bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional characterization of agonists at recombinant human 5‐HT2A, 5‐HT2B and 5‐HT2C receptors in CHO‐K1 cells
Authors: Porter R H P , Benwell K R , Lamb H , Malcolm C S , Allen N H , Revell D F , Adams D R , Sheardown M J
Published: January 2005

The goal of this study was to characterize the agonist pharmacology of human 5‐HT2A, 5‐HT2B and 5‐HT2C (VSV) receptors expressed in CHO‐K1 (Chinese hamster ovary) cells.

We used a fluorometric imaging plate reader (FLIPR) which allows rapid detection of rises in intracellular calcium levels upon the addition of agonists.

Stimulation of all three receptors by 5‐HT caused a robust concentration dependent increase in intracellular calcium levels. No such effect was observed from non‐transfected control CHO‐K1 cells.

The rank order of potency of agonists at the different receptor subtypes varied. Tryptamines, BW‐723C86, d‐norfenfluramine, Ro 60‐0175 and LSD exhibited the following rank order of potency; 5‐HT2B>5‐HT2C>5‐HT2A. Piperazines such as m‐Chlorophenylpiperazine (mCPP), ORG‐12962, MK‐212 and also ORG‐37684 exhibited a rank order of potency of 5‐HT2C>5‐HT2B>5‐HT2A. The phenylisopropylamines DOI and DOB had a rank order of 5‐HT2A>5‐HT2B>5‐HT2C.

Many agonists tested had partial agonist actions when compared to 5‐HT, and a wide range of relative efficacies were exhibited, which was cell line dependent. For example, mCPP had a relative efficacy of 65% at 5‐HT2C receptors but <25% at either 5‐HT2A or 5‐HT2B receptors.

Interpretation of literature values of functional assays using different cell lines, different receptor expression levels and different receptor isoforms, is complex. Species differences and the previous use of antagonist radioligands to characterize agonist potency in binding assays emphasizes the importance of studying agonists in the same experiment using the same assay conditions and parental cell lines.

British Journal of Pharmacology (1999) 128, 13–20; doi:10.1038/sj.bjp.0702751
A new experimental approach in endothelium‐dependent pharmacological investigations on isolated porcine coronary arteries mounted for impedance planimetry
Authors: Tankó László B , Mikkelsen Erich O , Simonsen Ulf
Published: January 2005

The aim of this study was to investigate whether the balloon‐based impedance planimetry technique could be a useful tool in endothelium‐dependent investigations.

Porcine large coronary arteries contracted with prostaglandin F2α (PGF2α, 10 μM) did not relax to bradykinin (0.1 nM–0.1 μM), but did relax to sodium nitroprusside (SNP, 10 μM). However, after eversion of the segments, bradykinin induced relaxations with pD2 values and maximal responses of 8.78±0.09 and 75±2% (n=6), respectively.

Incubation with captopril (1 μM) did not reveal a relaxation to bradykinin in the normal vessel configuration and had no influence on the concentration‐relaxation relationship in everted segments.

Lowering the luminal pressure in contracted segments from 131±5 mmHg (isometric, n=5) to 60 mmHg (isobaric, n=5) did not facilitate the action of bradykinin.

Eversion of segments did not influence the concentration‐response relationship for K+ (4.7–125 mM), PGF2α (0.3–30 μM), and SNP (30 nM–30 μM), although the time‐courses of responses were faster when the agents were added from the intimal compared to the adventitial side of the preparation.

In the same everted segment contracted with PGF2α, the concentration‐response relationship for bradykinin was not different under isometric and isobaric conditions.

These results indicate that, (1) reduced endothelium‐dependent relaxations to adventitially administered substances can be ascribed to a diffusion barrier in the vessel wall, while enzymatic degradation, luminal pressure and precontractile responses seem not to play a role, (2) impedance planimetry applied to everted cylindrical segments could be a useful experimental approach in pharmacological studies of endothelium‐dependent responses under isobaric and isometric conditions.

British Journal of Pharmacology (1999) 128, 165–173; doi:10.1038/sj.bjp.0702752
Hepatocyte growth factor regulates human trophoblast motility and invasion: a role for nitric oxide
Authors: Cartwright Judith E , Holden Desmond P , Whitley Guy St J
Published: January 2005

The expression of hepatocyte growth factor (HGF) is essential for normal placental development although its function is unknown. In this study we examined the effect of HGF on trophoblast cell motility and invasion of fibrin gels and investigated the possible role of nitric oxide (NO) in this process.

The human extravillous trophoblast cell line SGHPL‐4 express both the constitutive and inducible isoforms of nitric oxide synthase (NOS).

HGF significantly stimulates cell motility in monolayer culture, the invasion of fibrin gels and the production of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP).

Invasion, motility and cyclic GMP production were inhibited by Ng‐monomethyl‐L‐arginine (L‐NMMA).

Cell motility was also significantly inhibited by the inducible NOS specific inhibitor 1400 W.

Neither 8 Br‐cyclic GMP nor the NO donor spermine‐NO had any significant effect on basal trophoblast cell motility.

The data presented in this study demonstrate a direct effect of trophoblast‐derived NO synthesis on trophoblast cell function and support the idea that HGF is involved in the regulation of trophoblast invasion through mechanisms that involve the production of NO. However neither exogenous NO nor activation of cyclic GMP‐dependent pathways alone are sufficient to stimulate trophoblast cell motility.

British Journal of Pharmacology (1999) 128, 181–189; doi:10.1038/sj.bjp.0702757
P2Y2 receptor‐mediated inhibition of ion transport in distal lung epithelial cells
Authors: Ramminger S J , Collett A , Baines D L , Murphie H , McAlroy H L , Olver R E , Inglis S K , Wilson S M
Published: January 2005

Rat foetal distal lung epithelial cells were plated onto permeable supports where they became integrated into epithelial sheets that spontaneously generated short circuit current (ISC).

Apical ATP (100 μM) evoked a transient fall in ISC that was followed by a rise to a clear peak which, in turn, was succeeded by a slowly developing decline to a value below control. Apical UTP evoked an essentially identical response.

UDP and ADP were ineffective whilst ATP had no effect when added to the basolateral solution. These effects thus appear to be mediated by apical P2Y2 receptors.

The rising phase of the responses to ATP/UTP was selectively inhibited by anion transport inhibitors but persisted in the presence of amiloride, which abolished the inhibitory effects of both nucleotides. Thus, apical nucleotides appear to evoke a transient stimulation of anion secretion and sustained inhibition of Na+ absorption.

Basolateral isoprenaline (10 μM) elicited a rise in ISC but subsequent addition of apical ATP reversed this effect. Conversely, isoprenaline restored ISC to its basal level following stimulation with ATP. Apical P2Y2 receptors and basolateral β‐adrenoceptors thus allow their respective agonists to exert mutually opposing effects on ISC.

British Journal of Pharmacology (1999) 128, 293–300; doi:10.1038/sj.bjp.0702767
Pharmacological and molecular evidence for kinin B1 receptor expression in urinary bladder of cyclophosphamide‐treated rats
Authors: Belichard P , Luccarini J M , Defrêne E , Faye P , Franck R M , Duclos H , Paquet J L , Pruneau D
Published: January 2005

In the present study, we developed an experimental model of cystitis induced by cyclophosphamide (CYP). In order to characterize des‐Arg9‐BK‐induced contraction on the urinary bladder (UB) during the development of inflammation and to quantify kinin B1 receptor gene expression using a quantitative RT–PCR technique.

In the presence of peptidase inhibitors captopril (10 μM), DL‐thiorphan (1 μM) and DL‐2‐mercaptomethyl‐3‐guanidino‐ethylthiopropanoic acid (MERGEPTA 5 μM), bradykinin (BK) (0.3–3,000 nM) evoked a concentration‐dependent contraction of rat UB which was not different between the CYP‐ and vehicle‐treated groups. Unlike BK, des‐Arg9‐BK (0.3–100,000 nM) did not contract UB from vehicle‐treated rats but contracted vigorously bladder strips from CYP‐treated rats 14, 24 and 168 h after treatment. In UB of 24 h treated rat, the pD2 value of des‐Arg9‐BK was 7.3±0.1.

The cyclo‐oxygenase inhibitor indomethacin (3 μM) reduced by 30% the maximal response of des‐Arg9‐BK. Both the kinin B1 receptor antagonists des‐Arg9‐[Leu8]BK (10 μM) and des‐Arg10‐Hoe 140 (10 μM) produced a rightward shift of the concentration‐response curve to des‐Arg9‐BK yielding pKB values of 6.8±0.2 and 7.2±0.1, respectively, whilst the kinin B2 receptor antagonist Hoe 140 (1 μM) had no effect.

After CYP treatment, mRNA coding for the kinin B1 receptor appeared predominantly in UB. In this organ, the induction was progressive, reaching a maximum 48 h after CYP treatment.

In conclusion, the present study provides strong evidence for an induction of kinin B1 receptors in UB of CYP‐treated rats. This was associated at a molecular level with an increase in mRNA expression of the gene coding for the kinin B1 receptor. This kinin receptor displayed the whole features of a classical rat kinin B1 receptor.

British Journal of Pharmacology (1999) 128, 213–219; doi:10.1038/sj.bjp.0702769
Sulphonic acid derivatives as probes of pore properties of volume‐regulated anion channels in endothelial cells
Authors: Droogmans Guy , Maertens Chantal , Prenen Jean , Nilius Bernd
Published: January 2005

We have used the whole‐cell patch‐clamp technique to study the effects of 4‐sulphonic‐calixarenes and some other poly‐sulphonic acid agents, such as suramin and basilen blue, on volume‐regulated anion channel (VRAC) currents in cultured endothelial cells (CPAE cells).

The 4‐sulphonic‐calixarenes induced a fast inhibition at positive potentials but were ineffective at negative potentials. At small positive potentials, 4‐sulphonic‐calix[4]arene was a more effective inhibitor than 4‐sulphonic‐calix[6]arene and ‐calix[8]arene, which became more effective at more positive potentials.

Also suramin and basilen blue induced a voltage dependent current inhibition, reaching a maximum around +40 mV and declining at more positive potentials.

The voltage dependence of inhibition was modelled by assuming that these negatively charged molecules bind to a site inside VRAC that senses a fraction δ of the applied electrical field, ranging beween 0.16 to 0.32. 4‐Sulphonic‐calix[4]arene, suramin and basilen blue bind and occlude VRAC at moderate potentials, but permeate the channel at more positive potentials. 4‐Sulphonic‐calix[6]arene and ‐calix[8]arene however do not permeate the channel. From the structural information of the calixarenes, we estimate a lower and upper limit of 11*12 and 17*12 Å2 respectively for the cross‐sectional area of the pore.

British Journal of Pharmacology (1999) 128, 35–40; doi:10.1038/sj.bjp.0702770
Involvement of phospholipid signal transduction pathways in morphine tolerance in mice
Authors: Smith Forrest L , Lohmann Alka B , Dewey William L
Published: January 2005

Opioid tolerance involves an alteration in the activity of intracellular kinases such as cyclic AMP‐dependent protein kinase (PKA). Drugs that inhibit PKA reverse morphine antinociceptive tolerance. The hypothesis was tested that phospholipid pathways are also altered in morphine tolerance. Inhibitors of the phosphatidylinositol and phosphatidylcholine pathways were injected i.c.v. in an attempt to acutely reverse morphine antinociceptive tolerance.

Seventy‐two hours after implantation of placebo or 75 mg morphine pellets, mice injected i.c.v. with inhibitor drug were challenged with morphine s.c. for generation of dose‐response curves in the tail‐flick test. Placebo pellet‐implanted mice received doses of inhibitor drug having no effect on morphine's potency, in order to test for tolerance reversal in morphine pellet‐implanted mice. Injection of the phosphatidylinositol‐specific phospholipase C inhibitor ET‐18‐OCH3 significantly reversed tolerance, indicating a potential role for inositol 1,4,5‐trisphosphate (IP3) and protein kinase C (PKC) in tolerance. Alternatively, phosphatidylcholine‐specific phospholipase C increases the production of diacylglycerol and activation of PKC, without concomitant production of IP3. D609, an inhibitor of phosphatidylserine‐specific phospholipase C, also reversed tolerance. Heparin is an IP3 receptor antagonist. Injection of low molecular weight heparin also reversed tolerance. PKC was also examined with three structurally dissimilar inhibitors. Bisindolylmaleimide I, Go‐7874, and sangivamycin significantly reversed tolerance.

Chronic opioid exposure leads to changes in phospholipid metabolism that have a direct role in maintaining a state of tolerance. Evidence is accumulating that opioid tolerance disrupts the homeostatic balance of several important signal transduction pathways.

British Journal of Pharmacology (1999) 128, 220–226; doi:10.1038/sj.bjp.0702771
Inhibition of the human ether‐a‐go‐go‐related gene (HERG) potassium channel by cisapride: affinity for open and inactivated states
Authors: Walker B D , Singleton C B , Bursill J A , Wyse K R , Valenzuela S M , Qiu M R , Breit S N , Campbell T J
Published: January 2005

Cisapride is a prokinetic agent which has been associated with QT prolongation, torsades de pointes and cardiac arrest. The cellular mechanism for these observations is high affinity blockade of IKr (encoded by HERG).

In a chronic transfection model using CHO‐K1 cells, cisapride inhibited HERG tail currents after a step to +25 mV with similar potency at room and physiological temperatures (IC50 16.4 nM at 20–22°C and 23.6 nM at 37°C).

Channel inhibition exhibited time‐, voltage‐ and frequency‐dependence. In an envelope of tails test, channel blockade increased from 27±8% after a 120 ms depolarizing step to 50±4% after a 1.0 s step. These findings suggested affinity for open and/or inactivated channel states.

Inactivation was significantly accelerated by cisapride in a concentration‐dependent manner and there was a small (−7 mV) shift in the voltage dependence of steady state inactivation.

Channel blockade by cisapride was modulated by [K+]o, with a 26% reduction in the potency of channel blockade when [K+]o was increased from 1 to 10 mM.

In conclusion, HERG channel inhibition by cisapride exhibits features consistent with open and inactivated state binding and is sensitive to external potassium concentration. These features may have significant clinical implications with regard to the mechanism and treatment of cisapride‐induced proarrhythmia.

British Journal of Pharmacology (1999) 128, 444–450; doi:10.1038/sj.bjp.0702774
Prevention of the expression of inducible nitric oxide synthase by a novel positive inotropic agent, YS 49, in rat vascular smooth muscle and RAW 264.7 macrophages
Authors: Kang Young Jin , Koo Eui Bon , Lee Young Soo , Yun‐Choi Hye Sook , Chang Ki Churl
Published: January 2005

The effects of a novel positive inotropic isoquinoline compound, YS 49, on NO production and iNOS protein expression were investigated in cultured rat aortic vascular smooth muscle cells (RAVSMC) and RAW 264.7 cells exposed to lipopolysaccharide (LPS) plus interferon‐γ (IFN‐γ). In addition, the effects of YS 49 on vascular hyporeactivity in vitro and ex vivo, and on survival rate (mice) and serum NOx (rat) levels, were also investigated in LPS‐treated animals.

Pre‐ or co‐treatment of YS 49 with LPS plus IFN‐γ, concentration‐dependently reduced NO production in RAVSMC and RAW 264.7 cells (IC50 values, 22 and 30 μM, respectively). Although the inhibitory effect on NO production was reduced when YS 49 was applied 2 and 4 h after cytokine in RAW 264.7 cells, it was still statistically significant (P<0.05).

YS 49 reduced iNOS mRNA expression in LPS‐treated rat aorta in vitro, an effect which was associated with restoration of contractility to the vasoconstrictor, phenylephrine (PE), and reduction in L‐arginine‐induced relaxation.

Serum NOx levels were significantly (P<0.01) reduced by YS 49 (5 mg kg−1, i.p.) in LPS‐treated rats (10 mg kg−1, i.p.). Administration of YS 49 (10 and 20 mg kg−1) 30 min prior to LPS (10 mg kg−1) also significantly (P<0.01) increased the subsequent survival rates in mice.

Finally, expression of iNOS protein induced by LPS plus IFN‐γ in RAVSMC and RAW 264.7 cells was suppressed by YS 49, in a concentration‐dependent manner.

These data strongly suggest that YS 49 suppresses iNOS gene expression induced by LPS and/or cytokines in RAVSMC and RAW 264.7 cells at the transcriptional level. YS 49 could therefore be beneficial in septic shock and other diseases associated with iNOS over‐expression.

British Journal of Pharmacology (1999) 128, 357–364; doi:10.1038/sj.bjp.0702787
Regulation of cyclic AMP by extracellular ATP in cultured brain capillary endothelial cells
Authors: Anwar Zubeya , Albert Jennifer L , Gubby Sharon E , Boyle John P , Roberts Jonathon A , Webb Tania E , Boarder Michael R
Published: January 2005

In primary unpassaged rat brain capillary endothelial cell cultures (RBECs), using reverse‐transcriptase PCR with primers specific for P2Y receptor subtypes, we detected mRNA for P2Y2, P2Y4 and P2Y6, but not P2Y1 receptors.

None of the various nucleotides tested reduced forskolin elevated cyclic AMP levels in RBECs. ATP and ATPγS, as well as adenosine, enhanced cyclic AMP accumulation in the presence of forskolin.

Comparison of the concentration response curves to ATPγS with those for ATP and adenosine, at different incubation times, indicated that the response to purine nucleotides was not wholly dependent on conversion to adenosine. Adenosine deaminase abolished the response to adenosine but only reduced the response to ATP by about 50%. These results suggest the participation of a receptor responsive to nucleotides.

Isobutylmethylxanthine and 8‐sulphophenyltheophylline prevented the cyclic AMP response, while neither 8‐cyclopentyl‐1,3‐dipropylxanthine nor SCH58261 were effective antagonists. 2‐chloradenosine gave a robust response, but neither 2‐chloro‐N6‐cyclopentyladenosine nor CGS 21680 were agonists.

These results show that adenosine and ATP can elevate the cyclic AMP levels of brain endothelial cells by acting on receptors which have a pharmacology apparently distinct from known P2Y and adenosine receptors.

British Journal of Pharmacology (1999) 128, 465–471; doi:10.1038/sj.bjp.0702792
Effect of hypoxia alone or combined with inflammation and 3‐methylcholanthrene on hepatic cytochrome P450 in conscious rabbits
Authors: Kurdi J , Maurice H , El‐Kadi A O S , Ong H , Dalkara S , Bélanger P M , Du Souich P
Published: January 2005

To investigate the effect of moderate hypoxia alone or combined with an inflammatory reaction or after 3‐methylcholanthrene (3MC) pre‐treatment on cytochrome P450 (P450), conscious rabbits were exposed for 24 h to a fractional concentration of inspired O2 of 10% (mean PaO2 of 34 mmHg). Hypoxia decreased theophylline metabolic clearance (ClM) from 1.73±0.43 to 1.48±0.13 ml min−1 kg−1 (P<0.05), and reduced (P<0.05) the formation clearance of theophylline metabolites, 3‐methylxanthine (3MX), 1‐methyluric acid (1MU) and 1,3‐dimethyluric acid (1,3DMU). Hypoxia reduced the amount of CYP1A1 and 1A2 but increased CYP3A6 proteins.

Turpentine‐induced inflammatory reaction reduced (P<0.05) the formation clearance of 3MX, 1MU, and 1,3DMU, and diminished the amount of CYP1A1, 1A2 and 3A6 proteins. However, when combined with hypoxia, inflammation partially prevented the decrease in ClM, especially by impeding the reduction of 1,3DMU. The amount of CYP1A1 and 1A2 remained reduced but the amount of CYP3A6 protein returned to normal values.

Pre‐treatment with 3MC augmented the ClM by 114% (P<0.05) due to the increase in the formation clearance of 3MX, 1MU and 1,3DMU. 3MC treatment increased the amount of CYP1A1 and 1A2 proteins. Pre‐treatment with 3MC prevented the hypoxia‐induced decrease in amount and activity of the P450.

It is concluded that acute moderate hypoxia and an inflammatory reaction individually reduce the amount and activity of selected apoproteins of the P450. However, the combination of hypoxia and the inflammatory reaction restores P450 activity to near normal values. On the other hand, pre‐treatment with 3MC prevents the hypoxia‐induced depression of the P450.

British Journal of Pharmacology (1999) 128, 365–373; doi:10.1038/sj.bjp.0702795
Beta 3‐adrenoceptor stimulation induces vasorelaxation mediated essentially by endothelium‐derived nitric oxide in rat thoracic aorta
Authors: Trochu Jean‐Noël , Leblais Véronique , Rautureau Yohann , Bévérelli Fabrizio , Le Marec Hervé , Berdeaux Alain , Gauthier Chantal
Published: January 2005

The relaxant effects of isoprenaline may result from activation of another β‐adrenoceptor subtype in addition to β1 and β2. This study evaluated the role of a third β‐adrenoceptor subtype, β3, in β‐adrenoceptor‐induced relaxation of rat thoracic aorta by isoprenaline.

Isoprenaline produced a concentration‐dependent relaxation of phenylephrine pre‐contracted rings of the thoracic aorta (pD2=7.46±0.15; Emax=85.9±3.4%), which was partially attenuated by endothelium removal (Emax=66.5±6.3%) and administration of the nitric oxide (NO) synthase inhibitor, L‐NG‐monomethyl arginine (L‐NMMA) (Emax=61.3±7.9%).

In the presence of nadolol, a β1‐ and β2‐adrenoceptor antagonist, isoprenaline‐induced relaxation persisted (Emax=55.6±5.3%), but occurred at higher concentrations (pD2=6.71±0.10) than in the absence of nadolol and lasted longer.

Similar relaxant effects were obtained with two β3‐adrenoceptor agonists: SR 58611 (a preferential β3‐adrenoceptor agonist), and CGP 12177 (a partial β3‐adrenoceptor with β1‐ and β2‐adrenoceptor antagonistic properties). SR 58611 caused concentration‐dependent relaxation (pD2=5.24±0.07; Emax=59.5±3.7%), which was not modified by pre‐treatment with nadolol but antagonized by SR 59230A, a β3‐adrenoceptor antagonist. The relaxation induced by SR 58611 was associated with a 1.7 fold increase in tissue cyclic GMP content.

Both relaxation and the cyclic GMP increase induced by SR 58611 were greatly reduced by endothelium removal and in the presence of L‐NMMA.

We conclude that in the rat thoracic aorta, β3‐adrenoceptors are mainly located on endothelial cells, and act in conjuction with β1‐ and β2‐adrenoceptors to mediate relaxation through activation of an NO synthase pathway and subsequent increase in cyclic GMP levels.

British Journal of Pharmacology (1999) 128, 69–76; doi:10.1038/sj.bjp.0702797
The effects of endomorphin‐1 and endomorphin‐2 in CHO cells expressing recombinant μ‐opioid receptors and SH‐SY5Y cells
Authors: Harrison C , McNulty S , Smart D , Rowbotham D J , Grandy D K , Devi L A , Lambert D G
Published: January 2005

Endomorphin‐1 and ‐2 (E‐1/E‐2) have been proposed as endogenous ligands for the μ‐opioid receptor. The aims of this study are to characterize the binding of E‐1/E‐2 and the subsequent effects on cyclic AMP formation and [Ca2+]i levels in SH‐SY5Y and Chinese hamster ovary (CHO) cells expressing endogenous and recombinant μ‐opioid receptors.

E‐1 displaced [3H]‐diprenorphine ([3H]‐DPN) binding in CHOμ and SH‐SY5Y membranes with pKi values of 8.02±0.09 and 8.54±0.13 respectively. E‐2 displaced [3H]‐DPN binding in CHOμ and SH‐SY5Y cells with pKi values of 7.82±0.11 and 8.43±0.13 respectively. E‐1/E‐2 bound weakly to CHOδ and CHOκ membranes, with IC50 values of greater than 10 μM.

In CHOμ cells, E‐1/E‐2 inhibited forskolin (1 μM) stimulated cyclic AMP formation with pIC50 values of 8.03±0.16 (Imax=53.0±9.3%) and 8.15±0.24 (Imax=56.3±3.8%) respectively. In SH‐SY5Y cells E1/E2 inhibited forskolin stimulated cyclic AMP formation with pIC50 values of 7.72±0.13 (Imax=46.9±5.6%) and 8.11±0.31 (Imax=40.2±2.8%) respectively.

E‐1/E‐2 (1 μM) increased [Ca2+]i in fura‐2 loaded CHOμ cell suspensions in a thapsigargin sensitive and naloxone reversible manner. Mean increases observed were 106±28 and 69±6.7 nM respectively. In single adherent cells E‐1/E‐2 (1 μM) increased [Ca2+]i with a mean 340/380 ratio change of 0.81±0.09 and 0.40±0.08 ratio units respectively. E‐1/E‐2 failed to increase intracellular calcium in CHOδ, CHOκ and SH‐SY5Y cells.

These data show that E‐1/E‐2 bind with high affinity and selectivity to μ‐opioid receptors and modulate signal transduction pathways typical of opioids. This provides further evidence that these two peptides may be endogenous ligands at the μ‐opioid receptor.

British Journal of Pharmacology (1999) 128, 472–478; doi:10.1038/sj.bjp.0702798
The effect of body temperature on myocardial protection conferred by ischaemic preconditioning or the selective adenosine A1receptor agonist GR79236, in an anaesthetized rabbit model of myocardial ischaemia and reperfusion
Authors: Sheldrick A , Gray K M , Drew G M , Louttit J B
Published: January 2005

The cardioprotective effect of N‐[(1S, trans)‐2‐hydroxycyclopentyl]adenosine (GR79236), an adenosine A1receptor agonist, was compared with that produced by ischaemic preconditioning in an anaesthetized rabbit model of myocardial ischaemia and reperfusion. In addition, we examined the effect of different body core temperatures on GR79236‐ or ischaemic preconditioning‐induced cardioprotection when administered prior to ischaemia, and on cardioprotection induced by GR79236 administered 10 min prior to the onset of reperfusion.

When rabbits were subjected to 30 min occlusion of the left coronary artery, followed by 2 h reperfusion, GR79236 (3×10−8 mol kg−1i.v. (10.5 μg kg−1i.v.)) or ischaemic preconditioning (5 min ischaemia followed by 5 min reperfusion), administered or applied 10 min prior to the occlusion, significantly limited the development of infarction. The cardioprotective effect of ischaemic preconditioning was significantly greater than that seen after administration of GR79236. Pre‐treatment with the selective adenosine A1receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, 3.3×10−6 mol kg−1(1 mg kg−1i.v.)), prevented the cardioprotective effect of GR79236, but not that of ischaemic preconditioning.

Maintaining body core temperature at 38.5°C rather than at 37.0°C did not influence infarct size in control groups of rabbits, but reduced the cardioprotective effect of GR79236 when administered 10 min prior to occlusion or 10 min prior to the onset of reperfusion. The cardioprotective effect of ischaemic preconditioning was not temperature‐dependent.

In conclusion, myocardial protection conferred by GR79236 in anaesthetized rabbits is mediatedviaadenosine A1receptors. Myocardial protection can be conferred when GR79236 is administered before the onset of ischaemiaorreperfusion, and is reduced when body core temperature is maintained at 38.5°C rather than at 37.0°C. In contrast, myocardial protection conferred by ischaemic preconditioning is not reduced by adenosine A1receptor blockade, or by maintaining body core temperature at 38.5°C rather than at 37.0°C. These findings point to distinct differences in the mechanisms of induction of myocardial protection by adenosine A1receptor agonist and ischaemic preconditioning. They also highlight the need for careful control of body core temperature when investigating the phenomenon of cardioprotection.

British Journal of Pharmacology(1999)128, 385–395; doi:10.1038/sj.bjp.0702799
Inhibition of the current of heterologously expressed HERG potassium channels by imipramine and amitriptyline
Authors: Teschemacher Anja G , Seward Elizabeth P , Hancox Jules C , Witchel Harry J
Published: January 2005

Tricyclic antidepressants (TCAs) are associated with cardiovascular side effects including prolongation of the QT interval of the ECG. In this report we studied the effects of two TCAs (imipramine and amitriptyline) on ionic current mediated by cloned HERG potassium channels.

Voltage clamp measurements of HERG currents were made from CHO cells transiently transfected with HERG cDNA. HERG‐encoded potassium channels were inhibited in a reversible manner by both imipramine and amitriptyline. HERG tail currents (IHERG) following test pulses to +20 mV were inhibited by imipramine with an IC50 of 3.4±0.4 μM (mean±s.e.mean) and a Hill coefficient of 1.17±0.03 (n=5). 3 μM amitriptyline inhibited IHERG by 34±6% (n=3). The inhibition showed only weak voltage dependence.

Using an ‘envelope of tails’ comprised of pulses to +20 mV of varying durations, the τ of activation was found to be 155±30 ms for control and 132±26 ms for 3 μM imipramine (n=5). Once maximal channel activation was achieved after 320 ms (as demonstrated by maximal tail currents), further prolongation of depolarization did not increase imipramine‐mediated HERG channel inhibition.

Taking current measurements every second during a 10 s depolarizing pulse from −80 mV to 0 mV, block was observed during the first pulse in the presence of imipramine and the level of IHERG block was similar throughout the pulse (n=5).

A three pulse protocol (two depolarizing pulses to +20 mV separated by 20 ms at −80 mV) revealed that imipramine did not significantly alter the kinetics of IHERG inactivation. The τ of inactivation was 8±2 ms and 5.6±0.4 ms (n=5) in the absence and presence of 3 μM imipramine, respectively, and currents inactivated to a similar extent.

Our data are consistent with TCAs causing components of block of the HERG channel in both the closed and open states. Any component of open channel block occurs rapidly upon depolarization. Inhibition of IHERG by the prototype TCAs imipramine and amitriptyline may suggest a mechanism for QT prolongation associated with risks of arrhythmia and sudden death that accompany high concentrations of TCAs following overdose.

British Journal of Pharmacology (1999) 128, 479–485; doi:10.1038/sj.bjp.0702800
Pro‐oxidant challenge in vivo provokes the onset of NIDDM in the insulin resistant obese Zucker rat
Authors: Laight David W , Desai Kaushik M , Gopaul Nitin K , Änggård Erik E , Carrier Martin J
Published: January 2005

We investigated the ability of an acute pro‐oxidant challenge in vivo to deteriorate glycaemic control and insulin action in the obese Zucker rat, a model of insulin resistance associated with oxidant stress. In obese animals, the daily administration for 1 week of hydroquinone (HQ) in combination with L‐buthionine sulphoximine (BSO), elevated fasting plasma glycaemia and insulinaemia and markedly aggravated i.v. glucose‐stimulated hyperinsulinaemia without significantly affecting i.v. glucose tolerance, suggesting exacerbated insulin resistance. Intermediate effects on hyperinsulinaemia in obese animals were determined with HQ treatment alone while BSO treatment alone had no effect. In contrast, none of the pro‐oxidant treatments affected age‐matched, insulin sensitive, lean Zucker rats. Our data therefore demonstrate for the first time, a vulnerability to deterioration in insulin action in an established insulin resistant state following an environmental pro‐oxidative insult. This may have relevance in the conversion of insulin resistance syndrome (IRS) to non‐insulin dependent diabetes mellitus (NIDDM).

British Journal of Pharmacology (1999) 128, 269–271; doi:10.1038/sj.bjp.0702801
Curcumin inhibits Th1 cytokine profile in CD4+ T cells by suppressing interleukin‐12 production in macrophages
Authors: Kang B Y , Song Y J , Kim K ‐M , Choe Y K , Hwang S Y , Kim T S
Published: January 2005

Interleukin‐12 (IL‐12) plays a central role in the immune system by driving the immune response towards T helper 1 (Th1) type responses which are characterized by high IFN‐γ and low IL‐4 production. In this study we investigated the effects of curcumin, a natural product of plants obtained from Curcuma longa (turmeric), on IL‐12 production by mouse splenic macrophages and the subsequent ability of these cells to regulate cytokine production by CD4+ T cells.

Pretreatment with curcumin significantly inhibited IL‐12 production by macrophages stimulated with either lipopolysaccharide (LPS) or head‐killed Listeria monocytogenes (HKL).

Curcumin‐pretreated macrophages reduced their ability to induce IFN‐γ and increased the ability to induce IL‐4 in Ag‐primed CD4+ T cells. Addition of recombinant IL‐12 to cultures of curcumin‐pretreated macrophages and CD4+ T cells restored IFN‐γ production in CD4+ T cells.

The in vivo administration of curcumin resulted in the inhibition of IL‐12 production by macrophages stimulated in vitro with either LPS or HKL, leading to the inhibition of Th1 cytokine profile (decreased IFN‐γ and increased IL‐4 production) in CD4+ T cells.

These findings suggest that curcumin may inhibit Th1 cytokine profile in CD4+ T cells by suppressing IL‐12 production in macrophages, and points to a possible therapeutic use of curcumin in the Th1‐mediated immune diseases.

British Journal of Pharmacology (1999) 128, 380–384; doi:10.1038/sj.bjp.0702803
Functional expression of a cDNA encoding a human ecto‐ATPase
Authors: Mateo Jesús , Harden T Kendall , Boyer José L
Published: January 2005

The metabolism of extracellular nucleotides plays an important role in nucleotide signalling mediated by P2 receptors. The nucleotide sequence encoding a putative human ecto‐ATPase named CD39L1 was reported recently. However, the biological activity of this protein has not been established.

Based on the sequence of CD39L1 we isolated from mRNA from human ECV‐304 cells a sequence encoding a 495 amino acid protein that is identical to CD39L1, with the exception that this sequence contains a 23 amino acid stretch in the putative extracellular loop that is missing in CD39L1. Partial sequence of a genomic DNA clone indicates that the CD39L1 gene corresponds to an alternative spliced form of the human ecto‐ATPase.

Stable expression of isolated sequence in NIH‐3T3 mouse fibroblasts conferred a marked nucleotide hydrolytic activity consistent with the activity of an ecto‐ATPase.

The human ecto‐ATPase hydrolyzed all naturally occurring nucleoside triphosphates in a Ca2+‐ or Mg2+‐dependent manner. Nucleoside diphosphates were hydrolyzed at a rate approximately 5% of that of the corresponding triphosphates. The apparent Km and Vmax values were: 394±62 μM and 107±7 nmol Pi min−1 106 cells−1 for the hydrolysis of ATP, and 102±33 μM and 4±0.4 nmol Pi min−1 106 cells−1 for the hydrolysis of ADP, respectively.

In conclusion, we report here the cloning and functional expression of a human ecto‐ATPase. The study of the biochemical properties and the regulatory mechanisms of ecto‐ATPases of defined sequence will be valuable in the definition of their role in nucleotide signalling.

British Journal of Pharmacology (1999) 128, 396–402; doi:10.1038/sj.bjp.0702805
Cloning and preliminary pharmacological characterization of the anaphylatoxin C5a receptor in the rabbit
Authors: Bachvarov Dimcho R , Houle Steeve , Bachvarova Magdalena , Bouthillier Johanne , St‐Pierre Serge A , Fukuoka Yoshihiro , Ember Julia A , Marceau François
Published: January 2005

The rabbit receptor for C5a was cloned from a genomic library and found to be 79.5% identical to the human homologue, the highest degree of similarity found so far in nonprimate laboratory animals.

The rabbit C5a receptor stably expressed in RBL cells binds human 125I‐C5a (2 nM). Unlabelled C5a and the C‐terminal analogue N‐acetyl‐Tyr‐Ser‐Phe‐Lys‐Pro‐Met‐Pro‐Leu‐D‐Ala‐Arg (Ac‐YSFKPMPLaR) were found to be competitors of that binding, the peptide analogue retaining approximately 0.1% of the affinity of human C5a.

The order of potency human C5a>Ac‐YSFKPMPLaR was conserved in bioassays based on rabbits (relaxation of the isolated portal vein and pulmonary artery; acute in vivo neutropenia), but with a decreasing potency gap between the two compounds, a likely consequence of the resistance to peptidases of the analogue.

The molecular definition of the rabbit C5a receptor evidenced a high preservation degree of sequence and pharmacologic properties relative to the human ortholog receptor, thus defining a set of molecular tools for the investigation of the role of C5a in physiologic and pathologic models based on the rabbit (e.g. atherosclerosis, inflammation).

British Journal of Pharmacology (1999) 128, 321–326; doi:10.1038/sj.bjp.0702812
Vasoconstriction of intrapulmonary arteries to P2‐receptor nucleotides in normal and pulmonary hypertensive newborn piglets
Authors: McMillan M R , Burnstock G , Haworth S G
Published: January 2005

The vasoconstrictor responses of isolated intrapulmonary arteries (IPA) to P2‐receptor agonists was investigated during adaptation to extrauterine life in the normal piglet and the effect of pulmonary hypertension was studied following exposure of newborn animals to chronic hypobaric hypoxia (51 kPa) for 3 days.

At resting tone, α,β‐methyleneATP (α,β‐meATP) (P2X‐receptor agonist) contracted intrapulmonary arteries from adult, but not immature pigs, and repeated application desensitized the response.

Adenosine 5′‐triphosphate (ATP) induced endothelium‐independent relaxation at low concentrations at all ages, a variable contractile response to high concentrations developed by 3 days, becoming larger and consistent by 14 days of age.

Uridine 5′‐triphosphate (UTP) evoked a contractile response in normal intrapulmonary arteries from foetal to adult life, the magnitude of the response increasing with age. Endothelial removal and pre‐incubation with Nω‐nitro‐L‐arginine methyl ester (L‐NAME) (100 μM) increased the contractile response of adult vessels.

Pre‐incubation with α,β‐meATP (100 μM), increased the contractile response to UTP in both newborn and adult vessels. ATP‐induced relaxations were reduced in newborn vessels but there was no effect on the responses of adult vessels.

Responses to UTP, ATP and α,β‐meATP of intrapulmonary arteries from newborn piglets exposed to chronic hypobaric hypoxia for 3 days were normal.

In summary, UTP elicited marked vasoconstriction of porcine IPA at all ages. UTP and ATP responses were consistent with activation of the P2Y4‐receptor recently identified in vascular smooth muscle by others. α,β‐meATP induced a small vasoconstriction in the adult probably via the P2X1‐receptor. Responses remained normal in neonatal pulmonary hypertension.

British Journal of Pharmacology (1999) 128, 549–555; doi:10.1038/sj.bjp.0702814
Vasodilatation of intrapulmonary arteries to P2‐receptor nucleotides in normal and pulmonary hypertensive newborn piglets
Authors: McMillan M R , Burnstock G , Haworth S G
Published: January 2005

The vasodilator responses of isolated intrapulmonary arteries (IPA) to P2‐receptor agonists were investigated during adaptation to extrauterine life in the piglet. The effect of pulmonary hypertension on the normal response was determined after exposing newborn animals to chronic hypobaric hypoxia (51 kPa) for 3 days.

Adenosine 5′‐triphosphate (ATP), 2‐methylthioATP (2‐meSATP), adenosine 5‐O‐(2‐thiodiphos‐phate) (ADPβS) and uridine 5′‐triphosphate (UTP) induced a relaxation in normal newborn piglet IPA pre‐contracted with prostaglandin F2α (PGF2α). The relaxations were not affected by removal of the endothelium. The responses to ATP and ADPβS increased significantly with age.

The relaxation responses of IPA to ATP, 2‐meSATP and ADPβS continued to increase normally after birth in an hypoxic environment.

The results of the study show that vasodilatation of porcine intrapulmonary vessels to nucleotides increased during development from foetus to adult; that the vasodilatation to purines was mediated by P2Y‐receptors on the vascular smooth muscle rather than on the endothelium; and that the P2Y‐receptor mediated relaxation of IPA remained normal in the pulmonary hypertensive neonate.

British Journal of Pharmacology (1999) 128, 543–548; doi:10.1038/sj.bjp.0702815
Troglitazone and pioglitazone attenuate agonist‐dependent Ca2+ mobilization and cell proliferation in vascular smooth muscle cells
Authors: Asano Michiko , Nakajima Toshiaki , Iwasawa Kuniaki , Morita Toshihiro , Nakamura Fumitaka , Imuta Hiroyuki , Chisaki Keigo , Yamada Nobuhiro , Omata Masao , Okuda Yukichi
Published: January 2005

The effects of troglitazone and pioglitazone on agonist‐induced Ca2+ mobilization and cell proliferation were studied using fluorescent Ca2+ indicator fura‐2 AM and incorporation of [3H]‐thymidine in rat aortic smooth muscle cells. The patch clamp techniques were also employed.

Vasopressin and platelet‐derived growth factor‐BB (PDGF) caused a transient elevation in [Ca2+]i by Ca2+ mobilization from intracellular stores, followed by a sustained rise due to Ca2+ entry. Nicardipine partly inhibited the sustained phase, but La3+ completely abolished it.

Troglitazone and pioglitazone did not significantly affect the transient rise elicited by these agonists, but preferentially inhibited the sustained phase of [Ca2+]i.

Under voltage clamp conditions, troglitazone and pioglitazone inhibited voltage‐dependent L‐type Ca2+ current (ICa·L). They also inhibited nonselective cation channels (Icat) elicited by vasopressin in a concentration‐dependent manner. The half maximal inhibitory concentrations of troglitazone on ICa·L and Icat were 4.6 and 5.7 μM, respectively. On the other hand, nifedipine and nicardipine did not inhibit Icat.

Vasopressin and PDGF increased incorporation of [3H]‐thymidine, and nifedipine and nicardipine partly suppressed it. However, the inhibitory effects of La3+ and exclusion of extracellular Ca2+ were more potent than the Ca2+ blocking agents. Troglitazone and pioglitazone also inhibited it concentration‐dependently.

These results suggest that troglitazone and pioglitazone preferentially inhibited agonist (vasopressin and PDGF)‐induced Ca2+ entry and proliferation in rat vascular smooth muscle cells, where the inhibitory effects of thiazolidinediones on ICa·L and Icat might be partly involved. Thus, thiazolidinediones may exert hypotensive and antiatherosclerotic effects.

British Journal of Pharmacology (1999) 128, 673–683; doi:10.1038/sj.bjp.0702818
Activity profiles of dalargin and its analogues in μ‐, δ‐ and κ‐opioid receptor selective bioassays
Authors: Pencheva Nevena , Pospišek Jan , Hauzerova Linda , Barth Tomislav , Milanov Peter
Published: January 2005

To elucidate the structural features ensuring action of [D‐Ala2, Leu5]‐enkephalyl‐Arg (dalargin), a series of dalargin analogues were tested for their effectiveness in depressing electrically‐evoked contractions of the guinea‐pig myenteric plexus‐longitudinal muscle preparations (μ‐ and κ‐opioid receptors) and the vasa deferentia of the hamster (δ‐opioid receptors), mouse (μ‐, δ‐ and κ‐opioid receptors), rat (similar to μ‐opioid receptors) and rabbit (κ‐opioid receptors). The naloxone KB values in the myenteric plexus were also obtained.

[L‐Ala2]‐dalargin was 19 times less potent than dalargin, and its pharmacological activity was peptidase‐sensitive. The ratio of δ‐activity to μ‐activity for [L‐Ala2]‐dalargin was 6.78, and KB was 7.9 nM. This emphasizes the role that D‐configuration of Ala2 plays in determining the active folding of dalargin molecule as well as in conferring resistance to peptidases.

[Met5]‐dalargin was equipotent to dalargin in the myenteric plexus, but was more potent in the vasa deferentia of hamster and mouse (KB=5.5 nM). Leu5 and the interdependence of Leu5 and D‐Ala2 are of importance for the selectivity of dalargin for μ‐opioid receptors.

Dalarginamide was more potent and selective for μ‐opioid receptors than dalargin, whilst dalarginethylamide, though equipotent to dalarginamide in the myenteric plexus, was more potent at δ‐opioid receptors (KB=5.0 nM). [D‐Phe4]‐dalarginamide and N‐Me‐[D‐Phe4]‐dalarginamide were inactive indicating the contribution of L‐configuration of Phe4 to the pharmacological potency of dalargin.

N‐Me‐[L‐Phe4]‐dalarginamide possessed the highest potency and selectivity for μ‐opioid receptors (the ratio of δ‐activity to μ‐activity was 0.00053; KB=2.6 nM). The CONH2 terminus combined with the N‐methylation of L‐Phe4 increased the potency and selectivity of dalargin for μ‐opioid receptors.

British Journal of Pharmacology (1999) 128, 569–576; doi:10.1038/sj.bjp.0702825
Evidence that mast cell degranulation, histamine and tumour necrosis factor α release occur in LPS‐induced plasma leakage in rat skin
Authors: Iuvone Teresa , Van Den Bossche Rita , D'Acquisto Fulvio , Carnuccio Rosa , Herman Arnold G
Published: January 2005

In the present study we investigated the role of mast cells during inflammation in rat skin. As the release of several pro‐inflammatory mediators, such as histamine and tumour necrosis factor α (TNFα), occurs following mast cell activation we studied whether mast cell degranulation and the release of both histamine (H) and TNFα occurred in a model of lipopolysaccharide (LPS)‐induced plasma leakage in rat skin.

Plasma leakage in the rat skin was measured over a period of 2 h as the local accumulation of intravenous injection of 125I‐human serum albumin (125I‐HSA) in response to intradermal injection of LPS. LPS (10 μg site−1) produced an increase of plasma leakage (50.1±2.3 μl site−1) as compared to saline (9.0±3.2 μl site−1). Histological analysis of rat tissue showed that LPS induced a remarkable mast cell degranulation (59.8±2.1%) as compared to saline (13.5±2.2%).

Ketotifen (10−9–10−7 mol site−1), a well‐known mast cell‐membrane stabilizer, produced a dose‐related inhibition of LPS‐induced plasma leakage by 36±3.5%, 47±4.0%, 60±3.3% respectively. In addition, ketotifen (10−7 mol site−1) inhibited mast cell degranulation by 59.2±2.7%.

Chlorpheniramine maleate (CPM) (10−9–10−7 mol site−1), an H1 histamine receptor antagonist only partially inhibited LPS‐induced plasma leakage in rat skin (38±1.1% at the highest dose). Furthermore, CPM (10−7 mol site−1) did not prevent mast cell degranulation.

A polyclonal antibody against TNFα (1 : 500, 1 : 100, 1 : 50 v  v−1 dilution), locally injected, decreased LPS‐induced plasma leakage in the skin by 15±2.0%, 24±2.1% and 50±3.0% respectively.

Taken together these results suggest that LPS‐induced plasma leakage in rat skin is mediated, at least in part, by mast cell degranulation and by the release of histamine and TNFα from these cells.

British Journal of Pharmacology (1999) 128, 700–704; doi:10.1038/sj.bjp.0702828
Effects of a range of β2 adrenoceptor agonists on changes in intracellular cyclic AMP and on cyclic AMP driven gene expression in cultured human airway smooth muscle cells
Authors: Scott Mark G H , Swan Caroline , Jobson Timothy M , Rees Steve , Hall Ian P
Published: January 2005

The effects of the selective β2 adrenoceptor agonists salbutamol, terbutaline and salmeterol and the non‐selective β adrenoceptor agonist isoprenaline on [3H]‐cyclic AMP formation and cyclic AMP response element (CRE) driven luciferase expression, assessed using the construct p6CRE/luc, were studied in primary cultures of human airway smooth muscle (HASM) cells.

Optimal transfection conditions for transient expression of pGL3 Control were 4 μg DNA/well71 in a 6 well plate and 1.8 μl Transfectam/μg DNA. Expression was maximal at 48–72 h.

Salbutamol (maximum response 19%, EC50 0.6 μM), terbutaline (maximum response 38%, EC50 2.3 μM) and salmeterol (maximum response 18%, EC50 0.0012 μM) were all partial agonists for cyclic AMP formation compared with isoprenaline (EC50 0.08 μM). However, all of the β2 adrenoceptor agonists produced increases in CRE‐driven luciferase activity, in cultured HASM transfected with the vector p6CRE/luc, which were equivalent or greater (salmeterol) than those seen with isoprenaline.

Both salbutamol and salmeterol were more potent at increasing luciferase expression than in elevating cyclic AMP levels in these cells. The potency ratios (EC50 (cyclic AMP)/EC50 (LUC)) for the agents studied were isoprenaline: 0.2 fold, terbutaline: 3 fold, salbutamol: 24 fold, salmeterol: 38 fold.

These data suggest that important quantitative differences exist in the ability of β2 adrenoceptor agonists to increase whole cell cyclic AMP levels in airway smooth muscle and to drive gene expression via a CRE‐driven mechanism.

British Journal of Pharmacology (1999) 128, 721–729; doi:10.1038/sj.bjp.0702829