bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of soluble guanylyl cyclase by YC‐1 in aortic smooth muscle but not in ventricular myocardium from rat
Authors: Wegener Jörg W. , Gath Ingolf , Förstermann Ulrich , Nawrath Hermann
Published: January 2005

The effects of YC‐1 (3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzyl indazole), an activator of soluble guanylyl cyclase, on tension, levels of cyclic GMP and cyclic AMP, and cardiac L‐type Ca2+‐current (ICa(L)) were investigated in aortic smooth muscle and ventricular heart muscle from rat.

YC‐1 (0.1–30 μM) induced a concentration‐dependent relaxation in aortic rings precontracted with phenylephrine (3 μM). The relaxant effects of YC‐1 were reversed by 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (30 μM; ODQ), potentiated by zaprinast (10 μM) and antagonized by Rp‐8‐Br‐cGMPS (100 μM).

In ventricular heart muscle strips, YC‐1 (30 μM) exhibited no effects on force of contraction (Fc) in the absence or presence of either zaprinast (10 μM) or 3‐isobutyl‐1‐methylxanthine (30 μM). Fc was slightly increased by YC‐1 (30 μM) in the presence of isoprenaline (100 nM), but this effect was not influenced by ODQ (30 μM).

Cardiac ICa(L) was not significantly affected by YC‐1 (30 μM), either in the absence or presence of isoprenaline (30 nM).

In aortic rings, cyclic GMP levels were increased almost 3 fold by YC‐1 (30 μM); this effect was abolished by ODQ (30 μM). In isolated ventricular cardiomyocytes, cyclic GMP levels were not affected by YC‐1 (30 μM) but almost doubled by activation of particular guanylyl cyclase with atriopeptin II (100 nM).

YC‐1 (30 μM) did not increase cyclic AMP levels either in aortic rings or in ventricular cardiomyocytes. In contrast, isoprenaline (3 μM) increased cyclic AMP levels about two fold in both tissues. In cardiomyocytes, the effect of isoprenaline (3 μM) was slightly enhanced by YC‐1 (30 μM).

It is concluded that relaxation of smooth muscle preparations by YC‐1 is mediated mainly by activation of soluble guanylyl cyclase and subsequent increase in cyclic GMP levels. The failure of YC‐1 to affect cardiac Fc, levels of cyclic GMP, and ICa(L) suggests that soluble guanylyl cyclase is not influenced by YC‐1 in rat heart muscle or only barely present in this tissue.
Effect of fluoxetine on a neuronal, voltage‐dependent potassium channel (Kv1.1)
Authors: Tytgat J. , Maertens Ch , Daenens P.
Published: January 2005

Fluoxetine (Prozac) is widely used as an antidepressant drug and is assumed to be a selective 5‐hydroxytryptamine (5‐HT) reuptake inhibitor (SSRI). Claims that its beneficial psychotropic effects extend beyond those in treatment of depression have drawn clinical and popular attention to this compound, raising the question of whether there is anything exceptional about the supposed selective actions.

We have used the voltage clamp technique to study the effect of fluoxetine on a neuronal, voltage‐dependent potassium (K+) channel (RCK1; Kv1.1), expressed in Xenopus laevis oocytes. This channel subunit is abundantly expressed in the central nervous system and K+ channels containing this subunit are involved in the repolarization process of many types of neurones.

Blockade of the K+ currents by fluoxetine was found to be use‐ and dose‐dependent. Wash‐out of this compound could not be achieved. Fluoxetine did not affect the ion selectivity of this K+ channel, as the reversal potential was unaltered.

Slowing of both activation and deactivation kinetics of the channel by fluoxetine was observed, including tail current crossover upon repolarization.

Hodgkin‐Huxley type of models and more generalized Markov chain models were used to fit the kinetics of the data. Based upon a Markov kinetic scheme, our data can be interpreted to mean that blockade of fluoxetine consists of two components: a voltage‐independent occurring in the last closed, but available state of the channel, and a voltage‐dependent occurring in the open state.

This study describes the first biophysical working model for the mechanism of action of fluoxetine on a neuronal, voltage‐dependent K+ channel, RCK1. Although this channel is not very potently blocked by fluoxetine when expressed in oocytes, this study may help us to understand some of the clinical symptoms seen with elevated serum concentrations of this SSRI.
Comparative effects of the dual metallopeptidase inhibitor, MDL 100,240 and of enalaprilat on regional and on cardiac haemodynamics in conscious, hypertensive, transgenic ((mRen‐2)27) rats
Authors: Gardiner S M. , Kemp P A. , Brunner‐Ferber F. , Bennett T.
Published: January 2005

Heterozygous, male, hypertensive, transgenic ((mRen‐2)27) rats (350–450 g) were instrumented for the measurement of regional or cardiac haemodynamics (n = 16, in both groups). Animals were given continuous i.v. infusions of the angiotensin‐converting enzyme inhibitor, enalaprilat, or the dual metallopeptidase inhibitor, MDL 100,240 (both at 3 mg kg−1, 3 mg kg−1 h−1; n = 8 for regional and cardiac haemodynamics), for 32 h. Twenty four hours after the onset of infusion of enalaprilat or MDL 100,240, the bradykinin (B2)‐receptor antagonist, Hoe 140 (1 mg kg−1, i.v.), was given and measurements were continued for a further 8 h, to assess any possible involvement of bradykinin.

Over the first 8 h of infusion, both enalaprilat and MDL 100,240 had significant antihypertensive effects, accompanied by similar regional vasodilatations. However, the blood pressure lowering effect of MDL 100,240 (−54 ± 9 mmHg) was greater than that of enalaprilat (−38 ± 4 mmHg), because the former caused a significantly greater reduction in cardiac index.

Between 8–24 h after the onset of infusion, there was a reduction in the effect of enalaprilat on blood pressure, because cardiac index rose, with no further increase in total peripheral conductance. In contrast, the antihypertensive effect of MDL 100,240 persisted, in spite of a recovery in cardiac index, because there was further vasodilatation, particularly in the mesenteric and hindquarters vascular beds.

There were no apparent haemodynamic changes associated with the injection of Hoe 140, and over the following 8 h, the difference between the haemodynamic effects of enalaprilat and MDL 100,240 persisted; there was little evidence of suppression of the effects of either drug.

These results are more consistent with the antihypertensive effects of enalaprilat or MDL 100,240 in transgenic ((mRen‐2)27) rats being due to suppression of angiotensin II production, than due to inhibition of bradykinin degradation. The additional effects of MDL 100,240 may be accounted for by inhibition of the degradation of natriuretic peptides reducing cardiac output, initially, and decreasing vascular tone, subsequently. Alternatively, the additional increase in vascular conductance following treatment with MDL 100,240 may represent an autoregulatory response to the reduced pressure.
Scavenging of hydroxyl radicals but not of peroxynitrite by inhibitors and substrates of nitric oxide synthases
Authors: Rehman Almas , Whiteman Matthew , Halliwell Barry
Published: January 2005

The nitric oxide synthase inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME) is widely used to study the role of NO• in physiological and pathological processes, including its role in the generation of the cytotoxic species peroxynitrite (ONOO−) and of reactive oxygen radicals such as hydroxyl (OH•). Often l‐NAME is applied to tissues at mM concentrations. At such high concentrations, it might act as a free radical scavenger. A similar possibility might apply to the use of high levels of arginine to study the role of NO. in atherogenesis.

We therefore examined the rate of scavenging of OH• by l‐NAME and found that l‐NAME reacts more quickly with OH. than the established ‘OH. scavenger’ mannitol and the widely used ‘OH• trap’ salicylate. However, d‐NAME can scavenge OH• at rates equal to l‐NAME. Both l‐ and d‐arginine were also good OH• scavengers, comparable in effectiveness to mannitol.

Neither l‐NAME, d‐NAME, l‐arginine nor d‐arginine was able to inhibit ONOO−‐dependent nitration of tyrosine, suggesting that they are unlikely to be scavengers of ONOO−‐derived nitrating species.

Neither l‐NAME, d‐NAME, l‐arginine nor d‐arginine was able to inhibit the inactivation of α1‐antiproteinase by ONOO−, suggesting that they cannot prevent direct oxidations by peroxynitrite.

We conclude that l‐NAME has sufficient activity as an OH• scavenger to confound certain pharmacological experiments. However, this explanation of its biological effects can be ruled out if control experiments show that d‐NAME has no effect and that l‐arginine (also a free radical scavenger) antagonizes the action of l‐NAME.
Endothelin converting enzyme (ECE) activity in human vascular smooth muscle
Authors: Maguire Janet J. , Johnson Christopher M. , Mockridge James W. , Davenport Anthony P.
Published: January 2005

We have characterized the human smooth muscle endothelin converting enzyme (ECE) present in the media of the endothelium‐denuded human umbilical vein preparation.

Endothelin‐1 (ET‐1) and ET‐2 were potent constrictors of umbilical vein with EC50 values of 9.2 nm and 29.6 nm, respectively. ET‐1 was at least 30 times more potent than ET‐3 suggesting the presence of constrictor ETA receptors. Little or no response was obtained to the ETB‐selective agonist sarafotoxin 6c. These data suggest that endothelin‐mediated vasoconstriction is via ETA receptors in this preparation.

Autoradiographical visualization of endothelin receptors with subtype selective ligands confirmed the predominance of the ETA receptor in the media of umbilical vein. High density of binding was obtained with the ETA selective [125I]‐PD151242, with much lower levels detected with the ETB selective [125I]‐BQ3020.

Big ET‐1 (EC50=42.7 nm) and big ET‐2(1–38) (EC50=99.0 nm) were less potent than ET‐1 and ET‐2, respectively. Big ET‐2(1–38) was more potent than its isoform big ET‐2(1–37) with concentration–response curves to big ET‐2(1–37) incomplete at 300 nm. No response was obtained to big ET‐3 at concentrations up to 700 nm. The C‐terminal fragments, big ET‐1(22–38) and big ET‐2(22–38) were inactive.

Responses to ET‐1 were unaffected by either the neutral endopeptidase (NEP) inhibitor thiorphan (10−5 m) or by the dual NEP/ECE inhibitor phosphoramidon (10−5 m). Big ET‐1 was also unaffected by thiorphan but antagonized in a concentration‐dependent manner by phosphoramidon (10−5 m and 10−4 m).

Addition of all four big endothelin peptides to human umbilical vein preparations resulted in detectable amounts of ET‐IR in the bathing medium. Therefore, although big ET‐3 was functionally inactive this reflects the low potency of ET‐3 at the ETA receptor rather than the lack of ability of this smooth muscle ECE to convert big ET‐3 to ET‐3.

To conclude we have demonstrated the presence of a phosphoramidon‐sensitive ECE on the smooth muscle layer of the human umbilical vein which can convert big ET‐1, big ET‐2(1–37), big ET‐2(1–38) and big ET‐3 to their mature biologically active forms. The precise subcellular localization of this enzyme and its physiological relevance remains to be determined.
Antithrombotic effects of YM‐60828, a newly synthesized factor Xa inhibitor, in rat thrombosis models and its effects on bleeding time
Authors: Sato Kazuo , Kawasaki Tomihisa , Hisamichi Nami , Taniuchi Yuta , Hirayama Fukushi , Koshio Hiroyuki , Matsumoto Yuzo
Published: January 2005

The effects of YM‐60828, a newly synthesized factor Xa inhibitor, were investigated to analyse the relationship between its antithrombotic effects and its prolongation of template bleeding time in rats. YM‐60828 was compared with argatroban, heparin and dalteparin. All agents were intravenously administered as a bolus.

In ex vivo studies, YM‐60828 and argatroban prolonged both prothrombin time and activated partial thromboplastin time in a dose‐dependent manner, while heparin and dalteparin prolonged only activated partial thromboplastin time.

In a venous thrombosis model, all agents exerted antithrombotic effects in a dose‐dependent manner. The ID50 values of YM‐60828, argatroban, heparin and dalteparin were 0.0081 mg kg−1, 0.011 mg kg−1, 6.3 iu kg−1 and 4.7 iu kg−1, respectively.

In an arterio‐venous shunt model, all agents exerted antithrombotic effects in a dose‐dependent manner. The ID50 values of YM‐60828, argatroban, heparin and dalteparin were 0.010 mg kg−1, 0.011 mg kg−1, 10 iu kg−1 and 4.2  iu  kg−1, respectively.

In bleeding time studies, all agents prolonged template bleeding time in a dose‐dependent manner. ED2 values, the doses causing a 2 fold prolongation of bleeding time in the saline group, of YM‐60828, argatroban, heparin and dalteparin were 0.76 mg kg−1, 0.081 mg kg−1, 18 iu kg−1 and 25 iu kg−1, respectively.

The ratio (ED2/ID50) of YM‐60828 was more than 30 fold greater than that of heparin and more than 10 fold greater than those of argatroban and dalteparin.

These data show that YM‐60828 can exert its antithrombotic effects with little prolongation of bleeding time compared with the other currently used anticoagulant agents.

British Journal of Pharmacology (1998) 123, 92–96; doi:10.1038/sj.bjp.0701566
Differential modulation of rat neuronal nicotinic receptor subtypes by acute application of ethanol
Authors: Covernton Patrick J. O. , Connolly John G.
Published: January 2005

We have studied the effects of acute ethanol (EtOH) exposure on the agonist responses of rat neuronal nicotinic receptors expressed in Xenopus oocytes by means of voltage clamp techniques.

In some cells, agonist‐induced current responses with the α3β4 subunit combination could be either significantly potentiated or inhibited (range 25% to 237% of control response) by low ethanol concentrations (1–30 mm). At high ethanol concentrations (100–300 mm) robust potentiations were observed (range 135% to 305% of control).

The low EtOH concentration effects on the α3β4 subtype exhibited tolerance with repeated EtOH exposure.

In general, the α3β2, α4–1β2 and α4–1β4 subunit combinations were less sensitive to low concentrations of ethanol, but respectively showed potentiations of up to 178%, 226% and 154% at high EtOH concentrations.

The α7 homomeric receptor was also relatively insensitive at low EtOH concentrations. At high EtOH concentrations, potentiations, inhibitions or no alteration of control agonist response were observed (range 88% to 141% of control).

We conclude that all the neuronal nicotinic receptor subunit combinations tested here can be modulated by high concentrations of EtOH in a rapidly reversible manner. This modulation may underlie some of the behavioural effects of ethanol. The α3β4 subunit combination may be especially sensitive to modulation by low EtOH concentrations. This remarkable sensitivity and plasticity of nicotinic receptors may contribute to a process of mutual reinforcement in nicotine and alcohol addiction.
Release of the antioxidants ascorbate and urate from a nitrergically‐innervated smooth muscle
Authors: Lilley Elliot , Gibson Alan
Published: January 2005

The main object of the present study was to determine whether ascorbate, an antioxidant which has been shown to protect nitric oxide (NO) from attack by scavenger molecules, might be released from nitrergically‐innervated smooth muscle; ascorbate release from the rat anococcygeus was measured by use of h.p.l.c. with electrochemical detection.

Incubation of rat anococcygeus muscles in normal physiological salt solution (PSS; 30 min) resulted in release of ascorbate into the bathing medium (7.7±0.9 nmol g−1 tissue). This release was increased by 96% when muscles were incubated in high K+ (70 mm) PSS. The resting release of ascorbate was unaffected by tetrodotoxin (TTX; 1 μm), ω‐conotoxin GVIA (10 nm) or omission of calcium ions from the PSS (with addition of 0.2 mm EGTA), but all three procedures attenuated the increased release observed under depolarizing conditions. Resting release of ascorbate was unaffected by glutamate (100 μm), aspartate (100 μm), γ‐aminobutyric acid (100 μm) or carbachol (50 μm).

A second h.p.l.c. peak, which always preceded the ascorbate peak, was identified as urate. Urate release from the anococcygeus, following 30 min incubation in normal PSS, was 64.6±12.7 nmol g−1 tissue but, unlike ascorbate, urate release was unchanged in high K+ PSS. In functional experiments, urate (100–400 μm) partially protected NO (15 μm)‐induced relaxations of the rat anococcygeus from inhibition by 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (carboxy‐PTIO; 50 μm), but not from inhibition by hydroquinone or duroquinone (both 100 μm).

Muscles chemically sympathectomized with 6‐hydroxydopamine (6‐OHDA, 500 μm; 2 h) still exhibited release of ascorbate (2.5±0.4 nmol g−1 tissue) and urate (22.2±2.9 nmol g−1 tissue); in both cases the release was similar to that observed in time‐matched control tissues not exposed to 6‐OHDA. High K+ PSS produced a TTX‐sensitive increase in release of ascorbate, but not urate, from 6‐OHDA‐treated muscles.

The results demonstrate that significant amounts of ascorbate and urate are released from the rat anococcygeus muscle. Ascorbate, but not urate, release appears to be enhanced by activation of nerves which are resistant to 6‐OHDA pretreatment. Since both antioxidants can protect NO from attack by scavenger molecules, their release in nitrergically‐innervated tissues may be important for the provision of the correct redox environment to allow NO to fulfill its proposed neurotransmitter role.
Okadaic acid‐induced decrease in the magnitude and efficacy of the Ca2+ signal in pancreatic β cells and inhibition of insulin secretion
Authors: Sato Yoshihiko , Mariot Pascal , Detimary Philippe , Gilon Patrick , Henquin Jean‐Claude
Published: January 2005

Phosphorylation by kinases and dephosphorylation by phosphatases markedly affect the biological activity of proteins involved in stimulus‐response coupling. In this study, we have characterized the effects of okadaic acid, an inhibitor of protein phosphatases 1 and 2A, on insulin secretion. Mouse pancreatic islets were preincubated for 60 min in the presence of okadaic acid before their function was studied.

Okadaic acid dose‐dependently (IC50∼200 nM) inhibited insulin secretion induced by 15 mM glucose. At 0.5 μM, okadaic acid also inhibited insulin secretion induced by tolbutamide, ketoisocaproate and high K+, and its effects were not reversed by activation of protein kinases A or C.

The inhibition of insulin secretion did not result from an alteration of glucose metabolism (estimated by the fluorescence of endogenous pyridine nucleotides) or a lowering of the ATP/ADP ratio in the islets.

Okadaic acid treatment slightly inhibited voltage‐dependent Ca2+ currents in β cells (perforated patch technique), which diminished the rise in cytoplasmic Ca2+ (fura‐2 method) that glucose and high K+ produce in islets. However, this decrease (25%), was insufficient to explain the corresponding inhibition of insulin secretion (90%). Moreover, mobilization of intracellular Ca2+ by acetylcholine was barely affected by okadaic acid, whereas the concomitant insulin response was decreased by 85%.

Calyculin A, another inhibitor of protein phosphatases 1 and 2A largely mimicked the effects of okadaic acid, whereas 1‐norokadaone, an inactive analogue of okadaic acid on phosphatases, did not alter β cell function.

In conclusion, okadaic acid inhibits insulin secretion by decreasing the magnitude of the Ca2+ signal in β cells and its efficacy on exocytosis. The results suggest that, contrary to current concepts, both phosphorylation and dephosphorylation of certain β cell proteins may be involved in the regulation of insulin secretion.

British Journal of Pharmacology (1998) 123, 97–105; doi:10.1038/sj.bjp.0701578
Pharmacological analysis of G‐protein activation mediated by guinea‐pig recombinant 5‐HT1B receptors in C6‐glial cells: similarities with the human 5‐HT1B receptor
Authors: Pauwels Petrus J , Wurch Thierry , Palmier Christiane , Colpaert Francis C
Published: January 2005

The guinea‐pig recombinant 5‐hydroxytryptamine1B (gp 5‐HT1B) receptor stably transfected in rat C6‐glial cells was characterized by monitoring G‐protein activation in a membrane preparation with agonist‐stimulated [35S]‐GTPγS binding. The intrinsic activity of 5‐HT receptor ligands was compared with that determined previously at the human recombinant 5‐HT1B (h 5‐HT1B) receptor under similar experimental conditions.

Membrane preparations of C6‐glial/gp 5‐HT1B cells exhibited [3H]‐5‐carboxamidotryptamine (5‐CT) and [3H] ‐ N‐ [4‐methoxy‐3,4 ‐ methylpiperazin‐1‐yl) phenyl] ‐3 ‐ methyl ‐ 4‐(4 ‐ pyridinyl)benzamide (GR 125743) binding sites with a pKd of 9.62 to 9.85 and a Bmax between 2.1 to 6.4 fmol mg−1 protein. The binding affinities of a series of 5‐HT receptor ligands determined with [3H]‐5‐CT and [3H]‐GR 125743 were similar. Ligand affinities were comparable to and correlated (r2: 0.74, P<0.001) with those determined at the recombinant h 5‐HT1B receptor.

[35S]‐GTPγS binding to membrane preparations of C6‐glial/gp 5‐HT1B cells was stimulated by the 5‐HT receptor agonists that were being investigated. The maximal responses of naratriptan, zolmitriptan, sumatriptan, N‐methyl‐3‐[pyrrolidin‐2(R)‐ylmethyl]‐1H‐indol‐5‐ylmethylsulphonamide (CP122638), rizatriptan and dihydroergotamine were between 0.76 and 0.85 compared to 5‐HT. The potency of these agonists showed a positive correlation (r2: 0.72, P=0.015) with their potency at the recombinant h 5‐HT1B receptor. 1‐naphthylpiperazine, (±)‐cyanopindolol and (2′‐methyl‐4′‐(5‐methyl[1,2,4] oxadiazole‐3‐yl)biphenyl‐4‐carboxylic acid [4‐methoxy‐3‐(4‐methylpiperazin‐1‐yl)phenyl]amide (GR 127935) elicited an even smaller response (Emax: 0.32 to 0.63).

The ligands 1′‐methyl‐5‐(2′‐methyl‐4′‐(5‐methyl‐1,2,4‐oxadiazole‐3‐yl) biphenyl‐4‐carbonyl)‐2,3,6,7‐tetrahydrospiro [furo[2,3‐f]indole‐3‐spiro‐4′‐piperidine] (SB224289), methiothepin and ritanserin displayed inhibition of basal [35S]‐GTPγS binding at concentrations relevant to their binding affinity for the gp 5‐HT1B receptor. Methiothepin and SB224289 behaved as competitive antagonists at gp 5‐HT1B receptors; pA2 values were 9.74 and 8.73, respectively when 5‐HT was used as an agonist. These estimates accorded with the potencies measured in antagonism of zolmitriptan‐mediated inhibition of forskolin‐stimulated cyclic AMP formation. Ketanserin acted as a weak antagonist (pKB: 5.87) at gp 5‐HT1B receptors.

In conclusion, the recombinant gp 5‐HT1B receptor shares important pharmacological similarities with the recombinant h 5‐HT1B receptor. The finding that negative activity occurs at these receptors further suggests that SB224289, methiothepin and ritanserin are likely to be inverse agonists.

British Journal of Pharmacology (1998) 123, 51–62; doi:10.1038/sj.bjp.0701584
Modulation by adenine nucleotides of epileptiform activity in the CA3 region of rat hippocampal slices
Authors: Ross F M , Brodie M J , Stone T W
Published: January 2005

Hippocampal slices (450 μm) generate epileptiform bursts of an interictal nature when perfused with a zero magnesium medium containing 4‐aminopyridine (50 μM). The effect of adenine nucleotides on this activity was investigated.

ATP and adenosine depressed this epileptiform activity in a concentration‐dependent manner, with both purines being equipotent at concentrations above 10 μM.

Adenosine deaminase 0.2 u ml−1, a concentration that annuls the effect of adenosine (50 μM), did not significantly alter the depression of activity caused by ATP (50 μM).

8‐Cyclopentyl‐1, 3‐dimethylxanthine (CPT), an A1 receptor antagonist, enhanced the discharge rate significantly and inhibited the depressant effect of both ATP and adenosine such that the net effect of ATP or adenosine plus CPT was excitatory.

Several ATP analogues were also tested: α, β‐methyleneATP (α, β‐meATP), 2‐methylthioATP (2‐meSATP) and uridine triphosphate (UTP). Only α, β‐meATP (10 μM) produced an increase in the frequency of spontaneous activity which suggests a lack of involvement of P2Y or P2U receptors.

Suramin and pyridoxalphosphate‐6‐azophenyl‐2′, 4′‐disulphonic acid (PPADS), P2 receptor antagonists, failed to inhibit the depression produced by ATP (50 μM). The excitatory effect of α, β‐meATP (10 μM) was inhibited by suramin (50 μM) and PPADS (5 μM).

ATP therefore depresses epileptiform activity in this model in a manner which is not consistent with the activation of known P1 or P2 receptors, suggesting the involvement of a xanthine‐sensitive nucleotide receptor. The results are also indicative of an excitatory P2X receptor existing in the hippocampal CA3 region.

British Journal of Pharmacology (1998) 123, 71–80; doi:10.1038/sj.bjp.0701586
Preferential expression of the neuropeptide Y Y1 over the Y2 receptor subtype in cultured hippocampal neurones and cloning of the rat Y2 receptor
Authors: St‐Pierre Jacques‐André , Dumont Yvan , Nouel Dominique , Herzog Herbert , Hamel Edith , Quirion Rémi
Published: January 2005

Neuropeptide Y (NPY) and NPY receptors are most abundant in the hippocampal formation where they modulate cognitive functions. Expression of NPY receptors in rat cultured primary hippocampal cells was investigated in the present study by use of combined molecular, pharmacological and immunohistochemical approaches, including the cloning of the rat Y2 receptor described here for the first time.

More than 70% of the hippocampal neurones were endowed with [125I]‐[Leu31,Pro34]PYY Y1‐like receptor silver grain accumulations and Y1 receptor immunostaining. These radio‐ and immuno‐labelling signals were distributed over cell bodies and processes of bipolar, stellate and pyramidal‐like neuronal cells, as confirmed by neurone‐specific enolase and MAP‐2 staining.

Competition binding profiles revealed that specific [125I]‐[Leu31,Pro34]PYY binding was competitively displaced according to a ligand selectivity pattern prototypical of the Y1 receptor sub‐type with [Leu31,Pro34]substituted NPY/PYY analogues>>C‐terminal fragments=pancreatic polypeptides, with the non‐peptide antagonist BIBP3226 being most potent. This profile excludes the possible labelling by [125I]‐[Leu31,Pro34]PYY of the newly cloned Y4, Y5 and Y6 receptors.

The expression of the genuine Y1 receptor was confirmed by RT–PCR in hippocampal cultures. In contrast, negligible levels of Y2‐like/[125I]‐PYY3–36 binding were detected in these cultures in spite of the presence of its mRNA, as characterized by RT–PCR. The expression of both the Y1 and the Y2 receptor mRNAs was also noted in normal embryonic hippocampal tissues showing that signals expressed in cultured neurones were also present in utero.

Taken together, these results suggest that the Y1 receptor subtype may be of critical importance in the normal functioning of the rat hippocampus, especially during brain development and maturation.

British Journal of Pharmacology (1998) 123, 183–194; doi:10.1038/sj.bjp.0701589
Influence of dietary salts on the cardiovascular effects of low‐dose combination of ramipril and felodipine in spontaneously hypertensive rats
Authors: Mervaala Eero M A , Malmberg Lena , Teräväinen Terttu‐Liisa , Laakso Juha , Vapaatalo Heikki , Karppanen Heikki
Published: January 2005

In spontaneously hypertensive rat (SHR) we examined over a 4‐week period the influence of control low sodium diet, common salt‐enriched diet (sodium chloride 6% of the dry weight of the chow) and a novel mineral salt‐enriched diet (potassium‐, magnesium‐, and l‐lysine‐enriched mineral salt added at a 75% higher level of 10.5% to produce the same sodium chloride concentration of 6%) on the cardiovascular effects produced by a low‐dose combination of an angiotensin converting enzyme inhibitor ramipril (0.25 mg kg−1 day−1 in the food) and a calcium channel blocker felodipine (0.4 mg kg−1 day−1 subcutaneously via an osmotic minipump).

Common salt, but not the mineral salt, accelerated the development of hypertension and induced left ventricular and renal hypertrophy in SHR. Neither common salt nor mineral salt significantly affected heart rate.

The combination of ramipril and felodipine decreased systolic blood pressure and prevented the development of left ventricular hypertrophy effectively during the common salt diet without any significant effect on the heart rate. The cardiovascular effects of the drug combination were improved by the low sodium diet or by replacement of high common salt in the diet by mineral salt.

Responses of endothelium‐intact mesenteric arterial rings in vitro were examined at the end of the four‐week study. The combination of ramipril and felodipine markedly improved the endothelium‐dependent vascular relaxation responses to acetylcholine and enhanced the endothelium‐independent vascular relaxation responses to sodium nitroprusside in SHR on control and common salt diets. Replacement of common salt in the diet by mineral salt improved the endothelium‐dependent vascular relaxation responses to acetylcholine. The drug combination attenuated the α‐adrenoceptor‐mediated vascular contractile responses to noradrenaline during the common salt diet.

Ramipril and felodipine in combination increased plasma renin activity by 1.9–3.2 fold without affecting serum aldosterone levels.

Our findings suggest that the cardiovascular effect of the low‐dose combination of ramipril and felodipine was maintained during high salt intake. However, salt restriction or replacement of common salt in the diet by the potassium‐ and magnesium‐enriched mineral salt improved the cardiovascular effects of the drug combination. In the face of a high intake of sodium, a part of the beneficial cardiovascular effects of the drug combination is apparently mediated by improved endothelium‐dependent and endothelium‐independent vascular relaxation responses and attenuated α‐adrenoceptor‐mediated vascular contractile responses.

British Journal of Pharmacology (1998) 123, 195–204; doi:10.1038/sj.bjp.0701591
Distinct inhibitory effects of spinal endomorphin‐1 and endomorphin‐2 on evoked dorsal horn neuronal responses in the rat
Authors: Chapman Victoria , Diaz Alvaro , Dickenson Anthony H.
Published: January 2005

Intrathecal endomorphin‐1 and endomorphin‐2 (0.25–50 μg) dose‐relatedly reduced all components of electrical evoked C‐fibre responses of spinal neurones. These effects were partially reversed by naloxone. Endomorphin‐1, but not endomorphin‐2, dose‐relatedly reduced the Aβ‐fibre evoked responses. Peak inhibitory effects of endomorphin‐1 and −2 were at 15–20 min post‐administration. Thus spinal endomorphin‐2 had selective effects on noxious responses, whereas endomorphin‐1 was non‐selective.
Comparison of the effects of drugs on hyperexcitability induced in hippocampal slices by withdrawal from chronic ethanol consumption
Authors: Bailey C P , Molleman A , Little H J
Published: January 2005

The effects of drugs, previously demonstrated to have a range of effects on the behavioural signs of ethanol withdrawal hyperexcitability, were examined in area CA1 in isolated hippocampal slices prepared after withdrawal from chronic ethanol in vivo.

The decreases seen after the ethanol treatment in the thresholds for production of single and multiple population spikes were prevented when the dihydropyridine calcium channel antagonist, isradipine, was included in the perfusion medium at 4 μM.

Another dihydropyridine, felodipine, which had no activity against withdrawal signs in vivo, did not affect the changes in field potentials, at concentrations up to 10 μM.

Diltiazem, which increased withdrawal hyperexcitability in vivo, had no effect on the withdrawal changes in field potentials at 30 μM; higher concentrations affected the control slices.

The novel anticonvulsant, gabapentin, at 1 μM but not at 100 nM, significantly decreased the signs of withdrawal hyperexcitability in the hippocampal slices. When the CCKB antagonist, CI988, was added to the bathing medium, at 1 μM, there were small, but significant decreases in the withdrawal hyperexcitability.

The results showed that the actions of these drugs on the changes in the field potentials in isolated hippocampal slices were very similar to their previously demonstrated effects on the convulsive signs of ethanol withdrawal in vivo, but differences were seen in the corresponding comparison with anxiolytic actions in vivo.

British Journal of Pharmacology (1998) 123, 215–222; doi:10.1038/sj.bjp.0701596
Ethanol‐induced inhibition of a neuronal P2X purinoceptor by an allosteric mechanism
Authors: Li Chaoying , Peoples Robert W , Weight Forrest F
Published: January 2005

Ethanol inhibits a neuronal P2X purinoceptor by shifting the ATP concentration–response curve to the right in an apparently competitive manner. However, the underlying mechanism has not been determined. We investigated the effects of ethanol on the activation and deactivation time constants for ATP‐activated current in bullfrog dorsal root ganglion neurones. Ethanol decreased the time constant of deactivation of ATP‐gated ion channels without affecting the time constant of activation. The observations are not consistent with a competitive mechanism of inhibition by ethanol, but may be explained by an allosteric action of ethanol to decrease apparent agonist affinity. This represents a novel mechanism of action of ethanol on a neurotransmitter‐gated ion channel.

British Journal of Pharmacology (1998) 123, 1–3; doi:10.1038/sj.bjp.0701599
Efficacy of inverse agonists in cells overexpressing a constitutively active β2‐adrenoceptor and type II adenylyl cyclase
Authors: Stevens Patricia A , Milligan Graeme
Published: January 2005

Maximal stimulant output from the adenylyl cyclase cascade in neuroblastoma × glioma hybrid, NG108‐15, cells is limited by the levels of expression of isoforms of adenylyl cyclase. Stable expression in these cells of a constitutively active mutant (CAM) version of the human β2‐adrenoceptor resulted in higher basal adenylyl cyclase activity than following expression of the human wild type β2‐adrenoceptor. Isoprenaline acted as a full agonist in membranes from both wild type and CAM β2‐adrenoceptor expressing clones.

Expression of type II adenylyl cyclase resulted in a substantially elevated capacity of isoprenaline to stimulate [3H]‐forskolin binding, whereas in CAM β2‐adrenoceptor expressing cells the basal high affinity [3H]‐forskolin binding represented a markedly greater % of the maximal effect which could be produced by addition of isoprenaline, and the EC50 for isoprenaline was some 10 fold lower than in cells expressing the wild type β2‐adrenoceptor.

Further transfection of the CAM β2‐adrenoceptor expressing cells with type II adenylyl cyclase greatly increased both absolute basal and agonist‐stimulated levels of adenylyl cyclase activity.

Betaxolol, ICI 118,551, sotalol and timolol acted as inverse agonists with varying degrees of efficacy, whereas propranolol functioned as a neutral antagonist and alprenolol as a partial agonist.

Pretreatment of the CAM β2‐adrenoceptor and type II adenylyl cyclase expressing clones with the irreversible alkylating agent BAAM (1 μM) did not reduce the efficacy of isoprenaline but eliminated efficacy from all the inverse agonist ligands. This effect was dependent upon the concentration of BAAM employed, with half‐maximal effects being produced between 10 nM and 100 nM of the alkylating agent, which is similar to the concentrations required to prevent subsequent ligand access to some 50% of the CAM β2‐adrenoceptor population.

These data demonstrate that inverse agonist efficacy can be modulated by receptor availability and also indicate why in physiological systems, inverse agonism can be difficult to detect.

British Journal of Pharmacology (1998) 123, 335–343; doi:10.1038/sj.bjp.0701600
Interactions between P‐glycoprotein substrates and other cationic drugs at the hepatic excretory level
Authors: Smit Johan W. , Duin Erik , Steen Herman , Oosting Roelof , Roggeveld Jan , Meijer Dirk K F.
Published: January 2005

In the present study it was tested whether known P‐glycoprotein (P‐gp) substrates/MDR reversal agents interact with small (type 1) and bulky (type 2) cationic drugs at the level of biliary excretion in the rat isolated perfused liver model (IPRL). The studies were performed with model compounds tri‐n‐butylmethylammonium (TBuMA) (a relatively small type 1 organic cation), rocuronium (Roc) (a bulky type 2 organic cation) and the classical P‐gp substrate doxorubicin (Dox).

Inhibitors were given in a 4 fold molar excess to the substrate studied. To minimize an interaction of the substrates at the hepatic uptake level, the competing compounds were added when over 55% to 85% of the administered dose of the model compounds had been removed from the perfusate and taken up by the liver.

We found a mutual interaction between TBuMA and procainamidethobromide (PAEB), both type 1 cationic compounds during biliary excretion. Interestingly, type 2 compounds, such as rocuronium, clearly inhibited type 1 cationic drugs as well as Dox secretion into bile, whereas type 1 compounds did not significantly inhibit type 2 drug excretion into bile. The type 1 cations PAEB and TBuMA only moderately inhibited Dox biliary excretion. Dox did not inhibit the biliary excretion of the type 2 agent rocuronium whereas rocuronium reduced Dox biliary excretion by 50% compared to controls.

MDR substrates/reversal agents like verapamil, quinine, quinidine and vinblastine strongly reduced both type 1 and type 2 organic cation excretion into bile. Dox secretion into bile was also profoundly reduced by these drugs, vinblastine being the most potent inhibitor in general.

The lack of mutual inhibition observed in some combinations of substrates may indicate that major differences in affinity of the substrates for a single excretory system exist. Alternatively, multiple organic cation transport systems with separate substrate specificities may be involved in the biliary excretion of amphiphilic drugs. Furthermore, the present study revealed a clear positive correlation between the lipophilicity of the potential inhibitors studied and their respective inhibitory activity on the biliary excretion of the model drugs investigated.

Our data are compatible with a potential involvement of P‐glycoprotein in the hepatobiliary excretion of doxorubicin as well as of some type 1 and type 2 organic cations. Furthermore we postulate that the hydrophobic properties of the amphiphilic cationic drugs studied play a crucial role in the accommodation of these agents by P‐glycoprotein and/or other potential cationic drug carrier proteins in the canalicular membrane.

British Journal of Pharmacology (1998) 123, 361–370; doi:10.1038/sj.bjp.0701606
Effect of a monoclonal antibody against interleukin‐4 on collagen‐induced arthritis in mice
Authors: Yoshino Shin
Published: January 2005

The present study was undertaken to investigate the effect of a monoclonal antibody (11B11 mAb) against interleukin‐4 (IL‐4) on collagen‐induced arthritis (CIA) in mice.

11B11 mAb was daily injected intraperitoneally over a period of 10 days, commencing on the day of immunization with type II collagen (CII).

The results showed that the anti‐IL‐4 mAb markedly augmented both the incidence and the severity of CIA. The augmentation of the disease was associated with a significant increase in anti‐CII IgG2a antibody production, proliferative responses of lymph node cells to CII and interferon‐γ (IFN‐γ) secretion from the lymphoid cells. The production of anti‐CII IgG1 antibodies the secretion of IL‐4 was markedly reduced in the mAb‐treated mice.

Thus, the neutralization of IL‐4 by 11B11 mAb appears to be effective in augmenting CIA.

British Journal of Pharmacology (1998) 123, 237–242; doi:10.1038/sj.bjp.0701607
Effects of the α subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors
Authors: Matsuda K. , Buckingham S D. , Freeman J C. , Squire M D. , Baylis H A. , Sattelle D B.
Published: January 2005

Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (α4β2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co‐expression of a Drosophila melanogaster neuronal α subunit (SAD) with the chicken β2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)‐epibatidine, (−)‐nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two‐electrode, voltage‐clamp electrophysiology.

Imidacloprid alone of the 4 agonists behaved as a partial agonist on the α4β2 receptor; (+)‐epibatidine, (−)‐nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken β2 receptor, as was (−)‐nicotine, whereas (+)‐epibatidine and acetylcholine were full agonists.

The EC50 of imidacloprid was decreased by replacing the chicken α4 subunit with the Drosophila SAD α subunit. This α subunit substitution also resulted in an increase in the EC50 for (+)‐epibatidine, (−)‐nicotine and acetylcholine. Thus, the Drosophila (SAD) α subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs.

Imidacloprid acted as a weak antagonist of ACh‐mediated responses mediated by SADβ2 hybrid receptors and as a weak potentiator of ACh responses mediated by α4β2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the α subunit.

British Journal of Pharmacology (1998) 123, 518–524; doi:10.1038/sj.bjp.0701618
Effects of clozapine on the δ‐ and κ‐opioid receptors and the G‐protein‐activated K+ (GIRK) channel expressed in Xenopus oocytes
Authors: Kobayashi Toru , Ikeda Kazutaka , Kumanishi Toshiro
Published: January 2005

To investigate the effects of clozapine, an atypical antipsychotic, on the cloned μ‐, Δ‐ and κ‐opioid receptors and G‐protein‐activated inwardly rectifying K+ (GIRK) channel, we performed the Xenopus oocyte functional assay with each of the three opioid receptor mRNAs and/or the GIRK1 mRNA.

In the oocytes co‐injected with either the Δ‐ or κ‐opioid receptor mRNA and the GIRK1 mRNA, application of clozapine induced inward currents which were attenuated by naloxone, an opioid‐receptor antagonist, and blocked by Ba2+, which blocks the GIRK channel. Since the opioid receptors functionally couple to the GIRK channel, these results indicate that clozapine activates the Δ‐ and κ‐opioid receptors and that the inward‐current responses are mediated by the GIRK channel. The action of clozapine at the Δ‐opioid receptor was more potent and efficacious than that at the κ‐opioid receptor. In the oocytes co‐injected with the μ‐opioid receptor and GIRK1 mRNAs, application of clozapine (100 μM) did not induce an inward current, suggesting that clozapine could not activate the μ‐opioid receptor.

Application of clozapine caused a reduction of the basal inward current in the oocytes injected with the GIRK1 mRNA alone, but caused no current response in the uninjected oocytes. These results indicate that clozapine blocks the GIRK channel.

To test the antagonism of clozapine for the μ‐ and κ‐opioid receptors, we applied clozapine together with each selective opioid agonist to the oocytes co‐injected with either the μ‐ or κ‐opioid receptor mRNA and the GIRK1 mRNA. Each of the peak currents induced by each selective opioid agonist together with clozapine was almost equal to the responses to a selective opioid agonist alone. These results indicate that clozapine has no significant antagonist effect on the μ‐ and κ‐opioid receptors.

We conclude that clozapine acts as a Δ‐ and κ‐agonist and as a GIRK channel blocker. Our results suggest that the efficacy and side effects of clozapine under clinical conditions may be partly due to activation of the Δ‐opioid receptor and blockade of the GIRK channel.

British Journal of Pharmacology (1998) 123, 421–426; doi:10.1038/sj.bjp.0701621
Effects of the antihypertensive agent, cicletanine, on noradrenaline release and vasoconstriction in perfused mesenteric artery of SHR
Authors: Nasa Yoshihisa , Yoshida Hiroyuki , Urata Maki , Uchibayashi Kumiko , Tsunoda Yumi , Kamigata Keiko , Takeo Satoshi
Published: January 2005

The mechanism by which cicletanine (CIC) exerts its antihypertensive effects has not been fully elucidated. The present study was undertaken to examine the effects of in vivo and in vitro treatment with CIC on the pressor response and noradrenaline (NA) overflow during periarterial nerve stimulation (PNS) in perfused mesenteric arterial beds isolated from spontaneously hypertensive rats (SHR).

CIC at a dose of 50 mg kg−1 day−1 was administered orally to both SHR and normotensive Wistar‐Kyoto rats (WKY) from the 6th to 10th week of age. At the 10th week, the isolated mesenteric arterial bed was perfused with Krebs‐Henseleit buffer and changes in perfusion pressure and NA overflow during PNS were measured.

Chronic treatment with CIC suppressed the age‐related elevation of systemic blood pressure in SHR but not in WKY.

The PNS (20 Hz)‐induced mesenteric vasoconstrictor response and NA overflow were greater in SHR than in WKY. In the vasculature of SHR chronic treatment with CIC resulted in a significant attenuation of the vasoconstriction and the NA overflow during PNS, whereas it did not alter vasoconstrictor responses to bolus injections of KCl and phenylephrine.

Treatment with 30 μM CIC in vitro diminished the PNS‐induced vasoconstriction and NA overflow but not the NA‐ and KCl‐induced vasoconstriction in the vasculature of untreated SHR.

In the vasculature of SHR PNS‐induced NA overflow was attenuated by prostaglandin E2 (0.05 μM), whereas it was augmented by the cyclo‐oxygenase inhibitor diclofenac‐Na (30 μM). In the presence of diclofenac, in vitro treatment with CIC did not attenuate the NA overflow during PNS.

The results suggest that the antihypertensive effect of CIC in SHR is partially due to the presynaptic inhibition of NA release during sympathetic nerve activation. Transjunctional inhibition of NA release by prostaglandins may contribute to the inhibitory action of CIC on NA release in the vasculature of SHR.

British Journal of Pharmacology (1998) 123, 427–434; doi:10.1038/sj.bjp.0701622