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a Perinatal Research Centre, Department of Obstetrics and Gynecology, University of Alberta, Edmonton, Alberta, Canada T5H 3V9
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have measured OT and its extended forms using two antisera, one recognizing OT and its extended forms, the other recognizing only mature amidated OT. Uterine tissue concentrations of extended forms of OT were 5- to 30-fold greater than those of OT, and both increased progressively and significantly through late gestation. The ratio of OT to its extended forms did not change significantly. Antagonists of estrogen or progesterone receptors reduced concentrations of extended forms by > 90% and of OT by 50%, though the estrogen antagonist significantly prolonged gestation and the progesterone antagonist induced preterm delivery.
Using a muscle bath preparation, extended forms of OT were weak uterine stimulants and did not alter the OT concentration-response curves. Extended forms of OT were two to three orders of magnitude less able than OT to displace radiolabeled OT from late-gestational uterine binding sites.
We conclude that uterine carboxy-extended OT prohormones are regulated in part by estrogen and progesterone. However, these extended forms of OT have little direct biological activity and do not compete with OT for receptor binding. Their role in the process of parturition may be confined to acting as substrates for OT synthesis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The initial gene product from translation of OT mRNA is a peptide containing both OT and neurophysin I [7]. The tripeptide gly-lys-arg separates the nonapeptide OT from neurophysin I. In the hypothalamus, posttranslational processing of this large peptide involves cleavage of OT-gly-lys-arg from neurophysin I [8]. The basic amino acids arg and lys are then serially cleaved by carboxypeptidase B to yield OT-gly. The OT-gly is transformed by 
-amidating enzyme to mature, biologically active amidated OT. The intermediate forms containing 10, 11, or 12 amino acids are collectively referred to as carboxy-extended forms of OT, abbreviated OTX.
In the rat hypothalamus, the ability to process the initial translation product through to mature OT increases during late fetal and early neonatal development [8]. In fetal sheep plasma, the concentrations of OTX forms are 35-fold higher than that of OT at two thirds of the way through gestation [9]. However, by term, the concentration of OTX has declined to one half, and OT has increased several-fold, suggesting a similar pattern of maturation. The concentration of OTX forms still remains several-fold higher than that of OT. In human umbilical plasma, a similar pattern of maturation is noted [10].
The potential role of OTX forms in parturition has not been investigated. It is not known whether synthesis of OT within the uterus involves intermediate OTX forms, and the concentrations of these OT prohormones in uterine tissues have not been measured. Further, it is not known whether these OTX forms possess any oxytocic activity. In the present studies we hypothesized that these intermediates are synthesized in the rat uterus and that this is regulated by estrogen and progesterone. Further, we hypothesized that the OTX forms would have little biological activity but would compete with OT for OT receptors (OTR). Thus, parturition would not occur until the ratio of OT to OTX forms reached a critical level. The three objectives of the present experiments were 1) to measure the concentrations of OTX forms in late-gestational rat uterine tissues and calculate the ratio of OT to OTX with advancing gestation, 2) to determine the effects on OTX concentrations of maternal treatment with receptor antagonists of estrogen (tamoxifen) and progesterone (RU-486), and 3) to assess the ability of OTX forms in vitro to stimulate uterine contractions and compete with OT for binding to rat uterine OTR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All experiments were approved by the University of Alberta Animal Care committee. Time-mated, primigravid Sprague-Dawley rats (approximately 250 g each) were transferred from Charles River Canada (St-Constant, PQ, Canada) to our animal facility at Day 12 of pregnancy. Food and water were available ad libitum. The photoperiod was 12L:12D. Rats were killed via i.p. injection of Euthanyl (MTC Pharmaceuticals, Cambridge, ON, Canada) at 100 mg/kg BW. The uteri were removed immediately after animals were killed. Uterine tissues were frozen in liquid nitrogen and stored at -70°C until homogenization for further analysis. Since our interests concern paracrine interactions between the myometrium and endometrium, no attempt was made to separate these tissues for subsequent analyses.
Treatment Groups
The rats were divided into 5 groups. In this strain of rats, parturition usually occurs early in the afternoon of Day 22 (mating occurs on Day 0). The first control group (n = 37) consisted of normal untreated and uninstrumented animals. Subgroups of animals (4–6 in each subgroup) were killed on Days 13, 16, 19, 21, and 22 of pregnancy and after delivery of the first pup.
The first experimental group (n = 28) was treated with daily s.c. injections of the estrogen receptor antagonist tamoxifen, 200 µg/rat (Sigma Chemical Co., St. Louis, MO) in 0.4 ml oil, from Day 15 of pregnancy until death. This dosage was chosen since it has been shown to have maximal antagonistic effects with high estrogen levels as seen in late gestation [11], and we know that this dosage delays labor onset by 24 h [3]. Though tamoxifen may demonstrate agonistic properties in a low-estrogen environment, its properties are antagonistic in the presence of the high concentrations of estrogen in late gestation. Subgroups of animals were killed at pregnancy Day 19, 21, 21.5 (the evening of Day 21), and 22 and during labor (after delivery of the first pup). The control subgroups for this experimental group received daily injections with vehicle alone (0.4 ml oil). The rats were injected between 0900 and 1000 h, and animals were also killed at this time or, in the case of the 21.5-day groups, between 2100 and 2200 h. There were 4–6 animals at each time point in each experimental and control subgroup.
The second experimental group (n = 24) was treated with a single s.c. injection of the progesterone receptor antagonist RU-486 (2.5 mg/rat in 0.4 ml oil) at Day 15 of pregnancy. This causes parturition to occur at a mean of 27 ± 1.2 h after injection [4]. Animals (n = 5 in each subgroup) were killed at 0, 6, 12, or 24 after treatment and during labor (after delivery of first pup). In 4 of the RU-486-treated animals, delivery had not occurred by 48 h, and these animals were killed at that time. In all cases, there was vaginal bleeding suggesting that delivery was imminent. These animals were not included in the calculation of mean time to delivery after RU-486. The control subgroup (n = 5 in each subgroup) received only injections with vehicle (0.4 ml oil). The rats were injected between 0900 and 1000 h.
Synthetic Peptides
The carboxy-extended forms of OT were prepared by the Alberta Peptide Institute (Edmonton, AB, Canada) using an Applied Biosystems (Foster City, CA) model 430A Peptide Synthesizer or a Beckman (Palo Alto, CA) model 990 utilizing t-Boc N-protection and benzyl-type side-chain protection. The peptides were cleaved from the resin, and side-chain-protecting groups were removed with hydrogen fluoride. The cleaved peptides were purified by reversed-phase HPLC, and the purity of the peptide was confirmed by presence of a single peak. Amino acid composition was confirmed by both amino acid analysis and mass spectrometry.
Before use, we further confirmed purity of the synthesized peptides using reversed-phase HPLC. A Lichosorb (Hewlett-Packard, Palo Alto, CA) RP-18 column was used with a Hewlett-Packard Series 1050 HPLC and detector system. Solution A was 10 mM ammonium acetate, pH 4.2, and solution B was methanol with 0.15% acetic acid. Flow rate was 1 ml/min beginning with A:B at 95:5 in a linear gradient to A:B 65:35 over 25 min, then continuing isocratically to 45 min. Peptide peaks were detected at optical density of 230. The retention times for OT-gly-lys-arg (OT-GKR), OT-gly-lys (OT-GK), OT, and OT-gly (OT-G) were 32.5, 36.5, 37.5, and 42 min, respectively.
RIAs for OTX and OT
Uterine tissues were homogenized in acid buffer (5% formic acid, 10% trifluoroacetic acid, and 1% NaCl in 1.0 N HCl) and centrifuged at 1000 x g for 30 min. The supernatant treated with acid buffer was passed through C-18 Sep-Pak cartridges (Waters, Milford, MA). OT was eluted from the column with 75% acetonitrile in 0.01 M trifluoroacetic buffer. The extracts were dried and then reconstituted in RIA buffer containing 50 mM sodium phosphate, 10 mM ethylenediaminetetraacetic acid, and 0.2% gelatin at pH 7.2. The recovery of radiolabeled OT in this procedure is 80–90%.
The antiserum used to measure OTX forms was kindly supplied by Dr. Harold Gainer at the NICHHD (VA-17). This antiserum has been fully characterized and recognizes OT and all of the intermediate OTX forms [8]. The antiserum has highest affinity for OT-GKR (100%) and cross-reacts with OT-G, OT-GK, and OT at 43%, 41%, and 64%, respectively. Purified OT was used for the standard curve and 125I-OT as the tracer. The measurement unit is designated as OTtotal and expressed in picograms OT equivalents per gram tissue. No attempt was made to separate the individual OTX forms or measure each one separately. The main objective of these experiments was to determine the ratio of mature OT to the OTX forms, and expressing all measurements as "OT equivalents" facilitates this objective. An aliquot of the sample was added to the same RIA buffer as above with a final assay volume of 500 µl and a final antibody dilution of 1:40 000. The reaction was incubated for 48 h at 4°C; it was then quenched and the protein precipitated by adding 1.1 ml ethanol. Bound and free OT and OTX forms were separated by centrifugation at 1500 x g for 30 min at 4°C. The supernatant was aspirated and the pellet counted with a model 1275 Minigamma counter (LKB-Wallac Oy, Turku, Finland). The assay sensitivity was 3–4 fmol OT.
The antiserum used to measure OT was generously provided by Dr. A.P.F. Flint, University of Nottingham (antibody 242/1). We have described this RIA previously [3]. This antibody does not cross-react significantly with vasopressin or other known peptides [12]. We have confirmed that the antibody does not cross-react significantly with the carboxy-extended forms of OT. At 50% displacement of total bound radiolabeled OT, the cross-reactivity with the OTX forms was OT-G, < 0.05%; OT-GK, < 0.15%; and OT-GKR, < 0.05%. A 25- to 50-µl aliquot of tissue extract was incubated with 5000 cpm 125I-OT (New England Nuclear-DuPont, Boston, MA) and OT antibody 242/1 overnight at 4°C. The final antibody dilution was 1:100 000. After incubation with second antibody (rabbit anti-sheep serum) or normal sheep serum, the samples were centrifuged at 1500 x g for 15 min. The supernatant was aspirated, and the pellet was counted using a model 1275 Minigamma counter (LKB-Wallac Oy). The assay sensitivity was 2–3 fmol OT.
In the samples measured, the maximum ratio of OTX to OT was approximately 20-fold, so corrections were not made for cross-reactivity. Since the OT concentrations were to be expressed as a ratio to the OTX form concentrations, OT concentrations are expressed in picograms per gram tissue rather than the usual SI units. The molecular weight of OT is 1007.2, and 1 pg is approximately equal to 1 fmol. The ratio of OT to OTtotal was calculated by dividing the estimate obtained with the Flint 242/1 antiserum by that obtained using the Gainer VA-17 antiserum.
Maternal serum concentrations of OT and OTX forms also were measured using these antibodies and the method we have described previously [3].
Muscle Bath Preparation
The muscle bath preparation was established based on published methodology [13, 14]. Uteri from mature Sprague-Dawley rats were used. For experiments using nonpregnant tissues, rats were injected with estradiol (100 µg in 0.4 ml corn oil s.c.) daily for 3 days prior to experiments. Tissues from pregnant rats were obtained at Day 15, 20, or 22 of gestation. Uterine horns were removed immediately after death, and each horn was divided longitudinally into two halves. No attempt was made to separate circular from longitudinal muscle or to remove attached endometrium. For the pregnant animals, uterine tissues were freed from all fetuses, membranes, and placental tissues. For use in the muscle bath preparation, longitudinal muscle strips approximately 15 mm in length and 3 mm in width were excised. Each uterus yielded four such muscle strips. In the pregnant animals, an attempt was made to avoid implantation sites.
Muscle strips were tied at each end with silk thread and mounted vertically in separated jacketed organ baths each containing 20 ml Krebs buffer of the following composition: 118 µM NaCl; 4.7 µM KCl; 2.5 µM CaCl2; 1.2 µM KH2PO4; 0.59 µM MgSO4; 25 µM NaHCO3; 11.7 µM D-glucose, at pH 7.4, and constantly aerated with 95% O2:5% CO2 at 32°C. One end of each strip was anchored in the bath, and the other end was attached to an FT-03C Grass force-displacement transducer connected to a 7D polygraph system (Grass Instrument Co., Quincy, MA). Resting tension of 1 g was applied to each strip. This resting tension has been shown to develop maximum active tension in pregnant Sprague-Dawley rat myometrium [14]. Except for pregnancy Day 22 strips, muscle strips were equilibrated for 2 h with washing every 20 min, by which time muscle strips were almost completely quiescent. For Day 22 strips, 3 or 4 h was required to achieve muscle quiescence.
In preliminary experiments, uterine strips were compared from the tubal or the cervical end, or from the mesometrial or anti-mesometrial portion of the uterus. There were no systematic differences according to the anatomic site. For the remaining experiments, each uterine horn was bisected longitudinally and a strip was obtained from each segment. Four such strips were used in each experiment and were randomly allocated to be used for OT control, OT-G, OT-GK, or OT-GKR. For analyses, data from each strip were normalized to an OT control run on the same strip immediately prior to the experimental protocol.
To measure the response to OT or the carboxy-extended forms of OT, cumulative concentration-response curves were obtained using 5-min exposures at each concentration for OT and 10-min exposure for the carboxy-extended forms. Contractile activity was measured as the area under the tension trace integrated over the 5- or 10-min exposure period at each concentration. The concentration-response curves reached maximum stimulation in all cases by OT at 25 nM concentration. All data were normalized as a percentage of the tension developed by each strip in the presence of 25 nM OT. Concentration-response curves for OT were again constructed at the conclusion of each experiment to ensure that the muscle preparation was capable of responding. In all experiments, there was no difference in the OT response curves at the beginning and end of the experiment.
To measure the effects of the carboxy-extended forms of OT on the concentration-response curves for OT, strips were incubated with 4 cumulative concentrations of OT (0.39, 1.56, 6.25, and 25 nM) for 5-min exposures at each concentration. After a 20-min washout period, strips were incubated in one concentration of one of the carboxy-extended OT forms for 10 min, and the OT concentration-response curve was again constructed. Three concentrations (50, 400, and 1600 nM) of each of the carboxy-extended OT forms (OT-G, OT-GK, and OT-GKR) were tested in 3 or 4 different experiments using strips from different animals. Again, data were normalized and expressed as a percentage of maximal activity generated by OT.
Uterine OTR Binding
Binding of OT to its receptor in uterine tissues was measured using a modification of a published OT binding assay as we have previously described [3]. Briefly, uterine tissues were frozen in liquid nitrogen and stored at -70°C until homogenization. All products of conception were removed from the uterine wall, but no attempt was made to separate decidua from myometrium. Uterine tissues were homogenized in buffer containing 10 mM Tris, 1.5 mM EDTA, 5 mM sodium molybdate, and 1.0 mM monothioglycerol and centrifuged at 1000 x g for 15 min. The supernatant was centrifuged at 105 000 x g for 1 h, and the pellet was washed, resuspended in Tris buffer, and incubated for 1 h with 0.6 nM [3H]OT (New England Nuclear-DuPont) and increasing concentrations of nonradioactive OT (0.0–15.6 nM) in a final volume of 250 µl containing 25 mM Tris, 0.1% gelatin, and 5 mM MnCl2 at pH 7.4 for 1 h. Nonspecific binding was measured following the addition of 100 nM nonradioactive OT. Incubation was terminated by filtering the suspension through a glass microfiber filter (GF/C; Whatman, Springfield Mill, Maidstone, England) and rinsing with cold Tris buffer. The filters (carrying receptor-bound [3H]OT) were counted by scintillation spectrometry in 10 ml CytoScint (ICN, Irvine, CA). For the carboxy-extended forms of OT, binding was measured using concentrations of 40, 100, 200, 400, 800, and 2000 nM in the presence of the radiolabeled OT. After correction for nonspecific binding, data were expressed as the percentage of bound radiolabeled OT in the absence of nonradiolabeled OT.
Statistical Analysis
Data are presented in text and graphs as the mean ± SEM. The results for OT and OTX tissue concentrations were first analyzed by one-way ANOVA (Instat; GraphPad Software, San Diego, CA) to detect changes with advancing gestational age. Post hoc comparisons of the means were performed using the Tukey-Kramer Multiple Comparisons test. For the tamoxifen and RU-486 experiments, two-way ANOVA (Prism; GraphPad Software) was performed to detect differences between treatment groups. When between-treatment differences were found, differences between the experimental and control subgroups were sought using the two-tailed unpaired Student's t-test. If there was nonhomogeneity of variance, the Mann-Whitney U test was utilized. For comparison of the concentration-response curves for OT and the OTX forms from the muscle bath experiments, results were examined using two-way ANOVA. Differences were considered to be significant when a p value < 0.05 was obtained.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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OT concentrations in maternal serum through late gestation were extremely variable (62–645 pg/ml), and there was no significant change with advancing gestation. The serum measurements using the Gainer VA-17 antibody were never greater than the OT measurements using the Flint 242/1 antibody, suggesting that there were no OTX forms in the maternal circulation.
Treatment of pregnant rats with the estrogen receptor antagonist tamoxifen at a dose sufficient to significantly delay parturition was accompanied by a 90–98% reduction in OTtotal concentrations (Table 1). OT concentrations were decreased by approximately 50%. This resulted in a very significant increase in the OT:OTtotal ratio, which increased progressively from Day 19 of gestation onward. Despite the increase in the OT:OTtotal ratio, delivery in these animals occurred 27 ± 0.4 h later than in the control group (p < 0.01).
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Administration of the progesterone receptor antagonist RU-486 on Day 15 of gestation caused preterm parturition at 27 ± 1.2 h after the injection. There was a significant decrease in uterine concentrations of OTtotal compared to the value in controls at 24 h after RU-486 administration (Table 2). Tissue levels of OT remained unchanged. The ratio of OT:OTtotal was significantly elevated above control levels by 12 h after RU-486 administration and remained elevated until parturition.
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Both nonpregnant (n = 5) and pregnant (n = 4) myometrial strips in vitro demonstrated a concentration-response relationship with increasing OT concentrations (Fig. 2). Maximal responses were always achieved by 25 nM OT. Pregnant rat tissues were obtained from one animal at Day 15, one at Day 20, and two at Day 22 of gestation. There was no quantitative or qualitative difference among these tissues, so the results have been grouped in Figure 2. Although there was more variability with the pregnant tissues, the concentration at which half-maximal activity occurred was approximately 1–2 nM and was similar in pregnant and nonpregnant animals. The three carboxy-extended forms of OT appeared to have similar activities to each other and were approximately three orders of magnitude less potent than OT with respect to stimulation of uterine contractions. At the higher doses, all three OTX forms increased contractile frequency, but none increased the baseline tension as did OT. Again, there was more variability with the pregnant tissues, but the response was qualitatively similar to that of the nonpregnant myometrium. Even at the highest concentrations, which were more than two orders of magnitude greater than the concentrations of OT, the maximal activity of the OTX forms was only approximately half of that achieved with OT. By two-way ANOVA, there was no difference among the three OTX forms with either pregnant or nonpregnant tissues.
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Four-point concentration-response curves for OT (0.5, 1.5, 7.5, and 25 nM) were constructed in the absence or presence of each of the OTX forms at 50, 400, and 1600 nM. For these experiments, uteri from two pregnant (Day 22) and two nonpregnant rats were used. There were no apparent differences between the pregnant and nonpregnant tissues. The presence of OTX forms at any of these concentrations did not alter the concentration-response curve with OT (data not shown). By two-way ANOVA, there was no significant competitive inhibition between the OTX forms and OT.
The rat uterine binding data (Fig. 3) demonstrate that a concentration of 6–7 nM OT was required to displace 50% of radiolabeled OT from OTR in the late-gestation rat uterine membranes. In contrast, concentrations of > 1 µM of the OTX forms were required to displace half of the bound OT. Again, by two-way ANOVA, there were no significant differences among the OTX forms with respect to their ability to displace OT from its uterine receptor.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We elected not to attempt to separate endometrium from underlying myometrium in order to avoid unnecessary tissue manipulation. Further, since we are primarily interested in the paracrine relationships in uterine tissues, we preferred to study the intact tissues, both for the OTX concentration studies and the muscle bath experiments. We have not localized the precise tissue of origin of either OT or OTX forms. OT mRNA is localized predominantly in the endometrial layer of both the rat [1] and human [2]. Presumably the OTX forms are found in the same cells as the OT mRNA. However, we have not determined whether the OTX forms are ever secreted and thus have opportunity to interact with OTR. The rat appears to be different from sheep and humans in that the OTX forms do not appear in the rat circulation.
The results from the experiments using estrogen and progesterone receptor antagonists suggest that uterine concentrations of OTX forms are regulated by these sex steroids. Again, we have noted similar effects on OT mRNA concentrations [3, 4]. However, the reduction in mRNA was approximately 50–70% whereas the decrease in OTtotal was > 90%. This suggests that estrogen and progesterone may have independent effects on the concentrations of OT mRNA and of OTX, and it may indicate that they regulate both transcription and translation, though an effect on hormone clearance cannot be ruled out.
Contrary to our hypothesis, labor in the undisturbed control animals was not preceded by a significant increase in the ratio of OT to OTX forms. After treatment with the progesterone receptor antagonist RU-486, the ratio of OT:OTtotal was increased many-fold by 12 h, and this was accompanied by preterm labor and delivery. Whereas this would appear to support our initial hypothesis, the data from the experiments using the estrogen receptor antagonist tamoxifen suggest a different conclusion. In these experiments, the OT:OTtotal ratio increased significantly in the treated group. Despite this, the timing of parturition was delayed by 24 h. As we have noted previously [3, 4], parturition occurred in both pharmacologic models in the absence of an increase in uterine OT concentrations. Taken together, these findings suggest that the OTX forms do not compete with OT for receptor binding.
In the tamoxifen experiments, the concentration of OTX forms was decreased by more than 90% but OT concentrations were decreased only by 30–50%. Thus, in the presence of markedly reduced precursors, OT levels were relatively maintained in the face of the estrogen antagonist. One possible explanation for this is that a greater proportion of the precursor pool was being converted to mature OT. This would imply that the effects of estrogen may in fact inhibit further processing of the precursors. The maintenance of near-normal OT concentrations suggests that there may be some type of local feedback mechanism that could increase posttranslational processing when OTX forms are reduced. Another possible explanation is that the tissue half-life for OT is longer than for the OTX forms, resulting in tissue OT concentrations remaining relatively unchanged over the course of the experiments despite the marked decrease in precursor availability. Our previous studies have demonstrated active oxytocinase activity in these tissues, with no evidence of a change occurring at this time in the presence or absence of tamoxifen [15]. It is not known whether this oxytocinase activity also catabolizes the carboxy-extended forms of OT.
The muscle bath preparation provided a reliable measure of uterine activity. The concentration-response profiles with OT were very consistent. In preliminary experiments, rat uterine strips were obtained from rats at estrus. These preparations were quite active and required a long equilibration period in the muscle bath preparation before the background activity was low enough to allow the experiments to begin. As has been reported previously [13, 14], estradiol treatment to the nonpregnant animals for 2 days prior to experimentation reduced the background activity and allowed studies to be performed after an hour or two of equilibration. This period of estradiol treatment did not change the concentration-response profile of the uterine strips. The tissues obtained from the pregnant animals also demonstrated marked activity, and an equilibration period of approximately 4 h was required before experiments were commenced.
Myometrial concentrations of OTR increase significantly during late gestation in the rat [3, 16–18]. In the muscle bath experiments, the ED50 for OT in both pregnant and nonpregnant tissues is in the range of 1–2 nM, which is similar to that shown by Chan et al. [19]. This also is very similar to the Kd of OTR in both pregnant and nonpregnant uterine tissues [3, 17, 19] and close to tissue concentrations of OT near the onset of parturition. When the concentration-response curves were plotted using absolute tension rather than percentage of maximal stimulation, the curve for the pregnant tissues was shifted markedly to the left, indicating an increase in sensitivity. The maximal tension generated by the muscle strips from pregnant animals was approximately twice as much as for the tissues from nonpregnant rats. This may have been due to the hypertrophic or hyperplastic nature of the myometrium during gestation. We did not attempt to correct for the amount of muscle tissue mounted in the perfusion chambers.
The three carboxy-extended forms of OT demonstrated approximately similar concentration-response curves. Our concentration curves utilized concentrations up to 6400 nM. Endogenous tissue concentrations of the OTX forms rise to approximately 20 000 pg (corresponding to approximately 20 pmol)/g uterine tissue prior to delivery. This concentration may correspond to concentrations of 20 nM. Thus, it is likely that our concentration range greatly surpasses physiologic concentrations. Even at these high concentrations, it is not apparent that a maximal effect has been achieved, making it inappropriate to estimate an ED50. However, it is clear that this would be at least two to three orders of magnitude greater than for OT. Furthermore, even at pharmacologic concentrations, the uterine stimulation caused by the OTX forms is less than half that generated by maximal concentrations of OT. It is unlikely that the high concentrations of OTX forms caused down-regulation of OTR since there was a normal concentration-response curve for OT at the end of each experiment. These data suggest that the OTX forms have very little ability to directly stimulate uterine contractions.
The experiments demonstrating normal concentration-responses curves for OT in the presence of high concentrations of the OTX forms also suggest no interaction between OT and its extended forms. The original intent of these experiments was to determine pA2 values for each of the OTX forms, but this was impossible because of their failure to inhibit OT responses. In some instances, there appeared to be a slight increase in the contractile activity in the presence of the OTX forms at the lowest concentration of OT. This likely reflects the weak agonistic properties (Fig. 2) that are additive to OT.
The binding studies support this conclusion. OT displaced 50% of bound radiolabeled OT at approximately 5–10 nM, which is similar to the findings of Chan et al. [19]. In contrast, the OTX forms caused no displacement of radiolabeled OT until concentrations of 100 nM. The displacement curves for OTX were at least two orders of magnitude to the right of the OT curve. This is in close agreement with the low ability of the OTX forms to influence the physiologic effects of OT noted in the muscle bath experiments. Since OT and the OTX forms have similar amino terminals and differ only in the carboxy-terminals, these findings suggest that binding of OT to OTR is influenced by the carboxy-terminus of the OT molecule. This is compatible with recent evidence demonstrating that the specificity of OTR may depend upon interaction of the lateral chain of residue 8 of the OT molecule with the first extracellular loop of the G protein-linked OTR [20].
In summary, these experiments have demonstrated the presence of high concentrations of carboxy-extended forms of OT in the late-gestational rat uterus. The data support a role for estrogen and progesterone in the regulation of these concentrations. Since no other species has been studied to date, it is unclear whether these findings are specific to rat gestation or are generalizable across species. These are the first studies investigating the potential biological activities of the carboxy-extended forms of OT in the uterus. Although concentrations of these forms increase markedly in rat uterine tissues at the end of gestation, to levels 10- to 30-fold greater than those of OT, they have no biologically significant activity with respect to uterine contractions. In contrast to our hypothesis, OTX forms do not compete with OT for binding to OTR, and the ratio of OT to OTX forms appears to have no physiologic importance. It appears that the major physiologic role of the OTX forms is as a precursor pool for OT biosynthesis. Studies of the regulation of the processing of these prohormones to active mature OT may provide useful information regarding the control of uterine contractility in late gestation in the rat.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: B.F. Mitchell, University of Alberta, Department of Obstetrics and Gynecology, 205 CSC, 10240 Kingsway Ave., Edmonton, AB, Canada T5H 3V9. FAX: 403 477 4981; brymitch{at}gpu.srv.ualberta.ca
Accepted: July 4, 1998.
Received: February 16, 1998.
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