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Ghafourifar P, Farahani Z, Norooznezhad AH, Hantoushzadeh S, Azimzadeh M, Nabavian SM, Behzadian A, Allely QK. Insulin and myometrial contractility; Are there any links? A narrative review. Reprod Biol 2025; 25:100991. [PMID: 39798273 DOI: 10.1016/j.repbio.2024.100991] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2023] [Revised: 12/13/2024] [Accepted: 12/21/2024] [Indexed: 01/15/2025]
Abstract
Contrary to the evidence supporting the role for insulin in stimulating uterine contraction, only a limited number of studies have highlighted the inhibitory effect of insulin on myometrial contractions in human and rodent. A hypothetical narrative review of the current literature was conducted, revealing the current literature and shows the potential inhibitory effects of insulin on myometrial contractility. These inhibitory mechanisms include activation of adenylyl cyclase signaling pathways, an increase in cAMP production, a decrease in Ca2 + influx and cytosolic Ca2+, hyperpolarization of the cell membrane, and stimulation of NO synthesis. Altered oxytocin sensitivity, structural similarity to relaxin, modulating abscisic acid (ABA) effect, and synergistic interaction with progesterone, adiponectin, and leptin may also represent additional mechanisms for the inhibitory effects of insulin on myometrial contractions. The literature indicates that insulin exhibits inhibitory effects on myometrial contractility. Confirming such a conclusion through future studies may propose insulin as a possible uterine quiescent.
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Affiliation(s)
| | - Zahra Farahani
- Maternal, Fetal, and Neonatal Research Center, Family Health Research Institute, Tehran University of Medical Sciences, Tehran, Iran.
| | - Amir Hossein Norooznezhad
- Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.
| | - Sedigheh Hantoushzadeh
- Vali-E-Asr Reproductive Health Research Center, Family Health Research Institute, Tehran University of Medical Sciences, Tehran, Iran.
| | - Mansour Azimzadeh
- Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Putra, Selangor, Malaysia.
| | - Seyedeh Maedeh Nabavian
- Department of Obstetrics, Gynecology, and Perinatology, Hamadan University of Medical Sciences, Hamadan, Iran.
| | - Arezo Behzadian
- Maternal, Fetal, and Neonatal Research Center, Family Health Research Institute, Tehran University of Medical Sciences, Tehran, Iran.
| | - Quinn Kern Allely
- School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
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Dogan S, Walseth TF, Guvenc Tuna B, Uçar E, Kannan MS, Deshpande DA. CD38/cADPR-mediated calcium signaling in a human myometrial smooth muscle cell line, PHM1. IUBMB Life 2024; 76:1223-1233. [PMID: 39135342 PMCID: PMC11580371 DOI: 10.1002/iub.2904] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2024] [Accepted: 06/21/2024] [Indexed: 11/22/2024]
Abstract
Cyclic ADP-ribose (cADPR) has emerged as a calcium-regulating second messenger in smooth muscle cells. CD38 protein possesses ADP-ribosyl cyclase and cADPR hydrolase activities and mediates cADPR synthesis and degradation. We have previously shown that CD38 expression is regulated by estrogen and progesterone in the myometrium. Considering hormonal regulation in gestation, the objective of the present study was to determine the role of CD38/cADPR signaling in the regulation of intracellular calcium upon contractile agonist stimulation using immortalized pregnant human myometrial (PHM1) cells. Western blot, immunofluorescence, and biochemical studies confirmed CD38 expression and the presence of ADP-ribosyl cyclase (2.6 ± 0.1 pmol/mg) and cADPR hydrolase (26.8 ± 6.8 nmoles/mg/h) activities on the PHM1 cell membrane. Oxytocin, PGF2α, and ET-1 elicited [Ca2+]i responses, and 8-Br-cADPR, a cADPR antagonist significantly attenuated agonist-induced [Ca2+]i responses between 20% and 46% in average. The findings suggest that uterine contractile agonists mediate their effects in part through CD38/cADPR signaling to increase [Ca2+]i and presumably uterine contraction. As studies in humans are limited by the availability of myometrium from healthy donors, PHM1 cells form an in vitro model to study human myometrium.
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Affiliation(s)
- Soner Dogan
- Department of Medical Biology, School of MedicineYeditepe UniversityIstanbulTurkey
- Department of Veterinary and Biomedical SciencesUniversity of MinnesotaSt. PaulMinnesotaUSA
| | - Timothy F. Walseth
- Department of PharmacologyUniversity of MinnesotaMinneapolisMinnesotaUSA
| | - Bilge Guvenc Tuna
- Department of Biophysics, School of MedicineYeditepe UniversityIstanbulTurkey
| | - Eda Uçar
- Department of Medical Biology, School of MedicineYeditepe UniversityIstanbulTurkey
| | - Mathur S. Kannan
- Department of Veterinary and Biomedical SciencesUniversity of MinnesotaSt. PaulMinnesotaUSA
| | - Deepak A. Deshpande
- Department of Veterinary and Biomedical SciencesUniversity of MinnesotaSt. PaulMinnesotaUSA
- Center for Translational Medicine, Jane and Leonard Korman Lung CenterThomas Jefferson UniversityPhiladelphiaPennsylvaniaUSA
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Nieto-Del-Amor F, Ye-Lin Y, Monfort-Ortiz R, Diago-Almela VJ, Modrego-Pardo F, Martinez-de-Juan JL, Hao D, Prats-Boluda G. Automatic semantic segmentation of EHG recordings by deep learning: An approach to a screening tool for use in clinical practice. COMPUTER METHODS AND PROGRAMS IN BIOMEDICINE 2024; 254:108317. [PMID: 38996804 DOI: 10.1016/j.cmpb.2024.108317] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/15/2024] [Revised: 07/03/2024] [Accepted: 07/04/2024] [Indexed: 07/14/2024]
Abstract
BACKGROUND AND OBJECTIVE Preterm delivery is an important factor in the disease burden of the newborn and infants worldwide. Electrohysterography (EHG) has become a promising technique for predicting this condition, thanks to its high degree of sensitivity. Despite the technological progress made in predicting preterm labor, its use in clinical practice is still limited, one of the main barriers being the lack of tools for automatic signal processing without expert supervision, i.e. automatic screening of motion and respiratory artifacts in EHG records. Our main objective was thus to design and validate an automatic system of segmenting and screening the physiological segments of uterine origin in EHG records for robust characterization of uterine myoelectric activity, predicting preterm labor and help to promote the transferability of the EHG technique to clinical practice. METHODS For this, we combined 300 EHG recordings from the TPEHG DS database and 69 EHG recordings from our own database (Ci2B-La Fe) of women with singleton gestations. This dataset was used to train and evaluate U-Net, U-Net++, and U-Net 3+ for semantic segmentation of the physiological and artifacted segments of EHG signals. The model's predictions were then fine-tuned by post-processing. RESULTS U-Net 3+ outperformed the other models, achieving an area under the ROC curve of 91.4 % and an average precision of 96.4 % in detecting physiological activity. Thresholds from 0.6 to 0.8 achieved precision from 93.7 % to 97.4 % and specificity from 81.7 % to 94.5 %, detecting high-quality physiological segments while maintaining a trade-off between recall and specificity. Post-processing improved the model's adaptability by fine-tuning both the physiological and corrupted segments, ensuring accurate artifact detection while maintaining physiological segment integrity in EHG signals. CONCLUSIONS As automatic segmentation proved to be as effective as double-blind manual segmentation in predicting preterm labor, this automatic segmentation tool fills a crucial gap in the existing preterm delivery prediction system workflow by eliminating the need for double-blind segmentation by experts and facilitates the practical clinical use of EHG. This work potentially contributes to the early detection of authentic preterm labor women and will allow clinicians to design individual patient strategies for maternal health surveillance systems and predict adverse pregnancy outcomes.
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Affiliation(s)
- Félix Nieto-Del-Amor
- Centro de Investigación e Innovación en Bioingeniería, Universitat Politècnica de València (Ci2B), Valencia 46022, Spain
| | - Yiyao Ye-Lin
- Centro de Investigación e Innovación en Bioingeniería, Universitat Politècnica de València (Ci2B), Valencia 46022, Spain; BJUT-UPV Joint Research Laboratory in Biomedical Engineering, China
| | | | | | | | - Jose L Martinez-de-Juan
- Centro de Investigación e Innovación en Bioingeniería, Universitat Politècnica de València (Ci2B), Valencia 46022, Spain; BJUT-UPV Joint Research Laboratory in Biomedical Engineering, China
| | - Dongmei Hao
- Faculty of Environment and Life, Beijing University of Technology, Beijing International Science and Technology Cooperation Base for Intelligent Physiological Measurement and Clinical Transformation, Beijing 100124, China; BJUT-UPV Joint Research Laboratory in Biomedical Engineering, China
| | - Gema Prats-Boluda
- Centro de Investigación e Innovación en Bioingeniería, Universitat Politècnica de València (Ci2B), Valencia 46022, Spain; BJUT-UPV Joint Research Laboratory in Biomedical Engineering, China.
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Gárriz A, Morokuma J, Toribio D, Zoukhri D. Role of the adenylate cyclase/cyclic AMP pathway in oxytocin-induced lacrimal gland myoepithelial cells contraction. Exp Eye Res 2023; 233:109526. [PMID: 37290630 PMCID: PMC10527592 DOI: 10.1016/j.exer.2023.109526] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2022] [Revised: 05/03/2023] [Accepted: 06/06/2023] [Indexed: 06/10/2023]
Abstract
The aim of these studies was to investigate the involvement of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP) and its downstream effectors in oxytocin (OXT)-mediated lacrimal gland myoepithelial cell (MEC) contraction. Lacrimal gland MEC were isolated and propagated from alpha-smooth muscle actin (SMA)-GFP mice. RNA and protein samples were prepared to analyze G protein expression by RT-PCR and western blotting; respectively. Changes in intracellular cAMP concentration were measured using a competitive ELISA kit. To increase intracellular cAMP concentration, the following agents were used: forskolin (FKN, a direct activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of the phosphodiesterase that hydrolyzes cAMP), or a cell permeant cAMP analog, dibutyryl (db)-cAMP. In addition, inhibitors and selective agonists were used to investigate the role of cAMP effector molecules, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in OXT-induced MEC contraction. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software. The adenylate cyclase coupling G proteins, Gαs, Gαo, and Gαi, are expressed in lacrimal gland MEC at both the mRNA and protein levels. OXT increased intracellular cAMP in a concentration-dependent manner. FKN, IBMX and db-cAMP significantly stimulated MEC contraction. Preincubation of cells with either Myr-PKI, a specific PKA inhibitor or ESI09, an EPAC inhibitor, resulted in almost complete inhibition of both FKN- as well as OXT-stimulated MEC contraction. Finally, direct activation of PKA or EPAC using selective agonists triggered MEC contraction. We conclude that cAMP agonists modulate lacrimal gland MEC contraction via PKA and EPAC activation which also play a major role in OXT induced MEC contraction.
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Affiliation(s)
- Angela Gárriz
- Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, MA, USA
| | - Junji Morokuma
- Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, MA, USA
| | - Danny Toribio
- Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, MA, USA
| | - Driss Zoukhri
- Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, MA, USA; Department of Ophthalmology, Tufts University School of Medicine, Boston, MA, USA.
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Gárriz A, Aubry S, Wattiaux Q, Bair J, Mariano M, Hatzipetrou G, Bowman M, Morokuma J, Ortiz G, Hamrah P, Dartt DA, Zoukhri D. Role of the Phospholipase C Pathway and Calcium Mobilization in Oxytocin-Induced Contraction of Lacrimal Gland Myoepithelial Cells. Invest Ophthalmol Vis Sci 2021; 62:25. [PMID: 34812841 PMCID: PMC8626846 DOI: 10.1167/iovs.62.14.25] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Purpose We reported that oxytocin (OXT), added to freshly prepared lacrimal gland lobules, induced myoepithelial cell (MEC) contraction. In other systems, OXT activates phospholipase C (PLC) generating Inositol 1,4,5-trisphosphate (IP3) which increases intracellular calcium concentration ([Ca2+]i) causing contraction. The aim of the current study was to investigate the role of this pathway in OXT-induced contraction of MEC. Methods Tear volume was measured using the cotton thread method. Lacrimal gland MEC were isolated and propagated from α-smooth muscle actin (SMA)-green fluorescent protein (GFP) mice, in which MEC express GFP making them easily identifiable. RNA and protein samples were prepared for RT-PCR and Western blotting for G protein expression. Changes in [Ca2+]i were measured in Fura-2 loaded MEC using a ratio imaging system. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software. Results OXT applied either topically to surgically exposed lacrimal glands or delivered subcutaneously resulted in increased tear volume. OXT stimulated lacrimal gland MEC contraction in a dose-dependent manner, with a maximum response at 10-7 M. MEC express the PLC coupling G proteins, Gαq and Gα11, and their activation by OXT resulted in a concentration-dependent increase in [Ca2+]i with a maximum response at 10-6 M. Furthermore, the activation of the IP3 receptor to increase [Ca2+]i is crucial for OXT-induced MEC contraction since blocking the IP3 receptor with 2-APB completely abrogated this response. Conclusions We conclude that OXT uses the PLC/Ca2+ pathway to stimulate MEC contraction and increase lacrimal gland secretion.
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Affiliation(s)
- Angela Gárriz
- Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, Massachusetts, United States
| | - Salome Aubry
- Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, Massachusetts, United States
| | - Quentin Wattiaux
- Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, Massachusetts, United States
| | - Jeffrey Bair
- Schepens Eye Research Institute/Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States
| | - Michael Mariano
- Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, Massachusetts, United States
| | - Georgios Hatzipetrou
- Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, Massachusetts, United States
| | - Maytal Bowman
- Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, Massachusetts, United States
| | - Junji Morokuma
- Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, Massachusetts, United States
| | - Gustavo Ortiz
- Schepens Eye Research Institute/Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.,Department of Ophthalmology, Tufts University School of Medicine, Boston, Massachusetts, United States
| | - Pedram Hamrah
- Department of Ophthalmology, Tufts University School of Medicine, Boston, Massachusetts, United States
| | - Darlene A Dartt
- Schepens Eye Research Institute/Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States
| | - Driss Zoukhri
- Department of Comprehensive Care, Tufts University School of Dental Medicine, Boston, Massachusetts, United States.,Department of Ophthalmology, Tufts University School of Medicine, Boston, Massachusetts, United States
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de Sousa ÍA, de Meneses GMS, Cardoso JVM, Lopes PQ, de Sousa JA, Cavalcanti SMPG, da Silva Cavalcanti PM, Filho FC. Inhibitory effect of Pyr6 (an Orai channel blocker) on agonist-induced contractions in rat uterus. J Obstet Gynaecol Res 2021; 47:4306-4318. [PMID: 34571573 DOI: 10.1111/jog.15034] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2021] [Revised: 08/09/2021] [Accepted: 09/12/2021] [Indexed: 11/30/2022]
Abstract
AIM Both human and rat myometrium express stromal interaction molecule (STIM) and Orai/ transient receptor potential canonical (TRPC) proteins, which are components of plasma membrane Ca2+ store-operated channels. There are reports that these proteins mediate agonist-induced Ca2+ influx in cultured myometrial cells. In this study, we aimed to determine the effects of Pyr6, an Orai channel blocker, on different agonist-induced contractions in isolated segments of rat uterus. MAIN FINDINGS In Ca2+ -free Tyrode's solution, Pyr6 (3 μM) promoted a reduction in both the magnitude and frequency of Ca2+ (1 mM)-induced uterine contractions after the addition of carbachol (CCh, 100 μM), but not after the addition of oxytocin (OT, 150 nM). In Ca2+ (0.18 mM)-Tyrode's solution, Pyr6 completely relaxed uterine contractions induced by both CCh and cloprostenol (300 nM), but not those induced by either KCI (40-80 mM) or OT. The addition of Pyr6 abolished the oscillatory uterine contractions induced by Ca2+ after the addition of cyclopiazonic acid (CPA, 10 μM). When pre-incubated (5 min), Pyr6 reduced the magnitude of both CCh-induced phasic and tonic contractions. The addition of Pyr2 (3 μM), an Orai and TRPC channel blocker, abolished uterine contractions induced by CCh or OT. CONCLUSION Considering Pyr6 as an Orai channel blocker and its inhibitory effect on uterine contractions induced by CCh, CPA, and cloprostenol, we suggest that Orai channels are required for the maintenance of contractions induced by these agonists in rat uterus.
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Affiliation(s)
- Ícaro Araújo de Sousa
- Biophysics and Physiology Department, Health Sciences Center, Federal University of Piauí, Ininga, Teresina, Brazil
| | | | - José Victor Miranda Cardoso
- Biophysics and Physiology Department, Health Sciences Center, Federal University of Piauí, Ininga, Teresina, Brazil
| | - Pablo Queiroz Lopes
- Pharmacological Sciences Department, Health Sciences Center, Federal University of Paraíba, Cidade Universitária - Campus I. Castelo Branco, João Pessoa, Brazil
| | - Joubert Aires de Sousa
- Physiotherapy Department, Health Sciences Center, University of the State of Piauí, Teresina, Brazil
| | | | - Paulo Marques da Silva Cavalcanti
- Pharmacological Sciences Department, Health Sciences Center, Federal University of Paraíba, Cidade Universitária - Campus I. Castelo Branco, João Pessoa, Brazil
| | - Francisco Chagas Filho
- Biophysics and Physiology Department, Health Sciences Center, Federal University of Piauí, Ininga, Teresina, Brazil
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Salami SA, Salahdeen HM, Obafemi AE, Murtala BA. Uterine contractile activity and fetal outcome in rats treated with vitamin C during late gestational variable stress exposure. JOURNAL OF COMPLEMENTARY & INTEGRATIVE MEDICINE 2021; 18:745-751. [PMID: 33964203 DOI: 10.1515/jcim-2020-0276] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Subscribe] [Scholar Register] [Received: 08/07/2020] [Accepted: 11/11/2020] [Indexed: 11/15/2022]
Abstract
OBJECTIVES Stress responses vary throughout pregnancy and impact of late gestational variable stress (LGVS) with vitamin C supplementation on uterine contractility is barely explored. This study investigates fetal weight outcome and in-vitro uterine contractile responses to pharmacological agents during LGVS exposure. METHODS Twenty four nulliparous pregnant rats were divided into four groups of six. During gestation days 10-19, groups 1 & 2 received normal saline and vitamin C (10 mg/kg) respectively. Groups 3 and 4 were exposed to stress (sleep deprivation, predator exposure, immobility, rapid cage changes, noise, and foreign object) with group 4 concurrently supplemented with vitamin C (10 mg/kg). Serum cortisol, oxidative bio-markers, fetal weights and in-vitro contractile responses of excised uterine tissue to acetylcholine (Ach), oxytocin, calcium chloride (CaCl2), potassium chloride (KCl), diclofenac, and magnesium ions were determined. RESULTS Malondialdehyde activity and cortisol were significantly increased in variable stress only exposed group when compared with control and vitamin C supplemented groups. Fetal body weights, superoxide dismutase and catalase activity were significantly reduced in variable stress only exposed group. Significantly impaired contractile responses to Ach, CaCl2 & KCl in variable stress only exposed group were modulated in vitamin C supplemented groups. Impaired contractile response to oxytocin was however not reversed. Relaxation responses to diclofenac and magnesium ions were statistically unaltered across groups. CONCLUSIONS Impaired fetal weights and uterine contractile activity to Ach, CaCl2 and KCl during LGVS was modulated by vitamin C supplementation. Impaired oxytocin contractile activity was however unreversed.
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Affiliation(s)
- Shakiru A Salami
- Department of Physiology, Lagos State University College of Medicine, Ikeja, Lagos State, Nigeria
| | - Hussein M Salahdeen
- Department of Physiology, Lagos State University College of Medicine, Ikeja, Lagos State, Nigeria
| | - Abidemi E Obafemi
- Department of Physiology, Lagos State University College of Medicine, Ikeja, Lagos State, Nigeria
| | - Babatunde A Murtala
- Department of Physiology, Lagos State University College of Medicine, Ikeja, Lagos State, Nigeria
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Bafor EE, Prendergast C, Wray S. Justicia flava leaf extract potently relaxes pregnant human myometrial contractility: a lead plant for drug discovery of new tocolytic drugs. Exp Physiol 2020; 105:2033-2037. [PMID: 33094534 DOI: 10.1113/ep088819] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2020] [Accepted: 10/13/2020] [Indexed: 11/08/2022]
Abstract
NEW FINDINGS What is the central question of this study? Can Justicia flava leaf extract (JF) inhibit human myometrial contractility as was previously shown in mouse myometrium? What is the main finding and its importance? JF abolished human myometrial contractions and therefore presents as a lead plant in drug discovery studies involving drugs for preterm birth. ABSTRACT In the search for new potent therapies for preterm labour, Justicia flava leaf extract (JF) was previously shown to potently inhibit uterine contractility in both pregnant and non-pregnant mouse uterus. This study took the investigation a step further and investigated the activity of JF on pregnant human myometrial contractility. JF potently inhibited human myometrial contractility in a concentration-dependent manner. This pilot study provides evidence that JF should be further investigated as a lead plant in the drug discovery of new uterine relaxants.
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Affiliation(s)
- Enitome E Bafor
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Benin, Benin City, Edo State, Nigeria
| | - Clodagh Prendergast
- Department of Women and Children's Health, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool, UK
| | - Susan Wray
- Department of Women and Children's Health, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool, UK
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Efe OE, Lux KM, Emre Aydingöz S, Tuncer M. Relaxant effect of diallyl sulfide on nonpregnant rat uterus: Involvement of voltage-dependent calcium channels. J Obstet Gynaecol Res 2020; 47:88-95. [PMID: 32830389 DOI: 10.1111/jog.14421] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2020] [Revised: 06/23/2020] [Accepted: 07/25/2020] [Indexed: 11/30/2022]
Abstract
AIM We aimed to determine the effect and mechanism of action of diallyl sulfide (DAS), an active component of sulfur-containing foods such as garlic on rat uterine activity. METHODS Isometric tension changes in longitudinal uterine strips obtained from 20 female Sprague-Dawley rats (250-300 g) in estrus stage of estrous cycle were studied in isolated organ baths containing Krebs-Henseleit solution. RESULTS Diallyl sulfide (10-8 -10-6 M) caused a concentration-dependent relaxation on KCl (60 mM)-induced contractions and inhibited spontaneous peristaltic activity of uterine strips (P < 0.05). None of the following antagonists significantly changed the inhibitory effect of DAS on both KCl-precontracted uterine strips and spontaneous peristaltic activity of the uterus (P > 0.05): nitric oxide synthase inhibitor L-NAME (10-4 M), hydrogen sulfide-producing enzymes cystation β synthase and cystation γ-lyase inhibitors, aminooxyacetic acid (10-4 M) and propargylglycine (10-3 M) and nonselective cyclooxygenase inhibitor indomethacin (10-4 M). However, in calcium-free Krebs solution containing high KCl (30 mM), DAS significantly inhibited CaCl2 (10-5 -10-2 M)-induced uterine contractions in a concentration-dependent manner (P < 0.05). CONCLUSION Diallyl sulfide has a relaxing effect on KCl-contracted rat uterus strips and an inhibitory effect on spontaneous uterine activity, possibly by decreasing the calcium influx into the cytoplasm of uterine smooth muscle cells.
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Affiliation(s)
- Oğuzhan E Efe
- Department of Pharmacology, Baskent University Faculty of Medicine, Ankara, Turkey
| | - K Michael Lux
- Department of Pharmacology, Baskent University Faculty of Medicine, Ankara, Turkey
| | - Selda Emre Aydingöz
- Department of Pharmacology, Baskent University Faculty of Medicine, Ankara, Turkey
| | - Meral Tuncer
- Department of Pharmacology, Baskent University Faculty of Medicine, Ankara, Turkey
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10
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Tsuchiya H, Fujimura S, Fujiwara Y, Koshimizu TA. Critical role of V1a vasopressin receptor in murine parturition†. Biol Reprod 2020; 102:923-934. [PMID: 31836900 DOI: 10.1093/biolre/ioz220] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2019] [Revised: 08/16/2019] [Accepted: 12/09/2019] [Indexed: 11/15/2022] Open
Abstract
The precise mechanisms of the reproductive physiological processes, such as labor initiation, are poorly understood. Oxytocin (OT) is one of the well-known uterotonics and is clinically adopted as a medication to facilitate childbirth. Vasopressin (VP), a posterior pituitary hormone similar to OT, has also been proposed to be involved in the reproductive physiology. In this study, we found that a total deficiency of V1a receptor subtype (V1aR) in mice resulted in a reduced number of pups, delayed labor initiation, and increased post-delivery hemorrhage compared with those in wild-type mice. Among the VP receptor subtypes, only V1aR was found to be expressed in the murine uterus, and its distribution pattern was different from that of the oxytocin receptor (OTR); V1aR expression was mainly distributed in the circular myometrium, whereas OTR was strongly expressed in both the circular and longitudinal myometrium. The maximum contractile force of the circular myometrium, induced by VP or OT, was attenuated in the pregnant uterus of Avpr1a-deficient mice. Contrarily, while OT expression was decreased in the Avpr1a-deficient uterus, OTR expression was significantly increased. These results suggest that V1aR deficiency not only reduces the uterine contractile force but also perturbs the expression of genes responsible for the reproductive physiology. Therefore, V1aR is necessary to exert the maximum contraction of the circular myometrium to deliver pups. This study revealed an important role of V1aR in physiological contraction and term parturition in mice.
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Affiliation(s)
- Hiroyoshi Tsuchiya
- Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University, Shimotsuke, Tochigi, Japan
| | - Shyota Fujimura
- Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University, Shimotsuke, Tochigi, Japan
| | - Yoko Fujiwara
- Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University, Shimotsuke, Tochigi, Japan
| | - Taka-Aki Koshimizu
- Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University, Shimotsuke, Tochigi, Japan
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Bafor EE, Ukpebor F, Omoruyi O, Ochoyama E, Omogiade G, Ekufu J, Edrada-Ebel R. Tocolytic activity assessment of the methanol leaf extract of Justicia flava Vahl (Acanthaceae) on mouse myometrial contractility and preliminary mass spectrometric determination of secondary metabolites. JOURNAL OF ETHNOPHARMACOLOGY 2019; 243:112087. [PMID: 31310827 DOI: 10.1016/j.jep.2019.112087] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/07/2019] [Revised: 06/26/2019] [Accepted: 07/12/2019] [Indexed: 06/10/2023]
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE The leaves of Justicia flava are traditionally used in the South of Nigeria to prevent preterm births. AIM OF THE STUDY In this study, the activity of the methanol leaf extract of J. flava (JF) was investigated on uterine contractility in non-pregnant and pregnant isolated mouse tissues. MATERIAL AND METHODS The effects on spontaneous, oxytocin, and KCl-induced contractions were determined. The effects in calcium-free media were also determined. Possible mechanisms of activity were investigated using receptor and channel modulators. Mass spectrometric analysis was additionally performed on the leaf extract to identify secondary metabolites. RESULTS JF was observed to inhibit spontaneous, oxytocin and high KCl-induced uterine contractility. JF also inhibited contractions in Ca2+-free media. JF was found to exert its inhibitory effect via interaction with inositol triphosphate and ryanodine receptors and also through modulation of K+- channels. Lignans and alkaloids were identified with the lignans being the most abundant in JF. CONCLUSION JF has been shown to potently inhibit uterine contractions in non-pregnant and pregnant isolated mouse uterus. The inhibitory activity of JF has been shown to occur via blockade of extracellular and intracellular calcium entry and these effects may be due to the lignans identified in - JF. JF has therefore been shown in this study to be a lead plant in the discovery of new drugs with uterine inhibitory activity.
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Affiliation(s)
- Enitome E Bafor
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Benin, Benin City, Nigeria.
| | - Faith Ukpebor
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Benin, Benin City, Nigeria.
| | - Osemelomen Omoruyi
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Benin, Benin City, Nigeria
| | - Ejiro Ochoyama
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Benin, Benin City, Nigeria
| | - Glory Omogiade
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Benin, Benin City, Nigeria
| | - Jude Ekufu
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Benin, Benin City, Nigeria
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He L, Lee GT, Zhou H, Buhimschi IA, Buhimschi CS, Weiner CP, Mason CW. Expression, Regulation, and Function of the Calmodulin Accessory Protein PCP4/PEP-19 in Myometrium. Reprod Sci 2019; 26:1650-1660. [PMID: 30744532 DOI: 10.1177/1933719119828072] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
OBJECTIVE Calmodulin (CaM) plays a key role in the orchestration of Ca2+ signaling events, and its regulation is considered an important component of cellular homeostasis. The control of uterine smooth muscle function is largely dependent on the regulation of Ca2+ and CaM signaling. The objective of this study was to investigate the expression, function, and regulation of CaM regulatory proteins in myometrium during pregnancy. STUDY DESIGN Myometrium was obtained from nonpregnant women and 4 groups of pregnant women at the time their primary cesarean delivery: (i) preterm not in labor, (ii) preterm in labor with clinical and/or histological diagnosis of chorioamnionitis, (3) term not in labor; and (4) term in labor. The effect of perinatal inflammation on pcp4/pep-19 expression was evaluated in a mouse model of Ureaplasma parvum-induced chorioamnionitis. Human myometrial cells stably expressing wild-type and mutant forms of PCP4/PEP-19 were used in the evaluation of agonist-induced intracellular Ca2+ mobilization. RESULTS Compared to other CaM regulatory proteins, PCP4/PEP-19 transcripts were more abundant in human myometrium. The expression of PCP4/PEP-19 was lowest in myometrium of women with preterm pregnancy and chorioamnionitis. In the mouse uterus, pcp4/pep-19 expression was lower in late compared to mid-gestation and decreased in mice injected intra-amniotic with Ureaplasma parvum. In myometrial smooth muscle cells, tumor necrosis factor alpha and progesterone decreased and PCP4/PEP-19 promoter activity increased. Finally, the overexpression of PCP4/PEP-19 reduced agonist-induced intracellular Ca2+ levels in myometrial cells. CONCLUSION The decreased expression of PCP4/PEP-19 in myometrium contributes to a loss of quiescence in response to infection-induced inflammation at preterm pregnancy.
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Affiliation(s)
- Lily He
- Department of Obstetrics and Gynecology, Division of Research, University of Kansas School of Medicine, Kansas City, KS, USA
| | - Gene T Lee
- Maternal Fetal Medicine, Department of Obstetrics and Gynecology, University of Kansas School of Medicine, Kansas City, KS, USA.,The Center for Perinatal Research, University of Kansas School of Medicine, Kansas City, KS, USA
| | - Helen Zhou
- Department of Obstetrics and Gynecology, Division of Research, University of Kansas School of Medicine, Kansas City, KS, USA
| | - Irina A Buhimschi
- Department of Obstetrics and Gynecology, The Ohio State University, Columbus, OH, USA.,Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital and Department of Pediatrics, The Ohio State University College of Medicine, Columbus, OH, USA
| | - Catalin S Buhimschi
- Department of Obstetrics and Gynecology, The Ohio State University, Columbus, OH, USA.,Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital and Department of Pediatrics, The Ohio State University College of Medicine, Columbus, OH, USA
| | - Carl P Weiner
- Maternal Fetal Medicine, Department of Obstetrics and Gynecology, University of Kansas School of Medicine, Kansas City, KS, USA
| | - Clifford W Mason
- Department of Obstetrics and Gynecology, Division of Research, University of Kansas School of Medicine, Kansas City, KS, USA.,The Center for Perinatal Research, University of Kansas School of Medicine, Kansas City, KS, USA
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13
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Ge J, Han T, Li X, Shan L, Zhang J, Hong Y, Xia Y, Wang J, Hou M. S-adenosyl methionine regulates calcium channels and inhibits uterine smooth muscle contraction in rats with infectious premature delivery through the transient receptor protein 3/protein kinase Cβ/C-kinase-activated protein phosphatase-1 inhibitor of 17 kDa signaling pathway. Exp Ther Med 2018; 16:103-112. [PMID: 29896230 PMCID: PMC5995051 DOI: 10.3892/etm.2018.6164] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2017] [Accepted: 03/09/2018] [Indexed: 12/28/2022] Open
Abstract
The aim of the present study was to investigate the effects of S-adenosyl methionine (SAMe) on infectious premature inflammatory factors and uterine contraction, and to further explore its mechanism of action via the transient receptor protein 3 (TRPC3)/protein kinase Cβ (PKCβ)/C-kinase-activated protein phosphatase-1 inhibitor of 17 kDa (CPI-17) signaling pathway, following intervention by a TRPC3 inhibitor. A rat model of premature delivery induced by lipopolysaccharide (LPS) was established. Following treatment with SAMe and inhibiting TRPC3 expression, rat serum and uterus were isolated. Hematoxylin and eosin staining was used to observe the histopathological changes in the uterus. Uterine muscle strips in vitro were selected to measure the changes in muscle tension. ELISA was utilized to measure the changes in serum inflammatory factor and oxidative stress indexes. Immunohistochemistry, western blot assay and reverse transcription-quantitative polymerase chain reaction were applied to detect calcium channel protein expression in the uterus. Western blot analysis was employed to measure the expression of TRPC3/PKCβ/CPI-17 signaling pathway-related proteins. TRPC3 was highly expressed in the uterus of rat models of premature delivery induced by LPS. Following treatment with SAMe, inflammatory cell infiltration markedly reduced in the uterus and the tension of in vitro uterine muscle strips significantly decreased. SAMe treatment suppressed inflammatory reaction and oxidative stress, and diminished L-type and T-type calcium channel protein expression. TRPC3/PKCβ/CPI-17 signaling pathway-related protein expression was also reduced. When TRPC3 expression was suppressed, the effects of SAMe against inflammation and oxidative stress were diminished. TRPC3/PKCβ/CPI-17 signaling pathway-related protein expression significantly increased. SAMe was able to reduce inflammatory reaction and oxidative stress in the uterus of rat model of infectious premature delivery induced by LPS, prolong delivery time, reduce the mortality rate of offspring rats, and serve a therapeutic role. This effect is likely achieved via the regulation of uterine contractions and childbirth through the TRPC3/PKCβ/CPI-17 signaling pathway.
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Affiliation(s)
- Jing Ge
- Department of Maternity, General Hospital of Shenyang Military Area Command, Shenyang, Liaoning 110016, P.R. China
| | - Tao Han
- Department of Oncology, General Hospital of Shenyang Military Area Command, Shenyang, Liaoning 110016, P.R. China
| | - Xiaoqiu Li
- Department of Neurology, General Hospital of Shenyang Military Area Command, Shenyang, Liaoning 110016, P.R. China
| | - Lili Shan
- Department of Maternity, General Hospital of Shenyang Military Area Command, Shenyang, Liaoning 110016, P.R. China
| | - Jinhuan Zhang
- Department of Maternity, General Hospital of Shenyang Military Area Command, Shenyang, Liaoning 110016, P.R. China
| | - Yan Hong
- Department of Maternity, General Hospital of Shenyang Military Area Command, Shenyang, Liaoning 110016, P.R. China
| | - Yanqiu Xia
- Department of Maternity, General Hospital of Shenyang Military Area Command, Shenyang, Liaoning 110016, P.R. China
| | - Jun Wang
- Department of Maternity, General Hospital of Shenyang Military Area Command, Shenyang, Liaoning 110016, P.R. China
| | - Mingxiao Hou
- Department of Cardiothoracic Surgery, General Hospital of Shenyang Military Area Command, Shenyang, Liaoning 110016, P.R. China
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Idrizaj E, Garella R, Francini F, Squecco R, Baccari MC. Relaxin influences ileal muscular activity through a dual signaling pathway in mice. World J Gastroenterol 2018; 24:882-893. [PMID: 29491682 PMCID: PMC5829152 DOI: 10.3748/wjg.v24.i8.882] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/19/2017] [Revised: 11/14/2017] [Accepted: 11/27/2017] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the signaling pathways involved in the relaxin (RLX) effects on ileal preparations from mice through mechanical and electrophysiological experiments.
METHODS For mechanical experiments, ileal preparations from female mice were mounted in organ baths containing Krebs-Henseleit solution. The mechanical activity was recorded via force-displacement transducers, which were coupled to a polygraph for continuous recording of isometric tension. Electrophysiological measurements were performed in current- and voltage-clamp conditions by a microelectrode inserted in a single smooth muscle cell (SMC) of the ileal longitudinal layer. Both the membrane passive properties and inward voltage-dependent L-type Ca2+ currents were recorded using suitable solutions and voltage stimulation protocols.
RESULTS Mechanical experiments showed that RLX induced a decay of the basal tension and a reduction in amplitude of the spontaneous contractions. The effects of RLX were partially reduced by 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ) or 9-cyclopentyladenine mesylate (9CPA), inhibitors of guanylate cyclase (GC) and adenylate cyclase (AC), respectively, and were abolished in the concomitant presence of both drugs. Electrophysiological experiments demonstrated that RLX directly influenced the biophysical properties of ileal SMCs, decreasing the membrane conductance, hyperpolarizing the resting membrane potential, reducing the L-type calcium current amplitude and affecting its kinetics. The voltage dependence of the current activation and inactivation time constant was significantly speeded by RLX. Each electrophysiological effect of RLX was reduced by ODQ or 9CPA, and abolished in the concomitant presence of both drugs as observed in mechanical experiments.
CONCLUSION Our new findings demonstrate that RLX influences ileal muscle through a dual mechanism involving both GC and AC.
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Affiliation(s)
- Eglantina Idrizaj
- Department of Experimental and Clinical Medicine, Section of Physiological Sciences, University of Florence, Florence 50134, Italy
| | - Rachele Garella
- Department of Experimental and Clinical Medicine, Section of Physiological Sciences, University of Florence, Florence 50134, Italy
| | - Fabio Francini
- Department of Experimental and Clinical Medicine, Section of Physiological Sciences, University of Florence, Florence 50134, Italy
| | - Roberta Squecco
- Department of Experimental and Clinical Medicine, Section of Physiological Sciences, University of Florence, Florence 50134, Italy
| | - Maria Caterina Baccari
- Department of Experimental and Clinical Medicine, Section of Physiological Sciences, University of Florence, Florence 50134, Italy
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15
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Crowder CM, Meyer E, Fan TY, Weis VM. Impacts of temperature and lunar day on gene expression profiles during a monthly reproductive cycle in the brooding coral Pocillopora damicornis. Mol Ecol 2017; 26:3913-3925. [PMID: 28467676 DOI: 10.1111/mec.14162] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2016] [Revised: 04/14/2017] [Accepted: 04/17/2017] [Indexed: 12/15/2022]
Abstract
Reproductive timing in brooding corals has been correlated to temperature and lunar irradiance, but the mechanisms by which corals transduce these environmental variables into molecular signals are unknown. To gain insight into these processes, global gene expression profiles in the coral Pocillopora damicornis were examined (via RNA-Seq) across lunar phases and between temperature treatments, during a monthly planulation cycle. The interaction of temperature and lunar day together had the largest influence on gene expression. Mean timing of planulation, which occurred at lunar days 7.4 and 12.5 for 28- and 23°C-treated corals, respectively, was associated with an upregulation of transcripts in individual temperature treatments. Expression profiles of planulation-associated genes were compared between temperature treatments, revealing that elevated temperatures disrupted expression profiles associated with planulation. Gene functions inferred from homologous matches to online databases suggest complex neuropeptide signalling, with calcium as a central mediator, acting through tyrosine kinase and G protein-coupled receptor pathways. This work contributes to our understanding of coral reproductive physiology and the impacts of environmental variables on coral reproductive pathways.
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Affiliation(s)
- Camerron M Crowder
- Department of Integrative Biology, Oregon State University, Corvallis, OR, USA
| | - Eli Meyer
- Department of Integrative Biology, Oregon State University, Corvallis, OR, USA
| | - Tung-Yung Fan
- Institute of Marine Biology, National Dong Hwa University, Pingtung, Taiwan, R.O.C
| | - Virginia M Weis
- Department of Integrative Biology, Oregon State University, Corvallis, OR, USA
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16
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Functional involvement of protein kinase C, Rho-kinase and TRPC3 decreases while PLC increases with advancement of pregnancy in mediating oxytocin-induced myometrial contractions in water buffaloes ( Bubalus bubalis ). Theriogenology 2017; 92:176-189. [DOI: 10.1016/j.theriogenology.2016.12.008] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2016] [Revised: 12/06/2016] [Accepted: 12/06/2016] [Indexed: 11/20/2022]
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Molecular Basis of Oxytocin Receptor Signalling in the Brain: What We Know and What We Need to Know. Curr Top Behav Neurosci 2017; 35:3-29. [PMID: 28812263 DOI: 10.1007/7854_2017_6] [Citation(s) in RCA: 92] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Oxytocin (OT), a hypothalamic neuropeptide involved in regulating the social behaviour of all vertebrates, has been proposed as a treatment for a number of neuropsychiatric disorders characterised by deficits in the social domain. Over the last few decades, advances focused on understanding the social effects of OT and its role in physiological conditions and brain diseases, but much less has been done to clarify the molecular cascade of events involved in mediating such effects and in particular the cellular and molecular pharmacology of OT and its target receptor (OTR) in neuronal and glial cells.The entity and persistence of OT activity in the brain is closely related to the expression and regulation of the OTR expressed on the cell surface, which transmits the signal intracellularly and permits OT to affect cell function. Understanding the various signalling mechanisms mediating OTR-induced cell responses is crucial to determine the different responses in different cells and brain regions, and the success of OT and OT-derived analogues in the treatment of neurodevelopmental and psychiatric diseases depends on how well we can control such responses. In this review, we will consider the most important aspects of OT/OTR signalling by focusing on the molecular events involved in OT binding and coupling, on the main signalling pathways activated by the OTR in neuronal cells and on intracellular and plasma membrane OTR trafficking, all of which contribute to the quantitative and qualitative features of OT responses in the brain.
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18
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Ayres AW, Carr DW, McConnell DS, Lieberman RW, Smith GD. Expression and Intracellular Localization of Protein Phosphatases 2A and 2B, Protein Kinase A, A-Kinase Anchoring Protein (AKAP79), and Binding of the Regulatory (RII) Subunit of Protein Kinase A to AKAP79 in Human Myometrium. ACTA ACUST UNITED AC 2016. [DOI: 10.1016/s1071-55760300136-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Affiliation(s)
| | | | | | - Richard W. Lieberman
- Oregon Health & Sciences University, Veterans Affairs Medical Center, Portland, Oregon; Reproductive Science Program; Departments of Obstetrics and Gynecology, Pathology, and Physiology and Urology, University of Michigan, Ann Arbor, Michigan
| | - Gary D. Smith
- Oregon Health & Sciences University, Veterans Affairs Medical Center, Portland, Oregon; Reproductive Science Program; Departments of Obstetrics and Gynecology, Pathology, and Physiology and Urology, University of Michigan, Ann Arbor, Michigan; University of Michigan, 6428 Med Sci 1, 1301 E. Catherine Street, Ann Arbor, MI 48109-0617
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Ku CY, Babich L, Word RA, Zhong M, Ulloa A, Monga M, Sanborn BM. Expression of Transient Receptor Channel Proteins in Human Fundal Myometrium in Pregnancy. ACTA ACUST UNITED AC 2016; 13:217-25. [PMID: 16527499 DOI: 10.1016/j.jsgi.2005.12.007] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2005] [Indexed: 10/24/2022]
Abstract
OBJECTIVE Cation channels comprised of transient receptor potential (TrpC) proteins may play a role in signal-regulated calcium entry and calcium homeostasis in myometrium. The objective of this study was to determine the relative abundance of specific TrpC mRNAs expressed in human myometrium and determine if TrpC mRNA and protein concentrations differ in fundal myometrium before and after the onset of labor. METHODS A quantitative real-time polymerase chain reaction (Q-RT-PCR) procedure was developed for determining the concentration of TrpC mRNA expression in immortalized and primary human myometrial cells and myometrial fundus tissues from patients before and after the onset of labor. The corresponding TrpC proteins were detected by Western blot analysis and immunohistochemistry. RESULTS hTrpC1, 3, 4, 5, 6, and 7 mRNAs were expressed in two lines of immortalized human myometrial cells and in primary human myocytes. In all of these cells, hTrpC1 and hTrpC4 mRNAs were the most abundant, followed by hTrpC6. A similar distribution was observed in fundal myometrium samples from patients before and after the onset of labor. hTrpC4 mRNA was significantly lower after the onset of labor; there were no significant changes in the concentrations of other TrpC mRNAs. Immunohistochemistry identified hTrpC1, 3, 4, and 6 proteins in myometrial smooth muscle cells. Western blot analysis of myometrial membranes demonstrated no statistically significant changes in hTrpC1, 3, 4, and 6 proteins between samples collected before and after the onset of labor. CONCLUSIONS We have demonstrated that hTrpC1 and hTrpC4 are the most abundant TrpC mRNAs in human myometrium, with TrpC6 being the next most abundant. There was no increase in TrpC mRNA or protein in fundal myometrium with the onset of labor. Nonetheless, these isoforms may play significant roles in signal regulated calcium entry in human myometrium.
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Affiliation(s)
- Chun-Ying Ku
- Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523, USA.
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20
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Ku CY, Word RA, Sanborn BM. Differential Expression of Protein Kinase A, AKAP 79, and PP2B in Pregnant Human Myometrial Membranes Prior to and During Labor. ACTA ACUST UNITED AC 2016; 12:421-7. [PMID: 15914039 DOI: 10.1016/j.jsgi.2005.04.002] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2004] [Indexed: 11/29/2022]
Abstract
OBJECTIVE We have previously shown that the association of protein kinase A (PKA) with purified myometrial plasma membrane declined at the end of pregnancy in the rat. This study was designed to determine if a similar decline in PKA occurred in pregnant human myometrium. METHODS Myometrial plasma membranes were isolated from lower uterine segment tissues from not-in-labor (NIL) and in-labor (IL) patients undergoing cesarean delivery. Membrane proteins were subjected to Western blot analysis to detect PKA-catalytic (PKA-cat) and PKA-regulatory (PKA-reg) subunits, the PKA binding protein A-kinase anchoring protein 79 (AKAP79), protein phosphatase 2B (PP2B), and Galphaq, a guanosine triphosphate (GTP)-binding protein. Protein levels were expressed relative to caveolin-1, which was invariant between the two groups. RESULTS The amount of PKA-cat, PKA-reg, AKAP79, and PP2B in plasma membranes from myometrium of women in early labor decreased significantly compared with that in tissues from women not in labor. In contrast, Galphaq did not change. All proteins were localized to myometrial smooth muscle cells by immunohistochemistry. CONCLUSIONS Expression of PKA, PP2B, and AKAP79 is consistent with the presence of a functional AKAP-mediated signaling complex in pregnant human myometrial membranes. A small but significant decrease in PKA, AKAP79, and PP2B in myometrial tissues from women in labor may contribute to a decrease in negative feedback on and enhancement of contractant signals at term.
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Affiliation(s)
- Chun-Ying Ku
- Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523, USA.
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Talati C, Ramachandran N, Carvalho JCA, Kingdom J, Balki M. The Effect of Extracellular Calcium on Oxytocin-Induced Contractility in Naive and Oxytocin-Pretreated Human Myometrium In Vitro. Anesth Analg 2016; 122:1498-507. [DOI: 10.1213/ane.0000000000001264] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
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Elabd S, Sabry I. Two Birds with One Stone: Possible Dual-Role of Oxytocin in the Treatment of Diabetes and Osteoporosis. Front Endocrinol (Lausanne) 2015; 6:121. [PMID: 26322016 PMCID: PMC4530313 DOI: 10.3389/fendo.2015.00121] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/24/2015] [Accepted: 07/23/2015] [Indexed: 12/15/2022] Open
Abstract
Oxytocin (OT), a hormone most commonly associated with labor and lactation, may have a wide variety of physiological and pathological functions, which makes OT and its receptor potential targets for drug therapy. In this review, we highlight the newly discovered metabolic role of OT in diabetes and its complication, such as diabetic osteopathy. OT may have positive metabolic effects; this is based on the change in glucose metabolism, lipid profile, and insulin sensitivity. It may modify glucose uptake and insulin sensitivity both through direct and indirect effects. It may also cause regenerative changes in diabetic pancreatic islet cells. Moreover, it has an anabolic effect on the bone biology. So, the activation of the OT receptor pathway by infusion of OT, OT analogs, or OT agonists may represent a promising approach for the treatment of diabetes and some of its complications, including diabetic osteopathy.
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Affiliation(s)
- Seham Elabd
- Human Physiology Department, Medical Research Institute, Alexandria University, Alexandria, Egypt
- *Correspondence: Seham Elabd, Department of Human Physiology, Medical Research Institute, Alexandria University, 165, Horreya Avenue, Hadara, Alexandria, Egypt,
| | - Ismail Sabry
- Zoology Department, Faculty of Science, Alexandria University, Alexandria, Egypt
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Jorge S, Chang S, Barzilai JJ, Leppert P, Segars JH. Mechanical signaling in reproductive tissues: mechanisms and importance. Reprod Sci 2014; 21:1093-107. [PMID: 25001021 DOI: 10.1177/1933719114542023] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
The organs of the female reproductive system are among the most dynamic tissues in the human body, undergoing repeated cycles of growth and involution from puberty through menopause. To achieve such impressive plasticity, reproductive tissues must respond not only to soluble signals (hormones, growth factors, and cytokines) but also to physical cues (mechanical forces and osmotic stress) as well. Here, we review the mechanisms underlying the process of mechanotransduction-how signals are conveyed from the extracellular matrix that surrounds the cells of reproductive tissues to the downstream molecules and signaling pathways that coordinate the cellular adaptive response to external forces. Our objective was to examine how mechanical forces contribute significantly to physiological functions and pathogenesis in reproductive tissues. We highlight how widespread diseases of the reproductive tract, from preterm labor to tumors of the uterus and breast, result from an impairment in mechanical signaling.
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Affiliation(s)
- Soledad Jorge
- CRTP Scholars, NIH, Bethesda, MD, USA Yale University School of Medicine, New Haven, CT, USA
| | - Sydney Chang
- CRTP Scholars, NIH, Bethesda, MD, USA Duke University School of Medicine, Durham, NC, USA
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Arrowsmith S, Wray S. Oxytocin: its mechanism of action and receptor signalling in the myometrium. J Neuroendocrinol 2014; 26:356-69. [PMID: 24888645 DOI: 10.1111/jne.12154] [Citation(s) in RCA: 174] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/04/2014] [Revised: 03/14/2014] [Accepted: 03/28/2014] [Indexed: 12/17/2022]
Abstract
Oxytocin is a nonapeptide hormone that has a central role in the regulation of parturition and lactation. In this review, we address oxytocin receptor (OTR) signalling and its role in the myometrium during pregnancy and in labour. The OTR belongs to the rhodopsin-type (Class 1) of the G-protein coupled receptor superfamily and is regulated by changes in receptor expression, receptor desensitisation and local changes in oxytocin concentration. Receptor activation triggers a number of signalling events to stimulate contraction, primarily by elevating intracellular calcium (Ca(2+) ). This includes inositol-tris-phosphate-mediated store calcium release, store-operated Ca(2+) entry and voltage-operated Ca(2+) entry. We discuss each mechanism in turn and also discuss Ca(2+) -independent mechanisms such as Ca(2+) sensitisation. Because oxytocin induces contraction in the myometrium, both the activation and the inhibition of its receptor have long been targets in the management of dysfunctional and preterm labours, respectively. We discuss current and novel OTR agonists and antagonists and their use and potential benefit in obstetric practice. In this regard, we highlight three clinical scenarios: dysfunctional labour, postpartum haemorrhage and preterm birth.
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Affiliation(s)
- S Arrowsmith
- Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
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Salleh N, Ahmad VN. In-VITRo effect of Ficus deltoidea on the contraction of isolated rat's uteri is mediated via multiple receptors binding and is dependent on extracellular calcium. BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE 2013; 13:359. [PMID: 24330515 PMCID: PMC3866927 DOI: 10.1186/1472-6882-13-359] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/14/2013] [Accepted: 12/05/2013] [Indexed: 11/10/2022]
Abstract
BACKGROUND Ficus deltoidea, is a perennial herb that is used to assist labor, firm the uterus post-delivery and to prevent postpartum bleeding. In view of its claimed uterotonic action, the mechanisms underlying plant's effect on uterine contraction were investigated. METHODS Adult female SD rats were injected with 2 mg/kg 17β-oestradiol (E2) to synchronize their oestrous cycle. A day after injection, uteri were removed for in-vitro contraction studies. The dose dependent effect of Ficus deltoidea aqeous extract (FDA) on the tension produced by the isolated rat's uteri was determined. The effects of atropine (2×10(-8) M), atosiban (0.5 IU), THG113.31 (10 μM), oxodipine (0.25 mM), EDTA (1 mM), 2-amino-ethoxy-diphenylborate (2-APB) (40 mM) and thapsigargin (1 mM) on the maximum force of contraction (Emax) achieved following 2 mg/ml FDA administration were also investigated. RESULTS FDA induced in-vitro contraction of the isolated rat's uteri in a dose-dependent manner. Administration of atropine, atosiban and THG113.31 reduced the Emax with atosiban having the greatest effect. The Emax was also reduced following oxodipine and EDTA administration. There was no significant change observed following 2-APB administration. Thapsigargin, however, augmented Emax. CONCLUSIONS FDA-induced contraction of the isolated rat's uteri is mediated via multiple uterotonin receptors (muscarinic, oxytocin and prostaglandin F2α) and was dependent on the extracellular Ca2+. Contraction, however, was not dependent on the Ca2+ release from the internal stores. This in-vitro study provides the first scientific evidence on the claimed effect of Ficus Deltoidea on uterine contraction.
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Affiliation(s)
- Naguib Salleh
- Department of Physiology, Faculty of Medicine, University of Malaya, Lembah Pantai, Kuala Lumpur 50603, Malaysia
| | - Vivi Noryati Ahmad
- Department of Physiology, Faculty of Medicine, University of Malaya, Lembah Pantai, Kuala Lumpur 50603, Malaysia
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You X, Gao L, Liu J, Xu C, Liu C, Li Y, Hui N, Gu H, Ni X. CRH activation of different signaling pathways results in differential calcium signaling in human pregnant myometrium before and during labor. J Clin Endocrinol Metab 2012; 97:E1851-61. [PMID: 22869609 DOI: 10.1210/jc.2011-3383] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
CONTEXT Our previous study has demonstrated that CRH has differential effects on human uterine contractility before and after onset of labor. Intracellular Ca2+ concentration ([Ca2+]i) mobilization plays an important role in the control of uterine contraction. OBJECTIVE Our objective was to investigate the effects of CRH on [Ca2+]i homeostasis in laboring and nonlaboring myometrial cells and determine subsequent signaling involved in [Ca2+]i regulation by CRH. DESIGN The myometrial tissues were obtained from pregnant women who were undergoing or not undergoing labor at term. [Ca2+]i was determined by Ca2+ imaging system using the fluorescent dye fura-2-acetoxymethyl ester. Western blot analysis, ELISA, and RIA were used to determine the signaling pathways induced by CRH. RESULTS CRH induced Ca2+ transient in laboring cells, which was blocked by CRH receptor type 1 (CRHR1) antagonist antalarmin. CRHR1 knockdown impaired this effect of CRH. CRH activated Gi protein, decreased cAMP production, and induced phosphorylated phospholipase C-β3 and inositol-1,4,5-triphosphate production. Phospholipase C and inositol-1,4,5-triphosphate receptor inhibitors blocked the CRH-induced Ca2+ transient in laboring cells. CRH did not induce whereas antalarmin induced the Ca2+ transient in nonlaboring cells. Knockdown of CRHR1 impaired the effect of antalarmin. CRH acted on CRHR1 to activate Gs in nonlaboring cells. Forskolin blocked antalarmin-induced Ca2+ transient. CONCLUSIONS CRH acts on CRHR1 to activate different signaling pathways before and after onset of labor, thereby resulting in differential calcium signaling in response to CRH. The signaling pathways of CRHR1 might serve as a target for the development of new therapeutic strategies for preterm birth.
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Affiliation(s)
- Xingji You
- Department of Physiology, Second Military Medical University, and Department of Obsetrics and Gynecology, Changhai Hospital, 800 Xiangyin Road, Shanghai 200433, PR China
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Busnelli M, Saulière A, Manning M, Bouvier M, Galés C, Chini B. Functional selective oxytocin-derived agonists discriminate between individual G protein family subtypes. J Biol Chem 2011; 287:3617-29. [PMID: 22069312 DOI: 10.1074/jbc.m111.277178] [Citation(s) in RCA: 140] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
We used a bioluminescence resonance energy transfer biosensor to screen for functional selective ligands of the human oxytocin (OT) receptor. We demonstrated that OT promoted the direct engagement and activation of G(q) and all the G(i/o) subtypes at the OT receptor. Other peptidic analogues, chosen because of specific substitutions in key OT structural/functional residues, all showed biased activation of G protein subtypes. No ligand, except OT, activated G(oA) or G(oB), and, with only one exception, all of the peptides that activated G(q) also activated G(i2) and G(i3) but not G(i1), G(oA), or G(oB), indicating a strong bias toward these subunits. Two peptides (DNalOVT and atosiban) activated only G(i1) or G(i3), failed to recruit β-arrestins, and did not induce receptor internalization, providing the first clear examples of ligands differentiating individual G(i/o) family members. Both analogs inhibited cell proliferation, showing that a single G(i) subtype-mediated pathway is sufficient to prompt this physiological response. These analogs represent unique tools for examining the contribution of G(i/o) members in complex biological responses and open the way to the development of drugs with peculiar selectivity profiles. This is of particular relevance because OT has been shown to improve symptoms in neurodevelopmental and psychiatric disorders characterized by abnormal social behaviors, such as autism. Functional selective ligands, activating a specific G protein signaling pathway, may possess a higher efficacy and specificity on OT-based therapeutics.
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Affiliation(s)
- Marta Busnelli
- Consiglio Nazionale delle Ricerche Institute of Neuroscience, Via Vanvitelli 32, Milan 20143, Italy
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Lecarpentier ER, Claes VA, Timbely O, Arsalane A, Wipff JA, Hébert JLM, Michel FY, Lecarpentier YC. Mechanics and energetics of myosin molecular motors from nonpregnant human myometrium. J Appl Physiol (1985) 2011; 111:1096-105. [PMID: 21778420 DOI: 10.1152/japplphysiol.00414.2011] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Mechanical properties of spontaneously contracting isolated nonpregnant human myometrium (NPHM) were investigated throughout the whole continuum of load from zero load up to isometry. This made it possible to assess the three-dimensional tension-velocity-length (T-V-L) relationship characterizing the level of contractility and to determine crossbridge (CB) kinetics of myosin molecular motors. Seventy-seven muscle strips were obtained from hysterectomy in 42 nonpregnant patients. Contraction and relaxation parameters were measured during spontaneous mechanical activity. The isotonic tension-peak velocity (T-V) relationship was hyperbolic in 30 cases and nonhyperbolic in 47 cases. When the T-V relationship was hyperbolic, the Huxley formalism could be used to calculate CB kinetics and CB unitary force. At the whole muscle level and for a given isotonic load level, part of the V-L phase plane showed a common pathway, so that a given instantaneous length corresponded to only one possible instantaneous velocity, independent of time and initial length. At the molecular level, rate constants for CB attachment and detachment were dramatically low, ∼100 times lower than those of striated muscles, and ∼5 to 10 times lower than those of other smooth muscles. The CB unitary force was ∼1.4 ± 0.1 pN. NPHM shared similar basic contractile properties with striated muscles, reflected in the three-dimensional T-V-L relationship characterizing the contractile level. Low CB attachment and detachment rate constants made it possible to generate normal CB unitary force and normal muscle tension in NPHM, even though it contracted extremely slowly compared with other muscles.
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Brighton PJ, Rana S, Challiss RJ, Konje JC, Willets JM. Arrestins differentially regulate histamine- and oxytocin-evoked phospholipase C and mitogen-activated protein kinase signalling in myometrial cells. Br J Pharmacol 2011; 162:1603-17. [PMID: 21175586 DOI: 10.1111/j.1476-5381.2010.01173.x] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022] Open
Abstract
BACKGROUND AND PURPOSE The uterotonins oxytocin and histamine, mediate contractile signals through specific G protein-coupled receptors, a process which is tightly controlled during gestation to prevent preterm labour. We previously identified G protein-coupled receptor kinase (GRK)2 and GRK6 as respective cardinal negative regulators of histamine H(1) and oxytocin receptor signalling. GRK-mediated phosphorylation promotes arrestin recruitment, not only desensitizing receptors but activating an increasing number of diverse signalling pathways. Here we investigate potential roles that arrestins play in the regulation of myometrial oxytocin/histamine H(1) receptor signalling. EXPERIMENTAL APPROACH Endogenous arrestins2 and 3 were specifically depleted using RNA-interference in a human myometrial cell line and the consequences of this for G protein-coupled receptor-mediated signalling were assessed using Ca(2+) /inositol 1,4,5-trisphophate imaging and standard mitogen-activated protein kinase (MAPK) assays. KEY RESULTS Depletion of arrestin3, but not arrestin2 enhanced and prolonged H(1) receptor-stimulated Ca(2+) responses, whilst depletion of either arrestin increased oxytocin receptor responses. Arrestin3 depletion decreased H(1) receptor desensitization, whilst removal of either arrestin isoform was equally effective in preventing oxytocin receptor desensitization. Following arrestin3 depletion oxytocin-induced phospho-extracellular signal-regulated kinase1/2 signals were diminished and histamine-stimulated signals virtually absent, whereas depletion of arrestin2 augmented extracellular signal-regulated kinase1/2 responses to each agonist. Conversely, depletion of arrestin3 enhanced p38 signals to each agonist, whilst arrestin2 suppression increased oxytocin-, but not histamine-induced p38 MAPK responses. CONCLUSIONS AND IMPLICATIONS Arrestin proteins are key regulators of H(1) and oxytocin receptor desensitization, and play integral roles mediating uterotonin-stimulated MAPK-signalling. These data provide insights into the in situ regulation of these receptor subtypes and may inform pathophysiological functioning in preterm labour.
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Affiliation(s)
- Paul J Brighton
- Endocannabinoid Research Group, Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester Royal Infirmary, Leicester, UK
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Lowe NK, Corwin EJ. Proposed biological linkages between obesity, stress, and inefficient uterine contractility during labor in humans. Med Hypotheses 2011; 76:755-60. [PMID: 21382668 DOI: 10.1016/j.mehy.2011.02.018] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2010] [Revised: 02/07/2011] [Accepted: 02/07/2011] [Indexed: 10/18/2022]
Abstract
Cesarean delivery has reached epidemic proportions in contemporary western healthcare. For otherwise healthy first-time (nulliparous) women at term gestation with a single fetus in a head down position, the most common clinical diagnosis prompting cesarean delivery is dystocia, including clinical terms such as uterine dysfunction, failure to progress, arrest of dilation and/or arrest of descent of the fetal head. In 2006, the cesarean rate for this lowest risk population of childbearing women was 26% in the United States despite the goal of Healthy People 2010 to reduce this rate to 15% from a baseline of 18% in 1998. While multiple lines of evidence suggest that the nulliparous uterus is particularly vulnerable to a diagnosis of uterine dysfunction during labor, pathophysiologic explanations for this dysfunction have not been well described. The acute stress response has been implicated as one factor in this dysfunction for many years, while more recently the growing epidemic of adiposity among women of childbearing age has been suggested as an additional pathway by which myometrial cell function may be disrupted. Using both clinical and in vitro evidence, we hypothesize a combined model in which pathways of acute stress and changes associated with maternal adiposity, particularly exaggerated levels of cholesterol and leptin, may independently and synergistically impair the contractile apparatus of the myocyte leading to the clinical diagnosis of uterine dystocia and subsequent cesarean delivery.
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Affiliation(s)
- Nancy K Lowe
- Division of Women, Children, and Family Health, University of Colorado Denver, 13120 E. 19th Avenue, Mail Stop 288-18, Aurora, CO 80045, USA.
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Dörr J, Fecher-Trost C. TRP channels in female reproductive organs and placenta. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2011; 704:909-28. [PMID: 21290333 DOI: 10.1007/978-94-007-0265-3_47] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
TRP channel proteins are widely expressed in female reproductive organs. Based on studies detecting TRP transcripts and proteins in different parts of the female reproductive organs and placenta they are supposed to be involved in the transport of the oocyte or the blastocyte through the oviduct, implantation of the blastocyte, development of the placenta and transport processes across the feto-maternal barrier. Furthermore uterus contractility and physiological processes during labour and in mammary glands seem to be dependant on TRP channel expression.
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Affiliation(s)
- Janka Dörr
- Proteinfunktion Proteomics, Fachbereich Biologie, TU Kaiserslautern, D-67663 Kaiserslautern, Germany.
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Ku CY, Murtazina DA, Kim YS, Garfield RE, Sanborn BM. Changes in rat myometrial plasma membrane protein kinase A are confined to parturition. Reprod Sci 2010; 17:696-704. [PMID: 20457802 DOI: 10.1177/1933719110368869] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
We have previously shown that pregnant rat myometrial plasma membrane-associated cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) decreases prior to delivery, coincident with a decline in the inhibitory effect of cAMP on contractant-stimulated parameters. We now find that rat myometrial membrane-associated PKA concentrations in early to mid-pregnancy are equivalent to those in cycling rats. Following the decline associated with parturition, membrane PKA recovers within 1 to 2 days postpartum. Treatment with the antiprogestin onapristone caused a decrease in myometrial membrane PKA catalytic and regulatory subunits compared to untreated controls by 12 hours. This coincided temporally with recently reported increases in electrical and contractile activity. In unilaterally pregnant rats, the decline in plasma membrane PKA was observed in both nonpregnant and pregnant horns but was more rapid in the pregnant horns. These data indicate that the myometrial plasma membrane PKA pattern before and during most of pregnancy is not consistent with progesterone exerting a primary influence on PKA membrane localization. Rather, the fall in membrane PKA associated with parturition may contribute to or be influenced by the increased contractile and electrical activity of labor that is a consequence of the loss of progesterone influence and is not absolutely dependent on the presence of fetuses.
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Affiliation(s)
- Chun-Ying Ku
- Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA
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Chung D, Kim YS, Phillips JN, Ulloa A, Ku CY, Galan HL, Sanborn BM. Attenuation of canonical transient receptor potential-like channel 6 expression specifically reduces the diacylglycerol-mediated increase in intracellular calcium in human myometrial cells. Endocrinology 2010; 151:406-16. [PMID: 19940041 PMCID: PMC2803151 DOI: 10.1210/en.2009-0085] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
An increase in intracellular Ca(2+) ([Ca(2+)](i)) as a result of release of Ca(2+) from intracellular stores or influx of extracellular Ca(2+) contributes to the regulation of smooth muscle contractile activity. Human uterine smooth muscle cells exhibit receptor-, store-, and diacylglycerol (OAG)-mediated extracellular Ca(2+)-dependent increases in [Ca(2+)](i) (SRCE) and express canonical transient receptor potential-like channels (TRPC) mRNAs (predominantly TRPC1, -4, and -6) that have been implicated in SRCE. To determine the role of TRPC6 in human myometrial SRCE, short hairpin RNA constructs were designed that effectively targeted a TRPC6 mRNA reporter for degradation. One sequence was used to produce an adenovirus construct (TC6sh1). TC6sh1 reduced TRPC6 mRNA but not TRPC1, -3, -4, -5, or -7 mRNAs in PHM1-41 myometrial cells. Compared with uninfected cells or cells infected with empty vector, the increase in [Ca(2+)](i) in response to OAG was specifically inhibited by TC6sh1, whereas SRCE responses elicited by either oxytocin or thapsigargin were not changed. Similar findings were observed in primary pregnant human myometrial cells. When PHM1-41 cells were activated by OAG in the absence of extracellular Na(+), the increase in [Ca(2+)](i) was partially reduced. Furthermore, pretreatment with nifedipine, an L-type calcium channel blocker, also partially reduced the OAG-induced [Ca(2+)](i) increase. Similar effects were observed in primary human myometrial cells. These findings suggest that OAG activates channels containing TRPC6 in myometrial cells and that these channels act via both enhanced Na(+) entry coupled to activation of voltage-dependent Ca(2+) entry channels and a nifedipine-independent Ca(2+) entry mechanism to promote elevation of intracellular Ca(2+).
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Affiliation(s)
- Daesuk Chung
- Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523, USA
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Willets JM, Brighton PJ, Mistry R, Morris GE, Konje JC, Challiss RAJ. Regulation of oxytocin receptor responsiveness by G protein-coupled receptor kinase 6 in human myometrial smooth muscle. Mol Endocrinol 2009; 23:1272-80. [PMID: 19423652 PMCID: PMC5419184 DOI: 10.1210/me.2009-0047] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2009] [Accepted: 04/29/2009] [Indexed: 11/19/2022] Open
Abstract
Oxytocin plays an important role in the progression, timing, and modulation of uterine contraction during labor and is widely used as an uterotonic agent. We investigated the mechanisms regulating oxytocin receptor (OTR) signaling in human primary myometrial smooth muscle cells and the ULTR cell-line. Oxytocin produced concentration-dependent increases in both total [(3)H]inositol phosphate accumulation and intracellular Ca(2+) concentration ([Ca(2+)](i)); however, responses were greater and more reproducible in the ULTR cell line. Assessment of phospholipase C activity in single cells revealed that the OTR desensitizes rapidly (within 5 min) in the presence of oxytocin (100 nm). To characterize OTR desensitization further, cells were stimulated with a maximally effective concentration of oxytocin (100 nm, 30 sec) followed by a variable washout period and a second identical application of oxytocin. This brief exposure to oxytocin caused a marked decrease (>70%) in OTR responsiveness to rechallenge and was fully reversed by increasing the time period between agonist challenges. To assess involvement of G protein-coupled receptor kinases (GRKs) in OTR desensitization, cells were transfected with small interfering RNAs to cause specific > or =75% knockdown of GRKs 2, 3, 5, or 6. In both primary myometrial and ULTR cells, knockdown of GRK6 largely prevented oxytocin-induced OTR desensitization; in contrast, selective depletion of GRKs 2, 3, or 5 was without effect. These data indicate that GRK6 recruitment is a cardinal effector of OTR responsiveness and provide mechanistic insight into the likely in vivo regulation of OTR signaling in uterine smooth muscle.
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Affiliation(s)
- Jonathon M Willets
- Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, United Kingdom.
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Uh A, Simmons CF, Bresee C, Khoury N, Gombart AF, Nicholson RC, Kocak H, Equils O. MyD88 and TRIF mediate the cyclic adenosine monophosphate (cAMP) induced corticotropin releasing hormone (CRH) expression in JEG3 choriocarcinoma cell line. Reprod Biol Endocrinol 2009; 7:74. [PMID: 19615077 PMCID: PMC2720972 DOI: 10.1186/1477-7827-7-74] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/06/2009] [Accepted: 07/17/2009] [Indexed: 01/07/2023] Open
Abstract
BACKGROUND Classically protein kinase A (PKA) and transcription factor activator protein 1 (AP-1) mediate the cyclic AMP (cAMP) induced-corticotrophin releasing hormone (CRH) expression in the placenta. However enteric Gram (-) bacterial cell wall component lipopolysaccharide (LPS) may also induce-CRH expression via Toll like receptor (TLR)4 and its adaptor molecule Myd88. Here we investigated the role of MyD88, TRIF and IRAK2 on cAMP-induced CRH promoter activation in JEG3 cells in the absence of LPS/TLR4 stimulation. METHODS JEG3 cells were transfected with CRH-luciferase and Beta-galactosidase expression vectors and either empty or dominant-negative (DN)-MyD88, DN-TRIF or DN-IRAK2 vectors using Fugene6 (Roche). cAMP-induced CRH promoter activation was examined by using a luminometer and luciferase assay. Calorimetric Beta-galactosidase assays were performed to correct for transfection efficiency. Luciferase expression vectors of cAMP-downstream molecules, CRE and AP-1, were used to further examine the signaling cascades. RESULTS cAMP stimulation induced AP-1 and CRE promoter expression and led to dose-dependent CRH promoter activation in JEG3 cells. Inhibition of MyD88 signaling blocked cAMP-induced CRE and CRH promoter activation. Inhibition of TRIF signaling blocked cAMP-induced CRH but not CRE expression, while inhibition of IRAK2 did not have an effect on cAMP-induced CRH expression. CONCLUSION MyD88 and TRIF exert direct regulatory effect on cAMP-induced CRH promoter activation in JEG3 cells in the absence of infection. MyD88 most likely interacts with molecules upstream of IRAK2 to regulate cAMP-induced CRH expression.
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Affiliation(s)
- Andy Uh
- Ahmanson Department of Pediatrics, Room 4221, Steven Spielberg Pediatric Research Center, Burns and Allen Research Institute, Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Charles F Simmons
- Ahmanson Department of Pediatrics, Room 4221, Steven Spielberg Pediatric Research Center, Burns and Allen Research Institute, Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Catherine Bresee
- Samuel Oschin Comprehensive Cancer Institute Biostatistics Core, Cedars-Sinai Medical Center, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Nasif Khoury
- Ahmanson Department of Pediatrics, Room 4221, Steven Spielberg Pediatric Research Center, Burns and Allen Research Institute, Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Adrian F Gombart
- Linus Pauling Institute; Department of Biochemistry and Biophysics; ALS 2011, Oregon State University; Corvallis, OR 97331-7305, USA
| | - Richard C Nicholson
- Mothers and Babies Research Center, Hunter Medical Research Institute, John Hunter Hospital, Newcastle, Australia
| | - Hande Kocak
- Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA
| | - Ozlem Equils
- Ahmanson Department of Pediatrics, Room 4221, Steven Spielberg Pediatric Research Center, Burns and Allen Research Institute, Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
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Franova S, Janicek F, Visnovsky J, Dokus K, Zubor P, Sutovska M, Nosalova G. Utero-relaxant effect of PDE4-selective inhibitor alone and in simultaneous administration with beta2-mimetic on oxytocin-induced contractions in pregnant myometrium. J Obstet Gynaecol Res 2009; 35:20-5. [PMID: 19215543 DOI: 10.1111/j.1447-0756.2008.00839.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
BACKGROUND The objective of the study was to observe the effect of rolipram, the prototype phosphodiesterase 4 selective inhibitor, on oxytocin-induced contractions of human term myometrial strips, and compare the effect with salbutamol, beta(2)-adrenergic agonist, in single and the simultaneous application. METHODS Human myometrium was obtained from pregnant women in term that had a term delivery by the caesarian section. Myometrial strips were excised from the lower uterine segment and placed into an organ-bath with Krebs-Henseleit buffer. The mean peak amplitude of contraction (mN) of the myometrial smooth muscle to the doses of oxytocin (10(-6) mmol/L(-1)) with subsequent single administration of rolipram (10(-4) mmol/L(-1)), salbutamol (10(-4) mmol/L(1)) and simultaneous administration of rolipram and salbutamol (both 10(-4) mmol/L(-1)), was used as a parameter of myometrial reactivity. RESULTS Rolipram alone decreased the oxytocin-induced contractile amplitude to 47.98%, single salbutamol application resulted in amplitudinal decrease to 56.07%, and the combination of both compounds in their simultaneous administration resulted in the decrease of oxytocin-induced contractile amplitude to 29.1%. CONCLUSION Our data are consistent with previous studies of the enhanced efficiency of the beta(2)-adrenergic agonist, when administered together with the phosphodiesterase 4-inhibitor. Moreover we have shown that rolipram alone has a more profound effect on oxytocin-induced contractions than salbutamol alone.
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Affiliation(s)
- Sona Franova
- Department of Pharmacology, Jessenius Faculty of Medicine, Comenius University, Martin, Slovakia.
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Baston-Büst DM, Hess AP, Hirchenhain J, Krücken J, Wunderlich F, Krüssel JS, Friebe-Hoffmann UK. A possible ambivalent role for relaxin in human myometrial and decidual cells in vitro. Arch Gynecol Obstet 2009; 280:961-9. [PMID: 19319551 DOI: 10.1007/s00404-009-1046-8] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2009] [Accepted: 03/09/2009] [Indexed: 11/29/2022]
Abstract
PURPOSE Based on the reported tocolytic action of the hormone relaxin (RLX) in rodents, locally produced in reproductive tissues and the corpus luteum in mammals, the present study aimed to evaluate the influence of RLX on contraction-mediating cyclooxygenases-1 and -2 (COX) and the contractile prostaglandin PGE(2) in human myometrial and decidual cells. Primary cultured cells were obtained from uteri and placentas of term and preterm women undergoing elective caesarean section. METHODS In vitro culture of primary myometrial and decidual cells, immunocytochemistry, reverse transcription and real-time PCR, Western blot, ELISA. RESULTS We demonstrate for the first time an activating effect of RLX for human COX-1 and COX-2 in primary myometrial and decidual cells in vitro. CONCLUSIONS These effects might potentially contribute to birth-associated induction of contractions in vivo.
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Affiliation(s)
- Dunja M Baston-Büst
- Department of Obstetrics and Gynecology, Heinrich-Heine-University, Düsseldorf, Germany.
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Song J, Zhang X, Qi Z, Sun G, Chi S, Zhu Z, Ren J, Qiu Z, Liu K, Myatt L, Ma RZ. Cloning and characterization of a calcium-activated chloride channel in rat uterus. Biol Reprod 2009; 80:788-94. [PMID: 19144963 DOI: 10.1095/biolreprod.108.071258] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022] Open
Abstract
In a search for genes involved in regulation of uterine contractility, we cloned a novel calcium-activated chloride channel gene, named rat Clca4, from pregnant rat uterus. The gene shares approximately 83% and 70% nucleotide homology with mouse Clca6 and human CLCA4, respectively, and was expressed primarily in rat uterus. The transcripts were upregulated at Gestational Day 22 (prior to parturition), implying a functional involvement in parturition. Western blot analysis showed that rat CLCA4 protein was present in uterus, lung, and heart, but not in any other tissues examined. Confocal microscopy revealed that rat CLCA4 is localized in cell membrane and could not be removed by alkaline or PBS washing. Transient transfection of rat CLCA4-enhanced green fluorescent protein in Chinese hamster ovary cells resulted in production of characteristic Cl(-) currents that could be activated by Ca(2+) and ionomycin but inhibited by niflumic acid, a CLCA-channel blocker. The identification and characterization of rat Clca4 help decipher the contribution of Ca(2+)-activated Cl(-) conductance in myometrial contractility.
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Affiliation(s)
- Junfang Song
- Institute of Genetics and Developmental Biology and State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
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39
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Helguera G, Eghbali M, Sforza D, Minosyan TY, Toro L, Stefani E. Changes in global gene expression in rat myometrium in transition from late pregnancy to parturition. Physiol Genomics 2008; 36:89-97. [PMID: 19001510 DOI: 10.1152/physiolgenomics.00290.2007] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The process of parturition involves the complex interplay of factors that change the excitability and contractile activity of the uterus. We have compared the relative gene expression profile of myometrium from rats before parturition (21 days pregnant) and during delivery, using high-density DNA microarray. Of 8,740 sequences available in the array, a total of 3,782 were detected as present. From the sequences that were significantly altered, 59 genes were upregulated and 82 genes were downregulated. We were able to detect changes in genes described to have altered expression level at term, including connexin 43 and 26, cyclooxygenase 2, and oxytocin receptor, as well as novel genes that have been not previously associated with parturition. Quantitative real-time PCR on selected genes further confirmed the microarray data. Here we report for the first time that aquaporin5 (AQP5), a member of the aquaporin water channel family, was dramatically downregulated during parturition (approximately 100-fold by microarray and approximately 50-fold by real-time PCR). The emerging profile highlights biochemical cascades occurring in a period of approximately 36 h that trigger parturition and the initiation of myometrium reverse remodeling postpartum. The microarray analysis uncovered genes that were previously suspected to play a role in parturition. This regulation involves genes from immune/inflammatory response, steroid/lipid metabolism, calcium homeostasis, cell volume regulation, cell signaling, cell division, and tissue remodeling, suggesting the presence of multiple and redundant mechanisms altered in the process of birth.
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Affiliation(s)
- Gustavo Helguera
- Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine at University of California-Los Angeles, Los Angeles, CA 90095-7115, USA
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40
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Zhong M, Murtazina DA, Phillips J, Ku CY, Sanborn BM. Multiple signals regulate phospholipase CBeta3 in human myometrial cells. Biol Reprod 2008; 78:1007-17. [PMID: 18322273 DOI: 10.1095/biolreprod.107.064485] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
Phospholipase CB3 (PLCB3) serine(1105) (S(1105)), a substrate for multiple protein kinases, represents a potential point of convergence of several signaling pathways in the myometrium. To explore this hypothesis, the regulation of PLCB3-S(1105) phosphorylation (P-S(1105)) was studied in immortalized and primary human myometrial cells. 8-[4-chlorophenylthio] (CPT)-cAMP and calcitonin gene-related peptide (CALCA) transiently increased P-S(1105). Relaxin also stimulated P-S(1105); this effect was partially blocked by the protein kinase A (PRKA) inhibitor, Rp-8-CPT-cAMPS. Oxytocin, which stimulates Galphaq-mediated pathways, also rapidly increased P-S(1105), as did prostaglandin F2alpha and ATP. Oxytocin-stimulated phosphorylation was blocked by protein kinase C (PRKC) inhibitor Go6976 and by pretreatment overnight with a phorbol ester. Cypermethrin, a PP2B phosphatase inhibitor, but not okadaic acid, a PP1/PP2A inhibitor, prolonged the effect of CALCA on P-S(1105), whereas the reverse was the case for the oxytocin-stimulated increase in P-S(1105). PLCB3 was the predominant PLC isoform expressed in the myometrial cells and PLCB3 short hairpin RNA constructs significantly attenuated oxytocin-stimulated increases in intracellular calcium. oxytocin-induced phosphatidylinositol (PI) turnover was inhibited by CPT-cAMP and okadaic acid, but was enhanced by pretreatment with Go6976. CPT-cAMP inhibited oxytocin-stimulated PI turnover in the presence of overexpressed PLCB3, but not overexpressed PLCB3-S(1105)A. These data demonstrate that both negative crosstalk from the cAMP/PRKA pathway and a negative feedback loop in the oxytocin/G protein/PLCB pathway involving PRKC operate in myometrial cells and suggest that different protein phosphatases predominate in mediating P-S(1105) dephosphorylation in these pathways. The integration of multiple signal components at the level of PLCB3 may be important to its function in the myometrium.
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Affiliation(s)
- Miao Zhong
- Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523, USA
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41
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Cattaneo MG, Chini B, Vicentini LM. Oxytocin stimulates migration and invasion in human endothelial cells. Br J Pharmacol 2008; 153:728-36. [PMID: 18059319 PMCID: PMC2259201 DOI: 10.1038/sj.bjp.0707609] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2007] [Revised: 10/02/2007] [Accepted: 10/26/2007] [Indexed: 01/11/2023] Open
Abstract
BACKGROUND AND PURPOSE It has recently been reported that oxytocin is produced by some tumour cell types, and that oxytocin receptors, belonging to the G-protein-coupled receptor (GPCR) family, are expressed in a variety of cell types. Among these, human umbilical vein endothelial cells (HUVECs) respond to oxytocin with an increased proliferation, suggesting a possible role for the hormone in the regulation of angiogenesis. EXPERIMENTAL APPROACH We employed chemotaxis and chemoinvasion assays to characterize the effect of oxytocin on HUVEC motility, and immunoblot analysis to study its molecular mechanisms of action. KEY RESULTS We showed that oxytocin stimulates migration and invasion in HUVECs via oxytocin receptor activation. Searching for the molecular mechanism(s) responsible for oxytocin's pro-migratory effect, we identified the Gq coupling of oxytocin receptors and phospholipase C (PLC) as the main effectors of oxytocin's action in HUVECs. We also found that oxytocin stimulates the phosphorylation of endothelial nitric oxide synthase (eNOS) via the phosphatidylinositol-3-kinase (PI-3-K)/AKT pathway, and that the activation of PI-3-K and formation of nitric oxide (NO) are required for the pro-migratory effect of oxytocin. CONCLUSIONS AND IMPLICATIONS The ability of oxytocin to stimulate HUVEC motility and invasion suggests that the hormone can participate in physiopathological processes where activation of endothelial cells plays an important role, for example, in angiogenesis. Interestingly, both the AKT and eNOS phosphorylation induced by oxytocin receptor activation depended on PLC activity, thus suggesting the existence of a still undefined mechanism connecting PLC to the PI-3-K/AKT pathway, upon oxytocin stimulation.
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Affiliation(s)
- M G Cattaneo
- Department of Pharmacology, University of Milano Via Vanvitelli 32, Milano, Italy
| | - B Chini
- Consiglio Nazionale delle Ricerche, Institute of Neuroscience Via Vanvitelli 32, Milano, Italy
| | - L M Vicentini
- Department of Pharmacology, University of Milano Via Vanvitelli 32, Milano, Italy
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O'Brien M, Flynn D, Mullins B, Morrison JJ, Smith TJ. Expression of RHOGTPase regulators in human myometrium. Reprod Biol Endocrinol 2008; 6:1. [PMID: 18190708 PMCID: PMC2254629 DOI: 10.1186/1477-7827-6-1] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/17/2007] [Accepted: 01/11/2008] [Indexed: 01/24/2023] Open
Abstract
BACKGROUND RHOGTPases play a significant role in modulating myometrial contractility in uterine smooth muscle. They are regulated by at least three families of proteins, RHO guanine nucleotide exchange factors (RHOGEFs), RHOGTPase-activating proteins (RHOGAPs) and RHO guanine nucleotide inhibitors (RHOGDIs). RHOGEFs activate RHOGTPases from the inactive GDP-bound to the active GTP-bound form. RHOGAPs deactivate RHOGTPases by accelerating the intrinsic GTPase activity of the RHOGTPases, converting them from the active to the inactive form. RHOGDIs bind to GDP-bound RHOGTPases and sequester them in the cytosol, thereby inhibiting their activity. Ezrin-Radixin-Moesin (ERM) proteins regulate the cortical actin cytoskeleton, and an ERM protein, moesin (MSN), is activated by and can also activate RHOGTPases. METHODS We therefore investigated the expression of various RHOGEFs, RHOGAPs, a RHOGDI and MSN in human myometrium, by semi-quantitative reverse transcription PCR, real-time fluorescence RT-PCR, western blotting and immunofluorescence microscopy. Expression of these molecules was also examined in myometrial smooth muscle cells. RESULTS ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN mRNA and protein expression was confirmed in human myometrium at term pregnancy, at labour and in the non-pregnant state. Furthermore, their expression was detected in myometrial smooth muscle cells. It was determined that ARHGAP24 mRNA expression significantly increased at labour in comparison to the non-labour state. CONCLUSION This study demonstrated for the first time the expression of the RHOGTPase regulators ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN in human myometrium, at term pregnancy, at labour, in the non-pregnant state and also in myometrial smooth muscle cells. ARHGAP24 mRNA expression significantly increased at labour in comparison to the non-labouring state. Further investigation of these molecules may enable us to further our knowledge of RHOGTPase regulation in human myometrium during pregnancy and labour.
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Affiliation(s)
- Margaret O'Brien
- National Centre for Biomedical and Engineering Science, Orbsen Building, National University of Ireland Galway, University Road, Galway, Ireland
| | - David Flynn
- National Centre for Biomedical and Engineering Science, Orbsen Building, National University of Ireland Galway, University Road, Galway, Ireland
| | - Brian Mullins
- National Centre for Biomedical and Engineering Science, Orbsen Building, National University of Ireland Galway, University Road, Galway, Ireland
| | - John J Morrison
- National Centre for Biomedical and Engineering Science, Orbsen Building, National University of Ireland Galway, University Road, Galway, Ireland
- Department of Obstetrics and Gynaecology, National University of Ireland Galway, Clinical Science Institute, University College Hospital Galway, Newcastle Road, Galway, Ireland
| | - Terry J Smith
- National Centre for Biomedical and Engineering Science, Orbsen Building, National University of Ireland Galway, University Road, Galway, Ireland
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43
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Chevillard G, Derjuga A, Devost D, Zingg HH, Blank V. Identification of interleukin-1β regulated genes in uterine smooth muscle cells. Reproduction 2007; 134:811-22. [DOI: 10.1530/rep-07-0289] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
We analyzed the response of uterine smooth muscle cells to interleukin-1β (IL-1β). We first showed that PHM1-31 myometrial cells, our cellular model, are contractile. To determine the molecular mechanisms of uterine smooth muscle cell activation by proinflammatory cytokines, we performed genechip expression array profiling studies of PHM1-31 cells in the absence and the presence of IL-1β. In total, we identified 198 known genes whose mRNA levels are significantly modulated (> 2.0-fold change) following IL-1β exposure. We confirmed the expression changes for selected genes by independent mRNA and protein analysis. The group of genes induced by IL-1β includes transcription factors and inflammatory response genes such as nuclear factor of κ light polypeptide gene enhancer in B-cells (NFκB), pentraxin-related gene (PTX3), and tumor necrosis factor α-induced protein 3/A20 (TNFAIP3/A20). We also found up-regulation of chemokines like C-X-C motif ligand 3 (CXCL3) and extracellular matrix remodeling signaling molecules like tenascin C (TNC). Our data suggest that IL-1β elicits the rapid activation of a cellular network of genes particularly implicated in inflammatory response that may create a cellular environment favorable for myometrial cell contraction. Our results provide novel insights into the mechanisms of uterine smooth muscle cell regulation and possibly infection-induced preterm labor.
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44
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Chini B, Manning M. Agonist selectivity in the oxytocin/vasopressin receptor family: new insights and challenges. Biochem Soc Trans 2007; 35:737-41. [PMID: 17635137 DOI: 10.1042/bst0350737] [Citation(s) in RCA: 90] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The design and development of selective agonists acting at the OT (oxytocin)/AVP (vasopressin) receptors has been and continues to be a difficult task because of the great similarity among the different receptor subtypes as well as the high degree of chemical similarity between the active ligands. In recent decades, at least a thousand synthetic peptides have been synthesized and examined for their ability to bind to and activate the different OT/AVP receptors; an effort that has led to the identification of several receptor subtype-selective agonists in the rat. However, owing to species differences between rat and human AVP/OT receptors, these peptides do not exhibit the same selectivities in human receptor assays. Furthermore, the discovery of receptor promiscuity, which is the ability of a single receptor subtype to couple to several different G-proteins, has led to the definition of a completely new class of compounds, referred to here as coupling-selective ligands, which may activate, within a single receptor subtype, only a specific signalling pathway. Finally, the accumulating evidence that GPCRs (G-protein-coupled receptors) do not function as monomers, but as dimers/oligomers, opens up the design of another class of specific ligands, bivalent ligands, in which two agonist and/or antagonist moieties are joined by a spacer of the appropriate length to allow the simultaneous binding at the two subunits within the dimer. The pharmacological properties and selectivity profiles of these bivalent ligands, which remain to be investigated, could lead to highly novel research tools and potential therapeutic agents.
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Affiliation(s)
- B Chini
- CNR Institute of Neuroscience, Department of Pharmacology, University of Milan, via Vanvitelli 32, 20129 Milan, Italy.
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45
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Terzidou V. Preterm labour. Biochemical and endocrinological preparation for parturition. Best Pract Res Clin Obstet Gynaecol 2007; 21:729-56. [PMID: 17616441 DOI: 10.1016/j.bpobgyn.2007.05.001] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Preterm delivery is a common obstetric problem occurring in about 1 in 10 of all births. Preterm babies have a high risk of morbidity and mortality. Such births account for 75% of all major neonatal problems. At the other end of the spectrum, prolonged pregnancy is also a subject of concern because it too is associated with increased fetal morbidity and mortality. Despite extensive research, the mechanisms that control the length of human pregnancy and signal the onset of labour have not been fully determined. This chapter will discuss basic principles in the biology of parturition and the regulation of contraction-associated proteins including the oxytocin receptor. The major pathways regulating contractions and the transcriptional regulation of the main genes that are known to be involved in the onset of labour and parturition will be examined. Some new potentially therapeutic strategies for the biochemical management of preterm labour will be discussed.
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Affiliation(s)
- Vasso Terzidou
- Parturition Research Group, Faculty of Medicine, Imperial College London, Institute of Reproductive and Developmental Biology, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.
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46
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Abstract
A better understanding of the mechanisms that generate and modulate uterine contractility is needed if progress is to be made in the prevention or treatment of problems in labour. Dysfunctional labour describes the condition when uterine contractility is too poor to dilate the cervix, and it is the leading cause of emergency Caesarean sections. Recently, insight has been gained into a possible causal mechanism for dysfunctional labour. Study of the physiological mechanisms that produce excitation in the uterus, the subsequent Ca(2)(+) signals and biochemical pathway leading to contraction has underpinned this progress. In this review, I give an account of excitation-contraction signalling in the myometrium and explore the implications of recent findings concerning lipid rafts for these processes. I also discuss how changes of pH are fundamentally enmeshed in uterine activity and biochemistry and explore the effect that pH changes will have on human myometrium. Finally, I present the evidence that acidification of the myometrium is correlated with dysfunctional labour and suggest the processes by which it is occurring. It is only by gaining a better understanding of uterine physiology and pathophysiology that progress will be made and research findings translated into clinical benefit for women and their families.
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Affiliation(s)
- Susan Wray
- Department of Physiology, University of Liverpool, Crown Street, Liverpool L69 3BX, UK.
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47
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Srivastava A, Gupta PK, Knock GA, Aaronson PI, Mishra SK, Prakash VR. Effect of ceramide on the contractility of pregnant rat uterus. Eur J Pharmacol 2007; 567:159-65. [PMID: 17490636 DOI: 10.1016/j.ejphar.2007.04.013] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2006] [Revised: 04/02/2007] [Accepted: 04/03/2007] [Indexed: 11/27/2022]
Abstract
Ceramide and other sphingolipid mediators have emerged as a novel class of lipid second messengers in cell signaling. We assessed the effect of C(2)-ceramide (a membrane permeable analog of ceramide) on spontaneous and agonist-induced contractile responses of uterus, isolated from 19-day pregnant rats. Ceramide (3, 10 microM) moderately, but significantly inhibited the amplitude of spontaneous rhythmic contractions. However, a variable effect was seen on agonist-induced contractions. While 5-HT-induced contractions were markedly inhibited at 3 and 10 microM ceramide, oxytocin and carboprost (a PGF(2)alpha analogue)-induced contractions were not affected by the sphingolipid. Ceramide (10 microM) also markedly inhibited CaCl(2)-induced contractions elicited in K(+)-depolarized tissues. Further, in rabbit portal vein myocytes, which display robust L-type calcium channel current, ceramide inhibited the I(Ba) in a dose-dependent manner. Therefore, it is suggested that the inhibitory effect of ceramide on uterine contractility may involve a decrease in the influx of Ca(2+) through voltage-dependent L-type Ca(2+) channels, such that contractile responses that are primarily dependent on extracellular Ca(2+), like rhythmic and serotonin contractions, were inhibited by ceramide. Further study is required to establish the role of endogenous ceramide and other sphingolipids in regulating uterine tone during gestation and at term.
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Affiliation(s)
- Anuradha Srivastava
- Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India
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Sanborn BM. Hormonal signaling and signal pathway crosstalk in the control of myometrial calcium dynamics. Semin Cell Dev Biol 2007; 18:305-14. [PMID: 17627855 PMCID: PMC2000447 DOI: 10.1016/j.semcdb.2007.05.007] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2007] [Accepted: 05/03/2007] [Indexed: 10/23/2022]
Abstract
Understanding the basis for the control of myometrial contractant and relaxant signaling pathways is important to understanding how to manage myometrial contractions. Signaling pathways are influenced by the level of expression of the signals and signal pathway components, the location of these components in the appropriate subcellular environment, and covalent modification. Crosstalk between these pathways regulates the effectiveness of signal transduction and represents an important way by which hormones can regulate phenotype. This review deals primarily with signaling pathways that control Ca2+ entry and intracellular release, as well as the interplay between these pathways.
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Affiliation(s)
- Barbara M Sanborn
- Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1680, USA.
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49
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Yuan W, López Bernal A. Cyclic AMP signalling pathways in the regulation of uterine relaxation. BMC Pregnancy Childbirth 2007; 7 Suppl 1:S10. [PMID: 17570154 PMCID: PMC1892051 DOI: 10.1186/1471-2393-7-s1-s10] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
Studying the mechanism(s) of uterine relaxation is important and will be helpful in the prevention of obstetric difficulties such as preterm labour, which remains a major cause of perinatal mortality and morbidity. Multiple signalling pathways regulate the balance between maintaining relative uterine quiescence during gestation, and the transition to the contractile state at the onset of parturition. Elevation of intracellular cyclic AMP promotes myometrial relaxation, and thus quiescence, via effects on multiple intracellular targets including calcium channels, potassium channels and myosin light chain kinase. A complete understanding of cAMP regulatory pathways (synthesis and hydrolysis) would assist in the development of better tocolytics to delay or inhibit preterm labour. Here we review the enzymes involved in cAMP homoeostasis (adenylyl cyclases and phosphodiesterases) and possible myometrial substrates for the cAMP dependent protein kinase. We must emphasise the need to identify novel pharmacological targets in human pregnant myometrium to achieve safe and selective uterine relaxation when this is indicated in preterm labour or other obstetric complications.
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Affiliation(s)
- Wei Yuan
- University of Bristol, Clinical Science at South Bristol (Obstetrics and Gynaecology), St Michael's Hospital and Dorothy Hodgkin Building, Whitson Street, Bristol, BS1 3NY, UK
| | - Andrés López Bernal
- University of Bristol, Clinical Science at South Bristol (Obstetrics and Gynaecology), St Michael's Hospital and Dorothy Hodgkin Building, Whitson Street, Bristol, BS1 3NY, UK
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50
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Blanks AM, Shmygol A, Thornton S. Preterm labour. Myometrial function in prematurity. Best Pract Res Clin Obstet Gynaecol 2007; 21:807-19. [PMID: 17446138 DOI: 10.1016/j.bpobgyn.2007.03.003] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The primary function of the uterus during gestation is to harbour the growing conceptus in a largely quiescent environment. Upon maturation of the fetus to a point sufficient for extrauterine survival, the uterus must remodel itself sufficiently to generate forceful contractions during labour. During preterm delivery, the process of remodelling of the myometrium occurs early due to a number of different causes, although the underlying basis for myometrial contraction remains the same. This review summarises the anatomical, physiological and molecular basis for contraction. We describe the fibre structure of the human uterus and how this relates to the spread of electrical excitation during a contraction. The process of excitation within a single myometrial cell is described, as well as how this relates to contraction. We then focus on how excitation-contraction coupling is modulated by intercellular communication, pharmacomechanical-coupling and hormonal milieu. Lastly, we consider the actions of the commonly accepted uterine agonists oxytocin, prostaglandin F(2alpha), and prostaglandin E(2), and the tocolytic ritodrine.
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Affiliation(s)
- Andrew M Blanks
- Clinical Sciences Research Institute, University of Warwick, University Hospitals of Coventry and Warwickshire, Clifford Bridge Road, Coventry CV2 2DX, UK.
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