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1.  Oxytocin decreases colonic motility of cold water stressed rats via oxytocin receptors 
World Journal of Gastroenterology : WJG  2014;20(31):10886-10894.
AIM: To investigate whether cold water intake into the stomach affects colonic motility and the involvement of the oxytocin-oxytocin receptor pathway in rats.
METHODS: Female Sprague Dawley rats were used and some of them were ovariectomized. The rats were subjected to gastric instillation with cold (0-4 °C, cold group) or room temperature (20-25 °C, control group) saline for 14 consecutive days. Colon transit was determined with a bead inserted into the colon. Colonic longitudinal muscle strips were prepared to investigate the response to oxytocin in vitro. Plasma concentration of oxytocin was detected by ELISA. Oxytocin receptor expression was investigated by Western blot analysis. Immunohistochemistry was used to locate oxytocin receptors.
RESULTS: Colon transit was slower in the cold group than in the control group (P < 0.05). Colonic smooth muscle contractile response to oxytocin decreased, and the inhibitory effect of oxytocin on muscle contractility was enhanced by cold water intake (0.69 ± 0.08 vs 0.88 ± 0.16, P < 0.05). Atosiban and tetrodotoxin inhibited the effect of oxytocin on colonic motility. Oxytocin receptors were located in the myenteric plexus, and their expression was up-regulated in the cold group (P < 0.05). Cold water intake increased blood concentration of oxytocin, but this effect was attenuated in ovariectomized rats (286.99 ± 83.72 pg/mL vs 100.56 ± 92.71 pg/mL, P < 0.05). However, in ovariectomized rats, estradiol treatment increased blood oxytocin, and the response of colonic muscle strips to oxytocin was attenuated.
CONCLUSION: Cold water intake inhibits colonic motility partially through oxytocin-oxytocin receptor signaling in the myenteric nervous system pathway, which is estrogen dependent.
PMCID: PMC4138467  PMID: 25152590
Intragastric cold water stress; Colonic motility; Estradiol; Oxytocin; Oxytocin receptor; Irritable bowel syndrome
2.  Pentraxin 3 (PTX3) Expression in Allergic Asthmatic Airways: Role in Airway Smooth Muscle Migration and Chemokine Production 
PLoS ONE  2012;7(4):e34965.
Pentraxin 3 (PTX3) is a soluble pattern recognition receptor with non-redundant functions in inflammation and innate immunity. PTX3 is produced by immune and structural cells. However, very little is known about the expression of PTX3 and its role in allergic asthma.
Objectives and Methods
We sought to determine the PTX3 expression in asthmatic airways and its function in human airway smooth muscle cells (HASMC). In vivo PTX3 expression in bronchial biopsies of mild, moderate and severe asthmatics was analyzed by immunohistochemistry. PTX3 mRNA and protein were measured by real-time RT-PCR and ELISA, respectively. Proliferation and migration were examined using 3H-thymidine incorporation, cell count and Boyden chamber assays.
PTX3 immunoreactivity was increased in bronchial tissues of allergic asthmatics compared to healthy controls, and mainly localized in the smooth muscle bundle. PTX3 protein was expressed constitutively by HASMC and was significantly up-regulated by TNF, and IL-1β but not by Th2 (IL-4, IL-9, IL-13), Th1 (IFN-γ), or Th-17 (IL-17) cytokines. In vitro, HASMC released significantly higher levels of PTX3 at the baseline and upon TNF stimulation compared to airway epithelial cells (EC). Moreover, PTX3 induced CCL11/eotaxin-1 release whilst inhibited the fibroblast growth factor-2 (FGF-2)-driven HASMC chemotactic activity.
Our data provide the first evidence that PTX3 expression is increased in asthmatic airways. HASMC can both produce and respond to PTX3. PTX3 is a potent inhibitor of HASMC migration induced by FGF-2 and can upregulate CCL11/eotaxin-1 release. These results raise the possibility that PTX3 may play a dual role in allergic asthma.
PMCID: PMC3329534  PMID: 22529962
3.  CADM1 Is a Key Receptor Mediating Human Mast Cell Adhesion to Human Lung Fibroblasts and Airway Smooth Muscle Cells 
PLoS ONE  2013;8(4):e61579.
Mast cells (MCs) play a central role in the development of many diseases including asthma and pulmonary fibrosis. Interactions of human lung mast cells (HLMCs) with human airway smooth muscle cells (HASMCs) are partially dependent on adhesion mediated by cell adhesion molecule-1 (CADM1), but the adhesion mechanism through which HLMCs interact with human lung fibroblasts (HLFs) is not known. CADM1 is expressed as several isoforms (SP4, SP1, SP6) in HLMCs, with SP4 dominant. These isoforms differentially regulate HLMC homotypic adhesion and survival.
In this study we have investigated the role of CADM1 isoforms in the adhesion of HLMCs and HMC-1 cells to primary HASMCs and HLFs.
CADM1 overexpression or downregulation was achieved using adenoviral delivery of CADM1 short hairpin RNAs or isoform-specific cDNAs respectively.
Downregulation of CADM1 attenuated both HLMC and HMC-1 adhesion to both primary HASMCs and HLFs. Overexpression of either SP1 or SP4 isoforms did not alter MC adhesion to HASMCs, whereas overexpression of SP4, but not SP1, significantly increased both HMC-1 cell and HLMC adhesion to HLFs. The expression level of CADM1 SP4 strongly predicted the extent of MC adhesion; linear regression indicated that CADM1 accounts for up to 67% and 32% of adhesion to HLFs for HMC-1 cells and HLMCs, respectively. HLFs supported HLMC proliferation and survival through a CADM1-dependent mechanism. With respect to CADM1 counter-receptor expression, HLFs expressed both CADM1 and nectin-3, whereas HASMCs expressed only nectin-3.
Conclusion and Clinical Relevance
Collectively these data indicate that the CADM1 SP4 isoform is a key receptor mediating human MC adhesion to HASMCs and HLFs. The differential expression of CADM1 counter-receptors on HLFs compared to HASMCs may allow the specific targeting of either HLMC-HLF or HLMC-HASMC interactions in the lung parenchyma and airways.
PMCID: PMC3631237  PMID: 23620770
4.  Distinct phospholipase A2 enzymes regulate prostaglandin E2 and F2alpha production by bovine endometrial epithelial cells 
The rate-limiting step in prostaglandin (PG) biosynthesis is catalyzed by phospholipase A2 (PLA2) enzymes which hydrolyze arachidonic acid from membrane phospholipids. Despite their importance in uterine PG production, little is known concerning the specific PLA2 enzymes that regulate arachidonic acid liberation in the uterine endometrium. The objectives of this study were to evaluate the expression and activities of calcium-independent Group VI and Group IVC PLA2 (PLA2G6 and PLA2G4C) and calcium-dependent Group IVA PLA2 (PLA2G4A) enzymes in the regulation of bovine uterine endometrial epithelial cell PG production.
Bovine endometrial epithelial cells in culture were treated with oxytocin, interferon-tau and the PLA2G6 inhibitor bromoenol lactone, alone and in combination. Concentrations of PGF2alpha and PGE2 released into the medium were analyzed. Western blot analysis was performed on cellular protein to determine the effects of treatments on expression of PLA2G4A, PLA2G6 and PLA2G4C. Group-specific PLA2 activity assays were performed on cell lysates following treatment with oxytocin, interferon-tau or vehicle (control), alone and in combination. To further evaluate the role of specific PLA2 enzymes in uterine cell PG biosynthesis, cells were transfected with cDNAs encoding human PLA2G6 and PLA24C, treated as described above and PG assays performed.
Constitutive cell production of PGF2alpha was about two-fold higher than PGE2. Oxytocin stimulated production of both PGs but the increase of PGF2alpha was significantly greater. Interferon-tau diminished oxytocin stimulation of both PGs. The PLA2G6 inhibitor, bromoenol lactone, abolished oxytocin-stimulated production of PGF2alpha. Treatments had little effect on PLA2G4A protein expression. In contrast, oxytocin enhanced expression of PLA2G6 and this effect was diminished in the presence of interferon-tau. Expression of PLA2G4C was barely detectable in control and oxytocin treated cells but it was enhanced in cells treated with interferon-tau. Oxytocin stimulated PLA2 activity in assays designed to evaluate PLA2G6 activity and interferon-tau inhibited this response. In assays designed to measure PLA2G4C activity, only interferon-tau was stimulatory. Cells overexpressing PLA2G6 produced similar quantities of the two PGs and these values were significantly higher than PG production by non-transfected cells. Oxytocin stimulated production of both PGs and this response was inhibited by interferon-tau. Bromoenol lactone inhibited oxtocin stimulation of PGF2alpha production but stimulated PGE2 production, both in the absence and presence of oxytocin. Cells over-expressing PLA2G4C produced more PGE2 than PGF2alpha and interferon-tau stimulated PGE2 production.
Results from these studies indicate that oxytocin stimulation of uterine PGF2alpha production is mediated, at least in part, by up-regulation of PLA2G6 expression and activity. In addition to its known inhibitory effect on oxytocin receptor expression, interferon-tau represses oxytocin-stimulated PLA2G6 expression and activity and this contributes to diminished PGF2alpha production. Furthermore, endometrial cell PGE2 biosynthesis was associated with PLA2G4C expression and activity and interferon-tau was stimulatory to this process.
PMCID: PMC1868772  PMID: 17459165
5.  Regulation of IL-17A responses in human airway smooth muscle cells by Oncostatin M 
Respiratory Research  2015;16(1):14.
Regulation of human airway smooth muscle cells (HASMC) by cytokines contributes to chemotactic factor levels and thus to inflammatory cell accumulation in lung diseases. Cytokines such as the gp130 family member Oncostatin M (OSM) can act synergistically with Th2 cytokines (IL-4 and IL-13) to modulate lung cells, however whether IL-17A responses by HASMC can be altered is not known.
To determine the effects of recombinant OSM, or other gp130 cytokines (LIF, IL-31, and IL-6) in regulating HASMC responses to IL-17A, assessing MCP-1/CCL2 and IL-6 expression and cell signaling pathways.
Cell responses of primary HASMC cultures were measured by the assessment of protein levels in supernatants (ELISA) and mRNA levels (qRT-PCR) in cell extracts. Activation of STAT, MAPK (p38) and Akt pathways were measured by immunoblot. Pharmacological agents were used to assess the effects of inhibition of these pathways.
OSM but not LIF, IL-31 or IL-6 could induce detectable responses in HASMC, elevating MCP-1/CCL2, IL-6 levels and activation of STAT-1, 3, 5, p38 and Akt cell signaling pathways. OSM induced synergistic action with IL-17A enhancing MCP-1/CCL-2 and IL-6 mRNA and protein expression, but not eotaxin-1 expression, while OSM in combination with IL-4 or IL-13 synergistically induced eotaxin-1 and MCP-1/CCL2. OSM elevated steady state mRNA levels of IL-4Rα, OSMRβ and gp130, but not IL-17RA or IL-17RC. Pharmacologic inhibition of STAT3 activation using Stattic down-regulated OSM, OSM/IL-4 or OSM/IL-13, and OSM/IL-17A synergistic responses of MCP-1/CCL-2 induction, whereas, inhibitors of Akt and p38 MAPK resulted in less reduction in MCP-1/CCL2 levels. IL-6 expression was more sensitive to inhibition of p38 (using SB203580) and was affected by Stattic in response to IL-17A/OSM stimulation.
Oncostatin M can regulate HASMC responses alone or in synergy with IL-17A. OSM/IL-17A combinations enhance MCP-1/CCL2 and IL-6 but not eotaxin-1. Thus, OSM through STAT3 activation of HASMC may participate in inflammatory cell recruitment in inflammatory airway disease.
Electronic supplementary material
The online version of this article (doi:10.1186/s12931-014-0164-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4332894
Asthma; Cytokines; Chemokines; STAT signaling; Oncostatin M; Airway smooth muscle
6.  Role of Transient Receptor Potential C3 in TNF-α–Enhanced Calcium Influx in Human Airway Myocytes 
Previous studies have suggested that the proinflammatory cytokine, TNF-α, contributes to airway hyperresponsivness by altering airway smooth muscle (ASM) Ca2+ responses to agonist stimulation. The present study examined the effects of TNF-α on Ca2+ influx pathways in cultured human ASM cells (HASMCs). Proteins encoded by the transient receptor potential (TRP) gene family function as channels through which receptor-operated and store-operated Ca2+ entry (SOCE) occur. In the present study, the presence of TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 mRNA and protein expression was confirmed in cultured HASMCs using RT-PCR and Western blot analysis. TNF-α treatment significantly increased TRPC3 mRNA and protein levels in HASMCs as well as SOCE. TNF-α treatment also increased both the peak and plateau intracellular Ca2+ concentration responses in HASMCs elicited by acetylcholine and bradykinin. The effects of TNF-α treatment on SOCE and agonist-induced intracellular Ca2+ concentration responses were attenuated using small interfering RNA transfection, which knocked down TRPC3 expression. Thus, in inflammatory airway diseases, TNF-α treatment may result in increased myocyte activation due to altered Ca2+ influx pathways. These results suggest that TRPC3 may be an important therapeutic target in inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease.
PMCID: PMC2643259  PMID: 16574942
human airway smooth muscle; receptor-operated calcium entry; store-operated calcium entry; TNF-α; transient receptor potential channels
7.  An Interaction of Oxytocin Receptors with Metabotropic Glutamate Receptors in Hypothalamic Astrocytes 
Journal of neuroendocrinology  2009;21(12):1001-1006.
Hypothalamic astrocytes play a critical role in the regulation and support of many different neuroendocrine events, and are affected by estradiol. Both nuclear and membrane estrogen receptors (ERs) are expressed in astrocytes. Upon estradiol activation, membrane-associated ER (mER) signals through the type 1a metabotropic glutamate receptor (mGluR1a) to induce an increase of free cytoplasmic calcium concentration ([Ca2+]i). Since the expression of oxytocin receptors (OTRs) is modulated by estradiol, we tested if estradiol also influences oxytocin signaling. Oxytocin at 1 nM, 10 nM, and 100 nM induced a [Ca2+]i flux measured as a change in relative fluorescence (ΔF Ca2+ = 330 ± 17 relative fluorescent units (RFU), ΔF Ca2+ = 331 ± 22 RFU, and ΔF Ca2+ = 347 ± 13 RFU, respectively) in primary cultures of female post-pubertal hypothalamic astrocytes. Interestingly, OTRs interacted with mGluRs. The mGluR1a antagonist, LY 367385 (20 nM), blocked the oxytocin (1 nM)-induced [Ca2+]i flux (ΔF Ca2+ = 344 ± 19 vs. 127 ± 11 RFU, P < 0.001). Conversely, the mGluR1a receptor agonist, (RS)-3,5-dihydroxyphenyl-glycine (DHPG, 100 nM), increased the oxytocin (1 nM)-induced [Ca2+]i response (ΔF Ca2+ = 670 ± 31 RFU) compared with either compound alone (P < 0.001). Since both oxytocin and estradiol rapidly signal through the mGluR1a, we treated hypothalamic astrocytes sequentially with oxytocin and estradiol to determine whether stimulation with one hormone affected the subsequent [Ca2+]i response to the second hormone. Estradiol treatment did not change the subsequent [Ca2+]i flux to oxytocin (P > 0.05) and previous oxytocin exposure did not affect the [Ca2+]i response to estradiol (P > 0.05). Furthermore, simultaneous estradiol and oxytocin stimulation failed to yield a synergistic [Ca2+]i response. These results suggest that the OTR signals through the mGluR1a to release Ca2+ from intracellular stores and rapid, non-genomic estradiol stimulation does not influence OTR signaling in astrocytes.
PMCID: PMC2804744  PMID: 19807846
OTR; mGluR1a; [Ca2+]i; rapid cell signaling; mER
8.  Pro-inflammatory mechanisms of muscarinic receptor stimulation in airway smooth muscle 
Respiratory Research  2010;11(1):130.
Acetylcholine, the primary parasympathetic neurotransmitter in the airways, plays an important role in bronchoconstriction and mucus production. Recently, it has been shown that acetylcholine, by acting on muscarinic receptors, is also involved in airway inflammation and remodelling. The mechanism(s) by which muscarinic receptors regulate inflammatory responses are, however, still unknown.
The present study was aimed at characterizing the effect of muscarinic receptor stimulation on cytokine secretion by human airway smooth muscle cells (hASMc) and to dissect the intracellular signalling mechanisms involved. hASMc expressing functional muscarinic M2 and M3 receptors were stimulated with the muscarinic receptor agonist methacholine, alone, and in combination with cigarette smoke extract (CSE), TNF-α, PDGF-AB or IL-1β.
Muscarinic receptor stimulation induced modest IL-8 secretion by itself, yet augmented IL-8 secretion in combination with CSE, TNF-α or PDGF-AB, but not with IL-1β. Pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely normalized the effect of methacholine on CSE-induced IL-8 secretion, whereas PMA, a PKC activator, mimicked the effects of methacholine, inducing IL-8 secretion and augmenting the effects of CSE. Similar inhibition was observed using inhibitors of IκB-kinase-2 (SC514) and MEK1/2 (U0126), both downstream effectors of PKC. Accordingly, western blot analysis revealed that methacholine augmented the degradation of IκBα and the phosphorylation of ERK1/2 in combination with CSE, but not with IL-1β in hASMc.
We conclude that muscarinic receptors facilitate CSE-induced IL-8 secretion by hASMc via PKC dependent activation of IκBα and ERK1/2. This mechanism could be of importance for COPD patients using anticholinergics.
PMCID: PMC2955662  PMID: 20875145
9.  Reduction in TRPC4 expression specifically attenuates G-protein coupled receptor-stimulated increases in intracellular calcium in human myometrial cells 
Cell calcium  2009;46(1):73-84.
Canonical transient receptor potential (TRPC) proteins may play a role in regulating changes in intracellular calcium ([Ca2+]i). Human myometrium expresses TRPC4, TRPC1 and TRPC6 mRNAs in greatest relative abundance. Contributions of TRPC4 to increases in [Ca2+]i were assessed in PHM1-41 and primary human uterine smooth muscle (UtSMC) cells using short hairpin RNAs (shRNAs). Based on a reporter assay screen, one shRNA was selected to construct an adenoviral expression vector (TC4sh1). TC4sh1 induced both mRNA and protein TRPC4 knockdown in PHM1-41 cells without affecting expression of other TRPCs. Signal-regulated Ca2+ entry (SRCE), defined as a stimulus- and extracellular Ca2+-dependent increase in [Ca2+]i, was measured in PHM1-41 cells treated with oxytocin (G-protein coupled receptor (GPCR)-stimulated), thapsigargin (store depletion-simulated), and OAG (diacylglycerol-stimulated), using Fura-2. Cells infected with TC4sh1 exhibited attenuated oxytocin-, ATP- and PGF2α–mediated SRCE, but no change in thapsigargin- or OAG-stimulated SRCE. Similar results were obtained in primary uterine smooth muscle cells. Additionally, cells expressing TC4sh1 exhibited a significantly smaller increase in channel activity in response to oxytocin administration than did cells infected with empty virus. These data show that, in human myometrial cells, knockdown of endogenous TRPC4 specifically attenuates GPCR-stimulated, but not thapsigargin- or OAG-stimulated extracellular calcium-dependent increases in [Ca2+]i. These data imply that, in this cellular context, the mechanisms regulating extracellular Ca2+-dependent increases in [Ca2+]i are differentially affected by different signaling pathways.
PMCID: PMC2866636  PMID: 19523685
10.  Innate Immune Receptors in Human Airway Smooth Muscle Cells: Activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 Agonists 
PLoS ONE  2013;8(7):e68701.
Pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs), recognize microbial components and trigger a host defense response. Respiratory tract infections are common causes of asthma exacerbations, suggesting a role for PRRs in this process. The present study aimed to examine the expression and function of PRRs on human airway smooth muscle cells (HASMCs).
Expression of TLR, NLR and RLR mRNA and proteins was determined using real-time RT-PCR, flow cytometry and immunocytochemistry. The functional responses to ligand stimulation were investigated in terms of cytokine and chemokine release, cell surface marker expression, proliferation and proteins regulating the contractile state.
HASMCs expressed functional TLR2, TLR3, TLR4, TLR7 and NOD1. Stimulation with the corresponding agonists Pam3CSK4, poly(I:C), LPS, R-837 and iE-DAP, respectively, induced IL-6, IL-8 and GM-CSF release and up-regulation of ICAM-1 and HLA-DR, while poly(I:C) also affected the release of eotaxin and RANTES. The proliferative response was slightly increased by LPS. Stimulation, most prominently with poly(I:C), down-regulated myosin light chain kinase and cysteinyl leukotriene 1 receptor expression and up-regulated β2-adrenoceptor expression. No effects were seen for agonist to TLR2/6, TLR5, TLR8, TLR9, NOD2 or RIG-I/MDA-5.
Activation of TLR2, TLR3, TLR4, TLR7 and NOD1 favors a synthetic phenotype, characterized by an increased ability to release inflammatory mediators, acquire immunomodulatory properties by recruiting and interacting with other cells, and reduce the contractile state. The PRRs might therefore be of therapeutic use in the management of asthma and infection-induced disease exacerbations.
PMCID: PMC3701658  PMID: 23861935
11.  DNA Methylation of Specific CpG Sites in the Promoter Region Regulates the Transcription of the Mouse Oxytocin Receptor 
PLoS ONE  2013;8(2):e56869.
Oxytocin is a peptide hormone, well known for its role in labor and suckling, and most recently for its involvement in mammalian social behavior. All central and peripheral actions of oxytocin are mediated through the oxytocin receptor, which is the product of a single gene. Transcription of the oxytocin receptor is subject to regulation by gonadal steroid hormones, and is profoundly elevated in the uterus and mammary glands during parturition. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression of the oxytocin receptor in individuals with autism. Here, we hypothesized that transcription of the mouse oxytocin receptor is regulated by DNA methylation of specific sites in its promoter, in a tissue-specific manner. Hypothalamus-derived GT1-7, and mammary-derived 4T1 murine cell lines displayed negative correlations between oxytocin receptor transcription and methylation of the gene promoter, and demethylation caused a significant enhancement of oxytocin receptor transcription in 4T1 cells. Using a reporter gene assay, we showed that methylation of specific sites in the gene promoter, including an estrogen response element, significantly inhibits transcription. Furthermore, methylation of the oxytocin receptor promoter was found to be differentially correlated with oxytocin receptor expression in mammary glands and the uterus of virgin and post-partum mice, suggesting that it plays a distinct role in oxytocin receptor transcription among tissues and under different physiological conditions. Together, these results support the hypothesis that the expression of the mouse oxytocin receptor gene is epigenetically regulated by DNA methylation of its promoter.
PMCID: PMC3575498  PMID: 23441222
12.  The oxytocin/vasopressin receptor antagonist atosiban delays the gastric emptying of a semisolid meal compared to saline in human 
BMC Gastroenterology  2006;6:11.
Oxytocin is released in response to a meal. Further, mRNA for oxytocin and its receptor have been found throughout the gastrointestinal (GI) tract. The aim of this study was therefore to examine whether oxytocin, or the receptor antagonist atosiban, influence the gastric emptying.
Ten healthy volunteers (five men) were examined regarding gastric emptying at three different occasions: once during oxytocin stimulation using a pharmacological dose; once during blockage of the oxytocin receptors (which also blocks the vasopressin receptors) and thereby inhibiting physiological doses of oxytocin; and once during saline infusion.
Gastric emptying rate (GER) was assessed and expressed as the percentage reduction in antral cross-sectional area from 15 to 90 min after ingestion of rice pudding. The assessment was performed by real-time ultrasonography. At the same time, the feeling of satiety was registered using visual satiety scores.
Inhibition of the binding of endogenous oxytocin by the receptor antagonist delayed the GER by 37 % compared to saline (p = 0.037). In contrast, infusion of oxytocin in a dosage of 40 mU/min did not affect the GER (p = 0.610). Satiation scores areas in healthy subjects after receiving atosiban or oxytocin did not show any significant differences.
Oxytocin and/or vasopressin seem to be regulators of gastric emptying during physiological conditions, since the receptor antagonist atosiban delayed the GER. However, the actual pharmacological dose of oxytocin in this study had no effect. The effect of oxytocin and vasopressin on GI motility has to be further evaluated.
PMCID: PMC1434749  PMID: 16542457
13.  Oxytocin: an unexpected risk for cardiologic and broncho-obstructive effects, and allergic reactions in susceptible delivering women 
Oxytocin (Sintocynon) is considered an uncommon cause of severe allergic reactions during delivery. We have recently shown that allergic sensitization to latex might constitute an important predisposing risk factor for anaphylaxis after the first infusion of oxytocin during delivery.
Some oxytocin cardiovascular activities such as lowering blood pressure, negative cardiac inotropy and cronotropy, parasympathetic neuromodulation, vasodilatation etc. can induce significant side effects mimicking cardiac anaphylaxis, and constitute an additional differential diagnostic problem in delivering women with suspected or real allergic background. Finally, some ex vivo models have shown that oxytocin, under pro-inflammatory cytokines stimulation, such as those occurring in asthma, may induce contraction of smooth muscle and airway narrowing.
This background suggests that allergic sensitization to latex allergens constitutes a significant but underestimated risk factor for triggering severe systemic reactions after the infusion of oxytocin and, consequently, there is a need of particular attention in managing delivering women suffering from latex allergy and bronchial asthma. An accurate anamnestic, clinical and diagnostic evaluation, latex-free anesthesiological setting, use of oxytocin-alternative agents and, if necessary, a drug premedication are likely to reduce the risk of anaphylactic/broncho-obstructive reactions in these women.
PMCID: PMC4014859  PMID: 24139438
Anaphylaxis; Bronchial asthma; Delivery; Drug allergy; Heart; Hypersensitivity; Latex allergy; Oxytocin; Oxytocin allergy; Oxytocin and heart; Oxytocin side effects
14.  The Influence of Oxytocin Administration on Responses to Infant Faces and Potential Moderation by OXTR Genotype 
Psychopharmacology  2012;224(4):469-476.
Oxytocin is a neuropeptide that is associated with increases in social affiliative behaviors, particularly toward infants. However, no previous study has investigated healthy adults’ responses to infant faces following oxytocin administration. In addition, given that preliminary evidence suggests that a single nucleotide polymorphism (SNP) of the oxytocin receptor (OXTR) gene, rs53576, may influence behaviors associated with parental sensitivity, we assessed whether such responses vary according to OXTR rs53576 genotype.
The present study assessed the effects of intranasally administered oxytocin and OXTR genotype on human adults’ preferences for infant faces.
A double-blind, between-groups design was used, with 57 genotyped volunteers randomly assigned to receive intranasally administered oxytocin or placebo. Fifty minutes following the administration of oxytocin or placebo, participants viewed infants’ and adults’ faces showing neutral expressions and assessed how appealing they found each face.
Infants’ faces were more strongly preferred following oxytocin inhalation relative to placebo. When participants were separated according to genotype, this effect was only observed for participants homozygous for the rs53576G allele. Parallel effects were not seen for adults’ faces.
The present results are consistent with the hypothesis that acute oxytocin administration increases sensitivity to reward-relevant features of infants and/or reduces sensitivity to their aversive properties. The results also are consistent with suggestions of more efficient oxytocinergic function in rs53576G homozygotes.
PMCID: PMC3500580  PMID: 22763666
Oxytocin; OXTR; parental; faces; affiliation
15.  Advanced glycation end products promote human aortic smooth muscle cell calcification in vitro via activating NF-κB and down-regulating IGF1R expression 
Acta Pharmacologica Sinica  2013;34(4):480-486.
To investigate the effects of advanced glycation end products (AGEs) on calcification in human aortic smooth muscle cells (HASMCs) in vitro and the underlying mechanisms.
AGEs were artificially prepared. Calcification of HASMCs was induced by adding inorganic phosphate (Pi, 2 mmol/L) in the media, and observed with Alizarin red staining. The calcium content in the supernatant was measured using QuantiChrome Calcium Assay Kit. Expression of the related mRNAs and proteins was analyzed using real-time PCR and Western blot, respectively. Chromatin immunoprecipitation (ChIP) assay was used to detect the binding of NF-κB to the putative IGF1R promoter.
AGEs (100 μg/mL) significantly enhanced Pi-induced calcification and the levels of osteocalcin and Cbfα1 in HASMCs. Furthermore, the treatment decreased the expression of insulin-like growth factor 1 receptor (IGF1R). Over-expression of IGF1R in HASMCs suppressed the AGEs-induced increase in calcium deposition. When IGF1R expression was knocked down in HASMCs, AGEs did not enhance the calcium deposition. Meanwhile, AGEs time-dependently decreased the amounts of IκBα and Flag-tagged p65 in the cytoplasmic extracts, and increased the amount of nuclear p65 in HASMCs. In the presence of NF-κB inhibitor PDTC (50 μmol/L), the AGEs-induced increase in calcium deposition was blocked. Over-expression of p65 significantly enhanced Pi-induced mineralization, but suppressed IGF1R mRNA level. Knockdown of p65 suppressed the AGEs-induced increase in calcium deposition, and rescued the IGF1R expression. The ChIP analysis revealed that NF-κB bound the putative IGF1R promoter at position −230 to −219 bp. The inhibition of IGF1R by NF-κB was abolished when IGF1R reporter plasmid contained mutated binding sequence for NF-κB or an NF-κB reporter vector.
The results demonstrate that AGEs promote calcification of human aortic smooth muscle cells in vitro via activation of NF-κB and down-regulation of IGF1R expression.
PMCID: PMC4002782  PMID: 23416929
vascular calcification; advanced glycation end products; human aortic smooth muscle cell; osteocalcin; Cbfα1; insulin-like growth factor 1 receptor (IGF1R); nuclear factor-kappa B; PDTC
16.  Fibroblast activation protein is induced by inflammation and degrades type I collagen in thin-cap fibroatheromata 
European Heart Journal  2011;32(21):2713-2722.
Collagen degradation in atherosclerotic plaques with thin fibrous caps renders them more prone to rupture. Fibroblast activation protein (FAP) plays a role in arthritis and tumour formation through its collagenase activity. However, the significance of FAP in thin-cap human fibroatheromata remains unknown.
Methods and results
We detected enhanced FAP expression in type IV–V human aortic atheromata (n = 12), compared with type II–III lesions (n = 9; P < 0.01) and healthy aortae (n = 8; P < 0.01) by immunostaining and western blot analyses. Fibroblast activation protein was also increased in thin-cap (<65 µm) vs. thick-cap (≥65 µm) human coronary fibroatheromata (n = 12; P < 0.01). Fibroblast activation protein was expressed by human aortic smooth muscle cells (HASMC) as shown by colocalization on immunofluorescent aortic plaque stainings (n = 10; P < 0.01) and by flow cytometry in cell culture. Although macrophages did not express FAP, macrophage burden in human aortic plaques correlated with FAP expression (n = 12; R2= 0.763; P < 0.05). Enzyme-linked immunosorbent assays showed a time- and dose-dependent up-regulation of FAP in response to human tumour necrosis factor α (TNFα) in HASMC (n = 6; P < 0.01). Moreover, supernatants from peripheral blood-derived macrophages induced FAP expression in cultured HASMC (n = 6; P < 0.01), an effect abolished by blocking TNFα (n = 6; P < 0.01). Fibroblast activation protein associated with collagen-poor regions in human coronary fibrous caps and digested type I collagen and gelatin in vitro (n = 6; P < 0.01). Zymography revealed that FAP-mediated collagenase activity was neutralized by an antibody directed against the FAP catalytic domain both in HASMC (n = 6; P < 0.01) and in fibrous caps of atherosclerotic plaques (n = 10; P < 0.01).
Fibroblast activation protein expression in HASMC is induced by macrophage-derived TNFα. Fibroblast activation protein associates with thin-cap human coronary fibroatheromata and contributes to type I collagen breakdown in fibrous caps.
PMCID: PMC3205479  PMID: 21292680
Atherosclerosis; Antibodies; Collagen; Inflammation; Smooth muscle cells
17.  Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle 
Respiratory Research  2005;6(1):74.
Cigarette smoke is the leading risk factor for the development of chronic obstructive pulmonary disease (COPD) an inflammatory condition characterised by neutrophilic inflammation and release of proinflammatory mediators such as interleukin-8 (IL-8). Human airway smooth muscle cells (HASMC) are a source of proinflammatory cytokines and chemokines. We investigated whether cigarette smoke could directly induce the release of chemokines from HASMC.
HASMC in primary culture were exposed to cigarette smoke extract (CSE) with or without TNFα. Chemokines were measured by enzyme-linked immunosorbent assay (ELISA) and gene expression by real time polymerase chain reaction (PCR). Data were analysed using one-way analysis of variance (ANOVA) followed by Bonferroni's t test
CSE (5, 10 and 15%) induced IL-8 release and expression without effect on eotaxin or RANTES release. At 20%, there was less IL-8 release. TNFα enhanced CSE-induced IL-8 release and expression. However, CSE (5–30%) inhibited TNFα-induced eotaxin and RANTES production. The effects of CSE on IL-8 release were inhibited by glutathione (GSH) and associated with the induction of the oxidant sensing protein, heme oxygenase-1.
Cigarette smoke may directly cause the release of IL-8 from HASMC, an effect enhanced by TNF-α which is overexpressed in COPD. Inhibition of eotaxin and RANTES by cigarette smoke is consistent with the predominant neutrophilic but not eosinophilic inflammation found in COPD.
PMCID: PMC1187925  PMID: 16029496
18.  Sublingual Misoprostol versus Intramuscular Oxytocin for Prevention of Postpartum Hemorrhage in Uganda: A Double-Blind Randomized Non-Inferiority Trial 
PLoS Medicine  2014;11(11):e1001752.
In a double-blind randomized controlled trial, Esther Atukunda and colleagues evaluated whether sublingual misoprostol administered to women in labor was non-inferior to intramuscular oxytocin in preventing postpartum hemorrhage and reducing blood loss.
Please see later in the article for the Editors' Summary
Postpartum hemorrhage (PPH) is a leading cause of maternal death in sub-Saharan Africa. Although the World Health Organization recommends use of oxytocin for prevention of PPH, misoprostol use is increasingly common owing to advantages in shelf life and potential for sublingual administration. There is a lack of data about the comparative efficacy of oxytocin and sublingual misoprostol, particularly at the recommended dose of 600 µg, for prevention of PPH during active management of labor.
Methods and Findings
We performed a double-blind, double-dummy randomized controlled non-inferiority trial between 23 September 2012 and 9 September 2013 at Mbarara Regional Referral Hospital in Uganda. We randomized 1,140 women to receive 600 µg of misoprostol sublingually or 10 IU of oxytocin intramuscularly, along with matching placebos for the treatment they did not receive. Our primary outcome of interest was PPH, defined as measured blood loss ≥500 ml within 24 h of delivery. Secondary outcomes included measured blood loss ≥1,000 ml; mean measured blood loss at 1, 2, and 24 h after delivery; death; requirement for blood transfusion; hemoglobin changes; and use of additional uterotonics.
At 24 h postpartum, primary PPH occurred in 163 (28.6%) participants in the misoprostol group and 99 (17.4%) participants in the oxytocin group (relative risk [RR] 1.64, 95% CI 1.32 to 2.05, p<0.001; absolute risk difference 11.2%, 95% CI 6.44 to 16.1). Severe PPH occurred in 20 (3.6%) and 15 (2.7%) participants in the misoprostol and oxytocin groups, respectively (RR 1.33, 95% CI 0.69 to 2.58, p = 0.391; absolute risk difference 0.9%, 95% CI −1.12 to 2.88). Mean measured blood loss was 341.5 ml (standard deviation [SD] 206.2) and 304.2 ml (SD 190.8, p = 0.002) at 2 h and 484.7 ml (SD 213.3) and 432.8 ml (SD 203.5, p<0.001) at 24 h in the misoprostol and oxytocin groups, respectively. There were no significant differences between the two groups in any other secondary outcomes. Women in the misoprostol group more commonly experienced shivering (RR 1.91, 95% CI 1.65 to 2.21, p<0.001) and fevers (RR 5.20, 95% CI 3.15 to 7.21, p = 0.005).
This study was conducted at a regional referral hospital with capacity for emergency surgery and blood transfusion. High-risk women were excluded from participation.
Misoprostol 600 µg is inferior to oxytocin 10 IU for prevention of primary PPH in active management of labor. These data support use of oxytocin in settings where it is available. While not powered to do so, the study found no significant differences in rate of severe PPH, need for blood transfusion, postpartum hemoglobin, change in hemoglobin, or use of additional uterotonics between study groups. Further research should focus on clarifying whether and in which sub-populations use of oxytocin would be preferred over sublingual misoprostol.
Trial registration NCT01866241
Please see later in the article for the Editors' Summary
Editors' Summary
Every year, worldwide nearly 290,000 women die during pregnancy or labor or during the first six weeks after giving birth (the postpartum period). Almost all of these “maternal” deaths occur in low- or middle-income countries, and most are caused by a handful of preventable or treatable conditions—postpartum hemorrhage (severe bleeding from the uterus [womb] within 24 hours of childbirth), post-delivery infections, unsafe abortion, obstructive (difficult) labor, and blood pressure disorders during pregnancy. The leading cause of maternal deaths worldwide is postpartum hemorrhage, which is responsible for 25%–30% of all maternal deaths. Postpartum hemorrhage can be prevented by giving the mother an intramuscular injection of oxytocin, a hormone that stimulates uterine contractions and limits uterine bleeding, immediately after her child is born.
Why Was This Study Done?
Unfortunately, oxytocin needs to be kept cool, which limits its use in low- and middle-income countries, and, until recently, it was thought that only trained personnel could give intramuscular injections. Consequently, administration of misoprostol, a synthetic prostaglandin that has effects similar to those of oxytocin, has been proposed as an alternative way to prevent postpartum hemorrhage in resource-limited settings. Misoprostol is stable at room temperature, and because it can be given sublingually (beneath the tongue), it acts very quickly. However, the comparative efficacy of sublingual misoprostol and intramuscular oxytocin for the prevention of postpartum hemorrhage has not been established. Here, the researchers undertake a double-blinded, double-dummy randomized controlled non-inferiority trial to compare sublingual misoprostol and intramuscular oxytocin for the prevention of postpartum hemorrhage in Uganda, a country where there are more than 5,500 maternal deaths every year. A randomized controlled trial compares the outcomes of individuals assigned to different interventions through the play of chance. In a double-blinded trial, neither the researchers nor the participants know who is receiving which intervention. In this particular trial, double-blinding is achieved by giving a dummy (placebo) sublingual pill to the women assigned to the oxytocin group and a dummy injection to the women assigned to the misoprostol group, as well as their assigned treatments. A non-inferiority trial investigates whether one treatment is not worse than another treatment.
What Did the Researchers Do and Find?
The researchers measured blood loss over the first 24 hours after delivery in 1,140 women admitted to a regional referral hospital in Uganda. The women were given either sublingual misoprostol or intramuscular oxytocin at the currently recommended doses, along with matching placebos, immediately after the birth of their child. Postpartum hemorrhage (defined as the loss of more than 500 ml of blood within 24 hours of delivery; the trial's primary outcome) occurred in 28.6% and 17.4% of the women in the misoprostol and oxytocin groups, respectively (an absolute risk difference of 11.2%). Severe postpartum hemorrhage (loss of more than 1,000 ml of blood within 24 hours of delivery) occurred in 3.6% and 2.7% of participants in the misoprostol and oxytocin groups, respectively, but this difference was not statistically significant (it could have happened by chance). On average, women given misoprostol had lost slightly more blood by two and 24 hours after delivery than those given oxytocin. There were no significant differences between the groups in terms of death, the need for blood transfusion, or the use of additional drugs to prevent blood loss, but women given misoprostol experienced shivering and fever more often than those given oxytocin.
What Do These Findings Mean?
In their study protocol, the researchers specified that sublingual misoprostol would be deemed non-inferior to intramuscular oxytocin if the absolute risk difference for postpartum hemorrhage between the misoprostol and oxytocin treatment groups was less than 6% (the “non-inferiority” margin). These findings therefore indicate that sublingual misoprostol given at the recommended dose is inferior to intramuscular oxytocin for the prevention of postpartum hemorrhage in women undergoing an uncomplicated birth at a regional referral hospital in Uganda. Although several aspects of this study may affect the accuracy and generalizability of its findings (for example, women at high risk of birth complications were excluded from the study), the researchers conclude that oxytocin should remain the preferred agent for the prevention of postpartum hemorrhage where it is available. However, they note, sublingual misoprostol remains important for the prevention of postpartum hemorrhage where oxytocin is unavailable or its administration is not feasible.
Additional Information
Please access these websites via the online version of this summary at
The United Nations Children's Fund (UNICEF) provides information on maternal mortality; “Trends in Maternal Mortality: 1990 to 2013” is a recent WHO/UNICEF/UNFPA/World Bank publication that provides up-to-date information on maternal mortality worldwide
The World Health Organization provides information on maternal health (in several languages)
The Postpartum Hemorrhage Prevention and Treatment Website provides a forum for information sharing and learning between organizations and individuals working on the prevention and treatment of postpartum hemorrhage in developing countries; the website includes basic information about postpartum hemorrhage and links to additional resources
“Veil of Tears” contains personal stories (including stories about postpartum hemorrhage) from Afghanistan about loss in childbirth
“Maternal Death: The Avoidable Crisis” is a briefing paper published by Médecins Sans Frontières in 2012
More information about this trial is available
PMCID: PMC4219663  PMID: 25369200
19.  Variation in Oxytocin Receptor Density in the Nucleus Accumbens has Differential Effects on Affiliative Behaviors in Monogamous and Polygamous Voles 
Oxytocin receptors in the nucleus accumbens have been implicated in the regulation of alloparental behavior and pair bond formation in the socially monogamous prairie vole. Oxytocin receptor density in the nucleus accumbens is positively correlated with alloparenting in juvenile and adult female prairie voles, and oxytocin receptor antagonist infused into the nucleus accumbens blocks this behavior. Furthermore, prairie voles have higher densities of oxytocin receptors in the accumbens than non-monogamous rodent species, and blocking accumbal oxytocin receptors prevents mating-induced partner preference formation. Here we used adeno-associated viral vector gene transfer to examine the functional relationship between accumbal oxytocin receptor density and social behavior in prairie and meadow voles. Adult female prairie voles that over-express oxytocin receptor in the nucleus accumbens displayed accelerated partner preference formation after cohabitation with a male, but did not display enhanced alloparental behavior. However, partner preference was not facilitated in non-monogamous meadow voles by introducing oxytocin receptor into the nucleus accumbens. These data confirm a role for oxytocin receptor in the accumbens in the regulation of partner preferences in female prairie voles, and suggest that oxytocin receptor expression in the accumbens is not sufficient to promote partner preferences in non-monogamous species. These data are the first to demonstrate a direct relationship between oxytocin receptor density in the nucleus accumbens and variation in social attachment behaviors. Thus, individual variation in oxytocin receptor expression in the striatum may contribute to natural diversity in social behaviors.
PMCID: PMC2768419  PMID: 19193878
maternal; virus; preference; neuropeptide; cognition; autoradiography
20.  The Nonpeptide Oxytocin Receptor Agonist WAY 267,464: Receptor-Binding Profile, Prosocial Effects and Distribution of c-Fos Expression in Adolescent Rats 
Journal of neuroendocrinology  2012;24(7):1012-1029.
Previous research suggests that the nonpeptide oxytocin receptor (OTR) agonist WAY 267,464 may only partly mimic the effects of oxytocin in rodents. The present study further explored these differences and related them to OTR and vasopressin 1a receptor (V1aR) pharmacology and regional patterns of c-Fos expression. Binding data for WAY 267,464 and oxytocin were obtained by displacement binding assays on cellular membranes, while functional receptor data were generated by luciferase reporter assays. For behavioural testing, adolescent rats were tested in a social preference paradigm, the elevated plus-maze (EPM) and for locomotor activity changes following WAY 267,464 (10 and 100 mg/kg, i.p.) or oxytocin (0.1 and 1 mg/kg, i.p.). The higher doses were also examined for their effects on regional c-Fos expression. Results showed that WAY 267,464 had higher affinity (Ki) at the V1aR than the OTR (113 versus 978 nm). However, it had no functional response at the V1aR and only a weak functional effect (EC50) at the OTR (881 nm). This suggests WAY 267,464 is an OTR agonist with weak affinity and a possible V1aR antagonist. Oxytocin showed high binding at the OTR (1.0 nm) and V1aR (503 nm), with a functional EC50 of 9.0 and 59.7 nm, respectively, indicating it is a potent OTR agonist and full V1aR agonist. WAY 267,464 (100 mg/kg), but not oxytocin, significantly increased the proportion of time spent with a live rat, over a dummy rat, in the social preference test. Neither compound affected EPM behaviour, whereas the higher doses of WAY 267,464 and oxytocin suppressed locomotor activity. WAY 267,464 and oxytocin produced similar c-Fos expression in the paraventricular hypothalamic nucleus, central amygdala, lateral parabrachial nucleus and nucleus of the solitary tract, suggesting a commonality of action at the OTR with the differential doses employed. However, WAY 267,464 caused greater c-Fos expression in the medial amygdala and the supraoptic nucleus than oxytocin, and lesser effects in the locus coeruleus. Overall, our results confirm the differential effects of WAY 267,464 and oxytocin and suggest that this may reflect contrasting actions of WAY 267,464 and oxytocin at the V1aR. Antagonism of the V1aR by WAY 267,464 could underlie some of the prosocial effects of this drug either through a direct action or through disinhibition of oxytocin circuitry that is subject to vasopressin inhibitory influences.
PMCID: PMC3399775  PMID: 22420322
oxytocin; vasopressin; adolescent; social behaviour; anxiety
21.  Essential role of IFNβ and CD38 in TNFα-induced airway smooth muscle hyper-responsiveness 
Immunobiology  2008;213(6):499-509.
We recently identified autocrine interferon (IFN)β as a novel mechanism mediating tumor necrosis factor (TNF)α-induced expression of inflammatory genes in airway smooth muscle (ASM) cells, including CD38, known to regulate calcium signaling. Here, we investigated the putative involvement of IFNβ in regulating TNFα-induced airway hyper-responsiveness (AHR), a defining feature of asthma. Using our pharmacodynamic model to assess ex vivo AHR isolated murine tracheal rings, we found that TNFα-induced enhanced contractile responses to carbachol and bradykinin was abrogated by neutralizing anti-IFNβ antibody or in tracheal rings deficient in CD38. In cultured human ASM cells, where CD38 has been involved in TNFα-induced enhanced calcium signals to carbachol and bradykinin, we found that neutralizing anti-IFNβ prevented TNFα enhancing action only on carbachol responses but not to that induced by bradykinin. In a well-characterized model of allergic asthma (mice sensitized and challenged with Aspergillus fumigatus (Af)), we found heightened expression of both IFNβ and CD38 in the airways. Furthermore, allergen-associated AHR to methacholine, assessed by lung resistance and dynamic compliance, was completely suppressed in CD38-deficient mice, despite the preservation of airway inflammation. These data provide the first evidence that ASM-derived IFNβ and CD38 may play a significant role in the development of TNFα-associated AHR.
PMCID: PMC2587232  PMID: 18514752
Allergic asthma; Cytokine; Airway smooth muscle; Inflammation; Calcium signaling; Hypercontractility
22.  Mechanical Stretch Modulates MicroRNA 21 Expression, Participating in Proliferation and Apoptosis in Cultured Human Aortic Smooth Muscle Cells 
PLoS ONE  2012;7(10):e47657.
Stretch affects vascular smooth muscle cell proliferation and apoptosis, and several responsible genes have been proposed. We tested whether the expression of microRNA 21 (miR-21) is modulated by stretch and is involved in stretch-induced proliferation and apoptosis of human aortic smooth muscle cells (HASMCs).
Methods and Results
RT-PCR revealed that elevated stretch (16% elongation, 1 Hz) increased miR-21 expression in cultured HASMCs, and moderate stretch (10% elongation, 1 Hz) decreased the expression. BrdU incorporation assay and cell counting showed miR-21 involved in the proliferation of HASMCs mediated by stretch, likely by regulating the expression of p27 and phosphorylated retinoblastoma protein (p-Rb). FACS analysis revealed that the complex of miR-21 and programmed cell death protein 4 (PDCD4) participated in regulating apoptosis with stretch. Stretch increased the expression of primary miR-21 and pre-miR-21 in HASMCs. Electrophoretic mobility shift assay (EMSA) demonstrated that stretch increased NF-κB and AP-1 activities in HASMCs, and blockade of AP-1 activity by c-jun siRNA significantly suppressed stretch-induced miR-21 expression.
Cyclic stretch modulates miR-21 expression in cultured HASMCs, and miR-21 plays important roles in regulating proliferation and apoptosis mediated by stretch. Stretch upregulates miR-21 expression at least in part at the transcription level and AP-1 is essential for stretch-induced miR-21 expression.
PMCID: PMC3474731  PMID: 23082189
23.  Non-synonymous single-nucleotide variations of the human oxytocin receptor gene and autism spectrum disorders: a case–control study in a Japanese population and functional analysis 
Molecular Autism  2013;4:22.
The human oxytocin receptor (hOXTR) is implicated in the etiology of autism spectrum disorders (ASDs) and is a potential target for therapeutic intervention. Several studies have reported single-nucleotide polymorphisms (SNPs) of the OXTR gene associated with ASDs. These SNPs, however, reside outside the protein-coding region. Not much is known about genetic variations that cause amino acid substitutions that alter receptor functions.
Variations in the OXTR gene were analyzed in 132 ASD patients at Kanazawa University Hospital in Japan and 248 unrelated healthy Japanese volunteers by re-sequencing and real-time polymerase chain reaction-based genotyping. Functional changes in variant OXTRs were assessed by radioligand binding assay and measurements of intracellular free calcium concentrations ([Ca2+]i) and inositol 1,4,5-trisphosphate (IP3) levels.
Six subjects (4.5%) in the ASD group and two in the control group (0.8%) were identified as heterozygotes carrying the R376G variation (rs35062132; c.1126C>G); one individual from the ASD group (0.8%) and three members of the control group (1.2%) were found to be carrying R376C (c.1126C>T). The C/G genotype significantly correlated with an increased risk of ASDs (odds ratio (OR) = 5.83; 95% confidence interval (CI) = 1.16 to 29.33; P = 0.024, Fisher’s exact test). Consistently, the G allele showed a correlation with an increased likelihood of ASDs (OR = 5.73; 95% CI = 1.15 to 28.61; P = 0.024, Fisher’s exact test). The frequencies of the C/T genotype and the T allele in the ASD and control groups did not differ significantly. We also examined changes in agonist-induced cellular responses mediated by the variant receptors hOXTR-376G and hOXTR-376C. OXT-induced receptor internalization and recycling were faster in hOXTR-376G-expressing HEK-293 cells than in cells expressing hOXTR-376R or hOXTR-376C. In addition, the elevation in [Ca2+]i and IP3 formation decreased in the cells expressing hOXTR-376G and hOXTR-376C tagged with enhanced green fluorescent protein (EGFP), in comparison with the cells expressing the common-type hOXTR-376R tagged with EGFP.
These results suggest that the rare genetic variation rs35062132 might contribute to the pathogenesis of ASDs, and could provide a molecular basis of individual differences in OXTR-mediated modulation of social behavior.
PMCID: PMC3707786  PMID: 23815867
Autism spectrum disorders; Inositol-1,4,5-trisphosphate; Intracellular free calcium; Oxytocin; Oxytocin receptor; Single-nucleotide variation
24.  Newborn Analgesia Mediated by Oxytocin during Delivery 
The mechanisms controlling pain in newborns during delivery are poorly understood. We explored the hypothesis that oxytocin, an essential hormone for labor and a powerful neuromodulator, exerts analgesic actions on newborns during delivery. Using a thermal tail-flick assay, we report that pain sensitivity is two-fold lower in rat pups immediately after birth than 2 days later. Oxytocin receptor antagonists strongly enhanced pain sensitivity in newborn, but not in 2-day-old rats, whereas oxytocin reduced pain at both ages suggesting an endogenous analgesia by oxytocin during delivery. Similar analgesic effects of oxytocin, measured as attenuation of pain-vocalization induced by electrical whisker pad stimulation, were also observed in decerebrated newborns. Oxytocin reduced GABA-evoked calcium responses and depolarizing GABA driving force in isolated neonatal trigeminal neurons suggesting that oxytocin effects are mediated by alterations of intracellular chloride. Unlike GABA signaling, oxytocin did not affect responses mediated by P2X3 and TRPV1 receptors. In keeping with a GABAergic mechanism, reduction of intracellular chloride by the diuretic NKCC1 chloride co-transporter antagonist bumetanide mimicked the analgesic actions of oxytocin and its effects on GABA responses in nociceptive neurons. Therefore, endogenous oxytocin exerts an analgesic action in newborn pups that involves a reduction of the depolarizing action of GABA on nociceptive neurons. Therefore, the same hormone that triggers delivery also acts as a natural pain killer revealing a novel facet of the protective actions of oxytocin in the fetus at birth.
PMCID: PMC3080614  PMID: 21519396
GABA; neonate; oxytocin; pain
25.  Involvement of Prolactin-Releasing Peptide in the Activation of Oxytocin Neurones in Response to Food Intake 
Journal of Neuroendocrinology  2013;25(5):455-465.
Food intake activates neurones expressing prolactin-releasing peptide (PrRP) in the medulla oblongata and oxytocin neurones in the hypothalamus. Both PrRP and oxytocin have been shown to have an anorexic action. In the present study, we investigated whether the activation of oxytocin neurones following food intake is mediated by PrRP. We first examined the expression of PrRP receptors (also known as GPR10) in rats. Immunoreactivity of PrRP receptors was observed in oxytocin neurones and in vasopressin neurones in the paraventricular and supraoptic nuclei of the hypothalamus and in the bed nucleus of the stria terminalis. Application of PrRP to isolated supraoptic nuclei facilitated the release of oxytocin and vasopressin. In mice, re-feeding increased the expression of Fos protein in oxytocin neurones of the hypothalamus and bed nucleus of the stria terminalis. The increased expression of Fos protein in oxytocin neurones following re-feeding or i.p. administration of cholecystokinin octapeptide (CCK), a peripheral satiety factor, was impaired in PrRP-deficient mice. CCK-induced oxytocin increase in plasma was also impaired in PrRP-deficient mice. Furthermore, oxytocin receptor-deficient mice showed an increased meal size, as reported in PrRP-deficient mice and in CCKA receptor-deficient mice. These findings suggest that PrRP mediates, at least in part, the activation of oxytocin neurones in response to food intake, and that the CCK–PrRP–oxytocin pathway plays an important role in the control of the termination of each meal.
PMCID: PMC3664423  PMID: 23363338
oxytocin; PrRP; vasopressin; food intake; CCK

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