Characterization of mouse myometrial α1-AR subtypes by RT-PCR
We assessed the mRNA expression of the three α
1-AR subtypes (α
1a-, α
1b- and α
1d-AR) in late pregnant mouse myometrium and control tissues using the RT-PCR technique. As shown in
Fig. 1, only transcripts for α
1a-AR were amplified from the total RNA of mouse myometrium. In contrast, both α
1a- and α
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 α
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 α
1a-AR subtype in mouse myometrium and confirmed the co-expression of α
1a- and α
1b-AR subtypes in rat myometrium.
| Figure 1 RT-PCR for α1-AR subtypes from late pregnant mouse and rat myometria |
Characterization of myometrial PLCβ isoforms and Gαi/o proteins in late pregnant mouse myometrium
We performed immunoblotting studies using specific antibodies directed against the four isoforms of PLCβ (
Rebecchi & Pentyala, 2000) and the α-subunit of G
q and G
i/o families. Rat brain and pregnant myometrium, where the expression of different PLCβ isoforms and Gα 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 PLCβ
1and PLCβ
3 in mouse myometrial membranes and positive controls. In contrast, only a very faint band was observed for PLCβ
4 (130 kDa) in mouse myometrium as compared with controls. No signals were detected for PLCβ
2 in myometrium (data not shown), confirming the reported restricted expression of this isoform (
Rebecchi & Pentyala, 2000). To characterize the expression of PTX-sensitive (G
i/o) and -insensitive proteins (G
q/11) in mouse myometrium, we used different antibodies. The EC2 antibody recognized a signal of 40 kDa corresponding to Gα
o/i3 subunits and AS7 identified a 39 kDa band corresponding to Gα
i1/2 protein (
Fig. 2B). The QL antibody raised against Gα
q/11 stained a band of the appropriate molecular mass (42 kDa) in late pregnant mouse myometrium and controls (
Fig. 2B).
| Figure 2 Immunodetection of PLCβ isoforms and Gαi/o and Gαq/11 proteins in plasma membranes of late pregnant mouse myometrium |
These results demonstrated that mouse myometrium expresses two main PLCβ isoforms (PLCβ1 and PLCβ3) as well as Gαi/o and Gαq/11 proteins at late pregnancy. Since α1-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 PLCβ translocation to myometrial plasma membranes
Once activated, PLCβ
1 and PLCβ
3 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 α
1-AR and OTR recruits PLCβ
1 and/or PLCβ
3 to the plasma membrane of late pregnant mouse myometrium. As illustrated in
Fig. 3, Phe (10 μ
m) did not significantly increase either PLCβ
1 or PLCβ
3 translocation to the plasma membranes. In contrast, OT (10 μ
m) produced a statistically significant increase (about 80 % over control) in the level of plasma membrane PLCβ
1 and PLCβ
3 (
Fig. 3). It thus appeared that OTR activate both PLCβ
1 and PLCβ
3 whereas α
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 Ins
P production of myometrial strips in response to Phe and OT.
| Figure 3 Effects of phenylephrine and oxytocin on PLCβ1 and PLCβ3 translocation towards the plasma membrane |
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 Ins
P production with a mean EC
50 value of 2 n
m OT, in agreement with the affinity (
Kd) determined by [
3H]OT binding studies (1.8 ± 0.2 n
m). The maximal increase in Ins
P production (+130 % above basal) was reached with 100 n
m OT. Pretreatment of strips with PTX (300 ng ml
−1) for 2 h did not alter Ins
P production in response to OT (
Fig. 4B). Indeed, neither the EC
50 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 Ins
P production was observed, even at high concentrations of this agonist (
Fig. 4A).
| 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 |
Since a higher effect of α1-AR on PLC activity was observed in parturient rats (Limon-Boulez et al. 1997), we investigated whether the mouse α1-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.
| Figure 5 Contractile responses of uterine strips to phenylephrine, noradrenaline and oxytocin in late pregnant mouse |
Effects of Phe and OT on uterine contraction in late pregnant and parturient mice
Besides the PLC system, α
1-AR can activate other signalling transduction pathways to induce contraction (
Zhong & Minneman, 1999). It was therefore important to investigate whether or not α
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 β-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).
| Figure 6 Effects of propranolol pretreatment on phenylephrine- and noradrenaline-induced uterine relaxation |
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).