European Journal of Pharmacology (v.566, #1-3)

Coenzyme Q10 prevents high glucose-induced oxidative stress in human umbilical vein endothelial cells by Hiroshi Tsuneki; Naoto Sekizaki; Takashi Suzuki; Shinjiro Kobayashi; Tsutomu Wada; Tadashi Okamoto; Ikuko Kimura; Toshiyasu Sasaoka (1-10).
Hyperglycemia-induced oxidative stress plays a crucial role in the pathogenesis of vascular complications in diabetes. Although some clinical evidences suggest the use of an antioxidant reagent coenzyme Q10 in diabetes with hypertension, the direct effect of coenzyme Q10 on the endothelial functions has not been examined. In the present study, we therefore investigated the protective effect of coenzyme Q10 against high glucose-induced oxidative stress in human umbilical vein endothelial cells (HUVEC). HUVEC exposed to high glucose (30 mM) exhibited abnormal properties, including the morphological and biochemical features of apoptosis, overproduction of reactive oxygen species, activation of protein kinase Cβ2, and increase in endothelial nitric oxide synthase expression. Treatment with coenzyme Q10 strongly inhibited these changes in HUVEC under high glucose condition. In addition, coenzyme Q10 inhibited high glucose-induced cleavage of poly(ADP-ribose) polymerase, an endogenous caspase-3 substrate. These results suggest that coenzyme Q10 prevents reactive oxygen species-induced apoptosis through inhibition of the mitochondria-dependent caspase-3 pathway. Moreover, consistent with previous reports, high glucose caused upregulation of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in HUVEC, and promoted the adhesion of U937 monocytic cells. Coenzyme Q10 displayed potent inhibitory effects against these endothelial abnormalities. Thus, we provide the first evidence that coenzyme Q10 has a beneficial effect in protecting against the endothelial dysfunction by high glucose-induced oxidative stress in vitro.
Keywords: Coenzyme Q10; Endothelial cell; Apoptosis; Adhesion; Glucose; Diabetes;

Inhibition of the α9α10 nicotinic cholinergic receptor by neramexane, an open channel blocker of N-methyl-d-aspartate receptors by Paola V. Plazas; Jessica Savino; Sebastian Kracun; María E. Gomez-Casati; Eleonora Katz; Christopher G. Parsons; Neil S. Millar; Ana B. Elgoyhen (11-19).
In this study we report the effects of neramexane, a novel amino-alkyl-cyclohexane derivative that is a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, on recombinant rat α9α10 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes. We compared its effects with those of memantine, a well-studied pore blocker of NMDA receptors, currently used in therapeutics for the treatment of Alzheimer's disease. Our results indicate that both compounds block acetylcholine-evoked responses at micromolar concentrations with a rank order of potency of neramexane > memantine, P  < 0.05. Block by neramexane of acetylcholine responses was not overcome at high concentrations of the agonist, indicative of a non-competitive inhibition. The lack of interaction of neramexane with the ligand binding domain was confirmed by radioligand binding experiments in transfected tsA201 cells. Moreover, block did not involve an increase in desensitization kinetics, it was independent of the resting potential of the membrane at low concentrations of neramexane and slightly voltage-dependent at concentrations higher than 1 μM. Finally, clinically-relevant concentrations of neramexane blocked native α9α10-containing nicotinic acetylcholine receptors of rat inner hair cells, thus demonstrating a possible in vivo relevance in potentially unexplored therapeutic areas.
Keywords: Acetylcholine; Neramexane; N-methyl-d-aspartate receptor antagonist; Nicotinic acetylcholine receptors; Hair cells;

Platelet activation triggered by Chlamydia pneumoniae is antagonized by 12-lipoxygenase inhibitors but not cyclooxygenase inhibitors by Hanna Kälvegren; Johanna Andersson; Magnus Grenegård; Torbjörn Bengtsson (20-27).
Chlamydia pneumoniae is a respiratory pathogen that has been linked to cardiovascular disease. We have recently shown that C. pneumoniae activates platelets, leading to oxidation of low-density lipoproteins. The aim of the present study was to evaluate the inhibitory effects of different pharmacological agents on platelet aggregation and secretion induced by C. pneumoniae.Platelet interaction with C. pneumoniae was studied by analyzing platelet aggregation and ATP-secretion with Lumi-aggregometry.Platelet aggregation and ATP-secretion induced by C. pneumoniae was markedly inhibited by the NO-donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP), an effect that was counteracted by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Pre-treatment of platelets with the 12-lipoxygenase (12-LOX) inhibitors cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) and 5,6,7-trikydroxyflavone (baicalein) completely blocked the activation, whereas the cyclooxygenase (COX) inhibitors 2-acetyloxybenzoic acid (aspirin) and (8E)-8-[hydroxy-(pyridin-2-ylamino)methylidene]-9-methyl-10,10-dioxo-10$l^(6)thia-9-azabicyclo[4.4.0]deca-1,3,5-trien-7-one (piroxicam) had no inhibitory effects. Opposite to C. pneumoniae-induced activation, platelets stimulated by collagen were inhibited by the COX-inhibitors but were unaffected by the 12-LOX-inhibitors. The platelet activating factor (PAF) antagonist Ginkgolide B blocked the C. pneumoniae-induced platelet activation, whereas the responses to collagen were unaffected. Furthermore, the P2Y1 and P2Y12 purinergic receptor antagonists 2'-deoxy-N 6-methyladenosine 3',5'-bisphosphate (MRS2179) and N(6)-(2-methyl-thioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP (cangrelor) inhibited the aggregation and secretion caused by C. pneumoniae.It is well-known that the efficacy of COX inhibitors in the prevention and treatment of cardiovascular disease varies between different patients, and that patients with low responses to aspirin have a higher risk to encounter cardiovascular events. The findings in this study showing that platelets stimulated by C. pneumoniae are unaffected by COX inhibitors but sensitive to 12-LOX inhibitors, may thus be of importance in future management of atherosclerosis and thrombosis.
Keywords: Chlamydia pneumoniae; Platelet; Aggregation; Lipoxygenase; Cyclooxygenase;

Endothelin-1 decreases [Ca2+] i via Na+/Ca2+ exchanger in CHO cells stably expressing endothelin ETA receptor by Takahiro Horinouchi; Arata Nishimoto; Tadashi Nishiya; Lingyun Lu; Emi Kajita; Soichi Miwa (28-33).
Endothelin ETA receptor couples to Gq/11 protein that transduces a variety of receptor signals to modulate diverse cellular responses including Ca2+ mobilization. Stimulation of endothelin ETA receptor with endothelin-1 is generally believed to induce an increase in intracellular Ca2+ concentration ([Ca2+] i ) via Gq/11 protein. Here we provide the first convincing evidence that endothelin-1 elicited Gq/11 protein-dependent and -independent ‘decrease’ in [Ca2+] i via Na+/Ca2+ exchanger (NCX) in Chinese hamster ovary (CHO) cells stably expressing human endothelin ETA receptor. In the cells treated with 1 μM thapsigargin, an inhibitor of endoplasmic Ca2+ pump, that induces an increase in [Ca2+] i via capacitative Ca2+ entry, endothelin-1 induced a decrease in [Ca2+] i which was partially inhibited by YM-254890, a specific inhibitor of Gq/11, indicating that Gq/11-dependent and independent pathways are involved in the decrease. The endothelin-1-induced decrease in [Ca2+] i was markedly suppressed by 3′,4′-dichlorobenzamil hydrochloride, a potent NCX inhibitor, and also by a replacement of extracellular Na+ with Li+, which was not transported by NCX, indicating a major role of NCX operating in the forward mode in the endothelin-1-induced decrease in [Ca2+] i . Molecular approach with RT-PCR demonstrated the expression of mRNA for NCX1, NCX2 and NCX3. These results suggest that stimulation of endothelin ETA receptor with endothelin-1 activates the forward mode NCX through Gq/11-dependent and -independent mechanisms: the NCX exports Ca2+ out of the cell depending on Na+ gradient across the cell membrane, resulting in the decrease in [Ca2+] i .
Keywords: Endothelin-1; Endothelin receptor; Gq/11 protein; Na+/Ca2+ exchanger; Intracellular free Ca2+ concentration;

Is digitalis compound-induced cardiotoxicity, mediated through guinea-pig cardiomyocytes apoptosis? by Margarita Ramirez-Ortega; Gabriela Zarco; Vilma Maldonado; Jose F. Carrillo; Pilar Ramos; Guillermo Ceballos; Jorge Melendez-Zajgla; Noemí Garcia; Cecilia Zazueta; Jose Chanona; Jorge Suarez; Gustavo Pastelin (34-42).
Our aim in performing this study was to analyze in vivo the cell death mechanism induced by toxic doses of digitalis compounds on guinea-pig cardiomyocytes. We analyzed three study groups of five male guinea pigs each. Guinea pigs were intoxicated under anesthesia with ouabain or digoxin (at a 50–60% lethal dose); the control group did not receive digitalis. A 5-hours period elapsed before guinea pig hearts were extracted to obtain left ventricle tissue. We carried out isolation of mitochondria and cytosol, cytochrome c and caspase-3 and -9 determination, and electrophoretic analysis of nuclear DNA. TdT-mediated DUTP-X nick end labeling (TUNEL) reaction was performed in histologic preparations to identify in situ apoptotic cell death. Ultrastructural analysis was performed by electron microscopy. Electrophoretic analysis of DNA showed degradation into fragments of 200–400 base pairs in digitalis-treated groups. TUNEL reaction demonstrated the following: in the control group, < 10 positive nuclei per field; in the digoxin-treated group, 2–14 positive nuclei per field, while in the ouabain-treated group counts ranged from 9–30 positive nuclei per field. Extracts from ouabain-treated hearts had an elevation of cytochrome c in cytosol and a corresponding decrease in mitochondria; this release of cytochrome c provoked activation of caspase-9 and -3. Electron microscopy revealed presence of autophagic vesicles in cytoplasm of treated hearts. Toxic dosages of digitalis at 50–60% of the lethal dose are capable of inducing cytochrome c release from mitochondria, processing of procaspase-9 and -3, and DNA fragmentation; these observations are mainly indicative of apoptosis, although a mixed mechanism of cell death cannot be ruled out.
Keywords: Apoptosis; Myocytes; Ouabain; Digoxin; Caspase-3; Caspase-9; Cytochrome c;

4-hydroxy nimesulide effects on mitochondria and HepG2 cells. A comparison with nimesulide by Clayton S. Freitas; Daniel J. Dorta; Gilberto L. Pardo-Andreu; Cezar R. Pestana; Valeria G. Tudella; Fábio E. Mingatto; Sérgio A. Uyemura; Antonio C. Santos; Carlos Curti (43-49).
We previously reported that the nonsteroidal anti-inflammatory drug, nimesulide (N-[4-nitro-2-phenoxyphenyl]-methanesulfonamide), is an uncoupler and oxidizes NAD(P)H in isolated rat liver mitochondria, triggering mitochondrial Ca2+ efflux or, if this effect is inhibited, eliciting mitochondrial permeability transition (Mingatto et al., Br. J. Pharmacol. 131:1154–1160, 2000). We presently demonstrated that nimesulide's hydroxylated metabolite (4-hydroxy nimesulide) lacks the uncoupling property of the parent drug, while keeping its ability to oxidize mitochondrial NAD(P)H. In the presence of 10 μM Ca2+, low (5–50 μM) concentrations of 4-hydroxy nimesulide elicited mitochondrial permeability transition, as assessed by cyclosporin A-sensitive mitochondrial swelling, associated with mitochondrial Ca2+ efflux/membrane potential dissipation (Δψ), apparently occurring on account of the oxidation of mitochondrial protein thiols; no involvement of reactive oxygen species was observed. While nimesulide (0.5 or 1 mM, 30 h incubation) did not lead to significant HepG2 cell death, 4-hydroxy nimesulide caused a low extent (∼ 15%) of cell necrosis, partly prevented by cyclosporine A, suggesting the involvement of mitochondrial permeability transition. Both nimesulide and 4-hydroxy nimesulide caused NADPH oxidation and Δψ dissipation in HepG2 cells. Because such Δψ dissipation induced by the metabolite was almost completely inhibited by cyclosporine A, it probably results from the mitochondrial permeability transition. Therefore, mitochondrial permeability transition, in apparent association with NAD(P)H oxidation, constitutes the most probable cause of HepG2 cell death elicited by 4-hydroxy nimesulide.
Keywords: Nonsteroidal anti-inflammatory drugs; Nimesulide; 4-hydroxy nimesulide; Liver mitochondria; Mitochondrial permeability transition; Permeability transition pores;

Urine osmolality, cyclic AMP and aquaporin-2 in urine of patients under lithium treatment in response to water loading followed by vasopressin administration by Ingeborg Wilting; Ruben Baumgarten; Kris L.L. Movig; Jan van Laarhoven; Alfred J. Apperloo; Willem A. Nolen; Eibert R. Heerdink; Nine V.A.M. Knoers; Antoine C.G. Egberts (50-57).
Lithium is the drug that is most frequently associated with acquired nephrogenic diabetes insipidus (NDI). The exact mechanism of lithium-induced NDI in man is unknown. The aim of the present study was to investigate the kidney response to minimal and maximal stimulation of the kidney urine concentrating mechanism by measuring urine osmolality, and urine levels of cAMP and AQP-2 in urine of patients under long-term lithium treatment.Twenty patients under long-term lithium treatment were included. The kidney urinary 3′,5′-cyclic adenosine monophosphate (cyclic AMP), aquaporin-2 levels and urine osmolality were determined during a situation of minimal kidney urine concentrating activity (induced by water loading) and during a situation following maximal stimulation of kidney urine concentrating activity (induced by 1-desamino-8-d-arginine-vasopressin).Patients were classified as NDI, partial NDI and non-NDI based on maximal reached urine osmolality. The partial correlation (r) between urinary cyclic AMP levels (mol/l) and urine osmolality was 0.94 (P  < 0.001). No significant correlation was observed between urinary aquaporin-2 levels (mol/mol creatinine) and osmolality nor between urinary cyclic AMP and aquaporin-2 levels. The rise in urinary cyclic AMP but not aquaporin-2 levels upon 1-desamino-8-d-arginine-vasopressin administration after water loading significantly differed between the three categories, decreasing with increasing NDI category.In conclusion we found that in lithium-induced kidney urine concentrating deficit in man, the cyclic AMP generation in response to 1-desamino-8-d-arginine-vasopressin administration after water loading, is impaired. It remains to be elucidated whether principal cells, G-proteins or adenylate cyclase e.g. are the major targets for the mechanism underlying lithium-induced NDI in man.
Keywords: Lithium; Cyclic AMP; Aquaporin-2; Nephrogenic diabetes insipidus; Vasopressin;

Comparative effects of flavonoids on oxidant scavenging and ischemia-reperfusion injury in cardiomyocytes by Wei-Tien Chang; Zuo-Hui Shao; Jun-Jie Yin; Sangeeta Mehendale; Chong-Zhi Wang; Yimin Qin; Juan Li; Wen-Jone Chen; Chiang-Ting Chien; Lance B. Becker; Terry L. Vanden Hoek; Chun-Su Yuan (58-66).
Since flavonoids scavenge reactive oxygen species, they may potentially protect against ischemia/reperfusion injury. This study compared the scavenging capacity of specific flavonoids towards different reactive oxygen species. Whether the differential oxidant scavenging capacity correlated with their protective efficacy in ischemia/reperfusion injury of cardiomyocytes was determined. The free radical scavenging capacity of five flavonoids (wogonin, baicalin, baicalein, catechin and procyanidin B2) was analyzed using electron spin resonance spectrometry for 3 radicals: 1,1-diphenyl-2picrylhydrazyl (DPPH), superoxide and hydroxyl radical. A well-established chick cardiomyocyte model of ischemia (1 h)/reperfusion (3 h) was used to evaluate flavonoid-induced protection against ischemia/reperfusion injury in chronic treatment (pretreated 72 h and treated through ischemia/reperfusion) and acute treatment protocols (during ischemia/reperfusion or only at reperfusion). The cell viability was assessed by propidium iodide. The DPPH scavenging was most significant with catechin, followed by procyanidin B2, baicalein, baicalin, and wogonin. The superoxide scavenging was, similarly, most significant with catechin, followed by baicalein, procyanidin B2, and baicalin. For hydroxyl radical, only baicalein showed a significant scavenging capacity (> 50% reduction in ESR signal). For the cardiomyocyte studies, all flavonoids but wogonin showed protection against ischemia/reperfusion injury in the chronic treatment protocol. When flavonoids were administered only during ischemia/reperfusion, baicalein, procyanidin B2, and catechin significantly reduced cell death. If flavonoids were administered just at reperfusion, only baicalein and procyanidin B2 had protective effects, and the efficacy was less. Flavonoids possess specific but differential radical scavenging capacity, which, in conjunction with the timing of treatment, affects their protective efficacy in cardiomyocytes exposed to ischemia/reperfusion.
Keywords: Flavonoids; Reperfusion injury; Scavenging capacity; Superoxide; Hydroxyl radical;

Modulation of function of multidrug resistance associated-proteins by Kaempferia parviflora extracts and their components by Denpong Patanasethanont; Junya Nagai; Chie Matsuura; Kyoko Fukui; Khaetthareeya Sutthanut; Bung-orn Sripanidkulchai; Ryoko Yumoto; Mikihisa Takano (67-74).
In this study, the effects of extracts and flavone derivatives from the rhizome of Kaempferia parviflora on multidrug resistance associated-proteins (MRP)-mediated transport in A549 cells were examined. The cells employed express MRP1 and MRP2, but not P-glycoprotein. The cellular accumulation of calcein, an MRP substrate, was significantly increased by various MRP inhibitors without being affected by verapamil, a typical P-glycoprotein inhibitor. Ethanol and aqueous extracts from K. parviflora rhizome increased the accumulation of calcein and doxorubicin in A549 cells in a concentration-dependent manner. The inhibitory potency of the ethanol extract for MRP function was greater than that of the aqueous extract. Among six flavone derivatives isolated from K. parviflora rhizome, 5,7-dimethoxyflavone exhibited a maximal stimulatory effect on the accumulation of doxorubicin in A549 cells. The accumulation of doxorubicin was increased by four flavone derivatives without 5-hydroxy group, but not by the other two flavone derivatives with 5-hydroxy group. In addition, 5,7-dimethoxyflavone and 3,5,7,3′,4′-pentamethoxyflavone decreased resistance to doxorubicin in A549 cells. These findings indicate that extracts and flavone derivatives from the rhizome of K. parviflora suppress MRP function, and therefore may be useful as modulators of multidrug resistance in cancer cells.
Keywords: Multidrug resistance associated-protein; Kaempferia parviflora; Flavones; Multidrug resistance; A549 cells;

Treatment of pain with opioids is limited by their potential abuse liability. In an effort to develop analgesics without this side effect, a series of bivalent ligands containing a mu-opioid receptor agonist pharmacophore connected to a delta-opioid receptor antagonist pharmacophore through variable-length spacers (16–21 atoms) was synthesized. Members of this series [mu-opioid receptor (M)-delta-opioid receptor (D)-agonist (A)-antagonists (N): MDANs] are antinociceptive in the tail flick assay, but antinociceptive tolerance and physical dependence do not develop to ligands having spacers with 19–21 atoms. The current studies compared the rewarding properties of three bivalent ligands (MDAN-16, -19 and -21) and a mu-opioid receptor agonist (MA-19) to those of morphine in the conditioned place preference assay in mice after i.v. administration. Place preference developed to morphine and to MA-19, but not to the MDANs. The responses to MDAN-16 were highly variable, although place preference of borderline significance appeared to develop. Reinstatement was also evaluated after extinguishing morphine conditioned place preference; morphine and MA-19, but not the MDANs, reinstated morphine conditioned place preference. Taken together, these results suggest that the bivalents are less rewarding compared to morphine in opioid-naïve mice and do not induce reinstatement in previously morphine-preferring mice. The lack of a conditioned place preference response for MDAN-19 and -21, compared to the equivocal results with MDAN-16, suggests a minimum distance requirement between mu-opioid receptor and delta-opioid receptor recognition sites. This requirement may reflect the binding of MDAN-19 and -21 to mu-opioid receptor–delta-opioid receptor heterodimeric receptors that block reward but not antinociception.
Keywords: Opioid receptor; Heterodimer; Conditioned place preference; Antinociception; (Mice);

Neuroprotective effects of the novel polyethylene glycol-hemoglobin conjugate SB1 on experimental cerebral thromboembolism in rats by Hyeong-Jin Ji; Hee-Youl Chai; Sang-Soep Nahm; Junhee Lee; Gun Won Bae; Kwang Nho; Yun-Bae Kim; Jong-Koo Kang (83-87).
SunBio1 (SB1) is a novel polyethylene glycol-bovine hemoglobin conjugate. It is a small molecule that shows high oxygen-delivery capacity, and exhibits extended plasma half-life compared to hemoglobin alone, thus reducing renal toxicity. The aim of the present study was to evaluate potential neuroprotective effects of SB1 using a rat middle cerebral artery occlusion model. The middle cerebral artery of male Sprague–Dawley rats was occluded with a thrombotic blood clot and SB1 was administered via intra-arterial infusion 5 min after the operation. Brain tissue was harvested after 2 h, and cerebral infarct volumes were calculated from coronal sections stained with 2,3,5-triphenyltetrazolium chloride. Three to 6 days after the procedure, sub-groups of animals were subjected to an open field test and the Morris water maze to assess locomotor activity and learning/memory function. Thrombotic blood clots induced extensive brain infarction and edema; however, these were significantly reduced in SB1 treated animals. In addition, SB1 treatment increased locomotor activity in open field tests, and improved the learning/memory deficits caused by the thromboembolism. These results suggest that SB1 has neuroprotective effects against ischemic brain injury caused by thromboembolism.
Keywords: Hemoglobin; Polyethylene glycol conjugate; Stroke; Neuroprotection;

In the search for a selective delta-opioid receptor agonist, (−)-(1R,5R,9R)-5,9-dimethyl-2′-hydroxy-2-(6-hydroxyhexyl)-6,7-benzomorphan hydrochloride ((−)-NIH 11082) and the (+)-enantiomer were synthesized and tested. (−)-NIH 11082 displayed antinociceptive activity in the paraphenylquinone test (PPQ test) in male ICR mice [ED50  = 1.9 (0.7–5.3) mg/kg, s.c.] and showed little, if any, activity in the tail-flick and hot-plate assays. The (+)-enantiomer was essentially inactive indicating stereoselectivity. Opioid receptor subtype characterization studies indicated that naltrindole, a delta-opioid receptor antagonist, was potent versus the ED80 of (−)-NIH 11082 in the PPQ test [AD50  = 0.75 (0.26–2.20) mg/kg, s.c]. beta-Funaltrexamine and nor-binaltorphimine, selective mu- and kappa-receptor antagonists, respectively, were inactive versus the ED80 of (−)-NIH 11082. In rats with inflammation-induced pain, (−)-NIH 11082 produced antihyperalgesic effects that were attenuated by naltrindole. In morphine-dependent rhesus monkeys of both sexes, (−)-NIH 11082 neither substituted for morphine nor exacerbated withdrawal signs in the dose range of 4.0 to 32.0 mg/kg, s.c. Neither convulsions nor other overt behavioral signs were observed in any of the species tested. The results indicate that (−)-NIH 11082 has delta-opioid receptor properties.
Keywords: Selective delta opioid; (−)-(1R,5R,9R)-5,9-dimethyl-2′-hydroxy-2-(6-hydroxyhexyl)-6,7-benzomorphan hydrochloride ((−)-NIH 11082); Antinociception; Antihyperalgesia; (Mouse); (Rat); (Rhesus monkey);

Characterization of the antiparkinsonian effects of the new adenosine A2A receptor antagonist ST1535: Acute and subchronic studies in rats by Elisabetta Tronci; Nicola Simola; Franco Borsini; Nicoletta Schintu; Lucia Frau; Paolo Carminati; Micaela Morelli (94-102).
Antagonism of adenosine A2A receptor function has been proposed as an effective therapy in the treatment of Parkinson's disease. Thus, the study of new adenosine receptor antagonists is of great importance for the potential use of these drugs in clinical practice. The present study evaluated effects of the new preferential adenosine A2A receptor antagonist 2-butyl-9-methyl-8-(2H-1,2,3-triazol-2-yl)-9H-purin-6-ylamine (ST1535) in unilaterally 6-hydroxydopamine lesioned rats. Acute ST1535 dose-dependently potentiated contralateral turning behaviour induced by a threshold dose of l-3,4-dihydroxyphenylalanine (l-DOPA) (3 mg/kg i.p.), a classical test for antiparkinson drug screening. Subchronic (18 days, twice a day) ST1535 (20 mg/kg i.p.) +  l-DOPA (3 mg/kg i.p.) did not induce sensitization to turning behaviour or abnormal involuntary movements during the course of treatment, indicating a low dyskinetic potential of the drug. Moreover, while subchronic administration of a fully effective dose of l-DOPA (6 mg/kg i.p.) significantly increased GABA synthesizing enzyme glutamic acid decardoxylase (GAD67), dynorphin and enkephalin mRNA levels in the lesioned striatum, subchronic ST1535 (20 mg/kg i.p.) +  l-DOPA (3 mg/kg i.p.) did not modify any of these markers, although it induced a similar number of contralateral rotations at the beginning of treatment. Finally, acute administration of ST1535 (20 mg/kg i.p.) proved capable of reducing jaw tremors in tacrine model of Parkinson's disease tremor. Results showed that ST1535, in association with a low dose of l-DOPA, displayed antiparkinsonian activity similar to that produced by a full dose of l-DOPA without exacerbating abnormal motor side effects. Moreover, in agreement to other well characterized adenosine A2A receptor antagonists, ST1535 features antitremorigenic effects.
Keywords: Parkinson; Dyskinesia; Tremor; Dopamine; Neurodegenerative disease;

This research examines the effects of ethanolamine and other amino alcohols on the dynamics of acridine orange (AO), oxonol V, and [3H]-d-aspartic acid in synaptic preparations isolated from mammalian brain. Ethanolamine concentration-dependently enhanced AO release from synaptosomes. Similar effects were observed with methylethanolamine and dimethylethanolamine, but not choline. The enhancement of AO efflux by ethanolamine was independent of extrasynaptosomal calcium (in contrast to KCl-induced AO efflux), was unaffected by tetrodotoxin and did not involve depolarization of the synaptosomal plasma membrane. KCl was unable to release AO from synaptosomes following exposure to ethanolamine, however ethanolamine and other amino alcohols were found to enhance both basal and KCl-evoked release of [3H]-d-aspartic acid from synaptosomes. Using isolated synaptic vesicles we demonstrate that amino alcohols are able to 1) abolish the ATP-dependent intravesicular proton concentration (i.e. stimulate efflux of AO) in a similar way to carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2) increase the ATP-supported transvesicular membrane potential (i.e. quench oxonol V fluorescence) in contrast to CCCP and 3) enhance intravesicular uptake of [3H]-d-aspartic acid. These results suggest that positively charged, membrane impermeant amino alcohol species are generated within synaptic vesicles as they sequester protons. Cationic forms of these amino alcohols boost the transvesicular electrical potential which increases transmitter uptake into synaptic vesicles and facilitates enhancement of basal and evoked release of transmitter. Our data suggest a potential role for ethanolamine and related amino alcohols in the regulation of synaptic vesicle filling. These findings may also have relevance to neuropathophysiological states involving altered production of ethanolamine.
Keywords: Ethanolamine; Acridine orange; Oxonol V; [3H]-d-aspartate; Synaptosome; Synaptic vesicle; Mammalian brain;

Effects of imipramine on extracellular serotonin and noradrenaline concentrations in ACTH-treated rats by Yoshihisa Kitamura; Kozue Akiyama; Shinji Hashimoto; Kouhei Kitagawa; Hiromu Kawasaki; Kazuhiko Shibata; Kazuaki Shinomiya; Katsuya Suemaru; Hiroaki Araki; Toshiaki Sendo; Yutaka Gomita (113-116).
We investigated the effect of imipramine on extracellular serotonin (5-HT) and noradrenaline concentrations in the medial prefrontal cortex of rats treated with adrenocorticotropic hormone (ACTH) for 14 days using in vivo microdialysis. Chronic ACTH treatment did not affect basal extracellular 5-HT and noradrenaline concentrations compared with chronic saline treatment. Acute imipramine treatment plus chronic ACTH treatment significantly increased extracellular 5-HT concentrations, compared with imipramine treatment alone. 8-hydroxy-2-di-n-propylamino tetralin (8-OH-DPAT), a 5-HT1A receptors full agonist, caused a significant decrease in extracellular 5-HT concentrations. However, its inhibitory effect was attenuated by the treatment with ACTH for 14 days. These findings suggest that chronic treatment with ACTH enhances the increasing effect release of 5-HT by imipramine through the desensitization of somatodendritic 5-HT1A autoreceptors.
Keywords: Imipramine; 8-hydroxy-2-di-n-propylamino tetralin; 5-HT1A receptor; Microdialysis; Adrenocorticotropic hormone;

Synergistic antinociception by the cannabinoid receptor agonist anandamide and the PPAR-α receptor agonist GW7647 by Roberto Russo; Jesse LoVerme; Giovanna La Rana; Giuseppe D'Agostino; Oscar Sasso; Antonio Calignano; Daniele Piomelli (117-119).
The analgesic properties of cannabinoid receptor agonists are well characterized. However, numerous side effects limit the therapeutic potential of these agents. Here we report a synergistic antinociceptive interaction between the endogenous cannabinoid receptor agonist anandamide and the synthetic peroxisome proliferator-activated receptor-α (PPAR-α) agonist 2-(4-(2-(1-Cyclohexanebutyl)-3-cyclohexylureido)ethyl)phenylthio)-2-methylpropionic acid (GW7647) in a model of acute chemical-induced pain. Moreover, we show that anandamide synergistically interacts with the large-conductance potassium channel (KCa1.1, BK) activator isopimaric acid. These findings reveal a synergistic interaction between the endocannabinoid and PPAR-α systems that might be exploited clinically and identify a new pharmacological effect of the BK channel activator isopimaric acid.
Keywords: PPAR-α; Cannabinoid; Pain; Palmitoylethanolamide; Isopimaric acid; Formalin;

Chronic 3,4-methylenedioxymethamphetamine treatment suppresses cell proliferation in the adult mouse dentate gyrus by Kyung-Ok Cho; Seul-Ki Kim; Gyu Seek Rhee; Seung Jun Kwack; Dae Hyun Cho; Ki-Wug Sung; Seong Yun Kim (120-123).
We investigated the influence of chronic 3,4-methylenedioxymethamphetamine (MDMA) treatment on cell proliferation in the adult dentate gyrus. Mice were orally treated with MDMA (1.25 mg/kg–40 mg/kg) or saline for 30 days. To label dividing cells, mice were given 5-bromo-2′-deoxyuridine (BrdU) for 4 days from the day after the last administration of MDMA, and their brains were examined 24 h later. MDMA dose-dependently induced a decrease in the number of BrdU-positive cells in the male and female dentate gyrus. Our results suggest that chronic exposure to MDMA suppresses cell proliferation in the dentate gyrus.
Keywords: MDMA; Ecstasy; Cell proliferation; Dentate gyrus; Hippocampus;

Clinical evidence suggests that nicotine reduces anxiety in stressful situations. In the present study, we investigated the effect of nicotine on restraint-enhanced anxiety-like behavior, c-Fos expression, an index of neuronal activation in the brain, and plasma corticosterone. Two-hour restraint stress-enhanced anxiety-like behavior in the elevated plus-maze (EPM) and nicotine hydrogen tartrate (0.25 mg/kg, i.p.) attenuated the stress-induced changes. Pretreatment with the centrally acting nicotinic antagonist, mecamylamine (2 mg/kg), blocked nicotine's effects. In addition, restraint led to significant increases of c-Fos expression in several brain regions related to stress or anxiety including paraventricular hypothalamic nucleus (PVN), lateral hypothalamic area (LH), central amygdaloid nucleus (CeA), medial amygdaloid nucleus (MeA) and cingulate and retrosplenial cortices (Cg/RS), paraventricular thalamic nucleus (PVT), and basolateral amygdaloid nucleus (BLA). Nicotine attenuated the restraint-induced expression of c-Fos in the PVN, LH, CeA, MeA, and Cg/RS, while leaving the BLA and PVT unaffected. In contrast, nicotine did not reverse the increased levels of plasma corticosterone induced by restraint. These findings suggest that nicotine may modify the stress-induced behavioral changes via regulating the neuronal activation in selected brain regions rather than affecting hypothalamo-pituitary-adrenocortical axis hormone responses.
Keywords: Nicotine; Stress; Anxiety; c-Fos; Corticosterone;

NIH 11082 produces anti-depressant-like activity in the mouse tail-suspension test through a delta-opioid receptor mechanism of action by Pattipati S. Naidu; Aron H. Lichtman; Carey C. Archer; Everett L. May; Louis S. Harris; Mario D. Aceto (132-136).
The present study examined the effects of NIH 11082 ((−)-(1R,5R,9R)-5,9-dimethyl-2′-hydroxy-2-(6-hydroxyhexyl)-6,7-benzomorphan hydrochloride), a benzomorphan analogue, in the mouse tail-suspension, an assay used to detect anti-depressant agents. NIH 11082 significantly decreased immobility time during tail-suspension, with a comparable magnitude as the tricyclic anti-depressant desipramine. Importantly, NIH 11082 failed to elicit convulsions or other overt behavioral signs of toxicity. The delta-opioid receptor antagonist naltrindole (AD50  = 2.0 mg/kg), but not the non-selective mu-opioid receptor antagonist naltrexone or the kappa-opioid receptor antagonist nor-BNI, blocked the effects of NIH 11082 in the tail-suspension test. These results reinforce the notion that delta-opioid receptor agonists can produce significant effects in a behavioral model used to screen anti-depressant drugs.
Keywords: Delta-opioid; Anti-depressant; Naltrindole; Tail-suspension test; Naltrexone; Nor-binaltorphimine hydrochloride (nor-BNI);

Functional properties of a novel mutant of staphylokinase with platelet-targeted fibrinolysis and antiplatelet aggregation activities by Hongshan Chen; Wei Mo; Yanling Zhang; Huabo Su; Janying Ma; Ruiming Yao; Shaoheng Zhang; Junbo Ge; Houyan Song (137-144).
The present study was performed to characterize the functional properties of RGD-SAK, a novel mutant of staphylokinase (SAK). Biochemical analysis indicated that RGD-SAK maintained the similar structure and the fibrinolytic function of SAK. Measurement of platelet binding activity in vitro demonstrated that RGD-SAK had a much higher affinity with platelets than SAK. In vitro platelet-rich clot lysis assay demonstrated that the engineered mutant outperformed the non-manipulated SAK. The time required for 50% platelet-rich clot lysis was reduced significantly across different concentrations of RGD-SAK comparing with SAK. Meanwhile, RGD-SAK was found to inhibit ADP-induced platelet aggregation in a concentration-dependent manner while SAK had negligible effect on platelet aggregation. In concordance, further study in a porcine coronary balloon injury model demonstrated the efficacy of RGD-SAK for the lysis of platelet-rich coronary blood clots and for the prevention of reocclusion after thrombolysis. These results suggested that RGD-SAK may serve as a potential thrombolytic agent with platelet-targeted fibrinolysis and antiplatelet aggregation activities.
Keywords: Staphylokinase; RGD motif; Platelet-targeted fibrinolysis; Antiplatelet activity;

Increases in vanilloid TRPV1 receptor protein and CGRP content during endotoxemia in rats by María Luz Orliac; Roxana N. Peroni; Tamara Abramoff; Isabel Neuman; Ernesto J. Podesta; Edda Adler-Graschinsky (145-152).
The aim of the present study was to determine whether the transient receptor potential vanilloid (TRPV1) receptor protein as well as the calcitonin gene-related peptide (CGRP) content could be enhanced after the i.p. administration of 5 mg/kg lipopolysaccharide (LPS) to Sprague–Dawley rats. In tongue tissue, used as a representative model of TRPV1 receptors expression, there was a significant increase in the abundance of TRPV1 receptor protein 6 h after LPS administration. In mesenteric arteries, the density of the CGRP-positive nerves as well as the release of CGRP induced by 10 μM anandamide was also significantly increased 6 h after LPS administration. The relaxant responses induced by anandamide in mesenteric beds isolated from either untreated or LPS-treated rats were abolished after a 2 h exposure to 10 μM capsaicin. Moreover, anandamide-induced relaxations of untreated mesenteries were potentiated by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 0.1 μM), but not by its inactive analogue 4α-phorbol (0.1 μM). The potentiation of anandamide effects caused by the PKC activator was accompanied by a significant increase in the overflow of CGRP induced by anandamide in the untreated rats. It is proposed that the overexpression of the TRPV1 receptors and the increased content of CGRP could contribute to the enhancement of anandamide effects during the endotoxemic shock. An eventual phosphorylation event linked to the overflow of CGRP could also participate in the enhanced relaxation caused by anandamide in endotoxemia.
Keywords: Anandamide; TRPV1; CGRP; PKC; Vasorelaxation; Septic shock;

Pretreatment with eplerenone reduces stroke volume in mouse middle cerebral artery occlusion model by Jun Iwanami; Masaki Mogi; Shoko Okamoto; Xin-Yu Gao; Jian-Mei Li; Li-Juan Min; Ayumi Ide; Kana Tsukuda; Masaru Iwai; Masatsugu Horiuchi (153-159).
Eplerenone, a mineralocorticoid receptor antagonist, is reported to be effective to prevent end-stage cardiovascular damage induced by aldosterone. However, the effect of eplerenone on brain damage is not fully understood. Here, we investigated whether pretreatment with eplerenone attenuates stroke size in mice subjected to middle cerebral artery occlusion. Middle cerebral artery occlusion with a microfilament technique induced focal ischemia, to approximately 25% of the total area in a coronal section of the brain. Treatment with eplerenone at a dose of 1.67 mg/g chow significantly reduced the ischemic area, ischemic volume, and neurological deficit, without a blood pressure-lowering effect. Laser-Doppler flowmetry analysis showed a decrease in surface cerebral blood flow in the peripheral region after 1 h of middle cerebral artery occlusion. This decrease was smaller in mice treated with eplerenone. Superoxide production evaluated by staining with dihydroethidium was attenuated in the ischemic area of the brain in eplerenone-treated mice. Taken together, our findings suggest that eplerenone has a protective effect on ischemic brain damage, at least partly due to improvement of cerebral blood flow in the penumbra and reduction of oxidative stress.
Keywords: Stroke; Aldosterone; Eplerenone; Blood flow; Oxidative stress;

Flow- and acetylcholine-induced dilatation in small arteries from rats with renovascular hypertension — effect of tempol treatment by Frank Holden Christensen; Edgaras Stankevicius; Thomas Hansen; Malene Munk Jørgensen; Vanesa Lopez Valverde; Ulf Simonsen; Niels Henrik Buus (160-166).
We investigated whether renovascular hypertension alters vasodilatation mediated by nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) and the influence of the superoxide dismutase mimetic tempol on vasodilatation. One-kidney one-clip hypertensive Sprague–Dawley rats, treated with either vehicle or tempol (from weeks 5 to 10 after placement of the clip), and uninephrectomized control rats were investigated. In renal hypertensive rats systolic blood pressure increased to 171 ± 6 mmHg (n  = 10), while in tempol-treated rats systolic blood pressure remained normal (139 ± 7 mmHg, n  = 5). In isolated pressurized mesenteric small arteries NO-mediated dilatation was obtained by increasing flow rate and EDHF-mediated dilatation by acetylcholine. In arteries from hypertensive rats, flow-induced dilatation was blunted, as compared to normotensive and tempol-treated rats, while acetylcholine-induced dilatation remained normal. Measured by dihydroethidium staining there was an increased amount of superoxide in arteries from vehicle-treated rats, but not from tempol-treated rats. Expression by immunoblotting of endothelial NO synthase and the NAD(P)H oxidase subunit p47phox remained unaffected by high blood pressure and tempol treatment. Simultaneous measurements of NO-concentration and relaxation were performed in isolated coronary arteries from the same animals. As compared to vehicle-treated rats, both acetylcholine-induced relaxation and NO-concentration increased in arteries from tempol-treated animals, while only the relaxation was improved by the NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In conclusion renovascular hypertension selectively inhibits flow-induced NO-mediated vasodilatation, while EDHF-type vasodilatation remains unaffected, suggesting that high blood pressure leads to increased generation of superoxide contributing to decreased NO bioavailability. Furthermore, the abnormal endothelium function can be corrected by tempol treatment, but this seems to involve mechanisms partly independent of NO.
Keywords: Nitric oxide; Endothelium; Renovascular hypertension; Tempol; NO microsensor;

Extracellular nucleotides regulate ion transport, mucociliary clearance as well as inflammatory properties of the airway epithelium by acting on P2 receptors. Cyclooxygenase-2 (COX-2) is a key enzyme involved in the synthesis of prostaglandins during inflammation. In this study, using calcium imaging, DNA microarray experiments, real-time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and prostaglandin E2 (PGE2) measurement, we show for the first time that ATP, UTP or INS365 compound (P2Y2 receptor agonists) up-regulate COX-2 expression by ∼ 3-fold and enhance the release of PGE2 in human A549 airway epithelial cells. Our data suggest that P2Y receptors may represent putative targets in airway inflammatory diseases.
Keywords: P2Y receptor; Airway epithelium; COX-2; Prostaglandin E2; Cystic fibrosis; Extracellular nucleotide;

Intracellular calcium stores in β-escin skinned rat and guinea-pig bladders by N. Tugba Durlu-Kandilci; Alison F. Brading (172-180).
Intracellular Ca2+ stores in rat and guinea-pig bladders and taenia caecum were studied in β-escin skinned smooth muscle strips. 30 min of skinning with 40 μM and 80 μM β-escin were the best parameters found to obtain good calcium response curves (10− 7–10− 4 M) in rat and guinea pig, respectively. Calmodulin (1 μM) increased the calcium contractions significantly. pCa 6 was used to load intracellular stores and application of carbachol (50 μM) in all tissues then only contracted the tissues in the presence of guanosine-5′-triphosphate (GTP; 100 μM). Inositol triphosphate (IP3; 50 μM), applied after pCa 6, contracted all tissues. Carbachol added after IP3 or heparin (1 mg/ml) no longer caused a contraction in any of them. In bladders, caffeine (30 mM) but not ryanodine (5 μM) prevented the subsequent carbachol contraction. A slowly rising contraction with carbachol was elicited after caffeine (30 mM) or ryanodine (5 μM) in the taenia and after ryanodine in the bladders. Caffeine (30 mM) suppressed the calcium response curves in all tissues. Procaine (30 mM) blocked the carbachol (50 μM) contractions in bladders but not in taenia. These results suggest that calcium induced calcium release (CICR) and IP3 induced calcium release (IICR) release calcium from a common store in bladder but two different compartments in taenia.
Keywords: β-escin skinned; Bladder; Taenia caecum; Intracellular calcium store; Carbachol;

Role of cyclin-dependent kinase 5 in capsaicin-induced cough by Junzo Kamei; Shun-suke Hayashi; Yoshiki Takahashi; Chihiro Nozaki (181-184).
The role of cyclin-dependent kinase 5 (Cdk5) in the capsaicin-induced cough reflex was examined in mice. Pretreatment with inhaled roscovitine, a selective Cdk5 inhibitor, at concentrations of 0.3 to 3 mM inhibited the number of capsaicin-induced coughs in a concentration-dependent manner. Pretreatment with inhaled roscovitine, at a concentration of 3 mM also slightly but significantly inhibited the number of citric acid-induced coughs. The number of capsaicin-induced coughs was significantly reduced when C-fibers were desensitized by the pretreatment with capsaicin. The number of citric acid-induced coughs was slightly but significantly reduced in capsaicin-pretreated mice as compared with that in naive mice. Although the inhalation of roscovitine at a concentration of 3 mM significantly reduced the number of citric acid-induced coughs in naive mice to the level observed in capsaicin-pretreated mice, roscovitine had no effect on the number of citric acid-induced coughs in capsaicin-pretreated mice. These results suggest that Cdk5-dependent factors are involved in C-fiber-mediated cough signaling.
Keywords: Cyclin-dependent kinase 5 (Cdk5); Cough reflex; Capsaicin; TRPV1; Substance P;

Action of GLP-1 (7-36) amide and exendin-4 on Suncus murinus (house musk shrew) isolated ileum by Sze Wa Chan; Jufang He; Ge Lin; John A. Rudd; Kouichi Yamamoto (185-191).
Glucagon-like peptide-1 (GLP-1) receptor agonists have been reported to modulate gastrointestinal motility but the mechanism is essentially unknown. In the present studies, we investigated the potency and mechanism of action of GLP-1 receptor ligands on the isolated ileum of Suncus murinus, an insectivore used in anti-emetic research. Ileal segments were mounted in organ baths containing Kreb's solution. Cumulative concentration–response curves to GLP-1 (7-36) amide (0.1–300 nM) and exendin-4 (0.1–100 nM) were constructed in the absence and presence of exendin (9-39) amide (0.3–3 nM). GLP-1 (7-36) amide and exendin-4 induced concentration-dependent contractions yielding pEC50 values of 8.4 ± 0.2 and 8.4 ± 0.4, respectively. Exendin (9-39) antagonized the action of both agonists in a non-competitive reversible manner, with apparent pKB values of 9.5 and 9.7, respectively. Tetrodotoxin (1 μM), atropine (1 μM) and hexamethonium (500 μM) were used to determine the contractile mechanism of action of exendin-4. Tetrodotoxin and atropine significantly antagonized (P  < 0.01) the contractile action of exendin-4 (10 nM); hexamethonium (500 μM) had no action. These studies suggest that GLP-1 receptor agonists contract the ileum indirectly via postganglionic enteric neurones and an involvement of muscarinic receptors. These studies provide information relevant to the use of this species to estimate the therapeutic indexes of GLP-1 receptor agonists.
Keywords: GLP-1; Exendin-4; Exendin (9-39) amide; Suncus murinus; Ileum; Postganglionic enteric neurone; Nausea; Emesis; Diabetes;

Lycopene, quercetin and tyrosol prevent macrophage activation induced by gliadin and IFN-γ by Daniela De Stefano; Maria Chiara Maiuri; Vittorio Simeon; Gianluca Grassia; Antonio Soscia; Maria Pia Cinelli; Rosa Carnuccio (192-199).
Oxidative stress plays an important role in inflammatory process of celiac disease. We have studied the effect of the lycopene, quercetin and tyrosol natural antioxidants on the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expression in RAW 264.7 macrophages stimulated by gliadin in association with IFN-γ. The IFN-γ plus gliadin combination treatment was capable of enhancing iNOS and COX-2 gene expression and nuclear factor-κB (NF-κB), interferon regulatory factor-1 (IRF-1) and signal transducer and activator of transcription-1α (STAT-1α) activation induced by reactive oxygen species generation at 24 h. Lycopene, quercetin and tyrosol inhibited all these effects. The results here reported suggest that these compounds may represent non toxic agents for the control of pro-inflammatory genes involved in celiac disease.
Keywords: Celiac disease; Gliadin; Interferon-gamma; Transcription factors; RAW 264.7 macrophage;

Interactions between eotaxin and interleukin-5 in the chemotaxis of primed and non-primed human eosinophils by Gislaine G. Costa; Reginaldo M. Silva; Carla F. Franco-Penteado; Edson Antunes; Heloisa H.A. Ferreira (200-205).
This study was designed to understand the relationship between interleukin-5 and eotaxin in modulating the chemotaxis of eosinophils obtained from healthy subjects and subjects with allergic rhinitis. Chemotaxis of eosinophils from patients with allergic rhinitis toward interleukin-5 (0.25 ng/ml) was 78% higher than that of healthy subjects. Incubation of eosinophils with eotaxin (100 ng/ml) did not change the interleukin-5-induced chemotaxis of eosinophils from healthy subjects, but it reversed the enhanced chemotaxis seen in eosinophils from allergic patients. Chemotaxis of eosinophils from patients with allergic rhinitis toward eotaxin (100 ng/ml) was 65% higher than that of eosinophils from healthy subjects. Incubation of eosinophils with interleukin-5 (100 ng/ml) significantly increased the eotaxin-induced chemotaxis in both subject groups, but such increases were markedly higher for cells from patients with allergic rhinitis. Our finding that eotaxin inhibits the enhanced eosinophil chemotaxis toward interleukin-5 in primed cells suggests that this chemokine may downregulate eosinophil accumulation in the nasal mucosa of allergic patients.
Keywords: Allergic rhinitis; Eosinophil chemotaxis; Interleukin-5; Eotaxin; Fibronectin;

It has been found previously that hydroxycobalamine (vitamin B12b) amplifies significantly the cytotoxic effect of ascorbic acid (vitamin C) added to cells for а long period of time (48 h). However, according to pharmacokinetics, the concentration of vitamin C in vivo decreases to a physiological value within a short period of time (2–3 h) after the injection. Therefore, in this study we examined the cytotoxic effect of a short-time (up to 2 h) exposure of human larynx carcinoma HEp-2 cells to a combination of vitamins B12b and C (B12b  + C). The kinetics of the B12b  + C-caused extracellular oxidative burst in this time interval was also explored. Vitamin B12b combined with ascorbic acid provoked a rapid accumulation of extracellular hydrogen peroxide followed by intracellular oxidative stress, DNA single-strand breaks, and the initiation of apoptosis. The chelators of iron phenanthroline and desferrioxamine prevented B12b  + C-induced DNA single-strand breaks and cell death but not the accumulation of H2O2 in culture medium. The nonthiol antioxidants pyruvate and catalase were effective in preventing the prooxidant and cytotoxic effects of B12b  + C. Thiols, when added simultaneously with the combined vitamins, inhibited these effects only partially (N-acetylcysteine, GSH) or even amplified them (dithiothreitol). The results obtained point to the determining role of oxidative burst and iron-dependent DNA damage in the cytotoxic effect of short-time exposure to B12b  + C combination.
Keywords: Ascorbic acid; Vitamin B12b; Cytotoxicity; DNA single-strand break; Hydrogen peroxide; Oxidative stress; Tumor cell;

Amiodarone has anti-inflammatory and anti-oxidative properties: An experimental study in rats with carrageenan-induced paw edema by Zekai Halici; Gunnur Ozbakis Dengiz; Fehmi Odabasoglu; Halis Suleyman; Elif Cadirci; Mesut Halici (215-221).
Amiodarone is a widely used anti-arrhythmic agent. We have investigated alterations in the glutathione (GSH) level and the activities of anti-oxidative enzymes (superoxide dismutase, catalase, glutathione s-transferase and glutathione reductase) and myeloperoxidase, as marker of acute inflammation, following oral administration of amiodarone and diclofenac in rats with carrageenan-induced paw edema. In the present study, we found that 1) Amiodarone reduced the development of carrageenan-induced paw edema, to a greater degree than diclofenac; 2) Amiodarone and diclofenac alleviated increases in the activities of catalase and glutathione s-transferase enzymes resulting from edema; 3) Amiodarone and diclofenac ameliorated depressions in the GSH level and the activities of superoxide dismutase and glutathione reductase enzymes caused by carrageenan injection; and 4) All doses of amiodarone and diclofenac caused an amplification in myeloperoxidase activity resulting from induced paw edema. These results suggest that the anti-inflammatory effect of amiodarone on carrageenan-induced acute inflammation can be attributed to its ameliorating effect on the oxidative damage.
Keywords: Amiodarone; Carrageenan; Acute inflammation; Myeloperoxidase; Anti-oxidant enzyme;

Reduction of hERG potassium currents by hyperosmolar solutions by Fumie Yabuuchi; Rolf Beckmann; Erich Wettwer; Christa Hegele-Hartung; Jürgen F. Heubach (222-225).
We investigated the effects of hyperosmolar solutions on human ether-a-go-go related gene (hERG) potassium currents in chinese hamster ovary (CHO) cells. The addition of d-mannitol to the external solution caused cell shrinkage and reduced current amplitude. The effects were at least partially reversible. Exposure to 108 mM mannitol decreased current amplitude by 57 ± 13%. Major effects on current–voltage relations were not observed. Exposure to 308 mM mannitol reduced the current by 89 ± 5%, i.e. comparable to the block induced by 1 μM of the selective hERG channel blocker E-4031. We conclude that the investigation of hyperosmolar drug formulations requires control solutions of comparable osmolarity to separate specific drug effects from non-specific effects of hyperosmolarity.
Keywords: Patch-clamp technique; Human ether-a-go-go related gene; hERG; CHO cells; d-mannitol; Hyperosmolar solution;

Impaired arginine–vasopressin-induced aldosterone release from adrenal gland cells in mice lacking the vasopressin V1A receptor by Jun-ichi Birumachi; Masami Hiroyama; Yoko Fujiwara; Toshinori Aoyagi; Atsushi Sanbe; Akito Tanoue (226-230).
We examined aldosterone release in response to stimulation with arginine–vasopressin (AVP) using adrenal gland cells. AVP caused a significant increase in aldosterone release from the dispersed adrenal gland cells of wild-type mice (V1AR+/+) at concentrations from 0.1 μM to 1 μM. In contrast, AVP-induced aldosterone release was impaired in adrenal gland cells from mice lacking the vasopressin V1A receptor (V1AR−/−), while adrenocorticotropic hormone (ACTH)-induced aldosterone release in V1AR−/− mice was not significantly different from that in V1AR+/+ mice. In addition, a vasopressin V1A receptor-selective antagonist 1-[1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl]-3,4-dihydro-2(1H)-quinolinone (OPC-21268) potently inhibited AVP-induced aldosterone release. Thus, our study clearly demonstrates that AVP-induced aldosterone release from adrenal gland cells is mediated via the vasopressin V1A receptor in mice.
Keywords: Arginine–vasopressin; Vasopressin V1A receptor-deficient mice; Aldosterone; Adrenal gland cells;

Protective effect of Umbelliferone on membranous fatty acid composition in streptozotocin-induced diabetic rats by Balakrishnan Ramesh; Periyasamy Viswanathan; Kodukkur Viswanathan Pugalendi (231-239).
Umbelliferone (UMB), a natural antioxidant, is benzopyrone in nature, and it is present in the fruits of golden apple and bitter orange. Earlier we evaluated and reported the effect of Umbelliferone on antidiabetic, antioxidant and antihyperlipidemic properties, and this study was designed to evaluate the effect of Umbelliferone on membrane fatty acid composition and histopathology of liver and kidney of control and streptozotocin (STZ) diabetic rats. Male albino Wistar rats (180–200 g) were made diabetic by an intraperitonial administration of STZ (40 mg/kg). The control and diabetic rats were treated with Umbelliferone and glibenclamide dissolved in 10% dimethyl sulfoxide for 45 days. Diabetic rats had decreased insulin and increased glucose, and increased levels of thiobarbituric acid reactive substances, lipid hydroperoxides and conjugated dienes. The levels of palmitic, stearic and oleic acids increased and the levels of linolenic and arachidonic acids decreased in diabetic rats as compared with control rats. Thus, the saturated fatty acids and monounsaturated fatty acids increased and the polyunsaturated fatty acids decreased in diabetic rats. Diabetic rats had decreased liver weight and increased activities of alanine transaminase and aspartate transaminase; increased kidney weight and urine albumin, and decreased levels of urea, uric acid and creatinine in the urine. Histopathological studies of liver and kidney in diabetic rats showed fatty changes surrounding portal triad; enlargement of lining cells of tubules, fatty infiltration, large area of hemorrhage and lymphocyte infiltration. Treatment with Umbelliferone and glibenclamide reversed these changes to near normalcy. Our results showed that Umbelliferone has a protective effect on membrane fatty acid composition of liver and kidney as supported by antioxidant and antihyperlipidemic effects of Umbelliferone reported earlier as evidenced by improved histopathological changes, hepatic and nephritic markers, indicating recovery from the risk of diabetic complications.
Keywords: Diabetes mellitus; Streptozotocin; Umbelliferone; Fatty acid composition;