European Journal of Pharmacology (v.486, #2)

Involvement of Na+/H+ exchanger in hypoxia/re-oxygenation-induced neonatal rat cardiomyocyte apoptosis by He-Ying Sun; Ning-Ping Wang; Micheal E Halkos; Faraz Kerendi; Hajime Kin; Ruo-Xiang Wang; Robert A Guyton; Zhi-Qing Zhao (121-131).
Although increased Na+/H+ exchanger type-1 (NHE-1) activity has been implicated in the pathogenesis of myocardial infarction, the role of NHE-1 in induction of apoptosis, and the potential mechanisms involved have not been fully characterized. This study tested the hypothesis that NHE-1 activity is involved in hypoxia (H)/re-oxygenation (Re)-induced cardiomyocyte apoptosis by increasing mitochondrial Ca2+ ([Ca2+]m). Primary cultured neonatal rat cardiomyocytes were subjected to 4.5 h of H followed by 12 h of Re. Relative to H alone, the level of X-rhod-1 acetoxymethyl (AM)-labeled [Ca2+]m was increased, and the frequency of cell death (propidium iodide (PI) staining) and apoptotic cells (terminal deoxynucleotidyl transferase (TdT)-mediated-UTP nick end labeling [TUNEL]), confirmed by Annexin-V, were augmented at the end of Re, along with appearance of cytosolic cytochrome c, activation of caspase-3, and increased ratio of Bax and Bcl-2. Addition of cariporide (20 μmol/l), a well-known NHE-1 inhibitor, to cultured cells before H significantly reduced [Ca2+]m, the number of PI and TUNEL positive cells relative to the levels at end of Re, but did not completely eliminate these changes compared to Sham control. There was a strong trend for attenuation in increased levels of [Ca2+]m, and the number of PI and TUNEL positive cells when same dose of cariporide was added only at Re, but the difference in these variables did not reach significance. In contrast, the levels of [Ca2+]m and the number of PI and TUNEL positive cells were significantly reduced to a level comparable to Sham control when cariporide (20 μmol/l) was administered before H and during Re, respectively, associated with a reduction in cytosolic cytochrome c, caspase-3 activity and ratio of Bax and Bcl-2. In conclusion, these data suggest that NHE-1 is involved in induction of cardiomyocyte apoptosis during both H and Re through a [Ca2+]m-dependent manner, thereby resulting in activation of cytochrome c-caspase-3 signaling pathways.
Keywords: Apoptosis; Ca2+; Cytochrome c; Caspase-3; Hypoxia/re-oxygenation; Na+/H+ exchanger;

Y-26763: ATP-sensitive K+ channel activation and the inhibition of insulin release from human pancreatic β-cells by Karen E. Cosgrove; Susanne G. Straub; Philippa D. Barnes; Joanna Chapman; Geoffrey W. Sharp; Mark J. Dunne (133-139).
The effect of Y-26763 [(−)-(3S,4R)-4-(N-acetyl-N-hydroxyamino)-6-cyano-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-3-ol], a novel ATP-sensitive K+ (KATP) channel activator, was tested on insulin secretion from human pancreatic islets in vitro. Y-26763 was able to inhibit both glucose- and tolbutamide-induced insulin secretion from islets as assessed by radioimmunoassay. The mechanism for inhibition of insulin secretion was characterised using patch clamp electrophysiology on dispersed human pancreatic β-cells which express KATP channels comprised of Kir6.2 and SUR1, and the NES2Y human β-cell line, transfected with Kir6.2ΔC26. Y-26763 activated KATP channels in a reversible manner with a similar activity to diazoxide. This required the presence of hydrolysable nucleotides and appeared to be mediated by interaction of Y-26763 with SUR1 since: (a) tolbutamide was able to reverse the actions of Y-26763 and (b) Y-26763 failed to activate Kir6.2ΔC26 in the absence of SUR1. We conclude that Y-26763-induced inhibition of insulin release is dependent upon the activation of KATP channels in human β-cells.
Keywords: Y-26763; KATP channel; Insulin secretion; Human β-cell; NES2Y;

Expression of adhesion molecules by sphingosine 1-phosphate and histamine in endothelial cells by Ken Shimamura; Yuko Takashiro; Nobuteru Akiyama; Tetsuya Hirabayashi; Toshihiko Murayama (141-150).
We investigated the effects of sphingosine 1-phosphate and histamine on the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin, and their signaling pathways in human umbilical vein endothelial cells. Sphingosine 1-phosphate increased the mRNA and protein level of VCAM-1, and the mRNAs of E-selectin and ICAM-1. The effects of sphingosine 1-phosphate were inhibited by the pertussis toxin and the respective inhibitors (10 μM 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) for phosphoinositide-specific phospholipase C; 10 μM 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) for p38 mitogen-activated protein kinase (MAPK); 1 μM 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö6976) for the α form of protein kinase C (PKC-α)), but not by a PKC-δ inhibitor (1 μM rottlerin). Histamine, which alone showed no effect, enhanced the sphingosine 1-phosphate-induced expressions via histamine H1 receptor. The histamine response decreased by U73122 and rottlerin, but not by SB203580 and Gö6976. The effects of sphingosine 1-phosphate with and without histamine were abolished by the higher concentrations of PKC inhibitors and in the PKC-depleted cells. Sphingosine 1-phosphate and histamine alone stimulated phosphorylation of p38 MAPK in a phosphoinositide-specific phospholipase C-dependent but not in a PKCs-independent manner. These findings suggest that sphingosine 1-phosphate-induced expression of adhesion molecules was mediated by phosphoinositide-specific phospholipase C and preferentially by PKC-α and p38 MAPK, and the histamine response was mediated by phosphoinositide-specific phospholipase C and PKC-δ in human umbilical vein endothelial cells.
Keywords: Sphingosine 1-phosphate; Histamine; Protein kinase C; p38 MAPK; Adhesion molecule; Endothelial cell; (Human);

Enhanced antidepressant efficacy of σ1 receptor agonists in rats after chronic intracerebroventricular infusion of β-amyloid-(1–40) protein by Alexandre Urani; Pascal Romieu; François J. Roman; Kiyofumi Yamada; Yukihiro Noda; Hiroyuki Kamei; Hung Manh Tran; Taku Nagai; Toshitaka Nabeshima; Tangui Maurice (151-161).
Treatment of depressive symptoms in patients suffering from neurodegenerative disorders remains a challenging issue, since few available antidepressants present an adequate efficacy during pathological aging. Previous reports suggested that selective σ1 receptor agonists might constitute putative candidates. We here examined the pharmacological efficacy of igmesine and (+)-SKF-10,047 and the σ1 receptor-related neuroactive steroid dehydroepiandrosterone sulfate, in rats infused intracerebroventricularly during 14 days with the β-amyloid-(1–40) protein and then submitted to the conditioned fear stress test. Igmesine and (+)-SKF-10,047 significantly reduced the stress-induced motor suppression at 30 and 6 mg/kg, respectively, in β-amyloid-(40–1)-treated control rats. Active doses were decreased, to 10 and 3 mg/kg, respectively, in β-amyloid-(1–40)-treated animals. The dehydroepiandrosterone sulfate effect was also facilitated, both in dose (10 vs. 30 mg/kg) and intensity, in β-amyloid-(1–40)-treated rats. Neurosteroid levels were measured in several brain structures after β-amyloid infusion, in basal and stress conditions. Progesterone levels, both under basal and stress-induced conditions, were decreased in the hippocampus and cortex of β-amyloid-(1–40)-treated rats. The levels in pregnenolone, dehydroepiandrosterone and their sulfate esters appeared less affected by the β-amyloid infusion. The σ1 receptor agonist efficacy is known to be inversely correlated to brain progesterone levels, synthesized mainly by neurons that are mainly affected by the β-amyloid toxicity. The present study suggests that σ1 receptor agonists, due to their enhanced efficacy in a nontransgenic animal model, may alleviate Alzheimer's disease-associated depressive symptoms.
Keywords: σ1 Receptor; Neuro(active)steroid; β-Amyloid-(1–40) protein; Alzheimer's disease; Depression; Conditioned fear stress; (Rat);

Neurotrophic actions of the novel AMPA receptor potentiator, LY404187, in rodent models of Parkinson's disease by Michael J O'Neill; Tracey K Murray; Katherine Whalley; Mark A Ward; Caroline A Hicks; Sandra Woodhouse; David J Osborne; Phil Skolnick (163-174).
Recent developments in the molecular biology and pharmacology of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors has led to the discovery of selective, potent and systemically active AMPA receptor potentiators. These molecules enhance synaptic transmission and evidence suggests that they play important roles in plasticity and cognitive processes. Activation of AMPA receptors also increases neuronal activation and activity-dependent signalling, which may increase brain-derived neurotrophic factor (BDNF) expression and enhance cell proliferation in the brain. We therefore hypothesised that an AMPA receptor potentiator may provide neurotrophic effects in rodent models of Parkinson's disease. In the present studies we report that the potent and selective AMPA receptor potentiator, R,S-N-2-(4-(4-Cyanophenyl)phenyl)propyl 2-propanesulfonamide (LY404187), provides both functional, neurochemical and histological protection against unilateral infusion of 6-hydroxydopamine into the substantia nigra or striatum of rats. The compound also reduced 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity in mice. Interestingly, we were also able to observe large functional and histological effects when we delayed treatment until after cell death had occurred (3 or 6 days after 6-hydroxydopamine infusion), supporting a neurotrophic mechanism of action. In addition, LY404187 provided a dose-dependent increase in growth-associated protein-43 expression in the striatum. Therefore, we propose that AMPA receptor potentiators offer the potential of a new therapy to halt the progression and perhaps repair the degeneration in Parkinson's disease.
Keywords: Parkinson's disease; Neuroprotection; AMPA receptor potentiator; LY404187; 6-Hydroxydopamine; MPTP; GAP-43 (growth-associated protein-43); BDNF (brain-derived neurotrophic factor); (Rat);

Schizophrenia is marked by pronounced cognitive impairments in addition to the hallmark psychotic symptoms like hallucinations. Antipsychotic drugs can effectively reduce these hallucinations; however, the drugs have not resolved the cognitive impairment. Interestingly, nicotine, a drug commonly self-administered by people with schizophrenia, has been shown to significantly improve cognitive function of people with schizophrenia. The current study was conducted to determine the effect of typical (haloperidol) and atypical (clozapine and risperidone) antipsychotic drug treatment on sustained attention in rats performing a visual signal detection task. In addition, the interaction of haloperidol with chronic nicotine administration was assessed. Female Sprague–Dawley rats were injected subcutaneously with clozapine (0, 0.6, 1.25 and 2.5 mg/kg), risperidone (0, 0.025, 0.05 and 0.1 mg/kg) or haloperidol (0, 0.01, 0.02 and 0.04 mg/kg). In the second part of the study, the interaction of acute haloperidol (0, 0.005, 0.01 and 0.02 mg/kg) and chronic nicotine (5 mg/kg/day, for 4 weeks via osmotic minipump) was characterized. Clozapine, risperidone and haloperidol all caused dose-related impairments in percent hit performance. There was a significant linear dose-related impairment in percent hit caused by risperidone. All the doses of clozapine caused a significant impairment in percent hit at the higher luminance intensities in the visual signal detection task. The 0.01 and 0.02 mg/kg haloperidol doses caused significant decreases in percent hit. The 0.04 mg/kg haloperidol dose impaired performance of the task to the point that reliable choice accuracy measurements could not be made. Chronic nicotine infusion significantly diminished the impairing effects of haloperidol on performance during weeks 1–2. In summary, both typical and atypical antipsychotic drugs significantly impaired sustained attention in rats. Haloperidol was more detrimental than clozapine and risperidone. Chronic nicotine diminished the adverse effects of haloperidol on performance. This study establishes a paradigm to reliably determine the attentional impairment caused by antipsychotic drugs.
Keywords: Attention; Haloperidol; Clozapine; Risperidone; Cognition; Signal detection task;

Pre-weaning carvedilol treatment in spontaneously hypertensive rats by Erika I. Boesen; Gavin W. Lambert; Warwick P. Anderson; Michelle M. Kett (183-188).
Hypertension in spontaneously hypertensive rats (SHR) has been permanently abolished by aggressive treatment regimens targeted against the sympathetic nervous system and adrenal medulla, initiated during the pre-weaning period (guanethidine and nerve growth factor antiserum combined with either adrenal demedullation or prazosin treatment). To investigate the components of the sympatho-adrenal system involved, we treated pre-weaning SHR with the combined α1- and β-adrenoceptor antagonist carvedilol (60 mg/kg/day s.c.; postnatal days 1–21). Carvedilol treatment significantly blocked adrenoceptors during the treatment period, delayed development (eye opening), reduced growth, and reduced arterial pressure and heart rate. However, there was only modest attenuation of the subsequent development of hypertension at 10 weeks of age (mean arterial pressure 129.5±1.8 versus 136.1±1.6 mm Hg in vehicle-treated littermates; P<0.05). Thus pre-weaning carvedilol treatment slightly but significantly attenuated the development of SHR hypertension at 10 weeks, suggesting that the profound antihypertensive effects of pre-weaning sympatho-adrenal ablation are attributable to factors other than α1- and β-adrenoceptor-mediated effects of catecholamines during this period.
Keywords: Sympathetic nervous system; Andrenoceptor antagonist; Spontaneously hypertensive rat (SHR);

In vivo experimental approach for the risk assessment of fluoroquinolone antibacterial agents-induced long QT syndrome by Katsuyoshi Chiba; Atsushi Sugiyama; Takehiro Hagiwara; Shin-ichi Takahashi; Kiyoshi Takasuna; Keitaro Hashimoto (189-200).
The proarrhythmic effects of fluoroquinolone antibacterial agents, sitafloxacin, gatifloxacin and moxifloxacin, were compared using three in vivo models. In the halothane-anesthetized dogs (n=5), intravenous 10-min infusion of gatifloxacin and moxifloxacin (1–3 mg/kg) prolonged the ventricular effective refractory period and the repolarization period to a similar extent, whereas sitafloxacin (1–3 mg/kg) prolonged the former only. No significant change was detected in other cardiovascular parameters. In the chronic complete atrioventricular block dogs (n=4), oral administration of 100 mg/kg of gatifloxacin (2 of 4) and moxifloxacin (3 of 4) induced torsades de pointes, which was not observed by sitafloxacin. In the α-chloralose-anesthetized rabbits (n=5), intravenous 20-min infusion of 60 mg/kg of gatifloxacin induced torsades de pointes (1 of 5) in the presence of methoxamine infusion, which was not observed by sitafloxacin or moxifloxacin. Thus, the halothane-anesthetized model is suitable for assessing QT prolongation, whereas the chronic complete atrioventricular block model is sensitive for detecting torsadogenic action of drugs. The α-chloralose-anesthetized model is the simplest and least expensive method, but its sensitivity to detect proarrhythmic action may be less great.
Keywords: Fluoroquinolone; Monophasic action potential; QT interval; Torsades de pointes; Atrioventricular block; α1-Adrenoceptor stimulation;

Influence of the endothelium on ex vivo tolerance and metabolism of glyceryl trinitrate in rat aorta by Ivan S de la Lande; Tracey E Siebert; Catherine L Bennett; Irene Stafford; John D Horowitz (201-207).
The influence of the endothelium on glyceryl trinitrate metabolism and relaxation and the relationship to tolerance induced by transdermal glyceryl trinitrate was explored in rat aorta. Metabolism was assessed in artery segments incubated with glyceryl trinitrate (1.0 μM) for 2 min and the contents of 1,2- and 1,3-glyceryl dinitrate measured by gas chromatography. In non-tolerant arteries mean contents of glyceryl trinitrate, 1,2-glyceryl dinitrate and 1,3-glyceryl dinitrate were 3.2, 0.23 and 0.10 nmol/g, respectively; in tolerant arteries the content of 1,2-glyceryl dinitrate was reduced by approximately 60%. Endothelium removal or nitric oxide synthase (NOS) inhibition did not affect metabolite contents but increased the relaxant response to glyceryl trinitrate in the tolerant artery to an extent that tolerance was significantly attenuated. It is concluded that (i) tolerance is associated with depression of glyceryl trinitrate metabolism by an endothelium-independent mechanism and (ii) the endothelium contributes to tolerance by a mechanism which is independent of metabolism and may be linked with endothelial NOS.
Keywords: Aorta; Endothelium; Glyceryl trinitrate metabolism; Glyceryl dinitrate; Artery relaxation; Tolerance; NO (nitric oxide) synthase inhibition;

Differential effect of HERG blocking agents on cardiac electrical alternans in the guinea pig by Anthony A Fossa; Todd Wisialowski; Eric Wolfgang; Ellen Wang; Michael Avery; David L Raunig; Bernard Fermini (209-221).
Beat-to-beat alternations of the cardiac monophasic action potential, known as electrical alternans, were studied at drug concentrations that have known arrhythmogenic outcomes. Electrical alternans were elicited from the heart of anesthetized guinea pigs, both in the absence and presence of drugs that inhibit the delayed rectifier K+ channel encoded by the human ether a-go-go related-gene (HERG), and are associated with the fatal arrhythmia, Torsade de Pointes. Two other HERG inhibiting drugs not associated with Torsade de Pointes were also studied. At concentrations known to be proarrhythmic, E-4031 and bepridil increased mean alternans 10 and 40 ms at pacing frequencies ≤160 ms. Terfenadine and cisapride both increased mean alternans up to 20 and 21 ms, respectively, at pacing frequencies of ≤150 ms. On the other hand, verapamil and risperidone showed no increase in mean alternans while risperidone significantly reduced alternans at concentrations up to 74 times its therapeutic level. The magnitude of effect on rate-dependent alternans may allow the differentiation of proarrhythmia and non-arrhythmic HERG blockers at clinically relevant concentrations.
Keywords: Alternans; HERG; Torsade de Pointes; QT prolongation; Arrhythmia;

In circular smooth muscle strips of porcine gastric fundus, polyethylene-glycol-superoxide dismutase, a membrane-permeable analogue of endogenous copper/zinc (Cu/Zn) superoxide dismutase, reversed the inhibitory effect of the superoxide anion generator 6-anilino-5,8-quinolinedione (LY83583) on electrically induced nitrergic relaxations of fundic tissues which are depleted of the endogenous antioxidant Cu/Zn superoxide dismutase by diethyldithiocarbamate, to the same extent as exogenously added Cu/Zn superoxide dismutase. Addition of a second antioxidant together with Cu/Zn superoxide dismutase does not result in a higher degree of reversal of the inhibitory effect of LY83583. Depletion of either tissue glutathione or tissue catalase in combination with diethyldithiocarbamate does not increase the inhibitory action of LY83583 or the nitric oxide (NO)-scavenger hydroxocobalamin upon nitrergic relaxations (electrically induced or by exogenous NO) when compared to their action in the presence of diethyldithiocarbamate alone. In conclusion, these results demonstrate that endogenous Cu/Zn superoxide dismutase is the essential antioxidant responsible for safeguarding peripheral nitrergic neurotransmission, whereby extracellular protection of endogenous NO is most important.
Keywords: Polyethylene-glycol-superoxide dismutase; Cu/Zn superoxide dismutase; Diethyldithiocarbamate; 6-Anilino-5,8-quinolinedione; Nitrergic relaxation; Gastric fundus; (Pig);

Endogenous ligands of PPAR-γ reduce the liver injury in haemorrhagic shock by Marika Collin; Maha Abdelrahman; Christoph Thiemermann (233-235).
We demonstrate here for the first time that the novel, potent peroxisome proliferator-activated receptor (PPAR)-γ antagonist GW9662 (2-chloro-5-nitrobenzanilide) augments the degree of liver injury associated with haemorrhagic (haemorrhage for 90 min and resuscitation for 4 h), but not endotoxic (6 mg/kg E. coli endotoxin i.v. for 6 h) shock in the anaesthetised rat. Thus, endogenous ligands for PPAR-γ are released in haemorrhagic, but not endotoxic, shock in sufficient amounts to protect against injury.
Keywords: Shock; PPAR (peroxisome proliferator-activated receptor); GW9662;

Corrigendum to “Further evidence that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors” [Eur. J. Pharmacol. 477 (2003) 227–234] by Chiara Mioni; Daniela Giuliani; Maria Michela Cainazzo; Sheila Leone; Anna Iannone; Carla Bazzani; Paolo Grieco; Ettore Novellino; Aldo Tomasi; Alfio Bertolini; Salvatore Guarini (237).