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Isoniazid and rifampicin are first-line anti-tubercular drugs. In a recent paper, Shen et al. provided interesting findings that rifampicin exacerbated isoniazid toxicity in human hepatocytes but not in rat hepatocytes. The main conclusion was that the difference in cytochrome P450 2E1 (CYP2E1) induction by rifampicin between rat and human hepatocytes accounted for the difference in exacerbation of isoniazid hepatotoxicity by rifampicin. 4-Nitrophenol hydroxylase (4-NP) activity was the only probe of CYP2E1 activity used in the paper. The authors presented data showing that rifampicin enhanced 4-NP activity and CYP2E1 mRNA expression in human hepatocytes, but not in rat hepatocytes. However, CYP3A also makes a significant contribution to 4-NP activity in humans and rats, which has been confirmed by both CYP3A-specific inducer and inhibitors. Rifampicin is a strong inducer of human CYP3A; thus, the increase in 4-NP activity in human hepatocytes could be due to the induction of CYP3A. Rifampicin did not increase 4-NP activities in rat hepatocytes, which could reflect a lack of the induction of rat CYP3A by rifampicin. Additionally, more experiments are needed to support the conclusion that rifampicin increased CYP2E1 mRNA expression in human hepatocytes because of the small sample size and the limitations of semi-quantitative RT-PCR. The study by Shen et al. suggests that another drug-metabolizing enzyme rather than CYP2E1 could be involved in the aggravation of isoniazid toxicity by rifampicin in human hepatocytes. [ABSTRACT FROM AUTHOR]

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]

In the past few years, increasing interest has been directed to bioactive peptides of animal and plant origin: in particular, researchers have focused their attention on their mechanisms of action and potential role in the prevention and treatment of cancer, cardiovascular and infective diseases. We have developed a search strategy to identify these studies in PubMed (January 1980 to May 2016); particularly those papers presenting comprehensive reviews or meta-analyses, plus in vitro and in vivo studies and clinical trials on those bioactive peptides that affect cardiovascular diseases, immunity or cancer, or have antioxidant, anti-inflammatory and antimicrobial effects. In this review we have mostly focused on evidence-based healthy properties of bioactive peptides from different sources. Bioactive peptides derived from fish, milk, meat and plants have demonstrated significant antihypertensive and lipid-lowering activity in clinical trials. Many bioactive peptides show selective cytotoxic activity against a wide range of cancer cell lines in vitro and in vivo, whereas others have immunomodulatory and antimicrobial effects. Furthermore, some peptides exert anti-inflammatory and antioxidant activity, which could aid in the prevention of chronic diseases. However, clinical evidence is at an early stage, and there is a need for solid pharmacokinetic data and for standardized extraction procedures. Further studies on animals and randomized clinical trials are required to confirm these effects, and enable these peptides to be used as preventive or therapeutic treatments.Linked Articles: This article is part of a themed section on Principles of Pharmacological Research of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.11/issuetoc. [ABSTRACT FROM AUTHOR]

Background and purpose: Nitroxyl (HNO) is emerging as an important regulator of vascular tone as it is potentially produced endogenously and dilates conduit and resistance arteries. This study investigates the contribution of endogenous HNO to endothelium-dependent relaxation and hyperpolarization in resistance arteries. Experimental approach: Rat and mouse mesenteric arteries were mounted in small vessel myographs for isometric force and smooth muscle membrane potential recording. Key results: Vasorelaxation to the HNO donor, Angeli's salt, was attenuated in both species by the soluble guanylate cyclase inhibitor (ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one), the voltage-dependent K+ channel inhibitor, 4-aminopyridine (4-AP) and the HNO scavenger,l-cysteine. In mouse mesenteric arteries, nitric oxide (NO) synthase inhibition (withl-NAME, Nω-Nitro-L-arginine methyl ester) markedly attenuated acetylcholine (ACh)-mediated relaxation. Scavenging the uncharged form of NO (NO•) with hydroxocobalamin (HXC) or HNO withl-cysteine, or 4-AP decreased the sensitivity to ACh, and a combination of HXC andl-cysteine reduced ACh-mediated relaxation, as didl-NAME alone. ACh-induced hyperpolarizations were significantly attenuated by 4-AP alone and in combination withl-NAME. In rat mesenteric arteries, blocking the effects of endothelium-derived hyperpolarizing factor (EDHF) (charybdotoxin and apamin) decreased ACh-mediated relaxation 10-fold and unmasked a NO-dependent component, mediated equally by HNO and NO•, as HXC andl-cysteine in combination now abolished vasorelaxation to ACh. Furthermore, ACh-evoked hyperpolarizations, resistant to EDHF inhibition, were virtually abolished by 4-AP. Conclusions and implications: The factors contributing to vasorelaxation in mouse and rat mesenteric arteries are NO• = HNO > EDHF and EDHF > HNO = NO• respectively. This study identified HNO as an endothelium-derived relaxing and hyperpolarizing factor in resistance vessels. British Journal of Pharmacology (2009) 157, 540–550; doi:10.1111/j.1476-5381.2009.00150.x; published online 26 March 2009 This article is commented on by Martin, pp. 537–539 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]

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:F15063 is a high affinity D2/D3 antagonist, D4 partial agonist, and high efficacy 5-HT1A agonist, with little affinity (40-fold lower than for D2 receptors) at other central targets. Here, the profile of F15063 was evaluated in models of positive symptoms of schizophrenia and motor side-effects.Experimental approach:Rodent behavioural tests were based on reversal of hyperactivity induced by psychostimulants and on measures of induction of catalepsy and ‘serotonin syndrome’.Key results:F15063 potently (ED50s: 0.23 to 1.10 mg kg−1 i.p.) reversed methylphenidate-induced stereotyped behaviors, blocked d-amphetamine and ketamine hyperlocomotion, attenuated apomorphine-induced prepulse inhibition (PPI) deficits, and was active in the conditioned avoidance test. In mice, it reversed apomorphine-induced climbing (ED50 = 0.30 mg kg−1 i.p.). F15063, owing to its 5-HT1A agonism, did not produce (ED50 > 40 mg kg−1 i.p.) catalepsy in rats and mice, a behavior predictive of occurrence of extra-pyramidal syndrome (EPS) in man. This absence of cataleptogenic activity was maintained upon sub-chronic treatment of rats for 5 days at 40 mg kg−1 p.o. Furthermore, F15063 did not induce the ‘serotonin syndrome’ in rats (flat body posture and forepaw treading: ED50 >32 mg kg−1 i.p.).Conclusions and implications:F15063 conformed to the profile of an atypical antipsychotic, with potent actions in models of hyperdopaminergic activity but without inducing catalepsy. These data suggest that F15063 may display potent antipsychotic actions with low EPS liability. This profile is complemented by a favourable profile in rodent models of negative symptoms and cognitive deficits of schizophrenia (companion paper).British Journal of Pharmacology (2007) 151, 253–265. doi:10.1038/sj.bjp.0707159; published online 20 March 2007 [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: A major problem managing alcohol use disorder is the high vulnerability to relapse, even after long periods of abstinence. Chronic alcohol use dysregulates stress responsivity, rendering this system hyporesponsive and making individuals vulnerable to relapse. Orexin (hypocretin) plays a role in diverse physiological processes, including stress. Orexin neurons in the hypothalamus, project to the infralimbic cortex. This study asked does infralimbic cortex orexin transmission play a significant role in stress‐induced reinstatement of alcohol‐seeking behaviour in alcohol‐dependent rats. Experimental Approach: Male and female rats were trained to self‐administer 10% alcohol (3 weeks) and then made dependent via chronic intermittent alcohol vapour exposure. Following extinction (5 days·week−1 at 8 h abstinence for 10 sessions), rats received an intra‐ infralimbic cortex microinfusion of the OX1/2 antagonist TCS 1102 (15 μg/0.5 μl per side) and then tested for footshock stress‐induced reinstatement of alcohol seeking. In a separate cohort, orexin regulation of infralimbic cortex neuronal activity at the time of reinstatement was investigated using ex vivo electrophysiology. Key Results: TCS 1102 prevented reinstatement in dependent animals only. Moreover, Hcrtr mRNA expression in the hypothalamus and Hcrtr1/2 in the infralimbic cortex increased in alcohol‐dependent animals at the time of testing. Dependence dampened basal orexin/OX receptor influence over infralimbic cortex GABAergic synapses (using TCS 1102) allow for greater stimulated orexin effects. Conclusion and Implications: Infralimbic cortex transmission is implicate in stress‐induced reinstatement of alcohol‐seeking behaviour in subjects with a history of alcohol dependence and show maladaptive recruitment of infralimbic cortex transmission by alcohol dependence. [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: 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: 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: Ilexgenin A is a triterpenoid from ShanLv Cha with beneficial effects on metabolic homeostasis. We investigated whether ilexgenin A could inhibit hepatic de novo fatty acid synthesis via the interfering with SREBP1 maturation.Experimental Approach: The effects of Ilexgenin A on CRTC2 translocation and SREBP1 maturation were investigated in the liver of fasted mice and hepatocytes exposed to saturated fatty acids. The effect of Iilexgenin A on hepatic lipid accumulation was also observed in high-fat diet fed mice.Key Results: Sec23A and Sec31A are two subunits of COPII complex and their interaction is essential for the processing of SREBP1 maturation. Ilexgenin A activates AMPK by reducing cellular energy and preventing cytoplasmic CRTC2 to compete with Sec23A for binding to Sec31A under nutrient-rich conditions. Consequently, ilexgenin A impaired COPII-dependent SREBP1 maturation via disrupting Sec31A-Sec23A interaction, leading to the inhibition of de novo fatty acid synthesis in the liver. In contrast, mTORC1 phosphorylated Ser136 of CRTC2, facilitating the formation of Sec31A-Sec23A interaction to promote SREBP1 maturation, whereas this action was reversed by ilexgenin A in an AMPK-dependent manner. Ilexgenin A protected CRTC2 function and restrained hepatic lipogenic response in high fat diet-fed mice, providing in vivo evidence to support the beneficial effects of ilexgenin A on lipid metabolism.Conclusions and Implications: Ilexgenin A activated AMPK and restrained CRTC2 to the cytoplasm to prevent SREBP1 maturation via impairing COPII function in the liver. This suggests that CRTC2 might be a potential target for pharmacological intervention to prevent hepatic lipid deposition.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]

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]

Background and Purpose: While remifentanil can be used either during labour or fetal surgery, more should be known about the transplacental transfer of this opioid. The aim of this study was to investigate the placental transfer and haemodynamic effects of remifentanil after i.v. administration to pregnant ewes.Experimental Approach: Seven pregnant ewes received a continuous infusion of remifentanil (0.33 µg·kg⁻¹·min⁻¹) for 1 h, and maternal and fetal arterial blood samples were drawn at regular intervals during and up to 1 h after the discontinuation of the infusion. Haemodynamic parameters were monitored continuously. Blood gas samples were drawn at baseline and once during the infusion.Key Results: Peak maternal remifentanil plasma levels of 4.0 ± 0.9 ng·mL⁻¹ (mean ± SD) and peak fetal plasma levels of 0.4 ± 0.3 ng·mL⁻¹ were obtained. Remifentanil plasma levels dropped rapidly after the discontinuation of the infusion. The continuous infusion of remifentanil did not result in significant maternal or fetal haemodynamic changes.Conclusions and Implications: Remifentanil rapidly passes through the placenta of pregnant ewes and although remifentanil concentrations stay significantly lower in the fetus compared with those in the mother, remifentanil can be detected in significant amounts in the fetus. [ABSTRACT FROM AUTHOR]

Background and Purpose: Maternal infections are one of the main causes of adverse developmental outcomes including embryonic resorption and preterm labour. In this study a mouse model of inflammation-associated preterm delivery was developed, and used to study the relationship between nitric oxide (NO) and prostaglandins (PGs).Experimental Approach: The murine model of preterm labour was achieved by assaying different doses of bacterial lipopolysaccharides (LPS). Once established, it was used to analyse uterine levels of prostaglandins E(2) and F(2α) (by radioimmunoassay), cyclooxygenases (COX) and NOS proteins (by Western blot) and NO synthase (NOS) activity. Effects of inhibitors of COX and NOS on LPS-induced preterm labour were also studied. In vitro assays with a nitric oxide donor (SNAP) were performed to analyse the modulation of prostaglandin production by NO.Key Results: Lipopolysaccharide increased uterine NO and PG synthesis and induced preterm delivery. Co-administration of meloxicam, a cyclooxygenase-2 inhibitor, or aminoguanidine, an inducible NOS inhibitor, prevented LPS-induced preterm delivery and blocked the increase in PGs and NO. Notably, the levels of NO were found to determine its effect on PG synthesis; low concentrations of NO reduced PG synthesis whereas high concentrations augmented them.Conclusions and Implications: An infection-associated model of preterm labour showed that preterm delivery can be prevented by decreasing PG or NO production. NO was found to have a dual effect on PG synthesis depending on its concentration. These data contribute to the understanding of the interaction between NO and PGs in pregnancy and parturition, and could help to improve neonatal outcomes. [ABSTRACT FROM AUTHOR]

Background and Purpose: The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (K(iapp) 5.3 nM) and cathepsin G (K(iapp) 160.0 nM), and the cysteine peptidase cathepsin L (K(iapp) 0.2 nM). These three peptidases play important roles in the inflammatory process.Experimental Approach: We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte-endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by elisa.Key Results: Pretreatment of the animals with BbCI (2.5 mg·kg(-1)), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1β or tumour necrosis factor-α, however, did not change.Conclusions and Implications: Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles. [ABSTRACT FROM AUTHOR]

Background and Purpose: Insulin-induced Na(+) retention in the distal nephron may contribute to the development of oedema/hypertension in patients with type 2 diabetes. This response to insulin is usually attributed to phosphatidylinositol-3-kinase (PI3K)/serum and glucocorticoid-inducible kinase 1 (SGK1) but a role for protein kinase B (PKB) has been proposed. The present study therefore aimed to clarify the way in which insulin can evoke Na(+) retention.Experimental Approach: We examined the effects of nominally selective inhibitors of PI3K (wortmannin, PI103, GDC-0941), SGK1 (GSK650394A) and PKB (Akti-1/2) on Na(+) transport in hormone-deprived and insulin-stimulated cortical collecting duct (mpkCCD) cells, while PI3K, SGK1 and PKB activities were assayed by monitoring the phosphorylation of endogenous proteins.Key Results: Wortmannin substantially inhibited basal Na(+) transport whereas PI103 and GDC-0941 had only very small effects. However, these PI3K inhibitors all abolished insulin-induced Na(+) absorption and inactivated PI3K, SGK1 and PKB fully. GSK650394A and Akti-1/2 also inhibited insulin-evoked Na(+) absorption and while GSK650394A inhibited SGK1 without affecting PKB, Akti-1/2 inactivated both kinases.Conclusion and Implications: While studies undertaken using PI103 and GDC-0941 show that hormone-deprived cells can absorb Na(+) independently of PI3K, PI3K seems to be essential for insulin induced Na(+) transport. Akti-1/2 does not act as a selective inhibitor of PKB and data obtained using this compound must therefore be treated with caution. GSK650394A, on the other hand, selectively inhibits SGK1 and the finding that GSK650394A suppressed insulin-induced Na(+) absorption suggests that this response is dependent upon signalling via PI3K/SGK1. [ABSTRACT FROM AUTHOR]

Background and Purpose: Andrographolide is a diterpenoid lactone isolated from a traditional medicinal herb, Andrographis paniculata. It possesses potent anti-inflammatory activity. The present study examined potential therapeutic effects of andrographolide on cerebral ischaemia using a rat model with permanent middle cerebral artery occlusion (pMCAO).Experimental Approach: The MCA in rats was permanently occluded (by cautery), and 24 h later neurological effects were assessed with behavioural scores. Infarct volume and microglial activation were determined histologically. The p65 form of the transcription factor, nuclear factor-κB (NF-κB), was measured by Western blot, and cytokines by immunoassay of brain extracts.Key Results: Andrographolide, given i.p. 1 h after pMCAO, reduced infarct volume with a maximum reduction of approximately 50% obtained at 0.1 mg·kg(-1). Neurological deficits were also reduced by andrographolide, reflecting a correlation between infarct volume and neurological deficits. pMCAO was found to induce activation of microglia and elevate tumour necrosis factor (TNF)-α, interleukin (IL)-1β and prostaglandin (PG)E(2) in the ischaemic brain areas. Andrographolide (0.1 mg·kg(-1)) significantly attenuated or abolished these effects. In addition, andrographolide suppressed the translocation of p65 from cytosol to nucleus, indicating reduced NF-κB activation.Conclusions and Implications: Andrographolide exhibited neuroprotective effects, with accompanying suppression of NF-κB and microglial activation, and reduction in the production of cytokines including TNF-α and IL-1β, and pro-inflammatory factors such as PGE(2). Our findings suggest that andrographolide may have therapeutic value in the treatment of stroke. [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: 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: K(v)11.1 channels are involved in regulating cellular excitability in various tissues including brain, heart and smooth muscle. In these tissues, at least two isoforms, K(v)11.1a and K(v)11.1b, with different kinetics, are expressed. K(v)11.1 activators are potential therapeutic agents, but their effects have only been tested on the K(v)11.1a isoform. In this study, the effects of two different K(v)11.1 activators, NS1643 and RPR260243, were characterized on K(v)11.1a and K(v)11.1b channels.Experimental Approach: K(v)11.1a and K(v)11.1b channels were expressed in Xenopus laevis oocytes, and currents were measured using two-electrode voltage clamp. I/V curves and channel kinetics were measured before and after application of 30 µM NS1643 or 10 µM RPR260243.Key Results: NS1643 increased steady-state currents through Kv11.1b several fold more than through K(v)11.1a channels, without affecting EC(50) values. NS1643 increased activation rates and decreased rates of inactivation, recovery from inactivation and deactivation for both channels. Except for activation, where effect of NS1643 was comparable, relative changes were greater for Kv11.1b than for K(v)11.1a. RPR260243 increased steady-state currents only through Kv11.1a channels, but slowed the process of deactivation for both channels primarily by decreasing time constant of slow deactivation. This effect was greater on K(v)11.1b than on K(v)11.1a. Effects of both compounds on heteromeric K(v)11.1a/K(v)11.1b channels were similar to those on K(v)11.1a.Conclusions and Implications: Both NS1643 and RPR260243 displayed differential effects on K(v)11.1a and K(v)11.1b channels, the effects being relatively more pronounced on K(v)11.1b channels. This affirms the importance of testing the effect of K(v)11.1 activators on different channel isoforms. [ABSTRACT FROM AUTHOR]

Background and Purpose: The 5-HT(4) receptor may be a target for antidepressant drugs. Here we have examined the effects of the dual antidepressant, venlafaxine, on 5-HT(4) receptor-mediated signalling events.Experimental Approach: The effects of 21 days treatment (p.o.) with high (40 mg·kg(-1)) and low (10 mg·kg(-1)) doses of venlafaxine, were evaluated at different levels of 5-HT(4) receptor-mediated neurotransmission by using in situ hybridization, receptor autoradiography, adenylate cyclase assays and electrophysiological recordings in rat brain. The selective noradrenaline reuptake inhibitor, reboxetine (10 mg·kg(-1), 21 days) was also evaluated on 5-HT(4) receptor density.Key Results: Treatment with a high dose (40 mg·kg(-1)) of venlafaxine did not alter 5-HT(4) mRNA expression, but decreased the density of 5-HT(4) receptors in caudate-putamen (% reduction = 26 ± 6), hippocampus (% reduction = 39 ± 7 and 39 ± 8 for CA1 and CA3 respectively) and substantia nigra (% reduction = 49 ± 5). Zacopride-stimulated adenylate cyclase activation was unaltered following low-dose treatment (10 mg·kg(-1)) while it was attenuated in rats treated with 40 mg·kg(-1) of venlafaxine (% reduction = 51 ± 2). Furthermore, the amplitude of population spike in pyramidal cells of CA1 of hippocampus induced by zacopride was significantly attenuated in rats receiving either dose of venlafaxine. Chronic reboxetine did not modify 5-HT(4) receptor density.Conclusions and Implications: Our data indicate a functional desensitization of 5-HT(4) receptors after chronic venlafaxine, similar to that observed after treatment with the classical selective inhibitors of 5-HT reuptake. [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: 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: 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: Increased glutamatergic innervation of the substantia nigra pars reticulata (SNpr) and pars compacta (SNpc) may contribute to the motor deficits and neurodegeneration, respectively, in Parkinson's disease (PD). This study aimed to establish whether activation of pre-synaptic group III metabotropic glutamate (mGlu) receptors reduced glutamate release in the SN, and provided symptomatic or neuroprotective relief in animal models of PD.Experimental Approach: Broad-spectrum group III mGlu receptor agonists, O-phospho-l-serine (l-SOP) and l-2-amino-4-phosphonobutyrate (l-AP4), were assessed for their ability to inhibit KCl-evoked [(3)H]-d-aspartate release in rat nigral prisms or inhibit KCl-evoked endogenous glutamate release in the SNpr in vivo using microdialysis. Reversal of akinesia in reserpine-treated rats was assessed following intranigral injection of l-SOP and l-AP4. Finally, the neuroprotective effect of 7 days' supra-nigral treatment with l-AP4 was examined in 6-hydroxydopamine (6-OHDA)-lesioned rats.Key Results: l-SOP and l-AP4 inhibited [(3)H]-d-aspartate release by 33 and 44% respectively. These effects were blocked by the selective group III mGlu antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG). l-SOP also reduced glutamate release in the SNpr in vivo by 48%. Injection of l-SOP and l-AP4 into the SNpr reversed reserpine-induced akinesia. Following administration above the SNpc, l-AP4 provided neurochemical, histological and functional protection against 6-OHDA lesion of the nigrostriatal tract. Pretreatment with CPPG inhibited these effects.Conclusions and Implications: These findings highlight group III mGlu receptors in the SN as potential targets for providing both symptomatic and neuroprotective relief in PD, and indicate that inhibition of glutamate release in the SN may underlie these effects. [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: The present study evaluated the role of CB(2) receptors in the regulation of depressive-like behaviours. Transgenic mice overexpressing the CB(2) receptor (CB2xP) were challenged with different types of acute and chronic experimental paradigms to evaluate their response in terms of depressive-like behaviours.Experimental Approach: Tail suspension test (TST), novelty-suppressed feeding test (NSFT) and unpredictable chronic mild stress tests (CMS) were carried out in CB2xP mice. Furthermore, acute and chronic antidepressant-like effects of the CB(2) receptor-antagonist AM630 were evaluated by means of the forced swimming test (FST) and CMS, respectively, in wild-type (WT) and CB2xP mice. CB(2) gene expression, brain-derived neurotrophic factor (BDNF) gene and protein expressions were studied in mice exposed to CMS by real-time PCR and immunohistochemistry, respectively.Key Results: Overexpression of CB(2) receptors resulted in decreased depressive-like behaviours in the TST and NSFT. CMS failed to alter the TST and sucrose consumption in CB2xP mice. In addition, no changes in BDNF gene and protein expression were observed in stressed CB2xP mice. Interestingly, acute administration of AM630 (1 and 3 mg x kg(-1), i.p.) exerted antidepressant-like effects on the FST in WT, but not in CB2xP mice. Chronic administration of AM630 for 4 weeks (1 mg x kg(-1); twice daily, i.p.) blocked the effects of CMS on TST, sucrose intake, CB(2) receptor gene, BDNF gene and protein expression in WT mice.Conclusion and Implications: Taken together, these results suggest that increased CB(2) receptor expression significantly reduced depressive-related behaviours and that the CB(2) receptor could be a new potential therapeutic target for depressive-related disorders. [ABSTRACT FROM AUTHOR]

Background and Purpose: During migraine, trigeminal nerves may release calcitonin gene-related peptide (CGRP), inducing cranial vasodilatation and central nociception; hence, trigeminal inhibition or blockade of craniovascular CGRP receptors may prevent this vasodilatation and abort migraine headache. Several preclinical studies have shown that glutamate receptor antagonists affect the pathophysiology of migraine. This study investigated whether antagonists of NMDA (ketamine and MK801), AMPA (GYKI52466) and kainate (LY466195) glutamate receptors affected dural vasodilatation induced by alpha-CGRP, capsaicin and periarterial electrical stimulation in rats, using intravital microscopy.Experimental Approach: Male Sprague-Dawley rats were anaesthetized and the overlying bone was thinned to visualize the dural artery. Then, vasodilator responses to exogenous (i.v. alpha-CGRP) and endogenous (released by i.v. capsaicin and periarterial electrical stimulation) CGRP were elicited in the absence or presence of the above antagonists.Key Results: alpha-CGRP, capsaicin and periarterial electrical stimulation increased dural artery diameter. Ketamine and MK801 inhibited the vasodilator responses to capsaicin and electrical stimulation, while only ketamine attenuated those to alpha-CGRP. In contrast, GYKI52466 only attenuated the vasodilatation to exogenous alpha-CGRP, while LY466195 did not affect the vasodilator responses to endogenous or exogenous CGRP.Conclusions and Implications: Although GYKI52466 has not been tested clinically, our data suggest that it would not inhibit migraine via vascular mechanisms. Similarly, the antimigraine efficacy of LY466195 seems unrelated to vascular CGRP-mediated pathways and/or receptors. In contrast, the cranial vascular effects of ketamine and MK801 may represent a therapeutic mechanism, although the same mechanism might contribute, peripherally, to cardiovascular side effects. [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: A new class of heterotricyclic glutamate analogues recently was generated by incorporating structural elements of two excitotoxic marine compounds, kainic acid and neodysiherbaine A. Rather than acting as convulsants, several of these 'IKM' compounds markedly depressed CNS activity in mice. Here, we characterize the pharmacological profile of the series with a focus on the most potent of these molecules, IKM-159.Experimental Approach: The pharmacological activity and specificity of IKM compounds were characterized using whole-cell patch clamp recording from neurons and heterologous receptor expression systems, in combination with radioligand binding techniques.Key Results: The majority of the IKM compounds tested reduced excitatory synaptic transmission in neuronal cultures, and IKM-159 inhibited synaptic currents from CA1 pyramidal neurons in hippocampal slices. IKM-159 inhibited glutamate-evoked whole-cell currents from recombinant GluA2- and GluA4-containing alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors most potently, whereas kainate and NMDA receptor currents were not reduced by IKM-159. Antagonism of steady-state currents was agonist concentration dependent, suggesting that its mechanism of action was competitive, although it paradoxically did not displace [(3)H]-AMPA from receptor binding sites. IKM-159 reduced spontaneous action potential firing in both cultured hippocampal neurons in control conditions and during hyperactive states in an in vitro model of status epilepticus.Conclusions and Implications: IKM-159 is an AMPA receptor-selective antagonist. IKM-159 and related nitrogen heterocycles represent structurally novel AMPA receptor antagonists with accessible synthetic pathways and potentially unique pharmacology, which could be of use in exploring the role of specific populations of receptors in neurophysiological and neuropathological processes. [ABSTRACT FROM AUTHOR]

Background and Purpose: Previous studies demonstrated that intraplaque haemorrhage increased the contents of cholesterol and oxidants in atherosclerotic plaques. The present study was aimed to test the hypothesis that enhanced expression of haem oxygenase-1 (HO-1) may stabilize vulnerable plaques.Experimental Approach: Intravascular ultrasound (IVUS) was performed to identify three similar abdominal aortic plaques in each of 58 fat-fed New Zealand rabbits after aortic balloon injury. With the guidance of IVUS, 50 microL autologous erythrocytes (RBC) or normal saline (NS) were injected from adventitia into two of the pre-selected plaques, respectively, whereas the third plaque served as a blank control. All rabbits were randomly divided into two groups, receiving intraperitoneal injection of haemin and saline respectively.Key Results: Compared with NS or control plaques, RBC plaques had more macrophage infiltration and lipid content, thinner plaque fibrous cap, and higher expression of inflammatory factors and incidence of plaque rupture. RBC plaques in the haemin group had about a 50% lower incidence of plaque rupture than those in the control group.Conclusions and Implications: Haem oxygenase-1 may eliminate haem or other oxidants, exert unexpected anti-oxidative and anti-inflammatory effects and serve as a promising approach to the direct inhibition of erythrocyte-induced plaque instability. [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: Oenanthotoxin (OETX), a polyacetylenic alcohol from plants of the genus Oenanthe, has recently been identified as potent inhibitor of GABA-evoked currents. However, the effects of OETX on the inhibitory postsynaptic currents (IPSCs), as well as the pharmacological mechanism(s) underlying its effects on GABA(A) receptors, remain unknown. The purpose of this study was to elucidate the mechanism underlying the inhibition of GABAergic currents by OETX.Experimental Approach: Effects of OETX on GABAergic currents were studied using the patch clamp technique on rat cultured hippocampal neurons. Miniature IPSCs (mIPSCs) were recorded in the whole-cell configuration, while the current responses were elicited by ultrafast GABA applications onto the excised patches.Key Results: OETX potently inhibited both mIPSCs and current responses, but its effect was much stronger on synaptic currents. Analysis of the effects of OETX on mIPSCs and evoked currents disclosed a complex mechanism: allosteric modulation of both GABA(A) receptor binding and gating properties and a non-competitive, probably open channel block mechanism. In particular, OETX reduced the binding rate and nearly abolished receptor desensitization. A combination of rapid clearance of synaptic GABA and OETX-induced slowing of binding kinetics is proposed to underlie the potent action of OETX on mIPSCs.Conclusions and Implications: OETX shows a complex blocking mechanism of GABA(A) receptors, and the impact of this toxin is more potent on mIPSCs than on currents evoked by exogenous GABA. Such effects on GABAergic currents are compatible with the convulsions and epileptic-like activity reported for OETX. [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: P-glycoprotein (Pgp) efflux assays are widely used to identify Pgp substrates. The kidney cell lines Madin-Darby canine kidney (MDCK)-II and LLC-PK1, transfected with human MDR1 (ABCB1) are used to provide recombinant models of drug transport. Endogenous transporters in these cells may contribute to the activities of recombinant transporters, so that drug transport in MDR1-transfected cells is often corrected for the transport obtained in parental (wildtype) cells. However, expression of endogenous transporters may vary between transfected and wildtype cells, so that this correction may cause erroneous data. Here, we have measured the expression of endogenous efflux transporters in transfected and wildtype MDCK-II or LLC cells and the consequences for Pgp-mediated drug transport.Experimental Approach: Using quantitative real-time RT-PCR, we determined the expression of endogenous Mdr1 mRNA and other efflux transporters in wildtype and MDR1-transfected MDCK-II and LLC cells. Transcellular transport was measured with the test substrate vinblastine.Key Results: In MDR1-transfected MDCK cells, expression of endogenous (canine) Mdr1 and Mrp2 (Abcc2) mRNA was markedly lower than in wildtype cells, whereas MDR1-transfected LLC cells exhibited comparable Mdr1 but strikingly higher Mrp2 mRNA levels than wildtype cells. As a consequence, transport of vinblastine by human Pgp in efflux experiments was markedly underestimated when transport in MDR1-transfected MDCK cells was corrected for transport obtained in wildtype cells. This problem did not occur in LLC cells.Conclusions and Implications: Differences in the expression of endogenous efflux transporters between transfected and wildtype MDCK cells provide a potential bias for in vitro studies on Pgp-mediated drug transport. [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: 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: There are two important properties of receptor-ligand interactions: affinity (the ability of the ligand to bind to the receptor) and efficacy (the ability of the receptor-ligand complex to induce a response). Ligands are classified as agonists or antagonists depending on whether or not they have efficacy. In theory, it is possible to develop selective agonists based on selective affinity, selective intrinsic efficacy or both. This study examined the affinity and intrinsic efficacy of 31 beta-adrenoceptor agonists at the three human beta-adrenoceptors to determine whether the current agonists are subtype selective because of affinity or intrinsic efficacy.Experimental Approach: Stable clonal CHO-K1 cell lines, transfected with either the human beta(1), beta(2) or beta(3)-adrenoceptor, were used, and whole-cell [(3)H]-CGP 12177 radioligand binding and [(3)H]-cAMP accumulation were measured.Key Results: Several agonists were found to be highly subtype selective because of selective affinity (e.g. salmeterol and formoterol, for the beta(2)-adrenoceptor over the beta(1) or beta(3)), while others (e.g. isoprenaline) had little affinity-selectivity. However, the intrinsic efficacy of salmeterol, formoterol and isoprenaline was similar across all three receptor subtypes. Other ligands (e.g. denopamine for beta(1); clenbuterol, AZ 40140d, salbutamol for beta(2)) were found to have subtype-selective intrinsic efficacy. Several ligands appeared to activate two agonist conformations of the beta(1)- and beta(3)-adrenoceptors.Conclusions and Implications: There are agonists with subtype selectivity based upon both selective affinity and selective intrinsic efficacy. Therefore, there is scope to develop better selective agonists based upon both selective affinity and selective intrinsic efficacy. [ABSTRACT FROM AUTHOR]

Background and Purpose: Lopinavir is extensively metabolized by cytochrome P450 3A (CYP3A) and is considered to be a substrate for the drug transporters ABCB1 (P-glycoprotein) and ABCC2 (MRP2). Here, we have assessed the individual and combined effects of CYP3A, ABCB1 and ABCC2 on the pharmacokinetics of lopinavir and the relative importance of intestinal and hepatic metabolism. We also evaluated whether ritonavir increases lopinavir oral bioavailability by inhibition of CYP3A, ABCB1 and/or ABCC2.Experimental Approach: Lopinavir transport was measured in Madin-Darby canine kidney cells expressing ABCB1 or ABCC2. Oral lopinavir kinetics (+/- ritonavir) was studied in mice with genetic deletions of Cyp3a, Abcb1a/b and/or Abcc2, or in transgenic mice expressing human CYP3A4 exclusively in the liver and/or intestine.Key Results: Lopinavir was transported by ABCB1 but not by ABCC2 in vitro. Lopinavir area under the plasma concentration - time curve (AUC)(oral) was increased in Abcb1a/b(-/-) mice (approximately ninefold vs. wild-type) but not in Abcc2(-/-) mice. Increased lopinavir AUC(oral) (>2000-fold) was observed in cytochrome P450 3A knockout (Cyp3a(-/-)) mice compared with wild-type mice. No difference in AUC(oral) between Cyp3a(-/-) and Cyp3a/Abcb1a/b/Abcc2(-/-) mice was observed. CYP3A4 activity in intestine or liver, separately, reduced lopinavir AUC(oral) (>100-fold), compared with Cyp3a(-/-) mice. Ritonavir markedly increased lopinavir AUC(oral) in all CYP3A-containing mouse strains.Conclusions and Implications: CYP3A was the major determinant of lopinavir pharmacokinetics, far more than Abcb1a/b. Both intestinal and hepatic CYP3A activity contributed importantly to low oral bioavailability of lopinavir. Ritonavir increased lopinavir bioavailability primarily by inhibiting CYP3A. Effects of Abcb1a/b were only detectable in the presence of CYP3A, suggesting saturation of Abcb1a/b in the absence of CYP3A activity. [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: 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: Caffeine exacerbates the hyperthermia associated with an acute exposure to 3,4 methylenedioxymethamphetamine (MDMA, 'Ecstasy') in rats. The present study investigated the mechanisms mediating this interaction.Experimental Approach: Adult male Sprague-Dawley rats were treated with caffeine (10 mg x kg(-1); i.p.) and MDMA (15 mg x kg(-1); i.p.) alone and in combination. Core body temperatures were monitored before and after drug administration.Key Results: Central catecholamine depletion blocked MDMA-induced hyperthermia and its exacerbation by caffeine. Caffeine provoked a hyperthermic response when the catecholamine releaser d-amphetamine (1 mg x kg(-1)) was combined with the 5-HT releaser D-fenfluramine (5 mg x kg(-1)) or the non-selective dopamine receptor agonist apomorphine (1 mg x kg(-1)) was combined with the 5-HT(2) receptor agonist DOI (2 mg x kg(-1)) but not following either agents alone. Pretreatment with the dopamine D(1) receptor antagonist Schering (SCH) 23390 (1 mg x kg(-1)), the 5-HT(2) receptor antagonist ketanserin (5 mg x kg(-1)) or alpha(1)-adreno- receptor antagonist prazosin (0.2 mg x kg(-1)) blocked MDMA-induced hyperthermia and its exacerbation by caffeine. Co-administration of a combination of MDMA with the PDE-4 inhibitor rolipram (0.025 mg x kg(-1)) and the adenosine A(1/2) receptor antagonist 9-chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-C]quinazolin-5-amine 15943 (10 mg x kg(-1)) or the A(2A) receptor antagonist SCH 58261 (2 mg x kg(-1)) but not the A(1) receptor antagonist DPCPX (10 mg x kg(-1)) exacerbated MDMA-induced hyperthermia.Conclusions and Implications: A mechanism comprising 5-HT and catecholamines is proposed to mediate MDMA-induced hyperthermia. A combination of adenosine A(2A) receptor antagonism and PDE inhibition can account for the exacerbation of MDMA-induced hyperthermia by caffeine. [ABSTRACT FROM AUTHOR]

Background and Purpose: KMUP-1 is known to increase cGMP, enhance endothelial nitric oxide synthase (eNOS) and suppress Rho kinase (ROCK) expression in smooth muscle. Here, we investigated the mechanism of action of KMUP-1 on acute and chronic pulmonary artery hypertension (PAH) in rats.Experimental Approach: We measured pulmonary vascular contractility, wall thickening, eNOS immunostaining, expressions of ROCK II, RhoA activation, myosin phosphatase target subunit 1 (MYPT1) phosphorylation, eNOS, soluble guanylyl cyclase (sGC), protein kinase G (PKG) and phosphodiesterase 5A (PDE-5A), blood oxygenation and cGMP/cAMP, and right ventricular hypertrophy (RVH) in rats.Key Results: In rings of intact pulmonary artery (PA), KMUP-1 relaxed the vasoconstriction induced by phenylephrine (10 microM) or the thromboxane A(2)-mimetic U46619 (0.5 microM). In endothelium-denuded PA rings, this relaxation was reduced. In acute PAH induced by U46619 (2.5 microg x kg(-1) x min(-1), 30 min), KMUP-1 relaxed vasoconstriction by enhancing levels of eNOS, sGC and PKG, suppressing those of PDE-5A, RhoA/ROCK II activation and MYPT1 phosphorylation, and restoring oxygenation in blood and cGMP/cAMP in plasma. Incubating smooth muscle cells from PA (PASMCs) with KMUP-1 inhibited thapsigargin-induced Ca(2+) efflux and angiotensin II-induced Ca(2+) influx. In chronic PAH model induced by monocrotaline, KMUP-1 increased eNOS and reduced RhoA/ROCK II activation/expression, PA wall thickening, eNOS immunostaining and RVH. KMUP-1 and sildenafil did not inhibit monocrotaline-induced PDE-5A expression.Conclusion and Implications: KMUP-1 decreased PAH by enhancing NO synthesis by eNOS, with consequent cGMP-dependent inhibition of RhoA/ROCK II and Ca(2+) desensitization in PASMCs. KMUP-1 has the potential to reduce vascular resistance, remodelling and RVH in PAH. [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]