bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcitonin gene‐related peptide potentiates nicotinic acetylcholine receptor‐operated slow Ca2+ mobilization at mouse muscle endplates
Authors: Salim Safaa Y , Dezaki Katsuya , Tsuneki Hiroshi , Abdel‐Zaher Ahmed O , Kimura Ikuko
Published: January 2005

The involvement of calcitonin gene‐related peptide (CGRP) in the non‐contractile slow Ca2+ mobilization induced by prolonged nicotinic stimulation was investigated by measurement of [Ca2+]i levels in mouse single muscle cells (flexor digitorum brevis; FDB) loaded with a Ca2+ indicator fluo‐3 using confocal laser scanning microscopy.

CGRP (3–30 nM) potentiated acetylcholine (ACh, 1 μM)‐elicited slow Ca2+ mobilization in a concentration‐dependent manner.

The potentiation by CGRP of the slow Ca2+ component was greatly depressed by a competitive nicotinic antagonist (+)‐tubocurarine (5 μM). The Ca2+ channel blocker nitrendipine (1 μM) affected neither ACh responses nor the CGRP potentiation.

The slow Ca2+ component was completely abolished by reducing [Ca2+]0 from 2.5 to 0.25 mM whereas the fast component was not affected. The CGRP‐induced potentiation of slow Ca2+ signal was also depressed by decreasing [Ca2+]0.

Isoproterenol (30 μM) and 8‐bromo‐adenosine 3′,5′‐cyclic monophosphate (1 mM) potentiated the ACh‐elicited slow Ca2+ response. The potentiation by CGRP of the slow Ca2+ component was completely abolished by a protein kinase‐A inhibitor H‐89 (1 μM).

These findings indicate that CGRP potentiates the nicotinic ACh receptor‐operated slow Ca2+ signal via the activation of protein kinase‐A system at the skeletal muscle endplates.

British Journal of Pharmacology (1998) 125, 277–282; doi:10.1038/sj.bjp.0702058
SB‐224289–a novel selective (human) 5‐HT1B receptor antagonist with negative intrinsic activity
Authors: Selkirk J V , Scott C , Ho M , Burton M J , Watson J , Gaster L M , Collin L , Jones B J , Middlemiss D N , Price G W
Published: January 2005

Human 5‐HT1B (h5‐HT1B) and human 5‐HT1D (h5‐HT1D) receptors show remarkably similar pharmacology with few compounds discriminating the receptors. We report here on a novel compound, SB‐224289 (1′‐Methyl‐5‐[[2′‐methyl‐4′‐(5‐methyl‐1,2,4‐oxadiazol‐3‐yl)biphenyl‐4‐yl]carbonyl]‐2,3,6,7‐tetrahydrospiro [furo [2,3‐f]indole‐3,4′‐piperidine] oxalate), which has high affinity for h5‐HT1B receptors (pK1=8.16±0.06) and displays over 75 fold selectivity for the h5‐HT1B receptor over all other 5‐HT receptors including the h5‐HT1D receptor and all other receptors tested thus far.

Functional activity of SB‐224289 was measured in a [35S]GTPγS binding assay on recombinant h5‐HT1B and h5‐HT1D receptors expressed in Chinese Hamster Ovary (CHO) cells. SB‐224289 displayed negative intrinsic activity at both receptors with higher potency at h5‐HT1B receptors. SB‐224289 caused a rightward shift of agonist concentration response curves consistent with competitive antagonism and generated affinities comparable with those obtained from competition radioligand receptor binding studies.

SB‐224289 potentiated [3H]5‐HT release from electrically stimulated guinea‐pig cerebral cortical slices to the same extent as as the non‐selective 5‐HT1 antagonist methiothepin. SB‐224289 also fully reversed the inhibitory effect of exogenously superfused 5‐HT on electrically stimulated release.

Using SB‐224289 as a tool compound, we confirm that in guinea‐pig cerebral cortex the terminal 5‐HT autoreceptor is of the 5‐HT1B subtype.

British Journal of Pharmacology (1998) 125, 202–208; doi:10.1038/sj.bjp.0702059
Molecular cloning and functional characterization of a rat somatostatin sst2(b) receptor splice variant
Authors: Schindler Marcus , Kidd Emma J , Carruthers Alan M , Wyatt Mark A , Jarvie Emma M , Sellers Lynda A , Feniuk Wasyl , Humphrey Patrick P A
Published: January 2005

The mouse somatostatin (SRIF) sst2 receptor exists in two splice variants, sst2(a) and sst2(b), which differ in their intracellular carboxy‐termini only. The murine sst2(b) receptor was reported to be less prone to agonist‐induced desensitization as compared with the sst2(a) receptor. To determine whether a sst2(b) splice variant with similar functional characteristics exists in the rat, we have isolated a cDNA fragment from rat gastric mucosa encoding a sst2(b) receptor and expressed the full‐length protein in CHO‐K1 cells for functional characterization.

This study provides the first evidence for the occurrence in the rat of the sst2(b) receptor, which has a 15 amino acid carboxy‐terminus differing in composition to the 38 amino acid C‐terminus of the rat sst2(a) receptor.

In CHO‐K1 cells expressing rat recombinant sst2(a) or sst2(b) receptors, SRIF caused concentration‐dependent increases in extracellular acidification rates (EAR) with pEC50 values of 9.0 and 9.9, respectively. Pre‐treatment with pertussis toxin (Ptx) caused a rightward displacement of the SRIF concentration‐effect curves with pEC50 values of 8.3 (sst2(a)) and 8.4 (sst2(b)).

SRIF (3 pM–3 nM) also caused concentration‐dependent inhibition of forskolin‐stimulated cyclic AMP formation in CHO‐sst2(a) cells (pIC50 10.5) and CHO‐sst2(b) cells (pIC50 10.4). The degree of inhibition was less with higher concentrations of SRIF resulting in bell‐shaped concentration‐effect curves. Following pre‐treatment with Ptx, the inhibitory effect of SRIF was abolished and SRIF caused only increases in cyclic AMP formation.

Both the SRIF‐induced increases in EAR and inhibition of cyclic AMP formation were susceptible to agonist‐induced desensitization, but this was less apparent following pre‐treatment with Ptx.

This demonstrates that the operational characteristics of the recombinant rat sst2(a) and sst2(b) receptors are broadly similar. Both isoforms couple to Ptx‐sensitive as well as ‐insensitive G proteins and are equally prone to agonist‐induced desensitization.

British Journal of Pharmacology (1998) 125, 209–217; doi:10.1038/sj.bjp.0702064
A comparison of the effects on central 5‐HT function of sibutramine hydrochloride and other weight‐modifying agents
Authors: Heal D J , Cheetham S C , Prow M R , Martin K F , Buckett W R
Published: January 2005

Effects on 5‐HT function of sibutramine and its active metabolites, BTS 54 354 and BTS 54 505, were compared with fluoxetine, (+)‐fenfluramine and (+)‐amphetamine.

In vitro sibutramine weakly inhibited [3H]‐5‐HT uptake into brain synaptosomes. BTS 54 354, BTS 54 505 and fluoxetine were powerful [3H]‐5‐HT uptake inhibitors, whereas (+)‐fenfluramine and (+)‐amphetamine were very much weaker. Conversely, whilst sibutramine, its metabolites and fluoxetine did not release [3H]‐5‐HT from brain slices at 10−5M, (+)‐fenfluramine and (+)‐amphetamine concentration‐dependently increased [3H]‐5‐HT release.

Sibutramine and fluoxetine had no effect on 5‐hydroxytryptophan (5‐HTP) accumulation in either frontal cortex or hypothalamus at doses <10 mg kg−1. In contrast, (+)‐amphetamine (3 mg kg−1) reduced 5‐HTP in hypothalamus, whilst (+)‐fenfluramine (1 mg kg−1) decreased 5‐HTP in both regions.

Sibutramine (10 mg kg−1 i.p.) and fluoxetine (10 mg kg−1 i.p.) produced slow, prolonged increases of extracellular 5‐HT in the anterior hypothalamus. In contrast, (+)‐fenfluramine (3 mg kg−1 i.p.) and (+)‐amphetamine (4 mg kg−1 i.p.) induced rapid, short‐lasting increases in extracellular 5‐HT.

Only (+)‐fenfluramine (10 mg kg−1) altered 5‐HT2A receptors in rat frontal cortex when given for 14 days, producing a 61% reduction in receptor number and a 18% decrease in radioligand affinity.

These results show that sibutramine powerfully enhances central 5‐HT function via its secondary and primary amine metabolites; this effect, like that of fluoxetine, is almost certainly mediated through 5‐HT uptake inhibition. By contrast, (+)‐fenfluramine enhances 5‐HT function predominantly by increasing 5‐HT release. (+)‐Amphetamine, though weaker than (+)‐fenfluramine, also enhances 5‐HT function by release.

British Journal of Pharmacology (1998) 125, 301–308; doi:10.1038/sj.bjp.0702067
Actions of general anaesthetics and arachidonic acid pathway inhibitors on K+ currents activated by volatile anaesthetics and FMRFamide in molluscan neurones
Authors: Lopes C M B , Franks N P , Lieb W R
Published: January 2005

K+ currents activated by volatile general anaesthetics (IK(An)) and by the neuropeptide FMRFamide (IK(FMRFa)) were studied under voltage clamp in isolated identified neurones from the pond snail Lymnaea stagnalis.

IK(An) was activated by all the volatile anaesthetics studied. The maximal responses varied from agent to agent, with halothane≈sevoflurane>isoflurane>enflurane≈chloroform.

IK(An) was inhibited rather than activated by the n‐alcohols from hexanol to dodecanol and by the 6‐ and 8‐carbon cycloalcohols. The n‐alcohols exhibited a cutoff effect, with dodecanol being unable to half‐inhibit IK(An).

Unlike IK(An) which did not desensitize at reasonable halothane concentrations, IK(FMRFa) desensitized at most FMRFamide concentrations studied. This desensitization could be substantially removed by halothane. Nonetheless, both IK(An) and IK(FMRFa) had similar sensitivities to the potassium channel blockers tetraethylammonium and 4‐aminopyridine, consistent with both currents flowing through the same channels. Responses to low concentrations of halothane and FMRFamide showed synergy.

The phospholipase A2 inhibitor aristolochic acid inhibited IK(An), consistent with a role for arachidonic acid (AA). The lipoxygenase and cyclooxygenase inhibitor nordihydroguaiaretic acid blocked IK(FMRFa) but did not affect IK(An). IK(An) and IK(FMRFa) were little affected by the cyclooxygenase inhibitor indomethacin. These findings suggest that neither lipoxygenase nor cyclooxygenase pathways of AA metabolism are involved in the anaesthetic activation of IK(An).

Inhibitors of a third, cytochrome P450‐mediated, pathway of AA metabolism (clotrimazole and econazole) potently blocked IK(An), suggesting possible roles for certain cytochrome P450 isoforms in the activation of IK(An).

British Journal of Pharmacology (1998) 125, 309–318; doi:10.1038/sj.bjp.0702069
Blockade of 5‐Hydroxytryptamine and noradrenaline uptake by venlafaxine: a comparative study with paroxetine and desipramine
Authors: Béïque Jean‐Claude , de Montigny Claude , Blier Pierre , Debonnel Guy
Published: January 2005

Venlafaxine is an antidepressant agent which blocks in vitro the reuptake of both 5‐HT and NA. The present in vivo electrophysiological studies were undertaken, in the rat, to compare the effects of venlafaxine on 5‐HT and NA reuptake to those of the selective 5‐HT reuptake inhibitor paroxetine and the selective NA reuptake inhibitor desipramine.

Administered acutely, venlafaxine dose‐dependently prolonged the time required for a 50% recovery (RT50) of the firing activity of dorsal hippocampus CA3 pyramidal neurons from the suppression induced by microiontophoretic applications of 5‐HT and NA. Venlafaxine and paroxetine increased with a similar potency the RT50 values for 5‐HT, while desipramine was more potent than venlafaxine at increasing the RT50 values for NA. Moreover, venlafaxine demonstrated a greater potency at increasing the RT50 values for 5‐HT compared to that of NA.

A two‐day treatment with venlafaxine (delivered s.c. by osmotic minipumps) increased the RT50 values for both 5‐HT and NA applications. The RT50 values for 5‐HT were significantly increased at a dose of 10 mg kg−1 day−1, whereas those for NA were increased at a dose of 20 mg kg−1 day−1, consistent with the data obtained following the acute administration of venlafaxine.

Taken together, these results indicate that, in vivo, venlafaxine blocks both reuptake processes, with a potency greater for the 5‐HT than for the NA reuptake process. This dual action, combined with the differential potency of venlafaxine, might constitute the biological substratum responsible for its apparent unique clinical efficacy in major depression.

British Journal of Pharmacology (1998) 125, 526–532; doi:10.1038/sj.bjp.0702074
Interactions between inducible isoforms of nitric oxide synthase and cyclo‐oxygenase in vivo: investigations using the selective inhibitors, 1400W and celecoxib
Authors: Hamilton Lorna C , Warner Timothy D
Published: January 2005

Exposure of tissues to endotoxin (LPS) and/or cytokines leads to the induction of both inducible nitric oxide synthase (iNOS) and cyclo‐oxygenase‐2 (COX‐2). It has previously been reported that there is ‘cross‐talk’ between these two systems. However, such previous studies have been limited by the availability of highly selective inhibitors. Here we have investigated the interactions between iNOS and COX‐2 in vivo using 1400W, an iNOS‐selective inhibitor, and celecoxib, a COX‐2‐selective inhibitor.

Infusion of LPS to rats for 6 h caused a time‐dependent increase in the plasma concentrations of 6 keto‐prostaglandin F1α (6 keto‐PGF1α) and nitrite/nitrate (NO2/NO3), consistent with the induction of iNOS and COX‐2. Bolus injection of arachidonic acid (AA) at t=6 h resulted in a further increase of circulating levels of 6 keto‐PGF1α in LPS‐treated animals.

Treatment of rats with 1400W or the non‐selective NOS inhibitor NG‐monomethyl‐L‐arginine (L‐NMMA) inhibited the increase in plasma NO2/NO3 but were both without effect on the plasma concentration of 6 keto‐PGF1α before or after AA.

Treatment with the non‐steroidal anti‐inflammatory drugs (NSAIDs), A771726 or diclofenac, or with celecoxib significantly reduced the increase in circulating 6 keto‐PGF1α caused by LPS, and the large increase in 6 keto‐PGF1α following injection of AA. None of the COX inhibitors affected the increase in plasma NO2/NO3. Dexamethasone, however, significantly inhibited both the increase in 6 keto‐PGF1α and the increase in NO2/NO3.

In conclusion, the use of selective inhibitors does not support the concept of cross talk in vivo between iNOS and COX‐2.

British Journal of Pharmacology (1998) 125, 335–340; doi:10.1038/sj.bjp.0702077
P2X receptor‐mediated excitation of nociceptive afferents in the normal and arthritic rat knee joint
Authors: Dowd E , McQueen D S , Chessell I P , Humphrey P P A
Published: January 2005

We tested the hypothesis that functional P2X receptors are present on peripheral terminals of primary afferent articular nociceptors in the rat knee joint. Neural activity was recorded extracellularly from the medial articular nerve innervating the knee joint in rats anaesthetized with pentobarbitone.

The selective P2X receptor agonist, αβ methylene ATP (αβmeATP), and the endogenous ligand, ATP, caused a rapid short‐lasting excitation of a sub‐population of C and Aδ nociceptive afferent nerves innervating normal knee joints when injected intra‐arterially or intra‐articularly, and this effect was antagonized by the non‐selective P2 receptor antagonist PPADS.

Induction of a chronic (14–21 days) unilateral inflammatory arthritis of the knee joint using locally injected Freund's adjuvant neither increased or decreased responsiveness of joint nociceptors to αβmeATP or ATP.

Our results support the hypothesis that αβmeATP‐sensitive P2X receptors are expressed on peripheral nociceptive afferents in the rat knee joint suggesting that they may be involved in the initiation of nociception and pain.

British Journal of Pharmacology (1998) 125, 341–346; doi:10.1038/sj.bjp.0702080
Adaptive changes in the pharmacodynamics of midazolam in different experimental models of epilepsy: kindling, cortical stimulation and genetic absence epilepsy
Authors: Cleton A , Voskuyl R A , Danhof M
Published: January 2005

The objective of this investigation was to determine quantitatively whether experimental epilepsy is associated with a change in the pharmacodynamics of benzodiazepines in vivo. For that purpose the pharmacodynamics of midazolam were quantified by an integrated pharmacokinetic‐pharmacodynamic approach in three different models of experimental epilepsy: amygdala kindling, cortical stimulation and genetic absence epilepsy.

The time course of the EEG effect was determined in conjunction with the decline of drug concentrations after intravenous administration of 10 mg kg−1 midazolam. The pharmacokinetics of midazolam were most adequately described by a bi‐exponential equation. No influence of epilepsy on the pharmacokinetics of midazolam was observed.

The increase in β activity (11.5–30 Hz) of the EEG as derived by Fast Fourier Transformation analysis was used as pharmacodynamic endpoint. For each individual rat the increase in β activity was directly related to the concentration in blood on the basis of the sigmoidal Emax pharmacodynamic model. In all three models a significant reduction in the maximal effect was observed, in amygdala kindling 28%, in the cortical stimulation model 49% and in genetic absence epilepsy 37%. No differences in the other pharmacodynamic parameters, E0, EC50,u and Hill factor, were observed.

It is inferred that in three different models of epilepsy there is a similar change in GABAergic functioning which is associated with a significant reduction in the intrinsic activity of midazolam in vivo. These models provide therefore a useful basis for further studies on the mechanism of epilepsy‐induced changes in pharmacodynamics of anti‐epileptic drugs.

British Journal of Pharmacology (1998) 125, 615–620; doi:10.1038/sj.bjp.0702088
Mechanism of voltage‐ and use‐dependent block of class A Ca2+ channels by mibefradil
Authors: Aczél S , Kurka B , Hering S
Published: January 2005

The action of mibefradil was studied on wild type class A calcium (Ca2+) channels and various class A/L‐type channel chimaeras expressed in Xenopus oocytes. The mechanism of Ca2+ channel block by mibefradil was evaluated with two microelectrode voltage clamp.

Resting‐state dependent block (or initial block) of barium currents (IBa) through class A Ca2+ channels was concentration dependent with an IC50 value of 208±23 μM.

Mibefradil (50 μM) did not significantly affect the midpoint voltage of the steady‐state inactivation curve suggesting that inactivation does not promote Ca2+ channel block. Chimaeric class A/L‐type Ca2+ channels inactivating with faster or slower kinetics than wild type class A channels were equally well inhibited by mibefradil as wild type class A channels.

Frequent Ca2+ channel activation facilitated IBa inhibition by mibefradil (use‐dependent block). Recovery from use‐dependent block was voltage‐dependent, being slower at depolarized membrane potentials (τ=75±15 s at −70 mV, (n=6) vs τ=20±2 s at −100 mV, (n=6), P<0.05).

We suggest that use‐dependent block of class A Ca2+ channels by mibefradil occurs because of slow recovery from open channel block (SROB) and not because of drug binding to inactivated channels.

Voltage‐dependent slow recovery from open state‐dependent block provides a molecular basis for understanding the cardiovascular profile of mibefradil such as selectivity for vasculature and relative lack of negative inotropic effects.

British Journal of Pharmacology (1998) 125, 447–454; doi:10.1038/sj.bjp.0702092
Uncoupling of bradykinin‐induced phosphoinositide hydrolysis and Ca2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells
Authors: Yang Chuen‐Mao , Luo Shue‐Fen , Wu Wen‐Bin , Pan Shiow‐Lin , Tsai Yih‐Jeng , Chiu Chi‐Tso , Wang Chuan‐Chwan
Published: January 2005

Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Stimulation of TECs by bradykinin (BK) led to IPs formation and caused an initial transient [Ca2+]i peak in a concentration‐dependent manner.

Pretreatment of TECs with phorbol 12‐myristate 13‐acetate (PMA, 1 μM) for 30 min attenuated the BK‐induced IPs formation and Ca2+ mobilization. The maximal inhibition occurred after incubating the cells with PMA for 2 h.

The concentrations of PMA that gave half‐maximal (pEC50) inhibition of BK‐induced IPs accumulation and an increase in [Ca2+]i were 7.07 M and 7.11 M, respectively. Inactive phorbol ester, 4α‐phorbol 12,13‐didecanoate at 1 μM, did not inhibit these responses. Prior treatment of TECs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK‐induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC.

In parallel with the effect of PMA on the BK‐induced IPs formation and Ca2+ mobilization, the translocation and down‐regulation of PKC isozymes were determined. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC‐α, βI, βII, γ, δ, ε, Θ and ζ. With PMA treatment of the cells for various times, translocation of PKC‐α, βI, βII, γ, δ, ε and Θ from cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 6 h treatment caused a partial down‐regulation of these PKC isozymes. PKC‐ζ was not significantly translocated and down‐regulated at any of the times tested.

Treatment of TECs with 1 μM PMA for either 30 min or 6 h did not significantly change the KD and Bmax receptor for BK binding (control: KD=1.7±0.3 nM; Bmax=50.5±4.9 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK‐induced responses.

In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit independently both responses to BK. The translocation of pKC‐α, βI, βII, δ, ε, γ, and Θ induced by PMA caused an attenuation of BK‐induced IPs accumulation and Ca2+ mobilization in TECs.

British Journal of Pharmacology (1998) 125, 627–636; doi:10.1038/sj.bjp.0702094
Frequency‐dependent inhibition of neuronal activity by topiramate in rat hippocampal slices
Authors: Wu Shang‐Peng , Tsai Jing‐Jane , Gean Po‐Wu
Published: January 2005

Topiramate is a structurally novel anticonvulsant which was recently approved for adjunctive therapy in partial and secondarily generalized seizures. The present study was aimed at elucidating the mechanisms underlying the anticonvulsant efficacy of topiramate using intra‐ and extracellular recording techniques in the in vitro hippocampal slices.

When stimuli were delivered every 20 s, topiramate had no measurable effect on both field excitatory postsynaptic potentials (fEPSPs) and population spikes (PSs). However, increasing the stimulation frequency from 0.05–0.2 Hz, topiramate significantly decreased the slope of fEPSP and the amplitude of PS in a concentration‐dependent manner. The amplitude of presynaptic fiber volley was also reduced.

Topiramate did not affect the magnitude of paired‐pulse inhibition and monosynaptically evoked inhibitory postsynaptic potentials (IPSPs).

Sustained repetitive firing was elicited by injection of long duration (500 ms) depolarizing current pulses (500–800 pA). Superfusion with topiramate significantly reduced the number of action potentials evoked by a given current pulse.

After blockade of GABA receptors by bicuculline, burst firing which consisted of a train of several spikes riding on a large depolarizing wave termed paroxysmal depolarizing shift (PDS) was recorded. Application of topiramate reduced the duration of PDS and later spikes with less effect on the initial action potential.

These results suggest that frequency‐dependent inhibition of neuronal activity due to blockade of Na+ channels may account largely for the anticonvulsant efficacy of topiramate.

British Journal of Pharmacology (1998) 125, 826–832; doi:10.1038/sj.bjp.0702096
Enhanced acetylcholine induced relaxation in small mesenteric arteries from pregnant rats: an important role for endothelium‐derived hyperpolarizing factor (EDHF)
Authors: Gerber R T , Anwar M A , Poston L
Published: January 2005

Small mesenteric arteries from pregnant rats demonstrated greater sensitivity (pEC50 : P<0.001) and maximum relaxation (P<0.01) to acetylcholine (ACh) than those of control non‐pregnant animals.

Maximum relaxation, but not sensitivity, to ACh remained greater (P<0.01) in pregnant animals when evaluated in 25 mM KCl, which prevents relaxation dependent upon hyperpolarization. ACh induced relaxation in the presence of 25 mM KCl was completely inhibited in pregnant and non‐pregnant groups by Nω‐nitro L‐arginine methyl ester (L‐NAME, 100 μM), indomethacin (INDO, 10 μM) and oxadiazole quinoxalin (ODQ, 1 μM), suggesting pregnancy associated enhancement of dilator prostanoid and/or nitric oxide (NO) synthesis.

ACh induced relaxation in 5 mM KCl was only partially inhibited by a combination of Nω‐nitro L‐arginine methyl ester (L‐NAME, 100 μM), indomethacin (INDO, 10 μM) and oxadiazole quinoxalin (ODQ, 1 μM). The residual relaxation, which was greater in arteries from pregnant rats (maximum relaxation: P<0.01), was prevented by 25 mM KCl, indicating pregnancy associated enhanced synthesis/reduced degradation of a hyperpolarizing factor. Residual relaxation to ACh in 5 mM KCl was inhibited by the cytochrome P450 inhibitor, proadifen (1 μM) in the pregnant group (P<0.001).

Relaxation to spermine NONOate was similar in pregnant and non‐pregnant groups and totally inhibited by ODQ (in the presence of L‐NAME).

This study suggests that, in addition to enhanced endothelium dependent NO/dilator prostanoid synthesis, a hyperpolarizing factor may contribute to the vascular adaptation to pregnancy.

British Journal of Pharmacology (1998) 125, 455–460; doi:10.1038/sj.bjp.0702099
Effects of the Aconitum alkaloid songorine on synaptic transmission and paired‐pulse facilitation of CA1 pyramidal cells in rat hippocampal slices
Authors: Ameri Angela
Published: January 2005

The present study investigated the electrophysiological effects of songorine (1–100 μM), an alkaloid occurring in plants of the Aconitum genus, in rat hippocampal slices.

Songorine (10–100 μM) evoked a concentration‐dependent increase in the amplitude of the orthodromic population spike and in the slope of the field e.p.s.p. The enhancement was long‐lasting and was not reversed by up to 90 min of washout. Songorine failed to affect size and shape of the presynaptic fiber spike which represents the compound action potential of the Schaffer collaterals. This indicates that enhancement of the synaptic response is no consequence of an increased afferent excitability.

The antidromically evoked population spike was not affected by songorine at concentrations up to 100 μM suggesting that the enhancement of the orthodromic population spike and of the field e.p.s.p. was not due to an increase in pyramidal cell excitability.

The input‐output curve for the postsynaptic population spike was shifted to the left implying that a presynaptic fiber spike of the same size elicited a larger postsynaptic response, indicating a decrease in threshold for generation of the population spike.

The songorine‐evoked increase in excitability was not affected by the NMDA receptor antagonist, D‐AP5. However, the effect of songorine was completely abolished by the selective dopamine D2 receptor antagonist sulpiride (0.1 μM) as well as by haloperidol (10 μM) and was mimicked by application of the dopamine releaser, amantadine (100 mM). In contrast, the selective D1 receptor antagonist, SCH23390, did not block the action of songorine.

The results indicate that the plant alkaloid songorine enhances excitatory synaptic transmission which may be due to an agonistic action at D2 receptors.

British Journal of Pharmacology (1998) 125, 461–468; doi:10.1038/sj.bjp.0702100
Haemodynamic effects of a selective adenosine A2A receptor agonist, CGS 21680, in chronic heart failure in anaesthetized rats
Authors: Nekooeian Ali A , Tabrizchi Reza
Published: January 2005

Recently we demonstrated that the administration of an A2A adenosine receptor agonist, CGS 21680, to anaesthetized rats with acute heart failure (1 h post‐coronary artery ligation) resulted in an increase in cardiac output. In the present investigation, the effects of CGS 21680 on cardiac output, vascular resistance, heart rate, blood pressure and mean circulatory filling pressure (Pmcf) were investigated in anaesthetized rats with chronic heart failure (8 weeks post‐coronary artery ligation).

Experiments were conducted in five groups (n=6) of animals: sham‐operated vehicle‐treated (0.9% NaCl; 0.037 mL kg−1 min−1) animals in which the occluder was placed but not pulled to ligate the coronary artery; coronary artery‐ligated vehicle‐treated animals; and coronary artery‐ligated CGS 21680‐treated (0.1, 0.3 or 1.0 μg kg−1 min−1) animals.

Baseline blood pressure, cardiac output and rate of rise in left ventricular pressure (+dP/dt) were significantly reduced in animals with coronary artery ligation when compared to sham‐operated animals. Coronary artery ligation resulted in a significant increase in left ventricular end‐diastolic pressure, Pmcf and venous resistance when compared to sham‐operated animals.

Administration of CGS 21680 at 0.3 and 1.0 μg kg−1 min−1 significantly (n=6; P<0.05) increased cardiac output by 19±4% and 39±5%, and heart rate by 14±2% and 15±1%, respectively, when compared to vehicle treatment in coronary artery‐ligated animals. Administration of CGS 21680 also significantly reduced blood pressure and arterial resistance when compared to coronary artery‐ligated vehicle‐treated animals. Infusion of CGS 21680 also significantly reduced venous resistance when compared to vehicle‐treated coronary artery‐ligated animals.

The results show that heart failure is characterized by reduced cardiac output, and increased left ventricular end‐diastolic pressure, venous resistance and Pmcf. Acute treatment with CGS 21680 in animals with chronic heart failure decreased left ventricular end‐diastolic pressure and increased cardiac output. This increase in cardiac output was the result of reduced arterial and venous resistances and increased heart rate.

British Journal of Pharmacology (1998) 125, 651–658; doi:10.1038/sj.bjp.0702105
The signal transduction mechanism involved in kazinol B‐stimulated superoxide anion generation in rat neutrophils
Authors: Wang Jih P , Tsao Lo T , Raung Shue L , Lin Chun N
Published: January 2005

In this study, the underlying mechanism of stimulation of respiratory burst by kazinol B, a natural isoprenylated flavan, in rat neutrophils in vitro was investigated.

Kazinol B concentration‐dependently stimulated the superoxide anion (O2[dot over 2]−) generation, with a lag but transient activation profile, in neutrophils but not in a cell‐free system. The maximum response (13.2±1.4 nmol O2[dot over 2]− 10 min−1 per 106 cells) was observed at 10 μM kazinol B.

Pretreatment of neutrophils with phorbol 12‐myristate 13‐acetate (PMA) or formylmethionyl‐leucyl‐phenylalanine (fMLP) significantly enhanced the O2[dot over 2]− generation following the subsequent stimulation of cells with kazinol B.

Cells pretreated with EGTA or a protein kinase inhibitor staurosporine effectively attenuated the kazinol B‐induced O2[dot over 2]− generation. However, a p38 mitogen‐activated protein kinase (MAPK) inhibitor SB203580 and a phosphoinositide 3‐kinase (PI3K) inhibitor wortmannin had no effect on the kazinol B‐induced response.

Kazinol B significantly stimulated [Ca2+]i elevation in neutrophils, with a lag and slow rate of rise activation profile, and this response was attenuated by a phospholipase C (PLC) inhibitor U73122. Kazinol B also stimulated the inositol bis‐ and trisphosphate (IP2 and IP3) formation with a 1 min lag time.

The membrane‐associated PKC‐α and PKC‐θ but not PKC‐ι were increased following the stimulation of neutrophils with kazinol B. It was more rapid and sensitive in the activation of PKC‐θ than PKC‐α by kazinol B. Kazinol B partially inhibited the [3H]phorbol 12,13‐dibutyrate ([3H]PDB) binding to the neutrophil cytosolic PKC.

Neither the cellular mass of phosphatidic acid (PA) and phosphatidylethanol (PEt), in the presence of ethanol, nor the protein tyrosine phosphorylation were stimulated by kazinol B. In addition, the kazinol B‐induced O2[dot over 2]− generation remained relatively unchanged in cells pretreated with ethanol or a tyrosine kinase inhibitor genistein.

Collectively, these results indicate that the stimulation of the respiratory burst by kazinol B is probably mediated by the synergism of PKC activation and [Ca2+]i elevation in rat neutrophils.

British Journal of Pharmacology (1998) 125, 517–525; doi:10.1038/sj.bjp.0702106
Phosphorylation and IGF‐1‐mediated dephosphorylation pathways control the activity and the pharmacological properties of skeletal muscle chloride channels
Authors: De Luca Annamaria , Pierno Sabata , Liantonio Antonella , Camerino Claudia , Conte Camerino Diana
Published: January 2005

In the present study we tested the hypothesis that insulin‐like growth factor‐1 (IGF‐1) modulates resting chloride conductance (GCl) of rat skeletal muscle by activating a phosphatase and that the chloride channel, based on the activity of phosphorylating‐dephosphorylating pathways, has different sensitivity to specific ligands, such as the enantiomers of 2‐(p‐chlorophenoxy) propionic acid (CPP).

For this purpose GCl in EDL muscle isolated from adult rat was first lowered by treatment with 5 nM 4‐β‐phorbol 12,13 dibutyrate (4‐β‐PDB), presumably activating protein kinase C (PKC). The effects of IGF‐1 and of the enantiomers of CPP on GCl were then tested.

IGF‐1 (3.3 nM) had no effect of GCl on EDL muscle fibres in normal physiological solution, whereas it completely counteracted the 30% decrease of GCl induced by 4‐β‐PDB. No effects of IGF‐1 were observed on GCl lowered by the phosphatase inhibitor okadaic acid (0.25 μM).

Ceramide, reported to activate on okadaic acid‐sensitive phosphatase, mimicked the effects of IGF‐1. In fact, N‐acetyl‐sphingosine (2.5–5 μM), not very effective in control conditions, increased the GCl lowered by the phorbol ester, but not the GCl lowered by okadaic acid.

In the presence of 4‐β‐PDB, GCl was differently affected by the enantiomers of CPP. The S(−)‐CPP was remarkably less potent in producing the concentration‐dependent reduction of GCl, whereas the R(+)‐CPP caused an increase of GCl at all the concentrations tested.

In conclusion, the PKC‐induced lowering of GCl is counteracted by IGF‐1 through an okadaic acid sensitive phosphatase, and this effect can have therapeutic relevance in situations characterized by excessive channel phosphorylation. In turn the phosphorylation state of the channel can modulate the effects and the therapeutic potential of direct channel ligands.

British Journal of Pharmacology (1998) 125, 477–482; doi:10.1038/sj.bjp.0702107
Induction by interleukin‐1, tumor necrosis factor and lipopolysaccharides of histidine decarboxylase in the stomach and prolonged accumulation of gastric acid
Authors: Endo Yasuo , Kumagai Katsuo
Published: January 2005

Injection of interleukin‐1 (IL‐1) into pylorus‐ligated rats has been shown strongly to inhibit gastric secretion. However, in the present study, we found that an intraperitoneal injection of IL‐1 into intact (non‐pylorus‐ligated) fasted mice rapidly (within 30 min) induced an accumulation of gastric acid (‘early response’). When the dose of IL‐1 was larger, the accumulation lasted for a longer period.

Injection of IL‐1 also caused a later elevation of the activity of histidine decarboxylase (HDC), the histamine‐forming enzyme, in the stomach (‘later response’).

Cimetidine, an antagonist of histamine H2‐receptors, suppressed the accumulation of gastric acid in both the early and later periods. An irreversible inhibitor of HDC, α‐fluoromethylhistidine, partially inhibited the accumulation in the later period.

IL‐1, when injected 1 h after feeding in mice fasted overnight, markedly retarded gastric emptying.

Tumour necrosis factor (TNF) and lipopolysaccharide (LPS) or endotoxin from E. coli both had IL‐1‐like effects on the stomach, and their effects are presumably mediated by IL‐1.

These results support the idea that an inhibition of gastric emptying and an elevation of HDC activity in the stomach may explain the findings that a long‐lasting accumulation of gastric acid is induced by IL‐1 despite its potent inhibition of gastric acid secretion.

On the basis of these results, and in the light of the known actions of histamine, the possible roles of IL‐1 in gastric inflammation and ulceration are discussed.

British Journal of Pharmacology (1998) 125, 842–848; doi:10.1038/sj.bjp.0702108
The actions of some cannabinoid receptor ligands in the rat isolated mesenteric artery
Authors: White Richard , Robin Hiley C
Published: January 2005

The actions of a number of cannabinoid receptor ligands were investigated using the myograph‐mounted rat isolated mesenteric artery. Anandamide, CP 55,940, HU‐210, palmitoylethanolamide and WIN 55,212‐2 all caused concentration‐dependent relaxations of methoxamine‐precontracted vessels which were not affected by removal of the endothelium.

Precontracting vessels with 60 mM KCl instead of methoxamine greatly reduced the vasorelaxant effects of anandamide and palmitoylethanolamide. High K+ solution caused a modest decrease in the relaxant potency of CP 55,940 and HU‐210, and had no effect on relaxations induced by WIN 55,212‐2.

Relaxations of methoxamine‐induced tone by anandamide, CP 55,940 and HU‐210, but not palmitoylethanolamide and WIN 55,212‐2, were attenuated by the cannabinoid receptor antagonist, SR 141716A. Relaxation of vessels contracted with 60 mM KCl by CP 55,940 was also sensitive to SR 141716A.

Anandamide and CP 55,940 caused small but concentration‐dependent contractions in resting vessels in the absence of extracellular calcium. These were not sensitive to SR 141716A. Palmitoylethanolamide and WIN 55,212‐2 produced smaller contractions only at higher concentrations.

Anandamide and CP 55,940, but not palmitoylethanolamide and WIN 55,212‐2, caused concentration‐dependent inhibition of the phasic contractions induced by methoxamine in calcium‐free conditions, but only anandamide caused inhibition of contractions to caffeine under such conditions. These inhibitory effects were not antagonised by SR 141716A.

The present study provides the first detailed investigation of the actions of cannabinoid agonists on vascular smooth muscle. Our results show that these compounds exert both receptor‐dependent and ‐independent effects on agonist‐induced calcium mobilization in the rat isolated mesenteric artery.

British Journal of Pharmacology (1998) 125, 533–541; doi:10.1038/sj.bjp.0702111
Discovery of novel selective hypotensive vasopressin peptides that exhibit little or no functional interactions with known oxytocin/vasopressin receptors
Authors: Chan W Y , Wo N C , Stoev S , Cheng L L , Manning M
Published: January 2005

Arginine‐vasopressin (VP) has both vasoconstricting and vasodilating action. We report here the discovery of four novel selective hypotensive VP analogues: d(CH2)5[D‐Tyr(Et)2,Arg3,Val4]AVP; d(CH2)5[D‐Tyr(Et)2,Lys3,Val4]AVP and their iodinatable Tyr‐NH29 analogues.

Bioassays in rats for activities characteristic of neurohypophysial peptides showed that the four VP peptides possessed little or no V1a, V2 or oxytocin (OT) receptor agonistic or antagonistic activities.

In anaesthetized rats, these peptides (0.05–0.10 mg kg−1 i.v.) elicited a marked fall in arterial blood pressure.

Blockade of cholinoceptors, adrenoceptors and bradykinin B2 receptors, and inhibition of prostaglandin synthesis had little effect on their vasodepressor action.

Classical V1a, V2 and OT receptor antagonists did not block the vasodepressor response.

L‐NAME, 0.2 mg kg−1 min−1, markedly suppressed the hypotensive response to ACh but not the vasodepressor response to the hypotensive VP peptides. However, the duration of the vasodepressor response was shortened. Very high doses of L‐NAME attenuated both the vasodepressor response and the duration of action.

These findings indicate that the vasodepressor action of these VP peptides is independent of the peripheral autonomic, bradykinin and PG systems and is not mediated by the known classical OT/VP receptors. NO does not appear to have an important role in their vasodepressor action.

The discovery of these novel VP peptides could lead to the development of new tools for the investigation of the complex cardiovascular actions of VP and the introduction of a new class of hypotensive agents. The two iodinatable hypotensive VP peptides could be radiolabelled as potential markers for the localization of the receptor system involved.

British Journal of Pharmacology (1998) 125, 803–811; doi:10.1038/sj.bjp.0702114
Pharmacological and molecular characterization of P2X receptors in rat pelvic ganglion neurons
Authors: Zhong Yu , Dunn Philip M , Xiang Zhenghua , Bo Xuenong , Burnstock Geoffrey
Published: January 2005

The presence and characteristics of P2X receptors on neurons of the rat major pelvic ganglia (MPG) have been studied using whole cell voltage‐clamp, in situ hybridization and immunohistochemistry.

Rapid application of ATP (100 μM) to isolated rat MPG neurons induced moderately large inward currents (0.33–5.3 nA) in 39% of cells (108/277). The response to ATP occurred very rapidly, with an increase in membrane conductance, and desensitized slowly.

The concentration‐response curve for ATP yielded an EC50 of 58.9 μM. The agonist profile was ATP2MeSATP=ATPγS>BzATP, while α,β‐MeATP, β,γ‐MeATP, UTP and ADP were all inactive at concentrations up to 100 μM.

The response to ATP was antagonized by suramin (pA2=5.6), reactive blue‐2 (IC50=0.7 μM) and pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS).

Lowering the pH from 7.4 to 6.8 produced a marked potentiation (to 339% of control) of the responses to ATP (30 μM), while raising the pH to 8.0 attenuated the responses (to 20% of control). The EC50s for ATP were 28.8, 58.9 and 264 μM at pH 6.8, 7.4 and 8.0, respectively.

Co‐application of ATP with Zn2+ produced a marked enhancement of the responses to ATP, with an EC50 of 9.55 μM. In the presence of Zn2+ (30 μM), the EC50 for ATP was decreased to 4.57 μM.

In situ hybridization revealed that the P2X receptor transcripts levels in rat MPG neurons are P2X2>P2X4>P2X1, P2X3, P2X5 and P2X6. The immunohistochemical staining revealed a small number of neurons with strong P2X2 immunoreactivity.

In conclusion, our results indicate that there are P2X receptors present on MPG neurons. The pharmacological characteristics of these receptors, the in situ hybridization and immunohistochemical evidence are consistent with them being of the P2X2 subtype, or heteromultimers, with P2X2 being the dominant component.

British Journal of Pharmacology (1998) 125, 771–781; doi:10.1038/sj.bjp.0702118
Effects of intracerebroventricular leptin administration on food intake, body weight gain and diencephalic nitric oxide synthase activity in the mouse
Authors: Calapai Gioacchino , Corica Francesco , Allegra Alessandro , Corsonello Andrea , Sautebin Lidia , De Gregorio Tiziana , Di Rosa Massimo , Costantino Giuseppina , Buemi Michele , Caputi Achille P
Published: January 2005

Intracranial administration of leptin reduces both food intake and body weight gain in the mouse. Inhibitors of nitric oxide (NO) synthase produce similar effects.

To investigate the role of the brain L‐arginine/NO pathway in mediating this effect of leptin, we have evaluated food intake and body weight gain after daily (5 days) intracerebroventricular (i.c.v.) administration of leptin (0.5–2 μg) alone or in association with L‐arginine (10 μg). Moreover, we measured diencephalic nitric oxide synthase (NOS) activity after a single i.c.v. leptin (0.25–2 μg) injection and after consecutive doses of leptin (0.25–2 μg) over 5 days. The time course of the effect of leptin on NOS activity was also evaluated.

I.c.v. injected leptin (1 and 2 μg) significantly and dose‐dependently reduced food intake and body weight gain with respect to vehicle (food intake: 5.97±0.16 g 24 h−1 and 4.27±0.18 g 24 h−1, respectively, vs 8.05±0.34 g 24 h−1, P<0.001, n=6 for each group; body weight gain: −10.7±0.46% and −15.2±0.65%, respectively, vs 5.14±0.38%, P<0.001, n=6 for each group). This effect was antagonized by L‐arginine (food intake: 7.90±0.37 g 24 h; body weight gain: 5.11±0.31%, n=6). Diencephalic NOS activity was significantly reduced by the highest doses of leptin with respect to vehicle (vehicle: 0.90±0.04 nmol citrulline min−1 g−1 tissue; leptin 1 μg: 0.62±0.03 nmol citrulline min−1 g−1 tissue, P<0.001; leptin 2 μg: 0.44±0.03 nmol citrulline min−1 g−1 tissue, P<0.001, n=6 for each group). Similar results were obtained in animals treated with daily consecutive doses of leptin. The inhibitory effect appeared rapidly (within 30 min) and was long lasting (up to 12 h).

Our results suggest that the brain L‐arginine/NO pathway may be involved in the central effect of leptin on feeding behaviour and body weight gain in mice.

British Journal of Pharmacology (1998) 125, 798–802; doi:10.1038/sj.bjp.0702121
Effects of lysophosphatidylcholine on the production of interstitial adenosine via protein kinase C‐mediated activation of ecto‐5′‐nucleotidase
Authors: Sato Toshiaki , Obata Toshio , Yamanaka Yasumitsu , Arita Makoto
Published: January 2005

Adenosine plays a crucial role in the evolution of ischemic preconditioning. With the use of microdialysis techniques in in situ rat hearts, we assessed the activity of ecto‐5′‐nucleotidase (a key enzyme responsible for adenosine production), and examined the effects of lysophosphatidylcholine (LPC) on the production of interstitial adenosine.

The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rat hearts and perfused with Tyrode solution containing adenosine 5′‐monophosphate (AMP, 100 μM). With this system, the dialysate adenosine originates from the dephosphorylation of AMP, catalyzed by endogenous ecto‐5′‐nucleotidase. The level of dialysate adenosine is a measure of the ecto‐5′‐nucleotidase activity in vivo.

LPC at concentrations of 25 and 50 μM significantly increased the level of dialysate adenosine to 122.7±4.3% (n=4, P<0.05) and 158.6±7.2% (n=5, P<0.05) of the control, respectively. Chelerythrine (200 μM), a protein kinase C (PKC) inhibitor, completely abolished the increase of dialysate adenosine afforded by LPC (50 μM) (n=5).

These data provide the first evidence that LPC does increase the concentration of interstitial adenosine in rat hearts in situ, through the PKC‐mediated activation of endogenous ecto‐5′‐nucleotidase.

British Journal of Pharmacology (1998) 125, 493–498; doi:10.1038/sj.bjp.0702122
The actions of the cannabinoid receptor antagonist, SR 141716A, in the rat isolated mesenteric artery
Authors: White Richard , Robin Hiley C
Published: January 2005

The actions of the cannabinoid receptor antagonist, SR 141716A, were examined in rat isolated mesenteric arteries. At concentrations greater than 3 μM, it caused concentration‐dependent, but endothelium‐independent, relaxations of both methoxamine‐ and 60 mM KCl‐precontracted vessels.

SR 141716A (at 10 μM, but not at 1 μM) inhibited contractions to Ca2+ in methoxamine‐stimulated mesenteric arteries previously depleted of intracellular Ca2+ stores. Neither concentration affected the phasic contractions induced by methoxamine in the absence of extracellular Ca2+.

SR 141716A (10 μM) caused a 130 fold rightward shift in the concentration‐response curve to levcromakalim, a K+ channel activator, but had no effect at 1 μM.

SR 141716A (10 μM) attenuated relaxations to NS 1619 (which activates large conductance, Ca2+‐activated K+ channels; BKCa). The inhibitory effect of SR 141716A on NS 1619 was not significantly different from, and was not additive with, that caused by a selective BKCa inhibitor, iberiotoxin (100 nM). SR 141716A (1 μM) did not effect NS 1619 relaxation.

SR 141716A (10 μM) had no effect on relaxations to the nitric oxide donor S‐nitroso‐N‐acetylpenicillamine, or relaxations to carbachol in the presence of 25 mM KCl.

The results show that, at concentrations of 10 μM and above, SR 141716A causes endothelium‐independent vasorelaxation by inhibition of Ca2+ entry. It also inhibits relaxations mediated by K+ channel activation. This suggests that such concentrations of SR 141716A are not appropriate for investigation of cannabinoid receptor‐dependent processes.

British Journal of Pharmacology (1998) 125, 689–696; doi:10.1038/sj.bjp.0702127