European Journal of Pharmacology (v.635, #1-3)

Resveratrol, obesity and diabetes by Katarzyna Szkudelska; Tomasz Szkudelski (1-8).
Resveratrol belongs to the large group of biologically active substances found in plants. This compound is classified as phytoestrogen because of its ability to interact with estrogen receptor. Numerous beneficial effects of resveratrol described in the literature involve cardioprotective, anti-cancer, anti-inflammatory and antioxidant action. Recently, this broad spectrum of effects is enlarged by new data demonstrating a great potency of this compound in relation to obesity and diabetes. It is well established that resveratrol exerts beneficial effects in rodents fed a high-calorie diet. In some studies, resveratrol was reported to reduce body weight and adiposity in obese animals. The action of this compound involves favourable changes in gene expressions and in enzyme activities. The accumulating evidence also indicates the benefits of resveratrol in diabetes and diabetic complications. It is known that resveratrol affects insulin secretion and blood insulin concentration. In animals with hyperinsulinemia, resveratrol was found to reduce blood insulin. Moreover, numerous data indicate that in diabetic rats, resveratrol is able to reduce hyperglycemia. The mechanism of resveratrol's action is complex and is demonstrated to involve both insulin-dependent and insulin-independent effects. These data point to the potential possibility of use of resveratrol in preventing and/or treating both obesity and diabetes.
Keywords: Resveratrol; Prevention; Obesity; Diabetes;

The internalization of β2-adrenoceptors after agonist activation results in a desensitized and phosphorylated receptor that either resensitizes by recycling to the cell surface or becomes degraded by postendocytic sorting to lysosomes. The duration and physiological effects of agonists therefore depend on β2-adrenoceptor sorting, highlighting the importance of sorting signals. Dileucine motifs within other membrane proteins act as signals for endocytosis and/or postendocytic sorting, and the β2-adrenoceptor has a dileucine motif within helix 8 that might play a role in efficient receptor recycling and/or downregulation. β2-adrenoceptor internalization and sorting were studied in HEK293 cells stably expressing wild type or mutant dialanine L339A,L340A β2-adrenoceptors. The mutant β2-adrenoceptors showed a significantly lower initial rate of phosphorylation at the prominent G-protein coupled receptor kinase (GRK) sites Ser355 and 356 compared to wild type β2-adrenoceptors. Furthermore, the agonist-induced endocytic rate constant for L339A,L340A β2-adrenoceptors was reduced to ~ 25% that of wild type β2-adrenoceptors, which resulted in a similar reduction in agonist-induced downregulation. Internalized L339A,L340A β2-adrenoceptors recycled to the surface with a rate and extent similar to that of wild type β2-adrenoceptors. Therefore, although the role of L339,L340 in β2-adrenoceptor recycling or postendocytic sorting seems minimal, we conclude that L339,L340 is required for the initial high rate of phosphorylation by G-protein coupled receptor kinases at Ser355,356, which in turn is required for efficient β2-adrenoceptors endocytosis.
Keywords: β2-adrenoceptor; Sorting motif; G-protein coupled receptor kinase; Phosphorylation; Recycling; Endocytosis;

To determine the relationship between catechol ring modifications and the activity of caffeic acid phenethyl ester (CAPE) as a cytoprotective agent, six catechol ring-fluorinated CAPE derivatives were evaluated for their cytoprotective abilities, as well as for their antioxidant and heme oxygenase-1 (HO-1) inducing capacity in a human umbilical vein endothelial cell (HUVEC) model of oxidant stress. To ascertain the involvement of HO-1 induction in the cytoprotective effects of CAPE analogues, their ability to induce HO-1 at 20 µM was determined by reverse transcriptase polymerase chain reaction, western blotting and the use of HO-1 inhibitor tin protoporphyrin IX. There was significant induction of HO-1 by CAPE derivatives. Inhibition of HO-1 enzymatic activity resulted in reduced cytoprotection. Modification of the catechol ring of CAPE by introduction of fluorine at various positions resulted in dramatic changes in cytoprotective activity. The maintenance of at least one hydroxyl group on the CAPE catechol ring and the phenethyl ester portion was required for HO-1 induction. CAPE and its derivatives were screened for their ability to scavenge intracellular reactive oxygen species generated in HUVECs by measuring 5-(and-6)-chlormethyl-2′, 7′-dichlorodihydrofluorescein diacetate oxidation. The maintenance of 3, 4-dihydroxyl groups on the catechol ring was required for antioxidant activity, but antioxidant activity did not guarantee cytoprotection. Methylation or replacement of one hydroxyl group on the catechol ring of CAPE, however, provided both pro-oxidant and cytoprotective activities. These results indicate that the induction of HO-1 plays a more important role in the cytoprotective activity of CAPE derivatives than their direct antioxidant activity.
Keywords: Caffeic acid phenethyl ester; Fluorinated derivative; Cytoprotection; Oxidative stress; Human endothelial cell; Heme oxygenase-1; Structure–activity relationship;

Structural changes and inhibition of sucrase after binding of scopolamine by Dariush Minai-Tehrani; Negar Fooladi; Saeed Minoui; Zahra Sobhani-Damavandifar; Tooka Aavani; Soraya Heydarzadeh; Farnoosh Attar; Mina Ghaffari; Habibollah Nazem (23-26).
Scopolamine (hyoscine) is commonly used as an anticholinergic drug to relieve nausea, vomiting and dizziness of a motion sickness as well as recovery from anesthesia and surgery. Sucrase as a hydrolytic enzyme breaks down sucrose into its monomers, glucose and fructose. The aim of this study was to evaluate the effect of scopolamine on the activity and the structural changes of yeast sucrase. The results showed that binding of scopolamine to sucrase could inhibit the enzyme activity. A non-competitive inhibition was observed in different concentrations of scopolamine (0.6 to 3.6 mM). The study by circular dichroism measurement in far-UV showed that the absolute enzyme exhibited a flat negative trough, indicating the presence of α-helices and β-sheet structures in the enzyme. Binding of the inhibitor on the enzyme made a deeper trough at 218 nm, suggesting the increasing of β-sheet content of the enzyme. Fluorescence measurement showed that binding of scopolamine to free enzyme and enzyme–substrate complex increased the peak intensity at 350 nm and also induced red shift. Our findings suggest that scopolamine binds to the location other than the active site of enzyme and that the binding causes structural changes and inhibits the enzyme activity.
Keywords: Drug; Scopolamine; Enzyme; Inhibition; Sucrase;

Neuropeptide S and its receptor represent a novel neurotransmitter system mainly expressed in the brain. A single nucleotide polymorphism in the first extracellular loop (I107) increases the potency of neuropeptide S and has been identified for both the human neuropeptide S receptor short (A) and long (B) C-terminal forms. Preliminary human genetic studies link this polymorphism to asthma, panic disorders and altered sleep behavior. No polymorphism or splice variants have been reported for the rat neuropeptide S receptor, however it carries an isoleucine at position 107. To identify a suitable tracer for neuropeptide S receptor binding and investigate the role of specific amino acids within neuropeptide S we carried out mutagenesis of the peptide and assessed the ability of the mutations to stimulate calcium release in HEK293 cells expressing human neuropeptide S receptor variants (A, B, AI107, BI107) and rat neuropeptide S receptor. Replacement of threonine at position 8 by arginine and methionine at position 10 by tyrosine resulted in a mutant peptide slightly more potent on all neuropeptide S receptor variants compared to neuropeptide S and more importantly the iodinated mutant peptide was found to be a suitable tracer for binding studies with improved signal to noise ratio and stability compared to [125I–Y10] neuropeptide S. Replacement of serine at position 1 of neuropeptide S peptide by arginine resulted in a complete loss of potency for the neuropeptide S receptor (long and short form) but not for the I107 receptor variants (long and short) or rat neuropeptide S receptor.
Keywords: Neuropeptide S receptor; Neuropeptide S peptide; Mutagenesis; Radioligand binding; Intracellular calcium;

Radioligand binding characterization of the bradykinin B2 receptor in the rabbit and pig ileal smooth muscle by Stefania Meini; Paola Cucchi; Claudio Catalani; Francesca Bellucci; Paolo Santicioli; Sandro Giuliani; Carlo Alberto Maggi (34-39).
Several species-related differences have been reported in kinin B2 receptor pharmacology. The present study aimed to evaluate the affinity of the bradykinin B2 receptor antagonist MEN16132 for the rabbit and pig B2 receptor, and radioligand binding experiments using [3H]bradykinin and membranes of rabbit and pig ileum smooth muscle were conducted. The [3H]bradykinin binding was characterized by homologous displacement curves indicating Kd values of 0.65 and 0.33 nM in rabbit and pig, respectively. The B2 receptor specificity of [3H]bradykinin binding was shown by the low affinity (> µM) displayed by agonists ([desArg9]bradykinin and Lys[desArg9]bradykinin) and antagonists [Leu8,desArg9]bradykinin and Lys[Leu8,desArg9]bradykinin) selective for the B1 receptor. The affinity of MEN16132 and other antagonists was determined by inhibition curves (pKi values in the rabbit and pig assay, respectively): MEN16132 (10.4 and 10.3) and peptide compounds such as icatibant (10.1 and 9.9) and MEN11270 (10.3 and 10.1) displayed subnanomolar potency in both assays; the nonpeptide LF16-0687 (8.4 and 8.5) and FR173657 (8.2 and 9.1) exhibited a different affinity pattern, whereas WIN64338 displayed low affinity (5.7 and ≤ 5). Results are discussed focusing on comparisons with previous findings obtained in rabbit and pig vascular functional assays, but also with those obtained in analog guinea pig and mouse assays and at the human B2 receptor. An attempt to highlight differences which can undertake ligands selectivity across the species is presented. In conclusion, the present study indicates MEN16132 as the only nonpeptidic compound which displays an even subnanomolar affinity for the rabbit and pig B2 receptor.
Keywords: FR173657; Icatibant; LF16-0687; MEN16132; WIN64338;

Icariin exterts negative effects on human gastric cancer cell invasion and migration by vasodilator-stimulated phosphoprotein via Rac1 pathway by Yongping Wang; Huimin Dong; Meng Zhu; Yangwen Ou; Jie Zhang; Hesheng Luo; Ruoyu Luo; Junzhu Wu; Ming Mao; Xiaoheng Liu; Jingwei Zhang; Lei Wei (40-48).
Cellular movement is mainly orchestrated by actin-dependent cytoskeleton in which Rho GTPase Rac1 or vasodilator-stimulated phosphoprotein (VASP) closely collaborates. In the present in vitro study, we investigated the inhibitory effect and underlying molecular mechanism of icariin, a pure extract of the traditional Chinese medicine Herba epimedii, on the invasive and migration properties of human gastric cancer cell line BGC-823. At 50% growth-inhibiting concentration, icariin significantly suppressed tumor cells migration and invasion, which were traceable to down-regulation of Rac1 and VASP. Together with icariin, the selected siRNA targeting Rac1 or VASP reinforced these inhibitory effects. Rac1-siRNA-dependent down-regulation of Rac1 led to a large drop in VASP expression, whereas VASP-siRNA led to a slight fall in Rac1 expression, implying that the amount of Rac1 may influence VASP expression level. Moreover, transfection with Rac1 plasmids pcDNA3-EGFP-Rac1-Q61L led to the enhancement in expression level of both Rac1 and VASP. These results indicate that icariin exerts negative effects on tumor cell invasion and migration via the Rac1-dependent VASP pathway and may be a potential anti-cancer drug.
Keywords: Icariin; Human gastric cancer cell; Rac1; VASP (vasodilator-stimulated phosphoprotein); Cell migration; Cell invasion;

The L-, N-, and T-type triple calcium channel blocker benidipine acts as an antagonist of mineralocorticoid receptor, a member of nuclear receptor family by Hiromichi Kosaka; Kazunori Hirayama; Nobuyuki Yoda; Katsutoshi Sasaki; Tetsuya Kitayama; Hideaki Kusaka; Masahiro Matsubara (49-55).
Aldosterone-induced activation of mineralocorticoid receptor, a member of the nuclear receptor family, results in increased tissue damage such as vascular inflammation and cardiac and perivascular fibrosis. Benidipine, a long-lasting dihydropyridine calcium channel blocker, is used for hypertension and angina. Benidipine exhibits pleiotropic pharmacological features such as renoprotective and cardioprotective effects through triple blockade of L-, N-, and T-type calcium channels. However, the mechanism of additional beneficial effects on end-organ damage is poorly understood. Here, we examined the effects of benidipine and other calcium channel blockers on aldosterone-induced mineralocorticoid receptor activation using luciferase reporter assay system. Benidipine showed more potent activity than efonidipine, amlodipine, or azelnidipine. Benidipine depressed the response to higher concentrations of aldosterone, whereas pretreatment of eplerenone, a steroidal mineralocorticoid receptor antagonist, did not. Binding studies using [3H] aldosterone indicated that benidipine and other calcium channel blockers competed for binding to mineralocorticoid receptor. Benidipine and other calcium channel blockers showed antagonistic activity on Ser810 to Leu mutant mineralocorticoid receptor, which is identified in patients with early-onset hypertension. On the other hand, eplerenone partially activated the mutant. Results of analysis using optical isomers of benidipine indicated that inhibitory effect of aldosterone-induced mineralocorticoid receptor activation was independent of its primary blockade of calcium channels. These results suggested that benidipine directly inhibits aldosterone-induced mineralocorticoid receptor activation, and the antagonistic activity might contribute to the drug's pleiotropic pharmacological features.
Keywords: Benidipine; Calcium channel blocker; Mineralocorticoid receptor; Aldosterone;

Protective effect of all-trans retinoic acid on NMDA-induced neuronal cell death in rat retina by Kenji Sakamoto; Masahide Hiraiwa; Maki Saito; Tsutomu Nakahara; Yoji Sato; Taku Nagao; Kunio Ishii (56-61).
We histologically examined the effects of all-trans retinoic acid (ATRA) on neuronal injury induced by intravitreous injection of N-methyl-d-aspartic acid (NMDA) (200 nmol/eye). Treatment with ATRA for 7 days (15 mg/kg for the first two days and 10 mg/kg for the following five days, p.o.) reduced the decrease of cell number in the ganglion cell layer and the inner nuclear layer 7 days after NMDA injection. TUNEL staining 6 h after NMDA injection showed that treatment with ATRA (15 mg/kg, p.o.) 1 h prior to NMDA injection reduced the number of apoptotic cells in the ganglion cell layer and inner nuclear layer. The anti-apoptotic effect of ATRA was vanished by intravitreous injection of U0126, an extracellular signal-regulated kinase/mitogen-activated protein kinase kinase inhibitor (1 nmol/eye). These results suggest that ATRA has a protective effect, which is medicated by extracellular signal-regulated kinase pathway, on NMDA-induced apoptosis in the rat retina. ATRA may be useful as a therapeutic drug against retinal diseases that cause glutamate neurotoxicity.
Keywords: All-trans retinoic acid; Retina; N-methyl-d-aspartic acid; Extracellular signal-regulated kinase;

We have previously shown the involvement of central endothelin (ET) mechanisms in morphine analgesia and tolerance. Here we investigated the interaction of centrally administered endothelin ETA receptor antagonist, BMS182874, with DAMGO (µ opioid receptor agonist), SNC80 (δ opioid receptor agonist), U50,488H (κ opioid receptor agonist), and oxycodone (µ and κ opioid receptor agonist) towards antinociception, tolerance to antinociception and body temperature. Antinociception was determined using tail-flick latency method. BMS182874 (50 µg, i.c.v.) treatment alone did not produce analgesia or change in body temperature. However, BMS182874 significantly enhanced antinociception response of DAMGO (66.75%), SNC80 (62.40%), U50,488H (55.38%), and oxycodone (61.72%). Chronic treatment with DAMGO, SNC80, U50,488H or oxycodone, induced tolerance to antinociception. Treatment with BMS182874 restored antinociceptive effect in mice that were tolerant to DAMGO, SNC80, U50,488H as well as oxycodone. Antinociceptive response of DAMGO, SNC80, U50,488H, and oxycodone in tolerant mice treated with BMS182874 was significantly higher (44.55%, 37.48%, 43.02%, and 56.08%, respectively) compared to tolerant mice treated with vehicle. Body temperature decreased with DAMGO, SNC80, U50,488H, and oxycodone; tolerance did not develop to hypothermic effect and BMS182874 did not affect DAMGO, SNC80, U50,488H, or oxycodone induced changes in body temperature. Opioid-antagonist naloxone, completely blocked antinociceptive effect of DAMGO, SNC80, U50,488H or oxycodone and potentiation of antinociception by BMS182874. It is concluded that BMS182874 potentiated antinociception and restored antinociceptive effect in mice tolerant to µ, δ and κ selective, as well as a non-selective opioid receptor agonist. Therefore, endothelin ETA receptor antagonists could be useful in the restoration of antinociceptive effect during tolerance to opiates.
Keywords: Mu; Kappa; Delta; Opioid receptors; DAMGO; Oxycodone; SNC80; U50,488H; BMS182874; Antinociception; Tolerance; Endothelin;

Presynaptic kainate receptors increase GABAergic neurotransmission in rat periaqueductal gray neurons by Michiko Nakamura; Kyu-Hyung Choi; Sung-Keun Choi; Chung-Sik Do; Ju-Hye Jun; Hyung-Kook Kwon; So-Min Lee; Ryu-Jin Moon; Ki-Joung Yi; Il-Sung Jang (72-78).
Neurons within the periaqueductal gray (PAG) have been implicated in the central regulation of pain signals by affecting the descending inhibitory pathway. Here we report on the functional role of presynaptic kainate receptors within the PAG. Using a conventional whole-cell patch clamp technique, we recorded GABAergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) from mechanically isolated rat PAG neurons in the presence of 300 nM tetrodotoxin and 20 µM dl-2-amino-5-phosphonovaleric acid under voltage-clamp conditions. Kainic acid at a 10 µM concentration significantly increased the frequency of GABAergic mIPSCs without affecting their amplitude, suggesting that kainic acid acts presynaptically to enhance spontaneous GABA release. The kainic acid-induced increase in mIPSC frequency was completely blocked by CNQX, a selective AMPA/kainate receptor antagonist. While neither AMPA nor NMDA affected GABAergic mIPSC frequency, ATPA, a selective agonist of GluR5-containing kainate receptors, increased GABAergic mIPSC frequency in a concentration-dependent manner. The kainic acid-induced increase in mIPSC frequency was completely suppressed either in the presence of 100 µM Cd2+, a general voltage-dependent Ca2+ channel (VDCC) blocker, or in the Na+-free external solution. These results suggest that presynaptic kainate receptors have a low permeability to Ca2+, and that their activation elicits a presynaptic depolarization large enough to activate presynaptic VDCCs. Presynaptic kainate receptors on GABAergic nerve terminals appear to modulate GABAergic transmission, and in doing so may play an important role in the regulation of PAG neuron excitability.
Keywords: Kainate receptors; Presynaptic facilitation; GABAergic mIPSCs; PAG neurons; Patch clamp;

Effect of Corilagin on anti-inflammation in HSV-1 encephalitis and HSV-1 infected microglias by Yuan-Jin Guo; Lei Zhao; Xiao-Feng Li; Yuan-Wu Mei; Shu-Ling Zhang; Jun-Yan Tao; Yu Zhou; Ji-Hua Dong (79-86).
The aim of this explore is to study the anti-inflammatory effect of Corilagin in herpes simplex virus (HSV)-1 infected microglial cells and HSV-1 infected mouse brain. The cellular model was set with microglial cells stimulated by HSV-1 and divided respectively, into virus, astragalus polysaccharides (APS), Dexamethasone and Corilagin group. A normal control group consisting of uninfected microglial cells was also included. ELISA for measuring TNF-α, IL-1β and IL-10 and Greiss method for detecting NO secretion in supernatant, flow cytometry assay for examining apoptosis rate, expression of caspase-3, caspase-8, caspase-9 and caspase-12, and western-blot for measuring protein expression of cytochrome c were performed. The animal model was set up using Balb/c male mice that were intracranially inoculated with HSV-1. Animals were then divided in groups as described for the cellular model. Here, too a normal control group was included. HE staining was used to assay pathological changes in brain. As results, after Corilagin intervention, the release of TNF-α, IL-1β and NO from HSV-stimulated migroglia cells was significantly inhibited. Furthermore, Corilagin induced apoptosis of HSV-stimulated microglia through all the 3 known apoptotic pathways. The animal model treated with Corilagin also displayed significant decrease of herpes simplex encephalitis induced brain pathological changes. In conclusion, Corilagin has the potential to reduce HSV-1-induced inflammatory insult to the brain, and its mode of action is through the induction of apoptosis of microglias and reduction of cytokines production.
Keywords: Corilagin; Inflammation; Herpes simplex virus-1; Encephalitis; Apoptosis; Microglia;

The aim of the present study was the investigation of the mechanism, by which bradykinin B2 receptor stimulation evokes an increase of the cytosolic Ca2+ concentration in rat submucosal plexus. In ganglionic cells within the intact submucosal plexus, the Ca2+-response evoked by bradykinin was suppressed by Ni2+, suggesting that Ca2+ enters the cell through voltage-gated Ca2+ channels (Cav channels). Inhibition of Cav channel subtypes P, T and R with ω-agatoxin IVA, flunarizine, and SNX-482 did not affect the response to bradykinin. In contrast, verapamil, ω-conotoxin GVIA, and ω-conotoxin MVIIC attenuated the actions of bradykinin, indicating the involvement of the L-, N- and Q-subtypes of Cav channels. The combination of these three blockers had a strong inhibitory action on the bradykinin response. In order to study the mechanism of activation of Cav channels by bradykinin, isolated submucosal neurons in culture were used. Immunocytochemical stainings revealed that these neurons expressed the bradykinin B2 receptor, while the B1 receptor was absent. Isolated submucosal glial cells did not express the bradykinin B2 receptor. Whole-cell patch-clamp measurements of submucosal neurons showed that bradykinin induced a depolarisation of the membrane in average of 14 mV. The ionic mechanism underlying the depolarisation was identified with current measurements at two different membrane potentials (− 81 and 0 mV). The current associated to Na+ influx was not changed by bradykinin, whereas the current representing K+ outflux was reduced by 26%. The present results suggest that at submucosal neurons from the rat colon bradykinin induces a depolarisation by decreasing the K+ conductance, followed by activation of the Cav channels, which mediates the increase of the cytosolic Ca2+ concentration.
Keywords: Bradykinin; Bradykinin B2 receptor; Cell culture; Cytosolic Ca2+; Membrane potential; Patch-clamp; Submucosal neuron; Voltage-gated Ca2+ channel;

Beta-asarone protection against beta-amyloid-induced neurotoxicity in PC12 cells via JNK signaling and modulation of Bcl-2 family proteins by Chengchong Li; Guihua Xing; Miaoxian Dong; Li Zhou; Jiaming Li; Gang Wang; Dejia Zou; Rui Wang; Jicheng Liu; Yingcai Niu (96-102).
Neurodegenerative brain disorders such as Alzheimer's disease have been well investigated. However, significant methods for the treatment of the promotion and progression of Alzheimer's disease are unavailable to date. Apoptosis is a crucial pathway in neuronal loss in Alzheimer's disease patients. Thus, the suppression of apoptosis may be an effective therapeutic strategy for Alzheimer's disease. In this study, we evaluated the effect of β-asarone on β-amyloid (Aβ)-induced toxicity in cultured PC12 cells. Our data show significant induction of apoptosis in PC12 cells incubated with Aβ peptide, and this effect was reduced by β-asarone. Beta-asarone reduced Aβ-induced JNK activation. In addition, β-asarone attenuates Aβ-induced down-regulation of Bcl-w and Bcl-xL in a JNK-dependent manner, and subsequent inhibition mitochondrial release of cytochrome c and activation of caspase-3. Together, these findings indicate that Aβ-induced apoptosis of PC12 cells proceeds through mitochondrial pathway. Further, the JNK signaling cascade plays a role in regulating the anti-apoptotic effects of β-asarone. Thus, our results indicate that β-asarone might be a potentially therapeutic compound for Alzheimer's disease.
Keywords: Alzheimer's disease; Apoptosis; Beta-asarone; Beta-amyloid; C-Jun N-terminal kinase;

Protection in rats with heatstroke: Hyperbaric oxygen vs activated protein C therapy by Chao-Hung Yeh; Zhih-Cherng Chen; Chuan-Chih Hsu; Mao-Tsun Lin; Chien-Chang Chen (103-108).
The present study was attempted to evaluate the therapeutic effects of activated protein C and/or hyperbaric oxygen in an animal model of heatstroke. Sixty-eight minutes heat stress (43 °C) initiated, the anesthetized rats were randomized to several groups and administered: 1) no resuscitation (vehicle solution plus normabaric air, 2) intravenous activated protein C (1 mg in 1 ml of normal saline per kg of body weight), 3) hyperbaric oxygen (100% oxygen at 202 kpa for 17 min), and 4) intravenous activated protein C plus hyperbaric oxygen. Another group of rats exposed to room temperature (26 °C) was used as normothermic controls. Blood sampling was 0 min, 70 min, and 85 min after heat stress initiated. When the vehicle-treated rats underwent heat exposure, their survival time values found were to be 19–25 min. Resuscitation with activated protein C or hyperbaric oxygen significantly and equally improved survival during heatstroke (134–159 min). As compared with those of activated protein C or hyperbaric oxygen alone, combined activated protein C and hyperbaric oxygen significantly had higher survival time values (277–347 min). All vehicle-treated heatstroke animals displayed systemic response, hypercoagulable state, and hepatic and renal dysfunction. Combined activated protein C and hyperbaric oxygen therapy reduced these heatstroke reactions better than activated protein C or hyperbaric oxygen alone. The results indicate consequently, combined activated protein C and hyperbaric oxygen therapy heightens benefit in combating heatstroke reactions.
Keywords: Heatstroke; Protein C; Inflammation; Coagulation; Hyperbaric oxygen; Multiorgan dysfunction;

Dopa decarboxylase inhibitors are routinely used to potentiate the effects of L-DOPA in the treatment of Parkinson's disease. However, neither in clinical use nor in experimental models of Parkinson's disease have the timing and dose of dopa decarboxylase inhibitors been thoroughly explored. We now report on the choice of dopa decarboxylase inhibitors, dose and the time of dosing relationships of carbidopa, benserazide and L-α-methyl dopa (L-AMD) in potentiating the effects of L-DOPA in the 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)-treated common marmoset. Pre-treatment with benserazide for up to 3 h did not alter the motor response to L-DOPA compared to simultaneous administration with L-DOPA. There was some evidence of a relationship between carbidopa and benserazide dose and increased locomotor activity and the reversal of motor disability. But in general, commonly used dose levels of dopa decarboxylase inhibitors appeared to produce a maximal motor response to L-DOPA. In contrast, dyskinesia intensity and duration continued to increase with both carbidopa and benserazide dose. The novel dopa decarboxylase inhibitor, L-AMD, increased locomotor activity and improved motor disability to the same extent as carbidopa or benserazide but importantly this was accompanied by significantly less dyskinesia. This study shows that currently, dopa decarboxylase inhibitors may be routinely employed in the MPTP-treated primate at doses which are higher than those necessary to produce a maximal potentiation of the anti-parkinsonian effect of L-DOPA. This may lead to excessive expression of dyskinesia in this model of Parkinson's disease and attention should be given to the dose regimens currently employed.
Keywords: L-DOPA; Carbidopa; Benserazide; Motor disability; Dyskinesia; MPTP; Primate; Parkinson's disease;

The anti-fatigue effect of dicethiamine hydrochloride (DCET) was assessed and compared to that of thiamine hydrochloride (VB1HCl) in rats. The absorbability and tissue distribution of thiamine after oral administration of DCET and VB1HCl were also examined. To create fatigued animals, male SD rats were placed in plastic cages containing 1.5 cm of water for 5 consecutive days. The extent of fatigue was evaluated by a weight-loaded forced swimming test. After oral administration of DCET or VB1HCl to non-fatigued rats, blood and tissues were serially collected to determine the concentrations of thiamine and its phosphate esters. Pharmacokinetic analysis was performed to examine the thiamine profile in the body after administration of DCET or VB1HCl. Swimming time was significantly shorter for the fatigued vehicle group than the non-fatigued group. DCET (30 and 100 mg/kg) significantly prolonged the swimming time compared to the fatigued vehicle group. The anti-fatigue effect of VB1HCl (70.1 mg/kg) was not significant in our set of results. Both DCET and VB1HCl were rapidly absorbed into the circulating blood as thiamine and eventually became localized in the organs. Thiamine was distributed at higher concentrations to the blood, heart, thigh muscles, cerebellum, hippocampus, and thalamus after administration of DCET compared to VB1HCl. These results indicate that DCET is a vitamin B1 derivative that has excellent absorbability and transformability in tissues and suggest that DCET as an oral therapy may be useful against combined mental and physical fatigue, such as that often encountered in contemporary society.
Keywords: Fatigue; Thiamine; Dicethiamine; Absorption; Tissue distribution;

Mangiferin, a naturally occurring glucoxilxanthone improves long-term object recognition memory in rats by Gilberto L. Pardo Andreu; Natasha Maurmann; Gustavo Kellermann Reolon; Caroline B. de Farias; Gilberto Schwartsmann; René Delgado; Rafael Roesler (124-128).
Mangiferin (2-β-d-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone) is a xanthone widely distributed in higher plants showing antioxidative, antiviral, anticancer, antidiabetic, immunomodulatory, hepatoprotective, and analgesic effects. In the present study, we have investigated the effects of systemic administration of mangiferin on behavioral outcomes of neurological function in normal rats. A single intraperitoneal injection of mangiferin (10, 50, or 100 mg/kg body weight) enhanced novel object recognition (NOR) memory when given immediately post-training. The administration of mangiferin 6 h post-training did not affect NOR memory. There were no significant differences between groups in the total time exploring both objects, indicating that mangiferin did not affect locomotion or motivation. Mangiferin stimulated cell proliferation and induced a significant increase in the supernatant levels of nerve growth factor (NGF) and tumor necrosis factor (TNF)-α in vitro in human U138-MG glioblastoma cells. The results indicate that mangiferin enhances recognition memory through a mechanism that might involve an increase in neurotrophin and cytokine levels.
Keywords: Mangiferin; Memory; Object recognition; Nerve growth factor; Tumor necrosis factor;

The effects of daily administration of physiological saline of baclofen (1 and 4 mg/kg, i.p.) for 27 days were investigated on food intake and body weight in non-deprived rats in Experiment 1. Baclofen (1 and 4 mg/kg) significantly increased daily short-term food intake when measured at 30 min (F (2,15)  = 11.011, P  < 0.01) and 90 min (F (2,15)  = 7.3801, P  < 0.01) over the 27 day experimental period.. Tolerance did not develop to the short-term hyperphagic effects of baclofen. Baclofen (1 mg/kg) had no significant effects on body weight gain of the rats compared with controls. By contrast, baclofen (4 mg/kg) significantly (P  < 0.05) decreased the body weight gain of the animals. In Experiment 2, the effect of daily administration of baclofen (4 mg/kg, i.p.) for 24 days was investigated on 24 h food intake in rats measured after the first, eight, fifteenth and twenty second injections. The 24 h food intake of the animals was not significantly different from those of control rats on any of the measurement days (F (1,14)  = 1.602, ns). However, the body weight gain of the rats chronically treated with baclofen (4 mg/kg) was significantly reduced. (F (1,14)  = 14.011, P  < 0.01). The observations that chronic administration of baclofen (4 mg/kg) stimulates short-term food intake without affecting long term (24 h) feeding, but decreases body weight gain, suggest that baclofen may act through different mechanisms to influence food intake and body weight.
Keywords: GABAB receptor; Food intake; Baclofen; Chronic; Body weight gain; Tolerance;

Involvement of l-arginine–nitric oxide–cyclic guanosine monophosphate pathway in the antidepressant-like effect of bis selenide in the mouse tail suspension test by Cristiano R. Jesse; Ethel A. Wilhelm; Cristiani F. Bortolatto; João B.T. Rocha; Cristina W. Nogueira (135-141).
The present study investigated a possible antidepressant-like effect of bis selenide by using the forced swimming and the tail suspension tests. The involvement of the l-arginine–nitric oxide–cyclic guanosine monophosphate signaling pathway in the antidepressant-like action of bis selenide was investigated. Bis selenide, given by oral route at doses of 0.5–5 mg/kg, decreased the immobility time in the forced swimming and tail suspension tests. Pretreatment with l-arginine (750 mg/kg, intraperitoneal, i.p., a nitric oxide precursor), sildenafil (5 mg/kg, i.p., a phosphodiesterase 5 inhibitor) or S-nitroso-N-acetyl-penicillamine (25 μg/site, intracerebroventricular, i.c.v., a nitric oxide donor) reversed the reduction in the immobility time elicited by bis selenide (1 mg/kg, p.o.) in the tail suspension test. Bis selenide (0.1 mg/kg, p.o., a subeffective dose) produced a synergistic antidepressant-like effect with NG-nitro-l-arginine (0.3 mg/kg, i.p., an inhibitor of nitric oxide synthase) or 7-nitroindazole (25 mg/kg, i.p., a specific neuronal nitric oxide synthase inhibitor) in the tail suspension test. Pretreatment of animals with methylene blue (10 mg/kg, i.p., an inhibitor of nitric oxide synthase and soluble guanylate cyclase) or 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (30 pmol, i.c.v., a specific inhibitor of soluble guanylate cyclase), at subeffective doses, caused a synergistic effect with bis selenide in the tail suspension test. Bis selenide (1 mg/kg, p.o.), at an effective dose in the forced swimming and tail suspension tests, caused a significant decrease in the mouse cerebral nitrate/nitrite levels. The antidepressant-like effect of bis selenide in the tail suspension test is dependent on the inhibition of the l-arginine–nitric oxide–cyclic guanosine monophosphate pathway.
Keywords: Bis selenide; Selenium; Antidepressant; Oxide nitric; Cyclic guanosine monophosphate; Immobility;

Lipids and lipoproteins play an important role in the pathology of myocardial infarction. This manuscript reports the preventive effect of quercetin on lipids, lipoproteins and electrocardiogram in isoproterenol treated cardiotoxic male Wistar rats. Quercetin (10 mg/kg) was administered orally as pretreatment to Wistar rats daily for seven days. After pretreatment, rats were induced with myocardial infarction by subcutaneous injection of isoproterenol (100 mg/kg) at an interval of 24 h for two days. Quercetin pretreatment significantly (P  < 0.05) lowered ST-segment elevation and decreased the levels of lipid peroxidation products in plasma and heart in isoproterenol treated cardiotoxic rats. Quercetin pretreatment also significantly (P  < 0.05) reduced the levels of total cholesterol, triglycerides and free fatty acids in serum, heart and heart mitochondria and serum phospholipids in isoproterenol treated cardiotoxic rats. Significantly (P  < 0.05) increased levels of heart and heart mitochondria phospholipids were observed in quercetin pretreated isoproterenol treated cardiotoxic rats. It's pretreatment also significantly (P  < 0.05) reduced the levels of serum low-density lipoprotein and very low-density lipoprotein-cholesterol and significantly (P  < 0.05) increased serum high density lipoprotein-cholesterol in isoproterenol treated cardiotoxic rats. In addition, quercetin significantly (P  < 0.05) decreased the activity of 3-hydroxy-3-methyl glutaryl-Coenzyme A reductase in plasma and liver and significantly (P  < 0.05) increased the activity of liver lecithin cholesterol acyl transferase in isoproterenol treated cardiotoxic rats. In vitro study on total antioxidant activity clearly revealed the antioxidant property of quercetin. Thus, the antioxidant activity of quercetin inhibits lipid peroxidation and prevents accumulation of lipids, alterations in lipoproteins and electrocardiogram in isoproterenol treated cardiotoxic rats.
Keywords: Cardiotoxicity; Isoproterenol; Lipid; Lipoprotein; Quercetin;

This study characterised the effect of a hypercholesterolemic diet on the interactions of hormone receptors in the rabbit aorta, both in homologous desensitisation to angiotensin II and cross talk between α1-adrenoceptors and angiotensin AT1 receptors. Rabbits were fed either a normal chow or a diet containing 1% cholesterol for 6–7-weeks. Isometric contractions were measured in endothelium-intact or endothelium-removed aortic rings from control and hypercholesterolemic rabbits. Concentration response curves to angiotensin II or noradrenaline incubated with or without prazosin or losartan were performed. In another group, the resting potential was recorded at baseline and following angiotensin II or noradrenaline stimulation. Rabbits fed a hypercholesterolemic diet showed higher plasma levels of total cholesterol and LDL-cholesterol and impaired relaxation to acetylcholine. Homologous desensitisation to angiotensin II was found in endothelium-intact but not in endothelium-removed arteries. Cross talk between α1-adrenoceptors and angiotensin AT1 receptors was modified with respect to physiological conditions. In control rabbits, angiotensin II desensitised the noradrenaline response but noradrenaline did not modify the angiotensin II-response. However, in hypercholesterolemic rabbits, angiotensin II sensitised the noradrenaline-response and noradrenaline desensitised the angiotensin II-response. Furthermore, the resting potential remains hyperpolarised after noradrenaline stimulation in hypercholesterolemic rabbits. Modifications in homologous desensitisation to angiotensin II and cross talk between α1-adrenoceptors and angiotensin AT1 receptors suggest that hypercholesterolemia induces early tissue dysfunction by altering endothelial and smooth muscle cell regulatory properties. This may be one of the mechanisms by which hypercholesterolemia could be involved in the onset and progression of chronic vascular diseases such as hypertension and arteriosclerosis.
Keywords: Angiotensin II; Noradrenaline; Angiotensin AT1 receptor; α1 adrenoceptor; Cross talk; Endothelial dysfunction;

Heme oxygenase-1 and carbon monoxide promote neovascularization after myocardial infarction by modulating the expression of HIF-1α, SDF-1α and VEGF-B by Päivi Lakkisto; Ville Kytö; Hanna Forsten; Juha-Matti Siren; Heli Segersvärd; Liisa-Maria Voipio-Pulkki; Mika Laine; Kari Pulkki; Ilkka Tikkanen (156-164).
Heme oxygenase-1 (HO-1), a known cytoprotective enzyme implicated also in the cell cycle regulation and angiogenesis, exerts many of its beneficial effects through carbon monoxide (CO). We studied the roles of HO-1 and CO in cardiac regeneration after myocardial infarction. Prior to coronary artery ligation, male Wistar rats were given either cobolt protoporphyrin IX to induce HO-1 or CO-donor methylene chloride. Cardiac regeneration was assessed by immunohistochemistry and confocal microscopy. CO significantly increased the accumulation of c-kit+ stem/progenitor cells into the infarct area and induced formation of new coronary arteries by promoting a substantial differentiation of c-kit+ cells into vascular smooth muscle cells (c-kit+/GATA6+ cells). Furthermore, CO increased proliferation of cardiomyocytes in the infarct border area at 4 weeks post-infarction. This suggests proliferation of newly formed cardiomyocytes derived from c-kit+ cells as 10% of c-kit+ cells expressed early cardiac marker Nkx2.5. Increased expression of hypoxia-inducible factor-1α (HIF-1α), stromal cell derived factor-1α (SDF-1α) and vascular endothelial growth factor-B (VEGF-B) were found in the infarct areas of CO-donor pretreated hearts suggesting that these factors potentially promoted the migration of c-kit+ cells into the infarct area and subsequent vasculogenesis and myocardial regeneration by CO. HO-1 increased both capillary and vascular densities, while only a small increase of c-kit+ cells was found. HO-1 upregulated SDF-1α, but did not have effect on HIF-1α and VEGF-B. In conclusion, HO-1 and CO have differential roles and mechanisms of action in cardiac regeneration. Modulation of the HO-1/CO axis may provide a novel tool for the repair of cardiac injury.
Keywords: Carbon monoxide; Cardiac regeneration; Heme oxygenase-1; Myocardial infarction; Neovascularization;

Reversal of heparin-induced increases in aPTT in the rat by PM102, a novel heparin antagonist by Daniel J. Cushing; Warren D. Cooper; Marlene L. Cohen; Julie R.S. McVoy; Michael Sobel; Robert B. Harris (165-170).
Protamine is the only agent approved to reverse heparin-induced anticoagulation. Due to the significant adverse effects of protamine there is an important need for an alternative agent with an improved safety profile. The pharmacodynamics of PM102, a novel peptide-based heparin antagonist, was evaluated and compared to protamine in a rat model. Rats were dosed with intravenous heparin (50 U/kg) and 4 min later with protamine (0.25, 0.75 mg/kg single intravenous bolus) or PM102 (0.1, 0.3, 1, 3, 30 mg/kg single intravenous bolus). Blood samples were collected though 60 min for assessment of activated partial thromboplastin time (aPTT) and plasma concentration of PM102. Both doses of protamine markedly lowered the elevated aPTT to baseline values within 1 to 5 min after administration. PM102 (0.3–30 mg/kg) also rapidly and completely reversed heparin-induced increases in aPTT within 1 to 5 min. The effects of PM102 administered as an infusion over 10 min also reversed aPTT with similar potency to that observed for bolus administration. The onset of reversal with infusion was delayed relative to the same total dose given as a bolus; however, the maximum effect was similar. PM102 rapidly (T max 1–2.6 min) appeared in plasma after dosing. Concentrations of PM102 generally declined rapidly after reaching T max with a mean T 1/2 of 4 to 31 min. PM102 is a novel synthetic peptide that effectively reverses the anticoagulant effect of heparin. It's utility as a bolus injection as well as infusion, its rapid efficacy and its rapid clearance make this an ideal candidate for clinical development.
Keywords: Heparin; Protamine; Anticoagulation; PM102;

The plasma carnitine concentration regulates renal OCTN2 expression and carnitine transport in rats by Regula Schürch; Liliane Todesco; Katarina Novakova; Meike Mevissen; Bruno Stieger; Stephan Krähenbühl (171-176).
Previous findings in rats and in human vegetarians suggest that the plasma carnitine concentration and/or carnitine ingestion may influence the renal reabsorption of carnitine. We tested this hypothesis in rats with secondary carnitine deficiency following treatment with N-trimethyl-hydrazine-3-propionate (THP) for 2 weeks and rats treated with excess l-carnitine for 2 weeks. Compared to untreated control rats, treatment with THP was associated with an approximately 70% decrease in plasma carnitine and with a 74% decrease in the skeletal muscle carnitine content. In contrast, treatment with l-carnitine increased plasma carnitine levels by 80% and the skeletal muscle carnitine content by 50%. Treatment with l-carnitine affected neither the activity of carnitine transport into isolated renal brush border membrane vesicles, nor renal mRNA expression of the carnitine transporter OCTN2. In contrast, in carnitine deficient rats, carnitine transport into isolated brush border membrane vesicles was increased 1.9-fold compared to untreated control rats. Similarly, renal mRNA expression of OCTN2 increased by a factor of 1.7 in carnitine deficient rats, whereas OCTN2 mRNA expression remained unchanged in gut, liver or skeletal muscle. Our study supports the hypothesis that a decrease in the carnitine plasma and/or glomerular filtrate concentration increases renal expression and activity of OCTN2.
Keywords: Renal carnitine reabsorption; OCTN2; Secondary carnitine deficiency; N-trimethyl-hydrazine-3-propionate;

In patients with chronic obstructive pulmonary disease (COPD), mucociliary clearance of the respiratory tract is impaired due to enhanced mucus secretion and deterioration of normal ciliary activity. We investigated the effects of cyclic AMP-elevating agents with a different mode of action on ciliary beat frequency (CBF) in rat large central and small lateral airways by comparing the phosphodiesterase-4 (PDE4) inhibitors rolipram and roflumilast to the β2-adrenoceptor agonist terbutaline and the adenylyl cyclase activator forskolin. Rat precision-cut lung slices were prepared and effects of cyclic AMP-elevating agents on CBF were assessed for up to 4 h. In central airways a time- and concentration-dependent increase in CBF was seen for roflumilast (59 ± 4%, 1 µM, 60 min), rolipram (55 ± 4%, 1 µM, 60 min), terbutaline (64 ± 8%, 10 µM, 60 min) and forskolin (55 ± 8%, 100 µM, 60 min). Only roflumilast and rolipram increased CBF in lateral airways, with a similar time course and maximum efficacy (roflumilast 48 ± 5%, rolipram 54 ± 2%). Incubation of lateral airways with terbutaline (10 µM, + 11%) or forskolin (100 µM, + 1%) had negligible effects. As a major novel finding this study reveals that PDE4 inhibitors increased CBF in central as well as in lateral airways, while terbutaline and forskolin affected CBF in proximal airways only.
Keywords: Ciliary beat frequency; COPD; PDE4; Roflumilast;

Oral administration of ethanol with aspirin increases the concentration of salicylic acid in plasma and organs, especially the brain, in mice by Hideaki Kato; Kanji Yoshimoto; Masaki Kobayashi; Masaaki Sakabe; Hironao Funaki; Hiroshi Ikegaya (184-187).
Aspirin (acetylsalicylic acid) has been widely used as an over-the-counter drug to relieve pain throughout the world. In suicide attempts, aspirin is one of the most common drugs taken in large quantities. The concentration of salicylic acid shows a low-level distribution in the mouse brain, a site that may be critical regarding the expression of symptoms (e.g. hyperpnea, coma, convulsion and tremor) during acute aspirin toxicity. Therefore, it was suggested that sensitivity to salicylic acid concerning acute toxicity was higher in the brain than in other organs. Moreover, it is thought that it is common for aspirin and ethanol to be ingested at the same time. Therefore, the present study was designed to investigate the influence of ethanol on the distribution of salicylic acid, which is a primary metabolite of aspirin, and its related metabolite, salicyluric acid. The oral co-administration of aspirin (0.5 g/kg) and ethanol (2.5 g/kg; 10 ml/kg of 25% (w/v)) enhanced the concentrations of salicylic acid in the plasma and organs, especially in the brain, compared with the aspirin alone-treated group. On the other hand, ethanol did not influence the concentrations of salicyluric acid in the plasma and kidney compared with the aspirin alone-treated group. These results suggest that ethanol enhances aspirin absorption from the gastrointestinal tract but has no influence on its metabolism. Thus, it is dangerous to ingest the alcohol and aspirin at the same time, as this may exacerbate the acute toxicity of aspirin.
Keywords: Aspirin; Alcohol; Salicylic acid; Salicyluric acid; Tissue distribution; (Mouse);

Genipin protects lipopolysaccharide-induced apoptotic liver damage in d-galactosamine-sensitized mice by Seok-Joo Kim; Joon-Ki Kim; Dong-Ung Lee; Jong-Hwan Kwak; Sun-Mee Lee (188-193).
This study examined the effects of genipin, isolated from Gardenia jasminoides Ellis, on d-galactosamine (GalN) and lipopolysaccharide (LPS)-induced hepatic apoptosis and liver failure. Mice were given an intraperitoneal injection of genipin (25, 50, 100 and 200 mg/kg) 1 h before GalN (700 mg/kg)/LPS (10 μg/kg) administration. The survival rate of the genipin group was significantly higher than that of the control. Genipin markedly reduced the increases in serum aminotransferase activities and lipid peroxidation. The glutathione content decreased in GalN/LPS group, and this decrease was attenuated by genipin. Increases in serum tumor necrosis factor-α (TNF-α), which were observed in GalN/LPS-treated mice, were significantly reduced by genipin. Genipin attenuated the GalN/LPS-induced apoptosis of hepatocytes, as estimated by the caspase-3 and -8 activity assay, TNF-R1 associated death domain (TRADD) protein measurement and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. Moreover, increased cytosolic cytochrome c protein was reduced by genipin. After 3 h of GalN/LPS injection, nuclear phosphorylated c-Jun (p-c-Jun) level was significantly increased, whereas it was attenuated by genipin. Also, the increased nuclear level of nuclear factor-κB and the decreased cytosolic level of IκB-α protein were significantly attenuated by genipin. Our results suggest that genipin offers marked hepatoprotection against damage induced by GalN/LPS related with its antioxidative, anti-apoptotic activities, and inhibition of NF-κB nuclear translocation and nuclear p-c-Jun expression.
Keywords: Genipin; Galactosamine/lipopolysaccharide; Apoptosis; Fulminant hepatic failure;

The role of ATP-sensitive potassium channel on acute urinary retention and subsequent catheterization in the rat by Fumiya Ohmasa; Motoaki Saito; Shogo Shimizu; Sousuke Taniguchi; Fotios Dimitriadis; Itaru Satoh; Yukako Kinoshita; Keisuke Satoh (194-197).
We investigated the role of KATP channel on acute urinary retention (AUR) induced bladder dysfunction. Eight-week-old female Sprague–Dawley rats were divided into seven groups: a sham-operated control group, an AUR group, and five AUR groups treated with: two different KATP channel openers namely nicorandil (3 or 10 mg/kg), or cromakalim (100 or 300 μg/kg), or one KATP channel inhibitor namely glibenclamide (5 mg/kg). The drugs were administered 30 min before induction of AUR. After the urethra was obstructed with a clip, AUR was induced by intravesical infusion of 2.5 ml of saline via cystostomy. Following a 30 min obstruction the bladder was allowed to drain with a catheter in place for 60 min with real-time monitoring of intravesical pressure and blood flow. After the experimental period, the bladder function was assessed, using organ bath techniques (carbachol and 100 mM KCl). AUR increased the intravesical pressure and decreased the blood flow. The subsequent catheterization decreased the intravesical pressure and increased the blood flow. AUR group reduced significantly the contractile responses to both carbachol and KCl compared with the control group. Nicorandil and cromakalim but not glibenclamide prevented the bladder dysfunction after AUR suggesting that KATP channel openers may prevent the bladder dysfunction caused by AUR and subsequent catheterization.
Keywords: Acute urinary retention; Nicorandil; Cromakalim; Glibenclamide; Pharmacological preconditioning;

In recent in vitro reports, the IC50 value of ayanin (quercetin-3,7,4′-O-trimethylether) was 2.2 μM for inhibiting interleukin (IL)-4 production from purified basophils, and its therapeutic ratio was > 19. Therefore, we were interested in investigating the effects on ovalbumin induced airway hyperresponsiveness in vivo, and to clarify its potential for treating asthma. Ayanin (30–100 μmol/kg, orally (p.o.)) dose-dependently and significantly attenuated the enhanced pause (P enh) value induced by methacholine in sensitized and challenged mice. It also significantly suppressed the increases in total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils, and levels of cytokines, including IL-2, IL-4, IL-5, and tumor necrosis factor (TNF)-α in bronchoalveolar lavage fluid of these mice. However, at 100 μmol/kg, it significantly enhanced the level of interferon (IFN)-γ. In addition, ayanin (30–100 μmol/kg, p.o.) dose-dependently and significantly suppressed total and OVA-specific immunoglobulin (Ig)E levels in the serum and bronchoalveolar lavage fluid, and enhanced the IgG2a level in serum of these mice. In the present results, ayanin did not affect xylazine/ketamine-induced anesthesia, suggesting that ayanin has few or no adverse effects, such as nausea, vomiting, and gastric hypersecretion. In conclusion, the above results suggest that ayanin may have the potential for use in treating allergic asthma.
Keywords: Ayanin; Allergic asthma; Airway hyperresponsiveness; Cytokine; Phosphodiesterase inhibitor; PDE4H/PDE4L ratio;

Heterologous regulation of anion transporters by menthol in human airway epithelial cells by Masahiro Morise; Yasushi Ito; Tadakatsu Matsuno; Yoshitaka Hibino; Takefumi Mizutani; Satoru Ito; Naozumi Hashimoto; Masashi Kondo; Kazuyoshi Imaizumi; Yoshinori Hasegawa (204-211).
The present study concerns previously unreported effects of menthol, a cyclic terpene alcohol produced by the peppermint herb, on anion transporters in polarized human airway Calu-3 epithelia. Application of menthol (0.01–1 mM) attenuated transepithelial anion transport, estimated as short-circuit currents (I SC), after stimulation by forskolin (10 µM) but not before. In contrast, menthol potentiated forskolin-stimulated and -unstimulated apical Cl conductance, which reflected the cystic fibrosis transmembrane conductance regulator (CFTR: the cAMP-regulated Cl channel)-mediated conductance, without correlation to changes in cytosolic cAMP levels. These results indicate that menthol-induced attenuation of forskolin-induced I SC despite CFTR up-regulation was due to cAMP-independent inhibition of basolateral anion uptake, which is the rate-limiting step for transepithelial anion transport. Analyses of the responsible basolateral anion transporters revealed that forskolin increased both bumetanide (an inhibitor of the basolateral Na+–K+–2Cl cotransporter [NKCC1])- and DNDS (an inhibitor of basolateral HCO3 -dependent anion transporters [NBC1/AE2])-sensitive I SC in the control whereas only the former was prevented by the application of menthol. Neither the bumetanide- nor DNDS-sensitive component was, however, reduced by menthol without forskolin. These heterologous effects of menthol were reproduced by latrunculin B, an inhibitor of actin polymerization. F-actin staining showed that menthol prevented forskolin-stimulated rearrangements of actin microfilaments without affecting the distribution of forskolin-unstimulated microfilaments. Collectively, menthol functions as an activator of CFTR and prevents activation of NKCC1 without affecting NBC1/AE although all of these transporters are commonly cAMP-dependent. The heterologous effects may be mediated by the actin cytoskeleton, which interacts with CFTR and NKCC1.
Keywords: Airway epithelial cell; CFTR (cystic fibrosis transmembrane conductance regulator); NKCC1 (Na+–K+–2Cl); Short-circuit current; Menthol;

Novel p38 MAPK inhibitor ML3403 has potent anti-inflammatory activity in airway smooth muscle by Lenka Munoz; Emma E. Ramsay; Melanie Manetsch; Qi Ge; Christian Peifer; Stefan Laufer; Alaina J. Ammit (212-218).
SB203580 is the prototypical p38 MAPK inhibitor; however it cannot be used clinically due to liver toxicity. We developed a structural analogue of SB203580 – ML3403 – with equal in vitro and ex vivo p38α MAPK inhibition as SB203580, but with reduced activity towards liver cytochrome P450 enzymes. In addition, we developed a selective p38α MAPK inhibitor — CP41. The aim of this study is to compare the anti-inflammatory activity of ML3403 and CP41, with SB203580. We compare and contrast the ability of the p38 MAPK inhibitors to repress tumour necrosis factor α (TNFα)-induced interleukin 6 (IL-6) and interleukin 8 (IL-8) mRNA expression and protein secretion from airway smooth muscle cells. We also examined and compared the binding affinities of ML3403 and SB203580 to the active and inactive p38α MAPK. We demonstrate that ML3403 binds to both active and inactive p38 MAPK with high affinity and that it inhibits p38 MAPK-mediated airway smooth muscle synthetic function to an equivalent degree with SB203580. CP41 was not able to reduce IL-6 and IL-8 secretion in airway smooth muscle cells; a function of its higher IC50 against p38α MAPK when compared to SB203580 and ML3403. We show that p38 MAPK-mediated pro-inflammatory pathways in airway smooth muscle cells can be inhibited by ML3403. The anti-inflammatory activity is equivalent to the prototypical p38 MAPK inhibitor SB203580. Our results implicate a future pharmacotherapeutic strategy towards reducing inflammation in asthma and airway remodelling.
Keywords: ML3403; p38 MAPK; Inflammation; Asthma; Airway remodelling; Airway smooth muscle;

Glucosamine regulation of LPS-mediated inflammation in human bronchial epithelial cells by Yuh-Lin Wu; Yu Ru Kou; Hui-Ling Ou; Han-Yun Chien; Kun-Han Chuang; Han-Hsun Liu; Tzong-Shyuan Lee; Cheng-Yen Tsai; Meng-Lun Lu (219-226).
Inflammation is a complex process involving cytokine production to regulate host defense cascades in order to clear pathogenic agents. Upregulation of inflammatory cytokines, such as IL-6 and IL-8 by bacteria infection, occurs in pulmonary tissues and has been demonstrated to be critical to the lung inflammatory response. Glucosamine, primarily identified as an anti-arthritis supplement, has been also regarded as a potential anti-inflammatory agent. Thus we hypothesized that lipopolysaccharide (LPS) would activate IL-6 and IL-8 expressions in human primary bronchial epithelial cells and glucosamine could attenuate such an effect. The RT-PCR, real-time PCR, and ELISA analyses demonstrated that LPS-induced mRNAs encoding IL-6 and IL-8 and the subsequent secretion of IL-6 and IL-8 were inhibited by glucosamine treatment. MTT, alamarBlue, and annexin V apoptosis assays all suggested that this inhibition effect was not due to a cytotoxic effect mediated by glucosamine. Using the inhibitors of the MAP kinases and NFκB, it was revealed that p38, JNK and ERK, as well as NFκB, are all involved in LPS-induced IL-8 secretion; however only p38 is involved in LPS-induced IL-6 secretion. Immunoblot analysis further demonstrated that LPS-mediated phosphorylation of JNK and ERK, but not the LPS-induced NFκB translocation, was inhibited by glucosamine. Altogether, our results indicate that glucosamine can potently suppress LPS-induced inflammatory cytokine expression, at least in part via attenuation of MAPK activation.
Keywords: Inflammation; Bronchial epithelial cell; Glucosamine; MAP kinase;

Protective effect of amlodipine against osteoporosis in stroke-prone spontaneously hypertensive rats by Kentarou Ushijima; Yuwang Liu; Tomohiro Maekawa; Eiko Ishikawa; Yuya Motosugi; Hitoshi Ando; Shu-ichi Tsuruoka; Akio Fujimura (227-230).
Hypertensive patients have an increasing risk of osteoporosis. A recent case-controlled study has demonstrated that anti-hypertensive therapy reduced a risk of fracture in these patients. In this study, we investigated whether amlodipine protects against the reduction in bone density in stroke-prone spontaneously hypertensive rats (SHR-sp). Oral dosing of amlodipine (0.5 and 3.0 mg/kg/day) was started when SHR-sp were 3 months old, and continued for 3 months. At the end of the experiment, bone density of femur and serum concentrations of calcium, parathyroid hormone (PTH) and C-telopeptide of type I collagen (CTx), reflecting osteoclast activity, were measured. The bone density dose-dependently increased by the treatment with amlodipine. In addition, amlodipine reduced serum concentrations of calcium, PTH and CTx. This study showed that amlodipine prevents the reduction in bone density during the repeated dosing in SHR-sp. Amlodipine might exert its effect through a direct inhibition of osteoclast function and/or suppression of PTH secretion and subsequent inhibition of osteoclast activity.
Keywords: Amlodipine; Hypertension; Osteoclast; Osteoporosis; Parathyroid hormone;

Effect of the anti-diabetic drug metformin on bone mass in ovariectomized rats by Ying Gao; Yunfeng Li; Jing Xue; Yongqian Jia; Jing Hu (231-236).
Recent studies have reported bone loss in the patients with diabetes and a direct osteogenic effect of metformin on osteoblast-like cells in culture. In this study, we investigated the action of metformin on bone mass in ovariectomized (OVX) rats. Three months after either a sham surgery or bilateral ovariectomy, thirty-two female Sprague–Dawley rats were randomly assigned into four groups: (1) Sham group; (2) OVX group; (3) OVX + metformin (50 mg/kg/day) group; and (4) OVX + metformin (100 mg/kg/day) group. After 2 months of oral administration with or without metformin, tibiae were harvested for dual energy X-ray absorptiometry, micro-computed tomography (micro-CT) and histology analysis, while the bone marrow cells from tibiae were collected for measurement of the mRNAs expressions for three osteoblast genes and estrogen receptors alpha by the use of real-time RT-PCR. We found that the impaired bone density and quality induced by bilateral ovariectomy were significantly improved by the treatment of metformin (both 50 and 100 mg/kg/day), and this action could be partly mediated by regulating bone marrow cells development through induction of mechanisms regulating osteoblast markers core binding factor a1 and LDL receptor-related protein 5. These findings provide new evidence that the anti-diabetic drug metformin has a direct inhibition effect on bone loss in OVX rats, in addition to its well-documented osteogenic potency in vitro.
Keywords: Metformin; Ovariectomy; Bone density; Bone quality; Osteoblast markers;