bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of cyclosporin A and its vehicle on cardiac and skeletal muscle mitochondria: relationship to efficacy of the respiratory chain
Authors: Sanchez H , Zoll J , Bigard X , Veksler V , Mettauer B , Lampert E , Lonsdorfer J , Ventura‐Clapier R
Published: January 2005

Although cyclosporin (CsA) is considered to be the best immunosuppressive molecule in transplantation, it has been suspected to alter mitochondrial respiration of various tissues.

We evaluated the acute effect of CsA and its vehicle on maximal oxidative capacity (Vmax) of cardiac, soleus and gastrocnemius muscles of rats by an oxygraphic method in saponin skinned muscle fibres. The effects of Sandimmun (a formulation of CsA), vehicle of Sandimmun (cremophor and ethanol (EtOH)), CsA in EtOH and EtOH alone were tested. Increasing concentrations (5 – 20 – 50 – 100 μM) of CsA (or vehicles) were used.

Sandimmun profoundly altered the Vmax of all muscles. For example, at 20 μM, inhibition reached 18±3, 23±5, 45±5%, for heart, soleus and gastrocnemius respectively. There were only minor effects of CsA diluted in EtOH and EtOH alone on Vmax of cardiac muscle. Because the effects of vehicle on Vmax were similar or higher than those of Sandimmun, the inhibition of oxidative capacity could be entirely attributed to the vehicle for all muscles.

Next, we investigated the potential sites of action of the vehicle on the different complexes of the mitochondrial respiratory chain by using specific substrates and inhibitors. The vehicle affected mitochondrial respiration mainly at the level of complex I (∼−85% in skeletal muscles, and −32% in heart), but also at complex IV (∼−26% for all muscles).

The mechanism of action of the vehicle on the mitochondrial membrane and the implications for the clinical use of immunosuppressive drugs are discussed.

British Journal of Pharmacology (2001) 133, 781–788; doi:10.1038/sj.bjp.0704129
Reversal of cardiac and renal fibrosis by pirfenidone and spironolactone in streptozotocin‐diabetic rats
Authors: Miric Goran , Dallemagne Catherine , Endre Zoltan , Margolin Solomon , Taylor Stephen M , Brown Lindsay
Published: January 2005

Fibrosis leads to chronic impairment of cardiac and renal function and thus reversal of existing fibrosis may improve function and survival. This project has determined whether pirfenidone, a new antifibrotic compound, and spironolactone, an aldosterone antagonist, reverse both deposition of the major extracellular matrix proteins, collagen and fibronectin, and functional changes in the streptozotocin(STZ)‐diabetic rat.

Streptozotocin (65 mg kg−1 i.v.)‐treated rats given pirfenidone (5‐methyl‐1‐phenyl‐2‐[1H]‐pyridone; approximately 200 mg kg−1 day−1 as 0.2–2g 1−1 drinking water) or spironolactone (50 mg kg−1 day−1 s.c.) for 4 weeks starting 4 weeks after STZ showed no attenuation of the increased blood glucose concentrations and increased food and water intakes which characterize diabetes in this model.

STZ‐treatment increased perivascular and interstitial collagen deposition in the left ventricle and kidney, and surrounding the aorta. Cardiac, renal and plasma fibronectin concentrations increased in STZ‐diabetic rats. Passive diastolic stiffness increased in isolated hearts from STZ‐diabetic rats. Both pirfenidone and spironolactone treatment attenuated these increases without normalizing the decreased +dP/dtmax of STZ‐diabetic hearts.

Left ventricular papillary muscles from STZ‐treated rats showed decreased maximal positive inotropic responses to noradrenaline, EMD 57033 (calcium sensitizer) and calcium chloride; this was not reversed by pirfenidone or spironolactone treatment. STZ‐treatment transiently decreased GFR and urine flow rates in isolated perfused kidneys; pirfenidone but not spironolactone prevented the return to control values.

Thus, short‐term pirfenidone and spironolactone treatment reversed cardiac and renal fibrosis and attenuated the increased diastolic stiffness without normalizing cardiac contractility or renal function in STZ‐diabetic rats.

British Journal of Pharmacology (2001) 133, 687–694; doi:10.1038/sj.bjp.0704131
Characterization of ionotropic glutamate receptors in human lymphocytes
Authors: Lombardi Grazia , Dianzani Chiara , Miglio Gianluca , Canonico Pier Luigi , Fantozzi Roberto
Published: January 2005

The effect of L‐glutamate (Glu) on human lymphocyte function was studied by measuring anti‐CD3 monoclonal antibody (mAb) or phytohaemagglutinin (PHA)‐induced intracellular Ca2+ ([Ca2+]i) rise (Fura‐2 method), and cell proliferation (MTT assay).

Glu (0.001 – 100 μM) did not modify basal lymphocyte [Ca2+]i, but significantly potentiated the effects of anti‐CD3 mAb or PHA. Maximal [Ca2+]i rises over resting cells were: 165±8 and 247±10 nM at 3.0×10−2 mg ml−1 anti‐CD3 mAb; 201±4 and 266±9 nM at 5.0×10−2 mg ml−1 PHA, in the absence or presence of 1 μM Glu, respectively.

The Glu effect showed a bell‐shape concentration‐dependent relationship, with a maximum (+90±3% for anti‐CD3 mAb and +57±2% for PHA over Glu‐untreated cells) at 1 μM.

Non‐NMDA receptor agonists (1 μM) showed a greater efficacy (+76±2% for (S)‐AMPA; +78±4% for KA), if compared to NMDA (+46±2%), or Glu itself.

Ionotropic Glu receptor antagonists completely inhibited the effects of the corresponding specific receptor agonists (1 μM). The IC50 values calculated were: 0.9 μM for D‐AP5; 0.6 μM for (+)‐MK801; 0.3 μM for NBQX. Both NBQX and KYNA were able to abolish Glu effect. The IC50s calculated were: 3.4 μM for NBQX; 0.4 μM for KYNA.

Glu (0.1 – 1 mM) did not change the resting cell proliferation, whereas Glu (1 mM) significant inhibited (−27±4%) PHA (1.0×10−2 mg ml−1)‐induced lymphocyte proliferation at 72 h.

In conclusion, human lymphocytes express ionotropic Glu receptors functionally operating as modulators of cell activation.

British Journal of Pharmacology (2001) 133, 936–944; doi:10.1038/sj.bjp.0704134
Evidence that ET‐1, but not ET‐3 and S6b, ETA‐receptor mediated contractions in isolated rat mesenteric arteries are modulated by co‐activation of ETB receptors
Authors: Adner Mikael , Shankley Nigel , Edvinsson Lars
Published: January 2005

The effects of agonists with endothelin (ET) ETA‐receptor activity have been analysed in relation to their interaction with ETB receptors in rat mesenteric arteries.

ET‐1, sarafotoxin 6b (S6b) and ET‐3 induced large, slow‐onset and sustained contractions whereas S6c induced weak transient contractions. However, following pre‐contraction with U46619 and subsequent relaxation with forskolin, the effect of S6c was amplified, indicating a potential for powerful ETB‐receptor mediated contraction.

The selective ETA‐receptor antagonist, FR139317, produced parallel rightward shifts of ET‐1, S6b and ET‐3 concentration‐effect curves indicating that the contractions were mediated by ETA receptors. However, the corresponding FR139317 pKB values were significantly different between the agonists. As expected FR139317 had no effect on S6c responses.

Pre‐treatment with S6c to desensitize ETB receptors, increased ET‐1 potency and the pKB value for FR139317. In contrast, neither the potency of S6b and ET‐3 nor the pKB values for FR139317 estimated using these agonists were affected by ETB‐receptor desensitization.

Segments pre‐contracted with submaximal concentrations of S6b and ET‐3, but not ET‐1, rapidly relaxed following wash‐out or FR139317 administration.

The results indicate that the small contractile response to selective ETB receptor activation, barely detectable under standard bioassay conditions, is greatly amplified when adenylate cyclase activity is elevated. Moreover, the response to ETA receptor activation by ET‐1, but not ET‐3 and S6b, is significantly modified by co‐activation of ETB receptors. This interaction has a significant effect on the apparent affinity of ETA‐receptor selective antagonists when ET‐1 is used as agonist and decreases the potency of ET‐1.

British Journal of Pharmacology (2001) 133, 927–935; doi:10.1038/sj.bjp.0704135
Role of opioid receptors in neurogenic dural vasodilation and sensitization of trigeminal neurones in anaesthetized rats
Authors: Williamson D J , Shepheard S L , Cook D A , Hargreaves R J , Hill R G , Cumberbatch M J
Published: January 2005

Migraine headache is thought to be caused by a distension of meningeal blood vessels, the activation of trigeminal sensory neurones and the the development of a central sensitization within the trigeminal nucleus caudalis (TNC). It has been proposed that clinically effective 5‐HT1B/1D agonists act peripherally to inhibit the release of calcitonin gene‐related peptide (CGRP) and neurogenic dural vasodilation, and to attenuate nociceptive neurotransmission within the TNC. Since opioids are also effective anti‐migraine agents the present studies investigated the role of opioids within the trigemino‐vascular system in anaesthetised rats.

Electrical stimulation of the dura mater evoked neurogenic dural vasodilation which was significantly inhibited by morphine (1 mg kg−1) the selective μ‐opioid agonist DAGO (10 μg kg−1) and the mixed agonist/antagonist butorphanol (1 mg kg−1) but not by the κ‐ and δ‐opioid agonists (±) U50488H (100 μg kg−1) and DPDPE (1 mg kg−1). Morphine had no effect on CGRP‐evoked dural vasodilation.

In electrophysiological studies morphine (1 – 10 mg kg−1) significantly attenuated brainstem neuronal activity in response to electrical stimulation of the dura by 65% at 10 mg kg−1. Morphine (3 mg kg−1) also inhibited the TNC neuronal sensitization following CGRP‐evoked dilation.

The present studies have demonstrated that opioids block the nociceptive neurotransmission within the trigeminal nucleus caudalis and in addition inhibit neurogenic dural vasodilation via an action on μ‐opioid receptors located on trigeminal sensory fibres innervating dural blood vessels. These peripheral and central actions are similar to those of the ‘triptan’ 5‐HT1B/1D agonists and could account for the anti‐migraine actions of opioids.

British Journal of Pharmacology (2001) 133, 807–814; doi:10.1038/sj.bjp.0704136
A microdialysis study of the novel antiepileptic drug levetiracetam: extracellular pharmacokinetics and effect on taurine in rat brain
Authors: Tong X , Patsalos P N
Published: January 2005

Using a rat model which allows serial blood sampling and concurrent brain microdialysis sampling, we have investigated the temporal kinetic inter‐relationship of levetiracetam in serum and brain extracellular fluid (frontal cortex and hippocampus) following systemic administration of levetiracetam, a new antiepileptic drug. Concurrent extracellular amino acid concentrations were also determined.

After administration (40 or 80 mg kg−1), levetiracetam rapidly appeared in both serum (Tmax, 0.4 – 0.7 h) and extracellular fluid (Tmax, 2.0 – 2.5 h) and concentrations rose linearly and dose‐dependently, suggesting that transport across the blood‐brain barrier is rapid and not rate‐limiting. The serum free fraction (free/total serum concentration ratio; mean±s.e.mean range 0.93 – 1.05) was independent of concentration and confirms that levetiracetam is not bound to blood proteins.

The kinetic profiles for the hippocampus and frontal cortex were indistinguishable suggesting that levetiracetam distribution in the brain is not brain region specific. However, t1/2 values were significantly larger than those for serum (mean range, 3.0 – 3.3 h vs 2.1 – 2.3 h) and concentrations did not attain equilibrium with respect to serum.

Levetiracetam (80 mg kg−1) was associated with a significant reduction in taurine in the hippocampus and frontal cortex. Other amino acids were unaffected by levetiracetam.

Levetiracetam readily and rapidly enters the brain without regional specificity. Its prolonged efflux from and slow equilibration within the brain may explain, in part, its long duration of action. The concurrent changes in taurine may contribute to its mechanism of action.

British Journal of Pharmacology (2001) 133, 867–874; doi:10.1038/sj.bjp.0704141
Sirolimus, but not the structurally related RAD (everolimus), enhances the negative effects of cyclosporine on mitochondrial metabolism in the rat brain
Authors: Serkova Natalie , Jacobsen Wolfgang , Niemann Claus U , Litt Lawrence , Benet Leslie Z , Leibfritz Dieter , Christians Uwe
Published: January 2005

Clinical studies have shown enhancement of cyclosporine toxicity when co‐administered with the immunosuppressant sirolimus. We evaluated the biochemical mechanisms underlying the sirolimus/cyclosporine interaction on rat brain metabolism using magnetic resonance spectroscopy (MRS) and compared the effects of sirolimus with those of the structurally related RAD.

Two‐week‐old rats (25 g) were allocated to the following treatment groups (all n=6): I. control, II. cyclosporine (10 mg kg−1 d−1), III. sirolimus (3 mg kg−1 d−1), IV. RAD (3 mg kg−1 d−1), V. cyclosporine+sirolimus and VI. cyclosporine+RAD. Drugs were administered by oral gavage for 6 days. Twelve hours after the last dose, metabolic changes were assessed in brain tissue extracts using multinuclear MRS.

Cyclosporine significantly inhibited mitochondrial glucose metabolism (glutamate: 78±6% of control; GABA: 67±12%; NAD+: 76±3%; P<0.05), but increased lactate production. Sirolimus and RAD inhibited cytosolic glucose metabolism via lactate production (sirolimus: 81±3% of control, RAD: 69±2%; P<0.02). Sirolimus enhanced cyclosporine‐induced inhibition of mitochondrial glucose metabolism (glutamate: 60±4%; GABA: 59±8%; NAD+: 45±5%; P<0.02 versus cyclosporine alone). Lactate production was significantly reduced. In contrast, RAD antagonized the effects of cyclosporine (glutamate, GABA, and NAD+, not significantly different from controls).

The results can partially be explained by pharmacokinetic interactions: co‐administration increased the distribution of cyclosporine and sirolimus into brain tissue, while co‐administration with RAD decreased cyclosporine brain tissue concentrations. In addition RAD, but not sirolimus, distributed into brain mitochondria.

The combination of cyclosporine/RAD compares favourably to cyclosporine/sirolimus in regards to their effects on brain high‐energy metabolism and tissue distribution in the rat.

British Journal of Pharmacology (2001) 133, 875–885; doi:10.1038/sj.bjp.0704142
Inhibition of poly (ADP‐ribose) synthetase by gene disruption or inhibition with 5‐iodo‐6‐amino‐1,2‐benzopyrone protects mice from multiple‐low‐dose‐streptozotocin‐induced diabetes
Authors: Mabley Jon G , Suarez‐Pinzon Wilma L , Haskó György , Salzman Andrew L , Rabinovitch Alex , Kun Ernest , SzabÓ Csaba
Published: January 2005

Activation of poly(ADP‐ribose) synthetase (PARS, also termed polyADP‐ribose polymerase or PARP) has been proposed as a major mechanism contributing to β‐cell destruction in type I diabetes. In the present study, we have investigated the role of PARS in mediating the induction of diabetes and β‐cell death in the multiple‐low‐dose‐streptozotocin (MLDS) model of type I diabetes.

Mice genetically deficient in PARS were found to be less sensitive to MLDS than wild type mice, with a lower incidence of diabetes and reduced hyperglycemia.

A potent inhibitor of PARS, 5‐iodo‐6‐amino‐1,2‐benzopyrone (INH2BP), was also found to protect mice from MLDS and prevent β‐cell loss, in a dose‐dependent manner. Paradoxically, in the PARS deficient mice, the compound increased the onset of diabetes.

In vitro the cytokine combination; interleukin‐1β, tumor necrosis factor‐α and interferon‐γ inhibited glucose‐stimulated insulin secretion from isolated rat islets of Langerhans and decreased RIN‐5F cell viability. The PARS inhibitor, INH2BP, protected both the rat islets and the β‐cell line, RIN‐5F, from these cytokine‐mediated effects. These protective effects were not mediated by inhibition of cytokine‐induced nitric oxide formation.

Inhibition of PARS by INH2BP was unable to protect rat islet cells from cytokine‐mediated apoptosis.

Cytokines, peroxynitrite and streptozotocin were all shown to induce PARS activation in RIN‐5F cells, an effect suppressed by INH2BP.

The present study provides evidence for in vivo PARS activation contributing to β‐cell damage and death in the MLDS model of diabetes, and indicates a role for PARS activation in cytokine‐mediated depression of insulin secretion and cell viability in vitro.

British Journal of Pharmacology (2001) 133, 909–919; doi:10.1038/sj.bjp.0704156
Regional localization and developmental profile of acetylcholinesterase‐evoked increases in [3H]‐5‐fluorowillardiine binding to AMPA receptors in rat brain
Authors: Olivera Silvia , Rodriguez‐Ithurralde Daniel , Henley Jeremy M
Published: January 2005

In addition to its role in hydrolyzing the neurotransmitter acetylcholine, the synaptically enriched enzyme acetylcholinesterase (AChE) has been reported to play an important role in the development and remodelling of neural processes and synapses.

We have shown previously that AChE causes an increase in binding of the specific AMPA receptor ligand (S)‐[3H]‐5‐fluorowillardiine ([3H]‐FW) to rat brain membranes.

In this study we have used quantitative autoradiography to investigate the regional distribution and age‐dependence of AChE‐evoked increases in the binding of [3H]‐FW in rat brain.

Pretreatment of rat brain sections with AChE caused a marked enhancement of [3H]‐FW binding to many, but not all, brain areas. The increased [3H]‐FW binding was blocked by the specific AChE inhibitor BW 284c51.

The maximal potentiation of [3H]‐FW binding occurred at different developmental age‐points in different regions with a profile consistent with the peak periods for synaptogenesis in any given region.

In addition to its effects on brain sections, AChE also strongly potentiated [3H]‐FW binding to detergent solubilized AMPA receptors suggesting a direct action on the receptors themselves rather than an indirect effect on the plasma membrane.

These findings suggest that modulation of AMPA receptors could provide one molecular mechanism for the previously reported effects of AChE on synapse formation, synaptic plasticity and neurodegeneration.

British Journal of Pharmacology (2001) 133, 1055–1062; doi:10.1038/sj.bjp.0704167
A comparison of effects measured with isotonic and isometric recording: II. Concentration‐effect curves for physiological antagonists
Authors: Barlow R B , Bond Susan M , Grant Claire , McQueen D S , Yaqoob Zeenat
Published: January 2005

If one drug, B, antagonizes another, A, by producing the opposite physiological effect, the antagonist concentration‐effect curves should be affected by the recording system, which limits the range of agonist responses.

With pieces of isolated guinea‐pig ileum taken from adjacent parts of the same animal, one recorded isotonically, the other isometrically with the same load, the isotonic IC50 values for (−)isoprenaline opposing carbachol or histamine were lower than the isometric values (P<0.01) but there was a significant correlation between them (P<0.01): the isotonic curves were steeper (P<0.01) and there were wider shifts in IC50 before increasing the agonist reduced the maximum relaxation.

In similar experiments with pieces of rat uterus in oestrus from the same animal, the concentration‐effect curves for carbachol opposed by increasing concentrations of (−)isoprenaline or (−)adrenaline had slightly lower EC50 values with isometric recording but there was a significant correlation (P<0.01) with isotonic values. The antagonist effect (ratio of the EC50 relative to that for the control) was higher with isotonic recording (P<0.01 for (−)isoprenaline, P<0.025 for (−)adrenaline) and all (27) curves were steeper than the corresponding isometric curve (P<0.001).

The influence of the method of recording on the results is expected from the narrower operational window and smaller upper limit to relaxation with isotonic recording.

A way of obtaining measurements of IC50 against a standard agonist effect is suggested in an Appendix.

British Journal of Pharmacology (2001) 133, 1087–1095; doi:10.1038/sj.bjp.0704169
An alternative pathway for metabolism of leukotriene D4: effects on contractions to cysteinyl‐leukotrienes in the guinea‐pig trachea
Authors: Bäck Magnus , Kumlin Maria , Cotgreave Ian A , Dahlén Sven‐Erik
Published: January 2005

Contractions of guinea‐pig tracheal preparations to cysteinyl‐leukotrienes (LTC4, LTD4 and LTE4) were characterized in organ baths, and cysteinyl‐leukotriene metabolism was studied using radiolabelled agonists and RP‐HPLC separation.

In the presence of S‐hexyl GSH (100 μM) the metabolism of [3H]‐LTC4 into [3H]‐LTD4 was inhibited and the LTC4‐induced contractions were resistant to CysLT1 receptor antagonism but inhibited by the dual CysLT1/CysLT2 receptor antagonist BAY u9773 (0.3–3 μM) with a pA2‐value of 6.8±0.2.

In the presence of L‐cysteine (5 mM), the metabolism of [3H]‐LTD4 into [3H]‐LTE4 was inhibited and the LTD4‐induced contractions were inhibited by the CysLT1 receptor antagonist ICI 198,615 (1–10 nM) with a pA2‐value of 9.3±0.2. However, at higher concentrations of ICI 198,615 (30–300 nM) a residual contraction to LTD4 was unmasked, and this response was inhibited by BAY u9773 (1–3 μM).

In the presence of the combination of S‐hexyl GSH with L‐cysteine, the LTD4‐induced contractions displayed the characteristics of the LTC4 contractile responses, i.e. resistant to CysLT1 receptor antagonism, increased maximal contractions and slower time‐course. This qualitative change of the LTD4‐induced contraction was also observed in the presence of S‐decyl GSH (100 μM), GSH (10 mM) and GSSG (10 mM).

S‐hexyl GSH, S‐decyl GSH, GSH and GSSG all stimulated a formation of [3H]‐LTC4 from [3H]‐LTD4.

In conclusion, GSH and GSH‐related compounds changed the pharmacology of the LTD4‐induced contractions by stimulating the conversion of LTD4 into LTC4. Moreover, the results indicate that, in addition to the metabolism of LTC4 into LTD4 and LTE4, also the formation of LTC4 from LTD4 may regulate cysteinyl‐leukotriene function.

British Journal of Pharmacology (2001) 133, 1134–1144; doi:10.1038/sj.bjp.0704180
Antithrombotic efficacy of a novel factor Xa inhibitor, FXV673, in a canine model of coronary artery thrombolysis
Authors: Rebello Sam S , Bentley Ross G , Morgan Suzanne R , Kasiewski Charles J , Chu Valeria , Perrone Mark H , Leadley Robert J
Published: January 2005

We compared the antithrombotic efficacy of a potent factor Xa inhibitor, FXV673, to heparin and RPR109891, a GPIIb/IIIa antagonist, when used as adjunctive therapy in a canine model of rt‐PA‐induced coronary thrombolysis.

Thrombus formation was induced by electrolytic injury to stenosed coronary artery. After thrombotic occlusion, a 135 min infusion of saline (n=8), FXV673 (10, 30 or 100 μg kg−1+1, 3, or 10 μg kg−1 min−1, respectively; n=8 per dose), heparin (60 u kg−1+0.7 u kg−1 min−1, n=8), or RPR109891 (30 μg kg−1+0.45 μg kg−1 min−1, n=8), was initiated. Aspirin (5 mg kg−1, i.v.) was administered to all animals. Fifteen minutes after the start of drug infusion, rt‐PA was administered (100 μg kg−1+20 μg kg−1 min−1 for 60 min).

The incidence of reperfusion in the high dose FXV673 (8/8, 100%) was significantly greater than that in the heparin group (4/8, 50%), with a trend to faster reperfusion (23±5 min for FXV673 versus 41±11 min for heparin). Only 2/8 (25%) of the vessels reoccluded in the high dose FXV673 group, compared to 4/4 (100%) and 5/5 (100%) vessels in the heparin and RPR109891 groups, respectively (P<0.05).

Throughout the protocol, blood flow was higher in the FXV673 treated group compared to other groups. FXV673 enhanced vessel patency in a dose‐dependent manner.

Compared to vehicle and heparin groups, the thrombus mass was decreased by 60% in the high dose FXV673. FXV673, heparin and RPR109891 increased the bleeding time by 2.7, 1.7 and 4 fold, and APTT by 2.8, 2.7 and 1.2 fold, respectively.

In conclusion, FXV673 is more effective than heparin and at least as effective as RPR109891 when used as an adjunct during rt‐PA‐induced coronary thrombolysis.

British Journal of Pharmacology (2001) 133, 1190–1198; doi:10.1038/sj.bjp.0704182
High glucose induces cell death of cultured human aortic smooth muscle cells through the formation of hydrogen peroxide
Authors: Peiró Concepción , Lafuente Nuria , Matesanz Nuria , Cercas Elena , Llergo José L , Vallejo Susana , Rodríguez‐Mañas Leocadio , Sánchez‐Ferrer Carlos F
Published: January 2005

Alterations of the vessel structure, which is mainly determined by smooth muscle cells through cell growth and/or cell death mechanisms, are characteristic of diabetes complications. We analysed the influence of high glucose (22 mM) on cultured human aortic smooth muscle cell growth and death, as hyperglycaemia is considered one of the main factors involved in diabetic vasculopathy.

Growth curves were performed over 96 h in medium containing 0.5% foetal calf serum. Cell number increased by 2–4 fold over the culture period in the presence of 5.5 mM (low) glucose, while a 20% reduction in final cell number was observed with high glucose. Under serum‐free conditions, cell number remained constant in low glucose cultures, but a 40% decrease was observed in high glucose cultures, suggesting that high glucose may induce increased cell death rather than reduced proliferation. Reduced final cell number induced by high glucose was also observed after stimulation with 5 or 10% foetal calf serum.

The possible participation of oxidative stress was investigated by co‐incubating high glucose with different reactive oxygen species scavengers. Only catalase reversed the effect of high glucose. Intracellular H2O2 content, visualized with 2′,7′‐dichlorofluorescein and quantified by flow cytometry, was increased after high glucose treatment.

To investigate the cell death mechanism induced by high glucose, apoptosis and necrosis were quantified. No differences were observed regarding the apoptotic index between low and high glucose cultures, but lactate dehydrogenase activity was increased in high glucose cultures.

In conclusion, high glucose promotes necrotic cell death through H2O2 formation, which may participate in the development of diabetic vasculopathy.

British Journal of Pharmacology (2001) 133, 967–974; doi:10.1038/sj.bjp.0704184
Suppression of poly (ADP‐ribose) polymerase activation by 3‐aminobenzamide in a rat model of myocardial infarction: long‐term morphological and functional consequences
Authors: Liaudet Lucas , Szabó Éva , Timashpolsky Leonid , Virág László , Cziráki Attila , Szabó Csaba
Published: January 2005

Recent studies demonstrated that inhibition or genetic inactivation of the enzyme poly (ADP‐ribose) polymerase (PARP) is beneficial in myocardial reperfusion injury. PARP activation in the reperfused myocardium has been assumed, but not directly demonstrated. Furthermore, the issue whether pharmacological PARP inhibition affords long‐term functional benefit in the reperfused myocardium has not been explored. These questions were addressed in the present study.

In a rat model of myocardial ischemia (1 h) and reperfusion (up to 24 h), there was a marked and significant activation of PARP in the ischemic borderzone, as determined by poly(ADP‐ribose) (PAR) immunohistochemistry. PAR localized to the nuclei of myocytes and infiltrating mononuclear cells. In the core of the infarction, necrotic tissues and diffuse PAR staining were observed. PARP activation remained markedly detectable 24 h after reperfusion. The PARP inhibitor 3‐aminobenzamide (20 mg kg−1 intraperitoneally 10 min before reperfusion, and every 2 h thereafter for 6 h) markedly reduced the activation of the enzyme in myocytes.

3‐aminobenzamide significantly protected against myocardial morphological and functional alterations at 24 h post‐reperfusion. Notably, infarct size was reduced, circulating creatine kinase activity was attenuated, and myocardial contractility (dP dt−1) was restored by 3‐aminobenzamide.

Our results demonstrate a significant and prolonged activation of PARP in the reperfused myocardium, localizing to the necrotic area and the ischaemic borderzone. Furthermore, the studies demonstrate that PARP inhibition affords long‐term beneficial morphological and functional effects in the reperfused myocardium. These data strengthen the notion that pharmacological PARP inhibition is a viable novel experimental approach for protection against myocardial reperfusion injury.

British Journal of Pharmacology (2001) 133, 1424–1430; doi:10.1038/sj.bjp.0704185
Production of leukotrienes in a model of focal cerebral ischaemia in the rat
Authors: Ciceri Paola , Rabuffetti Monica , Monopoli Angela , Nicosia Simonetta
Published: January 2005

The aim of this work was to evaluate the role of leukotrienes in brain damage in vivo in a model of focal cerebral ischaemia in the rat, obtained by permanent occlusion of middle cerebral artery.

A significant (P<0.01) elevation of LTC4, LTD4 and LTE4 (cysteinyl‐leukotrienes) levels occurred 4 h after ischaemia induction in the ipsilateral cortices of ischaemic compared to sham‐operated animals (3998±475 and 897±170 fmol g−1 tissue, respectively, P<0.01).

The NMDA receptor antagonist MK‐801 and the adenosine A2A receptor antagonist SCH 58261 were administered in vivo at doses known to reduce infarct size and compared with the leukotriene biosynthesis inhibitor MK‐886.

MK‐886 (0.3 and 2 mg kg−1 i.v.) and MK‐801 (3 mg kg−1 i.p.) decreased cysteinyl‐leukotriene levels (−78%, P<0.05; −100%, P<0.01; −92%, P<0.01, respectively) 4 h after permanent occlusion of the middle cerebral artery, whereas SCH 58261 (0.01 mg kg−1 i.v.) had no significant effects.

MK‐886 (2 mg kg−1 i.v.) was also able to significantly reduce the cortical infarct size by 30% (P<0.05).

We conclude that cysteinyl‐leukotriene formation is associated with NMDA receptor activation, and that it represents a neurotoxic event, the inhibition of which is able to reduce brain infarct area in a focal ischaemic event.

British Journal of Pharmacology (2001) 133, 1323–1329; doi:10.1038/sj.bjp.0704189
Thalidomide impairment of trinitrobenzene sulphonic acid‐induced colitis in the rat–role of endothelial cell‐leukocyte interaction
Authors: Lienenlüke Bianca , Stojanovic Tomislav , Fiebig Thomas , Fayyazi Afshin , Germann Tieno , Hecker Markus
Published: January 2005

Immune response‐modulating drugs such as thalidomide may be of therapeutic value in the treatment of chronic inflammatory bowel diseases including Crohn's disease (CD). In the present study, we have investigated whether thalidomide exerts this effect by impairing endothelial cell‐leukocyte interaction through down‐regulation of the expression of pro‐inflammatory gene products in these cells.

Transient CD‐like colitis was induced in male Wistar rats by single enema with trinitrobenzene sulphonic acid (TNBS) in ethanol followed by macroscopic scoring, histology, intravital microscopy, RT–PCR and immunohistochemistry (IHC) analyses. Thalidomide or its analogue supidimide were administered in olive oil by intragastric instillation 6 h prior to the induction of colitis and then daily for one week.

Both thalidomide and supidimide (200 mg kg−1 d−1) significantly attenuated TNBS‐induced colitis as compared to vehicle‐treated control animals (44 and 37% inhibition, respectively), and this effect persisted for 7 days post cessation of thalidomide treatment (46% inhibition).

Moreover, thalidomide significantly reduced leukocyte sticking to postcapillary venular endothelial cells in the submucosa (by 45%), improved functional capillary density and perfusion, and attenuated endothelial interleukin‐8 expression, as judged by IHC analysis. According to RT–PCR analysis, both thalidomide and supidimide also significantly reduced vascular cell adhesion molecule‐1 mRNA expression in the affected part of the descending colon.

These findings suggest that thalidomide and one of its derivatives impairs CD‐like TNBS‐induced colitis in the rat by down‐regulating endothelial adhesion molecule and chemokine expression and, as a consequence, the interaction of these cells with circulating leukocytes.

British Journal of Pharmacology (2001) 133, 1414–1423; doi:10.1038/sj.bjp.0704193
Acute and chronic effects of desipramine and clorgyline on α2‐adrenoceptors regulating noradrenergic transmission in the rat brain: a dual‐probe microdialysis study
Authors: Mateo Y , Fernández‐Pastor B , Meana J J
Published: January 2005

The effects of desipramine (3 mg kg−1 i.p.) and clorgyline (1 mg kg−1 i.p.) on extracellular noradrenaline (NA) in the locus coeruleus (LC) and cingulate cortex were assessed in freely‐moving rats by dual‐probe microdialysis. Functional activities of α2‐adrenoceptors regulating NA release in the LC and cingulate cortex were determined by systemic (0.3 mg kg−1 i.p.) or local (0.1–100 μM) clonidine administration.

Extracellular NA was increased in the LC and cingulate cortex following acute desipramine but not clorgyline treatment. Systemic clonidine decreased NA similarly in desipramine‐, clorgyline‐, and saline‐treated animals, in both brain areas.

Long‐term (twice daily, 14 days) but not short‐term (twice daily, 7 days) desipramine, and long‐term clorgyline (once daily, 21 days) treatments increased NA (3 fold) in cingulate cortex but not in the LC. Following long‐term treatments, responses of NA to systemic clonidine were attenuated in the LC and cingulate cortex.

Clonidine perfusion by reverse dialysis into the cingulate cortex decreased local NA (−55±9%). The effect was attenuated by long‐term desipramine (−31±9%) and clorgyline (−10±2%) treatments.

Clonidine perfusion by reverse dialysis into the LC decreased NA in the LC (−89±2%) and in cingulate cortex (−52±12%). This effect was attenuated in the LC following long‐term desipramine (−72±4%) and clorgyline (−62±12%) treatments but it was not modified in the cingulate cortex (−57±10% and −68±6%, respectively).

These findings demonstrate that chronic desipramine or clorgyline treatments increase NA in noradrenergic terminal areas and desensitize α2‐adrenoceptors modulating local NA release at somatodendritic and terminal levels. However, somatodendritic α2‐adrenoceptors that control LC firing activity are not desensitized.

British Journal of Pharmacology (2001) 133, 1362–1370; doi:10.1038/sj.bjp.0704196
Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide
Authors: Jonsson Kent‐Olov , Vandevoorde Séverine , Lambert Didier M , Tiger Gunnar , Fowler Christopher J
Published: January 2005

The ability of a series of homologues and analogues of palmitoylethanolamide to inhibit the uptake and fatty acid amidohydrolase (FAAH)‐catalysed hydrolysis of [3H]‐anandamide ([3H]‐AEA) has been investigated.

Palmitoylethanolamide and homologues with chain lengths from 12–18 carbon atoms inhibited rat brain [3H]‐AEA metabolism with pI50 values of ∼5. Homologues with chain lengths eight carbon atoms gave <20% inhibition at 100 μM.

R‐palmitoyl‐(2‐methyl)ethanolamide, palmitoylisopropylamide and oleoylethanolamide inhibited [3H]‐AEA metabolism with pI50 values of 5.39 (competitive inhibition), 4.89 (mixed type inhibition) and 5.33 (mixed type inhibition), respectively.

With the exception of oleoylethanolamide, the compounds did not produce dramatic inhibition of [3H]‐WIN 55,212‐2 binding to human CB2 receptors expressed on CHO cells. Palmitoylethanolamide, palmitoylisopropylamide and R‐palmitoyl‐(2‐methyl)ethanolamide had modest effects upon [3H]‐CP 55,940 binding to human CB1 receptors expressed on CHO cells.

Most of the compounds had little effect upon the uptake of [3H]‐AEA into C6 and/or RBL‐2H3 cells. However, palmitoylcyclohexamide (100 μM) and palmitoylisopropylamide (30 and 100 μM) produced more inhibition of [3H]‐AEA uptake than expected to result from inhibition of [3H]‐AEA metabolism alone.

In intact C6 cells, palmitoylisopropylamide and oleoylethanolamide inhibited formation of [3H]‐ethanolamine from [3H]‐AEA to a similar extent as AM404, whereas palmitoylethanolamide, palmitoylcyclohexamide and R‐palmitoyl‐(2‐methyl)ethanolamide were less effective.

These data provide useful information upon the ability of palmitoylethanolamide analogues to act as ‘entourage’ compounds. Palmitoylisopropylamide may prove useful as a template for design of compounds that reduce the cellular accumulation and metabolism of AEA without affecting either CB1 or CB2 receptors.

British Journal of Pharmacology (2001) 133, 1263–1275; doi:10.1038/sj.bjp.0704199
Effects and mechanisms of emodin on cell death in human lung squamous cell carcinoma
Authors: Lee Hong‐Zin
Published: January 2005

Emodin (1,3,8‐trihydroxy‐6‐methylanthraquinone) is an active component from the root and rhizome of Rheum palmatum that has been reported to exhibit antitumour effects, but the mechanism is not known. The study investigated the effects and mechanisms of emodin‐induced cell death in human lung squamous carcinoma cell line CH27.

Emodin (50 μM)‐induced CH27 cell apoptosis was confirmed by cell morphological change, sub‐G1 formation in flow cytometry analysis, viability assay and degradation of focal adhesion kinase in this study.

Emodin‐induced apoptosis of CH27 cells does not involve modulation of endogenous Bcl‐XL protein expression, but appears to be associated with the increased expression of cellular Bak and Bax proteins. This study also demonstrated the translocation of Bak and Bax from cytosolic to particulate fractions.

This study has shown that emodin‐treated CH27 cells revealed the increases in the relative abundance of cytochrome c for the indicated time intervals in cytosolic fraction.

This study demonstrates that the activation of caspase‐3, caspase‐9 and caspase‐8 is an important determinant of apoptotic death induced by emodin.

These results suggested that emodin induces CH27 cell death by Bax death pathway and Fas pathway.

British Journal of Pharmacology (2001) 134, 11–20; doi:10.1038/sj.bjp.0704205
Vasorelaxant effects of a nitric oxide‐releasing aspirin derivative in normotensive and hypertensive rats
Authors: Muscará Marcelo N , Lovren Fina , McKnight Webb , Dicay Michael , Soldato Piero del , Triggle Christopher R , Wallace John L
Published: January 2005

Nonsteroidal anti‐inflammatory drugs have been reported to exacerbate hypertension and to interfere with the effectiveness of some anti‐hypertensive therapies. In this study, we tested the effects of a gastric‐sparing, nitric oxide‐releasing derivative of aspirin (NCX‐4016) on hypertension in rats.

Hypertension was induced by administering L‐NAME in the drinking water (400 mg l−1). Groups of rats were treated daily with aspirin, NCX‐4016 or vehicle.

NCX‐4016 significantly reduced blood pressure relative to the aspirin‐treated group over the 2‐week period of treatment. Aspirin and, to a lesser extent, NCX‐4016 suppressed whole blood thromboxane synthesis.

In anaesthetized rats, acute intravenous administration of NCX‐4016 caused a significant fall in mean arterial pressure in hypertensive rats, but was devoid of such effects in normotensive controls.

In vitro, NCX‐4016 relaxed phenylephrine‐pre‐contracted aortic rings obtained from both normotensive and hypertensive rats, and significantly reduced their responsiveness to the contractile effects of phenylephrine.

These results suggest that NCX‐4016 reduces blood pressure in hypertensive rats, not simply through the direct vasodilatory actions of the nitric oxide released by this compound, but also through possible interference with the effects of endogenous pressor agents. These properties, added to its anti‐thrombotic effects, suggest that NCX‐4016 may be a safer alternative to aspirin for use by hypertensive patients.

British Journal of Pharmacology (2001) 133, 1314–1322; doi:10.1038/sj.bjp.0704209
VEGF stimulation of endothelial cell PAF synthesis is mediated by group V 14 kDa secretory phospholipase A2
Authors: Bernatchez Pascal N , Winstead Michelle V , Dennis Edward A , Sirois Martin G
Published: January 2005

Vascular endothelial growth factor (VEGF) is a potent inducer of inflammation, and we have shown that this latter effect is mediated through endothelial cell (EC) PAF synthesis. Since the phospholipid remodelling pathway enzymes (CoA‐independent transacylase, CoA‐IT; phospholipase A2, PLA2; and lyso‐PAF acetyltransferase, lyso‐PAF‐AT) may participate in PAF synthesis, we assessed their contribution to VEGF‐induced PAF synthesis in bovine aortic EC (BAEC) and human umbilical vein EC (HUVEC).

VEGF enhanced BAEC and HUVEC PAF synthesis by up to 28 and 4 fold above basal levels respectively.

A pretreatment with a CoA‐IT and lyso‐PAF‐AT inhibitor (Sanguinarin; 500 nM) blocked VEGF‐induced PAF synthesis by 95%, a specific CoA‐IT inhibitor (SKF45905; 10 – 50 μM) was without effect, confirming the crucial role of the PLA2 and lyso‐PAF‐AT.

Treatment with secreted PLA2 (sPLA2) inhibitors which have been shown to inhibit both groups IIA and V sPLA2 (SB203347; 10 μM and LY311727; 100 μM) blocked EC PAF synthesis by up to 90%, whereas selective inhibition of group IIA sPLA2 (LY311727; 1 μM) had no significant effect.

RT – PCR and Western blot analyses demonstrated the presence of group V sPLA2 whereas group IIA sPLA2 was undetected in EC.

Treatment with cytosolic and calcium‐independent PLA2 inhibitors (Arachidonyl trifluoromethyl ketone, Bromoenol lactone, Methyl arachydonyl fluorophosphate, up to 50 μM) did not prevent but rather potentiated the VEGF effect on EC PAF synthesis.

These results provide evidence that with VEGF activation of EC cells, the group V sPLA2 provides substrate for EC PAF formation.

British Journal of Pharmacology (2001) 134, 197–205; doi:10.1038/sj.bjp.0704215
Noradrenaline‐induced changes in intracellular Ca2+ and tension in mesenteric arteries from diabetic rats
Authors: Chow Wing Lim , Zhang Lili , MacLeod Kathleen M
Published: January 2005

The purpose of this investigation was to determine whether enhanced contractile responses to noradrenaline (NA) of mesenteric arteries from rats with chronic streptozotocin‐induced diabetes are associated with increases in mean cytosolic [Ca2+]i.

[Ca2+]i was measured with fura 2‐AM, and was monitored simultaneously with tension in perfused endothelium‐denuded mesenteric arterial rings from 12 – 14 week diabetic rats and age‐ and gender‐matched control rats.

Basal [Ca2+]i (expressed as Rn, the normalized fura 2 ratio) was not significantly different in arteries from control and diabetic rats. Similarly, no differences between control and diabetic arteries in the tension or [Ca2+]i responses to 80 mM KCl in the presence of phentolamine were detected.

The rate of tension development, peak tension and integrated tension in response to 30 μM NA were all significantly greater in diabetic than control arteries. However, this was not associated with enhancement of the corresponding [Ca2+]i responses in the diabetic arteries.

Peak contractile responses to perfusion with both 0.3 and 3 μM NA, but peak [Ca2+]i only in response to 0.3 μM NA, were significantly greater in diabetic than control arteries.

NA (30 μM) produced a greater increase in both peak tension and [Ca2+]i in diabetic than control arteries perfused with Ca2+‐free solution containing 1 mM EGTA. Neither the rate nor the magnitude of NA‐induced Ca2+ influx appeared to be altered in the diabetic arteries.

The enhanced sustained contractile response of diabetic arteries to NA appears to be dissociated from increases in [Ca2+]i, and may be due to other factors, such as an increase in the Ca2+ sensitivity of the contractile proteins.

British Journal of Pharmacology (2001) 134, 179–187; doi:10.1038/sj.bjp.0704221
Dibutyryl‐cyclic GMP induces peripheral antinociception via activation of ATP‐sensitive K+ channels in the rat PGE2‐induced hyperalgesic paw
Authors: Soares A C , Duarte I D G
Published: January 2005

Using the rat paw pressure test, in which increased sensitivity is induced by intraplantar injection of prostaglandin E2, we studied the action of several K+ channel blockers in order to determine what types of K+ channels could be involved in the peripheral antinociception induced by dibutyrylguanosine 3 : 5′‐cyclic monophosphate (DbcGMP), a membrane permeable analogue of cyclic GMP.

DbcGMP elicited a dose‐dependent (50, 75, 100 and 200 μg paw−1) peripheral antinociceptive effect. The effect of the 100 μg dose of DbcGMP was considered to be local since only a higher dose (300 μg paw−1) produced antinociception in the contralateral paw.

The antinociceptive effect of DbcGMP (100 μg paw−1) was dose‐dependently antagonized by intraplantar administration of the sulphonylureas tolbutamide (20, 40 and 160 μg) and glibenclamide (40, 80 and 160 μg), selective blockers of ATP‐sensitive K+ channels.

Charybdotoxin (2 μg paw−1), a selective blocker of high conductance Ca2+‐activated K+ channels, and apamin (10 μg paw−1), a selective blocker of low conductance Ca2+‐activated K+ channels, did not modify the peripheral antinociception induced by DbcGMP.

Tetraethylammonium (2 mg paw−1), 4‐aminopyridine (200 μg paw−1) and cesium (800 paw−1), non‐selective voltage‐gated potassium channel blockers, also had no effect.

Based on this experimental evidence, we conclude that the activation of ATP‐sensitive K+ channels could be the mechanism by which DbcGMP induces peripheral antinociception, and that Ca2+‐activated K+ channels and voltage‐dependent K+ channels appear not to be involved in the process.

British Journal of Pharmacology (2001) 134, 127–131; doi:10.1038/sj.bjp.0704224
Block by gabapentin of the facilitation of glutamate release from rat trigeminal nucleus following activation of protein kinase C or adenylyl cyclase
Authors: Maneuf Yannick P , McKnight Alexander T
Published: January 2005

The effect of activation of protein kinase C (PKC) or adenylyl cyclase on release of glutamate has been investigated in a perfused slice preparation from the rat caudal trigeminal nucleus. Stimulation of PKC by phorbol 12‐myristate 13‐acetate (PMA) produced a concentration‐dependent increase in K+‐evoked release of [2H]‐glutamate (maximum increase 45%, EC50 11.8 nM), but in the presence of gabapentin (30 μM) the facilitation of release was blocked. The adenylyl cyclase activator forskolin (FSK) also induced a concentration‐dependent increase in K+‐evoked release of [3H]‐glutamate (maximum increase 36%, EC50 2.4 μM), and again this facilitatory effect was blocked by gabapentin (30 μM). We suggest that these results may be of relevance to the antihyperalgesic properties of gabapentin, in conditions where concomitant release of substance P and CGRP produces activation of PKC and adenylyl cyclase respectively.

British Journal of Pharmacology (2001) 134, 237–240; doi:10.1038/sj.bjp.0704227