bluuebunny/crossref_metadata_2025 huggingface dataset:
YC‐1 potentiates nitric oxide‐induced relaxation in guinea‐pig trachea
Authors: Hwang Tsong‐Long , Wu Chin‐Chung , Teng Che‐Ming
Published: January 2005

The effects of YC‐1 (3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzylindazole) on tension, levels of cyclic GMP and cyclic AMP were investigated in guinea‐pig trachea. We especially studied the combined effect of YC‐1 with exogenous or endogenous nitric oxide on these parameters.

YC‐1 at the concentration 3 or 10 μM, which caused only minor effect by itself, elicited concentration‐dependent potentiation of sodium nitroprusside (SNP)‐induced tracheal relaxation. This relaxation of YC‐1 with SNP was reversed by ODQ.

Relaxant responses to electric field stimulation (EFS) in the presence of indomethacin, atropine, guanethidine, α‐chymotrypsin and histamine were also markedly increased by YC‐1 (10 μM). In the presence of L‐NAME or ODQ, the relaxant effects to EFS were attenuated and the following addition of YC‐1 did not further enhance relaxation.

YC‐1 (10 μM) or SNP (0.3 μM) alone did not induce significant elevation of cyclic GMP levels in the presence of IBMX, whereas simultaneous application of both compounds markedly elevated the cyclic GMP accumulation. In contrast, the cyclic AMP levels were not altered even at the combination of YC‐1 and SNP. Additionally, YC‐1 also affected cyclic GMP metabolism, since it inhibited the activity of phosphodiesterase type V in human platelets.

YC‐1 (30 μM) did not scavenge superoxide anion and had no effect on the removal of superoxide anion by superoxide dismutase in a xanthine/xanthine oxidase system.

In conclusion, these results indicate that although YC‐1 elicits negligible relaxation of guinea‐pig trachea by itself, it can potentiate the relaxant responses of exogenous or endogenous NO. This synergistic response of YC‐1 is via the elevation of cyclic GMP contents.

British Journal of Pharmacology (1999) 128, 577–584; doi:10.1038/sj.bjp.0702830
Proteinase activated receptor 2: role of extracellular loop 2 for ligand‐mediated activation
Authors: Al‐Ani Bahjat , Saifeddine Mahmoud , Kawabata Atsufumi , Hollenberg Morley D
Published: January 2005

Rat proteinase‐activated receptor‐2 (PAR2) variants were stably expressed in rat KNRK cells: (a) wild‐type (wt)–PAR2; (b) PAR2PRR, with the extracellular loop 2 (EL‐2) sequence P231E232E233mutated to PRR and (c) PAR2NET, with the EL‐2 sequence, PEEV changed to NETL. Cell lines were evaluated for their sensitivity (calcium signalling) towards trypsin and the receptor‐activating peptides, SLIGRL‐NH2, SLIGEL‐NH2, trans‐cinnamoyl(tc)‐LIGRLO‐NH2, and SFLLR‐NH2.

SLIGEL‐NH2 exhibited low potency (1 : 200 relative to SLIGRL‐NH2) in wild‐type PAR2. Its activity was increased 5 fold in PAR2PRR, but it was inactive in PAR2NET.

In PAR2PRR, the potencies of SLIGRL‐NH2, tc‐LIGRLO‐NH2, and SFLLR‐NH2 were decreased by 80–100 fold. But, the potency of trypsin was decreased by only 7 fold.

In PAR2NET, highly homologous in EL‐2 with proteinase‐activated receptor‐1 (PAR1), the potency of the PAR1‐derived peptide, SFLLR‐NH2, was reduced by 100 fold compared with wt‐PAR2, whereas the potency of the PAR2‐derived AP, SLIGRL‐NH2 was reduced 10 fold. In contrast, the potency of trypsin in PAR2NET was almost the same as in wt‐PAR2.

We conclude that the acidic EL‐2 tripeptide, PEE, in PAR2 plays an important role in governing agonist activity.

The data obtained with the PEEV→NETL mutation suggested: (a) that SLIGRL‐NH2 and SFLLR‐NH2 interact in a distinct manner with PAR2 and (b) that SFLLR‐NH2 may interact differently with PAR2 than it does with PAR1.

The differential reductions in the potencies of SLIGRL‐NH2, compared with trypsin in the PAR2PRR and PAR2NET cell lines point to differences between the interactions of the trypsin‐revealed tethered ligand and the free receptor‐activating peptide with PAR2.

British Journal of Pharmacology (1999) 128, 1105–1113; doi:10.1038/sj.bjp.0702834
Reactive oxygen species generation and histamine release by activated mast cells: modulation by nitric oxide synthase inhibition
Authors: Brooks A C , Whelan C J , Purcell W M
Published: January 2005

We have examined the generation of intracellular reactive oxygen species (ROS) and release of histamine by rat peritoneal mast cells (RPMC) in response to stimulation with antigen (ovalbumin), compound 48/80, nerve growth factor (NGF) and substance P (SP).

We have also examined the effects of the non‐specific nitric oxide synthase inhibitor, L‐NAME (100 μM) upon the release of histamine and generation of intracellular ROS in response to the named secretagogues.

Ovalbumin (100–1000 μg ml−1), compound 48/80 (0.1–100 μg ml−1), NGF (0.1–100 μg ml−1), and SP (5–50 μM), caused a concentration‐dependent release of histamine from RPMC.

Ovalbumin (1 ng ml−1–0.1 μg ml−1), compound 48/80 (1–100 μg ml−1), NGF (1 pg ml−1–1 μg ml−1), and SP (0.005–50 μM) caused a concentration‐dependent generation of intracellular ROS by RPMC.

Pre‐incubation of RPMC with L‐NAME (100 μM) caused a significant enhancement of both histamine release and intracellular ROS from RPMC in response to ovalbumin, compound 48/80, NGF and SP.

Our data demonstrate that NGF, SP and ovalbumin are capable of causing intracellular ROS generation by RPMC at lower concentrations than those causing significant histamine release and we speculate that this may contribute to the activation of cytokine production.

The data also show that NO modulates histamine release, and ROS generation in response to the secretagogues used. This may have significance in pathologies where NO synthesis is decreased, leading to an increased activation of mast cells.

British Journal of Pharmacology (1999) 128, 585–590; doi:10.1038/sj.bjp.0702838
Tracheal relaxing effects and β2 adrenoceptor selectivity of S1319, a novel sponge‐derived bronchodilator agent, in isolated guinea‐pig tissues
Authors: Suzuki Hidefumi , Ueno Akihiro , Takei Masao , Sindo Kazutoshi , Miura Toru , Sakakibara Masayuki , Higa Tatsuo , Fukamachi Hiromi
Published: January 2005

S1319 (4‐hydroxy‐7‐[1‐(1‐hydroxy‐2‐methylamino)ethyl]‐1,3‐benzothiazol‐2(3H)‐one acetate), a novel non‐catecholamine β‐adrenoceptor agonist, has been compared with isoprenaline, salbutamol and formoterol for activity in vitro on a range of β‐adrenoceptor containing preparations from guinea‐pig.

S1319, like isoprenaline, salbutamol and formoterol, relaxed preparations of guinea‐pig trachea (contracted by histamine) in a concentration‐dependent manner. The relaxing activity of S1319 appeared to be more potent than that of isoprenaline and salbutamol, and similar to that of formoterol (pD2 values of 10.58±0.03 vs 7.60±0.01, 7.50±0.01 and 10.52±0.04, respectively), and was blocked by the β2‐adrenoceptor selective antagonist (ICI 118,551). The intrinsic activity of S1319 was close to 1.0.

In the β1‐adrenoceptor containing preparations, guinea‐pig right and left atria, a monophasic inotropic response of S1319 was observed. The pD2 value of S1319 for left atrial and right atrial inotropism was 6.70±0.15 and 7.81±0.01, respectively.

The selectivity ratio (trachea/left atrial inotropism) of S1319, formoterol, salbutamol and isoprenaline was 8523, 284, 4.8 and 0.45, respectively. The relative selectivity ratio of S1319 was 18743, 1858 and 30 times greater than that of isoprenaline, salbutamol and formoterol, respectively.

Relaxant responses of guinea‐pig trachea to S1319 declined rapidly when the agonist was washed from the tissues, with complete recovery within 30 min. The duration of action of S1319 was similar to that of isoprenaline and less than that of salbutamol and formoterol.

In summary, S1319, a sponge‐derived β‐adrenoceptor agonist, is a potent and selective β2‐adrenoceptor agonist with a short‐duration of action in isolated guinea‐pig tracheas.

British Journal of Pharmacology (1999) 128, 716–720; doi:10.1038/sj.bjp.0702839
Inhibition of NOS‐2 expression in macrophages through the inactivation of NF‐κB by andalusol
Authors: De Las Heras Beatriz , Navarro Antonio , Díaz‐Guerra María José , Bermejo Paloma , Castrillo Antonio , Boscá Lisardo , Villar Angel
Published: January 2005

Andalusol, ent‐6α,8α,18‐trihydroxy‐13(16),14‐labdadiene, is a naturally occurring diterpene, isolated from Sideritis foetens (Lamiaceae). This compound exhibited therapeutic activity when evaluated in in vivo models of paw and ear inflammation (Navarro et al., 1997: Z. Naturforsch., 52, 844‐849). The pharmacological effects of this diterpene have been analysed on the activation of the macrophage cell line J774 with lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ).

Incubation of J774 macrophages with andalusol (0.1–100 μM) inhibited the synthesis of nitrite caused by LPS (1 μg ml−1) in concentration and time‐dependent manners. The maximal inhibition was observed when andalusol was added 30 min before LPS stimulation and decreased progressively as the interval between andalusol and LPS challenge increased up to 14 h.

Incubation of J774 cells with LPS resulted in the expression of NOS‐2 protein (130 kDa) as identified by Western blot analysis. The levels of this enzyme decreased significantly in the presence of andalusol (IC50=10.5 μM), suggesting that this diterpene inhibited NOS‐2 expression.

Andalusol inhibited nuclear factor κB activation, a transcription factor necessary for NOS‐2 expression in response to LPS and IFN‐γ. This compound also inhibited the degradation of IκBα favouring the retention of the inactive NF‐κB complexes in the cytosol.

Related compounds to andalusol but lacking the polyol groups were less effective inhibiting NOS‐2 expression in LPS‐activated macrophages. The present findings provide a mechanism by which the anti‐inflammatory properties of this diterpene could be mediated.

British Journal of Pharmacology (1999) 128, 605–612; doi:10.1038/sj.bjp.0702844
Cyclic GMP‐associated apamin‐sensitive nitrergic slow inhibitory junction potential in the hamster ileum
Authors: Matsuyama Hayato , Thapaliya Sharada , Takewaki Tadashi
Published: January 2005

The mediators of non‐adrenergic, non‐cholinergic (NANC) inhibitory junction potentials (i.j.ps) in the circular smooth muscle cells of the hamster ileum were studied.

Electrical field stimulation (EFS; 0.5 ms duration, 15 V) of the intramural nerves with a train of five pulses at 20 Hz evoked a rapidly developing hyperpolarization (fast i.j.p.) followed by a sustained hyperpolarization (slow i.j.p.).

NG‐nitro‐L‐arginine methyl ester (L‐NAME; 50–200 μM) and NG‐nitro‐L‐arginine (L‐NNA; 50–200 μM), NO synthase inhibitors, inhibited or abolished the EFS‐induced fast and slow NANC i.j.ps. The effects of these NO synthase inhibitors were reversed by L‐arginine (5 mM) but not by D‐arginine (5 mM).

Exogenously applied nitric oxide (NO; 1–100 μM) induced concentration‐dependent hyperpolarizations.

Oxyhaemoglobin (5–50 μM), NO scavenger, inhibited only the slow i.j.p., and the NO‐induced hyperpolarization.

1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxaline‐1‐one (ODQ; 10 μM) and cystamine (10 mM), guanylate cyclase inhibitors, inhibited only the slow i.j.p. Zaprinast (100 μM), a phosphodiesterase type V inhibitor, enhanced the amplitude and duration of the slow i.j.p.

Apamin (100 nM), a small conductance Ca2+‐activated K+ channel blocker, inhibited only the slow i.j.p., and NO‐induced hyperpolarization. A high concentration of 8‐bromoguanosine 3′:5′‐cyclic monophosphate (8‐bromo‐cGMP; 1 mM)‐induced membrane hyperpolarization which was blocked by apamin.

These results suggest that NO, or a related compound, may be the inhibitory transmitter underlying the apamin‐sensitive NANC slow i.j.p. and cyclic GMP mediates the slow i.j.p. in the hamster ileum. It is also likely that NO, without involvement of guanylate cyclase is associated with the fast i.j.p.

British Journal of Pharmacology (1999) 128, 830–836; doi:10.1038/sj.bjp.0702851
Molecular and pharmacological characterization of a functional tachykinin NK3 receptor cloned from the rabbit iris sphincter muscle
Authors: Medhurst Andrew D , Hirst Warren D , Jerman Jeffery C , Meakin Jacqueline , Roberts Jennifer C , Testa Tania , Smart Darren
Published: January 2005

A functional tachykinin NK3 receptor was cloned from the rabbit iris sphincter muscle and its distribution investigated in ocular tissues.

Standard polymerase chain reaction (PCR) techniques were used to clone a full length rabbit NK3 receptor cDNA consisting of 1404 nucleotides. This cDNA encoded a protein of 467 amino acids with 91 and 87% homology to the human and rat NK3 receptors respectively.

In CHO‐K1 cells transiently expressing the recombinant rabbit NK3 receptor, the relative order of potency of NKB>>NKASP to displace [125I]‐[MePhe7]‐NKB binding and to increase intracellular calcium, together with the high affinity of NK3 selective agonists (e.g. senktide, [MePhe7]‐NKB) and antagonists (e.g. SR 142801, SB 223412) in both assays was consistent with NK3 receptor pharmacology. In binding and functional experiments, agonist concentration response curves were shallow (0.7–0.8), suggesting the possibility of multiple affinity states of the receptor.

Quantitative real time PCR analysis revealed highest expression of rabbit NK3 receptor mRNA in iris sphincter muscle, lower expression in retina and iris dilator muscle, and no expression in lens and cornea. In situ hybridization histochemistry revealed discrete specific localization of NK3 receptor mRNA in the iris muscle and associated ciliary processes. Discrete specific labelling of NK3 receptors with the selective NK3 receptor agonist [125I]‐[MePhe7]‐NKB was also observed in the ciliary processes using autoradiography.

Our study reveals a high molecular similarity between rabbit and human NK3 receptor mRNAs, as predicted from previous pharmacological studies, and provide the first evidence that NK3 receptors are precisely located on ciliary processes in the rabbit eye. In addition, there could be two affinity states of the receptor which may correspond to the typical and ‘atypical’ NK3 receptor subtypes previously reported.

British Journal of Pharmacology (1999) 128, 627–636; doi:10.1038/sj.bjp.0702854
Enhanced phenylephrine‐induced rhythmic activity in the atherosclerotic mouse aorta via an increase in opening of KCa channels: relation to Kv channels and nitric oxide
Authors: Jiang Jiahua , Thorén Peter , Caligiuri Giuseppina , Hansson Göran K , Pernow John
Published: January 2005

Mice lacking the apolipoprotein E and low density lipoprotein receptor genes (E°×LDLR°) develop atherosclerosis. The aim of this study was to investigate changes in endothelium‐dependent vasodilation and vasomotion in thoracic aortic rings of E°×LDLR° mice.

K+‐induced contractions of the aorta from E°×LDLR° mice were stronger than those from control mice. The sensitivity of E°×LDLR° aorta to phenylephrine (PE) was decreased but the maximal contractions were increased. Acetylcholine‐induced, but not sodium nitroprusside‐induced, relaxations of E°×LDLR° aorta was decreased.

PE induced rhythmic activity in both E°×LDLR° and control aorta but the amplitude was larger in E°×LDLR° than in control mice. PE‐induced rhythmic activity in both E°×LDLR° and control aorta was augmented by increase in extracellular Ca2+‐concentration, but was abolished by removal of the endothelium, the nitric oxide (NO) synthase inhibitor N‐nitro‐L‐arginine methyl ester, the guanylate cyclase inhibitor LY‐83583, high K+ solution and ryanodine.

4‐Aminopyridine, a voltage‐dependent potassium (KV) channel blocker, increased basal tension and induced rhythmic activity in E°×LDLR° aorta but not in control aorta.

The Ca2+‐activated potassium (KCa) channel blockers tetraethylammonium and charybdotoxin abolished PE‐induced rhythmic activity in E°×LDLR° aorta.

In conclusion, opening of Kv channels in E°×LDLR° mice aorta is reduced and it is susceptible to be depolarized resulting in Ca2+ entry. The vascular smooth muscle is then dependent on compensatory mechanisms to limit Ca2+‐entry. Such mechanisms may be decreased sensitivity to vasoconstrictors, or increased opening of KCa channels by NO via a cyclic GMP‐dependent mechanism.

British Journal of Pharmacology (1999) 128, 637–646; doi:10.1038/sj.bjp.0702855
Modulation of sibutramine‐induced increases in extracellular noradrenaline concentration in rat frontal cortex and hypothalamus by α2‐adrenoceptors
Authors: Wortley K E , Heal D J , Stanford S C
Published: January 2005

The effects of sibutramine (0.25–10 mg kg−1 i.p.) on extracellular noradrenaline concentration in the frontal cortex and hypothalamus of freely‐moving rats were investigated using microdialysis. The role of presynaptic α2‐adrenoceptors in modulating the effects of sibutramine in these brain areas was also determined.

Sibutramine induced an increase in extracellular noradrenaline concentration, the magnitude of which paralleled dose, in both brain areas. In the cortex, this increase was gradual and sustained, whereas in the hypothalamus it was more rapid and of shorter duration.

In both the cortex and hypothalamus, pretreatment of rats with the α2‐adrenoceptor antagonist RX821002 (3 mg kg−1 i.p.) potentiated increases in the accumulation of extracellular noradrenaline induced by sibutramine (10 mg kg−1 i.p.), by 7 and 10 fold respectively. RX821002 also reduced the latency of sibutramine to reach its maximum effect in the cortex, but not in the hypothalamus.

Infusion of RX821002 (1 μM) via the probe increased the accumulation of extracellular noradrenaline induced by sibutramine (10 mg kg−1 i.p.) in both brain areas. In the hypothalamus, the effects of RX821002 on the accumulation of noradrenaline induced by sibutramine were 2 fold greater than those in the cortex.

These findings support evidence that sibutramine inhibits the reuptake of noradrenaline in vivo, but that the accumulation of extracellular noradrenaline is limited by noradrenergic activation of presynaptic α2‐adrenoceptors. Furthermore, the data suggest that terminal α2‐adrenoceptors in the hypothalamus exert a greater inhibitory effect over the control of extracellular noradrenaline accumulation than do those in the cortex.

British Journal of Pharmacology (1999) 128, 659–666; doi:10.1038/sj.bjp.0702859
Evidence that histamine homologues discriminate between H3‐receptors in guinea‐pig cerebral cortex and ileum longitudinal muscle myenteric plexus
Authors: Harper E A , Shankley N P , Black J W
Published: January 2005

The binding of the selective histamine H3‐receptor agonist ([3H]‐R‐α‐methylhistamine) to sites in guinea‐pig cerebral cortex and ileum longitudinal muscle myenteric plexus has been characterized and a comparison made of the apparent affinities of a series of H3‐receptor ligands.

Saturation analysis suggested that [3H]‐R‐α‐methylhistamine labelled a homogeneous population of histamine H3‐receptors in guinea‐pig cerebral cortex (pKD=9.91±0.07; nH=1.07±0.03; n=5) and ileum longitudinal muscle myenteric plexus (pKD=9.75±0.21; nH=0.97±0.02; n=5). There was no significant difference in the estimated affinity of [3H]‐R‐α‐methylhistamine in the two tissues. The cerebral cortex had a significantly higher receptor density (3.91±0.37 fmol mg−1 tissue) than the ileum longitudinal muscle myenteric plexus (0.39±0.11 fmol mg−1).

Overall, the apparent affinities of compounds, classified as H3‐receptor ligands, in cerebral cortex and ileum longitudinal muscle myenteric plexus were well correlated (r=0.91, P<0.0001) and consistent with the cerebral cortex and ileum longitudinal muscle myenteric plexus expressing histamine H3‐receptor population(s) that are pharmacologically indistinguishable by the majority of histamine H3‐receptor ligands. However, it was evident that the homologues of histamine within this group of compounds could discriminate between the receptor populations in the two tissues. Thus, the estimated affinity of five imidazole unbranched alkylamines (histamine, homohistamine, VUF4701, VUF4732 and impentamine) were significantly higher in the guinea‐pig cerebral cortex than in the ileum longitudinal muscle myenteric plexus assay.

British Journal of Pharmacology (1999) 128, 751–759; doi:10.1038/sj.bjp.0702861
Effects of cyclopiazonic acid on contraction and intracellular Ca2+ in oesophageal striated muscle of normotensive and spontaneously hypertensive rats
Authors: Sekiguchi Fumiko , Shimamura Keiichi , Kawata Kyoko , Nakazawa Yumi , Saitoh Ritsuko , Yanagitani Yoshiko , Sunano Satoru
Published: January 2005

The effects of cyclopiazonic acid (CPA), a selective inhibitor of sarcoplasmic reticulum (SR) Ca2+‐ATPase, on twitch contraction and on the resting state of tension and intracellular Ca2+ level ([Ca2+]i) of the oesophageal striated muscle of stroke‐prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar Kyoto rats (WKY) were compared.

CPA (10 μM) augmented the twitch contraction of oesophageal striated muscle preparations from both SHRSP and WKY, reducing the rate of relaxation (−dT/dt), and thus resulting in the prolongation of the time to 80% relaxation. The effect was significantly smaller in the SHRSP preparations.

In the resting state, CPA caused a sustained elevation of [Ca2+]i. The elevation was greater in the WKY preparations. Tension development accompanied by the elevation was observed in WKY preparations, but not in SHRSP preparations.

The sustained elevation of [Ca2+]i induced by CPA was eliminated by the removal of extracellular Ca2+. Both the elevated [Ca2+]i and tension in the preparations from WKY were reduced by flufenamic acid (100 μM), mefenamic acid (100 μM), lanthanum (La3+, 100 μM), gadolinium (Gd3+, 100 μM) and SK&F 96365 (100 μM) but not by verapamil (10 μM).

Thapsigargin (3 μM), another SR Ca2+‐ATPase inhibitor, produced similar effects on basal tension to those of CPA, although it reduced the amplitude of twitch contraction.

These results suggest that in the rat oesophageal striated muscle, (1) CPA extends the sequestrating time of Ca2+ into the SR, (2) CPA induces a Ca2+ influx mediated through verapamil‐insensitive pathways, possibly nonselective cation channels, and (3) the mechanism of [Ca2+]i modulation due to CPA‐sensitive SR Ca2+‐ATPase is deteriorated in the oesophageal striated muscle from SHRSP as compared with WKY preparations.

British Journal of Pharmacology (1999) 128, 961–968; doi:10.1038/sj.bjp.0702867
Inhibition of vascular KATP channels by U‐37883A: a comparison with cardiac and skeletal muscle
Authors: Wellman G C , Barrett‐Jolley R , Köppel H , Everitt D , Quayle J M
Published: January 2005

The aim of this study was to investigate the selectivity of the ATP‐sensitive potassium (KATP) channel inhibitor U‐37883A (4‐morpholinecarboximidine‐N‐1‐adamantyl‐N′‐1‐cyclohexyl). Membrane currents through KATP channels were recorded in single muscle cells enzymatically isolated from rat mesenteric artery, cardiac ventricle and skeletal muscle (flexor digitorum brevis). KATP currents were induced either by cell dialysis with 0.1 mM ATP and 0.1 mM ADP, or by application of synthetic potassium channel openers (levcromakalim or pinacidil).

U‐37883A inhibited KATP currents in smooth muscle cells from rat mesenteric artery. Half inhibition of 10 μM levcromakalim‐induced currents occurred at a concentration of 3.5 μM.

Relaxations of rat mesenteric vessels caused by levcromakalim were reversed by U‐37883A. 1 μM levcromakalim‐induced relaxations were inhibited at a similar concentration of U‐37883A (half inhibition, 1.1 μM) to levcromakalim‐induced KATP currents.

KATP currents activated by 100 μM pinacidil were also studied in single myocytes from rat mesenteric artery, skeletal muscle and cardiac ventricle. 10 μM U‐37883A substantially inhibited KATP currents in vascular cells, but had little effect in skeletal or cardiac myocytes. Higher concentrations of U‐37883A (100 μM) caused a modest decrease in KATP currents in skeletal and cardiac muscle. The sulphonylurea KATP channel antagonist glibenclamide (10 μM) abolished currents in all muscle types.

The effect of U‐37883A on vascular inward rectifier (KIR) and voltage‐dependent potassium (KV) currents was also examined. While 10 μM U‐37883A had little effect on these currents, some inhibition was apparent at higher concentrations (100 μM) of the compound.

We conclude that U‐37883A inhibits KATP channels in arterial smooth muscle more effectively than in cardiac and skeletal muscle. Furthermore, this compound is selective for KATP channels over KV and KIR channels in smooth muscle cells.

British Journal of Pharmacology (1999) 128, 909–916; doi:10.1038/sj.bjp.0702868
Domains determining agonist selectivity in chimaeric VIP2 (VPAC2)/PACAP (PAC1) receptors
Authors: Lutz E M , MacKenzie C J , Johnson M , West K , Morrow J A , Harmar A J , Mitchell R
Published: January 2005

The VPAC2 and PAC1 receptors are closely related members of the Group II G protein‐coupled receptor family. At the VPAC2 receptor, VIP is equipotent to PACAP‐38 in stimulating cyclic AMP production, whereas at the PAC1 receptor PACAP‐38 is many fold more potent than VIP. In this study, domains which confer this selectivity were investigated by constructing four chimaeric receptors in which segments of the VPAC2 receptor were exchanged with the corresponding segment from the PAC1 receptor.

When expressed in COS 7 cells all the chimaeric receptors bound the common ligand [125I]PACAP‐27 and produced cyclic AMP in response to agonists.

Relative selectivity for agonists was determined primarily by the amino terminal extracellular domain of the PAC1 receptor and the VPAC2 receptor. The interchange of other domains had little effect on the potency of PACAP‐38 or PACAP‐27.

For chimaeric constructs with a PAC1 receptor amino terminal domain, the substitution of increasing portions of the VPAC2 receptor decreased the potency of VIP yet increased that of helodermin.

This suggests that the interaction of VIP/helodermin but not PACAP with the PAC1 receptor may be influenced (and differentially so) by additional receptor domains.

British Journal of Pharmacology (1999) 128, 934–940; doi:10.1038/sj.bjp.0702872
Elevated concentrations of morphine 6‐beta‐D‐glucuronide in brain extracellular fluid despite low blood–brain barrier permeability
Authors: Stain‐Texier Frédérique , Boschi Gabrielle , Sandouk Pierre , Scherrmann Jean‐Michel
Published: January 2005

This study was done to find out how morphine 6‐beta‐D‐glucuronide (M6G) induces more potent central analgesia than morphine, despite its poor blood–brain barrier (BBB) permeability. The brain uptake and disposition of these compounds were investigated in plasma and in various brain compartments: extracellular fluid (ECF), intracellular space (ICS) and cerebrospinal fluid (CSF).

Morphine or M6G was given to rats at 10 mg kg−1 s.c. Transcortical microdialysis was used to assess their distributions in the brain ECF. Conventional tissue homogenization was used to determine the distribution in the cortex and whole brain. These two procedures were combined to estimate drug distribution in the brain ICS. The blood and CSF pharmacokinetics were also determined.

Plasma concentration data for M6G were much higher than those of morphine, with Cmax and AUC 4–5 times more higher, Tmax shorter, and VZf−1 (volume of distribution) and CL f−1 (clearance) 4–6 times lower. The concentrations of the compounds in various brain compartments also differed: AUC values for M6G were lower than those of morphine in tissue and CSF and higher in brain ECF. AUC values in brain show that morphine levels were four times higher in ICS than in ECF, whereas M6G levels were 125 higher in ECF than in ICS.

Morphine entered brain cells, whereas M6G was almost exclusively extracellular. This high extracellular concentration, coupled with extremely slow diffusion into the CSF, indicates that M6G was predominantly trapped in the extracellular fluid and therefore durably available to bind at opioid receptors.

British Journal of Pharmacology (1999) 128, 917–924; doi:10.1038/sj.bjp.0702873
Diinosine pentaphosphate (IP5I) is a potent antagonist at recombinant rat P2X1 receptors
Authors: King B F , Liu M , Pintor J , Gualix J , Miras‐Portugal M T , Burnstock G
Published: January 2005

The antagonist activity of a series of diinosine polyphosphates (IpnI, where n=3, 4, 5) was assessed against ATP‐activated inward currents at rat P2X1–4 receptors expressed in Xenopus oocytes and studied under voltage‐clamp conditions.

Diinosine polyphosphates were prepared by the enzymatic degradation of their corresponding diadenosine polyphosphates (e.g., Ap5A into Ip5I) using 5′‐adenylic deaminase, and purified using reverse‐phase chromatography.

Against ATP‐responses at rP2X1 receptors, the potency order for antagonism was (pIC50): Ip5I (8.5)>Ip4I (6.3)>Ip3I (>4.5). Ip5I (10–100 nM) caused a concentration‐dependent rightwards displacement of the ATP concentration‐response curve without reducing the maximum ATP effect. However, the Schild plot was non‐linear which indicated Ip5I is not a competitive antagonist. Blockade by micromolar concentrations of Ip5I was not surmountable. Ip4I also behaved as a non‐surmountable antagonist.

Against ATP‐responses at rP2X3 receptors, the potency order for antagonism was (pIC50): Ip4I (6.0)>Ip5I (5.6)>Ip3I (>4.5). Blockade by Ip4I (pA2, 6.75) and Ip5I (pA2, 6.27) was surmountable at micromolar concentrations.

Diinosine polyphosphates failed to inhibit ATP‐responses at rP2X2 receptors, whereas agonist responses at rP2X4 were reversibly potentiated by Ip4I and Ip5I. None of the parent diadenosine polyphosphates behave as antagonists at rP2X1–4 receptors.

Thus, Ip5I acted as a potent and relatively‐selective antagonist at the rP2X1 receptor. This dinucleotide pentaphosphate represents a high‐affinity antagonist for the P2X1 receptor, at which it acts in a competitive manner at low (100 nM) concentrations but has more complex actions at higher (>100 nM) concentrations.

British Journal of Pharmacology (1999) 128, 981–988; doi:10.1038/sj.bjp.0702876
Role of L‐arginine in the deficiency of nitric oxide and airway hyperreactivity after the allergen‐induced early asthmatic reaction in guinea‐pigs
Authors: De Boer Jacob , Duyvendak Michiel , Schuurman Fineke E , Pouw F May H , Zaagsma Johan , Meurs Herman
Published: January 2005

Using a guinea‐pig model of allergic asthma, we investigated the role of L‐arginine limitation in the allergen‐induced deficiency of nitric oxide (NO) and airway hyperreactivity (AHR) after the early asthmatic reaction, by examining the effects of various concentrations of the NO synthase (NOS) substrate on the responsiveness to methacholine of isolated perfused tracheae from unchallenged (control) animals and from animals 6 h after ovalbumin challenge.

Preparations from ovalbumin‐challenged guinea‐pigs showed a 1.9 fold increase in the maximal response (Emax) to intraluminal (IL) administration of methacholine compared to controls (P<0.001). A similar 2.0 fold (P<0.05) increase in Emax to methacholine was observed in control airways incubated with the NOS inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME; 0.1 mM, IL), while L‐NAME had no further effect on the airways from ovalbumin‐challenged animals.

In control airways, extraluminal (EL) administration of 0.3, 1.0 and 5.0 mM L‐arginine all suppressed the Emax for methacholine by approximately 40% (P<0.01 all), whereas 5.0 mM D‐arginine (EL) had no effect.

L‐Arginine dose‐dependently reduced the AHR to methacholine in tracheae from ovalbumin‐challenged guinea‐pigs, the responsiveness being normalized in the presence of 5.0 mM L‐arginine. As in controls, 5.0 mM D‐arginine was without effect.

The results demonstrate that deficiency of endogenous NO contributes to the allergen‐induced AHR to methacholine after the early asthmatic reaction, which is reversed by exogenous administration of L‐arginine. This indicates that limitation of substrate may underly the reduced cNOS activity and subsequent AHR after the acute asthmatic response.

British Journal of Pharmacology (1999) 128, 1114–1120; doi:10.1038/sj.bjp.0702882
Effect of the umami peptides on the ligand binding and function of rat mGlu4a receptor might implicate this receptor in the monosodium glutamate taste transduction
Authors: Monastyrskaia Katherine , Lundstrom Kenneth , Plahl Doris , Acuna Gonzalo , Schweitzer Christophe , Malherbe Pari , Mutel Vincent
Published: January 2005

The effect of several metabotropic ligands and di‐ or tripeptides were tested on the binding of [3H]‐L(+)‐2‐amino‐4‐phosphonobutyric acid ([3H]‐L‐AP4) on rat mGlu4 receptor. For selected compounds, the functional activity was determined on this receptor using the guanosine‐5′[γ‐35S]‐thiotriphosphate [γ‐35S]‐GTP binding assay.

Using the scintillation proximity assay, [3H]‐L‐AP4 saturation analysis gave binding parameters KD and Bmax values of 150 nM and 9.3 pmoles mg−1 protein, respectively. The specific binding was inhibited concentration‐dependently by several mGlu receptor ligands, and their rank order of affinity was established.

Several peptides inhibited the [3H]‐L‐AP4 binding with the following rank order of potency: glutamate‐glutamate>glutamate‐glutamate‐leucine=aspartate ‐ glutamate>>glutamate ‐ glutamate‐aspartate>lactoyl‐glutamate>>aspartate‐aspartate. Aspartate‐phenylalanine‐methyl ester (aspartame) was inactive up to 1 mM and guanosine‐5′‐monophosphate and inosine‐5′‐monophosphate were inactive up to 100 μM.

The [γ‐35S]‐GTP binding functional assay was used to determine the agonist activities of the different compounds. For the rat mGlu4 agonists, L‐AP4 and L‐glutamate, the correlation between their occupancy and activation of the receptor was close to one. The peptides, Glu‐Glu, Asp‐Glu and Glu‐Glu‐Asp failed to stimulate the [γ‐35S]‐GTP binding at receptor occupancy greater than 80% and Glu‐Glu‐Leu appeared to be a weak partial agonist. These peptides did not elicit a clear dose‐dependent umami perception. However, Glu‐lac showed a good correlation between its potency to stimulate the [γ‐35S]‐GTP binding and its affinity for displacement of [3H]‐L‐AP4 binding. These data are in agreement with the peptide taste assessment in human subjects, which showed that the acid derivatives of glutamate had characteristics similar to umami.

British Journal of Pharmacology (1999) 128, 1027–1034; doi:10.1038/sj.bjp.0702885
Cardioprotective effects of N‐hydroxyguanidine PR5 in myocardial ischaemia and reperfusion in rats
Authors: Veveris Maris , Dambrova Maija , Cirule Helena , Meirena Dainuvite , Kalvinsh Ivars , Wikberg Jarl E S
Published: January 2005

The potential for the N‐hydroxyguanidine compound PR5 (N‐(3,4‐dimethoxy‐2‐chlorobenzylideneamino)‐N′‐hydroxyguanidine) as a cardioprotective agent in heart ischaemia and reperfusion injury was investigated using rat models.

Administration of 1–10 mg kg−1 of PR5 5 min before 10 min of left coronary artery occlusion, followed by 20 min reperfusion, strongly inhibited reperfusion burst of arrhythmias and markedly improved the survival of the animals (e.g. ventricular fibrillation incidence 93 vs 43% (P<0.05); mortality 47 vs 0% (P<0.05), for controls and for 3 mg kg−1 of PR5, respectively).

Administration of 3 mg kg−1 of PR5 1 min before reperfusion to rats subjected to 10 min occlusion, 20 min reperfusion was most effective in reducing arrhythmias and decreasing mortality (43 vs 0%, P<0.05), but effects were also seen when PR5 was administered 0, 1 and 5 min after start of reperfusion.

Coronary occlusion/reperfusion (10–20 min) increased malondialdehyde (MDA) of rat hearts (0.88±0.13 for sham vs 1.45±0.12 nmol mg−1 protein for ischaemic; P<0.05). In rats where 3 mg kg−1 PR5 were administered 1 min before reperfusion the increase was attenuated (MDA being 1.04±0.12; P<0.05 vs ischaemic).

PR5 caused a substantial reduction of the infarction size in rats subjected to 180 min left coronary artery occlusion, followed by 120 min of reperfusion; the necrotic zone being 326±32 mg for controls vs 137±21 mg for animals treated with 3×3 mg kg−1 of PR5 (P<0.01).

PR5 reduced the elevation of the ST‐segment of the ECGs, as well as caused pronounced attenuation of the rapid blood pressure drop seen at the start of reperfusion following coronary artery occlusion.

We conclude that the N‐hydroxyguanidine PR5 provides remarkable protection against ischaemia and reperfusion induced myocardial necrosis and life‐threatening arrhythmias. These effects of PR5 are discussed in relation to a recently discovered ability of N‐hydroxyguanidines to function as electron acceptors at the xanthine oxidase enzyme.

British Journal of Pharmacology (1999) 128, 1089–1097; doi:10.1038/sj.bjp.0702887
Effect of A2A adenosine receptor stimulation and antagonism on synaptic depression induced by in vitro ischaemia in rat hippocampal slices
Authors: Latini Serena , Bordoni Francesca , Corradetti Renato , Pepeu Giancarlo , Pedata Felicita
Published: January 2005

In the present study we investigated the role of A2A adenosine receptors in hippocampal synaptic transmission under in vitro ischaemia‐like conditions.

The effects of adenosine, of the selective A2A receptor agonist, CGS 21680 (2‐[p‐(2‐carboxyethyl)‐phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine), and of selective A2A receptor antagonists, ZM 241385 (4‐(2‐[7‐amino‐2‐(2‐furyl)‐{1,2,4}‐triazolo{2,3‐a}{1,3,5}triazin‐5‐ylamino]ethyl)phenol) and SCH 58261 (7‐(2‐phenylethyl)‐5‐amino‐2‐(2‐furyl)‐pyrazolo‐[4,3‐e]‐1,2,4‐triazolo[1,5‐c]pyrimidine), have been evaluated on the depression of field e.p.s.ps induced by an in vitro ischaemic episode.

The application of 2 min of in vitro ischaemia brought about a rapid and reversible depression of field e.p.s.ps, which was completely prevented in the presence of the A1 receptor antagonist DPCPX (1,3‐dipropyl‐8‐cyclopentylxanthine) (100 nM). On the other hand both A2A receptor antagonists, ZM 241385 and SCH 58261, by themselves did not modify the field e.p.s.ps depression induced by in vitro ischaemia.

A prolonged application of either adenosine (100 μM) or CGS 21680 (30, 100 nM) before the in vitro ischaemic episode, significantly reduced the synaptic depression. These effects were antagonized in the presence of ZM 241385 (100 nM).

SCH 58261 (1 and 50 nM) did not antagonize the effect of 30 nM CGS 21680 on the ischaemia‐induced depression.

These results indicate that in the CA1 area of the hippocampus the stimulation of A2A adenosine receptors attenuates the A1‐mediated depression of synaptic transmission induced by in vitro ischaemia.

British Journal of Pharmacology (1999) 128, 1035–1044; doi:10.1038/sj.bjp.0702888
‘Outside‐in’ signalling mechanisms underlying CD11b/CD18‐mediated NADPH oxidase activation in human adherent blood eosinophils
Authors: Lynch Oonagh T , Giembycz Mark A , Barnes Peter J , Hellewell Paul G , Lindsay Mark A
Published: January 2005

Incubation of human eosinophils in BSA‐coated tissue culture plates resulted in time‐dependent adhesion and attendant activation of the NADPH oxidase that were both inhibited (by >85%) by blocking antibodies raised against CD11b and CD18.

SB 203580, an inhibitor of p38 mitogen‐activated protein (MAP) kinase, did not influence adhesion but inhibited superoxide anion generation (pIC50=−6.57).

PP1, an inhibitor of the src‐family of protein tyrosine kinases, inhibited adhesion and CD11b/CD18‐mediated superoxide anion generation with similar potencies (pEC50s=−5.53 and −5.99 respectively) suggesting that inhibition of the NADPH oxidase was a direct consequence of blocking adhesion.

The protein kinase C (PKC) inhibitors Ro‐31 8220 (broad spectrum inhibitor), GF 109203X (inhibitor of conventional and novel isoforms) and Gö 6976 (inhibitor of conventional isoforms) suppressed adhesion‐dependent NADPH oxidase activation (pIC50s=−6.61, −6.05 and −4.89 respectively) without affecting adhesion. Based upon the selectivity of these drugs PKCδ and PKCε are implicated in the suppression of oxidant production.

Wortmannin, an inhibitor of phosphatidylinositol 3‐kinase (PtdIns 3‐kinase), abolished superoxide anion production in adherent eosinophils (pEC50=−9.06). Similarly, CD11b/CD18‐dependent adhesion was suppressed with the same potency (pEC50=−9.29) although the maximum effect did not exceed 50% implying that wortmannin also had an affect on those processes that govern adhesion‐driven oxidase activation.

PD 098059 and piceatannol, inhibitors of MAP kinase kinase‐1 and the syk tyrosine kinase respectively, had no effect on CD11b/CD18‐mediated adhesion or NADPH oxidase activation.

The results of this study demonstrate that human eosinophils adhere to BSA‐coated plastic by a CD11b/CD18‐dependent mechanism, which is responsible for activation of the NADPH oxidase. Although the signalling pathway(s) utilized by CD11b/CD18 is still to be elucidated, the data presented herein implicate p38 MAP kinase, novel PKCs and PtdIns 3‐kinase.

British Journal of Pharmacology (1999) 128, 1149–1158; doi:10.1038/sj.bjp.0702892
Cyclo‐oxygenase‐2: pharmacology, physiology, biochemistry and relevance to NSAID therapy
Authors: Mitchell Jane A , Warner Timothy D
Published: January 2005

Cyclo‐oxygenase is expressed in cells in two distinct isoforms. Cyclo‐oxygenase‐1 is present constitutively whilst cyclo‐oxygenase‐2 is expressed primarily after inflammatory insult. The activity of cyclo‐oxygenase‐1 and ‐2 results in the production of a variety of potent biological mediators (the prostaglandins) that regulate homeostatic and disease processes. Inhibitors of cyclo‐oxygenase include the nonsteroidal anti‐inflammatory drugs (NSAIDs) aspirin, ibuprofen and diclofenac. NSAIDs inhibit cyclo‐oxygenase‐2 at the site of inflammation, to produce their therapeutic benefits, as well as cyclo‐oxygenase‐1 in the gastric mucosa, which produces gastric damage. Most recently selective inhibitors of cyclo‐oxygenase‐2 have been developed and introduced to man for the treatment of arthritis. Moreover, recent epidemiological evidence suggests that cyclo‐oxygenase inhibitors may have important therapeutic relevance in the prevention of some cancers or even Alzheimer's disease. This review will discuss how the new advancements in NSAIDs research has led to the development of a new class of NSAIDs that has far reaching implications for the treatment of disease.

British Journal of Pharmacology (1999) 128, 1121–1132; doi:10.1038/sj.bjp.0702897
Effects of the gap junction uncoupler palmitoleic acid on the activation and repolarization wavefronts in isolated rabbit hearts
Authors: Dhein S , Krüsemann K , Schaefer T
Published: January 2005

The heart normally acts as an electrical syncytium coupled via gap junctional channels. Since closure of these channels has been considered arrhythmogenic, we wanted to elucidate, how activation and repolarization wavefronts are altered during progressive pharmacological gap junctional uncoupling.

We used the well known gap junction uncoupler palmitoleic acid (PA). The specificity of PA was tested in rabbit papillary muscles, which exhibited slowed conduction without affecting action potential morphology. We submitted isolated rabbit hearts (Langendorff‐technique) to increasing concentrations of palmitoleic acid (0.2, 1, 2, 5, 10, 20 μM), while 256 channel epicardial potential mapping was carried out.

In presence of PA activation recovery intervals (ARI) at the 256 electrodes became highly inhomogeneous with a dramatic increase in the dispersion of activation recovery intervals (from 6 to 35 ms, P>0.01; EC50=7 μM), while the mean ARI‐duration at 256 sites remained stable. PA led to marked alterations of the activation pattern, expressed as percentage of unchanged activation vectors (reduction from 32 to 10%, P<0.01, EC50=3.3 μM), to prolongation of atrioventricular conduction time (from 58 to 107 ms, P<0.01; EC50=8 μM) of total activation time (from 7 to 14 ms, P<0.05, EC50=11 μM) and of QRS‐complex‐duration.

In additional experiments the ventricle was paced via a bipolar electrode during the mapping procedure. From the isochrones longitudinal and transversal velocities were assessed showing that PA reduced transversal conduction velocity more distinctly than longitudinal.

With regard to maximum effects and EC50 values we conclude that gap junction uncoupling by PA mainly affects atrioventricular conduction, ARI‐dispersion and ventricular activation pattern. As important arrhythmogenic effects of uncoupling enhancement of dispersion with concomitant disturbation of the normal activation pattern and slowing of conduction might be considered.

British Journal of Pharmacology (1999) 128, 1375–1384; doi:10.1038/sj.bjp.0702902
Inhibition by genistein of the hyperpolarization‐activated cation current in porcine sino‐atrial node cells
Authors: Shibata Shigehiro , Ono Kyoichi , Iijima Toshihiko
Published: January 2005

The hyperpolarization‐activated cation current (If) was recorded in single pacemaker cells of the porcine sino‐atrial node, and the effects of genistein, an isoflavone inhibitor of tyrosine‐specific protein kinases was investigated by the whole‐cell patch clamp technique.

Genistein (20–500 μM) decreased If in a dose‐dependent manner with an IC50 value of 62.3 μM and a maximum inhibition of 45.3%.

The effect on If appeared without altering the half‐activation potential (control, −88.3±2.8 mV; genistein, −87.0±1.8 mV) and the slope factor (control, 8.0±0.3 mV; genistein, 8.6±0.7 mV) of the steady‐state activation curve. No significant voltage‐dependency was detected in the fully‐activated current‐voltage relation measured by the double‐pulse protocols.

The inactive form of genistein analogue, daidzein (500 μM) or genistin (200 μM), were without effect. If was not affected by another tyrosine kinase inhibitor, tryphostin‐47 (100 μM), but tyrphostin‐25 (100–200 μM) suppressed If in an irreversible manner.

Neither bath nor intracellular application of the tyrosine phosphatase inhibitor, orthovanadate, affected If, and subsequent application of genistein inhibited If significantly.

These data indicate that the inhibition of If by genistein is not mediated through tyrosine kinase inhibition but through nonselective block of the If channels.

British Journal of Pharmacology (1999) 128, 1284–1290; doi:10.1038/sj.bjp.0702903
Functional pharmacology of cloned heterodimeric GABAB receptors expressed in mammalian cells
Authors: Bräuner‐Osborne Hans , Krogsgaard‐Larsen Povl
Published: January 2005

In this study we report a new assay of heterodimeric γ‐amino‐butanoic acid subtype B (GABAB) receptors where either GABABR1a or GABABR1b are co‐expressed with GABABR2 and the chimeric G‐protein Gαq‐z5 in tsA cells. In this manner we obtained a robust response to GABAB agonists measured as increase in phosphoinositide hydrolysis.

We used this assay to characterize a number of commonly used GABAB receptor ligands. Both splice variants displayed the same rank order of agonist potency; 3‐aminopropyl(methyl)phosphinic acid (SKF‐97541)>GABA>(R)‐4‐amino‐3‐(4‐chlorophenyl)butanoic acid ((R)‐baclofen)>(RS)‐4‐amino‐3‐(5‐chloro‐2‐thienyl)butanoic acid (BCTG)>3‐aminopropylphosphonic acid (3‐APPA) and furthermore, the absolute agonist potency values were very close to each other.

3‐APPA was a partial agonist displaying maximal responses of 41 and 61% compared to GABA at GABABR1a and GABABR1b, respectively. The antagonist (RS)‐3‐amino‐2‐(4‐chlorophenyl)‐2‐hydroxypropylsulphonic acid (2‐OH‐saclofen) displayed KB values of 15 and 7.8 μM at GABABR1a and GABABR1b, respectively.

The rank order of agonist potency as well as the absolute ligand potencies correspond very well with those previously reported in different tissues, and this study thus provides a functional assay of cloned GABAB receptors which should be a valuable tool for further characterization of GABAB ligands. Finally, we can conclude that the functional pharmacological profiles of the two GABABR1 splice variants are very similar.

British Journal of Pharmacology (1999) 128, 1370–1374; doi:10.1038/sj.bjp.0702914