Vol. 274, Issue 6, L893-L900, June 1998
Pseudomonas pyocyanine alters
calcium signaling in human airway epithelial cells
Gerene M.
Denning1,
Michelle A.
Railsback1,
George T.
Rasmussen1,
Charles D.
Cox2, and
Bradley E.
Britigan1
Departments of 1 Internal
Medicine and 2 Microbiology,
Veterans Affairs Medical Center and The University of Iowa, Iowa
City, Iowa, 52242
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ABSTRACT |
Pseudomonas
aeruginosa, an opportunistic human pathogen, causes
both acute and chronic lung disease. P. aeruginosa exerts many of its pathophysiological
effects by secreting virulence factors, including pyocyanine, a
redox-active compound that increases intracellular oxidant stress.
Because oxidant stress has been shown to affect cytosolic
Ca2+ concentration
([Ca2+]c)
in other cell types, we studied the effect of pyocyanine on [Ca2+]c
in human airway epithelial cells (A549 and HBE). At lower
concentrations, pyocyanine inhibits inositol
1,4,5-trisphosphate formation and [Ca2+]c
increases in response to G protein-coupled receptor agonists. Conversely, at higher concentrations, pyocyanine itself increases [Ca2+]c.
The pyocyanine-dependent
[Ca2+]c
increase appears to be oxidant dependent and to result from increased
inositol trisphosphate and release of
Ca2+ from intracellular stores.
Ca2+ plays a central role in
epithelial cell function, including regulation of ion transport, mucus
secretion, and ciliary beat frequency. By disrupting
Ca2+ homeostasis, pyocyanine could
interfere with these critical functions and contribute to the
pathophysiological effects observed in
Pseudomonas-associated lung
disease.
oxidants; inositol phosphates; A549 cells; HBE cells; G
protein-coupled receptors
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INTRODUCTION |
THE GRAM-NEGATIVE bacterium
Pseudomonas aeruginosa causes acute
lung disease with high mortality in patients with hospital-acquired pneumonias (23) and is commonly associated with the chronic lung
disease observed in individuals with cystic fibrosis (9). Lung disease
is currently the leading cause of morbidity and mortality in cystic
fibrosis (26). During infection, P. aeruginosa secretes numerous virulence factors that
contribute to its pathophysiological effects. Among these factors is
the phenazine derivative pyocyanine (19), a redox-active compound that
increases intracellular oxidant stress. Currently, the mechanisms by
which these factors exert their effects are poorly understood.
Pyocyanine has been shown to have pathophysiological effects in
numerous cell types. Most, if not all, of these effects appear to be
due to increased intracellular oxidant formation and can be blocked by
exogenous addition of antioxidants. In airway epithelial cells,
pyocyanine inhibits ciliary beat frequency (16). This inhibition
correlates with decreased cellular levels of ATP and cAMP.
Our studies were designed to identify other potentially
pathophysiological effects by pyocyanine in human airway epithelial cells. Previous studies demonstrated that pyocyanine increases oxidant
formation in these cells (3, 10). Because oxidant stress has been shown
to affect Ca2+ homeostasis in
other cell types (8, 20, 24), we examined the effect of pyocyanine on
cytosolic Ca2+ concentration
([Ca2+]c)
using two human airway epithelial cell lines, A549 and HBE. We found
that pyocyanine increases
[Ca2+]c
under some conditions while inhibiting subsequent
[Ca2+]c
increases in response to G protein-coupled receptor agonists such as
the purinergic receptor agonist ATP. Additional studies were then
performed to explore the mechanisms that underlie these effects.
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MATERIALS AND METHODS |
Materials.
Buthionine sulfoximine (BSO), glutathione reductase, NADPH, human
placental collagen, N-acetylcysteine
(NAC), ATP, 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), and
carbamylcholine chloride (carbachol) were purchased from Sigma (St.
Louis, MO). Pyocyanine was generously provided by Dr. Cox.
Cell culture.
The human alveolar type II cell line A549 (American Type Culture
Collection CL-185) (18) was cultured in MEM with Earle's salts
(GIBCO BRL, Gaithersburg, MD)
supplemented with 10% fetal calf serum, 2 mM glutamine, and 500 U/ml
each of penicillin and streptomycin. Passages from 85 to 120 were used
in these studies. Stock but not experimental cultures of the human
bronchial epithelial cell line HBE (14) were cultured in
collagen-coated tissue cultureware ( passages
8-25) in the same medium. Experiments were performed on
cultures that were 80-90% confluent.
Spin trapping and electron paramagnetic
resonance.
Electron paramagnetic resonance (EPR) experiments were performed on
airway epithelial cells cultured in 24-well tissue culture plates.
Reaction mixture (0.5 ml/well) consisting of Hanks' balanced salt
solution containing 100 mM 5,5-dimethyl-1-pyrroline
N-oxide (DMPO; OMRF Spin Trap Source,
Oklahoma City, OK) and 100 µM diethylenetriamine pentaacetic acid
(DTPA; Aldrich Chemical, Milwaukee, WI) with or without the indicated
additions was placed on the cell monolayer. Samples were incubated for
15 min at room temperature, and then the reaction mixture was
transferred to a quartz flat cell. All spectra are the result of seven
signal-averaged scans and were obtained at room temperature using a
Bruker model ESP300 EPR spectrometer (Karlsruhe, Germany).
Instrument settings were as follows: microwave power, 20 mW; modulation
frequency, 100 kHz; receiver gain, 4 × 105; modulation amplitude, 0.501 G; time constant, 327.68 ms; sweep rate, 335.5 s.
Measurement of
[Ca2+]c.
Ca2+ measurements were performed
at the Cell Fluorescence Core Facility (Veterans Affairs Medical
Center, Iowa City, IA). Cells were cultured on collagen-coated 25-mm
round glass coverslips. Cells were loaded in complete medium with fura
2 by direct addition of the cell-permeant form, fura 2-AM (Molecular
Probes, Eugene, OR), to the culture dish containing the coverslip and
incubation for 30 min at 37°C. Cells were washed with
HEPES-buffered saline [in mM: 135 NaCl, 5 KOH, 10 HEPES, 1.2 CaCl2, 1.2 MgCl2, and 10 glucose
(HBS-G)], and measurements of the apparent
[Ca2+]c
were done in HBS-G using the Photoscan II spectrofluorometer (Photon
Technologies International, New Brunswick, NJ) with a Nikon microscope
(Nikon, Niles, IL). Final Ca2+
concentration ([Ca2+])
values were determined using a PTI software package from the ratio of
emission intensities [emission wavelength
(
em) 510 nm] for
excitation wavelengths (ex)
of 340 and 380 nm. Briefly, background fluorescence
intensities for each ex were
obtained using unloaded cells and were subtracted from the raw data.
The ratios of the corrected fluorescence intensities were then
converted to [Ca2+]
values using the formula
[Ca2+] = Kd · (R 
Rmin)/(Rmax R), where the maximum and minimum ratios
(Rmax and
Rmin, respectively) as well as the
apparent dissociation constant
(Kd) were
empirically derived from
[Ca2+] curves
generated using the instrument. In earlier work (6), we found that this
method gives similar values for basal
[Ca2+] as well as for
Ca2+ increases in response to
agonists as does the more laborious method that involves determining
Rmax and
Rmin using ionomycin followed by
EGTA (7).
Fluorescence measurements of intracellular oxidant
formation.
To measure intracellular oxidant formation, we used an
oxidant-sensitive dichlorodihydrofluorescein derivative (Iowa probe) generously provided by Dr. Stephen Hempel (Dept. of Internal Medicine, Veterans Affairs Medical Center and the Univ. of Iowa, Iowa City, IA). In cell-free studies (15), the Iowa probe behaved
identically to commercially available dichlorodihydrofluorescein
compounds (Molecular Probes). However, in cell culture studies, the
Iowa probe was significantly more sensitive in detecting oxidant
formation in response to pyocyanine as well as to other redox-active
compounds (S. Hempel, unpublished observations). Although the exact
mechanism by which these compounds detect oxidant formation is not
fully understood, reaction of these probes with
H2O2
appears to require peroxidase activity or iron (25).
For these studies, cells in 12-well tissue culture plates were washed
twice with warm HBS-G and preincubated at 37°C for 30 min in HBS-G
containing 5 µM Iowa probe. At the end of the preincubation period,
the indicated concentration of pyocyanine was added, and the cells were
incubated for 1 h at 37°C. To measure cell-associated fluorescence,
the cells were washed twice with ice-cold PBS and incubated on ice with
PBS containing 0.2% Triton X-100. The cell extract was removed from
the cells, and the relative fluorescence intensity of the extract
(ex, 485 nm;
em, 512 nm) was determined using a Gilford Fluoro IV spectrofluorometer (Ciba-Corning Diagnostics, Park Ridge, IL).
Altering cellular antioxidant capacity.
To reduce glutathione levels, cells were incubated with 100 µM BSO in
complete medium for 48 h. To measure glutathione, cells were washed
twice with ice-cold PBS, scraped into 0.01 N HCl, frozen overnight, and
thawed. Samples were centrifuged at 12,000 rpm for 5 min to remove
cellular debris, and total glutathione in the cell extract was
determined by assaying the rate of DTNB reduction in the presence of
NADPH and glutathione reductase (11). Oxidized glutathione
was measured in cell extracts treated for 1 h with 2-vinylpyridine
(Aldrich Chemical). Reduced glutathione was determined by subtracting
oxidized glutathione from total. Values were generated by comparison
with a reduced glutathione standard curve and were normalized to total
cell protein, measured using the micro bicinchoninic acid assay
(Pierce, Rockford, IL). For studies with NAC, cells were pretreated
with the indicated concentration of NAC for 2-4 h before
experiments were performed. NAC at these concentrations markedly
acidifies the medium. Thus all NAC-containing solutions were adjusted
to pH 7.3-7.5 before use.
Turnover of inositol phosphates.
Cells were seeded into six-well tissue culture plates, allowed to
attach overnight, and then cultured for 48 h in complete medium
containing 1 µCi/ml of
myo-[3H]inositol
(Amersham, Arlington Heights, IL). At the end of the labeling period,
turnover of inositol phosphates (IPs) was measured as
previously described (7). Briefly, cells were washed with HBS-G and
incubated in HBS-G for 20 min at 37°C. Cultures were then incubated
for 20 min with HBS-G containing 10 mM LiCl. Finally, cells were
stimulated with the indicated agonist for 20 min or with pyocyanine for
10 min and then agonist for 10 min. IPs were extracted overnight at
4°C with 0.5 M perchloric acid. The acid extract was neutralized
with 2.5 M KOH and 0.5 M HEPES (pH 7.4) and centrifuged to remove the
precipitate. The IP species were then collected using
anion-exchange column chromatography (Dowex AG, 1-8X, 100-200
mesh, formate form; Bio-Rad, Hercules, CA) as previously described
(22).
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RESULTS AND DISCUSSION |
Pyocyanine increases oxidant formation in A549 and
HBE cells.
Previous work in our laboratories (3) and by Gardner (10) using two
different assay techniques demonstrates that pyocyanine stimulates
oxidant formation in A549 cells. To verify these results in our A549
cultures and to determine whether pyocyanine increases superoxide anion
(· O
2) formation in HBE
cells, we used the spin-trapping agent DMPO and EPR spectroscopy.
Figure 1 shows pyocyanine-dependent
formation of the DMPO-· OH adduct (aN = aH = 14.9 G, where
aN and aH are the
splitting constants for nitrogen and hydrogen, respectively) in HBE
(Fig. 1A) and A549 (Fig.
1B) cells. Formation of
this adduct could result from a reaction between DMPO and either
· O2 or the hydroxyl radical
(· OH). Addition of superoxide dismutase markedly attenuates
or abolishes the signal (Fig. 1, C and
D), demonstrating that
· O2 is required for adduct formation. In addition, catalase has little or no effect (Fig. 1,
E and
F) on the signal. This result argues
against · OH formation, since its formation requires
H2O2
and hence is inhibited by catalase. Consistent with this conclusion is
our observation that the · OH scavenger DMSO, which reacts
with · OH to form the methyl radical (· CH3) and
subsequently DMPO-· CH3,
did not alter the response to pyocyanine (data not shown). Together,
these results indicate that the spectra shown in Fig. 1 reflect
pyocyanine-induced · O2
formation.
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Fig. 1.
Representative electron paramagnetic resonance (EPR) spectra from HBE
and A549 cells exposed to pyocyanine. Cells were exposed for 15 min at
room temperature to 80 µM pyocyanine alone
(A and
B), pyocyanine in presence of 3,000 U/ml of superoxide dismutase (C and
D), or pyocyanine in presence of
5,000 U/ml of catalase (E and
F). EPR spectra were subsequently
obtained as described in MATERIALS AND
METHODS using the 5,5-dimethyl-1-pyrroline
N-oxide (DMPO) spin-trapping system.
Spectra shown represent DMPO-· OH spin adduct. Similar
responses were observed in 2 other independent
experiments.
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Work by our laboratories (3) and others (13) suggests
that pyocyanine induces · O2
formation by accepting an electron from cellular NAD(P)H to form the
pyocyanine radical, which in turn reduces
O2 to form
· O2. This mechanism
suggests that pyocyanine will redox cycle, producing · O2 until these reducing
sources are consumed or until the pyocyanine is metabolized.
Pyocyanine increases
[Ca2+]c.
Previous studies (8, 24) show that oxidants increase
[Ca2+]c
in a variety of cell types. In early studies, we found that pyocyanine
causes an acute, transient increase in
[Ca2+]c.
A representative example of this response in A549 cells is shown in
Fig.
2A.
In these studies, the apparent basal
[Ca2+]c
values were ~100-150 nM for both A549 and HBE cells and were similar from experiment to experiment. In contrast, the magnitude of
the maximal
[Ca2+]c
increase in response to pyocyanine, although similar within an
experiment, varied considerably from experiment to experiment, ranging
from 50 to over 300 nM. Moreover, concentration-dependence studies
indicate that a threshold concentration of pyocyanine is required to
elicit the response. This threshold concentration also varied
considerably from experiment to experiment (ranging from 80 to 250 µM). Responses to other Ca2+
agonists in these same studies did not exhibit the same degree of
variability, suggesting that this is a specific characteristic of the
pyocyanine response. The basis for this variability remains unclear but
did not appear to depend on the preparation of pyocyanine used or on
the confluency state of the cells. We speculate that the variability
may reflect differences in uptake of pyocyanine, via a mechanism
currently unknown, or differences in the level of reducing sources
(NADPH, NADH). Further experiments will be necessary to test these and
other possibilities.
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Fig. 2.
Pyocyanine-dependent increases in cytosolic
Ca2+ concentration
([Ca2+]c).
A549 cells were loaded with fura 2 and stimulated with 300 µM
pyocyanine in presence (+; A) or
absence (; B) of
extracellular Ca2+.
C: cells were treated for 40 min with
2 µM thapsigargin (TSG) before loading with fura 2 and stimulation
with pyocyanine (added at 60 s).
Ca2+ measurements were performed
as described in MATERIALS AND METHODS.
Similar responses were observed in 2 other independent experiments.
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As a first step in determining the mechanism by which pyocyanine
increases
[Ca2+]c,
we designed experiments to identify the source of the
[Ca2+]c
increase. As shown in Fig. 2B, the
response to pyocyanine persists in the absence of extracellular
Ca2+. This suggests that
pyocyanine stimulates release of
Ca2+ from intracellular
Ca2+ stores. Consistent with this
conclusion, we found that depletion of these stores by treatment with
the endoplasmic reticulum
Ca2+-ATPase inhibitor thapsigargin
(TSG) abolishes the response to pyocyanine (Fig.
2C). Furthermore, the more transient
nature of the
[Ca2+]c
increase in the absence of extracellular
Ca2+ (Fig. 2, compare
A with
B) suggests that influx also
contributes to the pyocyanine-dependent increase in
[Ca2+]c.
However, the lack of a
[Ca2+]c
increase after TSG treatment (see Fig.
3C) suggests that this influx is
dependent on the stimulated release from stores. Similar results were
obtained with HBE cells (data not shown). These data indicate that
pyocyanine increases
[Ca2+]c
by stimulating release of Ca2+
from TSG-sensitive stores and that this release triggers
Ca2+ influx.
Pyocyanine stimulates IP turnover.
Release of Ca2+ from intracellular
Ca2+ stores can result from
formation of the second messenger inositol 1,4,5-trisphosphate (IP3). To determine whether
pyocyanine increases
[Ca2+]c
by increasing IP3, we used an
assay that measures turnover of IPs. This assay has been shown to be
the most sensitive measure of increased
IP3. Moreover, in previous work
(17), we found that results using this method accurately reflected
increased IP3 formation.
Using this approach, we found that pyocyanine increases IP turnover in
a concentration-dependent manner in both A549 and HBE cells. In three
independent experiments, the maximum increase observed in response to
pyocyanine ranged from 116 ± 6 to 169 ± 11% of control value
(means ± SD for triplicate samples). As a positive control in these
studies, we used the purinergic receptor agonist ATP. We observed that
1 mM ATP stimulated IP turnover in the range of 141 ± 10 to
206 ± 18% of control value in these experiments.
Moreover, as with our
[Ca2+]c
measurements, we observed that the minimum pyocyanine concentration required to stimulate IP turnover varied markedly. As stated above, the
basis for this variability remains unknown.
Cellular antioxidant capacity alters the response
to pyocyanine.
To determine whether oxidants contribute to the pyocyanine-dependent
increase in
[Ca2+]c,
we decreased the antioxidant capacity of the cells by decreasing intracellular levels of the thiol antioxidant glutathione. To do this,
cells were treated for 48 h with 100 µM BSO, an inhibitor of
-glutamylcysteine synthetase (21). Total glutathione
levels in control cells were 79 ± 7 and 42 ± 6 nmol/mg protein
(means ± SE; combined data from 6 independent experiments with
triplicate samples for each cell type) for A549 and HBE cells,
respectively. In each case, the majority (84-90%) of the
glutathione was in the reduced form. A 48-h treatment with BSO
decreased total glutathione levels by 80-98% (A549, 2.2 ± 0.44 ng/mg protein; HBE, 9.5 ± 3.0 ng/mg protein; means ± SE; combined data from two independent experiments with
triplicate samples for each cell type).
First, to determine whether BSO treatment affects pyocyanine-dependent
oxidant formation, we used a cell-permeable, oxidant-sensitive fluorescent probe. For these experiments, cells were pretreated for 48 h with or without BSO, washed with buffer, preincubated for 30 min with
5 µM probe, and then stimulated for 1 h with the indicated
concentration of pyocyanine. Cultures were then rapidly washed with
ice-cold PBS and permeabilized with 0.2% Triton X-100. Finally, the
relative fluorescence of the cell extract was determined. Figure
3 shows representative results from studies
with A549 cells. Similar results were seen with HBE cells (data not
shown). Under these conditions, pyocyanine increases cell-associated
probe fluorescence in a concentration-dependent manner (Fig.
3A, solid line) and BSO enhances
pyocyanine-dependent oxidation of the probe (Fig. 3A, dotted line).
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Fig. 3.
Effect of reducing glutathione on response to pyocyanine. A549 cells
were treated for 48 h with (dotted line) or without (solid line) 100 µM buthionine sulfoximine (BSO). A:
pyocyanine-dependent oxidant formation was then measured using an
oxidant-sensitive fluorescent probe as described in
MATERIALS AND METHODS. Values are
means ± SD of triplicate samples from a representative experiment.
Similar results were obtained in 2 other independent experiments.
B: pyocyanine-dependent changes in
[Ca2+]c
were measured in fura 2-loaded cells. Values are means of duplicate
samples from a representative experiment. A similar shift in
sensitivity after BSO treatment was observed for selected pyocyanine
concentrations in 2 other independent experiments. [Pyocyanine],
pyocyanine concentration.
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We then measured pyocyanine-dependent
[Ca2+]c
changes in control and BSO-treated cells (Fig.
3B). To do this, cells with or without BSO treatment were loaded with fura 2 and then individually placed in the spectrofluorometer. A baseline ratio was collected for
1-2 min. This ratio represents the apparent basal
[Ca2+]c
for the sample. Pyocyanine at the indicated concentration was then
added to the sample, and data were collected over the next 10 min. We
found that this time period was sufficient to observe maximum changes.
The peak ratio of the response was converted to
[Ca2+]c
and used to determine the increase over baseline. Consistent with a
role for oxidants in these changes, BSO treatment shifted the
pyocyanine dose-response curve to the left. The kinetics of Ca2+ release in response to
pyocyanine was not altered in BSO-treated cells. These data suggest
that glutathione plays a role in removal of pyocyanine-generated
oxidants and that oxidant formation contributes to the increase in
[Ca2+]c
in response to pyocyanine.
To assess further whether pyocyanine increases
[Ca2+]c
by increasing oxidant formation, we tested the effect of adding the
thiol antioxidant NAC. In these experiments, cells were preincubated for 2 h with 30 mM NAC. When compared with controls (A549, 92 ± 10 ng/mg protein; HBE, 36 ± 8 ng/mg protein; means ± SE), NAC had
no appreciable effect or slightly increased total glutathione levels
(A549, 110 ± 37 nmol/mg protein; HBE, 26 ± 5 nmol/mg protein; means ± SE). These values represent combined data from
two independent experiments with triplicate samples for each cell type.
As illustrated in Fig. 4 for HBE cells, NAC
inhibits the pyocyanine-dependent oxidation of the fluorescent probe
(Fig. 4A, dotted line). Control studies indicate that this inhibition is not due to NAC-dependent quenching of probe fluorescence. NAC also inhibits the
pyocyanine-dependent increase in
[Ca2+]c
(Fig. 4B). Similar results were
obtained with A549 cells (data not shown). These data provide further
evidence that oxidants mediate the pyocyanine-dependent
[Ca2+]c
increase.
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Fig. 4.
Effect of adding thiol antioxidant
N-acetylcysteine (NAC) on response to
pyocyanine. HBE cells were treated for 2 h with or without 30 mM NAC.
Measurements were then performed on control and NAC-treated cells to
determine oxidant formation (A;
fluorescent probe) and
[Ca2+]c
changes (B; fura 2) in response to
pyocyanine. Values are means ± SD of triplicate samples from a
representative experiment. Similar results were seen in a separate
independent experiment.
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Pyocyanine inhibits the response to
Ca2+ agonists.
Because pyocyanine itself increases
[Ca2+]c,
we wondered whether pyocyanine would affect the subsequent response to
Ca2+ agonists. The purinergic
receptor agonist ATP was used for these experiments, since it increases
[Ca2+]c
in both A549 (Fig.
5A) and
HBE (Fig. 5B) cells.
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Fig. 5.
Effect of pyocyanine on
[Ca2+]c
increase in response to Ca2+
agonists. Representative tracings from 2 to 4 independent experiments
for each cell type show an increase in
[Ca2+]c
in response to 1 mM ATP in HBE (A),
A549 (B), and HT-29
(C) cells.
D:
[Ca2+]c
increase in HT-29 cells in response to 100 µM carbachol.
E-H: corresponding tracings of
the same cell type and agonist first exposed to pyocyanine
(100-300 µM; first arrow) and then to agonist (second arrow).
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We found that pyocyanine inhibits the
[Ca2+]c
increases in response to ATP in both A549 and HBE cells (Fig. 5,
E and
F, respectively; second arrow).
Interestingly, this inhibition was often observed under conditions in
which pyocyanine itself had little or no effect on
[Ca2+]c
(Fig. 5, E and
F; first arrow). This latter
observation suggests that the inhibition is not simply due to depletion
of Ca2+ from hormone-sensitive
stores, a conclusion supported by our observation that pyocyanine does
not appreciably inhibit a subsequent [Ca2+]c
increase in response to TSG or ionomycin (data not shown). In addition,
these data suggest that the effect of pyocyanine on agonist-dependent
increases in
[Ca2+]c
is independent of the direct effect of pyocyanine on
[Ca2+]c
levels. NAC pretreatment prevents pyocyanine from inhibiting the
response to ATP (data not shown), suggesting that this pyocyanine effect is also mediated by oxidant formation.
The inhibitory effect by pyocyanine is not restricted to ATP or to
airway epithelial cells. Figure 5 also demonstrates that both ATP (Fig.
5C) and the muscarinic receptor
agonist carbachol (Fig. 5D) increase
[Ca2+]c
in the human intestinal epithelial cell line HT-29. Pyocyanine (Fig. 5,
G and
H; first arrow) inhibits the response
to both agonists (Fig. 5, G and
H; second arrow) in these cells. These
results suggest that pyocyanine inhibits signaling in response to
receptors that are coupled via G proteins to phosphatidylinositol
4,5-bisphosphate (PIP2)-specific
phospholipase C (PIP2-PLC).
Conversely, pyocyanine does not inhibit cytokine receptor-mediated
signaling, since we found that pyocyanine has no effect on
cytokine-dependent increases in polymeric IgA-receptor expression in
HT-29 cells (Denning, unpublished data) and synergizes with cytokines
in stimulating release of interleukin-8 by airway epithelial cells
(Denning, unpublished observations).
Pyocyanine inhibits IP turnover in response to
ATP.
To determine whether pyocyanine inhibits the
[Ca2+]c
increase in response to ATP by inhibiting agonist-dependent
IP3 formation, we measured the
effect of pyocyanine on ATP-stimulated turnover of IPs. In these
experiments, cells were labeled with
myo-[3H]inositol
for 48 h, washed, and incubated for 20 min in buffer with 10 mM LiCl.
The cells were then incubated for 10 min with or without pyocyanine and
finally for 10 min with or without 1 mM ATP. We found that pyocyanine
inhibits IP turnover in response to ATP. Moreover, inhibition of the
response to ATP can occur at pyocyanine concentrations that, within a
given experiment, do not by themselves stimulate an increase in IP
turnover. As an example of these results, we obtained values of 100 ± 11, 87 ± 2, 160 ± 27, and 86 ± 4% (means ± SD
for triplicate samples from a representative experiment) for control,
pyocyanine alone (200 µM), ATP alone, and pyocyanine followed by ATP,
respectively. Similar results were seen in two other independent
experiments. In studies in which we observed a pyocyanine-dependent
increase in IP turnover, no additional increase was observed with
subsequent exposure to ATP. These data further suggest that the effect
of pyocyanine on IP3 formation in
response to ATP is independent of its effect on
IP3 formation itself.
Activation of protein kinase C (PKC) has been shown to inhibit
signaling by G protein-coupled receptors linked to
PIP2-PLC (4). In addition, oxidant
stress has been shown to activate PKC (27). Thus, to determine whether
pyocyanine inhibits IP3 formation
in response to ATP by activating PKC, we tested the effect of the PKC
inhibitors staurosporine and bisindolylmaleimide. Results from these
experiments illustrate several points (Fig. 6). First, ATP stimulates IP turnover, and
inhibitor treatment has little or no effect on this response. Second,
activation of PKC by the phorbol ester phorbol 12-myristate 13-acetate
(PMA) inhibits the ATP response, and this inhibition is prevented by pretreating the cell with either inhibitor. Finally, pyocyanine inhibits IP turnover in response to ATP, but, in contrast to PMA, PKC
inhibitors do not block this effect. These data suggest that pyocyanine
does not exert its inhibitory effect by activating PMA-sensitive
isoforms of PKC. We cannot rule out the possibility, however, that the
ATP response is inhibited by pyocyanine through activation of
PMA-insensitive isoforms of PKC that are not inhibited by these
concentrations of PKC inhibitors.
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Fig. 6.
Studies with protein kinase C (PKC) inhibitors. A549 cells were
radiolabeled for 48 h with
myo-[3H]inositol
and then pretreated for 1 h with or without 300 nM PKC inhibitors
staurosporine (Staur) or bisindolylmaleimide (Bis). Inositol phosphate
(IP) turnover was then measured in control cells (data not shown), in
cells stimulated with 1 mM ATP, in cells pretreated for 10 min with 100 nM phorbol 12-myristate 13-acetate (PMA) before ATP stimulation, or in
cells pretreated for 10 min with 200 µM pyocyanine (Pyo) and then
stimulated with ATP. Values are means ± SD of triplicate samples.
Similar results were obtained in a separate independent experiment.
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Conclusions.
These studies are the first to examine the effect of
Pseudomonas pyocyanine on
[Ca2+]c
in human airway epithelial cells. We found that pyocyanine has two such
effects. Pyocyanine alone at higher concentrations increases
[Ca2+]c,
presumably by increasing IP3,
which stimulates Ca2+ release from
intracellular Ca2+ stores and a
subsequent influx of extracellular
Ca2+. Conversely, pyocyanine at
lower concentrations inhibits IP3 formation and the subsequent increase in
[Ca2+]c
in response to Ca2+ agonists.
Because of differences in the concentration dependence of these two
effects and because they reflect opposite effects on IP metabolism, it
follows that the two effects are independent and therefore must involve
different molecular mechanisms. However, both effects appear to share
the common feature of being mediated, at least in part, by
pyocyanine-generated oxidants.
These effects occur at concentrations ranging from 80 to 250 µM.
Pyocyanine has been detected at concentrations as high as 75-100
µM in sputum (28) and bronchoalveolar lavage fluid from patients with
Pseudomonas infections. With
consideration that dilution occurs as a result of lavage, it is
reasonable to assume that higher concentrations of pyocyanine are
present at the site of infection. Thus the concentrations used in our
studies are likely to be physiologically relevant.
The molecular mechanisms by which pyocyanine increases
IP3 and
[Ca2+]c
remain to be identified. Previous studies suggest several
possibilities. One of these involves oxidant-dependent activation of
protein tyrosine kinases (PTKs) with subsequent tyrosine
phosphorylation and activation of PLC-. As an example, oxidants
increase IP3 and
[Ca2+]c
in HL-60 cells by stimulating tyrosine phosphorylation and activation
of PLC-2 (2). Currently, however, two pieces of data argue against
this occurring in our system. First, the kinetics of the initial
[Ca2+]c
rise is slower (minutes) when tyrosine phosphorylation is involved, whereas the response to pyocyanine is rapid (seconds). Second, preliminary results indicate that the pyocyanine-dependent increase in
[Ca2+]c
is not inhibited by 300 µM PTK inhibitor genistein or by
concentrations of staurosporine (300 nM) that inhibit PTKs (5) (data
not shown). In parallel studies, these inhibitors prevented the
pyocyanine-dependent increase in interleukin-8 release, demonstrating
that pyocyanine does activate PTKs in these cells and that these
inhibitors are effective under our experimental conditions. Although
our data suggest that pyocyanine does not increase
[Ca2+]c
by stimulating tyrosine phosphorylation and activation of
PIP2-PLC, further studies will be
necessary to rigorously rule out this possibility.
Alternatively, pyocyanine could activate an isoform of
PIP2-PLC by a mechanism other than
tyrosine phosphorylation, or it could have a direct effect on IP
metabolism. Moreover, although it seems likely that the
[Ca2+]c
increase results from increased
IP3, on the basis of its
characteristic rapid onset, oxidants have been shown to have other
effects that may be relevant in this case. For example, oxidant stress
has been shown to inhibit the
Ca2+-ATPases in both endothelial
(12) and skeletal muscle (1) cells. If pyocyanine-induced oxidants
inhibit the endoplasmic reticulum
Ca2+-ATPase, then this might
contribute to the pyocyanine-dependent release of
Ca2+ from intracellular stores.
The molecular mechanism by which pyocyanine inhibits
IP3 formation and
[Ca2+]c
increases in response to hormonal agonists is also currently unknown.
We initially speculated that pyocyanine inhibits the response to ATP by
activating PKC, as is often illustrated by pretreating cells with PMA.
Our studies with PKC inhibitors suggest that if an isoform of PKC is
involved, it is likely to be PMA insensitive as well as relatively
insensitive to both staurosporine and bisindolylmaleimide. In addition
to being inhibited by PKC, however, signaling by G protein-coupled
receptors is inhibited by activation of receptor-associated kinases
(4). Of note with respect to our studies, these kinases are not
inhibited by inhibitors of PKC. It will be of interest to determine
whether these kinases are activated by pyocyanine and whether
activation of these kinases is responsible for the observed inhibition.
An alternative hypothesis is suggested by studies in human
neuroblastoma SH-SY5Y cells exposed to
H2O2
(17). In these cells, H2O2
inhibits IP3 formation in response
to carbachol. The authors provide evidence that PLC and
phosphoinositide metabolism are not inhibited but rather that oxidant
stress inhibits activation of the G proteins that couple to
PIP2-PLC. These results suggest that pyocyanine may inhibit G protein coupling to receptors through an
oxidant-dependent mechanism. If this is the case, however, then
pyocyanine-dependent increases in IPs must occur distal to or
independent of G protein-mediated events. Further studies will be
required to test these hypotheses.
Our work demonstrates that pyocyanine alters
Ca2+ homeostasis in human airway
epithelial cells, including inhibition of the response to
Ca2+ agonists. These agonists
regulate important epithelial cell functions including ion transport,
mucus secretion, and ciliary beat frequency. These functions, in turn,
influence mucociliary clearance. By altering
Ca2+ homeostasis in these cells,
pyocyanine could interfere with critical host defense mechanisms and
thereby contribute to the pathophysiological effects observed in
Pseudomonas-associated lung disease.
Antibiotic therapy has shown limited success in treating
Pseudomonas infections. Understanding
the mechanisms by which Pseudomonas virulence factors such as pyocyanine exert their effects may provide insight that will lead to more effective therapeutic approaches that
bypass antibiotic resistance mechanisms and that specifically target
this microorganism.
| |
ACKNOWLEDGEMENTS |
This work was supported in part by Veterans Affairs Merit Review
Grants (awarded to G. M. Denning and B. E. Britigan by the Office of
Research and Development, Medical Research Service, Department of
Veterans Affairs) as well as by National Institutes of Health Grant
AI-34954 and the Cystic Fibrosis Foundation.
| |
FOOTNOTES |
This work was performed during the tenure of B. E. Britigan as an
Established Investigator of the American Heart Association.
Address for reprint requests: G. M. Denning, Bldg. 3, Rm. 139, Veterans
Affairs Medical Center, Iowa City, IA 52246.
Received 2 June 1997; accepted in final form 2 February 1998.
| |
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