Carbachol-induced desensitization of PLC-
pathway in rat
myometrium: downregulation of
Gq
/G11
Sandrine
Lajat,
Simone
Harbon, and
Zahra
Tanfin
Signalisation et Régulations Cellulaires, Centre National de
la Recherche Scientifique, EP1088, Université Paris-Sud,
91405 Orsay Cedex, France
 |
ABSTRACT |
In the estrogen-treated rat myometrium, carbachol increased the
generation of inositol phosphates by stimulating the muscarinic receptor-Gq/G11-phospholipase
C-
3 (PLC-
3) cascade. Exposure to carbachol resulted in a rapid
and specific (homologous) attenuation of the subsequent muscarinic
responses in terms of inositol phosphate production, PLC-
3
translocation to membrane, and contraction. Refractoriness was
accompanied by a reduction of membrane muscarinic binding sites and an
uncoupled state of residual receptors. Protein kinase C (PKC) altered
the functionality of muscarinic receptors and contributed to the
initial period of desensitization. A delayed phase of the muscarinic
refractoriness was PKC independent and was associated with a
downregulation of
Gq
/G11
.
Atropine failed to induce desensitization as well as
Gq
/G11
downregulation, indicating that both events involve active occupancy of
the receptor. Prolonged exposure to
AlF
4 reduced subsequent AlF
4 as well as carbachol-mediated
inositol phosphate responses and similarly induced downregulation of
Gq
/G11
. Data suggest that a decrease in the level of
Gq
/G11
is subsequent to its activation and may account for
heterologous desensitization.
inositol phosphates; muscarinic receptors; desensitized receptors; phospholipase C-
3 translocation; myometrial contraction
 |
INTRODUCTION |
IN MANY INSTANCES, prolonged exposure of various
tissues to agonists at a G protein-linked receptor trigger a
counterregulatory process that attenuates receptor signaling, a
phenomenon commonly denoted as desensitization (2, 18). Desensitization
may be either agonist specific (homologous) or associated with
decreased responsiveness to other activators (heterologous). The direct uncoupling of the receptor from its respective G protein underlies the
rapid phase of desensitization and is mediated, at least in part, by
receptor phosphorylation. Two classes of serine/threonine kinases
phosphorylate G protein-coupled receptors: the G protein-coupled receptor kinases (GRKs) and the second-messenger-dependent protein kinases, PKA and PKC (2, 18, 25). Agonist-induced refractoriness may be
further associated with receptor sequestration and ultimately with
receptor recycling or degradation "downregulation." While regulation at the receptor level appears to be the predominant site of
desensitization of G protein-coupled receptors, there has been recently
increasing evidence that such regulation may also exist at the level of
the G proteins (21). It has been reported that chronic exposure to a G
protein-linked receptor can result in a reduction in the level of the G
protein that interacts specifically with this receptor. Agonist-induced
downregulation of cellular G
proteins has been observed for members
of the Gs (1, 21) and the
Gi (10, 21) families regulating
the stimulatory and inhibitory pathways of adenylyl cyclase and more
recently for Gq proteins coupled
to the phospholipase C (PLC) stimulatory cascade (12, 22).
It is well recognized that the generation of
D-myo-inositol
1,4,5-trisphosphate with the accompanying rise in cytosolic
Ca2+ is an important determinant
in uterine contractility. Many reports, including ours (7, 9, 20), have
demonstrated that the phosphoinositide-PLC transducing system can be
activated by agonists that induce contraction in different myometrial
preparations (15, 24). For most contractile agonists, the receptor PLC
coupling was insensitive to pertussis toxin (7, 9, 20). We have recently shown that in rat myometrium the responsiveness of the PLC
pathway, associated with receptor- and/or direct G
protein-mediated activation, increased during pregnancy. The
enhancement of PLC activity could not be ascribed to changes in the
amount of PLC-
3, the predominant PLC-
isoform expressed in rat
myometrium, but clearly coincided with the increase in the amount of
Gq
(16).
In the present study it is demonstrated that the PLC pathway can also
be negatively regulated. Exposure of the nonpregnant rat myometrium to
carbachol resulted in a dramatic decline in the ability of the
muscarinic agonist to increase the generation of inositol phosphates
and the tension in the desensitized tissue. Carbachol-induced refractoriness was associated with
1) both quantitative and functional
alterations of muscarinic receptors and
2) downregulation of
Gq
/G11
.
The decrease in Gq levels appears
to be subsequent to its sustained activation, whether direct or
receptor mediated, and may contribute to the development of
heterologous desensitization. Our observations provide additional
support for a pivotal role of Gq
levels in the control of agonist-mediated activation of the PLC-
pathway.
 |
MATERIALS AND METHODS |
Materials. Lithium chloride,
carbamylcholine chloride (carbachol),
-estradiol 3-benzoate,
oxytocin, phosphatidylinositol, leupeptin, aprotinin,
phenylmethylsulfonyl fluoride (PMSF), and phorbol 12,13-dibutyrate
(PDBu) were obtained from Sigma Chemical (St. Louis, MO). Endothelin-1
(ET-1) was from Neosystem (Strasbourg, France).
Myo-[2-3H]inositol
(10-20 Ci/mmol) was obtained from Amersham International (Les
Ulis, France). Ro-31-8220 was generously provided by Dr. D. Bradshaw (Roche, Hertfordshire, UK).
Anti-Gq
/G11
(QL) and anti-Gs
(RM1)
polyclonal antibodies and
[N-methyl-3H]scopolamine
methylchloride
([3H]NMS) at 80 Ci/mmol were obtained from New England Nuclear Product Division (DuPont
de Nemours, Les Ulis, France). The antibody directed against
-subunits of heterotrimeric G proteins was generously provided by
Dr. B. Rouot [Centre National de la Recherche Scientifique (CNRS), Montpellier, France]. PLC-
3 antibody was from Santa
Cruz Biotechnology. Silica gel plates were from Merck (Darmstad,
Germany), and AG1-X8 was from Bio-Rad (Ivry, France). Other chemicals
were of the highest grade commercially available.
Animals and tissue processing.
Immature female rats (Wistar, 5 wk old) were treated with 30 µg
estradiol for 2 days and used the following day. Rats were killed by
decapitation. Uteri were removed and the myometrium was prepared free
of endometrium (9, 16).
Tissue incubation and
[3H]inositol prelabeling
experiments.
Myometrial strips (~25 mg) were allowed to equilibrate for 25 min in
5 ml Krebs bicarbonate buffer (pH 7.4) containing (in mM) 117 NaCl, 4.7 KCl, 1.1 MgSO4, 1.2 KH2PO4,
24 NaHCO3, 0.8 CaCl2, and 1 glucose (gas phase
95% O2-5%
CO2) under constant agitation. Tissues were incubated with 7 µCi of
myo-[3H]inositol
(0.4 µM) in 1 ml of fresh buffer for 4 h as described (9, 16).
Incubations were continued for the time indicated without (control) or
with carbachol (pretreated). All tissues were then washed three times
with 10 ml of agonist-free nonradioactive Krebs buffer and
transferred into 1 ml of fresh buffer, during 5 min before the addition
of 10 mM LiCl. After 10 min, agonists to be tested were added at the
indicated concentration, and incubation was further continued for the
time indicated for the specific experiment. Reactions were stopped by
immersing the myometrial strips into 1.5 ml of cold 7% (wt/vol) TCA,
followed by homogenization and centrifugation at 10,000 g for 15 min at 4°C.
Measurement of
[3H]phosphoinositides.
The pellets obtained after centrifugation of the TCA homogenates were
washed with 0.5 ml of TCA to remove any residual
[3H]inositol.
Chloroform-methanol-12 M HCl (40:80:1, vol/vol/vol; 2.8 ml) was then
added and the phospholipids were extracted for 30 min at room
temperature. Chloroform (930 µl) and 0.1 N HCl (1,700 µl) were then
added, and two phases were obtained by centrifugation. The upper phase
was discarded, and the lower phase was dried under a stream of nitrogen
and used to determine the radioactivity incorporated into
[3H]inositol
phospholipids. In some experiments phosphatidylinositol (PtdIns),
phosphatidylinositol monophosphate
(PtdInsP), and phosphatidylinositol bisphosphate
(PtdInsP2) were
separated by TLC, and the associated radioactivity was determined as
previously described (7, 16).
Measurement of [3H]inositol
phosphates.
The TCA-soluble supernatants were extracted four times with 6 ml
diethyl ether, neutralized with Tris base, and applied to a column
(0.7 × 2 cm) of the anion-exchange resin (AG1-X8;
formate form, 200-400 mesh). Free inositol was eluted with 10 ml
water, and the individual inositol phosphates [inositol
trisphosphate (InsP3),
inositol bisphosphate
(InsP2), and
inositol monophosphate (InsP)]
were separated as described (9, 20). Alternatively, total inositol
phosphates (InsP3 + InsP2 + InsP) were eluted together in a
single step with 12 ml of 1 M ammonium formate/0.1 M formic acid. The
3H content of the various
fractions was determined by scintillation counting in Quicksafe A. Production of
[3H]inositol
phosphates was calculated as a percentage of radioactivity incorporated
into phosphoinositides obtained from the corresponding sample (16).
Method for recording uterine contractile
response. The contractile activity of isolated
myometrial strips was measured with an isometric transducing device.
The segments were loaded at a basal tension of 0.2-0.3
g and bathed at 37°C in 10 ml Krebs buffer under 95%
O2-5%
CO2. The contractile activity was
integrated during a 1-min exposure to the indicated agonist.
Membrane preparation. Myometrial
strips (100 mg) were homogenized with an Ultra-Turrax homogenizer in
0.6 ml buffer A containing 10 mM
Tris · HCl, 0.5 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM PMSF and were centrifuged for 5 min at 700 g. The
700-g supernatant (total homogenate)
was then centrifuged for 20 min at 100,000 g (Beckman TLC 100-4, fixed
rotor). The resulting supernatant represented the cytosolic fraction,
and the corresponding pellet was suspended in buffer
A at 4-6 mg protein/ml and constituted the
membrane or particulate fraction. Proteins were estimated using the
Lowry reagent (19).
Detergent-extracted proteins.
Myometrial strips (100 mg) were homogenized in 0.6 ml of cold
buffer A supplemented with 1% Triton
X-100 and 2 mM EGTA (16). After 30 min at 4°C, the lysates were
clarified by centrifugation at 10,000 g for 20 min, and the resulting
supernatant was used as detergent-extracted proteins.
Receptor binding assay. Tissues were
incubated in Krebs buffer with or without the addition of the indicated
agonist for various times depending on the experiment. Myometrial
strips were washed with hormone-free Krebs buffer, and membrane
preparations were obtained as described above. Membrane proteins
(150-300 µg) were incubated with the indicated concentration of
[3H]NMS for 1.5 h at 30°C in 50 mM Tris · HCl (pH
7.4), 10 mM MgCl2 in 1 ml final
volume. Triplicate 300-µl aliquots were filtered through Whatman GF/C
glass fiber filters, and the bound radioactivity was determined by
scintillation counting (16). Nonspecific binding was defined as the
amount of radioactivity bound to the filter when incubations were
performed in the presence of 1 µM atropine and was subtracted from
total binding to obtain specific binding. Under these conditions,
nonspecific binding represented <5% of total binding at
[3H]NMS concentrations
near its dissociation constant
(Kd) values. Binding data were analyzed using a nonlinear least-squares
curve-fitting program (Multifit program from Day Computing, Cambridge,
MA) to obtain IC50
values. Inhibition constant
(Ki) values
were calculated from IC50 values
by applying the equation of Cheng and Prusoff (4).
Immunologic analysis of
Gq
/G11
.
Protein samples were resolved by SDS-PAGE (10% wt/vol
acrylamide). Proteins were transferred to nitrocellulose and blocked as
described (16, 26). Primary antiserum QL (1:500 dilution) in 0.5%
nonfat dried milk in Tris-buffered saline (TBS; 20 mM Tris · HCl, pH 7.5, 500 mM NaCl) was then added and
left overnight at 4°C. Secondary antiserum (swine anti-rabbit IgG
coupled to horseradish peroxidase) was used at 1:2,000 dilution in
0.5% nonfat dried milk/TBS and left for 3 h at room temperature. The
immunoreactive bands were visualized using the enhanced
chemiluminescence (ECL) detection system (Amersham). Quantification of
the developed blots was performed using a Molecular Dynamics
Densitometer.
Immunologic analysis of PLC-
3.
Membrane proteins were resolved by SDS-PAGE (7.5% wt/vol acrylamide).
The separated proteins were transferred to a nitrocellulose sheet for
immunoblotting. The nitrocellulose sheet was blocked for 90 min at
37°C with 5% nonfat dried milk/TBS. Primary antiserum, anti-PLC-
3 (dilution 1:100) in 5% nonfat dried milk/TBS, was then
added and left for 1 h at room temperature. The immunoreactive bands
were visualized by the ECL detection system, after incubation with
swine anti-rabbit IgG coupled to horseradish peroxidase (dilution 1:2,000) for 60 min at room temperature.
Data analysis. The results are
expressed as means ± SE and were analyzed statistically using
Student's t-test.
P < 0.05 was considered significant.
 |
RESULTS |
Desensitization of the inositol phosphate response to
carbachol. When
[3H]inositol-prelabeled
myometrial strips that had been treated with 100 µM carbachol during
different times were washed to remove the agonist and challenged with
the muscarinic agonist for 15 min in the presence of LiCl, there was a
diminished response in terms of inositol phosphate accumulation,
compared with similarly treated tissues in carbachol-free medium (Fig.
1). Agonist-induced desensitization was
rapid, being significantly detectable (50%) at 15 min and maximal
(80% desensitization) at 60 min. Carbachol-induced desensitization was
also observed for individual inositol phosphates (InsP3,
InsP2, and
InsP), indicating that
desensitization occurred at the level of the
receptor-Gq-PLC cascade that
hydrolyses
PtdInsP2.

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Fig. 1.
Time course of carbachol-induced desensitization of inositol phosphate
responses in rat myometrium.
[3H]inositol-labeled
myometrial strips were incubated with 100 µM carbachol. At indicated
times, tissues were washed to remove the agonist and challenged with
100 µM carbachol ( ) and 0.1 µM oxytocin ( ) in the presence of
10 mM LiCl. After 15 min, incubations were stopped, and total
[3H]inositol
phosphates were eluted from AG1-X8 columns in a single step. Individual
[3H]inositol
phosphates, i.e., inositol monophosphate
(InsP), inositol bisphosphate
(InsP2), and
inositol trisphosphate
(InsP3) were
separated on AG1-X8 as described in MATERIALS AND
METHODS. Production of
[3H]inositol
phosphates was expressed as a percentage of maximal carbachol response
(total inositol phosphates, InsP,
InsP2, and
InsP3 = 34 ± 4, 29 ± 3, 3 ± 0.4, and 1.2 ± 0.2% of label in
phosphoinositides, respectively) and maximal oxytocin response in terms
of total inositol phosphates (69.3 ± 8% label in
phosphoinositides). Values are means ± SE of three different
experiments, each done in duplicate.
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Figure 1 further illustrates that the oxytocin-stimulated inositol
phosphate response was also reduced after carbachol treatment, albeit
with a differential time course: the onset of this heterologous desensitization was slower, not apparent before 15 min, and a maximal
decline was obtained after 60 min of exposure to carbachol. When
myometrial strips were treated with 100 µM carbachol during 60 min,
there was also a marked decrease in the ability of
AlF
4 to stimulate the generation
of inositol phosphates (63.4 ± 7 and 38.7 ± 5% of label in
phosphoinositides in control and carbachol-treated tissues,
respectively, n = 3). The attenuated
inositol phosphate responses were not brought about by a limiting
supply of the PLC substrate. Indeed, after 120 min of treatment with
carbachol, there was no significant alteration in the
[3H]inositol
incorporated into
PtdInsP2,
PtdInsP, and PtdIns (85.3 ± 7, 5.1 ± 0.5, and 8.5 ± 0.8% of total
[3H]phosphoinositides,
respectively) compared with the control (86.1 ± 4, 4.9 ± 0.4, and 8.7 ± 0.8% of
[3H]phosphoinositides,
respectively).
As indicated in Fig. 2, the magnitude of
the inositol phosphate refractory response was progressively larger
with increasing concentrations of carbachol during the initial 2-h
incubation period. It is interesting to note that the
concentration-dependent curve of carbachol-induced increases in
inositol phosphates was strikingly similar to the dose dependency of
carbachol-mediated inositol phosphate refractoriness, with virtually
the same half-maximal concentration
(EC50: 15.1 ± 1.4 and 10.2 ± 1.2 µM, respectively). In the presence of atropine, carbachol
failed to attenuate the subsequent inositol phosphate response. These
observations indicated that carbachol-induced desensitization was
triggered by receptor activation.

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Fig. 2.
Dose-response curves for carbachol (CB)-induced inositol phosphate
generation and carbachol-induced desensitization. For estimation of
inositol phosphate accumulation ( ),
[3H]inositol-labeled
myometrial strips were incubated for 10 min with 10 mM LiCl, followed
by 15-min incubation in presence of indicated concentrations of
carbachol. For desensitization experiments ( , ),
[3H]inositol-labeled
strips were initially exposed for 2 h to indicated concentrations of
carbachol added alone ( ) or combined ( ) with 0.1 µM atropine.
Myometrial strips were then washed and rechallenged with 100 µM
carbachol for 15 min. Production of
[3H]inositol phosphates was expressed as a
percentage of maximal carbachol response obtained in untreated tissues
(33.1 ± 3 of label in phosphoinositides). Values are means ± SE
of 3-4 different experiments.
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Absence of carbachol-mediated membrane association of
PLC-
3 in desensitized tissues.
It has been reported (15, 16) that PLC-
3 but not PLC-
1 or
PLC-
2 was immunodetected in detergent-extracted proteins derived
from estrogen-treated rat myometrium. It was further noted (experiments
not shown) that, under basal conditions, PLC-
3 was immunodetected
mainly in cytosol, with a minor amount (25-30%) consistently
detected in membranes. Experiments in Fig.
3 show that, after a 5-min carbachol
stimulation, there was a progressive and significant increase (2-fold)
of PLC-
3 associated with the membranes compared with control
tissues. These observations were reproduced in three separate
experiments described in the legend to Fig.
3B, which also illustrates that the
immunoreactive PLC-
3 associated with membranes derived from
stimulated and unstimulated preparations was related to the amount of
protein subjected to SDS-PAGE. Carbachol-induced increase of
membrane-associated PLC-
3 was prevented by atropine. Data in Fig. 3,
A and
B, further show that the ability of
carbachol to trigger translocation of PLC-
3 was markedly attenuated
in desensitized tissues after 2 h of pretreatment with the muscarinic
agonist.

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Fig. 3.
Effect of carbachol on level of membrane-associated phospholipase
C- 3 (PLC- 3) in control and desensitized tissues.
A: myometrial strips were incubated
for 2 h in absence or presence of 100 µM carbachol. Tisues were then
washed and further incubated for 3 and 5 min without or with 100 µM
carbachol. When used, atropine (0.1 µM) was added 5 min before
carbachol. Equal amounts (20 µg) of membrane proteins were resolved
by SDS-PAGE (7.5% wt/vol acrylamide) and were
immunoblotted in presence of PLC- 3 antibody (1:100 dilution).
B: myometrial strips were incubated 2 h in absence ( and lane 1; and
lane 2) or presence of 100 µM
carbachol ( and lane 3) and
stimulated for 5 min by carbachol ( and lane
2; and lane 3).
Membrane proteins (10, 20, and 30 µg) from each preparation were
resolved and immunoblotted with PLC- 3 antibody.
Top
(A and
B) is a representative immunoblot.
Immunoreactive bands from 3 separate experiments were quantified by
densitometric scanning. Data are means ± SE.
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Alteration of the contractile effect of carbachol in
desensitized myometrium. Muscarinic receptor-stimulated
myometrial contraction was previously shown to be associated with the
activation of PLC (17, 20). It was of interest to investigate to what
extent refractoriness of inositol phosphate response to carbachol might lead to alteration of the muscarinic physiological event, i.e., contraction. Figure 4 illustrates the
dose-dependent curves of carbachol-mediated contraction in a control
and a carbachol-desensitized myometrium. A 2-h exposure to carbachol
resulted in an attenuated contractile response to a subsequent
rechallenge to the agonist, as illustrated by a rightward shift in the
muscarinic dose-response curve
(EC50 = 3.1 ± 0.3 and 10.4 ± 0.9 µM for control and desensitized myometrium, respectively).
Maximal contraction could nevertheless be reached in the desensitized
tissue by increasing agonist concentration. This is not surprising,
considering that maximal activation of contraction (Fig. 4) required
fivefold less concentration of carbachol than did maximal
muscarinic-induced increase (Fig. 2) in inositol phosphates
(EC50 = 15.3 ± 1.3 µM). The
data are consistent with our previous observations (7, 17, 20), where a
small generation of
InsP3 triggered
by contractile agonists would be sufficient to activate contraction
maximally.

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Fig. 4.
Influence of carbachol-induced desensitization on muscarinic-mediated
myometrial contraction. Cumulative dose-response curves were obtained
with indicated concentrations of carbachol before (control) and after 2 h of carbachol pretreatment (desensitized). Isometric contractions were
recorded during 1-min exposure of loaded myometrial strips to indicated
concentrations of carbachol. Values were expressed as percentage of
maximal contractile response due to carbachol in control tissue. Values
are means ± SE of 3-4 independent experiments.
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Modulation in the number and properties of muscarinic
receptors in desensitized myometrial membranes.
[3H]NMS binding
studies on myometrial membrane preparations demonstrated the presence
of muscarinic receptors that are mostly of the
M3 receptor subtype, similar to
the M3 receptors coupled to the
activation of the PLC-
pathway (16). Table
1 illustrates the marked loss in
[3H]NMS binding sites
in membranes prepared from tissues exposed for 2 h to 100 µM
carbachol. The total number of
[3H]NMS binding sites
in postdesensitized membranes represented 60% of the initial number of
binding sites in membranes from untreated tissues (32 ± 5 vs. 56 ± 6 fmol/mg protein for control). Scatchard analysis of the data
(not shown) indicated that one class of
[3H]NMS binding sites
was characterized in control as well as in desensitized membranes, the
Kd of
[3H]NMS for binding to
control (0.38 ± 0.1 nM) and carbachol-treated membranes (0.40 ± 0.1 nM) being virtually identical. Figure 5 illustrates that the decline of muscarinic binding sites was rapid (25% after 15 min), with a maximal decline (40%) that was reached after 30 min of exposure of myometrial strips to carbachol. Of importance was the relationship between the time course of decrease in
[3H]NMS binding sites
and the decline in the inositol phosphate response triggered by the
muscarinic agonist (Fig. 1) in carbachol-treated tissues.

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Fig. 5.
Time course of decrease in muscarinic receptor density during
incubation of rat myometrium with carbachol. Tissues were incubated
with 100 µM carbachol. At indicated times, tissues were washed and
used for membrane preparation and for
[N-methyl-3H]scopolamine
methylchloride
([3H]NMS) binding
assay as described in MATERIALS AND
METHODS. Results were expressed as a percentage of
maximal [3H]NMS
binding (56 ± 6 fmol/mg protein) obtained in membranes from
untreated tissues. Values are means ± SE of 3 experiments.
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A fundamental property of receptors coupled to G proteins is the
ability of the agonist to allow formation of a high-affinity state of
the receptor, which is shifted to a lower-affinity state in the
presence of guanine nucleotides. Table 1 illustrates
that, in the control membranes, guanosine
5'-O-(3-thiotriphosphate) (GTP
S) decreased the
affinity of the receptors for carbachol (Ki = 0.5 ± 0.1 and 2.5 ± 0.2 µM, in the absence and presence of 100 µM
GTP
S, respectively). By contrast, in desensitized membranes, the
ability of the residual membrane receptors to form a high-affinity state seems to be impaired. Thus, in the absence of GTP
S, the Ki for carbachol
in the desensitized membranes was shifted to values fourfold greater
than that observed in control membranes, and GTP
S did not affect the
apparent affinity of the receptor for carbachol (2.0 ± 0.2 and 2.2 ± 0.3 µM in the absence and presence of GTP
S, respectively).
The data demonstrate that carbachol-induced desensitization was
associated with both a reduction in the number of muscarinic binding
sites and an uncoupled state of residual muscarinic receptors.
Role of PKC in carbachol-mediated stimulation and
desensitization. Figure
6 shows that, in the presence of 2 µM
PDBu, an exogenous activator of PKC, there was a marked and consistent
reduction (60%) in the generation of inositol phosphates triggered by
carbachol. The PDBu inhibitory effect was completely prevented by 10 µM Ro-31-8220, a selective PKC inhibitor (6), suggesting the
involvement of a PKC-mediated process. Under similar conditions, PDBu
failed to alter the production of inositol phosphates induced by ET-1 and by AlF
4. The findings
demonstrate that PKC did not exert any modulatory effect either at the
ET-1 receptor level or at the level of the G protein and PLC
activation. They rather indicate that PKC may elicit, presumably
through a phosphorylation reaction, a specific inhibition at the level
of the muscarinic receptor function.

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Fig. 6.
Specific inhibitory effect of phorbol 12,13-dibutyrate (PDBu) on
carbachol-mediated inositol phosphate generation.
[3H]inositol-labeled
myometrial strips were incubated for 10 min with 10 mM LiCl in absence
(open bars) or presence (hatched bars) of 2 µM PDBu. Tissues were
stimulated for an additional 15 min with 100 µM carbachol and 0.2 µM endothelin-1 (ET-1) or for 20 min with 20 mM NaF + 20 µM
AlCl3
(AlF 4). When used, Ro-31-8220 (10 µM) was added 5 min before PDBu (solid bar). Production of total
[3H]inositol
phosphates was expressed as percentage of maximal responses due to
carbachol, ET-1, and AlF 4, each added
alone (35 ± 4, 63.4 ± 7, and 65 ± 6% of label in
phosphoinositides, respectively). Values are means ± SE of 3-4
separate experiments.
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Because the activation of the PLC pathway by carbachol leads
to PKC activation, we examined a possible contribution of PKC in the
desensitization process triggered by carbachol. Figure 7 illustrates that the presence of
Ro-31-8220 caused a delay in the development of the process of
carbachol-induced desensitization. Thus a 15-min pretreatment with
carbachol plus Ro-31-8220 resulted in a barely detectable (10%)
attenuation of the subsequent muscarinic inositol phosphate response,
compared with a 50% attenuation after a pretreatment with carbachol
alone. However, with increasing time of pretreatment (60 min), the
extent of the attenuated carbachol response was identical whether
refractoriness was induced by carbachol alone or combined with
Ro-31-8220. These results demonstrate the involvement of a PKC-mediated
process in the initial mechanism of carbachol-induced refractoriness
and the involvement of another, PKC-independent process, in the
long-term desensitization triggered by carbachol.

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Fig. 7.
Differential time course of carbachol-induced refractoriness in absence
and presence of Ro-31-8220. Prelabeled
[3H]inositol
myometrial strips were incubated with 100 µM carbachol in absence or
presence of 10 µM Ro-31-8220. At indicated times, tissues were washed
and incubated for 10 min in presence of 10 mM LiCl before being
rechallenged for 15 min with 100 µM carbachol. Production of total
[3H]inositol
phosphates was expressed as percentage of carbachol response obtained
in untreated tissues. Values are means ± SE of 3 independent
experiments, each done in duplicate.
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|
Carbachol-mediated downregulation of
Gq
/G11
.
Our previous work (16) demonstrated the presence of
Gq
/G11
in rat myometrial membranes and its involvement in the PLC pathway. We
next examined the possible modulation of the
Gq
/G11
level in myometrial membranes derived from postdesensitized tissues. Figure 8A
displays an immunoblot of rat myometrial membranes obtained from
untreated and carbachol-pretreated tissues, resolved by SDS-PAGE. The
blot identified an apparent single band of 42 kDa, corresponding to
Gq
/G11
in all preparations. Figure 8 further illustrates that pretreatment of
myometrial strips with 100 µM carbachol led to a marked decrease at
the level of
Gq
/G11
associated with the membranes. Densitometric scanning of the immunoblot
indicated that, compared with control, the amount of
Gq
/G11
was decreased by 60-65% after 2 h of carbachol pretreatment, as
assayed with 10- and 20-µg loaded proteins. Figure
9 shows the time course of the
muscarinic-induced decrease in immunoreactive
Gq
/G11
in myometrial membranes. Compared with untreated tissue, a 15-, 30-, 60-, and 120-min treatment with 100 µM carbachol resulted in a 25 ± 3, 42 ± 5, 56 ± 6 and 66 ± 7% decrease in the level of
Gq
/G11
,
respectively, with a maximal reduction (73 ± 8%) being achieved by
4 h. When myometrial strips were exposed to carbachol for 4 h, the
half-maximal decrease in membrane-associated Gq
/G11
was observed at 30 µM and the maximal effect at 100 µM carbachol
(not shown). In the presence of atropine, carbachol failed to induce
the loss of membrane-associated
Gq
/G11
(Fig. 8A), indicating that the
process was triggered by muscarinic receptor activation. Results in
Fig. 8A also demonstrate that, in
membranes derived from carbachol-desensitized tissues, the amount of
Gs
isoforms (45 and 52 kDa)
that are not coupled to muscarinic receptor activation in rat
myometrium remained unchanged. Similarly, the amount of immunodetected
-subunits was not affected in the desensitized preparations.

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|
Fig. 8.
Sustained exposure to carbachol-induced downregulation of
membrane-associated
Gq /G11 .
A: myometrial strips were incubated
for 2 h without or with 100 µM carbachol. When used, atropine (0.1 µM) was added 5 min before carbachol. Equal amounts of membrane
proteins were resolved by SDS-PAGE (10% wt/vol
acrylamide) and then immunoblotted with
Gq /G11
antiserum (1:500 dilution),
anti-Gs (1:1,000 dilution), and
anti-G subunits (1:500 dilution).
B: tissues were incubated for 2 h in
absence or presence of 100 µM carbachol. Total homogenates (H),
cytosolic fraction (C), membrane proteins (M), and detergent-extacted
myometrial proteins (DE) were prepared as described in
MATERIALS AND METHODS. Equal amounts
of proteins (10, 25, 30, and 100 µg for M, DE, H, and C,
respectively) for each specific fraction derived from treated and
untreated tissues were subjected to SDS-PAGE. The resolved proteins
were probed by immunoblotting with
Gq /G11
antiserum (1:500 dilution). Data represent 1 of 3 separate
experiments.
|
|

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Fig. 9.
Time course of decrease in membrane-associated
Gq /G11
during carbachol treatment. After treatment of myometrial strips with
100 µM carbachol for various times, equal amounts of membrane
proteins (10 µg) were submitted to Western blot analysis in presence
of
Gq /G11
antibody. Immunoreactive bands were quantified by densitometric
scanning. Results were expressed as relative levels of
Gq /G11
compared with membranes from untreated control. Results are presented
as means ± SE from 3 independent experiments.
|
|
Data in Fig. 8B show that the
carbachol-induced decrease in membrane-associated
Gq
/G11
is not due to the transfer of G
proteins to cytoplasm. Indeed, no
significant signals were obtained by immunoblotting the corresponding
cytosolic protein fractions from either carbachol-treated or untreated
tissues. Furthermore, the decrease in membrane-associated
Gq
/G11
levels due to carbachol treatment was similarly observed in both total
myometrial homogenates (700-g
supernatants) and in detergent-extracted myometrial proteins. Taken
together, these results indicated that the decline of
Gq
/G11
levels observed in membranes derived from carbachol-desensitized tissues most probably reflects the downregulation of these proteins.
Experiments were then designed to evaluate the potential role of PKC in
the quantitative regulation of
Gq
/G11
.
Myometrial strips were incubated for 20 min in the presence of 2 µM
PDBu, conditions under which this agent was demonstrated to stimulate PKC activities (23) and to attenuate the carbachol-mediated inositol
phosphate response (Fig. 6). Myometrial strips were similarly incubated
with 2 µM PDBu for 2 h (a time period comparable with carbachol-induced desensitization). As shown in Table
2, short as well as prolonged PDBu
treatments were unable to affect the level of membrane-associated
Gq
/G11
,
indicating that agonist-induced downregulation of
Gq
/G11
did not occur subsequent to activation of phorbol ester-sensitive PKC
isoforms. The inability of Ro-31-8220 to prevent downregulation of
Gq
/G11
triggered by carbachol supports the contention that PKC did not
participate in this process.
AlF
4 induced
both desensitization of the PLC pathway and downregulation of
Gq
/G11
.
To investigate whether
Gq
/G11
downregulation is dependent or not on downregulation of muscarinic
receptors, we used AlF
4 that
activates G proteins by circumventing the need for receptor engagement.
Results in Fig.
10A
illustrate the increased production of inositol phosphates due to a
10-min stimulation by AlF
4. When
myometrial tissues were pretreated for 1 h with
AlF
4, there was a marked
attenuation of the inositol phosphate response (50 ± 6%) to a
subsequent challenge with AlF
4. A
1-h exposure to AlF
4 similarly led
to a subsequent refractory state of the myometrium to carbachol in
terms of inositol phosphate generation. The extent of such a
heterologous desensitization averaged 52 ± 6%. Heterologous
desensitization induced by AlF
4 indicated an alteration in the PLC pathway at a step distal to the
specific agonist receptor. Results in Fig.
10B further show that membranes
derived from AlF
4-pretreated tissues displayed an important decrease (72 ± 7%) in
Gq
/G11
levels. The data suggested that
Gq
/G11
downregulation occurred in a manner consecutive to its activation and
independent from any receptor participation.

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Fig. 10.
AlF 4 induced both desensitization of
[3H]inositol phosphate responses and downregulation of
Gq /G11 . A:
[3H]inositol-prelabeled myometrial strips were incubated
for 1 h in the presence or absence of 100 µM carbachol or
AlF 4 (20 mM NaF + 10 µM
AlCl3). Tissues were then washed and rechallenged for 15 min with carbachol or AlF 4. Production of
total [3H]inositol phosphates was expressed as percentage
of the responses to carbachol or AlF 4.
B: myometrial strips were incubated for 1 h in absence or presence of AlF 4 or
carbachol. Membrane proteins were prepared and
Gq /G11 immunologic procedure was
performed as described in Fig. 8. Immunoreactive bands were quantified
by densitometric scanning. Results were expressed as relative levels of
Gq /G11 compared with membranes from
untreated control. Results are presented as means ± SE from
3 independent experiments.
|
|
 |
DISCUSSION |
Our previous observations showed that, in rat myometrium, carbachol
stimulates the generation of inositol phosphates by activating muscarinic receptors that are coupled to PLC-
3 via
Gq/G11
proteins (16). Evidence is now presented that the myometrium is endowed with regulatory mechanisms that control the extent of its
responsiveness to the muscarinic agonist. Prolonged exposure of
myometrial strips to carbachol resulted in a progressive
desensitization of muscarinic receptor-mediated inositol phosphate
response. Refractoriness could be observed at the level of individual
inositol phosphates (InsP,
InsP2, and
InsP3),
indicating that desensitization occurred at the level of the receptor-G
protein-PLC cascade that hydrolyzes PtdInsP2. An
interesting indication of the muscarinic refractoriness was further
revealed at the level of PLC-
3, which is the predominant PLC-
isoform expressed in rat myometrium (15, 16). We found that, in rat
myometrial preparations, PLC-
3 was predominantly present in the
cytosol, as reported for other systems (8). Noteworthy was the
demonstration that during the earliest times of carbachol
treatment there was an increase in membrane-associated PLC-
3 that
correlates with the muscarinic-stimulated production of inositol
phosphates. Such an agonist-induced membrane translocation of PLC-
3
was markedly attenuated in the desensitized myometrium. The present
studies further demonstrated that prolonged exposure of myometrial
strips to carbachol, which causes desensitization of the muscarinic
response in terms of inositol phosphate production, is
similarly associated with a decrease in muscarinic receptor-mediated uterine contractions. The data are consistent with previous
interpretations for a major role of
InsP3 and the
accompanied release of intracellular Ca2+ in agonist-induced myometrial
contractions (7, 9, 13, 20).
Carbachol-evoked desensitization was found to be a time- and
dose-related phenomenon. A correlation was observed between muscarinic agonist concentrations that stimulate inositol phosphate generation and
those concentrations that trigger refractoriness, indicating that
carbachol-induced desensitization required active occupancy of the
receptor. The interpretation was strengthened by the observation that
atropine, a muscarinic antagonist, prevented carbachol-mediated desensitization. Kinetic analysis of desensitization triggered by
carbachol suggested the existence of two processes: a rapid, receptor-specific, homologous refractoriness that appeared as early as
10-30 min after exposure of the tissue to carbachol, followed by a
heterologous desensitization process that could be detected only after
prolonged (30 min) exposure to carbachol and that leads to an
attenuated response to oxytocin as well as to
AlF
4.
Many studies provide evidence that the initial phase of
agonist-mediated desensitization is subsequent to functional
and/or quantitative alteration of the receptor itself (2, 18,
25, 31). In this view, our binding studies with the hydrophilic ligand,
namely [3H]NMS,
demonstrated a decrease in muscarinic receptor density present at
the membrane surface as well as an uncoupling state of the residual
receptors in carbachol-postdesensitized myometrial preparations. Our
data could not provide any information about receptors that could be
internalized and/or downregulated, a phenomenon that has been
described for desensitized muscarinic receptors (18). A relationship
was noted between the time course of muscarinic receptor alteration and
the impairment of inositol phosphate response triggered by the
muscarinic agonist in carbachol-treated tissues.
Agonist-dependent phosphorylation of many G protein-coupled receptors
(2, 18, 25), including different muscarinic receptor subtypes (11, 31),
by GRK and/or second-messenger-activated kinases, is proposed
to be a key event in receptor desensitization. The data described here
for the myometrium imply that PKC, the kinase activated by signaling
pathways downstream of the receptor, exerts an inhibitory feedback
regulation at the muscarinic receptor function. Indeed, we demonstrate
that a short incubation with PDBu markedly reduced the generation of
inositol phosphates stimulated by carbachol. The effect is mediated by
PKC, since the selective inhibitor Ro-31-8220 was able to completely
prevent the action of PDBu. The failure of the phorbol ester to affect
the PLC pathway when activated by ET-1 and
AlF
4 clearly indicated that the
target for the PKC inhibitory effect was the muscarinic receptor itself
and not an element downstream of the receptor. This is in accord with
earlier observations that purified muscarinic receptors may act as in
vitro substrates for PKC (11) and that muscarinic receptor-mediated
production of inositol phosphates in numerous cell lines is
significantly affected by PKC activation (14, 28). The potential
contribution of PKC in the muscarinic desensitization was further
provided by the failure of carbachol, in the presence of Ro-31-8220, to
induce the early phase of inositol phosphate refractoriness. It is also
now well established that desensitization or inactivation of G
protein-coupled receptor signaling at the receptor level is mediated in
part by members of the GRKs (25). Muscarinic receptors have also been
shown to be agonist-dependent substrates for purified GRKs in vitro (11, 31). Furthermore, specific GRK subtypes have been demonstrated to
contribute to the desensitization in intact cells of at least two (11)
subclasses of muscarinic receptors. In view of these observations, the
contribution of GRKs in the negative regulation of the muscarinic
receptor function in the myometrium has to be retained. Although this
possibility was not investigated in the present study, the potential
activation of GRK2 by PKC is worth mentioning (5, 29). Thus the
combined effects of PKC, i.e., alterations at the muscarinic receptor
function and enhancement of GRK activities, may contribute to the
PKC-dependent process of the early phase of the muscarinic
desensitization.
Recently, it has been shown that G proteins represent an additional
target of agonist-induced desensitization (21). Chronic exposure to a G
protein-linked receptor agonist can frequently result in a decrease in
membrane level of the G protein that interacts with this receptor. Such
effects have been observed for members of the
Gs and
Gi (10, 21) families and more
recently for
Gq/G11 proteins coupled to PLC (12, 22). In the present study, we demonstrate
that sustained activation of myometrial strips with carbachol induced a
marked and selective decrease in the amount of membrane-associated
Gq/G11
-subunits without affecting the levels of
Gs
and G protein
-subunits. Evidence is further provided that the
reduction of
Gq
/G11
was subsequent to muscarinic receptor activation, since atropine by
itself was unable to cause any quantitative change in the amount of
Gq
/G11
but markedly attenuated the agonist-mediated effect. Our data argued
that the decline observed at the level of membrane-associated
Gq
/G11
corresponded most probably to a downregulation of these proteins,
consequent to their proteolytic degradation. Within the limits of the
immunologic assay, we were unable to detect any increase in cytosolic
levels of
Gq
/G11
in carbachol-exposed tissues. This is in line with current observations reporting that G protein
-subunits activated by a receptor are degraded considerably more rapidly than those in the inactive state
(30). Additionally, our observations support the contention that, in
rat myometrium, activation of PKC subsequent to muscarinic stimulation
did not contribute to downregulation of
Gq
/G11
. First, direct activation of PKC by PDBu failed to mimic
carbachol-induced downregulation of
Gq
/G11
,
and, second, the PKC inhibitor, Ro-31-8220, did not attenuate carbachol
regulation of
Gq
/G11
levels. Agonist-mediated downregulation of
Gq
/G11
,
independent of PKC activation, has similarly been reported for
angiotensin in vascular smooth muscle cells (12) and for
gonadotrophin-releasing hormone in
T3-1 pituitary cells (3). It
is reasonable to conclude that, in myometrium, downregulation of
Gq
/G11
may account for the PKC-independent process that developed during
long-term desensitization triggered by carbachol.
In rat myometrium, exposure to carbachol appeared to induce a negative
regulation at the level of both
Gq
/G11
and muscarinic receptors. A similarly concurrent regulation of the
receptor and the related G protein was observed in the prostacyclin
receptor-Gs
system (21) and
more recently in the muscarinic M1
receptor and the ATII
receptor-Gq/G11
system (12, 22). Although these observations may tend to suggest that a
functional interaction between a receptor and its cognate G protein is
required to trigger downregulation of each, recent reports provide
evidence that receptor downregulation was not a prerequisite process
for
Gq
/G11
downregulation (27). This is in line with our present data. Indeed
AlF
4, which directly activates G
proteins without any receptor engagement, was able to cause
heterologous refractoriness in terms of inositol phosphate production,
with a concomitant downregulation of
Gq
/G11
. Hence it is conceivable to postulate that downregulation of
Gq
/G11
is subsequent to its persistent activation whether direct or receptor mediated.
In conclusion, the present study illustrates that prolonged exposure of
myometrium to carbachol results in diverse negative regulatory
mechanisms operating at the level of both muscarinic receptors and
Gq/G11
proteins. Our data further suggest that the agonist-induced
Gq/G11
downregulation may be involved in part in mechanisms of long-term
desensitization of the
Gq/G11-mediated signaling system. Such a heterologous refractory state of the PLC/InsP3 pathway
would ultimately display a negative control on myometrial activities
triggered by diverse receptor-mediated contractile agonists. These
adaptive responses might be considered as an example of autoregulation
to protect the tissue from excessive fluctuations in local
concentrations of vasoactive agents that do occur in the vicinity of
the myometrium in certain physiological conditions.
 |
ACKNOWLEDGEMENTS |
We are grateful to Gisèle Thomas and Ginette Delarbre for
excellent technical assistance and for help with the figures.
 |
FOOTNOTES |
This work was supported by grants from the CNRS (Unité 0570) and
by a contribution from the Association de la Recherche contre le Cancer
(contrat no. 1355).
Address for reprint requests: Z. Tanfin, Signalisation et
Régulations Cellulaires, CNRS, EP1088, Bat 430, Université
Paris-Sud, 91405 Orsay Cedex, France.
Received 16 December 1997; accepted in final form 6 May 1998.
 |
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