HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mesonero, J. E. Articles by Harbon, S. Search for Related Content PubMed PubMed Citation Articles by Mesonero, J. E. Articles by Harbon, S. Agricola Articles by Mesonero, J. E. Articles by Harbon, S.

Differential Expression of Inositol 1,4,5-Trisphosphate Receptor Types 1, 2, and 3 in Rat Myometrium and Endometrium During Gestation¹

^a INSERM U442, Signalisation Cellulaire et Calcium, ^b Signalisation et Régulations Cellulaires, CNRS, IFR-FR 46, Université Paris-Sud, F-91405 Orsay Cedex, France

ABSTRACT

The regulation of the phospholipase C (PLC) and the expression of inositol 1,4,5-trisphosphate receptors (IP₃Rs) in terms of mRNA, proteins, and binding capacity were examined in the rat myometrium and endometrium at midgestation (Day 12) and at term (Day 21) comparatively to the estrogen-treated tissues (Day 0). In both uterine tissues, the production of inositol phosphates mediated by carbachol as well as by AlF₄^- was enhanced with advancing gestation. ³[H]IP₃ binding sites in membranes also increased during pregnancy (Day 21 > Day 12 > Day 0). The mRNAs encoding for three isoforms of IP₃R as well as their corresponding proteins, IP₃R-1, IP₃R-2, and IP₃R-3 were coexpressed, albeit to different extents, in the myometrium and endometrium. The expression of IP₃Rs increased with advancing gestation, except for IP₃R-2 that increased only in the endometrium at term. Thus, the pregnancy-related upregulation of the PLC cascade coincided with an increase in the expression of IP₃Rs. The difference noted between the two uterine tissues suggests that IP₃Rs may have cell-specific functions.

calcium, parturition, pregnancy, signal tranducers, signal transduction, uterus

INTRODUCTION

The uterus undergoes remarkable changes in growth and function during pregnancy. The biochemical and morphological changes that take place in the endometrium are thought to be a prerequisite for implantation and adequate development of the fetus. The function of the myometrium gradually changes during gestation. It is initially quiescent so as to maintain the growing fetus and then becomes active at term to expel the mature fetus. Cellular processes as diverse as cell motility, contraction, secretion, and cell proliferation are regulated by intracellular Ca²⁺ [1, 2], and this may be of crucial importance for uterine cells during gestation. The initial response of many cells to Ca²⁺-mobilizing receptor agonists is the activation of phospholipase C (PLC) catalyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) leading to an increase in inositol 1,4,5-trisphosphate (IP₃) production [3]. It is known that the generation of IP₃ and the associated increase in intracellular Ca²⁺ concentration is an important determinant of smooth muscle contractility [4]. Stimulation of the phosphoinositide–PLC pathway by contractile agonists that activate G-protein-coupled receptors has been demonstrated in various myometrial preparations [5–9].

IP₃ binds to an intracellular IP₃ receptor (IP₃R) located on the surface of intracellular Ca²⁺ stores, e.g., endoplasmic reticulum, and then releases Ca²⁺ into the cytoplasm [1, 10]. Thus, the IP₃R plays a central role in the conversion of IP₃ signals produced by external stimuli into intracellular Ca²⁺ signals. Molecular cloning studies have demonstrated the existence of at least three types of IP₃Rs, encoded by genes designated type 1 [11, 12], type 2 [13], and type 3 [14]. These three IP₃R types have different primary structures and tissue distributions, and their amino acid sequences are approximately 60–70% identical. IP₃Rs also differ in their affinity for IP₃, in the order IP₃R-2 > IP₃R-1 > IP₃R-3 [15].

We have recently shown that, in rat myometrium, the responsiveness of the PLCß₃ pathway associated with receptor and/or direct G-protein activation increased during gestation and coincided with the increase in the amount of G_q/G₁₁ [8]. Little is known about the IP₃ receptor isoforms present in the rat uterus and their possible regulation in relation to pregnancy and increases in the production of inositol phosphates. We addressed this question by assessing IP₃ binding capacity and the expression of IP₃R isoform genes in terms of mRNA and corresponding protein levels in the myometrium and endometrium at the middle (Day 12) and end (Day 21) of gestation compared to estrogen-primed rats (Day 0). We found that 1) the number of IP₃ binding sites increased in both tissues with advancing gestation; 2) IP₃R-1, -2, and -3 genes were coexpressed, albeit to different extents, in the nonpregnant myometrium and endometrium; and 3) the expression of IP₃R-1 and IP₃R-3 increased over the course of gestation, whereas the level of IP₃R-2 increased only in the endometrium at term.

MATERIALS AND METHODS

Materials

[³H]IP₃ (17–21 Ci/mmol) was obtained from New England Nuclear Product division (Dupont de Nemours, Les Ulis, France) and IP₃ from Calbiochem (La Jolla, CA). Myo-[2-³H]inositol (10–20 Ci/mmol), Hybond membranes, enhanced chemiluminescence (ECL) detection system, and the Megaprime DNA labeling kit were obtained from Amersham International (Les Ulis, France). AG1-X8 and Bradford reagent were from Bio-Rad (Ivry, France). Rabbit polyclonal antibody against a synthetic peptide corresponding to the 16 C-terminal residues of IP₃R-2 was prepared by Covalab (Oullins, France) [16]. Monoclonal antibody against IP₃R-3 was obtained from Transduction Laboratories (San Diego, CA) and rabbit polyclonal antibody against IP₃R-1 was obtained from Affinity Bioreagents (Goldon, CO). Antirabbit IgG and antimouse IgG antibodies were from Sanofi Diagnostics Pasteur (Marnes-La-Coquelte, France). A full-length cDNA encoding type 1 IP₃R (pCMVI-9) [12] and a rat type 2 partial cDNA (pCMVI-102) [13], were kindly provided by Dr. T.C. Südhof (Howard Hughes Medical Institute, Dallas, TX). cDNA (pCBG+IP₃R-3) [14] was obtained from Prof. G.I. Bell (Howard Hughes Medical Institute, University of Chicago). Lithium chloride, carbamoylcholine chloride (carbachol), ß-estradiol 3-benzoate, leupeptin, aprotinin, and PMSF were from Sigma Chemical Co. (St. Louis, MO). RNeasy Mini Kit and QIAshredder columns were from QIAGEN (Les Ulis, France).

Animals and Tissue Processing

Pregnant rats (Wistar) were used at Days 12 and 21 of gestation. Immature female rats (Wistar 5 wk old) were treated with 30 µg estradiol for 2 days and were used the following day. These estradiol-primed animals were considered as Day 0. Rats were killed by decapitation, and the uterine horns were quickly cleared of adhering fat and excised out longitudinally. After removal of fetuses, their sites of attachment, and placental tissues, myometrium was separated by stripping away the endometrium as previously described [5, 8]. Histological sections of myometrial preparations showed the samples to consist almost exclusively of longitudinal muscle. For each specific experiment we minimized variation by using tissues pooled from uteri from at least 12 (Day 0), 6 (Day 12), and 3 (Day 21) rats.

Determination of [³H]Inositol Phosphates

Myometrial and endometrial strips (~30 mg) were labeled with 5 µCi of myo-[³H]inositol (0.4 µM) [5, 8]. Tissues were washed three times with nonradioactive Krebs buffer and were incubated with 1 ml of fresh buffer, containing 10 mM LiCl before exposure to the agents indicated. Reactions were stopped by immersing the strips in 1.5 ml of cold 7% (w:v) trichloroacetic acid (TCA), followed by homogenization and centrifugation at 3000 x g for 20 min at 4°C. The TCA-soluble supernatants were extracted with diethylether, neutralized with Tris base, and applied to a column (0.7 x 2 cm) of anion-exchange AG1-X8. Total inositol phosphates (IP₃ + IP₂ + IP₁) were eluted together in a single step with 12 ml of 1 M ammonium formate/0.1 M formic acid. The [³H]phosphoinositides present in the TCA pellets were extracted by the method of Bligh and Dyer [17], essentially as previously described [6]. The ³H content of the various samples was determined by scintillation spectrometry. The production of [³H]inositol phosphates was expressed as a percentage of radioactivity incorporated into phosphoinositides obtained from the corresponding sample.

Western Blot Analysis

Endometrial and myometrial membranes were prepared as previously described [8] and stored at -80°C. Protein concentration was determined by the method of Bradford [18]. Protein samples were analyzed by SDS-PAGE in 5% acrylamide gels as described by Laemmli [19], and the proteins were then transferred to Hybond-ECL membranes that were then blocked 60 min in 5% nonfat dried milk in Tris-buffered saline (TBS; 20 mM Tris-HCl, pH 7.5, 500 mM NaCl). The blots were probed with rabbit polyclonal anti-IP₃R-1 antibody or rabbit polyclonal antibody against a synthetic peptide corresponding to the 16 C-terminal residues of IP₃R-2 [16], both used at 1:1000 dilution. Monoclonal antibody against IP₃R-3 was used at 1:2000 dilution. The primary antibodies against IP₃R-1 and IP₃R-2 were detected with an antirabbit IgG antibody coupled to horseradish peroxidase, and the primary antibody against IP₃R-3 was detected with an antimouse IgG antibody (dilution 1:5000), using the ECL detection system. In certain experiments, the blots were reprobed after stripping in 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM ß-mercaptoethanol for 60 min at 60°C. Nitrocellulose sheets were rinsed in TBS and then reblotted for 60 min in 5% nonfat dried milk in TBS before being sequentially probed with other antibodies. The developed blots were quantified by densitometry with a Molecular Dynamics Densitometer. We verified that the amplitude of the signal was proportional to the amount of proteins loaded on the gels, at least within the investigated range (10–50 µg). Thus, the binding of primary and secondary antibodies as well as the ECL reaction were not limiting.

Equilibrium [³H]IP₃ Binding Studies

For IP₃ binding studies, the myometrial and endometrial membrane fractions (300 µg) were incubated for 6 min on ice in 500 µl of ice-cold cytosol-like medium consisting of 110 mM KCl, 20 mM NaCl, 1 mM Na₂HPO₄, 1 mM EDTA, 25 mM HEPES/KOH, pH 7.5, supplemented with 1 mg/ml BSA and 1 nM [³H]IP₃ (20 Ci/mmol), with or without unlabeled IP₃, to give a final concentration of 1, 5, and 50 nM, as previously described [16, 20]. Nonspecific binding was determined in the presence of 5 µM IP₃. At the end of incubation, 400 µl of the sample was layered onto a Whatman GF/C glass-fiber filter and washed with 1 ml of ice-cold medium containing 250 mM sucrose, 10 mM Na₂HPO₄, at pH 8. Radioactivity retained on the filter was counted by scintillation spectrometry. IP₃ binding was expressed in each sample as femtomoles per milligram of protein.

cDNA Probes and Northern Blot Analysis

IP₃R-1 was detected with a 2.5-kilobase (kb) probe [12], obtained by digestion with BamHI (4126–6658) of the full-length rat cDNA encoding type 1 IP₃R (pCMVI-9). IP₃R-2 was detected with a 1.7-kb probe [13] obtained by digestion with BamHI and PstI (1804–3475) of the rat type 2 partial cDNA (pCMVI-102). IP₃R-3 was detected with a 1.9-kb probe [14] obtained by digestion with SacI (5247–7143) of the corresponding rat cDNA (pCBG+IP₃R-3). Probes were ³²P-labeled with the Megaprime DNA labeling kit.

Total RNA was extracted from isolated uterine tissues using the RNeasy Mini Kit and QIAshredder columns, according to the manufacturer's protocols. Samples of total RNA were denatured in 1 mM glyoxal [21] and fractionated by electrophoresis in 1% agarose gels. The RNA was then transferred to Hybond-N membranes and probed with the ³²P-labeled cDNA fragments as described elsewhere [21]. The membranes were washed in high-stringency conditions with a final 15-min wash in 0.1x standard saline citrate/0.1% SDS at 60°C. Radioactivity in the membrane was quantified with a PhosphoImager scanner using Imagequant 3.2 software (both from Molecular Dynamics) before the membrane was placed against x-ray film. RNA levels were standardized by reprobing the blots with 18S rRNA probes.

Data Analysis

The results are expressed as means ± SEM. Means were compared using Student's t-test. P < 0.05 was considered to be significant.

RESULTS

Increase in the Generation of [³H]Inositol Phosphates in Rat Myometrium and Endometrium During Gestation

We have previously demonstrated that pregnancy leads to a marked enhancement in phosphoinositide breakdown with an increased production of IP₃ in rat myometrium [8]. We found in this study that this is also the case for the endometrium (Table 1). In both myometrial and endometrial preparations, there was a basal level of inositol phosphates generation. Inositol phosphate levels were two and three times higher at Days 12 and 21, respectively, than at Day 0 for both tissues. Similarly, the induction by carbachol (100 µM) of inositol phosphate production increased during gestation (Day 21 > Day 12 > Day 0). At this concentration (100 µM), the muscarinic agonist caused a maximal inositol phosphate response in both tissues (data not shown). The pattern of the increase in inositol phosphate generation (Day 21 > Day 12 > Day 0) was similar if the stimulation was triggered by AlF₄^-, a direct activator of G-proteins.

IP₃ Binding Sites at Days 0, 12, and 21 in the Myometrium and Endometrium

Specific IP₃ binding sites were detected in membranes derived from Day 0, Day 12, and Day 21 myometrium and endometrium (Table 2). During pregnancy, the amount of IP₃ bound in both myometrial and endometrial membranes, as determinated in the presence of 1, 5, and 50 nM [³H]IP₃, increased significantly (Day 0 < Day 12 < Day 21). There was 50% more IP₃ binding at Day 21 than at Day 0 in both myometrium and endometrium. Homologous competition experiments (n = 4) performed in myometrial membranes revealed that IC₅₀ values (8.8 ± 3.5, 7.9 ± 1.2, and 9.9 ± 4.6 nM for Day 0, Day 12, and Day 21, respectively) were not significantly different. The resulting slope factors (0.70 ± 0.10, 0.69 ± 0.1, and 0.68 ± 0.06 for Day 0, Day 12, and Day 21, respectively) suggested the presence of a heterogeneous population of IP₃Rs in these preparations.

Pregnancy-Related Expression of IP₃R Proteins in Rat Myometrium and Endometrium

We used specific IP₃R-1, IP₃R-2, or IP₃R-3 antibodies to investigate the expression and possible quantitative changes in these receptors in rat myometrium and endometrium during gestation. We performed immunoblots with myometrial and endometrial membranes derived from various stages of gestation (0, 12, and 21 days), resolved by SDS-PAGE, and stained with specific antibodies (Fig. 1). The three types of IP₃R, all about 260 kDa in size, were present at day 0 and throughout gestation in both uterine tissues. Quantitative analysis of various blots indicated that IP₃R-1 was more strongly expressed (3.8 times higher levels, n = 3, P < 0.01) in myometrial than in endometrial membranes for Day 0 rats. IP₃R-1 levels were higher in both tissues during pregnancy than at Day 0. In the myometrium, IP₃R-1 protein levels were 1.5 and 3 times higher at Days 12 and 21, respectively, than at Day 0 (n = 3, P < 0.01). In the endometrium IP₃R-1 levels were 3 and 7 times higher at Days 12 and 21, respectively than at Day 0 (n = 3, P < 0.01). IP₃R-2 was detected in both myometrium and endometrium. The level of expression of this isoform did not change during pregnancy in the myometrium but was upregulated (2 times higher, n = 3, P < 0.01) at term in the endometrium. IP₃R-3 levels were upregulated during pregnancy in both endometrium and myometrium (2 and 4 times higher at Days 12 and 21, respectively, than at Day 0, n = 3, P < 0.01). The IP₃R-3 isoform was preferentially expressed in the endometrium. The above quantitative variations could be observed for different amounts of proteins up to 50 µg. Additionally, when the blots (Fig. 1) were reprobed with antibodies specific to calreticulin, a calcium-related protein, the immunologic signal remained identical for all myometrial and endometrial preparations, reflecting that the changes observed were specific for IP₃Rs (data not shown). Thus, in both the myometrium and the endometrium, the three types of IP₃R are expressed but are differentially regulated during the course of gestation.

View larger version (41K): [in this window] [in a new window]   FIG. 1. Immunodetection of IP3Rs in myometrium and endometrium during pregnancy. Membranes (20 µg) from isolated myometrium and endometrium at Days 0, 12, and 21 of gestation were separated by 5% SDS-PAGE. IP3R isoforms were detected with anti-IP3R-1, -IP3R-2, and -IP3R-3 antibodies as indicated. A typical Western blot for each IP3R is shown. Histograms (myometrium, open bars; endometrium, hatched bars) represent the densitometric quantification of each IP3R type from three blots derived from different membrane preparations (arbitrary units ± SEM relative to Day 0 myometrium = 100). *P < 0.01 significantly different from Day 0 myometrium values. #P < 0.01 significantly different from Day 0 endometrium values

Pregnancy-Related Expression of IP₃R mRNA in Rat Myometrium and Endometrium

We assessed the amount of mRNA present for each type of IP₃R by Northern blotting using specific probes obtained from the respective cDNAs cloned from rats (Fig. 2). Northern blots were carried out with 25 µg of total RNA from myometrium or endometrium obtained from Day 0 rats or from pregnant rats at Days 12 and 21 of gestation. We detected a single ~10-kb transcript for each type of IP₃R. IP₃R-1 mRNA levels at Day 0 were 4 times lower in the endometrium than in the myometrium (n = 3, P < 0.01). The pattern of IP₃R-1 mRNA expression during gestation differed in myometrium and endometrium. In the myometrium, IP₃R-1 mRNA levels were 3 times higher (n = 3, P < 0.01) at midgestation than Day 0 levels, and decreased to Day 0 levels at Day 21. However, in the endometrium, IP₃R-1 mRNA expression increased progressively throughout gestation, with levels 4 times higher at Day 21 than at Day 0 (n = 3, P < 0.01). IP₃R-2 mRNA expression was barely detectable in Day 0 myometrium, with no apparent change at Days 12 and 21. In the endometrium, IP₃R-2 mRNA levels were very low at Day 0 but consistently increased and were 2.5 times higher at the end of gestation (n = 3, P < 0.01). The IP₃R-3 isoform was detected in both myometrium and endometrium. At Day 0, the level of mRNA for this isoform in the endometrium was twice that in the myometrium, and the level of this mRNA progressively increased in both tissues during gestation, being 3 times higher at Day 21 (n = 3, P < 0.01).

View larger version (41K): [in this window] [in a new window]   FIG. 2. Differential expression of IP3R-1, IP3R-2, and IP3R-3 mRNAs in myometrium and endometrium during pregnancy. Northern blot analysis of 25 µg of total RNA extracted from rat myometrium and endometrium at Days 0, 12, and 21 of gestation probed with IP3R-1, IP3R-2, and IP3R-3 cDNA fragments as indicated. The blots were reprobed with 18S rRNA for quantification. A typical Northern blot of the IP3R mRNA types is shown. Histograms (myometrium, open bars; endometrium, hatched bars) represent the radioactive signal quantified from three individual blots derived from different RNA preparations (arbitrary units ± SEM relative to Day 0 myometrium = 100). *P < 0.01 significantly different from Day 0 myometrium values. #P < 0.01 significantly different from Day 0 endometrium values

DISCUSSION

We have previously reported that there is a progressive increase during gestation in the generation of inositol phosphates triggered by receptor and G-protein activation in the rat myometrium [8]. We demonstrate herein that the PIP₂/PLC signaling pathway is similarly upregulated in the endometrium throughout pregnancy (Day 21 > Day 12 > Day 0). In both uterine tissues, the higher levels of inositol phosphates at Days 12 and 21 of gestation were associated with an increase in the capacity for binding IP₃ to its receptors on the corresponding membrane preparations. Northern blot hybridization experiments demonstrated the presence in the myometrium and endometrium of three different mRNAs encoding for the three IP₃R (IP₃R-1, -2, and -3) isoforms. Immunoblot experiments with isoform-specific antibodies confirmed the presence of IP₃R-1, -2, and -3 proteins in both the myometrium and endometrium. The expression IP₃R-1 and IP₃R-3 increased during the course of gestation in both tissues, whereas the expression of IP₃R-2 increased only in the endometrium at term. There was a strong correlation between the levels of mRNA and protein except for IP₃R-1 in the myometrium. IP₃R-1 mRNA levels peaked at Day 12 and then declined at Day 21, whereas the corresponding protein level increased progressively (Day 21 > Day 12 > Day 0). Such a lack of correlation between mRNA and protein levels may reflect differences in half-time degradation for mRNA and proteins. IP₃R-1 appeared to be expressed preferentially in the myometrium, whereas IP₃R-3 levels were higher in the endometrium, and IP₃R-2 was poorly expressed in the myometrium as compared to the endometrium. These differences between the two uterine tissues suggest that IP₃ receptor isoforms have different cell-specific functions.

Several uterine functions are regulated during gestation. These include the cell proliferation that is associated with the hyperplasia of both uterine tissues, active cell death that increases as pregnancy progresses, the secretory function of the epithelial cells of the endometrium that contribute to the output of key regulatory factors necessary for the initiation and maintenace of pregnancy, and finally the coordinated muscle contraction needed to expel the fetus. All these functions are controlled by an increase in the cytosolic Ca²⁺ concentration in the target cells. The variety of the spatiotemporal pattern of the Ca²⁺ signaling has been implicated in this versatility of Ca²⁺-regulated functions. The frequency and the amplitude of the Ca²⁺ oscillations can be decoded to allow specific gene expression [22]. Using cell-permeant caged IP₃ ester, it was shown that Ca²⁺ spike frequency can optimize gene expression [23] or efficient activation of calmodulin-dependent protein kinase II [24]. Recent work demonstrated that specific Ca²⁺ signaling patterns can be encoded by differential expression of IP₃R subtypes [25]. Smooth muscle contraction is generally thought to be activated by IP₃-induced Ca²⁺ release from sarcoplasmic reticulum [1, 4]. Because IP₃R-1 levels are highest in the myometrium, it is likely that the type 1 IP₃R may be one of the suppliers of the Ca²⁺ required for activation of uterine contractions. This interpretation is supported by the upregulation of IP₃R-1 levels at midpregnancy, culminating near term. The simultaneous increase in IP₃ generation and IP₃R-1 expression near term may be of physiological relevance because this would make it possible for the myometrial cells to produce the appropriate coordinated contractions needed to expel the fetus. Worth considering are the reports demonstrating high levels of IP₃R-1 in human [26] and mouse [27] myometrium. IP₃R-3 is expressed and upregulated (Day 21 > Day 12 > Day 0) in both endometrial and myometrial cells. A potential role for IP₃R-3 in the Ca²⁺ regulation of myometrial contractility can similarly be retained. Ca²⁺ is also known to play a key role in smooth muscle cell proliferation, so it was not surprising that the amounts of uterine IP₃ receptors, mainly types 1 and 3, increased during gestation, which is associated with the proliferation and hyperplasia of both uterine tissues. The IP₃R-2 receptor was hardly detectable in the myometrium at Day 0 and throughout gestation; it was expressed in the endometrium where it was up-regulated at term, suggesting a functional role for this type of IP₃ receptor in the endometrium but its minimal role in controlling myometrial activites.

Pregnancy-related changes in the PLC signaling cascade in rat myometrium, operating at the level of G_q/G₁₁ have been reported [8]. This study demonstrates that the IP₃ receptor system is a further example of a key signaling component of the PLC pathway whose expression and binding capacities are affected by the hormonal status of the uterus. The increased amounts of IP₃ receptors and their mRNA levels in uterine tissues are probably due to changes in the estrogen/progesterone balance occurring during gestation. A potential regulation of the expression of IP₃ receptors by sex steroid hormones can thus be postulated. In line with this proposal is the recent report demonstrating the presence of a steroid element on the promotor of the gene for IP₃R-1 [28]. It has also recently been reported that in addition to sex steroids, the mechanical stretch imparted by the growing uterine content may contribute to differential control of gene expression in the pregnant uterus [29]. Our data concerning the differential expression of IP₃R in the endometrium and myometrium may allow the encoding of a different spatiotemporal pattern of Ca²⁺ signaling in the uterine cell types, resulting in the ultimate control of specific processes that occur in the uterus during gestation.

ACKNOWLEDGMENTS

We thank Dr. T.C. Südhof (Howard Hughes Medical Institute, Dallas), who kindly provided us with the rat IP₃R-1 and IP₃R-2 cDNA and Prof. G.I. Bell (Howard Hughes Medical Institute, University of Chicago) for providing the rat IP₃R-3 cDNA. We also thank G. Thomas for excellent technical assistance.

FOOTNOTES

First decision: 11 February 2000.

¹ J.E.M. is the recipient of "Poste Vert from INSERM" and "Conseil Régional d'Ile-de-France." The first two authors contributed equally to the work presented here.

² Correspondence: Jean Pierre Mauger, INSERM U442, Signalisation Cellulaire et Calcium, Université Paris-Sud, Bâtiment 443, F-91405 Orsay Cedex, France. FAX: 33 1 69 15 58 93; jean-pierre.mauger{at}ibaic.u-psud.fr

Accepted: March 28, 2000.

Received: December 31, 1999.

REFERENCES

1. Berridge MJ. Inositol trisphosphate and calcium signalling. Nature 1993; 361:315–325.[CrossRef][Medline]
2. Clapham DE. Calcium signaling. Cell 1995; 80:259–268.[CrossRef][Medline]
3. Exton JH. Phosphoinositide phospholipases and G proteins in hormone action. Annu Rev Physiol 1994; 56:349–369.[CrossRef][Medline]
4. Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 1994; 372:231–236.[CrossRef][Medline]
5. Goureau O, Tanfin Z, Marc S, Harbon S. Diverse prostaglandin receptors activate distinct signal transduction pathways in rat myometrium. Am J Physiol 1992; 263:C257–C265.
6. Dokhac L, Naze S, Harbon S. Endothelin receptor type A signals both the accumulation of inositol phosphates and the inhibition of cyclic AMP generation in rat myometrium: stimulation and desensitization. Mol Pharmacol 1994; 46:485–494.[Abstract]
7. Ku CY, Qian A, Wen Y, Anwer K, Sanborn BM. Oxytocin stimulates myometrial guanosine triphosphatase and phospholipase-C activities via coupling to Gq/11. Endocrinology 1995; 136:1509–1515.[Abstract]
8. Lajat S, Tanfin Z, Guillon G, Harbon S. Modulation of phospholipase C pathway and level of Gq/G11 in rat myometrium during gestation. Am J Physiol 1996; 271:C895–C904.
9. Bieber E, Stratman T, Sanseverino M, Sangueza J, Phillippe M. Phosphatidylinositol-specific phospholipase C isoform expression in pregnant and nonpregnat rat myometrial tissue. Am J Obstet Gynecol 1998; 178:848–854.[CrossRef][Medline]
10. Joseph SK. The inositol trisphosphate receptor family. Cell Signal 1996; 8:1–7.[CrossRef][Medline]
11. Furuichi T, Yoshikawa S, Miyawaki A, Wada K, Maeda N, Mikoshiba K. Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400. Nature 1989; 342:32–38.[CrossRef][Medline]
12. Mignery GA, Newton CL, Archer III BT, Südhof TC. Structure and expression of the rat inositol 1,4,5-trisphosphate receptor. J Biol Chem 1990; 265:12679–12685.[Abstract/Free Full Text]
13. Südhof TC, Newton CL, Archer III BT, Ushkaryov YA, Mignery GA. Structure of a novel InsP₃ receptor. EMBO J 1991; 10:3199–3206.[Medline]
14. Blondel O, Takeda J, Janssen H, Seino S, Bell GI. Sequence and functional characterization of a third inositol trisphosphate receptor subtype, IP₃R-3, expressed in pancreatic islets, kidney, gastrointestinal tract, and other tissues. J Biol Chem 1993; 268:11356–11363.[Abstract/Free Full Text]
15. Newton CL, Mignery GA, Südhof TC. Co-expression in vertebrate tissues and cell lines of multiple inositol 1,4,5-trisphosphate (InsP₃) receptors with distinct affinities for InsP₃. J Biol Chem 1994; 269:28613–28619.[Abstract/Free Full Text]
16. Picard L, Coquil JF, Mauger JP. Multiple mechanisms of regulation of the inositol 1,4,5-trisphosphate receptor by calcium. Cell Calcium 1998; 23:339–348.[CrossRef][Medline]
17. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959; 37:911–917.
18. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the preciple of protein-dye binding. Anal Biochem 1976; 72:248–254.[CrossRef][Medline]
19. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685.[CrossRef][Medline]
20. Pietri F, Hilly M, Mauger JP. Calcium mediates the interconversion between two states of the liver inositol 1,4,5-trisphosphate receptor. J Biol Chem 1990; 265:17478–17485.[Abstract/Free Full Text]
21. Thomas PS. Hybridization of denatured mRNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 1980; 77:5201–5205.[Abstract/Free Full Text]
22. Dolmetsch RE, Xu K, Lewis RS. Calcium oscillations increase the efficiency and specificity of gene expression. Nature 1998; 392:933–936.[CrossRef][Medline]
23. Li W, Llopis J, Whitney M, Zlokarnik G, Tsien RY. Cell-permeant caged InsP₃ ester shows that Ca²⁺ spike frequency can optimize gene expression. Nature 1998; 392:936–941.[CrossRef][Medline]
24. De Koninck P, Schulman H. Sensitivity of CaM kinase II to the frequency of Ca²⁺ oscillations. Science 1998; 279:227–230.[Abstract/Free Full Text]
25. Miyakawa T, Maeda A, Yamazawa T, Hirose K, Kurosaki T, Iino M. Encoding of Ca²⁺ signals by differential expression of IP₃ receptor subtypes. EMBO J 1999; 18:1303–1308.[CrossRef][Medline]
26. Morgan JM, De Smedt HD, Gillespie JI. Identification of three isoforms of the InsP₃ receptor in human myometrial smooth muscle. Pflueg Arch Eur J Physiol 1996; 431:697–705.[Medline]
27. Nakanishi S, Fujii A, Nakade S, Mikoshiba K. Immunohistochemical localization of inositol 1,4,5-trisphosphate receptors in non-neural tissues, with special reference to epithelia, the reproductive system, and muscular tissues. Cell Tissue Res 1996; 285:235–251.[CrossRef][Medline]
28. Kirkwood KL, Homick K, Dragon MB, Bradford PG. Cloning and characterization of the type I inositol 1,4,5-trisphosphate receptor gene promoter. Regulation by 17beta-estradiol in osteoblasts. J Biol Chem 1997; 272:22425–22431.[Abstract/Free Full Text]
29. Ou C-W, Orsino A, Lye SJ. Expression of Connexin-43 and Connexin-26 in the rat myometrium during pregnancy and labor is differentially regulated by mechanical and hormonal signals. Endocrinology 1997; 138:5398–5407.[Abstract/Free Full Text]

This article has been cited by other articles:

				O. Gomez, A. Romero, J. Terrado, and J. E Mesonero Differential expression of glucose transporter GLUT8 during mouse spermatogenesis Reproduction, January 1, 2006; 131(1): 63 - 70. [Abstract] [Full Text] [PDF]

				S. Dogan, T. A. White, D. A. Deshpande, M. P. Murtaugh, T. F. Walseth, and M. S. Kannan Estrogen Increases CD38 Gene Expression and Leads to Differential Regulation of Adenosine Diphosphate (ADP)-Ribosyl Cyclase and Cyclic ADP-Ribose Hydrolase Activities in Rat Myometrium Biol Reprod, March 1, 2002; 66(3): 596 - 602. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mesonero, J. E. Articles by Harbon, S. Search for Related Content PubMed PubMed Citation Articles by Mesonero, J. E. Articles by Harbon, S. Agricola Articles by Mesonero, J. E. Articles by Harbon, S.