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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]

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: 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]

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: 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: Certain 5-lipoxygenase (5-LO) inhibitors exhibit anti-carcinogenic activities against 5-LO overexpressing tumour types and cultured tumour cells. It has been proposed therefore that 5-LO products significantly contribute to tumour cell proliferation. To date, the relationship between the inhibitory mechanisms of 5-LO inhibitors, which vary widely, and tumour cell viability has not been evaluated. This study addresses the anti-proliferative and cytotoxic potency of a number of 5-LO inhibitors with different inhibitory mechanisms in 5-LO-positive and 5-LO-negative tumour cells.Experimental Approach: Cell viability was measured by the WST-1 assay; cell proliferation was assessed using the bromodeoxyuridine (BrdU) incorporation assay. Cell death was analysed by annexin V staining, Western blot analysis of PARP (poly ADP-ribose polymerase) cleavage and a cytotoxicity assay. 5-LO product formation was quantified by a 5-LO activity assay.Key Results: The common 5-LO inhibitors AA-861, Rev-5901 and MK-886 induced cytotoxic and anti-proliferative effects in 5-LO-positive Capan-2 pancreatic cancer cells; BWA4C and CJ-13,610 only caused anti-proliferative effects, while zileuton failed to impair cell viability. Moreover, the concentrations of the 5-LO inhibitors required to induce anti-proliferation and cytotoxicity highly exceeded those for suppression of 5-LO. Supplementation with mitogenic 5-LO products failed to protect Capan-2 cells from the effects of 5-LO inhibitors. Finally, the cytotoxic and anti-proliferative 5-LO inhibitors also potently reduced the viability of 5-LO-deficient tumour cell lines (HeLa, Panc-1 and U937).Conclusions and Implications: Certain 5-LO inhibitors cause cytotoxic and anti-proliferative effects independently of suppression of 5-LO activity. Thus, the role of 5-LO overexpression in tumour cell viability remains unclear and requires further elucidation. [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: 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: 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: 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: Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) pathway is frequently encountered in several human cancers including multiple myeloma (MM). Thus, agents that suppress STAT3 phosphorylation have a potential for treatment of MM. In the present report, we investigated whether thymoquinone (TQ), the main component isolated from the medicinal plant Nigella sativa, modulated the STAT3 signalling pathway in MM cells.Experimental Approach: The effect of TQ on both constitutive and IL-6-induced STAT3 activation, associated protein kinases, STAT3-regulated gene products involved in proliferation, survival and angiogenesis, cellular proliferation and apoptosis in MM cells, was investigated.Key Results: We found that TQ inhibited both constitutive and IL-6-inducible STAT3 phosphorylation which correlated with the inhibition of c-Src and JAK2 activation. Vanadate reversed the TQ-induced down-regulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that TQ can induce the expression of Src homology-2 phosphatase 2 that correlated with suppression of STAT3 activation. TQ also down-regulated the expression of STAT3-regulated gene products, such as cyclin D1, Bcl-2, Bcl-xL, survivin, Mcl-1 and vascular endothelial growth factor. Finally, TQ induced the accumulation of cells in sub-G1 phase, inhibited proliferation and induced apoptosis, as indicated by poly ADP ribose polymerase cleavage. TQ also significantly potentiated the apoptotic effects of thalidomide and bortezomib in MM cells.Conclusions and Implications: Our study has identified STAT3 signalling as a target of TQ and has thus raised its potential application in the prevention and treatment of MM and other cancers. [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: F15599, a novel 5-hydroxytryptamine (5-HT)(1A) receptor agonist with 1000-fold selectivity for 5-HT compared with other monoamine receptors, shows antidepressant and procognitive activity at very low doses in animal models. We examined the in vivo activity of F15599 at somatodendritic autoreceptors and postsynaptic 5-HT(1A) heteroreceptors.Experimental Approach: In vivo single unit and local field potential recordings and microdialysis in the rat.Key Results: F15599 increased the discharge rate of pyramidal neurones in medial prefrontal cortex (mPFC) from 0.2 microg x kg(-1) i.v and reduced that of dorsal raphe 5-hydroxytryptaminergic neurones at doses >10-fold higher (minimal effective dose 8.2 microg x kg(-1) i.v.). Both effects were reversed by the 5-HT(1A) antagonist (+/-)WAY100635. F15599 did not alter low frequency oscillations (approximately 1 Hz) in mPFC. In microdialysis studies, F15599 increased dopamine output in mPFC (an effect dependent on the activation of postsynaptic 5-HT(1A) receptors) with an ED(50) of 30 microg x kg(-1) i.p., whereas it reduced hippocampal 5-HT release (an effect dependent exclusively on 5-HT(1A) autoreceptor activation) with an ED(50) of 240 microg x kg(-1) i.p. Likewise, application of F15599 by reverse dialysis in mPFC increased dopamine output in a concentration-dependent manner. All neurochemical responses to F15599 were prevented by administration of (+/-)WAY100635.Conclusions and Implications: These results indicate that systemic administration of F15599 preferentially activates postsynaptic 5-HT(1A) receptors in PFC rather than somatodendritic 5-HT(1A) autoreceptors. This regional selectivity distinguishes F15599 from previously developed 5-HT(1A) receptor agonists, which preferentially activate somatodendritic 5-HT(1A) autoreceptors, suggesting that F15599 may be particularly useful in the treatment of depression and of cognitive deficits in schizophrenia. [ABSTRACT FROM AUTHOR]

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

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

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

Background and Purpose: 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: The aim of this study was to determine whether the apelin/APJ system is altered in human cardiovascular disease by investigating whether the expression of apelin or its receptor is altered at the protein level.Experimental Approach: Radioligand binding studies were used to determine apelin receptor density in human cardiac tissues. Apelin peptide levels in cardiovascular tissues were determined by radioimmunoassay. In vitro pharmacology was used to assess vasoactive properties of apelin in human coronary artery. Localization of apelin and its receptor in coronary artery was determined using immunohistochemistry.Key Results: Apelin receptor density was significantly decreased in left ventricle from patients with dilated cardiomyopathy or ischaemic heart disease compared with controls, but apelin peptide levels remained unchanged. Apelin was up-regulated in human atherosclerotic coronary artery and this additional peptide localized to the plaque, colocalizing with markers for macrophages and smooth muscle cells. Apelin potently constricted human coronary artery.Conclusions and Implications: We have detected changes in the apelin/APJ system in human diseased cardiac and vascular tissue. The decrease in receptor density in heart failure may limit the positive inotropic actions of apelin, contributing to contractile dysfunction. The contribution of the increased apelin levels in atherosclerotic coronary artery to disease progression remains to be determined. These data suggest a potential role for the apelin/APJ system in human cardiovascular disease. [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: Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration.Experimental Approach: We used the human endothelial cell line EA.hy926 to investigate the effect of nitric oxide (NO) donors on SK-1 expression, and on cell migration and tube formation.Key Results: We showed that exposure of EA.hy926 cells to Deta-NO (125-1000 microM) resulted in a time- and concentration-dependent up-regulation of SK-1 mRNA and protein expression, and activity with a first significant effect at 250 microM of Deta-NO. The increased SK-1 mRNA expression resulted from an enhanced SK-1 promoter activity. A similar effect was also seen with various other NO donors. In mechanistic terms, the NO-triggered effect occurred independently of cGMP, but involved the classical mitogen-activated protein kinase cascade because the MEK inhibitor U0126 abolished the NO-induced SK-1 expression. The effect of NO was also markedly reduced by the thiol-reducing agent N-acetylcysteine, suggesting a redox-dependent mechanism. Functionally, Deta-NO triggered an increase in the migration of endothelial cells in an adapted Boyden chamber assay, and also increased endothelial tube formation in a Matrigel assay. These responses were both abolished in cells depleted of SK-1.Conclusions and Implications: These data show that NO donors up-regulate specifically SK-1 expression and activity in human endothelial cells, and SK-1 in turn critically contributes to the migratory capability and tube formation of endothelial cells. Thus, SK-1 may be considered an attractive novel target to interfere with pathological processes involving angiogenesis. [ABSTRACT FROM AUTHOR]

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

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

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

Background and Purpose: 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: beta-Arrestins are critical scaffold proteins that shape spatiotemporal signalling from seven transmembrane domain receptors (7TMRs). Here, we study the association between neuropeptide Y (NPY) receptors and beta-arrestin2, using bimolecular fluorescence complementation (BiFC) to directly report underlying protein-protein interactions.Experimental Approach: Y1 receptors were tagged with a C-terminal fragment, Yc, of yellow fluorescent protein (YFP), and beta-arrestin2 fused with the complementary N-terminal fragment, Yn. After Y receptor-beta-arrestin association, YFP fragment refolding to regenerate fluorescence (BiFC) was examined by confocal microscopy in transfected HEK293 cells. Y receptor/beta-arrestin2 BiFC responses were also quantified by automated imaging and granularity analysis.Key Results: NPY stimulation promoted association between Y1-Yc and beta-arrestin2-Yn, and the specific development of BiFC in intracellular compartments, eliminated when using non-interacting receptor and arrestin mutants. Responses developed irreversibly and were slower than for downstream Y1 receptor-YFP internalization, a consequence of delayed maturation and stability of complemented YFP. However, beta-arrestin2 BiFC measurements delivered appropriate ligand pharmacology for both Y1 and Y2 receptors, and demonstrated higher affinity of Y1 compared to Y2 receptors for beta-arrestin2. Receptor mutagenesis combined with beta-arrestin2 BiFC revealed that alternative arrangements of Ser/Thr residues in the Y1 receptor C tail could support beta-arrestin2 association, and that Y2 receptor-beta-arrestin2 interaction was enhanced by the intracellular loop mutation H155P.Conclusions and Implications: The BiFC approach quantifies Y receptor ligand pharmacology focused on the beta-arrestin2 pathway, and provides insight into mechanisms of beta-arrestin2 recruitment by activated and phosphorylated 7TMRs, at the level of protein-protein interaction. [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: The major drug-metabolizing enzymes for the oxidation of oxycodone are CYP2D6 and CYP3A. A high interindividual variability in the activity of these enzymes because of genetic polymorphisms and/or drug-drug interactions is well established. The possible role of an active metabolite in the pharmacodynamics of oxycodone has been questioned and the importance of CYP3A-mediated effects on the pharmacokinetics and pharmacodynamics of oxycodone has been poorly explored.Experimental Approach: We conducted a randomized crossover (five arms) double-blind placebo-controlled study in 10 healthy volunteers genotyped for CYP2D6. Oral oxycodone (0.2 mg x kg(-1)) was given alone or after inhibition of CYP2D6 (with quinidine) and/or of CYP3A (with ketoconazole). Experimental pain (cold pressor test, electrical stimulation, thermode), pupil size, psychomotor effects and toxicity were assessed.Key Results: CYP2D6 activity was correlated with oxycodone experimental pain assessment. CYP2D6 ultra-rapid metabolizers experienced increased pharmacodynamic effects, whereas cold pressor test and pupil size were unchanged in CYP2D6 poor metabolizers, relative to extensive metabolizers. CYP2D6 blockade reduced subjective pain threshold (SPT) for oxycodone by 30% and the response was similar to placebo. CYP3A4 blockade had a major effect on all pharmacodynamic assessments and SPT increased by 15%. Oxymorphone C(max) was correlated with SPT assessment (rho(S)= 0.7) and the only independent positive predictor of SPT. Side-effects were observed after CYP3A4 blockade and/or in CYP2D6 ultra-rapid metabolizers.Conclusions and Implications: The modulation of CYP2D6 and CYP3A activities had clear effects on oxycodone pharmacodynamics and these effects were dependent on CYP2D6 genetic polymorphism. [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: There is high interindividual variability in the activity of drug-metabolizing enzymes catalysing the oxidation of oxycodone [cytochrome P450 (CYP) 2D6 and 3A], due to genetic polymorphisms and/or drug-drug interactions. The effects of CYP2D6 and/or CYP3A activity modulation on the pharmacokinetics of oxycodone remains poorly explored.Experimental Approach: A randomized crossover double-blind placebo-controlled study was performed with 10 healthy volunteers genotyped for CYP2D6 [six extensive (EM), two deficient (PM/IM) and two ultrarapid metabolizers (UM)]. The volunteers randomly received on five different occasions: oxycodone 0.2 mg x kg(-1) and placebo; oxycodone and quinidine (CYP2D6 inhibitor); oxycodone and ketoconazole (CYP3A inhibitor); oxycodone and quinidine+ketoconazole; placebo. Blood samples for plasma concentrations of oxycodone and metabolites (oxymorphone, noroxycodone and noroxymorphone) were collected for 24 h after dosing. Phenotyping for CYP2D6 (with dextromethorphan) and CYP3A (with midazolam) were assessed at each session.Key Results: CYP2D6 activity was correlated with oxymorphone and noroxymorphone AUCs and C(max) (-0.71 < Spearman correlation coefficient rhos < -0.92). Oxymorphone C(max) was 62% and 75% lower in PM than EM and UM. Noroxymorphone C(max) reduction was even more pronounced (90%). In UM, oxymorphone and noroxymorphone concentrations increased whereas noroxycodone exposure was halved. Blocking CYP2D6 (with quinidine) reduced oxymorphone and noroxymorphone C(max) by 40% and 80%, and increased noroxycodone AUC(infinity) by 70%. Blocking CYP3A4 (with ketoconazole) tripled oxymorphone AUC(infinity) and reduced noroxycodone and noroxymorphone AUCs by 80%. Shunting to CYP2D6 pathway was observed after CYP3A4 inhibition.Conclusions and Implications: Drug-drug interactions via CYP2D6 and CYP3A affected oxycodone pharmacokinetics and its magnitude depended on CYP2D6 genotype. [ABSTRACT FROM AUTHOR]

Background and Purpose: Natural and synthetic cannabinoids (CBs) induce deleterious or beneficial actions on neuronal survival. The Fas-associated protein with death domain (FADD) promotes apoptosis, and its phosphorylated form (p-FADD) mediates non-apoptotic actions. The regulation of Fas/FADD, mitochondrial apoptotic proteins and other pathways by CB receptors was investigated in the mouse brain.Experimental Approach: Wild-type, CB(1) and CB(2) receptor knock-out (KO) mice were used to assess differences in receptor genotypes. CD1 mice were used to evaluate the effects of CB drugs on canonical apoptotic pathways and associated signalling systems. Target proteins were quantified by Western blot analysis.Key Results: In brain regions of CB(1) receptor KO mice, Fas/FADD was reduced, but p-Ser191 FADD and the p-FADD/FADD ratio were increased. In CB(2) receptor KO mice, Fas/FADD was increased, but the p-FADD/FADD ratio was not modified. In mutant mice, cleavage of poly(ADP-ribose)-polymerase (PARP) did not indicate alterations in brain cell death. In CD1 mice, acute WIN55212-2 (CB(1) receptor agonist), but not JWH133 (CB(2) receptor agonist), inversely modulated brain FADD and p-FADD. Chronic WIN55212-2 induced FADD down-regulation and p-FADD up-regulation. Acute and chronic WIN55212-2 did not alter mitochondrial proteins or PARP cleavage. Acute, but not chronic, WIN55212-2 stimulated activation of anti-apoptotic (ERK, Akt) and pro-apoptotic (JNK, p38 kinase) pathways.Conclusions and Implications: CB(1) receptors appear to exert a modest tonic activation of Fas/FADD complexes in brain. However, chronic CB(1) receptor stimulation decreased pro-apoptotic FADD and increased non-apoptotic p-FADD. The multifunctional protein FADD could participate in the mechanisms of neuroprotection induced by CBs. [ABSTRACT FROM AUTHOR]

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

Background and Purpose: 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: 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]