bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacological evaluation of the role of cyclooxygenase isoenzymes on the micturition reflex following experimental cystitis in rats
Authors: Lecci Alessandro , Birder Lori A , Meini Stefania , Catalioto Rose‐Marie , Tramontana Manuela , Giuliani Sandro , Criscuoli Marco , Maggi Carlo A
Published: January 2005

Prostanoids, generated from cyclooxygenase (COX) isoenzymes, play a role in the physiological function of the lower urinary tract and are important mediators of inflammatory hyperalgesia. The present work evaluates the effects of the COX‐1/COX‐2 inhibitor dexketoprofen as well as of a selective COX‐2 inhibitor, NS‐398, on urodynamic function following endotoxin (LPS) or cyclophosphamide (CYP)‐induced inflammation of the urinary bladder.

The application of arachidonic acid (330 μg rat−1) onto the serosal surface of the urinary bladder in control rats elicited bladder contractions which could be blocked in a dose‐dependent manner by dexketoprofen (0.1–3 mg kg−1, i.v.) but not by NS‐398 (0.2–6 mg kg−1, i.v.).

Dexketoprofen (3 mg kg−1, i.v.) decreased the micturition frequency and increased the pressure threshold for triggering the micturition either when administered within 15 min or 3 h following surgery in control animals. NS‐398 (6 mg kg−1, i.v.) decreased the micturition frequency and increased the pressure threshold when administered 3 h but not 15 min following surgery.

Administration of LPS (2 mg kg−1, i.v., 90–120 min) increased both the micturition frequency and the pressure threshold for triggering the micturition reflex. Changes in urodynamic parameters induced by LPS were prevented by doses of either dexketoprofen (1 mg kg−1, i.v.) or NS‐398 (2 mg kg−1, i.v.) which were ineffective in control animals.

Pretreatment with CYP (150 mg kg−1, i.p., 48 h) increased the micturition frequency, pressure threshold, and the minimal intravesical pressure but decreased the mean amplitude of micturition contractions. In CYP‐treated rats, dexketoprofen (1 mg kg−1, i.v.) or NS‐398 (2 mg kg−1, i.v.) blocked the CYP‐induced urodynamic changes with exception of the micturition contraction amplitude.

These results indicate that COX‐1 may be involved in modulating the threshold for activating the micturition reflex in the normal rats and also demonstrates that inhibition of COX‐2 prevents or reverses the urodynamic changes associated with bladder inflammation induced either by surgery, LPS or CYP treatments.

British Journal of Pharmacology (2000) 130, 331–338; doi:10.1038/sj.bjp.0703309
CGRP2 receptor in the internal anal sphincter of the rat: implications for CGRP receptor classification
Authors: Wisskirchen F M , Marshall I
Published: January 2005

The CGRP receptor mediating relaxation of the rat internal anal sphincter (IAS) has been characterized using CGRP analogues, homologues, the antagonist CGRP8–37 and its analogues.

In isolated IAS strips, the spontaneously developed tone was concentration‐dependently relaxed by hα CGRP, hβ CGRP and rat β CGRP (pEC50 8.1±0.2, 8.3±0.1 and 8.4±0.2, respectively; 100% maximum response). Vasoactive intestinal polypeptide (VIP) was around 7 fold more potent than hα CGRP (pEC50 9.0±0.1; 100% maximum relaxation). [Cys(ACM2.7)] hα CGRP and salmon calcitonin were inactive (up to 10−5 M).

Hα CGRP8–37 (10−5 M) antagonized responses to hα CGRP (apparent pKB 5.7±0.3) and rat β CGRP (apparent pKB 5.8±0.2), but not to VIP. Hβ CGRP8–37 (10−5 M) was an antagonist against hα CGRP (apparent pKB 6.1±0.1). Hα CGRP8–37 analogues (10−5 M), with substitutions at the N‐terminus by either glycine8 or des‐NH2 valine8 or proline8, antagonized hα CGRP responses with similar affinities (apparent pKB 5.8±0.1, 5.8±0.1 and 5.5±0.1, respectively).

Peptidase inhibitors (amastatin, bestatin, captopril, phosphoramidon and thiorphan, 10−6 M each) did not increase the agonist potency of either hα CGRP or [Cys(ACM2,7)] hα CGRP, or the antagonist affinity of hα CGRP8–37 against hα CGRP or rat β CGRP.

These data demonstrate for the first time a CGRP receptor in the rat IAS for which hα CGRP 8–37 and its analogues have an affinity that is consistent with a CGRP2 receptor. However, there is a marked species difference as the antagonist has a 100 fold lower affinity in the rat than in the same tissue of the opossum (Chakder & Rattan, 1991).

British Journal of Pharmacology (2000) 130, 464–470; doi:10.1038/sj.bjp.0703315
Pharmacological characterization of neurogenic responses of the sheep isolated internal anal sphincter
Authors: Mundey M K , Jonas M , Worthley T , Scholefield J H , Wilson V G
Published: January 2005

The aim of the study was to establish the nature of the neurogenic responses of the sheep isolated anal sphincter.

Isolated strips of sheep internal anal sphincter develop intrinsic contractile tone following the application of stretch tension. On transmural stimulation (1–20 Hz, 10 V pulse strength, 0.5 ms pulse width, 1 s every 180 s) transient relaxations were observed.

The amplitude of the relaxations were frequency‐dependent reaching a maximal response at 10–20 Hz and were inhibited by tetrodotoxin (0.3 μM). Neither atropine (0.3 μM) nor phentolamine (1 μM) affected control responses.

The nitric oxide synthase inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μM) and the selective inhibitor of soluble guanylyl cyclase ODQ, (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one) (1 μM) completely inhibited the neurogenic relaxations and uncovered contractions that were abolished by 1 μM phentolamine and 0.1 μM prazosin. The effect of L‐NAME, but not that of ODQ, was partially reversed by the addition of L‐arginine (1 mM).

Sodium nitroprusside (10 nM–10 μM) caused concentration‐dependent inhibition of myogenic tone and this effect was significantly reduced by ODQ. Calcium‐free Krebs‐Henseleit solution also reduced myogenic tone by 85%.

Transmural electrical stimulation of the sheep isolated internal anal sphincter causes a transient relaxation of myogenic tone that appears to involve nitric oxide from non‐adrenergic, non‐cholinergic nerves and, to a lesser degree, noradrenaline from sympathetic nerves. The characteristics of the preparation compares well with that of human tissue and may prove to be a suitable animal based model for further studies.

British Journal of Pharmacology (2000) 130, 489–494; doi:10.1038/sj.bjp.0703322
Cyclo‐oxygenase‐2 enhances basic fibroblast growth factor‐induced angiogenesis through induction of vascular endothelial growth factor in rat sponge implants
Authors: Majima Masataka , Hayashi Izumi , Muramatsu Michiko , Katada Jun , Yamashina Shohei , Katori Makoto
Published: January 2005

Angiogenesis is reportedly enhanced by prostaglandins (PGs). In the present study, we investigated whether or not cyclo‐oxygenase (COX)‐2 mediated angiogenesis in chronic and proliferate granuloma.

In rat sponge implants, angiogenesis was gradually developed over a 14‐day experimental period as granuloma formed. This angiogenesis was enhanced by topical injections of human recombinant basic fibroblast growth factor (bFGF).

In sponge granuloma, mRNA of COX‐1 was constitutively expressed, whereas that of COX‐2 was increased with neovascularization in parallel with that of vascular endothelial growth factor (VEGF).

Topical injections of bFGF increased the expression of COX‐2 mRNA. bFGF‐stimulated angiogenesis was inhibited by indomethacin or selective COX‐2 inhibitors, NS‐398, nimesulide, and JTE‐522.

The levels of PGE2 and 6‐keto‐PGF1α in the sponge granuloma were increased with bFGF 13 fold and 9 fold, respectively, and these levels were markedly reduced by NS‐398.

The expression of VEGF mRNA in the granuloma was also enhanced by bFGF, and was reduced by NS‐398.

Topical injections of PGE2 and beraprost sodium, a PGI2 analogue, increased the expression of VEGF mRNA, with angiogenesis enhancement.

The enhanced angiogenesis by bFGF was significantly inhibited by topical injections of VEGF anti‐sense oligonucleotide.

These results suggested that COX‐2 may enhance bFGF‐induced neovascularization in sponge granuloma by PG‐mediated expression of VEGF, and that a COX‐2 inhibitor would facilitate the management of conditions involving angiogenesis.

British Journal of Pharmacology (2000) 130, 641–649; doi:10.1038/sj.bjp.0703327
Selective inhibitory effects of niflumic acid on 5‐HT‐induced contraction of the rat isolated stomach fundus
Authors: Scarparo H C , Santos G C M , Leal‐Cardoso J H , Criddle D N
Published: January 2005

The effects of niflumic acid (NFA), an inhibitor of calcium‐activated chloride currents ICl(Ca), were compared with the actions of the voltage‐dependent calcium channel (VDCC) blocker nifedipine on 5‐hydroxtryptamine (5‐HT)‐ and acetylcholine (ACh)‐induced contractions of the rat isolated fundus.

NFA (1–30 μM) elicited a concentration‐dependent inhibition of contractions induced by 5‐HT (10 μM) with a reduction to 15.5±6.0% of the control value at 30 μM. 1 μM nifedipine reduced 5‐HT‐induced contraction to 15.2±4.9% of the control, an effect not greater in the additional presence of 30 μM NFA.

In contrast, the contractile response to ACh (10 μM) was not inhibited by NFA in concentrations 100 μM, although this response was partly inhibited by nifedipine (1 μM) to 67.6±11.8% of the control value.

NFA (1–30 μM) did not affect contraction induced by either 20 mM or 60 mM KCl, suggesting that this drug was not acting via blockade of VDCCs or activation of potassium channels. In contrast, 3,5‐dichlorophenylamine‐2‐carboxylic acid and 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid were less selective in their inhibitory effects, inducing reductions of 60 mM KCl‐induced contraction at concentrations 10 μM.

Our results show that NFA can exert selective inhibitory effects on the chloride‐dependent 5‐HT‐induced contractions of the rat fundus. The data support the hypothesis that activation of Cl(Ca) channels leading to calcium entry via VDCCs is a mechanism utilized by 5‐HT, but not by ACh, to elicit contraction of the rat fundus.

British Journal of Pharmacology (2000) 130, 678–684; doi:10.1038/sj.bjp.0703331
Caspase inhibition and limitation of myocardial infarct size: protection against lethal reperfusion injury
Authors: Mocanu M M , Baxter G F , Yellon D M
Published: January 2005

Ischaemia‐reperfusion injury causes cell death by both necrosis and apoptosis. Caspase activation is a major event in apoptosis. We therefore examined the effect of caspase inhibitors during reperfusion upon myocardial infarction. Rat isolated hearts were subjected to 35 min coronary occlusion and 120 min reperfusion. Treatment groups were perfused with caspase inhibitors during early reperfusion. We assessed a non‐selective caspase inhibitor (Z‐VAD·fmk, 0.1 μM), a caspase‐8 inhibitor (Z‐IETD·fmk, 0.07 μM), a caspase‐9 inhibitor (Z‐LEHD·fmk, 0.07 μM) and a caspase‐3 inhibitor (Ac‐DEVD·cmk, 0.07 μM). All caspase inhibitors limited infarct size (infarct‐risk ratio per cent: control 38.5±2.6; Z‐VAD·fmk 24.6±3.4; Z‐LEHD·fmk 19.3±2.4; Z‐IETD·fmk 23.0±5.4; Ac‐DEVD·cmk 27.8±3.3; P<0.05 when compared with control value, 1‐way ANOVA). We conclude that caspase inhibition during early reperfusion protects myocardium against lethal reperfusion injury.

British Journal of Pharmacology (2000) 130, 197–200; doi:10.1038/sj.bjp.0703336
Thermodynamically distinct high and low affinity states of the A1 adenosine receptor induced by G protein coupling and guanine nucleotide ligation states of G proteins
Authors: Lorenzen Anna , Guerra Laura , Campi Franca , Lang Heidrun , Schwabe Ulrich , Borea Pier Andrea
Published: January 2005

The influence of the receptor‐G protein coupling state and the guanine nucleotide ligation state of the G protein on the binding mechanism of A1 adenosine receptor ligands has been investigated in [3H]‐1,3‐dipropyl‐8‐cyclopentylxanthine ([3H]‐DPCPX) binding studies in rat brain membranes. Thermodynamic parameters of binding of A1 adenosine receptor ligands of different intrinsic activities were determined in the absence or presence of GDP and compared to the binding mechanism after receptor‐G protein uncoupling.

In agreement with previous studies, it was found that xanthine and non‐xanthine antagonists showed an enthalpy‐ or enthalpy‐ and entropy‐driven binding mechanism under all conditions.

In contrast to antagonists, the binding mechanism of agonists was strongly affected by the G protein coupling state or the absence or presence of guanine nucleotides. Binding of full and partial agonists to the high‐affinity state of the A1 receptor was entropy‐driven in the absence of GDP, and a good correlation between intrinsic activities and the contribution of entropy was observed. In the absence of GDP, binding of full and partial agonists and antagonists to the high affinity state of the receptor was thermodynamically discriminated. In contrast, no such discrimination was found in the presence of GDP.

The binding mechanism of agonists to the low‐affinity state of the receptor was identical to that of antagonists only after uncoupling of the receptor from G proteins by pretreatment with N‐ethylmaleimide or guanosine‐5′‐(γ‐thio)‐triphosphate (GTPγS).

These results indicate the existence of two thermodynamically distinct high‐ and low‐affinity states of the A1 adenosine receptor.

British Journal of Pharmacology (2000) 130, 595–604; doi:10.1038/sj.bjp.0703339
Further evidence that 5‐HT‐induced relaxation of pig pulmonary artery is mediated by endothelial 5‐HT2B receptors
Authors: Glusa E , Pertz H H
Published: January 2005

The endothelial 5‐HT receptor mediating relaxation of pig pulmonary artery has been characterized using the selective 5‐HT2B receptor agonist BW 723C86 and a variety of structurally diverse 5‐HT receptor antagonists.

If arterial rings with intact endothelium were precontracted with prostaglandin F2α (3 μM), BW 723C86 caused concentration‐dependent relaxation with a pEC50=8.21±0.03 and Emax=89±4% relative to 5‐HT. The relaxant responses to BW 723C86 were inhibited by the 5‐HT2B receptor antagonist SB 204741, the 5‐HT2B/2C receptor antagonist SB 206553 and the antimigraine drug pizotifen, yielding pA2 values of 6.68, 7.20 and 8.32, respectively. The pA2 values against BW 723C86 were similar to those determined against 5‐HT.

The relaxant effect of 5‐HT was antagonized by a variety of 22 compounds of diverse chemical structures. Based on the calculated mean pA2 values the order of the most potent antagonists was ritanserin (9.38) > methysergide (8.86) > pizotifen (8.47) methiothepin (8.32) > LY 53857 (7.84) amoxapine (7.80) loxapine (7.73) metergoline (7.64) mianserin (7.51) rauwolscine (7.39). Compounds with weak blocking potency were yohimbine (6.37), spiperone (5.88) and ketanserin (5.85). Correlation analysis between the affinities of the antagonists in pig pulmonary artery and those from radioligand binding studies at human and rat 5‐HT2B receptors showed a highly significant correlation (r=0.95 and 0.84, P<0.002 and <0.005). Correlation with 5‐HT2C receptors was much lower (r=0.57, P=0.035), and no correlations were obtained with 5‐ht6 and 5‐HT7 receptors.

It is concluded that the 5‐HT receptor mediating endothelium‐dependent relaxation of pig pulmonary artery is of the 5‐HT2B subtype.

British Journal of Pharmacology (2000) 130, 692–698; doi:10.1038/sj.bjp.0703341
Anaesthetic agents inhibit gastrin‐stimulated but not basal histamine release from rat stomach ECL cells
Authors: Norlén P , Kitano M , Lindström E , Håkanson R
Published: January 2005

By mobilizing histamine in response to gastrin, the ECL cells in the oxyntic mucosa play a key role in the control of the parietal cells and hence of gastric acid secretion. General anaesthesia suppresses basal and gastrin‐ and histamine‐stimulated acid secretion. The present study examines if the effect of anaesthesia on basal and gastrin‐stimulated acid secretion is associated with suppressed ECL‐cell histamine secretion.

A microdialysis probe was implanted in the submucosa of the ventral aspect of the acid‐producing part of the stomach (32 rats). Three days later, ECL‐cell histamine mobilization was monitored 2 h before and 4 h after the start of intravenous infusion of gastrin (5 nmol kg−1 h−1). The rats were either conscious or anaesthetized. Four commonly used anaesthetic agents were given 1 h before the start of the experiments by intraperitoneal injection: chloral hydrate (300 mg kg−1), pentobarbitone (40 mg kg−1), urethane (1.5 g kg−1) and a mixture of fluanisone/fentanyl/midazolam (15/0.5/7.5 mg kg−1).

In a parallel series of experiments, basal‐ and gastrin‐induced acid secretion was monitored in six conscious and 25 anaesthetized (see above) chronic gastric fistula rats. All anaesthetic agents lowered gastrin‐stimulated acid secretion; also the basal acid output was reduced (fluanisone/fentanyl/midazolam was an exception).

Anaesthesia reduced gastrin‐stimulated but not basal histamine release by 55–80%. The reduction in gastrin‐induced acid response (70–95%) was strongly correlated to the reduction in gastrin‐induced histamine mobilization. The correlation is in line with the view that the reduced acid response to gastrin reflects impaired histamine mobilization.

Rat stomach ECL cells were purified by counter‐flow elutriation. Gastrin‐evoked histamine mobilization from the isolated ECL cells was determined in the absence or presence of anaesthetic agents in the medium. With the exception of urethane, they inhibited gastrin‐evoked histamine secretion dose‐dependently, indicating a direct effect on the ECL cells.

Anaesthetized rats are widely used to study acid secretion and ECL‐cell histamine release. The present results illustrate the short‐comings of such an approach in that a number of anaesthetic agents were found to impair not only acid secretion but also the secretion of ECL‐cell histamine – some acting in a direct manner.

British Journal of Pharmacology (2000) 130, 725–730; doi:10.1038/sj.bjp.0703347
[3H]‐Trimetazidine mitochondrial binding sites: regulation by cations, effect of trimetazidine derivatives and other agents and interaction with an endogenous substance
Authors: Morin Didier , Sapena Rosa , Elimadi Aziz , Testa Bernard , Labidalle Serge , Le Ridant Alain , Tillement Jean‐Paul
Published: January 2005

Trimetazidine, an antiischaemic drug, has been shown to restore impaired mitochondrial functions. Specific binding sites for [3H]‐trimetazidine have been previously detected in liver mitochondria. In the present study we confirm this observation and provide additional evidence for the involvement of these sites in the pharmacological effects of the drug.

Inhibition experiments using a series of trimetazidine derivatives revealed the presence of three classes of binding sites. An N‐benzyl substituted analogue of trimetazidine exhibited a very high affinity (Ki=7 nM) for one of these classes of sites.

Compounds from different pharmacological classes were evaluated for their ability to inhibit [3H]‐trimetazidine binding. Among the drugs tested pentazocine, ifenprodil, opipramol, perphenazine, haloperidol, and to a lower extent prenylamine, carbetapentane and dextromethorphan competed with high affinity, suggesting a similarity of high affinity [3H]‐trimetazidine sites with sigma receptors.

[3H]‐Trimetazidine binding was modulated by pH. Neutral trimetazidine had about 10 fold higher affinity than protonated trimetazidine for its mitochondrial binding sites. Various cations also affected [3H]‐trimetazidine binding. Ca2+ was the most potent inhibitor and totally suppressed the binding of [3H]‐trimetazidine to the sites of medium affinity.

An endogenous cytosolic ligand was able to displace [3H]‐trimetazidine from its binding sites. Its activity was not affected by boiling for 15 min, suggesting a non‐protein compound.

These data suggest that mitochondrial [3H]‐trimetazidine binding sites could have a physiological relevance and be involved in the antiischaemic effects of the drug.

British Journal of Pharmacology (2000) 130, 655–663; doi:10.1038/sj.bjp.0703348
Evidence that 2‐methylthioATP and 2‐methylthioADP are both agonists at the rat hepatocyte P2Y1receptor
Authors: Dixon C Jane
Published: January 2005

In the absence of selective antagonists, pharmacological characterization of P2Y receptor subtypes has relied heavily upon their distinct agonist profiles. 2‐methylthioADP (2‐MeSADP) is a selective agonist for the P2Y1receptor. The agonist action of 2‐MeSATP at the P2Y1receptor has recently been questioned.

The effects of both 2‐MeSADP and 2‐MeSATP have been studied on rat hepatocytes injected with the bioluminescent Ca2+indicator, aequorin. Single hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway.

The transients induced by 2‐MeSADP and 2‐MeSATP in the same cell were indistinguishable, indicating that they act at a common receptor. In contrast the transients evoked by ATP and UTP had very different profiles. Treatment of 2‐MeSATP with an ATP‐regenerating system to remove contaminating 2‐MeSADP did not abolish its agonist activity.

Application of the P2Y1antagonist, adenosine‐3′‐phosphate‐5′‐phosphate (A3P5P) inhibited the transients induced by both 2‐MeSADP and 2‐MeSATP. In contrast the transients induced by ATP and UTP were enhanced by the addition of A3P5P.

These results indicate that both 2‐MeSADP and 2‐MeSATP are agonists at the rat hepatocyte P2Y1receptor.

British Journal of Pharmacology(2000)130, 664–668; doi:10.1038/sj.bjp.0703350
Mechanisms underlying the attenuation of endothelium‐dependent vasodilatation in the mesenteric arterial bed of the streptozotocin‐induced diabetic rat
Authors: Makino Ayako , Ohuchi Kunihiro , Kamata Katsuo
Published: January 2005

Experiments were designed to investigate the mechanisms underlying the diabetes‐related impairment of the vasodilatations of the perfused mesenteric arterial bed induced by acetylcholine (ACh) and K+.

In streptozotocin (STZ)‐diabetic rats, the ACh‐induced endothelium‐dependent vasodilatation was attenuated. The dose‐response curves for ACh in control and diabetic rats were each shifted to the right by NG‐nitro‐L‐arginine (L‐NOARG) and by isotonic high K+ (60 mM). The ACh dose‐response curves under isotonic high K+ were not different between control and diabetic rats.

We also examined the vasodilatation induced by K+, which is a putative endothelium‐derived hyperpolarizing factor (EDHF). The mesenteric vasodilatation induced by a single administration of K+ was greatly impaired in STZ‐induced diabetic rats. Treatment with charybdotoxin plus apamin abolished the ACh‐induced vasodilatation but enhanced the K+‐induced response in controls and diabetic rats. After pretreatment with ouabain plus BaCl2, the ACh‐induced vasodilatation was significantly impaired and the K+‐induced relaxation was abolished in both control and diabetic rats.

The impairment of the endothelium‐dependent vasodilatation of the mesenteric arterial bed seen in STZ‐induced diabetic rats may be largely due to a defective vascular response to EDHF. It is further suggested that K+ is one of the endothelium‐derived hyperpolarizing factors and that the vasodilatation response to K+ is impaired in the mesenteric arterial bed from diabetic rats.

British Journal of Pharmacology (2000) 130, 549–556; doi:10.1038/sj.bjp.0703354
Exploration of the ligand binding site of the human 5‐HT4 receptor by site‐directed mutagenesis and molecular modeling
Authors: Mialet Jeanne , Dahmoune Yamina , Lezoualc'h Frank , Berque‐Bestel Isabelle , Eftekhari Pierre , Hoebeke Johan , Sicsic Sames , Langlois Michel , Fischmeister Rodolphe
Published: January 2005

Among the five human 5‐HT4 (h5‐HT4) receptor isoforms, the h5‐HT4(a) receptor was studied with a particular emphasis on the molecular interactions involved in ligand binding. For this purpose, we used site‐directed mutagenesis of the transmembrane domain. Twelve mutants were constructed with a special focus on the residue P4.53 of helix IV which substitutes in h5‐HT4 receptors the highly conserved S residue among the rhodopsin family receptors. The mutated receptors were transiently expressed in COS‐7 cells.

Ligand binding or competition studies with two h5‐HT4 receptor agonists, serotonin and ML10302 and two h5‐HT4 receptor antagonists, [3H]‐GR113808 and ML10375 were performed on wild type and mutant receptors. Functional activity of the receptors was evaluated by measuring the ability of serotonin to stimulate adenylyl cyclase.

Ligand binding experiments revealed that [3H]‐GR113808 did not bind to mutants P4.53A, S5.43A, F6.51A, Y7.43A and to double mutant F6.52V/N6.55L. On the other hand mutations D3.32N, S5.43A and Y7.43A appeared to promote a dramatic decrease of h5‐HT4(a) receptor functional activity. From these studies, S5.43 and Y7.43 clearly emerged as common anchoring sites to antagonist [3H]‐GR113808 and to serotonin.

According to these results, we propose ligand‐receptor complex models with serotonin and [3H]‐GR113808. For serotonin, three interaction points were selected including ionic interaction with D3.32, a stabilizing interaction of this ion pair by Y7.43 and a hydrogen bond with S5.43. [3H]‐GR113808 was also docked, based on the same type of interactions with S5.43 and D3.32: the proposed model suggested a possible role of P4.53 in helix IV structure allowing the involvement of a close hydrophobic residue, W4.50, in a hydrophobic pocket for hydrophobic interactions with the indole ring of [3H]‐GR113808.

British Journal of Pharmacology (2000) 130, 527–538; doi:10.1038/sj.bjp.0703356
Tumour necrosis factor‐α‐ and interleukin‐1β‐stimulated cell proliferation through activation of mitogen‐activated protein kinase in canine tracheal smooth muscle cells
Authors: Yang Chuen‐Mao , Luo Shue‐Fen , Wang Chuan‐Chwan , Chiu Chi‐Tso , Chien Chin‐Sung , Lin Chih‐Chung , Hsiao Li‐Der
Published: January 2005

The elevated levels of inflammatory cytokines such as tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) have been found in the fluid of airways in symptomatic asthmatics. These cytokines have been considered as mitogens to stimulate cell proliferation in tracheal smooth muscle cells (TSMCs). We therefore investigated the effects of TNF‐α and IL‐1β on cell proliferation and activation of p42/p44 mitogen‐activated protein kinase (MAPK) in these cells.

TNF‐α and IL‐1β induced [3H]‐thymidine incorporation in a time‐ and concentration‐dependent manner. The maximal stimulation of [3H]‐thymidine incorporation induced by TNF‐α and IL‐1β was seen 12 h after incubation with these cytokines.

In response to TNF‐α and IL‐1β, p42/p44 MAPK was activated with a concentration‐dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin did not change DNA synthesis and phosphorylation of MAPK induced by TNF‐α and IL‐1β. These responses were attenuated by a tyrosine kinase inhibitor herbimycin, a phosphatidyl choline (PC)‐phospholipase C (PLC) inhibitor D609, a phosphatidyl inositide (PI)‐PLC inhibitor U73122, a protein kinase C inhibitor staurosporine, and removal of Ca2+ by addition of BAPTA/AM plus EGTA.

TNF‐α‐ and IL‐1β‐induced [3H]‐thymidine incorporation and phosphorylation of p42/p44 MAPK was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses.

These results suggest that the mitogenic effects of TNF‐α and IL‐1β were mediated through the activation of MEK1/2 and p42/p44 MAPK pathway. TNF‐α‐ and IL‐1β‐mediated responses were modulated by PLC, Ca2+, PKC, and tyrosine kinase associated with cell proliferation in TSMCs.

British Journal of Pharmacology (2000) 130, 891–899; doi:10.1038/sj.bjp.0703359
Evidence for activation of the tissue kallikrein‐kinin system in nociceptive transmission and inflammatory responses of mice using a specific enzyme inhibitor
Authors: Emim José A da S , Souccar Caden , Castro Maria S de A , Godinho Rosely O , Cezari Maria H S , Juliano Luiz , Lapa Antonio J
Published: January 2005

The pharmacological activity of phenylacetyl‐Phe‐Ser‐Arg‐N‐(2,4‐dinitrophenyl)‐ethylenediamine (TKI), a tissue kallikrein specific inhibitor, was assessed using models of nociception and inflammation in mice.

Injection of TKI (13.6–136 μmol kg−1, i.p. or 41–410 μmol kg−1, s.c.) produced a dose‐related inhibition of the acetic acid‐induced writhes (by 37 to 85% or 34 to 80%, respectively). The antinociceptive activity of TKI (41 μmol kg−1, i.p.) was maximal after 30 min injection and lasted for 120 min. The effect was unaltered by pretreatment with naloxone (8.2 μmol kg−1, s.c.) or bilateral adrenalectomy.

TKI (41 and 136 μmol kg−1, i.p.) produced a dose‐related decrease of the late phase of formalin‐induced nociception by 79 and 98%, respectively. At 136 μmol kg−1, i.p., TKI also shortened the duration of paw licking in the early phase by 69%. TKI (41 and 136 μmol kg−1, i.p.) also reduced the capsaicin‐induced nociceptive response (by 51 to 79%).

TKI (41 μmol kg−1, i.p. or 410 μmol kg−1, s.c.) reduced the oedematogenic response, from the second to the fifth hour after carrageenin injection by 36 to 30% or by 47 to 39%, respectively.

Pretreatment with TKI (41 μmol kg−1, i.p.) reduced the capsaicin‐induced neurogenic inflammation in the mouse ear by 54%.

It is concluded that TKI presents antinociceptive and antiinflammatory activities mediated by inhibition of kinin formation by tissue kallikrein in mice. The results also indicate that the tissue kallikrein‐dependent pathway contributes to kinin generation in nociceptive and inflammatory processes in mice.

British Journal of Pharmacology (2000) 130, 1099–1107; doi:10.1038/sj.bjp.0703362
Multiple action sites of flufenamate on ion transport across the rat distal colon
Authors: Schultheiss G , Frings M , Hollingshaus G , Diener M
Published: January 2005

The antisecretory effects of flufenamate in the rat distal colon were investigated with the Ussing‐chamber and the patch‐clamp method as well as by measurements of the intracellular Ca2+ concentration using fura‐2‐loaded isolated crypts.

Flufenamate (5•10−4 mol l−1) suppressed the short‐circuit current (Isc) induced by carbachol (5•10−5 mol l−1), forskolin (5•10−6 mol l−1) and the Isc induced by the membrane‐permeable analogue of cyclic AMP, CPT–cyclic AMP (10−4 mol l−1).

Indomethacin (10−6–10−4 mol l−1) did not mimic the effect of flufenamate, indicating that the antisecretory effect of flufenamate is not related to the inhibition of the cyclo‐oxygenase.

When the basolateral membrane was depolarized by a high K+ concentration and a Cl− current was induced by a mucosally directed Cl− gradient, the forskolin‐stimulated Cl− current was blocked by flufenamate, indicating an inhibition of the cyclic AMP‐stimulated apical Cl− conductance.

When the apical membrane was permeabilized by the ionophore, nystatin, flufenamate decreased the basolateral K+ conductance and inhibited the Na+–K+‐ATPase.

Patch‐clamp experiments revealed a variable effect of flufenamate on membrane currents. In seven out of 11 crypt cells the drug induced an increase of the K+ current, whereas in the remaining four cells an inhibition was observed.

Experiments with fura‐2‐loaded isolated crypts indicated that flufenamate increased the basal as well as the carbachol‐stimulated intracellular Ca2+ concentration.

These results demonstrate that flufenamate possesses multiple action sites in the rat colon: The apical Cl− conductance, basolateral K+ conductances and the Na+–K+‐ATPase.

British Journal of Pharmacology (2000) 130, 875–885; doi:10.1038/sj.bjp.0703363
Contribution of β‐adrenoceptor subtypes to relaxation of colon and oesophagus and pacemaker activity of ureter in wildtype and β3‐adrenoceptor knockout mice
Authors: Oostendorp Jaap , Preitner Frédéric , Moffatt James , Jimenez Maria , Giacobino Jean Paul , Molenaar Peter , Kaumann Alberto Julio
Published: January 2005

The smooth muscle relaxant responses to the mixed β3‐, putative β4‐adrenoceptor agonist, (−)‐CGP 12177 in rat colon are partially resistant to blockade by the β3‐adrenoceptor antagonist SR59230A suggesting involvement of β3‐ and putative β4‐adrenoceptors. We now investigated the function of the putative β4‐adrenoceptor and other β‐adrenoceptor subtypes in the colon, oesophagus and ureter of wildtype (WT) and β3‐adrenoceptor knockout (β3KO) mice.

(−)‐Noradrenaline and (−)‐adrenaline relaxed KCl (30 mM)‐precontracted colon mostly through β1‐and β3‐adrenoceptors to a similar extent and to a minor extent through β2‐adrenoceptors. In colon from β3KO mice, (−)‐noradrenaline was as potent as in WT mice but the effects were mediated entirely through β1‐adrenoceptors. (−)‐CGP 12177 relaxed colon from β3KO mice with 2 fold greater potency than in WT mice. The maintenance of potency for (−)‐noradrenaline and increase for (−)‐CGP 12177 indicate compensatory increases in β1‐ and putative β4‐adrenoceptor function in β3KO mice.

In oesophagi precontracted with 1 μM carbachol, (−)‐noradrenaline caused relaxation mainly through β1‐and β3‐adrenoceptors. (−)‐CGP 12177 (2 μM) relaxed oesophagi from WT by 61.4±5.1% and β3KO by 67.3±10.1% of the (−)‐isoprenaline‐evoked relaxation, consistent with mediation through putative β4‐adrenoceptors.

In ureter, (−)‐CGP 12177 (2 μM) reduced pacemaker activity by 31.1±2.3% in WT and 31.3±7.5% in β3KO, consistent with mediation through putative β4‐adrenoceptors.

Relaxation of mouse colon and oesophagus by catecholamines are mediated through β1‐ and β3‐adrenoceptors in WT. The putative β4‐adrenoceptor, which presumably is an atypical state of the β1‐adrenoceptor, mediates the effects of (−)‐CGP 12177 in colon, oesophagus and ureter.

British Journal of Pharmacology (2000) 130, 747–758; doi:10.1038/sj.bjp.0703365
JTV‐519, a novel cardioprotective agent, improves the contractile recovery after ischaemia‐reperfusion in coronary perfused guinea‐pig ventricular muscles
Authors: Ito Ken‐ichiro , Shigematsu Sakuji , Sato Toshiaki , Abe Takako , Li Yulong , Arita Makoto
Published: January 2005

A newly synthesized benzothiazepine derivative, JTV‐519 (JT) has been reported to be cardioprotective. However, the precise mechanism underlying the cardioprotective effect of this drug is unknown.

Coronary‐perfused guinea‐pig ventricular muscles were subjected to 20‐min no‐flow ischaemia followed by 60‐min reperfusion (I/R). I/R significantly decreased the contraction in untreated preparations (control group, 34±4% of baseline value, n=6). Brief administration of JT (1.0 μM) prior to ischaemia significantly improved the postischaemic contractile recovery (63±5% of baseline value, n=4), as compared to the control group.

JT (1.0 μM) slightly prolonged action potential duration before ischaemia and induced conduction disturbance (2 : 1 block) after the initiation of ischaemia.

The cardioprotective effect of JT was antagonized by chelerythrine (CH, 5.0 μM), an inhibitor of protein kinase C (PKC) or by 5‐hydroxydecanoic acid (5‐HD, 400 μM), an inhibitor of mitochondrial ATP‐sensitive K+ (KATP) channels.

These results suggest that the protective effect of JT is due to the opening of mitochondrial KATP channels, which, in turn, is linked to PKC activation.

British Journal of Pharmacology (2000) 130, 767–776; doi:10.1038/sj.bjp.0703373
Role of loop D of the α7 nicotinic acetylcholine receptor in its interaction with the insecticide imidacloprid and related neonicotinoids
Authors: Matsuda Kazuhiko , Shimomura Masaru , Kondo Yumi , Ihara Makoto , Hashigami Kaori , Yoshida Naofumi , Raymond Valérie , Mongan Nigel P , Freeman John C , Komai Koichiro , Sattelle David B
Published: January 2005

The nitroguanidine insecticide imidacloprid along with a second generation of related compounds including nitenpyram, all nicotinic acetylcholine (ACh) receptor ligands, are used increasingly in many countries. Site‐directed mutagenesis and heterologous expression in Xenopus laevis oocytes have been deployed to investigate mutants (G189D and G189E) of the chicken α7 homomer‐forming nicotinic receptor subunit which are predicted to enhance the negative charge at the negative subsite (loop D) of the ACh binding site.

Xenopus oocytes expressing wild‐type α7 nicotinic receptors respond to imidacloprid with rapid inward currents. Imidacloprid and nitenpyram are partial agonists, whereas ACh, (−)‐nicotine and (+)‐epibatidine are full agonists.

Compared to wild‐type α7, the mutant G189D and G189E receptors are much less sensitive to the insecticides, whereas their sensitivity to (−)‐nicotine, ACh and (+)‐epibatidine is only slightly reduced. In contrast, G189N and G189Q mutants are sensitive not only to ACh, (−)‐nicotine and (+)‐epibatidine, but also to the two insecticides. Thus reduction of the insecticide‐sensitivity by the mutations G189D and G189E are attributed to an increase in negativity of loop D. Desnitro‐imidacloprid (DN‐IMI), an imidacloprid derivative lacking the nitro group is a potent agonist on the G189D and G189E mutants suggesting an important role of loop D in nicotinic receptor interactions with the nitro group of nitroguanidine insecticides.

British Journal of Pharmacology (2000) 130, 981–986; doi:10.1038/sj.bjp.0703374
Electrophysiological effects of endothelin‐1 and their relationship to contraction in rat renal arterial smooth muscle
Authors: Betts Luisa C , Kozlowski Roland Z
Published: January 2005

The electophysiological effects of endothelin‐1 (ET‐1) and their relationship to contraction remain unclear in the renal circulation. Using endotheliumdenuded arteries from the main branch of the renal artery proximal to the kidney of the rat, we have examined its effects on tension and conducted parallel patch‐clamp measurements using freshly isolated smooth muscle cells from this tissue.

Pharmacological experiments revealed that ET‐1 produced constriction of renal arteries dependent on the influx of extracellular Ca2+, mediated solely through ETA receptor stimulation.

Current‐clamp experiments revealed that renal arterial myocytes had a resting membrane potential of ∼32 mV, with the majority of cells exhibiting spontaneous transient hyperpolarizations (STHPs). Application of ET‐1 produced depolarization and in those cells exhibiting STHPs, either caused their inhibition or made them occur regularly.

Under voltage‐clamp conditions cells were observed to exhibit spontaneous transient outward currents (STOCs) inhibited by iberiotoxin. Application of voltage‐ramps revealed an outward current activated at ∼−30 mV, sensitive to both 4‐AP and TEA. Taken together these results suggest that renal arterial myocytes possess both delayed rectifying K+ (KV) and Ca2+‐activated K+ (BKCa) channels.

Under voltage‐clamp, ET‐1 attenuated the outward current and reduced the magnitude and incidence of STOCs: effects mediated solely as a consequence of ETA receptor stimulation.

Thus, in conclusion, activation of ETA receptors by ET‐1 causes inhibition of KV and BKCa channel activity, which could promote and/or maintain membrane depolarization. This effect is likely to favour L‐type Ca2+ channel activity providing an influx pathway for extracellular Ca2+ essential for contraction.

British Journal of Pharmacology (2000) 130, 787–796; doi:10.1038/sj.bjp.0703377
Inhibition of neuronal Ca2+ influx by gabapentin and subsequent reduction of neurotransmitter release from rat neocortical slices
Authors: Fink Klaus , Meder Wolfgang , Dooley David J , Göthert Manfred
Published: January 2005

Cytosolic calcium ion concentrations ([Ca2+]i) were measured in rat neocortical synaptosomes using fura‐2, and depolarization of synaptosomal membranes was induced by K+ (30 mM). The release of the endogenous excitatory amino acids glutamate and aspartate was evoked by K+ (50 mM) and determined by HPLC. The release of [3H]‐noradrenaline from rat neocortical synaptosomes or slices was evoked by K+ (15 and 25 mM) and measured by liquid scintillation counting.

Gabapentin produced a concentration‐dependent inhibition of the K+‐induced [Ca2+]i increase in synaptosomes (IC50=14 μM; maximal inhibition by 36%). The inhibitory effect of gabapentin was abolished in the presence of the P/Q‐type Ca2+ channel blocker ω‐agatoxin IVA, but not by the N‐type Ca2+ channel antagonist ω‐conotoxin GVIA.

Gabapentin (100 μM) decreased the K+‐evoked release of endogenous aspartate and glutamate in neocortical slices by 16 and 18%, respectively.

Gabapentin reduced the K+‐evoked [3H]‐noradrenaline release in neocortical slices (IC50=48 μM; maximal inhibition of 46%) but not from synaptosomes.

In the presence of the AMPA receptor antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and 2,3‐dioxo‐6‐nitro‐1,2,3,4‐tetrahydro[f]quinoxaline‐7‐sulphonamide (NBQX), gabapentin did not reduce [3H]‐noradrenaline release. Gabapentin did, however, cause inhibition in the presence of the NMDA receptor antagonist DL‐(E)‐2‐amino‐4‐methyl‐5‐phosphono‐3‐pentanoic acid (CGP 37849).

Gabapentin is concluded to reduce the depolarization‐induced [Ca2+]i increase in excitatory amino acid nerve terminals by inhibiting P/Q‐type Ca2+ channels; this decreased Ca2+ influx subsequently attenuates K+‐evoked excitatory amino acid release. The latter effect leads to a reduced activation of AMPA receptors which contribute to K+‐evoked noradrenaline release from noradrenergic varicosities, resulting in an indirect inhibition of noradrenaline release.

British Journal of Pharmacology (2000) 130, 900–906; doi:10.1038/sj.bjp.0703380
Regional haemodynamic effects of recombinant murine or human leptin in conscious rats
Authors: Gardiner S M , Kemp P A , March J E , Bennett T
Published: January 2005

Regional haemodynamic responses to recombinant murine or human leptin were assessed in conscious, chronically‐instrumented, male, Long‐Evans rats (350–450 g).

Human, but not murine, leptin caused a slight hindquarters vasoconstriction, but neither peptide had any effect on mean arterial blood pressure or heart rate.

In the presence of the β2‐adrenoceptor antagonist, ICI 118551, a hindquarters vasoconstrictor response to human leptin was not seen, and there was a tachycardia, as there was to murine leptin.

The nitric oxide synthase inhibitor, NG‐nitro‐L‐arginine methyl ester, (L‐NAME), did not influence the cardiovascular effects of murine or human leptin.

The results indicate that the previously reported sympathoexcitatory effects of murine leptin in anaesthetized rats are not manifest as regional haemodynamic changes in conscious rats, and this is not due to β2‐adrenoceptor‐mediated vasodilator mechanisms opposing any vasoconstrictor responses. Moreover, the ability of L‐NAME to unmask a pressor effect of murine leptin in anaesthetized rats may not be apparent in the conscious state.

British Journal of Pharmacology (2000) 130, 805–810; doi:10.1038/sj.bjp.0703381
Characterization of a novel VPAC1 selective agonist and identification of the receptor domains implicated in the carboxyl‐terminal peptide recognition
Authors: Van Rampelbergh Jean , Juarranz Maria‐Guillerma , Perret Jason , Bondue Antoine , Solano Rosa Maria , Delporte Christine , De Neef Philippe , Robberecht Patrick , Waelbroeck Magali
Published: January 2005

Vasoactive Intestinal Polypeptide (VIP) interacts with a high affinity to two subclasses of G protein coupled receptors named VPAC1 and VPAC2, and has a 3–10 fold preference for VPAC1 over VPAC2 receptors. Selective ligands for each receptor subclass were recently described. [R16]‐PACAP (1–23) and [L22]‐VIP are two selective VPAC1 agonists.

Chimaeric human VPAC2‐VPAC1 recombinant receptors expressed in CHO cells were used to identify the receptor domains implicated in these two selective ligands recognition.

The VPAC2 preference for [R16]‐PACAP (1–27) over [R16]‐PACAP (1–23) did not require the receptor's NH2‐terminus domain but involved the whole transmembrane domain.

In contrast, the selectivity of [L22]‐VIP depended only on the presence of the NH2 terminus and EC2 domains of the VPAC1 receptor.

The present data support the idea that in the GPCR‐B family of receptors the different selective ligands require different domains for their selectivity, and that the peptides carboxyl terminal sequence (amino acids 24–27) folds back on the transmembrane receptor domain, close to the peptides, aminoterminus.

British Journal of Pharmacology (2000) 130, 819–826; doi:10.1038/sj.bjp.0703384
Effects of 5‐aminoisoquinolinone, a water‐soluble, potent inhibitor of the activity of poly (ADP‐ribose) polymerase on the organ injury and dysfunction caused by haemorrhagic shock
Authors: McDonald Michelle C , Mota‐Filipe Helder , Wright James A , Abdelrahman Maha , Threadgill Michael D , Thompson Andrew S , Thiemermann Christoph
Published: January 2005

Poly (ADP‐ribose) synthetase (PARP) is a nuclear enzyme activated by strand breaks in DNA, which are caused inter alia by reactive oxygen species (ROS). Here we report on (i) a new synthesis of a water‐soluble and potent PARP inhibitor, 5‐aminoisoquinolinone (5‐AIQ) and (ii) investigate the effects of 5‐AIQ on the circulatory failure and the organ injury/dysfunction caused by haemorrhage and resuscitation in the anaesthetized rat.

Exposure of human cardiac myoblasts (Girardi cells) to hydrogen peroxide (H2O2, 3 mM for 1 h, n=9) caused a substantial increase in PARP activity. Pre‐treatment of these cells with 5‐AIQ (1 μM–1 mM, 10 min prior to H2O2) caused a concentration‐dependent inhibition of PARP activity (IC50: ∼0.01 mM, n=6).

Haemorrhage and resuscitation resulted (within 4 h after resuscitation) in a delayed fall in blood pressure (circulatory failure) as well as in rises in the serum levels of (i) urea and creatinine (renal dysfunction), (ii) aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma‐glutamyl‐transferase (γ‐GT) (liver injury and dysfunction), (iii) lipase (pancreatic injury) and (iv) creatine kinase (CK) (neuromuscular injury) (n=10).

Administration (5 min prior to resuscitation of 5‐AIQ) (0.03 mg kg−1 i.v., n=8, or 0.3 mg kg−1 i.v., n=10) reduced (in a dose‐related fashion) the multiple organ injury and dysfunction, but did not affect the circulatory failure, associated with haemorrhagic shock.

Thus, 5‐AIQ abolishes the multiple organ injury caused by severe haemorrhage and resuscitation.

British Journal of Pharmacology (2000) 130, 843–850; doi:10.1038/sj.bjp.0703391
Interaction between copper and zinc at GABAA receptors in acutely isolated cerebellar Purkinje cells of the rat
Authors: Sharonova Irina N , Vorobjev Vladimir S , Haas Helmut L
Published: January 2005

Nanomolar concentrations of Cu2+ induce a slowly reversible block of GABAA receptor‐mediated currents which can be removed by chelating substances.

The possible interaction of Cu2+ with the Zn2+ binding site on the GABAA receptor complex was studied in acutely isolated Purkinje cells using whole‐cell recording and a fast drug application system.

When Zn2+ was applied together with 2 μM GABA, the Zn2+‐induced block of GABA‐mediated currents was not additive to the Cu2+‐induced block. In the presence of 0.1 μM Cu2+ in the bath solution the degree of inhibition of GABA‐mediated responses by Zn2+ was strongly attenuated.

Preapplication of 100 μM Zn2+ during 10 s, terminated 1 s before exposure to 2 μM GABA did not affect the GABA current in Cu2+‐free solution, but relieved its block by 0.1 μM Cu2+. This effect of Zn2+ was concentration‐dependent with an EC50 of 72 μM.

When the Cu2+‐induced block was removed by histidine, preapplication of Zn2+ did not increase the GABA current, indicating that the relief of Cu2+ block by Zn2+ is the result of its ability to actively remove Cu2+ from the GABA receptor complex.

It is proposed that the inhibitory effects of Zn2+ and Cu2+ on GABA‐induced currents result from an action of these metal ions at distinct, but conformationally linked sites on the GABAA receptor protein. Under physiological conditions Zn2+ would liberate Cu2+ from the GABAA receptor, thus facilitating Cu2+ turnover and its binding by other endogenous chelating molecules.

British Journal of Pharmacology (2000) 130, 851–856; doi:10.1038/sj.bjp.0703392