bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the effects of [Phe1Ψ(CH2‐NH)Gly2]Nociceptin (1–13)NH2 in rat brain, rat vas deferens and CHO cells expressing recombinant human nociceptin receptors
Authors: Okawa Hirobumi , Nicol Beverley , Bigoni Raffaella , Hirst Robert A , Calo' Girolamo , Guerrini Remo , Rowbotham David J , Smart Darren , McKnight Alexander T , Lambert David G
Published: January 2005

Nociceptin(NC) is the endogenous ligand for the opioid receptor like‐1 receptor (NC‐receptor). [Phe1ΨC(CH2‐NH)Gly2]Nociceptin(1–13)NH2 ([F/G]NC(1–13)NH2) has been reported to antagonize NC actions in peripheral guinea‐pig and mouse tissues. In this study, we investigated the effects of a range of NC C‐terminal truncated fragments and [F/G]NC(1–13)NH2 on NC receptor binding, glutamate release from rat cerebrocortical slices (rCX), inhibition of cyclic AMP accumulation in CHO cells expressing the NC receptor (CHONCR) and electrically evoked contractions of the rat vas deferens (rVD).

In radioligand binding assays, a range of ligands inhibited [125I]‐Tyr14‐NC binding in membranes from rCX and CHONCR cells. As the peptide was truncated there was a general decline in pKi. [F/G]NC(1–13)NH2 was as potent as NC(1–13)NH2.

The order of potency for NC fragments to inhibit cyclic AMP accumulation in whole CHONCR cells was NCNH2NC=NC(1–13)NH2>NC(1–12)NH2>>NC(1–11)NH2. [F/G]NC(1–13)NH2 was a full agonist with a pEC50 value of 8.65.

NCNH2 and [F/G]NC(1–13)NH2 both inhibited K+ evoked glutamate release from rCX with pEC50 and maximum inhibition of 8.16, 48.5±4.9% and 7.39, 58.9±6.8% respectively.

In rVD NC inhibited electrically evoked contractions with a pEC50 of 6.63. Although [F/G]NC(1–13)NH2, displayed a small (instrinsic activity α=0.19) but consistent residual agonist activity, it acted as a competitive antagonist (pA2 6.76) in the rVD.

The differences between [F/G]NC(1–13)NH2 action on central and peripheral NC signalling could be explained if [F/G]NC(1–13)NH2 was a partial agonist with high strength of coupling in the CNS and low in the periphery. An alternative explanation could be the existence of central and peripheral receptor isoforms.

British Journal of Pharmacology (1999) 127, 123–130; doi:10.1038/sj.bjp.0702539
Comparison of the redox forms of nitrogen monoxide with the nitrergic transmitter in the rat anococcygeus muscle
Authors: Li Chun Guang , Karagiannis Joanna , Rand Michael J
Published: January 2005

A sustained tone was produced in rat isolated anococcygeus muscles with guanethidine and clonidine and relaxant responses were elicited by electrical stimulation of its nitrergic nerves and by the three redox forms of nitrogen monoxide.

The nitroxyl anion (NO−) was donated by dissociation of Angeli's salt; the free radical (NO•) was from an aqueous solution of nitric oxide gas; the nitrosonium cation (NO+) was donated by dissociation of nitrosonium tetrafluoroborate.

The concentrations producing approximately 50% relaxations of the anococcygeus muscle were 0.3 μM for Angeli's salt (nitroxyl), 0.5 μM for NO• and 100 μM for nitrosonium tetrafluoroborate. Nitrergic nerve stimulation at 1 Hz for 10 s produced equivalent relaxant responses.

The superoxide generator pyrogallol (100 μM) had no effect on responses to nitrergic nerve stimulation or Angeli's salt but significantly reduced responses to NO• and nitrosonium tetrafluoroborate.

The NO• scavenger carboxy‐PTIO (100 μM) had no effect on responses to nitrergic nerve stimulation or Angeli's salt but significantly reduced responses to NO• and nitrosonium tetrafluoroborate.

Hydroxocobalamin (30 μM) had no significant effect on responses to the nitrergic transmitter, enhanced the response to Angeli's salt, and significantly reduced responses to NO• and nitrosonium tetrafluoroborate.

The findings suggest that the nitroxyl anion donated by Angeli's salt is a better candidate than NO• to serve as the nitrergic transmitter in the rat anococcygeus muscle, although it still does not behave exactly like the transmitter.

British Journal of Pharmacology (1999) 127, 826–834; doi:10.1038/sj.bjp.0702540
Effects of (−)‐tertatolol, (−)‐penbutolol and (±)‐pindolol in combination with paroxetine on presynaptic 5‐HT function: anin vivomicrodialysis and electrophysiological study
Authors: Gartside S E , Clifford E M , Cowen P J , Sharp T
Published: January 2005

The antidepressant efficacy of selective serotonin reuptake inhibitors (SSRIs) might be enhanced by co‐administration of 5‐HT1Areceptor antagonists. Thus, we have recently shown that the selective 5‐HT1Areceptor antagonist, WAY 100635, blocks the inhibitory effect of an SSRI on 5‐HT cell firing, and enhances its ability to elevate extracellular 5‐HT in the forebrain. Here we determined whether the β‐adrenoceptor/5‐HT1Areceptor ligands (±)‐pindolol, (−)‐tertatolol and (−)‐penbutolol, interact with paroxetine in a similar manner.

Both (−)‐tertatolol (2.4 mg kg−1i.v.) and (−)‐penbutolol (2.4 mg kg−1i.v.) enhanced the effect of paroxetine (0.8 mg kg−1i.v.) on extracellular 5‐HT in the frontal cortex, whilst (±)‐pindolol (4 mg kg−1i.v.) did not. (−)‐Tertatolol (2.4 mg kg−1i.v.) alone caused a slight increase in 5‐HT however, (−)‐penbutolol (2.4 mg kg−1i.v.) alone had no effect.

In electrophysiological studies (−)‐tertatolol (2.4 mg kg−1i.v.) alone had no effect on 5‐HT cell firing but blocked the inhibitory effect of paroxetine. In contrast, (−)‐penbutolol (0.1–0.8 mg kg−1i.v.) itself inhibited 5‐HT cell firing, and this effect was reversed by WAY 100635 (0.1 mg kg−1i.v.). We have recently shown that (±)‐pindolol inhibits 5‐HT cell firingviaa WAY 100635‐sensitive mechanism.

Our data suggest that (−)‐tertatolol enhances the effect of paroxetine on forebrain 5‐HTviablockade of 5‐HT1Aautoreceptors which mediate paroxetine‐induced inhibition of 5‐HT cell firing. In comparison, the mechanisms by which (−)‐penbutolol enhances the effect of paroxetine on extracellular 5‐HT is unclear, since (−)‐penbutolol itself appears to have agonist properties at the 5‐HT1Aautoreceptor. Indeed, the agonist action of (±)‐pindolol at 5‐HT1Aautoreceptors probably explains its inability to enhance the effect of paroxetine on 5‐HT in the frontal cortex.

Overall, our data suggest that both (−)‐tertatolol and (−)‐penbutolol are superior to (±)‐pindolol in terms of enhancing the effect of an SSRI on extracellular 5‐HT. Both (−)‐tertatolol and (−)‐penbutolol are worthy of investigation for use as adjuncts to SSRIs in the treatment of major depression.

British Journal of Pharmacology(1999)127, 145–152; doi:10.1038/sj.bjp.0702546
Synergistic effect with Phe‐Gly‐Leu‐Met‐NH2 of the C‐terminal of substance P and insulin‐like growth factor‐1 on epithelial wound healing of rabbit cornea
Authors: Nakamura Masatsugu , Chikama Tai‐ichiro , Nishida Teruo
Published: January 2005

We previously reported that substance P and insulin‐like growth factor‐1 (IGF‐1) synergistically stimulate corneal epithelial wound healing in vitro and in vivo. We wished to identify which portion of the amino acid sequence of substance P might be responsible for this synergism.

Corneal epithelial migration was not affected by the addition of any one of the following factors: substance P; Phe‐Gly‐Leu‐Met‐NH2 (C‐terminal of substance P); Val‐Gly‐Leu‐Met‐NH2 (C‐terminal of neurokinin A, neurokinin B, and kassinin); Tyr‐Gly‐Leu‐Met‐NH2 (C‐terminal of physalaemin); Ile‐Gly‐Leu‐Met‐NH2 (C‐terminal of eledoisin); or Gly‐Leu‐Met‐NH2 (common C‐terminal of tachykinins).

In the presence of IGF‐1, only substance P and Phe‐Gly‐Leu‐Met‐NH2 were synergistic in stimulating corneal epithelial migration in a dose‐dependent fashion.

The combination of Phe‐Gly‐Leu‐Met‐NH2 and IGF‐1 did not affect the incorporation of [3H]‐thymidine into corneal epithelial cells.

Treatment with Phe‐Gly‐Leu‐Met‐NH2 and IGF‐1, but not with Phe‐Gly‐Leu‐Met‐NH2 or IGF‐1 alone, increased attachment of corneal epithelial cells to a fibronectin matrix.

The levels of α5 and β1 integrin were not affected by Phe‐Gly‐Leu‐Met‐NH2 or IGF‐1 alone, but they were significantly increased by the combination of Phe‐Gly‐Leu‐Met‐NH2 and IGF‐1.

Topical application of the same combination facilitated corneal epithelial wound closure in vivo.

These results demonstrated that Phe‐Gly‐Leu‐Met‐NH2, a sequence of 4 amino‐acids of the C‐terminal of substance P, is the minimum sequence necessary to produce the synergistic effects of substance P and IGF‐1 on corneal epithelial wound healing.

British Journal of Pharmacology (1999) 127, 489–497; doi:10.1038/sj.bjp.0702550
Characterization of [3H]‐heparin binding in human vascular smooth muscle cells and its relationship to the inhibition of DNA synthesis
Authors: Patel Mahendra K , Refson Jonathan S , Schachter Michael , Hughes Alun D
Published: January 2005

The glycosaminoglycan heparin inhibits vascular smooth muscle cell (VSMC) proliferation and migration, but the mechanism of its antiproliferative action remains unclear. Heparin has been reported to bind to high affinity cell surface sites on animal VSMC before undergoing receptor mediated endocytosis resulting in signal transduction into the cytoplasm and modulation of genes involved in proliferation. In this study, we have characterized the binding of [3H]‐heparin to human saphenous vein‐derived VSMC and examined whether there is any relationship between the affinity of [3H]‐heparin binding and the inhibitory effect of heparin and its structural analogues on DNA synthesis.

At 4°C [3H]‐heparin binding to human VSMC occurred in a specific, time and concentration‐dependent manner and was not influenced by the removal of calcium ions. Binding of the ligand appeared to occur to the cell surface and was both saturable and reversible. Kinetic and steady state data indicated a single class of binding sites.

The pharmacology of [3H]‐heparin binding was examined in displacement studies using unlabelled heparin and structural analogues. A comparison of the rank potencies of heparin, heparan sulphate fraction II, low molecular weight heparin and trehalose octasulphate showed that there was a marked discrepancy between their estimated affinities in the binding assays and their effect on DNA synthesis.

In summary, we have characterized the heparin binding site on human saphenous vein‐derived VSMC. Our findings suggest that the action of heparin and its analogues on DNA synthesis does not simply reflect an interaction with the cell‐associated heparin binding site defined in these studies, but may also be determined by the internalization and metabolism of the glycosaminoglycan(s).

British Journal of Pharmacology (1999) 127, 361–368; doi:10.1038/sj.bjp.0702559
Capsaicin‐insensitive sensory‐efferent meningeal vasodilatation evoked by electrical stimulation of trigeminal nerve fibres in the rat
Authors: Peitl Barna , Pethô Gábor , Pórszász Róbert , Németh József , Szolcsányi János
Published: January 2005

Antidromic vasodilatation and plasma extravasation to stimulation of the trigeminal ganglion or its perivascular meningeal fibres was investigated by laser‐Doppler flowmetry and 125I‐labelled bovin serum albumin in the dura mater and in exteroceptive areas (nasal mucosa, upper eyelid) of anaesthetized rats pretreated with guanethidine and pipecuronium.

Trigeminal stimulation at 5 Hz for 20 s elicited unilateral phasic vasodilatation in the dura and lasting response in the nasal mucosa. Resiniferatoxin (1–3 μg kg−1 i.v.), topical (1%) or systemic capsaicin pretreatment (300 mg kg−1 s.c. plus 1 mg kg−1 i.v.) did not inhibit the meningeal responses but abolished or strongly inhibited the nasal responses. Administration of vinpocetine (3 mg kg−1 i.v.) increased both basal blood flow and the dural vasodilatation to perivascular nerve stimulation.

Dural vasodilatation to trigeminal stimulation was not inhibited by the calcitonin gene‐related peptide‐1 receptor (CGRP‐1) antagonist hCGRP8–37 (15 or 50 μg kg−1 i.v). or the neurokinin‐1 receptor antagonist RP 67580 (0.1 mg kg−1 i.v.) although both antagonists inhibited the nasal response. Neither mucosal nor meningeal responses were inhibited by atropine (5 mg kg−1 i.v.), hexamethonium (10 mg kg−1 i.v.) or the vasoactive intestinal polypeptide (VIP) antagonist (p‐chloro‐D‐Phe6‐Leu17)VIP (20 μg kg−1 i.v.).

Plasma extravasation in the dura and upper eyelid elicited by electrical stimulation of the trigeminal ganglion was almost completely abolished in rats pretreated with resiniferatoxin (3 μg kg−1 i.v.).

It is concluded that in the rat meningeal vasodilatation evoked by stimulation of trigeminal fibres is mediated by capsaicin‐insensitive primary afferents, while plasma extravasation in the dura and upper eyelid and the vasodilatation in the nasal mucosa are mediated by capsaicin‐sensitive trigeminal fibres.

British Journal of Pharmacology (1999) 127, 457–467; doi:10.1038/sj.bjp.0702561
Subunit mutations affect ethanol actions on GABAA receptors expressed in Xenopus oocytes
Authors: Ueno Susumu , Wick Marilee J , Ye Qing , Harrison Neil L , Harris R Adron
Published: January 2005

Mutations of specific amino acids were introduced in transmembrane domains (TM) of GABAA receptor α2, β1 and γ2L subunits. The effects of these mutations on the action of ethanol were studied using the Xenopus oocyte expression system and two‐electrode voltage‐clamp recording techniques.

Mutant α2 subunits containing S270I (TM2) or A291W (TM3) made the receptor more sensitive to GABA, as compared to wild‐type α2β1γ2L receptor. The mutation S265I (TM2) of β1 and S280I (TM2) or S301W (TM3) in γ2L subunits did not alter apparent affinity of the receptor for GABA. M286W (TM3) in the β1 subunit resulted in a receptor that was tonically open.

Using an EC5 concentration of GABA, the function of the wild‐type receptor with α2β1γ2L subunits was potentiated by ethanol (50–200 mM). The mutations in TM2 or TM3 of the α2 subunit diminished the potentiation by ethanol. The action of ethanol was also eliminated with a mutation in the TM2 site of the β1 subunit. Ethanol produced significant inhibition of GABA responses in receptors containing the combination of α2 and β1 TM2 mutants with a wild‐type γ2L subunit. A small but significant reduction in the potentiation by ethanol was observed with γ2L TM2 and/or TM3 mutants.

From these results, we suggest that in heteromeric GABAA receptors composed of the α, β and γ subunits, ethanol may bind in a cavity formed by TM2 and TM3, and that binding to the α or β subunit may be more critical than the γ subunit.

British Journal of Pharmacology (1999) 127, 377–382; doi:10.1038/sj.bjp.0702563
Effects and interaction, of cariporide and preconditioning on cardiac arrhythmias and infarction in rat in vivo
Authors: Aye Nu Nu , Komori Sadayoshi , Hashimoto Keitaro
Published: January 2005

Although Na+‐H+ exchange (NHE) inhibitors are reported to protect the myocardium against ischaemic injury, NHE activation has also been proposed as a potential mechanism of ischaemic preconditioning‐induced protection. This study was performed to test any modifiable effect of cariporide, an NHE inhibitor, on cardioprotective effects of preconditioning.

Anaesthetized rats were subjected to 30 min of coronary artery occlusion and 150 min of reperfusion. The preconditioning (PC) was induced by 3 min of ischaemia and 10 min of reperfusion (1PC) or three episodes of 3 min ischaemia and 5 min reperfusion (3PC). Cariporide (0.3 mg kg−1) an NHE inhibitor, was administered 30 min (cari(30)) or 45 min (cari(45)) before coronary ligation (n=8–11 for each group).

Ventricular arrhythmias during 30 min ischaemia and infarct size (measured by triphenyltetrazolium (TTC) and expressed as a per cent area at risk (%AAR)) were determined. Cari(30) reduced ventricular fibrillation (VF) incidence and infarct size (from 45 to 0% and 34±4 to 9±2%; each P<0.05), whereas cari(45) did not. Likewise, 3PC reduced these variables (to 0% and 10±2%; P<0.05 in each case) whereas 1PC did not. Moreover, subthreshold preconditioning (1PC) and cariporide (cari(45)), when combined, reduced VF incidence and infarct size (to 0% and 15+3%; each P<0.05).

In conclusion, changes in NHE activity do not seem to be responsible for the cardioprotective action of ischaemic preconditioning. Protective effects of NHE inhibition and subthreshold preconditioning appear to act additively.

British Journal of Pharmacology (1999) 127, 1048–155; doi:10.1038/sj.bjp.0702566
Serotonin reuptake inhibitor, fluoxetine, dilates isolated skeletal muscle arterioles. Possible role of altered Ca2+ sensitivity
Authors: Pacher Pal , Ungvari Zoltan , Kecskemeti Valéria , Koller Akos
Published: January 2005

Inhibitors of serotonin reuptake in the central nervous system, such as fluoxetine, may also affect the function of vascular tissues. Thus, we investigated the effect of fluoxetine on the vasomotor responses of isolated, pressurized arterioles of rat gracilis muscle (98±4 μm in diameter at 80 mmHg perfusion pressure).

We have found that increasing concentrations of fluoxetine dilated arterioles up to 155±5 μm with an EC50 of 2.5±0.5×10−6 M.

Removal of the endothelium, application of 4‐aminopyridine (4‐AP, an inhibitor of aminopyridine sensitive K+ channels), or use of glibenclamide (an inhibitor of ATP‐sensitive K+ channels) did not affect the vasodilator response to fluoxetine.

In the presence of 10−6, 2×10−6 or 10−5 M fluoxetine noradrenaline (NA, 10−9–10−5 M) and 5‐hydroxytryptamine (5‐HT, 10−9–10−5 M)‐induced constrictions were significantly attenuated resulting in concentration‐dependent parallel rightward shifts of their dose‐response curves (pA2=6.1±0.1 and 6.9±0.1, respectively).

Increasing concentrations of Ca2+ (10−4–3×10−2 M) elicited arteriolar constrictions (up to ∼30%), which were markedly reduced by 2×10−6 M fluoxetine, whereas 10−5 M fluoxetine practically abolished these responses.

In conclusion, fluoxetine elicits substantial dilations of isolated skeletal muscle arterioles, a response which is not mediated by 4‐AP‐ and ATP‐sensitive K+ channels or endothelium‐derived dilator factors. The findings that fluoxetine had a greater inhibitory effect on Ca2+ elicited constrictions than on responses to NA and 5‐HT suggest that fluoxetine may inhibit Ca2+ channel(s) or interfere with the signal transduction by Ca2+ in the vascular smooth muscle cells.

British Journal of Pharmacology (1999) 127, 740–746; doi:10.1038/sj.bjp.0702571
Antiarrhythmic effect and its underlying ionic mechanism of 17β‐estradiol in cardiac myocytes
Authors: Nakajima Toshiaki , Iwasawa Kuniaki , Oonuma Hitoshi , Morita Toshihiro , Goto Atsuro , Wang Yuepeng , Hazama Hisanori
Published: January 2005

The effects of oestrogens on action potential and membrane currents were examined in single guinea‐pig atrial myocytes.

17β‐estradiol (3–10 μM) shortened the action potential duration without significant changes in the resting membrane potential. E‐4031 (1 μM) markedly prolonged the action potential duration and induced early afterdepolarization, and 17β‐estradiol (10 μM) abolished it.

When cells were perfused in isoproterenol‐containing solution, action potentials due to abnormal automaticity caused by membrane depolarization developed, and were also inhibited by 17β‐estradiol.

Under voltage clamp conditions, the voltage‐dependent Ca2+ currents consisted of both T‐(ICa.T) and L‐type (ICa.L). 17β‐estradiol reduced ICa.L concentration‐dependently, while it (10 μM) suppressed ICa.T only by approximately 10%. 17β‐estradiol did not affect time courses of ICa.L inactivation, but it shifted the steady‐state inactivation curve to more negative potentials.

17β‐estradiol (10 μM) did not affect the time‐dependent K+ current (IK), referred to as IKr and IKs, and inwardly rectifying K+ current. However, 17β‐estradiol (30 μM) or diethylstilbestrol (10 μM) inhibited K+ currents.

DES and ethinylestradiol (EES) also suppressed ICa.L, but testosterone and progesterone failed to inhibit ICa.L. The potency of the inhibitory effect on ICa.L was DES>EES>17β‐estradiol.

17β‐estradiol and DES also inhibited the cyclic AMP‐enhanced ICa.L, but cyclic GMP in the pipette or pretreatment of L‐NAME could not block the effects of oestrogen on ICa.L.

These results suggest that oestrogen specifically has antiarrhythmic effects, possibly by acting the L‐type Ca2+ channels. The antiarrhythmic effects of oestrogens may contribute to the cardioprotective actions of oestrogens.

British Journal of Pharmacology (1999) 127, 429–440; doi:10.1038/sj.bjp.0702576
Augmented acetylcholine‐induced, Rho‐mediated Ca2+ sensitization of bronchial smooth muscle contraction in antigen‐induced airway hyperresponsive rats
Authors: Chiba Yoshihiko , Takada Yuka , Miyamoto Shigeki , Mitsui‐Saito Minori , Karaki Hideaki , Misawa Miwa
Published: January 2005

Treatment with acetylcholine (ACh) of a β‐escin‐permeabilized intrapulmonary bronchial smooth muscle of the rat induced force when the Ca2+ concentration was clamped at 1 μM. The ACh‐induced Ca2+ sensitization of myofilaments was significantly greater in antigen‐induced airway hyperresponsive rats than in control rats. The ACh‐induced Ca2+ sensitization was completely blocked by treatment with Clostridium botulinum C3 exoenzyme, an inactivator of Rho family of proteins. Moreover, the protein level of RhoA in the intrapulmonary bronchi was significantly increased in the airway hyperresponsive rats. Thus, increased airway smooth muscle contractility observed in asthmatics may be related to augmented agonist‐induced, Rho‐mediated Ca2+ sensitization of myofilaments.

British Journal of Pharmacology (1999) 127, 597–600; doi:10.1038/sj.bjp.0702585
Modulation of haemostatic function and prevention of experimental thrombosis by red wine in rats: a role for increased nitric oxide production
Authors: Wollny Tomasz , Aiello Luca , Di Tommaso Donata , Bellavia Vincenzo , Rotilio Domenico , Donati Maria Benedetta , De Gaetano Giovanni , Iacoviello Licia
Published: January 2005

The effects of ethyl alcohol and wine (red and white) on haemostatic parameters and experimental thrombosis were studied in rats; NO was evaluated as a possible mediator of these effects.

We found that red wine (12% alcohol) supplementation (8.4±0.4 ml d−1 in drinking water, for 10 days) induced a marked prolongation of ‘template’ bleeding time (BT) (258±13 vs 132±13 s in controls; P<0.001), a decrease in platelet adhesion to fibrillar collagen (11.6±1.0 vs 32.2±1.3%; P<0.01) and a reduction in thrombus weight (1.45±0.33 vs 3.27±0.39 mg; P<0.01).

Alcohol‐free red wine showed an effect similar to red wine. In contrast, neither ethyl alcohol (12%) nor white wine (12% alcohol) affected these systems.

All these effects were also observed after red wine i.v. injection (1 ml kg−1 of 1 : 4 dilution) 15 min before the experiments.

The effects of red wine were prevented by the NO inhibitor, Nωnitro‐L‐arginine‐methyl ester (L‐NAME). L‐arginine, not D‐arginine, reversed the effect of L‐NAME on red wine infusion.

Red wine injection induced a 3 fold increase in total radical‐trapping antioxidant parameter values of rat plasma with respect to controls, while white wine and alcohol did not show any effect.

Our study provides evidence that red wine modulates primary haemostasis and prevents experimental thrombosis in rats, independently of its alcohol content, by a NO‐mediated mechanism.

British Journal of Pharmacology (1999) 127, 747–755; doi:10.1038/sj.bjp.0702586
Influence of S‐adenosyl‐L‐methionine on chronic mild stress‐induced anhedonia in castrated rats
Authors: Benelli A , Filaferro M , Bertolini A , Genedani S
Published: January 2005

S‐adenosyl‐L‐methionine (SAMe) is the most important methyl donor in the brain and is essential for polyamine synthesis. Methyl group deficiency in the brain has been implicated in depression; on the other hand, polyamines enhance phosphorylation processes, and phosphorylation of functional proteins in neurons is involved in the therapeutic mechanisms of antidepressants.

The effect of SAMe in an animal model of ‘depression’, the chronic mild stress‐induced anhedonia, was studied using long‐term castrated male and female Lister hooded rats.

Chronic daily exposure to an unpredictable sequence of mild stressors produced, within 3 weeks, a significant reduction of the consumption of a sucrose solution. SAMe (100, 200 or 300 mg kg−1 daily i.m.) while having no influence on sucrose intake in non‐stressed animals, dose‐dependently reinstated sucrose consumption within the first week of treatment, both in male and in female stressed rats. Imipramine (10 mg kg−1 daily i.p.) produced a similar effect after a 3 week treatment.

Similarly, a palatable food reward‐induced place preference conditioning was developed in SAMe (200 or 300 mg kg−1 daily i.m.)‐ and in imipramine (10 mg kg−1 daily i.p.)‐treated chronically stressed animals (males and females), whilst it could not be obtained in vehicle‐treated rats.

Moreover, the same doses of SAMe (but not of imipramine) restored the exploratory activity and curiosity for the environment (rearing), in the open‐field test.

While imipramine caused a blockade of the growth throughout the treatment, SAMe produced only a transient growth arrest during the first week of treatment.

These results show that SAMe reverses an experimental condition of ‘depression‐like’ behaviour in rats, the effect being more rapid and complete than that of imipramine, and without apparent side effects.

British Journal of Pharmacology (1999) 127, 645–654; doi:10.1038/sj.bjp.0702589
Extracellular adenosine concentrations during in vitro ischaemia in rat hippocampal slices
Authors: Latini Serena , Bordoni Francesca , Pedata Felicita , Corradetti Renato
Published: January 2005

The application of an ischaemic insult in hippocampal slices results in the depression of synaptic transmission, mainly attributed to the activation of A1 adenosine receptors by adenosine released in the extracellular space.

To estimate the concentration of endogenous adenosine acting at the receptor level during an ischaemic episode, we recorded field e.p.s.ps (fe.p.s.ps) from hippocampal slices, and evaluated the ability of the selective A1 receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), to reverse the fe.p.s.p. depression induced by in vitro ischaemia. A relationship between the IC50 of an antagonist and the endogenous concentration of a neurotransmitter has been used for pharmacological analysis.

The complete and reversible depression of fe.p.s.p. in the CA1 region induced by 5 min ischaemia was decreased in the presence of DPCPX (50–500 nM). 8‐Phenyltheophylline (10 μM) abolished the depression of fe.p.s.ps during the ischaemic period, while a small (peak effect 12±4%) decrease in fe.p.s.ps was observed during the initial phase of reperfusion.

In the time‐interval of maximal depression of fe.p.s.ps., IC50 and adenosine concentration changed as function of time with a good degree of correlation. The maximal value of adenosine concentration was 30 μM.

Our data provide an estimation of the adenosine concentration reached at the receptor level during an ischaemic episode, with a higher time discrimination (15 s) than that achieved with any biochemical approach. This estimation may be useful in order to establish appropriate concentrations of purinergic compounds to be tested for their pharmacological effects during an ischaemic episode.

British Journal of Pharmacology (1999) 127, 729–739; doi:10.1038/sj.bjp.0702591
Modulation of afferent‐evoked neurotransmission by 5‐HT3 receptors in young rat dorsal horn neurones in vitro: a putative mechanism of 5‐HT3 induced anti‐nociception
Authors: Khasabov S G , Lopez‐Garcia J A , Asghar A U R , King A E
Published: January 2005

The in vitro hemisected spinal cord from young rat was used to investigate the mechanism of serotoninergic modulation of primary afferent‐mediated synaptic transmission in the dorsal horn through activation of the 5‐HT3 receptor.

Dorsal root‐evoked excitatory post‐synaptic potentials (DR‐EPSPs) were recorded intracellularly from dorsal horn neurones. Extracellular recordings of the population primary afferent depolarization (PAD) and the dorsal rootevoked dorsal root reflex (DR‐DRR) were made from segmental dorsal roots.

5‐Hydroxytryptamine (5‐HT) and the selective 5‐HT3 receptor agonist 1‐(m‐chloro‐phenyl)‐biguanide hydrochloride (m‐ChPB) (10 and 50 μM) induced statistically significant reductions of the DR‐EPSP amplitude (P<0.001) and duration (P<0.001) in the majority of dorsal horn neurones. The 5‐HT3 receptor selective antagonists 3‐Tropanyl‐indole‐3‐carboxylate hydrochloride (Tropisetron, 10 μM) and N‐(1‐Azabicyclo[2.2.2]oct‐3‐yl)‐6‐chloro‐4‐methyl‐3‐oxo‐3,4‐dihydro‐2H‐1,4‐benzoxazine‐8‐carboxamide (Y‐25130, 10 μM) abolished m‐ChPB‐induced DR‐EPSP attenuation and partially blocked the 5‐HT effect.

m‐ChPB (50 μM)‐induced DR‐EPSP amplitude and duration attenuation was retained in the presence of the GABAA receptor antagonist bicuculline (30 μM), the GABAB receptor antagonist saclofen (50 μM) and the opioid receptor antagonist naloxone (50 μM).

Both 5‐HT and m‐ChPB (10 and 50 μM) induced a PAD but the mean peak amplitude of 5‐HT‐induced PAD was significantly greater than PAD to m‐ChPB (98.6±12 μV compared to 51.8±10 μV for 50 μM of agonist, respectively). Tropisetron partially reduced 5‐HT‐induced PAD and abolished m‐ChPB‐induced PAD. 5‐HT, but not m‐ChPB, significantly (P<0.001) reduced the peak amplitude of the DR‐DRR and this action of 5‐HT was unaffected by Tropisetron or Y‐25130.

These data provide experimental evidence for a putative cellular mechanism at the level of the dorsal horn for anti‐nociceptive effects of 5‐HT3 receptor activation. This 5‐HT3‐mediated modulation of sensory afferent transmission was evidently independent of inhibitory GABA‐ or opioid‐dependent interneuronal pathways. The extent to which the 5‐HT3 receptor could be involved in the operation of endogenous analgesia and sensory modulation by descending monoamine bulbo‐spinal pathways is discussed.

British Journal of Pharmacology (1999) 127, 843–852; doi:10.1038/sj.bjp.0702592
E5531, a synthetic non‐toxic lipid A derivative blocks the immunobiological activities of lipopolysaccharide
Authors: Kawata Tsutomu , Bristol John R , Rossignol Daniel P , Rose Jeffrey R , Kobayashi Seiichi , Yokohama Hiromitsu , Ishibashi Akira , Christ William J , Katayama Koichi , Yamatsu Isao , Kishi Yoshito
Published: January 2005

The major pathological responses to Gram‐negative bacterial sepsis are triggered by endotoxin or lipopolysaccharide. As endotoxin is shed from the bacterial outer membrane, it induces immunological responses that lead to release of a variety of cytokines and other cellular mediators. As part of a program aimed at developing a therapeutic agent for septic shock, we have developed E5531, a novel synthetic lipopolysaccharide antagonist.

As measured by release by tumour necrosis factor‐α, human monocytes or whole blood can be activated by lipopolysaccharide, lipid A, and lipoteichoic acid (from Gram‐positive bacteria). E5531 potently antagonizes activation by all these agents while itself being devoid of agonistic activity.

The inhibitory activity of E5531 was dependent on time of addition. When 10 nM E5531 was added simultaneously with lipopolysaccharide or 1–3 h before addition of lipopolysaccharide, production of tumour necrosis factor‐α was inhibited by more than 98%. The addition of E5531 1 h after lipopolysaccharide reduced the efficacy of E5531 by 47%.

Antagonistic activity of E5531 was specific for lipopolysaccharide as it was ineffective at inhibiting interferon‐γ mediated NO release of RAW 264.7 cells, phorbor 12‐myristate 13‐acetate stimulated superoxide anion production in human neutrophils, concanavalin A stimulated mitogenic activity in murine thymocytes and tumor necrosis factor‐α induced E‐selectin expression in human umbilical vein endothelial cells.

E5531 as well as MY4, an anti‐CD14 antibody, inhibited radiolabelled lipopolysaccharide binding in human monocytes.

These results support our contention that E5531 is a potent antagonist of lipopolysaccharide‐induced release of tumour necrosis factor‐α and other cellular mediators and may be an effective therapeutic agent for human septic shock due to Gram‐negative bacteria.

British Journal of Pharmacology (1999) 127, 853–862; doi:10.1038/sj.bjp.0702596
Relevance of benzyloxy group in 2‐indolyl methylamines in the selective MAO‐B inhibition
Authors: Pérez Virgili , Marco José Luis , Fernández‐Álvarez Eldiberto , Unzeta Mercedes
Published: January 2005

Previous studies with indolyl derivatives as monoamine oxidase (MAO) inhibitors have shown the relevance of the indole structure for recognition by the active site of this enzyme. We now report a new series of molecules with structural features which determine the selectivity of MAO inhibition.

A benzyloxy group attached at position 5 of the indole ring is critical for this selective behaviour. Amongst all of these benzyloxy‐indolyl methylamines, N‐(2‐propynyl)‐2‐(5‐benzyloxyindol)methylamine FA‐73 was the most potent MAO‐B ‘suicide’ inhibitor studied.

The Ki values for MAO‐A and MAO‐B were 800±60 and 0.75±0.15 nM, respectively. These data represent a selectivity value of 1066 for MAO‐B, being 48 times more selective than L‐deprenyl (Ki values of 376±0.032 and 16.8±0.1 nM for MAO A and MAO‐B, respectively). The IC50 values for dopamine uptake in striatal synaptosomal fractions from rats were 150±8 μM for FA‐73 and 68±10 μM for L‐deprenyl whereas in human caudate tissue the IC50 values were 0.36±0.015 μM for FA‐73 and 0.10±0.007 μM for L‐deprenyl. Moreover, mouse brain MAO‐B activity was 90% ex vivo inhibited by both compounds 1 h after 4 mg kg−1 adminstration, MAO‐A activity was not affected.

These novel molecules should provide a better understanding of the active site of monoamine oxidase and could be the starting point for the design of further selective, non‐amphetamine‐like MAO‐B inhibitors with therapeutic potential for the treatment of neurological disorders.

British Journal of Pharmacology (1999) 127, 869–876; doi:10.1038/sj.bjp.0702600
Pharmacological modulation of secondary mediator systems–cyclic AMP and cyclic GMP–on inflammatory hyperalgesia
Authors: Cunha F Q , Teixera M M , Ferreira S H
Published: January 2005

The objective of the present paper was to evaluate the relevance of neuronal balance of cyclic AMP and cyclic GMP concentration for functional regulation of nociceptor sensitivity during inflammation.

Injection of PGE2 (10–100 ng paw−1) evoked a dose‐dependent hyperalgesic effect which was mediated via a cyclic AMP‐activated protein kinase (PKA) inasmuch as hyperalgesia was blocked by the PKA inhibitor H89.

The PDE4 inhibitor rolipram and RP73401, but not PDE3 and PDE5 inhibitors potentiated the hyperalgesic effects of PGE2. The hyperalgesic effect of dopamine was also enhanced by rolipram. Moreover, rolipram significantly potentiated hyperalgesia induced by carrageenan, bradykinin, TNFα, IL‐1β, IL‐6 and IL‐8. This suggests that neuronal cyclic AMP mediates the prostanoid and sympathetic components of mechanical hyperalgesia. Moreover, in the neuron cyclic AMP is mainly metabolized by PDE4.

To examine the role of the NO/cyclic GMP pathway in modulating mechanical hyperalgesia, we tested the effects of the soluble guanylate cyclase inhibitor, ODQ. This substance counteracts the inhibitory effects of the NO donor, SNAP, on the hyperalgesia induced by PGE2.

The ODQ potentiated hyperalgesia induced by carrageenan, bradykinin, TNFα, IL‐1β, IL‐6 and IL‐8. In contrast, ODQ had no significant effect on the hyperalgesia induced by PGE2 and dopamine. This indicates that the hyperalgesic cytokines may activate soluble guanylate cyclase, which down‐regulate the ability of these substances to cause hyperalgesia. This event appears not to be mediated by prostaglandin or dopamine.

In conclusion, the results presented in this paper confirm an association between (i) hyperalgesia and elevated levels of cyclic AMP as well as (ii) antinociception and elevated levels of cyclic GMP. The intracellular levels of cyclic AMP that enhance hyperalgesia are controlled by the PDE4 isoform and appear to result in activation of protein kinase A whereas the intracellular levels of cyclic GMP results from activation of a soluble guanylate cyclase.

British Journal of Pharmacology (1999) 127, 671–678; doi:10.1038/sj.bjp.0702601
Protective effects of a superoxide dismutase mimetic and peroxynitrite decomposition catalysts in endotoxin‐induced intestinal damage
Authors: Salvemini Daniela , Riley Dennis P , Lennon Patrick J , Wang Zhi‐Qiang , Currie Mark G , Macarthur Heather , Misko Thomas P
Published: January 2005

The relative contributions of superoxide anion (O2−) and peroxynitrite (PN) were evaluated in the pathogenesis of intestinal microvascular damage caused by the intravenous injection of E. coli lipopolysaccharide (LPS) in rats. The superoxide dismutase mimetic (SODm) SC‐55858 and the active peroxynitrite decomposition catalysts 5,10,15,20‐tetrakis(2,4,6‐trimethyl‐3,5‐disulphonatophenyl)‐porphyrinato iron (III) and 5,10,15,20‐tetrakis(N‐methyl‐4′‐pyridyl)‐porphyrinato iron (III) (FeTMPS, FeTMPyP respectively) were used to assess the roles of O2− and PN respectively.

The intravenous injection of LPS elicited an inflammatory response that was characterized by a time‐dependent infiltration of neutrophils, lipid peroxidation, microvascular leakage (indicative of microvascular damage), and epithelial cell injury in both the duodenum and jejunum.

Administration of the SODm SC‐55858, FeTMPS or FeTMPyP at 3 h post LPS reduced the subsequent increase in microvascular leakage, lipid peroxidation and epithelial cell injury. Inactive peroxynitrite decomposition catalysts exhibited no protective effects. Only, SC‐55858 inhibited neutrophil infiltration.

Our results suggest that O2− and peroxynitrite play a significant role in the pathogenesis of duodenal and intestinal injury during endotoxaemia and that their removal by SODm and peroxynitrite decomposition catalysts offers a novel approach to the treatment of septic shock or clinical conditions of gastrointestinal inflammation. Furthermore, the remarkable protection of the intestinal epithelium by these agents suggests their use during chemo‐ and radiation therapy, cancer treatments characterized by gastrointestinal damage. Potential mechanisms through which these radicals evoke damage are discussed.

British Journal of Pharmacology (1999) 127, 685–692; doi:10.1038/sj.bjp.0702604
Induction of apoptosis in human mitogen‐activated peripheral blood T‐lymphocytes by the ether phospholipid ET‐18‐OCH3: Involvement of the Fas receptor/ligand system
Authors: Cabaner Christelle , Gajate Consuelo , Macho Antonio , Muñoz Eduardo , Modolell Manuel , Mollinedo Faustino
Published: January 2005

Activated T‐cells constitute a target for treatment of autoimmune diseases. We have found that the antitumour ether phospholipid 1‐O‐octadecyl‐2‐O‐methyl‐rac‐glycero‐3‐phosphocholine (ET‐18‐OCH3; edelfosine) induced dose‐ and time‐dependent apoptosis in human mitogen‐activated peripheral blood T‐lymphocytes, but not in resting T‐cells. T‐lymphocytes were stimulated with phytohemagglutinin and interleukin‐2 or with concanavalin A. Apoptosis was assessed by DNA fragmentation through cell cycle and TUNEL analyses, as well as through visualization of internucleosomal DNA fragmentation in agarose gels.

The ET‐18‐OCH3‐mediated apoptotic response in activated T‐lymphocytes was less intense than in human leukaemic T cell lines, such as Jurkat cells and Peer cells; namely about 25% apoptosis in activated T‐cells versus about 46–61% apoptosis in T leukaemic cells after 24 h treatment with 10 μM ET‐18‐OCH3.

The ET‐18‐OCH3 thioether analogue BM 41.440 (ilmofosine) showed a similar apoptotic capacity to that found with ET‐18‐OCH3 in activated T‐cells, whereas the phospholipid analogue hexadecylphosphocholine (miltefosine) failed to promote this response.

The uptake of [3H]‐ET‐18‐OCH3 was much larger in activated T‐cells than in resting lymphocytes.

Using a cytofluorimetric approach we have found that ET‐18‐OCH3 induced disruption of the mitochondrial transmembrane potential and production of reactive oxygen species in activated T‐cells, but not in resting lymphocytes.

ET‐18‐OCH3 induced an increase in Fas (APO‐1/CD95) ligand mRNA expression in activated T‐cells, and incubation with a blocking anti‐Fas (APO‐1/CD95) antibody partially inhibited the ET‐18‐OCH3‐induced apoptosis of activated T‐lymphocytes.

These results demonstrate that mitogen‐activated T‐cells, unlike resting lymphocytes, are able to take up significant amounts of ET‐18‐OCH3, and are susceptible to undergo apoptosis by the ether lipid via, in part, the Fas (APO‐1/CD95) receptor/ligand system. This ET‐18‐OCH3 apoptotic action can be of importance in the therapeutic action of this ether lipid in certain autoimmune diseases.

British Journal of Pharmacology (1999) 127, 813–825; doi:10.1038/sj.bjp.0702606
Comparative effects of several nitric oxide donors on intracellular cyclic GMP levels in bovine chromaffin cells: correlation with nitric oxide production
Authors: Ferrero Rut , Rodríguez‐Pascual Fernando , Miras‐Portugal Ma Teresa , Torres Magdalena
Published: January 2005

Sodium nitroprusside, S‐nitroso‐N‐acetyl‐D,L‐penicillamine, Spermine NONOate and DEA NONOate raised cyclic GMP levels in bovine chromaffin cells in a time and concentration dependent manner with different potencies, the most potent being DEA/NO with an EC50 value of 0.38±0.02 μM.

Measurements of NO released from these donors revealed that DEA/NO decomposed with a half‐life (t1/2) of 3.9±0.2 min. The t1/2 for SPER/NO was 37±3 min. SNAP decomposed more slowly (t1/2=37±4 h) and after 60 min the amount of NO produced corresponded to less than 2% of the total SNAP present. The rate of NO production from SNAP was increased by the presence of glutathione.

For DEA/NO and SPER/NO there was a clear correlation between nitric oxide production and cyclic GMP increases. Their threshold concentrations were 0.05 μM and maximal effective concentration between 2.5 and 5 μM.

For SNAP, threshold activation was seen at 1 μM, whereas full activation required a higher concentration (500–750 μM). The dose‐response for SNAP increases in cyclic GMP was shifted nearly two orders of magnitude lower in the presence of glutathione. At higher concentrations an inhibition of cyclic GMP accumulation was found. This effect was not observed with either the nitric oxide‐deficient SNAP analogue or other NO donors.

Although NO‐donors are likely to be valuable for studying NO functions, their effective concentrations and the amount of NO released by them are very different and should be assessed in each system to ensure that physiological concentrations of NO are used.

British Journal of Pharmacology (1999) 127, 779–787; doi:10.1038/sj.bjp.0702607
Intravascular ATP and coronary vasodilation in the isolated working rat heart
Authors: Korchazhkina Olga , Wright Gordon , Exley Christopher
Published: January 2005

Adenosine‐5′‐triphosphate (ATP) is a potent coronary vasodilator. Because of the efficient hydrolysis of ATP, adenosine‐5′‐diphosphate (ADP) and adenosine‐5′‐monophosphate (AMP) by ectonucleotidases located in the coronary endothelium ATP‐induced vasodilation may be mediated via both P1 (AMP and adenosine) and P2Y (ATP and ADP) receptors. We have used the change in total coronary resistance (TCR) induced by intravascular ATP in the isolated working rat heart to determine both the component of the vasodilation mediated via P2Y receptors and the identity of the subclass of receptor involved.

The dose response for ATP revealed a half maximal effect at an apparent ATP concentration of 0.08±0.009 μM. The response was saturated at apparent ATP concentrations greater than 0.23 μM. Contrary to much of the current literature, the perfusion of a 0.25 μM concentration of adenosine resulted in the identical response to an equimolar concentration of ATP suggesting a significant role for adenosine in coronary vasodilation.

The non‐selective P1 receptor antagonist 8‐(p‐Sulfophenyl)theophylline (8‐SPT) was used to show that the response to ATP was mediated via both P1 and P2Y receptors. Whilst 8‐SPT abolished the effect of adenosine it reduced the effect of ATP by only 50%. Thus, at a saturating concentration of ATP, P1 and P2Y receptors were shown to contribute equally to the observed vasodilation.

Uridine‐5′‐triphosphate (UTP), ADP and adenosine‐5′‐O‐thiotriphosphate (ATPγS) were used to characterize the component of coronary vasodilation that was mediated via P2Y receptors. UTP at 0.25 μM was ineffective and did not induce vasodilation. Perfusion with 0.25 μM ADP resulted in a vasodilation that was identical to 0.25 μM ATP. In the absence of 8‐SPT the perfusion of 0.25 μM ATPγS produced a vasodilation that was significantly (P<0.05) less than ATP. However, the vasodilation due to ATPγS, like that of adenosine, but unlike that of both ATP and ADP, was abolished in the presence of 8‐SPT. The ability of ADP to induce vasodilation combined with both the lack of response to UTP and the ability of 8‐SPT to abolish the vasodilation induced by ATPγS suggested very strongly that the component of ATP‐induced coronary vasodilation in the isolated working rat heart that was mediated via P2Y receptors was achieved by the action of ADP (and not ATP) at P2Y1 receptors.

These results suggest that the vasodilatory action of intravascular ATP in the coronary circulation should be attributed to the dual and equal activities of adenosine and ADP acting at P1 and P2Y1 receptors respectively.

British Journal of Pharmacology (1999) 127, 701–708; doi:10.1038/sj.bjp.0702610
Inverse agonist activities of β‐adrenoceptor antagonists in rat myocardium
Authors: Varma D R , Shen H , Deng X F , Peri K G , Chemtob S , Mulay S
Published: January 2005

Negative inotropic effects of several β‐adrenoceptor (βAR) antagonists on electrically‐stimulated right atria, left atria, right ventricles and left ventricular papillary muscles from reserpine‐treated rats were used as a measure of their inverse agonist activities.

β1AR antagonists acebutolol, atenolol and metoprolol, β2AR antagonist ICI‐181,551 and nonselective βAR antagonists alprenolol, nadolol, propranolol and timolol produced negative inotropic effects, which were most marked on the right atria.

The nonselective βAR antagonist pindolol did not exhibit inverse agonist activity but inhibited the negative inotropic activities of ICI‐118,551, atenolol and propranolol.

The negative inotropic effects of lidocaine, nifedipine and pentobarbitone were similar on all the four myocardial preparations.

The positive inotropic efficacy of salbutamol on right and left atria but not on right ventricles and papillary muscles was comparable to that of isoprenaline. The antagonist activity of ICI‐118,551 against isoprenaline was greater on right atria than on other cardiac regions.

β1AR proteins were expressed in all regions of the heart but of β2AR were primarily localized in the right atrium.

It is concluded that β2AR play a greater role in right atria than in other cardiac regions and almost all βAR antagonists behave as inverse agonists.

British Journal of Pharmacology (1999) 127, 895–902; doi:10.1038/sj.bjp.0702616