AJP - Endo Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 292: E1-E6, 2007. First published September 5, 2006; doi:10.1152/ajpendo.00529.2005
0193-1849/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/E1    most recent
00529.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Devost, D.
Right arrow Articles by Zingg, H. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Devost, D.
Right arrow Articles by Zingg, H. H.

Novel in vitro system for functional assessment of oxytocin action

Dominic Devost1,2 and Hans H. Zingg1,2,3

Departments of 1Medicine, 2Pharmacology and Therapeutics, and 3Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada

Submitted 2 November 2005 ; accepted in final form 28 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
One of the classical biological actions mediated by the posterior pituitary hormone oxytocin (OT) is contraction of the uterus at parturition. Moreover, premature activation of the OT system is thought to contribute to preterm labor, a major clinical problem in obstetrical practice. However, the molecular mechanisms linking activation of the OT receptor (OTR) to myometrial contractions are not fully understood. Here, we describe an in vitro system that should serve as a useful tool to study this question at a cellular level. The system consists of a collagen lattice contraction assay and two different human myometrial cell lines: a cell clone from a telomerase-immortalized human myometrial cell population (hTERT-C3) as well as a cell line derived from a primary culture of human myometrial cells (M11). Using this approach, we observed that 1 nM OT promoted an almost maximal effect on cell contraction in both cell lines tested. Furthermore, this dose-dependent, OT-induced contraction was antagonized by the specific OTR antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]OVT as well as the clinically used antagonist atosiban. This cell line-based contraction assay enables the application of molecular tools aimed at suppressing or overexpressing specific genes. It is also amenable to high-throughput testing approaches. Therefore, this system represents a powerful and improved experimental model that should facilitate the study of the molecular signal transduction pathways involved in the uterotonic actions of OT.

oxytocin receptor; myometrium; parturition; myometrial cell lines; mitogen-activated protein kinase; extracellular signal-regulated kinase 1/2

THE NONAPEPTIDE HORMONE OXYTOCIN (OT) has a wide spectrum of biological activities that are mediated through the OT receptor (OTR), a member of the heptahelical G protein-coupled receptor family (19). The OTR is primarily associated with the G protein G{alpha}q/11, and OTR activation results in a phospholipase C-mediated increase in intracellular calcium and inositol trisphosphate production (14). In addition, coupling of the OTR to G{alpha}i and Gh has also been observed (15, 2). The OTR is also linked to the MAP kinase pathway and is able to induce activation of ERK1/2 involving G{alpha}q, Gbeta/{gamma}, and transactivation of the epidermal growth factor receptor (18). One of the most recognized actions of OT is its capacity to induce uterine contraction at parturition. Indeed, at term, there is a marked increase in uterine OTR expression in all mammalian species studied, including the human (9, 11). OT is also used clinically to induce or augment labor and to prevent or treat uterine bleeding postpartum. Because mice that are deficient in OT deliver normally, the importance of the OT/OTR system for inducing parturition has been questioned (12, 17). However, the fact that the OT antagonist atosiban is able to delay preterm labor contractions in primates, including humans, argues in favor of an important role of the OT/OTR system in mediating uterine contractions preterm and possibly at term. In fact, atosiban is presently approved for clinical use in Europe to delay preterm labor.

Traditionally, the only bioassay to assess the effects of OT and of potential OT antagonists on uterine contractions consisted of freshly isolated uterine strips. For advanced studies on intracellular signaling mechanisms involved in mediating the contractile response in response to OTR activation, it is necessary to have the possibility to transfect cells with specific vectors that either overexpress or suppress specific genes in the target cells. This is difficult to achieve in freshly isolated tissues. Moreover, we wished to develop an assay system that allowed for the simultaneous analysis of a large number of samples and was possibly amenable to automated analysis. This prompted us to develop alternative in vitro systems using nontransformed, OT-responsive myometrial cell lines in combination with an in vitro cell contraction assay system. The present approach can be used to study the uterotonic activity of OT and any blockers thereof in a well-defined in vitro system of human myometrial cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue culture. Myometrial M11 cells were obtained from John A. Copland (Mayo Clinic College of Medicine, Jacksonville, FL). These cells were derived from dispersed primary human myometrial cells by repeated passage without the use of any immortalizing or transforming agent. M11 cells were maintained in DMEM high glucose (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Hyclone, Logan, UT), penicillin, and streptomycin. Myometrial human telomerase reverse transcriptase (hTERT)-HM cells were obtained from W. E. Rainey (5). hTERT-HM cells represent a cell population of human myometrial cells that was immortalized by transfection with an expression vector containing the hTERT. hTERT-C3 cells represent a subclone that we selected by serial dilution. This procedure maintains telomere length and endows the cells with an unlimited life span in culture (5). hTERT-C3 cells were maintained in DMEM-F-12 medium supplemented with 10% FBS and antibiotics. Both cell lines were cultured at 37°C in 5% CO2. Cells were trypsinized at 90% confluency and plated in T175 flask at a 1:5 dilution every 4–5 days.

Immunoblotting. The expression of cell markers was assessed by immunoblotting. Briefly, myometrial cells were plated onto 60-mm culture dishes and cultivated close to confluency on top of a collagen matrix. For monitoring MAP kinase activity, cells were serum starved for 2 days (M11 cells) or kept in DMEM-F-12 supplemented with only 0.5% FBS for 24 h (hTERT-C3 cells). Following OT treatment, cells were washed twice with ice-cold PBS and flash frozen in liquid nitrogen. Cells were lysed on ice with 250 µl of lysate buffer containing 25 mM HEPES-NaOH (pH 7.4), 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 10% glycerol, 1% Triton X-100, and a mix of protease inhibitors (1 µg/ml each aprotinin, pepstatin A, and leupeptin and 0.5 mM PMSF) and phosphatase inhibitors (50 mM NaF, 30 mM sodium pyrophosphate, and 1 mM sodium orthovanadate). Lysates were clarified by centrifugation at 15,000 g for 10 min at 4°C in a table top microcentrifuge. Total protein concentration was determined by a colorimetric assay using BSA as standard (BCA protein assay reagent kit; Pierce, Rockford, IL). Protein samples were denatured by boiling in Laemmli buffer [50 mM Tris·HCl (pH 6.8), 2% SDS, 10% glycerol, and 0.1 M beta-mercaptoethanol] for 5 min and subjected to SDS-PAGE and Western blotting. Immunodetection involved anti-smooth muscle {alpha}-actin (A-0307), anti-caldesmon (C-4562) (both from Sigma-Aldrich, St. Louis, MO), anti-phospho-ERK1/2 (9106s), and anti-pan-ERK1/2 (9102) (Cell Signaling Technology, Beverly, MA) antibodies in conjunction with an appropriate second horseradish peroxidase C-conjugated antibody and a chemiluminescence detection system (SuperSignal; Pierce).

Collagen gel retraction assay. The present in vitro system used to measure myometrial cell contraction in response to OT was developed on the basis of a technique originally described by Dallot et al. (6). Collagen type 1 from rat tail (Sigma) was resuspended overnight in 0.01 N HCl to prepare a 5 mg/ml stock solution and kept at 4°C until use. A working collagen solution was prepared by adding 5x PBS and 0.1 N NaOH to maintain the pH between 7.0 and 7.5 and diluted to 1.5 mg/ml with "starvation medium" (DMEM-F-12 supplemented with 0.5% FBS). The previous step was performed on ice to avoid premature collagen polymerization. Immediately thereafter, 0.5 ml of the ice-cold neutralized collagen solution was added to individual wells of a 24-well plate and left at 37°C for 1 h to allow gelling of the collagen. One millliter of starvation medium containing 25,000 myometrial cells was layered onto each collagen lattice, and cells were left to settle for 2 h at 37°C. To allow contraction, each collagen lattice was detached from the bottom of the well with a small spatula and left overnight at 37°C in absence or presence of different concentrations of OT (Sigma). Contraction was stopped by fixing the lattices in PBS-4% paraformaldehyde, and the lattice was kept at 4°C until analysis. To assess the surface area of the lattices, the liquid of each well was aspirated and the plate photographed using the Alpha Innotech Imaging System (Alpha Innotech, San Leandro, CA) equipped with an Olympus C-5060 digital camera. The surface area was determined quantitatively using the analysis software package supplied with the imaging system. For each OT concentration, collagen contraction was determined in triplicate or quadruplicate and expressed as percentage contraction. Percentage contraction was taken as the percentage of lattice size diminution relative to the size area of the well.

[3H]OT binding assay on cell membranes. The determination of OTR binding sites on cell membranes was assessed as described previously (10). Cells including the collagen lattices on which they were grown were harvested in ice-cold lysate buffer [15 mM Tris·HCl (pH 7.4), 2 mM MgCl2, and 0.3 mM EDTA] supplemented with protease inhibitors as above and lysed using a polytron (Tissue Tearor; Biospec Products). Membranes were pelleted and resuspended in ice-cold membrane buffer [50 mM Tris·HCl (pH 7.4), 3 mM MgCl2, and protease inhibitors] and centrifuged again. Washed pellets were resuspended in a small volume of membrane buffer, and membrane protein content was assessed by BCA. For the binding procedure, 20 µg of membranes were incubated with increasing concentrations of [3H]OT (PerkinElmer Life Sciences, Boston, MA) for 60 min at 30°C. Specific binding was determined as the difference between total and nonspecific binding in the absence and presence of 10 µM unlabeled OT, respectively. The dissociation constants (Kd) and the maximal receptor densities (Bmax) were obtained from the binding data using the software KELL (Biosoft). Data were represented graphically using GraphPad Prism version 4 (GraphPad Software, San Diego, CA).

Statistical data analysis. Experimental results were expressed as means ± SE. Student's t-test was used to determine statistical significance. A value of P < 0.05 was considered to be significant. For analysis of the dose-response curve of OT-induced contractions, including nonlinear sigmoidal, four-parameter logistic curve fitting, graphic representation, and ED50 calculation, the program Prism, by GraphPad Software, was used.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of human myometrial cell lines M11 and hTERT-C3. Two different human myometrial cell lines were used in the present study: a cultured primary myometrial cell line (M11) and a clonal cell line originating from a telomerase-immortalized myometrial cell population (hTERT-C3). To assess the cellular morphology, cells from both lines were cultured on a collagen matrix and photographed under phase-contrast optics. Both cell lines shared similar morphological characteristics that were typical of myometrial cells (Fig. 1A). Cells of both lines featured an elongated shape and a central nucleus. Compared with M11 cells, hTERT-C3 cells were smaller in size and often exhibited a bulky shape. To further determine the status of cell differentiation in both cell lines, the expression levels of typical smooth muscle differentiation markers were assessed by Western blotting. As shown in Fig. 1B, M11 cells as well as hTERT-C3 cells expressed two major smooth muscle cell markers: smooth muscle {alpha}-actin and hCaldesmon.


Figure 1
View larger version (48K):
[in this window]
[in a new window]

  		Fig. 1. A: phase contrast microphotograph of M11 or human telomerase reverse transcriptase (hTERT)-C3 cells grown on collagen lattices. Magnification, x400. B: immunodetection of the myometrial markers hCaldesmon (top) and smooth muscle {alpha}-actin (bottom) in total lysates from hTERT-C3 or M11 cells grown on collagen lattices.

 
The cell surface expression of OTR is another landmark of myometrial cells. To determine the level of OTR expression, the extent of [3H]OT binding was assessed. Scatchard plot analysis of [3H]OT binding to membranes derived from cells grown on collagen matrices revealed that both cell lines expressed large amounts of high affinity OTRs (Fig. 2). For M11 cells, we found a Kd of 1.62 ± 0.65 nM (n = 3) and a Bmax of 0.319 ± 0.101 pmol/mg protein (n = 3). For hTERT-C3 cells, we determined a Kd of 1.76 ± 0.3 nM (n = 3) and a Bmax of 0.705 ± 0.122 pmol/mg protein (n = 3). The binding of [3H]OT was also competed with by the presence of an excess of the specific OTR antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]OVT (OTA) (data not shown) (7).


Figure 2
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 2. M11 and hTERT-C3 cells express high-affinity oxytocin (OT) receptors. A: specific saturation equilibrium binding of [3H]OT to cell membranes from M11 cells grown on collagen lattices. {square}, Total binding; bullet, specific binding. B: Scatchard plot of binding data shown in A. C: specific saturation equilibrium binding of [3H]OT to cell membranes from hTERT-C3 cells grown on collagen lattices. {square}, Total binding; bullet, specific binding. D: Scatchard plot of binding data shown in C. Each data point represents the mean ± SE of triplicate determinations.

 
To assess the functionality of the demonstrated OTRs expressed on the myometrial cell lines, coupling to the MAP kinase pathway was determined using a phospho-ERK-specific antibody. Analysis by Western blotting demonstrated that OT application led to a dose-dependent increase in ERK1/2 phosphorylation (Fig. 3). The response was highly sensitive, and a marked increase could be detected at a dose as low as 0.1 nM for both cell lines. Taken together, these results indicate that both myometrial cell lines share many features of primary uterine smooth muscle cells, including the expression of specific and functional OTRs.


Figure 3
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 3. OT receptors (OTRs) present in M11 and hTERT-C3 cells are coupled to the MAP kinase pathway. Cells were growth arrested and stimulated with increasing concentrations of OT for 5 min. In some dishes, 100-fold excess of the OT antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]OVT (OTA) was added prior to OT addition. ERK1/2 activity was immunodetected in total cell lysates by Western blot using a specific phospho-ERK1/2 antibody. As a loading control, total MAP kinase immunoreactivity was assessed first by removing the phospho-ERK1/2 antibody by stripping the membrane and reprobing the latter with a total ERK1/2 antibody. The migration positions of ERK1 and ERK2 are indicated on the left.

 
OT-induced contraction of M11 and hTERT-C3 cells. As shown above, M11 and hTERT-C3 cells possess biochemical and morphological characteristics typical of myometrial cells. We were next interested to determine to what extent these cells also exhibited the prototypical physiological response of myometrial cells to OT stimulation, i.e., cellular contraction. To address this question, we developed an in vitro assay system consisting of a modified collagen lattice contraction assay that was based on a published procedure (6). In contrast to the published procedure, in which cells were imbedded in a collagen matrix, in the present system cells are layered on top of a preformed collagen matrix. This has the advantage of a better solute accessibility of the cells forming a monolayer on top of the matrix. Cellular contraction leads to a decrease in the diameter of the collagen matrix. Furthermore, if cells are layered on top of the matrix, cellular contraction also induces a concave shape of the matrix with raised borders. Both types of matrix deformation contribute additively to a decrease in matrix surface if viewed perpendicularly from above (Fig. 4A).


Figure 4
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 4. OT-induced contractions as assessed by the collagen lattice assay. A: representative collagen lattices. Lattices were prepared and covered by a layer of either hTERT-C3 cells (top) or M11 human myometrial cells (bottom), as described in MATERIALS AND METHODS. OT alone or in combination with the OT antagonist OTA was added at concentrations shown. Lattices are visible in the center of each well. B and C: quantitative determination of OT-induced contractions of hTERT-C3 (B) and M11 cells (C). OT and the specific OT-antagonist OTA were added as indicated. Each bar represents the mean ± SE of triplicate determinations. The experiments shown are representative of 3 independent experiments. *Significantly different from control (P < 0.05); +significantly different from treatment with OT alone (P < 0.05).

 
Using the present assay system, we observed a marked contraction exerted by hTERT-C3 cells in the absence of added OT (Fig. 4, A and B). This basal contraction was dependent on the presence of attached living myometrial cells and was absent from collagen lattices without cells (data not shown). Addition of a saturating concentration of OT led to a further contraction of hTERT-C3 cells (Fig. 4, A and B). This OT-induced contraction was fully antagonized by the OTR-specific antagonist OTA, supporting the idea that the OT-induced contraction in our assay occurred via an OTR-specific mechanism. Similar results were observed when M11 human myometrial cells were used (Fig. 4C). The addition of the OT antagonist OTA led to a relaxation of hTERT-C3 cells below the level of basal contraction (Fig. 4B). A similar trend was observed in M11 cells.

The basal as well as the OT-induced contractions were a hallmark of myometrial smooth muscle cells. This was clearly demonstrated when myometrial cells were replaced with human embryonic kidney-derived (HEK) 293 cells (Fig. 5). Using these noncontractile cells, neither basal nor OT-induced contractions were detectable.


Figure 5
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 5. Cell specificity of contraction. Basal and OT-induced contractions were assessed in lattices loaded with either hTERT-C3 cells or human embryonic kidney-derived (HEK) 293 cells as indicated. Each bar represents the mean ± SE of triplicate determinations. The experiments shown are representative of 3 independent experiments. *Value significantly different from control (P < 0.05).

 
An extended dose-response experiment of the uterotonic activity of OT on hTERT-C3 cells showed that 1 nM OT, an agonist concentration corresponding to the previously determined Kd of the OTR, induced an almost maximal contraction (Fig. 6). The logarithmic EC50 deduced from this experiment was –10.47 ± 0.258 (mean ± SE, n = 4). This result is in full agreement with values determined with preterm and term uterine strips, using a classical uterine strip bioassay (1).


Figure 6
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 6. Dose-response curve of OT-induced contractions of hTERT-C3 cells using the collagen lattice contraction assay. The percentage of OT-induced contraction has been plotted against the concentration of added OT. Results are expressed as means ± SE of 4 independent experiments performed in triplicate.

 
To further confirm that OT-induced contractions were mediated by the OTR, we tested the effect of the OTR selective agonist [Thr4,Gly7]OT (TGOT; Ref. 8). As shown in Fig. 7A, increasing amounts of TGOT led to a dose-dependent contraction. We next tested the effect of atosiban, an OT and vasopressin V1a antagonist in clinical use in Europe for the prevention of preterm labor. As demonstrated in Fig. 7B, the presence of a 100-fold excess of the antagonist totally blocked the contraction induced by 1 nM OT.


Figure 7
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 7. Effects of the selective OT agonist [Thr4,Gly7]OT (TGOT) (A) and the tocolytic agent atosiban (B) on hTERT-C3 cell contraction. A: increasing amounts of the OT agonist TGOT were added to each well as indicated. B: indicated amounts of OT were added to each well in absence or presence of 100 nM atosiban. Each bar represents the mean + SE of triplicate determinations. The experiments shown are representative of 3 independent experiments. *Significantly different from control (P < 0.05); +significantly different from treatment with OT alone (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Here, we report for the first time the use of an in vitro collagen lattice retraction assay for the study the uterotonic activity of OT. As an integral component of the assay system, we have characterized two novel nontransformed human myometrial cell lines with respect to their biochemical and physiological OT response characteristics. In the present in vitro contraction assay, cells were layered on top of a preestablished collagen lattice, whereas in the original approach cells were embedded within the lattice (6). With the present approach, less cells are needed per lattice (i.e., 25,000 vs. 150,000 cells). This is due to the fact that cells are concentrated in the same plane on the lattice surface rather than diluted in the entire volume of the collagen. The presumed higher level of cell-cell contacts also likely facilitates a concerted contraction of the entire layer. Moreover, we determined that leaving the cells on top of the collagen lattice for only 2 h was sufficient for the cells to settle down and be competent for OT-induced contraction. This is in contrast to the originally described procedure, where contraction was initiated only after 3 days in culture.

The use of clonal, immortalized, yet untransformed cells has notable advantages over a system using a heterogeneous cell population stemming from acutely isolated primary cells. Myometrial tissue is composed of different cell types, each differing with respect to the level of OTR expression (11), thus yielding an inhomogeneous population of primary cells. Using isolated cells from such a complex tissue may lead to a lack of consistency and reproducibility. The use of immortalized, yet nontransformed, myometrium cells like the ones used in this report thus has a distinct advantage over primary culture cells. An added benefit of the use of clonal cells is their almost infinite growth capacity, thus greatly facilitating the development of multiple consistent and reproducible assay systems. The present system should thus represent a valuable tool in the development of an integrative model of myometrial cells biology. The observations presented in this report were obtained using two different human myometrial cell lines: an established primary cultured cell line (M11) and a telomerase-immortalized myometrial cell clone (hTERT-C3). Our study shows that both cell lines exhibit phenotypes that are very similar to the primary cells from which they were derived in terms of morphology, OTR density, coupling of the activated OTR to the MAP kinase pathway, and the OT-induced contraction response curves. Overall, our data support the hTERT-C3 cell clone as a good model to study myometrial cells biology during the onset of labor, because this cell clone possesses several important characteristics of myometrial cells found at this stage of parturition. First, hTERT-C3 cells express similarly high levels of OTRs as the ones found in the myometrium at the onset of labor (3). Moreover, these receptors are also coupled to relevant signal transduction cascades, such as the activation of the MAP kinase ERK1/2 pathway. Finally, our data using the collagen retraction assay show a concentration-dependent response to OT as well as to the OTR-specific OT agonist TGOT that is indistinguishable from the one found with myometrial strips obtained from women at term (8, 13).

When compared with M11 cells, hTERT-C3 cells exhibited a stronger OT-induced contractile response (Fig. 4). hTERT-C3 cells also showed a higher OTR receptor density (Bmax) and an apparently higher level of smooth-muscle {alpha}-actin (Fig. 1). However, it remains to be determined whether any cause/effect relationships exist between these observations. Interestingly, in both cell lines tested, the presence of the OTR antagonist OTA inhibited not only OT-induced contractions but also reduced the basal, agonist-independent contraction. The effect of the antagonist on the basal uterine contraction can be explained either by its antagonistic activity per se on the uterotonic action of OT secreted in the medium by the cells themselves or by a possible reverse agonist activity on the OTR, as previously reported by Chini et al. (4).

In conclusion, in this report we describe a new experimental model to study the uterotonic action of OT in vitro directly on cells, using a collagen retraction assay in conjunction with two different human myometrial cell lines. This new approach should help to define, at the cellular level, the molecular events occurring in the myometrium at normal labor and should help to discover efficient approaches to counter the molecular cascades leading to preterm labor. (16)


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The work was supported by a grant from the Canadian Institutes for Health Research.


   ACKNOWLEDGMENTS
 
We wish to thank Marie-Eve Carrier for technical assistance and Michelle Carroll for help with microphotography. Atosiban was a generous gift from Ferring Pharmaceuticals, Copenhagen, Denmark.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. H. Zingg, Dept. of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montreal, QC, H3G 1Y6, Canada (e-mail: hans.zingg{at}mcgill.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Akerlund M, Bossmar T, Brouard R, Kostrzewska A, Laudanski T, Lemancewicz A, Serradeil-Le Gal C, Steinwall M. Receptor binding of oxytocin and vasopressin antagonists and inhibitory effects on isolated myometrium from preterm and term pregnant women. Br J Obstet Gynaecol 106: 1047–1053, 1999.[ISI][Medline]
  2. Baek KJ, Kwon NS, Lee HS, Kim MS, Muralidhar P, Im MJ. Oxytocin receptor couples to the 80 kDa Gh alpha family protein in human myometrium. Biochem J 315: 739–744, 1996.
  3. Bossmar T, Akerlund M, Fantoni G, Szamatowicz J, Melin P, Maggi M. Receptors for and myometrial responses to oxytocin and vasopressin in preterm and term human pregnancy: effects of the oxytocin antagonist atosiban. Am J Obstet Gynecol 171: 1634–1642, 1994.[ISI][Medline]
  4. Chini B, Albertazzi E, De Benedetti P, Fanelli F. Identification of a constitutively active mutant of the human oxytocin receptor. Adv Exp Med Biol 449: 367–369, 1998.[ISI][Medline]
  5. Condon J, Yin S, Mayhew B, Word RA, Wright WE, Shay JW, Rainey WE. Telomerase immortalization of human myometrial cells. Biol Reprod 67: 506–514, 2002.[Abstract/Free Full Text]
  6. Dallot E, Pouchelet M, Gouhier N, Cabrol D, Ferre F, Breuiller-Fouche M. Contraction of cultured human uterine smooth muscle cells after stimulation with endothelin-1. Biol Reprod 68: 937–942, 2003.[Abstract/Free Full Text]
  7. Elands J, Barberis C, Jard S, Tribollet E, Dreifuss JJ, Bankowski K, Manning M, Sawyer WH. 125I-labelled d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT: a selective oxytocin receptor ligand. Eur J Pharmacol 147: 197–207, 1988.[CrossRef][ISI][Medline]
  8. Elands J, Barberis C, Jard S. [3H]-[Thr4,Gly7]OT: a highly selective ligand for central and peripheral OT receptors. Am J Physiol Endocrinol Metab 254: E31–E38, 1988.[Abstract/Free Full Text]
  9. Fuchs AR, Fuchs F, Husslein P, Soloff MS. Oxytocin receptors in the human uterus during pregnancy and parturition. Am J Obstet Gynecol 150: 734–741, 1984.[ISI][Medline]
  10. Grazzini E, Durroux T, Payet MD, Bilodeau L, Gallo-Payet N, Guillon G. Membrane-delimited G protein-mediated coupling between V1a vasopressin receptor and dihydropyridine binding sites in rat glomerulosa cells. Mol Pharmacol 50: 1273–1283, 1996.[Abstract]
  11. Kimura T, Takemura M, Nomura S, Nobunaga T, Kubota Y, Inoue T, Hashimoto K, Kumazawa I, Ito Y, Ohashi K, Koyama M, Azuma C, Kitamura Y, Saji F. Expression of oxytocin receptor in human pregnant myometrium. Endocrinology 137: 780–785, 1996.[Abstract]
  12. Nishimori K, Young LJ, Guo Q, Wang Z, Insel TR, Matzuk MM. Oxytocin is required for nursing but is not essential for parturition or reproductive behavior. Proc Natl Acad Sci USA 93: 11699–11704, 1996.[Abstract/Free Full Text]
  13. Pierzynski PLA, Reinheimer T, Akerlund M, Laudanski T. Inhibitory effect of barusiban and atosiban on oxytocin-induced contractions of myometrium from preterm and term pregnant women. J Soc Gynecol Investig 11: 384–387, 2004.[Abstract/Free Full Text]
  14. Sanborn BM, Dodge K, Monga M, Qian A, Wang W, Yue C. Molecular mechanisms regulating the effects of oxytocin on myometrial intracellular calcium. Adv Exp Med Biol 449: 277–286, 1998.[ISI][Medline]
  15. Strakova Z, Soloff MS. Coupling of oxytocin receptor to G proteins in rat myometrium during labor: Gi receptor interaction. Am J Physiol Endocrinol Metab 272: E870–E876, 1997.[Abstract/Free Full Text]
  16. Wilson RJ, Allen MJ, Nandi M, Giles H, Thornton S. Spontaneous contractions of myometrium from humans, non-human primate and rodents are sensitive to selective oxytocin receptor antagonism in vitro. BJOG 108: 960–966, 2001.[CrossRef][Medline]
  17. Young WS 3rd, Shepard E, Amico J, Hennighausen L, Wagner KU, LaMarca ME, McKinney C, Ginns EI. Deficiency in mouse oxytocin prevents milk ejection, but not fertility or parturition. J Neuroendocrinol 8: 847–853, 1996.[CrossRef][ISI][Medline]
  18. Zhong M, Yang M, Sanborn BM. Extracellular signal-regulated kinase 1/2 activation by myometrial oxytocin receptor involves Galpha(q)Gbetagamma and epidermal growth factor receptor tyrosine kinase activation. Endocrinology 144: 2947–2956, 2003.[Abstract/Free Full Text]
  19. Zingg HH, Laporte SA. The oxytocin receptor. Trends Endocrinol Metab 14: 222–227, 2003.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 ReproductionHome page
G. Chevillard, A. Derjuga, D. Devost, H. H Zingg, and V. Blank
Identification of interleukin-1{beta} regulated genes in uterine smooth muscle cells
Reproduction, December 1, 2007; 134(6): 811 - 822.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
M. Kawamata, Y. Tonomura, T. Kimura, Y. Sugimoto, T. Yanagisawa, and K. Nishimori
Oxytocin-induced phasic and tonic contractions are modulated by the contractile machinery rather than the quantity of oxytocin receptor
Am J Physiol Endocrinol Metab, April 1, 2007; 292(4): E992 - E999.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/E1    most recent
00529.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Devost, D.
Right arrow Articles by Zingg, H. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Devost, D.
Right arrow Articles by Zingg, H. H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.