Respiratory Medicine Unit, City Hospital, Nottingham NG5 1PB, United Kingdom
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ABSTRACT |
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Although guanosine
3',5'-cyclic monophosphate (cGMP) acts as a relaxant second
messenger, the regulation of intracellular cGMP has not been
comprehensively studied in human airway smooth muscle. We studied the
production of cGMP by cultured human airway smooth muscle cells (HASMC)
after stimulation with activators of soluble guanylyl cyclase
[sodium nitroprusside (SNP) and
S-nitroso-N-acetylpenicillamine (SNAP)] and particulate guanylyl cyclase [atrial
natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type
natriuretic peptide (CNP), and Escherichia
coli heat stable enterotoxin (STa)]. cGMP was
measured by enzyme-linked immunosorbent assay. Both SNP
(106 to
10
3 M) and SNAP
(10
6 to
10
3 M) caused
concentration-dependent elevation of cGMP in the presence of the
nonselective phosphodiesterase (PDE) inhibitor
3-isobutyl-1-methylxanthine (10
3 M), with cGMP
increasing 6- and 15-fold in response to SNP and SNAP, respectively, at
the highest concentration tested
(10
3 M). The increases in
cGMP in response to SNP (5 × 10
5 M) and SNAP
(10
5 M) were inhibited by
hemoglobin (Hb; 5 × 10
5 M), a nitric oxide
scavenger, and methylene blue (MB; 5 × 10
4 M), an inhibitor of
guanylyl cyclase. cGMP accumulation after SNAP was abolished by both Hb
and MB. The response to SNP was inhibited by 79% with Hb and was
abolished with MB. ANP, BNP, and CNP
(10
9 to
10
5 M) + phosphoramidon
(10
6 M) caused a
concentration-dependent elevation in cGMP with an order of potency ANP > BNP > CNP. cGMP formation in the presence of the highest
concentration of the most potent natriuretic peptide (10
5 M ANP) was two- to
threefold greater than with the highest concentration of SNAP. The
increase in cGMP seen with natriuretic peptides was similar in the
presence or absence of phosphoramidon, a neutral endopeptidase (NEP)
inhibitor, suggesting that NEP is not playing a role in modulating the
effect of natriuretic peptides in HASMC. STa (400 IU/ml) had no effect
on cGMP levels. SNAP- and ANP-induced cGMP accumulation was increased
by the selective type V PDE inhibitors SKF-96231 and zaprinast,
suggesting that type V PDE is responsible for cGMP breakdown in HASMC.
These results suggest that cultured HASMC contain both soluble and
particulate guanylyl cyclases. The order of potency of the natriuretic
peptides ANP > BNP > CNP suggests that type A particulate
membrane-bound guanylate cyclase predominates.
nitric oxide; natriuretic peptides; guanosine 3',5'-cyclic monophosphate
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INTRODUCTION |
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AIRWAY SMOOTH MUSCLE relaxation is the dominant
mechanism of action of bronchodilator drugs in the treatment of asthma
and chronic obstructive pulmonary disease (30). The airway smooth muscle relaxant drugs in current use (-adrenoceptor agonists, theophyllines) act to elevate the intracellular second messenger adenosine 3',5'-cyclic monophosphate (cAMP; see Ref. 30).
Guanosine 3',5'-cyclic monophosphate (cGMP) is also a
relaxant second messenger in airway smooth muscle cells (8, 11, 12, 14,
15, 29, 48). This may be important physiologically as nitric oxide
(NO), which elevates cGMP, is the relaxant neurotransmitter of
nonadrenergic noncholinergic nerves (5, 28, 32, 48). Elevation of cGMP is also a potential target for pharmacological intervention.
cGMP is produced from GTP by the action of guanylyl cyclases. Guanylyl cyclases exist in two forms, a soluble form (GCs) and a particulate membrane-bound form (GCm; see Refs. 45, 47, and 50). Each form is activated by distinct agonists. GCs is activated by NO and NO donors, whereas GCm is a plasma membrane receptor for the natriuretic peptides and related hormones (45, 50). cGMP produced by guanylyl cyclases activates a group of distinct cGMP-dependent protein kinases, which have several sites of action in airway smooth muscle to bring about relaxation (8, 9, 21, 30, 33, 36). cGMP is metabolized to 5'-GMP by phosphodiesterases (PDE; see Refs. 32 and 46).
Agents that activate guanylyl cyclase have been shown to relax airway smooth muscle in vitro and bronchodilate in vivo. Nitrobronchodilators relax guinea pig (26), bovine (18), and human airway smooth muscle (14, 38, 48), atrial natriuretic peptide (ANP) relaxes guinea pig (10, 35), rat (10), and bovine (25) airway smooth muscle in vitro, and brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) relax guinea pig trachealis (43, 44). Studies with ANP in human airway smooth muscle in vitro have shown conflicting results, with some studies showing relaxation (2) and others showing no relaxation (7, 31). Inhaled NO has a weak bronchodilator effect in vivo (22), whereas ANP is a bronchodilator in both normal (23) and asthmatic subjects (1, 24).
Despite the potential importance of cGMP pathways in regulating airway smooth muscle tone, no previous studies have comprehensively examined the regulation of cGMP concentrations in human airway smooth muscle. The purpose of this study was to use a number of pharmacological tools to characterize the mechanisms responsible for cGMP formation and breakdown in cultured human airway smooth muscle cells (HASMC).
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MATERIALS AND METHODS |
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Materials. SKF-96231 was a gift from SmithKline Beecham Pharmaceuticals (Welwyn, UK). Unless otherwise stated, all other chemicals were obtained from Sigma-Aldrich (Poole, Dorset, UK). The peptides used were human 28-ANP and human 32-BNP and 22-CNP. All reagents were dissolved in Dulbecco's modified Eagle's medium (DMEM) with the exception of 3-isobutyl-1-methylxanthine (IBMX), zaprinast, and SKF-96231, which were first dissolved in ethanol to give a final concentration of 3% vol/vol ethanol in the bathing solution. The same concentration of ethanol was added to the bathing solutions of control experiments. All concentrations of reagents shown refer to the final concentration in the cell suspension.
Cell culture. Primary cultures of HASMC were prepared from explants of airway smooth muscle according to the method reported by Hall and co-workers (20) and Widdops et al. (49) with some modifications. Human trachea was obtained from postmortem individuals within 12 h of death. No patients had evidence of airway diseases as determined by history and pathological examination of the trachea and lungs. The tissue was transported to the laboratory in DMEM containing 10% fetal calf serum (FCS), penicillin G (50 U/ml), streptomycin (50 µg/ml), amphotericin B (2.5 µg/ml), and L-glutamine (4 mM). Tissue was then washed several times in the same medium. The trachealis muscle was then dissected free of epithelium and connective tissue under sterile conditions. Small (2 × 2-mm) explants of airway smooth muscle were then excised, and about 10 explants were placed in small petri dishes in DMEM containing FCS, antibiotics, amphotericin B, and L-glutamine. The explants were incubated in humidified 5% CO2-95% air at 37°C, and the medium was changed every 3 days. Smooth muscle cells were usually seen about 7 days later. When cells were near confluence, the explants were removed. Once confluent, cells were trypsinized with 0.25% trypsin and 0.02% EDTA in phosphate-buffered saline, centrifuged, and resuspended in the above medium. Next, the cells were counted and plated out in several 175-cm2 flasks and were grown to confluence. Cells were then detached with trypsin-EDTA, resuspended in 90% FCS-10% dimethyl sulfoxide at a density of 106 cells/ml, frozen in liquid nitrogen, and stored until required. Cells were thawed before use and were plated at a density of 2 × 104 cells/well in 12-well culture plates containing the above medium.
All experiments were performed in confluent HASMC that had been growth arrested for 24 h in serum-free DMEM containing insulin (6 µg/ml), transferin (5 µg/ml), and ascorbic acid (35 µg/ml). The culture medium was changed immediately before each experiment, and cells were incubated with the pharmacological agents or vehicle controls at 37°C for 2 h (unless otherwise stated) in humidified 5% CO2-95% air. Smooth muscle cultures were set up from tissue from three patients. Multiple wells of cells from one source were used for each experimental protocol to reduce variability.
Characterization of the HASMC. We
employed morphological and immunocytochemical staining to determine
whether the cultured cells had the characteristics of airway smoth
muscle cells. Under the light microscope, subconfluent HASMC were
spindle shaped with central oval nuclei, whereas the confluent cells
depicted the "hill-and-valley" appearance that is characteristic
of smooth muscle cells in culture. For the identification of the
markers of airway smooth muscle cell phenotype, the cells were plated in an eight-well chamber slide system, grown to confluence, growth arrested for 24 h, and fixed with methanol. The cells were then examined by standard immunocytochemical techniques using antibodies against smooth muscle cell-specific -actin (Sigma) and mature muscle
cell-specific desmin (Daco, High Wycombe, Bucks, UK). We demonstrated
that >95% of the cells were positively labeled. In contrast,
staining for the epithelial marker cytokeratin was negative.
cGMP measurement. cGMP was extracted
by adding 1 ml of ice-cold 0.1 M hydrochloric acid. Cells were removed
from culture wells mechanically. The resulting suspension was
freeze-dried (SB9; Lab Plant, Huddersfield, Yorkshire, UK) before the
measurement of cGMP content. cGMP was measured using a commercially
available enzyme-linked immunosorbent assay (ELISA) kit (RPN 226;
Amersham, Little Chalfont, Buckinghamshire, 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 using the method of Bradford (6). To test the
cross-reactivity of the assay with cAMP, experiments were performed
measuring cGMP after stimulation with isoproterenol (106 M), a
2-adrenoceptor agonist that
increases cAMP via adenylate cyclase. No increase in cGMP was seen
after stimulation with isoproterenol.
Statistical analyses. Results are shown as means ± SE. Between four and six wells of cells from a single aliquot were used for each experimental protocol. The significance of drug effects was assessed by one-way analysis of variance followed by unpaired Student's t-tests using SPSS (SPSS, Chicago, IL). A P value <0.05 was regarded as significant.
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RESULTS |
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Effect of IBMX on basal and stimulated cGMP
levels. Preliminary experiments were performed to
determine if PDE inhibition was necessary to show effects of guanylyl
cyclase activators in HASMC.
S-nitroso-N-acetylpenicillamine
(SNAP) was used as an NO donor and
GCs activator, and IBMX was used
as a nonselective PDE inhibitor. After 1 h of incubation, cGMP levels
in cells incubated with IBMX
(103 M; 102 ± 24 fmol/mg protein) did not differ significantly from cells incubated with
control (84 ± 12 fmol/mg protein;
P = 0.3, n = 4). IBMX
(10
3 M) increased cGMP
production in response to SNAP
(10
3 M). At 1 h, cGMP
levels in cells incubated with SNAP
(10
3 M) plus IBMX
(10
3 M) were 1,024 ± 135 fmol/mg protein compared with 329 ± 173 fmol/mg protein with
SNAP (10
3 M) alone
(P < 0.01, n = 4; Fig.
1). Similar experiments using ANP as a
GCm activator also showed that ANP
had no effect in the absence of IBMX. cGMP levels were 969 ± 201 fmol/mg protein in controls, 984 ± 118 in cells incubated with ANP
(10
6 M) alone, and 2,179 ± 239 in cells treated with ANP
(10
6 M) plus IBMX
(10
3 M;
P < 0.01, ANP + IBMX vs. ANP alone). In view of these results, all subsequent
experiments were carried out in the presence of IBMX
(10
3 M) unless otherwise
stated.
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Time course of cGMP production. cGMP
production in response to
103 M SNAP was measured at
0, 1, 20, 40, 60, 120, and 180 min. There was a time-dependent increase
in cGMP from 157 ± 12 fmol/mg protein at time
0 to 599 ± 191 fmol at 1 min
(P < 0.01, n = 6), 1,054 ± 142 fmol/mg at 20 min (P < 0.001, n = 6), 1,146 ± 271 fmol/mg protein at 40 min (P < 0.01, n = 6), 1,882 ± 216 fmol/mg
protein at 60 min (P < 0.001, n = 6), 3,493 ± 235 fmol/mg protein at 120 min
(P < 0.001, n = 6), and 4,260 ± 451 fmol/mg
protein at 180 min (P < 0.001, n = 6; Fig.
2).
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cGMP production in response to
105 M ANP,
10
5 M BNP, and
10
5 M CNP was measured at
1, 60, and 120 min. There was a time-dependent increase in cGMP for
each peptide. ANP (10
5 M)
increased cGMP levels from 170 ± 65 fmol/mg protein at
time 0 to 1,747 ± 148 fmol/mg
protein at 1 min (P < 0.001, n = 4), 3,920 ± 449 fmol/mg
protein at 60 min (P < 0.01, n = 4), and 12,520 ± 618 fmol/mg
protein at 120 min (P < 0.001, n = 4). BNP
(10
5 M) increased cGMP
levels from 170 ± 65 fmol/mg protein at time 0 to 729 ± 35 fmol/mg protein at 1 min
(P < 0.01, n = 4), 3,589 ± 434 fmol/mg
protein at 60 min (P < 0.01, n = 4), and 10,879 ± 652 fmol/mg
protein at 120 min (P < 0.001, n = 4). CNP
(10
5 M) increased cGMP
levels from 170 ± 65 fmol/mg protein at time 0 to 1,784 ± 171 fmol/mg protein at 1 min
(P < 0.001, n = 4), 3,337 ± 168 fmol/mg
protein at 60 min (P < 0.001, n = 4), and 4,604 ± 466 fmol/mg
protein at 120 min (P < 0.01, n = 4; Fig. 3).
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In view of these results, in all subsequent experiments, HASMC were incubated with the pharmacological agents/vehicle control for 2 h.
Effect of activators of GCs on cGMP
levels.
The NO donors sodium nitroprusside (SNP;
106 to
10
3 M) and SNAP
(10
6 to
10
3 M) were used to
activate GCs. Both SNP and SNAP
generated concentration-dependent elevations in cGMP levels. SNP
increased cGMP levels from a control of 418 ± 42 fmol/mg protein to
2,539 ± 175 fmol/mg protein at the highest concentration tested
[10
3 M;
P < 0.001, n = 5; half-maximal effective
concentration (EC50) = 1.05 × 10
4 M]. SNAP
increased cGMP levels from a control level of 283 ± 34 fmol/mg
protein to 4,369 ± 1,868 fmol/mg protein at the highest concentration tested (10
3
M; P < 0.05, n = 5;
EC50 = 1.1 × 10
4 M; Fig.
4). SNAP was more potent than SNP, with a
higher maximal response at
10
3 M
(P < 0.01).
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Effect of activators of GCm on cGMP
levels.
ANP, BNP, CNP (all 109 to
10
5 M), and
Escherichia coli heat stable toxin
(STa; 400 IU/ml) were used to activate
GCm in the presence of
phosphoramidon (10
6 M), a
neutral endopeptidase inhibitor. All three natriuretic peptides
produced significant elevations in cGMP levels (Fig. 6). cGMP levels increased from 78 ± 20 to 12,520 ± 618 fmol/mg protein with
10
5 M ANP
(n = 4, P < 0.001, EC50 = 1.0 × 10
7 M), from 278 ± 9 to
10,879 ± 652 fmol/mg protein with
10
5 M BNP
(n = 4, P < 0.001, EC50 = 1.8 × 10
7 M), and from 487 ± 126 to 4,604 ± 466 fmol/mg protein with
10
5 M CNP
(n = 4, P < 0.001, EC50 = 1.2 × 10
7 M; Fig. 6). To
determine whether enkephalinases were functionally active in HASMC, the
effects of all three natriuretic peptides were studied in the presence
and absence of phosphoramidon
(10
6 M), a neutral
endopeptidase inhibitor added concurrently. Phosphoramidon did not
alter the response to any of the peptides (all
P > 0.5, data not shown).
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DISCUSSION |
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Our results suggest that cultured HASMC contain both GCs and GCm. We performed experiments in growth-arrested cultured HASMC in their first and second passages. The advantage of using cell culture for these studies is that it is easier to manipulate the system experimentally than in studies in whole tissue. It also allows the study of individual cell types such as airway smooth muscle without confounding effects due to epithelial and inflammatory cells. Immunocytochemical stains confirmed that the cells we used had the characteristics of airway smooth muscle cells. The cGMP ELISA that we used is sensitive, reliable, and has little cross-reactivity with cAMP. We saw some differences in the baseline cGMP levels between experiments (which is most likely to represent a combination of experimental variability and intersubject variability between batches of cells), but the magnitude and direction of effect of NO donors and natriuretic peptides were consistent in all of the cells that we studied.
The time course of cGMP accumulation in response to both NO donors and natriuretic peptides showed a progressive increase over 2 h. We used a 2-h incubation in subsequent experiments to maximize the cGMP signal and to allow pharmacological characterization of the response with inhibitors. The time course of cGMP production in our experiments is similar to that seen in other cultured cells (15) and airway epithelial cells (39) in which measurements were made in the presence of PDE inhibitors. In experiments with bovine and human tracheal strips in the absence of PDE inhibitors, a more rapid response was seen, which waned with time (40, 48). The different time course is likely to be related to the use of PDE inhibitors.
In experiments using SNP and SNAP to activate GCs, we saw large increases in cGMP with both compounds, with SNAP being the more potent. The response to SNP and SNAP was inhibited by MB, an inhibitor of GCs (19), indicating involvement of GCs in the response. The response to SNP and SNAP was also inhibited by Hb, which acts as an NO scavenger (16), suggesting that these compounds act via NO release. Although no previous studies have looked at the effect of GCs activators on cGMP levels in cultured HASMC, there have been studies measuring cGMP production in whole airway tissue preparations from animal airways.
The increases in cGMP seen in our experiments with NO donors are broadly similar to those seen in previous studies in bovine (40), canine (51), and human (48) trachealis strips and intact human bronchi (14). The functional correlates of these findings have been studied in canine (51), guinea pig (42), and bovine (29, 40) trachealis in which SNP and other nitrovasodilators produced concentration-dependent relaxation that correlated with the cGMP elevation. In HASMC, NO donors also cause relaxation (48). Until recently, no endogenous mechanism for the control of airway tone was known to use the cGMP pathway in airway smooth muscle. However, several reports have shown that NO synthase inhibitors can inhibit the relaxant response to electrical field stimulation (28, 32), suggesting that NO is the neurotransmitter responsible for nonadrenergic noncholinergic airway relaxation.
We used ANP, BNP, CNP, and STa to activate GCm (50). All three natriuretic peptides increased cGMP in a concentration-dependent manner, whereas STa was without effect. Four classes of GCm that can be distinguished in part by their binding affinity for natriuretic peptides and related compounds have been described (50). Type A guanylyl cyclase preferentially binds ANP and BNP. Type B guanylyl cyclase preferentially binds CNP. Type C guanylyl cyclase is mainly found in the gastrointestinal tract and binds the STa as well as the endogenous peptide hormone guanylin. The specificity of the fourth member of this class, retinal guanylyl cyclase, has been less well characterized. The order of potency seen in our experiment (ANP > BNP > CNP) is consistent with the type A GCm subclass of GCm present in HASMC. The ANP that we used (human 28-ANP) is that which has previously been shown to have bronchodilator properties in humans (23, 24). The lack of response to STa suggests that C-type GCm is not expressed in these cells.
Early studies in human isolated bronchus were unable to show relaxation with ANP (7, 31). Candenas et al. (7) and Labat et al. (31) suggested that one possible explanation for this was that there are no ANP receptors on human airway smooth muscle and that the bronchodilator effect seen was an indirect one. Our result shows that this is clearly not the case. Subsequent studies by other authors have shown that ANP can relax HASMC and shift the concentration curve to methacholine to the right, suggesting that the increases in cGMP that we have seen are functionally important (2). None of these studies measured cGMP accumulation in response to natriuretic peptides.
ANP produces significant bronchodilation when given intravenously to both normal (23) and asthmatic subjects (1) and by inhalation in asthmatics (24). The effect of ANP was greater and more prolonged by the intravenous route, suggesting that peptidases present in airway epithelium may degrade it. Experiments in intact human tracheal preparations in vitro are consistent with this and have shown that the response to ANP can be potentiated by phosphoramidon, an inhibitor of neutral endopeptidase (2). In our experiments, we found no effect of phosphoramidon on cGMP levels produced by the natriuretic peptides. The most likely explanation for this is that neutral endopeptidase is functionally more important in airway epithelial or submucosal tissue and is not present in large quantities in airway smooth muscle. This is consistent with immunocytochemical studies showing a predominantly epithelial distribution of neutral endopeptidase (27).
The greater rise in cGMP seen in our experiments with the natriuretic peptides suggest that GCm is present in higher quantities than GCs in cultured HASMC. This contrasts with bovine trachea in which SNP produced a greater rise in cGMP than ANP (40). The relative effects of activators of GCs and GCm in our studies mirror the relative effects of these agents on human bronchomotor tone in vivo in which ANP causes moderate bronchodilation (1, 24), whereas the effects of NO donors have been less marked (22).
Cyclic nucleotides are broken down by PDE, of which there are at least seven subtypes that differ in their affinity for cAMP and cGMP (3, 34, 46). Anion exchange chromotography has revealed the presence of types II, III, IV, and V in human tracheal strips (17). Type V PDE is cGMP specific. Our results suggest that type V PDE is involved in cGMP degradation in HASMC, as SKF-96231 and zaprinast, which both selectively inhibit type V PDE (3, 46), increased SNAP- and ANP-induced cGMP accumulation. These results are consistent with functional studies in human tracheal strips showing that zaprinast relaxes spontaneous and methacholine-induced tone (4).
In conclusion, our study demonstrates that both GCs and GCm are present in HASMC. Pharmacological characterization shows that type A is the dominant form of GCm expressed. Type V PDE is likely to be responsible for cGMP degradation in HASMC. Cultured HASMC provide a useful model to study the regulation of guanylyl cyclases.
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ACKNOWLEDGEMENTS |
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We thank Rachel Tuck for typing the manuscript.
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FOOTNOTES |
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A. M. Hamad was supported by the Egyptian Ministry of higher education. S. Range was supported by the United Kingdom Medical Research Council.
Address for reprint requests: A. J. Knox, Respiratory Medicine Unit, City Hospital, Hucknall Rd., Nottingham NG5 1PB, UK.
Received 27 December 1996; accepted in final form 1 July 1997.
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