bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of vascular tone in isolated mesenteric arterial segments from hypertensive patients
Authors: Hutri‐Kähönen Nina , Kähönen Mika , Wu Xiumin , Sand Juhani , Nordback Isto , Taurio Jyrki , Pörsti Ilkka
Published: January 2005

Experimental hypertension is associated with several functional alterations of vascular endothelium and smooth muscle, but relatively few studies have examined the control of arterial tone in isolated vascular preparations from patients with essential hypertension. Therefore, we compared functional characteristics in vitro of distal ring segments of the mesenteric artery from 17 hypertensive and 22 normotensive humans.

Arterial constrictor responses induced by cumulative addition of Ca2+ in the presence of noradrenaline (NA) were more effectively inhibited by the Ca2+ entry blocker nifedipine (0.5 nM) in hypertensive than normotensive subjects (by 55.4±4.9, n=17 and 35.0±5.2%, n=22, respectively). Also the contractions elicited by high concentrations of KCl were more effectively inhibited by nifedipine in arterial rings from hypertensive than normotensive patients (by 38.9±3.7, n=17 and 20.2±4.6%, n=22, respectively). However, the concentration‐response curves of contractions to NA, serotonin and KCl in the absence of nifedipine were similar between the study groups.

The concentration‐response curves of endothelium‐dependent relaxations to acetylcholine and Ca2+ ionophore A23187, as well as of endothelium‐independent relaxations to the nitric oxide donor nitroprusside, β‐adrenoceptor agonist isoprenaline and K+ channel opener cromakalim did not show any differences between the groups. Moreover, the nitric oxide synthase inhibitor NG‐nitro‐L‐arginine methyl ester (0.1 mM) almost abolished the relaxations to acetylcholine and Ca2+ ionophore in both groups, indicating that these responses were largely mediated by nitric oxide. The function of arterial sodium pump was evaluated by relaxations elicited by the return of K+ upon contractions induced by K+‐free solution. The rate of K+‐relaxation was similar in hypertensive and normotensive arteries (for all these responses n=20–22 in the normotensive and 15–17 in the hypertensive group).

These results suggest abnormal function of voltage‐dependent Ca2+ channels in arterial smooth muscle of hypertensive patients, whereas vascular responses to endothelium‐dependent and ‐independent vasodilators and classical contractile agents were similar between hypertensive and normotensive subjects. The present findings support the view that blockade of voltage‐dependent Ca2+ channels is an effective means of reducing arterial tone in essential hypertension.

British Journal of Pharmacology (1999) 127, 1735–1743; doi:10.1038/sj.bjp.0702716
Comparison of changes in the extracellular concentration of noradrenaline in rat frontal cortex induced by sibutramine or d‐amphetamine: modulation by α2‐adrenoceptors
Authors: Wortley K E , Hughes Z A , 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 of halothane‐anaesthetized rats were compared with those of d‐amphetamine (1–3 mg kg−1, i.p.) using in vivo microdialysis. The role of presynaptic α2‐adrenoceptors in modulating the effects of these drugs on extracellular noradrenaline concentration were also investigated by pretreating rats with the selective α2‐adrenoceptor antagonist, RX821002.

Sibutramine induced a gradual and sustained increase in extracellular noradrenaline concentration. The dose‐response relationship was described by a bell‐shaped curve with a maximum effect at 0.5 mg kg−1. In contrast, d‐amphetamine induced a rapid increase in extracellular noradrenaline concentration, the magnitude of which paralleled drug dose.

Pretreatment with the α2‐adrenoceptor antagonist, RX821002 (dose 3 mg kg−1, i.p.) increased by 5 fold the accumulation of extracellular noradrenaline caused by sibutramine (10 mg kg−1) and reduced the latency of sibutramine to reach its maximum effect from 144–56 min.

RX821002‐pretreatment increased by only 2.5 fold the increase in extracellular noradrenaline concentration caused by d‐amphetamine alone (10 mg kg−1) and had no effect on the latency to reach maximum.

These findings support evidence that sibutramine acts as a noradrenaline uptake inhibitor in vivo and that the effects of this drug are blunted by indirect activation of presynaptic α2‐adreno‐ceptors. In contrast, the rapid increase in extracellular noradrenaline concentration induced by d‐amphetamine is consistent with this being mainly due to an increase in Ca2+‐independent release of noradrenaline.

British Journal of Pharmacology (1999) 127, 1860–1866; doi:10.1038/sj.bjp.0702720
Calcium channels involved in the inhibition of acetylcholine release by presynaptic muscarinic receptors in rat striatum
Authors: Doležal Vladimír , Tuček Stanislav
Published: January 2005

The mechanism of the inhibitory action of presynaptic muscarinic receptors on the release of acetylcholine from striatal cholinergic neurons is not known. We investigated how the electrically stimulated release of [3H]‐acetylcholine from superfused rat striatal slices and its inhibition by carbachol are affected by specific inhibitors of voltage‐operated calcium channels of the L‐type (nifedipine), N‐type (ω‐conotoxin GVIA) and P/Q‐type (ω‐agatoxin IVA).

The evoked release of [3H]‐acetylcholine was not diminished by nifedipine but was lowered by ω‐conotoxin GVIA and by ω‐agatoxin IVA, indicating that both the N‐ and the P/Q‐type (but not the L‐type) channels are involved in the release. The N‐type channels were responsible for approximately two thirds of the release. The release was >97% blocked when both ω‐toxins acted together.

The inhibition of [3H]‐acetylcholine release by carbachol was not substantially affected by the blockade of the L‐ or P/Q‐type channels. It was diminished but not eliminated by the blockade of the N‐type channels.

In experiments on slices in which cholinesterases had been inhibited by paraoxon, inhibition of [3H]‐acetylcholine release by endogenous acetylcholine accumulating in the tissue could be demonstrated by the enhancement of the release after the addition of atropine. The inhibition was higher in slices with functional N‐type than with functional P/Q‐type channels.

We conclude that both the N‐ and the P/Q‐type calcium channels contribute to the stimulation‐evoked release of acetylcholine in rat striatum, that the quantitative contribution of the N‐type channels is higher, and that the inhibitory muscarinic receptors are more closely coupled with the N‐type than with the P/Q‐type calcium channels.

British Journal of Pharmacology (1999) 127, 1627–1632; doi:10.1038/sj.bjp.0702721
The recombinant 5‐HT1A receptor: G protein coupling and signalling pathways
Authors: Raymond John R , Mukhin Yurii V , Gettys Thomas W , Garnovskaya Maria N
Published: January 2005

The 5‐hydroxytryptamine 5‐HT1A receptor was one of the first G protein coupled receptors whose cDNA and gene were isolated by molecular cloning methods. Transfection of the cDNA of this receptor into cells previously bearing no 5‐HT receptors has resulted in the acquisition of large amounts of information regarding potential signal transduction pathways linked to the receptor, correlations of receptor structure to its various functions, and pharmacological properties of the receptor. Transfection studies with the 5‐HT1A receptor have generated critical new information that might otherwise have been elusive. This information notably includes the discovery of unsuspected novel signalling linkages, the elucidation of the mechanisms of receptor desensitization, the refinement of models of the receptor pharmacophore, and the development of silent receptor antagonists, among others. The current review summarizes the most important studies of the recombinant 5‐HT1A receptor in the decade since the identificiation of its cDNA.

British Journal of Pharmacology (1999) 127, 1751–1764; doi:10.1038/sj.bjp.0702723
Glucocorticoids potently block tumour necrosis factor‐α‐ and lipopolysaccharide‐induced apoptotic cell death in bovine glomerular endothelial cells upstream of caspase 3 activation
Authors: Meßmer Udo K , Winkel Gundula , Briner Verena A , Pfeilschifter Josef
Published: January 2005

Endothelial cell damage in glomeruli and kidney arterioles appears to play a pivotal role in glomerular inflammatory diseases. Glomerular endothelial cells, a specialized microvascular cell type involved in the regulation of glomerular ultrafiltration, die by apoptosis in response to tumour necrosis factor‐α (TNF‐α), TNF‐α/basic fibroblast growth factor (bFGF), TNF‐α/cycloheximide, and bacterial lipopolysaccharide (LPS). Apoptotic cell death is characterized by extensive DNA cleavage, DNA ladder formation, and characteristic morphological alterations.

In search for apoptosis‐preventing signals, we identified glucocorticoids as potent death preventing factors. Co‐treatment of cells with 10 nM dexamethasone and TNF‐α, TNF‐α/bFGF, TNF‐α/cycloheximide, or LPS blocked roughly 90% of apoptotic cell death in glomerular endothelial cells.

Similarly to dexamethasone (TNF‐α‐ and LPS‐induced apoptosis are prevented with IC50 values of 0.8 and 0.9 nM, respectively), other synthetic and natural forms of glucocorticoids, such as fluocinolone, prednisolone, hydrocortisone, and corticosterone potently inhibited cell death with IC50 values of 0.2, 6, 50 and 1000 nM, for TNF‐α and 0.7, 8, 100 and 500 nM for LPS, respectively.

Apart from glucocorticoids, mineralocorticoids such as aldosterone also blocked TNF‐α/LPS‐induced apoptosis (IC50 ∼500 nM for TNF‐α and ∼500 nM for LPS), whereas sex hormones, i.e. β‐estradiol and testosterone remained without effect.

The protective effect of glucocorticoids (and mineralocorticoids) required glucocorticoid receptor binding as it could be antagonized by the glucocorticoid receptor antagonist RU‐486. Concerning TNF‐α and LPS signal transduction, we found that dexamethasone efficiently prevented TNF‐α‐ and LPS‐induced activation of caspase‐3‐like proteases. Therefore, we postulate inhibitory mechanisms upstream of terminal death pathways.

British Journal of Pharmacology (1999) 127, 1633–1640; doi:10.1038/sj.bjp.0702726
Effects of anion channel antagonists in canine colonic myocytes: comparative pharmacology of Cl−, Ca2+ and K+ currents
Authors: Dick Gregory M , Kong In Deok , Sanders Kenton M
Published: January 2005

Volume‐Sensitive, Outwardly Rectifying (VSOR) Cl− currents were measured in canine colonic myocytes by whole‐cell patch clamp. Decreasing extracellular osmolarity 50 milliosmoles l−1 activated current that was carried by Cl− and 5–7 times greater in the outward direction.

Niflumic acid, an inhibitor of Ca2+‐activated Cl− channels, did not inhibit VSOR Cl− current. Glibenclamide, an antagonist of CFTR, and anthracene‐9‐carboxylate (9‐AC) inhibited current less than 25% at 100 μM.

DIDS (4,4‐diisothiocyanato‐stilbene‐2,2′disulphonate) inhibited VSOR Cl− current more potently than SITS (4‐acetamido‐4′‐isothiocyanato‐stilbene‐2,2′‐disulphonate). IC50s were 0.84 and 226 μM, respectively.

VSOR Cl− current was strongly inhibited by tamoxifen ([Z]‐1‐[p‐dimethylaminoethoxy‐phenyl]‐1,2‐diphenyl‐1‐butene), an anti‐oestrogen compound (IC50=0.57 μM).

Gd3+ antagonized VSOR Cl− current more potently than La3+. The IC50 for Gd3+ was 23 μM. In contrast, 100 μM La3+ inhibited current only 35±7%.

Antagonists of VSOR Cl− current had non‐specific effects. These compounds blocked voltage‐dependent K+ and Ca2+ currents in colonic myocytes. Tamoxifen (10 μM) and DIDS (10 μM) inhibited L‐type Ca2+ current 87±7 and 31±5%, respectively. Additionally, in the presence of 300 nM charybdotoxin, tamoxifen (1 μM) and DIDS (10 μM) inhibited delayed rectifier K+ current 38±8 and 10±2%, respectively.

The pharmacology of VSOR Cl− channels overlaps with voltage‐dependent cation channels. DIDS and tamoxifen inhibited VSOR Cl− equally. However, because DIDS had much less effect on L‐type Ca2+ and delayed rectifier K+ channels than did tamoxifen, it might be useful in experiments to investigate the physiological and pathophysiological role of this conductance in whole tissues.

British Journal of Pharmacology (1999) 127, 1819–1831; doi:10.1038/sj.bjp.0702730
UTP‐preferring P2 receptor mediates inhibition of sodium transport in porcine thyroid epithelial cells
Authors: Bourke John , Abel Keith , Huxham Graham , Cooper Vanessa , Manley Simon
Published: January 2005

The effects of adenosine 5′‐triphosphate (ATP), uridine 5′‐triphosphate (UTP) and analogues on forskolin‐stimulated absorption of Na+ by porcine thyroid epithelial cells were analysed in cultures grown as confluent monolayers on permeable supports in Transwell Ussing chambers.

85% of the forskolin (10 μM)‐stimulated short‐circuit current was inhibited by phenamil (1 μM), which is a selective antagonist for epithelial type Na+ channels.

Phenamil‐sensitive current was inhibited in a dose dependent manner by nucleotides added to the apical compartment of Ussing chambers. In contrast, the phenamil‐resistant current, previously shown to represent anion secretion, was unaffected by nucleotides.

The order of potency (with EC50 values given in μM) was UTP (0.08)>>ATP (6.3)=uridine 5′‐diphosphate (UDP) (6.6)>2methyl‐thio‐adenosine‐5′‐triphosphate (2MeSATP) (84.5)>adenosine 5′‐diphosphate (ADP) (147.8)>α,β‐methylene ATP (>150)>>adenosine (>1000).

P2 receptors mediating inhibition of sodium absorption were present on the apical membrane of the cells since addition of UTP (1–1000 μM) to the basal compartment of the Ussing chambers had little effect while subsequent addition to the apical compartment produced a normal response.

Cibachron blue (Reactive blue 2) (1–100 μM), an antagonist at some P2 receptor subtypes, inhibited phenamil sensitive current in a dose dependent manner with half maximal inhibition occurring at 14.25 μM.

Suramin (100 μM), pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (100 μM) and pyridoxal 5′‐phosphate (P5P) (100 μM) showed only slight competitive antagonism against the response to UTP.

These results indicate that a UTP‐preferring P2 receptor located on the apical membrane of thyroid epithelial cells mediates inhibition of Na+ absorption.

British Journal of Pharmacology (1999) 127, 1787–1792; doi:10.1038/sj.bjp.0702733
L‐NAME inhibits Mg2+‐induced rat aortic relaxation in the absence of endothelium
Authors: Das Rapti , Kravtsov Gennadi M , Ballard Heather J , Kwan Chiu‐Yin
Published: January 2005

L‐NG‐nitro‐arginine methyl ester (L‐NAME; 100 μM), a nitric oxide synthase (NOS) inhibitor, reversed the relaxation induced by 3 μM acetylcholine (ACh) and 2–10 mM Mg2+ in endothelium‐intact (+E) rat aortic rings precontracted with 1 μM phenylephrine (PE). In PE‐precontracted endothelium‐denuded (−E) rat aorta, 3 μM ACh did not, but Mg2+ caused relaxation which was reversed by L‐NAME, but not by D‐NAME.

The concentration response profiles of L‐NAME in reversing the equipotent relaxation induced by 5 mM Mg2+ and 0.2 μM ACh were not significantly different.

L‐NAME (100 μM) also reversed Mg2+‐relaxation of −E aorta pre‐contracted with 20 mM KCl or 10 μM prostaglandin F2α (PGF2α). L‐NG‐monomethyl‐arginine (L‐NMMA; 100 μM) was also effective in reversing the Mg2+‐relaxation.

Addition of 0.2 mM Ni2+, like Mg2+, caused relaxation of PE‐pre‐contracted −E aorta, which was subsequently reversed by 100 μM L‐NAME.

Reversal of the Mg2+‐relaxation by 100 μM L‐NAME in PE‐precontracted −E aorta persisted following pre‐incubation with 1 μM dexamethasone or 300 μM aminoguanidine (to inhibit the inducible form of NOS, iNOS).

Pretreatment of either +E or −E aortic rings with 100 μM L‐NAME caused elevation of contractile responses to Ca2+ in the presence of 1 μM PE.

Our results suggest that L‐NAME exerts a direct action on, as yet, unidentified vascular smooth muscle plasma membrane protein(s), thus affecting its reactivity to divalent cations leading to the reversal of relaxation. Such an effect of L‐NAME is unrelated to the inhibition of endothelial NOS or the inducible NOS.

British Journal of Pharmacology (1999) 128, 493–499; doi:10.1038/sj.bjp.0702737
Effect of Na+/Ca2+ exchange inhibitor, KB‐R7943 on ouabain‐induced arrhythmias in guinea‐pigs
Authors: Watano Tomokazu , Harada Yoshimitsu , Harada Kengo , Nishimura Noriyasu
Published: January 2005

We investigated protective effects of KB‐R7943, a Na+/Ca2+ exchange (NCX) inhibitor, on ouabain‐induced tonotropy and arrhythmias in isolated whole atria and ouabain‐induced changes in electrocardiogram (ECG) in the guinea‐pig.

KB‐R7943 (10 and 30 μM) suppressed the tonotropic effect of ouabain, and prolonged the onset time of extra‐systole induced by ouabain in isolated atria.

The intravenous injection of KB‐R7943 (1 and 3 mg kg−1) significantly increased the doses of ouabain required to induce ventricular premature beats (VPB), ventricular tachycardia (VT), ventricular fibrillation (VF) and cardiac arrest (CA) in anaesthetized guinea‐pigs.

Lidocaine (Na+channel inhibitor) and R56865 (Na+ and Ca2+ overload inhibitor) also suppressed the ouabain‐induced tonotropic effect and extra‐systole in isolated atria, but Hoe‐694 (Na+/H+ exchange inhibitor) or diltiazem (Ca2+ channel inhibitor) did not affect them.

Lidocaine also increased the doses of ouabain required to induce VPB, VT, VF and CA in anaesthetized guinea‐pigs.

From these results, we conclude that KB‐R7943 suppresses ouabain‐induced arrhythmias through inhibition of the reverse‐mode NCX.

British Journal of Pharmacology (1999) 127, 1846–1850; doi:10.1038/sj.bjp.0702740
Inactivation block of the HERG human cardiac K+ channels by RP58866
Authors: Wang Huizhen , Shi Hong , Wang Zhiguo
Published: January 2005

RP58866 possesses a unique electrophysiological property: highly effective against various types of arrhythmias including ventricular fibrillation in animal models, noticeably those occurring during ischaemia with depolarized membrane due to elevated extracellular K+ concentrations. To understand the potential ionic mechanisms, we performed detailed studies on the effects of RP58866 on the HERG channels expressed in Xenopus oocytes, which are believed to be important compositions of the rapid component of delayed rectifier K+ current in the hearts.

RP58866 significantly inhibited the HERG channels in a concentration‐dependent manner, with ∼50% decrease in the current amplitude at a concentration of 1 μM.

RP58866 produced more pronounced inhibition with voltage protocols which favoured inactivation of the HERG channels. It caused substantial negative shift of the inactivation curves but did not alter the activation properties. The inhibition was considerably relieved by elevating [K+]o from 5–20 mM, which weakened the channel inactivation. More importantly, the potency was reduced by ∼100 fold on the mutated HERG channels (S631A) in which the C‐type inactivation was substantially weakened.

We conclude that blockade of the HERG channels by RP58866 is mainly associated with the binding of the drugs to the inactivated channels. This unique property of HERG blockade might explain some previously reported but unexplained observations: RP58866 maintains its efficacy in APD prolongation with depolarized membrane potential and in arrhythmias during ischaemia with manifested membrane depolarization.

British Journal of Pharmacology (1999) 127, 1899–1907; doi:10.1038/sj.bjp.0702741
Inducible expression of the kinin B1 receptor in the endotoxemic heart: mechanisms of des‐Arg9bradykinin‐induced coronary vasodilation
Authors: McLean Peter G , Perretti Mauro , Ahluwalia Amrita
Published: January 2005

We have investigated the role of kinin B1 receptor induction in the endotoxemic rat heart and elucidated the mechanisms underlying B1 receptor‐mediated coronary vasodilation. We also investigated the role of these receptors in endotoxin‐induced hypotension.

Endotoxin treatment induced cardiac B1 receptor mRNA expression and promoted a coronary vasodilation response to des‐Arg9bradykinin (DABK; ED50=149.4 pmol, n=9) ex vivo peaking at 6 h. The B1 receptor antagonist des‐Arg9‐[Leu8]‐BK (DALBK, 30 nM) significantly (P<0.05) inhibited the DABK‐induced response (pA2=8.4, n=5) whilst HOE140 (B2 receptor antagonist, 10 nM) was inactive (n=4).

Removal of the endothelium or infusion with indomethacin (5 μM), but not L‐NAME (300 μM) or ODQ (1 μM), inhibited (>85%, P<0.05, n=5) the DABK‐induced response. DABK caused a dose‐dependent release of the prostacyclin metabolite, 6‐keto‐PGF1a (Emax=0.3 ng ml−1, n=6).

In vitro perfusion of hearts with endotoxin (1 μg ml−1, n=6) or interleukin‐1β (5 ng ml−1, n=6) induced B1 receptor mRNA expression and promoted a time‐dependent vasodilation response to DABK.

Endotoxin treatment (6 h) in vivo promoted a hypotensive response to DABK (ED50=29.7 nmol kg−1, n=10) which was antagonised by DALBK (3–6 nmol kg−1 min−1, P<0.05, n=7). DALBK (3 nmol kg−1 min−1) and des‐Arg10HOE140 (B1 receptor antagonist, 30 nmol kg−1 min−1) produced a 5.3% (n=6, P<0.05) and 8.8% (n=5, P<0.05) reversal, respectively, of endotoxin‐induced hypotension.

In summary, we have shown that in endotoxemia activation of B1 receptors causes coronary vasodilation via endothelial prostacyclin release. Additionally, B1 receptor antagonists partially reversed endotoxin‐induced hypotension. Therefore activation of B1 receptors may have a role to play in the vascular changes associated with endotoxemia.

British Journal of Pharmacology (1999) 128, 275–282; doi:10.1038/sj.bjp.0702743
Inhibition of heterologously expressed cystic fibrosis transmembrane conductance regulator Cl− channels by non‐sulphonylurea hypoglycaemic agents
Authors: Cai Z , Lansdell K A , Sheppard D N
Published: January 2005

Hypoglycaemia‐inducing sulphonylureas, such as glibenclamide, inhibit cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels. In search of modulators of CFTR, we investigated the effects of the non‐sulphonylurea hypoglycaemic agents meglitinide, repaglinide, and mitiglinide (KAD‐1229) on CFTR Cl− channels in excised inside‐out membrane patches from C127 cells expressing wild‐type human CFTR.

When added to the intracellular solution, meglitinide and mitiglinide inhibited CFTR Cl− currents with half‐maximal concentrations of 164±19 μM and 148±36 μM, respectively. However, repaglinide only weakly inhibited CFTR Cl− currents.

To understand better how non‐sulphonylurea hypoglycaemic agents inhibit CFTR, we studied single channels. Channel blockade by both meglitinide and mitiglinide was characterized by flickery closures and a significant decrease in open probability (Po). In contrast, repaglinide was without effect on either channel gating or Po, but caused a small decrease in single‐channel current amplitude.

Analysis of the dwell time distributions of single channels indicated that both meglitinide and mitiglinide greatly decreased the open time of CFTR. Mitiglinide‐induced channel closures were about 3‐fold longer than those of meglitinide.

Inhibition of CFTR by meglitinide and mitiglinide was voltage‐dependent: at positive voltages channel blockade was relieved.

The data demonstrate that non‐sulphonylurea hypoglycaemic agents inhibit CFTR. This indicates that these agents have a wider specificity of action than previously recognized. Like glibenclamide, non‐sulphonylurea hypoglycaemic agents may inhibit CFTR by occluding the channel pore and preventing Cl− permeation.

British Journal of Pharmacology (1999) 128, 108–118; doi:10.1038/sj.bjp.0702748
trans‐Resveratrol inhibits calcium influx in thrombin‐stimulated human platelets
Authors: Dobrydneva Yuliya , Williams Roy L , Blackmore Peter F
Published: January 2005

The phytoestrogenic compound trans‐resveratrol (trans‐3,5,4′‐trihydroxystilbene) is found in appreciable quantities in grape skins and wine. It has been shown that both products rich in trans‐resveratrol and pure trans‐resveratrol inhibit platelet aggregation both in vivo and in vitro. However the mechanism of this action still remains unknown.

An essential component of the aggregation process in platelets is an increase in intracellular free Ca2+ ([Ca2+]i). Ca2+ must enter the cell from the external media through specific and tightly regulated Ca2+ channels in the plasma membrane. The objective of this study was to characterize what effect trans‐resveratrol had on the Ca2+ channels in thrombin stimulated platelets.

In this study we showed that trans‐resveratrol immediately inhibited Ca2+ influx in thrombin‐stimulated platelets with an IC50 of 0.5 μM. trans‐Resveratrol at 0.1, 1.0 and 10.0 μM produced 20±6, 37±6 and 57±4% inhibition respectively of the effect of thrombin (0.01 u  ml−1) to increase [Ca2+]i.

trans‐Resveratrol also inhibited spontaneous Ba2+ entry into Fura‐2 loaded platelets, with 0.1, 1.0 and 10.0 μM trans‐resveratrol producing 10±5, 30±5 and 50±7% inhibition respectively. This indicated that trans‐resveratrol directly inhibited Ca2+ channel activity in the platelets in the absence of agonist stimulation.

trans‐Resveratrol also inhibited thapsigargin‐mediated Ca2+ influx into platelets. This suggests that the store‐operated Ca2+ channels are one of the possible targets of trans‐resveratrol. These channels rely on the emptying of the internal Ca2+ stores to initiate influx of Ca2+ into the cell.

The phytoestrogens genistein, daidzein, apigenin and genistein‐glucoside (genistin) produced inhibitory effects against thrombin similar to those seen with trans‐resveratrol.

We conclude that trans‐resveratrol is an inhibitor of store‐operated Ca2+ channels in human platelets. This accounts for the ability of trans‐resveratrol to inhibit platelet aggregation induced by thrombin.

British Journal of Pharmacology (1999) 128, 149–157; doi:10.1038/sj.bjp.0702749
Selective β1‐adrenoceptor blockade enhances the activity of the stimulatory G‐protein in human atrial myocardium
Authors: Wang Tao , Plumpton Christopher , Brown Morris J
Published: January 2005

Chronic selective β1‐adrenoceptor (β1AR) blocker treatment enhances the sensitivity of β2‐adrenoceptor (β2AR) in human heart (Hall et al., 1990; 1991). To clarify the mechanism of the cross‐sensitization between β1AR and β2AR, we determined whether the stimulatory G‐protein (Gsα) function is increased in atria from β1AR‐blocker treated patients compared with non‐β‐blocked patients, and investigated whether this change is caused by an alteration of post‐translational modification of Gsα protein.

Gsα function was determined by reconstitution of human atrial Gsα into S49 cyc− cell membranes. In the reconstitution system, GTPγS stimulated cyclic AMP generation in a dose‐dependent manner. Upon 10−4 M GTPγS stimulation, Gsα activity in the β1AR‐blocker, atenolol, treated group (78.2±10.3 pmol cyclic AMP mg−1 min−1 10−3) was 65% higher than that in non‐β‐blocked patients (47.3±6.3 pmol cyclic AMP mg−1 min−1 10−3, n=15, P=0.02).

Isoelectric point (pI) values of Gsα were measured by two dimensional gel electrophoresis (2D‐E) and the amount of each isoform quantified by image analysis of a Western blot of the gel using specific antibody. Multiple isoforms of Gsα were detected by 2D‐E with different pI values. There were no significant differences between the groups of patients in either pI values or the proportions of the acidic isoforms of Gsα to the main basic form (n=12, P>0.05).

The results suggest that chronic β1AR‐blockade enhances Gsα function in human atrium, and this may account in part for the hypersensitivity of β2AR and other Gs‐coupled receptors during β1AR‐blockade. The increased Gsα function is unlikely to be caused directly by blockade of protein kinase A phosphorylation of Gsα protein.

British Journal of Pharmacology (1999) 128, 135–141; doi:10.1038/sj.bjp.0702750
Modulation by protease‐activated receptors of the rat duodenal motility in vitro: possible mechanisms underlying the evoked contraction and relaxation
Authors: Kawabata Atsufumi , Kuroda Ryotaro , Nishikawa Hiroyuki , Kawai Kenzo
Published: January 2005

The present study examined effects of agonist enzymes and receptor‐activating peptides for protease‐activated receptors (PARs) on duodenal motility in the rat, and also investigated possible mechanisms underlying the evoked responses.

Thrombin at 0.03–0.1 μM and the PAR‐1‐activating peptide SFLLR‐NH2 at 3–100 μM or TFLLR‐NH2 at 10–50 μM produced a dual action, relaxation followed by contraction of the duodenal longitudinal muscle. The PAR‐2‐activating peptide SLIGRL‐NH2 at 10–100 μM elicited only small contraction. Trypsin at 0.08 μM induced small contraction, or relaxation followed by contraction, depending on preparations. The PAR‐4‐activating peptide GYPGKF‐NH2 at 1000 μM exhibited no effect.

The contractile responses of the duodenal strips to TFLLR‐NH2 and to SLIGRL‐NH2 were partially attenuated by the L‐type calcium channel blocker nifedipine (1 μM), the protein kinase C inhibitor GF109203X (1 μM) and the tyrosine kinase inhibitor genistein (15 μM), but were resistant to indomethacin (3 μM) and tetrodotoxin (1–10 μM).

The relaxation of the preparations exerted by TFLLR‐NH2 was unaffected by indomethacin (3 μM), propranolol (5 μM), NG‐nitro‐L‐arginine methyl ester (100 μM) and tetrodotoxin (1–10 μM). This relaxation was resistant to either GF109203X (1 μM) or genistein (15 μM), but was, remarkably, attenuated by combined application of these two kinase inhibitors.

Apamin (0.1 μM), an inhibitor of calcium‐activated, small‐conductance potassium channels, but not charybdotoxin (0.1 μM), completely abolished the PAR‐1‐mediated duodenal relaxation, and significantly enhanced the PAR‐1‐mediated contraction.

These findings demonstrate that PAR‐1 plays a dual role, suppression and facilitation of smooth muscle motility in the rat duodenum, while PAR‐2 plays a minor excitatory role in the muscle, and that PAR‐4 is not involved in the duodenal tension modulation. The results also suggest that the contractile responses to PAR‐1 and PAR‐2 activation are mediated, in part, by activation of L‐type calcium channels, protein kinase C and tyrosine kinase, and that the relaxation response to PAR‐1 activation occurs via activation of apamin‐sensitive, but charybdotoxin‐insensitive, potassium channels, in which both protein kinase C and tyrosine kinase might be involved synergistically.

British Journal of Pharmacology (1999) 128, 865–872; doi:10.1038/sj.bjp.0702755
The cannabinoid CB1 receptor antagonist, SR141716A, selectively facilitates nociceptive responses of dorsal horn neurones in the rat
Authors: Chapman Victoria
Published: January 2005

The effect of spinal administration of the selective cannabinoid CB1 receptor antagonist, SR141716A, and the selective CB2 receptor antagonist, SR144528, on innocuous versus noxious evoked responses of dorsal horn neurones in the spinal cord of the anaesthetized rat was investigated. SR141716A (0.001–1 ng 50 μl−1) dose‐relatedly facilitated the non‐potentiated component of the electrical C‐fibre mediated neuronal response (120±6, 156±13, 192±33 and 192±31% of control respectively; n=6). In contrast, SR144528 (0.001–1 ng 50 μl−1) did not influence the non‐potentiated component of the C‐fibre evoked neuronal response (n=5). The electrical evoked Aβ‐fibre mediated neuronal responses were not influenced by SR141716A or SR144528. The results of this study provide evidence that tonic cannabinoid CB1 receptor activation, but not CB2 receptor activation, attenuates acute nociceptive transmission, at the level of the spinal cord. These results suggest a selective antinociceptive role of the endogenous cannabinoids at spinal CB1 receptors.

British Journal of Pharmacology (1999) 127, 1765–1767; doi:10.1038/sj.bjp.0702758
5‐CT stimulation of adenylyl cyclase activity in guinea‐pig hippocampus: evidence for involvement of 5‐HT7 and 5‐HT1A receptors
Authors: Thomas David R , Middlemiss Derek N , Taylor Stephen G , Nelson Paul , Brown Anthony M
Published: January 2005

A number of compounds, including the selective 5‐HT7 receptor antagonist SB‐258719, were investigated for their effect on [3H]‐5‐carboxamidotryptamine (5‐CT) radioligand binding and 5‐CT‐stimulated adenylyl cyclase activity in guinea‐pig hippocampal membranes, in order to confirm the presence of functionally coupled 5‐HT7 receptors in this tissue.

The [3H]‐5‐CT radioligand binding profile was consistent with binding predominantly to 5‐HT7 receptors. The affinity of SB‐258719 (pKi 7.2±0.1) was similar to its reported human 5‐HT7 receptor affinity.

In the adenylyl cyclase functional assay, 5‐CT was a potent and full agonist compared to 5‐HT, whereas 8‐hydroxy‐dipropylaminotetralin (8‐OH‐DPAT) was a partial agonist (intrinsic activity 0.4±0.1). The rank order of potency for agonists (5‐CT>5‐HT∼8‐OH‐DPAT) was consistent with activation of 5‐HT7 receptors. SB‐258719 (5  μM) and methiothepin (1 μM) surmountably antagonized the response to 5‐CT, consistent with competitive antagonism. The pKB for SB‐258719 (7.2±0.1) was in good agreement with its reported antagonist potency at the human cloned 5‐HT7 receptor.

In the functional assay, WAY‐100635 (100 nM) and cyanopindolol (1 μM) induced a biphasic 5‐CT response curve, consistent with selective antagonism of a component of the response to 5‐CT. The estimated pKB values for WAY‐100635 and cyanopindolol (9.6 and 8.4 respectively) were in good agreement with their reported 5‐HT1A receptor affinities.

The data are consistent with the presence of 5‐HT7 receptors in guinea‐pig hippocampus which are positively coupled to adenylyl cyclase. In addition, 5‐HT7 receptor‐mediated stimulation of adenylyl cyclase activity in this tissue appears to be augmented by a mechanism involving 5‐HT1A receptor activation.

British Journal of Pharmacology (1999) 128, 158–164; doi:10.1038/sj.bjp.0702759
Modulatory effects of adenosine on inhibitory postsynaptic potentials in the lateral amygdala of the rat
Authors: Heinbockel Thomas , Pape Hans‐Christian
Published: January 2005

Adenosine is a depressant in the central nervous system with pre‐ and postsynaptic effects. In the present study, intracellular recording techniques were applied to investigate the modulatory effects of adenosine on projection neurons in the lateral rat amygdala (LA), maintained as slices in vitro.

Adenosine reversibly reduced the amplitude of a fast inhibitory postsynaptic potential (IPSP) that was evoked by electrical stimulation of the external capsule and pharmacologically isolated by applying an N‐methyl‐D‐aspartate and non‐N‐methyl‐D‐aspartate receptor antagonist, DL‐(−)‐2‐amino‐5‐methyl‐4‐isoxazolepropionic acid and 6,7‐Dinitroquinoxaline‐2,3‐dione, respectively, and the γ‐aminobutyric acidB (GABAB) receptor antagonist CGP 35348. The postsynaptic potential that remained was abolished by locally applying bicuculline.

Adenosine reduced the amplitude of the fast IPSP on average by 40.3%. It had no significant effect on responses to exogenously applied GABA, on membrane potential or on input resistance, suggesting that the site of action was at presynaptic inhibitory interneurons in the LA.

The response to adenosine was mimicked by the selective adenosine A1 receptor agonist N6‐cyclohexyladenosine and blocked by the selective adenosine A1 receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine.

Neuronal responsiveness in the amygdala is largely controlled by inhibitory processes. Adenosine can presynaptically downregulate inhibitory postsynaptic responses and could exert dampening effects likely by depression of both excitatory and inhibitory neurotransmitter release.

British Journal of Pharmacology (1999) 128, 190–196; doi:10.1038/sj.bjp.0702761
Structural requirements of sulphonylureas and analogues for interaction with sulphonylurea receptor subtypes
Authors: Meyer Miriam , Chudziak Franz , Schwanstecher Christina , Schwanstecher Mathias , Panten Uwe
Published: January 2005

The structure‐activity relationship for hypoglycaemic sulphonylureas and analogues was examined. Binding affinities were compared using membranes from HIT‐T15 cells (β‐cell line) and from COS‐7 cells transiently expressing sulphonylurea receptor subtypes (SUR1, SUR2A and SUR2B). Inhibition of adenosine‐triphosphate‐sensitive K+ channels (KATP‐channels) was measured in mouse pancreatic β‐cells.

The tested compounds displayed similar binding affinities for SUR2A and SUR2B.

Meglitinide (benzoic acid derivative) bound to SUR1 and the SUR2 isoforms with similar affinities. Replacement of the carboxyl group of meglitinide by a methyl group significantly decreased the binding affinities for SUR1 and the SUR2 isoforms (>4 fold) and the potency to inhibit KATP‐channel activity of β‐cells (24 fold). Replacement of the carboxyl group of meglitinide by a sulphonylurea group significantly increased the affinities for SUR1 (5 fold) and the SUR2 isoforms (13–16 fold).

Glibenclamide bound to the SUR2 isoforms with 300–500 fold lower affinity than to SUR1. Exchanging the cyclohexyl ring of glibenclamide by a methyl group or removal of the lipophilic side chain of glibenclamide (5‐chloro‐2‐methoxy‐benzamidoethyl chain) markedly reduced but did not abolish the selectivity for SUR1.

In conclusion, interaction of sulphonylureas and acidic analogues with SUR1, SUR2A and SUR2B is favoured by the anionic group of these drugs. Hypoglycaemic sulphonylureas (e.g. glibenclamide) owe selectivity for SUR1 to lipophilic substitution on their urea group. Sulphonylureas without lipophilic substitution on the urea group could represent lead compounds for the development of SUR2‐selective drugs.

British Journal of Pharmacology (1999) 128, 27–34; doi:10.1038/sj.bjp.0702763
Receptors mediating CGRP‐induced relaxation in the rat isolated thoracic aorta and porcine isolated coronary artery differentiated by hα CGRP8–37
Authors: Wisskirchen F M , Gray D W , Marshall I
Published: January 2005

Receptors mediating CGRP‐induced vasorelaxation were investigated in rat thoracic aorta and porcine left anterior descending (LAD) coronary artery and anterior interventricular artery (AIA), using CGRP agonists, homologues and the antagonist hα CGRP8–37.

In the endothelium‐intact rat aorta, hα CGRP, hβ CGRP, rat β CGRP and human adrenomedullin caused relaxation with similar potencies. Compared with hα CGRP, rat amylin was about 25 fold less potent, while [Cys(ACM2,7)] hα CGRP and salmon calcitonin were at least 1000 fold weaker.

Hα CGRP8–37 (up to 10−5 M) did not antagonize responses to hα CGRP, hβ CGRP or rat β CGRP (apparent pKB <5). Peptidase inhibitors did not increase either the effect of hα CGRP or [Cys(ACM,2,7)] hα CGRP, while hα CGRP8–37 remained inactive. Endothelium‐dependent relaxation produced by hα CGRP was accompanied by increases in cyclic AMP and cyclic GMP, that were not inhibited by hα CGRP8–37 (10−5 M).

In porcine LAD and AIA, hα CGRP produced relaxation in an endothelium‐independent manner. Hα CGRP8‐37 competitively antagonized hα CGRP responses (pA2 6.3 and 6.7 (Schild slope 0.9±0.1, each), in LAD and AIA, respectively). In LAD artery, hα CGRP‐induced relaxation was accompanied by increases in cyclic AMP that were inhibited by hα CGRP8–37 (10−7–10−5 M).

In conclusion, the antagonist affinity for hα CGRP8–37 in porcine coronary artery is consistent with a CGRP1 receptor, while the lack of hα CGRP8–37 antagonism in rat aorta could suggest either a CGRP receptor different from CGRP1 and CGRP2 type, or a non‐CGRP receptor.

British Journal of Pharmacology (1999) 128, 283–292; doi:10.1038/sj.bjp.0702764
Alteration of glycogen and glucose metabolism in ischaemic and post‐ischaemic working rat hearts by adenosine A1 receptor stimulation
Authors: Fraser Heather , Lopaschuk Gary D , Clanachan Alexander S
Published: January 2005

Cardioprotection by adenosine A1 receptor activation limits infarct size and improves post‐ischaemic mechanical function. The mechanisms responsible are unclear but may involve alterations in myocardial glucose metabolism.

Since glycogen is an important source of glucose during ischaemia, we examined the effects of N6‐cyclohexyladenosine (CHA), an A1 receptor agonist, on glycogen and glucose metabolism during ischaemia as well as reperfusion.

Isolated working rat hearts were perfused with Krebs‐Henseleit solution containing dual‐labelled 5‐3H and 14C glucose and palmitate as energy substrates. Rates of glycolysis and glucose oxidation were measured directly from the production of 3H2O and 14CO2. Glycogen turnover was measured from the rate of change of [5‐3H and 14C]glucosyl units in total myocardial glycogen.

Following low‐flow (0.5 ml min−1) ischaemia (60 min) and reperfusion (30 min), left ventricular minute work (LV work) recovered to 22% of pre‐ischaemic values. CHA (0.5 μM) improved the recovery of LV work 2 fold.

CHA altered glycogen turnover in post‐ischaemic hearts by stimulating glycogen synthesis while having no effects on glycogen degradation. CHA also partially inhibited glycolysis. These changes accelerated the recovery of glycogen in CHA‐treated hearts and reduced proton production.

During ischaemia, CHA had no measurable effect on glycogen turnover or glucose metabolism. Glycogen phosphorylase activity, which was elevated after ischaemia, was inhibited by CHA, possibly in response to CHA‐induced inhibition of AMP‐activated protein kinase activity.

These results indicate that CHA‐induced cardioprotection is associated with alterations of glycogen turnover during reperfusion as well as improved metabolic coupling of glycolysis to glucose oxidation.

British Journal of Pharmacology (1999) 128, 197–205; doi:10.1038/sj.bjp.0702765
Interleukin‐1β (IL‐1β) inhibition: a possible mechanism for the anti‐inflammatory potency of liposomally conjugated methotrexate formulations in arthritis
Authors: Williams A S , Jones S G , Goodfellow R M , Amos N , Williams B D
Published: January 2005

Liposomes with conventional and long‐circulation times were employed as carriers for the methotrexate derivative MTX‐γ‐DMPE (MTX‐EPC and MTX‐PEG respectively), their mechanism of action was investigated in vitro and in vivo and their therapeutic efficacy assessed using the rat collagen‐induced arthritis (CIA) model.

At non‐toxic dose, both MTX‐EPC and MTX‐PEG inhibited the lipopolysaccharide (LPS) induced release of IL‐1β from activated rat peritoneal macrophages (rPMΦ) in a dose and time dependent manner. Free methotrexate (MTX) was not active in this respect. After a single intravenous injection (i.v.), and at equivalent doses, both free MTX (500 μg) and MTX‐EPC inhibited the LPS induced rise in plasma IL‐1β levels observed in MTX‐PEG and saline treated rats.

When used to treat established CIA, MTX‐EPC resulted in significantly lower clinical score (CS) (1.0±0.42 (P<0.001)) and hind paw diameter (HPD) (6.5±0.34 mm (P<0.001)) measurements than controls (3.0±0.26; 7.33±0.41 mm), after only two i.v. doses, and remained significantly lower for the entire experimental period. By day 24 both CS (2±0.61 (P<0.001)) and HPD (6.97±0.25 mm (P<0.002)) measurements had also become significantly lower in MTX‐PEG treated rats than in saline treated controls (3.62±0.17, 7.92±0.38 mm) and remained lower until day 30. Joint inflammation in MTX treated rats was completely ameliorated by day 20 but the health and well being of the animals was compromised and the experiment terminated at this time‐point.

Our results clearly demonstrate that both MTX‐EPC and MTX‐PEG liposomes have potential for development into therapeutic modalities for the treatment of inflammatory joint disease in man.

British Journal of Pharmacology (1999) 128, 234–240; doi:10.1038/sj.bjp.0702776
Inadequate ischaemia‐selectivity limits the antiarrhythmic efficacy of mibefradil during regional ischaemia and reperfusion in the rat isolated perfused heart
Authors: Farkas András , Qureshi Aasim , Curtis Michael J
Published: January 2005

Mibefradil was compared with (±)‐verapamil for effects on ischaemia‐ and reperfusion‐induced ventricular fibrillation (VF), and the role of ischaemia‐selective L‐channel block was examined. Langendorff perfused rat hearts (n=12/group) were used.

Neither drug at up to 100 nM reduced the incidence of VF during 30 min regional ischaemia. 300 and 600 nM (±)‐verapamil abolished VF (P<0.05); mibefradil was effective only at 600 nM (P<0.05). Reperfusion‐induced VF incidence was reduced only by 600 nM (±)‐verapamil (P<0.05). Both drugs at 100 nM increased coronary flow (P<0.05) with a similar potency and maximum effectiveness.

In separate hearts perfused with Krebs' solution containing 3 mM K+ (the same as that used for arrhythmia studies) neither drug at up to 600 nM affected ventricular contractility. With K+ raised to 6 mM, (±)‐verapamil 30 nM reduced developed pressure (P<0.05); mibefradil did so only at 600 nM (P<0.05). With K+ raised to 10 mM the effects of (±)‐verapamil were further increased (P<0.05) and mibefradil became active at 100 nM (P<0.05). Likewise both drugs impaired diastolic relaxation, with raised K+ exacerbating the effects and (±)‐verapamil being more potent and its effects more greatly exacerbated by K+. In contrast, when K+ was normal (3 mM), coronary flow was increased by each drug at 30 nM (P<0.05) indicating a marked vascular : myocardial selectivity.

In conclusion, mibefradil differed from (±)‐verapamil in its myocardial effects only in terms of its lower potency. As mibefradil is the more potent T‐channel blocker, the T‐channel is unlikely to represent the molecular target for these effects. The K+ elevations that occur in the ischaemic milieu determine the ability of both drugs to block myocardial L‐channels; this is sufficient to account for the drugs' actions on VF. Neither drug possesses sufficient selectivity for ischaemic myocardium versus blood vessels to permit efficacy (VF suppression without marked vasodilatation) and so inappropriate hypotension is likely to preclude the safe use of mibefradil (or similar analogue) in VF suppression, and explains the lack of clinical effectiveness of (±)‐verapamil.

British Journal of Pharmacology (1999) 128, 41–50; doi:10.1038/sj.bjp.0702778
Anti‐spasmogenic activity of isoenzyme‐selective phosphodiesterase inhibitors in guinea‐pig trachealis
Authors: Bernareggi Micaela M , Belvisi Maria G , Patel Hema , Barnes Peter J , Giembycz Mark A
Published: January 2005

The anti‐spasmogenic potential of SK&F 94120 (PDE3‐selective), rolipram (PDE4‐selective), zaprinast (PDE5‐selective), zardaverine (dual PDE3/4 inhibitor) and theophylline (non‐selective) was evaluated in guinea‐pig trachealis.

SK&F 94120 or rolipram (10 and 100 μM) antagonized histamine‐induced tension generation in a concentration‐dependent and non‐competitive manner whereas ACh‐induced contractions were unaffected. Similarly, SK&F 94120 and rolipram in combination were anti‐spasmogenic with respect to both contractile agonists to an extent that was greater than the effect of either drug alone. Identical results were obtained with zardaverine (1, 10 and 100 μM) and theophylline (100 μM and 1 mM).

Zaprinast protected guinea‐pig trachealis against histamine‐, but not ACh‐induced contractile responses in a manner that was indistinguishable from the results obtained with SK&F 94120. However, in contrast to the interaction between SK&F 94120 and rolipram, no further antagonism was seen when zaprinast and rolipram were used in combination.

Pre‐treatment of tissues with SNP (10 and 100 μM) antagonized histamine‐induced tension generation in a concentration‐dependent and non‐competitive manner. However, no further antagonism was produced when SNP and rolipram were used concurrently. Likewise, the protection afforded by a combination SNP and SK&F 94120 was no greater than that produced by SNP alone.

These results demonstrate that an inhibitor of PDE3 enhances the anti‐spasmogenic activity of rolipram but not drugs that elevate cyclic GMP mass. Moreover, the ability of SNP and zaprinast to protect guinea‐pig trachealis against histamine‐induced contractions apparently is not due to the inhibition of PDE3.

British Journal of Pharmacology (1999) 128, 327–336; doi:10.1038/sj.bjp.0702779