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ABSTRACT: Background:Studies from knockout mice suggest that perturbations in oviductal endocannabinoid levels, endocannabinoid receptors, or endocannabinoid degrading enzyme [fatty acid amide hydrolase (FAAH)] expression result in infertility secondary to physical trapping of embryos. Similar observations have been made in ectopic pregnant women together with a suggestion that the endocannabinoid receptor gene polymorphism 1359G/A (rs1049353) is associated with ectopic pregnancy. These observations led to the hypothesis that ectopic pregnancy is associated with a perturbation in levels of endocannabinoids and FAAH activity and that such changes are associated with impaired tubal function.Aims:The objective of the study was to quantify the plasma levels of endocannabinoids (anandamide, oleoylethanolamide, and palmitoylethanolamide) and evaluate blood endocannabinoid metabolizing enzyme activities FAAH and N-acyl-phosphatidyl-ethanolamine phospholipase D (NAPE-PLD) in ectopic pregnancy and normal pregnant controls and relate that to β-human chorionic gonadotropin (β-hCG) levels. Additionally, we wanted to examine the effect of endocannabinoids on cilia beat frequency in Fallopian tube epithelial cells ex vivo.Participants and Methods:Whole blood collected from ectopic and normal pregnancies was used for quantification of plasma endocannabinoid levels by ultra-HPLC-tandem mass spectrometry of FAAH and NAPE-PLD enzyme activities by radiometric assays, and β-hCG by immunoassay. Fallopian tube epithelial cells from healthy volunteers were treated with endocannabinoids and cilia beat frequency analyzed using a high-speed digital camera and CiliaFA software.Results:FAAH activity (P < .05) but not NAPE-PLD activity was significantly reduced in ectopic pregnancies. All 3 endocannabinoids levels were significantly higher (P < .05) in ectopic pregnancy. There was no correlation between endocannabinoids, enzyme activity, and β-hCG levels. Oleoylethanolamide (P < .05), but not methanandamide or palmitoylethanolamide, significantly decreased cilia beat frequency in Fallopian tube epithelial cells.Conclusion:Elevated endocannabinoid levels and reduced FAAH activity are associated with ectopic pregnancy and may modulate tubal function, suggesting dysfunctional endocannabinoid action in ectopic implantation. Oleoylethanolamide may play a critical role in embryo-tubal transport.
The Journal of clinical endocrinology and metabolism 01/2013; · 6.50 Impact Factor
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ABSTRACT: Ectopic pregnancy is associated with significant morbidity and mortality, but the molecular mechanisms underlying this condition remain unclear. Although the endocannabinoids, N-arachidonoylethanolamine (anandamide), N-oleoylethanolamine, and N-palmitoylethanolamine, are thought to play a negative role in ectopic pregnancy, their precise role(s) within the fallopian tube remains unclear. Anandamide activates cannabinoid receptors (CB1 and CB2) and, together with its degrading [e.g. fatty acid amide hydrolase (FAAH)] and synthesizing enzymes (e.g. N-acyl-phosphatidylethanolamine-specific phospholipase D), forms the endocannabinoid system. High anandamide levels are associated with tubal arrest of embryos in mice and may have a similar role in women.
The aims were to quantify the levels of the endocannabinoids and evaluate the expression of the modulating enzymes and the cannabinoid receptors in fallopian tubes of women with ectopic pregnancy compared to those of nonpregnant women.
We conducted a prospective study at the University Hospitals of the Leicester National Health Service Trust.
Fallopian tubes collected from women with ectopic pregnancy and nonpregnant women with regular menstrual cycles were used for quantification of endocannabinoids by ultra-HPLC tandem mass spectrometry, were fixed in formalin for immunohistochemistry, and had RNA extracted for RT-quantitative PCR or protein extracted for immunoblotting.
Anandamide, but not N-oleoylethanolamine and N-palmitoylethanolamine, levels were significantly higher in ectopic fallopian tubes. Endocannabinoid levels from isthmus to ampulla were not significantly different. Cannabinoid receptors and endocannabinoid modulating enzymes were localized in fallopian tube epithelium by immunohistochemistry and showed reduced CB1 and FAAH expression in ectopic pregnancy.
High anandamide levels and reduced expression of CB1 and FAAH may play a role in ectopic implantation.
The Journal of clinical endocrinology and metabolism 06/2012; 97(8):2827-35. · 6.50 Impact Factor
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ABSTRACT: RT-qPCR is commonly employed in gene expression studies in ectopic pregnancy. Most use RN18S1, β-actin or GAPDH as internal controls without validation of their suitability as reference genes. A systematic study of the suitability of endogenous reference genes for gene expression studies in ectopic pregnancy is lacking. The aims of this study were therefore to evaluate the stability of 12 reference genes and suggest those that are stable for use as internal control genes in fallopian tubes and endometrium from ectopic pregnancy and healthy non-pregnant controls. Analysis of the results showed that the genes consistently ranked in the top six by geNorm and NormFinder algorithms, were UBC, GAPDH, CYC1 and EIF4A2 (fallopian tubes) and UBC and ATP5B (endometrium). mRNA expression of NAPE-PLD as a test gene of interest varied between the groups depending on which of the 12 reference genes was used as internal controls. This study demonstrates that arbitrary selection of reference genes for normalisation in RT-qPCR studies in ectopic pregnancy without validation, risk producing inaccurate data and should therefore be discouraged.
International Journal of Molecular Sciences 01/2012; 13(3):2810-26. · 2.60 Impact Factor
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ABSTRACT: Overstimulation of endothelin type A (ET(A)) and nucleotide (P2Y) Gα(q)-coupled receptors in vascular smooth muscle causes vasoconstriction, hypertension, and, eventually, hypertrophy and vascular occlusion. G protein-coupled receptor kinases (GRKs) and arrestin proteins are sequentially recruited by agonist-occupied Gα(q)-coupled receptors to terminate phospholipase C signaling, preventing prolonged/inappropriate contractile signaling. However, these proteins also play roles in the regulation of several mitogen-activated protein kinase (MAPK) signaling cascades known to be essential for vascular remodeling. Here we investigated whether different arrestin isoforms regulate endothelin and nucleotide receptor MAPK signaling in rat aortic smooth muscle cells (ASMCs). When intracellular Ca(2+) levels were assessed in isolated ASMCs loaded with Ca(2+)-sensitive dyes, P2Y(2) and ET(A) receptor desensitization was attenuated by selective small-interfering (si)RNA-mediated depletion of G protein-coupled receptor kinase 2 (GRK2). Using similar siRNA techniques, knockdown of arrestin2 prevented P2Y(2) receptor desensitization and enhanced and prolonged p38 and ERK MAPK signals, while arrestin3 depletion was ineffective. Conversely, arrestin3 knockdown prevented ET(A) receptor desensitization and attenuated ET1-stimulated p38 and ERK signals, while arrestin2 depletion had no effect. Using Transwell assays to assess agonist-stimulated ASMC migration, we found that UTP-stimulated migration was markedly attenuated following arrestin2 depletion, while ET1-stimulated migration was attenuated following knockdown of either arrestin. These data highlight a differential arrestin-dependent regulation of ET(A) and P2Y(2) receptor-stimulated MAPK signaling. GRK2 and arrestin expression are essential for agonist-stimulated ASMC migration, which, as a key process in vascular remodeling, highlights the potential roles of GRK2 and arrestin proteins in the progression of vascular disease.
AJP Cell Physiology 12/2011; 302(5):C723-34. · 3.54 Impact Factor
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ABSTRACT: Mirena® has been shown to improve symptoms in women with minimal to moderate endometriosis. The precise mechanisms for this have not been thoroughly investigated. We investigate here one possible mechanism-alteration in the number of mast cells in the endometriotic tissue.
Tissues (endometrial, endometriotic and normal peritoneal biopsies) prospectively collected from twenty-eight women with laparoscopically confirmed minimal to moderate endometriosis before and 6 months after treatment with Mirena® were processed for immunohistochemistry for ER and PR expression followed by toluidine blue staining for mast cells. Photographs were obtained and the receptors and mast cells identified and quantified.
The mean (± SEM) age of the twenty-eight women was 31 (±7.2) (range 18-42) years. Eight of the endometrial biopsies were in the proliferative phase and twenty in the secretory phase. Six months after Mirena®, the number of mast cell expressed in the tissues decreased significantly in the eutopic (P=0.0358) and ectopic endometrium (P=0.0220) but not in the normal peritoneum (P>0.05). There were no ERs or PRs found in mast cells.
Mirena® causes a reduction in mast cell numbers in ectopic and eutopic endometrium in women undergoing symptomatic treatment of minimal to moderate endometriosis. This reduction could partly explain the efficacy of Mirena® in modulating pain in these women.
European journal of obstetrics, gynecology, and reproductive biology 12/2011; 159(2):439-42. · 1.97 Impact Factor
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ABSTRACT: Membrane potential is a key determinant of vascular tone and many vasodilators act through the modulation of ion channel currents [e.g. the ATP-sensitive potassium channel (K(ATP))] involved in setting the membrane potential. Adenylyl cyclase (AC) isoenzymes are potentially important intermediaries in such vasodilator signalling pathways. Vascular smooth muscle cells (VSMCs) express multiple AC isoenzymes, but the reason for such redundancy is unknown. We investigated which of these isoenzymes are involved in vasodilator signalling and regulation of vascular ion channels important in modulating membrane potential.
AC isoenzymes were selectively depleted (by >75%) by transfection of cultured VSMCs with selective short interfering RNA sequences. AC6 was the predominant isoenzyme involved in vasodilator-mediated cAMP accumulation in VSMCs, accounting for ∼60% of the total response to β-adrenoceptor (β-AR) stimulation. AC3 played a minor role in β-AR signalling, whereas AC5 made no significant contribution. AC6 was also the principal isoenzyme involved in β-AR-mediated protein kinase A (PKA) signalling (determined using the fluorescent biosensor for PKA activity, AKAR3) and the substantial β-AR/PKA-dependent enhancement of K(ATP) current. K(ATP) current was shown to play a vital role in setting the resting membrane potential and in mediating the hyperpolarization observed upon β-AR stimulation.
AC6, but not the closely related AC5, plays a principal role in vasodilator signalling and regulation of the membrane potential in VSMCs. These findings identify AC6 as a vital component in the vasodilatory apparatus central to the control of blood pressure.
Cardiovascular research 06/2011; 91(4):694-702. · 5.80 Impact Factor
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ABSTRACT: The endocannabinoid plays vital roles in several aspects of reproduction, including gametogenesis, fertilization and parturition. However, little is known regarding the presence or role of the endocannabinoid system in myometrial function. Here the presence of the endocannabinoid system and signalling properties of cannabinoid receptors were characterized.
Components of the endocannabinoid system were identified using qRT-PCR, immunohistochemical, immunoblotting and radioligand binding experiments. Cannabinoid receptor signalling pathways were characterized using standard MAPK and second messenger assays.
Primary myometrium expresses the endocannabinoid synthesizing enzyme N-acyl-phosphatidyl ethanolamine-specific phospholipase D, endocannabinoid degrading enzyme fatty acid amide hydrolase and cannabinoid CB(1) , but not CB(2) receptors or transient receptor potential vanilloid-type-1 channels. The CB(1) receptor ligand anandamide caused a Gα(i/o) -dependent inhibition of adenylate cyclase reducing intracellular cAMP levels, and Gα(i/o) , phosphoinositide-3-kinase, Src-kinase-dependent ERK activation. CB(1) receptor-generated signals declined following continual anandamide stimulation, possibly due to ligand metabolism since free anandamide concentrations declined during the experiment from 2.5 µM initially, to 500 nM after >30 min. However, identical loss of CB(1) receptor responsiveness occurred in the presence of the metabolically stable derivative methanandamide. Moreover, RNAi-mediated depletion of arrestin3 (a negative regulator of receptor signalling) prevented loss of CB(1) receptor activity, enhancing and prolonging ERK signals.
The myometrium has the capacity to synthesize, respond to and degrade endocannabinoids. Furthermore, reduced CB(1) receptor responsiveness occurs as a consequence of receptor desensitization, not agonist depletion and we identify a key role for arrestin3 in this process.
British Journal of Pharmacology 04/2011; 164(5):1479-94. · 4.41 Impact Factor
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ABSTRACT: The levonorgestrel (LNG) intrauterine system (LNG-IUS) has been shown to improve symptoms in women with minimal to moderate endometriosis. The precise mechanism for this is unknown. We hypothesized that this involves alteration in the expression of estrogen receptors (ER) and progesterone receptors (PR).
A prospective study of tissues obtained prospectively from 28 women with laparoscopically confirmed minimal to moderate endometriosis treated with LNG-IUS for 6 months. Endometrial and endometriotic biopsies obtained before and 6 months after treatment were processed and stained for ER-α, ER-β and PR expression by immunohistochemistry. Photographs were obtained and the receptors quantified.
The mean (±SD) age of the 28 women was 31±7.2 (range 18-42) years. Eight of them at initial biopsy were in the proliferative phase and 20 in the secretory phase. ER-α, ER-β and PR expression decreased significantly in the glandular (P<0.0001) and stromal (P<0.0001) compartments of the eutopic endometrium after treatment with LNG-IUS. Similarly, ER-α, ER-β and PR were significantly decreased in the stromal compartment of ectopic endometrium (P<0.0001), and significantly decreased in the ectopic glands of ER-α (P<0.0001), ER-β (P=0.0002) and PR (P=0.0064) expression.
The ameliorative effect of LNG-IUS on the symptoms of minimal to moderate endometriosis is likely modulated through a decrease in the expression of glandular and stromal ER-α, ER-β and PR in the ectopic endometrium.
European journal of obstetrics, gynecology, and reproductive biology 04/2011; 157(1):101-6. · 1.97 Impact Factor
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Jonathon M Willets
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ABSTRACT: The ability to assess whether individual proteins are involved in the signalling or regulation of G -protein-coupled receptor signalling is highly dependent on the pharmacological tools available. In the absence of appropriate pharmacological agents, alternative molecular approaches have been developed to alter either protein function or expression. This has included the use of mutants, for example catalytically inactive (kinase-dead) enzymes, which when overexpressed function as dominant negatives to inhibit endogenous enzyme function, and more latterly small (21-23 bp) interfering RNA dsRNA oligos, whose antisense strand is designed complementary to the target protein mRNA and which can be used to deplete target protein expression. Critically, the success of these approaches depends on the transfection efficiency, and the chosen experimental assay in the cell type studied. Therefore, three transfection techniques and their merits and drawbacks are described. In addition, one method of examining G protein-coupled receptor (GPCR) regulation, combining siRNA-mediated GRK depletion and imaging of fluorescent GPCR -signalling reporter biosensors in difficult-to-transfect cells is briefly described.
Methods in molecular biology (Clifton, N.J.) 01/2011; 746:99-112.
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ABSTRACT: prolonged P2Y-receptor signalling can cause vasoconstriction leading to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs) and arrestin proteins, preventing prolonged or inappropriate signalling. This study investigates whether GRKs and arrestins regulate uridine 5'-triphosphate (UTP)-stimulated contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs).
mesenteric arteries contracted in response to UTP challenge: When an EC(50) UTP concentration (30 µM, 5 min) was added 5 min before (R(1)) and after (R(2)) the addition of a maximal UTP concentration (R(max): 100 µM, 5 min), R(2) responses were decreased relative to R(1), indicating desensitization. UTP-induced P2Y-receptor desensitization of phospholipase C signalling was studied in isolated MSMCs transfected with an inositol 1,4,5-trisphosphate biosensor and/or loaded with Ca(2+)-sensitive dyes. A similar protocol (R(1)/R(2) = 10 µM; R(max) = 100 µM, applied for 30 s) revealed markedly reduced R(2) when compared with R(1) responses. MSMCs were transfected with dominant-negative GRKs or siRNAs targeting specific GRK/arrestins to probe their respective roles in P2Y-receptor desensitization. GRK2 inhibition, but not GRK3, GRK5, or GRK6, attenuated P2Y-receptor desensitization. siRNA-mediated knockdown of arrestin2 attenuated UTP-stimulated P2Y-receptor desensitization, whereas arrestin3 depletion did not. Specific siRNA knockdown of the P2Y(2)-receptor almost completely abolished UTP-stimulated IP(3)/Ca(2+) signalling, strongly suggesting that our study is specifically characterizing this purinoceptor subtype.
these new data highlight roles of GRK2 and arrestin2 as important regulators of UTP-stimulated P2Y(2)-receptor responsiveness in resistance arteries, emphasizing their potential importance in regulating vasoconstrictor signalling pathways implicated in vascular disease.
Cardiovascular research 01/2011; 89(1):193-203. · 5.80 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
British Journal of Pharmacology 12/2010; 162(7):1603-17. · 4.41 Impact Factor
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ABSTRACT: The endocannabinoids anandamide, palmitoylethanolamide and oleoylethanolamide have been detected in human seminal plasma and are bioactive lipids implicated in regulation of sperm motility, capacitation and acrosome reaction. Several methods exist for endocannabinoid quantification but none have been validated for measurement in human seminal plasma. We describe sensitive, robust, reproducible solid phase and isotope-dilution UHPLC-ESI-MS/MS methods for the extraction and quantification of anandamide, palmitoylethanolamide and oleoylethanolamide in human seminal plasma. Precision and accuracy were evaluated using pooled seminal plasma over a 4 day period. For all analytes, the inter- and intraday precision (CV%) was between 6.6-17.7% and 6.3-12.5%, respectively. Analyses were linear over the range 0.237-19nM for anandamide and oleoylethanolamide and 0.9-76nM for PEA. Limits of detection (signal-to-noise >3) were 50, 100 and 100fmol/mL and limits of quantification (signal-to-noise >10) were 100, 200 and 200fmol/mL, respectively for anandamide, palmitoylethanolamide and oleoylethanolamide. Anandamide and oleoylethanolamide were stable at -80°C for up to 4 weeks, but palmitoylethanolamide declined significantly. We assessed seminal plasma from 40 human donors with normozoospermia and found mean (inter-quartile range) concentrations of 0.21nM (0.09-0.27), 1.785nM (0.48-2.32) and 15.54nM (7.05-16.31) for anandamide, oleoylethanolamide and palmitoylethanolamide, respectively. Consequently, this UHPLC-ESI-MS/MS method represents a rapid, reliable and reproducible technique for the analysis of these endocannabinoids in fresh seminal plasma.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 10/2010; 878(31):3231-7. · 2.78 Impact Factor
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ABSTRACT: There is an increasing recognition that the endocannabinoid system is the crucial cytokine-hormone system regulating early human pregnancy. The synchronous development of the fertilized embryo and the endometrium to ensure timely implantation has been shown to be one of the pivotal steps to successful implantation. This development is thought to be regulated by a finely balanced relationship between various components of the endocannabinoid system in the endometrium, the embryo and the Fallopian tube. In addition, this system has also been shown to be involved in the regulation of the development and maturation of the gametes prior to fertilization. In this review, we will examine the evidence from animal and human studies to support the role of the endocannabinoid system in gametogenesis, fertilization, implantation, early pregnancy maintenance, and in immunomodulation of pregnancy. We will discuss the role of the cannabinoid receptors and the enzymes involved in the synthesis and degradation of the key endocannabinoid ligands (e.g., anandamide and 2-arachinoylglycerol) in early reproduction.
Pharmaceuticals. 01/2010;
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ABSTRACT: Prolonged endothelin (ET) receptor signalling causes vasoconstriction and can lead to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. Usually, G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs), preventing prolonged or inappropriate signalling. This study investigated whether GRKs regulate ET receptor contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs).
ET-1-stimulated phospholipase C (PLC) activity and changes in [Ca2+]i were assessed using confocal microscopy in rat MSMCs transfected with the pleckstrin-homology domain of PLCdelta1 (eGFP-PH) and loaded with Fura-Red. ET-1 applications (30 s) stimulated transient concentration-dependent eGFP-PH translocations from plasma membrane to cytoplasm and graded [Ca2+]i increases. ET-1-mediated PLC signalling was blocked by the type A endothelin receptor (ET(A)R) antagonist, BQ123. To characterize ET(A)R desensitization, cells were stimulated with a maximally effective concentration of ET-1 (50 nM, 30 s) followed by a variable washout period and a second identical application of ET-1. This brief exposure to ET-1 markedly decreased ET(A)R responsiveness to re-challenge, and reversal was incomplete even after increasing the time period between agonist challenges to 60 min. To assess GRK involvement in ET(A)R desensitization, MSMCs were co-transfected with eGFP-PH and catalytically inactive (D110A,K220R)GRK2, (D110A,K220R)GRK3, (K215R)GRK5, or (K215R)GRK6 constructs. (D110A,K220R)GRK2 expression significantly attenuated ET(A)R desensitization, whereas other constructs were ineffective. Small interfering RNA-targeted GRK2 depletion equally attenuated ET(A)R desensitization. Finally, immunocyotchemical data showed that ET(A)R activation recruited endogenous GRK2 from cytoplasm to membrane.
These studies identify GRK2 as a key regulator of ET(A)R responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.
Cardiovascular research 09/2009; 85(3):424-33. · 5.80 Impact Factor
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ABSTRACT: Accumulating evidence highlights the importance of the endocannabinoid anandamide (AEA) as a key mediator in reproductive physiology. Current data suggest potential roles for AEA in gametogenesis, fertilization, and parturition. AEA exerts its actions through two G protein-coupled receptors, termed cannabinoid receptor 1 (CB1), and 2 (CB2), and the ligand-gated transient receptor potential vanilloid receptor type 1 (TRPV1) ion channel. At present, the cellular mechanism(s) and consequences of AEA signaling in reproductive tissues, especially the myometrium, are poorly understood. Here, we examine the expression of CB1, CB2, and TRPV1 in the human myometrial smooth muscle cell-line (ULTR) and characterize intracellular signaling after stimulation with AEA. Radioligand binding analysis revealed a total CB receptor expression of 76 +/- 24 fmol/mg protein, with both quantitative PCR and competition binding studies indicating a negligible CB2 component. AEA caused Galpha(i/o)-dependent inhibition of adenylate cyclase to reduce intracellular cAMP levels. In addition, AEA caused a 2.5- to 3.5-fold increase in ERK activation, which was ablated by inhibition of Galpha(i/o), phosphoinositide-3-kinase and Src-kinase activities, but not by inhibition of Ca(2+)/calmodulin-dependent protein kinase or protein kinase C activities. TRPV1 channel activation with capsaicin failed to activate ERK. Consistent with these findings, the selective agonists, arachidonyl-2-chloroethylamide (CB1) and L759656 (CB2), and selective antagonists AM251 (CB1) and JTE907 (CB2), provided pharmacological evidence that the ERK signaling pathway is activated through endogenously expressed CB1. These findings provide an insight into myometrial AEA signaling, highlighting a potential role for endocannabinoids in the regulation of gene expression in myometrial smooth muscle cells.
Molecular Endocrinology 06/2009; 23(9):1415-27. · 4.54 Impact Factor
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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.
Molecular Endocrinology 05/2009; 23(8):1272-80. · 4.54 Impact Factor
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ABSTRACT: Arterial smooth muscle (ASM) contraction plays a critical role in regulating blood distribution and blood pressure. Vasoconstrictors activate cell surface receptors to initiate signaling cascades involving increased intracellular Ca(2+) concentration ([Ca(2+)](i)) and recruitment of protein kinase C (PKC), leading to ASM contraction, though the PKC isoenzymes involved vary between different vasoconstrictors and their actions. Here, we have used confocal microscopy of enhanced green fluorescence protein (eGFP)-labeled PKC isoenzymes to visualize PKC translocation in primary rat mesenteric ASM cells in response to physiological vasoconstrictors, with simultaneous imaging of Ca(2+) signaling. Endothelin-1, angiotensin II, and uridine triphosphate all caused translocation of each of the PKC isoenzymes alpha, delta, and epsilon; however, the kinetics of translocation varied between agonists and PKC isoenzymes. Translocation of eGFP-PKCalpha mirrored the rise in [Ca(2+)](i), while that of eGFP-PKCdelta or -epsilon occurred more slowly. Endothelin-induced translocation of eGFP-PKCepsilon was often sustained for several minutes, while responses to angiotensin II were always transient. In addition, preventing [Ca(2+)](i) increases using 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl) ester prevented eGFP-PKCalpha translocation, while eGFP-PKCdelta translocated more rapidly. Our results suggest that PKC isoenzyme specificity of vasoconstrictor actions occurs downstream of PKC recruitment and demonstrate the varied kinetics and complex interplay between Ca(2+) and PKC responses to different vasoconstrictors in ASM.
AJP Cell Physiology 11/2008; 295(6):C1590-601. · 3.54 Impact Factor
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ABSTRACT: Histamine stimulates uterine contraction; however, little is known regarding the mechanism or regulation of uterine histamine receptor signaling. Here we investigated the regulation of Galpha(q/11)-coupled histamine receptor signaling in human myometrial smooth muscle cells using the inositol 1,4,5-trisphosphate biosensor pleckstrin homology domain of phospholipase-delta1 tagged to enhanced green fluorescent protein and the Ca(2+)-sensitive dye Fluo-4. Histamine addition caused concentration-dependent increases in inositol 1,4,5-trisphosphate and [Ca(2+)](i) in the ULTR human uterine smooth muscle cell line and primary human myometrial cells. These effects were completely inhibited by the H(1) histamine receptor antagonist, diphenhydramine, and were unaffected by the H(2) histamine receptor antagonist, cimetidine. ULTR and primary myometrial cells were transfected with either dominant-negative G protein-coupled receptor kinases (GRKs) or small interfering RNAs targeting specific GRKs to assess the roles of this protein kinase family in H(1) histamine receptor desensitization. Dominant-negative GRK2, but not GRK5 or GRK6, prevented H(1) histamine receptor desensitization. Similarly, transfection with short interfering RNAs (that each caused >70% depletion of the targeted GRK) for GRK2, but not GRK3 or GRK6, also prevented H(1) histamine receptor desensitization. Our data suggest that histamine stimulates phospholipase C-signaling in myometrial smooth muscle cells through H(1) histamine receptors and that GRK2 recruitment is a key mechanism in the regulation of H(1) histamine receptor signaling in human uterine smooth muscle. These data provide insights into the in situ regulation of this receptor subtype and may inform pathophysiological functioning in preterm labor and other conditions involving uterine smooth muscle dysregulation.
Molecular Endocrinology 06/2008; 22(8):1893-907. · 4.54 Impact Factor
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ABSTRACT: To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-G(q/11)alpha-binding, catalytically-inactive (D110A,K220R)G protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCepsilon, but not Ca2+/diacylglycerol-sensitive eGFP-PKCbetaII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.
Journal of Neurochemistry 01/2008; 103(6):2268-80. · 4.06 Impact Factor
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ABSTRACT: A single asparagine-to-tyrosine point mutation in the human M muscarinic acetylcholine (mACh) receptor at residue 514 (N514Y) resulted in a marked increase (approximately 300%) in agonist-independent [3H]inositol phosphate ([3H]IPx) accumulation compared with the response observed for the wild-type (WT) receptor. All the antagonists tested were able to inhibit both the WT-M3 and (N514Y)M3 mACh receptor-mediated basal [3H]IPx accumulation in a concentration-dependent manner. However, significant differences in both potency and binding affinity were only seen for those antagonists that possess greater receptor affinity. Despite being transfected with equivalent amounts of cDNA, cells expressed the (N514Y)M3 mACh receptor at levels that were only 25 to 30% of those seen for the WT receptor. Differences in the ability of chronic antagonist exposure to up-regulate (N514Y)M3 mACh receptor expression levels were also seen, with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) producing only 50% of the receptor up-regulation produced by atropine or pirenzepine. Basal phosphorylation of the (N514Y)M3 mACh receptor was approximately 100% greater than that seen for the WT-M3 receptor. The ability of antagonists to decrease basal (N514Y)M3 mACh receptor phosphorylation revealed differences in inverse-agonist efficacy. Atropine, 4-DAMP, and pirenzepine all reduced basal phosphorylation to similar levels, whereas methoctramine, a full inverse agonist with respect to reducing agonist-independent [3H]IPx accumulation, produced no significant attenuation of basal receptor phosphorylation. This study shows that mACh receptor inverse agonists can exhibit differential signaling profiles, which are dependent on the specific pathway investigated, and therefore provides evidence that the molecular mechanism of inverse agonism is likely to be more complex than the stabilization of a single inactive receptor conformation.
Journal of Pharmacology and Experimental Therapeutics 07/2006; 317(3):1134-42. · 3.83 Impact Factor
