1 Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032; and 2 Department of Environmental Health Sciences, Division of Physiology, Johns Hopkins School of Public Health, Baltimore, Maryland 21205
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ABSTRACT |
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Cellular levels of cAMP are
an important determinant of airway smooth muscle tone. We have
previously shown that chronic (18 h) but not acute (30 min or 2 h)
pretreatment with the muscarinic receptor agonist carbachol resulted in
decreased adenylyl cyclase activity in response to GTP, isoproterenol,
or forskolin via a pathway blocked by the protein kinase C inhibitor
staurosporine. The present study was designed to determine if
carbachol-induced decreases in adenylyl cyclase activity were due to
regulatory events at the level of either Gs or adenylyl
cyclase. Detergent-solubilized Gs
from control or
carbachol-pretreated bovine airway smooth muscle had similar adenylyl
cyclase activity in response to either NaF or guanosine
5'-O-(3-thiotriphosphate) (GTP
S) when reconstituted into
S49 cyc
membranes that lack endogenous Gs
(carbachol pretreated: GTP
S, 93 ± 13% of control;
NaF/AlCl3, 99 ± 8.6% of control; n = 4). Exogenous Gs
solubilized from red blood cells failed
to restore normal adenylyl cyclase activity when reconstituted into
carbachol-pretreated bovine airway smooth muscle (carbachol pretreated:
GTP, 36 ± 10% of control; NaF/AlCl3, 54 ± 11%
of control; n = 4). [3H]forskolin
radioligand saturation binding assays revealed a decreased quantity of
total adenylyl cyclase protein after carbachol pretreatment (maximal
binding: 152 ± 40 and 107 ± 31 fmol/mg protein in control and carbachol-pretreated airway smooth muscle, respectively). These
results suggest that chronic activation of muscarinic receptors downregulates the expression of adenylyl cyclase protein in bovine airway smooth muscle.
muscarinic; [3H]forskolin; reconstitution; S49
cyc; bovine
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INTRODUCTION |
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AIRWAY SMOOTH MUSCLE
FROM asthmatic patients (4) and from animal models
of asthma (11) show reduced relaxation in response to
-adrenoceptor agonists. The distal effector targets of
-adrenoceptor stimulation are the adenylyl cyclase enzymes. This
enzyme family consists of at least nine subtypes that differ in their
responses to products of other second messenger pathways, including
calcium (6, 24, 28, 31), G protein
-subunits
(5, 27, 33), protein kinase A (13), and
protein kinase C (PKC; see Refs. 14, 15, 17). Evidence to date suggests
that reduced
-adrenoceptor responsiveness in asthmatic airway smooth
muscle is not only due to changes at the level of the
-adrenoceptor
but often involves more distal components of this
receptor-Gs
-adenylyl cyclase signaling cascade (3,
10).
The airway inflammatory component of asthma results in elevated levels of agonists such as histamine and bradykinin and increased release of acetylcholine, all of which activate airway smooth muscle receptors that couple to the Gq/PKC signaling pathway. It is known that PKC can acutely regulate the function of some adenylyl cyclase subtypes (14, 15, 17). Thus chronic activation of the Gq/PKC pathway is one possible mechanism by which airway smooth muscle relaxation in response to agonists that increase cAMP may be impaired in asthma.
We have previously shown that chronic but not acute activation of
muscarinic receptors results in downregulation of adenylyl cyclase
activity in response to GTP or forskolin, an effect blocked by the
protein kinase inhibitor staurosporine, but the site (i.e., Gs or adenylyl cyclase) of this effect was never fully
identified (23). Neither decreased numbers of cell surface
-adrenoceptors nor increased quantity of Gi
proteins
could account for carbachol's effect since they were not changed.
Because staurosporine blocked the effect of chronic carbachol
activation, we reasoned that chronic activation of the M3
muscarinic/Gq/PKC pathway might directly downregulate the
function and/or expression of the adenylyl cyclase protein. Direct
downregulation of adenylyl cyclase protein as opposed to decreased
activity has never been demonstrated in response to chronic muscarinic
receptor activation in any cell.
In our previous study, chronic muscarinic receptor activation reduced
subsequently measured forskolin-stimulated adenylyl cyclase activity
via a staurosporine-sensitive pathway. Although it is generally
believed that forskolin directly stimulates adenylyl cyclase activity,
the resulting level of activation is dependent upon the activation
state of Gs (26). Thus forskolin is not a
selective measure of adenylyl cyclase function only. Before more
directed mechanistic studies are planned to evaluate the potential for
direct regulation of the expression of specific subtypes of adenylyl
cyclases by chronic muscarinic receptor activation in airway smooth
muscle, one must unequivocally implicate adenylyl cyclase as the target
of muscarinic receptor-induced downregulation.
The present study was therefore designed to determine whether chronic
activation of muscarinic receptors regulates protein expression or
function at the level of either the Gs protein or the
adenylyl cyclase enzymes. We used complementary Gs
reconstitution studies to show that Gs
extracted from
chronic muscarinic receptor-activated airway smooth muscle had similar
function when reconstituted into S49 cyc
membranes and
that reconstitution of exogenous Gs
back into airway
smooth muscle failed to restore normal adenylyl cyclase activity after
chronic muscarinic receptor activation. Finally, we were able to
directly measure the total amount of adenylyl cyclase protein by
radioligand binding and show for the first time in any cell that
chronic muscarinic receptor activation leads to downregulation in the
function and expression of adenylyl cyclase protein.
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METHODS |
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Airway smooth muscle preparation and pretreatment protocol.
Bovine airway smooth muscle was isolated from tracheas obtained from a
local abattoir by previously described methods (23). Briefly, airway smooth muscle was removed from the trachea and finely
minced with razor blades and then was washed two times in serum-free
DMEM. These small airway smooth muscle fragments (~1 × 1 mm)
were incubated for the indicated period of time with the indicated
pretreatment in serum-free DMEM plus antibiotics (100 U/ml penicillin
G, 100 µg/ml streptomycin, 0.025 µg/ml amphotericin B, 100 U/ml
nystatin, and 1 µg/ml gentamicin) at 37°C in an atmosphere of 5%
CO2-95% air. Untreated control tissues were incubated for the same length of time in parallel experiments to compensate for any
loss of adenylyl cyclase activity due to incubation time. Airway smooth
muscle fragments were recovered at the same time from control and
pretreated groups after incubations and were washed three times in
serum-free cell culture media to remove excess effectors used for
pretreatments. Muscle was then homogenized using high-speed cutting
blades (Tissumizer Mark II; Tekmar-Dohrmann, Cincinnati, OH) at top
speed for 30 s on ice, filtered through one layer of Nitex mesh,
and frozen at 70°C for subsequent use in adenylyl cyclase,
[3H]forskolin radioligand binding, and protein assays.
Protein assays were performed immediately before adenylyl cyclase or
radioligand binding assays, and control and treated tissues were always
analyzed in parallel within the same binding or adenylyl cyclase assay.
Adenylyl cyclase assays in native bovine airway smooth muscle
membranes.
Adenylyl cyclase activity was determined in triplicate samples as
previously described (11), with minor modifications.
Briefly, cellular lysates (40 µg) prepared from airway smooth muscle
fragments that had been incubated for 18 h in the presence or
absence of 10 µM carbachol were incubated for 10 min at 30°C in 100 µl final volume containing 50 mM HEPES (pH 8.0), 50 mM NaCl, 0.4 mM
EGTA, 1 mg/ml BSA, 0.5 mM MgCl2, 0.1 mM
[-32P]ATP (0.1-0.3 mCi/µmol), 1 mM cAMP, 1 mM
dithiothreitol (DTT), 7 mM creatine phosphate, and 5 units creatine
phosphokinase. Other modulators were added as indicated in the text.
Assay tubes containing isoproterenol and/or PGE1 also
contained 10 µM GTP. Assay tubes containing 10 mM NaF also contained
100 µM AlCl3. Preliminary experiments confirmed the
linearity of adenylyl cyclase activity at the protein concentrations
and incubation times. cAMP was isolated by sequential chromatography
through Dowex and alumina (22) with column recoveries of
75-90%. Basal and stimulated adenylyl cyclase activities are
expressed as picomoles of cAMP synthesized per milligram membrane
protein for the 10-min incubation period. Preliminary experiments were
performed in the presence or absence of 100 µM atropine to confirm
that adenylyl cyclase activity was not affected by residual carbachol
used during pretreatment periods.
Preparation of cyc membranes.
Murine S49 lymphoma cyc
kinase+ cells were
grown in spinner flasks in DMEM with high glucose, 10% horse serum,
100 U/ml penicillin G, and 100 µg/ml streptomycin. Three liters of
cells at a density of 5 × 106 cells/ml were used for
membrane preparation. Cells were pelleted by centrifugation at 400 g for 15 min at 4°C and were resuspended in cold 0.1 M
HEPES, pH 7.4 (40 ml buffer/l of cells). Cells were disrupted by 30 strokes of a motor-driven Teflon pestle in a glass homogenizer.
Undisrupted cells and large organelles were removed by centrifugation
at 400 g for 15 min at 4°C, and the supernatant containing
cyc
cell membranes was stored in 1-ml aliquots at
70°C until used for reconstitution assays.
Preparation of red blood cell membranes.
Outdated human red blood cells (RBCs) were obtained from a local blood
bank and stored at 4°C. Fifty milliliters of packed RBCs were mixed
with 150 ml cold PBS (0.144 g/l KH2PO4, 9.0 g/l NaCl, and 0.795 g/l
Na2HPO4 · 7H2O), pH 7.4, and centrifuged at 5,900 g for 10 min at 4°C. The
supernatant was discarded, and cells were washed with 150 ml cold PBS,
centrifuged as above, and resuspended in 50 ml of 5 mM
Na2HPO4, pH 8.0, for hypotonic cell lysis.
Lysed cells were centrifuged at 25,000 g for 15 min at
4°C, after which the supernatant was gently aspirated and discarded. The RBC membrane pellet was repeatedly washed (4-5 times) with 5 mM Na2HPO4 until the pellet appeared white
(indicating removal of Hb), and membranes were frozen in 1-ml aliquots
at 70°C at 3-4 mg/ml.
Detergent extraction of Gs from human RBCs
or bovine airway smooth muscle.
Gs
was detergent solubilized from human RBCs or
bovine airway smooth muscle membranes by modifications of previously
published methods (7, 8). The day before the adenylyl
cyclase reconstitution assay, RBC or bovine airway smooth muscle
membranes were thawed and microcentrifuged at 14,000 g for
30 min at 4°C. The pellet was resuspended at 3 mg/ml in 1 ml of
TEM-Lubrol-DTT (final concentrations; 10 mM Tris, pH 7.5, 0.1 mM EDTA,
10 mM MgCl2, 0.2% Lubrol PX, and 1 mM DTT) and incubated
overnight at 4°C with gentle rocking. Microcentrifuge tubes were
centrifuged in an SS34 rotor (Sorvall) at 20,000 rpm [minimum radius
(rmin) g force of 14,300 g] for 30 min at 4°C, and the supernatant
containing solubilized RBC or bovine airway smooth muscle
Gs
was saved and divided into two aliquots. One aliquot
containing functional Gs
was kept on ice and labeled the
soluble extract. The second aliquot was heated to 65°C for 60 min to
inactive Gs
and was labeled heated extract (negative
control). Additional 50-µl aliquots from control and carbachol-pretreated samples were subjected to immunoblot analysis to
determine the content of Gs
.
Reconstitution of RBC Gs into bovine airway smooth
muscle membranes and measurement of adenylyl cyclase activity.
Assay tubes were assembled in triplicate in a final volume of 80 µl.
This reaction consisted of 20 µl of bovine airway smooth muscle
membranes (2 mg/ml), 10 µl of either soluble extract (containing functional Gs
) or heated extract (containing inactive
Gs
), and 50 µl of cold assay buffer (final
concentrations 50 mM Tris, pH 7.6, 1 mM EDTA, 1 mM DTT, 2 mM
MgCl2, 0.1 mM ATP, 0.2 mM cAMP, 6.7 mM creatine phosphate,
and 50 U/ml creatine phosphokinase) containing the indicated effectors
[achieving a final concentration of either 10 mM NaF/100 µM
AlCl3, 10 µM GTP, or 10 µM guanosine 5'-O-(3-thiotriphosphate) (GTP
S)]. Reconstitution of
solubilized RBC Gs
into control or carbachol-pretreated
bovine airway smooth muscle membranes was allowed to occur at 37°C
for 1 h, after which time 20 µl of [
-32P]ATP
(20-30 µCi/ml, 800 Ci/mmol) were added to each tube, and the
incubation continued for an additional 20 min. The reaction was stopped
by the addition of 100 µl stop buffer [50 mM HEPES, pH 7.5, 2 mM
ATP, 0.5 mM cAMP, 2% SDS, and 1 µCi/ml [3H]cAMP (37 Ci/mmol)], and newly synthesized [32P]cAMP was separated
from [
-32P]ATP using [3H]cAMP to monitor
column recovery by sequential column chromatography over Dowex and
alumina according to the method of Salomon et al. (22).
Preliminary experiments established that the time and temperature of
incubation resulted in linear increases in adenylyl cyclase activity
and that 10 µl/tube of soluble extract resulted in maximal adenylyl
cyclase activity for a given effector in both control and
carbachol-pretreated bovine airway smooth muscle membranes. Adenylyl
cyclase activity was expressed as picomole cAMP per milligram membrane
protein per 20 min, and activity in carbachol-pretreated membranes was
expressed as percentage of activity in control membranes. Positive
control experiments were performed in parallel using soluble and
heat-inactivated extracts of RBCs reconstituted into cyc
membranes to ensure successful reconstitution within each experiment.
Reconstitution of bovine airway smooth muscle Gs
into cyc
membranes and measurement of adenylyl cyclase
activity.
Assay tubes were assembled in triplicate in a final volume of 80 µl.
Each tube contained 10 µl of either soluble or heated-inactivated bovine airway smooth muscle detergent extract, 20 µg of
cyc
membranes, and 50 µl of cold assay buffer with
effectors (see above). Reconstitution of solubilized bovine airway
smooth muscle Gs
into cyc
membranes was
allowed to occur at 37°C for 1 h, after which 20 µl of
[
-32P]ATP (20-30 µCi/ml, 800 Ci/mmol) were
added to each tube and the incubation was continued for an additional
20 min. The reaction was terminated, and [32P]cAMP was
isolated exactly as described above. Preliminary experiments established that the time and temperature of incubation resulted in
linear increases in adenylyl cyclase activity. Adenylyl cyclase activity was expressed as picomole cAMP per milligram membrane protein
per 20 min, and adenylyl cyclase activity in cyc
membranes reconstituted with soluble extracts from carbachol-pretreated bovine airway smooth muscle membranes was expressed as percentage of
activity in cyc
membranes reconstituted with soluble
extracts from control membranes. Negative control experiments measured
adenylyl cyclase activity in cyc
membranes in the absence
of reconstitution to measure background adenylyl cyclase activity.
[3H]forskolin saturation radioligand binding. Bovine airway smooth muscle lysates (250 µg) from either control or carbachol-pretreated samples were incubated with [3H]forskolin (30.5 Ci/mmol; 4-120 nM) in duplicate in the presence or absence of 20 µM unlabeled forskolin in binding buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). Cytochalasin B (40 µM) was also included in binding buffer to prevent nonspecific binding of [3H]forskolin to the glucose transporter (20). Gpp(NH)p (100 µM) and NaF (10 mM) were also included in binding buffer to maximize affinity of adenylyl cyclase for forskolin (2, 18). All radioligand binding experiments were incubated in a final volume of 125 µl and were performed for 2 h at room temperature. Preliminary experiments confirmed that equilibrium binding had been achieved using this time and temperature of incubation and that linear increases in specific binding occurred over the range of protein concentrations used. Preliminary studies also confirmed that saturation of [3H]forskolin specific binding occurred at 40 nM [3H]forskolin. Binding assays were rapidly terminated by the addition of 5 ml of cold wash buffer (50 mM Tris, pH 7.5, 10 mM MgCl2) followed by immediate vacuum filtration through GF/C glass fiber filters. Tubes were rapidly washed an additional three times with 5 ml cold wash buffer. Incorporation of 3H was determined by submerging filters in 5 ml of scintillation cocktail and counting in a Beckman scintillation counter with an efficiency of 50-55%. Specific binding (total binding in the absence of unlabeled forskolin minus binding in the presence of unlabeled forskolin) was calculated after subtraction of control counts obtained from tubes lacking membranes. Specific binding was plotted as a function of radioligand and was fitted to a rectangular hyperbola equation. Total receptor numbers (Bmax) were determined from the y plateau values, and affinity (Kd) was determined from the midpoint on the y-axis relative to the y plateau values. Binding data were then subjected to Scatchard-Rosenthal transformation of the data.
Immunoblot analysis of Gs in soluble extracts of
bovine airway smooth muscle.
Fifty-microliter aliquots of Lubrol extracts of bovine airway smooth
muscle were subjected to immunoblot analysis as previously described
(12). Briefly, aliquots were mixed with an equal volume of
gel loading buffer and after boiling for 10 min were subjected to
electrophoresis through 10% polyacrylamide [10% total (T):0.1% crosslinker (C)]-SDS gels and transferred to polyvinylidene
difluoride (PVDF) membranes. After being blocked, PVDF membranes were
incubated for 2 h in Gs
primary antibody (RM/1,
1:1,000 dilution; NEN, Boston, MA). After two 15-min washes, PVDF
membranes were incubated for 2 h with goat anti-rabbit
IgG-horseradish peroxidase (1:3,000 dilution; Amersham, Arlington
Heights, IL). Bands were detected by enhanced chemiluminescence
according to the manufacturer's recommendations (Immunolite II;
Bio-Rad, Hercules, CA) with subsequent exposure to autoradiography
film. Band intensities were quantified with a scanner coupled to a
personal computer with MacBas 2.2 software.
Protein determination. Protein was assayed with the Pierce Chemical (Rockford, IL) BCA protein assay reagent, consisting of bicinchoninic acid and copper sulfate solutions (25). BSA was used as a standard.
Statistics. Unless otherwise noted, data are expressed as means ± SE. Data from adenylyl cyclase assays were analyzed by two-way ANOVA for treatments in the same tissues and one-way ANOVA for treatments between groups with Tukey-Kramer multiple comparisons. Data from forskolin binding assays were analyzed by two-tailed paired t-test. The null hypothesis was rejected when a P value < 0.05 was obtained.
Reagents. Except as noted, all reagents were purchased from Sigma (St. Louis, MO). All radioisotopes were purchased from NEN.
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RESULTS |
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Adenylyl cyclase activity was compared in homogenates of bovine
airway smooth muscle fragments after incubation for 18 h in the
presence or absence of 10 µM carbachol. As we have previously shown
(23), carbachol pretreatment resulted in significant
decreases in basal and GTP-, isoproterenol-, and forskolin-stimulated
adenylyl cyclase activity (Fig. 1).
Control basal activities of 37 ± 9.9 pmol cAMP · mg
protein1 · 10 min
1 were reduced by
carbachol pretreatment to 16 ± 5.7 pmol cAMP · mg
protein
1 · 10 min
1 (57% decrease).
Control GTP-stimulated activities of 51 ± 15 pmol
cAMP · mg protein
1 · 10 min
1 were reduced by carbachol pretreatment to
20 ± 5.5 pmol cAMP · mg
protein
1 · 10 min
1 (56% decrease).
Control isoproterenol-stimulated activities of 90 ± 17 pmol
cAMP · mg protein
1 · 10 min
1 were reduced by carbachol pretreatment to 33 ± 7.8 pmol cAMP · mg protein
1 · 10 min
1 (63% decrease). Control forskolin-stimulated
activities of 121 ± 23 pmol cAMP · mg
protein
1 · 10 min
1 were reduced by
carbachol-pretreatment to 41 ± 15 pmol cAMP · mg
protein
1 · 10 min
1 (66% decrease;
n = 4).
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To confirm that the carbachol-induced decreases in adenylyl cyclase
activity were not due to nonspecific cell toxicity resulting in an
overall decline in enzyme activity, a separate group of experiments was
performed in which carbachol was withdrawn after 18 h of
incubation and 100 µM atropine was added for an additional 48 h.
Recovery of adenylyl cyclase activities to control levels would suggest
that the cells were not nonspecifically injured by the 18-h carbachol
treatment. In these experiments (n = 3), homogenates
prepared from airway smooth muscle fragments that had received
carbachol for 18 h followed by 48 h of atropine had similar
adenylyl cyclase activity to those cells maintained in parallel
incubations without treatment for 66 h. Tissues treated for
18 h with carbachol followed by 48 h of atropine had basal activities 88 ± 5% of control, GTP-stimulated activities were 90 ± 18% of control, isoproterenol-stimulated activities were 104 ± 11% of control, and forskolin-stimulated activities were 125 ± 26% of control (Fig. 2).
Incubation in the presence of atropine alone had no significant effect
on adenylyl cyclase activity (data not shown).
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To determine where carbachol was having its effect in decreasing the
activity of the receptor-G protein-adenylyl cyclase cascade, we
performed a series of experiments to evaluate the quantity and function
of the Gs and adenylyl cyclase proteins. The function of
Gs
was evaluated by complimentary reconstitution assays.
In the first approach, Gs
was detergent solubilized from
control or carbachol-pretreated bovine airway smooth muscle and
reconstituted into a membrane system lacking endogenous
Gs
(i.e., cyc
), and its function was
assessed by stimulation with GTP
S and NaF/AlCl3. In a
second complementary approach, Gs
was solubilized from
human RBCs and reconstituted into control or carbachol-pretreated bovine airway smooth muscle to test for decreased Gs
function after carbachol pretreatment.
Activity levels of Gs from control and
carbachol-pretreated bovine airway smooth muscle reconstituted into
cyc
cell membranes were not different. Adenylyl cyclase
activity stimulated with 10 µM GTP
S was 93 ± 13% of
control, whereas activity stimulated with 10 µM NaF/AlCl3
was 99 ± 8.6% of control (n = 4; Fig.
3A). Control experiments
confirmed successful reconstitution of functional Gs
activity into cyc
membranes. The cyc
membranes had barely detectable adenylyl cyclase activity in the
presence of NaF/AlCl3 or GTP
s (2.4 ± 1.4 and
1.1 ± 0.9 pmol cAMP · mg
protein
1 · 10 min
1, respectively).
After reconstitution with Gs
extracted from control
bovine airway smooth muscle, adenylyl cyclase activity in response to
NaF/AlCl3 and GTP
s increased to 222 ± 51 and
372 ± 68 pmol cAMP · mg
protein
1 · 10 min
1, respectively
(Fig. 3B). The quantity of Gs
solubilized
from control and carbachol-pretreated bovine airway smooth muscle was not different, as detected by immunoblot analysis (Fig.
4).
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Gs was solubilized from human RBCs and reconstituted
into control or carbachol-pretreated bovine airway smooth muscle
homogenates in an attempt to restore normal adenylyl cyclase activity
to carbachol-pretreated muscle. However, decreases in basal and GTP-
and NaF/AlCl3-stimulated adenylyl cyclase activity
persisted in carbachol-pretreated bovine airway smooth muscle after
reconstitution. GTP- and NaF/AlCl3-stimulated activities
were 36 ± 10 and 54 ± 11% of control activities,
respectively (n = 4; Fig.
5A). Control experiments were
performed in parallel in cyc
membranes to ensure
successful reconstitution of RBC Gs
.
NaF/AlCl3-stimulated adenylyl cyclase activity was 9.3 ± 2.1 and 193 ± 42 in cyc
membranes in the absence
and presence of solubilized RBC Gs
, respectively
(n = 4; Fig. 5B).
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Radioligand binding assays were performed to measure the quantity of
total adenylyl cyclase enzyme in control and carbachol-pretreated bovine airway smooth muscle. A significant reduction in adenylyl cyclase protein was measured in carbachol-pretreated bovine airway smooth muscle compared with control (Bmax = 107 ± 31 and 152 ± 40 fmol/mg protein, respectively;
n = 5; P = 0.02; Fig.
6). Saturation of specific binding was
achieved at 40 nM of [3H]forskolin (Fig.
7A), and specific binding
(Bmax) and affinity values (Kd) were
determined from nontransformed data before Scatchard-Rosenthal transformations of the data were performed, which revealed a
single-affinity binding site (Fig. 7B). The affinity
(Kd) of [3H]forskolin for its
binding site was not different in control and carbachol-pretreated
bovine airway smooth muscle, with Kd values of
52 ± 8.0 and 48 ± 12 nM, respectively.
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DISCUSSION |
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In the current study, bovine airway smooth muscle pretreated with
carbachol for 18 h showed normal Gs function in
reconstitution assays but reduced adenylyl cyclase activity and reduced
amounts of total adenylyl cyclase protein. This study extends previous studies from our laboratory demonstrating that chronic carbachol pretreatment resulted in decreased adenylyl cyclase activity by implying that the site of regulation was at the level of adenylyl cyclase protein expression and not Gs
.
In the current study, reconstitution of Gs from an
exogenous source (human RBC) into carbachol-pretreated bovine airway
smooth muscle failed to restore normal adenylyl cyclase activities.
Additionally, Gs
extracted from carbachol-pretreated
bovine airway smooth muscle and reconstituted into a membrane
preparation lacking endogenous Gs
(i.e., S49
cyc
cells) had normal activity in this alternate membrane
environment compared with Gs
extracted from control
bovine airway smooth muscle. The results taken together suggest that
carbachol pretreatment was having an effect at the level of the
adenylyl cyclase enzyme itself. Therefore, radioligand binding assays
were performed to determine if quantitative changes occurred in
adenylyl cyclase protein. These studies confirmed a decrease in the
amount of total adenylyl cyclase protein after carbachol pretreatment
of bovine airway smooth muscle that likely accounts for the decrease in adenylyl cyclase activity in response to effectors acting at the receptor and Gs
protein levels of this signaling cascade.
Carbachol-induced decreases in adenylyl cyclase activity could not be due to loss of adenylyl cyclase enzyme activity during the 18-h incubation period, since control tissues incubated in parallel under identical conditions exhibited increased levels of adenylyl cyclase compared with carbachol-pretreated tissues. Carbachol-induced decreases in adenylyl cyclase activity were not due to nonspecific cellular toxicity induced by carbachol, since withdrawal of the carbachol and further incubation for 48 h in the presence of atropine allowed recovery of adenylyl cyclase activity to control levels.
Previous studies in a variety of cell lines agree with the current
study in that chronic activation of either PKC with phorbol esters or
of receptors potentially coupled to Gq leads to
decreased function of the receptor-Gs
-adenylyl cyclase
cascade. However, this is the first study in any cell to show a reduced
amount of adenylyl cyclase protein after chronic muscarinic receptor
activation. In the osteoblast-like cell line UMR-106, >6 h of
pretreatment with phorbol esters resulted in decreased adenylyl cyclase
activity in the presence of parathyroid hormone or forskolin,
suggesting PKC-mediated downregulation of adenylyl cyclase function
(16). In contrast, a study in canine cultured thyroid
cells showed that 18 h of phorbol ester pretreatment resulted in
decreased adenylyl cyclase activity in response to thyroid-stimulating
hormone but not forskolin (9), suggesting PKC cross talk
at a stimulatory receptor/Gs
level. However, in a
subsequent study, these authors showed that 2-4 h of carbachol
pretreatment (presumably working through a muscarinic
receptor-Gq
-PKC pathway in these cells) resulted in
decreased adenylyl cyclase activity in response to thyroid-stimulating
hormone, cholera toxin, GTP, and forskolin, again suggesting
cross-regulation at the level of adenylyl cyclase (21). In
none of these studies were Gs
reconstitution studies or
measurement of adenylyl cyclase protein performed. Perhaps these
studies differed regarding the apparent site of PKC cross-regulation because 1) phorbol ester may stimulate more than PKC (shown
to also stimulate mitogen-activated protein kinases; see Refs. 1 and
30), 2) the phorbol ester pretreatment was for a different time course than carbachol pretreatment, 3) different
isoforms of PKC may exist in the different cell types or be
activated/downregulated differently by phorbol esters, or 4)
activation of Gq
by receptor occupancy may couple to
other signaling pathways besides PKC.
The current study also agrees with a recently published study from our
laboratory using myometrial cells demonstrating that chronic treatment
of cultured rat myometrial cells with either oxytocin or phenylephrine
(both coupled to Gq) resulted in decreased adenylyl
cyclase activity in response to GTP, isoproterenol, NaF, MnCl2, or forskolin (suggesting cross-regulation at the
level of adenylyl cyclase) by a mechanism that was blocked by prior depletion of PKC by prolonged phorbol ester treatment
(19). Thus these studies in a variety of cell lines
coupled with the results of the present study suggest that
Gq
/PKC activation potentially cross-regulates several
levels of the receptor-Gs
-adenylyl cyclase cascade, but
the present study is the first study to demonstrate a downregulation of
adenylyl cyclase protein.
Classically, dual regulation of adenylyl cyclase activity has been
described when Gs- or Gi
-coupled
receptors are acutely activated. However, chronic activation of
Gi-coupled receptors is known to upregulate subsequently
measured adenylyl cyclase activity in many cell types and is known as
adenylyl cyclase superactivation. In studies evaluating the phenomenon
of adenylyl cyclase "superactivation," it is apparent that adenylyl
cyclase enzymes differ in both their acute and their chronic response
to Gi-coupled receptor activity. In COS-7 cells transfected
with various subtypes of adenylyl cyclase, patterns of responses to
acute or chronic Gi activation were seen that coincided
with previous subfamily groupings of the adenylyl cyclase family
(29). Subtypes I, V, VI,
and VIII were acutely inhibited by dopamine D2
receptor activation (coupling through Gi), but chronic
dopamine D2 receptor activation resulted in superactivation of these enzymes' activities. Subtypes II, IV,
and VII were activated by acute D2 receptor
activation but were inhibited after prolonged D2 receptor
occupancy. Conversely, type III was inhibited by either acute or chronic D2 receptor activation. Although these
findings are not directly linked to the current study in which
muscarinic receptor activation is involved, it suggests that different
subtypes of adenylyl cyclases differ in their responses to chronic
Gq/PKC activation, which may account for different effects
of chronic Gq/PKC stimulation found
in different cell types. Indeed, isoforms II,
III, V, and VII are activated by acute
treatments with phorbol esters (14, 15, 32), and
subtypes II and V are known to be phosphorylated
by PKC-
(15, 34). Perhaps a desensitization mechanism
exists for the downregulation of adenylyl cyclase subtypes sensitive to
PKC-mediated phosphorylation under chronic PKC activation conditions.
In summary, chronic activation of muscarinic receptors by carbachol in
airway smooth muscle downregulated the expression and function of
adenylyl cyclase proteins with no functional effect on
Gs. Reduced adenylyl cyclase content of airway smooth
muscle in response to chronic elevations of inflammatory agonists that couple to Gq in asthmatic airways may contribute to
impaired relaxation of asthmatic airway smooth muscle in response to
-agonists.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. W. Emala, Dept. of Anesthesiology, College of Physicians and Surgeons of Columbia Univ., 630 W. 168th St. PH 525, New York, NY 10032 (E-mail: cwe5{at}columbia.edu).
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. Section 1734 solely to indicate this fact.
Received 5 November 1999; accepted in final form 17 April 2000.
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