Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032
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ABSTRACT |
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Chronic inflammation is a characteristic feature
of asthma. Multiple inflammatory mediators are released within the
asthmatic lung, some of which may have detrimental effects on signal
transduction pathways in airway smooth muscle. The effects of tumor
necrosis factor (TNF)- on the expression and function of muscarinic
receptors and guanine nucleotide-binding protein (G
protein)
-subunits were examined in human airway smooth muscle
cells. Cultured human airway smooth muscle cells were incubated in
serum-free culture medium for 72 h in the presence and absence of 10 ng/ml of TNF-
, after which the cells were lysed and subjected to
electrophoresis and G
i-2,
Gq
, and
Gs
protein subunits were
detected by immunoblot analysis with specific antisera.
TNF-
treatment for 72 h significantly increased the expression of
G
i-2 and
Gq
proteins and enhanced carbachol (10
7 M)-mediated
inhibition of adenylyl cyclase activity and inositol phosphate
synthesis. These data provide new evidence demonstrating that TNF-
not only increases expression of
G
i-2 and
Gq
proteins but also augments
the associated signal transduction pathways that would facilitate
increased tone of airway smooth muscle.
cytokines; asthma; adenylyl cyclase; inositol phosphate
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INTRODUCTION |
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CHRONIC INFLAMMATION is a characteristic feature of
asthma. There is increasing evidence that proinflammatory cytokines,
which are released from lung macrophages (12), mast cells
(3, 11), eosinophils (6, 10), and epithelial cells (7), play an important role in producing and perpetuating airway inflammation. Several lines of evidence suggest that tumor necrosis factor (TNF)- is important in the pathogenesis of asthma. First, TNF-
is produced locally in the lung, in close proximity to the airway smooth muscle, and both TNF-
and mRNA for TNF-
are found in the bronchoalveolar lavage fluid and bronchial biopsies of asthmatic patients (4). Second,
levels of TNF-
detected in bronchoalveolar lavage fluid are
increased in symptomatic compared with asymptomatic asthmatic patients
(4), and TNF-
mRNA expression increases in allergic inflammation
(30). Third, inhaled TNF-
increases airway responsiveness to
serotonin in rodents (16) and to methacholine in normal volunteers (26), whereas the TNF-
receptor antagonist Ro-45-2081 prevents allergen-induced bronchial hyperresponsiveness in guinea pigs (21).
Evidence is now accumulating that proinflammatory cytokines play an
important role in regulating airway smooth muscle tone. TNF- impairs
-adrenoceptor-mediated airway smooth muscle relaxation in guinea pig
(29) and rabbit tracheae (14) and enhances thrombin- and
bradykinin-induced intracellular
Ca2+ release and inositol
phosphate turnover in cultured human airway smooth muscle cells (2).
A major determinant of airway smooth muscle tone is the concentration
of the second messenger cAMP, which is synthesized by the enzyme
adenylyl cyclase. Emala et al. (8) previously showed that
pretreatment of cultured canine airway smooth muscle cells with TNF-
for 72 h produced a specific impairment in the ability of
-adrenoceptors to stimulate adenylyl cyclase activity, with no
decrease in
-adrenoceptor number. These data provide one explanation for the impaired
-adrenoceptor-mediated relaxation seen in previous studies (14, 29). However, adenylyl cyclase is under dual regulation,
and an upregulation of the inhibitory pathway is an alternative or
additional possibility. TNF-
is known to modulate the expression of
guanine nucleotide-binding (G) protein
Gi, the G protein that inhibits
adenylyl cyclase. Reithmann et al. (20) reported that treatment with
TNF-
for 48 h upregulated Gi
protein in rat cardiomyocytes, whereas Scherzer et al. (24) reported that treatment with TNF-
for 10 min increased the expression of
G
i-2 and
G
i-3 proteins in
polymorphonuclear leukocytes.
Because TNF- alters many signaling pathways in airway smooth muscle
cells, it is unclear whether the functional effects of chronic TNF-
exposure result from G protein
-subunit upregulation. The
correlation between levels of G protein expression and changes in
signaling pathways appears to be cell and pathway specific. A
stoichiometric study (18) of G proteins suggested that an overabundance
of G proteins is available for transmitting cellular signals such that
increases or decreases in G protein expression have no effect on
downstream signaling. In contrast, multiple studies (1, 17, 19, 20, 23)
in a variety of cell types have shown a correlation between changes in
G protein expression and changes in the function of downstream
signaling pathways.
Although it is known that TNF- increases the expression of
Gi
protein in cardiomyocytes
and polymorphonuclear leukocytes and impairs
-adrenoceptor-mediated
relaxation of airway smooth muscle, it is not known whether this
upregulation of the inhibitory pathway of adenylyl cyclase by TNF-
occurs in human airway smooth muscle, whether expression of other G
protein
-subunits are altered by TNF-
, and, if so, whether this
upregulation is functionally important in the regulation of airway
smooth muscle tone.
Therefore, with the use of cultured human airway smooth muscle cells,
the aim of the present study was to evaluate the effects of chronic
TNF- exposure on the expression and function of muscarinic receptors, the G protein
-subunits that couple to these receptors, and the second messengers downstream from these G proteins.
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METHODS |
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Cell culture and TNF- treatment protocols.
Primary cultures of previously characterized (15, 28) human tracheal
smooth muscle cells were a kind gift from Dr. Ian Hall (Queens Medical Center, Nottingham, UK). These cells that express functionally coupled
M2 muscarinic receptors and low
levels of M3 muscarinic receptors
were grown in 75-cm2 cell culture
flasks containing culture medium (M-199 medium, 100 units/ml of penicillin G, 100 µg/ml of streptomycin, 0.25 µg/ml of
amphotericin B, and 10% fetal bovine serum) at 37°C in 5%
CO2-95% air. The cells were
plated on 24-well plates and incubated until they reached confluence.
Subsequently, the confluent cells in 24-well plates were incubated in
serum-free M-199 medium for 72 h [chosen because a previous study
(8) showed effects on adenylyl cyclase function at this time
point] in the presence and absence (control) of 10 ng/ml of human
recombinant TNF-
, with the medium and TNF-
being replaced every
24 h.
Immunoblot analysis. Expression of
Gi-2,
Gq
, and
Gs
proteins was determined by
immunoblot analysis. At the end of the treatment with TNF-
, the
cells were washed three times with serum-free medium, and the cells
from each group were lysed in 100 µl of sample buffer (62.5 mM Tris,
pH 6.8, 2% SDS, 10% glycerol, and 5%
-mercaptoethanol) for
immunoblot analysis. The remaining wells from each of the three groups
were harvested for cell viability quantification with trypan blue
staining. Thirty microliters of each sample were electrophoresed at
room temperature through 10% polyacrylamide gels at 80 V. The
separated proteins were transferred to polyvinylidene difluoride (PVDF)
membranes overnight at a constant voltage of 20 V at room temperature
in a transfer buffer (192 mM glycine, 25 mM Tris, and 10% methanol).
After transfer, the membranes were washed twice in Tris-buffered saline
(TBS; 20 mM Tris, pH 7.5, and 500 mM NaCl) and were incubated in TBS
containing 1% nonfat dry milk at room temperature for 90 min to block
nonspecific protein binding. G protein antisera AS/7, QL, and
RM/1 for G
i-2, Gq
and
Gs
proteins at 1:500, 1:250,
and 1:1,000 dilutions, respectively, in TBS-0.1% Tween 20 (TBS-T) containing 1% nonfat dry milk and 0.2% sodium
azide were added, and the PVDF membranes were incubated with gentle rocking at room temperature for 2-16 h. After
incubation with the G protein antisera, the PVDF membranes were washed
three times for 10 min each with TBS-T. The PVDF membranes were then incubated for 90 min at room temperature in goat anti-rabbit IgG conjugated to alkaline phosphatase at a 1:3,000 dilution in TBS-T containing 1% nonfat dry milk. After incubation with the goat anti-rabbit IgG, the PVDF membranes were washed three times for 10 min
each with TBS-T. The G proteins were detected with enhanced chemiluminescence immunoblotting detection reagents according to the
manufacturer's recommendations (ImmunoLite II, Bio-Rad, Hercules, CA),
with subsequent exposure to autoradiography film. The intensities of
the immunoblots were quantified with a scanner coupled to a personal
computer with MacBas 2.2 software.
To ensure that the band intensities that we measured fell within the
linear range of the autoradiographic film response, a series of control
exposures was performed. Increasing quantities of cellular protein were
subjected to immunoblot analysis as described above
with the primary antibody that recognizes
Gq protein. The relative band
intensities obtained were plotted against the increasing amount of
cellular protein analyzed. This yielded a standard curve of band
intensities, with a linear range against which band intensities from
control and TNF-
-treated cells were compared. All immunoblot band
intensities of G
i-2,
Gq
, or
Gs
protein that were analyzed for TNF-
-induced changes fell within the linear response range of
the film.
To determine whether TNF- altered the growth of cultured human
airway smooth muscle cells and therefore the amount of protein applied
to each lane of the gel, immunoblot measurements were corrected for
cell number. The arbitrary intensities of the immunoblots were log transformed.
Adenylyl cyclase assays. Adenylyl
cyclase activity was measured by the quantification of the synthesis of
[-32P]cAMP
from
[
-32P]ATP.
Assays were performed in 24-well plates. The cells were washed three
times with serum-free medium after TNF-
treatment. The cells were
immediately lysed in 100 µl of lysis buffer (10 mM HEPES, pH 8.0, 2 mM EDTA, and 100 µM phenylmethylsulfonyl fluoride) for 45 min at
37°C. Adenylyl cyclase assays were performed for 20 min at 30°C
in a total volume of 150 µl composed of 100 µl of lysed cells and
50 µl of assay buffer [final concentration: 0.5 mM
3-isobutyl-1-methylxanthine; 50 mM HEPES, pH 8.0; 50 mM NaCl; 0.4 mM
EGTA; 1 mM cAMP; 7 mM MgCl2; 0.1 mM ATP; 7 mM creatine phosphate; 50 units/ml of creatine phosphokinase;
0.1 mg/ml of BSA; 10 µCi/ml of
[
-32P]ATP (specific
activity 800 Ci/mmol); and the indicated effectors]. Preliminary
experiments confirmed the linearity of adenylyl cyclase activity at the
protein concentrations and incubation times used. The reactions were
terminated by the addition of 150 µl of stop buffer [50 mM
HEPES, pH 7.5, 2 mM ATP, 0.5 mM cAMP, 2% SDS, and 1 µCi/ml of
[3H]cAMP
(specific activity 25 Ci/mmol)].
[
-32P]cAMP was
recovered by sequential column chromatography (22). Recovery rates of
columns were 75-90%. To investigate whether increases in G
protein
-subunit expression were reflected in adenylyl cyclase
activity, we examined the inhibitory effect of carbachol
(10
7 M) on forskolin
(10
5 M)-stimulated adenylyl
cyclase activity in control and TNF-
-treated cells. Adenylyl cyclase
activity was corrected for cell number and is expressed as picomoles of
cAMP per milligram of protein per 20 min. Adenylyl cyclase activity in
response to carbachol is expressed as the percentage of inhibition of
forskolin-stimulated adenylyl cyclase activity.
Inositol phosphate assays.
[3H]inositol
phosphate formation was measured with a modification of the method of
Wedegaertner et al. (27). Assays were performed on confluent cells in
24-well plates. The medium was replaced with inositol-free Dulbecco's modified Eagle's medium (DMEM) containing 10 µCi/ml
myo-[3H]inositol
(specific activity 20 Ci/mmol) in the presence and absence of TNF-
on the day before the assay, 48 h after the beginning of TNF-
treatment. Inositol phosphate accumulation was measured in control or
TNF-
-treated cells under basal (unstimulated) and bradykinin-stimulated (0.1 µM) conditions. On the day of assay, the
cells were washed with DMEM containing 10 mM LiCl three times and were
incubated in 270 µl of the same buffer for 15 min at 37°C. Thirty
microliters of agonist or vehicle were then added, and the samples were
incubated for 30 min at 37°C. The reaction was terminated by the
addition of 330 µl of cold methanol. Then 660 µl of chloroform were
added, and the samples were transferred to an Eppendorf
tube. The phases of the samples were separated by centrifugation at 820 g for 10 min at 4°C. Four hundred
fifty microliters of the upper aqueous phase were transferred to a new glass tube. Three hundred microliters of cold 50 mM formic acid and one
hundred microliters of 3% ammonium hydroxide were added. Total
[3H]inositol
phosphates were finally separated from free
myo-[3H]inositol
by chromatography on Dowex AG1-8X columns. The 850-µl samples were loaded onto preequilibrated columns, and then 1 ml of 50 mM NH4OH was added. These 1.85-ml
fractions represented free inositol
([3H]inositol). After
a wash with 5 ml of 40 mM ammonium formate-0.2 M formic acid, the
[3H]inositol phosphate
fraction was eluted with 5 ml of 40 mM ammonium formate-0.2 M formic
acid twice and 2 ml of 4 M ammonium formate-0.1 M formic acid. Eluted
radioactivity was counted after addition of the scintillation cocktail.
Saturation radioligand binding. To
determine whether TNF- treatment for 72 h altered the number of
muscarinic receptors, the total muscarinic-receptor number was
quantitated in human airway smooth muscle cells in the presence and
absence of TNF-
, after which the cells were harvested from
75-cm2 flasks and incubated in a
saturating concentration (1 nM) of [1-3H]quinuclidinyl
benzilate
([3H]QNB; specific
activity 47 Ci/mmol) for 2 h at room temperature in a final volume of
250 µl of binding buffer (12.5 mM NaCl and 125 mM
KH2PO4,
pH 7.4) as previously described (9). Preliminary experiments confirmed
that saturation of specific binding was achieved with 1 nM
[3H]QNB. Nonspecific
binding was determined in the presence of 2 µM atropine. Binding
assays were terminated by filtration over GF/B glass fiber
filters followed by three washes with 5 ml of cold
normal saline (0.9% NaCl). The filters were then counted in 5 ml of
scintillation cocktail. Total muscarinic-receptor numbers were
determined from the number of specifically bound counts. Protein assays
were performed on sample aliquots, and muscarinic-receptor numbers are
expressed as femtomoles of muscarinic receptor per milligram of protein.
Trypan blue staining for cell count.
Because treatment with TNF- for 72 h may affect cell growth, the
immunoblot measurements and adenylyl cyclase activity were corrected
for cell number. Cells receiving either TNF-
or vehicle for 72 h
were washed with serum-free medium three times, after which the cells
were detached with the use of 100 µl of trypsin. Then 50 µl of
M-199 medium containing 10% BSA were added, and the cells were stained
with 50 µl of trypan blue. Five minutes after the cells were stained, an aliquot (5 × 10
4 ml) was counted with a hemocytometer.
Protein determination. Protein content was assayed with the Pierce Chemical (Rockford, IL) bicinchoninic acid protein assay reagent with BSA as a standard (25).
Materials. The primary cultures of
human airway smooth muscle cells used in this study have been
extensively characterized (15, 28) and were a kind gift from Dr. Ian
Hall (Queens Medical Center, Nottingham, UK). G protein antisera (AS/7,
QL, and RM/1), [-32P]ATP
(specific activity 800 Ci/mmol),
[3H]cAMP (specific
activity 25 Ci/mmol), and
myo-[3H]inositol
(specific activity 20 Ci/mmol) were obtained from NEN (Boston, MA).
Goat anti-rabbit IgG was obtained from Bio-Rad (Hercules, CA). PVDF
membranes were obtained from Millipore (Bedford, MA). [3H]QNB (specific
activity 47 Ci/mmol) was obtained from Amersham (Arlington Heights,
IL). Cell culture reagents were obtained from GIBCO BRL (Grand Island,
NY). All other reagents were obtained from Sigma (St. Louis, MO).
Statistics. All data are means ± SE. All data were analyzed by two-tailed paired Student's t-test. The null hypothesis was rejected when P < 0.05.
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RESULTS |
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Immunoblot analysis.
Immunoblot analysis was performed to
investigate the alterations in the expression of G protein -subunits after 72 h of TNF-
treatment (Fig.
1A).
Relative to control cells, TNF-
treatment significantly increased
the expression of G
i-2 protein
by 107% [
log(intensities of immunoblots) = 4.12 ± 0.173 and 4.41 ± 0.154 for control and TNF-
-treated cells,
respectively; P = 0.001;
n = 7 independent experiments]
and Gq
protein by 39% [
log(intensities of immunoblots) = 4.05 ± 0.201 and
4.18 ± 0.218 for control and TNF-
-treated cells, respectively;
P = 0.02;
n = 6 independent experiments; Fig.
1B]. In contrast, the expression of Gs
protein was not
significantly different between control and TNF-
-treated cells
[
log(intensities of immunoblots) = 4.15 ± 0.0475 and 4.18 ± 0.0447 for control and TNF-
-treated
cells, respectively; P = 0.56;
n = 4 independent experiments; Fig.
1B]. Intensities of the
immunoblots were corrected for cell numbers.
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Comparison of immunoblot band intensities is only accurate when
multiple factors relative to electrophoresis conditions and film
exposure are controlled for. All immunoblot comparisons were made with
control and TNF--treated samples that were subjected to analysis on
the same gels to control for potential variation in electrophoresis
conditions. Moreover, a standard curve of band intensities was made to
ensure that all measured band intensities fell within the linear range
of autoradiographic film responsiveness (Fig.
2A).
Increasing quantities of cellular protein generated a linear increase
in band intensity over a limited range (Fig. 2B). All bands analyzed in this
study fell within this linear range. The intensity of the single
lightest and single darkest bands analyzed in this study is indicated
in Fig. 2.
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Adenylyl cyclase assays. To
investigate whether TNF--induced increased expression of
G
i-2 protein altered inhibitory
effects on adenylyl cyclase, the effect of carbachol (which activates muscarinic receptors that couple to
G
i-2 protein, inhibiting adenylyl cyclase) on forskolin
(10
5 M)-stimulated adenylyl
cyclase activity was examined in control and TNF-
-treated cells.
Forskolin (105
M)-stimulated adenylyl cyclase activity was 37 ± 4.7 and 54 ± 8.7 pmol cAMP · mg
protein
1 · 20 min
1 for control and
TNF-
-treated cells, respectively (P = 0.09; n = 5 independent experiments performed in duplicate). In
control cells, carbachol (0.1-1.0 µM) significantly inhibited
forskolin-stimulated adenylyl cyclase activity (Fig.
3). Seventy-two hours of TNF-
pretreatment resulted in significantly greater adenylyl cyclase inhibition by carbachol
(10
7 M; Fig.
4). Inhibition of forskolin-stimulated
adenylyl cyclase activity by
10
7 M carbachol was 1.9 ± 4.8 and 31 ± 9.3% for control and TNF-
-treated cells,
respectively (P = 0.036;
n = 5 independent experiments performed in duplicate).
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Saturation radioligand binding. To
determine whether the enhanced inhibition of forskolin-stimulated
adenylyl cyclase activity by carbachol seen after TNF- treatment was
due to an increase in muscarinic-receptor number, total
muscarinic-receptor numbers were measured by saturation radioligand
binding. TNF-
treatment significantly decreased, not increased, the
muscarinic-receptor number (1,104 ± 236 and 548 ± 230 fmol/mg
protein for control and TNF-
-treated cells, respectively; Fig.
5; P = 0.0001; n = 3 independent experiments
performed in triplicate).
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Inositol phosphate assays. To
investigate whether the increase in the expression of
Gq results in an enhancement of
downstream inositol phosphate synthesis, the accumulation of total
inositol phosphate was examined in control and TNF-
-treated cells.
TNF-
pretreatment significantly increased the synthesis of inositol phosphate under basal conditions. Total inositol phosphate accumulation was 2,835 ± 520 and 4,095 ± 563 dpm for control and
TNF-
-treated cells, respectively (P = 0.012; n = 4 independent experiments performed in duplicate; Fig.
6, left). This represented a
148 ± 10.8% increase in basal inositol phosphate accumulation in
cells treated with TNF-
relative to control cells. TNF-
treatment also resulted in a marked augmentation in inositol phosphate
accumulation in response to 0.1 µM bradykinin (22,919 ± 5,307 and
33,256 ± 7,442 dpm for control and TNF-
-treated cells,
respectively; P = 0.024;
n = 4 independent experiments
performed in duplicate; Fig. 6, right). This represented a
148 ± 7.5% increase in bradykinin-stimulated inositol phosphate
accumulation relative to control cells.
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Cell count. Treatment with TNF- for
72 h resulted in a small decrease in total airway smooth muscle cell
numbers (23,000 ± 4,000/well and 19,000 ± 3,500/well for
control and TNF-
-treated cells, respectively;
P < 0.0001;
n = 12 independent experiments performed in duplicate)
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DISCUSSION |
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In the present study, treatment of cultured human airway smooth muscle
cells with TNF- for 72 h increased the expression of
G
i-2 and Gq
proteins, with no effect on
the expression of Gs
protein.
Moreover, TNF-
treatment enhanced carbachol-induced inhibition of
adenylyl cyclase activity and enhanced basal and bradykinin-induced
levels of inositol phosphate synthesis. These functional effects of
TNF-
were associated with decreased numbers of muscarinic receptors,
suggesting that the TNF-
-induced increase in
G
i-2 and
Gq
subunits resulted in
increased activity of their respective downstream second messenger pathways.
The results of the present study are consistent with those of previous
studies demonstrating increases in the expression of G protein
-subunits by TNF-
in other cell types. TNF-
increased G
i-2 subunit
expression in cultured rat cardiomyocytes (20) and in human
polymorphonuclear leukocytes (24). Although Amrani et al. (2) did not
measure G protein
-subunit expression, they reported enhanced
thrombin- and bradykinin-induced intracellular Ca2+ release and enhanced inositol
phosphate turnover in response to NaF in cultured human airway smooth
muscle cells treated with TNF-
for 24 h. An increase in
Gq
subunit expression, which
was observed in the present study, would be consistent with their findings. The findings of the present study are also consistent with
those of Hakonarson et al. (14), who reported that pretreatment of
rabbit tracheal smooth muscle for 24 h with the proinflammatory cytokine interleukin-1
impaired relaxation and enhanced
G
i-2 and
G
i-3 subunit expression.
Although isoproterenol-stimulated cAMP generation was decreased in the
cytokine-pretreated tissue, Gi
protein-activated inhibition of cAMP was not directly measured.
Adenylyl cyclase is under the dual regulation of a stimulatory
Gs protein and an inhibitory
Gi protein. Increases in the expression of
Gi-2 protein with no change in
Gs
protein would enhance the inhibitory pathway, leading to lower levels of adenylyl cyclase activity. In the present study, enhanced adenylyl cyclase inhibition by
carbachol was seen in TNF-
-treated cells. This enhanced inhibition of forskolin-stimulated adenylyl cyclase activity by TNF-
could not
be accounted for by an increase in muscarinic-receptor number because
we measured a decrease, not an increase, in muscarinic-receptor number
after TNF-
pretreatment. This indicates that the enhanced inhibition
of forskolin-stimulated adenylyl cyclase activity by carbachol in the
TNF-
-treated cells must be due to increased expression of the
G
i-2 subunit, resulting in
enhanced downstream cellular function.
In contrast to our study, TNF- did not decrease the expression of
M2 muscarinic-receptor protein in
HEL 299 cells, although a combination of TNF-
or
interleukin-1
led to downregulation of
M2 muscarinic receptors in these
cells (13). The difference between their report and our study most
likely is due to the cell type and/or species studied. Our
finding of enhanced carbachol inhibition of adenylyl cyclase with
decreased numbers of muscarinic receptors suggests that the expression
of the Gi protein rather than the
expression of the muscarinic receptor plays a more important role in
the ultimate inhibition of adenylyl cyclase. Indeed, a previous study
(23) reported that TNF-
enhanced a bradykinin-mediated increase in
phosphatidylinositol turnover despite a decrease in bradykinin-receptor number.
Stimulation of Gq protein
results in increased inositol phosphate synthesis and intracellular
Ca2+. TNF-
treatment increased
the expression of the Gq
subunit and enhanced basal and bradykinin-induced inositol phosphate
synthesis in the present study, which indicates that an increased level of Gq
protein resulted in
enhanced downstream second messenger function. This is consistent with
a study in human A-431 cells in which TNF-
decreased
bradykinin-receptor numbers yet still resulted in increased inositol
phosphate accumulation in response to bradykinin (23). The increased
basal inositol phosphate accumulation after TNF-
seen in the present
study, which correlates with increased Gq
protein, is also consistent
with increased basal inositol phosphate accumulation seen in a cell
line overexpressing a constitutively active mutant of
Gq
protein (5). Furthermore,
our data are also consistent with a previous study (2) in which TNF-
enhanced inositol phosphate turnover in response to NaF (which
activates G proteins at a site distal to a receptor), suggesting an
upregulation of Gq
protein and
function by TNF-
.
Previous studies (15, 28) in the cultured human airway
smooth muscle cells used in the present study have shown that carbachol produced only small increases in inositol phosphate synthesis because
M3 muscarinic-receptor expression
is downregulated when compared with
M3 muscarinic-receptor expression
in freshly dispersed airway smooth muscle cells or muscle tissue.
Although we did not examine the effect of carbachol on inositol
phosphate synthesis in the present study, it is possible that TNF-
could enhance carbachol-stimulated inositol phosphate synthesis in vivo
by upregulating the expression of
Gq
protein.
Several studies (16, 21, 29) have shown that TNF- contributes to
airway hyperresponsiveness. We demonstrated upregulation of G
i-2 and
Gq
subunit expression, enhanced
carbachol-induced inhibition of adenylyl cyclase activity and enhanced
synthesis of inositol phosphate after TNF-
pretreatment. The
enhanced carbachol-induced inhibition of adenylyl cyclase activity and
inositol phosphate synthesis are consistent with a functional role for
TNF-
in asthma. These results suggest that the upregulation of
G
i-2 and
Gq
subunit expression could be
one of the molecular mechanisms by which TNF-
contributes to airway
hyperresponsiveness. It is also possible, but less likely, that the
increased function of the downstream signaling pathways seen in the
present study was not due to an increase in G protein expression but to
a TNF-
-induced change in an other protein(s) important to the
function of these pathways.
A previous study (8) in cultured canine airway smooth muscle cells
showed that TNF- treatment for 72 h, but not for 24 h, impaired
-adrenoceptor-mediated airway smooth muscle relaxation, which is why
we chose 72 h of treatment for investigating the effect of TNF-
on
the expression of G protein
-subunits. The present study did not
evaluate the time course for TNF-
effects on
Gi
and
Gq
protein expression. Other
investigators have reported that TNF-
increased expression of G
protein
-subunits in human polymorphonuclear leukocytes after 10 min
of exposure (24) and in rat cardiomyocytes after 48 h of exposure (20).
The mechanism of the acute upregulation of
Gi protein is likely to be very
different from that seen in the present study. It is also possible that the upregulation of Gi and
Gq proteins may start earlier than after 72 h of treatment in cultured human airway smooth muscle cells.
Controversy exists over the ability of changes in G protein expression to affect the function of downstream signaling pathways. A study (18) of the stoichiometry of Gs protein signaling suggests that this protein may be present in great excess of receptors with which it couples. In this setting, severalfold changes in Gs protein expression would be expected to have no effect on downstream signaling pathways. In contrast, multiple functional studies (1, 17, 19, 20, 23) in a variety of cell types suggest that changes in G protein amounts correlate with functional changes in signaling pathways. These differences may be due to variable expression of protein components of signaling pathways in different cells or under different conditions.
In summary, treatment with TNF- for 72 h increased the expression of
G
i-2 and
Gq
but not of
Gs
proteins in cultured human airway smooth muscle cells. This increased expression of G protein
-subunits increased the activity of their respective downstream second messenger pathways, which could potentially lead to increases in
airway smooth muscle tone. These findings suggest that increased expression of G
i-2 and
Gq
proteins are additional
mechanisms by which TNF-
could contribute to airway
hyperresponsiveness in asthma.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ian Hall (Queens Medical Center, Nottingham, UK) for kindly providing the primary cultures of human airway smooth muscle cells used in this study.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute Grants HL-58519 and HL-62340 and a Research Grant from the American Lung Association (to C. W. Emala).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: K. Hotta, Dept. of Anesthesiology, College of Physicians & Surgeons of Columbia Univ., 630 West 168th St., PH 5, New York, NY 10032.
Received 13 July 1998; accepted in final form 13 November 1998.
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