bluuebunny/crossref_metadata_2025 huggingface dataset:
Tranilast inhibits protein kinase C‐dependent signalling pathway linked to angiogenic activities and gene expression of retinal microcapillary endothelial cells
Authors: Koyama Shinji , Takagi Hitoshi , Otani Atsushi , Suzuma Kiyoshi , Nishimura Kazuo , Honda Yoshihito
Published: January 2005

Tranilast, first developed as an anti‐allergic drug, has been reported to inhibit vascular endothelial growth factor (VEGF)‐induced angiogenesis and vasopermeability. To further clarify the inhibitory mechanism, we investigated the effects of tranilast on VEGF binding and subsequent intracellular signalling pathway linked to angiogenic activities and gene expression of bovine retinal microcapillary endothelial cells.

Tranilast significantly (P<0.01) inhibited VEGF, basic fibroblast growth factor (bFGF), and hypoxia conditioned media‐induced BREC proliferation in a dose dependent manner with IC50's of 22, 82 and 10 μM, respectively.

VEGF‐induced migration was also inhibited by tranilast in a dose dependent manner, with IC50 of 18 μM, and complete inhibition was observed at 300 μM (P<0.01). Tranilast suppressed VEGF‐induced tube formation in a dose dependent manner with maximum (46%) inhibition observed at 300 μM (P<0.05).

Tranilast inhibited phorbol myristate acetate (PMA)‐dependent stimulation of [3H]‐thymidine incorporation and VEGF‐ and PMA‐induced gene expression of integrin αv and c‐fos in BREC.

Tranilast suppressed VEGF‐ and PMA‐stimulated PKC activity in BREC.

Tranilast did not affect VEGF binding or VEGF‐induced phosphorylation of tyrosine residues of VEGF receptor‐ and phospholipase Cγ and their associated proteins.

These data suggest that tranilast might prove an effective inhibitor to prevent retinal neovascularization in ischaemic retinal diseases, and that its inhibitory effect might be through suppression of PKC‐dependent signal transduction in BREC.

British Journal of Pharmacology (1999) 127, 537–545; doi:10.1038/sj.bjp.0702564
Bradykinin down‐regulates LPS‐induced eosinophil accumulation in the pleural cavity of mice through type 2‐kinin receptor activation: a role for prostaglandins
Authors: Silva A R , Larangeira A P , Pacheco P , Calixto J B , Henriques M G M O , Bozza P T , Castro‐Faria‐Neto H C
Published: January 2005

The role of both exogenously administered and endogenously generated bradykinin (BK) on LPS‐induced eosinophil accumulation in the mice pleural cavity was investigated by means of treatment with BK selective receptor agonists/antagonists and captopril.

Intrathoracic (i.t.) injection of LPS (250 ng cavity−1) induced eosinophil influx at 24 h as previously described (Bozza et al., 1993). Pretreatment with the B1 receptor antagonist des‐Arg9‐[leu‐8]BK (0.025 and 0.25 nmol cavity−1) showed no effect on this phenomenon, whereas pretreatment with the B2 receptor antagonists, NPC 17731 (0.025 and 0.25 nmol cavity−1) or HOE 140 (2.5 nmol cavity−1), increased LPS‐induced eosinophil influx. Accordingly, pretreatment with captopril at 10 mg kg−1 i.p., inhibited eosinophil infiltration induced by LPS in the pleural cavity, suggesting that endogenous BK is down‐regulating LPS‐induced eosinophil accumulation.

BK administered at 15 and 25 nmol cavity−1, i.t. or i.p. also inhibited LPS‐induced eosinophil accumulation. BK alone had no effect on the basal number of leucocytes in the pleural or peritoneal cavity in doses up to 25 nmol cavity−1. Nevertheless, when injected at doses of 50 and 100 nmol cavity−1 BK induced leucocyte influx characterized by neutrophil and eosinophil accumulation at 24 h.

Similarly to what was observed with BK, a specific B2 receptor agonist, Tyr8BK, administered at 0.25 nmol cavity−1 i.p., significantly inhibited the eosinophil influx induced by LPS.

The mechanism by which B2 receptor agonists inhibit LPS‐induced eosinophil accumulation was investigated by pretreating the animals with indomethacin or a selective cyclooxygenase‐2 inhibitor, NS‐398. Pretreatment with either indomethacin or NS‐398 had no effect on eosinophil influx induced by LPS alone, but those drugs were able to restore the LPS‐induced eosinophil influx in Tyr8BK (0.25 nmol cavity−1) injected mice.

In conclusion, endogenously generated bradykinin seems to modulate, through activation of B2 receptors, eosinophil accumulation induced by LPS via a mechanism dependent on prostanoid synthesis.

British Journal of Pharmacology (1999) 127, 569–575; doi:10.1038/sj.bjp.0702567
Antibodies and antisense oligodeoxynucleotides to μ‐opioid receptors, selectively block the effects of μ‐opioid agonists on intestinal transit and permeability in mice
Authors: Pol Olga , Valle Lluís , Sánchez‐Blázquez Pilar , Garzón Javier , Puig Margarita M
Published: January 2005

We have studied the effects of μ and δ opioids on intestinal function (permeability, PER; gastrointestinal transit, GIT), and their antagonism after the intracerebroventricular (i.c.v.) administration of specific antibodies (ABs) or antisense oligodeoxynucleotides (ODN) to μ‐receptors (OR). Central versus peripheral site/s of action of subcutaneous (s.c.) μ‐opioids, were also assessed.

Male Swiss CD‐1 mice were used. GIT was measured with charcoal and PER by the passage of 51Cr‐EDTA from blood to lumen.

Morphine and fentanyl (i.c.v. and s.c.) inhibited GIT and PER in a dose‐related manner; they were more potent by i.c.v. route, both on GIT and PER (70 and 17 times for morphine and fentanyl). They also had a greater effect on GIT than PER (4.3 and 1.6 times). DPDPE had a lower potency than μ‐agonists in all experiments, and no dose‐response could be obtained after s.c. administration on GIT.

Pretreatment with i.c.v. ABs (24 h) or antisense ODN (5 days), decreased the effects (GIT and PER) of i.c.v. morphine and fentanyl, while those of DPDPE remained unchanged. The ABs did not alter the peripheral effects of μ‐opioids.

The results show that (i.c.v. or s.c.) μ opioids produce dose‐related inhibitions of PER and GIT, being more potent by the i.c.v. route. Delta‐opioids had a greater effect on PER than GIT, while the opposite occurred for μ‐agonists. Pretreatment with ABs or ODN to μ‐OR, blocked the central effects of μ (but not δ) agonists on GIT and PER.

British Journal of Pharmacology (1999) 127, 397–404; doi:10.1038/sj.bjp.0702570
Investigation of the role of 5‐HT1B and 5‐HT1D receptors in the sumatriptan‐induced constriction of porcine carotid arteriovenous anastomoses
Authors: De Vries Peter , Willems Edwin W , Heiligers Jan P C , Villalón Carlos M , Saxena Pramod R
Published: January 2005

It has previously been shown that the antimigraine drug sumatriptan constricts porcine carotid arteriovenous anastomoses via 5‐HT1‐like receptors, identical to 5‐HT1B/1D receptors. The recent availability of silent antagonists selective for the 5‐HT1B (SB224289) and 5‐HT1D (BRL15572) receptor led us to further analyse the nature of receptors involved.

In pentobarbitone‐anaesthetized, bilaterally vagosympathectomized pigs, sumatriptan (30, 100 and 300 μg kg−1, i.v.) dose‐dependently decreased carotid arteriovenous anastomotic conductance by up to 70±5%.

The dose‐related decreases in carotid arteriovenous anastomotic conductance by sumatriptan (30, 100 and 300 μg kg−1, i.v.) remained unchanged in animals treated (i.v.) with 1 mg kg−1 of BRL15572 (maximum decrease: 72±3%), but were significantly attenuated by 1 mg kg−1 (maximum decrease: 30±11%) and abolished by 3 mg kg−1 (maximum decrease: 3±7%) of SB224289. The highest dose of SB224289 did not attenuate the hypertension, tachycardia or increases in carotid blood flow induced by bolus injections of noradrenaline (0.1–3 μg kg−1, i.v.).

The results indicate that sumatriptan constricts porcine carotid arteriovenous anastomoses primarily via 5‐HT1B, but not via 5‐HT1D receptors.

British Journal of Pharmacology (1999) 127, 405–412; doi:10.1038/sj.bjp.0702572
Differential regulation of bradykinin receptor density, intracellular Ca2+, and prostanoid release in skin and foreskin fibroblasts. Effects of cell density and interleukin‐1α
Authors: Ochsenbein Regula M , Inaebnit Sven P , Luethy Christa M , Wiesmann Ulrich N , Oetliker Oskar H , Honegger Ulrich E
Published: January 2005

Bradykinin (BK) receptors, cytosolic Ca2+, and prostanoids were studied in human skin and foreskin fibroblasts.

Bmax values of BK receptors were higher in foreskin than in skin fibroblasts, increasing with cell densities in both cell types. IL‐1α‐dependent receptor induction was blocked by cycloheximide.

BK‐stimulated cytosolic Ca2+ elevation was higher in confluent than in non‐confluent cultures and larger in foreskin than in skin fibroblasts. Responses were not enhanced after IL‐1‐α‐induced up‐regulation of BK receptors.

Intrinsic prostanoid production was higher in foreskin than in skin fibroblasts at comparable cell densities. In foreskin, but not in skin fibroblasts, BK stimulation increased the release of PGE2 10 fold and that of 6‐oxo‐PGF1α 6–7 fold.

Preincubation with IL‐1α had a marked effect on prostanoid release in foreskin fibroblasts only. Subsequent BK stimulation increased the release of PGE2 and 6‐oxo‐PGF1α 7–10 fold in skin fibroblasts while this increase was only 30% in foreskin fibroblasts. Release of TXA2 reached values up to one third of the other prostanoids. The IL‐1α induced rise in BK‐stimulated PGE2 synthesis was fully abolished by specific inhibition of cyclo‐oxygenase 2.

IL‐1α sensitized BK‐stimulated prostanoid synthesis and modulated prostanoid patterns differently in fibroblasts from skin and foreskin. The IL‐1α effects on prostanoid release were not related to BK receptor numbers nor to the BK‐stimulated Ca2+ signal but appear to be due to induction of prostanoid synthesizing enzymes. Foreskin fibroblasts seem to be unique and significantly different from fibroblasts of other skin locations in respect to their response to inflammation‐associated kinins and cytokines.

British Journal of Pharmacology (1999) 127, 583–589; doi:10.1038/sj.bjp.0702578
Activation of outward K+ currents: effect of VIP in oesophagus
Authors: Jury Jennifer , Daniel Edwin E
Published: January 2005

Electrical field stimulations (EFS) of the opossum and canine lower oesophageal sphincters (OLOS and CLOS respectively) and opossum oesophageal body circular muscle (OOBCM) induce non‐adrenergic, non‐cholinergic (NANC) relaxations of any active tension and NO‐mediated hyperpolarization. VIP relaxes the OLOS and CLOS and any tone in OOBCM without major electrophysiological effects. These relaxations are not blocked by NOS inhibitors. Using isolated smooth muscle cells, we tested whether VIP acted through myogenic NO production.

Outward currents were similar in OOBCM and OLOS and NO increased them regardless of pipette Ca2+i, from 50–8000 nM. L‐NAME or L‐NOARG did not block outward currents in OLOS at 200 nM pipette Ca2+.

Outward currents in CLOS cells decreased at 200 nM pipette Ca2+ or less but NO donors still increased them. VIP had no effect on outward currents in cells from OOBCM, OLOS or CLOS under conditions of pipette Ca2+ at which NO donors increased outward K+ currents.

We conclude, VIP does not mimic electrophysiological effects of NO donors on isolated cells of OOBCM, OLOS or CLOS. VIP relaxes the OLOS and CLOS and inhibits contraction of OOBCM by a mechanism unrelated to release of myogenic NO or an increase in outward current.

Also, the different dependence of outward currents of OOBCM and OLOS on pipette Ca2+ from those of CLOS suggests that different K+ channels are involved and that myogenic NO production contributes to K+ channel activity in CLOS but not in OLOS or OOBCM.

British Journal of Pharmacology (1999) 127, 553–561; doi:10.1038/sj.bjp.0702579
Pharmacological evidence for the 5‐HT7 receptor mediating smooth muscle relaxation in canine cerebral arteries
Authors: Terrón José A , Falcón‐Neri Alicia
Published: January 2005

We investigated in the present study whether 5‐HT is able to exert direct relaxant responses in canine basilar and middle cerebral arteries via the 5‐HT7 receptor.

In arterial rings deprived of endothelium and pre‐contracted with prostaglandin F2α (2 μM), 5‐HT, 5‐carboxamidotryptamine (5‐CT), 5‐methoxytryptamine, sumatriptan or α‐methyl‐5‐HT produced further increase in tone and/or slight relaxation. Blockade of 5‐HT1B/1D and 5‐HT2A receptors with GR127935 (1 μM) and ketanserin (0.1 μM), respectively, antagonized the vasoconstrictor component of the response and unmasked a concentration‐dependent relaxation to 5‐HT, 5‐CT and 5‐methoxytryptamine; sumatriptan and α‐methyl‐5‐HT remained inactive as relaxant agonists. The rank order of agonist potency in both arteries was 5‐CT>5‐HT>5‐methoxytryptamine>>sumatriptanα‐methyl‐5‐HT.

In dog basilar artery, pre‐incubated with GR127935 (1 μM) and ketanserin (0.1 μM) and pre‐contracted with prostaglandin F2α (2 μM), the 5‐HT7 ligands, clozapine (1 μM), mesulergine (0.3 μM), methiothepin (3 nM), risperidone (3 nM), spiperone (1 μM) and LY215840 (10–100 nM), produced significant rightward shifts of the concentration‐response curves for 5‐HT and 5‐CT. Only methiothepin and risperidone reduced significantly the maximum relaxant response (Emax), whilst the other drugs behaved as competitive antagonists with affinity values (pKB) that significantly correlated with their binding affinity (pKi) at recombinant 5‐HT7 receptors.

These data disclosing the involvement of the 5‐HT7 receptor in cerebrovascular relaxation may be strongly relevant in the light of: (1) the involvement of 5‐HT in migraine; (2) the putative linkage between cephalovascular vasodilatation and migraine headache; and (3) the relatively high 5‐HT7 receptor affinity of migraine prophylactic 5‐HT antagonists.

British Journal of Pharmacology (1999) 127, 609–616; doi:10.1038/sj.bjp.0702580
In vivo pathway of thermal hyperalgesia by intrathecal administration of α,β‐methylene ATP in mouse spinal cord: Involvement of the glutamate‐NMDA receptor system
Authors: Tsuda Makoto , Ueno Shinya , Inoue Kazuhide
Published: January 2005

The aim of the present study is to characterize the role of the P2X receptor in spinal nociceptive processing in vivo. We investigated the mechanisms of the P2X receptor agonist α,β‐methylene ATP (α,βmeATP)‐induced modulation of acute nociceptive signalling in mouse spinal cord.

Intrathecal administration of α,βmeATP produced a significant and dose‐dependent thermal hyperalgesic response. This response was completely blocked by intrathecal pretreatment with the non‐selective P2 receptor antagonist, pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonate (PPADS) and the selective P2X1, P2X3 and P2X2+3 receptor antagonist, 2′,3′‐O‐(2,4,6‐trinitrophenyl)adenosine 5′‐triphosphate (TNP‐ATP). Pretreatment with α,βmeATP 15, 30 and 60 min prior to administration of a second dose of α,βmeATP diminished the α,βmeATP‐induced thermal hyperalgesia.

A potent agonist for the P2X1 receptor, β,γ‐methylene‐L‐ATP, did not show the hyperalgesic response, indicating that the P2X1 receptor is not involved in the spinal nociceptive pathway.

In fura‐2 experiments using mouse dorsal root ganglion (DRG) neurons, α,βmeATP (100 μM) increased intracellular Ca2+ ([Ca2+]i). This was not produced by a second application of α,βmeATP. The same DRG neurons also showed a marked [Ca2+]i increase in response to capsaicin (3 μM).

Intrathecal pretreatment with the Ca2+‐dependent exocytosis inhibitor, botulinum neurotoxin B, abolished the thermal hyperalgesia by α,βmeATP. Furthermore, thermal hyperalgesia was significantly inhibited by the N‐methyl‐D‐aspartate (NMDA) receptor antagonists, 2‐amino‐5‐phosphonopentanoate (APV), dizocilpine and ifenprodil.

These findings suggest that α,βmeATP‐induced thermal hyperalgesia may be mediated by the spinal P2X3 receptor subtype that causes unresponsiveness by repetitive agonist applications, and that α,βmeATP (perhaps through P2X3 receptors) may evoke spinal glutamate release which, in turn, leads to the generation of thermal hyperalgesia via activation of NMDA receptors.

British Journal of Pharmacology (1999) 127, 449–456; doi:10.1038/sj.bjp.0702582
Non‐NMDA glutamate receptors modulate capsaicin induced c‐fos expression within trigeminal nucleus caudalis
Authors: Mitsikostas Dimos D , Sanchez del Rio Margarita , Waeber Christian , Huang Zhihong , Cutrer F Michael , Moskowitz Michael A
Published: January 2005

We examined the effects of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)/kainate receptor antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and 1,2,3,4‐tetrahydro‐6‐nitro‐2,3‐dioxo‐benzol[f]quinoxaline‐7‐sulphonamide (NBQX), the kainate receptor antagonists γ‐(R‐)‐glutamylaminomethanesulphonic acid (GAMS) and 6,7,8,9‐tetrahydro‐5‐nitro‐1H‐benz[g]indole‐2,3‐dione‐3‐oxime (NS‐102), and the group III metabotropic glutamate receptor (mGluR) agonist 2‐amino‐4‐phosphono‐S‐butanoic acid (L‐AP4) on c‐fos‐like immunoreactivity (c‐fos LI) in trigeminal caudalis (Sp5C), lateral reticular (LRt), medullary reticular (Md) and solitary tract (Sol) nuclei, after intracisternal injection of capsaicin in urethane anaesthetized Sprague‐Dawley rats.

Few c‐fos labelled cells were observed within Sp5C in capsaicin‐vehicle treated animals. The number of positive c‐fos cells increased by 17 fold after intracisternal capsaicin (5 nmol) administration.

Pretreatment with CNQX (0.02, 0.1, 0.6, 3 and 15 mg kg−1) or NBQX (0.01, 0.1 and 1 mg kg−1), administered intraperitoneally 15 min before capsaicin, significantly reduced labelled cells within Sp5C by a maximum of 45 and 34%, respectively. The number of c‐fos LI cells within LRt, Md and Sol was not affected. Pretreatment with  L‐AP4 (1, 3 and 10 mg kg−1) decreased the number of Sp5C c‐fos LI cells by a maximum of 30%, whereas GAMS (1 and 10 mg kg−1) and NS‐102 (1 and 5 mg kg−1) did not show any significant effect.

These results suggest that blockade of AMPA receptors, but not kainate receptors, or the activation of group III mGluRs, decrease the response of Sp5C neurons to trigeminovascular activation. Thus, in addition to NMDA receptors, mGluRs and AMPA receptors may modulate cephalic pain and may provide a potential therapeutic target for antimigraine drugs.

British Journal of Pharmacology (1999) 127, 623–630; doi:10.1038/sj.bjp.0702584
Comparative effects of cyclo‐oxygenase and nitric oxide synthase inhibition on the development and reversal of spinal opioid tolerance
Authors: Powell Kelly J , Hosokawa Akiko , Bell Andrew , Sutak Maaja , Milne Brian , Quirion Remi , Jhamandas Khem
Published: January 2005

This study examined the effects of the COX inhibitors, ketorolac and ibuprofen, and the NOS inhibitor L‐NAME for their potential to both inhibit the development and reverse tolerance to the antinociceptive action of morphine.

Repeated administration of intrathecal morphine (15 μg), once daily, resulted in a progressive decline of antinociceptive effect and an increase in the ED50 value in the tailflick and paw pressure tests. Co‐administration of ketorolac (30 and 45 μg) or S(+) ibuprofen (10 μg) with morphine (15 μg) prevented the decline of antinociceptive effect and increase in ED50 value. Similar treatment with L‐NAME (100 μg) exerted weaker effects. Administration of S(+) but not R(−) ibuprofen (10 mg kg−1) had similar effects on systemic administration of morphine (15 mg kg−1).

Intrathecal or systemic administration of the COX or NOS inhibitors did not alter the baseline responses in either tests. Acute keterolac or S(+) ibuprofen also did not potentiate the acute actions of spinal or systemic morphine, but chronic intrathecal administration of these agents increased the potency of acute morphine.

In animals already tolerant to intrathecal morphine, subsequent administration of ketorolac (30 μg) with morphine (15 μg) partially restored the antinociceptive effect and ED50 value of acute morphine, reflecting the reversal of tolerance. Intrathecal L‐NAME (100 μg) exerted a weaker effect.

These data suggest that spinal COX activity, and to a lesser extent NOS activity, contributes to the development and expression of opioid tolerance. Inhibition of COX may represent a useful approach for the prevention as well as reversal of opioid tolerance.

British Journal of Pharmacology (1999) 127, 631–644; doi:10.1038/sj.bjp.0702587
Characterization of α1‐adrenoceptors expressed in a novel vascular smooth muscle cell line cloned from p53 knockout mice, P53LMAC01 (AC01) cells
Authors: Ohmi Kazuhiro , Shinoura Hitomi , Nakayama Yasuhisa , Goda Nobuhito , Tsujimoto Gozoh
Published: January 2005

We pharmacologically studied the α1‐adrenoceptor (AR) subtype(s) involved in receptor‐mediated signalling in a novel vascular smooth muscle cell line cloned from p53 knockout mice, P53LMAC01 (AC01) cells.

Radioligand binding studies with [125I]‐HEAT showed the existence of a homogeneous population of binding site with an affinity (Kd value) of 0.4 nM and a maximum number of binding sites (Bmax) of 100 fmol mg−1 protein. Catecholamines competed for [125I]‐HEAT binding stereospecifically and with the characteristic α1‐AR potency series.

Displacement curves for BMY‐7378 and KMD‐3213 best fitted a one‐site model with a pKi value (−log10 (equilibrium inhibition constant)) of 6.06 and 7.07, respectively.

Reverse transcription‐polymerase chain reaction (RT–PCR) assay detected α1B‐ and α1D‐AR, but not α1A‐AR transcript.

Chlorethylclonidine (CEC) treatment nearly abolished (−)noradrenaline (NA) (10 μM)‐induced inositol[1,4,5]trisphosphate (IP3) production, and BMY‐7378 inhibited the response with a Ki value of 0.3 nM, which value was similar to that obtained in the cells expressing α1D‐AR. In both AC01 cells and cells expressing α1D‐AR, BMY‐7378 protected α1‐ARs from CEC alkylation while it had little protective effect on CEC alkylation and NA‐induced IP3 production in cells expressing α1B‐AR.

The results indicate that AC01 cells contain predominantly α1B‐ARs and a small population of α1D‐ARs; however, phosphoinositide (PI)/Ca2+ signalling is mainly mediated through the minor population of α1D‐ARs, rather than the α1B‐ARs.

British Journal of Pharmacology (1999) 127, 756–762; doi:10.1038/sj.bjp.0702588
Tyrosine nitration in blood vessels occurs with increasing nitric oxide concentration
Authors: Amirmansour Charles , Vallance Patrick , Bogle Richard G
Published: January 2005

Experiments were designed to explore the effects of nitric oxide (NO) donors on generation of superoxide (O2.−) and peroxynitrite (ONOO−) in rabbit aortic rings.

Following inhibition of endogenous superoxide dismutase (SOD), significant basal release of O2.− was revealed (0.9±0.01×10−12 mol min−1 mg−1 tissue). Generation of O2.− increased in a concentration‐dependent manner in response to NADH or NADPH (EC50=2.34±1.18×10−4 and 6.21±1.79×10−3 M respectively, n=4). NADH‐stimulated O2.− chemiluminescence was reduced by approximately 85% in the presence of exogenous SOD (15×103 U ml−1).

Incubation of aortic rings with S‐nitrosoglutathione (GSNO; 1×10−5–3×10−3 M) or sodium nitroprusside (SNP; 1×10−8–1×10−3 M), resulted in a concentration‐dependent quenching of O2.− chemiluminescence which was proportional to NO release.

ONOO− formation was assessed indirectly by determining protein tyrosine nitration in rabbit aorta using a specific antibody against nitrotyrosine. Basally and in the presence of NADH, a single band was detected. Incubation of aortic rings with either GSNO (1×10−3 M) alone or GSNO with NADH resulted in the appearance of additional nitrotyrosine bands. Incubation of serum albumin with GSNO alone did not cause nitrotyrosine formation. In contrast, incubation with 3‐morpholinosydonomine (SIN‐1; 1×10−3 M, 10 min), resulted in marked nitration of albumin which was reduced by oxyhaemoglobin or SOD. Incubation of albumin with GSNO and pyrogallol, a O2.− generator, also resulted in protein nitration.

Addition of exogenous NO results in nitrotyrosine formation in rabbit aortic rings. Nitrotyrosine formation is likely to result from the reaction of exogenous NO and basal endogenous O2.− resulting in the formation of ONOO−. Formation of ONOO− and nitration of tyrosine residues potentially could lead to vascular damage and might represent unexpected adverse effects of long‐term nitrate therapy.

British Journal of Pharmacology (1999) 127, 788–794; doi:10.1038/sj.bjp.0702590
Endothelin‐1 enhances neutrophil adhesion to human coronary artery endothelial cells: role of ETA receptors and platelet‐activating factor
Authors: Zouki Christine , Baron Chantal , Fournier Alain , Filep János G
Published: January 2005

The potent coronary vasoconstrictor, endothelin‐1 (ET‐1) may also regulate neutrophil traffic into tissues. The aim of the present study was to characterize the endothelin receptors responsible and to investigate the underlying mechanisms.

ET‐1 (1 nM–1 μM) markedly enhanced attachment of human neutrophils to lipopolysaccharide‐, and to a lesser extent, to ET‐1‐activated human coronary artery endothelial cells (HCAEC). This can partially be blocked by monoclonal antibodies against E‐selectin, L‐selectin or CD18, whereas combination of the three antibodies inhibited adhesion by ∼83%. Increases in neutrophil adhesion evoked by ET‐1 were also blocked by the platelet‐activating factor (PAF) antagonists, BN 52021 (50 μM) and WEB 286 (10 μM).

ET‐1 downregulated the expression of L‐selectin and upregulated expression of CD11b/CD18 and CD45 on the neutrophil surface and induced gelatinase release with EC50 values of ∼2 nM. These actions of ET‐1 were almost completely prevented by the ETA receptor antagonist FR 139317 (1 μM) and the ETA/ETB receptor antagonist bosentan (10 μM), whereas the ETB receptor antagonist BQ 788 (1 μM) had no effect. ET‐1 slightly increased the expression of E‐selectin and ICAM‐1 on HCAEC, that was prevented by BQ 788, but not by FR 139317.

Receptor binding studies indicated the presence of ETB receptors (KD: 40 pM) on phosphoramidon‐treated HCAEC and the predominant expression of ETA receptors (KD: 38 pM) on neutrophils.

These results indicate that promotion by ET‐1 of neutrophil adhesion to HCAEC is predominantly mediated through activation of ETA receptors on neutrophils and subsequent generation of PAF.

British Journal of Pharmacology (1999) 127, 969–979; doi:10.1038/sj.bjp.0702593
NPY receptor subtype in the rabbit isolated ileum
Authors: Félétou Michel , Nicolas Jean‐Paul , Rodriguez Marianne , Beauverger Philippe , Galizzi Jean‐Pierre , Boutin Jean A , Duhault Jacques
Published: January 2005

The purpose of this work was to verify the hypothesis that the rabbit ileum is a selective preparation for the NPY Y5 receptor by using new selective antagonists recently synthesized. Spontaneous contractions of the rabbit isolated ileum were recorded and binding experiments were performed in cells expressing the human NPY Y1, Y2, Y4 or Y5 receptor subtype.

NPY analogues produced a concentration‐dependent transient inhibition of the spontaneous contractions of the rabbit ileum with the following order of potency hPP>rPP>PYY[Leu31,Pro34]‐NPY>NPY>>NPY13–36. Pre‐exposure to rPP, PYY, [Leu31,Pro34]‐NPY or NPY (but not NPY13–36) inhibited the effect of subsequent administration of hPP suggesting cross‐desensitization of the preparation. The apparent affinity of the various agonists studied was correlated to the affinity reported for the human Y4 receptor subtype (and to a lesser extent for the rat Y4 subtype) but not to the affinity for the Y5 receptor subtype.

BIBO 3304, a selective NPY Y1 receptor antagonist, and CGP 71683A, a selective NPY Y5 receptor antagonist, did not affect the response to hPP. JCF 109, another NPY Y5 receptor antagonist, produced an inhibition of the response to hPP but only at the highest dose tested (10 μM) which also, by itself, produced intrinsic inhibitory effects.

1229U91, a non‐selective ligand for Y1, Y2, Y4 and Y5 receptors with high affinity toward the Y1 and Y4 receptor subtypes, produced a concentration‐dependent transient inhibition of the spontaneous contractions of the rabbit ileum and a dose‐dependent inhibition of the response to hPP (apparent pKB: 7.2).

These results suggest that in the rabbit ileum, the NPY receptor involved in the inhibition of the spontaneous contractile activity is a NPY Y4 receptor subtype.

British Journal of Pharmacology (1999) 127, 795–801; doi:10.1038/sj.bjp.0702594
Nociceptin/orphanin FQ and nocistatin on learning and memory impairment induced by scopolamine in mice
Authors: Hiramatsu Masayuki , Inoue Kaori
Published: January 2005

Nociceptin, also known as orphanin FQ, is an endogenous ligand for the orphan opioid receptor‐like receptor 1 (ORL1) and involves in various functions in the central nervous system (CNS). On the other hand, nocistatin is recently isolated from the same precursor as nociceptin and blocks nociceptin‐induced allodynia and hyperalgesia.

Although ORL1 receptors which display a high degree of sequence homology with classical opioid receptors are abundant in the hippocampus, little is known regarding their role in learning and memory.

The present study was designed to investigate whether nociceptin/orphanin FQ and nocistatin could modulate impairment of learning and memory induced by scopolamine, a muscarinic cholinergic receptor antagonist, using spontaneous alternation of Y‐maze and step‐down type passive avoidance tasks in mice.

While nocistatin (0.5–5.0 nmol mouse−1, i.c.v.) administered 30 min before spontaneous alternation performance or the training session of the passive avoidance task, had no effect on spontaneous alternation or passive avoidance behaviours, a lower per cent alternation and shorter median step‐down latency in the retention test were obtained in nociceptin (1.5 and/or 5.0 nmol mouse−1, i.c.v.)‐treated normal mice.

Administration of nocistatin (1.5 and/or 5.0 nmol mouse−1, i.c.v.) 30 min before spontaneous alternation performance or the training session of the passive avoidance task, attenuated the scopolamine‐induced impairment of spontaneous alternation and passive avoidance behaviours.

These results indicated that nocistatin, a new biologically active peptide, ameliorates impairments of spontaneous alternation and passive avoidance induced by scopolamine, and suggested that these peptides play opposite roles in learning and memory.

British Journal of Pharmacology (1999) 127, 655–660; doi:10.1038/sj.bjp.0702595
Analysis of α1L‐adrenoceptor pharmacology in rat small mesenteric artery
Authors: Stam Wiro B , Van der Graaf Pieter H , Saxena Pramod R
Published: January 2005

To illuminate the controversy on α1A‐ or α1L‐adrenoceptor involvement in noradrenaline‐mediated contractions of rat small mesenteric artery (SMA), we have studied the effects of subtype‐selective α1‐adrenoceptor agonists and antagonists under different experimental conditions.

The agonist potency order in rat SMA was: A61603 >> SKF89748‐A > cirazoline > noradrenaline > ST‐587 > methoxamine. Prazosin antagonized all agonists with a low potency (pA2: 8.29–8.80) indicating the involvement of α1L‐ rather than α1A‐adrenoceptors.

The putative α1L‐adrenoceptor antagonist JTH‐601, but not the α1B‐adrenoceptor antagonist chloroethylclonidine (10 μM) antagonized noradrenaline‐induced contractions of SMA. The potency of the selective α1D‐adrenoceptor antagonist BMY 7378 against noradrenaline (pA2=6.16±0.13) and of the selective α1A‐adrenoceptor antagonist RS‐17053 against noradrenaline (pKB=8.35±0.10) and against the selective α1A‐adrenoceptor agonist A‐61603 (pKB=8.40±0.09) were too low to account for α1D‐ and α1A‐adrenoceptor involvement.

The potency of RS‐17053 (pKB/pA2's=7.72–8.46) was not affected by lowering temperature, changing experimental protocol or inducing myogenic tone via KCl or U46619.

Selective protection of a putative α1A‐adrenoceptor population against the irreversible action of phenoxybenzamine also failed to increase the potency of RS‐17053 (pA2=8.25±0.06 against A61603).

Combined concentration‐ratio analysis demonstrated that tamsulosin, which does not discriminate between α1A‐ and α1L‐adrenoceptors, and RS‐17053 competed for binding at the same site in the SMA.

In summary, data obtained in our experiments in rat SMA indicate that the α1‐adrenoceptor mediating noradrenaline‐induced contraction displays a distinct α1L‐adrenoceptor pharmacology. This study does not provide evidence for the hypothesis that α1L‐adrenoceptors represent an affinity state of the α1A‐adrenoceptor in functional assays. Furthermore, there is no co‐existing α1A‐adrenoceptor in the SMA.

British Journal of Pharmacology (1999) 127, 661–670; doi:10.1038/sj.bjp.0702598
Stimulant action of pituitary adenylate cyclase‐activating peptide on normal and drug‐compromised peristalsis in the guinea‐pig intestine
Authors: Heinemann Ákos , Holzer Peter
Published: January 2005

Pituitary adenylate cyclase‐activating peptide (PACAP) is known to influence the activity of intestinal smooth muscle. This study set out to examine the action of PACAP on normal and drug‐inhibited peristalsis and to shed light on its site and mode of action.

Peristalsis in isolated segments of the guinea‐pig small intestine was elicited by distension through a rise of the intraluminal pressure. Drug‐induced motility changes were quantified by alterations of the peristaltic pressure threshold at which aborally moving peristaltic contractions were triggered.

PACAP (1–30 nM) stimulated normal peristalsis as deduced from a concentration‐related decrease in the peristaltic pressure threshold (maximum decrease by 55%). The peptide's stimulant effect remained intact in segments pre‐exposed to apamin (0.5 μM), N‐nitro‐L‐arginine methyl ester (300 μM), naloxone (0.5 μM), atropine (1 μM) plus naloxone (0.5 μM) or hexamethonium (100 μM) plus naloxone (0.5 μM).

PACAP (10 nM) restored peristalsis blocked by morphine (10 μM), noradrenaline (1 μM) or N6‐cyclopentyladenosine (0.3 μM) and partially reinstated peristalsis blocked by Rp‐adenosine‐3′,5′‐cyclic monophosphothioate triethylamine (100 μM) but failed to revive peristalsis blocked by hexamethonium (100 μM) or atropine (1 μM). The peptide's spectrum of properistaltic activity differed from that of naloxone (0.5 μM) and forskolin (0.3 μM).

The distension‐induced ascending reflex contraction of the circular muscle was facilitated by PACAP (1–30 nM) which itself evoked transient nerve‐mediated contractions of intestinal segment preparations.

These data show that PACAP stimulates normal peristalsis and counteracts drug‐induced peristaltic arrest by a stimulant action on excitatory enteric motor pathways, presumably at the intrinsic sensory neurone level. The action of PACAP seems to involve multiple signalling mechanisms including stimulation of adenylate cyclase.

British Journal of Pharmacology (1999) 127, 763–771; doi:10.1038/sj.bjp.0702602
Characterization of β‐adrenoceptor mediated smooth muscle relaxation and the detection of mRNA for β1‐, β2‐ and β3‐adrenoceptors in rat ileum
Authors: Roberts S J , Papaioannou M , Evans B A , Summers R J
Published: January 2005

Functional and molecular approaches were used to characterize the β‐AR subtypes mediating relaxation of rat ileal smooth muscle.

In functional studies, (−)‐isoprenaline relaxation was unchanged by CGP20712A (β1‐AR antagonist) or ICI118551 (β2‐AR antagonist) but shifted by propranolol (pKB=6.69). (±)‐Cyanopindolol, CGP12177 and ICID7114 did not cause relaxation but antagonized (−)‐isoprenaline relaxation.

BRL37344 (β3‐AR agonist) caused biphasic relaxation. The high affinity component was shifted with low affinity by propranolol, (±)‐cyanopindolol, tertatolol and alprenolol. CL316243 (β3‐AR agonist) relaxation was unaffected by CGP20712A or ICI118551 but blocked by SR58894A (β3‐AR antagonist; pA2=7.80). Enhanced relaxation after exposure to forskolin and pertussis toxin showed that β3‐AR relaxation can be altered by manipulation of components of the adenylate cyclase signalling pathway.

The β1‐AR agonist RO363 relaxed the ileum (pEC50=6.18) and was blocked by CGP20712A. Relaxation by the β2‐AR agonist zinterol (pEC50=5.71) was blocked by SR58894A but not by ICI118551.

In rat ileum, β1‐, β2‐ and β3‐AR mRNA was detected. Comparison of tissues showed that β3‐AR mRNA expression was greatest in WAT>colon=ileum>cerebral cortex>soleus; β1‐AR mRNA was most abundant in cerebral cortex>WAT>ileum=colon>soleus; β2‐AR mRNA was expressed in soleus>WAT>ileum=colon>cerebral cortex.

These results show that β3‐ARs are the predominant β‐AR subtype mediating rat ileal relaxation while β1‐ARs may produce a small relaxation. The β2‐AR agonist zinterol produces relaxation through β3‐ARs and there was no evidence for the involvement of β2‐ARs in relaxation despite the detection of β2‐AR mRNA.

British Journal of Pharmacology (1999) 127, 949–961; doi:10.1038/sj.bjp.0702605
Failure of 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) to inhibit soluble guanylyl cyclase in rat ventricular cardiomyocytes
Authors: Wegener Jörg W , Closs Ellen I , Förstermann Ulrich , Nawrath Hermann
Published: January 2005

The effects of 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ), an inhibitor of soluble guanylyl cyclase (sGC), were investigated in aortic rings and ventricular cardiomyocytes from rats. The production of cyclic GMP was stimulated by NO•‐donors or carbachol. Additionally, the effects of ODQ were studied in cytosolic extracts from both tissues in which the cyclic GMP production was stimulated by S‐nitroso‐N‐acetylpenicillamine (SNAP).

In endothelium‐intact aortic rings, SNAP (100 μM), 2,2′‐(hydroxynitrosohydrazino)bis‐ethanamine (DETA NONOate; 100 μM), or carbachol (10 μM) increased cyclic GMP levels about 4 fold. These effects were abolished by ODQ (50 μM).

In cardiomyocytes, SNAP (100 μM), DETA NONOate (100 μM), or carbachol (10 μM) increased cyclic GMP levels about 2 fold. These effects were not affected by ODQ (50 μM).

In cytosolic extracts from aortic rings and cardiomyocytes, SNAP (100 μM) induced about 50 fold increases in cyclic GMP levels. ODQ (50 μM) reduced these effects by about 50%.

In extracts from cardiomyocytes, increases by SNAP (100 μM) of cyclic GMP levels were attenuated by myoglobin dependent on concentration: at 300 μM myoglobin, SNAP (100 μM) increased cyclic GMP levels only 3 fold. Inhibitory effects of ODQ (50 μM) were abolished by 300 μM myoglobin.

It is suggested that both NO• and ODQ can bind to myoglobin which, at high concentrations, can diminish their effects on sGC. Such a scavenger function of myoglobin could explain why NO• and ODQ exert only minor effects in cardiomyocytes (with high myoglobin content), but strong effects in aortic tissue (virtually devoid of myoglobin).

British Journal of Pharmacology (1999) 127, 693–700; doi:10.1038/sj.bjp.0702608
Molecular and pharmacological evidence for MT1 melatonin receptor subtype in the tail artery of juvenile Wistar rats
Authors: Ting K N , Blaylock N A , Sugden D , Delagrange P , Scalbert E , Wilson V G
Published: January 2005

In this study reverse transcriptase‐polymerase chain reaction (RT–PCR) has been used to identify mt1 and MT2 receptor mRNA expression in the rat tail artery. The contributions of both receptors to the functional response to melatonin were examined with the putative selective MT2 receptor antagonists, 4‐phenyl‐2‐propionamidotetraline (4‐P‐PDOT) and 2‐benzyl‐N‐pentanoyltryptamine. In addition, the action of melatonin on the second messenger cyclic AMP was investigated.

Using RT–PCR, mt1 receptor mRNA was detected in the tail artery from seven rats. In contrast MT2 receptor mRNA was not detected even after nested PCR.

At low concentrations of the MT2 selective ligands, neither 10 nM 4‐P‐PDOT (pEC50=8.70±0.31 (control) vs 8.73±0.16, n=6) nor 60 nM 2‐benzyl‐N‐pentanoyltryptamine (pEC50=8.53±0.20 (control) vs 8.83±0.38, n=6) significantly altered the potency of melatonin in the rat tail artery.

At concentrations non‐selective for mt1 and MT2 receptors, 4‐P‐PDOT (3 μM) and 2‐benzyl‐N‐pentanoyltryptamine (5 μM) caused a significant rightward displacement of the vasoconstrictor effect of melatonin. In the case of 4‐P‐PDOT, the estimated pKB (6.17±0.16, n=8) is similar to the binding affinity for mt1 receptor.

Pre‐incubation with 1 μM melatonin did not affect the conversion of [3H]‐adenine to [3H]‐cyclic AMP under basal condition (0.95±0.19% conversion (control) vs 0.92±0.19%, n=4) or following exposure to 30 μM forskolin (5.20±1.30% conversion (control) vs 5.35±0.90%, n=4).

Based on the above findings, we conclude that melatonin receptor on the tail artery belongs to the MT1 receptor subtype, and that this receptor is probably independent of the adenylyl cyclase pathway.

British Journal of Pharmacology (1999) 127, 987–995; doi:10.1038/sj.bjp.0702612
Ligand‐induced differentiation of glucocorticoid receptor (GR) trans‐repression and transactivation: preferential targetting of NF‐κB and lack of I‐κB involvement
Authors: Adcock Ian M , Nasuhara Yasuyuki , Stevens David A , Barnes Peter J
Published: January 2005

Glucocorticoids are highly effective in controlling chronic inflammatory diseases, such as asthma and rheumatoid arthritis, but the exact molecular mechanism of their anti‐inflammatory action remains uncertain. They act by binding to a cytosolic receptor (GR) resulting in activation or repression of gene expression. This may occur via direct binding of the GR to DNA (transactivation) or by inhibition of the activity of transcription factors such as AP‐1 and NF‐κB (transrepression).

The topically active steroids fluticasone propionate (EC50=1.8×10−11 M) and budesonide (EC50=5.0×10−11 M) were more potent in inhibiting GM‐CSF release from A549 cells than tipredane (EC50=8.3×10−10 M), butixicort (EC50=3.7×10−8 M) and dexamethasone (EC50=2.2×10−9 M). The anti‐glucocorticoid RU486 also inhibited GM‐CSF release in these cells (IC50=1.8×10−10 M).

The concentration‐dependent ability of fluticasone propionate (EC50=9.8×10−10 M), budesonide (EC50=1.1×10−9 M) and dexamethasone (EC50=3.6×10−8 M) to induce transcription of the β2‐receptor was found to correlate with GR DNA binding and occurred at 10–100 fold higher concentrations than the inhibition of GM‐CSF release. No induction of the endogenous inhibitors of NF‐κB, IκBα or I‐κBβ, was seen at 24 h and the ability of IL‐1β to degrade and subsequently induce IκBα was not altered by glucocorticoids.

The ability of fluticasone propionate (IC50=0.5×10−11 M), budesonide (IC50=2.7×10−11 M), dexamethasone (IC50=0.5×10−9 M) and RU486 (IC50=2.7×10−11 M) to inhibit a 3×κB was associated with inhibition of GM‐CSF release.

These data suggest that the anti‐inflammatory properties of a range of glucocorticoids relate to their ability to transrepress rather than transactivate genes.

British Journal of Pharmacology (1999) 127, 1003–1011; doi:10.1038/sj.bjp.0702613
The effect of site‐directed mutagenesis of two transmembrane serine residues on agonist‐specific coupling of a cloned human α2A‐adrenoceptor to adenylyl cyclase
Authors: Rudling Jane E , Kennedy Karen , Evans Peter D
Published: January 2005

The effects of substitution of the Ser200 and Ser204 residues with alanine on the signalling properties of the cloned human α2A‐adrenoceptor, stably expressed in Chinese hamster ovary (CHO) cell lines, have been investigated using noradrenaline and the structural isomers of octopamine.

The Ser→Ala200 or the Ser→Ala204 mutant forms of the α2A‐adrenoceptor, when expressed in cells in the absence of pertussis toxin pretreatment, are two orders of magnitude more sensitive to inhibition of cyclic AMP production by (±)‐para‐octopamine and (±)‐meta‐octopamine, respectively, than cells expressing the wild‐type receptor. Binding studies indicate that the effects are not due to an increased agonist affinity for the mutant receptors and that they are likely to be due to agonist‐mediated conformational changes in receptor structure.

After incubation with pertussis toxin, (±)‐meta‐octopamine (100 μM and above) produced a stimulation of cyclic AMP levels in cells expressing the Ser→Ala204 mutant form of the α2A‐adrenoceptor but showed no stimulation in cells expressing the Ser→Ala200 mutant receptor. Under these conditions (±)‐para‐octopamine did not produce any increases in cyclic AMP production in cells expressing either of the mutant receptor forms or the wild‐type receptor.

The results emphasise the importance of the Ser200 and Ser204 residues of the α2A‐adrenoceptor in exerting an inhibitory influence on the ability of (±)‐para‐octopamine and (±)‐meta‐octopamine respectively, to induce a receptor‐agonist conformation capable of inhibiting forskolin‐stimulation of cyclic AMP levels.

It is clear that Ser204 also prevents meta‐octopamine from generating a receptor‐agonist conformation that can increase cyclic AMP levels, emphasising the importance of this residue in the agonist‐specific coupling of this receptor to different second messenger systems.

British Journal of Pharmacology (1999) 127, 877–886; doi:10.1038/sj.bjp.0702614
Effect of protein kinase C and phospholipase A2 inhibitors on the impaired ability of human diabetic platelets to cause vasodilation
Authors: Oskarsson Helgi J , Hofmeyer Timothy G , Coppey Lawrence , Yorek Mark A
Published: January 2005

The aim of this study was to examine the mechanism of impaired platelet‐mediated endothelium‐dependent vasodilation in diabetes. Exposure of human platelets to high glucose in vivo or in vitro impairs their ability to cause endothelium‐dependent vasodilation. While previous data suggest that the mechanism for this involves increased activity of the cyclo‐oxygenase pathway, the signal transduction pathway mediating this effect is unknown.

Platelets from diabetic patients as well as normal platelets and normal platelets exposed to high glucose concentrations were used to determine the role of the polyol pathway, diacylglycerol (DAG) production, protein kinase C (PKC) activity and phospholipase A2 (PLA2) activity on vasodilation in rabbit carotid arteries.

We found that two aldose‐reductase inhibitors, tolrestat and sorbinil, caused only a modest improvement in the impairment of vasodilation by glucose exposed platelets. However, sorbitol and fructose could not be detected in the platelets, at either normal or hyperglycaemic conditions. We found that incubation in 17 mM glucose caused a significant increase in DAG levels in platelets. Furthermore, the DAG analog 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) caused significant impairment of platelet‐mediated vasodilation. The PKC inhibitors calphostin C and H7 as well as inhibitors of PLA2 activity normalized the ability of platelets from diabetic patients to cause vasodilation and prevented glucose‐induced impairment of platelet‐mediated vasodilation in vitro.

These results suggest that the impairment of platelet‐mediated vasodilation caused by high glucose concentrations is mediated by increased DAG levels and stimulation of PKC and PLA2 activity.

British Journal of Pharmacology (1999) 127, 903–908; doi:10.1038/sj.bjp.0702617