bluuebunny/crossref_metadata_2025 huggingface dataset:
Bisphenol A diglycidyl ether (BADGE) is a PPARγ agonist in an ECV304 cell line
Authors: Bishop‐Bailey David , Hla Timothy , Warner Timothy D
Published: January 2005

Peroxisome proliferator activated receptors (PPAR)s are nuclear transcription factors of the steroid receptor super‐family. One member, PPARγ, a critical transcription factor in adipogenesis, is expressed in ECV304 cells, and when activated participates in the induction of cell death by apoptosis. Here we describe a clone of ECV304 cells, ECV‐ACO.Luc, which stably expresses a reporter gene for PPAR activation. ECV‐ACO.Luc respond to the PPARγ agonists, 15‐deoxy‐Δ12,14 PGJ2, and ciglitizone, by inducing luciferase expression. Furthermore, using ECV‐ACO.Luc, we demonstrate that a newly described PPARγ antagonist, bisphenol A diglycidyl ether (BADGE) has agonist activities. Similar to 15‐deoxy‐Δ12,14 PGJ2, BADGE induces PPARγ activation, nuclear localization of the receptor, and induces cell death.

British Journal of Pharmacology (2000) 131, 651–654; doi:10.1038/sj.bjp.0703628
Caspase‐1‐inhibitor ac‐YVAD‐cmk reduces LPS‐lethality in rats without affecting haematology or cytokine responses
Authors: Mathiak Guenther , Grass Guido , Herzmann Thomas , Luebke Thomas , Zetina Cecilia Cu , Boehm Sebastian A , Bohlen Heribert , Neville Lewis F , Hoelscher Arnulf H
Published: January 2005

The effect of acetyl–tyrosyl‐valyl‐alanyl‐aspartyl–chloromethylketone (ac‐YVAD‐cmk), an irreversible caspase‐1 (IL‐1β converting enzyme, ICE) inhibitor on mortality, leukocyte and platelet counts and cytokine levels was investigated in a double‐blind rat model of endotoxaemia. Intravenous (i.v.) bolus administration of lipopolysaccharide (LPS) (25–75 mg kg−1, n=12 per group) to anaesthetized rats induced a dose dependent increase in mortality over 8 h (LD50=48 mg kg−1). During this period, animals became leukopenic and thrombocytopenic. Serum levels of IL‐β, IL‐6, and TNF‐α were highly elevated. Pretreatment of rats with ac‐YVAD‐cmk at a dose of 12.5 μmol kg−1 significantly reduced mortality from 83 to 33% using Log Rank analysis. However, ac‐YVAD‐cmk did not modify blood cell counts or cytokine profiles as compared with the LPS‐drug vehicle group. These data lay credence to the potential importance of caspase‐1‐inhibition in modifying the inflammatory response to endotoxin. Further investigations are warranted in understanding the relationship between caspase‐1 inhibition, cytokine production and animal survival in different experimental paradigms of sepsis.

British Journal of Pharmacology (2000) 131, 383–386; doi:10.1038/sj.bjp.0703629
Cyclo‐oxygenase‐1 and −2 contribution to endothelial dysfunction in ageing
Authors: Heymes Christophe , Habib Aida , Yang Di , Mathieu Eric , Marotte Françoise , Samuel JaneLyse , Boulanger Chantal M
Published: January 2005

Experiments were designed to investigate the role of cyclo‐oxygenase isoforms in endothelial dysfunction in ageing. Aortic rings with endothelium of aged and young (24 vs 4 month‐old) Wistar rats, were mounted in organ chambers for the recording of changes in isometric tension.

In young rats, acetylcholine (ACh) caused a complete relaxation which was not affected by indomethacin (0.3 μM), NS‐398 (a preferential COX‐2 inhibitor; 1 μM), SQ‐29548 (a thromboxane‐receptor antagonist; 1 μM), nor valeryl‐salicylate (VAS, a preferential inhibitor of COX‐1; 3 mM).

In aged rats, ACh caused a biphasic response characterized by a first phase of relaxation (0.01–1 μM ACh), followed by a contraction (3–100 μM ACh). Indomethacin, NS‐398 and SQ‐29548, but not VAS, augmented the first phase. Indomethacin, VAS, NS‐398 and SQ‐29548 decreased the contractions to high ACh concentrations. Then, the sensitivity to thromboxane receptor activation was investigated with U‐46619. The results show comparable EC50 values in young and aged rats.

In aged rats, the ACh‐stimulated release of prostacyclin, prostaglandin F2α and thromboxane A2 was decreased by either indomethacin, NS‐398, VAS or endothelium removal. However, in young animals, the ACh‐stimulated release of prostacyclin and prostaglandin F2α were smaller than in older animals and remained unaffected by NS‐398.

Aortic endothelial cells from aged – but not young – rats express COX‐2 isoform, while COX‐1 labelling was observed in endothelial cells from both young and aged rats.

These data demonstrate the active contribution of COX‐1 and −2 in endothelial dysfunction associated with ageing.

British Journal of Pharmacology (2000) 131, 804–810; doi:10.1038/sj.bjp.0703632
An orally active anti‐apoptotic molecule (CGP 3466B) preserves mitochondria and enhances survival in an animal model of motoneuron disease
Authors: Sagot Y , Toni N , Perrelet D , Lurot S , King B , Rixner H , Mattenberger L , Waldmeier P C , Kato A C
Published: January 2005

Apoptosis and mitochondrial dysfunction are thought to be involved in the aetiology of neurodegenerative diseases. We have tested an orally active anti‐apoptotic molecule (CGP 3466B) that binds to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) in an animal model with motoneuron degeneration, i.e. a mouse mutant with progressive motor neuronopathy (pmn).

In pmn/pmn mice, CGP 3466B was administered orally (10–100 nmol kg−1) at the onset of the clinical symptoms (2 weeks after birth). CGP 3466B slowed disease progression as determined by a 57% increase in life‐span, preservation of body weight and motor performance.

This improvement was accompanied by a decreased loss of motoneurons and motoneuron fibres as well as an increase in retrograde transport. Electron microscopic analysis showed that CGP 3466B protects mitochondria which appear to be selectively disrupted in the motoneurons of pmn/pmn mice.

The data support evaluation of CGP 3466B as a potential treatment for motor neuron disease.

British Journal of Pharmacology (2000) 131, 721–728; doi:10.1038/sj.bjp.0703633
In vivo transfer of antisense oligonucleotide against urinary kininase blunts deoxycorticosterone acetate‐salt hypertension in rats
Authors: Hayashi Izumi , Majima Masataka , Fujita Tomoe , Okumura Toshiaki , Kumagai Yuji , Tomita Naruya , Morishita Ryuichi , Higaki Jitsuo , Ogiwara Toshio
Published: January 2005

We have previously reported that the renal kallikrein‐kinin system suppressed the development of deoxycorticosterone acetate (DOCA)‐salt hypertension. Kinins were degraded in the kidney mainly by carboxypeptidase Y (CPY)‐like kininase. Blockade of renal kinin degradation may reduce hypertension in the developmental stage.

We constructed an antisense oligonucleotide against rat CPY homologue (5′‐CAT‐CTC‐TGC‐TTC‐CTT‐GTG‐TC‐3′, AS) and its randomized control oligonucleotide (5′‐TCC‐TTC‐CTG‐CTT‐GAG‐TTC‐CT‐3′, RC), and prepared an HVJ‐liposome complex that prolongs and increases the effectiveness of the antisense oligonucleotide. Antisense oligonucleotide was transfected (25 nmole rat−1, in terms of nucleotide) into the kidney from the renal artery.

Blood pressure was measured through a catheter inserted into the abdominal aorta. Mean blood pressure (MBP) in DOCA‐salt treated (for 2 weeks) Sprague Dawley strain rats was 130±3 mmHg (n=11), and was reduced significantly (P<0.05) more by AS transfection (122±4 mmHg, n=6) than by RC treatment (137±6 mmHg, n=5) 4 days after the transfection.

This reduction in MBP was accompanied by increased urinary sodium excretion (AS, 8.4±1.5 mmole day−1; RC, 4.6±0.5 mmole day−1, P<0.05) and a reduction in urinary CPY‐like kininase activity.

Ebelactone B (5 mg kg−1, twice a day, p.o.), an inhibitor for urinary CPY‐like kininase, also reduced MBP and induced natriuresis to the same degree as AS.

Lisinopril, an inhibitor for angiotensin converting enzyme (ACE) failed to reduce the elevated MBP.

These results suggest that CPY‐like kininase may have more contribution than ACE to degrade kinin in the kidney, and that knockdown of CPY‐like kininase in the kidney may partly prevent rat DOCA‐salt hypertension.

British Journal of Pharmacology (2000) 131, 820–826; doi:10.1038/sj.bjp.0703634
The interaction between components of the fibrinolytic system and GPIb/V/IX of platelets thrombus formation in mice
Authors: Matsuno Hiroyuki , Kozawa Osamu , Ueshima Shigeru , Matsuo Osamu , Collen Désiré , Uematsu Toshihiko
Published: January 2005

The interaction of fibrinolytic components with GPIb/V/IX of platelets on thrombus formation, was investigated in mice deficient in tissue type (tPA−/−), urokinase type plasminogen activator (uPA−/−) or plasminogen activator inhibitor‐1 (PAI‐1−/−) and in their wild type control (tPA+/+, uPA+/+, PAI‐1+/+).

A thrombus was induced in the murine carotid artery using a photochemical reaction. The times to occlusion after the initiation of endothelial injury in all wild type mice was within 12 min, and no significant changes in occlusion delay were observed in uPA−/− and tPA−/− mice compared to wild type mice, whereas that of PAI‐1 mice were significantly prolonged (16.9±2.9 min, P<0.05).

When high molecular weight aurintricarboxylic acid (ATA), an inhibitor of platelet glycoprotein Ib/V/IX, was administered, the time to occlusion was prolonged in a dose‐dependent manner in all types of mice. However, when this compound was injected in tPA−/− mice, the most significant changes were observed: i.e. the estimated ED50 was 20.2 times higher than that in tPA+/+ mice, but the estimated ED50 in uPA−/− mice was not changed as compared with that of wild type mice. On the other hand, when ATA was injected in PAI‐1−/− mice, the estimated ED50 was significantly decreased (P<0.05).

Platelet aggregation induced by botrocetin was not significantly different among all types of mice. The bleeding time was prolonged in a dose dependent‐manner when ATA was injected in all types of mice.

In conclusion, the antithrombotic effect of inhibition of platelet GPIb/V/IX is severely affected by the absence or presence of tPA‐production on thrombus formation and the inhibition of PAI‐1 could enhance this antithrombotic effect.

British Journal of Pharmacology (2000) 131, 858–864; doi:10.1038/sj.bjp.0703639
Increase in participation of vasoactive intestinal peptide in relaxation of the distal colon of Wistar rats with age
Authors: Takeuchi Tadayoshi , Negoro Tamotsu , Yamaji Michiru , Yamazaki Yasuko , Fujita Akikazu , Nishio Hideaki , Takewaki Tadashi , Takatsuji Koichi , Hata Fumiaki
Published: January 2005

Changes in participation of vasoactive intestinal peptide (VIP) in nonadrenergic noncholinergic (NANC) relaxation of longitudinal muscle of the distal colon with age were studied in 2‐ to 50‐week‐old Wistar rats in vitro.

The extent of the VIP‐mediated component of the relaxation induced by electrical field stimulation (EFS) was determined by the effect of VIP10–28, a VIP receptor antagonist. In 2‐week‐old rats, the extent of the VIP‐mediated component of the relaxation was scarce, about 10%, whereas the component gradually increase with age and reached the maximum extent 66% at 50‐week‐old.

Since our previous results suggest that VIP induces NANC relaxation via activation of charybdotoxin (ChTx, a blocker of large conductance Ca2+‐activated K+ channel)‐sensitive K+ channels with concomitant slow hyperpolarization in the muscle cells, we next studied whether ChTx‐sensitive component and slow hyperpolarization changes with age. Extent of ChTx‐sensitive component of the relaxation increased with age, showing a very similar pattern to VIP‐mediated one.

EFS induced monophasic inhibitory junction potentials (i.j.ps) in longitudinal muscle cells of the distal colon of 2‐ and 4‐week‐old. EFS also induced biphasic i.j.ps in many longitudinal muscle cells of 8‐ and 50‐week‐old: rapid and subsequent slow hyperpolarization. A VIP receptor antagonist selectively inhibited the slow hyperpolarization.

Exogenously added VIP induced no appreciable change in the membrane potential of longitudinal muscle cells of 2‐week‐old, whereas it induced slight slow hyperpolarization of the cell membrane in 4‐week‐old and magnitude of the hyperpolarization increased with age. On the other hand, relaxant response of the longitudinal muscle to exogenously added VIP was high in younger rats.

The present results suggest that the role of VIP in mediating NANC relaxation of longitudinal muscle of the Wistar rat distal colon is very little at neonatal stage, but it increases with age.

British Journal of Pharmacology (2000) 131, 942–948; doi:10.1038/sj.bjp.0703643
Molecular cloning, sequence analysis and pharmacological properties of the porcine 5‐HT1D receptor
Authors: Bhalla Pankaj , Sharma Hari S , Wurch Thierry , Pauwels Petrus J , Saxena Pramod R
Published: January 2005

A cDNA encoding the full‐length 5‐HT1D receptor derived from porcine cerebral cortex was amplified, cloned and sequenced, using guinea‐pig 5‐HT1D receptor coding sequence oligonucleotide primers in reverse transcription‐polymerase chain reaction (RT–PCR). The 5′ and 3′ ends of the porcine 5‐HT1D receptor cDNA were verified by inverse PCR.

Sequence analysis of porcine 5‐HT1D receptor cDNA revealed an open reading frame of 1134 nucleotides encoding a polypeptide of 377 amino acids having 92% homology with the human 5‐HT1D receptor and 88–90% homology with other species homologues.

The porcine 5‐HT1D receptor cDNA was further subcloned into a mammalian expression vector pcDNA3 and expressed in monkey Cos‐7 cells. Radioligand binding assays using either [3H]‐5‐CT or [3H]‐GR125743 on Cos‐7 cell membranes showed that pKi values of 14 serotonin ligands were highly correlated with those obtained with the human 5‐HT1D receptor. Nonetheless, a selective antagonist at the human 5‐HT1D receptor, BRL15572, only poorly recognized the porcine homologue.

Using membranes from cells co‐expressing the porcine 5‐HT1D receptor and rat GαilCys351 Ile protein, it was shown that 5‐HT and zolmitriptan increased, while ketanserin decreased basal [35S]‐GTPγS binding. The potency of zolmitriptan in the [35S]‐GTPγS binding assay (pEC50: 8.46±0.08) agreed with its affinity in displacing the radioligands [3H]‐5‐CT and [3H]‐GR125743 (pKi: 8.38±0.15 and 8.67±0.08, respectively).

In conclusion, we have established the cDNA sequence and pharmacology of the cloned porcine 5‐HT1D receptor. This information would be useful in exploring the role of divergent amino acid residues in the receptor‐ligand interaction as well as the role of 5‐HT1D receptor in pathophysiological processes relevant for novel drug discovery in diseases such as migraine.

British Journal of Pharmacology (2000) 131, 949–957; doi:10.1038/sj.bjp.0703645
Effects of mibefradil and nifedipine on arteriolar myogenic responsiveness and intracellular Ca2+
Authors: Potocnik Simon J , Murphy Timothy V , Kotecha Neela , Hill Michael A
Published: January 2005

Ca2+ entry mechanisms underlying spontaneous arteriolar tone and acute myogenic reactivity remain uncertain. These studies aimed to compare the effects of nifedipine and the putative T‐channel blocker, mibefradil, on arteriolar myogenic responsiveness and intracellular Ca2+ (Ca2+i).

First order cremaster muscle arterioles (1A) were isolated from rats, cannulated, pressurized to 70 mmHg in the absence of intraluminal flow, and mechanical responses studied by video microscopy. The Ca2+i was measured using fluorescence imaging of Fura 2 loaded arterioles.

Both nifedipine and mibefradil showed dose‐dependent inhibition of spontaneous myogenic tone (at 70 mmHg; pEC50 7.04±0.17 vs 6.65±0.20 respectively, n=6 for both, n.s.) and KCl‐induced vasoconstriction (at 70 mmHg; pEC50 6.93±0.38 vs 6.45±0.27 respectively, n=6 for both, n.s.).

In arterioles maintained at 50 mmHg, nifedipine (10−7 and 10−5 M) caused a concentration dependent reduction in Ca2+i, however, mibefradil (10−7 and 10−5 M) had no effect. Furthermore nifedipine significantly attenuated the increase in Ca2+i associated with an acute pressure step (50–120 mmHg) whereas mibefradil was considerably less effective.

Mibefradil (10−7 M) significantly attenuated contractile responses to 60 mM KCl without altering the KCl‐induced increase in Ca2+i, in contrast to nifedipine (10−7 M) which reduced both Ca2+i and contraction.

Membrane potential of arterioles with spontaneous myogenic tone (70 mmHg) was −41.5±1.0 mV. Nifedipine (10−7 or 10−5 M) had no effect on membrane potential, however mibefradil (10−5 M) caused significant depolarization.

In summary, both mibefradil and nifedipine inhibit arteriolar spontaneous tone and acute myogenic reactivity. While there may be overlap in the mechanisms by which these agents inhibit tone, differences in effects on membrane potential and intracellular Ca2+ levels suggest mibefradil exhibits actions other than blockade of Ca2+ entry in skeletal muscle arterioles.

British Journal of Pharmacology (2000) 131, 1065–1072; doi:10.1038/sj.bjp.0703650
Activation of phospholipase D by metabotropic glutamate receptor agonists in rat cerebrocortical synaptosomes
Authors: Shinomura T , Del Río E , Breen K C , Downes C P , McLaughlin M
Published: January 2005

The pharmacological profile of metabotropic glutamate receptor (mGluR) activation of phospholipase D (PLD), and the associated signalling pathways, were examined in rat cerebrocortical synaptosomes. The assay was conducted using a transphosphatidylation reaction in synaptosomes which were pre‐labelled with either [3H]‐arachidonic acid or [32P]‐orthophosphate.

The mGluR agonists (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD) and (RS)‐3,5‐dihydroxyphenylglycine (DHPG), both activated PLD, while phorbol 12,13‐dibutyrate (PDBu) treatment caused receptor‐independent activation of PLD and had an additive effect on 1S,3R‐ACPD induced PLD activity.

A protein kinase C (PKC) inhibitor, GF109203X, failed to antagonize mGluR receptor‐coupled PLD activity. We could not detect any increase in the products of PI (phosphoinositide)‐specific phospholipase C (PI‐PLC), inositol(1,4,5)trisphosphate or diacylglycerol, by 1S, 3R‐ACPD at 15 s. However, diacylglycerol increased monophasically in response to mGluR agonists and remained elevated for at least 15 min. Phosphatidic acid phosphohydrolase (PAP) activity, which converts PA to DAG, was present in the synaptosomes.

These data suggest that, in rat cerebrocortical synaptosomes, the 1S,3R‐ACPD‐sensitive mGluR is coupled to PLD through a mechanism that is independent of both PKC and PI‐PLC.

British Journal of Pharmacology (2000) 131, 1011–1018; doi:10.1038/sj.bjp.0703651
Effects of adenine nucleosides and nucleotides on neuromuscular transmission to the prostatic stroma of the rat
Authors: Preston Ashley , Lau Winnie A K , Pennefather Jocelyn N , Ventura Sabatino
Published: January 2005

The aim of this study was to investigate the effects of adenine nucleosides and nucleotides on contractility of the smooth muscle of rat prostate gland.

Nerve terminals within rat isolated prostatic tissues were electrically field stimulated (60 V, 0.5 ms, 10 Hz, 20 pulses every 60 s). Adenosine 5′‐triphosphate (ATP), adenosine 5′‐diphosphate (ADP), adenosine 5′‐monophosphate (AMP) and adenosine had no effect on baseline smooth muscle tone but concentration‐dependently inhibited electrically‐evoked contractile responses. The relative order of potency was ATP ≅ AMP ≅ adenosine>ADP.

The inhibition by ATP and adenosine of field stimulation‐induced contractions in the rat prostate was antagonized by 8‐phenyltheophylline (10 μM), but not by suramin (100 μM) and only slightly by reactive blue 2 (5 μM).

The adenosine metabolizing enzyme adenosine deaminase (0.1 unit ml−1) inhibited the inhibitory effects of ATP and adenosine. The P2 purinoceptor agonist 2‐methylthio ATP (10 nM–0.1 mM), had no effect on field stimulation‐induced contractions of the rat prostate.

ATP and adenosine did not modify the contractile responses of the rat prostate to exogenously added noradrenaline (10 μM).

Inhibitory concentration‐response curves to a number of adenosine analogues with differing stabilities and selectivities for the different adenosine receptors yielded a relative rank order of agonist potency of: N6‐cyclopentyladenosine (CPA)>N6‐cyclohexyladenosine (CHA) ≅ (−)‐N6‐(2‐phenylisopropyl)‐adenosine (R‐PIA) ≅ 5′‐(N‐ethylcarboxamido)‐adenosine (NECA)>(+)‐N6‐(2‐phenylisopropyl)‐adenosine (S‐PIA)>2‐p‐[2‐carboxyethyl]phenethyl‐amino‐5′‐N‐ethylcarboxamido‐adenosine (CGS 21680).

These results indicate that adenine nucleoside and nucleotide induced inhibition of electrically‐evoked contractions in the rat prostate occurs through activation of adenosine but not ATP receptors. The relative order of potency of adenosine analogues is consistent with activation of receptors of the A1‐adenosine receptor subtype. These receptors appear to be prejunctional.

British Journal of Pharmacology (2000) 131, 1073–1080; doi:10.1038/sj.bjp.0703652
Blockade and reversal of spinal morphine tolerance by peptide and non‐peptide calcitonin gene‐related peptide receptor antagonists
Authors: Powell Kelly J , Ma Weiya , Sutak Maaja , Doods Henri , Quirion Remi , Jhamandas Khem
Published: January 2005

This study examined the effects of the peptide CGRP receptor antagonist CGRP8‐37 and the newly‐developed non‐peptide CGRP receptor antagonist BIBN4096BS for their potential to both inhibit the development and reverse tolerance to the antinociceptive action of morphine.

Repeated administration of intrathecal morphine (15 μg), once daily, produced a progressive decline of antinociceptive effect and an increase in the ED50 value in the tailflick and paw pressure tests. Co‐administration of CGRP8‐37 (4 μg) or BIBN4096BS (0.05, 0.1 μg) with morphine (15 μg) prevented the decline of antinociceptive effect and increase in ED50 value in the tailflick test. Intrathecal administration of the CGRP receptor antagonists did not alter the baseline responses in either tests. Acute CGRP8‐37 also did not potentiate the acute actions of spinal morphine.

In animals rendered tolerant to intrathecal morphine, subsequent administration of CGRP8‐37 (4 μg) with morphine (15 μg) partially restored the antinociceptive effect and ED50 value of acute morphine, reflecting the reversal of tolerance.

Animals tolerant to intrathecal morphine expressed increased CGRP and substance P‐like immunostaining in the dorsal horn of the spinal cord. The increase in CGRP, but not substance P‐like immunostaining, was blocked by a co‐treatment with CGRP8‐37 (4 μg). In animals already tolerant to morphine, the increase in CGRP but not substance P‐like immunostaining was partially reversed by CGRP8‐37 (4 μg).

These data suggest that activation of spinal CGRP receptors contributes to both the development and expression of spinal opioid tolerance. CGRP receptor antagonists may represent a useful therapeutic approach for preventing as well as reversing opioid tolerance.

British Journal of Pharmacology (2000) 131, 875–884; doi:10.1038/sj.bjp.0703655
Effects of volatile solvents on recombinant N‐methyl‐D‐aspartate receptors expressed in Xenopus oocytes
Authors: Cruz Silvia L , Balster Robert L , Woodward John J
Published: January 2005

We have previously shown that toluene dose‐dependently inhibits recombinant N‐methyl‐D‐aspartate (NMDA) receptors at micromolar concentrations. This inhibition was rapid, almost complete and reversible. The NR1/2B combination was the most sensitive receptor subtype tested with an IC50 value for toluene of 0.17 mM.

We now report on the effects of other commonly abused solvents (benzene, m‐xylene, ethylbenzene, propylbenzene, 1,1,1‐trichlorethane (TCE) and those of a convulsive solvent, 2,2,2‐trifluoroethyl ether (flurothyl), on NMDA‐induced currents measured in Xenopus oocytes expressing NR1/2A or NR1/2B receptor subtypes.

All of the alkylbenzenes and TCE produced a reversible inhibition of NMDA‐induced currents that was dose‐ and subunit‐dependent. The NR1/2B receptor subtype was several times more sensitive to these compounds than the NR1/2A subtype.

The convulsant solvent flurothyl had no effect on NMDA responses in oocytes but potently inhibited ion flux through recombinant GABA receptors expressed in oocytes.

Overall, these results suggest that abused solvents display pharmacological selectivity and that NR1/2B NMDA receptors may be an important target for the actions of these compounds on the brain.

British Journal of Pharmacology (2000) 131, 1303–1308; doi:10.1038/sj.bjp.0703666
Effects of inhibition of calcium and potassium currents in guinea‐pig cardiac contraction: comparison of β‐caryophyllene oxide, eugenol, and nifedipine
Authors: Sensch Oliver , Vierling Wolfgang , Brandt Wolfgang , Reiter Melchior
Published: January 2005

To investigate the effects of the clove oil constituents β‐caryophyllene oxide and eugenol on the heart muscle, experiments were performed on isolated papillary muscles and on ventricular myocytes of the guinea‐pig. The results were compared with those obtained with the dihydropyridine, nifedipine.

All three substances exerted negative inotropic effects in heart muscle although with different potencies and different influences on the time course of the contraction curve.

They all reduced rested‐state contractions (RSCs) in the presence of isoprenaline which are thought to be due to the Ca2+ current (ICa).

β‐Caryophyllene oxide, eugenol and nifedipine inhibited the ICa in single cells from the guinea‐pig ventricle in a concentration‐dependent, reversible way, but with different potencies.

In addition to the ICa‐inhibiting effect, β‐caryophyllene oxide strongly inhibited and eugenol slightly inhibited the potassium current.

The action potential of papillary muscles at a 1 Hz contraction frequency was greatly shortened by nifedipine, slightly shortened by eugenol, but not changed by β‐caryophyllene oxide.

The inhibition of the potassium current by β‐caryophyllene oxide obviously prevents a shortening of the action potential due to the diminution of the ICa. Accordingly, the negative inotropic effect of β‐caryophyllene oxide is closely related to the inhibition of ICa. In contrast, eugenol and nifedipine, which shorten the action potential, exert stronger negative inotropic effects than expected from their influence on ICa.

The results show that the negative inotropic effect of a calcium channel blocker can be attenuated by an additional inhibition of potassium channels.

British Journal of Pharmacology (2000) 131, 1089–1096; doi:10.1038/sj.bjp.0703673
Endomorphin‐1 induced desensitization and down‐regulation of the recombinant μ‐opioid receptor
Authors: Harrison C , Rowbotham D J , Grandy D K , Lambert D G
Published: January 2005

Endomorphin‐1 (E1) is a peptide with high affinity and selectivity for the μ‐opioid receptor. The aim of this study was to determine if endomorphin‐1 caused desensitization and down‐regulation of the μ‐opioid receptor expressed in Chinese hamster ovary cells.

Following 10 μM E1 pre‐treatment, desensitization was assessed by measuring cyclic AMP inhibition, down‐regulation was assessed by [3H]‐diprenorphine ([3H]‐DPN) binding and immunoblotting.

Pre‐treatment of CHOμ cells with 10 μM E1 for 11 and 18 h caused significant reduction in cyclic AMP inhibition. (11 h=39.0±16.7%, 18 h 47.0±11.1% reduction).

At 18 h E1 pre‐treatment there was an enhancement (4.5 fold) of cyclic AMP production under forskolin stimulated conditions accompanied by a small rightward shift in the concentration‐response curve (pEC50 control=7.8±0.3, pEC50 E1=7.3±0.2) when cells were re‐challenged with E1.

In membranes prepared from untreated and 0.5 h E1 pre‐treated cells, addition of GTPγS produced a significant rightward shift in the concentration response curves for E1 displacement of [3H]‐DPN (0 h Ki control=7.86±0.11, GTPγS=7.37±0.15; 0.5 h Ki control=7.92±0.12, GTPγS=7.36±0.08) This was not observed in membranes prepared from cells that had been treated with E1 for 18 h (18 h Ki control=7.69±0.11, GTPγS=7.75±0.08).

In whole cells E1 treatment caused a rapid loss of cell surface receptors such that at 0.5 h there was a 30.5±1.5 reduction (this was unchanged for 18 h). In crude membranes a loss of receptors was also observed using radioligand binding or immuno‐blotting protocols.

These data show that E1 causes desensitization and down‐regulation of the rat μ‐opioid receptor expressed in CHO cells. However, these two responses appear temporally distinct.

British Journal of Pharmacology (2000) 131, 1220–1226; doi:10.1038/sj.bjp.0703683
p38 MAP kinase and MKK‐1 co‐operate in the generation of GM‐CSF from LPS‐stimulated human monocytes by an NF‐κB‐independent mechanism
Authors: Meja Koremu K , Seldon Paul M , Nasuhara Yasuyuki , Ito Kazuhiro , Barnes Peter J , Lindsay Mark A , Giembycz Mark A
Published: January 2005

The extent to which the p38 mitogen‐activated protein (MAP) kinase and MAP kinase kinase (MKK)‐1‐signalling pathways regulate the expression of granulocyte/macrophage colony‐stimulating factor (GM‐CSF) from LPS‐stimulated human monocytes has been investigated and compared to the well studied cytokine tumour necrosis factor‐α (TNFα).

Lipopolysaccharide (LPS) evoked a concentration‐dependent generation of GM‐CSF from human monocytes. Temporally, this effect was preceded by an increase in GM‐CSF mRNA transcripts and abolished by actinomycin D and cycloheximide.

LPS‐induced GM‐CSF release and mRNA expression were associated with a rapid and time‐dependent activation of p38 MAP kinase, ERK‐1 and ERK‐2.

The respective MKK‐1 and p38 MAP kinase inhibitors, PD 098059 and SB 203580, maximally suppressed LPS‐induced GM‐CSF generation by >90%, indicating that both of these signalling cascades co‐operate in the generation of this cytokine.

Electrophoretic mobility shift assays demonstrated that LPS increased nuclear factor κB (NF‐κB) : DNA binding. SN50, an inhibitor of NF‐κB translocation, abolished LPS‐induced NF‐κB : DNA binding and the elaboration of TNFα, a cytokine known to be regulated by NF‐κB in monocytes. In contrast, SN50 failed to affect the release of GM‐CSF from the same monocyte cultures.

Collectively, these results suggest that the generation of GM‐CSF by LPS‐stimulated human monocytes is regulated in a co‐operative fashion by p38 MAP kinase‐ and MKK‐1‐dependent signalling pathways independently of the activation of NF‐κB.

British Journal of Pharmacology (2000) 131, 1143–1153; doi:10.1038/sj.bjp.0703684
Involvement of 5‐HT3 receptors in the nucleus accumbens in the potentiation of cocaine‐induced behaviours in the rat
Authors: Herges Sonja , Taylor David A
Published: January 2005

The present study investigated the central effects of the selective serotonin reuptake inhibitor (SSRI) fluoxetine and the role of 5‐hydroxytryptamine3 (5‐HT3) receptors in the core of the nucleus accumbens (NAc) on cocaine‐induced behavioural changes in rats.

The 5‐HT3 receptor antagonist ondansetron (1 or 10 ng) was microinjected bilaterally into the core of the NAc 60 min prior to peripheral cocaine (15 mg kg−1, i.p.) administration followed by the assessment of locomotor activity, rearing activity and head bobs. Both doses of ondansetron attenuated cocaine's stimulatory effect on behaviours.

Fluoxetine (0.05 or 5 μg) microinjected bilaterally into the core of the NAc 30 min before peripheral administration of cocaine produced dose‐dependent biphasic effects on cocaine‐induced behaviours. Intra‐NAc administration of 0.05 μg fluoxetine resulted in a potentiation of cocaine‐induced behaviours, while the higher dose of the SSRI (5 μg) attenuated the stimulant effect of cocaine on behaviours.

To investigate a possible involvement of 5‐HT3 receptors in fluoxetine's facilitatory action, ondansetron (10 ng) was microinjected 30 min prior to fluoxetine (0.05 μg), which resulted in a significant attenuation of the facilitatory effect of fluoxetine on cocaine‐induced behaviours.

Thus, 5‐HT3 receptors in the core of the NAc appear to mediate stimulatory effects on cocaine‐induced locomotor activity, rears and head bobs, whereas the attenuation of cocaine‐induced behaviours by fluoxetine at the higher dose, suggests the involvement of a different 5‐HT receptor subtype.

British Journal of Pharmacology (2000) 131, 1294–1302; doi:10.1038/sj.bjp.0703687
The human prostanoid DP receptor stimulates mucin secretion in LS174T cells
Authors: Wright D Hamish , Ford‐Hutchinson Anthony W , Chadee Kris , Metters Kathleen M
Published: January 2005

This study demonstrates the localization of the prostaglandin (PG)D2 receptor (DP) within the mucous‐secreting globlet cells of the human colon by in situ hybridization, which suggests a role for DP in mucous secretion. Selective high affinity ligands were used, therefore, to evaluate DP regulation of mucous secretion in LS174T human colonic adenocarcinoma cells.

The expression of hDP in LS174T cells was confirmed at the mRNA level by reverse transcriptase‐polymerase chain reaction, and at the protein level by radioligand binding assays and signal transduction (cyclic AMP accumulation) assays. PGD2 and the highly selective DP‐specific agonist L‐644,698 ((4‐(3‐(3‐(3‐hydroxyoctyl)‐4‐oxo‐2‐thiazolidinyl) propyl) benzoic acid) (racemate)), but not PGE2 competed for [3H]‐PGD2‐specific binding to LS174T cell membranes (Ki values of 0.4 nM and 7 nM, respectively). The DP‐specific agonists PGD2, PGJ2, BW245C (5‐(6‐carboxyhexyl)‐1‐(3‐cyclohexyl‐3‐hydroxypropylhydantoin)), and L‐644,698 showed similar potencies in stimulating cyclic AMP accumulation (EC50 values: 45–90 nM) and demonstrated the expected rank order of potency. PGE2 also elicited cyclic AMP production in this cell line (EC50 value: 162 nM).

The activation of cyclic AMP production by PGD2 and L‐644,698, but not PGE2, was inhibited by the selective DP antagonist BW A868C. Thus, PGD2 and L‐644,698 act through hDP in LS174T cells. PGD2, L‐644,698 and PGE2 (an established mucin secretagogue) potently stimulated mucin secretion in LS174T cells in a concentration‐dependent manner (EC50<50 nM). However, BW A868C effectively antagonized only the mucin secretion mediated by PGD2 and L‐644,698 and not PGE2. These data support a role for the DP receptor in the regulation of mucous secretion.

British Journal of Pharmacology (2000) 131, 1537–1545; doi:10.1038/sj.bjp.0703688
Evidence from immunoneutralization and antisense studies that the inhibitory actions of glucocorticoids on growth hormone release in vitro require annexin 1 (lipocortin 1)
Authors: Taylor A D , Christian H C , Morris J F , Flower R J , Buckingham J C
Published: January 2005

Our previous studies have identified a role for annexin 1 as a mediator of glucocorticoid action in the neuroendocrine system. The present study centred on growth hormone (GH) and exploited antisense and immunoneutralization strategies to examine in vitro the potential role of annexin 1 in effecting the regulatory actions of glucocorticoids on the secretion of this pituitary hormone.

Rat anterior pituitary tissue responded in vitro to growth hormone releasing hormone, forskolin, 8‐Bromo‐cyclic adenosine 3′5′‐monophosphate (8‐Br‐cyclic AMP) and an L‐Ca2+ channel opener (BAY K8644) with concentration‐dependent increases GH release which were readily inhibited by corticosterone and dexamethasone.

The inhibitory actions of the steroids on GH release elicited by the above secretagogues were effectively reversed by an annexin 1 antisense oligodeoxynucleotide (ODN), but not by control (sense or scrambled) ODNs, as also were the glucocorticoid‐induced increases in annexin 1. Similarly, a specific anti‐annexin 1 monoclonal antibody quenched the corticosterone‐induced suppression of secretagogue‐evoked GH release while an isotype matched control antibody was without effect.

Transmission electron micrographs showed that the integrity and ultrastructural morphology of the pituitary cells were well preserved at the end of the incubation and unaffected by exposure to the ODNs, antibodies, steroids or secretagogues.

The results provide novel evidence for a role for annexin 1 as a mediator of the inhibitory actions of glucocorticoids on the secretion of GH by the anterior pituitary gland and suggest that its actions are effected at a point distal to the formation of cyclic AMP and Ca2+ entry.

British Journal of Pharmacology (2000) 131, 1309–1316; doi:10.1038/sj.bjp.0703694
RETRACTED: Cloricromene, a coumarine derivative, protects against collagen‐induced arthritis in Lewis rats
Authors: Cuzzocrea Salvatore , Mazzon Emanuela , Bevilaqua Carla , Costantino Giuseppina , Britti Domenico , Mazzullo Giuseppe , De Sarro Angela , Caputi Achille P
Published: January 2005

The aim of the present study was to investigate the effects of cloricromene, a coumarine derivative, in rats subjected to collagen‐induced arthritis.

Collagen‐induced arthritis (CIA) was induced in Lewis rats by an intradermal injection of 100 μl of the emulsion (containing 100 μg of bovine type II collagen) (CII) and complete Freund's adjuvant (CFA) at the base of the tail. On day 21, a second injection of CII in CFA was administered.

Lewis rats developed an erosive hind paw arthritis when immunized with CII in CFA. Macroscopic clinical evidence of CIA first appeared as peri‐articular erythema and oedema in the hind paws. The incidence of CIA was 100% by day 27 in the CII challenged rats and the severity of CIA progressed over a 35‐day period with radiographic evaluation revealing focal resorption of bone together with osteophyte formation in the tibiotarsal joint and soft tissue swelling.

The histopathology of CIA included erosion of the cartilage at the joint margins. Treatment of rats with cloricromene (10 mg kg−1 i.p. daily) starting at the onset of arthritis (day 23), delayed the development of the clinical signs at days 24–35 and improved histological status in the knee and paw.

Immunohistochemical analysis for iNOS, COX‐2, nitrotyrosine and for poly (ADP‐ribose) synthetase (PARS) revealed a positive staining in inflamed joints from collagen‐treated rats. The degree of staining for iNOS, COX‐2, nitrotyrosine and PARS were markedly reduced in tissue sections obtained from collagen‐treated rats, which had received cloricromene.

Radiographic signs of protection against bone resorption and osteophyte formation were present in the joints of cloricromene‐treated rat.

This study provides the first evidence that cloricromene, a coumarine derivative, attenuates the degree of chronic inflammation and tissue damage associated with collagen‐induced arthritis in the rat.

British Journal of Pharmacology (2000) 131, 1399–1407; doi:10.1038/sj.bjp.0703695
Morphine‐6β‐glucuronide has a higher efficacy than morphine as a mu‐opioid receptor agonist in the rat locus coeruleus
Authors: Osborne Peregrine B , Chieng Billy , Christie MacDonald J
Published: January 2005

The pharmacological properties of the active morphine metabolite, morphine‐6β‐D‐glucuronide (M6G), and the parent compound were compared in rat locus coeruleus neurons by electrophysiological recording in brain slices.

M6G and morphine activated potassium currents in voltage clamped neurons, which were blocked by the opioid receptor antagonist naloxone.

Both M6G and morphine behaved as partial agonists that produced maximal responses smaller than the system maximum, which was measured using [Met5]‐enkephalin. M6G produced a larger maximal response (78%) than morphine (62%), which we estimated was due to a 2–4 fold difference in the relative efficacy of the agonists.

3‐O‐methoxynaltrexone, which has been reported to behave as a selective antagonist of a M6G preferring receptor, was equally effective at blocking currents produced by M6G and the selective mu‐opioid receptor agonist DAMGO.

M6G currents were occluded by a prior application of morphine, and were reduced when mu‐opioid receptors were desensitized by using [Met5]‐enkephalin.

Morphine‐3β‐D‐glucuronide did not affect action potential firing or membrane currents in locus coeruleus neurons and had no effect on currents produced by M6G.

These results show that the relative efficacy of M6G is higher than morphine in locus coeruleus neurons, contrary to what has been shown using mu‐opioid receptors expressed in cell clones.

British Journal of Pharmacology (2000) 131, 1422–1428; doi:10.1038/sj.bjp.0703697
Modulation of ultraviolet‐induced hyperalgesia and cytokine upregulation by interleukins 10 and 13
Authors: Saadé Nayef E , Nasr Isam W , Massaad Cynthia A , Safieh‐Garabedian Bared , Jabbur Suhayl J , Kanaan Salim A
Published: January 2005

Exposure to midrange ultraviolet radiation (UVB) is known to produce skin inflammation similar to sunburn. The aim of this study was to characterize the hyperalgesia and cytokine upregulation induced by UVB and their modulation by antiinflammatory cytokines.

Acute exposure of the dorsal skin of mice to UVB (200, 250 and 300 mJ cm2) resulted in a dose‐dependent decrease in the latencies of the hot plate and tail flick tests, without evident signs of skin lesions.

The observed hyperalgesia displayed a biphasic temporal evolution with an acute phase (3–6 h) and a late (48–96 h) phase.

Exposure to UVB (300 mJ cm2) elicited significant upregulation of interleukin (IL)‐1β, tumour necrosis factor (TNF)‐α and nerve growth factor (NGF), determined by ELISA in the exposed skin. This upregulation was more important during the acute phase of hyperalgesia.

Daily treatment of mice, with i.p. injections of either IL‐10 or IL‐13 (1.5, 7.5 and 15 ng in 100 μl saline) produced a dose‐dependent attenuation of the UVB‐induced hyperalgesia.

Treatment with the highest doses of either IL‐10 or IL‐13, produced significant attenuation of the levels of the cytokines and NGF by UVB, with relatively more pronounced effects by IL‐13.

Acute exposure to moderate amounts of UVB results in a systemic hyperalgesia related to the upregulation of cytokine and NGF levels, since both were prevented by treatment with antiinflammatory cytokines.

British Journal of Pharmacology (2000) 131, 1317–1324; doi:10.1038/sj.bjp.0703699
Cardiovascular and autonomic effects of ω‐conotoxins MVIIA and CVID in conscious rabbits and isolated tissue assays
Authors: Wright Christine E , Robertson Alan D , Whorlow Sarah L , Angus James A
Published: January 2005

The effects of a novel N‐type voltage‐operated calcium channel antagonist, ω‐conotoxin CVID, were compared with ω‐conotoxin MVIIA on sympathetic‐evoked activation of right atria (RA), small mesenteric arteries (MA) and vasa deferentia (VD) isolated from the rat. Their effects were also compared on blood pressure and cardiovascular reflexes in conscious rabbits.

The pIC50 values for MVIIA and CVID, respectively, for inhibiting sympathetic‐evoked responses were equivalent in RA (8.7 and 8.7) and VD (9.0 and 8.7); however, in MA the values were 8.4 and 7.7. The cardiac to vascular (RA/MA) potency ratios, antilog (plog RA–plog MA), for MVIIA and CVID were 2 and 10. The offset rates for CVID and MVIIA were rapid, and peptide reapplication caused rapid onset of blockade, suggesting limited desensitization.

In the conscious rabbit, CVID and MVIIA (100 μg kg−1 i.v.) caused a similar fall in blood pressure and a tachycardia that rapidly reached maximum. Both peptides decreased the vagal‐ and sympathetic‐mediated components of the baroreflex, but had no effect on the vagal nasopharyngeal reflex. The orthostatic reflex to 90° tilt was blocked by MVIIA with sustained postural hypotension for 90 min after administration. In contrast, CVID caused postural hypotension at 30 min which recovered rapidly.

Neither CVID nor MVIIA (3 μg kg−1 i.t.) significantly altered cardiovascular variables or autonomic reflexes.

In conclusion, CVID appears to be relatively weak at inhibiting the reflex response to tilt consistent with its weaker inhibition of rat mesenteric artery constriction to perivascular nerve stimulation. This may point to subtype N‐type calcium channel selectivity.

British Journal of Pharmacology (2000) 131, 1325–1336; doi:10.1038/sj.bjp.0703701
Functional and desensitizing effects of the novel synthetic vanilloid‐like agent 12‐phenylacetate 13‐acetate 20‐homovanillate (PPAHV) in the perfused rat hindlimb
Authors: Griffiths Cory D , Vincent Michelle A , Szallasi Arpad , Colquhoun Eric Q , Geraghty Dominic P
Published: January 2005

In the present study, the effects of the novel vanilloid agonist, 12‐phenylacetate 13‐acetate 20‐homovanillate (PPAHV), on oxygen consumption (VO2) and vascular resistance (perfusion pressure, PP) were investigated in the constant flow, perfused rat hindlimb. The acute desensitizing properties of this novel synthetic agent were also examined.

Maximum stimulation of VO2 was produced by 0.2 μM PPAHV (ΔVO2, 0.83±0.06 μmol g−1 h−1) and was accompanied by mild vasoconstriction (increase in PP; 8.0±1.1 mmHg). The highest concentration of PPAHV tested (2 μM) caused inhibition of VO2 (ΔVO2, −2.73±0.51 μmol g−1 h−1) and strong vasoconstriction (ΔPP, 42.0±1.2 mmHg).

Capsazepine (10 μM) caused a parallel shift to the right of both VO2 and PP concentration‐response curves for PPAHV (pKb=5.00), indicative of competitive binding to vanilloid receptors.

The stimulation of VO2 produced by 0.2 μM PPAHV decreased, but was not completely abolished, after repeated infusion of PPAHV (change in VO2, first infusion, 0.66±0.18 μmol g−1 h−1; sixth infusion, 0.29±0.08 μmol g−1 h−1, P<0.05), an acute tachyphylactic response not previously seen with the repeated infusion of other vanilloid analogues. Conversely, the PP response to repeated PPAHV infusion increased (ΔPP, first infusion, 5.8±0.7 mmHg; sixth infusion, 9.0±0.6 mmHg, P<0.05).

In conclusion, PPAHV produces vasoconstriction and a biphasic effect on VO2 in the perfused rat hindlimb very similar to that induced by naturally occurring vanilloids. Both effects are blocked by the competitive antagonist capsazepine. Since, the metabolic response to low concentrations of PPAHV (stimulation of VO2) undergoes tachyphylaxis, the present data suggest that PPAHV desensitizes putative vanilloid receptors in the hindlimb.

British Journal of Pharmacology (2000) 131, 1408–1412; doi:10.1038/sj.bjp.0703702