European Journal of Pharmacology (v.644, #1-3)

Diabetic neuropathy is one of the most common complications in diabetes, and hyperalgesia and allodynia are serious symptoms of diabetic neuropathy. There are few therapeutic options available for the treatment of such diabetic painful neuropathy. While several reports have indicated that an abnormality of intracellular signaling molecules is involved in the pathogenesis of diabetic painful neuropathy, agents that affect these intracellular signaling molecules have failed to deliver convincing results in clinical trials. Recently, the small molecular G-protein RhoA has been shown to be involved in the pathogenesis of diabetic nephropathy. RhoA and its downstream kinase Rho kinase (ROCK) have been shown to modulate nociceptive transmission in the spinal cord. In this report, we provide a brief overview of the role of the RhoA/ROCK pathway in diabetic complications. We especially focus on the role of the spinal RhoA/ROCK pathway in the pathogenesis of diabetic painful neuropathy. Findings on the association between the spinal RhoA/ROCK pathway and diabetic painful neuropathy may lead to new strategies for its treatment.
Keywords: RhoA; Spinal cord; Diabetes mellitus; Neuropathic pain; Neuron;

Cyclosporin A induces hyperpermeability of the blood–brain barrier by inhibiting autocrine adrenomedullin-mediated up-regulation of endothelial barrier function by Shinya Dohgu; Noriko Sumi; Tsuyoshi Nishioku; Fuyuko Takata; Takuya Watanabe; Mikihiko Naito; Hideki Shuto; Atsushi Yamauchi; Yasufumi Kataoka (5-9).
Cyclosporin A, a potent immunosuppressant, can often produce neurotoxicity in patients, although its penetration into the brain is restricted by the blood–brain barrier (BBB). Brain pericytes and astrocytes, which are periendothelial accessory structures of the BBB, can be involved in cyclosporin A-induced BBB disruption. However, the mechanism by which cyclosporin A causes BBB dysfunction remains unknown. Here, we show that in rodent brain endothelial cells, cyclosporin A decreased transendothelial electrical resistance (TEER) by inhibiting intracellular signal transduction downstream of adrenomedullin, an autocrine regulator of BBB function. Cyclosporin A stimulated adrenomedullin release from brain endothelial cells, but did not affect binding of adrenomedullin to its receptors. This cyclosporin A-induced decrease in TEER was attenuated by exogenous addition of adrenomedullin. Cyclosporin A dose-dependently decreased the total cAMP concentration in brain endothelial cells. A combination of cyclosporin A (1 μM) with an adenylyl cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536; 10 μM), or a protein kinase A (PKA) inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89; 1 μM), markedly increased sodium fluorescein permeability in brain endothelial cells, whereas each drug alone had no effect. Thus, these data suggest that cyclosporin A inhibits the adenylyl cyclase/cyclic AMP/PKA signaling pathway activated by adrenomedullin, leading to impairment of brain endothelial barrier function.
Keywords: Blood–brain barrier; Cyclosporin A; Adrenomedullin; cAMP; Adenylyl cyclase; Permeability;

Mutational analysis of residues important for ligand interaction with the human P2Y12 receptor by Yingying Mao; Lili Zhang; Jianguo Jin; Barrie Ashby; Satya P. Kunapuli (10-16).
The P2Y12 receptor, a Gi protein-coupled receptor, plays a central role in platelet activation. In this study, we did a mutational analysis of residues possibly involved in the ligand interactions with the human P2Y12 receptor. Mutant receptors were stably expressed in CHO-K1 cells with an HA-tag at the N-terminus. Expression of wild-type and mutant receptors was confirmed by detecting the HA-tag on the cell membrane. Residues in transmembrane helical domains (TMs) 3, 5, 6, and 7, which are homologous to residues important for P2Y1 receptor activation and ligand recognition, were replaced by site-directed mutagenesis. ADP-induced inhibition of forskolin-stimulated cAMP levels in the presence or absence of antagonist AR-C69931MX were investigated for each of the mutant receptors. F104S and S288P significantly increased agonist-induced receptor function without affecting the antagonism by AR-C69931MX. Arg256 in TM6 and Arg 265 in extracellular loop 3 (EL3) are more important for antagonist recognition than effect on agonist-mediated receptor function. Compared to wild-type P2Y12 receptor, mutations in Arg 256 or/and Arg 265 significantly increased the sensitivity to antagonist AR-C69931MX. Our study shows that the cytosolic side of TM3 and the exofacial side of TM5 are critical for P2Y12 receptor function, which is different from P2Y1. Arg 256 in TM6 and Arg265 in EL3 appear to play a role in antagonist recognition rather than effects on agonist-induced receptor function.
Keywords: P2Y12; cyclic AMP; site-directed mutation;

Glycogen synthase kinase-3β negatively regulates TGF-β1 and Angiotensin II-mediated cellular activity through interaction with Smad3 by Fang Hua; Junlan Zhou; Jinwen Liu; Chuanjiang Zhu; Bing Cui; Heng Lin; Yuying Liu; Wen Jin; Hongzhen Yang; Zhuowei Hu (17-23).
Glycogen synthase kinase-3β (GSK3β) is a major negative modulator of cardiac hypertrophy. Here we report that GSK3β physically and functionally interacts with Smad3. The interaction between GSK3β and Smad3 may participate in the negative regulation of transforming growth factor β1 (TGF-β1) and Angiotensin II-induced transcription and apoptosis. GSK3β interacted directly with Smad3 to sequester it outside the nucleus and prevent its nuclear translocation. This resulted in the suppression of Smad3-mediated transcriptional activity and gene expression. GSK3β counteracted the pro-apoptotic effect of Smad3 and attenuated Angiotensin II-induced apoptosis in cardiac myocytes. Furthermore, stimulation of these cells with TGF-β1 and Angiotensin II led to the endogenous Smad3 disassociating from GSK3β and inactivating GSK3β by phosphorylation of its Ser9. These results uncovered a novel mechanism for the GSK3β negative regulation of TGF-β1/Smad3 and Angiotensin II/Smad3-mediated transcription and apoptosis by the identification of a crosstalk between GSK3β and Smad3 signal pathway.
Keywords: Transforming growth factor β1; Angiotensin II; Glycogen synthase kinase-3β; Smad3; Protein interaction;

Haloperidol causes cytoskeletal collapse in N1E-115 cells through tau hyperphosphorylation induced by oxidative stress: Implications for neurodevelopment by Gloria Benítez-King; Leonardo Ortíz-López; Graciela Jiménez-Rubio; Gerardo Ramírez-Rodríguez (24-31).
Haloperidol a typical antipsychotic commonly used in the treatment of schizophrenia causes neuronal damage and extrapiramidal symptoms after several years of treatment. These symptoms have been associated with increased levels of oxidative stress. Reactive oxygen species produce cytoskeletal collapse and an excessive phosphorylation of tau, a microtubule-associated protein that plays a key role in microtubule stabilization, and in growth cone and neurite formation, which are cytoskeletal phenotypes that participate in neurodevelopment. Thus, we hypothesized that haloperidol produces neurocytoskeletal disorganization by increasing free radicals and tau hyperphosphorylation, and consequently, the loss of neurodevelopmental cytoskeletal phenotypes, neurites and growth cones. The purpose of this work was the characterization of neuronal cytoskeletal changes caused by haloperidol in neuroblastoma N1E-115 cells. We also studied the mechanisms by which haloperidol causes cytoskeletal changes. The results showed that haloperidol at 100 μM caused a complete cytoskeleton collapse in the majority of the cells. Melatonin, a free radical scavenger, blocks tau hyperphosphorylation, and microtubule disorganization caused by haloperidol in a dose–response mode. Additionally, the indole blocks lipoperoxide formation in haloperidol treated cells. The results indicate that free radicals and tau hyperphosphorylation produced by haloperidol caused a cytoskeletal collapse and the lost of growth cones and neurites. These effects were blocked by melatonin. Data suggest that extrapiramidal symptoms in schizophrenic patients can be produced by cytoskeletal disorganization during adult brain neurodevelopment after prolonged haloperidol treatment that can be prevented by melatonin.
Keywords: Haloperidol; Cytoskeletal; Neurites; Growth cones; Neurodevelopment; Melatonin;

8-Prenylnaringenin is an inhibitor of multidrug resistance-associated transporters, P-glycoprotein and MRP1 by Olga Wesołowska; Jerzy Wiśniewski; Kamila Środa; Agnieszka Krawczenko; Aleksandra Bielawska-Pohl; Maria Paprocka; Danuta Duś; Krystyna Michalak (32-40).
Flavonoids with hydrophobic e.g. prenyl substituents might constitute the promising candidates for multidrug resistance (MDR) reversal agents. The interaction of 8-prenylnaringenin (8-isopentenylnaringenin), a potent phytoestrogen isolated from common hop (Humulus lupulus), with two multidrug resistance-associated ABC transporters of cancer cells, P-glycoprotein and MRP1, has been studied for the first time. Functional test based on the transport of fluorescent substrate BCECF revealed that the flavonoid strongly inhibited MRP1 transport activity in human erythrocytes (IC50  = 5.76 ± 1.80 μM). Expression of MDR-related transporters in drug-sensitive (LoVo) and doxorubicin-resistant (LoVo/Dx) human colon adenocarcinoma cell lines was characterized by RT-PCR and immunochemical methods and elevated expression of P-glycoprotein in resistant cells was found to be the main difference between these two cell lines. By means of flow cytometry it was shown that 8-prenylnaringenin significantly increased the accumulation of rhodamine 123 in LoVo/Dx cells. Doxorubicin accumulation in both LoVo and LoVo/Dx cells observed by confocal microscopy was also altered in the presence of 8-prenylnaringenin. However, the presence of the studied compound did not increase doxorubicin cytotoxicity to LoVo/Dx cells. It was concluded that 8-prenylnaringenin was not able to modulate MDR in human adenocarcinoma cell line in spite of the ability to inhibit both P-glycoprotein and MRP1 activities. To our best knowledge, this is the first report of 8-prenylnaringenin interaction with clinically important ABC transporters.
Keywords: Multidrug resistance; 8-prenylnaringenin; P-glycoprotein; Multidrug resistance-associated protein 1 MRP1; Flavonoid;

Differential inhibition of murine Bcrp1/Abcg2 and human BCRP/ABCG2 by the mycotoxin fumitremorgin C by Lucía González-Lobato; Rebeca Real; Julio G. Prieto; Ana I. Álvarez; Gracia Merino (41-48).
Breast Cancer Resistance Protein (ABCG2/BCRP) is an ATP-binding cassette transporter expressed in absorptive and excretory organs whose main physiological role is protection of cells against xenobiotics. In addition, ABCG2/BCRP expression has been linked to cellular resistance to anticancer drugs due to the acquisition of a multidrug resistance phenotype. Fumitremorgin C (FTC) is a mycotoxin described as a potent ABCG2/BCRP inhibitor that reverses multidrug resistance. However, little is known about its species-specificity. This issue is scientifically relevant since FTC is widely used to evaluate the in vitro role of BCRP. We compared the FTC-mediated inhibition of human BCRP and its murine orthologue, overexpressed in two independent cell lines, MDCKII and MEF3.8 transduced cell lines. Accumulation experiments, using mitoxantrone and chlorine e6 as substrates, revealed that although FTC inhibits both Bcrp1 and BCRP, the human transporter is more potently inhibited, resulting in significantly lower IC50 values. Transcellular transport of known Bcrp1/BCRP substrates, such as nitrofurantoin and mitoxantrone, was completely inhibited by FTC 1 μM in human BCRP-transduced cells but only moderately in murine Bcrp1-transduced cells. Finally, cytotoxicity assays using mitoxantrone and topotecan as substrates revealed that the EC90 values for FTC were always significantly lower in human BCRP-transduced cells. Altogether, these results indicate that human BCRP is more sensitive to inhibition by FTC than murine Bcrp1. This differential inhibition could have a great impact on the use of in vitro models of toxicity and pharmacological interaction for drug discovery and development involving FTC as Bcrp1/BCRP inhibitor.
Keywords: Fumitremorgin C; BCRP/ABCG2; Transport; Inhibition; Cytotoxicity;

The present study investigated the effect of per oral (p.o.) administration of butyl (2-phenylethynyl) selenide (1–50 mg/kg) on formalin-induced nociception in mice. The involvement of serotonergic, adenosinergic, muscarinic cholinergic and opioid mechanisms in the antinociceptive effect was also investigated. Butyl (2-phenylethynyl) selenide inhibited both neurogenic (at doses equal or higher than 10 mg/kg) and inflammatory (at doses equal or higher than 25 mg/kg) phases of the nociception caused by intraplantar ( injection of 2.5% formalin solution (20 μl), with ID50 values of 36.7 (29.28–46.0) and 20.37 (15.74–26.36) mg/kg, respectively. This compound reduced the formalin-induced paw oedema formation (55 ± 4%) at doses equal or higher than 25 mg/kg. The antinociceptive effect of compound (25 mg/kg, p.o.) was reversed by ondansetron (0.5 mg/kg, a 5-HT3 receptor antagonist) and caffeine (3 mg/kg, a nonselective adenosine receptor antagonist), but not by atropine (0.1 mg/kg, a non selective muscarinic antagonist), WAY100635 (0.1 mg/kg, a selective 5-HT1A receptor antagonist), ritanserin (1 mg/kg, a 5-HT2 receptor antagonist) and naloxone (1 mg/kg, a non selective opioid receptor antagonist). These results indicate that butyl (2-phenylethynyl) selenide produced antinociception in the formalin test through mechanisms that involve an interaction with serotonergic (5-HT3) and adenosinergic systems.
Keywords: Antinociception; Butyl (2-phenylethynyl) selenide; Selenium; Serotonergic; Adenosinergic; Formalin;

Peripheral mechanisms underlying the essential role of P2X7 receptors in the development of inflammatory hyperalgesia by Juliana Maia Teixeira; Maria Cláudia G. Oliveira; Carlos Amílcar Parada; Cláudia Herrera Tambeli (55-60).
Activation of P2X7 receptors by endogenous ATP contributes to the development of inflammatory hyperalgesia. Given the clinical importance of mechanical hyperalgesia in inflammatory states, we hypothesized that the activation of the P2X7 receptor by endogenous ATP contributes to carrageenan-induced mechanical hyperalgesia, and that this contribution is mediated by an indirect sensitization of the primary afferent nociceptors. Co-administration of the selective P2X7 receptor antagonist, A-438079, or the P2X7 receptor antagonist, oATP, with carrageenan blocked the mechanical hyperalgesia induced by carrageenan and significantly reduced the increased concentration of TNF-α, IL-6 and CINC-1, but not of IL-1β induced by carrageenan in the subcutaneous tissue of the rat's hind paw. We concluded that the activation of P2X7 receptors by endogenous ATP is essential to the development of the mechanical hyperalgesia induced by carrageenan in the subcutaneous tissue. It is suggested that this essential role of P2X7 receptors in the development of carrageenan-induced mechanical hyperalgesia is mediated by an indirect sensitization of the primary afferent nociceptors dependent on the previous release of TNF-α, IL-6 and CINC-1, but not of IL-1β.
Keywords: Mechanical inflammatory hyperalgesia; P2X7 receptor; ATP; Carrageenan; Cytokine;

Fasting hypometabolism and refeeding hyperphagia in rats: Effects of capsaicin desensitization of the abdominal vagus by András Garami; Márta Balaskó; Miklós Székely; Margit Solymár; Erika Pétervári (61-66).
Capsaicin-sensitive abdominal vagal fibers contribute to postprandial satiety and hypermetabolism. We hypothesized that the hypometabolic adaptation to fasting involves similar mechanisms and that blockade of such signals might enhance loss of body weight upon fasting. A low dosage of capsaicin (5 mg/kg) administered intraperitoneally desensitizes the local afferent vagal nerve endings for approximately three weeks without causing systemic desensitization or damaging the efferent fibers. Following such desensitization, male Wistar rats deprived of food for 120 h lost significantly (18.9 ± 0.4% vs. 15.8 ± 1.0%), i.e. 20% more weight than the controls. Based on the present results, this can only be explained by the demonstrated defective hypometabolic adaptation in desensitized animals. Other mechanisms do not seem to make up for this defective function. Upon refeeding following a period of fasting, in the first 0.5–3 h the food intake was significantly greater in capsaicin pretreated compared to the control group, demonstrating blockade of satiety as a sign of desensitization. The delayed gastrointestinal passage supported that vagal afferent nerve endings were in a desensitized state in these rats. In conclusion, local desensitization of the abdominal capsaicin-sensitive fibers attenuates the hypometabolic adaptation to food deprivation and the lack of fasting-induced activation of these fibers cannot be substituted by other fasting-dependent mechanisms. It is suggested that reports of low body weight in mice lacking the transient receptor potential vanilloid-1 channel and in rats with systemic capsaicin desensitization might be explained by a lasting absence of similar (vagus-mediated) hypometabolic processes, preventing weight gain or obesity.
Keywords: Capsaicin; Vagus; Fasting hypometabolism; Food intake; Body weight; TRPV1;

The acute administration of MDMA has been shown to promote glycogenolysis and increase the extracellular concentration of glucose in the striatum. In the present study the role of serotonergic and/or noradrenergic mechanisms in the MDMA-induced increase in extracellular glucose and glycogenolysis was assessed. The relationship of these responses to the hyperthermia produced by MDMA also was examined. The administration of MDMA (10 mg/kg, i.p.) resulted in a significant and sustained increase of 65–100% in the extracellular concentration of glucose in the striatum, as well as in the prefrontal cortex and hippocampus, and a 35% decrease in brain glycogen content. Peripheral blood glucose was modestly increased by 32% after MDMA treatment. Treatment of rats with fluoxetine (10 mg/kg, i.p.) significantly attenuated the MDMA-induced increase in extracellular glucose in the striatum but had no effect on MDMA-induced glycogenolysis or hyperthermia. Treatment with prazosin (1 mg/kg, i.p.) did not alter the glucose or glycogen responses to MDMA but completely suppressed MDMA-induced hyperthermia. Finally, propranolol (3 mg/kg, i.p.) significantly attenuated the MDMA-induced increase in extracellular glucose and glycogenolysis but did not alter MDMA-induced hyperthermia. The present results suggest that MDMA increases extracellular glucose in multiple brain regions, and that this response involves both serotonergic and noradrenergic mechanisms. Furthermore, β-adrenergic and α-adrenergic receptors appear to contribute to MDMA-induced glycogenolysis and hyperthermia, respectively. Finally, hyperthermia, glycogenolysis and elevated extracellular glucose appear to be independent, unrelated responses to acute MDMA administration.
Keywords: MDMA; Glycogen; Glucose; Hyperthermia; Microdialysis;

Endothelin antagonists are being investigated to prevent neuronal loss after cerebral ischemia. Acetaminophen has been tried in stroke patients to produce hypothermia so that injury following cerebral ischemia can be reduced. The aim of this study was to assess the effect of BQ123, an endothelin-A receptor antagonist, alone and in combination with acetaminophen on neurological outcome, oxidative stress and infarct volume in rats subjected to focal ischemia by occlusion of the middle cerebral artery. In normal rats, acetaminophen decreased, while BQ123 did not produce any change in body temperature, but rats treated with BQ123 and acetaminophen produced a significantly greater (41%) hypothermic response compared to acetaminophen group. In rats subjected to middle cerebral artery occlusion, neurologic deficit was observed; acetaminophen alone did not improve, but BQ123 alone and in combination with acetaminophen produced a significant improvement in neurological deficit. The level of malondialdehyde (MDA) increased and reduced glutathione (GSH) decreased in the brain following ischemia; acetaminophen did not but BQ123 alone and in combination with acetaminophen decreased MDA and increased GSH levels in ischemic rats. Cerebral ischemia produced significant infarction, the infarct volume decreased in response to BQ123 and its combination with acetaminophen. The infarct volume, MDA level and neurological deficit in ischemic rats significantly improved in rats treated with both BQ123 and acetaminophen compared to BQ123 alone. The results demonstrate that a combination of acetaminophen and BQ123 is more effective in reducing the neuronal damage following cerebral ischemia, and this combination may be worth investigating in stroke patients.
Keywords: Cerebral ischemia; Endothelin; Hypothermia; Oxidative stress; BQ123; Acetaminophen;

Amphetamine use is associated with dysphoric states, including heightened anxiety, that emerge within 24 h of withdrawal from the drug. Corticotropin-releasing factor increases serotonin release in the central nucleus of the amygdala, and this neurochemical circuitry may play a role in mediating fear and anxiety states. We have previously shown that chronic amphetamine treatment increases corticotropin-releasing factor receptor type-2 levels in the serotonergic dorsal raphe nucleus of the rat. Therefore, we hypothesized that chronic amphetamine treatment would enhance the amygdalar serotonergic response to corticotropin-releasing factor infused into the dorsal raphe nucleus. Male rats were injected once-daily with d-amphetamine (2.5 mg/kg i.p., or saline) for two weeks. Serotonin release within the central nucleus of the amygdala in response to intra-raphe infusion of corticotropin-releasing factor (100 ng) was measured 24 h after the last treatment in urethane-anesthetized (1.8 mg/kg, i.p.) rats using in vivo microdialysis. Rats pretreated with amphetamine showed significantly enhanced serotonin release in the central nucleus of the amygdala in response to corticotropin-releasing factor infusion when compared to saline pretreated rats. Furthermore, this enhanced response was blocked by the corticotropin-releasing factor type-2 receptor antagonist antisauvagine-30 (2 μg) infused into the dorsal raphe nucleus. These results suggest increased sensitivity to corticotropin-releasing factor as mediated by type-2 receptors following chronic amphetamine treatment, which may underlie dysphoric states observed during amphetamine withdrawal.
Keywords: Dorsal raphe nucleus; Central nucleus of the amygdala; CRF2 receptor; Microdialysis; (Rat);

In vivo pharmacology of the dopaminergic stabilizer pridopidine by Henrik Ponten; Johan Kullingsjö; Sören Lagerkvist; Peter Martin; Fredrik Pettersson; Clas Sonesson; Susanna Waters; Nicholas Waters (88-95).
Pridopidine (ACR16) belongs to a new pharmacological class of agents affecting the central nervous system called dopaminergic stabilizers. Dopaminergic stabilizers act primarily at dopamine type 2 (D2) receptors and display state-dependent behavioural effects. This article aims to give an overview of the preclinical neurochemical and behavioural in vivo pharmacological properties of pridopidine. Pridopidine was given s.c. to male Sprague–Dawley rats (locomotor, microdialysis and tissue neurochemistry) and i.p. to Swiss male mice (tail suspension test). Pridopidine dose-dependently increased striatal tissue levels of the dopamine metabolite 3,4-dihydroxyphenylalanin (ED50  = 81 μmol/kg), and prefrontal cortex dialysate levels of dopamine and noradrenaline as measured by high performance liquid chromatography. The agent reduced hyperlocomotion (d-amphetamine: ED50  = 54 μmol/kg; MK-801: ED50  = 40 μmol/kg), but preserved spontaneous locomotor activity, confirming state-dependent behavioural effects. In addition, pridopidine significantly reduced immobility time in the tail suspension test. We conclude that pridopidine state-dependently stabilizes psychomotor activity by the dual actions of functional dopamine D2 receptor antagonism and strengthening of cortical glutamate functions in various settings of perturbed neurotransmission. The putative restoration of function in cortico–subcortical circuitry by pridopidine is likely to make it useful for ameliorating several neurological and psychiatric disorders, including Huntington's disease.
Keywords: Pridopidine; ACR16; Dopamine; Dopaminergic stabilizer; Huntington's disease; Pharmacology;

Metabotropic glutamate mGlu5 receptor-mediated serine phosphorylation of NMDA receptor subunit NR1 in hippocampal CA1 region after transient global ischemia in rats by Norio Takagi; Shintaro Besshoh; Hirotsugu Morita; Mihoko Terao; Satoshi Takeo; Kouichi Tanonaka (96-100).
Phosphorylation of the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor has been implicated in the regulation of the receptor's ion channel. The contribution of metabotropic glutamate receptors to the NMDA receptor function after brain ischemia remains to be determined. Presently we investigated the effects of an antagonist of the metabotropic glutamate mGlu5 receptor on cell death and serine phosphorylation of the NR1 subunit of the NMDA receptor in the hippocampal CA1 region after transient global ischemia and sought to explore the mechanisms involved. Phosphorylation of serine residues at 890 and 896 of NR1 was increased predominantly in the deoxycholate (DOC)-insoluble fraction after transient global ischemia in rats; and the increase in the phosphorylation of S890, but not that of S896, of NR1 in this fraction was attenuated by the mGlu5 receptor antagonist. The administration of this antagonist also reduced the increase in the amount of protein kinase C (PKC)γ, but not that of PKCα, in the DOC-insoluble fraction. The results suggest that the mGlu5 receptor in the hippocampal CA1 region is involved in the phosphorylation of S890 of NR1 subunit via PKCγ following transient ischemia. As treatment with the mGlu5 receptor antagonist reduced cell death in the hippocampal CA1 region on day 3 after the start of the reperfusion, these changes in intracellular signaling through mGlu5 receptor may be linked to the pathogenesis of cerebral ischemia.
Keywords: Metabotropic glutamate receptor; NMDA receptor; Transient global ischemia; Phosphorylation;

Intranasal cocaine functions as reinforcer on a progressive ratio schedule in humans by William W. Stoops; Joshua A. Lile; Paul E.A. Glaser; Lon R. Hays; Craig R. Rush (101-105).
Cocaine dependence continues to be a worldwide public health concern. Although the majority of individuals reporting cocaine use do so via the intranasal route, relatively few laboratory experiments have examined the reinforcing effects of cocaine administered intranasally. The purpose of this experiment was to measure the reinforcing effects of intranasal cocaine using a progressive ratio schedule in which eight cocaine-using subjects chose between doses of cocaine (4 [placebo], 15, 30 and 45 mg) and an alternative reinforcer ($0.25). During each session, subjects first sampled the dose of cocaine available that day and then made six choices between that dose and money, which were available on concurrent progressive ratio schedules of responding. Break points for active cocaine doses were higher than those for placebo but no statistically significant active versus placebo dose effects were observed on subject-rated or physiological measures. These data demonstrate that intranasal cocaine functions as a reinforcer under a progressive ratio schedule in humans. Future research should test higher cocaine doses and larger values of the alternative reinforcer. These procedures may be useful for examining the influence of putative pharmacological and behavioral interventions on intranasal cocaine self-administration.
Keywords: Cocaine; Intranasal; Humans; Progressive ratio;

The antiepileptic drugs, phenobarbitone and carbamazepine are well known to cause cognitive impairment on chronic use. The increase in free radical generation has been implicated as one of the important mechanisms of cognitive impairment by antiepileptic drugs. Curcumin has shown antioxidant, anti-inflammatory and neuro-protective properties. Therefore, the present study was carried out to investigate the effect of chronic curcumin administration on phenobarbitone- and carbamazepine-induced cognitive impairment and oxidative stress in rats. Pharmacokinetic interactions of curcumin with phenobarbitone and carbamazepine were also studied. Vehicle/drugs were administered daily for 21 days to male Wistar rats. Passive avoidance paradigm and elevated plus maze test were used to assess cognitive function. At the end of study period, serum phenobarbitone and carbamazepine, whole brain malondialdehyde and reduced glutathione levels were estimated. The administration of phenobarbitone and carbamazepine for 21 days caused a significant impairment of learning and memory as well as an increased oxidative stress. Concomitant curcumin administration prevented the cognitive impairment and decreased the increased oxidative stress induced by these antiepileptic drugs. Curcumin co-administration did not cause any significant alteration in the serum concentrations of both phenobarbitone as well as carbamazepine. These results show that curcumin has beneficial effect in mitigating the deterioration of cognitive functions and oxidative damage in rats treated with phenobarbitone and carbamazepine without significantly altering their serum concentrations. The findings suggest that curcumin can be considered as a potential safe and effective adjuvant to phenobarbitone and carbamazepine therapy in preventing cognitive impairment associated with these drugs.
Keywords: Curcumin; Phenobarbitone; Carbamazepine; Learning; Memory; Lipid peroxidation; Glutathione;

Actions of thalidomide in producing vascular relaxations by Sai Wang Seto; Sotiria Bexis; P. Aiden McCormick; James R. Docherty (113-119).
We have investigated the cardiovascular actions of thalidomide in vivo and in vitro. Blood pressure was recorded in pentobarbitone anaesthetized rats. Isometric contractions were examined in rings of rat tail artery and aorta. Radioligand binding studies of α1A- and α1B-adrenoceptor sites were carried out in membranes of rat submandibular gland and spleen, respectively. In pentobarbitone anaesthetized rats, thalidomide and the T-type calcium channel blocker NNC55-0396 (both 1 mg/kg, i.v.) significantly reduced blood pressure. In rat tail artery, thalidomide (10–100 μM) produced relaxations of phenylephrine (1 μM) induced contractions. Also in tail artery, thalidomide (100 μM) significantly reduced the contraction to phenylephrine (1 μM), but not KCl (40 mM), produced by calcium restoration, and NNC55-0396 (100 μM) had similar actions to thalidomide. Glibenclamide (10 μM), calphostin C (1 μM) or SB203580 (1 μM) failed to affect the inhibitory actions of thalidomide, and thalidomide did not affect contractions to caffeine (10 mM). Ligand binding studies found no evidence for α1A- or α1B-adrenoceptor affinity of thalidomide, and functional studies in rat aorta found no evidence for α1D-adrenoceptor affinity. It is concluded that thalidomide has previously unreported vascular relaxant actions. Relaxant actions in vitro do not seem to involve α1-adrenoceptors, caffeine sensitive calcium stores, glibenclamide sensitive potassium channels, protein kinase C (PKC) or P38 mitogen activated protein kinase (P38 MAP kinase). However, actions of thalidomide resembled those of the T-type calcium channel blocker NNC 55-0396. Further study is necessary to establish the mode of action of thalidomide in causing relaxations.
Keywords: Thalidomide; Blood pressure; Hypotension; Postural hypotension; T-type calcium channels, NNC 55-0396; Mibefradil; α1-adrenoceptor;

Differential effects of four xylidine derivatives in the model of ischemia- and re-perfusion-induced arrhythmias in rats in vivo by Tadeusz F. Krzemiński; Katarzyna Mitręga; Michał Żorniak; Maurycy Porc (120-127).
The aim of our study was to find the most effective xylidine derivative, which reduced mortality, reduced incidence and duration of severe arrhythmias and had a beneficial influence on hemodynamic parameters in an in vivo setting. We compared the action of lidocaine, articaine, ropivacaine and mepivacaine in a dose 2.5 or 5 mg/kg/ml/h infused from 10 min before left anterior descending coronary artery occlusion until the end of the experiment. In the rat ischemia- and re-perfusion-induced arrhythmia models, the following parameters were measured or calculated: mortality index, ventricular fibrillation and tachycardia incidence and duration, systolic, diastolic and mean arterial blood pressure, heart rate and pressure rate product. Lidocaine produced the most significant reduction in mortality index (P  < 0.05) after both doses. At the higher dose, lidocaine and articaine shortened ventricular fibrillation and tachycardia duration (P  < 0.05–P  < 0.001), while ropivacaine prolonged them. A hypertensive effect was observed after a lower dose of lidocaine during occlusion and early re-perfusion as compared to others (P  < 0.05). Beneficial effects were mainly observed with lidocaine, which protected against sudden cardiac death. The novelty was lidocaine's dose independent protection against blood pressure drop in early re-perfusion, which could be linked to the effects observed on the other end-points. Articaine showed beneficial effects but they weren't as pronounced as that of lidocaine. Nevertheless, in the light of our results, articaine could supposedly be used as a substitute for lidocaine in patients with hypertension.
Keywords: Xylidine derivative; Ischemia and re-perfusion; Arrhythmia; Hemodynamics; (Rat);

Organ culture is an in vitro method for investigating cellular mechanisms involved in upregulation of vasocontractile G-protein coupled receptors. We hypothesize that mitogen-activated-protein kinase (MEK) and/or extracellular-signal-regulated kinase (ERK) specific inhibitors will attenuate the G-protein coupled receptor expression following organ culture.Rat cerebral arteries were incubated 48 h in the presence of MEK/ERK specific inhibitors U0126, PD98059, SL327, or AG126 for different time periods. Contractile responses by activation of endothelin receptor type A and type B, serotonin receptor 5-HT1B, prostanoid TP receptor, and angiotensin II receptor type 1 and type 2 were investigated. Results were verified by measurement of mRNA with real time PCR and by protein immunohistochemistry.Organ culture induced transcriptional upregulation of endothelin ETB receptor and of serotonin 5-HT1B receptor on translational level and increased respective contractions. The prostanoid TP receptor mediated contraction curve was left-wards shifted by organ culture. Organ culture was associated with elevated pERK1/2 in the vascular smooth muscle cells: the MEK1/2 inhibitor U0126 attenuated the endothelin ETB receptor mediated contraction at post-translational level or by changing the receptor affinities. The serotonin 5-HT1B receptor and prostanoid TP receptor mediated contractions were abolished by U0126. Administration of U0126 6 h after start of incubation blocked the receptor upregulation.In conclusion, MEK specific inhibitor U0126 is a potent inhibitor of G-protein coupled receptor alteration seen during organ culture. Given the ability to inhibit G-protein coupled receptor alteration at the clinically relevant time-point 6 h post incubation makes it an attractive therapeutic agent for in vivo studies.
Keywords: Organ culture; Cerebral arteries; Vascular smooth muscle cells; G-protein coupled receptor upregulation; MEK/ERK pathway; U0126;

Inhibitory effect of quercetin on matrix metalloproteinase 9 activity Molecular mechanism and structure–activity relationship of the flavonoid–enzyme interaction by Alejandra C. Saragusti; María G. Ortega; José L. Cabrera; Darío A. Estrin; Marcelo A. Marti; Gustavo A. Chiabrando (138-145).
Epidemiological studies have demonstrated an inverse association between the consumption of flavonoid-rich diets and the risk of atherosclerosis. In addition, an increased activity of the matrix metalloproteinase 9 (MMP-9) has been implicated in the development and progression of atherosclerotic lesions. Even though the relationship between flavonoid chemical structure and the inhibitory property on MMP activity has been established, the molecular mechanisms of this inhibition are still unknown. Herein, we first evaluated the inhibitory effect of quercetin on MMP-9 activity by zymography and a fluorescent gelatin dequenching assay, secondly we determined the most probable sites and modes of quercetin interaction with the MMP-9 catalytic domain by using molecular modelling techniques, and finally, we investigated the structure–activity relationship of the inhibitory effect of flavonoids on MMP-9 activity. We show that quercetin inhibited MMP-9 activity with an IC50 value of 22 μM. By using docking and molecular dynamics simulations, it was shown that quercetin interacted in the S1′ subsite of the MMP-9 active site. Moreover, the structure–activity relationship analysis demonstrated that flavonoid R3 –OH and R4 –OH substitutions were relevant to the inhibitory property against MMP-9 activity. In conclusion, our data constitute the first evidence about the quercetin and MMP-9 interaction, suggesting a mechanism to explain the inhibitory effect of the flavonoid on the enzymatic activity of MMP-9, which provides an additional molecular target for the cardioprotective activity of quercetin.
Keywords: Diet polyphenol; Docking; Flavonoid; Molecular dynamics; Zymography;

Peripheral benzodiazepine receptor ligand Ro5-4864 inhibits isoprenaline-induced cardiac hypertrophy in rats by Amardeep Jaiswal; Santosh Kumar; Rajesh Enjamoori; Sandeep Seth; Amit Kumar Dinda; Subir Kumar Maulik (146-153).
Oxidative stress plays a significant role in the pathogenesis of cardiac hypertrophy. Peripheral benzodiazepine receptors are ubiquitously expressed in various tissues, including the heart. Peripheral benzodiazepine receptors have been reported to be involved in the protection of cells against oxygen radical damage. The present study was designed to determine whether Ro5-4864 (a peripheral benzodiazepine receptor ligand) can inhibit isoprenaline-induced cardiac hypertrophy. Male Wistar rats (body weight 150–200 g) were administered, isoprenaline (5 mg/kg, body weight, subcutaneously) alone or along with Ro5-4864 (0.1 and 0.5 mg/kg, body weight, intraperitoneally) once daily for 14 days. Control rats received normal saline subcutaneously (1.0 ml/kg). Isoprenaline-induced changes in heart weight to body weight ratio, left ventricular wall thickness (M-mode echocardiography and gross morphometry) and myocyte size were significantly prevented by both the doses of Ro5-4864. Ro5-4864 also attenuated isoprenaline-induced increase in interstitial fibrosis, lipid peroxidation and changes in endogenous antioxidants (glutathione, superoxide dismutase and catalase). Isoprenaline-induced cardiac hypertrophy was associated with increased expression of β myosin heavy chain, which was also prevented by Ro5-4864. This is the first study to demonstrate a salutary effect of Ro5-4864 in experimental cardiac hypertrophy.
Keywords: Echocardiography; Oxidative stress; Fibrosis; Myosin heavy chain; Myocyte size;

Decreased vasoconstrictor responses in remote cerebral arteries after focal brain ischemia and reperfusion in the rat, in vitro by Anikó Kovács; Krisztina Móricz; Mihály Albert; Angéla Benedek; László G. Hársing; Gábor Szénási (154-159).
The effects of brain ischemia and reperfusion on smooth muscle function in remote cerebral and peripheral arteries are hardly known. Maximum vasoconstrictions (Emax) caused by 120 mmol/l KCl and 5-HT in endothelium-denuded ring preparations were measured in ischemic and control cerebral arteries of rats after a 1-h right middle cerebral artery occlusion followed by 0-min (I/NR) or 2–3-min (I/SR) reperfusion, and in peripheral arteries after I/SR. Surprisingly, vasoconstrictions to 5-HT and 120 mmol/l K+ were attenuated in remote brain vessels after I/SR, i.e. in the contralateral middle cerebral artery and the basilar artery, while I/NR depressed Emax of 5-HT and high KCl only in the ischemic middle cerebral artery. Pretreatment with N-(2-mercaptopropionyl) glycine (MPG, 100 mg/kg i.p.), a free radical scavenger, fully prevented the impairment of vasomotor function in the middle cerebral artery on both sides after I/SR. Moreover, vasomotor functions were normal in the coronary, renal and pulmonary arteries after I/SR. In conclusion, focal cerebral ischemia and reperfusion impaired vasoconstrictor responses in remote brain arteries of rats by a mechanism involving free radicals. The lack of similar effects in peripheral vessels indicates poor defence of brain arteries against remote injury caused by reactive oxygen species-dependent mechanisms.
Keywords: Ischemia; Middle cerebral artery; Remote effect; Reperfusion; Vascular dysfunction;

The present study was designed to investigate the cardioprotective effect of melatonin against isoproterenol induced myocardial infarction in rats by studying myocyte injury markers, antioxidant defense system, serum and heart lipid profile, inflammatory markers, electrocardiographic and histopathological changes. Male Sprague Dawley (SD) rats were randomly divided into four groups, namely control, melatonin, isoproterenol and melatonin + isoproterenol treated group. Melatonin treatment group received melatonin (10 mg/kg/day, i.p.) for 7 days. Myocardial infarction in rats was induced by isoproterenol administration (150 mg/kg, s.c.) at an interval of 24 h on 6th and 7th day. On 8th day ECG, gravimetric, biochemical and histopathological parameters were assessed. Isoproterenol administration showed changes in ECG pattern, including ST-segment elevation (diagnostic of myocardial infarction) increase in the serum levels of cardiac injury markers (creatine kinase-MB, lactate dehydrogenase, aspartate transaminase and alanine transaminase), decreased antioxidant defense system in the heart and altered lipid profile in the serum and heart. Isoproterenol administration also resulted in release of inflammatory markers and neutrophil infiltration along with histopathological changes. Melatonin pre-co-treatment prevented almost all the parameters of isoproterenol induced myocardial infarction in rats. The above finding was confirmed by the histopathological examination. In the baseline group (melatonin alone treated group) no significant change was observed. Results of the present study suggest that melatonin has a significant effect on the protection of the heart against isoproterenol induced myocardial infarction through maintaining endogenous antioxidant enzyme activities.
Keywords: Melatonin; Isoproterenol; Myocardial infarction; Antioxidant; Electrocardiography;

Positive influence of AT1 receptor antagonism upon the impaired celiprolol-induced vasodilatation in aorta from spontaneously hypertensive rats by Frédérique Sauvaget; Mohamed Yassine Mallem; Véronique Bucas; Marc Gogny; Jean-Claude Desfontis; Jacques Noireaud (169-175).
We evaluated celiprolol-induced vasodilatation in aorta taken from 12-week-old spontaneously hypertensive rats (SHR) and the effect of AT1 angiotensin II receptor antagonism on the vasodilatory action of celiprolol in Wistar Kyoto (WKY) rats and SHR. In WKY rats, the celiprolol-induced relaxation was greatly decreased in denuded aorta, and completely abolished in intact aorta by Nω-nitro-l-arginine methyl ester (l-NAME, 100 μM). In SHR, celiprolol-induced relaxation was reduced compared to WKY rats (E max (value obtained for the highest concentration, 300 μM) = 39.1 ± 3.78%, n  = 21 vs. 80.4 ± 3% in WKY rats, n  = 10; P  < 0.0001). Endothelium removal or pre-treatment with l-NAME did not alter celiprolol-induced relaxation in SHR. In both strains, relaxation to celiprolol was decreased in the presence of nadolol (a β12-adrenoceptor antagonist, 10 μM). N-[[3-[(2 S)-2-hydroxy-3-[[2-[4-[(phenylsulfonyl)amino] phenyl]ethyl]amino] propoxy]phenyl]methyl]-acetamide (L748337, a β3-adrenoceptor antagonist, 7 μM) had no effect. A 12-day treatment with candesartan cilexetil (an AT1 receptor antagonist, 0.37 or 1 mg/kg/day) reduced systolic blood pressure in both strains, but only improved relaxation to celiprolol in SHR, and only at the highest dose (E max  = 64.2 ± 3.9%, n  = 10, P  < 0.0001 vs. SHR control). In both strains, local aortic AT1 receptor antagonism with candesartan CV11974 (100 μM) had no effect. The endothelial β12 relaxation induced by celiprolol was therefore impaired in SHR aorta and AT1 receptor antagonism improved the response to celiprolol, in conjunction with a reduction in blood pressure. This work highlights the need to analyse the potential benefit of a combination of celiprolol/AT1 receptor antagonist in the treatment of hypertension.
Keywords: Celiprolol; β-Adrenergic receptor; Vasodilatation; SHR; Aorta; (Rat);

Doxycycline suppresses doxorubicin-induced oxidative stress and cellular apoptosis in mouse hearts by Hui-Chin Lai; Yueh-Chiao Yeh; Chih-Tai Ting; Wen-Lieng Lee; Hsiao-Wei Lee; Li-Chuan Wang; Kuo-Yang Wang; Hui-Chun Lai; Angie Wu; Tsun-Jui Liu (176-187).
Cardiac toxicity remains a serious yet unsolved complication of doxorubicin. This study was designed to examine whether doxycycline, a tetracycline-derived synthetic antibiotic with potential cytoprotective properties, could ameliorate this complication of doxorubicin. Male mice at 4-week of age were administrated with vehicle, doxorubicin (3 mg/kg intraperitoneally every other day at 3 doses), doxycycline (2.5 mg/kg intraperitoneally every other day for 3 doses), or doxycycline plus doxorubicin (each dose given 1 day post doxycycline). After 28 days, left ventricular geometric and systolic parameters were measured by transthoracic echocardiography, and hearts were harvested for extensive analyses regarding oxidative stress and cellular apoptosis. At 28 days, hearts of doxorubicin-treated mice were characterized by less weight compared with controls, also with remodeling and depressed systolic function of the left ventricle. Biochemical analyses disclosed that content of malondialdehyde was increased and activity of antioxidant enzymes, including superoxide dismutase and glutathione peroxidase, was decreased in these hearts. Both mitochondrion-dependent and endoplasmic reticulum stress-induced apoptotic pathways were also activated in the hearts of doxorubicin-treated mice as reflected by decreased Bcl-2/Bcl-XL and elevated Bax/Bad, p53/Apaf-1, endoplasmic reticulum glucose-related protein 78, C/EBP homologous protein, cytochrome c release from mitochondria, caspases-9/-3 cleavage, and cardiomyocyte apoptosis. In contrast, all the above left ventricular remodeling, systolic depressing, oxidative and pro-apoptotic actions of doxorubicin could be significantly alleviated by doxycycline pretreatment. Thus, doxycycline extensively counteracts multiple oxidative and apoptotic actions of doxorubicin in heart, hence may serve as an adjuvant agent to assuage the untoward cardiac effects of doxorubicin in clinical application.
Keywords: Doxorubicin; Doxycycline; Apoptosis; Oxidative stress; Heart;

Protease-activated receptor 1 antagonists prevent platelet aggregation and adhesion without affecting thrombin time by Florence Nadal-Wollbold; Arnaud Bocquet; Thierry Bourbon; Robert Létienne; Bruno Le Grand (188-194).
The aim of this study was to investigate the in vitro antithrombotic effects of two PAR1 antagonists, ER121958 and SCH203099 on both SFLLR-induced platelet adhesion and aggregation and on the thrombin time in human and guinea-pig platelets. ER121958 inhibited SFLLR-induced guinea-pig and human platelet adhesion with the IC50 values of 1.73 nM and 2.91 nM, respectively and SFLLR-induced guinea-pig and human platelet aggregation with the IC50 values of 2.74 nM and 11.9 nM, respectively. Similarly, SCH203099 exhibited a non competitive profile of inhibition on both SFLLR-induced guinea-pig and human platelet adhesion with the IC50 values of 93 nM and 127 nM, respectively or SFLLR-induced guinea-pig and human platelet aggregation with the IC50 values of 1.74 μM and 2.36 μM, respectively. These two antagonists failed to prolong the thrombin time. Altogether, these results highlighted the potent anti-platelets properties of both ER121958 and SCH203099 in an in vitro model of aggregation as well as in a static model of adhesion without any effect on the last step of coagulation cascade. Moreover, this work emphasized that guinea-pig is a suitable animal model to study the role of PAR1 antagonists since the magnitude of the effects of ER121958 and SCH203099 on both SFLLR-induced platelet adhesion and aggregation were similar in both species.
Keywords: PAR1 antagonist; Platelet; Aggregation; Adhesion; Thrombin time;

The brain-gut peptide neurotensin has complex effects on gastrointestinal smooth muscle. Our objective was to elucidate the mechanisms underlying neurotensin contractions in human colon. Discrete concentration response curves to neurotensin were obtained in strips of circular muscle and taenia coli from “normal” ascending and sigmoid colon segments, in the presence and absence of various pharmacological inhibitors. Potency of neurotensin in all regions was similar (pD2 ~ 7). Atropine and the selective muscarinic receptor antagonists, methoctramine and darifenacin, had no effect on neurotensin contractions. In ascending colon circular muscle, responses were enhanced by indomethacin (indicating inhibitory prostaglandin mechanisms) and by tetrodotoxin (TTX), hexamethonium and L-NAME, suggesting nicotinic and enteric inhibitory neurotransmission, with involvement of nitric oxide. In sigmoid circular muscle, neurotensin responses were also enhanced by TTX and hexamethonium, but were attenuated in the presence of mepyramine, MEN10627 and CP99994, suggesting inhibitory neuronal mechanisms and involvement of histamine and tachykinins, respectively; L-NAME and the GABAB receptor antagonist, CGP36742, were without effect. The transcripts of NTS1 and NTS3 receptors, but not NTS2 receptors, were detected in sigmoid colon circular muscle and taenia coli. No age and gender differences in NTS1 mRNA expression were found. In conclusion, neurotensin contracts circular muscle strips from ascending and sigmoid regions of the human colon via direct (muscle) and indirect (neuronal/non-neuronal mechanisms). The enteric mediators influenced by neurotensin are regionally specific. In taenia coli strips from both ascending and sigmoid colon, neurotensin contractions were unchanged in the presence of inhibitors, suggesting direct actions only.
Keywords: Neurotensin; Human colon; Neuronal; Indomethacin; Apamin; Nifedipine;

Angiostatins decrease in the kidney of newborn piglets after hypoxia-reoxygenation by Marwan Emara; Laila Obaid; Scott Johnson; David L. Bigam; Po-Yin Cheung (203-208).
Little is known about the expression of kidney angiostatin in the hypoxia and reoxygenation of neonates. In this study, we compared the effect of 21% and 100% reoxygenation on kidney levels of angiostatin and its related factors in newborn piglets subjected to hypoxia-reoxygenation. Newborn piglets were subjected to 2 h hypoxia followed by 1 h of reoxygenation with either 21% or 100% oxygen and observed for 4 days. There were 3 isoforms (38, 43 and 50 kDa) of angiostatins identified in the kidney tissue of newborn piglets with the 38 kDa being the major isoform (~ 60%). The 38 kDa, but not 43 and 50 kDa, angiostatin isoform correlated significantly with the levels of total angiostatin and plasminogen (r  = 0.95 and r  = 0.58, respectively). On day 4 of recovery in 100% hypoxic-reoxygenated group, there were decreases in kidney tissue levels of plasminogen, total angiostatin, angiostatin (38 and 43 kDa, but not 50 kDa), whereas no significant changes were found in the 21% hypoxic-reoxygenated group when compared to the sham-operated piglets with no hypoxia-reoxygenation. Both 21% and 100% hypoxic-reoxygenated groups did not show significant changes in kidney tissue levels of 50 kDa angiostatin, MMP-2, MMP-9 and HIF-1α. In comparison to 21% oxygen, neonatal resuscitation with 100% oxygen decreased the kidney tissue levels of plasminogen and angiostatin that may play a role in neonatal kidney injury and altered renal development in adulthood.
Keywords: Angiostatin; Hypoxia inducible factor-1α; Matrix metalloproteinases; Hypoxia-reoxygenation;

Azithromycin inhibits nontypeable Haemophilus influenzae-induced MUC5AC expression and secretion via inhibition of activator protein-1 in human airway epithelial cells by Nobuko Araki; Katsunori Yanagihara; Yoshitomo Morinaga; Koichi Yamada; Shigeki Nakamura; Yasuaki Yamada; Shigeru Kohno; Shimeru Kamihira (209-214).
Nontypeable Haemophilus influenzae (NTHi) is one of the most common pathogens in chronic airway infections and exacerbation. The hallmark of chronic respiratory diseases, including cystic fibrosis, diffuse panbronchiolitis and chronic obstructive pulmonary disease, is mucin overproduction. Prolonged macrolide antibiotic therapy at low doses is known to improve clinical outcome in patients with chronic respiratory diseases via anti-inflammatory effects. In this study, we investigated the effects of macrolide therapy on NTHi-induction of the MUC5AC mucin in human airway epithelial cells. A 15-membered macrolide, azithromycin, but not a 14-membered macrolide, clarithromycin, inhibited NTHi-induction of MUC5AC at both the mRNA and protein levels through selective suppression of activation of the transcription factor activator protein-1. Our findings suggest that each macrolide affects MUC5AC production in different ways and that azithromycin is more suitable for the treatment of NTHi-induced respiratory infection.
Keywords: Mucus; Mucin; Chronic obstructive pulmonary disease; Immunomodulatory effect; Macrolides;

Augmented venous responsiveness to leukotriene D4 in nasal septal mucosae of repeatedly antigen-challenged rats by Hiroyasu Sakai; Jun Enzaka; Michiko Sakai-Oshita; Yoshihiko Chiba; Miwa Misawa (215-219).
One possible mechanism of the nasal obstruction observed in allergic rhinitis is thought to be a dilatation of veins in nasal mucosa, although the exact mechanism(s) is not fully understood. An involvement of cysteinyl leukotrienes (CysLTs) in the nasal obstruction has also been suggested. In addition to the specific antigen-induced nasal symptoms, nasal hyperresponsiveness to non-specific stimuli is one of the characteristic features of patients with allergic rhinitis. Augmentation of LTD4-induced venodilatation (a part of nasal hyperresponsiveness) of nasal mucosae in antigen-challenged rats was investigated. The LTD4-induced venodilatation was significantly increased in antigen-challenged rats, although venodilatation by application of LTD4 was not induced in nasal mucosae of control rats. The LTD4-induced venodilatation was significantly inhibited by pretreatment with L-NMMA [an inhibitor of nitrix oxide synthase (NOS)]. Although mRNA of CysLT1 receptor of nasal mucosa was within control level, the LTD4-induced production of NOx in nasal cavity was augmented in repeatedly antigen challenge rats. In addition, the level of iNOS mRNA was also significantly augmented in nasal mucosae of repeatedly antigen-challenged rats. Interestingly, sodium nitroprusside (SNP; an NO donor)-induced venodilatation itself was significantly augmented in nasal mucosae of repeatedly antigen challenge rats. In conclusion, we here suggest that the sensitivity of venodilatation to LTD4 was augmented in nasal mucosae of challenged rats. Therein, not only increased NO production but also enhanced NO responsiveness might be involved in the development of nasal hyperresponsiveness in allergic rhinitis.
Keywords: Venodilatation; Cysteinyl leukotriene D4; Nasal mucosa; Nitric oxide; Nasal hyperresponsiveness;

7-hydroxyfrullanolide, a sesquiterpene lactone, inhibits pro-inflammatory cytokine production from immune cells and is orally efficacious in animal models of inflammation by Lyle C. Fonseca; Shruta S. Dadarkar; Aurelio S. Lobo; Ashish C. Suthar; Vijay S. Chauhan; Shanthi Chandrababu; Somesh D. Sharma; Nilesh M. Dagia; Muralidhara Padigaru (220-229).
A promising therapeutic approach to reduce pathological inflammation is to inhibit the increased production of pro-inflammatory cytokines (e.g., TNF-α, IL-6). In this study, we investigated the anti-inflammatory potential of 7-hydroxyfrullanolide (7HF). 7HF is an orally bioavailable, small molecule sesquiterpene lactone isolated from the fruit of Sphaeranthus indicus. 7HF significantly and dose-dependently diminished induced and spontaneous production of TNF-α and IL-6 from freshly isolated human mononuclear cells, synovial tissue cells isolated from patients with active rheumatoid arthritis and BALB/c mice. Oral administration of 7HF significantly protected C57BL/6J mice against endotoxin-mediated lethality. In the dextran sulfate sodium (DSS) model of murine colitis, oral administration of 7HF prevented DSS-induced weight loss, attenuated rectal bleeding, improved disease activity index and diminished shortening of the colon of C57BL/6J mice. Histological analyses of colonic tissues revealed that 7HF attenuated DSS-induced colonic edema, leukocyte infiltration in the colonic mucosa and afforded significant protection against DSS-induced crypt damage. 7HF was also significantly efficacious in attenuating carrageenan-induced paw edema in Wistar rats after oral administration. In the collagen-induced arthritis in DBA/1J mice, 7HF significantly reduced disease associated increases in articular index and paw thickness, protected against bone erosion and joint space narrowing and prominently diminished joint destruction, hyperproliferative pannus formation and infiltration of inflammatory cells. Collectively, these results provide evidence that 7HF-mediated inhibition of pro-inflammatory cytokines functionally results in marked protection in experimental models of acute and chronic inflammation.
Keywords: TNF-α; IL-6; Sesquiterpene lactone; Colitis; Rheumatoid arthritis; Sepsis;

Effects of sappanchalcone on the cytoprotection and anti-inflammation via heme oxygenase-1 in human pulp and periodontal ligament cells by Gil-Saeng Jeong; Dong-Sung Lee; Bin Li; Hwa-Jun Lee; Eun-Cheol Kim; Youn-Chul Kim (230-237).
Sappanchalcone has been demonstrated to possess several biological effects. However, the molecular mechanism underlying these effects is not fully understood. In this study, we examined the effects of sappanchalcone on hydrogen peroxide (H2O2)-induced cytotoxicity using human dental pulp (HDP) cells, and lipopolysaccharide (LPS)-induced inflammation using human periodontal ligament (HPDL) cells. Sappanchalone concentration proportionately increased heme oxygenase (HO)-1 protein expression and enzyme activity in both HDP and HPDL cells. It also protected HDP cells from H2O2-induced cytotoxicity and reactive oxygen species production. The cytoprotective effect of sappanchalcone was nullified by HO-1 inhibitor, Tin protoporphyrin (SnPP). Sappanchalcone is seen to inhibit LPS-stimulated nitric oxide (NO), prostaglandin E2 (PGE2), interlukine-1β (IL-1β), tumor necrosis factor-α (TNF-α), interlukine-6 (IL-6) and interlukine-12 (IL-12) release in addition to inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in HPDL cells. SnPP, a specific inhibitor of HO-1, partly blocked sappanchalcone mediated suppression of inflammatory mediator production, in LPS-stimulated HPDL cells. HDP and HPDL cells treated with sappanchalcone exhibited the transient activation of c-Jun NH2-terminal kinase (JNK) and NF-E2-related factor-2 (Nrf2). The expression of HO-1 protein by sappanchalcone was significantly reduced by pretreatment with JNK inhibitor. In conclusion, induction of HO-1 is an important cytoprotective mechanism by which sappanchalcone protects HDP cells from H2O2 and in addition it also exhibits anti-inflammatory effects in LPS-stimulated HPDL cells. Thus, sappanchalcone could potentially be a therapeutic approach for periodontal, pulpal and periapical inflammatory lesion.
Keywords: Sappanchalcone; Cytoprotection; Inflammation; Dental pulp cell; Periodontal ligament cell; Heme oxygenase-1;

New properties of the soybean trypsin inhibitor: Inhibition of human neutrophil elastase and its effect on acute pulmonary injury by Jannison K.C. Ribeiro; Dayse D.S. Cunha; Jacy M.S.L.L. Fook; Mauricio P. Sales (238-244).
Seeds from legumes including the Gilcine max are known to be a rich source of protease inhibitors. The soybean Kunitz trypsin inhibitors (SKTIs) have been well characterised and have been found to exhibit many biological activities. However their effects on inflammatory diseases have not been studied to date. In this study, SKTI was purified using anion exchange chromatography using a Resource Q column. The purified protein was able to inhibit human neutrophil elastase (HNE) and bovine trypsin. Purified SKTI inhibited HNE with an IC50 value of 8 μg or 0.3 nM. At this concentration SKTI showed neither cytotoxic nor haemolytic effects on human blood cell populations. SKTI showed no deleterious effects on organs, blood cells or the hepatic enzymes ALT and AST in the mouse model of acute systemic toxicity. Human neutrophils incubated with SKTI released less HNE than control neutrophils when stimulated with PAF or fMLP (83.1% and 70% respectively). These results showed that SKTI affected both pathways of elastase release by PAF and fMLP stimuli, suggesting that SKTI is an antagonist of fMLP/PAF receptors. In an in vivo mouse model of LPS acute lung injury, SKTI significantly suppressed the inflammatory effects caused by elastase in a dose-dependent manner. Histological sections stained by hematoxylin/eosin confirmed this decrease in inflammation. These results showed that SKTI could be used as a pharmacological agent for the therapy of many inflammatory diseases.
Keywords: Kunitz; Skti; Neutrophil elastase; Acute lung injury;

Nimesulide improves the disease modifying anti-rheumatic profile of methotrexate in mice with collagen-induced arthritis by Ahmed M. Al-Abd; Julia J. Inglis; Salwa M. Nofal; Amani E. Khalifa; Richard O. Williams; Wafaa I. El-Eraky; Ashraf B. Abdel-Naim (245-250).
Methotrexate is a disease modifying anti-rheumatic drug that is widely used for the treatment of rheumatoid arthritis. Nimesulide is a non-steroidal anti-inflammatory drug which is frequently used as adjuvant therapy for symptomatic alleviation of rheumatoid arthritis. In this study, we have evaluated the potential influence of nimesulide on the disease modifying anti-rheumatic properties of methotrexate using the collagen-induced arthritis model. Mice were immunized with collagen type II for the induction of arthritis and treated with methotrexate (2.5 mg/kg) twice a week, nimesulide (20 mg/kg) every other day or a combination of both drugs. Treatment started one week after the onset of arthritis until day 40. An arthritic index was used to compare the severity of arthritis between different treatments. In addition, articular hyperalgesia, joint stiffness, radiological deterioration and intra-articular leucocytic infiltration were evaluated. Methotrexate alone showed modest but significant analgesic and anti-inflammatory effects, and the effects of nimesulide were comparable. On the other hand, nimesulide significantly improved the disease modifying anti-rheumatic profile of methotrexate in terms of arthritic index and joint mobility. Furthermore, although nimesulide failed to show any radiological evidence of articular protection, it significantly improved methotrexate-induced joint protection as judged by X-ray analysis.
Keywords: Methotrexate; Nimesulide; Rheumatoid arthritis; Collagen-induced arthritis;

Involvement of cholecystokinin in baseline and post-prandial whole body insulin sensitivity in rats by Barna Peitl; Róbert Döbrönte; László Drimba; Réka Sári; Angelika Varga; József Németh; Tamás Pázmány; Zoltán Szilvássy (251-256).
The objective of the study was to investigate the role of cholecystokinin (CCK) on the food-induced insulin sensitization phenomenon in healthy Long Evans Tokushima Otsuka (LETO) and Otsuka Long Evans Tokushima Fatty (OLETF) rats. Whole body insulin sensitivity determined by hyperinsulinaemic euglycaemic glucose clamping and the rapid insulin sensitivity test served as endpoints. Determinations were done in both fasted and re-fed animals. The involvement of CCK in post-prandial insulin sensitization was assessed by using proglumide, a CCK receptor blocker, by assessment of hypothalamic CCK-1/CCK-2 receptor expression by rt-PCR technique and by plasma insulin immunoreactivity determinations by means of radioimmunoassay as pharmacological, genetic and analytical approaches, respectively. The body weight of the OLETF rats and the amount of food consumed much exceeded those seen with LETO rats. The post-prandial increase in insulin sensitivity was marked in LETO, but not in OLETF rats. Intravenous proglumide attenuated post-prandial insulin sensitivity in LETO rats, with no effect in OLETF rats. Nevertheless, baseline insulin sensitivity was much lower in OLETF than in LETO rats. Treatment with rosiglitazone increased baseline insulin sensitivity of OLETF rats and evoked an increase in CCK-1 receptor gene expression in LETO rats. The results provide evidence for the involvement of CCK receptors in adjustment of both fasting and post-prandial insulin sensitivity. The data obtained with OLETF rats strongly suggest the predominant role of CCK-1 receptors.
Keywords: Insulin sensitivity; Otsuka Long Evans Tokushima Fatty rat; CCK-1 receptor; Proglumide; Hyperinsulinaemic euglycaemic glucose clamping; Rapid insulin sensitivity test;

Absence of somatostatin SST2 receptor internalization in vivo after intravenous SOM230 application in the AR42J animal tumor model by Beatrice Waser; Renzo Cescato; Maria-Luisa Tamma; Helmut R. Maecke; Jean Claude Reubi (257-262).
Among clinically relevant somatostatin functions, agonist-induced somatostatin receptor subtype 2 (sst2) internalization is a potent mechanism for tumor targeting with sst2 affine radioligands such as octreotide. Since, as opposed to octreotide, the second generation multi-somatostatin analog SOM230 (pasireotide) exhibits strong functional selectivity, it appeared of interest to evaluate its ability to affect sst2 internalization in vivo. Rats bearing AR42J tumors endogenously expressing somatostatin sst2 receptors were injected intravenously with SOM230 or with the [Tyr3, Thr8]-octreotide (TATE) analog; they were euthanized at various time points; tumors and pancreas were analyzed by immunohistochemistry for the cellular localization of somatostatin sst2 receptors. SOM230-induced sst2 internalization was also evaluated in vitro by immunofluorescence microscopy in AR42J cells. At difference to the efficient in vivo sst2 internalization triggered by intravenous [Tyr3, Thr8]-octreotide, intravenous SOM230 did not elicit sst2 internalization: immunohistochemically stained sst2 in AR42J tumor cells and pancreatic cells were detectable at the cell surface at 2.5 min, 10 min, 1 h, 6 h, or 24 h after SOM230 injection while sst2 were found intracellularly after [Tyr3, Thr8]-octreotide injection. The inability of stimulating sst2 internalization by SOM230 was confirmed in vitro in AR42J cells by immunofluorescence microscopy. Furthermore, SOM230 was unable to antagonize agonist-induced sst2 internalization, neither in vivo, nor in vitro. Therefore, SOM230 does not induce sst2 internalization in vivo or in vitro in AR42J cells and pancreas, at difference to octreotide derivatives with comparable sst2 binding affinities. These characteristics may point towards different tumor targeting but also to different desensitization properties of clinically applied SOM230.
Keywords: Somatostatin receptor; Internalization; Tumor targeting; SOM230; Octreotide; Functional selectivity; Desensitization;

Pharmacological concentrations of biotin reduce serum triglycerides and the expression of lipogenic genes by Elena Larrieta; Fidel Velasco; Paz Vital; Teresita López-Aceves; María Luisa Lazo-de-la-Vega-Monroy; Alberto Rojas; Cristina Fernandez-Mejia (263-268).
Besides its role as a carboxylase prosthetic group, biotin regulates gene expression and has a wide repertoire of effects on systemic processes. Several studies have shown that pharmacological concentrations of biotin reduce hypertriglyceridemia. The molecular mechanisms by which pharmacological concentrations of biotin affect lipid metabolism are largely unknown. The present study analyzed the effects of pharmacological doses of biotin on triglyceridemia, insulin sensitivity and on mRNA expression of various lipogenic genes. Three-week-old male BALB/cAnN Hsd mice were fed a biotin-control or a biotin-supplemented diet (1.76 or 97.7 mg of free biotin/kg diet, respectively) over a period of eight weeks. Serum triglyceride concentrations, insulin and glucose tolerance and mRNA abundance of various lipogenic genes were investigated. The biotin-supplemented group showed 35% less serum triglycerides than control mice. In the liver, we found a significant (P  < 0.05) reduction of mRNA levels of SREBP1-c, glucose transporter-2, phosphofructokinase-1, pyruvate kinase, acetyl-CoA carboxylase and fatty acid synthase, while glucose-6-phosphate dehydrogenase expression increased. No changes in glucokinase, stearoyl-CoA desaturase-1, FoxO1 or PPAR-gamma expression were observed. In adipose tissue, we found a decreased expression of SREBP1c, glucose-6-phosphate deshydrogenase, acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase-1, phosphofructokinase-1 and PPAR-gamma, but no changes in FoxO1 expression. Moreover, the group fed a biotin-supplemented diet showed a significant decrease in adipose tissue weight. No differences in insulin sensitivity or serum insulin concentrations were observed between groups. Our results indicate that pharmacological concentrations of biotin decrease serum tryglyceride concentrations and lipogenic gene expression in liver and adipose tissues.
Keywords: Biotin supplementation; Triglyceride; Lipogenesis;