bluuebunny/crossref_metadata_2025 huggingface dataset:
The interaction of substituted benzamides with brain benzodiazepine binding sites in vitro
Authors: Horton Roger W. , Lowther Sandra , Chivers Jane , Jenner Peter , Marsden C. David , Testa Bernard
Published: July 2012

The interaction of substituted benzamides with brain benzodiazepine (BDZ) binding sites was examined by their ability to displace [3H]‐flunitrazepam ([3H]‐FNm) from specific binding sites in bovine cortical membranes in vitro.

Clebopride, Delagrange 2674, Delagrange 2335 and BRL 20627 displayed concentration‐dependent displacement of [3H]‐FNM with IC50 values of 73 nM, 132 nM, 7.7 μm and 5.9 μm, respectively. Other substituted benzamides including metoclopramide, sulpiride, tiapride, sultopride and cisapride were inactive at 10−5 m.

Inhibition by clebopride and Delagrange 2674 of [3H]‐FNM binding was apparently competitive and readily reversible.

In the presence of γ‐aminobutyric acid (GABA), the ability of diazepam and Delagrange 2674 to displace [3H]‐Ro 15–1788 binding was increased 3.6 and 1.6 fold respectively, compared to the absence of GABA, while ethyl β‐carboline‐3‐carboxylate (βCCE) and clebopride were less potent in the presence of GABA.

Diazepam was 30 fold less potent at displacing [3H]‐Ro 15–1788 in membranes that had been photoaffinity labelled with FNM than in control membranes, whereas the potency of βCCE did not differ. Clebopride and Delagrange 2674 showed a less than two fold loss of potency in photoaffinity labelled membranes.

The pattern of binding of clebopride and Delagrange 2674 in these in vitro tests is similar to that found previously with partial agonists or antagonists at BDZ binding sites.

Clebopride and Delagrange 2674 inhibited [3H]‐FNM binding with similar potency in rat cerebellar and hippocampal membranes, suggesting they have no selectivity for BDZ1 and BDZ2 binding sites.

Clebopride and Delagrange 2674 are structurally dissimilar to other BDZ ligands and represent another chemical structure to probe brain BDZ binding sites.
The mechanism of action of calcium antagonists on arrhythmias in early myocardial ischaemia: studies with nifedipine and DHM9
Authors: Curtis M.J. , Walker M.J.A.
Published: July 2012

Nifedipine and DHM9 (carboxymethyl methyl 1,4‐dihydro‐2,6‐dimethyl‐4‐(3‐nitrophenyl)−3,5‐pyridinedicarboxylate) were studied for their effects on arrhythmias resulting from regional myocardial ischaemia in conscious rats, and for their effects on left ventricular developed pressure in vitro.

Nifedipine possessed antiarrhythmic activity at a high dose of 10 mg kg−1 i.v., but not at 0.5 or 2 mg kg−1. Ventricular fibrillation (VF), tachycardia (VT), and ventricular premature beats (VPB) were all attenuated to a similar degree; nifedipine did not have a selectivity of action for high frequency arrhythmias.

Before coronary occlusion, the three doses of nifedipine reduced arterial blood pressure by a similar magnitude, indicating a similar (maximal) degree of systemic vasodilatation. The reductions in blood pressure were accompanied by reflex tachycardia. Heart rate and blood pressure did not correlate with the incidence or severity of arrhythmias.

DHM9 had no influence on arrhythmias, haemodynamic variables or the ECG, even at 20 mg kg−1 i.v.

Nifedipine concentration‐dependently reduced contractility in perfused paced (5 Hz) rat ventricles in vitro. Raising the concentration of K+ in the perfusion fluid from 3 to 10 mequiv.1−1 increased the potency (‐log10 EC50) of nifedipine up to four fold, and caused a significant depression in excitability.

DHM9 at up to 3 × 10‐5 m had no significant influence on ventricular contractility in vitro.

The results provided indirect evidence in support of the hypothesis that calcium antagonists inhibit ischaemia‐induced arrhythmias by virtue of inhibition of the slow inward current (Isi) in the ischaemic ventricular myocardium.
Prostaglandin E2 inhibits and indomethacin and aspirin enhance, A23187‐stimulated leukotriene B4 synthesis by rat peritoneal macrophages
Authors: Elliott Graham R. , Lauwen Adrianus P.M. , Bonta Ivan L.
Published: July 2012

The calcium ionophore, A23187, stimulated leukotriene B4 (LTB4), thromboxane B2 (TxB2) and prostaglandin E2 (PGE2) synthesis by 4 day carrageenin‐elicited rat peritoneal macrophages.

At concentrations of 2 × 10−7‐2 × 10−5 m indomethacin and aspirin enhanced A23187‐stimulated LTB4 synthesis and inhibited PGE2 and TXB2 formation.

PGE2 inhibited A23187‐stimulated LTB4 and TXB2 formation as well as the augmentation of LTB4 release caused by aspirin and indomethacin. However, PGE2 was ineffective when the cells were challenged with arachidonic acid (AA).

Dibutyryl adenosine 3′: 5′‐cyclic monophosphate (db‐cyclic AMP) partially inhibited A23187‐stimulated LTB4 production.

Our results suggest that PGE2 inhibits macrophage LTB4 synthesis by limiting the availability of AA. Indomethacin and aspirin, possibly by removing the regulatory effect of PGE2, promote the synthesis of the pro‐inflammatory LTB4.
The human astrocytoma cell line 1321 N1 contains M2‐glandular type muscarinic receptors linked to phosphoinositide turnover
Authors: Kunysz Elizabeth A. , Michel Anton D. , Whiting Roger L. , Woods Kris
Published: July 2012

Muscarinic receptors present in the human astrocytoma cell line 1321 N1 were characterized in radioligand binding studies and in functional studies of carbachol‐stimulated phosphatidylinositol (PI) turnover.

In radioligand binding studies the muscarinic receptor in intact cells could be labelled using [3H]‐N‐methylscopolamine ([3H]‐NMS) but not by [3H]‐pirenzepine. In the intact cells these receptors displayed low pirenzepine affinity (pKi = 6.83) indicating that they were not of the M1 subtype. Furthermore, the 1321 N1 muscarinic receptors displayed low affinity for the two M2‐cardiac selective ligands methoctramine (pKi = 5.82) and AF‐DX 116 (pKi = 6.29). This pharmacology was consistent with the 1321 N1 cells containing a single population of muscarinic receptors that displayed a similar pharmacology to the M2‐receptor present in exocrine gland tissue.

The M2‐gland nature of the receptors was further indicated in the functional studies where antagonist affinities were determined from their ability to antagonize carbachol‐stimulated PI turnover in 1321 N1 cells. pA2 values for pirenzepine (7.31), methoctramine (6.10) and AF‐DX 116 (6.52) were similar to those determined in the binding studies.

From these studies we conclude that 1321 N1 astrocytoma cells contain an M2‐gland muscarinic receptor which mediates muscarinic receptor‐mediated stimulation of PI turnover in these cells.
5‐HT1 agonists reduce 5‐hydroxytryptamine release in rat hippocampus in vivo as determined by brain microdialysis
Authors: Sharp Trevor , Bramwell Steven R. , Grahame‐Smith David G.
Published: July 2012

An intracerebral perfusion method, brain microdialysis, was used to assess changes of 5‐hydroxytryptamine (5‐HT) release in the ventral hippocampus of the chloral hydrate‐anaesthetized rat in response to systemic administration of a variety of 5‐HT1 receptor agonists.

A stable output of reliably detectable endogenous 5‐HT was measured in dialysates collected from ventral hippocampus with the 5‐HT reuptake inhibitor, citalopram, present in the perfusion medium.

Under these conditions the putative 5‐HT1A agonist 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT) caused a dose‐dependent (5–250 μg kg−1, s.c.) reduction of 5‐HT in hippocampal dialysates.

Similarly, the putative 5‐HT1A agonists gepirone (5 mg kg−1, s.c.), ipsapirone (5 mg kg−1, s.c.) and buspirone (5 mg kg−1, s.c.) markedly reduced levels of 5‐HT in hippocampal perfusates whereas their common metabolite 1‐(2‐pyrimidinyl) piperazine (5 mg kg−1, s.c.), which does not bind to central 5‐HT1A recognition sites, had no effect.

5‐Methoxy‐3‐(1,2,3,6‐tetrahydro‐4‐pyridinyl)‐lH‐indole (RU 24969), a drug with reported high affinity for brain 5‐HT1B binding sites, also produced a dose‐dependent (0.25–5 mg kg−1, s.c.) decrease of hippocampal 5‐HT output.

These data are direct biochemical evidence that systemically administered putative 5‐HT1A and 5‐HT1B agonists markedly inhibit 5‐HT release in rat ventral hippocampus in vivo.
The mechanism of action and pharmacological specificity of the anticonvulsant NMDA antagonist MK‐801: a voltage clamp study on neuronal cells in culture
Authors: Halliwell Robert F. , Peters John A. , Lambert Jeremy J.
Published: July 2012

Some possible molecular mechanisms of action of the anxiolytic, anticonvulsant and neuroprotective agent MK‐801 have been examined in ‘whole‐cell’ voltage clamp recordings performed on rat hippocampal and cortical neurones, bovine adrenomedullary chromaffin cells and N1E‐115 neuroblastoma cells maintained in cell culture.

Transmembrane currents recorded from rat hippocampal and cortical neurones in response to locally applied N‐methyl‐d‐aspartate (NMDA) were antagonized by MK‐801 (0.1–3.0 μm). Blockade was use‐dependent, and little influenced by transmembrane potential. MK‐801 (3 μm) had no effect on currents evoked by kainate (100 μm).

The antagonism of NMDA‐induced currents by MK‐801 was only slowly and incompletely reversed when the cell membrane potential was clamped at −60mV during washout. Prolonged applications of NMDA at +40, but not −60 mV during washout, markedly accelerated recovery from block.

In contrast to MK‐801, ketamine (10 μm) blocked NMDA‐induced currents in a voltage‐dependent manner. Blockade increased with membrane hyperpolarization and was completely reversible upon washout.

MK‐801 (1–10 μm) produced a voltage‐ and concentration‐dependent block of membrane currents elicited by ionophoretically applied acetylcholine (ACh) recorded from bovine chromaffin cells. The block was readily reversible upon washout.

γ‐Aminobutyric acidA (GABAA) receptor‐mediated chloride currents of chromaffin cells were unaffected by MK‐801 (1–100 μm). In contrast, such currents were potentiated by diazepam (1 μm). MK‐801 (100 μmm) had no effect on currents evoked by GABA on hippocampal neurones.

MK‐801 (10 μm) had little effect on membrane currents recorded from N1E‐115 neuroblastoma cells in response to ionophoretically applied 5‐hydroxytryptamine (5‐HT). Such currents were antagonized by the 5‐HT3 receptor antagonist GR 38032F (1 nm) and also by MK‐801 at high concentration (100 μm).

Voltage‐activated, tetrodotoxin‐sensitive, sodium currents of chromaffin cells were unaffected by 10 μm MK‐801. However, at a relatively high concentration (100 μm), MK‐801 reduced the amplitude of such currents to approximately 77% of control.

The relevance of the present results to the central actions of MK‐801 is discussed.
Evidence for an intracellular action of platelet‐activating factor in bovine cultured aortic endothelial cells
Authors: Stewart Alastair G. , Dubbin Philip N. , Harris Trudi , Dusting Gregory J.
Published: July 2012

Bovine culture endothelial cells (BAECs) generate platelet‐activating factor (Paf) following activation by bradykinin (Bk 0.1 nm), the ionophore, A23187 (3 μm), and ATP (10 μm), but Paf is not released from the cells. These stimuli also elicit generation of prostacyclin (PGI2). The specific and competitive Paf receptor antagonists, WEB 2086 (0.1–1.0 μm) and CV 6209 (0.01–0.1 μm), inhibited Bk‐, A23187‐ and, to a lesser extent, ATP‐induced PGI2 generation but had no effect on basal PGI2 generation. These data suggest a role for intracellular Paf in signal transduction.
Acute stress and GABAergic function in the rat brain
Authors: Losada Matilde E. Otero
Published: July 2012

The function of the γ‐aminobutyric acid (GABA)‐ergic system in certain areas of the rat brain was investigated after acute (5 min) exposure to immobilization stress.

The activities of glutamate decarboxylase and GABA‐transaminase, GABA concentrations, GABA turnover in vivo and uptake of [3H]‐GABA were measured.

After 5 min of immobilization stress, GABA concentrations and [3H]‐GABA uptake were reduced, and GABA turnover stimulated in the olfactory bulbs. In contrast the uptake of [3H]‐GABA was increased in the corpus striatum after 5 min of immobilization stress.

None of the parameters measured was significantly altered by acute immobilization stress in the frontal cortex, hippocampus or medio‐basal hypothalamus.

These findings show that the olfactory bulbs and the corpus striatum are sensitive to the effects of acute stress. Since GABA in the olfactory bulbs is involved in the development of aggression and increased emotional state, it follows that neurochemical changes induced by acute stress might underlie some behavioural manifestations observed after stress.
Effects of peptidases on non‐adrenergic, non‐cholinergic inhibitory responses of tracheal smooth muscle: a comparison with effects on VIP‐ and PHI‐induced relaxation
Authors: Ellis James L. , Farmer Stephen G.
Published: July 2012

The effects of peptidase enzymes on non‐adrenergic, non‐cholinergic (NANC) inhibitory responses of guinea‐pig trachea to electrical field stimulation (EFS), and on relaxations induced by vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) have been examined.

α‐Chymotrypsin reduced both the magnitude and, particularly, the duration of the inhibitory response to EFS, whereas papain reduced only the magnitude. Aprotinin, a peptidase inhibitor prevented the effects of α‐chymotrypsin but was without effect on papain.

α‐Chymotrypsin and papain both abolished relaxant responses to exogenous VIP and PHI. The action of α‐chymotrypsin was prevented by aprotinin, whereas that of papain was not affected.

The peptidases were without effect on concentration‐response curves to methacholine or to isoprenaline. It was also observed that, in the absence of the peptidases, aprotinin had no effect on inhibitory responses either to EFS or to exogenous VIP and PHI.

It is suggested that neuropeptides, possibly VIP and PHI, released during EFS of guinea‐pig trachea, partly mediate NANC relaxations, and that their action may be inhibited by peptidases. However, the lack of effect of aprotonin alone, on responses to EFS, suggests that, if endogenous peptidases are important in terminating the action of neuropeptides, they are resistant to the effect of this particular peptidase inhibitor. It is further suggested that neurogenic relaxation of guinea‐pig trachea is also partly mediated by a substance, possibly non‐peptide, other than VIP or PHI.
The effect of glucocorticosteroids on in vitro motility of the ureter of the sheep
Authors: Angelo‐Khattar Maria , Thulesius Olav , Cherian Thomas
Published: July 2012

The effects of three glucocorticosteroids, hydrocortisone, methylprednisolone and dexamethasone, as well as two non‐steroidal anti‐inflammatory agents (NSAIDs) indomethacin and diclofenac sodium were tested in vitro on the spontaneously rhythmic contracting ureteral preparation of the sheep.

The NSAIDs and the steroids methylprednisolone (10−7‐10−4m) and dexamethasone (10−8‐10−4m), dose‐dependently inhibited ureteral motility. Hydrocortisone caused a cessation of spontaneous contractions only at the high concentration of 10−4 m.

Pretreatment of ureteral strips with the protein synthesis inhibitor cycloheximide (10−6m) abolished the inhibitory action of the corticosteroids on peristalsis, consistent with the suggestion that the action of steroids on the ureter is mediated via the synthesis of the anti‐phospholipase protein, lipocortin.

The potency of the steroids in descending order was found to be dexamethasone > methylprednisolone > hydrocortisone.
Effects of capsaicin desensitization on the stimulatory effect of kinins, prostaglandins, biogenic amines and various drugs in guinea‐pig isolated atria
Authors: Bernoussi Abderrahman , Rioux Francis
Published: July 2012

A simple desensitization protocol was set up using capsaicin and isolated, spontaneously beating atria of guinea‐pigs to assess the possible participation of cardiac, capsaicin‐sensitive, substance P (SP)− and calcitonin gene‐related peptide (CGRP)‐containing sensory nerve fibres, in the cardiac stimulatory effects of bradykinin (Bk), kallidin (Kd), 5‐hydroxytryptamine (5‐HT), histamine, prostaglandin E2 (PGE2), prostaglandin E1 (PGE1), prostaglandin F2α, (PGF2α), adrenaline (Ad), glucagon, nicotine and angiotensin II (AII).

The positive chronotropic and inotropic effects of Bk, Kd and 5‐HT were markedly reduced in capsaicin‐desensitized atria compared to control. The percentage inhibition of the chronotropic and inotropic responses to the three agonists seemed to be inversely related to the concentration of agonist used and to vary also with the type of cardiac effect produced by the drug (for Bk the percentage inhibition was: 36–81% (chronotropic effect) and 62–86% (inotropic effect); for Kd: 61–78% (chronotropic effect) and 53–77% (inotropic effect); for 5‐HT: 25–66% (chronotropic effect) and 40–64% (inotropic effect)).

The positive chronotropic and inotropic effects of histamine, PGE1, PGE2, PGF2α, glucagon and AII had similar amplitudes in capsaicin‐desensitized and control atria.

The positive chronotropic and inotropic effects of Ad and nicotine were differentially affected by capsaicin desensitization. The inotropic effects of 7.5 × 10−7 and 7.5 × 10−6 m Ad were reduced by 41 and 27% respectively, in capsaicin‐desensitized atria compared to control. The chronotropic effects of 1.54 × 10−5 and 6.17 × 10−5 m nicotine were inhibited by 57 and 26% respectively, by capsaicin desensitization. On the other hand, the chronotropic effect of Ad and the inotropic action of nicotine were of similar amplitude in capsaicin‐desensitized and control atria.

These results were taken as an indication that a substantial part of the chronotropic and inotropic effects of Bk, Kd or 5‐HT in guinea‐pig atria, unlike those of histamine, PGE1, PGE2 PGF2α, glucagon and AII, might be the result of stimulation of capsaicin‐sensitive, SP‐ and CGRP‐ containing sensory nerve fibres. The slight, differential inhibition of the chronotropic and inotropic effects of Ad and nicotine by capsaicin desensitization suggests a minor contribution by cardiac, capsaicin‐sensitive sensory nerve fibres to the effects of nicotine and Ad in guinea‐pig atria.
Contractile effects of substance P and other tachykinins on the mouse isolated distal colon
Authors: Fontaine Jeanine , Lebrun Philippe
Published: July 2012

Substance P (SP), physalaemin, eledoisin and kassinin induced concentration‐related contractions of the longitudinal muscle of the mouse distal colon. The responses were not antagonized by atropine (1.5 × 10−7 m), mepyramine (2.5 × 10−7 m), methysergide (5 × 10−7 m), timolol (10−6 m), phentolamine (10−6m) or naloxone (4 × 10−7m). They were enhanced by tetrodotoxin (TTX, 1.5 × 10−7 m). These observations indicate that the contractile responses to the tachykinins result from a direct activation of smooth muscle cells.

The contractile activity provoked by SP and physalaemin was inhibited by nifedipine (a Ca2+‐entry blocker) and was abolished in Ca2+ ‐free EGTA solution. Such data suggest that the myogenic effects of SP and physalaemin are mainly dependent on their ability to promote Ca2+ influx.

Eledoisin and kassinin evoked a contractile response in the absence of external Ca2+ and their myogenic activity was, to some extent, resistant to the inhibitory effect of nifedipine. This may indicate that an additional process, probably the release of an intracellularly bound Ca2+ store, participates in the mechanism by which eledoisin and kassinin contract the mouse distal colon.

After desensitization of the mouse distal colon to SP, the contractile activity provoked by SP or physalaemin was totally abolished whilst the responses evoked by eledoisin and kassinin were barely affected. These observations and other experimental findings indirectly support the assumption that the mouse distal colon could possess different tachykinin‐binding sites.
Analysis of the hyperpolarizing effect of catecholamines on canine cardiac Purkinje fibres
Authors: Neto Francisco Riccioppo , Sperelakis Nicholas
Published: July 2012

The hyperpolarization induced by catecholamines on barium‐depolarized (0.2–0.8 mm BaCl) canine cardiac Purkinje fibres, in vitro, was studied by use of conventional microelectrode recordings of transmembrane electrical potentials.

Noradrenaline, adrenaline and isoprenaline hyperpolarized Purkinje fibres in a concentration‐dependent manner from a threshold concentration around 5 nm. The three catecholamines were shown to be approximately equipotent. Tachyphylaxis was observed when the interval between catecholamine applications was less than 15 min.

Atenolol (10 μm) blocked the hyperpolarization reversibly and theophylline (0.5 mm) potentiated it.

Tetrodotoxin (5 μm) did not affect the hyperpolarization induced by isoprenaline. Acetylcholine and histamine, up to 10 μm, were not effective in hyperpolarizing Purkinje fibres.

Low extracellular potassium concentrations (zero and 1 mm) did not affect the hyperpolarization, but high extracellular potassium concentrations (10–20 mm), markedly reduced the effect of isoprenaline (100 nm).

Reduction of the extracellular sodium concentration produced a roughly proportional reduction in the isoprenaline‐induced hyperpolarization. The hyperpolarization was reversibly blocked in 34 mm sodium Tris‐Tyrode solution.

The hyperpolarization was not reduced in Tyrode solution containing 0.6 mm calcium, but was drastically reduced in zero‐calcium Tyrode solution. This effect was reversible.

Addition of verapamil (5–10 μm) diminished the hyperpolarization, in a concentration‐dependent manner. This effect was partially reversed after washing.

Ouabain (0.7–1 μm) significantly reduced the isoprenaline‐induced hyperpolarization, but 2,4‐dinitrophenol (0.2 mm) did not affect it.

Caesium chloride (20 mm) abolished the hyperpolarization. The blockade was only partially reversed upon washing.

It is suggested that the hyperpolarization induced by a short exposure to catecholamines is mainly due to an increase in potassium permeability (PK). A mechanism involving calcium‐dependent potassium channels might underlie the increase in PK.
Effect of phorbol ester and pertussis toxin on the enhancement of noradrenaline release by angiotensin II in mouse atria
Authors: Musgrave Ian F. , Majewski Henryk
Published: July 2012

Mouse atria were incubated with [3H]‐noradrenaline, and the outflow of radioactivity due to electrical field stimulation (5 Hz, 60 s) was used as an index of noradrenaline release. Angiotensin II (0.01 and 0.1 μm) significantly enhanced the stimulation‐induced (S‐I) outflow of radioactivity.

Phorbol 12‐myristate 13‐acetate (0.001, 0.03, 0.1 and 1.0 μm), a protein kinase C activating phorbol ester, significantly enhanced the S‐I outflow of radioactivity. When angiotensin II (0.1 μm) was present with the concentration of phorbol 12‐myristate 13‐acetate that was maximally effective in increasing the S‐I outflow (0.1 μm), the enhancement of S‐I outflow produced by angiotensin II was maintained.

Polymyxin B (70 μm), an inhibitor of protein kinase C, significantly inhibited the S‐I outflow. Polymyxin B also inhibited the enhancement of the S‐I outflow produced by angiotensin II (0.1 μm).

In another series of experiments mice were injected with pertussis toxin (1.5 μg per mouse), 4 days before their atria were removed. The effectiveness of pertussis toxin pretreatment was determined indirectly using carbachol. Carbachol caused a concentration‐dependent fall in both the rate and force of beating of isolated spontaneously beating atria from mice pretreated with vehicle. This effect of carbachol was not seen with atria from mice pretreated with pertussis toxin.

Pertussis toxin pretreatment did not alter the enhancement of the S‐I outflow of radioactivity produced by angiotensin II (0.01 and 0.1 μm).

These results suggest that angiotensin II receptor modulation of noradrenaline release is not mediated through either a pertussis toxin sensitive guanine nucleotide‐binding protein or activation of protein kinase C.
Swim‐stress‐induced antinociception in young rats
Authors: Jackson Helen C. , Kitchen Ian
Published: July 2012

Opioid and non‐opioid mechanisms have been implicated in the phenomenon of stress‐induced antinociception in adult rodents. We have studied stress‐induced antinociception in developing rats and characterized differences in the neurochemical basis of this effect in pre‐ and post‐weanling animals.

Twenty and 25 day old rats were stressed using warm water (20°C) swimming for 3 or 10 min periods and antinociception was assessed by the tail immersion test (50°C).

A 3 min swim in 20 and 25 day old rats produced marked antinociception which was blocked by naloxone, Mr 1452, 16‐methyl cyprenorphine and levallorphan but not Mr 1453 or N‐methyl leval‐lorphan. The δ‐opioid receptor antagonist ICI 174,864 attenuated stress‐induced antinociception in 25 day old rats but was without effect in 20 day old animals.

A 10 min swim in 25 day old rats produced antinociception which was non‐opioid in nature. In contrast, antinociception was not observed in 20 day old rats after a 10 min swim‐stress.

Pretreatment of animals with dexamethasone blocked 3 min swim‐stress antinociception in 20 and 25 day old animals but had no effect on antinociception induced by a 10 min swim.

Swim‐stress‐induced antinociception can be observed in young rats and dissociated into opioid and non‐opioid types dependent on the duration of swimming stress. The non‐opioid type appears to develop more slowly and cannot be observed in preweanling rats. The opioid type is a predominantly μ‐receptor phenomenon in preweanling animals but δ‐receptor components are observable in postweanling rats.
Development of cholinergic nerve transmission in the chick oesophagus
Authors: Miyazaki Hideto , Taneike Tetsuro , Ohga Akira
Published: July 2012

The onset and development of cholinergic mechanisms in the smooth muscle of the chick oesophagus were studied by estimating the changes in mechanical response and biochemical parameters between 9 days of incubation and 7 days after hatching.

Transmural and vagal nerve stimulation first evoked contraction in the oesophagus at 10 days and 11 days of incubation, respectively. These contractions were inhibited by atropine (1–2 μm) and potentiated by physostigmine (0.2 μm). On the other hand, hexamethonium (200 μm) had an inhibitory effect on vagal nerve stimulation but not on transmural nerve stimulation.

The relative amplitude of contraction induced by both vagal nerve and transmural stimulations compared to high K+ (80 mm)‐induced contractions, progressively increased with age in embryos up to 19 days of incubation.

The activity of choline acetyltransferase (ChAT), an enzyme synthesizing acetylcholine (ACh), also gradually increased in the oesophagus during the period from 9 days to 19 days of incubation, which was similar to the change in the nerve‐mediated contraction. On the other hand, the cholinesterase activity reached a maximum at 13 days of incubation and decreased until 7 days after hatching.

The contractile response to ACh and binding sites of [3H]‐quinuclidinyl benzilate ([3H]‐QNB) were observed in the oesophagus at 9 days of incubation. The maximum response produced by ACh (300 μm) tended to be greater in early stages (9–13 days of incubation) than in later stages. The sensitivity estimated from pD2 values increased up to 15 days of incubation. During the embryonic period, the number of muscarinic receptors estimated from the binding of [3H]‐QNB changed very little.

These results suggest that in the chick oesophagus, extrinsic and intrinsic cholinergic innervation start to function at 10 days and 11 days of incubation, respectively and continue to develop progressively up to the time of hatching. It seems likely that the functional and biochemical maturation of receptive mechanisms on the smooth muscle precede those of cholinergic innervation.
Modulation of phospholipase A2 activity in human fibroblasts
Authors: Solito Egle , Parente Luca
Published: July 2012

Human embryonic skin fibroblasts (HSF) incubated overnight with either human recombinant interleukin‐1α (rIL‐1α) or interleukin‐1β (rIL‐1β) released large amounts of prostaglandin E2 (PGE2).

rIL‐1β, bradykinin (Bk) and arachidonic acid (AA) significantly stimulated PGE2 release from HSF incubated overnight in the presence of either interleukin.

Hydrocortisone inhibited the PGE2 release induced by rIL‐1β and Bk, but not by AA.

The steroid inhibitory effect was reversed by actinomycin D as well as by an anti‐lipocortin monoclonal antibody.

The results suggest that in HSF, rIL‐1β is able to stimulate both cyclo‐oxygenase and phospholipase A2 (PLA2) activity.

The stimulation of PLA2 activity by rIL‐1β is inhibited by hydrocortisone, probably via induction of lipocortin‐like proteins.
Variable, voltage‐dependent, blocking effects of nitrendipine, verapamil, diltiazem, cinnarizine and cadmium on adrenomedullary secretion
Authors: López Manuela G. , Moro María A. , Castillo Carlos F. , Artalejo Cristina R. , García Antonio G.
Published: July 2012

Catecholamine release from cat adrenal glands perfused at a high rate (4 ml min−1) at 37°C with modified Krebs solutions lacking Ca and containing 1.2 mm K (hyperpolarizing solution) or 118 mm K (depolarizing solution) was triggered by 10‐s pulses of Ca (0.5 mm) in the presence of 118 mm K. Hyperpolarized glands released 1280 ± 135 ng per pulse and depolarized glands 831 ± 98 ng per pulse (n = 29).

While the dihydropyridine Ca channel blocker nitrendipine inhibited secretion in hyperpolarized glands with an IC50 of 214 nm, in depolarizing conditions the drug was much more potent (IC50 = 0.99 nm). In contrast, the inorganic Ca channel blocker cadmium inhibited secretion with the same potency both in hyperpolarized or depolarized glands.

Cinnarizine, diltiazem and verapamil exhibited intermediate degrees of voltage‐dependence in blocking secretion. The IC50 ratios between hyperpolarized and depolarized glands were 215, 36, 19, 8 and 0.76 respectively for nitrendipine, cinnarizine, diltiazem, verapamil and cadmium. Because the experimental design (strong depolarization in the absence of Ca) favours the highest opening probability of Ca channels, it seems that these drugs bind preferentially to their receptors when these channels are in their open state.

Variable voltage‐dependent effects of the five Ca channel blockers on adrenomedullary catecholamine release suggests different sites and mechanisms of action on, or near L‐type Ca channels in chromaffin cells. In addition, these findings might help to explain why these drugs exhibit tissue selectivity and why they act differently in normal polarized as compared to ischaemic depolarized cells.
Role of selective cyclic GMP phosphodiesterase inhibition in the myorelaxant actions of M&B 22,948, MY‐5445, vinpocetine and 1‐methyl‐3‐isobutyl‐8‐(methylamino)xanthine
Authors: Souness John E. , Brazdil Roman , Diocee Baljeet K. , Jordan Roy
Published: July 2012

The mechanism by which M&B 22,948, MY‐5445, vinpocetine and 1‐methyl‐3‐isobutyl‐8‐(methylamino)xanthine (MIMAX), which have been described as selective cyclic GMP phosphodiesterase (PDE) inhibitors, relax rat aorta was investigated.

Three cyclic nucleotide PDEs were identified in the soluble fraction of rat aorta; a Ca2+‐insensitive form exhibiting substrate selectivity for cyclic GMP (cGMP PDE), a Ca2+/calmodulin‐stimulated form which also preferentially hydrolyzed cyclic GMP (Ca2+ PDE), and a form demonstrating substrate selectivity for cyclic AMP (cAMP PDE).

M&B 22,948 and MIMAX inhibited cGMP PDE (Ki = 0.16 μm and 0.43 μm, respectively) and Ca2+ PDE (Ki = 9.9 μm and 0.55 μm, respectively), but exhibited weak activity against cAMP PDE (Ki = 249 μm and 42 μm, respectively). MY‐5445 selectivity inhibited cGMP PDE (Ki = 1.3 μm) and vinpocetine selectively inhibited Ca2+ PDE (Ki = 14 μm).

M&B 22,948 and MIMAX induced dose‐dependent increases in the accumulation of cyclic GMP, but not cyclic AMP, in rat aorta pieces. These effects were greatly reduced by endothelial denudation and by methylene blue (5 μm) which blocks the actions of endothelium‐derived relaxant factor. MY‐5445 and vinpocetine had no effect on rat aorta cyclic GMP or cyclic AMP accumulation.

All four compounds caused dose‐related relaxation of 5‐hydroxytryptamine (10 μm) contracted, endothelium‐intact rat aorta, the effects of M&B 22,948 and MIMAX being greatly reduced by methylene blue (5 μm). Methylene blue also caused 10 fold and 100 fold rightward shifts in the dose‐response curves of MY‐5445 and vinpocetine, respectively.

The results are consistent with the smooth muscle relaxant actions of M&B 22,948 and MIMAX, but not vinpocetine and MY‐5445, being mediated through a mechanism involving inhibition of cyclic GMP hydrolysis.
A patch‐clamp study of effects of dihydroavermectin on Ascaris muscle
Authors: Martin R.J. , Pennington A.J.
Published: July 2012

Effects of bath‐application of the anti‐parasitic agent 22,23 dihydroavermectin B1a (DHAVM 20 fM to 0.2 μm) on outside‐out and inside‐out patches from collagenase‐treated muscle membranes of Ascaris suum were examined.

DHAVM was tested as a γ‐aminobutyric acid (GABA) agonist by application to outside‐out patches. DHAVM failed to open characteristic GABA channels even when high concentrations (20 nm to 0.2 μm) were used. These high concentrations of DHAVM also failed to potentiate opening of channels activated by application of 3–4 μm GABA; instead they acted to depress GABA‐activated channel currents by reducing mean conductances and Popen.

In outside‐out patches, low concentrations of DHAVM (1 pM to 100 pM) produced opening of 0–13 channels, the number depending on the patch. The progressive opening of channels gave rise to a ‘Staircase’ effect. The conductance of these channels was 9–15 pS and open times were long (greater than 100 mS). Ion substitution experiments showed these channels to be permeable to Cl. The channels were not blocked by 20 μm picrotoxin. There was a long delay (greater than 15 s) between DHAVM application and channel opening; this delay and lipophilic nature of DHAVM suggested a site of action in the lipid phase of the membrane.

The effect of bath‐application of low concentrations of DHAVM on inside‐out patches was investigated under low‐Ca conditions (to avoid Ca‐activated Cl channels). DHAVM (20 fM to 1 nm) did not produce opening of Cl channels but produced opening after a long delay (mean 2.5 minutes) of noisy cation‐selective channels which had conductances of 5–30 pS.

The actions of DHAVM are discussed.
Effects of potassium channel toxins from Leiurus quinquestriatus hebraeus venom on responses to cromakalim in rabbit blood vessels
Authors: Strong P.N. , Weir Sheila W. , Beech D.J. , Hiestand P. , Kocher H.P.
Published: July 2012

The effects of fractionated Leiurus quinquestriatus hebraeus venom on cromakalim‐induced 86Rb+ efflux in rabbit aortic smooth muscle were examined.

Crude venom (0.1–30 μg ml−1) produced a concentration‐dependent decrease of 1 μm cromakalim‐induced 86Rb+ response. The maximum blocking activity attainable was approximately 60%.

Fractionation of crude venom by gel permeation chromatography and subsequent chromatography on a cation ion‐exchange column, produced two fractions (X and XI), active in the 86Rb+ blocking assay.

Fraction XII contained charybdotoxin (∼85% pure). After a final high performance liquid chromatography (h.p.l.c.) purification step, the purified toxin failed to inhibit the cromakalim‐stimulated 86Rb+ efflux although it was a potent inhibitor of A23187‐induced K+ flux in human erythrocytes and the large conductance calcium‐activated potassium channel in rabbit portal vein smooth muscle.

Subsequent purification of fraction X by h.p.l.c. yielded a minor peak which contained 86Rb+ blocking activity. This subfraction was also capable of inhibiting apamin‐sensitive, angiotensin II‐stimulated K+ flux in guinea‐pig hepatocytes.

It is concluded that the potassium channel opened by cromakalim in rabbit aortic smooth muscle is not blocked by charybdotoxin but by another distinct toxin in the venom of Leiurus quinquestriatus hebraeus.
An electrophysiological comparison of a novel class Ic antiarrhythmic agent, NIK‐244 (ethacizin) and flecainide in canine ventricular muscle
Authors: Satoh Hiroyasu , Ishii Masaaki , Hashimoto Keitaro
Published: July 2012

Electrophysiological effects of NIK‐244 (ethacizin), a novel class I antiarrhythmic drug, were compared with flecainide in canine ventricular muscle by use of conventional microelectrode techniques.

At concentrations of 10−6 m. or higher, NIK‐244 depressed the maximum rate of rise of depolarization () significantly in a concentration‐dependent manner. Also, the resting potential was depolarized at 10−5 m. NIK‐244 did not have any effect on the other action potential parameters or on the effective refractory period.

Flecainide significantly decreased at 3 × 10−6 m. or higher and the resting potential was depolarized at 10−5 m. Like NIK‐244, flecainide did not affect other action potential parameters.

NIK‐244 and flecainide caused a use‐dependent block of , and the rates of onset of inhibition at 3 Hz stimulation were 0.014 ± 0.002 AP−1 at 2 × 10−6 m. NIK‐244 and 0.021 ± 0.012 AP−1 at 10−5 m. flecainide. Under the same conditions, the time constants of the recovery from use‐dependent block were 27.1 ± 13.3 s and 12.2 ± 2.5 s for NIK‐244 and flecainide, respectively.

These results suggest that NIK‐244, like flecainide, should be classified as a slow kinetic drug and as Ic.
Differences between the α2‐adrenoceptor in rat submaxillary gland and the α2A‐ and α2B‐adrenoceptor subtypes
Authors: Michel Anton D. , Loury Dana N. , Whiting Roger L.
Published: July 2012

The α2‐adrenoceptors of rat submaxillary gland, labelled with [3H]‐rauwolscine, were characterized by use of a range of subtype selective ligands and were compared to rabbit spleen α2A‐adrenoceptors and rat kidney α2B‐adrenoceptors.

In rat submaxillary gland, [3H]‐rauwolscine labelled an apparently homogeneous population of binding sites with relatively low affinity (Kd = 11.65 nm) compared to the affinity in rat kidney (Kd = 2.18 nm) and rabbit spleen (Kd = 4.64 nm).

In competition studies using 16 ligands the α2‐adrenoceptors in rat submaxillary gland appeared to differ from both the α2A‐adrenoceptor of rabbit spleen (r = 0.62) and also the α2B‐adrenoceptor of rat kidney (r = 0.28).

The affinity data obtained with benoxathian, imiloxan and WB 4101 indicated the presence of an α2B‐adrenoceptor in rat submaxillary gland. However, data for chlorpromazine, oxymetazoline, spiroxatrine and xylometazoline indicated that submaxillary gland α2‐adrenoceptors were of the α2A subtype. The affinity estimate for prazosin in rat submaxillary gland was intermediate between its affinity at the α2A‐ and α2B‐adrenoceptors while affinity estimates for idazoxan and phentolamine in rat submaxillary gland were greater than those obtained at either the α2A‐ or α2B‐adrenoceptor.

These data indicate that rat submaxillary gland α2‐adrenoceptors differ from the α2A‐ and α2B‐adrenoceptors found in rabbit spleen and rat kidney, respectively.
Cycloleucine blocks NMDA responses in cultured hippocampal neurones under voltage clamp: antagonism at the strychnine‐insensitive glycine receptor
Authors: Hershkowitz Norman , Rogawski Michael A.
Published: July 2012

Radioligand binding studies have demonstrated that the neutral amino acid cycloleucine may act as a competitive antagonist at the glycine modulatory site on the N‐methyl‐D‐aspartate (NMDA) receptor complex. In the present study, we examined the effects of cycloleucine on NMDA‐evoked inward current responses in dissociated hippocampal neuronal cultures using the whole cell voltage‐clamp technique.

In the presence of 1 μm glycine, cycloleucine caused a reversible, dose‐dependent inhibition of NMDA responses with an IC50 of 24 μm. An increase in glycine to 100 μm resulted in a shift to the right of the cycloleucine concentration‐effect curve (IC50, 1.4 mm). However, with cycloleucine concentrations ≤ 100 μm, a fraction of the block could not be overcome by glycine even at concentrations as high as 1 mm.

The cycloleucine block was unaffected by shifts in the holding potential (‐60 to +60mV), and there was no effect of cycloleucine on the reversal potential of the NMDA‐evoked current.

Cycloleucine failed to effect kainic acid‐ and quisqualic acid‐evoked currents at concentrations which inhibited NMDA responses.

We conclude that cycloleucine is a potent and selective antagonist of NMDA‐receptor mediated responses. Although this effect occurs in part via competitive antagonism at the glycine modulatory site, the cycloleucine block cannot be completely reversed by glycine indicating an interaction with an additional site on the receptor‐channel complex.
Convulsions induced by centrally administered NMDA in mice: effects of NMDA antagonists, benzodiazepines, minor tranquilizers and anticonvulsants
Authors: Moreau Jean‐Luc , Pieri Lorenzo , Prud'hon Bernadette
Published: July 2012

Convulsions were induced reproducibly by intracerebroventricular injection of N‐methyl‐D‐aspartic acid (NMDA) to conscious mice.

Competitive (carboxypiperazine‐propylphosphonic acid, CPP; 2‐amino‐7‐phosphonoheptanoic acid, AP7) and non‐competitive (MK801; phencyclidine, PCP; thienylcyclohexylpiperidine, TCP; dextrorphan; dextromethorphan) NMDA antagonists prevented NMDA‐induced convulsions.

Benzodiazepine receptor agonists and partial agonists (triazolam, diazepam, clonazepam, Ro 16–6028), classical anticonvulsants (diphenylhydantoin, phenobarbitone, sodium valproate) and meprobamate were also found to prevent NMDA‐induced convulsions.

Flumazenil (a benzodiazepine receptor antagonist) and the GABA agonists THIP and muscimol (up to subtoxic doses) were without effect.

Flumazenil reversed the anticonvulsant action of diazepam, but not that of MK801.

Results obtained in this model differ somewhat from those described in a seizure model with systemic administration of NMDA. An explanation for this discrepancy is offered.

This model is a simple test for assessing the in vivo activity of NMDA antagonists and also expands the battery of chemically‐induced seizure models for characterizing anticonvulsants not acting at NMDA receptors.
GABAB‐related activity involved in synaptic processing of somatosensory information in S1 cortex of the anaesthetized cat
Authors: Kaneko T. , Hicks T.P.
Published: July 2012

The possible role of GABAB receptor mechanisms in information processing in primary somatosensory (S1) cortex was assessed by use of extracellular recording combined with microiontophoretic methods from 161 neurones in anaesthetized, paralysed cats.

Baclofen‐induced suppressions of cell responses were reversible and stereoselective, the (+)‐isomer being inactive and the (−)−isomer having two to three times the apparent potency of γ‐aminobutyric acid (GABA). The responses measured were threshold to natural stimulation of receptive fields (RFs), responsiveness to thalamic electrical stimulation, change in RF size and magnitude of firing elicited by iontophoretic glutamate.

The action of GABA always was mimicked by muscimol or 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP) but not always by (−)−baclofen; in certain cases (−)−baclofen enhanced neuronal responses while the opposite occurred with GABA or with the other GABAA agonists. The elevation of response thresholds by (−)−baclofen was relatively stronger in peripheral than in central subregions of cutaneous RFs, by contrast with the action of muscimol which was relatively non‐selective as to the area in which it was effective.

Glutamate‐induced and thalamically‐evoked cortical responses as well as spontaneous activity were differentially sensitive to the suppressant effects of muscimol and (−)−baclofen.

Bicuculline methiodide reversibly blocked THIP‐ and muscimol‐induced suppressions of tactile‐ (air puffer)‐induced S1 responses but spared those produced by (−)−baclofen. Phaclofen and δ‐amino‐n‐valeric acid were essentially inactive as blockers of (−)−baclofen‐induced effects and in fact often acted as (−)−baclofen‐like agonists, phaclofen being considerably weaker than δ‐amino‐n‐valeric acid in this respect.

The range of suppressant effects produced by GABA as well as by muscimol and THIP, considered in conjunction with the actions of bicuculline methiodide, suggest that the effects observed by ejected GABA are likely to be due principally to GABAA processes, those mediated by GABAB receptors largely being masked. However, GABAB mechanisms are extant and do appear to be active, probably presynaptically and probably at sites distal to the soma.
The role of α‐ and β‐adrenoceptor subtypes in mediating the effects of catecholamines on fasting glucose and insulin concentrations in the rat
Authors: John G.W. , Doxey J.‐C. , Walter D.S. , Reid J.L.
Published: July 2012

The role of α‐ and β‐adrenoceptor subtypes in the regulation of plasma glucose and immunoreactive insulin (IRI) levels has been investigated in normal conscious fasted rats by employing selective agonists and antagonists.

Adrenaline (0.2 mg kg−1)‐induced hyperglycaemia was abolished by the selective α2‐adrenoceptor antagonist idazoxan (1.0 mg kg−1), unaltered by non‐selective β‐adrenoceptor blockade (propranolol, 1.0 mg kg−1) and potentiated by the selective α1‐adrenoceptor antagonist prazosin (0.3 mg kg−1). Adrenaline increased plasma IRI levels in the presence of idazoxan but not in the presence of either prazosin or propranolol.

The selective α2‐adrenoceptor agonists UK 14304 (0.1 and 0.3 mgkg−1) and BHT‐920 (0.2 and 0.5 mg kg−1) elicited dose‐dependent hyperglycaemic responses, but did not alter plasma IRI levels. UK 14304 (0.1 mg kg−1)‐evoked hyperglycaemia was blocked by idazoxan but not by prazosin.

The selective α1‐adrenoceptor agonists methoxamine (0.3 mg kg−1) and phenylephrine (0.3 mg kg−1) failed to modify either plasma glucose or IRI levels.

Isoprenaline (0.2 mg kg−1) elicited hyperglycaemic and insulinotropic responses which were attenuated by propranolol (1.0 mg kg−1) and the selective β2‐adrenoceptor antagonist ICI 118551 (1.0 mg kg−1), but not by the β1‐selective antagonists atenolol (1.0 mg kg−1) and betaxolol (1.0 mg kg−1).

None of the antagonists per se affected basal plasma glucose or IRI concentrations, except prazosin (1.0 mg kg−1).

The results indicate that adrenoceptors do not appear to be involved in regulating basal plasma glucose and IRI concentrations in the fasted rat. However, the effects of catecholamines on these parameters are mediated by α2‐ and β2‐adrenoceptors, whereas α1‐ or β1‐adrenoceptors do not appear to be involved.