bluuebunny/crossref_metadata_2025 huggingface dataset:
Human P2Y2 receptor polymorphism: identification and pharmacological characterization of two allelic variants
Authors: Janssens Rodolphe , Paindavoine Pascale , Parmentier Marc , Boeynaems Jean‐Marie
Published: January 2005

In the process of cloning the human P2Y2 receptor in order to establish 1321N1 cell lines expressing this receptor, we detected a gene polymorphism characterized by an arginine 334 to cysteine 334 transition.

The frequency distribution of the polymorphism was studied in a European population. We observed that 66% of the tested persons are homozygotes R/R, 29% are heterozygotes R/C and 5% are homozygotes C/C. The frequency of the R allele was 0.8 versus 0.2 for the C allele.

We stably expressed each form of the human P2Y2 receptor into 1321N1 cells and isolated clones by limiting dilution. The effects of nucleotides and antagonists on inositol trisphosphate accumulation and cyclic AMP formation were compared between the two cell lines.

The time‐courses of inositol trisphosphate accumulation as well as concentration‐response curves characterizing the effects of UTP, ATP, AP4A and ATPγS were mostly similar, except for slight kinetic differences (slower time‐course with the 334C form).

The sensitivity to pertussis toxin of inositol trisphosphates accumulation was critically dependent on the agonist concentration and stimulation duration, suggesting the involvement of a Gi,0 protein during the early stimulation by low nucleotide concentrations. No inhibition of cyclic AMP accumulation could be detected. These properties were observed with both polymorphic receptors.

British Journal of Pharmacology (1999) 127, 709–716; doi:10.1038/sj.bjp.0702619
Characterization of central and peripheral effects of septide with the use of five tachykinin NK1 receptor antagonists in the rat
Authors: Cellier Eric , Barbot Lionel , Iyengar Smriti , Couture Réjean
Published: January 2005

Effects of two tachykinin NK1 receptor selective agonists (septide and [Sar9, Met(O2)11]SP) were compared on the increases in mean arterial pressure (MAP), heart rate (HR) and motor behaviour following intracerebroventricular (i.c.v.) administration in unanaesthetized rat, and on the vascular permeability increases to intradermal (i.d.) injection in the anaesthetized rat. Moreover, five tachykinin NK1 receptor selective antagonists (LY303870, LY306740, LY303241, SR140333 and RP67580) were tested against the two agonists to compare their pharmacological profile.

[Sar9, Met(O2)11]SP and septide (10–100 pmol per rat, i.c.v.) were equipotent in increasing MAP and HR, yet they had dissimilar time‐course. Both agonists increased dose‐dependently face washing and sniffing while [Sar9, Met(O2)11]SP was the sole to produce grooming. septide was more potent than [Sar9, Met(O2)11]SP (6.5–650 pmol) in increasing vascular permeability.

For most centrally mediated responses, LY303870 and RP67580 were significantly more potent in inhibiting septide than [Sar9, Met(O2)11]SP. In some parameters, greater blockade was achieved when antagonists (particularly LY306740) were given 1 h instead of 10 min prior to i.c.v. septide.

All antagonists except LY303241 blocked dose‐dependently the increases in vascular permeability to equipotent doses of [Sar9, Met(O2)11]SP and septide. LY303870 and LY306740 were more potent against septide.

The antagonism afforded by LY303870, LY306740 and LY303241 was stereoselective and only SR140333 was found to cause central and peripheral non specific effects.

The data confirm a distinct pharmacological profile for septide in vivo. RP67580 and LY306740 are currently the most valuable tachykinin NK1 receptor antagonists for in vivo studies in rat.

British Journal of Pharmacology (1999) 127, 717–728; doi:10.1038/sj.bjp.0702620
Studies on the role of serotonin receptor subtypes in the effect of sibutramine in various feeding paradigms in rats
Authors: Grignaschi G , Fanelli E , Scagnol I , Samanin R
Published: January 2005

The effect of the 5‐hydroxytryptamine (5‐HT) and noradrenaline (NA) reuptake inhibitor sibutramine was studied in food deprived, neuropeptide Y (NPY)‐ or muscimol‐injected rats.

Sibutramine dose‐dependently reduced feeding caused by food‐deprivation (ED50=5.1±0.8 mg kg−1) or by NPY injection into the paraventricular nucleus of the hypothalamus (ED50=6.0±0.5 mg kg−1). The increase in food intake caused by muscimol injected into the dorsal raphe was not modified by sibutramine (1–10 mg kg−1).

The hypophagic effect of 5.1 mg kg−1 sibutramine in food‐deprived rats was studied in rats pretreated with different serotonin receptor antagonists. Metergoline (non‐selective, 0.3 and 1.0 mg kg−1), ritanserin (5‐HT2A/2C, 0.5 and 1.0 mg kg−1) and GR127935 (5‐HT1B/1D, 0.5 and 1.0 mg kg−1) did not modify the hypophagic effect of sibutramine, while SB206553 (5‐HT2B/2C, 5 and 10 mg kg−1) slightly but significantly reduced it (Fint(2.53)=3.4; P<0.05).

The reduction in food intake caused by 6.0 mg kg−1 sibutramine in NPY‐injected rats was not modified by GR127935 (1.0 mg kg−1).

The results suggest that, with the possible exception of a partial involvement of 5‐HT2B/2C receptors in sibutramine's hypophagia in food‐deprived rats, 5‐HT1 and 5‐HT2 receptor subtypes do not play an important role in the hypophagic effect of sibutramine, at least in the first 2 h after injection.

British Journal of Pharmacology (1999) 127, 1190–1194; doi:10.1038/sj.bjp.0702633
Influence of the endothelium, nitric oxide and serotonergic receptors on coronary vasomotor responses evoked by ergonovine in conscious dogs
Authors: Karila‐Cohen Daniel , Delpy Eric , Dubois‐Randé Jean‐Luc , Puybasset Louis , Hittinger Luc , Giudicelli Jean‐François , Berdeaux Alain
Published: January 2005

The respective contributions of coronary vascular endothelium, nitric oxide (NO) and serotonergic receptors to the effects of ergonovine on large and small coronary arteries were investigated in conscious dogs.

In seven dogs with an endothelium intact, ergonovine (30–1000 μg, i.v.) induced a biphasic response on large coronary artery with an early and transient vasodilatation (up to +2.9±0.5% from 3310±160 μm, P<0.01) followed by a sustained vasoconstriction (down to −4.9±0.5%, P<0.001) which occurred simultaneously with a sustained increase in coronary blood flow (CBF) (up to +100±26% from 28±4 ml min−1, P<0.001). After endothelium removal (balloon angioplasty), the ergonovine‐induced vasodilatation was abolished and vasoconstriction potentiated (−6.4±0.9% after vs −4.9±0.5% before endothelium removal, P<0.01).

After blockade of NO synthesis by Nω‐nitro‐L‐arginine (30 mg kg−1) in four other dogs, the early vasodilatation induced by ergonovine was abolished but the delayed vasoconstriction as well as the increase in CBF remained unchanged.

Both ketanserin and methiothepin (0.3 mg kg−1) abolished the early vasodilatation and reduced the delayed vasoconstriction induced by ergonovine. Ketanserin decreased and methiothepin abolished the reduction in coronary resistance induced by ergonovine.

Thus, the complex interactions between vascular endothelium and serotonergic receptors to ergonovine‐induced constriction of large coronary arteries might explain the induction of coronary spasms in patients with endothelial dysfunction.

British Journal of Pharmacology (1999) 127, 1039–1047; doi:10.1038/sj.bjp.0702635
The contribution of intracellular Ca2+release to contraction in human bladder smooth muscle
Authors: Masters J G , Neal D E , Gillespie J I
Published: January 2005

The importance of Ca2+ release from the sarcoplasmic reticulum (SR) in excitation contraction (EC) coupling in human detrusor muscle remains controversial. In this paper the contribution of Ca2+ release to agonist induced contraction is assessed.

Dose response curves to carbachol (0.01–10 μM) were constructed before and after exposure to 200 nM Thapsigargin (Tg). Tg pre‐treatment reduced the force of contraction at all agonist concentrations however, the reduction was dose dependent. At 0.1 μM the contractions were reduced to 14.5±7% (mean±s.e.mean) of controls (n=8) while at 10 μM the contractions were only reduced to 92±3% of controls (n=10).

The role of external Ca2+ was examined by measuring the magnitude of contraction to low and high doses of agonist in the presence and absence of external Ca2+. With (0.1–0.3 μM) carbachol the contractions in nominally Ca2+ free media were 4±4% of controls (n=7) whilst with (1–10 μM) carbachol the contractions were 36±8% of controls (n=7) suggesting that at low agonist concentrations the release of Ca2+ has a requirement for external Ca2+.

Pre‐treatment of muscle strips with the Ca2+ channel blocking agent diltiazem reduced the contractile responses to carbachol. Contractions induced by 0.1 μM were reduced to 29±11% (P<0.05) of controls while those activated by 10 μM were reduced to 86±6% (P=0.1) of controls (n=4) suggesting the Ca2+ influx needed to activate internal store release at low agonist stimulation is through L‐type Ca2+ channels.

These observations confirm the importance of thapsigargin sensitive intracellular Ca2+ store release in the activation of contraction of detrusor smooth muscle and suggest the overall contribution of this store depends upon the magnitude of the agonist stimulation.

British Journal of Pharmacology (1999) 127, 996–1002; doi:10.1038/sj.bjp.0702640
Subtype‐selective inhibition of [methyl‐3H]‐N‐methylscopolamine binding to muscarinic receptors by α‐truxillic acid esters
Authors: Lysíková Michaela , Fuksová Květa , Elbert Tomáš , Jakubík Jan , Tuček Stanislav
Published: January 2005

Seven esters of α‐truxillic acid have been synthesized: bis‐3‐piperidylpropyl ester and its quaternary bis‐N‐ethyl derivative, bis‐N‐diethylaminopropyl ester and its quaternary bis‐N‐methyl derivative, and bis‐4‐piperidylbutyl ester and its quaternary bis‐N‐methyl and bis‐N‐ethyl derivatives.

All esters inhibited the specific binding of muscarinic receptor antagonist [methyl‐3H]‐N‐methylscopolamine ([3H]‐NMS) to muscarinic receptors in membranes of CHO cell lines stably expressing the human gene for the M1, M2, M3 or M4 subtype of muscarinic receptors. All esters displayed the highest potency at the M2 and the lowest potency at the M3 receptor subtype.

In experiments performed on the M2 muscarinic receptor subtype, the affinity between the receptors and the esters was greatly increased when the concentration of ions was diminished. The highest affinities were found for the tertiary bis‐3‐piperidylpropyl and bis‐4‐piperidylbutyl aminoesters (equilibrium dissociation constants of 52 and 179 pM, respectively, in the low ionic strength medium).

All investigated esters slowed down the dissociation of [3H]‐NMS from the M2 muscarinic receptor subtype. [3H]‐NMS dissociation from the M1, M3 and M4 muscarinic receptor subtypes was investigated in experiments with the bis‐4‐piperidylbutyl aminoester and also found to be decelerated.

It is concluded that the esters of α‐truxillic acid act as M2‐selective allosteric modulators of muscarinic receptors and that, by their potency, the tertiary bis‐3‐piperidylpropyl and bis‐4‐piperidylbutyl aminoesters surpass the other known allosteric modulators of these receptors.

British Journal of Pharmacology (1999) 127, 1240–1246; doi:10.1038/sj.bjp.0702646
Prejunctional angiotensin receptors involved in the facilitation of noradrenaline release in mouse tissues
Authors: Cox S L , Trendelenburg A U , Starke K
Published: January 2005

The effect of angiotensin II, angiotensin III, angiotensin IV and angiotensin‐(1–7) on the electrically induced release of noradrenaline was studied in preparations of mouse atria, spleen, hippocampus, occipito‐parietal cortex and hypothalamus preincubated with [3H]‐noradrenaline. The prejunctional angiotensin receptor type was investigated using the non‐selective receptor antagonist saralasin (AT1/AT2) and the AT1 and AT2 selective receptor antagonists losartan and PD 123319, respectively.

In atrial and splenic preparations, angiotensin II (0.01 nM–0.1 μM) and angiotensin III (0.01 and 0.1 nM–1 μM) increased the stimulation‐induced overflow of tritium in a concentration‐dependent manner. Angiotensin IV, only at high concentrations (1 and 10 μM), enhanced tritium overflow in the atria, while angiotensin‐(1–7) (0.1 nM–10 μM) was without effect in both preparations.

In preparations of hippocampus, occipito‐parietal cortex and hypothalamus, none of the angiotensin peptides altered the evoked overflow of tritium.

In atrial and splenic preparations, saralasin (0.1 μM) and losartan (0.1 and 1 μM), but not PD 123319 (0.1 μM), shifted the concentration‐response curves of angiotensin II and angiotensin III to the right.

In conclusion, in mouse atria and spleen, angiotensin II and angiotensin III facilitate the action potential induced release of noradrenaline via a prejunctional AT1 receptor. Only high concentrations of angiotensin IV are effective in the atria and angiotensin‐(1–7) is without effect in both preparations. In mouse brain areas, angiotensin II, angiotensin III, angiotensin IV and angiotensin‐(1–7) do not modulate the release of noradrenaline.

British Journal of Pharmacology (1999) 127, 1256–1262; doi:10.1038/sj.bjp.0702652
Quantitation of extracellular UTP using a sensitive enzymatic assay
Authors: Lazarowski Eduardo R , Harden T Kendall
Published: January 2005

The wide distribution of the uridine nucleotide‐activated P2Y2, P2Y4 and P2Y6 receptors suggests a role for UTP as an important extracellular signalling molecule. However, direct evidence for UTP release and extracellular accumulation has been addressed only recently due to the lack of a sensitive assay for UTP mass. In the present study, we describe a method that is based on the uridinylation of [14C]‐glucose‐1P by the enzyme UDP‐glucose pyrophosphorylase which allows quantification of UTP in the sub‐nanomolar concentration range.

The UTP‐dependent conversion of [14C]‐glucose‐1P to [14C]‐UDP‐glucose was made irreversible by including the pyrophosphate scavenger inorganic pyrophosphatase in the reaction medium and [14C]‐glucose‐1P and [14C]‐UDP‐glucose were separated and quantified by HPLC. Formation of [14C]‐UDP‐glucose was linearly observed between 1 and 300 nM UTP. The reaction was highly specific for UTP and was unaffected by a 1000 fold molar excess of ATP over UTP.

Release of UTP was measured with a variety of cells including platelets and leukocytes, primary airway epithelial cells, rat astrocytes and several cell lines. In most resting attached cultures, extracellular UTP concentrations were found in the low nanomolar range (1–10 nM in 0.5 ml medium bathing 2.5 cm2 dish). Up to a 20 fold increase in extracellular UTP levels was observed in cells subjected to a medium change. Extracellular UTP levels were 10–30% of the ATP levels in both resting and mechanically‐stimulated cultured cells. In unstirred platelets, a 1 : 100 ratio UTP/ATP was observed. Extracellular UTP and ATP increased 10 fold in thrombin‐stimulated platelets.

Detection of UTP in nanomolar concentrations in the medium bathing resting cultures suggests that constitutive release of UTP may provide a mechanism of regulation of the basal activity of uridine nucleotide sensitive receptors.

British Journal of Pharmacology (1999) 127, 1272–1278; doi:10.1038/sj.bjp.0702654
Multiple effector pathways regulate the insulin secretory response to the imidazoline RX871024 in isolated rat pancreatic islets
Authors: Mourtada Mirna , Chan Sue L F , Smith Stephen A , Morgan Noel G
Published: January 2005

When isolated rat islets were cultured for 18 h prior to use, the putative imidazoline binding site ligand, RX871024 caused a dose‐dependent increase in insulin secretion at both 6 mM and 20 mM glucose. By contrast, a second ligand, efaroxan, was ineffective at 20 mM glucose whereas it did stimulate insulin secretion in response to 6 mM glucose.

Exposure of islets to RX871024 (50 μM) for 18 h, resulted in loss of responsiveness to this reagent upon subsequent re‐exposure. However, islets that were unresponsive to RX871024 still responded normally to efaroxan.

The imidazoline antagonist, KU14R, blocked the insulin secretory response to efaroxan, but failed to prevent the stimulatory response to RX871024.

By contrast with its effects in cultured islets, RX871024 inhibited glucose‐induced insulin release from freshly isolated islets. Efaroxan did not inhibit insulin secretion under any conditions studied.

In freshly isolated islets, the effects of RX871024 on insulin secretion could be converted from inhibitory to stimulatory, by starvation of the animals.

Inhibition of insulin secretion by RX871024 in freshly isolated islets was prevented by the cyclo‐oxygenase inhibitors indomethacin or flurbiprofen. Consistent with this, RX871024 caused a marked increase in islet PGE2 formation. Efaroxan did not alter islet PGE2 levels.

The results suggest that RX871024 exerts multiple effects in the pancreatic β‐cell and that its effects on insulin secretion cannot be ascribed only to interaction with a putative imidazoline binding site.

British Journal of Pharmacology (1999) 127, 1279–1287; doi:10.1038/sj.bjp.0702656
Suc‐[Glu9, Ala11,15]‐endothelin‐1 (8–21), IRL 1620, identifies two populations of ETB receptors in guinea‐pig bronchus
Authors: Mazzoni Maria R , Breschi M Cristina , Ceccarelli Francesca , Lazzeri Nicola , Giusti Laura , Nieri Paola , Lucacchini Antonio
Published: January 2005

The pharmacological properties of endothelin receptors (ETR) were investigated in guinea‐pig bronchus by comparing binding and functional results.

In binding assays, both the ETB agonists, endothelin‐3 (ET‐3) and N‐suc‐[Glu9,Ala11,15]ET‐1(8–21) (IRL 1620), and the antagonist, N‐cis‐2,6‐dimethylpiperidinocarbonyl‐L‐γ‐methylleucyl‐D‐1‐methoxycarbonyltryptophanyl‐D‐norleucine (BQ 788), showed biphasic inhibition curves of [125I]‐endothelin‐1 (ET‐1) binding to bronchus membranes prepared from intact or epithelium‐deprived tissue. IRL 1620 did not completely displace specifically [125I]‐ET‐1 bound to these tissue preparations. In the presence of the ETA‐selective antagonist, cyclo(‐D‐Trp‐D‐Asp‐L‐Pro‐D‐Val‐L‐Leu) (BQ 123, 1 μM), IRL 1620 displacement curves were shallow but a complete inhibition was reached at a concentration of 1 μM. Both curves were better represented by two‐site models. In addition, BQ 788 competition curves became monophasic when binding experiments were performed in the presence of 1 μM BQ 123. The non‐selective agonist, ET‐1, and BQ 123 inhibited [125I]‐ET binding to bronchus membranes in dose‐dependent fashions with monophasic curves.

The contracting activity of IRL 1620 (0.55 nM–1.6 μM) was tested on multiple‐ring bronchial preparations pretreated with peptidase and cyclo‐oxygenase inhibitors. BQ 788 shifted IRL1620 concentration‐response curves to the right while BQ 123 did not influence bronchial responsiveness. In addition, a potentiation of the maximal response to the agonist was observed in BQ 788 treated bronchial rings. This effect was abolished by tissue pretreatment with Nω‐nitro‐L‐argininemethylester (L‐NAME) or epithelium removal but not by pretreatment with atropine or iberiotoxin.

Our results demonstrate that guinea‐pig bronchus contains two populations of ETB receptors with different affinities for the ETB‐selective agonist, IRL 1620. One ETB receptor population appears to activate bronchial muscle contraction while another on epithelial cells causes muscle relaxation through the release of nitric oxide (NO).

British Journal of Pharmacology (1999) 127, 1406–1414; doi:10.1038/sj.bjp.0702672
Antagonism of the mGlu5 agonist 2‐chloro‐5‐hydroxyphenylglycine by the novel selective mGlu5 antagonist 6‐methyl‐2‐(phenylethynyl)‐pyridine (MPEP) in the thalamus
Authors: Salt T E , Binns K E , Turner J P , Gasparini F , Kuhn R
Published: January 2005

Our previous work has shown that Group I mGlu receptors participate in thalamic sensory processing in vivo. However, unequivocal demonstration of mGlu5 participation has not been possible due to the lack of specific ligands. We have therefore made a preliminary study of the in vivo actions of the agonist (R,S)‐2‐Chloro‐5‐hydroxyphenylglycine [CHPG] and the novel mGlu5 antagonist 6‐methyl‐2‐(phenylethynyl)‐pyridine [MPEP] in order to characterize their suitability for functional studies. Iontophoretically administered MPEP selectively antagonized excitatory responses of single rat thalamic neurones to CHPG compared to the broad‐spectrum mGlu agonist (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylate. In contrast, the established mGlu1 and mGlu5 antagonist (S)‐4‐carboxyphenylglycine reduced responses to both agonists. These findings are the first demonstration of an in vivo action of CHPG and its antagonism by a selective mGlu5 antagonist. Furthermore MPEP appears to be a good tool for functional studies of mGlu5.

British Journal of Pharmacology (1999) 127, 1057–1059; doi:10.1038/sj.bjp.0702677
Spontaneously hypertensive rats cholinergic hyper‐responsiveness: central and peripheral pharmacological mechanisms
Authors: Lazartigues E , Brefel‐Courbon C , Tran M A , Montastruc J L , Rascol O
Published: January 2005

The mechanisms and the subtypes of muscarinic receptors implicated in the cardiovascular effects of physostigmine were investigated in conscious normotensive and spontaneously hypertensive rats.

Intravenous (i.v.) physostigmine (50 μg kg−1) induced in both strains a long pressor response, accompanied by a bradycardia. This pressor response was larger in spontaneously hypertensive (+41±6 mmHg) than in Wistar‐Kyoto (+25±2 mmHg) rats (P<0.05).

Pretreatment with atropine sulphate (0.4 mg kg−1 i.v.), completely abolished the physostigmine‐induced pressor response in both normotensive and hypertensive rats. In both strains, the physostigmine pressor response was significantly reduced by the systemic administration of either an α1‐adrenoceptor antagonist (prazosin, 1 mg kg−1) or a V1A‐vasopressin receptor antagonist (AVPX, 20 μg kg−1). This physostigmine pressor effect was completely abolished in both strains when both antagonists were administered concomitantly.

In WKY rats, the pressor response to physostigmine (50 μg kg−1 i.v.) was inhibited in a dose‐dependent manner by i.c.v. administration of atropine (ID50=3.70 nmoles), the M1 receptor antagonist pirenzepine (ID50=10.71 nmoles), the M2 receptor antagonist methoctramine (ID50=4.31 nmoles), the M3 receptor antagonist p‐F‐HHSiD (ID50=60.52 nmoles) and the M4 receptor antagonist tropicamide (ID50=214.20 nmoles). In the hypertensive strain, the ID50 were found to be significantly higher for atropine (7.34 nmoles), pirenzepine (21.60 nmoles) and p‐F‐HHSiD (139.50 nmoles) (P<0.05).

The present results indicate that physostigmine acts in normotensive and spontaneously hypertensive rats, through stimulation of both central M2 and M1 cholinoceptors to induce a rise in blood pressure mediated by an increase in plasma vasopressin and sympathetic outflow. Moreover, our results suggest that some modifications of the M1 receptor subtypes in terms of expression or affinity could be responsible for the hyper‐responsiveness of the hypertensive strain to cholinomimetic agents.

British Journal of Pharmacology (1999) 127, 1657–1665; doi:10.1038/sj.bjp.0702678
Atriopeptin, sodium azide and cyclic GMP reduce secretion of aqueous humour and inhibit intracellular calcium release in bovine cultured ciliary epithelium
Authors: Shahidullah Mohammad , Wilson William S
Published: January 2005

This study examined the involvement of cyclic GMP, protein kinase G and intracellular Ca2+ movements in the modulation of aqueous humour formation.

Using the bovine arterially‐perfused eye preparation, drug effects on intraocular pressure and aqueous humour formation rate were measured by manometry and fluorescein dilution, respectively. Drug effects on intracellular [Ca2+] were determined by fura‐2 fluorescence ratio technique in non‐transformed, cultured ciliary epithelium.

Intra‐arterial injection of atriopeptin (50 pmol) or sodium azide (10 nmol) produced significant reduction in aqueous humour formation (>38%). This was blocked by selective inhibition (KT‐5823) of protein kinase G, but not by selective inhibition (KT‐5720) of protein kinase A. Reductions of intraocular pressure produced by atriopeptin or azide were almost completely blocked by KT‐5823.

ATP (100 μM) caused rapid, transient increase in intracellular Ca2+ followed by a slow decline and prolonged plateau. This response showed concentration‐dependent inhibition by atriopeptin, azide or 8‐bromo cyclic GMP, and this inhibition of the rapid (peak) Ca2+ increase was enhanced by zaprinast (100 μM; phosphodiesterase inhibitor). KT‐5823 blocked the suppression of the peak Ca2+ response but not suppression of the plateau.

Arterial perfusion of ATP (0.1–100 μM) produced a concentration‐dependent decrease in aqueous humour formation.

Aqueous humour formation in the bovine eye can be manipulated through cyclic GMP, operating via protein kinase G. Close parallels appear when Ca2+ movements are modified by similar manipulations of cyclic GMP, suggesting that Ca2+ transients may play an important role in aqueous humour formation and that interplay occurs between cyclic GMP and Ca2+.

British Journal of Pharmacology (1999) 127, 1438–1446; doi:10.1038/sj.bjp.0702681
Spinal effect of a neuropeptide FF analogue on hyperalgesia and morphine‐induced analgesia in mononeuropathic and diabetic rats
Authors: Courteix Christine , Coudoré‐Civiale Marie‐Ange , Privat Anne‐Marie , Zajac Jean‐Marie , Eschalier Alain , Fialip Joseph
Published: January 2005

1DMe, a neuropeptide FF (NPFF) analogue, has been shown to produce antinociception and to enhance morphine analgesia in rats after intrathecal administration. To determine whether 1DMe could correct hyperalgesia and restore morphine efficacy in mononeuropathic (MN) and diabetic (D) rats we examined the spinal effect of 1DMe in MN and D rats without and after spinal blockade of μ‐ and δ‐opioid receptors with CTOP and naltrindole, respectively. The influence of 1DMe on morphine‐induced antinociception was assessed in the two models using isobolographic analysis.

Whereas 1DMe intrathecally injected (0.1, 1, 7.5 μg rat−1) was ineffective in normal (N) rats, it suppressed mechanical hyperalgesia (decrease in paw pressure‐induced vocalisation thresholds) in both MN and D rats. This effect was completely cancelled by CTOP (10 μg rat−1) and naltrindole (1 μg rat−1) suggesting that it requires the simultaneous availability of μ‐ and δ‐opioid receptors.

The combinations of morphine : 1DMe (80.6 : 19.4% and 99.8 : 0.2%, in MN and D rats, respectively) followed by isobolographic analysis, showed a superadditive interaction, relative to the antinociceptive effect of single doses, in D rats only. In N rats, the combination of morphine : 1DMe (0.5 mg kg−1, i.v.: 1 μg rat−1, i.t., ineffective doses) resulted in a weak short‐lasting antinociceptive effect.

These results show a different efficacy of 1DMe according to the pain model used, suggesting that the pro‐opioid effects of the NPFF in neuropathic pain are only weak, which should contribute to hyperalgesia and to the impaired efficacy of morphine.

British Journal of Pharmacology (1999) 127, 1454–1462; doi:10.1038/sj.bjp.0702682
Involvement of 5‐HT1B receptors in collar‐induced hypersensitivity to 5‐hydroxytryptamine of the rabbit carotid artery
Authors: Geerts Inge S , Matthys Katelijne E , Herman Arnold G , Bult Hidde
Published: January 2005

In humans intimal thickening is a prerequisite of atherosclerosis. Application of a silicone collar around the rabbit carotid artery induces an intimal thickening but in addition it increases the sensitivity to the vasoconstrictor action of serotonin (5‐hydroxytryptamine, 5‐HT). The 5‐HT receptors involved in collar‐induced hypersensitivity to 5‐HT were investigated using several agonists and antagonists.

One week after placement of collars around both carotid arteries of anaesthetized rabbits, rings (2 mm width) from inside (=collar) and outside (=sham) the collars were mounted in organ baths (10 ml) for isometric force measurements at 6 g loading tension.

Collared rings were more sensitive to the contractile effect of 5‐HT (7.6 fold) and 5‐carboxamidotryptamine (31 fold, 5‐CT, 5‐HT1 agonist) in cumulative concentration response curves. Sumatriptan (5‐HT1B/1D agonist) caused concentration‐dependent constrictions in collared rings only.

Collar placement did not significantly alter pA2 values (Schild regression) or apparent pKb values (non‐linear regression) of spiperone and methysergide (mixed 5‐HT2A/5‐HT1 antagonists) or ketanserin and ritanserin (5‐HT2A antagonists), indicating unchanged binding characteristics of the 5‐HT2A receptor. However, the reduced slope of the Schild regression pointed to a heterogeneous receptor population in collared rings.

In contrast, the apparent pKb value of methiothepin (5‐HT1B antagonist) was significantly reduced by collar placement, and its antagonism shifted from non‐surmountable in sham rings to surmountable in collared segments.

Taken together, this study demonstrates that the serotonergic receptor involved in the hypersensitivity to 5‐HT of rabbit collared carotid artery is a 5‐HT1B receptor subtype.

British Journal of Pharmacology (1999) 127, 1327–1336; doi:10.1038/sj.bjp.0702684
Antagonist activities of mecamylamine and nicotine show reciprocal dependence on beta subunit sequence in the second transmembrane domain
Authors: Webster J Christopher , Francis Michael M , Porter Julia K , Robinson Gillian , Stokes Clare , Horenstein Ben , Papke Roger L
Published: January 2005

We show that a portion of the TM2 domain regulates the sensitivity of beta subunit‐containing rat neuronal nicotinic AChR to the ganglionic blocker mecamylamine, such that the substitution of 4 amino acids of the muscle beta subunit sequence into the neuronal beta4 sequence decreases the potency of mecamylamine by a factor of 200 and eliminates any long‐term effects of this drug on receptor function.

The same exchange of sequence that decreases inhibition by mecamylamine produces a comparable potentiation of long‐term inhibition by nicotine.

Inhibition by mecamylamine is voltage‐dependent, suggesting a direct interaction of mecamylamine with sequence elements within the membrane field. We have previously shown that sensitivity to TMP (tetramethylpiperidine) inhibitors is controlled by the same sequence elements that determine mecamylamine sensitivity. However, inhibition by bis‐TMP compounds is independent of voltage.

Our experiments did not show any influence of voltage on the inhibition of chimeric receptors by nicotine, suggesting that the inhibitory effects of nicotine are mediated by binding to a site outside the membrane's electric field.

An analysis of point mutations indicates that the residues at the 6′ position within the beta subunit TM2 domain may be important for determining the effects of both mecamylamine and nicotine in a reciprocal manner. Single mutations at the 10′ position are not sufficient to produce effects, but 6′ 10′ double mutants show more effect than do the 6′ single mutants.

British Journal of Pharmacology (1999) 127, 1337–1348; doi:10.1038/sj.bjp.0702686
Mutation at the putative GABAA ion‐channel gate reveals changes in allosteric modulation
Authors: Thompson Sally‐Anne , Smith Maria Z , Wingrove Peter B , Whiting Paul J , Wafford Keith A
Published: January 2005

We have mutated a conserved leucine in the putative membrane‐spanning domain to serine in human GABAA β2 and investigated the actions of a number of GABAA agonists, antagonists and modulators on human α1β2ΔL259Sγ2s compared to wild type α1β2γ2s GABAA receptors, expressed in Xenopus oocytes.

The mutation resulted in smaller maximum currents to γ‐aminobutyric acid (GABA) compared to α1β2γ2s receptors, and large leak currents resulting from spontaneous channel opening. As reported, this mutation significantly decreased the GABA EC50 (110 fold), and reduced desensitization. Muscimol and the partial agonists 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP) and piperidine‐4‐sulphonic acid (P4S) also displayed a decrease in EC50.

In addition to competitively shifting GABA concentration response curves, the antagonists bicuculline and SR95531 both inhibited the spontaneous channel activity on α1β2ΔL259Sγ2s receptors, with different degrees of maximum inhibition.

The effects of a range of allosteric modulators, including benzodiazepines and anaesthetics were examined on a submaximal GABA concentration (EC20). Compared to wild type, none of these modulators potentiated the EC20 response of α1β2ΔL259Sγ2s receptors, however they all directly activated the receptor in the absence of GABA.

To conclude, the above mutation resulted in receptors which exhibit a degree of spontaneous activity, and are more sensitive to agonists. Benzodiazepines and other agents modulate constitutive activity, but positive modulation of GABA is lost. The competitive antagonists bicuculline and SR95531 can also act as allosteric channel modulators through the same GABA binding site.

British Journal of Pharmacology (1999) 127, 1349–1358; doi:10.1038/sj.bjp.0702687
Involvement of thromboxane A2 in the endothelium‐dependent contractions induced by myricetin in rat isolated aorta
Authors: Jiménez Rosario , Andriambeloson Emile , Duarte Juan , Andriantsitohaina Ramaroson , Jiménez José , Pérez‐Vizcaino Francisco , Zarzuelo Antonio , Tamargo Juan
Published: January 2005

The present study was undertaken to analyse the mechanism of the contractile response induced by the bioflavonoid myricetin in isolated rat aortic rings.

Myricetin induced endothelium‐dependent contractile responses (maximal value=21±2% of the response induced by 80 mM KCl and pD2=5.12±0.03). This effect developed slowly, reached a peak within 6 min and then declined progressively.

Myricetin‐induced contractions were almost abolished by the phospholipase A2 (PLA2) inhibitor, quinacrine (10 μM), the cyclo‐oxygenase inhibitor, indomethacin (10 μM), the thromboxane synthase inhibitor, dazoxiben (100 μM), the putative thromboxane A2 (TXA2)/prostaglandin endoperoxide receptor antagonist, ifetroban (3 μM). These contractions were abolished in Ca2+‐free medium but were not affected by the Ca2+ channel blocker verapamil (10 μM).

In cultured bovine endothelial cells (BAEC), myricetin (50 μM) produced an increase in cytosolic free calcium ([Ca2+]i) which peaked within 1 min and remained sustained for 6 min, as determined by the fluorescent probe fura 2. This rise in [Ca2+]i was abolished after removal of extracellular Ca2+ in the medium.

Myricetin (50 μM) significantly increased TXB2 production both in aortic rings with and without endothelium and in BAEC. These increases were abolished both by Ca2+‐free media and by indomethacin.

Taken together, these results suggests that myricetin stimulates Ca2+ influx and subsequently triggers the activation of the PLA2 and cyclo‐oxygenase pathways releasing TXA2 from the endothelium to contract rat aortic rings. The latter response occurs via the activation of Tp receptors on vascular smooth muscle cells.

British Journal of Pharmacology (1999) 127, 1539–1544; doi:10.1038/sj.bjp.0702694
Effects of exercise training on responsiveness of the mesenteric arterial bed to phenylephrine and KCl in male rats
Authors: Jansakul C , Hirunpan P
Published: January 2005

We aimed to determine whether there are any changes in responsiveness of the mesenteric arterial beds to phenylephrine (Phe) and KCl in exercise‐trained rats, and whether vascular endothelium and/or vascular smooth muscle play a role in these changes.

Adult male rats were subjected to a swimming schedule every day for 28–33 days. Studies were performed in vitro using Krebs perfused mesenteric arterial beds.

Maximum perfusion pressure responses to KCl and Phe of the mesenteric arterial beds from exercise‐trained rats were significantly lower than those from sedentary controls. However, these differences disappeared after blocking the nitric oxide synthase by NG‐nitro‐L‐arginine (L‐NOARG).

3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propanesulphonate (CHAPS, 3 mg ml−1, 2 min infusion) caused a significant increase in maximum perfusion pressure responses to KCl to the same extent in both exercise‐trained and sedentary control rats. CHAPS caused about a 4.5 fold leftward shift of the curve with no change in maximum response to Phe for the mesenteric arterial beds from sedentary control rats, but not for those obtained from exercise‐trained rats. However, these differences were abolished in the presence of L‐NOARG.

Indomethacin did not alter the dose‐response curves to KCl or Phe in either swimming or control groups.

These results suggest that there was a lower vascular responsiveness to KCl and Phe in exercise‐trained rats at rest. The decrease in reactivities to KCl or decrease in sensitivity to Phe after having endothelium impairment by CHAPS of the mesenteric arterial beds of exercise‐trained rats were due to an increase in both spontaneous release and upregulation of phenylephrine‐stimulated release of nitric oxide from both the vascular endothelium and the vascular smooth muscle cells, and may not be a consequence of an increase in vasodilator prostaglandins by the vascular bed.

British Journal of Pharmacology (1999) 127, 1559–1566; doi:10.1038/sj.bjp.0702697
Nociceptin‐induced scratching, biting and licking in mice: involvement of spinal NK1 receptors
Authors: Sakurada Tsukasa , Katsuyama Sou , Sakurada Shinobu , Inoue Makoto , Tan‐No Koichi , Kisara Kensuke , Sakurada Chikai , Ueda Hiroshi , Sasaki Jun
Published: January 2005

Intrathecal (i.t.) injection of nociceptin at small doses (fmol order) elicited a behavioural response consisting of scratching, biting and licking in conscious mice. Here we have examined the involvement of substance P‐containing neurons by using i.t. injection of tachykinin neurokinin (NK)1 receptor antagonists and substance P (SP) antiserum.

Nociceptin‐induced behavioural response was evoked significantly 5–10 min after i.t. injection and reached a maximum at 10–15 min. Dose‐dependency of the induced response showed a bell‐shaped pattern from 0.375–30.0 fmol, and the maximum effect was observed at 3.0 fmol.

The behavioural response elicited by nociceptin (3.0 fmol) was dose‐dependently inhibited by intraperitoneal (i.p.) administration of morphine.

The NK1 receptor antagonists, CP‐96,345, CP‐99,994 and sendide, inhibited nociceptin‐induced behavioural response in a dose‐dependent manner. A significant antagonistic effect of [D‐Phe7, D‐His9]SP (6–11), a selective antagonist for SP receptors, was observed against nociceptin‐induced response. The NK2 receptor antagonist, MEN‐10376, had no effect on the response elicited by nociceptin.

Pretreatment with SP antiserum resulted in a significant reduction of the response to nociceptin. No significant reduction of nociceptin‐induced response was detected in mice pretreated with NKA antiserum.

The N‐methyl‐D‐aspartate (NMDA) receptor antagonists, dizocilpine (MK‐801) and D(−)‐2‐amino‐5‐phosphonovaleric acid (APV) (D‐APV), and L‐NG‐nitro arginine methyl ester (L‐NAME), a nitric oxide (NO) synthase inhibitor, failed to inhibit nociceptin‐induced behavioural response.

The present results suggest that SP‐containing neurons in the mouse spinal cord may be involved in elicitation of scratching, biting and licking behaviour following i.t. injection of nociceptin.

British Journal of Pharmacology (1999) 127, 1712–1718; doi:10.1038/sj.bjp.0702698
Anisatin modulation of the γ‐aminobutyric acid receptor‐channel in rat dorsal root ganglion neurons
Authors: Ikeda Tomoko , Ozoe Yoshihisa , Okuyama Emi , Nagata Keiichi , Honda Hiroshi , Shono Toshio , Narahashi Toshio
Published: January 2005

Anisatin, a toxic, insecticidally active component of Sikimi plant, is known to act on the GABA system. In order to elucidate the mechanism of anisatin interaction with the GABA system, whole‐cell and single‐channel patch clamp experiments were performed with rat dorsal root ganglion neurons in primary culture.

Repeated co‐applications of GABA and anisatin suppressed GABA‐induced whole‐cell currents with an EC50 of 1.10 μM. No recovery of currents was observed after washout with anisatin‐free solution.

However, pre‐application of anisatin through the bath had no effect on GABA‐induced currents. The decay phase of currents was accelerated by anisatin. These results indicate that anisatin suppression of GABA‐induced currents requires opening of the channels and is use‐dependent.

Anisatin suppression of GABA‐induced currents was not voltage dependent.

Picrotoxinin attenuated anisatin suppression of GABA‐induced currents. [3H]‐EBOB binding to rat brain membranes was competitively inhibited by anisatin. These data indicated that anisatin bound to the picrotoxinin site.

At the single‐channel level, anisatin did not alter the open time but prolonged the closed time. The burst duration was reduced and channel openings per burst were decreased indicating that anisatin decreased the probability of openings.

British Journal of Pharmacology (1999) 127, 1567–1576; doi:10.1038/sj.bjp.0702700
Effects of propranolol treatment on left ventricular function and intracellular calcium regulation in rats with postinfarction heart failure
Authors: Litwin Sheldon E , Katz Sarah E , Morgan James P , Douglas Pamela S
Published: January 2005

Chronic treatment with beta‐adrenergic blocking agents can improve survival in patients with heart failure. The mechanisms underlying the beneficial effects and whether these effects are generalizable to ischaemic heart failure are unresolved.

We performed echocardiographic‐Doppler examinations in rats (n=28) 1 and 6 weeks after myocardial infarction (MI) or sham surgery. Rats were randomized to no treatment or propranolol (500 mg/l in drinking water) after the first echocardiogram. Isometric contractions and intracellular Ca transients were recorded simultaneously in noninfarcted left ventricular (LV) papillary muscles.

Untreated MI rats had significant LV dilatation (10.6±0.4* vs 8.9±0.3 mm, MI vs control), impaired systolic function (fractional shortening=11±2* vs 38±2%), and a restrictive LV diastolic filling pattern. MI rats receiving propranolol had similar LV chamber sizes (10.6±0.5 mm) and systolic function (13±2%). The propranolol treated animals had higher LV end‐diastolic pressures (27±2* vs 20±3 mmHg) and a more restricted LV diastolic filling pattern (increased ratio of early to late filling velocities and more rapid E wave deceleration rate). Contractility of papillary muscles from untreated MI rats was depressed (1.6±0.3 vs 2.4±0.5 g mm−2). In addition, Ca transients were prolonged and the inotropic response to isoproterenol was blunted. Propranolol treatment did not improve force development (1.6±0.3 g mm−2) or the duration of Ca transients during isoproterenol stimulation.

Chronic propranolol treatment in rats with postinfarction heart failure did not improve LV remodeling or systolic function. LV diastolic pressures and filling patterns were worsened by propranolol. Treatment also did not produce appreciable improvement in contractility, intracellular Ca regulation or beta‐adrenergic responsiveness in the noninfarcted myocardium.

British Journal of Pharmacology (1999) 127, 1671–1679; doi:10.1038/sj.bjp.0702701
Pharmacological characterization of β‐adrenoceptors mediating relaxation of the rat urinary bladder in vitro
Authors: Longhurst Penelope A , Levendusky Mark
Published: January 2005

Isoproterenol relaxed KCl‐precontracted rat bladder strips with a pD2 of 7.21 leaving a residual contractile response of 3.2% after 30 μM. The selective β1‐agonist, T‐0509 (pD2 : 6.24, 10.1% residual contraction after 100 μM), β2‐agonist, terbutaline (pD2 : 5.43, 13.7% residual contraction after 100 μM), and β3‐agonists, BRL 37344A (pD2 : 6.60, 17.3% residual contraction after 100 μM), and SR 58611A (pD2 : 5.15, 34.0% residual contraction after 100 μM), also relaxed bladder strips.

The relaxant response to isoproterenol was weakly but significantly antagonized by 1 μM propranolol which produced a 3 fold shift of the concentration‐response curve to the right, and significantly antagonized by the β1‐selective antagonist, metoprolol (10 μM, 3 fold shift), and the β2‐selective antagonist, butoxamine (100 μM, 6 fold shift). A combination of 10 μM metoprolol and 100 μM butoxamine caused a 15 fold shift of the concentration‐response curve for isoproterenol to the right. Incubation with the β3‐antagonist, SR 59230A (1 μM), caused a 6 fold shift of the concentration response curve for isoproterenol to the right.

The non‐conventional partial agonist, CGP 12177A, weakly relaxed KCl‐precontracted bladder strips (pD2 : 3.31, 51.3% residual contraction after 300 μM); the relaxation was resistant to blockade by 1 or 10 μM propranolol.

In the presence of 200 μM propranolol, CGP 12177A (20 μM) or SR 59230A (10 μM) antagonized surmountably the relaxant effects of BRL 37344A.

The data suggest that rat urinary bladder body contains β1, β2, and β3‐adrenoceptors, all of which mediate relaxation.

British Journal of Pharmacology (1999) 127, 1744–1750; doi:10.1038/sj.bjp.0702709
Effects of nociceptin and nocistatin on antidromic vasodilatation in hairless skin of the rat hindlimb in vivo
Authors: Häbler H ‐J , Timmermann L , Stegmann J ‐U , Jänig W
Published: January 2005

We tested whether nociceptin (NCE), the endogenous ligand of the opioid receptor‐like 1 (ORL1) receptor, and nocistatin (NST), which reverses central NCE effects when applied intrathecally (i.t.), affect small‐diameter afferent fibre‐mediated vasodilatation in rat hairless skin.

Female Wistar rats were vagotomized. Ongoing sympathetic vasoconstrictor activity was abolished by bilateral section of the lumbar sympathetic trunk between ganglia L2 and L3. Sensory axons were selectively stimulated in the dorsal root L5 by 20 electrical impulses supramaximal for activating C‐fibres at 1 Hz. Blood flow was measured on the plantar skin of the left hind paw in the L5 dermatome using laser Doppler flowmetry.

NCE injected intravenously (i.v.) as single boluses (1, 10 and 100 nmol kg−1) 7–8 min before dorsal root stimulation (n=6) dose‐dependently decreased blood pressure and local vascular resistance and suppressed antidromic vasodilatation maximally by 47% (P<0.01). When NCE was injected 2 min before stimulation (n=3), antidromic vasodilatation was reduced by 64% after NCE (1 nmol kg−1) and totally, or almost totally, abolished after the two higher doses.

NST (1–100 nmol kg−1 i.v., n=6) was without significant effect on blood pressure and cutaneous vascular resistance. Applied 5 (n=6) or 2 min (n=3) before stimulation it also did not affect antidromic vasodilatation. NST (100 nmol kg−1 i.v.) applied shortly before an equimolar dose of NCE did not antagonize NCE effects on vascular resistance, blood pressure and antidromic vasodilatation (n=4).

In conclusion, NCE inhibits antidromic vasodilatation, a component of neurogenic inflammation, in rat skin while NST is without effect. NST, at the small‐diameter sensory ending, is not an effective antagonist of NCE.

British Journal of Pharmacology (1999) 127, 1719–1727; doi:10.1038/sj.bjp.0702712