bluuebunny/crossref_metadata_2025 huggingface dataset:
Depression of A and C fibre‐evoked segmental reflexes by morphine and clonidine in the in vitro spinal cord of the neonatal rat
Authors: Faber E. S. L. , Chambers J. P. , Brugger F. , Evans R. H.
Published: August 2006

Population synaptic responses of motoneurones were recorded from a ventral root following electrical stimulation of the corresponding lumbar dorsal root in neonatal rat hemisected spinal cord preparations in vitro. Two levels of electrical stimulation were used to elicit dorsal root compound action potentials that contained either an A fibre component alone or both A and C fibre components. The effects of centrally acting analgesics and an N‐methyl‐d‐aspartate (NMDA) receptor antagonist were tested on synaptic responses produced by these two levels of stimulation.

At stimulus intensities below four times threshold (T) there was no C fibre component in the dorsal root compound action potential. Responses to a single pulse at 3T (the low intensity excitatory postsynaptic potential (e.p.s.p.)), a train of five pulses at 2T (the train e.p.s.p.) and a single supramaximal pulse (the high intensity e.p.s.p.) were used to compare the depressant actions of morphine, clonidine and the competitive NMDA antagonist CGP40116 (d‐(E)‐2‐amino‐4‐methyl‐5‐phosphono‐pentenoic acid). The train e.p.s.p. (mean half‐time to decay 5±0.6 s, n=6) had a similar profile to the high intensity e.p.s.p. (mean half‐time to decay 6.8±0.7, n=8).

The monosynaptic compound action potential of motoneurones (MSR) was resistant to all three drugs irrespective of the intensity of dorsal root stimulation. The low intensity e.p.s.p., the train e.p.s.p. and the high intensity e.p.s.p. were depressed by all three drugs. The EC50 values for depression by morphine were 79±1 nm (n=8) for the high intensity e.p.s.p. and 99±1 nm (n=4) for the low intensity e.p.s.p. The corresponding values for clonidine were 25±1 nm (n=8) and 9±1 nm (n=4) and those for CGP40116 were 860±1.3 nm (n=4) and 76±1.1 nm (n=4).

The depressant profile of the NMDA antagonist, having the least depressant activity on the C fibre‐mediated response, was different from that of the two analgesics. CGP40116 (3 μm) depressed the high intensity e.p.s.p. to 62±8%, the low intensity e.p.s.p. to 22±4% and the train e.p.s.p. to 16±2% of control values.

The depressant actions of morphine were fully reversed by naloxone (1 μm) and those of clonidine were fully reversed by atipamezole (1 μm).

These results show that, in contrast to previous findings, activation of primary afferent C fibres in dorsal roots is not required for generation of morphine‐ or clonidine‐sensitive synaptic responses in ventral roots of this in vitro preparation.

British Journal of Pharmacology (1997) 120, 1390–1396; doi:10.1038/sj.bjp.0701064
Nitric oxide synthase and cyclo‐oxygenase pathways in the inflammatory response induced by zymosan in the rat air pouch
Authors: PayÁ Miguel , GarcÍA PASTOR Pablo , Coloma Julio , JosÉ ALCARAZ M.
Published: August 2006

We have studied the participation of nitric oxide (NO) in an animal model of inflammation, the rat air pouch stimulated with zymosan.

Saline or zymosan was injected into 6‐day rat air pouches at different time points and measurements were made of cell migration, levels of nitrite/nitrate (NO2−/NO3−), prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and secretory phospholipase A2 (sPLA2) in exudates. Nitric oxide synthase (NOS) activity was determined in high speed supernatants from cells present in pouch exudates. Western blot analysis was also performed on these samples.

Zymosan injection induced a time‐dependent increase in leukocyte infiltration, NO2−/NO3− levels and cellular NOS activity that reached a peak by 8 h. Western blot analysis showed the same time course for induction of NOS protein. Colchicine administration to rats inhibited cellular infiltration and decreased the levels of NO metabolites and cellular NOS activity zymosan‐injected air pouch at 8 h. NOS activity was present in polymorphonuclear leukocytes (PMNs) and monocytes, but not in the lymphocytes present in exudates. This enzyme is calcium‐independent and needs NADPH for activity. PGE2 levels in exudates showed a time course inverse to that of NOS activity and NO metabolites, with maximum levels of PGE2 observed at 4 h after zymosan injection.

Administration of NG‐nitro‐l‐arginine methyl ester (l‐NAME) or aminoguanidine to rats significantly reduced cellular NOS activity, NO2−/NO3− levels and chemiluminescence, whereas they were without effect on cell migration and degranulation, eicosanoid levels and sPLA2 activity.

Treatment of animals with dexamethasone inhibited cellular NOS activity, NO2−/NO3− levels, chemiluminescence and the increase in the levels of PGE2 and LTB4, with only a weak effect on elastase release.

Administration of the selective cyclo‐oxygenase‐2 (COX‐2) inhibitor NS398 to rats strongly reduced PGE2 levels in exudates without affecting NO metabolites or NOS activity at 4 h after zymosan injection.

Our data indicate that NOS is induced in the zymosan‐stimulated rat air pouch model of inflammation. This enzyme is expressed in the cells migrating into the air pouch and caused an increased production of NO metabolites in exudates. The results also suggest the presence of an earlier phase in which eicosanoids play the main role, with participation of COX‐2 activity, and a later phase mediated by NO. The endogenous release of NO does not modify prostaglandin biosynthesis in this in vivo model.

British Journal of Pharmacology (1997) 120, 1445–1452; doi:10.1038/sj.bjp.0701073
Differential effects of diadenosine phosphates on purinoceptors in the rat isolated perfused kidney
Authors: Van Der Giet Markus , Khattab Mohammed , Börgel Jan , Schlüter Hartmut , Zidek Walter
Published: August 2006

The activation of various purinoceptors in rat renal vasculature by P1,P2‐diadenosine pyrophosphate (Ap2A), P1,P3‐diadenosine triphosphate (Ap3A), P1,P4‐diadenosine tetraphosphate (Ap4A), P1,P5‐diadenosine pentaphosphate (Ap5A), P1,P6‐diadenosine hexaphosphate (Ap6A) was studied by measuring their effects of perfusion pressure of a rat isolated perfused kidney.

The vasoconstrictive response to Ap5A was completely due to P2X purinoceptor activation, that to Ap4A and Ap6 was P2X purinoceptor mediated to a large extent, as evidenced by the inhibitory effects of suramin and pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid tetrasodium (PPADS).

The vasoconstrictive effects of Ap2A and Ap3A were mostly due to stimulation of A1‐receptors, as shown by the inhibitory effect of 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX).

The vasoconstrictive response to Ap6A was partially insensitive to A1 and P2X purinoceptor blockers.

In raised tone preparations Ap2A and Ap3A evoked vasodilatation, which was blocked by the A2 receptor blocker, 3,7‐dimethyl‐1‐propargylxanthine (DMPX).

In raised tone preparations Ap4A evoked vasodilatation when the P2‐purinoceptors were blocked by suramin.

The activation of different purinoceptor subtypes by diadenosine phosphates critically depends on the number of phosphate groups.

British Journal of Pharmacology (1997) 120, 1453–1460; doi:10.1038/sj.bjp.0701074
Regulation of the resting potential of rabbit pulmonary artery myocytes by a low threshold, O2‐sensing potassium current*
Authors: Osipenko Oleg N. , Mark Evans A. , Gurney Alison M.
Published: August 2006

The contributions of specific K+ currents to the resting membrane potential of rabbit isolated, pulmonary artery myocytes, and their modulation by hypoxia, were investigated by use of the whole‐cell, patch‐clamp technique.

In the presence of 10 μm glibenclamide the resting potential (−50±4 mV, n=18) was unaffected by 10 μm tetraethylammonium ions, 200 nm charybdotoxin, 200 nm iberiotoxin, 100 μm ouabain or 100 μm digitoxin. The negative potential was therefore maintained without ATP‐sensitive (KATP) or large conductance Ca2+‐sensitive (BKCa) K channels, and without the Na+‐K+ATPase.

The resting potential, the delayed rectifier current (IK(V)) and the A‐like K+ current (IK(A)) were all reduced in a concentration‐dependent manner by 4‐aminopyridine (4‐AP) and by quinine.

4‐AP was equally potent at reducing the resting potential and IK(V), 10 mm causing depolarization from −44 mV to −22 mV with accompanying inhibition of IK(V) by 56% and IK(A) by 79%. In marked contrast, the effects of quinine on resting potential were poorly correlated with its effects on both IK(A) and IK(V). At 10 mm, quinine reduced IK(V) and IK(A) by 47% and 38%, respectively, with no change in the resting potential. At 100 μm, both currents were almost abolished while the resting potential was reduced <50%. Raising the concentration to 1 mm had little further effect on IK(A) or IK(V), but essentially abolished the resting potential.

Reduction of the resting potential by quinine was correlated with inhibition of a voltage‐gated, low threshold, non‐inactivating K+ current, IK(N). Thus, 100 μm quinine reduced both IK(N) and the resting potential by around 50%.

The resting membrane potential was the same whether measured after clamping the cell at −80 mV, or immediately after a prolonged period of depolarization at 0 mV, which inactivated IK(A) and IK(V), but not IK(N).

When exposed to a hypoxic solution, the O2 tension near the cell fell from 125±6 to 14±2 mmHg (n=20), resulting in a slow depolarization of the myocyte membrane to −35±3 mV (n=16). The depolarization occurred without a change in the amplitude of IK(V) or IK(A), but it was accompanied by 60% inhibition of IK(N) at 0 mV.

Our findings suggest that the resting potential of rabbit pulmonary artery myocytes depends on IK(N), and that inhibition of IK(N) may mediate the depolarization induced by hypoxia.

British Journal of Pharmacology (1997) 120, 1461–1470; doi:10.1038/sj.bjp.0701075
Ocular inflammation induced by electroconvulsive treatment: contribution of nitric oxide and neuropeptides mobilized from C‐fibres
Authors: Wang Zun‐Yi , Waldeck Kristian , Grundemar Lars , Håkanson Rolf
Published: August 2006

Electroconvulsive treatment (ECT) of rabbits produced ocular inflammation consisting of conjunctival hyperaemia, miosis and protein extravasation into the aqueous humour, reflected by the so‐called aqueous flare response (AFR); the maximal reduction in pupil size was 3.8±0.1 mm (s.e. of mean, n=16) while the maximal AFR was 28.1±2.8 (arbitrary units).

ECT also caused release of substance P (SP), pituitary adenylate cyclase‐activating peptide (PACAP)‐27, ‐38 and calcitonin gene‐related peptide (CGRP). The concentrations of SP and CGRP in the aqueous humour of normal, untreated eyes were 10.6±1.4 and 117.4±12.4 pmol l−1, respectively, while the concentrations of PACAP‐27 and ‐38 were below the detection limit. After ECT the concentrations of SP, PACAP‐27, ‐38 and CGRP were 65.0±9.6, 46.9±8.4, 50.2±5.4 and 1109.9±133.1 pmol l−1, respectively (s.e. of mean, n=12). Conceivably, ECT evoked an antidromic activation of sensory neurones in the trigeminal ganglion with the consequent release of neuropeptides from C‐fibres in the uvea and the development of neurogenic inflammation.

Rabbits received the nitric oxide (NO) synthase inhibitor, NG‐nitro‐l‐arginine methyl ester (l‐NAME, 200 mg kg−1, i.v.). This pretreatment inhibited the ECT‐evoked conjunctival hyperaemia, miosis and AFR; under these circumstances the maximal reduction in pupil size was 1.9±0.1 mm while the maximal AFR was 2.7±0.9 (n=16). l‐NAME also inhibited the ECT‐evoked release of SP, PACAP‐27, ‐38 and CGRP into the aqueous humour; the concentrations of SP and CGRP were 13.2±1.5 and 204.8±33.5 pmol l−1, respectively, while PACAP‐27 and ‐38 were below the detection limit (n=12).

The ECT‐evoked miosis was also inhibited by pretreatment with the tachykinin receptor antagonist d‐Pal9 spantide II (90 nmol, intravitreal injection); under these circumstances the maximal reduction in pupil size was only 0.7±0.03 mm, indicating an important role for SP in the miotic response. Pretreatment of the eye with capsaicin, which is known to cause functional ablation of C‐fibres, inhibited the conjunctival hyperaemia, miosis and AFR by 4050%; the maximal reduction in pupil size being 2.2±0.2 mm and the maximal AFR 13.8±2.1 (arbitrary units) (n=8).

The results suggest (1) that ECT evokes ocular inflammation through antidromic C‐fibre activation; (2) that SP contributes to the ECT‐evoked miosis; and (3) that NO contributes to the antidromic C‐fibre activation and possibly to the vascular responses mediated by the C‐fibre transmitters.

British Journal of Pharmacology (1997) 120, 1491–1495; doi:10.1038/sj.bjp.0701083
The in vitro pulmonary vascular effects of FK409 (nitric oxide donor): a study in normotensive and pulmonary hypertensive rats
Authors: Wanstall Janet C , Kaye Jacqueline A , Gambino Agatha
Published: August 2006

Vasorelaxant responses to the nitric oxide (NO) donor, FK409 ((±)‐(E)‐4‐ethyl‐2‐[(E)‐hydroxyimino]‐5‐nitro‐3‐hexenamide), were evaluated on precontracted isolated ring preparations of main pulmonary artery and intralobar pulmonary artery from rats.

On main pulmonary artery FK409 fully reversed the precontractions. Responses were inhibited by methylene blue but were independent of the endothelium. The potency (−log EC50) of FK409 was the same on preparations contracted with noradrenaline (7.62) or the thromboxane‐mimetic, U44619 (7.63).

On intralobar pulmonary artery FK409 caused only 80% reversal of the precontraction and was 2 fold less potent than on main pulmonary artery. These differences in maximum response and potency between main and intralobar arteries are in keeping with previous findings with other NO donors.

Pulmonary hypertension was induced in rats by chronic exposure to hypoxia (10% O2) for 1 or 4 weeks. Main pulmonary arteries from 1 week hypoxic rats had inherent tone and showed spontaneous contractile activity. In these arteries FK409 reversed not only the precontraction induced by noradrenaline but also the inherent tone. However, FK409 was 17 fold less potent than in control arteries, reflecting previous findings with other NO donors. Main pulmonary arteries from 4 week hypoxic rats had minimal inherent tone and were quiescent and FK409 was 4.5 fold less potent than in control arteries. In intralobar pulmonary arteries from 4 week hypoxic rats FK409 was 12 fold less potent than in controls.

Treatment of arteries with either (a) in vitro hypoxic conditions (PO2 of solution in organ bath <10 mmHg) or (b) superoxide dismutase (SOD; 150 u ml−1) together with catalase (1200 u ml−1) significantly increased the potency of FK409 in preparations from hypoxic rats but had no effect on the potency in control preparations. Neither SOD nor catalase, alone, nor the nitric oxide synthase inhibitor, NG‐nitro‐L‐arginine methyl ester, had any effect on the potency of FK409 in preparations from control or hypoxic rats.

It is concluded that the reduction in potency of FK409 seen in pulmonary arteries from rats with chronic hypoxic pulmonary hypertension may be due in part to the presence of one or more reactive oxygen species (either hydroxyl or superoxide plus hydrogen peroxide).

British Journal of Pharmacology (1997) 121, 280–286; doi:10.1038/sj.bjp.0701105
Central involvement of kinin B1 and B2 receptors in the febrile response induced by endotoxin in rats
Authors: Coelho M M , Oliveira C R , Pajolla G P , Calixto J B , Pelá I R
Published: August 2006

The effect of central injection of selective kinin B1 and B2 receptor antagonists on the febrile response induced by endotoxin (E. coli lipopolysaccharide, LPS) in rats was investigated.

Intracerebroventricular (i.c.v.) injection of a selective B2 receptor antagonist (Hoe‐140, 8 nmol) reduced the early (0–2 h), but increased the late phase (4–6 h) of the febrile response induced by intravenous (i.v.) injection of LPS (0.5 μg kg−1).

Co‐administration of Hoe‐140 (8 nmol, i.c.v.) with LPS (0.5 μg kg−1, i.v.), followed 2.5 h later by the i.c.v. injection of a selective B1 receptor antagonist [des‐Arg9‐Leu8]‐bradykinin (BK, 8 nmol), significantly reduced the febrile response induced by LPS throughout the whole experimental period.

Intravenous injection of Hoe‐140 (1 mg kg−1) significantly reduced the febrile response induced by LPS (0.5 μg kg−1, i.p.).

Pretreatment (24 h) with LPS (0.5 μg kg−1, i.v.) reduced the febrile response induced by BK or [Tyr8]‐BK (both, 5 nmol, i.c.v.), but markedly increased the febrile response induced by [des‐Arg9]‐BK (5 nmol, i.c.v.). The response induced by [des‐Arg9]‐BK in LPS‐pretreated rats was significantly inhibited by co‐injection of [des‐Arg9‐Leu8]‐BK (15 nmol, i.c.v.).

The results suggest that kinins are involved in the induction of LPS‐induced fever and that central B2 and B1 receptors are activated during the initial and late phase of this response, respectively. The results also suggest that downregulation and/or desensitization of B2 receptors and induction and/or upregulation of B1 receptors in LPS‐pretreated animals may have a significant pathophysiologcal role in the induction and maintenance of fever. These observations may be specifically important in the case of chronic inflammatory conditions, because the BK metabolite [des‐Arg9]‐BK, so far considered an inactive metabolite, acquires an active and relevant role with the progressive expression of B1 receptors that occurs in such states.

British Journal of Pharmacology (1997) 121, 296–302; doi:10.1038/sj.bjp.0701110
Effects of phosphodiesterase inhibitors on human lung mast cell and basophil function
Authors: Weston Marie C , Anderson Nicola , Peachell Peter T
Published: August 2006

The non‐hydrolysable cyclic AMP analogue, dibutyryl (Bu2)‐cyclic AMP, inhibited the stimulated release of histamine from both basophils and human lung mast cells (HLMC) in a dose‐dependent manner. The concentrations required to inhibit histamine release by 50% (IC50) were 0.8 and 0.7 mM in basophils and HLMC, respectively. The cyclic GMP analogue, Bu2‐cyclic GMP, was ineffective as an inhibitor of histamine release in basophils and HLMC.

The non‐selective phosphodiesterase (PDE) inhibitors, theophylline and isobutyl‐methylxanthine (IBMX) inhibited the IgE‐mediated release of histamine from both human basophils and HLMC in a dose‐dependent fashion. IBMX and theophylline were more potent inhibitors in basophils than HLMC. IC50 values for the inhibition of histamine release were, 0.05 and 0.2 mM for IBMX and theophylline, respectively, in basophils and 0.25 and 1.2 mM for IBMX and theophylline in HLMC.

The PDE 4 inhibitor, rolipram, attenuated the release of both histamine and the generation of sulphopeptidoleukotrienes (sLT) from activated basophils at sub‐micromolar concentrations but was ineffective at inhibiting the release of histamine and the generation of both sLT and prostaglandin D2 (PGD2) in HLMC. Additional PDE 4 inhibitors, denbufylline, Ro 20–1724, RP 73401 and nitraquazone, were all found to be effective inhibitors of mediator release in basophils but were ineffective in HLMC unless high concentrations (1 mM) were employed.

Neither 8‐methoxymethyl IBMX (PDE 1 inhibitor), zaprinast (PDE 5 inhibitor) nor a range of PDE 3 inhibitors (siguazodan, SKF 94120, SKF 95654) were effective inhibitors of mediator release from either basophils or HLMC.

In basophils, rolipram acted to potentiate the inhibitory effects of the adenylate cyclase activator, forskolin, whereas in HLMC, rolipram failed to potentiate the inhibitory effects of forskolin.

Extracts of purified HLMC and basophils hydrolysed cyclic AMP. IBMX (100 μM) inhibited the PDE activity in basophil extracts by 67±7% (P<0.0001) and in HLMC extracts by 63±9% (P<0.0005). The hydrolysis of cyclic AMP by basophil extracts was inhibited by the selective PDE inhibitors (all at 10 μM), rolipram (56±8%, P<0.0001) and the mixed PDE 3/4 inhibitor, Org 30029 (47±9%, P<0.01), whereas 8‐methoxymethyl IBMX, siguazodan and zaprinast were ineffective. In HLMC, rolipram, Org 30029, 8‐methoxymethyl IBMX, siguazodan and zaprinast all inhibited the hydrolysis of cyclic AMP by extracts to a significant (P<0.05) and similar extent (approximately 25% inhibition at 10 μM).

In total, these data suggest that modulation of the PDE 4 isoform can regulate basophil responses whereas an association of the PDE 4 isoform with the regulation of HLMC function remains uncertain.

British Journal of Pharmacology (1997) 121, 287–295; doi:10.1038/sj.bjp.0701115
A site‐directed mutagenesis study on the conserved alanine residue in the distal third intracellular loops of cholecystokininB and neurotensin receptors
Authors: Wang Hung‐Li
Published: August 2006

An alanine residue at the C‐terminal tail of the third intracellular loop is highly conserved among various Gq protein‐coupled receptors including rat cholecystokininB (CCKB) and neurotensin receptors. To investigate the functional significance of the conserved alanine in the activation of Gq proteins and phospholipase C (PLC) by CCKB and neurotensin receptors, the alanine residue was mutated in the present study. Subsequently, the ability of resulting mutant receptors to activate PLC was investigated by measuring the formation of inositol phosphates (IP) in COS‐7 cells and recording Ca2+‐activated chloride currents from Xenopus oocytes.

Site‐directed mutagenesis was performed to mutate alanine at position 332 of rat CCKB receptor to glutamate. When the (A332E) mutant receptor was expressed in COS‐7 cells and Xenopus oocytes, the efficacy and the potency of sulphated cholecystokinin octapeptide (CCK‐8) to stimulate polyphosphoinositide hydrolysis in COS‐7 cells and evoke calcium‐dependent Cl− currents in oocytes were not significantly affected.

Alanine residue at position 302 of rat neurotensin receptor was also mutated to glutamate. When expressed in COS‐7 cells and Xenopus oocytes, the resulting (A302E) mutant receptor was strongly defective in stimulating phosphatidylinositol turnover in COS‐7 cells and evoking Ca2+‐dependent chloride currents in oocytes.

In summary, the present study demonstrates that alanine residue at the C‐terminus of third cytoplasmic domain is required for the full activation of Gq proteins and PLC by neurotensin receptors. However, in contrast to other Gq protein‐coupled receptors, alanine at the distal third intracellular loop does not play a significant role in CCKB receptor activation of PLC.

British Journal of Pharmacology (1997) 121, 310–316; doi:10.1038/sj.bjp.0701117
NO‐dependent and NO‐independent IL‐1 production by a human colonic epithelial cell line under inflammatory stress
Authors: Vallette Geneviève , Jarry Anne , Lemarre Philippe , Branka Jean‐Eric , Laboisse Christian L
Published: August 2006

The present study was designed to investigate, in an in vitro model of the human intestinal barrier, the ability of epithelial cells to produce interleukin‐1 (IL‐1), the cellular mechanisms involved in IL‐1 release, and the intracellular signalling pathways involved in IL‐1 up‐regulation during inflammatory stress.

This study was based on the human colonic epithelial cell line HT29‐Cl.16E, maintained as polarized monolayers on filters mounted in culture chambers and treated with various proinflammatory cytokines (interferon γ (IFNγ), tumour necrosis factor α (TNFα), IL‐1β) alone or in combination.

IL‐1 production, restricted to IL‐1α, was induced by the combination of IFNγ/TNFα. When IL‐1β was added to IFNγ/TNFα, it led to an additional production of IL‐1α. IL‐1α release was associated with cell damage, as shown by the correlation between lactate dehydrogenase (LDH) release and extracellular IL‐1 production, and was not accounted for by a secretory mechanism.

Both IFNγ/TNFα and IFNγ/TNFα/IL‐1β induced inducible nitric oxide synthase (iNOS) expression as shown by quantitation of NO2−/NO3− by use of the Griess reagent, quantitation of cells scoring positive with an anti‐iNOS antibody and detection of mRNAs coding for iNOS by RT–PCR. The use of NG‐monomethyl‐L‐arginine (L‐NMMA), an inhibitor of NOS, led to the demonstration of two distinct signalling pathways in IL‐1 production by HT29‐Cl.16E cells, one dependent on NO (L‐NMMA‐sensitive) under treatment with IFNγ/TNFα/IL‐1β, and the other independent of NO (L‐NMMA‐insensitive) under treatment with IFNγ/TNFα.

Moreover, we examined whether a redox‐based mechanism could be responsible for the apparent discrepancy between NO production and NO implication in IL‐1 production under IFNγ/TNFα and IFNγ/TNFα/IL‐1β treatments. Experiments with cysteine, which acts as a powerful reductant, suggest that the nitrosonium character of NO is involved in the NO‐dependent pathway in IL‐1 production.

British Journal of Pharmacology (1997) 121, 187–192; doi:10.1038/sj.bjp.0701118
Effect of L‐dopa alone and with benserazide on the spontaneous activity of striatal neurones in normal and 6‐hydroxydopamine‐lesioned rats
Authors: Chang W Y , Webster R A
Published: August 2006

The effects of L‐dopa methylester (LDME), an analogue of levodopa, on the spontaneous activity of dopamine sensitive neurones in the rat striatum, after 6‐hydroxydopamine induced degeneration of the nigrostriatal tract were compared with those in unlesioned animals both in the absence and presence of benserazide, a peripheral DOPA decarboxylase inhibitor (PDI).

Studies were performed at 5–7 days post lesion (group 1 animals), at 21 days (group 2) when denervation supersensitivity was evident by contralateral turning to apomorphine and at the same time but following 7 days dosing with LDME plus benserazide (group 3).

In unlesioned animals, LDME alone inhibited spontaneous firing by some 45% over 60 min including a marked but transient early phase which was still present in all lesioned animals even though the later inhibition was significantly reduced in group 1 and 3 animals.

When given after benserazide in unlesioned animals LDME still produced a similar level of overall inhibition but without the early phase. The lesion reduced the overall inhibition, except in group 2 animals, and after chronic dosing (group 3) it was almost absent.

It is proposed that since the early inhibition with LDME alone is still seen after lesion of the nigrostriatal tract but not after the PDI benserazide, it is caused by peripherally formed dopamine and that as the delayed inhibition with LDME alone and after benserazide are all reduced by nigrostriatal lesions, as is its amphetamine like ipsilateral turning, that this depends on locally (striatal) synthesized dopamine.

This study also shows that chronic levodopa/PDI treatment reduces the compensating increased activity of surviving dopaminergic neurones and the functional supersensitivity to dopamine suggests that the long term administration of levodopa may reduce its own utilization and activity in the striatum and in the treatment of Parkinson's Disease.

British Journal of Pharmacology (1997) 121, 331–337; doi:10.1038/sj.bjp.0701133
An examination of deoxyadenosine 5′(α‐thio)triphosphate as a ligand to define P2Y receptors and its selectivity as a low potency partial agonist of the P2Y1 receptor
Authors: Schachter Joel B , Kendall Harden T
Published: August 2006

The functional activity of deoxyadenosine 5′(α‐thio)triphosphate (dATPαS) was assessed at the cloned human P2Y1 receptor stably expressed in 1321N1 human astrocytoma cells and transiently expressed in Cos‐7 cells.

Cells expressing the receptor responded to adenine nucleotides with an increase in [3H]‐inositol phosphate accumulation. Half‐maximal responses were obtained at approximately 30 nM for 2‐methylthioadenosine‐5′‐triphosphate (2MeSATP), 300 nM for dATPαS, and 1000 nM for adenosine 5′‐triphosphate (ATP). dATPαS produced a maximal response that was only 37±4% of that produced by ATP or 2MeSATP. dATPαS also competitively antagonized the phospholipase C response to 2MeSATP with a KB of 644±14 nM. Thus dATPαS acts as a low potency partial agonist at P2Y1 receptors.

The selectivity of dATPαS for P2Y1 receptors was determined by examining its capacity to activate P2Y2, P2Y4 and P2Y6 receptors also stably expressed in 1321N1 cells. Although dATPαS was a partial agonist at P2Y1 receptors it was a full agonist at P2Y2 receptors, albeit with a potency that was two orders of magnitude lower than at P2Y1 receptors. No agonist or antagonist activity was observed at P2Y4 and P2Y6 receptors.

Although [35S]‐dATPαS bound to a relatively high density (ca 10 pmol mg−1 protein) of binding sites in membranes from 1321N1 or Cos‐7 cells expressing the P2Y1 receptor, no difference in the total density of sites was observed between membranes from wild‐type, empty vector‐transfected, or P2Y1 receptor‐expressing cells. Moreover, adenine nucleotide analogues inhibited [35S]‐dATPαS binding with an order of potency that differed markedly from that for the accumulation of inositol phosphates in intact transfected P2Y1 receptor‐expressing cells. Saturation binding experiments demonstrated multiple affinity states for [35S]‐dATPαS binding in wild‐type Cos‐7 cell membranes. These data from 1321N1 and Cos‐7 cells suggest that cellular membranes exhibit a large number of high affinity binding sites for [35S]‐dATPαS that are not related to P2Y receptor subtypes.

British Journal of Pharmacology (1997) 121, 338–344; doi:10.1038/sj.bjp.0701136
Induction by low Na+ or Cl− of cocaine sensitive carrier‐mediated efflux of amines from cells transfected with the cloned human catecholamine transporters†
Authors: Pifl Christian , Agneter Ernst , Drobny Helmut , Reither Harald , Singer Ernst A
Published: August 2006

COS‐7 cells transfected with the cDNA of the human dopamine transporter (DAT cells) or the human noradrenaline transporter (NAT cells) were loaded with [3H]‐dopamine or [3H]‐noradrenaline and superfused with buffers of different ionic composition.

In DAT cells lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H‐efflux. Cocaine (10 μM) or mazindol (0.3 μM) blocked the efflux at low Na+, but not at 0 Na+. Lowering the Cl− concentration to 0, 5 or 10 mM resulted in an increased efflux, which was blocked by cocaine or mazindol. Desipramine (0.1 μM) was without effect in all the conditions tested.

In NAT cells, lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H‐efflux, which was blocked by cocaine or mazindol. Desipramine produced a partial block, its action being stronger at 5 or 10 mM Na+ than at 0 mM Na+. Efflux induced by 0, 5 or 10 mM Cl− was completely blocked by all three uptake inhibitors.

In cross‐loading experiments, 5 mM Na+‐ or 0 Cl−‐induced efflux was much lower from [3H]‐noradrenaline‐loaded DAT, than NAT cells and was sensitive to mazindol, but not to desipramine. Efflux from [3H]‐dopamine‐loaded NAT cells elicited by 5 mM Na+ or 0 Cl− was blocked by mazindol, as well as by desipramine.

Thus, cloned catecholamine transporters display carrier‐mediated efflux of amines if challenged by lowering the extracellular Na+ or Cl−, whilst retaining their pharmacological profile. The transporters differ with regard to the ion dependence of the blockade of reverse transport by uptake inhibitors.

British Journal of Pharmacology (1997) 121, 205–212; doi:10.1038/sj.bjp.0701137
Effect of copper on nitric oxide synthase and guanylyl cyclase activity in the rat isolated aorta
Authors: Plane Frances , Wigmore Sarah , Angelini Gianni D , Jeremy Jamie Y
Published: August 2006

The potential role of copper (Cu2+) in modulating the activity of nitric oxide synthase (NOS) and guanylyl cyclase (GC) was investigated by use of diethyldithiocarbamic acid (DEDCA), a high affinity Cu2+ chelator.

DEDCA 100 μM inhibited sodium nitroprusside (SNP; 0.005–10 μM)‐evoked relaxation of rat isolated aortic rings precontracted with 3 μM phenylephrine (PE). A lower concentration of DEDCA (10 μM) did not significantly attenuate SNP‐evoked responses but did inhibit relaxation to the endothelium‐dependent dilator, A23187 (0.01–10 μM).

The presence of 100 μM Cu2+, but not 100 μM Fe2+, alone enhanced A23187‐ and SNP‐evoked relaxation of aortae precontracted with PE.

The inhibitory effect of DEDCA on SNP‐ and A23187‐induced relaxation was reversed by equimolar concentrations of Cu2+ but not Fe2+, indicating that DEDCA does not act via removal of haem‐iron from the NOS and GC complexes.

Superoxide dismutase (30 μ ml−1) was without effect on the inhibition of DEDCA relaxation induced by either SNP or A23187 in aortae precontracted with PE.

When assessed by radioimmunoassay, DEDCA inhibited SNP‐ and A23187‐stimulated cyclic GMP formation with IC50 values of 0.5 μM and 50 μM, respectively.

These data demonstrate that Cu2+ plays a role in controlling NOS and GC activity in the rat aorta.

British Journal of Pharmacology (1997) 121, 345–350; doi:10.1038/sj.bjp.0701144
Attenuation of p53 expression and Bax down‐regulation during phorbol ester mediated inhibition of apoptosis
Authors: Meßmer Udo K. , Brüne Bernhard
Published: August 2006

Nitric oxide (NO) caused apoptotic cell death in murine RAW 264.7 macrophages. Associated with apoptotic morphology we observed p53 up‐regulation and increased Bax expression. 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), a protein kinase C (PKC) activator potently blocked NO‐induced apoptosis. To gain insights into the mechanisms involved we investigated the effect of TPA on apoptotic conveying proteins such as p53 and Bax.

TPA (100 nM) attentuated p53 up‐regulation elicited by the NO‐releasing compounds, S‐nitrosoglutathione (1 mM) and sodium nitroprusside (1 mM), and suppressed p53 protein accumulation in response to endogenously generated NO. Hence, TPA appeared to lower the steady state p53 level following its up‐regulation by NO.

Mezerein, a stage 2 tumour promoter and PKC activating agent was equally active to TPA. Moreover, two potent PKC inhibitors, staurosporine (10 nM) and Gö 6976 (50 nM), reversed the inhibitory effect of TPA. However, bisinoylmaleimide I (up to 500 nM) was ineffective.

By extending the studies, we revealed a TPA‐mediated p53 down‐regulation in response to etoposide (50 μM), mitomycin C (5 μg ml−1) and actinomycin D (2 μg ml−1).

With the notion that TPA suppressed apoptotic DNA fragmentation in p53 antisense expressing cells as well, we searched for additional inhibitory actions of TPA. As well as affecting p53, TPA elicited a rapid decline of the steady state level of Bax within 30 min.

We concluded that down‐regulation of two classical apoptotic promoting proteins contributes to the anti‐apoptotic action of mezerein and TPA.

British Journal of Pharmacology (1997) 121, 625–634; doi:10.1038/sj.bjp.0701156
Modulation by low sodium intake of glomerular response to cicletanine and atrial natriuretic factor
Authors: Kalinowski Leszek , Szczepanska‐Konkel Miroslawa , Jankowski Maciej , Angielski Stefan
Published: August 2006

The aim of the study was to investigate whether cicletanine (CIC), as a potential inhibitor of cyclic GMP phosphodiesterase, is able to restore glomerular response to atrial natriuretic factor (ANF) in rats under conditions of diet deprived of sodium. We examined the effects of CIC on glomerular filtration rate (GFR), natriuresis and nephrogenous cyclic GMP excretion in response to ANF and the effects of both agents on intracapillary volume and cyclic GMP accumulation in isolated glomeruli of rats on normal and low sodium diets.

CIC (0.25 mg min−1 kg−1 BW) of ANF (0.5 μg min−1 BW) alone, given in pharmacological doses, increased Cin significantly in normal sodium rats, whereas the effect of each agent was blunted in low sodium diet rats. Pretreatment with CIC restored the increase in Cin in response to ANF infusion in low sodium diet rats. In rats on either diet, there were no differences in the extent of diuresis and natriuresis induced by CIC or ANF alone. In contrast to FENa, combined effects of both agents on V and UNaV in rats on normal and low sodium diets were observed.

In normal sodium diet rats, CIC 10−4 M or ANF 10−6 M alone inhibited angiotensin II 10−6 M (AII)‐induced decrease in intracapillary volume reflected by the glomerular [3H]‐inulin space (GIS). In contrast, CIC or ANF alone did not inhibit AII‐induced decrease in GIS in low sodium diet rats. Both agents given together inhibited AII‐induced decrease in GIS in low sodium diet rats.

CIC both alone and in combination with ANF increased nephrogenous cyclic GMP excretion and cyclic GMP accumulation in isolated glomeruli of rats on normal and low sodium diets. In rats on either diet, CIC abolished the difference in ANF‐stimulated increase in nephrogenous cyclic GMP excretion and cyclic GMP accumulation in glomeruli.

These results suggest that CIC and ANF alone induce relaxation of glomeruli and a resultant increase in glomerular filtration rate in normal sodium diet rats; in contrast, these effects are blunted in the low sodium diet rats. CIC restores glomerular response to ANF in low sodium diet rats, apparently involving inhibition of the cyclic GMP phosphodiesterase.

British Journal of Pharmacology (1997) 121, 635–642; doi:10.1038/sj.bjp.0701160
Characterization of endothelium‐derived relaxing factors released by bradykinin in human resistance arteries
Authors: Ohlmann P. , Martínez M. C. , Schneider F. , Stoclet J. C. , Andriantsitohaina R.
Published: August 2006

Relaxing factors released by the endothelium and their relative contribution to the endothelium‐dependent relaxation produced by bradykinin (BK) in comparison with different vasodilator agents were investigated in human omental resistance arteries.

BK produced an endothelium‐dependent relaxation of arteries pre‐contracted with the thromboxane A2 agonist, U46619. The B2 receptor antagonist, Hoe 140 (0.1, 1 and 10 μM), produced a parallel shift to the right of the concentration‐response curve to BK with a pA2 of 7.75.

Neither the cyclo‐oxygenase inhibitor, indomethacin (10 μM) alone, the nitric oxide synthase inhibitor, Nω‐nitro‐L‐arginine methyl ester (L‐NAME, 300 μM) alone, the nitric oxide scavenger, oxyhaemoglobin (Hb, 10 μM) alone, nor the combination of L‐NAME plus Hb affected the concentration‐response curve to BK. Conversely, the combination of indomethacin with either L‐NAME or Hb attenuated but did not abolish the BK‐induced relaxation. By contrast, the relaxations produced by the Ca2+ ionophore, calcimycin (A23187), and by the inhibitor of sarcoplasmic reticulum Ca2+‐ATPase, thapsigargin (THAPS), were abolished in the presence of indomethacin plus L‐NAME. Also, the presence of indomethacin plus L‐NAME produced contraction of arteries with functional endothelium.

The indomethacin plus L‐NAME resistant component of BK relaxation was abolished in physiological solution (PSS) containing 40 mM KCl and vice versa. However, in the presence of KCl 40 mM, indomethacin plus L‐NAME did not affect the nitric oxide donor, S‐N‐acetylpenicillamine‐induced relaxation.

The indomethacin plus L‐NAME resistant component of the relaxation to BK was significantly attenuated by the K+ channel blocker tetrabutylammonium (TBA, 1 mM). However, it was not affected by other K+ channel blockers such as apamin (10 μM), 4‐aminopyridine (100 μM), glibenclamide (10 μM), tetraethylammonium (10 mM) and charybdotoxin (50 nM).

In the presence of indomethacin plus L‐NAME, the relaxation produced by BK was not affected by the phospholipase A2 inhibitor, quinacrine (10 μM) or by the inhibitor of cytochrome P450, SKF 525a (10 μM). Another cytochrome P450 inhibitor, clotrimazole (10 μM) which also inhibits K+ channels, inhibited the relaxation to BK.

These results show that BK induces endothelium‐dependent relaxation in human small omental arteries via multiple mechanisms involving nitric oxide, cyclo‐oxygenase derived prostanoid(s) and another factor (probably an endothelium‐derived hyperpolarizing factor). They indicate that nitric oxide and cyclo‐oxygenase derivative(s) can substitute for each other in producing relaxation and that the third component is not a metabolite of arachidonic acid, formed through the cytochrome P‐450 pathway, in these arteries.

British Journal of Pharmacology (1997) 121, 657–664; doi:10.1038/sj.bjp.0701169
The effect of R 15.7/HO, an anti‐CD18 antibody, on the late airway response and airway hyperresponsiveness in an allergic rabbit model
Authors: El‐Hashim A. Z. , Jacques C. A. , Herd C. M. , Lee T. H. , Page C. P.
Published: August 2006

The effects of a mouse (IgG1 fraction) anti‐CD 18 neutralizing antibody (R15.7) on allergen‐induced late airway response (LAR), airway hyperresponsiveness (AHR) and cellular recruitment were investigated in an allergic rabbit model.

Litter‐matched NZW rabbits immunized within 24 h of birth with Alternaria tenuis (i.p.) and subsequently exposed to the allergen (i.p.) for the first 3 months of life were challenged with inhaled allergen as adult rabbits. Lung function in terms of dynamic compliance (Cdyn; ml cmH2O−1) and total lung resistance (RL; cmH2O−1 s−1) was monitored for 6 h following the allergen challenge. On day 16, separate groups of rabbits were pretreated with either control antibody (a non‐binding mouse IgG1, 1 mg kg−1, i.v.) or R15.7 (1 mg kg−1, i.v.) and 1 h later all were challenged with Alternaria tenuis and lung function monitored thereafter. Airway responsiveness to inhaled histamine was assessed by measuring RL and Cdyn 24 h before and after allergen challenge and bronchoalveolar lavage (BAL) was also performed 24 h before and after allergen challenge.

Pretreatment of rabbits with the control antibody had no effect on the LAR as measured by AUC (Cdyn, 0–6 h). However, the magnitude of the LAR following treatment with R15.7 was significantly reduced when compared to LAR demonstrated on 1st challenge (P<0.001) or to that of the control group on both challenges (P<0.01).

In control antibody pretreated rabbits allergen induced a significant 3.4 fold reduction in the PC50 response to inhaled histamine in terms of RL changes (P<0.05) and a significant 2.1 fold reduction in PC35 response to inhaled histamine in terms of Cdyn changes (P<0.05). However, in anti‐CD 18 antibody pretreated rabbits there was no significant change in responsiveness to histamine 24 h following allergen, as assessed by either RL PC50 or Cdyn PC35.

Allergen challenge induced a significant increase in eosinophil and neutrophil numbers (P<0.05) in rabbits pre‐treated with control antibody, whereas treatment with R15.7 significantly inhibited this increase in the numbers of both cell types.

This study demonstrates that the neutralization of CD‐18 molecules reduces allergen‐induced infiltration of both eosinophils and neutrophils into the airways and abolishes the accompanying LAR and AHR. These results provide evidence to support a role for CD‐18 adhesion molecules in the transmigration of inflammatory cells into airways.

British Journal of Pharmacology (1997) 121, 671–678; doi:10.1038/sj.bjp.0701176
Pharmacological characterization of endothelin‐induced rat pulmonary arterial dilatation
Authors: Higashi Tooru , Ishizaki Takeshi , Shigemori Kazuo , Yamamura Takaki , Nakai Tuguhiko
Published: August 2006

The aim of study was to characterize endothelin (ET)‐induced vasodilatation in isolated extrapulmonary rat arteries (EPA) and in intrapulmonary arteries (IPA) preconstricted with 1 μM phenylephrine.

The ET‐3 (1 nM–100 nM)‐ and ET‐1 (10 nM–100 nM)‐induced transient vasodilatations in EPA were more potent than those in IPA. The vasodilatation induced by ET‐3 (100 nM) was larger than that induced by ET‐1 (100 nM).

Both the ETB antagonist, BQ788 (3 μM) and or endothelium denudation, but not the ETA antagonist, BQ123 (3 μM), abolished the vasodilatation induced by ET‐1 or ET‐3 (100 nM each) in EPA and in IPA. The ATP‐sensitive K+channel blocker, glibenclamide (20 μM) and the nitric oxide synthase inhibitor, NG‐monomethyl‐L‐arginine (L‐NMMA, 1 mM) suppressed the ET‐induced vasodilatation in EPA and in IPA.

We conclude that the vasodilatation induced by endothelins is markedly reduced in rat isolated IPA, and suggest that the endothelial ETB‐mediated vasodilatation varies depending on rat pulmonary arterial regions. Furthermore, ETB‐mediated vasodilatation involves activation of ATP‐sensitive K+ channels and of nitric oxide synthase in rat isolated EPA and IPA.

British Journal of Pharmacology (1997) 121, 782–786; doi:10.1038/sj.bjp.0701177
Vasodilator effects of adrenomedullin on small pulmonary arteries and veins in anaesthetized cats
Authors: Shirai Mikiyasu , Shimouchi Akito , Ikeda Soichiro , Ninomiya Ishio , Sunagawa Kenji , Kangawa Kenji , Matsuo Hisayuki
Published: August 2006

This study was conducted to determine adrenomedullin (AM) action sites in the pulmonary vascular bed and the relation between its vasodilator effects and vascular tone. Moreover, an examination was made into whether calcitonin gene‐related peptide (CGRP) receptors mediate pulmonary vasodilatations induced by AM. To this end, we directly measured internal diameter (i.d.) changes in small pulmonary arteries and veins (100–1100 μm i.d.) by use of an X‐ray televison system on the in vivo cat lung.

Under control (resting vascular tone) conditions, AM injections into the left main pulmonary artery caused dose‐related i.d. increases in both small arteries and veins. The mean i.d. increase of the 100–1100 μm arteries (4±1, 11±2, and 17±2% with 0.01, 0.1, and 1 nmol kg−1 AM, respectively) was significantly larger than that for the veins (1±1, 5±2, and 7±2% with 0.01, 0.1 and 1 nmol kg−1 AM, respectively) whatever the injected dose of AM.

When unilobar hypoxia (5% O2) had decreased the i.d. of the 100–1100 μm arteries and veins by 16±3 and 6±3%, respectively, AM (0.1 nmol kg−1) was able to induce significantly larger i.d. increases in the arteries (28±3%) and veins (11±3%) than those under control conditions.

The AM‐induced i.d. response pattern in the serially connected pulmonary arteries was quite different from that induced by CGRP; AM caused a greater increase in smaller vessels (100–500 μm) than in larger vessels (500–1100 μm). In the case of CGRP, a greater increase was observed in the larger vessels.

CGRP8–37 (100 nmol kg−1, i.v., followed by a continuous infusion of 0.2 nmol kg−1 min−1) had no significant effect on the i.d. increase induced by AM (0.1 nmol kg−1) in any serial segments of the arteries and veins.

The results indicate that, in the cat, AM induces greater vasodilatation in small pulmonary arteries and lesser vasodilatation in small veins, the maximum dilatation being in the more peripheral arterial segment (100–500 μm). The vasodilator effect of AM was enhanced when vascular tone was elevated. The data suggest that the AM‐induced pulmonary vasodilatation is not mediated by CGRP receptors but by its own specific receptor.

British Journal of Pharmacology (1997) 121, 679–686; doi:10.1038/sj.bjp.0701178