bluuebunny/crossref_metadata_2025 huggingface dataset:
Actions of adenosine A1 and A2 receptor antagonists on CFTR antibody‐inhibited β‐adrenergic mucin secretion response
Authors: Pereira M M C , Lloyd Mills C , Dormer R L , McPherson M A
Published: January 2005

The cystic fibrosis gene protein, the cystic fibrosis transmembrane conductance regulator (CFTR) acts as a chloride channel and is a key regulator of mucin secretion. The mechanism by which 3‐isobutyl‐1‐methylxanthine (IBMX) corrects the defect in CFTR mediated β‐adrenergic stimulation of mucin secretion has not been determined. The present study has investigated the actions of adenosine A1 and A2 receptor antagonists to determine whether ability to stimulate mucin secretion correlates with correction of CFTR antibody inhibited β‐adrenergic response and whether excessive cyclic AMP rise is required.

CFTR antibodies were introduced into living rat submandibular acini by hypotonic swelling. Following recovery, mucin secretion in response to isoproterenol was measured.

The adenosine A1 receptor antagonist, 8 cyclopentyltheophylline (CPT) was a less potent stimulator of mucin secretion than was the A2 receptor antagonist dimethylpropargylxanthine (DMPX). A concentration of CPT close to the Ki for A1 receptor antagonism (10 nM) did not stimulate mucin secretion.

DMPX, although a potent stimulator of mucin secretion, did not correct CFTR antibody inhibited mucin secretion.

CPT corrected defective CFTR antibody inhibited mucin secretion at a high (1 mM) concentration, suggesting a mechanism other than adenosine receptor antagonism.

DMPX potentiated the isoproterenol induced cyclic AMP rise, whereas CPT did not.

Correction of the defective CFTR mucin secretion response did not correlate with ability to stimulate mucin secretion and did not require potentiation of β‐adrenergic induced increases in cyclic AMP. This affords real promise for the development of a selective drug treatment for cystic fibrosis.

British Journal of Pharmacology (1998) 125, 697–704; doi:10.1038/sj.bjp.0702128
Nicotine administration stimulates the in vivo N‐methyl‐D‐aspartate receptor/nitric oxide/cyclic GMP pathway in rat hippocampus through glutamate release
Authors: Fedele Ernesto , Varnier Giorgia , Antonia Ansaldo Maria , Raiteri Maurizio
Published: January 2005

The in vivo effects of nicotine on the nitric oxide (NO) synthase/cyclic GMP pathway of the adult rat hippocampus have been investigated by monitoring the levels of extracellular cyclic GMP during microdialysis in conscious unrestrained animals.

Intraperitoneal (i.p.) administration of nicotine caused elevation of cyclic GMP levels which was prevented by mecamylamine. The effect of nicotine was abolished by local infusion of the NO synthase inhibitor NG‐nitro‐L‐arginine (L‐NOARG) or by the soluble guanylyl cyclase blocker 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxaline‐1‐one (ODQ).

Local administration of the NMDA receptor antagonists cis‐4‐(phosphonomethyl)‐2‐piperidinecarboxylic acid (CGS19755) and dizocilpine (MK‐801) inhibited by about 60% the nicotine‐induced elevation of cyclic GMP. Nicotine was able to stimulate cyclic GMP outflow also when administered directly into the hippocampus; the effect was sensitive to mecamylamine, L‐NOARG, ODQ or MK‐801.

Nicotine, either administered i.p. or infused locally, produced augmentation of glutamate and aspartate extracellular levels, whereas the outflows of γ‐aminobutyric acid (GABA) and glycine remained unaffected. Following local administration of high concentrations of nicotine, animals displayed symptoms of mild excitation (sniffing, increased motor and exploratory activity) during the first 20–40 min of infusion, followed by wet dog shake episodes; these behavioural effects were prevented by mecamylamine or MK‐801, but not by L‐NOARG or by ODQ.

It is concluded that (a) nicotine stimulates the production of NO and cyclic GMP in the hippocampus; (b) this occurs, at least in part, through release of glutamate/aspartate and activation of NMDA receptors. Modulation of the NMDA receptor/NO synthase/cyclic GMP pathway may be involved in the cognitive activities of nicotine.

British Journal of Pharmacology (1998) 125, 1042–1048; doi:10.1038/sj.bjp.0702130
Effects of nitric oxide on proliferation and differentiation of rat brown adipocytes in primary cultures
Authors: Nisoli Enzo , Clementi Emilio , Tonello Cristina , Sciorati Clara , Briscini Luca , Carruba Michele O
Published: January 2005

In the present work, we study the effect of NO on the proliferation and differentiation of brown fat cells in primary cultures.

Brown fat precursor cells isolated from rat brown adipose tissue were cultured for 8 days until confluence and treated daily with the NO donating agents, S‐nitroso‐acetyl penicillamine (SNAP) or S‐nitroso‐L‐glutathione (GSNO). Both agents (300 μM) decreased cell proliferation approximately 8 fold on day 8. The inhibitory effect of NO was unlikely to be due to cytotoxicity since (i) cells never completely lost their proliferation capacity even after 8 days of exposure to repeated additions of SNAP or GSNO, and (ii) the inhibitory effect was reversible after removal of the media containing NO donors.

Daily treatment with nitric oxide synthase inhibitors, such as NG‐nitro‐L‐arginine methyl ester (L‐NAME, 300 μM), led to the stimulation of cell proliferation by 44±5%, n=3, suggesting that NO, endogenously produced in brown adipocytes, may be involved in modulating cell growth.

Daily treatment with both SNAP or GSNO induced significant mitochondriogenesis, measured as the mitochondrial conversion of 3‐[4,5‐dimethylthiazol‐2‐yl‐]‐2,5‐diphenyl tetrazolium bromide (MTT) to formazan, whilst daily treatment with L‐NAME was without effect.

The inhibition of cell proliferation by NO donors was accompanied by the expression of two genes coding for peroxisome proliferator activated receptor‐γ and uncoupling protein‐1, which are upregulated during differentiation.

Increasing cyclic GMP in cells by 8‐bromo‐cyclic GMP (100–1000 μM) did not reproduce the observed NO effects on either cell number or gene expression. On the other hand, chronic treatment with the inhibitor of the NO‐stimulated guanylyl cyclase, 1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one (ODQ), reduced the expression of peroxisome proliferator activated receptor‐γ and uncoupling protein‐1.

British Journal of Pharmacology (1998) 125, 888–894; doi:10.1038/sj.bjp.0702131
A partial involvement of histamine in ultrasonically nebulized distilled water‐induced bronchoconstriction in guinea‐pigs
Authors: Fujimura Masaki , Amemiya Tokunao , Myou Shigeharu , Ishiura Yoshihisa , Sasaki Shigeki , Matsuda Tamotsu
Published: January 2005

Inhalation of ultrasonically nebulized distilled water (UNDW) can induce bronchoconstriction only in asthmatics, but mechanisms of the response are not well known. We recently reported a guinea‐pig model of UNDW‐induced bronchoconstriction (UNDW‐IB) in which UNDW induces bronchoconstriction when UNDW is inhaled 20 min after a challenge with aerosolized ovalbumin (OA) in passively sensitized, anaesthetized and artificially ventilated guinea‐pigs.

To elucidate the role of histamine in the UNDW‐IB, we examined the effects of antihistamines, diphenhydramine hydrochloride (DH) and chlorpheniramine maleate (CP), and measured histamine content in bronchoalveolar lavage fluid (BALF) in the animal model.

DH in doses of 0.1, 1.0 and 10 mg kg−1 and CP in doses of 0.01, 0.1 and 1.0 mg kg−1 administered intravenously 15 min after the OA challenge partially reduced the UNDW‐IB at 1 and 2 min after the UNDW inhalation in a dose‐dependent manner. Histamine content in BALF recovered 10 min after the UNDW inhalation following the OA provocation was significantly increased compared with that after saline inhalation and before the UNDW inhalation following the OA challenge.

Intravenous atropine in a dose of 0.5 mg kg−1 or inhaled disodium cromoglycate in concentrations of 1 and 10 mg ml−1 did not alter the UNDW‐IB.

These results suggest that histamine is involved in part in the UNDW‐IB in our animal model.

British Journal of Pharmacology (1998) 125, 902–908; doi:10.1038/sj.bjp.0702135
Contributions of cysteine 114 of the human D3 dopamine receptor to ligand binding and sensitivity to external oxidizing agents
Authors: Alberts Glen L , Pregenzer Jeffrey F , Bin Im Wha
Published: January 2005

Cysteine 114 (C114) of the human dopamine D3 receptor is located at the helical face of transmembrane segment III (TMIII) near aspartate 110, a counterion for the amine group of catecholamines. The contributions of C114 to receptor function were investigated here using site‐directed mutagenetis of C114 to serine.

The C114S mutant, as expressed in Sf‐9 cells, bound aminotetralin antagonists (UH‐232 and AJ‐76) and several agonists ((−)3‐PPP, apomorphine, pramipexole and quinpirole) with markedly lower affinities as compared to the wild type D3 receptor, but bound other structurally diverse dopaminergic ligands with only minor changes in affinity. Because an N‐propyl substituent is the only common structural feature among most affected ligands, we propose that the mutation alters ‘a propyl cleft’ on the receptor. The mutation hardly affected quinpirole‐dependent [35S]‐GTPγS binding, suggesting C114 plays a minimal role in receptor‐G‐protein coupling.

N‐Ethylmaleimide(NEM), a sulfhydryl modifying agent, blocked ligand binding to the D3 receptor, but not to the C114S mutant. We infer that C114 is the primary residue on the D3 receptor vulnerable to external oxidizing agents. Dopamine D2long and D42 receptors contain highly homologous TMIII sequences including an equivalent cysteine residue. However, only the D2long receptor, not the D42 receptor, displayed NEM sensitivity similar to that of the D3 receptor.

We conclude that C114 is critical for high affinity interactions between the D3 receptor and ligands containing an N‐propyl substituent, and unlike its counterpart in the D42 receptor, is highly susceptible to external oxidizing agents.

British Journal of Pharmacology (1998) 125, 705–710; doi:10.1038/sj.bjp.0702136
Lipopolysaccharide enhances neurogenic plasma exudation in guinea‐pig airways
Authors: Kuo H ‐P , Hwang K ‐H , Lin H ‐C , Wang C ‐H , Liu C ‐Y , Lu L ‐C
Published: January 2005

Lipopolysaccharide (LPS) is implicated in many pulmonary and airway inflammatory diseases. Tachykinins released from nerve endings increase vascular permeability. In this study, we have assessed the enhancement by LPS of tachykinin‐mediated plasma exudation in guinea‐pig airways, and examined the role of oxidants as well as leukocyte adherence.

LPS (100 μg kg−1, i.v.) was administered 0–3 h before bilateral electrical stimulation of the cervical vagus nerves in animals anaesthetized with urethane and ventilated. Vagal stimulation increased vascular permeability in the airways. LPS enhanced the vagally‐mediated plasma exudation with the peak effect at 1 h after LPS administration. LPS alone induced no significant plasma exudation. LPS also enhanced exogenous substance P (10−8 mol kg−1, i.v.)‐induced plasma exudation.

The NK‐1 receptor antagonist L‐732,138 abolished vagally‐induced plasma exudation and significantly inhibited the enhancement by LPS. Pretreatment with superoxide dismutase (SOD, 5000 U kg−1, i.p.) did not affect the vagally‐induced plasma exudation, but inhibited the LPS‐enhanced neurogenic plasma leakage. The LPS‐enhanced vagally‐induced plasma exudation was not completely inhibited by either L‐732,138 or SOD pretreatment alone, but was blocked by the combination of both pretreatments.

Neutrophil depletion by cyclophosphamide alone did not influence vagally‐induced plasma exudation, but significantly inhibited the LPS‐enhanced response.

In conclusion, we have demonstrated LPS enhanced neurogenic plasma exudation by augmenting the response to tachykinins, partly through NK‐1 receptors, to directly increase vascular permeability or to enhance leukocyte adhesion‐mediated endothelial cell injury. Tachykinins released from nerve endings may contribute to endotoxin‐related airway inflammatory responses.

British Journal of Pharmacology (1998) 125, 711–716; doi:10.1038/sj.bjp.0702138
Effect of endothelin‐1 (1‐31) on extracellular signal‐regulated kinase and proliferation of human coronary artery smooth muscle cells
Authors: Yoshizumi Masanori , Kim Shokei , Kagami Shoji , Hamaguchi Akinori , Tsuchiya Koichiro , Houchi Hitoshi , Iwao Hiroshi , Kido Hiroshi , Tamaki Toshiaki
Published: January 2005

We have previously found that human chymase cleaves big endothelins (ETs) at the Tyr31‐Gly32 bond and produces 31‐amino acid ETs (1‐31), without any further degradation products. In this study, we investigated the effect of synthetic ET‐1 (1‐31) on the proliferation of cultured human coronary artery smooth muscle cells (HCASMCs).

ET‐1 (1‐31) increased [3H]‐thymidine incorporation and cell numbers to a similar extent as ET‐1 at 100 nM. This ET‐1 (1‐31)‐induced [3H]‐thymidine uptake was not affected by phosphoramidon, an inhibitor of ET‐converting enzyme. It was, however, inhibited by BQ123, an endothelin ETA receptor antagonist, but not by BQ788, an endothelin ETB receptor antagonist.

By using an in‐gel kinase assay, we demonstrated that ET‐1 (1‐31) activated extracellular signal‐regulated kinase 1/2 (ERK1/2) in a concentration‐dependent manner (100 pM to 1 μM) in HCASMCs. ET‐1 (1‐31)‐induced ERK1/2 activation was inhibited by BQ123, but not by BQ788 and phosphoramidon. Inhibition of protein kinase C (PKC) and ERK kinase also caused a reduction of ET‐1 (1‐31)‐induced ERK1/2 activation, whereas tyrosine kinase inhibition had little effect.

Gel‐mobility shift analysis revealed that the ERK1/2 activation was followed by an increase in transcription factor activator protein‐1 DNA binding activity in HCASMCs.

Our results strongly suggest that ET‐1 (1‐31) itself stimulates HCASMC proliferation probably through endothelin ETA or ETA‐like receptors. The underlining mechanism of cell growth by ET‐1 (1‐31) may be explained in part by PKC‐dependent ERK1/2 activation. Since human chymase has been proposed to play a role in atherosclerosis, ET‐1 (1‐31) may be one of the mediators.

British Journal of Pharmacology (1998) 125, 1019–1027; doi:10.1038/sj.bjp.0702141
Comparative desensitization of the human 5‐HT2A and 5‐HT2C receptors expressed in the human neuroblastoma cell line SH‐SY5Y
Authors: Briddon S.J. , Leslie R.A. , Elliott J.M.
Published: January 2005

We have used previously characterized clones of the human neuroblastoma cell line, SH‐SY5Y, constitutively expressing either the human 5‐HT2A or 5‐HT2C receptor to compare their desensitization profiles after exposure to 5‐HT.

5‐HT stimulated [3H]‐inositol phosphate ([3H]‐IPx) production at both the 5‐HT2C (pEC50=8.03±0.15) and 5‐HT2A receptors (pEC50=7.15±0.08), with maximal responses occurring after exposure to 1 μM and 10 μM 5‐HT, respectively.

Exposure of cells to maximally effective concentrations of 5‐HT caused time‐ and concentration‐dependent desensitization of [3H]‐IPx formation. The 5‐HT2A response desensitized slower (t1/2=110 min) and with lower sensitivity than that of the 5‐HT2C receptor (t1/2=12.5 min). In each case, desensitization was blocked by co‐administration of a specific receptor antagonist. Following exposure to 10 μM 5‐HT for 2 h, both receptors exhibited extensive desensitization, with subsequent responses to 5‐HT reduced by more than 80%.

5‐HT stimulated Ins(1,4,5)P3 production with a potency similar to that for [3H]‐IPx production at each receptor. In both cases Ins(1,4,5)P3 levels peaked rapidly then returned to basal level within a short time. This peak consistently occurred earlier for the 5‐HT2C receptor (5 s) than for the 5‐HT2A receptor (20 s).

Prior exposure of SH‐SY5Y/5‐HT2C cells to 5‐HT (1 μM/15 min) caused a significant decrease in the 5‐HT‐stimulated peak in Ins(1,4,5)P3 levels whereas no such change occurred in SH‐SY5Y/5‐HT2A cells following exposure to 10 μM 5‐HT for 15 min.

These results indicate that the human 5‐HT2A and 5‐HT2C receptors both exhibit desensitization at the level of inositol phosphate formation when expressed in the same cellular environment, with the 5‐HT2C receptor being more sensitive to 5‐HT‐mediated desensitization than the 5‐HT2A receptor.

British Journal of Pharmacology (1998) 125, 727–734; doi:10.1038/sj.bjp.0702145
Characterization of receptors mediating contraction of the rat isolated small mesenteric artery and aorta to arginine vasopressin and oxytocin
Authors: Stam Wiro B , Van der Graaf Pieter H , Saxena Pramod R
Published: January 2005

The exact nature of the receptor subtype(s) involved in the action of arg‐vasopressin (AVP) on the rat aorta and small mesenteric artery (SMA) is controversial. Therefore, we have studied the effects of the selective V1A receptor antagonists, OPC 21268 and SR 49059, and the oxytocin (OT) receptor antagonist, atosiban, on the AVP‐ and OT‐induced contractions of the two vessels.

AVP and OT displayed similar intrinsic activities in the rat aorta and SMA, but AVP was ∼130 fold and ∼500 fold more potent than OT, respectively. In the rat aorta, Hill slopes (nH) were similar for OT and AVP. However, in rat SMA, the OT concentration‐effect (E/[A]) curve was significantly steeper than the AVP E/[A] curve (nH,=3.3±0.20, 2.3±0.15; P<0.001).

In the aorta OPC 21268, SR 49059 and atosiban competitively antagonized the AVP and OT E/[A] curves. Except for atosiban and SR 49059 against AVP, competitive antagonism was also observed in the SMA. Atosiban caused concentration‐dependent steepening of the AVP E/[A] curve, whereas SR 49059 decreased the upper asymptote.

Schild analysis yielded affinities indicative of V1A receptor involvement in both vessels: pKB/pA2=9.20–9.48, 7.56–7.71 and 6.19–6.48 for SR 49059, OPC 21268 and atosiban, respectively.

Neither AVP nor OT relaxed U46619 pre‐contracted aorta or SMA in the presence of SR 49059, suggesting no interference of a vasodilatory component.

Despite predominant involvement of V1A receptors in both vessels, the different Hill slopes of AVP and OT E/[A] curves as well as the steepening of the AVP E/[A] curves by atosiban are indicative of receptor heterogeneity in the rat SMA.

British Journal of Pharmacology (1998) 125, 865–873; doi:10.1038/sj.bjp.0702149
Pharmacodynamic interaction between phenytoin and sodium valproate changes seizure thresholds and pattern
Authors: Paschoa O. E. Della , Kruk M. R. , Hamstra R , Voskuyl R. A. , Danhof M
Published: January 2005

In this study we used cortical stimulation to assess the effects of phenytoin (PHT), sodium valproate (VPA), and their interaction on total motor seizure and on the constituent elements of the seizure.

PHT (40 mg kg−1) was administered as an intravenous bolus infusion to animals receiving either a continuous infusion of VPA or saline. VPA plasma concentration was maintained at levels that produced no detectable anticonvulsant effect.

Analysis of ictal components (eyes closure, jerk, gasp, forelimb, clonus, and hindlimb tonus) and their durations revealed both qualitative and quantitative differences in drug effects.

The anticonvulsant effect is represented by the increase in the duration of the stimulation required to reach a given seizure threshold. PHT significantly increased the duration of the stimulation and of the motor seizure. This increase was greatly enhanced by VPA. In addition, ictal component analysis revealed that the combination of PHT and VPA causes the reduction of a specific seizure component (JERK).

Neither the free fraction of PHT nor the biophase equilibration kinetics changes in the presence of VPA. It is concluded that the synergism may be due to a pharmacodynamic rather than a pharmacokinetic interaction.

British Journal of Pharmacology (1998) 125, 997–1004; doi:10.1038/sj.bjp.0702155
The action of calcium channel blockers on recombinant L‐type calcium channel α1‐subunits
Authors: Morel Nicole , Buryi Vitali , Feron Olivier , Gomez Jean‐Pierre , Christen Marie‐Odile , Godfraind Théophile
Published: January 2005

CHO cells expressing the α1C‐a subunit (cardiac isoform) and the α1C‐b subunit (vascular isoform) of the voltage‐dependent L‐type Ca2+ channel were used to investigate whether tissue selectivity of Ca2+ channel blockers could be related to different affinities for α1C isoforms.

Inward current evoked by the transfected α1 subunit was recorded by the patch‐clamp technique in the whole‐cell configuration.

Neutral dihydropyridines (nifedipine, nisoldipine, (+)‐PN200‐110) were more potent inhibitors of α1C‐b‐subunit than of α1C‐a‐subunit. This difference was more marked at a holding potential of −100 mV than at −50 mV. SDZ 207‐180 (an ionized dihydropyridine) exhibited the same potency on the two isoforms.

Pinaverium (ionized non‐dihydropyridine derivative) was 2 and 4 fold more potent on α1C‐a than on α1C‐b subunit at Vh of −100 mV and −50 mV, respectively. Effects of verapamil were identical on the two isoforms at both voltages.

[3H]‐(+)‐PN 200‐110 binding experiments showed that neutral dihydropyridines had a higher affinity for the α1C‐b than for the α1C‐a subunit. SDZ 207‐180 had the same affinity for the two isoforms and pinaverium had a higher affinity for the α1C‐a subunit than for the α1C‐b subunit.

These results indicate marked differences among Ca2+ channel blockers in their selectivity for the α1C‐a and α1C‐b subunits of the Ca2+ channel.

British Journal of Pharmacology (1998) 125, 1005–1012; doi:10.1038/sj.bjp.0702162
Effects of chronic application of propranolol on β‐adrenergic signal transduction in heart ventricles from myopathic BIO TO2 and control hamsters
Authors: Witte Klaus , Schnecko Anke , Hauth Daniela , Wirzius Sandra , Lemmer Björn
Published: January 2005

In human congestive heart failure β‐adrenoceptor antagonists improve exercise tolerance and cardiac contractility. These beneficial effects are thought to reflect an up‐regulation of cardiac β‐adrenoceptors, involving mainly the β1‐subtype. In the present study we evaluated the functional contribution of β‐adrenoceptor subtypes to stimulation of adenylyl cyclase in an animal model of dilated cardiomyopathy, and compared the effects of treatment with propranolol on cardiac β‐adrenergic signal transduction in myopathic and control hamsters.

Cardiomyopathic BIO TO2 hamsters and BIO F1B controls aged 270 days were used. In the treatment study, hamsters received drinking water with or without propranolol 40 mg kg−1 d−1 for 4 weeks prior to sacrifice. Density and subtype distribution of β‐adrenoceptors were determined in radioligand binding studies. Functional contributions of β‐adrenoceptors were evaluated by subtype‐selective stimulation of adenylyl cyclase. Cardiac G‐protein content was determined by immunoblotting.

Compared to BIO F1B controls, myopathic hamsters showed increases in cardiac total β‐ and β2‐adrenoceptor density, Gsα and Giα content. In BIO TO2 ventricles, β1‐adrenoceptors were almost completely uncoupled from adenylyl cyclase stimulation despite an unchanged density. Treatment of hamsters with propranolol resulted in increased density of β1‐adrenoceptors in both strains, but had no effect on their functional efficacy. Moreover, β2‐adrenergic stimulation of adenylyl cyclase was even reduced in propranolol‐treated animals, which could not be explained by changes in cardiac G‐protein content.

Cardiomyopathic BIO TO2 hamsters showed functional uncoupling of cardiac β1‐adrenoceptors, which could not be normalized by propranolol and, therefore, is unlikely to be solely due to agonist‐dependent desensitization. The paradoxical reduction in β2‐adrenergic efficiency in propranolol‐treated myopathic and control hamsters deserves further investigation.

British Journal of Pharmacology (1998) 125, 1033–1041; doi:10.1038/sj.bjp.0702165
Characterization of protein serine/threonine phosphatase activities in human lung mast cells and basophils
Authors: Peirce Matthew J. , Munday Michael R. , Peachell Peter T.
Published: January 2005

The serine/threonine protein phosphatase (PP) inhibitors, okadaic acid and calyculin, attenuated the IgE‐mediated release of histamine from human lung mast cells (HLMC) and basophils in a dose‐dependent manner whereas an alternative PP inhibitor, microcystin, was ineffective. Calyculin was more potent than okadaic acid in both cell types. The concentration required to inhibit by 50% (IC50) the release of histamine was 15 (HLMC) and 50 nM(basophils) for calyculin and 200 (HLMC) and 300 nM(basophils) for okadaic acid.

Lysates of purified HLMC and basophils dephosphorylated radiolabelled glycogen phosphorylase, a substrate for both PP1 and PP2A. The PP activity in lysates of both cell types was inhibited in a dose‐dependent fashion by the PP inhibitors with the following rank order of activity, calyculin (approximate IC50; 0.02–0.1 nM)microcystin (0.1 nM)>okadaic acid (70 nM).

The PP1‐selective inhibitor, inhibitor‐2 (I‐2), attenuated the dephosphorylation of glycogen phosphorylase in lysates of both HLMC and basophils. I‐2 (20 nM) inhibited the glycogen phosphorylase PP activity by 71±3% and 49±13% in HLMC and basophil extracts, respectively. There were, approximately, 6 fold greater levels of I‐2‐sensitive activity in HLMC than in basophils. Qualitatively similar results were obtained with an alternative PP1‐selective inhibitor, inhibitor‐1 (I‐1).

Lysates derived from HLMC and basophils dephosphorylated radiolabelled casein which is a PP2A‐restricted substrate. HLMC lysates contained, approximately, 2.5 fold higher levels of casein PP activity than basophil lysates.

These data indicate that HLMC and basophils both contain PP1 and PP2A. The data suggest that, on a per cell basis, HLMC have higher levels of both PP1 and PP2A. Moreover, the ratio of PP1 to PP2A is higher in HLMC than in basophils.

British Journal of Pharmacology(1998)125, 1095–1101; doi:10.1038/sj.bjp.0702169
LU 73068, a new non‐NMDA and glycine/NMDA receptor antagonist: pharmacological characterization and comparison with NBQX and L‐701,324 in the kindling model of epilepsy
Authors: Potschka H , Löscher W , Wlaź P , Behl B , Hofmann H P , Treiber H‐J , Szabo L
Published: January 2005

The aim of this study was to assess whether a drug which combines an antagonistic action at both NMDA and non‐NMDA receptors offers advantages for treatment of epileptic seizures compared to drugs which antagonize only one of these ionotropic glutamate receptors.

The novel glutamate receptor antagonist LU 73068 (4,5‐dihydro‐1‐methyl‐4‐oxo‐7‐trifluoromethyl‐imidazo[1,2a]quinoxaline‐2‐carbonic acid) binds with high affinity to both the glycine site of the NMDA receptor (Ki 185 nm) and to the AMPA receptor (Ki 158 nm). Furthermore, binding experiments with recombinant kainate receptor subunits showed that LU 73068 binds to several of these subunits, particularly to rGluR7 (Ki 104 nm) and rGluR5 (Ki 271 nm). In comparison, the prototype non‐NMDA receptor antagonist NBQX (2,3‐dihydroxy‐6‐nitro‐7‐sulphamoyl‐benzo[f]quinoxaline) binds with high affinity to AMPA receptors only.

Both NBQX and LU 73068 were about equieffective after i.p. injection in mice to block lethal convulsions induced by AMPA or NMDA.

In the rat amygdala kindling model of temporal lobe epilepsy, LU 73068 dose‐dependently increased the focal seizure threshold (afterdischarge threshold, ADT). When rats were stimulated with a current 20% above the individual control ADT, LU 73068 completely blocked seizures with an ED50 of 4.9 mg kg−1.

Up to 20 mg kg−1, only moderate adverse effects, e.g. slight ataxia, were observed.

NBQX, 10 mg kg−1, and the glycine/NMDA site antagonist L‐701,324 (7‐chloro‐4‐hydroxy‐3‐(3‐phenoxy)phenyl‐quinoline‐2(1H)one), 2.5 or 5 mg kg−1, exerted no anticonvulsant effects in kindled rats when administered alone, but combined treatment with both drugs resulted in a significant ADT increase.

The data indicate that combination of glycine/NMDA and non‐NMDA receptor antagonism in a single drug is an effective means of developing a potent and effective anticonvulsant agent.

British Journal of Pharmacology (1998) 125, 1258–1266; doi:10.1038/sj.bjp.0702172
Characterization of the endothelin receptor subtype mediating epithelium‐derived relaxant nitric oxide release from guinea‐pig trachea
Authors: Emanueli Costanza , Ricciardolo Fabio , Vergnani Luciana , Bertrand Claude , Ricci Franco , Manzoli Nadia , Folkerts Gert , Nijkamp Frans P. , Geppetti Pierangelo
Published: January 2005

The endothelin (ET) receptor subtype that mediates niric oxide (NO)‐dependent airway relaxation in tracheal tube preparations precontracted with carbachol and pretreated with indomethacin was investigated. The release of NO induced by ET from guinea‐pig trachea using a recently developed porphyrinic microsensor was also measured.

ET‐1 (1 pM–100 nM) contracted tracheal tube preparations pretreated with the NO‐synthase inhibitor, L‐NMMA, and relaxed, in an epithelium‐dependent manner, preparations pretreated with the inactive enantiomer D‐NMMA. The effect of L‐NMMA was reversed by L‐Arg, but not by D‐Arg.

The selective ETB receptor agonists, IRL 1620 or sarafotoxin S6c, both (1 pM–100 nM) contracted tracheal tube preparations in a similar manner either after treatment with D‐NMMA or with L‐NMMA. In the presence of the ETA receptor antagonist, FR139317 (10 μM), ET‐1 administration resulted in a contraction that was similar after either L‐NMMA or D‐NMMA. In the presence of the ETB receptor antagonist, BQ788 (1 μM), ET‐1 relaxed and contracted tracheas pretreated with D‐NMMA and L‐NMMA, respectively.

Exposure of tracheal segments to ET‐1 (1–1000 nM) caused a concentration‐dependent increase in NO release that was reduced by L‐NMMA. IRL1620 (1 μM) did not cause any significant NO release. FR139317 (10 μM), but not, BQ788 (1 μM), inhibited the NO release induced by ET‐1.

These results demonstrate that in the isolated guinea‐pig trachea activation of ETB receptors results in a contractile response, whereas activation of ETA receptors cause both a contraction, and an epithelium‐dependent relaxation that is mediated by NO release.

British Journal of Pharmacology (1998) 125, 963–968; doi:10.1038/sj.bjp.0702174
Venous versus arterial actions of diethylamine/nitric oxide (DEA/NO) complex and S‐nitroso‐N‐acetylpenicillamine (SNAP) in vivo
Authors: Ng Sylvia S W , Pang Catherine C Y
Published: January 2005

We studied the effects of diethylamine/NO complex (DEA/NO) and S‐nitroso‐N‐acetylpenicillamine (SNAP), relative to those of sodium nitroprusside (SNP) and nitroglycerin (NTG), on mean arterial pressure (MAP), mean circulatory filling pressure (MCFP), arterial resistance (Ra), venous resistance (Rv), heart rate (HR), cardiac output (CO) and stroke volume (SV) in groups of Inactin‐anaesthetized rats pre‐treated with i.v. mecamylamine (3.7 μmol kg−1) and noradrenaline (6.8 nmol kg−1 min−1). Doses of each that reduced MAP by 30%, 80% and the lowest dose that maximally reduced MAP were examined to allow a comparison of the compounds' dilator actions at equivalent effective depressor doses.

DEA/NO (4, 32 and 256 μg kg−1 min−1), SNAP (4, 32 and 256 μg kg−1 min−1) and SNP (8, 32 and 128 μg kg−1 min−1) caused similar dose‐dependent reductions in MAP and Ra, and increases in CO and SV. NTG (0.2, 0.8 and 6.4 μg kg−1 min−1) dose‐dependently reduced Ra, and increased CO and SV, but lowered MAP only at the highest dose.

DEA/NO, SNAP and SNP but not NTG lowered MCFP with efficacy: DEA/NO>SNAP>SNP. All four drugs reduced Rv with efficacy: DEA/NO ≈amp; SNAP>SNP ≈amp; NTG.

Therefore, all compounds lowered Ra and Rv. DEA/NO, SNAP and SNP but not NTG reduced MCFP. The pharmacological profiles of DEA/NO and SNAP resemble SNP more than NTG.

British Journal of Pharmacology (1998) 125, 1247–1251; doi:10.1038/sj.bjp.0702175
Learning impairments induced by glutamate blockade using dizocilpine (MK‐801) in monkeys
Authors: Harder J. A. , Aboobaker A. A. , Hodgetts T. C. , Ridley R. M.
Published: January 2005

This study investigated the effects of dizocilpine (MK‐801) on learning ability in a non‐human primate. Acquisition and reversal learning of visual discrimination tasks and acquisition of visuo‐spatial discrimination tasks were assessed in marmosets using the Wisconsin General Test Apparatus. Dizocilpine impaired acquisition of visuo‐spatial (conditional) tasks requiring spatial responses to coloured objects, and perceptually difficult visual discrimination tasks in which stimulus objects are painted black. Dizocilpine did not, however, impair either acquisition or reversal of a simple visual discrimination task using easily discriminated, coloured objects.

Motor effects of dizocilpine treatment, which have been seen in other primates, were examined by observation of the marmosets in their home cages, using both an automated locomotor activity monitor and ‘blind’, subjective counting of the number of abnormal movements in a given time period. Locomotor activity, assessed using the automated monitor, was not significantly affected at any of the doses tested. Incoordination, assessed by human observation of abnormal movements, was significantly increased only at a dose of 30 μg kg−1 i.m., which was twice the highest dose used to assess the effects of dizocilpine on cognition.

We have, therefore, found an effect of dizocilpine on acquisition and reversal of some types of cognitive task, at a dose which does not cause significant motor effects. This demonstration of a cognitive deficit associated with glutamatergic blockade in a primate may be useful in understanding the contribution of glutamatergic dysfunction to cognitive decline in neurodegenerative disease, especially Alzheimer's disease.

British Journal of Pharmacology (1998) 125, 1013–1018; doi:10.1038/sj.bjp.0702178
Characterization of the relaxant action of urocortin, a new peptide related to corticotropin‐releasing factor in the rat isolated basilar artery
Authors: Schilling L , Kanzler Ch , Schmiedek P , Ehrenreich H
Published: January 2005

In addition to its well established neuroendocrine and neurotransmitter effects, corticotropin releasing factor (CRF) exerts a potent vasorelaxant action. Recently, a CRF‐related peptide, urocortin, has been identified in the mammalian brain. In the present study, the cerebral vasomotor action of this peptide and the mechanism underlying its relaxant effect are characterized.

Ring segments obtained from the rat basilar artery were used for measurement of isometric force. The relaxant action of urocortin, CRF and sauvagine was studied in segments with a functionally intact endothelium.

In segments precontracted with prostaglandin F2α, urocortin, CRF and sauvagine induced concentration‐related relaxation. The order of potency was as follows (pD2±s.e.m. given in brackets): urocortin (9.32±0.07) > sauvagine (9.08±0.08) > CRF (7.50±0.07). Complete relaxation was achieved with each agonist. Relaxation was not affected by removal of the endothelium but was markedly attenuated in segments precontracted with 50 mm K+ Krebs solution. The relaxant effect of urocortin was inhibited by astressin in an apparently competitive manner. A pA2 value of 7.52 was estimated for astressin. Inhibition of urocortin‐induced relaxation was also observed in the presence of the adenylate cyclase inhibitor SQ22536 (pD2 in the presence of 300 μm SQ22536, 9.36±0.05) and the K+ channel blockers tetraethylammonium (10 mm; pD2, 8.65±0.07), iberiotoxin (100 nm; pD2, 8.88±0.08) and apamin (10 nm; pD2, 8.94±0.07). However, the inhibitory actions of SQ22536 and apamin or iberiotoxin were not additive.

The results suggest that urocortin induces relaxation of cerebral arteries by activating CRF‐R2 receptors present in the vascular wall. Relaxation appears to be mediated by adenylate cyclase stimulation and activation of Ca2+‐dependent K+ channels.

British Journal of Pharmacology (1998) 125, 1164–1171; doi:10.1038/sj.bjp.0702182
Analysis of an H1 receptor‐mediated, zinc‐potentiated vasoconstrictor action of the histidyl dipeptide carnosine in rabbit saphenous vein
Authors: O'Dowd Anne , Miller David J
Published: January 2005

The contractile action of the dipeptide carnosine (β‐alanyl‐l‐histidine), active as a Zn·carnosine complex (Zn·Carn), was investigated in isolated rings of rabbit saphenous vein (RSV) and was found to be antagonized by the H1 antagonist mepyramine.

Mepyramine‐sensitive, histamine‐induced contractures in RSV, were smaller (73±0.1%) and less well sustained than carnosine‐induced contractures.

Schild plot values for mepyramine antagonism were, for carnosine‐induced contractures; pA2=7.97±0.12, slope=1.33±0.06 (r = 0.793) and for histamine‐induced contractures; pA2=8.48±0.07, slope=0.63±0.05, r = 0.957).

Serotonergic antagonists methiothepin and ketanserin, antagonize both carnosine‐ and histamine‐induced contractures in RSV, probably reflecting coincidental inhibition at the H1‐receptor.

Carnosine, with Zn present, can inhibit the H1‐specific binding of [3H]‐mepyramine to isolated guinea‐pig cerebellar membranes (log IC50s−2.78±0.02, −3.93±0.03 and −4.64±0.03 at 10, 30 and 80 μm Zn respectively; values corrected for the Zn‐specific inhibition which has a logIC50 of −4.20).

In the radioligand binding assay, the effect of carnosine can be described as a function of Zn·Carn concentration with an apparent logIC50 of −5.61. This value is consistent with that obtained from the functional studies on RSV.

Histamine‐induced contractures have an indomethacine‐sensitive component (27.2±8.3% of control response), not apparent with carnosine‐induced contractures.

Like histamine, carnosine evoked an H2‐mediated (cimetidine‐sensitive) relaxation in the presence of mepyramine, but was less potent (10.8±3.1% residual tension at 10 mm carnosine compared with 13.4±7.5% at 0.1 mm histamine).

Carnosine, like mepyramine, can ‘reveal’ the H2‐mediated relaxation of histamine providing further evidence that carnosine binds at the H1 receptor.

We conclude that carnosine can act at the smooth muscle H1‐receptor to provoke vasoconstriction and that it also has the potential to act at H1‐receptors in CNS.

British Journal of Pharmacology (1998) 125, 1272–1280; doi:10.1038/sj.bjp.0702184
Evidence that platelets promote tube formation by endothelial cells on matrigel
Authors: Pipili‐Synetos E , Papadimitriou E , Maragoudakis M E
Published: January 2005

The involvement of platelets in neovascularization was investigated in the matrigel tube formation assay, an in vitro model of angiogenesis.

Platelets promoted the formation of capillary‐like structures (expressed as relative tube area) number‐ and time‐dependently. Relative tube area increased from 0.98±0.02 (n = 8) in the presence of 6.25×104, to 3.21±0.12 (n = 8) in the presence of 106 platelets/well compared to 0.54±0.04 (n = 8) in their absence. This increase was unaffected by acetyl salicylic acid (ASA), apyrase, and hirudin. Photographs from representative experiments, showed that platelets adhered along the differentiating endothelium.

Addition of α‐thrombin (0.1–1 i.u. ml−1), the nitric oxide (NO) donor sodium nitroprusside (SNP; 1–100 μm) or the NO synthase inhibitor, l‐NG‐arginine‐methylester (l‐NAME, 30–300 μm) to the assay, had no effect on tube formation compared to that seen with platelets alone.

Neuraminidase (0.01 i.u./107 platelets), which strips sialic acid residues from membrane glycoproteins, abolished the promoting effect of platelets on tube formation. The relative tube area in the presence of neuraminidase‐treated platelets was 0.81±0.03 (n = 8), in the presence of untreated platelets 1.69±0.09, P < 0.001 (n = 8) and in the absence of platelets, 0.80±0.04 (n = 8). The tetrapeptide Arg‐Gly‐Asp‐Ser (RGDS; 20–200 μm) which inhibits von Willebrand factor, fibrinogen and fibronectin‐mediated adhesion, had no effect on the promoting effect of platelets on tube formation.

These results indicate that platelets promote angiogenesis in vitro. This effect is largely independent from activation by α‐thrombin, is not modified by manipulating NO and prostaglandin metabolism and proceeds possibly through adhesion of the platelets to the differentiating endothelium.

British Journal of Pharmacology (1998) 125, 1252–1257; doi:10.1038/sj.bjp.0702191
Distribution of the perivascular nerve Ca2+ receptor in rat arteries
Authors: Wang Yanlin , Bukoski Richard D.
Published: January 2005

We recently showed that perivascular sensory nerves of mesenteric branch arteries express a receptor for extracellular Ca2+ (CaR), and reported data indicating that this CaR mediates relaxation induced by physiologic levels of Ca2+. We have now tested whether the perivascular sensory nerve CaR‐linked dilator system is a local phenomenon restricted to the mesentery, or is present in other circulations.

Vessels from the mesenteric, renal, coronary, and cerebral circulations were studied. Immunocytochemical analysis was performed using anti‐CaR and anti‐neural cell adhesion molecule (NCAM) antibodies. Wire myography was used to assess contracation and relaxation.

Although perivascular nerves of all arteries stained for CaR protein, there were regional differences. A morphometric method used to estimate CaR positive nerve density revealed the following rank order: mesenteric branch artery>basilar artery=renal interlobar artery>main renal trunk artery>left anterior descending coronary artery.

Vessels from the mesentery, renal, coronary, and cerebral circulations showed nerve‐dependent relaxation in response to electrical field stimulation (EFS) when precontracted with serotonin in the presence of guanethidine. The degree of Ca2+‐induced relaxation of mesenteric, renal, and cerebral arteries positively correlated with the magnitude of EFS‐induced relaxation. In contrast, coronary arteries contracted at Ca2+ levels between 1.5 and 3 mmol L−1, and relaxed to a small degree to 5 mmol L−1 Ca2+.

Thus, a functional perivascular sensory nerve CaR‐linked dilator system is present to varying degrees in the mesenteric, renal, and cerebral circulations, but only to a very limited extent in the coronary circulation.

British Journal of Pharmacology (1998) 125, 1397–1404; doi:10.1038/sj.bjp.0702195
Contractile responses elicited by hydrogen peroxide in aorta from normotensive and hypertensive rats. Endothelial modulation and mechanism involved
Authors: Rodríguez‐Martínez M Angeles , García‐Cohen Edith C , Baena Ana B , González Rita , Salaíces Mercedes , Marín Jesús
Published: January 2005

The present study analyses the influence of hypertension and endothelium on the effect induced by hydrogen peroxide (H2O2) on basal tone in aortic segments from normotensive Wistar‐Kyoto (WKY) and spontaneously hypertensive rats (SHR) of 6‐month‐old, as well as the possible mechanisms involved.

Single (1 mm) or cumulative (100 nm–10 mm) concentrations of H2O2 produced a transient contraction or a concentration‐dependent increase of basal tone, respectively, in segments from WKY and SHR. In both cases, the contractions were higher in intact segments from hypertensive than from normotensive rats, and increased by endothelium removal in both strains. Catalase (1000 u ml−1, a H2O2 scavenger) abolished the contraction elicited by 1 mm H2O2 in both strains.

Superoxide dismutase (SOD, 150 u ml−1) and dimethylsulphoxide (DMSO, 7 mm), scavengers of superoxide anions and hydroxyl radicals, respectively, did not alter H2O2‐induced contractions in intact segments from both strains. However, l‐NG‐nitroarginine methyl ester (l‐NAME, 100 μm, a nitric oxide synthase inhibitor) increased the response to H2O2 in normotensive rats, although the increase was less than that produced by endothelium removal.

Incubation of segments with 1 mm H2O2 for 15 min and subsequent washout reduced the contractile responses induced by 75 mm KCl in intact segments from SHR and in endothelium‐denuded segments from both strains; this effect being prevented by catalase (1000 u ml−1).

Indomethacin (10 μm, a cyclo‐oxygenase inhibitor) and SQ 29,548 (10 μm, a prostaglandin H2/thromboxane A2 receptor antagonist) practically abolished the contractions elicited by H2O2 in normotensive and hypertensive rats.

We conclude that: (1) the oxidant stress induced by H2O2 produces contractions mediated by generation of a product of the cyclo‐oxygenase pathway, prostaglandin H2 or more probably thromboxane A2, in normotensive and hypertensive rats; (2) oxygen‐derived free radicals are not involved in the effect of H2O2; (3) in normotensive rats, endothelium protects against H2O2‐mediated injury to contractile machinery, determined by the impairment of KCl‐induced contractions; and (4) endothelial nitric oxide has a protective role on the contractile effect induced by H2O2, that is lost in hypertension.

British Journal of Pharmacology (1998) 125, 1329–1335; doi:10.1038/sj.bjp.0702200
Functional effects of the muscarinic receptor agonist, xanomeline, at 5‐HT1 and 5‐HT2 receptors
Authors: Watson J. , Brough S. , Coldwell M. C. , Gager T. , Ho M. , Hunter A. J. , Jerman J. , Middlemiss D. N. , Riley G. J. , Brown A. M.
Published: January 2005

Xanomeline [3(3‐hexyloxy‐1,2,5‐thiadiazol‐4‐yl)‐1,2,5,6‐tetrahydro‐1‐methylpyridine] has been reported to act as a functionally selective muscarinic partial agonist with potential use in the treatment of Alzheimer's disease. This study examined the functional activity of xanomeline at 5‐HT1 and 5‐HT2 receptors in native tissue and/or human cloned receptors.

Xanomeline had affinity for muscarinic receptors in rat cortical membranes where the ratio of the displacement affinity of [3H]‐Quinuclidinyl benzilate vs that of [3H]‐Oxotremorine‐M was 16, indicative of partial agonist activity. Radioligand binding studies on human cloned receptors confirmed that xanomeline had substantial affinity for M1, M2, M3, M4, M5 receptors and also for 5‐HT1 and 5‐HT2 receptor subtypes.

Carbachol and xanomeline stimulated basal [35S]‐GTPγS binding in rat cortical membranes with micromolar affinity. The response to carbachol was attenuated by himbacine and pirenzepine with pA2 of 8.2, 6.9 respectively consistent with the response being mediated, predominantly, via M2 and M4 receptors. Xanomeline‐induced stimulation of [35S]‐GTPγS binding was inhibited by himbacine with an apparent pKb of 6.3, was not attenuated by pirenzepine up to 3 μm and was inhibited by the selective 5‐HT1A antagonist WAY100635 with an apparent pKb of 9.4. These data suggest the agonist effect of xanomeline in this tissue is, in part, via 5‐HT1A receptors. Similar studies on human cloned receptors confirmed that xanomeline is an agonist at human cloned 5‐HT1A and 5‐HT1B receptors.

In studies using the fluorescent cytoplasmic Ca2+ indicator FLUO‐3AM, xanomeline induced an increase in cytoplasmic Ca2+ concentration in SH‐SY5Y cells expressing recombinant human 5‐HT2C receptors. Atropine antagonized this response, consistent with mediation via endogenously‐expressed muscarinic receptors. In the presence of atropine, xanomeline antagonized 5‐HT‐induced cytoplasmic changes in Ca2+ concentration in cells expressing h5‐HT2A, h5‐HT2B and h5‐HT2C receptors with potencies similar to its affinity at these receptors.

These studies indicate that xanomeline is a potent agonist at 5‐HT1A and 5‐HT1B receptors and an antagonist at 5‐HT2 receptor subtypes.

British Journal of Pharmacology (1998) 125, 1413–1420; doi:10.1038/sj.bjp.0702201