bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacological analysis of the calcium‐dependence of μ‐receptor agonism
Authors: Dougall I.G. , Leff P.
Published: July 2012

A number of studies in isolated tissues have shown that μ‐opioid‐receptor‐mediated agonism can be augmented or potentiated with reduction in extracellular calcium concentration ([Ca2+]o). These effects have been ascribed to alterations in post‐receptor coupling but the nature of the changes involved have not been quantitatively elucidated.

In this paper, logistic curve‐fitting and operational model‐fitting (Black & Leff, 1983) were used to analyse the effects of variations in [Ca2+]o on the μ‐receptor‐mediated effects of [d‐Ala2, MePhe4, Glyol5]enkephalin (DAGOL) in the isolated, coaxially‐stimulated ileum of the guinea‐pig. At each value of [Ca2+]o, the effects of irreversible receptor alkylation by β‐chlornaltrexamine (β‐CNA) were also investigated.

From these analyses it is concluded that with reduction in [Ca2+]o the efficacy of DAGOL in this system is increased and the sensitivity of the transducer relation is also enhanced, the latter indicating a trend towards positive co‐operativity. Reduction of [Ca2+]o also appeared to produce a reduction in agonist affinity.

Experimental manipulation of [Ca2+]o may provide a useful means of enhancing μ‐agonist efficacy, allowing detection of agonism in compounds with low intrinsic efficacies. However, the accompanying change in co‐operativity of the transducer relation must be considered when quantifying agonism under these conditions.
Effects of theophylline, choleragen and loperamide on rabbit ileal fluid and electrolyte transport in vitro
Authors: Ahsan M.A. , Ilundain Ana , Naftalin R.J. , Sandhu Bhupinda K. , Smith P.M.
Published: July 2012

The effects of theophylline and cholera toxin on water and anion movements across rabbit ileum in vitro and the reversal of these effects by the opiate action of loperamide have been investigated. Water movement across the mucosal and serosal surfaces of the tissue was measured continuously by a high resolution method.

Theophylline caused an increase in short circuit current and reversed the direction of net Cl− movement, due mainly to a decrease in mucosal‐serosal flux. It also caused a rapid, but transient, reversal in the direction of fluid movement across the mucosal surface. Fluid outflow across the serosal surface was decreased but not reversed. Cholera toxin caused a slow inhibition of water movement across both mucosal and serosal surfaces.

Theophylline increased the exit rate of 77Br across the mucosal surface and decreased the exit rate of 77Br across the serosal surface. Theophylline increased the exit rate of 3H‐labelled mannitol across the mucosal surface.

Loperamide reversed the effects of theophylline and cholera toxin on water flow across the mucosal and serosal surfaces and on net transepithelial Cl− flux; it also increased the rate of 77Br exit across the serosal surface of theophylline‐treated tissue. These effects of loperamide could be reversed by naloxone.

The hydraulic conductivity, Lp of the serosal surface was measured directly by determining the osmotic flow generated by low concentrations of polyethylene glycol (mol. wt. 20000 and 90000). Theophylline reduced the Lp by 57%. Loperamide added to theophylline‐treated tissues increased the Lp by 340%. This effect was reversed by naloxone.

These results indicate that modulation of intestinal smooth muscle tone affects transepithelial ion and water flows in vitro. The increase in tone induced by secretagogues increases ion and water reflux via wide shunt channels in the mucosa and thereby reduces net absorption. The increased net fluid and electrolyte absorption induced by loperamide results from the opiate‐dependent inhibition of acetylcholine release from intrinsic ganglia which reduces smooth muscle tone and thereby enhances the fluid and electrolyte conductance of the submucosal layers.
Sensory neuropeptide effects in human skin
Authors: Fuller R.W. , Conradson T.‐B. , Dixon C.M.S. , Crossman D.C. , Barnes P.J.
Published: July 2012

Neuropeptides released from sensory nerves may account for cutaneous flare and wheal following local trauma. In 28 normal subjects we have studied the effects of four sensory neuropeptides given by intradermal injection on the forearm or back.

All peptides caused a flare distant from the site of injection, presumably due to an axon reflex. Substance P (SP) was the most potent (geometric mean dose causing 50% of maximum flare, 4.2 pmol). Neurokinin A (NKA) was the next most potent with neurokinin B (NKB) and calcitonin gene‐related peptide (CGRP) the least. The distant flare response to SP, NKA and NKB was maximal at 5 min and disappeared within 2 h.

CGRP caused a local erythema over the site of injection at doses above 0.5 pmol which at higher doses lasted for up to 12 h.

SP, NKA and NKB caused wheals at doses above 5 pmol with SP and NKB being the most potent. CGRP (up to 250 pmol) did not consistently cause wheal formation. There was no significant effect of coinjection of CGRP upon the response to SP although there was a tendency for an enhancement of the wheal response.

The H1‐histamine antagonist terfenadine (60 mg orally) significantly inhibited the wheal and distant flare response to histamine (5 nmol) and NKA, but not that caused by NKB. The distant flare of CGRP was also reduced but the local erythema was unaltered.

Aspirin (600 mg orally) significantly inhibited the distant flare response to SP, NKA and CGRP, but not that caused by NKB or histamine; the local erythema induced by CGRP was unaffected by aspirin. Aspirin also inhibited the wheal formed by NKA but not the wheal induced by the other substances.

These results suggest that tachykinins cause a distant flare response partially via the release of histamine and cyclo‐oxygenase products, but cause a wheal by a direct effect on the skin microvasculature. The order of potency SP > NKB > NKA suggests that an SPp or NKl receptor is involved in the wheal response. CGRP by contrast has a direct vasodilator effect which is very prolonged.
Inhibition by quinine of endothelium‐dependent relaxation of rabbit aortic strips
Authors: Gebremedhin D. , Hadházy P. , Magyar K.
Published: July 2012

The effects of quinine sulphate, tetramethylammonium chloride (TMA) and tetraethylammonium chloride (TEA) (all blockers of the Ca2+‐activated K+ channels) on the relaxations induced by acetylcholine (ACh), calcium ionophore A23187 and sodium nitrite were studied in helical strips of rabbit aorta.

The strips were contracted to a moderate stable tone with phenylephrine (10−7 m). ACh (4 × 10−9 to 10−6 m) as well as A23187 (10−8 to 3 × 10−7 m) reduced this tone in a concentration‐and endothelium‐dependent manner.

Pretreatment of the tissues with quinine (2.5 × 10−5 to 10−4 m) for 60 min produced a concentration‐dependent inhibition of the relaxation induced by ACh. Also 90 min incubation of the strips with TMA (3 × 10−3 to 6.5 × 10−2 m) or TEA (10−3 to 3 × 10−2 m) inhibited the ACh‐evoked relaxation in a manner similar to quinine.

Quinine (10−4 m, 60 min), TMA (6.5 × 10−2 m, 90 min) or TEA (3 × 10−2 m, 90 min) produced 5 to 10 fold reductions in the relaxant EC50 values of A23187 and ACh and depressed (by 40 to 95%) the maximal relaxations to the ionophore and ACh.

On a molar basis, quinine was more effective than the two tetraalkylammonium ions in reducing the endothelium‐dependent relaxations of the aortic strips induced by ACh or A23187. The inhibitory actions were reversible after 60 to 90 min washout.

Exposure of the strips to either quinine (10−4 m, 60 min), TMA (6.5 × 10−2 m, 90 min) or TEA (3 × 10−2 m, 90 min), however, did not influence significantly the relaxations evoked by sodium nitrite, a direct smooth muscle relaxant.

These results suggest that stimulation of the Ca2+‐activated K+ channels could be, at least partially, responsible for the endothelium‐dependent relaxations induced by ACh or A23187. Their activation might not be required for the endothelium‐independent relaxant effects of sodium nitrite.
The inhibition of release of endothelium‐derived relaxant factor by manoalide, a potent inhibitor of phospholipase A2
Authors: Long Clive J. , Sarau Henry M. , Berkowitz Barry A.
Published: July 2012

The inhibitory action of manoalide on vascular relaxation was characterized. Manoalide was a potent inhibitor of endothelium‐dependent relaxations in the isolated aorta of the rabbit. Responses to acetylcholine (ACh), A23187 and substance P were reduced by manoalide in a dose‐dependent manner whilst those to nitroglycerin were unaffected.

Repeated washing of manoalide‐treated tissues did not restore the relaxant response to ACh, indicating an irreversible action of manoalide. Scanning electron microscopic studies revealed that the endothelium remained intact on manoalide‐treated tissues.

Rabbit aortae from which the endothelium had been removed relaxed in response to perfusion with ACh when delivered via an upstream endothelium‐bearing tissue, indicating release of an endothelium‐derived relaxant factor (EDRF). Incubation of the tissue without endothelium with manoalide (100 nm; 30 min) or inclusion of manoalide in the superfusate at a point just distal to the endothelium bearing tissue did not reduce the relaxant potency of EDRF.

Contractile responses of the guinea‐pig isolated ileum to ACh were not affected by manoalide and, furthermore, binding of [3H]‐quinuclidinyl benzilate to striatal membranes was not reduced by manoalide except at very high concentrations.

Manoalide therefore appears to inhibit vascular relaxation with a selectivity directed towards that mediated by EDRF. A direct antagonism of neither cholinoceptors nor EDRF receptors occurs and it is suggested that manoalide acts at a site within the endothelium to inhibit the synthesis and/or release of EDRF. Based upon these and previous data the possibility that EDRF is lipid‐like or controlled by an arachidonic acid metabolite must continue to be considered.
The effect of apomorphine on exocytosis and metabolic burst of polymorphonuclear leukocytes
Authors: Elferink Jan G.R.
Published: July 2012

In rabbit polymorphonuclear leukocytes (PMNLs) apomorphine at 10–100 μm inhibits fMet‐Leu‐Phe and A23187‐induced exocytosis, and the phorbol myristate acetate‐and fMet‐Leu‐Phe‐induced activation of the metabolic burst. The secretory response was not restored by washing the cells after pretreatment with apomorphine.

The inhibitory effect of apomorphine was not prevented by the dopamine receptor antagonists haloperidol and pimozide, nor did dopamine itself inhibit fMet‐Leu‐Phe‐induced exocytosis. It therefore seems unlikely that effects are mediated via dopamine receptors. However, sulphydryl reagents reduced the inhibitory effect of apomorphine, suggesting that it may depend upon interaction with susceptible sulphydryl groups, the intactness of which is required for exocytosis and other functions of PMNLs.
Dopamine receptor‐mediated spinal antinociception in the normal and haloperidol pretreated rat: effects of sulpiride and SCH 23390
Authors: Barasi S. , Ben‐Sreti M.M. , Clatworthy A.L. , Duggal K.N. , Gonzalez J.P. , Robertson J. , Rooney K.F. , Sewell R.D.E.
Published: July 2012

Nociceptive tail flick latencies (TFL) were recorded in response to noxious thermal stimuli applied to lightly anaesthetized rats. The effects of intrathecally administered dopamine receptor agonists alone and combined with dopamine receptor antagonists were examined upon the TFL. Experiments were repeated on animals made supersensitive to dopamine following withdrawal from 28 day administration of haloperidol.

In untreated animals the D2‐receptor agonist LY171555 and apomorphine produced an increase in TFL. In contrast, the D1‐receptor agonist SKF 38393 had no significant effect on TFL.

Following haloperidol‐induced dopamine‐supersensitivity, SKF 38393 produced an increase in TFL. In contrast, LY 171555 and apomorphine had minimal effects on TFL in this preparation.

In animals not treated with haloperidol, the dopamine receptor antagonists SCH 23390 and (±)‐sulpiride both blocked the increase in TFL produced by the D2‐agonists.

SCH 23390 and (±)‐sulpiride also blocked the increase in TFL produced by SKF 38393 in haloperidol‐supersensitized animals.

The antinociceptive action of intrathecally administered dopamine agonists appears to be mediated via D2‐receptors. Whether the antinociception produced by SKF 38393 is exclusively contingent upon the activation of D1‐receptors in the dopamine‐supersensitive animal is as yet unresolved.
Antagonism between (–)‐N6‐phenylisopropyl‐adenosine and the calcium channel facilitator Bay K 8644, on guinea‐pig isolated atria
Authors: Caparrotta L. , Fassina G. , Froldi G. , Poja R.
Published: July 2012

Antagonism between (–)‐N6‐phenylisopropyladenosine (PIA) and the dihydropyridine calcium channel facilitator Bay K 8644 was investigated in guinea‐pig spontaneously beating or electrically driven isolated atria, taken from normal and from reserpine‐treated animals.

PIA (3–100 nm) produced a dose‐dependent decrease in contractile tension and frequency in spontaneously beating atria being more effective in reserpinized preparations.

Bay K 8644 (5–200 nm) produced an increase in contractile tension in both normal and reserpinized atria. In electrically driven left atria the positive inotropic effect of Bay K 8644 was similar to that in spontaneously beating preparations. The positive chronotropic effect of Bay K 8644 was slight and variable.

PIA produced a rightward parallel shift of the concentration‐response curves for the positive inotropic effects of Bay K 8644 in all experimental conditions. In spontaneously beating atria from normal guinea‐pigs, the Schild regression plot was linear and its slope near to unity; pA2 of PIA 8.63 ± 0.05 (IC50 2.35 ± 0.25 nm). In electrically driven atria the antagonism by PIA of the effects of Bay K 8644 was apparently competitive, and the IC50 of PIA was 18.6 ± 0.4 nM. PIA antagonized the positive chronotropic effect of Bay K 8644 in spontaneously beating preparations, both from normal and from reserpine‐treated animals.

Carbachol did not modify the positive inotropic effects of Bay K 8644.

These data indicate that PIA may interact with Bay K 8644 at the level of the slow calcium channels, and may decrease the transmembrane calcium flux into the cell.
Lack of involvement of α‐adrenoceptors in sympathetic neural vasoconstriction in the hindquarters of the rabbit
Authors: Hirst G.D.S. , Lew M.J.
Published: July 2012

The hypothesis that sympathetic nerves in arterial blood vessels activate excitatory receptors distinct from α‐adrenoceptors was investigated in vivo in the rabbit.

In anaesthetized, ganglion‐blocked rabbits, graded stimulation of the lumbar sympathetic nerve chains caused graded hind limb vasoconstriction. The responses to single pulses and short trains of stimuli were unaffected by benextramine (10 mg kg‐1) and the longer trains were enhanced. Phenoxybenzamine (5 mg kg−1) slightly reduced the responses to short trains of stimuli and did not affect the responses to long trains. The dose‐response curve to intra‐arterial noradrenaline (after β‐adrenoceptor blockade) was shifted rightwards about ten fold by benextramine (10 mg kg−1) and by phenoxybenzamine (5 mg kg−1).

In conscious rabbits the vasoconstriction caused by the nasopharyngeal reflex initiated by smoke inhalation was unaffected by benextramine (10 mg kg−1).

Small mesenteric arteries (<250 μm) taken from untreated rabbits responded to noradrenaline with a threshold concentration of about 1 μm. Similar tissues from benextramine (10 mg kg−1)‐treated rabbits were unresponsive to noradrenaline at concentrations up to 300 μm. However, these tissues were able to respond to potassium and angiotensin II. Aortic ring segments taken from the same rabbits were only about ten fold less sensitive to noradrenaline than segments from control rabbits.

These results are in accord with the hypothesis that sympathetic nerves activate non‐α‐receptors in the vasculature of the rabbit.
A pharmacological study of the rabbit saphenous artery in vitro: a vessel with a large purinergic contractile response to sympathetic nerve stimulation
Authors: Burnstock Geoffrey , Warland Jennifer J.I.
Published: July 2012

Mechanical responses to transmural electrical stimulation were recorded in isolated transverse ring preparations of rabbit saphenous artery. Electrical stimulation for a period of 1 s produced a rapid monophasic contraction and, for a period of 1 min, a biphasic contraction consisting of a rapid constriction followed by a slower sustained constriction. All contractions were abolished in the presence of tetrodotoxin (1 μg ml−1) or guanethidine (4 μm).

After desensitization of the P2‐purinoceptor with α,β‐methylene ATP, contractions to electrical stimulation for 1 s were reduced significantly at all frequencies tested: responses evoked by stimulation at 4 Hz were usually almost completely inhibited, whereas those evoked by stimulation at 64 Hz were only partially inhibited. On the other hand, in the presence of the α‐adrenoceptor antagonist, prazosin, neurogenic contractions were only partially reduced: at 4 Hz there was no significant reduction in the neurogenic contractions while at 32 and 64 Hz, contractions were reduced by an average of 20 and 28% respectively. Usually all contractions were abolished by a combination of the two drugs.

Prazosin antagonized contractions of the vessel to exogenously applied noradrenaline but not to ATP, whereas desensitization of the P2‐purinoceptor with α,β‐methylene ATP blocked responses to ATP but not those to noradrenaline. The concentration‐response curve to histamine was not affected by treatment of the vessel with prazosin, or after desensitization of the P2‐purinoceptor with α,β‐methylene ATP.

These results suggest that noradrenaline and ATP are co‐released from sympathetic nerves supplying the rabbit saphenous artery, both substances being involved in the mechanical contractions of this tissue. Further, the ratio of ATP to noradrenaline involved in these mechanical contractions is dependent upon the frequency of stimulation, but at all frequencies tested the purinergic component is greater than the adrenergic component.
Electrical changes produced by injury to the rat myocardium in vitro and the protective effects of certain antiarrhythmic drugs
Authors: Northover B.J.
Published: July 2012

Glass microelectrodes were used to record intracellular electrical activity from rat isolated and superfused atrial myocardium during external electrical stimulation.

After 2 h in normal oxygenated physiological salt solution the muscle was exposed for 30 min to a superfusate simulating the composition of extracellular fluid during myocardial ischaemia (SI). This fluid contained lactate (20 mm), a raised potassium concentration (7 mm), no glucose and a pH lowered to 6.4, and was gassed with N2 in place of O2 (hypoxia).

During SI the diastolic threshold voltage for stimulation increased, the speed of action potential conduction between the right and left atria slowed, and both the effective and functional refractory periods of the right atrium shortened, as did the duration of the right atrial action potential.

The only component of SI which separately caused electrical changes similar to those of the full simulation was hypoxia.

Addition to the superfusate of verapamil (0.5 μg ml−1), sulphinpyrazone (1–20 μg ml−1) or indomethacin (10–20 μg ml−1) attenuated many of the Sl‐induced electrical changes, although indomethacin was much less effective than the other two drugs.

Lowering the calcium concentration of the superfusate from 2 mm to 0.5 mm protected against the Si‐induced electrical changes that were inhibitable with sulphinpyrazone and verapamil.
Suppression and potentiation of 5‐hydroxytryptophan‐induced hypoglycaemia by α‐monofluoromethyldopa: correlation with the accumulation of 5‐hydroxytryptamine in the liver
Authors: Endo Yasuo
Published: July 2012

Experiments were done to examine whether the accumulation of 5‐hydroxytryptamine (5‐HT) in the liver is responsible for the hypoglycaemia induced in mice by 5‐hydroxytryptophan (5‐HTP) and lipopolysaccharides (LPS).

(±)‐α‐Monofluoromethyldopa (FMD), a potent irreversible inhibitor of aromatic amino acid decarboxylase, suppressed the 5‐HTP‐induced accumulation of 5‐HT in the liver at a dose of 2 mg kg−1 or more, but potentiated the accumulation at a lower dose of 0.4 mg kg−1. Corresponding to these effects, the hypoglycaemic response was prevented by the higher doses of FMD and potentiated by the lower dose. These contrasting effects of FMD were explicable by the amounts of 5‐HTP entering the liver.

In contrast, FMD did not prevent either the hypoglycaemia or the accumulation of 5‐HT in the liver induced by LPS.

These results further support the hypothesis that the accumulation of 5‐HT in the liver is causally related to the hypoglycaemia induced by 5‐HTP and indicate that the LPS‐induced 5‐HT accumulation in the liver is not derived from stimulation of 5‐HT synthesis. It is still not clear whether the accumulation of 5‐HT in the liver is involved in the hypoglycaemic response to LPS.
Paf‐acether‐induced death in mice: involvement of arachidonate metabolites and β‐adrenoceptors
Authors: Criscuoli Marco , Subissi Alessandro
Published: July 2012

Intravenous Paf‐acether (Paf, 15–80 μg kg−1) killed conscious Swiss mice in a dose‐dependent manner, without causing platelet aggregation in the lung micro vasculature, or pulmonary oedema.

Propranolol (0.01–10 mg kg−1, i.p.) potentiated the effects of an LD20 of Paf dose‐dependently, while the β1‐adrenoceptor selective antagonist, metoprolol, was three orders of magnitude less potent in this respect. Salbutamol (1 mg kg−1, i.p.) provided complete protection against an LD80 of Paf.

High doses of indomethacin, aspirin, benoxaprofen and FPL 55712 given i.p. failed to inhibit the effects of an LD80 of Paf, while BW 755C (50–100 mg kg−1) exerted a dose‐dependent protection and benzydamine (50 mg kg−1) and nordihydroguaiaretic acid (200 mg kg−1) were partially active. Dexamethasone (1–5 mg kg−1, s.c.) exerted a dose‐dependent protection, when administered at least 4h before Paf.

In mice anaesthetized with urethane, Paf (1–30 μg kg−1) produced hypotension which was not clearly dose‐related. The effects of the highest dose were also tested on the resistance of the lungs to inflation and found to produce bronchoconstriction.

It may be concluded that pharmacological manipulation of β2‐adrenoceptors modulates Paf‐induced death in mice, while arachidonate metabolites of the cyclo‐oxygenase pathway and peptidoleukotrienes do not appear to be involved. However, lipoxygenase products, distinct from peptidoleukotrienes, may play a role in this phenomenon. It is suggested that bronchoconstriction, probably associated with cardiovascular effects, is a major determinant of the acute toxicity of Paf in mice.
Functional and direct binding studies using subtype selective muscarinic receptor antagonists
Authors: Kunysz E.L. , Michel Anton D. , Whiting Roger L.
Published: July 2012

Muscarinic receptor antagonists were examined in direct binding studies on guinea‐pig cardiac and cortical muscarinic receptors. Pirenzepine, dicyclomine and hexahydroadiphenine were shown to be selective ligands for the putative M1‐muscarinic receptor.

Functional affinity estimates of the muscarinic ligands studied was determined from their ability to inhibit carbachol‐stimulated inositol phosphate (IP) accumulation in guinea‐pig cortical slices.

The affinity estimates for the inhibition of muscarinic agonist‐stimulated IP accumulation were better correlated with affinity estimates obtained from binding studies on the M1 than the M2 muscarinic receptor.

These data provide additional evidence, both from direct binding and functional studies, for the presence of M1 and M2 muscarinic receptor subtypes.
Endothelium‐dependent hyperpolarization of canine coronary smooth muscle
Authors: Feletou Michel , Vanhoutte Paul M.
Published: July 2012

Experiments were designed to determine whether endothelium‐dependent relaxing factor(s) released by acetylcholine from the canine femoral artery influences the membrane potential of coronary arterial smooth muscle.

The membrane potential was recorded in small canine coronary arteries (internal diameter ≤ 500 μm; without endothelium) by means of intracellular microelectrodes. The organ bath also contained a strip of left descending coronary artery without endothelium in which isometric force was measured to bioassay relaxing factors(s) as well as segments of femoral artery with endothelium, which served as the source of endothelium‐derived relaxing factor(s).

Acetylcholine induced endothelium‐dependent, transient hyperpolarizations and relaxations that were not affected by indomethacin.

Inhibition of the sodium‐potassium pump by ouabain or potassium‐free solution did not inhibit the relaxation to acetylcholine but prevented the corresponding hyperpolarization.

Activation of the sodium‐potassium pump of the smooth muscle cells by readmission of potassium ions after incubation in potassium‐free solution caused relaxation and marked hyperpolarization.

These results suggest that endothelium‐derived relaxing factor(s) induces hyperpolarization of vascular smooth muscle of the canine coronary artery, possibly by activation of sodium‐potassium pumping, but that this effect on the cell membrane may only partially explain endothelium‐dependent relaxations evoked by acetylcholine.
Mechanisms of action of lipoxygenase and cytochrome P‐450‐mono‐oxygenase inhibitors in blocking endothelium‐dependent vasodilatation
Authors: Förstermann Ulrich , Alheid Ute , Frölich Jürgen C. , Mülsch Alexander
Published: July 2012

Acetylcholine, ionophore A23187 and melittin induced endothelium‐dependent relaxations of preconstricted strips of rabbit aorta. These relaxations are likely to be mediated by endothelium‐derived relaxing factor (EDRF).

Relaxations in response to acetylcholine (1 μm) were inhibited by the following lipoxygenase inhibitors, with the approximate IC50 values indicated in parentheses: gossypol (1.5 μm), nordi‐hydroguairetic acid (NDGA, 5 μm), AA 861 (20 μm), phenidone (30 μm), quercetin (40 μm), BW 755C (300 μm), and piriprost (500 μm); with cirsiliol 50% inhibition was not achieved. Acetylcholine‐induced relaxations were also blocked by the cytochrome P‐450‐mono‐oxygenase inhibitors proadifen (SKF 525A, 4 μm), metyrapone (300 μm), and cimetidine (300 μm); 7,8 benzoflavone had no effect up to 100 μm.

The more potent inhibitors were also tested against relaxations induced by A23187 (0.1 μm) and melittin (1 μm) and produced partial inhibition of these relaxations.

The mechanism of action of the more potent inhibitors was investigated in a bioassay system. EDRF was produced in columns filled with cultured human endothelial cells. The factor was bioassayed with endothelium denuded segments of rabbit femoral artery. When added to effluent of the column, NDGA, AA861, proadifen and metyrapone inhibited the EDRF‐induced vasodilatation, whereas gossypol had no effect. Gossypol, however, blocked EDRF production when infused through the column.

The more potent inhibitors were also tested to determine their effect on purified soluble guanylate cyclase. While gossypol, NDGA and proadifen had no appreciable effects, basal and nitroprusside (50 μm)‐stimulated guanylate cyclase activity was inhibited by AA861 and metyrapone.

These data suggest that many of the above compounds inhibit EDRF by mechanisms other than lipoxygenase‐ or cytochrome P‐450‐mono‐oxygenase inhibition.
Pentoxifylline does not reduce infarct size in a canine model of acute myocardial infarction
Authors: Campbell Colin A. , Clavenna Carl F. , Wynne Joshua , Kloner Robert A.
Published: July 2012

The effect of the haemorrheological agent pentoxifylline was investigated in a canine model of acute myocardial infarction, induced by occlusion of the left anterior descending coronary for 6 h. Thirty minutes post‐occlusion the dogs were randomized to receive either distilled water or pentoxifylline (0.3 mg kg−1 min−1 for 1 h followed by 0.15 mg kg−1 min−1 for 4.5 h) intravenously.

At 6 h post‐occlusion the in vivo area at risk was determined with monastral blue dye and the area of necrosis was determined with triphenyltetrazolium chloride. The area at risk was 16.5 ± 1.3% in the control group (n = 10) and 17.2 ± 1.8% in the pentoxifylline treated group (n = 10; NS). The area of necrosis was 12.3 ± 1.9% in the control group and 11.9 ± 2.2% in the pentoxifylline treated group (NS). The area of necrosis expressed as a percentage of the area at risk was 69.3 ± 7.7% in the control group and 63.6 ± 7.4% in the pentoxifylline treated group (NS).

Pentoxifylline had no significant effects on heart rate, systolic or diastolic blood pressure. Regional myocardial blood flow, measured by the radioactive microsphere technique, was not significantly different between the groups.

Thus, pentoxifylline does not reduce infarct size in this model of acute myocardial infarction and does not enhance coronary collateral blood flow.
A modified mouse air pouch model for evaluating the effects of compounds on granuloma induced cartilage degradation
Authors: Bottomley K.M.K. , Griffiths R.J. , Rising T.J. , Steward A.
Published: July 2012

Employing rat femoral head cartilage implanted in a 6 day old mouse air pouch, the effects of inflammatory stimuli (i.e. cotton pellets, carrageenan, zymosan) on the loss of proteoglycan and collagen and granuloma formation have been studied.

Wrapping of the cartilage in cotton resulted in granuloma formation with accelerated loss of proteoglycan and collagen over the 14 day implantation period. The amount of loss increased with increasing weight of cotton.

The effects of different classes of anti‐rheumatic drugs on granuloma formation and proteoglycan and collagen loss from cotton wrapped femoral head cartilage in the mouse air pouch have been studied.

Non‐steroidal anti‐inflammatory drugs (NSAIDs) had no influence on granuloma formation, but in general accelerated the rates of proteoglycan and collagen loss.

Dexamethasone and prednisolone significantly reduced granuloma formation and had a marked protective effect on cartilage breakdown.

Of the slow acting anti‐rheumatic drugs examined, only gold sodium thiomalate (GSTM) and dapsone significantly decreased cartilage loss, with an accompanying modest decrease in granuloma formation.

The immunosuppressants cyclophosphamide and methotrexate, but not azathioprine, reduced cartilage degradation, but had no effect on granuloma formation.

The results for the different classes of anti‐inflammatory and anti‐rheumatic drugs are discussed in relation to their effects in other animal models and their reported therapeutic activities in man. It is concluded that the mouse air pouch method as described offers advantages as an animal model over existing procedures to predict therapeutic efficacy in man.
Lipophilicity and pharmacodynamics of cardiotonic steroids in guinea‐pig isolated heart muscle preparations
Authors: Dzimiri Nduna , Fricke Uwe
Published: July 2012

The inhibitory potency of cardiotonic steroids on myocardial Na+‐K+‐ATPase increases with increasing lipophilicity. Employing the inotropic action on guinea‐pig isolated left atria, the relationship between lipophilicity, chemical structure and the pharmacodynamic properties of cardiac glycosides was further examined. Nineteen digitoxigenin‐and digoxigenin‐derivatives, whose lipophilic nature and inhibitory effects on the myocardial Na+‐K+‐ATPase have been previously investigated, were tested.

All steroids exhibited positive inotropic effects which varied with the lipophilicity of these drugs. The dependence of the relationship between these two parameters on structural transformations of the steroids showed qualitatively very close parallelism to that between lipophilicity and their inhibitory effects on myocardial Na+‐K+‐ATPase.

The positive inotropic effects and the inhibitory effects on myocardial Na+‐K+‐ATPase also correlated very well, exhibiting similar patterns in their respective correlations with the individual lipophilicity parameters.

It is inferred that (a) the positive inotropic effects of the cardiac glycosides vary with their lipophilicity, exhibiting trends similar to those shown for their inhibitory effects on myocardial Na+‐K+‐ATPase, (b) this interdependence is secondary to the influence of structural changes, particularly on the steroid nucleus, and (c) both the lipophilic nature and pharmacodynamic behaviour of the cardiac glycosides almost exclusively depend on the steroid nucleus itself.
Quinidine blocks cardiac sodium channels during opening and slow inactivation in guinea‐pig papillary muscle
Authors: Hondeghem Luc M. , Matsubara Tetsu
Published: July 2012

In order to quantify the time‐and voltage‐dependent block of sodium channels by quinidine, we voltage clamped guinea‐pig papillary muscles and measured the maximum upstroke velocity () of the cardiac action potential.

Quinidine reduces presumably by blocking cardiac sodium channels. In therapeutic concentrations, quinidine causes a small amount of tonic block. Upon depolarization of the cardiac cell membrane, a use‐dependent block develops.

A slow component of use‐dependent block has time‐and voltage‐dependence similar to that of slow inactivation, develops for the duration of the depolarization or until a steady state is reached.

In addition, closely associated with the action potential upstroke, a fraction of the channels blocks very quickly. This represents block of activated or open channels.

Near the normal resting potential, channels recover from block with a time constant of 3 to 8 s. At more negative membrane potentials recovery from block occurs slightly faster, while at more positive potentials recovery from block proceeds somewhat more slowly.

In terms of the modulated receptor hypothesis, quinidine has a low affinity for the rested state, avidly blocks open sodium channels, but does not bind significantly to inactivated channels. In addition, quinidine blocks channels as they exhibit slow inactivation.
Studies on the mechanism by which tryptophan efflux from isolated synaptosomes is stimulated by depolarization
Authors: Collard K.J. , Wilkinson L.S. , Lewis D.J.
Published: July 2012

The efflux and influx of tryptophan across the synaptosomal plasma membrane has been studied under a variety of experimental conditions, in order to examine the mechanism by which depolarization enhances the efflux of tryptophan from superfused synaptosomes.

Efflux of [3H]‐tryptophan from preloaded superfused synaptosomes was found to be enhanced by K+ depolarization in a Ca2+ and dose‐dependent manner. In contrast, [3H]‐phenylalanine efflux was only poorly stimulated by depolarization and only by very high concentrations of K+.

Tryptophan efflux was also enhanced by decreasing the extracellular Na+ concentration, but this effect was not dependent on extracellular Ca2+.

Influx of [3H]‐tryptophan into synaptosomes was stimulated by extracellular Na+ removal, but the uptake of [3H]‐phenylalanine was unaffected by this procedure.

Both the induced influx and efflux of tryptophan observed under these experimental conditions was inhibited by immobilizing the plasma membrane carrier with parachlorophenylalanine. This implied that both the enhanced influx and efflux arose as a consequence of the activation of the membrane tryptophan carrier, the direction of the observed effect being dependent upon the manner in which the experiments were conducted.

The relationship between depolarization, the activation of the membrane tryptophan carrier and the significance of this to the in vivo situation is discussed.
Subtypes of muscarinic receptor on cholinergic nerves and atrial cells of chicken and guinea‐pig hearts
Authors: Jeck D. , Lindmar R. , Löffelholz K. , Wanke M.
Published: July 2012

Electrically driven chicken and guinea‐pig atria were used to investigate the negative inotropic effects of the muscarinic agonists methacholine and acetylcholine (ACh). The release of ACh from isolated hearts into the perfusate in response to (preganglionic) vagal or (pre‐and postganglionic) field stimulation was bioassayed on the guinea‐pig ileum or determined by labelling with [3H]‐choline.

Concentration‐response curves for the negative inotropic effect of methacholine were shifted to the right by pirenzepine in various concentrations (0.03 to 10 μmol l−1). The pA2 values were 7.76 in chicken atria and 6.53 in guinea‐pig atria. Pirenzepine and atropine antagonized the negative inotropic response to 0.3 μmol l−1 ACh. The half‐maximally effective concentrations (IC50) of pirenzepine (Pz) and atropine were 40 and 5.4 nmol l−1 in chicken atria and 330 and 3.5 nmol l−1, respectively, in guinea‐pig atria. Thus, the respective potency ratios (IC50Pz/IC50atropine) were 7.4 and 94.3 in the two species.

Pirenzepine in low concentrations increased the release of unlabelled and 3H‐labelled ACh from isolated hearts evoked by vagal and field stimulation only in chicken, but not in guinea‐pigs. The half‐maximally‐effective concentration of pirenzepine was about 30 nmol l−1 in the chicken heart, whereas, in the guinea‐pig heart, an increased release was observed at 300 nmol l−1.

(+)‐Tubocurarine [(+)‐Tc; 100 μmol l−1] reduced the release of ACh evoked by (preganglionic) vagal stimulation to a (+)‐Tc‐resistant release of about 30%. The time‐course of the neuronal release of [3H]‐ACh was markedly altered: the onset was delayed and the termination was extended beyond the period of stimulation (1 min or 5 s) by several seconds. The (+)‐Tc‐resistant release was nearly abolished by 30 nmol l−1 pirenzepine.

In conclusion, the pre‐and post‐synaptic muscarinic receptors of the parasympathetic neuroeffector junction of the heart both belong to the M1‐subtype in the chicken and to an M2‐subtype in the guinea‐pig. Block of the nicotinic ganglionic transmission in the chicken heart by (+)‐Tc unmasked a muscarinic transmission, which presumably was mediated through M1‐receptors stimulating a low and prolonged postganglionic release of ACh.
Relative affinities of drugs acting at cholinoceptors in displacing agonist and antagonist radioligands: the NMS/Oxo‐M ratio as an index of efficacy at cortical muscarinic receptors
Authors: Freedman S.B. , Harley E.A. , Iversen L.L.
Published: July 2012

Radioligand binding assays using [3H]‐N‐methylscopolamine (NMS) and [3H]‐oxotremorine M (Oxo‐M) have been devised to predict the efficacy of test compounds at muscarinic receptors in rat cerebral cortex.

Muscarinic antagonists, including non‐selective and both M1‐ and M2‐selective compounds, displayed similar affinity for both binding assays.

Full agonists such as carbachol and muscarine possessed a ratio of potencies against the antagonist versus the agonist ligand (NMS/Oxo‐M ratio) of > 4000.

Compounds which have been shown previously to display partial agonist activity in functional assays e.g. pilocarpine and RS86 had intermediate NMS/Oxo‐M ratios of 100–150. A second group of compounds which included oxotremorine had somewhat higher ratios (500–1400).

The ratio of affinity constants for the two assays predicted the ability of agonists to stimulate cortical phosphatidyl‐inositol turnover.

These results suggest that the NMS/Oxo‐M ratio may be a useful prediction of efficacy for novel compounds acting at cortical muscarinic receptors.
Bradykinin analogues: differential agonist and antagonist activities suggesting multiple receptors
Authors: Braas Karen M. , Manning Donald C. , Perry David C. , Snyder Solomon H.
Published: July 2012

Bradykinin analogues with specific antagonist activity in several bioassays were evaluated for effects on [3H]‐bradykinin receptor binding sites and inositol phosphate production in neuroblastoma N1E‐115 cells. The analogues varied in their affinities for bradykinin receptors in guinea‐pig ileum and N1E‐115 cell membranes, in their effects on uterine and ileal contractions and in their agonist or antagonist activity on phosphoinositide turnover in N1E‐115 cells. These tissue specific effects suggest the presence of multiple bradykinin receptor subtypes.
Clonidine effects on disposition of xenobiotics in rats: inhibited elimination of flow‐limited but not extraction‐limited agents
Authors: Ben‐Zvi Zvi , Hurwitz Aryeh
Published: July 2012

The α2‐adrenoceptor agonist, clonidine, reduces the hepatobiliary clearance of the anionic dye, sulphobromophthalein (BSP) in rodents. We now compare the effects of clonidine on BSP elimination with its effects on disposition of compounds which are metabolized by hepatic microsomal mixed function oxidases.

BSP, 100 mg kg−1 was administered i.v. to rats at 4h after s.c. saline or clonidine, 0.2 mg kg−1. Thirty min later, plasma BSP levels were 121.4 ± 2.25 μg ml−1 in saline‐treated rats, while in clonidine‐treated rats they were 631.5 ± 141.0 μg ml−1. Clonidine raised hepatic BSP levels from 256.0 ± 28.9 μg g−1 tissue to 568.5 ± 86.5 μg g−1.

Acute administration of clonidine (0.2 mg kg−1 s.c.) or repeated clonidine dosing (0.2 mg kg−1, s.c. twice daily for 10 days) did not affect the disposition of intravenously administered [14C]‐antipyrine (15 mg kg−1).

Activities of the P450 mixed function oxidase enzymes, aniline hydroxylase and aminopyrine N‐demethylase, were identical in liver microsomes from saline‐treated rats and in microsomes from rats given single or multiple s.c. doses of clonidine (0.2 mg kg−1).

Addition of clonidine or other 2‐substituted imidazoles at concentrations up to 2 μm did not affect the activities of aniline hydroxylase or of aminopyrine N‐demethylase in suspensions of rat liver microsomes. Other substituted imidazoles, including cimetidine, clotrimazole and metronidazole, at concentrations of 0.2 μm or higher, inhibited the activities of these microsomal enzymes.

Clonidine slowed BSP elimination, which is probably hepatic blood flow‐limited, but not the extraction‐limited elimination of antipyrine, which is metabolized by hepatic microsomal enzymes.
Evidence that mCPP may have behavioural effects mediated by central 5‐HT1C receptors
Authors: Kennett G.A. , Curzon G.
Published: July 2012

The effects of 1‐(3‐chlorophenyl)piperazine (mCPP) and 1‐[3‐(trifluoromethyl)phenyl] piperazine (TFMPP) on activity of rats in a novel cage, and on the rotorod and elevated bar co‐ordination tests was examined.

Peripherally administered mCPP and TFMPP dose‐dependently reduced locomotion, rearing, and feeding scores but not grooming of freely fed rats placed in a novel observation cage. Yawning behaviour was increased. Similar effects were also observed after injection of mCPP into the 3rd ventricle.

Co‐ordination on a rotating drum of both untrained and trained rats was impaired following mCPP but co‐ordination on an elevated bar was not.

The hypoactivity induced by mCPP was opposed by three antagonists with high affinity for the 5‐hydroxytryptamine (5‐HT1C) site; metergoline, mianserin, cyproheptadine and possibly also by a fourth antagonist mesulergine. Metergoline, mianserin and cyproheptadine also opposed the reduction in feeding scores. However, neither effect of mCPP was antagonized by the 5‐HT2‐receptor antagonists ketanserin or ritanserin, the 5‐HT3‐receptor antagonist ICS 205–930, the 5‐HT1A and 5‐HT1B‐receptor antagonists (—)‐pindolol, (—)‐propranolol and (±)‐cyanopindolol or the 5‐HT1A‐, 5‐HT2‐ and dopamine receptor antagonist spiperone. The specific α2‐adrenoceptor antagonist idazoxan was also without effect.

Hypoactivity induced by TFMPP was similarly antagonized by mianserin but unaffected by (±)‐cyanopindolol.

These results suggest that the hypoactivity is mediated by central 5‐HT1C‐receptors and that mCPP and possibly TFMPP may be 5‐HT1C‐receptor agonists.

As mianserin, cyproheptadine and mesulergine in the absence of mCPP did not increase locomotion but increased the number of feeding scores, the activation of 5‐HT1C‐receptors may be of physiological importance in the control of appetite. The possible relevance of these results to the therapeutic and side‐effects of clinically used antidepressants (particularly trazodone and mianserin) and anorexigenic drugs is discussed.
The origin of the anticholinesterase‐induced repetitive activity of the phrenic nerve‐diaphragm preparation of the rat in vitro
Authors: Ferry C.B.
Published: July 2012

Action potentials have been recorded in contracting muscle cells of the phrenic nerve‐diaphragm preparation from rats. After the organophosphorous anticholinesterase, ecothiopate, some cells fired repetitive action potentials. In 0.1 mm [Mg2+]o the repetitive activity was generated presynaptically or postsynaptically, and in 1 mm [Mg2+]o, probably only postsynaptically.

The repetitive action potentials in muscle were generated ectopically about 0.2 mm away from the usual site.

In mm [Mg2+]o, spontaneous action potentials in muscle were generated presynaptically. These were often followed by repetitive action potentials generated either presynaptically or postsynaptically.

The initiation of centripetal action potentials in the phrenic nerve was coincident with the repetitive firing in the muscle.

The results are discussed in relation to the manner of generation of repetitive activity in nerve and muscle after anticholinesterases.
Comparison of the effects of some muscarinic agonists on smooth muscle function and phosphatidylinositol turnover in the guinea‐pig taenia caeci
Authors: Gardner A.L. , Choo L.K. , Mitchelson F.
Published: July 2012

The effects of the muscarinic agonists acetylcholine (ACh), carbachol (CCh), AHR‐602, and McN‐A‐343 on contractility and on inositol phosphate accumulation in the presence of lithium were compared in the taenia of the guinea‐pig caecum.

Compared to CCh, ACh was a full agonist for contraction but AHR‐602 and McN‐A‐343 were partial agonists producing 80–85% of the maximal response to CCh. Similar to previous findings with CCh, tonic contractions produced by AHR‐602 and McN‐A‐343 were less sensitive to inhibition by nifedipine or verapamil than tonic contractions to ACh.

CCh and ACh produced similar increases in inositol phosphate accumulation and the effect of CCh (0.1 mm) was inhibited by atropine (IC50 8.5 nm) and pirenzepine (IC50 450 nm). The accumulation of inositol phosphates in the presence of AHR‐602 or McN‐A‐343 was not significantly different (P > 0.05) from basal levels.

A concentration of 0.2 mm AHR‐602 produced a parallel shift of the concentration‐response curve to CCh on inositol phosphate accumulation. The IC50 value for inhibition of CCh (0.1 mm) was > 50 fold higher than the EC50 value for contraction produced by the partial agonist. McN‐A‐343 (20 μm) produced a flattening of the concentration‐response curve to CCh for inositol phosphate accumulation.

The results suggest that the increase in phosphatidylinositol turnover produced by muscarinic agonists, like the contractile response, involves an M2‐muscarinic receptor. AHR‐602 and McN‐A‐343 are partial agonists for the contractile response and while producing no significant increase in phosphatidylinositol turnover inhibit the response to CCh.