bluuebunny/crossref_metadata_2025 huggingface dataset:
[125I‐His9]‐Ghrelin, a novel radioligand for localizing GHS orphan receptors in human and rat tissue; up‐regulation of receptors with atherosclerosis
Authors: Katugampola Sidath D , Pallikaros Zakos , Davenport Anthony P
Published: January 2005

Ghrelin is the recently identified endogenous ligand for the cloned growth hormone secretagogue receptor (GHS‐R). We have characterized for the first time the binding of human [125I‐His9]‐ghrelin to normal human and rat tissue and demonstrated expression of this ‘orphan’ receptor that has previously been predicted to exist from mRNA. Furthermore, we have discovered that [125I‐His9]‐ghrelin density is significantly increased in atherosclerosis.

[125I‐His9]‐Ghrelin bound to non‐diseased human heart (left ventricle) with an association rate constant (kobs) of 0.16±0.004 min−1, a dissociation rate constant of 0.068±0.0005 min−1 (kinetically derived KD of 0.1 nM; n=5 individuals±s.e.mean), a KD of 0.43±0.08 nM and Bmax of 7.8±0.9 fmol mg−1 protein (n=6 individual±s.e.mean).

Specific [125I‐His9]‐ghrelin binding was to the human vasculature including aorta, coronary, pulmonary, arcuate arteries in the kidney and saphenous veins. In rat tissues, binding sites were also localized to the vasculature in peripheral tissues as well as the granular layer of the cerebellum in the CNS.

[125I‐His9]‐Ghrelin binding was significantly up‐regulated (3 – 4 fold) in both atherosclerotic coronary arteries and saphenous vein grafts with advanced intimal thickening, compared with normal vessels (P<0.05).

Our results suggest that the native receptor for [125I‐His9]‐ghrelin may be widely distributed in the human cardiovascular system. Furthermore, changes in the density of this proposed ghrelin receptor implicates this new transmitter system in the development of atherosclerosis and may therefore represent a novel therapeutic target in the treatment of cardiovascular disease.

British Journal of Pharmacology (2001) 134, 143–149; doi:10.1038/sj.bjp.0704228
The mechanisms involved in the long‐lasting neuroprotective effect of fluoxetine against MDMA (‘ecstasy’)‐induced degeneration of 5‐HT nerve endings in rat brain
Authors: Sanchez V , Camarero J , Esteban B , Peter M J , Green A R , Colado M I
Published: January 2005

It has been reported that co‐administration of fluoxetine with 3,4‐methylenedioxymethamphetamine (MDMA, ‘ecstasy’) prevents MDMA‐induced degeneration of 5‐HT nerve endings in rat brain. The mechanisms involved have now been investigated.

MDMA (15 mg kg−1, i.p.) administration produced a neurotoxic loss of 5‐HT and 5‐hydroxyindoleacetic acid (5‐HIAA) in cortex, hippocampus and striatum and a reduction in cortical [3H]‐paroxetine binding 7 days later. Fluoxetine (10 mg kg−1, i.p., ×2, 60 min apart) administered concurrently with MDMA or given 2 and 4 days earlier provided complete protection, and significant protection when given 7 days earlier. Fluvoxamine (15 mg kg−1, i.p., ×2, 60 min apart) only produced neuroprotection when administered concurrently.

Fluoxetine (10 mg kg−1, ×2) markedly increased the KD and reduced the Bmax of cortical [3H]‐paroxetine binding 2 and 4 days later. The Bmax was still decreased 7 days later, but the KD was unchanged. [3H]‐Paroxetine binding characteristics were unchanged 24 h after fluvoxamine (15 mg kg−1, ×2).

A significant cerebral concentration of fluoxetine plus norfluoxetine was detected over the 7 days following fluoxetine administration. The fluvoxamine concentration had decreased markedly by 24 h.

Pretreatment with fluoxetine (10 mg kg−1, ×2) failed to alter cerebral MDMA accumulation compared to saline pretreated controls.

Neither fluoxetine or fluvoxamine altered MDMA‐induced acute hyperthermia.

These data demonstrate that fluoxetine produces long‐lasting protection against MDMA‐induced neurodegeneration, an effect apparently related to the presence of the drug and its active metabolite inhibiting the 5‐HT transporter. Fluoxetine does not alter the metabolism of MDMA or its rate of cerebral accumulation.

British Journal of Pharmacology (2001) 134, 46–57; doi:10.1038/sj.bjp.0704230
Apstatin, a selective inhibitor of aminopeptidase P, reduces myocardial infarct size by a kinin‐dependent pathway
Authors: Wolfrum S , Richardt G , Dominiak P , Katus H A , Dendorfer A
Published: January 2005

Inhibitors of the angiotensin converting enzyme (ACE) have been shown to exert their cardioprotective actions through a kinin‐dependent mechanism. ACE is not the only kinin degrading enzyme in the rat heart.

Since aminopeptidase P (APP) has been shown to participate in myocardial kinin metabolism to the same extent as ACE, the aims of the present study were to investigate whether (a) inhibition of APP leads to a reduction of myocardial infarct size in a rat model of acute ischaemia and reperfusion, (b) reduction of infarct size is mediated by bradykinin, and (c) a combination of APP and ACE inhibition leads to a more pronounced effect than APP inhibition alone.

Pentobarbital‐anaesthetized rats were subjected to 30 min left coronary artery occlusion followed by 3 h reperfusion. The APP inhibitor apstatin, the ACE‐inhibitor ramiprilat, or their combination were administered 5 min before ischaemia. Rats receiving HOE140, a specific B2 receptor antagonist, were pretreated 5 min prior to enzyme inhibitors. Myocardial infarct size (IS) was determined by tetrazolium staining and expressed as percentage of the area at risk (AAR).

IS/AAR% was significantly reduced in rats that received apstatin (18±2%), ramiprilat (18±3%), or apstatin plus ramiprilat (20±4%) as compared with those receiving saline (40±2%), HOE (43±3%) or apstatin plus HOE140 (49±4%).

Apstatin reduces IS in an in vivo model of acute myocardial ischaemia and reperfusion to the same extent than ramiprilat. Cardioprotection achieved by this selective inhibitor of APP is mediated by bradykinin. Combined inhibition of APP and ACE did not result in a more pronounced reduction of IS than APP‐inhibition alone.

British Journal of Pharmacology (2001) 134, 370–374; doi:10.1038/sj.bjp.0704236
Effects of imidazoline antihypertensive drugs on sympathetic tone and noradrenaline release in the prefrontal cortex
Authors: Szabo Bela , Fritz Thomas , Wedzony Krzysztof
Published: January 2005

The aim of the present study was to compare the effects of the centrally acting antihypertensive drugs rilmenidine, moxonidine, clonidine and guanabenz on sympathetic tone with their effects on noradrenaline release in the cerebral cortex. In particular, the hypothesis was tested that rilmenidine and moxonidine, due to their high affinity for sympatho‐inhibitory imidazoline I1 receptors and low affinity for α2‐adrenoceptors, lower sympathetic tone without causing an α2‐adrenoceptor‐mediated inhibition of cerebrocortical noradrenaline release.

In rats anaesthetized with urethane, blood pressure and heart rate were measured and the concentration of noradrenaline in arterial blood plasma was determined. The release of noradrenaline in the medial prefrontal cortex was estimated by microdialysis. Intravenous administration of rilmenidine (30, 100, 300 and 1000 μg kg−1), moxonidine (10, 30, 100 and 300 μg kg−1), clonidine (1, 3, 10 and 30 μg kg−1) and guanabenz (1, 3, 10 and 30 μg kg−1) led to dose‐dependent hypotension and bradycardia; the plasma noradrenaline concentration also decreased. After the two highest doses, all four drugs lowered noradrenaline release in the prefrontal cortex. At doses eliciting equal hypotensive and sympatho‐inhibitory responses, rilmenidine and moxonidine inhibited cerebral cortical noradrenaline release at least as much as clonidine and guanabenz.

The results show that rilmenidine and moxonidine lower cerebrocortical noradrenaline release at doses similar to those which cause sympatho‐inhibition. This effect was probably due to an α2‐adrenoceptor‐mediated inhibition of the firing of locus coeruleus neurons and, in addition, to presynaptic inhibition of noradrenaline release at the level of the axon terminals in the cortex. The results argue against the hypothesis that rilmenidine and moxonidine, due to their selectivity for sympatho‐inhibitory I1 imidazoline receptors, do not suppress noradrenergic neurons in the central nervous system.

British Journal of Pharmacology (2001) 134, 295–304; doi:10.1038/sj.bjp.0704237
Nitroglycerin‐induced relaxation of anorectal smooth muscle: evidence for apparent lack of tolerance development in the anaesthetized rat
Authors: Wang Ellen Q , Soda David M , Fung Ho‐Leung
Published: January 2005

Recent studies indicate that nitroglycerin (NTG) can produce beneficial clinical effects in healing anal fissures through the relaxation of the internal anal sphincter. The in vivo relaxation effects of NTG on the anorectal smooth muscle have not been studied and it is not known whether this tissue may also exhibit pharmacological tolerance toward NTG.

We have developed an in vivo procedure in the anaesthetized rat that permits continual monitoring of anorectal pressure after intravenous (i.v.) and intra‐rectal application of NTG. The relaxant effects of NTG were quantified via the area‐under‐the‐contraction‐waveforms vs time curve (AUEC).

AUEC decreased significantly after intra‐rectal bolus doses of NTG (5 – 25 μg), in a dose‐ and time‐dependent manner. Sustained relaxation effects on anorectal pressure were also observed after continuous intra‐rectal infusions of NTG.

Two‐hours of i.v. NTG infusion led to a significant reduction in the mean arterial blood pressure (MAP) response toward a i.v. NTG (30 μg) bolus challenge. In contrast, relaxation of the anorectal pressure toward the challenge dose was not altered after NTG infusion.

In isolated tissues, cyclic GMP accumulation was significantly decreased after NTG pre‐incubation in the rat aorta but not in the rat anorectal smooth muscle and anal sphincter.

These results indicate that the relaxation response toward NTG was not diminished in the anorectum under conditions that produced vascular tolerance. Thus, NTG causes significant and sustained in vivo relaxation of anorectal smooth muscle in the anaesthetized rat without evidence of tolerance development.

British Journal of Pharmacology (2001) 134, 418–424; doi:10.1038/sj.bjp.0704239
Adenosine A2A receptor antagonists are potential antidepressants: evidence based on pharmacology and A2A receptor knockout mice
Authors: Yacoubi Malika El , Ledent Catherine , Parmentier Marc , Bertorelli Rosalia , Ongini Ennio , Costentin Jean , Vaugeois Jean‐Marie
Published: January 2005

Adenosine, an ubiquitous neuromodulator, and its analogues have been shown to produce ‘depressant’ effects in animal models believed to be relevant to depressive disorders, while adenosine receptor antagonists have been found to reverse adenosine‐mediated ‘depressant’ effect.

We have designed studies to assess whether adenosine A2A receptor antagonists, or genetic inactivation of the receptor would be effective in established screening procedures, such as tail suspension and forced swim tests, which are predictive of clinical antidepressant activity.

Adenosine A2A receptor knockout mice were found to be less sensitive to ‘depressant’ challenges than their wildtype littermates. Consistently, the adenosine A2A receptor blockers SCH 58261 (1 – 10 mg kg−1, i.p.) and KW 6002 (0.1 – 10 mg kg−1, p.o.) reduced the total immobility time in the tail suspension test.

The efficacy of adenosine A2A receptor antagonists in reducing immobility time in the tail suspension test was confirmed and extended in two groups of mice. Specifically, SCH 58261 (1 – 10 mg kg−1) and ZM 241385 (15 – 60 mg kg−1) were effective in mice previously screened for having high immobility time, while SCH 58261 at 10 mg kg−1 reduced immobility of mice that were selectively bred for their spontaneous ‘helplessness’ in this assay.

Additional experiments were carried out using the forced swim test. SCH 58261 at 10 mg kg−1 reduced the immobility time by 61%, while KW 6002 decreased the total immobility time at the doses of 1 and 10 mg kg−1 by 75 and 79%, respectively.

Administration of the dopamine D2 receptor antagonist haloperidol (50 – 200 μg kg−1 i.p.) prevented the antidepressant‐like effects elicited by SCH 58261 (10 mg kg−1 i.p.) in forced swim test whereas it left unaltered its stimulant motor effects.

In conclusion, these data support the hypothesis that A2A receptor antagonists prolong escape‐directed behaviour in two screening tests for antidepressants. Altogether the results support the hypothesis that blockade of the adenosine A2A receptor might be an interesting target for the development of effective antidepressant agents.

British Journal of Pharmacology (2001) 134, 68–77; doi:10.1038/sj.bjp.0704240
Real‐time analysis of dopamine: antagonist interactions at recombinant human D2long receptor upon modulation of its activation state
Authors: Pauwels Petrus J , Tardif Stéphanie , Wurch Thierry , Colpaert Francis C
Published: January 2005

Antipsychotic drugs may mediate their therapeutic effects not only by preventing the binding of dopamine but also by decreasing the propensity of the dopamine receptor to assume an active R* state. Ligand‐mediated activation and blockade of the recombinant human D2long receptor was investigated in CHO‐K1 cells upon modulation of its R* state.

Both the Ala371Lys (A371K) and Thr372Arg (T372R) D2long receptor mutants could be activated in a ligand‐dependent manner via a chimeric Gαq/o protein, and more efficaciously so than with the promiscuous Gα15 protein.

Dopamine and partial agonists (Emax: lisuride>>(+)‐UH 232≃bromerguride) displayed dissimilar Ca2+ kinetic properties at wild‐type and mutant receptors. A371K and T372R D2long receptor mutants demonstrated an attenuated and enhanced maximal response to these partial agonists, respectively.

Dopamine antagonists were unable to block the transient high‐magnitude Ca2+ phase at the wild‐type D2long receptor upon simultaneous exposure to antagonist and dopamine, while full blockade of the low‐magnitude Ca2+ phase did occur at a later time (onset‐time: haloperidol<bromerguride<(+)‐butaclamol). A similar, though more efficacious, antagonist profile was also found at the A371K mutant receptor. Conversely, the blockade of the low‐magnitude Ca2+ phase was attenuated (haloperidol) or almost absent [(+)‐butaclamol and bromerguride] at the T372R mutant receptor.

In conclusion, mutagenesis of the Ala371 and Thr372 positions affects in an opposite way the ligand‐dependent activation and blockade of the D2long receptor. The observed attenuation of dopamine‐mediated Ca2+ signal generation with different decay‐times may underlie distinct properties of the dopaminergic ligands.

British Journal of Pharmacology (2001) 134, 88–97; doi:10.1038/sj.bjp.0704243
The plasma protein extravasation induced by adenosine and its analogues in the rat dorsal skin: evidence for the involvement of capsaicin sensitive primary afferent neurones and mast cells
Authors: Esquisatto Laura C M , Costa Soraia K P , Camargo Enilton A , Ribela Maria T C P , Brain Susan D , Nucci Gilberto de , Antunes Edson
Published: January 2005

The contribution of sensory neurons and mast cells to the oedema evoked by adenosine A1 (N6‐cyclopentyladenosine, CPA, 3 – 30 nmol site−1), A2 (5′N‐ethylcarboxamidoadenosine, NECA, 1 – 10 nmol site−1) and A3 receptor agonists (N6‐[3‐iodobenzyl]‐N‐methyl‐5′‐carboxiamidoadenosine, IB‐MECA, 0.01 – 3 nmol site−1) was investigated in the rat skin microvasculature, by the extravascular accumulation of intravenously‐injected (i.v.) 125I‐albumin.

Intradermal (i.d.) injection of adenosine and analogues induced increased microvascular permeability in a dose‐dependent manner (IB‐MECA > NECA > CPA > adenosine). The non‐selective adenosine receptor antagonist theophylline (5 – 50 nmol site−1) markedly inhibited adenosine, CPA or NECA but not IB‐MECA‐induced plasma extravasation. The A1 receptor antagonist 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX, 0.3 – 3 μmol kg−1, i.v.) significantly reduced CPA‐induced plasma extravasation whereas responses to adenosine, NECA or IB‐MECA were unchanged. The A2 receptor antagonist 3,7‐dymethyl‐1‐proprargylxanthine (DMPX, 0.5 – 50 nmol site−1) significantly reduced NECA‐induced plasma extravasation without affecting responses to adenosine, CPA and IB‐MECA.

The tachykinin NK1 receptor antagonist (S)‐1‐[2‐[3‐(3,4‐dichlorphenyl)‐1 (3‐isopropoxyphenylacetyl) piperidin‐3‐yl] ethyl]‐4‐phenyl‐1 azaniabicyclo [2.2.2]octane chloride (SR140333), but not the NK2 receptor antagonist (S)‐N‐methyl‐N[4‐acetylamino‐4‐phenyl piperidino)‐2‐(3,4‐dichlorophenyl)butyl]‐benzamide (SR48968), significantly inhibited the plasma extravasation evoked by higher doses of adenosine (100 nmol site−1), CPA (100 nmol site−1), NECA (1 nmol site−1) and IB‐MECA (0.1 – 1 nmol site−1). In rats treated with capsaicin to destroy sensory neurons, the response to higher doses of adenosine, CPA and NECA, but not IB‐MECA, was significantly inhibited.

The effects of adenosine and analogues were largely inhibited by histamine and 5‐hydroxytryptamine (5‐HT) antagonists and by compound 48/80 pretreatment.

In conclusion, our results provide evidence that adenosine A1 and A2, but not A3, receptor agonists may function as cutaneous neurogenic pro‐inflammatory mediators; acting via microvascular permeability‐increasing mechanisms that can, depending on dose of agonist and purine receptor under study, involve the tachykinin NK1 receptor and mast cell amines.

British Journal of Pharmacology (2001) 134, 108–115; doi:10.1038/sj.bjp.0704245
Influence of aldosterone on collagen synthesis and proliferation of rat cardiac fibroblasts
Authors: Rombouts Krista , Wielant Annemie , Hellemans Karine , Schuppan Detlef , Geerts Albert
Published: January 2005

Previous in vivo studies in men and experimental animal models have shown that hyperaldosteronemia is correlated with cardiac fibrosis due to increased total collagen synthesis. As yet, it is unclear whether aldosterone has direct pro‐fibrogenic effect on cardiac fibroblasts, the fibrogenic effector cell in the myocardium, and if so which procollagens specifically are synthesized at higher rates.

The present study aims at establishing whether de novo collagen synthesis by cardiac fibroblasts is enhanced following exposure for 2×24 h to pharmacological (10−7 – 10−8 M), near‐physiological (10−9 M) or physiological (10−10 – 10−11 M) aldosterone concentrations. During the last 24 h, cells were metabolically labelled with [35S]‐methionine/[35S]‐cysteine. Labelled procollagens were immunoprecipitated quantitatively using antibodies against specific procollagens. Contrary to expectations, 10−7 M aldosterone inhibited significantly de novo synthesis of procollagens type I and IV (−35% and −42%, respectively). For procollagen type III, only a tendency towards inhibition was observed. At lower concentrations of aldosterone (10−8 – 10−10 M), synthesis of procollagens type I, III or IV was unaffected.

Cellular DNA synthesis under influence of aldosterone was evaluated by measuring BrdU incorporation. Cells were treated with aldosterone, while BrdU was added during the last 16 h of treatment. Aldosterone had no demonstrable effect on cellular proliferation.

Reverse transcription‐polymerase chain reaction (RT – PCR) clearly demonstrated the presence of mineralocorticoid receptor mRNA in cardiac fibroblasts.

In spite of the expression of the mineralocorticoid receptor by cultured cardiac fibroblasts, the pro‐fibrogenic effect of aldosterone as observed in vivo, is not likely to be due to a direct effect of this hormone in cardiac fibroblasts.

British Journal of Pharmacology (2001) 134, 224–232; doi:10.1038/sj.bjp.0704247
Effects of combining three different antiplatelet agents on platelets and leukocytes in whole blood in vitro
Authors: Zhao Lian , Bath Philip , Heptinstall Stan
Published: January 2005

Antiplatelet drugs have been demonstrated to reduce the incidence of recurrent events in patients with symptomatic vascular disease. However, there is no experimental data indicating the effects of these agents when given together on platelets and leukocytes. We investigated the ability of aspirin (an inhibitor of cyclo‐oxygenase), dipyridamole (an inhibitor of phospodiesterases and adenosine uptake) and AR‐C69931 (a direct acting P2T antagonist with effects similar to those of clopidogrel which can be used in vitro) when used alone or in combination to inhibit platelet and leukocyte function.

Measurements of platelet and leukocyte function were performed in blood taken from normal volunteers, and the inhibitory effects of aspirin (100 μmol l−1), dipyridamole (10 μmol l−1) and AR‐C66931 (100 nmol l−1) were determined. Platelet aggregation was induced by stirring blood with and without adenosine diphosphate (ADP) or platelet activating factor (PAF) and measured by platelet counting. Platelet P‐selectin expression, platelet‐leukocyte conjugate formation, and leukocyte activation were determined by flow cytometry.

Dipyridamole, AR‐C69931, dipyridamole and AR‐C69931, dipyridamole and aspirin, AR‐C69931 and aspirin, and all three agents together inhibited platelet aggregation induced by stirring, ADP and PAF (P<0.01). However, it was only the combination of all three agents inhibited P‐selectin expression (P<0.01). Similarly, it was the combination of all three antiplatelet agents that most consistently inhibited platelet‐monocyte and platelet‐neutrophil conjugate formation and monocyte and neutrophil activation.

Since both platelets and leukocytes are thought to contribute to arterial thrombosis and atherosclerosis, it is possible that combinations of different antiplatelet agents with different mechanisms of action may afford better protection than individual or pairs of agents used on their own.

British Journal of Pharmacology (2001) 134, 353–358; doi:10.1038/sj.bjp.0704248
Bupivacaine effects on hKv1.5 channels are dependent on extracellular pH
Authors: Longobardo M , González T , Caballero R , Delpón E , Tamargo J , Valenzuela C
Published: January 2005

Bupivacaine‐induced cardiotoxicity increases in hypoxic and acidotic conditions. We have analysed the effects of R(+)bupivacaine on hKv1.5 channels stably expressed in Ltk− cells using the whole‐cell patch‐clamp technique, at three different extracellular pH (pHo), 6.5, 7.4 and 10.0.

Acidification of the pHo from 7.4 to 6.5 decreased 4 fold the potency of R(+)bupivacaine to block hKv1.5 channels. At pHo 10.0, the potency of the drug increased ∼2.5 fold.

Block induced by R(+)bupivacaine at pHo 6.5, 7.4 and 10.0, was voltage‐ and time‐dependent in a manner consistent with an open state block of hKv1.5 channels.

At pHo 6.5, but not at pHo 7.4 or 10.0, R(+)bupivacaine increased by 95±3 % (n=6; P<0.05) the hKv1.5 current recorded at −10 mV, likely due to a drug‐induced shift of the midpoint of activation (ΔV=−8.5±1.4 mV; n=7).

R(+)bupivacaine development of block exhibited an ‘instantaneous’ component of block at the beginning of the depolarizing pulse, which averaged 12.5±1.8% (n=5) and 4.6±1.6% (n=6), at pHo 6.5 and 7.4, respectively, and that was not observed at pHo 10.0.

It is concluded that: (a) alkalinization of the pHo increases the potency of block of R(+)bupivacaine, and (b) at pHo 6.5, R(+)bupivacaine induces an ‘agonist effect’ of hKv1.5 current when recorded at negative membrane potentials.

British Journal of Pharmacology (2001) 134, 359–369; doi:10.1038/sj.bjp.0704251
Distinction between relaxations induced via prostanoid EP4 and IP1 receptors in pig and rabbit blood vessels
Authors: Jones Robert L , Chan Kam‐ming
Published: January 2005

Our study shows that the prostacyclin analogues AFP‐07 and cicaprost are moderately potent agonists for prostanoid EP4 receptors, in addition to being highly potent IP1 receptor agonists. Both activities were demonstrated on piglet and rabbit saphenous veins, which are established EP4 preparations.

On piglet saphenous vein, PGE2 was 6.1, 24, 96, 138, 168 and 285 times respectively more potent than AFP‐07, cicaprost, PGI2, iloprost, carbacyclin and TEI‐9063 in causing relaxation. Another prostacyclin analogue taprostene did not induce maximum relaxation (21 – 74%), and did not oppose the action of PGE2. The EP4 receptor antagonist AH 23848 (30 μM) blocked relaxant responses to PGE2 (dose ratio=8.6±1.3, s.e.mean) to a greater extent than cicaprost (4.9±0.7) and AFP‐07 (3.8±0.8), had variable effects on TEI‐9063‐induced relaxation (3.7±1.5), and had no effect on taprostene responses (<2.0).

On rabbit saphenous vein, AH 23848 blocked the relaxant actions of PGE2, AFP‐07, cicaprost, iloprost and carbacyclin to similar extents.

AFP‐07, cicaprost and TEI‐9063 showed high IP1 relaxant potency on piglet carotid artery, rabbit mesenteric artery and guinea‐pig aorta, with AFP‐07 confirmed as the most potent IP1 agonist reported to date. AH 23848 did not block cicaprost‐induced relaxation of piglet carotid artery. EP3 contractile systems in these preparations can confound IP1 agonist potency estimations.

Caution is urged when using AFP‐07 and cicaprost to characterize IP1 receptors in the presence of EP4 receptors. Taprostene may be a lead to a highly selective IP1 receptor agonist.

British Journal of Pharmacology (2001) 134, 313–324; doi:10.1038/sj.bjp.0704252
Hydrogen peroxide‐mediated inhibition of lipopolysaccharide‐stimulated inhibitory kappa B kinase activity in rat aortic smooth muscle cells
Authors: Torrie Lindsay J , MacKenzie Christopher J , Paul Andrew , Plevin Robin
Published: January 2005

In rat aortic smooth muscle cells (RASMC), exposure to lipopolysaccharide (LPS) resulted in NF‐κB‐DNA binding, degradation of IκB‐α, ‐β and ‐ε and increased activity of both α and β isoforms of inhibitory kappa B kinase (IKK).

Expression of dominant‐negative (DN)‐IKK‐α, IKK‐β and NF‐κB‐inducing kinase (NIK) abolished LPS‐stimulated NF‐κB reporter activity, suggesting that activation of a NIK/IKK‐dependent pathway is indispensable for NF‐κB activation by LPS in this cell type.

The tyrosine phosphatase inhibitor, pervanadate, abolished LPS‐stimulated NF‐κB‐DNA‐binding activity. However, the effect of pervanadate was shown to be mediated by excess hydrogen peroxide (H2O2) present in the reaction mix. Preincubation of RASMC with H2O2 inhibited LPS‐stimulated IKK kinase activity and downstream NF‐κB‐DNA binding activity.

H2O2 also strongly stimulated p38 MAP kinase activity in RASMCs. Effective inhibition of this pathway using SB203580 did not reverse the effects of H2O2 on LPS‐stimulated IKK/NF‐κB signalling.

These studies show that hydrogen peroxide‐mediated inhibition of LPS‐stimulated NF‐κB activation in RASMC occurs upstream of IKK. The inhibitory effect of H2O2 is not due to tyrosine phosphatase inhibition, it is mediated by H2O2 through a mechanism which is independent of any cross‐talk involving MAP kinase homologues.

British Journal of Pharmacology (2001) 134, 393–401; doi:10.1038/sj.bjp.0704259
Antisense knockdown of inducible nitric oxide synthase inhibits the relaxant effect of VIP in isolated smooth muscle cells of the mouse gastric fundus
Authors: Dick J M C , Van Molle W , Libert C , Lefebvre R A
Published: January 2005

Our previous results showed that the non‐selective nitric oxide synthase (NOS) inhibitor L‐NG‐nitroarginine (L‐NOARG) and the selective inducible NOS (iNOS) inhibitor N‐(3‐(acetaminomethyl)‐benzyl)acetamidine (1400W) inhibited the relaxant effect of vasoactive intestinal polypeptide (VIP) in isolated smooth muscle cells of the mouse gastric fundus, suggesting the involvement of iNOS. The identity of the NOS isoform involved in the VIP‐induced relaxation in isolated smooth muscle cells of the mouse gastric fundus was now further investigated by use of antisense oligodeoxynucleotides (aODNs) to iNOS.

Incubation of isolated smooth muscle cells with fluorescein isothiocyanate (FITC)‐labelled aODNs showed that nuclear accumulation occurs quickly and reaches saturation after 60 min. The in vivo intravenous administration of aODNs to iNOS, 24 and 12 h before murine tumour necrosis factor alpha (mTNFα) challenge, significantly reduced the nitrite levels induced by the mTNFα challenge.

Intravenous administration of aODNs to iNOS in mice, 24 and 12 h before isolation of the gastric smooth muscle cells, decreased the inhibitory effect of the NOS inhibitors L‐NOARG and 1400W on the relaxant effect of VIP, whereas neither saline nor sODNs had any influence.

Preincubation of the isolated smooth muscle cells with aODNs almost abolished the inhibitory effect of L‐NOARG and 1400W on the VIP‐induced relaxation, whereas sODNs failed.

These results illustrate that the inhibitory effect of NOS inhibitors in isolated smooth muscle cells of the mouse gastric fundus is due to inactivation of iNOS. iNOS, probably induced by the isolation procedure of the smooth muscle cells, seems involved in the relaxant effect of VIP in isolated gastric smooth muscle cells.

British Journal of Pharmacology (2001) 134, 425–433; doi:10.1038/sj.bjp.0704262
Synthesis and characterization of a quinolinonic compound activating ATP‐sensitive K+ channels in endocrine and smooth muscle tissues
Authors: Becker B , Antoine M‐H , Nguyen Q‐A , Rigo B , Cosgrove K E , Barnes P D , Dunne M J , Pirotte B , Lebrun P
Published: January 2005

Original quinolinone derivatives structurally related to diazoxide were synthesized and their effects on insulin secretion from rat pancreatic islets and the contractile activity of rat aortic rings determined.

A concentration‐dependent decrease of insulin release was induced by 6‐chloro‐2‐methylquinolin‐4(1H)‐one (HEI 713). The average IC50 values were 16.9±0.8 μM for HEI 713 and 18.4±2.2 μM for diazoxide.

HEI 713 increased the rate of 86Rb outflow from perifused pancreatic islets. This effect persisted in the absence of external Ca2+ but was inhibited by glibenclamide, a KATP channel blocker. Inside‐out patch‐clamp experiments revealed that HEI 713 increased KATP channel openings.

HEI 713 decreased 45Ca outflow, insulin output and cytosolic free Ca2+ concentration in pancreatic islets and islet cells incubated in the presence of 16.7 or 20 mM glucose and extracellular Ca2+. The drug did not affect the K+(50 mM)‐induced increase in 45Ca outflow.

In aortic rings, the vasorelaxant effects of HEI 713, less potent than diazoxide, were sensitive to glibenclamide and to the extracellular K+ concentration.

The drug elicited a glibenclamide‐sensitive increase in 86Rb outflow from perifused rat aortic rings.

Our data describe an original compound which inhibits insulin release with a similar potency to diazoxide but which has fewer vasorelaxant effects.

Our results suggest that, in both aortic rings and islet tissue, the biological effects of HEI 713 mainly result from activation of KATP channels ultimately leading to a decrease in Ca2+ inflow.

British Journal of Pharmacology (2001) 134, 375–385; doi:10.1038/sj.bjp.0704266
Effects of adenosine receptor agonists and antagonists in a genetic animal model of primary paroxysmal dystonia
Authors: Richter Angelika , Hamann Melanie
Published: January 2005

Recent studies have shown beneficial effects of an adenosine A2A receptor agonist in dtsz mutant hamsters, an animal model of paroxysmal dystonia, in which stress and consumption of coffee can precipitate dystonic attacks. This prompted us to examine the effects of adenosine receptor agonists and antagonists on severity of dystonia in dtsz hamsters in more detail.

The non‐selective adenosine A1/A2A receptor antagonists, caffeine (10 – 20 mg kg−1 i.p.) and theophylline (10 – 30 mg kg−1 s.c.), worsened the dystonia in dtsz hamsters.

Aggravation of dystonia was also caused by the selective adenosine A1/A2A antagonist CGS 15943 (9‐chloro2‐2‐furyl)[1,2,4]triazolo[1,5‐c]quinazolin‐5‐amine) at a dose of 30 mg kg−1 i.p. and by the adenosine A1 antagonist DPCPX (8‐cyclopentyl‐1,3‐dipropylxanthine; 20 – 30 mg kg−1 i.p.), while the A2 antagonist DMPX (3,7‐dimethyl‐1‐propargylxanthine; 2 – 4 mg kg−1 i.p.) and the highly selective A2A antagonist ZM 241385 (4‐(2‐[7‐amino‐2‐(2‐furyl)[1,2,4]triazolo[2,3‐a][1,3,5]triazin‐5‐ylamino]ethyl)phenol; 2 – 5 mg kg−1 i.p.) failed to exert any effects on dystonia.

In contrast to the antagonists, both the adenosine A1 receptor agonist CPA (N6‐cyclopentyladenosine; 0.1 – 1.0 mg kg−1 i.p.) and the A2A agonist CGS 21680 (2p‐(2carboxyethylphen‐ethylamino‐5′‐N‐ethylcarboxamindoadenosine; 0.1 – 2.0 mg kg−1 i.p.) exerted a striking improvement of dystonia.

These data suggest that the precipitating effects of methylxanthines are, at least in part, related to their adenosine receptor antagonistic action.

Although adenosine receptor agonists can be regarded as interesting candidates for the therapy of paroxysmal dystonia, adverse effects may limit the therapeutic potential of adenosine A1 agonists, while beneficial effects of the adenosine A2A agonist CGS 21680 were already found at well tolerated doses.

British Journal of Pharmacology (2001) 134, 343–352; doi:10.1038/sj.bjp.0704268
Pharmacological evidence for capacitative Ca2+ entry in cannulated and pressurized skeletal muscle arterioles
Authors: Potocnik Simon J , Hill Michael A
Published: January 2005

Arteriolar myogenic tone shows a marked dependency on extracellular Ca2+. The contribution played by mechanisms such as intracellular Ca2+ release and capacitative entry, however, are less certain. The present studies aimed to demonstrate functional evidence for involvement of such mechanisms in myogenic tone and reactivity.

Single cremaster arterioles were denuded of endothelium, pressurized under no‐flow conditions and loaded with fura 2‐AM for measurement of changes in intracellular Ca2+ [Ca2+]i. The cell permeable, putative, IP3 receptor antagonist 2APB (2 aminoethoxydiphenyl borate) was used to determine the possible role of IP3 receptor‐mediated mechanisms in arteriolar myogenic tone and reactivity.

Arterioles dilated in response to increasing concentrations of 2APB (1 – 300 μM) without a concomitant change in global [Ca2+]i. Also 2APB (50 μM) completely inhibited the myogenic constriction in response to a step change in luminal pressure (50 – 120 mmHg) with no apparent effect on pressure‐mediated increases in [Ca2+]i. 2APB markedly attenuated the constrictor response and [Ca2+]i increase stimulated by phenylephrine but not KCl.

Capacitative Ca2+ influx in arterioles was demonstrated either by re‐addition of extracellular [Ca2+] following pre‐treatment with 1 or 10 μM nifedipine in Ca2+ free buffer or exposure of vessels to thapsigargin (1 μM) to induce store depletion. In both cases 2APB inhibited the increase in [Ca2+]i. Capacitative Ca2+ entry showed an inverse relationship with intraluminal pressure over the range 10 – 120 mmHg.

Consistent with an effect on a Ca2+ entry pathway, 2APB had no effect on intracellular (caffeine releasable) Ca2+ stores while decreasing the rate of Mn2+ quench of fura 2 fluorescence.

The results provide functional evidence for capacitative Ca2+ entry in intact arteriolar smooth muscle. The effectiveness of 2APB in inhibiting both non‐voltage gated Ca2+ entry and responsiveness to an acute pressure step is consistent with the involvement of an axis involving IP3‐mediated and or capacitative Ca2+ entry mechanisms in myogenic reactivity. Given the lack of effect of 2APB on pressure‐induced changes in global [Ca2+]i it is suggested that such mechanisms participate on a localized level to couple the myogenic stimulus to contraction.

British Journal of Pharmacology (2001) 134, 247–256; doi:10.1038/sj.bjp.0704270
Pharmacological modulation of monovalent cation currents through the epithelial Ca2+ channel ECaC1
Authors: Nilius Bernd , Prenen Jean , Vennekens Rudi , Hoenderop Joost G J , Bindels René J M , Droogmans Guy
Published: January 2005

The recent identification of the epithelial Ca2+ channel, ECaC1, represents a major step forward in our knowledge of renal Ca2+ handling. ECaC1 constitutes the rate‐limiting apical Ca2+ entry mechanism of active, transcellular Ca2+ reabsorption. This unique highly selective Ca2+ channel shares a low but significant homology with transient receptor potential (TRP) channels and vanilloid receptors (VR).

We have studied the pharmacological modulation of currents through ECaC1 heterologously expressed in HEK 293 cells. Monovalent cation currents were measured by use of the whole cell patch clamp technique in cells dialysed with 10 mM BAPTA or 10 mM EGTA to prevent the fast Ca2+ dependent inactivation of ECaC1.

Several modulators were tested, including inorganic cations, putative store‐operated Ca2+ entry (SOC) blockers, the vanilloid receptor (VR‐1) blocker capsazepine, protein tyrosine kinase blockers, calmodulin antagonists and ruthenium red.

Ruthenium red and econazole appeared to be the most effective inhibitors of currents through ECaC1, with IC50 values of 111 nM and 1.3 μM, respectively, whereas the selective SOC inhibitor, SKF96365, was nearly ineffective.

The divalent cation current block profile for ECaC1 is Pb2+=Cu2+>Zn2+>Co2+>Fe2+ with IC50 values between 1 and approximately 10 μM.

In conclusion, ECaC activity is effectively inhibited by various compounds including ruthenium red, antimycotic drugs and divalent cations, which might be useful tools for pharmacological manipulation and several disorders related to Ca2+ homeostasis could benefit from such developments.

British Journal of Pharmacology (2001) 134, 453–462; doi:10.1038/sj.bjp.0704272
Common mechanisms of inhibition for the Na+/glucose (hSGLT1) and Na+/Cl−/GABA (hGAT1) cotransporters
Authors: Hirayama Bruce A , Díez‐Sampedro Ana , Wright Ernest M
Published: January 2005

Electrophysiological methods were used to investigate the interaction of inhibitors with the human Na+/glucose (hSGLT1) and Na+/Cl−/GABA (hGAT1) cotransporters. Inhibitor constants were estimated from both inhibition of substrate‐dependent current and inhibitor‐induced changes in cotransporter conformation.

The competitive, non‐transported inhibitors are substrate derivatives with inhibition constants from 200 nM (phlorizin) to 17 mM (esculin) for hSGLT1, and 300 nM (SKF89976A) to 10 mM (baclofen) for hGAT1. At least for hSGLT1, values determined using either method were proportional over 5‐orders of magnitude.

Correlation of inhibition to structure of the inhibitors resulted in a pharmacophore for glycoside binding to hSGLT1: the aglycone is coplanar with the pyranose ring, and binds to a hydrophobic/aromatic surface of at least 7×12Å. Important hydrogen bond interactions occur at five positions bordering this surface.

In both hSGLT1 and hGAT1 the data suggests that there is a large, hydrophobic inhibitor binding site ∼8Å from the substrate binding site. This suggests an architectural similarity between hSGLT1 and hGAT1. There is also structural similarity between non‐competitive and competitive inhibitors, e.g., phloretin is the aglycone of phlorizin (hSGLT1) and nortriptyline resembles SKF89976A without nipecotic acid (hGAT1).

Our studies establish that measurement of the effect of inhibitors on presteady state currents is a valid non‐radioactive method for the determination of inhibitor binding constants. Furthermore, analysis of the presteady state currents provide novel insights into partial reactions of the transport cycle and mode of action of the inhibitors.

British Journal of Pharmacology (2001) 134, 484–495; doi:10.1038/sj.bjp.0704274
Assessment of agonism at G‐protein coupled receptors by phosphatidic acid and lysophosphatidic acid in human embryonic kidney 293 cells
Authors: Alderton Forbes , Sambi Balwinder , Tate Rothwelle , Pyne Nigel J , Pyne Susan
Published: January 2005

Several different molecular species of phosphatidic acid (PA) bind to a G‐protein coupled receptor (GPCR) to induce activation of the p42/p44 mitogen‐activated protein kinase (p42/p44 MAPK) pathway in HEK 293 cells. PA is active at low nanomolar concentrations and the response is sensitive to pertussis toxin (which uncouples GPCRs from Gi/o). The de‐acylated product of PA, lysophosphatidic acid (LPA), which binds to members of the endothelial differentiation gene (EDG) family of receptors also stimulated p42/p44 MAPK in a pertussis toxin sensitive manner, but with an ∼100 – 1000 fold lower potency compared with the different molecular species of PA. RT – PCR using gene‐specific primers showed that HEK 293 cells express EDG2 and PSP24, the latter being a lipid binding GPCR out with the EDG cluster. We conclude that PA is a novel high potency GPCR agonist.

British Journal of Pharmacology (2001) 134, 6–9; doi:10.1038/sj.bjp.0704278
Effects of local cytochalasin D delivery on smooth muscle cell migration and on collar‐induced intimal hyperplasia in the rabbit carotid artery
Authors: Bruijns Richard H J , Bult Hidde
Published: January 2005

Smooth muscle cell (SMC) migration has been implicated in neointima formation after angioplasty. Therefore, we investigated whether cytochalasin D, a fungal metabolite that inhibits actin filament formation, suppressed SMC migration and collar‐induced intimal hyperplasia in the rabbit carotid artery.

To establish effective concentrations, contractions of carotid artery rings to phenylephrine were determined after incubation with cytochalasin D (10−8 – 10−6 M) for 30 min or 3 days. In vitro cell migration was studied using carotid artery explants and a modified Boyden chamber with SMCs isolated from the rabbit aorta. The in vivo effect was tested after infusion of 10−8 – 10−4 M cytochalasin D into collars placed around the left carotid artery; collars placed around the right artery served as controls.

Contractions to phenylephrine decreased after 30 min or 3 days exposure to 10−7 and 10−6 M cytochalasin D; the effect was partly reversible. These concentrations also inhibited cellular outgrowth and SMC migration in the in vitro assays.

Immunohistochemistry showed that local delivery of 10−5 or 10−4 M cytochalasin D for 2 weeks suppressed collar‐induced α‐SMC actin expression in the intima by 68% and 84% respectively. However, the cross‐sectional area of the intima was not reduced due to an influx of T‐lymphocytes and macrophages.

It is concluded that cytochalasin D suppressed SMC contractility and migration in vitro. Although perivascular infusion of cytochalasin D inhibited collar‐induced SMC migration from media to intima in vivo as well, the intimal hyperplasia was not reduced due to concomitant development of an inflammatory response.

British Journal of Pharmacology (2001) 134, 473–483; doi:10.1038/sj.bjp.0704281
Role of multidrug resistance protein 2 (MRP2) in glutathione‐bimane efflux from Caco‐2 and rat renal proximal tubule cells
Authors: Terlouw Sylvie A , Masereeuw Rosalinde , Van Den Broek Petra H H , Notenboom Sylvia , Russel Frans G M
Published: January 2005

The multidrug resistance protein 2 (MRP2) has been shown to play an important role in the transport of glutathione conjugates in the liver. Its importance in renal excretion, however, is still uncertain and other organic anion transporters may be involved. The objective of the present study was to characterize glutathione conjugate efflux from rat kidney proximal tubule cells (PTC), and to determine the contribution of Mrp2.

We used isolated PTC in suspension, as well as grown to monolayer density. For comparison, transport characteristics were also determined in the human intestinal epithelial cell line Caco‐2, an established model to study MRP2‐mediated transport. The cells were loaded with monochlorobimane (MCB) at 10°C. MCB enters the cells by simple diffusion and is conjugated with glutathione to form the fluorescent glutathione‐bimane (GS‐B).

In primary cultures of rat PTC, no indications for a transporter‐mediated mechanism were found. The efflux of GS‐B from Caco‐2 cells and freshly isolated PTC was time‐ and temperature‐dependent. Furthermore, GS‐B transport in both models was inhibited by chlorodinitrobenzene (CDNB), with an inhibitory constant of 46.8±0.9 μM in freshly isolated PTC. In Caco‐2 cells, the inhibitory potency of CDNB was approximately 20 fold higher. Finally, efflux of GS‐B from freshly isolated PTC from Mrp2‐deficient (TR−) rats was studied. As compared to normal rat PTC, transport characteristics were not different.

We conclude that in freshly isolated rat PTC glutathione conjugate excretion is mediated by other organic anion transporters rather than by Mrp2.

British Journal of Pharmacology (2001) 134, 931–938; doi:10.1038/sj.bjp.0704284
Determination of effects of antiepileptic drugs on SNAREs‐mediated hippocampal monoamine release using in vivo microdialysis
Authors: Murakami Takuya , Okada Motohiro , Kawata Yuko , Zhu Gan , Kamata Akihisa , Kaneko Sunao
Published: January 2005

To elucidate possible mechanisms underlying the effects of carbamazepine (CBZ), valproate (VPA) and zonisamide (ZNS) on neurotransmitter exocytosis, the interaction between these three antiepileptic drugs (AEDs) and botulinum toxins (BoNTs) on basal, Ca2+‐ and K+‐evoked release of dopamine (DA) and serotonin (5‐HT) were determined by microdialysis in the hippocampus of freely moving rats.

Basal release of monoamine was decreased by pre‐microinjection of the syntaxin inhibitor, BoNT/C, but only weakly affected by the synaptobrevin inhibitor, BoNT/B. Ca2+‐evoked release was inhibited by BoNT/C selectively. K+‐evoked release was reduced by BoNT/B predominantly and BoNT/C weakly.

Perfusion with low and high concentrations of CBZ and ZNS increased and decreased basal monoamine release, respectively. Perfusion with VPA increased basal 5‐HT release concentration‐dependently, whereas basal DA release was affected by VPA biphasic concentration‐dependently, similar to CBZ and ZNS. This stimulatory action of AEDs on basal release was inhibited by BoNT/C predominantly.

Ca2+‐evoked monoamine release was increased by low concentrations of CBZ, ZNS and VPA, but decreased by high concentrations. These effects of the AEDs on Ca2+‐evoked release were inhibited by BoNT/C, but not by BoNT/B.

K+‐evoked monoamine release was reduced by AEDs concentration‐dependently. The inhibitory effect of these three AEDs on K+‐evoked release was inhibited by BoNT/B, but not by BoNT/C,

These findings suggest that the therapeutic‐relevant concentration of CBZ, VPA and ZNS affects exocytosis of DA and 5‐HT, the enhancement of syntaxin‐mediated monoamine release during resting stage, and the inhibition of synaptobrevin‐mediated release during depolarizing stage.

British Journal of Pharmacology (2001) 134, 507–520; doi:10.1038/sj.bjp.0704285
Selective inhibition of thapsigargin‐induced contraction and capacitative calcium entry in mouse anococcygeus by trifluoromethylphenylimidazole (TRIM)
Authors: Gibson Alan , Fernandes Filomena , Wallace Pat , McFadzean Ian
Published: January 2005

This study examined the effects of trifluoromethylphenylimidazole (TRIM) on tone, and calcium entry, in mouse anococcygeus stimulated by either thapsigargin (Tg; 100 nM) which activates capacitative calcium entry (CCE), or high K (60 mM) which activates voltage‐operated calcium channels. TRIM (1 – 333 μM) produced concentration‐related relaxation of Tg‐induced tone (EC50, 42 μM) but was much less effective against high K. In single smooth muscle cells loaded with FURA‐2, TRIM reduced the increase in fluorescence ratio produced by Tg but had no effect on that produced by high K. The relaxations of Tg‐induced tone, and reduction in fluorescence ratio, were obtained in the presence of L‐NG‐nitroarginine and were thus independent of nitric oxide synthase inhibition; further, TRIM had no discernible effect on nitrergic responses. TRIM provides a novel drug for the selective inhibition of CCE and a template for the development of more potent inhibitors.

British Journal of Pharmacology (2001) 134, 233–236; doi:10.1038/sj.bjp.0704286