Search

Background and Purpose: Although exogenous nitric oxide (NO) clearly modifies platelet function, the role and the source of endogenous NO in vivo remain undefined. In addition, endothelial NO synthase (NOS-3) critically regulates vessel tone but its role in modulating platelet function is unclear. In this paper we have investigated the roles of endogenous NO and NOS-3 in regulating platelet function in vivo and determined the functional contribution made by platelet-derived NO.Experimental Approach: We used a mouse model for directly assessing platelet functional responses in situ in the presence of an intact vascular endothelium with supporting in vitro and molecular studies.Key Results: Acute NOS inhibition by N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME) enhanced platelet aggregatory responses to thrombin and platelets were shown to be regulated primarily by NO sources external to the platelet. Elevation of endogenous NOS inhibitors to mimic effects reported in patients with cardiovascular diseases did not enhance platelet responses. Platelet responsiveness following agonist stimulation was not modified in male or female NOS-3(-/-) mice but responses in NOS-3(-/-) mice were enhanced by L-NAME.Conclusions and Implications: Platelets are regulated by endogenous NO in vivo, primarily by NO originating from the environment external to the platelet with a negligible or undetectable role of platelet-derived NO. Raised levels of endogenous NOS inhibitors, as reported in a range of diseases were not, in isolation, sufficient to enhance platelet activity and NOS-3 is not essential for normal platelet function in vivo due to the presence of bioactive NO following deletion of NOS-3. [ABSTRACT FROM AUTHOR]

Background and Purpose: Cystic fibrosis (CF) is a debilitating disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for a Cl-/HCO3 - channel. F508del, the most common CF-associated mutation, causes both gating and biogenesis defects in the CFTR protein. This paper describes the optimization of two fluorescence assays, capable of measuring CFTR function and cellular localization, and their use in a pilot drug screen.Experimental Approach: HEK293 cells expressing YFP-F508del-CFTR, in which halide sensitive YFP is tagged to the N-terminal of CFTR, were used to screen a small library of compounds based on the VX-770 scaffold. Cells expressing F508del-CFTR-pHTomato, in which a pH sensor is tagged to the fourth extracellular loop of CFTR, were used to measure CFTR plasma membrane exposure following chronic treatment with the novel potentiators.Key Results: Active compounds with efficacy ~50% of VX-770, micromolar potency, and structurally distinct from VX-770 were identified in the screen. The F508del-CFTR-pHTomato assay suggests that the hit compound MS131A, unlike VX-770, does not decrease membrane exposure of F508del-CFTR.Conclusions and Implications: Most known potentiators have a negative influence on F508del-CFTR biogenesis/stability, which means membrane exposure needs to be monitored early during the development of drugs targeting CFTR. The combined use of the two fluorescence assays described here provides a useful tool for the identification of improved potentiators and correctors. The assays could also prove useful for basic scientific investigations on F508del-CFTR, and other CF-causing mutations. [ABSTRACT FROM AUTHOR]

Background and purpose: Vascular ATP-sensitive potassium (KATP) channels are activated by cyclic AMP elevating vasodilators through protein kinase A (PKA). Direct channel phosphorylation is a critical mechanism, though the phosphatase opposing these effects is unknown. Previously, we reported that calcineurin, a Ca2+-dependent phosphatase, inhibits KATP channels, though neither the site nor the calcineurin isoform involved is established. Given that the type-2 regulatory (RII) subunit of PKA is a substrate for calcineurin we considered whether calcineurin regulates channel activity through interacting with PKA. Experimental approach: Whole-cell recordings were made in HEK-293 cells stably expressing the vascular KATP channel (KIR6.1/SUR2B). The effect of intracellular Ca2+ and modulators of the calcineurin and PKA pathway on glibenclamide-sensitive currents were examined. Key results: Constitutively active calcineurin Aα but not Aβ significantly attenuated KATP currents activated by low intracellular Ca2+, whereas calcineurin inhibitors had the opposite effect. PKA inhibitors reduced basal KATP currents and responses to calcineurin inhibitors, consistent with the notion that some calcineurin action involves inhibition of PKA. However, raising intracellular Ca2+ (equivalent to increasing calcineurin activity), almost completely inhibited KATP channel activation induced by the catalytic subunit of PKA, whose enzymatic activity is independent of the RII subunit. In vitro phosphorylation experiments showed calcineurin could directly dephosphorylate a site in Kir6.1 that was previously phosphorylated by PKA. Conclusions and implications: Calcineurin Aα regulates KIR6.1/SUR2B by inhibiting PKA-dependent phosphorylation of the channel as well as PKA itself. Such a mechanism is likely to directly oppose the action of vasodilators on the KATP channel. British Journal of Pharmacology (2009) 157, 554–564; doi:10.1111/j.1476-5381.2009.00221.x; published online 7 May 2009 This article is commented on by Tammaro, pp. 551–553 of this issue and 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]

Striking QT prolongation and the morphologically distinctive ventricular tachycardia torsades de pointes can occur in up to 5% of patients treated with certain antiarrhythmic drugs. This adverse drug reaction also occurs, albeit far less frequently, during therapy with a range of drugs not used for cardiovascular indications; examples include certain antibiotics, antipsychotics and antihistamines. The common mechanism for drug-induced torsades de pointes is inhibition of a specific repolarizing potassium current, I(Kr). The key question facing clinicians, regulators and those who develop drugs is why torsades de pointes only occurs in some patients exposed to I(Kr) block. This paper reviews the clinical, cellular, molecular and genetic features of the arrhythmia that may provide an answer to this question and proposes future studies in this area. [ABSTRACT FROM AUTHOR]

Background and Purpose: The human kinome consists of roughly 500 kinases, including 150 that have been proposed as therapeutic targets. Protein kinases regulate an array of signalling pathways that control metabolism, cell cycle progression, cell death, differentiation and survival. It is not surprising, then, that new kinase inhibitors developed to treat cancer, including sorafenib, also exhibit cardiotoxicity. We hypothesized that sorafenib cardiotoxicity is related to its deleterious effects on specific cardiac metabolic pathways given the critical roles of protein kinases in cardiac metabolism.Experimental Approach: FVB/N mice (10 per group) were challenged with sorafenib or vehicle control daily for 2 weeks. Echocardiographic assessment of the heart identified systolic dysfunction consistent with cardiotoxicity in sorafenib-treated mice compared to vehicle-treated controls. Heart, skeletal muscle, liver and plasma were flash frozen and prepped for non-targeted GC-MS metabolomics analysis.Key Results: Compared to vehicle-treated controls, sorafenib-treated hearts exhibited significant alterations in 11 metabolites, including markedly altered taurine/hypotaurine metabolism (25-fold enrichment), identified by pathway enrichment analysis.Conclusions and Implications: These studies identified alterations in taurine/hypotaurine metabolism in the hearts and skeletal muscles of mice treated with sorafenib. Interventions that rescue or prevent these sorafenib-induced changes, such as taurine supplementation, may be helpful in attenuating sorafenib-induced cardiac injury. [ABSTRACT FROM AUTHOR]

Background and Purpose: Chronic pain is often a symptom of knee osteoarthritis (OA) for which current analgesics are either inadequate or are associated with serious side effects. The endocannabinoid system may offer alternative targets for pain relief. We evaluated the effects of a potent and selective monoacylglycerol (MAG) lipase inhibitor (MJN110) on OA pain behaviour, spinal mechanisms of action and joint histopathology in the rat.Experimental Approach: Intra-articular injection of monosodium iodoacetate (MIA) models OA pain and mimics clinical joint pathology. Effects of MJN110 on MIA-induced weight-bearing asymmetry and lowered paw withdrawal thresholds (PWTs), changes in spinal gene expression and brain levels of relevant lipids were determined.Key Results: Acute MJN110 (5 mg·kg-1 ) significantly reversed MIA-induced weight-bearing asymmetry (MIA/vehicle: 68 ± 6 g; MIA/MJN110: 35 ± 4 g) and lowered ipsilateral PWTs (MIA/vehicle: 7 ± 0.8 g; MIA/MJN110: 11 ± 0.6 g), via both CB1 and CB2 receptors. Repeated treatment with MJN110 (5 mg·kg-1 ) resulted in anti-nociceptive tolerance. A lower dose of MJN110 (1 mg·kg-1 ) acutely inhibited pain behaviour, which was maintained for 1 week of repeated administration but had no effect on joint histology. MJN110 significantly inhibited expression of membrane-associated PGE synthase-1 in the ipsilateral dorsal horn of the spinal cord of MIA rats, compared with vehicle-treated MIA rats. Both doses of MJN110 significantly elevated brain levels of the endocannabinoid 2-arachidonoylglycerol.Conclusions and Implications: Our data support further assessment of the therapeutic potential of MAG lipase inhibitors for the treatment of OA pain. [ABSTRACT FROM AUTHOR]

Background and Purpose: Recently pentoxifylline, a non-selective phosphodiesterase inhibitor and adenosine receptor antagonist, has attracted much interest for the treatment of the increased vascular resistance and endothelial dysfunction in pre-eclampsia. We therefore investigated the placental transfer, vascular effects and anti-inflammatory actions of pentoxifylline in healthy and pre-eclamptic human placentas.Experimental Approach: The placental transfer and metabolism of pentoxifylline were studied using ex vivo placenta perfusion experiments. In wire myography experiments with chorionic plate arteries, pentoxifyllines vasodilator properties were investigated, focusing on the cGMP and cAMP pathways and adenosine receptors. Its effects on inflammatory factors were also studied in placental explants.Key Results: Pentoxifylline transferred from the maternal to foetal circulation, reaching identical concentrations. The placenta metabolized pentoxifylline into its active metabolite lisofylline (M1), which was released into both circulations. In healthy placentas, pentoxifylline potentiated cAMP- and cGMP-induced vasodilation, as well as causing vasodilation by adenosine A1 antagonism and via NO synthase and PKG. Pentoxifylline also reduced inflammatory factors secretion. In pre-eclamptic placentas, we observed that its vasodilator capacity was preserved, however not via NO-PKG but likely through adenosine signalling. Pentoxifylline neither potentiated vasodilation through cAMP and cGMP, nor suppressed the release of inflammatory factors from these placentas.Conclusion and Implications: Pentoxifylline is transferred across and metabolized by the placenta. Its beneficial effects on the NO pathway and inflammation are not retained in pre-eclampsia, limiting its application in this disease, although it could be useful for other placenta-related disorders. Future studies might focus on selective A1 receptor antagonists as a new treatment for pre-eclampsia. [ABSTRACT FROM AUTHOR]

Background and Purpose: Pathological angiogenesis is a major cause of irreversible blindness in individuals with neovascular age-related macular degeneration (nAMD). Macrophages and microglia (MΦ) contribute to aberrant ocular angiogenesis. However, the role of glucose metabolism of MΦ in nAMD is still undefined. Here, we have investigated the involvement of glycolysis, driven by the kinase/phosphatase PFKFB3, in the development of choroidal neovascularization (CNV).Experimental Approach: CNV was induced in mice with laser photocoagulation. Choroid/retinal pigment epithelium (RPE) complexes and MΦ were isolated for analysis by qRT-PCR, western blot, flow cytometry, immunostaining, metabolic measurements and angiogenesis assays.Key Results: MΦ accumulated within the CNV of murine nAMD models and expressed high levels of glycolysis-related enzymes and M1/M2 polarization markers. This phenotype of hyper-glycolytic and activated MΦ was replicated in bone marrow-derived macrophages stimulated by necrotic RPE in vitro. Myeloid cell-specific knockout of PFKFB3, a key glycolytic activator, attenuated pathological neovascularization in laser-induced CNV, which was associated with decreased expression of MΦ polarization markers and pro-angiogenic factors, along with decreased sprouting of vessels in choroid/RPE complexes. Mechanistically, necrotic RPE increased PFKFB3-driven glycolysis in macrophages, leading to activation of HIF-1α/HIF-2α and NF-κB, and subsequent induction of M1/M2 markers and pro-angiogenic cytokines, finally promoting macrophage reprogramming towards an angiogenic phenotype to facilitate development of CNV. The PFKFB3 inhibitor AZ67 also inhibited activation of HIF-1α/HIF-2α and NF-κB signalling and almost completely prevented laser-induced CNV in mice.Conclusions and Implications: Modulation of PFKFB3-mediated macrophage glycolysis and activation is a promising strategy for the treatment of nAMD. [ABSTRACT FROM AUTHOR]

Background and Purpose: The pathogenesis of osteoarthritis implicates a low-grade inflammation associated to the innate immune system activation. Toll like receptor (TLR) stimulation triggers the release of inflammatory mediators, which aggravate osteoarthritis. We studied the preventive effect of 6-shogaol, a potential TLR4 inhibitor, on the treatment of experimental knee osteoarthritis.Experimental Approach: Osteoarthritis was induced in C57BL6 mice by surgical section of the medial meniscotibial ligament, which received 6-shogaol for eight weeks. Cartilage damage, inflammatory mediator presence and disease markers were assessed in joint tissues by immunohistochemistry. Computational modelling was used to predict binding modes of 6-shogaol into the TLR4/MD2 receptor and its permeability across cellular membranes. Employing LPS-stimulated chondrocytes and MAPK assay, we elucidated 6-shogaol action mechanisms.Key Results: 6-Shogaol treatment prevented articular cartilage lesions, synovitis and the presence of pro-inflammatory mediators, and disease markers in osteoarthritis animals. Molecular modelling studies predicted 6-shogaol interaction with the TLR4/MD-2 heterodimer in an antagonist conformation through its binding into the MD-2 pocket. In cell culture, we confirmed that 6-shogaol reduced LPS-induced TLR4 inflammatory signalling pathways. Besides, MAPK assay demonstrated that 6-shogaol directly inhibits the ERK1/2 phosphorylation activity.Conclusion and Implications: 6-Shogaol evoked a preventive action on cartilage and synovial inflammation in osteoarthritis mice. 6-shogaol effect may take place not only by hindering the interaction between TLR4 ligands and the TLR4/MD-2 complex in chondrocytes, but also through inhibition of ERK phosphorylation, implying a pleiotropic effect on different mediators activated during osteoarthritis, which proposes it as an attractive drug for osteoarthritis treatments. [ABSTRACT FROM AUTHOR]

Background and Purpose: Macrophage-rich atherosclerotic arteries are highly active in glycolysis. PFKFB3, a key glycolytic enzyme, has emerged as a potential therapeutic target in atherosclerosis. Small-molecule inhibitors of PFKFB3, such as 3PO and PFK158, have demonstrated efficacy in hampering atherogenesis in preclinical models. However, genetic studies elucidating the role of Pfkfb3 in atherogenesis need to be conducted to validate pharmacological findings and to unveil potential pharmacological side effects.Experimental Approach: Apoe-/- mice with global heterozygous or myeloid cell-specific Pfkfb3 deficiency were fed a Western diet (WD), after which atherosclerosis development was determined. Monocyte subsets in atherosclerotic mice and patients were examined by flow cytometry. Monocyte infiltration was assayed by a Ly6Chi monocyte-specific latex labelling procedure. In situ efferocytosis was assessed on mouse aortic root sections. Additionally, metabolic status, macrophage motility, efferocytosis, and involved mechanisms were analysed in peritoneal macrophages.Key Results: Global heterozygous or myeloid cell-specific Pfkfb3 deficiency reduced atherogenesis in Apoe-/- mice. Mechanistic studies showed that PFKFB3 controlled the proliferation and infiltration of proinflammatory monocytes. Moreover, PFKFB3 expression was associated with inflammatory monocyte expansion in patients with atherosclerotic coronary artery disease. Surprisingly, homozygous loss of Pfkfb3 impaired macrophage efferocytosis and exacerbated atherosclerosis in Apoe-/- mice. Mechanistically, PFKFB3-driven glycolysis was shown to be essential for actin polymerization, thus aiding the efferocytotic function of macrophages.Conclusion and Implications: Collectively, these findings suggest the existence of a double-edged sword effect of myeloid PFKFB3 on the pathogenesis of atherosclerosis and highlight the need for caution in developing anti-atherosclerotic strategies that target PFKFB3. [ABSTRACT FROM AUTHOR]

Background and Purpose: Vascular tone is regulated by the relative contractile state of vascular smooth muscle cells (VSMCs). Several integrins directly modulate VSMC contraction by regulating calcium influx through L-type voltage-gated Ca2+ channels (VGCCs). Genetic variants in ITGA9, which encodes the α9 subunit of integrin α9β1, and SVEP1, a ligand for integrin α9β1, associate with elevated blood pressure; however, neither SVEP1 nor integrin α9β1 has reported roles in vasoregulation. We determined whether SVEP1 and integrin α9β1 can regulate VSMC contraction.Experimental Approach: SVEP1 and integrin binding were confirmed by immunoprecipitation and cell binding assays. Human induced pluripotent stem cell-derived VSMCs were used in in vitro [Ca2+ ]i studies, and aortas from a Svep1+/- knockout mouse model were used in wire myography to measure vessel contraction.Key Results: We confirmed the ligation of SVEP1 to integrin α9β1 and additionally found SVEP1 to directly bind to integrin α4β1. Inhibition of SVEP1, integrin α4β1 or α9β1 significantly enhanced [Ca2+ ]i levels in isolated VSMCs to Gαq/11 -vasoconstrictors. This response was confirmed in whole vessels where a greater contraction to U46619 was seen in vessels from Svep1+/- mice compared to littermate controls or when integrin α4β1 or α9β1 was inhibited. Inhibition studies suggested that this effect was mediated via VGCCs, PKC and Rho A/Rho kinase dependent mechanisms.Conclusions and Implications: Our studies reveal a novel role for SVEP1 and the integrins α4β1 and α9β1 in reducing VSMC contractility. This could provide an explanation for the genetic associations with blood pressure risk at the SVEP1 and ITGA9 loci. [ABSTRACT FROM AUTHOR]

Background and Purpose: Over past decades, targeted therapies and immunotherapy have improved survival and reduced the morbidity of patients with BRAF-mutated melanoma. However, drug resistance and relapse hinder overall success. Therefore, there is an urgent need for novel compounds with therapeutic efficacy against BRAF-melanoma. This prompted us to investigate the antiproliferative profile of a tachykinin-peptide from the Octopus kaurna, Octpep-1 in melanoma.Experimental Approach: We evaluated the cytotoxicity of Octpep-1 by MTT assay. Mechanistic insights on viability and cellular damage caused by Octpep-1 were gained via flow cytometry and bioenergetics. Structural and pharmacological characterization was conducted by molecular modelling, molecular biology, CRISPR/Cas9 technology, high-throughput mRNA and calcium flux analysis. In vivo efficacy was validated in two independent xerograph animal models (mice and zebrafish).Key Results: Octpep-1 selectively reduced the proliferative capacity of human melanoma BRAFV600E -mutated cells with minimal effects on fibroblasts. In melanoma-treated cells, Octpep-1 increased ROS with unaltered mitochondrial membrane potential and promoted non-mitochondrial and mitochondrial respiration with inefficient ATP coupling. Molecular modelling revealed that the cytotoxicity of Octpep-1 depends upon the α-helix and polyproline conformation in the C-terminal region of the peptide. A truncated form of the C-terminal end of Octpep-1 displayed enhanced potency and efficacy against melanoma. Octpep-1 reduced the progression of tumours in xenograft melanoma mice and zebrafish.Conclusion and Implications: We unravel the intrinsic anti-tumoural properties of a tachykinin peptide. This peptide mediates the selective cytotoxicity in BRAF-mutated melanoma in vitro and prevents tumour progression in vivo, providing a foundation for a therapy against melanoma. [ABSTRACT FROM AUTHOR]

Background and Purpose: Ischaemia is known to cause massive neuronal depolarization, termed anoxic depolarization (AD), due to energy failure and loss of membrane ion gradients. The neuromodulator adenosine accumulates extracellularly during ischaemia and activates four metabotropic receptors: A1 , A2A , A2B and A3 . Striatal medium spiny neurons (MSNs) express high levels of A2A receptors and are particularly vulnerable to ischaemic insults. A2A Receptor blockade reduces acute striatal post-ischaemic damage but the cellular mechanisms involved are still unknown.Experimental Approach: We performed patch-clamp recordings of MSNs in rat striatal slices subjected to oxygen and glucose deprivation (OGD) to investigate the effects of A2A receptor ligands or ion channel blockers on AD and OGD-induced ionic imbalance, measured as a positive shift in Erev of ramp currents.Key Results: Our data indicate that the A2A receptor antagonist SCH58261 (10 μM) significantly attenuated ionic imbalance and AD appearance in MSNs exposed to OGD. The K+ channel blocker Ba2+ (2 mM) or the Na+ channel blocker tetrodotoxin (1 μM) exacerbated and attenuated, respectively, OGD-induced changes. Spontaneous excitatory post-synaptic current (sEPSC) analysis in MSNs revealed that the A2A receptor agonist CGS21680 (1 μM) prevented OGD-induced decrease of sEPSCs within the first 5 min of the insult, an effect shared by the K+ channel blocker Ba2+ , indicating facilitated glutamate release.Conclusion and Implications: Adenosine, released during striatal OGD, activates A2A receptors that may exacerbate OGD-induced damage through K+ channel inhibition. Our results could help to develop A2A receptor-selective therapeutic tools for the treatment of brain ischaemia. [ABSTRACT FROM AUTHOR]

Background and Purpose: Before advancing to clinical trials, new drugs are screened for their pro-arrhythmic potential using a method that is overly conservative and provides limited mechanistic insight. The shortcomings of this approach can lead to the mis-classification of beneficial drugs as pro-arrhythmic.Experimental Approach: An in silico-in vitro pipeline was developed to circumvent these shortcomings. A computational human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model was used as part of a genetic algorithm to design experiments, specifically electrophysiological voltage clamp (VC) protocols, to identify which of several cardiac ion channels were blocked during in vitro drug studies. Such VC data, along with dynamically clamped action potentials (AP), were acquired from iPSC-CMs before and after treatment with a control solution or a low- (verapamil), intermediate- (cisapride or quinine) or high-risk (quinidine) drug.Key Results: Significant AP prolongation (a pro-arrhythmia marker) was seen in response to quinidine and quinine. The VC protocol identified block of IKr (a source of arrhythmias) by all strong IKr blockers, including cisapride, quinidine and quinine. The protocol also detected block of ICaL by verapamil and Ito by quinidine. Further demonstrating the power of the approach, the VC data uncovered a previously unidentified If block by quinine, which was confirmed with experiments using a HEK-293 expression system and automated patch-clamp.Conclusion and Implications: We developed an in silico-in vitro pipeline that simultaneously identifies pro-arrhythmia risk and mechanism of ion channel-blocking drugs. The approach offers a new tool for evaluating cardiotoxicity during preclinical drug screening. [ABSTRACT FROM AUTHOR]

Background and Purpose: Recent evidence links brain insulin resistance with neurodegenerative diseases, where hyperphosphorylated tau protein contributes to neuronal cell death. In the present study, we aimed to evaluate if d-pinitol inositol, which acts as an insulin sensitizer, affects the phosphorylation status of tau protein.Experimental Approach: We studied the pharmacological effect of d-pinitol on insulin signalling and tau phosphorylation in the hippocampus of Wistar and Zucker rats. To this end, we evaluated by western blotting the Akt pathway and its downstream proteins as being one of the main insulin-mediator pathways. Also, we explored the functional status of additional kinases phosphorylating tau, including PKA, ERK1/2, AMPK and CDK5. We utilized the 3xTg mouse model as a control for tauopathy, since it carries tau mutations that promote phosphorylation and aggregation.Key Results: Surprisingly, we discovered that oral d-pinitol treatment lowered tau phosphorylation significantly, but not through the expected kinase GSK-3 regulation. An extensive search for additional kinases phosphorylating tau revealed that this effect was mediated through a mechanism dependent on the reduction of the activity of the CDK5, affecting both its p35 and p25 subunits. This effect disappeared in leptin-deficient Zucker rats, uncovering that the association of leptin deficiency, obesity, dyslipidaemia and hyperinsulinaemia abrogates d-pinitol actions on tau phosphorylation. The 3xTg mice confirmed d-pinitol effectiveness in a genetic AD-tauopathy.Conclusion and Implications: The present findings suggest that d-pinitol, by regulating CDK5 activity through a decrease of CDK5R1, is a potential drug for developing treatments for neurological disorders such as tauopathies. [ABSTRACT FROM AUTHOR]

The rupture of unstable atherosclerotic plaques is the major cause of cardiovascular mortality and morbidity. Despite significant limitations in our understanding and ability to identify unstable plaque pathology and prevent plaque rupture, most atherosclerosis research utilises preclinical animal models exhibiting stable atherosclerosis. Here, we introduce the tandem stenosis (TS) mouse model that reflects plaque instability and rupture, as seen in patients. The TS model involves dual ligation of the right carotid artery, leading to locally predefined unstable atherosclerosis in hypercholesterolaemic mice. It exhibits key characteristics of human unstable plaques, including plaque rupture, luminal thrombosis, intraplaque haemorrhage, large necrotic cores, thin or ruptured fibrous caps and extensive immune cell accumulation. Altogether, the TS model represents an ideal preclinical tool for improving our understanding of human plaque instability and rupture, for the development of imaging technologies to identify unstable plaques, and for the development and testing of plaque-stabilising treatments for the prevention of atherosclerotic plaque rupture. LINKED ARTICLES: This article is part of a themed issue on Preclinical Models for Cardiovascular disease research (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.5/issuetoc. [ABSTRACT FROM AUTHOR]

Unlabelled: Glucokinase activators (GKAs) represent one of the leading hopes for the next generation of type 2 diabetes (T2D) therapeutics, showing efficacy in reducing blood glucose and HbA1c levels in animal models of T2D and short-term human trials. While the hypoglycaemic risks of GCK activation in pancreatic beta-cells have long been appreciated, the hepatic effects of GKAs have generally been perceived to be without significant side effect. In this issue of the British Journal of Pharmacology, De Ceuninck et al. report that acute and chronic GKA treatment of normoglycaemic and hyperglycaemic rodent models results in significant accumulation of triglycerides in the liver. This suggests GKA-mediated activation of hepatic glucose uptake and suppression of endogenous glucose production may come at a significant cost; namely, the development of hepatic steatosis. This raises important questions regarding the safety of GKAs and emphasizes that both plasma and hepatic lipid profiles should be carefully monitored in on-going and future studies of these molecules.Linked Article: This article is a commentary on De Ceuninck et al., pp. 339-353 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2012.02184.x. [ABSTRACT FROM AUTHOR]

Background and Purpose: Endocannabinoids have both anti-inflammatory and neuroprotective properties against harmful stimuli. We previously demonstrated that the endocannabinoid 2-arachidonoylglycerol (2-AG) protects hippocampal neurons by limiting the inflammatory response via a CB(1) receptor-dependent MAPK/NF-κB signalling pathway. The purpose of the present study was to determine whether PPARγ, an important nuclear receptor, mediates 2-AG-induced inhibition of NF-κB phosphorylation and COX-2 expression, and COX-2-enhanced miniature spontaneous excitatory postsynaptic currents (mEPSCs).Experimental Approach: By using a whole-cell patch clamp electrophysiological recording technique and immunoblot analysis, we determined mEPSCs, expression of COX-2 and PPARγ, and phosphorylation of NF-kB in mouse hippocampal neurons in culture.Key Results: Exogenous and endogenous 2-AG-produced suppressions of NF-κB-p65 phosphorylation, COX-2 expression and excitatory synaptic transmission in response to pro-inflammatory interleukin-1β (IL-1β) and LPS were inhibited by GW9662, a selective PPARγ antagonist, in hippocampal neurons in culture. PPARγ agonists 15-deoxy-Δ(12,14) -prostaglandin J(2) (15d-PGJ(2)) and rosiglitazone mimicked the effects of 2-AG on NF-κB-p65 phosphorylation, COX-2 expression and mEPSCs, and these effects were eliminated by antagonism of PPARγ. Moreover, exogenous application of 2-AG or elevation of endogenous 2-AG by inhibiting its hydrolysis with URB602 or JZL184, selective inhibitors of monoacylglycerol lipase (MAGL), prevented the IL-1β- and LPS-induced reduction of PPARγ expression. The 2-AG restoration of the reduced PPARγ expression was blocked or attenuated by pharmacological or genetic inhibition of the CB(1) receptor.Conclusions and Implications: Our results suggest that CB(1) receptor-dependent PPARγ expression is an important and novel signalling pathway in endocannabinoid 2-AG-produced resolution of neuroinflammation in response to pro-inflammatory insults. [ABSTRACT FROM AUTHOR]

Given that cardiovascular safety liabilities remain a major cause of drug attrition during preclinical and clinical development, adverse drug reactions, and post-approval withdrawal of medicines, the Medical Research Council Centre for Drug Safety Science hosted a workshop to discuss current challenges in determining, understanding and addressing 'Cardiovascular Toxicity of Medicines'. This article summarizes the key discussions from the workshop that aimed to address three major questions: (i) what are the key cardiovascular safety liabilities in drug discovery, drug development and clinical practice? (ii) how good are preclinical and clinical strategies for detecting cardiovascular liabilities? and (iii) do we have a mechanistic understanding of these liabilities? It was concluded that in order to understand, address and ultimately reduce cardiovascular safety liabilities of new therapeutic agents there is an urgent need to: • Fully characterize the incidence, prevalence and impact of drug-induced cardiovascular issues at all stages of the drug development process. • Ascertain the predictive value of existing non-clinical models and assays towards the clinical outcome. • Understand the mechanistic basis of cardiovascular liabilities; by addressing areas where it is currently not possible to predict clinical outcome based on preclinical safety data. • Provide scientists in all disciplines with additional skills to enable them to better integrate preclinical and clinical data and to better understand the biological and clinical significance of observed changes. • Develop more appropriate, highly relevant and predictive tools and assays to identify and wherever feasible to eliminate cardiovascular safety liabilities from molecules and wherever appropriate to develop clinically relevant and reliable safety biomarkers. [ABSTRACT FROM AUTHOR]

Background and Purpose: Alarin is a recently discovered member of the galanin peptide family encoded by a splice variant of galanin-like peptide (GALP) mRNA. Galanin and GALP regulate energy homeostasis and reproduction. We therefore investigated the effects of alarin on food intake and gonadotrophin release.Experimental Approach: Alarin was administered into the third cerebral ventricle (i.c.v.) of rats, and food intake or circulating hormone levels were measured. The effect of alarin on the hypothalamo-pituitary-gonadal axis was investigated in vitro using hypothalamic and anterior pituitary explants, and immortalized cell lines. Receptor binding assays were used to determine whether alarin binds to galanin receptors.Key Results: The i.c.v. administration of alarin (30 nmol) to ad libitum fed male rats significantly increased acute food intake to 500%, and plasma luteinizing hormone (LH) levels to 170% of responses to saline. In vitro, 100 nM alarin stimulated neuropeptide Y (NPY) and gonadotrophin-releasing hormone (GnRH) release from hypothalamic explants from male rats, and 1000 nM alarin increased GnRH release from GT1-7 cells. In vivo, pretreatment with the GnRH receptor antagonist cetrorelix prevented the increase in plasma LH levels observed following i.c.v. alarin administration. Receptor binding studies confirmed alarin did not bind to any known galanin receptor, or compete with radiolabelled galanin for hypothalamic binding sites.Conclusions and Implications: These results suggest alarin is a novel orexigenic peptide, and that it increases circulating LH levels via hypothalamic GnRH. Further work is required to identify the receptor(s) mediating the biological effects of alarin. [ABSTRACT FROM AUTHOR]

Background and Purpose: Ethanol is known to have both pre-synaptic and post-synaptic effects at a range of loci in the mammalian nervous system, including the neuromuscular junction. However, the effects of ethanol on evoked synaptic transmission have not been previously studied at the mouse neuromuscular junction. Here, we report on the effects of ethanol on evoked neuromuscular transmission and the interaction of ethanol with non-depolarizing blocking drugs.Experimental Approach: Electrophysiological techniques to measure synaptic potentials and synaptic currents were employed in this study.Key Results: Ethanol (≥100 mM) produced increases in the amplitudes of both spontaneous and evoked synaptic events. Under conditions in which neuromuscular transmission was blocked by (+)-tubocurarine, ethanol (12-100 mM) produced greater increases in evoked response amplitude than in spontaneous response amplitude recorded in the absence of (+)-tubocurarine. Ethanol (100 mM) did not affect evoked neurotransmitter release in low-calcium/high-magnesium solutions. With respect to the clinically used neuromuscular blocking drugs, ethanol (100 mM) interfered with the blocking action of vecuronium, but not cisatracurium.Conclusions and Implications: Under these conditions, the stimulant effect of ethanol on neuromuscular transmission is exclusively on the post-junctional elements, both to enhance transmission through nicotinic receptors and also via interactions with neuromuscular blocking agents. These actions of ethanol on neuromuscular transmission may affect the dosage of neuromuscular blockers required in patients who have imbibed significant amounts of alcohol. [ABSTRACT FROM AUTHOR]

Background and Purpose: The glycerol-based lysophospholipid lysophosphatidylinositol (LPI) is an endogenous agonist of the G-protein-coupled receptor 55 (GPR55) exhibiting cannabinoid receptor-like properties in endothelial cells. To estimate the contribution of GPR55 to the physiological effects of LPI, the GPR55-dependent and -independent electrical responses in this cell type were investigated.Experimental Approach: Applying small interference RNA-mediated knock-down and transient overexpression, GPR55-dependent and -independent effects of LPI on cytosolic free Ca(2+) concentration, membrane potential and transmembrane ion currents were studied in EA.hy296 cells.Key Results: In a GPR55-dependent, GDPbetaS and U73122-sensitive manner, LPI induced rapid and transient intracellular Ca(2+) release that was associated with activation of charybdotoxin-sensitive, large conductance, Ca(2+)-activated, K(+) channels (BK(Ca)) and temporary membrane hyperpolarization. Following these initial electrical reactions, LPI elicited GPR55-independent long-lasting Na(+) loading and a non-selective inward current causing sustained membrane depolarization that depended on extracellular Ca(2+) and Na(+) and was partially inhibited by Ni(2+) and La(3+). This inward current was due to the activation of a voltage-independent non-selective cation current. The Ni(2+) and La(3+)-insensitive depolarization with LPI was prevented by inhibition of the Na/K-ATPase by ouabain.Conclusions and Implications: LPI elicited a biphasic response in endothelial cells of which the immediate Ca(2+) signalling depends on GPR55 while the subsequent depolarization is due to Na(+) loading via non-selective cation channels and an inhibition of the Na/K-ATPase. Thus, LPI is a potent signalling molecule that affects endothelial functions by modulating several cellular electrical responses that are only partially linked to GPR55. [ABSTRACT FROM AUTHOR]

Background and Purpose: Bone morphogenetic proteins (BMPs) were first identified through their role in inducing bone and cartilage formation, but many other important functions have since been ascribed to BMPs, including dorsoventral patterning, angiogenesis and tissue homeostasis. Using dorsomorphin and LDN193189, selective small molecule inhibitors of BMP signalling, we investigated the role of BMP signalling in early vascular patterning in zebrafish.Experimental Approach: The effects of dorsomorphin and LDN193189 on vascular endothelial growth factor-a (VEGF) and BMP signalling in developing zebrafish and in human pulmonary artery endothelial cells were determined using confocal microscopy, Western blotting and quantitative PCR.Key Results: We showed that dorsomorphin, similar to the VEGF inhibitor SU5416, strongly inhibits intersegmental vessel formation in zebrafish and that this is due to inhibition of VEGF activation of VEGF receptor 2 (VEGFR2), leading to reduced VEGF-induced phospho-ERK (extracellular regulated kinase) 1/2 and VEGF target gene transcription. These effects occurred at concentrations of dorsomorphin that block BMP signalling. We also showed that LDN193189, an analogue of dorsomorphin, more potently blocks BMP signalling but has no effect on VEGF signalling in zebrafish and does not disrupt early vascular patterning.Conclusions and Implications: Dorsomorphin inhibits both BMP and VEGF signalling, whereas LDN193189 is a more selective BMP antagonist. Results obtained in cardiovascular studies using dorsomorphin need to be interpreted with caution, and use of LDN193189 would be preferable due to its selectivity. Our data also suggest that BMP signalling is dispensable for early patterning of intersegmental vessels in zebrafish. [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: Carboxylesterases (CEs) metabolize a wide range of xenobiotic substrates including heroin, cocaine, meperidine and the anticancer agent CPT-11. In this study, we have purified to homogeneity human liver and intestinal CEs and compared their ability with hydrolyse heroin, cocaine and CPT-11.Experimental Approach: The hydrolysis of heroin and cocaine by recombinant human CEs was evaluated and the kinetic parameters determined. In addition, microsomal samples prepared from these tissues were subjected to chromatographic separation, and substrate hydrolysis and amounts of different CEs were determined.Key Results: In contrast to previous reports, cocaine was not hydrolysed by the human liver CE, hCE1 (CES1), either as highly active recombinant protein or as CEs isolated from human liver or intestinal extracts. These results correlated well with computer-assisted molecular modelling studies that suggested that hydrolysis of cocaine by hCE1 (CES1), would be unlikely to occur. However, cocaine, heroin and CPT-11 were all substrates for the intestinal CE, hiCE (CES2), as determined using both the recombinant protein and the tissue fractions. Again, these data were in agreement with the modelling results.Conclusions and Implications: These results indicate that the human liver CE is unlikely to play a role in the metabolism of cocaine and that hydrolysis of this substrate by this class of enzymes is via the human intestinal protein hiCE (CES2). In addition, because no enzyme inhibition is observed at high cocaine concentrations, potentially this route of hydrolysis is important in individuals who overdose on this agent. [ABSTRACT FROM AUTHOR]

Background and Purpose: Toll-like receptor 4 (TLR4) expressed on spinal microglia and astrocytes has been suggested to play an important role in the regulation of pain signalling. The purpose of the present work was to examine the links between TLR4, glial activation and spinal release of prostaglandin E(2) (PGE(2)) and tumour necrosis factor (TNF), and the role these factors play in TLR4-induced tactile allodynia.Experimental Approach: Toll-like receptor 4 was activated by intrathecal (i.t.) injection of lipopolysaccharide (LPS) and KDO(2)-Lipid A (KDO(2)) to rats. Tactile allodynia was assessed using von Frey filaments and cerebrospinal fluid collected through spinal dialysis and lumbar puncture. PGE(2) and TNF levels were measured by mass spectometry and elisa. Minocycline and pentoxifylline (glia inhibitors), etanercept (TNF-blocker) and ketorolac (COX-inhibitor) were given i.t. prior to injection of the TLR4-agonists, in order to determine if these agents alter TLR4-mediated nociception and the spinal release of PGE(2) and TNF.Key Results: Spinal administration of LPS and KDO(2) produced a dose-dependent tactile allodynia, which was attenuated by pentoxifylline, minocycline and etanercept but not ketorolac. Both TLR4 agonists induced the spinal release of PGE(2) and TNF. Intrathecal pentoxifylline blunted PGE(2) and TNF release, while i.t. minocycline only prevented the spinal release of TNF. The release of PGE(2) induced by LPS and KDO(2) was attenuated by i.t. administration of ketorolac.Conclusions and Implications: Activation of TLR4 induces tactile allodynia, which is probably mediated by TNF released by activated spinal glia. [ABSTRACT FROM AUTHOR]

Background and Purpose: Transient receptor potential ankyrin 1 (TRPA1) channels are expressed by primary afferent neurones and activated by irritant chemicals including allyl isothiocyanate (AITC). Here we investigated whether intracolonic AITC causes afferent input to the spinal cord and whether this response is modified by mild colitis, morphine or a TRPA1 channel blocker.Experimental Approach: One hour after intracolonic administration of AITC to female mice, afferent signalling was visualized by expression of c-Fos in laminae I-II(o) of the spinal dorsal horn at sacral segment S1. Mild colitis was induced by dextran sulphate sodium (DSS) added to drinking water for 1 week.Key Results: Relative to vehicle, AITC (2%) increased expression of c-Fos in the spinal cord. Following induction of mild colitis by DSS (2%), spinal c-Fos responses to AITC, but not vehicle, were augmented by 41%. Colonic inflammation was present (increased myeloperoxidase content and disease activity score), whereas colonic histology, locomotion, feeding and drinking remained unchanged. Morphine (10 mg.kg(-1)) or the TRPA1 channel blocker HC-030031 (300 mg.kg(-1)) inhibited the spinal c-Fos response to AITC, in control and DSS-pretreated animals, whereas the response to intracolonic capsaicin (5%) was blocked by morphine but not HC-030031.Conclusions and Implications: Activation of colonic TRPA1 channels is signalled to the spinal cord. Mild colitis enhanced this afferent input that, as it is sensitive to morphine, is most likely of a chemonociceptive nature. As several irritant chemicals can be present in chyme, TRPA1 channels may mediate several gastrointestinal pain conditions. [ABSTRACT FROM AUTHOR]

Background and Purpose: The sarcoplasmic reticulum (SR) releases Ca(2+) via inositol 1,4,5-trisphosphate receptors (IP(3)R) in response to IP(3)-generating agonists. Ca(2+) release subsequently propagates as Ca(2+) waves. To clarify the role of IP(3) production in wave generation, the contribution of a key enzyme in the production of IP(3) was examined using a phosphoinositide-specific phospholipase C (PI-PLC) inhibitor, U-73122.Experimental Approach: Single colonic myocytes were voltage-clamped in whole-cell configuration and cytosolic Ca(2+) concentration ([Ca(2+)](cyto)) measured using fluo-3. SR Ca(2+) release was evoked either by activation of IP(3)Rs (by carbachol or photolysis of caged IP(3)) or ryanodine receptors (RyRs; by caffeine).Key Results: U-73122 inhibited carbachol-evoked [Ca(2+)](cyto) transients. The drug also inhibited [Ca(2+)](cyto) increases, evoked by direct IP(3)R activation (by photolysis of caged IP(3)) and RyR activation (by caffeine), which do not require PI-PLC activation. U-73122 also increased steady-state [Ca(2+)](cyto) and slowed the rate of Ca(2+) removal from the cytoplasm. An inactive analogue of U-73122, U-73343, was without effect on either IP(3)R- or RyR-mediated Ca(2+) release.Conclusions and Implications: U-73122 inhibited carbachol-evoked [Ca(2+)](cyto) increases. However, the drug also reduced Ca(2+) release when evoked by direct activation of IP(3)R or RyR, slowed Ca(2+) removal and increased steady-state [Ca(2+)](cyto). These results suggest U-73122 reduces IP(3)-evoked Ca(2+) transients by inhibiting the SR Ca(2+) pump to deplete the SR of Ca(2+) rather than by inhibiting PI-PLC. [ABSTRACT FROM AUTHOR]

Background and Purpose: Pilsicainide, an anti-arrhythmic drug used in Japan, is described as a pure sodium channel blocker. We examined the mechanisms by which it is able to block open channels, because these properties may be especially useful to reduce hyperexcitability in pathologies characterized by abnormal sodium channel opening.Experimental Approach: The effects of pilsicainide on various heterologously expressed human sodium channel subtypes and mutants were investigated using the patch clamp technique.Key Results: Pilsicainide exhibited tonic and use-dependent effects comparable to those of mexiletine and flecainide on hNav1.4 channels. These use-dependent effects were abolished in the mutations F1586C and Y1593C within segment 6 of domain IV, suggesting that the interaction of pilsicainide with these residues is critical for its local anaesthetic action. Its affinity constants for closed channels (K(R)) and channels inactivated from the closed state (K(I)) were high, suggesting that its use-dependent block (UDB) requires the channel to be open for it to reach a high-affinity blocking site. Accordingly, basic pH, which slightly increased the proportion of neutral drug, dramatically decreased K(R) and K(I) values. Effects of pilsicainide were similar on skeletal muscle hNav1.4, brain hNav1.1 and heart hNav1.5 channels. The myotonic R1448C and G1306E hNav1.4 mutants were more and less sensitive to pilsicainide, respectively, due to mutation-induced gating modifications.Conclusions and Implications: Although therapeutic concentrations of pilsicainide may have little effect on resting and closed-state inactivated channels, it induces a strong UDB due to channel opening, rendering the drug ideally suited for inhibition of high-frequency action potential firing. [ABSTRACT FROM AUTHOR]

Background and Purpose: Current therapies for muscular dystrophy are based on corticosteroids. Significant side effects associated with these therapies have prompted several studies aimed at identifying possible alternative strategies. As inflammation and defects of nitric oxide (NO) generation are key pathogenic events in muscular dystrophies, we have studied the effects of combining the NO donor isosorbide dinitrate (ISDN) and the non-steroidal anti-inflammatory drug ibuprofen.Experimental Approach: alpha-Sarcoglycan-null mice were treated for up to 8 months with ISDN (30 mg.kg(-1)) plus ibuprofen (50 mg.kg(-1)) administered daily in the diet. Effects of ISDN and ibuprofen alone were assessed in parallel. Drug effects on animal motility and muscle function, muscle damage, inflammatory infiltrates and cytokine levels, as well as muscle regeneration including assessment of endogenous stem cell pool, were measured at selected time points.Key Results: Combination of ibuprofen and ISDN stimulated regeneration capacity, of myogenic precursor cells, reduced muscle necrotic damage and inflammation. Muscle function in terms of free voluntary movement and resistance to exercise was maintained throughout the time window analysed. The effects of ISDN and ibuprofen administered separately were transient and significantly lower than those induced by their combination.Conclusions and Implications: Co-administration of NO and ibuprofen provided synergistic beneficial effects in a mouse model of muscular dystrophy, leading to an effective therapy. Our results open the possibility of immediate clinical testing of a combination of ISDN and ibuprofen in dystrophic patients, as both components are approved for use in humans, with a good safety profile. [ABSTRACT FROM AUTHOR]

Background and Purpose: SKF96365 (SKF), originally identified as a blocker of receptor-mediated calcium entry, is widely used diagnostically, as a blocker of transient receptor potential canonical type (TRPC) channels. While SKF has been used as a tool to define the functional roles of TRPC channels in various cell and tissue types, there are notable overlapping physiological and pathophysiological associations between TRPC channels and low-voltage-activated (LVA) T-type calcium channels. The activity of SKF against T-type Ca channels has not been previously explored, and here we systematically investigated the effects of SKF on recombinant and native voltage-gated Ca channel-mediated currents.Experimental Approach: Effects of SKF on recombinant Ca channels were studied under whole-cell patch clamp conditions after expression in HEK293 cells. The effect of SKF on cerebellar Purkinje cells (PCs) expressing native T-type Ca channels was also assessed.Key Results: SKF blocked recombinant Ca channels, representative of each of the three main molecular genetic classes (Ca(V)1, Ca(V)2 and Ca(V)3) at concentrations typically utilized to assay TRPC function (10 microM). Particularly, human Ca(V)3.1 T-type Ca channels were more potently inhibited by SKF (IC(50) approximately 560 nM) in our experiments than previously reported for similarly expressed TRPC channels. SKF also inhibited native Ca(V)3.1 T-type currents in a rat cerebellar PC slice preparation.Conclusions and Implications: SKF was a potent blocker of LVA T-type Ca channels. We suggest caution in the interpretation of results using SKF alone as a diagnostic agent for TRPC activity in native tissues. [ABSTRACT FROM AUTHOR]

Background and Purpose: The overexpression of epidermal growth factor receptor (EGFR) and its mutated variant EGFRvIII occurs in 50% of glioblastoma multiforme. We developed antibody fragments against EGFR/EGFRvIII for molecular imaging and/or therapeutic targeting applications.Experimental Approach: An anti-EGFR/EGFRvIII llama single-domain antibody (EG(2)) and two higher valency format constructs, bivalent EG(2)-hFc and pentavalent V2C-EG(2) sdAbs, were analysed in vitro for their binding affinities using surface plasmon resonance and cell binding studies, and in vivo using pharmacokinetic, biodistribution, optical imaging and fluorescent microscopy studies.Key Results: Kinetic binding analyses by surface plasmon resonance revealed intrinsic affinities of 55 nM and 97 nM for the monovalent EG(2) to immobilized extracellular domains of EGFR and EGFRvIII, respectively, and a 10- to 600-fold increases in apparent affinities for the multivalent binders, V2C-EG(2) and EG(2)-hFc, respectively. In vivo pharmacokinetic and biodistribution studies in mice revealed plasma half-lives for EG(2), V2C-EG(2) and EG(2)-hFc of 41 min, 80 min and 12.5 h, respectively, as well as a significantly higher retention of EG(2)-hFc compared to the other two constructs in EGFR/EGFRvIII-expressing orthotopic brain tumours, resulting in the highest signal in the tumour region in optical imaging studies. Time domain volumetric optical imaging fusion with high-resolution micro-computed tomography of microvascular brain network confirmed EG(2)-hFc selective accumulation/retention in anatomically defined tumour regions.Conclusions: Single domain antibodies can be optimized for molecular imaging applications by methods that improve their apparent affinity and prolong plasma half-life and, at the same time, preserve their ability to penetrate tumour parenchyma. [ABSTRACT FROM AUTHOR]

Background and Purpose: In vitro studies demonstrate that cannabinoid CB(1) receptors subserve activity-dependent suppression of inhibition in the neocortex. To examine this mechanism in vivo, we assessed the effects of local changes in CB(1) receptor activity on somatosensory cortex neuronal activation by whisker movement in rats.Experimental Approach: Laser Doppler flowmetry and c-Fos immunohistochemistry were used to measure changes in local blood flow and neuronal activation, respectively. All drugs were applied directly to the cranium above the whisker barrel fields of the primary somatosensory cortex.Key Results: The CB(1) receptor agonist WIN55212-2 potentiated the hyperaemia induced by whisker movement and this potentiation was occluded by bicuculline. The CB(1) receptor antagonists, rimonabant and AM251, inhibited hyperaemic responses to whisker movement; indicating that activation of endogenous CB(1) receptors increased during whisker movement. Whisker movement-induced expression of c-Fos protein in neurons of the whisker barrel cortex was inhibited by rimonabant. Movement of the whiskers increased the 2-arachidonoylglycerol content in the contralateral, compared to the ipsilateral, sensory cortex.Conclusions and Implications: These results support the hypothesis that endocannabinoid signalling is recruited during physiologically relevant activation of the sensory cortex. These data support the hypothesis that the primary effect of CB(1) receptor activation within the activated whisker barrel cortex is to inhibit GABA release, resulting in disinhibition of neuronal activation. These studies provide physiological data involving endocannabinoid signalling in activity-dependent regulation of neuronal activation and provide a mechanistic basis for the effects of cannabis use on sensory processing in humans. [ABSTRACT FROM AUTHOR]

Background and Purpose: Increased circulating levels of L-alpha-lysophosphatidylinositol (LPI) are associated with cancer and LPI is a potent, ligand for the G-protein-coupled receptor GPR55. Here we have assessed the modulation of breast cancer cell migration, orientation and polarization by LPI and GPR55.Experimental Approach: Quantitative RT-PCR was used to measure GPR55 expression in breast cancer cell lines. Cell migration and invasion were measured using a Boyden chamber chemotaxis assay and Cultrex invasion assay, respectively. Cell polarization and orientation in response to the microenvironment were measured using slides containing nanometric grooves.Key Results: GPR55 expression was detected in the highly metastatic MDA-MB-231 breast cancer cell line. In these cells, LPI stimulated binding of [(35)S]GTPgammaS to cell membranes (pEC(50) 6.47 +/- 0.45) and significantly enhanced cell chemotaxis towards serum. MCF-7 cells expressed low levels of GPR55 and did not migrate or invade towards serum factors. When GPR55 was over-expressed in MCF-7 cells, serum induced a robust migratory and invasive response, which was further enhanced by LPI and prevented by siRNA to GPR55. The physical microenvironment has been identified as a key factor in determining breast tumour cell metastatic fate. LPI endowed MDA-MB-231 cells with the capacity to detect shallow (40 nm deep) grooved slides and induced marked cancer cell polarization on both flat and grooved surfaces.Conclusions and Implications: LPI and GPR55 play a role in the modulation of migration, orientation and polarization of breast cancer cells in response to the tumour microenvironment. [ABSTRACT FROM AUTHOR]

Background and Purpose: Accumulating recent evidence suggests that cannabinoid-1 (CB(1)) receptor activation may promote inflammation and cell death and its pharmacological inhibition is associated with anti-inflammatory and tissue-protective effects in various preclinical disease models, as well as in humans.Experimental Approach: In this study, using molecular biology and biochemistry methods, we have investigated the effects of genetic deletion or pharmacological inhibition of CB(1) receptors on inflammation, oxidative/nitrosative stress and cell death pathways associated with a clinically relevant model of nephropathy, induced by an important chemotherapeutic drug cisplatin.Results: Cisplatin significantly increased endocannabinoid anandamide content, activation of p38 and JNK mitogen-activated protein kinases (MAPKs), apoptotic and poly (ADP-ribose)polymerase-dependent cell death, enhanced inflammation (leucocyte infiltration, tumour necrosis factor-alpha and interleukin-1beta) and promoted oxidative/nitrosative stress [increased expressions of superoxide-generating enzymes (NOX2(gp91phox), NOX4), inducible nitric oxide synthase and tissue 4-hydroxynonenal and nitrotyrosine levels] in the kidneys of mice, accompanied by marked histopathological damage and impaired renal function (elevated creatinine and serum blood urea nitrogen) 3 days following its administration. Both genetic deletion and pharmacological inhibition of CB(1) receptors with AM281 or SR141716 markedly attenuated the cisplatin-induced renal dysfunction and interrelated oxidative/nitrosative stress, p38 and JNK MAPK activation, cell death and inflammatory response in the kidney.Conclusions and Implications: The endocannabinoid system through CB(1) receptors promotes cisplatin-induced tissue injury by amplifying MAPK activation, cell death and interrelated inflammation and oxidative/nitrosative stress. These results also suggest that inhibition of CB(1) receptors may exert beneficial effects in renal (and most likely other) diseases associated with enhanced inflammation, oxidative/nitrosative stress and cell death. [ABSTRACT FROM AUTHOR]

Background and Purpose: Although GPR55 is potently activated by the endogenous lysophospholipid, L-alpha-lysophosphatidylinositol (LPI), it is also thought to be sensitive to a number of cannabinoid ligands, including the prototypic CB1 receptor antagonists AM251 and SR141716A (Rimonabant). In this study we have used a range of functional assays to compare the pharmacological activity of selected cannabinoid ligands, AM251, AM281 and SR141716A with LPI in a HEK293 cell line engineered to stably express recombinant, human GPR55.Experimental Approach: We evaluated Ca(2+) signalling, stimulation of extracellular signal regulated kinase (ERK1/2) mitogen activated kinase MAP-kinases, induction of transcriptional regulators that are downstream of GPR55, including nuclear factor of activated T cells (NFAT), nuclear factor-kappaB (NF-kappaB) and cAMP response element binding protein (CREB), as well as receptor endocytosis. In addition, we assessed the suitability of a novel, label-free assay for GPR55 ligands that involves optical measurement of dynamic mass redistribution following receptor activation.Key Results: GPR55 linked to a range of downstream signalling events and that the activity of GPR55 ligands was influenced by the functional assay employed, with differences in potency and efficacy observed.Conclusions and Implications: Our data help to resolve some of the issues surrounding the pharmacology of cannabinoid ligands at GPR55 and highlight some differences in effector coupling associated with distinct GPR55 ligands. [ABSTRACT FROM AUTHOR]

Background and Purpose: The endocannabinoid-like molecule N-oleoylethanolamine (OEA) is found in the small intestine and regulates food intake and promotes weight loss. The principal aim of the present study was to evaluate the vascular effects of OEA.Experimental Approach: Perfused isolated mesenteric arterial beds were pre-contracted with methoxamine or high potassium buffers and concentration-response curves to OEA were constructed. Combinations of inhibitors to block nitric oxide production, sensory nerve activity, cyclooxygenase activity, potassium channels, chloride channels and gap junctions, and a cannabinoid CB(1) receptor antagonist, were used during these experiments. The effects of OEA on caffeine-induced contractions in calcium-free buffer were also assessed. Isolated thoracic aortic rings were used as a comparison.Key Results: OEA caused concentration-dependent vasorelaxation in rat isolated mesenteric arterial beds and thoracic aortic rings, with a greater maximal response in mesenteric vessels. This relaxation was sensitive to inhibition of sensory nerve activity and endothelial removal in both preparations. The cyclooxygenase inhibitor indomethacin reversed the effects of capsaicin pre-treatment in perfused mesenteric arterial beds and indomethacin alone enhanced vasorelaxation to OEA. The OEA-induced vasorelaxation was inhibited by a CB(1) receptor antagonist only in aortic rings. In mesenteric arteries, OEA suppressed caffeine-induced contractions in calcium-free buffer.Conclusions and Implications: The vasorelaxant effects of OEA are partly dependent on sensory nerve activity and a functional endothelium in the vasculature. In addition, vasorelaxation to OEA is enhanced following cyclooxygenase inhibition. OEA may also interfere with the release of intracellular calcium in arterial preparations. [ABSTRACT FROM AUTHOR]

Background and Purpose: Some neurosteroids, notably 3alpha-hydroxysteroids, positively modulate GABA(A) receptors, but sulphated steroids negatively modulate these receptors. Recently, other lipophilic amphiphiles have been suggested to positively modulate GABA receptors. We examined whether there was similarity among the actions of these agents and the mechanisms of neurosteroids. Significant similarity would affect theories about the specificity of steroid actions.Experimental Approach: Xenopus laevis oocytes were challenged with Triton X-100, octyl-beta-glucoside, capsaicin, docosahexaenoic acid and sodium dodecyl sulphate (SDS), along with different GABA concentrations.Key Results: These compounds have both positive and negative effects on GABA currents, which can be accentuated according to the degree of receptor activation. A low GABA concentration (1 microM) promoted potentiation and a high concentration (20 microM) promoted inhibition of current, except for SDS that inhibited function even at low GABA concentrations. Amphiphile inhibition was characterized by enhanced apparent desensitization and by weak voltage dependence, similar to pregnenolone sulphate antagonism. We then tested amphiphile effects on mutated receptor subunits that are insensitive to negative (alpha1V256S) and positive (alpha1Q241L or alpha1N407A/Y410F) steroid modulation. Negative regulation by amphiphiles was nearly abolished in alpha1V256S-mutated receptors, but potentiation was unaffected. In alpha1Q241L- or alpha1N407A/Y410F-mutated receptors, potentiation by amphiphiles remained intact.Conclusions and Implications: Structurally diverse amphiphiles have antagonist actions at GABA(A) receptors very similar to those of sulphated neurosteroids, while the potentiating mechanisms of these amphiphiles are distinct from those of neurosteroid-positive modulators. Thus, such antagonism at GABA(A) receptors does not have a clear pharmacophore requirement. [ABSTRACT FROM AUTHOR]

Background and Purpose: Limited data on the brain penetration of potential stroke treatments have been cited as a major weakness contributing to numerous failed clinical trials. Thus, we tested whether interleukin-1 receptor antagonist (IL-1RA), established as a potent inhibitor of brain injury in animals and currently in clinical development, reaches the brain via a clinically relevant administration route, in experimental stroke.Experimental Approach: Male, Sprague-Dawley rats [either naïve or exposed to middle cerebral artery occlusion (MCAo)] were given a single s.c. dose of IL-1RA (100 mg*kg(-1)). The pharmacokinetic profile of IL-1RA was assessed in plasma and CSF up to 24 h post-administration. Brain tissue distribution of administered IL-1RA was assessed using immunohistochemistry. In a separate experiment, the neuroprotective effect of the single s.c. dose of IL-1RA in MCAo was assessed versus a placebo control group.Key Results: A single s.c. dose of IL-1RA reduced damage caused by MCAo by 33%. This dose resulted in sustained, high concentrations in plasma and CSF, penetrated brain tissue exclusively in areas of blood-brain barrier breakdown and co-localized with morphologically viable neurones. CSF concentrations did not reflect massive parenchymal infiltration of IL-1RA in MCAo animals compared to naïve.Conclusions and Implications: These data are the first to show that a potential treatment for stroke, IL-1RA, rapidly reaches salvageable brain tissue via an administration route that is clinically relevant. This allows confidence that IL-1RA, as a candidate for further clinical development, is able to confer its protective actions both peripherally and centrally. [ABSTRACT FROM AUTHOR]

Background and Purpose: The functional characterization of secreted peptides can provide the basis for the development of novel therapeutic agents. Augurin is a recently identified secreted peptide of unknown function expressed in multiple endocrine tissues, and in regions of the brain including the hypothalamus. We therefore investigated the effect of hypothalamic injection of augurin on the hypothalamo-pituitary-adrenal (HPA) axis in male Wistar rats.Experimental Approach: Augurin was given as a single injection into the third cerebral ventricle (i.c.v.) or into the paraventricular nucleus (iPVN) of the hypothalamus. Circulating hormone levels were then measured by radioimmunoassay. The effect of augurin on the release of hypothalamic neuropeptides was investigated ex vivo using hypothalamic explants. The acute effects of iPVN augurin on behaviour were also assessed.Key Results: i.c.v. injection of augurin significantly increased plasma ACTH and corticosterone, compared with vehicle-injected controls, but had no effect on other hypothalamo-pituitary axes hormones. Microinjection of lower doses of augurin into the PVN caused a similar increase in plasma ACTH and corticosterone, without significant alteration in behavioural patterns. Incubation of hypothalamic explants with increasing doses of augurin significantly elevated corticotrophin-releasing factor (CRF) and arginine vasopressin release. In vivo, peripheral injection of a CRF(1/2) receptor antagonist prevented the rise in ACTH and corticosterone caused by i.c.v. augurin injection.Conclusions and Implications: These data suggest that augurin stimulates the release of ACTH via the release of hypothalamic CRF. Pharmacological manipulation of the augurin system may therefore be a novel target for regulation of the HPA axis. [ABSTRACT FROM AUTHOR]

Background and Purpose: Recent findings suggest that the noxious gas H(2)S is produced endogenously, and that physiological concentrations of H(2)S are able to modulate pain and inflammation in rodents. This study was undertaken to evaluate the ability of endogenous and exogenous H(2)S to modulate carrageenan-induced synovitis in the rat knee.Experimental Approach: Synovitis was induced in Wistar rats by intra-articular injection of carrageenan into the knee joint. Sixty minutes prior to carrageenan injection, the rats were pretreated with indomethacin, an inhibitor of H(2)S formation (DL-propargylglycine) or an H(2)S donor [Lawesson's reagent (LR)].Key Results: Injection of carrageenan evoked knee inflammation, pain as characterized by impaired gait, secondary tactile allodynia of the ipsilateral hindpaw, joint swelling, histological changes, inflammatory cell infiltration, increased synovial myeloperoxidase, protein nitrotyrosine residues, inducible NOS (iNOS) activity and NO production. Pretreatment with LR or indomethacin significantly attenuated the pain responses, and all the inflammatory and biochemical changes, except for the increased iNOS activity, NO production and 3-NT. Propargylglycine pretreatment potentiated synovial iNOS activity (and NO production), and enhanced macrophage infiltration, but had no effect on other inflammatory parameters.Conclusions and Implications: Whereas exogenous H(2)S delivered to the knee joint can produce a significant anti-inflammatory and anti-nociceptive effect, locally produced H(2)S exerts little immunomodulatory effect. These data further support the development and use of H(2)S donors as potential alternatives (or complementary therapies) to the available anti-inflammatory compounds used for treatment of joint inflammation or relief of its symptoms. [ABSTRACT FROM AUTHOR]

Background and Purpose: Neuronal ion channels are key targets of general anaesthetics and alcohol, and binding of these drugs to pre-existing and relatively specific sites is thought to alter channel gating. However, the underlying molecular mechanisms of this action are still poorly understood. Here, we investigated the neuronal Shaw2 voltage-gated K(+) (K(v)) channel to ask whether the inhalational anaesthetic halothane and n-alcohols share a binding site near the activation gate of the channel.Experimental Approach: Focusing on activation gate mutations that affect channel modulation by n-alcohols, we investigated n-alcohol-sensitive and n-alcohol-resistant K(v) channels heterologously expressed in Xenopus oocytes to probe the functional modulation by externally applied halothane using two-electrode voltage clamping and a gas-tight perfusion system.Key Results: Shaw2 K(v) channels are reversibly inhibited by halothane in a dose-dependent and saturable manner (K(0.5)= 400 microM; n(H)= 1.2). Also, discrete mutations in the channel's S4S5 linker are sufficient to reduce or confer inhibition by halothane (Shaw2-T330L and K(v)3.4-G371I/T378A respectively). Furthermore, a point mutation in the S6 segment of Shaw2 (P410A) converted the halothane-induced inhibition into halothane-induced potentiation. Lastly, the inhibition resulting from the co-application of n-butanol and halothane is consistent with the presence of overlapping binding sites for these drugs and weak binding cooperativity.Conclusions and Implications: These observations strongly support a molecular model of a general anaesthetic binding site in the Shaw2 K(v) channel. This site may involve the amphiphilic interface between the S4S5 linker and the S6 segment, which plays a pivotal role in K(v) channel activation. [ABSTRACT FROM AUTHOR]

Background and Purpose: Molecular mechanisms underlying the links between dietary intake of flavonoids and reduced cardiovascular disease risk are only partially understood. Key events in the pathogenesis of cardiovascular disease, particularly thrombosis, are inhibited by these polyphenolic compounds via mechanisms such as inhibition of platelet activation and associated signal transduction, attenuation of generation of reactive oxygen species, enhancement of nitric oxide production and binding to thromboxane A(2) receptors. In vivo, effects of flavonoids are mediated by their metabolites, but the effects and modes of action of these compounds are not well-characterized. A good understanding of flavonoid structure-activity relationships with regard to platelet function is also lacking.Experimental Approach: Inhibitory potencies of structurally distinct flavonoids (quercetin, apigenin and catechin) and plasma metabolites (tamarixetin, quercetin-3'-sulphate and quercetin-3-glucuronide) for collagen-stimulated platelet aggregation and 5-hydroxytryptamine secretion were measured in human platelets. Tyrosine phosphorylation of total protein, Syk and PLCgamma2 (immunoprecipitation and Western blot analyses), and Fyn kinase activity were also measured in platelets. Internalization of flavonoids and metabolites in a megakaryocytic cell line (MEG-01 cells) was studied by fluorescence confocal microscopy.Key Results: The inhibitory mechanisms of these compounds included blocking Fyn kinase activity and the tyrosine phosphorylation of Syk and PLCgamma2 following internalization. Principal functional groups attributed to potent inhibition were a planar, C-4 carbonyl substituted and C-3 hydroxylated C ring in addition to a B ring catechol moiety.Conclusions and Implications: The structure-activity relationship for flavonoids on platelet function presented here may be exploited to design selective inhibitors of cell signalling. [ABSTRACT FROM AUTHOR]

Background and Purpose: In adults, neurogenesis persists in the hippocampus and the subventricular zone (SVZ), and this is important for learning and memory. Inhibitors of COX-2 suppress ischaemia-induced neurogenesis in the hippocampus. Here, we have determined the effects of COX-2 inhibitors on neurogenesis throughout the normal adult mouse brain.Experimental Approach: Young adult mice were treated with COX-2 inhibitors, and the proliferation of neural progenitor cells was measured in the SVZ and hippocampus. In addition, the local uptake of lentiviral vectors in the rostral migratory stream enabled the formation of new neurons in the olfactory bulb (OB) to be assessed.Key Results: The COX-2 inhibitor meloxicam suppressed progenitor cell proliferation in the SVZ and hippocampus. A significant decrease in the appearance of new neurons in the OB was also observed. Similar effects on progenitor proliferation in the SVZ were seen with nimesulide. The absence of COX-2 expression in the proliferating progenitors in vivo, and the lack of effect of the COX-2 inhibitors on the growth rate of a cultured progenitor cell line, suggest that the effect is indirect. The specific expression of COX-2 in resting microglia that closely associate with the proliferating progenitor cells provides for a possible site of action.Conclusions and Implications: Treatment with a COX-2 inhibitor results in a substantial inhibition of adult neurogenesis. Studies on human tissues are warranted in order to determine if this effect extends to humans, and whether inhibition of neurogenesis should be considered as an adverse effect of these drugs. [ABSTRACT FROM AUTHOR]

Background and Purpose: Big endothelin-1 (ET-1) circulates in plasma but does not bind to ET receptors until converted to ET-1 by smooth muscle converting enzymes. We hypothesized that tissue-specific conversion of [(18)F]-big ET-1 to [(18)F]-ET-1 could be imaged dynamically in vivo within target organs as binding to ET receptors.Methods: [(18)F]-big ET-1 conversion imaged in vivo following infusion into rats using positron emission tomography (PET).Key Results: [(18)F]-big ET-1 was rapidly cleared from the circulation (t(1/2)= 2.9 +/- 0.1 min). Whole body microPET images showed highest uptake of radioactivity in three major organs. In lungs and liver, time activity curves peaked within 2.5 min, then plateaued reaching equilibrium after 10 min, with no further decrease after 120 min. Phosphoramidon did not alter half life of [(18)F]-big ET-1 but uptake was reduced in lung (42%) and liver (45%) after 120 min, consistent with inhibition of enzyme conversion and reduction of ET-1 receptor binding. The ET(A) antagonist, FR139317 did not alter half-life of [(18)F]-big ET-1 (t(1/2)= 2.5 min) but radioactivity was reduced in all tissues except for kidney consistent with reduction in binding to ET(A) receptors. In kidney, however, the peak in radioactivity was higher but time to maximum accumulation was slower ( approximately 30 min), which was increased by phosphoramidon, reflecting renal excretion with low conversion and binding to ET receptors.Conclusions and Implications: A major site for conversion was within the vasculature of the lung and liver, whereas uptake in kidney was more complex, reflecting excretion of [(18)F]-big ET-1 without conversion to ET-1. [ABSTRACT FROM AUTHOR]

Background and Purpose: Pharmacological analysis of synergism or functional antagonism between different receptors commonly assumes that interacting receptors are located in the same cells. We have now investigated the distribution of alpha-adrenoceptors, beta-adrenoceptors and cannabinoid-like (GPR55) receptors in the mouse arteries.Experimental Approach: Fluorescence intensity from vascular tissue incubated with fluorescent ligands (alpha(1)-adrenoceptor ligand, BODIPY-FL-prazosin, QAPB; beta-adrenoceptor ligand, TMR-CGP12177; fluorescent angiotensin II; a novel diarylpyrazole cannabinoid ligand (Tocrifluor 1117, T1117) was measured with confocal microscopy. Small mesenteric and tail arteries of wild-type and alpha(1B/D)-adrenoceptor-KO mice were used.Key Results: T1117, a fluorescent form of the cannabinoid CB(1) receptor antagonist AM251, was a ligand for GPR55, with low affinity for CB(1) receptors. In mesenteric arterial smooth muscle cells, alpha(1A)-adrenoceptors were predominantly located in different cells from those with beta-adrenoceptors, angiotensin receptors or cannabinoid-like (GPR55) receptors. Cells with beta-adrenoceptors predominated at arterial branches. Endothelial cells expressed beta-adrenoceptors, alpha-adrenoceptors and cannabinoid-like receptors. Only endothelial alpha-adrenoceptors appeared in clusters. Adventitia was a rich source of G protein-coupled receptors (GPCRs), particularly fibroblasts and nerve tracts, where Schwann cells bound alpha-adrenoceptor, beta-adrenoceptor and CB-receptor ligands, with a mix of separate receptor locations and co-localization.Conclusions and Implications: Within each cell type, each GPCR had a distinctive heterogeneous distribution with limited co-localization, providing a guide to the possibilities for functional synergism, and suggesting a new paradigm for synergism in which interactions may be either between cells or involve converging intracellular signalling processes. [ABSTRACT FROM AUTHOR]

Background and Purpose: Sphingosine-1-phosphate and its receptors may be involved in vascular smooth muscle cell (VSMC) proliferation following vascular injury. Here, we evaluate the effect of d-erythro-N,N-dimethylsphingosine (DMS), a sphingosine kinase (SK) inhibitor, on VSMC proliferation, apoptosis and neointimal formation.Experimental Approach: Growth responses in vitro to fetal calf serum (FCS) were measured by [(3)H]-thymidine incorporation and extracellular signal-regulated kinase-1/2 (ERK-1/2) activation in quiescent primary cultures of porcine VSMC in the presence and absence of various concentrations of the SK inhibitor DMS. In vivo treatment with DMS was delivered with a local endoluminal catheter, following balloon injury of coronary arteries. The artery intimal formation was investigated by angiography, myography and histomorphometry.Key Results: In vitro experiments indicated that DMS induced a dose-dependent reduction in [(3)H]-thymidine incorporation and ERK-1/2 activation via a protein kinase C (PKC) independent mechanism with an IC(50) value of 12 +/- 6 and 15 +/- 10 microM respectively. DMS also reduced Akt signalling. Four weeks following in vivo delivery of DMS, complete functional endothelial regeneration was observed in all treatment groups, with significant reduction of intimal formation (vehicle 23.7 +/- 4.6% vs. DMS infusion 8.92 +/- 2.9%, P < 0.05).Conclusions and Implications: Taken together, these results demonstrate that local administration of the SK inhibitor, DMS, reduced neointimal formation, and this effect could involve inhibition of ERK-1/2 and Akt signalling, and modulation of smooth muscle growth. [ABSTRACT FROM AUTHOR]

Background and Purpose: We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A(2B) adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5'-(N-ethylcarboxamido)-adenosine.Experimental Approach: The trafficking of the wild type A(2B) adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy.Key Results: The wild type A(2B) adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln(325)-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu(330)-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln(325)-stop, Ser(326)-stop and Phe(328)-stop receptors. Following internalization, the wild type A(2B) adenosine receptor recycled rapidly to the cell surface, whereas the Gln(325)-stop receptor did not recycle.Conclusions and Implications: Deletion of the COOH-terminus of the A(2B) adenosine receptor beyond Leu(330) switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A(2B) adenosine receptor following prolonged agonist addition. [ABSTRACT FROM AUTHOR]

Background and Purpose: Changes in tissue P-glycoprotein (P-gp) activity during pregnancy could affect the pharmacokinetics and thus the efficacy and toxicity of many drugs. Therefore, using positron emission tomography (PET) imaging, we tested whether gestational age affects tissue P-gp activity in the pregnant non-human primate, Macaca nemestrina.Experimental Approach: Mid-gestational (day 75 +/- 13, n= 7) and late-gestational (day 150 +/- 10, n= 5) age macaques were imaged after administration of a prototypic P-gp substrate, (11)C-verapamil (13.7-75.4 MBq.kg(-1)), before and during intravenous infusion of a P-gp inhibitor, cyclosporin A (CsA) (12 or 24 mg.kg(-1).h(-1)). Accumulation of radioactivity in the fetal liver served as a reporter of placental P-gp activity. P-gp activity was expressed as CsA-induced percent change in the ratio of the area (0-9 min) under the (11)C-radioactivity concentration-time curve in the tissue (AUC(tissue)) to that in the maternal plasma (AUC(plasma)).Key Results: The CsA-induced change in AUC(fetal liver)/AUC(maternal)(plasma) of (11)C-radioactivity significantly increased from mid- (35 +/- 25%) to late gestation (125 +/- 66%). Likewise, the CsA-induced change in AUC(maternal brain)/AUC(plasma) increased from mid- (172 +/- 80%) to late gestation (337 +/- 148%). The AUC ratio for the other maternal tissues was not significantly affected. Neither the CsA blood concentrations nor the level of circulating (11)C-verapamil metabolites were significantly affected by gestational age.Conclusions and Implications: P-gp activity at the blood-brain barrier and the placental barrier in the macaque increased with gestational age. If replicated in humans, the exposure of the fetus and maternal brain to P-gp substrate drugs, and therefore their efficacy and toxicity, will change during pregnancy. [ABSTRACT FROM AUTHOR]