bluuebunny/crossref_metadata_2025 huggingface dataset:
Inactivation of platelet‐derived growth factor‐BB following modification by ADP‐ribosyltransferase
Authors: Saxty Barbara A , Yadollahi‐Farsani Masoud , Upton Paul D , Johnstone Stephen R , MacDermot John
Published: January 2005

Arginine‐specific ADP‐ribosyltransferase (ART1) is expressed on the surface of a number of cell types, and catalyses the transfer of ADP‐ribose from NAD+ to target proteins. We investigated whether extracellular proteins such as growth factors may serve as substrates for this enzyme, with subsequent alteration in their biological activity. Experiments were performed with rat skeletal muscle membranes and V79 Chinese hamster lung fibroblasts with doxycycline‐inducible expression of human ART.

From a panel of growth factors, platelet‐derived growth factor‐BB (PDGF‐BB) was found to be the best substrate for ART1, whereas the structural homologue PDGF‐AA was not a substrate. Under conditions of maximum labelling 5 mol ADP‐ribose was incorporated per mol of PDGF‐BB.

Purified (ADP‐ribosyl)‐PDGF‐BB did not stimulate a mitogenic or chemotactic response in human pulmonary smooth muscle cells, and showed a reduced capacity to bind to PDGF receptors in competition binding experiments, when compared to unmodified PDGF‐BB.

PDGF‐dependent [3H‐methyl]‐thymidine incorporation was measured in the ART1‐transfected fibroblast cell line at physiological concentrations of PDGF‐BB, and without addition of extracellular NAD+. Fibroblasts expressing human ART1 at the cell surface showed reduced mitogenic responses to PDGF‐BB, but not to PDGF‐AA. This loss of mitogenic response in cells expressing ART1 activity was reversed by the addition of agmatine (an ART1 substrate).

In conclusion, we propose that PDGF‐BB‐dependent signalling may be regulated by post‐translational modification of the growth factor by ART1 at the cell surface. This has been demonstrated in membranes of rat skeletal muscle, and the reaction confirmed in ART1‐transfected fibroblasts.

British Journal of Pharmacology (2001) 133, 1219–1226; doi:10.1038/sj.bjp.0704187
Nebivolol, bucindolol, metoprolol and carvedilol are devoid of intrinsic sympathomimetic activity in human myocardium
Authors: Brixius Klara , Bundkirchen Andreas , Bölck Birgit , Mehlhorn U , Schwinger Robert H G
Published: January 2005

The present study investigated whether or not there may be differences in the direct cardiac actions of the novel, highly β1‐selective adrenoceptor antagonist nebivolol (NEB) in comparison to metoprolol (MET), bisoprolol (BIS), carvedilol (CAR) and bucindolol (BUC) in human myocardium (n=9).

The rank order of β1‐selectivity as judged by competition experiments to 3H‐CGP 12.1777 in the presence of CGP 207.12 A (300 nmol l−1, Kiβ2) or ICI 118.551 (50 nmol l−1, Kiβ1) were NEB(Kiβ2/Kiβ1: 40.7)>BIS(15.6)>MET(4.23)>CAR(0.73)>BUC(0.49).

The rank order of the negative inotropic potency of the β‐adrenoceptor antagonists measured in left ventricular trabeculae (dilated cardiomyopathy, DCM) as judged by the concentration needed to induce a 50% decrease in isoprenaline (1 μmol l−1)‐stimulated force (IC50) was: MET (0.6 μmol l−1)>CAR (4.1 μmol l−1)>NEB (7.0 μmol l−1).

NEB, BUC, MET and CAR did not not exert an intrinsic sympathomimetic activity (ISA) as determined by measurements of force development in forskolin (0.3 μmol l−1) pre‐treated left ventricular trabeculae, nor by measuring adenylate cyclase activity in forskolin (0.3 μmol l−1)‐stimulated assays (crude membranes). This also holds true for radioligand binding assays with or without guanine nucleotide guanyl‐5′‐yl imidodiphosphate (Gpp(NH)p).

Although all studied β‐adrenoceptor antagonists lack intrinsic sympathomimetic activity (ISA), they differ in the β1‐selectivity as well as in their direct negative inotropic action. These differences as well as the mode of extracardiac action may have an impact on outcome of patients treated with β‐adrenoceptor antagonists.

British Journal of Pharmacology (2001) 133, 1330–1338; doi:10.1038/sj.bjp.0704188
The cholecystokinin analogues JMV‐180 and CCK‐8 stimulate phospholipase C through the same binding site of CCKA receptor in rat pancreatic acini
Authors: Sarri Elisabet , Ramos Belén , Salido Ginés M , Claro Enrique
Published: January 2005

This study was designed to address the controversy related to the involvement of phospholipase C in the signalling pathway linked to CCKA receptor stimulation by the cholecystokinin analogue JMV‐180, a full agonist for amylase release, in rat pancreatic acini.

JMV‐180 was shown to stimulate phospholipase C by measuring the incorporation of [32P]‐orthophosphoric acid ([32P]‐Pi) into phosphatidic acid (PtdOH) and phosphatidylinositol (PtdIns). Both responses elicited by JMV‐180 were time and concentration dependent. Maximal effects elicited by JMV‐180 were 39.08±0.72 and 8.02±0.40% for the labelling of [32P]‐PtdIns and [32P]‐PtdOH, respectively, as compared to the maximal effects of CCK‐8, a full agonist of the CCKA receptor.

[32P]‐Pi incorporation into PtdOH and PtdIns was sensitive to lithium, demonstrating that both responses are a consequence of phospholipase C activation. However, since lithium blocks the phosphoinositide cycle by an uncompetitive mechanism, its effect was only apparent at high concentrations of CCK‐8 (>10 pM), which elicited stimuli above 20 and 60% of the maximal [32P]‐PtdOH and [32P]‐PtdIns labelling, respectively.

JMV‐180 inhibited the incorporation of [32P]‐Pi into PtdOH and PtdIns as stimulated by CCK‐8, down to its own maximal effect. The estimated IC50 values for the inhibition curves were not significantly different from those calculated assuming the same single binding site for both agonists. These results indicated that the well established role of JMV‐180 as a partial agonist for CCKA receptor‐linked signalling responses, also applies for the stimulation of phospholipase C.

The comparison of CCK‐8 and JMV‐180 dose‐response curves of amylase release to those of PtdIns and PtdOH labelling with [32P]‐Pi showed the existence of an amplification mechanism between phospholipase C and amylase release for both agonists.

In conclusion, we show that JMV‐180, as well as CCK‐8, stimulate phospholipase C upon interaction with the same binding site at the CCKA receptor in rat pancreatic acini.

British Journal of Pharmacology (2001) 133, 1227–1234; doi:10.1038/sj.bjp.0704190
Role of nitric oxide and superoxide in allergen‐induced airway hyperreactivity after the late asthmatic reaction in guinea‐pigs
Authors: Boer Jacob de , Meurs Herman , Flendrig Leonard , Koopal Miranda , Zaagsma Johan
Published: January 2005

In the present study, the roles of nitric oxide (NO) and superoxide anions (O2−) in allergen‐induced airway hyperreactivity (AHR) after the late asthmatic reaction (LAR) were investigated ex vivo, by examining the effects of the NO synthase inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME) and superoxide dismutase (SOD) on the responsiveness to methacholine of isolated perfused guinea‐pig treacheae from unchallenged (control) animals and from animals 24 h after ovalbumin challenge.

At 24 h after allergen challenge, the animals developed AHR in vivo, as indicated by a mean 2.63±0.54 fold (P<0.05) increase in sensitivity to histamine inhalation.

Compared to unchallenged controls, tracheal preparations from the ovalbumin‐challenged guinea‐pigs displayed a significant 1.8 fold (P<0.01) increase in the maximal response (Emax) to methacholine, both after intraluminal (IL) and extraluminal (EL) administration of the agonist. No changes were observed in the sensitivity (pEC50) to the agonist. Consequently, the ΔpEC50 (EL‐IL), as a measure of epithelial integrity, was unchanged.

In the presence of L‐NAME (100 μM, IL), tracheae from control guinea‐pigs showed a 1.6 fold (P<0.05) increase in the Emax of IL methacholine. By contrast, the Emax of IL methacholine was significantly decreased in the presence of 100 u ml−1 EL SOD (54% of control, P<0.01).

Remarkably, the increased responsiveness to IL methacholine at 24 h after allergen challenge was reversed by L‐NAME to control (P<0.01), and a similar effect was observed with SOD (P<0.01).

The results indicate that both NO and O2− are involved in the tracheal hyperreactivity to methacholine after the LAR, possibly by promoting airway smooth muscle contraction through the formation of peroxynitrite.

British Journal of Pharmacology (2001) 133, 1235–1242; doi:10.1038/sj.bjp.0704191
Identification of the dopamine autoreceptor in the guinea‐pig retina as D2 receptor using novel subtype‐selective antagonists
Authors: Weber Bernd , Schlicker Eberhard , Sokoloff Pierre , Stark Holger
Published: January 2005

Dopamine release in the retina is subject to modulation via autoreceptors, which belong to the D2 receptor family (encompassing the D2, D3 and D4 receptors). The aim of the present study was to determine the receptor subtype (D2 vs D3) involved in the inhibition of dopamine release in guinea‐pig retinal discs, using established (haloperidol, (S)‐nafadotride) and novel dopamine receptor antagonists (ST‐148, ST‐198).

hD2L and hD3 receptors were expressed in CHO cells and the pKi values determined in binding studies with [125I]‐iodosulpride were: haloperidol 9.22 vs 8.54; ST‐148 7.85 vs 6.60; (S)‐nafadotride 8.52 vs 9.51; ST‐198 6.14 vs 7.92.

The electrically evoked tritium overflow from retinal discs preincubated with [3H]‐noradrenaline (which represents quasi‐physiological dopamine release) was inhibited by the dopamine receptor agonists B‐HT 920 (talipexole) and quinpirole (maximally by 82 and 71%; pEC50 5.80 and 5.83). The concentration‐response curves of these agonists were shifted to the right by haloperidol (apparent pA2 8.69 and 8.23) and ST‐148 (7.52 and 7.66). (S)‐Nafadotride 0.01 μM and ST‐198 0.32 μM did not affect the concentration‐response curve of B‐HT 920.

The dopamine autoreceptor in the guinea‐pig retina can be classified as a D2 receptor. ST‐148 and ST‐198 show an improved selectivity for D2 and D3 receptors when compared to haloperidol and (S)‐nafadotride, respectively.

British Journal of Pharmacology (2001) 133, 1243–1248; doi:10.1038/sj.bjp.0704192
Human colonic anti‐secretory activity of the potent NK1 antagonist, SR140333: assessment of potential anti‐diarrhoeal activity in food allergy and inflammatory bowel disease
Authors: Moriarty Derek , Goldhill Jon , Selve Norma , O'Donoghue Diarmuid P , Baird Alan W
Published: January 2005

This in vitro study was designed to determine the potential use of the NK1 antagonist, SR140333 as an anti‐diarrhoeal treatment for food allergy or inflammatory bowel disease. The effect of various immune and neuronal stimuli on human colonic substance P (SP) release and the effect of SR140333 on subsequently stimulated mucosal ion transport was investigated.

Submucosal and sensory nerve fibre stimulation using electrical field stimulation (1 ms/7 Hz/7 V) and capsaicin (50 μM) respectively, mast cell activation by anti‐IgE (1/250 dilution) and granulocyte stimulation using fMLP (50 μM) each released SP and evoked a secretory response.

SP and the NK1 selective agonist, Sar‐SP (0.1–1000 nM) stimulated an increase in colonic secretion which was antagonized by SR140333 (pD′2=6.7 and 7.25 versus SP and Sar‐SP respectively).

SR140333, at a concentration that blocked NK1‐mediated secretion (500 nM), also reduced the secretory response to both αIgE and capsaicin. This suggests a pathophysiologic role for NK1 receptors.

Capsaicin evoked SP release was increased in tissue taken from Crohn's disease but not ulcerative colitis patients. The response to SP was however reduced by 70 and 89% respectively.

Mast cells and sensory afferents contribute to allergic diarrhoea. Since SR140333 reduced the secretory response to mast cell and afferent stimulation this compound may be particularly useful in reducing the symptoms of food allergy.

British Journal of Pharmacology (2001) 133, 1346–1354; doi:10.1038/sj.bjp.0704194
Mutational analysis of the human vasoactive intestinal peptide receptor subtype VPAC2: role of basic residues in the second transmembrane helix
Authors: Vertongen P , Solano R M , Perret J , Langer I , Robberecht P , Waelbroeck M
Published: January 2005

We investigated the role of two conserved basic residues in the second transmembrane helix arginine 172 (R172) and lysine 179 (K179) of the VPAC2 receptor.

Vasoactive intestinal polypeptide (VIP) activated VPAC2 receptors with an EC50 value of 7 nM, as compared to 150, 190 and 4000 nM at R172L, R172Q and K179Q‐VPAC2 receptors, respectively. It was inactive at K179I mutated VPAC2 receptors. These results suggested that both basic residues were probably implicated in receptor recognition and activation.

The VPAC2‐selective VIP analogue, [hexanoyl‐His1]‐VIP (C6‐VIP), had a higher affinity and efficacy as compared to VIP at the mutated receptors.

VIP, Asn3‐VIP and Gln3‐VIP activated adenylate cyclase through R172Q receptors with EC50 values of 190, 2 and 2 nM, respectively, and through R172L receptors with EC50 values of 150, 12 and 8 nM, respectively. Asn3‐VIP and Gln3‐VIP behaved as partial agonists at the wild type receptor, with Emax values (in per cent of VIP) of 75 and 52%, respectively. In contrast, they were more efficient than VIP (Emax values of 150 and 150% at the R172Q VPAC2 receptors, and of 400 and 360% at the R172L receptors, respectively). These results suggested that the receptor's R172 and the ligand's aspartate 3 are brought in close proximity in the active ligand‐receptor complex.

The K179I and K179Q mutated receptors had a lower affinity than the wild‐type receptors for all the agonists tested in this work: we were unable to identify the VIP amino acid(s) that interact with K179.

British Journal of Pharmacology (2001) 133, 1249–1254; doi:10.1038/sj.bjp.0704195
Diazepam increases the hypothalamic‐pituitary‐adrenocortical (HPA) axis activity by a cyclic AMP‐dependent mechanism
Authors: Vargas M Luisa , Abella Cristina , Hernandez Jesus
Published: January 2005

Previous studies in this laboratory have shown that diazepam behaves as a phosphodiesterase 4 (PDE 4) inhibitor. It has been reported that PDE‐4 inhibitors activate the hypothalamic‐pituitary‐adrenocortical (HPA) axis in the rat. In the present study we have examined whether activation of the cyclic AMP‐dependent protein kinase (PKA) is involved in the effect of diazepam on basal HPA axis activity.

Acute systemic administration of diazepam (10 mg kg−1 i.p.) was found to increase the basal HPA axis activity, increasing the plasma concentrations of corticotrophin (ACTH) and corticosterone 30 min post injection. Diazepam also elevated cyclic AMP content of the hypothalamus.

Pretreatment of the animals with dexamethasone (1 mg kg−1 s.c.) for 3 days completely abolished the effect of diazepam on HPA axis activity.

The antagonists of central and peripheral benzodiazepine receptors, flumazenil (10 mg kg−1 i.p.) and PK 11195 (5 mg kg−1 i.p.) did not affect the diazepam induced increase of HPA axis activity nor did they have an effect per se.

The increase in ACTH and corticosterone levels was significantly reduced by the cyclic AMP‐dependent protein kinase (PKA) inhibitor, H‐89, given either subcutaneously (5 mg kg−1 s.c.) or intracerebroventricularly (i.c.v.; 28 μg in 10 μl).

The results indicate that diazepam can stimulate basal HPA axis activity in the rat by a cyclic AMP‐dependent PKA mediated pathway.

British Journal of Pharmacology (2001) 133, 1355–1361; doi:10.1038/sj.bjp.0704201
Effects of the novel selective endothelin ETA receptor antagonist, SB 234551, on the cardiovascular responses to endotoxaemia in conscious rats
Authors: Gardiner S M , March J E , Kemp P A , Bennett T
Published: January 2005

In conscious, freely moving, male, Long Evans rats, regional haemodynamic responses to exogenous endothelin‐1 (ET‐1; 25, 50 and 250 pmol kg−1 i.v.) were assessed in the presence of vehicle, or the selective ETA‐receptor antagonist, SB 234551. On the following day, the effects of SB 234551 on the haemodynamic responses to lipopolysaccharide (LPS) infusion (150 μg kg−1 h−1, i.v.) were determined.

When SB 234551 was given i.v. by primed infusion at a dose of 0.3 mg kg−1 bolus, 0.3 mg kg−1 h−1 infusion, it caused selective inhibition of the vasoconstrictor effects of exogenous endothelin‐1, whereas at a dose of 1 mg kg−1, 1 mg kg−1 h−1, SB 234551 also inhibited some of the vasodilator effects of endothelin‐1.

Infusion of LPS, in the presence of vehicle, caused a short‐lived (1–2 h) hypotension, tachycardia, and vasodilatation in renal, superior mesenteric and hindquarters vascular beds. Thereafter, blood pressure, heart rate and mesenteric vascular conductance returned to baseline values, but renal vasodilatation persisted, and there was vasoconstriction in the hindquarters.

In the presence of SB 234551 (0.3 mg kg−1, 0.3 mg kg−1 h−1), the early (1–2 h) cardiovascular responses to LPS infusion were unaffected, but the subsequent recovery of mean arterial blood pressure was impaired, due to developing vasodilatation in the mesenteric and, to a lesser extent, hindquarters, vascular beds. SB 234551 had no effect on the renal haemodynamic responses to LPS infusion.

The results confirm an important, regionally‐selective, vasoconstrictor role for endogenous endothelin in this model of endotoxaemia.

British Journal of Pharmacology (2001) 133, 1371–1377; doi:10.1038/sj.bjp.0704202
Mechanism of trypsin‐induced contraction in the rat myometrium: the possible involvement of a novel member of protease‐activated receptor
Authors: Shintani Yoshinobu , Hirano Katsuya , Nakayama Tetsuzo , Nishimura Junji , Nakano Hitoo , Kanaide Hideo
Published: January 2005

The mechanism of trypsin‐induced contraction in the rat myometrium was investigated using front‐surface fluorimetry on fura‐PE3‐loaded strips. The expression of protease‐activated receptors (PARs) in the rat myometrium was determined by reverse transcription polymerase chain reaction (RT–PCR).

In non‐pregnant rats, 10 μM trypsin developed a force of up to 30.5±5.1% of that obtained during the 40 mM K+‐depolarization‐induced contraction. In pregnant rats, the maximal level of the cytosolic Ca2+ concentration and tension obtained with 3 μM trypsin was 143.2±6.0% and 63.2±7.9%, respectively. The depletion of the extracellular Ca2+ abolished the trypsin‐induced contraction.

Trypsin‐induced contraction was abolished by the pre‐treatment of a serine protease inhibitor. PAR1‐activating peptide (PAR1‐AP) caused a potent contraction of the myometrium, while neither PAR2‐AP nor PAR4‐AP induced any contraction.

RT–PCR analysis detected the expression of PAR1 mRNA. However, neither PAR2 nor PAR4 mRNA was detected in the rat myometrium.

Once the strips were stimulated with thrombin, the subsequent application of thrombin failed to induce any contraction, while trypsin induced a contraction similar to that observed without the pre‐stimulation with thrombin. Once the strip was stimulated with trypsin, neither trypsin nor thrombin induced any contraction. The response to PAR1‐AP remained after the pre‐stimulation with thrombin and trypsin.

In conclusion, PAR1 was the only known receptor for trypsin expressed in the rat myometrium, but it was suggested to be cleaved and inactivated by trypsin. Trypsin was thus suggested to contract the rat myometrium via a novel type of PAR, which might be upregulated during pregnancy.

British Journal of Pharmacology (2001) 133, 1276–1285; doi:10.1038/sj.bjp.0704206
Evidence that the anti‐spasmogenic effect of the β‐adrenoceptor agonist, isoprenaline, on guinea‐pig trachealis is not mediated by cyclic AMP‐dependent protein kinase
Authors: Spicuzza Lucia , Belvisi Maria G , Birrell Mark A , Barnes Peter J , Hele David J , Giembycz Mark A
Published: January 2005

The spasmolytic and anti‐spasmogenic activity of β‐adrenoceptor agonists on airways smooth muscle is thought to involve activation of the cyclic AMP/cyclic AMP‐dependent protein kinase (PKA) cascade. Here we have tested the hypothesis that PKA mediates the anti‐spasmogenic activity of isoprenaline and other cyclic AMP‐elevating agents in guinea‐pig isolated trachea by utilizing a number of cell permeant cyclic AMP analogues that act as competitive ‘antagonists’ of PKA.

Anion‐exchange chromatography of guinea‐pig tracheae resolved two peaks of PKA activity that corresponded to the type I (∼5%) and type II (∼93%) isoenzymes.

Pre‐treatment of tracheae with zardaverine (30 μM), vasoactive intestinal peptide (VIP) (1 μM) and the non‐selective activator of PKA, Sp‐8‐CPT‐cAMPS (10 μM), produced a non‐parallel rightwards shift in the concentration‐response curves that described acetylcholine (ACh)‐induced tension generation. The type II‐selective PKA inhibitor, Rp‐8‐CPT‐cAMPS (300 μM), abolished this effect.

Pre‐treatment of tracheae with Sp‐8‐Br‐PET‐cGMPS (30 μM) produced a non‐parallel rightwards shift of the concentration‐response curves that described ACh‐induced tension generation. The selective cyclic GMP‐dependent protein kinase (PKG) inhibitor, Rp‐8‐pCPT‐cGMPS (300 μM), abolished this effect.

Pre‐treatment of tracheae with isoprenaline (1 μM) produced a 10 fold shift to the right of the ACh concentration‐response curve by a mechanism that was unaffected by Rp‐8‐Br‐cAMPS (300 μM, selective inhibitor of type I PKA), Rp‐8‐CPT‐cAMPS (300 μM) and Rp‐8‐pCPT‐cGMPS (300 μM).

We conclude that the anti‐spasmogenic activity of Sp‐8‐CPT‐cAMPS, zardaverine and VIP in guinea‐pig trachea is attributable to activation of the cyclic AMP/PKA cascade whereas isoprenaline suppresses ACh‐induced contractions by a mechanism(s) that is independent of PKA and PKG.

British Journal of Pharmacology (2001) 133, 1201–1212; doi:10.1038/sj.bjp.0704213
A novel tachykinin NK2 receptor antagonist prevents motility‐stimulating effects of neurokinin A in small intestine
Authors: Lördal Mikael , Navalesi Giovanni , Theodorsson Elvar , Maggi Carlo A , Hellström Per M
Published: January 2005

MEN 11420 (nepadutant) is a potent, selective and competitive antagonist of tachykinin NK2 receptors.

The objective of the present study was to assess the capability of the drug to antagonize the stimulatory effects of neurokinin A (NKA) on gastrointestinal motility, as well as to change the fasting migrating motor complex (MMC).

Thirty‐four male volunteers were randomized to treatment with either placebo or MEN 11420 in a double‐blinded manner. Effects of MEN 11420 (8 mg intravenously) were evaluated as changes in phases I, II and III of MMC, as well as contraction frequency, amplitude and motility index during baseline conditions and during stimulation of motility using NKA (25 pmol kg−1 min−1 intravenously).

NKA preceded by placebo increased the fraction of time occupied by phase II, increased contraction frequency, amplitude and motility index.

MEN 11420 effectively antagonized the motility‐stimulating effects of NKA. MEN 11420 reduced the phase II‐stimulating effect of NKA. In addition, the stimulatory effect of NKA on contraction frequency and amplitude, as well as motility index were inhibited by MEN 11420. MEN 11420 did not affect the characteristics of MMC during saline infusion.

Plasma levels of MEN 11420 peaked during the first hour after infusion and decreased to less than half during the first 2 h.

In conclusion, intravenous MEN 11420 effectively inhibited NKA‐stimulated, but not basal gastrointestinal motility, and was well tolerated by all subjects.

British Journal of Pharmacology (2001) 134, 215–223; doi:10.1038/sj.bjp.0704217
Effects of ACE inhibition and angiotensin II type 1 receptor blockade on cardiac function and G proteins in rats with chronic heart failure
Authors: Yoshida Hiroyuki , Takahashi Masaya , Tanonaka Kouichi , Maki Toshiyuki , Nasa Yoshihisa , Takeo Satoshi
Published: January 2005

Inhibition of the renin‐angiotensin system (RAS) improves symptoms and prognosis in heart failure. The experimental basis for these benefits remains unclear. We examined the effects of inhibition of ACE or blockade of angiotensin II type 1 (AT1) receptor on the haemodynamics, cardiac G‐proteins, and collagen synthesis of rats with coronary artery ligation (CAL), a model in which chronic heart failure (CHF) is induced.

Rats were orally treated with the ACE inhibitor trandolapril (3 mg kg−1 day−1) or the AT1 receptor blocker L‐158809 (1 mg kg−1 day−1) from the 2nd to 8th week after CAL. CAL resulted in decreases in the left ventricular systolic pressure and its positive and negative dP/dt, an increase in the left ventricular end‐diastolic pressure, and the rightward shift of the left ventricular pressure‐volume curve. Long‐term treatment with either drug improved these signs of CHF to a similar degree.

Cardiac Gsα and Gqα protein levels decreased, whereas the level of Giα protein increased in the animals with CHF. Long‐term treatment with trandolapril or L‐158809 attenuated the increase in the level of cardiac Giα protein of the animals with CHF without affecting Gsα and Gqα protein levels. Cardiac collagen content of the failing heart increased, whose increase was blocked by treatment with either drug.

Exogenous angiotensin I stimulated collagen synthesis in cultured cardiac fibroblasts, whose stimulation was attenuated by either drug.

These results suggest that blockade of the RAS, at either the receptor level or the synthetic enzyme level, may attenuate the cardiac fibrosis that occurs after CAL and thus affect the remodelling of the failing heart.

British Journal of Pharmacology (2001) 134, 150–160; doi:10.1038/sj.bjp.0704219
NO‐induced relaxation of labouring and non‐labouring human myometrium is not mediated by cyclic GMP
Authors: Buxton Iain L O , Kaiser Robert A , Malmquist Nicholas A , Tichenor Stephen
Published: January 2005

In myometrial strips from near‐term non‐labouring human uterus, addition of oxytocin (OT) evoked dose‐dependent (10 – 3000 nM) phasic contractions that were antagonized by atosiban (1 μM) and relaxed by addition of the nitric oxide donor S‐nitroso L‐cysteine (Cys‐NO). In near‐term labouring myometrium, however, addition of OT was ineffective at raising additional tone.

In both labouring and non‐labouring tissue, Cys‐NO mediated relaxation of spontaneous or OT‐induced contractions (IC50=1 μM) was unaffected by prior addition of the guanylyl cyclase (GC) inhibitors ODQ (1H‐[1,2,4]oxadiazolo[4,3,‐α]quinoxalin‐1‐one; 1 μM), or methylene blue (MB; 10 μM).

Elevation of intracellular cyclic GMP accompanying 30 μM Cys‐NO addition in non‐labouring tissue (7.5 fold) or in labouring tissues (2.5 fold) was completely blocked in tissues that had been pre‐treated with ODQ or MB.

Charybdotoxin (ChTx), iberiotoxin (IbTx) and kaliotoxin (KalTx) all shifted the Cys‐NO inhibition curve to the right and reduced the degree of relaxation produced by maximal Cys‐NO treatment (100 μM in non‐labouring tissue; in labouring tissue, KalTx prevented Cys‐NO mediated relaxation in both stimulated and unstimulated tissue.

Addition of the NO‐donor S‐nitroso N‐acetyl penicillamine (SNAP) produced a dose‐dependent relaxation of pregnant myometrium while 3‐morpholinosyndonimine (SIN‐1) did not. The failure of SIN‐1 to relax OT‐induced contractions was not due to a failure of the donor to stimulate myometrial GC.

We demonstrate that despite the ability of NO to stimulate myometrial GC in pregnant uterine muscle, relaxations are independent of cyclic GMP action. Effects of K+‐channel inhibitors suggests that NO‐induced relaxation in human uterine smooth muscle may be subserved by direct or indirect activation of one or more calcium‐activated K+‐channels.

British Journal of Pharmacology (2001) 134, 206–214; doi:10.1038/sj.bjp.0704226
A xanthine‐based KMUP‐1 with cyclic GMP enhancing and K+ channels opening activities in rat aortic smooth muscle
Authors: Wu Bin‐Nan , Lin Rong‐Jyh , Lin Chiu‐Yin , Shen Kuo‐Pyng , Chiang Lien‐Chai , Chen Ing‐Jun
Published: January 2005

KMUP‐1 (1, 3, 5 mg kg−1, i.v.), a xanthine derivative, produced dose‐dependent sustained hypotensive and short‐acting bradycardiac effects in anaesthetized rats. This hypotensive effect was inhibited by pretreatment with glibenclamide (5 mg kg−1, i.v.).

In endothelium‐intact or denuded aortic rings preconstricted with phenylephrine, KMUP‐1 caused a concentration‐dependent relaxation. This relaxation was reduced by endothelium removal, the presence of NOS inhibitor L‐NAME (100 μM) and sGC inhibitors methylene blue (10 μM) and ODQ (1 μM).

The vasorelaxant effects of KMUP‐1 was attenuated by pretreatment with various K+ channel blockers TEA (10 mM), glibenclamide (1 μM), 4‐AP (100 μM), apamin (1 μM) and charybdotoxin (ChTX, 0.1 μM).

Increased extracellular potassium levels (30 – 80 mM) caused a concentration‐related reduction of KMUP‐1‐induced vasorelaxations. Preincubation with KMUP‐1 (1, 10, 100 nM) increased the ACh‐induced maximal vasorelaxations mediated by endogenous NO release, and enhanced the potency of exogenous NO‐donor SNP.

The vasorelaxant responses of KMUP‐1 (0.01, 0.05, 0.1 μM) together with a PDE inhibitor IBMX (0.5 μM) had an additive action. Additionally, KMUP‐1 (100 μM) affected cyclic GMP metabolism since it inhibited the activity of PDE in human platelets.

KMUP‐1 induced a dose‐related increase in intracellular cyclic GMP levels in rat A10 vascular smooth muscle (VSM) cells, but not cyclic AMP. The increase in cyclic GMP content of KMUP‐1 (0.1 – 100 μM) was almost completely abolished in the presence of methylene blue (10 μM), ODQ (10 μM), and L‐NAME (100 μM).

In conclusion, these results indicate that KMUP‐1 possesses the following merits: (1) stimulation of NO/sGC/cyclic GMP pathway and subsequent elevation of cyclic GMP, (2) K+ channels opening, and (3) inhibition of PDE or cyclic GMP breakdown. Increased cyclic GMP display a prominent role in KMUP‐1‐induced VSM relaxations.

British Journal of Pharmacology (2001) 134, 265–274; doi:10.1038/sj.bjp.0704231
Modulation of eotaxin formation and eosinophil migration by selective inhibitors of phosphodiesterase type 4 isoenzyme
Authors: Silva Patrícia M R , Alves Alessandra C , Serra Magda F , Pires Ana Lucia A , Silva Juliane P , Barreto Emiliano O , Cordeiro Renato S B , Jose Peter J , Teixeira Mauro M , Lagente Vincent , Martins Marco A
Published: January 2005

This study was undertaken to investigate the possible contribution of the blockade of eotaxin generation to the anti‐eosinophilotactic effect of phosphodiesterase (PDE) type 4 inhibitors. In some experiments, the putative synergistic interaction between PDE type 4 inhibitors and the β2‐agonist salbutamol was also assessed.

Sensitized guinea‐pigs aerosolized with antigen (5% ovalbumin, OVA) responded with a significant increase in eotaxin and eosinophil levels in the bronchoalveolar lavage fluid (BALF) at 6 h. Eosinophil recruitment was inhibited by both PDE type 4 inhibitors rolipram (5 mg kg−1, i.p.) and RP 73401 (5 mg kg−1, i.p.) treatments. In contrast, only rolipram inhibited eotaxin production.

Sensitized rats intrapleurally challenged (i.pl.) with antigen (OVA, 12 μg cavity−1) showed a marked eosinophil infiltration at 24 h, preceded by eotaxin generation at 6 h. Intravenous administration of a rabbit anti‐mouse eotaxin antibody (0.5 mg kg−1) significantly reduced allergen‐evoked eosinophilia in this model.

Local pretreatment with rolipram (40 μg cavity−1) or RP 73401 (40 μg cavity−1) 1 h before challenge reduced eosinophil accumulation evaluated in the rat pleural effluent, but only the former was active against eotaxin generation. The inhibitors of PDE type 3 (SK&F 94836) and type 5 (zaprinast) failed to alter allergen‐evoked eosinophil recruitment in rats.

Local injection of β2‐agonist salbutamol (20 μg cavity−1) inhibited both eosinophil accumulation and eotaxin production following pleurisy. The former was better inhibited when salbutamol and rolipram were administered in combination.

Treatment with rolipram and RP 73401 dose‐dependently inhibited eosinophil adhesion and migrationin vitro. These effects were clearly potentiated by salbutamol at concentrations that had no effect alone.

Our findings indicate that although rolipram and RP 73401 are equally effective in inhibiting allergen‐induced eosinophil infiltration only the former prevents eotaxin formation, indicating that PDE 4 inhibitors impair eosinophil accumulation by mechanisms independent of eotaxin production blockade.

British Journal of Pharmacology(2001)134, 283–294; doi:10.1038/sj.bjp.0704233
Neuronal nitric oxide synthase (NOS) regulates leukocyte‐endothelial cell interactions in endothelial NOS deficient mice
Authors: Sanz Maria‐Jesus , Hickey Michael J , Johnston Brent , McCafferty Donna‐Marie , Raharjo Eko , Huang Paul L , Kubes Paul
Published: January 2005

The present study was designed to examine the possible role of neuronal nitric oxide synthase (nNOS) in regulation of leukocyte – endothelial cell interactions in the absence of endothelial nitric oxide synthase (eNOS), using intravital microscopy of the cremasteric microcirculation of eNOS−/− mice.

Baseline leukocyte rolling and adhesion revealed no differences between wild‐type and eNOS−/− mice in either the cremasteric or intestinal microcirculations.

Superfusion with L‐NAME (100 μM) caused a progressive and significant increase in leukocyte adhesion in both wild‐type and eNOS−/− mice, without detecting differences between the two strains of mice.

Superfusion with 7‐nitroindazole (100 μM), a selective inhibitor of nNOS, had no effect on leukocyte adhesion in wild‐type animals. However, it increased leukocyte adhesion significantly in eNOS−/− mice, which was reversed by systemic L‐arginine pre‐administration.

Stimulation of the microvasculature with H2O2 (100 μM) induced a transient elevation in leukocyte rolling in wild‐type mice. Conversely, the effect persisted during the entire 60 min of experimental protocol in eNOS−/− mice either with or without 7‐nitroindazole.

Semi‐quantitative analysis by RT – PCR of the mRNA for nNOS levels in eNOS−/− and wild‐type animals, showed increased expression of nNOS in both brain and skeletal muscle of eNOS−/− mice.

In conclusion, we have demonstrated that leukocyte‐endothelial cell interactions are predominantly modulated by eNOS isoform in postcapillary venules of normal mice, whereas nNOS appears to assume the same role in eNOS−/− mice. Interestingly, unlike eNOS there was insufficient NO produced by nNOS to overcome leukocyte recruitment elicited by oxidative stress, suggesting that nNOS cannot completely compensate for eNOS.

British Journal of Pharmacology (2001) 134, 305–312; doi:10.1038/sj.bjp.0704234
Dependency of detrusor contractions on calcium sensitization and calcium entry through LOE‐908‐sensitive channels
Authors: Jezior James R , Brady Jeffrey D , Rosenstein Daniel I , McCammon Kurt A , Miner Amy S , Ratz Paul H
Published: January 2005

The subcellular mechanisms regulating stimulus‐contraction coupling in detrusor remain to be determined. We used Ca2+‐free solutions, Ca2+ channel blockers, cyclopiazonic acid (CPA), and RhoA kinase (ROK) inhibitors to test the hypothesis that Ca2+ influx and Ca2+ sensitization play primary roles.

In rabbit detrusor, peak bethanechol (BE)‐induced force was inhibited 90% by incubation for 3 min in a Ca2+‐free solution. By comparison, a 20 min incubation of rabbit femoral artery in a Ca2+‐free solution reduced receptor‐induced force by only 5%.

In detrusor, inhibition of sarcoplasmic reticular (SR) Ca2+ release by 2APB, or depletion of SR Ca2+ by CPA, inhibited BE‐induced force by only 27%. The CPA‐insensitive force was abolished by LaCl3. By comparison, 2APB inhibited receptor‐induced force in rabbit femoral artery by 71%.

In the presence of the non‐selective cation channel (NSCC) inhibitor, LOE‐908, BE did not produce an increase in [Ca2+]i but did produce weak increases in myosin phosphorylation and force.

Inhibitors of ROK‐induced Ca2+ sensitization, HA‐1077 and Y‐27632, inhibited BE‐induced force by ∼50%, and in combination with LOE‐908, nearly abolished force.

These data suggest that two principal muscarinic receptor‐stimulated detrusor contractile mechanisms include NSCC activation, that elevates [Ca2+]i and ROK activation, that sensitizes cross bridges to Ca2+.

British Journal of Pharmacology (2001) 134, 78–87; doi:10.1038/sj.bjp.0704241
Effects of pH on the inhibition of fatty acid amidohydrolase by ibuprofen
Authors: Holt Sandra , Nilsson Jonas , Omeir Romelda , Tiger Gunnar , Fowler Christopher J
Published: January 2005

British Journal of Pharmacology (2001) 134, 451; doi:10.1038/sj.bjp.0704257
Ezetimibe selectively inhibits intestinal cholesterol absorption in rodents in the presence and absence of exocrine pancreatic function
Authors: Van Heek Margaret , Farley Constance , Compton Douglas S , Hoos Lizbeth , Davis Harry R
Published: January 2005

Ezetimibe potently inhibits the transport of cholesterol across the intestinal wall, thereby reducing plasma cholesterol in preclinical animal models of hypercholesterolemia. The effect of ezetimibe on known absorptive processes was determined in the present studies.

Experiments were conducted in the hamster and/or rat to determine whether ezetimibe would affect the absorption of molecules other than free cholesterol, namely cholesteryl ester, triglyceride, ethinylestradiol, progesterone, vitamins A and D, and taurocholic acid. In addition, to determine whether exocrine pancreatic function is involved in the mechanism of action of ezetimibe, a biliary anastomosis model, which eliminates exocrine pancreatic function from the intestine while maintaining bile flow, was established in the rat.

Ezetimibe reduced plasma cholesterol and hepatic cholesterol accumulation in cholesterol‐fed hamsters with an ED50 of 0.04 mg kg−1. Utilizing cholesteryl esters labelled on either the cholesterol or the fatty acid moiety, we demonstrated that ezetimibe did not affect cholesteryl ester hydrolysis and the absorption of fatty acid thus generated in both hamsters and rats. The free cholesterol from this hydrolysis, however, was not absorbed (92 – 96% inhibition) in the presence of ezetimibe. Eliminating pancreatic function in rats abolished hydrolysis of cholesteryl esters, but did not affect the ability of ezetimibe to block absorption of free cholesterol (−94%). Ezetimibe did not affect the absorption of triglyceride, ethinylestradiol, progesterone, vitamins A and D, and taurocholic acid in rats.

Ezetimibe is a potent inhibitor of intestinal free cholesterol absorption that does not require exocrine pancreatic function for activity. Ezetimibe does not affect the absorption of triglyceride as a pancreatic lipase inhibitor (Orlistat) would, nor does it affect the absorption of vitamin A, D or taurocholate, as a bile acid sequestrant (cholestyramine) would.

British Journal of Pharmacology (2001) 134, 409–417; doi:10.1038/sj.bjp.0704260
The role of the hydrophilic Asn230 residue of the μ‐opioid receptor in the potency of various opioid agonists
Authors: Pil Joost , Tytgat Jan
Published: January 2005

To investigate the effect of the hydrophilic Asn amino acid at position 230 of the human μ‐opioid receptor (hMOR230) on the potency of various agonists, we mutated this residue to Thr and Leu (hMORN230T and hMORN230L respectively).

Taking advantage of the functional coupling of the opioid receptor with the heteromultimeric G‐protein‐coupled inwardly rectifying K+ (GIRK1/GIRK2) channel, either the wild type hMOR or one of the mutated receptors (hMORN230L or hMORN230T) were functionally coexpressed with GIRK1/GIRK2 channels and a regulator of G‐protein signalling (RGS4) in Xenopus laevis oocytes.

The two‐microelectrode voltage clamp technique was used to measure the opioid receptor‐activated GIRK1/GIRK2 channel responses. The potency of [D‐Ala2,N‐MePhe4,Gly5‐ol]‐enkephalin (DAMGO), remained unaffected as measured via hMORN230T and hMORN230L, while the potency of fentanyl and morphine significantly increased via these mutated receptors.

Our results are indicative for the existence of hydrophobic interactions between a methyl‐group of the side chain of Thr or Leu on the one hand and the piperidine‐ring of fentanyl and the hexene‐ring of morphine on the other. The mutations also had no influence on the potency of morphine‐6‐glucuronide (M6G) and morphine‐3‐glucuronide (M3G).

We conclude that the hydrophilic side chain of Asn in position 230 is not involved in the formation of a H‐bond with the aliphatic alcohol of morphine and that an enhancement of the potency of morphine and fentanyl can be explained by mutating this residue towards more hydrophobic amino acids.

British Journal of Pharmacology (2001) 134, 496–506; doi:10.1038/sj.bjp.0704263
A1‐, A2A‐ and A3‐subtype adenosine receptors modulate intraocular pressure in the mouse
Authors: Avila Marcel Y , Stone Richard A , Civan Mortimer M
Published: January 2005

Despite the potential importance of the mouse in studying the pharmacology of aqueous dynamics, measurement of intraocular pressure (IOP) in its very small eye has been problematic. Utilizing a novel servo‐null electrophysiologic approach recently applied to the mouse, we have identified a diversity of adenosine‐receptor mechanisms in modulating IOP in this species. We report the first evidence that A3 receptors increase IOP in any species, and verify in the mouse reports with larger mammals that A1 receptors lower and A2A receptors increase IOP.

British Journal of Pharmacology (2001) 134, 241–245; doi:10.1038/sj.bjp.0704267
P2YAC−‐receptor agonists enhance the proliferation of rat C6 glioma cells through activation of the p42/44 mitogen‐activated protein kinase
Authors: Claes Patrik , Grobben Bert , Van Kolen Kristof , Roymans Dirk , Slegers Herman
Published: January 2005

Extracellularly added P1,P3‐di(adenosine‐5′) triphosphate (Ap3A), P1,P4‐di(adenosine‐5′) tetraphosphate (Ap4A), ATP, ADP, AMP and adenosine are growth inhibitory for rat C6 glioma cells. Analysis of nucleotide hydrolysis and the use of nucleotidase inhibitors demonstrated that the latter inhibition is due to hydrolysis of the nucleotides to adenosine.

Agonists of the P2YAC−‐receptor enhance the growth of C6 cells if their hydrolysis to adenosine is inhibited by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS). In these conditions, the potency to stimulate cell growth parallels the ranking of the receptor agonists, i.e. 2‐methylthioadenosine‐5′‐diphosphate (2MeSADP)>Ap3A>Ap4A. ATP and ADP are still hydrolysed in the presence of PPADS and have no proliferative effect on C6 cells.

The enhanced growth is due to a P2YAC−‐receptor‐mediated activation of p42/44 mitogen‐activated protein kinase (MAPK) as shown by immunoblotting and protein kinase assays for active MAPK and the use of the MAPK/extracellular signal‐regulated kinase kinase (MEK) inhibitor PD98059.

The UTP‐induced enhancement of the growth of C6 cells is due to activation of MAPK by a PPADS sensitive nucleotide receptor.

In conclusion, the effect of nucleotides on the growth of C6 cells is determined by ecto‐nucleotidases and by activation of nucleotide receptors. Hydrolysis of nucleotides to adenosine induces growth inhibition while inhibition of the hydrolysis of agonists of the P2YAC−‐receptor enhances cell growth by activation of MAPK.

British Journal of Pharmacology (2001) 134, 402–408; doi:10.1038/sj.bjp.0704271
Increased contractile response to 5‐hydroxytryptamine1‐receptor stimulation in pulmonary arteries from chronic hypoxic rats: role of pharmacological synergy
Authors: MacLean Margaret R , Morecroft Ian
Published: January 2005

5‐Hydroxytryptamine (5‐HT)1‐receptor‐induced contraction is enhanced, or uncovered, by elevated vascular tone in many arteries including pulmonary arteries. In hypoxia‐induced pulmonary hypertension, the endogenous tone of pulmonary arteries is elevated and this may contribute to increased 5‐HT1‐receptor‐induced contraction. Here we investigate the influence of vascular tone induced by endothelin‐1 (ET‐1), neuropeptide Y (NPY), KCl, 4‐aminopyridine (inactivator of Kv channels, 4‐AP) or the calcium ionophore A23187 on contractile responses to the 5‐HT1‐receptor agonist 5‐carboxamidotryptamine (5‐CT) in small muscular pulmonary arteries from control rats and rats exposed to chronic hypoxia. The influence of the nitric oxide synthase inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μM) was also studied. These conditions were chosen to mimic those that influence pulmonary vascular tone in hypoxia‐induced pulmonary hypertension.

In control rat small pulmonary arteries, only high concentrations of 5‐CT (>1 μM) induced vasoconstriction. Tone induced by NPY, 4‐AP and A23187 had no effect on responses to 5‐CT whilst responses to 5‐CT were increased by ET‐1‐ and KCl‐induced tone. In the presence of L‐NAME these responses to 5‐CT were enhanced further.

Responses to 5‐CT were enhanced 3 – 4 fold in small pulmonary arteries from hypoxia‐exposed, pulmonary hypertensive rats and neither L‐NAME nor increasing tone with NPY, 4‐AP, A23187, ET‐1 or KCl had any further effect on responses to 5‐CT.

The results suggest that inhibition of nitric oxide synthase combined with KCl‐ or ET‐1‐induced vascular tone potentiates responses to 5‐HT1‐receptor‐induced contraction in pulmonary arteries in a synergistic fashion and this mimics the effects of chronic hypoxic exposure.

British Journal of Pharmacology (2001) 134, 614–620; doi:10.1038/sj.bjp.0704273
Effects of potassium channel and Na+‐Ca2+ exchange blockers on the responses of slowly adapting pulmonary stretch receptors to hyperinflation in flecainide‐treated rats
Authors: Matsumoto Shigeji , Nishikawa Toshimi , Yoshida Shinki , Ikeda Mizuho , Tanimoto Takeshi , Saiki Chikako , Takeda Mamoru
Published: January 2005

The effects of K+ channel blockers, such as 4‐aminopyridine (4‐AP) and tetraethylammonium (TEA), and a reverse‐mode Na+ – Ca2+ exchange blocker, 2‐[2‐[4‐(4‐nitrobenzyloxyl) phenyl] ethyl] isothiourea methanesulphonate (KB‐R7943), on the responses of slowly adapting pulmonary stretch receptor activity to hyperinflation (inflation volume=3 tidal volumes) were investigated in anaesthetized, artificially ventilated, unilaterally vagotomized rats after pretreatment with a Na+ channel blocker flecainide. The administration of flecainide (9 mg kg−1) at a dose greater than that which abolished 50 μg kg−1 veratridine‐induced SAR stimulation also inhibited hyperinflation‐induced stimulation of SARs.

In flecainide‐treated animals, administration of 4‐AP (0.7 and 2 mg kg−1) stimulated SAR activity during normal inflation and also caused a partial blockade of hyperinflation‐induced SAR inhibition.

The discharges of SARs during normal inflation in flecainide‐treated animals were not significantly altered by administration of either TEA (2 and 7 mg kg−1) or KB‐R7943 (1 and 3 mg kg−1), but both K+ channel and Na+‐Ca2+ exchange blockers partially attenuated hyperinflation‐induced SAR inhibition.

These results suggest that hyperinflation‐induced SAR inhibition in the presence of flecainide (9 mg kg−1) involves the activation of several K+ conductance pathways.

British Journal of Pharmacology (2001) 134, 682–690; doi:10.1038/sj.bjp.0704277