bluuebunny/crossref_metadata_2025 huggingface dataset:
Cerebral vasoconstriction produced by vasopressin in conscious goats: role of vasopressin V1 and V2 receptors and nitric oxide
Authors: Fernández Nuria , Martínez María Angeles , García‐Villalón Angel Luis , Monge Luis , Diéguez Godofredo
Published: January 2005

To examine the role of vasopressin V1 and V2 receptors, nitric oxide and prostanoids in the cerebrovascular effects of arginine vasopressin, cerebral blood flow was electromagnetically measured in awake goats.

In 16 animals, vasopressin (0.03 – 1 μg), injected into the cerebral circulation, caused increments of resting cerebrovascular resistance which ranged from 18% (0.03 μg, P<0.01) to 79% (1 μg, P<0.01). Desmopressin (0.03 – 1 μg, four goats) did not affect significantly cerebrovascular resistance.

The cerebrovascular resistance increases by vasopressin were reduced significantly by the antagonist for vasopressin V1 receptors d(CH2)5Tyr(Me)‐AVP in a rate depending way (five (six goats) and 15 (four goats) μg min−1), and by the mixed antagonist for vasopressin V1 and V2 receptors desGly‐d(CH2)5‐D‐Tyr(Et)Val‐AVP (5 μg min−1, four goats), and they were not significantly affected by the antagonist for vasopressin V2 receptors d(CH2)5, D‐Ile2, Ile4‐AVP (5 μg min−1, four goats).

The inhibitor of nitric oxide synthesis Nw‐nitro‐L‐arginine methyl ester (L‐NAME, 47 mg kg−1 i.v., five goats) augmented cerebrovascular resistance by 130% (P<0.01), and for 24 h after this treatment the cerebrovascular effects of vasopressin were potentiated.

The inhibitor of cyclo‐oxygenase meclofenamate (6 mg kg−1 i.v., five goats) did not modify significantly resting haemodynamic variables measured or the cerebrovascular effects of vasopressin.

Therefore, the vasopressin‐induced cerebral vasoconstriction may be mediated by vasopressin V1 receptors, without involvement of vasopressin V2 receptors, and may be modulated by nitric oxide but not by prostanoids.

British Journal of Pharmacology (2001) 132, 1837–1844; doi:10.1038/sj.bjp.0704034
Mobilization of rat stomach ECL‐cell histamine in response to short‐ or long‐term treatment with omeprazole and/or YF 476 studied by gastric submucosal microdialysis in conscious rats
Authors: Konagaya T , Bernsand M , Norlén P , Håkanson R
Published: January 2005

Mobilization of histamine from the ECL cells was monitored by gastric submucosal microdialysis in conscious rats. The ECL cells are known to operate under gastrin control and the purpose of the present study was to examine their in situ response to short‐term (12 h) as well as long‐term (28 days) hypergastrinaemia, induced by treatment with the proton pump inhibitor omeprazole.

Hypergastrinaemia promptly raised the histamine concentration in the microdialysate. The effect was prevented by CCK2 receptor blockade (YF476). On day 7 of omeprazole treatment the microdialysate histamine concentration reached a peak, five times higher than before treatment. Subsequently (14 and 28 days), less histamine was mobilized.

Gastrin infusion (4 h) raised the microdialysate histamine concentration in a dose‐dependent manner in fasted rats and freely fed rats and in rats treated with omeprazole for a week. However, while fasted and fed rats responded to low doses of gastrin, the omeprazole‐treated rats required large doses of gastrin to respond.

When the amount of histamine mobilized was related to the serum gastrin concentration the following EC50 values could be calculated: fasted rats 2.3×10−10 M, freely fed rats 2.5×10−10 M, omeprazole‐treated rats 8.7×10−10 M. The maximal histamine responses in the three groups were 18.4 pmol 4 h−1±0.8, 21.9 pmol 4 h−1±1.2 and 68.0 pmol 4 h−1±3.5, respectively.

The results suggest that ECL cells, exposed to a high gastrin concentration for a week, respond with a shift in the receptor‐ligand binding affinity from high to low. Apparently, CCK2 receptors of the ECL cells are subject to dynamic changes with respect to ligand‐binding affinity.

British Journal of Pharmacology (2001) 133, 37–42; doi:10.1038/sj.bjp.0704037
Inhibition of m3 muscarinic acetylcholine receptors by local anaesthetics
Authors: Hollmann Markus W , Ritter Carsten H , Henle Philipp , De Klaver Manuela , Kamatchi Ganesan L , Durieux Marcel E
Published: January 2005

Muscarinic m1 receptors are inhibited by local anaesthetics (LA) at nM concentrations. To elucidate in more detail the site(s) of LA interaction, we compared these findings with LA effects on m3 muscarinic receptors.

We expressed receptors in Xenopus oocytes. Using two‐electrode voltage clamp, we measured the effects of lidocaine, QX314 (permanently charged) and benzocaine (permanently uncharged) on Ca2+‐activated Cl−‐currents (ICl(Ca)), elicited by acetyl‐β‐methylcholine bromide (MCh). We also characterized the interaction of lidocaine with [3H]‐quinuclydinyl benzylate ([3H]‐QNB) binding to m3 receptors. Antisense‐injection was used to determine the role of specific G‐protein α subunits in mediating the inhibitory effects of LA. Using chimeric receptor constructs we investigated which domains of the muscarinic receptors contribute to the binding site for LA.

Lidocaine inhibited m3‐signalling in a concentration‐dependent, reversible, non‐competitive manner with an IC50 of 370 nM, approximately 21 fold higher than the IC50 (18 nM) reported for m1 receptors. Intracellular inhibition of both signalling pathways by LA was similar, and dependent on the Gq‐ protein α subunit. In contrast to results reported for the m1 receptor, the m3 receptor lacks the major extracellular binding site for charged LA. The N‐terminus and third extracellular loop of the m1 muscarinic receptor molecule were identified as requirements to obtain extracellular inhibition by charged LA.

British Journal of Pharmacology (2001) 133, 207–216; doi:10.1038/sj.bjp.0704040
Murine ventricular L‐type Ca2+ current is enhanced by zinterol viaβ1‐adrenoceptors, and is reduced in TG4 mice overexpressing the human β2‐adrenoceptor
Authors: Heubach Jürgen F , Graf Eva M , Molenaar Peter , Jäger Andreas , Schröder Frank , Herzig Stefan , Harding Sian E , Ravens Ursula
Published: January 2005

The functional coupling of β2‐adrenoceptors (β2‐ARs) to murine L‐type Ca2+ current (ICa(L)) was investigated with two different approaches. The β2‐AR signalling cascade was activated either with the β2‐AR selective agonist zinterol (myocytes from wild‐type mice), or by spontaneously active, unoccupied β2‐ARs (myocytes from TG4 mice with 435 fold overexpression of human β2‐ARs). Ca2+ and Ba2+ currents were recorded in the whole‐cell and cell‐attached configuration of the patch‐clamp technique, respectively.

Zinterol (10 μM) significantly increased ICa(L) amplitude of wild‐type myocytes by 19±5%, and this effect was markedly enhanced after inactivation of Gi‐proteins with pertussis‐toxin (PTX; 76±13% increase). However, the effect of zinterol was entirely mediated by the β1‐AR subtype, since it was blocked by the β1‐AR selective antagonist CGP 20712A (300 nM). The β2‐AR selective antagonist ICI 118,551 (50 nM) did not affect the response of ICa(L) to zinterol.

In myocytes with β2‐AR overexpression ICa(L) was not stimulated by the activated signalling cascade. On the contrary, ICa(L) was lower in TG4 myocytes and a significant reduction of single‐channel activity was identified as a reason for the lower whole‐cell ICa(L). The β2‐AR inverse agonist ICI 118,551 did not further decrease ICa(L). PTX‐treatment increased current amplitude to values found in control myocytes.

In conclusion, there is no evidence for β2‐AR mediated increases of ICa(L) in wild‐type mouse ventricular myocytes. Inactivation of Gi‐proteins does not unmask β2‐AR responses to zinterol, but augments β1‐AR mediated increases of ICa(L). In the mouse model of β2‐AR overexpression ICa(L) is reduced due to tonic activation of Gi‐proteins.

British Journal of Pharmacology (2001) 133, 73–82; doi:10.1038/sj.bjp.0704045
The properties of ryanodine‐sensitive Ca2+ release in mouse gastric smooth muscle cells
Authors: Tokutomi Yoshiko , Tokutomi Naofumi , Nishi Katsuhide
Published: January 2005

Under voltage‐clamped conditions, gastric smooth muscle cells of BALB/c mice developed spontaneous (STOCs) and caffeine‐ (ICAF) and carbachol‐induced (ICCh) transient outward currents.

In fura‐2 microscopic measurements of intracellular Ca2+ concentration ([Ca2+]i), caffeine and carbachol (CCh) provoked similar transient [Ca2+]i elevations.

Both ICCh and CCh‐induced [Ca2+]i elevation of single smooth muscle cells occurred in an ‘all‐or‐nothing’ fashion in contrast to the reproducible caffeine responses.

On the basis of the suppression of STOCs and ICAF by nicardipine, tetraethylammonium and iberiotoxin, but not by charybdotoxin nor apamin, it was suggested that both currents were generated by large conductance type Ca2+‐activated K+ channels.

In measurements of isometric tension, caffeine produced relaxation of gastric smooth muscle strips in a concentration‐dependent manner (0.1 – 3 mM). The concentration‐dependent relaxation with caffeine was mimicked by dibutyryl cyclic AMP which produced potentiation of contraction triggered by 50 mM KCl.

At caffeine concentrations >3 mM, a transient contraction followed by relaxation was provoked as the quasi maximal response to caffeine. In the quasi maximal response, caffeine acted as a potent relaxant in smooth muscle strips precontracted with 50 mM KCl or 3 μM CCh.

The relaxation with caffeine was significantly accelerated in those strips precontracted with KCl or CCh. All these results suggest that ryanodine‐sensitive Ca2+ release, which is triggered by caffeine, is an important modifier of Ca2+ homeostasis in the cytoplasm and the contractility of gastric smooth muscle cells of mice.

British Journal of Pharmacology (2001) 133, 125–137; doi:10.1038/sj.bjp.0704048
Depressor and regionally‐selective vasodilator effects of human and rat urotensin II in conscious rats
Authors: Gardiner S M , March J E , Kemp P A , Davenport A P , Bennett T
Published: January 2005

The regional haemodynamic effects of rat or human urotensin II (U‐II) 3, 30, 300 and 3000 pmol kg−1, i.v.) were assessed in separate groups of conscious, unrestrained, male, Sprague‐Dawley rats (n=8 in each). Rat and human U‐II had similar effects. At a dose of 3 pmol kg−1, neither peptide had any significant action, while at a dose of 30 pmol kg−1, there was a transient mesenteric vasodilatation (significant only for rat U‐II). At doses of 300 and 3000 pmol kg−1, there were dose‐dependent tachycardias, and mesenteric and hindquarters hyperaemic vasodilatations. Thus, in conscious rats, the predominant cardiovascular action of rat and human U‐II is vasodilatation. This is in contrast to recent findings with human U‐II in non‐human primates, but is consistent with effects on human isolated resistance vessels.

British Journal of Pharmacology (2001) 132, 1625–1629; doi:10.1038/sj.bjp.0704051
Effects of yohimbine on the antinociceptive and place conditioning effects of opioid agonists in rodents
Authors: Morales Lydia , Perez‐Garcia Carmen , Alguacil Luis F
Published: January 2005

The pharmacological modulation of opioid actions by drugs acting on heterologous mechanisms could be useful to overcome some of the main problems associated with the use of opiate agonists. Based on previous findings on the interactions between yohimbine and opioid drugs, we have further studied the effects of yohimbine on the antinociceptive and positive‐negative reinforcing effects of morphine (μ opioid receptor‐preferring agonist), U‐50,488 (κ agonist) and SNC80 (δ agonist).

Pretreatment with yohimbine completely blocked the antinociception provided by the three opioid agonists in the mouse tail‐immersion test.

A similar blockade of SNC80 and U‐50,488‐induced antinociception was observed with yohimbine in the mouse hot plate test at the same doses. In this paradigm, the effect of the κ agonist was very slight and the actions of yohimbine rather variable.

In place conditioning experiments with SD (Sprague – Dawley) male rats, yohimbine alone was inactive but it limited the preference induced by morphine and SNC80 and the aversive effect of U‐50,488. Impaired novelty preference was also observed with the combination of yohimbine and U‐50,488.

It is concluded that yohimbine tends to limit opioid antinociception and the addictive potential of μ and δ opioid agonists. More selective drugs could help to understand the mechanisms involved in these actions.

British Journal of Pharmacology (2001) 133, 172–178; doi:10.1038/sj.bjp.0704057
IL‐17‐induced cytokine release in human bronchial epithelial cells in vitro: role of mitogen‐activated protein (MAP) kinases
Authors: Laan Martti , Lötvall Jan , Chung K Fan , Lindén Anders
Published: January 2005

Recent data indicate that interleukin (IL)‐17 may contribute to neutrophilic airway inflammation by inducing the release of neutrophil‐mobilizing cytokines from airway cells. The aim of this study was to evaluate the role of mitogen activated protein kinases in IL‐17 induced release of IL‐8 and IL‐6 in bronchial epithelial cells.

Transformed human bronchial epithelial cells (16HBE) were stimulated with either IL‐17 or vehicle. Both groups were treated either with SB202190 (inhibitor of p38 MAP kinase), PD98059 (inhibitor of extracellular‐signal‐regulated kinase [ERK] pathway), Ro‐31‐7549 (protein kinase C [PKC] inhibitor), LY 294002 (a phosphatidylinositol 3‐kinase [PI 3‐kinase] inhibitor) or vehicle. IL‐6 and IL‐8 levels were measured in conditioned media by ELISA.

The IL‐17‐induced release of IL‐6 and IL‐8 was concentration‐dependently inhibited by SB202190 and by PD98059 in bronchial epithelial cells without affecting cell proliferation or survival.

Ro‐31‐7549 and LY294002 had no significant effect on IL‐17‐induced IL‐6 or IL‐8 release in bronchial epithelial cells.

Taken together, these data indicate a role for p38 and ERK kinase pathways in IL‐17‐induced release of neutrophil‐mobilizing cytokines in human bronchial epithelial cells. These mechanisms constitute potential pharmacotherapeutical targets for inhibition of the IL‐17‐mediated airway neutrophilia.

British Journal of Pharmacology (2001) 133, 200–206; doi:10.1038/sj.bjp.0704063
Drug‐disease interactions: reduced β‐adrenergic and potassium channel antagonist activities of sotalol in the presence of acute and chronic inflammatory conditions in the rat
Authors: Kulmatycki Kenneth M , Abouchehade Kassem , Sattari Saeed , Jamali Fakhreddin
Published: January 2005

Inflammation may influence response to pharmacotherapy.

We investigated the effect of inflammation on response to sotalol, a β‐adrenergic receptor and potassium channel antagonist. Racemic sotalol (40 mg kg−1) was administered to healthy, acutely (interferonα 2a‐induced) and chronically (Mycobacterium butyricum‐induced adjuvant arthritis) inflamed male Sprague‐Dawley rats (n=4 – 6/group). Another group of interferon‐treated rats received 3 mg kg−1 of anti‐TNF antibody infliximab. Electrocardiogram (ECG) recorded and plasma sotalol concentration monitored for 6 h. The study was repeated in acutely inflamed rats following administration of stereochemically pure individual sotalol enantiomers [40 mg kg−1 S (potassium channel blocker) or 20 mg kg−1 R (β‐adrenergic/potassium channel blocker)].

Chronic arthritis was readily evident. Acute arthritis was associated with elevated segmented neutrophils and increased plasma nitrite and tumour necrosis factor (TNF) concentrations. Sotalol affected ECG in all rats. In both inflamed groups, however, response to sotalol in prolongation of QT interval (potassium channel sensitivity) was reduced. The effect of PR interval (β‐adrenergic activity) was also reduced following administration of the racemate and R‐enantiomer. No significant differences in pharmacokinetics were observed between control and inflamed rats.

Infliximab reduced nitrite and TNF concentrations and reversed the effect of acute inflammation on both PR and QT intervals.

The reduced electrocardiographic responses to sotalol is likely due to the influence of inflammation on the action of the drug on both β‐adrenergic and potassium channel receptors secondary to over‐expression of pro‐inflammatory cytokines and/or nitric oxide.

Our observation may have therapeutic consequences in all conditions where inflammatory mediators are increased.

British Journal of Pharmacology (2001) 133, 286–294; doi:10.1038/sj.bjp.0704067
A new HMG‐CoA reductase inhibitor, rosuvastatin, exerts anti‐inflammatory effects on the microvascular endothelium: the role of mevalonic acid
Authors: Stalker Timothy J , Lefer Allan M , Scalia Rosario
Published: January 2005

Recent studies have reported that hydroxymethylglutaryl coenzyme A (HMG‐CoA) reductase inhibitors have vasculoprotective effects independent of their lipid‐lowering properties, including anti‐inflammatory actions. We used intravital microscopy of the rat mesenteric microvasculature to examine the effects of rosuvastatin, a new HMG‐CoA reductase inhibitor, on leukocyte‐endothelium interactions induced by thrombin.

Intraperitoneal administration of 0.5 and 1.25 mg kg−1 rosuvastatin 18 h prior to the study, significantly and dose‐dependently attenuated leukocyte rolling, adherence, and transmigration in the rat mesenteric microvasculature superfused with 0.5 u ml‐1 thrombin. This protective effect of rosuvastatin was reversed by intraperitoneal injection of 25 mg kg−1 mevalonic acid 18 h before the study.

Immunohistochemical detection of the endothelial cell adhesion molecule P‐selectin showed a 70% decrease in endothelial cell surface expression of P‐selectin in thrombin‐stimulated rats given 1.25 mg kg−1 rosuvastatin. In addition, rosuvastatin enhanced release of nitric oxide (NO) from the vascular endothelium as measured directly in rat aortic segments. Moreover, rosuvastatin failed to attenuate leukocyte‐endothelium interactions in peri‐intestinal venules of eNOS−/− mice.

These data indicate that rosuvastatin exerts important anti‐inflammatory effects via inhibition of endothelial cell adhesion molecule expression, and that this protective action of rosuvastatin requires release of nitric oxide by the vascular endothelium. These data also demonstrate that the mechanism of the non‐lipid lowering actions of HMG‐CoA reductase inhibitors in vivo may be due to reduced formation or availability of mevalonic acid within endothelial cells.

British Journal of Pharmacology (2001) 133, 406–412; doi:10.1038/sj.bjp.0704070
Antigenicity and immunogenicity of sulphamethoxazole: demonstration of metabolism‐dependent haptenation and T‐cell proliferation in vivo
Authors: Naisbitt Dean J , Gordon S Fraser , Pirmohamed Munir , Burkhart Christoph , Cribb Alistair E , Pichler Werner J , Park B Kevin
Published: January 2005

Sulphamethoxazole has been associated with the occurrence of hypersensitivity reactions. There is controversy as to whether the immune response is metabolism‐dependent or ‐independent. We have therefore investigated the site of antigen formation and the nature of the drug signal presented to the immune system in vivo.

Male Wistar rats were dosed with sulphamethoxazole, sulphamethoxazole hydroxylamine or nitroso sulphamethoxazole. Antigen formation on cell surfaces was determined by flow cytometry using a specific anti‐sulphamethoxazole antibody. Immunogenicity was determined by assessment of ex vivo T‐cell proliferation.

Administration of nitroso sulphamethoxazole, but not sulphamethoxazole or sulphamethoxazole hydroxylamine, resulted in antigen formation on the surface of lymphocytes, splenocytes and epidermal keratinocytes, and a strong proliferative response of splenocytes on re‐stimulation with nitroso sulphamethoxazole. Rats dosed with sulphamethoxazole or sulphamethoxazole hydroxylamine did not respond to any of the test compounds.

CD4+ or CD8+ depleted cells responded equally to nitroso sulphamethoxazole. The proliferative response to nitroso sulphamethoxazole was seen even after pulsing for only 5 min, and was not inhibited by glutathione. Responding cells produced IFN‐γ, but not IL‐4.

Haptenation of cells by sulphamethoxazole hydroxylamine was seen after depletion of glutathione by pre‐treating the rats with diethyl maleate. Splenocytes from the glutathione‐depleted sulphamethoxazole hydroxylamine‐treated rats responded weakly to nitroso sulphamethoxazole, but not to sulphamethoxazole or sulphamethoxazole hydroxylamine.

Dosing of rats with sulphamethoxazole produced a cellular response to nitroso sulphamethoxazole (but not to sulphamethoxazole or its hydroxylamine) when the animals were primed with complete Freund's adjuvant.

These studies demonstrate the antigenicity of nitroso sulphamethoxazole in vivo and provide evidence for the role of drug metabolism and cell surface haptenation in the induction of a cellular immune response in the rat.

British Journal of Pharmacology (2001) 133, 295–305; doi:10.1038/sj.bjp.0704074
Temporal variation in endotoxin‐induced vascular hyporeactivity in a rat mesenteric artery organ culture model
Authors: O'Brien A J , Wilson A J , Sibbald R , Singer M , Clapp L H
Published: January 2005

Endotoxin‐induced vascular hyporeactivity to phenylephrine (PE) is well described in rodent aorta, but has not been investigated in smaller vessels in vitro.

Segments of rat superior mesenteric artery were incubated in culture medium with or without foetal bovine serum (10%) for 6, 20 or 46 h in the presence or absence of bacterial lipopolysaccharide (LPS; 1 – 100 μg ml−1).

Contractions to PE were measured with or without nitric oxide synthase (NOS) inhibitors: L‐NAME (300 μM), aminoguanidine (AMG; 400 μM) 1400W (10 μM) and GW273629 (10 μM); the guanylyl cyclase inhibitor, ODQ (3 μM); the COX‐2 inhibitor, NS‐398 (10 μM). Contractile responses to the thromboxane A2 mimetic, U46619 were also assessed.

In the presence of serum, LPS induced hyporeactivity at all time points. In its absence, hyporeactivity only occurred at 6 and 20 h.

L‐NAME and AMG fully reversed hyporeactivity at 6 h, whereas they were only partially effective at 20 h and not at all at 46 h. In contrast partial reversal of peak contraction was observed with 1400W (62% at 46 h), GW273629 (57% at 46 h) and ODQ (75% at 46 h). COX‐2 inhibition produced no reversal.

In contrast to PE, contractions to U46619 were substantially less affected by LPS.

We describe a well‐characterized reproducible model of LPS‐induced hyporeactivity, which is largely mediated by the NO‐cyclic GMP‐dependent pathway. Importantly, long‐term (2‐day) production of NO via iNOS is demonstrated. Moreover, conventional doses of L‐NAME and AMG became increasingly ineffective over time. Thus, the choice of inhibitor merits careful consideration in long‐term models.

British Journal of Pharmacology (2001) 133, 351–360; doi:10.1038/sj.bjp.0704079
Differential prevention of morphine amnesia by antisense oligodeoxynucleotides directed against various Gi‐protein α subunits
Authors: Galeotti Nicoletta , Ghelardini Carla , Bartolini Alessandro
Published: January 2005

The effect of the i.c.v. administration of pertussis toxin (PTX) and antisense oligodeoxynucleotide directed against the α subunit of different Gi‐proteins (anti‐Giα1, anti‐Giα2, anti‐Giα3) on amnesia induced by morphine was evaluated in the mouse passive avoidance test.

The administration of morphine (6 – 10 mg kg−1 i.p.) immediately after the training session produced amnesia that was prevented by PTX (0.25 μg per mouse i.c.v.) administered 7 days before the passive avoidance test.

Anti‐Giα1 (6.25 μg per mouse i.c.v.) and anti‐Giα3 (12.5 μg per mouse i.c.v.), administered 18 and 24 h before the training session, prevented the morphine amnesia. By contrast, pretreatment with anti‐Giα2 (3.12 – 25 μg per mouse i.c.v.) never modified the impairment of memory processes induced by morphine.

At the highest effective doses, none of the compounds used impaired motor coordination, as revealed by the rota rod test, nor modified spontaneous motility and inspection activity, as revealed by the hole board test.

These results suggest the important role played by Gi1 and Gi3 protein subtypes in the transduction mechanism involved in the impairment of memory processes produced by morphine.

British Journal of Pharmacology (2001) 133, 267–274; doi:10.1038/sj.bjp.0704081
CXC chemokines, MIP‐2 and KC, induce P‐selectin‐dependent neutrophil rolling and extravascular migration in vivo
Authors: Zhang Xiao Wei , Liu Qing , Wang Yusheng , Thorlacius Henrik
Published: January 2005

The purpose of this study was to examine the impact of CXC chemokines, i.e. macrophage inflammatory protein‐2 (MIP‐2) and KC, on leukocyte‐endothelium interactions in detail and to evaluate the role of P‐selectin by use of intravital microscopy in the mouse cremaster muscle.

Administration of MIP‐2 and KC provoked a dose (5 – 500 ng)‐ and time (0 – 4 h)‐dependent increase in leukocyte rolling, adhesion and tissue recruitment. Neutrophils comprised more than 92% of the leukocyte response. Pretreatment with an antibody directed against P‐selectin (RB40.34) significantly inhibited MIP‐2‐ and KC‐induced leukocyte rolling by more than 96%. This marked decrease in rolling abolished firm adhesion and extravascular accumulation of neutrophils (>89% reduction), suggesting that CXC chemokines induce P‐selectin‐dependent rolling, which in turn apparently is a precondition for the subsequent stationary adhesion and extravasation of neutrophils.

Moreover, the extravascular recruitment of leukocytes was evaluated in whole‐mounts of the cremaster muscle without preceding intravital microscopy. Using this approach, it was again observed that MIP‐2‐ and KC‐induced neutrophil accumulation was completely dependent onP‐selectin function. In contrast to the CXC chemokines, administration of the classical chemoattractant formyl‐methionyl leucyl phenylalanine (fMLP) did not provoke extravascular tissue accumulation of neutrophils.

We could not detect gene expression of CXCR2 in murine endothelial cells, whereas neutrophils were positive, indicating that the stimulatory effect of CXC chemokines on leukocyte‐endothelium interactions is not a direct effect on the endothelium but rather an indirect effect via activation of an intermediary tissue cell. However, challenge with MIP‐2 and KC did not increase the number of degranulated mast cells.

In conclusion, our data demonstrate that CXC chemokines induce all steps in the extravasation process of leukocytes, including rolling, adhesion and transmigration in vivo. Moreover, these results show that P‐selectin plays a critical role in MIP‐2 and KC provoked neutrophil recruitment as a critical mediator of initial leukocyte rolling. Additionally, our study suggest that a restricted action of MIP‐2 and KC on neutrophils is far too simplistic to explain the complex mechanisms of action of CXC chemokines on neutrophil infiltration in vivo.

British Journal of Pharmacology (2001) 133, 413–421; doi:10.1038/sj.bjp.0704087
Acute effect of the dual angiotensin‐converting enzyme and neutral endopeptidase 24‐11 inhibitor mixanpril on insulin sensitivity in obese Zucker rat
Authors: Arbin V , Claperon N , Fournié‐Zaluski M ‐C , Roques B P , Peyroux J
Published: January 2005

The aim of this study was to determine whether acute dual angiotensin‐converting enzyme (ACE)/neutral endopeptidase 24‐11 (NEP) inhibition could improve whole body insulin‐mediated glucose disposal (IMGD) more than ACE inhibition alone and whether this effect was mediated by the kinin‐nitric oxide (NO) pathway activation.

We therefore compared in anaesthetized obese (fa/fa) Zucker rats (ZOs) the effects of captopril (2 mg kg−1, i.v.+2 mg kg−1 h−1), retrothiorphan (25 mg kg−1, i.v. +25 mg  kg−1 h−1), a selective NEP inhibitor, and mixanpril (25 mg kg−1, i.v.+25 mg kg−1 h−1), a dual ACE/NEP inhibitor, on IMGD using hyperinsulinaemic euglycaemic clamp technique. The role of the kinin‐NO pathway in the effects of mixanpril was tested using a bradykinin B2 receptor antagonist (Hoe‐140, 300 μg kg−1) and a NO‐synthase inhibitor (Nω‐nitro‐L‐arginine methyl ester, L‐NAME, 10 mg kg−1 i.v. +10 mg kg−1 h−1) as pretreatments.

Insulin sensitivity index (ISI) was lower in ZO controls than in lean littermates. Increases in ISI were observed in captopril‐ and retrothiorphan‐treated ZOs. In mixanpril‐treated ZOs, ISI was further increased, compared to captopril‐ and retrothiorphan‐treated ZOs.

In ZOs, Hoe‐140 and L‐NAME alone did not significantly alter and slightly reduced the ISI respectively. Hoe‐140 and L‐NAME markedly inhibited the ISI improvement induced by mixanpril.

These results show that in obese insulin‐resistant Zucker rats, under acute conditions, NEP or ACE inhibition can improve IMGD and that dual ACE/NEP inhibition improves IMGD more effectively than does either single inhibition. This effect is linked to an increased activation of the kinin‐NO pathway.

British Journal of Pharmacology (2001) 133, 495–502; doi:10.1038/sj.bjp.0704098
HET0016, a potent and selective inhibitor of 20‐HETE synthesizing enzyme
Authors: Miyata Noriyuki , Taniguchi Kazuo , Seki Takayuki , Ishimoto Tsuyoshi , Sato‐Watanabe Mariko , Yasuda Yoshiko , Doi Mariko , Kametani Shunichi , Tomishima Yasumitsu , Ueki Tomokazu , Sato Masakazu , Kameo Kazuya
Published: January 2005

The present study examined the inhibitory effects of N‐Hydroxy‐N′‐(4‐butyl‐2‐methylphenyl)‐formamidine (HET0016) on the renal metabolism of arachidonic acid by cytochrome P450 (CYP) enzymes. HET0016 exhibited a high degree of selectivity in inhibiting the formation of 20‐hydroxy‐5,8,11,14‐eicosatetraenoic acid (20‐HETE) in rat renal microsomes. The IC50 value averaged 35±4 nM, whereas the IC50 value for inhibition of the formation of epoxyeicosatrienoic acids by HET0016 averaged 2800±300 nM. In human renal microsomes, HET0016 potently inhibited the formation of 20‐HETE with an IC50 value of 8.9±2.7 nM. Higher concentrations of HET0016 also inhibited the CYP2C9, CYP2D6 and CYP3A4‐catalysed substrates oxidation with IC50 values of 3300, 83,900 and 71,000 nM. The IC50 value for HET0016 on cyclo‐oxygenase activity was 2300 nM. These results indicate that HET0016 is a potent and selective inhibitor of CYP enzymes responsible for the formation of 20‐HETE in man and rat.

British Journal of Pharmacology (2001) 133, 325–329; doi:10.1038/sj.bjp.0704101
Electrophysiological effects of dronedarone (SR 33589), a noniodinated amiodarone derivative in the canine heart: comparison with amiodarone
Authors: Varró András , Takács János , Németh Miklós , Hála Ottó , Virág László , Iost Norbert , Baláti Beáta , Ágoston Márta , Vereckei András , Pastor Gilbert , Delbruyère Martine , Gautier Patrick , Nisato Dino , Papp Julius Gy
Published: January 2005

The electrophysiological effects of dronedarone, a new nonionidated analogue of amiodarone were studied after chronic and acute administration in dog Purkinje fibres, papillary muscle and isolated ventricular myocytes, and compared with those of amiodarone by applying conventional microelectrode and patch‐clamp techniques.

Chronic treatment with dronedarone (2×25 mg−1 kg−1 day p.o. for 4 weeks), unlike chronic administration of amiodarone (50 mg−1 kg−1 day p.o. for 4 weeks), did not lengthen significantly the QTc interval of the electrocardiogram or the action potential duration (APD) in papillary muscle. After chronic oral treatment with dronedarone a small, but significant use‐dependent Vmax block was noticed, while after chronic amiodarone administration a strong use‐dependent Vmax depression was observed.

Acute superfusion of dronedarone (10 μM), similar to that of amiodarone (10 μM), moderately lengthened APD in papillary muscle (at 1 Hz from 239.6±5.3 to 248.6±5.3 ms, n=13, P<0.05), but shortened it in Purkinje fibres (at 1 Hz from 309.6±11.8 to 287.1±10.8 ms, n=7, P<0.05).

Both dronedarone (10 μM) and amiodarone (10 μM) superfusion reduced the incidence of early and delayed afterdepolarizations evoked by 1 μM dofetilide and 0.2 μM strophantidine in Purkinje fibres.

In patch‐clamp experiments 10 μM dronedarone markedly reduced the L‐type calcium current (76.5±0.7 %, n=6, P<0.05) and the rapid component of the delayed rectifier potassium current (97±1.2 %, n=5, P<0.05) in ventricular myocytes.

It is concluded that after acute administration dronedarone exhibits effects on cardiac electrical activity similar to those of amiodarone, but it lacks the ‘amiodarone like’ chronic electrophysiological characteristics.

British Journal of Pharmacology (2001) 133, 625–634; doi:10.1038/sj.bjp.0704106
The synthetic cannabinoid WIN55,212‐2 attenuates hyperalgesia and allodynia in a rat model of neuropathic pain
Authors: Bridges Daniel , Ahmad Kamran , Rice Andrew S C
Published: January 2005

The analgesic properties of the synthetic cannabinoid WIN55,212‐2 were investigated in a model of neuropathic pain. In male Wistar rats, bilateral hind limb withdrawal thresholds to cold, mechanical and noxious thermal stimuli were measured. Following this, unilateral L5 spinal nerve ligation was performed. Seven days later, sensory thresholds were reassessed and the development of allodynia to cold and mechanical stimuli and hyperalgesia to a noxious thermal stimulus confirmed.

The effect of WIN55,212‐2 (0.1 – 5.0 mg kg−1, i.p.) on the signs of neuropathy was then determined; there was a dose related reversal of all three signs of painful neuropathy at doses which did not generally alter sensory thresholds in the contralateral unligated limb. This effect was prevented by co‐administration of the CB1 receptor antagonist SR141716a, but not by co‐administration of the CB2 receptor antagonist SR144528, suggesting this action of WIN55,212‐2 is mediated via the CB1 receptor. Administration of SR141716a alone had no affect on the observed allodynia and hyperalgesia, which does not support the concept of an endogenous analgesic tone.

These data indicate that cannabinoids may have therapeutic potential in neuropathic pain, and that this effect is mediated through the CB1 receptor.

British Journal of Pharmacology (2001) 133, 586–594; doi:10.1038/sj.bjp.0704110
Generation of 8‐epi‐prostaglandin F2α in isolated rat kidney glomeruli by a radical‐independent mechanism
Authors: Klein Thomas , Neuhaus Katrin , Reutter Felix , Nüsing Rolf M
Published: January 2005

Isoprostanes comprise a group of free radical‐catalyzed products of arachidonic acid. However, there is recent evidence pointing towards an enzyme‐dependent formation of isoprostanes.

With the use of isolated rat glomeruli we addressed the mechanisms of isoprostane generation. Synthesis of prostanoids and isoprostanes, including 8‐epi‐PGF2α, was studied under conditions favouring radical formation.

Cultured glomeruli formed different prostanoids including 8‐epi‐PGF2α. Upon LPS challenge cyclo‐oxygenase (COX)‐2 expression was enhanced, and this was paralleled by a 2–9‐fold increase in prostanoid formation, including isoprostanes. Addition of COX‐isoform unselective inhibitors (diclofenac, indomethacin) or a selective inhibitor (NS‐398) suppressed the synthesis of prostanoids, 8‐epi‐PGF2α and total isoprostane fraction; however, inhibition of the latter was less pronounced.

Antioxidants such as butylated hydroxytoluene (BHT), nordihydroguaiaretic acid (NDGA), or dimethylurea exhibited an only minimal inhibitory effect on 8‐epi‐PGF2α synthesis. Moreover, ROS‐generating drugs (menadione, methylviologen) or NADPH‐driven radical formation were unable to cause the generation of significant amounts of 8‐epi‐PGF2α by rat glomeruli. In contrast, the total isoprostane fraction could be increased by menadione addition.

These data provide further evidence for a radical‐independent, but COX‐dependent formation of 8‐epi‐PGF2α in renal tissue. Regarding the other isoprostanes, both radicals and COX enzymes contribute to their formation. Based on our data we assume that elevated release of vasoactive 8‐epi‐PGF2α has to be expected under conditions when the prostanoid system in the kidney is stimulated, e.g. under inflammatory conditions. Regarding renal oxidative injuries, the usefulness of 8‐epi‐PGF2α as a representative marker molecule of oxidative stress has to be questioned.

British Journal of Pharmacology (2001) 133, 643–650; doi:10.1038/sj.bjp.0704111
Blockade by ruthenium red of tissue factor‐initiated coagulation
Authors: Chu Arthur J , Wang Zhen‐Guo , Nwobi Obioma I , Beydoun Salwa
Published: January 2005

The ability of ruthenium red (RuR) to inhibit tissue factor (TF)‐initiated blood coagulation was demonstrated at the protein and cellular levels as well as in human plasma.

In a single‐stage clotting assay, RuR concentration‐dependently inhibited rabbit brain thromboplastin (rbTF)‐induced coagulation and offset bacterial endotoxin (LPS)‐induced monocytic TF (mTF) hypercoagulation; the IC50s were estimated at 7.5 and 12.3 μM, respectively.

A 15‐min preincubation of RuR with rbTF or monocyte suspension resulted in the pronounced inhibition with a significantly lowered IC50 at 1.8 or 7.7 μM for rbTF or mTF procoagulation, respectively. The differences in IC50s between rbTF and mTF without or with the preincubation indicated that TF was a primary target for RuR action.

The effect of RuR on the physiological function of TF in FVII activation was demonstrated by the proteolytic cleavage of FVII zymogen to its active forms of serine protease on Western blotting analyses. RuR readily blocked TF‐catalyzed FVII activation (diminished FVIIa formation), thus down regulating the initiation of blood coagulation.

Inclusion of RuR into human plasma samples in vitro significantly prolonged prothrombin time, indicating the depressed coagulation. FVII activity was inhibited by 30–60% depending on the dose; as a result, FX activity also decreased. However, RuR showed no effect on thrombin time. Thus, RuR inhibited FVII activation to block the initiation of coagulation.

British Journal of Pharmacology (2001) 133, 659–664; doi:10.1038/sj.bjp.0704116
Dominant role of L‐ and P‐selectin in mediating CXC chemokine‐induced neutrophil migration in vivo
Authors: Miotla Jadwiga M , Ridger Victoria C , Hellewell Paul G
Published: January 2005

The role of selectins in neutrophil emigration in response to the CXC chemokines KC and MIP‐2 was investigated in wild type and P‐selectin deficient mice.

Intrapleural injection of KC or MIP‐2 induced a rapid and specific neutrophil accumulation. Emigration 2 h after KC or MIP‐2 was reduced 83 – 88% by anti‐L‐selectin mAb and 53 – 63% by anti‐P‐selectin mAb. Co‐administration of anti‐L‐ and P‐selectin mAbs abolished neutrophil migration induced by either chemokine.

An anti‐E‐selectin mAb tested alone did not affect KC‐induced neutrophil migration after 2 or 4 h. Moreover, anti‐E‐selectin did not have an additive inhibitory effect on KC‐induced neutrophil migration compared with P‐selectin blockade alone. This was found when neutrophil migration was measured at 2 and 4 h after KC.

Despite a blood neutrophilia, neutrophil migration at 2 and 4 h after KC was markedly smaller (by approximately 90%) in P‐selectin deficient mice compared with wild type animals. Responses at both time points were not decreased further in animals given E‐selectin mAb but were reduced to the PBS control level in the presence of anti‐L‐selectin.

In vitro study of cultured murine endothelial cells demonstrated that KC can directly increase cell surface P‐selectin expression.

These data suggest that CXC chemokine‐induced neutrophil accumulation is dependent on both neutrophil L‐selectin and a rapid upregulation of endothelial P‐selectin but there is no evidence for E‐selectin induction.

British Journal of Pharmacology (2001) 133, 550–556; doi:10.1038/sj.bjp.0704118
Identification and quantification of phosphodiesterase 4 subtypes in CD4 and CD8 lymphocytes from healthy and asthmatic subjects
Authors: Landells L J , Szilagy C M , Jones N A , Banner K H , Allen J M , Doherty A , O'Connor B J , Spina D , Page C P
Published: January 2005

In the present study, for the first time, PDE4 subtypes were identified and semi‐quantified in both CD4 and CD8 lymphocytes from healthy and asthmatic individuals.

CD4 and CD8 lymphocytes from healthy and mild asymptomatic asthmatic subjects (receiving β‐agonist therapy only) were isolated from peripheral venous blood using appropriate antibody coated paramagnetic beads. PDE4 subtypes and β‐actin were identified by digoxigenin (DIG)‐labelling reverse transcriptase‐polymerase chain reaction and semi‐quantified by DIG‐detection enzyme‐linked immunosorbance assay.

In CD4 and CD8 lymphocytes PDE4A, PDE4B and PDE4D were detected, with no significant differences observed between healthy and asthmatic groups. In CD8 lymphocytes, enzyme subtype expression was lower and showed more intersubject variability.

In functional studies investigating the effects of various PDE inhibitors on PHA‐induced proliferation of mononuclear cells from healthy and asthmatic subjects, CDP840 (0.03–10 μM), rolipram (0.1–10 μM) and theophylline (10 μM–1 mM) inhibited PHA‐induced proliferation of mononuclear cells from healthy and asthmatic subjects in a concentration‐dependent manner, although no significant difference was observed between the groups investigated.

In additional studies, total monocyte cyclic AMP PDE activity was investigated in cells isolated from asthmatic subjects both prior to and 24 h after allergen challenge. Total monocyte cyclic AMP PDE activity remained unaffected following challenge of asthmatic subjects with either house dust mite or cat dander and was inhibited in a concentration‐dependent manner by rolipram (0.01–100 μM) both before and after allergen challenge.

British Journal of Pharmacology (2001) 133, 722–729; doi:10.1038/sj.bjp.0704120
The mechanism for the contraction induced by leukotriene C4 in guinea‐pig taenia coli
Authors: Ieiri Satoshi , Nishimura Junji , Hirano Katsuya , Suita Sachiyo , Kanaide Hideo
Published: January 2005

The mechanism underlying the LTC4‐induced contraction of guinea‐pig taenia coli was determined using the simultaneous measurements of [Ca2+]i and force in whole muscle preparations. Additional experiments were performed in receptor coupled permeabilized preparation. For comparison purposes, the contraction which was induced by a typical G‐protein mediated agonist, carbachol was also characterized.

LTC4 induced a contraction in the guinea‐pig taenia coli in a concentration‐dependent manner. The maximal response was obtained at 100 nM and the EC50 value was 5.4±1.9 nM.

Both LTC4 and carbachol induced increases in [Ca2+]i and force. The maximum force induced by 100 nM LTC4 was significantly smaller than that induced by 10 μM carbachol, although an increase in [Ca2+]i produced by both agonists was similar. In the permeabilized preparations, carbachol, but not LTC4, induced an additional force development at a fixed Ca2+ concentration.

LTC4 induced no increase in [Ca2+]i and force in the Ca2+‐free solution, while carbachol induced transient increases in both [Ca2+]i and force in a Ca2+‐free solution.

Both diltiazem and SK&F 96365 significantly inhibited the LTC4− and carbachol‐induced increases in [Ca2+]i and force in normal PSS. The inhibitory pattern of [Ca2+]i by these drugs was also similar.

We thus conclude that LTC4 induces the contraction of the guinea‐pig taenia coli mainly through Ca2+ influx via both the diltiazem‐sensitive and SK&F 96365‐sensitive Ca2+ channels, without affecting either the Ca2+‐sensitivity or the intracellular Ca2+ release. These results indicated that the mechanism underlying the LTC4‐induced contraction differs greatly from that for conventional G‐protein mediated agonists, such as carbachol.

British Journal of Pharmacology (2001) 133, 529–538; doi:10.1038/sj.bjp.0704122
Y‐27632, an inhibitor of Rho‐kinase, antagonizes noradrenergic contractions in the rabbit and human penile corpus cavernosum
Authors: Rees Rowland W , Ralph David J , Royle Michael , Moncada Salvador , Cellek Selim
Published: January 2005

We have examined the effect of an inhibitor of Rho‐kinase, (+)‐(R)‐trans‐4‐(1‐aminoethyl)‐N‐(4‐pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y‐27632), on the contractions elicited by noradrenergic nerve stimulation and by phenylephrine in the human and rabbit penile corpus cavernosum. In both tissues, after treatment with scopolamine (10 μM) and NG‐nitro‐L‐arginine methyl ester (L‐NAME; 300 μM), electrical field stimulation (EFS) elicited noradrenergic contractions. These contractions were inhibited by Y‐27632 in a concentration‐dependent manner. The compound caused concentration‐dependent relaxation of phenylephrine‐contracted tissues, which were treated with scopolamine (10 μM), guanethidine (10 μM) and L‐NAME (300 μM). These results suggest that Rho‐kinase is involved in noradrenergic contractile pathway in the cavernosal smooth muscle of the penis.

British Journal of Pharmacology (2001) 133, 455–458; doi:10.1038/sj.bjp.0704124
The effect of the beta2 adrenoceptor gene Thr164Ile polymorphism on human adipose tissue lipolytic function
Authors: Hoffstedt Johan , Iliadou Anastasia , Pedersen Nancy L , Schalling Martin , Arner Peter
Published: January 2005

A rare β2‐adrenoceptor gene polymorphism, Thr164Ile, has been described that impairs receptor function when transfected into cell lines. We investigated whether the polymorphism influences native receptor function by studying lipolysis in freshly isolated subcutaneous fat cells from 236 apparently healthy subjects.

Twelve subjects were heterozygous for the 164Ile variant. The fat cells of Ile carriers displayed a 6 fold increase (P=0.02) in the lipolytic EC50 of terbutaline (a selective β2‐adrenoceptor agonist), but no change in the lipolytic action of dobutamine (a selective β1‐adrenoceptor agonist), compared with the Thr carriers. Maximum adrenoceptor agonist stimulated lipolysis did not differ between Thr and Ile carriers.

The influence of two other polymorphisms (Arg16Gly and Gln27Glu) in the β2‐adrenoceptor gene was considered. Six 164Ile carriers also carried the 16Gly and 27Glu alleles. The latter combination occurred among 105 of the 164Thr carriers. For the 16Gly27Glu subgroup, the EC50 of terbutaline was about 10 fold higher in 164Ile as than in 164Thr carriers (P=0.02) but there was no difference between genotypes in maximum terbutaline action. There was no difference between groups in dobutamine action.

In conclusion, the 164Ile variant of the β2‐adrenoceptor is associated with a decreased native adipocyte receptor function, as evidenced by a marked increase in the half maximal effective concentration of the lipolytic action of a selective β2‐adrenoceptor agonist. This suggests that genetic variance in the β2‐adrenoceptor gene might be important for catecholamine function in humans, at least as far as adipocyte lipolysis is concerned.

British Journal of Pharmacology (2001) 133, 708–712; doi:10.1038/sj.bjp.0704125