bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation by extracellular ATP and UTP of the stress‐activated protein kinase cascade in rat renal mesangial cells
Authors: Huwiler Andrea , Van Rossum Gerda , Wartmann Markus , Pfeilschifter Josef
Published: August 2006

Extracellular adenosine 5′‐triphosphate (ATP) and uridine 5′‐triphosphate (UTP) have been shown to activate a nucleotide receptor (P2U receptor) in rat mesangial cells that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases C and D, respectively. This is followed by an increased activity of the mitogen‐activated protein kinase cascade and cell proliferation. Here we show that ATP and UTP potently stimulate the stress‐activated protein kinase pathway and phosphorylation of the transcription factor c‐Jun.

Both nucleotides stimulated a rapid (within 5 min) and concentration‐dependent activation of stress‐activated protein kinases as measured by the phosphorylation of c‐Jun in a solid phase kinase assay.

When added at 100 μm the rank order of potency of a series of nucleotide analogues for stimulation of c‐Jun phosphorylation was UTP>ATP=UDP=ATPγS>2‐methylthio‐ATP>βγ‐imido‐ATP= ADP>AMP=UMP=adenosine=uridine. Activation of stress‐activated protein kinase activity by ATP and UTP was dose‐dependently attenuated by suramin.

Down‐regulation of protein kinase C‐α, ‐δ and ‐ε isoenzymes by 24 h treatment of the cells with 12‐O‐tetradecanoylphorbol 13‐acetate did not inhibit ATP‐ and UTP‐induced activation of c‐Jun phosphorylation. Furthermore, the specific protein kinase C inhibitors, CGP 41251 and Ro 31–8220, did not inhibit nucleotide‐stimulated c‐Jun phosphorylation, suggesting that protein kinase C is not involved in ATP‐ and UTP‐triggered stress‐activated protein kinase activation.

Pretreatment of the cells with pertussis toxin or the tyrosine kinase inhibitor, genistein, strongly attenuated ATP‐ and UTP‐induced c‐Jun phosphorylation. Furthermore, N‐acetyl‐cysteine completely blocked the activation of stress‐activated protein kinase in response to extracellular nucleotide stimulation.

In summary, these results suggest that ATP and UTP trigger the activation of the stress‐activated protein kinase module in mesangial cells by a pathway independent of protein kinase C but requiring a pertussis toxin – sensitive G‐protein and tyrosine kinase activation.

British Journal of Pharmacology (1997) 120, 807–812; doi:10.1038/sj.bjp.0700979
The duration of action of non‐β2‐adrenoceptor mediated responses to salmeterol
Authors: Nials A T , Coleman R A , Johnson M , Vardey C J
Published: August 2006

To investigate further the mechanism of the long duration of action of the selective β2‐adrenoceptor agonist, salmeterol, we have determined the duration of action of some responses to salmeterol which are not mediated throughβ2‐adrenoceptors.

In the presence of propranolol (1 μm), salmeterol (1–30 μm) caused concentration‐related relaxation of superfused, pre‐contracted strips of guinea‐pig gastric fundus. On washing the tissues, these relaxant responses were rapidly lost, the time to 50% recovery being approximately 30 min.

In human neutrophils, salmeterol (1–100 μm) caused concentration‐related inhibition of FMLP‐induced O2− release. Propranolol (1 μm) had little or no effect on the inhibitory activity of salmeterol. Washing the cells twice over a 40 min period caused a marked reduction of the inhibitory activity of salmeterol.

In guinea‐pig superfused trachea, in the absence of propranolol, infusions of (RS)‐salmeterol (10–30 nm) and the less potent (S)‐enantiomer of salmeterol (300–3000 nm) inhibited electrically‐induced contractile responses. When the infusion was stopped, there was no recovery from the inhibitory responses within 200 min. In the presence of propranolol (1 μm), infusions of (RS)‐salmeterol (10 μm) and (S)‐salmeterol (10–100 μm) also inhibited the contractile responses, but, in contrast, on stopping the infusions differences were observed in recovery times. Thus no appreciable recovery was observed from the responses to (RS)‐salmeterol, whereas a rapid loss of inhibition was observed on stopping the infusion of (S)‐salmeterol, the time to 50% recovery being 30–35 min.

These relatively short‐lasting effects of salmeterol which are not mediated through β2‐adrenoceptors, contrast with the persistence of the responses which are mediated through β2‐adrenoceptors seen in a variety of tissues, but are similar to the rate of dissociation of salmeterol observed from artificial membranes. These observations suggest that the sustained agonist activity of salmeterol is peculiar to responses mediated by β2‐adrenoceptors.

British Journal of Pharmacology (1997) 120, 961–967; doi:10.1038/sj.bjp.0700986
The inhibitory effect of the antipsychotic drug haloperidol on HERG potassium channels expressed in Xenopus oocytes
Authors: Suessbrich H , Schönherr R , Heinemann S H , Attali B , Lang F , Busch A E
Published: August 2006

The antipsychotic drug haloperidol can induce a marked QT prolongation and polymorphic ventricular arrhythmias. In this study, we expressed several cloned cardiac K+ channels, including the human ether‐a‐go‐go related gene (HERG) channels, in Xenopus oocytes and tested them for their haloperidol sensitivity.

Haloperidol had only little effects on the delayed rectifier channels Kv1.1, Kv1.2, Kv1.5 and IsK, the A‐type channel Kv1.4 and the inward rectifier channel Kir2.1 (inhibition <6% at 3 μm haloperidol).

In contrast, haloperidol blocked HERG channels potently with an IC50 value of approximately 1 μm. Reduced haloperidol, the primary metabolite of haloperidol, produced a block with an IC50 value of 2.6 μm.

Haloperidol block was use‐ and voltage‐dependent, suggesting that it binds preferentially to either open or inactivated HERG channels. As haloperidol increased the degree and rate of HERG inactivation, binding to inactivated HERG channels is suggested.

The channel mutant HERG S631A has been shown to exhibit greatly reduced C‐type inactivation which occurs only at potentials greater than 0 mV. Haloperidol block of HERG S631A at 0 mV was four fold weaker than for HERG wild‐type channels. Haloperidol affinity for HERG S631A was increased four fold at +40 mV compared to 0 mV.

In summary, the data suggest that HERG channel blockade is involved in the arrhythmogenic side effects of haloperidol. The mechanism of haloperidol block involves binding to inactivated HERG channels.

British Journal of Pharmacology (1997) 120, 968–974; doi:10.1038/sj.bjp.0700989
The induction of nitric oxide‐mediated relaxation of human isolated pulmonary arteries by PACAP
Authors: Cardell Lars Olaf , Hjert Ola , Uddman Rolf
Published: August 2006

The effects of pituitary adenylate cyclase‐activating peptide (PACAP) and vasoactive intestinal peptide (VIP) were analysed in human isolated circular segments of pulmonary arteries. Guinea‐pig pulmonary arteries were used for comparison. The responses obtained were analysed in relation to the vascular endothelium and the nitric oxide (NO) synthase inhibitor NG‐monomethyl l‐arginine (l‐NMMA).

PACAP and VIP induced concentration‐dependent relaxations of precontracted pulmonary arteries. The maximal dilator response (Imax,%) and the potency (pEC50 value) were the same for both peptides, and there were no differences in the effects obtained on human and guinea‐pig segments. PACAP and VIP were both more potent that acetylcholine (ACh).

Removal of the vascular endothelium abolished the PACAP induced dilator response in pulmonary arteries from both species. The VIP induced dilatation was unaffected, whereas the response to ACh was abolished. l‐NMMA given before PACAP inhibited the dilatation. Furthermore, l‐NMMA also reversed the dilatation already induced by PACAP and excess concentrations of l‐arginine restored the dilator response of the l‐NMMA treated arteries.

PACAP is a potent dilator of human pulmonary arteries. Although the dilator effect seems to be similar in amplitude to the one induced by VIP, the present results suggest differences in the underlying mechanisms of action (endothelium‐dependency) between the two peptides.

British Journal of Pharmacology (1997) 120, 1096–1100; doi:10.1038/sj.bjp.0700992
Differential block of nicotinic synapses on B versus C neurones in sympathetic ganglia of frog by α‐conotoxins MII and ImI
Authors: Tavazoie Sohail F , Tavazoie Masoud F , McIntosh J Michael , Olivera Baldomero M , Yoshikami Doju
Published: August 2006

The effects of two new acetylcholine receptor antagonists, α‐conotoxin MII and α‐conotoxin ImI, on nicotinic synaptic transmission in the 10th paravertebral sympathetic ganglion of the leopard frog (Rana pipiens) were examined. The preganglionic nerve was electrically stimulated (at low frequency, 1 min−1, to avoid use‐dependent changes) while compound action potentials of B and C neurones were monitored from the postganglionic nerve.

α‐Conotoxins MII and ImI, at low micromolar concentrations, reversibly blocked both B and C waves. α‐Conotoxin MII blocked the C wave more effectively than the B wave, whereas the potency of α‐conotoxin ImI was opposite that of MII. The observation that nicotinic antagonists can differentially block synaptic transmission of B versus C neurones with opposite selectivities strongly suggests that these neurones possess distinct nicotinic receptors.

In addition to fast and slow B waves described by others, C waves with two temporally distinguishable components were present in our recordings. Each α‐conotoxin affected fast and slow B waves similarly. Likewise, toxins did not discriminate between the two components of C waves. This suggests that all neurones within each major class (B or C) may have the same nicotinic receptors.

Synthetic forms of α‐conotoxins MII and ImI were used in the present study. Their ease of synthesis and their specificities should make these toxins useful probes to investigate the various subtypes of neuronal nicotinic acetylcholine receptors.

British Journal of Pharmacology (1997) 120, 995–1000; doi:10.1038/sj.bjp.0700993
Mechanism of nitric oxide‐induced contraction in the rat isolated small intestine
Authors: Lefebvre R A , Barthó L
Published: August 2006

The contractile response to nitric oxide (NO) in ral ileal myenteric plexus‐longitudinal muscle strips was pharmacologically analysed.

NO (10−7 m) induced only contraction while 10−6 m NO induced contraction followed by relaxation. Methylene blue (up to 10−4 m) did not affect the NO‐induced contractions but significantly reduced the relaxation evoked by 10−6 m NO. Administration of 8‐bromo‐cyclic GMP (10−6–10−4 m) only induced relaxation.

Sodium nitroprusside (SNP; 10−7–10−5 m) induced concentration‐dependent contractions per se; the contractile response to NO, administered within 10 min after SNP, was concentration‐dependently reduced. The guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) content of the tissues was not increased during contractions with 10−8 m NO and 10−6 m SNP; it was increased by a factor of 2 during contraction with 10−7 m NO, and by a factor of 12 during relaxation with 3×10−6 m NO.

The NO‐induced contractions were not affected by ryanodine (3×10−5 m) but were concentration‐dependently reduced by nifedipine (10−8–10−7 m) and apamin (3×10−9–3×10−8 m).

These results suggest that cyclic GMP is not involved in the NO‐induced contraction in the rat small intestine. The NO‐induced contraction is related to extracellular Ca2+ influx through L‐type Ca2+ channels, that might be activated in response to the closure of Ca2+‐dependent K+ channels.

British Journal of Pharmacology (1997) 120, 975–981; doi:10.1038/sj.bjp.0700996
Attenuation of morphine withdrawal symptoms by subtype‐selective metabotropic glutamate receptor antagonists
Authors: Fundytus Marian E , Ritchie Jennifer , Coderre Terence J
Published: August 2006

We have previously shown that chronic antagonism of group I metabotropic glutamate receptors (mGluRs), in the brain, attenuates the precipitated morphine withdrawal syndrome in rats. In the present investigation we assessed the effects of chronic antagonism of group II and III mGluRs on the severity of withdrawal symptoms in rats treated chronically with subcutaneous (s.c.) morphine.

Concurrently with s.c. morphine we infused intracerebroventricularly (i.c.v.) one of a series of phenylglycine derivatives selective for specific mGluR subtypes. Group II mGluRs (mGluR2,3), which are negatively coupled to adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) production, were selectively antagonized with 2s, 1′s, 2′s‐2‐methyl‐2‐(2′‐carboxycyclopropyl) glycine (MCCG). Group III mGluRs (mGluR4,6,7 and 8), which are also negatively linked to cyclic AMP production, were selectively antagonized with α‐methyl‐l‐amino‐4‐phosphonobutanoate (MAP4). The effects of MCCG and MAP4 were compared with α‐methyl‐4‐carboxyphenylglycine (MCPG), which non‐selectively antagonizes group II mGluRs, as well as group I mGluRs (mGluR1,5) which are positively coupled to phosphatidylinositol (PI) hydrolysis.

Chronic i.c.v. administration of both MCCG and MAP4 significantly decreased the time spent in withdrawal, MCPG and MCCG reduced the frequency of jumps and wet dog shakes and attenuated the severity of agitation.

Acute i.c.v. injection of mGluR antagonists just before the precipitation of withdrawal failed to decrease the severity of abstinence symptoms. Rather, acute i.c.v. injection of MCCG significantly increased the time spent in withdrawal.

Our results suggest that the development of opioid dependence is affected by mGluR‐mediated PI hydrolysis and mGluR‐regulated cyclic AMP production.

British Journal of Pharmacology (1997) 120, 1015–1020; doi:10.1038/sj.bjp.0701000
Tachykinin receptors in the guinea‐pig isolated oesophagus: a complex system
Authors: Kerr K P , Mitchelson F , Coupar I M
Published: August 2006

The tachykinin receptors mediating contraction of isolated longitudinal strips of the guinea‐pig oesophageal body were characterized with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) as well as the analogues, [Sar9,Met(O2)11]SP, [Nle10]NKA(4–10) and [MePhe7]NKB, selective for NK1, NK2 and NK3, receptors, respectively. Experiments were performed both in the absence and presence of a cocktail of peptidase inhibitors, captopril (1 μm), thiorphan (1 μm) and amastatin (20 μm), in order to determine whether membrane bound proteases are important in the metabolism of tachykinins in this preparation.

All agonists produced concentration‐dependent contractile effects. The presence of the peptidase inhibitors shifted the concentration‐response curves of SP, [Nle10]NKA(4–10) and [MePhe7]NKB significantly leftwards and the concentration‐response curve of NKB was shifted significantly rightwards. However, the EC50 values were significantly different only for [Nle10]NKA(4–10) and NKB.

In the presence of the peptidase inhibitors, the EC50 values of the selective agonists, [MePhe7]NKB (0.6 nm) and [Nle10]NKA(4–10) (66 nm) indicated the presence of both tachykinin NK3 and NK2 receptors. [MePhe7]NKB produced less than 50% of the maximal response obtained with the other agonists. Since [Sar9,Met(O2)11]SP produced a small response in the nanomolar concentration range in about 30% of the preparations tested, it is possible that some NK1 receptors were also present.

Assuming competitive antagonism, the NK2‐selective antagonist SR 48,968 (30 nm) gave apparent pKB values of 8.13 and 8.65 for [Nle10]NKA(4–10) in the absence and presence of peptidase inhibitors, respectively, supporting the presence of NK2 receptors.

The NK3‐selective antagonist SR 142,801 (0.1 μm), suppressed responses to low (0.1–10 nm) concentrations of [MePhe7]NKB. These contractile responses to [MePhe7]NKB were also abolished by atropine (0.6 μm) suggesting that this response was mediated via cholinergic nerves.

It is concluded that the guinea‐pig oesophagus is a complex system which has both NK2 and NK3 receptors and possibly some NK1 receptors as well.

British Journal of Pharmacology (1997) 120, 1021–1028; doi:10.1038/sj.bjp.0701001
Enhancement of the response to purinergic agonists in P2Y1 transfected 1321N1 cells by antagonists suramin and PPADS
Authors: Brown Colin A , Charlton Steven J , Boarder Michael R
Published: August 2006

We have previously shown that both suramin and pyridoxal‐phosphate‐6‐azophenyl‐2′, 4′ disulphonic acid (PPADS) act as antagonists at transfected P2Y1 receptors. Here we show that under certain experimental conditions these two P2 antagonists can enhance the response to agonists acting at these receptors.

The expression of either P2Y1 or P2Y2 receptors in 1321N1 human astrocytoma cells results, on a change of medium, in an elevation of basal (no added agonist) accumulation of [3H]‐inositol(poly)phosphates([3H]‐InsPx) compared to cells not expressing these receptors. This elevation is much greater in P2Y1 transfectants than in P2Y2 transfectants.

Both PPADS and suramin reduced this basal level of [3H]‐InsPx accumulation in the P2Y1 expressing cells.

When a protocol was used which required changing the culture medium, antagonists were added at a concentration which reduced the basal accumulation by about 50%, there was a significant stimulation in response to increasing concentrations of 2‐methylthioadenosine 5′‐triphosphate (2MeSATP), in the absence of antagonists there was no significant effect of the agonist.

However, when 2MeSATP was added in the absence of a change of medium and with no antagonist present, there was a several fold increase in [3H]‐InsPx accumulation. These results show that a release of endogenous agonist activity (possibly ATP/ADP) from the P2Y1 expressing cells can create conditions in which a response to an agonist such as 2MeSATP can only be seen in the presence of a competitive antagonist.

British Journal of Pharmacology (1997) 120, 1049–1052; doi:10.1038/sj.bjp.0701010
The effect of the selectin binding polysaccharide fucoidin on eosinophil recruitment in vivo
Authors: Teixeira Mauro M , Hellewell Paul G
Published: August 2006

In order to accumulate at sites of inflammation, leukocytes initially roll on endothelial cells of post‐capillary venules before becoming firmly attached. This process of rolling is mediated by selectins which bind to carbohydrate counter‐ligands present on the surface of both leukocytes and endothelial cells. The polysaccharide fucoidin has been previously shown to inhibit leukocyte rolling in the mesenteric circulation and to reduce neutrophil accumulation in the skin and meninges in experimental inflammation.

In the present study we have assessed the effects of fucoidin on eosinophil function in vitro and eosinophil accumulation at sites of inflammation in guinea‐pig skin.

At concentrations of up to 1200 μg ml−1, fucoidin inhibited phorbol myristate acetate (PMA)‐induced eosinophil homotypic aggregation by up to 60% but had no inhibitory effect on PMA‐induced eosinophil adhesion to serum‐coated plates.

Fucoidin effectively reduced the binding of the anti‐L‐selectin mAb MEL‐14 to guinea‐pig eosinophils. Binding of a P‐selectin‐IgG chimera to eosinophils was also partially inhibited by fucoidin, but binding of an anti‐CD18 or an anti‐VLA‐4 mAb were unaffected.

When given systemically to guinea‐pigs, fucoidin suppressed 111In‐labelled eosinophil recruitment to sites of allergic inflammation. 111In‐labelled eosinophil accumulation induced by platelet‐activating factor (PAF) and zymosan‐activated plasma (as a source of C5a des Arg) was also inhibited.

These results demonstrate a role for fucoidin‐sensitive selectins in mediating eosinophil recruitment in vivo.

British Journal of Pharmacology (1997) 120, 1059–1066; doi:10.1038/sj.bjp.0701024
Sources of Ca2+ in relation to generation of acetylcholine‐induced endothelium‐dependent hyperpolarization in rat mesenteric artery
Authors: Fukao Mitsuhiro , Hattori Yuichi , Kanno Morio , Sakuma Ichiro , Kitabatake Akira
Published: August 2006

The aim of the present study was to identify the sources of Ca2+ contributing to acetylcholine (ACh)‐induced release of endothelium‐derived hyperpolarizing factor (EDHF) from endothelial cells of rat mesenteric artery and to assess the pathway involved. The changes in membrane potentials of smooth muscles by ACh measured with the microelectrode technique were evaluated as a marker for EDHF release.

ACh elicited membrane hyperpolarization of smooth muscle cells in an endothelium‐dependent manner. The hyperpolarizing response was not affected by treatment with 10 μm indomethacin, 300 μm NG‐nitro‐l‐arginine or 10 μm oxyhaemoglobin, thereby indicating that the hyperpolarization is not mediated by prostanoids or nitric oxide but is presumably by EDHF.

In the presence of extracellular Ca2+, 1 μm ACh generated a hyperpolarization composed of the transient and sustained components. By contrast, in Ca2+‐free medium, ACh produced only transient hyperpolarization.

Pretreatment with 100 nm thapsigargin and 3 μm cyclopiazonic acid, endoplasmic reticulum Ca2+‐ATPase inhibitors, completely abolished ACh‐induced hyperpolarization. Pretreatment with 20 mm caffeine also markedly attenuated ACh‐induced hyperpolarization. However, the overall pattern and peak amplitude of hyperpolarization were unaffected by pretreatment with 1 μm ryanodine.

In the presence of 5 mm Ni2+ or 3 mm Mn2+, the hyperpolarizing response to ACh was transient, and the sustained component of hyperpolarization was not observed. On the other hand, 1 μm nifedipine had no effect on ACh‐induced hyperpolarization.

ACh‐induced hyperpolarization was nearly completely eliminated by 500 nm U‐73122 or 200 μm 2‐nitro‐4‐carboxyphenyl‐N,N‐diphenylcarbamate, inhibitors of phospholipase C, but was unchanged by 500 nm U‐73343, an inactive form of U‐73122. Pretreatment with 20 nm staurosporine, an inhibitor of protein kinase C, did not modify ACh‐induced hyperpolarization.

These results indicate that the ACh‐induced release of EDHF from endothelial cells of rat mesenteric artery is possibly initiated by Ca2+ release from inositol 1,4,5‐trisphosphate (IP3)‐sensitive Ca2+ pool as a consequence of stimulation of phospholipid hydrolysis due to phospholipase C activation, and maintained by Ca2+ influx via a Ni2+‐ and Mn2+‐sensitive pathway distinct from L‐type Ca2+ channels. The Ca2+‐influx mechanism seems to be activated following IP3‐induced depletion of the pool.

British Journal of Pharmacology (1997) 120, 1328–1334; doi:10.1038/sj.bjp.0701027
Direct cardiac and vascular actions of adrenomedullin in conscious sheep
Authors: Parkes David G , May Clive N
Published: August 2006

Adrenomedullin (ADM) is a recently characterized circulating hormone which affects haemodynamic, renal and pituitary function in mammals. We have shown previously that in sheep, ADM produces vasodilatation together with increases in cardiac output and contractility. However, whether these effects are direct or mediated by autonomic reflexes is unclear. The present study examined the cardiovascular actions of an intravenous infusion of ADM in conscious, chronically instrumented sheep with either sympathetic, parasympathetic or autonomic ganglion blockade, to determine the role of the autonomic nervous system in mediating these cardiovascular changes.

Human ADM (1–52) was infused for 60 min at 2 μg kg−1 h−1 following: (1) saline control, (2) combined α/β‐adrenoceptor (sympathetic) blockade (proporanolol 0.4 mg kg−1 h−1+phentolamine 0.15 mg kg−1 h−1 for 20 h), (3) muscarinic (parasympathetic) blockade (methscopolamine 0.05 mg kg−1 h−1 for 20 h) or (4) ganglion blockade (hexamethonium 3 mg kg−1 h−1 for 4 h). Measurements were made of mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), stroke volume (SV), total peripheral conductance (TPC), maximal aortic flow (Fmax) and maximal rate of change of aortic flow (dF/dt).

ADM reduced MAP by 3±1 mmHg, and increased CO (1.2±0.2 l min−1), HR (14±2 beats min−1), TPC (21±3 ml min−1 mmHg−1), Fmax (2.3±0.8 l min−1) and dF/dt (86±21 l min−1 s−1) in normal sheep. In animals with α/β blockade, similar changes were observed with ADM. However, during muscarinic blockade, the increases in HR (32±4 beats min−1), CO (2.1±0.4 l min−1), TPC (31±4 ml min−1 mmHg−1), Fmax (4.0±0.6 l min−1), and dF/dt (150±12 l min−1 s−1) produced by ADM were enhanced. During ganglion blockade, ADM produced a greater reduction in MAP (−10±2 mmHg) compared to controls (−3±1 mmHg). However, there was no increase in HR. The changes in CO, TPC and contractility were similar to those observed in control animals.

These results suggest that the vasodilator effects of ADM on the periphery and its ability to increase CO and cardiac contractility are not mediated by the autonomic nervous system, but are probably the result of direct actions of ADM on the heart and vasculature.

British Journal of Pharmacology (1997) 120, 1179–1185; doi:10.1038/sj.bjp.0701034
Activation of endothelin ETA receptors masks the constrictor role of endothelin ETB receptors in rat isolated small mesenteric arteries
Authors: Mickley Emma J. , Gray Gillian A. , Webb David J.
Published: August 2006

Endothelin‐1 (ET‐1) produces constriction of the rat mesenteric vascular bed in vivo via ETA and ETB receptor subtypes. The aim of this study was to investigate the relative roles of these receptor subtypes in rat isolated, endothelium‐denuded, small mesenteric arteries, under pressure, by use of ET‐1; the ETA receptor antagonist, BQ‐123; the ETB receptor selective agonist, sarafotoxin S6c (SRTX S6c); the ETB receptor selective antagonist, BQ‐788; and the ETA/ETB antagonist, TAK‐044.

In 3rd generation mesenteric arteries, ET‐1 (10−1310−7 m) produced concentration‐dependent contractions (pD2 9.86). SRTX S6c (10−1210−7 m) also induced concentration‐dependent contractions in 53% of arteries studied, although the Emax was much less than that obtained with ET‐1 (10.7±2.9% vs 101.9±2.6% of the 60 mm KCl‐induced contraction).

Neither ETB receptor desensitization, by a supra‐maximal concentration of SRTX S6c (10−7 m), nor incubation with BQ‐788 (3×10−8 m), had any significant effect on the ET‐1 concentration‐response curve, although both treatments tended to enhance rather than inhibit responses to ET‐1.

In the presence of BQ‐123 (10−6 m), responses to low concentrations of ET‐1 (up to 10−10 m) were unaffected but responses to concentrations of ET‐1 above 10−10 m were significantly inhibited.

SRTX S6c desensitization followed by incubation with BQ‐123 (10−6 m) or co‐incubation with BQ‐788 (3×10−8 m) and BQ‐123 caused inhibition of responses to all concentrations of ET‐1, resulting in a rightward shift of the ET‐1 concentration‐response curve. The same effect was obtained by incubation with TAK‐044 (10−8 m and 3×10−7 m).

Thus, responses of rat small mesenteric arteries to ET‐1 are mediated by both ETA and ETB receptors. The relative role of ETB receptors is greater than that predicted by the small responses to SRTX S6c or by resistance of ET‐1‐induced contraction to ETB receptor desensitization or BQ‐788. The effect of ETB receptor desensitization or blockade is only revealed in the presence of ETA receptor blockade, suggesting the presence of a ‘crosstalk’ mechanism between the receptors. These results support the concept that dual receptor antagonists, like TAK‐044, may be required to inhibit completely constrictor responses to ET‐1.

British Journal of Pharmacology (1997) 120, 1376–1382; doi:10.1038/sj.bjp.0701036
Sites of action of IL‐1 in the development of fever and cytokine responses to tissue inflammation in the rat
Authors: Miller Andrew J. , Hopkins Stephen J. , Luheshi Giamal N.
Published: August 2006

The objective of the present study was to determine the sites of action of the cytokine, interleukin‐1 (IL‐1), in the febrile response to local inflammation in the rat, by comparing the importance of IL‐1 in the local tissues, the circulation and the brain. This was achieved by injecting lipopolysaccharide (LPS, 100 μg kg−1) into a subcutaneous air pouch and testing the effects of blocking IL‐1 action with the human recombinant interleukin‐1 receptor antagonist (IL‐1ra) injected either into the air pouch, intraperitoneally (1 mg kg−1, 0+1 h, i.p.), or intracerebroventricularly (200 μg/rat, 0+1 h, i.c.v.).

To investigate the effect of IL‐1ra on fever and the induction of local and circulating cytokines (IL‐1 and IL‐6), separate experiments were performed in which groups of animals were killed 1.5, 3 or 5 h after LPS injection. Plasma and pouch fluid samples were collected for bioassay of IL‐1 and IL‐6.

Injection of LPS into the air pouch significantly increased (1.5°C) body temperature, local (air pouch) concentrations of bioactive IL‐1 and IL‐6, and circulating bioactive IL‐6, compared to saline‐treated controls.

Injection of IL‐1ra into the pouch significantly attenuated LPS fever (P<0.001). This decrease in body temperature was associated with significant inhibition of local IL‐1 bioactivity 1.5 (96%), 3 (84%) and 5 h (72%), and in bioactive IL‐6 in pouch lavage fluid 1.5 (45%) and 5 h (35%), after LPS injection. The concentration of bioactive IL‐6 in the plasma was significantly reduced (39%) at 3 h, when temperature was approaching the maximal value.

Both systemic (i.p.) and central (i.c.v.) administration of IL‐1ra significantly attentuated LPS fever (P<0.05). However, it had no effect on either local concentrations of bioactive IL‐1 or IL‐6, or circulating IL‐6, at any of the sample points.

These data suggest that IL‐1 is released locally, at the site of tissue inflammation and that it is an important mediator of the febrile response to local inflammation. The results also indicate that IL‐1 produced locally may contribute to the production of IL‐6 which is released into the circulation, and that IL‐1 has important actions in the generation of fever at other sites, including the brain.

British Journal of Pharmacology (1997) 120, 1274–1279; doi:10.1038/sj.bjp.0701049
Contribution of α‐adrenoceptors to depolarization and contraction evoked by continuous asynchronous sympathetic nerve activity in rat tail artery
Authors: Brock J. A. , Mclachlan E. M. , Rayner S. E.
Published: August 2006

The effects of continuous but asynchronous nerve activity induced by ciguatoxin (CTX‐1) on the membrane potential and contraction of smooth muscle cells have been investigated in rat proximal tail arteries isolated in vitro. These effects have been compared with those produced by the continuous application of phenylephrine (PE).

CTX‐1 (0.4 nm) and PE (10 μm) produced a maintained depolarization of the arterial smooth muscle that was almost completely blocked by α‐adrenoceptor blockade. In both cases, the depolarization was more sensitive to the selective α2‐adrenoceptor antagonist, idazoxan (0.1 μm), than to the selective α1‐adrenoceptor antagonist, prazosin (0.01 μm).

In contrast, the maintained contraction of the tail artery induced by CTX‐1 (0.2 nm) and PE (2 and 10 μm) was more sensitive to prazosin (0.01) μm, than to idazoxan (0.01 μm). In combination, these antagonists almost completely inhibited contraction to both agents.

Application of the calcium channel antagonist, nifedipine (1 μm), had no effect on the depolarization induced by either CTX‐1 or PE but maximally reduced the force of the maintained contraction to both agents by about 50%.

We conclude that the constriction of the tail artery induced by CTX‐1, which mimics the natural discharge of postganglionic perivascular axons, is due almost entirely to α‐adrenoceptor activation. The results indicate that neuronally released noradrenaline activates more than one α‐adrenoceptor subtype. The depolarization is dependent primarily on α2‐adrenoceptor activation whereas the contraction is dependent primarily on α1‐adrenoceptor activation. The links between α‐adrenoceptor activation and the voltage‐dependent and voltage‐independent mechanisms that deliver Ca2+ to the contractile apparatus appear to be complex.

British Journal of Pharmacology (1997) 120, 1513–1521; doi:10.1038/sj.bjp.0701055
Sequential development of angiotensin receptors and angiotensin I converting enzyme during angiogenesis in the rat subcutaneous sponge granuloma
Authors: Walsh David A. , Hu De‐En , Wharton John , Catravas John D. , Blake David R. , Fan Tai‐Ping D.
Published: August 2006

The vasoconstrictor peptide antiotensin II (AII) can stimulate angiogenesis, an important process in wound healing, tumour growth and chronic inflammation. To elucidate mechanisms underlying AII‐enhanced angiogenesis, we have studied a subcutaneous sponge granuloma model in the rat by use of 133Xe clearance, morphometry and quantitative in vitro autoradiography.

When injected directly into the sponge, AII (1 nmol day−1) increased 133Xe clearance from, and fibrovascular growth in sponge granulomas, indicating enhanced angiogenesis 6 to 12 days after implantation. This AII‐enhanced angiogenesis was inhibited by daily doses (100 nmol/sponge) of the specific but subtype non‐selective AII receptor antagonist (Sar1, Ile8)AII, and by the selective non‐peptide AT1 receptor antagonists losartan and DuP 532. In contrast, AII‐enhanced neovascularization was not inhibited by the AT2 receptor antagonist PD123319, nor was it mimicked by the AT2 receptor agonist CGP42112A (each at 100 nmol/sponge day−1).

AI (1 nmol/sponge day−1), the angiotensin converting enzyme (ACE) inhibitors captopril (up to 100 μg/sponge day−1) and lisinopril (40 μg/sponge day−1), or AII receptor antagonists did not affect angiogenesis in the absence of exogenous AII.

[125I]‐(Sar1, Ile8)AII binding sites with characteristics of AT1 receptors were localized to microvessels and to non‐vascular cells within the sponge stroma from 4 days after implantation, and were at higher density than in skin throughout the study.

[125I]‐(Sar1, Ile8)AII binding sites with characteristics of AT2 receptors were localized to non‐vascular stromal cells, were of lower density and appeared later than did AT1 sites.

The ACE inhibitor [125I]‐351A bound to sites with characteristics of ACE, 14 days after sponge implantation. [125I]‐351A bound less densely to sponge stroma than to skin.

We propose that AII can stimulate angiogenesis, acting via AT1 receptors within the sponge granuloma. AT1 and AT2 receptors and ACE develop sequentially during microvascular maturation, and the role of the endogenous angiotensin system in angiogenesis will depend on the balanced local expression of its various components. Pharmacological modulation of this balance may provide novel therapeutic approaches in angiogenesis‐dependent diseases.

British Journal of Pharmacology (1997) 120, 1302–1311; doi:10.1038/sj.bjp.0701062
The involvement of pertussis toxin‐sensitive G proteins in the post receptor mechanism of central I1‐imidazoline receptors
Authors: Takada Koji , Hayashi Yukio , Kamibayashi Takahiko , Mammoto Tadanori , Yamatodani Atsushi , Kitamura Seiji , Yoshiya Ikuto
Published: August 2006

To elucidate the possible involvement of pertussis toxin (PTX)‐sensitive G proteins in the post receptor mechanism of α2‐adrenoceptors and imidazoline receptors, we examined the effect of pretreatment of the central nervous system with PTX on the antidysrhythmic effect of dexmedetomidine, a selective α2‐adrenoceptor agonist, and rilmenidine, a selective I1‐imidazoline receptor agonist on halothane‐adrenaline dysrhythmias in rats.

Dexmedetomidine (0, 1.0, 2.0, 5.0 μg kg−1 min−1, i.v.) and rilmenidine (0, 1.0, 3.0, 10, 20 μg kg−1, i.v.) prevented the genesis of halothane‐adrenaline dysrhythmias in a dose‐dependent fashion. Both idazoxan (10, 20 μg kg−1, intracerebroventricularly (i.c.v.)), an α2‐adrenoceptor antagonist with high affinity for imidazoline receptors, and rauwolscine, (40 μg kg−1, i.c.v.), an α2‐adrenoceptor antagonist with low affinity for imidazoline receptors inhibited the action of dexmedetomidine (5.0 μg kg−1 min−1, i.v.), but the inhibitory potency of idazoxan was much greater than that of rauwolscine. While the pretreatment with PTX (0.1, 0.5, 1.0 μg kg−1, i.c.v.) did not change the dysrhythmogenecity of adrenaline, this treatment completely blocked the antidysrhythmic property of rilmenidine (20 μg kg−1, i.v.) as well as dexmedetomidine (5.0 μg kg−1 min−1, i.v.).

It is suggested that central I1‐imidazoline receptors as well as α2‐adrenoceptors may be functionally coupled to PTX‐sensitive G proteins.

British Journal of Pharmacology (1997) 120, 1575–1581; doi:10.1038/sj.bjp.0701090
Pharmacological evidence that the activation of the Na+‐Ca2+ exchanger protects C6 glioma cells during chemical hypoxia
Authors: Amoroso Salvatore , De Maio Matteo , Russo Giovanni M , Catalano Annalisa , Bassi Antonella , Montagnani Stefania , Di Renzo Gianfranco , Annunziato Lucio
Published: August 2006

In C6 glioma cells exposed to chemical hypoxia a massive release of lactate dehydrogenase (LDH) occurred at 3 and 6 h, coupled with an increased number of propidium‐iodide positive dead cells.

Extracellular Na+ removal, which activates the Na+‐Ca2+ exchanger as a Na+ efflux pathway and prevents Na+ entrance, significantly reduced LDH release and the number of propidium iodide positive C6 cells.

During chemical hypoxia, in the presence of extracellular Na+ ions, a progressive increase of [Ca2+]i occurred; in the absence of extracellular Na+ ions [Ca2+]i was enhanced to a greater extent.

The blockade of the Na+‐Ca2+ exchanger by the amiloride derivative 5‐(N‐4‐chlorobenzyl)‐2′,4′‐dimethylbenzamil (CB‐DMB), lanthanum (La3+) and the Ca2+ chelator EGTA, completely reverted the protective effect exerted by the removal of Na+ ions on C6 glioma cells exposed to chemical hypoxia.

The inhibition of the Na+‐Ca2+ antiporter enhanced chemical hypoxia‐induced LDH release when C6 glioma cells were incubated in the presence of physiological concentrations of extracellular Na+ ions (145 mM), suggesting that the blockade of the Na+‐Ca2+ antiporter during chemical hypoxia can lead to increased cell damage.

Collectively, these results suggest that activation of the Na+‐Ca2+ exchanger protects C6 glioma cells exposed to chemical hypoxia, whereas its pharmacological blockade can exacerbate cellular injury.

British Journal of Pharmacology (1997) 121, 303–309; doi:10.1038/sj.bjp.0701092
Mechanism of enhanced vasoconstrictor hormone action in vascular smooth muscle cells by cyclosporin A
Authors: Lo Russo Alexandre , Passaquin Anne‐Catherine , Rüegg Urs T
Published: August 2006

The use of the immunosuppressive drug cyclosporin A (CsA) is limited by two major side effects, nephrotoxicity and hypertension, which are caused by drug‐induced local vasoconstriction. We have recently shown that CsA potentiates the contraction of isolated resistance arteries to vasoconstrictor hormones and increases the calcium response to these agents in vascular smooth muscle cells (VSMC). The goal of the present study was to investigate further the molecular mechanism(s) involved in these effects.

Stimulation of VSMC with [Arg]8vasopressin (AVP) induced a concentration‐dependent increase in total inositol phosphates (InsP) and cellular calcium response (as measured by 45Ca2+ efflux). Preincubation of VSMC with CsA increased both InsP formation and 45Ca2+ efflux.

The potentiating effect of CsA on AVP‐elicited InsP formation and 45Ca2+ efflux was inhibited by co‐incubation with the protein synthesis inhibitors actinomycin D and cycloheximide, indicating that CsA acted on gene expression.

Binding experiments with [3H]‐AVP on VSMC showed that CsA increased the number of AVP receptors by about two fold without affecting receptor affinity. Actinomycin D completely blocked this increase.

These results demonstrate for the first time that incubation of VSMC with CsA increases the expression of AVP receptors, resulting in a potentiation of InsP formation and calcium response upon stimulation with AVP. This effect of CsA is likely to occur with other vasoconstrictor hormone receptors as well and could be a key mechanism in the induction of vasoconstriction, and subsequent drug‐induced nephrotoxicity and hypertension.

British Journal of Pharmacology (1997) 121, 248–252; doi:10.1038/sj.bjp.0701102
Selective induction of cyclo‐oxygenase‐2 activity in the permanent human endothelial cell line HUV‐EC‐C: biochemical and pharmacological characterization
Authors: Miralpeix Montserrat , Camacho Mercedes , López‐Belmonte Jesús , Canalías Francesca , Beleta Jordi , M Palacios José , Vila Luis
Published: August 2006

Cyclo‐oxygenase (COX), the enzyme responsible for the conversion of arachidonic acid (AA) to prostaglandin H2 (PGH2), exists in two forms, termed COX‐1 and COX‐2 which are encoded by different genes. COX‐1 is expressed constitutively and is known to be the site of action of aspirin and other non‐steroidal anti‐inflammatory drugs. COX‐2 may be induced by a series of pro‐inflammatory stimuli and its role in the development of inflammation has been claimed.

Endothelial cells are an important physiological source of prostanoids and the selective induction of COX‐2 activity has been described for finite cultures of endothelial cells, but not for permanent endothelial cell lines.

The HUV‐EC‐C line is a permanent endothelial cell line of human origin. We have determined the COX activity of these cells under basal conditions and after its exposure to two different stimuli, phorbol 12‐myristate 13‐acetate (PMA) and interleukin‐1β (IL‐1β).

Both PMA and IL‐1β produced dose‐ and time‐dependent increases of the synthesis of the COX‐derived eicosanoids. These increases were maximal after the treatment with 10 nM PMA for 6 to 9 h. Under these conditions, the main eicosanoid produced by the cells was PGE2.

The increase of COX activity by PMA or IL‐1β correlated with an increase of the enzyme's apparent Vmax, whilst the affinity for the substrate, measured as apparent Km, remained unaffected.

Treatment of the cells with PMA induced a time‐dependent increase in the expression of both COX‐1 and COX‐2 mRNAs. Nevertheless, this increase was reflected only as an increase of the COX‐2 isoenzyme at the protein level.

The enzymatic activity of the PMA‐induced COX was measured in the presence of a panel of enzyme inhibitors, and the IC50 values obtained were compared with those obtained for the inhibition of human platelet COX activity, a COX‐1 selective assay. Classical non‐steroidal anti‐inflammatory drugs (NSAIDs) inhibited both enzymes with varying potencies but only the three compounds previously shown to be selective COX‐2 inhibitors (SC‐58125, NS‐398 and nimesulide) showed higher potency towards the COX of PMA‐treated HUV‐EC‐C.

Overall, it appears that the stimulation of the HUV‐EC‐C line with PMA selectively induces the COX‐2 isoenzyme. This appears to be a suitable model for the study of the physiology and pharmacology of this important isoenzyme, with a permanent endothelial cell line of human origin.

British Journal of Pharmacology (1997) 121, 171–180; doi:10.1038/sj.bjp.0701112
Differential modulation of AMPA receptor mediated currents by Evans Blue in postnatal rat hippocampal neurones
Authors: Schürmann Britta , Wu Xueqing , Dietzel Irmgard D , Leßmann Volkmar
Published: August 2006

The modulation of non‐N‐methyl‐D‐aspartate (NMDA) receptor‐mediated whole cell currents and of glutamatergic synaptic transmission by purified Evans Blue (EB) was investigated in rat cultured postnatal hippocampal neurones by use of patch clamp recordings and a fast drug application system.

Three different groups of neurones could be distinguished with respect to the type of modulation obtained with 10 μM EB: EB was either a predominant inhibitor of desensitization (13% of the neurones), a predominant inhibitor of current amplitudes (42%) or a mixed inhibitor of both properties (45%). Both effects were not use‐dependent and reached maximal levels after 30 s of pre‐equilibration with the diazo dye.

Dose‐response curves obtained from glutamate activated whole cell currents yielded an IC50 value for EB of 13.3 μM (Hill coefficient: 1.3) for the inhibition of desensitization, and an IC50 value of 10.7 μM (Hill coefficient: 1.2) for the inhibition of current amplitudes.

Chicago acid SS (100 μM) which is one of the synthesis precursors of EB had no effect on current amplitudes of glutamate activated whole cell currents but was a weak inhibitor of desensitization in all hippocampal neurones investigated, irrespective of the type of modulation obtained with EB in the same neurone.

Oxidatively modified EB (the so‐called VIMP (10 μM)) had no effect on the kinetics but was a partial inhibitor of glutamate‐activated whole cell currents in all hippocampal neurones investigated.

EB (10 μM) inhibited the amplitudes of non‐NMDA receptor mediated autaptic currents to the same extent (to 39±19% of control) as observed for glutamate activated whole cell currents (to 41±17% and 56±20%). However, the decay of the autaptic responses remained uninfluenced upon EB application, indicating that either receptor desensitization does not dominate the time course of the synaptic response or that the non‐NMDA receptors sensitive to modulation of desensitization by EB are not present in the postsynaptic membrane.

In conclusion, EB differentially modulates α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) receptor gating in different subsets of neurones. Upon identification of the cellular determinants for the differential modulation (e.g. AMPA receptor subunit composition) EB could become a useful tool to investigate receptor subtypes during electrophysiological recordings.

British Journal of Pharmacology (1997) 121, 237–247; doi:10.1038/sj.bjp.0701125
Inhibition by nociceptin of the light‐evoked release of ACh from retinal cholinergic neurones
Authors: Neal M. J. , Cunningham J. R. , Paterson S. J. , Mcknight A. T.
Published: August 2006

The retina possesses cholinergic amacrine cells which release acetylcholine (ACh) in response to flickering light. Using an eye‐cup preparation in anaesthetized rabbits we found that when the retina was exposed to nociceptin, the light‐evoked release of ACh was reduced in a concentration‐dependent manner (IC50 = 100 nm), the maximum effect being 60% inhibition. Opioid receptors were not involved in the inhibitory effect of nociceptin because its action was not blocked by naloxone (1 μm) and furthermore μ‐opioids enhanced the light‐evoked release of ACh. Using rabbit retina homogenates we found that the retina possessed a substantial number of high‐affinity binding sites for [3H]‐nociceptin indicating the presence of ORLI‐receptors. Since [des‐Phe1]‐nociceptin, which has no affinity for the ORLI‐receptor, had no effect on the light‐evoked release of ACh it is unlikely that the action of nociceptin was simply non‐specific. We conclude that the inhibitory effect of nociceptin on retinal ACh release involves activation of the ORLI receptors.

British Journal of Pharmacology (1997) 120, 1399–1400; doi:10.1038/sj.bjp.0701135
Endothelin receptors and their cellular signal transduction mechanism in human cultured prostatic smooth muscle cells
Authors: Saita Yuji , Koizumi Tomonobu , Yazawa Hidenori , Morita Takashi , Takenaka Toichi , Honda Kazuo
Published: August 2006

Endothelin (ET) receptors, and their cellular signal transduction mechanism, were characterized in a primary culture of human prostatic smooth muscle cells (HP cell).

[125I]‐ET‐1 and [125I]‐ET‐3 binding studies revealed that both ETA and ETB receptors were present in the HP cells, and the ratio of ETA to ETB receptors was 1.4:1.

Analysis of ET receptor mRNA by reverse transcription‐polymerase chain reaction also demonstrated that HP cells express both ETA and ETB receptors.

ET‐1 and ET‐3 increased intracellular free Ca2+ concentration ([Ca2+]i) in the HP cells in a concentration‐dependent manner. Use of subtype selective antagonists BQ‐123 and BQ‐788, indicated that both ETA and ETB receptors were coupled to an increase in [Ca2+]i.

Pretreatment of the cells with pertussis toxin resulted in a significant but partial attenuation of the [Ca2+]i increase mediated through the ETA and ETB receptors. However, sensitivity to pertussis toxin (PTX) was significantly different between them.

In conclusion, HP cells possess ETA and ETB receptors. Further, these two endothelin receptor subtypes evoke an increase in [Ca2+]i possibly via the action of different GTP‐binding proteins.

British Journal of Pharmacology (1997) 121, 687–694; doi:10.1038/sj.bjp.0701179