bluuebunny/crossref_metadata_2025 huggingface dataset:
Contractile 5‐HT1B receptors in human cerebral arteries: pharmacological characterization and localization with immunocytochemistry
Authors: Nilsson Torun , Longmore Jenny , Shaw David , Olesen Inger Jansen , Edvinsson Lars
Published: January 2005

The cerebrovascular receptor(s) that mediates 5‐hydroxytryptamine (5‐HT)‐induced vasoconstriction in human cerebral arteries (HCA)has proven difficult to characterize, yet these are essential in migraine. We have examined 5‐HT receptor subtype distribution in cerebral blood vessels by immunocytochemistry with antibodies selective for human 5‐HT1B and human 5‐HT1D receptors and also studied the contractile effects of a range of 5‐HT receptor agonists and antagonists in HCA.

Immunocytochemistry of cerebral arteries showed dense 5‐HT1B receptor immunoreactivity (but no 5‐HT1D receptor immunoreactivity) within the smooth muscle wall of the HCA. The endothelial cell layer was well preserved and weak 5‐HT1B receptor immunoreactivity was present.

Pharmacological experiments on HCA with intact endothelium showed that 5‐carboxamidotryptamine was significantly more potent than α‐methyl‐5‐HT, 2‐methyl‐5‐HT and 5‐HT in causing vasoconstriction. The 5‐HT1B/1D receptor agonists naratriptan, sumatriptan, zolmitriptan and 181C91 (N‐desmethyl zolmitriptan), all induced equally strong contractions and with similar potency as 5‐HT. The maximum contractile response was significantly less for avitriptan and dihydroergotamine. There was a significant correlation between vasoconstrictor potency and 5‐HT1B‐ and 5‐HT1D‐receptor affinity, but not with 5‐HT1A‐, 5‐ht1F or 5‐HT2‐ receptor affinity.

The 5‐HT1B/1D‐receptor antagonist GR 55562 (10−7–10−6 M) inhibited the contractile responses to sumatriptan and 5‐CT in a competitive manner with a pKB value for GR 55562 of 7.4. Furthermore, ketanserin (10−7 M), prazosin (10−7 M), and sulpiride (10−7 M) were devoid of significant antagonistic activity of 5‐HT‐induced contraction in the HCA.

The results are compatible with the hypothesis that the 5‐HT1B receptors play a major role in 5‐HT‐induced vasoconstriction in HCA.

British Journal of Pharmacology (1999) 128, 1133–1140; doi:10.1038/sj.bjp.0702773
Endothelium‐derived hyperpolarizing factor but not NO reduces smooth muscle Ca2+ during acetylcholine‐induced dilation of microvessels
Authors: Bolz Steffen‐Sebastian , De Wit Cor , Pohl Ulrich
Published: January 2005

We hypothesized that nitric oxide (NO) and the endothelium‐dependent hyperpolarizing factor (EDHF) may dilate microvessels by different cellular mechanisms, namely Ca2+‐desensitization versus decrease in intracellular free calcium.

Effects of acetylcholine (ACh) and the NO donors sodium nitroprusside (SNP, 0.1–10 μmol l−1) and S‐Nitroso‐N‐acetyl‐D,L‐penicillamine (SNAP, 0.01–10 μmol l−1) on intracellular calcium ([Ca2+]i, fura 2) and vascular diameter (videomicroscopy) were studied in isolated resistance arteries from hamster gracilis muscle (194±12 μm) pretreated with indomethacin and norepinephrine. Membrane potential changes were determined using 1,3‐dibutylbarbituric acid trimethineoxonol (DiBAC4(3)).

ACh (0.1 and 1 μmol l−1)‐induced dilations were associated with a [Ca2+]i decrease (by 13±3 and 32±4%) and hyperpolarization of vascular smooth muscle (VSM, by 12±1% at 1 μmol l−1 ACh). Nω‐nitro‐L‐arginine (L‐NA, 30 μmol l−1) partially inhibited the dilation but did not affect VSM [Ca2+]i decreases or hyperpolarization. In contrast, the KCa channel inhibitors tetrabutylammonium (TBA, 1 mmol l−1) and charybdotoxin (ChTX, 1 μmol l−1) abolished the ACh‐induced [Ca2+]i decrease and the hyperpolarization in VSM while a significant dilation remained (25 and 40%). This remaining dilation was abolished by L‐NA. ChTX did not affect [Ca2+]i increase and hyperpolarization in endothelial cells. SNP‐ or SNAP‐induced dilations were not associated with decreases in VSM [Ca2+]i or hyperpolarization although minor transient decreases in VSM [Ca2+]i were observed at high concentrations.

These data suggest that ACh‐induced dilations in microvessels are predominantly mediated by a factor different from NO and PGI2, presumably EDHF. EDHF exerts dilation by activation of KCa channels and a subsequent decrease in VSM [Ca2+]i, NO dilates the microvessels in a calcium‐independent manner.

British Journal of Pharmacology (1999) 128, 124–134; doi:10.1038/sj.bjp.0702775
Influence of redox compounds on nitrovasodilator‐induced relaxations of rat coronary arteries
Authors: Murphy Michael E
Published: January 2005

Various classes of nitrovasodilators release nitric oxide (NO) through distinct reaction pathways, many of which involve endogenous reductants and/or oxidants. This study examined relaxations of isolated rat coronary arteries induced by spermine NONOate (SPNO), 3‐morpholinosydnonimine (SIN‐1), nitroprusside (NP), S‐nitroso‐N‐acetylpenicillamine (SNAP) and nitroglycerin (NTG) in order to assess whether their potency was influenced by any of six redox compounds: 1 mM ascorbate, 1 mM dehydroascorbate, 0.1 mM dithiothreitol, 10 μM diamide, 0.1 mM ferrocyanide, and 0.1 mM ferricyanide.

Only SPNO spontaneously generated NO at measurable levels. These levels were decreased by the presence of ascorbate and dithiothreitol, which likewise decreased the potency of SPNO.

The potency of SIN‐1 was unaffected by any redox compound except ferricyanide, which increased the potency not only of SIN‐1, but also of other nitrovasodilators and NO‐independent vasodilators.

The potency of NP was decreased by two structurally similar multivalent anions, ferrocyanide and ferricyanide, suggesting that NP metabolism requires ionic binding to tissue.

SNAP lost its potency in solutions containing ascorbate or dehydroascorbate. SNAP potency was also decreased by the glutathione oxidant, diamide, and by ferrocyanide and ferricyanide, suggesting that glutathione and ionic binding may be required for NO release.

NTG appeared to relax arteries via two pathways. One required only low concentrations of NTG and a labile endogenous factor that was preserved by dithiothreitol and eliminated by ferricyanide. A distinct second pathway required higher concentrations of NTG.

These distinct attributes of nitrovasodilator metabolism may underlie differences in regional specificity or tolerance development, and therefore might eventually be exploited in the development and use of nitrovasodilators.

British Journal of Pharmacology (1999) 128, 435–443; doi:10.1038/sj.bjp.0702777
Recovery of dopamine neuronal transporter but lack of change of its mRNA in substantia nigra after inactivation by a new irreversible inhibitor characterized in vitro and ex vivo in the rat
Authors: Do Régo Jean‐Claude , Syringas Maria , Leblond Bertrand , Costentin Jean , Bonnet Jean‐Jacques
Published: January 2005

In vitro, the ability of DEEP‐NCS {1‐[2‐(diphenylmethoxy)ethyl]‐4‐[2‐(4‐isothiocyanatophenyl)ethyl]‐piperazine} to inhibit [3H]‐dopamine uptake by rat striatal synaptosomes was concentration‐dependent and inversely related to the protein concentration. This inhibition was irreversible and resulted from changes in Vmax and KM. DEEP‐NCS was less potent on noradrenaline, serotonin and choline transport.

One day after intrastriatal injections of DEEP‐NCS (100 and 1000 pmol) in 20% dimethylsulphoxide, moderate decreases in the ex vivo dopamine uptake were observed in synaptosomes obtained from striatum injected with DEEP‐NCS or solvent, and the contralateral uninjected striatum.

In similar conditions, 300 pmol DEEP‐NCS in 45% 2 hydroxypropyl‐γ‐cyclodextrin–0.5% dimethylsulphoxide solution sub‐totally reduced ex vivo dopamine uptake and mazindol binding, and moderately decreased choline and serotonin transport. These reductions were specific to DEEP‐NCS‐injected striata. A clomipramine pretreatment (16 mg kg−1 i.p. 1 h before) was performed in following experiments, since it reduced the DEEP‐NCS‐elicited decrease in serotonin uptake without affecting other indices.

One day after intrastriatal injection, DEEP‐NCS elicited similar dose‐dependent decreases in ex vivo dopamine uptake and mazindol binding (ID50=6.9‐8 ng striatum−1). Changes in KM and Vmax for ex vivo dopamine transport produced by DEEP‐NCS disappeared according to similar time‐courses.

The t½ for transporter recovery was 6.1 days. This value should correspond to its actual turnover rate in vivo, since no change in transporter mRNA level was observed in substantia nigra ipsilateral to 300 pmol DEEP‐NCS‐injected striatum.

The results indicate that DEEP‐NCS behaves as a potent, quite selective, irreversible inhibitor of the DAT, in vitro and in vivo. Its use in vivo suggests that the physiological half‐life of the rat striatal DAT is close to 6 days.

British Journal of Pharmacology (1999) 128, 51–60; doi:10.1038/sj.bjp.0702784
Nepalolide A inhibits the expression of inducible nitric oxide synthase by modulating the degradation of IκB‐α and IκB‐β in C6 glioma cells and rat primary astrocytes
Authors: Wang Chuen‐Neu , Shiao Young‐Ji , Lin Yun‐Lian , Chen Chieh‐Fu
Published: January 2005

The effects of nepalolide A on the expression of inducible nitric oxide synthase (iNOS) caused by incubation with lipopolysaccharide/interferon‐γ (LPS/IFN‐γ) or tumour necrosis factor‐α/interleukin‐1β/IFN‐γ (TNF‐α/IL‐1β/IFN‐γ, mixed cytokines) in C6 glioma cells and primary astrocytes of rat were investigated. The mechanisms by which nepalolide A confers its effect on iNOS expression were also elucidated.

Treatment with LPS/IFN‐γ and mixed cytokines for 24 h elicited the induction of iNOS activity as determined by nitrite accumulation in the culture medium and assay of enzyme activity. Nepalolide A at 10 μM abrogated the LPS/IFN‐γ‐ and mixed cytokines‐mediated induction of iNOS by more than 90% in C6 glioma cells, and by 80% for mixed cytokines‐induced induction of iNOS in primary astrocytes. The effect of nepalolide A (2–10 μM) was concentration‐dependent.

The inhibition of iNOS induction by nepalolide A was attributed to decreases in the content of iNOS protein and the level of iNOS mRNA, as measured by immunoblotting and reverse transcriptase‐polymerase chain reaction.

Electrophoretic mobility shift assay was used to evaluate the effect of nepalolide A on the activation of nuclear factor‐κB (NF‐κB). Results showed that nepalolide A diminished the LPS/IFN‐γ‐mediated association of NF‐κB with consensus oligonucleotide in a concentration‐dependent manner. The activation of NF‐κB by mixed cytokines was modulated both in the extent of activation and in its time‐course by nepalolide A.

The ability of nepalolide A to inhibit NF‐κB activation was further confirmed by studies on the degradation of the inhibitor of NF‐κB, IκB, as measured by immunoblotting.

The present study demonstrates that the attenuation of NF‐κB activation by nepalolide A was mediated by blockade of the degradation of IκB, leading to suppression of the expression of iNOS.

British Journal of Pharmacology (1999) 128, 345–356; doi:10.1038/sj.bjp.0702785
Potent depression of stimulus evoked field potential responses in the medial entorhinal cortex by serotonin
Authors: Schmitz Dietmar , Gloveli Tengis , Empson Ruth M , Heinemann Uwe
Published: January 2005

The entorhinal cortex (EC), main input structure to the hippocampus, gets innervated by serotonergic terminals from the raphe nuclei and expresses 5‐HT‐receptors at high density. Using extra‐ and intracellular recording techniques we here investigated the effects of serotonin on population and cellular responses within the EC.

Stimulation in the lateral entorhinal cortex resulted in complex field potential responses in the superficial EC. The potentials are composed of an early antidromic and a late orthodromic component reflecting the efferent and afferent circuitry.

Serotonin (5‐HT) reduced synaptic potentials of the stimulus evoked extracellular field potential at all concentrations tested (0.1–100 μM; 59%‐depression by 10 μM serotonin), while the antidromic response was not significantly changed by up to 50 μM 5‐HT. Depression of field potential responses by serotonin was associated with a significant increase in paired‐pulse facilitation from 1.15 to 1.88.

The effects of serotonin on field potential responses were mimicked by 5‐HT1A‐receptor agonists (8‐OH‐DPAT, 5‐CT) and partially prevented by the 5‐HT1A‐receptor antagonist (S‐UH‐301). Moreover, the 5‐HT1A‐receptor antagonist WAY100635 reduced the effect of 5‐CT.

Fenfluramine, a serotonin releaser, mimics the effects of serotonin on stimulus‐evoked field potential responses, indicating that synaptically released serotonin can produce the changes in reactivity to afferent stimulation.

Depression of isolated AMPA‐receptor mediated EPSCs by serotonin as well as fenfluramine was associated with an increase in paired pulse facilitation, indicating a presynaptic locus of action.

We conclude that physiological concentrations of serotonin potently suppresses excitatory synaptic transmission in the superficial entorhinal cortex by a presynaptic mechanism.

British Journal of Pharmacology (1999) 128, 248–254; doi:10.1038/sj.bjp.0702788
The influence of allopurinol on kidney haemodynamic and excretory responses to renal ischaemia in anaesthetized rats
Authors: Hestin Dominique , Johns Edward J
Published: January 2005

This study examined the impact of allopurinol on the renal functional responses to a 30 min period of ischaemia in anaesthetized rats.

Immediately on reperfusion, blood pressure rose transiently, while renal blood flow remained stable throughout at control values. Glomerular filtration rate was decreased by some 90% over the first and 80% over the sixth hour (P<0.001).

Allopurinol, 50 or 100 mg kg−1, had no effect on the blood pressure or renal blood flow responses over the 6 h reperfusion period but glomerular filtration decreased by 60% initially, and to less than 30% of basal at 6 h.

Urine flow and absolute sodium excretion increased 2–3 fold in the first 2 h but decreased thereafter. Fractional sodium excretion was 30 times higher for the first 2 h but decreased reaching some 10 fold higher at 6 h. In the presence of allopurinol, urine flow and absolute sodium excretion increased by 5–6 fold in the first 2 h, and fell by half by 6 h which was greater than in the vehicle group (P<0.01). Fractional sodium excretion increased 20 fold in the allopurinol animals in the first 2 h period, but fell at a faster rate (P<0.01) than in untreated rats.

Potassium excretion decreased (P<0.05) by one half for the 6 h reperfusion period but in the allopurinol animals it was minimally altered.

Allopurinol largely ameliorated the decrease in kidney haemodynamic and excretory function following an ischeamic period for the initial few hours of reperfusion.

British Journal of Pharmacology (1999) 128, 255–261; doi:10.1038/sj.bjp.0702789
Inhibitory effect of nociceptin on [3H]‐5‐HT release from rat cerebral cortex slices
Authors: Siniscalchi Anna , Rodi Donata , Beani Lorenzo , Bianchi Clementina
Published: January 2005

The effect of nociceptin (NC) on 5‐hydroxytryptamine (5‐HT) release was studied in rat cerebral cortex slices preincubated with [3H]‐5‐HT and electrically stimulated (3 Hz, for 2 min) at the 45th (St1) and the 75th (St2) min of superfusion.

NC (0.1–3 μM), present in the medium from the 70th min onward, concentration‐dependently reduced electrically evoked [3H]‐5‐HT efflux (pEC50=6.54, Emax −54%). The inhibition was not antagonized by naloxone (1 μM) ruling out the involvement of opioid receptors.

Phe1ψ(CH2‐NH2)Gly2]NC(1‐13)NH2, which acts as an opioid‐like receptor (ORL1) antagonist at the peripheral level, behaved as a partial agonist in cerebral cortex slices i.e. it inhibited [3H]‐5‐HT efflux when added before St2, however, when present in the medium throughout the whole experiment, [Phe1ψ(CH2‐NH2)Gly2]NC(1‐13)NH2 prevented the action of NC added at the 70th min.

The non‐selective ORL1 receptor antagonist, naloxone benzoylhydrazone (3 μM), in the presence of 10 μM naloxone, did not modify the St2/St1 ratio but completely abolished the NC effect.

These findings demonstrate that NC inhibits 5‐HT release from rat cerebral cortex slices via ORL1 receptors, suggesting its involvement in central processes mediated by 5‐HT.

British Journal of Pharmacology (1999) 128, 119–123; doi:10.1038/sj.bjp.0702793
Inhibition of uptake, steady‐state currents, and transient charge movements generated by the neuronal GABA transporter by various anticonvulsant drugs
Authors: Eckstein‐Ludwig Ursula , Fei Jian , Schwarz Wolfgang
Published: January 2005

We have expressed the GABA transporter (GAT1) of mouse brain in Xenopus oocytes and have investigated the effects of four antiepileptic drugs, tiagabine (TGB), vigabatrin (VGB), gabapentin (GBP) and valproate (VAL), on GAT1 transporter function by measurements of 3H‐labelled GABA uptake and GAT1‐mediated currents.

Not only TGB, a well‐known inhibitor of GAT1‐mediated transport, but also the other drugs efficiently inhibit the uptake of [3H]‐GABA by GAT1. Inhibition at 50% is obtained for VGB, TGB, GBP, and VAL at concentrations of about 1 nM, 1 μM, 50 μM and 100 μM, respectively.

However, GAT1‐mediated steady‐state and transient currents are nearly unaffected by VGB, GBP, and VAL at even five times higher concentrations. Only TGB blocks the uptake and steady‐state and transient currents at micromolar concentrations.

VGB exhibits a complex interaction with GAT1; at concentrations about 1 nM, the inhibition of uptake is released, but at millimolar concentrations the uptake is inhibited again, and also the GAT1‐mediated current is finally inhibited at these concentrations with a KI value of 0.5 mM. The concentration dependency of inhibition of uptake can be explained by two interaction sites with different affinities, a blocking site and a transport site.

The differences in effects of VAL, GBP, and VGB on uptake and currents can be attributed to the fact that GAT1 has the capability to operate in an electrogenic mode without uptake of GABA. We suggest that inhibition occurs only when GAT1 operates in the GABA‐uptake mode.

The inhibition of GABA uptake by these four drugs will result in an elevation of the GABA concentration in the synaptic cleft, which will enhance synaptic inhibition and thereby contribute to their antiepileptic effects.

British Journal of Pharmacology (1999) 128, 92–102; doi:10.1038/sj.bjp.0702794
21‐Aminosteroids prevent the down‐regulation of hepatic cytochrome P450 induced by hypoxia and inflammation in conscious rabbits
Authors: Galal Ahmed , Du Souich Patrick
Published: January 2005

This study was conducted to assess whether a 21‐aminosteroid, U74389G, could prevent the down‐regulation of hepatic cytochrome P450 (P450) induced by acute moderate hypoxia or an inflammatory reaction.

The rabbits of two groups (n=6 per group) were subjected to acute moderate hypoxia (PaO2∼35 mmHg), one pre‐treated with U74389G (3 mg kg−1 i.v. every 6 h, for 48 h). The rabbits of two other groups received 5 ml of turpentine s.c., one of them being pre‐treated with U74389G (3 mg kg−1 i.v. every 6 h, for 72 h). The kinetics of theophylline (2.5 mg kg−1) were assessed to evaluate the activity of the P450. Once the rabbits were sacrificed, the P450 content and the amount of thiobarbituric acid reactive substances (TBARS), a marker of lipid peroxidation, were estimated in the liver.

Compared with control rabbits, hypoxia and inflammation increased theophylline plasma concentrations, as a result of a decrease in theophylline systemic clearance (P<0.05). Both experimental conditions reduced hepatic content of P450 by 40–50% (P<0.05) and increased the amount of hepatic TBARS by around 50% (P<0.05). Pre‐treatment with U74389G prevented the hypoxia‐ and inflammation‐induced decrease in theophylline systemic clearance, the down‐regulation of hepatic P450, and the increase in liver TBARS.

It is concluded that in the rabbit, U74389G prevents hepatic P450 depression produced by acute moderate hypoxia and a turpentine‐induced inflammatory reaction, possibly by eliciting a radical quenching antioxidant activity.

British Journal of Pharmacology (1999) 128, 374–379; doi:10.1038/sj.bjp.0702796
The role of P‐glycoprotein in blood‐brain barrier transport of morphine: transcortical microdialysis studies in mdr1a (−/−) and mdr1a (+/+) mice
Authors: Xie Rujia , Hammarlund‐Udenaes Margareta , De Boer Albertus G , De Lange Elizabeth C M
Published: January 2005

The aim of this study was to investigate whether blood‐brain barrier transport of morphine was affected by the absence of mdr1a‐encoded P‐glycoprotein (Pgp), by comparing mdr1a (−/−) mice with mdr1a (+/+) mice.

Mdr1a (−/−) and (+/+) mice received a constant infusion of morphine for 1, 2 or 4 h (9 nmol/min/mouse). Microdialysis was used to estimate morphine unbound concentrations in brain extracellular fluid during the 4 h infusion. Two methods of estimating in vivo recovery were used: retrodialysis with nalorphine as a calibrator, and the dynamic‐no‐net‐flux method.

Retrodialysis loss of morphine and nalorphine was similar in vivo. Unbound brain extracellular fluid concentration ratios of (−/−)/(+/+) were 2.7 for retrodialysis and 3.6 for the dynamic‐no‐net‐flux at 4 h, with corresponding total brain concentration ratios of (−/−)/(+/+) being 2.3 for retrodialysis and 2.6 for the dynamic‐no‐net‐flux. The total concentration ratios of brain/plasma were 1.1 and 0.5 for mdr1a (−/−) and (+/+) mice, respectively.

No significant differences in the pharmacokinetics of the metabolite morphine‐3‐glucoronide were observed between (−/−) and (+/+) mice.

In conclusion, comparison between mdr1a (−/−) and (+/+) mice indicates that Pgp participates in regulating the amount of morphine transport across the blood‐brain barrier.

British Journal of Pharmacology (1999) 128, 563–568; doi:10.1038/sj.bjp.0702804
Cannabinoid agonists and antagonists discriminated by receptor binding in rat cerebellum
Authors: Griffin Graeme , Wray Emma J , Martin Billy R , Abood Mary E
Published: January 2005

The effect of allosteric regulators on the binding affinity of a number of cannabinoid receptor ligands of varying efficacy in the rat cerebellum was investigated.

Radioligand ([3H]‐SR141716A) competition curves were constructed in the presence or absence of sodium ions, magnesium ions and guanine nucleotides.

It was found that the presence of these allosteric regulators did not affect the affinity of the two antagonists used but did cause a significant decrease in the affinity of full and partial agonists.

This reduction in affinity ranged from a 3.67 fold rightward shift of the displacement curve of a mixed agonist/antagonist (3‐(6‐cyano‐2‐hexynyl)‐delta‐8‐tetrahydrocannabinol‐O‐823) to a 38 fold rightward shift for 3‐(1,1‐dimethyl‐6‐dimethylcarboxamide)‐delta‐8‐tetrahydrocannabinol (O‐1125), a full agonist.

In summary, the results of this study suggest a simple method for the inference of functional data using the classical radioligand binding assay.

British Journal of Pharmacology (1999) 128, 684–688; doi:10.1038/sj.bjp.0702806
The molecular interaction of the high affinity reversal agent XR9576 with P‐glycoprotein
Authors: Martin Catherine , Berridge Georgina , Mistry Prakash , Higgins Christopher , Charlton Peter , Callaghan Richard
Published: January 2005

The kinetics and nature of equilibrium binding were used to characterize the molecular interaction of the anthranilic acid derivative [3H]‐XR9576 with the multidrug resistance P‐glycoprotein (P‐gp). XR9576 displayed specific high‐affinity binding to P‐gp (Bmax=275 pmol mg−1, Kd=5.1 nM). The transport substrates [3H]‐vinblastine and [3H]‐paclitaxel displayed 4 fold and 20 fold lower affinity respectively for P‐gp. The duration of action of XR9576 with P‐gp was increased in comparison to that of vinblastine which displayed a slower rate of association and a faster dissociation rate.

The relative affinities of several modulators and transport substrates to interact with P‐gp were determined from displacement drug equilibrium binding assays. Vinblastine and paclitaxel could only fractionally displace [3H]‐XR9576 binding, displaying Ki values significantly different from their measured Kd values. This suggests a non‐competitive interaction between XR9576 and the P‐gp substrates vinblastine and paclitaxel.

XR9576 was shown to be a potent modulator of P‐gp mediated [3H]‐vinblastine and [3H]‐paclitaxel transport as it increased the steady‐state accumulation of these cytotoxics in CHrB30 cells to levels observed in non‐P‐gp‐expressing AuxB1 cells (EC50=487±50 nM). This inhibition of drug transport is not mediated through competition for transport since [3H]‐XR9576 accumulation was not influenced by P‐gp expression or function.

These results demonstrate that the P‐gp modulator XR9576 exhibits greater selectivity, duration of inhibition and potency of interaction with this transporter than any other reported modulators. Several lines of evidence suggest that XR9576 inhibits P‐gp function by binding at a site which is distinct from the site of interaction of transport substrates. The two sites may be classified as serving modulatory or transport functions.

British Journal of Pharmacology (1999) 128, 403–411; doi:10.1038/sj.bjp.0702807
Different receptors mediating the inhibitory action of exogenous ATP and endogenously released purines on guinea‐pig intestinal peristalsis
Authors: Heinemann Ákos , Shahbazian Anaid , Barthó Loránd , Holzer Peter
Published: January 2005

Adenosine 5′‐triphosphate (ATP) is an enteric neurotransmitter which acts at purine receptors on intestinal nerve and muscle. This study set out to shed light on the receptor mechanisms by which exogenous and endogenous ATP influences intestinal peristalsis.

Peristalsis in isolated segments of the guinea‐pig small intestine was triggered by a perfusion‐induced rise of the intraluminal pressure. Motor changes were quantified by alterations of the peristaltic pressure threshold (PPT) at which propulsive muscle contractions were elicited.

ATP (3 μM) increased PPT and abolished peristalsis at concentrations of 100–300 μM. Adenosine 5′‐O‐2‐thiodiphosphate (ADPβS, 3–100 μM) was more potent, whereas α,β‐methylene ATP (α,β‐meATP, 3–100 μM) was less potent, than ATP in depressing peristalsis.

8‐Phenyltheophylline (10 μM) attenuated the anti‐peristaltic effect of 10 and 30 μM ATP but not that of higher ATP concentrations. Apamin (0.5 μM) counteracted the ability of ATP, ADPβS and α,β‐meATP to enhance PPT. Suramin (300 μM) and pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS, 150 μM) antagonized the inhibitory effect of α,β‐meATP on peristalsis but did not alter the effect of ATP and ADPβS.

PPADS (50–150 μM) reduced PPT by as much as 50%. This stimulant effect on peristalsis was prevented by suramin (300 μM) but left unaltered by apamin (0.5 μM) and NG‐nitro‐L‐arginine methyl ester (300 μM).

These data show that exogenous and endogenous ATP inhibits intestinal peristalsis via different apamin‐sensitive purinoceptor mechanisms. Exogenous ATP depresses peristalsis mostly via suramin‐ and PPADS‐insensitive P2 receptors, whereas endogenous purines act via P2 receptors sensitive to both suramin and PPADS.

British Journal of Pharmacology (1999) 128, 313–320; doi:10.1038/sj.bjp.0702808
Long‐term deprivation of oestrogens by ovariectomy potentiates β‐amyloid‐induced working memory deficits in rats
Authors: Yamada Kiyofumi , Tanaka Tomoko , Zou Li‐Bo , Senzaki Kouji , Yano Kohji , Osada Takashi , Ana Olariu , Ren Xiuhai , Kameyama Tsutomu , Nabeshima Toshitaka
Published: January 2005

In the present study, we examined whether deprivation of oestrogens by ovariectomy could modify learning and memory deficits caused by a continuous intracerebroventricular (i.c.v.) infusion of amyloid β‐peptide (Aβ), the major constituent of senile plaques in AD.

Neither long‐term (3 months) nor short‐term (1 month), deprivation of oestrogens by ovariectomy caused a significant impairment in spatial learning and memory in a water maze and spontaneous alternation behaviour in a Y‐maze.

A continuous i.c.v. infusion of Aβ‐(1‐42) caused spatial learning and memory deficits in both ovariectomized and sham‐operated rats.

The Aβ‐induced working memory deficits were significantly potentiated in ovariectomized rats compared with sham‐operated rats when mnemonic ability was examined 3 months after ovariectomy.

These results suggest that long‐term deprivation of oestrogens induced by ovariectomy increases susceptibility to memory deficits produced by Aβ‐(1‐42) in rats.

British Journal of Pharmacology (1999) 128, 419–427; doi:10.1038/sj.bjp.0702811
Ser203 as well as Ser204 and Ser207 in fifth transmembrane domain of the human β2‐adrenoceptor contributes to agonist binding and receptor activation
Authors: Sato Takayuki , Kobayashi Hiroyuki , Nagao Taku , Kurose Hitoshi
Published: January 2005

We examined the contribution of Ser203 of the human β2‐adrenoceptor (β2‐AR) to the interaction with isoprenaline. The affinity of (−)‐isoprenaline was reduced by substitution of an alanine for Ser203, as well as for Ser204 and Ser207. An (−)‐isoprenaline derivative with only one hydroxyl group, at the meta‐position, showed reduced affinity for wild‐type β2‐AR and S207A‐β2‐AR and even lower affinities for S203A‐β2‐AR and S204A‐β2‐AR. By contrast, an (−)‐isoprenaline derivative with only a para‐hydroxyl group showed reduced affinity for wild‐type β2‐AR but the serine to alanine mutations did not cause further decreases. The EC50 value for cyclic AMP generation in response to (−)‐isoprenaline was increased, by about 120 fold, for each alanine‐substituted β2‐AR mutant. These results suggest that Ser203 of the human β2‐AR is important for both ligand binding and receptor activation.

British Journal of Pharmacology (1999) 128, 272–274; doi:10.1038/sj.bjp.0702813
Constitutive activity of the δ‐opioid receptor expressed in C6 glioma cells: identification of non‐peptide δ‐inverse agonists
Authors: Neilan Claire L , Akil Huda , Woods James H , Traynor John R
Published: January 2005

G‐protein coupled receptors can exhibit constitutive activity resulting in the formation of active ternary complexes in the absence of an agonist. In this study we have investigated constitutive activity in C6 glioma cells expressing either the cloned δ‐(OP1) receptor (C6δ), or the cloned μ‐(OP3) opioid receptor (C6μ).

Constitutive activity was measured in the absence of Na+ ions to provide an increased signal. The degree of constitutive activity was defined as the level of [35S]‐GTPγS binding that could be inhibited by pre‐treatment with pertussis toxin (PTX). In C6δ cells the level of basal [35S]‐GTPγS binding was reduced by 51.9±6.1 fmols mg−1 protein, whereas in C6μ and C6 wild‐type cells treatment with PTX reduced basal [35S]‐GTPγS binding by only 10.0±3.5 and 8.6±3.1 fmols mg−1 protein respectively.

The δ‐antagonists N,N‐diallyl‐Tyr‐Aib‐Aib‐Phe‐Leu‐OH (ICI 174,864), 7‐benzylidenenaltrexone (BNTX) and naltriben (NTB), in addition to clocinnamox (C‐CAM), acted as δ‐opioid receptor inverse agonists. Naloxone, buprenorphine, and naltrindole were neutral antagonists. Furthermore, naltrindole blocked the reduction in [35S]‐GTPγS binding caused by the inverse agonists. The inverse agonists did not inhibit basal [35S]‐GTPγS binding in C6μ or C6 wild‐type cell membranes.

Competition binding assays in C6δ cell membranes revealed a leftward shift in the displacement curve of [3H]‐naltrindole by ICI 174,864 and C‐CAM in the presence of NaCl and the GTP analogue, GppNHp. There was no change in the displacement curve for BNTX or NTB under these conditions.

These data confirm the presence of constitutive activity associated with the δ‐opioid receptor and identify three novel, non‐peptide, δ‐opioid inverse agonists.

British Journal of Pharmacology (1999) 128, 556–562; doi:10.1038/sj.bjp.0702816
Thapsigargin‐induced endothelium‐dependent triphasic regulation of vascular tone in the porcine renal artery
Authors: Ihara Eikichi , Hirano Katsuya , Nishimura Junji , Nawata Hajime , Kanaide Hideo
Published: January 2005

To elucidate the role of thapsigargin‐induced Ca2+ entry in endothelial cells in the regulation of vascular tone, changes in Ca2+ and force of smooth muscle were simultaneously monitored in fura‐2‐loaded strips of porcine renal artery.

During phenylephrine‐induced sustained contraction, thapsigargin caused an endothelium‐dependent triphasic response; an initial relaxation, a subsequent transient contraction, and a sustained relaxation. The initial relaxation and the contraction were associated with a decrease and an increase in [Ca2+]i, respectively. There was no apparent [Ca2+]i decrease during the sustained relaxation. Thapsigargin‐induced responses were observed at 10−8 M and higher concentrations, with the maximum response observed at 10−6 M.

The transient contraction was inhibited by a cyclo‐oxygenase inhibitor (10−5 M indomethacin), a thromboxane A2 (TXA2)/prostagrandin H2 (PGH2) receptor antagonist (10−5 M ONO‐3708), and a TXA2 synthase inhibitor (10−5 M OKY‐046).

During the phenylephrine‐induced contraction in the presence of indomethacin, thapsigargin caused an initial, but not a sustained relaxation, in the presence of Nω‐nitro‐L‐arginine methylester (L‐NAME). During the contraction induced by phenylephrine plus 40 mM K+‐depolarization in the presence of indomethacin, thapsigargin induced both a transient and a sustained relaxation. However, these relaxations were completely abolished in the presence of L‐NAME.

Thapsigargin caused a large Ca2+ elevation in cultured endothelial cells of the renal artery. The concentration‐response relation was thus similar to that for force development in the arterial strips.

In conclusion, thapsigargin‐induced Ca2+ entry in endothelial cells led to triphasic changes in the tone of the porcine renal artery. The endothelium‐dependent contraction was mediated mainly by TXA2. Nitric oxide and hyperpolarizing factor are both involved in the initial relaxation. However, a sustained relaxation was observed which mainly depended on nitric oxide.

British Journal of Pharmacology (1999) 128, 689–699; doi:10.1038/sj.bjp.0702821
Autoradiographical and behavioural effects of a chronic infusion of antisense to the α2D‐adrenoceptor in the rat
Authors: Robinson E S J , Nutt D J , Hall L , Jackson H C , Hudson A L
Published: January 2005

The aims of this study were, firstly to use receptor autoradiography to investigate the effect of antisense oligonucleotides to the α2D‐adrenoceptor on receptor binding and, secondly to measure behavioural and physiological parameters to determine whether the chronic antisense infusion had any effect on α2‐adrenoceptor function in vivo.

A 3 day infusion of antisense to the α2D‐adrenoceptor significantly reduced specific [3H]‐RX821002 binding in the septum (20–30%) and anterior hypothalamic area (20–30%). β‐Adrenoceptor expression was unaffected in those brain areas examined, indicating the antisense knockdown was specific to the α2‐adrenoceptors.

On the second day of the infusion, the hypothermic response to UK 14,304 was significantly attenuated in the antisense‐treated group compared with both vehicle and mismatch controls. The effect was fully reversible and a similar decrease in body temperature was observed in all the treatment groups 4 days after the end of infusion.

During the second day of the infusion, the effects of UK 14,304 on behaviour were reduced in the antisense‐treated rats, but were not significantly lower than those of the vehicle and mismatch, UK 14,304 controls. These trends were not observed 4 days after the end of the infusion.

In conclusion, antisense has been shown to selectively knockdown α2‐adrenoceptor expression in specific brain areas. The consequence of this knockdown is a significant attenuation of UK 14,304‐induced hypothermia and a reduction in its sedative actions. These changes were fully reversed 4 days after the end of the infusion.

British Journal of Pharmacology (1999) 128, 515–522; doi:10.1038/sj.bjp.0702823
Catechol‐O‐methyltransferase activity in CHO cells expressing norepinephrine transporter
Authors: Percy Elodie , Kaye David M , Lambert Gavin W , Gruskin Sara , Esler Murray D , Du Xiao‐Jun
Published: January 2005

We examined the existence of catecholamine metabolizing enzymes (catechol‐O‐methyltransferase, COMT, and monoamine oxidase, MAO) in CHO cells transfected with norepinephrine (NE) transporter (NET) cDNA.

NET activity was studied by incubating cells with [3H]‐NE (0.5 μCi ml−1, 20 min) in a Na+ containing medium. Incubation with [3H]‐NE lead to [3H] accumulation at 47797±4864 d.p.m. per well. Specific inhibitors of NET abolished this uptake.

During post‐uptake incubation, [3H] leaked rapidly from cells and the extracellular phase comprised 89% of total radioactivity within 40 min. Both [3H] retention and [3H]‘leakage’ were largely unaffected by inhibitors for MAO. In contrast, COMT inhibitors, U‐0521 and Ro 41‐0960, dose‐dependently increased intracellular [3H]‐NE retention with a maximal increase of 4.5 fold. The EC50 for Ro 41‐0960 was 139‐times lower than that of U‐0521. U‐0521 largely inhibited [3H]‘leakage’ and doubled the apparent Vmax for [3H]‐NE uptake.

Addition of U‐0521 during uptake incubation increased intracellular NE content by 8 fold. Normetanephrine, the COMT‐dependent metabolite of NE, was formed in large quantities during post‐uptake incubation. U‐0521 significantly inhibited the formation of NMN with an equal preservation of intracellular NE.

CHO cells expressing NET possess COMT activity, which is responsible for the metabolism of NE to form lipophilic metabolite normetanephrine. The apparent ‘properties’ of the NET function expressed in CHO cells changed, after inhibition of COMT, in such a way closer to that described in the native neuronal preparations.

British Journal of Pharmacology (1999) 128, 774–780; doi:10.1038/sj.bjp.0702831
On the role of 5‐HT1B/1D receptors in modulating transmission in a spinal reflex pathway in the decerebrated rabbit
Authors: Ogilvie J , Wigglesworth M , Appleby L , Kingston T O W , Clarke R W
Published: January 2005

In decerebrated rabbits, the selective 5‐HT1B/1D receptor antagonist GR 127,935 had no significant effects on reflexes evoked in medial gastrocnemius motoneurones by electrical stimulation of the sural nerve, or on arterial blood pressure or heart rate when given by the intrathecal (up to 543 nmol cumulative) or intravenous (up to 1.8 μmol cumulative) routes.

In decerebrated, spinalized rabbits, intrathecal GR 127,935 in doses of up to 543 nmol, had no effect on the sural‐gastrocnemius reflex. Furthermore, this drug failed to alter enhancement of the sural‐gastrocnemius reflex induced by 8‐hydroxy‐2‐(di‐n‐propyl)aminotetralin (8‐OH‐DPAT), given at 300 nmol kg−1 i.v.

In decerebrated, spinalized rabbits, the selective 5‐HT1B/1D receptor agonists L‐694,247 (cumulative doses of 2–243 nmol kg−1 i.v.) and L‐741,604 (cumulative doses of 3–307 nmol kg−1 i.v.), each caused the sural‐gastrocnemius reflex to increase to 140% of pre‐drug levels, and arterial blood pressure to rise by about 10 mmHg. Subsequent administration of GR 127,935 at 0.9–1.8 μmol kg−1 reversed the pressor effect of the agonists but not the increase in reflexes. The 5‐HT1A receptor antagonist WAY‐100,635 (185 nmol kg−1 i.v.) also failed to reverse the increase in reflexes, but the 5‐HT1B/1D/5‐HT2/5‐HT7 ligand ritanserin (1.6 μmol kg−1 i.v.) restored reflexes to pre‐drug control values after L‐741,604 (it was not tested against L‐694,247).

These data indicate that 5‐HT1B/1D receptors do not significantly modulate transmission in the sural‐gastrocnemius reflex pathway, and that the enhancement of reflexes by 8‐OH‐DPAT and L‐741,604 is probably mediated by 5‐HT7 receptors.

British Journal of Pharmacology (1999) 128, 781–787; doi:10.1038/sj.bjp.0702832
Anti‐atherosclerotic effects of an angiotensin converting enzyme inhibitor and an angiotensin II antagonist in Cynomolgus monkeys fed a high‐cholesterol diet
Authors: Miyazaki Mizuo , Sakonjo Hiroshi , Takai Shinji
Published: January 2005

We investigated the relationship between angiotensin II formation and the development of atherosclerotic lesions in the aorta of monkeys (Macaca fascicularis) fed a high‐cholesterol (4% cholesterol and 6% corn oil) diet for 6 months, and studied the effects of an angiotensin converting enzyme (ACE) inhibitor, trandolapril (10 mg kg−1 per day, p.o.), and an angiotensin II type 1 receptor antagonist, 2‐butyl‐4‐(methylthio)‐1‐[[2′[[[(propylamino)carbonyl]amino]sulfonyl](1,1′‐biphenyl)‐4‐yl]methyl]‐1H‐imidazole‐5‐carboxylate (HR 720; 20 mg kg−1 per day, p.o.).

The level of low‐density lipoprotein was significantly increased by the cholesterol diet, whereas that of high‐density lipoprotein was significantly decreased. The relative areas of the atherosclerotic lesions in the thoracic aorta in the normal and cholesterol‐diet groups were 1.3±0.3 and 64±10%, respectively.

Plasma renin and ACE activities showed no differences between the normal and cholesterol‐diet groups. ACE activity and the concentration of angiotensin II were significantly increased in the aorta of the cholesterol‐fed monkeys.

Trandolapril and HR 720 decreased significantly the area of the atherosclerotic lesions in the thoracic aorta of cholesterol‐fed monkeys, but not the mean blood pressure and the levels of low‐density and high‐density lipoproteins.

In plasma and aorta, trandolapril, but not HR 720, decreased significantly the ACE activities in the cholesterol‐fed monkeys, while both of these drugs decreased significantly the angiotensin II levels.

In conclusion, blockade of angiotensin II function in vascular tissues by trandolapril or HR 720 may play an important role in preventing the development of atherosclerotic lesions.

British Journal of Pharmacology (1999) 128, 523–529; doi:10.1038/sj.bjp.0702833
Characterization of chemokine CCR3 agonist‐mediated eosinophil recruitment in the Brown‐Norway rat
Authors: Kudlacz Elizabeth M , Whitney Carrie A , Andresen Catharine J , Umland John P , Cheng John B
Published: January 2005

The ability of various C‐C chemokines to elicit tissue eosinophil infiltration following intradermal injection or peripheral blood eosinophilia following intravenous injection were compared in the Brown‐Norway rat.

Eotaxin (0.1–3 μg site−1) of human and murine origin produced equivalent, dose‐dependent increases in eosinophil peroxidase activity in rat dermis 4 h post‐injection.

Human eotaxin‐2 was equipotent with human eotaxin in terms of dermal eosinophil recruitment. Other human CCR3 agonists, such as MCP‐3, RANTES and MCP‐4 failed to increase dermal eosinophil peroxidase activity at doses up to 1 μg site−1 whereas the latter did produce a small effect at 3 μg site−1.

Consistent with observations in vivo, human eotaxin displaced [125I]‐eotaxin from rat spleen membranes more potently (IC50=2 nM) than did MCP‐4 (IC50=500 nM). RANTES did not compete with the radiolabelled chemokine at concentrations up to 1 μM.

Human eotaxin (5 μg) administered intravenously increased circulating eosinophils ∼3 fold whereas MCP‐4 (5 μg, i.v.) increased circulating monocytes ∼3 fold without affecting eosinophil numbers.

Dexamethasone pretreatment inhibited eotaxin‐induced dermal eosinophil influx only at a steroid dose (0.1 mg kg−1, s.c.) which significantly reduced circulating eosinophil numbers. The steroid also reduced eosinophilia in peripheral blood resulting from systemic eotaxin administration (5 μg, i.v.).

These data suggest differences in rat CCR3 relative to other species as surmised from a distinctive rank order of chemokine potency. In addition to its chemotactic effects eotaxin, but not MCP‐4, promotes eosinophil recruitment into the circulation. One of the mechanisms by which glucocorticoids, such as dexamethasone, acutely inhibits eotaxin‐induced dermal eosinophil influx is to diminish the circulating numbers of these cells available for tissue recruitment.

British Journal of Pharmacology (1999) 128, 788–794; doi:10.1038/sj.bjp.0702835
Stimulatory effect of exogenous diadenosine tetraphosphate on insulin and glucagon secretion in the perfused rat pancreas
Authors: Silvestre Ramona A , Rodríguez‐Gallardo Jovita , Egido Eva M , Marco José
Published: January 2005

Diadenosine triphosphate (AP3A) and diadenosine tetraphosphate (AP4A) are released by various cells (e.g. platelets and chromaffin cells), and may act as extracellular messengers. In pancreatic B‐cells, AP3A and AP4A are inhibitors of the ATP‐regulated K+ channels, and glucose increases intracellular levels of both substances.

We have studied the effect of exogenous AP3A and AP4A on insulin and glucagon secretion by the perfused rat pancreas.

AP3A did not significantly modify insulin or glucagon release, whereas AP4A induced a prompt, short‐lived insulin response (∼4 fold higher than basal value; P<0.05) in pancreases perfused at different glucose concentrations (3.2, 5.5 or 9 mM). AP4A‐induced insulin release was abolished by somatostatin and by diazoxide. These two substances share the capacity to activate ATP‐dependent K+ channels, suggesting that these channels are a potential target for AP4A in the B‐cell.

AP4A stimulated glucagon release at both 3.2 and 5.5 mM glucose. This effect was abolished by somatostatin.

The results suggest that extracellular AP4A may play a physiological role in the control of insulin and glucagon secretion.

British Journal of Pharmacology (1999) 128, 795–801; doi:10.1038/sj.bjp.0702837
Effect of pre‐exposure to vasoconstrictors on isoprenaline‐induced relaxation in rat aorta: involvement of inducible nitric oxide synthase
Authors: Eckly‐Michel Anita , Keravis Thérèse , Boudjemaa Najette , Lugnier Claire
Published: January 2005

The aim of this study was to determine whether a brief (30 min) episode of contractile receptor stimulation could affect the degree of a subsequent vasorelaxation. Therefore, concentration–relaxation curves of the rat aorta to isoprenaline were compared before and after exposure of the tissue to noradrenaline (100 μM) or prostaglandin F2α (PGF2α, 100 μM).

Exposure to noradrenaline enhanced the second maximal relaxant effect of isoprenaline (from 20–95% relaxation). This effect was not due to significant differences in precontraction levels and was not modified by the presence of the endothelium. Treatment with PGF2α mimicked the actions of noradrenaline on subsequent vasorelaxation to isoprenaline.

Before exposure to noradrenaline (100 μM), forskolin (10 μM) did not produce any significant relaxation of the rat aorta. After exposure to noradrenaline, forskolin caused a concentration‐dependent relaxation with a maximal effect of more than 90% in rings with and without endothelium suggesting that the change in vasorelaxation to isoprenaline occured downstream from the β‐adrenoceptor.

The increase in relaxation due to exposure to noradrenaline was markedly attenuated by treatment with a protein synthase inhibitor (cycloheximide), a nitric oxide (NO) synthase inhibitor (L‐NG‐nitroarginine methyl ester, L‐NAME) and an inhibitor of the activation of soluble guanylyl cyclase (methylene blue).

Western blot analysis showed an increase of inducible NO synthase (iNOS) in aortic rings exposed to noradrenaline or PGF2α.

Together, these findings suggest that pretreatment of rat aorta with noradrenaline or PGF2α could induce vascular NOS which would in turn result in an increase in isoprenaline‐induced vasorelaxation, this increase occuring downstream from receptor activation. Such a mechanism might participate in cardioprotection during preconditioning induced by noradrenaline.

British Journal of Pharmacology (1999) 128, 591–596; doi:10.1038/sj.bjp.0702840