bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of tumour necrosis factor and reversal of endotoxin‐induced shock by U‐83836E, a ‘second generation’ lazaroid in rats
Authors: Altavilla Domenica , Squadrito Francesco , Serrano Micaela , Campo Giuseppe M. , Squadrito Giovanni , Arlotta Mariarita , Urna Giuseppe , Sardella Aurora , Saitta Antonino , Caputi Achille P.
Published: January 2005

Antioxidants can exert protective effects in endotoxic shock by either a reduction of the oxidant damage or attenuation of Tumour Necrosis Factor (TNF‐α) production.

Lazaroids are a family of compounds that inhibit lipid peroxidation. Besides, they can also reduce TNF‐α. U‐83836E is a new lazaroid lacking the glucocorticoid ring.

Aim of our study was to investigate the effect of U‐83836E on TNF‐α production either in vivo or in vitro. Endotoxic shock was produced in male rats by a single intravenous (i.v.) injection of 20 mg kg−1 of S. enteritidis lipopolysaccharide (LPS). LPS administration reduced survival rate (0% survival, 72 h after endotoxin administration), decreased mean arterial blood pressure, increased serum and macrophage TNF‐α and enhanced plasma malonylaldehyde (MAL) levels. Furthermore aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE 1 nm–10 μm).

Treatment with U‐83836E (7.5, 15 and 30 mg kg−1, i.v.) 5 min after endotoxin challenge significantly protected against LPS induced lethality (90% survival rate and 80% survival rate 24 h and 72 h after LPS injection respectively, following the highest dose of the drug), reduced hypotension, blunted plasma MAL, decreased serum and macrophage TNF‐α and restored the hyporeactivity of aortic rings to control values. In vitro LPS stimulation (50 μg ml−1 for 4 h) significantly increased cytokine production in macrophages (MΦ) harvested from untreated normal rats. Pretreatment with pertussis toxin (PT; 0.1, 1 and 10 ng ml−1 4 h before LPS) significantly increased TNF‐α production. PT effects on these LPS responses were correlated with a PT mediated ADP ribosylation of a 41 kDa protein. U‐83836E (50 μm) reduced, in a dose dependent manner, LPS induced TNF‐α production and inhibited the PT effects on cytokine production and on ADP ribosylation of the protein.

Our data suggest that lazaroids may affect the early events associated with LPS receptor mediated activation of a G protein in LPS induced TNF‐α production. These molecular events may explain, at least in part, the in vivo inhibition of cytokine production and reversal of endotoxic shock.

British Journal of Pharmacology (1998) 124, 1293–1299; doi:10.1038/sj.bjp.0701944
Evidence from microdialysis and synaptosomal studies of rat cortex for noradrenaline uptake sites with different sensitivities to SSRIs
Authors: Hughes Zoë A. , Stanford S. Clare
Published: January 2005

Microdialysis of the frontal cortex of freely‐moving rats and uptake of [3H]noradrenaline into cortical synaptosomes were used to evaluate changes in efflux of noradrenaline in vivo and uptake of [3H]noradrenaline in vitro, respectively, induced by the selective serotonin reuptake inhibitors (SSRIs), fluoxetine and citalopram, and the tricyclic antidepressant, desipramine.

Noradrenaline efflux was increased during local infusion into the cortex of each of these drugs. All three agents also inhibited synaptosomal uptake of [3H]noradrenaline; this inhibition was unaffected by a substantial (50%) lesion of central 5‐hydroxytrytaminergic neurones induced by intracerebroventricular infusion of 5,7‐DHT (150 μg).

A noradrenergic lesion (70%), induced by pretreatment with the selective neurotoxin, N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP‐4, 40 mg kg−1 i.p.), 5 days earlier, abolished the increase in noradrenaline efflux caused by local infusion of fluoxetine. In contrast, the desipramine‐induced increase in efflux was greater than in non‐lesioned rats whereas the effect of citalopram on noradrenaline efflux was unaffected by DSP‐4 pretreatment.

The combined results of all these experiments suggest that there could be more than one, functionally distinct, noradrenaline uptake site in rat frontal cortex which can be distinguished by their different sensitivities to desipramine and the SSRIs, fluoxetine and citalopram.

British Journal of Pharmacology (1998) 124, 1141–1148; doi:10.1038/sj.bjp.0701947
Inhibition of excitatory amino acid‐activated currents by trichloroethanol and trifluoroethanol in mouse hippocampal neurones
Authors: Peoples Robert W. , Weight Forrest F.
Published: January 2005

The effects of the active metabolite of chloral derivative sedative‐hypnotic agents, 2,2,2‐trichloroethanol (trichloroethanol), and its analog 2,2,2‐trifluoroethanol (trifluoroethanol), were studied on ion current activated by the excitatory amino acids N‐methyl‐d‐aspartate (NMDA) and kainate in mouse hippocampal neurones in culture using whole‐cell patch‐clamp recording.

Both trichloroethanol and trifluoroethanol inhibited excitatory amino acid‐activated currents in a concentration‐dependent manner. Trichloroethanol inhibited NMDA‐ and kainate‐activated currents with IC50 values of 6.4 and 12 mm, respectively, while trifluoroethanol inhibited NMDA‐ and kainate‐activated currents with IC50 values of 28 and 35 mm, respectively.

Both trichloroethanol and trifluoroethanol appeared to be able to inhibit excitatory amino acid‐activated currents by 100 per cent.

Concentration‐response analysis of NMDA‐ and kainate‐activated current revealed that trichloro‐ethanol decreased the maximal response to both agonists without significantly affecting their EC50 values.

Both trichloroethanol and trifluoroethanol inhibited excitatory amino acid‐activated currents more potently than did ethanol. The inhibitory potency of trichloroethanol and trifluoroethanol appears to be associated with their increased hydrophobicity.

The observation that trichloroethanol inhibits excitatory amino acid‐activated currents at anaesthetic concentrations suggests that inhibition of excitatory amino acid receptors may contribute to the CNS depressant effects of chloral derivative sedative‐hypnotic agents.

British Journal of Pharmacology (1998) 124, 1159–1164; doi:10.1038/sj.bjp.0701949
Role of endogenous nitric oxide in allergen‐induced airway responses in guinea‐pigs
Authors: Iijima Hiroaki , Uchida Yoshiyuki , Endo Takeo , Xiang Anbo , Shirato Manabu , Nomura Akihiro , Hasegawa Shizuo
Published: January 2005

Endogenous nitric oxide (NO) can be detected in exhaled air and accumulates in inflamed airways. However its physiological role has not been fully elucidated. In this study, we investigated a role for endogenous NO in allergen‐induced airway responses. Sensitised guinea‐pigs were treated with NG‐nitro‐l‐arginine methyl ester l‐NAME (2.0 mm) or aminoguanidine (AG) (2.0 mm) 30 min before the allergen challenge, and 3 and 4 h after the challenge. Alternatively, l‐arginine (2.4 mm) treatment was performed 30 min before, and 2 and 3 h after the challenge. In all groups, ovalbumin (OVA) challenge (2 mg ml−1 for 2 min) was performed, and airway responses, NO production, infiltration of inflammatory cells, plasma exudation and histological details were examined.

Allergen‐challenged animals showed an immediate airway response (IAR) and a late airway response (LAR), which synchronised with an increase in exhaled NO. Treatment with l‐NAME and AG did not affect IAR while they significantly blocked LAR (72% and 80% inhibition compared to vehicle) and production of NO (35% and 40% inhibition). On the other hand, treatment with l‐arginine did not affect IAR but potentiated LAR (74% augmentation).

In bronchoalveolar lavage (BAL) fluid, allergen‐induced increases in eosinophils were reduced by 48% for l‐NAME treatment compared to vehicle, and increased by 56% for l‐arginine treatment.

Treatment with l‐NAME significantly decreased airway microvascular permeability to both Monastral blue (MB) and Evans blue (EB) dye (50.6% and 44% inhibition).

We conclude that allergen‐induced LAR is closely associated with NO production, and that NO plays a critical role in inflammatory cell infiltration and plasma exudation in the allergic condition.

British Journal of Pharmacology (1998) 124, 1019–1028; doi:10.1038/sj.bjp.0701951
Prostaglandins inhibit secretion of histamine and pancreastatin from isolated rat stomach ECL cells
Authors: Lindström Erik , Håkanson Rolf
Published: January 2005

The present study examines the effect of naturally occurring prostanoids and prostaglandin (PG) congeners on gastrin‐ and pituitary adenylate cyclase‐activating peptide (PACAP)‐evoked histamine and pancreastatin secretion from isolated rat stomach ECL cells.

ECL cells (75–85% purity) were isolated from rat stomach using pronase digestion followed by repeated counter‐flow elutriation and cultured for 48 h before secretion experiments. The release of histamine and pancreastatin was determined by radioimmunoassay.

None of the PGs tested stimulated the release of either histamine or pancreastatin.

PGE1 and PGE2 inhibited both gastrin‐ and PACAP‐evoked histamine and pancreastatin secretion (IC50 = 1–2×10−10 m). Most other naturally occuring prostanoids and PG congeners had no or little inhibitory effect. The PGE analogues misoprostol and sulprostone were more potent (IC50 = 0.9×10−11 m and 2×10−11 m respectively) than PGE1 and PGE2. The rank order of potency was misoprostol>sulprostone>PGE1 = PGE2, suggesting the involvement of the so‐called EP3 receptor.

The effects of PGs on the stomach ECL cells may be direct or indirect, for instance through the stimulated release of somatostatin from contaminating D cells (2–3%). However, the amount of somatostatin in the cell culture after 48 h was below the limit of detection, and somatostatin immunoneutralization did not prevent misoprostol from inhibiting secretion from the ECL cells.

The misoprostol‐induced inhibition was reversed by pertussis toxin suggesting the involvement of G‐protein subunits Gα0 and/or Gαi.

In view of the potency by which PGE1, PGE2, misoprostol and sulprostone inhibited the stimulated release of histamine and pancreastatin, we suggest that the ECL cells represent a primary target for prostaglandins acting via an EP3 receptor in the oxyntic mucosa.

The results suggest that the clinically useful effect of misoprostol as an anti‐ulcer drug reflects its ability to inhibit stomach ECL‐cell histamine secretion.

British Journal of Pharmacology (1998) 124, 1307–1313; doi:10.1038/sj.bjp.0701953
Selective inhibition of M‐type potassium channels in rat sympathetic neurons by uridine nucleotide preferring receptors
Authors: Boehm Stefan
Published: January 2005

UTP and UDP depolarize rat superior cervical ganglion neurons and trigger noradrenaline release from these cells. The present study investigated the mechanisms underlying this excitatory action of uridine nucleotides by measuring whole‐cell voltage‐dependent K+ and Ca2+ currents.

Steady‐state outward (holding) currents measured in the amphotericin B perforated‐patch configuration at a potential of −30 mV were reduced by 10 μm UTP in a reversible manner, but steady‐state inward (holding) currents at −70 mV were not affected. This action of UTP was shared by the muscarinic agonist oxotremorine‐M. In current‐voltage curves between −20 and −100 mV, UTP diminished primarily the outwardly rectifying current components arising at potentials positive to −60 mV.

Slow relaxations of muscarinic K+ currents (IM) evoked by hyperpolarizations from −30 to −55 mV were also reduced by 10 μm UTP (37% inhibition) and oxotremorine‐M (81% inhibition). In contrast, transient K+‐currents, delayed rectifier currents, fast and slow Ca2+‐dependent K+ currents, as well as voltage‐dependent Ca2+ currents were not altered by UTP.

In conventional (open‐tip) whole‐cell recordings, replacement of GTP in the pipette by GDPβS abolished the UTP‐induced inhibition of IM, whereas replacement by GTPγS rendered it irreversible.

The UTP‐induced reduction of IM was half maximal at 1.5 μm with a maximum of 37% inhibition; UDP was equipotent and equieffective, while ADP was less potent (half maximal inhibition at 29 μm). ATP had no effect at 30 μm.

The inhibition of IM induced by 10 μm UTP was antagonized by pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) at 30 μm and by reactive blue 2 at 10 μm, but not by suramin at concentrations up to 30 μm.

These results show that rat superior cervical ganglion neurons possess uridine nucleotide preferring P2Y receptors which inhibit KM channels. This effect presumably forms the basis of the excitatory action of uridine nucleotides in rat sympathetic neurons.

British Journal of Pharmacology (1998) 124, 1261–1269; doi:10.1038/sj.bjp.0701956
4‐Oxystilbene compounds are selective ligands for neuronal nicotinic αBungarotoxin receptors
Authors: Gotti C. , Balestra B. , Moretti M. , Rovati G. E. , Maggi L. , Rossoni G. , Berti F. , Villa L. , Pallavicini M. , Clementi F.
Published: January 2005

Starting from the structure of an old 4‐oxystilbene derivate with ganglioplegic activity (MG624), we synthesized two further derivates (F2 and F3) and two stereoisomers of F3 (F3A and F3B), and studied their selective effect on neuronal nicotinic acetylcholine receptor (AChR) subtypes.

MG 624, F3, F3A and F3B inhibited of 125I‐αBungarotoxin (αBgtx) binding to neuronal chick optic lobe (COL) membranes, with nM affinity, but inhibited 125I‐αBgtx binding to TE671 cell‐expressed muscle‐type AChR only at much higher concentrations.

We immobilized the α7, β2 and β4 containing chick neuronal nicotinic AChR subtypes using anti‐subunit specific antibodies. MG 624, F3, F3A and F3B inhibited 125I‐αBgtx binding to the α7‐containing receptors with nm affinity, but inhibited 3H‐Epi binding to β2‐containing receptors only at very high concentrations (more than 35 μm); their affinity for the β4‐containing receptors was ten times more than for the β2‐containing subtype.

Both MG624 and F3 compounds inhibited the ACh evoked currents in homomeric oocyte‐expressed chick α7 receptors with an IC50 of respectively 94 and 119 nm.

High doses of both MG 624 and F3 depressed the contractile response to vagus nerve stimulation in guinea pig nerve‐stomach preparations although at different IC50s (49.4 vs 166.2 μm) The effect of MG624 on rat nerve‐hemidiaphragm preparations was 33 times less potent than that of F3 (IC50 486 vs 14.5 μm).

In conclusion, MG624 and F3 have a high degree of antagonist selectivity for neuronal nicotinic αBgtx receptors containing the α7 subunit.

British Journal of Pharmacology (1998) 124, 1197–1206; doi:10.1038/sj.bjp.0701957
Effects of antagonists at the human recombinant P2X7 receptor
Authors: Chessell I. P. , Michel A. D. , Humphrey P. P. A.
Published: January 2005

We have used whole‐cell patch clamping methods to examine the properties of the recombinant human P2X7 (P2Z) receptor stably expressed in HEK‐293 cells.

In an extracellular solution with lowered concentrations of divalent cations (zero Mg2+ and 0.5 mm Ca2+), both ATP and the nucleotide analogue, 2′‐ and 3′‐O‐(4‐benzoylbenzoyl)‐adenosine 5′‐triphosphate (Bz‐ATP) evoked concentration‐dependent whole‐cell inward currents with maxima of 4658±671 and 5385±990 pA, respectively, at a holding potential of −90 mV. Current‐voltage relationships determined using 100 μm Bz‐ATP reversed at −2.7±3.1 mV, and did not display significant rectification.

Repeated applications of 300 μm Bz‐ATP produced inward currents with similar rise‐times (approx. 450 ms, 5–95% current development) but with progressively slower 95–5% decay times, with the eighth application of this agonist yielding a decay time of 197% of the first application.

Concentration‐effect curves to ATP and Bz‐ATP produced estimated EC50 values of 780 and 52.4 μm, respectively. Consecutive concentration‐effect curves to Bz‐ATP produced curves with similar maxima and EC50 values.

The non‐selective P2 antagonists, pyridoxal‐phosphate‐6‐azophenyl‐, 2′,4′‐disulphonic acid (PPADS) and suramin, both produced concentration‐dependent increases in maximal inward currents to Bz‐ATP, with IC50 concentrations of approximately 1 μm and 70 μm, respectively. The profile of antagonism produced by PPADS was not that of a competitive antagonist.

The isoquinolene derivatives 1‐(N,O‐bis[5‐isoquinolinesulphonyl]‐N‐methyl‐l‐tyrosyl)‐4‐phenylpiperazine (KN‐62) and calmidazolium both produced antagonism which was not competitive, with IC50 concentrations of approximately 15 and 100 nm, respectively. HMA (5‐(N,N‐hexamethylene)‐ amiloride) was also an effective antagonist at a concentration of 10 μm. The group IIb metal, copper, also displayed antagonist properties at the human P2X7 receptor, reducing the maximum response to Bz‐ATP by about 50% at a concentration of 1 μm.

These data demonstrate that the human recombinant P2X7 receptor displays functional behaviour which is similar to the recombinant rat P2X7 receptor, but has a distinct pharmacological profile with respect to agonist and antagonist sensitivity.

British Journal of Pharmacology (1998) 124, 1314–1320; doi:10.1038/sj.bjp.0701958
Different receptors for angiotensin II at pre‐ and postjunctional level of the canine mesenteric and pulmonary arteries
Authors: Guimarães S. , Paiva M. Q. , Moura D.
Published: January 2005

This investigation was undertaken to compare pre‐ and postjunctional receptors involved in the responses of the canine mesenteric and pulmonary arteries to angiotensin II.

In the mesenteric artery, angiotensin II caused an enhancement of tritium overflow evoked by electrical stimulation (EC30% = 5 nm), the maximal effect representing an increase by about 45%. Postjunctionally, angiotensin II caused concentration‐dependent contractions (pD2 = 8.57). Saralasin antagonized both pre‐ and postjunctional effects of angiotensin II, but it was more potent at post‐ than at prejunctional level (pA2 of 9.51 and 8.15, respectively), while losartan antagonized exclusively the postjunctional effects of angiotensin II (pA2 = 8.15). PD123319 had no antagonist effect either pre‐ or postjunctionally.

In the pulmonary artery, angiotensin II also caused an enhancement of the electrically‐evoked tritium overflow (EC30% = 1.54 nm), its maximal effect increasing tritium overflow by about 80%. Postjunctionally, angiotensin II caused contractile responses (pD2 = 8.52). As in the mesenteric artery, saralasin antagonized angiotensin II effects at both pre‐ and postjunctional level and it was more potent postjunctionally (pA2 of 9.58 and 8.10, respectively). Losartan antagonized only the postjunctional effects of angiotensin II (pA2 = 7.96) and PD123319 was ineffective.

It is concluded that in both vessels: (1) pre‐ and postjunctional receptors belong to a different subtype, since they are differently antagonized by the same antagonists; (2) postjunctional receptors belong to AT1 subtype, since they are blocked by losartan but not by AT2 antagonists; (3) prejunctional receptors apparently belong to neither AT1 or AT2 subtype since they are blocked by neither AT1 nor AT2 antagonists.

British Journal of Pharmacology (1998) 124, 1207–1212; doi:10.1038/sj.bjp.0701959
In vitro characterization of tachykinin NK2‐receptors modulating motor responses of human colonic muscle strips
Authors: Croci Tiziano , Aureggi Giulio , Manara Luciano , Emonds‐Alt Xavier , Le Fur Gerard , Maffrand Jean‐Pierre , Mukenge Sylvain , Ferla Gianfranco
Published: January 2005

Human in vitro preparations of transverse or distal colonic circular smooth muscle were potently and dose‐dependently contracted by neurokinin A (EC50, 4.9 nm), the tachykinin NK2‐receptor selective agonist [β‐Ala8]neurokinin A (4–10) ([β‐Ala8]NKA (4–10)) (EC50, 5.0 nm), neurokinin B (EC50, 5.3 nm) and substance P (EC50, 160 nm), but not by the tachykinin NK1‐receptor selective agonist [Sar9Met(O2)11] substance P, or the NK3‐receptor selective agonists, senktide and [MePhe7] neurokinin B. No regional differences between transverse and distal colon were observed in response to [β‐Ala8]NKA (4–10).

Atropine (1 μm) and tetrodotoxin (1 μm) did not significantly inhibit responses to [β‐Ala8]NKA (4–10), neurokinin A, substance P or neurokinin B.

The newly developed non‐peptide antagonists for tachykinin NK2‐receptors SR 48968, SR 144190 and its N‐demethyl (SR 144743) and N,N‐demethyl (SR 144782) metabolites, were used to challenge agonist responses, as appropriate. SR 144190 and the metabolites all potently and competitively antagonized the response to [β‐Ala8]NKA (4–10), with similar potency (Schild plot pA2 values 9.4, 9.4 and 9.3, slope = 1). SR 48968 antagonism was not competitive: the Schild plot slope was biphasic with a high (X intercept∼9.3) and a low (X intercept 8.4, slope 1.6) affinity site. Co‐incubation of SR 48968 (10, 100 nm) and SR 144782 (10 nm) produced additive effects; in this experimental condition, SR 48968 apparent affinity (pKB) was 8.2. In addition, SR 144782 (0.1 μm) antagonized responses to neurokinin A, substance P and neurokinin B, with pKB consistent with its affinity for tachykinin NK2‐receptors. The potent and selective NK1 and NK3‐receptor antagonists, SR 140333 and SR 142801 (both 0.1 μm), failed to inhibit contractions induced by SP or NKB.

In conclusion, the in vitro mechanical responses of circular smooth muscle preparations from human colon are strongly consistent with the presence of non‐neuronal tachykinin NK2‐receptors, but not tachykinin NK1‐ or NK3‐receptors. Our findings with SR 48968 suggest the existence of two tachykinin NK2‐receptor subtypes, that it seems to distinguish, unlike SR 144190 and its metabolites. However, the precise nature of SR 48968 allotopic antagonism remains to be elucidated, since allosteric effects at the tachykinin NK2‐receptor might well account for the complexity of the observed interaction.

British Journal of Pharmacology (1998) 124, 1321–1327; doi:10.1038/sj.bjp.0701960
Impaired IL‐1β‐induced neutrophil accumulation in tachykinin NK1 receptor knockout mice
Authors: Ahluwalia Amrita , De Felipe Carmen , O'Brien John , Hunt Stephen P. , Perretti Mauro
Published: January 2005

Tachykinin NK1 receptors play an important role in the development of neurogenic inflammatory responses. We have used the murine air‐pouch model to investigate whether the neurogenic component of the cellular inflammatory response to interleukin‐1β (IL‐1β, 10 ng into the air‐pouch) is altered in NK1 receptor knockout mice compared to wild type controls. Air‐pouches were washed following a 4 h IL‐1β treatment, the wash collected and neutrophil number estimated using a Neubauer haemocytometer. The response to IL‐1β was significantly attenuated in NK1 receptor +/− (40% reduction) and −/− mice (62% reduction) compared to wild type controls (+/+), whilst the response to cytokine‐induced neutrophil chemoattractant (CINC, 0.3 μg) was unaffected. The response to substance P (7.5 nmol) was attenuated by approximately 50% in both NK1 receptor +/− and −/− mice compared to wild type controls. In conclusion NK1 receptors play a significant role in the cellular response to IL‐1β in a model of inflammation.

British Journal of Pharmacology (1998) 124, 1013–1015; doi:10.1038/sj.bjp.0701978
U937 cells deprived of endogenous annexin 1 demonstrate an increased PLA2 activity
Authors: Solito Egle , Raguenes‐Nicol Celine , De Coupade Catherine , Bisagni‐Faure Anne , Russo‐Marie Françoise
Published: January 2005

Annexin 1 (An 1), a phospholipid and calcium binding protein, is strongly expressed in differentiated U 937 cells. In attempting to correlate the expression of An 1 with phospholipase A2 (PLA2) activity, U 937 cells were stably transfected both with a Sense and Antisense cDNA for An 1. PLA2 activity was measured by Flow cytometry analysis utilizing the bis‐Bodipy‐C11‐PC fluorescent probe.

U 937 cells stably transfected with the sense or antisense vectors were differentiated for 24 h with phorbol 12‐myristate 13‐acetate (PMA, 6 ng ml−1). Both in undifferentiated and differentiated cells, the Antisense clone (36.4 AS) showed consistently higher PLA2 activity than the control Sense clone (15 S).

Since the fluorescent probe measures the total PLA2 activity, we used two different stimuli, PMA: (100 ng ml−1) or lipopolysaccharide (LPS, 10 ng ml−1), and two different inhibitors, to discriminate the PLA2 involved (namely arachidonyl trifluoromethyl ketone or AACOCF3, which is specific for the cytosolic PLA2, and SB 203347 specific for the secretory PLA2).

In the Antisense clone the inhibitory effect of AACOCF was stronger [68%, P<0.025] than in the Sense, which may reflect the lower endogenous level of An 1 present in the cells. On the contrary, the inhibitory effect of SB 203347 [60% of inhibition] was identical in both clones.

Since cPLA2 activity is correlated with its phosphorylation, Western and shift blot analysis were performed. They did not show any significative difference between the phosphorylated and non phosphorylated form of the enzyme in both the differentiated or not, Sense and Antisense clones. Furthermore the tyrosine phosphorylation analysis of An 1 showed that less than 10% of An 1 was phosphorylated irrespective of PMA presence or absence.

From the pattern of inhibition observed, we propose that the endogenous unphosphorylated form of An 1 may act intracellularly to block the activity of a cytosolic PLA2.

British Journal of Pharmacology (1998) 124, 1675–1683; doi:10.1038/sj.bjp.0701991
Anti‐nociceptive responses produced by human putative counterpart of nocistatin
Authors: Minami Toshiaki , Okuda‐Ashitaka Emiko , Nishiuchi Yuji , Kimura Terutoshi , Tachibana Shinro , Mori Hidemaro , Ito Seiji
Published: January 2005

b‐nocistatin is a heptadecapeptide produced from bovine prepronociceptin and blocks the induction of hyperalgesia and touch‐evoked pain (allodynia) by intrathecal administration of nociceptin or prostaglandin E2 (PGE2). Human prepronociceptin may generate a 30‐amino acid peptide different in length from b‐nocistatin. Here, we examine whether the human putative counterpart of nocistatin (h‐nocistatin) possessed the same biological activities as b‐nocistatin. Simultaneous intrathecal injection of h‐nocistatin in mice blocked the induction of allodynia by nociceptin and PGE2 in a dose‐dependent manner with ID50 values of 329 pg kg−1 and 16.6 ng kg−1, respectively. h‐nocistatin was about 10 times less potent than b‐nocistatin. h‐nocistatin also attenuated the nociceptin‐ and PGE2‐induced hyperalgesia. These results demonstrate that h‐nocistatin is biologically active and may be involved in the processing of pain at the spinal level in humans.

British Journal of Pharmacology (1998) 124, 1016–1018; doi:10.1038/sj.bjp.0701995
Presynaptic and postsynaptic actions of halothane at glutamatergic synapses in the mouse hippocampus
Authors: Kirson Eilon D , Yaari Yoel , Perouansky Misha
Published: January 2005

Whole‐cell patch‐clamp recordings in adult mouse hippocampal slices were used to test the mechanism by which the volatile anesthetic halothane inhibits glutamate receptor‐mediated synaptic transmission. Non‐N‐methyl‐D‐aspartate (nonNMDA) and NMDA receptor‐mediated currents in CA1 pyramidal cells were pharmacologically isolated by bath application of D,L‐2‐amino‐5‐phosphonovaleric acid (APV; 100 μM) or 6‐cyano‐7‐nitro‐quinoxaline‐2,3‐dione (CNQX; 5 μM), respectively.

Halothane blocked both nonNMDA and NMDA receptor‐mediated excitatory postsynaptic currents (EPSCs) to a similar extent (IC50 values of 0.66 and 0.57 mM, respectively).

Partial blockade of the EPSCs by lowering the extracellular concentration of calcium ([Ca2+]o), but not by application of CNQX (1 μM), was accompanied by an increase in paired‐pulse facilitation (PPF). Halothane‐induced blockade of the EPSCs also was associated with an increase in PPF.

The effects of halothane on α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) and NMDA receptor‐mediated currents induced by agonist iontophoresis, were compared. AMPA‐induced currents were blocked with an IC50 of 1.7 mM. NMDA‐induced currents were significantly less sensitive to halothane (IC50 of 5.9 mM).

The effect of halothane on iontophoretic AMPA dose‐response curves was tested. Halothane suppressed the maximal response to AMPA without affecting its EC50, suggesting a noncompetitive mechanism of inhibition.

All effects of halothane were reversible upon termination of the exposure to the drug.

These data suggest that halothane blocks central glutamatergic synaptic transmission by presynaptically inhibiting glutamate release and postsynaptically blocking the AMPA subtype of glutamate receptors.

British Journal of Pharmacology (1998) 124, 1607–1614; doi:10.1038/sj.bjp.0701996
Characterization of an atypical muscarinic cholinoceptor mediating contraction of the guinea‐pig isolated uterus
Authors: Boxall Donna K , Ford Anthony P D W , Choppin Agnes , Nahorski Stefan R , Challiss R A John , Eglen Richard M
Published: January 2005

In many smooth muscle tissues a minor M3‐muscarinic acetylcholine (mACh) receptor population mediates contraction, despite the presence of a larger M2‐mACh receptor population. However, this is not the case for guinea‐pig uterus where radioligand binding and functional studies exclude a dominant role for M3‐mACh receptors.

Using tissue from animals pre‐treated with diethylstilboestrol, estimates of antagonist affinity were made before and after selective alkylation procedures, together with estimates of agonist affinity to characterise the mACh receptor population mediating carbachol‐induced contraction of guinea‐pig isolated uterus.

Antagonist affinity estimates made at `protected' receptors were not significantly different from those made in untreated tissues. However all estimations were significantly different from those reported in guinea‐pig ileum and atria. The rank order of affinities were atropine>zamifenacin=tripitramine>methoctramine. Carbachol‐induced contractions were insensitive to the M4‐selective muscarinic toxin MTx‐3, or PD102807 (0.1 μM) ruling out a role for M4‐mACh receptors.

The agonist affinity value for L‐660,863, a putative `M2‐selective' agonist of 5.44±0.30 (n=6) was significantly different from that reported in guinea‐pig atria. In contrast, the pKA value for carbachol (4.22±0.17; n=8) agrees with that reported for guinea‐pig ileum.

Carbachol‐induced contractions were insensitive to pertussis toxin although carbachol‐induced inhibition of forskolin‐stimulated cyclic AMP production was attenuated, ruling out the involvement of Gi‐proteins in contraction.

Radioligand binding studies revealed a KD for N‐[3H]‐methylscopolamine of 0.12±0.05 nM and a Bmax of 147±18 fmol mg protein−1. Antagonist affinity estimates made using competition binding studies supported previous data suggesting the presence of a homogenous population of M2‐mACh receptors.

These data suggest a small population of mACh receptors with an atypical operational profile which can not be distinguished using radioligand binding studies may mediate carbachol‐induced contraction of guinea‐pig isolated uterus.

British Journal of Pharmacology (1998) 124, 1615–1622; doi:10.1038/sj.bjp.0702002
Metabolic response to various β‐adrenoceptor agonists in β3‐adrenoceptor knockout mice: Evidence for a new β‐adrenergic receptor in brown adipose tissue
Authors: Preitner Frédéric , Muzzin Patrick , Revelli Jean‐Pierre , Seydoux Josiane , Galitzky Jean , Berlan Michel , Lafontan Max , Giacobino Jean‐Paul
Published: January 2005

The β3‐adrenoceptor plays an important role in the adrenergic response of brown and white adipose tissues (BAT and WAT). In this study, in vitro metabolic responses to β‐adrenoceptor stimulation were compared in adipose tissues of β3‐adrenoceptor knockout and wild type mice. The measured parameters were BAT fragment oxygen uptake (MO2) and isolated white adipocyte lipolysis.

In BAT of wild type mice (−)‐norepinephrine maximally stimulated MO2 4.1±0.8 fold. Similar maximal stimulations were obtained with β1‐,β2‐ or β3‐adrenoceptor selective agonists (dobutamine 5.1±0.3, terbutaline 5.3±0.3 and CL 316,243 4.8±0.9 fold, respectively); in BAT of β3‐adrenoceptor knockout mice, the β1‐ and β2‐responses were fully conserved.

In BAT of wild type mice, the β1/β2‐antagonist and β3‐partial agonist CGP 12177 elicited a maximal MO2 response (4.7±0.4 fold). In β3‐adrenoceptor knockout BAT, this response was fully conserved despite an absence of response to CL 316,243. This unexpected result suggests that an atypical β‐adrenoceptor, distinct from the β1‐, β2‐ and β3‐subtypes and referred to as a putative β4‐adrenoceptor is present in BAT and that it can mediate in vitro a maximal MO2 stimulation.

In isolated white adipocytes of wild type mice, (−)‐epinephrine maximally stimulated lipolysis 12.1±2.6 fold. Similar maximal stimulations were obtained with β1‐, β2‐ or β3‐adrenoceptor selective agonists (TO509 12±2, procaterol 11±3, CL 316,243 11±3 fold, respectively) or with CGP 12177 (7.1±1.5 fold). In isolated white adipocytes of β3‐adrenoceptor knockout mice, the lipolytic responses to (−)epinephrine, to the β1‐, β2‐, β3‐adrenoceptor selective agonists and to CGP 12177 were almost or totally depressed, whereas those to ACTH, forskolin and dibutyryl cyclic AMP were conserved.

British Journal of Pharmacology (1998) 124, 1684–1688; doi:10.1038/sj.bjp.0702007
Inhibitors of the activity of poly (ADP‐ribose) synthetase reduce the cell death caused by hydrogen peroxide in human cardiac myoblasts
Authors: Bowes Joanne , Piper Julie , Thiemermann Christoph
Published: January 2005

Poly (ADP‐ribose) synthetase (PARS) is a nuclear enzyme activated by strand breaks in DNA which are caused by reactive oxygen species (ROS). Inhibitors of PARS activity reduce the degree of reperfusion injury of the heart in vivo and in vitro. Here we investigate the role of PARS in the cell death of human cardiac myoblasts caused by hydrogen peroxide.

Exposure of human cardiac myoblasts to hydrogen peroxide caused a time‐ and concentration‐dependent reduction in mitochondrial respiration (cell injury), an increase in cell death (LDH release), as well as an increase in PARS activity.

The PARS inhibitors 3‐aminobenzamide (3 mM), 1,5‐dehydroxyisoquinoline (300 μM) or nicotinamide (3 mM) attenuated the cell injury and death as well as the increase in PARS activity caused by hydrogen peroxide (3 mM; 4 h for cell injury/death, 60 min for PARS activity) in human cardiac myoblasts. In contrast, the inactive analogues 3‐aminobenzoic acid (3 mM) or nicotinic acid (3 mM) were without effect.

The iron chelator deferoxamine (1–10 mM) caused a concentration‐dependent reduction in the cell injury and death caused by hydrogen peroxide in these human cardiac myoblasts.

Thus, the cell injury/death caused by hydrogen peroxide in human cardiac myoblasts is secondary to the formation of hydroxyl radicals and due to an increase in PARS activity. We therefore propose that activation of PARS contributes to the cell injury/cell death associated with oxidant stress in the heart.

British Journal of Pharmacology (1998) 124, 1760–1766; doi:10.1038/sj.bjp.0702009
L‐687,414, a low efficacy NMDA receptor glycine site partial agonist in vitro, does not prevent hippocampal LTP in vivo at plasma levels known to be neuroprotective
Authors: Priestley Tony , Marshall George R , Hill Raymond G , Kemp John A
Published: January 2005

N‐methyl‐D‐aspartic acid (NMDA) receptors are known to play a key role in the induction phase of long‐term potentiation (LTP) at certain hippocampal synapses and to represent some component of spatial learning in animals. The ability of NMDA receptor antagonists (or gene knockout) to impair LTP has led to the suggestion that the therapeutic use of such antagonists may impair cognitive function in humans. The present study compares the effects on LTP of NMDA receptor ion channel block by MK‐801 and glycine‐site antagonism by 3R(+)cis‐4‐methyl‐pyrrollid‐2‐one (L‐687,414).

In vitro experiments using rat cortical slices revealed L‐687,414 to be ∼3.6 fold more potent than its parent analogue, R(+)HA‐966 at antagonizing NMDA‐evoked population depolarizations (apparent Kbs: 15 μM and 55 μM, respectively).

Whole‐cell voltage‐clamp experiments using rat cultured cortical neurones revealed L‐687,414 to shift to the right the concentration‐response relationship for NMDA‐evoked inward current responses (pKb=6.2±0.12). L‐687,414 affinity for the glycine site on the NMDA receptor complex was also determined from concentration‐inhibition curves, pKi=6.1±0.09. In the latter experiments, L‐687,414 and R(+)HA‐966 were unable to completely abolish inward current responses suggesting each compound to be a low efficacy partial agonist (estimated intrinsic activity=∼10 and 20% of glycine, respectively).

L‐687,414 and MK‐801 were compared for their effects on NMDA receptor‐dependent LTP in the dentate gyrus of anaethestized rats following high frequency stimulation of the medial perforant path (mPP) afferents. Control rats, administered saline (0.4 ml kg−1 followed by 0.0298 ml min−1), showed a robust augmentation of the population e.p.s.p. risetime (LTP) recorded in the dentate hilus following tetanic stimulation of the mPP. LTP was effectively abolished in a separate group of rats treated with an MK‐801 dosing regimen (0.12 mg kg−1 i.v. followed by 1.8 μg kg−1 h−1) known to produce maximal neuroprotection in a rat stroke model but, by contrast, remained largely intact in a third group of animals given a similarly neuroprotective L‐687,414 treatment (28 mg kg−1 i.v. followed by 28 mg kg−1 h−1).

These experiments suggest that a low level of intrinsic activity at the glycine site may be sufficient to support NMDA receptor‐dependent LTP but in circumstances where there is likely to be an excessive NMDA receptor activation the agonism associated with a low efficacy partial agonist, such as L‐687,414, is dominated by the antagonist properties. Thus, an NMDA receptor partial agonist profile may offer a therapeutic advantage over neutral antagonists by permitting an acceptable level of `normal' synaptic transmission whilst curtailing excessive receptor activation.

British Journal of Pharmacology (1998) 124, 1767–1773; doi:10.1038/sj.bjp.0702010
Flunitrazepam rapidly reduces GABAA receptor subunit protein expression via a protein kinase C‐dependent mechanism
Authors: Johnston Jonathan D. , Price Sally A. , Bristow David R.
Published: January 2005

Acute flunitrazepam (1 μm) exposure for 1 h reduced GABAA receptor α1 (22±4%, mean±s.e.mean) and β2/3 (21±4%) subunit protein levels in cultured rat cerebellar granule cells. This rapid decrease in subunit proteins was completely prevented by bisindolymaleimide 1 (1 μm), an inhibitor of protein kinase C, but not by N‐[2‐((p‐bromocinnamyl)amino)ethyl]‐5‐isoquinolinesulfonamide (H‐89, 4.8 μm), an inhibitor of protein kinases A and G. These results suggest the existence of a benzodiazepine‐induced mechanism to rapidly alter GABAA receptor protein expression, that appears to be dependent on protein kinase C activity.

British Journal of Pharmacology (1998) 124, 1338–1340; doi:10.1038/sj.bjp.0702012
Action potential shortening through the putative β4‐adrenoceptor in ferret ventricle: comparison with β1‐ and β2‐adrenoceptor‐mediated effects
Authors: Lowe Martin D. , Grace Andrew A. , Vandenberg Jamie I. , Kaumann Alberto J.
Published: January 2005

The electrophysiological responses to (−)‐CGP 12177 ((−)‐4‐(3‐tertiarybutylamino‐2‐hydroxypropoxy) benzimidazol‐2‐one), an agonist for the putative β4‐adrenoceptor, were investigated on isolated perfused ferret hearts paced at 100 min−1 and compared to those of (−)‐noradrenaline and (−)‐adrenaline, mediated through β1‐ and β2‐adrenoceptors respectively. The three agonists decreased ventricular monophasic action potential duration but prolonged the action potential plateau; β3‐adrenoceptor‐selective agonists had no effect. (−)‐CGP 12177 was the most potent, but (−)‐noradrenaline the most efficacious; both agonists caused ventricular extra‐systoles. Because only (−)‐noradrenaline but not (−)‐CGP 12177 elicited shortening of the refractory period, the mechanism of arrhythmias mediated through β1‐ and putative β4‐adrenoceptors may be different.

British Journal of Pharmacology (1998) 124, 1341–1344; doi:10.1038/sj.bjp.0702013
Analgesic and sedative concentrations of lignocaine shunt tonic and burst firing in thalamocortical neurones
Authors: Schwarz Stephan K W , Puil Ernest
Published: January 2005

The effects of lignocaine [lidocaine] HCl (0.6 μM–1 mM) on the membrane electrical properties and action potential firing of neurones of the ventral posterolateral (VPL) nucleus of the thalamus were investigated using whole cell recording techniques in rat brain slices in vitro.

Bath application of lignocaine reversibly decreased the input resistance (Ri) of VPL neurones. This effect was observed at low, clinically sedative and analgesic concentrations (i.e., maximal amplitude at 10 μM) whereas higher concentrations (300 μM–1 mM) had no effect on Ri.

Lignocaine (10–100 μM) depolarized VPL neurones up to 14 mV in a reversible manner.

Consistent with a decreased Ri, low concentrations of lignocaine shunted the current required for spike generation in the tonic pattern. Lignocaine increased the threshold amplitude of current required for firing and decreased the tonic firing frequency, without concomitant elevation of the voltage threshold for firing or reduction in the maximal rate of rise (dV/dtmax) of spikes.

Low concentrations of lignocaine shunted low threshold spike (LTS) burst firing evoked either from hyperpolarized potentials or as rebound bursts on depolarization from prepulse‐conditioned potentials.

Higher concentrations of lignocaine (300 μM–1 mM), not associated with a decrease in Ri, elevated the voltage threshold for firing and reduced the dV/dtmax of spikes in a concentration‐dependent fashion.

In conclusion, low concentrations of lignocaine shunted tonic and burst firing in VPL neurones by decreasing Ri, a mechanism not previously described for local anaesthetics in the CNS. We suggest that a decreased resistance in thalamocortical neurones contributes to the sedative, analgesic, and anaesthetic properties of systemic lignocaine in vivo.

British Journal of Pharmacology (1998) 124, 1633–1642; doi:10.1038/sj.bjp.0702015
Alternative pathways of angiotensin II production in the human saphenous vein
Authors: Borland Julie A A , Chester Adrian H , Morrison Karen A , Yacoub Magdi H
Published: January 2005

The aim of our study was to demonstrate the existence, location and functional importance of an alternative angiotensin II‐forming pathway other than angiotensin converting enzyme (ACE) in the human saphenous vein (SV).

Vascular reactivity studies using an in vitro organ bath technique showed that the SV (n=20) produced similar maximum contractions in response to angiotensin I (41.5±5.4 mN) compared to those observed to angiotensin II (46.7±10.9 mN). The response to angiotensin I could be significantly inhibited (P<0.05) by incubation with the AT1 receptor antagonist losartan (1 μM).

Prior incubation of segments of SV with either captopril (1 μM) (n=6), quinaprilat (1 μM) (n=7), or the chymase inhibitor soya bean trypsin inhibitor (SBTI) (10 μM) (n=7) singularly failed to have any inhibitory effect on the response to angiotensin I. However when vessel segments (n=7) were co‐incubated with quinaprilat (1 μM) and SBTI (10 μM), the SV exhibited a rightward shift in curve profile to angiotensin I and a markedly reduced maximum response 12.5±2.4 mN, when compared to control (30.4±7.6 mN), quinaprilat (24.5±9.4 mN), and SBTI (31.6±10.7 mN) on their own.

An immunohistochemical technique employing streptavidin biotin peroxidase localised ACE to both endothelial cells and smooth muscle cells while chymase was confined to mast cells in the adventitia of the vessel wall.

In conclusion, our results demonstrate the existence of an alternative angiotensin I converting pathway to that of ACE, involving chymase. Therefore, there is the capacity for a continuation of angiotensin II formation. in the presence of ACE inhibition.

British Journal of Pharmacology (1998) 125, 423–428; doi:10.1038/sj.bjp.0702018
Potent inhibition of both the acute and delayed emetic responses to cisplatin in piglets treated with GR205171, a novel highly selective tachykinin NK1 receptor antagonist
Authors: Grélot Laurent , Dapzol Julien , Estève Eric , Frugière Alain , Bianchi Armand L , Sheldrick Robert L G , Gardner Christopher J , Ward Peter
Published: January 2005

The effects of GR205171, a selective tachykinin NK1 receptor antagonist, were investigated on both the acute and delayed phases of cisplatin‐induced nausea‐like behaviour and vomiting in the conscious piglet.

Animals receiving cisplatin (5.5 mg kg−1, i.v.) were observed for 60 h. Fifteen min prior to cisplatin infusion (T0−15 min), eight piglets acting as controls received an intravenous injection of saline solution (1 ml kg−1), whereas experimental animals received a single i.v. administration of GR205171 (1 ml kg−1) at a dose of 0.01 (n=8), 0.03 (n=8), 0.1 (n=8), 0.3 (n=16) or 1.0 (n=13) mg kg−1. In eight additional piglets, GR205171 (1 mg kg−1) was administered 15 min before the onset of the delayed phase (T16−15 min). A further five piglets received GR205171 (1 mg kg−1) every 6 h throughout the experiment.

The latencies of the first emetic episode (EE) and nausea‐like behavioural episode (NE) increased in all experimental groups treated at T0−15 min, and the total number of both EE and NE during the 60 h was reduced in a dose‐dependent manner. In piglets treated at T0−15 min with GR205171 1 mg kg−1, eight out of 13 (62%) did not vomit throughout the experiment. Animals treated with GR205171 (1 mg kg−1) at T16−15 min exhibited an acute response to cisplatin but did not vomit during the delayed phase. The greatest inhibition of both nausea‐like behaviour and vomiting was observed in piglets receiving multiple injections of GR205171.

These results demonstrate the long‐lasting anti‐emetic effects of GR205171, and confirm the key role of substance P within the emetic reflex.

British Journal of Pharmacology (1998) 124, 1643–1650; doi:10.1038/sj.bjp.0702019
Effect of chronic hypoxia on adrenoceptor responses of ovine foetal umbilical vessels
Authors: Zhang Lubo , Hu Xiangqun , Longo Lawrence D
Published: January 2005

The effects of chronic hypoxia on α1‐adrenoceptor‐mediated contractions were investigated in foetal umbilical vessels obtained from near‐term (∼140 day gestation) pregnant sheep maintained near sea level (∼300 m) and at high altitude (3820 m) from 30 day gestation.

Chronic hypoxia significantly decreased contractile sensitivity of the umbilical vein to noradrenaline (pD2: 6.22±0.19 vs 5.67±0.09) and reduced the maximum response by 43%. Noradrenaline‐induced contraction of the umbilical artery was abolished. In contrast, contractions to KCl were not affected by chronic hypoxia.

In umbilical vein, the apparent dissociation constant (KA) of noradrenaline to α1‐adrenoceptors was increased from 0.54±0.06 μM in control animals to 1.35±0.14 μM in chronically hypoxic animals. In accordance, radioligand binding of agonist showed high and low affinity binding sites for noradrenaline in both normoxic and chronically hypoxic tissues. Addition of GTPγS (100 μM) abolished apparent high affinity binding sites. Whereas proportional binding sites were not changed by chronic hypoxia, the apparent high affinity of noradrenaline was significantly decreased (pKi: 7.80±0.17 vs 7.20±0.16).

Chronic hypoxia significantly decreased α1‐adrenoceptor density (fmol mg protein−1) in umbilical vein (24.6±3.2 vs 12.3±3.1) and the artery (7.1±0.4 vs 3.1±0.9) with no change in [3H]‐prazosin binding affinity. There was a linear correlation of the maximum contractions to noradrenaline and α1‐adrenoceptor density.

We conclude that chronically hypoxic‐induced depression in contractions of ovine foetal umbilical vessels to noradrenaline is mediated predominantly by decreases in α1‐adrenoceptor density and the agonist binding affinity.

British Journal of Pharmacology (1998) 125, 136–142; doi:10.1038/sj.bjp.0702022