies are needed to determine the mechanisms involved in HASMCs.
Further evidence for dissociation between the effect of GC activators
on cGMP formation and proliferation comes from our studies with ANP. We
used human ANP-(128) as an activator of particulate GC because Hamad
et al. (21) previously showed it to be the most potent natriuretic
peptide at elevating cGMP in HASMCs. Although we found that human
ANP-(1
28), like SNAP, had antiproliferative effects, it produced a
smaller maximum effect. This difference in efficacy contrasts with
relative abilities to elevate cGMP (ANP > SNAP) (21). This paradox
suggests that either additional cGMP-independent mechanisms are
involved with SNAP or compartmentalization of cGMP pools enables cGMP
generated by soluble GC to have a greater antiproliferative effect than
that generated by particulate GC. The potentiation of the antimitogenic
effect of human ANP-(1
28) by zaprinast suggests that the
antiproliferative effect of human ANP-(1
28) was mediated, at least in
part, by cGMP because zaprinast alone had no antiproliferative effect.
To investigate further cGMP-independent effects of ANP, we extended our
studies of ANP to compare human ANP-(1
28) with rat ANP-(104
126).
Biochemical studies have shown two categories of ANP receptors, one of
which is GC linked (GC-A), whereas the second category is termed the ANP clearance receptor, named for its ability to mediate the cellular uptake (plasma clearance) of ANP (35). Human ANP-(1
28) can bind to
both types of receptors, whereas rat ANP-(104
126) binds selectively
to ANP clearance receptors without elevating cGMP. Rat ANP-(104
126)
had a small antiproliferative effect, suggesting that additional
cGMP-independent mechanism may contribute to the antiproliferative
effects of ANPs. We confirmed that rat ANP-(104
126) had no effect on
cGMP levels in our cells. These results are consistent with previous
reports in rat aortic smooth muscle cells (6, 7) showing that ANP can
exert antiproliferative effects in a cGMP-independent manner through
the clearance receptors. It is also interesting that, in contrast to NO
donors and 8-bromo-cGMP, ANP inhibited serum-induced proliferation to a
greater extent than thrombin-induced proliferation, providing further
evidence that the mechanisms involved in their antiproliferative
effects are different.
The effects of SNAP and SNP in our studies were greater than those of
ANP or 8-bromo-cGMP, which were only partially effective at inhibiting
thymidine incorporation. The magnitude of these partial effects is
comparable to the effects reported with other antiproliferative agents
in ASM cells such as
2-adrenoceptor agonists and
cAMP mimetics, corticosteroids, interleukin-4, and heparin (23, 30, 40,
57, 60).
Our results aid our understanding of the hyperplastic changes occurring in ASM in asthma. Disruption and loss of the bronchial epithelium is a well-described histological feature of asthma (32). Because this epithelium is an important source of NO, its loss in asthma could therefore lead to the removal of an important inhibitory paracrine mechanism and contribute to the increase in ASM bulk, which characterizes this condition. Epithelial loss would also deplete other antimitogenic protective factors such as PGE2 (12). We have also considered whether the antiproliferative effect of ANP might have a physiological role. The lung is capable of synthesizing ANP (47), and both types of ANP receptors have been characterized and localized throughout the lung. The specific binding sites per gram of tissue weight or per whole organ in rats were found to be greater in the lung than in other tissues including the kidney (19). In the heart, where more is known about the functions of ANP, ANP release is stretch stimulated (11). Similarly, Springall et al. (56) suggested that stretch of rat pulmonary veins stimulates ANP release. Preliminary data support a similar stretch-dependent mechanism for ANP release in tracheal muscle because sustained stretch led to increased cGMP, but not cAMP, levels in the trachealis muscle of anesthetized sheep (46). Stretch-dependent release of ANP from ASM, occurring with deep inspiration, could lead to cGMP elevation, with subsequent inhibition of ASM tone and cell proliferation.
In conclusion, we have shown for the first time that NO donors and ANP can inhibit HASMC proliferation in culture. The fact that these effects were mimicked by a cell-permeable cGMP analog and potentiated by a cGMP-specific PDE inhibitor suggests that the effects are partly related to an elevation in cGMP. However, the fact that the relative potencies of SNAP and ANP do not reflect their ability to generate cGMP suggests that additional cGMP-independent effects are involved. The effects of NO and ANP may be important in developing strategies for the prevention of airway remodeling in chronic asthma.
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ACKNOWLEDGEMENTS |
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A. M. Hamad was supported by the Egyptian Ministry of Higher Education.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. J. Knox, Division of Respiratory Medicine, Clinical Science Bldg., City Hospital, Hucknall Rd., Nottingham NG5 1PB, UK (E-mail: Alan.knox{at}nottingham.ac.uk).
Received 16 December 1998; accepted in final form 1 July 1999.
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