bluuebunny/crossref_metadata_2025 huggingface dataset:
5‐HT1A receptor agonist‐antagonist binding affinity difference as a measure of intrinsic activity in recombinant and native tissue systems
Authors: Watson J , Collin L , Ho M , Riley G , Scott C , Selkirk J V , Price G W
Published: January 2005

It has been reported that radiolabelled agonist : antagonist binding affinity ratios can predict functional efficacy at several different receptors. This study investigates whether this prediction is true for recombinant and native tissue 5‐HT1A receptors.

Saturation studies using [3H]‐8‐OH‐DPAT and [3H]‐MPPF revealed a single, high affinity site (KD∼1 nM) in HEK293 cells expressing human 5‐HT1A receptors and rat cortex. In recombinant cells, [3H]‐MPPF labelled 3–4 fold more sites than [3H]‐8‐OH‐DPAT suggesting the presence of more than one affinity state of the receptor. [3H]‐Spiperone labelled a single, lower affinity site in HEK293 cells expressing h5‐HT1A receptors but did not bind to native tissue 5‐HT1A receptors. These data suggest that, in transfected HEK293 cells, human 5‐HT1A receptors exist in different affinity states but in native rat cortical tissue the majority of receptors appear to exist in the high agonist affinity state.

Receptor agonists inhibited [3H]‐MPPF binding from recombinant 5‐HT1A receptors in a biphasic manner, whereas antagonists and partial agonists gave monophasic inhibition curves. All compounds displaced [3H]‐8‐OH‐DPAT and [3H]‐spiperone binding in a monophasic manner. In rat cortex, all compounds displaced [3H]‐MPPF and [3H]‐8‐OH‐DPAT in a monophasic manner.

Functional evaluation of compounds, using [35S]‐GTPγS binding, produced a range of intrinsic activities from full agonism, displayed by 5‐HT and 5‐CT to inverse agonism displayed by spiperone.

[3H]‐8‐OH‐DPAT : [3H]‐MPPF pKi difference correlated well with functional intrinsic activity (r=0.86) as did [3H]‐8‐OH‐DPAT : [3H]‐spiperone pKi difference with functional intrinsic activity (r=0.96).

Thus agonist : antagonist binding affinity differences may be used to predict functional efficacy at human 5‐HT1A receptors expressed in HEK293 cells where both high and low agonist affinity states are present but not at native rat cortical 5‐HT1A receptors in which only the high agonist affinity state was detectable.

British Journal of Pharmacology (2000) 130, 1108–1114; doi:10.1038/sj.bjp.0703394
Inhibition of mitochondrial proton F0F1‐ATPase/ATP synthase by polyphenolic phytochemicals
Authors: Zheng Jianbiao , Ramirez Victor D
Published: January 2005

Mitochondrial proton F0F1‐ATPase/ATP synthase synthesizes ATP during oxidative phosphorylation. In this study, we examined the effects of several groups of polyphenolic phytochemicals on the activity of the enzyme.

Resveratrol, a stilbene phytoalexin that is present in grapes and red wine, concentration‐dependently inhibited the enzymatic activity of both rat brain and liver F0F1‐ATPase/ATP synthase (IC50 of 12–28 μM).

Screening of other polyphenolic phytochemicals using rat brain F0F1‐ATPase activity resulted in the following ranking potency (IC50 in parenthesis): piceatannol (8 μM)>resveratrol (19 μM)=(−)epigallocatechin gallate (17 μM)>(−)epicatechin gallate, curcumin (45 μM)>genistein=biochanin A=quercetin=kaempferol=morin (55–65 μM)>phloretin=apigenin=daidzein (approx. 100 μM). Genistin, quercitrin, phloridzin, (+)catechin, (+)epicatechin, (−)epicatechin and (−)epigallocatechin had little effect at similar concentrations. Tannic acid, theaflavins (tea extract) and grape seed proanthocyanidin extract (GSPE) had IC50 values of 5, 20 and 30 μg ml−1, respectively. Several monophenolic antioxidants and non‐phenolic compounds were ineffective at concentrations of 210 μM or higher.

The inhibition of F0F1‐ATPase by resveratrol and genistein was non‐competitive in nature.

The effects of polyphenolic phytochemicals were additive.

Both resveratrol and genistein had little effect on the Na+/K+‐ATPase activity of porcine cerebral cortex, whereas quercetin had similar inhibitory potency as for F0F1‐ATPase.

In conclusion, the ATP synthase is a target for dietary phytochemicals. This pharmacological property of these phytochemicals should be included in the examination of their health benefits as well as potential cytotoxicity.

British Journal of Pharmacology (2000) 130, 1115–1123; doi:10.1038/sj.bjp.0703397
On the regulation of ischaemia‐induced glutamate efflux from rat cortex by GABA; in vitro studies with GABA, clomethiazole and pentobarbitone
Authors: Nelson R M , Green A R , Lambert D G , Hainsworth A H
Published: January 2005

Prisms of adult rat cortex were maintained in vitro in either aerobic conditions (control) or conditions simulating an acute ischaemic challenge (hypoxia with no added glucose).

Endogenous glutamate efflux increased with time in ischaemic conditions, being 2.7 fold higher than control efflux at 45 min. Returning prisms to control solution after 20 min of simulated ischaemia resulted in glutamate efflux returning to near‐control values. Endogenous GABA efflux in ischaemic conditions also increased, being 4.5 fold higher than control efflux at 45 min.

Ischaemia‐induced glutamate efflux was not accompanied by increased lactate dehydrogenase efflux and was unaltered by omitting calcium from the extra‐cellular solution and adding EGTA (0.1 mM).

Both GABA and the GABA‐mimetic clomethiazole inhibited ischaemia‐induced glutamate efflux, with IC50 values of 26 and 24 μM respectively. The maximum inhibition by either drug was 60–70%. Bicuculline (10 μM) abolished the inhibitory effect of GABA (100 μM) but not clomethiazole (100 μM). Picrotoxin (100 μM) abolished the action of both GABA and clomethiazole.

Pentobarbitone inhibited glutamate efflux at 100–300 μM (maximal inhibition: 39%). Bicuculline (10 μM) abolished this effect.

These data suggest that ischaemia‐induced glutamate efflux from rat cerebral cortex is calcium‐independent and not due to cell damage up to 45 min. The inhibitory effect of GABA, clomethiazole and pentobarbitone on ischaemia‐induced glutamate efflux appears to be mediated by GABAA receptors. The results suggest that clomethiazole, unlike pentobarbitone, is able to activate the GABAA receptor‐linked chloride channel directly and not merely potentiate the effect of endogenous GABA.

British Journal of Pharmacology (2000) 130, 1124–1130; doi:10.1038/sj.bjp.0703398
Different intrinsic activities of bucindolol, carvedilol and metoprolol in human failing myocardium
Authors: Maack C , Cremers B , Flesch M , Höper A , Südkamp M , Böhm M
Published: January 2005

Clinical studies have shown different effects of β‐blockers on the β‐adrenergic system, tolerability and outcome in patients with heart failure.

The study examines β‐adrenoceptor‐G‐protein coupling and intrinsic activity of bucindolol, carvedilol and metoprolol in human ventricular myocardium.

Radioligand binding studies ([125I]‐Iodocyanopindolol) were performed in membrane preparations of human failing and nonfailing myocardium. Functional experiments were carried out in isolated muscle preparations of human left ventricular myocardium from failing hearts.

Bucindolol and carvedilol bound non‐selectively to β1‐ and β2‐adrenoceptors and exerted guanine nucleotide modulatable binding. Metoprolol was 35‐fold β1‐selective and lacked guanine nucleotide modulatable binding.

All β‐blockers antagonized isoprenaline‐induced enhancement of contractility.

In preparations in which the coupling of the stimulatory G‐protein to adenylate cyclase was facilitated by forskolin, bucindolol increased force of contraction in three and decreased it in five experiments. Carvedilol increased force in one and decreased it in six experiments. Metoprolol decreased force in all experiments by 89.4±2.2% (P<0.01 metoprolol vs carvedilol and bucindolol). The negative inotropic effect of metoprolol was antagonized by bucindolol.

It is concluded that differences in intrinsic activity can be detected in human myocardium and have an impact on cardiac contractility. In human ventricular myocardium, bucindolol displays substantially higher intrinsic activity than metoprolol and carvedilol. Bucindolol can behave as partial agonist or partial inverse agonist depending on the examined tissue.

Differences in intrinsic activity may contribute to differences in β‐adrenoceptor regulation and possibly to differences in tolerability and outcomes of patients with heart failure.

British Journal of Pharmacology (2000) 130, 1131–1139; doi:10.1038/sj.bjp.0703400
Apocynin inhibits peroxynitrite formation by murine macrophages
Authors: Muijsers R B R , Van Den Worm E , Folkerts G , Beukelman C J , Koster A S , Postma D S , Nijkamp F P
Published: January 2005

Peroxynitrite (ONOO−) the highly reactive coupling product of nitric oxide and superoxide, has been implicated in the pathogenesis of an increasing number of (inflammatory) diseases. At present, however, selective peroxynitrite antagonizing agents with therapeutic potential are not available. Therefore, the NADPH‐oxidase inhibitor apocynin (4‐hydroxy‐3‐methoxy‐acetophenone) was tested for its ability to inhibit peroxynitrite formation in vitro

The murine macrophage cell‐line J774A.1, stimulated with IFNγ/LPS, was used as a model. Conversion of 123‐dihydrorhodamine (123‐DHR) to its oxidation product 123‐rhodamine was used to measure peroxynitrite production.

Stimulated peroxynitrite formation could be completely inhibited by apocynin, by the superoxide scavenger TEMPO as well as by the nitric oxide synthase inhibitor aminoguanidine. Apocynin and aminoguanidine specifically inhibited superoxide and nitric oxide formation respectively as confirmed by measuring lucigenin enhanced chemiluminescence and nitrite accumulation.

It is concluded that J774A.1 macrophages produce significant amounts of peroxynitrite, which is associated with nitric oxide production and NADPH‐oxidase dependent superoxide formation. The NADPH‐oxidase inhibitor apocynin proved to be a potent inhibitor of both superoxide and peroxynitrite formation by macrophages, which may be of future therapeutic significance in a wide range of inflammatory disorders.

British Journal of Pharmacology (2000) 130, 932–936; doi:10.1038/sj.bjp.0703401
Murine myeloid progenitor responses to GM‐CSF and eosinophil precursor responses to IL‐5 represent distinct targets for downmodulation by prostaglandin E2
Authors: Gaspar Elsas Maria Ignez C , Joseph Danielle , Lintomen Letícia , Maximiano Elisabeth S , Bodstein Michael , Elsas P Xavier , Vargaftig B Boris
Published: January 2005

Because Prostaglandin E2 (PGE2) and dibutiryl cyclic AMP (dbcAMP) modulate the production and effects of haemopoietic cytokines in allergy, we examined their ability to modulate responses of myeloid progenitors to GM‐CSF, and of eosinophil precursors to IL‐5.

The ability of PGE2, dbcAMP, rolipram, forskolin, dbcGMP and PGD2, to modulate the responses to GM‐CSF and IL‐5 in colony formation (progenitor) and eosinophil differentiation (precursor) assays using bone‐marrow from nonsensitized or from intranasally‐challenged, ovalbumin‐sensitized mice of five strains was studied.

PGE2 (10−7 M) inhibited GM‐CSF‐stimulated colony formation in bone‐marrow from BP‐2 mice. This effect was duplicated by dbcAMP (0.3–1×10−6 M), Rolipram (10−5 M) and forskolin (3×10−5 M), but not Prostaglandin D2 (10−6 M). Inhibition affected similarly all myeloid colony types. Progenitors from sensitized and challenged BP‐2 mice were also inhibited by PGE2 and cyclic AMP. PGE2 inhibited progenitors from C57BL/10, CBA/J and A/J, but not BALB/c mice. However, BALB/c progenitors were sensitive to dbcAMP and Forskolin (10−4 M). In contrast, in precursor assays, PGE2 (10−7–10−9 M) blocked responses to IL‐5 in bone‐marrow from BP‐2 and BALB/c mice, either naïve or sensitized and challenged, to a similar extent. PGD2 (10−6 M) was ineffective, as was PGE2 (10−7 M), if added after 48 h of culture.

In conclusion, PGE2 inhibits the responses of bone‐marrow myeloid progenitors to GM‐CSF and of eosinophil precursors to IL‐5, in naïve or ovalbumin sensitized and challenged mice. These effects are duplicated by cyclic AMP‐elevating agents. In the BALB/c strain, the resistance of progenitors, but not precursors, to PGE2 inhibition, indicates these developmental stages are separate targets for PGE2 modulation.

British Journal of Pharmacology (2000) 130, 1362–1368; doi:10.1038/sj.bjp.0703403
Blockade of phencyclidine‐induced effects by a nitric oxide donor
Authors: Bujas‐Bobanovic M , Bird D C , Robertson H A , Dursun S M
Published: January 2005

Phencyclidine (PCP) is widely used as an animal model of schizophrenia. The aim of this study was to better understand the role of nitric oxide (NO) in the mechanism of action of PCP and to determine whether positive NO modulators may provide a new approach to the treatment of schizophrenia.

The effects of the NO donor, sodium nitroprusside (SNP), were studied in PCP‐treated rats. Following drug administration, behavioural changes and the expression of c‐fos, a metabolic marker of functional pathways in the brain, were simultaneously monitored.

Acute PCP (5 mg kg−1, i.p.) treatment induced a complex behavioural syndrome, consisting of hyperlocomotion, stereotyped behaviours and ataxia. Treatment with SNP (2–6 mg kg−1, i.p.) by itself produced no effect on any behaviour studied but completely abolished PCP‐induced behaviour in a dose‐ and time‐dependent manner.

PCP had differential regional effects on c‐fos expression in rat brain, suggesting regionally different patterns of neuronal activity. The most prominent immunostaining was observed in the cortical regions. Pre‐treatment with SNP blocked PCP‐induced c‐fos expression at doses similar to those that suppress PCP‐induced behavioural effects.

These results implicate the NO system in the mechanism of action of PCP. The fact that SNP abolished effects of PCP suggests that drugs targeting the glutamate‐NO system may represent a novel approach to the treatment of PCP‐induced psychosis and schizophrenia.

British Journal of Pharmacology (2000) 130, 1005–1012; doi:10.1038/sj.bjp.0703406
Characteristics of the Ca2+‐dependent inhibition of cyclic AMP accumulation by histamine and thapsigargin in human U373 MG astrocytoma cells
Authors: Wong M‐P Mabel , Cooper Dermot M F , Young Kenneth W , Young J Michael
Published: January 2005

Histamine, acting on H1‐receptors, caused a Ca2+‐dependent inhibition of forskolin‐ and isoprenaline‐induced cyclic AMP accumulation in monolayers of human U373 MG cells (IC50 1.3±0.3 μM, maximum inhibition 66±3%). The inhibition was not reversed by the protein kinase inhibitor K‐252A.

Thapsigargin also inhibited cyclic AMP accumulation (IC50 6.0±0.3 nM, maximum inhibition 72±1%). In the absence of extracellular Ca2+ 5 μM thapsigargin caused only a 12±2% inhibition of cyclic AMP accumulation.

The inhibitory effect of 100 nM thapsigargin on forskolin‐stimulated cyclic AMP accumulation was blocked by La3+ (best‐fit maximum inhibition 81±4%, IC50 125±8 nM). In contrast, the inhibitory action of 10 μM histamine was much less sensitive to reversal by 1 μM La3+ (33±5% reversal, compared with 78±6% reversal of the inhibition by thapsigargin measured concurrently). However, in the presence of both thapsigargin and histamine the inhibition of cyclic AMP accumulation was reversed by 1 μM La3+ to the same extent as the inhibition by thapsigargin alone.

Thapsigargin (5 μM)+1 μM La3+ caused only a 20±1% inhibition of histamine‐stimulated phosphoinositide hydrolysis.

There was no indication from measurement of intracellular Ca2+ of any persistent La3+‐insensitive Ca2+ entry component activated by histamine.

The results provide evidence that Ca2+ entry is required for the inhibition by histamine and thapsigargin of drug‐induced cyclic AMP accumulation in U373 MG astrocytoma cells. The differential sensitivity of the inhibitory action of the two agents to block by La3+ suggests that more than one pathway of Ca2+ entry is involved.

British Journal of Pharmacology (2000) 130, 1021–1030; doi:10.1038/sj.bjp.0703411
D2 dopamine receptors enable Δ9‐tetrahydrocannabinol induced memory impairment and reduction of hippocampal extracellular acetylcholine concentration
Authors: Nava F , Carta G , Battasi A M , Gessa G L
Published: January 2005

The systemic administration of Δ9‐tetrahydrocannabinol (2.5–7.5 mg kg−1) reduced hippocampal extracellular acetylcholine concentration and impaired working memory in rats.

Both effects were antagonized not only by the CB1 cannabinoid receptor antagonist SR141716A (0.5 mg kg−1, i.p.) but also unexpectedly by the D2 dopamine receptor antagonist S(−)‐sulpiride (5, 10 and 25 mg kg−1, i.p.). Conversely, Δ9‐tetrahydrocannabinol‐induced memory impairment and inhibition of hippocampal extracellular acetylcholine concentration were potentiated by the subcutaneous administration of the D2 dopamine receptor agonist (−)‐quinpirole (25 and 500 μg kg−1). The inhibition of hippocampal extracellular acetylcholine concentration and working memory produced by the combination of (−)‐quinpirole and Δ9‐tetrahydrocannabinol was suppressed by either SR141716A or S(−)‐sulpiride.

Our findings suggest that impairment of working memory and inhibition of hippocampal extracellular acetylcholine concentration are mediated by the concomitant activation of D2 dopamine and CB1 cannabinoid receptors, and that D2 dopamine receptor antagonists may be useful in the treatment of the cognitive deficits induced by marijuana.

British Journal of Pharmacology (2000) 130, 1201–1210; doi:10.1038/sj.bjp.0703413
Effect of amygdala kindling on the central nervous system effects of tiagabine: EEG effects versus brain GABA levels
Authors: Cleton A , Altorf B A , Voskuyl R A , Danhof M
Published: January 2005

The objective of this investigation was to determine the influence of amygdala kindling on the pharmacodynamics of tiagabine in vivo, using quantitative EEG parameters and extracellular GABA concentrations as pharmacodynamic endpoints. In integrated pharmacokinetic/pharmacodynamic (PK/PD) studies the time course of these effects was determined in conjunction with plasma concentrations following intravenous administration of 10 mg kg−1. An ‘effect compartment’ model was used to derive individual concentration–effect relationships.

Tiagabine produced an increase in the amplitude of the 11.5–30 Hz frequency band of the EEG. The relationship between concentration and EEG effect was non‐linear and described by the Hill equation. In kindled rats the EC50 was reduced to 291 ng ml−1 from the original value of 521 ng ml−1 in controls. The values of all other parameters were unchanged.

In kindled rats the baseline extracellular GABA concentration was increased to 1.58 μM from 0.74 μM in controls. The relationships between tiagabine concentration and extracellular GABA concentration were again non‐linear and described by the Hill equation. No differences were observed between kindled rats and controls. In the synaptoneurosmal preparation in vitro no changes in the functioning of the GABA transporter were observed.

It is concluded that unlike the situation with midazolam, there is no resistance to the EEG effect of tiagabine in the kindling model of experimental epilepsy. The observed shift in the concentration–EEG effect relationship to lower concentrations can presumably be explained by the increase in the baseline GABA levels.

British Journal of Pharmacology (2000) 130, 1037–1044; doi:10.1038/sj.bjp.0703417
Inhibition of NFκB‐mediated pro‐inflammatory gene expression in rat mesangial cells by the enolized 1,3‐dioxane‐4,6‐dione‐5‐carboxamide, CGP‐43182
Authors: Scholz‐Pedretti Kirsten , Eberhardt Wolfgang , Rupprecht Gerhard , Beck Karl‐Friedrich , Spitzer Silke , Pfeilschifter Josef , Kaszkin Marietta
Published: January 2005

CGP‐43182 has been described as a potent inhibitor of group IIA secreted phospholipase A2 (group IIA sPLA2) activity in vitro. In rat mesangial cells, inhibition of group IIA sPLA2 activity by CGP‐43182 results in a 70% reduction of cytokine‐stimulated prostaglandin E2 biosynthesis, suggesting that group IIA sPLA2 participates in arachidonic acid release and eicosanoid formation. Under these conditions the cytosolic phospholipase A2 is not affected.

In mesangial cells, in addition to inhibition of catalytic activity, the membrane‐permeant CGP‐43182 completely blocked interleukin 1β (IL1β)‐stimulated group IIA sPLA2 gene expression.

A further action of CGP‐43182 was a complete inhibition of cyclo‐oxygenase‐2 gene expression, resulting in a drastic reduction of prostaglandin formation in mesangial cells.

Moreover, CGP‐43182 completely blocked IL1β‐induced gene expression of the inducible nitric oxide synthase, leading to an inhibition of cytokine‐stimulated nitric oxide formation.

In contrast, the stimulatory effect of the cell‐permeant cyclic AMP‐analogue, dibutyryl‐cAMP, on the induction of these enzymes was not inhibited by CGP‐43182. These data indicate that CGP‐43182 interferes with IL1β‐ but not cyclic AMP‐activated transcriptional regulation.

By studying components of the upstream transcription machinery, we observed an inhibition of NFκB activation by CGP‐43182 in IL1β‐treated cells. Moreover, we observed that CGP‐43182 prevented the phosphorylation and proteolytic degradation of the endogenous NFκB inhibitor, IκB, a process necessary for NFκB activation.

From our data, we propose that CGP‐43182 is a potent anti‐inflammatory drug useful for preventing the consequences of a concerted action of cytokine‐stimulated pro‐inflammatory genes mediated by NFκB.

British Journal of Pharmacology (2000) 130, 1183–1190; doi:10.1038/sj.bjp.0703419
Stimulus‐specific alteration of the relationship between cytosolic Ca2+ transients and nitric oxide production in endothelial cells ex vivo
Authors: Mizuno Osamu , Kobayashi Sei , Hirano Katsuya , Nishimura Junji , Kubo Chiharu , Kanaide Hideo
Published: January 2005

To investigate the quantitative relationship between elevation in the intracellular Ca2+ concentration ([Ca2+]i) and nitric oxide (NO) production, the changes in [Ca2+]i and NO production were determined in parallel, using fluorimetry of fura‐2 and 2,3‐diaminonaphthalene, respectively, in endothelial cells ex vivo of pig aortic valves.

The extent of [Ca2+]i elevation was quantitatively assessed by two parameters: the level of peak [Ca2+]i elevation and the area under the [Ca2+]i curve during treatment (the integrated [Ca2+]i elevation). The amount of NO production was expressed as a percentage of that obtained with 10 μM ATP for 3 min.

ATP, bradykinin, thrombin, and ionomycin were used as stimulation to induce NO production, and all these caused [Ca2+]i increases and NO production in a concentration‐dependent manner.

The relationships between the peak [Ca2+]i and NO production or between the integrated [Ca2+]i elevation and NO production were well described by a straight line. However, the slope value of the linear relationship in both cases varied with the type of stimulation, with thrombin giving the greatest value, followed by ATP, bradykinin and ionomycin.

These data suggest that in endothelial cells ex vivo: (1) [Ca2+]i elevation regulates NO production, but (2) the peak [Ca2+]i elevation‐ or the integrated [Ca2+]i elevation‐NO production relationships varies depending on the type of agonists. Our results thus demonstrate the presence of the agonists‐dependent modulation of the relationship between [Ca2+]i elevation and NO production in endothelial cells ex vivo.

British Journal of Pharmacology (2000) 130, 1140–1146; doi:10.1038/sj.bjp.0703420
Comparison of enalapril and valsartan in cyclosporine A‐induced hypertension and nephrotoxicity in spontaneously hypertensive rats on high‐sodium diet
Authors: Lassila Markus , Finckenberg Piet , Pere Anna‐Kaisa , Krogerus Leena , Ahonen Juhani , Vapaatalo Heikki , Nurminen Marja‐Leena
Published: January 2005

We compared the effects of the angiotensin converting enzyme (ACE) inhibitor enalapril and the angiotensin AT1 receptor antagonist valsartan in cyclosporine A (CsA)‐induced hypertension and nephrotoxicity in spontaneously hypertensive rats (SHR).

SHR (8–9 weeks old) on high‐sodium diet were given CsA (5 mg kg−1d −1 s.c.) for 6 weeks. The rats were treated concomitantly either with enalapril (30 mg kg−1d −1 p.o.) or valsartan (3 or 30 mg kg−1 d −1 p.o.). To evaluate the role of bradykinin in the action of enalapril, some rats received a bradykinin B2 receptor antagonist icatibant (HOE 140, 500 μg kg−1 d −1 s.c.) during the last 2 weeks of enalapril treatment.

Blood pressure was recorded every second week by tail cuff method. Renal function was measured by serum creatinine, creatinine clearance and urinary excretion of proteins at the end of the experiment. The activity of the renal kallikrein‐kinin system was estimated by urinary kallikrein excretion.

CsA caused hypertension, impaired renal function and induced morphological nephrotoxicity with glomerular damage and interstitial fibrosis.

Enalapril and the lower dose of valsartan attenuated the CsA‐induced hypertension to the same extent, while the higher dose of valsartan totally abolished it. Icatibant did not reduce the antihypertensive effect of enalapril. Urinary kallikrein excretion was similar in all groups. Enalapril and valsartan equally prevented the CsA‐induced deterioration of kidney function and morphology.

The renin‐angiotensin but not the kallikrein‐kinin system plays a crucial role in CsA‐toxicity during high intake of sodium in SHR.

British Journal of Pharmacology (2000) 130, 1339–1347; doi:10.1038/sj.bjp.0703422
On the mechanism of ADP‐induced alteration of sulphonylurea sensitivity in cardiac ATP‐sensitive K+ channels
Authors: Miyamura Akihiro , Kakei Masafumi , Ichinari Kotaro , Okamura Midori , Oketani Naoya , Tei Chuwa
Published: January 2005

To study the mechanism of regulation of sulphonylurea sensitivity in ATP‐sensitive K+ (KATP) channels, we used the inside‐out patch clamp technique in guinea‐pig ventricular myocytes.

In the absence of nucleotides, the half maximal concentration of tolbutamide inhibition of KATP channels (IC50) was 0.4 mM, and it decreased to 0.1 mM when 0.1 mM ATP was added.

Increasing the ADP concentration from 0 to 0.1 and 0.3 mM in the absence of ATP shifted the IC50 from 0.4 to 5.3 and 11.4 mM, respectively. Increasing the ADP concentration further to 1 and 3 mM conversely reduced the IC50 to 9.5 and 4.4 mM, respectively.

In the absence of Mg2+ and ADP, the IC50 was calculated to 16.6 mM which was found to be less, 12.3, 5.1 and 2.5 mM, respectively, when the ADP concentration was increased to 0.1, 0.3 and 1 mM.

The IC50s for tolbutamide obtained at various concentrations of ADP in the presence of Mg2+ were best fitted by equations reflecting a model that assumed two binding sites for ADP; one is a high affinity site that reduces the sensitivity to the sulphonylurea, while the other is a low affinity site that increases such sensitivity. Dissociation constants calculated for ADP to sites 1 and 2 were 2.6 μM and 46.7 mM, respectively. In the absence of Mg2+, data were fitted by equations corresponding to a single site model (site 2); the dissociation constant for ADP was 25.0 mM.

It is concluded that ADP modifies tolbutamide sensitivity by binding to two sites. The high affinity site is strongly Mg2+‐dependent, whereas the low affinity site is Mg2+‐independent.

British Journal of Pharmacology (2000) 130, 1411–1417; doi:10.1038/sj.bjp.0703423
The emetic and anti‐emetic effects of the capsaicin analogue resiniferatoxin in Suncus murinus, the house musk shrew
Authors: Andrews P L R , Okada F , Woods A J , Hagiwara H , Kakaimoto S , Toyoda M , Matsuki N
Published: January 2005

In Suncus murinus the ultrapotent capsaicin analogue resiniferatoxin (RTX) induced an emetic response in the dose range 1–1000 μg kg−1, s.c. The latency was inversely related to dose and ranged from 41.2±4.4 min. (1 μg kg−1, s.c.) to 2.7±0.6 min. (1000 μg kg−1, s.c.).

The emetic response to RTX (10 or 100 μg kg−1, s.c.) was blocked or markedly reduced by pre‐treatment with RTX (100 μg kg−1, s.c.), 8‐OH‐DPAT (100 μg kg−1, s.c.), morphine (2 mg kg−1, s.c.), neonatal capsaicin (100 mg kg−1, s.c.) and the NK1 receptor antagonist CP‐99,994 (10–20 mg kg−1, s.c.) but not by the 5‐HT3 receptor antagonist tropisetron (200 μg kg−1, s.c.).

RTX (100 μg kg−1, s.c.) induced c‐fos‐like immunoreactivity in the area postrema and parts of the nucleus tractus solitarius. This pattern is consistent with the proposal that the emetic effect is mediated via one or both of these structures and an involvement of substance P is discussed.

RTX (10 and 100 μg kg−1, s.c.) had broad‐spectrum antiemetic effects in Suncus as indicated by its ability to block or markedly reduce the emetic response to motion (1 Hz, 4 cm lateral, 10 min.), cisplatin (20 mg kg−1, i.p.), intragastric copper sulphate (40 mg kg−1, p.o.), nicotine (10 mg kg−1, s.c.) and RTX (100 μg kg−1, s.c.) itself.

It is proposed that the site of the anti‐emetic effect is in the nucleus tractus solitarius and mechanisms involving the modulation of substance P release are discussed.

The general utility of Suncus for investigations of vanilloid receptors is reviewed in the light of the exquisite sensitivity of the emetic reflex in this species to resiniferatoxin.

British Journal of Pharmacology (2000) 130, 1247–1254; doi:10.1038/sj.bjp.0703428
Effect of furanocoumarin derivatives in grapefruit juice on the uptake of vinblastine by Caco‐2 cells and on the activity of cytochrome P450 3A4
Authors: Ohnishi Ayako , Matsuo Hirotami , Yamada Shiho , Takanaga Hitomi , Morimoto Satoshi , Shoyama Yukihiro , Ohtani Hisakazu , Sawada Yasufumi
Published: January 2005

The presence of inhibitors of drug efflux transporters, such as P‐glycoprotein (P‐gp), in grapefruit juice (GFJ) was confirmed based on the uptake of [3H]‐vinblastine (VBL) by Caco‐2 cells.

The uptake of [3H]‐VBL by Caco‐2 cells was significantly increased by the ethyl acetate extract of GFJ as well as by cyclosporin A. The extract was separated on a Cosmosil column and the eluate with 60% methanol increased [3H]‐VBL uptake, while the activity to inhibit CYP3A4 was greatest in the 70 and 80% eluates.

These results show that the major inhibitor of efflux transport of VBL is different from that of CYP3A4.

Further separation of the 60% methanol eluate afforded dihydroxybergamottin (DHBG). Both ethyl acetate extract of GFJ and DHBG increased steady‐state [3H]‐VBL uptake by LLC‐GA5‐COL300 cells. Besides DHBG, other furanocoumarins contained in GFJ, such as bergamottin, FC726, bergaptol and bergapten, increased the steady‐state uptake of [3H]‐VBL by Caco‐2 cells.

The order of inhibitory potency of these compounds was FC726>DHBG>bergamottin>bergapten>bergaptol. While, the IC50 values for inhibition of CYP3A4 were 0.075, 0.45, 1.0, 1.0 and >20 μM, respectively. Bergaptol specifically inhibited VBL efflux.

DHBG was thus identified as a candidate for inhibitors of VBL transport, together with other furanocoumarins. Moreover, partly involvement of the P‐gp inhibition was suggested.

Therefore, the inhibition of efflux transport of drugs as well as of drug metabolism by CYP3A4 could be an important cause of drug‐GFJ interaction.

British Journal of Pharmacology (2000) 130, 1369–1377; doi:10.1038/sj.bjp.0703433
Inhibition by ethanol of rat P2X4 receptors expressed in Xenopus oocytes
Authors: Xiong Keming , Li Chaoying , Weight Forrest F
Published: January 2005

The effect of ethanol on the function of P2X4 receptors expressed in Xenopus oocytes was studied using two‐electrode voltage‐clamp recording.

The amplitude of current activated by 1 μM ATP was decreased by ethanol in a concentration‐dependent manner over the concentration range 1–500 mM. The concentration of ethanol that produced 50% inhibition (IC50) of current activated by 1 μM ATP was 58 mM.

Ethanol inhibition of ATP‐activated current was not dependent on membrane potential from −60 to +20 mV, and ethanol did not change the reversal potential of ATP‐activated current.

Ethanol, 50 mM, shifted the ATP concentration‐response curve to the right, increasing the EC50 for ATP from 9.1 to 16.0 μM, but did not reduce the maximal response to ATP.

The results suggest that ethanol may inhibit P2X4 receptors by decreasing the apparent affinity of the binding site for ATP.

Since the P2X4 receptor is the most abundant P2X subunit in the brain, these receptors could be important effectors of ethanol action in the central nervous system.

British Journal of Pharmacology (2000) 130, 1394–1398; doi:10.1038/sj.bjp.0703439
Voltage‐dependent blockade of normal and mutant muscle sodium channels by benzylalcohol
Authors: Haeseler G , Mamarvar M , Bufler J , Dengler R , Hecker H , Aronson J K , Piepenbrock S , Leuwer M
Published: January 2005

We studied the effects of benzylalcohol on heterologously expressed wild type (WT), paramyotonia congenita (R1448H) and hyperkalaemic periodic paralysis (M1360V) mutant α‐subunits of human skeletal muscle sodium channels.

Benzylalcohol blocked rested channels at −150 mV membrane potential, with an ECR50 of 5.3 mM in wild type, 5.1 mM in R1448H, and 6.2 mM in M1360V. When blockade was assessed at −100 mV, the ECR50 was reduced in R1448H (2 mM) compared with both wild type (4.3 mM; P<0.01) and M1360V (4.3 mM).

Membrane depolarization before the test depolarization significantly promoted benzylalcohol‐induced sodium channel blockade. The values of KD for the fast‐inactivated state derived from benzylalcohol‐induced shifts in steady‐state availability curves were 0.66 mM in wild type and 0.58 mM in R1448H. In the presence of slow inactivation induced by 2.5 s depolarizing prepulses, the ECI50 for benzylalcohol‐induced current inhibition was 0.59 mM in wild type and 0.53 mM in R1448H.

Recovery from fast inactivation was prolonged in the presence of drug in all clones.

Benzylalcohol induced significant frequency‐dependent block at stimulating frequencies of 10, 50, and 100 Hz in all clones.

Our results clearly show that benzylalcohol is an effective blocker of muscle sodium channels in conditions that are associated with membrane depolarization. Mutants that enter voltage‐dependent inactivation at more hyperpolarized membrane potentials compared with wild type are more sensitive to inhibitory effects at the normal resting potential.

British Journal of Pharmacology (2000) 130, 1321–1330; doi:10.1038/sj.bjp.0703447
NO‐naproxen modulates inflammation, nociception and downregulates T cell response in rat Freund's adjuvant arthritis
Authors: Cicala Carla , Ianaro Angela , Fiorucci Stefano , Calignano Antonio , Bucci Mariarosaria , Gerli Roberto , Santucci Luca , Wallace John L , Cirino Giuseppe
Published: January 2005

Anti‐inflammatory non steroidal drugs releasing NO (NO‐NSAIDs) are a new class of anti‐inflammatory drugs to which has been added an NO‐releasing moiety. These compounds have been shown to retain the anti‐inflammatory, analgesic and antipyretic activity of the parent compound but to be devoid of gastrointestinal (GI) toxicity.

Freund's adjuvant (FA) arthritis was induced in rats by a single intraplantar injection into the right hindpaw of 100 μl of mycobacterium butirricum (6 mg ml−1). The effect of equimolar doses of naproxen (1, 3 and 10 mg kg−1) and NO‐naproxen (1.5, 4.5 and 16 mg kg−1) was evaluated using two dosage regimen protocols: (i) preventive, starting oral administration of the drugs at the time of induction of arthritis and for the following 21 days (day 1–21); (ii) therapeutic, starting oral administration of the drugs 7 days after adjuvant injection and for the following 14 days (day 7–21).

Hindpaw swelling (days 3, 7, 11, 14, 17, 21) and nociception (days 15 and 21) were measured. On day 22 rats were sacrificed, draining lymph nodes were removed and T cells isolated. In vitro proliferation of T cells following stimulation with concanavalin A (0.5–5 μg ml−1) was measured using a tritiated thymidine incorporation assay. IL‐2 receptor expression on T cells was measured by FACS analysis.

Naproxen and NO‐naproxen showed similar activity in reducing oedema formation in the non‐injected (controlateral) hindpaw. Both drugs showed anti‐nociceptive effect. NO‐naproxen was anti‐nociceptive at a dose of 4.5 mg kg−1 while naproxen showed the same extent of inhibition only at a dose of 10 mg kg−1.

T cells were isolated and characterized by FACS analysis. Stimulation of isolated T cells with concanavallin A in vitro caused a significant increase in thymidine uptake. NO‐naproxen at a dose of 4.5 mg kg−1 inhibited T cell proliferation to the same extent as 10 mg kg−1 of naproxen.

Inhibition of T cell proliferation was well correlated with reduced IL‐2 receptor expression on T cells. In addition, NO‐naproxen reduced both IL‐1β and TNFα plasma levels whilst naproxen reduced IL‐1β levels only.

In conclusion, both naproxen and NO‐naproxen reduce inflammation and nociception associated with arthritis. In addition NO‐naproxen interferes to a larger extent with cellular mechanism involved in T cell activation in rat adjuvant arthritis indicating that introduction of the NO moiety in the naproxen structure increases the effect at the level of the immune system.

British Journal of Pharmacology (2000) 130, 1399–1405; doi:10.1038/sj.bjp.0703449
Characterization of SB‐271046: A potent, selective and orally active 5‐HT6 receptor antagonist
Authors: Routledge Carol , Bromidge Steven M , Moss Stephen F , Price Gary W , Hirst Warren , Newman Helen , Riley Graham , Gager Tracey , Stean Tania , Upton Neil , Clarke Stephen E , Brown Anthony M , Middlemiss Derek N
Published: January 2005

SB‐271046, potently displaced [3H]‐LSD and [125I]‐SB‐258585 from human 5‐HT6 receptors recombinantly expressed in HeLa cells in vitro (pKi 8.92 and 9.09 respectively). SB‐271046 also displaced [125I]‐SB‐258585 from human caudate putamen and rat and pig striatum membranes (pKi 8.81, 9.02 and 8.55 respectively).

SB‐271046 was over 200 fold selective for the 5‐HT6 receptor vs 55 other receptors, binding sites and ion channels.

In functional studies on human 5‐HT6 receptors SB‐271046 competitively antagonized 5‐HT‐induced stimulation of adenylyl cyclase activity with a pA2 of 8.71.

SB‐271046 produced an increase in seizure threshold over a wide‐dose range in the rat maximal electroshock seizure threshold (MEST) test, with a minimum effective dose of 0.1 mg kg−1 p.o. and maximum effect at 4 h post‐dose. The level of anticonvulsant activity achieved correlated well with the blood concentrations of SB‐271046 (EC50 of 0.16 μM) and brain concentrations of 0.01–0.04 μM at Cmax.

These data, together with the observed anticonvulsant activity of other selective 5‐HT6 receptor antagonists, SB‐258510 (10 mg kg−1, 2–6 h pre‐test) and Ro 04‐6790 (1–30 mg kg−1, 1 h pre‐test), in the rat MEST test, suggest that the anticonvulsant properties of SB‐271046 are likely to be mediated by 5‐HT6 receptors.

Overall, these studies demonstrate that SB‐271046 is a potent and selective 5‐HT6 receptor antagonist and is orally active in the rat MEST test. SB‐271046 represents a valuable tool for evaluating the in vivo central function of 5‐HT6 receptors.

British Journal of Pharmacology (2000) 130, 1606–1612; doi:10.1038/sj.bjp.0703457
Effect of venotropic drugs on the respiratory activity of isolated mitochondria and in endothelial cells
Authors: Janssens Dominique , Delaive Edouard , Houbion Andrée , Eliaers François , Remacle José , Michiels Carine
Published: January 2005

Several drugs used in the treatment of chronic peripheral ischaemic and venous diseases, i.e. aescine, Cyclo 3, Ginkor Fort, hydroxyethylrutosides, naftidrofuryl, naphthoquinone and procyanidolic oligomers, were tested on the mitochondrial respiratory activity.

The results show that all these drugs protected human endothelial cells against the hypoxia‐induced decrease in ATP content. In addition, they all induced a concentration‐dependent increase in respiratory control ratio (RCR) of liver mitochondria pre‐incubated with the drugs for 60 min.

The drugs were divided into two groups according to their effects. The first group (A), comprising aescine, Ginkor Fort, naftidrofuryl and naphthoquinone, increased RCR by decreasing state 4 respiration rate. The second group of drugs (B), comprising hydroxyethylrutosides, procyanidolic oligomers and Cyclo 3, increased RCR by increasing state 3 respiration rate. The drugs of group A were able to prevent the inhibition of complexes I and III respectively by amytal and antimycin A while the first two drugs of group B increased adenine nucleotide translocase activity. Cyclo 3 inhibited the carbonylcyanide m‐chlorophenyl hydrazone (mCCP)‐induced uncoupling of mitochondrial respiration. None of these seven drugs could protect complexes IV and V, respectively, from inhibition by cyanide and oligomycin.

When tested on endothelial cells the drugs of group A, in contrast to group B, prevented the decrease in ATP content induced by amytal or antimycin A.

The present results suggest that the protective effects on mitochondrial respiration activity by these venotropic drugs may explain their protective effect on the cellular ATP content in ischaemic conditions and some of their beneficial therapeutic effect in chronic vascular diseases.

British Journal of Pharmacology (2000) 130, 1513–1524; doi:10.1038/sj.bjp.0703461
Orphanin FQ/nociceptin and [Phe1Ψ(CH2‐NH)Gly2] nociceptin(1‐13)‐NH2 stimulate gastric motor function in anaesthetized rats
Authors: Krowicki Zbigniew K , Kapusta Daniel R , Hornby Pamela J
Published: January 2005

Orphanin FQ/nociceptin (OFQ/N) is a preferred endogenous ligand for the orphan opioid receptor‐like‐1 receptor. This peptide has been reported to increase intestinal, but not gastric, motor activity.

In the present study, OFQ/N (0.6–60 nmol kg−1 i.v.) increased intragastric pressure and antral contractility and, as expected, decreased blood pressure in anaesthetized rats.

The gastric motor effects of OFQ/N (6 nmol kg−1) were not affected by inhibition of nitric oxide synthase or opioid receptor blockade.

OFQ/N (6 nmol kg−1) evoked gastric motor increases and hypotension were not affected by prior administration of its derivative [Phe1Ψ(CH2‐NH)Gly2]nociceptin‐(1‐13)‐NH2 unless the pseudopepotide was administered shortly (5 min) prior to OFQ/N. This putative antagonist (6–300 nmol kg−1) alone increased antral motility with approximately 100 fold lower potency than OFQ/N.

Neither bilateral vagotomy nor spinal cord transection altered OFQ/N‐evoked increases in intragastric pressure and antral contractility.

In conclusion, OFQ/N induces gastric motor excitation in addition to its known effects to increase intestinal motility. The gastric responses to OFQ/N are not dependent on ‘classical’ opioid receptor activation or nitric oxide, similar to the case for the intestines. The primary site of action of OFQ/N on the stomach is probably via enteric nerves, since central descending vagal or sympathetic pathways are not necessary for OFQ/N to increase gastric motility. The gastric motor effects of the derivative [Phe1Ψ(CH2‐NH)Gly2]nociceptin‐(1‐13)‐NH2 are similar to OFQ/N, although with lower potency. The effects of the derivative as a partial agonist or antagonist in different experimental paradigms may reflect tissue OFQ/N receptor reserve.

British Journal of Pharmacology (2000) 130, 1639–1645; doi:10.1038/sj.bjp.0703463
Inhibition of HERG potassium channels by the antimalarial agent halofantrine
Authors: Tie H , Walker B D , Singleton C B , Valenzuela S M , Bursill J A , Wyse K R , Breit S N , Campbell T J
Published: January 2005

Halofantrine is a widely used antimalarial agent which has been associated with prolongation of the ‘QT interval’ of the electrocardiogram (ECG), torsades de pointes and sudden death. Whilst QT prolongation is consistent with halofantrine‐induced increases in cardiac ventricular action potential duration, the cellular mechanism for these observations has not been previously reported.

The delayed rectifier potassium channel, IKr, is a primary site of action of drugs causing QT prolongation and is encoded by the human‐ether‐a‐go‐go‐related gene (HERG). We examined the effects of halofantrine on HERG potassium channels stably expressed in Chinese hamster ovary (CHO‐K1) cells.

Halofantrine blocked HERG tail currents elicited on repolarization to −60 mV from +30 mV with an IC50 of 196.9 nM. The therapeutic plasma concentration range for halofantrine is 1.67–2.98 μM.

Channel inhibition by halofantrine exhibited time‐, voltage‐ and use‐dependence. Halofantrine did not alter the time course of channel activation or deactivation, but inactivation was accelerated and there was a 20 mV hyperpolarizing shift in the mid‐activation potential of steady‐state inactivation. Block was enhanced by pulses that render channels inactivated, and channel blockade increased with increasing duration of depolarizing pulses.

We conclude that HERG channel inhibition by halofantrine is the likely underlying cellular mechanism for QT prolongation. Our data suggest preferential binding of halofantrine to the open and inactivated channel states.

British Journal of Pharmacology (2000) 130, 1967–1975; doi:10.1038/sj.bjp.0703470
FK506 potently inhibits T cell activation induced TNF‐α and IL‐1β production in vitro by human peripheral blood mononuclear cells
Authors: Sakuma Shozo , Kato Yasuko , Nishigaki Fusako , Sasakawa Tatsuya , Magari Katsue , Miyata Susumu , Ohkubo Yoshitaka , Goto Toshio
Published: January 2005

The aim of this study was to elucidate the in vitro inhibitory potency of FK506 on production of the inflammatory cytokines, tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β, with a view to assessing this immunosuppressive agent as a potential anti‐rheumatic drug.

We employed an in vitro model which produces TNF‐α and IL‐1β through T cell activation. Human peripheral blood mononuclear cells (PBMC) were cultured with immobilized anti‐CD3/CD28 monoclonal antibody in this model.

FK506 inhibited anti‐CD3/CD28 induced TNF‐α and IL‐1β production at concentrations less than 1 ng ml−1. Flow cytometric analysis of intracellular TNF‐α and IL‐1β positive cells showed that FK506 potently suppresses inflammatory cytokine production from CD14+ monocytes as well as from T cells.

Cyclosporin A (CsA) and dexamethasone (DEX) also inhibited the anti‐CD3/CD28 induced cytokine production, but were less potent than FK506. FK506 and CsA, but not DEX, specifically inhibited anti‐CD3/CD28 induced inflammatory cytokine production without affecting the lipopolysaccaride (LPS) induced effect. Methotrexate (MTX) was completely inactive for suppressing cytokine production under either condition.

Anti‐CD3/CD28 stimulated PBMC culture supernatants were found to enhance the expression of adhesion molecules in human vascular endothelial cells. FK506, CsA and DEX led to the suppression of adhesion molecule expression probably by inhibiting cytokine production from PBMC.

The inhibitory potency of agents on TNF‐α and IL‐1β production was compared with cytotoxicity and FK506 was not cytotoxic at concentrations several orders of magnitude greater than those required for cytokine inhibition.

These results strongly suggest that FK506 may be most effective to specifically prevent T cell activation mediated inflammatory cytokine production in a clinical setting.

British Journal of Pharmacology (2000) 130, 1655–1663; doi:10.1038/sj.bjp.0703472