bluuebunny/crossref_metadata_2025 huggingface dataset:
A redox‐based mechanism for nitric oxide‐induced inhibition of DNA synthesis in human vascular smooth muscle cells
Authors: Bundy Ruth E , Marczin Nándor , Chester Adrian H , Yacoub Magdi
Published: January 2005

The current study explored potential redox mechanisms of nitric oxide (NO)‐induced inhibition of DNA synthesis in cultured human and rat aortic smooth muscle cells.

Exposure to S‐nitrosothiols, DETA‐NONOate and NO itself inhibited ongoing DNA synthesis and S phase progression in a concentration‐dependent manner, as measured by thymidine incorporation and flow cytometry. Inhibition by NO donors occurred by release of NO, as detected by chemiluminescence and judged by the effects of NO scavengers, haemoglobin and cPTIO.

Co‐incubation with redox compounds, N‐acetyl‐L‐cysteine, glutathione and L‐ascorbic acid prevented NO inhibition of DNA synthesis. These observations suggest that redox agents may alternatively attenuate NO bioactivity extracellularly, interfere with intracellular actions of NO on the DNA synthesis machinery or restore DNA synthesis after established inhibition by NO.

Recovery of DNA synthesis after inhibition by NO was similar with and without redox agents suggesting that augmented restoration of DNA synthesis is an unlikely mechanism to explain redox regulation.

Study of extracellular interactions revealed that all redox agents potentiated S‐nitrosothiol decomposition and NO release.

Examination of intracellular NO bioactivity showed that as opposed to attenuation of NO inhibition of DNA synthesis by redox agents, there was no inhibition (potentiation in the presence of ascorbic acid) of soluble guanylate cyclase (sGC) activation judged by cyclic GMP accumulation in rat cells.

These data provide evidence that NO‐induced inhibition of ongoing DNA synthesis is sensitive to redox environment. Redox processes might protect the DNA synthesis machinery from inhibition by NO, in the setting of augmented liberation of biologically active NO from NO donors.

British Journal of Pharmacology (2000) 129, 1513–1521; doi:10.1038/sj.bjp.0703240
GABAB receptor‐mediated effects on vagal pathways to the lower oesophageal sphincter and heart
Authors: Blackshaw L Ashley , Smid Scott D , O'Donnell Tracey A , Dent John
Published: January 2005

GABAB receptors influencing vagal pathways to the lower oesophageal sphincter and heart were investigated.

In urethane‐anaesthetized ferrets, the GABAB agonist baclofen (7 μmol kg−1 i.v.) increased basal lower oesophageal sphincter (LOS) pressure. This was reversed by antagonism with CGP35348 (100 μmol kg−1 i.v.). Baclofen's effect was abolished by vagotomy, suggesting a central action, yet it was ineffective when given centrally (3–6 nmol i.c.v.).

Peripheral vagal stimulation (10 Hz, 5 s duration) caused LOS inhibition, followed by excitation, then prolonged inhibition. Bradycardia was also evoked during stimulation. Bradycardia and LOS responses were abolished after chronic supranodose vagotomy, indicating that they were due to stimulation of vagal pre‐ganglionic neurones, not antidromic stimulation of afferents.

Baclofen (1–10 μmol kg−1) reduced bradycardia and enhanced LOS excitation, which was also seen in animals pretreated with atropine (400 μg kg−1 i.v.) and guanethidine (5 mg kg−1 i.v.), but not in those pretreated with L‐NAME (100 mg kg−1 i.v.).

Effects of baclofen (7 μmol kg−1 i.v.) on vagal stimulation‐induced LOS and cardiac responses were unchanged by the GABAB antagonists CGP35348 or CGP36742 (up to 112 μmol kg−1 i.v.), but were reversed by CGP62349 (ED50 37 nmol kg−1 i.v.) or CGP54626 (ED50 100 nmol kg−1 i.v.).

Responses of isolated LOS strips to electrical stimulation, capsaicin, NK‐1, NK‐2 and nicotinic receptor agonists were all unaffected by baclofen (200 μ M).

We conclude that baclofen reduces vagal output at two peripheral sites: one presynaptically on pre‐ganglionic neurones (CGP35348‐insensitive), and another (CGP35348‐sensitive) that could not be identified. This demonstrates heterogeneity of GABAB receptors through differential sensitivity to antagonists.

British Journal of Pharmacology (2000) 130, 279–288; doi:10.1038/sj.bjp.0703244
Antihyperalgesic effects of δ opioid agonists in a rat model of chronic inflammation
Authors: Fraser Graeme L , Gaudreau Geneviève‐Anne , Clarke Paul B S , Ménard Daniel P , Perkins Martin N
Published: January 2005

Opioid receptors in the brain activate descending pain pathways to inhibit the nociceptive response to acute noxious stimuli. The aim of the present study was to clarify the role of supraspinal opioid receptors in modulating the nociceptive response to persistent inflammation in rats.

Subcutaneous administration of 50 μl of complete Freund's Adjuvant (CFA) into the plantar surface of the hindpaw induced a significant decrease in paw withdrawal latency to thermal stimuli (P<0.01) at 24 h post‐injection.

Intracerebroventricular (i.c.v.) administration of the μ opioid receptor agonists, DAMGO and morphine, and the δ opioid receptor agonists, deltorphin II and SNC80, significantly reversed the hyperalgesic response associated with peripheral inflammation in a dose‐dependent manner (P<0.0001).

The μ and δ agonists also significantly attenuated the antinociceptive response to acute thermal stimulation in rats (P<0.001). However, deltorphin II and SNC80 were less potent, and in the case of SNC80 less efficacious, in modulating the response to acute thermal nociception in comparison to hyperalgesia associated with persistent inflammation.

These results indicate that μ and δ opioid receptors in the brain modulate descending pain pathways to attenuate the nociceptive response to acute thermal stimuli in both normal and inflamed tissues. The heightened response to δ agonists in the hyperalgesia model suggests that δ opioid receptors in the brain are promising targets for the treatment of pain arising from chronic inflammation.

British Journal of Pharmacology (2000) 129, 1668–1672; doi:10.1038/sj.bjp.0703248
Role of kynurenines in the neurotoxic actions of kainic acid
Authors: Behan W M H , Stone T W
Published: January 2005

The neurotoxic actions of kainic acid can be partly suppressed by antagonists acting at N‐methyl‐D‐aspartate (NMDA) receptors. The present study examined the possible role of endogenous components of the kynurenine pathway to this phenomenon.

Administration of kainate (2 nmols) into the hippocampus of anaesthetized rats produced damage in the CA1 and CA3 regions. The involvement of NMDA receptors was confirmed by the ability of dizocilpine (1 mg kg−1) to reduce cell loss in the CA1 region from 92 to 42%.

The co‐administration of m‐nitrobenzoylalanine (20 nmols into the hippocampus), an inhibitor of kynurenine hydroxylase and kynureninase, together with a systemic injection of the compound (100 mg kg−1, i.p.), afforded some protection against kainate, reducing cell loss from 91 to 48%. Protection was not exerted against damage by quinolinic acid or NMDA, excluding a direct interaction between m‐nitrobenzoylalanine and NMDA receptors.

The protective effect of m‐nitrobenzoylalanine was not prevented by glycine, which would be expected to reverse protection caused by an elevation in the levels of endogenous kynurenic acid, arguing against a major role for increased levels of kynurenic acid.

The results indicate that inhibition of the kynurenine pathway offers protection against kainate‐induced damage. One possible mechanism for the protection is that an increased production of quinolinic acid in the brain, possibly from glial cells and macrophages activated by the initial kainate insult, normally contributes to the local activation of NMDA receptors and thus to kainate‐induced cerebral insults. This generation of endogenous quinolinic acid would be suppressed by m‐nitrobenzoylalanine.

British Journal of Pharmacology (2000) 129, 1764–1770; doi:10.1038/sj.bjp.0703250
Enhanced relaxation of porcine coronary arteries after acute exposure to a physiological level of 17β‐estradiol involves non‐genomic mechanisms and the cyclic AMP cascade
Authors: Teoh Hwee , Man Ricky Y K
Published: January 2005

The present study extends our previous finding that the endothelium‐independent relaxation in porcine coronary artery rings is enhanced after short‐term (20 min) exposure to a physiological concentration (1 nM) of 17β‐estradiol and demonstrates that this effect may be attributable to activation of the cyclic AMP pathway.

Isometric tension was recorded in isolated rings of porcine coronary arteries.

Relaxation by levcromakalim and sodium nitroprusside, but not bradykinin and calcium ionophore A23187, were significantly potentiated following 20 min treatment with 1 nM 17β‐estradiol. This enhancing effect was insensitive to the transcriptional and translational inhibitors, actinomycin D and cycloheximide respectively and absent following repeated washing of the rings prior to construction of relaxation‐response curves.

The potentiating actions of 1 nM 17β‐estradiol on endothelium‐independent relaxation were mimicked by the cyclic AMP analogue 8‐Bromo‐cyclic AMP and the protein kinase A activator Sp‐cyclic AMPS but not by the cyclic GMP analogue 8‐Bromo‐cyclic GMP. The modulatory effect of 17β‐estradiol was increased in the presence of the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine.

The cyclic AMP‐dependent protein kinase A inhibitor Rp‐cyclic AMPS, but not the cyclic GMP antagonist Rp‐8‐Bromo‐cyclic GMPS, effectively inhibited the enhancing effects 1 M 17β‐estradiol had on the relaxation responses of levcromakalim and sodium nitroprusside.

These data support our earlier findings that physiologically relevant concentrations of 17β‐estradiol can acutely modify vasorelaxation in vitro. Furthermore, we report that this short‐term effect of 17β‐estradiol on vasorelaxation appears to be mediated via non‐genomic pathways and involves the cyclic AMP cascade.

British Journal of Pharmacology (2000) 129, 1739–1747; doi:10.1038/sj.bjp.0703252
ATP‐induced [Ca2+]i changes and depolarization in GH3 cells
Authors: Chung Hae Sook , Park Kyu Sang , Cha Seung Kyu , Kong In Deok , Lee Joong Woo
Published: January 2005

Extracellular ATP is a neurotransmitter and mediates a variety of responses. In the endocrine system, there are data suggesting a physiological role for ATP in Ca2+ signalling and hormone secretion. However, the ATP receptor subtype involved has not been clearly elucidated in GH3 cells, a rat anterior pituitary cell line.

BzATP‐ and ATP‐induced [Ca2+]i responses had EC50 values of 18 and 651 μM, respectively. The maximal response to ATP was only 59±8% of that for BzATP. The BzATP‐induced [Ca2+]i increase was dependent upon the extracellular Ca2+ concentration. Preincubation with oxidized ATP (oATP) nearly abolished the ATP‐ and BzATP‐induced [Ca2+]i increases.

Both BzATP and ATP induced depolarization in GH3 cells, with EC50 values of 31 μM and 1 mM, respectively. The maximal depolarization to BzATP and ATP were 152±21 and 146±16% of that elicited by 30 mM KCl.

The rank order of agonist potency for [Ca2+]i and depolarization responses was BzATP ≫ ATP >2‐MeSATP and purine derivatives such as ADP, AMP, adenosine were ineffective. Neither UTP nor α, β‐methylene ATP showed any effect.

In low‐divalent conditions BzATP evoked non‐desensitizing inward currents, which were reversed at ∼0 mV. This nonselective cationic conductance was increased by repeated applications of BzATP and the cells became very permeable to NMDG. Longer applications (30 min) of BzATP stimulated ethidium bromide influx in low divalent conditions, suggesting increased permeability to larger molecules. We also identified the existence of P2X7 mRNA on GH3 cells by using reverse transcriptase (RT)‐polymerase chain reaction (PCR).

These results suggest that the GH3 cells have an endogenous P2X7 receptor and purinergic stimulation may play a potential role in neuroendocrine modulation on these cells.

British Journal of Pharmacology (2000) 130, 1843–1852; doi:10.1038/sj.bjp.0703253
An improved in vitro bioassay for the study of 5‐HT4 receptors in the human isolated large intestinal circular muscle
Authors: Prins N H , Shankley N P , Welsh N J , Briejer M R , Lefebvre R A , Akkermans L M A , Schuurkes J A J
Published: January 2005

Recently, it was demonstrated that 5‐HT induces relaxation of human colon circular muscle through activation of 5‐HT4 receptors and 5‐HT7 receptors. The aim of the current study was to develop a new in vitro bioassay of human colon that would facilitate the pharmacological analysis of 5‐HT responses mediated solely by 5‐HT4 receptors.

Contracting circular muscle strips with KCl (80 mM) yielded a stable contractile tension and, in contrast to muscarinic cholinoceptor agonists and histamine, a profound reduction of spontaneous contractility. This allowed the establishment of reproducible, fully‐defined, agonist concentration‐response curves by cumulative dosing. Under these conditions, 5‐HT induced a concentration‐dependent relaxation (pEC50 7.31, Hill slope 0.91).

Neither methysergide (10 μM) nor granisetron (1 μM) affected the 5‐HT‐induced relaxation, suggesting that 5‐HT1, 5‐HT2, 5‐HT3, 5‐ht5, 5‐HT6 or 5‐HT7 receptors are not involved. The lack of effect of tetrodotoxin (0.3 μM) indicated a direct effect of 5‐HT on the smooth muscle.

The selective 5‐HT4 receptor antagonists GR 113808, GR 125487 and RS 39604 competitively antagonized the 5‐HT‐induced relaxation (pKB 9.43, 10.12 and 8.53, respectively). SB 204070 (1 nM) produced a rightward shift (pA2 10.34) and depression of the 5‐HT curve. These affinity estimates are similar to those previously reported for 5‐HT4 receptors.

The selective 5‐HT4 receptor agonists, prucalopride and R076186, induced relaxations (pEC50 7.50 and 7.57, respectively), that were blocked by GR 113808 (3 nM), yielding pA2 estimates of 9.31 and 9.21, respectively.

To summarise, in KCl (80 mM)‐contracted muscle strips, 5‐HT induces relaxation through activation of a homogeneous smooth muscle 5‐HT4 receptor population. This new bioassay allows the focused, pharmacological characterization of human colonic 5‐HT4 receptors in vitro.

British Journal of Pharmacology (2000) 129, 1601–1608; doi:10.1038/sj.bjp.0703254
Suppression of apoptosis by glucocorticoids in glomerular endothelial cells: effects on proapoptotic pathways
Authors: Meßmer Udo K , Winkel Gundula , Briner Verena A , Pfeilschifter Josef
Published: January 2005

Tumour necrosis factor‐α (TNF‐α)‐ and lipopolysaccharide (LPS)‐induced apoptosis of bovine glomerular endothelial cells is now recognized as an important part in the pathogenesis of glomerulonephritis characterized by early mitochondrial cytochrome c release, mitochondrial permeability transition, Bak protein upregulation, Bcl‐XL protein downregulation and caspase‐3 activation.

Co‐treatment of cells with 10 nM dexamethasone and TNF‐α or LPS blocked roughly 90% of apoptotic cell death in glomerular endothelial cells. The action of glucocorticoids could be documented in that they prevented all apoptotic markers such as DNA laddering, DNA fragmentation measured by the diphenylamine assay as well as morphological alterations.

To mechanistically elucidate the action of glucocorticoids we evaluated whether glucocorticoids elicit a time‐dependent effect. For dexamethasone, to maximally inhibit DNA fragmentation a preincubation period was not required. Even if dexamethasone was supplemented 6 h following TNF‐α or LPS we observed a maximal inhibitory effect.

Concerning its influence on TNF‐α and LPS signal transduction, we found that dexamethasone only partially prevented cytochrome‐c‐release as a first sign of apoptotic cell death but efficiently blocked mitochondrial permeability transition. Moreover, TNF‐α‐ and LPS‐induced Bak upregulation, Bcl‐XL‐downregulation, and the activation of caspase‐3‐like proteases, measured fluorometrically using DEVD‐AMC and PARP cleavage, were efficiently blocked by dexamethasone.

We postulate that glucocorticoids exert their inhibitory action upstream of the terminal death pathways but downstream of primary receptor mediated signals by blocking pro‐apoptotic signals pre‐ and/or post cytochrome c release and mitochondrial signalling.

British Journal of Pharmacology (2000) 129, 1673–1683; doi:10.1038/sj.bjp.0703255
The hypocretins are weak agonists at recombinant human orexin‐1 and orexin‐2 receptors
Authors: Smart D , Jerman J C , Brough S J , Neville W A , Jewitt F , Porter R A
Published: January 2005

The pharmacology of the orexin‐like peptides, hypocretin‐1 and hypocretin‐2, was studied in Chinese hamster ovary (CHO) cells stably expressing orexin‐1 (OX1) or orexin‐2 (OX2) receptors by measuring intracellular calcium ([Ca2+]i) using Fluo‐3AM. Orexin‐A and orexin‐B increased [Ca2+]i in CHO‐OX1 (pEC50=7.99±0.05 and 7.00±0.10 respectively, n=8) and CHO‐OX2 (pEC50=8.30±0.05 and 8.21±0.07 respectively, n=5). However, hypocretin‐1 and hypocretin‐2 were markedly less potent, with pEC50 values of 5.31±0.04 and 5.41±0.04 respectively in CHO‐OX2 cells (n=5). In CHO‐OX1 cells 10 μM hypocretin‐1 only elicited a 37.5±3.4% response whilst 10 μM hypocretin‐2 elicited a 18.0±2.1% response (n=8). Desensitisation of OX1 or OX2 with orexin‐A (100 nM) abolished the response to orexin‐A (10 nM) and the hypocretins (10 μM), but not to UTP (3 μM). In conclusion, the hypocretins are only weak agonists at the orexin receptors.

British Journal of Pharmacology (2000) 129, 1289–1291; doi:10.1038/sj.bjp.0703257
Involvement of sensory nerves in vasodilator responses to acetylcholine and potassium ions in rat hepatic artery
Authors: Högestätt Edward D , Johansson Rebecka , Andersson David A , Zygmunt Peter M
Published: January 2005

In the presence of ouabain (1 mM), acetylcholine and KCl (5 mM) evoked endothelium‐independent relaxations in rat hepatic arteries. Treatment with capsaicin (10 μM), scopolamine (1 μM) or CGRP8–37 (3 μM) prevented these relaxations. Acetylcholine‐induced relaxations in intact arterial segments in the presence of indomethacin (10 μM) and NG‐nitro‐L‐arginine (0.3 mM) were only partially inhibited by ouabain plus BaCl2 (30 μM). However, ouabain plus BaCl2 almost abolished such relaxations in capsaicin‐pre‐treated preparations. In arteries without endothelium, the neurosecretagogue α‐latrotoxin (1 nM) induced complete relaxations, which were abolished by CGRP8–37 or pre‐treatment with capsaicin. α‐Latrotoxin also induced a smooth muscle hyperpolarization (12±2 mV), which was abolished by CGRP8–37. The ability of ouabain to disclose a CGRP‐mediated neurogenic relaxation must be considered when this agent is used as a pharmacological tool. The results further suggest that CGRP is a nerve‐derived hyperpolarizing factor in the rat hepatic artery.

British Journal of Pharmacology (2000) 130, 27–32; doi:10.1038/sj.bjp.0703258
Selective block of late Na+ current by local anaesthetics in rat large sensory neurones
Authors: Baker Mark D
Published: January 2005

The actions of lignocaine and benzocaine on transient and late Na+ current generated by large diameter (50 μm) adult rat dorsal root ganglion neurones, were studied using patch‐clamp techniques.

Both drugs blocked whole‐cell late Na+ current in a concentration‐dependent manner. At 200 ms following the onset of a clamp step from −110 to −40 mV, the apparent K for block of late Na+ current by lignocaine was 57.8±15 μM (mean±s.e.mean, n=4). The value for benzocaine was 24.9±3.3 μM, (mean±s.e.mean, n=3).

The effect of lignocaine on transient current, in randomly selected neurones, appeared variable (n=8, half‐block from ∼50 to 400 μM). Half‐block by benzocaine was not attained, but both whole‐cell (n=11) and patch data suggested a high apparent K,>250 μM. Transient current always remained after late current was blocked.

The voltage‐dependence of residual late current steady‐state inactivation was not shifted by 20 μM benzocaine (n=3), whereas 200 μM benzocaine shifted the voltage‐dependence of transient current steady‐state inactivation by −18.7±5.9 mV (mean±s.e.mean, n=4).

In current‐clamp, benzocaine (250 μM) could block subthreshold, voltage‐dependent inward current, increasing the threshold for eliciting action potentials, without preventing their generation (n=2).

Block of late Na+ current by systemic local anaesthetic may play a part in preventing ectopic impulse generation in sensory neurones.

British Journal of Pharmacology (2000) 129, 1617–1626; doi:10.1038/sj.bjp.0703261
Central and peripheral cannabinoid modulation of gastrointestinal transit in physiological states or during the diarrhoea induced by croton oil
Authors: Izzo Angelo A , Pinto Luisa , Borrelli Francesca , Capasso Raffaele , Mascolo Nicola , Capasso Francesco
Published: January 2005

We have evaluated the effect of cannabinoid drugs, administered intraperitoneally (i.p.) or intracerebroventricularly (i.c.v.) on upper gastrointestinal transit in control and in croton oil‐treated mice.

The cannabinoid agonists, WIN 55,212‐2 (2–239 nmol mouse−1) and cannabinol (24–4027 nmol mouse−1), decreased while the CB1 antagonist SR141716A (2–539 nmol mouse−1) increased transit in control mice. WIN 55,212‐2, cannabinol and SR141716A had lower ED50 values when administered i.c.v., than when administered i.p. The CB2 antagonist SR144528 (52 nmol mouse−1, i.p.) was without effect.

During croton oil (0.01 ml mouse−1, p.o.)‐induced diarrhoea, the ED50 values of i.p.‐injected WIN 55,212‐2 and cannabinol (but not SR141716A) were significantly decreased (compared to control mice). However, the ED50 values of WIN 55,212‐2 were similar after i.p. or i.c.v. administration.

The inhibitory effects of WIN 55,212‐2 and cannabinol were counteracted by SR141716A (16 nmol mouse−1, i.p.) but not by SR144528 (52 nmol mouse−1, i.p.) both in control and croton‐oil treated mice.

Ganglionic blockade with hexamethonium (69 nmol mouse−1, i.p.) did not modify the inhibitory effect of i.p.‐injected cannabinoid agonists either in control or in croton‐oil treated mice.

The lower ED50 values of cannabinoid drugs after i.c.v. administration suggest a central (CB1) site of action. However, a peripheral site of action is suggested by the lack of effect of hexamethonium. In addition, croton oil‐induced diarrhoea enhances the effect of cannabinoid agonists by a peripheral mechanism.

British Journal of Pharmacology (2000) 129, 1627–1632; doi:10.1038/sj.bjp.0703265
Loss of muscarinic antinociception by antisense inhibition of M1 receptors
Authors: Ghelardini Carla , Galeotti Nicoletta , Bartolini Alessandro
Published: January 2005

The effect on cholinergic analgesia of inactivation of the M1 gene by an antisense oligodeoxyribonucleotide (aODN) was investigated in the mouse hot plate test. Mice received a single intracerebroventricular (i.c.v.) injection of anti‐M1 aODN (0.3, 1.0 or 2.0 nmol per injection), degenerate ODN (dODN) or vehicle on days 1, 4 and 7.

A dose‐dependent inhibition of the antinociception induced by the muscarinic agonists oxotremorine (0.1 mg kg−1 s.c.) and McN‐A‐343 (30 μg per mouse i.c.v.) and the cholinesterase inhibitor physostigmine (0.2 mg kg−1 s.c.) was observed 24 h after the last i.c.v. injection of aODN. Time‐course experiments revealed that, after the end of the aODN treatment, sensitivity to analgesic drugs progressively appeared reaching the normal range at 96 h.

The anti‐M1 aODN was selective against muscarinic antinociception since the enhancement of pain threshold produced by morphine and baclofen were not affected by the above‐mentioned treatment. dODN, used as control, did not affect muscarinic antinociception.

Binding studies evidenced a selective reduction of M1 receptor levels in the hippocampus of aODN‐treated mice.

Neither aODN, dODN nor vehicle produced any behavioural impairment of mice as revealed by the rota‐rod and Animex experiments.

These results indicate that activation of M1 muscarinic receptor subtype is fundamental to induce central cholinergic analgesia in mice.

British Journal of Pharmacology (2000) 129, 1633–1640; doi:10.1038/sj.bjp.0703268
Glucocorticoids act within minutes to inhibit recruitment of signalling factors to activated EGF receptors through a receptor‐dependent, transcription‐independent mechanism
Authors: Croxtall Jamie D , Choudhury Qam , Flower Rod J
Published: January 2005

Recruitment to activated tyrosine kinase growth factor receptors of Grb2 and p21ras leads to downstream activation of the kinases Raf, MAPK/Erk kinase (Mek) and, subsequently, extracellular signal‐regulated kinase (Erk). Activated Erk phosphorylates specific serine residues within cytosolic phospholipase A2 (PLA2), promoting enzyme translocation to membranes and facilitating liberation of arachidonic acid (AA).

In the A549 human adenocarcinoma cell line dexamethasone inhibited epidermal growth factor (EGF)‐stimulated cytosolic PLA2 (cPLA2) activation and AA release by blocking the recruitment of Grb2 to the activated EGF receptor (EGF‐R) through a glucocorticoid receptor (GR)‐dependent (RU486‐sensitive), transcription‐independent (actinomycin‐insensitive), mechanism.

The dexamethasone‐induced block of Grb2 recruitment was parallelled by changes in phosphorylation status and subcellular localization of lipocortin 1 (LC1) and an increase in the amount of the tyrosine phosphoprotein co‐localized with EGF‐R.

Like dexamethasone, peptides containing E‐Q‐E‐Y‐V from the N‐terminal domain of LC1 also blocked ligand‐induced association of Grb2, p21ras and Raf.

Our results point to an unsuspected rapid effect of glucocorticoids, mediated by occupation of GR but not by changes in gene transcription, which is brought about by competition between LC1 and Grb2 leading to a failure of recruitment off signalling factors to EGF‐R

British Journal of Pharmacology (2000) 130, 289–298; doi:10.1038/sj.bjp.0703272
Induction of skeletal muscle contracture and calcium release from isolated sarcoplasmic reticulum vesicles by sanguinarine
Authors: Hu C M , Cheng H W , Cheng Y W , Kang J J
Published: January 2005

The benzophenanthrine alkaloid, sanguinarine, was studied for its effects on isolated mouse phrenic‐nerve diaphragm preparations. Sanguinarine induced direct, dose‐dependent effects on muscle contractility.

Sanguinarine‐induced contracture was partially inhibited when the extracellular Ca2+ was removed or when the diaphragm was pretreated with nifedipine. Depletion of sarcoplasmic reticulum (SR) internal calcium stores completely blocked the contracture.

Sanguinarine induced Ca2+ release from the actively loaded SR vesicles was blocked by ruthenium red and dithiothreitol (DTT), consistent with the ryanodine receptor (RyR) as the site of sanguinarine action.

Sanguinarine altered [3H]‐ryanodine binding to the RyR of isolated SR vesicles, potentiating [3H]‐ryanodine binding at lower concentrations and inhibiting binding at higher concentrations. All of these effects were reversed by DTT, suggesting that sanguinarine‐induced Ca2+ release from SR occurs through oxidation of critical SH groups of the RyR SR calcium release channel.

British Journal of Pharmacology (2000) 130, 299–306; doi:10.1038/sj.bjp.0703279
Correlation of leukocyte adhesiveness, adhesion molecule expression and leukocyte‐induced contraction following balloon angioplasty
Authors: Kennedy Simon , McPhaden Allan R , Wadsworth Roger M , Wainwright Cherry L
Published: January 2005

The aim of this study was to examine the changes in leukocyte adhesion and leukocyte‐induced contraction in balloon‐injured rabbit subclavian artery and to correlate these changes with vessel morphology and expression of adhesion molecules on the injured arteries.

Rabbits were anaesthetized and their left subclavian arteries were injured by balloon inflation and withdrawal followed by sacrifice at 2, 24, 48 h or 8 days after injury. The left and right subclavian arteries were removed and leukocytes were isolated from autologous rabbit blood. Leukocyte‐induced contraction was measured in 5‐HT precontracted artery rings and leukocyte adhesion was measured using 51Cr‐labelled leukocytes. Immunocytochemistry using paraffin‐embedded tissue was employed to detect changes in the expression of adhesion molecules on injured arteries.

Autologous leukocytes caused a contraction of rabbit subclavian artery rings, which was prevented by L‐NAME (10−3 M). Balloon‐induced injury abolished the contractile response to leukocytes, which correlated with loss of carbachol‐induced relaxation

Balloon injury markedly enhanced the adhesiveness of the subclavian artery for leukocytes, most notably at 24 and 48 h after injury (1.7 and 1.8 fold respectively). Increased leukocyte adhesion at these two time points correlated with an upregulation of E‐selectin, P‐selectin and VCAM‐1 expression on the remaining endothelium of the injured artery.

Vessel morphology revealed that balloon inflation had induced an infiltration of inflammatory cells into the vessel wall, the greatest increase being seen at 24 h after injury.

It is concluded that an increase in the expression of E‐selectin, P‐selectin and VCAM‐1 following balloon‐induced injury leads to enhanced leukocyte adhesion and migration into the injured vessel.

British Journal of Pharmacology (2000) 130, 95–103; doi:10.1038/sj.bjp.0703282
Cisplatin‐induced early and delayed emesis in the pigeon
Authors: Tanihata Sachiko , Igarashi Hiroaki , Suzuki Masami , Uchiyama Toshimitsu
Published: January 2005

Intravenously injected cisplatin at a dose of 4 mg kg−1 induced early and delayed emesis in all pigeons without occurrence of lethality during a 72 h observation period. The early emetic response occurred with a latency of 81.3±8.0 min (n=15) and reached a peak at 2–3 h, and decreased gradually within 8 h after injection. Then the delayed emetic response, whose peak was found between 10 to 23 h, lasted up to 48 h. The emetic response markedly declined after 48 h.

Reserpine markedly reduced monoamine levels in both brain and intestine and completely abolished the early and delayed emesis. Dexamethasone markedly reduced not only the early but also the delayed emetic responses. p‐Chlorophenylalanine decreased the level of serotonin in brain and intestine without affecting noradrenaline and dopamine and partly reduced the early emetic response, but did not affect delayed emesis.

Bilateral vagotomy prolonged the latency time to the onset of early emesis, and reduced the emetic responses in both the early and delayed phases.

The above results suggest that the cisplatin‐induced early emesis in the pigeon is partially mediated via the vagal nerve and reserpine‐sensitive monoaminergic systems including the serotonergic system; the delayed emesis is associated with monoaminergic but not the serotonergic systems.

British Journal of Pharmacology (2000) 130, 132–138; doi:10.1038/sj.bjp.0703283
Non‐NF‐κB elements are required for full induction of the rat type II nitric oxide synthase in vascular smooth muscle cells
Authors: Zhang Hanfang , Teng Xingwu , Snead Connie , Catravas John D
Published: January 2005

We have investigated the role of the NF‐κB binding sites and other promoter elements beyond NF‐κB in iNOS induction in rat vascular smooth muscle cells (SMC).

Rat aortic SMC transfected with iNOS promoter constructs with either mutation or deletion of the downstream NF‐κB site exhibited about 50% reduction in promoter activity in response to a cytokine mixture, whereas either mutation or deletion of the upstream NF‐κB site reduced promoter activity by 90%, suggesting that the latter site is the most important, and that co‐existence of two NF‐κB sites is necessary for iNOS induction.

Nuclear NF‐κB activity was robustly induced by TNF‐α. However, TNF‐α alone did not induce iNOS promoter activity, protein expression, or nitrite production, indicating that NF‐κB activation alone is not sufficient for iNOS induction.

The construct up to −890 bp, containing the downstream NF‐κB site, exhibited little response to cytokines. The construct up to −1.0 kb, containing the two NF‐κB sites exhibited only 22% of full promoter activity. The regions −1001 to −1368 bp and −2 to −2.5 kb contributed an additional 43 and 22% promoter activity, respectively.

Internal deletion or reversal of the orientation of −1001 to −1368 bp in the full promoter resulted in 40% reduction in promoter activity.

These data suggest that the co‐existence of two NF‐κB sites is essential for core promoter activity, but that full induction of the rat SMC iNOS gene requires other elements located between −1.0 to −1.37 and −2.0 to −2.5 kb of the promoter.

British Journal of Pharmacology (2000) 130, 270–278; doi:10.1038/sj.bjp.0703284
Ecto‐nucleotide pyrophosphatase modulates the purinoceptor‐mediated signal transduction and is inhibited by purinoceptor antagonists
Authors: Grobben Bert , Claes Patrik , Roymans Dirk , Esmans Edgard L , Van Onckelen Harry , Slegers Herman
Published: January 2005

The effect of ecto‐nucleotide pyrophosphatase (ecto‐NPPase; EC 3.6.1.9) on the ATP‐ and ADP‐mediated receptor activation was studied in rat C6 glioma cells. The P2‐purinoceptor antagonists pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) and reactive blue (RB2) are potent inhibitors (IC50=12±3 μM) of the latter enzyme. 4,4′‐diisothiocyanatostilbene‐2,2′ disulfonic acid (DIDS), 5′‐phosphoadenosine 3′‐phosphate (PAP) and suramin were less potent inhibitors with an IC50 of 22±4, 36±7 and 72±11 μM respectively.

P1‐purinoceptor antagonists CGS 15943, cyclo‐pentyl theophylline (CTP) and theophylline did not affect the activity of the ecto‐NPPase.

ATP‐ and ADP‐mediated P2Y1‐like receptor activation inhibited the (−)‐isoproterenol‐induced increase of intracellular cyclic AMP concentration. PPADS, an ineffective P2Y‐antagonist in C6, potentiated the ATP and ADP effect approximately 3 fold due to inhibition of nucleotide hydrolysis by the ecto‐NPPase.

We conclude that ecto‐NPPase has a modulatory effect on purinoceptor‐mediated signalling in C6 glioma cell cultures.

British Journal of Pharmacology (2000) 130, 139–145; doi:10.1038/sj.bjp.0703289
Role of L‐arginine in the vascular actions and development of tolerance to nitroglycerin
Authors: Abou‐Mohamed Gamal , Kaesemeyer Wayne H , Caldwell Ruth B , Caldwell Robert W
Published: January 2005

The goal of this work was to test the role of nitric oxide synthase (NOS) and its substrate L‐arginine in development of tolerance to nitroglycerin's (GTN) vasodilator actions.

GTN's effects on NOS activity and NO formation were tested in cultured bovine aortic endothelial cells (BAECs). The arginine to citrulline conversion assay showed that GTN stimulated NOS basal activity in BAECs by ∼40%, comparable with acetylcholine (ACh)‐treated controls. Both effects were blocked by L‐NMMA. Photometric assays showed that both GTN and ACh‐stimulated NO formation. Both effects were potentiated by L‐arginine and inhibited by L‐NAME. L‐NAME inhibited ACh responses ∼80% compared with ∼40% for GTN responses.

The aortic ring assay showed that 2 h pretreatment with GTN caused substantial tolerance to GTN's vasodilating effects as evidenced by a 38 fold rightward shift of the concentration‐relaxation curve. In contrast to D‐arginine, addition of L‐arginine substantially inhibited this effect, reducing the rightward shift to 4.4 fold of control values. GTN tolerance was associated with a 40% reduction in L‐arginine tissue levels. GTN had a biphasic effect on BAEC uptake of L‐arginine, stimulating uptake at 5 and 15 min, and suppressing uptake after 1 and 4 h

In summary, acute GTN treatment stimulates endothelial NOS activity in producing NO and increases cellular uptake of L‐arginine. Prolonged GTN exposure reduces GTN's vasodilator actions, decreases L‐arginine tissue levels and depresses BAECs uptake of L‐arginine. Supplementation of L‐arginine reduces development of GTN tolerance. These data indicate that GTN tolerance depends in part on activation of the NOS pathway.

British Journal of Pharmacology (2000) 130, 211–218; doi:10.1038/sj.bjp.0703293
Activation of I2‐imidazoline receptors enhances supraspinal morphine analgesia in mice: a model to detect agonist and antagonist activities at these receptors
Authors: Sánchez‐Blázquez Pilar , Boronat M Assumpció , Olmos Gabriel , García‐Sevilla Jesús A , Garzón Javier
Published: January 2005

This work investigates the receptor acted upon by imidazoline compounds in the modulation of morphine analgesia. The effects of highly selective imidazoline ligands on the supraspinal antinociception induced by morphine in mice were determined.

Intracerebroventricular (i.c.v.) or subcutaneous (s.c.) administration of ligands selective for the I2‐imidazoline receptor, 2‐BFI, LSL 60101, LSL 61122 and aganodine, and the non selective ligand agmatine, increased morphine antinociception in a dose‐dependent manner. Neither moxonidine, a mixed I1‐imidazoline and α2‐adrenoceptor agonist, RX821002, a potent α2‐adrenoceptor antagonist that displays low affinity at I2‐imidazoline receptors, nor the selective non‐imidazoline α2‐adrenoceptor antagonist RS‐15385‐197, modified the analgesic responses to morphine.

Administration of pertussis toxin (0.25 μg per mouse, i.c.v.) 6 days before the analgesic test blocked the ability of the I2‐imidazoline ligands to potentiate morphine antinociception.

The increased effect of morphine induced by I2‐imidazoline ligands (agonists) was completely reversed by idazoxan and BU 224. Identical results were obtained with IBI, which alkylates I2‐imidazoline binding sites. Thus, both agonist and antagonist properties of imidazoline ligands at the I2‐imidazoline receptors were observed.

Pre‐treatment (30 min) with deprenyl, an irreversible inhibitor of monoamine oxidase B (IMAO‐B), produced an increase of morphine antinociception. Clorgyline, an irreversible IMAO‐A, given 30 min before morphine did not alter the effect of the opioid. At longer intervals (24 h) a single dose of either clorgyline or deprenyl reduced the density of I2‐imidazoline receptors and prevented the I2‐mediated potentiation of morphine analgesia.

These results demonstrate functional interaction between I2‐imidazoline and opioid receptors. The involvement of Gi‐Go transducer proteins in this modulatory effect is also suggested.

British Journal of Pharmacology (2000) 130, 146–152; doi:10.1038/sj.bjp.0703294
Role of uptake inhibition and autoreceptor activation in the control of 5‐HT release in the frontal cortex and dorsal hippocampus of the rat
Authors: Hervás Ildefonso , Queiroz Claudio M T , Adell Albert , Artigas Francesc
Published: January 2005

Using brain microdialysis, we compared the relative role of 5‐hydroxytryptamine (5‐HT; serotonin) blockade and somatodendritic 5‐HT1A and/or terminal 5‐HT1B autoreceptor activation in the control of 5‐HT output.

Fluoxetine (10 mg kg−1 i.p.) doubled the 5‐HT output in frontal cortex and dorsal hippocampus. The 5‐HT1A receptor antagonist WAY 100635, (0.3 mg kg−1 s.c.) potentiated the effect of fluoxetine only in frontal cortex (to ∼500 % of baseline).

Methiothepin (10 mg kg−1 s.c.) further enhanced the 5‐HT rise induced by fluoxetine+WAY 100635, to 835±179% in frontal cortex and 456±24% in dorsal hippocampus. Locally applied, methiothepin potentiated the fluoxetine‐induced 5‐HT rise more in the former area.

The selective 5‐HT1B receptor antagonist SB‐224289 (4 mg kg−1 i.p.) enhanced the effect of fluoxetine (10 mg kg−1 i.p.) in both areas. As with methiothepin, SB‐224289 (4 mg kg−1 i.p.) further enhanced the 5‐HT increase produced by fluoxetine+WAY 100635 more in frontal cortex (613±134%) than in dorsal hippocampus (353±59%).

Locally applied, fluoxetine (10–300 μM; EC50=28–29 μM) and citalopram (1–30 μM; EC50=1.0–1.4 μM) increased the 5‐HT output two to three times more in frontal cortex than in dorsal hippocampus.

These data suggest that the comparable 5‐HT increase produced by systemic fluoxetine in frontal cortex and dorsal hippocampus results from a greater effect of reuptake blockade in frontal cortex that is offset by a greater autoreceptor‐mediated inhibition of 5‐HT release. As a result, 5‐HT autoreceptor antagonists preferentially potentiate the effect of fluoxetine in frontal cortex.

British Journal of Pharmacology (2000) 130, 160–166; doi:10.1038/sj.bjp.0703297
Apparent species differences in the kinetic properties of P2X7 receptors
Authors: Hibell A D , Kidd E J , Chessell I P , Humphrey P P A , Michel A D
Published: January 2005

Apparent species differences in the responses of recombinant P2X7 receptors to repeated application of 2′‐ and 3′‐O‐(4‐benzoylbenzoyl)‐ATP (BzATP) have been investigated.

Repeated application of 100 μM BzATP resulted in a progressive increase in current magnitude (current growth) at mouse and human, but not rat P2X7 receptors.

Current growth was thought to reflect progressive dilation of the P2X7 ion‐channel to a pore permeable to large molecules (MW<900), suggesting that channel dilation was not occurring at the rat P2X7 receptor. However, 100 μM BzATP produced a rapid influx of YO‐PRO‐1 (MW375) in cells expressing rat or human P2X7 receptors.

There were, however, species differences in agonist potency such that 100 μM BzATP was a supra‐maximal concentration at rat, but not human or mouse, P2X7 receptors. Importantly, when sub‐maximal concentrations of BzATP or ATP were examined, current growth occurred at rat P2X7 receptors.

The rate of current growth and YO‐PRO‐1 accumulation increased with agonist concentration and appeared more rapid at rat and human, than at mouse P2X7 receptors.

The potency of BzATP and ATP was 1.5–10 fold lower in naïve cells than in cells repeatedly exposed to ATP.

This study demonstrates that current growth occurs at mouse, rat and human P2X7 receptors but only when using sub‐maximal concentrations of agonist. Previously, current growth was thought to reflect the progressive increase in pore diameter of the P2X7 receptor ion channel, however, the results of this study suggest a progressive increase in agonist potency may also contribute.

British Journal of Pharmacology (2000) 130, 167–173; doi:10.1038/sj.bjp.0703302
Identification of the adenylyl cyclase‐activating 5‐hydroxytryptamine receptor subtypes expressed in the rat submandibular gland
Authors: Bourdon D M , Camden J M , Landon L A , Levy F O , Turner J T
Published: January 2005

Serotonin (5‐hydroxytryptamine, 5‐HT) has been shown to increase cyclic AMP production in dispersed cell aggregates from the major salivary glands of the rat. The goal of the present study was to identify the 5‐HT receptor subtypes that mediate these effects in rat submandibular glands (SMG).

Among the 5‐HT receptor subtypes identified in the rat, 5‐HT4(a,b), 5‐HT6 and 5‐HT7(a,b,c) activate adenylyl cyclase (AC). We used subtype specific primers to screen rat SMG by reverse transcription‐PCR. Results indicate the presence of mRNA for 5‐HT4(b) and 5‐HT7(a) but not for 5‐HT4(a), 5‐HT6 and 5‐HT7(b,c).

In dispersed SMG cells, 5‐carboxyamidotryptamine (5‐CT), a 5‐HT7 receptor agonist, stimulated cyclic AMP synthesis with higher potency (EC50=27±5 nM) but lower efficacy than 5‐HT, suggesting a 5‐HT7 component and an additional component in the response to 5‐HT. The 5‐HT7 contribution was further supported by antagonism of the 5‐CT effect by metergoline, a 5‐HT7 antagonist, which exhibited an affinity (Ki=50 nM) similar to that obtained at the cloned 5‐HT7 receptor.

In the presence of a maximally effective concentration of 5‐CT, 5‐HT produced an additional increase in cyclic AMP production that was inhibited by the 5‐HT4 receptor antagonist, GR113808, suggesting that the second component of cyclic AMP production is mediated by 5‐HT4 receptors.

These findings indicate the presence in rat SMG of both 5‐HT4(b) and 5‐HT7(a) receptors positively coupled to AC.

British Journal of Pharmacology (2000) 130, 104–108; doi:10.1038/sj.bjp.0703303
Involvement of the cyclic AMP system in the switch from tolerance into supersensitivity to the antinociceptive effect of the opioid sufentanil
Authors: Hurlé María A , Goirigolzarri Isabel , Valdizán Elsa M
Published: January 2005

We have previously demonstrated that chronic and simultaneous treatment of rats with the μ‐opioid receptor agonist sufentanil and the Ca2+ channel blocker nimodipine, not only prevented tolerance development, but the animals became supersensitive to the antinociceptive effect of the opioid. The focus of the present work was to determine the possible involvement of cross interactions between the adenylyl cyclase pathway and L‐type voltage‐sensitive Ca2+‐channels, in modulating the switch from opioid tolerance into supersensitivity.

The modulatory effect of sufentanil on adenylyl cyclase activity was determined by measuring cyclic AMP production in slices from the cortex of rats rendered tolerant or supersensitive to the antinociceptive effect of the opioid. Tolerance was induced by chronic infusion of sufentanil, at a rate of 2 μg h−1, for 7 days. Supersensitivity was induced by concurrent infusion of sufentanil (2 μg h−1) and nimodipine (1 μg h−1) for 7 days. Antinociception was evaluated by the tail‐flick test.

Tolerance to the analgesic effect of sufentanil was associated with a significant reduction in the response of adenylyl cyclase to forskolin. Furthermore, the effect of the opioid on forskolin‐induced cyclic AMP accumulation was abolished. On the other hand, supersensitivity to the analgesic effect of the opioid was associated with an increase in both, the adenylyl cyclase response to forskolin, and the opioid inhibition of cyclic AMP production.

We suggest that sustained L‐type Ca2+ channel blockade may result in changes in the adenylyl cyclase effector system triggered by μ‐opioid receptor activation, leading to the switch from opioid tolerance into supersensitivity.

British Journal of Pharmacology (2000) 130, 174–180; doi:10.1038/sj.bjp.0703304
Inhibition of pulmonary eosinophilia and airway hyperresponsiveness in allergic mice by rolipram: involvement of endogenously released corticosterone and catecholamines
Authors: Kung T T , Crawley Y , Luo B , Young S , Kreutner W , Chapman R W
Published: January 2005

This study investigates the role of adrenal‐derived catecholamines and corticosterone on the inhibition by rolipram, a phosphodiesterase (PDE)‐4 inhibitor, of pulmonary eosinophilia and airway hyperresponsiveness (AHR) in allergic mice.

The following experimental groups were studied in mice sensitized and challenged with ovalbumin (OVA): normal, adrenalectomized, propranolol (β‐adrenoceptor antagonist) and metyrapone (corticosterone synthesis inhibitor) treated. These interventions were studied both in the absence and in the presence of rolipram. Eosinophil numbers in the bronchoalveolar lavage (BAL) and AHR to methacholine were measured 24 h after OVA challenge.

Treatment of sensitized mice with rolipram (0.3–10 mg kg−1, p.o.), inhibited pulmonary eosinophilia and the AHR to methacholine in OVA‐challenged mice.

Adrenalectomy increased the number of eosinophils in the BAL of OVA‐challenged mice but had no effect on AHR to methacholine. Adrenalectomy attenuated both the rolipram‐induced inhibition of BAL eosinophilia and AHR to methacholine in OVA challenged mice. Propranolol (10 mg kg−1, p.o.) had no effect on the inhibition of eosinophilia by rolipram but attenuated the inhibition of AHR to methacholine in OVA challenged mice. On the other hand, metyrapone (10 mg kg−1, p.o.) attenuated the inhibition of eosinophilia by rolipram but had no effect on the inhibition of AHR to methacholine in OVA challenged mice. Metyrapone‐treatment alone increased the number of eosinophils in the BAL of OVA‐challenged mice.

These results identify an important role for adrenal‐derived catecholamines and corticosterone on the inhibition of pulmonary eosinophilia and AHR by rolipram in allergic mice.

British Journal of Pharmacology (2000) 130, 457–463; doi:10.1038/sj.bjp.0703308