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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 9 3087-3092
Copyright © 1997 by The Endocrine Society

Original Studies

Activation of Adenylyl Cyclase in Human Myometrial Smooth Muscle Cells by Neuropeptides1

M. Linette Casey, Jesse Smith, Grady Alsabrook and Paul C. MacDonald

The Cecil H. and Ida Green Center for Reproductive Biology Sciences and Departments of Obstetrics-Gynecology and Biochemistry, The University of Texas Southwestern Medical School at Dallas, Texas 75235

Address all correspondence and requests for reprints to: M. Linette Casey, The Cecil H. and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9051. E-mail: casey{at}grnctr.swmed.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The levels of intracellular cAMP in human myometrial smooth muscle cells in serum-free medium, or medium that contained FBS (1%, vol/vol), were determined after treatment with the homologous peptides, calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), and amylin, without or with added isobutylmethylxanthine (IBMX). These cells were sensitive to CGRP, responding in a dose-dependent manner, with maximal levels of cAMP being attained with 5 nM CGRP in the presence of IBMX (1 mM). In the absence of IBMX, the level of cAMP attained in cells treated with CGRP (5 nM) (675.3 ± 58.8 pmol•mg protein•15 min; mean ± SEM, n = 3) was approximately 90x that in nontreated cells (7.5 ± 0.4 pmol•mg protein•15 min). The level of cAMP in myometrial cells treated with CGRP (5 nM) + IBMX (1 mM), 1998 ± 420 pmol•mg protein•15 min, was 29x that in cells treated with IBMX alone (69.2 ± 10.2). The maximum level of cAMP achieved by treatment with ADM + IBMX was similar to that with CGRP + IBMX, but the dose of ADM required (1 µM) was approximately 200x that of CGRP. Amylin amide also caused an increase in cAMP but with considerably less potency; at a concentration of 500 nM, amylin amide + IBMX effected a 2.3-fold increase in cAMP relative to IBMX alone. CGRP8–37, an antagonist of CGRP via the CGRP1 receptor, inhibited the action of CGRP, ADM, and amylin in myometrial cells. Treatment with [cys(ACM)2–7]-CGRP, a CGRP2 receptor agonist, did not cause an increase in the levels of cAMP in these cells. These findings are indicative that CGRP, ADM, and amylin act via that the CGRP1 receptor in human myometrial cells. Vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide also caused a dose-dependent increase in cAMP in myometrial cells. The findings of this study are indicative that multiple neuropeptides, acting by way of heptahelical receptors linked to the G{alpha}s-subunit of the G-proteins, may contribute to the maintenance of uterine quiescence during some period of human pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN myometrium is, inherently, a contractile tissue; yet during approximately the first 36–38 weeks of pregnancy, the myometrium is maintained in a remarkable state of contractile refractoriness. This long interval of myometrial tranquillity, referred to as uterine phase 0 of parturition (1, 2, 3), is so effective as to be a source of wonderment to all who reflect on this poorly understood period of pregnancy. Phase 0 of parturition is not a static state, however; rather, this phase of parturition must accommodate multiple, highly diverse transitions. For example, the early days and weeks of pregnancy are dominated by an endometrial period during which blastocyst invasion of the endometrium and early embryonic development are confined to the decidua. Rapid uterine growth during the first 14–16 weeks of pregnancy is characterized by appreciably greater expansion of the uterine cavity than of the conceptus (4). At about 16 weeks gestation, the amnionic sac fills the uterine cavity (4); and thereafter, as the fetus grows and the volume of the amniotic fluid increases, profound uterine stretching is obliged. Each of these periods of uterine accommodation (endometrial invasion and embryonic growth, rapid uterine growth, and myometrial stretch) may oblige different or supplemental processes to maintain myometrial contractile refractoriness.

A definition of the processes that safeguard myometrial quiescence at all periods of uterine phase 0 is fundamental to an understanding of the initiation of parturition at term and preterm. Comparatively less research, however, has been conducted to define the mechanisms that promote myometrial relaxation during human pregnancy compared with that devoted to a search for some factor(s) (viz., a uterotonin) that causes the onset of labor. In part, this may have been the case because it has been presumed that the actions of progesterone were dominant in effecting myometrial quiescence throughout phase 0 of parturition in all mammals. It is reasonably clear that the endometrial/decidual period of early embryonic development is dependent on the actions of progesterone. Removal of the corpus luteum of human pregnancy before 8 weeks gestation results in abortion; and, the administration of a progesterone receptor antagonist in early human pregnancy also is effective in causing abortion.

Presently, however, the concept of an exclusive or preeminent role for progesterone in mediating uterine tranquillity throughout gestation, at least in some mammalian species, must be questioned. In primates, viz., great apes, old world monkeys, and women (5, 6), guinea pigs (6), and armadillos (7, 8), progesterone withdrawal does not precede the initiation of parturition. Moreover, the administration of progesterone late in pregnancy does not forestall the timely onset of labor in these mammals; and, the administration of a progesterone receptor antagonist to primates late in pregnancy does not cause the orderly initiation of parturition and the onset of labor as it does in some species (9, 10). In the horse and the elephant, the levels of progesterone in maternal plasma are very low or undetectable (11, 12, 13). Therefore, it is likely that other factors, possibly in concert with progesterone, are operative to promote myometrial quiescence during various periods of phase 0. A multicomponent fail-safe system can be envisioned in which some components of the system are redundant, a common occurrence in the evolution of the physiological adaptations of pregnancy (3).

A sizable number of ligands that act via seven transmembrane-spanning (heptahelical) receptors linked to the G{alpha}s-subunit of the heterotrimeric G-proteins are functional in the human myometrium. The ligands for these receptors are neuropeptides, hormones, and autocoids; several of these ligands are present in increased concentration in maternal plasma or are produced in situ by nerves supplying the myometrium or in myometrial cells directly, or in contiguous tissues/cells. Activation of these receptors by binding to specific ligands activates the G{alpha}s-GTP signaling system, leading to activation of K+ channels and adenylyl cyclase, processes that promote myometrial relaxation (14). Therefore, the G{alpha}s-linked myometrial heptahelical receptors may constitute one component (possibly in cooperation with progesterone) to maintain uterine quiescence. In addition, evidence has been presented that the nitric oxide-guanylyl cyclase-guanosine 3'5'-cyclic monophosphate (cGMP) system also may be involved in promoting the uterine quiescence of pregnancy (15, 16).

This study was conducted to examine the response of human myometrial cells in culture to treatment with neuropeptides that nominally act by way of linkage to G{alpha}s, viz., calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), amylin, and vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and culture of human myometrial smooth muscle cells

Myometrial tissue was obtained from uteri of ovulatory women after hysterectomy, placed in culture medium, and immediately transported to the laboratory. Informed consent for the use of these tissues was obtained in writing from each woman before the surgical procedure. The consent forms used and the experimental protocols were approved by the Institutional Review Board of this university.

Myometrial smooth muscle cells were isolated and maintained in culture as previously described (17). The cells were maintained in culture in Ham’s F12/DMEM (F12:DMEM, 1:1, vol/vol) that contained FBS (10%, vol/vol), penicillin G (100 U/mL), streptomycin sulfate (100 µg/mL), and amphotericin B (0.25 µg/mL) plated in plastic 75-cm2 culture flasks at a density of approximately 100,000 cells/cm2 and maintained at 37 C in a humidified atmosphere of air and CO2 (5%) until confluent (7–10 days after plating). The culture medium was changed every 72 h. Confluent, first-passage cells were used for all experiments. At the time confluence was attained, the culture medium was changed; and 24 h thereafter, the cells were preincubated for 24 h in serum-free F12:DMEM that contained BSA (1%, vol/vol) or medium that contained FBS (1%, vol/vol) before the medium was changed to that which contained the test agents.

RIA of intracellular cAMP

Intracellular cAMP was quantified by RIA using a rabbit polyclonal antibody generously provided by Dr. William E. Rainey (The University of Texas Southwestern Medical School, Dallas, TX). Myometrial cells (in 24-well plates) were incubated with test agents for 15 min. After treatment, the culture medium was removed and ice-cold ethanol (65% in water) was added to each well. The plates (cells and ethanol) were stored at -80 C until sonication. Total cell protein in an aliquot of the cell sonicate was quantified by the bioinchoninic acid method (18). cAMP was quantified in an aliquot of a 10,000 x g (30 min at 4 C) supernatate of the sonicate by RIA. The cAMP standards (3.125–1600 fmol/tube) and samples were acetylated by the addition of trietylamine/acetic anhydride (2:1, vol/vol; 5 µL/tube). [125I]Succinyl cAMP was used as tracer. cAMP bound to antibody was recovered by use of magnetic beads coated with goat antirabbit IgG, and radioactivity was quantified in a gamma spectrometer.

Data for cAMP (measured in duplicate for each well of cells) was normalized to cell protein (quantified in triplicate). Each treatment condition was evaluated in triplicate or quadruplicate. Comparisons were made using ANOVA and Dunnett’s post hoc test. Data presented are representative of studies conducted on at least three occasions with cells isolated from the myometrium of different women.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Levels of cAMP in human myometrial cells after treatment with IBMX and forskolin in various concentrations

Confluent myometrial smooth muscle cells were treated with IBMX (0–1 mM) for 15 min. A highly significant correlation (r2 = 0.977) was found between the dose of IBMX and the intracellular level of cAMP (Fig. 1AGo). These cells also were treated with forskolin (0.1–250 µM) + IBMX (1 mM) for 15 min, and a dose-dependent increase in intracellular cAMP was observed (Fig. 1BGo).



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  		Figure 1. cAMP levels in myometrial cells treated with IBMX (A) or forskolin (B). A, Confluent cells were treated for 15 min with IBMX (5, 15, 50, 100, 150, 500, and 1000 µM); intracellular cAMP was quantified in replicates of 3–6 wells of cells and normalized to level of total cellular protein. Data presented (mean ± SEM) were compiled from 42 experiments and represent fold change in cAMP levels (after normalization for protein). Each point represents data from 1–42 experiments: n = 1 for 5, 15, 150 µM; n = 2 for 50 and 500 µM; n = 42 for 100 µM; and n = 19 for 1 mM. First-order regression analysis of data yields r2 = 0.977. B, Confluent cells were treated for 15 min with forskolin in various concentrations ± IBMX (100 µM). Data are mean ± SEM for replicates of four from myometrial cells from uterus of one woman.

 
Activation of adenylyl cyclase by CGRP, ADM, and amylin

Treatment of myometrial cells with CGRP, ADM, or amylin + IBMX caused a dose-dependent increase in the intracellular levels of cAMP. Among these agents, CGRP was the most effective. In the absence of IBMX, the level of cAMP after 15 min treatment with CGRP (675.3 ± 58.8 pmol•mg protein•15 min; mean ± SEM; n = 3), which was approximately 90x that in nontreated cells (7.5 ± 0.4, n = 3). CGRP effected a 29-fold (n = 7 experiments) increase in cAMP after 15 min treatment in the presence of IBMX (1 mM) compared with that elicited by IBMX treatment alone (Fig. 2AGo). Pretreatment for 5 min with CGRP8–37 (100 nM), an antagonist of CGRP via the CGRP1 receptor, attenuated the action of CGRP by approximately 87% (Fig. 2BGo). [Cys(ACM)2–7]-CGRP, which acts primarily via the CGRP2 receptor, was ineffective in increasing cAMP levels in myometrial cells (Fig. 2CGo). These findings are indicative that the CGRP1, but not the CGRP2, receptor is expressed in human myometrial cells.



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  		Figure 2. Activation of adenylyl cyclase by CGRP via CGRP1 receptor. A, Dose-dependent increase in intracellular cAMP in response to CGRP (0.1 nM–100 nM) + IBMX (1 mM). Data are presented from one of six similar experiments in which myometrial cells were treated with CGRP (at various concentrations) + IBMX (1 mM) for 15 min. B, Attenuation of CGRP (5 nM) action by CGRP8–37, a CGRP1 receptor antagonist (C-A). C, Lack of activation of adenylyl cyclase by [Cys(ACM)2–7]-CGRP (DAMC), a CGRP2 receptor agonist. All data are presented as mean ± SEM for replicates of three wells of myometrial cells treated identically. Data for dose-response studies are presented on logarithmic x-axes.

 
ADM treatment of myometrial smooth muscle cells also caused a striking dose-dependent increase in the intracellular levels of cAMP; but at similar doses, ADM was about 0.5% as effective as CGRP (Fig. 3AGo). The action of ADM was inhibited by CGRP8–37, indicative that ADM also is active in human myometrial cells via the CGRP1 receptor (Fig. 3BGo).



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  		Figure 3. Activation of adenylyl cyclase by ADM. A, Cells were treated with IBMX (1 mM) + ADM, at concentrations of 5 nM, 50 nM, 500 nM, or 5 µM, or with IBMX (1 mM) + CGRP (5 nM) + ADM (50 nM). Data are compared with those for cells treated with vehicle only (Ctl), IBMX (1 mM) only, or IBMX (1 mM) + CGRP (5 nM). B, Cells were treated with IBMX (1 mM) + ADM at concentrations of 100 nM, 300 nM, or 1 µM. In both A and B, data for dose-response studies are represented on logarithmic x-axes.

 
Amylin, at a concentration of 500 nM, + IBMX caused a significant increase (2.4-fold, P < 0.001) in the accumulation of cAMP in the culture medium (compared with IBMX alone), but the effectiveness of this agent was considerably less than that of CGRP + IBMX (51.1-fold in this study) (Fig. 4AGo). At concentrations of 50 nM and lower, amylin did not cause an increase in cAMP accumulation. The CGRP1 receptor antagonist prevented the increase in cAMP caused by amylin (1 µM) (Fig. 4BGo).



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  		Figure 4. Activation of adenylyl cyclase by amylin. A, Confluent myometrial cells were treated with IBMX (1 mM) + amylin at concentrations of 0.5, 5, 50, and 500 nM. Data for dose-response studies are represented on a logarithmic x-axis and are compared with those for cells treated with vehicle only (Ctl), IBMX (1 mM) only, or IBMX (1 mM) + CGRP (5 nM). B, Cells were treated with IBMX (1 mM) or IBMX (1 mM) + amylin (1 µM) in absence or presence of CGRP1 receptor antagonist (CGRP8–37, 1 µM). Treatment with CGRP8–37 was for 5 min before addition of IBMX ± amylin and was continued during incubation with IBMX ± amylin. Data are mean ± SEM for replicates of three.

 
Activation of adenylyl cyclase in myometrial smooth muscle cells by VIP and PACAP

VIP caused a dose-dependent increase in cAMP accumulation in myometrial cells. With VIP treatment at concentrations of 50 and 100 nM [in the presence of IBMX (1 mM)], the increase in cAMP accumulation was significantly greater than that in cells treated with IBMX alone (3.6- and 2.8-fold, respectively, P < 0.05, ANOVA). In this study, CGRP (5 nM) + IBMX (1 mM) caused a 15.4-fold increase in cAMP accumulation (Fig. 5Go).



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  		Figure 5. Activation of adenylyl cyclase by VIP. Confluent myometrial cells were treated for 15 min with VIP at concentrations of 0.1, 1, 5, 10, 50, and 100 nM in presence of IBMX (1 mM). Data (mean ± SEM) are compared with nontreated cells (control, Ctl), cells treated with IBMX (1 mM) alone, and cells treated with CGRP (5 nM) + IBMX (1 mM). Levels of cAMP were significantly (P < 0.05, ANOVA) greater than IBMX alone in cells treated with IBMX (1 mM) + VIP at concentrations of 50 and 100 nM.

 
PACAP also caused a dose-dependent increase in cAMP accumulation in myometrial cells. The increase in cAMP accumulation with PACAP, at concentrations of 1, 10, 100, and 1000 nM [in the presence of IBMX (1 mM)] was significantly greater than that in cells treated with IBMX alone [3.5-, 5.7-, 8.2-, 6.5-fold, respectively; P < 0.05, ANOVA (Fig. 6Go)].



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  		Figure 6. Activation of adenylyl cyclase by PACAP. Confluent myometrial cells were treated for 15 min with PACAP at concentrations of 0.1, 1, 10, 100, and 1000 nM in presence of IBMX (1 mM). Data (mean ± SEM) are compared with nontreated cells (control, Ctl), cells treated with IBMX (1 mM) alone, and cells treated with CGRP (5 nM) + IBMX (1 mM). Levels of cAMP were significantly (P < 0.05, ANOVA) greater than IBMX alone in cells treated with IBMX (1 mM) + PACAP at concentrations of 10, 100, or 1000 nM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Treatment of human myometrial smooth muscle cells in culture with the structurally homologous neuropeptides, CGRP, ADM, and amylin, caused a dose-dependent increase in the intracellular levels of cAMP. The response of myometrial cells to CGRP was striking, with maximal levels of cAMP being similar to those attained with forskolin (10 µM) + IBMX. In the absence of IBMX, the increase in cAMP in myometrial cells in response to CGRP (5 nM) treatment for 15 min was approximately 90x that in nontreated cells. In the presence of IBMX, the maximal response to ADM was similar to that of CGRP, but the dose of ADM required was approximately 200x that of CGRP. Amylin was appreciably less effective than CGRP or ADM. Human myometrial cells also responded to VIP and PACAP with a dose-dependent increase in the levels of intracellular cAMP, with maximal levels being approximately one-fifth those attained with CGRP.

Fragments and analogs of CGRP are used to characterize the CGRP receptor subtypes. CGRP8–37 is an effective antagonist specific for the CGRP1 receptor (19), and [cys(ACM)2–7]-CGRP is an agonist specific for the CGRP2 receptor (20), albeit less potent than CGRP1–37. The actions of CGRP, ADM, and amylin in myometrial cells was attenuated when CGRP8–37 was present. Treatment of myometrial cells with [cys(ACM)2–7]-CGRP did not effect a significant increase in the intracellular levels of cAMP. These findings are indicative that CGRP, ADM, and amylin are operative in human myometrial smooth muscle cells by way of the CGRP1 receptor. Differences in the potency of these ligands in stimulating adenylyl cyclase may derive from the induction by each of the distinct conformational changes in the receptor and hence variations in the efficiency in coupling with G{alpha}s.

CGRP, the most potent endogenously produced smooth muscle relaxant discovered, is present in the circulation, and the plasma levels of this neuropeptide are increased to approximately 50–150 pM during human pregnancy (21, 22, 23). The concentrations of VIP in plasma of nonpregnant and pregnant women (~5 pM) are similar (24). Immunoreactive CGRP, VIP, and PACAP are present in myometrial tissues of many nonpregnant mammalian species, including humans (24, 25, 26, 27, 28) and experimental animals (29, 30, 31, 32, 33, 34). CGRP also is present in the uterine cervix during early human pregnancy (35).

During pregnancy, there may be a decrease in the levels of these neuropeptides in the myometrium, at least at term. CGRP is not demonstrable in myometrium of term pregnant guinea pigs (29); and a reduction in the concentration of VIP in myometrial tissue obtained during pregnancy at term has been observed in several species, viz., the human, rat, and guinea pig (29, 36). The concentration of VIP in myometrium of pregnant women at term (<0.1 pmol/g wet wt) is significantly less than that in the uterus of nonpregnant women (1.6 pmol/g wet wt) (24). The levels of PACAP in myometrial tissue of pregnant women at term are lower than those in myometrial tissue of nonpregnant women (27). In part, the decrease in the concentration of neuropeptides in the uterus during pregnancy may be the result of myometrial hypertrophy without an actual decrease in total uterine content. There also is appreciable evidence, however, from the study of several species, that a functional denervation of the myometrium occurs during the course of pregnancy (37, 38, 39). There are few data, however, to establish when in gestation the loss of nerve tissue-derived neuropeptides occurs.

CGRP, VIP, and PACAP are known to act on myometrial smooth muscle tissue obtained from multiple nonpregnant species to inhibit spontaneous or induced contractions. CGRP acts on myometrial tissue of nonpregnant women to activate K+ channels and to suppress spontaneous contractions (25, 40). CGRP also acts on myometrial tissue of pregnant women at term (not in labor) to inhibit spontaneous and oxytocin-induced contractions, and the dose of CGRP required is appreciably less than that required to inhibit contractions of myometrial tissue of nonpregnant women and pregnant women at term in labor (40). CGRP also acts on myometrial tissue of nonpregnant rats (25, 41, 42) to inhibit contractions. VIP promotes relaxation of myometrial tissue of nonpregnant (24) and midtrimester pregnant women (31). VIP also inhibits contraction of strips of myometrium from a number of mammalian species (32, 33, 43, 44, 45). PACAP also inhibits spontaneous contraction of myometrial tissues of nonpregnant women (28). Treatment of myometrial tissues obtained during pregnancy at term with VIP and PACAP, however, is ineffective in inhibiting contractions (24, 27). There is, however, a paucity of data concerning the expression of the neuropeptide heptahelical receptors linked to G{alpha}s at any time during pregnancy.

Myometrial responsiveness also has been demonstrated for a number of other agents that are presumed to act by way G{alpha}s-linked heptahelical receptors: for example, catecholamines (e.g. ß-adrenergic agents), protein hormones [e.g. PTH-related protein (46, 47), CRH (48, 49), human CG (50), relaxin (51), and prostanoids (52, 53, 54)]. It seems likely, therefore, that there are multiple systems, involving a variety of agents, that could act, possibly in concert with progesterone, to provide a fail-safe means of ensuring myometrial quiescence during phase 0 of parturition.

By contrast, it also has been demonstrated that there are a large number of ligands that are functional by way of heptahelical receptors linked to G{alpha}i- or G{alpha}q/11-subunits in the myometrium, especially near term. The G{alpha}i- and G{alpha}q/11-subunits act to inhibit adenylyl cyclase and to activate phospholipase C, respectively, favoring an increase in intracellular Ca2+, thereby promoting myometrial contractions. Among those acting via G{alpha}i and G{alpha}q/11 are the following: oxytocin, PGE2, PGF2{alpha}, histamine, serotonin, angiotensin-II, substance P, galanin, endothelin, platelet-activating factor, neurotensin, neuropeptide Y, and {alpha}-adrenergic agents.

As long as G{alpha}s-linked receptors that activate K+ channels and adenylyl cyclase dominate, uterine quiescence should prevail. If this myometrial phenotype were abandoned (by processes not defined), the G{alpha}i- and G{alpha}q/11-linked heptahelical receptor phenotype should prevail as the uterus is prepared for labor (phase 1 of parturition), leading to the myometrial contractions of active labor, i.e. phase 2 of parturition. Therefore, an important determinant of the functional state of the myometrium may be the heptahelical receptor-G{alpha}-subunit-effector enzyme phenotype of the myometrium.


   Acknowledgments
 
The authors gratefully acknowledge the editorial assistance of Ms. Rosemary Bell and Ms. Kathy Loppnow.


   Footnotes
 
1 This work was supported in part by United States Public Health Service Grant 5-P50-HD11149. Back

Received March 17, 1997.

Revised June 12, 1997.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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