bluuebunny/crossref_metadata_2025 huggingface dataset:
Paired pulse analysis of ATP and noradrenaline release from sympathetic nerves of rat tail artery and mouse vas deferens: effects of K+ channel blockers
Authors: Msghina Mussie , Gonon Francois , Stjärne Lennart
Published: January 2005

The paired pulse stimulus paradigm – two pulses of equal strength delivered at variable interpulse intervals – was used to study the release of ATP and noradrenaline (NA) from post ganglionic sympathetic nerves of rat tail artery and mouse vas deferens.

Excitatory junction currents (EJCs) were used to measure the release of ATP, and differential pulse amperometry to measure that of NA.

At interpulse intervals of 0.1–1 s paired pulse stimulation caused an increase in the size of the second EJC, both in rat tail artery and mouse vas deferens. As the interpulse interval was increased to 10 s or more, the two EJCs became of equal size.

In both preparations the K+ channel blockers tetraethylammonium (TEA, 20 mM) and 4‐aminopyridine (4‐AP, 1 mM) prolonged the duration of the nerve terminal spike and greatly amplified the first EJC of the pair.

In the presence of TEA and 4‐AP in rat tail artery paired pulse stimulation caused a dramatic depression of the second EJC without markedly affecting the nerve terminal spike. The depression of the second EJC decreased with increasing interpulse intervals, and also when external Ca2+ was reduced to 0.2 mM. In mouse vas deferens, TEA and 4‐AP caused only a modest depression of the second EJC.

In rat tail artery in the presence of TEA and 4‐AP paired pulse stimulation caused a depression of the NA oxidation current evoked by the second pulse, which was similar in magnitude and time course to that of the EJC. Similar TEA and 4‐AP induced depression of the second pulse response was also observed when the purinergic and noradrenergic components of the contractile response were investigated.

The results show that in rat tail artery K+ channel blockers cause a dramatic paired pulse depression of the release of ATP and NA. The similarity in the depression of the EJC, the NA oxidation current, and the purinergic and noradrenergic components of the contractile response is compatible with the hypothesis that ATP and NA are released in parallel from the same neuronal sources.
Release of immunoreactive substance P in the brain stem upon stimulation of the cranial dura mater with low pH – inhibition by the serotonin (5‐HT1) receptor agonist CP 93,129
Authors: Messlinger Karl , Ebersberger Andrea , Schaible Hans‐Georg
Published: January 2005

The therapeutical benefit of serotonin (5‐HT1) receptor agonists in the treatment of migraine headache has been attributed to their inhibitory effect on the release of pro‐inflammatory neuropeptides from trigeminal afferents within the cranial meninges. The effect of 5‐HT1 receptor agonists on the release of neuropeptides from central afferent terminals has not been examined so far. In the present study in the rat we therefore measured the effect of the 5‐HT1B receptor agonist CP 93,129 on the stimulation‐evoked release of immunoreactive substance P (ir‐SP) in the spinal trigeminal nucleus.

To measure release of ir‐SP, microprobes coated with antibody to substance P were inserted into the medulla oblongata at the level of the obex. The ipsilateral parietal dura mater encephali was exposed and stimulated with acid phosphate buffered Tyrode solution (pH 5.8). This chemical stimulus increased the release of ir‐SP in the medullary dorsal horn.

Systemic (i.v.) administration of CP 93,129 (460 nmol kg−1) prior to stimulation suppressed the stimulation‐evoked increase of release of ir‐SP. Local administration of CP 93,129 (10 μM) to the dorsal surface of the medulla had no significant inhibitory effect on the release.

It is concluded that systemically applied 5‐HT1 receptor agonists reduce the stimulation‐evoked release of substance P from the central endings of meningeal afferents in the spinal trigeminal nucleus (medullary dorsal horn). This inhibitory effect may contribute to the antinociceptive effect of 5‐HT1 receptor agonists in migraine.
JTT‐501, a novel oral antidiabetic agent, improves insulin resistance in genetic and non‐genetic insulin‐resistant models
Authors: Shibata Tsutomu , Matsui Kenichi , Yonemori Fumihiko , Wakitani Korekiyo
Published: January 2005

We investigated whether JTT‐501 (4‐[4‐[2‐(5‐methyl‐2‐phenyl‐4‐oxazolyl)ethoxy]benzyl]‐3,5‐isoxazolidinedione) would improve insulin resistance in genetic (Zucker fatty rats) and non‐genetic (high‐fat fed rats) rodent models of obesity.

JTT‐501 (10–100 mg kg−1 day−1) was administered orally to Zucker fatty rats for 7–21 days. In the high‐fat fed rat model, JTT‐501 (100 mg kg−1 day−1) was administered orally for 7 days. In both models, JTT‐501 improved metabolic abnormalities by enhancing insulin action during the glucose tolerance test and the euglycaemic‐hyperinsulinaemic clamp study. In ex vivo assays, JTT‐501 ameliorated the impaired insulin‐sensitive glucose oxidation and lipid synthesis in peripheral tissues. Furthermore, JTT‐501 enhanced insulin receptor autophosphorylation in hindlimb muscle.

JTT‐501 reduced serum leptin concentrations in both models, but did not affect body weight or epididymal fat weight.

Our observations indicate that JTT‐501 improves the metabolic abnormalities in both genetic and non‐genetic insulin‐resistant models by enhancing insulin action in peripheral tissues. These effects of JTT‐501 are due, at least in part, to enhanced insulin receptor autophosphorylation. In addition, JTT‐501 is able to reduce serum leptin concentrations in hyperleptinaemia of the insulin‐resistant model. We expect JTT‐501 to show promise for treating non‐insulin dependent diabetes mellitus patients with insulin resistance.
Inhibition of endothelium‐dependent hyperpolarization by endothelial prostanoids in guinea‐pig coronary artery
Authors: Yajima Kazuhiro , Nishiyama Makoto , Yamamoto Yoshimichi , Suzuki Hikaru
Published: January 2005

In smooth muscle of the circumflex coronary artery of guinea‐pig, acetylcholine (ACh, 10−6 M) produced an endothelium‐dependent hyperpolarization consisting of two components. An initial component that occurs in the presence of ACh and a slow component that developed after ACh had been withdrawn. Each component of the hyperpolarization was accompanied by an increase in membrane conductance.

Indomethacin (5×10−6 M) or diclofenac (10−6 M), both inhibitors of cyclooxygenase, abolished only the slow hyperpolarization. The initial hyperpolarization was not inhibited by diclofenac nor by nitroarginine, an inhibitor of nitric oxide synthase.

Both components of the ACh‐induced hyperpolarization were abolished in the presence of atropine (10−6 M) or high‐K solution ([K+]0=29.4 mM).

The interval between ACh‐stimulation required to generate an initial hyperpolarization of reproducible amplitude was 20 min or greater, but it was reduced to less than 5 min after inhibiting cyclooxygenase activity. Conditioning stimulation of the artery with substance P (10−7 M) also caused a long duration (about 20 min) inhibition of the ACh‐response.

The amplitude of the hyperpolarization generated by Y‐26763, a K+‐channel opener, was reproducible within 10 min after withdrawal of ACh.

Exogenously applied prostacyclin (PGI2) hyperpolarized the membrane and reduced membrane resistance in concentrations over 2.8×10−9M.

At concentrations below threshold for hyperpolarization and when no alteration of membrane resistance occurred, PGI2 inhibited the initial component of the ACh‐induced hyperpolarization.

It is concluded that endothelial prostanoids, possibly PGI2, have an inhibitory action on the release of endothelium‐derived hyperpolarizing factor.

British Journal of Pharmacology (1999) 126, 1–10; doi:10.1038/sj.bjp.0702254
Stimulation of locus coeruleus neurons by non‐I1/I2‐type imidazoline receptors: an in vivo and in vitro electrophysiological study
Authors: Ugedo Luisa , Pineda Joseba , Ruiz‐Ortega José A. , Martín‐Ruiz Raúl
Published: January 2005

Imidazoline binding sites have been reported to be present in the locus coeruleus (LC). To investigate the role of these sites in the control of LC neuron activity, we studied the effect of imidazolines using in vivo and in vitro single‐unit extracellular recording techniques.

In anaesthetized rats, local (27 pmoles) and systemic (1 mg kg−1, i.v.) administrations of 2‐(2‐benzofuranyl)‐2‐imidazoline (2‐BFI), a selective I‐imidazoline receptor ligand, increased the firing rate of LC cells (maximal increase: 22 ± 5%, P<0.001 and 16 ± 7%, P<0.001 respectively). Chronic pretreatment with the irreversible monoamine oxidase inhibitor clorgyline (3 mg kg−1, i.p., every 12 h for 14 days) abolished this effect.

In rat midpontine brain slices containing the LC, bath application (1 mM) of the imidazolines 2‐BFI, 2‐(4,5‐dihydroimidaz‐2‐yl)‐quinoline (BU224), idazoxan, efaroxan, phentolamine and (2‐2‐methoxy‐1,4‐benzodioxan‐2‐yl)‐2‐imidazoline (RX821002) reversibly stimulated LC cells. The maximal effect was ∼90% except for RX821002 and efaroxan which induced smaller maximal effects (∼58% and ∼35% respectively). Simultaneous application of idazoxan and 2BFI did not lead to additive effects.

Bath application of the α2‐adrenoceptor antagonists, yohimbine (1–10 μM) and N‐ethoxycarbonyl‐1,2‐dihydroquinoline (EEDQ) (10 μM), failed to modify LC activity. The irreversible blockade of α2‐adrenoceptors with EEDQ (10 μM) did not alter the effect of idazoxan or that of efaroxan. Previous application of clorgyline (10 μM) did not modify the excitatory effect of 2‐BFI or efaroxan.

Changes in the pH of the bathing solution (6.84–7.84) did not influence the effect caused by idazoxan. Bath application of 2‐BFI (1 mM) reversed the inhibition induced by diazoxide (300 μM), an ATP‐sensitive K+ channel opener, whereas application of glibenclamide (3 μM), an ATP‐sensitive K+ channel blocker, partially blocked the effect of 2‐BFI.

This study shows that imidazoline compounds stimulate the firing rate of LC neurons. This effect is not mediated by α2‐adrenoceptors nor by I1 or I2‐imidazoline receptors but involves a different subtype of imidazoline receptor. Our results indicate that this receptor is located extracellularly and modulates ATP‐sensitive K+ channels.
Effects of Ca2+ concentration and Ca2+ channel blockers on noradrenaline release and purinergic neuroeffector transmission in rat tail artery
Authors: Brock James A , Cunnane Thomas C
Published: January 2005

The effects of Ca2+ concentration and Ca2+ channel blockers on noradrenaline (NA) and adenosine 5′‐triphosphate (ATP) release from postganglionic sympathetic nerves have been investigated in rat tail arteries in vitro. Intracellularly recorded excitatory junction potentials (e.j.ps) were used as a measure of ATP release and continuous amperometry was used to measure NA release.

Varying the extracellular Ca2+ concentration similarly affected the amplitudes of e.j.ps and NA‐induced oxidation currents evoked by trains of ten stimuli at 1 Hz.

The N‐type Ca2+ blocker, ω‐conotoxin GVIA (ω‐CTX GVIA, 0.1 μM) reduced the amplitudes of both e.j.ps (evoked by trains of ten stimuli at 1 Hz) and NA‐induced oxidation currents (evoked by trains of ten stimuli at 1 Hz and 50 stimuli at 10 Hz) by about 90%.

The ω‐CTX GVIA resistant e.j.ps and NA‐induced oxidation currents evoked by trains of 50 stimuli at 10 Hz were abolished by the non‐selective Ca2+ channel blocker, Cd2+ (0.1 mM), and were reduced by ω‐conotoxin MVIIC (0.5 μM) and ω‐agatoxin IVA (40 nM).

Nifedipine (10 μM) had no inhibitory effect on ω‐CTX GVIA resistant e.j.ps and NA‐induced oxidation currents.

Thus both varying Ca2+ concentration and applying Ca2+ channel blockers results in similar effects on NA and ATP release from postganglionic sympathetic nerves. These findings are consistent with the hypothesis that NA and ATP are co‐released together from the sympathetic nerve terminals.

British Journal of Pharmacology (1999) 126, 11–18; doi:10.1038/sj.bjp.0702256
NMDA receptor activation modulates evoked release of substance P from rat spinal cord
Authors: Malcangio Marzia , Fernandes Karin , Tomlinson David R.
Published: January 2005

The possible modulation exerted by glutamate on substance P (SP) release from the rat spinal cord has been investigated. The N‐methyl‐D‐aspartate (NMDA) receptor agonist, NMDA (1 μM), increased SP basal outflow by 46.5 ± 10.9% (n=3, P<0.01) without changing the evoked release of the peptide. Conversely, NMDA antagonists but not 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) inhibited both electrically‐evoked and capsaicin‐induced release of SP. In particular, D‐2‐amino‐5‐phosphonopentanoate (D‐AP5; 50 μM) inhibited electrically‐evoked and capsaicin‐induced release of SP by 93 ± 2.4% and 93.2 ± 3.8% (n=12, P<0.01), respectively. Functional pharmacological evidence is provided for glutamate exerting a positive feedback on SP release evoked by C fibre stimulation via NMDA receptor activation.
Acetylcholine‐induced membrane potential changes in endothelial cells of rabbit aortic valve
Authors: Ohashi Masuo , Satoh Kohichi , Itoh Takeo
Published: January 2005

Using a microelectrode technique, acetylcholine (ACh)‐induced membrane potential changes were characterized using various types of inhibitors of K+ and Cl− channels in rabbit aortic valve endothelial cells (RAVEC).

ACh produced transient then sustained membrane hyperpolarizations. Withdrawal of ACh evoked a transient depolarization.

High K+ blocked and low K+ potentiated the two ACh‐induced hyperpolarizations. Charybdotoxin (ChTX) attenuated the ACh‐induced transient and sustained hyperpolarizations; apamin inhibited only the sustained hyperpolarization. In the combined presence of ChTX and apamin, ACh produced a depolarization.

In Ca2+‐free solution or in the presence of Co2+ or Ni2+, ACh produced a transient hyperpolarization followed by a depolarization. In BAPTA‐AM‐treated cells, ACh produced only a depolarization.

A low concentration of A23187 attenuated the ACh‐induced transient, but not the sustained, hyperpolarization. In the presence of cyclopiazonic acid, the hyperpolarization induced by ACh was maintained after ACh removal; this maintained hyperpolarization was blocked by Co2+.

Both NPPB and hypertonic solution inhibited the membrane depolarization seen after ACh washout. Bumetanide also attenuated this depolarization.

It is concluded that in RAVEC, ACh produces a two‐component hyperpolarization followed by a depolarization. It is suggested that ACh‐induced Ca2+ release from the storage sites causes a transient hyperpolarization due to activation of ChTX‐sensitive K+ channels and that ACh‐activated Ca2+ influx causes a sustained hyperpolarization by activating both ChTX‐ and apamin‐sensitive K+ channels. Both volume‐sensitive Cl− channels and the Na+‐K+‐Cl− cotransporter probably contribute to the ACh‐induced depolarization.

British Journal of Pharmacology (1999) 126, 19–26; doi:10.1038/sj.bjp.0702262
Long term orexigenic effect of a novel melanocortin 4 receptor selective antagonist
Authors: Skuladottir Gudrun V , Jonsson Logi , Skarphedinsson Jon O , Mutulis Felikss , Muceniece Ruta , Raine Amanda , Mutule Ilze , Helgason Johannes , Prusis Peteris , Wikberg Jarl E S , Schiöth Helgi B
Published: January 2005

We designed and synthesized several novel cyclic MSH analogues and tested their affinities for cells expressing the MC1, MC3, MC4 and MC5 receptors.

One of the substances HS028 (cyclic [AcCys11, dichloro‐D‐phenylalanine14, Cys18, Asp‐NH222]‐β‐MSH11–22) showed high affinity (Ki of 0.95nM) and high (80 fold) MC4 receptor selectivity over the MC3 receptor. HS028 thus shows both higher affinity and higher selectivity for the MC4 receptor compared to the earlier first described MC4 receptor selective substance HS014.

HS028 antagonised a α‐MSH induced increase in cyclic AMP production in transfected cells expressing the MC3 and MC4 receptors, whereas it seemed to be a partial agonist for the MC1 and MC5 receptors.

Chronic intracerebroventricularly (i.c.v.) administration of HS028 by osmotic minipumps significantly increased both food intake and body weight in a dose dependent manner without tachyphylaxis for a period of 7 days.

This is the first report demonstrating that an MC4 receptor antagonist can increase food intake and body weight during chronic administration providing further evidence that the MC4 receptor is an important mediator of long term weight homeostasis.

British Journal of Pharmacology (1999) 126, 27–34; doi:10.1038/sj.bjp.0702264
Cytokine‐mediated inflammatory hyperalgesia limited by interleukin‐4
Authors: Cunha F Q , Poole S , Lorenzetti B B , Veiga F H , Ferreira S H
Published: January 2005

The effect of IL‐4 on responses to intraplantar (i.pl.) carrageenin, bradykinin, TNFα, IL‐1β, IL‐8 and PGE2 was investigated in a model of mechanical hyperalgesia in rats. Also, the cellular source of the IL‐4 was investigated.

IL‐4, 30 min before the stimulus, inhibited responses to carrageenin, bradykinin, and TNFα, but not responses to IL‐1β, IL‐8 and PGE2.

IL‐4, 2 h before the injection of IL‐1β, did not affect the response to IL‐1β, whereas IL‐4, 12 or 12+2 h before the IL‐1β, inhibited the hyperalgesia (−30%, −74%, respectively).

In murine peritoneal macrophages, murine IL‐4 for 2 h before stimulation with LPS, inhibited (−40%) the production of IL‐1β but not PGE2. Murine IL‐4 (for 16 h before stimulation with LPS) inhibited LPS‐stimulated PGE2 but not IL‐1β.

Anti‐murine IL‐4 antibodies potentiated responses to carrageenin, bradykinin and TNFα, but not IL‐1β and IL‐8, as well as responses to bradykinin in athymic rats but not in rats depleted of mast cells with compound 40/80.

These data suggest that IL‐4 released by mast cells limits inflammatory hyperalgesia. During the early phase of the inflammatory response the mode of action of the IL‐4 appears to be inhibition of the production TNFα, IL‐1β and IL‐8. In the later phase of the response, in addition to inhibiting the production of pro‐inflammatory cytokines, IL‐4 also may inhibit the release of PGs.

British Journal of Pharmacology (1999) 126, 45–50; doi:10.1038/sj.bjp.0702266
Interaction of amylin with calcitonin gene‐related peptide receptors in the microvasculature of the hamster cheek pouch in vivo
Authors: Hall Judith M , Brain Susan D
Published: January 2005

This study used intravital microscopy to investigate the receptors stimulated by amylin which shares around 50% sequence homology with the vasodilator calcitonin gene‐related peptide (CGRP) in the hamster cheek pouch microvasculature in vivo.

Receptor agonists dilated arterioles (diameters 20–40 μm). The −log of the concentrations (±s.e.mean; n=8) causing 50% increase in arteriole diameter were: human βCGRP (10.8±0.3), human αCGRP (10.8±0.4), rat αCGRP (10.4±0.3). Rat amylin and the CGRP2 receptor selective agonist [Cys(ACM2,7]‐human αCGRP were 100 fold less potent (estimates were 8.5±0.4 and 8.2±0.3 respectively).

The GCRP1 receptor antagonist, CGRP8–37 (300 nmol kg−1; i.v.) reversibly inhibited the increase in diameter evoked by human αCGRP (0.3 nM) from 178±22% to 59±12% (n=8; P<0.05) and by rat amylin (100 nM) from 138±23% to 68±24% (n=6; P<0.05). CGRP8–37 did not inhibit vasodilation evoked by substance P (10 nM; n=4; P>0.05).

The amylin receptor antagonist, amylin8–37 (300 nmol kg−1; i.v.) did not significantly inhibit the increase in diameter evoked by human αCGRP (0.3 nM) which was 112±26% in the absence, and 90±29% in the presence of antagonist (n=4; P<0.05); nor that evoked by rat amylin (100 nM) which was 146±23% in the absence and 144±32% in the presence of antagonist (n=4; P>0.05).

The agonist profile for vasodilatation and the inhibition of this dilatation by CGRP8–37, although not the amylin8–37 indicates that amylin causes vasodilatation through interaction with CGRP1 receptors in the hamster cheek pouch.

British Journal of Pharmacology (1999) 126, 280–284; doi:10.1038/sj.bjp.0702272
Specific inhibition of ADP‐induced platelet aggregation by clopidogrel in vitro
Authors: Weber Artur‐Aron , Reimann Stephanie , Schrör Karsten
Published: January 2005

The thienopyridine clopidogrel is a specific inhibitor of ADP‐induced platelet aggregation ex vivo. No direct effects of clopidogrel (100 μM) on platelet aggregation in vitro have been described so far.

Possible in vitro antiaggregatory effects (turbidimetry) of clopidogrel were studied in human platelet‐rich plasma and in washed platelets.

Incubation of platelet‐rich plasma with clopidogrel (100 μM) for up to 8 h did not result in any inhibition of ADP (6 μM)‐induced platelet aggregation.

Incubation of washed platelets with clopidogrel resulted in a time‐ (maximum effects after 30 min) and concentration‐dependent (IC50 1.9±0.3 μM) inhibition of ADP (6 μM)‐induced platelet aggregation. Clopidogrel (30 μM) did not inhibit collagen (2.5 μg ml−1)‐, U46619 (1 μM)‐ or thrombin (0.1 u ml−1)‐induced platelet aggregation. The inhibition of ADP‐induced aggregation by clopidogrel (30 μM) was insurmountable indicating a non‐equilibrium antagonism of ADP actions. The R enantiomer SR 25989 C (30 μM) was significantly less active than clopidogrel (30 μM) in inhibiting platelet aggregation (32±5 % vs 70±1 % inhibition, P<0.05, n=5).

In washed platelets, clopidogrel (30 μM) did not significantly reverse the inhibition of prostaglandin E1 (1 μM)‐induced platelet cyclic AMP formation by ADP (6 μM).

The antiaggregatory effects of clopidogrel were unchanged when the compound was removed from the platelet suspension. However, platelet inhibition by clopidogrel was completely abolished when albumin (350 mg ml−1) was present in the test buffer.

It is concluded that clopidogrel specifically inhibits ADP‐induced aggregation of washed platelets in vitro without hepatic bioactivation. Inhibition of ADP‐induced platelet aggregation by clopidogrel in vitro occurs in the absence of measurable effects on the reversal of PGE1‐stimulated cyclic AMP by ADP.

British Journal of Pharmacology (1999) 126, 415–420; doi:10.1038/sj.bjp.0702276
Rat somatostatin sst2(a) and sst2(b) receptor isoforms mediate opposite effects on cell proliferation
Authors: Alderton Forbes , Fan Tai‐Ping D. , Schindler Marcus , P A Humphrey Patrick
Published: January 2005

We have investigated the actions of somatostatin (SRIF) and angiopeptin on cell proliferation of CHO‐K1 cells expressing the recently cloned rat sst2(b) receptor (CHOsst2(b)) and compared these to their effects in cells expressing the sst2(a) receptor (CHOsst2(a)). In contrast to the sst2(a) receptor, the sst2(b) receptor did not mediate inhibition of bFGF (10 ng ml−1)‐stimulated re‐growth and cell proliferation. Rather, SRIF (0.1–1000 nM) and angiopeptin (0.1–1000 nM) stimulated basal re‐growth and proliferation of CHOsst2(b) cells in a concentration‐dependent manner (estimated pEC50 values of 7.8 and 7.9, respectively). The opposite effects of SRIF on cell proliferation mediated through the two sst2 receptor isoforms were both abolished by 18 h pre‐treatment with pertussis toxin. The proliferative effect via the sst2(b) receptor was also abolished by the tyrosine kinase inhibitor, genistein. In conclusion, the present study shows that the rat sst2(a) and sst2(b) receptor splice variants mediate opposite effects on cell proliferation.
NMDA receptor characterization and subunit expression in rat cultured mesencephalic neurones
Authors: Allgaier C , Scheibler P , Müller D , Feuerstein T J , Illes P
Published: January 2005

NMDA‐induced changes in free intracellular Ca2+ concentration ([Ca2+]i) were determined in individual cultured rat mesencephalic neurones by the fura‐2 method. mRNA expression encoding NMDA receptor subunits (NR1, NR2A‐D) was examined by RT–PCR.

NMDA (1–100 μM, plus 10 μM glycine) induced a concentration‐dependent increase in [Ca2+]i (EC50=5.7 μM). The effect of NMDA was virtually insensitive to tetrodotoxin (0.3 μM) and nitrendipine (1 μM), but dependent on extracellular Ca2+. 5,7‐Dichlorokynurenic acid (10 μM), a specific antagonist at the glycine binding site on the NMDA receptor, abolished the NMDA response.

Memantine, an open‐channel blocker, and ifenprodil, a preferential non‐competitive NR1/NR2B receptor antagonist diminished the NMDA effect with an IC50 value of 0.17 and 1 μM, respectively. Ethanol at 50 and 100 mM caused about 25 and 45%‐inhibition, respectively.

Agarose gel analysis of the PCR products followed by ethidium bromide fluorescence or CSPD chemiluminescence detection revealed an almost exclusive expression of the NR1 splice variants lacking exon (E) 5 and E22. The 3′ splice form without both E21 and E22 exceeded that containing E21 by approximately 4 fold. The relative amounts of NR2A, NR2B, NR2C corresponded to approximately 1:2:1. NR2D mRNA was also detectable.

In conclusion, mesencephalic neurones bear ethanol‐sensitive NMDA receptors which might be involved in the development of ethanol dependence and withdrawal. The high affinity of NMDA to this receptor, its sensitivity to ifenprodil and memantine may suggest that the mesencephalic NMDA receptor comprises the NR1 splice variant lacking E5, NR2B, and NR2C, respectively.

British Journal of Pharmacology (1999) 126, 121–130; doi:10.1038/sj.bjp.0702284
Formal analysis of electrogenic sodium, potassium, chloride and bicarbonate transport in mouse colon epithelium
Authors: Cuthbert A W , Hickman M E , MacVinish L J
Published: January 2005

The mammalian colonic epithelium carries out a number of different transporting activities simultaneously, of which more than one is increased following activation with a single agonist. These separate activities can be quantified by solving a set of equations describing these activities, provided some of the dependent variables can be eliminated. Using variations in the experimental conditions, blocking drugs and comparing wild type tissues with those from transgenic animals this has been achieved for electrogenic ion transporting activity of the mouse colon.

Basal activity and that following activation with forskolin was measured by short circuit current in isolated mouse colonic epithelia from normal and cystic fibrosis (CF) mice.

Using amiloride it is shown that CF colons show increased electrogenic sodium absorption compared to wild type tissues. CF mice had elevated plasma aldosterone, which may be responsible for part or all of the increased sodium absorbtion in CF colons.

The derived values for electrogenic chloride secretion and for electrogenic potassium secretion were increased by 13 and 3 fold respectively by forskolin, compared to basal state values for these processes.

The loop diuretic, frusemide, completely inhibited electrogenic potassium secretion, but apparently only partially inhibited electrogenic chloride secretion. However, use of bicarbonate‐free solutions and acetazolamide reduced the frusemide‐resistant current, suggesting that electrogenic bicarbonate secretion accounts for the frusemide‐resistant current.

It is argued that the use of tissues from transgenic animals is an important adjunct to pharmacological analysis, especially where effects in tissues result in the activation of more than one sort of response.

British Journal of Pharmacology (1999) 126, 358–364; doi:10.1038/sj.bjp.0702290
Identification of a region of the C‐terminal domain involved in short‐term desensitization of the prostaglandin EP4 receptor
Authors: Bastepe Murat , Ashby Barrie
Published: January 2005

The prostaglandin EP4 receptor, which couples to stimulation of adenylyl cyclase, undergoes rapid agonist‐induced desensitization when expressed in CHO‐K1 cells.

Truncation of the 488‐amino acid receptor at residue 350 removes the carboxy‐terminal domain and abolishes desensitization.

To further delineate residues involved in desensitization, the receptor was truncated at position 408, 383 or 369. Receptors truncated at position 408 or 383 underwent PGE2‐induced desensitization, whereas the receptor truncated at position 369 displayed sustained activity, indicating that the essential residues for desensitization lie between 370 and 383.

The six serines in the 14‐amino acid segment between residues 370 and 383 were mutated to alanine, retaining the entire C‐terminal domain. Desensitization was absent in cells expressing this mutant.

The results indicate involvement of serines located between 370 and 382 in rapid desensitization of the EP4 receptor.

British Journal of Pharmacology (1999) 126, 365–371; doi:10.1038/sj.bjp.0702291
[3H]‐Mesulergine labels 5‐HT7 sites in rat brain and guinea‐pig ileum but not rat jejunum
Authors: Hemedah Maggie , Coupar Ian M , Mitchelson Fred J
Published: January 2005

The primary aim of this investigation was to determine whether binding sites corresponding to the 5‐HT7 receptor could be detected in smooth muscle of the rat jejunum. Binding studies in rat brain (whole brain minus cerebellum) and guinea‐pig ileal longitudinal muscle were also undertaken in order to compare the binding characteristics of these tissues. Studies were performed using [3H]‐mesulergine, as it has a high affinity for 5‐HT7 receptors.

In the rat brain and guinea‐pig ileum, pKD values for [3H]‐mesulergine of 8.0±0.04 and 7.9±0.11 (n=3) and Bmax values of 9.9±0.3 and 21.5±4.9 fmol mg−1 protein were obtained respectively, but no binding was detected in the rat jejunum. [3H]‐mesulergine binding in the rat brain and guinea‐pig ileum was displaced with the agonists 5‐carboxamidotryptamine (5‐CT)>5‐hydroxytryptamine (5‐HT)5‐methoxytryptamine (5‐MeOT)>sumatriptan and the antagonists risperidoneLSDmetergoline>ritanserin>>pindolol.

Despite the lack of [3H]‐mesulergine binding in the rat jejunum, functional studies undertaken revealed a biphasic contractile response to 5‐HT which was partly blocked by ondansetron (1 μM). The residual response was present in over 50% of tissues studied and was found to be inhibited by risperidone>LSD>metergoline>mesulergine=ritanserin>pindolol, but was unaffected by RS 102221 (3 μM), cinanserin (30 nM), yohimbine (0.1 μM) and GR 113808 (1 μM). In addition, the agonist order of potency was 5‐CT>5‐HT>5‐MeOT>sumatriptan.

In conclusion, binding studies performed with [3H]‐mesulergine were able to detect 5‐HT7 sites in rat brain and guinea‐pig ileum, but not in rat jejunum, where a functional 5‐HT7‐like receptor was present.

British Journal of Pharmacology (1999) 126, 179–188; doi:10.1038/sj.bjp.0702293
Reduction of sodium deoxycholic acid‐induced scratching behaviour by bradykinin B2 receptor antagonists
Authors: Hayashi Izumi , Majima Masataka
Published: January 2005

Subcutaneous injection of sodium deoxycholic acid into the anterior of the back of male ddY mice elicited dose‐dependent scratching of the injected site with the forepaws and hindpaws.

Up to 100 μg of sodium deoxycholic acid induced no significant increase in vascular permeability at the injection site as assessed by a dye leakage method.

Bradykinin (BK) B2 receptor antagonists, FR173657 and Hoe140, significantly decreased the frequency of scratching induced by sodium deoxycholic acid.

Treatment with aprotinin to inhibit tissue kallikrein reduced the scratching behaviour induced by sodium deoxycholic acid, whereas treatment with soybean trypsin inhibitor to inhibit plasma kallikrein did not.

Although injection of kininase II inhibitor, lisinopril together with sodium deoxycholic acid did not alter the scratching behaviour, phosphoramidon, a neutral endopeptidase inhibitor, significantly increased the frequency of scratching.

Homogenates of the skin excised from the backs of mice were subjected to gel‐filtration column chromatography followed by an assay of kinin release by trypsin from each fraction separated. Less kinin release from the fractions containing kininogen of low molecular weight was observed in the skin injected with sodium deoxycholic acid than in normal skin.

The frequency of scratching after the injection of sodium deoxycholic acid in plasma kininogen‐deficient Brown Norway Katholiek rats was significantly lower than that in normal rats of the same strain, Brown Norway Kitasato rats.

These results indicate that BK released from low‐molecular‐weight kininogen by tissue kallikrein, but not from high‐molecular‐weight kininogen by plasma kallikrein, may be involved in the scratching behaviour induced by the injection of sodium deoxycholic acid in the rodent.

British Journal of Pharmacology (1999) 126, 197–204; doi:10.1038/sj.bjp.0702296
Accelerated intimal hyperplasia and increased endogenous inhibitors for NO synthesis in rabbits with alloxan‐induced hyperglycaemia
Authors: Masuda Hiroshi , Goto Moritaka , Tamaoki Satoru , Azuma Hiroshi
Published: January 2005

We examined whether endogenous inhibitors of NO synthesis are involved in the augmentation of intimal hyperplasia in rabbits with hyperglycaemia induced by alloxan.

Four weeks after the endothelial denudation of carotid artery which had been performed 12 weeks after alloxan, the intimal hyperplasia was greatly augmented with hyperglycaemia. The degree of hyperplasia was assessed using three different parameters of histopathological findings as well as changes in luminal area and intima : media ratio.

There were positive and significant correlations between intima : media ratio, plasma glucose, and concentrations of NG‐monomethyl‐L‐arginine (L‐NMMA) and NG, NG‐dimethyl‐L‐arginine (ADMA) in endothelial cells, that is, the intima : media ratio became greater as plasma glucose and endothelial L‐NMMA and ADMA were increased. Furthermore, endothelial L‐NMMA and ADMA were increased in proportion to the increase in plasma glucose.

In contrast, there were inverse and significant correlations between cyclic GMP production by carotid artery strips with endothelium and plasma glucose, between cyclic GMP production and endothelial L‐NMMA and ADMA, and between the intima : media ratio and cyclic GMP production.

Exogenously applied L‐NMMA and ADMA inhibited cyclic GMP production in a concentration‐dependent manner. IC50 values were determined to be 12.1 μM for the former and 26.2 μM for the latter. The cyclic GMP production was abolished after the deliberate removal of endothelium from the artery strips.

These results suggest that the augmentation of intimal hyperplasia with hyperglycaemia is closely related to increased accumulation of L‐NMMA and ADMA with hyperglycaemia, which would result in an accelerated reduction in NO production/release by endothelial cells.

British Journal of Pharmacology (1999) 126, 211–218; doi:10.1038/sj.bjp.0702298
Modulation of acute and chronic inflammatory processes by cacospongionolide B, a novel inhibitor of human synovial phospholipase A2
Authors: Pastor Pablo García , De Rosa Salvatore , De Giulio Alfonso , Payá Miguel , Alcaraz M José
Published: January 2005

Cacospongionolide B is a novel marine metabolite isolated from the sponge Fasciospongia cavernosa. In in vitro studies, this compound inhibited phospholipase A2 (PLA2), showing selectivity for secretory PLA2 (sPLA2) versus cytosolic PLA2 (cPLA2), and its potency on the human synovial enzyme (group II) was similar to that of manoalide.

This activity was confirmed in vivo in the 8 h zymosan‐injected rat air pouch, on the secretory enzyme accumulating in the pouch exudate. Cacospongionolide B, that is bioavailable when is given orally, reduced the elevated levels of sPLA2 present in paw homogenates of rats with adjuvant arthritis.

This marine metabolite showed topical anti‐inflammatory activity on the mouse ear oedema induced by 12‐O‐tetradecanoylphorbol acetate (TPA) and decreased carrageenin paw oedema in mice after oral administration of 5, 10 or 20 mg kg−1.

In the mouse air pouch injected with zymosan, cacospongionolide B administered into the pouch, induced a dose‐dependent reduction in the levels of eicosanoids and tumour necrosis factor α (TNFα) in the exudates 4 h after the stimulus. It also had a weak effect on cell migration.

The inflammatory response of adjuvant arthritis was reduced by cacospongionolide B, which did not significantly affect eicosanoid levels in serum, paw or stomach homogenates and did not induce toxic effects.

Cacospongionolide B is a new inhibitor of sPLA2 in vitro and in vivo, with anti‐inflammatory properties in acute and chronic inflammation. This marine metabolite was active after oral administration and able to modify TNFα levels, and may offer an interesting approach in the search for new anti‐inflammatory agents.

British Journal of Pharmacology (1999) 126, 301–311; doi:10.1038/sj.bjp.0702302
Bronchoconstrictor effect of thrombin and thrombin receptor activating peptide in guinea‐pigs in vivo
Authors: Cicala Carla , Bucci Mariarosaria , De Dominicis Gianfranco , Harriot Pat , Sorrentino Ludovico , Cirino Giuseppe
Published: January 2005

Several thrombin cellular effects are dependent upon stimulation of proteinase activated receptor‐1 (PAR‐1) localized over the cellular surface. Following activation by thrombin, a new N‐terminus peptide is unmasked on PAR‐1 receptor, which functions as a tethered ligand for the receptor itself. Synthetic peptides called thrombin receptor activating peptides (TRAPs), corresponding to the N‐terminus residue unmasked, reproduce several thrombin cellular effects, but are devoid of catalytic activity. We have evaluated the bronchial response to intravenous administration of human α‐thrombin or a thrombin receptor activating peptide (TRAP‐9) in anaesthetized, artificially ventilated guinea‐pigs.

Intravenous injection of thrombin (100 u kg−1) caused bronchoconstriction that was recapitulated by injection of TRAP‐9 (1 mg kg−1). Animal pretreatment with the thrombin inhibitor Hirulog™ (10 mg kg−1  i.v.) prevented thrombin‐induced bronchoconstriction, but did not affect bronchoconstriction induced by TRAP‐9. Both agents did not induce bronchoconstriction when injected intravenously to rats.

The bronchoconstrictor effect of thrombin and TRAP‐9 was subjected to tolerance; however, in animals desensitized to thrombin effect, TRAP‐9 was still capable of inducing bronchoconstriction, but not vice versa.

Depleting animals of circulating platelets prevented bronchoconstriction induced by both thrombin and TRAP‐9.

Bronchoconstriction was paralleled by a biphasic change in arterial blood pressure, characterized by a hypotensive phase followed by a hypertensive phase. Thrombin‐induced hypotension was not subject to tolerance and was inhibited by Hirulog™; conversely, hypertension was subject to tolerance and was not inhibited by Hirulog™. Hypotension and hypertension induced by TRAP‐9 were neither subject to tolerance nor inhibited by Hirulog™.

Our results indicate that thrombin causes bronchoconstriction in guinea‐pigs through a mechanism that requires proteolytic activation of its receptor and the exposure of the tethered ligand peptide. Platelet activation might be triggered by the thrombin effect.

British Journal of Pharmacology (1999) 126, 478–484; doi:10.1038/sj.bjp.0702303
Anaphylactic bronchoconstriction in BP2 mice: interactions between serotonin and acetylcholine
Authors: Eum Seok‐Yong , Norel Xavier , Lefort Jean , Labat Carlos , Vargaftig B Boris , Brink Charles
Published: January 2005

Immunized BP2 mice developed an acute bronchoconstriction in vivo and airway muscle contraction in vitro in response to ovalbumin (OA) and these contractions were dose dependent.

Methysergide or atropine inhibited OA‐induced bronchoconstriction in vivo and airway muscle contraction in vitro.

Neostigmine potentiated the OA‐induced bronchoconstriction in vivo and airway muscle contraction in vitro of BP2 mice. This potentiation was markedly reduced by the administration of methysergide or atropine and when the two antagonists were administered together, the responses were completely inhibited.

Neostigmine also potentiated the serotonin (5‐HT)‐ and acetylcholine (ACh)‐induced bronchoconstriction and this potentiation was significantly reversed by atropine.

These results indicate that OA provokes a bronchoconstriction in immunized BP2 mice by stimulating the release of 5‐HT, which in turn acts via the cholinergic mediator, ACh.

British Journal of Pharmacology (1999) 126, 312–316; doi:10.1038/sj.bjp.0702304
Taurine modulates IKr but not IKs in guinea‐pig ventricular cardiomyocytes
Authors: Satoh Hiroyasu
Published: January 2005

Effects of taurine on the delayed rectifier K+ current (IK) in isolated guinea‐pig ventricular cardiomyocytes were examined at different intracellular Ca2+ concentration ([Ca2+]i), using whole‐cell voltage and current clamp techniques. Experiments were performed at 36°C.

Addition of taurine (10–20 mM) decreased the action potential duration (APD) at pCa 8, but increased the APD at pCa 6. Taurine (20 mM) enhanced IK at 70 mV by 22.4±3.1% (n=6, P<0.01) at pCa 8, whereas taurine inhibited the IK by 27.1±2.7% (n=6, P<0.01) at pCa 6. These responses behaved in a concentration‐dependent manner.

The IK is composed of the rapid and slow components (IKr and IKs). When [Ca2+]i was pCa 6, taurine at 20 mM reduced the tail current of IKr at 70 mV by 16.5±2.7% (n=5, P<0.05) and that of IKs at 70 mV by 27.1±2.8% (n=6, P<0.01). In contrast, at pCa 8, the tail currents of IKr and IKs at 70 mV were enhanced by 13.4±3.2% (n=7, P<0.05) and by 22.4±3.1% (n=7, P<0.01), respectively. The voltages of half‐maximum activation (V1/2) for IKr and IKs were not modified by taurine.

Addition of E‐4031 (5 μM) to taurine had a complete blockade of the tail current of IKr, but not IKs. The remained tail current (IKs) in the presence of E‐4031 (5 μM) was not affected by taurine (20 mM), but was blocked by 293B (30 μM).

These results indicate that taurine modulates IKr but not IKs, depending on [Ca2+]i, resulting in regulation of the APD.

British Journal of Pharmacology (1999) 126, 87–92; doi:10.1038/sj.bjp.0702308
Improvement by nefiracetam of β‐amyloid‐(1‐42)‐induced learning and memory impairments in rats
Authors: Yamada Kiyofumi , Tanaka Tomoko , Mamiya Takayoshi , Shiotani Tadashi , Kameyama Tsutomu , Nabeshima Toshitaka
Published: January 2005

We have previously demonstrated that continuous i.c.v. infusion of amyloid β‐peptide (Aβ), the major constituent of senile plaques in the brains of patients with Alzheimer's disease, results in learning and memory deficits in rats.

In the present study, we investigated the effects of nefiracetam [N‐(2,6‐dimethylphenyl)‐2‐(2‐oxo‐1‐pyrrolidinyl) acetamide, DM‐9384] on Aβ‐(1–42)‐induced learning and memory deficits in rats.

In the Aβ‐(1–42)‐infused rats, spontaneous alternation behaviour in a Y‐maze task, spatial reference and working memory in a water maze task, and retention of passive avoidance learning were significantly impaired as compared with Aβ‐(40–1)‐infused control rats.

Nefiracetam, at a dose range of 1–10 mg kg−1, improved learning and memory deficits in the Aβ‐(1–42)‐infused rats when it was administered p.o. 1 h before the behavioural tests.

Nefiracetam at a dose of 3 mg kg−1 p.o. increased the activity of choline acetyltransferase in the hippocampus of Aβ‐(1–42)‐infused rats.

Nefiracetam increased dopamine turnover in the cerebral cortex and striatum of Aβ‐(1–42)‐infused rats, but failed to affect the noradrenaline, serotonin and 5‐hydroxyindoleacetic acid content.

These results suggest that nefiracetam may be useful for the treatment of patients with Alzheimer's disease.

British Journal of Pharmacology (1999) 126, 235–244; doi:10.1038/sj.bjp.0702309
Role of K+ channels in A2A adenosine receptor‐mediated dilation of the pressurized renal arcuate artery
Authors: Prior H M , Yates M S , Beech D J
Published: January 2005

Adenosine A2A receptor‐mediated renal vasodilation was investigated by measuring the lumenal diameter of pressurized renal arcuate arteries isolated from the rabbit.

The selective A2A receptor agonist CGS21680 dilated the arteries with an EC50 of 130 nM. The CGS21680‐induced vasodilation was, on average, 34% less in endothelium‐denuded arteries.

The maximum response and the EC50 for CGS21680‐induced vasodilation in endothelium‐intact arteries were not significantly affected by incubation with the K+ channel blockers apamin (100 nM), iberiotoxin (100 nM), 3,4‐diaminopyridine (1 mM), glibenclamide (1 μM) or Ba2+ (10 μM). However, a cocktail mixture of these blockers did significantly inhibit the maximum response by almost 40%, and 1 mM Ba2+ alone or 1 mM Ba2+ in addition to the cocktail inhibited the maximum CGS21680‐response by 58% and about 75% respectively.

CGS21680‐induced vasodilation was strongly inhibited when the extracellular K+ level was raised to 20 mM even though the dilator response to 1 μM levcromakalim, a KATP channel opener drug, was unaffected.

CGS21680‐induced vasodilation was inhibited by 10 μM ouabain, an inhibitor of Na+/K+‐ATPase, but ouabain had a similar inhibitory effect on vasodilation induced by 30 nM nicardipine (a dihydropyridine Ca2+ antagonist) or 1 μM levcromakalim.

The data suggest that K+ channel activation does play a role in A2A receptor‐mediated renal vasodilation. The inhibitory effect of raised extracellular K+ levels on the A2A response may be due to K+‐induced stimulation of Na+/K+‐ATPase.

British Journal of Pharmacology (1999) 126, 494–500; doi:10.1038/sj.bjp.0702310
Endothelium‐dependent hyperpolarization in resting and depolarized mammary and coronary arteries of guinea‐pigs
Authors: Hammarström Anna K M , Parkington Helena C , Tare Marianne , Coleman H A
Published: January 2005

The membrane potential responses in guinea‐pig coronary and mammary arteries attributable to endothelium‐derived nitric oxide (NO), prostaglandin (PG) and hyperpolarizing factor (EDHF), and to exogenous NO and the prostacyclin analogue, iloprost, were compared at rest and when depolarized with the thromboxane analogue, U46619.

In the coronary artery, stimulation of the endothelium with acetylcholine (ACh) evoked hyperpolarization attributable to NO and a PG with similar pD2s at rest and in the presence of U46619. However, in depolarized tissues, the pD2 of the response attributed to EDHF required a 10 fold lower concentration of ACh compared with at rest.

In the mammary artery, lower concentrations of ACh were required to evoke NO‐ and EDHF‐dependent hyperpolarizations in depolarized mammary artery compared with at rest, while PG‐dependent hyperpolarization did not occur until the concentration of ACh was increased some 10 fold both at rest and in U46619.

The smooth muscle of the coronary artery of guinea‐pigs was some 4 fold more sensitive to exogenous NO and iloprost than was the mammary artery.

In conclusion, the membrane potential response in arteries at rest, that is, in the absence of constrictor, may be extrapolated to events in the presence of constrictor when NO and PG are under study. However, the sensitivity to ACh and the magnitude of the hyperpolarization attributed to EDHF obtained in tissues at rest may underestimate these parameters in depolarized tissues.

British Journal of Pharmacology (1999) 126, 421–428; doi:10.1038/sj.bjp.0702313