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Increased potency of 1-adrenergic receptors to induce inositol phosphates production correlates with the up-regulation of 1d/Gh/phospholipase C1 signaling pathway in term rat myometrium

CNRS UMR 7079, Laboratoire de Physiologie et Physiopathologie, 75231 Paris Cedex 05, France and¹ INRA UMR 1054, Unité de Neuro-Gastroentérologie and Nutrition, 31931 Toulouse Cedex 09, France

Correspondence should be addressed to S Mhaouty-Kodja who is now at CNRS UMR 7148, Collège de France, 11 place Marcelin Berthelot, 75231 Paris Cedex 05, France; Email: sakina.mhaouty-kodja{at}college-de-france.fr

	Abstract

Top Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
In the present study, we studied the potential regulation by rat myometrial 1-adrenergic receptors (1-AR) of the newly identified Gh protein/phospholipase C1 (PLC1) signaling pathway and compared myometrial inositol phosphates (InsP) production and activity of the uterine circular muscle in response to 1-AR activation between mid-pregnancy and term. For this, we quantified the level of rat myometrial 1-AR coupling to Gh protein by photoaffinity-labeling, the cytosolic amount of PLC1 enzyme by immunoblotting, and the expression level of 1-AR subtypes by RT-PCR. The results showed an increased level of 1-AR/Gh protein coupling and the amount of PLC1 at term (+147 and +65% respectively, versus mid-pregnancy). This was correlated with an up-regulation of 1d-AR subtype (+70% versus mid-pregnancy). Incubation of myometrial strips with phenylephrine (Phe), a global 1-agonist, increased InsP production in a dose-dependent manner at both mid-pregnancy and term, but with an enhanced potency (tenfold decrease in EC₅₀ value) at term. Phe also dose-dependently induced contraction of the circular muscle at both mid-pregnancy and term. However, unlike InsP response, no amelioration of potency was observed at term. Similar results were obtained with the endogenous agonist norepinephrine. Our results show, for the first time, that rat myometrial 1d-AR/Gh/PLC1 signaling pathway is up-regulated at term. This is associated with an increased potency of 1-AR to elicit InsP production but not uterine contraction at this period. It is thus hypothesized that 1-AR, through activation of Gh/PLC1 system, are not primarily involved in the initiation of labor but may rather regulate responses such as myometrial cell proliferation or hypertrophy.

	Introduction

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It is well established that catecholamines (norepinephrine, epinephrine) regulate uterine contractility. The first evidence came from Rüsse and Marshall's works, which showed that norepinephrine, through activation of -adrenergic receptors (-AR), enhances the effects of uterotonic agents by improving the excitability of the uterine muscle in the guinea pig (Russe & Marshall 1970). Moreover, Legrand and Maltier have reported evidences for a noradrenergic transmission in the control of both pregnancy and parturition in rat. For instance, administration of propranolol, a β-AR antagonist, advanced pregnancy (Maltier & Cavaillé 1975). In contrast, administration to near term rats of prazosin, a powerful -AR antagonist, reduced myometrial excitability and delayed parturition (Legrand & Maltier 1986).

Norepinephrine signals through nine different G-protein-coupled ARs divided into three subfamilies, namely 1 (1a, 1b, 1d), 2 (2a, 2b, 2c), and β (β1, β2, β3). Using specific radioligands, we have shown that β- and 2-AR are co-localized in the longitudinal muscle, whereas 1-AR are expressed in the circular layer of rat myometrium (Legrand et al. 1991, 1993). Co-activation of β- and 2-AR increases myometrial cAMP production at mid-pregnancy (Mhaouty et al. 1995), thereby resulting in a relaxation of the longitudinal uterine smooth muscle. Near term, progesterone withdrawal associated with increase in estradiol concentrations trigger desensitization of β-adrenergic signaling pathway (Cohen-Tannoudji et al. 1991) and 2-AR shift to inhibitors of adenylyl cyclase (Mhaouty et al. 1995). These events probably participate in the initiation of labor.

Activation of global 1-AR triggers phospholipase C (PLC)-mediated degradation of phosphatidyl-inositol in plasma membrane preparations from late pregnant rat and human myometrium (Breuiller-Fouche et al. 1991, Limon-Boulez et al. 1997). Different mammalian PLC isoforms have been described including four β (PLCβ 1–4), two (1–2), four (1–4), and . Among the different subfamilies of PLCs, PLCβ members are triggered by G-protein-coupled receptors. In many smooth muscle cell types, stimulation of the Gq/PLCβ system is intimately linked to contraction through inositol phosphates (InsP₃)-dependent liberation of Ca²⁺ from intracellular stores. In line with these data, we have shown that the up-regulation of rat myometrial PLCβ1 and PLCβ3 and Gq protein correlates well with the increased uterine responsiveness to oxytocin and carbachol at term (Houdeau et al. 2005). In pregnant and parturient rat myometrium, we have also shown that stimulation of 1-AR results in the activation of Gh protein pathway (Dupuis et al. 2004). Gh protein is a bifunctional enzyme with both transglutaminase and GTPase activities (Nakaoka et al. 1994). By virtue of its GTP binding/GTPase activity, Gh protein acts as a signaling molecule. Nevertheless, unlike the Gq subunit, Gh protein does not interact with the members of the PLCβ family but selectively activates PLC1 enzyme (Das et al. 1993, Feng et al. 1996, Baek et al. 2001). The role of the Gh/PLC1 signaling pathway in smooth muscle cells remains to be clearly identified. PLC1 is, however, known to be induced during the cell cycle or by stress responses (Yagisawa et al. 2006).

Therefore, in light of all these data, the present work was undertaken to compare the level of rat myometrial 1-AR/Gh coupling, the expression pattern of 1-AR subtypes and PLC1 enzyme, and the potency of 1-AR to induce both myometrial InsP production and uterine contraction of the circular muscle between mid-pregnancy and term.

	Results

Top Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Quantification of Gh/PLC1 signaling components in mid-pregnant and parturient rat
We recently reported that Gh protein, a new class of GTP-binding proteins, functionally interacts with the rat myometrial 1-AR (Dupuis et al. 2004). In order to determine whether Gh protein plays an important role in the transduction of rat myometrial 1-AR, we compared by photoaffinity labeling studies 1-AR/Gh coupling between mid-pregnancy and term. Figure 1A illustrates the net incorporation of [³²P]GTP into Gh protein following activation of 1-AR by 100 µM Phe. Our results show that 1-AR/Gh coupling occurred on day 12 of pregnancy and significantly increased at term (2.47- and 2.1-fold increases at term versus days 12 and 15 of pregnancy respectively; P<0.05). We also quantified, by immunoblotting, the cytosolic amount of PLC1 enzyme, the known target of 1-AR/Gh coupling. Results illustrated in Fig. 1B show that the amount of PLC1 increases progressively from day 12 to term when it finally reaches a maximal level (+65% at term versus day 12 of pregnancy; P<0.05).

View larger version (14K): [in this window] [in a new window]   Figure 1 (A) Quantification of [-32P]GTP-photolabeled Gh protein following stimulation of 1-AR by 100 µM phenylephrine at days 12 (D12), 15 (D15), and 20 (D20) of pregnancy and at term (T). Results are expressed as percentage of basal photolabeled Gh (without phenylephrine) and are means±S.E.M. of three independent experiments done in duplicate. aP<0.05 versus D12 and D15. (B) Upper panel: representative western blots for PLC1 at pregnancy on days 12 (D12), 15 (D15), and 20 (D20) of pregnancy and at term (T); lower panel: quantitative data are expressed as percentage of term and are means±S.E.M. of four independent experiments. aP<0.05 versus D12 and D15; bP<0.05 versus D12, D15, and D20.

Correlation between rat myometrial 1d- expression and 1-AR/Gh coupling
It is well known that Gh protein selectively interacts with 1b- and 1d- but not with 1a-subtype (Chen et al. 1996). We thus characterized the expression of rat myometrial 1-adrenergic subtypes using RT-PCR technique. Our results show that the three 1-subtypes are present in rat myometrium but exhibit a differential expression during the course of pregnancy (Fig. 2A). Indeed, 1a- and 1d-transcripts were detected from day 12 of pregnancy until term with a significant lower level of expression (–69%, P<0.05) for 1a- and a progressive increase (+70%, P<0.05) for 1d- between day 12 of pregnancy and term (Fig. 2B). In contrast, detectable levels of 1b-mRNAs were observed only from day 15 of pregnancy, with a significant decreased expression at term (–50% versus day 15, P<0.05).

View larger version (16K): [in this window] [in a new window]   Figure 2 Myometrial mRNA expression of 1-AR subtypes during pregnancy. (A) Total RNA isolated from rat myometrium at days 12 (D12), 15 (D15), and 20 (D20) of pregnancy and at term (T) was reverse-transcribed and amplified for 1a-, 1b-, and 1d-AR subtypes. (B) The average results from three independent experiments are expressed as percentage of T. aP<0.05 versus previous stages of pregnancy; bP<0.05 versus D12 and D15.

View larger version (11K): [in this window] [in a new window]   Figure 3 A study of correlation between the amount of 1-AR-coupled Gh protein and the expression of 1b (A), 1d (B), and PLC1 (C) in pregnant rat myometrium.

Myometrial InsP response to the activation of 1-AR in mid-pregnant and term rat
Myometrial strips from mid-pregnant or parturient rats were incubated with increasing concentrations of Phe, a global 1-AR agonist. As basal InsP production significantly increases at term (Houdeau et al. 2005), Phe-induced InsP accumulation was expressed as the percentage of its corresponding basal in order to evaluate the net responses due to the specific activation of 1-AR. Results illustrated in Fig. 4A show that Phe dose-dependently enhanced InsP production at both mid-pregnancy and term. The E_max was unchanged between day 15 of pregnancy and term, while the EC₅₀ value was significantly decreased at term (P<0.05, Table 1) as evidenced by the leftward shift in the dose–response curve. Pre-treatment of myometrial strips with 100 µM prazosin, an 1-AR antagonist, completely blocked InsP production in response to maximally effective Phe (1 µM) in both pregnant and term rats (Fig. 5). This indicates that InsP response is due to the specific activation of 1-AR at both stages of pregnancy.

View larger version (13K): [in this window] [in a new window]   Figure 4 (A) Myometrial inositol phosphates (InsP) production in response to increasing concentrations (0.1 nM–1 mM) of phenylephrine (Phe) at day 15 of pregnancy (D15) and at term (T). (B) Contractile activity of the circular layer of myometrium in response to increasing concentrations (0.1 nM–1 mM) of phenylephrine (Phe) at D15 of pregnancy and term. (C) A comparison of the effect of increasing concentrations of Phe and norepinephrine (NE) on contractile activity of the circular layer of myometrium at term. Results are expressed as the percentage of basal InsP production (A) or spontaneous contraction (B and C) and are means±S.E.M. of three to six experiments done in triplicate. aP<0.05 versus the same concentration at D15.

View larger version (11K): [in this window] [in a new window]   Figure 5 Effect of an 1-adrenergic antagonist prazosin (Praz, 100 µM) on myometrial inositol phosphates (InsP) production elicited by 1 µM phenylephrine (Phe) at day 15 of pregnancy (D15) and at term (T). Results are expressed as the percentage of basal InsP production without Phe or Praz and are means±S.E.M. of three independent experiments. aP<0.05 versus basal InsP response; bP<0.05 versus InsP production in the presence of Phe.

Uterine contraction induced by rat myometrial 1-AR at mid- pregnancy and term
Since PLC activation is generally linked to uterine contraction, we compared, for the first time, InsP production and contraction of the circular layer of smooth muscle uterus in response to 1-AR activation. We have previously reported that 1-AR are mainly located in the uterine circular muscle (Legrand et al. 1991). This was confirmed by our recent functional studies, which showed that Phe specifically enhances the activity of the uterine circular muscle and has no effect on the longitudinal layer (Mhaouty-Kodja et al. 2004). We thus studied the contractile activity of the circular muscle layer in response to increasing concentrations of Phe. All uterine strips exhibited spontaneous rhythmic contractions a few minutes after being mounted on the bath (data not shown). As illustrated in Fig. 1B, Phe elicited a dose-dependent increase in uterine activity at both mid-pregnancy and term. An analysis of the sigmoid dose–response curves revealed no changes in the potency and maximal response for Phe between mid-pregnancy and term (Fig. 4B, Table 1). When challenged with the endogenous agonist norepinephrine, uterine strips exhibited a dose-dependent contraction (Fig. 4C) with an EC₅₀ value at the micromolar range (2±0.4 µM) similar to that obtained for Phe. Again, no change was observed in the maximal response (not shown).

Altogether, these results show that the up-regulation of 1-AR/Gh protein/PLC1 and the amelioration of InsP response at term are not primarily linked to contractile activity of the circular muscle.

	Discussion

Top Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
We report, in the present study, an up-regulation of 1-AR/Gh protein/PLC1 signaling pathway in term rat myometrium. Indeed, both the level of 1-AR/Gh protein coupling and the amount of PLC1 significantly increased at this period. The enhanced 1-AR/Gh interaction could be explained by the induction of Gh expression at both mRNAs and protein levels as reported previously (Dupuis et al. 2004). In addition, we also showed that, among the Gh-protein-coupled 1-AR, the expression of 1b-AR was down-regulated, whereas that of 1d-AR was significantly enhanced at term. Interestingly, we found a good temporal correlation between 1-AR/Gh coupling and 1d-AR expression during the course of pregnancy. The regulatory factors underlying this up-regulation need to be determined. It is, however, interesting to note that we did not find any effect of progesterone or estradiol on Gh expression (data not shown).

We also report that the up-regulation of 1-AR/Gh protein/PLC1 signaling pathway correlates well with the increased potency of 1-AR to induce InsP production (a tenfold lower EC₅₀ value) at term. This is in agreement with the increased Phe-stimulated PLC activity reported in plasma membrane preparations from term rat myometrium (Limon-Boulez et al. 1997). Surprisingly, this was not associated with an amelioration of uterine contraction in response to either Phe or the endogenous agonist norepinephrine. Parallel experiments where InsP production and contractile activity of the circular muscle were measured in response to oxytocin indicated a higher potency for this agonist to elicit both responses at term (Houdeau et al. 2005). This increased term sensitivity to oxytocin correlated well with the up-regulation of OTR, Gq protein, and PLCβ1/PLCβ3 enzymes as shown previously (Houdeau et al. 2005). Indeed, progesterone withdrawal and the increase in estradiol concentrations at the end of pregnancy induce expression of all these signaling molecules (Houdeau et al. 2005). As shown by our previous pharmacological studies (Limon-Boulez et al. 1997) and confirmed by the present study, rat myometrium expresses a heterogeneous population of 1-AR. The three 1-AR subtypes (1a, 1b, and 1d) are present during the second half of pregnancy although they exhibit different expression patterns. Previous studies attempted to assign different roles to 1-AR subtypes in the regulation of uterine contraction (Ducza et al. 2002, Mihalyi et al. 2003). It is, however, clear from the present study that, in physiological conditions where the three 1-AR subtypes are co-activated by their endogenous ligand norepinephrine, contraction of the circular muscle is induced at term but not as potently as does oxytocin. Indeed, the EC₅₀ values calculated for norepinephrine and phenylephrine are at the micromolar range compared with the 20 nM obtained for oxytocin under similar experimental conditions (Houdeau et al. 2005). It thus appears that rat myometrial 1-AR are not efficiently linked to the classical Gq/PLCβ system and consequently to uterine contraction. In line with this finding, we have previously shown that the Gq/PLCβ-coupled 1a-AR subtype undergoes uncoupling at term (Limon-Boulez et al. 1997). This, together with the presently reported down-regulation of 1b-AR expression and the emergence of the 1d-AR/Gh/PLC1 signaling pathway, strongly suggests that 1-AR preferentially signal through Gh/PLC1 at term.

In myometrium, the physiological meaning of the emergence of 1d-AR/Gh/PLC1 signaling pathway in the late pregnant rat myometrium remains to be determined. However, it is interesting to note that the plasma membrane 1-AR/Gh interaction takes place at mid-pregnancy and progressively increases until term. One may then postulate that this signaling pathway participates to the myometrial cell hypertrophy or proliferation that occurs during this period. Indeed, Gh protein was shown to mediate 1-AR-induced hepatocyte proliferation (Wu et al. 2000) and activation of extracellular signal-regulated kinases in neonatal rat cardiomyocytes (Lee et al. 2003). In agreement with these results, we have recently shown that Gh protein participates to the 1-AR-mediated regulation of smooth muscle cell proliferation (Dupuis et al. 2004). Alternatively, the 1/Gh/PLC1 system may also play a role in the preparation of uterus to labor by regulating the expression of some contraction-associated proteins. For instance, the directed overexpression of Gh protein in cardiomyocytes resulted in the up-regulation of cyclo-oxygenase-2, prostaglandin F2 receptors, and Gi proteins (Zhang et al. 2003). Mice lacking tissue transglutaminase II/Gh protein do not seem to exhibit any alteration in the pregnancy or parturition processes. However, it is possible that compensatory mechanisms taking place during development mask the real importance of myometrial Gh protein. One way to answer this question in vivo is to induce the conditional deletion of transglutaminase II/Gh in pregnant myometrium by using the smooth muscle-inducible CreERT system as was recently described by Döring et al. (2006).

In conclusion, the present work describes the up-regulation of different transduction components involved in the newly identified 1/Gh/PLC1 signaling pathway in rat myometrium between mid-pregnancy and term. The increased 1-AR/Gh coupling as well as 1d-AR and PLC1 expression correlates well with the increased potency of 1-AR to induce myometrial InsP production but not uterine contraction at term. This lead us to hypothesize that myometrial 1d-AR, through activation of Gh/PLC1 system, are not primarily involved in the initiation of labor but may either induce the expression of contraction-associated proteins during the phase of uterine preparation to labor and/or regulate other uterine responses like myometrial cell hypertrophy or proliferation.

	Materials and Methods

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Animals
Sprague–Dawley rats were obtained from Janvier (Le Genest, France). The females were caged with males overnight and successful mating was determined by the presence of spermatozoa in the vaginal smear (day 1 of pregnancy). Animals were killed by cervical dislocation at days 12, 15, 20 of pregnancy or at term during the expulsion of fetoplacental units, following the guidelines laid down by the NIH Guide. The uterine horns were quickly isolated, cut open lengthwise and the fetoplacental units removed. The myometrium was then freed of the adherent endometrium except for uterine tension studies.

RT-PCR
Using SuperScript Reverse Transcriptase kit (Gibco BRL Life Technologies), 5 µg total RNA was reverse-transcribed and the resulting cDNA was stored at –80 °C. PCR amplification was performed with 1/20 volume of each RT reaction using specific upstream and downstream primers for 1a-, 1b-, and 1d-AR subtypes and the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously (Mhaouty-Kodja et al. 2001, Dupuis et al. 2004). For semi-quantitative analysis, PCR cycle profiles were conducted and the cycle number (25 cycles) was chosen from the linear portion of the curve. The PCR products were separated by electrophoresis on ethidium bromide containing 2% agarose gel.

Preparation of myometrial fractions
Rat myometrium was homogenized in buffer A (50 mM Tris (pH 7.3), 100 mM NaCl, 2 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 0.5 mM dithiothreitol, 0.32 M sucrose) for Western blot analysis, and in buffer B (10 mM HEPES (pH 7.5), 250 mM sucrose, 5 mM EGTA) for photoaffinity labeling experiments supplemented with a cocktail of protease inhibitors (Sigma–Aldrich). After 10 min centrifugation at 4 °C, the supernatants were collected and submitted to 100 000 g centrifugation at 4 °C for 1 h to separate plasma membranes from cytosol. The pellet containing plasma membranes was resuspended in a homogenization buffer and the protein concentration of plasma membrane and cytosolic fractions was determined according to Bradford (1976) with BSA as standard. Samples were stored at –80 °C until use.

Western blot analysis
Cytosolic samples (20 µg protein) from rat myometrium were separated by 7.5% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Perkin–Elmer Life Sciences, Paris, France). The blots were blocked overnight at 4 °C in Tris-buffered saline (TBS) containing 5% non-fat-dried milk and incubated for 1 h at room temperature with monoclonal anti-PLC1 (Upstate Biotechnology, Euromedex, Souffelweyersheim, France) diluted 1:500. Incubation with secondary anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA, USA; 1:10 000 dilution) was carried out for 45 min at room temperature. Immunoreactive bands were visualized by the chemiluminescence detection system (Amersham Pharmacia Biotech) and quantified by densitometric scanning followed by computer analysis using the NIH image 1.62 program (http://rsb.info.nih.gov/nih-image/download.html).

Photoaffinity labeling
Photoaffinity labeling of Gh with [-³²P]GTP (3000 Ci/mmol, Perkin–Elmer Life Sciences) was carried out as described previously (Dupuis et al. 2004). Briefly, plasma membrane fractions (200 µg protein) were incubated with 10 µCi [-³²P]GTP for 10 min at 30 °C in a labeling buffer in the absence or presence of 100 µM Phe (Sigma–Aldrich). Reactions were stopped in ice and the samples were irradiated with u.v. light (254 nm) for 20 min and solubilized with Laemmli solution for 1 h at room temperature. The samples were then subjected to SDS-PAGE in 7.5% gels and transferred to the PVDF membranes. The membranes were first autoradiographed and then immunodetected with a monoclonal anti-Gh diluted 1:500 from NeoMarkers (Interchim, Monluçon, France). The obtained levels of labeled Gh were normalized with the corresponding amount of immunodetected protein. Densitometric scanning followed by computer analysis using NIH image 1.62 program was used to determine the levels of labeled and immunodetected Gh protein.

Measurement of myometrial InsP accumulation
InsP production was measured as described previously (Mhaouty-Kodja et al. 2001). Myometrial strips were incubated at 37 °C for 4 h with 7 µCi myo-[³H]inositol (10–25 Ci/mmol, Perkin–Elmer Life Sciences) in 1 ml Krebs bicarbonate buffer. Increasing concentrations (0.1 nM–1 mM) of Phe were added after 10 min incubation of the myometrial strips with 10 mM LiCl (Sigma–Aldrich) in Krebs bicarbonate buffer. Assays were stopped 15 min later by freezing the strips in liquid N₂. When used, 100 µM prazosin was added 15 min before the addition of 100 µM Phe.

Strips were homogenized in 7% trichloroacetic acid and the obtained supernatant was extracted with diethyl ether, neutralized with Tris-base, and then chromatographed over an anion-exchange resin (AG1-X8, Bio-Rad Laboratories). Total InsP eluted with 1 M ammonium formate/0.1 M formic acid (Sigma–Aldrich) were counted by liquid scintillation in a 1214 Rack-beta spectrometer (LKB, Turku, Finland) for tritium.

Uterine contraction studies
Uterine strips, 4 mm long, were prepared from pregnant rats and mounted in organ baths containing 8 ml Krebs solution as described previously (Mhaouty-Kodja et al. 2001). We measured isometric contractions of the circular muscle using a Bioscience UF1 tension transducer (Phymep, Paris, France) under 0.7 g resting force. A 30 min equilibration period was allowed before adding cumulative doses of Phe (0.1 nM–1 mM). The concentration–response curves were recorded by computerized calculation of the integral under the tension/time curve for 3 min using Prism 4 software (Graph Pad, San Diego, CA, USA) for sigmoidal dose–response (nonlinear regression fit). Isometric changes in tissue tension and maximal effects (E_max) were expressed as a percentage above the spontaneous activity in the absence of agonists, and potency as EC₅₀.

Statistical analysis
Results are expressed as means±S.E.M. Statistical significance was assessed by Student's t-test for unpaired data. P<0.05 was considered significant.

	Acknowledgements

Top Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Footnotes

 
M Dupuis is now at Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany

Received 19 July 2007
First decision 7 September 2007
Accepted 1 October 2007

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