bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of the reactivity of the guinea‐pig isolated trachealis by respiratory epithelium: Effects of cooling
Authors: Lamport Sheila J. , Fedan Jeffrey S.
Published: July 2012

Examination has been made of the effects of epithelium removal on the reactivity of guinea‐pig trachealis to methacholine at 37°C and 22°C, and on responses to activation of the Na+/K+‐pump by abrupt temperature increase from 22°C to 37°C.

At 37°C, epithelium removal increased the sensitivity of isolated tracheal strips to methacholine without affecting the maximum isometric contractile response. Epithelium removal resulted at 22°C in a decrease in sensitivity to methacholine, i.e. an effect opposite to that seen at 37°C. While the maximum response of intact strips to methacholine was enhanced at 22°C, the maximum response of denuded preparations was decreased.

The increase in sensitivity to methacholine at 37°C after epithelium removal was mimicked in intact preparations by indomethacin (1 μm). Indomethacin did not mimic the decrease in methacholine sensitivity and maximum response caused by epithelium removal at 22°C.

Following incubation at 22°C, abrupt increase in temperature to 37°C elicited relaxation in both epithelium‐containing and epithelium‐denuded tracheal strips. In epithelium‐containing preparations the relaxation was more pronounced and followed by contraction. Ouabain (1 μm) converted the relaxation of denuded preparations to contraction, but was ineffective in intact strips. The relaxation of intact strips was, however, inhibited by a greater ouabain concentration (10 μm).

These findings indicate that the modulatory effect of the epithelium is temperature‐dependent. In cooled preparations, the epithelium enhances reactivity. At 37°C, an epithelium‐derived factor reduces reactivity, and this may partially be due to activation of the electrogenic Na+/K+‐pump.
Phaclofen‐insensitive presynaptic inhibitory action of (±)‐baclofen in neonatal rat motoneurones in vitro
Authors: Wang M.Y. , Dun N.J.
Published: July 2012

Intracellular recordings were made from antidromically identified motoneurones in transverse spinal cord slices from neonatal (12–16 day) rats.

Superfusion of (±)‐baclofen (0.5–50 μm) reduced the excitatory postsynaptic potentials (e.p.s.ps) and inhibitory postsynaptic potentials (i.p.s.ps) evoked by dorsal root or dorsal root entry zone stimulation in a concentration‐dependent manner; the calculated EC50 was 2.4 μm. Baclofen in comparable concentrations also reversibly eliminated spontaneously occurring e.p.s.ps and i.p.s.ps.

(−)‐Baclofen was more effective as compared to baclofen in reducing the synaptic responses, whereas (+)‐baclofen at concentrations as high as 50 μm was ineffective.

Baclofen (< 5 μm) attenuated the synaptic responses without causing a significant change of passive membrane properties and depolarizations induced by exogenously applied glutamate. In addition to synaptic depression, baclofen (> 5 μm) caused a hyperpolarization associated with decreased membrane resistance in some of the motoneurones; the glutamate responses were also attenuated.

Baclofen reversibly depressed the spike after‐hyperpolarization of the motoneurones.

GABA (1–10 mm) depressed synaptic transmission and depolarized or hyperpolarized motoneurones. While potentiated by the uptake inhibitor nipecotic acid, the synaptic depressant effect of GABA was not antagonized by bicuculline.

The synaptic depressant effect of baclofen was neither blocked by GABAA antagonists bicuculline and picrotoxin (10–50 μm) nor by the GABAB antagonist phaclofen (0.1–1 mm).

It is suggested that baclofen depresses excitatory and inhibitory transmission in rat motoneurones by primarily a presynaptic mechanism in reducing the liberation of chemical transmitters from nerve endings via a phaclofen‐insensitive GABAB receptor.
The effects of epithelium removal on the actions of cholinomimetic drugs in opened segments and perfused tubular preparations of guinea‐pig trachea
Authors: Small R.C. , Good D.M. , Dixon J.S. , Kennedy I.
Published: July 2012

Isolated segments of guinea‐pig trachea or perfused tracheal tubes were arranged for the recording of trachealis tension changes in Krebs solution containing indomethacin (2.8 μm).

In opened tracheal segments, epithelium removal caused modest (2–3 fold) potentiation of the effects of acetylcholine (ACh) and methacholine (MeCh) but failed to potentiate carbachol (CCh), bethanechol (BeCh), oxotremorine or KCl.

Pretreatment with ecothiopate potentiated effects of ACh and MeCh but not of CCh or BeCh. Removal of epithelium in ecothiopate‐treated tissue potentiated effects of ACh and MeCh but not of CCh or BeCh.

Guinea‐pig ileum challenged with ACh was used as a bioassay system for cholinesterase activity. Scrapings of tracheal epithelium did not hydrolyse ACh.

Histochemical staining revealed no fibres positive for acetylcholinesterase or pseudocholinesterase in the tracheal epithelium. However, the underlying tissues contained acetylcholinesterase‐positive nerve fibres and the trachealis muscle itself stained positively for pseudocholinesterase activity.

Neither tetrodotoxin (3 μm) nor hexamethonium (500 μm) modified the ability of epithelium removal to potentiate ACh.

In perfused tracheal tubes where spasmogens were added to the luminal perfusate, epithelium removal potentiated effects of ACh (31 fold), CCh (10 fold), oxotremorine (2 fold) and KCl.

In perfused tracheal tubes where spasmogens were added to the Krebs solution superfusing the adventitial surface of the tissue, epithelium removal significantly reduced the potency of CCh, oxotremorine and KCl.

It is concluded that the selectivity and magnitude of the potentiation of cholinomimetics caused by epithelium removal depends on the route by which the cholinomimetic agent gains access to the trachealis muscle. The potentiation of acetic acid esters of choline seen in opened tracheal segments does not reflect the loss of epithelial cholinesterase activity and does not depend on the activity of nervous reflex arcs in the tracheal wall. The reduced potency of adventitially‐applied cholinomimetics and KCl seen in epithelium‐denuded tissue strongly suggests that the epithelium can moderate trachealis sensitivity to cholinomimetic agents not only by releasing epithelium‐derived relaxing factor but also by acting as a barrier to drug diffusion.
Effects of metabolic inhibitors on endothelium‐dependent and endothelium‐independent vasodilatation of rat and rabbit aorta
Authors: Weir Cameron J. , Gibson Ian F. , Martin William
Published: July 2012

Basal release of endothelium‐derived relaxing factor (EDRF) rendered endothelium‐containing rings of rat aorta 4.7 fold less sensitive to the contractile actions of phenylephrine and depressed the maximum response when compared with endothelium‐denuded rings. The responsiveness and maximum response to phenylephrine was, however, similar in rings of rabbit aorta with or without endothelium.

Rotenone (1 nm‐0.1 μm), an inhibitor of oxidative phosphorylation, induced a profound, irreversible blockade of phenylephrine‐induced tone in endothelium‐containing and endothelium‐denuded rings of rat aorta, but induced only slight inhibition of tone in rings of rabbit aorta.

2‐Deoxy glucose (10 mm), an inhibitor of glycolysis, had no effect on phenylephrine‐induced contraction in endothelium‐denuded rings of rat aorta, but inhibited reversibly the endothelium‐dependent depression of contraction in endothelium containing rings. 2‐Deoxy glucose had no effect on phenylephrine‐induced contraction in rings of rabbit aorta with or without endothelium.

Rotenone (0.1 μm) inhibited acetylcholine‐induced, endothelium‐dependent relaxation of phenylephrine‐contracted rings or rat and rabbit aorta. In endothelium‐denuded rings of rat aorta, relaxation induced by glyceryl trinitrate of isoprenaline was also inhibited, but relaxation induced by 8‐bromo cyclic GMP or dibutyryl cyclic AMP was not. Relaxation induced by verapamil on KCl‐contracted, endothelium‐denuded rings of rat aorta was also unaffected.

2‐Deoxy glucose (10 mm) inhibited acetylcholine‐induced, endothelium‐dependent relaxation of phenylephrine‐contracted rings of rat and rabbit aorta. In endothelium‐denuded rings of rat aorta, relaxation induced by glyceryl trinitrate and by isoprenaline was also inhibited, but relaxation induced by 8‐bromo cyclic GMP or dibutyryl cyclic AMP was not. Relaxation induced by verapamil on KCl‐contracted, endothelium‐denuded rings of rat aorta was also unaffected.

These data suggest that in rabbit and in rat aorta, rotenone inhibits acetylcholine‐induced relaxation by inhibiting EDRF production, and by depressing smooth muscle sensitivity to EDRF, respectively. They further suggest that 2‐deoxy glucose inhibits acetylcholine‐induced relaxation in both tissues by depressing the sensitivity to EDRF, probably as a result of reduced synthesis of cyclic GMP. The additional possibility that 2‐deoxy glucose inhibits EDRF production warrants further investigation.

The blockade by 2‐deoxy glucose of the endothelium‐dependent depression of phenylephrine‐induced tone in rat aorta probably reflects blockade of the actions of spontaneously released EDRF.
Contractile 5‐HT1 receptors in human isolated pial arterioles: correlation with 5‐HT1D binding sites
Authors: Hamel Edith , Bouchard Denis
Published: July 2012

The 5‐hydroxytryptamine (5‐HT) receptor responsible for inducing vasoconstriction in human isolated pial arterioles has been pharmacologically characterized.

Of several 5‐HT agonists tested, 5‐carboxamidotryptamine (5‐CT) was the most potent and the rank order of agonist potency can be summarized as: 5‐CT > 5‐HT > RU 24969 = α‐methyl‐5‐HT = methysergide ≫ MDL 72832 = 2‐methyl‐5‐HT ≫ 2‐dipropylamino‐8‐hydroxy‐1,2,3,4‐tetrahydro‐naphthalene (8‐OH‐DPAT). With few exceptions, the maximal contractile responses of these agonists were comparable to that induced by 5‐HT.

A correlation analysis performed between the agonists vascular potency (pD2 values) and their affinities (pKD values) published at various subtypes of 5‐HT binding sites showed a positive significant correlation with rat cortical 5‐HT1B (r = 0.86; P < 0.01) and human caudate 5‐HT1D (r = 0.98; P < 0.005) subtypes.

Selective antagonists at 5‐HT2 (ketanserin, mianserin, MDL 11939) and 5‐HT3 (MDL 72222) sites were totally devoid of inhibitory activity on the 5‐HT‐induced contraction, an observation which agreed with the agonist data and further excluded activation of these receptors. In contrast, the 5‐HT1‐like/5‐HT2 antagonist methiothepin and the non‐selective 5‐HT1D compound metergoline inhibited with high affinity the contraction induced by 5‐HT with respective pA2 values of 8.55 ± 0.16 and 6.88 ± 0.05. This contractile response was, however, insensitive to 5‐HT1B (propranolol) and 5‐HT1C (mesulergine, mianserin) antagonists.

It is concluded that a 5‐HT1‐like receptor, which shares strong similarities with the 5‐HT1D binding sites identified in human caudate membranes, is mediating the vasocontractile action of 5‐HT in human pial arterioles.
Properties of 5‐hydroxytryptamine3 receptor‐gated currents in adult rat dorsal root ganglion neurones
Authors: Robertson Brian , Bevan Stuart
Published: July 2012

Responses to 5‐hydroxytryptamine (5‐HT) were examined on rat dorsal root ganglion (DRG) neurones maintained in tissue cultures, by use of whole cell recording techniques.

5‐HT (usually 10 μm) evoked a depolarization associated with an increase in membrane conductance in 40% of DRG neurones. There was a considerable variation in the size and persistence of this response between different batches of cells.

The 5‐HT response was mimicked by applying the agonists 2‐methyl‐5‐HT (10 μm) and phenylbiguanide (10 μm). Responses were blocked by ICS 205–930 (100 nm), but not by methysergide (0.1–1.0 μm).

5‐HT currents could be carried by sodium and caesium ions, but not by choline ions. The amplitude and duration of the 5‐HT responses were dependent on the concentration of divalent cations in the extracellular solution: both became greater when calcium and magnesium concentrations were decreased.

Staurosporine, a putative antagonist of protein kinases, inhibited responses to 5‐HT.
Amantadine and sparteine inhibit ATP‐regulated K‐currents in the insulin‐secreting β‐cell line, HIT‐T15
Authors: Ashcroft Frances M. , Kerr Amanda J. , Gibson John S. , Williams Beatrice A.
Published: July 2012

The effects of pharmacological agents that potentiate insulin release were studied on ATP‐regulated K‐currents (K‐ATP currents) in the insulin‐secreting β‐cell line HIT‐T15 by use of patch‐clamp methods.

The tricyclic drug, 1‐adamantanamine (amantadine), reversibly inhibited both whole‐cell currents (with a Ki of 120 μm) and single channel currents in inside‐out patches. This effect was principally due to an increase in a long closed state which reduced the channel open probability. The related compound, 1‐adamantanol, in which the amino group is substituted by a hydroxyl one, did not inhibit K‐ATP currents substantially.

The alkaloid, sparteine, reversibly inhibited both whole‐cell K‐ATP currents (Ki = 171 μm) and single channel currents in inside‐out patches.

The results suggest that sparteine and amantadine can block the K‐ATP channel from either side of the membrane and support the idea that at least part of the stimulatory effect of these agents on insulin secretion results from inhibition of this channel.
Differential effects of l‐arginine on the inhibition by NG‐nitro‐l‐arginine methyl ester of basal and agonist‐stimulated EDRF activity
Authors: Randall Michael D. , Griffith Tudor M.
Published: July 2012

An isolated, buffer‐perfused rabbit ear preparation was used to investigate the influence of NG‐nitro‐l‐arginine methyl ester (l‐NAME) on endothelium‐dependent vasodilatation and modulation of vasoconstrictor responses and vascular conductance.

Acetylcholine (0.55 pmol‐1.6 nmol) caused dose‐related vasodilatation of preparations constricted by the combination of 5‐hydroxytryptamine and histamine (both 1 μm), with an ED50 = 31.1 ± 7.8 pmol and a maximum dilatation of 69.9 ± 4.3%. In the presence of 10 μm l‐NAME the dose‐response for vasodilator effects was shifted significantly (P < 0.001) to the right (ED50 = 3.07 ± 1.18 nmol) and there was a significant (P < 0.01) depression of the maximum response (Rmax = 44.3 ± 4.0%). The higher concentration of 100 μm l‐NAME completely abolished vasodilatation to acetylcholine. l‐Arginine (10 mm) did not reverse the inhibitory actions of l‐NAME at either concentration.

l‐NAME 100 μm, augmented vascular tone induced by 1 μm 5‐hydroxytryptamine and 1 μm histamine, thus altering the characteristics of both pressure/flow and conductance/flow relationships such that conductance was reduced at all flow rates. The augmentation of constrictor tone was reversed in a concentration‐dependent manner by l‐arginine (10 μm–10 mm) and the effect of l‐NAME on the conductance/flow relationships was similarly reversed by 10 mm l‐arginine. The augmentation of tone was endothelium‐dependent as it did not occur following functional destruction of the endothelium by perfusion of the vascular bed with the detergent CHAPS (0.3%) for 150 s.

In conclusion, l‐NAME is a potent inhibitor of agonist‐induced endothelium‐dependent vasodilatation. l‐NAME reduces vascular conductance in pharmacologically constricted preparations and this emphasizes the important role of EDRF in vascular regulation. The ability of l‐arginine to reverse l‐NAME‐induced inhibition of basal EDRF activity but not l‐NAME‐induced inhibition of agonist‐induced endothelium‐dependent relaxations suggests that there is pharmacological heterogeneity in the mechanisms responsible for the conversion of l‐arginine to EDRF.
The cardioprotective effect of γ‐glutamylcysteine ethyl ester during coronary reperfusion in canine hearts
Authors: Nishinaka Yasuto , Kitahara Shigehisa , Sugiyama Satoru , Yokota Mitsuhiro , Saito Hidehiko , Ozawa Takayuki
Published: July 2012

The cardioprotective effect of γ‐glutamylcysteine ethyl ester was investigated on ischaemia‐reperfusion‐induced myocardial damage in anaesthetized dogs.

Open chest anaesthetized dogs were divided into four groups: 2 h occlusion of the left anterior descending coronary artery (LAD); 2 h LAD occlusion followed by 1 h reperfusion; 2 h LAD occlusion followed by 1 h reperfusion with administration of γ‐glutamylcysteine ethyl ester (10 mg kg−1 just before reperfusion); 2 h LAD occlusion followed by 1 h reperfusion with administration of GSH (the reduced form of glutathione, 10 mg kg−1 just before reperfusion).

After occlusion or reperfusion, heart mitochondria were prepared from the normal area and the occluded or the reperfused area, and mitochondrial function (rate of oxygen consumption in State III, and respiratory control index) was measured polarographically.

Mitochondrial GSH and GSSG (the oxidized form of glutathione) concentrations, and activities of glutathione peroxidase and glutathione reductase were measured.

Two h of LAD occlusion induced mitochondrial dysfunction with depletion of mitochondrial GSH concentration. One h of reperfusion after 2 h LAD occlusion induced significant mitochondrial dysfunction associated with a marked depletion of mitochondrial GSH concentration.

γ‐Glutamylcysteine ethyl ester reduced mitochondrial dysfunction and depletion of mitochondrial GSH concentration after 2 h LAD occlusion and 1 h reperfusion. In contrast, GSH did not prevent depletion of mitochondrial GSH concentration and mitochondrial dysfunction after 2 h LAD occlusion followed by 1 h reperfusion.

The activities of glutathione peroxidase and glutathione reductase did not change significantly in each group.

One h of reperfusion after 2 h occlusion of LAD induced ventricular arrhythmias. γ‐Glutamylcysteine ethyl ester markedly reduced the development of reperfusion arrhythmias, whilst GSH showed no protective effect.

γ‐Glutamylcysteine ethyl ester maintained mitochondrial GSH concentration, prevented reperfusion myocardial damage, and reduced reperfusion arrhythmias.
Depression of miniature endplate potential frequency by acetylcholine and its analogues in frog
Authors: Nikolsky E.E. , Bukharaeva E.A. , Strunsky E.G. , Vyskočil F.
Published: July 2012

Acetylcholine (ACh), 7.5 × 10−5 m, and carbachol, 5 × 10−6m (CCh) depressed the frequency of miniature endplate potentials (m.e.p.ps) in the frog (Rana temporaria) sartorious neuromuscular junction with active acetylcholinesterase to about 50–55% of the controls.

A similar depression was produced by the nicotinic agonists, nicotine, suberyldicholine and tetramethylammonium.

The muscarinic agonists, oxotremorine, methylfurmethide and methacholine were without effect on m.e.p.p. frequency. The muscarinic antagonist, atropine and the nicotinic antagonist, (+)‐tubocurarine, had no effect on the depression of m.e.p.p. frequency evoked by CCh.

The ganglionic blockers, benzhexonium and IEM‐1119, were also without effect on the CCh‐evoked depression of m.e.p.p. frequency.

Pretreatment of muscles with anticholinesterases did not prevent the CCh‐induced drop in m.e.p.p. frequency.

The effect of CCh was proportionally the same as in the controls in preparations where the m.e.p.p. frequency was changed by elevation of K+ and in the presence of theophylline, noradrenaline, dibutyryl adenosine 3′:5′‐cyclic monophosphate (db cyclic AMP) and db cyclic GMP.

An inhibitor of Na+,K+‐ATPase, ouabain, 5 × 10−5 mol l−1, prevented or reversed the depression of m.e.p.p. frequency by CCh. However, the depression was present in a nominally K+‐free medium. Insulin and adrenaline, which are considered to be Na+,K+‐ATPase activators, were without effect on depression of m.e.p.p. frequency.

The depression of m.e.p.p. frequency by 5 × 10−6 m CCh was the same at temperatures between 5 and 30°C with a Q10 near to 1.0. When threshold amounts of CCh were used (6 × 10−7 and 3 × 10−7 m), the depression was less at higher temperatures.

The receptive structures responsible for the CCh (or ACh)‐evoked depression of m.e.p.p. frequency differ pharmacologically from muscarinic, nicotinic ganglionic and neuromuscular junction ACh‐receptors as well as from the synaptic cholinesterase, in contrast to previous reports (Duncan & Publicover, 1979). The low temperature‐dependence points to the possibility that physical rather than biochemical processes are limiting in this presynaptic effect of cholinomimetics.;
Estimation of the efficacy and affinity of the β2‐adrenoceptor agonist salmeterol in guinea‐pig trachea
Authors: Dougall I.G. , Harper D. , Jackson D.M. , Leff P.
Published: July 2012

We have attempted to estimate the affinity and efficacy of the new β2‐adrenoceptor agonist salmeterol in the guinea‐pig isolated trachea and to compare these estimates with those obtained for salbutamol.

The kinetics of the relaxation to salmeterol were considerably slower than those to salbutamol and the responses were poorly defined. Nevertheless, in experiments employing a cumulative curve design both salmeterol and salbutamol exhibited partial agonist behaviour. Operational model‐fitting of these data indicated that the intrinsic efficacies of the two agonists were indistinguishable but that salmeterol had an affinity (pKA = 7.4) some 30 fold greater than salbutamol (pKA = 5.9).

Single dose experiments, using maximally effective concentrations of salmeterol or salbutamol were carried out in an attempt to quantify more reliably the relative efficacies of the two agonists. In these experiments salbutamol was found to have an efficacy (2.5) approximately 3 fold greater than that of salmeterol (0.8).

Full/partial agonist interaction studies were carried out to provide another estimate of the affinity of salmeterol. Isoprenaline and adrenaline were employed as full agonists in these experiments and the affinity (pKB) estimates obtained for salmeterol against these agonists were 7.3 and 7.4 respectively. These affinity estimates were similar to the pKA value (7.4) obtained by operational model‐fitting.

Salmeterol has slightly lower efficacy (3 fold) but markedly greater affinity (30 fold) than salbutamol at β2‐receptors. The slightly lower efficacy of salmeterol is unlikely to have clinical implications for this drug as a bronchodilator β2‐adrenoceptor agonist in asthma.;
Multiple effects of ryanodine on intracellular free Ca2+ in smooth muscle cells from bovine and porcine coronary artery: modulation of sarcoplasmic reticulum function
Authors: Wagner‐Mann Colette , Hu Qicheng , Sturek Michael
Published: July 2012

The effects of ryanodine and caffeine on intracellular free Ca2+ concentration ([Ca2+]i) were studied by use of fura‐2 microfluorometry in single smooth muscle cells freshly dispersed from bovine and porcine coronary artery.

Bovine and porcine cells demonstrated similar sensitivities to 10 min of exposure to ryanodine in physiological salt solution (PSS), as determined by comparable dose‐dependent decreases in the subsequent [Ca2+]i transient induced by 5 mm caffeine.

Ryanodine (10 μm) caused a significant increase in [Ca2+]i to a plateau level 27 ± 3% and 38 ± 4% above baseline [Ca2+]i (baseline [Ca2+]i = [Ca2+]i at 0 min) in porcine and bovine cells, respectively, when bathed in PSS. In bovine cells the time required to reach ½ the plateau level was only 3 min versus 6 min for porcine cells.

The ryanodine‐induced plateau increase in [Ca2+]i was 35 ± 5% above baseline for bovine cells bathed in 0 Ca PSS (PSS including 10 μm EGTA with no added Ca2+), but only 7 ± 3% above baseline in porcine cells during 10 min exposure to 10 μm ryanodine. In bovine cells [Ca2+]i showed proportional increases when extracellular Ca2+ was increased from the normal 2 mm Ca2+ PSS to 5 and 10 mm.

Cells pretreated with caffeine in 0 Ca PSS, which depleted the caffeine‐sensitive sarcoplasmic reticulum Ca2+ store, showed no increase in [Ca2+]i when challenged with 10 μm ryanodine. The ryanodine‐associated increase in [Ca2+]i, which was sustained in 0 Ca PSS during the 10 min ryanodine exposure in cells not pretreated with caffeine, suggests that ryanodine releases Ca2+ from the sarcoplasmic reticulum, but also inhibits Ca2+ efflux.

Intracellular free Ba2+ ([Ba2+]i) was measured with fura‐2 microfluorometry to define further the Ca2+ efflux pathway inhibited by ryanodine; specifically, Ba2+ is not transported by the Ca2+ pump, but will substitute for Ca2+ in Na+‐Ca2+ exchange. In porcine cells pretreated with caffeine in 0 Ca PSS to deplete the caffeine‐sensitive sarcoplasmic reticulum Ca2+ store, depolarization with 80 mm K+ in 2 mm external Ba2+ caused a 100 ± 6% increase in fura‐2 fluorescence ([Ba2+]i). During the 17.5 min 0 Ca PSS recovery from depolarization, exposure to 10 μm ryanodine inhibited the removal of [Ba2+]i by 69 ± 3% when compared with control (0 Ca PSS without ryanodine).

It was concluded that in bovine and porcine smooth muscle cells: (a) ryanodine (≥ 10 μm) releases Ca2+ from the sarcoplasmic reticulum; (b) ryanodine (≥ 10 μm) decreases Ca2+ efflux, probably by inhibition of Na+‐Ca2+ exchange; (c) the sarcoplasmic reticulum Ca2+ store may be larger in bovine than in porcine smooth muscle cells; thus, porcine cells have a relatively greater reliance on Ca2+ influx to increase [Ca2+]i.
Effects of BRL 38227 on neurally‐mediated responses in the guinea‐pig isolated bronchus
Authors: Good D.M. , Clapham J.C. , Hamilton T.C.
Published: July 2012

In guinea‐pig isolated bronchus treated with indomethacin (2.8 μm), electrical field stimulation (EFS; 10 Hz, 0.5 ms, 60–70 V, for 10 s) evoked a tetrodotoxin (3 μm)‐sensitive, biphasic contraction comprising a rapid, atropine (1 μm)‐sensitive cholinergic response succeeded by a slowly developing, capsaicin (10 μm)‐sensitive, non‐adrenergic, non‐cholinergic excitatory (NANCe) response.

BRL 38227 (0.3–3 μm), salmeterol (0.003–3 μm) and ketotifen (1.0–300 μm) each produced concentration‐dependent inhibition of both NANCe and cholinergic responses to EFS in guinea‐pig isolated bronchus.

Substance P (SP; 1 μm) and neurokinin A (NKA; 0.07 μm) produced contractions equivalent in magnitude to the NANCe response to EFS, which were inhibited by salmeterol (1 μm), but not by BRL 38227 (3 μm) or ketotifen (100 μm).

Acetylcholine (ACh; 6 μm) was equi‐effective with the electrical activation of cholinergic neurones. BRL 38227 (3 μm) slightly inhibited responses to ACh (6 μm). Salmeterol (1 μm) and ketotifen (100 μm) markedly inhibited responses to ACh (6 μm).

In bronchial rings pre‐contracted with ACh (100 μm), BRL 38227 (0.1– 30 μm), salmeterol (0.001–3 μm) and ketotifen (0.1–100 μm) each produced concentration‐dependent relaxation. Unlike ketotifen, BRL 38227 and salmeterol only partially (18.8 ± 2.1% and 51.8 ± 3.9% respectively) reversed the ACh‐induced contraction.

The (+)‐analogue of BRL 38227, BRL 38226 (0.3–100 μm), was without effect on responses to EFS and had no effect on the inhibition caused by BRL 38227. The K+‐channel activators pinacidil (3.0–30 μm) and RP 52891 (3.0–30 μm) exerted similar inhibitory actions on responses to EFS as BRL 38227, but were less potent. Glibenclamide (0.1–1.0 μm) and phentolamine (3 μm) antagonized the inhibitory effects of BRL 38227 on responses to EFS.

It is concluded that BRL 38227 and ketotifen can inhibit NANCe neuroeffector transmission at concentrations exerting little or no inhibitory effects on responses to exogenously applied tachykinins. By contrast, in addition to suppressing NANCe responses to EFS, salmeterol also markedly inhibits responses to SP and NKA. At concentrations markedly suppressing cholinergic neuroeffector transmission, BRL 38227 has only minor effects on responses to exogenously‐applied ACh. Salmeterol and ketotifen both depress responses to ACh within the concentration‐range over which they inhibit cholinergic responses to EFS. The inhibitory effects of BRL 38227 on responses to EFS exhibit stereo‐specificity and may involve the opening of a neuronal K+‐channel. This K+‐channel is glibenclamide‐ and phentolamine‐sensitive and appears similar to the smooth muscle K+‐channel which is modulated by BRL 38227.
Endothelium‐dependent increase in vascular sensitivity to phenylephrine in long‐term streptozotocin diabetic rat aorta
Authors: Chang Kyoung S.K. , Stevens Wendell C.
Published: July 2012

The effect of short‐ and long‐term streptozotocin (STZ)‐induced diabetes (12 and 52 weeks) on the vascular response to phenylephrine was examined in the isolated thoracic aorta with and without intact endothelium from diabetic, age matched control rats and diabetic rats treated with insulin.

Twelve weeks after induction of diabetes, aortae with intact endothelium demonstrated no changes either in sensitivity (defined as pD2) or contractility (defined as the maximal developed tension per aortic tissue wet weight) to phenylephrine.

In contrast, 52 weeks after induction of diabetes, aortae with intact endothelium demonstrated an increased sensitivity to phenylephrine while contractility to phenylephrine was not changed. Insulin treatment partially corrected the increased sensitivity to phenylephrine observed in diabetic rat aorta.

Removal of endothelium abolished the difference in phenylephrine sensitivity between diabetic and control aortae at 52 weeks.

Pretreatment of intact aortae with methylene blue, an inhibitor of endothelium‐derived relaxing factor (EDRF), abolished the difference in phenylephrine sensitivity between control and diabetic rat aortae at 52 weeks, while pretreatment with indomethacin, an inhibitor of cyclo‐oxygenase, had no effect. These results suggest that decreases in production or release of EDRF might be responsible for the increased vascular sensitivity to phenylephrine observed in long‐term STZ diabetic rats.

Acetylcholine‐induced relaxation, which is EDRF‐dependent, was less in diabetic rat aortae with intact endothelium at 52 weeks, but not at 12 weeks. These results further support the theory that decreases in capacity of the endothelium to synthesize or release EDRF may occur in long‐term STZ diabetic rats.
Lack of effect of potassium channel openers on ATP‐modulated potassium channels recorded from rat ventromedial hypothalamic neurones
Authors: Sellers A.J. , Boden P.R. , Ashford M.L.J.
Published: July 2012

Single neuronal cells were freshly isolated from the ventromedial hypothalamic nuclei (VMHN) of the rat brain. Currents through ATP‐modulated and large conductance (160 and 250 pS) calcium‐activated potassium channels were recorded by the cell‐attached and excised inside‐out patch techniques.

BRL38227 (lemakalim; 30–90 μm) applied to the superfusing medium produced no change in firing rate of isolated glucose‐receptive VMHN neurones in cell‐attached recordings.

BRL38227, at concentrations of between 30–100 μm applied to the intracellular (cytoplasmic) aspect of inside‐out patches, had no effect on the activity of ATP‐sensitive K+ channels in the absence of ATP or in the presence of a sub‐maximal inhibitory concentration (3 mm) of ATP. Cromakalim, pinacidil, minoxidil sulphate and diazoxide also produced no effect under these conditions.

The potassium channel openers (KCO's) were tested on ATP‐activated potassium channels recorded from a further subpopulation of VMHN neurones. Application of BRL38227 (up to and including 100 μm) to this channel in inside‐out patches either in the absence of ATP or when activated by 5 mm ATP had no effect on channel activity. Identical results were obtained with cromakalim and pinacidil.

BRL38227 had no effect on either of the large conductance (250 pS and 160 pS) calcium‐activated potassium channels in VMHN neurones.

Intracellular recordings were made from glucose‐receptive VMHN neurones in rat brain slices. Cromakalim (50 μm) or diazoxide (60 μm) did not alter the firing rate or passive membrane properties of these neurones demonstrated to be sensitive to tolbutamide (0.1 mm).

These results show that the KCO's tested in this study have no effect either on VMHN neurones contained in brain slices or on the activity of any of the ATP‐modulated potassium channels under isolated patch conditions associated with these neurones.
l‐Leucyl‐l‐arginine, naltrindole and d‐arginine block antinociception elicited by l‐arginine in mice with carrageenin‐induced hyperalgesia
Authors: Kawabata Atsufumi , Nishimura Yumiko , Takagi Hiroshi
Published: July 2012

Intraplantar injection of carrageenin into the mouse hind paw produced hyperalgesia when measured by the paw pressure test (Randall & Selitto method).

Subcutaneous administration of l‐arginine (100–1,000 mg kg−1), a possible precursor of kyotorphin which is an endogenous analgesic neuropeptide, inhibited carrageenin‐induced hyperalgesia in a dose‐dependent manner. This effect was blocked by subcutaneous administration of naloxone, naltrindole, a selective δ‐opioid receptor antagonist (enkephalin antagonist), and d‐arginine.

Intracerebroventricular administration of l‐leucyl‐l‐arginine inhibited the antinociceptive effect of systemically administered l‐arginine in hyperalgesic mice.

Intracerebroventricular administration of l‐arginine (3 and 30 μg per mouse) and kyotorphin (300 ng − 3 μg per mouse) produced antinociception in hyperalgesic mice. The antinociceptive effects of l‐arginine but not kyotorphin were blocked by intracerebroventricular administration of d‐arginine.

These results suggest that l‐arginine‐induced antinociception is mediated by activation of ‘kyotorphinergic’ nerves followed by activation of the ‘opioidergic’ (possible ‘enkephalinergic’) nerves in the central nervous system.
Characterization of endothelium‐dependent relaxations resistant to nitro‐l‐arginine in the porcine coronary artery
Authors: Nagao Tetsuhiko , Vanhoutte Paul M.
Published: July 2012

Previous studies, demonstrated that endothelium‐dependent relaxations which are resistant to nitro‐l‐arginine (an inhibitor of nitric oxide synthase) are accompanied by membrane hyperpolarization in the porcine coronary artery. The present experiments were designed to characterize further this type of endothelium‐dependent relaxation in response to bradykinin by measuring isometric force in isolated rings of that artery. The experiments were performed in the presence of indomethacin to rule out vasoactive prostanoids.

Bradykinin induced comparable endothelium‐dependent relaxations of proximal and distal rings of porcine coronary arteries contracted with prostaglandin F2α in the presence of nitro‐l‐arginine.

Bradykinin and SIN 1 (a donor of nitric oxide) reduced contractions induced by prostaglandin F2α in an additive fashion in the presence of nitro‐l‐arginine.

Bradykinin (in the presence of nitro‐l‐arginine) relaxed the tissues contracted with tetraethylammonium, prostaglandin F2α, phorbol 12, 13‐diacetate or endothelin, with similar pD2 values.

The time course of the relaxations induced by bradykinin (in the presence of nitro‐l‐arginine) and UK 14304 (an α2‐adrenoceptor agonist, in the absence of the inhibitor of nitric oxide synthase) were comparable.

These results suggest that, in the porcine coronary artery, nitro‐l‐arginine‐resistant relaxations (a) are distributed similarly in the proximal and distal parts of the artery, (b) contribute to inhibition of vascular smooth muscle with nitric oxide in an additive fashion, (c) occur during contractions induced by various contractile agents and (d) do not precede those mediated by nitric oxide.
Subclassification of release‐regulating α2‐autoreceptors in human brain cortex
Authors: Raiteri Maurizio , Bonanno Giambattista , Maura Guido , Pende Mario , Andrioli Gian Carlo , Ruelle Antonio
Published: July 2012

Release‐regulating α2‐autoreceptors in human brain were characterized pharmacologically in cortical slices from patients undergoing neurosurgery to remove subcortical tumours; the slices were prelabelled with [3H]‐noradrenaline ([3H]‐NA) and stimulated electrically (3 Hz, 2 ms, 24 mA) under superfusion conditions.

The stimulus‐evoked tritium overflow was almost totally Ca2+‐dependent and tetrodotoxin‐sensitive.

Clonidine and oxymetazoline 0.01 to 1 μm inhibited in a concentration‐dependent manner the evoked overflow of tritium. The two drugs were equipotent (EC50 = 0.03 μm) and their maximal effect was approx. 45%. Phenylephrine and methoxamine, up to 1 μm, did not affect tritium overflow.

Yohimbine (0.01–0.1 μm) shifted the concentration‐response curve of clonidine to the right. The calculated pA2 value was 8.29.

Prazosin and 2‐[2‐[4‐(o‐methoxyphenyl)piperazine‐1‐yl]ethyl]‐4,4‐dimethyl‐1,3(2H,4H)‐isoquinolinedione (AR‐C 239), tested at 0.3 μm, did not modify the concentration‐response curve of clonidine.

The effect of clonidine was antagonized by (+)‐mianserin (pA2 = 7.74), but not by up to 0.3 μm of the (−)‐enantiomer. The concentration‐response curve of clonidine was shifted to the right by the novel α2‐adrenoceptor antagonist, 5‐chloro‐4‐(1‐butyl‐1,2,5,6‐tetrahydropyridin‐3‐yl)‐thiazole‐2‐amine (Z)‐2‐butenedioate (1:1) salt (ORG 20350) (pA2 = 7.55).

Yohimbine, (+)‐mianserin and ORG 20350, but not prazosin and (−)‐mianserin, increased the electrically‐evoked tritium overflow, suggesting that autoreceptors may be tonically activated by endogenous NA.

Desipramine (1 μm) increased evoked tritium overflow from human cortex slices. The effect of clonidine (0.01 − 1 μm) on the evoked overflow of tritium was reduced in presence of 1 μm desipramine.

It is proposed that autoregulation of NA release can occur in human cerebral cortex. The process involves activation of α2‐adrenoceptors which may be either the α2A or the α2D subtype.
The antagonism of benzodiazepine withdrawal effects by the selective cholecystokininB receptor antagonist CI‐988
Authors: Singh Lakhbir , Field Mark J. , Vass Caroline A. , Hughes John , Woodruff Geoffrey N.
Published: July 2012

The ability of a selective cholecystokininB (CCKB) receptor antagonist, CI‐988, to block benzodiazepine withdrawal effects was examined in mice. The discontinuation of twice daily administration of diazepam (1 mg kg−1, i.p.) induced withdrawal anxiogenesis and a proconvulsant effect. In contrast, no such effects were seen following withdrawal from similar administration of CI‐988. However, CI‐988 dose‐dependently (0.001–1.0 mg kg−1, s.c.) antagonized both the anxiogenesis and the proconvulsant effect following diazepam‐withdrawal.
One way cross tolerance between cromakalim and salbutamol in the uterus of the rat in vivo
Authors: Downing Sandra J. , Hollingsworth Michael
Published: July 2012

Cross tolerance between the potassium (K+) channel opener, cromakalim and the β2‐adrenoceptor agonist, salbutamol, was investigated in the uterus of the non‐pregnant rat in vivo. Uterine sensitivity to salbutamol was similar in both vehicle‐treated and cromakalim‐tolerant rats. In salbutamol‐tolerant rats, uterine responses to cromakalim were markedly decreased compared with saline‐infused rats, such that maximum inhibition of uterine contractions was less than 40%.

Propranolol treatment and salbutamol tolerance each produced similar reductions in sensitivity of the uterus to salbutamol of approximately 10 fold. The same dose of propranolol did not influence uterine sensitivity to cromakalim, which suggests that the relaxant action of cromakalim is not due to a direct or indirect activation of uterine β2‐adrenoceptors.

Salbutamol produced a marked (11.7 fold) increase in uterine adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) concentrations measured ex vivo, which was completely inhibited by propranolol pretreatment, but was unaffected by glibenclamide pretreatment. Cromakalim did not increase uterine cyclic AMP concentrations, suggesting that stimulation of adenylate cyclase is not significant in the uterine relaxant action of cromakalim.

The lack of propranolol antagonism of cromakalim and of cromakalim‐induced changes in uterine cyclic AMP concentrations suggests that the cross tolerance observed between salbutamol and cromakalim may be at the level of K+‐channels.
Platelet‐activating factor relaxes ferret tracheal smooth muscle and reduces transepithelial potential difference in vitro
Authors: Webber S.E. , Morikawa T. , Widdicombe J.G.
Published: July 2012

The effects of platelet activating factor (PAF) were examined on the smooth muscle tone, mucus volume, lysozyme and albumin outputs and potential difference (PD) across the ferret tracheal wall.

PAF (0.1–10 μm) had no direct effect on mucus volume, lysozyme or albumin output from the ferret trachea. PAF produced concentration‐dependent relaxations of the tracheal smooth muscle and reductions in PD across the tracheal wall. There was no change in the histological appearance of the trachea after exposure to PAF.

The PAF‐induced smooth muscle relaxation was not affected by FPL55712, a combination of mepyramine and cimetidine, or by a combination of the oxygen free‐radical scavengers catalase and superoxide dismutase (SOD); but was abolished by indomethacin or the PAF‐receptor antagonist WEB2086.

The PAF‐induced reduction in PD was not affected by indomethacin, FPL55712 or mepyramine and cimetidine, but was prevented by catalase and SOD, and by WEB2086.

We conclude that PAF relaxes ferret tracheal smooth muscle in vitro by receptor‐mediated release of a bronchodilator prostaglandin, possibly PGE2. PAF also reduces PD across the trachea suggesting changes in epithelial function; however, there is no histological epithelial damage after PAF. The reduction in PD with PAF is probably produced by receptor‐mediated release of oxygen free‐radicals. The cellular source of these free‐radicals and of the dilator prostaglandin is unclear.
Cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca2+‐pump, reduces Ca2+‐dependent K+ currents in guinea‐pig smooth muscle cells
Authors: Suzuki Masanori , Muraki Katsuhiko , Imaizumi Yuji , Watanabe Minora
Published: July 2012

Effects of cyclopiazonic acid (CPA), a specific inhibitor of the Ca2+‐ATPase in sarcoplamic reticulum (SR), on membrane ionic currents were examined in single smooth muscle cells freshly isolated from ileal longitudinal strips and urinary bladder of the guinea‐pig.

Under whole‐cell clamp, CPA (1–10 μm) reduced peak outward current elicited by depolarization in a concentration‐dependent manner. The concentration of CPA required for 50% decrease in the peak outward current was ∼ 3 μm in ileal cells under these conditions. The current reduced by CPA recovered by more than 70% after washout.

The transient outward current elicited by application of 5 mm caffeine at a holding potential of — 50 mV in Ca2+ free solution was almost abolished, when the preceding Ca2+‐loading of the cell in a solution containing 2.2 mm Ca2+ was performed in the presence of 3 μm CPA.

When the Ca2+‐dependent K+ current (IK‐Ca) and Ca2+ current (ICa) were inhibited by addition of Ca2+, the remaining delayed rectifier type K+ current was not affected by 10 μm CPA. When outward currents were blocked by replacement of K+ by Cs+ in the pipette solution, the remaining ICa was not affected by 10 μm CPA.

CPA (10 μm) did not affect the conductance of single maxi Ca2+‐dependent K+ channels or the Cd2+‐dependence of their open probability in both inside‐ and outside‐out configurations.

6 These results indicate that IK‐Ca is selectively and strongly suppressed by CPA. Its effects may be attributed to a decrease in Ca2+‐uptake into SR, resulting in a decrease in Ca2+‐induced Ca2+ release which is triggered by Ca2+ entering through voltage‐dependent Ca2+ channels and therefore less activation of these K channels.

7 CPA may be extremely valuable pharmacological tool for investigating intracellular Ca2+ mobilization and ionic currents regulated by intracellular Ca2+.
The actions of nitric oxide donors in the prevention or induction of injury to the rat gastric mucosa
Authors: Lopez‐Belmonte J. , Whittle B.J.R. , Moncada S.
Published: July 2012

The protective or damaging actions on the gastric mucosa, of locally infused nitrovasodilators that donate nitric oxide (NO), have been investigated in the pentobarbitone‐anaesthetized rat.

Local intra‐arterial infusion of endothelin‐1 (ET‐1; 5 pmol kg−1 min−1 for 10 min) induced extensive, macroscopically apparent, haemorrhagic injury to the rat gastric mucosa. This damage was dose‐dependently reduced by concurrent local intra‐arterial infusion of glyceryl trinitrate (GTN; 10–40 μg kg−1 min−1) which liberates NO on metabolic transformation, or the nitrosothiol, S‐nitroso‐N‐acetyl‐penicillamine (SNAP, 2.5–10 μg kg−1 min−1) which spontaneously liberates NO.

Local infusion of higher doses of SNAP (20 and 40 μg kg−1 min−1, i.a.) did not, however, significantly protect against mucosal injury induced by ET‐1.

Furthermore, local infusion alone of these higher doses of SNAP, as well as sodium nitroprusside (10–40 μg kg−1 min−1, i.a.) which also spontaneously liberates NO, induced significant mucosal injury, as assessed macroscopically and confirmed by histology.

Local infusion of these higher doses of SNAP and nitroprusside reduced systemic arterial blood pressure (BP), but this was not correlated with the extent of mucosal injury.

Furthermore, local infusion of GTN (10–40 μg kg−1 min−1, i.a.) alone, which also reduced BP, failed to induce gastric mucosal damage.

These findings suggest that exogenous NO can protect the rat gastric mucosa from damage induced by the vasoconstrictor peptide ET‐1, which may reflect local microcirculatory interactions. However, the unregulated release of high levels of NO within the microvasculature induces mucosal injury.
Mediation by the same muscarinic receptor subtype of phasic and tonic contractile activities in the rat isolated portal vein
Authors: Pfaffendorf M. , Van Zwieten P.A.
Published: July 2012

The effects of several agonists on the phasic and tonic contractile responses to muscarinic receptor stimulation have been investigated in the rat portal vein in vitro.

Neither chemical denervation with 6‐hydroxydopamine nor the presence of the α1‐adrenoceptor antagonist, prazosin, influenced the spontaneous or the stimulated myogenic activity of the portal vein.

Indomethacin and NG‐nitro‐l‐arginine were used to investigate the influence of vasoactive factors in this preparation. They slightly increased the frequency and the amplitude of the spontaneous myogenic activity of the portal vein, respectively. NG‐nitro‐l‐arginine but not indomethacin enhanced the maximal phasic response to carbachol. Both indomethacin and NG‐nitro‐l‐arginine failed to influence the tonic response to carbachol.

Muscarinic agonists increased phasic activity according to the rank order of potency: acetylcholine > muscarine > methacholine > carbachol > aceclidine > bethanechol. These effects were superimposed on a sustained contracture at higher concentrations. Oxotremorine was more potent than arecoline in increasing the mechanical phasic activity, without inducing a sustained contracture. Pilocarpine and McN A 343 were weak agonists, producing submaximal effects only on phasic activity.

The muscarinic antagonists AF‐DX 116, 4‐diphenylacetoxy‐N‐methylpiperidine (4‐DAMP), P‐fluorohexahydrosiladiphenidol (pFHHSiD) and pirenzepine antagonized the phasic and tonic mechanical responses to carbachol. Although the tonic contracture was slightly more sensitive to all antagonists studied, the rank order of potency: 4‐DAMP > pFHHSiD > pirenzepine > AF‐DX 116 was the same for both types of response, which is indicative of a M3‐receptor subtype.

The tonic contractile response of the rat portal vein to carbachol was more susceptible to partial receptor inactivation with propylbenzilylcholine mustard than the phasic contractile response. The dissociation constants (KA) obtained from an analysis according to Furchgott & Bursztyn (1967) were found to be 4.32 ± 0.31 μm for the phasic and 3.56 ± 0.21 μm for the tonic type of carbachol‐induced response, respectively. Since the EC50‐values for both carbachol‐induced effects were different (phasic 0.232 ± 0.02 μm; tonic 2.75 ± 0.1 μm) the phasic type of response appears to involve a large receptor reserve.