Modulation of human airway smooth muscle proliferation by type
3 phosphodiesterase inhibition
Charlotte K.
Billington,
Sunil K.
Joseph,
Caroline
Swan,
Mark G. H.
Scott,
Timothy M.
Jobson, and
Ian P.
Hall
Division of Therapeutics, University Hospital, Nottingham NG7 2UH,
United Kingdom
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ABSTRACT |
Elevation in cell
cAMP content can inhibit mitogenic signaling in cultured human airway
smooth muscle (HASM) cells. We studied the effects of the type
3-selective phosphodiesterase inhibitor siguazodan, the type
4-selective phosphodiesterase inhibitor rolipram, and the nonselective
inhibitor 3-isobutyl-1-methylxanthine (IBMX) on proliferation of
cultured HASM cells. At concentrations selective for the type 3 phosphodiesterase isoform, siguazodan inhibited both
[3H]thymidine
incorporation (IC50 2 µM) and the increase in cell number (10 µM; 64%
reduction) induced by platelet-derived growth factor-BB (20 ng/ml).
These effects were mimicked by IBMX. At concentrations selective for
type 4 phosphodiesterase inhibition, rolipram was without effect. A
20-min exposure to siguazodan and rolipram did not increase whole cell
cAMP levels. However, in HASM cells transfected with a cAMP-responsive
luciferase reporter (p6CRE/Luc), increases in cAMP-driven luciferase
expression were seen with siguazodan (3.9-fold) and IBMX (16.5-fold).
These data suggest that inhibition of the type 3 phosphodiesterase
isoform present in airway smooth muscle results in inhibition of
mitogenic signaling, possibly through an increase in cAMP-driven gene expression.
mitogenesis; adenosine 3',5'-cyclic monophosphate; transfection; luciferase
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INTRODUCTION |
PRIMARY CULTURES of human airway smooth muscle cells
provide a useful system for studying the regulation of mitogenic
responses in human airway smooth muscle (13). These cells have been
shown to respond to a number of mitogens, including both the AB and BB
forms of platelet-derived growth factor (PDGF), thrombin, histamine, mast cell tryptase, endothelin, and epidermal growth factor (3, 11, 15,
21, 23). An increase in airway smooth muscle mass is
observed in the airways of patients with chronic asthma (9) and is
believed to contribute to the irreversible component of airway
obstruction seen in chronic asthma when airway remodeling has occurred.
Hence it is important to gain an understanding of the mechanisms
underlying control of airway smooth muscle cell number.
Although there are extensive data in the literature
regarding the effects of potential mitogens on the proliferative
responses of these cells (3, 11, 15, 21, 23), less is known about the
regulation of mitogenic signaling in airway smooth muscle. A previous
study (29) suggested that agents that elevate cAMP are
able to inhibit mitogenic signaling; for example, salbutamol, isoproterenol, and the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) are able to inhibit tritiated thymidine incorporation in response to mitogens such as PDGF in human
airway smooth muscle cells in culture. In preliminary studies, it has
been demonstrated that this effect is likely to be cAMP mediated
because microinjection of the catalytic subunit of protein kinase A
into individual airway smooth muscle cells inhibited 5-bromo-2'-deoxyuridine incorporation in response to mitogenic stimulation (20).
Breakdown of cAMP within airway smooth muscle cells is dependent on the
activity of phosphodiesterase isoenzymes (2). Airway smooth muscle
cells have been shown to express a number of isoforms of
phosphodiesterase (7, 28, 30). The predominant forms responsible for
the physiological control of cAMP levels in this tissue appear to be
members of the type 3 and type 4 isoform families (30, 31). The aim of
the present study was, therefore, to investigate the ability of
selective inhibitors of these phosphodiesterase isoenzymes to modulate
mitogenic signaling in cultured human airway smooth muscle cells. A
preliminary account of these data has been presented (16).
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MATERIALS AND METHODS |
Culture of human airway smooth muscle cells. Primary
cultures of human airway smooth muscle cells were prepared from
explants of trachealis muscle obtained from individuals without
respiratory disease within 12 h of death as previously described (6).
Hall and Kotlikoff (13) and others (10, 22) have
extensively characterized the phenotype of these cells, which retain
many properties of acutely isolated airway smooth muscle cells. A
segment of trachea was removed from immediately above the carina. A
strip of the trachealis ~2 × 1 cm was then dissected clear from
the surrounding tissue and transported to the laboratory in DMEM
containing penicillin G (200 U/ml), streptomycin (200 µg/l), and
amphotericin B (0.5 µg/l). The tissue was washed several times in 10 ml of DMEM containing antibiotics and antifungal agents at double the above concentrations. The overlying mucosa was dissected free from the
airway smooth muscle under sterile conditions. Small (0.2 × 0.2-cm) explants of the airway muscle were then excised, and ~15
explants were placed in each of several 60-mm Petri dishes. After the
explants adhered, DMEM containing antibiotics, amphotericin B, 10%
fetal calf serum (FCS), and glutamine (2 mM) was added to just cover
the explants. The medium was changed each day for the first 3 days to
reduce the incidence of fungal infection. Smooth muscle cell growth
usually occurred ~7-10 days after the explants were placed in
culture. When growth commenced, the cultures were supplemented with
fresh DMEM containing 10% FCS and 2 mM glutamine approximately every 3 days. When the cells were approaching confluence in some parts of the
vessel, the explants were removed, and 24 h later, the cells were
harvested by trypsinization. Cells from an individual dish or flask
were then plated in one 75-cm2
flask and grown to confluence. When confluent, each flask was split
into four new flasks. Antibiotics and amphotericin B were not added to
the medium used for all subsequent passages after this stage
(passage 2). Cells for
experiments were seeded in 24-well (for cAMP, thymidine incorporation,
and cell counts) or 6-well (for transfection) plates unless otherwise
stated. All primary cell cultures from each donor were examined with
standard immunocytochemical techniques with anti-smooth muscle actin
antibody (1:100 dilution; Sigma) to confirm the presence of smooth
muscle type cells. Primary cell cultures used for the experiments
described in this study showed >95% of cells staining for smooth
muscle actin. Cells from preparations from four individuals were used.
Determination of cAMP responses.
Accumulation of
[3H]cAMP was measured
by a modification of a previously described method (26). In brief,
confluent monolayers of cells plated in 24-well plates were labeled
with [3H]adenine (2 µCi/well) for 2 h in DMEM at 37°C in an incubator constantly
gassed with air-5% CO2. At the
end of this period, the cells were washed three times with 1 ml of
Hanks-HEPES buffer and allowed to rewarm to 37°C for 20 min in the
presence or absence of phosphodiesterase inhibitors. At the end of this
period, the agonists were added for 20 min before the reactions were
terminated by the addition of 50 µl of concentrated HCl. The cells
were then stored at
20°C.
[3H]cAMP was
determined by column chromatography after the cells were rethawed as
previously described (8, 12). Aliquots of [14C]cAMP were added
to each sample, and the counts obtained from this recovery marker were
used to correct for variations in recovery from each column. In
addition, a 100-µl aliquot was taken from each well of the plate
after the reactions were stopped and counted for tritium to correct for
variations in the number of cells per well.
[3H]thymidine incorporation.
Subconfluent cultures (70-90%) of human airway smooth muscle
cells in 24-well plates were washed and then incubated in 1 ml of DMEM
containing 0.1% FCS and 2 mM glutamine for 48 h to growth arrest the
cells. Agonists were then added for 24 h.
[3H]thymidine (1 µCi/well) was added for the final 16 h of the incubation. At the end
of this period, the supernatant was aspirated, and the cells were
washed twice with PBS before being fixed with methanol-glacial acetic
acid (3:1) for at least 1 h at room temperature. Two further washes
with methanol-water (4:1) were performed before the cells were lysed
with 1 ml of 1 M NaOH (adapted from Ref. 5). Nine hundred microliters
of the supernatant were transferred to a scintillation vial along with
10 ml of scintillation fluid (Packard, Meriden, CT) and counted on an
LKB scintillation counter (efficiency ~30%), the results being
expressed as disintegrations per minute or as a multiple
of stimulation over the control value.
Determination of cell number.
Subconfluent cultures (30-40%) of human airway smooth muscle
cells grown in 24-well plates were washed twice in DMEM, then put into
1 ml of DMEM containing 1% FCS for 24 h to induce partial growth
arrest. Phosphodiesterase inhibitors and/or growth factors were
added in 5- or 10-µl aliquots (time
0), and the cells were incubated at 37°C and 5%
CO2 for 48-120 h, after which
the medium was aspirated and the cells were washed twice in PBS. One
milliliter of trypsin-EDTA was added, and the cells were incubated at
37°C for 5-10 min until the cells could be seen to be in
suspension. The cells were harvested by gentle but repeated suspension
with a pipette and transferred to a clear plastic tube containing 10 ml
of Isoton (Coulter Electronics, Luton, UK). Preliminary experiments
demonstrated that human airway smooth muscle cells have a diameter in
suspension of 15-25 µm, and so a lower size limit of 15 µm was
used for Coulter counting; setting a limit below this leads to
inclusion of cell debris in the counts. The number of cells in a
500-µl sample was then established in duplicate for each well with
the Coulter counter. One triplicate of wells was taken from each plate
for cell counting before the addition of agents (i.e., at
time 0); the counts from these wells were taken as baseline, and all results are expressed as a percentage of this control value.
Constructs used for transfection
experiments. Transfections were performed with two
constructs. pGL3 (Promega) was used as a control vector to assess
transfection efficiency; this construct (the pGL3 control vector)
contains the gene for firefly luciferase under the control of an SV40
promoter and enhancer. p6CRE/luc was obtained from S. Rees (Glaxo
Wellcome, Stevenage, UK). This construct contains the gene for firefly
luciferase under the control of the minimal herpes simplex
virus thymidine kinase promoter together with six cAMP
response elements (CREs). In preliminary experiments in cultured human
airway smooth muscle cells, Scott et al. (27) showed that this
construct is responsive to a range of agents known to elevate cAMP
levels. Plasmid for transfection experiments was purified from
large-scale cultures with a column method (Qiagen Megaprep).
Transfection of airway smooth muscle
cells. Human airway smooth muscle cells grown in
six-well plates were transfected at 70-80% confluency with
methods recently standardized by Scott et al. (27) to provide optimal
levels of expression. Immediately before transfection, the medium was
aspirated from the cells and replaced with 5 ml of fresh medium
(containing 10% FCS). DNA and Transfectam reagent (Promega) were
briefly vortexed and then incubated at room temperature for 10 min to
facilitate the binding of DNA to liposomes before being added to the
cells. Four micrograms of DNA and 1.8 µl Transfectam/µg DNA were
used for each well of a six-well plate. DNA was left in contact with
the cells for the whole transfection period unless otherwise stated.
Measurement of luciferase activity.
Luciferase activity was measured in lysates of cultured human airway
smooth muscle cells with a commercially available kit (Promega) as
described in the product information but with minor modifications.
Medium was aspirated from the wells, and the cells were washed twice
with 1 ml of phosphate-buffered saline solution. Three hundred
microliters of lysis buffer were then added to each well, and the cells
were incubated for 10-15 min at room temperature. Any cell debris
together with the lysis buffer was then removed from the individual
wells of each six-well plate. The lysates were spun at 13,000 rpm for
30 s to pellet large cell debris. Twenty microliters of the supernatant
were then assayed for luciferase activity in a Turner luminometer; measurement was made for 30 s after an initial delay of 10 s after addition of the substrate. Protein concentrations in the cell lysate
supernatant were determined with a miniaturized Bradford assay with
96-well plates read in a plate reader. All luciferase activities were
then corrected for protein content to normalize for variation in cell
number and lysis efficiency between experiments.
Materials.
[2,8-3H]adenine (26 µCi/mmol) and
[8-14C]cAMP (42.4 µCi/mmol) were purchased from Amersham (Little Chalfont, UK). The
firefly luciferase vector pGL3 (used as a control), Transfectam reagent, and luciferase assay kits were obtained from Promega UK.
p6CRE/Luc was a gift from S. Rees (Glaxco Wellcome). Siguazodan was a
gift from Dr. T. Torphy (SmithKline Beecham, King of Prussia, PA).
Rolipram was obtained from Calbiochem (Nottingham, UK). All other
chemicals were obtained from Sigma (Poole, UK). Relevant vehicle
controls were included in all experiments involving rolipram and
siguazodan, for which stock solutions were made as follows: 10 mM
rolipram in 10% DMSO and 20 mM siguazodan in 100% DMSO. The
antibodies used for immunocytochemistry were anti-smooth muscle
-actin (Sigma) and mouse IgG whole molecule (host goat;
Sigma). Plasticware was obtained from Costar (High Wycombe, UK).
Data analysis and statistics.
EC50 values for PDGF-BB and
IC50 values for the
phosphodiesterase inhibitors were defined in each individual experiment
and used to calculate mean values. Each data point in individual
experiments was calculated from the mean of triplicate determinations.
Statistical analysis of the data was performed with paired or unpaired
t-tests, Dunnett's test, Tukey's
honestly significant difference test, or analysis of variance as
appropriate. Data were log transformed where appropriate. All values
are means ± SE of n separate experiments.
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RESULTS |
Induction of [3H]thymidine
incorporation by PDGF-BB.
Initial experiments were performed to confirm that PDGF-BB is able to
induce [3H]thymidine
incorporation into cultured human airway smooth muscle cells. Figure
1 shows the concentration-response
relationship for PDGF-BB-induced thymidine incorporation in these
cells. The maximum increase in stimulation was observed with 100 ng/ml
of PDGF-BB, and the response was increased 6.3 ± 1.4-fold compared with the basal value (P < 0.05;
n = 4). The
EC50 for this response was 8.3 ± 1.5 ng/ml (n = 4).

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Fig. 1.
[3H]thymidine
incorporation in cultured human airway smooth muscle cells in response
to a range of concentrations of platelet-derived growth factor
(PDGF)-BB. control, Response in absence of PDGF-BB. Each point is mean ± SE of multiple of increase over basal value from 4 experiments;
in each experiment, response to each concentration was determined in
triplicate.
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Inhibition of PDGF-BB-stimulated
[3H]thymidine incorporation by
phosphodiesterase inhibitors.
We next studied the potential of phosphodiesterase inhibitors to
inhibit PDGF-BB-stimulated thymidine incorporation in cultured human
airway smooth muscle cells. The agents chosen were the type 3 (siguazodan)- and type 4 (rolipram)-selective phosphodiesterase inhibitors together with the nonselective phosphodiesterase inhibitor IBMX, all of which have been shown to be effective relaxant agents in
this tissue and also to inhibit the type 3 and type 4 phosphodiesterase activities present in trachealis homogenates in previous studies (24,
25, 30). Vehicle (DMSO, highest concentration 0.6%) was included at
the same concentration in all relevant incubations, although no
significant effect of vehicle alone was observed on thymidine
incorporation. Siguazodan, rolipram, and IBMX produced a
concentration-related inhibition of PDGF-BB-induced
[3H]thymidine
incorporation (Fig. 2). The maximum
inhibition seen and the apparent
IC50 for these responses are shown
in Table 1. Interestingly, IBMX was only
effective at inhibiting thymidine incorporation at concentrations of
100 µM and above, and the effect observed with IBMX was less than
that with the isoenzyme-selective phosphodiesterase inhibitors. We
attempted to study higher concentrations of IBMX but found that
prolonged incubation with these higher concentrations of IBMX produced
apparent cytotoxicity with marked cell detachment. Rolipram produced
significant inhibition of thymidine incorporation only at
concentrations > 10 µM. The combination of siguazodan and rolipram
was not significantly more effective than the additive effects of
either compound alone (Table 1). In addition, we studied the response
to rolipram and siguazodan in combination with the
2-adrenoceptor
agonist isoproterenol (1 µM; Fig.
3). Isoproterenol alone induced a 63 ± 12% inhibition of the response to PDGF-BB
(P < 0.05; n = 4). Siguazodan (10 µM) in addition to isoproterenol gave a 66 ± 25% reduction in the thymidine response compared with the effect of
isoproterenol alone on the PDGF-BB response
(P < 0.05;
n = 4). Rolipram (10 µM) did not
significantly increase the inhibitory effect of isoproterenol (30 ± 19% reduction; P > 0.05;
n = 4). We also examined the effect of
rolipram and siguazodan (both 10 µM) on the thymidine response to
thrombin (100 U/ml; response 5.2 ± 0.4-fold over basal value; n = 3) to define whether the effects
of siguazodan were mitogen specific. Siguazodan produced a 46 ± 5%
inhibition of the thrombin response (P < 0.05; n = 3). No significant
inhibition was seen with rolipram at this concentration.

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Fig. 2.
Effects of preincubation with a range of concentrations of siguazodan
([siguazodan]; A),
rolipram ([rolipram];
B), and IBMX ([IBMX];
C) on PDGF-BB-induced
[3H]thymidine
incorporation. Values are means ± SE of response induced by 20 ng/ml of PDGF-BB in presence of phosphodiesterase inhibitor as a
percentage of response to 20 ng/ml of PDGF-BB alone from 5-14
separate experiments.
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Fig. 3.
Effects of 2-adrenoceptor
agonist isoproterenol (I; 1 µM), type 3 phosphodiesterase inhibitor
siguazodan (S; 10 µM), and type 4-selective phosphodiesterase
inhibitor rolipram (R; 10 µM) on
[3H]thymidine
incorporation induced by PDGF-BB (P) alone and in combination. con,
Response in absence of PDGF-BB. Data are means ± SE of
response to PDGF-BB (20 ng/ml; n = 4 experiments/group). * Significant inhibition compared with
relevant paired value (see text), P < 0.05.
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Inhibition of PDGF-BB-stimulated cell proliferation by
phosphodiesterase inhibitors. The effect of these
agents on cell number was studied by incubating cells with the relevant
agents and then performing Coulter counting (Fig.
4). Cells were initially placed in 1% FCS
for 24 h before the agents were added, and cell counts were performed
at that stage and 48, 72, or 120 h after the addition. One percent FCS
(rather than 0.1% FCS) was used because with the longer incubation
required to study changes in cell number (compared with thymidine
incorporation), significant cell death was observed with lower levels
of FCS. In the presence of 1% FCS but in the absence of other added
mitogens, the cell number slowly increased (1.8 ± 0.2-fold compared
with the starting cell number; P < 0.05; n = 12). The addition of PDGF-BB
(20 ng/ml) produced an additional 1.8 ± 0.1-fold increase in cell
number (P < 0.05;
n = 12) compared with 1% FCS alone.
In keeping with the data obtained from the [3H]thymidine
incorporation assay, no significant effect of vehicle alone on the
changes in cell number was observed (data not shown). After incubation
for 3-5 days, significant inhibition of PDGF-BB-driven increases
in cell number was seen with siguazodan and IBMX but not with rolipram
(10 µM siguazodan: 48 ± 7% inhibition,
P < 0.05, n = 3; 50 µM IBMX: 81 ± 26% inhibition, P < 0.05, n = 4; 10 µM rolipram: 15 ± 5%
inhibition, not significant, n = 3;
Fig. 4).

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Fig. 4.
Effects of type 3-selective phosphodiesterase inhibitor siguazodan (10 µM) and type 4-selective phosphodiesterase inhibitor rolipram (10 µM) alone and in combination on cell number after 120 h of incubation
with 1% FCS and 20 ng/ml of PDGF-BB. Data are means ± SE
of percent increase in cell number compared with initial count
determined in 4 separate experiments. * Significant inhibition of
PDGF-BB response, P < 0.05.
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Siguazodan and IBMX induce an increase in cAMP-driven
luciferase activity in human airway smooth muscle
cells. To determine the effect of these agents on cell
cAMP content, we used two approaches. First, we measured cAMP levels at
an early time point after the addition of phosphodiesterase inhibitors.
As a positive control, we used isoproterenol, which Hall et al. (14)
previously showed induces cAMP formation via
2-adrenoceptor stimulation in
these cells. The overall change in cAMP levels with phosphodiesterase inhibitors at a single time point (20 min) is small in these cells; as
previously reported (14), we observed very small
responses to rolipram, siguazodan, and IBMX in the
absence of other agonists (Table 2), with
only the response to IBMX being significant. PDGF-BB (20 ng/ml)
produced no significant change in cAMP levels itself. The apparent lack
of effect of phosphodiesterase inhibitors was not due to the inability
of the assay to detect change because the response to isoproterenol (1 µM; 6.6-fold) seen in these experiments was similar to that
previously reported by Hall et al. (14). In addition, we also measured
cAMP with a radioimmunoassay and were again unable to demonstrate
significant cAMP responses to the isoform-selective phosphodiesterase
inhibitors used in this study (data not shown) despite the control
levels of cAMP recorded in these cells with this assay being above the
lower limit of detection of the assay determined with cAMP standards.
One possible explanation for this apparent discrepancy is that it is
the integrated cAMP response over time that is relevant for
antimitogenic signaling. In the proliferation experiments,
phosphodiesterase inhibitors were present throughout the experiment
and prolonged phosphodiesterase inhibition would be expected to have
occurred. Therefore, we studied the ability of these agents to modulate
cAMP over longer time periods by utilizing a luciferase reporter
construct under the control of six CREs (27) to provide an integrated
readout for cAMP changes during the time course of the experiment. The
luciferase response in cell lysates transfected with a range of agents
capable of elevating cell cAMP content is shown in Fig.
5. The response of primary cultures of
human airway smooth muscle cells transfected with this construct and
stimulated with phosphodiesterase inhibitors is shown in Table
3. It can be seen that when measured with
an assay that assesses the integrated change in cAMP levels over a
longer time period, larger effects were observed, which reached significance with siguazodan and IBMX
(P < 0.05;
n = 11). None of these agents appeared
to alter transfection efficiency per se because in a parallel series of
experiments, the level of expression of a control (i.e., nonresponsive
to cAMP) luciferase vector (pGL3) was not altered by exposure to any of
the agents studied (data not shown).

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Fig. 5.
Effects of -adrenoceptor agonist isoproterenol (ISO; 10 µM),
PGE2 (1 µM), and adenylyl
cyclase activator forskolin (FSK; 10 µM) on cAMP-driven increases in
luciferase activity over 24 h in human airway smooth muscle cells
transfected with p6CRE/Luc. Data are means ± SE determined in 6 separate experiments. * Significant increase in luciferase
activity compared with control value,
P < 0.05.
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Table 3.
Effects of PDE inhibitors on cAMP-driven increases in luciferase
activity over 24 h in HASM cells transfected with p6CRE/Luc
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DISCUSSION |
These results demonstrate that nonselective and isoform-selective
phosphodiesterase inhibitors are able to modulate proliferative responses of human airway smooth muscle cells in culture. These cells
provide a useful model for studying proliferative responses to mitogens
likely to be important in airway remodeling (10, 22). We were able to
demonstrate effects by looking at a measure of DNA synthesis (thymidine
incorporation) and also at total cell number, suggesting that the
inhibition of PDGF-BB-driven DNA synthesis directly results in a
reduction in cell number. In general, the effects on DNA synthesis of
the agents studied reflected the effects seen on cell number, although
it is interesting that IBMX appeared to have a greater effect on cell
number than on thymidine incorporation. Cell number is dependent on the
balance between cell proliferation, cell survival, and cell death; in
the present study, we did not examine the ability of phosphodiesterase
inhibitors to modulate cell death, and it is possible that the
quantitative differences observed were due to effects on survival.
However, we had to employ slightly different assay conditions (i.e.,
1% FCS) for the cell number studies because over the longer time
period of these experiments, we found that cells seeded in 0.1% FCS
declined in number, presumably due to low background rates of
apoptosis; this may also account for the quantitative differences seen
in the two sets of experiments. At the concentrations of agents used,
no cytotoxicity was observed, although at very high concentrations of
IBMX (>100 µM), cytotoxicity was seen after prolonged (>24-h)
incubation (data not shown). We also observed qualitatively similar
effects of these agents when thrombin was used to induce DNA synthesis
in these cells and when epidermal growth factor was used to increase
cell number (data not shown), suggesting that the effects of siguazodan
are not due to antagonism of the PDGF-BB receptor.
The effects of the type 3 phosphodiesterase inhibitor siguazodan on
both DNA synthesis and proliferation occurred at concentrations likely
to be selective for this isoform; the
IC50 for siguazodan inhibition of
PDGF-BB-driven increases in thymidine incorporation was 1.8 µM, and
inhibition of PDGF-BB-driven increases in cell number was observed with
10 µM siguazodan (Fig. 4). In contrast, although rolipram produced
inhibition of PDGF-BB-induced
[3H]thymidine
incorporation, this effect was only observed at concentrations unlikely
to be selective for binding to either the high- or low-affinity sites
on the type 4 phosphodiesterase isoform.
In keeping with the data on DNA synthesis, no effect of rolipram was
observed on PDGF-BB-induced increases in cell number at the highest
concentration likely to be selective for the type 4 isoform (Fig. 4).
Hence it seems likely that the type 3 isoform is the more important
target for inhibiting airway smooth muscle proliferation.
At least seven families of phosphodiesterase isoenzymes exist, each
containing multiple isoforms and splice variants (1, 2). Airway smooth
muscle contains a range of phosphodiesterase isoenzymes. In previous
studies, Hall et al. (14) and others (7, 28, 30) have shown that
inhibitors of the type 4 (cAMP-selective) and, to a lesser extent, the
type 3 (cGMP-inhibited, cAMP-selective) phosphodiesterase families
appear to have the greatest effect on cell cAMP content and tissue
tone. Another study (30) has shown that although other
phosphodiesterase isoforms are present in airway smooth muscle, the
type 3 and type 4 isoenzymes are physiologically the most important in
controlling cAMP breakdown in these cells. However, there is
controversy regarding the mechanism of action of phosphodiesterase
inhibitors in airway cells. Given the relatively small change in total
cell cAMP content seen with these agents in the absence of other
activators of adenylyl cyclase, it has been suggested that the
physiological effects of these agents are due to effects other than
those directly related to changes in whole cell cAMP levels. In the
present study, we were unable to demonstrate significant changes in
whole cell cAMP levels assayed at a single time point after the
addition of a type 3 phosphodiesterase inhibitor alone and only
observed a small change after the addition of the nonselective
inhibitor IBMX despite using two different, sensitive assays for cAMP
formation. These data suggest that the physiological effects of these
agents are unlikely to be due to changes in whole cell
cAMP levels at early time points. However, two explanations could
account for this anomaly. The first is that a small change in cAMP
levels that is sustained may be important for the action of these
drugs. The second is that there may be a larger change in cAMP levels
in a cell compartment that will not be detected by whole cell lysate cAMP assays but that may be functionally relevant.
To investigate this apparent disparity between cAMP levels and the
physiological effects of phosphodiesterase inhibitors further, we used
a different approach that provides an integrated readout for changes in
cell cAMP content over longer time periods. This approach involves
transfection of primary cultures of human airway smooth muscle cells
with a reporter construct that contains the gene for firefly luciferase
under the control of six CREs (27). First, we demonstrated that when
transiently expressed in cultured human airway smooth muscle cells,
this construct enables changes in cAMP-driven luciferase expression to
be studied after the elevation of cell cAMP content with a range of
agonists acting through different mechanisms (Fig. 5). By using this
approach, we believed that we should be able to detect effects mediated
through either of the two mechanisms described above. When we assessed
cAMP-driven luciferase expression as an integrated readout for cAMP
elevation in these cells, significant responses to siguazodan and IBMX
were observed. These data suggest that integrated or subcellular
changes in cAMP levels in the absence of a detectable change in whole cell cAMP levels may be important for the effects of cAMP on mitogenic signaling.
It is also clear that cAMP inhibits mitogenic signaling in these cells
when other mechanisms (e.g., direct receptor stimulation) are used to
elevate cAMP levels. For example, inhibition of DNA synthesis has been
observed when the catalytic subunit of protein kinase A was
microinjected directly into airway smooth muscle cells but not when
inactive (boiled) catalytic subunit was microinjected (20). In
addition, inhibition of thymidine incorporation into cultured airway
smooth muscle cells has previously been observed when cell cAMP content
has been elevated by other agents including
2-adrenoceptor agonists such as
salbutamol and isoproterenol and also vasoactive intestinal peptide
(20, 29). We observed similar effects to those previously described
with isoproterenol in the present study and, in addition, were able to
show that the effects of phosphodiesterase inhibitors on thymidine
incorporation were additive to the effects of isoproterenol. There are
still, however, some quantitative differences in the magnitude of
responses observed and the changes in whole cell cAMP seen with
different agonists. Disparity between direct changes in cell cAMP
content and physiological effects has been previously noted in airway smooth muscle (e.g., Ref. 33). One possible explanation might be that
under some conditions, increases in cell cAMP content may actually
enhance proliferative signaling, for example, when
-hexosaminidase
is used as an agonist (17). We attempted to study the role of cAMP in
more detail by using the protein kinase A inhibitor H-89 (4) to reverse
the effects of siguazodan on PDGF-BB-driven thymidine incorporation.
Interestingly, in these experiments, H-89 (100 nM) itself produced a
small inhibition of PDGF-BB-induced thymidine incorporation (7.8 ± 4.2%), which made interpretation of the effects of H-89 on the
response to siguazodan difficult.
Assuming that cAMP is important in the effect of phosphodiesterase
inhibition leaves the question of the site of action of protein kinase
A. This remains to be determined, but phosphorylation of Raf-1 may be
one potential mechanism inhibiting mitogenic signaling driven through,
e.g., MAP kinase pathways. In addition, cAMP may directly alter the
expression of genes important in mitogenic signaling by the activation
of CRE binding proteins binding to CREs in target genes. Our data with
the p6CRE/Luc construct demonstrate that this effect occurs with
inhibition of the type 3 phosphodiesterase isoform present in airway
smooth muscle. Elevation in cell cAMP content has also been observed to
inhibit mitogenic signaling in a range of other myofibroblast cell
types including vascular smooth muscle cells and renal mesangial cells
in primary culture; in both these cell types, inhibition of type 3 and
type 4 phosphodiesterase isoforms was effective in inhibiting
mitogenesis (18, 19, 32).
In conclusion, therefore, this study demonstrates that siguazodan, a
selective inhibitor of the type 3 phosphodiesterase isoenzyme, is able
to inhibit both DNA synthesis and increases in cell number in response
to the mitogen PDGF-BB in cultured human airway smooth muscle cells. In
contrast, the type 4-selective phosphodiesterase inhibitor rolipram
only produced inhibition of DNA synthesis at concentrations of drug
unlikely to be selective for type 4 phosphodiesterase inhibition.
Administration of type 3 phosphodiesterase inhibitors to patients with
airway diseases such as asthma may therefore potentially prevent some
of the airway remodeling that leads, in part, to the irreversible
component of the airflow obstruction observed in some patients with
chronic asthma.
 |
ACKNOWLEDGEMENTS |
This work was funded by the National Asthma Campaign.
 |
FOOTNOTES |
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 reprint requests to I. P. Hall.
Received 6 February 1998; accepted in final form 10 November 1998.
 |
REFERENCES |
1.
Beavo, J. A.
Multiple isozymes of cyclic nucleotide phosphodiesterase.
Adv. Second Messenger Phosphoprotein Res.
22:
1-38,
1988[Medline].
2.
Beavo, J. A.,
and
D. H. Reifsnyder.
Primary sequence of cyclic nucleotide phosphodiesterase isozymes and the design of selective inhibitors.
Trends Pharmacol. Sci.
11:
150-155,
1990[Medline].
3.
Brown, J. K.,
C. A. Jones,
G. H. Caughey,
and
I. P. Hall.
Mast cell tryptase is the dominant mitogenic service protease for human and dog airway smooth muscle cells (Abstract).
Am. J. Respir. Crit. Care Med.
155:
A905,
1997.
4.
Chijiwa, T.,
A. Mishima,
M. Hagiwara,
M. Sano,
K. Hayashi,
T. Inoue,
K. Naito,
T. Toshioka,
and
H. Hidaka.
Inhibition of forskolin-induced neurite outgrowth, and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), in PC12D pheochromocytoma cells.
J. Biol. Chem.
265:
5267-5272,
1989[Abstract/Free Full Text].
5.
Danielpour, D.,
L. L. Dart,
K. C. Flanders,
A. B. Roberts,
and
M. B. Sporn.
Immunodetection and quantitation of the two forms of transforming growth factor (TGF-
1 and TGF-
2) secreted by cells in culture.
J. Cell. Physiol.
138:
79-86,
1989[Medline].
6.
Daykin, K.,
S. Widdop,
and
I. P. Hall.
Control of histamine induced inositol phospholipid hydrolysis in cultured human tracheal smooth muscle cells.
Eur. J. Pharmacol.
246:
135-140,
1993[Medline].
7.
De Boer, J.,
A. J. Philpott,
G. M. Van Amsterdam,
M. Shahid,
J. Zaagsma,
and
C. D. Nicholson.
Human bronchial cyclic nucleotide phosphodiesterase isoenzymes: biochemical and pharmacological analysis using selective inhibitors.
Br. J. Pharmacol.
106:
1028-1034,
1992[Abstract].
8.
Donaldson, J.,
S. J. Hill,
and
A. M. Brown.
Kinetic studies on the mechanism by which histamine H1 receptors potentiate cyclic AMP accumulation in guinea pig cerebral cortical slices.
Mol. Pharmacol.
33:
626-633,
1988[Abstract].
9.
Dunhill, M. S.,
G. R. Masserella,
and
J. A. Anderson.
A comparison of the quantitative anatomy of the bronchi in normal subjects, in status asthmaticus, in chronic bronchitis, and in emphysema.
Thorax
24:
179-190,
1969.
10.
Durand-Arczynska, W.,
N. Marmy,
and
J. Durand.
Caldesmon, calponin and
-smooth muscle actin expression in subcultured smooth muscle cells from human airways.
Histochemistry
100:
465-471,
1993[Medline].
11.
Grunstein, M. M.,
J. P. Noveral,
S. M. Rosenberg,
R. A. Anbar,
and
N. A. Pawlowski.
Role of endothelin-1 in regulating proliferation of cultured rabbit airway smooth muscle cells.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L317-L324,
1992[Abstract/Free Full Text].
12.
Hall, I. P.,
J. Donaldson,
and
S. J. Hill.
Inhibition of histamine stimulated inositol phospholipid hydrolysis by agents which increase cyclic AMP levels in bovine tracheal smooth muscle.
Br. J. Pharmacol.
97:
603-613,
1989[Abstract].
13.
Hall, I. P.,
and
M. Kotlikoff.
Use of cultured airway myocytes for study of airway smooth muscle.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L1-L11,
1995[Abstract/Free Full Text].
14.
Hall, I. P.,
S. Widdop,
P. Townsend,
and
K. Daykin.
Control of cyclic AMP content in primary cultures of human tracheal smooth muscle cells.
Br. J. Pharmacol.
107:
422-428,
1992[Abstract].
15.
Hirst, S. J.,
P. J. Barnes,
and
C. H. C. Twort.
Quantifying proliferation of cultured human and rabbit airway smooth muscle cells in response to serum and platelet-derived growth factor.
Am. J. Respir. Cell Mol. Biol.
7:
574-581,
1992[Medline].
16.
Joseph, S. K.,
T. M. Jobson,
and
I. P. Hall.
The type IV phosphodiesterase inhibitor rolipram inhibits DNA synthesis in primary cultures of human airway smooth muscle cells (Abstract).
Br. J. Pharmacol.
120:
10P,
1997.
17.
Lew, D. B.,
C. Nebigil,
and
K. U. Malik.
Dual regulation by cAMP of
-hexosaminidase-induced mitogenesis in bovine tracheal myocytes.
Am. J. Respir. Cell Mol. Biol.
7:
614-619,
1992[Medline].
18.
Matousovic, K.,
J. P. Grande,
C. S. Chini,
E. N. Chini,
and
T. P. Dousa.
Inhibitors of cyclic nucleotide phosphodiesterase isozymes type III and IV suppress mitogenesis of rat mesangial cells.
J. Clin. Invest.
96:
401-410,
1995[Medline].
19.
Pan, X.,
E. Arauz,
J. J. Krzanowski,
D. F. Fitzpatrick,
and
J. B. Polson.
Synergistic interactions between selective pharmacological inhibitors of phosphodiesterase isozyme families PDE III and PDE IV to attenuate proliferation of rat vascular smooth muscle cells.
Biochem. Pharmacol.
48:
827-835,
1994[Medline].
20.
Panettieri, R. A.,
M. D. Cohen,
and
G. Bilgen.
Airway smooth muscle proliferation is inhibited by microinjection of the catalytic subunit of cAMP dependent protein kinase A (Abstract).
Am. Rev. Respir. Dis.
147:
A252,
1993.
21.
Panettieri, R. A.,
I. P. Hall,
and
R. K. Murray.
Thrombin increases cytosolic calcium and induces human airway smooth muscle cell proliferation.
Am. J. Respir. Cell Mol. Biol.
13:
205-213,
1995[Abstract].
22.
Panettieri, R. A.,
R. K. Murray,
L. R. DePalo,
P. A. Yadvish,
and
M. I. Kotlikoff.
A human airway smooth muscle cell line that retains physiological responsiveness.
Am. J. Physiol.
256 (Cell Physiol. 25):
C329-C335,
1989[Abstract/Free Full Text].
23.
Panettieri, R. A.,
P. A. Yadvish,
A. M. Kelly,
N. A. Rubenstein,
and
M. I. Kotlikoff.
Histamine stimulates proliferation of airway smooth muscle and induces c-fos expression.
Am. J. Physiol.
259 (Lung Cell. Mol. Physiol. 3):
L365-L371,
1990[Abstract/Free Full Text].
24.
Qian, Y.,
E. Naline,
J.-A. Karlsson,
D. Raeburn,
and
C. Advenier.
Effects of rolipram and siguazodan on the human isolated bronchus and their interaction with isoprenaline and sodium nitroprusside.
Br. J. Pharmacol.
109:
774-778,
1993[Abstract].
25.
Rabe, K. F.,
H. Tenor,
G. Dent,
C. Schudt,
S. Liebig,
and
H. Magnussen.
Phosphodiesterase isozymes modulating inherent tone in human airways: identification and characterization.
Am. J. Physiol.
264 (Lung Cell. Mol. Physiol. 8):
L458-L464,
1993[Abstract/Free Full Text].
26.
Ruck, A.,
D. A. Kendall,
and
S. J. Hill.
Alpha and beta adrenergic regulation of cyclic AMP accumulation in cultured rat astrocytes.
Biochem. Pharmacol.
42:
59-69,
1991[Medline].
27.
Scott, M. G. H.,
P. Hill,
S. Rees,
S. Brown,
M. Lee,
and
I. P. Hall.
Control of gene expression by elevation of cell cAMP content in primary cultures of human airway smooth muscle cells (HASM) transfected with a cAMP-responsive reporter construct (Abstract).
Br. J. Pharmacol.
120:
9P,
1997.
28.
Shahid, M.,
R. G. Van Amsterdam,
J. De Boer,
R. E. Tenfberge,
C. D. Nicholson,
and
J. Zaagsma.
The presence of five cyclic nucleotide phosphodiesterase isoenzyme activities in bovine tracheal smooth muscle and the functional effects of selective inhibitors.
Br. J. Pharmacol.
104:
471-477,
1991[Abstract].
29.
Tomlinson, P. R.,
J. W. Wilson,
and
A. G. Stewart.
Salbutamol inhibits the proliferation of human airway smooth muscle cells grown in culture: relationship to elevated cAMP levels.
Biochem. Pharmacol.
49:
1809-1819,
1995[Medline].
30.
Torphy, T. J.,
M. Burman,
L. B. F. Huang,
and
S. S. Tucker.
Inhibition of the low Km cyclic AMP phosphodiesterase in intact canine trachealis by SK&F 94836: mechanical and biochemical responses.
J. Pharmacol. Exp. Ther.
246:
843-850,
1988[Abstract].
31.
Torphy, T. J.,
and
B. J. Undem.
Phosphodiesterase inhibitors: new opportunities for the treatment of asthma.
Thorax
46:
512-523,
1991[Medline].
32.
Tsuboi, Y.,
S. J. Shankland,
J. P. Grande,
H. J. Walker,
R. J. Johnson,
and
T. P. Dousa.
Suppression of mesangial proliferative glomerulonephritis development in rats by inhibitors of cAMP phosphodiesterase isozymes types III and IV.
J. Clin. Invest.
98:
262-270,
1996[Abstract/Free Full Text].
33.
Zhou, H. L.,
S. J. Newsholme,
and
T. J. Torphy.
Agonist-related differences in the relationship between cAMP content and protein kinase activity in canine trachealis.
J. Pharmacol. Exp. Ther.
261:
1260-1267,
1992[Abstract].
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