bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of cyclic GMP production in cultured endothelial cells of the pig by bradykinin, adenosine diphosphate, calcium ionophore A23187 and nitric oxide
Authors: Boulanger Chantal , Schini Valerie B. , Moncada Salvador , Vanhoutte Paul M.
Published: July 2012

The effects of bradykinin, adenosine diphosphate, calcium ionophore A23187 and nitric oxide on the production of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) and guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) were investigated in cultured aortic endothelial cells of the pig.

Bradykinin (10−7m), adenosine diphosphate (3 × 10−5m), nitric oxide (2 × 10−6m) and A23187 (10−6m) stimulated the production of cyclic GMP. This stimulation reached a maximum within 1 min and declined rapidly with the first three agonists whereas that induced by A23187 was long‐lasting.

These concentrations of bradykinin, A23187 and nitric oxide had no effect on cyclic AMP production. However, adenosine diphosphate (3 × 10−5m) slightly but significantly enhanced its production by about 1.7 fold.

The basal content of cyclic GMP in endothelial cells was significantly reduced by haemoglobin (10−5m, a scavenger of endothelium‐derived relaxing factor(s) and methylene blue (10−5m, an inhibitor of the activation of soluble guanylate cyclase) and was significantly enhanced by superoxide dismutase (500 u ml−1, a scavenger of superoxide anions). The basal content of cyclic GMP was not affected by NG‐monomethyl‐l‐arginine (10−5m, a specific inhibitor of the formation of nitric oxide from l‐arginine) and was slightly but significantly increased by its d‐enantiomer, NG‐monomethyl‐d‐arginine.

The production of cyclic GMP stimulated by bradykinin, adenosine diphosphate, A23187 and nitric oxide was inhibited by haemoglobin (10−5m) and methylene blue (10−5m) but was unaffected by superoxide dismutase (500 u ml−1).

The production of cyclic GMP stimulated by bradykinin, adenosine diphosphate or A23187, but not that stimulated by nitric oxide, was significantly reduced by NG‐monomethyl‐l‐arginine (10−5m). The production of cyclic GMP evoked by nitric oxide, but not that induced by the other three agents, was enhanced significantly by NG‐monomethyl‐d‐arginine by about 1.5 fold.

These data indicate that the endothelium‐dependent vasodilators bradykinin, adenosine diphosphate and A23187 activate the production of cyclic GMP in endothelial cells via the synthesis of nitric oxide, which in turn stimulates the soluble guanylate cyclase.
Reversible and selective antagonism by suramin of ATP‐activated inward current in PC12 phaeochromocytoma cells
Authors: Nakazawa Ken , Fujimori Kannosuke , Takanaka Akira , Inoue Kazuhide
Published: July 2012

The effects of suramin on an adenosine 5′‐triphosphate (ATP)‐ activated inward current were investigated in PC12 phaeochromocytoma cells with whole‐cell voltage‐clamp techniques.

Suramin (30 to 300 μm) inhibited the ATP‐activated current in a dose‐dependent manner. The inhibitory effects were reversible and competitive.

Suramin also suppressed the current activated by adenosine 5′‐0‐(3‐thio triphosphate) but did not affect the current activated by nicotine. Suramin did not affect the suppression of a K current induced by methacholine.

The results suggest that suramin antagonizes the ATP‐receptor‐operated membrane current reversibly and selectively.
The pharmacokinetics of γ‐glutamyl‐l‐dopa in normal and anephric rats and rats with glycerol‐induced acute renal failure
Authors: Boateng Yaw A. , Barber Harold E. , MacDonald Thomas M. , Petrie James C. , Lee Michael R. , Whiting Paul H.
Published: July 2012

The pharmacokinetics of γ‐glutamyl‐l‐dopa (gludopa) and its metabolite, l‐dopa, have been studied in normal rats at three dose levels of gludopa: 2 mg kg−1, 5 mg kg−1 and 7.5 mg kg−1. The extent of metabolism in normal rats, and the pharmacokinetics in anephric rats and rats with glycerol‐induced acute renal failure (ARF) were also studied at a gludopa dose of 2 mg kg−1.

Gludopa was extensively metabolised to l‐dopa with only about 10% of an injected dose being excreted unchanged. Normal rats had a rapid gludopa clearance of 50.9 ± 9.6 ml min−1 kg−1 and elimination rate constant of 2.99 ± 0.27 h−1. The mean residence time and half‐life were 20.9 ± 1.4 and 14.4 ± 1.0 min, respectively. The apparent volume of distribution at steady state was 1.05 ± 0.181 kg−1.

No statistically significant differences were found in the main pharmacokinetic parameters between ARF and controls for either gludopa or its metabolite l‐dopa.

In anephric rats and controls the kidneys were found to contribute about 68.5% and 67.2% to the elimination of gludopa and the metabolite l‐dopa, respectively.

These results confirm that gludopa is an efficient pro‐drug for l‐dopa, and that the kidneys are the major site of gludopa metabolism. It seems likely that the renal specificity of gludopa persists in ARF.
The role of cyclic AMP and its protein kinase in mediating acetylcholine release and the action of adenosine at frog motor nerve endings
Authors: Hirsh Jody K. , Silinsky Eugene M. , Solsona Carles S.
Published: July 2012

The importance of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) and its protein kinase (protein kinase A, PKA) in promoting acetylcholine (ACh) release was studied at frog motor nerve endings. The effects of cyclic AMP‐dependent protein phosphorylation on the action of adenosine receptor agonists were also investigated.

Cyclic AMP was delivered to a local region of the cytoplasm just beneath the plasma membrane of motor nerve endings using phospholipid vesicles (liposomes) as a vehicle. Cyclic AMP in liposomes produced a parallel reduction in the mean level of evoked ACh release (m) and spontaneous ACh release (miniature endplate potential frequency; m.e.p.p.f) in most experiments. These inhibitory effects of cyclic AMP on quantal ACh release resemble the action of adenosine.

The effects of global increases in cytoplasmic cyclic AMP concentrations using lipophilic cyclic AMP analogues were generally different from those observed with cyclic AMP. 8‐(4‐Chlorophenylthio) cyclic AMP (CPT cyclic AMP) produced approximately two fold increases in m and m.e.p.p.f. Dibutyryl cyclic AMP (db cyclic AMP) also increased m and m.e.p.p.f, with the effect on m being smaller and more variable.

All three cyclic AMP analogues reduced the effects of adenosine receptor agonists on spontaneous and evoked ACh release.

The roles of protein phosphorylation in mediating ACh release and the inhibitory effects of adenosine were studied with the protein kinase inhibitor H7. H7 (30–100 μm) produced no consistent effect on evoked or spontaneous ACh release. At these concentrations, however, H7 exerted an unfortunate inhibitory action on the nicotinic ACh receptor/ion channel.

H7 prevented the increases in spontaneous ACh release produced by CPT cyclic AMP (250 μm). Thus H7 is likely to inhibit PK A in frog motor nerve endings.

H7 did not alter the inhibitory effect of adenosine on evoked and spontaneous ACh release.

The results suggest: (i) that the adenylyl cyclase‐cyclic AMP‐PK A system is compartmentalized within the motor nerve terminal, (ii) that phosphorylation does not play a major role in ACh release and (iii) the cyclic AMP‐PK A system modulates rather than mediates the inhibitory effects of adenosine.
Pharmacological studies of human erectile tissue: characteristics of spontaneous contractions and alterations in α‐adrenoceptor responsiveness with age and disease in isolated tissues
Authors: Christ George J. , Maayani Saul , Valcic Mira , Melman Arnold
Published: July 2012

The pathophysiology of impotence related to vascular smooth muscle dysfunction in the male corpus cavernosum was studied on human isolated erectile tissue (HET). Studies were conducted on 140 sections of HET obtained from 38 male patients undergoing surgery for implantation of penile prostheses to correct underlying erectile dysfunction.

Spontaneous myotonic oscillations were characteristic of greater than 90% of all HET preparations at 37°C. These spontaneous oscillations were markedly attenuated by indomethacin, BW755C, nifedipine, removal of extracellular Ca2+, or lower temperatures (≤ 32°C), but were not sensitive to inhibition by atropine, phentolamine or tetrodotoxin. Our data suggest that the oscillations may, at least in part, result from the generation and/or release of a stable cyclo‐oxygenase product and a consequent increase in transmembrane Ca2+ influx.

The phenylephrine‐induced contractions in HET may be reliably assayed up to 24 h after surgical removal, without significant alterations in the EC50, maximum response (Emax) or slope index of the steady‐state concentration‐response curve to phenylephrine.

The competitive and surmountable nature of the antagonism of phenylephrine‐induced contractions by prazosin and yohimbine allowed calculation of antagonist dissociation constants. The calculated pKb values for prazosin and yohimbine, respectively, were 9.47 ± 0.49 and 5.54 ± 0.22. The rank order of agonist potency in HET was: noradrenaline = phenylephrine ≫ clonidine. These data indicate the presence of a population of membrane receptors that are predominantly of the α1‐adrenoceptor subtype.

The entire patient population was stratified on a decennial basis into five age groups, and each age group was subsequently subdivided into diabetic and nondiabetic diagnostic categories. With respect to the steady‐state phenylephrine concentration‐response curves, a Winer two‐factor analysis of variance revealed a significant effect of age on the calculated pEC50 value, as well as a significant age‐diagnosis interaction. A post hoc statistical analysis for unpaired samples yielded significant differences between pEC50 values for diabetic and nondiabetic patients in age groups 41–50 and 61–70 years. In addition, a Winer two‐factor analysis of variance also detected a significant effect of age on the calculated Emax value.

In conclusion, our studies demonstrate that spontaneous contractions in HET are likely to be mediated by the generation and release of a stable cyclo‐oxygenase product. Furthermore, the results of both agonist and antagonist studies are consistent with the presence of a homogeneous α1‐adrenoceptor population. Lastly, the responsiveness of isolated HET to phenylephrine was shown to be altered by both age and disease.
The effectiveness of α2‐adrenoceptor activation increases from the distal to the proximal part of the veins of canine limbs
Authors: Guimaraes Serafim , Nunes Jose P.
Published: July 2012

The effectiveness of α1‐ and α2‐adrenoceptor activation was compared at different levels of the saphenous and cephalic vein of the dog in vitro.

Helically cut strips were used to determine concentration‐response curves to phenylephrine, noradrenaline, UK‐14, 304 (5‐bromo‐6‐(imidazoline‐2‐ylamino)‐quinoxaline) and B‐HT 920 (2‐amino‐6‐allyl‐5,6,7,8‐tetra‐hydro‐4H‐(thiazo)‐4,5‐d‐azepine). The effect of prazosin and yohimbine on these curves was also studied.

At the distal level, the maximum response to UK‐14,304 amounted to 33 and 50% of those to noradrenaline in the saphenous and cephalic vein, respectively, while at the proximal level the maximum response to UK‐14,304 amounted to 72 and 78% of those to noradrenaline, in the saphenous and cephalic vein, respectively.

In both vessels, the results obtained with B‐HT 920 were very similar to those for UK‐14,304.

The pD2 values for UK‐14,304 — which were identical at the three levels of both vessels — and the pA2 values for the antagonism exerted by either prazosin or yohimbine against the responses to UK‐14,304 indicate that the α2‐adrenoceptors are identical at the different levels of both vessels.

These results show that the effectiveness of α2‐adrenoceptor stimulation increases from the distal to the proximal regions of canine limb veins. Apparently, this is due to a greater density of α2‐adrenoceptors in the proximal regions.

Yohimbine is much more potent against phenylephrine distally than proximally in both vessels. However, after 30 nm phenoxybenzamine — a concentration which eliminates the vast majority of α1‐adrenoceptors without affecting α2‐adrenoceptors — yohimbine became equally potent at both levels, suggesting that the difference existing before phenoxybenzamine depended on α1‐adrenoceptors. Hence it is concluded that α1‐adrenoceptors in distal and proximal portions may differ.
GABAA receptor‐mediated stimulation of non‐adrenergic non‐cholinergic neurones in the dog ileocolonic junction
Authors: Boeckxstaens Guy E. , Pelckmans Paul A. , Rampart Marc , Ruytjens Isabelle F. , Verbeuren Tony J. , Herman Arnold G. , Van Maercke Yvan M.
Published: July 2012

The inhibitory effects of γ‐aminobutyric acid (GABA), the GABAA receptor agonist homotaurine and the GABAB receptor agonist (±)‐baclofen were investigated on circular muscle strips of the dog terminal ileum and ileocolonic junction.

In the presence of atropine, GABA and homotaurine induced concentration‐dependent relaxations, similar to the non‐adrenergic non‐cholinergic (NANC)‐mediated relaxations evoked by electrical stimulation or by acetylcholine. The ileocolonic junction was more sensitive to GABA and homotaurine than the ileum. (±)‐Baclofen had no effect. Cross desensitization only occurred between GABA and homotaurine.

The GABAA receptor antagonist bicuculline shifted the concentration‐response curves to GABA and homotaurine to the right. The maximal relaxation to GABA remained unaffected.

GABA‐induced relaxations were not inhibited by timolol, guanethidine, domperidone, hexamethonium and desensitization to ATP, but were abolished by tetrodotoxin.

Bicuculline, and pretreatment with GABA or (±)‐baclofen had no effect on the NANC‐evoked relaxations to electrical stimulation and acetylcholine.

In conclusion, GABA stimulates GABAA receptors located on inhibitory NANC neurones in the dog ileocolonic junction. Our results suggest that it is unlikely that GABA is the final inhibitory NANC neurotransmitter.
Adverse effects on rat cardiac function ex vivo after repeated administration of the benzodiazepine partial inverse agonist, FG7142
Authors: Stanford S.C. , Gettins D. , Littel H.J.
Published: July 2012

The Langendorff preparation was used to investigate functional changes in rat heart one week after the last of a course of repeated injections of the benzodiazepine inverse agonist, FG7142 (20 mg kg−1 i.p; three times weekly for five weeks).

Under these conditions, FG7142 caused a statistically significant reduction in both cardiac basal tension and the inotropic effect of noradrenaline at doses giving 50 and 100% of the maximum response.

Basal heart rate, basal coronary perfusion pressure and the effects of noradrenaline ex vivo on these parameters were all unaffected by repeated administration of FG7142.

FG7142 had no intrinsic effects on cardiac function when administered in vitro.

We discuss mechanisms which could underlie the effects of FG7142 on cardiac tension ex vivo and consider the possibility that this action may be related to the anxiogenic or proconvulsant actions of this drug.
Structure‐activity studies on bradykinin and related peptides: agonists
Authors: Rhaleb Nour‐Eddine , Drapeau Guy , Dion Stéphane , Jukic Daniela , Rouissi Noureddine , Regoli Domenico
Published: July 2012

Bradykinin, kallidin, T‐kinin, [Hyp3]‐bradykinin and several analogues were prepared by solid‐phase synthesis and purified by high performance liquid chromatography.

The various peptides were tested for their abilities to relax the dog carotid and renal arteries, or to contract the rabbit jugular vein and aorta, in order to measure their activities on BK2 (the first three preparations) or BK1 (the rabbit aorta) receptors. The dog renal artery without endothelium was also used as a BK1 receptor system.

T‐kinin was found to be less active than bradykinin, while the replacement of Pro3 with Hyp favoured BK2 receptor occupation. [Hyp3,Tyr(Me)8]‐BK was found to be a selective BK2 receptor agonist.

Amidation or methylation of the C‐terminal carboxyl decreased activity, while extension of the N‐terminal with Sar or d‐Arg increased affinity and selectivity for BK1 (Sar) and affinity for BK2 (d‐Arg) receptors. Acetylation of N‐terminal amide brought affinity down to 10% or less.

Replacement of the peptide bonds Phe8‐Arg9 to protect from kininase I and II, decreased affinities slightly, but was incompatible with additional changes at the N‐terminal or in the peptide bond Gly4‐Phe5.

Substitution of C‐terminal Phe in desArg9‐BK (the BK1 receptor stimulant) with d‐Phe increased potency and selectivity for BK1 receptors while protecting from carboxypeptidases. Sar[d‐Phe8]desArg9‐BK was found to be a potent and selective BK1 receptor agonist.
The effects of siguazodan, a selective phosphodiesterase inhibitor, on human platelet function
Authors: Murray Kenneth J. , England Paul J. , Hallam Trevor J. , Maguire Joanne , Moores Kitty , Reeves Martin L. , Simpson Alec W. M. , Rink Timothy J.
Published: July 2012

The effects of siguazodan (SK&F 94836) a selective phosphodiesterase (PDE) inhibitor with inotropic and vasodilator activity, were studied on human platelets.

Siguazodan selectively inhibited the major cyclic AMP‐hydrolysing PDE in human platelet supernatants. The inhibited enzyme has been variously termed cyclic GMP‐inhibited PDE or PDE‐III.

In platelet‐rich plasma (PRP), siguazodan inhibited U46619‐induced aggregation more potently than that induced by ADP and collagen. Treatment of the PRP with aspirin had no effect on the potency of siguazodan.

In washed platelets, siguazodan increased cyclic AMP levels and reduced cytoplasmic free calcium ([Ca2+]i). ADP decreased the ability of siguazodan to raise cyclic AMP and this may explain its lower potency in inhibiting responses to ADP.

Siguazodan has anti‐platelet actions over the same concentration range that it is an inotrope and vasodilator.
Voltage‐dependent modulation of rat sensory neurone calcium channel currents by G protein activation: effect of a dihydropyridine antagonist
Authors: Scott R.H. , Dolphin A.C.
Published: July 2012

The ability of a depolarizing prepulse to increase the rate of activation of IBa has been examined in cultured sensory neurones of the rat. Both in control neurones and in the presence internally of the guanine nucleotide analogue, guanosine 5′‐O‐3‐thiotriphosphate (GTPγS) which markedly slows the rate of activation of IBa and reduces its amplitude, a depolarizing prepulse increased the rate of activation of IBa, but did not increase its amplitude measured at the end of the 100 ms voltage step. The calcium channel antagonist (−)‐202–791, which we have previously shown to increase the amplitude of IBa in the presence of GTPγS, did not occlude the response to a depolarizing prepulse, suggesting that the mechanism of action of (−)‐202–791 is not to disrupt the interaction of the channels with activated G proteins.
5‐Hydroxytryptamine and arachidonic acid metabolites modulate extensive platelet activation induced by collagen in cats in vivo
Authors: De Clerck F. , Loots W. , Somers Y. , Beetens J. , Wouters L. , Wynants J. , Janssen P.A.J.
Published: July 2012

The pathways contributing to the platelet adhesion/aggregation reaction elicited by collagen microfibrils, administered to cats in vivo, were analysed.

The intra‐aortic infusion of collagen (100 μg kg−1 in 1 min) caused an extensive activation of platelets, as evidenced by the time‐dependent drop of free platelet numbers in whole blood, and the increases of 5‐hydroxyindoles (5‐HI), 5‐hydroxytryptamine (5‐HT) and thromboxane B2 (TXB2) levels in plasma, prepared from effluent venous blood sampled from the inferior caval vein.

5‐HT2 receptor blockade with ketanserin (0.63 mg kg−1 i.v., 10 min) and cyclo‐oxygenase inhibition with aspirin (10 mg kg−1 i.v., 10 min) slightly attenuated the peak reduction of free platelets in whole blood in response to collagen without affecting changes in plasma 5‐HI. Aspirin, but not ketanserin, reduced the collagen‐induced changes in plasma TXB2, prostaglandin E2 (PGE2) and 6K‐PGF1α.

Dual TXA2 synthetase inhibition/TXA2‐prostaglandin endoperoxide receptor antagonism with ridogrel (5 mg kg−1 i.v., 10 min) halved the drop in free platelets, reduced the release of platelet 5‐HI, inhibited the increase in plasma TXB2 and elevated that of 6K‐PGF1α and PGE2 in response to collagen.

Combined treatment with ketanserin and aspirin reduced the collagen‐induced drop of free platelets and the release of platelet 5‐HI to a similar extent as ridogrel alone; plasma prostanoids were affected as with aspirin alone.

Combined administration of ketanserin and ridogrel virtually eliminated the collagen‐induced platelet adhesion/aggregation response and release of 5‐HI; prostanoids were affected as with ridogrel alone.

The results indicate that the interplay between 5‐HT and arachidonic acid metabolites is causally involved in the platelet reaction to activation induced by collagen in cats in vivo.
Regulation of the synthesis and metabolism of striatal dopamine after disruption of nerve conduction in the medial forebrain bundle
Authors: Commissiong John W. , Slimovitch Caroline , Toffano Gino
Published: July 2012

After physical (knife‐cut) or chemically‐mediated (tetrodotoxin 300 nm, 1.5 μl; 1.0 μl min−1) interruption of nerve conduction in the nigrostriatal tract, there was a marked increase in the synthesis and metabolism of dopamine in the isolated dopaminergic nerve terminals of the striatum. The effect peaked at 4 h post‐transection, at which time 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were increased by 300% and 700% respectively (DOPAC: 27 ± 13 vs 80 ± 17 nmol g−1; HVA: 6.66 ± 3.57 vs 54 ± 18 nmol g−1). The increases in dopamine content and metabolism are secondary to an increase in the rate of synthesis on the lesioned side, versus the intact, control side.

In both experimental situations, haloperidol (1.0 mg kg−1, i.p.) retained its known ability to induce a significant increase in DOPAC and HVA in the striatum, despite the interruption of nerve conduction in the nigrostriatal tract.

Six days after cutting the left nigrostriatal tract, dopamine in the left striatum was reduced to <5% of the control value, and DOPAC and HVA were not detectable. In the denervated, left striatum, the synthesis of dopamine (from injected l‐DOPA), and its metabolism to DOPAC and HVA, occurred to the same degree as in the intact right side. In these DOPA‐treated rats, haloperidol (1.0 mg kg−1, i.p.) caused a further increase in DOPAC and HVA in the intact striatum, but not in the denervated striatum.

Under non‐stressful conditions, using a combination of anaesthetic treatments, electrical stimulation (400 μA, 0.4 ms, 15 Hz, 15 min) of the nigrostriatal tract did not increase DOPAC or HVA in the striatum on the stimulated side.

It is concluded (a) that there is a significant presynaptic, and/or local circuit mechanism capable of activating the synthesis and metabolism of dopamine in the isolated, striatal, dopaminergic nerve terminals. Furthermore, haloperidol can act directly on the striatal, dopaminergic nerve terminal, to cause an increase in the synthesis and metabolism of striatal dopamine. (b) After degeneration of the striatal dopaminergic nerves, the denervated striatum retains the ability to synthesize (from l‐DOPA) and metabolize dopamine, to the same degree as the intact, innervated, contralateral striatum. (c) When stress is minimized, and release of dopamine is induced by electrical stimulation of the medial forebrain bundle, the catabolism of dopamine (to DOPAC and HVA) during the release‐uptake cycle may not be a significant factor under physiological conditions. (d) When dopamine synthesis is increased in the striatum, the normal blood concentration of tyrosine is adequate to sustain the increased synthesis, and precursor availability is not a limiting factor. (e) These results suggest that some of the basic concepts about the neurochemical/neurophysiological regulation of monoaminergic neurones may require further re‐evaluation.
The actions of human atrial natriuretic factor on hepatic arterial and portal vascular beds of the anaesthetized dog
Authors: Withrington P.G. , Dhume V.G. , Croxton R. , Gerbes A.L.
Published: July 2012

The vascular actions of atrial natriuretic factor (ANF) have been assessed with other vasoactive agents on the hepatic arterial and portal vascular beds of the anaesthetized dog.

Intra‐arterial bolus injections of ANF (0.1–50 nmol) caused graded increases in hepatic arterial blood flow representing a vasodilatation of relatively short duration. Vasoconstriction was never observed.

The maximum increase in hepatic arterial blood was the same for ANF and isoprenaline (Iso) i.e. approximately 60–70% increase over control flow.

On a molar basis, ANF was less potent than Iso although over the higher dose range (10−9−10−7 mol) its vasodilator activity exceeded that of the endogenous vasodilator adrenaline.

Intraportal bolus injections (1.0–50 nmol) of ANF did not alter portal inflow resistance since no changes in portal inflow pressure occurred when the portal circuit was perfused at constant inflow volume.

This differential action of ANF on the hepatic arterial and portal vascular beds may provide a change in total liver blood flow in favour of the arterial component.

ANF, by altering hepatic haemodynamics to favour formation of trans‐sinusoidal fluid exchange, may provide a temporary expansion of the extravascular fluid reservoir to buffer any increased venous pressure. However, chronically elevated plasma levels of ANF would encourage the formation of ascitic fluid.
Acetylcholinesterase activity in regions of mouse brain following acute and chronic treatment with a benzodiazepine inverse agonist
Authors: Appleyard M.E. , Taylor S.C. , Little H.J.
Published: July 2012

Chronic administration of the benzodiazepine inverse agonist FG 7142 has previously been shown to induce seizure activity in mice. In the present study we have investigated the effects of acute and chronic treatment with FG 7142 in mice on the levels of acetylcholinesterase activity in cortex, hippocampus, midbrain and striatum. We have also investigated the effects of acute and chronic stress in the form of handling (vehicle‐injection) on acetylcholinesterase levels.

A single dose of FG 7142 produced a marked elevation of total acetylcholinesterase activities in the hippocampus and midbrain when compared with vehicle‐injected control levels, but the levels were not different from those in unhandled animals.

Acute stress, in the form of vehicle‐injection produced decreases in cortical and hippocampal soluble acetylcholinesterase activity but FG 7142 had no effect upon these stress‐induced changes.

Total cortical and hippocampal acetylcholinesterase activities were increased by 56% and 16% respectively in the chronic FG 7142‐treated mice that exhibited seizure activity (compared with vehicle‐injected controls).

Soluble acetylcholinesterase activity in the midbrain was decreased to 82% of control levels only in animals that had undergone FG 7142‐induced kindling. Smaller or no changes in acetylcholinesterase activity in the midbrain were observed in chronically FG 7142‐treated animals that exhibited no seizure activity.

Mice that did not demonstrate seizure activity in response to chronic FG 7142 treatment showed alterations in the soluble acetylcholinesterase activities of the hippocampus and midbrain.

It is concluded that chronic treatment with the benzodiazepine inverse agonist FG 7142 produces alterations in the acetylcholinesterase activities of various brain regions, in a manner related to the kindling that can be produced by this treatment.

Chronic mild stress, in the form of repeated handling (vehicle injection), induced changes in brain activity with decreases in total activity occurring in the cortex and hippocampus, and an increase in soluble acetylcholinesterase activity occurring in the midbrain.

All these stress‐induced changes appeared to be prevented by administration of FG 7142 at the time of the stress. It would appear therefore that FG 7142 can prevent the effects of chronic stress on brain acetylcholinesterase activity.
Suramin is a slowly‐equilibrating but competitive antagonist at P2x‐receptors in the rabbit isolated ear artery
Authors: Leff P. , Wood B.E. , O'Connor S.E.
Published: July 2012

The antagonist dynamics of suramin were investigated at P2x‐receptors in isolated rings of endothelium‐denuded ear artery from New Zealand White (NZW) rabbits.

α,β‐Methylene adenosine 5′‐triphosphate (ATP) concentration‐effect curves were constructed cumulatively in a paired curve design in the absence and presence of increasing concentrations of suramin, incubated for 45 min. The slope of the resulting Schild plot was significantly greater than unity (1.50 ± 0.08).

Assuming that slow equilibration by suramin explains the steep Schild plot, further experiments were conducted using short (15 min) and long (3 h) incubation times. The resulting Schild plot slopes were 1.66 ± 0.36 and 1.06 ± 0.13 respectively confirming the assumption. However, after 3 h incubation, suramin also caused depression of α,β‐methylene ATP curves.

In an attempt to minimize the depressant effect of suramin, a kinetic study was designed to calculate the minimum incubation times for each concentration of suramin used in the Schild analysis to achieve effectively complete equilibrium. Theoretically fractional occupancy for the antagonist is given by (r − 1)/r, where r is the dose‐ratio. A plot of (r − 1)/r against time allowed the apparent ‘on’ and ‘off’ rate constants to be calculated.

With the resulting rate constant estimates, an optimised antagonism study was carried out in which incubation times were chosen such that > 95% occupancy by suramin could be achieved without agonist curve depression at each concentration of suramin used.

Under these conditions, suramin fulfilled all criteria for simple competition: parallel rightward displacement of α,β‐methylene ATP curves and a Schild plot slope of unity (1.00 ± 0.09). The resulting pKB estimate was 4.79 ± 0.05. This estimate of affinity was shown to be independent of the agonist used in another experiment in which l‐β,γ‐methylene ATP was employed (pKB = 5.17).

Under the same conditions, suramin was found to have no effect on KCl‐induced contractions and only slight effects on phenylephrine‐ and histamine‐induced responses.

This analysis provides the first evidence that suramin is a genuine competitive P2x‐receptor antagonist.
The effects of 5‐HT on articular sensory receptors in normal and arthritic rats
Authors: Birrell G.J. , McQueen D.S. , Iggo A. , Grubb B.D.
Published: July 2012

The effects of intra arterial (i.a.) injections of 5‐hydroxytryptamine (5‐HT, 1–100 μg) on the discharge of (a) identified articular high threshold mechanoreceptors and (b) unidentified chemosensitive receptors in the ankle joint have been studied electrophysiologically in anaesthetized normal and arthritic rats. Recordings were made from a fine branch of the medial plantar nerve.

5‐HT increased the mechanical responsiveness of high threshold nociceptive mechanoreceptors with C and Aδ fibre afferents in both normal and adjuvant‐arthritic rats. Receptors in arthritic joints were more sensitive to 5‐HT than were those from normal joints.

5‐HT produced a complex response from both types of articular receptors following i.a. injection. Two separate components were identified: (a) a fast transient burst of activity was obtained within 10 s of this injection in 66% of units from normal animals and 45% from arthritics, followed by (b) a delayed slow longer‐lasting excitation seen in 62% of the units examined from normals and 77% of units from arthritic rats.

Increased mechanoreceptor resonsiveness produced by 5‐HT was reduced or abolished by the 5‐HT3 receptor antagonists studied (MDL 72222, ICS 205–930, or GR 38032F, in single doses of 100 μg kg−1, i.a.).

Fast excitation showed marked tachyphylaxis and was antagonized by MDL 72222, ICS 205–930 or GR 38032F. It was unaffected by ketanserin (100 μg kg−1, i.a.). Delayed excitation was reduced or abolished by ketanserin but was unaffected by the 5‐HT3‐receptor antagonists.

Administration of MDL 72222, ICS 205–930 or GR 38032F caused short lasting (< 5 min) reductions in background activity from both types of unit recorded in arthritic rats, as well as in normal rats in which activity had increased following administration of 5‐HT. Ketanserin caused similar reductions in background activity in chemosensitive units, but had no effect on mechanoreceptors.

At least two types of receptor are involved in the actions of 5‐HT on articular sensory receptors with fine afferent fibres. Increased mechano‐responsiveness involves a 5‐HT3‐receptor as does fast excitation. Delayed excitation probably involves a 5‐HT2‐receptor. Endogenous 5‐HT appears not to play a crucial role in sensitization of high threshold mechanoreceptors in this model of chronic inflammation and arthritis, although its local release may potentiate the actions of other inflammatory mediators on sensory receptors in the ankle joint.
Endothelin and a Ca2+ ionophore raise cyclic GMP levels in a neuronal cell line via formation of nitric oxide
Authors: Reiser Georg
Published: July 2012

The vasoconstrictor peptide endothelin‐1 caused a fast, transient rise in guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) levels in a neuronal cell line (mouse neuroblastoma x rat glioma hybrid cells 108CC15). The mechanism of activation of guanylate cyclase by endothelin‐1 was investigated. The endothelin‐1‐induced rise depended on the release of internal Ca2+.

The stimulation of cyclic GMP synthesis induced by endothelin‐1 was suppressed after preincubating the cells in medium containing haemoglobin (IC50 3 μm). Similarly, pretreatment of the cells with the l‐arginine analogues, l‐canavanine (IC50 60 μm) or NG‐monomethyl‐l‐arginine (IC50 2.5 μm), inhibited the cyclic GMP response to endothelin‐1. Therefore, endothelin‐1 activates guanylate cyclase most probably via formation of nitric oxide, which is released from l‐arginine.

The Ca2+ ionophore ionomycin induced a transient rise in cyclic GMP levels, which was also suppressed by preincubation in the presence of either haemoglobin or the l‐arginine analogues l‐canavanine or NG‐monomethyl‐l‐arginine. Therefore, we conclude that ionomycin can activate guanylate cyclase by a mechanism involving nitric oxide formation, similar to that induced by endothelin‐1.

The alkaloid veratridine, which activates Na+ channels and also causes influx of Ca2+ induced a transient rise of cyclic GMP levels in the neuronal cell line. This stimulation was blocked by pretreating the cells with l‐canavanine, NG‐monomethyl‐l‐arginine or haemoglobin.

Loading the cells with the Ca2+ chelator BAPTA suppresed the cyclic GMP response to application of endothelin‐1, ionomycin, or veratridine. Thus, in the neuronal cell line a rise in cytosolic Ca2+ activity seems to be sufficient to stimulate the nitric oxide forming enzyme which synthesizes the activator of soluble guanylate cyclase.
Electrophysiological and arrhythmogenic effects of platelet activating factor during normal perfusion, myocardial ischaemia and reperfusion in the guinea‐pig
Authors: Flores Nicholas A. , Sheridan Desmond J.
Published: July 2012

Platelet activating factor (PAF) is often used to study the effects of platelet activation. While direct myocardial electrophysiological effects of PAF have been described in superfused myocardial tissue, little is known about its actions on the whole heart.

The cellular electrophysiological and arrhythmogenic effects of PAF (10−11 m, 10−10 m and 10−9 m) were studied during normal perfusion, global myocardial ischaemia and reperfusion in Langendorff‐perfused guinea‐pig hearts at 32°C.

PAF (10−9 m) increased the incidence of ventricular fibrillation during ischaemia and reduced action potential duration (APD) during normal perfusion and early myocardial ischaemia (10−9 m and 10−10 m). PAF also reduced refractory period (RP) during normal perfusion (10−9 m) and early ischaemia (10−9 m and 10−10 m). PAF prevented recovery of APD (10−9 m) and RP (10−9 m and 10−10 m) during reperfusion. PAF at a concentration of 10−11 m had no electrophysiological effects.

PAF (10−9 m) increased the QRS width of the electrocardiogram during late ischaemia while 10−10 m PAF raised pacing threshold during late ischaemia.

Perfusion pressure was increased, and developed tension decreased by 10−9 m PAF.

These results demonstrate that PAF has direct myocardial electrophysiological effects in the whole heart which occur during normal perfusion and are capable of augmenting the effects of myocardial ischaemia, but are independent of the presence of platelets.
Bradykinin‐induced plasma exudation in guinea‐pig airways: involvement of platelet activating factor
Authors: Rogers Duncan F. , Dijk Sas , Barnes Peter J.
Published: July 2012

We studied the effect of bradykinin on plasma exudation in the airways of the anaesthetized guinea‐pig in vivo. Tissue content of extravasated Evans blue dye was used as an index of protein exudation in the larynx, trachea, main bronchi and intrapulmonary airways (i.p.a.).

Bradykinin increased the content of Evans blue in all tissues studied in a dose‐related manner. The response was greatest in the main bronchi and i.p.a., less in the trachea and least in the larynx. A dose of 47 nmol kg−1 was the lowest tested which caused significant (P > 0.001) plasma exudation with increases in leakage above control values of 256% in the larynx, 405% in the trachea, 394% in the main bronchi and 485% in intrapulmonary airways.

Leakage was significantly (P > 0.05) increased above control values by 1 min after bradykinin (47 nmol kg−1) in the main bronchi and intrapulmonary airways and was maximal in all airways 5 min after bradykinin. Although reduced by 15 min, the tissue content of dye was still significantly (P > 0.05) increased 2 h after bradykinin.

The prolonged tissue dye retention was due to a later phase of slow and maintained exudation preventing full clearance of dye after the initial response.

The initial phase of leakage was partially attenuated by the platelet activating factor (PAF) receptor antagonists WEB 2086 or BN 52021, by indomethacin or by inhibiting sensory nerve activation by opioid anaesthesia: it was not affected by mepyramine and cimetidine nor by the sulphidopeptide leukotriene receptor antagonists FPL 55712 or ICI 198,615. Adrenoceptor blockade of the anti‐leakage effects of endogenously‐released catecholamines significantly (P > 0.05) enhanced leakage.

The later phase of plasma leakage was completely inhibited by the PAF antagonists.

We conclude that, in guinea‐pig airways in vivo, the initial phase of bradykinin‐induced plasma exudation is mediated in part by PAF, sensory nerves and prostaglandins, whereas the later, prolonged phase of leakage is mediated exclusively by PAF. If bradykinin is generated in asthma, its potent and prolonged effects on plasma leakage may contribute significantly to airway oedema and may be involved in the development of bronchial hyperresponsiveness.
Effect of blockade of noradrenaline re‐uptake on evoked tritium overflow from mouse vasa deferentia and rat cortex slices
Authors: El‐Mas M. , Hughes I.E.
Published: July 2012

In tissues previously incubated with [3H]‐noradrenaline exposure to cocaine (0.1 to 10 μm) or desmethylimipramine (0.01 to 1 μm) produced a concentration‐dependent increase (up to 2 fold) in electrically evoked (3 Hz, 2 ms, 20 mA, 120 s every 20 min) fractional overflow of tritium from rat brain cortex slices but not from mouse vas deferens (2.5 Hz, 2 ms, 400 mA, for 90s every 14 min).

Yohimbine and idazoxan (0.01 to 1 μm) increased fractional evoked overflow of tritium by up to 2 fold; in the presence of these drugs, cocaine (10 μm) produced an increase in both tissues (up to 3.5 fold over control).

In brain slice an increase in stimulation frequency (0.1, 0.5, 1, 3 and 6 Hz) decreased fractional evoked overflow of tritium per pulse but cocaine (10 μm) produced a significant enhancement at each frequency except 6 Hz. In vas deferens fractional tritium overflow per pulse changed little with increasing frequency and cocaine produced no effect.

In both tissues fractional evoked overflow of tritium was dependent on the stimulation current; cocaine (10 μm) increased fractional evoked overflow from brain slice at every current tested but was without effect in vas deferens.

Chromatographic separation of the released tritium showed there was little difference in the proportions of [3H]‐noradrenaline and 3H‐metabolites overflowing from the tissues. Cocaine increased the proportion of [3H]‐noradrenaline and decreased the proportion of [3H]‐DOPEG overflowing both at rest and during stimulation.

In brain slice an increase in electrically evoked overflow was produced by cocaine (10 μm) whether total tritium overflow (1.8 fold), overflow of [3H]‐noradrenaline (1.8 fold) or overflow of unlabelled noradrenaline (1.8 fold) was measured. Evoked overflow from vas deferens was unaffected when assessed by any of these three methods.

The mechanism responsible for this differential effect of cocaine is unclear but may involve differences in the physical relationship between release sites, reuptake sites and presynaptic autoreceptors.
The booster injection of antigen during active sensitization of guinea‐pig modifies the anti‐anaphylactic activity of the PAF antagonist WEB 2086
Authors: Desquand S. , Lefort J. , Dumarey C. , Vargaftig B.B.
Published: July 2012

Serum IgG levels of sensitized guinea‐pigs bled at various times after the booster injection were evaluated and its capacity to sensitize passively lung strips from normal guinea‐pigs assessed. Following the booster injection, both serum IgG and the ability to sensitize passively lung strips increased during the first week and decreased slowly thereafter.

The PAF antagonist WEB 2086 (3 mg kg−1, i.v.) blocked the anaphylactic bronchoconstriction induced by intravenous administration of ovalbumin (1 mg kg−1) when guinea‐pigs were challenged 2 and 4 days after the booster injection, but became ineffective when tested in guinea‐pigs challenged 7, 28 and 56 days after the booster injection.

The ability of WEB 2086 to reduce anaphylactic bronchoconstriction of guinea‐pigs challenged 2 and 4 days after the booster injection was unrelated to either the selective involvement of one type of immunoglobulin, low IgG titres in sera or a reduced sensitizing capacity.

The booster injection, which accounts for the loss of efficacy of WEB 2086 from the fourth day thereafter, probably operates as a PAF‐independent inflammatory challenge.

The protocol for immunisation and the day of experiment after the booster injection determines the sensitivity of the anaphylactic bronchoconstriction to inhibition by PAF antagonists.
NaF and guanine nucleotides modulate adenylate cyclase activity in NG108‐15 cells by interacting with both Gs and Gi
Authors: Kelly Eamonn , Keen Mary , Nobbs Peter , MacDermot John
Published: July 2012

NaF (10 mm) produced a 2–3 fold increase in adenylate cyclase activity in homogenates of NG108‐15 cells incubated in the presence of 1 μm GTP. Higher concentrations of NaF suppressed adenylate cyclase activity.

In the presence of the adenosine receptor agonist 5′‐(N‐ethyl)‐carboxamidoadenosine (NECA; 100 μm) or the prostacyclin receptor agonist iloprost (10 nm), NaF produced a much smaller increase in adenylate cyclase activity, whereas in the presence of a saturating concentration of iloprost (1 μm), NaF only inhibited adenylate cyclase activity.

Similarly, Gpp(NH)p activated basal adenylate cyclase activity, and inhibited 1 μm iloprost‐activated enzyme activity. In the presence of 10 μm forskolin, NaF or Gpp(NH)p increased adenylate cyclase activity synergistically. Analysis of concentration‐effect curves indicated that NaF (2 mm) or Gpp(NH)p (100 μm) increased the potency with which forskolin activated adenylate cyclase, whilst reducing the maximum activation of adenylate cyclase by iloprost.

Opiate receptors mediate inhibition of adenylate cyclase, and the opiate agonist morphine (100 μm) reduced the capacity of NaF or Gpp(NH)p to inhibit iloprost‐activated adenylate cyclase. Unexpectedly, pertussis toxin treatment enhanced the ability of NaF or Gpp(NH)p to inhibit iloprost‐activated adenylate cyclase.

In the absence of GTP, NaF and Gpp(NH)p remained able both to activate basal adenylate cyclase and to be synergistic with forskolin in activating the enzyme. In contrast the ability of NaF and Gpp(NH)p to inhibit iloprost‐activated adenylate cyclase was substantially lost in the absence of added GTP.

These results suggest that NaF modulates adenylate cyclase activity in NG108‐15 cell membranes by interacting with the α subunits of both Gs and Gi regulatory proteins. The effects of NaF and Gpp(NH)p are critically dependent on the prior mode and extent of activation or inhibition of this transmembrane signalling pathway. This simple system may be of use in assessing alterations in Gs‐Gi interaction following manipulations such as hormone receptor desensitization.
Evaluation of the bronchodilator properties of Ro 31–6930, a novel potassium channel opener, in the guinea‐pig
Authors: Paciorek P.M. , Cowlrick I.S. , Perkins R.S. , Taylor J.C. , Wilkinson G.F. , Waterfall J. F.
Published: July 2012

Ro 31–6930 (0.001‐0.3 μm), cromakalim (0.03‐3.0 μm), salbutamol (0.001‐0.3 μm) and theophylline (0.3–100 μm) evoked dose‐related reductions in guinea‐pig spontaneous tracheal tone with IC50 values of 0.044, 0.20, 0.021 and 21.0 μm respectively. All four agents also relaxed tone supported by betahistine, carbachol, 5‐hydroxytryptamine (5‐HT), leukotriene D4 (LTD4), U46619 and prostaglandin D2 (PGD2). The order of potency of tracheal relaxants was always salbutamol > Ro 31–6930 > cromakalim > theophylline.

All four agents evoked dose‐related reductions in 5‐HT‐ and histamine‐induced bronchoconstriction in pithed vagotomised guinea‐pigs. The dose of Ro 31–6930 producing 50% inhibition of a 5‐HT bronchoconstriction was 11.6 μg kg−1 and the dose producing 50% inhibition of a histamine bronchoconstriction was 4.4 μg kg−1. Salbutamol was approximately 4–5 times more potent than Ro 31–6930 whilst cromakalim was approximately 10 times less potent than Ro 31–6930 as a bronchodilator. Theophylline was markedly less potent than any of the other agents.

Ro 31–6930, cromakalim, salbutamol and theophylline each protected conscious guinea‐pigs from histamine‐induced respiratory distress. Ro 31–6930 and salbutamol were each effective at oral doses of 1.0 and 3.0 mg kg−1 whilst cromakalim was effective at oral doses of 3.0 and 10.0 mg kg−1. Theophylline showed activity only at 300 mg kg−1 p.o.

Ro 31–6930 is a novel potassium channel opener which is a potent relaxant of guinea‐pig tracheal smooth muscle in vitro and a bronchodilator in vivo.