bluuebunny/crossref_metadata_2025 huggingface dataset:
Adenosine receptor expression and function in rat striatal cholinergic interneurons
Authors: Preston Zoe , Lee Kevin , Widdowson Leon , Freeman Tom C , Dixon Alistair K , Richardson Peter J
Published: January 2005

Cholinergic neurons were identified in rat striatal slices by their size, membrane properties, sensitivity to the NK1 receptor agonist (Sar9, Met(O2)11) Substance P, and expression of choline acetyltransferase mRNA. A1 receptor mRNA was detected in 60% of the neurons analysed, and A2A receptor mRNA in 67% (n=15).

The A1 receptor agonist R‐N6‐(2‐phenylisopropyl)adenosine (R‐PIA) hyperpolarized cholinergic neurons in a concentration dependent manner sensitive to the A1 antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, 100 nM).

In dual stimulus experiments, the A2A receptor antagonist 8‐(3‐chlorostyryl)caffeine (CSC, 500 nM) decreased release of [3H]‐acetylcholine from striatal slices (S2/S1 0.78±0.07 versus 0.95±0.05 in control), as did adenosine deaminase (S2/S1 ratio 0.69±0.05), whereas the A1 receptor antagonist DPCPX (100 nM) had no effect (S2/S1 1.05±0.14).

In the presence of adenosine deaminase the adenosine A2A receptor agonist 2‐p‐((carboxyethyl)phenylethylamino)‐5′‐N‐ethylcarboxamidoadenosine (CGS21680, 10 nM) increased release (S2/S1 ratio 1.03±0.05 versus 0.88±0.05 in control), an effect blocked by the antagonist CSC (500 nM, S2/S1 0.68±0.05, versus 0.73±0.08 with CSC alone). The combined superfusion of bicuculline (10 μM), saclofen (1 μM) and naloxone (10 μM) had no effect on the stimulation by CGS21680 (S2/S1 ratio 0.99±0.04).

The A1 receptor agonist R‐PIA (100 nM) inhibited the release of [3H]‐acetylcholine (S2/S1 ratio 0.70±0.03), an effect blocked by DPCPX (S2/S1 ratio 1.06±0.07).

It is concluded that both A1 and A2A receptors are expressed on striatal cholinergic neurons where they are functionally active.

British Journal of Pharmacology (2000) 130, 886–890; doi:10.1038/sj.bjp.0703366
Increased defecation during stress or after 5‐hydroxytryptophan: selective inhibition by the 5‐HT4 receptor antagonist, SB‐207266
Authors: Sanger G J , Yoshida M , Yahyah M , Kitazumi K
Published: January 2005

5‐HT4 receptor antagonism pr2events the ability of exogenous 5‐HT or 5‐HTP to sensitize the intestinal peristaltic reflex and increase the rate of defecation, generally without affecting non‐stimulated intestinal function. In this study we confirmed the ability of the selective 5‐HT4 receptor antagonist SB‐207266 1–1000 μg kg−1 p.o., to prevent the increase in defecation evoked over a 60 min period by 5‐HTP 10 mg kg−1 s.c. in conscious mice, in the absence of an apparent constipating action.

The role of endogenous 5‐HT in the mechanisms of increased defecation and/or diarrhoea was then investigated in conscious, fed rats. This was evoked by 180 min exposure to restraint stress, which increased both the number and mean weight of formed, faecal pellets excreted over the entire time period.

SB‐207266 1–1000 μg kg−1 p.o. (dosed 30 min before restraint) did not affect the increase in defecation evoked during the first 60 min of restraint stress, but significantly and dose‐dependently reduced or prevented the increased defecation during the remaining 120 min of the experiment; this action occurred in the absence of an apparent constipating action of SB‐207266.

In fasted rats exposed to restraint stress, watery diarrhoea developed and although there was a tendency for SB‐207266 1–1000 μg kg−1 p.o. (dosed 30 min before restraint) to reduce the incidence of diarrhoea, this inhibition was not complete.

We conclude that selective 5‐HT4 receptor antagonism prevents disruptions in defecation behaviours caused by exogenous or endogenous enteric 5‐HT and that this activity is not accompanied by a concomitant suppression of activity (constipation‐like) within the intestine itself.

British Journal of Pharmacology (2000) 130, 706–712; doi:10.1038/sj.bjp.0703367
Antagonist effects on human P2X7 receptor‐mediated cellular accumulation of YO‐PRO‐1
Authors: Michel A D , Kaur R , Chessell I P , Humphrey P P A
Published: January 2005

We have examined the interaction of P2 antagonists with the human P2X7 receptor by studying their effect on 2′ and 3′‐O‐benzoyl‐benzoyl‐ATP (DbATP) stimulated cellular accumulation of the fluorescent, DNA binding dye, YO‐PRO‐1 (MW=375Da).

In suspensions of HEK293 cells expressing human recombinant P2X7 receptors, DbATP produced time and concentration‐dependent increases in YO‐PRO‐1 fluorescence. This response presumably reflects YO‐PRO‐1 entry through P2X7 receptor channels and binding to nucleic acids. When studies were performed in a NaCl‐free, sucrose‐containing buffer, full concentration‐effect curves to DbATP could be constructed.

The P2 antagonists, pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) and periodate oxidized ATP (oATP), reduced the potency of DbATP and decreased its maximum response. 1‐[N,O‐bis(1,5‐isoquinolinesulphonyl)‐N‐methyl‐L‐tyrosyl]‐4‐phenylpiperazine (KN62) and its analogue, KN04, reduced the potency of DbATP. Schild slopes for KN62 and KN04 were shallow and exhibited a plateau at concentrations of compound greater than 1 μM, indicating that these compounds were not competitive antagonists.

Calmidazolium and a monoclonal antibody to human P2X7 receptors attenuated DbATP‐stimulated YO‐PRO‐1 accumulation but they were not competitive antagonists and only produced 2–3 fold decreases in the potency of DbATP.

The effects of PPADS and KN62 were partially reversible whereas those of oATP were not. PPADS protected cells against the irreversible antagonist effects of oATP suggesting a common site of action. In contrast KN62 was not effective suggesting that it may bind at a different site to oATP and PPADS.

This study has demonstrated that P2X7 receptor function can be quantified by measuring DbATP stimulated YO‐PRO‐1 accumulation and has provided additional information about the interaction of P2 receptor antagonists with the human P2X7 receptor.

British Journal of Pharmacology (2000) 130, 513–520; doi:10.1038/sj.bjp.0703368
Betaxolol, a β1‐adrenoceptor antagonist, reduces Na+ influx into cortical synaptosomes by direct interaction with Na+ channels: comparison with other β‐adrenoceptor antagonists
Authors: Chidlow Glyn , Melena José , Osborne Neville N
Published: January 2005

Betaxolol, a β1‐adrenoceptor antagonist used for the treatment of glaucoma, is known to be neuroprotective in paradigms of ischaemia/excitotoxicity. In this study, we examined whether betaxolol and other β‐adrenoceptor antagonists interact directly with neurotoxin binding to sites 1 and 2 of the voltage‐sensitive sodium channel (Na+ channel) in rat cerebrocortical synaptosomes.

Betaxolol inhibited specific [3H]‐batrachotoxinin‐A 20‐α‐benzoate ([3H]‐BTX‐B) binding to neurotoxin site 2 in a concentration‐dependent manner with an IC50 value of 9.8 μM. Comparison of all the β‐adrenoceptor antagonists tested revealed a potency order of propranolol>betaxolol∼levobetaxolol>levobunolol∼carteololtimolol>atenolol.

None of the drugs caused a significant inhibition of [3H]‐saxitoxin binding to neurotoxin receptor site 1, even at concentrations as high as 250 μM.

Saturation experiments showed that betaxolol increased the KD of [3H]‐BTX‐B binding but had no effect on the Bmax. The association kinetics of [3H]‐BTX‐B were unaffected by betaxolol, but the drug significantly accelerated the dissociation rate of the radioligand. These findings argue for a competitive, indirect, allosteric mode of inhibition of [3H]‐BTX‐B binding by betaxolol.

Betaxolol inhibited veratridine‐stimulated Na+ influx in rat cortical synaptosomes with an IC50 value of 28.3 μM. Carteolol, levobunolol, timolol and atenolol were significantly less effective than betaxolol at reducing veratridine‐evoked Na+ influx.

The ability of betaxolol to interact with neurotoxin site 2 of the Na+ channel and inhibit Na+ influx may have a role in its neuroprotective action in paradigms of excitotoxicity/ischaemia and in its therapeutic effect in glaucoma.

British Journal of Pharmacology (2000) 130, 759–766; doi:10.1038/sj.bjp.0703369
Regulation of human D1, D2(long), D2(short), D3and D4dopamine receptors by amiloride and amiloride analogues
Authors: Hoare S R J , Coldwell M C , Armstrong D , Strange P G
Published: January 2005

The modulatory effects of the allosteric effectors methylisobutylamiloride (MIA), benzamil and amiloride have been examined at human D1, D2, D3and D4dopamine receptors. The subtype selectivity and the mechanism of action of this allosteric regulation was examined.

In radioligand dissociation experiments each modulator accelerated dissociation from all four receptor subtypes indicating allosteric regulation. MIA displayed selectivity for the D3subtype for acceleration of radioligand dissociation.

In equilibrium binding (pseudo‐competition) experiments the three compounds inhibited radioligand binding at the four receptor subtypes. Inhibition curves for D1, D2(short), D2(long)and D3receptors were described by Hill coefficients exceeding unity and data were fitted best by a model that assumes binding of modulator to both the primary and allosteric binding sites of the receptor (the allosteric/competitive model).

At the D4subtype, Hill coefficients of unity described the binding data for amiloride and benzamil, consistent with competitive inhibition. The Hill coefficient for MIA at the D4subtype was less than unity and data could be fitted well by the allosteric/competitive model, but it was not possible to define unambiguously the modulatory mechanism. For this effect a better definition of the mechanism could be obtained by simultaneous analysis of data obtained in the presence of a range of concentrations of a purely competitive ligand.

MIA reduced the potency with which dopamine stimulated [35S]‐GTPγS binding at the D2receptor. The effects of MIA could be described by the allosteric/competitive model with effects of MIA to inhibit the binding of dopamine but not its ability to induce a response.

British Journal of Pharmacology(2000)130, 1045–1059; doi:10.1038/sj.bjp.0703370
Citrulline does not relax isolated rat and rabbit vessels
Authors: Marx Stephen , Vedernikov Yuri , Saade George R , Garfield Robert E
Published: January 2005

The study was prompted by the report of Ruiz E. & Tejerina T., 1998 describing endothelium‐independent relaxation by L‐citrulline via activation of particulate guanylate cyclase.

We compared the effects of L‐citrulline and L‐arginine in isolated aortic rings of rats and in isolated aortic, carotid and femoral artery rings of rabbits.

No significant relaxation to either L‐citrulline or L‐arginine was found in the concentration range of 10−12 to 10−3 M, while 3‐morpholinosydnonimine hydrochloride (SIN‐1, 10−6 M) relaxed vascular tissues.

This study does not support the conclusion that L‐citrulline has direct vasorelaxing action on vascular smooth muscle.

British Journal of Pharmacology (2000) 130, 713–716; doi:10.1038/sj.bjp.0703372
Endogenous calcitonin gene‐related peptide (CGRP) mediates adrenergic‐dependent vasodilation induced by nicotine in mesenteric resistance arteries of the rat
Authors: Shiraki Hinako , Kawasaki Hiromu , Tezuka Satoko , Nakatsuma Akira , Kurosaki Yuji
Published: January 2005

The mechanisms underlying vasodilator effect of nicotine on mesenteric resistance blood vessels and the role of calcitonin gene‐related peptide (CGRP)‐containing (CGRPergic) vasodilator nerves were studied in the rat.

Mesenteric vascular beds isolated from Wistar rats were perfused with Krebs solution, and perfusion pressure was measured with a pressure transducer.

In preparations with intact endothelium and contracted by perfusion with Krebs solution containing methoxamine, perfusion of nicotine (1–100 μM) for 1 min caused a concentration‐dependent vasodilator response without vasoconstriction.

The nicotine‐induced vasodilation was markedly inhibited by hexamethonium (nicotinic cholinoceptor antagonist, 10 μM) and blocked by guanethidine (adrenergic neuron blocker, 5 μM).

Either denervation by cold storage (4°C for 72 h) or adrenergic denervation by 6‐hydroxydopamine (toxin for adrenergic neurons, 2 mM for 20 min incubation, twice) blocked the nicotine‐induced vasodilation.

Neither endothelium removal with perfusion of sodium deoxycholate (1.80 mg ml−1, for 30 s) nor treatment with Nω‐nitro‐L‐arginine (nitric oxide synthase inhibitor, 100 μM), atropine (muscarinic cholinoceptor antagonist, 10 nM) or propranolol (β‐adrenoceptor antagonist, 100 nM) affected the nicotine‐induced vasodilation.

In preparations without endothelium, treatment with capsaicin (depleting CGRP‐containing sensory nerves, 1 μM) or human CGRP[8–37] (CGRP receptor antagonist, 0.5 μM) markedly inhibited the nicotine‐induced vasodilation.

These results suggest that, in the mesenteric resistance artery of the rat, nicotine induces vasodilation, which is independent of the function of the endothelium and is involved in activation of CGRPergic nerves. It is also suggested that nicotine stimulates presynaptic nicotinic cholinoceptors on adrenergic nerves to release adrenergic neurotransmitters, which then act on CGRPergic nerves to release endogenous CGRP from the nerve.

British Journal of Pharmacology (2000) 130, 1083–1091; doi:10.1038/sj.bjp.0703376
Effects of sodium on agonist efficacy for G‐protein activation in μ‐opioid receptor‐transfected CHO cells and rat thalamus
Authors: Selley Dana E , Cao Cheng‐Chun , Liu Qixu , Childers Steven R
Published: January 2005

Sodium ions inhibit spontaneous Gi/Go‐coupled receptor activity and promote agonist‐induced responses in vitro. The effects of sodium on the relative efficacy of opioid agonists for G‐protein activation was measured by guanosine‐5′‐O‐(γ‐35S)‐triphosphate ([35S]‐GTPγS) binding in membranes from two μ‐opioid receptor‐containing systems: CHO cells stably transfected with mouse μ receptors (mMOR‐CHO cells) and rat thalamus.

NaCl inhibited basal [35S]‐GTPγS binding in both systems, and this effect was partially mimicked by KCl. In mMOR‐CHO membranes, net [35S]‐GTPγS binding stimulated by partial but not full agonists was inhibited by NaCl with a potency that was inversely proportional to agonist efficacy. Monovalent cations were required for agonist‐stimulated [35S]‐GTPγS binding in this system, and increasing NaCl concentrations magnified relative efficacy differences among agonists.

In thalamic membranes, which contain a lower receptor:G‐protein ratio than mMOR‐CHO cells, similar monovalent cation effects were observed, with two exceptions: (1) [35S]‐GTPγS binding stimulated by both full and partial agonists was inhibited by NaCl; and (2) monovalent cations were not required to observe agonist‐stimulated [35S]‐GTPγS binding.

Basal [35S]‐GTPγS binding stimulated by the absence of monovalent cations resembled that of agonist‐stimulated binding and was blocked by pretreatment of mMOR‐CHO cells with pertussis toxin.

These results indicate that sodium inhibits spontaneous and agonist‐occupied μ receptor‐mediated G‐protein activation in a manner inversely proportional to the efficacy of the agonist, and that spontaneous μ receptor activity and the relative efficacy of partial agonists acting at these receptors are both increased by increases in the stoichiometric ratio of receptors:G‐proteins.

British Journal of Pharmacology (2000) 130, 987–996; doi:10.1038/sj.bjp.0703382
Opposite effects of flurbiprofen and the nitroxybutyl ester of flurbiprofen on apoptosis in cultured guinea‐pig gastric mucous cells
Authors: Johal Kamaljit , Hanson Peter J
Published: January 2005

The nitric oxide (NO)‐donating nitroxybutyl ester of flurbiprofen (NO‐flurbiprofen), shows reduced gastro‐intestinal toxicity relative to flurbiprofen. NO may exert either pro‐ or anti‐apoptotic effects, while non‐steroidal anti‐inflammatory drugs may induce apoptosis. The aim of the present work was therefore to compare the effects of flurbiprofen and NO‐flurbiprofen on apoptosis in guinea‐pig gastric mucous cells.

Apoptotic activity was assessed by assay of caspase activity and from the fragmentation and condensation of nuclei.

Incubation with flurbiprofen for 24 h produced a concentration‐dependent induction of apoptosis in cells attached to the culture plate (caspase 3‐like activity increased by 257% at 500 μM), while NO‐flurbiprofen inhibited basal apoptosis (caspase 3‐like activity decreased by 71% at 500 μM).

Caspase activity and nuclear fragmentation were substantially increased in cells that had spontaneously detached from the culture plate. NO‐flurbiprofen inhibited caspase activity (55% at 500 μM) but not nuclear fragmentation in these detached cells.

NO flurbiprofen inhibited the activation of apoptosis by 25 μM C6‐ceramide in cells attached to the culture plate.

Inhibition of caspase activity by NO‐flurbiprofen was detectable after 6 h of incubation with intact cells, but by contrast with the NO‐donor S‐nitrosyl‐N‐acetyl‐penicillamine, was not demonstrable with cell homogenates.

Activation of caspase 3‐like activity by flurbiprofen was slow (>6 h incubation needed) and was inhibited by cycloheximide.

The presence of a nitroxybutyl ester moiety on flurbiprofen prevents the pro‐apoptotic activity of the parent compound and may contribute to the reduced gastro‐intestinal toxicity of NO‐flurbiprofen.

British Journal of Pharmacology (2000) 130, 811–818; doi:10.1038/sj.bjp.0703383
Role of A2A‐adenosine receptor activation for ATP‐mediated coronary vasodilation in guinea‐pig isolated heart
Authors: Erga Knut Ståle , Seubert Christoph N , Liang Hui‐Xiu , Wu Lin , Shryock John C , Belardinelli Luiz
Published: January 2005

Adenosine‐5′‐triphosphate (ATP) and adenosine are potent coronary vasodilators. ATP is rapidly converted to adenosine by ectonucleotidases. We examined whether coronary vasodilation caused by exogenous ATP is mediated by P2 receptor activation or by A2A‐adenosine receptor activation.

Effects of interventions on coronary conductance were determined by measuring coronary perfusion pressure in guinea‐pig isolated hearts perfused at a constant flow of 10 ml min−1.

ATP and adenosine both caused sustained, concentration‐dependent increases of coronary conductance. Maximal responses to both agonists were equivalent. The values of pD2 (±s.e.mean) for ATP and adenosine were 6.68±0.04 and 7.06±0.05, respectively. Adenosine was significantly more potent than ATP (P<0.0001, n=10).

The values of pIC50 for the selective A2A‐adenosine receptor antagonist SCH58261 to antagonize equivalent responses to ATP and adenosine were 8.28±0.08 and 8.28±0.06 (P=0.99, n=6), respectively.

The non‐selective adenosine receptor antagonists xanthine amine congener (XAC) and CGS15943 antagonized similarly the equivalent vasodilations caused by ATP (pIC50 values 7.48±0.04 and 7.45±0.06, respectively) and adenosine (pIC50 values 7.37±0.13 and 7.56±0.11).

In contrast to ATP and adenosine, the two P2 agonists 2‐methylthio‐ATP and uridine‐5′‐triphosphate failed to cause stable increases of coronary conductance, caused desensitization of vasodilator responses, and were not antagonized by SCH 58261, 8‐parasulphophenyltheophylline, or XAC.

Glibenclamide attenuated coronary vasodilations caused by ATP and adenosine by 88 and 89%, respectively, but failed to attenuate those caused by 2‐methylthio‐ATP.

These results strongly suggest that sustained, submaximal coronary vasodilation caused by exogenous ATP is entirely mediated by adenosine acting upon A2A‐adenosine receptors.

British Journal of Pharmacology (2000) 130, 1065–1075; doi:10.1038/sj.bjp.0703386
The involvement of intracellular Ca2+ in 5‐HT1B/1D receptor‐mediated contraction of the rabbit isolated renal artery
Authors: Hill P B , Dora K A , Hughes A D , Garland C J
Published: January 2005

5‐Hydroxytryptamine1B/1D (5‐HT1B/1D) receptor coupling to contraction was investigated in endothelium‐denuded rabbit isolated renal arteries, by simultaneously measuring tension and intracellular [Ca2+], and tension in permeabilized smooth muscle cells.

In intact arterial segments, 1 nM–10 μM 5‐HT failed to induce contraction or increase the fura‐2 fluorescence ratio (in the presence of 1 μM ketanserin and prazosin to block 5‐HT2 and α1‐adrenergic receptors, respectively). However, in vessels pre‐exposed to either 20 mM K+ or 30 nM U46619, 5‐HT stimulated concentration‐dependent increases in both tension and intracellular [Ca2+].

1 nM–10 μM U46619 induced concentration‐dependent contractions. In the presence of nifedipine (0.3 and 1 μM) the maximal contraction to U46619 (10 μM) was reduced by around 70%. The residual contraction was abolished by the putative receptor operated channel inhibitor, SKF 96365 (2 μM).

With 0.3 μM nifedipine present, 100 nM U46619 evoked similar contraction to 30 nM U46619 in the absence of nifedipine, but contraction to 5‐HT (1 nM–10 μM) was abolished.

In permeabilized arterial segments, 10 mM caffeine, 1 μM IP3 or 100 μM phenylephrine, each evoked transient contractions by releasing Ca2+ from intracellular stores, whereas 5‐HT had no effect. In intact arterial segments pre‐stimulated with 20 mM K+, 5‐HT‐evoked contractions were unaffected by 1 μM thapsigargin, which inhibits sarco‐ and endoplasmic reticulum calcium‐ATPases.

In vessels permeabilized with α‐toxin and then pre‐contracted with Ca2+ and GTP, 5‐HT evoked further contraction, reflecting increased myofilament Ca2+‐sensitivity.

Contraction linked to 5‐HT1B/1D receptor stimulation in the rabbit renal artery can be explained by an influx of external Ca2+ through voltage‐dependent Ca2+ channels and sensitization of the contractile myofilaments to existing levels of Ca2+, with no release of Ca2+ from intracellular stores.

British Journal of Pharmacology (2000) 130, 835–842; doi:10.1038/sj.bjp.0703387
Participation of kinins in the captopril‐induced inhibition of intimal hyperplasia caused by interruption of carotid blood flow in the mouse
Authors: Emanueli Costanza , Salis Maria Bonaria , Figueroa Carlos , Chao Julie , Chao Lee , Gaspa Leonardo , Capogrossi Maurizio C , Madeddu Paolo
Published: January 2005

In the rat balloon injury model, angiotensin‐converting enzyme (ACE) inhibitors prevent vascular remodelling by inhibiting angiotensin II generation and kinin breakdown. We investigated if ACE inhibition also prevents the structural vascular responses to disruption of carotid artery blood flow and if kinin potentiation plays a role in such a protection.

Morphometric analysis of the structural alterations caused by ligation of the left carotid artery was performed 14 days after surgery in J129Sv wild‐type mice (B2+/+) drinking normal tap water or water containing captopril (120 mg kg−1 per day). In addition, the effect of captopril on vascular remodelling was tested in B2+/+ given the bradykinin (BK) B1 receptor antagonist des‐Arg9‐[Leu8]‐BK (DALBK, 50 nmol kg−1 per day, intraperitoneally) or the BK B2 receptor antagonist D‐Arg,[Hyp3,Thi5D‐Tic7,Oic8]‐BK (icatibant, 1 μmol kg−1 per day, intraperitoneally), and in B2 receptor gene knockout mice (B2−/−).

Interruption of blood flow resulted in carotid artery intimal hyperplasia and media thickening in untreated B2+/+, these responses being partially suppressed by captopril. The inhibition of intimal thickening exerted by captopril was reduced in B2+/+ given DALBK or icatibant (P<0.05 for both comparisons) as well as in B2−/− (P<0.05). Neither antagonism of kinin receptors nor disruption of the B2 receptor gene altered the suppressive effect of captopril on media thickening. The protection of vascular wall structure was independent of the reduction in blood pressure by captopril.

These results demonstrate that kinins participate in the inhibitory effect of captopril on intimal hyperplasia via B1 and B2 receptor signalling. Our findings may have important implications in treating vascular remodelling evoked by altered shear stress conditions.

British Journal of Pharmacology (2000) 130, 1076–1082; doi:10.1038/sj.bjp.0703388
Deoxycorticosterone acetate‐salt hypertensive rats display gender‐related differences in ETB receptor‐mediated vascular responses
Authors: Passaglia Rita C A Tostes , David Flavia Lucia , Fortes Zuleica B , Nigro Dorothy , Scivoletto Regina , Catelli de Carvalho Maria Helena
Published: January 2005

Male DOCA‐salt rats exhibit vasoconstriction upon ETB activation. Because hypertension is less severe in female than male DOCA rats, we hypothesized that female DOCA rats would display attenuated ETB vasoconstrictor responses.

Uninephrectomized Wistar rats received DOCA and drinking water containing NaCl/KCl. Control rats received vehicle and tap water. Systolic blood pressure was higher in male vs female DOCA rats. Responses to endothelin‐1 (ET‐1), IRL‐1620, an ETB agonist, and acetylcholine were evaluated in isolated aortas and in vivo in the mesenteric microcirculation.

Endothelium‐denuded aortas from male, but not female, DOCA rats displayed increased sensitivity to ET‐1. IRL‐1620 contracted aortas from male DOCA rats, but not control or female DOCA aortas. Noradrenaline‐constricted and endothelium‐intact aortas from male, but not female, DOCA rats displayed increased relaxation to IRL‐1620 compared to control aortas.

In vivo, increased vasoconstriction to ET‐1 was observed in male and female DOCA rats. IRL‐1620 induced vasodilation in control rats, but vasoconstriction in male DOCA rats. There were minimal changes in diameter in vessels from female DOCA rats.

The initial fall in blood pressure induced by ET‐1 and IRL‐1620 was attenuated in male DOCA rats. Bosentan, a mixed ETA/ETB receptor antagonist, lowered blood pressure in male and female DOCA rats, but a greater and marked decrease occurred in the male DOCA group.

The gender‐related differences in ET‐1/ETB‐mediated effects both in the vasculature and blood pressure suggest that sex‐related functional up‐regulation of ETB receptors may play a role in the more severe hypertension in male DOCA hypertensive rats.

British Journal of Pharmacology (2000) 130, 1092–1098; doi:10.1038/sj.bjp.0703389
Endothelial dysfunction in diabetes
Authors: De Vriese An S , Verbeuren Tony J , Van de Voorde Johan , Lameire Norbert H , Vanhoutte Paul M
Published: January 2005

Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease. The endothelium controls the tone of the underlying vascular smooth muscle through the production of vasodilator mediators. The endothelium‐derived relaxing factors (EDRF) comprise nitric oxide (NO), prostacyclin, and a still elusive endothelium‐derived hyperpolarizing factor (EDHF). Impaired endothelium‐dependent vasodilation has been demonstrated in various vascular beds of different animal models of diabetes and in humans with type 1 and 2 diabetes. Several mechanisms of endothelial dysfunction have been reported, including impaired signal transduction or substrate availibility, impaired release of EDRF, increased destruction of EDRF, enhanced release of endothelium‐derived constricting factors and decreased sensitivity of the vascular smooth muscle to EDRF. The principal mediators of hyperglycaemia‐induced endothelial dysfunction may be activation of protein kinase C, increased activity of the polyol pathway, non‐enzymatic glycation and oxidative stress. Correction of these pathways, as well as administration of ACE inhibitors and folate, has been shown to improve endothelium‐dependent vasodilation in diabetes. Since the mechanisms of endothelial dysfunction appear to differ according to the diabetic model and the vascular bed under study, it is important to select clinically relevant models for future research of endothelial dysfunction.

British Journal of Pharmacology(2000)130, 963–974; doi:10.1038/sj.bjp.0703393
Sensitivity of Kir6.2‐SUR1 currents, in the absence and presence of sodium azide, to the KATP channel inhibitors, ciclazindol and englitazone
Authors: McKay N G , Kinsella J M , Campbell C M , Ashford M L J
Published: January 2005

Two electrode voltage clamp and single channel recordings were used to investigate the actions of various ATP‐sensitive K+ (KATP) channel inhibitors on cloned KATP channels, expressed in Xenopus oocytes and HEK 293 cells.

Oocytes expressing Kir6.2 and SUR1 gave rise to inwardly rectifying K+ currents following bath application of 3 mM sodium azide. Inside‐out recordings from non‐azide treated oocytes demonstrated the presence of KATP channels which were activated by direct application of 3 mM azide and 0.1 mM Mg‐ATP.

Tolbutamide inhibited azide‐induced macroscopic Kir6.2‐SUR1 currents, recorded from Xenopus oocytes, with an IC50 value similar to native KATP channels. Ciclazindol and englitazone also inhibited these currents in a concentration‐dependent manner, but with relative potencies substantially less than for native KATP channels.

Single channel currents recorded from inside‐out patches excised from oocytes expressing Kir6.2‐SUR1 currents were inhibited by tolbutamide, Mg‐ATP, englitazone and ciclazindol, in the absence of azide, with potencies similar to native KATP channels. In the presence of azide, Kir6.2‐SUR1 currents were inhibited by englitazone and tolbutamide but not ciclazindol.

Single channel currents derived from Kir6.2Δ26, expressed in HEK 293 cells, were inhibited by ciclazindol and englitazone irrespective of the absence or presence of SUR1.

In conclusion, heterologously expressed Kir6.2 and SUR1 recapitulate the pharmacological profile of native pancreatic β‐cell KATP channels. However, currents induced by azide exhibit a substantially reduced sensitivity to ciclazindol. It is likely that ciclazindol and englitazone inhibit KATP currents by interaction with the Kir6.2 subunit.

British Journal of Pharmacology (2000) 130, 857–866; doi:10.1038/sj.bjp.0703395
Mechanisms of nitric oxide‐independent relaxations induced by carbachol and acetylcholine in rat isolated renal arteries
Authors: Jiang Fan , Li Chun Guang , Rand Michael J
Published: January 2005

In rat isolated renal artery segments contracted with 0.1 μM phenylephrine and in the presence of the NO synthase inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME), carbachol and acetylcholine produced endothelium‐dependent relaxations. The mechanisms underlying these relaxations were studied.

These relaxations were not affected by ODQ (1H‐[1,2,4]oxadiazolo[4,3,‐a]quinoxalin‐1‐one) or indomethacin. In arteries contracted with 20–30 mM K+, L‐NAME‐resistant relaxations induced by carbachol and acetylcholine were virtually absent.

The Na+‐K+ ATPase inhibitor ouabain reduced these relaxations in a concentration‐dependent manner.

In K+‐free media, addition of K+ (5 mM) produced 90.5±3.9% (n=3) relaxation of phenylephrine‐induced tone. This relaxation was endothelium‐independent and ouabain‐sensitive.

Tetraethylammonium (TEA), charybdotoxin (ChTX) and iberiotoxin (IbTX) reduced the sensitivity of carbachol‐induced relaxations, but did not change the maximal response. These relaxations were not altered by 4‐aminopyridine (4‐AP), glibenclamide or apamin. Acetylcholine (1 μM)‐induced relaxation was reduced by ChTX, but not by TEA or IbTX.

The cytochrome P450 inhibitor miconazole, but not 17‐octadecynoic acid, reduced the sensitivity of carbachol‐induced relaxations, without changing the maximal response.

In conclusion, in rat isolated renal arteries, acetylcholine and carbachol produced a non‐NO/non‐PGI2 relaxation which is mediated by an endothelium‐derived hyperpolarizing factor (EDHF). This factor does not appear to be a cytochrome P450 metabolite. The inhibition by ouabain of these relaxations suggests the possible involvement of Na+‐K+ ATPase activation in EDHF responses, although other mechanisms cannot be totally ruled out.

British Journal of Pharmacology (2000) 130, 1191–1200; doi:10.1038/sj.bjp.0703408
Mechanisms underlying the neurokinin A‐induced contraction of the pregnant rat myometrium
Authors: Shintani Yoshinobu , Nishimura Junji , Niiro Naohisa , Hirano Katsuya , Nakano Hitoo , Kanaide Hideo
Published: January 2005

Using fura‐PE3 fluorimetry and α‐toxin permeabilization, the characteristics of the contractile responses to neurokinin A (NKA) were determined in the pregnant rat myometrium.

NKA induced contractions in rat myometrium in a concentration‐dependent manner. There were no significant differences in the maximum contractions and EC50 values between the pregnant and non‐pregnant myometrium, however, the contraction of only the former was greatly enhanced in the presence of phosphoramidon (PPAD), an endopeptidase inhibitor.

In the pregnant myometrium, NKA induced sustained increases in [Ca2+]i and tension in normal physiological saline solution, while only small transient increases in [Ca2+]i and tension were observed in Ca2+‐free solution.

Both diltiazem (10 μM) and SK‐F 96365 (10 μM) significantly inhibited the NKA‐induced elevations of [Ca2+]i and tension. The effects were additive when these drugs were used together.

NKA induced a significant leftward shift of the [Ca2+]i‐tension curve obtained by changing the external Ca2+ (0–2.5 mM) during depolarization with high K+ solution. This Ca2+‐sensitizing effect by NKA was also observed in the α‐toxin permeabilized myometrium.

These results indicated that in the pregnant rat myometrium: (1) the responsiveness to NKA increased, although it was masked by the increase in the endopeptidase activity; (2) NKA induced contractions of the myometrium by increasing both [Ca2+]i and the myofilament Ca2+ sensitivity and (3) The NKA‐induced [Ca2+]i elevation was partly due to the intracellular Ca2+ release and mainly due to the Ca2+ influx, which was thought to be through both voltage dependent calcium channels and non‐specification channels.

British Journal of Pharmacology (2000) 130, 1165–1173; doi:10.1038/sj.bjp.0703410
Potassium does not mimic EDHF in rat mesenteric arteries
Authors: Doughty Joanne M , Boyle John P , Langton Philip D
Published: January 2005

K+ has been proposed to be EDHF in small arteries. We compared ACh‐stimulated, EDHF‐mediated dilatation/relaxation with raised [K+]o in rat mesenteric arteries.

In pressurized arteries, ACh (10 μM) dilated all arteries. Raising [K+]o from 5.88 to 10.58 mM only dilated 30% of arteries. Ba2+ (30 μM) did not affect dilatation to ACh, but abolished 40% of dilatations to raised [K+]o.

If [K+]o was lowered to 1.18 mM, restoring [K+]o to 5.88 mM produced dilatation which was depressed by Ba2+ or ouabain (1 mM). Combined application of Ba2+ and ouabain abolished dilatation. In 1.18 mM K+, dilatation to ACh was depressed by ouabain, but not by Ba2+. Combined application of Ba2+ and ouabain depressed dilatation further. Gap junction inhibitors (Gap‐27; 300 μM and 18‐α‐glycyrrhetinic acid; 100 μM) also depressed dilatation to ACh.

In arteries mounted isometrically, ACh (1 μM) relaxed endothelium intact (+E), but not endothelium denuded (−E) arteries. Raising [K+]o from 5.9–10.9 mM failed to relax all arteries. When [K+]o was lowered to 1 mM, raising [K+]o to 6 mM produced relaxation. In −E arteries, relaxation was unaffected by Ba2+ but abolished by ouabain. In +E arteries, Ba2+ depressed and ouabain abolished relaxation. In +E arteries, with 1 mM K+, ACh relaxation was depressed by ouabain but not Ba2+. The combined application of Ba2+ and ouabain further depressed relaxation.

In summary, both EDHF and raised [K+]o dilate/relax rat mesenteric arteries, though sensitivities to barium and ouabain differ. K+ may be a relaxing factor in this tissue, but its characteristics differ from EDHF. Gap junction inhibitors depress EDHF, implying an important role for myo‐endothelial gap junctions.

British Journal of Pharmacology (2000) 130, 1174–1182; doi:10.1038/sj.bjp.0703412
Leukaemia inhibitory factor (LIF) upregulates excitatory non‐adrenergic non‐cholinergic and maintains cholinergic neural function in tracheal explants
Authors: Knight D A , D'Aprile A C , Spalding L J , Goldie R G , Thompson P J
Published: January 2005

The effect of leukaemia inhibitory factor (LIF) in modulating cholinergic and sensory nerve function was examined using guinea‐pig tracheal explants. Specific LIF receptors (LIFR) were immunolocalized to both cholinergic and sensory nerves. Release of SP in culture was not influenced by LIF. Similarly, maximum contraction to carbachol (Cmax) was not influenced by LIF. After 3 h, maximum (Emax) eNANC‐induced contraction in controls was 32±2.5% of Cmax. In LIF‐treated preparations, Emax was enhanced to 50±4.5% Cmax (P<0.05). Cholinergic nerve‐induced contractions after 3 h incubation with LIF were similar to control. After 24 h, control Emax was 25±4.5% Cmax (58% smaller than Emax at 3 h). In contrast, in LIF‐treated preparations, Emax was 37±2.5% Cmax, (24% smaller than at 3 h, P<0.05). This did not appear to be due to the effect of LIF on muscarinic M2 receptor expression or function. Thus LIF appears to differentially influence the function of airway nerves and thus may provide an important link between the immune and neural systems.

British Journal of Pharmacology (2000) 130, 975–979; doi:10.1038/sj.bjp.0703415
Nitric oxide mediated modulation of norepinephrine transport: identification of a potential target for S‐nitrosylation
Authors: Kaye David M , Gruskin Sara , Smith A Ian , Esler Murray D
Published: January 2005

Carrier mediated uptake (uptake‐1) transport of norepinephrine (NE) plays a key role in the regulation of sympathetic neurotransmission. Recent investigations indicate that nitric oxide (NO) may modulate uptake‐1 activity, possibly in a cyclic GMP independent manner.

Carrier mediated transport of [3H‐NE] and [3H‐dopamine, DA] was examined in CHO cells transfected with cDNA for the NE and DA transporters (NET, DAT) respectively.

While exposure to the NO donor S‐nitroso‐N‐acetylpenicillamine (100 μM, SNAP) significantly reduced [3H‐NE] uptake (P<0.001), no effect on [3H‐DA] transport was apparent.

Comparison of the amino acid sequences for NET and DAT identified cysteine residue 351 in NET, which was not present in DAT. Site‐directed mutagenesis of Cys 351 to Ser produced a functional NET that was resistant to the inhibitory effects of SNAP.

The presence of SNAP mediated nitrosylation of the cysteine residue in an 8‐mer model peptide based around Cys 351 in NET was confirmed by both biochemical and mass spectroscopic means.

These data indicate the potential regulatory role for NO in modulating sympathetic neurotransmission, and further confirm the importance of non‐cyclic GMP dependent mechanisms in mediating the actions of NO.

British Journal of Pharmacology (2000) 130, 1060–1064; doi:10.1038/sj.bjp.0703416
Effect of cytochrome P‐450 inhibitors econazole, bifonazole and clotrimazole on prostanoid formation
Authors: Köfeler Harald C , Fauler Günter , Windischhofer Werner , Leis Hans J
Published: January 2005

The present study was carried out to clarify the effect of the imidazole antimycotics econazole, bifonazole and clotrimazole on prostanoid biosynthesis. Osteoblast‐like MC3T3‐E1 cells stimulated by endothelin‐1, melittin, ionomycin or arachidonic acid showed diminished prostaglandin E2 (PGE2) production upon pretreatment with econazole. Following pretreatment with bifonazole, stimulation with ionomycin or arachidonic acid also resulted in decreased PGE2 formation. Clotrimazole inhibited ionomycin but not arachidonic acid stimulated PGE2 synthesis in MC3T3‐E1 cells.

The results observed in osteoblast‐like UMR‐106 cells pretreated with econazole, bifonazole or clotrimazole and stimulated by arachidonic acid were similar with the exception of clotrimazole which was a more effective inhibitor of PGE2 biosynthesis than in MC3T3‐E1 cells.

Upon treatment with arachidonic acid thromboxane B2 (TXB2) production in human platelets was abolished completely at concentrations of the three imidazole antimycotics higher than 5 μM (IC50<1 μM).

These data were confirmed by a direct assay using purified ram seminal vesicle prostaglandin H2 synthase‐1 (PGHS‐1), which clearly showed inhibitory properties of econazole (IC50 4.7±2.3 μM), bifonazole (IC50 9.4±0.8 μM) and clotrimazole (IC50 4.4±0.6 μM).

Summarizing, these results indicate an inhibitory effect of econazole, bifonazole and clotrimazole on PGHS‐1, varying in its potency dependent on the cell system used. In addition TXB2 formation is affected at doses even lower than those needed to suppress PGE2 biosynthesis.

British Journal of Pharmacology (2000) 130, 1241–1246; doi:10.1038/sj.bjp.0703427
Sodium reabsorption in thick ascending limb of Henle's loop: effect of potassium channel blockade in vivo
Authors: Huang Dan Y , Osswald Hartmut , Vallon Volker
Published: January 2005

Based on previous in vitro studies, inhibition of K+ recycling in thick ascending limb (TAL) is expected to lower Na+ reabsorption through (i) reducing the luminal availability of K+ to reload the Na+‐2Cl−‐K+ cotransporter and (ii) diminishing the lumen positive transepithelial potential difference which drives paracellular cation transport.

This issue was investigated in anaesthetized rats employing microperfusion of Henle's loop downstream from late proximal tubular site with K+‐free artificial tubular fluid in nephrons with superficial glomeruli.

The unselective K+ channel blocker Cs+ (5–40 mM) dose‐dependently increased early distal tubular delivery of fluid and Na+ with a maximum increase of ∼20 and 185%, respectively, indicating predominant effects on water‐impermeable TAL.

The modest inhibition of Na+ reabsorption in response to the 15 mM of Cs+ but not the enhanced inhibition by 20 mM Cs+ was prevented by luminal K+ supplementation. Furthermore, pretreatment with 20 mM Cs+ did not attenuate the inhibitory effect of furosemide (100 μM) on Na+‐2Cl−‐K+ cotransport.

Neither inhibitors of large (charybdotoxin 1 μM) nor low (glibenclamide 250 μM; U37883A 100 μM) conductance K+ channels altered loop of Henle fluid or Na+ reabsorption.

The intermediate conductance K+ channel blockers verapamil and quinine (100 μM) modestly increased early distal tubular Na+ but not fluid delivery, indicating a role for this K+ channel in Na+ reabsorption in TAL. As observed for equieffective concentrations of Cs+ (15 mM), Na+ reabsorption was preserved by K+ supplementation.

The results indicate that modest inhibition of K+ channels lowers the luminal availability of K+ and thus transcellular Na+ reabsorption in TAL. More complete inhibition lowers paracellular Na+ transport probably by reducing or even abolishing the lumen positive transepithelial potential difference. Under the latter conditions, transcellular Na+ transport may be restored by paracellular K+ backleak.

British Journal of Pharmacology (2000) 130, 1255–1262; doi:10.1038/sj.bjp.0703429
Mutational analysis of the interaction of the N‐ and C‐terminal ends of angiotensin II with the rat AT1A receptor
Authors: Costa‐Neto Claudio M , Miyakawa Ayumi A , Oliveira Laerte , Hjorth Siv A , Schwartz Thue W , Paiva Antonio C M
Published: January 2005

The role of different residues of the rat AT1A receptor in the interaction with the N‐ and C‐terminal ends of angiotensin II (AngII) was studied by determining ligand binding and production of inositol phosphates (IP) in COS‐7 cells transiently expressing the following AT1A mutants: T88H, Y92H, G196I, G196W and D278E.

G196W and G196I retained significant binding and IP‐production properties, indicating that bulky substituents in position 196 did not affect the interaction of AngII's C‐terminal carboxyl with Lys199 located three residues below.

Although the T88A mutation did not affect binding, the T88H mutant had greatly decreased affinity for AngII, suggesting that substitution of Thr88 by His might hinder binding through an indirect effect.

The Y92H mutation caused loss of affinity for AngII that was much less pronounced than that reported for Y92A, indicating that His in that position can fulfil part of the requirements for binding.

Replacing Asp278 by Glu caused a much smaller reduction in affinity than replacing it by Ala, indicating the importance of Asp's β‐carboxyl group for AngII binding.

Mutations in residues Thr88, Tyr92 and Asp278 greatly reduced affinity for AngII but not for Sar1 Leu8‐AngII, suggesting unfavourable interactions between these residues and AngII's aspartic acid side‐chain or N‐terminal amino group, which might account for the proposed role of the N‐terminal amino group of AngII in the agonist‐induced desensitization (tachyphylaxis) of smooth muscles.

British Journal of Pharmacology (2000) 130, 1263–1268; doi:10.1038/sj.bjp.0703430
The MAP kinase inhibitors, PD098059, UO126 and SB203580, inhibit IL‐1β‐dependent PGE2 release via mechanistically distinct processes
Authors: Newton Robert , Cambridge Lisa , Hart Lorraine A , Stevens David A , Lindsay Mark A , Barnes Peter J
Published: January 2005

In common with human bronchial epithelial cells, pulmonary A549 cells release prostaglandin (PG) E2 in response to pro‐inflammatory cytokines. We have therefore used these cells to examine the effect of the selective mitogen activated protein (MAP) kinase inhibitors; PD098059, a mitogen activated and extracellular regulated kinase kinase (MEK) 1 inhibitor, UO126, a dual MEK1 & MEK2 inhibitor, and SB203580, a p38 MAP kinase inhibitor in the IL‐1β‐dependent release of PGE2.

Following IL‐1β treatment the extracellular regulated kinases (ERKs) and the p38 MAP kinases were rapidly phosphorylated.

PD09059, UO126 and SB203580 prevented IL‐1β‐induced PGE2 release at doses that correlated closely with published IC50 values. Small or partial effects at the relevant doses were observed on induction of cyclo‐oxygenase (COX) activity or COX‐2 protein suggesting that the primary effects were at the level of arachidonate availability.

Neither PD098059 nor SB203580 showed any effect on IL‐1β‐induced arachidonate release. We therefore speculate that the MEK1/ERK and p38 kinase cascades play a role in the functional coupling of arachidonate release to COX‐2.

In contrast, UO126 was highly effective at inhibiting IL‐1β‐dependent arachidonate release, implicating MEK2 in the activation of the PLA2 that is involved in IL‐1β‐dependent PGE2 release.

We conclude that the MEK1, MEK2 and p38 MAP kinase inhibitors, PD098059, UO126 and SB203580, are highly potent in respect of inflammatory PG release. Finally, we conclude that these inhibitors act via mechanistically distinct processes, which may have anti‐inflammatory benefits.

British Journal of Pharmacology (2000) 130, 1353–1361; doi:10.1038/sj.bjp.0703431
Involvement of central opioid systems in human interferon‐α induced immobility in the mouse forced swimming test
Authors: Makino Mitsuhiro , Kitano Yutaka , Komiyama Chika , Hirohashi Masaaki , Takasuna Kiyoshi
Published: January 2005

We investigated the mechanism by which human interferon‐α (IFN‐α) increases the immobility time in a forced swimming test, an animal model of depression.

Central administration of IFN‐α (0.05–50 IU per mouse, i.cist.) increased the immobility time in the forced swimming test in mice in a dose‐dependent manner.

Neither IFN‐β nor ‐γ possessed any effect under the same experimental conditions.

Pre‐treatment with an opioid receptor antagonist, naloxone (1 mg kg−1, s.c.) inhibited the prolonged immobility time induced by IFN‐α (60 KIU kg−1, i.v. or 50 IU per mouse. i.cist.).

Peripheral administration of naloxone methiodide (1 mg kg−1, s.c.), which does not pass the blood–brain barrier, failed to block the effect of IFN‐α, while intracisternal administration of naloxone methiodide (1 nmol per mouse) completely blocked.

The effect of IFN‐α was inhibited by a μ1‐specific opioid receptor antagonist, naloxonazine (35 mg kg−1, s.c.) and a μ1/μ2 receptor antagonist, β‐FNA (40 mg kg−1, s.c.). A selective δ‐opioid receptor antagonist, naltrindole (3 mg kg−1, s.c.) and a κ‐opioid receptor antagonist, nor‐binaltorphimine (20 mg kg−1, s.c.), both failed to inhibit the increasing effect of IFN‐α.

These results suggest that the activator of the central opioid receptors of the μ1‐subtype might be related to the prolonged immobility time of IFN‐α, but δ and κ‐opioid receptors most likely are not involved.

British Journal of Pharmacology (2000) 130, 1269–1274; doi:10.1038/sj.bjp.0703432