bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin‐1α and tumour necrosis factor‐α modulate airway smooth muscle DNA synthesis by induction of cyclo‐oxygenase‐2: inhibition by dexamethasone and fluticasone propionate
Authors: Vlahos Ross , Stewart Alastair G
Published: January 2005

Previous studies have established that glucocorticoids inhibit airway smooth muscle DNA synthesis. The effects of a combination of the pro‐inflammatory cytokines, interleukin‐1α (IL‐1α) and tumour necrosis factor‐α (TNF‐α) on the inhibition of DNA synthesis by glucocorticoids in human cultured airway smooth muscle have now been investigated, since these cytokines are chronically expressed in asthmatic airways.

Thrombin (0.3 u ml−1) and basic fibroblast growth factor (bFGF, 300 pM) stimulated increases in DNA synthesis which were concentration‐dependently inhibited by dexamethasone (1–1000 nM).

The cytokine mixture, comprising IL‐1α (0.01 and 0.1 pM) and TNF‐α (3 and 30 pM), directly evoked increases in DNA synthesis which were attenuated by dexamethasone. However, the cytokine mixture prevented responses to bFGF or thrombin.

Paradoxically, in the presence of the cytokine mixture and bFGF, dexamethasone (1–1000 nM) concentration‐dependently increased DNA synthesis. Furthermore, neither dexamethasone (100 nM) nor fluticasone propionate (1 nM) inhibited DNA synthesized in response to bFGF/cytokine mixture combination and dexamethasone was similarly inactive against the thrombin/cytokine mixture.

The levels of prostaglandin E2 (PGE2), an established inhibitor of airway smooth muscle DNA synthesis, remained below the limits of assay detection (0.05 nM) under basal conditions or following stimulation with either thrombin or bFGF. In contrast, the cytokine mixture alone, and in the presence of thrombin or bFGF, induced biologically active levels of PGE2. Dexamethasone (100 nM), the non‐selective cyclo‐oxygenase (COX) inhibitor indomethacin (3 μM) or the selective COX‐2 inhibitor L‐745,337 (0.3 μM) completely inhibited synthesis of PGE2.

Neither indomethacin (3 μM) nor L‐745,337 (0.3 μM) influenced thrombin‐ or bFGF‐induced DNA synthesis. However, each COX inhibitor enhanced DNA synthesis in cytokine‐treated cells.

In unstimulated airway smooth muscle cells, COX‐1, but not COX‐2 protein was detectable by Western blotting. The induction of COX‐2 protein by the cytokine mixture was attenuated by dexamethasone (100 nM), whereas the level of COX‐1 protein was unaffected by either the cytokines or by dexamethasone.

Cytokine‐induced, COX‐2‐dependent eicosanoid production inhibits DNA synthesis. The paradoxical increase in DNA synthesis observed in glucocorticoid treated airway smooth muscle stimulated by cytokine/bFGF combinations may be explained by the ability of glucocorticoids to repress COX‐2 induction and prevent cytokine‐induction of the DNA synthesis inhibitor, PGE2.

British Journal of Pharmacology (1999) 126, 1315–1324; doi:10.1038/sj.bjp.0702424
Effects of intravenous administration of prostacyclin on regional blood circulation in awake rats
Authors: Raczka Ewa , Quintana Antonio
Published: January 2005

The effects of intravenous infusion of prostacyclin (PGI2, 0.1, 0.2, 0.3 and 1.0 μg kg−1 min−1 lasting 5 min) on regional blood flow and regional vascular resistance have been studied in awake rats using the radioactive microsphere method.

The control values of blood flow to the heart, kidney, small intestine, hind limb muscle, pericranial skin and brain as well as the corresponding vascular resistance were not modified by an i.v. infusion (0.1 ml min−1) of Tris‐buffer (the vehicle of PGI2).

The i.v. infusion of PGI2 produced graded dose‐dependent decreases of MAP (r=0.87, P<0.001; ED20=0.73 [0.13–2.55] μg kg−1 min−1) as well as decreases of vascular resistance in the heart (r=0.83, P<0.001; ED30=0.17 [0.09–0.31] μg kg−1 min−1), pericranial skin (r=0.88, P<0.001; ED30=0.28 [0.18–0.43] μg kg−1 min−1) and small intestine (r=0.74, P<0.001; ED30=0.21 [0.11–0.39] μg kg−1 min−1), which led to dose‐related increases of blood flow to these territories.

On the contrary, PGI2 increased vascular resistance in skeletal muscle (r=0.73, P<0.001; ED30=0.20 [0.10–0.39] μg kg−1 min−1) with corresponding reductions in blood flow. The low doses reduced renal blood flow but there were no significant changes during the high ones. Cerebral vessels did not dilate during any infusion of PGI2 and cerebral blood flow decreased as MAP fell (r=0.56, P<0.01).

We conclude that, in awake rats, the coronary vessels are extremely sensitive to the vasodilating effect of PGI2 and that the mesenteric vessels and those of the pericranial skin are very responsive too. Moreover, autoregulation is inefficient to maintain cerebral blood flow during infusion of PGI2.

British Journal of Pharmacology (1999) 126, 1325–1332; doi:10.1038/sj.bjp.0702426
Effects of propafenone on K currents in human atrial myocytes
Authors: Seki Akiko , Hagiwara Nobuhisa , Kasanuki Hiroshi
Published: January 2005

The class Ic anti‐arrhythmic agent, flecainide is known to inhibit the transient outward K current (Ito) selectively in human atrium. We studied the effects of propafenone, another class Ic anti‐arrhythmic agent, on K currents in human atrial myocytes using a whole‐cell voltage‐clamp method.

Propafenone inhibited both Ito and the sustained or ultra‐rapid delayed rectifier K current (Isus or Ikur) evoked by depolarization pulses. The concentration for half‐maximal inhibition (IC50) was 4.9 μM for Ito and 8.6 μM for Isus. Propafenone blocked Ito and Isus in a voltage‐ and use‐independent fashion and accelerated the inactivation time constant of Ito [from 28.3 to 6.7 ms at 10 μM propafenone].

The steady‐state inactivation curve for Ito was unaffected by propafenone. Propafenone did not affect the initial current at depolarizing potentials, but it did produce a block that increased as a function of time after depolarization (time constant of 3.4 ms). This suggests that propafenone preferentially blocked Ito in the open state.

Propafenone had no significant effect on the rate at which Ito recovered from inactivation at −80 mV suggesting that propafenone dissociates rapidly from the channel.

The steady‐state activation curve for Isus was not affected by propafenone. Propafenone slowed the time course of the onset of the Isus tail current. This suggests that propafenone blocked Isus in the open state.

The present results suggest that, unlike flecainide, propafenone blocks both Ito and Isus in human atrial myocytes in the open state at clinically relevant concentrations.

British Journal of Pharmacology (1999) 126, 1153–1162; doi:10.1038/sj.bjp.0702428
The vasoconstrictor effect of 8‐epi prostaglandin F2α in the hypoxic rat heart
Authors: Kromer Brendan M , Tippins John R
Published: January 2005

8‐epi prostaglandin (PG) F2α, a vasoconstrictor isoprostane, is synthesized under conditions of oxidative stress. This study was undertaken to investigate the vasoconstrictor effect of 8‐epi PGF2α in the coronary circulation before and after a period of oxidative stress.

The effects of the isoprostane 8‐epi PGF2α and the thromboxane mimetic U46619 were compared in the isolated rat heart perfused in the Langendorff mode at a constant pressure of 80 mmHg.

In normal hearts U46619 caused a dose‐related reduction in coronary flow (ED50 4.7±2.2 nmol). In contrast, 8‐epi PGF2α had no effect.

After reducing perfusion pressure to 20 mmHg for 30 min and reperfusing at 80 mmHg, the dose‐response curve to U46619 was unaffected. In contrast, 8‐epi PGF2α caused a dose‐dependent drop in coronary flow (ED50 52.6±12.7 nmol), producing a similar maximal reduction to U46619.

Similarly, after perfusion with xanthine and xanthine oxidase for either 15 or 30 min there was little change in the response to U46619 in comparison to control hearts. In contrast, 8‐epi PGF2α caused a reduction in coronary flow similar to that produced by U46619, the magnitude of the response being related to the length of xanthine/xanthine oxidase perfusion.

Responses to both U46619 and 8‐epi PGF2α after xanthine/xanthine oxidase perfusion were blocked by the selective thromboxane receptor antagonist SQ29548 10−7 M.

These results show that oxidative stress in the isolated perfused rat heart reveals a potent vasoconstrictor effect of the isoprostane 8‐epi PGF2α by an action on the thromboxane receptor.

The data also suggest that, since 8‐epi PGF2α is a partial agonist at the thromboxane receptor, thromboxane receptor reserve is increased by oxidative stress.

British Journal of Pharmacology (1999) 126, 1171–1174; doi:10.1038/sj.bjp.0702433
Induction of cyclo‐oxygenase‐2 expression by methyl arachidonyl fluorophosphonate in murine J774 macrophages: roles of protein kinase C, ERKs and p38 MAPK
Authors: Lin Wan‐W , Chen Bing‐C
Published: January 2005

Methyl arachidonyl fluorophosphonate (MAFP), an inhibitor of phospholipase A2 (PLA2), has been widely used to assess the roles of PLA2 in various cell functions. Here, we report on a novel action of this compound at concentrations similar to those used for PLA2 inhibition.

The murine macrophage J774 released a large amount of prostaglandin E2 (PGE2) by MAFP (1–30 μM), which was abolished by indomethacin and NS‐398 but not by valeryl salicylate, and results from increased cyclo‐oxygenase‐2 (COX‐2) protein levels and gene expression.

This PGE2 release was blocked by inhibitors of tyrosine kinase (genistein), protein kinase C (PKC) (Ro 31–8220, Go 6976 or LY 379196), mitogen‐activated protein kinase kinase (MEK) (PD 098059) or p38 mitogen‐activated protein kinase (MAPK) (SB 203580).

Consistent with these results, MAFP caused membrane translocation of PKCβI and βII isoforms and activated extracellular signal‐regulated kinase (ERK) and p38 MAPK.

In accordance with these effects of MAFP, PKC activator phorbol 12‐myristate 13‐acetate (PMA) increased PGE2 release and caused activation of PKCβ, ERKs and p38 MAPK.

This is the first report that the PLA2 inhibitor, MAFP, can induce COX‐2 gene expression and PGE2 synthesis via the PKC‐, ERK‐ and p38 MAPK‐dependent pathways. Thus, the use of MAFP as a PLA2 inhibitor should be treated with caution.

British Journal of Pharmacology (1999) 126, 1419–1425; doi:10.1038/sj.bjp.0702436
Cytosolic Ca2+ movements of endothelial cells exposed to reactive oxygen intermediates: Role of hydroxyl radical‐mediated redox alteration of cell‐membrane Ca2+ channels
Authors: Az‐ma Toshiharu , Saeki Noboru , Yuge Osafumi
Published: January 2005

The mode of action of reactive oxygen intermediates in cysosolic Ca2+ movements of cultured porcine aortic endothelial cells exposed to xanthine/xanthine oxidase (X/XO) was investigated.

Cytosolic Ca2+ movements provoked by X/XO consisted of an initial Ca2+ release from thapsigargin‐sensitive intracellular Ca2+ stores and a sustained Ca2+ influx through cell‐membrane Ca2+ channels. The Ca2+ movements from both sources were inhibited by catalase, cell‐membrane permeable iron chelators (o‐phenanthroline and deferoxamine), a •OH scavenger (5,5‐dimethyl‐1‐pyrroline‐N‐oxide), or an anion channel blocker (disodium 4, 4′‐diisothiocyano‐2, 2′‐stilbenedisulphonic acid), suggesting that •O2− influx through anion channels was responsible for the Ca2+ movements, in which •OH generation catalyzed by intracellular transition metals (i.e., Haber‐Weiss cycle) was involved.

After an initial Ca2+ elevation provoked by X/XO, cytosolic Ca2+ concentration decreased to a level higher than basal levels. Removal of X/XO slightly enhanced the Ca2+ decrease. Extracellular addition of sulphydryl (SH)‐reducing agents, dithiothreitol or glutathione, after the removal of X/XO accelerated the decrement. A Ca2+ channel blocker, Ni2+, abolished the sustained increase in Ca2+, suggesting that Ca2+ influx through cell‐membrane Ca2+ channels was extracellularly regulated by the redox state of SH‐groups.

The X/XO‐provoked change in cellular respiration was inhibited by Ni2+ or dithiothreitol as well as inhibitors of Haber‐Weiss cycle, suggesting that Ca2+ influx was responsible for •OH‐mediated cytotoxicity. We concluded that intracellular •OH generation was involved in the Ca2+ movements in endothelial cells exposed to X/XO. Cytosolic Ca2+ elevation was partly responsible for the oxidants‐mediated cytotoxicity.

British Journal of Pharmacology (1999) 126, 1462–1470; doi:10.1038/sj.bjp.0702438
Inhibitory effect of 4‐aminopyridine on responses of the basilar artery to nitric oxide
Authors: Sobey Christopher G , Faraci Frank M
Published: January 2005

Voltage‐dependent K+ channels are present in cerebral arteries and may modulate vascular tone. We used 200 μM 4‐aminopyridine (4‐AP), thought to be a relatively selective inhibitor of voltage‐dependent K+ channels at this concentration, to test whether activation of these channels may influence baseline diameter of the basilar artery and dilator responses to nitric oxide (NO) and cyclic GMP in vivo.

Using a cranial window in anaesthetized rats, topical application of 4‐AP to the basilar artery (baseline diameter=240±5 μm, mean±s.e.mean) produced 10±1% constriction. Sodium nitroprusside (a NO donor), acetylcholine (which stimulates endothelial release of NO), 8‐bromo cyclic GMP (a cyclic GMP analogue), cromakalim (an activator of ATP‐sensitive K+ channels) and papaverine (a non‐NO, non‐K+ channel‐related vasodilator) produced concentration‐dependent vasodilator responses that were reproducible.

Responses to 10 and 100 nM nitroprusside were inhibited by 4‐AP (20±4 vs 8±2% and 51±5 vs 33±5%, respectively, n=10; P<0.05). Responses to acetylcholine and 8‐bromo cyclic GMP were also partially inhibited by 4‐AP. In contrast, 4‐AP had no effect on vasodilator responses to cromakalim or papaverine. These findings suggest that NO/cyclic GMP‐induced dilator responses of the basilar artery are selectively inhibited by 4‐aminopyridine.

Responses to nitroprusside were also markedly inhibited by 10 μM 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (an inhibitor of soluble guanylate cyclase; 16±4 vs 1±1% and 44±7 vs 7±1%; n=10; P<0.05).

Thus, dilator responses of the rat basilar artery to NO appear to be mediated by activation of soluble guanylate cyclase and partially by activation of a 4‐aminopyridine‐sensitive mechanism. The most likely mechanism would appear to be activation of voltage‐dependent K+ channels by NO/cyclic GMP.

British Journal of Pharmacology (1999) 126, 1437–1443; doi:10.1038/sj.bjp.0702439
Kinetics of rate‐dependent shortening of action potential duration in guinea‐pig ventricle; effects of IK1 and IKr blockade
Authors: Williams Beatrice A , Dickenson David R , Beatch Gregory N
Published: January 2005

The kinetics of shortening of action potential duration (APD) following an increase in pacing rate, from 2 to 3.3 Hz, was characterized in guinea‐pig ventricular preparations. Terikalant (RP62719), an inhibitor of the inwardly rectifying K+ current (IK1), and dofetilide, a specific inhibitor of the rapidly activating delayed‐rectifier current (IKr), were applied to determine the effect of inhibition of these ion currents on slow APD shortening.

Action potentials were recorded from isolated guinea‐pig ventricular myocytes using the perforated‐patch patch‐clamp technique, and monophasic action potentials were recorded from Langendorff‐perfused guinea‐pig ventricles using a contact epicardial probe.

Under control conditions, after an increase in pacing rate, APD immediately decreased, and then shortened slowly with an exponential time course. In ventricular myocytes, the time constant of this exponential shortening was 28±4 s and the amount of slow shortening was 21.9±0.9 ms (n=8) for an increase in rate from 2 to 3.3 Hz. Similar values were observed in Langendorff‐perfused ventricles.

Terikalant dose‐dependently increased APD and the increase was enhanced by rapid pacing (‘positive’ rate‐dependence). The drug dose‐dependently decreased the time constant of shortening and amount of slow APD shortening. In contrast, dofetilide, an inhibitor of IKr, which shows ‘reverse’ rate‐dependent APD widening, had no significant effect on the time constant or amount of slow shortening.

These observations suggest that IK1 plays a role in rate‐dependent shortening of APD. The results appear to support the hypothesis that ‘reverse’ rate‐dependent effects of IKr blockers are due to these drugs not affecting the ion current(s) mediating intrinsic rate‐dependent slow shortening of APD.

British Journal of Pharmacology (1999) 126, 1426–1436; doi:10.1038/sj.bjp.0702443
Dural vasodilation causes a sensitization of rat caudal trigeminal neurones in vivo that is blocked by a 5‐HT1B/1D agonist
Authors: Cumberbatch M J , Williamson D J , Mason G S , Hill R G , Hargreaves R J
Published: January 2005

Migraine headache pain is thought to result from an abnormal distention of intracranial, extracerebral blood vessels and the consequent activation of the trigeminal nervous system. Migraine is also often accompanied by extracranial sensory disturbances from facial tissues. These experiments investigate whether meningeal dilation produces central sensitization of neurones that receive convergent input from the face.

Single unit extracellular activity was recorded from the trigeminal nucleus caudalis of anaesthetized rats in response to either noxious stimulation of the dura mater, innocuous stimulation of the vibrissae or to a transient dilation of the meningeal vascular bed.

Rat α‐CGRP (calcitonin gene‐related peptide; 1 μg kg−1, i.v.) caused a dilation of the middle meningeal artery and facilitated vibrissal responses by 36±7%.

The 5‐HT1B/1D agonist, L‐741,604 (3 mg kg−1, i.v.), inhibited responses to noxious stimulation of the dura mater (16±7% of control) and, in a separate group of animals, blocked the CGRP‐evoked facilitation of vibrissal responses.

L‐741,604 (3 mg kg−1, i.v.) also inhibited responses to innocuous stimulation of the vibrissa (14±10% of control) with neurones that received convergent input from the face and from the dura mater, but not with cells that received input only from the face (70±12% of control).

These data show that dilation of meningeal blood vessels causes a sensitization of central trigeminal neurones and a facilitation of facial sensory processing which was blocked by activation of pre‐synaptic 5‐HT1B/1D receptors.

Sustained dural blood vessel dilation during migraine may cause a sensitization of trigeminal neurones. This may underlie some of the symptoms of migraine, such as the headache pain and the extracranial allodynia. Inhibition of this central sensitization may therefore offer a novel strategy for the development of acute and/or prophylactic anti‐migraine therapies.

British Journal of Pharmacology (1999) 126, 1478–1486; doi:10.1038/sj.bjp.0702444
Investigation of stretching behaviour induced by the selective 5‐HT6 receptor antagonist, Ro 04–6790, in rats
Authors: Bentley Jane C , Bourson Anne , Boess Frank G , Fone Kevin C F , Marsden Charles A , Petit Nadine , Sleight Andrew J
Published: January 2005

The present study examined the effects of the selective 5‐HT6 receptor antagonist 4‐amino‐N‐(2, 6 bis‐methylamino‐pyrimidin‐4‐yl)‐benzene sulphonamide (Ro 04–6790) on locomotor activity and unconditioned behaviour in male Sprague Dawley rats (230–300 g).

In non‐quantified behavioural observations, animals treated with Ro 04–6790 (3, 10 or 30 mg kg−1, i.p) showed no overt behavioural signs except a dose‐dependent reduction in locomotor activity and a behavioural syndrome of stretching, yawning and chewing. The latter behaviour was most pronounced between 30 and 90 min following the administration of Ro 04–6790.

Detailed analysis of the stretching and yawning behaviour showed that Ro 04–6790 (3, 10 or 30 mg kg−1, i.p.) dose‐dependently induced stretching. The number of stretches observed following treatment with either Ro 04–6790 (10 mg kg−1 i.p.) or Ro‐04‐6790 (30 mg kg−1, i.p.) was significantly greater than that observed in saline‐treated rats. The yawning behaviour, however, was not dose‐dependent nor was the number of yawns in any of the drug treated groups significantly greater than in those treated with saline.

Pretreatment (30 min) with the non‐selective muscarinic antagonists scopolamine (0.1, 0.3 or 1 mg kg−1, i.p.) and atropine (0.3, 1 or 3 mg kg−1, s.c.) but not methylatropine (1, 3 or 10 mg kg−1, s.c) significantly inhibited stretching induced by Ro 04–6790 (30 mg kg−1, i.p.).

The dopamine D2‐like receptor antagonist, haloperidol (0.03, 0.1 or 0.3 mg kg−1, s.c.) given at the same time as Ro 04–6790 (30 mg kg−1, i.p.) had no effect on the stretching induced by the 5‐HT6 antagonist.

These data suggest that systemic injection of the 5‐HT6 antagonist, Ro 04–6790, produces a stretching behaviour that appears to be mediated by an increase in cholinergic neurotransmission in the CNS and which could be a useful functional correlate for 5‐HT6 receptor blockade. There is no evidence for dopamine D2‐like receptor involvement in this behaviour.

British Journal of Pharmacology (1999) 126, 1537–1542; doi:10.1038/sj.bjp.0702445
Characterization of the binding of a novel radioligand to CCKB/gastrin receptors in membranes from rat cerebral cortex
Authors: Harper E A , Shankley N P , Black J W
Published: January 2005

We have investigated the binding of a novel radiolabelled CCKB/gastrin receptor ligand, [3H]‐JB93182 (5[[[(1S)‐[[(3,5‐dicarboxyphenyl)amino]carbonyl]‐2‐phenylethylamino]‐carbonyl]‐6‐[[(1‐adamantylmethyl) amino]carbonyl]‐indole), to sites in rat cortex membranes.

The [3H]‐JB93182 was 97% radiochemically pure as assessed by reverse‐phase HPLC (RP‐HPLC) and was not degraded by incubation (150 min) with rat cortex membranes.

Saturation analysis indicated that [3H]‐JB93182 labelled a homogeneous population of receptors in rat cortex membranes (pKD=9.48±0.08, Bmax=3.61∼plusmn;0.65 pmol g−1 tissue, nH=0.97±0.02, n=5). The pKD was not significantly different when estimated by association‐dissociation analysis (pKD=9.73±0.11; n=10).

In competition studies, the low affinity of the CCKA receptor antagonists, L‐364,718; SR27897 and 2‐NAP, suggest that, under the assay conditions employed, [3H]‐JB93182 (0.3 nM) does not label CCKA receptors in the rat cortex.

The affinity estimates obtained for reference CCKB/gastrin receptor antagonists were indistinguishable from one of the affinity values obtained when a two site model was used to interpret [125I]‐BH‐CCK8S competition curves obtained in the same tissue (Harper et al., 1999).

This study provides further evidence for the existence of two CCKB/gastrin sites in rat cortex. [3H]‐JB93182 appears to label selectively sites previously designated as gastrin‐G1 and therefore it may be a useful compound for the further discrimination and characterization of these putative receptor subtypes.

British Journal of Pharmacology (1999) 126, 1504–1512; doi:10.1038/sj.bjp.0702447
Rapid actions of calcitriol and its side chain analogues CB1093 and GS1500 on intracellular calcium levels in skeletal muscle cells: a comparative study
Authors: Vazquez Guillermo , Sellés Juana , De Boland Ana Russo , Boland Ricardo
Published: January 2005

The ability of synthetic analogues of the secosteroid hormone 1α,25‐dihydroxy‐vitamin‐D3 [calcitriol, CT; 1,25(OH)2D3] to exert non‐genomic (rapid) effects on target cells has been scarcely studied. To evaluate the pharmacological potential of the CT side‐chain analogues CB1093 and GS1500, we compared their fast effects on intracellular calcium concentration ([Ca2+]i) in chick skeletal muscle cells with those elicited by the natural hormone.

Both analogues, similarly to CT, specifically induced rapid (30–60 s) and sustained rises in [Ca2+]i levels. CB1093 and GS1500 were more potent than the natural hormone at concentrations as low as 10−13 M (4.5 fold stimulation) and 10−12 M (2.5 fold), respectively, whereas higher concentrations (10−9–10−8 M) of CT were more effective than the analogues in elevating [Ca2+]i. Cyclic AMP was markedly increased by both analogues pointing for a role of this messenger in the fast actions of the synthetic compounds.

In Ca2+ free medium CT and analogues elicited a transient elevation in [Ca2+]i. The PLC inhibitors U73122 (2 μM) and neomycin (0.5 mM), as well as depletion of intracellular stores with thapsigargin (1 μM), completely prevented CB1093/GS1500‐dependent changes in [Ca2+]i suggesting that, similarly to CT, these analogues mobilized Ca2+ from an IP3/thapsigargin‐sensitive store.

The voltage‐dependent calcium channel (VDCC) blocker nifedipine (2 μM) reduced by 50–60% the influx phase of the [Ca2+]i response to CB1093 and GS1500, indicating that VDCC contributed partially to Ca2+ entry. The Ca2+ readdition protocol suggested that analogue‐dependent activation of a SOC entry pathway accounted, to the same extent as for CT, for the remaining non‐VDCC mediated Ca2+ influx.

British Journal of Pharmacology (1999) 126, 1815–1823; doi:10.1038/sj.bjp.0702451
Involvement of CB1 cannabinoid receptors in the EDHF‐dependent vasorelaxation in rabbits
Authors: Niederhoffer Nathalie , Szabo Bela
Published: January 2005

It was recently suggested that an endogenous cannabinoid could represent an endothelium‐derived hyperpolarizing factor (EDHF). The aim of the present study was to clarify whether CB1 cannabinoid receptors are involved in the nitric oxide (NO)‐ and prostanoid‐independent vasodilation produced by acetylcholine in rabbits.

Pithed rabbits received indomethacin. Noradrenaline was infused to raise blood pressure, and vasodilation was elicited by bolus injections of acetylcholine. The NO‐synthase inhibitor Nω‐nitro‐L‐arginine methylester inhibited the acetylcholine‐evoked vasodilation by about 40%. The remaining vasodilation was unaffected by the CB1 cannabinoid receptor antagonist SR141716A, but was inhibited by the potassium channel blocker tetraethylammonium. In addition, the mixed CB1/CB2 cannabinoid receptor agonist WIN55212‐2 did not elicit vasodilation.

No CB1 cannabinoid receptors were involved in the prostanoid‐ and NO‐independent vasodilation produced by acetylcholine. An exogenous cannabinoid also did not cause vasodilation. Therefore, it is unlikely that an endogenous cannabinoid serves as an EDHF acting at smooth muscle CB1 cannabinoid receptors in the rabbit.

British Journal of Pharmacology (1999) 126, 1383–1386; doi:10.1038/sj.bjp.0702452
Effects of dexamethasone on airway hyper‐responsiveness to the adenosine A1 receptor agonist cyclo‐pentyl adenosine in an allergic rabbit model
Authors: El‐Hashim Ahmed Z , Banner Katharine H , Paul William , Page Clive P
Published: January 2005

New Zealand White (NZW) rabbits were immunized within 24 h of birth with Alternaria tenuis in aluminium hydroxide (Al (OH)3) (i.p.) or sham immunized (saline plus Al (OH)3 i.p.) and subsequently injected with the allergen (i.p.) or sham‐immunized for the next 3 months. At 3 months of age, baseline airway responsiveness was assessed using cyclo‐pentyl adenosine (CPA). Bronchoalveolar lavage (BAL) was performed in all animals and samples of peripheral blood were collected from some animals for estimation of dexamethasone levels. In some animals, blood was collected at the end of the experiment and cellular function was assessed by measurement of ex vivo proliferation of mononuclear cells in response to phytohaemagglutinin (PHA).

Allergen immunization significantly increased baseline airway responsiveness to inhaled CPA (P<0.05) in comparison with sham‐immunized animals, at 3 months after immunization. Dexamethasone (0.5 mg kg−1 day−1) treatment for 1 month did not modify this established airway hyper‐responsiveness to CPA. Dexamethasone treatment did not affect either total or differential cell numbers in BAL fluid during the 4 week period, although significant plasma levels of dexamethasone were achieved in dexamethasone treated animals.

Treatment of rabbits with dexamethasone (0.1 mg kg−1 i.p.), 6 h prior to each allergen injection from the neonatal stage, significantly reduced baseline airway hyper‐responsiveness to CPA measured at 3 months (P<0.05). There was no significant difference in either total or differential cell numbers in BAL fluid, or any difference in mitogen‐induced proliferation of mononuclear cells between dexamethasone and vehicle treated rabbits.

These results suggest that introduction of glucocorticosteroids in early life can prevent baseline airway hyper‐responsiveness to inhaled CPA in allergic rabbits. However, once established, such underlying airway hyper‐responsiveness is difficult to resolve, even with prolonged treatment with glucocorticosteroids.

British Journal of Pharmacology (1999) 126, 1513–1521; doi:10.1038/sj.bjp.0702455
Interaction of amiodarone and triiodothyronine on the expression of β‐adrenoceptors in brown adipose tissue of rat
Authors: Adli Houria , Bazin Raymond , Perret Gérard Y
Published: January 2005

This study was undertaken to evaluate in vivo the influence of amiodarone on the effects of triiodothyronine (T3) in brown adipose tissue (BAT) which are independent of thyroid hormone synthesis and of the conversion of thyroxine (T4) to T3. Thyroidectomized rats were given a replacement dose of T3 (0.5 mg kg−1 p.o. daily for 3 days) with or without amiodarone (50 mg kg−1 p.o. daily for 1 week).

As assessed by RT–PCR, treatment of thyroidectomized rats with T3 caused a 2 fold decrease in β3‐adrenoceptor (β3‐AR) mRNA levels and a 2 fold increase in β1‐AR mRNA levels.

Binding studies using [3H]‐CGP 12177 as a ligand showed that treatment of thyoidectomized rats with T3 resulted in a 70% decrease in β3‐AR number and in an 80% increase in β1‐AR in BAT membranes.

T3‐treatment abolished the increase in BAT adenylyl cyclase (AC) activity induced by CGP12177 in thyroidectomized rats. It also decreased the amount of Gi protein (ADP‐ribosylation) by 30%.

At variance with the literature on the heart, amiodarone administration did not inhibit the positive effect of T3 on β1‐AR expression in BAT in thyroidectomized rats. However, it antagonized the effect of T3 on β3‐AR number, but not on AC activity or on Gi expression.

These results indicate that the effects of thyroid hormones on the responsiveness of BAT to catecholamines involves both receptor and post‐receptor mechanisms, they also suggest that interaction between amiodarone and thyroid hormones is highly tissue‐specific and depends on the β‐AR subtype.

British Journal of Pharmacology (1999) 126, 1455–1461; doi:10.1038/sj.bjp.0702456
Functional comparison of muscarinic partial agonists at muscarinic receptor subtypes hM1, hM2, hM3, hM4 and hM5 using microphysiometry
Authors: Wood Martyn D , Murkitt Karen L , Ho Michael , Watson Jeannette M , Brown Frank , Hunter A Jacqueline , Middlemiss Derek N
Published: January 2005

This study describes the pharmacological comparison of the muscarinic partial agonists sabcomeline, xanomeline and milameline at human cloned muscarinic receptor subtypes (hM1–5).

Radioligand binding studies at the hM1–5 muscarinic receptor subtypes were compared with functional studies using microphysiometry using carbachol as the standard full agonist.

In binding assays none of the compounds studied displayed preferential affinity for the M1,3,4 or M5 subtypes although carbachol was less potent at hM1 than hM3,4,5.

In functional studies, all of the compounds studied displayed similar levels of efficacy across the muscarinic receptors with the exception of M3, where there was a large apparent receptor reserve and the compounds behaved essentially as full agonists.

Sabcomeline was the most potent agonist in functional studies but also showed the lowest efficacy. In terms of potency, xanomeline showed some selectivity for M1 over M2 receptors and milameline showed some selectivity for M2 over M1 receptors.

These results show the value of microphysiometry in being able to compare receptor pharmacology across subtypes irrespective of the signal transduction pathway.

None of the partial agonists showed functional selectivity for M1 receptors, or indeed any muscarinic receptor, in the present study.

British Journal of Pharmacology (1999) 126, 1620–1624; doi:10.1038/sj.bjp.0702463
Relationship between NaF‐ and thapsigargin‐induced endothelium‐dependent hyperpolarization in rat mesenteric artery
Authors: Fukao Mitsuhiro , Hattori Yuichi , Sato Atsushi , Liu Ming‐Yue , Watanabe Hiroshi , Kim Tran Quang , Kanno Morio
Published: January 2005

In isolated rat mesenteric artery with endothelium, NaF caused slowly developing hyperpolarization. The hyperpolarizing effect was unchanged in the presence of NG‐nitro‐L‐arginine (L‐NOARG) and indomethacin, but was markedly reduced by high K+. In Ca2+‐free medium or in the presence of Ni2+, NaF failed to produce hyperpolarization.

NaF‐induced hyperpolarization was substantially unaffected by deferoxamine, an Al3+ chelator, okadaic acid and calyculin A, phosphatase inhibitors, and preincubation with pertussis toxin, suggesting that neither the action of fluoroaluminates as a G protein activator nor inhibition of phosphatase activity contributes to the hyperpolarizing effect.

The selective inhibitors of the Ca2+‐pump ATPase of endoplasmic reticulum, thapsigargin and cyclopiazonic acid, elicited hyperpolarization, whose properties were very similar to those of NaF. When intracellular Ca2+ stores had been depleted with these inhibitors, NaF no longer generated hyperpolarization.

In Ca2+‐free medium, NaF (or thapsigargin) caused a transient increase in the cytosolic Ca2+ concentration ([Ca2+]i) in cultured porcine aortic endothelial cells, and subsequent application of thapsigargin (or NaF) failed to increase [Ca2+]i.

In arterial rings precontracted with phenylephrine, NaF produced endothelium‐dependent relaxation followed by sustained contraction even in the presence of L‐NOARG and indomethacin. The relaxant response was abolished by high K+ or cyclopiazonic acid.

These results indicate that NaF causes endothelium‐dependent hyperpolarization, thereby leading to smooth muscle relaxation of rat mesenteric artery. This action appears to be mediated by the promotion of Ca2+ influx into endothelial cells that can be triggered by the emptying of intracellular Ca2+ stores, as proposed for those of thapsigargin and cyclopiazonic acid.

British Journal of Pharmacology (1999) 126, 1567–1574; doi:10.1038/sj.bjp.0702466
An investigation into the structural determinants of cannabinoid receptor ligand efficacy
Authors: Griffin Graeme , Wray Emma J , Rorrer William K , Crocker Peter J , Ryan William J , Saha Bijali , Razdan Raj K , Martin Billy R , Abood Mary E
Published: January 2005

A number of side‐chain analogues of Δ8‐THC were tested in GTPγS binding assay in rat cerebellar membranes. O‐1125, a saturated side‐chain compound stimulated GTPγS binding with an Emax of 165.0%, and an EC50 of 17.4 nM.

O‐1236, O‐1237 and O‐1238, three‐enyl derivatives containing a cis carbon‐carbon double bond in the side‐chain, stimulated GTPγS binding, acting as partial agonists with Emax values ranging from 51.3–87.5% and EC50 values between 4.4 and 29.7 nM.

The stimulatory effects of O‐1125, O‐1236, O‐1237 and O‐1238 on GTPγS binding were antagonized by the CB1 receptor antagonist SR 141716A. The KB values obtained ranged from 0.11–0.21 mM, suggesting an action at CB1 receptors.

Five‐ynyl derivatives (O‐584, O‐806, O‐823, O‐1176 and O‐1184), each containing a carbon‐carbon triple bond in the side‐chain, did not stimulate GTPγS binding and were tested as potential cannabinoid receptor antagonists.

Each ‐ynyl compound antagonized the stimulatory effects of four cannabinoid receptor agonists on GTPγS binding. The KB values obtained, all found to be in the nanomolar range, did not differ between agonists or from cerebellar binding affinity.

In conclusion, alterations of the side‐chain of the classical cannabinoid structure may exert a large influence on affinity and efficacy at the CB1 receptor.

Furthermore, this study confirms the ability of the GTPγS binding assay to assess discrete differences in ligand efficacies which potentially may not be observed using alternative functional assays, thus providing a unique tool for the assessment of the molecular mechanisms underlying ligand efficacies.

British Journal of Pharmacology (1999) 126, 1575–1584; doi:10.1038/sj.bjp.0702469
In vivo effects of new inhibitors of catechol‐O‐methyl transferase
Authors: Rivas Emilia , De Ceballos Maria L , Nieto Ofelia , Fontenla José Angel
Published: January 2005

The effects of two new synthetic compounds showing in vitro catechol‐O‐methyl transferase (COMT) inhibitor properties were studied in vivo and compared with the effects of nitecapone and Ro‐41‐0960.

QO IA (3‐(3‐hydroxy‐4‐methoxy‐5‐nitrobenzylidene)‐2,4‐pentanedione), QO IIR ([2‐(3,4‐dihydroxy‐2‐nitrophenyl)vinyl]phenyl ketone), nitecapone and Ro‐41‐0960 (30 mg kg−1, i.p.) were given to reserpinized rats 1 h before the administration of L‐DOPA/carbidopa (LD/CD, 50 : 50 mg kg−1, i.p.). Locomotor activity was assessed 1 h later. All the COMT inhibitors (COMTI), with the exception of QO IA, markedly potentiated LD/CD reversal of reserpine‐induced akinesia. Similar results were obtained when the COMTI were coadministered with LD/CD. The effect of compound QO IIR was dose‐dependent (7.5–30 mg kg−1, i.p.).

The COMTI (30 mg kg−1, i.p.) potentiated LD/CD reversal of both catalepsy and hypothermia of reserpinized mice.

QO IIR, nitecapone and Ro‐41‐0960 (30 mg kg−1, i.p.) reduced striatal 3‐methyl‐DOPA (3‐OMD) levels and increased dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) levels. Compound QO IA was devoid of any effect on striatal amine levels. In contrast to the other inhibitors, Ro‐41‐0961 reduced HVA levels as well. The effect of QO IIR on striatal amine levels was dose‐dependent (7.5–60 mg kg−1, i.p.)

These results suggest that the new compound QO IIR is an effective peripherally acting COMT inhibitor in vivo.

British Journal of Pharmacology (1999) 126, 1667–1673; doi:10.1038/sj.bjp.0702474
The antifungal antibiotic clotrimazole potently inhibits L‐type calcium current in guinea‐pig ventricular myocytes
Authors: Thomas George P , Karmazyn Morris , Zygmunt Andrew C , Antzelevitch Charles , Narayanan Njanoor
Published: January 2005

The antimycotic agent clotrimazole (CLT) is a promising potential therapeutic agent for a variety of diseases including cancer. Although it is known that CLT alters calcium homeostasis in many cell types, its cardiac effects are virtually unknown. We investigated the effects of CLT on L‐type calcium current (ICa,L) and action potentials in guinea‐pig ventricular myocytes. CLT (5, 25 and 50 μM) inhibited basal ICa,L by 16, 59 and 93%, respectively. The inhibitory effect of CLT was rapid and the peak effect was attained within 3 min. At a concentration of 25 μM, the inhibitory effect of CLT was partially reversible whereas the response to 50 μM CLT persisted following drug withdrawal. CLT abbreviated action potential duration at 50 and 90% of repolarization and suppressed the plateau significantly. These results indicate that CLT may have important cardiac effects at concentrations used to induce the antiproliferative action of the drug.

British Journal of Pharmacology (1999) 126, 1531–1533; doi:10.1038/sj.bjp.0702475
UK‐78,282, a novel piperidine compound that potently blocks the Kv1.3 voltage‐gated potassium channel and inhibits human T cell activation
Authors: Hanson Douglas C , Nguyen Angela , Mather Robert J , Rauer Heiko , Koch Kevin , Burgess Laurence E , Rizzi James P , Donovan Carol B , Bruns Matthew J , Canniff Paul C , Cunningham Ann C , Verdries Kimberly A , Mena Edward , Kath John C , Gutman George A , Cahalan Michael D , Grissmer Stephan , Chandy K George
Published: January 2005

UK‐78,282, a novel piperidine blocker of the T lymphocyte voltage‐gated K+ channel, Kv1.3, was discovered by screening a large compound file using a high‐throughput 86Rb efflux assay. This compound blocks Kv1.3 with a IC50 of ∼200 nM and 1 : 1 stoichiometry. A closely related compound, CP‐190,325, containing a benzyl moiety in place of the benzhydryl in UK‐78,282, is significantly less potent.

Three lines of evidence indicate that UK‐78,282 inhibits Kv1.3 in a use‐dependent manner by preferentially blocking and binding to the C‐type inactivated state of the channel. Increasing the fraction of inactivated channels by holding the membrane potential at −50 mV enhances the channel's sensitivity to UK‐78,282. Decreasing the number of inactivated channels by exposure to ∼160 mM external K+ decreases the sensitivity to UK‐78,282. Mutations that alter the rate of C‐type inactivation also change the channel's sensitivity to UK‐78,282 and there is a direct correlation between τh and IC50 values.

Competition experiments suggest that UK‐78,282 binds to residues at the inner surface of the channel overlapping the site of action of verapamil. Internal tetraethylammonium and external charybdotoxin do not compete UK‐78,282's action on the channel.

UK‐78,282 displays marked selectivity for Kv1.3 over several other closely related K+ channels, the only exception being the rapidly inactivating voltage‐gated K+ channel, Kv1.4.

UK‐78,282 effectively suppresses human T‐lymphocyte activation.

British Journal of Pharmacology (1999) 126, 1707–1716; doi:10.1038/sj.bjp.0702480
JTT‐608 restores impaired early insulin secretion in diabetic Goto‐Kakizaki rats
Authors: Ohta Takeshi , Furukawa Noboru , Komuro Goro , Yonemori Fumihiko , Wakitani Korekiyo
Published: January 2005

We investigated the pharmacological effects of a new antidiabetic agent, JTT‐608, in comparison with the sulphonylurea tolbutamide, in Goto‐Kakizaki (GK) rats, a genetic model of non‐obese insulin‐dependent diabetes mellitus (NIDDM).

In isolated perfused pancreas from GK rats, JTT‐608 (200 μM) enhanced 11.1 mM glucose‐stimulated insulin secretion in the first and second phases, but had little effect on insulin secretion at 2.8 mM glucose. In contrast, tolbutamide (100 μM) markedly stimulated insulin secretion at 2.8 mM glucose and enhanced the second phase of insulin secretion but not the first phase at 11.1 mM glucose.

In vivo JTT‐608 also enhanced early insulin secretion only with glucose‐loading. In contrast, tolbutamide enhanced insulin secretion both with and without glucose‐loading.

JTT‐608 (10–100 mg kg−1) improved oral glucose tolerance with enhanced insulin secretion in a meal tolerance test (MTT). In comparison with tolbutamide, JTT‐608 improved glucose tolerance more efficiently in GK rats than in Wistar rats.

We conclude that in diabetic GK rats JTT‐608 suppressed postprandial glucose excursions with enhanced glucose‐stimulated insulin secretion, especially the first phase of insulin secretion.

British Journal of Pharmacology (1999) 126, 1674–1680; doi:10.1038/sj.bjp.0702481
5‐Hydroxytryptamine stimulation of phospholipase D activity in the rabbit isolated mesenteric artery
Authors: Hinton J M , Adams D , Garland C J
Published: January 2005

The involvement of phospholipase D (PLD) in the 5‐hydroxytryptamine 5‐HT1B/5‐HT1D‐signalling pathway was assessed in the rabbit isolated mesenteric artery.

RT–PCR analysis of mesenteric smooth muscle cells revealed a strong signal corresponding to mRNA transcript for the 5‐HT1B receptor. The PCR fragment corresponded to the known sequence for the 5‐HT1B receptor. No signal corresponding to 5‐HT1D mRNA was detected.

Neither 5‐HT (3 μM) nor KCl (45 mM) individually stimulated any significant increase in the smooth muscle concentration of [33P]‐PtdBut to reflect PLD activity. However, in the presence of KCl (45 mM), 5‐HT evoked a concentration‐dependent increase in [33P]‐PtdBut, to a maximum of 84% with 5‐HT (3 μM).

[33P]‐PtdBut accumulation evoked by 5‐HT in the presence of KCl was abolished in nominally calcium‐free Krebs‐Henseleit Buffer (KHB) or with the selective protein kinase C inhibitor, Ro‐31 8220 (10 μM, 20 min).

5‐HT (3 μM) in the presence of KCl (45 mM) failed to increase either the accumulation of [33P]‐phosphatidic acid in the presence of butanol, or total [3H]‐inositol phosphates ([3H]‐InsP) in the presence of LiCl (10 mM).

5‐HT (0.1–1 μM) abolished forskolin (1 μM) stimulated increases in cyclic AMP (15 fold increase), an action which was pertussis toxin‐sensitive.

Therefore, in the presence of raised extracellular potassium 5‐HT can stimulate PLD via 5‐HT1B receptors in the rabbit mesenteric artery. This action requires extracellular calcium and the activation of protein kinase C. These characteristics are identical to the profile for 5‐HT1B/5‐HT1D‐receptor evoked contraction in vascular smooth muscle cells, suggesting a role for PLD in this response to 5‐HT.

British Journal of Pharmacology (1999) 126, 1601–1608; doi:10.1038/sj.bjp.0702484
The involvement of noradrenergic transmission in the morphine‐induced locomotor hyperactivity in mice withdrawn from repeated morphine treatment
Authors: Airio Juha , Ahtee Liisa
Published: January 2005

Our previous studies suggest that in addition to the cerebral dopaminergic systems the noradrenergic ones have a crucial role in the morphine‐induced behavioural sensitization in mice. Therefore the effects of α2‐adrenoceptor antagonist, idazoxan (1 and 3 mg kg−1, i.p.) on morphine‐induced locomotor hyperactivity as well as on morphine‐induced changes in cerebral noradrenaline (NA) and striatal dopamine (DA) metabolism were studied in mice withdrawn for 3 days from 5 day repeated morphine treatment. The concentrations of NA, free 3‐methoxy‐4‐hydroxyphenylethylene glycol (MOPEG), DA, 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3‐methoxytyramine (3‐MT) were determined.

Acute morphine (10 mg kg−1, s.c.) increased locomotor activity in control and in morphine‐withdrawn mice; idazoxan alone did not alter the activity. Idazoxan pretreatment did not alter the locomotor hyperactivity induced by acute morphine in control mice but potentiated it in morphine‐withdrawn mice.

Acute morphine elevated MOPEG less but increased DOPAC and HVA more clearly in morphine‐withdrawn mice than in controls, and decreased 3‐MT only in controls. Idazoxan alone did not alter the NA or DA metabolite concentrations in control mice, but elevated MOPEG as well as DOPAC in morphine‐withdrawn mice.

In control mice idazoxan enhanced acute morphine's elevating effect on MOPEG. In withdrawn mice idazoxan counteracted the tolerance so that acute morphine elevated MOPEG in these mice to about similar level as in controls.

Idazoxan pretreatment abolished the HVA increasing effect of acute morphine both in control and withdrawn mice. In control mice idazoxan enhanced morphine's elevating effect on DOPAC and abolished morphine's decreasing effect on 3‐MT. Idazoxan did not alter morphine's effects on DOPAC or 3‐MT concentrations in withdrawn mice.

Our results show that in morphine‐withdrawn mice idazoxan pretreatment reveals the morphine‐induced locomotor sensitization. This most probably occurs by overcoming the tolerance towards the acute morphine‐induced increase of cerebral NA turnover and release. It is suggested that in mice the cerebral noradrenergic in addition to the dopaminergic systems are major determinants of the behavioural sensitization to morphine.

British Journal of Pharmacology (1999) 126, 1609–1619; doi:10.1038/sj.bjp.0702485