bluuebunny/crossref_metadata_2025 huggingface dataset:
Antimuscarinic action of liriodenine, isolated from Fissistigma glaucescens, in canine tracheal smooth muscle
Authors: Lin Chien‐Huang , Yang Chuen‐Mao , Ko Feng‐Nien , Wu Yang‐Chang , Teng Che‐Ming
Published: July 2012

The antimuscarinic properties of liriodenine, isolated from Fissistigma glaucescens, were compared with methoctramine (cardioselective M2 antagonist) and 4‐diphenylacetoxy‐N‐methylpiperidine (4‐DAMP, smooth muscle selective M3 antagonist) by radioligand binding tests, functional tests and measurements of second messenger generation in canine cultured tracheal smooth muscle cells.

Liriodenine, pirenzepine, methoctramine and 4‐DAMP displaced [3H]‐N‐methyl scopolamine ([3H]‐NMS) binding in a concentration‐dependent manner with Ki values of 2.2 ± 0.4 × 10−6, 3.3 ± 0.7 × 10−7, 8.9 ± 2.3 × 10−8 and 2.3 ± 0.6 × 10−9 m, respectively. The curves for competitive inhibition of [3H]‐NMS with liriodenine, methoctramine and 4‐DAMP were best fitted according to a two site model of binding, but pirenzepine was best fitted according to a model with one site.

Liriodenine and 4‐DAMP displayed a high affinity for blocking tracheal contraction (pKB = 5.9 and 9.1, respectively) and inositol phosphate formation (pKh = 6.0 and 8.9, respectively), but a low affinity for antagonism of cyclic AMP inhibition (pKB = 4.7 and 7.8, respectively).

Methoctramine blocked cyclic AMP inhibition with a high affinity (pKB = 7.4), but it antagonized tracheal contraction and inositol phosphate formation with a low affinity (pKh = 6.1 and 6.0, respectively).

In conclusion, both M2 and M3 muscarinic receptor subtypes coexist in canine tracheal smooth muscle and are coupled to the inhibition of cyclic AMP formation and phosphoinositide breakdown, respectively. The antimuscarinic characteristics of liriodenine are similar to those of 4‐DAMP. It may act as a selective M3 receptor antagonist in canine tracheal smooth muscle.
Involvement of tyrosine kinase in the induction of cyclo‐oxygenase and nitric oxide synthase by endotoxin in cultured cells
Authors: Akarasereenont P. , Mitchell J.A. , Appleton I. , Thiemermann C. , Vane J.R.
Published: July 2012

Cyclo‐oxygenase (COX) and nitric oxide synthase (NOS) are two enzymes which have distinct cytokine‐inducible isoforms (COX‐2 and iNOS). Many cytokine receptors have an intracellular tyrosine kinase domain. Here we have used the tyrosine kinase inhibitors, erbstatin and genistein, to investigate the potential role of tyrosine kinase activation in the induction on COX‐2 and iNOS caused by endotoxin (lipopolysaccharide; LPS) in bovine aortic endothelial cells (BAEC) and J774.2 macrophages.

The main COX metabolites, 6‐oxo‐prostaglandin F1α (6‐oxo‐PGF1α) (for BAEC) and PGF2α (for 774.2 macrophages) were measured by radioimmunossay: (i) accumulation of COX metabolites from endogenous arachidonic acid was measured at 24 h after addition of LPS (1 μg ml−1); (ii) in experiments designed to measure ‘COX activity’, COX metabolites generated by BAEC or J774.2 macrophages activated with LPS were assayed (at 12 h after LPS administration) after incubation of the washed cells with exogenous arachidonic acid (30 μg for 15min). Western blot analysis with a specific antibody to COX‐2 was used to determine the expression of COX‐2 protein caused by LPS in cell extracts. Accumulation of nitrite (measured by the Griess reaction) was used as an indicator of NO formation and, hence, iNOS activity.

Erbstatin (0.05 to 5 μg ml−1) or genistein (0.5 to 50 μg ml−1) caused a dose‐dependent inhibition of the accumulation of COX metabolites in the supernatant of BAEC or J774.2 macrophages activated with LPS. Erbstatin or genistein also caused a dose‐dependent inhibition of ‘COX activity’ in both cell types. Western blot analysis showed that erbstatin (5 μg ml−1) or genistein (50μg ml−1) inhibited the expression of COX‐2 protein in BAEC and J774.2 macrophages activated with LPS (1 μg ml−1 for 24 h).

Erbstatin or genistein also caused a dose‐dependent inhibition of nitrite accumulation in J774.2 macrophages activated with LPS (1 μg ml−1 for 24 h). In contrast to J774.2 macrophages, BAEC stimulated with LPS (1 μg ml−1 for 24 h) did not produce detectable amounts (> 1μm) of nitrite.

These results suggest that tyrosine phosphorylation is part of the signal transduction mechanism that mediates (i) the induction of COX‐2 and iNOS elicited by LPS in J774.2 macrophages, and (ii) the induction of COX‐2 by LPS in BAEC.
Two populations of muscarinic binding sites in the chick heart distinguished by affinities for ligands and selective inactivation
Authors: Jakubik Jan , Tuĉek Stanislav
Published: July 2012

By measuring the binding of N‐[3H‐methyl]‐scopolamine ([3H]‐NMS) and of unlabelled subtype‐specific muscarinic antagonists, two populations of muscarinic binding sites can be distinguished in the membranes of cardiac ventricles taken from 1‐day‐old chicks. One of them, corresponding to approximately 80% of [3H]‐NMS binding sites, has higher affinities for AF‐DX116 (pKi = 6.42) and methoct‐ramine (pKi = 7.33); the rate of [3H]NMS dissociation from these sites is fast. The other population, corresponding to approximately 20% of [3H]‐NMS binding sites, has lower affinities for AF‐DX116 (pKi = 5.00) and methoctramine (pKi = 6.19); the rate of [3H]‐NMS dissociation from these sites is slow. Both populations have high affinities for pirenzepine, but the affinity of the former (major) population is lower (pKi = 7.99) than that of the latter (minor) population (pKi = 10.14).

Since it has been shown earlier that two mRNAs for muscarinic receptors are expressed in the chick heart, one of them close to the genetically defined m2 and the other to the m4 subtype, we propose that the major population of binding sites with high affinities for AF‐DX116 and methoctramine and the lower affinity for pirenzepine represents the M2‐like receptors, while the minor population represents the M4‐like receptors.

It proved possible to obtain isolated samples of either population by selectively protecting the M2‐like sites with AF‐DX116 and the M4‐like sites with pirenzepine, and by inactivating the unprotected sites with benzilylcholine mustard. The properties of the isolated populations corresponded to those derived from the analysis of [3H]‐NMS binding to the original mixed population.

Alcuronium exerted positive allosteric action on the binding of [3H]‐NMS both to the M2‐like and the M4‐like population and severely slowed down [3H]‐NMS dissociation from them; its affinity for the M2‐like sites was 3–10 times higher.
Endogenous expression of histamine H1 receptors functionally coupled to phosphoinositide hydrolysis in C6 glioma cells: regulation by cyclic AMP
Authors: Peakman Marie‐Claire , Hill Stephen J.
Published: July 2012

The effects of histamine receptor agonists and antagonists on phospholipid hydrolysis in rat‐derived C6 glioma cells have been investigated.

Histamine H1 receptor‐stimulation caused a concentration‐dependent increase in the accumulation of total [3H]‐inositol phosphates in cells prelabelled with [3H]‐myo‐inositol. The rank order of agonist potencies was histamine (EC50 = 24 μm) > Nα‐methylhistamine (EC50 = 31 μm) > 2‐thiazolylethylamine (EC50 = 91 μm).

The response to 0.1 mm histamine was antagonized in a concentration‐dependent manner by the H1‐antagonists, mepyramine (apparent Kd = 1 nm) and (+)‐chlorpheniramine (apparent Kd = 4 nm). In addition, (−)−chlorpheniramine was more than two orders of magnitude less potent than its (+)‐stereoisomer.

Elevation of intracellular cyclic AMP accumulation with forskolin (10 μm, EC50 = 0.3 μm), isoprenaline (1 μm, EC50 = 4 nm) or rolipram (0.5 μm), significantly reduced the histamine‐mediated (0.1 μm) inositol phosphate response by 37%, 43% and 26% respectively. In contrast, 1,9‐dideoxyforskolin did not increase cyclic AMP accumulation and had no effect on the phosphoinositide response to histamine.

These data indicate the presence of functionally coupled, endogenous histamine H1 receptors in C6 glioma cells. Furthermore, the results also indicate that H1 receptor‐mediated phospholipid hydrolysis is inhibited by the elevation of cyclic AMP levels in these cells.
Role of endothelin‐1 and the ETA receptor in the maintenance of deoxycorticosterone acetate‐salt‐induced hypertension
Authors: Fujita Katsuya , Matsumura Yasuo , Kita Satomi , Miyazaki Yohko , Hisaki Kazuhiro , Takaoka Masanori , Morimoto Shiro
Published: July 2012

To search for a possible role for endothelin‐1 (ET‐1) in deoxycorticosterone acetate (DOCA)‐salt‐induced hypertension, we examined changes in concentration of ET‐1 in vascular and renal tissue in DOCA‐salt hypertensive rats and evaluated the antihypertensive effect of the ETA receptor antagonist, FR139317.

There was an increase in aortic immunoreactive‐ET (IR‐ET) concentrations in association with hypertension‐induced treatment. There were no significant changes in ET‐1 levels in the kidney with DOCA‐salt treatment.

In DOCA‐salt hypertensive rats, a significant correlation (r = 0.83, P <0.01) was found between aortic IR‐ET concentrations and systolic blood pressure.

High‐performance liquid chromatography analysis of the aortic extract from DOCA‐salt rats revealed one major component corresponding to the elution position of synthetic ET‐1.

The intravenous bolus injection of FR139317 (10 mg kg−1) produced a slight decrease in blood pressure in the control rats and in the DOCA‐salt hypertensive rat, FR139317 had a more pronounced hypotensive effect.

We propose that ET‐1 production in vascular tissues is increased in DOCA‐salt hypertensive rats. In addition, our study indicates the pathophysiological importance of increased endogenous ET‐1 in the maintenance of DOCA‐salt‐induced hypertension, through interaction of the peptide with ETA receptors.
Blockade of motion‐ and cisplatin‐induced emesis by a 5‐HT2 receptor agonist in Suncus murinus
Authors: Okada Fumihiko , Saito Hiroshi , Matsuki Norio
Published: July 2012

The effects of (±)‐1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane (DOl), a 5‐hydroxytryptamine 5‐HT2A/5‐HT2C receptor agonist, on motion‐ and cisplatin‐induced emesis were studied in Suncus murinus. Subcutaneous injection of DOl, 30 min prior to the emetic stimuli, dose‐dependently blocked the emesis induced by motion sickness and cisplatin (20 mg kg−1, i.p.) with estimated ID50 values of 640 and 780 μg kg−1, respectively.

α‐Methyl‐5‐hydroxytryptamine (α‐Me‐5‐HT), a peripheral 5‐HT2A/5‐HT2C receptor agonist, had no effect on motion‐ and cisplatin‐induced emesis.

The antiemetic effects of DOl on motion‐ and cisplatin‐induced emesis were attenuated by pre‐administration of ketanserin, a selective 5‐HT2A receptor antagonist.

The present results suggest an inhibitory role for central 5‐HT2 receptors in the emetic reflex mechanism and that a 5‐HT2 receptor agonist may be a useful tool to investigate the involvement of 5‐HT receptors in the emetic reflex.
Pharmacology of a non‐selective ETA and ETB receptor antagonist, TAK‐044 and the inhibition of myocardial infarct size in rats
Authors: Watanabe Toshifumi , Awane Yumiko , Ikeda Shota , Fujiwara Shuji , Kubo Kazuki , Kikuchi Takashi , Kusumoto Keiji , Wakimasu Mitsuhiro , Fujino Masahiko
Published: July 2012

The aims of the present study were to characterize the pharmacological profile of a new endothelin (ET) receptor antagonist, TAK‐044 and to consider whether it limits the extension of myocardial infarct size in rats.

Binding of [125I]‐ET‐1 to ET receptors on rabbit ventricular and cerebellar membrane fractions was inhibited by TAK‐044 with IC50 values of 3.8 nm and 130 nm, respectively.

It inhibited ET‐1, ET‐2 and ET‐3‐induced vasoconstriction of porcine isolated coronary arteries in a competitive (ET‐1, ET‐2) and a non‐competitive (ET‐3) manner.

In the rat in vivo, the ET‐1‐induced blood pressure changes including transient hypotension followed by sustained hypertension, were inhibited by TAK‐044 (0.1–10 mg kg−1, i.v.) in a dose‐dependent manner.

Acute myocardial infarction induced by 1 h coronary occlusion followed by 24 h reperfusion in rats caused an infarct size of 60 ± 2% (n = 12) of the area‐at‐risk by weight.

Intravenous injection of TAK‐044 10 min before coronary occlusion reduced the infarct size in a dose‐dependent manner: 32% and 54% reductions at 1 and 3 mg kg−1, respectively.

TAK‐044 administered 10 min before or 1 h after reperfusion (1 mg kg−1, i.v.) showed similar inhibitory effects: 34% and 23% reductions, respectively.

We conclude that TAK‐044 is an ETA/ETB receptor antagonist which shows strong inhibitory effects on the extension of myocardial infarct size after coronary artery occlusion‐reperfusion in rats.
The effect of morphine on formalin‐evoked behaviour and spinal release of excitatory amino acids and prostaglandin E2 using microdialysis in conscious rats
Authors: Malmberg Annika B. , Yaksh Tony L.
Published: July 2012

In the present study, the object was to examine the effects of morphine on spinal release in vivo of excitatory amino acids (EAA), prostaglandin E2 (PGE2), and a marker for nitric oxide (NO) synthesis, citrulline (Cit), evoked by a protracted noxious stimulus produced by the injection of formalin into the paw. Spinal release was monitored in conscious rats using a microdialysis probe implanted into the subarachnoid space with the active site placed at the level of the lumbar enlargement. In split dialysate samples, EAAs were measured by high performance liquid chromatography (h.p.l.c.) and PGE2 was determined by radioimmunoassay.

Resting concentrations in nmol ml−1 for the amino acids (mean ± s.e.mean, n = 21) were: 4.8 ± 0.4 for glutamate (Glu), 0.8 ± 0.1 for aspartate (Asp), 8.8 ± 0.8 for taurine (Tau), 24 ± 3 for glycine (Gly), 19 ± 3 for serine (Ser), 5.2 ± 0.8 for asparagine (Asn), 64 ± 4 for glutamine (Gln) and 5.2 ± 0.4 for Cit. Mean basal release for PGE2 was 12 ± 1 pmol ml−1.

Subcutaneous (s.c.) injection of 5% formalin evoked a biphasic flinching behaviour (phase 1: 0–9 min and phase 2: 10–60 min) of the injected paw. Corresponding to phase 1 behaviour, there was a significant increase (50–100%) in spinal levels of Glu, Asp, Tau, Gly, Cit and PGE2, but not Ser, Asn and Gln. A significant (P<0.01) second phase increase in release was observed only for Cit and PGE2. However, Glu and Asp levels were increased by approximately 45%.

Injection of morphine sulphate (3 mg kg−1, s.c.) had no effect on resting release, but produced a significant suppression of the formalin‐evoked behaviour and release of Glu, Asp, Tau, Gly, Cit and PGE2. The effect of morphine was reversed by pretreatment with 1 mg kg−1 naloxone. Naloxone by itself did not change the release or behaviour of the formalin test.

This study demonstrates that both spinal EAA and PGE2 release patterns correlate with behavioural nociceptive responses in the formalin test and that morphine suppresses the formalin‐evoked behaviours and spinal release. The reversal by naloxone of the morphine effect indicates mediation via an opioid receptor.
Copper chelation‐induced reduction of the biological activity of S‐nitrosothiols
Authors: Gordge M.P. , Meyer D.J. , Hothersall J. , Neild G.H. , Payne N.N. , Noronha‐Dutra A.
Published: July 2012

The effect of copper on the activity of the S‐nitrosothiol compounds S‐nitrosocysteine (cysNO) and S‐nitrosoglutathione (GSNO) was investigated, using the specific copper chelator bathocuproine sul‐phonate (BCS), and human washed platelets as target cells.

Chelation of trace copper with BCS (10 μm) in washed platelet suspensions reduced the inhibition of thrombin‐induced platelet aggregation by GSNO; however, BCS had no significant effect on the anti‐aggregatory action of cysNO. BCS inhibited cyclic GMP generation in response to both cysNO and GSNO.

The effect of BCS was rapid (within 30 s), and could be abolished by increasing the platelet concentration to 500 times 109 1−1.

In BCS‐treated platelet suspensions, the addition of Cu2+ ions (0.37–2.37 μm) led to a restoration of both guanylate cyclase activation and platelet aggregation inhibition by GSNO.

The anti‐aggregatory activity of GSNO was reduced in a concentration‐dependent manner by the copper (I)‐specific chelators BCS and neocuproine, and to a smaller extent by desferal. No effect was observed with the copper (II) specific chelator, cuprizone, the iron‐specific chelator, bathophenanthroline sulphonate, or the broader‐specificity copper chelator, d‐penicillamine.

In both BCS‐treated and ‐untreated platelet suspensions, cys NO was more potent than GSNO as a stimulator of guanylate cyclase. In BCS‐treated platelet suspensions there was no significant difference between the anti‐aggregatory potency of cysNO and GSNO; however, in untreated suspensions, GSNO was significantly more potent than cysNO. Thus, when copper was available, GSNO produced a greater inhibition of aggregation than cysNO, despite being a less potent activator of guanylate cyclase.

The breakdown of cysNO and GSNO was measured spectrophotometrically by decrease in absor‐bance at 334 nm. In Tyrode buffer, cysNO (10 μm) broke down at a rate of 3.3 μm min−1. BCS (10 μm) reduced this to 0.5 μm min−1. GSNO, however, was stable, showing no fall in absorbance over a period of 7 min even in the absence of BCS.

We conclude that copper is required for the activity of both cysNO and GSNO, although its influence on anti‐aggregatory activity is only evident with GSNO. The stimulatory effect of copper is unlikely to be explained solely by catalysis of S‐nitrosothiol breakdown. The enhancement by copper of th anti‐aggregatory activity of GSNO, relative to cysNO, suggests that copper may be required for biological activity of GSNO which is independent of guanylate cyclase stimulation.
Differential effects of OPC‐18790, amrinone and dobutamine on cardiac function and energy metabolism in the guinea‐pig isolated ischaemic heart
Authors: Itoh Shuji , Mori Toyoki , Tominaga Michiaki , Ishikawa Makoto , Koga Keiko , Yabuuchi Youichi
Published: July 2012

The effects of OPC‐18790, a novel positive inotropic agent, on cardiac function and myocardial energy metabolism in the guinea‐pig isolated heart with ischaemia were studied by 31P‐magnetic resonance spectroscopy (MRS) and compared with those of amrinone and dobutamine.

Cardiac ischaemia was induced by intracoronary infusion of 15 μm microspheres to reduce coronary perfusion flow (CPF) by 50%. Microsphere embolisation caused a 40% decrease in left ventricular systolic pressure (LVSP), cardiac contractility measured by peak of ventricular pressure development (LVdP/dt) and slightly reduced heart rate. There was also a decrease in ATP and creatine phosphate (PCr) by 20%, an increase in inorganic phosphate (Pi) by 25% and an acidic shift of intracellular pH in the ischaemic heart.

In the ischaemic heart, OPC‐18790, amrinone and dobutamine were applied at concentrations which increased LVdP/dt by about 60%. These compounds increased LVP by 15% to 30% and increased CPF by about 10%. Amrinone and dobutamine but not OPC‐18790 increased heart rate. When these drugs produced the haemodynamic changes described above, amrinone and dobutamine reduced ATP and PCr, increased Pi and produced further intracellular acidosis, whereas, OPC‐18790 did not change these parameters.

Cardiac pacing at 285 beats min−1 produced decreases in LVP, LVdP/dt and CPF by about 30%, 20%, 5%, respectively and an increase in Pi, decreases in PCr and ATP, and intracellular acidosis.

These results suggest that degradation of high energy phosphate compounds closely relates to increase in heart rate in the ischaemic heart. Positive inotropic agents without chronotropic action seem to be beneficial in support of the ischaemic heart.
Characterization of [3H]‐imidazenil binding to rat brain membranes
Authors: Lipartiti Maria , Arban Roberto , Fadda Emanuela , Zanotti Adriano , Giusti Pietro
Published: July 2012

1 The binding of [3H]‐imidazenil, an imidazobenzodiazepine carboxamide, to rat cerebellar membranes was characterized at different temperatures.

2 Specific binding was linear with tissue concentrations and reached maximum after 90, 30 and 5 min incubation at 0, 21 and 37°, respectively. The binding was of high affinity, specific and saturable; non linear regression and Scatchard analysis of the data was compatible with the presence of a single population of receptor sites with Bmax of 0.74 ± 0.020, 0.90 ± 0.011 and 1.0 ± 0.036 pmol mg−1 protein at 0, 21 and 27°, respectively. Binding affinity decreased with increasing temperature: Kd were 0.29 ± 0.051 nM (0°), 1.0 ± 0.080 nM (21°) and 2.4 ± 0.38 nM (37°).

3 At all tested temperatures, [3H]‐imidazenil binding was reversible and the Kd calculated from the dissociation and association rate constants approximated the equilibrium Kd.

4 In the presence of γ‐aminobutyric acid (GABA), Kd increased 4 fold at 0°, whereas Bmax increased, albeit slightly, at all temperatures.

5 Benzodiazepines (BZDs), imidazopyridines and methyl‐β‐carboline‐3‐carboxylate (βCCM) were effective inhibitors of [3H]‐imidazenil binding. Conversely, GABAA antagonists, barbiturates, picrotoxin and peripheral BZD receptor ligands were devoid of any activity.

6 Comparing [3H]‐imidazenil to [3H]‐flumazenil binding in various brain areas, similar densities of recognition sites as well as like regional differences in the distribution of binding sites for both radioligands were observed (cortex=striatum>cerebellum>spinal cord).

7 The present results indicate that [3H]‐imidazenil specifically binds to the BZD sites of GABAA receptors. Furthermore, the effects of GABA and temperature differentiate imidazenil from classical BZDs. It is suggested that the characteristics of imidazenil binding may be relevant to the in vivo pharmacology of the drug.
Participation of NMDA and non‐NMDA excitatory amino acid receptors in the mediation of spinal reflex potentials in rats: an in vivo study
Authors: Farkas S. , Ono H.
Published: July 2012

1 The effect of various intravenously administered excitatory amino acid (EAA) antagonists on the dorsal root stimulation‐evoked, short latency (up to 10 ms) spinal root reflex potentials of chloralose‐urethane anaesthetized C1 spinal rats was studied, in order to gain information on the involvement of non‐NMDA (AMPA/kainate; AMPA = α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionate) and NMDA (N‐methyl‐d‐aspartate) receptors in their mediation. The competitive non‐NMDA antagonist, 2,3‐dihydroxy‐6‐nitro‐7‐sulphamoyl‐benzo(F)quinoxaline (NBQX; 1–32 mg kg−1), the non‐competitive non‐NMDA antagonist, 1‐(amino)phenyl‐4‐methyl‐7,8‐methylendioxy‐5H‐2,3‐benzodiazepine (GYKI 52466; 0.5–8 mg kg−1), the competitive NMDA antagonist 3‐((±)‐2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid (CPP, 2‐8mg kg−1) and two non‐competitive NMDA antagonists: MK‐801 (0.5–2 mg kg−1) and ketamine (2–32 mg kg−1) were used as pharmacological tools.

2 Validating the applied pharmacological tools regarding selectivity at the applied doses, their effects were tested on direct (electrical) as well as on synaptic excitability of motoneurones evoked by intraspinal stimulation. Furthermore, their effect was investigated on the responses elicited by microion‐tophoretic application of EAA agonists (AMPA, kainate and NMDA) into the motoneurone pool, where the extracellular field potential evoked by antidromic stimulation of the ventral root was recorded to detect the effects of EAA agonists.

3 NBQX and GYKI 52466 were able to abolish completely the mono‐, di‐ and polysynaptic ventral root reflexes (MSR, DSR, PSR) and the synaptic excitability of motoneurones, while hardly influencing direct excitability of motoneurones. They markedly attenuated AMPA and kainate responses whilst having little or no effect on NMDA responses.

4 Apparently ‘supramaximal’ doses of CPP and MK‐801 slightly inhibited MSR (by about 10%) moderately reduced DSR and PSR (by about 20–30%) and did not influence excitability of motoneurones. They selectively blocked responses to NMDA.

5 Ketamine dose‐dependently inhibited MSR, DSR and PSR. Nevertheless, diminution of none of the responses exceeded 50%. It reduced both direct and synaptic excitability of motoneurones, thus displaying a local anaesthetic‐like effect, which may contribute to its reflex inhibitory action. It depressed responses to NMDA whilst having negligible effects on responses to AMPA and kainate.

6 We conclude that non‐NMDA receptors play a substantial role in the mediation of MSR, DSR and PSR, while NMDA receptors contribute little to this. Neither MSR nor PSR is mediated exclusively by non‐NMDA or NMDA receptors, respectively.

7 The drugs investigated in this study, with the exception of ketamine, proved to be useful tools for elucidation of the involvement of EAA receptors in various processes in vivo
Acute withdrawal after bremazocine and the interaction between μ‐ and κ‐opioid receptors in isolated gut tissues
Authors: Valeri P. , Morrone L.A. , Romanelli L. , Amico M.C.
Published: July 2012

1 This study was undertaken to investigate whether, after a brief exposure of guinea‐pig isolated ileum and rabbit jejunum to bremazocine, a κ‐opioid agonist also possessing antagonist activity at μ‐opioid receptors, the addition of opioid antagonists produced withdrawal contractures. Our aim was to verify in these tissues the existence of an interaction between the μ‐ and κ‐opioid systems.

2 In guinea‐pig ileum preparations previously exposed for 5 min to bremazocine at 5.7 times 10−7 m and 5.7 times 10−8 m, naloxone (5 times 10−7 m) elicited no response whereas in tissues exposed to a lower bremazocine concentration (5.7 times 10−9 m), naloxone (5 times 10−7 m) and the selective κ‐opioid antagonist, nor‐binaltorphimine (3.4 times 10−8 m) both produced a strong contracture.

3 Bremazocine (5.7 times 10−7 m) administered to guinea‐pig isolated ileum, previously exposed for 5 min to morphine (10−7 m), induced a withdrawal contracture. In contrast, lower bremazocine concentrations (1.4 and 7.1 times 10−8 m) did not elicit a withdrawal contracture.

4 Naloxone (5 times 10−7 m), added to the bath after a 5 min exposure of guinea‐pig ileum to morphine (10−7 m), elicited the characteristic withdrawal contracture. Bremazocine (1.4–7.1 times 10−8 m) added 1 min before naloxone (5 times 10−7 m) inhibited the naloxone withdrawal contracture in a dose‐related way whereas naloxone 5 times 10−8 m added 1 min before naloxone 5 times 10−7m, did not affect the withdrawal response.

5 In the rabbit jejunum, bremazocine (1.4–7.1 times 10−8 m) caused a decrease in amplitude in the spontaneous tissue activity. In tissues exposed to these bremazocine concentrations, naloxone (5 times 10−7 m) elicited a marked contracture. A similar contracture occurred when nor‐binaltorphimine (3.4 times 10−8 m) was added in place of naloxone. These effects were dose‐related to the bremazocine concentration. The specific κ‐agonist, U‐50,488H (5 times 10−8 m), elicited the same effects as bremazocine.

6 These findings show that stimulation of κ‐opioid receptors induces a state of dependence that is not prevented by blocking the μ‐opioid system. The observation that low bremazocine concentrations inhibit the morphine‐induced withdrawal contractures, indicates an interaction between the μ‐ and κ‐opioid system in guinea‐pig isolated ileum, similar to that observed in the whole animal.
An electrophysiological investigation of the properties of a murine recombinant 5‐HT3 receptor stably expressed in HEK 293 cells
Authors: Gill Catherine H. , Peters John A. , Lambert Jeremy J.
Published: July 2012

1 The pharmacological and biophysical properties of a recombinant 5‐HT3 receptor have been studied by use of patch‐clamp techniques applied to HEK 293 cells stably transfected with the murine 5‐HT3 R‐A cDNA.

2 At a holding potential of −60 mV, 77% of cells investigated responded to ionophoretically applied 5‐HT with an inward current. Such currents were unaffected by methysergide (1 μm), or ketanserin (1 μm), but were antagonized in a concentration‐dependent and reversible manner by the selective 5‐HT3 receptor antagonist, ondansetron (IC50 = 440 pm) and the non‐selective antagonists (+)‐tubocurarine (IC50 = 1.8 nm) and metoclopramide (IC50 50 nm).

3 The 5‐HT‐induced current reversed in sign (E5‐HT) at approximately – 2 mV and exhibited inward rectification. The influence of extra‐ and intracellular ion substitutions upon E5‐HT indicates the 5‐HT‐evoked current to be mainly mediated by a mixed monovalent cation conductance.

4 Calcium and magnesium (0.1–10 nm) produced a concentration‐dependent, voltage‐independent, inhibition of the 5‐HT‐induced response. Zinc (0.3–300 μm) exerted a biphasic effect with low concentrations enhancing, and high concentrations depressing, the 5‐HT‐evoked current.

5 Fluctuation analysis of inward currents evoked by a low (1 μm) concentration of 5‐HT suggests the current to be mediated by the opening of channels with a conductance of 420 fS.

6 The pharmacological and biophysical properties of the 5‐HT3 R‐A are similar to those previously described for 5‐HT3 receptors native to murine neuroblastoma cell lines, with the exception that the function of the recombinant receptor was enhanced by low concentrations of zinc. This observation suggests that the properties of the native receptor are not completely represented by the 5‐HT3 R‐A subunit alone.
Potentiation by endothelin‐1 of 5‐hydroxytryptamine responses in aortae from streptozotocin‐diabetic rats: a role for thromboxane A2
Authors: James Gail M. , Hodgson Wayne C.
Published: July 2012

1 We have previously reported maximum responses to 5‐hydroxytryptamine (5‐HT) are diminished in endothelium‐intact and ‐denuded aortae from rats with streptozotocin‐induced diabetes of 2‐weeks duration.

2 In the present study, the thromboxane A2/prostaglandin H2 (TP) receptor antagonist GR32191B (1 μm) significantly reduced maximum responses to 5‐HT in endothelium‐intact aortae from both control and diabetic rats. In the presence of GR32191B, maximum responses to 5‐HT, in endothelium‐intact aortae from diabetic rats, were still significantly reduced compared to those obtained in aortae from controls.

3 GR32191B (1 μm) had no significant effect on maximum responses to 5‐HT in endothelium‐denuded aortae from either control or diabetic rats.

4 Interaction between 5‐HT (0.1 μm‐0.1 mm) and threshold concentrations of endothelin‐1 (ET‐1) or the thromboxane (Tx)A2‐mimetic, U46619, were examined in endothelium‐intact and ‐denuded aortae from control and 2‐week streptozotocin‐diabetic rats.

5 Maximum responses to 5‐HT in the presence of a threshold concentration of ET‐1 (3 nm), in endothelium‐intact aortae from diabetic rats, were not significantly different from those of control rats.

6 Maximum responses to 5‐HT in the combined presence of ET‐1 (3 nm) and GR32191B (1 μm), in endothelium‐intact aortae from diabetic rats, were significantly reduced compared to those obtained in aortae from controls.

7 Maximum responses to 5‐HT in the presence of ET‐1 (3 nm) in endothelium‐denuded aortae from diabetic rats were significantly reduced compared to those from controls.

8 Maximum responses to 5‐HT in the presence of a threshold concentration of U46619 (20 or 30 nm), in endothelium‐intact aortae from diabetic rats, were not significantly different from responses of controls.

9 Maximum responses to 5‐HT in the presence of a threshold (5–20 nm) concentration of U46619, in endothelium‐denuded aortae from diabetic rats, were not significantly different from responses of controls.

10 The results of the present study indicate that endothelial‐derived TxA2 contributes to the contractile response to 5‐HT in aortae from control and diabetic rats. Endothelial‐derived TxA2 also appears to play a role in the potentiation of 5‐HT responses by ET‐1 in aortae from diabetic rats.
Enhancement of cyclic AMP accumulation mediated by 5‐HT after chronic amitriptyline treatment in NG 108‐15 cells
Authors: Shimizu Masami , Nishida Akira , Fukuda Hiroyuki , Saito Hiroshi , Yamawaki Shigeto
Published: July 2012

1 The effects of chronic in vitro administration of amitriptyline, a tricyclic antidepressant, on 5‐hydroxytryptamine (5‐HT) receptor‐mediated adenylyl cyclase activity was studied in the neuroblastoma x glioma hybrid cell line, NG 108‐15.

2 Treatment of NG 108–15 cells with 8 μm amitriptyline for 3 days increased forskolin‐stimulated (0.1 μm) adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) accumulation. Addition of 5‐HT (0.1–100 μm) increased forskolin‐stimulated cyclic AMP accumulation in amitriptyline‐treated cells in a concentration‐dependent manner. However, 5‐HT did not affect forskolin‐stimulated cyclic AMP accumulation in untreated cells.

3 The 5‐HT4 receptor agonist, 5‐methoxytryptamine, significantly enhanced forskolin‐stimulated cyclic AMP accumulation in amitriptyline‐treated cells. In contrast, amitriptyline treatment failed to modify 8‐hydroxy‐2‐(di‐n‐propylamine) tetralin‐induced inhibition of forskolin‐stimulated cyclic AMP accumulation.

4 Pretreatment of cells with pertussis toxin did not affect the 5‐HT‐induced enhancement of cyclic AMP accumulation.

5 The 5‐HT‐induced enhancement of cyclic AMP accumulation in amitriptyline‐treated cells was attenuated by the 5‐HT4 receptor antagonists, GR 113808 and ICS 205–930, with relatively low potency. However, spiperone, SCH 23390, and pindolol were completely ineffective against this 5‐HT‐induced enhancement.

6 Chronic treatment with amitriptyline did not modify the cyclic AMP production stimulated by prostaglandin E1 or cholera toxin. This treatment also had no effect on GTPγS‐, NaF‐, and Mn2+‐stimulated cyclic AMP accumulation in isolated cell membranes.

7 Chronic treatment with the 5‐HT receptor antagonists, pindolol or ICS 205–930, did not inhibit the 5‐HT‐induced enhancement of cyclic AMP accumulation.

8 Chronic treatment with other antidepressant drugs, imipramine, mianserin or paroxetine, elicited the 5‐HT‐induced enhancement of cyclic AMP accumulation.

9 Taken together, these results suggest that chronic amitriptyline treatment of NG 108–15 cells causes 5‐HT to enhance forskolin‐stimulated cyclic AMP accumulation by enhancing 5‐HT receptor‐mediated stimulation of adenylyl cyclase and not by reducing 5‐HT‐mediated inhibition of adenylyl cyclase. The 5‐HT‐induced enhancement of cyclic AMP accumulation in amitriptyline‐treated cells may result from changes at the level of the 5‐HT receptor rather than at the level of Gs proteins or adenylyl cyclase. It is unlikely that this enhancement of cyclic AMP accumulation is caused by long‐term antagonism of the 5‐HT receptor by amitriptyline.
Coronary vasoconstriction in vitro in the hearts of polyarthritic rats: effectiveness of in vivo treatment with the endothelin receptor antagonist SB 209670
Authors: Warner Timothy D. , Willis Dean , Moore Adrian R. , Vane John R. , Klemm Peter
Published: July 2012

Here we demonstrate that perfused hearts removed from polyarthritic rats develop a pronounced coronary vasoconstriction ex vivo. This vasoconstriction is almost entirely blocked by in vivo pretreatment of the rats with the endothelin receptor antagonist, SB 209670. Thus, inflammatory states may be associated with an increased activity of the endothelin system, leading to vascular dysfunction and vasoconstriction.
Functional dopamine D2 receptors on rat vagal afferent neurones
Authors: Lawrence Andrew J. , Krstew Elena , Jarrott Bevyn
Published: July 2012

1 In the present study in vitro electrophysiology and receptor autoradiography were used to determine whether rat vagal afferent neurones possess dopamine D2 receptors.

2 Dopamine (10–300 μm) elicited a temperature‐ and concentration‐dependent depolarization of the rat isolated nodose ganglion preparation. When applied to the tissue 15 min prior to agonist, raclopride (10 μm), clozapine (10 μm) or a mixture of raclopride and clozapine (10 μm each) all produced a threefold parallel shift to the right of the dopamine concentration‐response curve. In contrast, SCH 23390 (100 nM), phentolamine and propranolol (1 μm each) failed to antagonize the dopamine‐mediated depolarization.

3 [125I]‐NCQ 298 (0.5 nM), a D2 selective radioligand, bound topographically to sections of rat brainstem. Densitometric quantification of autoradiograms revealed 93.8 ± 0.5% specific binding of this salicylamide radioligand, as determined by raclopride (10 μm, n = 10 animals). Binding was highest in the nucleus tractus solitarius (NTS), particularly the medial and gelatinous subnuclei. In addition, specific binding was also observed in the interpolar spinal trigeminal nucleus and the inferior olive.

4 Unilateral nodose ganglionectomy caused a 36.6 ± 3.0% reduction in specific binding in the denervated NTS compared to the contralateral NTS. Furthermore, the loss of binding was confined to the dorsal aspect of the medial subnucleus of the NTS. Sham surgery had no effect on the binding of [125I]‐NCQ 298 in rat brainstem.

5 The present data provide evidence for the presence of functionally relevant dopamine D2 receptors on both the soma and central terminals of rat vagal afferent neurones. In addition, the majority of D2 receptors in the rat NTS appear to be located postsynaptically with respect to vagal terminals, and are presumably located either on ascending glossopharyngeal terminals, descending terminals from higher brain regions or on neuronal cell bodies within the NTS.
Selective modification of rat hepatic microsomal fatty acid chain elongation and desaturation by fibrates: relationship with peroxisome proliferation
Authors: Alegret Marta , Cerqueda Enric , Ferrando Rosa , Vazquez Manuel , Sánchez Rosa M. , Adzet Tomás , Merlos Manuel , Laguna Juan C.
Published: July 2012

1 The time‐course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl‐, palmitoleoyl‐ and γ‐linolenoyl‐CoA elongase, Δ‐9, Δ‐6 and Δ‐5 desaturase activities, and microsomal electron transport chains, as well as the correlation with the peroxisomal proliferation phenomenon have been studied in male Sprague‐Dawley rats.

2 As reported in our previous work, the three drugs behave as peroxisomal proliferators (the order of potency was BFB> CFB ≤ GFB) and induced a clear reduction in both plasma cholesterol and triglyceride levels.

3 Palmitoyl‐CoA elongation activity was increased by the three drugs (BFB = GFB> CFB), whereas palmitoleoyl‐CoA elongation activity was only enhanced by GFB. Elongation activity was not modified by fibrates when γ‐linolenoyl‐CoA was used as substrate. These results are in accordance with the existence of three different elongation systems for saturated, mono‐ and polyunsaturated fatty acids.

4 Δ‐9, Δ‐6 and Δ‐5 desaturase activities were increased by the three fibrates, with an order of potency BFB>CFB = GFB for Δ‐9 and Δ‐5, and GFB>BFB = CFB for Δ‐6.

5 Of the enzyme activities integrated in the microsomal electron transport chains, NADH cytochrome b5 reductase was not affected by fibrate treatment, NADPH cytochrome c reductase activity was enhanced (BFB = GFB> CFB), whereas NADH cytochrome c reductase activity was reduced by CFB and BFB.

6 The increase in Δ‐9 and Δ‐5 desaturase activities was highly dependent on the peroxisomal proliferation phenomena, whereas the increase in Δ‐6 desaturase activity and the decrease in NADH cytochrome c reductase was mainly independent. The modifications of palmitoyl‐CoA elongase and NADPH cytochrome c reductase activities, as well as plasma lipid levels, were partially correlated with peroxisomal β‐oxidation, but the r2 values obtained point to the existence of additional independent mechanisms.

7 As man is assumed to be a species refractory to peroxisomal proliferation, only those fibrate effects not absolutely related to this phenomenon are expected to appear after fibrate therapy.
Blockade by polyamine NMDA antagonists related to ifenprodil of NMDA‐induced synthesis of cyclic GMP, increases in calcium and cytotoxicity in cultured neurones
Authors: Beart Philip M. , Schousboe Arne , Frandsen Aase
Published: July 2012

1 Antagonists acting at the polyamine site of the N‐methyl‐D‐aspartate (NMDA) subtype of glutamate receptor, including a number of heterocyclic aminoalcohols related to ifenprodil, were investigated to establish their functional interaction at the NMDA receptor and their neuroprotective profile.

2 In murine cultured neocortical neurones, NMDA (100μm)‐stimulated production of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) was blocked by N‐l([thienyl]‐cyclohexyl)‐piperidine (1 μm) and by the nitric oxide (NO) synthase inhibitor NG‐nitro‐L‐arginine (100 μm). Ifenprodil and structurally related heterocyclic aminoalcohols inhibited in a concentration‐dependent manner the NMDAstimulated, NO‐dependent production of cyclic GMP; rank potency order was: ifenprodil > 2309 BT > tibalosine > threo‐tibalosine > 840S.

3 All of the polyamine NMDA antagonists blocked NMDA (300 μm)‐stimulated increases in intracellular calcium concentrations as measured by changes in the fluorescence of pre‐loaded fluo‐3‐acetoxy methyl ester. Rank potency order was: ifenprodil > 2309 BT > 840S > tibalosine > threotibalosine.

4 In a series of experiments to evaluate the effectiveness of the polyamine NMDA antagonists as blockers of NMDA‐induced cytotoxicity, all of the drugs were found to inhibit the leakage of lactate dehydrogenase after the exposure of the murine neocortical cultures to NMDA (100 μm, 5 h). Rank potency order was: 2309 BT > ifenprodil > tibalosine > threo‐tibalosine > 840S.

5 These results provide direct evidence that polyamine NMDA antagonists produce a functional blockade of the NMDA receptor complex. The heterocyclic aminoalcohols described herein, like ifenprodil, block NMDA‐mediated elevation of intracellular NO and calcium, two key events in the excitotoxic cascade, and are cytoprotective.
Effects of acute and chronic ethanol on cyclic AMP accumulation in NG108‐15 cells: differential dependence of changes on extracellular adenosine
Authors: Kelly E. , Williams RJ. , Harrison P.K.
Published: July 2012

1 This study investigated the effects of acute and chronic ethanol on basal, agonist‐ and forskolin‐stimulated cyclic AMP formation in NG108‐15 mouse neuroblastoma x rat glioma hybrid cells, and examined the role of changes in extracellular adenosine concentrations on the effects observed.

2 NG108‐15 cells incubated acutely with ethanol (1–200 mM) displayed concentration‐dependent increases in basal and iloprost‐stimulated (300 nM; a prostanoid IP receptor agonist) cyclic AMP accumulation but a concentration‐dependent decrease in forskolin‐stimulated (10 μm) accumulation.

3 Cells treated chronically with ethanol (200 mM) for 48 h displayed increases over control in basal, iloprost‐ (0.001–10 μm) and forskolin (0.01–100 μm)‐stimulated cyclic AMP formation. However, chronic ethanol did not affect [3H]‐iloprost binding to cell membranes.

4 Inclusion of adenosine deaminase (ADA; 1 unit ml−1) during the incubation period to measure cyclic AMP accumulation completely abolished the increase in basal accumulation following chronic ethanol, but did not affect the increase in iloprost stimulation. On the other hand ADA partially reversed the increase in forskolin stimulation following chronic ethanol, but even in the presence of high concentrations of ADA (5 units ml−1) the forskolin stimulation remained elevated above control.

5 Cells treated chronically with the adenosine receptor agonist 5′‐(N‐ethylcarboxamido)‐adenosine (NEC A; 10 μm for 48 h) displayed a reduction in subsequent NEC A‐ and forskolin‐stimulated cyclic AMP accumulation, but iloprost stimulation was not affected. ADA included acutely during the incubation period to measure cyclic AMP accumulation abolished the reduction in forskolin but not NECA stimulation produced by the chronic NECA pretreatment.

6 We have previously noted that ethanol inhibits NG108‐15 cell proliferation and alters cell morphology. To mimic this, cells were incubated in the absence of foetal calf serum for 48 h. Following this time, basal, iloprost‐ and forskolin‐stimulated cyclic AMP formation was enhanced over that in cells grown in the presence of serum.

7 These results indicate that chronic ethanol enhances cyclic AMP formation in intact NG108‐15 cells by more than one mechanism: one involves increased extracellular adenosine concentrations and the other a change in the transduction system beyond the receptor, possibly involving the adenylyl cyclase enzyme. Furthermore the ethanol‐induced changes in cyclic AMP accumulation may relate to alterations in NG108‐15 cell growth and development.
Inhibition of serum‐induced proliferation of bovine tracheal smooth muscle cells in culture by heparin and related glycosaminoglycans
Authors: Kilfeather S.A. , Tagoe S. , Perez A.C. , Okona‐Mensa K. , Matin R. , Page C.P.
Published: July 2012

1 The effect of heparin and related glycosaminoglycans on bovine airway smooth muscle proliferation has been investigated.

2 Foetal bovine serum stimulated division of bovine trachealis smooth muscle cells in a concentration‐dependent fashion at concentrations between 1 and 30%.

3 Heparin (0.1–100 μg ml‐1), heparan sulphate (0.1–100 μg ml−1) and fragmin (0.1–100 μg ml−1) inhibited smooth muscle division in a concentration‐dependent fashion between 0.1–100 μg ml−1. A heparin disaccharide did not exhibit inhibition of division at 100 μg ml−1.

4 Dextran sulphate at molecular weights of 5 × 103 and 5 × 105 concentration‐dependently inhibited division between 0.1–100 μg ml−1. Dextran without sulphation did not exhibit inhibition of division at 100 μg ml−1.

5 The magnitude of inhibition of proliferation did not reach 100% for any compounds examined at concentrations up to 100 μg ml−1 during incubations for 5 and 14 days. IC50 values for inhibition of proliferation ranged between 1–5 μg ml−1.

6 These findings suggest that heparin and related glycosaminoglycans inhibit bovine airway smooth muscle cell division.
Characterization of histamine H3 receptors regulating acetylcholine release in rat entorhinal cortex
Authors: Arrang J.M. , Drutel G. , Schwartz J.‐C.
Published: July 2012

1 The pharmacological properties and location of H3 receptors modulating acetylcholine release have been investigated in non‐superfused slices and synaptosomes of rat entorhinal cortex preloaded with [3H]‐choline.

2 (R)α‐methylhistamine, an H3‐receptor agonist, potently inhibited the K+‐evoked tritium release from slices, an effect antagonized by thioperamide, an H3‐receptor antagonist, with nanomolar potency.

3 The K+‐evoked tritium release from synaptosomes remained unaltered in the presence of the potent and selective H3‐receptor agonists, imetit and (R)α‐methylhistamine, suggesting that H3 receptors modulating acetylcholine release are not presynaptically located on cholinergic nerve terminals.

4 Phenylbutanoylhistamine and phenylpropylhistamine, two H3‐receptor antagonists of moderate potency, failed to antagonize the inhibitory effects of (R)α‐methylhistamine observed in slices. Unexpectedly, both compounds when used alone, inhibited tritium release from slices and synaptosomes with micromolar potency and to the same extent (by approximately 50% when added at a final concentration of 200 μm). This inhibitory effect did not involve H1, H2 or H3 receptors and was not mediated by an unknown histamine receptor site, since histamine used at a high concentration neither reproduced nor antagonized the effect of phenylbutanoylhistamine. It remained unaltered in the presence of scopolamine and was neither mimicked nor antagonized by vasoactive intestinal peptide, previously shown to be colocalized with acetylcholine in some neurones.

5 It is concluded that acetylcholine release in rat entorhinal cortex is modulated by H3 receptors presumably not located on cholinergic axon terminals. It remains to be established whether these H3 receptors belong to a receptor subtype different from those previously described since the potency of phenylbutanoylhistamine and phenylpropylhistamine as H3‐receptor antagonists was probably greatly underestimated by additional properties of both drugs.