bluuebunny/crossref_metadata_2025 huggingface dataset:
21‐NO‐prednisolone is a novel nitric oxide‐releasing derivative of prednisolone with enhanced anti‐inflammatory properties
Authors: Paul‐Clark Mark , Del Soldato Piero , Fiorucci Stefano , Flower Roderick J , Perretti Mauro
Published: January 2005

Anti‐inflammatory effects of a novel derivative of the glucocorticoid prednisolone were investigated. NCX‐1015 (prednisolone 21‐[(4′‐nitrooxymethyl)benzoate]) incubation in human platelet‐rich plasma produced a time (0–60 min) and concentration (3–300 μM) dependent release of nitrite, that was mirrored by accumulation of cyclic guanosine monophosphate in the human platelets. Intraperitoneal injection of NCX‐1015 to mice (up to 27.7 μmol kg−1) produced nitrite accumulation in the peritoneal cavity maximal at 60 min.

NCX‐1015 dose‐dependently induced the steroid sensitive cell surface marker CD163 in human peripheral blood mononuclear cells (PBMCs). NCX‐1015 was more potent than prednisolone in inducing CD163. Similarly, lipopolysaccharide induced interleukin‐1β release from these cells was inhibited by NCX‐1015 with higher potency than prednisolone.

In the zymosan peritonitis model, NCX‐1015 was more active than prednisolone in suppressing neutrophil extravasation (ED50 of 5.5 and 25.8 μmol kg−1, respectively), nitrite accumulation (ED50 of 1.38 and 22.2 μmol kg−1, respectively) and release of the chemokine KC (ED50 of 5.5 and 27.7 μmol kg−1, respectively) as determined at the 4 h time‐point. No differences were measured for the levels of interleukin‐1β or prostaglandin E2. NCX‐1015 administered orally was also found to be equally active. Co‐administration of the nitric oxide donors NOC‐18 ((z)‐1‐[(2‐aminoethyl)‐N‐(2‐aminoethyl)amino] diazen‐1‐ium‐1, 2‐diolate; 7.9 μmol kg−1) or sodium nitroprusside (13.8 μmol kg−1) with prednisolone resulted in an additive anti‐migratory action.

In a chronic model of granulomatous tissue inflammation, administration of NCX‐1015 (13.9 μmol kg−1) from day 1 (i.e. after induction of inflammation) was more effective than prednisolone in reducing the granuloma dry weight, and this was associated to a lower anti‐angiogenic effect.

In conclusion we show that NCX‐1015 is more potent than prednisolone in controlling several, though not all, parameters of acute and chronic inflammation, and propose that this effect may be due to a co‐operation between the steroid moiety and nitric oxide or related species released in biological fluids. Whereas this aspect needs to be further clarified, we propose NCX‐1015 as the first member of a novel class of anti‐inflammatory compounds, the nitro‐steroids.

British Journal of Pharmacology (2000) 131, 1345–1354; doi:10.1038/sj.bjp.0703704
Endothelial dysfunction in atherosclerotic mice: improved relaxation by combined supplementation with L‐arginine‐tetrahydrobiopterin and enhanced vasoconstriction by endothelin
Authors: Jiang Jiahua , Valen Guro , Tokuno Shinichi , Thorén Peter , Pernow John
Published: January 2005

Mice lacking the apolipoprotein E and low density lipoprotein receptor genes (E°xLDLR°) develop atherosclerosis and endothelial dysfunction. The aim of this study was to characterize the roles of L‐arginine and tetrahydrobiopterin (BH4) for endothelium‐dependent relaxation and the changes in the vasoconstrictor response to endothelin‐1 (ET‐1) in thoracic aortic rings of E°xLDLR° mice.

Histological examination revealed severe atherosclerosis of the thoracic aorta of E°xLDLR° mice. Relaxations induced by acetylcholine (Ach), but not that to sodium nitroprusside, were significantly impaired in E°xLDLR° mice compared to control mice indicating attenuated endothelium‐dependent relaxations.

Preincubation with the nitric oxide (NO) substrate L‐arginine did not affect, whereas the co‐factor for NO synthase, BH4, slightly improved the relaxations induced by Ach. Combined preincubation with L‐arginine and BH4 induced a pronounced enhancement of Ach‐induced relaxations in E°xLDLR° mice. The relaxations induced by Ach in E°xLDLR° mice in the presence of L‐arginine and BH4 were not different from those observed in control mice.

Preincubation with superoxide dismutase did not affect Ach‐induced relaxations in aorta from E°xLDLR° mice.

The contractile response to ET‐1 was enhanced in E°xLDLR° mouse aorta. The contractions were abolished by the ETA receptor antagonist LU 135252. The ETB receptor agonist sarafotoxin 6c did not induce contractions or relaxations.

It is concluded that endothelial dysfunction of E°xLDLR° mouse aorta is reversed by combined administration of L‐arginine and BH4. In addition, the ETA receptor‐mediated vasoconstriction by ET‐1 is enhanced in E°xLDLR° mice.

British Journal of Pharmacology (2000) 131, 1255–1261; doi:10.1038/sj.bjp.0703705
Effects of diclofenac, aceclofenac and meloxicam on the metabolism of proteoglycans and hyaluronan in osteoarthritic human cartilage
Authors: Blot Laurent , Marcelis Annette , Devogelaer Jean‐Pierre , Manicourt Daniel‐Henri
Published: January 2005

Since nonsteroidal anti‐inflammatory drugs (NSAIDs) may impair the ability of the chondrocyte to repair its damaged extracellular matrix, we explored the changes in the metabolism of newly synthesized proteoglycan and hyaluronan (HA) molecules produced by aceclofenac, diclofenac and meloxicam in human osteoarthritic (OA) cartilage.

Explants were sampled from the medial femoral condyle and were classified by use of the Mankin's histological‐histochemical grading system. Cartilage specimens exhibited moderate (M) OA in 20 subjects and had severe (S) OA in 20.

Cartilage explants were pulsed with [‐3H]‐glucosamine and chased in the absence or in the presence of 0.3–3 μg ml−1 of either aceclofenac, diclofenac or meloxicam. After papain digestion, the labelled chondroitin sulphate ([‐3H]‐proteoglycans) and [‐3H]‐HA molecules present in the tissue and media were purified by anion‐exchange chromatography.

In cartilage with MOA and SOA, the metabolic balance of proteoglycan and HA was unaffected by diclofenac. In contrast, and in a dose‐dependent manner, aceclofenac and meloxicam both increased the synthesis of proteoglycans and HA in explants with MOA and SOA; these two NSAIDs also reduced significantly the net loss of [‐3H]‐proteoglycans and [‐3H]‐HA molecules from cartilage explants.

The data obtained in short‐term in vitro cultures indicate that, at the concentrations found in synovial fluid, aceclofenac and meloxicam may exert a favourable effect on the overall metabolism of proteoglycans and HA in cartilage with MOA and SOA.

British Journal of Pharmacology (2000) 131, 1413–1421; doi:10.1038/sj.bjp.0703710
Distinct pharmacology of rat and human histamine H3 receptors: role of two amino acids in the third transmembrane domain
Authors: Ligneau X , Morisset S , Tardivel‐Lacombe J , Gbahou F , Ganellin C R , Stark H , Schunack W , Schwartz J ‐C , Arrang J ‐M
Published: January 2005

Starting from the sequence of the human histamine H3 receptor (hH3R) cDNA, we have cloned the corresponding rat cDNA. Whereas the two deduced proteins show 93.5% overall homology and differ only by five amino acid residues at the level of the transmembrane domains (TMs), some ligands displayed distinct affinities. Thioperamide and ciproxifan were about 10 fold more potent at the rat than at the human receptor, whereas FUB 349 displayed a reverse preference. Histamine, (R)α‐methylhistamine, proxyfan or clobenpropit were nearly equipotent at H3 receptors of both species. The inverse discrimination patterns of ciproxifan and FUB 349 were partially changed by mutation of one amino acid (V122A), and fully abolished by mutation of two amino acids (A119T and V122A), in TM3 of the rH3R located in the vicinity of Asp114 purported to salt‐link the ammonium group of histamine. Therefore, these two residues appear to be responsible for the distinct pharmacology of the H3R in the two species.

British Journal of Pharmacology (2000) 131, 1247–1250; doi:10.1038/sj.bjp.0703712
Selective mGluR5 antagonists MPEP and SIB‐1893 decrease NMDA or glutamate‐mediated neuronal toxicity through actions that reflect NMDA receptor antagonism
Authors: O'Leary Deirdre M , Movsesyan Vilen , Vicini Stefano , Faden Alan I
Published: January 2005

The metabotropic glutamate receptors (mGluRs) are a family of G‐protein linked receptors that can be divided into three groups (group I, II and III). A number of studies have implicated group I mGluR activation in acute neuronal injury, but until recently it was not possible to pharmacologically differentiate the roles of the two individual subunits (mGluR1 and mGluR5) in this group.

We investigated the role of mGluR5 in acute NMDA and glutamate mediated neurodegeneration in cultured rat cortical cells using the mGluR5 antagonists MPEP and SIB‐1893, and found that they provide significant protection at concentrations of 20 or 200 μM.

These compounds act as effective mGluR5 antagonists in our cell culture system, as indicated by the ability of SIB‐1893 to prevent phosphoinositol hydrolysis induced by the specific mGluR5 agonist, (RS)‐2‐chloro‐5‐hydroxyphenylglycine (CHPG).

However, they also significantly reduce NMDA evoked current recorded from whole cells voltage clamped at −60 mV, and significantly decrease the duration of opening of NMDA channels recorded in the outside out patch configuration.

This suggests that although MPEP and SIB‐1893 are effective mGluR5 antagonists, they also act as noncompetitive NMDA receptor antagonists. Therefore, the neuroprotective effects of these compounds are most likely mediated through their NMDA receptor antagonist action, and caution should be exercised when drawing conclusions about the roles of mGluR5 based on their use.

British Journal of Pharmacology (2000) 131, 1429–1437; doi:10.1038/sj.bjp.0703715
Mechanism of action of the hypnotic zolpidem in vivo
Authors: Crestani Florence , Martin James R , Möhler Hanns , Rudolph Uwe
Published: January 2005

Zolpidem is a widely used hypnotic agent acting at the GABAA receptor benzodiazepine site. On recombinant receptors, zolpidem displays a high affinity to α1‐GABAA receptors, an intermediate affinity to α2‐ and α3‐GABAA receptors and fails to bind to α5‐GABAA receptors. However, it is not known which receptor subtype is essential for mediating the sedative‐hypnotic action in vivo. Studying α1(H101R) mice, which possess zolpidem‐insensitive α1‐GABAA receptors, we show that the sedative action of zolpidem is exclusively mediated by α1‐GABAA receptors. Similarly, the activity of zolpidem against pentylenetetrazole‐induced tonic convulsions is also completely mediated by α1‐GABAA receptors. These results establish that the sedative‐hypnotic and anticonvulsant activities of zolpidem are due to its action on α1‐GABAA receptors and not on α2‐ or α3‐GABAA receptors.

British Journal of Pharmacology (2000) 131, 1251–1254; doi:10.1038/sj.bjp.0703717
The role of M2‐muscarinic receptors in mediating contraction of the pig urinary bladder in vitro
Authors: Yamanishi Tomonori , Chapple Christopher R , Yasuda Kosaku , Chess‐Williams Russell
Published: January 2005

In urinary bladder, M2‐muscarinic receptors predominate, but it is the smaller population of M3‐receptors which mediate detrusor contraction. This study examines the M2 : M3 ratio and the role of M2‐receptors in contraction of pig urinary bladder.

Competition experiments with [3H]‐QNB determined the ratio of M2 : M3. In functional studies, affinity values (pKB) for 4‐DAMP, darifenacin and methoctramine were calculated. Similar experiments were performed on tissues following selective M3‐inactivation (incubation with 40 nM 4‐DAMP mustard in the presence of 1 μM methoctramine to protect M2‐receptors), precontraction with 50 mM KCl and relaxation with isoprenaline (30 μM) or forskolin (1 μM).

In competition binding, displacement of [3H]‐QNB by 4‐DAMP, darifenacin and methoctramine best fitted a two‐site model suggesting a predominant (70–80%) population of M2‐receptors.

On normal detrusor in vitro, 4‐DAMP and methoctramine caused surmountable antagonism of responses to carbachol with pKB values of 9.37±0.07 and 6.05±0.05 respectively. Darifenacin caused unsurmountable antagonism, the apparent pKB value being 8.61±0.10.

In tissues where the M3‐receptors had been inactivated and cyclic AMP levels elevated, 4‐DAMP and darifenacin were less potent, with apparent pKB values of 8.72±0.08 and 6.74±0.07. In contrast, methoctramine was more potent, the apparent pKB value increasing significantly to 6.86±0.06.

These data suggest that the pig bladder possesses a similar muscarinic receptor population to the human bladder and that the M3‐receptor subtype mediates contraction of the normal detrusor muscle. However an involvement of M2‐receptors in contraction can be observed following pharmacological manipulation of the receptor population.

British Journal of Pharmacology (2000) 131, 1482–1488; doi:10.1038/sj.bjp.0703719
In vivo modulation of vagal‐identified dorsal medullary neurones by activation of different 5‐Hydroxytryptamine2 receptors in rats
Authors: Sévoz‐Couche Caroline , Spyer K Michael , Jordan David
Published: January 2005

In in vivo experiments, DOI (a 5‐HT2 receptor agonist), MK‐212 (a 5‐HT2C receptor agonist), and BW‐723C86 (a 5‐HT2B receptor agonist) were applied by ionophoresis to neurones in the rat nucleus tractus solitarius (NTS) receiving vagal afferent input.

The majority of the putative ‘monosynaptically’ vagal activated cells were inhibited by both MK‐212 (4/6) and DOI (2/4), but unaffected by BW‐723C86 (12/14). In contrast, ‘polysynaptically’ activated NTS cells were excited by both BW‐723C86 (13/19) and DOI (9/10). Inactive ‘intermediate’ cells were inhibited by BW‐723C86 (9/12), MK‐212 (5/6) and DOI (3/4), whilst active cells of this group were excited by BW‐723C86 (7/13) and DOI (5/5).

The selective 5‐HT2B receptor antagonist LY‐202715 significantly reduced the excitatory actions of BW‐723C86 on ‘intermediate’ and ‘polysynaptic’ cells (13/13), but not the inhibitory effects observed on inactive Group 2 cells (n=5) whereas the selective 5‐HT2C receptor antagonist RS‐102221 reversed the inhibitory effects of MK‐212 and DOI on ‘monosynaptic and ‘intermediate’ neurones.

Cardio‐pulmonary afferent stimulation inhibited two of four putative ‘monosynaptically’ activated calls and all four inactive intermediate cells. These were also inhibited by DOI and MK‐212. In contrast, cardio‐pulmonary afferents excited all five active intermediate cells and all six putative ‘polysynaptically’ activated NTS cells, while all were also previously excited by BW‐723C86 and/or DOI.

In conclusion, these data demonstrate that neurones in the NTS are affected differently by 5‐HT2 receptor ligands, in regard of their vagal postsynaptic location, the type of cardio‐pulmonary afferent they receive and the different 5‐HT2 receptors activated.

British Journal of Pharmacology (2000) 131, 1445–1453; doi:10.1038/sj.bjp.0703722
The role of the Ca2+ regulatory sites of skeletal troponin C in modulating muscle fibre reactivity to the Ca2+ sensitizer bepridil
Authors: Kischel P , Bastide B , Potter J D , Mounier Y
Published: January 2005

The Ca2+‐sensor protein troponin C (TnC) exerts a key role in the regulation of muscle contraction, and constitutes a target for Ca2+ sensitizer compounds, such as bepridil, known to increase its apparent Ca2+ affinity. Moreover, bepridil has been reported to exert a differential effect in slow and fast skeletal muscle fibres, which express the slow/cardiac and fast TnC isoform, respectively.

The role of the TnC isoform in establishing the differential effect of bepridil was assessed in slow soleus and fast tibialis rat skinned fibres, by extraction of endogenous TnC and consecutive reconstitution with either slow or fast recombinant TnC. A mutant (VG2), lacking the regulatory site II, was also used to distinguish the role of each regulatory site.

Fast tibialis fibres reconstituted with cardiac TnC exhibited a typical slow bepridil reactivity, while slow soleus fibres reincorporated with fast TnC displayed a typically fast reactivity to bepridil. These results indicated that the differential effect of bepridil in slow and fast fibres is related to the TnC isoform predominantly expressed in a fibre.

Experiments with the VG2 mutant demonstrated that BPD can achieve an increase in the Ca2+ affinity in the absence of a functional site II. Thus, site I was necessary for the BPD effect to be independent of the Ca2+ concentration. Moreover, the amplitude of the reinforcement in the Ca2+ affinity, induced by the binding of bepridil to the TnC molecule, is dependent on the number of functional regulatory sites, the larger affinity reinforcement being detected when only one regulatory site (either site I or II) is functional.

British Journal of Pharmacology (2000) 131, 1496–1502; doi:10.1038/sj.bjp.0703727
Nifedipine suppresses neointimal thickening by its inhibitory effect on vascular smooth muscle cell growth via a MEK‐ERK pathway coupling with Pyk2
Authors: Hirata Akihiko , Igarashi Masahiko , Yamaguchi Hiroshi , Suwabe Akira , Daimon Makoto , Kato Takeo , Tominaga Makoto
Published: January 2005

The aim of this study was to determine whether nifedipine could suppress an atherogenic process such as balloon‐injured intimal thickening in vivo and the proliferation of vascular smooth muscle cells (VSMC) in vitro.

First, we examined the in vivo effect of nifedipine to determine whether it could suppress intimal thickening induced by balloon catheterization. Sprague‐Dawley (SD) rats were divided into three groups (L, nifedipine 0.3 mg kg−1 day−1; H, nifedipine 3 mg kg−1 day−1; C, no nifedipine), and Alzet® osmotic pumps were implanted in their backs for continuous administration. The neointimal layers were completely occupied by proliferated VSMC, and the area ratios of neointima/media treated with nifedipine significantly decreased dose‐dependently compared to those of the control. Neither blood pressure nor lipid levels changed among the three groups.

We next evaluated the in vitro effect of nifedipine on the proliferation of cultured rat VSMC. Nifedipine decreased the values of [3H]‐thymidine incorporation and total cellular protein content as well as the levels of phosphorylated extracellular signal‐regulated protein kinase (ERK) 1/2, mitogen‐activated protein kinase kinase (MEK) 1/2, and even the phosphorylation of Pyk2, in dose‐dependent fashions. In addition, nifedipine suppressed the levels of proliferative cell nuclear antigen (PCNA) dose‐dependently in both VSMC and balloon‐injured thoracic aortae.

These results indicate that nifedipine has an inhibitory effect on intimal thickening by attenuating intimal VSMC proliferation, suggesting that nifedipine could be effective for preventing the progression of atherosclerotic plaque as in restenosis after angioplasty.

British Journal of Pharmacology (2000) 131, 1521–1530; doi:10.1038/sj.bjp.0703730
Collar‐induced elevation of mRNA and functional activity of 5‐HT1B receptor in the rabbit carotid artery
Authors: Geerts Inge S , De Meyer Guido R Y , Bult Hidde
Published: January 2005

Hypersensitivity to serotonin (5‐HT) develops in rabbit collared carotid arteries. Previous data demonstrated the involvement of 5‐HT1‐like receptors which are not active in normal carotid arteries. This study investigated the interaction in the rabbit carotid artery between 5‐HT and a moderate tone as this can uncover functional 5‐HT1‐like receptors. Furthermore, the expression of messenger RNA (mRNA) and protein of 5‐HT1B, 5‐HT1D and 5‐HT2A receptors was addressed.

Silicone collars were placed around the carotid arteries of male New Zealand White rabbits for 1 week. Rings from inside (=collar) and outside (=sham) the collar were either mounted in isolated organ baths for isometric force measurements or frozen in liquid nitrogen to isolate total RNA or proteins which were subsequently analysed by respectively reverse transcriptase‐polymerase chain reaction and Western blot analysis.

In sham and collared rings concentration‐response curves (CRC's) to 5‐HT were monophasic. Only in collared segments the presence of a 5‐HT2A antagonist (spiperone or ketanserin, 0.1 μM) revealed a biphasic CRC which was even more pronounced when a moderate tone was induced by KCl pointing to functional 5‐HT1‐like receptors.

The rabbit carotid artery constitutively expressed 5‐HT1B and 5‐HT2A mRNA, not 5‐HT1D mRNA. Manipulation of the carotid artery increased the 5‐HT1B mRNA level. Collar placement raised it even further. The 5‐HT2A mRNA level remained unchanged. All the anti‐5‐HT receptor antibodies tested resulted in variable, non specific patterns with multiple bands.

In conclusion, collar placement elevates mRNA expression and activity of the 5‐HT1B receptor in the rabbit carotid artery.

British Journal of Pharmacology (2000) 131, 1723–1731; doi:10.1038/sj.bjp.0703732
Spontaneous release of large packets of noradrenaline from sympathetic nerve terminals in rat mesenteric arteries in vitro
Authors: Brock James A , Dunn William R , Boyd Nicola S F , Wong Danny K Y
Published: January 2005

Continuous amperometry was used to monitor noradrenaline (NA) release from sympathetic nerves supplying rat mesenteric arteries in vitro. During electrical stimulation the amplitude of oxidation currents evoked by successive stimuli varied over a small range, with occasional events of larger amplitude. In the absence of stimulation, spontaneous oxidation currents (s.o.cs) were recorded. The frequency of s.o.cs was increased by α‐latrotoxin (1 nM). This agent also increased the frequency of spontaneous excitatory junction potentials (s.e.j.ps), which monitor the packeted release of adenosine 5′ triphosphate (ATP). The frequency of s.o.cs recorded 20–25 min after applying α‐latrotoxin was about four times the control value, but that of s.e.j.ps was about 30 times the control value. The findings suggest that continuous amperometry can detect the spontaneous packeted release of NA, probably from large dense‐cored vesicles. In contrast, s.e.j.ps monitor spontaneous release of neurotransmitter (ATP) from a different store, most likely the small dense‐cored vesicles.

British Journal of Pharmacology (2000) 131, 1507–1511; doi:10.1038/sj.bjp.0703733
Effect of protease‐activated receptor (PAR)‐1, −2 and −4‐activating peptides, thrombin and trypsin in rat isolated airways
Authors: Chow Jean M , Moffatt James D , Cocks Thomas M
Published: January 2005

Mechanisms of relaxation and contraction to protease‐activated receptor‐ (PAR) tethered ligand peptides (SFLLRN/TFLLR, SLIGRL and GYPGKF (all C‐terminally amidated) for PAR1, PAR2 and PAR4, respectively) and enzymes (thrombin and trypsin) were investigated in isolated segments of rat trachea, main and first order intrapulmonary bronchi.

In airway segments previously exposed to SLIGRL, SFLLRN caused contractions that were potentiated by indomethacin, but were independent of mast cell degranulation. Contractions to TFLLR in the intrapulmonary bronchi were similarly potentiated by indomethacin.

SLIGRL caused epithelium‐dependent relaxations which were unaffected by NG‐nitro‐L‐arginine, 1‐H‐oxodiazol‐[1,2,4]‐[4,3‐a]quinoxaline‐1‐one or zinc‐protoporphyrin‐IX but were abolished by haemoglobin in all three regions of the airways. Relaxations to SLIGRL were markedly attenuated by indomethacin only in the main and intrapulmonary bronchi.

GYPGKF caused epithelium‐dependent relaxations in all three regions of the airway which were only significantly inhibited by indomethacin in the intrapulmonary bronchi.

In general, thrombin and trypsin failed to cause any response in the airways tested.

Intense PAR2‐immunoreactivity was observed on airway epithelium. PAR1‐immunoreactivity was faint on airway epithelium and smooth muscle, but was prevalent in mast cells.

These findings indicate that PAR2 and possibly PAR4 present on rat airway epithelia mediate smooth muscle relaxation via cyclo‐oxygenase‐dependent and ‐independent mechanisms. PAR1‐mediated contractions were most likely due to activation of smooth muscle receptors. The general failure of thrombin and trypsin to cause responses which may have been due to endogenous protease inhibitors, highlights the need for caution in assessing pathophysiological roles for PARs if only enzymes are used to activate PARs.

British Journal of Pharmacology (2000) 131, 1584–1591; doi:10.1038/sj.bjp.0703738
Angiotensin‐converting enzyme (ACE) inhibition attenuates insulin‐like growth factor‐I (IGF‐I) induced cardiac fibroblast proliferation
Authors: Van Eickels Martin , Vetter Hans , Grohé Christian
Published: January 2005

The effects of angiotensin‐converting enzyme (ACE) inhibition and angiotensin type 1 (AT1) receptor blockade on insulin‐like growth factor‐I (IGF‐I) induced proliferation and immediate‐early‐gene expression of neonatal rat cardiac fibroblasts were investigated. Moreover the role of the IGF‐I receptor (IGF‐IR) in this process was evaluated.

IGF‐I (10−9–10−7 M) stimulated neonatal rat cardiac fibroblast growth in a dose‐dependent fashion (maximum: 3.5±0.1 fold, 10−7 M), as determined by 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation. ACE inhibition or AT1 receptor blockade attenuated the IGF‐I (10−7 M) induced neonatal rat cardiac fibroblast growth in a concentration‐dependent fashion (moexiprilat: 50±2%, enalaprilat: 31±2%, CV11974; 58±1%, all: 10−7 M).

IGF‐I stimulated cellular growth was accompanied by an upregulation of the immediate early genes c‐Fos (2.4±0.3 fold), Egr‐1 (4.7±1.1 fold) and Sp1 (6.2±0.7 fold). IGF‐I induced expression was completely inhibited by ACE inhibition or AT1 receptor blockade.

Stimulation with IGF‐I or Ang II (10−7 M) increased IGF‐IR expression 5.7±0.5 fold and 3.6±0.5 fold respectively. The IGF‐I induced overexpression of the IGF‐IR was reduced by ACE inhibition with moexiprilat (10−7 M) by 79±7% and by AT1 receptor blockade with CV11974 (10−7 M) by 79±5%.

These data demonstrate that the mitogenic action of IGF‐I in neonatal rat cardiac fibroblasts is in part mediated by activation of the renin‐angiotensin system (RAS) with subsequent upregulation of IGF‐IR expression. This observation has important implications for the treatment of cardiac diseases with ACE inhibitors alone and their combination with IGF‐I or growth hormone.

British Journal of Pharmacology (2000) 131, 1592–1596; doi:10.1038/sj.bjp.0703740
Lack of run‐down of smooth muscle P2X receptor currents recorded with the amphotericin permeabilized patch technique, physiological and pharmacological characterization of the properties of mesenteric artery P2X receptor ion channels
Authors: Lewis C J , Evans R J
Published: January 2005

Immunoreactivity for P2X1, P2X4 and P2X5 receptor subtypes was detected in the smooth muscle cell layer of second and third order rat mesenteric arteries immunoreactivity, for P2X2, P2X3, P2X6 and P2X7 receptors was below the level of detection in the smooth muscle layer.

P2X receptor‐mediated currents were recorded in patch clamp studies on acutely dissociated mesenteric artery smooth muscle cells. Purinergic agonists evoked transient inward currents that decayed rapidly in the continued presence of agonist (τ∼200 ms). Standard whole cell responses to repeated applications of agonist at 5 min intervals ran down. Run‐down was unaffected by changes in extracellular calcium concentration, intracellular calcium buffering or the inclusion of ATP and GTP in the pipette solution.

Run‐down was overcome and reproducible responses to purinergic agonists were recorded using the amphotericin permeabilized patch recording configuration.

The rank order of potency at the P2X receptor was ATP=2 methylthio ATP>α,β‐methylene ATP>CTP=l‐β,γ‐methylene ATP. Only ATP and 2meSATP were full agonists. The P2 receptor antagonists suramin and PPADS inhibited P2X receptor‐mediated currents with IC50s of 4 μM and 70 nM respectively.

These results provide further characterization of artery P2X receptors and demonstrate that the properties are dominated by a P2X1‐like receptor phenotype. No evidence could be found for a phenotype corresponding to homomeric P2X4 or P2X5 receptors or to heteromeric P2X1/5 receptors and the functional role of these receptors in arteries remains unclear.

British Journal of Pharmacology (2000) 131, 1659–1666; doi:10.1038/sj.bjp.0703744
Relationships between the Na+/K+ pump and ATP and ADP content in mouse pancreatic islets: effects of meglitinide and glibenclamide
Authors: Elmi Adrian , Idahl Lars‐Åke , Sehlin Janove
Published: January 2005

We have previously demonstrated that both D‐glucose and glibenclamide stimulate the Na+/K+ pump and suggested that this may be part of the membrane repolarization process, following the primary depolarization by these agents. The aim of this study was to investigate whether the non‐sulphonylurea meglitinide (HB 699) exerts similar effects as glibenclamide or glucose on the islet Na+/K+ pump and if effects of meglitinide or glibenclamide on this pump activity is paralleled by changes in islet ATP content and/or ATP/ADP ratio.

The acyl‐amino‐alkyl benzoic acid derivative, meglitinide, stimulated the islet ouabain‐sensitive portion of 86Rb+ influx (Na+/K+ pump) by 53%, while the ouabain‐resistant portion was inhibited by 70%. The stimulatory effect was not additive to that caused by D‐glucose, suggesting that both agents may activate the Na+/K+ pump via the same mechanism.

Glibenclamide (10 μM) decreased the islet ATP and ADP content as well as the ATP/ADP ratio at 0 mM glucose. These effects were no longer observed at 10 mM glucose.

Meglitinide (10 or 50 μM) lowered the islet ATP and ADP content at 0 mM glucose without affecting the ATP/ADP ratio. At 10 mM glucose, however, 10 μM of the drug reduced the islet ATP content but not the ATP/ADP ratio, while 50 μM of the drug, besides lowering the ATP content, also reduced the ATP/ADP ratio.

Diazoxide (0.5 mM) increased the islet ATP content in the absence of glucose, an effect not seen in the presence of 10 mM glucose.

The rate of glucose oxidation at 1, 10 or 20 mM of the sugar was not affected by glibenclamide (0.1–10 μM) and at 10 or 20 mM of the sugar not affected by meglitinide (1–100 μM).

These results suggest that glibenclamide and meglitinide lower the islet ATP level by indirectly activating the β‐cell Na+/K+ pump, which is a major consumer of ATP in the islets, while diazoxide increases the ATP level due to inhibition of the pump.

British Journal of Pharmacology (2000) 131, 1700–1706; doi:10.1038/sj.bjp.0703745
Suppression of adjuvant arthritis of rats by a novel matrix metalloproteinase‐inhibitor
Authors: Hamada Tetsuo , Arima Nobuyuki , Shindo Miki , Sugama Kazushige , Sasaguri Yasuyuki
Published: January 2005

BAY 12‐9566 (4‐[4‐(chlorophenyl)phenyl]‐4‐oxo‐2S‐(phenylthiomethyl) butanoic acid) is a newly developed, synthetic matrix metalloproteinase (MMP) inhibitor (MMPI) that selectively inhibits MMP‐2, MMP‐3 and MMP‐9 isozymes. We study the effect of BAY 12‐9566 on inflammation and cartilage destruction in adjuvant‐induced arthritis (AA) in rats.

Rats were injected with adjuvant and treated for 21 days with vehicle, Indomethacin or BAY 12‐9566. AA was assessed: by measuring arthritic index, paw volume, urinary pyridinoline (Pyr) and deoxypyridinoline (Dpyr); by examining joint inflammation; and by microscopic morphometry of articular cartilages.

Oral treatment of rats for 22 days with 50 mg kg−1 body weight/d BAY 12‐9566 showed decreased AA as determined by improvement in body weight gain (P<0.01), arthritic index (P<0.05) and swelling of paws contralateral to the adjuvant injection site (P<0.05). Neutrophil infiltration and collagen degradation were also significantly lower (P<0.01) in this treatment group. Cartilage destruction was successfully suppressed (P<0.01) in rats treated with either 50 mg kg−1 body weight/d BAY 12‐9566 or 1 mg kg−1 body weight/d Indomethacin.

These results indicate that BAY 12‐9566 successfully suppressed inflammation and cartilage destruction in rats with AA. Moreover, these results also suggested that MMP‐2, MMP‐3 and MMP‐9 are involved in arthritic diseases such as rheumatoid arthritis.

British Journal of Pharmacology (2000) 131, 1513–1520; doi:10.1038/sj.bjp.0703751
Mechanisms of vasorelaxation induced by eicosapentaenoic acid (20:5n‐3) in WKY rat aorta
Authors: Engler Mary B , Engler Marguerite M , Browne Amanda , Sun Yi‐Ping , Sievers Richard
Published: January 2005

The vasorelaxant activity of eicosapentaenoic acid (EPA, 20:5n‐3), the omega‐3 polyunsaturated fatty acid, was investigated in isolated Wistar Kyoto (WKY) rat aortae by measuring isometric tension.

Eicosapentaenoic acid (1–100 μM) relaxed rat aortae contracted with high K+ (80 mM) or noradrenaline (NA, 1 μM) in a concentration‐dependent manner. Contractions induced by Bay K 8644 or increasing concentrations of calcium were unaffected by EPA.

The relaxant effect of EPA (3–100 μM) was significantly inhibited by indomethacin (10 μM), the cyclo‐oxygenase inhibitor, but not by the nitric oxide (NO) synthesis inhibitor, Nω‐nitro‐L‐arginine methyl ester hydrochloride (L‐NAME, 100 μM). Removal of the endothelium did not alter EPA‐induced relaxations.

In Ca2+‐free, EGTA 2 mM solution, EPA (10–30 μM significantly inhibited NA‐sustained contractions. Incubation with EPA (5, 10 μM) diminished both NA‐induced (1 μM) phasic and sustained contractions.

The vasorelaxant effects of EPA (30 μM) on NA‐induced (1 μM) contractions were significantly inhibited by the K+ channel blocker, glibenclamide (10 μM), but not tetraethylammonium (1 mM). Moreover, indomethacin and glibenclamide combined significantly inhibited EPA‐induced (1–100 μM) responses.

These results indicate EPA exerts its endothelium‐independent vasorelaxant effects in WKY rat aortae through production of prostanoids which activate K+ATP channels. Inhibition of Ca2+ mobilization from intracellular pools and influx through the non‐L‐type, but not the L‐type, Ca2+ channel are also possible mechanisms action of EPA's.

British Journal of Pharmacology (2000) 131, 1793–1799; doi:10.1038/sj.bjp.0703754
Ivermectin excretion by isolated functionally intact brain endothelial capillaries
Authors: Nobmann Stephanie , Bauer Björn , Fricker Gert
Published: January 2005

Functionally intact brain endothelial capillaries were isolated from porcine brain. p‐Glycoprotein was localized at the lumenal membrane of intact capillaries by immunohistochemistry using a murine monoclonal antibody and a secondary FITC fluorescent labelled anti‐mouse IgG. Western blot staining of p‐glycoprotein in isolated endothelial cells confirmed the immunohistochemistry.

Excretion of the fluorescent labelled anthelmintic drug Ivermectin (BODIPY‐Ivermectin) was studied in the isolated brain endothelial capillaries. Drug accumulation in the capillary lumen was visualized by fluorescence confocal laser scanning microscopy and was measured by image analysis. Secretion of BODIPY‐Ivermectin into the capillary lumen exhibited characteristics of specific and energy‐dependent transport. Steady state lumenal fluorescence intensity averaged 1.6 times cellular fluorescence and was reduced 3 – 4 times below cellular levels when metabolism was inhibited by NaCN.

BODIPY‐Ivermectin secretion was inhibited in a concentration‐dependent manner by unlabeled Ivermectin. In addition, lumenal but not cellular fluorescence intensity was significantly decreased when capillaries were incubated with PSC‐833, Cyclosporin A or Verapamil, all inhibitors of p‐glycoprotein. Conversely, unlabelled Ivermectin reduced the p‐glycoprotein (Pgp)‐mediated secretion of a fluorescent derivative of Verapamil, (BODIPY‐Verapamil).

BODIPY‐Ivermectin secretion was not affected in the presence of Leucotriene C4(LTC4), a potent inhibitor of multidrug resistance related protein (mrp)‐mediated transport processes. In addition, excretion of Fluorescein‐Methotrexate, an mrp‐substrate, was not inhibited by Ivermectin.

Uptake experiments with isolated porcine brain capillary cells showing increased cellular uptake of BODIPY‐Ivermectin in the presence of unlabelled drug or PSC‐833 supported the findings of a Pgp interaction in intact capillaries.

The data are consistent with BODIPY‐Ivermectin and Ivermectin being transported across the lumenal membrane of brain capillaries. For the first time Pgp‐interaction of Ivermectin at the blood brain barrier is demonstrated on a cellular level in an intact vascular tissue.

British Journal of Pharmacology(2001)132, 722–728; doi:10.1038/sj.bjp.0703762
Differences in action potential and early afterdepolarization properties in LQT2 and LQT3 models of long QT syndrome
Authors: Studenik Christian R , Zhou Zhengfeng , January Craig T
Published: January 2005

Long OT syndrome has many causes from both acquired and congenital disorders. For the congenital disorders, their presentation and disease course are not identical. We studied two pharmacological models of long QT syndrome (LQT) to identify differences in cellular electrophysiological properties that may account for this. LQT2 was simulated by suppression of the rapidly activating delayed rectifier potassium current (IKr) with the drug E‐4031, and LQT3 was simulated by slowing of the sodium current (INa) decay with the toxin ATX II.

Single rabbit ventricular cell action potentials were studied using the amphotericin B perforated patch clamp technique. Action potential and early afterdepolarization (EAD) properties were rigorously defined by the frequency power spectra obtained with fast Fourier transforms.

The E‐4031 (n=43 myocytes) and ATX II (n=50 myocytes) models produced different effects on action potential and EAD properties. The major differences are that ATX II, compared with E‐4031, caused greater action potential prolongation, more positive plateau voltages, lower amplitude EADs with less negative take‐off potentials, greater time to the EAD peak voltage, and longer duration EADs. Despite causing greater action potential prolongation, the incidence of EAD induction was much less with the ATX II model (28%) than with the E‐4031 model (84%). Thus these two pharmacological models have strikingly different cellular electrophysiological properties.

Our findings provide cellular mechanisms that may account for some differences in the clinical presentation of LQT2 and LQT3.

British Journal of Pharmacology (2001) 132, 85–92; doi:10.1038/sj.bjp.0703770
Combined blockade of endothelin‐1 and thromboxane A2 receptors against postischaemic contractile dysfunction in rat hearts
Authors: Hornstein Pius S , Zaugg Christian E , Zhu Peili , Allegrini Peter R , Buser Peter T
Published: January 2005

Endothelin‐1 (ET‐1) may play a role in myocardial ischaemia/reperfusion injury because both the release and vasoconstrictor effect of ET‐1 are increased after ischaemia. Since the increased vasoconstrictor effect of ET‐1 can be mediated by ET‐1‐induced release of thromboxane A2 (TXA2), the aim of this study was to test whether combined blockade of ET and TXA2 receptors protects the coronary flow, contractile performance, and cardiac energy metabolism during ischaemia and reperfusion.

Bosentan (antagonist for ETA and ETB receptors, 1 μM based on concentration‐response curves of ET‐1), SQ 30,741 (antagonist of TXA2 receptors, 0.1 μM), or the combination thereof was administered to isolated perfused rat hearts undergoing 15 min of global ischaemia and 60 min of reperfusion.

Neither bosentan or SQ 30,741 alone, nor the combination thereof, improved the incomplete postischaemic recovery of coronary flow, left ventricular developed pressure, phosphocreatine, or ATP. However, they attenuated ischaemia‐induced acidosis but this did not translate into a measurable effect on haemodynamic or metabolic variables.

Thus, combined blockade of ET and TXA2 receptors does not protect the coronary flow, contractile performance, and cardiac energy metabolism during ischaemia and reperfusion in isolated perfused rat hearts. This finding suggests that neither ET‐1 nor ET‐1‐induced release of TXA2 play a major role in the postischaemic recovery of the cardiac contractile function and energy metabolism.

British Journal of Pharmacology (2001) 132, 234–240; doi:10.1038/sj.bjp.0703773
Multiple P2X receptors on guinea‐pig pelvic ganglion neurons exhibit novel pharmacological properties
Authors: Zhong Yu , Dunn Philip M , Burnstock Geoffrey
Published: January 2005

Application of ATP and α,β‐methylene ATP (αβmeATP) to voltage‐clamped guinea‐pig pelvic neurons produced three types of inward currents. A fast‐desensitizing response was present in 5% (25/660) of neurons, 70% gave slowly‐desensitizing currents, and the remainder had biphasic responses.

Slowly‐desensitizing responses were characterized pharmacologically. The response to αβmeATP 100 μM was 46±27% (range 0 – 100%) of that evoked by ATP 100 μM in the same cell. Cross‐desensitization indicated the presence of αβmeATP‐sensitive and ‐insensitive receptors.

The concentration‐response curve for αβmeATP had an EC50 of 55 μM, and a Hill coefficient of 0.99, while at the αβmeATP‐insensitive receptor, ATP had an EC50 of 73 μM, with a Hill coefficient of 1.78.

The response to αβmeATP was blocked by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), suramin and Cibacron blue. However, the αβmeATP‐insensitive receptor was inhibited by PPADS, but not by the other two antagonists.

2′‐ (or 3′‐) O‐trinitrophenyl‐ATP was 10 times more potent in inhibiting responses to αβmeATP than to ATP (at the αβmeATP‐insensitive receptor).

Lowering extracellular pH potentiated responses to αβmeATP and ATP, while raising pH attenuated them.

Co‐application of Zn2+ (3 – 300 μM) inhibited the responses to αβmeATP and ATP, with IC50 values of 286 and 60 μM, respectively.

In conclusion, unlike rat and mouse pelvic ganglion neurons, which only express P2X2 homomers, at least three distinct P2X receptors are present in guinea‐pig pelvic neurons, probably homomeric P2X2, P2X3 and heteromeric P2X2/3 receptors. However, some of the novel pharmacological properties observed suggest that the guinea‐pig P2X receptor subtypes may differ from their rat orthologues.

British Journal of Pharmacology (2001) 132, 221–233; doi:10.1038/sj.bjp.0703778
Function of cyclo‐oxygenase‐1 and cyclo‐oxygenase‐2 in the ductus arteriosus from foetal lamb: differential development and change by oxygen and endotoxin
Authors: Coceani F , Ackerley C , Seidlitz E , Kelsey L
Published: January 2005

Prenatal patency of the ductus arteriosus is maintained mainly by prostaglandin(PG) E2. Here we have examined the relative importance of cyclo‐oxygenase‐1 (COX1) and cyclo‐oxygenase‐2 (COX2) for PGE2 formation in the foetal lamb ductus (0.65 gestation onwards).

Using fluorescence microscopy and immunogold staining, COX1 appeared more abundant than COX2 in endothelial and smooth muscle cells, and this difference was greater before‐term. Inside muscle cells, COX1 and COX2 immunoreactivity was located primarily in the perinuclear region. Endotoxin, given to the lamb in utero (∼0.1 μg kg−1), caused COX2 upregulation, while an opposite effect with disappearance of the enzyme followed endotoxin treatment in vitro (100 ng ml−1). COX1 immunoreactivity remained virtually unchanged with either treatment; however, this isoform as well as any induced COX2 migrated towards the outer cytoplasm.

The COX2 inhibitor L‐745,337 (1 – 10 μM) contracted the isolated ductus at term, the response being almost as high as that to indomethacin (dual COX1/COX2 inhibitor) over the same dose‐range. Conversely, L‐745,337 was relatively less effective in the premature.

Pretreatment of the premature in vivo with endotoxin enhanced the contraction of the ductus to L‐745,337, while in vitro endotoxin had a variable effect.

The premature ductus exhibited a stronger contraction to L‐745,337 following exposure to oxygen. On the other hand, the oxygen contraction, which is modest before‐term, was enhanced by L‐745,337.

We conclude that COX1 and COX2 develop unevenly in the ductus. While both enzymes contribute to PGE2 formation at term, COX1 is the major isoform in the premature. COX2, however, may acquire greater importance before‐term following physiological and pathophysiological stimuli.

British Journal of Pharmacology (2001) 132, 241–251; doi:10.1038/sj.bjp.0703779
α1B adrenergic receptors in gonadotrophin‐releasing hormone neurones: relation to Transport‐P
Authors: Al‐Damluji S , Shen W B , White S , Barnard E A
Published: January 2005

Peptidergic neurones accumulate amines via an unusual uptake process, designated Transport‐P. [3H]‐prazosin binds to α1 adrenoceptors on these cells and is displaceable by unlabelled prazosin in concentrations up to 10−7 M. However, at greater concentrations of prazosin, there is a paradoxical accumulation of [3H]‐prazosin which we have attributed to Transport‐P. Uptake of prazosin via Transport‐P is detectable at 10−10 M prazosin concentration, is linear up to 10−7 M and at greater concentrations becomes non‐linear. In contrast, in noradrenergic neurones, noradrenaline uptake is linear and saturates above 10−7 M. In noradrenergic neurones and in non‐neuronal cells, there is no uptake of prazosin in concentrations up to 10−6 M, suggesting that Transport‐P is a specialised function of peptidergic neurones.

Using a mouse peptidergic (gonadotrophin‐releasing hormone, GnRH) neuronal cell line which possesses Transport‐P, we have studied the interaction of α1 adrenoceptors with Transport‐P. Polymerase chain reactions and DNA sequencing of the products demonstrated that only the α1B sub‐type of adrenoceptors is present in GnRH cells.

In COS cells transfected with α1b adrenoceptor cDNA and in DDT1 MF‐2 cells which express native α1B adrenoceptors, [3H]‐prazosin was displaced by unlabelled prazosin in a normal equilibrium process, with no prazosin paradox in concentrations up to 10−6 M. In DDT1 MF‐2 cells, [3H]‐prazosin was displaced likewise by a series of α1 adrenergic agonists, none of which increased the binding of [3H]‐prazosin. Hence, the prazosin paradox is not due to some function of α1 adrenoceptors, such as internalization of ligand‐receptor complexes.

In neurones which possess Transport‐P, transfection with α1b adrenoceptor cDNA resulted in over‐expression of α1B adrenoceptors, but the prazosin paradox was unaltered. Thus, α1 adrenoceptors and Transport‐P mediate distinct functions in peptidergic neurones.

British Journal of Pharmacology (2001) 132, 336–344; doi:10.1038/sj.bjp.0703781
Effects of tyrosine kinase inhibitors on cell death induced by sodium fluoride and pertussis toxin in the pancreatic β‐cell line, RINm5F
Authors: Elliott Jim , Scarpello John H B , Morgan Noel G
Published: January 2005

Sodium fluoride causes apoptosis of pancreatic β‐cells and this response is enhanced by pre‐treatment with pertussis toxin. In the present study, tyrosine kinase inhibitors were used to investigate the mechanisms of action of NaF and pertussis toxin in the β‐cell line, RINm5F.

Exposure of RINm5F cells to low concentrations of genistein or tyrphostin A25 resulted in significant inhibition of cell death induced by 5 mM NaF. Higher concentrations (>25 μM) were cytotoxic in the absence of NaF but, paradoxically, the combination of genistein and NaF induced less cell death than when each agent was used alone.

The increase in cell death induced by 100 μM genistein was markedly inhibited by ciprofloxacin, a drug which binds to topoisomerase II. Etoposide (which inhibits topoisomerase II but has no effect on tyrosine kinase activity) also caused an increase in RINm5F cell death. Neither etoposide nor ciprofloxacin altered the response to 5 mM NaF.

Pertussis toxin markedly enhanced the extent of RINm5F cell death induced by NaF and this effect was completely prevented by 25 μM genistein. The inhibition caused by genistein was not affected by ciprofloxacin but was reproduced by a structurally dissimilar tyrosine kinase inhibitor, herbimycin A.

The results demonstrate that RINm5F β‐cells express a pertussis toxin sensitive pathway that is anti‐apoptotic. The activity of this pathway is most evident in cells exposed to pro‐apoptotic stimuli where the effects of pertussis toxin can be blocked by inhibitors of tyrosine kinase enzymes. A genistein‐sensitive tyrosine kinase does not appear to be involved in RINm5F cell survival under basal conditions.

British Journal of Pharmacology (2001) 132, 119–126; doi:10.1038/sj.bjp.0703783