bluuebunny/crossref_metadata_2025 huggingface dataset:
QT analysis: a complex answer to a ‘simple’ problem
Authors: Li Lang , Desai Mehul , Desta Zeruesenay , Flockhart David
Published: August 2004

Prolongation of the QT interval on a surface electrocardiogram is a biomarker for a potentially life‐threatening arrhythmia. It is used by drug developers and regulatory agencies as a measure of drug safety. Heart rate or RR interval (the inverse of heart rate) correction of the QT interval is necessary because of the QT interval shortening that accompanies physiologic decreases in the RR interval. When a drug alters the RR interval, it is important to distinguish a QT change that is due to a drug effect versus an artefact of a heart rate change. A two‐step off‐drug subject‐specific QT correction analysis is discussed. At the first step, a linear mixed model based only on the placebo (off‐drug) RR/QT data produces a correction coefficient that can be applied to a generic formula, and QT intervals are corrected for heart rate on both placebo and treatment period data using that formula. At step two, the heart rate corrected QT interval (QTc) is then compared between placebo and treatment groups at a pre‐specified heart rate (usually 60 bpm) based on another linear mixed model. This two‐step QT analysis implicitly assumes the slope of log(QT) versus log(RR) is unchanged by drug. Practically, it is important to understand how much this assumption can bias the QT prolongation estimates if it is not valid. We propose a one‐step off–on‐drug subject‐specific QT correction analysis that would pool placebo and treatment period RR/QT data and derive different subject specific coefficients for the treatment and placebo data based on a linear mixed model, which can avoid the unchanged slope assumption. It is also a known unbiased and the most efficient method. The applications of both methods are demonstrated through the QT analysis of haloperidol, a neuroleptic known to prolong QTc. Both theoretical and empirical results show that, although the two‐step off‐drug QT correction analysis is biased, the bias is small in the case of haloperidol (0.1–0.2ms). The two‐step off‐drug QT correction analysis is shown to be almost as efficient as our one‐step off‐ and on‐drug QT analysis. Copyright © 2004 John Wiley & Sons, Ltd.
Practical recommendations to minimize software capability evaluation risks
Authors: Saiedian Hossein
Published: August 2004

Many organizations, and, in particular, the US Department of Defense, find the Capability Maturity Model's (CMM) software capability evaluation (SCE) to be a useful tool for evaluating the maturity and capability of a number of competing software contractors and thus selecting a prospective contractor. The evaluation commonly takes place during the acquisition phase of a software project in order to focus on identifying and reducing risks associated with quality, scheduling, and budget. When an unbiased evaluation is performed, the information revealed can help determine potential risks in selecting a particular candidate contractor. However, there are many ways to manipulate the outcome of the SCE by the evaluation team or the candidate contractor. It is important that the potential flaws and shortcomings in the SCE be identified and eliminated to ensure a viable evaluation. This article briefly discusses the concerns associated with and flaws in conducting SCEs and proposes a number of practical recommendations to eliminate or substantially reduce them. Copyright © 2004 John Wiley & Sons, Ltd.
Software quality improvement in France telecom research center
Authors: Moreau Bernard , Lassudrie Claire , Nicolas Bertrand , I'Homme Odile , d'Anterroches Cécile , Gall Gilbert Le
Published: August 2004

The needs and business goals of France Telecom R&D are centered around achieving enhanced customer satisfaction and greater competitiveness. These key management concerns become drivers that initiate software process improvement with goals of higher software quality, lower development and maintenance costs, shorter time to market, and increased predictability and control of software products and processes.

Software process improvement is considered as a continuous process; France Telecom R&D moves continually around a stabilization cycle, Standardize Do Check Act (SDCA) and an improvement cycle, Plan Do Check Act(PDCA). Within these cycles, in a series of steps of specific improvement, actions such as introducing new or improved practices into software processes are accomplished.

The software process model is based on the ISO/IEC 12207 standard ‘Software Life‐Cycle Processes’, and the assessment method uses the ISO/IEC 15504 standard ‘Software process Assessment’. Some new processes are defined and used in the area of software engineering. Copyright © 2004 John Wiley & Sons, Ltd.
Towards software excellence—informal self‐assessment for software developers
Authors: Daily K. , Dresner D.
Published: August 2004

‘Towards Software Excellence’ (TSE) is a web‐based facility established by the UK's National Computing Centre (NCC), a not‐for‐profit organisation dedicated to helping IT users and providers implement effective information technology. TSE provides software developers with a simple and easy‐to‐use means of self‐assessing their development processes. Its users can understand the practices expected by TSE, identify the ‘strengths and weaknesses’ of their current practice, and then use this knowledge to improve.

TSE is especially suited to small‐ and medium‐sized enterprises (SMEs) and development teams or groups within larger organisations. It can provide a first step towards more formal Capability Maturity Model (CMM) or SPICE‐based assessments. TSE's definition of best practice is based on the ISO15504 standard. Once registered, users can log on to the website as many times as they wish to complete their assessment—they don't have to enter all the information at once or cover all processes and practices. Explanations of TSE's best practices can be examined and a ‘gap analysis’ report for the user's assessment can be produced.

With financial support from the UK government and promotional support from trade and professional associations, over 1500 users have performed TSE assessments during 2002. As well as helping its users to identify process improvements, the TSE initiative has started to create overall profiles of the strengths and weaknesses of the practices declared by its users.

Analysis of the initial set of TSE assessments has identified the generally best and worst performing processes and practices of its users and also those practices that have generated the most and least interest. For example, good performing topics include problem resolution and project management, while low performing topics include verification and measurement. TSE users are most interested in assessing the performance of their development and acquisition practices and least interested in assessing their improvement and legal practices. As use builds up, it is hoped that regular comparison of overall strengths and weaknesses of development practice declared by users will become possible, and the general improvement of process capability for software development can be tracked. Copyright © 2004 John Wiley & Sons, Ltd.
Evaluating the SPICE rating scale with regard to the internal consistency of capability measures
Authors: Jung Ho‐Won , Hunter Robin
Published: August 2004

The international SPICE (Software Process Improvement and Capability dEtermination) project mounted a series of trials to validate the emerging international standard ISO/IEC 15504 against the goals and requirements defined at the start of the project and to verify the consistency and usability of its component parts. This study aims to evaluate the SPICE rating scale with regard to the internal consistency (reliability) of capability measures. For this purpose, this study analyzed 691 process instances assessed during the Phase 2 SPICE Trials (between September 1996 and June 1998). Our findings indicate that the current four‐category rating scale gave a higher internal consistency of capability level than two‐ or three‐category rating scales. Since the current version of ISO/IEC 15504, due to become an International Standard scheduled for publication (Part 2) in 2003, still uses the four‐category rating scale, the results in this article are still valid for the current version. Copyright © 2004 John Wiley & Sons, Ltd.
Genetics and variability in opioid response
Authors: Stamer Ulrike M. , Bayerer Bettina , Stüber Frank
Published: August 2004

The human genome project has revealed data on genomic variation which may influence the pharmacological responses. In pain therapy, the genetic background influencing the efficacy of opioid therapy is of special interest. Screening for variations in expression of drug metabolizing enzymes has been suggested as a potential tool for improving patient therapy. CYP2D6 genetic variability is supposed to be a major factor of adverse drug reaction, possibly influencing hospital stay and total costs. Further candidate genes involved in pain perception, pain processing and pain management like opioid receptors, transporters and other targets of pharmacotherapy are under investigation. Aspects of genetic differences influencing efficacy, side effects and adverse outcome of pharmacotherapy will be of importance for future pain management.
Effects of the Antiandrogen Flutamide on the Expression of Protein Kinase C Isoenzymes in LNCaP and PC3 Human Prostate Cancer Cells
Authors: Montalvo Leire , Carmena María J. , Bolaños Oscar , Rodríguez-Henche Nieves , Sánchez-Chapado Manuel , Prieto Juan C.
Published: August 2004

Flutamide is a nonsteroidal antiandrogen that is frequently used for total androgen blockage in the treatment of advanced prostate cancer. We investigated the effect of this antiandrogen on the expression of protein kinase C (PKC) isoenzymes (α, β1, ∊, ζ) that are involved in cell growth, apoptosis and neoplastic transformation. Androgen-dependent (LNCaP) and independent (PC3) human prostate cancer cells were cultured in a medium that contained fetal bovine serum (FBS) or charcoal-stripped serum (CSS) and treated with 10 μM flutamide. The expression of PKC isoenzymes and the androgen receptor (AR) were analyzed by Western blot and RT-PCR, respectively. Serum steroids differentially regulate the expression of PKC isoenzymes in LNCaP and PC3 cells. Flutamide up-regulated the expression of α, β1 and ζ, but not ∊, PKC isoenzymes in CSS-LNCaP cells. These results were not homogeneously reproduced in the presence of androgens. We observed an opposite effect of flutamide, compared to CSS, on PKCβ1 isoform expression in CSS-LNCaP suggesting that this antiandrogen exerts an agonistic effect. In PC3 cells flutamide potentiated the expression of the four PKC isoenzymes in almost all conditions tested (FBS- and CSS-cultured cells). Such effect of flutamide in PC3 cells is independent of AR since no expression of AR was detected. These results provide new evidence on antagonistic/agonistic responses of prostate cancer cells to antiandrogen drugs that are widely used in therapy and show that flutamide can elicit responses in prostate cancer cells that do not express AR.
Heparin normalizes allergen‐induced nitric oxide deficiency and airway hyperresponsiveness
Authors: Maarsingh Harm , De Boer Jacob , Kauffman Henk F , Zaagsma Johan , Meurs Herman
Published: August 2004

It has been established that polycations cause airway hyperresponsiveness (AHR) to methacholine by inducing a deficiency of constitutive nitric oxide synthase (cNOS)‐derived bronchodilating nitric oxide (NO). Since a deficiency of cNOS‐derived NO also contributes to allergen‐induced AHR after the early asthmatic reaction (EAR) and since this AHR is associated with the release of polycationic proteins from infiltrated eosinophils in the airways, we hypothesized that endogenous polycations underlie or at least contribute to the allergen‐induced NO deficiency and AHR.

Using a guinea‐pig model of allergic asthma, we addressed this hypothesis by examining the effect of the polyanion heparin, acting as a polycation antagonist, on the responsiveness to methacholine of isolated perfused tracheae from unchallenged control animals and from animals 6 h after ovalbumin challenge, that is, after the EAR.

A 2.0‐fold AHR (P<0.001) to intraluminal administration of methacholine was observed in airways from allergen‐challenged animals compared to control. Incubation of these airways with 250 U ml−1 heparin completely normalized the observed hyperresponsiveness (P<0.001), whereas the responsiveness to methacholine of airways from unchallenged control animals was not affected.

The effect of heparin on airways from allergen‐challenged guinea‐pigs was dose‐dependently (0.1 and 1.0 mM) reversed by the NOS inhibitor L‐NAME (P<0.01).

These results indicate that endogenous (presumably eosinophil‐derived) polycations are involved in allergen‐induced NO deficiency and AHR after the EAR, probably by inhibition of L‐arginine transport.

British Journal of Pharmacology (2004) 142, 1293–1299. doi:10.1038/sj.bjp.0705848
Effects of dietary lectins on ion transport in epithelia
Authors: Kunzelmann Karl , Sun J , Schreiber R , König Jens
Published: August 2004

Phytohemagglutinins are widely distributed in common food items. They constitute a heterogeneous group of proteins, which are often resistant to proteolysis in the gastrointestinal tract. Upon binding to the luminal membrane of intestinal cells, they can interfere with digestive, protective or secretory functions of the intestine.

Phytohemagglutinins present in red kidney beans and jackbeans have been shown to induce diarrhea and hypersecretion in human airways, but the underlying mechanisms remain obscure. We examined how agglutinins from wheat germ (WGA), soy bean (SBA), red kidney beans (Pha‐E, Pha‐L), and jackbeans (Con‐A) affect ion transport in mouse airways and large intestine using Ussing chamber techniques.

We found that Pha‐E, Pha‐L, and Con‐A but not WGA and SBA inhibit electrogenic Na+ absorption dose dependently in both colon and trachea. The inhibitory effects of Con‐A on Na+ absorption were suppressed by the sugar mannose, by inhibition of phospholipase C (PLC) and protein kinase C (PKC).

Thus, nutritional phytohemagglutinins block salt absorption in a PLC‐ and PKC‐dependent manner, probably by inhibition of the epithelial Na+ channel (ENaC). This effect may be therapeutically useful in patients suffering from cystic fibrosis.

British Journal of Pharmacology (2004) 142, 1219–1226. doi:10.1038/sj.bjp.0705857
Indomethacin potentiates acetylcholine‐induced vasodilation by increasing free radical production
Authors: De Angelis Antonella , Rinaldi Barbara , Capuano Annalisa , Rossi Francesco , Filippelli Amelia
Published: August 2004

We studied the effects of indomethacin on endothelium‐dependent and ‐independent vascular relaxation in rat thoracic aortic rings and its role in superoxide anion (O2−) production.

We measured isometric force changes in response to acetylcholine (Ach, 1 nM–0.1 mM), sodium nitroprusside (SNP, 0.1 nM–0.1 μM; a nitric oxide (NO) donor) and cromakalim (1 nM–0.1 mM; a KATP‐channel opener) in aorta rings contracted with norepinephrine (NE, 0.1 μM). Indomethacin (10 μM; 20 min) significantly increased Ach‐induced vasodilation (EC50 decreased from 8.99 μM to 16 nM). The free radical scavengers superoxide dismutase and 4‐hydroxy‐2,2,6,6‐tetramethylpiperidine‐N‐oxyl completely reverted these effects. Indomethacin did not affect SNP‐ or cromakalim‐induced vasodilation.

Neither acetylsalicylic acid (ASA, 5–100 μM; 15 min) nor ketoprofen (1−100 μM; 15 min) affected Ach, SNP and cromakalim concentration–response curves.

Incubation of the aorta with Ach (1 μM) rapidly and markedly increased intracellular NO fluorescence in the aorta endothelium. Indomethacin did not affect Ach‐induced NO production.

We measured intracellular O2− in the aorta endothelium with dihydroethidium (DHE) dye. Indomethacin significantly increased O2− fluorescence versus controls. Neither ASA nor ketoprofen affected O2− fluorescence.

Nitrotyrosine staining was increased in indomethacin‐treated aorta sections exposed to Ach, which indicates endogenous formation of peroxynitrite. It was low in aorta sections exposed to Ach alone or with ASA or ketoprofen.

We cannot judge if indomethacin‐induced endothelium‐dependent vasodilation damages or protects the cardiovascular system. Here, we show that indomethacin acts on the cardiovascular system regardless of cyclooxygenase inhibition.

British Journal of Pharmacology (2004) 142, 1233–1240. doi:10.1038/sj.bjp.0705877
Methyllycaconitine, α‐bungarotoxin and (+)‐tubocurarine block fast ATP‐gated currents in rat dorsal root ganglion cells
Authors: Lalo Ulyana , Pankratov Yuri , Krishtal Oleg , Alan North R
Published: August 2004

The effects of nicotinic acetylcholine receptor antagonists were studied on currents evoked by application of ATP to rat isolated dorsal root ganglion cells, and human embryonic kidney 293 cells expressing rat P2X3 and P2X2/3 receptors.

The rapidly desensitising (within 100 ms) current in dorsal root ganglion cells was inhibited by methyllycaconitine, α‐bungarotoxin and (+)‐tubocurarine (concentrations giving half‐maximal inhibition were approximately 40, 60 and 800 nM, respectively), but not by hexamethonium (100 μM) or mecamylamine (100 μM). The sustained (>250 ms) current in dorsal root ganglion cells was inhibited by (+)‐tubocurarine (80% by 10 μM), but not by methyllycaconitine (200 nM), α‐bungarotoxin (200 nM), mecamylamine (100 μM) or hexamethonium (100 μM).

Rapidly desensitising currents evoked by α,βmethylene‐ATP in human embryonic kidney cells expressing P2X3 receptors were inhibited by methyllycaconitine and α‐bungarotoxin, at concentrations similar to those effective in dorsal root ganglion cells.

The results indicate that some nicotinic acetylcholine receptor antagonists are potent blockers of P2X receptors on neurons, particularly the homo‐oligomeric P2X3 receptor. This finding suggests that these drugs should be used with care to discriminate between P2X and neuronal acetylcholine receptor types.

British Journal of Pharmacology (2004) 142, 1227–1232. doi:10.1038/sj.bjp.0705878
Ranolazine: Ion‐channel‐blocking actions and in vivo electrophysiological effects
Authors: Schram Gernot , Zhang Liming , Derakhchan Katayoun , Ehrlich Joachim R , Belardinelli Luiz , Nattel Stanley
Published: August 2004

Ranolazine is a novel anti‐ischemic drug that prolongs the QT interval. To evaluate the potential mechanisms and consequences, we studied: (i) Ranolazine's effects on HERG and IsK currents in Xenopus oocytes with two‐electrode voltage clamp; (ii) effects of ranolazine, compared to D‐sotalol, on effective refractory period (ERP), QT interval and ventricular rhythm in a dog model of acquired long QT syndrome; and (iii) effects on selected native currents in canine atrial myocytes with whole‐cell patch‐clamp technique.

Ranolazine inhibited HERG and IsK currents with different potencies. HERG was inhibited with an IC50 of 106 μmol l−1, whereas the IC50 for IsK was 1.7 mmol l−1.

D‐Sotalol caused reverse use‐dependent ERP and QT interval prolongation, whereas ranolazine produced modest, nonsignificant increases that plateaued at submaximal doses. Neither drug affected QRS duration. D‐Sotalol had clear proarrhythmic effects, with all D‐sotalol‐treated dogs developing torsades de pointes (TdP) ventricular tachyarrhythmias, of which they ultimately died. In contrast, ranolazine did not generate TdP.

Effects on IKr and IKs were similar to those on HERG and IsK. Ranolazine blocked ICa with an IC50 of ∼300 μmol l−1. INa was unaffected.

We conclude that ranolazine inhibits IKr by blocking HERG currents, inhibits ICa at slightly larger concentrations, and has modest and self‐limited effects on the QT interval. Unlike D‐sotalol, ranolazine does not cause TdP in a dog model. The greater safety of ranolazine may be due to its ability to inhibit ICa at concentrations only slightly larger than those that inhibit IKr, thus producing offsetting effects on repolarization.

British Journal of Pharmacology (2004) 142, 1300–1308. doi:10.1038/sj.bjp.0705879
Evidence for gender differences in electrophysiological properties of canine Purkinje fibres
Authors: Abi‐Gerges Najah , Small Ben G , Lawrence Chris L , Hammond Tim G , Valentin Jean‐Pierre , Pollard Chris E
Published: August 2004

Women are more prone to develop torsades de pointes, a rare life‐threatening polymorphic ventricular tachycardia, than are men during administration of medicines that have the potential to block IKr (rapid delayed rectifier cardiac K+ current) and to prolong the QT interval. Blockade of IKr, hypokalaemia and extreme bradycardia were used to evaluate whether there are gender differences in cardiac repolarisation in canine Purkinje fibres (PFs). Microelectrode techniques were employed to measure action potential (AP) parameters in PFs from adult female and male dogs.

Under control conditions, fibres from female animals in normal or low K+ conditions exhibited significantly longer AP durations at 50% (APD50) and 90% (APD90) of repolarisation as compared with APDs of fibres from male animals.

Gender‐related difference to rate adaptation was also present in APD90 of fibres from female animals compared to males.

At a stimulation rate of 0.2 Hz, but not at 1.0 Hz, dofetilide elicited a significantly higher increase in APD90, incidence of early afterdepolarisations, triggered and sustained‐triggered activities (TAs) in fibres from female animals compared to males in either normal or low K+ conditions. The sustained TAs were reversed by raising the concentration of [K+]0 in Purkinje preparations from both male (one out of one) and female (12 out of 12) dogs.

In conclusion, our data provide experimental evidence pointing to gender differences in canine AP repolarisation. PFs from female dogs can be used in safety pharmacology studies as a sensitive model for evaluating the potential proarrhythmic events in vitro of a new medicinal product.

British Journal of Pharmacology (2004) 142, 1255–1264. doi:10.1038/sj.bjp.0705880
Efficacy in CB1 receptor‐mediated signal transduction
Authors: Howlett Allyn C
Published: August 2004

CB1 receptor cellular signal transduction is dependent on the expression of G proteins to which the receptor couples, the potential for precoupling of particular G proteins to the receptors either by scaffolding mechanisms or colocalization in lipid raft domains, and the effector mechanisms that these transducer molecules regulate. This discourse will evaluate studies of efficacy for CB1 receptor‐Gi/o activation at the molecular level. Evidence for brain regional differences in CB1 receptor signal transduction efficacy and agonist selectivity for G proteins will be summarized. The possibility that CB1 receptors interact with Gs or Gq will be evaluated, and questions with regard to the constitutive activity and G protein sequestration will be posed.

British Journal of Pharmacology (2004) 142, 1209–1218. doi:10.1038/sj.bjp.0705881
Role of different brain structures in the behavioural expression of WIN 55,212‐2 withdrawal in mice
Authors: Castañé Anna , Maldonado Rafael , Valverde Olga
Published: August 2004

We have evaluated several responses induced by the cannabinoid agonist WIN 55,212‐2 related to its addictive properties, including rewarding effects and the development of physical dependence in mice. Moreover, we have studied the specific involvement of several brain regions with high density of CB1 cannabinoid receptors, such as striatum, hippocampus, amygdala and cerebellum, in the behavioural expression of SR 141716A‐precipitated WIN 55,212‐2 withdrawal.

The systemic administration of the CB1 receptor antagonist SR 141716A (10 mg kg−1, s.c.) precipitated behavioural signs of withdrawal in mice chronically treated with WIN 55,212‐2 (1 and 2 mg kg−1, intraperitoneal (i.p.)), revealing the development of physical dependence.

The microinjection of SR 141716A (1.5 and 3 μg) into the cerebellum induced severe manifestations of abstinence in mice dependent on WIN 55,212‐2 (1 mg kg−1, i.p.). Out of 10 signs evaluated, seven were statistically significant: wet dog shakes, body tremor, paw tremor, piloerection, mastication, genital licks and sniffing. When the cannabinoid antagonist was administered into the hippocampus and the amygdala, a moderate but significant withdrawal syndrome was also observed. However, no signs of abstinence were induced when SR 141716A was microinjected into the striatum.

WIN 55,212‐2 produced rewarding effects in the place‐conditioning paradigm in mice pre‐exposed to a priming injection of the drug.

These results show a reliable behavioural model to reveal rewarding effects and physical dependence induced by the repeated administration of WIN 55,212‐2 in mice. The cerebellum and to a lesser extent the hippocampus and the amygdala participate in the behavioural expression of cannabinoid withdrawal.

British Journal of Pharmacology (2004) 142, 1309–1317. doi:10.1038/sj.bjp.0705882
Matrix metalloproteinase expression and activity in human airway smooth muscle cells
Authors: Elshaw Shona R , Henderson Neil , Knox Alan J , Watson Susan A , Buttle David J , Johnson Simon R
Published: August 2004

Airway remodelling is a feature of chronic asthma comprising smooth muscle hypertrophy and deposition of extracellular matrix (ECM) proteins. Matrix metalloproteinases (MMPs) breakdown ECM, are involved in tissue remodelling and have been implicated in airway remodelling. Although mesenchymal cells are an important source of MMPs, little data are available on airway smooth muscle (ASM) derived MMPs. We therefore investigated MMP and tissue inhibitor of metalloproteinase (TIMP) production and activity in human ASM cells.

MMPs and TIMPs were examined using quantitative real‐time RT–PCR, Western blotting, zymography and a quench fluorescence (QF) assay of total MMP activity.

The most abundant MMPs were pro‐MMP‐2, pro‐ MMP‐3, active MMP‐3 and MT1‐MMP. TIMP‐1 and TIMP‐2 expression was low in cell lysates but high in conditioned medium. High TIMP secretion was confirmed by the ability of ASM‐conditioned medium to inhibit recombinant MMP‐2 in a QF assay. Thrombin increased MMP activity by activation of pro‐MMP‐2 independent of the conventional smooth muscle thrombin receptors PAR 1 and 4.

In conclusion, ASM cells express pro‐MMP‐2, pro and active MMP‐3, MMP‐9 and MT1‐MMP. Unstimulated cells secrete excess TIMP 1 and 2, preventing proteolytic activity. MMP‐2 can be activated by thrombin which may contribute to airway remodelling.

British Journal of Pharmacology (2004) 142, 1318–1324. doi:10.1038/sj.bjp.0705883
Synergistic action between inhibition of P2Y12/P2Y1 and P2Y12/thrombin in ADP‐ and thrombin‐induced human platelet activation
Authors: Nylander Sven , Mattsson Christer , Ramström Sofia , Lindahl Tomas L
Published: August 2004

The objective of this study was to investigate if there is a synergistic effect of a combination of P2Y12 and P2Y1 inhibition and P2Y12 and thrombin inhibition, on ADP‐ and thrombin‐induced platelet activation, respectively. The rationale being that these combinations will cause a concurrent inhibition of both Gαq and Gαi signalling.

Blood from healthy volunteers was preincubated with AR‐C69931MX, a reversible P2Y12 antagonist; MRS2179, a reversible P2Y1 antagonist; or melagatran, a direct reversible thrombin inhibitor; alone or in various combinations prior to activation with ADP or thrombin. Platelet function in whole blood was assessed by flow cytometry using the antibody PAC‐1 to estimate the expression of active αIIbβ3 (the fibrinogen receptor GPIIb/IIIa). A synergistic effect was evaluated by comparing the concentrations in the different combinations with those of corresponding equipotent concentrations of each single inhibitor alone. The equipotent single concentrations were experimentally obtained from concentration response curves performed in parallel.

A synergistic effect regarding inhibition of ADP‐induced platelet activation (10 μM) was obtained with different combinations of AR‐C69931MX and MRS2179.

Inhibition of thrombin‐induced platelet activation (2 nM) with combinations of AR‐C69931MX and the thrombin inhibitor melagatran did also result in a strong synergistic effect.

To our knowledge, this is the first time that data supporting a synergistic effect has been published for the inhibitor combinations described.

Whether this synergistic effect in vitro also results in an improved antithrombotic effect in vivo with or without an increased risk of bleeding remains to be studied in well‐conducted clinical studies.

British Journal of Pharmacology (2004) 142, 1325–1331. doi:10.1038/sj.bjp.0705885
Triazine dyes are agonists of the NAADP receptor
Authors: Billington Richard A , Bak Judit , Martinez‐Coscolla Ana , Debidda Marcella , Genazzani Armando A
Published: August 2004

NAADP has been shown to be a potent calcium‐releasing second messenger in a wide variety of cell types to date. However, research has been hampered by a lack of pharmacological agents, with which to investigate NAADP‐induced calcium release, and by the molecular identity of its cellular target protein being unknown.

In the present paper, the sea urchin egg model was used to investigate whether triazine dyes, which can act as nucleotide mimetics, can bind to the NAADP receptor, induce Ca2+ release and be used for affinity chromatography of the receptor.

Indeed, all the triazine dyes tested (Reactive Red 120 (RR120), Reactive Green 19 (RG19), Reactive Green 5 (RG5), Cibacron Blue 3GA and Reactive Yellow 86) displayed micromolar affinities, except for Reactive Orange 14. Furthermore, unlike NAADP, RR120, RG19 and RG5 did not bind in an irreversible manner.

The compound that displayed the highest affinity, RR120, was tested in a 45Ca2+ efflux assay. This compound released Ca2+ via the NAADP receptor, as shown by the ability of subthreshold NAADP concentrations to inhibit this release. Furthermore, heparin and ruthenium red were unable to block RR120‐induced Ca2+ release.

We have also shown that RG5 and RG19, immobilised on resins, retain the ability to bind to the receptor, and that this interaction can be disrupted by high salt concentrations. As a proof of principle, we have shown that this can be used to partially purify the NAADP receptor by at least 75‐fold.

In conclusion, triazine dyes interact with the NAADP receptor, and this could be exploited in future to create a new generation of pharmacological tools to investigate this messenger and, in combination with other techniques, to purify the receptor.

British Journal of Pharmacology (2004) 142, 1241–1246. doi:10.1038/sj.bjp.0705886
Increased wakefulness, motor activity and decreased theta activity after blockade of the 5‐HT2B receptor by the subtype‐selective antagonist SB‐215505
Authors: Kantor Sandor , Jakus Rita , Balogh Brigitta , Benko Anita , Bagdy Gyorgy
Published: August 2004

Serotonin‐2 receptor antagonists, like ritanserin, greatly enhance deep slow wave sleep (SWS‐2) and low‐frequency EEG power in humans and rodents. 5‐HT2A and 5‐HT2C receptors may be involved in these effects, but the role of the 5‐HT2B receptor is still unclear.

To investigate the role of the 5‐HT2B receptor in regulation of the sleep–wake cycle, the subtype‐selective antagonist SB‐215505 (0.1, 0.3 and 1.0 mg kg−1 i.p.) was administered to Sprague–Dawley rats at light onset (beginning of passive phase). EEG, EMG and motor activity were recorded during the subsequent 8 h.

SB‐215505 dose‐dependently increased wakefulness (W) at the expense of the intermediate stage of sleep, paradoxical sleep (PS) and SWS‐2 in the first hour. Parallel to increased W, significantly increased motor activity was found. Spectral analysis of the EEG in W showed a dose‐dependent decrease in power density in the 3–8 Hz frequency range (maximum effect at 6 Hz). In light slow wave sleep and SWS‐2, the drug reduced low‐frequency (<8 Hz) EEG power, suggesting decreased sleep intensity after SB‐215505 treatment. In PS, the drug dose‐dependently decreased EEG power solely in the theta (6–9 Hz) band, primarily affecting the peak power value (7 Hz).

The well‐known SWS‐2 enhancing effect of 5‐HT2 receptor antagonists is mediated by 5‐HT2A and/or 5‐HT2C receptors. In contrast, blockade of 5‐HT2B receptors increases motor activity and W along with decreased theta activity during W and PS. Activation of 5‐HT2B receptors may contribute to initiation of sleep and to theta generation during W and PS under physiological conditions.

British Journal of Pharmacology (2004) 142, 1332–1342. doi:10.1038/sj.bjp.0705887
The NAADP receptor: commentary on Billington et al.
Authors: Galione A , Parrington J , Dowden J
Published: August 2004

NAADP is a recently described calcium‐mobilizing messenger. First discovered as a potent calcium‐releasing molecule in sea urchin eggs, its actions have now been reported in several mammalian cell types. In the sea urchin egg, NAADP‐sensitive calcium release channels appear distinct from inositol trisphosphate or ryanodine receptors, and are mainly localized to acidic compartments. In this study, Billington et al. extend the pharmacology of the putative NAADP receptor utilizing molecules unrelated to NAADP itself. This work may provide an important step in developing selective NAADP receptor modulators that will help define the role of NAADP in cell signalling.

British Journal of Pharmacology (2004) 142, 1203–1207. doi:10.1038/sj.bjp.0705888
Effects of cannabinoid receptor‐2 activation on accelerated gastrointestinal transit in lipopolysaccharide‐treated rats
Authors: Mathison Ronald , Ho Winnie , Pittman Quentin J , Davison Joseph S , Sharkey Keith A
Published: August 2004

The biological effects of cannabinoids (CB) are mediated by CB1 and CB2 receptors. The role of CB2 receptors in the gastrointestinal tract is uncertain. In this study, we examined whether CB2 receptor activation is involved in the regulation of gastrointestinal transit in rats.

Basal and lipopolysaccharide (LPS)‐stimulated gastrointestinal transit was measured after instillation of an Evans blue‐gum Arabic suspension into the stomach, in the presence of specific CB1 and CB2 agonists and antagonists, or after treatment with inhibitors of mediators implicated in the transit process.

In control rats a CB1 (ACEA; 1 mg kg−1), but not a CB2 (JWH‐133; 1 mg kg−1), receptor agonist inhibited basal gastrointestinal transit. The effects of the CB1 agonist were reversed by the CB1 antagonist AM‐251, which alone increased basal transit. LPS treatment increased gastrointestinal transit. This increased transit was reduced to control values by the CB2, but not the CB1, agonist. This inhibition by the CB2 agonist was dose dependent and prevented by a selective CB2 antagonist (AM‐630; 1 mg kg−1).

By evaluating the inhibition of LPS‐enhanced gastrointestinal transit by different antagonists, the effects of the CB2 agonist (JWH‐133; 1 mg kg−1) were found to act via cyclooxygenase, and to act independently of inducible nitric oxide synthase (NOS) and platelet‐activating factor. Interleukin‐1β and constitutive NOS isoforms may be involved in the accelerated LPS transit.

The activation of CB2 receptors in response to LPS is a mechanism for the re‐establishment of normal gastrointestinal transit after an inflammatory stimulus.

British Journal of Pharmacology (2004) 142, 1247–1254. doi:10.1038/sj.bjp.0705889
Cannabinoids and intestinal motility: welcome to CB2 receptors
Authors: Izzo Angelo A
Published: August 2004

Δ9‐Tetrahydrocannabinol (the active ingredient of marijuana), as well as endogenous and synthetic cannabinoids, exert many biological functions by activating two types of cannabinoid receptors, CB1 receptors (expressed by central and peripheral neurons) and CB2 receptors (that occur mainly in immune cells). Convincing evidence has accumulated in recent years that cannabinoids inhibit gastric and intestinal motility through activation of enteric CB1 receptors. However, a report in this issue of British Journal of Pharmacology has highlighted the possibility that CB2 receptors in the rat intestine could contribute to reducing the increase of intestinal motility induced by an endotoxic inflammation. By minimizing the adverse psychotropic effects associated with brain cannabinoid receptors, the CB2 receptor represents a new molecular target for the treatment of motility disorders associated with intestinal inflammation.

British Journal of Pharmacology (2004) 142, 1201–1202. doi:10.1038/sj.bjp.0705890
Effects of phosphodiesterase inhibitors on interleukin‐4 and interleukin‐13 generation from human basophils
Authors: Eskandari Nahid , Wickramasinghe Thulani , Peachell Peter T
Published: August 2004

The aim of the present study was to determine whether inhibition of cyclic nucleotide phosphodiesterase (PDE) modulates the stimulated generation of the cytokines, interleukin‐4 (IL‐4) and IL‐13, from human basophils. This was addressed by evaluating the effects of both nonselective and selective inhibitors of PDEs on the generation of cytokines from basophils.

The nonselective PDE inhibitors, isobutyl‐methylxanthine (IBMX) and theophylline, attenuated the IgE‐mediated generation of IL‐4 and IL‐13 and, also, the release of histamine from basophils.

The effects of the isoform‐selective inhibitors, 8‐methoxymethyl‐IBMX (PDE 1 inhibitor), siguazodan (PDE3 inhibitor), rolipram (PDE4 inhibitor), denbufylline (PDE4 inhibitor), Org 30029 (mixed PDE3 and 4 inhibitor) and zaprinast (PDE5 inhibitor), were studied. Of these selective compounds, only rolipram, denbufylline and Org 30029 inhibited the IgE‐dependent generation of IL‐4, IL‐13 and histamine from basophils to a statistically significant (P<0.05) degree.

The effects of isoform‐selective inhibitors on basophils activated by IL‐3 were evaluated. The IL‐3‐induced generation of IL‐4, IL‐13 and histamine was inhibited to a statistically significant (P<0.05) extent, only by compounds that act as inhibitors of PDE4.

These data suggest that inhibition of PDE4 can regulate the generation of cytokines from human basophils.

British Journal of Pharmacology (2004) 142, 1265–1272. doi:10.1038/sj.bjp.0705892
Dopamine D3 receptor‐mediated inhibition of Na+/H+ exchanger activity in normotensive and spontaneously hypertensive rat proximal tubular epithelial cells
Authors: Pedrosa Rui , Gomes Pedro , Zeng Chunyu , Hopfer Ulrich , Jose Pedro A , Soares‐da‐Silva Patrício
Published: August 2004

This study evaluated the response of the Na+/H+ exchanger (NHE) to dopamine D1‐ and D2‐like receptor stimulation in immortalized renal proximal tubular epithelial cells and freshly isolated renal proximal tubules from the spontaneously hypertensive rat (SHR) and their normotensive controls (Wistar Kyoto rats; WKY).

Stimulation of D1‐like receptors with SKF 38393 attenuated NHE activity in WKY cells (IC50=151 nM), but not in SHR cells. Stimulation of D2‐like receptors with quinerolane (IC50=120 nM) attenuated NHE activity in SHR cells, but not in WKY cells. Forskolin was equipotent in SHR and WKY cells in inhibiting NHE activity. The effect of SKF 38393 was abolished by overnight treatment of WKY cells with cholera toxin (CTX, 500 ng ml−1), but not with pertussis toxin (PTX, 100 ng ml−1). The effect of quinerolane (1 μM) was abolished by overnight treatment of SHR cells with PTX, but not with CTX.

The D3 receptor agonist 7‐OH‐DPAT (IC50=0.8 μM) attenuated NHE activity in SHR cells only. This effect was abolished by S‐sulpiride and by overnight treatment with PTX. The D4 receptor agonist RBI 257 did not affect NHE activity.

The 7‐OH‐DPAT inhibited NHE activity in freshly isolated renal proximal tubules from 4‐ and 12‐week‐old SHR and 12‐week‐old WKY, but not in freshly isolated renal proximal tubules from 4‐week‐old WKY.

It is concluded that D3 receptors coupled to a Gi/o protein play a role in the handling of tubular Na+, namely through inhibition of the NHE activity, this being of particular relevance in the SHR, which fail to respond to D1‐like dopamine receptor stimulation.

British Journal of Pharmacology (2004) 142, 1343–1353. doi:10.1038/sj.bjp.0705893
Cellular actions of somatostatin on rat periaqueductal grey neurons in vitro
Authors: Connor Mark , Bagley Elena E , Mitchell Vanessa A , Ingram Susan L , Christie MacDonald J , Humphrey Patrick P A , Vaughan Christopher W
Published: August 2004

Functional studies indicate that the midbrain periaqueductal grey (PAG) is involved in the analgesic actions of somatostatin; however, the cellular actions of somatostatin in this brain region are unknown. In the present study, whole‐cell patch clamp recordings were made from rat PAG neurons in vitro. In 93% of acutely isolated neurons, somatostatin inhibited Ca2+‐channel currents. This effect was mimicked by the sst‐2 selective agonist BIM‐23027, but not by the sst‐1 and sst‐5 selective agonists CH‐275 and L‐362855. In brain slices, 81% of neurons responded to somatostatin (300 nM) with an increase in K+ conductance that reversed polarity at −114 mV. A greater proportion of somatostatin‐sensitive neurons (93%) than somatostatin‐insensitive neurons (53%) responded to the opioid agonist met‐enkephalin (10 μM). Somatostatin also reduced the amplitude of evoked GABAA‐mediated inhibitory postsynaptic currents (IPSCs). The actions of somatostatin in brain slices were mimicked by BIM‐23027, but not by CH‐275. Somatostatin had a variable effect on the rate of spontaneous miniature IPSCs in normal external potassium solutions. In high external potassium solutions, somatostatin reduced the rate of miniature IPSCs in all neurons, and this inhibition was abolished by addition of Cd2+ (30 μM). Somatostatin had no effect on the amplitude of miniature IPSCs. These results indicate that somatostatin acts via sst‐2 receptors to directly inhibit a subpopulation of PAG neurons by activating a potassium conductance and inhibits GABA release within PAG via a presynaptic Ca2+‐dependent mechanism. Thus, like opioids, somatostatin has the potential to exert pre‐ and postsynaptic disinhibitory effects within the PAG.

British Journal of Pharmacology (2004) 142, 1273–1280. doi:10.1038/sj.bjp.0705894
Intestinal Na+–K+–ATPase activity and molecular events downstream of interferon‐γ receptor stimulation
Authors: Magro Fernando , Fraga Sónia , Ribeiro Tomé , Soares‐da‐Silva Patrício
Published: August 2004

This study evaluated the effects of human interferon‐γ (IFN‐γ) on Na+–K+–ATPase activity and the intracellular signaling pathways involved in human intestinal epithelial Caco‐2 cells.

Na+–K+–ATPase activity was determined as the difference between total and ouabain‐sensitive ATPase. p38 MAP kinase activity was analyzed by Western blotting using the p38 MAP kinase assay kit. Total and phosphorylated STAT1 protein levels were detected using the PhosphoPlus® Stat1.

IFN‐γ decreased Na+–K+–ATPase activity in a time‐ and concentration‐dependent manner. The IFN‐γ‐induced decrease in Na+–K+–ATPase activity was accompanied by no changes in the abundance of α1 subunit Na+–K+–ATPase. Downregulation of protein kinase C (PKC) with phorbol‐12,13‐dibutyrate (PDBu) prevented the inhibitory effect of IFN‐γ on Na+–K+–ATPase activity. Inhibition of Raf‐1, mitogen‐activated protein kinase kinase (MAPKK/MEK), p38 MAPK and STAT1 with, respectively, GW 5074, PD 98059, SB 203580 and epigallocatechin gallate prevented inhibition of Na+–K+–ATPase activity by IFN‐γ.

Treatment with IFN‐γ markedly increased the expression of total and phospho‐STAT1, this being accompanied by activation of p38 MAPK. Activation of phospho‐STAT1 by IFN‐γ was almost abolished by epigallocatechin gallate and markedly reduced by SB 203580, but insensitive to downregulation of PKC.

The increase in short circuit current (Isc) by 1.0 and 2.5 μg ml−1 amphotericin B was markedly attenuated in IFN‐γ‐treated cells. However, the inhibitory effect of PDBu on the amphotericin B‐induced increase in Isc was of similar magnitude in vehicle‐ and IFN‐γ‐treated cells.

It is concluded that IFN‐γ markedly attenuates Na+–K+–ATPase activity. The transduction mechanisms set into motion by IFN‐γ involve the activation of PKC downstream STAT1 phosphorylation and Raf‐1, MEK, ERK2 and p38 MAPK pathways, in a complex sequence of events.

British Journal of Pharmacology (2004) 142, 1281–1292. doi:10.1038/sj.bjp.0705895
Anandamide acts as a vasodilator of dural blood vessels in vivo by activating TRPV1 receptors
Authors: Akerman S , Kaube H , Goadsby P J
Published: August 2004

Migraine pathophysiology is believed to involve the release of neuropeptides via the activation of trigeminal afferents that innervate the cranial vasculature.

Anandamide, the endogenous ligand to the cannabinoid receptor, is able to inhibit neurogenic dural vasodilatation, calcitonin gene‐related peptide (CGRP)‐induced and nitric oxide‐induced dural vessel dilation in the intravital microscopy model. In an in vitro setting anandamide is also able to activate the vanilloid type 1 (TRPV1) receptor and cause vasodilation, via the release of CGRP.

In this study we used intravital microscopy to study whether anandamide behaves as a TRPV1 receptor agonist in the trigeminovascular system. We examined if anandamide‐induced dural vasodilation involves CGRP release that can be reversed by the CGRP receptor antagonist, CGRP8–37, and whether like capsaicin the anandamide effect could be reversed by the TRPV1 receptor antagonist, capsazepine.

Anandamide 1 (19±9%, n=12), 3 (29±5%, n=37), 5 (74±7%, n=13) and 10 mg kg−1 (89±18%, n=6) was able to cause a dose‐dependent increase in dural vessel diameter. Capsazepine (3 mg kg−1, t5=6.2, P<0.05) and CGRP8–37 (300 μg kg−1, t6=11.1, P<0.05) attenuated the anandamide‐induced dural vessel dilation when compared to control (Student's paired t‐test). AM251 (3 mg kg−1), a cannabinoid type 1 (CB1) receptor antagonist, was unable to reverse this anandamide‐induced dilation.

The study demonstrates that anandamide acts as a TRPV1 receptor agonist in the trigeminovascular system, activating TRPV1 receptors that promote CGRP release and cause vasodilation independent of any action at the CB1 receptor. Anandamide has been shown previously to inhibit trigeminovascular neurons and prevent vasodilation, through an action at CB1 receptors.

British Journal of Pharmacology (2004) 142, 1354–1360. doi:10.1038/sj.bjp.0705896