bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the purinergic and noradrenergic components in the potentiation by endothelin‐1 of the sympathetic contraction of the rabbit central ear artery during cooling
Authors: García‐Villalón A. L. , Padilla J. , Monge L. , Fernández N. , Gómez B. , Diéguez G.
Published: January 2005

To examine the role of the purinergic and noradrenergic components in the potentiation of endothelin‐1 on the vascular response to sympathetic nerve stimulation, we recorded the isometric response of isolated segments, 2 mm long, from the rabbit central ear artery to electrical field stimulation (1–8 Hz) under different conditions, at 37°C and during cooling (30°C).

Electrical field stimulation produced frequency‐dependent contraction, which was reduced during cooling (about 60% for 8 Hz). Both at 37°C and 30°C, phentolamine (1 μM) or blockade of α1‐adrenoceptors with prazosin (1 μM) reduced, whereas blockade of α2‐adrenoceptors with yohimbine (1 μM) increased, the contraction to electrical field stimulation. This contraction was increased after desensitization of P2‐receptors with α,β‐methylene adenosine 5′‐triphosphate (α,β‐meATP, 3 μM) at 37°C but not at 30°C, and was not modified by blockade of P2‐receptors with pyridoxalphosphate‐6‐azophenyl‐2,4′‐disulphonic acid (PPADS, 30 μM) at either temperature.

Endothelin‐1 (1, 3 and 10 nM) at 37°C did not affect, but at 30°C it potentiated in a concentration‐dependent manner the contraction to electrical field stimulation (from 28±6 to 134±22%, for 8 Hz). At 37°C, endothelin‐1 in the presence of phentolamine or prazosin, but not in that of yohimbine, α,β‐meATP or PPADS, potentiated the contraction to electrical stimulation. At 30°C, phentolamine or yohimbine reduced, prazosin or PPADS did not modify and α,β‐meATP slightly increased the potentiation by endothelin‐1 of the response to electrical stimulation.

The arterial contraction to ATP (2 mM) and the α2‐adrenoceptor agonist BHT‐920 (10 μM), but not that to (−)‐noradrenaline (1 μM), was potentiated by endothelin‐1 at both 37°C and 30°C.

These results in the rabbit central ear artery suggest that the sympathetic response: (a) at 37°C, could be mediated mainly by activation of α1‐adrenoceptors, with low participation of P2‐receptors, (b) is diminished during cooling, probably by a reduction in the participation of α1‐adrenoceptors, and in this condition the response could be mediated in part by P2‐receptors, and (c) is potentiated by endothelin‐1 during cooling, probably by increasing the response of both postjunctional α2‐adrenoceptors and P2‐receptors.

British Journal of Pharmacology (1997) 122, 172–178; doi:10.1038/sj.bjp.0701359
Stretch‐induced contraction of rabbit isolated pulmonary artery and the involvement of endothelium‐derived thromboxane A2
Authors: Nakayama Koichi , Ueta Kiichiro , Tanaka Yoshio , Tanabe Yoshiyuki , Ishi Kunio
Published: January 2005

The mechanism of stretch‐induced contraction of the intrapulmonary artery of rabbit was studied with special regard to the endothelium‐dependence and production of prostanoids.

Isolated intrapulmonary artery of rabbits in ring form produced contraction when stretched slowly up to 180% of its initial muscle length (=100%) at a rate of 0.44 mm s−1, with a stimulus period of 5 min.

The stretch‐induced contraction was attenuated by the mechanical removal of the endothelium, inhibitors of cyclo‐oxygenase such as aspirin and indomethacin, [1S‐[1α,2α(Z),3α,4α]]‐7‐[3‐[[2‐[(phenylamino) carbonyl] hydrazino]methyl]‐7‐oxabicyclo[2.2.1]hept‐2‐yl]‐5‐heptenoic acid (SQ 29,548), which is a thromboxane A2/prostaglandin H2 receptor antagonist, or by ozagrel, an inhibitor of thromboxane A2 synthase.

Biochemical assay indicated that the production of thromboxane B2, a stable metabolite of thromboxane A2, was increased 17 times in response to stretch only when the endothelium was intact. The production of thromboxane B2 was also inhibited by aspirin or ozagrel.

The production of 6‐keto prostaglandin F1α, a stable metabolite of prostacyclin, was also increased in response to stretch in the preparation with intact endothelium. However, ozagrel showed no apparent effect on the production of 6‐keto prostaglandin F1α.

These results suggest that a mechanical stimulus like stretch can act on endothelial cells of rabbit pulmonary artery to cause contraction by activation of arachidonic acid metabolism via the cyclo‐oxygenase pathway and subsequent release of thromboxane A2 and/or an increase in the ratio of thromboxane A2/prostacyclin.

British Journal of Pharmacology (1997) 122, 199–208; doi:10.1038/sj.bjp.0701362
Angiotensin II responses of vascular smooth muscle cells from hypertensive rats: enhancement at the level of p42 and p44 mitogen activated protein kinase
Authors: Wilkie Neil , Ng Leong L. , Boarder Michael R.
Published: January 2005

Stimulation of the AT1 receptor by angiotensin II (AII) gives a larger mitogenic response in vascular smooth muscle cells from spontaneously hypertensive rats (SHR) compared to those from normotensive (WKY) controls. Here we investigated whether the p42 and p44 mitogen activated protein kinase (MAPK) pathway is differentially regulated in these cells by AT1 receptors.

We showed that there is a similar level of p42 and p44 MAPK immunoreactivity in the SHR and WKY derived cells.

However, by use of an antiserum specific for the tyrosine phosphorylated form of MAPK, and an assay with a nonapeptide MAPK substrate, we showed that AII (100 nM)‐stimulated phosphorylation and activation of p42mapk and p44mapk are enhanced in the SHR derived cells.

This increased MAPK activity in SHR derived cells was also seen on protein kinase C activation with 100 nM phorbol myristate acetate (PMA). The size and time course of the response to PMA was the same as that to AII in each cell type.

The protein kinase C inhibitor Ro 31–8220 attenuated the early (2 min) phase of AII stimulation of MAPK activity and the entire stimulation caused by PMA. At longer times of AII stimulation both p42mapk and p44mapk were activated by an Ro 31–8220‐insensitive mechanism.

Agonist or PMA stimulation of MAPK activity was inhibited by the tyrosine kinase inhibitor genistein. AII stimulated tyrosine protein phosphorylation to a greater degree in SHR than WKY cells.

These results show that the MAPK response of SHR derived cells is increased over that of WKY cells by mechanisms independent of the enhanced stimulation of phospholipase C; amplification at the level of sequential protein kinase C and tyrosine kinase steps leads to the enhanced responsiveness of MAPK in the SHR derived cells.

British Journal of Pharmacology (1997) 122, 209–216; doi:10.1038/sj.bjp.0701366
Negative chronotropic actions of endothelin‐1 on rabbit sinoatrial node pacemaker cells
Authors: Tanaka Hideo , Habuchi Yoshizumi , Yamamoto Taku , Nishio Manabu , Morikawa Junichiro , Yoshimura Manabu
Published: January 2005

The effects of endothelin‐1 (ET‐1) on sinoatrial (SA) node preparations of the rabbit heart were studied by means of whole‐cell clamp techniques.

ET‐1 at 1 nM slowed the spontaneous beating activity and rendered half of the cells quiescent. At a higher concentration of 10 nM, the slowing and cessation of spontaneous activity were accompanied by hyperpolarization.

In voltage‐clamp experiments, ET‐1 decreased the basal L‐type Ca2+ current (ICa(L)) dose‐dependently with a half‐maximal inhibitory concentration (EC50) of 0.42 nM and maximal inhibitory response (Emax) of 49.5%. The delayed rectifying K+ current (IK) was also reduced by 33.2±11.1% at 1 nM. In addition, an inwardly rectifying K+ current was activated by ET‐1 at higher concentrations (EC50=4.8 nM). These ET‐1‐induced changes in membrane currents were abolished by BQ485 (0.3 μM), a highly selective ETA receptor antagonist.

When ICa(L) was inhibited by ET‐1 (1 nM), subsequent application of 10 μM ACh showed no additional decrease in ICa(L), suggesting the involvement of cyclic AMP in the effects of ET‐1 on ICa(L). In contrast, 1 nM ET‐1 further decreased ICa(L) in the presence of 10 μM ACh, suggesting that ET‐1 activates some additional mechanism(s) which inhibit ICa(L). The ET‐1‐induced ICa(L) inhibition was abolished by protein kinase A inhibitory peptide (PKI, 20 μM) or H‐89 (5 μM). However, the ICa(L) inhibition was not affected by methylene blue (10 μM), suggesting a minor role for cyclic GMP in the effect of ET‐1 under basal conditions.

ET‐1 failed to inhibit ICa(L) when the pipette contained GDPβS (200 μM). However, incubation of the cells with pertussis toxin (PTX, 5 μg ml−1, >6 h) only reduced the ET‐1‐induced inhibition to 21.5±9.5%, whereas it abolished the inhibitory effect of ACh on ICa(L).

Intracellular perfusion of 8‐bromo cyclicAMP (8‐Br cyclicAMP, 500 μM) attenuated, but did not abolish the inhibitory effect of ET‐1 on ICa(L). This 8‐Br cyclicAMP‐resistant component (17.5±14.4%, n=20) was not affected by combined application of 8‐Br cyclicAMP with 8‐bromo cyclicGMP (500 μM), ryanodine (1 μM) or phorbol‐12‐myristate‐13‐acetate (TPA; 50 nM).

In summary, ET‐1 exerts negative chronotropic effects on the SA node via ETA‐receptors. ET‐1 inhibits both ICa(L) and IK, and increases background K+ current. The inhibition of ICa(L) by ET‐1 is mainly due to reduction of the cyclicAMP levels via PTX‐sensitive G protein, but some other mechanism(s) also seems to be operative.

British Journal of Pharmacology (1997) 122, 321–329; doi:10.1038/sj.bjp.0701370
Effects of increased extracellular glutamate levels on the local field potential in the brain of anaesthetized rats
Authors: Obrenovitch T. P. , Urenjak J. , Zilkha E.
Published: January 2005

It is generally considered that glutamate‐mediated transmission can be altered from a physiological to neurotoxic action when extracellular glutamate levels become excessive subsequent to impaired uptake and/or excessive release. However, high extracellular glutamate does not consistently correlate with neuronal dysfunction and death in vivo. The purpose of this study was to examine in situ the local depolarizations, as indicated by negative shifts of the extracellular field (d.c.) potential, produced by local inhibition of high‐affinity glutamate uptake, with or without co‐application of exogenous glutamate, in three brain regions of anaesthetized rats.

Microdialysis probes incorporating an electrode were used to apply exogenous glutamate and/or its uptake inhibitor L‐trans‐pyrrolidine‐2,4‐dicarboxylate (L‐trans‐PDC), and to monitor the resulting changes in extracellular glutamate and d.c. potential at the sites of application within the cortex, striatum and hippocampus.

Perfusion of 1 to 10 mM L‐trans‐PDC markedly and concentration‐dependently increased extracellular glutamate levels (by up to 1700% of basal level in the parietal cortex). Despite their large magnitude, glutamate changes were associated with minor negative shifts of the d.c. potential (<2 mV), which were not suppressed by the N‐methyl‐D‐aspartate (NMDA)‐channel blocker, dizocilpine (MK‐801, 2 mg kg−1, i.v.), or the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic acid (AMPA)/kainate‐receptor antagonist, 6‐nitro‐7‐sulphamoylbenzo(f)quinoxaline‐2,3‐dione (NBQX, 30 mg kg−1, i.p.). L‐trans‐PDC had virtually identical concentration‐dependent effects on dialysate glutamate in the hippocampus and striatum, but those induced in the cortex were around 40% larger (P<0.002). In contrast, the associated depolarizations were around twice as large in the striatum and cortex as in the hippocampus (P<0.002). Finally, co‐application of L‐trans‐PDC did not enhance the d.c. potential changes evoked by perfusion of 5 or 20 mM glutamate.

As the neurotoxic potency of glutamate agonists is considered to be linked to excessive opening of glutamate‐operated ion channels, these results challenge the notion that high extracellular glutamate levels may be the key to excitotoxicity in neurological disorders. In particular, they do not support the hypothesis that high extracellular glutamate causes the sudden negative shifts of the d.c. potential associated with ischaemia (i.e. anoxic depolarization), traumatic brain injury and spreading depression. Impaired uptake and excessive release of glutamate may well lead to excitotoxicity, but only at the synaptic level, not by spreading through the interstitial fluid.

British Journal of Pharmacology (1997) 122, 372–378; doi:10.1038/sj.bjp.0701372
Potent and voltage‐dependent block by philanthotoxin‐343 of neuronal nicotinic receptor/channels in PC12 cells
Authors: Liu Min , Nakazawa Ken , Inou Kazuhide , Ohno Yasuo
Published: January 2005

Block by philanthotoxin‐343 (PhTX‐343), a neurotoxin from wasps, of ionic currents mediated through neuronal nicotinic acetylcholine (ACh) receptor/channels was characterized in rat phaeochromocytoma PC12 cells, by use of whole cell voltage‐clamp techniques.

In the cells held at −60 mV, PhTX‐343 at 0.1 and 1 μM inhibited an inward current activated by 100 μM ACh. The current inhibition was relieved by depolarizing steps, and augmented at negative potentials, suggesting that PhTX‐343 blocks the channel in a voltage‐dependent manner. Joro spider toxin‐3 (JSTX‐3) also exerted voltage‐dependent inhibition of ACh‐activated currents in a similar concentration range, but argiotoxin636 did not affect the currents.

Analysis of the current decay during hyperpolarizing steps indicated that the current inhibition by 100 nM PhTX‐343 develops in an order of several hundreds of milliseconds. On the other hand, the recovery from the current inhibition during depolarizing steps developed in an order of about 100 ms.

The results suggest that PhTX‐343 blocks neuronal nicotinic receptor channels in PC12 cells at concentrations lower than those required for channel block in non‐mammalian cells, and the block exhibits clear voltage‐dependence. Estimated from the voltage‐dependence, the binding site of PhTX‐343 may be located near the outer mouth of the channel.

British Journal of Pharmacology (1997) 122, 379–385; doi:10.1038/sj.bjp.0701373
Effect of acute and chronic tramadol on [3H]‐5‐HT uptake in rat cortical synaptosomes
Authors: Giusti Pietro , Buriani Alessandro , Cima Lorenzo , Lipartiti Maria
Published: January 2005

Tramadol hydrochloride is a centrally acting opioid analgesic, the efficacy and potency of which is only five to ten times lower than that of morphine. Opioid, as well as non‐opioid mechanisms, may participate in the analgesic activity of tramadol.

[3H]‐5‐hydroxytryptamine (5‐HT) uptake in rat isolated cortical synaptosomes was studied in the presence of tramadol, desipramine, fluoxetine, methadone and morphine. Methadone and tramadol inhibited synaptosomal [3H]‐5‐HT uptake with apparent Kis of 0.27±0.04 and 0.76±0.04 μM, respectively. Morphine essentially failed to inhibit [3H]‐5‐HT uptake (Ki 0.50±0.30 M).

Methadone, morphine and tramadol were active in the hot plate test with ED50s of 3.5, 4.3 and 31 mg kg−1, respectively. At the highest tested dose (80 mg kg−1) tramadol produced only 77±5.3% of the maximal possible effect.

When [3H]‐5‐HT uptake was examined in synaptosomes prepared from rats 30 min after a single dose of morphine, methadone or tramadol, only tramadol (31 mg kg−1, s.c., equal to the ED50 in the hot plate test) and methadone (35 mg kg−1, s.c., equal to the ED90 in the hot plate test) decreased uptake.

Animals were chronically treated for 15 days with increasing doses of tramadol or methadone (5 to 40 mg kg−1 and 15 to 120 mg kg−1, s.c., respectively). Twenty‐four hours after the last drug injection, a challenge dose of methadone (35 mg kg−1, s.c.) or tramadol (31 mg kg−1, s.c.) was administered. [3H]‐5‐HT uptake was not affected in synaptosomes prepared from rats chronically‐treated with methadone, whereas chronic tramadol was still able to reduce this parameter by 42%.

Rats chronically‐treated with methadone showed a significant increase in [3H]‐5‐HT uptake (190%) 72 h after drug withdrawal. In contrast, [3H]‐5‐HT uptake in rats chronically‐treated with tramadol (110%) did not differ significantly from control animals.

These results further support the hypothesis that [3H]‐5‐HT uptake inhibition may contribute to the antinociceptive effects of tramadol. The lack of tolerance development of [3H]‐5‐HT uptake, together with the absence of behavioural alterations after chronic tramadol treatment, suggest that tramadol has an advantage over classical opioids in the treatment of pain disorders.

British Journal of Pharmacology (1997) 122, 302–306; doi:10.1038/sj.bjp.0701374
Interaction of cyclosporin derivatives with the ATPase activity of human P‐glycoprotein
Authors: Watanabe Toru , Kokubu Noriko , Charnick Steven B. , Naito Mikihiko , Tsuruo Takashi , Cohen Dalia
Published: January 2005

P‐glycoprotein, a 170–180 kDa membrane glycoprotein that mediates multidrug resistance, hydrolyses ATP to efflux a broad spectrum of hydrophobic agents. In this study, we analysed the effects of three MDR reversing agents, verapamil, cyclosporin A and [3′‐keto‐Bmt]‐[Val*]‐cyclosporin (PSC 833), on the adenosine triphosphatase (ATPase) activity of human P‐glycoprotein.

P‐glycoprotein was immunoprecipitated with a monoclonal antibody (MRK‐16) and the P‐glycoprotein‐MRK‐16‐Protein A‐Sepharose complexes obtained were subjected to a coupled enzyme ATPase assay.

While verapamil activated the ATPase, the cyclosporin derivatives inhibited both the substrate‐stimulated and the basal P‐glycoprotein ATPase. No significant difference was observed between PSC 833 and cyclosporin A on the inhibition of basal P‐glycoprotein ATPase activity. PSC 833 was more potent than cyclosporin A for the substrate‐stimulated activity.

Kinetic analysis indicated a competitive inhibition of verapamil‐stimulated ATPase by PSC 833.

The binding of 8‐azido‐[α‐32P]‐ATP to P‐glycoprotein was not altered by the cyclosporin derivatives, verapamil, vinblastine and doxorubicin, suggesting that the modulation by these agents of P‐glycoprotein ATPase cannot be attributed to an effect on ATP binding to P‐glycoprotein.

The interaction of the cyclosporin derivatives with ATPase of P‐glycoprotein might present an alternative and/or additional mechanism of action for the modulation of P‐glycoprotein function.

British Journal of Pharmacology (1997) 122, 241–248; doi:10.1038/sj.bjp.0701377
Characterization of A2A adenosine receptors in human lymphocyte membranes by [3H]‐SCH 58261 binding
Authors: Varani Katia , Gessi Stefania , Dalpiaz Alessandro , Ongini Ennio , Andrea Borea Pier
Published: January 2005

The present study describes for the first time the characterization of the adenosine A2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [3H]‐5‐amino‐7‐(2‐phenylethyl)‐2‐(2‐furyl)‐pyrazolo [4,3‐e]‐1,2,4 triazolo [1,5‐c] pyrimidine, ([3H]‐SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies.

Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 0.85 nM and 35 fmol mg−1 protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [3H]‐SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A2A‐adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments.

Thermodynamic data indicate that [3H]‐SCH 58261 binding to human lymphocytes is entropy and enthalpy‐driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A2A‐adenosine receptors.

It is concluded that in human lymphocyte membranes [3H]‐SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A2A‐receptor. The presence of A2A‐receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses.

British Journal of Pharmacology (1997) 122, 386–392; doi:10.1038/sj.bjp.0701378
The effect of 15‐HPETE on airway responsiveness and pulmonary cell recruitment in rabbits
Authors: Riccio M. M. , Matsumoto T. , Adcock J. J. , Douglas G. J. , Spina D. , Page C. P.
Published: January 2005

In the present study we have investigated the effect of 15‐hydroperoxyeicosatetraenoic acid (15‐HPETE) and 15‐hydroxyeicosatetraenoic acid (15‐HETE) on airway responsiveness to inhaled histamine in rabbits in vivo.

15‐HPETE increased airway responsiveness to histamine 24 h after tracheal instillation and this was associated with a cellular infiltration consisting mainly of neutrophils, as measured by bronchoalveolar lavage. The airway hyperresponsiveness induced by 15‐HPETE was still present 72 h after tracheal instillation of 15‐HPETE, but had returned to baseline values one week post challenge. The number of neutrophils in bronchoalveolar lavage remained significantly elevated compared to pre‐challenge levels. In contrast to 15‐HPETE, the major metabolite 15‐HETE, failed to alter airway hyperresponsiveness to histamine despite the recruitment of neutrophils into the lung, suggesting that the effect of 15‐HPETE was not secondary to the generation of this metabolite nor dependent on the influx of neutrophils.

Both capsaicin and atropine but not the peripherally acting μ‐opioid receptor agonist, BW443C (H‐Tyr‐D‐Arg‐Gly‐Phe(4‐NO2)‐Pro‐NH4), attenuated 15‐HPETE‐induced hyperresponsiveness. The increased cellular infiltration induced by 15‐HPETE was only attenuated by capsaicin.

The results of the present study suggest that the release of 15‐HPETE into the airways could contribute to sensitization of afferent nerve endings analogous to the hyperalgesia induced by this mediator in skin.

British Journal of Pharmacology (1997) 122, 249–256; doi:10.1038/sj.bjp.0701379
A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis
Authors: Guatimosim C. , Romano‐Silva M. A. , Cruz J. S. , Beirão P. S. L. , Kalapothakis E. , Moraes‐Santos T. , Cordeiro M. N. , Diniz C. R. , Gomez M. V. , Prado M. A. M.
Published: January 2005

The aim of the present experiments was to investigate the pharmacological action of a toxin from the spider Phoneutria nigriventer, Tx3‐3, on the function of calcium channels that control exocytosis of synaptic vesicles.

Tx3‐3, in confirmation of previous work, diminished the intracellular calcium increase induced by membrane depolarization with KCl (25 mM) in rat cerebrocortical synaptosomes. The toxin was very potent (IC50 0.9 nM) at inhibiting calcium channels that regulate calcium entry in synaptosomes. In addition, Tx3‐3 blocked the exocytosis of synaptic vesicles, as measured with the fluorescent dye FM1‐43.

Using ω‐toxins that interact selectively with distinct neuronal calcium channels, we investigated whether the target of Tx3‐3 overlaps with known channels that mediate exocytosis. The results indicate that the main population of voltage‐sensitive calcium channels altered by Tx3‐3 can also be inhibited by ω‐agatoxin IVA, an antagonist of P/Q calcium channels. ω‐conotoxin GVIA, which inhibits N type calcium channels did not decrease significantly the entry of calcium or exocytosis of synaptic vesicles in depolarized synaptosomes.

It is concluded that Tx3‐3 potently inhibits ω‐agatoxin IVA‐sensitive calcium channels, which are involved in controlling exocytosis in rat brain cortical synaptosomes.

British Journal of Pharmacology (1997) 122, 591–597; doi:10.1038/sj.bjp.0701381
Effect of chronic treatment with the GABA transaminase inhibitors γ‐vinyl GABA and ethanolamine O‐sulphate on the in vitro GABA release from rat hippocampus
Authors: Qume M. , Fowler L. J.
Published: January 2005

The effects of 2, 8 and 21 day oral treatment with the specific γ‐aminobutyric acid transaminase (GABA‐T) inhibitors γ‐vinyl GABA (GVG) and ethanolamine O‐sulphate (EOS) on brain GABA levels, GABA‐T activity, and basal and stimulated GABA release from rat cross‐chopped brain hippocampal slices was investigated.

Treatment with GABA‐T inhibitors lead to a reduction in brain GABA‐T activity by 65–80% compared with control values, with a concomitant increase in brain GABA content of 40–100%.

Basal hippocampal GABA release was increased to 250–450% of control levels following inhibition of GABA‐T activity. No Ca2+ dependence was observed in either control or treated tissues.

GVG and EOS administration led to a significant elevation in the potassium stimulated release of GABA from cross‐chopped hippocampal slices compared with that of controls. Although stimulated GABA release from control tissues was decreased in the presence of a low Ca2+ medium, GVG and EOS treatment abolished this Ca2+ dependency.

GABA compartmentalization, Na+ and Cl− coupled GABA uptake carriers and glial release may provide explanations for the loss of the Ca2+ dependency of stimulated GABA release observed following GVG and EOS treatment.

Administration of GABA‐T inhibitors led to increases in both basal and stimulated hippocampal GABA release. However, it is not clear which is the most important factor in the anticonvulsant activity of these drugs, the increased GABA content ‘leaking’ out of neurones and glia leading to widespread inhibition, or the increase in stimulated GABA release which may occur following depolarization caused by an epileptic discharge.

British Journal of Pharmacology (1997) 122, 539–545; doi:10.1038/sj.bjp.0701383
Role of ATP in fast excitatory synaptic potentials in locus coeruleus neurones of the rat
Authors: Nieber Karen , Poelchen Wolfgang , Illes Peter
Published: January 2005

Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). The pressure application of α,β‐methylene ATP (α,β‐meATP) caused reproducible depolarizations which were depressed by suramin (30 μM) and abolished by suramin (100 μM). Pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS; 10, 30 μM) also concentration‐dependently inhibited the α,β‐meATP‐induced depolarization, although with a much slower time‐course than suramin. Almost complete inhibition developed with 30 μM PPADS. Reactive blue 2 (30 μM) did not alter the effect of α,β‐meATP, while reactive blue 2 (100 μM) slightly depressed it.

Pressure‐applied (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) also depolarized LC neurones. Kynurenic acid (500 μM) depressed and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 50 μM) abolished the response to AMPA. Suramin (100 μM) potentiated the AMPA effect.

Pressure‐applied noradrenaline hyperpolarized LC neurones. Suramin (100 μM) did not alter the effect of noradrenaline.

Focal electrical stimulation evoked biphasic synaptic potentials consisting of a fast depolarization (p.s.p.) followed by a slow hyperpolarization (i.p.s.p.). A mixture of D(−)‐2‐amino‐5‐phosphonopentanoic acid (AP‐5; 50 μM), CNQX (50 μM) and picrotoxin (100 μM) depressed both the p.s.p. and the i.p.s.p. Under these conditions suramin (100 μM) markedly inhibited the p.s.p., but did not alter the i.p.s.p. In the combined presence of AP‐5 (50 μM), CNQX (50 μM), picrotoxin (100 μM), strychnine (0.1 μM), tropisetron (0.5 μM) and hexamethonium (100 μM), a high concentration of suramin (300 μM) almost abolished the p.s.p. without changing the i.p.s.p.

In the presence of kynurenic acid (500 μM) and picrotoxin (100 μM), PPADS (30 μM) depressed the p.s.p. Moreover, the application of suramin (100 μM) to the PPADS (30 μM)‐containing medium failed to cause any further inhibition. Neither PPADS (30 μM) nor suramin (100 μM) altered the i.p.s.p.

It was concluded that the cell somata of LC neurones are endowed with excitatory P2‐purinoceptors. ATP may be released either as the sole transmitter from purinergic neurones terminating at the LC or as a co‐transmitter of noradrenaline from recurrent axon collaterals or dendrites of the LC neurones themselves.

British Journal of Pharmacology (1997) 122, 423–430; doi:10.1038/sj.bjp.0701386
Influence of ETR‐p1/f1 antisense peptide on endothelin‐induced constriction in rat renal arcuate arteries
Authors: Wu Xiaochun , Richards Nicholas T. , Johns Edward J. , Kohsaka Takeo , Nakamura Akio , Okada Hidechika
Published: January 2005

This study set out to examine the endothelin receptor subtypes mediating vasoconstriction in the rat renal arcuate artery. This was done in isolated vessels 120–200 μm in diameter, incubated with a selective agonist and the novel ‘antisense’ peptide to part of the human endothelinA receptor.

Groups of vessels (n=6) were incubated with increasing concentrations of endothelin‐1 (ET‐1), from 1 to 100 nM, which caused a 65% maximal contraction at the highest dose with an pEC50 of 8.16±0.11 M. By contrast, in six other vessels sarafotoxin 6c over the same dose range gave a minimal contraction (around 5% of maximum).

Preincubation of six vessels with the antisense peptide ETR p1/f1 at 1 μM had no effect on the ET‐1 induced vasoconstriction, in terms of displacement of the concentration‐response curve or the maximal tension achieved by the agonist. In the six vessels exposed to 4 μM ETR p1/f1, there was a significant shift of the concentration‐response curve and a lower pEC50 at 7.78±0.09 M (P<0.05). At the highest concentrations of ETR p1/f1, there was a marked suppression of all responses to ET‐1, which at the maximal concentrations tested, 0.1 μM, only reached some 10% of the maximal achievable contraction.

Increasing ET‐1 concentrations up to 2 μM in vessels incubated with 40 μM ETR‐p1/f1 showed that the blockade could be overcome and that the relationship was shifted to the right (P<0.001) by approximately one log unit with a pEC50 of 7.13±0.11 M. A Schild plot of the data indicated the antagonist to be acting competitively at a single population of receptors.

At the highest concentrations tested, 40 μM, ETR‐p1/f1 had no effect on noradrenaline‐induced contractions, indicating a lack of non‐specific actions.

Together, these data suggest that at the rat renal arcuate artery the endothelinA receptor is the predominant functional receptor mediating contraction. Furthermore, this study has shown the potential usefulness of this novel type of ‘antisense’ peptide in blocking receptor activation.

British Journal of Pharmacology (1997) 122, 316–320; doi:10.1038/sj.bjp.0701388
Effect of the molecular polymorphisms of human paraoxonase (PON1) on the rate of hydrolysis of paraoxon
Authors: Mackness Bharti , Mackness Michael I. , Arrol Sharon , Turkie Wajdi , Durrington Paul N.
Published: January 2005

The hydrolysis of organophosphate pesticides (OP) and nerve gases by serum paraoxonase (PON1) is an important factor determining their toxicity to mammals including man. The PON1 gene contains 2 polymorphic sites at amino acid positions 55 (L→M) and 192 (G→A, classically defined as the A and B genotypes) which result in several alloenzymes of PON1 in human serum.

The 192 polymorphism has previously been shown to affect PON1 activity. We have investigated the effect of both polymorphisms on the hydrolysis of paraoxon by serum from 279 healthy human subjects.

The 55 polymorphism significantly influenced PON1 activity. MM homozygotes had over 50% less activity towards paraoxon compared to the LL and LM genotypes regardless of the 192 genotype (P<0.001).

Multiple regression analysis indicated that the 192 polymorphism, 55 polymorphism and serum PON1 concentration were responsible for 46, 16 and 13% of the variation in PON1 activity, respectively (all P<0.001). None of the other parameters investigated significantly affected PON1 activity.

Therefore both PON1 polymorphisms affect the hydrolysis of paraoxon. AA/MM and AB/MM individuals may be potentially more susceptible to OP intoxication.

Genotyping individuals for both PON1 polymorphisms may provide a method for identifying those individuals at most risk of OP poisoning. The effect of PON1 polymorphisms on activity may also explain why some Gulf War Veterans have developed Gulf War Syndrome and some have not.

British Journal of Pharmacology (1997) 122, 265–268; doi:10.1038/sj.bjp.0701390
Homocysteine as a modulator of platelet‐derived growth factor action in vascular smooth muscle cells: a possible role for hydrogen peroxide
Authors: Nishio Eisuke , Watanabe Yasuhiro
Published: January 2005

Homocysteine is an independent risk factor for cardiovascular disease. The mechanisms by which elevated plasma concentrations of homocysteine are related to the pathogenesis of atherosclerosis are not fully understood. Therefore, we examined the effect of homocysteine on cell replication of rat cultured vascular smooth muscle cells (VSMCs) at concentrations similar to those observed in clinical studies.

The incorporation of [3H]‐thymidine was used as a marker of mitosis. Homocysteine (250–500 μM) was a weak mitogen as compared to platelet‐derived growth factor‐BB (PDGF‐BB, 1 nM) and serum (10%), but it potentiated the mitogenic effect of PDGF‐BB four fold at 500 μM. This enhancement of mitogenesis was blunted by the addition of the scavenging enzyme catalase or the antioxidant N‐acetyl‐L‐cysteine.

Furthermore, stimulation of VSMC with homocysteine (25–500 μM) decreased the glutathione peroxidase activity of the cells to 50% of control at 500 μM. Inversely, homocysteine enhanced the superoxide dismutase (SOD) activity to 137% of control at 500 μM, but it had no effect on the catalase activity.

Homocysteine decreased the activity of bovine purified liver cytosolic glutathione peroxidase in a time‐ and dose‐dependent manner. The maximum decrease was 50%.

In summary, homocysteine has a weak mitogenic effect on VSMC, but it dramatically enhances the mitogenic response of PDGF‐BB, presumably by disturbing the activity of antioxidant enzymes.

British Journal of Pharmacology (1997) 122, 269–274; doi:10.1038/sj.bjp.0701391
Differential effect of dexamethasone on interleukin 1β‐ and cyclic AMP‐triggered expression of GTP cyclohydrolase I in rat renal mesangial cells
Authors: Plüss Christoph , Werner Ernst R. , Wachter Helmut , Pfeilschifter Josef
Published: January 2005

Endogenous synthesis of tetrahydrobiopterin (BH4) is an essential requirement for cytokine‐stimulated nitric oxide (NO) synthesis in rat mesangial cells. GTP cyclohydrolase I, the rate‐limiting enzyme in BH4 synthesis, is expressed in renal mesangial cells in response to two principal classes of activating signals. These two groups of activators comprise inflammatory cytokines such as interleukin (IL)‐1β and agents that elevate cellular levels of cyclic AMP.

We examined the action of the potent anti‐inflammatory drug dexamethasone on GTP cyclohydrolase I induction in response to IL‐1β and a membrane‐permeable cyclic AMP analogue, N6, O‐2′‐dibutyryladenosine 3′‐5′‐phosphate (Bt2cyclic AMP).

Nanomolar concentrations of dexamethasone markedly attenuated IL‐1β‐induced GTP cyclohydrolase I mRNA steady state level as well as IL‐1β‐induced GTP cyclohydrolase I protein expression and enzyme activity. In contrast, dexamethasone did not inhibit Bt2cyclic AMP‐triggered increase in GTP cyclohydrolase I mRNA level and protein expression, and low (1 nM) or high (1 and 10 μM) doses of dexamethasone consistently increased Bt2cyclic AMP‐induced GTP cyclohydrolase activity.

In summary, these results suggest that glucocorticoids act at several levels, critically dependent on the stimulus used, to control GTP cyclohydrolase I expression.

British Journal of Pharmacology (1997) 122, 534–538; doi:10.1038/sj.bjp.0701395
Potentiation by vasopressin of adrenergic vasoconstriction in the rat isolated mesenteric artery
Authors: Noguera I. , Medina P. , Segarra G. , Martínez M. C. , Aldasoro M. , Vila J. M. , Lluch S.
Published: January 2005

The aim of the present study was to investigate in rat mesenteric artery rings whether low concentrations of vasopressin could modify the contractile responses to noradrenaline and electrical stimulation of perivascular nerves.

Vasopressin (10−10–10−7 M) caused concentration‐dependent contractions (pD2=8.36±0.09). The V1‐receptor antagonist d(CH2)5Tyr(Me)AVP (10−9–10−8 M) produced parallel rightward shifts of the control curve for vasopressin. Schild analysis yielded a pA2 value of 9.83 with a slope of 1.10±0.14.

Vasopressin (3×10 −10 and 10−9 M) caused concentration‐dependent potentiation of the contractions elicited by electrical stimulation (2–8 Hz; 0.2 ms duration for 30 s) and produced leftward shifts of the concentration‐response curve for noradrenaline. The V1‐receptor antagonist induced concentration‐dependent inhibitions of potentiation induced by vasopressin. The selective V1‐receptor agonist [Phe*, Orn8]‐vasotocin (3×10 −10 and 10−9 M) induced potentiation of electrical stimulation‐evoked responses which was also inhibited in the presence of the V1 antagonist (10−8 M). In contrast, the V2‐receptor agonist deamino‐8‐D‐arginine vasopressin (desmopressin 10−8–10−7 M) did not modify the electrical stimulation‐induced responses and the V2‐receptor antagonist [d(CH2)5, D‐Ile*, Ile4, Arg8]‐vasopressin (10−8–10−7 M) did not affect the potentiation evoked by vasopressin.

In artery rings contracted by 10−6 M noradrenaline in the presence of 10−6 M guanethidine and 10−6 M atropine, electrical stimulation (2, 4 and 8 Hz) produced frequency‐dependent relaxations which were unaffected by 10−9 M vasopressin but abolished by 10−6 M tetrodotoxin.

Vasopressin also potentiated contractions elicited by KCl and contractions induced by addition of CaCl2 to KCl depolarized vessels. The augmenting effects were inhibited by the V1 antagonist.

In the presence of the calcium antagonist nifedipine (10−6 M), vasopressin failed to enhance the contractile responses to electrical stimulation, noradrenaline and KCl.

The results demonstrate that low concentrations of vasopressin strongly potentiate the contractions to adrenergic stimulation and KCl depolarization. This effect appears to be mediated by V1 receptor stimulation which brings about an increase in calcium entry through dihydropyridine‐sensitive calcium channels.

British Journal of Pharmacology (1997) 122, 431–438; doi:10.1038/sj.bjp.0701397
Cyclo‐oxygenase isozymes in mucosal ulcerogenic and functional responses following barrier disruption in rat stomachs
Authors: Hirata Takuya , Ukawa Hideki , Yamakuni Hisashi , Kato Shinichi , Takeuchi Koji
Published: January 2005

We examined the effects of selective and nonselective cyclo‐oxygenase (COX) inhibitors on various functional changes in the rat stomach induced by topical application of taurocholate (TC) and investigated the preferential role of COX isozymes in these responses.

Rat stomachs mounted in ex vivo chambers were perfused with 50 mM HCl and transmucosal potential difference (p.d.), mucosal blood flow (GMBF), luminal acid loss and luminal levels of prostaglandin E2 (PGE2) were measured before, during and after exposure to 20 mM TC.

Mucosal application of TC in control rats caused a reduction in p.d., followed by an increase of luminal acid loss and GMBF, and produced only minimal damage in the mucosa 2 h later. Pretreatment with indomethacin (10 mg kg−1, s.c.), a nonselective COX‐1 and COX‐2 inhibitor, attenuated the gastric hyperaemic response caused by TC without affecting p.d. and acid loss, resulting in haemorrhagic lesions in the mucosa. In contrast, selective COX‐2 inhibitors, such as NS‐398 and nimesulide (10 mg kg−1, s.c.), had no effect on any of the responses induced by TC and did not cause gross damage in the mucosa.

Luminal PGE2 levels were markedly increased during and after exposure to TC and this response was significantly inhibited by indomethacin but not by either NS‐398 or nimesulide. The expression of COX‐1‐mRNA was consistently detected in the gastric mucosa before and after TC treatment, while a faint expression of COX‐2‐mRNA was detected only 2 h after TC treatment.

Both NS‐398 and nimesulide significantly suppressed carrageenan‐induced rat paw oedema, similar to indomethacin.

These results confirmed a mediator role for prostaglandins in the gastric hyperaemic response following TC‐induced barrier disruption, and suggest that COX‐1 but not COX‐2 is a key enzyme in maintaining ‘housekeeping’ functions in the gastric mucosa under both normal and adverse conditions.

British Journal of Pharmacology (1997) 122, 447–454; doi:10.1038/sj.bjp.0701399
Inhibitory effects of (±)‐propranolol on excitation‐contraction coupling in isolated soleus muscles of the rat
Authors: Ha Tuyen N. V. , Fryer Martin W.
Published: January 2005

The effect of a β‐adrenoceptor antagonist, propranolol, was investigated on excitation‐contraction coupling in small, intact bundles of soleus muscle fibres from the rat.

(±)‐Propranolol significantly inhibited twitch and tetanic tension with IC50 values of 6.7 μM and 3.5 μM, respectively.

(+)‐Propranolol (which has 100 times less β‐blocking activity than the (±) form) was approximately one third as effective as the (±) form at inhibiting isometric tension.

(±)‐Propranolol (20 μM) had no significant effect on the amplitude of caffeine contractures, suggesting that it did not directly inhibit Ca2+ release from the sarcoplasmic reticulum.

The resting membrane potential measured after 15 min perfusion with 20 μM (±)‐propranolol was not significantly different from control. However, this concentration of (±)‐propranolol significantly reduced both the peak amplitude and the maximum rate of rise of the action potential. Both effects were only partially reversible after extensive washing.

(±)‐Propranolol perfusion caused a modest reduction in the amplitude of sub‐maximal K+ contractures at concentrations (5  μM) that markedly depressed tetanic tension.

The results indicate that (±)‐propranolol can decrease isometric tension independently of β‐receptor occupation by (i) reducing the amplitude and rate of rise of the action potential and (ii) by directly inhibiting excitation‐contraction coupling. The relatively low IC50 for the ‘membrane‐stabilizing’ action of propranolol on tetanic tension (3.5 μM), combined with the ability of the drug to accumulate gradually in biological membranes, may contribute to a peripheral component of the tremorolytic and fatigue‐inducing actions of propranolol on skeletal muscle.

British Journal of Pharmacology (1997) 122, 463–468; doi:10.1038/sj.bjp.0701405
Nitroglycerin‐inhibited whole blood aggregation is partially mediated by calcitonin gene‐related peptide–a neurogenic mechanism
Authors: Booth Brian P. , Nolan Timothy D. , Fung Ho‐Leung
Published: January 2005

The role of the vasculature and calcitonin gene‐related peptide (CGRP) in nitroglycerin (NTG)‐mediated platelet inhibition was studied.

In vitro incubations of CGRP in whole blood induced a dose‐dependent inhibition of platelet aggregation with an IC50 of 62.1 nM.

The platelet inhibition induced by CGRP was blocked by co‐incubation of 0.53 μM CGRP8‐37, as well as 30 μM NG‐nitro‐monomethyl‐L‐arginine (L‐NMMA).

In a separate group of experiments, 100 nM NTG in rat whole blood (WB) induced platelet inhibition of 6.0±1.3% (mean±s.d.), which was enhanced to 77.6±3.5% by the addition of rat aortic tissue (AT) (P<0.001). The inclusion of CGRP8–37 with NTG and AT in WB reduced platelet inhibition to 31.6±6.8% (P<0.01). Incubation of WB and AT with 30 μM L‐NMMA reduced NTG‐induced inhibition of platelet aggregation to 26.4±4.2% (P<0.001).

It is concluded that vascular tissue contributes to the antiplatelet mechanism of action of NTG. Furthermore, NTG apparently evokes the release of CGRP from vascular tissue and this neuropeptide contributes to the antiplatelet actions of NTG.

The antiplatelet activity of CGRP in whole blood is mediated primarily through the activation of nitric oxide synthase.

British Journal of Pharmacology (1997) 122, 577–583; doi:10.1038/sj.bjp.0701408
Selective antagonism of the GABAA receptor by ciprofloxacin and biphenylacetic acid
Authors: Green M. A. , Halliwell R. F.
Published: January 2005

Previous studies have shown that ciprofloxacin and biphenylacetic acid (BPAA) synergistically inhibit γ‐aminobutyric acid (GABA)A receptors. In the present study, we have investigated the actions of these two drugs on other neuronal ligand‐gated ion channels.

Agonist‐evoked depolarizations were recorded from rat vagus and optic nerves in vitro by use of an extracellular recording technique.

GABA (50 μM)‐evoked responses, in the vagus nerve in vitro, were inhibited by bicuculline (0.3–10 μM) and picrotoxin (0.3–10 μM), with IC50 values and 95% confidence intervals (CI) of 1.2 μM (1.1–1.4) and 3.6 μM (3.0–4.3), respectively, and were potentiated by sodium pentobarbitone (30 μM) and diazepam (1 μM) to (mean±s.e.mean) 168±18% and 117±4% of control, respectively. 5‐Hydroxytryptamine (5‐HT; 0.5 μM)‐evoked responses were inhibited by MDL 72222 (1 μM) to 10±4% of control; DMPP (10 μM)‐evoked responses were inhibited by hexamethonium (100 μM) to 12±5% of control, and αbMeATP (30 μM)‐evoked responses were inhibited by PPADS (10 μM) to 21±5% of control. Together, these data are consistent with activation of GABAA, 5‐HT3, nicotinic ACh and P2X receptors, respectively.

Ciprofloxacin (10–3000 μM) inhibited GABAA‐mediated responses in the vagus nerve with an IC50 (and 95% CI) of 202 μM (148–275). BPAA (1–1000 μM) had little or no effect on the GABAA‐mediated response but concentration‐dependently potentiated the effects of ciprofloxacin by up to 33,000 times.

Responses mediated by 5‐HT3, nicotinic ACh and P2X receptors in the vagus nerve and strychnine‐sensitive glycine receptors in the optic nerve were little or unaffected by ciprofloxacin (100 μM), BPAA (100 μM) or the combination of these drugs (both at 100 μM).

GABA (1 mM)‐evoked responses in the optic nerve were inhibited by bicuculline with an IC50 of 3.6 μM (2.8–4.5), a value not significantly different from that determined in the vagus nerve. Ciprofloxacin also inhibited the GABA‐evoked response with an IC50 of 334 μM (256–437) and BPAA (100 μM) potentiated these antagonist effects. However, the magnitude of the synergy was 48 times less than that seen in the vagus nerve.

These data indicate that ciprofloxacin and BPAA are selective antagonists of GABAA receptors, an action that may contribute to their excitatory effects in vivo. Additionally, our data suggest that the molecular properties of GABAA receptors in different regions of the CNS influence the extent to which these drugs synergistically inhibit the GABAA receptor.

British Journal of Pharmacology (1997) 122, 584–590; doi:10.1038/sj.bjp.0701411