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Biology of Reproduction 60, 322-329 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Effect of Oxytocin Receptor and ß2-Adrenoceptor Blockade on Myometrial Oxytocin Receptors in Parturient Rats1

T. Engstrøm2,a,b, P. Bratholma, H. Vilhardtb, and Niels J. Christensena

a Department of Internal Medicine and Endocrinology, Herlev Hospital, University of Copenhagen, 2730 Herlev, Denmark b Department of Medical Physiology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been proposed that the rise in myometrial oxytocin receptor (OTR) concentrations at term triggers parturition. In the present study, we have shown that in vivo infusion of the ß2-adrenoceptor (ß2AR) antagonist ICI-118.551 in late pregnant rats prevents the rise in myometrial OTR binding normally seen during delivery. A reduced contractile responsiveness of uterine strips isolated from rats in labor when challenged with oxytocin (OT) and a slight shortening of gestation accompanied this effect. OTR mRNA levels were, however, unaltered after the treatment, suggesting that the effect of ß2AR blockade on myometrial OTR was posttranscriptional or due to influences on extra-myometrial tissue.

Infusion of the OTR antagonist atosiban down-regulated OTR binding sites in the parturient myometrium and resulted in an impaired contractile response to OT without affecting gestational length. OTR gene expression did not change, as seen from unchanged OTR mRNA values. Neither atosiban nor ICI-118.551 infusions alone changed fetal mortality. A significant increase in the incidence of fetal deaths was found, however, when rats were treated with a combination of atosiban and ICI-118.551. This treatment also down-regulated myometrial OTR and weakened the contractile response to OT, but it did not change gestational length.

We conclude that the timing and onset of a normal parturition as well as a favorable outcome seem to be independent of a rise in OTR. This fact cannot exclude the possibility that an increase in OTR is of importance in the genesis of preterm labor. We suggest that ß2 stimulation up-regulates OTR during delivery. This effect may partly be responsible for the tachyphylaxis seen after the use of ß2 agonists to control preterm labor. We further suggest that OTR stimulation up-regulates OTR during labor. The OTR down-regulation seen after atosiban treatment adds to the direct relaxing effect of atosiban on the myometrium. In view of this, atosiban may prove to be a more useful tocolytic than the traditionally used ß2 agonists.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The precise trigger of parturition remains unknown. It is, however, well established that the concentration of myometrial oxytocin receptors (OTR) increases dramatically at term [1, 2]. It has therefore been proposed that a rise in the number of OTR in itself initiates parturition [3]. Indeed OTR concentrations in rat myometrium are highest when myometrial sensitivity to oxytocin (OT) is maximal [4], and in the bovine uterus OTR increased at term with a further, marked increase at parturition [5].

The precise mechanisms behind the induction of OTR have not been fully elucidated. A number of steroids regulate the expression of OTR [6–8]. Other hormones including OT itself influence OTR levels in nonpregnant rats [9] and ewes [10]. We recently reported that in vivo infusion in nonpregnant estrogen-primed rats of the ß2-adrenoceptor (ß2AR) agonist isoproterenol enhanced the number and function of myometrial OTR [11]. This finding is supported by other studies in which cAMP-dependent mechanisms induced OTR expression in rabbit amnion [12, 13]. In addition, Bale and Dorsa [14] recently described a cAMP response element within the rat OTR gene.

It is an apparent contradiction that ß2AR stimulation should induce myometrial OTR since the direct effect of ß2AR activation in this tissue is relaxation [15]. The effect may, however, contribute to explaining the well-known diminished responsiveness to ß2 stimulants that follows sustained ß2AR activation [16], and it would provide new insight as to the pharmacodynamics of ß2 agonists in the treatment of preterm labor.

The purpose of the present study was to test the hypothesis that endogenous ß2 stimulation contributes to the amply demonstrated [1, 17] up-regulation of OTR during parturition in rats. We further wanted to investigate whether OT itself participates in this regulation. We have measured the effects of late pregnancy treatments with the OT antagonist atosiban and the ß2AR antagonist ICI-118.551 on OTR binding, OTR mRNA levels, and OT-induced uterine contractility. To evaluate the physiological relevance of the treatments employed, these receptor characteristics were related to gestational length and fetal survival.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Animals were maintained under controlled conditions in the Panum Institute Animal House. Food and water were freely available. Individual female rats (200–220 g) were placed in a cage with a male rat for 24 h. Absence of the vaginal plug at the end of the period was taken as a sign that copulation had taken place. Some rats were left untreated, and at Days 7, 14, and 21 of pregnancy as well as during labor and 5 days postpartum, they were anesthetized with CO2 and decapitated. The abdomen was opened longitudinally, and the uterine horns were removed. Endometrial tissue was carefully scraped off, and the remaining myometrium was saved for isolation of mRNA or preparation of plasma membranes. Some rats from the labor group were treated with OTR and ß2AR antagonists from Day 18 of pregnancy. On this day they were anesthetized with a mixture of Dormicum (1.25 mg; Hoffman-La-Roche, Basel, Switzerland) and Hypnorm (0.4 ml; Janssen Chimica, Geel, Belgium). An incision in the lower abdominal wall was made, and an osmotic mini-pump (Alzet, Palo Alto, CA) was introduced in the abdominal cavity. The pumps had pumping rates of 1 µl/h and were filled with the OT antagonist atosiban (5 mg/ml; Ferring Pharmaceuticals, Malmö, Sweden) or saline. The atosiban dosage was chosen since it has previously been shown to affect myometrial OTR binding in rats [9]. When corrected for body weight, this dosage was also comparable with the minimal dosage able to totally inhibit human premature uterine contractions [18]. Other rats from the labor group received twice daily from Day 18 of pregnancy 1.5 mg of the ß2AR antagonist ICI-118.551 (Biomol, Plymouth Meeting, PA) by means of i.p. injections [19]. The dosage of ICI-118.551 was chosen since it has previously been shown to affect ß2AR density in female rats [20]. To ensure that ICI-118.551 did indeed block myometrial ß2ARs, we performed initial experiments in which the relaxing in vitro response to the ß agonist isoproterenol was examined with or without the inclusion of ICI-118.551 in the organ chamber. In these experiments, ICI-118.551 was shown to reduce the maximal effect of isoproterenol by 60%. Yet other rats from the labor group were given treatments with both atosiban and ICI-118.551 in the same doses as indicated above. All treated and untreated rats from the labor group were decapitated when signs of labor were apparent, which was defined as the point of time immediately following delivery of the first pup. The gestational length was calculated. The numbers of dead fetuses were counted and related to the total offspring.

Primers and Construction of Internal mRNA Standard

The primers used for OTR mRNA detection were sense primer: 5' GGG ACG TCA ATG CGC CCA AGG AA 3' (nucleotides [nt] 2816–2838) and antisense primer: 5' ACC AAT AGA CAC CTA ATG CA 3' (nt 3921–3940). The primers used for ß2AR detection were sense primer: 5' TCT TCG AAA ACC TAT GGG AAC GGC 3' (nt 1036–1059) and antisense primer: 5' GGA TGT GCC CCT TCT GCA AAA TCT 3' (nt 1355–1378).

Basic Local Alignment Search Tool (Blast) [21] was used to search all nonredundant databases (GenBank+EMBL+DDBJ+PDB) for sequence homology. No homology with any known product other than the actual receptors was found.

The amplified DNA fragments consisted of 342 base pairs (bp) (ß2AR) and 375 bp (OTR). The exact identities of the polymerase chain reaction (PCR) products were confirmed by sequencing [11]. The relevant peaks were collected from the HPLC effluent, and 1/100 volume of 30% acetic acid was added to adjust pH. After addition of 50 µg glycogen, the PCR products were precipitated with an equal volume of isopropanol, washed with 70% ethanol, and dried. Sequencing was performed by the Applied Biosystems dRhodamin sequencing kit (Perkin Elmer, Allerød, Denmark) using Applied Biosystems ABI 310 apparatus for separation and fluorescence detection.

A PCR-MIMIC construction kit (Clontech, Palo Alto, CA) was used to construct a 240-bp internal standard DNA [22]. The internal standard RNA was constructed mainly as described by Faure et al. [23]. A composite primer, comprising 37 nt of bacteriophage T7 RNA-polymerase promoter followed by the sequence of our usual sense primer, was used for PCR amplification of the 240-bp internal standard DNA PCR product. The resulting 277-bp product was re-amplified using our antisense primer and a primer consisting of the initial 23 nt of the T7 RNA-polymerase promoter region. After HPLC purification, the re-amplified product was used for production of RNA by in vitro transcription (riboprobe; Promega, Madison, WI). The resulting RNA standard was quantitated by UV detection at 260 nM (Gene-quant; Pharmacia, Stockholm, Sweden). Subsequently, the RNA standard underwent reverse transcription (RT) in order to verify that the resulting product was indistinguishable from the internal DNA standard.

RT-PCR

Polyadenylated mRNA from homogenized rat myometrium was isolated using the PolyATract system 1000 (Promega) [22]. Reverse transcription was performed in a mixture consisting of 250 µM dNTP, 40 U MMLV-RT (Promega), 31.2 U RNA-guard, 200 pmol antisense primer, and 5 µl internal standard RNA in Promega RT buffer. Incubations were carried out for 60 min at 37°C, and the resulting cDNA was used immediately or stored at -80°C.

PCR was carried out with 5 µl cDNA, 37.5 µM of each dNTP, 1.0 U Taq polymerase (Pharmacia), and 40 pmol of both sense primer and antisense primer in PCR buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, pH 9.0). Amplification took place in a Perkin Elmer Model 460 thermocycler. Cycling parameters were as follows: 95°C for 2 min followed by 27 cycles consisting of 90 sec at 94°C, 60°C for 2 min, and 72°C for 2 min (ß2AR), or by 27 cycles consisting of 90 sec at 94°C, 56°C for 45 sec, and 70°C for 2 min (OTR). PCR products were used immediately or stored at -80°C.

Quantitation of PCR products was carried out by means of HPLC using a TSK DEAE-NPR column (TosoHaas, Montgomeryville, PA) [22]. After chromatography, PCR products were UV-detected at 254 nm. The areas of the 240-bp PCR products of the internal standards represented 0.024 (ß2AR) and 0.050 (OTR) amol, respectively. Hence the amount of the PCR products could be quantitated relative to these standards, and they were finally related to tissue wet weight. The average value of the uterine horns of each animal was calculated to represent the amount of specific mRNA.

The concentration of specific receptor mRNA was in the range of attomoles expressed per milligram of wet tissue weight. Coefficients of variation for isolation of mRNA-(RT-PCR)-HPLC were 14% (OTR) and 6.7% (ß2AR).

In Vitro Examination of Contractile Force ofMyometrial Strips

In vitro contractility was measured basically as described previously [24]. One uterine horn was opened longitudinally, and a middle segment measuring 5 mm was mounted in an isometric myograph connected to a Grass force transducer, the resting tension being 1.5 g. The strip was placed in an organ bath containing 8 ml of Munsick's buffer, pH 7.4 [25], and allowed to rest for 30 min. Every 10 min during this period, the buffer was refreshed in order to wash out any antagonist potentially remaining in the tissue specimen. Before stimulation with OT, the specimens were further washed five times. The temperature was kept at 30°C, and the solution was constantly aerated with 5% CO2 in O2. Contractile responsiveness to OT was measured using OT doses in the range of 0.63 x 10-10–0.13 x 10-6 M. After each stimulation, the amplitude of the contractile response was recorded, and the organ bath was subsequently washed three times with buffer. Responses were expressed as a percentage of the contraction induced by 50 mM KCl and were plotted against the logarithm to the agonist concentration. The contractile responses to KCl were in the range of 3.37 ± 0.85 to 7.49 ± 0.53 g among treated and untreated rats in labor. No significant differences between the groups were found, indicating that the effect of antagonist treatment on OT-elicited contractions was specific. To rule out any direct effect on OT-induced contractility of theoretically remaining ICI-118.551 in the specimens, experiments were performed in which 10 µM ICI-118.551 was included in the organ chamber during OT stimulation. Dose-response curves obtained from these experiments were not different from those obtained in the absence of the ß2 antagonist.

In order to evaluate ß2AR function following atosiban treatment, the tissue was precontracted with 50 mM KCl, and the relaxing effect of isoproterenol over a range of 10-10 to 4 x 10-6 M was examined. The addition of isoproterenol was done in a cumulative manner to obtain increasing concentrations, and phentolamine (Regitin; Novartis Pharma, Bern, Switzerland) at a concentration of 1 µg/ml was present in the incubation medium to block adrenergic {alpha}-receptors. The effect of each dose of isoproterenol was allowed to level off before addition of the next. It was ensured that the contractile response to KCl in itself did not significantly decrease during the period of the experiment.

Binding of 3H-OT to Isolated MyometrialPlasma Membranes

Preparation of rat myometrial plasma membranes was performed by subcellular fractionation [24]. Subsequent binding of 3H-OT was carried out as previously described [9]. Plasma membranes were incubated for 60 min with concentrations of 3H-OT varying from 0.42 to 24.20 nM (Amersham, Arlington Heights, IL; specific activity 35.0 Ci/mmol). The assay was performed at room temperature and initiated by the addition of plasma membranes. At the end of the incubation period, bound ligand was separated from unbound by filtration. Specific binding of 3H-OT was calculated by subtraction of binding in the presence of a 1000-fold excess of unlabeled OT and was finally related to protein content measured according to Lowry et al. [26].

To secure that ICI-118.551 had no affinity for the OTR, studies were performed in which 3H-OT was bound at concentrations of 6.05 nM and 12.10 nM in the presence and absence of 10 µM ICI-118.551. 3H-OT binding was not affected. We further wanted to exclude the possibility that residual peptide was present in the plasma membrane preparations after mini-pump infusions. Plasma membranes were therefore incubated with approximately 40 000 cpm of 3H-OT corresponding to 6.05 nM. After washing procedures identical to those used during plasma membrane isolation, less than 0.1% of added cpm resided in the membranes.

Data Analysis

A computer program (Fig.P.; Biosoft, Cambridge, UK) was used for data analysis. A four-parameter nonlinear curve-fitting model was used to evaluate myometrial responsiveness to OT. Maximal contraction (Emax) and the agonist concentration giving half this effect (EC50) were obtained from curve-fits of individual dose-response curves using the equation: E = Emin + (Emax - Emin) / (1 + (([OT]/EC50)-P)). Specific binding of 3H-OT to isolated myometrial plasma membranes was calculated by subtracting nonspecific binding from the total binding. Specific binding data from saturation binding experiments were analyzed using the equation: specific binding = [3H-OT] x Bmax / ([3H-OT] + KD). Subsequently maximal specific binding (Bmax) and dissociation constants (KD) were obtained from linear regression of individual double reciprocal plots following the equation: 1/specific binding = 1/Bmax + KD/Bmax x 1/[3H-OT].

One-way ANOVA or Kruskal-Wallis tests were used to compare multiple groups. Significant effects among individual means were subsequently separated using post-hoc tests for multiple comparison (Student-Neuman-Keuls or Dunn's method). The effects of treatment with atosiban, ICI-118.551, or both on mortality rate among the offspring were evaluated by means of chi-square analysis. Multiple comparisons among individual groups were done using chi-square analysis with Bonferroni's correction. Comparison of ß2AR mRNA between Day 21 of pregnancy and labor was done by Student's t-test. A p value < 0.05 was considered statistically significant. Results are presented as mean ± SEM unless otherwise stated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Binding of 3H-OT to Isolated MyometrialPlasma Membranes

Specific binding of 3H-OT to isolated plasma membranes was saturable and time-dependent. Equilibrium was attained after 15 min. Double reciprocal plots revealed binding sites of only a single affinity, and they were used to calculate KD and Bmax values. Bmax values changed significantly during pregnancy (Fig. 1, p < 0.001). A 3-fold increase during labor compared to Day 21 was observed (1.43 ± 0.22 vs. 0.46 ± 0.27 fmol/µg protein, p < 0.05). Five days postpartum, Bmax was reduced by 97% (0.04 ± 0.01 fmol/µg protein, p < 0.05) when compared to that at labor. Dissociation constants were in the nanomolar range (1.57 ± 0.21 nM in the labor group), and they remained unaltered across pregnancy as a whole. Any of the three treatments caused Bmax to decrease (22 ± 2.7%, 45 ± 10%, and 46 ± 7.3% of vehicle for atosiban, ICI-118.551/atosiban, and ICI-118.5511, respectively, p < 0.001, Fig. 1). Multiple comparisons of means showed that these values were significantly reduced (p < 0.05) when compared with those of the vehicle group or the untreated labor group (p < 0.05). Bmax in the untreated labor group was not statistically different from Bmax in the vehicle-treated labor group (1.42 ± 0.22 vs. 1.05 ± 0.14 fmol/µg protein). No significant differences were found between Bmax in the atosiban, atosiban/ICI-118.551, and the ICI-118.551 groups. KD values remained unchanged after any treatment.



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  		FIG. 1. Maximal specific binding of 3H-OT to isolated myometrial plasma membranes. Membranes were isolated from untreated rats at different gestational ages and from rats in labor treated with saline, atosiban (5 µg/h), ICI-118.551 (1.5 mg twice daily), or both atosiban and ICI-118.551 (same doses as individual treatments) from Day 18 of pregnancy. Different letters indicate statistical differences among rats in labor. Values are mean ± SEM, n = 4–7.

Messenger RNA

ß2AR mRNA increased 4-fold from Day 21 of pregnancy until labor (0.03 ± 0.01 vs. 0.11 ± 0.02 amol/mg wet tissue, p = 0.016). ß2AR mRNA from rats in labor was not affected by atosiban treatment, indicating that OTR blockade did not influence ß2AR gene transcription (0.37 ± 0.12 vs. 0.23 ± 0.05 amol/mg wet tissue).

The concentration of OTR mRNA changed across pregnancy (p < 0.001). Values from animals in labor showed a 15-fold increase compared to Day 21 (1.22 ± 0.33 vs. 0.08 ± 0.04 amol/mg wet tissue, p < 0.05). Five days postpartum, the level of OTR mRNA was reduced by 90% (0.13 ± 0.05 amol/mg wet tissue, p < 0.05), when compared to that at labor (Fig. 2). Treatment with atosiban, ICI-118.551, or both did not change OTR mRNA values when compared to those of the untreated labor group or the vehicle labor group (Fig. 2), nor were the values of the vehicle labor group different from those of the untreated labor group.



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  		FIG. 2. Myometrial OTR mRNA from untreated rats at different gestational ages and from rats treated with saline, atosiban (5 µg/h), ICI-118.551 (1.5 mg twice daily), or both atosiban and ICI-118.551 (same doses as individual treatments) from Day 18 of pregnancy. Values are mean ± SEM, n = 4–6. Values among rats in labor are not statistically different, as indicated by identical letters.

Contractile Responsiveness of Isolated Uterine Strips Challenged with OT

Stimulation of isolated uterine strips with OT revealed sigmoid dose-response curves. Emax-values across pregnancy varied from 77.0 ± 5.41 to 117 ± 9.95% of the potassium-induced response (Table 1, p < 0.05). An increase during labor when compared to Day 21 was observed (117 ± 9.95% vs. 107 ± 14.1%). This difference, however, was not significant. Five days postpartum, Emax reached 72% of the labor level (77.0 ± 5.41 vs. 117 ± 9.95%, p < 0.05). EC50 from OT-stimulated uteri reached its lowest value during labor, indicating an increased sensitivity of the myometrium to OT at this time (Table 1). Differences among EC50 values, however, were not statistically significant. Treatment with either atosiban, ICI-118.551, or both resulted in significant decreases in Emax values of approximately 30% (p < 0.001; Fig. 3 and Table 1). Multiple comparisons among means revealed statistical differences between the vehicle labor group and all of the treated groups, and between the untreated labor group and all the treated groups (p < 0.05). No differences were found between specific treatments or between the vehicle labor group and the untreated labor group. Significant differences between mean EC50 values were not found (Table 1).


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  		TABLE 1. EC50 and Emax values of isolated uterine strips challenged with OT.*



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  		FIG. 3. Contractile responsiveness of isolated uterine strips challenged with OT. Strips were isolated from rats in labor treated with saline (circles), atosiban (open squares, 5 µg/h), ICI-118.551 (stars, 1.5 mg twice daily), or both atosiban and ICI-118.551 (solid squares, same doses as individual treatments) from Day 18 of pregnancy. Values are mean ± SEM, n = 3–5.

Neither Emax nor EC50 of isoproterenol-induced relaxation of isolated strips from rats in labor were affected by atosiban treatment, indicating that the interaction between OTR and ß2AR function was unilateral (Emax = 95.0 ± 2.65% vs. 92.1 ± 3.73%, EC50 = 1.73 ± 0.67 nM vs. 1.42 ± 0.18 nM).

Gestational Length and Fetal Outcome

Gestational length among treated rats is shown in Figure 4. The length of pregnancy in controls was 22.8 ± 0.17 days. Treatment with atosiban or ICI-118.551 resulted in gestational ages of 23.3 ± 0.38 and 21.9 ± 0.64 days, respectively, whereas treatment with both compounds resulted in a mean delivery time inbetween these two groups (22.4 ± 0.63 days). Overall differences were statistically significant (p = 0.002). When all pair-wise multiple comparisons were done, statistically significant differences were found only between the atosiban and ICI-118.551 group (p < 0.05), between the vehicle and ICI-118.551 group (p < 0.05), and between the atosiban and the atosiban + ICI-118.551 group (p < 0.05).



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  		FIG. 4. Length of gestational periods of delivering rats treated with saline (vehicle), atosiban (5 µg/h), ICI-118.551 (1.5 mg twice daily), or both atosiban and ICI-118.551 (same doses as individual treatments) from Day 18 of pregnancy. Points with different letters are statistically different. Values are mean ± SEM, n = 5–6.

In Table 2, the pup mortality is shown. Dams treated with vehicle alone gave birth to only living pups. The overall effect of the treatments was increased incidence of stillborn fetuses (p < 0.001). This difference, however, was found only between the vehicle group and the combined treatment group when multiple comparisons were performed with Bonferroni's correction (p = 0.012). The combined treatment with atosiban and ICI-118.551 was associated with the largest number of deaths even though this treatment did not influence gestational length. In this group, one parent rat experienced uterine rupture.


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  		TABLE 2. Fetal outcome following the treatments indicated.*


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The regulation of myometrial OTR is thought to play a pivotal role in the process of parturition [1]. Indeed, a rise in myometrial OTR concentrations has been proposed to be the stimulus that transforms the myometrium from a quiescent to an active and contractile state [3]. In the present study, we have shown that the rise in myometrial OTR binding during labor is paralleled by increased OTR gene expression and enhanced uterine contractility, as indicated by the increased Emax values of isolated strips challenged with OT. Further, we have for the first time shown that ß2AR blockade during late pregnancy blunts this up-regulation of OTR. The effect of ß2AR blockade was accompanied by a reduced maximal contractile response of isolated uterine strips to OT, indicating the loss of active OTR. Previously we have demonstrated that ß2AR stimulation in the nonpregnant estrogen-dominated rat myometrium induces the formation of OTR [11]. A similar effect on OTR concentrations was found by Jeng et al. [12] when cultured rabbit amnion cells were incubated with isoproterenol. This effect was partly abolished when the cells were cotreated with the ß2AR blocking agent propranolol. The present data showed that ß2AR mRNA values increased during labor compared to Day 21 of pregnancy. In addition Suzuki et al. [27] have shown that maternal plasma catecholamines increased in women in labor. Both observations indicate that a high ß2-adrenergic tone is imposed on the myometrium during labor, thereby probably providing the basis for the induction of OTR formation. A proposed role of ß2AR in the up-regulation of OTR constitutes an apparent contradiction since ß2AR agonists are widely used as tocolytics. Several studies, however, have shown that continuous exposure of the myometrium to tocolytics of this kind results in a loss of responsiveness to subsequent ß2AR activation [28, 29]. Indeed, preterm delivery of fetuses was not prevented in rats infused with salbutamol [28]; and in sheep in preterm labor, continuous infusion of ritodrine produced only a transient inhibition of uterine contractions [30]. This loss of responsiveness, known as desensitization, involves receptor uncoupling [31], receptor internalization [32], and finally receptor degradation. On the basis of the present results, we suggest that the failure of ß2 agonists to induce tocolysis may additionally involve the induction of OTR expression.

Bale and Dorsa recently described a cAMP response element within the OTR gene [14]. The unaltered levels of OTR mRNA following ß2AR blockade in the present studies, however, suggest that the influence on OTR function may be posttranscriptional unless mRNA stability is changed by the treatment, too. In rat C6 glioma cells, Huang et al [33] showed that lactate dehydrogenase A subunit messenger RNA stability was increased in response to cAMP. Thus in our study, the unchanged high level of OTR mRNA following ICI-118.551 treatment may reflect partly degraded mRNA unable to serve as a template for protein synthesis. In addition, mechanisms independent of gene expression appear to be responsible for the decreased OTR function. Isoproterenol stimulation has been described, via protein kinase A (PKA), to enhance the responsiveness to IP3 of intracellular calcium in guinea pig hepatocytes [34]. This effect is thought to be mediated by the phosphorylation of IP3 receptors by PKA [35]. In addition, cAMP potentiates the stimulation of phospholipase C by vasopressin in rat hepatocytes [36]. ß2AR blockade in the present study might block such enhancing effects of cAMP on OTR function.

Regulation of myometrial OTR involves other factors—in particular, gonadal steroids [37–40] and OT itself. Down-regulation of OTR by OT in nonpregnant myometrial tissue is described for both humans [41–43] and rats [9]. Adachi and Oku [41] found an OT-induced decrease in surface OTR in human myometrial monolayer culture. This effect was suppressed by concanavalin A, suggesting that internalization was the main mechanism of down-regulation. OTR mRNA remained unchanged in estrogen primed nonpregnant rat myometrium after in vivo infusion of OT, indicating that OTR gene expression is not involved in OT-induced OTR down-regulation (unpublished data). The present studies demonstrate a decline in OTR binding in parturient rats following atosiban infusion. Moreover, the effect on OTR binding was paralleled by a reduction in maximal contractility of isolated uterine strips challenged with OT, indicating that the effect of atosiban may involve loss of plasma membrane-associated receptors, e.g., by compartmentalization. Alternatively, phosphorylation of the receptors may have occurred, leaving them unable to bind OT and subsequently evoke contraction. As indicated by Kimura et al. [44] a number of possible phosphorylation sites are available in the OTR. Since OTR mRNA levels remained unchanged, atosiban treatment does not seem to alter transcription rates unless mRNA half-lives are changed by the treatment, too.

The question arises whether the effect of atosiban on OTR, found in the present study, is due to the receptor-ligand interaction in itself. If so, the observed down-regulation would be comparable to that seen following OT exposure in vitro [41, 43]. Engstrøm et al. [9], however, showed an up-regulation of OTR after atosiban infusion in nonpregnant rats, and Phaneuf et al. [42] demonstrated that the OT-induced desensitization in cultured human myometrial cells was prevented by co-incubation with atosiban. On the basis of these results, we suggest that OT in vivo participates in the up-regulation of OTR in the myometrium during labor and in this way plays a different role from that in nonpregnant myometrium. In accordance with our results, Randolph and Fuchs [45] found no evidence for down-regulation of OTR during continuous infusion of OT in rats in labor. On the contrary, they observed enhanced myometrial sensitivity to OT after pulsatile infusion of OT, which might indicate up-regulation of OTR by OT during labor.

In our present study, OTR blockade by atosiban in rats in late pregnancy did not postpone the onset of delivery, and it did not significantly increase fetal mortality despite a reduced number of functional OTR. The onset and timing of parturition, therefore, do not appear to be dependent on an increase in OTR expression, at least not as a single factor. Considering the dramatic increase in OTR expression seen during normal labor, this is a surprising finding. It has been shown that transgenic mice that have the OT gene disrupted deliver their litters at term [46, 47]. Chan and Chen [48] found the gestational length to be unaltered by OTR blockade in vivo, and Schellenberg [49] reported a similar finding in guinea pigs. We are not aware, however, of any study showing that a normal onset and a favorable outcome of parturition can be obtained without an increase in OTR concentrations. In this context, it is important to note that our studies demonstrated a decrease in OTR binding in vitro after in vivo infusion of atosiban, and this effect was paralleled by a decreased maximal contractility of isolated uterine strips challenged with OT in vitro. Because of the extensive washing procedures before the in vitro experiments, it appears unlikely that these changes reflect effects of residual atosiban in the myometrial strips and plasma membranes. Thus the OTR system was impaired by long-term atosiban treatment.

A rise in the concentration of OTR has been suggested as an important etiological factor in preterm labor [1, 50]. Indeed, atosiban has been shown to effectively inhibit premature uterine contractions with only minimal side effects [50]. The present data indicate that the tocolytic effect of atosiban may not merely be a direct effect on OTR but may also include reduced OTR binding after prolonged exposure to atosiban.

Treatment with the ß2AR antagonist ICI-118.551 reduced the gestational length significantly. This finding was accompanied by a slight but insignificant increased incidence of fetal mortality. The onset of parturition remained unchanged when both ICI-118.551 and atosiban were administered from Day 18 of pregnancy, although a significant rise in pup mortality rate was observed in this group. Several mechanisms may account for the effect of the combined treatment on fetal outcome. All dead pups in any of the treated groups were found in utero, and therefore local intrauterine factors seem to play an important role. Stimulation of ß2AR present in fetal vasculature in the placenta causes vasodilatation [51]. Treatment with an ß2 antagonist might therefore reduce placental blood flow by placental vasoconstriction and thereby increase the risk of intrauterine demise. Activation of OTR is involved in the dilatation of cervix uteri before delivery [52]. Thus blockade of cervical OTR by atosiban may prolong parturition because of inadequate cervical ripening and may thereby aggravate the effect of ß2AR blockade on placental vessels. A toxic effect on the fetuses of the combined administration of atosiban and ICI-118.551 cannot be excluded. However, according to Greig et al. [53], atosiban transfer from late gestation pregnant sheep to their fetuses is negligible, and in women placental transfer of atosiban was found to be minimal [54]. Similar studies have not been performed with ICI-118.551, but even if this compound crosses the placenta freely, the present data show no significant increase in pup mortality when ICI-118.551 was administered alone. When all of these results are considered, the combined effect of the two compounds on fetal mortality appears not to be due to toxicity.

In conclusion, we have shown that ß2AR blockade prevents the rise in myometrial OTR that normally precedes parturition, without affecting OTR gene expression. An impaired contractile responsiveness in vitro accompanied this effect. Atosiban treatment reduced OTR concentrations, but this reduction was found not to be essential for the timing and onset of normal labor nor for the outcome of parturition. On the other hand, a proper function of both OTR and ß2AR was essential for a normal propagation and a normal outcome of parturition since blockade of both receptors increased pup mortality. The precise mechanism behind this effect of combined treatment needs to be elucidated. The effect of ß2AR blockade on OTR suggests that the development of tachyphylaxis to ß2 agonist during the treatment of preterm labor may involve up-regulation of OTR. On the other hand, the effect of atosiban on OTR indicates that the tocolytic effect of this compound consists of both a direct competitive inhibition of uterine contractions and a down-regulation of OTR. Thus atosiban may prove to be a more useful tocolytic than ß2 agonists.


   ACKNOWLEDGMENTS
 
We thank technicians Gurli Habekost, Jakob Utzon-Frank, and Vibeke Voxen for their helpful assistance.


   FOOTNOTES
 
1 The present study was supported by The Danish Hospital Foundation for Medical Research (Region of Copenhagen, The Faeroe Islands, and Greenland), The Danish Biotechnology Programme, Brødrene Hartmanns Foundation, The Danish Medical Research Council, The Novo Nordisk Foundation, The Beckett Foundation, Jacob Madsen&Hustru Olga Madsens Foundation, The Danish Medical Association Research Fund, and Ove Villiam Buhl Olesen & ægtefælle Edith Buhl Olesens Foundation. Back

2 Correspondence. FAX: 45 4488 3659; engstrom{at}mfi.ku.dk Back

Accepted: September 11, 1998.

Received: February 23, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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