bluuebunny/crossref_metadata_2025 huggingface dataset:
Glimepiride block of cloned β‐cell, cardiac and smooth muscle KATP channels
Authors: Song Dae‐Kyu , Ashcroft Frances M
Published: January 2005

We examined the effect of the sulphonylurea glimepiride on three types of recombinant ATP‐sensitive potassium (KATP) channels.

KATP channels share a common pore‐forming subunit, Kir6.2, which associates with different sulphonylurea receptor isoforms (SUR1 in β‐cells, SUR2A in heart and SUR2B in smooth muscle).

Kir6.2 was coexpressed with SUR1, SUR2A or SUR2B in Xenopus oocytes and macroscopic KATP currents were recorded from giant inside‐out membrane patches. Glimepiride was added to the intracellular membrane surface.

Glimepiride inhibited Kir6.2/SUR currents by interaction with two sites: a low‐affinity site on Kir6.2 (IC50=∼400 μM) and a high‐affinity site on SUR (IC50=3.0 nM for SUR1, 5.4 nM for SUR2A and 7.3 nM for SUR2B). The potency of glimepiride at the high‐affinity site is close to that observed for glibenclamide (4 nM for SUR1, 27 nM for SUR2A), which has a similar structure.

Glimepiride inhibition of Kir6.2/SUR2A and Kir6.2/SUR2B currents, but not Kir6.2/SUR1 currents, reversed rapidly.

Our results indicate that glimepiride is a high‐affinity sulphonylurea that does not select between the β‐cell, cardiac and smooth muscle types of recombinant KATP channel, when measured in inside‐out patches. High‐affinity inhibition is mediated by interaction of the drug with the sulphonylurea receptor subunit of the channel.

British Journal of Pharmacology (2001) 133, 193–199; doi:10.1038/sj.bjp.0704062
Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats
Authors: Duarte Juan , Pérez‐Palencia Raquel , Vargas Felix , Angeles Ocete Maria , Pérez‐Vizcaino Francisco , Zarzuelo Antonio , Tamargo Juan
Published: January 2005

The effects of an oral daily dose (10 mg kg−1) of the flavonoid quercetin for 5 weeks in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rats (WKY) were analysed.

Quercetin induced a significant reduction in systolic (−18%), diastolic (−23%) and mean (−21%) arterial blood pressure and heart rate (−12%) in SHR but not in WKY rats.

The left ventricular weight index and the kidney weight index in vehicle‐treated SHR were significantly greater than in control WKY and these parameters were significantly reduced in quercetin‐treated SHR in parallel with the reduction in systolic blood pressure.

Quercetin had no effect on the vasodilator responses to sodium nitroprusside or to the vasoconstrictor responses to noradrenaline or KCl but enhanced the endothelium‐dependent relaxation to acetylcholine (Emax=58±5%vs 78±5%, P<0.01) in isolated aortae.

The 24 h urinary isoprostane F2α excretion and the plasma malonyldialdehyde (MDA) levels in SHR rats were increased as compared to WKY rats. However, in quercetin‐treated SHR rats both parameters were similar to those of vehicle‐treated WKY.

These data demonstrate that quercetin reduces the elevated blood pressure, the cardiac and renal hypertrophy and the functional vascular changes in SHR rats without effect on WKY. These effects were associated with a reduced oxidant status due to the antioxidant properties of the drug.

British Journal of Pharmacology (2001) 133, 117–124; doi:10.1038/sj.bjp.0704064
Short‐ and long‐term influences of calcitonin gene‐related peptide on the synthesis of acetylcholinesterase in mammalian myotubes
Authors: Da Costa Valter Luiz , Lapa Antonio José , Godinho Rosely O
Published: January 2005

The present study analyses the short‐ (15 min – 2 h) and long‐term (24 – 48 h) influences of calcitonin gene‐related peptide (CGRP) on acetylcholinesterase (AChE) expression in the rat cultured skeletal muscle and the signal transduction events underlying CGRP actions.

To assess the effect of CGRP on AChE synthesis, myotubes were pre‐exposed to the irreversible AChE inhibitor diisopropyl fluorophosphate (DFP) and treated with CGRP or forskolin, an adenylyl cyclase (AC) activator. Treatment of myotubes with 1 – 100 nM CGRP for 2 h increased by up to 42% the synthesis of catalytically active AChE with a parallel increase in the intracellular cyclic AMP.

The stimulation of AChE synthesis induced by CGRP was mimicked by direct activation of AC with 3 – 30 μM forskolin. In contrast, pre‐treatment of cultures with 100 nM CGRP for 20 h reduced by 37% the subsequent synthesis of AChE, resulting in a 15% decrease in total AChE activity after 48 h CGRP treatment.

Moreover, 24 h treatment of myotubes with 100 nM CGRP reduced by 54% the accumulation of cyclic AMP induced by a subsequent CGRP treatment.

These findings indicate that, in skeletal muscle cells, CGRP modulates the AChE expression in a time‐dependent manner, initially stimulating the enzyme synthesis through a cyclic AMP‐dependent mechanism. The decreased AChE synthesis observed after long‐term CGRP treatment suggests that CGRP signalling system is subject to desensitization or down‐regulation, that might function as an important adaptative mechanism of the muscle fibre in response to long‐term changes in neuromuscular transmission.

British Journal of Pharmacology (2001) 133, 229–236; doi:10.1038/sj.bjp.0704069
Inhibition of calcium channels by opioid‐ and adenosine‐receptor agonists in neurons of the nucleus accumbens
Authors: Chieng Billy , Bekkers John M
Published: January 2005

The pharmacological effects of opioid‐ and adenosine‐receptor agonists on neural signalling were investigated by measuring drug actions on barium current flowing through calcium channels in acutely‐dissociated neurons of the rat nucleus accumbens (NAc).

Under whole‐cell voltage clamp, opioids acted via μ, but not δ or κ, receptors to partially inhibit barium current. Mean inhibition was 35±2% (±s.e.mean, n=33) for methionine‐enkephalin and 37±1% (n=65) for the selective μ receptor agonist DAMGO, both measured at saturating agonist concentrations in neurons with diameter 20 μm. EC50 for DAMGO was 100 nM. Perfusion of naloxone reversed the current inhibition by DAMGO.

Adenosine also partially inhibited barium current in these neurons. Mean inhibition was 28±2% (n=29) for adenosine and 33±3% (n=27) for the selective A1 receptor agonist N6CPA, both at saturating concentrations in neurons with diameter 20 μm. EC50 for N6CPA was 34 nM. Adenosine inhibition was reversed by perfusion of an A1 receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine, while the selective A2A receptor agonist, CGS 21680, had no effect.

Inhibition by opioids and adenosine was mutually occlusive, suggesting a converging pathway onto calcium channels.

These actions involved a G‐protein‐coupled mechanism, as demonstrated by the partial relief of inhibition by strong depolarization and by the application of N‐ethylmaleimide or GTP‐γ‐S.

Inhibition of barium current by opioids had their greatest effect in large neurons, that is, in presumed interneurons. In contrast, opioid inhibition in neurons with diameter 15 μm was 11±2% (n=26) for methionine‐enkephalin and 11±4% (n=17) for DAMGO, both measured at saturating agonist concentrations. Adenosine inhibition in neurons with diameter 15 μm was 22±5% (n=9).

These results implicate the interneurons as a locus for the modulation of the excitability of projection neurons in the NAc during the processes of addiction and withdrawal.

British Journal of Pharmacology (2001) 133, 337–344; doi:10.1038/sj.bjp.0704072
Respective contributions of α‐adrenergic and non‐adrenergic mechanisms in the hypotensive effect of imidazoline‐like drugs
Authors: Bruban Véronique , Feldman Josiane , Greney Hugues , Dontenwill Monique , Schann Stephan , Jarry Christian , Payard Marc , Boutin Jean , Scalbert Elizabeth , Pfeiffer Bruno , Renard Pierre , Vanhoutte Paul , Bousquet Pascal
Published: January 2005

The hypotensive effect of imidazoline‐like drugs, such as clonidine, was first attributed to the exclusive stimulation of central α2‐adrenoceptors (α2ARs).

However, a body of evidence suggests that non‐adrenergic mechanisms may also account for this hypotension.

This work aims (i) to check whether imidazoline‐like drugs with no α2‐adrenergic agonist activity may alter blood pressure (BP) and (ii) to seek a possible interaction between such a drug and an α2ARs agonist α‐methylnoradrenaline (α‐MNA).

We selected S23515 and S23757, two imidazoline‐like drugs with negligible affinities and activities at α2ARs but with high affinities for non‐adrenergic imidazoline binding sites (IBS).

S23515 decreased BP dose‐dependently (−27±5% maximal effect) when administered intracisternally (i.c.) to anaesthetized rabbits. The hypotension induced by S23515 (100 μg kg−1 i.c.) was prevented by S23757 (1 mg kg−1 i.c.) and efaroxan (10 μg kg−1 i.c.), while these compounds, devoid of haemodynamic action by themselves, did not alter the hypotensive effect of α‐MNA (3 and 30 μg kg−1 i.c.). Moreover, the α2ARs antagonist rauwolscine (3 μg kg−1 i.c.) did not prevent the effect of S23515.

Finally, whilst 3 μg kg−1 of S23515 or 0.5 μg kg−1 of α‐MNA had weak hypotensive effects, the sequential i.c. administration of these two drugs induced a marked hypotension (−23±2%).

These results indicate that an imidazoline‐like drug with no α2‐adrenergic properties lowers BP and interacts synergistically with an α2ARs agonist.

British Journal of Pharmacology (2001) 133, 261–266; doi:10.1038/sj.bjp.0704080
Characteristics of the pulse waveform during altered nitric oxide synthesis in the rabbit
Authors: Weinberg P D , Habens F , Kengatharan M , Barnes S E , Matz J , Änggård E E , Carrier M J
Published: January 2005

Nitrovasodilators produce characteristic changes in the shape of the peripheral pulse wave. Similar changes might also be caused by alteration of endogenous NO activity, which would allow such activity to be assessed in vivo.

We investigated whether manipulation of the NO pathway influences the pulse waveform, and the mechanisms involved. The pulse wave in the ear of normal rabbits was examined by reflectance photoplethysmography before and during infusion of vasoactive agents. Pulse wave velocity was assessed by using an additional sensor on the rear foot.

A diastolic peak was observed in the ear pulse; its timing was consistent with it being a reflection of the systolic peak from the lower body. The height of the dicrotic notch marking the start of this diastolic wave was decreased by acetylcholine or an NO donor, and further decreased by a phosphodiesterase type V inhibitor. The acetylcholine‐induced decreases were blocked by inhibiting NO synthesis with NG‐nitro‐L‐arginine methyl ester (L‐NAME) but were unaffected by the inactive enantiomer D‐NAME.

These data demonstrate that NO influences the height of the notch in the pulse wave. Heart rate and blood pressure were altered during acetylcholine or L‐NAME infusion, but there were no changes in pulse wave amplitude or velocity, or in the timing of the diastolic peak or dicrotic notch. The slope of the pulse wave between the systolic peak and notch changed substantially. These effects are most convincingly explained by changes in wave reflection, not only from the lower body but also from more proximal sites.

British Journal of Pharmacology (2001) 133, 361–370; doi:10.1038/sj.bjp.0704084
[125I]‐S36057: a new and highly potent radioligand for the melanin‐concentrating hormone receptor
Authors: Audinot Valérie , Lahaye Chantal , Suply Thomas , Beauverger Philippe , Rodriguez Marianne , Galizzi Jean‐Pierre , Fauchère Jean‐Luc , Boutin Jean A
Published: January 2005

Shortened, more stable and weakly hydrophobic analogues of melanin‐concentrating hormone (MCH) were searched as candidates for radioiodination. Starting from the dodecapeptide MCH6 – 17, we found that: (1) substitution of Tyr13 by a Phe residue; (2) addition of a 3‐iodo‐Tyr residue at the N‐terminus; and (3) addition of a hydrophilic spacer 8‐amino‐3,6‐dioxyoctanoyl between the 3‐iodo‐Tyr and MCH6 – 17 (compound S36057), led to an agonist more potent than MCH itself in stimulating [35S]‐GTPγS binding at membranes from HEK293 cells stably expressing the human MCH receptor.

Specific binding of [125I]‐S36057 was found in HEK293 and CHO cell lines stably expressing the human MCH receptor. This radioligand recognized a similar number of binding sites (ca. 800 fmol mg−1) than [125I]‐[3‐iodo Tyr13]‐MCH.

However, the KD for [125I]‐S36057 obtained from saturation studies (0.037 nM) or from binding kinetics (0.046 nM) was at least 10 fold higher to that of [125I]‐[3‐iodo Tyr13]‐MCH (0.46 nM).

Affinities determined for a series of MCH analogues were similar with both radioligands, S36057 being the most potent compound tested (Ki=0.053 nM).

Finally, [125I]‐S36057 also potently labelled the MCH receptor in membranes from whole rat brain (KD 0.044 nM, Bmax=11 fmol mg−1).

In conclusion, [125I]‐S36057 is a more potent and more stable radioligand than [125I]‐[3‐iodo Tyr13]‐MCH that will represent a reliable tool for binding assays in the search of novel MCH ligands. It should also provide great help for autoradiographic studies of the MCH receptor distribution in the central nervous system.

British Journal of Pharmacology (2001) 133, 371–378; doi:10.1038/sj.bjp.0704085
Endothelium‐dependent relaxation induced by cathepsin G in porcine pulmonary arteries
Authors: Glusa Erika , Adam Christine
Published: January 2005

Serine proteinases elicit profound cellular effects in various tissues mediated by activation of proteinase‐activated receptors (PAR). In the present study, we investigated the vascular effects of cathepsin G, a serine proteinase that is present in the azurophil granules of leukocytes and is known to activate several cells that express PARs.

In prostaglandin F2α (3 μM)‐precontracted rings from porcine pulmonary arteries with intact endothelium, cathepsin G caused concentration‐dependent relaxant responses (pEC50=9.64±0.12). The endothelium‐dependent relaxant effect of cathepsin G could also be demonstrated in porcine coronary arteries (pEC50=9.23±0.07). In pulmonary arteries the cathepsin G‐induced relaxation was inhibited after blockade of nitric oxide synthesis by L‐NAME (200 μM) and was absent in endothelium‐denuded vessels. Bradykinin‐ and cathepsin G‐induced relaxant effects were associated with a 5.7 fold and 2.4 fold increase in the concentration of cyclic GMP, respectively.

Compared with thrombin and trypsin, which also produced an endothelium‐dependent relaxation in pulmonary arteries, cathepsin G was 2.5 and four times more potent, respectively. Cathepsin G caused only small homologous desensitization. In cathepsin G‐challenged vessels, thrombin was still able to elicit a relaxant effect.

The effects of cathepsin G were blocked by soybean trypsin inhibitor (IC50=0.043 μg ml−1), suggesting that proteolytic activity is essential for induction of relaxation. Recombinant acetyl‐eglin C proved to be a potent inhibitor (IC50=0.14 μg ml−1) of the cathepsin G effect, whereas neither indomethacin (3 μM) nor the thrombin inhibitor hirudin (5 ATU ml−1) elicited any inhibitory activity. Due to their polyanionic structure defibrotide (IC50=0.11 μg ml−1), heparin (IC50=0.48 μg ml−1) and suramin (IC50=1.85 μg ml−1) diminished significantly the relaxation in response to the basic protein cathepsin G.

In conclusion, like thrombin and trypsin, cathepsin G is able to induce endothelium‐dependent vascular relaxation. It can be released from activated leukocytes at sites of vascular injury and inflammation and, therefore, sufficiently high concentrations might be reached locally in the vascular space to induce vasodilatation.

British Journal of Pharmacology (2001) 133, 422–428; doi:10.1038/sj.bjp.0704089
Vascular actions of MDMA involve α1 and α2‐adrenoceptors in the anaesthetized rat
Authors: McDaid John , Docherty James R
Published: January 2005

We have investigated the effects of methylenedioxymethamphetamine (MDMA, ‘ecstasy’), i.v., on diastolic blood pressure (DBP) in pithed and pentobarbitone anaesthetized rats.

In pithed rats, the non‐selective 5‐HT receptor antagonist methiothepin (0.1 mg kg−1) and the α2‐adrenoceptor antagonists methoxyidazoxan and yohimbine (1 mg kg−1) showed significant α1‐adrenoceptor antagonist potency, but methiothepin did not show α2‐adrenoceptor antagonist potency. MDMA (1 and 5 mg kg−1) produced pressor responses which were significantly reduced by the α1‐adrenoceptor antagonist prazosin (0.1 mg kg−1), yohimbine (1 mg kg−1) or methiothepin (0.1 mg kg−1), but not by the 5‐HT2 receptor antagonist ritanserin (1 mg kg−1).

In anaesthetized rats, antagonists revealed two phases with three components to the effects of MDMA (5 mg kg−1) on DBP: an initial pressor response, a later pressor component at 1 min, the sustained depressor response. Methoxyidazoxan, methiothepin or the combination ritanserin/prazosin significantly reduced the initial pressor response, although neither of the latter compounds alone had any effect.

The pressor response to MDMA (5 mg kg−1) at 1 min was converted to a depressor response by prazosin and to a lesser extent methiothepin and methoxyidazoxan.

The depressor response to MDMA (5 mg kg−1) was significantly reduced by methoxyidazoxan (0.1 mg kg−1), and by the noradrenaline re‐uptake blocker cocaine 10 mg kg−1 but not 1 mg kg−1. However, the most marked reduction in the depressor response was produced by the combination of methoxyidazoxan and cocaine.

It is concluded that the initial pressor response to MDMA (5 mg kg−1) in anaesthetized rats involves α2‐ and possibly α1‐adrenoceptors and 5‐HT2 receptors, the pressor component at 1 min is largely α1‐adrenoceptor mediated, and the sustained depressor response involves α2‐adrenoceptors.

British Journal of Pharmacology (2001) 133, 429–437; doi:10.1038/sj.bjp.0704094
Protection of vascular wall function in insulin‐resistant rats from copper oxidative stress
Authors: O'Brien Sheila F , Davidge Sandra T , Zhang Yunlong , Russell James C
Published: January 2005

The effects of oxidative stress on vascular function in the insulin‐resistant state were assessed in mesenteric resistance arteries of obese, insulin‐resistant (cp/cp) and lean, normal (+/?) JCR : LA‐cp rats.

Nitric oxide‐mediated relaxation of noradrenaline‐contracted arteries in response to acetylcholine was impaired after 2 h of incubation with Cu2+ in both genotypes, with or without the continuing presence of Cu2+. Relaxation was enhanced on initial exposure to Cu2+, and post‐incubation removal of the Cu2+ resulted in a greater impairment of relaxation. Arteries from cp/cp rats were less impaired in function by Cu2+ incubation than were those of +/? controls.

Sodium nitroprusside‐mediated relaxation was impaired by exposure to Cu2+, with an accompanying increase in EC50.

The impairment in acetylcholine‐mediated relaxation in the arteries from both cp/cp and +/? rats was completely inhibited by co‐incubation with copper‐zinc superoxide dismutase and catalase, confirming that the impairment associated with Cu2+ incubation was due to oxidative stress.

The impairment appears to involve both smooth muscle and the endothelium.

The cp/cp rats showed greater resistance to the effects of oxidative stress on arterial function, possibly due to an adaptation to oxidative stress on arterial function associated with the insulin‐resistant state.

British Journal of Pharmacology (2001) 133, 477–484; doi:10.1038/sj.bjp.0704095
The C6‐2B glioma cell P2YAC receptor is pharmacologically and molecularly identical to the platelet P2Y12 receptor
Authors: Jin Jianguo , Tomlinson Wendy , Kirk Ian P , Kim Young B , Humphries Robert G , Kunapuli Satya P
Published: January 2005

P2Y receptor activation in many cell types leads to phospholipase C activation and accumulation of inositol phosphates, while in blood platelets, C6‐2B glioma cells, and in B10 microvascular endothelial cells a P2Y receptor subtype, which couples to inhibition of adenylyl cyclase, historically termed P2YAC, (P2TAC or P2T in platelets) has been identified. Recently, this receptor has been cloned and designated P2Y12 in keeping with current P2 receptor nomenclature.

Three selective P2T receptor antagonists, with a range of affinities, inhibited ADP‐induced aggregation of washed human or rat platelets, in a concentration‐dependent manner, with a rank order of antagonist potency (pIC50, human: rat) of AR‐C78511 (8.5 : 9.1)>AR‐C69581 (6.2 : 6.0)>AR‐C70300 (5.4 : 5.1). However, these compounds had no effect on ADP‐induced platelet shape change.

All three antagonists had no significant effect on the ADP‐induced inositol phosphate formation in 1321N1 astrocytoma cells stably expressing the P2Y1 receptor, when used at concentrations that inhibit platelet aggregation.

These antagonists also blocked ADP‐induced inhibition of adenylyl cyclase in rat platelets and C6‐2B cells with identical rank orders of potency and overlapping concentration – response curves.

RT – PCR and nucleotide sequence analyses revealed that the C6‐2B cells express the P2Y12 mRNA.

These data demonstrate that the P2YAC receptor in C6‐2B cells is pharmacologically identical to the P2TAC receptor in rat platelets.

British Journal of Pharmacology (2001) 133, 521–528; doi:10.1038/sj.bjp.0704114
Disturbance of the prejunctional modulation of cholinergic neurotransmission during chronic granulomatous inflammation of the mouse ileum
Authors: De Man Joris G , Moreels Tom G , De Winter Benedicte Y , Bogers Johannes J , Van Marck Eric A , Herman Arnold G , Pelckmans Paul A
Published: January 2005

The effect of chronic granulomatous inflammation of the intestine was studied on the prejunctional modulation of cholinergic nerve activity in the mouse ileum.

Contractions to carbachol (0.01–0.3 μM) and to electrical field stimulation (EFS, 0.25–8 Hz) of enteric neurons were higher in inflamed ileum as compared to control ileum. However, when the neurally‐mediated contractions to EFS were expressed as percentage of the direct smooth muscle contraction to carbachol, the responses to EFS were similar in control and inflamed ileum.

Atropine (1 μM) abolished all contractions to EFS and carbachol in control and inflamed ileum. DMPP (3–30 μM), a nicotinic receptor agonist, induced concentration‐dependent contractions that were more pronounced in inflamed ileum as compared to control ileum. Hexamethonium (100 μM), a nicotinic receptor blocker, significantly inhibited the contractions to EFS in inflamed ileum but not in control ileum.

In control ileum, histamine (10–100 μM) and the histamine H1 receptor agonist HTMT (3–10 μM) inhibited the contractions to EFS concentration‐dependently without affecting the contractions to carbachol. The inhibitory effect of histamine and HTMT was prevented by the histamine H1 antagonist mepyramine (5–10 μM) but not by the H2‐ and H3‐receptor antagonists cimetidine and thioperamide (both 10 μM). In chronically inflamed ileum however, histamine (10–100 μM) and HTMT (3–10 μM) failed to inhibit the contractions to EFS. The histamine H2 and H3 receptor agonists dimaprit and R(−)‐α‐methylhistamine did not affect the contractions to EFS in control and inflamed ileum.

The α2‐receptor agonist UK 14.304 (0.01–0.1 μM) inhibited the contractions to EFS in control and inflamed ileum without affecting the contractions to carbachol. The effect of UK 14.304 was reversed by the α2‐receptor antagonist yohimbine (1 μM). The inhibitory effect of UK 14.304 on contractions to EFS was of similar potency in control and inflamed ileum.

Our results suggest that the prejunctional modulation of cholinergic nerve activity by nicotinic and histaminic H1 receptors is disturbed during chronic intestinal inflammation whereas the modulation by α2‐receptors is preserved. Such a disturbance of cholinergic nerve activity may contribute to the motility disturbances that are often observed during chronic intestinal diseases in humans.

British Journal of Pharmacology (2001) 133, 695–707; doi:10.1038/sj.bjp.0704115
Physiological antagonism of endothelin‐1 in human conductance and resistance coronary artery
Authors: Wiley Katherine E , Davenport Anthony P
Published: January 2005

The ability of four endogenous vasodilators, nitric oxide (NO; 0.01 – 30 μM), atrial (ANP), brain (BNP) and C‐type (CNP) natriuretic peptide (0.1 – 300 nM), to reverse endothelin‐1 (ET‐1; 10 nM) constrictions in human resistance and conductance coronary arteries (CA) in vitro was investigated.

ET‐1 (0.1 – 300 nM) constricted resistance CA more potently than conductance CA (P<0.05; EC50 values 2.98 nM (95% CI: 1.49 – 5.95 nM and 8.58 (4.72 – 15.6 nM) respectively)).

The NO‐donor diethylamine NONOate fully reversed the ET‐1 constriction in conductance CA (EMAX 127±9.16%), however only partial reversal was observed in resistance CA (EMAX 78.8±8.13; P<0.05). The soluble guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (100 μM) reduced the maximum response to diethylamine NONOate to 76.9±14.4% in conductance CA (P<0.05), but had no effect on resistance CA (EMAX 77.2±18.4%).

There was no difference between responses to ANP in conductance and resistance CA (EC50 values 4.25 nM (0.84 – 21.4 nM) and 18.4 nM (2.92 – 116 nM), EMAX 53.1±14.7% and 48.6±11.8% respectively).

BNP was a more potent vasodilator of conductance than resistance CA. In conductance CA the mean EC50 value was 2.4 nM (0.74 – 7.75 nM), EMAX 54.5±14.9%. Concentration‐response curves to BNP were incomplete in resistance CA.

Concentration‐response curves to CNP were incomplete in both conductance and resistance CA.

The greater potency of ET‐1 in resistance vessels may exacerbate the effects of increased circulating levels of the peptide in disease. Only NO could fully reverse ET‐1 mediated constrictions in conductance CA, and none of the dilators tested could completely counteract constrictions in resistance CA.

British Journal of Pharmacology (2001) 133, 568–574; doi:10.1038/sj.bjp.0704119
In vivo pharmacology of BIIR 561 CL, a novel combined antagonist of AMPA receptors and voltage‐dependent Na+ channels
Authors: Wienrich M , Brenner M , Löscher W , Palluk R , Pieper M , Potschka H , Weiser T
Published: January 2005

Glutamate receptors of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) subtype and voltage‐gated Na+ channels are associated with diseases of the central nervous system characterized by neuronal over‐excitation as in epilepsy or cerebral ischaemia. In animal models, AMPA receptor antagonists and Na+ channel blockers provide protection in these conditions.

Dimethyl‐{2‐[2‐(3‐phenyl‐[1,2,4]oxadiazol‐5‐yl)‐phenoxyl]‐ethyl}‐amine hydrochloride (BIIR 561 CL) combines both, AMPA receptor – and Na+ channel blocking properties in one molecule. Here, BIIR 561 CL was investigated in vivo.

BIIR 561 CL protected mice against AMPA‐induced toxicity with an ED50 value of 4.5 mg kg−1 following subcutaneous (s.c.) administration. A 0.1% solution of BIIR 561 CL provided local anaesthesia in the corneal reflex test in rabbits. In mice, the compound prevented tonic seizures in the maximal electroshock (MES) model with an ED50 value of 3.0 mg kg−1 s.c. In amygdala‐kindled rats, BIIR 561 CL inhibited seizures at doses of 3 and 11 mg kg−1 following intraperitoneal (i.p.) injection.

The data show that the combination of blocking AMPA receptor‐ and voltage‐gated Na+ channels in one molecule induces effective protection in animal models of neuronal over‐excitation.

British Journal of Pharmacology (2001) 133, 789–796; doi:10.1038/sj.bjp.0704132
Role of cyclic nucleotides in vasodilations of the rat thoracic aorta induced by adenosine analogues
Authors: Hourani Susanna M O , Boon Katherine , Fooks Helen M , Prentice Deborah J
Published: January 2005

Although adenosine analogues such as 5′‐N‐ethylcarboxamidoadenosine (NECA) relax the rat thoracic aorta in a partially endothelium‐dependent manner via adenosine A2A receptors, others such as N6‐R‐phenylisopropyladenosine (R‐PIA) act via an endothelium‐independent, antagonist‐insensitive mechanism. The role of cyclic nucleotides in these relaxations was investigated in isolated aortic rings using inhibitors of adenylate and guanylate cyclases as well as subtype‐selective phosphodiesterase inhibitors.

The adenylate cyclase inhibitor 9‐(tetrahydro‐2‐furanyl)‐9H‐purin‐6‐amine (SQ 22536; 100 μM) significantly inhibited responses to NECA, but not responses to R‐PIA. The type IV (cyclic AMP‐selective) phosphodiesterase inhibitor 4‐[(3‐butoxy‐4‐methoxyphenyl)methyl]‐2‐imidazolidinone (RO 20‐1724; 30 μM) significantly enhanced responses to NECA and to a lesser extent those to R‐PIA.

The guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3a]quinoxalin‐1‐one (ODQ; 100 μM) significantly inhibited responses to NECA and acetylcholine but not responses to R‐PIA. The selective phosphodiesterase V (cyclic GMP‐selective) inhibitors, zaprinast (10 μM) and 4‐{[3′,4′‐(methylenedioxy)benzyl]amino}‐6‐methoxyquinazoline (MMQ; 1 μM), had no significant effect on responses to either NECA or R‐PIA, but enhanced responses to acetylcholine.

These results are consistent with the effects of NECA being via activation of endothelial receptors to release NO which stimulates guanylate cyclase, as well as smooth muscle receptors coupled to stimulation of adenylate cyclase. The lack of effect of zaprinast and MMQ on responses to NECA are likely to be due to simultaneous activation of both adenylate and guanylate cyclases in the smooth muscle, as cyclic AMP reduces the sensitivity of phosphodiesterase V to inhibitors. These results also suggest that the effects of R‐PIA are via neither of these mechanisms.

British Journal of Pharmacology (2001) 133, 833–840; doi:10.1038/sj.bjp.0704140
Pharmacological evidence for the activation of potassium channels as the mechanism involved in the hypotensive and vasorelaxant effect of dioclein in rat small resistance arteries
Authors: Côrtes Steyner F , Rezende Bruno A , Corriu Catherine , Medeiros Isac A , Teixeira Mauro M , Lopes Miguel José , Lemos Virgínia S
Published: January 2005

The hypotensive and vasorelaxant effect of dioclein in resistance mesenteric arteries was studied in intact animals and isolated vessels, respectively.

In intact animals, initial bolus administration of dioclein (2.5 mg kg−1) produced transient hypotension accompanied by an increase in heart rate. Subsequent doses of dioclein (5 and 10 mg kg−1) produced hypotensive responses with no significant change in heart rate. NG‐nitro‐L‐arginine methyl ester (L‐NAME) did not affect the hypotensive response.

In endothelium‐containing or ‐denuded vessels pre‐contracted with phenylephrine, dioclein (5 and 10 mg kg−1 produced a concentration‐dependent vasorelaxation (IC50=0.3±0.06 and 1.6±0.6 μM, respectively) which was not changed by 10 μM indomethacin. L‐NAME (300 μM) produced a shift to the right.

Dioclein was without effect on contraction of vessels induced by physiological salt solution (PSS) containing 50 mM KCl and the concentration dependence of dioclein's effect on phenylephrine induced contraction was shifted to the right in vessels bathed in PSS containing 25 mM KCl.

Tetraethylammonium (10 mM) and BaCl2 (1 mM) increased the IC50 for dioclein‐induced vasorelaxation without affecting the maximal response (Emax). Charybdotoxin (100 nM), 4‐aminopyridine (1 mM) and iberiotoxin (100 nM) increased the IC50 and reduced the Emax. Apamin (1 μM) reduced the Emax without affecting the IC50.

Dioclein produced a hyperpolarization in smooth muscle of mesenteric arteries with or without endothelium (7.7±1.4 mV and 12.3±3.6 mV, respectively).

In conclusion dioclein lowered arterial pressure probably through a decrease in peripheral vascular resistance. The underling mechanism implicated in the vasorelaxant effect of dioclein appears to be the opening of KCa and Kv channels and subsequent membrane hyperpolarization.

British Journal of Pharmacology (2001) 133, 849–858; doi:10.1038/sj.bjp.0704147
Molecular cloning, pharmacological properties and tissue distribution of the porcine 5‐HT1B receptor
Authors: Bhalla Pankaj , Sharma Hari S , Ma Xiaoqian , Wurch Thierry , Pauwels Petrus J , Saxena Pramod R
Published: January 2005

Using a combination of RT – PCR and inverse‐PCR techniques, we amplified, cloned and sequenced a full‐length porcine 5‐HT1B receptor cDNA derived from porcine cerebral cortex. Sequence analysis revealed 1170 bp encoding an open reading frame of 390 amino acids showing a 95% similarity with the human 5‐HT1B receptor.

The recombinant porcine 5‐HT1B cDNA was expressed in monkey Cos‐7 cells and its pharmacological profile was determined by radioligand binding assay using [3H]‐GR125743. The affinities of several agonists (L694247>ergotamine 5‐carboxamidotryptamine=dihydroergotamine=5‐HT>CP122638=zolmitriptan>sumatriptan) and putative antagonists (GR127935>methiothepin>SB224289>>ritanserin>ketanserin BRL15572) correlated highly with those described for the recombinant human 5‐HT1B receptor.

In membranes obtained from cells co‐expressing the porcine 5‐HT1B receptor and a mutant GαoCys351Ile protein, 5‐HT and zolmitriptan increased, while the 5‐HT1B receptor antagonist SB224289 decreased basal [35S]‐GTPγS binding, thus showing inverse agonism. The potency of zolmitriptan in the [35S]‐GTPγS binding assay (pEC50: 7.64±0.04) agreed with its affinity in displacing the antagonist [3H]‐GR125743 (pKi: 7.36±0.07).

The 5‐HT1B receptor mRNA was observed by RT‐PCR in several blood vessels, cerebral cortex, cerebellum and trigeminal ganglion. In situ hybridization performed in frontal cerebral cortex sections revealed the expression of 5‐HT1B receptor mRNA in pyramidal cells.

In conclusion, we have cloned and established the amino acid sequence, ligand binding profile and location of the porcine 5‐HT1B receptor. This information may be useful in exploring the role of 5‐HT1B receptor in pathophysiological processes relevant for novel drug discovery in diseases such as migraine.

British Journal of Pharmacology (2001) 133, 891–901; doi:10.1038/sj.bjp.0704150
Angiogenesis gene therapy to rescue ischaemic tissues: achievements and future directions
Authors: Emanueli Costanza , Madeddu Paolo
Published: January 2005

Ischaemic diseases are characterized by an impaired supply of blood resulting from narrowed or blocked arteries that starve tissues of needed nutrients and oxygen. Coronary‐atherosclerosis induced myocardial infarction is one of the leading causes of mortality in developed countries. Ischaemic disease also affects the lower extremities. Considerable advances in both surgical bypassing and percutaneous revascularization techniques have been reached. However, many patients cannot benefit from these therapies because of the extension of arterial occlusion and/or microcirculation impairment. Consequently, the need for alternative therapeutic strategies is compelling. An innovative approach consists of stimulating collateral vessel growth, a natural host defence response that intervenes upon occurrence of critical reduction in tissue perfusion (Isner & Asahara, 1999). This review will debate the relevance of therapeutic angiogenesis for promotion of tissue repair. The following issues will receive attention: (a) vascular growth patterns, (b) delivery systems for angiogenesis gene transfer, (c) achievements of therapeutic angiogenesis in myocardial and peripheral ischaemia, and (d) future directions to improve effectiveness and safety of vascular gene therapy.

British Journal of Pharmacology (2001) 133, 951–958; doi:10.1038/sj.bjp.0704155
Absence of ligand‐induced regulation of kinin receptor expression in the rabbit
Authors: Sabourin Thierry , Guay Katline , Houle Steeve , Bouthillier Johanne , Bachvarov Dimcho R , Adam Albert , Marceau François
Published: January 2005

The induction of B1 receptors (B1Rs) and desensitization or down‐regulation of B2 receptors (B2Rs) as a consequence of the production of endogenous kinins has been termed the autoregulation hypothesis. The latter was investigated using two models based on the rabbit: kinin stimulation of cultured vascular smooth muscle cells (SMCs) and in vivo contact system activation (dextran sulphate intravenous injection, 2 mg kg−1, 5 h).

Rabbit aortic SMCs express a baseline population of B1Rs that was up‐regulated upon interleukin‐1β treatment ([3H]‐Lys‐des‐Arg9‐BK binding or mRNA concentration evaluated by RT–PCR; 4 or 3 h, respectively). Treatment with B1R or B2R agonists failed to alter B1R expression under the same conditions.

Despite consuming endogenous kininogen (assessed using the kinetics of immunoreactive kinin formation in the plasma exposed to glass beads ex vivo) and producing hypotension mediated by B2Rs in anaesthetized rabbits, dextran sulphate treatment failed to induce B1Rs in conscious animals (RT–PCR in several organs, aortic contractility). By contrast, lipopolysaccharide (LPS, 50 μg kg−1, 5 h) was an effective B1R inducer (kidney, duodenum, aorta) but did not reduce kininogen reserve.

We tested the alternate hypothesis that endogenous kinin participate in LPS induction of B1Rs. Kinin receptor antagonists (icatibant combined to B‐9858, 50 μg kg−1 of each) failed to prevent or reduce the effect of LPS on B1R expression. Dextran sulphate or LPS treatments did not persistently down‐regulate vascular B2Rs (jugular vein contractility assessed ex vivo).

The kinin receptor autoregulation hypothesis is not applicable to primary cell cultures derived from a tissue known to express B1Rs in a regulated manner (aorta). The activation of the endogenous kallikrein‐kinin system is ineffective to induce B1Rs in vivo in an experimental time frame sufficient for B1R induction by LPS.

British Journal of Pharmacology (2001) 133, 1154–1162; doi:10.1038/sj.bjp.0704158
Vasorelaxant effect of nitric oxide releasing steroidal and nonsteroidal anti‐inflammatory drugs
Authors: Keeble J , Al‐Swayeh O A , Moore P K
Published: January 2005

The effect of several nitric oxide releasing‐non‐steroidal anti‐inflammatory drugs (NO‐NSAID) and nitroprednisolone on blood vessel relaxation in vitro and in vivo was studied. Nitroflurbiprofen (NOF; EC50, 688.8±93.8 μM), nitroaspirin (NOA; EC50, 57.9±6.5 μM), nitroparacetamol (NOPARA; EC50, 71.5±14.6 μM) and nitroprednisolone (EC50, 15.1±1.4 μM) caused concentration‐related relaxation of noradrenaline (NA)‐contracted rat aortic rings. All NO releasing compounds tested were approximately three orders of magnitude less potent than sodium nitroprusside (SNP, EC50, 35.7±3.5 nM).

The vasorelaxant effect of NOF and NOPARA in the rat aorta was potentiated by zaprinast (5 μM) and reduced by ODQ (5 μM). Flurbiprofen and paracetamol (100 μM) caused minimal (<10%) relaxation of the rat aorta and did not affect the response to SNP. The effect of NOF was unchanged in the presence of L‐NAME (100 μM; EC30, 181.8±35.1 μM cf. EC30, 125.1±17.0 μM, P>0.05) but increased by removal of the endothelium (EC30, 164.3±26.3 μM cf. EC50, 688.8±93.8 μM, P<0.05).

NOF (0.1–50 μM) produced a small but not concentration‐related vasodilation of the NA‐preconstricted (i.e. ‘high tone’) perfused rat mesentery preparation (cf. SNP, EC30, 4.4±0.7 μM). In contrast, NOF (1–100 μM) produced concentration‐related vasodilation of the ‘high tone’ perfused rat kidney with an EC50 of 33.1±4.4 μM.

Neither NOF (74 mg kg−1, i.p.) nor NOA (91.9 mg kg−1, i.p.) nor equimolar doses of flurbiprofen (50 mg kg−1, i.p.) or aspirin (50 mg kg−1, i.p.) affected mean arterial blood pressure (MAP) or heart rate (HR) of pentobarbitone‐anaesthetized rats over a 1 h period.

NO‐NSAID relax blood vessels in vitro by an NO‐dependent mechanism. The absolute vasorelaxant effect of NO releasing drug varies greatly with the choice of compound and between blood vessel preparations.

British Journal of Pharmacology (2001) 133, 1023–1028; doi:10.1038/sj.bjp.0704161
A comparison of effects measured with isotonic and isometric recording: I. Concentration‐effect curves for agonists
Authors: Barlow R B , Bond Susan M , Grant Claire , McQueen D S , Yaqoob Zeenat
Published: January 2005

Concentration‐effect curves were obtained with carbachol tested on isolated preparations of guinea‐pig ileum taken from adjacent sites in the same animal, one recorded isotonically, the other isometrically: similar experiments were made with histamine as agonist and with carbachol on rat uterus (in oestrus).

The position and steepness of the curves was expressed as the values of [EC50] and the exponent, P: with carbachol or histamine on guinea‐pig ileum the curves were significantly steeper with isotonic recording (P<0.02, sign test) and displaced towards lower concentrations (P<0.005) but there were significant correlations (P<0.05) between values obtained with tissues from the same animal.

The curves for carbachol on the rat uterus were very steep: with isotonic recording the exponent (often eight or more) was consistently higher than with isometric (P<0.001): there was no significant displacement but there was a significant correlation (P<0.05) between values of [EC50] obtained with tissues from the same animal.

Although the results obtained by the two methods are different, they are correlated.

These effects are to be expected because with isotonic recording there can be no change in length until the tension exceeds the load and the tissue bulk sets an upper limit to shortening: the range within which an effect can be measured (the ‘operational window’) is smaller. The observed effects on [EC50] and P have been reproduced with theoretical data.

British Journal of Pharmacology (2001) 133, 1081–1086; doi:10.1038/sj.bjp.0704168
Functional characterization of coronary vascular adenosine receptors in the mouse
Authors: Flood Amanda , Headrick John P
Published: January 2005

Coronary responses to adenosine agonists were assessed in perfused mouse and rat hearts. The roles of nitric oxide (NO) and ATP‐dependent K+ channels (KATP) were studied in the mouse.

Resting coronary resistance was lower in mouse vs rat, as was minimal resistance (2.2±0.1 vs 3.8±0.2 mmHg ml−1 min−1 g−1). Peak hyperaemic flow after 20–60 s occlusion was greater in mouse.

Adenosine agonists induced coronary dilation in mouse, with pEC50s of 9.4±0.1 for 2‐[p‐(2‐carboxyethyl)phenethylamino]‐5′‐N‐ethyl carboxamidoadenosine (CGS21680, A2A‐selective agonist), 9.3±0.1 for 5′‐N‐ethylcarboxamidoadenosine (NECA, A1/A2 agonist), 8.4±0.1 for 2‐chloroadenosine (A1/A2 agonist), 7.7±0.1 for N6‐(R)‐(phenylisopropyl)adenosine (R‐PIA, A1/A2B selective), and 6.8±0.2 for adenosine. The potency order (CGS21680=NECA>2‐chloroadenosine>R‐PIA>adenosine) supports A2A adenosine receptor‐mediated dilation in mouse coronary vessels. 0.2–2 μM of the A2B‐selective antagonist alloxazine failed to alter CGS21680 or 2‐chloroadenosine responses.

pEC50s in rat were 6.7±0.2 for CGS21680, 7.3±0.1 for NECA, 7.6±0.1 for 2‐chloroadenosine, 7.2±0.1 for R‐PIA, and 6.2±0.1 for adenosine (2‐chloroadenosine>NECA=R‐PIA>CGS21680> adenosine), supporting an A2B adenosine receptor response.

NO‐synthase antagonism with 50 μM NG‐nitro L‐arginine (L‐NOARG) increased resistance by ∼25%, and inhibited responses to CGS21680 (pEC50=9.0±0.1), 2‐chloroadenosine (pEC50=7.3±0.2) and endothelial‐dependent ADP, but not sodium nitroprusside (SNP). KATP channel blockade with 5 μM glibenclamide increased resistance by ∼80% and inhibited responses to CGS21680 in control (pEC50=8.3±0.1) and L‐NOARG‐treated hearts (pEC50=7.3±0.1), and to 2‐chloroadenosine in control (pEC50=6.7±0.1) and L‐NOARG‐treated hearts (pEC50=5.9±0.2).

In summary, mouse coronary vessels are more sensitive to adenosine than rat vessels. A2A adenosine receptors mediate dilation in mouse coronary vessels vs A2B receptors in rat. Responses in the mouse involve a sensitive NO‐dependent response and KATP‐dependent dilation.

British Journal of Pharmacology (2001) 133, 1063–1072; doi:10.1038/sj.bjp.0704170
Vascular pharmacology of BIIE0246, the first selective non‐peptide neuropeptide Y Y2 receptor antagonist, in vivo
Authors: Malmström Rickard E
Published: January 2005

BIIE0246, a recently introduced non‐peptide neuropeptide Y (NPY) Y2 receptor antagonist, was pharmacologically characterized in vivo, on vascular responses evoked in the anaesthetized pig.

The NPY Y2 receptor agonist N‐acetyl[Leu28Leu31]NPY(24‐36) evoked dose‐dependent vasoconstriction in spleen. These vascular responses were potently and dose‐dependently antagonized by BIIE0246. Significant inhibition was seen already at 1 nmol kg−1, whereas at 100 nmol kg−1 of BIIE0246 these responses were completely abolished. The ID50 value for this antagonism was 2.1 nmol kg−1.

Peptide YY (PYY) evoked dose‐dependent vasoconstriction in both kidney and spleen, vascular responses mediated by the NPY Y1 receptor and both NPY Y1 and Y2 receptors, respectively. Only the splenic response was inhibited by BIIE0246, the effect of which reached significance at 1 nmol kg−1. Already 30 min after the last dose of BIIE0246 there was a significant recovery of the PYY‐evoked splenic vasoconstriction, and a further 60 min later, this response was no longer significantly inhibited compared to control.

BIIE0246 (100 nmol kg−1) did not affect renal and splenic vasoconstrictor responses either to the NPY Y1 receptor agonist [Leu31Pro34]NPY, the α1‐adrenoceptor agonist phenylephrine, the P2X1‐purinoceptor agonist α,β‐methylene ATP or angiotensin II, demonstrating both selectivity and specificity for the NPY Y2 receptor in vivo.

It is concluded that BIIE0246 is a highly potent and selective NPY Y2 receptor antagonist, albeit with rather short duration of action, in vivo. BIIE0246 thus represents the first interesting tool for studies on NPY Y2 receptor‐mediated transmission in vivo.

British Journal of Pharmacology (2001) 133, 1073–1080; doi:10.1038/sj.bjp.0704171
2,5‐Di‐t‐butyl‐1,4‐benzohydroquinone (BHQ) inhibits vascular L‐type Ca2+channelviasuperoxide anion generation
Authors: Fusi Fabio , Saponara Simona , Gagov Hristo , Sgaragli Giampietro
Published: January 2005

The aim of the present study was to investigate the effects of 2,5‐di‐t‐butyl‐1,4‐benzohydroquinone (BHQ), an inhibitor of the sarco‐endoplasmic reticulum Ca2+‐ATPase (SERCA), on the whole‐cell voltage‐dependent L‐type Ca2+current (ICa(L)) of freshly isolated smooth muscle cells from the rat tail artery using the patch‐clamp technique.

BHQ, added to the perfusion solution, reduced ICa(L)in a concentration‐ (IC50=66.7 μM) and voltage‐dependent manner. This inhibition was only partially reversible.

BHQ shifted the voltage dependence of the steady‐state inactivation curve to more negative potentials by 7 mV in the mid‐potential of the curve, without affecting the activation curve as well as the time course of ICa(L)inactivation.

Preincubation of the cells either with 10 μMcyclopiazonic acid, a SERCA inhibitor, or with 3 mMdiethyldithiocarbamate, an inhibitor of intracellular superoxide dismutase (SOD), did not modify BHQ inhibition of ICa(L). On the contrary, this effect was no longer evident when SOD (250 u ml−1) was added to the perfusion medium.

Either in the presence or in the absence of cells, BHQ gave rise to superoxide anion formation, which was markedly inhibited by the addition of SOD.

These results indicate that, at micromolar concentrations, BHQ inhibits vascular ICa(L)by giving rise to the formation of superoxide anion which in turn impairs the channel function.

British Journal of Pharmacology(2001)133, 988–996; doi:10.1038/sj.bjp.0704183