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Background and purpose: There are interactions between endothelin-1 (ET-1) and endothelial vascular injury in hyperhomocysteinemia (HHcy), but the underlying mechanisms are poorly understood. Here we evaluated the effects of HHcy on the endothelin system in rat carotid arteries. Experimental approach: Vascular reactivity to ET-1 and ETA and ETB receptor antagonists was assessed in rings of carotid arteries from normal rats and those with HHcy. ETA and ETB receptor expression was assessed by mRNA (RT-PCR), immunohistochemistry and binding of [125I]-ET-1. Key results: HHcy enhanced ET-1-induced contractions of carotid rings with intact endothelium. Selective antagonism of ETA or ETB receptors produced concentration-dependent rightward displacements of ET-1 concentration response curves. Antagonism of ETA but not of ETB receptors abolished enhancement in HHcy tissues. ETA and ETB receptor gene expressions were not up-regulated. ETA receptor expression in the arterial media was higher in HHcy arteries. Contractions to big ET-1 served as indicators of endothelin-converting enzyme activity, which was decreased by HHcy, without reduction of ET-1 levels. ET-1-induced Rho-kinase activity, calcium release and influx were increased by HHcy. Pre-treatment with indomethacin reversed enhanced responses to ET-1 in HHcy tissues, which were reduced also by a thromboxane A2 receptor antagonist. Induced relaxation was reduced by BQ788, absent in endothelium-denuded arteries and was decreased in HHcy due to reduced bioavailability of NO. Conclusions and implications: Increased ETA receptor density plays a fundamental role in endothelial injury induced by HHcy. ET-1 activation of ETA receptors in HHcy changed the balance between endothelium-derived relaxing and contracting factors, favouring enhanced contractility. British Journal of Pharmacology (2009) 157, 568–580; doi:10.1111/j.1476-5381.2009.00165.x; published online 9 April 2009 This article is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: [ABSTRACT FROM AUTHOR]

Background and Purpose: Cannabinoid CB(1) receptor antagonists reduce food intake and body weight, but clinical use in humans is limited by effects on the CNS. We have evaluated a novel cannabinoid antagonist (AM6545) designed to have limited CNS penetration, to see if it would inhibit food intake in rodents, without aversive effects.Experimental Approach: Cannabinoid receptor binding studies, cAMP assays, brain penetration studies and gastrointestinal motility studies were carried out to assess the activity profile of AM6545. The potential for AM6545 to induce malaise in rats and the actions of AM6545 on food intake and body weight were also investigated.Key Results: AM6545 binds to CB(1) receptors with a K(i) of 1.7 nM and CB(2) receptors with a K(i) of 523 nM. AM6545 is a neutral antagonist, having no effect on cAMP levels in transfected cells and was less centrally penetrant than AM4113, a comparable CB(1) receptor antagonist. AM6545 reversed the effects of WIN55212-2 in an assay of colonic motility. In contrast to AM251, AM6545 did not produce conditioned gaping or conditioned taste avoidance in rats. In rats and mice, AM6545 dose-dependently reduced food intake and induced a sustained reduction in body weight. The effect on food intake was maintained in rats with a complete subdiaphragmatic vagotomy. AM6545 inhibited food intake in CB(1) receptor gene-deficient mice, but not in CB(1)/CB(2) receptor double knockout mice.Conclusions and Implications: Peripherally active, cannabinoid receptor antagonists with limited brain penetration may be useful agents for the treatment of obesity and its complications. [ABSTRACT FROM AUTHOR]

Background and Purpose: F15599, a novel 5-hydroxytryptamine (5-HT)(1A) receptor agonist with 1000-fold selectivity for 5-HT compared with other monoamine receptors, shows antidepressant and procognitive activity at very low doses in animal models. We examined the in vivo activity of F15599 at somatodendritic autoreceptors and postsynaptic 5-HT(1A) heteroreceptors.Experimental Approach: In vivo single unit and local field potential recordings and microdialysis in the rat.Key Results: F15599 increased the discharge rate of pyramidal neurones in medial prefrontal cortex (mPFC) from 0.2 microg x kg(-1) i.v and reduced that of dorsal raphe 5-hydroxytryptaminergic neurones at doses >10-fold higher (minimal effective dose 8.2 microg x kg(-1) i.v.). Both effects were reversed by the 5-HT(1A) antagonist (+/-)WAY100635. F15599 did not alter low frequency oscillations (approximately 1 Hz) in mPFC. In microdialysis studies, F15599 increased dopamine output in mPFC (an effect dependent on the activation of postsynaptic 5-HT(1A) receptors) with an ED(50) of 30 microg x kg(-1) i.p., whereas it reduced hippocampal 5-HT release (an effect dependent exclusively on 5-HT(1A) autoreceptor activation) with an ED(50) of 240 microg x kg(-1) i.p. Likewise, application of F15599 by reverse dialysis in mPFC increased dopamine output in a concentration-dependent manner. All neurochemical responses to F15599 were prevented by administration of (+/-)WAY100635.Conclusions and Implications: These results indicate that systemic administration of F15599 preferentially activates postsynaptic 5-HT(1A) receptors in PFC rather than somatodendritic 5-HT(1A) autoreceptors. This regional selectivity distinguishes F15599 from previously developed 5-HT(1A) receptor agonists, which preferentially activate somatodendritic 5-HT(1A) autoreceptors, suggesting that F15599 may be particularly useful in the treatment of depression and of cognitive deficits in schizophrenia. [ABSTRACT FROM AUTHOR]

Background and Purpose: Excessive production of nitric oxide (NO) by inducible NO synthase (iNOS) is thought to underlie the vascular dysfunction, systemic hypotension and organ failure that characterize endotoxic shock. Plasma levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are raised in animal models and humans with endotoxic shock and correlate with the associated cardiovascular dysfunction. Since both NO and natriuretic peptides play important roles in cardiovascular homeostasis via activation of guanylate cyclase-linked receptors, we used mice lacking natriuretic peptide receptor (NPR)-A (NPR1) to establish if natriuretic peptides contribute to the cardiovascular dysfunction present in endotoxic shock.Experimental Approach: Wild-type (WT) and NPR-A knockout (KO) mice were exposed to lipopolysaccharide (LPS) and vascular dysfunction (in vitro and in vivo), production of pro-inflammatory cytokines, and iNOS expression and activity were evaluated.Key Results: LPS-treated WT animals exhibited a marked fall in mean arterial blood pressure (MABP) whereas NPR-A KO mice maintained MABP throughout. LPS administration caused a greater suppression of vascular responses to the thromboxane-mimetic U46619, ANP, acetylcholine and the NO-donor spermine-NONOate in WT versus NPR-A KO mice. This differential effect on vascular function was paralleled by reduced pro-inflammatory cytokine production, iNOS expression and activity (plasma [NO(x)] and cyclic GMP).Conclusions and Implications: These observations suggest that NPR-A activation by natriuretic peptides facilitates iNOS expression and contributes to the vascular dysfunction characteristic of endotoxic shock. Pharmacological interventions that target the natriuretic peptide system may represent a novel approach to treat this life-threatening condition. [ABSTRACT FROM AUTHOR]

Background and Purpose: P2Y receptors evoke Ca(2+) signals in vascular smooth muscle cells and regulate contraction and proliferation, but the roles of the different P2Y receptor subtypes are incompletely resolved.Experimental Approach: Quantitative PCR was used to define expression of mRNA encoding P2Y receptor subtypes in freshly isolated and cultured rat aortic smooth muscle cells (ASMC). Fluorescent indicators in combination with selective ligands were used to measure the changes in cytosolic free [Ca(2+)] in cultured ASMC evoked by each P2Y receptor subtype.Key Results: The mRNA for all rat P2Y receptor subtypes are expressed at various levels in cultured ASMC. Four P2Y receptor subtypes (P2Y1, P2Y2, P2Y4 and P2Y6) evoke Ca(2+) signals that require activation of phospholipase C and comprise both release of Ca(2+) from stores and Ca(2+) entry across the plasma membrane.Conclusions and Implications: Combining analysis of P2Y receptor expression with functional analyses using selective agonists and antagonists, we isolated the Ca(2+) signals evoked in ASMC by activation of P2Y1, P2Y2, P2Y4 and P2Y6 receptors. [ABSTRACT FROM AUTHOR]

Background and Purpose: Gram-negative bacteria contain ligands for Toll-like receptor (TLR) 4 and nucleotide oligomerization domain (NOD) 1 receptors. Lipopolysaccharide (LPS) activates TLR4, while peptidoglycan products activate NOD1. Activation of NOD1 by the specific agonist FK565 results in a profound vascular dysfunction and experimental shock in vivo.Experimental Approach: Here, we have analysed a number of pharmacological inhibitors to characterize the role of key signalling pathways in the induction of NOS2 following TLR4 or NOD1 activation.Key Results: Vascular smooth muscle (VSM) cells expressed NOD1 mRNA and protein, and, after challenge with Escherichia coli or FK565, NOS2 protein and activity were induced. Macrophages had negligible levels of NOD1 and were unaffected by FK565, but responded to E. coli and LPS by releasing increased NO and expression of NOS2 protein. Classic pharmacological inhibitors for NF-kappaB (SC-514) and mitogen-activated protein kinase (SB203580, PD98059) signalling pathways inhibited responses in both cell types regardless of agonist. While TLR4-mediated responses in macrophages were specifically inhibited by the pan-caspase inhibitor z-VAD-fmk and the PKC inhibitor Gö6976, NOD1-mediated responses in VSM cells were inhibited by the Rip2 inhibitor PP2.Conclusions and Implications: Our findings suggest a selective role for NOD1 in VSM cells, and highlight NOD1 as a potential novel therapeutic target for the treatment of vascular inflammation. [ABSTRACT FROM AUTHOR]

Background and Purpose: Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration.Experimental Approach: We used the human endothelial cell line EA.hy926 to investigate the effect of nitric oxide (NO) donors on SK-1 expression, and on cell migration and tube formation.Key Results: We showed that exposure of EA.hy926 cells to Deta-NO (125-1000 microM) resulted in a time- and concentration-dependent up-regulation of SK-1 mRNA and protein expression, and activity with a first significant effect at 250 microM of Deta-NO. The increased SK-1 mRNA expression resulted from an enhanced SK-1 promoter activity. A similar effect was also seen with various other NO donors. In mechanistic terms, the NO-triggered effect occurred independently of cGMP, but involved the classical mitogen-activated protein kinase cascade because the MEK inhibitor U0126 abolished the NO-induced SK-1 expression. The effect of NO was also markedly reduced by the thiol-reducing agent N-acetylcysteine, suggesting a redox-dependent mechanism. Functionally, Deta-NO triggered an increase in the migration of endothelial cells in an adapted Boyden chamber assay, and also increased endothelial tube formation in a Matrigel assay. These responses were both abolished in cells depleted of SK-1.Conclusions and Implications: These data show that NO donors up-regulate specifically SK-1 expression and activity in human endothelial cells, and SK-1 in turn critically contributes to the migratory capability and tube formation of endothelial cells. Thus, SK-1 may be considered an attractive novel target to interfere with pathological processes involving angiogenesis. [ABSTRACT FROM AUTHOR]

Background and Purpose: Previous work has shown that N(G)-monomethyl-l-arginine (l-NMMA) paradoxically inhibits basal, but not ACh-stimulated activity of nitric oxide in rat aorta. The aim of this study was to determine if the endogenously produced agent, asymmetric N(G), N(G)-dimethyl-l-arginine (ADMA), also exhibits this unusual selective blocking action.Experimental Approach: The effect of ADMA on basal nitric oxide activity was assessed by examining its ability to enhance phenylephrine (PE)-induced tone in endothelium-containing rings. Its effect on ACh-induced relaxation was assessed both in conditions where ADMA greatly enhanced PE tone and where tone was carefully matched with control tissues at a range of different levels.Key Results: ADMA (100 microM) potentiated PE-induced contraction, consistent with inhibition of basal nitric oxide activity. Higher concentrations (300-1000 microM) had no greater effect. Although ADMA (100 microM) also appeared to block ACh-induced relaxation when it enhanced PE tone to maximal levels, virtually no block was seen at intermediate levels of tone in the presence of ADMA. Even ADMA at 1000 microM had no effect on the maximal relaxation to ACh, although it produced a small (two- to threefold) reduction in sensitivity. ADMA and l-NMMA, like l-arginine (all at 1000 microM), protected ACh-induced relaxation against blockade by l-NAME (30 microM).Conclusions and Implications: In the rat aorta, ADMA, like l-NMMA, blocks basal activity of nitric oxide, but has little effect on that stimulated by ACh. Further studies are required to explain these seemingly anomalous actions of ADMA and l-NMMA. [ABSTRACT FROM AUTHOR]

Background and Purpose: AE9C90CB (N- [(1R, 5S, 6R)-3-azabicyclo [3.1.0] hex-6-ylmethyl]-2-hydroxy-N-methyl-2, 2-diphenylacetamide), a novel muscarinic receptor antagonist, was synthesized for the treatment of overactive bladder. Here we describe the in vitro and in vivo profiles of AE9C90CB for action in bladder over salivary gland and compare it with four agents already in clinical use (tolterodine, oxybutynin, solifenacin and darifenacin).Experimental Approach: Radioligand binding assay and isolated tissue-based functional assay were used to evaluate affinity, potency, and receptor subtype selectivity of compounds. Inhibition of carbachol-induced increase in intravesicular pressure and salivary secretion were measured in anaesthetized rabbits to assess the functional selectivity.Key Results: In vitro radioligand binding study using human recombinant muscarinic receptors showed that AE9C90CB had greater affinity for M(3) muscarinic receptors with pKi of 9.90 +/- 0.11 and was 20-fold more selective for M(3) than for M(2) muscarinic receptors. AE9C90CB exhibited an unsurmountable antagonism on rat bladder strips (pK(B), 9.13 +/- 0.12). In anaesthetized rabbits after intravenous administration, AE9C90CB dose dependently inhibited carbachol-induced increase in intravesicular pressure and salivary secretion, and exhibited functional selectivity for urinary bladder over salivary gland which was ninefold better than that of oxybutynin.Conclusions and Implications: We have identified AE9C90CB, a compound exhibiting moderate selectivity for M(3) over M(2) receptors but greater selectivity for urinary bladder over salivary gland than oxybutynin, tolterodine, solifenacin and darifenacin. Therefore, AE9C90CB may be a promising compound for the treatment of overactive bladder with reduced potential to cause dry mouth than currently available antimuscarinic drugs. [ABSTRACT FROM AUTHOR]

Background and Purpose: Growing evidence implicates NF-kappaB as an important contributor to metastasis and increased chemoresistance of melanoma. Here, we report the effects of parthenolide on either untreated, cisplatin- or TNFalpha-treated melanoma cell lines A375, 1205Lu and WM793, exhibiting different levels of constitutive NF-kappaB activity.Experimental Approach: Electrophoretic mobility shift assay was used to assess changes in NF-kappaB activity, and real-time PCR to evaluate expression of NF-kappaB-regulated genes. Cell cycle arrest and apoptosis were assessed by flow cytometry. Cell death was also visualized by fluorescence microscopy. Migration was determined by scratch assay and invasiveness by Matrigel assay.Key Results: Parthenolide suppressed both constitutive and induced NF-kappaB activity in melanoma cells. This was accompanied by down-regulation of cancer-related genes, with NF-kappaB-binding sites in their promoters, including: Bcl-X(L), survivin, cyclin D1, interleukin 8 and matrix metalloproteinase 9. When the various effects of 6 microM parthenolide were compared, apoptosis associated with loss of mitochondrial membrane potential was most efficiently induced in 1205Lu cells, cell cycle arrest in G(0)/G(1) phase was observed in WM793 cells, and high metastatic potential was markedly reduced in A375 cells. These findings not only reflected differences between melanoma cell lines in basal expression of NF-kappaB-regulated genes, but also suggested other parthenolide targets involved in cell cycle progression, migration, invasiveness and survival.Conclusions: Inhibition of constitutive and therapeutically induced NF-kappaB pathway by parthenolide might be useful in the treatment of melanoma, although the diversity of changes induced in melanoma cells with different genetic backgrounds indicate context-dependent poly-pharmacological properties of this compound. [ABSTRACT FROM AUTHOR]

Background and Purpose: Population studies have revealed that treatment with the anti-diabetic drug metformin is significantly associated with reduced cancer risk, but the underlying mode of action has not been elucidated. The aim of our study was to determine the effect of metformin on tumour invasion and migration, and the possible mechanisms, using human fibrosarcoma HT-1080 cells.Experimental Approach: We employed invasion, migration and gelatin zymography assays to characterize the effect of metformin on HT-1080 cells. Transient transfection assays were performed to gene promoter activities, and immunoblot analysis to study its molecular mechanisms of action.Key Results: Metformin inhibited migration and invasion by HT-1080 cells at sub-toxic concentrations. In these cells, metformin also suppressed phorbol-12-myristate-13-acetate (PMA)-enhanced levels of matrix metalloproteinases-9 (MMP-9) protein, mRNA and transcription activity through suppression of activator protein-1 (AP-1) activation. In addition, metformin strongly repressed the PMA-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and protein kinase C(PKC)alpha, whereas the phosphorylation of p38 mitogen-activated protein kinase was not affected by metformin. Metformin decreased the PMA-induced Ca(2+) influx. Furthermore, treatment with an intracellular Ca(2+) chelator (BAPTA-AM) or a selective calmodulin antagonist (W7) markedly decreased PMA-induced MMP-9 secretion and cell migration, as well as activation of ERK and JNK/AP-1.Conclusions and Implications: Metformin inhibited PMA-induced invasion and migration of human fibrosarcoma cells via Ca(2+)-dependent PKCalpha/ERK and JNK/AP-1-signalling pathways. Metformin therefore has the potential to be a potent anti-cancer drug in therapeutic strategies for fibrosarcoma metastasis. [ABSTRACT FROM AUTHOR]

Background and Purpose: Arteries from hypertensive subjects are reportedly hyperresponsive to 5-hydroxytryptamine (5-HT), but it remains unclear whether this is true in chronic type 2 diabetes. We have assessed responses to 5-HT shown by mesenteric arteries from type 2 diabetic ob/ob mice (27-32 weeks old) and have identified the molecular mechanisms involved.Experimental Approach: Contractions of mesenteric rings to 5-HT were examined in vitro. Activation of mesenteric RhoA, Rho kinase and Src was measured by Western blotting or by modified enzyme-linked immunosorbent assay.Key Results: Concentration-dependent contractions to 5-HT were greater in mesenteric rings from the ob/ob than in those from the age-matched control ('Lean') group. In each group, there was no significant change in the 5-HT-induced contractions after inhibition of nitric oxide synthase (with N(G)-nitro-L-arginine), of cyclooxygenase (with indomethacin) or of protein kinase C (with chelerythrine). However inhibition of the MEK/ERK pathway (with PD98059) decreased the response to 5-HT. Although the diabetes-related enhancement of the 5-HT response was preserved with each of these inhibitors, enhancement was abolished by a Rho kinase inhibitor (Y27632) and by Src kinase inhibitors (PP1 analogue or Src kinase inhibitor I). 5-HT-induced activation of RhoA, Rho kinase and Src kinase in mesenteric arteries was greater in the ob/ob than in the Lean group, but the expression of RhoA, Rho kinase isoforms and Src did not differ between these groups.Conclusions and Implications: These results suggest that the enhancement of 5-HT-induced contraction in mesenteric arteries from ob/ob mice may be attributable to increased activation of RhoA/Rho kinase and Src kinase. [ABSTRACT FROM AUTHOR]

Background and Purpose: Recent studies suggest that the effects of cyclooxygenase-2 (COX-2) inhibition are mediated by cannabinoid receptor activation. However, some non-steroidal anti-inflammatory drugs inhibit the enzyme fatty acid amide hydrolase, which regulates levels of some endocannabinoids. Whether COX-2 directly regulates levels of endocannabinoids in vivo is unclear. Here, the effect of the COX-2 inhibitor nimesulide, which does not inhibit fatty acid amide hydrolase, on spinal nociceptive processing was determined. Effects of nimesulide on tissue levels of endocannabinoids and related compounds were measured and the role of cannabinoid 1 (CB(1)) receptors was determined.Experimental Approach: Effects of spinal and peripheral administration of nimesulide (1-100 microg per 50 microL) on mechanically evoked responses of rat dorsal horn neurones were measured, and the contribution of the CB(1) receptor was determined with the antagonist AM251 (N-(piperidin-1-yl)-5-(-4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide), in anaesthetized rats. Effects of nimesulide on spinal levels of endocannabinoids and related compounds were quantified using liquid chromatography-tandem mass spectrometry.Key Results: Spinal, but not peripheral, injection of nimesulide (1-100 microg per 50 microL) significantly reduced mechanically evoked responses of dorsal horn neurones. Inhibitory effects of spinal nimesulide were blocked by the CB(1) receptor antagonist AM251 (1 microg per 50 microL), but spinal levels of endocannabinoids were not elevated. Indeed, both anandamide and N-oleoylethanolamide (OEA) were significantly decreased by nimesulide.Conclusions and Implications: Although the inhibitory effects of COX-2 blockade on spinal neuronal responses by nimesulide were dependent on CB(1) receptors, we did not detect a concomitant elevation in anandamide or 2-AG. Further understanding of the complexities of endocannabinoid catabolism by multiple enzymes is essential to understand their contribution to COX-2-mediated analgesia. [ABSTRACT FROM AUTHOR]

Background and Purpose: In various models vagus nerve activation has been shown to ameliorate intestinal inflammation, via nicotinic acetylcholine receptors (nAChRs) expressed on immune cells. As the alpha7 nAChR has been put forward to mediate this effect, we studied the effect of nicotine and two selective alpha7 nAChR agonists (AR-R17779, (-)-spiro[1-azabicyclo[2.2.2] octane-3,5'-oxazolidin-2'-one and GSK1345038A) on disease severity in two mouse models of experimental colitis.Experimental Approach: Colitis was induced by administration of 1.5% dextran sodium sulphate (DSS) in drinking water or 2 mg 2,4,6-trinitrobenzene sulphonic acid (TNBS) intrarectally. Nicotine (0.25 and 2.50 micromol.kg(-1)), AR-R17779 (0.6-30 micromol.kg(-1)) or GSK1345038A (6-120 micromol.kg(-1)) was administered daily by i.p. injection. After 7 (DSS) or 5 (TNBS) days clinical parameters and colonic inflammation were scored.Key Results: Nicotine and both alpha7 nAChR agonists reduced the activation of NF-kappaB and pro-inflammatory cytokines in whole blood and macrophage cultures. In DSS colitis, nicotine treatment reduced colonic cytokine production, but failed to reduce disease parameters. Reciprocally, treatment with AR-R17779 or GSK1345038A worsened disease and led to increased colonic pro-inflammatory cytokine levels in DSS colitis. The highest doses of GSK1345038A (120 micromol.kg(-1)) and AR-R17779 (30 micromol.kg(-1)) ameliorated clinical parameters, without affecting colonic inflammation. Neither agonist ameliorated TNBS-induced colitis.Conclusions and Implications: Although nicotine reduced cytokine responses in vitro, both selective alpha7 nAChR agonists worsened the effects of DSS-induced colitis or were ineffective in those of TNBS-induced colitis. Our data indicate the need for caution in evaluating alpha7 nAChR as a drug target in colitis. [ABSTRACT FROM AUTHOR]

Background and Purpose: The uterine pathophysiology underlying inflammatory conditions such as chorioamnionitis remains largely unclear. As we have shown that beta(3)-adrenoceptors act as regulators of myometrial inflammation, we wanted to investigate the potential role of beta(3)-adrenoceptors in preventing uterine remodelling induced by inflammation.Experimental Approach: The consequences of human chorioamnionitis on myometrial remodelling were characterized by Sirius Red staining and metalloproteinase (MMP) expression, and compared with the effects of incubating human myometrial samples with Escherichia coli lipopolysaccharide (LPS) in vitro. We also assessed the effect of SAR150640, a selective beta(3)-adrenoceptor agonist, on the production and activity of MMPs.Key Results: Chorioamnionitis was associated with a 46% decrease in total collagen, as well as over-expression of MMP2 (+61%) and MMP9 (+84%); both effects were reproduced by incubation with LPS (10 microg x mL(-1), 48 h). LPS-induced over-expression of MMP2 and MMP9 in normal human myometrium was paralleled by an overactivity of the proteins. Both over-expression and overactivity were prevented by the beta(3)-adrenoceptor agonist SAR150640 in a concentration-dependent manner. SAR150640, by itself, did not exhibit any effect on MMP production in control tissues.Conclusions and Implications: This study shows that inflammation was associated with an intense remodelling of human myometrium, a process likely to be explained by MMP activation. Our study emphasizes the potential therapeutic relevance of beta(3)-adrenoceptor agonists to the treatment of preterm labour and other uterine inflammatory conditions. [ABSTRACT FROM AUTHOR]

Background and Purpose: It is well known that adenine-based purines exert multiple effects on pain transmission. However, less attention has been given to the potential effects of guanine-based purines on pain transmission. The aim of this study was to investigate the effects of intraperitoneal (i.p.) and oral (p.o.) administration of guanosine on mice pain models. Additionally, investigation into the mechanisms of action of guanosine, its potential toxicity and cerebrospinal fluid (CSF) purine levels were also assessed.Experimental Approach: Mice received an i.p. or p.o. administration of vehicle (0.1 mM NaOH) or guanosine (up to 240 mg x kg(-1)) and were evaluated in several pain models.Key Results: Guanosine produced dose-dependent antinociceptive effects in the hot-plate, glutamate, capsaicin, formalin and acetic acid models, but it was ineffective in the tail-flick test. Additionally, guanosine produced a significant inhibition of biting behaviour induced by i.t. injection of glutamate, AMPA, kainate and trans-ACPD, but not against NMDA, substance P or capsaicin. The antinociceptive effects of guanosine were prevented by selective and non-selective adenosine receptor antagonists. Systemic administration of guanosine (120 mg x kg(-1)) induced an approximately sevenfold increase on CSF guanosine levels. Guanosine prevented the increase on spinal cord glutamate uptake induced by intraplantar capsaicin.Conclusions and Implications: This study provides new evidence on the mechanism of action of the antinociceptive effects after systemic administration of guanosine. These effects seem to be related to the modulation of adenosine A(1) and A(2A) receptors and non-NMDA glutamate receptors. [ABSTRACT FROM AUTHOR]

Background and Purpose: Hyperglycaemia is known to reduce nitric oxide (NO) bioavailability by modulating endothelial NO synthase (eNOS) activity, and polyphenols are believed to have cardiovascular benefit. One possible mechanism could be through interaction with eNOS.Experimental Approach: The effects of the oligomerized polyphenol oligonol on eNOS phosphorylation status and activity were examined in porcine aortic endothelial cells cultured in high glucose concentrations.Key Results: Exposure to high glucose concentrations strongly inhibited eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 in bradykinin (BK)-stimulated cells. These inhibitory effects of high glucose were significantly prevented by treatment with oligonol. Akt and p38 mitogen-activated protein kinase (MAPK) were activated in BK-stimulated cells. High glucose inhibited Akt activation but enhanced p38 MAPK activation, both of which were reversed by oligonol treatment. The phosphatidylinositol 3-kinase inhibitor wortmannin blocked the reversal by oligonol of phosphorylation at Ser-1177, but not dephosphorylation at Thr-495, in BK-stimulated cells exposed to high glucose. The effect of oligonol on BK dephosphorylation under high glucose was mimicked by protein kinase C (PKC) epsilon-neutralizing peptides. These data suggest that the effects of oligonol on high glucose-induced attenuation of eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation may be regulated by Akt activation and PKCepsilon inhibition respectively. Oligonol also prevented high glucose-induced attenuation of BK-stimulated NO production.Conclusions and Implications: Oligonol prevented the impairment of eNOS activity induced by high glucose through reversing altered eNOS phosphorylation status. This mechanism may underlie the beneficial cardiovascular health effects of this oligomerized polyphenol. [ABSTRACT FROM AUTHOR]

Background and Purpose: Activation of the proteinase-activated receptor-2 (PAR-2) induces scratching behaviour in mice. Here, we have investigated the role of kinin B(1) and B(2) receptors in the pruritogenic response elicited by activators of PAR-2.Experimental Approach: Scratching was induced by an intradermal (i.d.) injection of trypsin or the selective PAR-2 activating peptide SLIGRL-NH(2) at the back of the mouse neck. The animals were observed for 40 min and their scratching response was quantified.Key Results: I.d. injection of trypsin or SLIGRL-NH(2) evoked a scratching behaviour, dependent on PAR-2 activation. Mice genetically deficient in kinin B(1) or B(2) receptors exhibited reduced scratching behaviour after i.d. injection of trypsin or SLIGRL-NH(2). Treatment (i.p.) with the non-peptide B(1) or B(2)receptor antagonists SSR240612 and FR173657, respectively, prevented the scratching behaviour caused by trypsin or SLIGRL-NH(2). Nonetheless, only treatment i.p. with the peptide B(2)receptor antagonist, Hoe 140, but not the B(1)receptor antagonist (DALBK), inhibited the pruritogenic response to trypsin. Hoe 140 was also effective against SLIGRL-NH(2)-induced scratching behaviour when injected by i.d. or intrathecal (i.t.) routes. Also, the response to SLIGRL-NH(2) was inhibited by i.t. (but not by i.d.) treatment with DALBK. Conversely, neither Hoe 140 nor DALBK were able to inhibit SLIGRL-NH(2)-induced scratching behaviour when given intracerebroventricularly (i.c.v.).Conclusions and Implications: The present results demonstrated that kinins acting on both B(1) and B(2) receptors played a crucial role in controlling the pruriceptive signalling triggered by PAR-2 activation in mice. [ABSTRACT FROM AUTHOR]

Background and Purpose: The endocannabinoid system and the cannabinoid CB(1) receptor have been identified in human sperm, and it is well known that endocannabinoids have pronounced adverse effects on male and female reproduction. In order to elucidate further the pathophysiological role of the endocannabinoid system in male fertility, we investigated the activity of the CB(1) receptor antagonist rimonabant (SR141716) on the fertilizing ability of human sperm.Experimental Approach: We evaluated in vitro the effects of rimonabant on motility, survival, capacitation, acrosin activity and metabolism of human sperm. Particularly, capacitation was studied by using three different approaches: intracellular free Ca(2+) content assay, cholesterol efflux assay and protein tyrosine phosphorylation analysis.Key Results: Rimonabant significantly increased sperm motility and viability through the induction of pAkt and pBcl2, key proteins of cell survival and metabolism, and it induced acrosome reaction and capacitation as well. Rimonabant reduced the triglyceride content of sperm, while enhancing lipase and acyl-CoA dehydrogenase activities, implying an overall lipolytic action in these cells. Rimonabant also affected sperm glucose metabolism by decreasing phosphorylation of glycogen synthase kinase 3 and increasing glucose-6-phosphate dehydrogenase activity, suggesting a role in inducing sperm energy expenditure. Intriguingly, agonism at the CB(1) receptor, with an anandamide analogue or a selective inhibitor of fatty acid amide hydrolase, produced opposing effects on human sperm functions.Conclusions and Implications: Our data suggest that blockade of the CB(1) receptor by rimonabant induces the acquisition of fertilizing ability and stimulates energy expenditure in human sperm. [ABSTRACT FROM AUTHOR]

Background and Purpose: The aim of this study was to investigate the effect of chronic treatment with antihypertensive and non-antihypertensive doses of zofenopril on cardiovascular changes in spontaneously hypertensive rats (SHR).Experimental Approach: Male SHR were treated with 0.5 or 10 mg kg(-1) per day of zofenopril (Z(0.5) and Z(10)) for 3 months. SHR and Wistar-Kyoto rats (WKY) receiving vehicle were used as controls. Systolic blood pressure was measured using the tail cuff method. Left ventricular weight/body weight ratio was calculated as cardiac hypertrophy index. Angiotensin converting enzyme (ACE) activity was determined in plasma and tissues by a fluorimetric method. Vascular reactivity was evaluated on aortic rings by acetylcholine and sodium nitroprusside relaxations. Effects on vascular structure were assessed by lumen diameter, wall thickness and medial cross-sectional area determination. Superoxide anion generation was quantified using lucigenin-amplified chemiluminescence in aorta.Results: Long-term daily administration of zofenopril (10 mg kg(-1)) to SHR reduced blood pressure to WKY values, decreased cardiac hypertrophy, improved the acetylcholine-induced relaxant response and reversed the vascular remodelling. ACE inhibition and antioxidant activity were involved in these effects. 0.5 mg kg(-1) per day of zofenopril slightly modified blood pressure and the other effects were weaker.Conclusions and Implications: Antihypertensive effects of chronic treatment with zofenopril were accompanied by recovery of endothelial function and improvement of cardiovascular structure. Low-dose zofenopril had little effect on blood pressure, with some benefits on cardiovascular structure and function. Inhibition of ACE and antioxidant activity were involved in these effects. [ABSTRACT FROM AUTHOR]

Background and Purpose: Advanced glycation end products (AGEs) and endothelial progenitor cells (EPCs) play key roles in pathogenesis of diabetes-related vascular complications. AGEs can induce dysfunction in EPCs. The peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists are widely used in the treatment of type 2 diabetes, and it remains unknown if they could attenuate EPC dysfunction induced by AGEs.Experimental Approach: EPCs isolated from healthy adults were cultured with various concentrations of AGEs (0, 50, 100 and 200 mg L(-1)) with or without rosiglitazone (10 nM), antibody for the receptors for AGE-human serum albumin (anti-receptor for advanced glycation end products (RAGE); 50 microg mL(-1)), phosphatidylinositol-3-kinase (PI3K) inhibitor (LY294002, 5 microM), nitric oxide (NO) synthase inhibitor (L-N(G)-nitro-arginine methyl ester (L-NAME), 100 microM) or sodium nitroprusside (SNP, 25 microM). Proliferation, apoptosis, cell adhesion, migration and NO production in EPCs were assessed, and expressions of endothelial NO synthase (eNOS) and Akt were determined.Key Results: Number, proliferation/migration capacities, eNOS and Akt phosphorylation as well as NO synthesized by EPCs were increased by rosiglitazone and reduced by AGEs. AGEs promoted while rosiglitazone reduced EPC apoptosis. The AGE-induced effects were significantly ameliorated by pre-incubation with rosiglitazone, RAGE antibody and SNP. The beneficial effects of rosiglitazone could be blocked by pretreatment with L-NAME and LY294002.Conclusions and Implications: The PPARgamma agonist rosiglitazone increased EPC function and attenuated EPC dysfunction induced by AGEs via upregulating the Akt-eNOS signal pathways of EPCs. [ABSTRACT FROM AUTHOR]

Background and Purpose: The effects of hydrogen peroxide (H(2)O(2)) on uterine smooth muscle are not well studied. We have investigated the effect and the mechanism of action of exogenous hydrogen peroxide on rat uteri contractile activity [spontaneous and calcium ion (Ca(2+))-induced] and the effect of such treatment on anti-oxidative enzyme activities.Experimental Approach: Uteri were isolated from virgin Wistar rats and suspended in an organ bath. Uteri were allowed to contract spontaneously or in the presence of Ca(2+) (6 mM) and treated with H(2)O(2) (2 microM-3 mM) over 2 h. Anti-oxidative enzyme activities (manganese superoxide dismutase-MnSOD, copper-zinc superoxide dismutase-CuZnSOD, catalase-CAT, glutathione peroxidase-GSHPx and glutathione reductase-GR) in H(2)O(2)-treated uteri were compared with those in uteri immediately frozen after isolation or undergoing spontaneous or Ca(2+)-induced contractions, without treatment with H(2)O(2). The effect of inhibitors (propranolol, methylene blue, L-NAME, tetraethylamonium, glibenclamide and 4-aminopyridine) on H(2)O(2)-mediated relaxation was explored.Key Results: H(2)O(2) caused concentration-dependent relaxation of both spontaneous and Ca(2+)-induced uterine contractions. After H(2)O(2) treatment, GSHPx and MnSOD activities were increased, while CuZnSOD and GR (In Ca(2+)-induced rat uteri) were decreased. N(omega)-nitro-L-arginine methyl ester antagonized the effect of H(2)O(2) on Ca(2+)-induced contractions. H(2)O(2)-induced relaxation was not affected by propranolol, potentiated by methylene blue and antagonized by tetraethylamonium, 4-aminopyridine and glibenclamide, with the last compound being the least effective.Conclusions and Implications: H(2)O(2) induced dose-dependent relaxation of isolated rat uteri mainly via changes in voltage-dependent potassium channels. Decreasing generation of reactive oxygen species by stimulation of anti-oxidative pathways may lead to new approaches to the management of dysfunctional uteri. [ABSTRACT FROM AUTHOR]

Background and Purpose: Buthus martensi Karsch (BmK) AS is a scorpion polypeptide toxin, said to target the voltage-gated sodium channels (VGSCs). However, the mechanism of action of BmK AS on the VGSCs has yet to be defined.Experimental Approach: We examined the electrophysiological effects of BmK AS in a wide dose range on the rat brain-type VGSC alpha-subunit, rNav1.2a, heterologously expressed in Xenopus oocytes and on the VGSCs endogenously expressed in the dorsal root ganglion neuroblastoma ND7-23 cell line.Key Results: In the oocytes, BmK AS depolarized the voltage dependence of activation and inactivation of rNav1.2a at 0.1 and 500 nM whereas these parameters were hyperpolarized at 1 nM. In ND7-23 cells, BmK AS hyperpolarized the voltage dependence of activation and inactivation at 0.1, 1 and 100 nM but not 10 nM. BmK AS also hyperpolarized the voltage dependence of recovery from inactivation at 0.1 and 100 nM and slowed the recovery kinetics at all concentrations, but the effects of 1 and 10 nM were relatively smaller than those at 0.1 and 100 nM. Moreover, the inactivation of VGSCs was potentiated by 10 nM BmK AS in both systems, whereas it was inhibited by 0.1 or 100 nM BmK AS in the oocytes or ND7-23 cells respectively.Conclusions and Implications: BmK AS modulated the VGSCs in a unique U-shaped dose-dependent manner, which could be due to the opposing effects of binding to two distinct receptor sites on the VGSCs. [ABSTRACT FROM AUTHOR]

Background and Purpose: Here we have examined the effects of the novel peptide antagonist N-[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indol-5-yl]aminocarbonyl]-glycinyl-L-lysinyl-L-phenylalanyl-N-benzhydrylamide (K-14585) on proteinase-activated receptor (PAR)(2)-mediated intracellular signalling events.Experimental Approach: Using NCTC2544 cells expressing PAR(2), we assessed the effects of K-14585 on PAR(2)-mediated [(3)H] inositol phosphate accumulation, MAP kinase activation, p65 NFkappaB phosphorylation and DNA binding and IL-8 production.Key Results: Pretreatment with K-14585 (5 microM) inhibited [(3)H] inositol phosphate levels stimulated by PAR(2)-activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH) in PAR(2)-expressing NCTC2544 cells. K-14585 pretreatment did not influence PAR(2)-mediated extracellular regulated kinase activation but inhibited p38 MAP kinase phosphorylation. At a higher concentration (30 microM), K-14585 alone stimulated p38 MAP kinase activation. These effects were replicated in EAhy926 cells, endogenously expressing PAR(2), but not in parental or PAR(4)-expressing NCTC2544 cells, suggesting these effects were PAR(2)-dependent. SLIGKV-mediated stimulation of p38 MAP kinase phosphorylation was substantially reduced by the G(q/11) inhibitor YM-254890, without affecting K-14585-mediated phosphorylation. Pretreatment with K-14585 inhibited PAR(2)-mediated p65 NFkappaB phosphorylation and NFkappaB-DNA binding. K-14585 (30 microM) alone stimulated comparable NFkappaB reporter activity to SLIGKV-OH. K-14585 inhibited SLIGKV-stimulated IL-8 production, but given alone increased IL-8. While SLIGKV-induced IL-8 formation was reduced by both SB203580 and YM-254890, the response to K-14585 was sensitive to SB203580 but not YM-254890.Conclusions and Implications: These data reveal that K-14585 has a duality of action functioning both as an antagonist and agonist due to either partial agonist actions or possible agonist-directed signalling. The data also suggest two modes of p38 MAP kinase activation emanating from PAR(2), one G(q/11)-dependent and the other G(q/11)-independent. [ABSTRACT FROM AUTHOR]

Background and Purpose: In previous work, 10 pM BRL37344 and 10 pM clenbuterol stimulated glucose uptake in mouse soleus muscle. Ten nM BRL37344 also stimulated uptake but 100 nM clenbuterol inhibited uptake. Antagonist studies suggested that the opposite effects of 10 nM BRL37344 and 100 nM clenbuterol are mediated by the beta(2)-adrenoceptor. BRL37344 and clenbuterol have been studied in muscles that lack beta(3)-, beta(2)- or all three beta-adrenoceptors. Effects of beta-adrenoceptor antagonists on responses to the agonists have been studied further using muscles from wild-type mice.Experimental Approach: Soleus muscles of wild-type or beta-adrenoceptor knockout mice were incubated with 2-deoxy[1-(14)C]-glucose, and beta-adrenoceptor ligands. Formation of 2-deoxy[1-(14)C]-glucose-6-phosphate was measured.Key Results: Concentration-response relationships were similar for BRL37344 and clenbuterol in normal muscle and muscle lacking beta(3)-adrenoceptors. Ten pM BRL37344 and clenbuterol stimulated glucose uptake in muscle lacking beta(2)-adrenoceptors or all three beta-adrenoceptors, but 10 nM BRL37344 did not stimulate uptake in either case, and 100 nM clenbuterol stimulated, rather than inhibited, uptake in muscle lacking beta(2)-adrenoceptors. One hundred nM clenbuterol also stimulated glucose uptake in normal muscle when beta(2)-adrenoceptors were blocked with ICI118551, and this was not prevented by antagonism of beta(1)- or beta(3)-adrenoceptors.Conclusions and Implications: Ten nM BRL37344 and 100 nM clenbuterol have opposite effects on glucose uptake but both effects are mediated by the beta(2)-adrenoceptor - apparently an example of agonist-directed signalling. Ten pM BRL37344, 10 pM clenbuterol and 100 nM clenbuterol in the presence of ICI118551 stimulate glucose uptake via beta-adrenoceptor-independent mechanisms, demonstrating unknown properties for the agonists. [ABSTRACT FROM AUTHOR]

Background and Purpose: Earlier we reported that 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG), an oxidatively modified guanine nucleoside, exerted anti-inflammatory activity through inactivation of the GTP binding protein, Rac. In the present study, the effects of 8-oxo-dG were investigated on responses to antigen challenge in sensitized mice, as Rac is also involved at several steps of the immune process including antigen-induced release of mediators from mast cells.Experimental Approach: Mice were sensitized and challenged with ovalbumin without or with oral administration of 8-oxo-dG during the challenge. Effects of 8-oxo-dG were assessed by measuring lung function, cells and cytokines in broncho-alveolar lavage fluid (BALF) and serum levels of antigen-specific IgE. Rac activity in BALF cells was also measured.Key Results: 8-oxo-dG inhibited the increased airway resistance and decreased lung compliance of sensitized and challenged mice to the levels of non-sensitized control mice and lowered the increased leukocytes particularly, eosinophils, in BALF. Furthermore, 8-oxo-dG suppressed allergy-associated immune responses, such as raised anti- ovalbumin IgE antibody in serum, increased expression of CD40 and CD40 ligand in lung, increased interleukin-4, -5, -13, interferon-gamma and tumour necrosis factor-alpha in BALF and mRNA levels of these cytokines in BALF cells, dose-dependently. The corresponding purine, 8-oxo-guanine, showed no effects in the same experiments. Finally, 8-oxo-dG, but not 8-oxo-guanine, inhibited the increased Rac activity in sensitized and challenged mice.Conclusion and Implications: 8-Oxo-dG had anti-allergic actions that might be mediated by Rac inactivation. This compound merits further evaluation of its therapeutic potential in allergic asthma. [ABSTRACT FROM AUTHOR]

Background and Purpose: Prostaglandin F(2alpha) (PGF(2alpha)) is implicated in the pathogenesis of inflammatory bowel disease and colorectal cancer. This study investigates the effects of PGF(2alpha) on electrophysiological parameters in isolated human colonic mucosa.Experimental Approach: Ion transport was measured as changes in short-circuit current across human colonic epithelia mounted in Ussing chambers. Colonic crypts were isolated by calcium chelation and cyclic adenosine monophosphate (cAMP) was measured by ELISA.Key Results: PGF(2alpha) stimulated chloride secretion in a concentration-dependent manner with an EC(50) of 130 nM. The PGF(2alpha) induced increase in chloride secretion was inhibited by AL8810 (10 microM), a specific PGF(2alpha) receptor antagonist. In addition, PGF(2alpha) (1 microM) significantly increased levels of cAMP in isolated colonic crypts.Conclusions and Implications: PGF(2alpha) stimulated chloride secretion in samples of human colon in vitro through a previously unrecognizd cAMP-mediated mechanism. These findings have implications for inflammatory states. [ABSTRACT FROM AUTHOR]

Background and Purpose: The association between torcetrapib and its off-target effects on blood pressure suggested a possible class-specific effect. The effects of dalcetrapib (RO4607381/JTT-705) and torcetrapib on haemodynamics and the renin-angiotensin-aldosterone system (RAAS) were therefore assessed in a rat model.Experimental Approach: Arterial pressure (AP) and heart rate were measured by telemetry in normotensive and spontaneously hypertensive rats (SHR) receiving torcetrapib 10, 40 or 80 mg kg(-1) day(-1); dalcetrapib 100, 300 or 500 mg(-1) kg day(-1); or vehicle (placebo) for 5 days. Expression of RAAS genes in adrenal gland, kidney, aorta and lung from normotensive rats following 5 days' treatment with torcetrapib 40 mg kg(-1) day(-1), dalcetrapib 500 mg kg(-1) day(-1) or vehicle was measured by quantitative polymerase chain reaction.Key Results: Torcetrapib transiently increased mean AP in normotensive rats (+3.7 +/- 0.1 mmHg), whereas treatment in SHR resulted in a dose-dependent and sustained increase [+6.5 +/- 0.6 mmHg with 40 mg kg(-1) day(-1) at day 1 (P < 0.05 versus placebo)], which lasted over the treatment period. No changes in AP or heart rate were observed with dalcetrapib. Torcetrapib, but not dalcetrapib, increased RAAS-related mRNAs in adrenal glands and aortas.Conclusions and Implications: In contrast to torcetrapib, dalcetrapib did not increase blood pressure or RAAS-related gene expression in rats, suggesting that the off-target effects of torcetrapib are not a common feature of all compounds acting on cholesteryl ester transfer protein. [ABSTRACT FROM AUTHOR]

Background and Purpose: Stimulation of vascular beta-adrenoceptors causes vasodilatation through activation of adenylyl cyclase (AC) and plasma membrane potassium channels, and beta-adrenoceptors have been linked to activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase in various cell lines. However, how these findings relate to functional responses in intact tissues is largely unknown. The aim of this study, therefore, was to investigate the role of ERK in beta-adrenoceptor-induced vasodilatation.Experimental Approach: Segments of porcine coronary artery were mounted in a Mulvany wire myograph and bathed in Krebs-Henseleit buffer gassed with 95% O(2)/5% CO(2) and maintained at 37 degrees C. Tissues were pre-contracted with the thromboxane mimetic U46619, endothelin-1 or KCl. Cumulative concentration-response curves to beta-adrenoceptor agonists or forskolin were then carried out in the absence or presence of the mitogen-activated protein kinase kinase (MEK) inhibitors PD98059 (10 or 50 microM) or U0126 (10 microM).Key Results: PD98059 caused a concentration-dependent leftward shift in response to isoprenaline (pEC(50) control, 7.5 +/- 0.1; 50 microM PD98059, 8.1 +/- 0.1: P < 0.05). Inhibition of MEK also enhanced the maximum relaxation seen with salbutamol, but not the responses to the beta(1)-adrenoceptor selective agonist xamoterol or the AC activator forskolin. There was no enhancement of the relaxations to beta-adrenoceptor agonists after inhibition of ERK activation in tissues pre-contracted with KCl or treated with the K(+) channel blocker tetraethylammonium.Conclusions and Implications: These data indicate that ERK inhibits beta(2)-adrenoceptor-mediated vasodilatation through a mechanism which may involve inactivation of plasma membrane potassium channels. [ABSTRACT FROM AUTHOR]

Background and Purpose: Several agonists of the alpha7 nicotinic acetylcholine receptor (nAChR) have been developed for treatment of cognitive deficits. However, agonist efficacy in vivo is difficult to reconcile with rapid alpha7 nAChR desensitization in vitro; and furthermore, the correlation between in vitro receptor efficacy and in vivo behavioural efficacy is not well delineated. The possibility that agonists of this receptor actually function in vivo as inhibitors via desensitization has not been finally resolved.Experimental Approach: Two structurally related alpha7 nAChR agonists were characterized and used to assess the degree of efficacy required in a behavioural paradigm.Key Results: NS6784 activated human and rat alpha7 nAChR with EC(50)s of 0.72 and 0.88 microM, and apparent efficacies of 77 and 97% respectively. NS6740, in contrast, displayed little efficacy at alpha7 nAChR (<2% in oocytes, < or =8% in GH4C1 cells), although its agonist-like properties were revealed by adding a positive allosteric modulator of alpha7 nAChRs or using the slowly desensitizing alpha7V274T receptor. In mouse inhibitory avoidance (IA) memory retention, NS6784 enhanced performance as did the 60% partial agonist A-582941. In contrast, NS6740 did not enhance performance, but blocked effects of A-582941.Conclusions and Implications: Collectively, these findings suggest that a degree of alpha7 nAChR agonist efficacy is required for behavioural effects in the IA paradigm, and that such behavioural efficacy is not due to alpha7 nAChR desensitization. Also, a partial agonist of very low efficacy for this receptor could be used as an inhibitor, in the absence of alpha7 nAChR antagonists with favourable CNS penetration. [ABSTRACT FROM AUTHOR]

Background and Purpose: Extracellular nucleotides play a crucial role in the regulation of vascular tone and blood flow. Stimulation of endothelial cell P2Y1 receptors evokes concentration-dependent full dilatation of resistance arteries. However, this GPCR can desensitize upon prolonged exposure to the agonist. Our aim was to determine the extent and nature of P2Y1 desensitization in isolated and pressurized rat small mesenteric arteries.Experimental Approach: The non-hydrolyzable selective P2Y1 agonist ADPbetaS (3 microM) was perfused through the lumen of arteries pressurized to 70 mmHg. Changes in arterial diameter and endothelial cell [Ca(2+)](i) were obtained in the presence and absence of inhibitors of protein kinase C (PKC).Key Results: ADPbetaS evoked rapid dilatation to the maximum arterial diameter but faded over time to a much-reduced plateau closer to 35% dilatation. This appeared to be due to desensitization of the P2Y1 receptor, as subsequent endothelium-dependent dilatation to acetylcholine (1 microM) remained unaffected. Luminal treatment with the PKC inhibitors BIS-I (1 microM) or BIS-VIII (1 microM) tended to augment concentration-dependent dilatation to ADPbetaS (0.1-3 microM) and prevented desensitization. Another PKC inhibitor, Gö 6976 (1 microM), was less effective in preventing desensitization. Measurements of endothelial cell [Ca(2+)](i) in pressurized arteries confirmed the P2Y1 receptor but not M(3) muscarinic receptor desensitization.Conclusions and Implications: These data demonstrate for the first time the involvement of PKC in the desensitization of endothelial P2Y1 receptors in pressurized rat mesenteric arteries, which may have important implications in the control of blood flow by circulating nucleotides. [ABSTRACT FROM AUTHOR]

Background and Purpose: This study investigated the role of central sympathetic activity and related mitogen-activated protein kinase (MAPK) signalling in the cardiovascular effects of ethanol in a model of acute renal failure (ARF).Experimental Approach: The effects of pharmacological interventions that inhibit peripheral or central sympathetic activity or MAPK on the cardiovascular actions of ethanol in rats with ARF induced by glycerol were evaluated.Key Results: Glycerol (50%, 10 mL.kg(-1), i.m.) caused progressive increases and decreases in blood pressure (BP) and heart rate (HR) respectively. Subsequent i.v. ethanol (0.25 or 1 g.kg(-1)) elicited dose-related changes in BP (decreases) and HR (increases). These effects were replicated after intracisternal (i.c.) administration of ethanol. Blockade of nicotinic cholinoceptors (nAChR, hexamethonium, 20 mg.kg(-1)) or alpha(1)-adrenoceptors (prazosin, 1 mg.kg(-1)) attenuated cardiovascular effects of ethanol. Ethanol hypotension was also attenuated after the centrally acting sympatholytic drug moxonidine (selective I(1)-site agonist, 100 microg.kg(-1) i.v.), but not guanabenz (selective alpha(2)-receptor agonist, 30 microg.kg(-1), i.v.), suggesting involvement of central circuits of I(1) sites in ethanol-evoked hypotension. Selective blockade I(1) sites (efaroxan) but not alpha(2) (yohimbine) adrenoceptors abolished the hypotensive response to ethanol. Intracisternal administration of PD98059 or SB203580, inhibitors of extracellular signal-regulated kinase (ERK 1/2) and p38 MAPK, respectively, reduced the hypotensive action of moxonidine or ethanol. When used simultaneously, the two MAPK inhibitors produced additive attenuation of ethanol hypotension.Conclusions and Implications: Sympathoinhibitory pathways of central I(1)-sites and downstream ERK/p38 MAPK signalling were involved in the hypotensive action of ethanol in ARF. [ABSTRACT FROM AUTHOR]

Background and Purpose: ABC multidrug transporters (MDR-ABC proteins) cause multiple drug resistance in cancer and may be involved in the decreased anti-cancer efficiency and modified pharmacological properties of novel specifically targeted agents. It has been documented that ABCB1 and ABCG2 interact with several first-generation, small-molecule, tyrosine kinase inhibitors (TKIs), including the Bcr-Abl fusion kinase inhibitor imatinib, used for the treatment of chronic myeloid leukaemia. Here, we have investigated the specific interaction of these transporters with nilotinib, dasatinib and bosutinib, three clinically used, second-generation inhibitors of the Bcr-Abl tyrosine kinase activity.Experimental Approach: MDR-ABC transporter function was screened in both membrane- and cell-based (K562 cells) systems. Cytotoxicity measurements in Bcr-Abl-positive model cells were coupled with direct determination of intracellular TKI concentrations by high-pressure liquid chromatography-mass spectrometry and analysis of the pattern of Bcr-Abl phosphorylation. Transporter function in membranes was assessed by ATPase activity.Key Results: Nilotinib and dasatinib were high-affinity substrates of ABCG2, and this protein mediated an effective resistance in cancer cells against these compounds. Nilotinib and dasatinib also interacted with ABCB1, but this transporter provided resistance only against dasatinib. Neither ABCB1 nor ABCG2 induced resistance to bosutinib. At relatively higher concentrations, however, each TKI inhibited both transporters.Conclusions and Implications: A combination of in vitro assays may provide valuable preclinical information for the applicability of novel targeted anti-cancer TKIs, even in multidrug-resistant cancer. The pattern of MDR-ABC transporter-TKI interactions may also help to understand the general pharmacokinetics and toxicities of new TKIs. [ABSTRACT FROM AUTHOR]

Background and Purpose: Fentanyl overdose deaths have reached "epidemic" levels in North America. Death in opioid overdose invariably results from respiratory depression. In the present work, we have characterized how fentanyl depresses respiration, and by comparing fentanyl with heroin and morphine, the active breakdown product of heroin, we have sought to determine the factors, in addition to high potency, that contribute to the lethality of fentanyl.Experimental Approach: Respiration (rate and tidal volume) was measured in awake, freely moving mice by whole body plethysmography.Key Results: Intravenously administered fentanyl produced more rapid depression of respiration than equipotent doses of heroin or morphine. Fentanyl depressed both respiratory rate and tidal volume. Fentanyl did not depress respiration in μ-opioid receptor knockout mice. Naloxone, the opioid antagonist widely used to treat opioid overdose, reversed the depression of respiration by morphine more readily than that by fentanyl, whereas diprenorphine, a more lipophilic antagonist, was equipotent in reversing fentanyl and morphine depression of respiration. Prolonged treatment with morphine induced tolerance to respiratory depression, but the degree of cross tolerance to fentanyl was less than the tolerance to morphine itself.Conclusion and Implications: We propose that several factors (potency, rate of onset, lowered sensitivity to naloxone, and lowered cross tolerance to heroin) combine to make fentanyl more likely to cause opioid overdose deaths than other commonly abused opioids. Lipophilic antagonists such as diprenorphine may be better antidotes than naloxone to treat fentanyl overdose. [ABSTRACT FROM AUTHOR]

Background and Purpose: Endothelium-derived vasoconstriction is a hallmark of vascular dysfunction in hypertension. In some cases, an overproduction of endothelium-derived prostacyclin (PGI2 ) can cause contraction rather than relaxation. Relaxin is well known for its vasoprotective actions, but the possibility that this peptide could also reverse endothelium-derived vasoconstriction has never been investigated. We tested the hypothesis that short-term relaxin treatment mitigates endothelium-derived vasoconstriction in spontaneously hypertensive rats (SHR).Experimental Approach: Male Wistar Kyoto rats (WKY) and SHR were subcutaneously infused with either vehicle (20 mmol·L-1 sodium acetate) or relaxin (13.3 μg·kg-1 ·hr-1 ) using osmotic minipumps for 3 days. Vascular reactivity to the endothelium-dependent agonist ACh was assessed in vitro by wire myography. Quantitative PCR and LC-MS were used to identify changes in gene expression of prostanoid pathways and PG production, respectively.Key Results: Relaxin treatment ameliorated hypertension-induced endothelial dysfunction by increasing NO-dependent relaxation and reducing endothelium-dependent contraction. Notably, short-term relaxin treatment up-regulated mesenteric PGI2 receptor (IP) expression, permitting PGI2 -IP-mediated vasorelaxation. In the aorta, reversal of contraction was accompanied by suppression of the hypertension-induced increase in prostanoid-producing enzymes and reduction in PGI2 -evoked contractions.Conclusions and Implications: Relaxin has region-dependent vasoprotective actions in hypertension. Specifically, relaxin has distinct effects on endothelium-derived contracting factors and their associated vasoconstrictor pathways in mesenteric arteries and the aorta. Taken together, these observations reveal the potential of relaxin as a new therapeutic agent for vascular disorders that are associated with endothelium-derived vasoconstriction including hypertension. [ABSTRACT FROM AUTHOR]

Background and Purpose: In patients with pulmonary hypertension (PH) associated with lung disease and/or hypoxia (Group III), decreased pulmonary vascular tone and tissue hypoxia is therapeutically beneficial. PGE2 and PGI2 induce potent relaxation of human bronchi from non-PH (control) patients via EP4 and IP receptors, respectively. However, the effects of PGE2 /PGI2 and their mimetics on human bronchi from PH patients are unknown. Here, we have compared relaxant effects of several PGI2 -mimetics approved for treating PH Group I with several PGE2 -mimetics, in bronchial preparations derived from PH Group III and control patients.Experimental Approach: Relaxation of bronchial muscle was assessed in samples isolated from control and PH Group III patients. Expression of prostanoid receptors was analysed by western blot and real-time PCR, and endogenous PGE2 , PGI2 , and cAMP levels were determined by ELISA.Key Results: Maximal relaxations induced by different EP4 receptor agonists (PGE2 , L-902688, and ONO-AE1-329) were decreased in human bronchi from PH patients, compared with controls. However, maximal relaxations produced by PGI2 -mimetics (iloprost, treprostinil, and beraprost) were similar for both groups of patients. Both EP4 and IP receptor protein and mRNA expressions were significantly lower in human bronchi from PH patients. cAMP levels significantly correlated with PGI2 but not with PGE2 levels.Conclusion and Implications: The PGI2 -mimetics retained maximal bronchodilation in PH Group III patients, whereas bronchodilation induced by EP4 receptor agonists was decreased. Restoration of EP4 receptor expression in airways of PH Group III patients with respiratory diseases could bring additional therapeutic benefit. [ABSTRACT FROM AUTHOR]

Background and Purpose: CR4056 is a first-in-class imidazoline-2 (I2 ) receptor ligand characterized by potent analgesic activity in different experimental animal models of pain. In a recent phase II clinical trial, CR4056 effectively reduced pain in patients with knee osteoarthritis. In the present study, we investigated the effects of CR4056 on PKCε translocation in vitro and on PKCε activation in vivo in dorsal root ganglia (DRG) neurons.Experimental Approach: Effects of CR4056 on bradykinin-induced PKCε translocation were studied in rat sensory neurons by immunocytochemistry. PKCε activation was investigated by immunohistochemistry analysis of DRG from complete Freund's adjuvant-treated animals developing local hyperalgesia. The analgesic activity of CR4056 was tested on the same animals.Key Results: CR4056 inhibited PKCε translocation with very rapid and long-lasting activity. CR4056 decreased hyperalgesia and phospho-PKCε immunoreactivity in the DRG neurons innervating the inflamed paw. The effect of CR4056 on PKCε translocation was blocked by pertussis toxin, implying that the intracellular pathways involved Gi proteins. The inhibition of PKCε translocation by CR4056 was independent of the α2 -adrenoeceptor and, surprisingly, was also independent of idazoxan-sensitive I2 binding sites. The I2 agonist 2BFI had no effect alone but potentiated the activity of low concentrations of CR4056.Conclusions and Implications: Our results demonstrate that CR4056 shares the ability to inhibit PKCε translocation with other analgesics. Whether the inhibition of PKCε involves binding to specific subtype(s) of I2 receptors should be further investigated. If so, this would be a new mode of action of a highly specific I2 receptor ligand. [ABSTRACT FROM AUTHOR]

Background and Purpose: The TNF family ligands, B cell activating factor of the TNF family (BAFF, also known as B lymphocyte stimulator, BLyS) and a proliferation-inducing ligand (APRIL), share the transmembrane activator and calcium-modulator and cyclophilin ligand (CAML)-interactor (TACI) as one of their common receptors. Atacicept, a chimeric recombinant TACI/IgG1-Fc fusion protein, inhibits both ligands. TACI and APRIL also bind to proteoglycans and to heparin that is structurally related to proteoglycans. It is unknown whether the portion of TACI contained in atacicept can bind directly to proteoglycans, or indirectly via APRIL, and whether this could interfere with the anti-coagulant properties of heparin.Experimental Approach: Binding of atacicept and APRIL to proteoglycan-positive cells was measured by FACS. Activities of heparin and atacicept were measured with activated factor Xa inhibition and cell-based assays. Effects of heparin on circulating atacicept was monitored in mice.Key Results: Atacicept did not bind to proteoglycan-positive cells, but when complexed to APRIL could do so indirectly via APRIL. Multimers of atacicept obtained after exposure to cysteine or BAFF 60-mer bound directly to proteoglycans. Atacicept alone, or in complex with APRIL, or in a multimeric form did not interfere with heparin activity in vitro. Conversely, heparin did not influence inhibition of BAFF and APRIL by atacicept and did not change circulating levels of atacicept.Conclusions and Implications: Lack of detectable interference of APRIL-bound or free atacicept on heparin activity makes it unlikely that atacicept at therapeutic doses will interfere with the function of heparin in vivo. [ABSTRACT FROM AUTHOR]

Background and Purpose: Depression and anxiety are common causes of disability, and innovative tools and potential pharmacological targets are actively sought for prevention and treatment. Therapeutic strategies targeting the relaxin-3 peptide or its primary endogenous receptor, RXFP3, for the treatment of major depression and anxiety disorders have been limited by a lack of compounds with drug-like properties. We proposed that a hydrocarbon-stapled mimetic of relaxin-3, when administered intranasally, might be uniquely applicable to the treatment of these disorders.Experimental Approach: We designed a series of hydrocarbon-stapled relaxin-3 mimetics and identified the most potent compound using in vitro receptor binding and activation assays. Further, we assessed the effect of intranasal delivery of relaxin-3 and the lead stapled mimetic in rat models of anxiety and depression.Key Results: We developed an i,i+7 stapled relaxin-3 mimetic that manifested a stabilized α-helical structure, proteolytic resistance, and confirmed agonist activity in receptor binding and activation in vitro assays. The stapled peptide agonist enhanced food intake after intracerebral infusion in rats, confirming in vivo activity. We showed that intranasal delivery of the lead i,i+7 stapled peptide or relaxin-3 had orexigenic effects in rats, indicating a potential clinically translatable route of delivery. Further, intranasal administration of the lead i,i+7 stapled peptide exerted anxiolytic and antidepressant-like activity in anxiety- and depression-related behaviour paradigms.Conclusions and Implications: Our preclinical findings demonstrate that targeting the relaxin-3/RXFP3 receptor system via intranasal delivery of an i,i+7 stapled relaxin-3 mimetic may represent an effective treatment approach for depression, anxiety, and related neuropsychiatric disorders. [ABSTRACT FROM AUTHOR]

Background and Purpose: Osteoarthritic pain is a chronic disabling condition lacking effective treatment. Continuous use of opioid drugs during osteoarthritic pain induces tolerance and may result in dose escalation and abuse. Sigma-1 (σ1) receptors, a chaperone expressed in key areas for pain control, modulates μ-opioid receptor activity and represents a promising target to tackle these problems. The present study investigates the efficacy of the σ1 receptor antagonist E-52862 to inhibit pain sensitization, morphine tolerance, and associated electrophysiological and molecular changes in a murine model of osteoarthritic pain.Experimental Approach: Mice received an intra-knee injection of monoiodoacetate followed by 14-day treatment with E-52862, morphine, or vehicle, and mechanical sensitivity was assessed before and after the daily doses.Key Results: Monoiodoacetate-injected mice developed persistent mechanical hypersensitivity, which was dose-dependently inhibited by E-52862. Mechanical thresholds assessed before the daily E-52862 dose showed gradual recovery, reaching complete restoration by the end of the treatment. When repeated treatment started 15 days after knee injury, E-52862 produced enhanced short-term analgesia, but recovery to baseline threshold was slower. Both a σ1 receptor agonist and a μ receptor antagonist blocked the analgesic effects of E-52862. An acute, sub-effective dose of E-52862 restored morphine analgesia in opioid-tolerant mice. Moreover, E-52862 abolished spinal sensitization in osteoarthritic mice and inhibited pain-related molecular changes.Conclusion and Implications: These findings show dual effects of σ1 receptor antagonism alleviating both short- and long-lasting antinociception during chronic osteoarthritis pain. They identify E-52862 as a promising pharmacological agent to treat chronic pain and avoid opioid tolerance. [ABSTRACT FROM AUTHOR]

Background and Purpose: Salinomycin is a well-known inhibitor of human cancer stem cells (CSCs). However, the molecular mechanism(s) by which salinomycin targets colorectal CSCs is poorly understood. Here, we have investigated underlying antitumour mechanisms of salinomycin in colorectal cancer cells and three tumour models.Experimental Approach: The inhibitory effect of salinomycin on the Wnt/β-catenin pathway was analysed with the SuperTopFlash reporter system. The mRNA expression of Wnt target genes was evaluated with real-time PCR. Effects of salinomycin on β-catenin/TCF4E interaction were examined using co-immunoprecipitation and an in vitro GST pull-down assay. Cell proliferation was determined by BrdU incorporation and soft agar colony formation assay. The stemness of the cells was assessed by sphere formation assay. Antitumour effects of salinomycin on colorectal cancers was evaluated with colorectal CSC xenografts, APCmin/+ transgenic mice, and patient-derived colorectal tumour xenografts.Key Results: Salinomycin blocked β-catenin/TCF4E complex formation in colorectal cancer cells and in an in vitro GST pull-down assay, thus decreasing expression of Wnt target genes. Salinomycin also suppressed the transcriptional activity mediated by β-catenin/LEF1 or β-catenin/TCF4E complex and exhibited an inhibitory effect on the sphere formation, proliferation, and anchorage-independent growth of colorectal cancer cells. In colorectal tumour xenografts and APCmin/+ transgenic mice, administration of salinomycin significantly reduced tumour growth and the expression of CSC-related Wnt target genes including LGR5.Conclusions and Implications: Our study suggested that salinomycin could suppress the growth of colorectal cancer by disrupting the β-catenin/TCF complex and thus may be a promising agent for colorectal cancer treatment. [ABSTRACT FROM AUTHOR]

Background and Purpose: Much of the opioid epidemic arose from abuse of prescription opioid drugs. This study sought to determine if the combination of a cannabinoid with an opioid could produce additive or synergistic effects on pain, allowing reduction in the opioid dose needed for maximal analgesia.Experimental Approach: Pain was assayed using the formalin test in mice and the carrageenan assay in rats. Morphine and two synthetic cannabinoids were tested: WIN55,212-2 (WIN), which binds to both CB1 and CB2 receptors, and possibly TRPV1 channels; and GP1a, which has activity at CB2 receptors and is reported to inhibit fatty acid amide hydrolase, thus raising levels of endogenous cannabinoids.Key Results: Morphine in combination with WIN in the formalin test gave synergistic analgesia. Studies with selective antagonists showed that WIN was acting through CB1 receptors. Morphine in combination with GP1a in the formalin test was sub-additive. In the carrageenan test, WIN had no added effect when combined with morphine, but GP1a with morphine showed enhanced analgesia. Both WIN and Gp1a used alone had analgesic activity in the formalin pain test, but not in the carrageenan pain test.Conclusions and Implications: The ability of a cannabinoid to produce an additive or synergistic effect on analgesia when combined with morphine varies with the pain assay and may be mediated by CB1 or CB2 receptors. These results hold the promise of using cannabinoids to reduce the dose of opioids for analgesia in certain pain conditions. [ABSTRACT FROM AUTHOR]

Background and Purpose: The microRNA miR-29b-3p shows important roles in regulating apoptosis and inflammation. However, its effects on intestinal ischaemia/reperfusion (II/R) injury have not been reported. Here we have investigated the functions of miR-29b-3p on II/R injury on order to find drug targets to treat the injury.Experimental Approach: Two models - in vitro hypoxia/reoxygenation (H/R) of IEC-6 cells; in vivo, II/R injury in C57BL/6 mice were used. Western blotting and dual-luciferase reporter assays were used and mimic and siRNA transfection tests were applied to assess the effects of miR-29b-3p on II/R injury via targeting TNF receptor-associated factor 3 (TRAF3).Key Results: The H/R procedure decreased cell viability and promoted inflammation and apoptosis in IEC-6 cells, and the II/R procedure also promoted intestinal inflammation and apoptosis in mice. Expression levels of miR-29b-3p were decreased in H/R-induced cells and II/R-induced intestinal tissues of mice compared with control group or sham group, which directly targeted TRAF3. Decreased miR-29b-3p level markedly increased TRAF3 expression via activating TGF-α-activated kinase 1 phosphorylation, increasing NF-κB (p65) levels to promote inflammation, up-regulating Bcl2-associated X expression, and down-regulating Bcl-2 expression to trigger apoptosis. In addition, the miR-29b-3p mimetic and TRAF3 siRNA in IEC-6 cells markedly suppressed apoptosis and inflammation to alleviate II/R injury via inhibiting TRAF3 signallimg.Conclusions and Implications: The miR-29b-3p played a critical role in II/R injury, via targeting TRAF3, which should be considered as a significant drug target to treat the disease. [ABSTRACT FROM AUTHOR]

Background and Purpose: Vascular endothelial growth factor A (VEGF-A) is a key mediator of angiogenesis. A striking feature of the binding of a fluorescent analogue of VEGF165 a to nanoluciferase-tagged VEGF receptor 2 (VEGFR2) in living cells is that the BRET signal is not sustained and declines over time. This may be secondary to receptor internalisation. Here, we have compared the binding of three fluorescent VEGF-A isoforms to VEGFR2 in cells and isolated membrane preparations.Experimental Approach: Ligand-binding kinetics were monitored in both intact HEK293T cells and membranes (expressing nanoluciferase-tagged VEGFR2) using BRET between tagged receptor and fluorescent analogues of VEGF165 a, VEGF165 b, and VEGF121 a. VEGFR2 endocytosis in intact cells expressing VEGFR2 was monitored by following the appearance of fluorescent ligand-associated receptors in intracellular endosomes using automated quantitative imaging.Key Results: Quantitative analysis of the effect of fluorescent VEGF-A isoforms on VEGFR2 endocytosis in cells demonstrated that they produce a rapid and potent translocation of ligand-bound VEGFR2 into intracellular endosomes. NanoBRET can be used to monitor the kinetics of the binding of fluorescent VEGF-A isoforms to VEGFR2. In isolated membrane preparations, ligand-binding association curves were maintained for the duration of the 90-min experiment. Measurement of the koff at pH 6.0 in membrane preparations indicated shorter ligand residence times than those obtained at pH 7.4.Conclusions and Implications: These studies suggest that rapid VEGF-A isoform-induced receptor endocytosis shortens agonist residence times on the receptor (1/koff ) as VEGFR2 moves from the plasma membrane to the intracellular endosomes. [ABSTRACT FROM AUTHOR]

Background and Purpose: Emerging evidence indicates that hypertension is mediated by immune mechanisms. We hypothesized that exposure to Porphyromonas gingivalis antigens, commonly encountered in periodontal disease, can enhance immune activation in hypertension and exacerbate the elevation in BP, vascular inflammation and vascular dysfunction.Experimental Approach: Th1 immune responses were elicited through immunizations using P. gingivalis lysate antigens (10 μg) conjugated with aluminium oxide (50 μg) and IL-12 (1 μg). The hypertension and vascular endothelial dysfunction evoked by subpressor doses of angiotensin II (0.25 mg·kg-1 ·day-1 ) were studied, and vascular inflammation was quantified by flow cytometry and real-time PCR.Key Results: Systemic T-cell activation, a characteristic of hypertension, was exacerbated by P. gingivalis antigen stimulation. This translated into increased aortic vascular inflammation with enhanced leukocyte, in particular, T-cell and macrophage infiltration. The expression of the Th1 cytokines, IFN-γ and TNF-α, and the transcription factor, TBX21, was increased in aortas of P. gingivalis/IL-12/aluminium oxide-immunized mice, while IL-4 and TGF-β were unchanged. These immune changes in mice with induced T-helper-type 1 immune responses were associated with an enhanced elevation of BP and endothelial dysfunction compared with control mice in response to 2 week infusion of a subpressor dose of angiotensin II.Conclusions and Implications: These results support the concept that Th1 immune responses induced by bacterial antigens such as P. gingivalis can increase sensitivity to subpressor pro-hypertensive insults such as low-dose angiotensin II, thus providing a mechanistic link between chronic infection, such as periodontitis, and hypertension.Linked Articles: This article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc. [ABSTRACT FROM AUTHOR]

Background and Purpose: Numerous claims are made for cannabis' therapeutic utility upon human seizures, but concerns persist about risks. A potential confounder is the presence of both Δ9 -tetrahydrocannabinol (THC), variously reported to be pro- and anticonvulsant, and cannabidiol (CBD), widely confirmed as anticonvulsant. Therefore, we investigated effects of prolonged exposure to different THC/CBD cannabis extracts on seizure activity and associated measures of endocannabinoid (eCB) system signalling.Experimental Approach: Cannabis extract effects on in vivo neurological and behavioural responses, and on bioanalyte levels, were measured in rats and dogs. Extract effects on seizure activity were measured using electroencephalography telemetry in rats. eCB signalling was also investigated using radioligand binding in cannabis extract-treated rats and treatment-naïve rat, mouse, chicken, dog and human tissue.Key Results: Prolonged exposure to cannabis extracts caused spontaneous, generalized seizures, subserved by epileptiform discharges in rats, but not dogs, and produced higher THC, but lower 11-hydroxy-THC (11-OH-THC) and CBD, plasma concentrations in rats versus dogs. In the same rats, prolonged exposure to cannabis also impaired cannabinoid type 1 receptor (CB1 receptor)-mediated signalling. Profiling CB1 receptor expression, basal activity, extent of activation and sensitivity to THC suggested interspecies differences in eCB signalling, being more pronounced in a species that exhibited cannabis extract-induced seizures (rat) than one that did not (dog).Conclusions and Implications: Sustained cannabis extract treatment caused differential seizure, behavioural and bioanalyte levels between rats and dogs. Supporting radioligand binding data suggest species differences in eCB signalling. Interspecies variations may have important implications for predicting cannabis-induced convulsions from animal models.Linked Articles: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc. [ABSTRACT FROM AUTHOR]

Background and Purpose: Abdominal aortic aneurysm (AAA) is a degenerative disease affecting human health, but there are no safe and effective medications for AAA therapy. Cycloastragenol (CAG), derived from Astragali Radix, has various pharmacological effects. However, whether CAG can protect against AAA remains elusive. In this study, we investigated whether CAG has an inhibitory effect on AAA and its related mechanism.Experimental Approach: The AAA mouse model was induced by incubating the abdominal aorta with elastase. CAG was administered by gavage at different doses beginning on the same day or 14 days after inducing AAA to explore its preventive or therapeutic effects respectively. The preventive effects of CAG on AAA were verified in another AAA mouse model induced by angiotensin II in ApoE-/- mouse. In vitro experiments were implemented on rat vascular smooth muscle cells (VSMCs) stimulated by TNF-α.Key Results: Compared to the control AAA model group, CAG (125 mg·kg-1 body weight day-1 ) reduced the incidence of AAA, the dilatation of aorta and elastin degradation in media in both mouse models of AAA. CAG suppressed the inflammation, oxidation, phenotype switch and apoptosis in TNF-α-stimulated VSMCs, ameliorated the expression and activity of MMPs and decreased the activation of the ERK/JNK signalling pathway. CAG also inhibited the degradation of elastin in TNF-α-stimulated VSMCs.Conclusion and Implications: CAG presents protective effects against AAA through down-regulation of the MAPK signalling pathways and thus attenuates inflammation, oxidation, VSMC phenotype switch and apoptosis and the expression of MMPs as well as increasing elastin biosynthesis. [ABSTRACT FROM AUTHOR]

Background and Purpose: Atopic dermatitis (AD) is a multifactorial skin condition with complex interactions of innate and adaptive immune responses. There are several existing therapies for AD, including topical glucocorticosteroids, emollients, phototherapies, calcineurin inhibitors and immunosuppressants, such as cyclosporine A. Although these therapies reduce inflammation, they also cause serious side effects. Therefore, it is necessary to develop new therapeutic approaches for AD treatment without side effects. There are several studies on natural materials or toxins, such as herbs, ginseng extract and snake venom, for AD treatment. However, treatment of AD with bee venom and its major component, melittin has rarely been studied.Experimental Approach: Effects of bee venom and melittin were studied in a model of AD in vivo induced by 1-chloro-2,4-dinitrobenzene (DNCB) in female Balb/c mice and in cultures of human keratinocytes, stimulated by TNF-α/IFN-γ. The potential pharmacological effects of bee venom and melittin on these in vivo and in vitro AD-like skin disease models were studied.Key Results: Bee venom and melittin exhibited potent anti-atopic activities, shown by decreased AD-like skin lesions, induced by DNCB in mice. In vitro studies using TNF-α/IFN-γ-stimulated human keratinocytes showed that bee venom and melittin inhibited the increased expression of chemokines, such as CCL17 and CCL22, and pro-inflammatory cytokines, including IL-1β, IL-6 and IFN-γ, through the blockade of the NF-κB and STAT signalling pathways.Conclusions and Implications: Our results suggest that bee venom and melittin would be suitable for epicutaneous application, as topical administration is often appropriate for the treatment of AD. [ABSTRACT FROM AUTHOR]

Background and Purpose: Enhanced late sodium current (late INa ) in heart failure and long QT syndrome type 3 is proarrhythmic. This study investigated the antiarrhythmic effect and mode of action of the selective and potent late INa inhibitor GS-458967 (GS967) against Torsades de Pointes arrhythmias (TdP) in the chronic atrioventricular block (CAVB) dog.Experimental Approach: Electrophysiological and antiarrhythmic effects of GS967 were evaluated in isolated canine ventricular cardiomyocytes and CAVB dogs with dofetilide-induced early afterdepolarizations (EADs) and TdP, respectively. Mapping of intramural cardiac electrical activity in vivo was conducted to study effects of GS967 on spatial dispersion of repolarization.Key Results: GS967 (IC50 ~200nM) significantly shortened repolarization in canine ventricular cardiomyocytes and sinus rhythm (SR) dogs, in a concentration and dose-dependent manner. In vitro, despite addition of 1μM GS967, dofetilide-induced EADs remained present in 42% and 35% of cardiomyocytes from SR and CAVB dogs, respectively. Nonetheless, GS967 (787±265nM) completely abolished dofetilide-induced TdP in CAVB dogs (10/14 after dofetilide to 0/14 dogs after GS967), while single ectopic beats (sEB) persisted in 9 animals. In vivo mapping experiments showed that GS967 significantly reduced spatial dispersion of repolarization: cubic dispersion was significantly decreased from 237±54ms after dofetilide to 123±34ms after GS967.Conclusion and Implications: GS967 terminated all dofetilide-induced TdP without completely suppressing EADs and sEB in vitro and in vivo, respectively. The antiarrhythmic mode of action of GS967, through the reduction of spatial dispersion of repolarization, seems to predominantly impede the perpetuation of arrhythmic events into TdP rather than their initiating trigger. [ABSTRACT FROM AUTHOR]

Background and Purpose: Conditions such as hypertension and renal allograft rejection are accompanied by chronic, agonist-independent, signalling by angiotensin II AT1 receptors. The current treatment paradigm for these diseases entails the preferred use of inverse agonist AT1 receptor blockers (ARBs). However, variability in the inverse agonist activities of common biphenyl-tetrazole ARBs for the active state of AT1 receptors often leads to treatment failure. Therefore, characterization of robust inverse agonist ARBs for the active state of AT1 receptors is necessary.Experimental Approach: To identify the robust inverse agonist for active state of AT1 receptors and its molecular mechanism, we performed site-directed mutagenesis, competition binding assay, inositol phosphate production assay and molecular modelling for both ground-state wild-type AT1 receptors and active-state N111G mutant AT1 receptors.Key Results: Although candesartan and telmisartan exhibited weaker inverse agonist activity for N111G- compared with WT-AT1 receptors, only eprosartan exhibited robust inverse agonist activity for both N111G- and WT- AT1 receptors. Specific ligand-receptor contacts for candesartan and telmisartan are altered in the active-state N111G- AT1 receptors compared with the ground-state WT-AT1 receptors, suggesting an explanation of their attenuated inverse agonist activity for the active state of AT1 receptors. In contrast, interactions between eprosartan and N111G-AT1 receptors were not significantly altered, and the inverse agonist activity of eprosartan was robust.Conclusions and Implications: Eprosartan may be a better therapeutic option than other ARBs. Comparative studies investigating eprosartan and other ARBs for the treatment of diseases caused by chronic, agonist-independent, AT1 receptor activation are warranted. [ABSTRACT FROM AUTHOR]