Departments of 1 Obstetrics and Gynecology and 2 Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75235
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ABSTRACT |
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Although it is well known that progesterone alters uterine contractility and plays an important role in maintenance of pregnancy, the biochemical mechanisms by which progesterone alters uterine contractility in human gestation are less clear. In this investigation we sought to identify progesterone-induced adaptations in human myometrial smooth muscle cells that may alter Ca2+ signaling in response to contractile agents. Cells were treated with vehicle or the progesterone analog medroxyprogesterone acetate (MPA) for 5 days, and intracellular free Ca2+ concentration ([Ca2+]i) was quantified after treatment with oxytocin (OX) or endothelin (ET)-1. OX- and ET-1-induced increases in [Ca2+]i were significantly attenuated in cells pretreated with MPA in a dose-dependent manner. Progesterone receptor antagonists prevented the attenuated Ca2+ transients induced by MPA. ETA and ETB receptor subtypes were expressed in myometrial cells, and treatment with MPA resulted in significant downregulation of ETA and ETB receptor binding. MPA did not alter ionomycin-stimulated increases in [Ca2+]i and had no effect on inositol trisphosphate-dependent or -independent release of Ca2+ from internal Ca2+ stores. We conclude that adaptations of Ca2+ homeostasis in myometrial cells during pregnancy may include progesterone-induced modification of receptor-mediated increases in [Ca2+]i.
progesterone receptor; endothelin receptors; uterus; endothelin; oxytocin; antiprogestin; medroxyprogesterone acetate
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INTRODUCTION |
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IN MOST SPECIES at full term, progesterone withdrawal, together with increasing levels of estrogen, leads to increased expression of a number of genes believed to be important in the onset of parturition (6, 21, 42). It has also been suggested that the contractile phenotype of uterine smooth muscle is increased by estradiol treatment and decreased by progesterone (8, 39). Although it is well known that progesterone alters uterine contractility and plays an important role in maintenance of pregnancy in most species (5), the biochemical mechanisms by which progesterone alters uterine contractility in human gestation are less clear.
The effects of progesterone on uterine smooth muscle appear to be multiple. Progesterone may promote uterine relaxation by nongenomic and genomic mechanisms. Nongenomic effects of acute progesterone exposure include inhibition of transmembrane Ca2+ entry, release of Ca2+ from intracellular stores (19), and membrane hyperpolarization with subsequent activation of K+ channels (27). Recently, the list of nongenomic effects of progesterone has been expanded to include direct inhibition of oxytocin (OX) binding to the rat, but not the human, OX receptor (11). Most effects of progesterone, however, are mediated through its specific binding to nuclear hormone receptors with subsequent changes in expression of target genes. Specifically, progesterone inhibits expression of connexin43 (10, 21), modulates OX receptor density (16, 34), decreases estradiol-induced increases in cGMP-dependent protein kinase (39), and decreases the expression of interleukin-8 in myometrial and cervical stromal cells (15). The effects of progesterone on intracellular Ca2+ homeostasis in intact myometrial cells have not been systematically evaluated.
Previously, we reported that treatment of human myometrial smooth muscle cells in culture with endothelin (ET)-1 resulted in marked increases in intracellular free Ca2+ concentration ([Ca2+]i) and the extent of myosin light chain phosphorylation (41). ET-1 is a member of a family of sarafotoxin-like peptides and appears to be an important endogenous modulator of uterine contractility (26, 40, 41). The agonist and its receptors have been identified in the human uterus (30). ET-1 and OX increase [Ca2+]i by at least two mechanisms. Both agonists bind to plasma membrane receptors, resulting in influx of extracellular Ca2+ and inositol trisphosphate (IP3)-mediated release of Ca2+ from intracellular stores (9, 24). In myocytes, this transient elevation of [Ca2+]i is quickly reversed by high-efficiency plasma membrane and sarcoplasmic reticulum Ca2+ pumps and an Na+/Ca2+ exchange mechanism (19).
In this investigation we sought to identify progesterone-induced adaptations in OX- and ET-1-mediated Ca2+ signaling in myometrial smooth muscle cells. Treatment of human myometrial smooth muscle cells in culture with the progesterone analog medroxyprogesterone acetate (MPA) resulted in significant decreases in ET-1- and OX-mediated increases in [Ca2+]i. This effect was reversed by the specific progesterone receptor antagonist ZK-98299. Treatment with MPA also resulted in a significant decrease in ET receptor binding. Progestin treatment had no effect on IP3-dependent or -independent release of Ca2+ from the sarcoplasmic reticulum. These results suggest that human myometrial smooth muscle cells in primary culture are responsive to progesterone with decreases in OX and ET receptor density. Thus one mechanism by which progesterone promotes uterine relaxation during pregnancy may be to decrease binding of contractile agents with resulting decreases in [Ca2+]i.
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MATERIALS AND METHODS |
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Human myometrial smooth muscle cells. Human uterine tissue was obtained from nonpregnant women at the time of hysterectomy for reasons other than endometrial or myometrial disease. Consent for the use of tissue was obtained from the women undergoing surgery according to a protocol approved by the Institutional Review Board. The tissue was transported to the laboratory in Hanks' balanced salt solution that contained 25 mM HEPES and 0.1875% sodium bicarbonate, pH 7.4. Fresh tissues were minced and incubated for 16 h at 34°C in a solution that contained collagenase B (1 mg/ml) and DNase (0.15 mg/ml) to disperse the smooth muscle cells. Thereafter, the smooth muscle cell fraction was purified by a discontinuous Percoll density gradient (1.03/1.06) for 20 min at 2,000 g. Smooth muscle cells were collected and diluted with DMEM buffered with 25 mM HEPES and 0.1875% sodium bicarbonate, pH 7.4. The medium was supplemented with fetal bovine serum (10% by volume), penicillin (200 U/ml), streptomycin (200 µg/ml), and sodium pyruvate (1 mM) and centrifuged (700 g for 10 min). The cell pellet was washed and resuspended in DMEM and centrifuged, and the final pellet was resuspended in DMEM that contained 10% fetal bovine serum. Myocyte viability was >90% as determined by trypan blue staining. The cells were plated at 5 × 105 cells/cm2 with the culture medium changed every other day until confluency (2-4 days). The cells were treated with MPA for 5 days, including serum-free medium 24 h before experimental protocols. For comparison, smooth muscle cells from rabbit aorta were prepared in an identical fashion, except elastase (1 mg/ml) was added to the tissue digest medium, and cells were dispersed within 4 h.
Determination of
[Ca2+]i
in myometrial cells.
Smooth muscle cells were plated on glass coverslips (9 × 35 mm) as described above. Cells were loaded with fura 2 by
incubation in culture medium that contained 5 µM fura 2-AM for 30 min. Coverslips were washed three times and incubated for an additional
15 min in buffer containing (in mM) 4.8 KCl, 130 NaCl, 1.0 MgCl2, 1.5 CaCl2, 1.0 Na2HPO4,
15 glucose, and 10 HEPES (pH 7.4) supplemented with 0.1% (wt/vol)
human serum albumin. Experiments were completed within 1 h of loading.
Fura 2-containing cells were rinsed with albumin-free buffer, and
coverslips were mounted in a cuvette equipped with an electronically
controlled, mini-motorized Teflon rotor (Instech Laboratories, Horsham,
PA). Fura 2 fluorescence was recorded with a fluorescence
spectrophotometer (model 650-10S, Perkin-Elmer, Norwalk, CT) at
excitation wavelength of 340 nm, and emission was monitored at 510 nm.
Measurements were corrected for autofluorescence and extracellular
fluorescence.
[Ca2+]i
was calculated according to the formula:
[Ca2+]i = Kd(F Fmin)/(Fmax
F), where
F is the experimentally determined fluorescence,
Fmax the maximum
fluorescence in the presence of 50 µM ionomycin, and
Fmin the minimum
fluorescence in the presence of 12 mM EGTA plus 20 mM Tris base (22).
The Ca2+ dissociation constant
(Kd) for fura 2 is 224 nM (12).
Ca2+ fluxes
in permeabilized myocytes.
IP3-induced
Ca2+ release from internal stores
was quantified as described previously (31) with modification. Briefly,
confluent primary cultures of myometrial cells in 35-mm dishes were
rinsed three times with Hanks' balanced salt solution and
permeabilized for 10 min in the medium containing 120 mM KCl,
2 mM MgCl2, 1 mM
ATP, 1 mM EGTA, 30 mM imidazole-HCl (pH 6.8), and saponin (20 µg/ml).
Cells were washed with buffer without saponin for 40 min. Thereafter,
intracellular Ca2+ stores were
loaded for 30 min at 25°C in buffer containing (in mM)
120 KCl, 10 NaN3, 5 MgCl2, 5 ATP, 0.44 EGTA,
0.08 45 + 40CaCl2,
and 30 imidazole-HCl (pH 6. 8). Loading of intracellular Ca2+ stores was terminated by
replacing loading medium with efflux medium containing (in mM) 120 KCl,
5 NaN3, 2 MgCl2, 1 ATP, 1 EGTA, and 30 imidazole (pH 6.8). Concentration of free
Ca2+ in the efflux medium was
brought to 4 × 109 M. The cells were rinsed for 3 min with 1-ml aliquots of efflux medium.
The first two aliquots were discarded, representing
45Ca2+
not taken up by the stores. Subsequent aliquots were placed in scintillation vials, and radioactivity was determined by scintillation counting. Permeabilized cells were treated with
IP3 and ionomycin at specified
time points. Nonspecific
45Ca2+
efflux before treatment was subtracted from the sum of aliquots obtained during 5 min after treatment with test agents to quantify Ca2+ release from intracellular
stores as nanomoles of Ca2+ per
milligram of protein per 5 min. Cells were then removed from the
dishes, and protein concentration was determined by the Lowry method.
Radioligand binding studies and ET receptor subtype determination.
ET receptor binding characteristics and subtype expression were
determined in confluent myocytes treated identically to those used for
analysis of
[Ca2+]i.
Confluent myocytes in short-term primary culture were treated for 4 days with vehicle (0.1% ethanol) or
107 M MPA in 12- or 24-well
plates. Control and MPA-treated cells were placed in serum-free medium
24 h before radioligand binding assays. Thereafter, cells were washed
twice with prewarmed serum-free, steroid-free DMEM supplemented with
0.3% BSA (binding buffer). ET receptor binding and subtype assays were
performed in a total volume of 1 ml of binding buffer. Specific binding
of 125I-labeled ET-1 reached
equilibrium by 60 min; therefore, 60-min incubations were used in all
binding studies. Incubations were conducted at 37°C and terminated
by four rapid 1-ml washes of ice-cold binding buffer. The cells were
dislodged and dissolved by incubation with 0.1 M NaOH for 20 min at
25°C. The protein concentration for cells in each well was
determined by a modification of the methods of Lowry. The effluent was
transferred to scintillation vials, and the radioactivity was
determined with a scintillation counter (Packard Instruments, Downers
Grove, IL) with an efficiency of 83% for
125I.
Chemicals.
Fura 2-AM was obtained from Molecular Probes (Eugene, OR), ionomycin
and IP3 from Calbiochem
(Alexandria, Australia), and 45CaCl2
and 125I-labeled ET-1 from Dupont
NEN (Boston, MA). The ET receptor antagonists FR-139317 and IRL-1038
were generously provided by Dr. M. Yanagisawa (University of Texas
Southwestern Medical Center at Dallas). The progesterone receptor
antagonist
11-(4-dimethylamino-phenyl)-17
-hydroxy-17
-(3-hydroxypropyl)-13
-methyl-4,9-gonadien-3-one (ZK-98299) was a gift from Schering (Berlin, Germany). All other compounds were purchased from Sigma Chemical (St. Louis, MO).
Statistical analysis.
Values are means ± SE. For values with equal variances, multiple
comparisons were conducted with ANOVA followed by Student-Newman-Keuls test. Nonparametric comparisons were used for data with disparate variances (Kruskal-Wallis analysis followed by
2 approximation).
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RESULTS |
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Effect of progesterone on
[Ca2+]i
in myometrial cells.
To study the effect of progesterone on intracellular
Ca2+ homeostasis in human uterine
smooth muscle, myometrial smooth muscle cells in short-term primary
culture were used as a model system. Cells were treated with
107 M MPA for 5 days, and
intracellular Ca2+ transients in
response to contractile agents and
Ca2+ ionophore were quantified
with fura 2. The synthetic derivative of progesterone, MPA, was
utilized in these studies, because these cells metabolize progesterone
to 3
-hydroxy-5
-pregnan-20-one, 3
-hydroxy-5
-pregnan-20-one,
and 5
-pregnane-3,20-dione (38; unpublished observations). Relatively
long preincubation times were utilized to recapitulate results obtained
in studies utilizing steroid hormones to modulate uterine contractility
in vivo (15, 16, 20, 23). Myometrial cells responded to 20 nM OX and 10
7 M ET-1 with marked
increases in cytoplasmic Ca2+
(Fig. 1). As expected,
progestin pretreatment resulted in significant attenuation of
OX-induced Ca2+ transients. These
results confirmed that these cells were responsive to OX and
progesterone in vitro in a manner analogous to those in vivo.
Short-term incubations (<1 h) with
10
7 M progesterone or
10
7 M MPA or simple
inclusion of the steroid in the buffer did not alter
[Ca2+]i
responses in these cells.
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Effect of antiprogestins on
[Ca2+]i
in myometrial cells.
We used progesterone receptor antagonists to test the hypothesis that
inhibition of OX and ET-1 responses involved progesterone receptors.
Myometrial cells were treated with 2 × 108 M MPA, progesterone
receptor antagonists (RU-486 or ZK-98299, 10
6 M), MPA + RU-486, or
MPA + ZK-98299 (Table 1, Fig.
4). Intracellular Ca2+ transients in response to OX
and ET-1 were inhibited by MPA. Although differences between OX and
ET-1 responses in control and RU-486-treated cells did not reach
statistical significance, these responses tended to be decreased in
RU-486-treated cells (Table 1). RU-486 partially prevented
MPA-induced inhibition of OX and ET-1 responses (Table 1). It is likely
that the mixed agonist-antagonist properties of RU-486 resulted in
these complex responses (7, 26, 36). The "pure" progesterone
receptor antagonist ZK-98299 alone resulted in small, but significantly enhanced, OX responses, whereas ET-1 responses were unaffected by
ZK-98299 (Fig. 4). Similar to RU-486, ZK-98299 completely prevented MPA-induced inhibition of OX- and ET-1-induced
Ca2+ transients (Fig. 4).
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Effect of progesterone on intracellular
Ca2+ stores.
The finding that progesterone altered receptor-mediated
increases in
[Ca2+]i
but not ionophore-mediated responses suggested that release of
Ca2+ from
IP3-dependent stores may be
altered in progestin-treated cells. To test this possibility,
Ca2+ efflux in response to
IP3 and ionomycin was quantified
in MPA-treated and control cells (Table 2).
IP3 (0.4 and 4 µM) resulted in
release of Ca2+ from intracellular
stores in control and MPA-treated cells. Additional intracellular
Ca2+ was released from
IP3-insensitive stores after
treatment with 10 µM ionomycin.
IP3-sensitive and total internal
Ca2+ stores were similar in
control and MPA-treated cells (Table 2). These results indicate that
the amount of intracellular Ca2+
stores and IP3-mediated release of
intracellular Ca2+ are not altered
in progestin-treated cells.
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Effects of progesterone on ET receptor binding characteristics and
subtype expression.
The effect of progesterone on specific binding of ET-1 in intact
myometrial smooth muscle cells was determined. LIGAND analysis of the
displacement of 125I-labeled ET-1
by unlabeled ET-1 demonstrated a one-site model for receptors in
primary cultured myometrial smooth muscle cells (P < 0.05, r > 0.93). Representative Scatchard
plots of specific binding data for myometrial smooth muscle cells from
nonpregnant women cultured in the absence or presence of MPA are
illustrated in Fig.
5A. The
slopes of the two treatment groups did not differ, reflecting similar
Kd
values: 0.13 ± 0.02 (control) compared with 0.14 ± 0.03 nM
(MPA). Specific binding at
Kd
for ET receptors in myometrial cells exceeded 81%. Hill coefficients
ranged from 0.77 to 0.99 and from 0.87 to 0.96 in control and
progestin-treated cells, respectively, indicating that cooperativity
was not involved in the
125I-labeled ET-1 binding to these
cells.
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DISCUSSION |
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Progesterone-induced myometrial quiescence is essential to the maintenance of most, if not all, mammalian pregnancies (5). In a number of systems the effects of progesterone are antagonistic to those of estrogen (9, 10, 14, 18, 28). Progesterone inhibition of estrogen action is complex, involving downregulation of estrogen receptors, induction of estrogen metabolism (35), or transcriptional repression of estradiol-responsive genes (37). Progesterone, however, does not inhibit estrogen responses universally. For example, uterine OX gene expression is synergistically regulated by estradiol and progesterone (20). In this investigation we evaluated the effect of progesterone on Ca2+ homeostasis in myometrial smooth muscle cells in short-term primary culture.
Previous studies have demonstrated that progesterone decreases OX binding in rabbit, rat, human, and ovine uterine smooth muscle cells (8, 9, 16, 20, 28, 34). Thus we used OX effects as a control for progesterone responsiveness. Although there is no doubt that estrogen and progesterone receptor concentrations varied considerably among the tissues obtained from women in different phases of the menstrual cycle, progesterone responsiveness was remarkably consistent in the cultured cells (plated at high density and used within 7-10 days). During the course of this investigation, 24 primary cultures of human myometrial cells were treated with MPA. With the exception of two cell cultures, all were responsive to progestin treatment with inhibition of OX responsiveness. Cells in passage 1 or >10 days in primary culture were not responsive to progesterone, presumably because of loss of expression of progesterone receptors, as has been described for other uterine cells (33).
Progesterone-induced attenuation of OX responses was prevented by progesterone receptor antagonists. ZK-98299 alone resulted in small, but significant, enhancement of OX responses, whereas ET-1 responses were unaffected. Antiprogestins have been shown to increase uterine sensitivity to OX in a number of in vivo and in vitro studies (2, 3, 6). Increased OX sensitivity is believed to be due to prostaglandin-mediated increases in OX receptors (6, 42), although other mechanisms are clearly possible (3). Thus the enhanced OX sensitivity in myometrial cells treated with ZK-98299 may be consequent to increased prostaglandin production in antiprogestin-treated cells. To our knowledge, expression of ET receptors is not regulated by prostaglandins. Thus the finding that ZK-98299 enhanced sensitivity to oxytocin, but not ET-1, is not surprising. In addition, antiprogestins may alter OX receptor-associated G proteins or other processes associated with G protein coupling that are distinct from those associated with ETA receptors.
Recently, Grazzini et al. (11) reported that progesterone, R-5020, and
RU-486 inhibited OX binding to rat OX receptors. Progesterone, R-5020,
and RU-486 did not inhibit OX binding to human OX receptors on cell
membranes. In the present study we confirmed and extended these
findings. Short-term incubations (<1 h) with
107 M progesterone or
10
7 M MPA did not alter
[Ca2+]i
responses in intact human myometrial smooth muscle cells. Inhibition of
OX and ET-1 responses, however, was observed after treatment with MPA
for 5 days. Thus the results of this study suggest that progesterone,
acting through its nuclear hormone receptor, attenuates OX- and
ET-1-induced increases in
[Ca2+]i
through downregulation of human OX and ET receptors. The role of
progesterone receptors in this process is indicated by the findings
that 1) progesterone receptor
antagonists precluded the diminished ET-1 response,
2) progesterone did not alter ET-1
responsiveness in cells deficient in progesterone receptors, and
3) other steroid hormones did not
alter ET-1 responses.
The data reported here demonstrate that progesterone decreases ETA and ETB receptor density proportionately and decreases the acute response to ET-1 in myometrial smooth muscle cells from nonpregnant women. This effect of progesterone does not appear to be limited to myometrium. Recently, others have reported that progesterone also inhibits ET-mediated luteinizing hormone secretion in cultured rat pituitary cells (28). In vivo, progesterone inhibits estradiol-induced increases in ET receptors in rabbit myometrium (21); however, estradiol did not directly alter ET receptors in myometrial smooth muscle cells in culture (21). Recently, Osada and co-workers (30) reported that ET-1-induced contractile force is increased in human myometrium during pregnancy and that this effect was mediated by increased ETA receptors. The reported increases in ETA receptors in myometrium from pregnant women in late gestation may reflect increased estradiol levels during human pregnancy, decline in progesterone responsiveness at full term, or other processes, such as uterine hypertrophy and myometrial stretch, that were not manifest in the current study.
Progesterone has no effect on IP3-inducible Ca2+ release from the sarcoplasmic reticulum or on total intracellular Ca2+ stores. Thus fundamental mechanisms of intracellular Ca2+ stores are not affected by activation of progesterone receptors. Progesterone attenuates OX- (9), prostaglandin- (25), and ET-receptor binding (present study). Thus one mechanism of progesterone-induced myometrial quiescence during pregnancy may be progesterone-mediated decreases in the binding of contractile stimulants to their G protein-linked transmembrane receptors. The function and cellular distribution (internalization/recycling) of many G protein-linked receptors are regulated by phosphorylation-dephosphorylation processes (for review see Ref. 32). Although the role of progesterone in modulating these events is not known, progesterone may downregulate certain G protein-linked receptors via a common mechanism, e.g., G protein-related kinases.
In summary, these studies provide evidence that intracellular Ca2+ homeostasis in human myometrial cells is directly regulated by progesterone. Downregulation of ET and OX receptors by progesterone may result in alterations of many cellular signaling pathways that converge to maintain uterine quiescence during pregnancy. These effects may result in decreased uterine contractility, remodeling of the extracellular matrix (43), and modulation of adenylate cyclase activity (17) during uterine hypertrophy and growth.
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ACKNOWLEDGEMENTS |
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The authors thank Drs. Jim Stull, Kris Kamm, and William Hurd for thoughtful discussions, Dawn Singh for excellent technical assistance, Dr. Susan Cox for invaluable assistance in tissue procurement, Dr. M. Yanagisawa for providing ET receptor antagonists, and Dr. L. Missiaen for the program to compute free Ca2+ concentration.
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FOOTNOTES |
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This investigation was supported by National Institute of Child Health and Human Development Grants HD-11149 and HD-30497 (to R. A. Word) and HD-08783 (to B. E. Cox).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: R. A. Word, Dept. of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9032.
Received 28 May 1998; accepted in final form 23 October 1998.
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