bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential modulation of endothelin ligand‐induced contraction in isolated tracheae from endothelin B (ETB) receptor knockout mice
Authors: Hay Douglas W P , Douglas Stephen A , Ao Zhaohui , Moesker Rodney M , Self Glenn J , Rigby Paul J , Luttmann Mark A , Goldie Roy G
Published: January 2005

The role of endothelin B (ETB) receptors in mediating ET ligand‐induced contractions in mouse trachea was examined in ETB receptor knockout animals.

Autoradiographic binding studies, using [125I]‐ET‐1, confirmed the presence of ETA receptors in tracheal and bronchial airway smooth muscle from wild‐type (+/+) and homozygous recessive (−/−) ETB receptor knockout mice. In contrast, ETB receptors were not detected in airway tissues from (−/−) mice.

In tracheae from (+/+) mice, the rank order of potencies of the ET ligands was sarafotoxin (Stx) S6c>ET‐1>ET‐3; Stx S6c had a lower efficacy than ET‐1 or ET‐3. In tissues from (−/−) mice there was no response to Stx S6c (up to 0.1 μM), whereas the maximum responses and potencies of ET‐1 and ET‐3 were similar to those in (+/+) tracheae. ET‐3 concentration‐response curve was biphasic in (+/+) tissues (via ETA and ETB receptor activation), and monophasic in (−/−) preparations (via stimulation of only ETA receptors).

In (+/+) preparations SB 234551 (1 nM), an ETA receptor‐selective antagonist, inhibited the secondary phase, but not the first phase, of the ET‐3 concentration‐response curve, whereas A192621 (100 nM), an ETB receptor‐selective antagonist, had the opposite effect. In (−/−) tissues SB 234551 (1 nM), but not A192621 (100 nM), produced a rightward shift in ET‐3 concentration‐response curves.

The results confirm the significant influence of both ETA and ETB receptors in mediating ET‐1‐induced contractions in mouse trachea. Furthermore, the data do not support the hypothesis of atypical ETB receptors. In this preparation ET‐3 is not an ETB receptor‐selective ligand, producing contractions via activation of both ETA and ETB receptors.

British Journal of Pharmacology (2001) 132, 1905–1915; doi:10.1038/sj.bjp.0703957
Heterogeneous increases of cytoplasmic calcium: distinct effects on down‐regulation of cell surface sodium channels and sodium channel subunit mRNA levels
Authors: Shiraishi Seiji , Shibuya Izumi , Uezono Yasuhito , Yokoo Hiroki , Toyohira Yumiko , Yamamoto Ryuichi , Yanagita Toshihiko , Kobayashi Hideyuki , Wada Akihiko
Published: January 2005

Long‐term (12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca2+ ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA)] caused a time‐ and concentration‐dependent reduction of cell surface [3H]‐saxitoxin (STX) binding capacity, but did not change the KD value. In A23187‐ or TG‐treated cells, veratridine‐induced 22Na+ influx was reduced (with no change in veratridine EC50 value) while it was enhanced by α‐scorpion venom, β‐scorpion venom, or Ptychodiscus brevis toxin‐3, like in nontreated cells.

The A23187‐ or TG‐induced decrease of [3H]‐STX binding was diminished by BAPTA‐AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca2+ ([Ca2+]i) to a greater extent than that observed with TG. 2,5‐Di‐(t‐butyl)‐1,4‐benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca2+]i, without any effect on [3H]‐STX binding.

Reduction in [3H]‐STX binding capacity induced by A23187 or TG was attenuated by Gö6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na+ channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans‐Golgi network), A23187 or TG accelerated the reduction of [3H]‐STX binding capacity.

Six hours treatment with A23187 lowered Na+ channel α‐ and β1‐subunit mRNA levels, whereas TG had no effect.

These results suggest that elevation of [Ca2+]i caused by A23187, TG or DBHQ exerted differential effects on down‐regulation of cell surface functional Na+ channels and Na+ channel subunit mRNA levels.

British Journal of Pharmacology (2001) 132, 1455–1466; doi:10.1038/sj.bjp.0703960
Brain α2‐adrenoceptors in monoamine‐depleted rats: increased receptor density, G coupling proteins, receptor turnover and receptor mRNA
Authors: Ribas Catalina , Miralles Antonio , Busquets Xavier , García‐Sevilla Jesús A
Published: January 2005

This study was designed to assess the molecular and cellular events involved in the up‐regulation (and receptor supersensitivity) of brain α2‐adrenoceptors as a result of chronic depletion of noradrenaline (and other monoamines) by reserpine.

Chronic reserpine (0.25 mg kg−1 s.c., every 48 h for 6 – 14 days) increased significantly the density (Bmax values) of cortical α2‐adrenoceptor agonist sites (34 – 48% for [3H]‐UK14304, 22 – 32% for [3H]‐clonidine) but not that of antagonist sites (11 – 18% for [3H]‐RX821002). Competition of [3H]‐RX821002 binding by (−)‐adrenaline further indicated that chronic reserpine was associated with up‐regulation of the high‐affinity state of α2‐adrenoceptors.

In cortical membranes of reserpine‐treated rats (0.25 mg kg−1 s.c., every 48 h for 20 days), the immunoreactivities of various G proteins (Gαi1/2, Gαi3, Gαo and Gαs) were increased (25 – 34%). Because the high‐affinity conformation of the α2‐adrenoceptor is most probably related to the complex with Gαi2 proteins, these results suggested an increase in signal transduction through α2‐adrenoceptors (and other monoamine receptors) induced by chronic reserpine.

After α2‐adrenoceptor alkylation, the analysis of receptor recovery (Bmax for [3H]‐UK14304) indicated that the increased density of cortical α2‐adrenoceptors in reserpine‐treated rats was probably due to a higher appearance rate constant of the receptor (Δr=57%) and not to a decreased disappearance rate constant (Δk=7%).

Northern‐ and dot‐blot analyses of RNA extracted from the cerebral cortex of saline‐ and reserpine‐treated rats (0.25 mg kg−1, s.c., every 48 h for 20 days) revealed that reserpine markedly increased the expression of α2a‐adrenoceptor mRNA in the brain (125%). This transcriptional activation of the receptor gene expression appears to be the cellular mechanism by which reserpine induces up‐regulation in the density of brain α2‐adrenoceptors.

British Journal of Pharmacology (2001) 132, 1467–1476; doi:10.1038/sj.bjp.0703963
Lysosphingolipid receptor‐mediated diuresis and natriuresis in anaesthetized rats
Authors: Bischoff Angela , Heringdorf Dagmar Meyer zu , Jakobs Karl H , Michel Martin C
Published: January 2005

Lysosphingolipids such as sphingosine‐1‐phosphate (SPP) and sphingosylphosphorylcholine (SPPC) can act on specific G‐protein‐coupled receptors. Since SPP and SPPC cause renal vasoconstriction, we have investigated their effects on urine and electrolyte excretion in anaesthetized rats.

Infusion of SPP (1 – 30 μg kg−1 min−1) for up to 120 min dose‐dependently but transiently (peak after 15 min, disappearance after 60 min) reduced renal blood flow without altering endogenous creatinine clearance. Nevertheless, infusion of SPP increased diuresis, natriuresis and calciuresis and, to a lesser extent, kaliuresis. These tubular lysosphingolipid effects developed more slowly (maximum after 60 – 90 min) and also abated more slowly upon lysosphingolipid washout than the renovascular effects.

Infusion of SPPC, sphingosine and glucopsychosine (3 – 30 μg kg−1 min−1 each) caused little if any alterations in renal blood flow but also increased diuresis, natriuresis and calciuresis and, to a lesser extent, kaliuresis.

Pretreatment with pertussis toxin (10 μg kg−1 3 days before the acute experiment) abolished the renovascular and tubular effects of 30 μg kg−1 min−1 SPP.

These findings suggest that lysosphingolipids are a hitherto unrecognized class of endogenous modulators of renal function. SPP affects renovascular tone and tubular function via receptors coupled to Gi‐type G‐proteins. SPPC, sphingosine and glucopsychosine mimic only the tubular effects of SPP, and hence may act on distinct sites.

British Journal of Pharmacology (2001) 132, 1925–1933; doi:10.1038/sj.bjp.0703969
Docosahexaenoic acid improves long‐term potentiation attenuated by phospholipase A2 inhibitor in rat hippocampal slices
Authors: Fujita Shinichi , Ikegaya Yuji , Nishikawa Masazumi , Nishiyama Nobuyoshi , Matsuki Norio
Published: January 2005

We investigated the possible involvement of phospholipase A2 (PLA2) and its products in long‐term potentiation (LTP) in the CA1 neurotransmission of rat hippocampal slices.

Inhibitors of Ca2+‐independent PLA2 (iPLA2) prevented the induction of LTP without affecting the maintenance phase of LTP whereas Ca2+‐dependent PLA2 inhibitors were virtually ineffective, which suggests a pivotal role of iPLA2 in the initiation of LTP.

We then investigated the effect of docosahexaenoic acid (DHA) and arachidonic acid (AA) on BEL (bromoenol lactone, an iPLA2‐inhibitor) ‐impaired LTP, and found that either DHA or AA abolished the effect of BEL. However, DHA did not restore BEL‐attenuated LTP when applied after the tetanus. DHA per se affected neither the induction nor maintenance of LTP. Linoleic acid had no effects, either.

These results suggest that DHA is crucial for the induction of LTP and that endogenously released DHA during tetanus is sufficient to trigger the formation of LTP.

British Journal of Pharmacology (2001) 132, 1417–1422; doi:10.1038/sj.bjp.0703970
Functional characterization of rat submaxillary gland muscarinic receptors using microphysiometry
Authors: Meloy Trena D , Daniels Donald V , Hegde Sharath S , Eglen Richard M , Ford Anthony P D W
Published: January 2005

Muscarinic cholinoceptors (MChR) in freshly dispersed rat salivary gland (RSG) cells were characterized using microphysiometry to measure changes in acidification rates. Several non‐selective and selective muscarinic antagonists were used to elucidate the nature of the subtypes mediating the response to carbachol.

The effects of carbachol (pEC50=5.74±0.02 s.e.mean; n=53) were highly reproducible and most antagonists acted in a surmountable, reversible fashion. The following antagonist rank order, with apparent affinity constants in parentheses, was noted: 4‐DAMP (8.9)=atropine (8.9)>tolterodine (8.5)>oxybutynin (7.9)>S‐secoverine (7.2)>pirenzepine (6.9)>himbacine (6.8)>AQ‐RA 741 (6.6)>methoctramine (5.9).

These studies validate the use of primary isolated RSG cells in microphysiometry for pharmacological analysis. These data are consistent with, and extend, previous studies using alternative functional methods, which reported a lack of differential receptor pharmacology between bladder and salivary gland tissue.

The antagonist affinity profile significantly correlated with the profile at human recombinant muscarinic M3 and M5 receptors. Given a lack of antagonists that discriminate between M3 and M5, definitive conclusion of which subtype(s) is present within RSG cells cannot be determined.

British Journal of Pharmacology (2001) 132, 1606–1614; doi:10.1038/sj.bjp.0703971
Effect of vanilloid drugs on gastrointestinal transit in mice
Authors: Izzo Angelo A , Capasso Raffaele , Pinto Luisa , Carlo Giulia Di , Mascolo Nicola , Capasso Francesco
Published: January 2005

We have studied the effect of capsaicin, piperine and anandamide, drugs which activate vanilloid receptors and capsazepine, a vanilloid receptor antagonist, on upper gastrointestinal motility in mice.

Piperine (0.5 – 20 mg kg−1 i.p.) and anandamide (0.5 – 20 mg kg−1 i.p.), dose‐dependently delayed gastrointestinal motility, while capsaicin (up to 3 mg kg−1 i.p.) was without effect. Capsazepine (15 mg kg−1 i.p.) neither per se affected gastrointestinal motility nor did it counteract the inhibitory effect of both piperine (10 mg kg−1) and anandamide (10 mg kg−1).

A per se non effective dose of SR141716A (0.3 mg kg−1 i.p.), a cannabinoid CB1 receptor antagonist, counteracted the inhibitory effect of anandamide (10 mg kg−1) but not of piperine (10 mg kg−1). By contrast, the inhibitory effect of piperine (10 mg kg−1) but not of anandamide (10 mg kg−1) was strongly attenuated in capsaicin (75 mg kg−1 in total, s.c.)‐treated mice.

Pretreatment of mice with NG‐nitro‐L‐arginine methyl ester (25 mg kg−1 i.p.), yohimbine (1 mg kg−1, i.p.), naloxone (2 mg kg−1 i.p.), or hexamethonium (1 mg kg−1 i.p.) did not modify the inhibitory effect of both piperine (10 mg kg−1) and anandamide (10 mg kg−1).

The present study indicates that the vanilloid ligands anandamide and piperine, but not capsaicin, can reduce upper gastrointestinal motility. The effect of piperine involves capsaicin‐sensitive neurones, but not vanilloid receptors, while the effect of anandamide involves cannabinoid CB1, but not vanilloid receptors.

British Journal of Pharmacology (2001) 132, 1411–1416; doi:10.1038/sj.bjp.0703975
Intracellular Angiotensin II and cell growth of vascular smooth muscle cells
Authors: Filipeanu Catalin M , Henning Robert H , De Zeeuw Dick , Nelemans Adriaan
Published: January 2005

We recently demonstrated that intracellular application of Angiotensin II (Angiotensin IIintr) induces rat aorta contraction independent of plasma membrane Angiotensin II receptors. In this study we investigated the effects of Angiotensin IIintr on cell growth in A7r5 smooth muscle cells.

DNA‐synthesis was increased dose‐dependently by liposomes filled with Angiotensin II as measured by [3H]‐thymidine incorporation at high (EC50=27±6 pM) and low (EC50=14±5 nM) affinity binding sites with increases in Emax of 58±4 and 37±4% above quiescent cells, respectively. Cell growth was corroborated by an increase in cell number.

Extracellular Angiotensin II (10 pM – 10 μM) did not modify [3H]‐thymidine incorporation.

Growth effects of Angiotensin IIintr mediated via high affinity sites were inhibited by liposomes filled with 1 μM of the non‐peptidergic antagonists losartan (AT1‐receptor) or PD123319 (AT2‐receptor) or with the peptidergic agonist CGP42112A (AT2‐receptor). Emax values were decreased to 30±3, 29±4 and 4±2%, respectively, without changes in EC50. The Angiotensin IIintr effect via low affinity sites was only antagonized by CGP42112A (Emax=11±3%), while losartan and PD123319 increased Emax to 69±4%. Intracellular applications were ineffective in the absence of Angiotensin IIintr.

Neither intracellular nor extracellular Angiotensin I (1 μM) were effective.

The Angiotensin IIintr induced growth response was blocked by selective inhibition of phosphatidyl inositol 3‐kinase (PI‐3K) by wortmannin (1 μM) and of the mitogen‐activated protein kinase (MAPK/ERK) pathway by PD98059 (1 μM) to 61±14 and 4±8% of control, respectively.

These data demonstrate that Angiotensin IIintr induces cell growth through atypical AT‐receptors via a PI‐3K and MAPK/ERK ‐sensitive pathway.

British Journal of Pharmacology (2001) 132, 1590–1596; doi:10.1038/sj.bjp.0703984
Social isolation modifies nicotine's effects in animal tests of anxiety
Authors: Cheeta Survjit , Irvine Elaine , File Sandra E
Published: January 2005

These experiments determined whether the housing conditions of rats influenced the effects of nicotine in two animal tests of anxiety, social interaction and elevated plus‐maze tests.

In animals housed singly for 7 days, (−)nicotine (0.025 mg kg−1 s.c.) was ineffective, but 0.05, 0.1 and 0.25 mg kg−1 (s.c.) significantly increased the time spent in social interaction, without changing locomotor activity, thus indicating anxiolytic actions. (−)Nicotine (0.45 mg kg−1 s.c.) significantly reduced social interaction, indicating an anxiogenic effect.

However, in group‐housed animals, (−)nicotine (0.025 mg kg−1 s.c.) had a significant anxiolytic effect in the social interaction test, but 0.01, 0.05, 0.1, 0.25 and 0.45 mg kg−1 were ineffective. (−)Nicotine (1 mg kg−1) reduced motor activity and social interaction in the group‐housed animals.

In the elevated plus‐maze, the time‐course and the dose‐response curve to nicotine were investigated. In both singly‐ and group‐housed rats, (−) nicotine (0.1 – 0.45 mg kg−1 s.c.) decreased the per cent entries into, and per cent time spent on, the open arms, indicating anxiogenic effects.

The housing condition influenced the time course, with significant effects at 5 and 30 min after injection in group‐housed rats, and significant effects at 30 and 60 min in singly‐housed rats.

In the social interaction test there was no difference in the scores of the first and last rats removed from group cages, whereas the order of removal from the cages did affect the scores in the elevated plus‐maze.

These results provide further evidence that the two animal tests model distinct states of anxiety, and show how social isolation powerfully modifies both anxiolytic and anxiogenic effects of nicotine.

British Journal of Pharmacology (2001) 132, 1389–1395; doi:10.1038/sj.bjp.0703991
Characteristics of AMPA receptor‐mediated responses of cultured cortical and spinal cord neurones and their correlation to the expression of glutamate receptor subunits, GluR1‐4
Authors: Dai Wei‐Min , Egebjerg Jan , Lambert John D C
Published: January 2005

Electrophysiological recordings have been used to characterize responses mediated by AMPA receptors expressed by cultured rat cortical and spinal cord neurones. The EC50 values for AMPA were 17 and 11 μM, respectively.

Responses of cortical neurones to AMPA were inhibited competitively by NBQX (pKi=6.6). Lower concentrations of NBQX (1 μM) also potentiated the plateau responses of spinal cord neurones to AMPA, which could be attributed to a depression of desensitization to AMPA.

GYKI 52466 inhibited responses of spinal cord neurones to AMPA to about twice the extent of responses of cortical neurones.

Blockade of AMPA receptor desensitization by cyclothiazide (CTZ) potentiated responses of spinal cord neurones (6.8 fold) significantly more than responses of cortical neurones (4.8 fold). Responses of cortical neurones to KA were potentiated 3.5 fold by CTZ, while responses of spinal cord neurones were unaffected.

Ultra‐fast applications of AMPA to outside‐out patches showed responses of spinal cord neurones desensitized by 97.5% and exhibit marked inward rectification, whereas cortical neurones desensitized by 91% and exhibited slight outward rectification. The time constants of deactivation and desensitization were about twice as fast in spinal cord than cortical neurones.

In cortical neurones, single‐cell RT – PCR showed GluR2 and GluR1 accounted for 91% of all subunits and were expressed together in 67% of neurones, predominantly as the flip variants (78%). GluR2 was detected alone in 24% of neurones. GluR3 and GluR4 were present in only 14 and 29% of neurones, respectively. For spinal cord neurones, GluR4o was detected in 81% of neurones, whereas predominantly flop versions of GluR1, 2 and 3 were detected in 38, 13 and 13% of neurones, respectively. These expression patterns are related to the respective pharmacological and mechanistic properties.

British Journal of Pharmacology (2001) 132, 1859–1875; doi:10.1038/sj.bjp.0703993
Novel histamine H3‐receptor antagonists and partial agonists with a non‐aminergic structure
Authors: Nickel Tobias , Bauer Ulrich , Schlicker Eberhard , Kathmann Markus , Göthert Manfred , Sasse Astrid , Stark Holger , Schunack Walter
Published: January 2005

We determined the affinities of eight novel histamine H3‐receptor ligands (ethers and carbamates) for H3‐receptor binding sites and their agonistic/antagonistic effects in two functional H3‐receptor models. The compounds differ from histamine in that the ethylamine chain is replaced by a propyloxy chain; in the three ethers mentioned below (FUB 335, 373 and 407), R is n‐pentyl, 3‐methylbutyl and 3,3‐dimethylbutyl, respectively.

The compounds monophasically inhibited [3H]‐Nα‐methylhistamine binding to mouse cerebral cortex membranes (pKi 7.51 – 9.53).

The concentration‐response curve of histamine for its inhibitory effect on the electrically evoked [3H]‐noradrenaline overflow from mouse cortex slices was shifted to the right by these compounds (apparent pA2 6.61 – 8.00). Only FUB 373 and 407 inhibited the evoked overflow by themselves (intrinsic activities 0.3 and 0.4); these effects were counteracted by the H3‐receptor antagonist clobenpropit.

[35S]‐GTPγS binding to mouse cortex membranes was stimulated by the H3‐receptor agonist (R)‐α‐methylhistamine in a manner sensitive to clobenpropit. Among the novel compounds only FUB 373 and 407 stimulated [35S]‐GTPγS binding (intrinsic activities 0.6 and 0.4).

In conclusion, the novel compounds are partial H3‐receptor agonists (FUB 373 and 407) or H3‐receptor antagonists; comparison with FUB 335 shows that the transition from antagonist to agonist is caused by a slight structural change. A protonated N atom in the side chain is not necessary for agonism at H3 receptors, proposing a receptor‐ligand interaction different from that of classical agonists.

British Journal of Pharmacology (2001) 132, 1665–1672; doi:10.1038/sj.bjp.0704013
The nitric oxide donor pentaerythritol tetranitrate can preserve endothelial function in established atherosclerosis
Authors: Hacker Andreas , Müller Senta , Meyer Wilfried , Kojda Georg
Published: January 2005

Recent results suggested that long‐term treatment with a low dose of the organic nitrate pentaerythritol tetranitrate (PETN, 6 mg kg−1 per day) for 16 weeks slightly decreases aortic superoxide production in normal rabbits. We sought to determine if PETN can preserve endothelium dependent relaxation (EDR) in atherosclerotic rabbits.

Three groups of 9 – 10 New Zealand White rabbits received a cholesterol chow (0.75%) for 16 weeks. One group (CHOL16) served as control and two groups were fed for another 16 weeks a cholesterol‐chow without (CHOL32) or with 6 mg PETN kg−1 per day (PETN32).

Isolated aortic rings of CHOL16 showed a typical impairment of EDR with a maximal relaxation at 1 μM acetylcholine of 28±16%. In CHOL32‐rings EDR was completely impaired. In striking contrast, EDR in PETN32 (24±15%) was similar to that of CHOL16 indicating a protective effect of PETN on endothelial function. Vascular superoxide production measured with the lucigenin method was not different between the groups.

Aortic lesion formation in PETN32 was smaller than in CHOL32 (P<0.008). The onset of copper‐induced LDL‐oxidation (lag‐time) after 16 weeks of cholesterol feeding (214±9 min) was reduced in CHOL32 (168±24 min, P=0.035) but not in PETN32 (220±21 min). This indicates prevention of increased LDL oxidation by PETN.

The halfmaximal effective vasodilator concentrations of PETN (in −logM) were identical in CHOL16 (7.9±0.1), CHOL32 (7.6±0.2) and PETN32 (7.7±0.2). Similar results were obtained with S‐nitroso‐N‐acetyl‐D,L‐penicillamine.

These data suggest that PETN can reduce the progression of lesion formation, endothelial dysfunction and of LDL‐oxidation in established atherosclerosis.

British Journal of Pharmacology (2001) 132, 1707–1714; doi:10.1038/sj.bjp.0704021
Structural requirements for voltage‐dependent block of muscle sodium channels by phenol derivatives
Authors: Haeseler G , Piepenbrink A , Bufler J , Dengler R , Aronson J K , Piepenbrock S , Leuwer M
Published: January 2005

We have studied the effects of four different phenol derivatives, with methyl and halogen substituents, on heterologously expressed human skeletal muscle sodium channels, in order to find structural determinants of blocking potency.

All compounds blocked skeletal muscle sodium channels in a concentration‐dependent manner. The methylated phenol 3‐methylphenol and the halogenated phenol 4‐chlorophenol blocked sodium currents on depolarization from −100 mV to 0 mV with IC50 values of 2161 and 666 μM respectively. Methylation of the halogenated compound further increased potency, reducing the IC50 to 268 μM in 2‐methyl‐4‐chlorophenol and to 150 μM in 3,5‐dimethyl‐4‐chlorophenol.

Membrane depolarization before the test depolarization increased sodium channel blockade. When depolarizations were started from −70 mV or when a 2.5 s prepulse was introduced before the test pulse inducing slow inactivation, the IC50 was reduced more than 3 fold in all compounds. The values of KD for the fast‐inactivated state derived from drug‐induced shifts in steady‐state availability curves were 14 μM for 3,5‐dimethyl‐4‐chlorophenol, 19 μM for 2‐methyl‐4‐chlorophenol, 26 μM for 4‐chlorophenol and 115 μM for 3‐methylphenol.

All compounds accelerated the current decay during depolarization and slowed recovery from fast inactivation. No relevant frequency‐dependent block after depolarizing pulses applied at 10, 50 and 100 Hz was detected for any of the compounds.

All the phenol derivatives that we examined are effective blockers of skeletal muscle sodium channels, especially in conditions that are associated with membrane depolarization. Blocking potency is increased by halogenation and by methylation with increasing numbers of methyl groups.

British Journal of Pharmacology (2001) 132, 1916–1924; doi:10.1038/sj.bjp.0704024
Signal transduction pathways involved in kinin B2 receptor‐mediated vasodilation in the rat isolated perfused kidney
Authors: Bagaté Karim , Grima Michèle , Imbs Jean‐Louis , Jong Wybren De , Helwig Jean‐Jacques , Barthelmebs Mariette
Published: January 2005

The signal transduction pathways involved in kinin B2 receptor‐related vasodilation were investigated in rat isolated perfused kidneys. During prostaglandin F2α or KCl‐induced constriction, the vasodilator response to a selective B2 receptor agonist, Tyr(Me)8bradykinin (Tyr(Me)8BK), was assessed.

Tyr(Me)8BK produced a concentration‐ and endothelium‐dependent relaxation that was decreased by about 30 – 40% after inhibition of nitric oxide (NO) synthase by NG‐nitro‐L‐arginine (L‐NOARG) or of cyclo‐oxygenase by indomethacin; a greater decrease (about 40 – 50%) was observed after concomitant inhibition of the two pathways.

High extracellular K+ diminished Tyr(Me)8BK‐induced relaxation by about 75% suggesting a major contribution of endothelium‐derived hyperpolarization. The residual response was almost completely suppressed by NO synthase and cyclo‐oxygenase inhibition. The K+ channel inhibitors, tetrabutylammonium (non‐specific) and charybdotoxin (specific for Ca2+‐activated K+ channel), suppressed Tyr(Me)8BK‐induced relaxation resistant to L‐NOARG and indomethacin.

Inhibition of cytochrome P450 (clotrimazole or 7‐ethoxyresorufin) decreased the NO/prostanoids‐independent relaxation to Tyr(Me)8BK by more than 60%, while inhibition of the cannabinoid CB1 receptor (SR 141716A) had only a moderate effect.

Acetylcholine induced a concentration‐dependent relaxation with characteristics nearly similar to the response to Tyr(Me)8BK. In contrast, the relaxation elicited by sodium nitroprusside was potentiated in the absence of NO (L‐NOARG or removal of endothelium) but remained unchanged otherwise.

These results indicate that the activation of kinin B2 receptors in the rat isolated kidney elicits an endothelium‐dependent vasorelaxation, mainly dependent on the activation of charybdotoxin‐sensitive Ca2+‐activated K+ channels. In addition, cytochrome P450 derivatives appear to be involved.

British Journal of Pharmacology (2001) 132, 1735–1742; doi:10.1038/sj.bjp.0704027
Characterization of palmitoylethanolamide transport in mouse Neuro‐2a neuroblastoma and rat RBL‐2H3 basophilic leukaemia cells: comparison with anandamide
Authors: Jacobsson Stig O P , Fowler Christopher J
Published: January 2005

The endogenous cannabinoid receptor agonist anandamide (AEA) and the related compound palmitoylethanolamide (PEA) are inactivated by transport into cells followed by metabolism by fatty acid amide hydrolase (FAAH). The cellular uptake of AEA has been characterized in detail, whereas less is known about the properties of the PEA uptake, in particular in neuronal cells. In the present study, the pharmacological and functional properties of PEA and AEA uptake have been investigated in mouse Neuro‐2a neuroblastoma and, for comparison, in rat RBL‐2H3 basophilic leukaemia cells.

Saturable uptake of PEA and AEA into both cell lines were demonstrated with apparent KM values of 28 μM (PEA) and 10 μM (AEA) in Neuro‐2a cells, and 30 μM (PEA) and 9.3 μM (AEA) in RBL‐2H3 cells. Both PEA and AEA uptake showed temperature‐dependence but only the AEA uptake was sensitive to treatment with Pronase and phenylmethylsulfonyl fluoride.

The AEA uptake was inhibited by AM404, 2‐arachidonoylglycerol (2‐AG), R1‐ and S1‐methanandamide, arachidonic acid and olvanil with similar potencies for the two cell types. PEA, up to a concentration of 100 μM, did not affect AEA uptake in either cell line. AEA, 2‐AG, arachidonic acid, R1‐methanandamide, Δ9‐THC, and cannabidiol inhibited PEA transport in both cell lines. The non‐steroidal anti‐inflammatory drug indomethacin inhibited the AEA uptake but had very weak effects on the uptake of PEA.

From these data, it can be concluded that PEA is transported in to cells both by passive diffusion and by a facilitated transport that is pharmacologically distinguishable from AEA uptake.

British Journal of Pharmacology (2001) 132, 1743–1754; doi:10.1038/sj.bjp.0704029
Sibutramine reduces feeding, body fat and improves insulin resistance in dietary‐obese male Wistar rats independently of hypothalamic neuropeptide Y
Authors: Brown Michael , Bing Chen , King Peter , Pickavance Lucy , Heal David , Wilding John
Published: January 2005

We studied the effects of the novel noradrenaline and serotonin (5‐HT) reuptake inhibitor sibutramine on feeding and body weight in a rat model of dietary obesity, and whether it interacts with hypothalamic neuropeptide Y (NPY) neurones.

Chow‐fed and dietary‐obese (DIO) male Wistar rats were given sibutramine (3 mg kg−1 day−1 p.o.) or deionized water for 21 days.

Sibutramine decreased food intake throughout the treatment period in both dietary‐obese rats (P<0.0001) and lean rats (P<0.0001). Weight gain was reduced so that final body weight was 10% lower in dietary‐obese (P<0.005) and 8% lower in lean (P<0.05) rats versus their untreated controls. Plasma leptin concentration was lower in sibutramine‐treated dietary‐obese rats (P<0.05), and in treated lean rats (P<0.05). Using the homeostasis model assessment (HOMA) as a measure of insulin resistance, untreated DIO rats were significantly more insulin resistant than controls (P<0.005), and this was corrected by sibutramine treatment (P<0.05). Neither hypothalamic NPY mRNA nor NPY peptide levels in a number of hypothalamic nuclei were significantly altered by sibutramine compared to untreated controls.

The hypophagic and anti‐obesity effects of sibutramine in dietary‐obese Wistar rats appear not to be mediated by inhibition of ARC NPY neurones.

British Journal of Pharmacology (2001) 132, 1898–1904; doi:10.1038/sj.bjp.0704030
Contribution of α2 receptor subtypes to nerve injury‐induced pain and its regulation by dexmedetomidine
Authors: Malmberg Annika B , Hedley Linda R , Jasper Jeffrey R , Hunter John C , Basbaum Allan I
Published: January 2005

There is evidence that noradrenaline contributes to the development and maintenance of neuropathic pain produced by trauma to a peripheral nerve. It is, however, unclear which subtype(s) of α adrenergic receptors (AR) may be involved. In addition to pro‐nociceptive actions of AR stimulation, α2 AR agonists produce antinociceptive effects.

Here we studied the contribution of the α2 AR subtypes, α2A, α2B and α2C to the development of neuropathic pain. We also examined the antinociceptive effect produced by the α2 AR agonist dexmedetomidine in nerve‐injured mice.

The studies were performed in mice that carry either a point (α2A) or a null (α2B and α2C) mutation in the gene encoding the α2 AR. To induce a neuropathic pain condition, we partially ligated the sciatic nerve and measured changes in thermal and mechanical sensitivity.

Baseline mechanical and thermal withdrawal thresholds were similar in all mutant and wild‐type mice; and, after peripheral nerve injury, all mice developed comparable hypersensitivity (allodynia) to thermal and mechanical stimulation.

Dexmedetomidine reversed the allodynia at a low dose (3 μg kg−1, s.c.) and produced antinociceptive effects at higher doses (10 – 30 μg kg−1) in all groups except in α2A AR mutant mice. The effect of dexmedetomidine was reversed by intrathecal, but not systemic, injection of the α2 AR antagonist RS 42206.

These results suggest that neither α2A, α2B nor α2C AR is required for the development of neuropathic pain after peripheral nerve injury, however, the spinal α2A AR is essential for the antinociceptive effects of dexmedetomidine.

British Journal of Pharmacology (2001) 132, 1827–1836; doi:10.1038/sj.bjp.0704032
The effects of the statins lovastatin and cerivastatin on signalling by the prostanoid IP‐receptor
Authors: Lawler Orlaith A , Miggin Sinead M , Kinsella B Therese
Published: January 2005

The prostanoid‐IP receptor may be unique among G protein coupled receptors in that it is isoprenylated. In this study, we investigated the effects of the statins lovastatin and cerivastatin on signalling by the mouse (m) IP and the human (h) IP receptors, over‐expressed in human embryonic kidney (HEK) 293 cells and by the hIP receptor, endogenously expressed in human erythroleukaemia cells.

Both statins significantly reduced IP receptor‐mediated cyclic AMP generation and intracellular calcium ([Ca2+]i) mobilization in a time and concentration dependent manner but had no effect on signalling by the non‐isoprenylated β2 adrenergic receptor or by the human prostanoid‐TP receptor isoforms.

Cerivastatin (IC50, 50 – 90 nM) was significantly more potent than lovastatin (IC50, 0.80 – 4.2 μM) in inhibiting IP receptor signalling.

Whereas IC50 values indicated that the hIP receptor was significantly more sensitive than the mIP receptor to the statins, the extent of inhibition of cyclic AMP generation by the mIP receptor was significantly greater than that of the hIP receptor to either statin, even at the highest concentrations used.

Pretreatment with either statin significantly reduced IP receptor mediated desensitization of signalling by the h.TPα, but not by the h.TPβ, receptor isoform.

These data generated in whole cells point to the possibility that statin therapy may interfere with IP receptor signalling in vivo; such interference may be extenuated under conditions where circulating statin levels are elevated and may account, in part, for some of the pleiotropic affects of the statins not attributed solely to their lipid lowering properties.

British Journal of Pharmacology (2001) 132, 1639–1649; doi:10.1038/sj.bjp.0704033
Functional characterization of a human receptor for neuropeptide FF and related peptides
Authors: Kotani Masato , Mollereau Catherine , Detheux Michel , Le Poul Emmanuel , Brézillon Stéphane , Vakili Jalal , Mazarguil Honoré , Vassart Gilbert , Zajac Jean‐Marie , Parmentier Marc
Published: January 2005

Neuropeptides FF (NPFF) and AF (NPAF) are involved in pain modulation and opioid tolerance. These peptides were known to act through uncharacterized G protein‐coupled receptors (GPCR). We describe here, using an aequorin‐based assay as screening tool, that an orphan GPCR, previously designated HLWAR77, is a functional high affinity receptor for NPFF and related peptides. This receptor is further designated as NPFFR.

Binding experiments were performed with a new radioiodinated probe, [125I]‐EYF, derived from the EFW‐NPSF sequence of the rat NPFF precursor. Chinese hamster ovary (CHO) cell membranes expressing NPFFR bound [125I]‐EYF with a Kd of 0.06 nM. Various NPFF analogues and related peptides inhibited [125I]‐EYF specific binding with the following rank order (Ki): human NPAF (0.22 nM), SQA‐NPFF (0.29 nM), NPFF (0.30 nM), 1DMe (0.31 nM), EYW‐NPSF (0.32 nM), QFW‐NPSF (0.35 nM), 3D (1.12 nM), Met‐enk‐RF‐NH2 (3.25 nM), FMRF‐NH2 (10.5 nM) and NPSF (12.1 nM).

The stimulatory activity of the same set of peptides was measured by a functional assay based on the co‐expression of NPFFR, Gα16 and apoaequorin. The rank order of potency was consistent with the results of the binding assay.

Membranes from NPFFR expressing CHO cells bound GTPγ[35S] in the presence of SQA‐NPFF. This functional response was prevented by pertussis toxin treatment, demonstrating the involvement of Gi family members.

SQA‐NPFF inhibited forskolin induced cyclic AMP accumulation in recombinant CHO cells in a dose dependent manner. This response was abolished as well by pertussis toxin pre‐treatment.

RT – PCR analysis of human tissues mRNA revealed that expression of NPFFR was mainly detected in placenta, thymus and at lower levels in pituitary gland, spleen and testis.

British Journal of Pharmacology (2001) 133, 138–144; doi:10.1038/sj.bjp.0704038
Dual action of ZD6169, a novel K+ channel opener, on ATP‐sensitive K+ channels in pig urethral myocytes
Authors: Teramoto Noriyoshi , Yunoki Takakazu , Takano Makoto , Yonemitsu Yoshikazu , Masaki Ichiro , Sueishi Katsuo , Brading Alison F , Ito Yushi
Published: January 2005

The effects of ZD6169, a novel K+ channel opener, on both membrane and unitary currents in pig urethra were investigated using patch‐clamp techniques. Its effect was also examined on currents in inside‐out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K+ channel (Kir6.2) subunits (Kir6.2ΔC36) which form ATP‐sensitive K+ channels (KATP channels).

In current‐clamp mode, ZD6169 (10 μM) induced a concentration‐dependent membrane hyperpolarization. Higher concentrations (30 μM) caused a transient membrane hyperpolarization, followed by a gradual membrane depolarization. On removal of ZD6169, an after hyperpolarization was observed.

In conventional voltage‐clamp configuration, at −50 mV in symmetrical 140 mM K+ conditions, ZD6169 (100 μM) caused a transient inward current which gradually decayed. Removal of ZD6169 evoked a much larger amplitude K+ current with a similar time course.

ZD6169 produced an inward glibenclamide‐sensitive K+ current, demonstrating a bell‐shaped concentration‐response relationship.

In cell‐attached configuration in symmetrical 140 mM K+ conditions, ZD6169 (30 μM) activated an KATP channel which was reversibly suppressed by application of glibenclamide. In contrast, ZD6169 (100 μM) inhibited the activity of the levcromakalim‐induced KATP channels.

ZD6169 (100 μM) had no significant effect on the channel activity of Kir6.2ΔC36 in inside‐out configuration, although cibenzoline greatly suppressed the channel activity.

These results demonstrate that ZD6169 possesses a dual effect on the activity of the KATP channel; activating at low concentration and inhibiting at higher concentration.

British Journal of Pharmacology (2001) 133, 154–164; doi:10.1038/sj.bjp.0704042
The role of α1‐adrenoceptors and 5‐HT1A receptors in the control of the micturition reflex in male anaesthetized rats
Authors: Conley Rachel K , Williams Timothy J , Ford Anthony P D W , Ramage Andrew G
Published: January 2005

The effects of the α1‐adrenoceptor antagonists doxazosin (0.1 – 2 mg kg−1), RS‐100329 (α1A; 0.01 – 1 mg kg−1), RS‐513815 (Ro 151‐3815, α1B; 0.3 – 3 mg kg−1) and BMY 7378 (α1D; 0.1 – 1 mg kg−1), the 5‐HT1A receptor agonist, 8‐OH‐DPAT (0.03 – 0.3 mg kg−1) and antagonist WAY‐100635 (0.03 – 0.3 mg kg−1) were investigated (i.v.) on the ‘micturition reflex’ in the urethane anaesthetized male rat.

Reflex‐evoked urethra contractions were most sensitive to the inhibitory action of RS‐100329, followed by doxazosin, BMY 7378 and WAY‐100635 and then RS‐513815. The maximum inhibition was 66, 63, 54, 46 and 22% at doses of 0.3, 0.5, 0.3, 0.3 and 3 mg kg−1 respectively.

BMY 7378 and 8‐OH‐DPAT decreased, while WAY‐100635 increased, the pressure threshold to induce bladder contraction. WAY‐100635 (0.01 mg kg−1) blocked the effects of BMY 7378 (1 mg kg−1) on bladder pressure and volume threshold.

Doxazosin, RS‐100329 and BMY 7378 had a similar potency in inducing a fall in arterial blood pressure while WAY‐100635 only caused a fall at the highest dose.

Therefore, reflex‐evoked urethral contraction involves the activation of α1A/1D‐adrenoceptors, as BMY 7378 and RS‐100329 are similarly potent in attenuating this effect. The ability of WAY‐100635 to attenuate this contraction may suggest that 5‐HT1A receptors are also involved. However, as this inhibition occurred at the highest dose of WAY‐100635, which also caused a fall in arterial blood pressure; this effect is considered to be due to blockade of α1‐adrenoceptors not 5‐HT1A receptors. Nevertheless the initiation of the ‘micturition reflex’ involves the activation of 5‐HT1A receptors.

British Journal of Pharmacology (2001) 133, 61–72; doi:10.1038/sj.bjp.0704043
Agonist and antagonist activities on human NPFF2 receptors of the NPY ligands GR231118 and BIBP3226
Authors: Mollereau Catherine , Gouardères Christine , Dumont Yvan , Kotani Masato , Detheux Michel , Doods Henri , Parmentier Marc , Quirion Rémi , Zajac Jean‐Marie
Published: January 2005

Neuropeptide FF (NPFF) is a part of a neurotransmitter system acting as a modulator of endogenous opioid functions. At this time, no non‐peptide or peptide NPFF‐antagonists have been discovered. Here, we demonstrate that Neuropeptide Y (NPY) ligands, in fact possess significant ability to interact with the human NPFF2 receptors. NPY Y1 antagonist BIBP3226 and mixed Y1 antagonist/Y4 agonist GR231118 are able to displace with low affinity, 50 – 100 nM, the specific binding on NPFF receptors expressed in CHO cells as well as in rat dorsal spinal cord, an affinity however superior to those determined against Y2, Y4 or Y5 receptors. Furthermore, BIBP3226 which is unable to inhibit the forskolin‐stimulated cyclic AMP production mediated by NPFF2 receptors, antagonizes the effect of NPFF, revealing the first antagonist of NPFF receptors. These properties of NPY ligands on Neuropeptide FF receptors must be considered when evaluating pharmacological activities of these drugs.

British Journal of Pharmacology (2001) 133, 1–4; doi:10.1038/sj.bjp.0704049
Involvement of accumulated endogenous NOS inhibitors and decreased NOS activity in the impaired neurogenic relaxation of the rabbit proximal urethra with ischaemia
Authors: Masuda Hitoshi , Tsujii Toshihiko , Okuno Tetsuo , Kihara Kazunori , Goto Moritaka , Azuma Hiroshi
Published: January 2005

We examined the effect of ischaemia on the neurogenic and nitric oxide (NO)‐mediated urethral relaxation.

Rabbits were divided into control and urethral ischaemia (UI) groups, which was prepared by the partial occlusion of bilateral iliac arteries using blood vessel occluders.

Neurogenic and NO‐mediated proximal urethral relaxation induced by electrical field stimulation (EFS) was greatly impaired in the UI group, while relaxation by sodium nitroprusside (SNP) as a NO donor showed no difference between the two groups. Pretreatment with L‐arginine significantly improved but did not normalize the impaired relaxation in the UI group. Not only basal level, but also stimulated production of cyclic GMP with EFS, were significantly decreased in the UI group.

The tissue contents of NG‐methyl‐L‐arginine (L‐NMA) and asymmetric NG, NG‐dimethyl‐L‐arginine (ADMA) in the proximal urethra were increased following ischaemia. While L‐arginine and symmetric NG, N′G‐dimethyl‐L‐arginine (SDMA) contents remained unchanged. Exogenously applied authentic L‐NMA and ADMA (1 – 100 μM) concentration‐dependently inhibited the EFS‐induced urethral relaxation in the control group. The inhibition with L‐NMA and ADMA was undetectable in the presence of 3 mM L‐arginine.

The Ca2+‐dependent NOS activity in the urethra from the UI group was significantly lower than that from the control group and was not restored by an addition of 3 mM L‐arginine.

These results suggest that the impaired neurogenic and NO‐mediated urethral relaxation with ischaemia is closely related to the increased accumulation of L‐NMA and ADMA and decreased NOS activity, which would result in an accelerated reduction in NO production/release.

British Journal of Pharmacology (2001) 133, 97–106; doi:10.1038/sj.bjp.0704050
Role of bradykinin and eNOS in the anti‐ischaemic effect of trandolapril
Authors: Cargnoni Anna , Comini Laura , Bernocchi Palmira , Bachetti Tiziana , Ceconi Claudio , Curello Salvatore , Ferrari Roberto
Published: January 2005

Angiotensin converting enzyme (ACE) inhibitors are under study in ischaemic heart diseases, their mechanism of action being still unknown.

The anti‐ischaemic effect of trandolapril and the possible involvement of a bradykinin‐modulation on endothelial constitutive nitric oxide synthase (eNOS) in exerting this effect, were investigated.

Three doses of trandolapril, chronically administered in vivo, were studied in isolated perfused rat hearts subjected to global ischaemia followed by reperfusion.

Trandolapril has an anti‐ischaemic effect. The dose of 0.3 mg kg−1 exerted the best effect reducing diastolic pressure increase during ischaemia (from 33.0±4.5 to 14.0±5.2 mmHg; P<0.05 vs control) and reperfusion (from 86.1±9.4 to 22.2±4.1 mmHg; P<0.01 vs control), improving functional recovery, counteracting creatine phosphokinase release and ameliorating energy metabolism after reperfusion.

Trandolapril down‐regulated the baseline developed pressure.

Trandolapril increased myocardial bradykinin content (from 31.8±6.1 to 54.8±7.5 fmol/gww; P<0.05, at baseline) and eNOS expression and activity in aortic endothelium (both P<0.01 vs control) and in cardiac myocytes (from 11.3±1.5 to 17.0±2.0 mUOD μg protein−1 and from 0.62±0.05 to 0.80±0.06 pmol mg prot−1 min−1; both P<0.05 vs control).

HOE 140 (a bradykinin B2 receptor antagonist) and NOS inhibitors counteracted the above‐reported effects.

There was a negative correlation between myocyte's eNOS up‐regulation and myocardial contraction down‐regulation.

Our findings suggest that the down‐regulation exerted by trandolapril on baseline cardiac contractility, through a bradykinin‐mediated increase in NO production, plays a crucial role in the anti‐ischaemic effect of trandolapril by reducing energy breakdown during ischaemia.

British Journal of Pharmacology (2001) 133, 145–153; doi:10.1038/sj.bjp.0704052
Transfection of annexin 1 in monocytic cells produces a high degree of spontaneous and stimulated apoptosis associated with caspase‐3 activation
Authors: Solito Egle , De Coupade Catherine , Canaider Silvia , Goulding Nicolas J , Perretti Mauro
Published: January 2005

Transfection of the pre‐monomyelocytic U937 cell line with a plasmid coding for full‐length annexin 1 (ANX1, 347 amino acid) leads to cell death by promoting apoptosis. In addition, over‐expression of the N‐terminal and the first domain of the protein (144 amino acids, clone ANX1‐S), which does not contain the Ca2+ binding sites, gives susceptibility to cell apoptosis following activation by either 5 ng ml−1 tumour necrosis factor (TNF)‐α or 1 – 40 μg ml−1 etoposide. This was demonstrated by using the fluorescent labelled annexin V, cell cycle and nuclear staining analyses.

Transfection with an empty plasmid (clone CMV) or with a plasmid carrying the cDNA antisense for ANX1 (clone ANX1‐AS) did not alter U937 cells to the degree of apoptosis promoted by either stimulant.

Treatment of CMV U937 cells with TNF‐α increased ANX1 mRNA and protein expression in a time‐dependent manner, with maximal increases at 3 and 6 h, respectively.

Clone ANX1‐S showed higher constitutive (more than 2 fold) and activated caspase‐3 activity, associated with higher phospholipase A2 (PLA2) activity (in the region of +50 – 100%), whereas expression of cytosolic PLA2 Bax and Bcl‐2 were similar in all cell clones, as determined by Western blotting.

In conclusion, this study demonstrates a complex regulatory role of cell apoptosis for ANX1, at least with regards to cells of the myelo‐monocytic lineage.

British Journal of Pharmacology (2001) 133, 217–228; doi:10.1038/sj.bjp.0704054