bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacological differences between the human and rat vanilloid receptor 1 (VR1)
Authors: McIntyre Peter , McLatchie Linda M , Chambers Anne , Phillips Elsa , Clarke Melanie , Savidge Jonathan , Toms Christy , Peacock Marcus , Shah Kirti , Winter Janet , Weerasakera Natasha , Webb Mike , Rang Humphrey P , Bevan Stuart , James Iain F
Published: January 2005

Vanilloid receptors (VR1) were cloned from human and rat dorsal root ganglion libraries and expressed in Xenopus oocytes or Chinese Hamster Ovary (CHO) cells.

Both rat and human VR1 formed ligand gated channels that were activated by capsaicin with similar EC50 values. Capsaicin had a lower potency on both channels, when measured electrophysiologically in oocytes compared to CHO cells (oocytes: rat=1.90±0.20 μM; human=1.90±0.30 μM: CHO cells: rat=0.20±0.06 μM; human=0.19±0.08 μM).

In CHO cell lines co‐expressing either rat or human VR1 and the calcium sensitive, luminescent protein, aequorin, the EC50 values for capsaicin‐induced responses were similar in both cell lines (rat=0.35±0.06 μM, human=0.53±0.03 μM).

The threshold for activation by acidic solutions was lower for human VR1 channels than that for rat VR1 (EC50 pH 5.49±0.04 and pH 5.78±0.09, respectively).

The threshold for heat activation was identical (42°C) for rat and human VR1.

PPAHV was an agonist at rat VR1 (EC50 between 3 and 10 μM) but was virtually inactive at the human VR1 (EC50>10 μM).

Capsazepine and ruthenium red were both more potent at blocking the capsaicin response of human VR1 than rat VR1.

Capsazepine blocked the human but not the rat VR1 response to low pH. Capsazepine was also more effective at inhibiting the noxious heat response of human than of rat VR1.

British Journal of Pharmacology (2001) 132, 1084–1094; doi:10.1038/sj.bjp.0703918
HMG‐CoA reductase inhibitors and P‐glycoprotein modulation
Authors: Bogman Katrijn , Peyer Anne‐Kathrin , Török Michael , Küsters Ernst , Drewe Jürgen
Published: January 2005

Five 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitors (statins), (e.g. atorvastatin, fluvastatin, lovastatin, pravastatin and simvastatin), were investigated for their ability to reverse P‐glycoprotein (P‐gp) mediated rhodamine 123 (R123) transport in a murine monocytic leukaemia cell line that over‐expresses the multi‐drug resistance protein 1a/b (mdr1a/1b).

P‐gp modulation was studied by a fluorimetric assay and confocal microscopy by means of R123 efflux and uptake experiments, respectively.

Atorvastatin acid, methyl ester and lactone, lovastatin lactone and simvastatin lactone inhibited R123 transport in a concentration‐dependent manner. Lovastatin acid, simvastatin acid, fluvastatin and pravastatin did not show a significant inhibition of the R123 transport in our cell system. Atorvastatin methyl ester and lactone showed the highest affinities for P‐gp and results were comparable for both methods.

In conclusion, monitoring of R123 transport in living cells by confocal microscopy in addition to fluorimetric assay is a sensitive tool to study P‐gp affinity in drug screening that is especially useful for early phases of drug development.

British Journal of Pharmacology (2001) 132, 1183–1192; doi:10.1038/sj.bjp.0703920
Inhibitory effect of 2,3‐butanedione monoxime (BDM) on Na+/Ca2+exchange current in guinea‐pig cardiac ventricular myocytes
Authors: Watanabe Yasuhide , Iwamoto Takahiro , Matsuoka Isao , Ohkubo Satoko , Ono Tomoyuki , Watano Tomokazu , Shigekawa Munekazu , Kimura Junko
Published: January 2005

The effect of 2,3‐butanedione monoxime (BDM), a ‘chemical phosphatase’, on Na+/Ca2+exchange current (INCX) was investigated using the whole‐cell voltage‐clamp technique in single guinea‐pig cardiac ventricular myocytes and in CCL39 fibroblast cells expressing canine NCX1.

INCXwas identified as a current sensitive to KB‐R7943, a relatively selective NCX inhibitor, at 140 mMNa+and 2 mMCa2+in the external solution and 20 mMNa+and 433 nMfree Ca2+in the pipette solution.

In guinea‐pig ventricular cells, BDM inhibitedINCXin a concentration‐dependent manner. The IC50value was 2.4 mMwith a Hill coefficients of 1. The average time for 50% inhibition by 10 mMBDM was 124±31 s (n=5).

The effect of BDM was not affected by 1 μMokadaic acid in the pipette solution, indicating that the inhibition was notviaactivation of okadaic acid‐sensitive protein phosphatases.

Intracellular trypsin treatmentviathe pipette solution significantly suppressed the inhibitory effect of BDM, implicating an intracellular site of action of BDM.

PAM (pralidoxime), another oxime compound, also inhibitedINCXin a manner similar to BDM.

Isoprenaline at 50 μMand phorbol 12‐myristate 13‐acetate (PMA) at 8 μMdid not reverse the inhibition ofINCXby BDM.

BDM inhibitedINCXin CCL39 cells expressing NCX1 and in its mutant in which its three major phosphorylatable serine residues were replaced with alanines.

We conclude that BDM inhibitsINCXbut the mechanism of inhibition is not by dephosphorylation of the Na+/Ca2+exchanger as a ‘chemical phosphatase’.

British Journal of Pharmacology(2001)132, 1317–1325; doi:10.1038/sj.bjp.0703926
Inhibition of aggregation of rabbit and human platelets induced by adrenaline and 5‐hydroxytryptamine by KB‐R7943, a Na+/Ca2+ exchange inhibitor
Authors: Takano Shizuko , Kimura Junko , Ono Tomoyuki
Published: January 2005

We investigated the effect of KB‐R7943, a Na+/Ca2+ exchange inhibitor, on the aggregation response induced by adrenaline and 5‐hydroxytryptamine (5‐HT), alone or in combination in human and rabbit platelets in the presence or absence of ouabain.

KB‐R7943 inhibited aggregation induced by the combination of adrenaline and 5‐HT in a concentration‐dependent manner. The IC50 values of KB‐R7943 were 4.2±2.0 or 3.0±0.7 μM with washed rabbit platelets with or without ouabain pretreatment, respectively.

In platelet‐rich human plasma, the aggregation was biphasic. The IC50 value of KB‐R7943 was 17.2±4.4 μM for the first phase aggregation.

KB‐R7943 did not inhibit the first phase of aggregation induced by adrenaline alone, or the monophasic aggregation induced by 5‐HT alone.

The aggregation of rabbit platelets depended on the presence of K+ in the medium, and K+‐dependent and K+‐independent Ca2+ influx were observed in resting platelets. Ouabain treatment increased only the K+‐dependent but not the K+‐independent Ca2+ influx.

KB‐R7943 inhibited K+‐dependent Ca2+ influx with or without ouabain pretreatment, but not K+‐independent Ca2+ influx.

From these results, we conclude that KB‐R7943 inhibits the adrenaline plus 5‐HT induced aggregation of rabbit and human platelets by inhibiting K+‐dependent Na+/Ca2+ exchange (NCKX). Our results suggest that NCKX plays an important role in platelet aggregation.

British Journal of Pharmacology (2001) 132, 1383–1388; doi:10.1038/sj.bjp.0703928
Investigation of mechanisms that mediate reactive hyperaemia in guinea‐pig hearts: role of KATP channels, adenosine, nitric oxide and prostaglandins
Authors: Kingsbury M P , Robinson H , Flores N A , Sheridan D J
Published: January 2005

Reactive hyperaemia is a transient vasodilatation following a brief ischaemic period. ATP‐dependent K+ (KATP) channels may be important in mediating this response, however it is unclear whether mitochondrial KATP channels contribute to this in the heart.

We examined the involvement of KATP channels and the relative role of mitochondrial channels as mediators of coronary reactive hyperaemia and compared them to mechanisms involving NO, prostaglandins and adenosine in the guinea‐pig isolated heart.

Reactive hyperaemic vasodilatation (peak vasodilator response and flow debt repayment) were assessed after global zero‐flow ischaemia (5 – 120 s) in the presence of nitro‐L‐arginine methyl ester (L‐NAME, 10−5 M, n=9), 8‐phenyltheophylline (8‐PT, 10−6 M, n=12) and indomethacin (10−5 M, n=12). Glibenclamide (10−6 M, n=12) a non‐selective KATP channel inhibitor and 5‐hydroxy‐decanoic acid (5‐HD, 10−4 M, n=10) a selective mitochondrial KATP channel inhibitor were also used. The specificity of the effects of glibenclamide and 5‐HD (n=6 each) were confirmed using pinacidil (38 nmol – 10 μmol) and diazoxide (42 nmol – 2 μmol).

Glibenclamide was most effective in blocking the hyperaemic response (by 87%, P<0.001) although 5‐HD and 8‐PT also had a marked effect (40% inhibition, P<0.001 and 32%, P<0.001, respectively). L‐NAME and indomethacin had little effect.

Perfusion with L‐NAME and glibenclamide significantly reduced baseline coronary flow (22%, P<0.01 and 33%, P<0.01) while 8‐PT, indomethacin and 5‐HD had no effect.

KATP channels are the major mediators of the coronary reactive hyperaemic response in the guinea‐pig. Although mitochondrial KATP channels contribute, they appear less important than sarcolemmal channels.

British Journal of Pharmacology (2001) 132, 1209–1216; doi:10.1038/sj.bjp.0703929
Gα14 links a variety of Gi‐ and Gs‐coupled receptors to the stimulation of phospholipase C
Authors: Ho Maurice K C , Yung Lisa Y , Chan Joy S C , Chan Jasmine H P , Wong Cecilia S S , Wong Yung H
Published: January 2005

The bovine Gα14 is a member of the Gq subfamily of G proteins that can regulate phospholipase Cβ isoforms but the extent to which Gα14 recognizes different receptor classes is not known.

Gα14 was cotransfected with a variety of receptors in COS‐7 cells, and agonist‐induced stimulation of phospholipase C was then measured.

Activation of the type 2 but not type 1 somatostatin receptor in cells coexpressing Gα14 stimulated the accumulation of inositol phosphates; functional expression of both subtypes of somatostatin receptors was determined by the ability of somatostatin to inhibit cyclic AMP accumulation.

Among the three opioid receptors (μ, δ, and κ), only the δ receptor was capable of stimulating IP formation when coexpressed with Gα14 in COS‐7 cells.

A panel of Gi‐ and Gs‐linked receptors was screened for their ability to stimulate IP accumulation via Gα14. The adenosine A1, complement C5a, dopamine D1, D2 and D5, formyl peptide, luteinizing hormone, secretin, and the three subtypes of melatonin (mt1, MT2, and Xenopus) receptors were all incapable of activating Gα14, while the α2‐ and β2‐adrenoceptors were able to do so.

Gα14‐mediated stimulation of phospholipase Cβ was agonist dose‐dependent. These data demonstrate that although Gα14 can interact with different classes of receptors, it is much less promiscuous than Gα15 or Gα16.

British Journal of Pharmacology (2001) 132, 1431–1440; doi:10.1038/sj.bjp.0703933
Protective effect of a PAR2‐activating peptide on histamine‐induced bronchoconstriction in guinea‐pig
Authors: Cicala C , Spina D , Keir S D , Severino B , Meli R , Page C P , Cirino G
Published: January 2005

Protease activated receptor‐2 (PAR2) is a seven transmembrane domain G protein coupled receptor proteolytically activated. PAR2, together with other PARs, can be also activated by peptides mimicking the sequence of the receptor tethered ligand. We have evaluated the effect of systemic administration of a peptide activating PAR2 (PAR2‐AP, SLIGRL) on histamine‐induced increase in lung resistances in the guinea‐pig.

Intravenous administration of PAR2‐AP (1 mg kg−1) significantly inhibited histamine‐induced increase in lung resistance in a time‐dependent fashion that was not abolished by indomethacin or vagotomy.

Bronchoprotective effect of PAR2‐AP was not reversed by the cyclo‐oxygenase inhibitor, indomethacin, the nitric oxide synthetase inhibitor, L‐NAME, nor by the non‐selective beta‐antagonist, propranolol.

Indomethacin augmented the bronchoconstriction to histamine which was inhibited by PAR2‐AP. Furthermore, in vagotomized animals, the bronchial hyper‐responsiveness to histamine was significantly reduced, and in these circumstances, PAR2‐AP still retained the capacity to provide bronchoprotection against histamine.

PAR2‐AP also produced a modest reduction in histamine‐induced protein leakage in trachea and upper bronchi.

Our results indicated that PAR2 might have a bronchoprotective role in the guinea‐pig in vivo independent of prostaglandin or nitric oxide release.

British Journal of Pharmacology (2001) 132, 1229–1234; doi:10.1038/sj.bjp.0703934
Mechanism of CGRP‐induced relaxation in rat intramural coronary arteries
Authors: Sheykhzade Majid , Berg Nyborg Niels C
Published: January 2005

This study investigates the mechanism of CGRP‐induced relaxation in intramural coronary arteries by determining the effect of CGRP on cytosolic Ca2+ concentration ([Ca2+]i) using FURA‐2 technique.

CGRP concentration‐dependently (10 pM – 100 nM) decreased the [Ca2+]i and tension of coronary arteries precontracted with either U46619 or BAY K 8644, and also of resting coronary arteries in PSS. In 36 mM K+‐depolarized arteries, CGRP reduced only the tension without affecting the [Ca2+]i.

In 300 nM U46619‐ precontracted arteries, pretreatment with 10 μM thapsigargin significantly (P<0.05) attenuated the CGRP‐induced reduction in the tension (but not [Ca2+]i).

In 300 nM U46619‐precontracted arteries, pretreatment with either 100 nM charybdotoxin or 100 nM iberiotoxin or 10 nM felodipine significantly (P<0.05) attenuated the CGRP‐induced reduction in both [Ca2+]i and tension. In contrast, 1 μM glibenclamide did not affect the CGRP‐induced responses in these coronary arteries.

In resting coronary arteries, only pretreatment with the combination of 1 μM glibenclamide and 100 nM charybdotoxin attenuated the CGRP‐induced decrease in the [Ca2+]i and tension, suggesting a different mechanism of action for CGRP in resting coronary arteries.

We conclude that CGRP relaxes precontracted rat coronary arteries via three mechanisms: (1) a decrease in [Ca2+]i by inhibiting the Ca2+ influx through membrane hyperpolarization mediated partly by activation of the large conductance Ca2+‐activated potassium channels, (2) a decrease in [Ca2+]i presumably by sequestrating cytosolic Ca2+ into thapsigargin‐sensitive Ca2+ storage sites and (3) a decrease in the Ca2+‐sensitivity of the contractile apparatus. In resting coronary arteries, however, there seems to be an interplay between different types of K+ channels.

British Journal of Pharmacology (2001) 132, 1235–1246; doi:10.1038/sj.bjp.0703936
GLC‐3: a novel fipronil and BIDN‐sensitive, but picrotoxinin‐insensitive, L‐glutamate‐gated chloride channel subunit from Caenorhabditis elegans
Authors: Horoszok Lucy , Raymond Valérie , Sattelle David B , Wolstenholme Adrian J
Published: January 2005

We report the cloning and expression of a novel Caenorhabditis elegans polypeptide, GLC‐3, with high sequence identity to previously cloned L‐glutamate‐gated chloride channel subunits from nematodes and insects.

Expression of glc‐3 cRNA in Xenopus oocytes resulted in the formation of homo‐oligomeric L‐glutamate‐gated chloride channels with robust and rapidly desensitizing currents, an EC50 of 1.9±0.03 mM and a Hill coefficient of 1.5±0.1. GABA, glycine, histamine and NMDA all failed to activate the GLC‐3 homo‐oligomer at concentrations of 1 mM. The anthelminthic, ivermectin, directly and irreversibly activated the L‐glutamate‐gated channel with an EC50 of 0.4±0.02 μM.

The GLC‐3 channels were selective for chloride ions, as shown by the shift in the reversal potential for L‐glutamate‐gated currents after the reduction of external Cl− from 107.6 to 62.5 mM.

Picrotoxinin failed to inhibit L‐glutamate agonist responses at concentrations up to 1 mM. The polycyclic dinitrile, 3,3‐bis‐trifluoromethyl‐bicyclo[2,2,1]heptane‐2,2‐dicarbonitrile (BIDN), completely blocked L‐glutamate‐induced chloride currents recorded from oocytes expressing GLC‐3 with an IC50 of 0.2±0.07 μM. The phenylpyrazole insecticide, fipronil, reversibly inhibited L‐glutamate‐gated currents recorded from the GLC‐3 receptor with an IC50 of 11.5±0.11 μM.

In this study, we detail the unusual antagonist pharmacology of a new GluCl subunit from C. elegans. Unlike all other native and recombinant nematode GluCl reported to date, the GLC‐3 receptor is insensitive to picrotoxinin, but is sensitive to two other channel blockers, BIDN and fipronil. Further study of this receptor may provide insights into the molecular basis of non‐competitive antagonism by these compounds.

British Journal of Pharmacology (2001) 132, 1247–1254; doi:10.1038/sj.bjp.0703937
Disturbance of peristalsis in the guinea‐pig isolated small intestine by indomethacin, but not cyclo‐oxygenase isoform‐selective inhibitors
Authors: Shahbazian Anaid , Schuligoi Rufina , Heinemann Akos , Peskar Bernhard A , Holzer Peter
Published: January 2005

Since the cyclo‐oxygenase (COX) isoform‐nonselective inhibitor indomethacin is known to modify intestinal motility, we analysed the effects of COX‐1 and COX‐2 inhibition on intestinal peristalsis.

Peristalsis in isolated segments of the guinea‐pig small intestine was triggered by a rise of the intraluminal pressure and recorded via the pressure changes associated with peristalsis.

The COX‐1 inhibitor SC‐560, the COX‐2 inhibitor NS‐398 (both at 0.1  – 1 μM) and the isoform‐nonselective inhibitors flurbiprofen (0.01 – 10 μM) and piroxicam (0.1 – 50 μM) were without major influence on peristalsis, whereas indomethacin and etodolac (0.1 – 10 μM) disturbed the regularity of peristalsis by causing nonpropulsive circular muscle contractions.

Radioimmunoassay measurements showed that SC‐560, NS‐398, indomethacin and etodolac (each at 1 μM) suppressed the release of 6‐keto‐prostaglandin F1α (6‐keto‐PGF1α) from the intestinal segments.

Reverse transcription – polymerase chain reaction tests revealed that, relative to glyceraldehyde‐3 phosphate dehydrogenase ribonucleic acid, the expression of COX‐1 mRNA increased by a factor of 2.0 whereas that of COX‐2 mRNA rose by a factor of 7.9 during the 2 h experimental period.

Pharmacological experiments indicated that the action of indomethacin to disturb intestinal peristalsis was unrelated to inhibition of L‐type calcium channels, adenosine triphosphate‐sensitive potassium channels or phosphodiesterase type IV.

These results show that selective inhibition of COX‐1 and COX‐2 does not grossly alter peristaltic motor activity in the guinea‐pig isolated small intestine and that the effect of indomethacin to disturb the regular pattern of propulsive motility in this species is unrelated to COX inhibition.

British Journal of Pharmacology (2001) 132, 1299–1309; doi:10.1038/sj.bjp.0703940
Platelet GPIIb/IIIa binding characteristics of small molecule RGD mimetic: distinct binding profile for Roxifiban
Authors: Mousa Shaker A , Bozarth Jeffrey M , Naik Ulhas P , Slee Andrew
Published: January 2005

A number of non‐peptide orally active RGD mimetic prodrug such as Orbofiban, Sibrafiban, SR121566, Roxifiban and others entered into the clinical evaluation stage. Some of these agents were terminated and some are still in clinical trials.

The present study examined the platelet GPIIb/IIIa binding profiles for the active form of Roxifiban, Sibrafiban, SR121566 and Orbofiban using 3H‐Roxifiban active form (XV459), 3H‐DMP728, 125I‐Echistatin, and 125I‐Fibrinogen.

Either DMP728, Orbofiban, Sibrafiban, SR121566 or Roxifiban active form as well as other RGD mimetic bind to the same binding site (s) on human platelets as evident from the competitive inhibition of binding of each other to human platelet. Additionally, Roxifiban active form competed with FITC labeled GPIIb/IIIa antagonist cyclic RGD peptidomimetic (XL086) as demonstrated using confocal microscopy technique.

Roxifiban active form (XV459) demonstrated the highest potency in inhibiting 3H‐XV459, 3H‐DMP728, 125I‐Echistatin, and 125I‐Fibrinogen binding to human platelets as compared to the others.

Structure activity relationship within the isoxazoline Roxifiban series showed that substituent at the α‐carbon next to the carboxy terminal represents an exosite for the affinity binding to human platelets leading to slow platelet dissociation rate.

These data indicated a distinct binding profile for Roxifiban (high affinity to both activated and resting platelets associated with a relatively slow Koff) as compared to others. These differences might determine the pharmacodynamics and pharmackokinetics of the different GPIIb/IIIa antagonists.

British Journal of Pharmacology (2001) 133, 331–336; doi:10.1038/sj.bjp.0703943
Chronic systemic administration of salmeterol to rats promotes pulmonary β2‐adrenoceptor desensitization and down‐regulation of Gsα
Authors: Finney Paul A , Donnelly Louise E , Belvisi Maria G , Chuang Tsu‐Tshen , Birrell Mark , Harris Andrew , Mak Judith C W , Scorer Carol , Barnes Peter J , Adcock Ian M , Giembycz Mark A
Published: January 2005

The aim of the present study was to examine the effects of chronic infusion of the long‐acting agonist salmeterol on pulmonary β2‐adrenoceptor function in Sprague‐Dawley rats in vivo and to elucidate the molecular basis of any altered state.

Systemic administration of rats with salmeterol for 7 days compromised the ability of salmeterol and prostaglandin E2 (PGE2), given acutely by the intravenous route, to protect against ACh‐induced bronchoconstriction when compared to rats treated identically with vehicle.

β1‐ and β2‐adrenoceptor density was significantly reduced in lung membranes harvested from salmeterol‐treated animals, which was associated with impaired salmeterol‐ and PGE2‐induced cyclic AMP accumulation ex vivo.

Three variants of Gsα that migrated as 42, 44 and 52 kDa peptides on SDS polyacrylamide gels were detected in lung membranes prepared from both groups of rats but the intensity of each isoform was markedly reduced in rats that received salmeterol.

The activity of cytosolic, but not membrane‐associated, G‐protein receptor‐coupled kinase was elevated in the lung of salmeterol‐treated rats when compared to vehicle‐treated animals.

The ability of salmeterol, administered systemically, to protect the airways of untreated rats against ACh‐induced bronchoconstriction was short‐acting (toff ∼45 min), which contrasts with its long‐acting nature when given to asthmatic subjects by inhalation.

These results indicate that chronic treatment of rats with salmeterol results in heterologous desensitization of pulmonary Gs‐coupled receptors. In light of previous data obtained in rats treated chronically with salbutamol, we propose that a primary mechanism responsible for this effect is a reduction in membrane‐associated Gsα. The short‐acting nature of salmeterol, when administered systemically, and the reduction in β‐adrenoceptor number may be due to metabolism to a biologically‐active, short‐acting and non‐selective β‐adrenoceptor agonist.

British Journal of Pharmacology (2001) 132, 1261–1270; doi:10.1038/sj.bjp.0703946
Evidence for a non‐adrenoceptor, imidazoline‐mediated contractile response to oxymetazoline in the porcine isolated rectal artery
Authors: Minyan Wang , Dunn W R , Blaylock N A , Chan S L F , Wilson V G
Published: January 2005

Imidazoline derivatives are known to elicit responses through both α2‐adrenoceptor and non‐adrenoceptor, imidazoline sites, though as yet there are no examples of the latter on vascular smooth muscle. In the presence of 0.3 μM prazosin, neither UK‐14304 (0.01 – 3 μM) nor oxymetazoline (0.01 – 30 μM) caused a significant contraction of the porcine isolated rectal artery, a preparation with a low density of α2‐adrenoceptors. In the presence of a combination of U46619 and forskolin, however, both agonists produced concentration‐dependent contractions. Pretreatment with phenoxybenzamine (3 μM) abolished responses to UK‐14304, but left those elicited by oxymetazoline largely unaffected. The putative I3 imidazoline antagonist 2‐(2,3 dihydro‐2‐benzofuranyl)‐2‐imidazole (KU‐14R, 10 μM) caused a 6 fold rightward displacement of the phenoxybenzamine‐insensitive concentration – response curve to oxymetazoline. Our data indicates that non‐adrenoceptor, imidazoline sites, pharmacologically similar to the I3 imidazoline site on islet cells, mediate vasoconstriction in the porcine isolated rectal artery.

British Journal of Pharmacology (2001) 132, 1359–1363; doi:10.1038/sj.bjp.0703949
Thrombin‐induced p38 mitogen‐activated protein kinase activation is mediated by epidermal growth factor receptor transactivation pathway
Authors: Kanda Yasunari , Mizuno Katsushige , Kuroki Yasutomi , Watanabe Yasuhiro
Published: January 2005

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMC) and has been implicated its pathogenic role in vascular remodelling. However, the signalling pathways by which thrombin mediates its mitogenic response are not fully understood.

We have previously reported that thrombin activates p38 mitogen‐activated protein kinase (p38 MAPK) by a tyrosine kinase‐dependent mechanism, and that p38 MAPK has a role in thrombin‐induced mitogenic response in rat VSMC.

In the present study, we examine the involvement of epidermal growth factor (EGF) receptor in thrombin‐induced p38 MAPK activation. We found that thrombin induced EGF receptor tyrosine phosphorylation (transactivation) in A10 cells, a clonal VSMC cell line. A selective inhibitor of EGF receptor kinase (AG1478) inhibited the p38 MAPK activation in a dose‐dependent manner, whereas it had no effect on the response to platelet‐derived growth factor (PDGF). EGF receptor phosphorylation induced by thrombin was inhibited by BAPTA‐AM and GF109203X, which suggest a requirement for intracellular Ca2+ increase and protein kinase C.

We next examined the effect of AG1478 on thrombin‐induced DNA synthesis. AG1478 inhibited thrombin‐induced DNA synthesis in a dose‐dependent manner. In contrast, PDGF‐induced DNA synthesis was not affected by AG1478.

In conclusion, these data suggest that the EGF receptor transactivation and subsequent p38 MAPK activation is required for thrombin‐induced proliferation of VSMC.

British Journal of Pharmacology (2001) 132, 1657–1664; doi:10.1038/sj.bjp.0703952
Nociceptin inhibits cough in the guinea‐pig by activation of ORL1 receptors
Authors: McLeod Robbie L , Parra Leonard E , Mutter Jennifer C , Erickson Christine H , Carey Galen J , Tulshian Deen B , Fawzi Ahmad B , Smith‐Torhan April , Egan Robert W , Cuss Francis M , Hey John A
Published: January 2005

We studied the central and peripheral antitussive effect of ORL1 receptor activation with nociceptin/orphanin FQ in conscious guinea‐pigs. In guinea‐pig cough studies, nociceptin/orphanin FQ (10, 30, and 90 μg) given directly into the CNS by an intracerebroventricular (i.c.v.) route inhibited cough elicited by capsaicin exposure by approximately 23, 29 and 52%, respectively. The antitussive activity of nociceptin/orphanin FQ (90 μg, i.c.v.) was blocked by the selective ORL1 antagonist [Phe1γ(CH2‐NH)Gly2]nociceptin‐(1‐13)‐NH2 (180 μg, i.c.v.) and J113397 (10 mg kg−1, i.p.) but not by the opioid antagonist, naltrexone (3 mg kg−1, i.p.). Furthermore, intravenous (i.v.) nociceptin/orphanin FQ (1.0 and 3.0 mg kg−1) also inhibited cough approximately by 25 and 42%, respectively. These findings indicate that selective ORL1 agonists display the potential to inhibit cough by both a central and peripheral mechanism, and potentially represent a novel therapeutic approach for the treatment of cough.

British Journal of Pharmacology (2001) 132, 1175–1178; doi:10.1038/sj.bjp.0703954
Effects of specific inhibition of cyclo‐oxygenase‐1 and cyclo‐oxygenase‐2 in the rat stomach with normal mucosa and after acid challenge
Authors: Gretzer Britta , Maricic Nenad , Respondek Michael , Schuligoi Rufina , Peskar Brigitta M
Published: January 2005

Effects of the cyclo‐oxygenase (COX)‐1 inhibitor SC‐560 and the COX‐2 inhibitors rofecoxib and DFU were investigated in the normal stomach and after acid challenge.

In healthy rats, neither SC‐560 nor rofecoxib (20 mg kg−1 each) given alone damaged the mucosa. Co‐treatment with SC‐560 and rofecoxib, however, induced severe lesions comparable to indomethacin (20 mg kg−1) whereas co‐administration of SC‐560 and DFU (20 mg kg−1 each) had no comparable ulcerogenic effect 5 h after dosing.

SC‐560 (20 mg kg−1) inhibited gastric 6‐keto‐prostaglandin (PG) F1α by 86±5% and platelet thromboxane (TX) B2 formation by 89±4% comparable to indomethacin (20 mg kg−1). Rofecoxib (20 mg kg−1) did not inhibit gastric and platelet eicosanoids.

Intragastric HCl elevated mucosal mRNA levels of COX‐2 but not COX‐1. Dexamethasone (2 mg kg−1) prevented the up‐regulation of COX‐2.

After acid challenge, SC‐560 (5 and 20 mg kg−1) induced dose‐dependent injury. Rofecoxib (20 mg kg−1), DFU (5 mg kg−1) and dexamethasone (2 mg kg−1) given alone were not ulcerogenic but aggravated SC‐560‐induced damage. DFU augmented SC‐560 damage 1 but not 5 h after administration whereas rofecoxib increased injury after both treatment periods suggesting different time courses.

Gastric injurious effects of rofecoxib and DFU correlated with inhibition of inflammatory PGE2.

The findings show that in the normal stomach lesions only develop when both COX‐1 and COX‐2 are inhibited. In contrast, during acid challenge inhibition of COX‐1 renders the mucosa more vulnerable suggesting an important role of COX‐1 in mucosal defence in the presence of a potentially noxious agent. In this function COX‐1 is supported by COX‐2. In the face of pending injury, however, COX‐2 cannot maintain mucosal integrity when the activity of COX‐1 is suppressed.

British Journal of Pharmacology (2001) 132, 1565–1573; doi:10.1038/sj.bjp.0703955
Role of endothelin and vasopressin in DOCA‐salt hypertension
Authors: Yu Ming , Gopalakrishnan V , Robert McNeill J
Published: January 2005

The relative roles of endothelin (ET) and vasopressin (AVP) in the regulation of blood pressure (BP), cardiac output (CO) and total peripheral resistance (TPR) were investigated in the early stages (24 – 31 days) of development of hypertension in the conscious deoxycorticosterone acetate (DOCA)‐salt hypertensive rat model.

BP was recorded with radiotelemetry devices and CO with ultrasonic transit‐time probes. TPR was calculated from the BP and CO recordings. The contributions of endogenous ET and AVP were studied by infusing [d(CH2)51,O‐Me_Tyr2,Arg8]‐vasopressin, a V1‐receptor antagonist, and bosentan, a mixed ETA/ETB receptor antagonist (Study 1). Vascular responsiveness was estimated from the changes in TPR evoked by i.v. infusions of ET‐1 and AVP (Study 2).

In study 1, infusion of bosentan reduced TPR and BP dramatically in DOCA‐salt hypertensive rats but not in SHAM control rats, and this effect was greater when the AVP system had been blocked. In contrast, the V1 receptor antagonist alone failed to change TPR and BP in DOCA‐salt hypertensive rats. However, subsequent infusion of the V1 receptor antagonist during the plateau phase of the response in bosentan pretreated DOCA‐salt hypertensive rats led to significant decreases in both BP and TPR.

In study 2, TPR and BP responses to ET‐1, but not AVP, were greater in DOCA‐salt rats than in control rats. CO responses to ET‐1 or AVP were similar in the two groups.

The results suggest that both ET and AVP play a role in the maintenance of TPR and BP; when one system is blocked the other compensates. However, the magnitude of the contribution to the hypertensive state appears greater for ET than for AVP. Enhanced vascular responses to ET appear to contribute to this greater role.

British Journal of Pharmacology (2001) 132, 1447–1454; doi:10.1038/sj.bjp.0703958
Chronic vascular toxicity of doxorubicin in an organ‐cultured artery
Authors: Murata Takahisa , Yamawaki Hideyuki , Hori Masatoshi , Sato Koichi , Ozaki Hiroshi , Karaki Hideaki
Published: January 2005

We investigated the chronic effects of doxorubicin (DXR) on morphological and functional changes in the rabbit mesenteric artery using an organ culture system.

In arteries cultured with 0.3 μM DXR for 7 days, the contractions induced by noradrenaline, but not those induced by endothelin‐1 or high K+, were strongly inhibited. This reaction was followed by a decrease in the induction of the α1A‐adrenoceptor without any change in the mRNA level. Inhibition of noradrenaline‐induced contractions by DXR was attenuated by superoxide dismutase, and α1A‐adrenoceptor protein expression recovered.

In the arteries cultured with 1 μM DXR for 7 days, contractions induced by endothelin‐1 or high K+ and absolute force in the permeabilized muscles were also inhibited. Morphological examinations revealed the existence of concentrated nuclei and terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling (TUNEL)‐positive smooth muscle cells, and internucleosomal DNA fragmentation was also detected, indicating the induction of apoptosis.

In the arteries cultured with 10 μM DXR for 7 days, nuclear swelling, karyolysis and random DNA fragmentation indicative of necrosis were observed, and muscle contractility was abolished.

These results suggest that 0.3 μM DXR selectively down‐regulates the α1A‐adrenoceptor protein expression, resulting in a decrease in the noradrenaline‐induced contraction. This down‐regulation may be at least partly due to the production of a superoxide radical. DXR also caused a decrease in muscle contractility followed by apoptotic changes at 1 μM and necrotic changes at 10 μM. These changes might be responsible for the disturbance of the circulatory system that is often observed during the course of repetitive chemotherapy.

British Journal of Pharmacology (2001) 132, 1365–1373; doi:10.1038/sj.bjp.0703959
Mechanism of airway hyperresponsiveness to adenosine induced by allergen challenge in actively sensitized Brown Norway rats
Authors: Hannon J P , Tigani B , Williams I , Mazzoni L , Fozard J R
Published: January 2005

We have explored the role of allergen sensitization and challenge in defining the response of the airways of the Brown Norway (BN) rat to adenosine.

In naïve animals or in rats sensitized to ovalbumin (OA) adenosine induced only weak bronchoconstrictor responses. Challenge of sensitized animals with OA induced a marked airway hyperresponsiveness to adenosine which was not seen with methacholine or bradykinin.

The augmented bronchoconstrictor response to adenosine was not affected by acute bivagotomy or atropine nor mimicked by an i.v. injection of capsaicin. It was, however, blocked selectively by disodium cromoglycate methysergide or ketanserin and reduced in animals treated sub‐chronically with compound 48/80.

The augmented response to adenosine was associated with increases in the plasma concentrations of both histamine and 5‐hydroxytryptamine (5‐HT), which were attenuated by pretreatment with disodium cromoglycate, and degranulation of mast cells in the lung.

Parenchymal strips from lungs removed from sensitized rats challenged with OA gave augmented bronchoconstrictor responses to adenosine relative to strips from sensitized animals challenged with saline. Responses were inhibited by methysergide and disodium cromoglycate.

These data demonstrate a marked augmentation of the bronchoconstrictor response to adenosine in actively sensitized BN rats challenged with OA. The augmented response is primarily a consequence of mast cell activation, leading to the release of 5‐HT, which in turn induces bronchoconstriction. Our data further suggest the involvement of a discrete lung‐based population of mast cells containing and releasing mainly 5‐HT and brought into play by prior exposure to allergen.

British Journal of Pharmacology (2001) 132, 1509–1523; doi:10.1038/sj.bjp.0703961
Effects of mitiglinide (S 21403) on Kir6.2/SUR1, Kir6.2/SUR2A and Kir6.2/SUR2B types of ATP‐sensitive potassium channel
Authors: Reimann Frank , Proks Peter , Ashcroft Frances M
Published: January 2005

We have investigated the mechanism of action of the novel anti‐diabetic agent mitiglinide (S 21403) on Kir6.2/SUR1, Kir6.2/SUR2A and Kir6.2/SUR2B types of ATP‐sensitive potassium (KATP) channel. These possess a common pore‐forming subunit, Kir6.2, and different regulatory sulphonylurea receptor (SUR) subunits. It is believed that they correspond to native KATP channels in pancreatic β‐cells, heart and non‐vascular smooth muscle, respectively.

Kir6.2 was coexpressed with SUR1, SUR2A or SUR2B in Xenopus oocytes and macroscopic currents were recorded in giant inside‐out membrane patches. Mitiglinide was added to the intracellular membrane surface.

Mitiglinide inhibited Kir6.2/SUR currents at two sites: a low‐affinity site on Kir6.2 and a high‐affinity site on SUR. Low‐affinity inhibition was similar for all three types of KATP channel but high‐affinity inhibition was greater for Kir6.2/SUR1 currents (IC50, 4 nM) than for Kir6.2/SUR2A or Kir6.2/SUR2B currents (IC50, 3 and 5 μM, respectively).

Inhibition of Kir6.2/SUR1 currents was only slowly reversible on the time scale of electrophysiological experiments.

Kir6.2/SUR1‐S1237Y currents, which previously have been shown to lack high affinity tolbutamide inhibition, resembled Kir6.2/SUR2 currents in being unaffected by 100 nM but blocked by 10 μM mitiglinide.

Our results show that mitiglinide is a high‐affinity drug that shows a 1000 fold greater affinity for the β‐cell type than the cardiac and smooth muscle types of KATP channel, when measured in excised patches.

British Journal of Pharmacology (2001) 132, 1542–1548; doi:10.1038/sj.bjp.0703962
Endothelium‐dependent vasorelaxation independent of nitric oxide and K+ release in isolated renal arteries of rats
Authors: Jiang Fan , Dusting Gregory J
Published: January 2005

We investigated whether K+ can act as an endothelium‐derived hyperpolarizing factor (EDHF) in isolated small renal arteries of Wistar‐Kyoto rats.

Acetylcholine (0.001 – 3 μM) caused relaxations that were abolished by removal of the endothelium. However, acetylcholine‐induced relaxations were not affected by the nitric oxide (NO) synthase inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μM), by L‐NAME plus the soluble guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3,‐a]quinoxalin‐1‐one (ODQ, 1 μM) or by L‐NAME plus the cyclo‐oxygenase inhibitor indomethacin (10 μM). In rings precontracted with high‐K+(60 mM) physiological salt solution in the presence of L‐NAME, acetylcholine‐induced relaxations were abolished.

L‐NAME‐resistant relaxations were abolished by the large‐conductance Ca2+‐activated K+ channel inhibitor charybdotoxin plus the small‐conductance Ca2+‐activated K+ channel inhibitor apamin, while the inward rectifier K+ channel inhibitor Ba2+ or the gap junction inhibitor 18α‐glycyrrhetinic acid had no effect. Acetylcholine‐induced relaxation was unchanged by ouabain (10 μM) but was partially inhibited by a higher concentration (100 μM).

In half of the tissues tested, K+(10 mM) itself produced L‐NAME‐resistant relaxations that were blocked by ouabain (10 μM) and partially reduced by charybdotoxin plus apamin, but not affected by 18α‐glycyrrhetinic acid or Ba2+. However, K+ did not induce relaxations in endothelium‐denuded tissues.

In conclusion, acetylcholine‐induced relaxations in this tissue are largely dependent upon hyperpolarization mechanisms that are initiated in the endothelium but do not depend upon NO release. K+ release cannot account for endothelium‐dependent relaxation and cannot be an EDHF in this artery. However, K+ itself can initiate endothelium‐dependent relaxations via a different pathway from acetylcholine, but the mechanisms of K+‐induced relaxations remain to be clarified.

British Journal of Pharmacology (2001) 132, 1558–1564; doi:10.1038/sj.bjp.0703965
Tedisamil and dofetilide‐induced torsades de pointes, rate and potassium dependence
Authors: Barrett Terrance D , Hennan James K , Fischbach Peter S , O'Neill Brian P , Driscoll Edward M , Lucchesi Benedict R
Published: January 2005

Tedisamil is a bradycardiac agent that prolongs the QT interval of the ECG and prevents cardiac arrhythmias. Given this profile, tedisamil might be expected to have proarrhythmic actions similar to Class III antiarrhythmic drugs. To address this question, the actions of dofetilide and tedisamil were examined in rabbit isolated hearts in which bradycardia was induced by AV ablation.

The QT interval was prolonged in a reverse rate‐dependent fashion by dofetilide (3 and 30 nM) and tedisamil (0.3 and 3 μM).

Torsades de pointes was observed in 1/7 hearts treated with 3 nM dofetilide and 0/7 hearts treated with 0.3 μM tedisamil. The incidence of torsades de pointes was increased to 5/7 in hearts treated with 30 nM dofetilide and to 7/7 in hearts treated with 3 μM tedisamil (both P<0.05 vs control).

The actions of 30 nM dofetilide and 3 μM tedisamil were also examined in hearts paced at 50, 100, 200 and 50 beats min−1 successively. Both drugs caused torsades de pointes in 5/5 hearts paced at 50 beats min−1; however, the incidence was reduced to 0/5 during pacing at 200 beats min−1. Thus, drug‐induced proarrhythmia was bradycardia‐dependent.

Drug‐induced prolongation of the interval between the peak and end of the T‐wave (QTa‐e) was reverse rate‐dependent and was associated with the occurrence of torsades de pointes (r=0.91, P<0.01).

The results suggest that tedisamil, like dofetilide, presents a risk for development of torsades de pointes.

British Journal of Pharmacology (2001) 132, 1493–1500; doi:10.1038/sj.bjp.0703967
Serum constituents can affect 2′‐& 3′‐O‐(4‐benzoylbenzoyl)‐ATP potency at P2X7 receptors
Authors: Michel A D , Xing M , Humphrey P P A
Published: January 2005

2′‐& 3′‐O‐(4‐benzoylbenzoyl)‐ATP (BzATP) is the prototypic agonist for P2X7 receptors. In this study we demonstrate that bovine serum albumin (BSA) can affect the potency of BzATP at P2X receptors.

BzATP potency (pEC50) to stimulate ethidium accumulation in cells expressing recombinant P2X7 receptors varied between 6.5 and 4, depending upon the species orthologue studied and ionic conditions employed. BSA (0.1 – 1 mg ml−1) and foetal bovine serum (FBS, 1 – 10% v v−1) inhibited responses to BzATP but only when the BzATP pEC50 exceeded 5.

BSA did not block ATP‐stimulated ethidium accumulation, suggesting its effects were independent of P2X7 receptor blockade.

BSA did not cause breakdown of nucleotides, although FBS (10% v v−1) exhibited appreciable nucleotidase activity and caused significant breakdown of ATP.

In the presence of BSA, lipids such as 11‐((5‐dimethylaminonaphthalene‐1‐sulphonyl)amino)undecanoic acid (DAUDA) and arachidonic acid (AA) markedly increased BzATP potency. Lipids had no affect on ATP potency in the presence of BSA and had little effect on responses to BzATP in the absence of BSA.

These results suggested that the reduction in BzATP potency by BSA was due to BzATP binding to BSA and that lipids prevented this binding. Consistent with this hypothesis, BzATP inhibited binding of the fluorescent lipid, DAUDA, to BSA.

In conclusion, BSA and lipids can markedly affect BzATP potency at P2X7 receptors but this is probably a consequence of BzATP binding to BSA. This finding has important implications when using BzATP in vivo or in the presence of albumin.

British Journal of Pharmacology (2001) 132, 1501–1508; doi:10.1038/sj.bjp.0703968
The effect of ABT‐702, a novel adenosine kinase inhibitor, on the responses of spinal neurones following carrageenan inflammation and peripheral nerve injury
Authors: Suzuki Rie , Stanfa Louise C , Kowaluk Elizabeth A , Williams Michael , Jarvis Michael F , Dickenson Anthony H
Published: January 2005

Adenosine (ADO) receptor activation modulates sensory transmission in the dorsal horn. Little is known about the circumstances underlying release of the purine. The present study was conducted to investigate the effect of a novel and potent non‐nucleoside adenosine kinase (AK) inhibitor, ABT‐702, on the responses of dorsal horn neurones to selected peripheral stimuli. ABT‐702 is orally effective to reduce behavioural signs of nociception in models of acute, inflammatory, and neuropathic pain.

Electrophysiological recordings were made from wide dynamic range (WDR) neurones in halothane‐anaesthetized rats. ABT‐702 was given subcutaneously following either carrageenan inflammation or peripheral nerve injury (L5/L6 spinal nerve ligation). Comparisons were made between carrageenan and uninjected control animals, and similarly between spinal nerve ligated (SNL) and sham operated animals.

ABT‐702 produced inhibition of the postdischarge, wind‐up and C‐fibre evoked responses in both carrageenan and nerve‐injured animals. Furthermore, the mechanical and thermal evoked responses were similarly reduced in SNL rats. Overall, ABT‐702 produced a significantly greater inhibition of these responses in SNL rats as compared to sham controls. Similarly ABT‐702 tended to produce greater effects after carrageenan inflammation, however this did not reach significance.

Protection of endogenous adenosine by ABT‐702 therefore produces a marked inhibition of the noxious evoked neuronal activity in inflamed and neuropathic rats. Our results demonstrate a plasticity in the endogenous adenosine‐mediated inhibitory system following SNL and provide a possible basis for the use of this compound for the treatment of neuropathic and other persistent pain states.

British Journal of Pharmacology (2001) 132, 1615–1623; doi:10.1038/sj.bjp.0703972
Tetrapentylammonium block of chloramine‐T and veratridine modified rat brain type IIA sodium channels
Authors: Ghatpande A S , Rao S , Sikdar S K
Published: January 2005

Tetrapentylammonium (TPeA) block of rat brain type IIA sodium channel α subunit was studied using whole cell patch clamp. Results indicate that TPeA blocks the inactivating brain sodium channel in a potential and use‐dependent manner similar to that of the cardiac sodium channel. Removal of inactivation using chloramine‐T (CT) unmasks a time‐dependent block by TPeA consistent with slow blocking kinetics. On the other hand, no time dependence is observed when inactivation is abolished by modification with veratridine. TPeA does not bind in a potential‐dependent fashion to veratridine‐modified channels and does not significantly affect gating of veratridine‐modified channels suggesting that high affinity binding of TPeA to the brain sodium channel is lost after veratridine modification.

British Journal of Pharmacology (2001) 132, 1755–1760; doi:10.1038/sj.bjp.0703973
Heterogeneity of neuronal nicotinic acetylcholine receptors in 5‐HT‐containing chemoreceptor cells of the chicken aorta
Authors: Ito Shigeo , Ohta Toshio , Kasai Yohei , Yonekubo Kazuki , Nakazato Yoshikazu
Published: January 2005

The effects of nicotinic agonists and antagonists on whole‐cell currents and 5‐hydroxytryptamine (5‐HT) release were studied in order to characterize nicotinic ACh receptors on the 5‐HT‐containing chemoreceptor cells of the chicken aorta.

ACh, nicotine and dimethylphenylpiperazinium (DMPP) evoked concentration‐dependent inward currents accompanied by increases in current noise at a holding potential of −70 mV. The peak amplitude of the current response to DMPP was 50% larger than that to either nicotine or ACh.

Hexamethonium, α – bungarotoxin (α – BTX) and methyllycaconitine decreased nicotine‐induced inward currents in a concentration‐dependent manner. Although hexamethonium (0.1 mM) abolished the current response to nicotine (30 μM), a high concentration (1 μM) of α – BTX decreased it only by about 30% of the control response. Methyllycaconitine (0.1 μM) decreased the current response to nicotine to the same extent as did α – BTX whilst a high concentration (10 μM) abolished the response.

ACh, nicotine and DMPP caused concentration‐dependent increases in 5‐HT output from the thoracic aorta which effect was blocked by hexamethonium (0.1 mM). Pre‐treatment with α – BTX (1 μM) for 30 min reduced the output of 5‐HT induced by ACh to 70% of the control response.

It is suggested that neuronal nicotinic ACh receptors, sensitive and insensitive to α – BTX, are present on the chemoreceptor cells of the chicken aorta, the activation of which causes the release of 5‐HT.

British Journal of Pharmacology (2001) 132, 1934–1940; doi:10.1038/sj.bjp.0703978