bluuebunny/crossref_metadata_2025 huggingface dataset:
Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide
Authors: Ciucci Alessandra , Palma Carla , Manzini Stefano , Werge Thomas M
Published: January 2005

The binding modalities of substance P and neurokinin A on the wild type and Gly166 to‐Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.

On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high‐affinity binding constant (determined in homologous binding experiments). The Gly166 to‐Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.

On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.

Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one‐site model was unable to accommodate all the experimental data, whereas a two‐site model provided a dramatically better description.

These two receptor‐sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.

The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.

The binding modalities of septide‐like ligands (i.e. neurokinin B, SP(6–11), SP‐methyl ester) are affected similarly to neurokinin A and are better resolved into two sites. The mutation leaves the affinity of these ligands for the two receptor forms unchanged, but increases the fraction of high‐affinity sites. On the other hand, the binding of non‐peptide and peptide antagonists (SR140.333 and FK888) behaved similarly to substance P with a single high affinity site that is unaffected by the mutation.

These findings may suggest that the NK1 receptor exists in two different forms with similar affinity for substance P and NK1 antagonists, but with a high and a low affinity for neurokinin A and septide‐like ligands. Hence, the Gly166 in the NK1 receptor would seem to control the distribution between a pan‐reactive form and a substance P‐selective form of the receptor.

British Journal of Pharmacology (1998) 125, 393–401; doi:10.1038/sj.bjp.0702070
Andrographolide suppresses the expression of inducible nitric oxide synthase in macrophage and restores the vasoconstriction in rat aorta treated with lipopolysaccharide
Authors: Chiou Wen‐Fei , Lin Jin‐Jung , Chen Chieh‐Fu
Published: January 2005

We investigated whether andrographolide, a diterpenoid lactone found at Andrographis paniculata, influences the induction of the inducible nitric oxide synthase (iNOS) in RAW264.7 cells activated by bacterial endotoxin (LPS), as well as in the rats with endotoxic shock and in aortic rings treated with LPS.

Incubation of RAW264.7 cells with andrographolide (1 to 50 μM) inhibited the LPS (1 μg ml−1)‐induced nitrite accumulation in concentration‐ and time‐dependent manners. Maximum inhibition was observed when andrographolide was added together with LPS and decreased progressively as the interval between andrographolide and LPS was increased to 20 h.

Western blot analysis demonstrated that iNOS expression was markedly attenuated in the presence of andrographolide for 6–24 h, suggesting that andrographolide inhibited iNOS protein induction.

Thoracic aorta incubation with LPS (300 ng ml−1) for 5 h in vitro exhibited a significant decrease in the maximal contractile response to phenylephrine (10−9–10−5 M). Andrographolide (30 μM) restored the contractile response to control level.

In anaesthetized rats, LPS (10 mg kg−1, i.v.) caused a fall in mean arterial blood pressure (MAP) from 116±4 to 77±5 mmHg. The pressor effect of phenylephrine (10 μg ml−1, i.v.) was also significantly reduced at 30, 60, 120 and 180 min after LPS injection. In contrast, animals pretreated with andrographolide (1 mg kg−1, i.v., 20 min prior to LPS) maintained a significantly higher MAP when compared to LPS‐rats given with vehicle. Administration of andrographolide 60 min after LPS caused a increase in MAP and significantly reversed the reduction of the pressor response to phenylephrine.

Our results indicated that andrographolide inhibits nitrite synthesis by suppressing expression of iNOS protein in vitro. And, this inhibition of iNOS synthesis may contribute to the beneficial haemodynamic effects of andrographolide in endotoxic shock.

British Journal of Pharmacology (1998) 125, 327–334; doi:10.1038/sj.bjp.0702073
Essential role for endothelin ETB receptors in fever induced by LPS (E. coli) in rats
Authors: Fabricio Aline S C , Silva Carlos A A , Rae Giles A , D'Orléans‐Juste Pedro , Souza Glória E P
Published: January 2005

The influence of endothelin receptor antagonists on febrile responses to E. coli lipopolysaccharide (LPS), interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α) and endothelin‐1 (ET‐1) was assessed in conscious rats.

Intravenous (i.v.) LPS (5.0 μg kg−1) markedly increased rectal temperature to a peak of 1.30°C over baseline at 2.5 h. Pretreatment with the mixed endothelin ETA/ETB receptor antagonist bosentan (10 mg kg−1, i.v.) or the selective endothelin ETB receptor antagonist BQ‐788 (N‐cis‐2,6‐dimethyl‐piperidinocarbonyl‐L‐γ‐methylleucyl‐D‐1‐methoxycarboyl‐D‐norleucine; 3 pmol, into a lateral cerebral ventricle–i.c.v.) reduced the peak response to LPS to 0.90 and 0.75°C, respectively. The selective endothelin ETA receptor antagonist BQ‐123 (cyclo[D‐Trp‐D‐Asp‐Pro‐D‐Val‐Leu]; 3 pmol, i.c.v.) was ineffective.

Increases in temperature caused by IL‐1β (180 fmol, i.c.v.), TNF‐α (14.4 pmol, i.c.v.) or IL‐1β (150 pmol kg−1, i.v.) were unaffected by BQ‐788 (3 pmol, i.c.v.).

Central injection of endothelin‐1 (0.1 to 3 fmol, i.c.v.) caused slowly‐developing and long‐lasting increases in rectal temperature (starting 2 h after administration and peaking at 4–6 h between 0.90 and 1.15°C) which were not clearly dose‐dependent. The response to endothelin‐1 (1 fmol, i.c.v.) was prevented by BQ‐788, but not by BQ‐123 (each at 3 pmol, i.c.v.). Intraperitoneal pretreatment with the cyclo‐oxygenase inhibitor indomethacin (2 mg kg−1), which partially reduced LPS‐induced fever, did not modify the hyperthermic response to endothelin‐1 (3 fmol, i.c.v.).

Therefore, central endothelin(s) participates importantly in the development of LPS‐induced fever, via activation of a prostanoid‐independent endothelin ETB receptor‐mediated mechanism possibly not situated downstream from IL‐1β or TNF‐α in the fever cascade.

British Journal of Pharmacology (1998) 125, 542–548; doi:10.1038/sj.bjp.0702075
Bradykinin‐evoked sensitization of neuropeptide release from afferent neurons in the guinea‐pig lung
Authors: Schuligoi R , Peskar B A , Donnerer J , Amann R
Published: January 2005

It has been shown that bradykinin (BK) causes sensitization of airway sensory neurons and an enhancement of the cough reflex in guinea‐pigs. In the present study, the guineapig isolated perfused lung was used to investigate the possible enhancement by BK of histamine‐evoked neuropeptide release from peripheral terminals of primary afferent neurons, and to determine the contribution of cyclooxygenase products of arachidonate metabolism to this effect.

The lung was perfused with oxygenated physiological salt solution containing peptidase inhibitors (thiorphan, bestatin and captopril, 1 μM each). BK and histamine were added to the perfusate for 10 and 5 min, respectively.

BK alone (0.1 μM) evoked the release of 10.35±2.4 fmol immunoreactive calcitonin gene‐related peptide (CGRP), histamine alone (100 μM) evoked the release of 12.7±1.6 fmol CGRP. Stimulation with 100 μM histamine in the presence of 0.1 μM BK (added 5 min before histamine and present during histamine) evoked the release of 67.1±5.3 fmol CGRP.

Prostaglandin (PG) release was stimulated by BK (418±71 pmol 15‐keto‐13,14‐dihydro‐PGF2α and 345±59 pmol 6‐keto‐PGF1α), and, to a lesser extent, by histamine (36.1±7.4 pmol 15‐keto‐13,14‐dihydro‐PGF2α, and 24.6±3.9 pmol 6‐keto‐PGF1α). Prostaglandin release induced by histamine in the presence of BK was not significantly higher than with BK alone.

Indomethacin (5 μM) as well as the bradykinin B2 receptor antagonist HOE140 (icatibant, 1 μM) inhibited prostaglandin release following stimulation with histamine in combination with BK. CGRP release evoked by histamine in combination with BK was attenuated by indomethacin and HOE140 to 22.1±7.8 fmol and 16.4±3.8 fmol, respectively, significantly less than the value obtained in control experiments (67.1±5.3 fmol).

The results suggest that BK‐induced stimulation of prostaglandin synthesis results in facilitation of histamine–evoked release of pro‐inflammatory neuropeptides from afferent neurons, a mechanism that probably becomes relevant during inflammation, and that can be blocked by a bradykinin B2 receptor antagonist.

British Journal of Pharmacology (1998) 125, 388–392; doi:10.1038/sj.bjp.0702079
Subtype selectivity of the novel nonpeptide neuropeptide Y Y1 receptor antagonist BIBO 3304 and its effect on feeding in rodents
Authors: Wieland H A , Engel W , Eberlein W , Rudolf K , Doods H N
Published: January 2005

The novel Y1‐selective argininamide derivative BIBO 3304 ((R)‐N‐[[4‐(aminocarbonylaminomethyl)phenyl]methyl]‐N2‐(diphenylacetyl)‐argininamide trifluoroacetate) has been synthesized and was examined for its subtype selectivity, its in vitro antagonistic properties and its food intake inhibitory properties.

BIBO 3304 displayed subnanomolar affinity for both the human and the rat Y1 receptor (IC50 values 0.38±0.06 nM and 0.72±0.42 nM, respectively). The inactive enantiomer of BIBO 3304 (BIBO 3457) had low affinity for both the human and rat Y1 receptor subtype (IC50>1000 nM). BIBO 3304 showed low affinity for the human Y2 receptor, human and rat Y4 receptor as well as for the human and rat Y5 receptor (IC50 values >1000 nM).

30 μg BIBO 3304 administered into the paraventricular nucleus inhibited the feeding response induced by 1 μg NPY as well as the hyperphagia induced by a 24 h fast implying a role for Y1 receptors in NPY mediated feeding. The inactive enantiomer had no effect.

BIBO 3304 inhibits neither the galanin nor the noradrenaline induced orexigenic response, but it blocked feeding behaviour elicited by both [Leu31, Pro34]NPY and NPY (3–36) suggesting an interplay between different NPY receptor subtypes in feeding behavior.

The present study reveals that BIBO 3304 is a subtype selective nonpeptide antagonist with subnanomolar affinity for the Y1 receptor subtype that significantly inhibits food intake induced by application of NPY or by fasting.

British Journal of Pharmacology (1998) 125, 549–555; doi:10.1038/sj.bjp.0702084
Metabolism of the vitamin D3 analogue EB1089 alters receptor complex formation and reduces promoter selectivity
Authors: Quack Marcus , Mørk Hansen Christina , Binderup Ernst , Kissmeyer Anne‐Marie , Carlberg Carsten
Published: January 2005

1α,25‐dihydroxyvitamin3 (VD) is a nuclear hormone that has important cell regulatory functions but also a strong calcemic effect. EB1089 is a potent antiproliferative VD analogue, which has a modified side chain resulting in increased metabolic stability and a selective functional profile. Since EB1089 is considered for potential systemic application, it will be investigated to what extent its recently identified metabolites (hydroxylated at positions C26 and C26a) contribute to biological profile of the VD analogue.

Limited protease digestion analysis demonstrated that EB1089 is able to stabilize the high affinity ligand binding conformation of the VDR, starting at concentrations of 0.1 nM and affecting up to 80% of all receptor molecules. The metabolites EB1445 and EB1470 showed to be 100 fold less potent than EB1089, whereas the remaining three metabolites (EB1435, EB1436 and EB1446) showed a clearly reduced ability to stabilize the high affinity ligand binding conformation. Interestingly, at pharmacological concentrations all EB1089 metabolites stabilized a second, apparently lower affinity conformation to a much higher extent than EB1089.

In reporter gene assays all metabolites showed lower potency than EB1089. Moreover, the preference of EB1089 for activation of VDR binding to sites formed by inverted palindromic arrangements spaced by nine nucleotide (IP9‐type VD response elements) appeared to be reduced (with EB1445 and EB1470) or completely lost (with EB1435, EB1436 and EB1446). The ranking of EB1089 and its metabolites that was obtained by limited protease digestion and reporter gene assays was confirmed by an analysis of their antiproliferative effect in breast cancer cells.

The potency and selectivity of the EB1089 metabolites in mediating gene regulatory effects was found to be drastically reduced in comparison to the parent compound suggesting that the contribution of the metabolites to the biological effect of EB1089 is minor. However, the compounds showed to be interesting tools for understanding the selective biological profile of EB1089.

British Journal of Pharmacology (1998) 125, 607–614; doi:10.1038/sj.bjp.0702086
Ramipril‐induced delayed myocardial protection against free radical injury involves bradykinin B2 receptor‐NO pathway and protein synthesis
Authors: Jin Zhu‐Qiu , Chen Xiu
Published: January 2005

The aim of the present study was to examine whether ramipril induces delayed myocardial protection against free radical injuries ex vivo and to determine the possible role of the bradykinin B2–nitric oxide (NO) pathway, prostaglandins(PGs) and protein synthesis in this delayed adaptive response.

Rats were pretreated with ramipril (10 or 50 μg kg−1, i.v.) and hearts were isolated after 24, 48 and 72 h. Langendorff hearts were subjected to 1,1‐diphenyl‐2‐picryl‐hydrazyl (DPPH) free radical‐induced injury.

Left ventricular developed pressure (LVDP) and its maximal increase velocity (+dP/dtmax), coronary flow (CF), heart rate (HR), lactate dehydrogenase (LDH) in coronary effluent and thiobarbituric acid reactive substances (TBARS) in the myocardium were measured.

The results showed that in the DPPH control group, 20 min after free radical‐induced injury, LVDP, +dP/dtmax, CF, HR declined, whereas TBARS and LDH increased significantly. The above cardiac function parameters were significantly improved in RAM‐pretreated rats after 24 and 48 h.

Pretreatment with HOE 140, the selective bradykinin B2 receptor antagonist, NG‐nitro‐L‐arginine, the NO synthase inhibitor, and actinomycin D, the RNA transcription inhibitor, prior to ramipril injection abolished the beneficial effects of ramipril at 24 h while indomethacin, a cyclooxygenase inhibitor, pretreatment had no effect on ramipril‐induced delayed protection.

In conclusion, ramipril induces delayed myocardial protection against free radical injury in the rat heart. This delayed protection was sustained for 48 h, is associated with the bradykinin B2 receptor–NO pathway and depends on protein but not prostaglandin synthesis.

British Journal of Pharmacology (1998) 125, 556–562; doi:10.1038/sj.bjp.0702089
Potentiation of barbiturate‐induced alterations in presynaptic noradrenergic function in rat frontal cortex by imidazol(in)e α2‐adrenoceptor agonists
Authors: Dalley J W , Parker C A , Wülfert E , Hudson A L , Nutt D J
Published: January 2005

In order to resolve the extent to which presynaptic noradrenergic mechanisms contribute to the anaesthetic‐sparing effects of α2‐adrenoceptor agonists in vivo microdialysis was used to investigate the combined effects of sodium pentobarbitone and imidazol(in)e α2‐adrenoceptor agonists on extracellular levels of noradrenaline (NA) in the rat frontal cortex.

Dialysate levels of NA were markedly reduced by the addition of TTX (2 μM) or by the removal of calcium in the perfusate. These data imply that dialysate NA levels are ultimately dependent on exocytotic release mechanisms from afferent coeruleo‐cortical neurones.

Systemic administration of sodium pentobarbitone (85 mg kg−1, i.p.) induced general anaesthesia and reduced NA levels by 92% after 30 min. The restoration of basal levels 90 min later was closely associated with a return of the corneal blink reflex.

Basal NA levels in conscious animals were not affected by an intravenous infusion of equally radioactive solutions of either imidazoline (clonidine) or imidazole (mivazerol) α2‐adrenoceptor agonists. The dose rate employed for each compound was 2 μg kg−1 h−1 over 2 h.

The co‐administration of intravenous clonidine or mivazerol, each at 2 μg kg−1 h−1 for 2 h, with sodium pentobarbitone (85 mg kg−1, i.p.), produced a marked and prolonged reduction in NA efflux. After 2 h, NA levels remained suppressed by 95% (clonidine) and 80% (mivazerol) and animals remained deeply anaesthetized.

The accumulation of tritium in brain tissue was 42–73% lower across all brain regions examined after [3H]‐mivazerol administration than after [3H]‐clonidine administration. Sodium pentobarbitone did not alter the accumulation of tritium in brain tissue after the administration of either α2‐adrenoceptor agonist.

These data demonstrate that α2‐adrenoceptor agonists potentiate the inhibitory effects of sodium pentobarbitone on extracellular levels of NA in the frontal cortex. Further studies will be necessary to establish a causal role of noradrenergic mechanisms in the potentiation of anaesthesia by selective α2‐adrenoceptor agonists.

British Journal of Pharmacology (1998) 125, 441–446; doi:10.1038/sj.bjp.0702091
Vascular smooth muscle cell hypertrophy induced by glycosylated human oxyhaemoglobin
Authors: Peiró Concepción , Angulo Javier , Rodríguez‐Mañas Leocadio , Llergo José L , Vallejo Susana , Cercas Elena , Sánchez‐Ferrer Carlos F
Published: January 2005

Nonenzymatic protein glycosylation is a possible mechanism contributing to oxidative stress and vascular disease in diabetes. In this work, the influence of 14%‐glycosylated human oxyhaemoglobin (GHHb), compared to the non‐glycosylated protein (HHb), was studied on several growth parameters of rat cultured vascular smooth muscle cells (VSMC). A role for reactive oxygen species was also analysed.

Treatment of VSMC for 48 h with GHHb, but not with HHb, increased planar cell surface area in a concentration dependent manner. The threshold concentration was 10 nM, which increased cell size from 7965±176 to 9411±392 μm2. Similarly, only GHHb enhanced protein content per well in VSMC cultures.

The planar surface area increase induced by 10 nM GHHb was abolished by superoxide dismutase (SOD; 50–200 u ml−1), deferoxamine (100 nM–100 μM), or dimethylthiourea (1 mM), while catalase (50–200 u ml−1) or mannitol (1 mM) resulted in a partial inhibition of cell size enhancement.

When a known source of oxygen free radicals was administered to VSMC, the xanthine/xanthine oxidase system, the results were analogous to those produced by GHHb. Indeed, enhancements of cell size were observed, which were inhibited by SOD, deferoxamine, or catalase.

These results indicate that, at low concentrations, GHHb induces hypertrophy in VSMC, this effect being mediated by superoxide anions, hydrogen peroxide, and/or hydroxyl radicals. Therefore, glycosylated proteins can have a role in the development of the structural vascular alterations associated to diabetes by enhancing oxidative stress.

British Journal of Pharmacology (1998) 125, 637–644; doi:10.1038/sj.bjp.0702097
Endogenous asymmetrical dimethylarginine and hypertension associated with puromycin nephrosis in the rat
Authors: Sato Jun , Masuda Hiroshi , Tamaoki Satoru , Hamasaki Hidehisa , Ishizaka Kazuhiro , Matsubara Osamu , Azuma Hiroshi
Published: January 2005

The present experiments were designed to investigate the role of asymmetrical NG,NG‐dimethyl‐L‐arginine (ADMA) in causing hypertension associated with the focal and segmental glomerulosclerosis (FSGS) produced by a single bolus of puromycin aminonucleoside (PAN) and successive injection of protamine for 7 days in rats which had undergone unilateral nephrectomy.

After the unilateral nephrectomy, and administering PAN and protamine, histological examinations of the kidney revealed a typical FSGS, that is, evident abnormalities including segmental mesangial proliferation, obliteration of glomerular capillary lumens and adhesions between the glomerulus and Bowman's capsule could be observed. Changes in the glomerular epithelial cells consisted of the swelling with bleb formation.

In the FSGS rats, urine volume and urinary protein were significantly (P<0.05 and P<0.005) increased throughout 4‐week experimental period, while the creatinine clearance was significantly (P<0.005) and transiently decreased, and recovered 4 weeks later. These changes were associated with the sustained elevation of the systolic blood pressure.

ADMA levels in aortic endothelial cells, plasma and urine were significantly (P<0.05 and P<0.005) increased in the FSGS rats, but the level in the kidney remained unchanged.

The basal level and net production of cyclic GMP in the aortic vessel wall with endothelium when stimulated by norepinephrine and acetylcholine were significantly (P<0.05 and P<0.01) attenuated in the FSGS rats.

There were significant and positive correlations between systolic blood pressure (y) and ADMA levels (x) in endothelial cells (y=4.43x+122.2, r=0.979, P<0.0001), plasma (y=0.10x+71.9, r=0.921, P<0.001) and urine (y=0.48x+126.9, r=0.699, P<0.005), but not significant in the kidney (y=0.06x+102.7, r=0.252, NS).

These findings suggest that ADMA as an endogenous inhibitor of NO synthesis may play an important role for the pathogenesis in the hypertension associated with the experimental FSGS in the rat.

British Journal of Pharmacology (1998) 125, 469–476; doi:10.1038/sj.bjp.0702101
Increased response to big endothelin‐1 in atherosclerotic human coronary artery: functional evidence for up‐regulation of endothelin‐converting enzyme activity in disease
Authors: Maguire Janet J , Davenport Anthony P
Published: January 2005

Overproduction of the potent vasoconstrictor peptide endothelin‐1 (ET‐1) is implicated in the pathogenesis of coronary artery disease. In endothelium‐denuded human coronary arteries the response to big ET‐1 was significantly enhanced in atherosclerotic arteries (coronary artery disease, CAD; n=7) with an EC50 value of 96 nM (57–161 nM, 95% C.I.) compared to 274 nM (205–365 nM) in non‐diseased arteries (dilated cardiomyopathy, DCM; n=10) (Mann‐Whitney U‐test, P<0.05). Higher levels of immunoreactive endothelin (ET) could be detected by radioimmunoassay in bathing medium taken from CAD arteries than from DCM arteries (2.8±0.5 nM, n=5 vs 1.1±0.2 nM, n=7) (Student's two‐tailed t‐test, P<0.05). There were no differences in responses of arteries from either group to ET‐1 (EC50 10 nM, CAD vs 14 nM, DCM). The enhanced response of atherosclerotic human coronary arteries to big ET‐1 appears to be due to up‐regulation of endothelin‐converting enzyme (ECE) activity rather than to an augmented response of the arteries to ET‐1. This non‐endothelial ECE may therefore be an important therapeutic target in coronary artery disease.

British Journal of Pharmacology (1998) 125, 238–240; doi:10.1038/sj.bjp.0702102
EB1089, a synthetic analogue of vitamin D, induces apoptosis in breast cancer cells in vivo and in vitro
Authors: James Sharon Y. , Mercer Elizabeth , Brady Matthew , Binderup Lise , Colston Kay W.
Published: January 2005

Effects of the synthetic vitamin D analogue EB1089 on indices of apoptosis in cultured human breast cancer cells and in nitrosomethylurea‐induced rat mammary tumours in vivo were investigated.

At a dose of 0.5 μg kg−1 body weight, EB1089 caused significant inhibition of tumour progression over the 28 day treatment period in the absence of a significant increase in serum calcium concentration. Higher doses of EB1089 (1 and 2.5 μg kg−1) produced substantial regression of the experimental tumours which was accompanied by a striking change in the histological appearance of tumours consistent with induction of tumour cell death.

Fragmentation of genomic DNA is a characteristic feature of apoptosis. With the terminal transferase (TdT) assay, 3′ DNA breaks indicative of DNA fragmentation were detected histochemically in mammary tumour cells from animals treated with EB1089 (2.5 μg kg−1) for 14 days.

Effects of the vitamin D analogue on induction of apoptosis were examined in vitro using the MCF‐7 human breast cancer cell line. Using the TUNEL method, positive nuclear staining indicative of DNA fragmentation was detected in cells treated for 4 days with 10 nM EB1089. Apoptosis was also quantitated using a cell death ELISA which revealed a time and dose dependent induction of apoptosis by EB1089.

The effects of EB1089 on the expression of two oncoproteins which may regulate apoptosis, bcl‐2 and bax were examined by Western analysis. In MCF‐7 cell cultures treated with 1,25(OH)2D3 or EB1089 (1×10−8 M), bcl‐2 protein levels were decreased in a time‐dependent manner relative to control levels. In contrast bax protein was not markedly regulated by these compounds. Densitometric analyses indicate that the vitamin D compounds lower the bcl‐2/bax ratio favouring increased susceptibility of MCF‐7 cells to undergo apoptosis.

These results suggest that the synthetic vitamin D analogue EB1089 may promote tumour regression by inducing active cell death.

British Journal of Pharmacology (1998) 125, 953–962; doi:10.1038/sj.bjp.0702103
Expression of cyclo‐oxygenase‐2 in human airway smooth muscle is associated with profound reductions in cell growth
Authors: Belvisi Maria G. , Saunders Michael , Yacoub Magdi , Mitchell Jane A.
Published: January 2005

It is now accepted that uncontrolled proliferation of human airway smooth muscle (HASM) cells contributes, in many cases, to the chronic stages of asthma. However, the physiological and pathophysiological processes regulating cell growth and division in the airway are not clear. We have recently shown that the immediate early gene, cyclo‐oxygenase‐2, is induced by cytokines in HASM cells. Since cyclo‐oxygenase metabolites, such as prostaglandin (PG) E2 have been shown to modulate HASM cell growth, we have investigated any autocrine action of endogenously released cyclo‐oxygenase‐1/2 products on the proliferative responses in these cells.

HASM cells were cultured from healthy tissue obtained at lung or heart/lung transplantation. HASM cell proliferation was measured by [3H]‐methyl thymidine uptake by cells and by cell counts. Cyclo‐oxygenase‐2 expression was measured by Western blot analysis and activity measured by the release of PGE2, by radioimmunoasay.

HASM cells proliferated in response to foetal calf serum, a response that was greatly inhibited when cyclo‐oxygenase‐2 was induced with either interleukin‐1β plus tumour necrosis factor‐α or interleukin‐1β, tumour necrosis factorα plus interferonγ (each at 10 ng ml−1). The inhibitory effect of cytokines on HASM cell proliferation was reversed in a concentration dependent manner by either the mixed cyclo‐oxygenase‐1/‐2 inhibitor, indomethacin or the selective cyclo‐oxygenase‐2 inhibitor, L‐745,337 (each at 10 μM).

PGE2 or the stable analogue of prostacyclin, cicaprost concentration‐dependently (0.1 pmol to 1 μM) inhibited serum induced proliferation of HASM cells. By contrast, the TP receptor agonist, U46619 stimulated proliferation of HASM cells when cells were cultured without but not with serum. Other cyclo‐oxygenase products, PGD2, PGF2α had no effect on cellular proliferation at concentrations up to 1 μM.

These observations illustrate a profound inhibitory effect of cyclo‐oxygenase‐2 induction on HASM cell proliferation, possibly via IP or EP receptor activation. Cyclo‐oxygenase‐2 induction has, thus far, been associated with the pro‐inflammatory responses of plasma exudation and oedema formation and is assumed to be an enzyme worthy of selective inhibition in many disease states. However, our observations suggest that cyclo‐oxygenase‐2 can have an anti‐inflammatory, anti‐proliferative function in the airways. These observations may have importance in the use and development of therapies for airway disease such as asthma.

British Journal of Pharmacology (1998) 125, 1102–1108; doi:10.1038/sj.bjp.0702104
Mg2+ and ATP dependence of KATP channel modulator binding to the recombinant sulphonylurea receptor, SUR2B
Authors: Hambrock Annette , Löffler‐Walz Cornelia , Kurachi Yoshihisa , Quast Ulrich
Published: January 2005

The binding of modulators of the ATP‐sensitive K+ channel (KATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular KATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 37°C using the tritiated KATP channel opener, [3H]‐P1075.

Binding of [3H]‐P1075 required the presence of Mg2+ and ATP. MgATP activated binding with EC50 values of 10 and 3 μM at free Mg2+ concentrations of 3 μM and 1 mM, respectively. At 1 mM Mg2+, binding was lower than at 3 μM Mg2+.

[3H]‐P1075 saturation binding experiments, performed at 3 mM ATP and free Mg2+ concentrations of 3 μM and 1 mM, gave KD values of 1.8 and 3.4 nM and BMAX values of 876 and 698 fmol mg−1, respectively.

In competition experiments, openers inhibited [3H]‐P1075 binding with potencies similar to those determined in rings of rat aorta.

Glibenclamide inhibited [3H]‐P1075 binding with Ki values of 0.35 and 2.4 μM at 3 μM and 1 mM free Mg2+, respectively. Glibenclamide enhanced the dissociation of the [3H]‐P1075‐SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas.

It is concluded that an MgATP site on SUR2B with μM affinity must be occupied to allow opener binding whereas Mg2+ concentrations 10 μM decrease the affinities for openers and glibenclamide. The properties of the [3H]‐P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.

British Journal of Pharmacology (1998) 125, 577–583; doi:10.1038/sj.bjp.0702109
Mechanism of block by tedisamil of transient outward current in human ventricular subepicardial myocytes
Authors: Wettwer Erich , Himmel Herbert M , Amos Gregory J , Li Qi , Metzger Franz , Ravens Ursula
Published: January 2005

Tedisamil is a new antiarrhythmic drug with predominant class III action. The aim of the present study was to investigate the blocking pattern of the compound on the transient outward current (Ito) in human subepicardial myocytes isolated from explanted left ventricles. Using the single electrode whole cell voltage clamp technique, Ito was analysed after appropriate voltage inactivation of sodium current and block of calcium current.

Tedisamil reduced the amplitude of peak Ito, but did not affect the amplitude of non‐inactivating outward current. The drug accelerated the apparent rate of Ito inactivation. The reduction in time constant of Ito inactivation depended on drug concentration, the apparent IC50 value was 4.4 μM.

Tedisamil affected Ito amplitude in a use‐dependent manner. After 2 min at −80 mV, maximum block of Ito was reached after 4–5 clamp steps either at the frequency of 0.2 or 2 Hz, indicating that the block was not frequency‐dependent in an experimentally relevant range. Recovery from block was very slow and proceeded with a time constant of 12.1±1.8 s. Also in the presence of drug, a fraction of channels recovered from inactivation with a similar time constant as in control myocytes (i.e. 81±40 ms and 51±8 ms, respectively, n.s.).

From the onset of fractional block of Ito by tedisamil during the initial 60 ms of a clamp step, we calculated k1=9×106 mol−1 s−1 for the association rate constant, and k2=23 s−1 for the dissociation rate constant. The resulting apparent KD was 2.6 μM and is similar to the IC50 value.

The effects of tedisamil on Ito could be simulated by assuming a four state channel model where the drug binds to the channel in an open (activated) conformation. It is concluded that in human subepicardial myocytes tedisamil is an open channel blocker of Ito and that this effect probably contributes to the antiarrhythmic potential of this drug.

British Journal of Pharmacology (1998) 125, 659–666; doi:10.1038/sj.bjp.0702110
Disruption of synchronous gamma oscillations in the rat hippocampal slice: A common mechanism of anaesthetic drug action
Authors: Faulkner H J , Traub R D , Whittington M A
Published: January 2005

At the molecular level much progress has been made towards elucidating the mechanisms of action of general and dissociative anaesthetics. However, little is known about how these molecular actions may lead to disruption of cognitive function.

A promising physiological correlate of cognitive function is the ability of spatially separate areas of the brain to synchronize firing patterns via mutual inhibitory, gamma‐frequency (20–80 Hz) electrical oscillations. Here we examine the effects of five different anaesthetic/hypnotic agents with different primary mechanisms of action on these oscillations in the hippocampus.

Gamma oscillations were elicited simultaneously at two sites at either end of area CA1 by tetanic stimulation. Such oscillations are synchronous between these areas even when separated by up to c. 4 mm in control conditions.

Agents which act directly on GABAA receptor‐mediated inhibition had different effects on synchronous gamma oscillations. Thiopental (10–200 μm) markedly disrupted the oscillation and resulting synchrony whereas the benzodiazepines diazepam and temazepam (0.05–1.0 μM) had little effect.

The opiate morphine (10–200 μM) and dissociative agent ketamine (10–100 μM) had a different profile of effects on gamma oscillations. However, as with thiopental, both agents markedly disrupted between site synchrony. These three agents demonstrated this effect at aqueous concentrations relevant to anaesthetic ED50.

Using the hippocampus as a model neuronal network we propose that, despite differing primary mechanisms of action, anaesthetics may disrupt cognitive function by interfering with the mechanism of generation of synchronous firing patterns between spatially separate areas of the brain.

British Journal of Pharmacology (1998) 125, 483–492; doi:10.1038/sj.bjp.0702113
Analysis of the influence of nucleotidases on the apparent activity of exogenous ATP and ADP at P2Y1 receptors
Authors: Vigne Paul , Philippe Breittmayer Jean , Frelin Christian
Published: January 2005

ADP is a potent agonist of rat and human P2Y1 purinoceptors. ATP is a weak competitive antagonist. This study analyses the situation in which P2Y1 receptors are exposed to ATP in the presence of exogenous ecto‐nucleotidases (apyrases) that have high or low ATPase/ADPase activity ratio.

Rat brain capillary endothelial cells of the B10 clone express P2Y1 receptors that couple to intracellular Ca2+ mobilization. They have low endogenous ecto‐ATPase and ecto‐ADPase activities.

ATP did not raise intracellular Ca2+ in B10 cells. Addition of apyrases III or VII (1 u ml−1) to ATP treated cells induced large intracellular Ca2+ transients. Apyrases had no action in the absence of ATP.

A 1 u ml−1 apyrase III solution generated 20 μM ADP from 0.1 mM ATP within 15 s. This concentration of ADP was sufficient to produce maximal activation of P2Y1 receptors.

ATP was a full agonist of P2Y1 receptors in the presence of 1 u ml−1 apyrase III. Dose response curves for the apparent actions of ATP were bell shaped in the presence of 0.1 u ml−1 apyrase III. Apyrase III did not alter ADP dose response curves when coincubated with ADP for 15 s.

Apyrase VII (1 u ml−1) shifted dose response curves for the actions of ADP to larger concentrations. It induced a bell shaped ATP dose response curve.

Results suggest that ATPDases prevent P2Y1 receptor activation by degrading ADP but may contribute to P2Y1 receptor activation by generating ADP from ATP.

British Journal of Pharmacology (1998) 125, 675–680; doi:10.1038/sj.bjp.0702115
Recovery by ascorbate of impaired nitric oxide‐dependent relaxation resulting from oxidant stress in rat aorta
Authors: Dudgeon Sinead , Benson David P , MacKenzie Andrew , Paisley‐Zyszkiewicz Karen , Martin William
Published: January 2005

In this study we investigated the ability of ascorbate to protect nitric oxide from destruction by superoxide anion.

Ascorbate produced concentration‐dependent relaxation of rings of rat aorta, comprising two components: the first, seen at 1–300 μM, reached a maximum of 45.3±2.8%, and was abolished by endothelial removal or treatment with L‐NAME (100 μM), demonstrating involvement of nitric oxide. The second occurred at concentrations of 1 mM and above and was associated with falls in the pH of the bathing fluid.

Pretreatment with ascorbate at concentrations up to 3 mM had no effect on the relaxation to acetylcholine (10 nM–10 μM) on endothelium‐containing rings or adenosine (0.1 μM–3 mM) on endothelium‐denuded rings.

An oxidant stress was applied to aortic rings, comprising inhibition of endogenous Cu/Zn superoxide dismutase by diethyldithiocarbamate (0.1 mM) followed by generation of superoxide anion by hypoxanthine (0.1 mM/xanthine oxidase (16 u ml−1). This reduced maximal acetylcholine‐induced relaxation from 96.7±1.3% to 42.4±3.5% (P<0.001). Treatment with ascorbate (30 μM–3 mM) reversed this blockade in a concentration‐dependent manner.

Our findings show that ascorbate has the ability to protect nitric oxide from destruction by superoxide anion. This action is seen with ascorbate at levels normally present in plasma, suggesting that this antioxidant may exert a tonic protective effect on nitric oxide within the vasculature.

British Journal of Pharmacology (1998) 125, 782–786; doi:10.1038/sj.bjp.0702120
Decline in the expression of copper/zinc superoxide dismutase in the kidney of rats with endotoxic shock: Effects of the superoxide anion radical scavenger, tempol, on organ injury
Authors: Leach Mary , Frank Stefan , Olbrich Antje , Pfeilschifter Josef , Thiemermann Christoph
Published: January 2005

Endotoxaemia causes an enhanced formation of reactive oxygen species (ROS) which contribute to the multiple organ dysfunction syndrome (MODS) in septic shock. Here we investigate (i) the effects of endotoxin on the expression of two isoforms of superoxide dismutase (SOD), namely Cu/Zn‐SOD (cytosol) and Mn‐SOD (mitochondria) in the rat kidney, and (ii) the effects of the radical scavenger tempol on the MODS caused by lipopolysaccharide (LPS, E. coli, 6 mg kg−1 i.v.) in the rat.

Endotoxaemia resulted in a rapid, but transient, decline in the expression of both mRNA and protein of Cu/Zn‐SOD as well as an increase in the expression of the mRNA of Mn‐SOD in the kidney. Endotoxaemia for 6 h also caused hypotension, acute renal dysfunction, hepatocellular injury, pancreatic injury and an increase in the plasma levels of nitrite/nitrate.

Pretreatment of rats with tempol (100 mg kg−1 i.v. bolus injection, 15 min prior to LPS followed by an infusion of 30 mg kg−1 i.v., n=9) did not affect the circulatory failure, but attenuated the renal dysfunction and the hepatocellular injury/dysfunction caused by LPS. Tempol did not affect the rise in nitrite/nitrate caused by endotoxin.

These results imply that an enhanced formation of ROS (including superoxide anions) in conjunction with inadequate defences against such ROS contributes to the injury and dysfunction of the kidney and the liver in endotoxic shock.

British Journal of Pharmacology (1998) 125, 817–825; doi:10.1038/sj.bjp.0702123
Effect of DMPPO, a phosphodiesterase type 5 inhibitor, on hypoxic pulmonary hypertension in rats
Authors: Eddahibi Saadia , Raffestin Bernadette , Le Monnier de Gouville Anne‐Charlotte , Adnot Serge
Published: January 2005

Cyclic guanosine 3′–5′‐monophosphate (cyclic GMP) is the second messenger of important physiologically active mediators controlling the pulmonary vascular tone. To potentiate the effects of cyclic GMP on the pulmonary vasculature, we used DMPPO, a new selective PDE‐5 inhibitor, and examined its action in a rat model of hypoxic pulmonary hypertension.

Levels of cyclic GMP measured during baseline conditions at 5 and 60 min of perfusion were similar in the perfusate of isolated lungs from normoxic and chronically hypoxic rats and did not differ with time. Pretreatment with DMPPO (1 μM) induced a larger increase in cyclic GMP concentration in the perfusate from chronically hypoxic rat lungs (319±36 at 5 min to 1821±83 pmol ml−1 at 60 min) than in normoxic rat lungs (329±20 to 1281±127 pmol ml−1, P<0.05).

In isolated lungs preconstricted with U‐46619, pretreatment with DMPPO (1 μM) potentiated the vasodilator effects of atrial natriuretic peptide (100 pM–10 nM) and sodium nitroprusside (1 pM–10 nM), but did not alter vasodilation to isoproterenol.

In conscious rats previously exposed to 15 days hypoxia and studied under 10% O2, DMPPO (0.01, 0.05 and 0.1 mg kg−1, i.v. bolus) caused a dose‐dependent decrease in pulmonary arterial pressure (Pap) with no change in systemic artery pressure (Sap) and cardiac output.

Continuous infusion of DMPPO (0.1 mg kg−1 h−1 i.v. by osmotic pumps) in rats exposed to 10% O2 during 2‐weeks reduced the Pap (P<0.05) and the degree of muscularization of pulmonary vessels at the alveolar wall (P<0.01) and alveolar duct levels (P<0.05) despite no significant change in right ventricular hypertrophy.

These results suggest that cyclic GMP phosphodiesterase inhibition may selectively dilate pulmonary circulation during chronic hypoxia.

British Journal of Pharmacology (1998) 125, 681–688; doi:10.1038/sj.bjp.0702124
Desensitization of ETA endothelin receptor‐mediated negative chronotropic response in right atria–species difference and intracellular mechanisms
Authors: Ono Kageyoshi , Sakamoto Aiji , Masaki Tomoh , Satake Motoyoshi
Published: January 2005

Desensitization of ETA endothelin receptor (ETAR) was compared between the rat and guinea‐pig with regard to negative chronotropic response (NC) in the right atria (RA).

ET‐1 (100 nM) produced distinct NC in the presence of BQ788 (300 nM), and positive chronotropic response (PC) in the presence of BQ123 (1 μM) in both species, showing that ETAR and ETB endothelin receptor (ETBR) mediate NC and PC, respectively.

Repetitive applications of ET‐1 (50 nM) desensitized PC, and the second application only induced a strong NC in both species. Later applications of ET‐1 produced virtually no response in the rat RA, whereas they produced BQ123‐sensitive NCs repetitively in guinea‐pig RA, exhibiting marked species difference in desensitization of ETAR‐mediated NC.

Pretreatment with staurosporine (100 nM) prevented desensitization of ETAR in the rat RA altogether. However, phorbol 12‐myristate 13‐acetate (PMA, 300 nM) failed to induce, but rather hampered, desensitization of ETAR.

Partial amino acid sequencing of ETARs, spanning from the 2nd through the 4th intracellular loops, revealed that all the potential Ser/Thr phosphorylation sites, including a protein kinase C (PKC) site, are conserved among guinea‐pigs, rats, rabbits, bovines and humans.

In guinea pig RA, pretreatment with okadaic acid (1 μg ml−1) and PMA did not facilitate desensitization of ETAR whereas these agents successfully desensitized ETAR during combined stimulation of β‐adrenoceptor and ETAR by isoproterenol (300 nM) and ET‐1 (100 nM).

These results suggest that species differences in desensitization of ETAR are not caused by differences in the site(s) of, but caused by differences in the environment for phosphorylation of the receptor. Desensitization of ETAR appears to require phosphorylation of the receptor by PKC as well as a kinase stimulated by β‐adrenoceptor activation.

British Journal of Pharmacology (1998) 125, 787–797; doi:10.1038/sj.bjp.0702125
The role of A3 adenosine receptors in central regulation of arterial blood pressure
Authors: Stella Luigi , de Novellis Vito , Marabese Ida , Berrino Liberato , Maione Sabatino , Filippelli Amelia , Rossi Francesco
Published: January 2005

Pharmacological studies have suggested that A3 receptors are present on central neurons. Recently this adenosine receptor subtype has been identified in the rat and its presence in the central nervous system has been confirmed.

In this study we investigated the effects of acute intracerebroventricular (i.c.v.) injections of N6‐2‐(4‐aminophenyl)‐ethyladenosine (APNEA), a non‐selective A3 adenosine receptor agonist, on arterial blood pressure (ABP) and heart rate (HR), after treatment with 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), a selective antagonist of A1 adenosine receptors.

Anaesthetized rats, after DPCPX (12 μg−1 kg i.c.v.), were treated with APNEA (0.4–4 μg kg−1 i.c.v.) resulting in a transitory and dose‐dependent decrease in arterial blood pressure without a change in heart rate. APNEA also induced hypotensive responses after i.c.v. pretreatment with aminophylline, at a dose of 20 μg kg−1. In contrast, pretreatment 48 h before, with 4 μg kg−1 i.c.v. of pertussis toxin reduced the hypotensive effect induced by APNEA. Administration of APNEA at a higher dose (20 μg kg−1 i.c.v.), after DPCPX, induced a decrease in ABP of −66±5.4 mmHg and after 3 min a decrease in heart rate of −62±6.0 beats min−1. Transection of the spinal cord abolished this significant fall in ABP, but not the decrease of HR.

These results suggest that a population of A3‐receptors is present in the CNS, whose activation induces a decrease in blood pressure with no change of heart rate.

British Journal of Pharmacology (1998) 125, 437–440; doi:10.1038/sj.bjp.0702126
Investigation of oxidant stress and vasodepression to glyceryl trinitrate in the obese Zucker rat in vivo
Authors: Laight David W , Kengatharan K M , Gopaul Nitin K , Änggård Erik E , Carrier Martin J
Published: January 2005

We examined the relationship between oxidant stress and the vasodepressor activity of glyceryl trinitrate (GTN) in vivo, including rapid GTN tolerance development, in 13‐week old obese and age‐matched lean Zucker rats which had been maintained for 4 weeks on either control diet or diets enriched with the lipophilic, chain‐breaking antioxidants vitamin E (0.5% w w−1) or probucol (0.5% w w−1) or the superoxide anion scavenger tiron (1% w v−1 in drinking water).

The basal plasma level of the isoprostane 8‐epi‐PGF2α, an in vivo marker of lipid peroxidation, was elevated by approximately 5 fold in the obese Zucker rat and markedly reduced by dietary lipophilic antioxidants and depressed by dietary tiron.

Vasodepression to bolus does GTN (0.1–100 μg kg−1 i.v.), but not endothelium‐dependent vasodepression to bolus dose acetylcholine (ACh, 0.02–2.0 μg kg−1 i.v.), was impaired in obese animals and completely restored by dietary antioxidants.

Nitrate tolerance developed in vivo during a 1 h infusion of GTN (40 μg kg−1 min−1 i.v.) appeared more severe in obese animals. However, rapid nitrate tolerance was not affected by dietary antioxidants in either the obese or lean Zucker rat.

We therefore provide evidence that elevated oxidant stress in the obese Zucker rat is associated with an impairment in nitrate vasodepressor activity. However, our data are not consistent with either a role for oxidant stress in rapid nitrate tolerance development in the anaesthetized Zucker rat or the aggravation of this tolerance by pre‐existing oxidant stress.

British Journal of Pharmacology (1998) 125, 895–901; doi:10.1038/sj.bjp.0702132