bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of μ‐ and κ‐, but not δ‐, opioid receptors in the peristaltic motor depression caused by endogenous and exogenous opioids in the guinea‐pig intestine
Authors: Shahbazian Anaid , Heinemann Akos , Schmidhammer Helmut , Beubler Eckhard , Holzer‐Petsche Ulrike , Holzer Peter
Published: January 2005

Opiates inhibit gastrointestinal propulsion, but it is not clear which opioid receptor types are involved in this action. For this reason, the effect of opioid receptor – selective agonists and antagonists on intestinal peristalsis was studied.

Peristalsis in isolated segments of the guinea‐pig small intestine was triggered by a rise of the intraluminal pressure and recorded via the intraluminal pressure changes associated with the peristaltic waves.

μ‐Opioid receptor agonists (DAMGO, morphine), κ‐opioid receptor agonists (ICI‐204,448 and BRL‐52,537) and a δ‐opioid receptor agonist (SNC‐80) inhibited peristalsis in a concentration‐related manner as deduced from a rise of the peristaltic pressure threshold (PPT) and a diminution of peristaltic effectiveness.

Experiments with the δ‐opioid receptor antagonists naltrindole (30 nM) and HS‐378 (1 μM), the κ‐opioid receptor antagonist nor‐binaltorphimine (30 nM) and the μ‐opioid receptor antagonist cyprodime (10 μM) revealed that the antiperistaltic effect of ICI‐204,448 and BRL‐52,537 was mediated by κ‐opioid receptors and that of morphine and DAMGO by μ‐opioid receptors. In contrast, the peristaltic motor inhibition caused by SNC‐80 was unrelated to δ‐opioid receptor activation.

Cyprodime and nor‐binaltorphimine, but not naltrindole and HS‐378, were per se able to stimulate intestinal peristalsis as deduced from a decrease in PPT.

The results show that the neural circuits controlling peristalsis in the guinea‐pig small intestine are inhibited by endogenous and exogenous opioids acting viaμ‐ and κ‐, but not δ‐, opioid receptors.

British Journal of Pharmacology (2002) 135, 741–750; doi:10.1038/sj.bjp.0704527
Cyclooxygenase‐1 derived prostaglandins are involved in the maintenance of renal function in rats with cirrhosis and ascites
Authors: López‐Parra Marta , Clària Joan , Planagumà Anna , Titos Esther , Masferrer Jaime L , Woerner B Mark , Koki Alane T , Jiménez Wladimiro , Altuna Rosario , Arroyo Vicente , Rivera Francisca , Rodés Joan
Published: January 2005

The maintenance of renal function in decompensated cirrhosis is highly dependent on prostaglandins (PGs). Since PG synthesis is mediated by cyclooxygenase‐1 and −2 (COX‐1 and COX‐2), the present study was designed to examine which COX isoform is involved in this phenomenon.

Renal COX‐1 and COX‐2 protein expression and distribution were analysed by Western blot and immunohistochemistry in nine rats with carbon tetrachloride‐induced cirrhosis and ascites and 10 control animals. The effects of placebo and selective COX‐1 (SC‐560) and COX‐2 (celecoxib) inhibitors on urine flow (V), urinary excretion of sodium (UNaV) and PGE2 (UPGE2V), glomerular filtration rate (GFR), renal plasma flow (RPF), the diuretic and natriuretic responses to furosemide and renal water metabolism were assessed in 88 rats with cirrhosis and ascites.

COX‐1 protein levels were found to be unchanged in kidneys from cirrhotic rats. In contrast, these animals showed enhanced renal COX‐2 protein expression which was focally increased in the corticomedullary region. Although UPGE2V was equally reduced by SC‐560 and celecoxib, only SC‐560 produced a significant decrease in UNaV, GFR and RPF and a pronounced impairment in the diuretic and natriuretic responses to furosemide in rats with cirrhosis and ascites. Neither SC‐560 nor celecoxib affected renal water metabolism in cirrhotic rats.

These results indicate that despite abundant renal COX‐2 protein expression, the maintenance of renal function in cirrhotic rats is mainly dependent on COX‐1‐derived prostaglandins.

British Journal of Pharmacology (2002) 135, 891–900; doi:10.1038/sj.bjp.0704528
Pulmonary hypertension secondary to left ventricular dysfunction: the role of nitric oxide and endothelin‐1 in the control of pulmonary vascular tone
Authors: Deuchar Graeme A , Docherty Andrew , MacLean Margaret R , Hicks Martin N
Published: January 2005

Using an in vivo model of pulmonary hypertension (PHT) secondary to left ventricular dysfunction (LVD), the pulmonary arterial response to the nitric oxide synthase (NOS) blocker L‐NAME (30 μmol.min−1 i.v.) and the subsequent responses to cumulatively administered endothelin‐1 (ET‐1) (0.001 – 4 nmol.kg−1 i.v.) or big ET‐1 (0.1 – 2.0 nmol.kg−1 i.v.) were studied. Additionally, the effect of the non‐selective ET‐1 receptor antagonist, SB209670, was investigated.

Eight weeks after coronary artery ligation or sham operation, rabbits demonstrated increased mean pulmonary arterial pressure (PAP) accompanied by right ventricular hypertrophy.

Blockade of NOS caused a greater increase in basal PAP (increased by 7.7±1.1 mmHg c.f. 3.8±1.0 mmHg in controls, P<0.05) and uncovered a greater pulmonary pressor response to exogenous ET‐1 in rabbits with PHT (increased by 10.2±2.3 mmHg c.f. 4.9±1.0 mmHg in controls, P<0.05).

Big ET‐1 evoked a pulmonary pressor effect, in both groups of rabbits, that was increased following blockade of NOS and was more potent in rabbits with PHT.

The non‐selective ET‐1 receptor antagonist, SB209670, reduced basal PAP (from 16.9 mmHg to 15.9 mmHg, P<0.05) in rabbits with PHT and blocked the response to ET‐1 in the presence of L‐NAME.

In conclusion, the results demonstrate that basal NO activity masks a pulmonary pressor response to exogenously administered ET‐1. An increased responsiveness to ET‐1 was shown in the pulmonary arterial bed of rabbits with PHT secondary to LVD, implicating a pathophysiological role for ET‐1 in this model.

British Journal of Pharmacology (2002) 135, 1060–1068; doi:10.1038/sj.bjp.0704529
A flavonoid‐rich diet increases nitric oxide production in rat aorta
Authors: Benito S , Lopez D , Sáiz M P , Buxaderas S , Sánchez J , Puig‐Parellada P , Mitjavila M T
Published: January 2005

Red wine intake is associated with a low risk of cardiovascular disease. This effect has been partly attributed to the action of polyphenolic compounds, which decrease the oxidation of plasma low density lipoproteins. Moreover, nitric oxide (•NO) is a vasodilator and polyphenolic compounds induce endothelium‐dependent vasorelaxation in vitro.

Here we studied whether a diet rich in dealcoholated red wine (DRW) increases acetylcholine‐induced vasorelaxation and whether ingestion of DRW‐, quercetin‐ or catechin‐rich diets modifies the •NO‐cyclic guanosine‐3′,5′‐monophosphate (cyclic GMP) pathway and superoxide anion (O2.−) release in aorta in a resting state in rats fed semi‐purified diets containing either 35% (v w−1) DRW, 0.3% (w w−1) quercetin or 0.3% (w w−1) catechin for 10 days.

•NO‐mediated vasorelaxation induced by acetylcholine was greater in rats fed the DRW‐rich diet than in those that received the control diet.

Expression of endothelial •NO synthase (eNOS) was similar in the four dietary groups. The aortic rings of rats fed either the DRW‐, quercetin‐, or catechin‐rich diets showed higher NOS activity, •NO production and cyclic GMP content than those of rats fed the control diet. No changes were observed in O2.− production.

In summary, diets rich in either DRW, quercetin or catechin induced endothelium‐dependent vasorelaxation in rat aorta in a resting state through the enhancement of •NO production, without modifying O2.− generation, thus the bioavailability of •NO was increased. The increase in the •NO‐cyclic GMP pathway explains the beneficial effect of flavonoids at vascular level.

British Journal of Pharmacology (2002) 135, 910–916; doi:10.1038/sj.bjp.0704534
Effects of ketanserin and DOI on spontaneous and 5‐HT‐evoked peristalsis of the pig ureter in vivo
Authors: Hauser D S , Mevissen M , Weiss R , Portier C J , Scholtysik G , Studer U E , Danuser H
Published: January 2005

The influence of 5‐hydroxytryptamine (5‐HT) receptor agonists and antagonists on the ureter motility was investigated in vivo on intact ureters of anaesthetized pigs. Drugs were administered intravenously or topically.

5‐HT induced a dose‐dependent increase in the frequency of ureter contractions in anaesthetized pigs when given intravenously (0.0001 – 1 mg kg−1; ED50 0.066 mg kg−1) or topically (0.001 – 1 mg ml−1; EC50 0.043 mg ml−1). Significant increases in heart rate and blood pressure were observed when the drug was given intravenously but not topically.

The 5‐HT2A agonist, DOI (1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane) increased the frequency of ureteral contractions in a dose‐dependent manner (1 – 300 μg kg−1 i.v.). Calculation of ED50 indicated this compound to be about 1.5 times more potent with an efficacy of 23% compared to 5‐HT.

The 5‐HT2A/2C antagonist, ketanserin (0.5 mg kg−1) and the 5‐HT2C antagonist, methysergide (1 mg kg−1) antagonized the 5‐HT‐induced ureter peristalsis when given intravenously. Contraction amplitude, blood pressure and heart rate were not affected by the antagonists.

Intravenous (0.0001 – 1 mg kg−1) and topical (0.0001 – 1 μg ml−1) ketanserin significantly decreased the frequency of spontaneous ureteral contractions to about 30% of controls, which could be partly reversed by 5‐HT (0.3 mg kg−1 i.v.). The contraction amplitude, contractions of the contralateral, saline perfused ureter, heart rate and mean arterial blood pressure were not affected.

Thus, contractility of porcine ureter is mediated by 5‐HT2 receptors. Their antagonists ketanserin and methysergide seem to be promising drugs for treatment of acute ureteric colic or in preparing the ureter for ureteroscopy.

British Journal of Pharmacology (2002) 135, 1026–1032; doi:10.1038/sj.bjp.0704536
Attenuation of cardiac fibrosis by pirfenidone and amiloride in DOCA‐salt hypertensive rats
Authors: Mirkovic Stevo , Seymour Anne‐Marie L , Fenning Andrew , Strachan Anna , Margolin Solomon B , Taylor Stephen M , Brown Lindsay
Published: January 2005

This study has administered pirfenidone (5‐methyl‐1‐phenyl‐2‐[1H]‐pyridone) or amiloride to attenuate the remodelling and associated functional changes, especially an increased cardiac stiffness, in DOCA‐salt hypertensive rats.

In control rats, the elimination half‐life of pirfenidone following a single intravenous dose of 200 mg kg−1 was 37 min while oral bioavailability at this dose was 25.7%. Plasma pirfenidone concentrations in control rats averaged 1.9±0.1 μg ml−1 over 24 h after 14 days' administration as a 0.4% mixture in food.

Pirfenidone (approximately 250 – 300 mg kg−1 day−1 as 0.4% in food) and amiloride (1 mg kg−1 day−1 sc) were administered for 2 weeks starting 2 weeks post‐surgery. Pirfenidone but not amiloride attenuated ventricular hypertrophy (2.69±0.09, UNX 2.01±0.05. DOCA‐salt 3.11±0.09 mg kg−1 body wt) without lowering systolic blood pressure.

Collagen deposition was significantly increased in the interstitium after 2 weeks and further increased with scarring of the left ventricle after 4 weeks; pirfenidone and amiloride reversed the increases and prevented further increases. This accumulation of collagen was accompanied by an increase in diastolic stiffness constant; both amiloride and pirfenidone reversed this increase.

Noradrenaline potency (positive chronotropy) was decreased in right atria (neg log EC50: control 6.92±0.06; DOCA‐salt 6.64±0.08); pirfenidone but not amiloride reversed this change. Noradrenaline was a more potent vasoconstrictor in thoracic aortic rings (neg log EC50: control 6.91±0.10; DOCA‐salt 7.90±0.07); pirfenidone treatment did not change noradrenaline potency.

Thus, pirfenidone and amiloride reverse and prevent cardiac remodelling and the increased cardiac stiffness without reversing the increased vascular responses to noradrenaline.

British Journal of Pharmacology (2002) 135, 961–968; doi:10.1038/sj.bjp.0704539
Pharmacological characterization of the chemokine receptor, CCR5
Authors: Mueller Anja , Mahmoud Nasir G , Goedecke Marc C , McKeating Jane A , Strange Philip G
Published: January 2005

We investigated the effects of a number of naturally occurring chemokines (MIP‐1α, MIP‐1β, RANTES, MCP‐2, MCP‐3, MCP‐4) on different processes linked to the chemokine receptor CCR5 in recombinant CHO cells expressing the receptor at different levels.

Internalization of CCR5 following chemokine treatment was studied and MIP‐1α, MIP‐1β and RANTES (50 nM) were able to induce internalization (∼50%) of the receptor. Internalization due to MCP‐2, MCP‐3 and MCP‐4 was less (∼20%).

Phosphorylation of CCR5 following chemokine treatment was studied and MIP‐1α, MIP‐1β and RANTES (50 nM) were able to induce phosphorylation of CCR5 whereas the other chemokines did not induce CCR5 phosphorylation.

MIP‐1α, MIP‐1β, RANTES and MCP‐2 were able to stimulate [35S]‐GTPγS binding, an index of receptor/G protein activation, whereas MCP‐3 and MCP‐4 had no effect in this assay. MCP‐2 was a partial agonist (∼80%) compared to MIP‐1α, MIP‐1β and RANTES, which gave similar maximal stimulations in this assay.

MIP‐1α, MIP‐1β, RANTES, MCP‐2 and MCP‐4 were able to stimulate increases in intracellular calcium ions via activation of CCR5 whereas MCP‐3 was without effect.

It is concluded that different chemokines interacting with CCR5 mediate different patterns of cellular responses.

British Journal of Pharmacology (2001) 135, 1033–1043; doi:10.1038/sj.bjp.0704540
Divergent effects of vitamin C on relaxations of rabbit aortic rings to acetylcholine and NO‐donors
Authors: Saram K de , McNeill K L , Khokher S , Ritter J M , Chowienczyk P J
Published: January 2005

Vitamin C may influence NO‐dependent relaxation independently of effects on oxidant stress.

We investigated effects of vitamin C (0.1 – 10 mmol l−1) on relaxation of pre‐constricted rabbit aortic rings to acetylcholine (ACh), authentic NO and the NO‐donors glyceryl trinitrate (GTN), nitroprusside (NP) and S‐nitroso‐N‐acetyl‐penicillamine (SNAP). DETCA (2 – 6 mmol l−1), a cell permeable inhibitor of endogenous Cu‐Zn superoxide dismutase (SOD) was used to increase intracellular superoxide anion (O2−).

Vitamin C reduced the response to ACh (71±7% inhibition of maximum relaxation at 10 mmol l−1) and inhibited relaxation to authentic NO. Vitamin C inhibited relaxation to GTN but potentiated relaxations to NP and SNAP, causing a parallel shift to a lower concentration range of the log dose‐response curve by approximately one log unit at the highest dose.

Vitamin C increased the concentration of NO in bath solution (plus EDTA, 1.0 mmol l−1) following the addition of SNAP from 53±14 to 771±101 nmol l−1 over the range 0.1 – 3.0 mmol l−1.

DETCA inhibited relaxation to ACh (71±9% inhibition of maximum relaxation). This inhibition was abolished by a cell permeable SOD mimetic, but not by vitamin C. DETCA inhibited relaxation to SNAP but not that to NP nor to GTN.

Vitamin C inhibits endothelium‐dependent relaxations of rabbit aortic rings to ACh and authentic NO and does not reverse impaired relaxation resulting from increased intracellular oxidant stress. Vitamin C potentiates relaxation to the NO‐donors NP and SNAP by a mechanism that could involve release of NO from nitrosothiols.

British Journal of Pharmacology (2002) 135, 1044–1050; doi:10.1038/sj.bjp.0704541
Specific [3H]‐guanosine binding sites in rat brain membranes
Authors: Traversa Ugo , Bombi Giulia , Iorio Patrizia Di , Ciccarelli Renata , Werstiuk Eva S , Rathbone Michel P
Published: January 2005

Extracellular guanosine has diverse effects on many cellular components of the central nervous system, some of which may be related to its uptake into cells and others to its ability to release adenine‐based purines from cells. Yet other effects of extracellular guanosine are compatible with an action on G‐protein linked cell membrane receptors.

Specific binding sites for [3H]‐guanosine were detected on membrane preparations from rat brain. The kinetics of [3H]‐guanosine binding to membranes was described by rate constants of association and dissociation of 2.6122×107 M−1 min−1and 1.69 min−1, respectively.

A single high affinity binding site for [3H]‐guanosine with aKDof 95.4±11.9 nMand Bmaxof 0.57±0.03 pmol mg−1protein was shown.

This site was specific for guanosine, and the order of potency in displacing 50 nM[3H]‐guanosine was: guanosine=6‐thio‐guanosine>inosine>6‐thio‐guanine>guanine. Other naturally occurring purines, such as adenosine, hypoxanthine, xanthine caffeine, theophylline, GDP, GMP and ATP were unable to significantly displace the radiolabelled guanosine. Thus, this binding site is distinct from the well‐characterized receptors for adenosine and purines.

The addition of GTP produced a small concentration‐dependent decrease in guanosine binding, suggesting this guanosine binding site was linked to a G‐protein.

Our results therefore are consistent with the existence of a novel cell membrane receptor site, specific for guanosine.

British Journal of Pharmacology(2002)135, 969–976; doi:10.1038/sj.bjp.0704542
Cyclosporin A‐induced free radical generation is not mediated by cytochrome P‐450
Authors: Krauskopf Alexandra , Buetler Timo M , Nguyen Nathalie S D , Macé Katherine , Ruegg Urs T
Published: January 2005

Reactive oxygen species (ROS) have been proposed to play a role in the side effects of the immunosuppressive drug cyclosporin A (CsA).

The aim of this study was to investigate whether cytochrome P‐450 (CYP) dependent metabolism of CsA could be responsible for ROS generation since it has been suggested that CsA may influence the CYP system to produce ROS.

We show that CsA (1 – 10 μM) generated antioxidant‐inhibitable ROS in rat aortic smooth muscle cells (RASMC) using the fluorescent probe 2,7‐dichlorofluorescin diacetate.

Using cytochrome c as substrate, we show that CsA (10 μM) did not inhibit NADPH cytochrome P‐450 reductase in microsomes prepared from rat liver, kidney or RASMC.

CsA (10 μM) did not uncouple the electron flow from NADPH via NADPH cytochrome P‐450 reductase to the CYP enzymes because CsA did not inhibit the metabolism of substrates selective for several CYP enzymes that do not metabolize CsA in rat liver microsomes.

CsA (10 μM) did not generate more radicals in CYP 3A4 expressing immortalized human liver epithelial cells (T5‐3A4 cells) than in control cells that do not express CYP 3A4.

Neither diphenylene iodonium nor the CYP 3A inhibitor ketoconazole were able to block ROS formation in rat aortic smooth muscle or T5‐3A4 cells.

These results demonstrate that CYP enzymes do not contribute to CsA‐induced ROS formation and that CsA neither inhibits NADPH cytochrome P‐450 reductase nor the electron transfer to the CYP enzymes.

British Journal of Pharmacology (2002) 135, 977–986; doi:10.1038/sj.bjp.0704544
Prevention of a hypoxic Ca2+i response by SERCA inhibitors in cerebral arterioles
Authors: Guibert C , Flemming R , Beech D J
Published: January 2005

The aim of the study was to investigate the mechanism of a novel effect of hypoxia on intracellular Ca2+ signalling in rabbit cerebral arteriolar smooth muscle cells, an effect that was resistant to the L‐type Ca2+ channel antagonist methoxyverapamil (D600).

[Ca2+]i of smooth muscle cells in intact arteriolar fragments was measured using the Ca2+‐indicator dye fura‐PE3. Hypoxia (PO2 10 – 20 mmHg) lowered basal [Ca2+]i but did not inhibit Ca2+ entry pathways measured by Mn2+‐quenching of fura‐PE3.

The effect of hypoxia was completely prevented by thapsigargin or cyclopiazonic acid, selective inhibitors of sarcoplasmic reticulum Ca2+ ATPase (SERCA). Since these inhibitors do not block Ca2+ extrusion or uptake via the plasma membrane, the data indicate that the effect of hypoxia depends on a functional sarcoplasmic reticulum.

Because actions of nitric oxide (NO) on vascular smooth muscle are also prevented by SERCA inhibitors it was explored whether the effect of hypoxia occurred via modulation of endogenous NO release. Residual NOS‐I and NOS‐III were detected by immunostaining, and there were NO‐dependent effects of NOS inhibitors on Ca2+i‐signalling. Nevertheless, inhibition of endogenous NO production did not prevent the effect of hypoxia on [Ca2+]i.

The experiments reveal a novel nitric oxide‐independent effect of hypoxia that is prevented by SERCA inhibitors.

British Journal of Pharmacology (2002) 135, 927–934; doi:10.1038/sj.bjp.0704547
The 5‐series F2‐isoprostanes possess no vasomotor effects in the rat thoracic aorta, the human internal mammary artery and the human saphenous vein
Authors: Marlière Stéphanie , Cracowski Jean‐Luc , Durand Thierry , Chavanon Olivier , Bessard Janine , Guy Alexandre , Stanke‐Labesque Françoise , Rossi Jean‐Claude , Bessard Germain
Published: January 2005

Among the F2‐isoprostanes, the 15‐ and the 5‐series are currently used as markers of lipid peroxidation in vascular diseases. 15‐F2t‐IsoP (also named iPF2α‐III) exerts a vasoconstriction in most vessels, whereas no data is available concerning 5‐F2t‐IsoP (also named iPF2α‐VI), which is more abundant in plasma.

The aim of this study was to determine whether 5‐F2t‐IsoP possess any vascular effects on various vessels including the isolated rat thoracic aorta, the human internal mammary artery and the saphenous vein.

In organ baths, 5‐F2t‐IsoP and its 5‐epimer did not affect the basal tone of any vessel, unlike 15‐F2t‐IsoP. These compounds possessed no antagonist effects on 15‐F2t‐IsoP‐induced contractions, No dilator effect was observed in comparison with sodium nitroprusside and acetylcholine on the rat aorta.

In conclusion, we show that unlike 15‐F2t‐IsoP, 5‐F2t‐IsoP and its 5‐epimer possess no vasomotor effects and as such are unlikely to be involved in the pathogenesis of vascular diseases. Further studies are required to test whether these mediators may have effects on systems not being measured in the current study.

British Journal of Pharmacology (2002) 135, 1276–1280; doi:10.1038/sj.bjp.0704558
Mechanisms of action of proteinase‐activated receptor agonists on human platelets
Authors: Chung Ada W Y , Jurasz Paul , Hollenberg Morley D , Radomski Marek W
Published: January 2005

We studied the activation of human platelets by thrombin and proteinase activated receptor (PAR)‐activating peptides (PAR‐APs) [SFLLRNPNDKYEPF‐amide (TRAP), TFLLR‐amide (PAR1AP) and AYPGKF‐amide (PAR4AP)].

PAR agonist‐induced platelet aggregation, glycoprotein (GP) Ib and GPIIb/IIIa surface expression and ADP release were measured by light aggregometry, flow cytometry and chemiluminescence.

Aggregation inhibitors, including prostacyclin (PGI2), nitric oxide‐releasing agent (S‐nitroso‐glutathione, GSNO), aspirin, apyrase, and phenanthroline were used to study the susceptibility of PAR agonist‐induced aggregation to pharmacological inhibition.

Thrombin was the most potent platelet agonist, followed by PAR1AP, TRAP and PAR4AP.

The aggregatory potencies of PAR‐APs were not modified by the aminopeptidase inhibitor, amastatin.

Subthreshold concentrations of PAR1AP potentiated the effects of PAR4AP to stimulate maximal aggregation.

Both PGI2 and GSNO reduced PAR agonist‐induced aggregation and diminished GPIIb/IIIa up‐regulation.

PAR agonist‐induced aggregation was aspirin‐insensitive indicating a minor role for TXA2.

In contrast, phenanthroline and apyrase significantly enhanced the anti‐aggregatory effects of aspirin against thrombin‐, PAR1AP‐ and TRAP‐induced aggregation suggesting the involvement of ADP‐ and MMP‐2‐dependent pathways.

PAR4AP‐induced aggregation (but not PAR1AP‐induced aggregation) was entirely ADP‐dependent (abolished by apyrase) and resistant to phenanthroline (MMP‐2‐independent).

Thus, the mechanisms of PAR1 and 4‐induced platelet aggregation are distinct and depend differentially on their ability to interact with pathways of aggregation, along with the subsequent activation of GPIIb/IIIa receptors.

British Journal of Pharmacology (2002) 135, 1123–1132; doi:10.1038/sj.bjp.0704559
Multiple pathways for fluoroquinolone secretion by human intestinal epithelial (Caco‐2) cells
Authors: Lowes Simon , Simmons Nicholas L
Published: January 2005

Human intestinal epithelial Caco‐2 cells, T84 cells, and MDCKII cells transfected with human MDR1, were used to investigate the mechanistic basis of transintestinal fluoroquinolone secretion.

The fluoroquinolone grepafloxacin was secreted across Caco‐2 monolayers by a saturable process (Vmax=16.9±3.4 nmol.cm−2.h−1). Net secretion was reduced by 2‐deoxyglucose/azide treatment to reduce intracellular ATP.

Grepafloxacin inhibited [14C]‐ciprofloxacin (100 μM) secretion across Caco‐2 monolayers (K0.5=0.8 mM), and concurrently increased the cellular accumulation of ciprofloxacin from the basal medium, indicating inhibition of export across the apical membrane.

The unconjugated bile acid, cholic acid, was secreted across Caco‐2 monolayers, and this secretion was sensitive to inhibition by the MRP‐selective inhibitor MK‐571, suggesting MRP2 involvement. Secretion of cholic acid (10 μM) across the apical membrane was also inhibited by grepafloxacin (K0.5=0.3 mM), but not by ciprofloxacin.

In MDCKII‐MDR1 monolayers, net secretion of grepafloxacin was increased by 3.5 fold compared with untransfected controls. Neither ciprofloxacin nor cholic acid showed net secretion in either MDCKII or MDCKII‐MDR1 monolayers, showing that in contrast to grepafloxacin, neither are substrates for MDR1.

In T84 monolayers, which express MDR1 but not MRP2, neither ciprofloxacin nor cholic acid was secreted, whilst the Vmax for grepafloxacin secretion was lower than in Caco‐2 cells, which express both MDR1 and MRP2.

In conclusion, the transepithelial secretion of grepafloxacin is mediated by both MRP2 and MDR1, whereas ciprofloxacin is a substrate for neither. Grepafloxacin also competes for the ciprofloxacin‐sensitive pathway, which remains to be elucidated.

British Journal of Pharmacology (2002) 135, 1263–1275; doi:10.1038/sj.bjp.0704560
Extracellular signal‐regulated kinase plays an essential role in endothelin‐1‐induced homotypic adhesion of human neutrophil granulocytes
Authors: József Levente , Khreiss Tarek , Fournier Alain , Chan John S D , Filep János G
Published: January 2005

Endothelin‐1 (ET‐1) stimulates integrin‐dependent adhesion of neutrophil granulocytes to endothelial cells, one of the early key events in acute inflammation. However, the signalling pathway(s) of ET‐1‐stimulated neutrophil adhesive responses has not been elucidated. Previous studies indicated that extracellular signal‐regulated kinase (ERK) activation could mediate rapid responses of neutrophil granulocytes to various stimuli. In this study, we investigated the role of ERK signalling in human neutrophil granulocytes challenged with ET‐1.

ET‐1 rapidly down‐regulated the expression of L‐selectin and up‐regulated the expression of CD11b/CD18 on the neutrophil surface. Concomitantly, ET‐1 induced homotypic adhesion (aggregation) of neutrophils, that was blocked by a monoclonal antibody to CD18.

ET‐1, through ETA receptors, evoked activation of Ras and subsequent phosphorylation of Raf‐1, mitogen‐activated protein kinase kinase (MAPK/ERK kinase) and ERK 1/2. ERK activation by ET‐1 was rapid, concordant with the kinetics of ET‐1‐stimulated neutrophil aggregation.

Neutrophil responses to ET‐1 were markedly attenuated by the MAPK/ERK kinase inhibitor PD98059, whereas inhibitors of p38 MAPK, tyrosine kinases and phosphatidylinositol 3‐kinase had no detectable effects. We have observed a tight correlation between neutrophil ERK activation and homotypic adhesion.

These data indicate an essential role for ERK in mediating ET‐1‐stimulated adhesive responses of human neutrophil granulocytes.

British Journal of Pharmacology (2002) 135, 1167–1174; doi:10.1038/sj.bjp.0704561
Protease‐activated receptor‐2 (PAR‐2) in the rat gastric mucosa: immunolocalization and facilitation of pepsin/pepsinogen secretion
Authors: Kawao Naoyuki , Sakaguchi Yuriko , Tagome Ai , Kuroda Ryotaro , Nishida Shozo , Irimajiri Kiyohiro , Nishikawa Hiroyuki , Kawai Kenzo , Hollenberg Morley D , Kawabata Atsufumi
Published: January 2005

Agonists of protease‐activated receptor‐2 (PAR‐2) trigger neurally mediated mucus secretion accompanied by mucosal cytoprotection in the stomach. The present study immunolocalized PAR‐2 in the rat gastric mucosa and examined if PAR‐2 could modulate pepsin/pepsinogen secretion in rats.

PAR‐2‐like immunoreactivity was abundant in the deep regions of gastric mucosa, especially in chief cells.

The PAR‐2 agonist SLIGRL‐NH2, but not the control peptide LSIGRL‐NH2, administered i.v. repeatedly at 0.3 – 1 μmol kg−1, four times in total, significantly facilitated gastric pepsin secretion, although a single dose produced no significant effect.

The PAR‐2‐mediated gastric pepsin secretion was resistant to omeprazole, NG‐nitro‐L‐arginine methyl ester (L‐NAME) or atropine, and also to ablation of sensory neurons by capsaicin.

Our study thus provides novel evidence that PAR‐2 is localized in mucosal chief cells and facilitates gastric pepsin secretion in the rats, most probably by a direct mechanism.

British Journal of Pharmacology (2002) 135, 1292–1296; doi:10.1038/sj.bjp.0704562
Probucol preserves endothelial function by reduction of the endogenous nitric oxide synthase inhibitor level
Authors: Jiang Jun‐Lin , Li Nian‐sheng , Li Yuan‐Jian , Deng Han‐Wu
Published: January 2005

Oxide low‐density lipoprotein (ox‐LDL) is believed to play an important role in early events of atherogenesis, and asymmetric dimethylarginine (ADMA) is associated with the development of endothelial dysfunction. The present study examined the effect of a single injection of native low‐density lipoprotein (LDL) on endothelium function and the serum level of ADMA and the effect of probucol on endothelium function and ADMA level in rats.

Endothelial injury was induced by intravenous injection of LDL at the dose of 2, 4, or 6 mg kg−1 for 24, 48, or 72 h, and vasodilator responses to acetylcholine in the aortic rings and serum levels of ADMA, nitrite/nitrate (NO) and malondialdehyde (MDA) were determined.

Pretreatment with LDL markedly reduced endothelium‐dependent relaxation in a concentration‐dependent manner. Inhibition of vasodilator responses to acetylcholine by LDL was abolished in the presence of L‐arginine (3×10−4 M). Serum levels of ADMA and MDA were significantly elevated in the rats pretreated with LDL, while serum level of nitrite/nitrate was markedly decreased.

Pretreatment with probucol significantly improved endothelium‐dependent relaxation, decreased concentrations of ADMA and MDA and increased nitrite/nitrate level in the rats treated with LDL. A similar effect was seen in the rats pretreated with an antioxidant vitamin E.

These results suggest that a single injection of native LDL causes endothelial dysfunction by elevation of ADMA levels and that the protective effect of probucol on endothelial cells is related to reduction of ADMA concentration.

British Journal of Pharmacology (2002) 135, 1175–1182; doi:10.1038/sj.bjp.0704563
Platelet‐activating factor drives eotaxin production in an allergic pleurisy in mice
Authors: Klein André , Pinho Vanessa , Alessandrini Ana Letícia , Shimizu Takao , Ishii Satoshi , Teixeira Mauro M
Published: January 2005

The activation of eosinophils via G‐protein‐coupled seven transmembran receptors play a necessary role in the recruitment of these cells into tissue. The present study investigates a role for PAF in driving eotaxin production and eosinophil recruitment in an allergic pleurisy model in mice.

The intrapleural injection of increasing doses of PAF (10−11 to 10−9 moles per cavity) induced a dose‐ and PAF receptor‐dependent recruitment of eosinophils 48 h after stimulation.

Intrapleural injection of PAF induced the rapid (within 1 h) release of eotaxin into the pleural cavity of mice and an anti‐eotaxin antibody effectively inhibited PAF‐induced recruitment of eosinophils.

Eosinophil recruitment in the allergic pleurisy was markedly inhibited by the PAF receptor antagonist UK‐74,505 (modipafant, 1 mg kg−1). Moreover, recruitment of eosinophils in sensitized and challenged PAF receptor‐deficient animals was lower than that observed in wild‐type animals.

Blockade of PAF receptors with UK‐74,505 suppressed by 85% the release of eotaxin in the allergic pleurisy.

Finally, the injection of a sub‐threshold dose of PAF and eotaxin cooperated to induce eosinophil recruitment in vivo.

In conclusion, the production of PAF in an allergic reaction could function in multiple ways to facilitate the recruitment of eosinophils  –  by facilitating eotaxin release and by cooperating with eotaxin to induce greater recruitment of eosinophils.

British Journal of Pharmacology (2002) 135, 1213–1218; doi:10.1038/sj.bjp.0704570
5‐HT2B receptors play a key role in mediating the excitatory effects of 5‐HT in human colon in vitro
Authors: Borman R A , Tilford N S , Harmer D W , Day N , Ellis E S , Sheldrick R L G , Carey J , Coleman R A , Baxter G S
Published: January 2005

5‐Hydroxytryptamine (5‐HT) is known to produce a number of different effects in the gastrointestinal tract of various species, and has been proposed to play a key role in a number of intestinal disorders in man, including irritable bowel syndrome (IBS), although the receptors involved have yet to be established. The aim of the present study was to investigate the distribution and function of 5‐HT2B receptors in human colon, and to establish their possible role in the aetiology of IBS.

The distribution of 5‐HT2B receptor mRNA and protein were investigated by quantitative RT – PCR, Western analysis and immunocytochemistry. High levels of both mRNA and protein for 5‐HT2B receptors were found throughout the human gastrointestinal tract, and in particular in colon, where 5‐HT2B receptors were found predominantly in the longitudinal and circular smooth muscle layers within the muscularis externa, and in the myenteric nerve plexus lying between these two layers.

Electrical field stimulation of longitudinal muscle preparations of human colon mounted in organ baths resulted in neuronally‐mediated contractile responses, that were significantly potentiated by application of 5‐HT (up to 10−7 M), with a pEC50 of 8.2±0.1 (n=49 donors). The response to 5‐HT was inhibited by a number of selective 5‐HT2B receptor antagonists.

This study has shown for the first time that, in contrast to animal studies, the excitatory effects of 5‐HT in human colon are mediated by 5‐HT2B receptors. It is proposed that these receptors contribute to the putative 5‐HT‐induced colonic smooth muscle hypersensitivity associated with IBS.

British Journal of Pharmacology (2002) 135, 1144–1151; doi:10.1038/sj.bjp.0704571
Effect of SEA0400, a novel inhibitor of sodium‐calcium exchanger, on myocardial ionic currents
Authors: Tanaka Hikaru , Nishimaru Kazuhide , Aikawa Tokiko , Hirayama Wataru , Tanaka Yoshio , Shigenobu Koki
Published: January 2005

The effects of 2‐[4‐[(2,5‐difluorophenyl) methoxy]phenoxy]‐5‐ethoxyaniline (SEA0400), a newly synthesized Na+‐Ca2+ exchanger (NCX) inhibitor, on the NCX current and other membrane currents were examined in isolated guinea‐pig ventricular myocytes and compared with those of 2‐[2‐[4‐(4‐nitrobenzyloxy) phenyl]ethyl]isothiourea (KB‐R7943). SEA0400 concentration‐dependently inhibited the NCX current with a 10 fold higher potency than that of KB‐R7943; 1 μM SEA0400 and 10 μM KB‐R7943 inhibited the NCX current by more than 80%. KB‐R7943, at 10 μM, inhibited the sodium current, L‐type calcium current, delayed rectifier potassium current and inwardly rectifying potassium current by more than 50%, but SEA0400 (1 μM) had no significant effect on these currents. These results indicate that SEA0400 is a potent and highly selective inhibitor of NCX, and would be a powerful tool for further studies on the role of NCX in the heart and the therapeutic potential of its inhibition.

British Journal of Pharmacology (2002) 135, 1096–1100; doi:10.1038/sj.bjp.0704574
Naloxone prevents cell‐mediated immune alterations in adult mice following repeated mild stress in the neonatal period
Authors: Loizzo Alberto , Loizzo Stefano , Lopez Luisa , D'Amore Antonio , Renzi Paolo , Spampinato Santi , Di Carlo Simonetta , Bacosi Antonella , Zuccaro Piergiorgio , Pacifici Roberta
Published: January 2005

Mild stress plus mild pain (solvent injection) applied daily to neonatal mice induces hormonal, behavioural and metabolic changes perduring in the adult life.

We investigated whether daily mild stress to neonatal mice induces also long‐term defined changes of immune response, and whether immune changes are prevented through repeated administration of the opioid antagonist naloxone.

Mild stress plus solvent injection administered from birth to the 21st postnatal day causes not only behavioural and metabolic changes, but also long‐term (up to 110 days of life) splenocytes modifications, consisting in: increased release of the Th‐1 type cytokines interleukin‐2 (IL‐2) (from an average of 346 to 788 pg ml−1), interferon‐γ (from 1770 to 3942) and tumour necrosis factor‐α (from 760 to 1241); decreased release of the Th‐2 type cytokines IL‐4 (from 49.1 to 28.4) and IL‐10 (from 1508 to 877). Moreover, enhanced natural killer‐cell activity; enhanced proliferative splenocytes properties in resting conditions and following phytohemoagglutinin and concanavalin‐A stimulation are observed. Immunological, behavioural and metabolic changes are prevented by the opioid antagonist (−)naloxone (1 mg kg−1 per day s.c., administered instead of solvent) but not by the biologically inactive enantiomorph (+)naloxone.

In conclusion, endogenous opioid systems sensitive to naloxone are involved in long‐lasting enhancement of the Th‐1 type cytokines and cell‐mediated immunological response caused by repeated mild stress administered postnatally.

British Journal of Pharmacology (2002) 135, 1219–1226; doi:10.1038/sj.bjp.0704577
Characterization of the discriminable stimulus produced by 2‐BFI: effects of imidazoline I2‐site ligands, MAOIs, β‐carbolines, agmatine and ibogaine
Authors: MacInnes Nicholas , Handley Sheila L
Published: January 2005

The molecular nature and functions of the I2 subtype of imidazoline binding sites are unknown but evidence suggests an association with monoamine oxidase (MAO). Rats can distinguish the selective imidazoline I2‐site ligand 2‐BFI from vehicle in drug discrimination, indicating functional consequences of occupation of these sites. We have used drug discrimination to investigate the nature of the discriminable stimulus, especially in relation to MAO inhibition.

Following training to distinguish 2‐BFI 7 mg kg−1 i.p. from saline vehicle in two‐lever operant‐chambers, male Hooded Lister rats underwent sessions where test substances were given instead and the proportion of lever presses on the 2‐BFI‐associated lever (substitution) recorded.

2‐BFI; its cogeners BU216, BU224, BU226 and LSL60101; the reversible MAO‐A inhibitors moclobemide and RO41‐1049; the β‐carbolines harmane, norharmane and harmaline which also reversibly inhibit MAO‐A, and the anti‐addictive substance ibogaine exhibited potent, dose‐dependent substitution for 2‐BFI.

Agmatine, and LSL60125 substituted at one dose only. The reversible MAO‐B inhibitors lazabemide and RO16‐1649; the σ2‐site ligand SKF10,047 and the I2A‐site ligand, amiloride, failed to substitute. The irreversible inhibitor of MAO, deprenyl, substituted for 2‐BFI while clorgyline did not.

These results suggest imidazoline I2 site ligands produce a common discriminable stimulus that appears associated with reversible inhibition of MAO‐A rather than MAO‐B, possibly through increases in extracellular concentration of one or more monoamines. Ibogaine exhibits a commonality in its subjective effects with those of I2‐site ligands.

British Journal of Pharmacology (2002) 135, 1227–1234; doi:10.1038/sj.bjp.0704579
Kainic acid‐induced neuronal cell death in cerebellar granule cells is not prevented by caspase inhibitors
Authors: Verdaguer Ester , García‐Jordà Elvira , Jiménez Andrés , Stranges Alessandra , Sureda Francesc X , Canudas Anna M , Escubedo Elena , Camarasa Jordi , Pallàs Mercè , Camins Antoni
Published: January 2005

We examined the role of non‐NMDA receptors in kainic acid (KA)‐induced apoptosis in cultures of rat cerebellar granule cells (CGCs). KA (1 – 500 μM) induced cell death in a concentration‐dependent manner, which was prevented by NBQX and GYKI 52466, non‐NMDA receptor antagonists. Moreover, AMPA blocked KA‐induced excitotoxicity, through desensitization of AMPA receptors.

Similarly, KA raised the intracellular calcium concentration of CGCs, which was inhibited by NBQX and GYKI 52466. Again, AMPA (100 μM) abolished the KA (100 μM)‐induced increase in intracellular calcium concentration.

KA‐induced cell death in CGCs had apoptotic features, which were determined morphologically, by DNA fragmentation, and by expression of the prostate apoptosis response‐4 protein (Par‐4).

KA (500 μM) slightly (18%) increased caspase‐3 activity, which was strongly enhanced by colchicine (1 μM), an apoptotic stimulus. However, neither Z‐VAD.fmk, a pan‐caspase inhibitor, nor the more specific caspase‐3 inhibitor, Ac‐DEVD‐CHO, prevented KA‐induced cell death or apoptosis. In contrast, both drugs inhibited colchicine‐induced apoptosis.

The calpain inhibitor ALLN had no effect on KA or colchicine‐induced neurotoxicity.

Our findings indicate that colchicine‐induced apoptosis in CGCs is mediated by caspase‐3 activation, unlike KA‐induced apoptosis.

British Journal of Pharmacology (2002) 135, 1297–1307; doi:10.1038/sj.bjp.0704581
Characterization of the human fMLP receptor in neutrophils and in Xenopus oocytes
Authors: Wittmann Sigrid , Fröhlich Dieter , Daniels Stephen
Published: January 2005

N‐formyl peptides (e.g. fMLP; N‐formyl‐L‐methionyl‐L‐leucyl‐phenylalanine) are potent mediators for inflammatory reactions. We report functional expression in Xenopus oocytes of human fMLP‐R98 cDNA, without co‐expression of the promiscuous G‐protein subunit, Gα‐16.

Stimulation of voltage‐clamped oocytes (−70 mV) with fMLP produced a dose‐dependent biphasic inward current with fast and slow components. Analysis using GTP‐γ‐S and cholera and pertussis toxins suggested these currents are mediated by an endogenous G‐protein of the Gq family.

The fast current reversed at −25 mV and was blocked by SITS (4‐acetamido‐4′‐isothiocyanatostilbene‐2,2′‐disulphonic acid), suggesting the current is carried by Cl−. The slow current showed weak inward rectification, was Ca2+‐dependent and blocked by Cd2+, 4‐AP (4‐aminopyridine) and haloperidol, suggesting activation of a mixed population of cation channels.

Comparative experiments with human neutrophils using flow cytometric analysis showed that the proportion of neutrophils activated by fMLP was reduced in the presence of SITS, in the absence of external calcium and in the presence of Cd2+, TEA (tetraethylammonium) and haloperidol but not 4‐AP. In addition, the oxidative burst from activated neutrophils was reduced by SITS and by the absence of external calcium but not by Cd2+, TEA, 4‐AP or haloperidol.

We suggest that in human neutrophils activation by fMLP is dependent on store‐operated calcium influx that appears to be regulated by Cl− channels and linked, in part, to non‐selective cation channels.

British Journal of Pharmacology (2002) 135, 1375–1382; doi:10.1038/sj.bjp.0704592
Postnatal changes in β‐adrenoceptors in the lung and the effect of hypoxia induced pulmonary hypertension of the newborn
Authors: Hislop Alison A , Mak Judith C W , Kelly David , Reader Jayne A , Barnes Peter J , Haworth Sheila G
Published: January 2005

β‐adrenoceptor activation leads to pulmonary vasodilatation. The increase in circulating catecholamines at birth may assist the postnatal fall in vascular resistance by their activation. To study β1‐ and β2‐adrenoceptors during postnatal adaptation, we used [125I]‐iodocyanopindolol (ICYP) binding to lung membranes and sections to quantify and locate the binding sites in piglets from birth to 14 days of age and compared them with those in adult pigs. In addition, pulmonary hypertension was induced in newborn piglets by hypobaric hypoxia.

In lung membranes the equilibrium dissociation constant (Kd) did not change with age for total β‐adrenoceptors or for β2‐adrenoceptors, but there was a significant increase in maximum binding sites (Bmax) between birth and 3 days of age. On tissue sections, Bmax increased between 3 days and adulthood with no change in Kd.

Binding sites of β1‐ and β2‐adrenoceptors were localized to the bronchial epithelium, to endothelium of extra‐ and intra‐pulmonary arteries and to lung parenchyma. Total β‐adrenoceptor density increased with age at all locations (P<0.05 – 0.01). At birth intrapulmonary arteries showed no binding, β2‐adrenoceptors appeared on day 1 and increased up to 14 days of age. β1‐adrenoceptors appeared by 3 days of age and increased with age.

Hypobaric hypoxia from birth led to attenuation in the normal postnatal increase in receptor number, but hypoxia from 3 – 6 days did not decrease receptor density.

The normal postnatal increase in β‐adrenoceptors suggests a potential for catecholamine induced dilatation in the lung during adaptation which is attenuated in pulmonary hypertension.

British Journal of Pharmacology (2002) 135, 1415–1424; doi:10.1038/sj.bjp.0704597
Neostigmine interactions with non steroidal anti‐inflammatory drugs
Authors: Miranda Hugo F , Sierralta Fernando , Pinardi Gianni
Published: January 2005

The common mechanism of action of non‐steroidal anti‐inflammatory drugs (NSAIDs) is the inhibition of the enzyme cyclo‐oxygenase (COX), however, this inhibition is not enough to completely account for the efficacy of these agents in several models of acute pain.

It has been demonstrated that cholinergic agents can induce antinociception, but the nature of the interaction between these agents and NSAIDs drugs has not been studied. The present work evaluates, by isobolographic analysis, the interactions between the cholinergic indirect agonist neostigmine (NEO) and NSAIDs drugs, using a chemical algesiometric test.

Intraperitoneal (i.p.) or intrathecal (i.t.) administration of NEO and of the different NSAIDs produced dose‐dependent antinociception in the acetic acid writhing test of the mouse.

The i.p. or i.t. co‐administration of fixed ratios of ED50 fractions of NSAIDs and NEO, resulted to be synergistic or supra‐additive for the combinations ketoprofen (KETO) and NEO, paracetamol (PARA) and NEO) and diclofenac (DICLO) and NEO administered i.p. However, the same combinations administered i.t. were only additive. In addition, the combinations meloxicam (MELO) and NEO and piroxicam (PIRO) and NEO, administered either i.p. or i.t., were additive.

The results suggest that the co‐administration of NEO with some NSAIDs (e.g. KETO, PARA or DICLO) resulted in a synergistic interaction, which may provide evidence of supraspinal antinociception modulation by the increased acetylcholine concentration in the synaptic cleft of cholinergic interneurons. The interaction obtained between neostigmine and the NSAIDs could carry important clinical implications.

British Journal of Pharmacology (2002) 135, 1591–1597; doi:10.1038/sj.bjp.0704599
Commentary on Neostigmine Interactions with Non steroidal Anti‐inflammatory Drugs by Miranda et al.
Authors: Tallarida Ronald J
Published: January 2005

The article by Miranda et al. presents data showing significant synergism between NEO and certain NSAIDs administered i.p. in the mouse. Their conclusions follow from isobolar analysis accompanied by statistical confidence limits that show significant differences between the additive (expected) and experimental potencies. This commentary discusses the features of the graphical method of analysis and points out other experimental designs and the methods used to analyse them.

British Journal of Pharmacology (2002) 135, 1589–1590; doi:10.1038/sj.bjp.0704600