bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulators of internal Ca2+ stores and the spontaneous electrical and contractile activity of the guinea‐pig renal pelvis
Authors: Lang R J , Hashitani H , Keller S , Takano H , Mulholland E L , Fukuta H , Suzuki H
Published: January 2005

The role of internal Ca2+ stores in the generation of the rhythmic electrical and contractile activity in the guinea‐pig proximal renal pelvis was examined using intracellular microelectrode and muscle tension recording techniques.

Ryanodine (30 μM) transiently increased contraction amplitude, while caffeine (0.5 – 3 mM) reduced contraction amplitude and frequency. Contractility was also reduced by 2‐aminoethoxy‐diphenylborate (2‐APB 60 μM), xestospongin C (1 μM), U73122 (5 μM) and neomycin (4 mM), blockers of IP3‐dependent release from Ca2+ stores.

60 mM K+ saline‐evoked contractions were reduced by caffeine (1 mM), U73122 (5 μM) and neomycin (4 mM), but little affected by ryanodine or 2‐APB (60 μM).

Spontaneous action potentials consisting of an initial spike followed by a long plateau were recorded (frequency 8.6±1.0 min−1) in small urothelium‐denuded strips of proximal renal pelvis.

Action potential discharge was blocked in 75 and 35% of cells by 2‐APB (60 μM) and caffeine (1 mM), respectively. In the remaining cells, only a truncation of the plateau phase was observed.

Cyclopiazonic acid (CPA 10 μM for 10 – 180 min), blocker of CaATPase, transiently increased contraction frequency and amplitude. Action potential durations were increased 3.6 fold. Contraction amplitude and frequency slowly declined during a prolonged (>60 min) CPA exposure.

We conclude that the action potential in caffeine‐sensitive cells and the shoulder component of caffeine‐insensitive action potential arise from the entry of Ca2+ through Ca2+ channels. The inhibitory actions of modulators of internal Ca2+ release were partially explained by a blockade of Ca2+ entry.

British Journal of Pharmacology (2002) 135, 1363–1374; doi:10.1038/sj.bjp.0704609
Anthrapyridones, a novel group of antitumour non‐cross resistant anthraquinone analogues. Synthesis and molecular basis of the cytotoxic activity towards K562/DOX cells
Authors: Tarasiuk J , Stefańska B , Plodzich I , Tkaczyk‐Gobis K , Seksek O , Martelli S , Garnier‐Suillerot A , Borowski E
Published: January 2005

Multidrug resistance (MDR) to antitumour agents, structurally dissimilar and having different intracellular targets, is the major problem in cancer therapy. MDR phenomenon is associated with the presence of membrane proteins which belong to the ATP‐binding cassette family transporters responsible for the active drug efflux leading to the decreased intracellular accumulation.

The search of new compounds able to overcome MDR is of prime importance.

Recently we have synthesized a new family of anthrapyridone compounds. The series contained derivatives modified with appropriate hydrophobic or hydrophylic substituents at the side chain.

The interaction of these derivatives with erythroleukemia K562 sensitive and K562/DOX resistant (overexpressing P‐glycoprotein) cell lines has been examined. The study was performed using a spectrofluorometric method which allows to continuously follow the uptake and efflux of fluorescent molecules by living cells.

It was demonstrated that the increase in the lipophilicity of anthrapyridones favoured the very fast cellular uptake exceeding the rate of P‐gp dependent efflux out of the cell. For these derivatives, very high accumulation (the same for sensitive and resistant cells) was observed and the in vitro biological data confirmed that these compounds exhibited comparable cytotoxic activity towards sensitive and P‐gp resistant cell line. In contrast, anthrapyridones modified with hydrophylic substituents exhibited relatively low kinetics of cellular uptake.

For these derivatives decreased accumulation in resistant cells was observed and the in vitro biological data demonstrated that they were much less active against P‐gp resistant cells in comparison to sensitive cells.

We also studied, using confocal microscopy, the intracellular distribution of anthrapyridones in NIH‐3T3 cells. Our data showed that these compounds were strongly accumulated in the nucleus and lysosomes.

British Journal of Pharmacology (2002) 135, 1513–1523; doi:10.1038/sj.bjp.0704611
2‐Chloroadenosine but not adenosine induces apoptosis in rheumatoid fibroblasts independently of cell surface adenosine receptor signalling
Authors: Koshiba Masahiro , Kosaka Hidekazu , Nakazawa Takashi , Hayashi Nobuhide , Saura Ryuichi , Kitamura Noriko , Kumagai Shunichi
Published: January 2005

The apoptotic effect of adenosine and its analogues was studied in fibroblast‐like synoviocytes derived from rheumatoid arthritis patients (RA‐FLSs). Evoked cell death was quantitatively examined by assessing DNA fragmentation using an enzyme‐liked immunosorbent assay and by measuring phosphatidylserine exposure through flow cytometric analysis of annexin V binding.

Exposing cells for 24 h to 2‐chloroadenosine (2‐CADO), a nonspecific, adenosine deaminase (ADA)‐resistant, adenosine receptor (AdoR) agonist, induced DNA fragmentation, and thus apoptosis, in RA‐FLSs at concentrations 50 μM. By contrast, incubation with adenosine for up to 72 h did not evoke DNA fragmentation, even in the presence of ADA inhibitor coformycin and nucleoside transporter inhibitor nitrobenzylmercaptopurin (NBMPR). Transcription of all four AdoR isoforms was detected in RA‐FLSs; nevertheless selective AdoR agonists similarly failed to induce DNA fragmentation.

DNA fragmentation evoked by 2‐CADO was inhibited by NBMPR and by 5′‐iodotubercidin, an adenosine kinase inhibitor, but not by xanthine amine congener, an A1 and A2 receptor antagonist, or by selective AdoR antagonists.

The nonspecific caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp fluoromethyl ketone abolished the apoptotic effect of 2‐CADO.

These results suggest that 2‐CADO induces apoptosis in RA‐FLSs independently of AdoR‐mediated signalling. Instead, 2‐CADO, but not adenosine, is taken up into RA‐FLSs via human equilibrative nucleoside transporter‐1, where it is phosphorylated by adenosine kinase. The resultant phospho‐2‐CADO induces DNA fragmentation by activating a caspase pathway.

British Journal of Pharmacology (2002) 135, 1477–1486; doi:10.1038/sj.bjp.0704612
Enhanced neuronal damage by co‐administration of quinolinic acid and free radicals, and protection by adenosine A2A receptor antagonists
Authors: Behan W M H , Stone T W
Published: January 2005

Quinolinic acid may be an important endogenous excitotoxin, but its concentrations in brain are low. We have therefore attempted to determine whether its neurotoxicity can be increased by the simultaneous presence of free radicals.

Quinolinic acid was injected into the hippocampus of anaesthetized rats at doses of 40 and 80 nmols which produced little neuronal loss, and 120 nmols which produced over 90% neuronal loss.

A mixture of xanthine and xanthine oxidase, a known source of free radical reactive oxygen species, also generated little damage alone, but killed over 80% of CA1 neurons when combined with 80 nmols of quinolinic acid. Similarly, the nitric oxide donor S‐nitroso‐N‐acetylpenicillamine (SNAP) potentiated the damage produced by quinolinic acid.

The glutamate antagonist 5,7‐dichlorokynurenic acid prevented the damage produced by 120 nmols of quinolinic acid, but not that produced by quinolinic acid plus xanthine/xanthine oxidase, indicating that damage was not simply the result of free radical enhancement of NMDA receptor activation.

Three chemically dissimilar antagonists at adenosine A2A receptors prevented the damage caused by quinolinic acid and xanthine/xanthine oxidase or by quinolinic acid plus SNAP.

It is concluded that reactive oxygen species can potentiate the neurotoxicity of quinolinic acid. The site of interaction is probably distal to the NMDA receptor. Blockade of adenosine A2A receptors can protect against this combined damage, suggesting potential value in the prevention of brain damage.

British Journal of Pharmacology (2002) 135, 1435–1442; doi:10.1038/sj.bjp.0704613
Inhibition by glucocorticoids of the interleukin‐1β‐enhanced expression of the mast cell growth factor SCF
Authors: Silva Carla Alexandra Da , Kassel Olivier , Mathieu Eric , Massard Gilbert , Gasser Bernard , Frossard Nelly
Published: January 2005

Stem cell factor (SCF) is a major mast cell growth factor that promotes differentiation and chemotaxis of mast cells and inhibits their apoptosis.

We evaluated the effect of interleukin (IL)‐1β, a major pro‐inflammatory cytokine, on the constitutive expression of SCF and studied the effects of two glucocorticoids, budesonide and dexamethasone, on the IL‐1β‐enhanced SCF expression.

Human lung fibroblasts in culture were serum‐starved for 48 h and treated with IL‐1β, budesonide and/or RU486. SCF cDNA was quantified after total RNA reverse transcription by on‐line fluorescent polymerase chain reaction. SCF protein was quantified by ELISA.

IL‐1β induced an increase in SCF mRNA (+91% at 2.5 h) and protein production (+32%) by human lung fibroblasts in culture (P<0.001).

Budesonide inhibited IL‐1β‐induced SCF mRNA expression (−68%) at 2.5 h and even more so at 10 h (−192%) (P<0.001). The expression of SCF protein also decreased by 3.5‐fold at 10 h. Results were similar with dexamethasone. The glucocorticoid antagonist RU486 cancelled the effects induced by the glucocorticoids.

Increased SCF mRNA levels were associated with increased stability of this mRNA as measured after treatment with actinomycin D (1.9‐fold at 2.5 h). Budesonide decreased this IL‐1β‐enhanced stability by about 1.5‐fold (P<0.001).

We conclude that in ‘inflammatory’ conditions, mimicked in vitro by IL‐1β, glucocorticoid treatment inhibits expression of the mast cell growth factor SCF. The reduced number and activation of mast cells observed in the bronchi of asthmatic patients treated by glucocorticoids may be due in part to this effect.

British Journal of Pharmacology (2002) 135, 1634–1640; doi:10.1038/sj.bjp.0704617
Ascorbate blocks endothelium‐derived hyperpolarizing factor (EDHF)‐mediated vasodilatation in the bovine ciliary vascular bed and rat mesentery
Authors: McNeish Alister J , Wilson William S , Martin William
Published: January 2005

The effects of ascorbate were assessed on vasodilatation mediated by endothelium‐derived hyperpolarizing factor (EDHF) in the ciliary vascular bed of the bovine isolated perfused eye and in the rat isolated perfused mesenteric arterial bed.

In the bovine eye, EDHF‐mediated vasodilator responses induced by acetylcholine or bradykinin were powerfully blocked when ascorbate (50 μM) was included in the perfusion medium for at least 120 min; with acetylcholine a normally‐masked muscarinic vasoconstrictor response was also uncovered.

The blockade of EDHF‐mediated vasodilatation by ascorbate was time‐dependent (maximum blockade at 120 min) and concentration‐dependent (10 – 150 μM).

Ascorbate (50 μM) also blocked acetylcholine‐induced, EDHF‐mediated vasodilator responses in the rat mesenteric arterial bed in a time‐dependent manner (maximum blockade at 180 min).

The ability of ascorbate to block EDHF‐mediated vasodilatation is likely to result from its reducing properties, since this action was mimicked in the bovine eye by two other reducing agents, namely, N‐acetyl‐L‐cysteine (1 mM) and dithiothreitol (100 μM), but not by the redox‐inactive analogue, dehydroascorbate (50 μM).

In conclusion, concentrations of ascorbate present in normal plasma block EDHF‐mediated vasodilator responses in the bovine eye and rat mesentery. The mechanism and physiological consequences of this blockade remain to be determined.

British Journal of Pharmacology (2002) 135, 1801–1809; doi:10.1038/sj.bjp.0704623
Gastric antisecretory role and immunohistochemical localization of cannabinoid receptors in the rat stomach
Authors: Adami Maristella , Frati Paolo , Bertini Simone , Kulkarni‐Narla Anjali , Brown David R , Caro Giuseppe de , Coruzzi Gabriella , Soldani Giulio
Published: January 2005

The role of cannabinoid (CB) receptors in the regulation of gastric acid secretion was investigated in the rat by means of functional experiments and by immunohistochemistry.

In anaesthetized rats with lumen‐perfused stomach, the non selective CB‐receptor agonist WIN 55,212‐2 (0.30 – 4.00 μmol kg−1, i.v.) and the selective CB1‐receptor agonist HU‐210 (0.03 – 1.50 μmol kg−1, i.v.), dose‐dependently decreased the acid secretion induced by both pentagastrin (30 nmol kg−1 h−1) and 2‐deoxy‐D‐glucose (1.25 mmol kg−1, i.v.). By contrast, neither WIN 55,212‐2 (1 – 4 μmol kg−1, i.v.) nor HU‐210 (0.03 – 1.50 μmol kg−1, i.v.) did modify histamine‐induced acid secretion (20 μmol kg−1 h−1). The selective CB2‐receptor agonist JWH‐015 (3 – 10 μmol kg−1, i.v.) was ineffective.

The gastric antisecretory effects of WIN 55,212‐2 and HU‐210 on pentagastrin‐induced acid secretion were prevented by the selective CB1‐receptor antagonist SR141716A (0.65 μmol kg−1, i.v.) and unaffected by the selective CB2‐receptor antagonist SR144528 (0.65 – 2 μmol kg−1, i.v.).

Bilateral cervical vagotomy and ganglionic blockade with hexamethonium (10 mg kg−1, i.v., followed by continuous infusion of 10 mg kg−1 h−1) significantly reduced, but not abolished, the maximal inhibitory effect of HU‐210 (0.3 μmol kg−1, i.v.) on pentagastrin‐induced acid secretion; by contrast, pretreatment with atropine (1 mg kg−1, i.v.) did not modify the antisecretory effect of HU‐210.

Immunoreactivity to the CB1 receptor was co‐localized with that of the cholinergic marker choline acetyltransferase in neural elements innervating smooth muscle, mucosa and submucosal blood vessels of rat stomach fundus, corpus and antrum. In contrast, CB2 receptor‐like immunoreactivity was not observed.

These results indicate that gastric antisecretory effects of cannabinoids in the rat are mediated by suppression of vagal drive to the stomach through activation of CB1 receptors, located on pre‐ and postganglionic cholinergic pathways. However, the ineffectiveness of atropine in reducing the effect of HU‐210 suggests that the release of non cholinergic excitatory neurotransmitters may be regulated by CB1 receptors.

British Journal of Pharmacology (2002) 135, 1598–1606; doi:10.1038/sj.bjp.0704625
Endothelial dysfunction in aging animals: the role of poly(ADP‐ribose) polymerase activation
Authors: Pacher Pál , Mabley Jon G , Soriano Francisco G , Liaudet Lucas , Komjáti Katalin , Szabó Csaba
Published: January 2005

Recent work has demonstrated the production of reactive oxygen and nitrogen species in the vasculature of aging animals. Oxidant induced cell injury triggers the activation of nuclear enzyme poly(ADP ribose) polymerase (PARP) leading to endothelial dysfunction in various pathophysiological conditions (reperfusion, shock, diabetes). Here we studied whether the loss of endothelial function in aging rats is dependent upon the PARP pathway within the vasculature. Young (3 months‐old) and aging (22 months‐old) Wistar rats were treated for 2 months with vehicle or the PARP inhibitor PJ34. In the vehicle‐treated aging animals there was a significant loss of endothelial function, as measured by the relaxant responsiveness of vascular rings to acetylcholine. Treatment with PJ34, a potent PARP inhibitor, restored normal endothelial function. There was no impairment of the contractile function and endothelium‐independent vasodilatation in aging rats. Furthermore, we found no deterioration in the myocardial contractile function in aging animals. Thus, intraendothelial PARP activation may contribute to endothelial dysfunction associated with aging.

British Journal of Pharmacology (2002) 135, 1347–1350; doi:10.1038/sj.bjp.0704627
Acute cerebral focal ischaemia alters the adrenergic and NANC responses in the bisected rat vas deferens
Authors: Boselli C , Cozzi A , Rozza A , Favalli L , Govoni F , Govoni S
Published: January 2005

Disturbances of the autonomic nervous system are common in right hemisphere stroke patients, including a marked decline in male sexual functions. There is a lack of information on the influence of stroke on male secondary sex organs such as the vas deferens.

This study investigates the effect of right brain focal ischaemia on the adrenergic and purinergic responses in isolated epididymal and prostatic portions of rat vas deferens.

In both epididymal and prostatic portions the concentration‐response curves to noradrenaline are flattened resulting in a reduction (up to 67 – 76%) of the maximum contractile response in the tissue from ischaemic rats compared to the controls. In the prostatic portion from ischaemic rats the concentration‐response curve to α,β‐methylene ATP was also depressed.

The first purinergic and the second delayed adrenergic phase to single pulse was not modified by brain ischaemia. In contrast both phasic and tonic components of the electrically induced contractions by trains of stimuli at high frequencies (2 – 30 Hz) were significantly depressed in the epididymal and prostatic portions from ischaemic rats.

These results demonstrate an autonomic imbalance at the level of male sexual secondary organs which may contribute to sexual impairment after stroke.

British Journal of Pharmacology (2002) 135, 1723–1732; doi:10.1038/sj.bjp.0704628
Effects of a phosphodiesterase IV inhibitor rolipram on microsphere embolism‐induced defects in memory function and cerebral cyclic AMP signal transduction system in rats
Authors: Nagakura Akira , Niimura Makiko , Takeo Satoshi
Published: January 2005

The effects of treatment with rolipram, a specific phosphodiesterase IV inhibitor, on learning and memory function and on the cyclic AMP/PKA/CREB signal transduction system were examined in rats with microsphere embolism (ME)‐induced cerebral ischaemia.

Sustained cerebral ischaemia was induced by the injection of 900 microspheres (48 μm in diameter) into the right hemisphere of the rat brain. The animals were treated once daily with 3 mg kg−1 rolipram i.p. from 6 h after the onset of the operation for consecutive 10 days.

Microsphere‐embolized rats showed prolongation of the escape latency in the water maze task starting from day 7 after the operation and lasting for 3 consecutive days. Treatment with rolipram reduced the escape latency.

ME decreased the cyclic AMP content, cytosolic PKA Cβ level, and nuclear PKA Cα and Cβ levels, as well as reduced the pCREB level and the DNA‐binding activity of CREB in the cerebral cortex and hippocampus on day 10 after the operation. These alterations were attenuated by treatment with rolipram.

These results suggest that ME‐induced failure in learning and memory function may be mediated by dysfunction of the cyclic AMP/PKA/CREB signal transduction system, that rolipram may ameliorate ME‐induced impairment of learning and memory function, and that the drug effect may be partly attributed to activation of the cyclic AMP/PKA/CREB signal transduction system.

British Journal of Pharmacology (2002) 135, 1783–1793; doi:10.1038/sj.bjp.0704629
Pharmacologic and autoradiographic evidence for an up‐regulation of kinin B2 receptors in the spinal cord of spontaneously hypertensive rats
Authors: Cloutier Frank , Buck Hudson de Sousa , Ongali Brice , Couture Réjean
Published: January 2005

The effects of intrathecally (i.t.) injected kinin B1 and B2 receptor agonists and antagonists were measured on mean arterial pressure (MAP) and heart rate (HR) of conscious unrestrained spontaneously hypertensive rats (SHR of 16 weeks old) and age‐matched normotensive Wistar Kyoto (WKY). Quantitative in vitro autoradiographic studies were also performed on the thoracic spinal cord of both strains with specific radioligands for B2 receptors, [125I]‐HPP‐Hoe 140, and B1 receptors, [125I]‐HPP‐[des‐Arg10]‐Hoe140.

Bradykinin (BK) (0.81 – 810 pmol) increased MAP dose‐dependently with increases or decreases of HR. The pressor response to BK was significantly greater in SHR. The cardiovascular response to 8.1 pmol BK was reversibly blocked by 81 pmol Hoe 140 (B2 antagonist) but not by 81 – 810 pmol [des‐Arg10]‐Hoe 140 (B1 antagonist) in both strains.

The B1 receptor agonist, des‐Arg9‐BK (8100 pmol) produced either no effects or increased MAP with variable effects on HR. These responses were similar in both strains and were reversibly blocked by 81 pmol Hoe 140. Inhibition with 8100 pmol [des‐Arg10]‐Hoe 140 was not specific to B1 agonist‐mediated responses.

[125I]‐HPP‐Hoe 140 specific binding sites were predominantly located to superficial laminae of the dorsal horn and were significantly higher in SHR. Low levels of [125I]‐HPP‐[des‐Arg10]‐HOE 140 specific binding sites were found in all laminae of both strains.

It is concluded that the hypersensitivity of the cardiovascular response to BK is due to an increased number of B2 receptors in the spinal cord of SHR and that B1 receptors are unlikely involved in spinal cardiovascular regulation in SHR.

British Journal of Pharmacology (2002) 135, 1641–1654; doi:10.1038/sj.bjp.0704632
Pharmacological characterization of F‐180: a selective human V1a vasopressin receptor agonist of high affinity
Authors: Andrés Miriam , Trueba Miguel , Guillon Gilles
Published: January 2005

The pharmacological properties of F‐180, a vasopressin (VP) structural analogue, were determined on CHO cells expressing the different human vasopressin and oxytocin (OT) receptor subtypes. Binding experiments revealed that F‐180 exhibited a high affinity for the human V1a receptor subtype (Ki=11 nM) and was selective for this receptor subtype.

Functional studies performed on CHO cells expressing human V1a receptors indicate that similarly to AVP, F‐180 can stimulate the accumulation of inositol phosphate. The activation constant (Kact) for both F‐180 and AVP was 1.7 nM. F‐180 was also an agonist for the human V2 and V1b receptor subtypes and an antagonist for the human OT receptor.

Since marked species pharmacological differences for vasopressin receptors have been described, we studied the properties of F‐180 on various mammalian species. F‐180 showed high affinity and good selectivity for human and bovine V1a receptors, but weak affinity and non selective properties for rat V1a receptors.

To assess the functional properties of F‐180 on a native biological model, we performed studies on primary cultures of cells from bovine zona fasciculata (ZF). As AVP, F‐180 stimulated inositol phosphate accumulation and cortisol secretion with similar efficiency.

In conclusion, we demonstrate that F‐180 is the first selective V1a agonist described for human and bovine vasopressin receptors. Therefore F‐180 can be used as a powerful pharmacological tool to characterize the actions of vasopressin that are mediated by V1a receptor subtypes.

British Journal of Pharmacology (2002) 135, 1828–1836; doi:10.1038/sj.bjp.0704634
Kainate receptor‐mediated apoptosis in primary cultures of cerebellar granule cells is attenuated by mitogen‐activated protein and cyclin‐dependent kinase inhibitors
Authors: Giardina Sarah F , Beart Philip M
Published: January 2005

Previous studies have suggested that neuronal apoptosis is the result of an abortive attempt to re‐enter the cell cycle, and more recently the cyclin‐dependent (CDKs) and the mitogen‐activated protein (MAP) kinases, two superfamilies of kinases that influence and control cell cycle progression, have been implicated in neuronal apoptosis.

Here, to examine whether CDK/MAPK related pathways are involved in excitotoxicity, we studied the actions of various kinase inhibitors on apoptosis induced by the ionotropic glutamate (Glu) receptor agonist, kainate (KA), in primary cultures of murine cerebellar granule cells (CGCs).

KA‐mediated neurotoxicity was concentration‐dependent, as determined by a cell viability assay monitoring the reduction of 3‐(4,5‐dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), and largely apoptotic in nature, as shown by morphological examination and labelling of DNA fragmentation in situ using terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP digoxigenin nick‐end labelling (TUNEL).

KA‐mediated neurotoxicity and apoptosis was completely attenuated by the mixed CDK and MAP kinase inhibitor, olomoucine, in a concentration‐dependent manner (50 – 600 μM), and partially by roscovitine (1 – 100 μM), a more selective CDK inihibitor.

The p38 MAP kinase inhibitor, SB203580 (1 – 100 μM), partially attenuated KA receptor‐mediated apoptosis, as did the MAP kinase kinase inhibitors PD98509 (1 – 100 μM) and U0126 (1 – 100 μM).

These findings provide new evidence for a complex network of interacting pathways involving CDK/MAPK that control apoptosis downstream of KA receptor activation in excitotoxic neuronal cell death.

British Journal of Pharmacology (2002) 135, 1733–1742; doi:10.1038/sj.bjp.0704636
17‐β‐oestradiol‐induced vasorelaxation in vitro is mediated by eNOS through hsp90 and akt/pkb dependent mechanism
Authors: Bucci Mariarosaria , Roviezzo Fiorentina , Cicala Carla , Pinto Aldo , Cirino Giuseppe
Published: January 2005

The L‐arginine‐NO pathway has been implicated in the vasorelaxant effect of 17‐β‐oestradiol. Here we have addressed the involvement of two distinct activation steps of endothelial nitric oxide synthase (eNOS) in the 17‐β‐oestradiol‐induced vasorelaxant effect on rat aortic rings.

Rat aortic rings contracted with phenylephrine (PE) 1 μM relaxed in a concentration related fashion to 17‐β‐oestradiol water soluble cyclodextrin‐encapsulated (E2) only when endothelium was present. The pure anti‐oestrogen of E2 receptor ICI 182,780 (20 μM) significantly inhibited E2‐induced vasorelaxation.

Geldanamycin (10 μM), a specific inhibitor of heat shock protein 90 (hsp90) and Nω‐nitro‐L‐arginine‐methyl ester (L‐NAME, 100 μM), a nitric oxide synthase inhibitor, significantly inhibited E2‐induced vasorelaxation.

Incubation of rat aortic rings up to 6 h with LY 294002 (25 μM), a specific inhibitor of PI(3)K akt/pkb pathway reduced E2‐induced vasorelaxation.

Incubation of rat isolated aorta with E2, induced prostacyclin (PGI2) release. PGI2 levels, measured as 6‐keto PGF1α, were abolished by ibuprofen (10 μM), both L‐NAME and GA did not influence basal or E2‐stimulated PGI2 confirming the specificity of these two compounds on eNOS pathway.

In conclusion, we demonstrate that E2 interaction with its receptor is followed by a vasorelaxant effect in rat aortic rings mediated by eNOS activation through both hsp90 and akt/pkb dependent mechanisms.

British Journal of Pharmacology (2002) 135, 1695–1700; doi:10.1038/sj.bjp.0704641
Muscarinic agonist potencies at three different effector systems linked to the M2 or M3 receptor in longitudinal smooth muscle of guinea‐pig small intestine
Authors: Okamoto H , Prestwich S A , Asai S , Unno T , Bolton T B , Komori S
Published: January 2005

The abilities of muscarinic agonists (arecoline, bethanechol, carbachol, McN‐A343, methacholine, pilocarpine) to inhibit isoprenaline‐induced cyclic AMP production in chopped fragments (via M2 receptors), and to evoke cationic current (Icat) (via M2 receptors) or calcium store release (via M3 receptors) in enzyme‐dispersed, single voltage‐clamped cells from longitudinal smooth muscle of the guinea‐pig small intestine were examined.

All muscarinic agonists (1 – 300 μM) examined inhibited isoprenaline (1 μM)‐induced accumulation of cyclic AMP, the IC50 varying from 52 to 248 μM. However, their relative potencies to evoke this M2 effect were not significantly correlated with their ability to evoke Icat, also a M2 effect, whether or not calcium stores were depleted; pilocarpine and McN‐A343 inhibited the Icat response to carbachol.

Muscarinic agonists (concentration 300 or 1000 μM), except pilocarpine and McN‐A343 which were ineffective, evoked Ca2+‐activated K+ current (IK‐Ca) resulting from Ca2+ store release (M3 effect). Their effectiveness was tested by estimating residual stored calcium by subsequent application of caffeine (10 mM). The relative potencies to evoke Ca2+ store release (M3) and for Icat activation (M2) were closely correlated (P<0.001).

These data might be explained if M2‐mediated adenylyl cyclase inhibition and Icat activation involve different G proteins, or involve different populations of M2 receptors. The observed correlation of agonist potency between Icat activation and Ca2+ store release supports the proposal (Zholos & Bolton, 1997) that M3 activation can potentiate M2‐cationic channel coupling through Ca2+‐independent mechanisms.

British Journal of Pharmacology (2002) 135, 1765–1775; doi:10.1038/sj.bjp.0704642
Neuropeptide Y increases 4‐aminopyridine‐sensitive transient outward potassium current in rat ventricular myocytes
Authors: Heredia M P , Fernández‐Velasco M , Benito G , Delgado C
Published: January 2005

The modulation of 4‐aminopyridine sensitive transient outward potassium current (4‐AP Ito) by neuropeptide Y (NPY) (100 nM) in rat ventricular myocytes was examined using the whole cell voltage‐clamp technique.

Continuous exposure to NPY (100 nM) for 3 – 6 h significantly increased 4‐AP Ito density. The stimulation of 4‐AP Ito density by NPY was concentration‐dependent (EC50=10 nM).

In the presence of BIBP 3226, an NPY receptor antagonist that binds selectively to NPY Y1‐receptors, the effect of NPY on 4‐AP Ito density was maintained. However, in the presence of BIIE 0246, a highly selective non‐peptide NPY Y2‐receptor antagonist, NPY was unable to increase 4‐AP Ito density.

The effect of NPY on 4‐AP Ito density was prevented by pretreatment with 500 ng ml−1 pertussis toxin (PTX) and by the specific protein kinase C (PKC) inhibitor, calphostin C (100 nM).

Thus, short term exposure to NPY induces an increase of 4‐AP Ito density in rat ventricular myocytes mediated by Y2‐receptors and involving the action of PKC via a PTX‐sensitive signalling cascade.

British Journal of Pharmacology (2002) 135, 1701–1706; doi:10.1038/sj.bjp.0704643
Aponecrotic, antiangiogenic and antiproliferative effects of a novel dextran derivative on breast cancer growth in vitro and in vivo
Authors: Benedetto Mélanie Di , Starzec Anna , Colombo Bruno M , Briane Dominique , Perret Gérard Y , Kraemer Michel , Crépin Michel
Published: January 2005

1 Since the sodium phenylacetate (NaPa) was reported to enhance the inhibitory effect of carboxymethyl benzylamide dextran (CMDB) on the breast cancer growth, we performed the esterification of CMDB with NaPa to obtain a new drug carrying the characteristics of these two components. A new molecule, phenylacetate carboxymethyl benzylamide dextran, was named NaPaC.

We investigated in vitro and in vivo the effects of NaPaC on MCF‐7ras cell growth as well as its apoptotic and antiangiogenic effects in comparison to NaPa and CMDB. In addition, we assessed in vitro the antiproliferative effects of these drugs on other breast cancer cells, including MDA‐MB‐231, MDA‐MB‐435 and MCF‐7.

In vitro, NaPaC inhibited MCF‐7ras cell proliferation by 40% at concentration lower than that of CMDB and NaPa (12 μM vs 73 μM and 10 mM). IC50s were 6 and 28 μM for NaPaC and CMDB, respectively. The similar results were obtained for three other breast cancer cell lines. NaPaC reduced the DNA replication and induced cell recruitment in G0/G1 phase more efficiently than its components. Moreover, it induced a cell death at concentration 1000‐fold lower than NaPa.

In vivo, CMDB (150 mg kg−1) and NaPa (40 mg kg−1) inhibited the MCF‐7ras tumour growth by 37 and 57%, respectively, whereas NaPaC (15 mg kg−1) decreased tumour growth by 66% without toxicity.

NaPa or CMDB reduced the microvessel number in tumour by 50% after 7 weeks of treatment. NaPaC had the same effect after only 2 weeks. After 7 weeks, it generated a large necrosis area without detectable microvessels. In vitro, NaPaC inhibited human endothelial cell proliferation more efficiently than CMDB or NaPa. NaPaC interacts with vascular endothelial growth factor as observed by affinity electrophoresis.

NaPaC acts like NaPa and CMDB but in more potent manner than components used separately. Its antiproliferative, aponecrotic and anti‐angiogenic actions make it a good candidate for a new anti‐cancer drug.

British Journal of Pharmacology (2002) 135, 1859–1871; doi:10.1038/sj.bjp.0704645
In vitro and in vivo pharmacology of a structurally novel Na+‐H+ exchange inhibitor, T‐162559
Authors: Kusumoto Keiji , Igata Hideki , Abe Akemi , Ikeda Shota , Tsuboi Ayako , Imamiya Eikoh , Fukumoto Shoji , Shiraishi Mitsuru , Watanabe Toshifumi
Published: January 2005

We investigated the inhibitory effects of a non‐acylguanidine Na+‐H+ exchange (NHE) inhibitor, T‐162559 ((5E,7S)‐[7‐(5‐fluoro‐2‐methylphenyl)‐4‐methyl‐7,8‐dihydro‐5(6H)‐quinolinylideneamino] guanidine dimethanesulphonate), on NHE‐1, and its cardioprotective effect against ischaemia and reperfusion injury in rats and rabbits.

T‐162559 inhibited human platelet NHE‐1 in a concentration‐dependent manner, with an IC50 value of 13±3 nmol l−1, making it 16 and three times more potent than cariporide IC50: 209±75 nmol l−1, P<0.01) and eniporide (IC50: 40±11 nmol l−1, P=0.066), respectively. T‐162559 also inhibited rat NHE‐1 with an IC50 value of 14±2 nmol l−1, which was five and three times lower than that of cariporide (IC50: 75±7 nmol l−1, P<0.01) and eniporide (IC50: 44±2 nmol l−1, P<0.01), respectively.

T‐162559 inhibited, in a concentration‐dependent manner, the reduction in cardiac contractility, progression of cardiac contracture, and increase in lactate dehydrogenase release after global ischaemia and reperfusion in perfused rat hearts. The inhibitory effects of T‐162559 were observed at a lower concentration range (10 – 100 nmol l−1) than with cariporide and eniporide. T‐162559 did not alter basal cardiac contractility or coronary flow after reperfusion, suggesting that it exerts direct cardioprotective effects on the heart.

Intravenous administration of T‐162559 (0.03 and 0.1 mg kg−1) significantly inhibited the progression of myocardial infarction induced by left coronary artery occlusion and reperfusion in rabbits; the infarct size normalized by area at risk was 74±6% in the vehicle group, and 47±5% and 51±7% in the T‐162559‐0.03 mg kg−1 and T‐162559‐0.1 mg kg−1 groups (both P<0.05), respectively.

These results indicate that the new structural NHE‐1 inhibitor T‐162559 is more potent than cariporide and eniporide and possesses a cardioprotective effect against ischaemia and reperfusion injury in rat and rabbit models.

British Journal of Pharmacology (2002) 135, 1995–2003; doi:10.1038/sj.bjp.0704647
Complex regional haemodynamic effects of anandamide in conscious rats
Authors: Gardiner S M , March J E , Kemp P A , Bennett T
Published: January 2005

Experiments were carried out in conscious, chronically instrumented, male, Sprague‐Dawley rats to delineate the regional haemodynamic effects of the putative endogenous cannabinoid, anandamide, (0.075 – 3 mg kg−1), and to dissect some of the mechanisms involved.

At all doses of anandamide, there was a significant, short‐lived increase in mean arterial blood pressure associated with vasoconstriction in renal, mesenteric and hindquarters vascular beds.

The higher doses (2.5 and 3 mg kg−1), caused an initial, marked bradycardia accompanied, in some animals, by a fall in arterial blood pressure which preceded the hypertension. In addition, after the higher doses of anandamide, the hindquarters vasoconstriction was followed by vasodilatation.

Although some of the effects described above resembled those of 5‐HT (25 μg kg−1), the bradycardia and hypotensive actions of the latter were abolished by the 5HT3‐receptor antagonist, azasetron, whereas those of anandamide were generally unaffected.

None of the cardiovascular actions of anandamide were influenced by the CB1‐receptor antagonist, AM 251, but its bradycardic effect was sensitive to atropine, and its hindquarters vasodilator action was suppressed by the β2‐adrenoceptor antagonist, ICI 118551.

The results differ, in several aspects, from those previously reported in anaesthetized animals, and underscore the important impact anaesthesia can have on responses to anandamide.

British Journal of Pharmacology (2002) 135, 1889–1896; doi:10.1038/sj.bjp.0704649
Mouse β3a‐ and β3b‐adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signalling pathways
Authors: Hutchinson Dana S , Bengtsson Tore , Evans Bronwyn A , Summers Roger J
Published: January 2005

This study characterizes the mouse β3a‐adrenoceptor (AR) and the splice variant of the β3‐AR (β3b‐AR) expressed in Chinese hamster ovary cells (CHO‐K1).

Stable clones with high (∼1200), medium (∼500) or low receptor expression (∼100 fmol mg protein−1) were determined by saturation binding with [125I]‐(−)‐cyanopindolol. Competition binding studies showed no significant differences in affinity of β‐AR ligands for either receptor.

Several functional responses of each receptor were measured, namely extracellular acidification rate (EAR; cytosensor microphysiometer), cyclic AMP accumulation, and Erk1/2 phosphorylation. The β3‐AR agonists BRL37344, CL316243, GR265162X, L755507, SB251023, the non‐conventional partial β‐AR agonist CGP12177 and the β‐AR agonist (−)‐isoprenaline caused concentration‐dependent increases in EAR in cells expressing either splice variant. CL316243 caused concentration‐dependent increases in cyclic AMP accumulation and Erk1/2 phosphorylation in cells expressing either receptor.

PTX treatment increased maximum EAR and cyclic AMP responses to CL316243 in cells expressing the β3b‐AR but not in cells expressing the β3a‐AR at all levels of receptor expression.

CL316243 increased Erk1/2 phosphorylation with pEC50 values and maximum responses that were not significantly different in cells expressing either splice variant. Erk1/2 phosphorylation was insensitive to PTX or H89 (PKA inhibitor) but was inhibited by LY294002 (PI3Kγ inhibitor), PP2 (c‐Src inhibitor), genistein (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor).

The adenylate cyclase activators forskolin or cholera toxin failed to increase Erk1/2 levels although both treatments markedly increased cyclic AMP accumulation in both β3a‐ or β3b‐AR transfected cells.

These results suggest that in CHO‐K1 cells, the β3b‐AR, can couple to both Gs and Gi to stimulate and inhibit cyclic AMP production respectively, while the β3a‐AR, couples solely to Gs to increase cyclic AMP levels. However, the increase in Erk1/2 phosphorylation following receptor activation is not dependent upon coupling of the receptors to Gi or the generation of cyclic AMP.

British Journal of Pharmacology (2002) 135, 1903–1914; doi:10.1038/sj.bjp.0704654
Regulation of IL‐13 synthesis in human lymphocytes: implications for asthma therapy
Authors: Pahl Andreas , Zhang Meixia , Kuss Hildegard , Szelenyi Istvan , Brune Kay
Published: January 2005

IL‐13 is an important mediator in inflammatory diseases such as asthma. IL‐13 is mainly produced by T cells. However, signalling pathways leading to induction of this cytokine are not well‐characterized. We analysed the regulation of IL‐13 in human peripheral blood mononuclear cells and CD4+ T cells.

Cyclosporine (CsA) and FK‐506 inhibited IL‐13 synthesis, when cells were stimulated by TPA/ionomycin. However, stimulation by α‐CD3/α‐CD28 led to an enhanced IL‐13 synthesis.

NF‐κB inhibitor N‐tosyl‐L‐lysine chloromethylketone (TLCK) inhibited IL‐13 synthesis more effectively after TPA/ionomycin stimulation. After α‐CD3/α‐CD28 stimulation, only 300 μM TLCK inhibited IL‐13 synthesis. Dexamethasone inhibited IL‐13 equally effective after α‐CD3/α‐CD28 and TPA/ionomycin stimulation.

p38 MAPK inhibitor SB203580 inhibited IL‐13 synthesis only partially. MEK inhibitor U0126 inhibited TPA/ionomycin induced IL‐13 synthesis very effectively, whereas α‐CD3/α‐CD28 stimulated IL‐13 induction was resistant to this drug.

These results were confirmed in purified CD4+ T cells. In difference to PBMCs α‐CD3/α‐CD28 stimulated IL‐13 synthesis was effectively inhibited by CsA, FK‐506 and U0126.

Therefore U0126 was tested in an animal model of allergic asthma. We could demonstrate for the first time that inhibition of the MEK – ERK cascade is a therapeutic option for asthma. Intraperitoneal administration of 10 mg kg−1 U0126 reduced lung eosinophilia in ovalbumin‐challenged Brown Norway rats by 44%.

These results demonstrate that different signalling pathways are involved in regulating IL‐13 synthesis in primary human T cells. Characterizing highly potent inhibitors of IL‐13 synthesis can be exploited to identify new drugs to treat immunological diseases such as asthma.

British Journal of Pharmacology (2002) 135, 1915–1926; doi:10.1038/sj.bjp.0704656
Direct visualization of ligand‐protein interactions using atomic force microscopy
Authors: Neish Calum S , Martin Ian L , Henderson Robert M , Edwardson J Michael
Published: January 2005

Streptavidin is a 60‐kDa tetramer which binds four molecules of biotin with extremely high affinity (KA∼1014 M−1). We have used atomic force microscopy (AFM) to visualize this ligand‐protein interaction directly.

Biotin was tagged with a short (152‐basepair; 50‐nm) DNA rod and incubated with streptavidin. The resulting complexes were then imaged by AFM. The molecular volume of streptavidin calculated from the dimensions of the protein particles (105±3 nm3) was in close agreement with the value calculated from its molecular mass (114 nm3). Biotinylation increased the apparent size of streptavidin (to 133±2 nm3), concomitant with an increase in the thermal stability of the tetramer.

Images of streptavidin with one to four molecules of DNA‐biotin bound were obtained. When two ligands were bound, the angle between the DNA rods was either acute or obtuse, as expected from the relative orientations of the biotin binding sites. The ratio of acute : obtuse angles (1 : 3) was lower than the expected value (1 : 2), indicating a degree of steric hindrance in the binding of the DNA‐biotin. The slight under‐representation of higher occupancy states supported this idea.

Streptavidin with a single molecule of DNA‐biotin bound was used to tag biotinylated β‐galactosidase, a model multimeric enzyme.

The ability to image directly the binding of a ligand to its protein target by AFM provides useful information about the nature of the interaction, and about the effect of complex formation on the structure of the protein. Furthermore, the use of DNA‐biotin/streptavidin tags could potentially shed light on the architecture of multi‐subunit proteins.

British Journal of Pharmacology (2002) 135, 1943–1950; doi:10.1038/sj.bjp.0704660
Activity profile of calpains I and II in chronically infarcted rat myocardium – influence of the calpain inhibitor CAL 9961
Authors: Sandmann Steffen , Prenzel Freerk , Shaw Lee , Schauer Roland , Unger Thomas
Published: January 2005

The calpains have been proposed to be activated following cardiac ischaemia and to contribute to myocyte damage after myocardial infarction (MI). In this study, the activity of calpains I and II in the infarcted and non‐infarcted rat myocardium and the action of the selective calpain inhibitor, CAL 9961, has been investigated.

MI was induced by permanent ligation of the left coronary artery. One, 3, 7 and 14 days post MI, the enzymes calpain I and II were separated from homogenates of the interventricular septum (IS) and left ventricular free wall (LVFW) by chromatography on DEAE‐Sepharose. The activity of the calpains was measured in sham‐operated and MI animals chronically treated with placebo or CAL 9961 (15 mg kg−1 d−1 s.c.) in a synthetic substrate assay. Treatment was started 3 days before MI induction.

Calpain I activity reached highest values in IS 14 days post MI, whereas maximum activity of calpain II was measured in LVFW 3 days post MI. In experiments in vitro, CAL 9961 completely inhibited both calpains. In vivo, chronic treatment of MI animals with CAL 9961 partially prevented the increase in calpain I activity in IS and reduced calpain II activity in LVFW to sham levels.

Our findings demonstrate that calpains I and II are activated after MI, however, both enzymes differ in their regional and temporal activation within the infarcted myocardium. Chronic inhibition of these enzymes with CAL 9961 might limit the calpain‐induced myocardial damage and preserve cardiac structural integrity post MI.

British Journal of Pharmacology (2002) 135, 1951–1958; doi:10.1038/sj.bjp.0704661
Xestospongin C, a novel blocker of IP3 receptor, attenuates the increase in cytosolic calcium level and degranulation that is induced by antigen in RBL‐2H3 mast cells
Authors: Oka Tatsuya , Sato Koichi , Hori Masatoshi , Ozaki Hiroshi , Karaki Hideaki
Published: January 2005

We evaluated the role of the cross‐linking of FcεRI‐mediated inositol 1,4,5‐triphosphate (IP3) in the increase in cytosolic Ca2+ level ([Ca2+]i) using xestospongin C, a selective membrane permeable blocker of IP3 receptor, in RBL‐2H3 mast cells.

In the cells sensitized with anti‐dinitrophenol (DNP) IgE, DNP‐human serum albumin (DNP‐HSA) and thapsigargin induced degranulation of β‐hexosaminidase and a sustained increase in [Ca2+]i. Xestospongin C (3 – 10 μM) inhibited both of these changes that were induced by DNP‐HSA without changing those induced by thapsigargin.

In the absence of external Ca2+, DNP‐HSA induced a transient increase in [Ca2+]i. Xestospongin C (3 – 10 μM) inhibited this increase in [Ca2+]i.

In the cells permeabilized with β‐escin, the application of IP3 decreased Ca2+ in the endoplasmic reticulum (ER) as evaluated by mag‐fura‐2. Xestospongin C (3 – 10 μM) inhibited the effect of IP3.

After the depletion of Ca2+ stores due to stimulation with DNP‐HSA or thapsigargin, the addition of Ca2+ induced capacitative calcium entry (CCE). Xestospongin C (3 – 10 μM) inhibited the DNP‐HSA‐induced CCE, whereas it did not affect the thapsigargin‐induced CCE.

These results suggest that FcεRI‐mediated generation of IP3 contributes to Ca2+ release not only in the initial phase but also in the sustained phase of the increase in [Ca2+]i, resulting in prolonged Ca2+ depletion in the ER. The ER Ca2+ depletion may subsequently activate CCE to achieve a continuous [Ca2+]i increase, which is necessary for degranulation in the RBL‐2H3 mast cells. Xestospongin C may inhibit Ca2+ release and consequently may attenuate degranulation.

British Journal of Pharmacology (2002) 135, 1959–1966; doi:10.1038/sj.bjp.0704662
The involvement of smooth muscle P2X receptors in the prolonged vasorelaxation response to purine nucleotides in the rat mesenteric arterial bed
Authors: Ralevic V
Published: January 2005

ATP and adenine dinucleotides can elicit three different types of vasomotor response in the rat mesenteric arterial bed; vasocontraction, rapid relaxation (which may be masked by contraction) and slow and prolonged vasorelaxation. Contraction is mediated by smooth muscle P2X receptors and rapid relaxation by endothelial P2Y receptors. The mechanism of prolonged relaxation is, however, controversial.

In the present study, bolus injection of doses of α,β‐methylene ATP (α,β‐meATP; 5 pmol – 0.5 μmol; P2X receptor agonist) in methoxamine‐preconstricted rat isolated mesenteric arterial beds, mimicked the action of ATP, causing contraction (Rmax 76±9 mmHg) followed by prolonged relaxation (78±11%; t1/2 14.6±1.5 min). KCl also elicited a biphasic response (Rmax contraction 73±8 mmHg; Rmax prolonged relaxation 70±6%; t1/2 7.7±1.9 min).

P2X receptor desensitization caused by perfusion with α,β‐meATP (10 μM) abolished contraction and prolonged relaxation to doses of α,β‐meATP (50 nmol). Rapid relaxation (32±7%; t1/2 32±2 s) was revealed, which was abolished by removal of the endothelium using distilled water.

Sodium deoxycholate treatment blocked contractile and prolonged relaxation responses to α,β‐meATP, ATP and KCl, whilst distilled water treatment had no significant effect on either phase of the biphasic responses.

These data indicate that smooth muscle P2X receptors are involved in both phases of the biphasic response (contraction followed by prolonged relaxation) to purine nucleotides in the rat isolated mesenteric arterial bed. Caution should be applied when using sodium deoxycholate to remove the endothelium because of possible damage caused by the detergent to receptors and/or the vascular smooth muscle.

British Journal of Pharmacology (2002) 135, 1988–1994; doi:10.1038/sj.bjp.0704663