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BOR - Papers in Press, published online ahead of print October 6, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.033779
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BIOLOGY OF REPRODUCTION 72, 416–422 (2005)
DOI: 10.1095/biolreprod.104.033779
© 2005 by the Society for the Study of Reproduction, Inc.

Progesterone Upregulates Calcitonin Gene-Related Peptide and Adrenomedullin Receptor Components and Cyclic Adenosine 3'5'-Monophosphate Generation in Eker Rat Uterine Smooth Muscle Cell Line1

Chandrasekhar Thota, and Chandrasekhar Yallampalli2

Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas 77555-1062


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM), two potent smooth-muscle relaxants, have been shown to cause uterine relaxation. Both CGRP- and AM-binding sites in the uterus increase during pregnancy and decrease at labor and postpartum. These changes in binding sites appear to be related to the changes in calcitonin receptor-like receptor (CRLR), receptor activity-modified protein 1 (RAMP1), RAMP2, and RAMP3 mRNA levels. It is not clear, however, whether the changes in the receptor components occur in the myometrial cells and whether the steroid hormones can directly alter these receptor components in the muscle cells. In addition, the mechanism of CGRP and AM signaling in the rat myometrium is not well understood. Therefore, we examined the mRNA expression of CGRP- and AM-receptor components, G protein G{alpha}s, CGRP, and AM stimulation of cAMP and cGMP, and the effects of progesterone on these parameters in the Eker rat uterine myometrial smooth-muscle cell line (ELT3). ELT3 cells expressed CGRP- and AM-receptor components CRLR, RAMP1, RAMP2, and RAMP3. Expression of CRLR and RAMP1 mRNA increased with progesterone treatment and decreased with estradiol-17ß treatment. However, RAMP2 and RAMP3 mRNA expressions were unaltered by both progesterone and estradiol. Progesterone increased (P < 0.05) G{alpha}s expression and augmented CGRP- and AM-induced increases in cAMP levels. In uterine smooth-muscle cells, the antagonist to G{alpha}s protein NF449 decreased basal as well as CGRP- and AM-stimulated cAMP levels. None of the cell treatments affected cyclic GMP production. Our results suggest that the progesterone-stimulated increases in CGRP and AM receptors, G{alpha}s protein levels, and cAMP generation in the myometrial cells may be responsible for increased uterine relaxation sensitivity to CGRP and AM during pregnancy.

cyclic adenosine monophosphate, neuropeptides, progesterone, signal transduction, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulation of uterine contractions is critical for the maintenance of pregnancy. The uterus remains quiescent during gestation, and at term, the coordinated expression of contraction-associated proteins [1–4] in the uterus causes spontaneous contractions leading to parturition. Progesterone concentrations increase early in pregnancy and remain high throughout gestation, with a decrease occurring at term. In the rat, uterine quiescence during gestation appears to be related to elevated progesterone levels, and a decrease in progesterone at term induces uterine contractility leading to parturition. In addition, a surge in serum estradiol levels that occur at term in rats has been implicated in parturition. Other factors help maintain uterine quiescence during pregnancy. The uterus generates nitric oxide (NO) and both the production of NO and the relaxation sensitivity of the uterus to NO are elevated during pregnancy and decreased at term [5–7]. Dong et al. and Upton et al. [8, 9] have suggested that calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are involved in the maintenance of uterine quiescence during pregnancy. The CGRP levels in plasma are increased during pregnancy and decreased at labor [10]; CGRP has been shown to inhibit myometrial contractions in rats [8], human [11], and mice [12] during pregnancy but not during labor. Thus, CGRP appears to require progesterone to play an active physiological role during gestation [8]. Circulatory levels of AM are elevated during pregnancy and decrease to nonpregnant levels postpartum [9]. Adrenomedullin has been reported to inhibit spontaneous and bradykinin-induced [13] and galanin-induced [9] uterine contractions in rats.

Calcitonin receptor-like receptor (CRLR), in association with receptor activity-modifying protein 1 (RAMP1), forms a CGRP-A receptor, while CRLR, in association with RAMP2, forms an AM receptor. During pregnancy, messenger RNA expression of CRLR (the common receptor for both CGRP and AM) and RAMP1 and RAMP2 (receptor components for CGRP and AM, respectively) are increased [14]. Binding sites for both CGRP [15] and AM [9] increase during pregnancy. When treated with progesterone, nonpregnant ovariectomized rats show an increase in mRNA expression of CGRP- and AM-receptor components [14] and CGRP-binding sites [15] in the uterus. The progesterone antagonist RU486 decreases mRNA expression of CRLR, RAMP1, RAMP2 [14], and CGRP-binding sites [15] in the uterus of pregnant rats. Progesterone may regulate CGRP and AM receptors and possibly the second messengers in the uterus during pregnancy.

The mechanism of action of CGRP and AM in rat uterine tissue is not fully understood. Swiss 3T3 cells transfected with RAMP1 and CRLR have shown that G proteins are involved in the CGRP signaling [16]. G proteins consist of Gs and Gi families that stimulate and inhibit adenyl cyclase, respectively [17]. In humans, Gs isoforms are increased in the uterus during pregnancy [18, 19]; in late pregnant rats, progesterone treatment increased Gs isoforms [20]. Calcitonin gene-related peptide also induces smooth-muscle or vascular relaxation through activation of guanylate cyclase, ATP activated K channels [21], and modulation of potassium channels via adenylate cyclase-activated cyclic adenosine monophosphate-protein kinase A pathway [22] in different tissues. Adrenomedullin stimulates cAMP accumulation and calcium mobilization in aortic endothelial cells [23], which suggests that different mechanisms help mediate both CGRP and AM actions. We hypothesize that CGRP and AM exert effects binding to receptors and signaling through G{alpha}s, cAMP, and cGMP in the smooth-muscle cells of the myometrium. These effects are regulated by steroid hormones. In this study, we used the Eker rat uterine smooth-muscle cell line (ELT3), a well-established myometrial tumor cell line expressing receptors for both estrogen and progesterone. We used this cell line to 1) examine the mRNA expression of CGRP and AM receptors; 2) examine the involvement of the G{alpha}s protein and the secondary messengers (cAMP and cGMP) in the signaling mechanism of CGRP and AM; and 3) assess the regulation of CRLR, RAMP1, RAMP2, and RAMP3 mRNA expression, G{alpha}s protein, cAMP, and cGMP by steroid hormones.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture

Eker rat uterine smooth-muscle cells, a gift from Cheryl L. Walker, University of Texas M.D. (Anderson Cancer Center, Smithville, TX; see [24] for the generation and characterization of cells), were cultured in Dulbecco modified Eagles medium (Life Technologies, Rockville, MD) containing high (4.5 g/L) glucose, 1 mM sodium pyruvate (Sigma Chemical Co., St. Louis, MO), 100 U/ml penicillin, 100 ng/ml streptomycin (Sigma Chemical Co.) and 10% fetal bovine serum (Life Technologies) in a 75-cm2 culture flask. These cell cultures were maintained at 37°C in a humidified atmosphere of air and CO2 (5%). After they reached confluency, the cells were serum starved for 24 h in a medium containing 0.5% BSA (Sigma Chemical Co.) and then treated with the test reagents.

Isolation of Total RNA and Reverse Transcription

Uterine smooth-muscle cells grown in 60-mm dishes were treated with progesterone (10 nM; Sigma Chemical Co.), estradiol-17ß (1 nM; Sigma Chemical Co.), or vehicle for 24 h. Our pilot studies have shown that the maximal effects were observed using these concentrations for 24 h of treatment (data not shown). Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) as described previously [14]. For reverse transcription (RT), 2 µg of total RNA was briefly mixed with a master mix containing 3.0 nmol of random primer (Invitrogen), 200 µM dNTP (Sigma Chemical Co.), 10 U avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI), and 5 U RNase inhibitor (Invitrogen). These mixtures were placed in a thermal cycler for one cycle at 28°C for 15 min, 42°C for 30 min, 99°C for 5 min, and 4°C for 5 min. The cDNA was stored at –20°C.

Polymerase Chain Reaction

Polymerase chain reaction (PCR) of the cDNA was initiated using the primers designed for CRLR and RAMP1 [25], RAMP2, and RAMP3 [14]. Primers used for the 18S gene were obtained from Ambion, Inc. (Austin, TX). The PCRs were performed using 2.5 µl of cDNA. Complimentary DNA was mixed with a master mix containing appropriate primers, and the PCRs were carried out in a Gene amp PCR System 9700 (Perkin-Elmer, Norwalk, CT) as described previously [14]. Reactions were terminated by a 7-min elongation step at 72°C. The total cycle number was chosen for each gene from the linear portion of their respective curves (data not shown). The PCR products were visualized on 1.6% (w/v) agarose gels containing 0.5 mg/ml ethidium bromide (Sigma Chemical Co.) and run in 0.5x (Tris/boric acid/EDTA; Sigma Chemical Co.) buffer at 100 volts. The DNA signals were imaged under ultraviolet light and analyzed using the Fluorchem digital imaging system (Alpha Innotech Corp., San Leandro, CA). The levels of expression of CRLR, RAMP1, RAMP2, and RAMP3 were calculated as a ratio of their respective 18S values. The identity of the amplified sequences was verified by sequence analysis of PCR products, which showed 100% homology to the published sequences. Total RNA was taken as a negative control, and PCR was performed using the four different gene primers mentioned earlier. A signal was not detectable for any of the genes when total RNA was used and the product was run on an agarose gel (data not shown).

Western Immunoblot Analysis of G Protein G{alpha}s

Uterine smooth-muscle cells grown in 60-mm dishes were treated with 10 nM progesterone for 24 h. Whole-cell lysate was made using 2% SDS buffer (62.5 nM Tris, 2% SDS, 10% glycerol; Sigma Chemical Co.) containing 1x protease inhibitor cocktail (Roche, Indianapolis, IN). Cells collected in a vial were subjected to brief sonication and centrifuged at 10 000 rpm for 5 min at 4°C. The supernatant was aliquoted and stored at –80°C.

Polyacrylamide Gel Electrophoresis and Western Analysis

Equal amounts of protein (10 µg) were resolved on a 10% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane by electroblotting. Membranes, blocked with 10% nonfat milk in 20 mM Tris saline buffer with 0.095% Tween 20 (TTBS) were incubated in G{alpha}s antibody (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA). After being incubated with goat anti-rabbit IgG coupled with horseradish peroxidase (1:5000; Amersham Pharmacia Biotechnology, Piscataway, NJ) diluted in 10% nonfat milk, immunoreactive proteins were detected using an ECL Western blotting detection kit (Amersham Pharmacia Biotechnology). Immunodetected bands on Hyperfilm ECL were scanned using the Fluorchem digital imaging system (Alpha Innotech Corp). Blots were reprobed for ß tubulin using rabbit polyclonal antibody (1:1500; Santa Cruz Biotechnology) and anti-rabbit HRP-conjugate (1:5000; Amersham Pharmacia Biotechnology). G{alpha}s was expressed as a ratio of their respective ß-tubulin values.

Radioimmunoassay of cAMP and cGMP

To assess the dose-response of CGRP and AM on cAMP stimulation, cells cultured in a 35-mm well plates were initially stimulated with five doses of CGRP (ranging between 0.1 nM and 1000 nM) and four doses of AM (ranging between 1.0 nM and 1000 nM) for two time periods (5 and 15 min) in the presence of 100 µM phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX; Sigma Chemical Co.). We studied how progesterone affects the ability of CGRP and AM to stimulate cAMP and cGMP by pretreating the cells with progesterone (10 nM) for 24 h before stimulation with CGRP (100 nM for 5 min) or AM (10 nM for 5 min) in the presence of IBMX. Cells treated with IBMX alone served as the control.

To study the specific effects of G{alpha}s on cAMP generation, cells pretreated with progesterone (10 nM) for 24 h were incubated with two concentrations (1 and 100 µM) of G{alpha}s antagonist, NF449 (Calbiochem-Novabiochem Corp., San Diego, CA). After 25 min of incubation with NF449, IBMX (100 µM) was added. After IBMX was added, the cells were incubated with CGRP (100 nM) or AM (10 nM) for 5 min. Reactions were terminated by replacing the medium with ice-cold ethanol (65% [v/v] in water) and freezing the cells at –80°C. Supernatant obtained after brief sonication and centrifugation of cells was concentrated in a speed vacuum pump and reconstituted in a 500-µl assay buffer.

Both cAMP and cGMP were quantified using cAMP 125I and cGMP 125I assay systems (Amersham Pharmacia Biotechnology), respectively, as described by the supplier. The cAMP and cGMP standards (2–128 fmol/ tube) and samples were acetylated by adding triethylamine/acetic anhydride (2:1 [v/v] 5 µl/tube). Labeled cAMP and cGMP bound to their respective antibodies were recovered by using magnetic beads coated with goat anti-rabbit IgG, and radioactivity was quantified in a gamma counter. Cyclic AMP and GMP are presented as picomoles/million cells.

Statistical Analysis

Results are shown as the mean ± SEM of four independent experiments. Differences between two groups were evaluated with Student t-test; differences among multiple groups were evaluated with one-way analysis of variance followed by a Tukey multiple comparisons test. A P value less than 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Messenger RNA Expression of CGRP-A Receptor and AM Receptor Components and Steroid Hormone Effect on CRLR, RAMP1, RAMP2, and RAMP3

Progesterone treatment increased CRLR and RAMP1 (P < 0.01) mRNA expression compared with vehicle-treated control cells (Fig. 1). Both RAMP2 and RAMP3 mRNA expression did not differ from the control values. Estradiol-17ß treatment, however, decreased both CRLR (P < 0.05) and RAMP1 (P < 0.01) mRNA expression. Estradiol-17ß treatment had no effect on RAMP2 and RAMP3 mRNA expression in these cells.



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  		FIG. 1. Expression of CRLR, RAMP1, RAMP2, and RAMP3 in Eker rat uterine smooth-muscle cells treated with progesterone (P4, 10 nM) and estradiol-17ß (E2, 1 nM) for 24 h. Top) Samples analyzed for mRNA expression of CRLR, RAMP1, RAMP2, and RAMP3 by RT-PCR technique. Bottom) Densitometric analysis of respective PCR products for all components and the data are presented as a ratio, relative to that of 18S. Bars represent the mean ± SEM from four replicates in each group. Groups with asterisks are significantly (* P < 0.05 and ** P < 0.01) different

Western Immunoblot Analysis of G{alpha}s Protein

Western blot analysis of protein obtained from vehicle-treated cells demonstrated the presence of G{alpha}s in ELT3 cells. Expression of G{alpha}s protein increased with progesterone treatment (<0.05) compared with the vehicle-treated control (Fig. 2).



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  		FIG. 2. Expression of G{alpha}s in Eker rat uterine smooth-muscle cells treated with progesterone (P4, 10 nM) for 24 h. Top) Two representative samples from Western blotting for G{alpha}s and ß-tubulin. Bottom) Densitometric analysis of the protein in samples from untreated cells and cells treated with P4 and expressed relative to ß-tubulin. Bars represent the mean ± SEM from four replicates. Group with asterisks is significantly (* P < 0.05) different from control

Effects of CGRP and AM on cAMP and cGMP Generation in Smooth-Muscle Cells: Modulation by Progesterone

Both CGRP and AM dose-dependently increased intracellular cAMP concentrations when measured at 5- and 15-min intervals in the presence of IBMX (Figs. 3 and 4). The increases in cAMP levels were significant at both incubation times with a minimum concentration of 100 nM (P < 0.01) and 10 nM (P < 0.001) for CGRP and AM, respectively. Pretreatment with progesterone (10 nM) further augmented the cAMP production (P < 0.001) stimulated by CGRP (Fig. 5) and AM (Fig. 6) in these cells. Neither CGRP (Fig. 7) nor AM (Fig. 8) stimulated cGMP generation in these cells, and progesterone pretreatment did not alter the cGMP generation in response to CGRP or AM. These cells responded well to sodium nitroprusside, a nitric oxide donor, and increased cGMP (P < 0.001), confirming the ability of these cells to increase cGMP with appropriate stimuli.



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  		FIG. 3. Cyclic AMP dose-response curve in ELT3 cells treated with different doses of calcitonin gene-related peptide (CGRP). Cells were stimulated with five different doses of CGRP (0.1, 1, 10, 100, and 1000 nM) in the presence of IBMX (100 µM) for two time intervals (5 and 15 min). Bars represent the mean ± SEM from four replicates for each concentration used at each time. Groups with asterisks are significantly (** P < 0.01 and *** P < 0.001) different from control in the presence of IBMX



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  		FIG. 4. Cyclic AMP dose-response curve in ELT3 cells treated with different doses of adrenomedullin (AM). Cells were stimulated with four different doses of AM (1, 10, 100, and 1000 nM) in the presence of IBMX (100 µM) for two time intervals (5 and 15 min). Bars represent the mean ± SEM from four replicates for each concentration used at each time. Groups with asterisks are significantly (*** P < 0.001) different from control in the presence of IBMX



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  		FIG. 5. Calcitonin gene-related peptide (CGRP) stimulation of cAMP in ELT3 cells in the absence and presence of progesterone (P4). Cells were pretreated with P4 (10 nM) for 24 h and stimulated for 5 min with CGRP (100 nM). All stimulatory treatments were done in the presence of IBMX (100 µM). Bars represent the mean ± SEM from four replicates. Groups with different letters at the top of the bars are significantly (P < 0.001) different



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  		FIG. 6. Adrenomedullin (AM) stimulation of cAMP in ELT3 cells in the absence and presence of progesterone (P4). Cells were treated with P4 (10 nM) for 24 h and stimulated for 5 min with AM (10 nM). All stimulatory treatments were done in the presence of IBMX (100 µM). Bars represent the mean ± SEM from four replicates. Groups with different letters at the top of the bars are significantly (P < 0.001) different



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  		FIG. 7. Calcitonin gene-related peptide (CGRP) stimulation of cGMP in ELT3 cells in the absence and presence of progesterone (P4). Cells were treated with P4 (10 nM) for 24 h and stimulated for 5 min with CGRP (100 nM). All stimulatory treatments were done in the presence of IBMX (100 µM). Bars represent the mean ± SEM from four replicates. Groups with different letters at the top of the bars are significantly (P < 0.001) different



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  		FIG. 8. Adrenomedullin (AM) stimulation of cGMP in ELT3 cells in the absence and presence of progesterone (P4). Cells were treated with P4 (10 nM) for 24 h and stimulated for 5 min with AM (10 nM). All stimulatory treatments were done in the presence of IBMX (100 µM). Bars represent the mean ± SEM from four replicates. Groups with different letters at the top of the bars are significantly (P < 0.001) different

Effects of G{alpha}s Antagonist NF449 on cAMP Generation by CGRP and AM

Involvement of G protein, G{alpha}s, in cAMP generation in ELT3 cells stimulated with CGRP and AM was tested using the G{alpha}s-specific antagonist NF449. G{alpha}s antagonist at both 1 and 100 µM concentrations decreased (P < 0.01) basal cAMP production in ELT3 cells. The cAMP generation in response to CGRP (Fig. 9) and AM (Fig. 10) was attenuated by NF449 at 100 µM (P < 0.001) but not at the 1 µM concentration.



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  		FIG. 9. Inhibition of cAMP in ELT3 cells by G{alpha}s antagonist NF449 in the absence and presence of calcitonin gene-related peptide (CGRP). Cells pretreated with progesterone (P4, 10 nM) for 24 h were treated with NF449 (1 and 100 µM) for 25 min and cAMP generation to the absence (–) or presence (+) of CGRP (100 nM) for 5 min was assessed. All stimulatory treatments were done in the presence of IBMX (100 µM). Bars represent the mean ± SEM from four replicates. Groups with different letters at the top of the bars are significantly (P < 0.05–0.001) different



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  		FIG. 10. Inhibition of cAMP in ELT3 cells by G{alpha}s antagonist, NF449, in the absence and presence of adrenomedullin (AM). Cells pretreated with progesterone (P4, 10 nM) for 24 h were treated with NF449 (1 and 100 µM) for 25 min, and cAMP generation in the absence (–) or presence (+) of AM (10 nM) for 5 min was assessed. All stimulatory treatments were done in the presence of IBMX (100 µM). Bars represent the mean ± SEM from four replicates. Groups with different letters at the top of the bars are significantly (P < 0.05–0.001) different


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated that rat myometrial smooth-muscle cells (ELT3) express mRNA for CRLR, a common receptor component for both CGRP and AM. These cells also express mRNA for RAMP1, RAMP2, and RAMP3, which are specific for CGRP and AM binding. Expression of CRLR and RAMP1 mRNA increased with progesterone and decreased with estradiol-17ß treatments. RAMP2 and RAMP3 mRNA expressions were not affected by either of the steroid hormone treatments. G{alpha}s protein expression in myometrial cells increased with the progesterone treatment. Both CGRP and AM dose-dependently stimulated cAMP production. Progesterone pretreatment augmented these effects, and the G{alpha}s antagonist NF449 caused a decrease in both CGRP and AM stimulated and unstimulated cAMP production in ELT3 cells. The cGMP generated was not affected by any of the treatments. These data suggest that progesterone regulates the expression of CRLR (a common receptor component of CGRP-A and AM), RAMP1 (specific receptor component for CGRP-A), stimulatory G protein G{alpha}s, and cAMP in Eker rat myometrial smooth-muscle cells. These data further suggest that cAMP is a secondary messenger in the CGRP and AM signaling in uterine smooth-muscle cells.

Previously, we have reported [8, 11] that CGRP causes a substantial relaxation in both rat and human uterine tissues obtained during pregnancy. Uterine quiescence appears to be related to elevated levels of progesterone and low levels of estradiol observed during gestation. The CGRP-induced relaxation effects decrease during labor in both species [8, 11], indicating decreased relaxation sensitivity to CGRP. This is attributed to low levels of progesterone and a surge of estradiol at term in rats. We have shown that the decrease in CGRP sensitivity appeared to be related to lower levels of CGRP binding sites in the uterus [15]. Many CGRP binding sites in the uterus were upregulated by progesterone and downregulated by antiprogesterone, RU486 [15]. Furthermore, the changes in CGRP binding sites in the uterus caused by pregnancy, labor, and steroid hormone treatments appear to be related to the changes in CRLR and RAMP1 mRNA levels [14]. The effects of AM on uterine relaxation are similar to those caused by CGRP. AM inhibited spontaneous, bradykinin-induced [13] and galanin-induced [9] rat uterine contractions. Changes in the AM binding sites observed in the rat uterus during pregnancy and postpartum [9] may also be related to changes in CRLR and RAMP2 mRNA levels [14]. It is unclear, however, if the changes in these receptor components occur in myometrial cells and whether steroid hormone can directly alter these components on the muscle cells.

Our studies have demonstrated the expression of CRLR, RAMP1, RAMP2, and RAMP3 for the first time in isolated rat myometrial smooth-muscle cells. This expression suggests that the effects of CGRP and AM on the uterus occur at the level of myometrial smooth-muscle cells. Expression of CRLR and RAMP1 mRNA in smooth-muscle cells increased with progesterone treatment and decreased with estradiol treatment. These results demonstrate steroid hormone effects on receptor components in smooth-muscle cells and suggest a direct action of these steroid hormones. Therefore, we speculate that steroid hormone-induced changes in CGRP and AM receptor levels in full-thickness rat uterus [14] may reflect the changes in the myometrial smooth-muscle cells.

Although RAMP2 and RAMP3 levels in ELT3 cells were unaffected by steroid hormones in our study, progesterone increased the CRLR levels. Koller et al. [26] suggest that the amino acid sequence TRNKIMIT corresponding with residues 14–20 of N terminus of the mouse CRLR is required for a functional mCRLR/RAMP2 AM receptor. Therefore, an increase in CRLR after progesterone treatment may be sufficient to provide increased AM receptivity without changes in RAMP2 or RAMP3 levels. This speculation is supported by the reported increases in binding sites for AM [9] in pregnant rats and the increase in cAMP in response to AM treatments in the smooth-muscle cells in our study. These observations, together with the reported increase in circulating AM levels during pregnancy, suggest a role for AM-induced uterine relaxation during pregnancy.

Uterine smooth-muscle cells showed abundant expression of G{alpha}s protein in the present study. G{alpha}s protein expression was reported to increase during pregnancy in the human myometrium [18, 19] and in late pregnant rats treated with progesterone [20]. These reports as well as the increases in myometrial cell G{alpha}s in response to progesterone observed in this study suggest that progesterone upregulates G{alpha}s expression in myometrium during pregnancy.

Both CGRP and AM stimulated cAMP generation in ELT3 cells, and these effects were dose dependent. Our results suggest the involvement of cAMP as a signaling messenger molecule for both CGRP and AM in uterine smooth-muscle cells from rats. Similar increases in cAMP in response to CGRP were previously reported for human myometrial smooth-muscle cells [27], longitudinal muscles of guinea pig ileum [28], and rat aortic vascular smooth-muscle cells [29]. Adrenomedullin was reported to increase cAMP in vascular smooth-muscle cells [30], iris sphincter smooth-muscle [31], and human myometrial smooth-muscle cells [27]. Results from our study and the available literature suggest that cAMP is the secondary messenger in smooth-muscle relaxation and vasodilatory functions of CGRP and AM. Although basal cAMP levels in muscle cells were unaffected, CGRP- and AM-stimulated cAMP levels were elevated by pretreatment with progesterone. Therefore, elevated progesterone levels during pregnancy may increase the CGRP- and AM-stimulated cAMP, which results in relaxation sensitivity. Progesterone treatment also increases receptor components for both CGRP and AM and G{alpha}s protein in ELT3 cells, further supporting the involvement of progesterone in CGRP- and AM-induced uterine relaxation during pregnancy.

Involvement of cGMP as a second messenger to CGRP and AM in uterine smooth-muscle cells has not been demonstrated previously, and our study suggests that cGMP may not be involved. While none of the treatments in our study altered cGMP levels, stimulation with sodium nitroprusside caused a significant increase in cGMP response in the uterine smooth-muscle cells. This increase confirmed the ability of these cells to increase cGMP with appropriate stimuli. Our current findings, along with the reported [32] downregulation of cGMP in pregnant rat uterus, suggest that cGMP is not a candidate for cell signaling of CGRP and AM in the smooth-muscle cells of the rat uterus.

The G{alpha}s antagonist NF449 inhibited basal cAMP levels both at 1 and 100 µM concentrations used in our study. However, the stimulatory responses to CGRP and AM were inhibited by NF449 at 100 µM. Similar decreases in cAMP production with NF449 treatment was reported in S49 Cyc– membranes successfully reconstituted with recombinant G{alpha}s-s [33]. These results further confirm the involvement of G{alpha}s protein in CGRP- and AM-induced cAMP generation in uterine smooth-muscle cells.

In summary, we have demonstrated the expression of CGRP-A and AM receptor components CRLR, RAMP1, RAMP2, and RAMP3 in isolated myometrial smooth-muscle (ELT3) cells from rats. Regulating these receptor components with steroid hormones produced results that support our previous findings from in vivo studies: increases with progesterone and decreases with estradiol. Further, progesterone enhances cAMP generation through G{alpha}s protein in response to both CGRP and AM in uterine smooth-muscle cells. These studies suggest that the progesterone-induced increases in CGRP and AM receptor components, G{alpha}s protein levels, and cAMP generation in the rat myometrial cells may mediate enhanced uterine relaxation sensitivity to CGRP and AM during pregnancy.


   ACKNOWLEDGMENTS
 
We would like to thank Cheryl Welch for typing and the Publications, Grants, and Manuscripts Office in the Obstetrics and Gynecology Department for editorial assistance.


   FOOTNOTES
 
1 Supported by the National Institutes of Health through grants HL-58144, HL-72650, and HD-40828. Back

2 Correspondence: Chandrasekhar Yallampalli, Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1062. FAX: 409 747 0475; chyallam{at}utmb.edu Back

Received: 30 June 2004.

First decision: 26 July 2004.

Accepted: 21 September 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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