bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibitory effects of cyclic AMP elevating agents on lipopolysaccharide (LPS)‐induced microvascular permeability change in mouse skin
Authors: Irie Kaoru , Fujii Emiko , Ishida Hiroyasu , Wada Keiji , Suganuma Taiyo , Nishikori Tomohiro , Yoshioka Toshimasa , Muraki Takamura
Published: January 2005

Anti‐inflammatory effects of cyclic AMP elevating agents were examined in a mouse model of lipopolysaccharide (LPS)‐induced microvascular permeability change.

Vascular permeability on the back skin was measured by the local accumulation of Pontamine sky blue (PSB) after subcutaneous injection of LPS (400 μg site−1) from Salmonella typhimurium.

Dye leakage in the skin was significantly increased 2 h after injection of LPS. This LPS‐induced dye leakage was suppressed by phosphodiesterase inhibitors, including pentoxifylline (160 mg kg−1), milrinone (5 – 10 mg kg−1), rolipram (0.5 – 10 mg kg−1) and zaprinast (5 – 10 mg kg−1). The dye leakage was also inhibited by β‐adrenoceptor agonists, including isoproterenol (0.5 – 5 mg kg−1) and salbutamol (0.05 – 5 mg kg−1), an adenylate cyclase activator, forskolin (5 mg kg−1), and a cell permeable cyclic AMP analogue, 8‐bromo‐cyclic AMP (8‐Br‐cAMP, 10 mg kg−1).

LPS caused a transient increase in serum TNF‐α level peaking at 1 h after the injection. This increase in serum TNF‐α was completely blocked by a pretreatment with pentoxifylline (160 mg kg−1), milrinone (5 mg kg−1), rolipram (1 mg kg−1), zaprinast (10 mg kg−1), salbutamol (0.5 mg kg−1), forskolin (1 mg kg−1) and 8‐Br‐cAMP (10 mg kg−1).

LPS caused an increase in serum IL‐1α level peaking at 3 h after injection. This increase in serum IL‐1α was not significantly suppressed by the cyclic AMP elevating agents.

Our study suggests that cyclic AMP elevating agents attenuate LPS‐induced microvascular permeability change by suppressing TNF‐α up regulation.

British Journal of Pharmacology (2001) 133, 237–242; doi:10.1038/sj.bjp.0704073
Synergistic protective effect of caspase inhibitors and bFGF against brain injury induced by transient focal ischaemia
Authors: Ma Jianya , Qiu Jianhua , Hirt Lorenz , Dalkara Turgay , Moskowitz Michael A
Published: January 2005

We tested the hypothesis that combined use of trophic factors and caspase inhibitors increases brain resistance to ischaemia in mice.

Intracerebroventricular administration of bFGF (>10 ng) 30 min after MCA occlusion decreased infarct size and neurological deficit in a dose‐dependent manner following 2 h ischemia and reperfusion (20 h).

Combined administration of the subthreshold doses of bFGF (3 ng) and caspase inhibitors (z‐VAD.FMK, 27 ng or z‐DEVD.FMK, 80 mg) reduced infarct volume by 60%, and reduced neurological deficit.

Treatment with a subthreshold dose of bFGF (3 ng) extended the therapeutic window for z‐DEVD.FMK (480 ng) from 1 to 3 h after reperfusion.

Caspase‐3 activity in the ischaemic brain was increased 30 min and 2 h after reperfusion but, was significantly reduced in bFGF‐treated animals by 29 and 16%, respectively. Caspase‐3 activity was not reduced by a direct bFGF effect because addition of bFGF (10 nM – 2 μM) did not decrease recombinant caspase‐3 activity, in vitro.

Our data show that combining caspase inhibitors and bFGF lengthens the treatment window for the second treatment, plus lowers the dosage requirements for neuroprotection. These findings are important because low doses of caspase inhibitors or bFGF reduce the possibility of side effects plus extend the short treatment window for ischaemic stroke.

British Journal of Pharmacology (2001) 133, 345–350; doi:10.1038/sj.bjp.0704075
Mechanisms of leukotriene D4‐induced constriction in human small bronchioles
Authors: Snetkov V A , Hapgood K J , McVicker C G , Lee T H , Ward J P T
Published: January 2005

We examined the mechanisms underlying leukotriene D4‐ (LTD4) induced constriction of human small (300 – 500 μm i.d.) bronchioles, and the effect of LTD4 on ion currents and Ca2+ transients in smooth muscle cells (SMC) isolated from these bronchioles.

LTD4 caused a concentration‐dependent bronchoconstriction with an EC50=0.58±0.05 nM (n=7) which was not easily reversible upon washout. This bronchoconstriction was entirely dependent on extracellular Ca2+.

Blockade of L‐type Ca2+ channels with nifedipine (10 μM) reduced LTD4 response by 39±2% (n=8), whilst La3+, Gd3+ and SK&F 96,365 abolished LTD4‐induced bronchoconstriction completely and reversibly, suggesting the majority of Ca2+ entry was via non‐selective cation channels.

Antagonists of PI‐PLC (U73,122 and ET‐18‐OCH3), PLD (propranolol) and PKC (cheleretrine and Ro31‐8220) were without any effect on LTD4‐induced bronchoconstriction, whilst the PC‐PLC inhibitor D609 caused complete relaxation. Inhibition of protein tyrosine kinase with tyrphostin A23 (100 μM) caused about 50% relaxation, although the inactive analogue tyrphostin A1 was without effect.

In freshly isolated SMC from human small bronchioles LTD4 caused a slow increase of intracellular Ca2+ concentration, with a consequent rise of the activity of large conductance Ca2+‐dependent K+ channels and the amplitude of depolarization‐induced outward whole‐cell current. Again, no effect of LTD4 could be observed in the absence of extracellular Ca2+.

We conclude that LTD4 causes constriction of these small bronchioles primarily by activating Ca2+ entry via non‐voltage gated channels, possibly by a PC‐PLC mediated pathway.

British Journal of Pharmacology (2001) 133, 243–252; doi:10.1038/sj.bjp.0704076
Detection of receptor ligands by monitoring selective stabilization of aRenillaluciferase‐tagged, constitutively active mutant, G‐protein‐coupled receptor
Authors: Ramsay Douglas , Bevan Nicola , Rees Stephen , Milligan Graeme
Published: January 2005

The wild‐type β2‐adrenoceptor and a constitutively active mutant of this receptor were C‐terminally tagged with luciferase from the sea pansyRenilla reniformis.

C‐terminal addition ofRenillaluciferase did not substantially alter the levels of expression of either form of the receptor, the elevated constitutive activity of the mutant β2‐adrenoceptor nor the capacity of isoprenaline to elevate cyclic AMP levels in intact cells expressing these constructs.

Treatment of cells expressing constitutively active mutant β2‐adrenoceptor‐Renillaluciferase with antagonist/inverse agonist ligands resulted in upregulation of levels of this polypeptide which could be monitored by the elevated luciferase activity.

The pEC50for ligand‐induced luciferase upregulation and ligand affinity to bind the receptor were highly correlated.

Similar upregulation could be observed following sustained treatment with agonist ligands.

These effects were only observed at a constitutively active mutant of the β2‐adrenoceptor. Co‐expression of the wild‐type β2‐adrenoceptor C‐terminally tagged with the luciferase fromPhotinus pyralisdid not result in ligand‐induced upregulation of the levels of activity of this luciferase.

Co‐expression of the constitutively active mutant β2‐adrenoceptor‐Renillaluciferase and an equivalent mutant of the α1b‐adrenoceptor C‐terminally tagged with green fluorescent protein allowed pharmacological selectivity of adrenoceptor antagonists to be demonstrated.

This approach offers a sensitive and convenient means, which is amenable to high throughput analysis, to monitor ligand binding to a constitutively active mutant receptor.

As no prior knowledge of receptor ligands is required this approach may be suitable to identify ligands at orphan G protein‐coupled receptors.

British Journal of Pharmacology(2001)133, 315–323; doi:10.1038/sj.bjp.0704077
The broad‐spectrum anti‐emetic activity of AS‐8112, a novel dopamine D2, D3 and 5‐HT3 receptors antagonist
Authors: Yoshikawa Takashi , Yoshida Naoyuki , Oka Makoto
Published: January 2005

The anti‐emetic and pharmacological profile of AS‐8112 ((R)‐5‐bromo‐N‐(1‐ethyl‐4‐methylhexahydro‐1H‐1,4‐diazepin‐6‐yl)‐2‐methoxy‐6‐methylamino‐3‐pyridinecarboxamide·2 fumarate), a novel and potent dopamine D2, D3 and 5‐hydroxytryptamine‐3 (5‐HT3) receptors ligand, was investigated in the present study.

In guinea‐pig isolated colon, AS‐8112 produced a rightward shift of the concentration‐response curves of 2‐methyl‐5HT, a 5‐HT3 receptor agonist (pA2 value of 7.04). Other 5‐HT3 receptor antagonists also produced such a shift in the following antagonistic‐potency order: granisetron> ondansetron=AS‐8112>>metoclopramide.

In mice, AS‐8112 (1.0 – 3.0 mg kg−1 s.c.) potently inhibited hypothermia induced by the dopamine D3 receptor agonist; R(+)‐7‐OH‐DPAT (R(+)‐7‐hydroxy‐2‐(N,N‐di‐n‐propylamino)tetraline) (0.3 mg kg−1 s.c.). Domperidone and haloperidol, which have affinity for dopamine D3 receptor, also inhibited R(+)‐7‐OH‐DPAT‐induced hypothermia.

In ferrets or dogs, AS‐8112 dose‐dependently inhibited emesis induced by R(+)‐7‐OH‐DPAT, apomorphine, morphine or cisplatin with ID50 values of 2.22 μg kg−1 s.c., 10.5 μg kg−1 s.c., 14.2 μg kg−1 i.v. and 17.6 μg kg−1 i.v., respectively. Moreover, oral administration of AS‐8112 significantly inhibited emesis induced by these emetogens. AS‐8112 (0.3 mg kg−1 i.v.) significantly inhibited emesis induced by cyclophosphamide and doxorubicin.

In conclusion, AS‐8112 is a potent dopamine D2, D3 and 5‐HT3 receptors antagonist, and a novel anti‐emetic agent with a broad‐spectrum of anti‐emetic activity. These results suggest that this compound is worthy of clinical investigation.

British Journal of Pharmacology (2001) 133, 253–260; doi:10.1038/sj.bjp.0704078
Influence of the 5‐HT2C receptor antagonist SB242,084 on behaviour produced by the 5‐HT2 agonist Ro60‐0175 and the indirect 5‐HT agonist dexfenfluramine
Authors: Higgins G A , Ouagazzal A M , Grottick A J
Published: January 2005

Ro60‐0175 has been described as a selective agonist at the 5‐HT2C receptor, yet it has only 10‐ fold higher affinity at the 5‐HT2C compared to the 5‐HT2A subtype, and equivalent affinity for the 5‐HT2B receptor.

The selective 5‐HT2C receptor antagonist SB242,084 (0.5 mg kg−1 i.p.), blocked the hypoactivity and penile grooming induced by Ro60‐0175 (1 mg kg−1 s.c.). The combination of SB242,084 (0.5 mg kg−1 i.p.) and Ro60‐0175 (3 – 10 mg kg−1) produced a completely different pattern of behaviours including wet‐dog shakes, hyperactivity and back muscle contractions. These latter effects were blocked by the selective 5‐HT2A receptor antagonist MDL100,907 (0.5 mg kg−1 i.p.), but not the 5‐HT2B receptor antagonist SB215,505 (3 mg kg−1 p.o.).

The indirect 5‐HT releaser/reuptake inhibitor dexfenfluramine (1 – 10 mg kg−1 i.p.) produced a mild increase in locomotor activity, penile grooming, and occasional back muscle contractions and wet‐dog shakes. Pre‐treatment with SB242,084 (0.5 mg kg−1), blocked the incidence of penile grooming, and markedly potentiated both the dexfenfluramine‐induced hyperactivity, the incidence of back muscle contractions, and to a lesser extent wet‐dog shakes. Some toxicity was also evident in animals treated with dexfenfluramine (10 mg kg−1)/SB242,084 (0.5 mg kg−1), but not in any other treatment groups.

The hyperactivity and toxicity produced by the dexfenfluramine (10 mg kg−1)/SB242,084 (0.5 mg kg−1) combination was replicated in a further study, and hyperthermia was also recorded. Both hyperthermia and toxicity were blocked by MDL100,907 (0.5 mg kg−1) but not SB215,505 (3 mg kg−1). An attenuation of the hyperlocomotor response was also observed following MDL100,907.

These findings suggest that 5‐HT2C receptor activation can inhibit the expression of behaviours mediated through other 5‐HT receptor subtypes.

British Journal of Pharmacology (2001) 133, 459–466; doi:10.1038/sj.bjp.0704082
Effect of 1‐week treatment with erythropoietin on the vascular endothelial function in anaesthetized rabbits
Authors: Noguchi Katsuhiko , Yamashiro Satoshi , Matsuzaki Toshihiro , Sakanashi Mayuko , Nakasone Junko , Miyagi Kanako , Sakanashi Matao
Published: January 2005

Chronic administration of erythropoietin (EPO) is often associated with hypertension in animals and humans. The aim of this study was to estimate whether 1‐week treatment with EPO can affect the vascular endothelial function.

Rabbits were given with EPO (400 iu kg−1 s.c.) or saline each other day for 1 week. Hypotensive responses to intravenously given acetylcholine (ACh), endothelium‐independent nitric oxide donors (NOC7, nitroprusside and nitroglycerin) and prostaglandin I2 were tested before and after administration of NG‐nitro‐L‐arginine methyl ester (L‐NAME), a specific nitric oxide synthase inhibitor, under pentobarbitone anaesthesia.

Blood haemoglobin concentration in EPO group was significantly higher than that in control group, whereas baseline values of aortic pressure, heart rate and femoral vascular resistance were similar. The dose of ACh (172 ng kg−1) requiring for a 15 mmHg hypotension from the baseline in EPO group was apparently higher than that (55 ng kg−1) in control group. On the contrary, hypotensive responses to NOC7, nitroprusside, nitroglycerin and prostaglandin I2 were comparable between two groups. The extent of ACh‐induced hypotension did not correlate with haemoglobin concentration.

L‐NAME significantly inhibited the ACh‐induced vasodilating response in control group but did not in EPO group.

In another set of rabbits, the same treatment with EPO also decreased vasodilating responses to carbachol, bradykinin and substance P besides ACh as compared with control group.

These results indicate that 1‐week treatment with EPO selectively attenuates depressor responses to endothelium‐dependent vasodilators in anaesthetized rabbits, most likely due to inhibition of endothelial nitric oxide synthase.

British Journal of Pharmacology (2001) 133, 395–405; doi:10.1038/sj.bjp.0704083
The vascular activity of some isoflavone metabolites: implications for a cardioprotective role
Authors: Chin‐Dusting Jaye P F , Fisher Lisa J , Lewis Tamara V , Piekarska Anna , Nestel Paul J , Husband Alan
Published: January 2005

Legume‐derived isoflavones such as genistein, diadzein and equol have been associated with a reduction in risk of cardiovascular disease. In the current study, we explore the vascular activity of several isoflavone metabolites namely dihydrodaidzein, cis and trans‐tetrahydrodaidzein and dehydroequol for potential cardioprotective properties.

Rat isolated aortic rings were used. 17β‐oestradiol, equol, and all four of the metabolites studied significantly antagonized contractile responses to noradrenaline.

The direct vasodilatory action of these compounds were examined and in contrast to 17β‐oestradiol, the vasodilatory effect of which was demonstrated to be endothelium independent, the dilatory action of all four compounds could be inhibited by endothelium denudation.

Further, the dilatory action of both dihydrodaidzein and cis‐tetrahydrodaidzein were inhibited by the nitric oxide synthase inhibitor, Nω‐nitro‐L‐arginine (NOLA), by the soluble guanylate cyclase inhibitor, 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) and by 40 mM KCl. Dilatory responses to dehydroequol and trans‐tetrahydrodaidzein, on the other hand, were inhibited by 40 mM KCL but not by NOLA nor ODQ.

Finally, we examined the protective potential of these compounds in inhibiting endothelium damage by oxidized low density lipoprotein (ox‐LDL). Trans‐tetrahydrodaidzein was at least 10 fold more potent than 17β‐oestradiol in protecting against ox‐LDL induced damage.

We conclude that the isoflavone metabolites, dihydrodaidzein, cis‐ and trans‐tetrahydrodaidzein and dehydroequol, may potentially represent a novel series of cardioprotective therapeutics.

British Journal of Pharmacology (2001) 133, 595–605; doi:10.1038/sj.bjp.0704088
Inhibition of nitric oxide synthase abrogates lipopolysaccharides‐induced up‐regulation of L‐arginine uptake in rat alveolar macrophages
Authors: Hammermann Rainer , Stichnote Christina , Closs Ellen Ildicho , Nawrath Hermann , Racké Kurt
Published: January 2005

It was tested whether the inducible nitric oxide synthase (iNOS) pathway might be involved in lipopolysaccharides‐(LPS)‐induced up‐regulation of L‐arginine transport in rat alveolar macrophages (AMΦ).

AMΦ were cultured in absence or presence of LPS. Nitrite accumulation was determined in culture media and cells were used to study [3H]‐L‐arginine uptake or to isolate RNA for RT – PCR.

Culture in presence of LPS (1 μg ml−1, 20 h) caused 11 fold increase of nitrite accumulation and 2.5 fold increase of [3H]‐L‐arginine uptake.

The inducible NO synthase (iNOS) inhibitor 2‐amino‐5,6‐dihydro‐6‐methyl‐4H‐1,3‐thiazine (AMT) present alone during culture had only marginal effects on [3H]‐L‐arginine uptake. However, AMT present during culture additionally to LPS, suppressed LPS‐induced nitrite accumulation and LPS‐stimulated [3H]‐L‐arginine uptake in the same concentration‐dependent manner. AMT present only for the last 30 min of the culture period had similar effects on [3H]‐L‐arginine uptake. AMT present only during the uptake period also inhibited LPS‐stimulated [3H]‐L‐arginine uptake, but with lower potency.

The inhibitory effect of AMT could not be opposed by the NO releasing compound DETA NONOate.

LPS caused an up‐regulation of the mRNA for the cationic amino acid transporter CAT‐2B, and this effect was not affected by AMT.

AMT (100 μM) did not affect L‐arginine transport studied by electrophysiological techniques in Xenopus laevis oocytes expressing either the human cationic amino acid transporter hCAT‐1 or hCAT‐2B.

In conclusion, iNOS inhibition in rat AMΦ abolished LPS‐activated L‐arginine uptake. This effect appears to be caused by reduced flow of L‐arginine through the iNOS pathway.

British Journal of Pharmacology (2001) 133, 379–386; doi:10.1038/sj.bjp.0704091
Characterization of EP receptor subtypes responsible for prostaglandin E2‐induced pain responses by use of EP1 and EP3 receptor knockout mice
Authors: Minami Toshiaki , Nakano Hiroyuki , Kobayashi Takuya , Sugimoto Yukihiko , Ushikubi Fumitaka , Ichikawa Atsushi , Narumiya Shuh , Ito Seiji
Published: January 2005

Prostaglandin E2 (PGE2) is known to be the principal pro‐inflammatory prostanoid and play an important role in nociception. To identify PGE receptor (EP) subtypes that mediate pain responses to noxious and innocuous stimuli, we studied them by use of EP1 and EP3 knockout (EP1−/− and EP3−/−) mice.

PGE2 could induce mechanical allodynia in EP1+/+, EP3+/+ and EP3−/− mice, but not in EP1−/− mice. N‐methyl‐D‐aspartate (NMDA), the substrate of nitric oxide (NO) synthase L‐arginine, or the NO donor sodium nitroprusside administered intrathecal (i.t.) could induce allodynia in EP3−/− and EP1−/− mice. Activation of EP1 receptors appears to be upstream, rather than downstream, of NMDA receptor activation and NO production in the PGE2‐induced allodynia.

Although PGE2 produced thermal hyperalgesia over a wide range of dosages from 50 pg to 0.5 μg kg−1 in EP3+/+ mice, it showed a monophasic hyperalgesic action at 5 ng kg−1 or higher doses in EP3−/− mice. The selective EP3 agonist, ONO‐AE‐248, induced hyperalgesia at 500 pg kg−1 in EP3+/+ mice, but not in EP3−/− mice.

Saline‐injected EP1−/− mice showed hyperalgesia, which was reversed by i.t. PGE2 in a dose‐dependent manner.

There was no significant difference in the formalin‐induced behaviours between EP1−/− or EP3−/− mice and the cognate wild‐type mice.

These results demonstrate that spinal EP1 receptors are involved in the PGE2‐induced allodynia and that spinal EP3 receptors are involved in the hyperalgesia induced by low doses of PGE2. However, the formalin‐induced pain cannot be ascribed to a single EP receptor subtype EP1 or EP3.

British Journal of Pharmacology (2001) 133, 438–444; doi:10.1038/sj.bjp.0704092
Dexamethasone inhibits TNF‐α‐induced apoptosis and IAP protein downregulation in MCF‐7 cells
Authors: Messmer Udo K , Pereda‐Fernandez Carmen , Manderscheid Markus , Pfeilschifter Josef
Published: January 2005

Exposure of human mammary carcinoma cell line MCF‐7 to TNF‐α leads to apoptotic cell death within 24 h. In search for apoptosis‐preventing signals, we identified glucocorticoids as potent death‐preventing compounds. Ten nM dexamethasone provided a significant protective effect whereas 100 nM dexamethasone roughly blocked 80 – 90% of TNF‐α‐induced apoptosis.

Surprisingly, dexamethasone exerted a protective effect even when supplied several hours after TNF‐α. This points to a powerful inhibition of even advanced apoptotic processes by dexamethasone.

To further pinpoint the anti‐apoptotic glucocorticoid action, we investigated the expression levels of several members of the inhibitors of apoptosis (IAPs) family of proteins in response to TNF‐α and dexamethasone. IAP proteins directly block caspase protease activities including caspase‐3, caspase‐7, and caspase‐9. Exposure of MCF‐7 cells to TNF caused an extensive downregulation of cIAP1, cIAP2, and XIAP protein levels. The decline of the IAP protein levels temporally paralleled the appearance of apoptotic DNA fragments which started 12 – 14 h following TNF‐α addition and maximal effects were seen within 24 h.

Coincubation of cells with TNF‐α and dexamethasone potently blocked cIAP1, cIAP2, and XIAP downregulation.

TNF‐α‐mediated IAP protein downregulation was not affected by proteasome inhibitors like lactacystin, ALLN or ALLM, whereas it was blocked by the broad‐spectrum caspase inhibitor Z‐VAD‐fmk which also prevented TNF‐α‐induced apoptotic cell death. These data suggest that inhibition of IAP downregulation mediated by a caspase proteolytic activity constitutes the anti‐apoptotic action of glucocorticoids in MCF‐7 carcinoma cells.

British Journal of Pharmacology (2001) 133, 467–476; doi:10.1038/sj.bjp.0704093
Cyclic AMP elevating agents and nitric oxide modulate angiotensin II‐induced leukocyte‐endothelial cell interactions in vivo
Authors: Alvarez Angeles , Piqueras Laura , Blazquez Maria‐Amparo , Sanz Maria‐Jesus
Published: January 2005

Angiotensin (Ang‐II) is a key molecule in the development of cardiac ischaemic disorders and displays proinflammatory activity in vivo. Since intracellular cyclic nucleotides elevating agents have proved to be effective modulators of leukocyte recruitment, we have evaluated their effect on Ang‐II‐induced leukocyte‐endothelial cell interactions in vivo using intravital microscopy within the rat mesenteric microcirculation.

Pretreatment with iloprost significantly inhibited (1 nM) Ang‐II‐induced increase in leukocyte rolling flux, adhesion and emigration at 60 min by 96, 92 and 90% respectively, and returned leukocyte rolling velocity to basal levels. Pretreatment with salbutamol or co‐superfusion with forskolin exerted similar effects.

When theophylline was administered, leukocyte rolling flux, adhesion and emigration elicited by Ang‐II were significantly attenuated by 81, 89 and 71% respectively. Rolipram administration caused similar reduction of Ang‐II‐induced leukocyte responses.

Co‐superfusion of Ang‐II with the NO‐donor, spermine‐NO, or 8‐Br‐cyclic GMP, or pretreatment with a transdermal nytroglycerin patch, resulted in a significant reduction of the leukocyte‐endothelial cell interactions elicited by Ang‐II.

Salbutamol preadministration did not modify leukocyte‐endothelial cell interactions elicited by either L‐NAME or L‐NAME+Ang‐II, indicating that the inhibitory leukocyte effects caused by cyclic AMP‐elevating agents are mediated through NO release.

In conclusion, we have provided evidence that cyclic AMP elevating agents and NO donors, are potent inhibitors of Ang‐II‐induced leukocyte‐endothelial cell interactions. Thus, they could constitute a powerful therapeutical tool in the control of the leukocyte recruitment characteristic of the vascular lesions that occur in cardiovascular disease states where Ang‐II plays a critical role.

British Journal of Pharmacology (2001) 133, 485–494; doi:10.1038/sj.bjp.0704096
Involvement of the NH2 terminal domain of catecholamine transporters in the Na+ and Cl−‐dependence of a [3H]‐dopamine uptake
Authors: Syringas Maria , Janin François , Giros Bruno , Costentin Jean , Bonnet Jean‐Jacques
Published: January 2005

The ionic dependence of the [3H]‐dopamine uptake was studied in transfected cells expressing the human neuronal transporter for dopamine (hDAT) or noradrenaline (hNET), and chimeric transporters resulting from the symmetrical exchange of the region from the NH2 terminal through the first two transmembrane domains (cassette I). Chimera A is formed by hDAT comprising cassette I from hNET, whereas chimera B corresponds to the reverse construct.

The appearance or the intensity of a Cl−‐independent component of transport was linked to the presence of the COOH terminal part of hNET in both monoclonal and polyclonal Ltk− cells (Cl− substituted by isethionate and NO3−, respectively), and in transiently transfected COS‐7 cells.

Cassette I was also involved in the Cl−‐dependence because the transport activity of polyclonal Ltk− cells expressing A was partly Cl−‐independent and because Ltk− cells expressing transporters containing cassette I of hDAT displayed higher KmCl− values than cells expressing the reverse constructs.

In monoclonal Ltk− cell lines, KmNa+ values and biphasic vs monophasic dependence upon Na+ concentrations differentiate transporters containing cassette I of hNET from those containing cassette I of hDAT. In COS‐7 cells, the exchange of cassette I produced a significant change in Hill number values.

In Na+‐dependence studies, exchange of the COOH terminal part significantly modified Hill number values in both Ltk− and COS‐7 cells.

Hill number values close to two were found for hNET and hDAT when sucrose was used as substitute for NaCl.

The NH2 terminal part of the transporters bears some of the differences in the Na+ and Cl−‐dependence of the uptake that are observed between hDAT and hNET. Present results also support a role of the COOH terminal part in the ionic dependence.

British Journal of Pharmacology (2001) 133, 387–394; doi:10.1038/sj.bjp.0704097
Ergolide, sesquiterpene lactone from Inula britannica, inhibits inducible nitric oxide synthase and cyclo‐oxygenase‐2 expression in RAW 264.7 macrophages through the inactivation of NF‐κB
Authors: Whan Han Jeung , Gon Lee Byeong , Kee Kim Yong , Woo Yoon Jong , Kyoung Jin Hye , Hong Sungyoul , Young Lee Hoi , Ro Lee Kang , Woo Lee Hyang
Published: January 2005

We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) and cyclo‐oxygenase‐2 (COX‐2) by ergolide, sesquiterpene lactone from Inula britannica.

iNOS activity in cell‐free extract of LPS/IFN‐γ‐stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its inhibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN‐γ‐stimulated RAW 264.7 macrophages in a concentration‐dependent manner. However, its inhibitory effect does not result from direct inhibition of the catalytic activity of NOS.

Ergolide markedly decreased the production of prostaglandin E2 (PGE2) in cell‐free extract of LPS/IFN‐γ‐stimulated RAW 264.7 macrophages in a concentration‐dependent manner, without alteration of the catalytic activity of COX‐2 itself.

Ergolide decreased the level of iNOS and COX‐2 protein, and iNOS mRNA caused by stimulation of LPS/IFN‐γ in a concentration‐dependent manner, as measured by Western blot and Northern blot analysis, respectively.

Ergolide inhibited nuclear factor‐κB (NF‐κB) activation, a transcription factor necessary for iNOS and COX‐2 expression in response to LPS/IFN‐γ. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF‐κB as well as IκB‐α degradation. In addition, these effects were completely blocked by treatment of cysteine, indicating that this inhibitory effect of ergolide could be mediated by alkylation of NF‐κB itself or an upstream molecule of NF‐κB.

Ergolide also directly inhibited the DNA‐binding activity of active NF‐κB in LPS/IFN‐γ‐pretreated RAW 264.7 macrophages.

These results demonstrate that the suppression of NF‐κB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF‐κB resulted from blockade of the degradation of IκB and the direct modification of active NF‐κB, leading to the suppression of the expression of iNOS and COX‐2, which play important roles in inflammatory signalling pathway.

British Journal of Pharmacology (2001) 133, 503–512; doi:10.1038/sj.bjp.0704099
Regional haemodynamic responses to the cannabinoid agonist, WIN 55212‐2, in conscious, normotensive rats, and in hypertensive, transgenic rats
Authors: Gardiner S M , March J E , Kemp P A , Bennett T
Published: January 2005

Regional haemodynamic responses to the cannabinoid agonist, WIN 55212‐2 (5 – 250 μg kg−1 i.v.) were assessed in conscious, normotensive, Hannover, Sprague‐Dawley (HSD) rats, and in hypertensive, transgenic ((mRen‐2)27) (abbreviated to TG) rats.

In HSD rats, WIN 55212‐2 caused pressor, and renal and mesenteric vasoconstrictor effects, with a hindquarters vasodilator effect occurring only at the highest dose. In TG rats, the effects of the cannabinoid agonist were qualitatively similar to those seen in HSD rats, except there was no hindquarters vasodilatation.

In both strains of rat, in the presence of losartan, pentolinium and a vasopressin (V1‐receptor) antagonist, the pressor and vasoconstrictor effects of WIN 55212‐2 were abolished, but the hindquarters vasodilator response was enhanced (HSD rats) or was seen only in that circumstance (TG rats). Under these conditions, both strains of rat showed a modest fall in blood pressure, together with mesenteric vasodilatation.

In additional experiments in normotensive SD rats from Charles River (CRSD), it was shown that, in the presence of the V1‐receptor antagonist alone, or losartan alone, or the two antagonists together, the cardiovascular effects of WIN 55212‐2 (50 or 150 μg kg−1) were not attenuated. Hence, the effects described above were likely due to pentolinium.

There were no consistent differences between HSD and TG rats in their haemodynamic responses to methoxamine or noradrenaline, indicating the two strains were not likely to differ markedly in their responsiveness to any putative sympathetic activation induced by WIN 55212‐2.

Collectively, the results indicate that the predominant cardiovascular effects of WIN 55212‐2 in conscious HSD and TG rats (i.e., pressor and vasoconstrictor actions) can be attributed largely to indirect, pentolinium‐sensitive mechanisms, which appear to differ little in the normotensive and hypertensive state, at least in conscious animals. Under the conditions of our experiments, signs of cannabinoid‐induced vasodilatation were modest.

British Journal of Pharmacology (2001) 133, 445–453; doi:10.1038/sj.bjp.0704100
Platelet‐activating factor and endotoxin activate CCAAT/enhancer binding protein in rat small intestine
Authors: Wang Hao , Qu Xiaowu , De Plaen Isabelle G , Hsueh Wei
Published: January 2005

The transcription factor family CCAAT/enhancer binding proteins (C/EBP) is involved in inflammation via the regulation of the gene expression of various pro‐inflammatory cytokines and proteins. PAF and endotoxin (lipopolysaccharide, LPS) are known agents causing intestinal inflammation and injury. In this study, we examined the binding activity of C/EBP isoforms in rat small intestine in response to PAF (1.5 μg kg−1, i.v.) or LPS (5 mg kg−1, i.v.).

We found that C/EBP is constitutively active in normal small intestine, mainly as C/EBP‐α and β (C/EBP‐β>α). Both C/EBP‐α and β are localized in the intestinal epithelial cells: C/EBP‐α mainly in the crypts, and C/EBP‐β in both villi and crypts, as well as in some lamina propria cells. Only minute amounts of C/EBP‐δ were found.

PAF rapidly upregulates the binding activity of C/EBP‐α and β within 30 min. The increase in C/EBP‐α is prominent in the crypt cells, whereas the change of C/EBP‐β is more widespread. LPS also increases the binding activity of C/EBP‐α and β, and the response is slower than PAF. PAF synergizes with LPS to markedly activate all three subunits. The increase in C/EBP‐α is transient, whereas the other two have a sustained elevation until 120 min.

After challenge with PAF (but not LPS), small amounts of nuclear factor ‐κB (NF‐κB) p50 and p65 subunits are found in the C/EBP‐DNA binding complex, indicating cross‐dimerization of the two transcription families. Pretreatment of rats with pyrrolidine dithiocarbamate (PDTC) suppresses LPS‐, but not PAF‐, induced NF‐κB and C/EBP binding activity, and significantly increases the C/EBP‐δ subunit in LPS‐ or PAF‐induced C/EBP complex.

These results suggest that PAF and LPS activate intestinal C/EBP in vivo, probably via different pathways.

British Journal of Pharmacology (2001) 133, 713–721; doi:10.1038/sj.bjp.0704102
Nitric oxide‐ and nitric oxide donors‐induced relaxation and its modulation by oxidative stress in piglet pulmonary arteries
Authors: López‐López José Gustavo , Pérez‐Vizcaíno Francisco , Cogolludo Angel L , Ibarra Manuel , Zaragozá‐Arnáez Francisco , Tamargo Juan
Published: January 2005

Inhaled nitric oxide (iNO) is widely used in the treatment of pulmonary hypertension while inhaled NO donors have been suggested as an alternative therapy. The differential susceptibility to inactivation by oxidative stress and oxyhaemoglobin of NO and two NO donors, sodium nitroprusside (SNP) and S‐nitroso‐N‐acetyl‐penicillamine (SNAP) were analysed in isolated endothelium‐denuded pulmonary arteries from 2‐week‐old piglets stimulated with U46619.

NO, SNAP and SNP relaxed the arteries (pIC30=7.73±0.12, 7.26±0.17 and 6.43±0.13, respectively) but NO was not detected electrochemically in the bath after the addition of SNP and only at concentrations at which SNAP produced more than 50% relaxation.

The sGC inhibitor ODQ (10−6 M) or the sarcoplasmic Ca2+‐ATPase thapsigargin (2×10−6 M) markedly inhibited the relaxation induced by NO, SNAP and SNP.

Addition of oxyhaemoglobin (3×10−7 M) or diethyldithiocarbamate (1 mM) markedly inhibited NO‐ (pIC30=6.88±0.07 and 6.92±0.18, respectively), weakly inhibited SNAP‐ and had no effect on SNP‐induced relaxation. Xanthine oxidase (5 mu ml−1) plus hypoxanthine (10−4 M) markedly inhibited NO‐ (pIC30=6.96±0.12) but not SNAP‐ or SNP‐induced relaxation.

Superoxide dismutase (SOD), MnCl2, diphenileneiodonium and exposing the luminal surface of the rings outwards (inversion) potentiated the relaxant responses of NO (pIC30=8.52±0.16, 8.23±0.11, 8.01±0.11 and 8.20±0.10, respectively). However, SOD did not modify the NO detected by the electrode and had no effect on SNAP‐ or SNP‐induced relaxation.

Therefore, the kinetics and local distribution of NO release of NO donors influence the susceptibility to the scavenging effects of oxyhaemoglobin and superoxide.

British Journal of Pharmacology (2001) 133, 615–624; doi:10.1038/sj.bjp.0704103
Pharmacological discrimination between effects of carbamazepine on hippocampal basal, Ca2+‐ and K+‐evoked serotonin release
Authors: Kawata Yuko , Okada Motohiro , Murakami Takuya , Kamata Akihisa , Zhu Gang , Kaneko Sunao
Published: January 2005

To elucidate mechanisms of hippocampal serotonin release and possible mechanisms of clinical action of carbamazepine (CBZ), we determined interaction between antagonists of N‐type (ω‐conotoxin GVIA:GVIA), P‐type (ω‐agatoxin IVA:IVA) Ca2+ channels, Na+ channel (tetrodotoxin: TTX) and CBZ on hippocampal basal, Ca2+‐ and K+‐evoked serotonin releases, using microdialysis in freely moving rats.

Basal release was reduced by TTX, GVIA and IVA (GVIA>IVA). Ca2+‐evoked release was reduced by GVIA but unaffected by TTX and IVA. K+‐evoked release was reduced by TTX, GVIA and IVA (GVIA<IVA).

TTX inhibited actions of IVA and GVIA on respective basal and K+‐evoked releases, without affecting Ca2+‐evoked release.

Perfusion with 100 μM CBZ (estimated‐concentration in hippocampal tissue: 19±2 μM) enhanced basal and Ca2+‐evoked releases, but reduced K+‐evoked release, whereas 1000 μM CBZ (estimated‐concentration in hippocampal tissue: 188±16 μM) reduced three types of releases.

Under condition of pretreatment with 100 and 1000 μM CBZ, TTX unaffected basal and K+‐evoked releases. Under condition of pretreatment with 100 μM CBZ, IVA and GVIA unaffected basal and K+‐evoked releases, respectively, but GVIA reduced basal, Ca2+‐evoked releases and IVA also reduced K+‐evoked release. Under condition of pretreatment with 1000 μM CBZ, GVIA unaffected three types of releases, and IVA unaffected basal release but reduced K+‐evoked release.

These findings contribute towards the possible mechanisms of concentration‐dependent antiepileptic action of CBZ, which possibly inhibits Na+ channel related neurotransmitter release mechanisms during K+‐evoked stage, and simultaneously enhances N‐type Ca2+ channel related basal serotonin release at the resting stage.

British Journal of Pharmacology (2001) 133, 557–567; doi:10.1038/sj.bjp.0704104
Differential cardiovascular responses to blockade of nNOS or iNOS in rostral ventrolateral medulla of the rat
Authors: Chan Samuel H H , Wang Ling‐Lin , Wang Shu‐Huei , Chan Julie Y H
Published: January 2005

We investigated the contribution of neuronal or inducible nitric oxide synthase (nNOS or iNOS) at the rostral ventrolateral medulla (RVLM) to central cardiovascular regulation by endogenous nitric oxide (NO), using Sprague‐Dawley rats anaesthetized and maintained with propofol.

Microinjection bilaterally into the RVLM of a NO trapping agent, carboxy‐2‐phenyl‐4,4,5,5‐tetramethylimidazoline‐l‐oxy‐l‐3‐oxide (10, 50 or 100 nmoles) resulted in significant hypotension and bradycardia.

Similar application of a selective antagonist of nNOS, 7‐nitroindazole (1, 2.5 or 5 pmoles), or selective antagonists of iNOS, aminoguanidine (125, 250 or 500 pmoles), N6‐(l‐iminoethyl)‐L‐lysine (250 pmoles) or S‐methylisothiourea (250 pmoles), induced respectively a reduction or an enhancement in systemic arterial pressure, heart rate and power density of the vasomotor components in the spectrum of arterial blood pressure signals, the experimental index for sympathetic neurogenic vasomotor tone.

Both hypotension and bradycardia induced by the NO precursor, L‐arginine (100 nmoles), were significantly blunted when aminoguanidine (250 pmoles) was co‐microinjected bilaterally into the RVLM. On the other hand, co‐administered 7‐nitroindazole (2.5 pmoles) was ineffective.

Whereas low doses of S‐nitro‐N‐acetylpenicillamine (0.25 or 0.5 nmoles) elicited hypertension and tachycardia, high doses of this non‐nitrate NO donor (5 nmoles) induced hypotension and bradycardia.

Reverse transcription – polymerase chain reaction analysis revealed that both iNOS and nNOS mRNA were expressed in the ventrolateral medulla.

We conclude that the prevalence of nNOS over iNOS activity at the RVLM and the associated dominance of sympathoexcitation over sympathoinhibition may underlie the maintenance of sympathetic vasomotor outflow and stable systemic arterial pressure by the endogenous NO.

British Journal of Pharmacology (2001) 133, 606–614; doi:10.1038/sj.bjp.0704105
Role of protein kinase C in the reduction of infarct size by N‐methyl‐1‐deoxynojirimycin, an α‐1,6‐glucosidase inhibitor
Authors: Arai Masazumi , Minatoguchi Shinya , Kumada Hirokazu , Uno Yoshihiro , Nishida Yoshio , Hashimoto Kazuaki , Wang Ningyuan , Takemura Genzou , Fujiwara Takako , Higashioka Masaya , Kuwano Keiichi , Fujiwara Hisayoshi
Published: January 2005

Preischaemic treatment with N‐methyl‐1‐deoxynojirimycin (MOR‐14), an α‐1,6‐glucosidase inhibitor, attenuates glycogenolysis and lactate accumulation during ischaemia and markedly reduces infarct size in rabbit hearts. In the present study, we have investigated whether protein kinase C (PKC), a principal mediator of ischaemic preconditioning, is also involved in the cardioprotective effect of MOR‐14.

To assess the effect of PKC inhibition on infarct size in MOR‐14‐treated hearts, 38 rabbits were subjected to 30 min of ischaemia followed by 48 h of reperfusion. Infarct size, as a per cent of area at risk, was significantly smaller in rabbits administered 100 mg kg−1 of MOR‐14 10 min before ischaemia (17±2%, n=10), than in a control group (46±5%, n=10). This beneficial effect of MOR‐14 was abolished when 5 mg kg−1 of chelerythrine, a PKC inhibitor, was given 10 min prior to MOR‐14 injection (39±4%, n=10), although chelerythrine alone did not alter infarct size (43±4%, n=8). Further, chelerythrine had no effect on MOR‐14‐induced attenuation of glycogen breakdown and lactate accumulation in hearts excised at 30 min of ischaemia.

Immunoblot analysis of PKC in homogenates of Langendorff‐perfused rabbit hearts revealed that MOR‐14 significantly increased levels of PKC‐ε in the particulate fraction at 20 and 30 min of ischaemia and in the cytosolic fraction at 30 min of ischaemia.

Taken as a whole, our data suggest that PKC acts downstream of the inhibition of glycogenolysis by MOR‐14 to reduce infarct size. Thus, activation of PKC is a more direct mediator of the cardioprotection afforded by MOR‐14 than is inhibition of glycogenolysis.

British Journal of Pharmacology (2001) 133, 635–642; doi:10.1038/sj.bjp.0704107
Tolbutamide stimulation of pancreatic β‐cells involves both cell recruitment and increase in the individual Ca2+ response
Authors: Jonkers Françoise C , Guiot Yves , Rahier Jacques , Henquin Jean‐Claude
Published: January 2005

Individual pancreatic β‐cells are functionally heterogeneous. Their sensitivity to glucose is variable, so that the proportion of active cells increases with the glucose concentration (recruitment).

We have investigated whether sulphonylureas also recruit β‐cells, by measuring cytoplasmic Ca2+ ([Ca2+]i) – the triggering signal of insulin secretion – in single cells and clusters of cells prepared from mouse islets.

In 4 mM glucose, the threshold concentration of tolbutamide inducing a [Ca2+]i rise was variable (5 – 50 μM). The proportion of responsive cells and clusters therefore increased with the tolbutamide concentration, to reach a maximum of 90% of the cells and 100% of the clusters. This recruitment occurred faster when the glucose concentration was increased from 4 to 5 mM (EC50 of ∼14 and ∼4 μM tolbutamide respectively).

Within responsive clusters little recruitment was observed; when a cluster was active, all or nearly all cells were active probably because of cell coupling. Thus, tolbutamide‐induced [Ca2+]i oscillations were synchronous in all cells of each cluster, whereas there was no synchrony between clusters or individual cells.

Independently of cell recruitment, tolbutamide gradually augmented the magnitude of the [Ca2+]i rise in single cells and clusters. This increase occurred over a broader range of concentrations than did recruitment (EC50 of ∼50 and 25 μM tolbutamide at 4 and 5 mM glucose respectively).

Tolbutamide (10 μM) accelerated the recruitment of single cells and clusters brought about by increasing glucose concentrations (range of 3 – 7 mM instead of 4 – 10 mM glucose), and potentiated the amplification of the individual responses that glucose also produced.

In conclusion, both metabolic (glucose) and pharmacologic (sulphonylurea) inhibition of K+‐ATP channels recruits β‐cells to generate a [Ca2+]i response. However, the response is not of an all‐or‐none type; it increases in amplitude with the concentration of either glucose or tolbutamide.

British Journal of Pharmacology (2001) 133, 575–585; doi:10.1038/sj.bjp.0704108
The effects of flecainide on ATP‐sensitive K+ channels in pig urethral myocytes
Authors: Yunoki Takakazu , Teramoto Noriyoshi , Naito Seiji , Ito Yushi
Published: January 2005

The effects of the antiarrhythmic drug flecainide on levcromakalim‐induced hyperpolarization, macroscopic and unitary K+ currents in pig urethra were investigated using patch‐clamp techniques. The effects of flecainide were also examined on currents in inside‐out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K+ channel (Kir6.2) subunits (i.e. Kir6.2ΔC36) which form ATP‐sensitive K+ channels (KATP channels).

In current‐clamp mode, application of flecainide (100 μM) caused a significant depolarization after the membrane potential had been hyperpolarized by levcromakalim.

In voltage‐clamp experiments, the levcromakalim‐induced outward current was suppressed by 300 μM flecainide in quasi‐physiological K+ conditions (Ki=51 μM). In contrast, approximately 20% of the levcromakalim‐induced inward current still remained even after application of 300 μM flecainide in symmetrical 140 mM K+ conditions (Ki=51 μM). In contrast, approximately 20% of the levcromakalim‐induced inwar=126 μM).

In cell‐attached configuration, the channel activity of the levcromakalim‐induced KATP channels was reversibly inhibited by flecainide (30 μM) at −50 mV. Their activity was also suppressed by either disopyramide or cibenzoline.

Flecainide reversibly inhibited the channel activity of Kir6.2ΔC36 expressed in COS7 cells using inside‐out configuration.

Inhibitory effects of flecainide on the levcromakalim‐induced currents became more potent when the value of external pH increased, although this slightly reduced the proportion of drug molecules carrying a positive charge.

These results suggest that flecainide inhibits channel activity through blocking the pore site of the KATP channel in pig urethra.

British Journal of Pharmacology (2001) 133, 730–738; doi:10.1038/sj.bjp.0704109
Straight‐chain alcohols exhibit a cutoff in potency for the inhibition of recombinant glutamate receptor subunits
Authors: Akinshola B Emmanuel
Published: January 2005

The effects of n‐alcohols (methanol to 1‐decanol) on kainate‐activated AMPA receptor subunit GluR1 and GluR3 ion currents were studied in Xenopus oocytes using the two‐electrode voltage‐clamp recording technique.

For short‐chain alcohols from methanol to 1‐hexanol, potency for inhibition of GluR1 and GluR3 receptor‐mediated current increased in proportion to the chain length or hydrophobicity of the alcohol.

The IC50 values of these alcohols for GluR1 were: methanol, 702 mM; ethanol, 170 mM; 1‐propanol, 69 mM; 1‐butanol, 20 mM; 1‐pentanol, 17 mM; and 1‐hexanol, 10 mM. For GluR3, IC50 values were: methanol, 712 mM; ethanol, 238 mM; 1‐propanol, 50 mM; 1‐butanol, 32 mM; 1‐pentanol, 13 mM; and 1‐hexanol, 7 mM.

For long‐chain alcohols, 1‐heptanol was less potent than 1‐hexanol (estimated IC50: 19 mM for GluR1 and 18 mM for GluR3), 1‐octanol had little effect only on GluR3, and 1‐nonanol and 1‐decanol did not significantly inhibit both GluR1 and GluR3 responses.

The observations indicate that straight‐chain n‐alcohols exhibit a cutoff in their potency for inhibition of the function of non‐NMDA glutamate receptor subunits, GluR1 and GluR3. The cutoff in potency of n‐alcohols for inhibition of non‐NMDA glutamate receptor function is consistent with the interpretation that alcohols affect the function of these receptor‐channels by interacting with an alcohol binding site of specific dimensions on the receptor protein.

British Journal of Pharmacology (2001) 133, 651–658; doi:10.1038/sj.bjp.0704112