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Reproductive Endocrinology |
Institut für Pharmakologie und Toxikologie der Universität Würzburg, Würzburg (S.E., M.J.L.); Frauenklinik (W.Z.), Mannheim; Medizinische Klinik und Poliklinik der Universität Essen (J.K., M.C.M., O.-E.B.), Essen; and Institut für Pharmakologie und Toxikologie der Universität Halle (O.-E.B.), Germany
Address all correspondence and requests for reprints to: O.-E. Brodde, Institut für Pharmakologie und Toxikologie, Martin-Luther-Universität Halle-Wittenberg, Magdeburger Strasse 4, D-06097 Halle, Germany.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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2-adrenergic receptor densities were similar.
Gs and Gi G-protein -subunit densities were
unaltered as assessed by Western blotting and pertussis toxin-catalyzed
[32P]ADP-ribosylation. ß-Adrenergic receptor kinase
(ßARK) activity, as determined using bovine rhodopsin as the
substrate, was the same in the two groups. Adenylyl cyclase activities
in the presence of guanine nucleotides, NaF, forskolin, or
Mn++ were also not altered by fenoterol treatment. The
messenger RNA (mRNA) concentrations of ß2-adrenergic
receptors, ßARK-I and glyceraldehyde-3-phosphate dehydrogenase (as a
reference), as determined by quantitative PCR, were unaffected by
fenoterol treatment. We conclude that tocolysis with fenoterol results
in a selective down-regulation of myometrial ß-adrenergic receptors,
which is not associated with a reduction in the respective mRNA
concentrations or alterations of 2-adrenergic receptors,
Gs and Gi -subunits, or ßARK activity or
mRNA. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Agonist-stimulation of ß-adrenergic receptors causes receptor desensitization in many cells and tissues (see Refs. 6 and 7 for reviews). Desensitization of agonist-stimulated ß-adrenergic receptors has also been observed in the myometrium. Lye et al. (8) induced preterm labor in sheep with mifepristone (RU486) and observed that iv ritodrine initially inhibited labor contractions but lost its effects within 16 h. This loss of efficacy was accompanied by a reduced cAMP-response to ß-adrenergic receptor stimulation and by reduced densities of ß-adrenergic receptors in the myometria. Other studies have also observed desensitization of myometrial ß-adrenergic receptors in sheep on prolonged agonist infusion (9, 10). In the human myometrium, ß-adrenergic receptor agonists have been shown to reduce the number of ß-adrenergic receptors, which were mainly of the ß2-subtype (11, 12). However, nothing is known about the underlying mechanisms leading to this decrease in receptor number in the myometrium.
ß-Adrenergic receptor desensitization can occur by multiple biochemical mechanisms. The two most important mechanisms are the rapid uncoupling between receptors and their G-proteins caused by phosphorylation of the receptors by the ß-adrenergic receptor kinases (ßARK) (13), followed by binding of the inhibitor protein ß-arrestin (14), and the much slower reduction of the receptor number (down-regulation) that evolves over many hours (15). When the different isoforms of ß-adrenergic receptors are expressed in identical cell lines, these desensitization mechanisms are most pronounced for the ß2-subtype, less pronounced for the ß1-subtype, and largely nonexistent for the ß3-subtype (16, 17).
The present study was undertaken to investigate the alterations that
tocolysis might cause in the myometrial ß-adrenergic receptor system,
including not only the receptors themselves, but also the
ß-adrenergic receptor kinase and the stimulatory and inhibitory
G-proteins as well as the Gi-protein coupled
2-adrenergic receptors. We report that a reduction of
the ß2-adrenergic receptor number, without a concomitant
reduction of the corresponding messenger RNA (mRNA), is the only
alteration found in response to long-term tocolysis with fenoterol.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We studied myometrial biopsies obtained from 28 patients during
Caesarean section after at least 5 days of fenoterol administration
(n = 14) or from untreated control patients (n = 14). The
studies had been approved by the ethical committee of the medical
faculty, and all patients gave informed written consent. The clinical
data of these patients are given in Table 1
. All
patients in the fenoterol group received fenoterol at the indicated
doses, and all but one received MgSO4 to enhance tocolytic
effects, and metoprolol to block ß1-adrenergic receptors.
None of the patients from the control group received catecholamines or
ß-adrenergic receptor antagonists. The average gestational age was 4
weeks higher in the control group because for ethical reasons tocolytic
treatment was given to all women of low gestational age. Myometrial
specimens of 0.5–3 g were removed from the free wall of the uterus at
the time of the Caesarean section and placed in liquid nitrogen
immediately after removal and stored at -80 C. Tissue procurement was
the same for all biopsies. Unfortunately, the amount of available
tissue did not allow to quantify each parameter in all patients.
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Crude membrane fractions were prepared from a fraction of the
frozen tissue samples as previously described (12, 18). Briefly, a
piece of frozen tissue was thawed and homogenized with an Ultra-Turrax
(Janke & Kunkel, Staufen, Germany) in ice-cold buffer (0.25
M sucrose, 5 mM Tris-HCl, 1 mM
EDTA, pH 7.4). The homogenate was centrifuged at 600 x
g for 15 min, the supernatant filtered through four layers
of medical gauze, and centrifuged at 50,000 x g for 20
min at 4 C. The resulting pellets were resuspended in 10 mM
Tris-HCl, 154 mM NaCl buffer, p.H 7.4, that contained 0.55
mM ascorbic acid to yield a protein concentration of 0.5–1
mg/mL (for ß-adrenergic receptor assessment) or in 50 mM
Tris-HCl, 0.5 mM EDTA buffer, pH 7.4, to yield a protein
concentration of 2–3 mg/mL (for 2-adrenergic receptor
assessment). The protein concentration was assessed by the method of
Bradford (19) using bovine immunoglobulin G as standard.
ß2-Adrenergic receptors were quantitated by
(-)[125I]-iodo-cyanopindolol (ICYP, SA 2200 Ci/mmol, New
England Nuclear, Dreieich, Germany) binding with six concentrations
ranging from 5–200 pM; nonspecific binding was defined as
binding in the presence of 1 µM (±)-CGP 12177
(4-(3-tertiarybutylamino-2-hydroxypropoxyl-benzimidazole-2-on).
The number of myometrial 2-adrenergic receptors was
assessed by [3H]rauwolscine (SA 80 Ci/mmol, New England
Nuclear) binding using six concentrations ranging from 0.5–10
nM; nonspecific binding was defined by the presence of 10
µM phentolamine.
Quantification of G-protein -subunits
G-protein -subunits were quantified by immunoblotting and
pertussis toxin-catalyzed ADP-ribosylation as previously described in
detail (20). Briefly, -subunits of Gs and Gi
were detected using the antisera RM/1 and AS/7 (New England Nuclear),
respectively, at a 1:500 dilution, followed by quantitation on the
blots with [125I]-protein A (SA 8.5 µCi/µg, New
England Nuclear). Pertussis toxin-catalyzed ADP-ribosylation was
performed by incubating crude myometrial membranes (50 µg) with
activated pertussis toxin in the presence of [32P]-NAD
(SA 30 Ci/mmol) for 60 min at 30 C. Proteins were resolved by SDS-PAGE,
and the ADP-ribosylated bands of 39–41 kilodaltons were excised and
quantitated by Cerenkov counting.
Determination of ßARK activity
Enzymatic activity of ßARK was measured as previously described (21, 22). Frozen myometrial tissue (100 mg) was homogenized for 30 sec with a polytron device in 1 mL 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, and centrifuged for 10 min at 200,000 x g. The protein concentration in the supernatants was determined according to Bradford (19). Urea-treated rod outer segments containing >95% rhodopsin were prepared from bovine retinae as the substrate for ßARK. Aliquots (50 µg protein) of these cytosolic preparations were incubated with 500 pmol rhodopsin in the same buffer containing 10 mM MgCl2 and 0.3 mM [32P]ATP in a total volume of 60 µL. 32P incorporation into rhodopsin was determined by separation of the reaction mixture by SDS-PAGE and quantitation of the radioactivity in the rhodopsin band with a phosphoimager (Fuji, Tokyo, Japan).
Determination of mRNA concentrations
RNA from the frozen tissue samples was prepared by a shortened version of the protocol of Chomczinski and Sacchi (23) essentially as recently described (24, 25). The purity of the RNA was checked by measuring the ratio of the absorbance at 260 and 280 nm and was 1.8–2.0 in all cases. Aliquots (500 ng) of RNA were reverse transcribed into complementary DNA (cDNA) using random hexamers and Superscript II reverse transcriptase (GIBCO/BRL, Gaithersburg, MD) as described elsewhere (24). In all experiments, reactions containing no reverse transcriptase were done as negative controls.
Sense and antisense oligonucleotide primer pairs were synthesized to
match the sequences of the human ß2-adrenergic receptor
(26), human ßARK-I (21) and human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH (27). Details about the primers and the expected
PCR products are given in Table 2. PCR was done with the
transcript obtained from 50 ng RNA using 0.5 µM of the
respective primers, 1.25 U Taq polymerase (Perkin Elmer,
Norwalk, CT), 200 µM desoxynucleotides plus 0.3 µCi
[-32P]deoxycytidine triphosphate, 1.5 mM
MgCl2, 50 mM KCl, 10 mM Tris-HCl,
pH 8.3, in a volume of 50 µL. Amplifications were done in a
Perkin-Elmer model 480 thermal cycler with denaturation at 94 C for 1
min (3 min in first cycle), annealing for 1 min at the temperatures
indicated in Table 2, and an extension at 72 C for 1 min (10 min in
last cycle). Multiple samples from different biopsies were assayed for
each gene using a single master reaction mixture. The PCR products were
isolated by electrophoresis on a 10% polyacrylamide gel. After drying
on a gel dryer, the incorporated radioactivity was determined using a
phosphoimager (Fuji).
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Adenylyl cyclase assays
Adenylyl cyclase activity was assessed as described by Salomon
et al. (28) with minor modifications as detailed elsewhere
(29). Two different incubation conditions were employed. For Gpp(NH)p,
NaF, and forskolin activation, membranes (30–40 µg protein) were
incubated for 10 min at 30 C in a final volume of 100 µL containing
40 mM HEPES buffer, pH 7.4, 5 mM
MgCl2, 1 mM EDTA, 500 µM
[-32P]ATP, 100 µM cAMP, and an ATP
regenerating system (5 mM phosphocreatine and 50 U/mL
creatine phosphokinase, buffer A). For Mn++ activation,
membranes were incubated in buffer A without Mg++. The
reaction was stopped by adding 0.8 mL 50 mM Tris-HCl buffer
(pH 7.4 at 25 C) containing 40 mM ATP and 1.4
mM cAMP. [3H]cAMP (5,000–10,000 cpm) was
then added to monitor the recovery of [32P]cAMP (28).
Statistical analysis
Data are expressed as mean ± SEM. Comparison between the two different groups was performed by two-tailed unpaired t tests. A P value <0.05 was considered significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The high-affinity antagonist radioligand ICYP was used to
quantitate ß-adrenergic receptors in human myometrial membranes,
85.5% of which are of the ß2-subtype (12). The number of
binding sites for ICYP was 6.3 ± 0.6 fmol/mg protein in the
control group but only 2.8 ± 0.4 fmol/mg protein in patients who
had been treated with fenoterol (P < 0.01, Fig. 1A). On the other hand, ß-adrenergic receptor number
in the control and in the tocolysis group was not significantly related
to the gestational age (r2 0.2344 and 0.0497, respectively;
P > 0.05 in both groups; Fig. 1B). The number of
2-adrenergic receptors was not significantly changed in
the fenoterol treatment group (Fig. 1A).
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Gs and Gi -subunits were assayed by
quantitative Western blotting using the RM/1 or AS/7 antiserum,
respectively, in conjunction with [125I]-protein A
detection. The densities of both classes of -subunits were
unaffected by fenoterol treatment (Table 3). Likewise,
the concentration of substrates for pertussis toxin-catalyzed
ADP-ribosylation, which corresponds to the -subunits of the
Go/Gi-family, were not different between the
two groups of patients (Table 3). These results indicate that G-protein
densities in human myometrium were not affected by tocolytic
treatment.
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, Gpp(NH)p-, NaF-,
forskolin-, and Mn++-induced adenylyl cyclase activation
was not affected by fenoterol treatment, indicating that the functional
activities of Gs, Gi, and the catalytic unit of
adenylyl cyclase were not altered.
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To test whether the ß-adrenergic receptor down-regulation is
[as in heart failure (24, 33)], accompanied by increased ßARK
expression, we measured the total ßARK activity in cytosolic
preparations from the myometria. These assays were done using rhodopsin
as the substrate, which can be phosphorylated not only by the
predominant kinase, ßARK-I, but also by the other related members of
this kinase family (13). Rhodopsin phosphorylation was readily obtained
with cytosolic preparations from both control and fenoterol-treated
patients (Fig. 3, inset). Quantitative
analysis of these assays revealed almost identical activities in the
two groups of patients (Fig. 3).
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To determine the steady state concentrations of the mRNAs for the
ß2-adrenergic receptor and also for ßARK-I,
quantitative RT-PCR were carried out with RNA prepared from human
myometria. GAPDH, a housekeeping gene widely used for standardization
in such experiments, was chosen as an endogenous control. The absolute
values of the expression of GAPDH in these biopsies were very similar
in both groups (data not shown). In agreement with our earlier data on
myocardium (24, 25, 33), this indicates that this mRNA is not affected
by ß-adrenergic stimulation and can therefore be used for
normalization. Figure 4A shows the PCR products obtained
for the ß2-adrenergic receptor, GAPDH, and ßARK-I. In
all cases, a single well-defined product of the appropriate size was
obtained, which could be used for quantification by phosphoimaging.
Such quantifications are shown in Fig. 4B and demonstrate exponential
amplification between cycles 30 and 34 for ßARK-I, cycles 21 and 25
for GAPDH, and cycles 28 and 34 for ß2-adrenergic
receptor. Consequently, 32 cycles were chosen for the amplification of
ßARK, 30 for the ß2-adrenergic receptor, and 23 for
GAPDH. The efficiencies of amplification were almost identical, which
allows the use of the GAPDH-product for normalization.
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). Likewise, in spite of the marked reduction of the
ß2-adrenergic receptor expression, fenoterol treatment
had no effect on their mRNA (Fig. 5). This suggests that the
down-regulation of the receptors occurs on the protein rather than the
mRNA concentration.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The present study was undertaken with the hypothesis that chronic
activation of myometrial ß2-adrenergic receptors with
ß2-adrenergic receptor agonists might cause similar
changes as those observed for cardiac ß1-adrenergic
receptors in chronic heart failure, and that the ensuing
desensitization of the ß2-adrenergic receptor system
might be a reason for the limited usefulness of this tocolytic
therapeutic regimen. According to standard medical practice in Germany,
it is not considered acceptable to withhold ß2-adrenergic
agonist treatment in women in preterm labor. Moreover, a
ß1-selective antagonist (e.g.
metoprolol) and MgSO4 are added regularly to minimize
concomitant stimulation of cardiac ß1-adrenoceptors and
to enhance tocolytic efficacy, respectively. Therefore, the women
receiving the ß2-adrenergic agonist differ from the
control patients of our study in three additional ways,
i.e. gestational age, metoprolol treatment, and
MgSO4 treatment. Although we cannot rule out that any of
these additional factors has affected the outcome of our study, we feel
that the fenoterol treatment had the dominant effect for the following
reasons: to our knowledge MgSO4 has never been shown to
affect ß-adrenergic receptor function or desensitization, and no
mechanism to this effect is known. Although treatment with
ß1-selective antagonists such as metoprolol can
up-regulate ß1-adrenergic receptors, the density of
ß2-adrenergic receptors was not affected in such studies
(45). A more serious problem is whether different gestational ages
might have contributed to the observed data in our study. For example,
it has been observed that human myometrial ß-adrenergic receptors
undergo functional uncoupling towards the end of pregnancy,
i.e. in gestational weeks 39–40 (46). However,
we feel that this cannot explain the reduction of ß-adrenergic
receptors seen in our study for several reasons. First, gestational age
and myometrial ß-adrenergic receptor density were not significantly
correlated within the control or tocolysis group. Second,
ß-adrenergic receptor density was also lower in the tocolysis group
in the subset of patients with similar gestational ages in both groups
(cf. Fig. 1B
). Third, the data by Litime et al.
(46) imply functional uncoupling towards the end of pregnancy, which,
if anything, would result in an understimation of the desensitization
in tocolysis patients with a smaller gestational age compared with
control term women. Therefore, it appears that the
ß2-adrenergic agonist treatment indeed is the most likely
cause of our observations. Confirmation in a study design with random
allocation of tocolytic treatment would be useful, but that is not
considered ethically acceptable in Germany.
The overall pattern of changes in the present study was quite different
from that observed for ß1-adrenergic receptors in heart
failure: we found that tocolysis with fenoterol caused an isolated
decrease of receptor densities, without a concomitant decrease of the
corresponding mRNA. Neither the activity or mRNA concentration of
ßARK-I nor the concentrations or functional activities of the
relevant G-protein -subunits were altered. Furthermore, we found no
evidence for a cross-regulation of 2-adrenergic
receptors.
A possible reason for this discrepancy might lie in the fact that
whereas heart failure develops over very long periods of time,
tocolysis in our patients was only performed for days, and that the
other changes observed in heart failure might appear only later.
However, application of ß-adrenergic receptor agonists to rats for 4
days has been reported to cause not only down-regulation of cardiac
ß-adrenergic receptors, but also receptor uncoupling as well as
up-regulation of Gi -subunits (43, 44). Furthermore,
rapidly evolving experimental models of heart failure in dogs have been
reported to decrease ß-adrenergic receptor number und coupling,
Gs -subunit expression, and adenylyl cyclase (for
references see Ref.34). Thus, the cardiac ß-adrenergic receptor
system of several species reacts to changes in ß-adrenergic
stimulation with an array of changes in a matter of days. This suggests
that the different patterns of changes in the cardiac and myometrial
ß-adrenergic receptor systems are not caused by the relatively short
duration of ß-adrenergic receptor activation during tocolysis. On the
other hand, similar to the myometrium (present study), we recently
found that a 2-week treatment of healthy volunteers with the
ß2-adrenergic receptor agonist terbutaline markedly
reduced lymphocyte ß2-adrenergic receptor number but did
not affect lymphocyte Gi-densities (47), and that treatment
of the human neuroblastoma cell line, SK-N-MC, with isoprenaline for
24 h caused a marked decrease in ß1-adrenergic
receptor number but did not change Gi-densities (48);
extending the incubation to 4 days also did not lead to any changes in
Gi (our unpublished observations).
A second possible explanation for these differences is that whereas the
cardiac ß-adrenergic receptors are mostly of the
ß1-subtype (39), the ß2-subtype
predominates in the myometrium (11, 12). However, when expressed in the
same cell line, the ß2-subtype has been shown to be more
readily regulated than the ß1-subtype (16). Furthermore,
cross-regulation between ß-adrenergic receptors and Gi
-subunits and 2-adrenergic receptors has been
demonstrated much better for the ß2- than for the
ß1-subtype (49, 50). And finally, alterations in
Gi -subunit expression are thought to be cAMP-mediated
(42), and both the ß1- and the ß2-subtype
are coupled to increases in cAMP. Thus, there is no reason to assume
that in a tissue expressing ß2-adrenergic receptors the
regulatory response to ß-receptor agonists would be more limited than
in a tissue expressing the ß1-subtype.
Taken together, the most reasonable explanation for the different types of regulation of myometrial and myocardial ß-adrenergic receptors is the hypothesis that there are tissue or organ-specific factors that determine the pattern of receptor desensitization. Such tissue-specific factors might be the expression of specific regulatory proteins, such as the receptor kinases, phosducins, or mRNA-regulatory proteins (51, 52, 53).
It is interesting to note that tocolysis causes a reduction of myometrial ß2-adrenergic receptor densities without a concomitant reduction of the corresponding mRNA. In many studies on isolated cells, a similar time course for the agonist-induced down-regulation of ß2-adrenergic receptors and their mRNA has been observed, and this mRNA reduction has been suggested to be an essential mechanism of receptor down-regulation (54, 55, 56, 57). This may indeed be true for cell culture lines, because a continuous synthesis is necessary to maintain receptor densities in a rapidly dividing cell population. However, in the tissues of intact animals, ß-adrenergic receptors have a half-life of many days to several weeks (58). Thus, reduced mRNA concentrations should not be able to induce substantial down-regulation of the receptors themselves in a matter of a few days (7), and in fact, we did not even find any reduction in myometrial ß2-adrenergic receptor mRNA after tocolysis. The down-regulation of these receptors by tocolysis must therefore be caused by degradation of the receptors themselves.
In summary, our study indicates that tocolytic treatment with fenoterol caused only a single change in the myometrial ß2-adrenergic receptor system: an isolated down-regulation of the receptors that was most likely caused by direct degradation of the receptors. Comparison of these changes with those observed in the cardiac ß-adrenergic receptor systems underlines the importance of tissue specific factors in receptor regulation. It appears likely that the down-regulation of myometrial ß-adrenergic receptors limits the usefulness of ß-adrenergic receptor agonists as tocolytic agents.
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Footnotes |
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Received June 12, 1996.
Revised November 6, 1996.
Accepted January 1, 1997.
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2. J Biol Chem. 265:14784–14790.1-adrenergic receptor mRNA levels. J Biol Chem. 266:2233–2238.
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C. Rouget, M. Bardou, M. Breuiller-Fouche, C. Loustalot, H. Qi, E. Naline, T. Croci, D. Cabrol, C. Advenier, and M. J. Leroy {beta}3-Adrenoceptor Is the Predominant {beta}-Adrenoceptor Subtype in Human Myometrium and Its Expression Is Up-Regulated in Pregnancy J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1644 - 1650. [Abstract] [Full Text] [PDF]
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) and nontreated pregnant women (
). For
details see Patients and Methods. Ordinate: myometrial
ß-adrenergic receptor density in femtomoles ICYP specifically bound
per milligrams protein; abscissa: gestational age of patients in
weeks.
