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Journal of Physiology (2001), 536.1, pp. 123-131
© Copyright 2001 The Physiological Society
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ABSTRACT |
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1-adrenergic receptors (1-AR) are involved in mouse uterine contraction at parturition. Myometrial phospholipase C (PLC) activity and uterine contraction were measured in response to noradrenaline (NA), the specific 1-AR agonist phenylephrine (Phe) and oxytocin (OT).
1a-AR subtype in late pregnant mouse myometrium. We also detected, by immunoblotting studies, PLC
1, PLC3 and different -subunits of pertussis toxin-insensitive (Gq/11) and -sensitive G proteins (Go/i3, Gi1/2) .
1 and PLC3 to myometrial plasma membranes. The OT-induced InsP response was not altered by pertussis toxin (300 ng ml-1, 2 h pretreatment), suggesting the involvement of a member of the Gq family.
1-AR are linked to myometrial PLC activation and uterine contraction in late pregnant and parturient mouse. This discrepancy between mouse and other mammals could be attributed to the 1-AR subtype expressed in myometrium at this time.
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INTRODUCTION |
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Catecholamines (adrenaline, noradrenaline) play an important role in the regulation of mammalian uterine contractility. The type of regulation depends on hormonal state and involves activation of myometrial adrenergic receptors (-, 1-, and 2-AR) (Marshall, 1981). During the course of pregnancy, -AR mediate the inhibitory effect exerted by catecholamines on uterine contraction through activation of adenylyl cyclase (Wray, 1993). Near term, -AR dominance is replaced by an excitatory effect which seems to involve -AR. In particular, the contribution of 1-AR has been well documented by the fact that administration of prazosin (an 1-AR antagonist) to late pregnant rats decreased uterine electromyographic activity and delayed parturition (Legrand & Maltier, 1986). In human and rat myometria, 1-AR are linked to PLC (Breuiller-Fouche et al. 1991; Limon-Boulez et al. 1997) via activation of Gq/11 protein (Limon-Boulez et al. 1997). It is now well established that inositol 1,4,5-trisphosphate produced via PLC-induced degradation of phosphatidyl inositol biphosphate mobilizes calcium from internal stores. The following increase in intracellular free calcium activates myosin light chain kinase, the key enzyme which promotes the interaction between actin and myosin, resulting in contraction. It is of interest that 1-AR are more effective at increasing PLC activity at term than during pregnancy in the rat (Limon-Boulez et al. 1997), indicating an increased sensitivity of the myometrium to the stimulant action of catecholamines near term. Using pharmacological studies, we identified 1a- and 1b-AR subtypes in rat myometrium. However, only the 1b-AR subtype seems to mediate the effects of catecholamines near term. Indeed, an 1b-AR-specific antagonist completely inhibited the Phe-induced PLC response at parturition. In addition, as measured by competition studies of Gpp(NH)p with an 1-AR-specific radioligand, the G-protein-coupled state of the 1b-AR subtype increases at parturition compared with pregnancy. The resulting enhancement of 1b-AR coupling to the PLC pathway can be explained, at least in part, by the upregulation of transduction entities operating downstream from receptors such as the Gq protein and PLC3 isoform (Cohen-Tannoudji et al. 1995; Lajat et al. 1996).
In mouse, little is known about the signalling pathways involved in the initiation of uterine contractions at term. In particular, the role of the 1-adrenergic pathway in parturition has not been studied. This information is important in this model since genetically altered mice are used intensively to delineate the role of many factors in the switch of uterine activity at term (Kimura et al. 1999). Therefore, the aim of this study was to investigate whether activation of the 1-adrenergic signalling pathway contributes to uterine contraction in mouse at term. For this purpose, we first characterized the 1-adrenergic subtypes expressed in mouse myometrium. We also determined the types of transduction entities that could potentially mediate PLC responses. Indeed, no data are available on the types of PLC isoforms and G subunits expressed in late pregnant mouse myometrium. We also compared the effects of two adrenergic agonists, i.e. Phe and NA, on PLC translocation and InsP production. To our knowledge, this is the first study on the PLC pathway in mouse myometrium. Finally, we measured uterine contractions in response to increasing concentrations of Phe and NA. We used as control OT, a known uterine contractant which seems to utilize the same signal pathway as 1-AR to induce uterine contraction in rat and human (Sanborn et al. 1998).
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METHODS |
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Materials
myo-[2-3H]Inositol (10-25 Ci mmol-1) and [3H]OT (30-60 Ci mmol-1) were purchased from NEN Life Science Products. Rabbit polyclonal antibodies directed against the carboxyl termini of PLC1/2/4 were obtained from Santa Cruz Biotechnology and the -subunit of Gio/3 (EC2), Gi1/2 (AS7) and Gq/11 (QL) proteins were from NEN Life Science Products. Rabbit polyclonal anti-carboxyl terminal of PLC3 was kindly provided by Dr Z. Tanfin. Phenylephrine, OT, NA and pertussis toxin (PTX) were from Sigma, enhanced chemiluminescence reagent from Amersham Pharmacia Biotech, and AG1-X8 resin from Biorad. Krebs bicarbonate buffer contained (mM): NaCl 117; KCl 4.7; MgSO4 1.1; KH2PO4 1.2; NaHCO3 24; CaCl2 0.8; glucose 1; pH 7.4.
Animals and tissues
C57BL/6J mice were obtained from a colony bred and maintained at the animal house of the University Pierre et Marie Curie. They were housed three per cage with free access to food and water, in a 12:12 h light-dark cycle. Day 1 of pregnancy corresponds to the day on which a plug of semen is found in the vagina. In our breeding colony, parturition occurs between 00.00 and 08.00 h on day 20 for 70 % of mice. Pregnant Sprague-Dawley rats were obtained from Janvier (Le Genest, France). Animals were killed by cervical dislocation at late pregnancy (day 19) or during the expulsion of fetoplacental units (parturition or term), following the guidelines laid down by the French/European ethical committee. The uterine horns were quickly isolated, cut open lengthwise and the fetoplacental units removed. For RT-PCR, plasma membrane preparations, PLC translocation and InsP production studies, the myometrium was freed of adherent endometrium.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA from mouse and rat myometria or rat brain was prepared using an RNA-PLUS kit (Bioprobe). Five micrograms of total RNA were reverse-transcribed using a kit from GibcoBRL Life Technologies and the resulting complementary DNA (cDNA) was stocked at -80 °C. Polymerase chain reaction (PCR) amplification was performed with specific upstream and downstream primers previously described for 1a-, 1b- and 1d-AR subtypes (Cavalli et al. 1997). A 1/20 volume of each RT reaction was amplified using a kit from GibcoBRL Life Technologies. Reaction cycles (40 times) were one denaturation step for 2 min at 94 °C, followed by annealing for 1 min at 56 °C and extension for 2 min at 72 °C. The PCR products were separated by electrophoresis on an ethidium bromide-containing 2 % agarose gel. Control PCR reactions performed on non-transcribed RNA indicated no contamination of the RNA preparations with genomic DNA.
Myometrial plasma membrane preparations
Crude membranes were prepared from mouse and rat myometria or rat brain as described previously (Mhaouty et al. 1995). Plasma membrane preparations were resuspended in homogenization buffer for immunoblotting studies and in buffer A (25 mM Tris, 10 mM MgCl2, pH 7.5) for [3H]OT binding studies. Protein concentration was determined according to Bradford (1976) with bovine serum albumin (BSA) as standard. Samples were stored at -80 °C until use.
Immunoblotting
Fifty micrograms of myometrial plasma membranes were subjected to SDS-PAGE in 7.5 % gels and transferred to polyvinylidene difluoride (PVDF) membranes (NEN Life Science Products). The PVDF membranes were blocked overnight at 4 °C in 5 % non-fat dried milk-Tris-buffered saline (TBS) and incubated for 1 h at room temperature with anti-PLC (diluted 1:200) or anti-G protein (diluted 1:1000) prepared in 5 % non-fat dried milk-TBS. After 45 min incubation at room temperature with secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit antibody, Jackson) diluted 1:10000, immunoreactive bands were visualized by the chemiluminescence detection system.
PLC translocation studies
Myometrial strips (50 mg) were incubated at 37 °C in Krebs bicarbonate buffer in the presence of 5 % CO2-95 % O2. After 30 min equilibration, 10 µM Phe or OT was added to the buffer. Reactions were stopped 1 min later by adding a large volume of cold EDTA (10 mM). Strips were then frozen in liquid N2 until plasma membrane preparations and immunoblotting studies with specific PLC antibodies. The amount of immunoreactivity was determined by densitometric scanning and computer analysis of the immunobands using the NIH image 1.62 programme.
Myometrial InsP production
Myometrial production of InsP was measured as described previously (Lajat et al. 1996). Briefly, myometrial strips (20 mg) were incubated at 37 °C for 4 h with 7 µCi myo-[3H]inositol (0.4 µM) in 1 ml of Krebs bicarbonate buffer in the presence of 5 % CO2-95 % O2. Increasing concentrations of Phe or OT were added after 10 min incubation of myometrial strips with 10 mM LiCl in Krebs bicarbonate buffer. Assays were stopped 15 min later by freezing the strips in liquid N2. When used, PTX (300 ng ml-1) was added 2 h before the addition of myo-[3H]inositol and the incubations were continued as described above.
[3H]InsP was measured as follows. Strips were homogenized in 7 % trichloroacetic acid and the obtained supernatant extracted with diethyl ether, neutralized with Tris-base and then chromatographed over anion-exchange resin (AG1-X8). Total InsP eluted with 1 M ammonium formate-0.1 M formic acid were counted by liquid scintillation in a 1214 Rack-beta spectrometer (LKB) for tritium.
[3H]OT binding studies
[3H]OT binding experiments were performed as previously described (Engstrom et al. 1988). Briefly, 70 µg of myometrial plasma membranes were incubated for 1 h at room temperature in buffer A supplemented with BSA 0.1 % in the presence of eight different concentrations of [3H]OT (0.4 nM to 24 nM). Non-specific binding was measured in the presence of 10 µM unlabelled OT. Incubations were stopped by adding 5 ml of Tris-HCl ice-cold buffer followed by rapid cold filtration over GF/C glass fibre filters (Whatman). Radioactivity was counted by liquid scintillation in a 1214 Rack-beta spectrometer (LKB). All assays were performed in duplicate. Specific binding was calculated as the difference between total and non-specific binding. Maximal specific binding (Bmax) and receptor affinity (Kd) were determined from regression analysis of Scatchard plots.
Isometric contraction measurements
Uterine strips 4 mm in length were prepared from late pregnant or parturient mice and mounted in tissue baths containing 8 ml of Krebs bicarbonate buffer bubbled continuously with 5 % CO2-95 % O2 and warmed to 30 °C. Depending on the orientation of the strips, we measured tissue tension of the circular inner or longitudinal outer layer of uterine muscle using a Bioscience UF1 tension transducer (Phymep, Paris). Strips were washed twice with Krebs buffer and allowed to equilibrate for 30 min under 0.7 g resting force. Responses to cumulative doses of Phe, NA or OT (0.1 nM to 10 µM) were examined either in the absence or in the presence of the -AR antagonist propranolol (10 µM, 10 min pre-incubation). All drugs used represented 1:1000 of total volume. The concentration-response curves were recorded by computerized calculation of the integral under the tension-time curve for 3 min. The contractile response to Phe, NA or OT was expressed as the percentage of the spontaneous activity in the absence of drugs.
Statistical studies
Results are expressed as means ± standard error of the mean (S.E.M.). Statistical significance was assessed by Student's t test for paired and unpaired data. A P value less than 0.05 was considered to be significant.
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RESULTS |
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Characterization of mouse myometrial 1-AR subtypes by RT-PCR
We assessed the mRNA expression of the three 1-AR subtypes (1a-, 1b- and 1d-AR) in late pregnant mouse myometrium and control tissues using the RT-PCR technique. As shown in Fig. 1, only transcripts for 1a-AR were amplified from the total RNA of mouse myometrium. In contrast, both 1a- and 1b-AR mRNAs were detected in late pregnant rat myometrium, which is in agreement with our previous pharmacological studies (Limon-Boulez et al. 1997). The 1d-AR transcripts were not detected in mouse and rat myometria whereas a specific signal was seen in rat brain known to express this subtype (Lomasney et al. 1991). Thus, these results indicated the expression of 1a-AR subtype in mouse myometrium and confirmed the co-expression of 1a- and 1b-AR subtypes in rat myometrium.
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Figure 1. RT-PCR for Sequences of | ||
Characterization of myometrial PLC isoforms and Gi/o proteins in late pregnant mouse myometrium
We performed immunoblotting studies using specific antibodies directed against the four isoforms of PLC (Rebecchi & Pentyala, 2000) and the -subunit of Gq and Gi/o families. Rat brain and pregnant myometrium, where the expression of different PLC isoforms and G subunits have been already reported (Cohen-Tannoudji et al. 1995; Ku et al. 1995), were used as controls. Figure 2A shows the presence, at the expected molecular mass (150 kDa), of PLC1and PLC3 in mouse myometrial membranes and positive controls. In contrast, only a very faint band was observed for PLC4 (130 kDa) in mouse myometrium as compared with controls. No signals were detected for PLC2 in myometrium (data not shown), confirming the reported restricted expression of this isoform (Rebecchi & Pentyala, 2000). To characterize the expression of PTX-sensitive (Gi/o) and -insensitive proteins (Gq/11) in mouse myometrium, we used different antibodies. The EC2 antibody recognized a signal of 40 kDa corresponding to Go/i3 subunits and AS7 identified a 39 kDa band corresponding to Gi1/2 protein (Fig. 2B). The QL antibody raised against Gq/11 stained a band of the appropriate molecular mass (42 kDa) in late pregnant mouse myometrium and controls (Fig. 2B).
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Figure 2. Immunodetection of PLC Representative immunoblots for PLC | ||
These results demonstrated that mouse myometrium expresses two main PLC isoforms (PLC1 and PLC3) as well as Gi/o and Gq/11 proteins at late pregnancy. Since 1-AR are also expressed in mouse myometrium, as shown above, we investigated whether they couple these characterized proteins to increase PLC activity.
Effects of Phe and OT on PLC translocation to myometrial plasma membranes
Once activated, PLC1 and PLC3 are rapidly translocated, in the first minutes following receptor activation, towards the plasma membrane (Lajat et al. 1998). We took advantage of this property to determine whether activation of 1-AR and OTR recruits PLC1 and/or PLC3 to the plasma membrane of late pregnant mouse myometrium. As illustrated in Fig. 3, Phe (10 µM) did not significantly increase either PLC1 or PLC3 translocation to the plasma membranes. In contrast, OT (10 µM) produced a statistically significant increase (about 80 % over control) in the level of plasma membrane PLC1 and PLC3 (Fig. 3). It thus appeared that OTR activate both PLC1 and PLC3 whereas 1-AR do not seem to be linked to the PLC pathway in late pregnant mouse myometrium. In order to verify this hypothesis, we measured the InsP production of myometrial strips in response to Phe and OT.
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Figure 3. Effects of phenylephrine and oxytocin on PLC Strips from late pregnant mouse myometrium were incubated in the absence (basal) or presence of 10 µM phenylephrine (Phe) or oxytocin (OT). Plasma membranes were then prepared and studied for PLC | ||
Effects of Phe and OT on myometrial InsP production in late pregnant and parturient mice
Myometrial strips obtained from late pregnant mice were exposed to increasing concentrations of Phe or OT. As illustrated in Fig. 4A, OT elicited a dose-dependent increase in total InsP production with a mean EC50 value of 2 nM OT, in agreement with the affinity (Kd) determined by [3H]OT binding studies (1.8 ± 0.2 nM). The maximal increase in InsP production (+130 % above basal) was reached with 100 nM OT. Pretreatment of strips with PTX (300 ng ml-1) for 2 h did not alter InsP production in response to OT (Fig. 4B). Indeed, neither the EC50 nor the maximal response were affected by PTX pretreatment. In contrast with results obtained with OT, when myometrial strips were incubated with Phe, no increase in InsP production was observed, even at high concentrations of this agonist (Fig. 4A).
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Figure 4. Inositol phophate production in response to phenylephrine and oxytocin and effect of pertussis toxin on oxytocin effect in late pregnant and parturient mouse myometrium A, myometrial inositol phosphate (InsP) production of strips from late pregnant mice in response to increasing concentrations of phenylephrine (Phe) or oxytocin (OT) (0.1 nM to 10 µM). B, myometrial InsP production in response to increasing concentrations of OT in the absence or presence of pertussis toxin (PTX) (300 ng ml-1, 2 h pretreatment) at late pregnancy. Mean basal InsP production was 170 ± 15 c.p.m. (mg tissue)-1 in the absence or presence of PTX. C, myometrial InsP production in response to increasing concentrations of OT, Phe and NA at parturition. Mean basal InsP production was 260 c.p.m. (mg tissue)-1. Responses were expressed as a percentage of basal InsP production and results are the means of four independent experiments (A and B) and two independent experiments (C). *P < 0.05; **P < 0.01; ***P < 0.001 versus basal. | ||
Since a higher effect of 1-AR on PLC activity was observed in parturient rats (Limon-Boulez et al. 1997), we investigated whether the mouse 1-adrenergic pathway could be activated in the last hours of pregnancy. We then tested the effects of increasing concentrations of Phe or OT on myometrial strips obtained from parturient mice. As shown in Fig. 4C, again only OT was able to increase myometrial InsP production, with an EC50 value in the range of that reported above for late pregnant mouse. In contrast, whatever the concentration of Phe used, no increase in InsP production was observed (Fig. 5). We tested the effect of the endogenous agonist NA on the myometrial InsP response in parturient mice. As shown in Fig. 4C, stimulation of myometrial strips with increasing concentrations of NA had no effect on the InsP response.
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Figure 5. Contractile responses of uterine strips to phenylephrine, noradrenaline and oxytocin in late pregnant mouse A, representative recordings of uterine tissue tension in the presence of oxytocin (OT), phenylephrine (Phe) and noradrenaline (NA). OT increased both the frequency and amplitude of uterine tension in a dose-dependent manner whereas Phe and NA induced relaxation at high concentrations. B, average results from four independent experiments done in triplicate. Data are represented as a percentage of spontaneous contraction in the absence of drugs. *P < 0.05; **P < 0.01; ***P < 0.001 versus spontaneous contraction. | ||
Effects of Phe and OT on uterine contraction in late pregnant and parturient mice
Besides the PLC system, 1-AR can activate other signalling transduction pathways to induce contraction (Zhong & Minneman, 1999). It was therefore important to investigate whether or not 1-AR activation has an effect on uterine contraction in late pregnant and parturient mouse. Since myometrium contains an inner circular and an outer longitudinal layer, we measured contractions of both smooth muscles in the presence of increasing concentrations of Phe, NA or OT. We observed similar patterns of responses between both myometrial layers for all tested drugs. The results obtained for the circular layer are described below.
All uterine strips studied exhibited spontaneous contractions a few minutes after being mounted in the bath. Addition of increasing concentrations of OT increased both the frequency and amplitude of contractions in all studied preparations (Fig. 5A and 5B). Analysis of the sigmoid dose-response curves obtained (Fig. 5B) revealed a mean EC50 value of 70 nM and maximal stimulation at a concentration of 1 µM OT. In the presence of Phe, we did not observe any increase in the uterine contractile response at any of the concentrations tested (Fig. 5A and 5B). Rather, a significant decrease was observed at high concentrations of Phe (30 % and 51 % decrease at 1 µM and 10 µM, respectively). When challenged with NA, a significant decrease in uterine contraction was also noted (Fig. 5A and 5B) but with a lower mean EC50 value compared with Phe (0.1 µM for NA vs. 1.5 µM for Phe). Pre-incubation of uterine strips with a -AR antagonist (propranolol 10 µM, 10 min pre-incubation) had by itself no effect on tissue tension but abolished relaxation induced by both Phe and NA (Fig. 6A and 6B).
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Figure 6. Effects of propranolol pretreatment on phenylephrine- and noradrenaline-induced uterine relaxation Uterine strips were pre-incubated in the absence or presence of the | ||
Similar responses to OT and adrenergic agonists were observed in parturient mice (data not shown). At this time, however, the mean EC50 calculated for OT was eight-fold lower than that reported above (about 9 nM). In addition, Phe-induced uterine relaxation was attenuated (10 % of decrease at parturition vs. 51 % at late pregnancy for 1 µM Phe).
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DISCUSSION |
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In the present study, we investigated whether activation of the myometrial 1-adrenergic signalling pathway may contribute to uterine contraction in mouse near parturition. Our data indicate that, in contrast to OT, catecholamines are not linked to the myometrial PLC pathway and uterine contraction. This discrepancy between mouse and other mammals (rat and human) is discussed below.
Characterization of myometrial 1-AR subtypes, G proteins and PLC isoforms in late pregnant mouse
Using the RT-PCR technique, we reported the expression of 1-AR in mouse myometrium. In particular, 1a-AR seems to be the predominant subtype at the end of pregnancy. In contrast, both 1a- and 1b-AR were detected in rat at this time, suggesting differences in 1-AR subtype expression between mouse and rat. Using immunoblotting studies, we also detected two major PLC isoforms, i.e. PLC1 and PLC3 in late pregnant mouse myometrium. PLC2 was not found either in mouse or rat myometrium, which is in agreement with the specific expression of this isoform in haematopoeitic cells (Rebecchi & Pentyala, 2000). A similar result has been reported for late pregnant rat (Lajat et al. 1996; Dodge et al. 1999) and the PHM1-41 cell line derived from human pregnant myometrium (Dodge & Sanborn, 1998). In addition to PLC1 and PLC3, late pregnant rat myometrium also expresses PLC4, as shown in our study. In contrast, only a very low expression of this isoform was detected in mouse myometrium, suggesting specific differences in the expression level of myometrial PLC4 between rat and mouse at late pregnancy. Using specific antibodies, we also detected several -subunits of heterotrimeric G proteins in late pregnant mouse myometrium, i.e. Go/i3, Gi1/2 and Gq/11. In particular, the expression of Gq/11 presents a good candidate for PLC1 and PLC3 activation (Rhee & Bae, 1997). Nonetheless, a role for Go/i3 and Gi1/2 cannot be excluded since PLC3 can also integrate signals from Gi/o proteins (Rhee & Bae, 1997).
Myometrial OT pathway in late pregnant and parturient mouse myometrium
Oxytocin elicited a dose-dependent contraction of late pregnant mouse uterus, confirming previous data (Suzuki & Kuriyama, 1975). In rat and human myometria, OT induces contraction via activation of PLC (Sanborn et al. 1998). For instance, OT-induced contraction can be prevented by the selective PLC antagonist U-73122 in rat (Wassdal et al. 1998). We showed, in the present report, that OT also increases myometrial InsP production in late pregnant mouse. Furthermore, we demonstrated that two PLC isoforms (PLC1 and PLC3) are activated by OTR, suggesting the interaction of OTR with both isoforms to increase InsP production near term. We also showed that this response was mediated via a PTX-insensitive G protein, probably Gq/11 protein which is concomitantly expressed in myometrium at this time. As was found previously (Suzuki & Kuriyama, 1975), we observed an increased sensitivity of mouse uterus to OT at parturition since the contraction-response curve was shifted to the left by a factor of eight. This could be explained, at least in part, by a significant increase in myometrial OTR density since specific [3H]OT-bound sites increased three-fold at term (513 ± 115 fmol (mg protein)-1 at day 19 vs. 1381 ± 205 fmol (mg protein)-1 at term). Consistent with this result, an upregulation at the level of OTR transcripts was reported by several groups in mouse myometrium near term (Russell & Leng, 1998). It remains to be determined whether Gq/11 protein, PLC1 and PLC3 are also upregulated in mouse myometrium at this time, as shown in late pregnant rat (Cohen-Tannoudji et al. 1995; Lajat et al. 1996).
1-Adrenergic pathway in late pregnant and parturient mouse myometrium
In contrast to OT, Phe and NA failed to contract the circular and longitudinal layers of late pregnant or parturient mouse uterus. Rather, high concentrations of both agonists decreased uterine contraction. This effect seems to involve -AR since it was blocked by pre-incubation of uterine strips with propranolol. The implication of -AR in such a response is further substantiated by the fact that Phe was less efficient than NA at inducing uterine relaxation (our results), consistent with the known higher selectivity of Phe for 1-AR. These results indicated the presence of functional -AR in both circular and longitudinal layers of pregnant mouse myometrium. As previously reported (Cruz et al. 1990), -AR seem to induce uterine relaxation during pregnancy. Interestingly, -AR-induced uterine relaxation was attenuated in parturient mice (data not shown), and this may be due to desensitization of the -adrenergic pathway which takes place in the myometrium of various species near term (Russell & Leng, 1998).
Our results further demonstrated that 1-AR are not linked to uterine contraction in late pregnant and parturient mouse. Firstly, any effect of Phe or NA on tissue tension was unmasked when -AR-induced uterine relaxation was blocked by propranolol. Secondly, at parturition when -AR-induced relaxation was attenuated, we did not observe any effect of Phe on uterine tension. Thus, it seems that the uterine action of catecholamines does not switch to an 1-excitatory effect in mouse at term. This distinguishes mouse from both rat and human, for which an increased sensitivity of the uterus to 1-AR activation at term has been reported (Legrand & Maltier, 1986; Pennefather et al. 1993). The higher uterine responsiveness to 1-AR activation in parturient rat was found to be associated with an increase in 1-AR coupling to the Gq/11-PLC system (Limon-Boulez et al. 1997). Interestingly, and in line with the uterine contraction studies described above, we found no evidence of 1-AR coupling to the PLC pathway in late pregnant mouse myometrium. In fact, neither of the two main PLC isoforms (PLC1 and PLC3) was recruited to plasma membranes in response to Phe. Furthermore, there was no InsP production in response to increasing concentrations of Phe and NA. These findings cannot be explained by an absence and/or lesion in distal elements in the signalling machinery. Firstly, Gq/11 and PLC1/3 are expressed in late pregnant mouse myometrium. Secondly, OTR activated the PLC pathway and induced uterine contraction under similar conditions.
Our interpretation of these data is that myometrial 1a-AR, which seems to be the predominant 1-AR subtype at late pregnancy, is not linked to the myometrial PLC pathway and uterine contraction in mouse. Interestingly, although two 1-AR subtypes (1a- and 1b-) have been identified using pharmacological (Limon-Boulez et al. 1997) and molecular tools (the present study) in rat myometrium, only the 1b- subtype has been found to be linked to the PLC pathway at parturition (Limon-Boulez et al. 1997). Consistent with this result, Hrometz et al. (1999) reported that the 1a-AR subtype can be expressed but does not contribute to contractile regulation in some vascular smooth muscle cells. Taken together, all these observations lead us to suggest that when present, the 1b-AR subtype regulates uterine contraction whereas the 1a-AR subtype regulates other cellular processes in myometrial cells. During pregnancy, the myometrium undergoes important phenotypic changes that permit growth of fetoplacental units. This consists of myometrial cell hyperplasia and hypertrophy, as has been reported previously (Alexandrova & Soloff, 1980; Engstrom et al. 1997). It is of interest that 1-AR have been shown to regulate both cell growth and hypertrophy via activation of mitogen-activated protein kinase pathways in smooth muscle cells (Zhong & Minneman, 1999). Whether or not the 1a-AR subtype controls such responses in pregnant mouse myometrium remains to be determined.
Differences between 1a- and 1b-AR subtypes in terms of their coupling to phosphatidylinositol hydrolysis and mitogen-activated protein kinase pathways have been observed also in neonatal rat cardiac myocytes (Wenham et al. 1997). However, in contrast to late pregnant myometrium, the 1a-AR subtype appeared to couple PLC whereas activation of mitogen-activated protein kinase involved the 1b-AR subtype. This suggests that, as for 2-AR (Duzic et al. 1992; Duzic & Lanier, 1992), 1-AR coupling is not only subtype- but also cell type-specific. Further investigations will be needed to evaluate whether this specificity could rely on the type of heterotrimeric G proteins and effector molecules activated by 1-AR and/or unknown accessory proteins that regulate the interaction between these entities.
In conclusion, our results indicate that, in contrast to other mammals, catecholamines are not linked to the myometrial PLC pathway and uterine contraction in late pregnant mouse. This discrepancy could be attributed to the 1-AR subtype expressed in myometrium at this time. From the present study and our previous data, we propose the existence of a relationship between myometrial 1b-AR expression and regulation of uterine contraction. Future characterizations of myometrial 1-AR subtypes involved in uterine contraction of other mammals will further test this hypothesis.
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Acknowledgements
We thank Dr Z. Tanfin for providing the anti-PLC3, Dr I. Limon-Boulez for critical reading and M. T. Robin for illustration of the manuscript, and P. Thouvenot for taking care of the animals.
Corresponding author
S. Mhaouty-Kodja: Université Pierre et Marie Curie, Laboratoire de Physiologie de la Reproduction, Bât A 6ème étage, 4 place jussieu, 75252 Paris Cedex 05, France.
Email: Sakina.Mhaouty-Kodja{at}snv.jussieu.fr
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