European Journal of Pharmacology (v.549, #1-3)

Selection of new chemical entities with decreased potential for adverse drug reactions by Kevin B. Park; Emma Dalton-Brown; Charlotte Hirst; Dominic P. Williams (1-8).
Adverse drug reactions, such as hepatotoxicity, blood dyscrasias and hypersensitivity are a major obstacle for the use and the development of new medicines. Many forms of organ-directed toxicity can arise from the bioactivation of drugs to so-called chemically reactive metabolites, which can modify tissue macromolecules. It is well established that the toxicities of model hepatotoxins, such as acetaminophen, furosemide, bromobenzene and methapyrilene can be correlated with the generation of chemically reactive metabolites, which can be detected by measurement of the irreversible binding of radiolabelled material to hepatic protein and/or the detection of stable phase II metabolites such as glutathione conjugates. The basic chemistry of the reaction of such metabolites with model nucleophiles is relatively well understood. A major challenge is to define how certain reactive intermediates may chemically modify critical proteins and how modification of specific amino acids may alter protein function which in turn may affect cell signalling, regulation, defence, function and viability. This in turn will determine whether or not bioactivation will result in a particular form of drug-induced injury. It is now clear that even relatively simple reactive intermediates can react in a discriminative manner with particular cellular proteins and even with specific amino acids within those proteins. Therefore both non-covalent, as well as covalent bonds will be important determinants of the target protein for a particular reactive metabolite. Mammalian cells have evolved numerous defence systems against reactive intermediates. Sensitive redox proteins such as Nrf-2 recognize oxidative stress and electrophilic agents. This is achieved by chemical modification of cysteine groups within keap-1, which normally forms an inactive heterodimer with Nrf-2. Modification of keap-1 releases Nrf-2 that translocates to the nucleus and effects gene transcription of a number of genes involved in the detoxication of chemically reactive metabolites. Diminution of protein function can occur by either covalent modification of nucleophilic amino acids (e.g. cysteine, lysine, histidine etc.) or oxidation of thiols, which can be reversible or irreversible. In the case of acetaminophen, more than 30 target proteins have been identified and for several of them, corresponding alterations in protein function have been defined in the context of tissue necrosis. Alternatively, protein modification may induce signalling systems which initiate cell death, an immune response or to an altered tissue genotype.
Keywords: Bioactivation; Hepatotoxicity; Reactive metabolite;

The novel histone deacetylase inhibitor BML-210 exerts growth inhibitory, proapoptotic and differentiation stimulating effects on the human leukemia cell lines by Jurate Savickiene; Veronika-Viktorija Borutinskaite; Grazina Treigyte; Karl-Eric Magnusson; Ruta Navakauskiene (9-18).
Histone deacetylase inhibitors have a potent role in the strategy for the treatment of leukemias. BML-210 (N-(2-Aminophenyl)-N′ phenyloctanol diamine) is the novel histone deacetylase inhibitor, and its mechanism of action has not been characterized. In this study, we examined the in vitro effects of BML-210 on the human leukemia cell lines (NB4, HL-60, THP-1, and K562). We found that BML-210 inhibits the growth of all cell lines and promotes apoptosis in a dose- and time-dependent manner. BML-210 alone induces HL-60 and K562 cell differentiation (up to 30%) to granulocytes and erythrocytes, respectively, and in combination with differentiation agents — all-trans retinoic acid and hemin, markedly potentates it. Those treatments cause G1 arrest and histone H4 acetylation, affects transcription factor NF-κB and Sp1 binding activity to their consensus sequences, the p21 or the FasL promoters, and influences expression of Sp1, NF-κB, p21 and FasL. These findings suggest that BML-210 could be a promising antileukemic agent to induce apoptosis and to modulate differentiation through the modulation of histone acetylation and gene expression.
Keywords: Apoptosis; Differentiation; Histone deacetylase inhibitor; Leukemia; Transcription factors;

Neuroprotective effects of galanthamine and tacrine against glutamate neurotoxicity by Yuki Takada-Takatori; Toshiaki Kume; Mitsuhiro Sugimoto; Hiroshi Katsuki; Tetsuhiro Niidome; Hachiro Sugimoto; Takeshi Fujii; Susumu Okabe; Akinori Akaike (19-26).
We examined the mechanisms of the neuroprotective effects of two central-type acetylcholinesterase inhibitors, galanthamine and tacrine, on nitric oxide-mediated glutamate neurotoxicity using primary cultures from the cerebral cortex of fetal rats. Galanthamine and tacrine showed prominent protective effects against glutamate neurotoxicity. Mecamylamine, a nicotinic acetylcholine receptor antagonist, but not scopolamine, a muscarinic acetylcholine receptor antagonist, inhibited the protective effects of these inhibitors on glutamate neurotoxicity. Furthermore, dihydro-β-erythroidine, an α4-nicotinic receptor antagonist, and methyllycaconitine, an α7-nicotinic receptor antagonist, inhibited the neuroprotective effects of galanthamine but not tacrine. Next, we investigated the site of action where galanthamine and tacrine prevent glutamate neurotoxicity. Both these acetylcholinesterase inhibitors prevented glutamate- and ionomycin-induced neurotoxicity, but only tacrine prevented S-nitrosocysteine-induced neurotoxicity. These results suggest that galanthamine and tacrine protect cortical neurons from glutamate neurotoxicity via different mechanisms.
Keywords: Acetylcholinesterase; Alzheimer's disease; Cortical culture; Glutamate; Nicotinic acetylcholine receptor; Neurotoxicity;

In vitro and in vivo antitumour effects of novel, orally active bile acid-conjugated platinum complexes on rat hepatoma by Cecilia Barbara; Paola Orlandi; Guido Bocci; Anna Fioravanti; Antonello Di Paolo; Gianfranco Natale; Mario Del Tacca; Romano Danesi (27-34).
(NH3)2Pt(triacid) and (PPh3)2Pt(dehydrocholate)2 are novel bile acid-conjugated platinum complexes administered by oral route. The aims of the present study were to evaluate their in vitro cytotoxic activities on rat hepatoma cell line N1–S1, the in vivo antitumour effects in a syngeneic and orthotopic rat hepatoma model and the drug-related toxicities. Cisplatin, carboplatin and mitoxantrone were used as control drugs. In vitro experiments showed a concentration- and time-dependent antiproliferative activity of bile-conjugated platinum complexes. (NH3)2Pt(triacid) had similar effects on cell growth of cisplatin and carboplatin (e.g. at 48 h, IC50 0.7 ± 0.05 μM vs. 0.63 ± 0.28 μM and 1.1 ± 0.3 μM, respectively; mean ± S.D.). (NH3)2Pt(triacid) was able to inhibit tumour growth in a dose-dependent extent, reaching the maximum inhibitory effect at the 80 mg/kg dose (1.95 ± 0.5 g vs. 13.85 ± 3.9 g of control tumour weight). By contrast, despite the promising in vitro antiproliferative activity, (PPh3)2Pt(dehydrocholate)2 showed no significant in vivo antitumour effect. The toxicity profile of (NH3)2Pt(triacid) resulted favourable with minimal loss of weight and no gastrointestinal or neurological symptoms. Instead, (PPh3)2Pt(dehydrocholate)2 showed dose-dependent signs of severe weight loss and neurological alterations. In conclusion (NH3)2Pt(triacid) is a tolerable and active platinum derivative endowed by a preclinical antitumour activity by oral route.
Keywords: Hepatoma; Platinum complex; Bile acid; Cisplatin; Carboplatin; Mitoxantrone;

Pranlukast, a cysteinyl leukotriene receptor 1 antagonist, protects mice against brain cold injury by Xiao-Dong Qian; Er-Qing Wei; Lei Zhang; Wen-Wen Sheng; Meng-Ling Wang; Wei-Ping Zhang; Zhong Chen (35-40).
We have reported the neuroprotective effect of cysteinyl leukotriene receptor 1 (CysLT1) antagonists on cerebral ischemia. Here, we further determined the protective effect of pranlukast, a CysLT1 receptor antagonist, on brain cold injury in mice. Brains were injured by placing a cooled metal probe on the skull surface for 30 s. We found that pranlukast significantly reduced cold-induced lesion volume (0.3 mg/kg) and the percentage increase in lesioned hemisphere volume (0.03–0.3 mg/kg) 24 h after injury, but did not show any effect 72 h after injury. Pranlukast also significantly inhibited neuron loss 24 h (0.1 mg/kg) and 72 h (0.1–0.3 mg/kg) after injury, and decreased the density of degenerated neurons 24 h (0.01–0.3 mg/kg) and 72 h (0.03–0.3 mg/kg) after injury. In addition, pranlukast (0.1–0.3 mg/kg) significantly reduced endogenous IgG exudation both 24 h and 72 h after injury. Thus, this study indicates the protective effect of pranlukast on brain cold injury.
Keywords: Leukotriene receptor antagonist; Pranlukast; Cold injury; Brain oedema; Blood–brain barrier; Neuroprotection;

High permeability of the anionic form restricts accumulation of indomethacin by cultured gastric surface epithelial cells exposed to low apical pH by Klairi M. Kavvada; James G. Murray; Vanessa A. Moore; Alan G.A. Coombes; Peter J. Hanson (41-49).
The ‘ion-trapping’ hypothesis suggests that the intracellular concentration of acidic non-steroidal anti-inflammatory drugs in gastric epithelial cells could be much higher than in the gastric lumen, and that such accumulation could contribute to their gastrotoxicity. Our aim was to examine the effect of the pH of the apical medium on the apical to basal transfer of the acidic drug indomethacin (pK a 4.5) across a gastric mucous epithelial cell monolayer, and to determine whether indomethacin accumulated in cells exposed to a low apical pH. Guinea-pig gastric mucous epithelial cells were grown on porous membrane culture inserts (Transwells®) for 72 h. Transfer and accumulation of [14C] indomethacin were assessed by scintillation counting. Transfer of [3H]mannitol and measurement of trans-epithelial electrical resistance were used to assess integrity of the monolayer. Distribution of [14C] urea was used to estimate the intracellular volume of the monolayer. The monolayer was not disrupted by exposure of the apical face to media of pH ≥ 3, or by indomethacin. Transfer of indomethacin (12 μM) to the basal medium increased with decreasing apical medium pH. The apparent permeability of the undissociated acid was estimated to be five times that of the anion. The intracellular concentration of indomethacin was respectively 5.3, 4.1 and 4.3 times that in the apical medium at pH 5.5, 4.5 and 3.0. In conclusion, this study represents the first direct demonstration that indomethacin accumulates in gastric epithelial cells exposed to low apical pH. However, accumulation of indomethacin was moderate and the predictions of the ion-trapping hypothesis were not met, probably due to the substantial permeability of anionic indomethacin across membranes.
Keywords: Stomach; Gastric; Mucous epithelial cell; Indomethacin; Non-steroidal anti-inflammatory drug; Cyclooxygenase inhibitor;

Nerve growth factor (NGF) differentiated pheochromocytoma PC12 cells exposed to 1-methyl-4-phenylpyridinium (MPP+) toxin were used as an in vitro pharmacological model of Parkinson's disease to examine the neuroprotective effects of 4-hydroxy-2,2,6,6-tetramethyl piperidine-n-oxyl (Tempol), a free radical scavenger and a superoxide dismutase-mimetic compound. MPP+-induced PC12 cell death was measured 72 h after exposure to 1.5 mM MPP+ by the release of lactate dehydrogenease, caspase-3 activation and stimulation of survival and stress mitogen-activated protein kinases. Exposure of PC12 cells to MPP+ activated ERK1 and ERK2 (forty-fold over control after 72 h), JNK1 and JNK2 (fourfold after 48 h) and p-38α (tenfold after 24 h). Pretreatment of PC12 cells with 500 μM Tempol, 1 h before induction of the MPP+ insult, reduced by 70% the release of LDH into the medium, inhibited caspase-3 activity by 30% and improved by 33% mitochondrial function, effects correlated with a 70% reduction in ERK1 and ERK2 phosphorylation activity. These findings support the neuroprotective effect of Tempol in the MPP+-induced PC12 cell death model and its use as a potential drug for treatment of Parkinson's disease.
Keywords: Parkinson's disease; PC12 cells; Tempol; MAPK (mitogen-activated protein kinases); Neurotoxicity; Neuroprotection;

We investigated the role of Ca2+-independent phospholipase A2 (iPLA2) as well as cytosolic phospholipase A2 (cPLA2) in hypoxia-inducible factor-1 (HIF-1)-dependent gene expression. An inhibitor of both iPLA2 and cPLA2, methyl arachidonyl fluorophosphonate (MAFP), prevented hypoxia-induced erythropoietin mRNA expression without affecting HIF-1α accumulation in Hep3B cells. The DNA-binding of HIF-1α was suppressed by MAFP as confirmed by luciferase reporter gene assays with the hypoxia response element. Translocation of HIF-1α to the nucleus assessed by its presence in the nuclear extracts of cells exposed to hypoxia, was diminished by MAFP. However, hypoxia-dependent gene expression was not affected in mesangial cells obtained from cPLA2α null mice. Furthermore, a specific iPLA2 inhibitor, bromoenol lactone, suppressed erythropoietin mRNA expression and HIF-1α translocation to the nucleus under hypoxic conditions. Thus, iPLA2, but not cPLA2α, may play an important role in regulating the transport of HIF-1α to the nucleus.
Keywords: Hypoxia-inducible factor-1α; Ca2+-independent phospholipase A2; Hypoxia; Bromoenol lactone;

Involvement of μ-opioid receptors in antinociception and inhibition of gastrointestinal transit induced by 7-hydroxymitragynine, isolated from Thai herbal medicine Mitragyna speciosa by Kenjiro Matsumoto; Yoshio Hatori; Toshihiko Murayama; Kimihito Tashima; Sumphan Wongseripipatana; Kaori Misawa; Mariko Kitajima; Hiromitsu Takayama; Syunji Horie (63-70).
7-Hydroxymitragynine, a constituent of the Thai herbal medicine Mitragyna speciosa, has been found to have a potent opioid antinociceptive effect. In the present study, we investigated the mechanism of antinociception and the inhibitory effect on gastrointestinal transit of 7-hydroxymitragynine, and compared its effects with those of morphine. When administered subcutaneously to mice, 7-hydroxymitragynine produced antinociceptive effects about 5.7 and 4.4 times more potent than those of morphine in the tail-flick (ED50  = 0.80 mg/kg) and hot-plate (ED50  = 0.93 mg/kg) tests, respectively. The antinociceptive effect of 7-hydroxymitragynine was significantly blocked by the μ12-opioid receptor antagonist β-funaltrexamine hydrochloride (β-FNA) and the μ1-opioid receptor-selective antagonist naloxonazine in both tests. Thus, 7-hydroxymitragynine acts predominantly on μ-opioid receptors, especially on μ1-opioid receptors. Isolated tissue studies further supported its specificity for the μ-opioid receptors. Further, 7-hydroxymintragynine dose-dependently (ED50  = 1.19 mg/kg, s.c.) and significantly inhibited gastrointestinal transit in mice, as morphine does. The inhibitory effect was significantly antagonized by β-FNA pretreatment, but slightly antagonized by naloxonazine. The ED50 value of 7-hydroxymitragynine on gastrointestinal transit was larger than its antinociceptive ED50 value. On the other hand, morphine significantly inhibits gastrointestinal transit at a much smaller dose than its antinociceptive dose. These results suggest that μ-opioid receptor mechanisms mediate the antinociceptive effect and inhibition of gastrointestinal transit. This compound induced more potent antinociceptive effects and was less constipating than morphine.
Keywords: 7-Hydroxymitragynine; Antinociception; Gastrointestinal transit; μ-Opioid receptor; Morphine;

Altered hippocampal gene expression 2 days after general anesthesia in rats by Deborah J. Culley; Rustam Y. Yukhananov; Zhongcong Xie; Reddy R. Gali; Rudolph E. Tanzi; Gregory Crosby (71-78).
We profiled changes in gene expression in the hippocampus 2 days after a 4 h general anesthetic with isoflurane and nitrous oxide. Eighteen month old Fisher 344 rats were anesthetized for 4 h with 1.2% isoflurane and 70% nitrous oxide (N  = 9) whereas control rats breathed 30% oxygen for 4 h (N  = 9). Rats were sacrificed 48 h later and RNA extracted from the hippocampus for gene expression profiling. Three gene arrays were used per group, with samples prepared by pooling RNA from three rats. Differentially expressed genes were analyzed based on a weighted error statistical model. Microarray results for 6 differentially expressed genes were verified with reverse transcriptase polymerase chain reaction. Compared to unanesthetized controls, 297 genes were differentially expressed 2 days following anesthesia (P  < 0.05). Of these, 113 are named genes; 64% were up-regulated and 36% were down-regulated. The majority of differentially expressed genes are implicated in cell stress and replication, signal transduction, transcription, protein biosynthesis, cell structure, and metabolism. The correlation between fold changes on array and reverse transcriptase polymerase chain reaction was good (R 2  = 0.85) for the 6 genes examined with both methods. These results demonstrate that in rats general anesthesia is associated with persistent changes in hippocampal gene expression, suggesting that recovery of the brain from anesthesia is considerably slower than generally recognized.

Improgan is a non-opioid analgesic which does not act at known histamine or cannabinoid receptors. Because improgan antinociception is blocked by low doses of a cannabinoid CB1 antagonist, the present experiments determined if development of cannabinoid tolerance in mice would alter improgan antinociception. Twice-daily injections of Δ9-tetrahydrocannabinol (THC, 10 mg/kg, s.c.) for 3.5 days induced 47–54% and 42–56% reductions in cannabinoid (WIN 55,212-2, 20 μg, i.c.v.) and improgan (30 μg, i.c.v.) antinociception, respectively, as compared with responses from vehicle-treated groups. Because improgan lacks cannabinoid-like side effects in rats, and does not act directly on cannabinoid CB1 receptors, the finding that development of cannabinoid tolerance reduces improgan antinociception suggests that this drug may release endocannabinoids, or activate novel cannabinoid sites. Either possibility offers the potential for developing new types of analgesics.
Keywords: Improgan; Analgesia; Pain; Cannabinoid; Cannabinoid CB1 receptor; Tetrahydrocannabinol;

The central dopamine system plays a prominent role in the effect of psychostimulants such as methamphetamine, cocaine and nicotine. l-3,4-Dihydroxyphenylalanine (DOPA), a precursor of dopamine, has been proposed as a neurotransmitter in the central nervous system. We have studied the effects of these psychostimulants on the release of DOPA and dopamine from the nucleus accumbens shell in conscious rats using in vivo microdialysis. Methamphetamine and cocaine increase the extracellular levels of dopamine. The effect of methamphetamine (1 mg/kg s.c.) on the release of dopamine was almost comparable to that of cocaine (10 mg/kg i.p.). However, methamphetamine increases, but cocaine decreases the extracellular levels of DOPA. In a behavioral study, methamphetamine (1 mg/kg s.c.) induced chewing, walking and sniffing behavior. Cocaine (10 mg/kg i.p.) produces weak effects on these behavioral parameters, when compared to the effects of methamphetamine (1 mg/kg s.c.). The behavioral changes produced by methamphetamine are suppressed by DOPA cyclohexyl ester (30 mg/kg i.p.), a competitive DOPA antagonist. Endogenous DOPA in the nucleus accumbens thus appears to be in involved in the behavioral responses to these psychomotor stimulants.
Keywords: DOPA; Dopamine; Release; Cocaine; Methamphetamine; Nicotine; Behavior; Nucleus accumbens; Microdialysis;

Anti-arrhythmic and cardio-protective effects of furnidipine in a rat model: A dose response study by Tadeusz F. Krzemiński; Joanna Grzyb; Maurycy P. Porc; Shyam S. Chatterjee (91-97).
Protective effects of acute oral or intravenous doses of furnidipine against ischemia and re-perfusion-induced arrhythmias and creatine kinase release were studied in a rat model for cardiac ischemia and re-perfusion. Transient cardiac ischemia was induced by occluding the left coronary descending artery of anaesthetized rats for 7 min, and re-perfusion period studied was 15 min. Pre-treatment period for oral doses (1, 5 or 10 mg/kg) was 1 h, whereas that for the intravenous ones (1.25, 2.5, 5 or 10 μg/kg) was 10 min. After both routes of administration, significant protective effects of furnidipine on creatine kinase release were observed after the two lowest doses only. In contrast, its higher dosages were more effective in preventing re-perfusion-induced mortality, arrhythmias and hypotensive episodes, and for transiently lowering arterial blood pressure before initiation of ischemia. These observations suggest potential uses of furnidipine for preventing re-perfusion triggered lethal arrhythmias. Efforts to evaluate therapeutic potential of low dose furnidipine as a cardio-protective agent seem warrantable.
Keywords: Furnidipine; Ischemia and re-perfusion; Arrhythmias; Cardio-protection; (Rat);

Effects of G-CSF on cardiac remodeling and arterial hyperplasia in rats by Yuxin Li; Noboru Fukuda; Shin-Ichiro Yokoyama; Yoshiaki Kusumi; Kazuhiro Hagikura; Taro Kawano; Tadateru Takayama; Taro Matsumoto; Aya Satomi; Junko Honye; Hideo Mugishima; Masako Mitsumata; Satoshi Saito (98-106).
Although granulocyte colony-stimulating factor (G-CSF) has been shown to prevent cardiac remodeling after acute myocardial infarction, the mechanism and safety of G-CSF treatment acute myocardial infarction remain controversial. The purpose of the present study was to investigate in a rat model the mechanisms underlying the beneficial effect of G-CSF in acute myocardial infarction and to determine whether G-CSF treatment aggravates vascular remodeling of injured artery after acute myocardial infarction. Sprague–Dawley rats received transplanted bone marrow cells from green fluorescent protein (GFP) transgenic rats. Acute myocardial infarction was induced by ligation of the left coronary artery. After 24 h, the right carotid artery was injured with a balloon catheter. G-CSF (100 μg/kg/day) or saline was injected subcutaneously for 5 consecutive days after induction of acute myocardial infarction. G-CSF treatment significantly improved left ventricle function and reduced infarct size in rats with acute myocardial infarction. Expression of mRNA for the angiogenic cytokines was significantly higher in the infarction border area in the G-CSF group than in the control group. The surviving cardiomyocytes in infarction area were more in the G-CSF group. GFP-positive cells were gathered in the infarction border area in both groups; G-CSF did not increase cardiac homing of GFP-positive bone marrow cells in contrast to control group. Most GFP-positive cells were CD68-positive (macrophages). It was difficult to find bone marrow-derived cardiomyocytes in the infarcted area. G-CSF treatment inhibited neointima formation and increased reendothelialization of the injured artery. GFP-positive cells were identified most in the adventitia of the injured artery. A few cells in the neointima and reendothelialization were GFP positive. In conclusion, administration of G-CSF appears to be effective for treatment of left ventricular remodeling after acute myocardial infarction and does not aggravate vascular remodeling. The effect of G-CSF on cardiac and vascular remodeling may occur mainly through a direct action on the heart and arteries.
Keywords: Cytokine; Coronary disease; Infarction; Stem cell; Restenosis;

Prostacyclin therapy increases right ventricular capillarisation in a model for flow-associated pulmonary hypertension by Mirjam E. van Albada; Rolf M.F. Berger; Marnix Niggebrugge; Richard van Veghel; Adri H. Cromme-Dijkhuis; Regien G. Schoemaker (107-116).
Pulmonary hypertension, and consequently right ventricular failure, complicates several congenital heart defects. Although intervention in the prostacyclin–thromboxane ratio is known to improve outcome, the underlying mechanism is not clear. Therefore, effects of acetyl salicylic acid and iloprost are studied in an animal model for flow-associated pulmonary hypertension. Male Wistar rats with flow-associated pulmonary hypertension, an aortocaval shunt in addition to monocrotaline induced pulmonary hypertension, were treated with low-dose aspirin (25 mg/kg/day) or iloprost (72 μg/kg/day). Effects on pulmonary hemodynamics and pulmonary vascular remodeling as well as right ventricular hemodynamics and remodeling were evaluated. Ninety percent (n  = 7 / 8) of the untreated pulmonary hypertensive rats developed dyspnea and pleural fluid, whereas this was seen in 50% (n  = 4 / 8, ns) and 10% (n  = 1 / 8, P  < 0.05 vs. untreated animals) of the aspirin and iloprost-treated rats, respectively. This could not be attributed to changes in pulmonary artery pressure, wall–lumen ratio of the pulmonary vasculature or right ventricular hypertrophy. However, both therapies restored reduced right ventricular capillary to myocyte ratio in pulmonary hypertensive rats (0.95 ± 0.10 in untreated rats vs. 1.38 ± 0.18 in control animals; P  < 0.05, and 1.32 ± 0.11 in aspirin-treated and 1.29 ± 0.9 in iloprost-treated rats; both P  < 0.05 vs. non-treated animals), which was associated with improved right ventricular contractility (iloprost). Thus, interventions in the prostacyclin–thromboxane metabolism improve outcome in rats with flow-associated pulmonary hypertension. However, these effects may be attributed to effects on cardiac rather than on pulmonary vascular remodeling.
Keywords: Pulmonary circulation; Heart failure; Histopathology; Cyclo-oxygenase; Angiogenesis;

Intermedin1–53 protects the heart against isoproterenol-induced ischemic injury in rats by Yue-Xia Jia; Jing-Hui Yang; Chun-Shui Pan; Bin Geng; Jing Zhang; Yang Xiao; Jing Zhao; Helen Gerns; Jun Yang; Jaw-Kang Chang; Jin Kun Wen; Chao-Shu Tang; Yong-Fen Qi (117-123).
Intermedin is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family peptide, which has vasodilatory and hypotensive actions identical to those of adrenomedullin and CGRP. Cleavage sites located between 2 basic amino acids at Arg93–Arg94 result in the production of prepro-intermedin95–147, namely intermedin1–53. The bioactive action of intermedin1–53 and its physiological significance are unclear. In this work, we aimed to explore the effects of intermedin1–53 on acute myocardial injury induced by isoproterenol. Myocardial ischemia injury in rats was induced by subcutaneous injection of a high dose of isoproterenol, and the therapeutic effect of intermedin1–53 was observed. Plasma lactate dehydrogenase activity, myocardial and plasma malondialdehyde content were higher in the isoproterenol group than that in controls. Isoproterenol-treated rats showed lower maximal rate of increase and decrease of left-ventricle pressure development (± left-ventricle dp/dt max) and higher left-ventricle end-diastolic pressure (all P  < 0.01), which suggested severe heart failure and myocardial injury. Semi-quantitative RT-PCR analysis showed that the gene expression of calcitonin receptor-like receptor and receptor-activity-modifying protein (RAMP)1, RAMP2 and RAMP3 in ventricular myocardia were up-regulated by 79% (P  < 0.01), 48% (P  < 0.01), 31% (P  < 0.05) and 130% (P  < 0.01), respectively, compared with controls. In myocardial sarcolemmal membranes, the maximum binding capacity for [125I]-intermedin1–53 was increased by 118% (P  < 0.01) in the isoproterenol group compared with controls. Rats treated with low dosage intermedin1–53 (5 nmol/kg/day, 2 days) showed 21% (P  < 0.05) higher myocardial cAMP content, 18% and 31% higher + left-ventricle dp/dt max and -left-ventricle dp/dt max respectively, 288% lower left-ventricle end-diastolic pressure (all P  < 0.01), and attenuated myocardial lactate dehydrogenase leakage and malondialdehyde formation (all P  < 0.01). Treatment with high dosage intermedin1–53 (20 nmol/kg/day, 2 days) gave better results than that with low dosage intermedin1–53. These results suggest that the intermedin receptor system was up-regulated in isoproterenol-induced myocardial ischemic injury and intermedin1–53 might play a pivotal cardioprotective role in such injury.
Keywords: Intermedin; Isoproterenol; Calcitonin receptor-like receptor; Receptor-activity-modifying protein; cAMP;

The direct antioxidative and anti-inflammatory effects of peroxisome proliferator-activated receptors ligands are associated with the inhibition of angiotensin converting enzyme expression in streptozotocin-induced diabetic rat aorta by Hiroe Toba; Shunsuke Miki; Takahiro Shimizu; Akiko Yoshimura; Riyako Inoue; Naoki Sawai; Rie Tsukamoto; Masahumi Murakami; Yosuke Morita; Yusuke Nakayama; Miyuki Kobara; Tetsuo Nakata (124-132).
Peroxisome proliferator-activated receptors (PPARs) are expressed on vascular tissue. To investigate the direct vasoprotective effects of PPARγ and PPARα ligands, pioglitazone (3 mg/kg/day) and bezafibrate (10 mg/kg/day) were given by gavage to streptozotocin-induced diabetic rats for 4 weeks. Streptozotocin (65 mg/kg, i.p.) significantly increased NADPH oxidase, vascular call adhesion molecule-1 (VCAM-1), and osteopontin mRNA levels in the aorta, as determined by reverse transcription (RT)-polymerase chain reaction (PCR). Immunohistochemical analysis revealed that the expression of osteopontin protein was also enhanced in the streptozotocin-injected rat aorta. Pioglitazone or bezafibrate attenuated the streptozotocin-induced increase in the expression of NADPH oxidase and VCAM-1 mRNA. The enhanced expression of osteopontin gene and protein induced by streptozotocin was suppressed by pioglitazone, whereas treatment with bezafibrate had no effect on the expression of osteopontin. We also demonstrated that pioglitazone or bezafibrate prevented the streptozotocin-induced increase in angiotensin converting enzyme (ACE) gene and protein content, by the means of RT-PCR and Western blotting. On the other hand, the treatment of pioglitazone or bezafibrate in the present study did not affect glucose tolerance, serum insulin or lipid level in streptozotocin-induced diabetic rats. These results suggest that the direct anti-oxidant and anti-inflammatory effects of PPARs ligands in the aorta of streptozotocin-induced diabetic rats were not likely to have been mediated by the normalization of glucose or lipid metabolism, but instead these salutary effects appear to have been associated with the inhibition of the expression of ACE. In addition, pioglitazone appeared to be more effective on the suppression of osteopontin expression compared with bezafibrate.
Keywords: Peroxisome proliferator-activated receptors; Diabetes; Angiotensin converting enzyme;

Effects of postconditioning of adenosine and acetylcholine on the ischemic isolated rat ventricular myocytes by Jun Lu; Wei-Jin Zang; Xiao-Jiang Yu; Bing Jia; Alzbeta Chorvatova; Lei Sun (133-139).
In this study, protective effects of adenosine and acetylcholine-induced postconditioning were investigated on the contractile function of the ischemic isolated rat ventricular myocytes. A video-based edge-detection system was used to monitor single ventricular myocytes contraction. Adenosine and acetylcholine were administrated for 6 min before ischemia as preconditioning, or 15 min after ischemia as postconditioning. Adenosine and acetylcholine receptor antagonists and mitoKATP inhibitor were used to analyze pathways underlying the effects on postconditioning. Results: (1) The peak shortening of ischemic heart cells was improved by both adenosine and acetylcholine during preconditioning (84.72 ± 5.34% and 68.61 ± 8.10% vs. control: 8.43 ± 5.35% of the pre-ischemia value), as well as postconditioning (76.47 ± 7.87% and 57.48 ± 6.97% vs. control: 8.43 ± 5.35% of the pre-ischemia value) and the effects of preconditioning and postconditioning were comparable. More datum in the normal text. (2) Observed effects of adenosine and acetylcholine postconditioning were missing in the presence of adenosine A1 receptor and muscarinic M2 receptor antagonists, respectively. (3) Adenosine and acetylcholine-induced postconditioning was also blocked by mitoKATP antagonist. These results suggest that both adenosine and acetylcholine protect the contractile function of ischemic heart cells to a similar extent during preconditioning and postconditioning. The postconditioning of adenosine and acetylcholine is relative to the adenosine A1 and muscarinic M2 receptors, respectively. MitoKATP is implicated in the postconditioning of both acetylcholine and adenosine.
Keywords: Acetylcholine; Adenosine; Ischemia; Myocyte; Postconditioning;

MEN15596, a novel nonpeptide tachykinin NK2 receptor antagonist by Cecilia Cialdai; Manuela Tramontana; Riccardo Patacchini; Alessandro Lecci; Claudio Catalani; Rose-Marie Catalioto; Stefania Meini; Claudio Valenti; Maria Altamura; Sandro Giuliani; Carlo Alberto Maggi (140-148).
The pharmacological profile of MEN15596 or (6-methyl-benzo[b]thiophene-2-carboxylic acid [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide), a novel potent and selective tachykinin NK2 receptor antagonist endowed with oral activity, is described. At the human recombinant tachykinin NK2 receptor, MEN15596 showed subnanomolar affinity (pK i 10.1) and potently antagonized (pK B 9.1) the neurokinin A-induced intracellular calcium release. MEN15596 selectivity for the tachykinin NK2 receptor was assessed by binding studies at the recombinant tachykinin NK1 (pK i 6.1) and NK3 (pK i 6.4) receptors, and at a number of 34 molecular targets including receptors, transporters and ion channels. In isolated smooth muscle preparations MEN15596 showed a marked species selectivity at the tachykinin NK2 receptor with the highest antagonist potency in guinea-pig colon, human and pig bladder (pK B 9.3, 9.2 and 8.8, respectively) whereas it was three orders of magnitude less potent in the rat and mouse urinary bladder (pK B 6.3 and 5.8, respectively). In agreement with binding experiments, MEN15596 showed low potency in blocking selective NK1 or NK3 receptor agonist-induced contractions of guinea-pig ileum preparations (pA2  ≤ 6). In anaesthetized guinea-pigs, MEN15596 inhibited in a dose-related and persistent manner colon contractions induced by the selective tachykinin NK2 receptor agonist, [βAla8]neurokinin A(4–10) (3 nmol/kg i.v.), either after intravenous (ED50 0.18 μmol/kg), intraduodenal (ED50 3.16 μmol/kg) or oral administration (10–30 μmol/kg) without affecting, at 3 μmol/kg, i.v., the colonic contractions produced by the NK1 receptor selective agonist [Sar9]substance P sulfone (3 nmol/kg i.v.). In addition MEN15596 was effective in inhibiting bronchoconstriction produced by i.v. administration of [βAla8]neurokinin A(4–10). Overall the results indicate that MEN15596 is a potent and selective tachykinin NK2 receptor antagonist possessing high affinity and potency for guinea-pig, pig and human receptor, long duration of action in in vivo experiments and good oral bioavailability.
Keywords: Tachykinins; Neurokinin A; NK2 receptor antagonist; Oral activity; Colon contraction; Gastrointestinal disorders; Bronchoconstriction;

Effects of 3-aminobenzamide, an inhibitor of poly (ADP-ribose) polymerase, in a mouse model of acute pancreatitis induced by cerulein by Emanuela Mazzon; Tiziana Genovese; Rosanna Di Paola; Carmelo Muià; Concetta Crisafulli; Giuseppe Malleo; Emanuela Esposito; Rosaria Meli; Edoardo Sessa; Salvatore Cuzzocrea (149-156).
Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA, plays an important role in the colon injury associated with experimental colitis. The aim of the present study was to examine the effects of 3-aminobenzamide (3-AB), an inhibitor of PARP activity, in the development of acute pancreatitis caused by cerulein in mice. Intraperitoneal injection of cerulein in mice resulted in severe, acute pancreatitis characterized by oedema, neutrophil infiltration and necrosis and elevated serum levels of amylase and lipase. Infiltration of pancreatic and lung tissue with neutrophils (measured as increase in myeloperoxidase activity) was associated with enhanced expression of the intercellular adhesion molecule-1 (ICAM-1) and P-selectin. Immunohistochemical examination demonstrated a marked increase in the staining (immunoreactivity) for transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF) in the pancreas of cerulein-treated mice in comparison to sham-treated mice. Acute pancreatitis in vehicle-treated mice was also associated with a significant mortality (40% survival at 5 days after cerulein administration). In contrast, (1) the degree of pancreatic inflammation and tissue injury (histological score), (2) upregulation/formation of ICAM-1 and P-selectin, (4) neutrophils infiltration and (5) the expression of TGF-β and VEGF was markedly reduced in pancreatic tissue obtained from cerulein-treated mice which have been treated with 3-AB. These findings provide the evidence that PARP inhibition reduce the degree of pancreas injury caused by acute pancreatitis induced by cerulein administration.
Keywords: PARP [Poly (ADP-ribose) polymerase]; Pancreatitis; Adhesion molecules; Inflammation; Neutrophil infiltration;

β-endorphin differentially affects inflammation in two inbred rat strains by Stanislava Stanojević; Katarina Mitić; Vesna Vujić; Vesna Kovačević-Jovanović; Mirjana Dimitrijević (157-165).
It has been shown that inflammation of rat paws elicits accumulation of opioid peptide β-endorphin-containing immune cells in the inflamed subcutaneous tissue, contributing to immunocyte-produced pain suppression. However, the possible mechanisms involved in the pharmacological application of β-endorphin in rat paw inflammation have not been investigated. The present study was set up to explore the effects of intraplantar injection of β-endorphin on Concanavalin A-induced paw edema in two inbred rat strains, Albino Oxford (AO) and Dark Agouti (DA). Both high dose-induced suppression and low dose-induced potentiation of edema development in AO and DA rats, respectively, were blocked with antagonists specific for δ (naltrindole) and κ (nor-binaltorphimine) opioid receptors. β-endorphin in vitro decreased phagocytosis and increased nitric oxide (NO) production in air pouch granulocytes obtained from AO rats. However, in cells from DA rat strain β-endorphin modulated both phagocytosis and NO production in a concentration-dependent manner. It could be concluded that the strain-dependent opposing effects of β-endorphin on paw inflammation are mediated through δ and κ opioid receptors and probably involve changes in the production of reactive oxygen species by inflammatory cells. Our results point to the importance of genotype for pharmacological manipulations and the development of inflammation.
Keywords: Paw edema; β-endorphin; Granulocytes; Phagocytosis; NO (nitric oxide) production; μ, δ, κ opioid receptors; AO (Albino Oxford) rat; DA (Dark Agouti) rat;

Cryptotanshinone inhibits cyclooxygenase-2 enzyme activity but not its expression by Dao-Zhong Jin; Lin-Lin Yin; Xin-Quan Ji; Xing-Zu Zhu (166-172).
Cyclooxygenase-2 (COX-2) is a key enzyme that catalyzes the biosynthesis of prostaglandins from arachidonic acid and plays a critical role in some pathologies including inflammation, neurodegenerative diseases and cancer. Cryptotanshinone is a major constituent of tanshinones, which are extracted from the medicinal herb Salvia miltiorrhiza Bunge, and has well-documented antioxidative and anti-inflammatory effects. This study confirmed the remarkable anti-inflammatory effect of cryptotanshinone in the carrageenan-induced rat paw edema model. Since the action of cryptotanshinone on COX-2 has not been previously described, in the present study, we further examined the effect of cryptotanshinone on cyclooxygenase activity in the exogenous arachidonic acid-stimulated insect sf-9 cells, which highly express human COX-2 or human COX-1, and on cyclooxygenases expression in human U937 promonocytes stimulated by lipopolysaccharide (LPS) plus phorbolmyristate acetate (PMA). Cryptotanshinone reduced prostaglandin E2 synthesis and reactive oxygen species generation catalyzed by COX-2, without influencing COX-1 activity in cloned sf-9 cells. In PMA plus LPS-stimulated U937 cells, cryptotanshinone had negligible effects on the expression of COX-1 and COX-2, at either a mRNA or protein level. These results demonstrate that the anti-inflammatory effect of cryptotanshinone is directed against enzymatic activity of COX-2, not against the transcription or translation of the enzyme.
Keywords: Cryptotanshinone; Cyclooxygenase-2; Prostaglandin E2; Reactive oxygen species;

Aldose reductase inhibitor zopolrestat restores allergic hyporesponsiveness in alloxan-diabetic rats by Vinicius F. Carvalho; Emiliano O. Barreto; Magda F. Serra; Renato S.B. Cordeiro; Marco A. Martins; Zuleica Bruno Fortes; Patrícia M.R. e Silva (173-178).
This study was undertaken to investigate the role of the aldose reductase in the refractoriness of diabetic rats to allergic inflammation. Wistar rats were actively sensitized with a mixture of Al(OH)3 plus ovalbumin and intrapleurally challenged with ovalbumin, 14 days later. Diabetes was induced by intravenous injection of alloxan into fasted rats, 7 days before sensitization, and the aldose reductase inhibitor zopolrestat was administered after 3 days of diabetes induction, once a day during 18 consecutive days. The treatment with zopolrestat restored antigen-induced protein extravazation and mast cell degranulation in the pleural cavity of diabetic sensitized rats. Zopolrestat also significantly reversed the suppression in the increase of total and specific levels of serum immunoglobulin E (IgE) noted in sensitized animals under conditions of diabetes. In addition, we noted that the drop in the pleural mast cell numbers as well as the increase in serum corticosterone levels in diabetic rats were inhibited by the drug. Our findings show that zopolrestat restored the hyporesponsiveness of diabetic rats to antigen provocation, in parallel with impairment of alloxan-induced mast cell depletion and hypercorticolism, indicating that polyol pathway activity seems to play an important role in these phenomena.
Keywords: Diabetes; Allergy; Aldose reductase; Mast cells;

Verapamil-sensitive Ca2+ channel regulation of Th1-type proliferation of splenic lymphocytes induced by Walker 256 tumor development in rats by Giovanna R. Degasperi; Karina G. Zecchin; Jiri Borecký; Maria A. Cruz-Höfling; Roger F. Castilho; Lício A. Velloso; Fernando Guimarães; Anibal E. Vercesi (179-184).
Recently, we demonstrated that verapamil, an L-type Ca2+ channel blocker, inhibits the activation of splenic lymphocytes during Walker 256 ascitic tumor development in adult rats. In the present study we have analyzed the changes in spleen size, splenic lymphocyte proliferation, white pulp organization and relative size as well as food intake, and levels of blood haemoglobin in Walker 256 tumor bearing rats. These rats displayed a spleen enlargement associated with a significant increase in white pulp area and TCD8+ lymphocyte proliferation. Levels of interferon-gamma, but not of interleukin-10, were elevated in tumor bearing rats, indicating a Th1-type immune response. These manifestations were accompanied by reduced food intake and anaemia. Treatment of tumor bearing rats with verapamil avoided spleen enlargement and increased expression of cytokines, as well as the splenic TCD8+ lymphocyte proliferation. In addition, verapamil treatment promoted an exacerbation of the anorexia and anaemia caused by Walker tumor development. No such effect was observed in control rats treated with verapamil. Taken together, these findings suggest that verapamil inhibits the immune response to cancer, resulting in an increase of the systemic effects induced by Walker 256 tumor.
Keywords: Cancer; Immune response; L-type Ca2+ channel; Verapamil; Walker 256 tumor;

YM440, a novel hypoglycemic agent, protects against nephropathy in Zucker fatty rats via plasma triglyceride reduction by Ryosuke Nakano; Eiji Kurosaki; Akiyoshi Shimaya; Satoru Kajikawa; Masayuki Shibasaki (185-191).
The novel hypoglycemic agent, YM440 ((Z)-1,4-bis{4-[(3,5-dioxo-1,2,4-oxadiazolidin-2-yl) methyl] phenoxy}but-2-ene) is a ligand of the peroxisome proliferator-activated receptor, (PPAR) γ. YM440 has been shown to counteract insulin resistance in diabetic rodent models. However, it is not clear whether this compound has a significant effect on hyperlipidemia in vivo. Hyperlipidemia has been reported to be a risk factor for the early development of renal disease. The aim of this study is to examine the effects of chronic treatment with YM440 on hyperlipidemia and renal injury in obese Zucker fatty (ZF) rats. Treatment of 8-week-old ZF rats with YM440 (100 mg/kg/day) for 16 weeks decreased plasma triglyceride and cholesterol concentrations. YM440 markedly reduced the rate of progression of both albuminuria and proteinuria. YM440 normalized urinary N-acetyl-β-d-glucosaminidase (NAG) activity, which is a marker for renal proximal tubular damage, and ameliorated the rise in systolic blood pressure compared to the vehicle control. YM440 also blocked the development of nephromegaly. Histological analyses revealed that both glomerular area expansion and tubular cast accumulation gradually lessened in YM440-treated ZF rats. Regression analyses between the plasma triglyceride levels and the renal parameters (urinary protein excretion and albumin excretion) indicated that the renal parameters correlated positively with the plasma triglyceride levels. In conclusion, the hypolipidemic effects of YM440 prevent renal injury in ZF rats. YM440 might be useful for preventing the early development of diabetic nephropathy in subjects with type 2 diabetes by ameliorating metabolic control problems.
Keywords: Insulin resistance; Diabetic nephropathy; Peroxisome proliferator-activated receptor γ; YM440; Zucker fatty rat; Hyperlipidemia;

Author index (193-195).

Keyword index (196-200).