bluuebunny/crossref_metadata_2025 huggingface dataset:
Blockade of HERG channels by the class III antiarrhythmic azimilide: mode of action
Authors: Busch A E , Eigenberger B , Jurkiewicz N K , Salata J J , Pica A , Suessbrich H , Lang F
Published: January 2005

The class III antiarrhythmic azimilide has previously been shown to inhibit IKs and IKr in guinea‐pig cardiac myocytes and IKs (minK) channels expressed in Xenopus oocytes. Because HERG channels underly the conductance IKr in human heart, the effects of azimilide on HERG channels expressed in Xenopus oocytes were the focus of the present study.

In contrast to other well characterized HERG channel blockers, azimilide blockade was reverse use‐dependent, i.e., the relative block and apparent affinity of azimilide decreased with an increase in channel activation frequency. Azimilide blocked HERG channels at 0.1 and 1 Hz with IC50 s of 1.4 μM and 5.2  μM respectively.

In an envelope of tail test, HERG channel blockade increased with increasing channel activation, indicating binding of azimilide to open channels.

Azimilide blockade of HERG channels expressed in Xenopus oocytes and IKr in mouse AT‐1 cells was decreased under conditions of high [K+]e, whereas block of slowly activating IKs channels was not affected by changes in [K+]e.

In summary, azimilide is a blocker of cardiac delayed rectifier channels, IKs and HERG. Because of the distinct effects of stimulation frequency and [K+]e on azimilide block of IKr and IKs channels, we conclude that the relative contribution of block of each of these cardiac delayed rectifier channels depends on heart frequency. [K+]e and regulatory status of the respective channels.

British Journal of Pharmacology (1998) 123, 23–30; doi:10.1038/sj.bjp.0701575
Glutamate release in human cerebral cortex and its modulation by 5‐hydroxtryptamine acting at h 5‐HT1D receptors
Authors: Maura Guido , Marcoli Manuela , Tortarolo Massimo , Andrioli Gian Carlo , Raiteri Maurizio
Published: January 2005

The release of glutamic acid and its modulation by 5‐hydroxytryptamine (5‐HT) in the human brain has been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery to reach deeply sited tumours.

The Ca2+‐dependent K+ (15 mM)‐evoked overflow of glutamate was inhibited by 5‐HT in a concentration‐dependent manner (EC50=2.9 nM; maximal effect ≃50%). The inhibition caused by 5‐HT was antagonized by the 5‐HT1/5‐HT2 receptor antagonist methiothepin. The 5‐HT1B/5‐HT1D receptor agonist sumatriptan mimicked 5‐HT (EC50=6.4 nM; maximal effect ≃50%); the effect of sumatriptan was also methiothepin‐sensitive. Selective 5‐HT1A receptor antagonists could not prevent the inhibition of glutamate release by 5‐HT.

The 5‐HT1B/5‐HT1D receptor ligand GR 127935 and the 5‐HT2C/5‐HT1B/5‐HT1D receptor ligand metergoline were unable to prevent the 5‐HT effect; instead they inhibited glutamate release, their effects being abolished by methiothepin. Some 5‐HT1A receptor antagonists also displayed intrinsic agonist activity.

The effect of sumatriptan was prevented by ketanserin, a drug known to display much higher affinity for recombinant h 5‐HT1D than for h 5‐HT1B receptors.

We propose that neocortical glutamatergic nerve terminals in human brain cortex possess release‐inhibiting presynaptic heteroreceptors that appear to belong to the h 5‐HT1D subtype.

British Journal of Pharmacology (1998) 123, 45–50; doi:10.1038/sj.bjp.0701581
Effect of nitroglycerin and long‐term electrical stimulation on nitrergic relaxation in the pig gastric fundus
Authors: Lefebvre R A , Vandekerckhove K
Published: January 2005

The effect of incubation with the nitric oxide (NO) donor nitroglycerin and of long‐term electrical stimulation on relaxations induced by non‐adrenergic, non‐cholinergic nerve stimulation, exogenous NO, vasoactive intestinal polypeptide (VIP) and lemakalim was investigated in the pig gastric fundus.

In physiological salt solution containing 10−6 M atropine and 4×10−6 M guanethidine, electrical field stimulation (40 V, 0.1 ms, 0.5–8 Hz) for periods of 10 s at 5 min intervals (train stimulation) and administration of NO (2×10−6–10−4 M) at 5 min intervals (NO boli) induced frequency‐ and concentration‐dependent transient relaxations, respectively. Continuous electrical field stimulation with stepwise increase of the frequency (0.5–8 Hz; cumulative stimulation) induced frequency‐dependent sustained relaxations. VIP (10−7 M), lemakalim (10−5 M) and an infusion of NO induced a sustained relaxation.

Pretreatment for 30 min with 5×10−4 M nitroglycerin reduced the relaxations induced by train and cumulative stimulation, but also the relaxant responses to NO, both when given in boli or as an infusion. The relaxations to VIP and lemakalim were not influenced by pretreatment with nitroglycerin.

Long‐term electrical stimulation at 4 Hz for 40 min induced a sustained relaxation of the tissues. Administration of 3×10−4 M NG‐nitro‐L‐arginine methyl ester after 10, 20 or 30 min reversed the relaxation to a similar extent (approximately 70%). Previous long‐term electrical stimulation at 4 Hz for 30 min did not affect the responses to stimulation, NO and VIP.

These results illustrate that nitroglycerin can induce a postjunctional tolerance to nitrergic stimuli in the pig gastric fundus but evidence for a prejunctional inhibition of neuronal NO synthase by NO was not obtained.

British Journal of Pharmacology (1998) 123, 143–149; doi:10.1038/sj.bjp.0701582
Involvement of 5‐hydroxytryptamine7 receptors in inhibition of porcine myometrial contractility by 5‐hydroxytryptamine
Authors: Kitazawa Takio , Kubo Osamu , Satoh Masami , Taneike Tetsuro
Published: January 2005

5‐Hydroxytryptamine (5‐HT; 1 nM–100 μM) concentration‐dependently inhibited the amplitude and frequency of spontaneous contractions in longitudinal and circular muscles of the porcine myometrium. The circular muscle (EC50; 68–84 nM) was more sensitive than the longitudinal muscle (EC50; 1.3–1.44 μM) to 5‐HT. To characterize the 5‐HT receptor subtype responsible for inhibition of myometrial contractility, the effects of 5‐HT receptor agonists on spontaneous contractions and of 5‐HT receptor antagonists on inhibition by 5‐HT were examined in circular muscle preparations.

Pretreatment with tetrodotoxin (1 μM), propranolol (1 μM), atropine (1 μM), guanethidine (10 μM) or L‐NAME (100 μM) failed to change the inhibition by 5‐HT, indicating that the inhibition was due to a direct action of 5‐HT on the smooth muscle cells.

5‐CT, 5‐MeOT and 8‐OH‐DPAT mimicked the inhibitory response of 5‐HT, and the rank order of the potency was 5‐CT>5‐HT>5‐MeOT>8‐OH‐DPAT. On the other hand, oxymethazoline, α‐methyl‐5‐HT, 2‐methyl‐5‐HT, cisapride, BIMU‐1, BIMU‐8, ergotamine and dihydroergotamine had almost no effect on spontaneous contractions, even at 10–100 μM.

Inhibition by 5‐HT was not decreased by either pindolol (1 μM), ketanserin (1 μM), tropisetron (10 μM), MDL72222 (1 μM) or GR113808 (10 μM), but was antagonized by the following compounds in a competitive manner (with pA2 values in parentheses): methiothepin (8.05), methysergide (7.92), metergoline (7.4), mianserin (7.08), clozapine (7.06) and spiperone (6.86).

Ro 20‐1724 (20 μM) and rolipram (10 μM) significantly enhanced the inhibitory response of 5‐HT, but neither zaprinast (10 μM) nor dipyridamole (10 μM) altered the response of 5‐HT.

5‐HT (1 nM–1 μM) caused a concentration‐dependent accumulation of intracellular cyclic AMP in the circular muscle.

From the present results, the 5‐HT receptor, which is functionally correlated with the 5‐HT7 receptor, mediates the inhibitory effect of 5‐HT on porcine myometrial contractility. This inhibitory response is probably due to an increase in intracellular cyclic AMP through the activation of adenylate cyclase that is positively coupled to 5‐HT7 receptors.

British Journal of Pharmacology (1998) 123, 173–182; doi:10.1038/sj.bjp.0701583
Mediation of irregular spiking activity by multiple neurokinin‐receptors in the small intestine of the rat
Authors: Lördal Mikael , Bränström Robert , Hellström Per M
Published: January 2005

We have studied the small intestinal myoelectric response to the natural tachykinins substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and the neurokinin‐receptor selective agonists substance P methyl esther (SPME), [β‐Ala8]neurokinin A 4‐10, and senktide in conscious rats.

The effects of the agonists were studied before and after administration of the selective neurokinin 2 (NK2)‐receptor antagonist MEN 10,627.

Under basal conditions SP, NKA, NKB, as well as the selective NK1‐receptor agonist SPME, the NK2‐receptor agonist [β‐Ala8]NKA 4–10, and the NK3‐receptor agonist senktide, disrupted the interdigestive rhythm with regularly recycling migrating myoelectric complexes and induced a phase II‐like irregular spiking activity.

MEN 10,627 given alone did not affect the interdigestive rhythm.

MEN 10,627 inhibited the response to [β‐Ala8]NKA 4–10 but not to SP, SPME, NKA, NKB or senktide.

It is concluded that not only NK2 receptors, but also other receptors, such as NK1 and NK3 receptors, may mediate the motility‐stimulating action of different tachykinins in vivo.

It is further concluded that MEN 10,627 exerts a selective NK2‐receptor antagonism, and may be a valuable tool for assessing the functional role of NK2‐receptors in gastrointestinal physiology.

British Journal of Pharmacology (1998) 123, 63–70; doi:10.1038/sj.bjp.0701585
Characterization of [125I]‐PD164333, an ETA selective non‐peptide radiolabelled antagonist, in normal and diseased human tissues
Authors: Davenport Anthony P , Kuc Rhoda E , Ashby Michael J , Patt William C , Doherty Annette M
Published: January 2005

We have synthesized a new low molecular weight, non‐peptide radioligand, [125I]‐PD164333, an analogue of the orally active butenolide antagonists of the endothelin ETA receptor.

Analysis of saturation binding assays demonstrated that [125I]‐PD164333 bound with high affinity to a single population of receptors (n3 individuals ±s.e.mean) in human aorta (KD=0.26±0.08 nM; Bmax=8.8±3.95 fmol mg‐1 protein), left ventricle from the heart (KD=0.16±0.02 nM; Bmax=34.2± 3.02 fmol mg‐1 protein) and kidney (KD=1.24±0.16 nM; Bmax=125.3±35.07 fmol mg‐1 protein). In each case Hill slopes were close to unity.

In kinetic experiments, the binding of [125I]‐PD164333 to ETA receptors in sections of heart was time‐dependent and rapid at 23°C. The data were fitted to a one site model, with an association rate constant (K1 of 2.66±0.213×108 M‐1 min‐1, and a half‐time for association of 11 min. The binding was reversible at 23°C: analysis of the data indicated [125I]‐PD164333 dissociated from a single site, with a dissociation rate constant of 0.0031±0.0004 min‐1, a half‐time for dissociation of 216 min and a KD calculated from these kinetic data of 0.01 nM.

Unlabelled PD164333 inhibited the binding of [125I]‐ET‐1 to left ventricle (which expresses both subtypes) in a biphasic manner with a KDETA of 0.99±0.32 nM and KDETB of 2.41±0.22 μM, giving a selectivity of 2500 fold. ETA‐selective ligands competed monophasically for [125I]‐PD164333 binding in left ventricle, a one site fit was preferred to a two site model giving similar nanomolar affinities: BQ123, KD=3.93 ±0.18 nM; FR139317 KD=3.53±0.69 nM. In contrast, the ETB selective agonists, BQ3020 and sarafotoxin S6c (1 μM) did not inhibit binding.

In human isolated saphenous vein, unlabelled PD164333 was a functional antagonist, producing parallel rightward shifts of the endothelin‐1 (ET‐1) concentration‐response curve (pA2=8.84) and a slope of unity.

In the human brain, autoradiography revealed high levels of [125I]‐PD164333 binding to the pial arteries of the cerebral cortex and to the numerous smaller intercerebral vessels penetrating the underlying grey and white matter. Conduit and resistance vessels contributing to the control of blood pressure from the heart, kidney, lungs and adrenal also displayed high densities of binding. In diseased vessels, binding of [125I]‐PD164333 was confined to the medial layer of both coronary arteries with advanced atherosclerotic lesions or occluded saphenous vein grafts. In contrast, little or no binding was detected in the proliferated smooth muscle of the intimal layer or occluded lesion.

These results show [125I]‐PD164333 is a specific, high affinity, reversible non‐peptide radioligand for human ETA receptors, which will facilitate the further characterization of this subtype, in vitro and in vivo.

British Journal of Pharmacology (1998) 123, 223–230; doi:10.1038/sj.bjp.0701597
Studies on the effects of anandamide in rat hepatic artery
Authors: Zygmunt Peter M. , Högestätt Edward D. , Waldeck Kristian , Edwards Gillian , Kirkup Anthony J. , Weston Arthur H.
Published: January 2005

The effects of anandamide on K+ currents and membrane potential have been examined in freshly‐isolated smooth muscle cells from rat hepatic artery and the results compared with the effects of this arachidonic acid derivative on tension and membrane potential changes in segments of whole artery.

In the presence of 0.3 mm l‐NOARG and 10 μm indomethacin, anandamide (0.1–100 μm) and endothelium‐derived hyperpolarizing factor (EDHF; liberated by acetylcholine, 0.01–10 μm) each relaxed endothelium‐intact segments of hepatic artery precontracted with phenylephrine. These effects of anandamide, but not those of EDHF, were antagonized by the cannabinoid receptor antagonist, SR141716A (3 μm).

The relaxant effects of anandamide were unaffected by a toxin combination (apamin plus charybdotoxin, each 0.3 μm) which abolishes EDHF relaxations and were essentially unchanged in endothelium‐denuded arteries. The relaxant effects of anandamide in endothelium‐intact arteries were significantly reduced in a physiological salt solution containing 30 mm KCl and abolished when the K+ concentration was raised to 60 mm.

Anandamide (10 μm), acetylcholine (1 μm, via release of EDHF) and levcromakalim (10 μm) each markedly hyperpolarized the membrane potential of the smooth muscle cells of endothelium‐intact arteries. However, when the endothelium was removed, the hyperpolarizing effects of both anandamide (10 μm) and acetylcholine were essentially abolished whereas those of levcromakalim (10 μm) were unaffected.

Under voltage‐clamp conditions, anandamide (10 μm) abolished spontaneous transient outward currents (STOCs) in freshly‐isolated single hepatic artery cells held at 0 mV but had no effect on the holding current at this potential. In current‐clamp mode, the spontaneous hyperpolarizing potentials which corresponded to the STOCs were abolished with no significant change in basal membrane potential.

Anandamide (10 μm) abolished the iberiotoxin‐sensitive K+ current (IBK(Ca)) produced by caffeine and the corresponding hyperpolarizations generated by this xanthine derivative in current‐clamp mode. In contrast, anandamide had no effect on IBK(Ca) generated on exposure to NS1619 (30 μm).

It was concluded that anandamide is not EDHF in the rat hepatic artery. Anandamide‐induced hyperpolarization is exerted indirectly and requires the presence of the endothelium. Anandamide also acts on the smooth muscle cells to inhibit processes which require functional intracellular calcium stores. This direct action seems more important than membrane hyperpolarization in relaxing phenylephrine‐contracted vessels.
Investigation of the effects of platelet‐activating factor (PAF) on ion transport and prostaglandin synthesis in human colonic mucosa in vitro
Authors: Borman R A , Jewell R , Hillier K
Published: January 2005

We have investigated the effects of platelet‐activating factor (PAF), an endogenous mediator of inflammation, on ion transport and prostaglandin synthesis in the human isolated colon.

Application of PAF to the serosal surface of human colonic mucosa induced a marked, concentration‐dependent increase in ion transport. Mucosal application was without effect.

The secretory response to PAF was significantly inhibited by prior application of a specific PAF receptor antagonist WEB 2170, indicating that the response is dependent on PAF receptor activation.

The response to PAF was attenuated by prior application of indomethacin or piroxicam, implicating products of the cyclo‐oxygenase pathway in the response.

The response to PAF was attenuated by the loop diuretic bumetanide, indicating an involvement of chloride ion secretion in the response.

Addition of PAF to the serosal surface induced a significant increase in serosal prostaglandin E2 (PGE2), but not 6‐oxo‐PGF1α release. There was no effect on mucosal application of PAF.

In summary, we have shown that PAF is a potent secretagogue in isolated preparations of human colon and that the response is dependent on a specific PAF receptor, cyclo‐oxygenase products and bumetanide‐sensitive chloride ion transport.

British Journal of Pharmacology (1998) 123, 231–236; doi:10.1038/sj.bjp.0701602
Endothelium‐dependent sensory NANC vasodilatation: involvement of ATP, CGRP and a possible NO store
Authors: Kakuyama Masahiro , Vallance Patrick , Ahluwalia Amrita
Published: January 2005

Non‐adrenergic non‐cholinergic (NANC) vasodilator nerves regulate tone in certain vascular beds. We have investigated the mechanisms of the NANC dilator response in the isolated small mesenteric artery of the rabbit by use of the tension myograph.

Small second or third order (150–300 μm in diameter) arteries of the rabbit mesenteric bed were mounted in a Mulvany tension myograph. Responses to electrical field stimulation (EFS) and exogenous vasodilators were investigated.

EFS (0.5–16 Hz, 10 V, 0.3 ms for 5 s), in the presence of guanethidine (5 μM) and atropine (1 μM) produced frequency‐dependent relaxation of small arteries. Pretreatment with tetrodotoxin (1 μM) abolished the relaxation and desensitization with capsaicin (10 μM) strongly inhibited the relaxation.

Pretreatment with a P2Y‐purinoceptor antagonist, basilen blue (3 μM) or a human calcitonin gene‐related peptide (hCGRP) receptor antagonist, hCGRP8–37 (1 μM) suppressed the NANC relaxation by approximately 40–60 % in each case and combined pretreatment almost abolished the relaxation.

The EFS‐induced relaxation was suppressed by endothelium‐removal, pretreatment with the soluble guanylyl cyclase inhibitor ODQ (1 μM) and the NO scavenger oxyhaemoglobin (OxyHb; 20 μM) but not by NO synthase inhibitors NG‐nitro‐L‐arginine methyl ester (L‐NAME; 300 μM) or NG‐nitro‐L‐arginine (L‐NOARG; 300 μM). Combined pretreatment with ODQ and CGRP8–37 almost abolished the relaxation.

A P2Y‐purinoceptor agonist, 2‐methylthio ATP, produced endothelium‐dependent relaxation which was inhibited by L‐NAME and ODQ (1 μM), whilst hCGRP produced endothelium‐independent and ODQ‐insensitive relaxation.

Ultraviolet light (320 nm, 5 shots over 20 s) produced relaxation that was blocked by both OxyHb and ODQ but not by NG‐monomethyl‐L‐arginine (L‐NMMA, 300 μM).

The present study suggests that EFS‐induced NANC relaxation of the mesenteric small artery of the rabbit is mediated mainly by capsaicin‐sensitive sensory C‐fibres and that both ATP and CGRP are involved. The action of ATP released by EFS appears to be endothelium‐dependent and involve activation of soluble guanylyl cyclase, but is resistant to inhibitors of NO synthase. The response to CGRP is endothelium‐independent. These results show that ATP and CGRP account fully for the NANC relaxation of this vessel type and that the endothelium is involved in NANC‐induced relaxation. The endothelium‐dependent part of the response is consistent with the release of NO, either from NO synthase, incompletely inhibited by the NO synthase inhibitors, or by some preformed stores.

British Journal of Pharmacology (1998) 123, 310–316; doi:10.1038/sj.bjp.0701610
Effect of dietary phenolic compounds on apoptosis of human cultured endothelial cells induced by oxidized LDL
Authors: Vieira Otilia , Escargueil‐Blanc Isabelle , Meilhac Olivier , Basile Jean‐Pierre , Laranjinha Joao , Almeida Leonor , Salvayre Robert , Nègre‐Salvayre Anne
Published: January 2005

Oxidized low density lipoproteins (LDL) are toxic to cultured endothelial cells. Mildly oxidized LDL, characterized by relatively low levels of TBARS and only minor modifications of apoB, were obtained by using 2 experimental model systems of oxidation, namely oxidation by u.v. radiation or ferrylmyoglobin (a two electron oxidation product from the reaction of metmyoglobin with H2O2).

Toxic concentrations of mildly oxidized LDL induce apoptosis (programmed cell death) of cultured endothelial cells, as shown by typical morphological features, by the in situ TUNEL procedure and by DNA fragmentation revealed on gel electrophoresis. This apoptosis is calcium‐dependent and subsequent to the intense and sustained cytosolic [Ca2+]i peak elicited by oxidized LDL.

Five naturally occurring phenolic compounds present in food and beverages were able to prevent, in a concentration‐dependent manner, the apoptosis of endothelial cells induced by oxidized LDL. Among the compounds tested, caffeic acid was the most effective. Under the conditions used, the protective effect of caffeic acid (IC50 8.3±2.1 μmol  l−1) in the prevention of apoptosis induced by oxidized LDL was significantly higher than that of the other compounds tested (IC50s were 12.4±3.2, 14.1±4.1, 20.4±4.4 and 72.6±9.2 μmol  l−1 for ferulic, protocatechuic, ellagic and p‐coumaric acids, respectively).

The anti‐apoptotic effect of caffeic acid results from the addition of two effects, (i) the antioxidant effect which prevents LDL oxidation and subsequent toxicity (‘indirect’ protective effect); (ii) a ‘direct’ cytoprotective effect, acting at the cellular level.

Effective concentrations of caffeic acid acted at the cellular level by blocking the intense and sustained cytosolic [Ca2+]i rise elicited by oxidized LDL.

In conclusion, phenolic acids (caffeic and ferulic acids being the most potent of the compounds tested under the conditions used) exhibit a potent cytoprotective effect of cultured endothelial cells against oxidized LDL. In addition to antioxidant effect delaying LDL oxidation, caffeic acid acts as a cytoprotective agent, probably by blocking the intracellular signalling triggered by oxidized LDL and culminating in the sustained calcium rise which is involved in oxidized LDL‐induced apoptosis.

British Journal of Pharmacology (1998) 123, 565–573; doi:10.1038/sj.bjp.0701624
Pharmacological characterization of the cysteinyl‐leukotriene antagonists CGP 45715A (iralukast) and CGP 57698 in human airways in vitro
Authors: Capra Valérie , Bolla Manlio , Angelo Belloni Pier , Mezzetti Maurizio , Carlo Folco G. , Nicosia Simonetta , Enrico Rovati G.
Published: January 2005

Cysteinyl‐leukotrienes (cysteinyl‐LTs) are important mediators in the pathogenesis of asthma. They cause bronchoconstriction, mucus hypersecretion, increase in microvascular permeability, plasma extravasation and eosinophil recruitment.

We investigated the pharmacological profile of the cysteinyl‐LT antagonists CGP 45715A (iralukast), a structural analogue of LTD4 and CGP 57698, a quinoline type antagonist, in human airways in vitro, by performing binding studies on human lung parenchyma membranes and functional studies on human isolated bronchial strips.

Competition curves vs [3H]‐LTD4 on human lung parenchyma membranes demonstrated that: (a) both antagonists were able to compete for the two sites labelled by [3H]‐LTD4; (b) as in all the G‐protein coupled receptors, iralukast and CGP 57698 did not discriminate between the high and the low affinity states of the CysLT receptor labelled by LTD4 (Ki1=Ki2=16.6 nM±36% CV and Ki1= Ki2=5.7 nM±19% CV, respectively); (c) iralukast, but not CGP 57698, displayed a slow binding kinetic, because preincubation (15 min) increased its antagonist potency.

In functional studies: (a) iralukast and CGP 57698 antagonized LTD4‐induced contraction of human bronchi, with pA2 values of 7.77±4.3% CV and 8.51±1.6% CV, respectively, and slopes not significantly different from unity; (b) the maximal LTD4 response in the presence of CGP 57698 was actually increased, thus clearly deviating from apparent simple competition.

Both antagonists significantly inhibited antigen‐induced contraction of human isolated bronchial strips in a concentration‐dependent manner, lowering the upper plateau of the anti‐IgE curves.

In conclusion, the results of the present in vitro investigation indicate that iralukast and CGP 57698 are potent antagonists of LTD4 in human airways, with affinities in the nanomolar range, similar to those obtained for ICI 204,219 and ONO 1078, two of the most clinically advanced CysLT receptor antagonists. Thus, these compounds might be useful drugs for the therapy of asthma and other allergic diseases.

British Journal of Pharmacology (1998) 123, 590–598; doi:10.1038/sj.bjp.0701636
Altered active but not passive properties of mesenteric resistance arteries from the vitamin E‐deprived rat
Authors: Davidge Sandra T , Gandley Robin E , McLaughlin Margaret K
Published: January 2005

We tested the hypothesis that lowering antioxidant protection through dietary vitamin E deprivation would alter active and passive mechanical properties in resistance arteries of the rat. Specifically, we hypothesized that vascular tone in isolated mesenteric arteries of the vitamin E‐deprived rats would be altered due to impaired endothelial influences of nitric oxide and/or prostaglandins.

Lumen diameter and wall thickness were measured in pressurized arteries (≈amp;250 μm diameter) from control (n=9) and vitamin E deprived (n=9) Sprague‐Dawley female rats by use of a dimension analysing system.

Treatment with a cyclo‐oxygenase inhibitor (meclofenamate) did not affect the basal vascular tone in either group. Treatment with a nitric oxide synthase inhibitor (NG‐methyl‐L‐arginine) caused a significant increase in basal tone only in the vitamin E‐deprived rats (% tone: 6.2±1.1 vs 1.2±0.3%; P<0.05). When tone was induced to 25% of the initial diameter with phenylephrine, treatment with the nitric oxide synthase inhibitor resulted in a greater potentiated tone in the vitamin E‐deprived rats compared to the controls (26.5±2.7 vs 16.4±3.4%; P<0.05); suggesting a greater nitric oxide affect in the vessels from the vitamin E‐deprived rats. Meclofenamate treatment in the induced tone arteries significantly relaxed (−17.4±4.0%; P<0.05) only the arteries from the vitamin E‐deprived rats, indicating that a vasoconstrictor was modifying tone. The passive characteristics of distensibility and stress‐strain relationship were not different between the two groups of rats.

In summary, vitamin E deprivation in the rat enhanced the modulation of vascular tone by both the nitric oxide and cyclo‐oxygenase pathways but did not alter passive characteristics of mesenteric arteries.

British Journal of Pharmacology (1998) 123, 275–280; doi:10.1038/sj.bjp.0701639
A new selective antagonist of the nociceptin receptor
Authors: Guerrini Remo , Calo Girolamo , Rizzi Anna , Bigoni Raffaella , Bianchi Clementina , Salvadori Severo , Regoli Domenico
Published: January 2005

[Phe1Ψ(CH2‐NH)Gly2]NC(1‐13)NH2 has been tested in the electrically stimulated guinea pig ileum and mouse vas deferens, two nociceptin sensitive preparations. The new compound showed per se little or no effect in the two tissues, but it displaced to the right the concentration‐response curves of nociceptin in a concentration‐dependent manner. Schild analyses of the data indicated a competitive type of antagonism and pA2 values of 7.02 and 6.75 in the guinea‐pig ileum and the mouse vas deferens, respectively. At 10 μM [Phe1Ψ(CH2‐NH)Gly2]NC(1‐13)NH2 does not modify either the inhibitory effect of deltorphin I (the selective δ opioid receptor agonist) in the mouse vas deferens or that of dermorphine (the selective μ opioid receptor agonist) in the guinea‐pig ileum. The present findings indicate that [Phe1Ψ(CH2‐NH)Gly2]NC(1‐13)NH2 is a selective antagonist of the nociceptin receptor.

British Journal of Pharmacology (1998) 123, 163–165; doi:10.1038/sj.bjp.0701640
Effects of pyrethroid molecules on rat nerves in vitro: potential to reverse temperature‐sensitive conduction block of demyelinated peripheral axons
Authors: Lees George
Published: January 2005

Prolongation of action potentials by cooling or pharmacological treatment can restore conduction in demyelinated axons. We have assessed the ability of pyrethroids (in vitro) to modify action potential kinetics and to reverse conduction block in lesioned peripheral nerve.

Fast Na+ currents were isolated in mammalian neuroblastoma (NIE115). Pyrethroids (4 μM) concurrently slowed inactivation and produced a spectrum of pronounced tail currents: s‐bioallethrin (duration 12.2±7 ms), permethrin (24.2±3 ms) and deltamethrin (2230±100 ms).

Deltamethrin (5 μM) effected a slowly developing depression of compound action potential (CAP) amplitude in peroneal nerve trunks (P<0.05). Permethrin produced no net effect on CAP amplitude, area or repolarization time.

s‐Bioallethrin (5 μM) enhanced CAP area, time for 90% repolarization and induced regenerative activity in a subpopulation of axons.

Tibial nerve trunks were demyelinated by lysolecithin (2 μl) injection: 6–14 days later, slowly‐conducting axons in the CAP (and peri‐axonal microelectrode recordings) were selectively blocked by warming to 37°C.

At 37°C, s‐bioallethrin (45 min, 5 μM) produced much greater after‐potentials in lesioned nerves than in uninjected controls: area (P<0.05) and relative amplitude ratios (P<0.0001) were significantly altered.

In 3 of 4 cells (single‐unit recording), s‐bioallethrin restored conduction through axons exhibiting temperature‐dependent block by raising blocking temperature (by 1.5 to >3°C) and reducing refractory period.

s‐Bioallethrin induced temperature‐dependent regenerative activity only in a sub‐population of axons even after prolonged superfusion (>1 h).

It was concluded that pyrethroids differentially alter Na+ current kinetics and action potential kinetics. The effects of s‐bioallethrin are consistent with reversal of conduction block by demyelinated axons but regenerative/ectopic firing even in normal cells is likely to underpin its acknowledged neurotoxic actions and severely limit the clinical potential of this and related molecules.

British Journal of Pharmacology (1998) 123, 487–496; doi:10.1038/sj.bjp.0701644
Structural basis for specificity and potency of xanthine derivatives as activators of the CFTR chloride channel
Authors: Chappe V. , Mettey Y. , Vierfond J M. , Hanrahan J W. , Gola M. , Verrier B. , Becq F.
Published: January 2005

On the basis of their structure, we compared the ability of 35 xanthine derivatives to activate the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel stably expressed in chinese hamster ovary (CHO) cells using the cell‐attached patch clamp and iodide efflux techniques.

Activation of CFTR channels was obtained with 3‐mono, 1,3‐di or 1,3,7‐tri‐substituted alkyl xanthine derivatives (enprofylline, theophylline, aminophylline, IBMX, DPMX and pentoxifylline). By contrast, xanthine derivatives substituted at the C8‐ or N9‐position failed to open CFTR channels.

The CFTR chloride channel activity was blocked by glibenclamide (100 μM) but not by DIDS (100 μM).

Activation of CFTR by xanthines was not mimicked by the calcium ionophore A23187, adenosine, UTP, ATP or the specific phosphodiesterase inhibitors rolipram, Ro 20‐1724 and milrinone. In addition, we found no correlation between the effect of xanthines on CFTR and on the cellular cyclic AMP or ATP levels.

We then synthesized a series of 3,7‐dimethyl‐1‐alkyl xanthine derivatives; among them, 3,7‐dimethyl‐1‐propyl xanthine and 3,7‐dimethyl‐1‐isobutyl xanthine both activated CFTR channels without increasing the intracellular cyclic AMP level, while the structurally related 3,7‐dimethyl‐1‐(2‐propenyl) xanthine and 3,7‐dimethyl‐1‐(oxiranyl methyl) xanthine were inactive.

Our findings delineate a novel function for xanthine compounds and identify the molecular features that enable xanthine activation of CFTR. These results may be useful in the development of new molecules for studying the pharmacology of chloride channels.

British Journal of Pharmacology (1998) 123, 683–693; doi:10.1038/sj.bjp.0701648
Relaxant effects of NKH477, a new water‐soluble forskolin derivative, on guinea‐pig tracheal smooth muscle: the role of Ca2+‐activated K+ channels
Authors: Satake K. , Takagi K. , Kodama I. , Honjo H. , Toyama J. , Shibata S.
Published: January 2005

Mechanisms underlying the bronchorelaxant action of NKH477, a newly developed water‐soluble forskolin derivative, were investigated in guinea‐pig isolated tracheal smooth muscle.

In muscles precontracted with 3 μM histamine, NKH477 (1 nM–1 μM) caused a concentration‐dependent decrease of isometric tension, resulting in a complete relaxation at 300 nM. The EC50 for the relaxation was 32.6±4.3 nM (n=6).

In the presence of 30 or 90 nM iberiotoxin (IbTX), a selective blocker of the large‐conductance Ca2+‐activated K+ (BKCa) channel, the relaxing action of NKH477 on the histamine‐induced contraction was inhibited, giving rise to a parallel shift of the concentration‐response curves; the EC50 of NKH477 was increased to 131.4±20.4 nM at 30 nM IbTX (n=4), and 125.3±12.2 nM at 90 nM IbTX (n=4).

Pretreatment of muscles with 30 mM tetraethylammonium (TEA) caused a similar rightward shift of the concentration‐response curve to NKH477 with an increase of the EC50 to 139.8±18.4 nM (n=5). In contrast, the relaxing action of NKH477 was unaffected by 10 μM glibenclamide, an ATP‐sensitive K+ channel blocker, or by 100 nM apamin, a blocker of small conductance Ca2+‐activated K+ channels.

In muscles pretreated with 1 μM nifedipine, a blocker of the voltage‐dependent Ca2+ channel (VDC), 30–90 nM IbTX did not affect the relaxant effects of NKH477 on the histamine‐induced contraction.

In muscles precontracted by a K+‐rich (40 mM) solution, NKH477 caused only minimal relaxation (19.8±1.7%, n=4) even at the highest concentration (1 μM).

In experiments to measure the ratio of fura‐2 fluorescence signals (R340/380) as an index of the intracellular Ca2+ concentration ([Ca2+]i), the application of 100 nM NKH477 or 200 nM isoprenaline to the preparation precontracted by 3 μM histamine resulted in a decrease in [Ca2+]i in association with a decrease in tension. The reduction of [Ca2+]i and tension by NKH477 was 47.0±5.6% and 62.8±7.0%, respectively (n=5), and that with isoprenaline 60.6±7.4% and 67.4±6.4%, respectively (n=5). These effects of NKH477 and isoprenaline on [Ca2+]i and tension were inhibited by 30 nM IbTX. The inhibitory action of IbTX was abolished in the presence of 1 μM nifedipine.

These results suggest that the bronchorelaxant action of NKH477 may result, at least in part, from activation of BKCa channels, which may cause a hyperpolarization of smooth muscle cell membranes and a secondary decrease in Ca2+ influx through VDCs, leading to a decrease in [Ca2+]i.

British Journal of Pharmacology (1998) 123, 753–761; doi:10.1038/sj.bjp.0701655
Comparison of relaxin receptors in rat isolated atria and uterus by use of synthetic and native relaxin analogues
Authors: Tan Y Y. , Wade J D. , Tregear G W. , Summers R J.
Published: January 2005

The receptors for relaxin in the rat atria and uterus were investigated and compared by use of a series of synthetic and native relaxin analogues. The assays used were the positive chronotropic and inotropic effects in rat spontaneously beating, isolated right atrium and electrically driven left atrium and the relaxation of K+ precontracted uterine smooth muscle.

Relaxin analogues with an intact A‐ and B‐chain were active in producing powerful chronotropic and inotropic effects in the rat isolated atria at nanomolar concentrations. Single‐chain analogues and structural homologues of relaxin such as human insulin and sheep insulin‐like growth factor I had no agonist action and did not antagonize the effect of the B29 form of human gene 2 relaxin.

Shortening the B‐chain carboxyl terminal of human gene 1 (B2–29) relaxin to B2–26 reduced the activity of the peptide and removal of another 2 amino acid residues (B2–24) abolished the activity. This suggests that the B‐chain length may be important for determination of the activity of relaxin. More detailed studies are needed to determine the effect of progressive amino acid removal on the structure and the bioactivity of relaxin.

Porcine prorelaxin was as active as porcine relaxin on a molar basis, suggesting that the presence of the intact C‐peptide did not affect the binding of the prorelaxin to the receptor to produce functional responses.

Relaxin caused relaxation of uterine longitudinal and circular smooth muscle precontracted with 40 mM K+. The pEC50 values for human gene 2 and porcine relaxins were lower than those in the atrial assay, but rat relaxin had similar pEC50 values in both atrial and uterine assays. Rat relaxin was significantly less potent than either human gene 2 or porcine relaxin in the atrial assay, but in the uterine assay they were equipotent. The results suggest that the relaxin receptor or the signalling pathway in rat atria may differ from that in the uterus.

British Journal of Pharmacology (1998) 123, 762–770; doi:10.1038/sj.bjp.0701659
Stimulation of nitric oxide release from rat spinal cord by prostaglandin E2
Authors: Sakai Masato , Minami Toshiaki , Hara Naoki , Nishihara Isao , Kitade Hiroaki , Kamiyama Yasuo , Okuda Kazuyuki , Takahashi Hakuo , Mori Hidemaro , Ito Seiji
Published: January 2005

We recently demonstrated that intrathecal administration of prostaglandin E2 (PGE2) and PGF2α induced allodynia through a pathway that includes the glutamate receptor and nitric oxide (NO)‐generating systems from pharmacological studies. In order to clarify the involvement of NO in prostaglandin‐induced allodynia, we measured NO released from rat spinal cord slices by a chemiluminescence method.

PGE2 stimulated NO release from both dorsal and ventral regions all along the spinal cord. PGE2 stimulated the release within 10 min and increased it in a time‐dependent manner.

The PGE2‐induced NO release was observed at 100 nM–10 μM. PGF2α stimulated the release at concentrations higher than 1 μM, but PGD2 (up to 10 μM) did not enhance it.

17‐Phenyl‐ω‐trinor PGE2 (EP1>EP3) and sulprostone (EP1<EP3) were as potent as PGE2, but PGE1 was less potent, in stimulating NO release. While M&B 28767 (EP3) did not enhance the release, butaprost (EP2) stimulated it at 1 μM. The PGE2‐evoked release was blocked by ONO‐NT‐012, a bifunctional EP1 antagonist/EP3 agonist.

The PGE2‐evoked release was Ca2+‐dependent and blocked by MK‐801 (NMDA receptor antagonist) and L‐NAME (NO synthase inhibitor). The release was also inhibited by PGD2 and dibutyryl‐cyclic AMP.

The present study demonstrated that PGE2 stimulates NO release in the rat spinal cord by activation of NMDA receptors through the EP1 receptor, and supports our previous findings that the NO‐generating system is involved in the PGE2‐induced allodynia.

British Journal of Pharmacology (1998) 123, 890–894; doi:10.1038/sj.bjp.0701661
Suppression of nitric oxide formation by tyrosine kinase inhibitors in murine N9 microglia
Authors: Lockhart B P. , Cressey K C. , Lepagnol J M.
Published: January 2005

Microglial cells represent the first line of defence in the brain against infection and damage. However, under conditions of chronic inflammation and neurodegeneration, excessive activation of microglia can contribute to the neurodegenerative process by releasing a cornucopia of potentially cytotoxic substances including the cytotoxic free radical nitric oxide (NO). Although the cell signalling events implicated in NO formation in peripheral macrophages are well defined, events occurring in the phenotypically homologous cerebral microglial cell are not yet fully characterized.

In the present study, a cloned murine microglial cell line (N9), stimulated with combined lipopolysaccharide/interferon‐γ (LPS/IFN) incubation, was shown to produce a significant increase in NO formation, as measured by medium nitrite levels, during 8–72 h exposure.

LPS/IFN‐stimulated NO production was partially inhibited with the nitric oxide synthase (NOS) competitive antagonists; Nω‐nitro‐L‐arginine methyl ester and Nω‐nitro‐L‐arginine. The ability of the selective inducible (iNOS) inhibitor, aminoguanidine, but not the selective ‘neuronal‐type’ constitutive (cNOS) inhibitor 7‐nitroindazole, to inhibit NO production suggested a primary role of iNOS in this response and was confirmed by immunolabelling of activated cells with a specific iNOS antibody.

A series of tyrosine kinase inhibitors, herbimycin A, genestein, tyrphostins, AG‐126, AG‐556 and the tyrosine phosphatase inhibitors, sodium orthovanadate and phenylarsine oxide, significantly attenuated LPS/IFN‐mediated NO production. The serine/threonine kinase inhibitors, staursporine (protein kinase C), H‐9 (cyclic GMP/cyclic AMP‐dependent kinase) or serine/threonine phosphatase inhibitors, cyclosporin A (phosphatase 2B) and okadaic acid (phosphatase 1/2A), reduced NO formation by an apparent cytostatic mechanism, as determined by cellular reduction of 3‐(4,5‐dimethylthiazol‐2‐yi)‐2,5‐diphenyl‐tetrazolium bromide (MTT).

The present results suggest that the co‐ordinated activation of protein tyrosine kinases/phosphatases, and proximal signalling events implicating the interplay between serine‐threonine kinases/phosphatases, is intricately linked with inflammatory mediated mechanisms of iNOS activation in microglial cells by regulating the activation of the transcription factor NFκB.

British Journal of Pharmacology (1998) 123, 879–889; doi:10.1038/sj.bjp.0701664
Inhibitory action of insulin‐sensitizing agents on calcium channels in smooth muscle cells from resistance arteries of guinea‐pig
Authors: Nakamura Yoshito , Ohya Yusuke , Onaka Uran , Fujii Koji , Abe Isao , Fujishima Masatoshi
Published: January 2005

The actions of troglitazone, pioglitazone, metformin and bezafibrate, agents that improve insulin‐resistance, on voltage‐dependent Ca2+ channels in arterial smooth muscle cells were examined by use of the conventional and nystatin‐perforated whole‐cell clamp methods. Single cells were freshly isolated from resistance mesenteric arteries of guinea‐pigs. The actions of these agents on 77 mM K+‐induced contraction of the isolated arteries were also examined with the use of isometric tension recording.

The thiazolidinedione derivatives, troglitazone and pioglitazone, inhibited whole‐cell Ca2+ currents in a dose‐dependent manner with dissociation constants of 3.0 μM and 44.9 μM and Hill coefficients of 0.61 and 0.68, respectively. These two agents inhibited the 77 mM K+‐induced contraction with similar potencies as those inhibiting the Ca2+ currents. Metformin and bezafibrate had no apparent effects on the Ca2+ current or high K+‐induced contraction.

The inhibitory action of troglitazone on Ca2+ currents was not affected by the command potential, the holding potential, or the stimulation frequency, suggesting that its mode of the action differs from that of known organic Ca2+ channel antagonists.

The inhibitory action of troglitazone on Ca2+ currents was not affected by the addition of insulin to, or the removal of glucose from, the solutions.

In conclusion, the thiazolidinedione derivatives directly inhibited the voltage‐dependent Ca2+ channels in a different manner from that of organic Ca2+ channel antagonists. This inhibitory action on Ca2+ channels was not a common feature of insulin‐sensitizing agents.

British Journal of Pharmacology (1998) 123, 675–682; doi:10.1038/sj.bjp.0701669
Inhibition of glycosphingolipid synthesis by threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP) and the modulation of IL‐1β‐stimulated expression of inducible nitric oxide synthase in rat aortic smooth muscle cells
Authors: Weber Artur‐Aron , Seul Claudia , Harth Volker , Ko Yon , Seewald Stefan , Vetter Hans , Sachinidis Agapios
Published: January 2005

The composition of glycosphingolipids is altered in atherosclerotic tissue. In order to study the possible modulation of interleukin‐1β (IL‐1β)‐induced expression of inducible nitric oxide synthase (iNOS) by endogenously synthesized glycosphingolipids, we investigated rat aortic vascular smooth muscle cells (VSMC) grown in the presence of the inhibitor of glycosphingolipid synthesis, threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP).

Depletion of glycosphingolipids by PDMP (20–30 μM) was demonstrated by thin‐layer chromatography of D‐[1−14C]‐galactose‐ or L‐[‐U14C]‐serine‐labelled glycosphingolipids. Nitrite generation was measured by the diaminonaphthalene assay, nitric oxide was determined by the oxyhaemoglobin technique and iNOS protein was detected by immunocytochemistry.

In VSMC grown in the presence of PDMP, the glycosphingolipid content was reduced by 30–50%. In PDMP‐treated VSMC, IL‐1β (3 u ml−1)‐stimulated release of nitrite (135±4 nmol mg−1 protein 48 h−1) was significantly increased as compared to IL‐1β‐stimulated control cells (40±3 nmol mg−1 protein 48 h−1; n=6, P<0.001). Similarly, IL‐1β (3 u ml−1, 36 h)‐stimulated release of nitric oxide was higher in PDMP‐treated VSMC (6.1±0.5 nmol mg−1 protein h−1) as compared to untreated cells (2.0±0.6 nmol mg−1 protein h−1; n=3, P<0.01). These findings were confirmed by the demonstration of increased expression of iNOS protein (14.9±1.2% vs 6.4±0.2%; n=4, P<0.001), as shown by immunocytochemistry.

Evidence is presented that endogenous glycosphingolipids are important modulators of cytokine‐induced iNOS expression. In view of an altered glycosphingolipid profile in atherosclerotic arteries, these mechanisms might be of relevance for the pathogenesis of atherosclerosis and restenosis subsequent to vessel injury.

British Journal of Pharmacology (1998) 123, 906–910; doi:10.1038/sj.bjp.0701674
Histamine‐induced biphasic macromolecular leakage in the microcirculation of the conscious hamster: evidence for a delayed nitric oxide‐dependent leakage
Authors: Gimeno G , Carpentier P H. , Desquand‐Billiald S , Hanf R , Finet M
Published: January 2005

Late effects (up to 3 h) of intravenously‐injected histamine on FITC‐dextran extravasation were investigated in the conscious hamster, by use of computer‐assisted image analysis of fluorescence distribution in a microscopic window of dorsal skin fold preparations. This analysis allowed measurement of local (skin) and general (all organs) extravasations caused by a bolus injection of histamine (1 mg kg−1, i.v.)

Histamine doses higher than 0.01 mg kg−1 caused biphasic local and general extravasations. Initial phases developed fully within 15 min (for local) and 60 min (for general) and were followed by late phases beginning 90 min after histamine injection. Although the initial and late phases of histamine‐induced extravasations had differential apparent reactivities to the autacoid, all the effects of histamine on the microcirculation (1 mg kg−1) were inhibited by pyrilamine (1 mg kg−1, i.v.) but not by cimetidine (1 mg kg−1, i.v.).

Pretreatment with NG‐monomethyl‐L‐arginine (L‐NMMA, 30 mg kg−1, i.v.) or NG‐nitro‐L‐arginine methyl ester (L‐NAME, 100 mg kg−1, i.v.) did not affect the initial phases but did prevent the late phases of local and general extravasations triggered by 1 mg kg−1 histamine. The inhibitory effects of L‐NAME were reversed by L‐arginine (30 mg kg−1) but not by D‐arginine (30 mg kg−1) according to the enantioselectivity of nitric oxide synthase (NOS). A late NO‐mediated venular dilatation occurred in response to plasma histamine.

A low dose of aminoguanidine (1 mg kg−1, i.v.), a selective inhibitor of the inducible isoform of NOS (iNOS), mimicked the inhibitory effects of L‐NAME on the late phases of histamine‐induced macromolecular extravasations and venular dilatation.

Pretreatment with dexamethasone (1 mg kg−1, i.v.) prevented both the initial and late phases of histamine‐induced extravasations. Fucoidan (1 or 25 mg kg−1, i.v.) prevented the late phases without affecting initial phases, consistent with a role for leukocytes adhesion in the development of the late NO‐mediated effects of histamine.

We conclude that intravenous injection of histamine triggers a biphasic inflammatory cascade via initial activation of H1 receptors which induces a late NO‐mediated PMN‐dependent extravasation process.

British Journal of Pharmacology (1998) 123, 943–951; doi:10.1038/sj.bjp.0701676