bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential affinity of dihydroimidazoquinoxalines and diimidazoquinazolines to the α1β2γ2 and α6β2γ2 subtypes of cloned GABAA receptors
Authors: Im Wha Bin , Im Haesook K. , Pregenzer Jeffrey F. , Hamilton Beverly J. , Carter Donald B. , Jacobsen E. Jon , TenBrink Ruth E. , VonVoigtlander Philip F.
Published: July 2012

In this study, we compared two series of newly discovered ligands for their selectivity to benzodiazepine sites in the α1β2γ2 and the α6β2γ2 subtypes of cloned γ‐aminbutyric acidA (GABAA) receptors, the latter being unique in not interacting with classical benzodiazepines.

The prototype compounds, U‐85575 (12‐chloro‐5‐(5‐cyclopropyl‐1′,2′,4′‐oxadiazol‐3′‐yl)‐2,3‐dihydro‐diimidazo[1,5‐a;1,2‐c]quinazoline), and U‐92330 (5‐acetyl‐3‐(5′‐cyclopropyl‐1′,2′,4′‐oxadiazole‐3′‐yl)‐7‐chloro‐4,5‐dihydro[1,5‐a]quinoxaliné), appear to share an overlapping recognition site with classical benzodiazepines on the GABAA receptor, because their potentiation of GABA‐mediated Cl− currents in both subtypes were sensitive to Ro 15–1788, a classical benzodiazepine antagonist.

Minor changes in the ring substituents of the drugs reduced their affinity to the α6β2γ2 subtype more pronouncedly than to the α1β2γ2 subtype. The diimidazoquinazoline containing a 2‐methyl group which projected below the plane of the rigid ring showed a markedly lower affinity to the α6β2γ2 subtype as compared to its stereoisomer having the methyl group above the plane of the ring. Also, the dihydroimidazoquinoxalines containing the 5‐benzoyl group showed a lower affinity to the α6β2γ2 subtype than the 5‐acetyl counterpart. In particular, the 5‐benzoyl analogue containing a 6‐fluoro group showed no interaction with the α6β2γ2 subtype even at the concentration of 10 μm, probably due to stabilization of the benzoyl group in the out‐of‐plane region by the steric and electrostatic effects of the 6‐fluoro group.

We propose that the benzodiazepine site of the α6β2γ2 subtype shares overlapping regions with that of the α1β2γ2 subtype, but has a sterically restricted out‐of‐plane region, which may be also incompatible with the 5‐phenyl group of classical benzodiazepines.
Ionomycin‐induced acetylcholine release and its inhibition by adenosine at frog motor nerve endings
Authors: Hunt Jarnes M. , Silinsky Eugene M.
Published: July 2012

Acetylcholine (ACh) evoked secretion by the calcium ionophore, ionomycin, was studied at frog motor nerve endings.

Bath application of ionomycin stimulated an irreversible increase in the rate of spontaneous, quantal ACh release in the presence of extracellular Ca2+. In contrast, local application of ionomycin stimulated a rapid, reversible acceleration of spontaneous ACh release.

The magnitude of the secretory response to ionomycin was dependent both upon the concentration of ionophore and the concentration of extracellular Ca2+.

Adenosine or 2‐chloroadenosine inhibited ionomycin‐stimulated ACh release with the same potency and efficacy observed previously for these adenosine analogues as inhibitors of ACh secretion evoked by nerve impulses.

These results support the conclusion that adenosine receptor activation inhibits quantal ACh secretion at a site distal to that of Ca2+ entry at frog motor nerve endings.
Stimulated eosinophils and proteinases augment the transepithelial flux of albumin in bovine bronchial mucosa
Authors: Herbert Carolyn A. , Edwards David , Boot John R. , Robinson Clive
Published: July 2012

The apical to basolateral transmucosal flux of albumin has been measured in isolated sheets of bovine bronchial and tracheal mucosa. Under resting conditions the net unidirectional flux in the bronchial mucosa was not significantly different from that measured previously for the basolateral to apical vector. In contrast, the apical to basolateral flux in the tracheal mucosa was significantly lower than that measured in the opposite direction.

Addition of guinea‐pig peritoneal eosinophils to the apical side of the tissues had no significant effect on the transmucosal flux of albumin in either the bronchial or tracheal mucosa.

When eosinophils were stimulated with the ionophore A23187 or by opsonic adherence to tissues treated with a guinea‐pig anti‐bovine airway epithelium antibody, the bronchial mucosal sheets that had been exposed showed a significant increase in the transmucosal flux of albumin. However, tissues from the tracheal mucosa were resistant to the effects of stimulated eosinophils.

Histologically, sheets of mucosa from bovine main bronchi that had been exposed to stimulated eosinophils were characterized by epithelial injury consisting of loss of columnar epithelium from the underlying basal cell layer and biomatrix. Much less evidence of cellular injury was observed in tracheal tissues.

Bacterial collagenases applied to the apical side of the sheets were shown to increase the permeability of the bronchial mucosa to albumin and to produce histological changes that had similarities with the pattern of damage produced by stimulated eosinophils.

These observations demonstrate that the ability of eosinophils to injure the bronchial mucosa is independent of the side of the tissue on which they are present. Furthermore, key aspects of the injury process may be reproduced, at least in part, by metalloproteinases.
Characterization of the adenosine receptors of the rat superior cervical ganglion
Authors: Connolly G.P. , Stone T.W. , Brown F.
Published: July 2012

Adenosine analogues caused hyperpolarization and inhibition of the depolarizing response to muscarine of the rat isolated superior cervical ganglion (SCG) measured by a ‘grease gap’ recording technique. The receptors mediating these responses have been characterized by use of a range of selective adenosine analogues and adenosine receptor antagonists.

In decreasing order of potency N6‐cyclopentyladenosine (CPA), 2‐chloroadenosine (2CA), adenosine, 2‐phenylaminoadenosine (PAA), caused concentration‐dependent hyperpolarizations whilst N6‐(9‐fluorenylmethyl)adenosine (PD 117,413) was inactive at up to 100 μm.

The order of potency of adenosine analogues in depressing depolarization caused by a submaximal concentration of muscarine (100 nm) was: CPA > R‐PIA = 2C A > NEC A > S‐PIA > BZA > adenosine > PAA, where R‐ and S‐PIA = R(−)‐ and S(+)‐N6‐(2‐phenylisopropyl)adenosine, NECA = 5′N‐ethylcarboxamidoadenosine and BZA = N6‐benzyladenosine. PD 117,413 was inactive at concentrations up to 100 μm. The maximum inhibitions of the muscarine‐induced depolarization by CPA, 2CA, NECA and BZA were similar. R‐PIA, S‐PIA and PAA produced similar maximal inhibitions which were significantly smaller than those produced by CPA.

Hyperpolarizations caused by adenosine were antagonized by the P1‐purinoceptor selective antagonist 1,3‐dimethyl‐8‐phenylxanthine (8PT) and by the selective A1‐adenosine receptor antagonist, 1,3‐dipropyl‐8‐(4‐((2aminoethyl)amino)carbonylmethyloxyphenyl)xanthine (XAC). Hyperpolarizations caused by CPA, adenosine and PAA were antagonized by the A1‐selective antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) but not by the A2‐selective antagonist, 3,7‐dimethyl‐1‐propargylxanthine (DMPX).

Inhibition of the muscarinic‐induced depolarization by CPA was antagonized by 8PT and DPCPX but not by DMPX.

It is concluded that the neurones of the rat SCG possess P1‐purinoceptors of the A1‐adenosine receptor subtype which mediate hyperpolarization and inhibition of depolarization caused by muscarine.
Serum corticosterone, interleukin‐1 and tumour necrosis factor in rat experimental endotoxaemia: comparison between Lewis and Wistar strains
Authors: Perretti M. , Duncan G.S. , Flower R.J. , Peers S.H.
Published: July 2012

Circulating corticosterone, interleukin‐1 (IL‐1) and tumour necrosis factor‐α (TNFα) activities in serum of Lewis and Wistar rats were measured following injection of lipopolysaccharide (LPS). IL‐1 was measured as ‘lymphocyte activation factor’ (LAF) activity following precipitation of inhibitory activity with polyethylene glycol. TNFα activity was measured as cytotoxic activity.

Compared to the Wistar, the Lewis rat had higher circulating LAF and TNF activities following LPS, and release of both cytokines was prolonged in this strain.

Corticosterone increases in response to LPS were less in the Lewis than in the Wistar rat following the initial peak at 1 h; basal corticosterone was lower in the Lewis rat.

Adrenalectomized Lewis rats had even greater amounts of circulating LAF and TNF activities following LPS than did intact animals; the effect of adrenalectomy was not however mimicked by acute treatment with the steroid receptor antagonist, RU486, suggesting that endogenous corticosteroids did not acutely control cytokine release.

Although in vivo administration of anti‐murine IL‐1α antiserum significantly lowered LAF activity of serum, circulating corticosterone in response to LPS was not affected. Similarly, treatment with anti‐murine TNFα monoclonal antibody (mAb) abrogated TNF activity without affecting corticosterone, suggesting that other mediators may be responsible for corticosterone release following LPS.

This ‘overproduction’ of inflammatory cytokines together with lower circulating corticosterone may contribute to the susceptibility of the Lewis rat to diseases such as adjuvant arthritis or experimental allergic encephalomyelitis.
Characterization and pharmacological modulation of antigen‐induced peritonitis in actively sensitized mice
Authors: Zuany‐Amorim Cláudia , Leduc Dominique , Vargaftig B. Boris , Pretolani Marina
Published: July 2012

The intraperitoneal (i.p.) injection of 1 or 10 μg ovalbumin to sensitized Balb/c mice led to an acute histamine release, firstly evidenced 1 min after the challenge and returning to basal levels 30 min thereafter. This phenomenon was unaccompanied by protein extravasation. A dose‐dependent increase in the amounts of immunoreactive leukotriene (LT) C4 and LTB4 was observed in the peritoneal washing from sensitized mice 6 h after 1 or 10 μg ovalbumin administration. In separate experiments, the i.p. administration of 1 mg activated zymosan to non‐immunized mice was followed by a marked protein extravasation, and by immunoreactive LTC4 and LTB4, but not histamine, release in mouse peritoneum 1 h after its injection.

Mediator release in the mice peritoneal cavity was concomitant with a transient neutrophil infiltration, which peaked at 6 h and returned to basal levels thereafter. An intense eosinophil accumulation starting at 24 h, peaking at 48 h and returning to basal values at 164 h, was also observed.

Ovalbumin (1 μg)‐induced eosinophilia, observed at 24 h, was reduced by the pretreatment of the animals with dexamethasone (1 mg kg−1, s.c.) or with the 5‐lipoxygenase inhibitor, BWA4C (20 mg kg−1, s.c.), whereas indomethacin (2 mg kg−1, s.c.) and the platelet‐activating factor (PAF)‐antagonist SR 27417 (10 mg kg−1, s.c.) were ineffective. These results indicate that metabolites of arachidonic acid of lipoxygenase pathway, but not cyclo‐oxygenase derivatives or PAF, mediate antigen‐induced eosinophil accumulation in the mouse peritoneum.

The histamine H1 receptor antagonist drug, cetirizine (15–30 mg kg−1, s.c.) markedly reduced ovalbumin‐induced eosinophil accumulation under conditions where terfenadine was ineffective, suggesting that the effect of cetirizine was not related to the inhibition of the H1 receptor effects of histamine.

The immunosuppressive agent, FK‐506 (1–2 mg kg−1, s.c.) and the protein synthesis inhibitor, cylcoheximide, when administered either in situ (0.06 ng/cavity) or systemically (5 mg kg−1, s.c.), prevented antigen‐induced eosinophil accumulation in the mouse peritoneum, contributing to the concept that substances (probably cytokines) originating from lymphocytes may be involved in the modulation of the eosinophilotactic response in this model.

The results of the present study indicate that the i.p. administration of ovalbumin to actively sensitized mice induced late eosinophil accumulation in the peritoneal cavity. This phenomenon, which may be in part mediated by the release of lipoxygenase metabolites and/or by newly generated factors, such as T‐lymphocytes‐derived eosinophilotactic cytokines, offers an interesting tool to investigate the mechanism of action of anti‐allergic and anti‐inflammatory drugs.
Characterization of EP‐receptor subtypes involved in allodynia and hyperalgesia induced by intrathecal administration of prostaglandin E2 to mice
Authors: Minami Toshiaki , Nishihara Isao , Uda Rumiko , Ito Seiji , Hyodo Masayoshi , Hayaishi Osamu
Published: July 2012

Intrathecal (i.t.) administration of prostaglandin E2 (PGE2) to conscious mice induced allodynia, a state of discomfort and pain evoked by innocuous tactile stimuli, and hyperalgesia as assessed by the hot plate test. We characterized prostaglandin E receptor subtypes (EP1–3) involved in these sensory disorders by use of 7 synthetic prostanoid analogues.

Sulprostone (EP1 < EP3) induced allodynia over a wide range of dosages from 50 pg to 5 μg kg−1. The maximal allodynic effect was observed at 5 min after i.t. injection, and the response gradually decreased over the experimental period of 50 min. This sulprostone‐induced allodynia showed a time course similar to that induced by PGE2.

17‐Phenyl‐ω‐trinor PGE2 (EP1 > EP3) and 16,16‐dimethyl PGE2 (EP1 = EP2 = EP3) were as potent as PGE2 in inducing allodynia, and more potent than sulprostone. Butaprost (EP2), 11‐deoxy PGE1 (EP2 = EP3), MB 28767 (EP3), and cicaprost (prostaglandin I2 (IP‐) receptor) induced allodynia, but with much lower scores. 13,14‐Dihydro‐15‐keto PGE2, a metabolite of PGE2, did not induce allodynia.

16,16‐Dimethyl PGE2 as well as PGE2 induced hyperalgesia over a wide range of dosages (16,16‐dimethyl PGE2: 5 pg–0.5 μg kg−1 PGE2: 50 pg–0.5 μg kg−1) with two apparent peaks at 0.5 ng kg−1 and 0.5 μg kg−1. Sulprostone (EP1 < EP3) and 17‐phenyl‐ω‐trinor PGE2 (EP1 > EP3) showed a bell‐shaped hyperalgesia at lower doses of 5 pg–5 ng kg−1 and 50 pg–50 ng kg−1, respectively. MB 28767 (EP3) showed a monophasic hyperalgesic action over a wide range of dosages at 50 pg–5 μg kg−1. Butaprost (EP2) induced hyperalgesia at doses higher than 50 ng kg−1.

These results demonstrate that PGE2 may exert allodynia through the EP1‐receptor and hyperalgesia through EP2‐ and EP3‐receptors in the mouse spinal cord.
Characterization of the effects of 2‐methylthio‐ATP and 2‐chloro‐ATP on brain capillary endothelial cells: similarities to ADP and differences from ATP
Authors: Vigne Paul , Feolde Erick , Breittmayer Jean Philippe , Frelin Christian
Published: July 2012

Brain capillary endothelial cells responded to 2‐methylthio‐ATP (2MeSATP) by large increases in [Ca2+]i (EC50 = 27 nm) that were partially dependent on the presence of extracellular Ca2+ and that were not associated with a measurable production of inositol phosphates.

2‐chloro‐ATP (2ClATP) raised [Ca2+]i in a biphasic manner. At low concentrations, intracellular Ca2+ mobilization was not associated with a measurable production of inositol phosphates. At concentrations >30 μm, 2ClATP activated phospholipase C.

The actions of 2ClATP, 2MeSATP and ADP on [Ca2+]i were additive to those of ATP and UTP. Non‐additive actions of 2MeSATP and of low concentrations of ADP or of 2ClATP were observed.

Cross desensitizations of the actions of ADP, 2MeSATP and 2ClATP were observed. None of them desensitized cells to the action of ATP.

It is concluded that 2MeSATP and low concentrations of 2ClATP and ADP induce intracellular Ca2+ mobilization by acting via an atypical P2y purinoceptor that is not coupled to phospholipase C. At high concentrations, 2C1ATP also activates phospholipase C and further increases [Ca2+]i probably by acting on P2u purinoceptors.
Lack of correlation of hypotensive effects with prevention of cardiac hypertrophy by perindopril after ligation of rat coronary artery
Authors: Chiba Kiyoshi , Moriyama Sachiko , Ishigai Yutaka , Fukuzawa Akiko , Irie Kiyoshi , Shibano Toshiro
Published: July 2012

The present study was designed to test the hypothesis that beneficial effects of angiotensin converting enzyme (ACE)inhibitors are independent of a fall in blood pressure in rat experimental heart failure following coronary ligation.

The animals were assigned randomly to six groups; sham operation, controls subjected to coronary ligation (control), coronary ligation plus chronic treatment with ACE inhibitors at non‐ and hypotensive doses; perindopril (0.2 or 2 mg kg−1 day−1) or enalapril (2 or 20 mg kg−1 day−1) for three weeks starting one week after the ligation.

Systemic blood pressure was measured every week during the experiments. At the end of the treatments, cardiac function and heart weight (an index of myocardial hypertrophy) were determined. In the other animals, ACE activities in plasma and tissues including heart, kidney, lung and blood vessels were measured.

In the controls, cardiac ACE activity, weight of right ventricle and left ventricular end‐diastolic pressure (LVEDP) were higher compared to those in the sham‐operated animals four weeks after the coronary ligation. However, ACE activities were not changed in plasma, kidney, lung and aorta by ligation of the coronary artery.

The chronic treatment with perindopril at a dose of 0.2 mg kg−1 day−1 inhibited the increase in ACE activity in cardiac tissue and suppressed the right ventricular hypertrophy without affecting systemic haemodynamics. In contrast, enalapril at a dose of 20 mg kg−1 day−1, but not 2 mg kg−1 day−1, prevented the development of the right ventricular hypertrophy. Enalapril at 20 mg kg−1 day−1 also lowered systemic blood pressure.

There is no significant correlation between systemic blood pressure and right ventricular hypertrophy at the end of the treatment with perindopril (r = 0.06) or enalapril (r = 0.1).

These findings demonstrate that perindopril, an ACE inhibitor, prevents cardiac hypertrophy without affecting systemic blood pressure in the rat with heart failure after coronary ligation, and suggest that selective augmentation of ACE activity in cardiac tissue is involved in the progression of hypertrophy in this model.
The effect of allosteric antagonists in modulating muscarinic M2‐receptor function in guinea‐pig isolated trachea
Authors: Spina D. , Minshall E. , Goldie R.G. , Page C.P.
Published: July 2012

We have assessed the influence of a range of synthetic cationic polypeptides with putative inhibitory actions at prejunctional muscarinic M2‐receptors on electrical field stimulation‐induced contraction of guinea‐pig isolated tracheal preparations. Electrical field stimulation of epithelium‐denuded guinea‐pig trachea resulted in frequency‐dependent contractile responses. As expected, tracheal smooth muscle sensitivity to electrical field stimulation was increased in tissues pretreated with the muscarinic M2‐receptor antagonist, gallamine. In contrast, gallamine did not significantly alter the contractile potency to acetylcholine.

Unlike gallamine, the synthetic cationic polypeptides, poly‐l‐arginine, poly‐l‐lysine, poly‐d‐lysine, the cationic dye ruthenium red and the anionic polysaccharide, heparin, failed to increase significantly tracheal smooth muscle sensitivity to electrical field stimulation.

Poly‐l‐arginine, ruthenium red and heparin had no effect on the contractile response to exogenously applied methacholine.

These data are consistent with the concept that in guinea‐pig tracheal smooth muscle, gallamine is an allosteric antagonist of guinea‐pig tracheal muscarinic M2‐receptors, whereas the various cationic polypeptides and the polyanion, heparin, are not.
Lack of effect of microinjection of noradrenaline or medetomidine on stimulus‐evoked release of substance P in the spinal cord of the cat: a study with antibody microprobes
Authors: Lang C.W. , Hope P.J. , Grubb B.D. , Duggan A.W.
Published: July 2012

Experiments were performed on barbiturate anaesthetized, spinalized cats to investigate the effect of microinjected noradrenaline or medetomidine on the release of immunoreactive substance P in the dorsal spinal cord following peripheral nerve stimulation. The presence of immunoreactive substance P was assessed with microprobes bearing C‐terminus‐directed antibodies to substance P.

Noradrenaline or medetomidine were microinjected into the grey matter of the spinal cord, near microprobe insertion sites, at depths of 2.5, 2.0, 1.5 and 1.0 mm below the spinal cord surface with volumes of approximately 0.125 μl and a concentration of 10−3m.

In the untreated spinal cord, electrical stimulation of the ipsilateral tibial nerve (suprathreshold for C‐fibres) elicited release of immunoreactive substance P which was centred in and around lamina II. Neither noradrenaline nor medetomidine administration in the manner described produced significant alterations in this pattern of nerve stimulus‐evoked release.

In agreement with recent ultrastructural studies these results do not support a control of substance P release by catecholamines released from sites near to the central terminals of small diameter primary afferent fibres.
Lack of importance of NO in β‐adrenoceptor‐mediated relaxation of large epicardial canine coronary arteries
Authors: Béa Marie‐Luce , Ghaleh Bijan , Giudicelli Jean‐François , Berdeaux Alain
Published: July 2012

This study was designed to test the role of endothelium and the l‐arginine/NO pathway in the relaxation of canine large coronary arteries to the β‐adrenoceptor agonist, isoprenaline. Relaxation of left circumflex (LCX) and left anterior descending (LAD) coronary arteries were measured in organ baths after contraction with the thromboxane analogue, U46619, either in absence or in presence of endothelium and in LCX arteries after pretreatment with NG‐nitro‐l‐arginine‐methyl‐ester (l‐NAME). LAD arteries with and without endothelium relaxed identically to isoprenaline but their maximal relaxation was smaller than corresponding LCX arteries with endothelium. A slight but non significant difference was observed in LCX arteries with endothelium as compared to rings without endothelium or pretreated with l‐NAME. No difference was observed in the relaxation of LCX arteries to forskolin in arteries with or without endothelium. These results suggest that endothelium is not essential and that NO is not directly involved in the relaxation of large coronary arteries induced by isoprenaline.
Sensitivity of G‐protein involved in endothelin‐1‐induced Ca2+ influx to pertussis toxin in porcine endothelial cells in situ
Authors: Aoki Hiroki , Kobayashi Sei , Nishimura Junji , Kanaide Hideo
Published: July 2012

We designed a new method to determine quantitatively the intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ, using front‐surface fluorometry and fura‐2‐loaded porcine aortic valvular strips. Using this method, we investigated the characteristics of the G‐protein involved in endothelin‐1 (ET‐1)‐induced changes in [Ca2+]i of endothelial cells in situ.

Endothelial cells were identified by specific uptake of acetylated‐low density lipoprotein labelled with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl‐indocarbocyanine perchlorate (DiI‐Ac‐LDL). Double staining with DiI‐Ac‐LDL and fura‐2 showed that the valvular strip was covered with a monolayer of endothelial cells and that the cellular component which contributed to the fura‐2 fluorescence, [Ca2+]i signal, was exclusively endothelial cells.

ET‐1 (10−7 m) induced an elevation of [Ca2+]i consisting of two components: the first was a rapid and transient elevation to reach a peak, followed by a second, sustained elevation (the second phase). The first phase was composed of extracellular Ca2+‐independent and ‐dependent components, while the second phase was exclusively extracellular Ca2+‐dependent. The extracellular Ca2+‐independent component of the first phase was due to the release of Ca2+ from intracellular storage sites. The second phase and part of the first phase of [Ca2+]i elevation were attributed to the influx of extracellular Ca2+. The Ca2+ influx component was completely inhibited by 10−3 m Ni2+ but was not affected by 10−5 m diltiazem.

Pertussis toxin (IAP) markedly inhibited the extracellular Ca2+‐dependent elevation of [Ca2+]i, but had no effect on the extracellular Ca2+‐independent elevation of [Ca2+]i caused by ET‐1 (10−7 m).

Bradykinin (10−7 m) or ATP (10−5 m) elevated [Ca2+]i and these responses also consisted of extracellular Ca2+‐independent and extracellular Ca2+‐dependent components. IAP had no effect on either component of the [Ca2+]i elevation induced by bradykinin or ATP.

From these findings we conclude that, in porcine endotheliel cells in situ, ET‐1 elevates [Ca2+]i as a result of a Ca2+ influx component from the extracellular space and release of intracelluarly stored Ca2+. The Ca2+ influx is regulated by an IAP‐sensitive G‐protein, while the release of Ca2+ from the intracellular store is not.
Effect of duration of ischaemia on reduction of myocardial infarct size by inhibition of neutrophil accumulation using an anti‐CD18 monoclonal antibody
Authors: Williams F.M. , Kus M. , Tanda K. , Williams T.J.
Published: July 2012

Neutrophil accumulation is a characteristic feature of the inflammatory response in myocardial tissue which has undergone a period of ischaemia. The aim of this study was to examine whether inhibition of myocardial neutrophil infiltration, using an antibody to the CD18 leukocyte adhesion molecule, was effective in reducing infarct size in anaesthetized rabbits.

Anaesthetized rabbits underwent coronary artery occlusion (CAO) for periods of 30 or 45 min followed by reperfusion for 3 h. Animals were treated intravenously 10 min prior to reperfusion with IB4, a monoclonal antibody to CD18 (1 mg kg−1) or saline (1 ml kg−1). In one group undergoing 45 min CAO, a control antibody, OKMI (1 mg kg−1) was given.

Following either 30 or 45 min of CAO, administration of IB4 resulted in a <75% inhibition in neutrophil accumulation in the area at risk myocardium (AR) compared with control animals.

With the 30 min occlusion period, IB4 significantly reduced myocardial infarct size, 27.2 ± 3.2% vs 67.4 ± 5.6% in the saline control group (n = 5 P < 0.01). In contrast, IB4 did not reduce infarct size following a 45 min period of ischaemia.

In the same animals administration of IB4 significantly inhibited oedema formation in skin elicited by intradermal administration of the neutrophil chemoattractant f‐Met‐Leu‐Phe, but had no effect on coronary microvascular plasma protein leakage in the AR.

Our results indicate that infiltrating neutrophils exacerbate tissue injury following a relatively short, 30 min period of myocardial ischaemia in the rabbit. However, protection with IB4 was no longer seen if the period of CAO was extended to 45 min. The results in this model suggest neutrophils are not a major determinant of tissue injury following more than a very short period of ischaemia.
Modulation by central postsynaptic α2‐adrenoceptors of the jaw‐opening reflex induced by orofacial stimulation in rats
Authors: García‐Vallejo Pascual , Barturen Fernando , García‐Sevilla Jesús A.
Published: July 2012

The modulation by α2‐adrenoceptors of the jaw‐opening reflex (digastric electromyographic responses) elicited by orofacial electrical stimulation (OF‐JOR) in pentobarbitone anaesthetized rats was investigated.

Increasing doses of clonidine (0.1–1000 μg kg−1, i.v.) reduced, in a dose‐dependent manner until abolition, the amplitude and duration of the OF‐JOR and increased the latency to onset. The sum of amplitudes of the reflex was the most sensitive parameter to the inhibitory effects of clonidine (ED50 = 13.9 μg kg−1).

Pretreatment with the α2‐adrenoceptor antagonist, idazoxan (0.03–1 mg kg−1, i.v.), caused a dose‐dependent shift (1.5 to 37 fold) to the right of the dose‐response curve for clonidine without significant change of maximum inhibitory effect, in a manner compatible with competitive antagonism (ED50B = 29.0 μg kg−1). Pretreatment with yohimbine (0.3 mg kg−1, i.v.) also antagonized the inhibitory effect of clonidine on the OF‐JOR. In contrast, the α2‐adrenoceptor antagonist ARC‐239 (0.3 mg kg−1, i.v.) did not antagonize the effect of clonidine on the reflex.

In rats pretreated with reserpine (5 mg kg−1, s.c., 18 h) the OF‐JOR was not modified, but the potency of clonidine in inhibiting the reflex was potentiated (ED50 value decreased to 6.8 μg kg−1) without a significant change of maximum inhibitory effect.

Increasing doses of amphetamine (0.1–3000 μg kg−1, i.v.) caused a dose‐related, but partial, inhibition of the OF‐JOR (IC50 = 135 μg kg−1; Emax = 67%). Pretreatment with idazoxan (0.1 mg kg−1, i.v.) induced a nine fold shift to the right of the dose‐response curve for amphetamine, while treatment with the depleting drug α‐methyl‐p‐tyrosine (150 mg kg−1daily, i.p., for 14 days) abolished the inhibitory effect of this indirect adrenoceptor agonist on the OF‐JOR.

Morphine (0.1–3000 μg kg−1, i.v.) also reduced the OF‐JOR in a dose‐dependent manner (ED50 value about 325 μg kg−1) but, in contrast to clonidine, it failed to inhibit the reflex fully (Emax = 48%). As expected, pretreatment with the opioid antagonist naloxone (1 mg kg−1, i.v.) abolished the inhibitory effect of morphine on the OF‐JOR, while it did not alter that of clonidine.

Chronic, but not acute, pretreatment with idazoxan (3 mg kg−1daily, i.p. for 14 days) led to a marked potentiation of the inhibitory effect of clonidine on the OF‐JOR (ED50 value decreased to 4.2 μg kg−1), without a significant change of maximum inhibitory effect.

Together the results indicate that clonidine evokes a potent inhibition of the OF‐JOR in rats through the activation of postsynaptic α2‐adrenoceptors. It is suggested that this functional response represents a simple and useful in vivo model for studying various regulatory mechanisms of central α2‐adrenoceptors.
The pharmacological properties of Y‐23684, a benzodiazepine receptor partial agonist
Authors: Yasumatsu Hiroshi , Morimoto Yasuto , Yamamoto Yoshinobu , Takehara Shuzo , Fukuda Takemi , Nakao Tohru , Setoguchi Michihide
Published: July 2012

The pharmacological properties of a benzodiazepine receptor (BZR) partial agonist, Y‐23684 were investigated in comparison with those of diazepam, a conventional BZR full agonist.

Y‐23684 and diazepam showed high and selective affinity for the BZR with Ki values of 41 and 5.8 nm, respectively.

In contrast to diazepam, variability was noted in the anticonvulsive potency of Y‐23684 depending on convulsants (bicuculline, pentylenetetrazol and maximal electrical shock). Y‐23684 produced the most potent protective effect against bicuculline in rats and mice with ED50s of 1.3 and 1.2 mg kg−1, respectively.

In rat conflict models (Geller‐Seifter and water‐lick tests), Y‐23684 produced an antipunishment action at doses 2–4 times lower than diazepam. In contrast to diazepam, Y‐23684 did not affect unpunished responding up to 50 mg kg−1in the Geller‐Seifter test.

In other rat models of anxiety (social interaction and elevated plus‐maze tests), Y‐23684 was as efficacious as and ten fold more potent than diazepam. In a mouse model of anxiety (exploration (light/dark box) test), Y‐23684 was as efficacious and two fold less potent as diazepam. In these paradigms, Y‐23684 showed a selective anxiolytic profile over a wide dose‐range without loss of efficacy and sedative action.

The impairment of motor coordination (rotarod) and potentiation of CNS depressants (ethanol and hexobarbitone) by Y‐23684 was much weaker than that of diazepam.

These results suggest that Y‐23684 would be a potent and selective anxiolytic agent in man with less side‐effects than conventional BZ‐anxiolytics.
IgE‐receptor activated chloride uptake in relation to histamine secretion from rat mast cells
Authors: Friis U.G. , Johansen T. , Hayes N.A. , Foreman J.C.
Published: July 2012

Antigen‐stimulated histamine secretion from rat peritoneal mast cells was inhibited when extracellular chloride was replaced by either isethionate or gluconate anions, but the histamine release still remained quite substantial.

Rat peritoneal mast cells take up 36Cl and the uptake reaches a steady state after 60 min incubation with the isotope. At steady state, the intracellular chloride level in the cells was calculated to be 29 ± 11.5 mm.

The chloride uptake in mast cells was exponential with a rate constant of 0.036 min−1in resting cells. When the cells were stimulated with antigen, and rate constant for chloride uptake increased to 0.90 min−1: an increase of 25 fold. Under identical experimental conditions histamine release increased 3 fold.

The rate of chloride uptake in either resting cells or in antigen‐stimulated cells was not changed when the extracellular medium was nominally calcium‐free but histamine release was almost completely inhibited in the absence of extracellular calcium.

The putative chloride channel blocker DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid) 0.3 to 30 μm, produced a concentration‐related inhibition of antigen‐stimulated histamine secretion but DIDS (30 μm) did not inhibit the antigen‐stimulated increase of chloride uptake.

The cyclic AMP analogue, dibutyryl cyclic AMP (1 mm) produced a delayed increase in chloride uptake in resting mast cells but neither dibutyryl cyclic AMP nor 8‐bromo cyclic AMP per se induced any histamine secretion.

Ouabain (1 mm) which inhibits the Na+/K+ATPase in rat peritoneal mast cells, failed to affect the uptake of chloride in resting mast cells.

The Na/K/2Cl‐cotransport inhibitor, furosemide (0.7 mm), slowed the unstimulated chloride uptake in resting mast cells and abolished the increased antigen‐induced chloride uptake when added together with antigen. In contrast, spontaneous and antigen‐induced histamine release were unaffected by the presence of furosemide. However, when furosemide was added to the cell suspension 5 min before stimulation, furosemide was without effect on the antigen‐induced chloride uptake.

In addition to the chloride uptake mediated by chloride channels which may be related to the mechanism of histamine secretion, crosslinking of the high affinity membrane receptors for IgE is followed by a fast chloride uptake that is likely to occur through a furosemide‐sensitive Na/K/2Cl‐cotransporter.
Pulmonary effects of type V cyclic GMP specific phosphodiesterase inhibition in the anaesthetized guinea‐pig
Authors: Turner Nicholas C. , Dolan John S. , Grimsditch David , Lamb Janine , Worby Angela , Murray Kenneth J. , Coates William J. , Warrington Brian H.
Published: July 2012

We have investigated the bronchodilator potential of type V phosphodiesterase (PDE V) inhibitors in anaesthetized ventilated guinea‐pigs using the potent and selective PDE V inhibitor, SK&F 96231. We have compared its activity to that of salbutamol, the PDE III inhibitors, siguazodan and SK&F 95654 and to the PDE IV inhibitor rolipram.

Administered as an i.v. infusion SK&F 96231 (0.6 and 1 mg kg−1min−1, i.v.) caused a slowly developing inhibition of histamine (100 nmol kg−1, i.v.)‐induced bronchoconstriction and elevated tracheal cyclic GMP levels in the anaesthetized guinea‐pig. SK&F 96231 (0.1 and 0.3 mg kg−1min−1, i.v.) was without effect on histamine‐induced bronchoconstriction. In the presence of a sub‐threshold infusion of SNP (0.1 μmol kg−1min−1, i.v.) there was a marked enhancement of SK&F 96231‐induced inhibition of histamine responses such that at infusion rates that were ineffective alone, SK&F 96231 caused a > 50% inhibition of histamine responses. The stimulation of tracheal cyclic GMP accumulation by SK&F 96231 was also potentiated.

Administered directly into the airway, SK&F 96231 (300 μg in 5 mg lactose carrier) was largely without effect on histamine‐induced bronchoconstriction (4.9 ± 1.9% inhibition). In the presence of SNP (0.1 μmol kg−1min−1, i.v.) or isosorbide dinitrate (200 μg administered by insufflation into the trachea) there was a marked potentiation of the inhibitory activity of SK&F 96231 (40 ± 4% and 62 ± 1.8% respectively).

Salbutamol and rolipram (3–300 μg by insufflation) caused a dose‐related inhibition of histamine responses with a maximum of 91 ± 2% and 59 ± 10% respectively. The PDE III inhibitor, siguazodan, was without effect on histamine responses but they were reduced (27.7 ± 4.8% at 300 μg) by SK&F 95654. There was a marked enhancement of the inhibitory activity of rolipram in the presence of SK&F 95654.

We conclude that SK&F 96231 has weak anti‐spasmogenic activity in the guinea‐pig in vivo, we suggest that this is primarily a consequence of a low endogenous guanylate cyclase activity in the airway. The potentiation of the anti‐spasmogenic activity of SK&F 96231 by SNP suggests that a combination of PDE V inhibitor and guanylate cyclase agonist might provide significant bronchodilator activity.

We have established that PDE IV inhibitors are bronchodilators when administered directly into the airway of anaesthetized guinea‐pigs but that PDE III inhibitors are only weakly active. The marked enhancement of the inhibitory activity of rolipram by the PDE III inhibitor, SK&F 95654, indicates that inhibitors of both PDE III and PDE IV might offer greater potential as bronchodilators than inhibitors of either isoenzyme alone.
Effect of a 5‐lipoxygenase inhibitor, ZM 230487, on cutaneous allergic inflammation in the guinea‐pig
Authors: Teixeira Mauro M. , Hellewell Paul G.
Published: July 2012

Leukotrienes have potent biological effects in vitro and in vivo and are found in tissue and in biological fluids in various pathological conditions including allergic diseases. Leukotriene B4 (LTB4) is a potent stimulus for eosinophil accumulation and activation and there is much interest in determining its importance in mediating the accumulation of eosinophils at sites of allergic inflammation in vivo. In this study, we investigated the effects of a potent 5‐lipoxygenase inhibitor, ZM 230487, on the accumulation of eosinophils and on local oedema formation in cutaneous inflammation in the guinea‐pig.

The i.d. injection of increasing concentrations of arachidonic acid (AA) led to a dose‐dependent accumulation of 111In‐eosinophils but oedema formation was only significant at the top dose of AA tested (3 × 10−8mol per site). Co‐injection of ZM 230487 with AA inhibited 111In‐eosinophil accumulation up to 99% but the small oedema response to AA was only partially inhibited. AA‐induced oedema formation was only effectively inhibited when a combination of a PAF antagonist, an antihistamine and ZM 230487 was used.

Local administration of the cyclo‐oxygenase inhibitor, ibuprofen, partially inhibited AA‐induced oedema formation suggesting that vasodilator prostaglandins may be released following i.d. injection of AA. AA‐induced 111In‐eosinophil accumulation was also partially inhibited by ibuprofen.

PAF‐induced 111In‐eosinophil accumulation was partially suppressed by local administration of ZM 230487. In contrast, LTB4‐induced 111In‐eosinophil accumulation was enhanced by ZM 230487. These data suggest that locally‐released leukotrienes may modulate mediator‐induced eosinophil accumulation. ZM 230487 had no effect on PAF‐or LTB4‐induced oedema formation.

ZM 230487 significantly inhibited the accumulation of 111In‐eosinophils, but did not affect local oedema formation, in a passive cutaneous anaphylaxis (PCA) reaction. However, the PAF antagonist WEB 2086 either alone or in combination with ZM 230487 had no effect on 111In‐eosinophil accumulation or oedema formation in the PCA reaction.

In conclusion, it appears that a product of 5‐lipoxygenase, probably LTB4, is important for the accumulation of 111In‐eosinophils, but not local oedema formation, in the PCA reaction in guinea‐pig skin. These data support a major role for LTB4 in allergic inflammation in the guinea‐pig and make this animal (and the PCA model) suitable for studying the effects of inhibitors of leukotriene synthesis or action in vivo.
Ischaemia/reperfusion selectively attenuates coronary vasodilatation to an adenosine A2‐ but not to an A1‐agonist in the dog
Authors: Cox Bryan F. , Greenland Brett D. , Perrone Mark H. , Merkel Linda A.
Published: July 2012

The effects of myocardial ischaemia/reperfusion were tested on the coronary vasorelaxant responses to agonists selective for the A1 and A2 adenosine receptor subtypes in the dog. The left anterior descending (LAD) coronary artery was occluded distal to the first diagonal branch. The occlusion was maintained for 1 h, followed by 1 h of reperfusion.

In the first series of experiments, LAD and circumflex arteries were excised and contracted with prostaglandin F2α (PGF2α). Ischaemia/reperfusion did not significantly alter the vasorelaxation produced by either sodium nitroprusside (endothelium‐independent) or acetylcholine (endothelium‐dependent). The A1 selective agonist, cyclopentyladenosine (CPA), produced coronary vasorelaxation in both normally perfused vessels and vessels subjected to ischaemia/reperfusion. In contrast, the relaxation produced by the A2‐selective agonist N6‐{2‐(3,5‐dimethoxyphenyl)‐2‐(2‐methylphenyl) ethyl} adenosine (DPMA) was significantly attenuated by ischaemia/reperfusion (14 fold shift in EC50).

In the second series of experiments, coronary blood flow was increased by administration of the A1 and A2 agonists before and after ischaemia/reperfusion of the LAD in anaesthetized dogs. Both compounds dose‐dependently increased coronary blood flow. The slopes of the dose‐response functions to CPA or DPMA were not significantly altered in the normally perfused circumflex vascular bed. Similarly, the CPA dose‐response function in the LAD was unaltered by ischaemia/reperfusion. However, the slope of the coronary vasodilator response to the A2 agonist was significantly reduced following ischaemia/reperfusion of the LAD.

We conclude that ischaemia/reperfusion reduces responsiveness to an adenosine A2 receptor subtype agonist, but not an A1 receptor subtype agonist. These data confirm the independent nature of A1 and A2‐mediated coronary vasodilatation.
Effect of anti‐fungal imidazoles on mRNA levels and enzyme activity of inducible nitric oxide synthase
Authors: Bogle R.G. , Whitley G.St.J. , Soo S.‐C. , Johnstone A.P. , Vallance P.
Published: July 2012

Experiments were performed to examine the effects of anti‐fungal imidazole compounds (clotrimazole, econazole and miconazole) on the induction of nitric oxide (NO) synthase and subsequent production of NO in the cultured murine monocyte/macrophage cell line J774 using a specific cDNA probe for inducible NO synthase mRNA and by monitoring nitrite production.

Stimulation of J774 cells with lipopolysaccharide (LPS, 10 μg ml−1) resulted in the induction of NO synthase activity as determined by nitrite accumulation in the culture medium (48 ± 3 nmol per 106cells over 24 h). Production of nitrite was inhibited by co‐incubation of cells with LPS (10 μg ml−1) and either dexamethasone (10 μm) or NG‐monomethyl‐l‐arginine (l‐NMMA; 0.1 mm), however, only l‐NMMA was an effective inhibitor of nitrite production when added after induction of NO synthase had occurred.

Co‐incubation of J774 cells with LPS (10 μg ml−1) and either clotrimazole, econazole or miconazole (1–10 μm) resulted in a concentration‐dependent inhibition of nitrite production over the subsequent 24 h without any evidence for a cytotoxic effect. However, addition of these imidazoles after induction of NO synthase did not inhibit nitrite production.

Messenger RNA for inducible NO synthase was not detected in unstimulated J774 cells. Treatment with LPS (10 μg ml−1) for 4 h resulted in significant expression of mRNA for inducible NO synthase which was not altered in the presence of econazole (10 μm) but was reduced significantly by dexamethasone (10 μm).

These results demonstrate that anti‐fungal imidazoles inhibit the production of nitric oxide by cultured J774 cells by a mechanism which appears to differ from that of dexamethasone and substrate‐type inhibitors of NO synthase. Furthermore, the presence of mRNA for NO synthase does not indicate the presence of functionally active NO synthase.
Differential effects of ω‐conotoxin GVIA and tetrodotoxin on vasoconstrictions evoked by electrical stimulation and nicotinic receptor stimulation in canine isolated, perfused splenic arteries
Authors: Ren Lei‐Ming , Nakane Tokio , Chiba Shigetoshi
Published: July 2012

The effects of ω‐conotoxin GVIA (ω‐CgTX) and tetrodotoxin (TTX) on vasoconstrictions induced by acetylcholine (ACh) and nicotine were investigated and compared with those induced by periarterial electrical stimulation in the isolated and perfused canine splenic arteries.

ACh and nicotine at doses of 0.01 to 1 μmol constricted the splenic artery, dose‐dependently. ACh induced consistent responses, but the vasoconstrictor responses to nicotine became significantly smaller with repeated administration of nicotine.

Periarterial electrical stimulation produced a vasoconstriction that was abolished by either TTX (30 nmol) or ω‐CgTX (3 nmol), but the vasoconstrictor response to nicotine was not significantly affected by the same doses of TTX and ω‐CgTX. Inhibitions by TTX and ω‐CgTX of ACh‐induced vasoconstrictions were small but statistically significant, showing that the percentage inhibition was less than 15%. TTX and ω‐CgTX did not affect the vasoconstrictor responses to exogenous noradrenaline (NA).

ACh did not produce any vasoconstriction in the preparations treated either with α‐adrenoceptor antagonists (10 μm bunazosin and 10 μm midaglizole) or with 30 μm guanethidine. NA‐induced responses were abolished by α‐adrenoceptor antagonists, but not affected by guanethidine treatment.

Vascular responses to ACh were completely inhibited by 1 μmol hexamethonium. In the preparations treated with 100 nmol nicotine, ACh did not produce any vasoconstriction. However, the NA‐induced vasoconstriction was affected by neither hexamethonium nor nicotine treatment.

Atropine (1 μm) significantly inhibited but did not abolish the vasoconstrictor responses to ACh. The vascular responses to nicotine and NA were also significantly inhibited by atropine treatment.

These results indicate that (1) ACh constricts the splenic artery through the activation of presynaptic nicotinic receptors present on the sympathetic nerves; (2) differential effects of TTX and ω‐CgTX on the vascular responses to ACh and nicotine, and to electrical stimulation suggest that the receptor‐operated ion channels are mainly responsible for NA release induced by nicotinic receptor stimulation, but N‐type VOCCs are responsible for that by electrical stimulation; (3) atropine may have an inhibitory action on nicotine‐related responses, in addition to its inhibitory action on NA.
Investigation of the specificity of FK 888 as a tachykinin NK1 receptor antagonist
Authors: Wang Zun‐Yi , Tung Steven R. , Strichartz Gary R. , Håkanson Rolf
Published: July 2012

A recently described peptide tachykinin (NK1) receptor antagonist, FK 888, was found to inhibit the electrically‐evoked, tachykinin‐mediated contractile responses of the rabbit iris sphincter in a concentration‐dependent manner; the pIC50 value was 6.6 ±0.08.

Contractions induced by a selective NK1 receptor agonist, [Sar9, Met(O2)11]substance P, were inhibited competitively by FK 888; the pKB value was 7.1.

FK 888 (1 nm‐100 μm) was without effect on the electrically‐evoked, cholinergic response of the rabbit iris sphincter and the electrically‐evoked, sympathetic response of the guinea‐pig vas deferens. The contractions of the rabbit iris sphincter, induced by either carbachol (10 nm‐30 μm) or noradrenaline (0.1–100 μm), were not affected by 10 μm FK 888.

FK 888 (1–30 μm) did not induce histamine release from rat peritoneal mast cells.

FK 888 (33 and 333 μm) was without effect on the electrically‐evoked action potentials of the frog sciatic nerve. Thus, FK 888 is a moderately high affinity and selective tachykinin (NK1) receptor antagonist.