European Journal of Pharmacology (v.651, #1-3)

Pharma–nutrition interface: The gap is narrowing by Niki A. Georgiou; Johan Garssen; Renger F. Witkamp (1-8).
The interaction between pharmacology and nutrition science is on the rise. Nutritional status is considered one of the important determinants of health and disease and several diseases of our time have a clear link with lifestyle factors including the diet. There is also increasing realization that a continuum between health and disease often exists without strict boundaries. Understanding the subtle interactions between genes, environment and homeostatic processes is the key in finding effective ways to prevent, treat or manage disease. Both pharmacologists and nutritionists are recognizing that most of the low hanging fruit has been picked, and that the one disease–one target–one drug (or nutrient) concept will provide fewer successes than it did in the past. Instead, complex multi-factorial diseases require multi-pathway understanding and multi-targeting approaches which will often result in compound combinations. Therapeutic synergy between foods and drugs does not necessarily mean that both have the same primary target. There are also examples of nutritional products that effectively contribute to the therapeutic regimen by improving the patients' general condition or by reducing side-effects of drugs. Examples of conditions and diseases that are highlighted in this review include the metabolic syndrome with its co-morbidities, immune-related diseases and HIV. With the aging population there are other fields emerging, including CNS-related diseases and cancer, where we will likely see an increased synergy between the two disciplines that seemed to have lost contact since the times of Hippocrates.
Keywords: Nutrition; Food-Pharma; Immunopharmacology; Obesity; Inflammation;

Cannabinoid CB1 receptor ligand binding and function examined through mutagenesis studies of F200 and S383 by Doree F. Sitkoff; Ning Lee; Bruce A. Ellsworth; Qi Huang; Liya Kang; RoseAnn Baska; Yanting Huang; Chongqing Sun; Annapurna Pendri; Mary F. Malley; Raymond P. Scaringe; Jack Z. Gougoutas; Patricia H. Reggio; William R. Ewing; Mary Ann Pelleymounter; Kenneth E. Carlson (9-17).
The cannabinoid CB1 G protein-coupled receptor has been shown to be a regulator of food consumption and has been studied extensively as a drug target for the treatment of obesity. To advance understanding of the receptor's three-dimensional structure, we performed mutagenesis studies at human cannabinoid CB1 receptor residues F200 and S383 and measured changes in activity and binding affinity of compounds from two recently discovered active chemotypes, arylsulfonamide agonists and tetrahydroquinoline-based inverse agonists, as well as literature compounds. Our results add support to previous findings that both agonists and inverse agonists show varied patterns of binding at the two mutated residue sites, suggesting multiple subsites for binding to the cannabinoid CB1 receptor for both functional types of ligands. We additionally find that an F200L mutation in the receptor largely restores binding affinity to ligands and significantly decreases constitutive activity when compared to F200A, resulting in a receptor phenotype that is closer to the wild-type receptor. The results downplay the importance of aromatic stacking interactions at F200 and suggest that a bulky hydrophobic contact is largely sufficient to provide significant receptor function and binding affinity to cannabinoid CB1 receptor ligands.
Keywords: Cannabinoid receptor; G protein-coupled receptor; Mutagenesis; Rimonabant; Constitutive activity; Aromatic stacking;

Inhibition of cell survival, invasion, tumor growth and histone deacetylase activity by the dietary flavonoid luteolin in human epithelioid cancer cells by Samir Attoub; Ahmed H. Hassan; Barbara Vanhoecke; Rabah Iratni; Takashi Takahashi; Anne-Marie Gaben; Marc Bracke; Salma Awad; Anne John; Hamda Ahmed Kamalboor; Mahmood Ahmed Al Sultan; Kholoud Arafat; Christian Gespach; Georg Petroianu (18-25).
Phytochemical compounds and histone deacetylase (HDAC) inhibitors are emerging as a new generation of anticancer agents with limited toxicity in cancer patients. We investigated the impact of luteolin, a dietary flavonoid, on survival, migration, invasion of cancer cells in vitro, and tumor growth in vivo. Luteolin (25–200 μM) decreased the viability of human cancer cell lines originating from the lung (LNM35), colon (HT29), liver (HepG2) and breast (MCF7/6 and MDA-MB231-1833). Luteolin effectively increased the sub-G1 (apoptotic) fraction of cells through caspase-3 and -7 dependent pathways. We provide evidence that luteolin at sub-lethal/non-toxic concentrations inhibited the invasive potential of LNM35, MCF-7/6 and MDA-MB231-1833 cancer cells using Matrigel as well as the chick heart and Oris invasion assays. Moreover, we demonstrate for the first time that luteolin is a potent HDAC inhibitor that potentiates the cytotoxicity of cisplatin in LNM35 cells and decreases the growth of LNM35 tumor xenografts in athymic mice after intraperitoneal injection (20 mg/kg/day for 18 days) Thus, luteolin, in combination with standard anticancer drugs such as cisplatin, may be a promising HDAC inhibitor for the treatment of lung cancer.
Keywords: Lung and breast cancer; Luteolin; Viability; Invasion; Caspase-3; Histones H3/H4;

Anti-proliferative effect of Kv1.3 blockers in A549 human lung adenocarcinoma in vitro and in vivo by Soo Hwa Jang; Seon Young Choi; Pan Dong Ryu; So Yeong Lee (26-32).
Voltage-gated potassium (Kv) channels are widely expressed in the plasma membranes of numerous cells and contribute to a variety of cellular functions in both excitable neuronal cells and non-excitable epithelial cells. Recently, it has been demonstrated that Kv channels are associated with the proliferation of several types of cancer cells. In the present study, we investigated the effects of suppression of Kv1.3 expression on cell proliferation and cell cycle progression in human lung adenocarcinoma, A549 cells. Treatment with margatoxin (MgTX), a selective blocker of Kv1.3 or short hairpin RNA (shRNA) against Kv1.3, significantly blocked A549 cells' proliferation. In addition, selective inhibition of Kv1.3 significantly increased expression level of p21Waf1/Cip1 and significantly decreased the expression level of Cdk4 and cyclin D3. We also applied the MgTX into a xenograft model using nude mice, and MgTX caused a reduction of tumor volume when it was injected into the tumor tissues. These results suggest that Kv1.3 may serve as a novel therapeutic target for lung adenocarcinoma therapy.
Keywords: Voltage-gated K channel; Cell proliferation; Cell cycle progression; G1–S transition; Margatoxin; shRNA;

DHF-18, a new synthetic flavonoid, induced a mitochondrial-mediated apoptosis of hepatocarcinoma cells in vivo and in vitro by Lin-bo Zhang; Lei Qiang; Fei-hong Chen; Tian Wu; Jing-jing Rong; Qing Zhao; Mei-juan Zou; Zhen Yang; Qi-dong You; Zhi-yu Li; Yu-lin Wu; Qing-long Guo (33-40).
A new synthetic flavonoid DHF-18, synthesized with a piperazine substitution, has been recently found to show potent anti-tumor activities both in vivo and in vitro. In this study, we demonstrated that DHF-18 significantly inhibited tumor growth in mice inoculated with Heps hepatoma cells without evident toxicity. After the treatment of 40 mg/kg DHF-18, the inhibitory rate of tumor weight was 53.69%. To investigate whether apoptosis induction contributed to the anti-tumor effects of DHF-18, DAPI (diamidino-phenyl-indole) staining and Annexin V/PI (Propidium iodide) double staining were performed in our tests. The data showed that DHF-18 could induce the apoptosis cell death in HepG2 cells. Moreover, the apparent increase of intracellular reactive oxygen species levels and the reduction of mitochondria ΔΨm were both observed in HepG2 cells after DHF-18 treatment. Meanwhile, the transposition of apoptotic inducing factor (AIF) from mitochondria to nuclei, the release of cytochrome c from mitochondria and the activation of caspase-3, -9 were also detected, indicating that DHF-18 may induce apoptosis through a mitochondrial-mediated pathway. Additionally, DHF-18 decreased the expression of Bcl-2 protein, whereas the levels of Bax and Bak were found to increase after DHF-18 treatment. Moreover, the activation of caspase-8, the increase of TNF-R1 (Tumor necrosis factor receptor) and Bid were found. Taken together, our results suggested that DHF-18 may induce HeG2 cells apoptosis through a mitochondrial-dependent and independent pathway.
Keywords: Flavonoid; Apoptosis; Caspase cascade; Mitochondria;

Metformin reduces cisplatin-mediated apoptotic death of cancer cells through AMPK-independent activation of Akt by Kristina Janjetovic; Ljubica Vucicevic; Maja Misirkic; Urosh Vilimanovich; Gordana Tovilovic; Nevena Zogovic; Zoran Nikolic; Svetlana Jovanovic; Vladimir Bumbasirevic; Vladimir Trajkovic; Ljubica Harhaji-Trajkovic (41-50).
Metformin is an antidiabetic drug with anticancer properties, which mainly acts through induction of AMP-activated protein kinase (AMPK). In the present study we investigated the influence of metformin on the in vitro anticancer activity of the well-known chemotherapeutic agent cisplatin. Cell viability was determined by MTT and LDH release assay, oxidative stress and apoptosis (caspase activation, DNA fragmentation, and phosphatidylserine exposure) were assessed by flow cytometry, while activation of AMPK and Akt was analyzed by immunoblotting. Although metformin reduced the number of tumour cells when applied alone, it surprisingly antagonized the cytotoxicity of cisplatin towards U251 human glioma, C6 rat glioma, SHSY5Y human neuroblastoma, L929 mouse fibrosarcoma and HL-60 human leukemia cell lines. Only in B16 mouse melanoma cells metformin augmented the cytotoxicity of cisplatin. In U251 glioma cells metformin suppressed cisplatin-induced apoptotic cell death through inhibition of oxidative stress and caspase activation. The observed cytoprotection was apparently AMPK-independent, as metformin did not further increase cisplatin-induced AMPK activation in U251 cells and other pharmacological AMPK activators failed to block cisplatin-mediated apoptosis. On the other hand, metformin induced Akt activation in cisplatin-treated cells and Akt inhibitor 10-DEBC hydrochloride or phosphoinositide 3-kinase/Akt inhibitor LY294002 abolished metformin-mediated antioxidant and antiapoptotic effects. In conclusion, the antidiabetic drug metformin reduces cisplatin in vitro anticancer activity through AMPK-independent upregulation of Akt survival pathway. These data warrant caution when considering metformin for treatment of diabetic cancer patients receiving cisplatin or as a potential adjuvant in cisplatin-based chemotherapeutic regimens.
Keywords: Metformin; Cisplatin; Cancer; Apoptosis; AMPK; Akt;

The effect of psychotropic drugs on cytochrome P450 2D (CYP2D) in rat brain by Anna Haduch; Ewa Bromek; Władysława A. Daniel (51-58).
The aim of the study was to investigate the influence of selected antidepressants and neuroleptics on the protein level and activity of cytochrome P450 2D (CYP2D) in rat brain. The obtained results showed that imipramine, fluoxetine, nefazodone, thioridazine and perazine, added to brain microsomes of control rats, inhibited CYP2D activity to a lower extent (Ki  = 255–485 μM) than when added to liver microsomes (Ki  = 1–45 μM), which may result from their stronger affinity for liver CYP2D2 (Ki  = 2.7 and 1.25 μM for imipramine and fluoxetine, respectively) than for brain CYP2D4 (Ki  = 25 and 10 μM for imipramine and fluoxetine, respectively), as well as from their high non-specific binding in brain microsomes. Two-week treatment with fluoxetine evoked decreases in the level and activity of CYP2D in the striatum and the nucleus accumbens. In contrast, fluoxetine increased CYP2D expression in the cerebellum, while nefazodone considerably enhanced the activity (but not the protein level) of CYP2D in the truncus cerebri. Imipramine and mirtazapine (active in the liver) did not affect brain CYP2D. Chronic thioridazine decreased CYP2D activity in the substantia nigra and nucleus accumbens, but significantly increased that activity in the striatum and cerebellum. Clozapine significantly enhanced CYP2D activity in the truncus cerebri. In conclusion, psychotropics influence CYP2D in the brain, but their effect is different than in the liver and depends on the cerebral structure. The observed psychotropics–brain CYP2D interactions may be important for the metabolism of neurosteroids and monoaminergic neurotransmitters, and for the local biotransformation of drugs.
Keywords: Brain and liver microsome; cDNA-expressed CYP2D; Antidepressant; Neuroleptic; Chronic treatment; (Rat CYP2D);

Astrocytic glutamate transporter-dependent neuroprotection against glutamate toxicity: An in vitro study of maslinic acid by Yisong Qian; Teng Guan; Xuzhen Tang; Longfei Huang; Menghao Huang; Yunman Li; Hongbin Sun; Rong Yu; Fan Zhang (59-65).
The astrocytic glutamate transporters GLAST/EAAT1 and GLT-1/EAAT2 are crucial for the removal of glutamate from the synaptic cleft and are essential for maintaining a low concentration of extracellular glutamate in the brain. Enhanced transporter expression is neuroprotective. In the present study, we tested the neuropotective effects of maslinic acid, a natural product from the Olea europaea plant, on cultures of primary neurons from the cerebral cortex. Studies showed that astrocyte-conditioned medium from maslinic acid-treated astrocytes dose-dependently promoted neuron survival during glutamate toxicity by enhancing extracellular glutamate clearance. Real-time PCR and western blot analysis revealed that maslinic acid pre-treatment significantly increased the expression of GLAST and GLT-1 at the protein and mRNA levels. In addition, this neuroprotection was abolished by the glutamate transporter inhibitor, L-Threohydroxy aspartate (THA), in a co-culture of astrocytes and neurons. These findings suggest that maslinic acid regulates the extracellular glutamate concentration by increasing the expression of astrocytic glutamate transporters, which may, in turn, provide neuroprotection.
Keywords: Astrocyte; GLAST; GLT-1; Maslinic acid; Glutamate;

Involvement of mouse μ-opioid receptor splice variants in the spinal antinociception induced by the dermorphin tetrapeptide analog amidino-TAPA by Hirokazu Mizoguchi; Chizuko Watanabe; Takayuki Higashiya; Satoshi Takeda; Kaori Moriyama; Akihiko Yonezawa; Takumi Sato; Takaaki Komatsu; Tsukasa Sakurada; Shinobu Sakurada (66-72).
The involvement of the mouse μ-opioid receptor (mMOR-1) splice variants in the antinociceptive effect of intrathecally (i.t.) administered N α-amidino-Tyr-D-Arg-Phe-β-Ala (amidino-TAPA) and [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) was investigated in mice by monitoring the recovery from acute antinociceptive tolerance to amidino-TAPA and DAMGO. A single i.t. pretreatment with DAMGO produced an acute antinociceptive tolerance, which peaked at 2 h and disappeared within 5 h after the pretreatment. In contrast, a single i.t. pretreatment with amidino-TAPA produced an acute antinociceptive tolerance, which disappeared within 3 h after the pretreatment. The concomitant i.t. pretreatment with an antisense oligodeoxynucleotide (ODN) for exon-1, exon-12, exon-13 or exon-14 of mMOR-1 maintained the acute antinociceptive tolerance to amidino-TAPA for 24 h after the pretreatment. On the other hand, the concomitant i.t. pretreatment with an antisense ODN for exon-1 of mMOR-1, but not an antisense ODN for exon-12, exon-13 or exon-14 of mMOR-1, maintained the acute antinociceptive tolerance to DAMGO for 24 h after the pretreatment. The present results suggest that the spinal antinociception of amidino-TAPA is partially mediated through the activation of the amidino-TAPA-sensitive and DAMGO-insensitive mMOR-1 splice variants MOR-1J, MOR-1K and MOR-1L, which contain the sequence encoded by exon-12, exon-13 and exon-14, respectively.
Keywords: Antinociception; Acute tolerance; mu-Opioid receptor; Opioid peptides; Splice variants; Spinal cord;

Aripiprazole protects cortical neurons from glutamate toxicity by Vuk Koprivica; Karen Regardie; Christina Wolff; Raymond Fernalld; Janelle Johnson Murphy; Junichi Kambayashi; Tetsuro Kikuchi; Shaun Jordan (73-76).
Neurodegeneration is thought to be a component of schizophrenia pathology, and some antipsychotics appear to slow degenerative changes in patients. Aripiprazole, the first partial dopamine D2 receptor agonist approved for the treatment of schizophrenia, is suggested to be neuroprotective based on non-clinical studies using transformed cell lines and in vivo stress and lesion paradigms. However, aripiprazole-induced neuroprotection has not been studied in a neuronal glutamate toxicity assay, which may model aspects of neurodegeneration occurring in schizophrenia. This study examined whether therapeutically relevant concentrations of aripiprazole protect rat embryonic cortical neurons from glutamate toxicity in biochemical and high-content imaging assays. Aripiprazole inhibited glutamate-induced neurotoxicity by 40% in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, in contrast to risperidone and olanzapine, which had little neuroprotective activity. This neuroprotective effect of aripiprazole was not mediated by the activation of serotonin 5-HT1A or dopamine D2 receptors, Akt or glycogen-synthase kinase-3β signaling (GSK-3β), or through the inhibition of poly-ADP ribose polymerase (PARP). Further experiments are required to determine the biochemical nature of aripiprazole-induced neuroprotection and whether any such activity might have clinical relevance.
Keywords: Aripiprazole; Neuron; Neuroprotection; Glutamate; Serotonin; Dopamine; PARP; Akt; GSK-3β;

Effects of n-hexanal on dopamine release in the striatum of living rats by Hironari Kako; Yoko Kobayashi; Hidehiko Yokogoshi (77-82).
Green odor is present in many green leaves, vegetables, and fruits and is composed of four 6-carbon straight-chain alcohols, n-hexanol, (E)-2-hexenol, (Z)-3-hexenol, and (E)-3-hexenol, and four aldehydes, n-hexanal, (E)-2-hexenal, (Z)-3-hexenal, and (E)-3-hexenal. It has been reported that certain green odor compounds enhance dopamine release from rat brain striatal slices and rat pheochromocytoma cells (PC12 cells). It is well known that intracellular Ca2+ levels regulate dopamine release. The amount of dopamine released by n-hexanal-treated PC12 cells decreased in cells pretreated with a membrane-permeable Ca2+ chelator. In this study, the effect of n-hexanal on dopamine release in the brain striatum of living rats was studied using an in vivo brain microdialysis system. Local stimulation with n-hexanal diluted in Ringer's solution to 0.01%, 0.05%, or 0.1% enhanced dopamine release in a concentration-dependent manner. The amount of dopamine released with 0.01% n-hexanal administration significantly declined when either extracellular or intracellular Ca2+ levels decreased. Furthermore, the extracellular dopamine concentration increased with perfusion of nomifensine, an inhibitor of dopamine uptake into cells. When nomifensine was co-perfused with n-hexanal into the striatum, extracellular dopamine release increased further. Accordingly, the concentration of dopamine metabolite and the ratio of dopamine metabolite to dopamine decreased after treatment with n-hexanal. These responses were similar to those seen with KCl stimulation. These data suggest that n-hexanal stimulates dopamine release but does not inhibit dopamine uptake in the brain striatum of living rats, and that dopamine release associated with n-hexanal is regulated by both extracellular and intracellular Ca2+ levels.
Keywords: n-Hexanal; Green odor; Dopamine; Microdialysis; Dopamine uptake;

Diabetic rats show reduced cardiac–somatic reflex evoked by intrapericardial capsaicin by Xiao-Hua Liu; Chao Qin; Jian-Qing Du; Yan Xu; Na Sun; Jing-Shi Tang; Qiang Li; Robert D. Foreman (83-88).
Painless myocardial infarction is a serious complication of diabetes. The present study examined whether cardiac nociception was altered in the streptozotocin-induced diabetic rat model by assessing intrapericardial capsaicin-evoked electromyography (EMG) responses in the spinotrapezius muscle. Somatic sensitivities to mechanical and thermal stimulation of the skin were also determined. Intrapericardial administration of capsaicin evoked a concentration-dependent EMG response, which was reproducible with repeated administration. However, the capsaicin-induced EMG responses were different in streptozotocin-induced diabetic rats and controls. Intrapericardial capsaicin produced fewer EMG responses, which were delayed and reduced in streptozotocin-treated rats compared to controls. Pretreatment with capsazepine, a TRPV1 antagonist, significantly decreased capsaicin-evoked EMG activity in both streptozotocin-treated and control rats. In addition, streptozotocin-treated rats showed a decreased paw withdrawal threshold in response to mechanical stimulation but no change in response to radiant heat stimulation. These results suggest that streptozotocin-induced diabetic rats develop somatic mechanical hypersensitivity (allodynia), but reduced cardiac nociception. Decreased TRPV1 function may contribute to the reduction of cardiac nociception in the diabetic rat.
Keywords: Diabetes; Visceral neuropathy; Cardiac nociception; Angina pectoris;

Buprenorphine-induced hyperalgesia in the rat by Elzbieta P. Wala; Joseph R. Holtman (89-95).
In addition to analgesia opioids may also enhance pain sensitivity. Opioid-induced hyperalgesia, typically associated with potent mu-opioid agonists (e.g. fentanyl, morphine, and heroin), may be of clinical importance due to the possible counteraction of analgesia and/or paradoxical enhancement of a pre-existing pain condition during opioid therapy. Buprenorphine, a potent opioid analgesic, has a complex pharmacology on mu and kappa receptors. Buprenorphine has a better analgesia/toxicity profile (a ceiling effect for respiratory depression, less potential for abuse) compared to typical mu-opioids. Little is known about buprenorphine-induced hyperalgesia. Potentially, a lack of hyperalgesia with these other characteristics could make buprenorphine a more desirable opioid for management of chronic pain. Responsiveness to high and ultra-low doses of buprenorphine was examined following acute and repeated administration in a rat model of thermal nociception (the tail-flick test). Buprenorphine produced a dose-related antinociception. Loss of efficacy (tolerance) followed by enhanced pain sensitivity occurred with repeated dosing of buprenorphine. Delayed hyperalgesia, seen in association with antinociceptive tolerance, was blocked by the NMDA receptor antagonist, ketamine. Buprenorphine (ultra-low dose) resulted in immediate hyperalgesia, which was also reversed by ketamine, in a dose-related fashion. No tolerance to hyperalgesia was seen with repeated dosing of low-dose buprenorphine. The antinociceptive effect of buprenorphine was diminished in rats, which previously exhibited hyperalgesia with buprenorphine. In summary, bimodal properties of buprenoprhine were separately demonstrated: pronociceptive at ultra-low dose and antinociceptive at higher doses. An NMDA-receptor mechanism was involved in hyperalgesia with buprenorphine.
Keywords: Buprenorphine; Antinociception; Hyperalgesia; Tolerance; NMDA-receptor antagonist;

Structural and pharmacological analysis of O-2050, a putative neutral cannabinoid CB1 receptor antagonist by Jenny L. Wiley; Christopher S. Breivogel; Anu Mahadevan; Roger G. Pertwee; Maria Grazia Cascio; Daniele Bolognini; John W. Huffman; D. Matthew Walentiny; Robert E. Vann; Raj K. Razdan; Billy R. Martin (96-105).
Rimonabant, the prototypic antagonist of cannabinoid CB1 receptors, has been reported to have inverse agonist properties at higher concentrations, which may complicate its use as a tool for mechanistic evaluation of cannabinoid pharmacology. Consequently, recent synthesis efforts have concentrated on discovery of a neutral antagonist using a variety of structural templates. The purpose of this study was to evaluate the pharmacological properties of the putative neutral cannabinoid CB1 receptor antagonist O-2050, a sulfonamide side chain analog of Δ8-tetrahydrocannabinol. O-2050 and related sulfonamide cannabinoids exhibited good affinity for both cannabinoid CB1 and CB2 receptors. While the other sulfonamide analogs produced cannabinoid agonist effects in vivo (e.g., activity suppression, antinociception, and hypothermia), O-2050 stimulated activity and was inactive in the other two tests. O-2050 also decreased food intake in mice, an effect that was reminiscent of that produced by rimonabant. Unlike rimonabant, however, O-2050 did not block the effects of cannabinoid agonists in vivo, even when administered i.c.v. In contrast, O-2050 antagonized the in vitro effects of cannabinoid agonists in [35S]GTPγS and mouse vas deferens assays without having activity on its own in either assay. Further evaluation revealed that O-2050 fully and dose-dependently substituted for Δ9-tetrahydrocannabinol in a mouse drug discrimination procedure (a cannabinoid agonist effect) and that it inhibited forskolin-stimulated cyclic AMP signaling with a maximum efficacy of approximately half that of the full agonist CP55,940 [(-)-cis-3-[2-hydroxy-4(1,1-dimethyl-heptyl)phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol]. Together, these results suggest that O-2050 is not a viable candidate for classification as a neutral cannabinoid CB1 receptor antagonist.
Keywords: Cannabinoid; CB1 receptor; Neutral antagonist; O-2050; Structure–activity relationship;

Tipepidine enhances the antinociceptive-like action of carbamazepine in the acetic acid writhing test by Kazuaki Kawaura; Risa Miki; Yuri Urashima; Sokichi Honda; Ahmed M. Shehata; Fumio Soeda; Tetsuya Shirasaki; Kazuo Takahama (106-108).
Several antidepressants have been used to treat severe pain in clinics. Recently, we reported that the centrally acting non-narcotic antitussive (cough suppressant drug), tipepidine produces an antidepressant-like effect in the forced swimming test, although the mechanism of action appears to be quite different from that of known antidepressants. In the present study, we investigated whether a combination of tipepidine and carbamazepine acts synergistically to induce an antinociceptive effect in the acetic acid-induced writhing test in mice. Prior to studying the combination of tipepidine and carbamazepine, the analgesic action of tipepidine alone was also examined in mice. Tipepidine at 5–40 mg/kg i.p. significantly reduced the number of writhes induced by acetic acid in mice. Carbamazepine at 20 mg/kg i.p. also significantly reduced the writhing reaction. Furthermore, co-administration of carbamazepine (5 and 10 mg/kg, i.p.) and tipepidine (2.5 mg/kg i.p.) significantly decreased the number of writhes induced by acetic acid. This finding suggests that a combination of carbamazepine and tipepidine may be a new strategy for the treatment of neuropathic pain such as what occurs in trigeminal neuralgia, because the use of carbamazepine is often limited by its adverse effects and by reduction of its analgesic efficacy by microsomal enzyme induction.
Keywords: Tipepidine; Antitussives; G-protein coupled inwardly rectifying potassium ion channels; Antinociceptive-like action; Carbamazepine;

Studies have demonstrated that clonidine (α2-adrenoceptor and imidazoline receptor agonist) and BMS182874 (endothelin ETA receptor antagonist) potentiate morphine and oxycodone analgesia. Agmatine, an endogenous clonidine-like substance, enhances morphine analgesia. However, its effect on oxycodone analgesia and its interaction with endothelin ETA receptor antagonists are not known. The present study was performed to determine the effect of agmatine on morphine and oxycodone analgesia and the involvement of α 2 -adrenoceptors, imidazoline receptors, opioid receptors, and endothelin receptors. Antinociception at various time intervals was determined by the tail-flick latency method in mice. Agmatine produced dose-dependent increase in tail-flick latency, while BMS182874 did not produce any change over the 360-min observation period. Agmatine significantly potentiated morphine as well as oxycodone analgesia which was not altered by BMS182874. BMS182874 pretreatment did not increase the analgesic effect produced by agmatine alone. Agmatine-induced potentiation of morphine and oxycodone analgesia was blocked by idazoxan (imidazoline receptor/α2-adrenoceptor antagonist) and yohimbine (α2-adrenoceptor antagonist). BMS182874-induced potentiation of morphine or oxycodone analgesia was not affected by yohimbine. However, idazoxan blocked BMS182874-induced potentiation of oxycodone but not morphine analgesia. This is the first report demonstrating that agmatine potentiates not only morphine but also oxycodone analgesia in mice. Potentiation of morphine and oxycodone analgesia by agmatine appears to involve α2-adrenoceptors, imidazoline receptors, and opioid receptors. In addition, imidazoline receptors may be involved in BMS182874-induced potentiation of oxycodone but not morphine analgesia. It is concluded that agmatine may be used as an adjuvant in opiate analgesia.
Keywords: Agmatine; Endothelin ETA receptor; Morphine; Oxycodone; α 2 -Adrenoceptor; Imidazoline receptor; Idazoxan; Yohimbine; Naloxone; Antinociception;

Effect of dextromethorphan on human Kv1.3 channel activity: Involvement of C-type inactivation by Jun-Ho Lee; Sun-Hye Choi; Tae-Joon Shin; Byung-Hwan Lee; Sung-Hee Hwang; Hyoung-Chun Kim; Seung-Yeol Nah (122-127).
Dextromethorphan exhibits neuroprotective effects against inflammation-mediated neurodegeneration. However, relatively little is known regarding the molecular mechanism for this inflammation-mediated neuroprotection. Human Kv1.3 channels, one of the voltage-gated potassium channels, are widely expressed in the immune and nervous systems. Activation of human Kv1.3 channels causes neuroglia-mediated neurodegeneration. Agents that inhibit human Kv1.3 channel activity have been developed as novel drugs for immunosuppression. In the present study, we investigated the effects of dextromethorphan on human Kv1.3 or Kv1.2 channel activity heterologously expressed in Xenopus laevis oocytes. The channel currents were measured with the two-electrode voltage clamp technique. Activation of both channels induced outward peak and steady-state currents. Dextromethorphan treatment induced a slight inhibition of peak currents in human Kv1.2 and Kv1.3 channels, whereas dextromethorphan profoundly inhibited the steady-state currents of human Kv1.3 channels compared to Kv1.2 channel currents. Dextromethorphan's action on steady-state currents of human Kv1.3 channels was in a concentration-dependent manner. The half-maximal inhibitory concentration (IC50) on steady-state currents of human Kv1.3 channels was 12.8 ± 1.6 μM. Dextromethorphan also accelerated the C-type inactivation rate, increased the current decay rate, and inhibited currents in a use-dependent manner. These results indicate that dextromethorphan accelerates C-type inactivation of human Kv1.3 channels and acts as an open-channel blocker. These results further suggest the possibility that dextromethorphan-mediated acceleration of C-type inactivation of human Kv1.3 channels might be one of the cellular bases of dextromethorphan-mediated protection against inflammation-mediated neurodegeneration.
Keywords: Dextromethorphan; Kv1.3 channel; C-type inactivation; Xenopus oocyte;

The effects of abnormalities of glucose homeostasis on the expression and binding of muscarinic receptors in cerebral cortex of rats by Antony Sherin; Kumar T. Peeyush; George Naijil; Mohan Sobhana Nandhu; Sadanandan Jayanarayanan; Paul Jes; Cheramadathikudiyil Skaria Paulose (128-136).
Glucose homeostasis in humans is an important factor for the functioning of nervous system. Both hypo and hyperglycemia contributes to neuronal functional deficit. In the present study, effect of insulin induced hypoglycemia and streptozotocin induced diabetes on muscarinic receptor binding, cholinergic enzymes; AChE, ChAT expression and GLUT3 in the cerebral cortex of experimental rats were analysed. Total muscarinic, muscarinic M1 receptor showed a significant decrease and muscarinic M3 receptor subtype showed a significant increased binding in the cerebral cortex of hypoglycemic rats compared to diabetic and control. Real-Time PCR analysis of muscarinic M1, M3 receptor subtypes confirmed the receptor binding studies. Immunohistochemistry of muscarinic M1, M3 receptors using specific antibodies were also carried out. AChE and GLUT3 expression up regulated and ChAT expression down regulated in hypoglycemic rats compared to diabetic and control rats. Our results showed that hypo/hyperglycemia caused impaired glucose transport in neuronal cells as shown by altered expression of GLUT3. Increased AChE and decreased ChAT expression is suggested to alter cortical acetylcholine metabolism in experimental rats along with altered muscarinic receptor binding in hypo/hyperglycemic rats, impair cholinergic transmission, which subsequently lead to cholinergic dysfunction thereby causing learning and memory deficits. We observed a prominent cholinergic functional disturbance in hypoglycemic condition than in hyperglycemia. Hypoglycemia exacerbated the neurochemical changes in cerebral cortex induced by hyperglycemia. These findings have implications for both therapy and identification of causes contributing to neuronal dysfunction in diabetes.
Keywords: Hypoglycemia; Diabetes; Cerebral cortex; Muscarinic receptor; GLUT3;

Up-regulation of Cav1.2 subunit via facilitating trafficking induced by Vps34 on morphine-induced place preference in mice by Masahiro Shibasaki; Kazuhiro Kurokawa; Koji Mizuno; Seitaro Ohkuma (137-145).
In the present study, we investigated the role of Vps34, a kinase classified into PI 3-kinase class III, in the brain of mouse after repeated in vivo treatment with morphine. The intracerebroventricular (i.c.v.) administration of nifedipine caused a dose-dependent inhibition of morphine-induced place preference. The protein levels of Vps34 in the frontal cortex including the cingulate cortex and the limbic forebrain including the nucleus accumbens significantly increased in morphine-induced place preference. I.c.v. administration of PI 3-kinase inhibitors such as wortmannin and LY294002 inhibited morphine-induced place preference in a dose-dependent manner. Cav1.2 protein level of L-type voltage-dependent calcium channels significantly increased in the frontal cortex and the limbic forebrain of the morphine psychologically dependent mice, which was significantly inhibited by PI 3-kinase inhibitors. Similar changes in the expression of Cav1.2 and Vps34 proteins are found in the primary culture of cerebral cortical neurons during short-term exposure to morphine. Under such conditions, facilitated translocation of Cav1.2 to the plasma membrane remarkably increased by the treatment of the cultured neurons with morphine when measuring protein level of Cav1.2, and such changes in Cav1.2 were also suppressed by PI 3-kinase inhibitors. These findings indicate a critical role of Vps34 in the up-regulation of Cav1.2 in the neuronal plasma membrane and the development of morphine-induced place preference.
Keywords: L-type voltage-dependent calcium channel α1C subunit (Cav1.2); Morphine; Place preference; Trafficking; Vps34;

Opioid receptor agonist Eribis peptide 94 reduces infarct size in different porcine models for myocardial ischaemia and reperfusion by Lars O. Karlsson; Lars Grip; Erik Bissessar; Irina Bobrova; Thomas Gustafsson; Mohammad Kavianipour; Jacob Odenstedt; Gerhard Wikström; Adrian T. Gonon (146-151).
Eribis peptide 94 (EP 94) is a novel enkephalin analog, thought to interact with the μ- and δ-opioid receptors. The purpose of the present study was to examine the cardioprotective potential of EP 94 in two clinically relevant porcine models of myocardial ischaemia and reperfusion, and to investigate if such an effect is associated with an increased expression of endothelial nitric oxide synthase (eNOS). Forty-one anesthetized pigs underwent 40 min of coronary occlusion followed by 4 h of reperfusion. In Protocol I, balloon occlusion of the left anterior descending artery was performed with concurrent intravenous administration of (A) vehicle (n = 7), (B) EP 94 (1 ug/kg) after 5, 12, 19 and 26 min of ischaemia (n = 4) or (C) EP 94 (1 ug/kg) after 26, 33, 40 min of ischaemia (n = 6). In Protocol II, open-chest pigs were administered (D) vehicle (n = 6) or (E) 0.2 ug/kg/min of EP 94 (n = 6) through an intracoronary infusion into the jeopardized myocardium, started after 30 min of ischaemia and maintained for 15 min. The hearts were stained and the protein content of eNOS measured. EP 94 reduces infarct size when administered both early and late during ischaemia compared with vehicle (infarct size group A 61.6 ± 2%, group B 50.2 ± 3% and group C 49.2 ± 2%, respectively, P < 0.05), as well as when infused intracoronary (infarct size group D 82.2 ± 3.9% and group E 61.2 ± 2.5% respectively, P < 0.01). Phosphorylated eNOS Ser1177 in relation to total eNOS was significantly increased in the group administered EP 94, indicating activation of nitric oxide production.
Keywords: Reperfusion injury; Myocardial ischaemia; Nitric oxide; Opioid receptor; Porcine;

The role of endogenous nitric oxide in regulating platelet function in vivo is incompletely understood. The enzymic and anatomic sources of bioactive NO remain unclear and the consequences of the differences in endothelial function between males and females to platelet responsiveness are not known. We employed a mouse model of platelet thromboembolism to assess platelet aggregation in vivo along with supporting in vitro studies to investigate these issues. Pharmacological nitric oxide synthase (NOS) inhibition protracted the duration of thromboembolic responses to ADP (adenosine diphosphate) and enhanced in vivo platelet aggregation following activation of the coagulation cascade. Collagen induced in vivo platelet aggregation was enhanced in female eNOS−/− mice and the NOS inhibitor L-NAME (Nω-Nitro-l-arginine methyl ester hydrochloride) potentiated collagen induced thromboembolism although selective iNOS and nNOS antagonists had no effect. None of the NOS inhibitors tested had significant effects on platelet aggregation in isolated whole blood. In conclusion, endogenous NO derived from eNOS in the vascular endothelium is a critical regulator of platelet function in vivo in both males and females with negligible roles of iNOS and nNOS. Despite the expression of NOS enzymes in circulating blood elements, there is no evidence of a functional role of endogenous NO from these cells in regulating platelets. eNOS and its up- and down-stream mediators are therefore potential anti-thrombotic targets.
Keywords: Animal model; Endothelium; Nitric oxide; Platelet; Thrombosis;

Genistein stimulates duodenal HCO3 secretion through PI3K pathway in mice by Biguang Tuo; Guorong Wen; Penghong Song; Jingyu Xu; Xuemei Liu; Ursula Seidler; Hui Dong (159-167).
Genistein has been proposed as a promising pharmacotherapeutic for cystic fibrosis. We recently found that genistein stimulates murine duodenal HCO3 secretion through cystic fibrosis transmembrane conductance regulator (CFTR). The aim of the present study was to determine the intracellular signal pathways involved in genistein-stimulated duodenal HCO3 secretion. Murine duodenal mucosal HCO3 secretion was examined in vitro in Ussing chambers by the pH-stat technique. The results showed that neither cAMP-dependent signal pathway inhibitors MDL-12330A and KT-5720, nor cGMP signal pathway inhibitors NS2028 and KT5823, nor calcium signal pathway inhibitors verapamil and W-13, altered genistein-stimulated duodenal HCO3 secretion. In calcium-free solution, genistein-stimulated duodenal HCO3 secretion was not altered either. Vanadate, an inhibitor of protein tyrosine phosphatase, only partially inhibited genistein-stimulated duodenal HCO3 secretion. However, both wortmannin and LY294002, two structurally and mechanistically distinct phosphatidylinositol 3-kinase (PI3K) inhibitors, markedly inhibited genistein-stimulated duodenal HCO3 secretion. Genistein increased duodenal mucosal PI3K activity and induced the phosphorylation of Akt, a signaling molecule downstream of PI3K, which was again inhibited by wortmannin. Estrogen receptor antagonist, ICI182,780, also markedly inhibited genistein-stimulated duodenal HCO3 secretion and genistein-induced PI3K activity increase in duodenal mucosa. These results demonstrate that genistein stimulates duodenal HCO3 secretion mainly through estrogen receptor and PI3K-dependent pathway. These findings contribute to the understanding of the molecular mechanism of genistein-induced anion secretion and further pharmacotherapeutic development and use of genistein or related substances in the treatment of diseases of epithelial tissues.
Keywords: Genistein; CFTR; Phosphatidylinositol 3-kinase; Duodenum; Bicarbonate secretion;

Berberine is an isoquinoline alkaloid, occurring in nature as the main constituent of several plants with medicinal use in kidney stone disease. This work was undertaken to evaluate its antiurolithic potential and explore the possible underlying mechanism(s). Berberine was tested in vitro for the antioxidant effect and in vivo for diuretic and antiurolithic effects on an animal model of calcium oxalate urolithiasis. Berberine exhibited concentration-dependent (50–150 μg/ml) antioxidant effect against ferrous-ascorbate induced lipid peroxidation in rat kidney homogenate with potency slightly higher than the reference antioxidant, butylated hydroxytoluene. In Wistar rats, berberine (5–20 mg/kg) increased urine output accompanied by increased pH and Na+ and K+ excretion and decreased Ca2+ excretion, similar to hydrochlorothiazide. In an animal model of calcium oxalate urolithiasis developed in male Wistar rats by adding 0.75% ethylene glycol in drinking water, berberine (10 mg/kg) prevented as well as eliminated calcium oxalate crystal deposition in renal tubules and protected against deleterious effects of lithogenic treatment including weight loss, impaired renal function and oxidative stress, manifested as increased malondialdehyde and protein carbonyl contents, depleted GSH and decreased antioxidant enzyme activities of the kidneys. In naïve rats, berberine (10 mg/kg) increased urine volume and pH and decreased Ca2+ excretion. Results of this study suggest the presence of antiurolithic effects in berberine against calcium oxalate stones mediated through a combination of antioxidant, diuretic, urinary alkalinizing and hypocalciuric effects. These data invite future studies on berberine to establish its efficacy for clinical use.
Keywords: Berberine; Nephrolithiasis; Antioxidant; Diuretic; Animal model;

Lipopolysaccharide increases Na+,K+-pump, but not ENaC, expression in guinea-pig airway epithelium by Michael W. Dodrill; Donald H. Beezhold; Terence Meighan; Michael L. Kashon; Jeffrey S. Fedan (176-186).
Earlier, we found in functional experiments that lipopolysaccharide (LPS; 4 mg/kg; i.p.) hyperpolarized the epithelium by stimulating the transepithelial transport of Na+ in guinea-pig tracheal epithelium. Epithelial sodium channel (ENaC) activity and Na+,K+-pump activity were increased. In this study, we hypothesized that LPS increases the expression of ENaC and the Na+,K+-pump in the epithelium and investigated the levels of transcription and protein abundance. Using qPCR, the effects of LPS on the transcription of αENaC, α1 Na+,K+-pump, COX-2, eNOS, iNOS, IL-1β, and TNF-α were measured at 3 and 18 h. In the epithelium, LPS increased the transcription of COX-2, IL-1β, and, to a nonsignificant extent, TNF-α at 3 h, but not at 18 h. In alveolar macrophages, TNF-α, and, to a nonsignificant extent, COX-2 and IL-1β were up-regulated at 3 h, but not at 18 h. Even though LPS stimulated the transcription of some genes, αENaC and α1 Na+,K+-ATPase transcription were not affected. The expressions of α-, β-, and γ-ENaC and α1 Na+,K+-pump from the tracheal epithelium and kidney cortex/medulla were investigated by western blotting. All three ENaC subunits were detected as cleavage fragments, yet LPS had no effect on their expression. LPS increased the expression of the α1 subunit and the α1, α2, and α3 subunits, collectively, of the Na+,K+-pump. Taken together, these data indicate that LPS increases Na+ transport downstream of the genetic level, in part, by stimulating the expression of the Na+,K+-pump.
Keywords: Endotoxin; Lung; Airway epithelium; Epithelial sodium channel (ENaC); Sodium, Potassium ATPase (Na+,K+-ATPase);

Ulcerative colitis involves complicated etiology and presents diverse symptoms including intestine inflammation, bowel pain and diarrhea. Anti-inflammatory drugs are the mainstay in patient care, accompanied with antidiarrhea and analgesic agents used as symptomatic treatment. A classic traditional Chinese medicine formula, Fructus Mume pill (FMP), showed remarkable therapeutic efficacy in treating ulcerative colitis. However, since it contains many herbs and countless chemicals, the underlying mechanism is not clear. In this study, we selected three alkaloids from FMP, namely, berberine, hypaconitine and skimmianine to study the individual drug effect and compare these results with the BHS combination on: 1) The recovery of ulcerative colitis rats induced by trinitrobenzene-sulfonic acid. 2) Mice with xylene-induced acute exudative edema and acetic acid-induced writhing. 3) Gastrointestinal transit inhibition, and 4) the response of HT29 cells after treatment with lipopolysaccharide. We found that the compound hypaconitine showed a potent analgesic effect, while skimmianine acted as an antidiarrhea agent and the component berberine was the key agent exerting anti-inflammatory effect. However, since berberine killed the commensal bacteria and induced lipopolysaccharide release, it could at the same time aggravate colon inflammation. The three-alkaloid combination BHS produced complementary and synergistic effects in colon inflammation recovery, relieving acetic acid-induced bowel pain and xylene-induced acute exudative edema. BHS also decreased lipopolysaccharide production and enhanced the therapeutic efficacy. It is hoped that this study will lay the foundation to further dissect and understand the FMP formula to improve the treatment with simplified and well defined drug combinations for this dreadful disease.
Keywords: Ulcerative colitis; Traditional Chinese medicine; Drug combination; Synergism; Alkaloid;

Histamine H4 receptor antagonism inhibits allergen-specific T-cell responses mediated by human dendritic cells by Kristina Lundberg; Sissela Broos; Lennart Greiff; Carl A.K Borrebaeck; Malin Lindstedt (197-204).
Dendritic cells are potential targets in allergy therapy as they, under the influence of their microenvironment, regulate T-cell responses. Histamine has been shown to promote Th2 polarization by dendritic cells. However, neither the mechanism nor the functionality of the different histamine receptors in this process has been fully elucidated. The aim of the present study was to identify factors involved in histamine-mediated dendritic cell activation as well as to study dendritic cell expression of histamine H1 and H4 receptors and their influence on allergen-specific T-cell responses in grass pollen allergy. Assessment of dendritic cell gene regulation by histamine using mRNA microarrays demonstrated that histamine alters many immunoregulatory genes of which the majority are novel in this context. Additionally, immunocytochemical stainings showed protein expression of histamine H1 and H4 receptors on dendritic cells from healthy and allergic donors. Furthermore, histamine H1 and H4 receptor antagonists (pyrilamine/N-(4-methoxybenzyl)-N′,N′-dimethyl-N-pyridin-2-ylethane-1,2-diamine and JNJ7777120/1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine, respectively) were shown to influence histamine-induced dendritic cell maturation. Interestingly, JNJ7777120 inhibited dendritic cells’ capacity to induce allergen-specific T-cell proliferation. In conclusion, H4 receptor antagonism suppressed DC-induced, allergen-specific T-cell responses in humans and might thus inhibit allergic responses. This finding indicates that the H4 receptor is a potential treatment target in human allergic conditions.
Keywords: Dendritic cell; Transcriptional activity; Th2 cell; Histamine; Histamine receptor; Allergy;

Jaceosidin inhibits contact hypersensitivity in mice via down-regulating IFN-γ/STAT1/T-bet signaling in T cells by Ye Yin; Yang Sun; Liyun Gu; Wei Zheng; Fangyuan Gong; Xingxin Wu; Yan Shen; Qiang Xu (205-211).
In the present study, we aimed to investigate the immunosuppressive activity of jaceosidin, a flavone isolated from Artemisia vestita, on T lymphocytes both in vitro and in vivo, and further explore its potential molecular mechanism. Jaceosidin exerted a significant inhibition on the T cell proliferation and activation induced by concanavalin A (Con A) in a concentration-dependent manner and it also inhibited the secretion of the proinflammatory cytokines such as IL-2, TNF-α and IFN-γ of activated T cells. Further study showed that jaceosidin down-regulated STAT1 activation and T-bet expression in activated T cells. Moreover, in order to investigate the immunosuppressive effect of jaceosidin in vivo, the picryl chloride (PCl)-induced ear contact dermatitis model was performed on BALB/c mice. Jaceosidin significantly ameliorated PCl-induced ear swelling in a dose-dependent manner, which was due to its inhibition of the STAT1/T-bet signaling pathway. In summary, these findings suggest that jaceosidin exerts its immunosuppressive effect both in vitro and in vivo through inhibiting T cell proliferation and activation, which is closely associated with its potent down-regulation of the IFN-γ/STAT1/T-bet signaling pathway.
Keywords: Jaceosidin; Contact hypersensitivity; STAT1 [signal transducer and activator of transcription factor 1]; T-bet;

Adenosine A2A receptor agonist (CGS-21680) prevents endotoxin-induced effects on nucleotidase activities in mouse lymphocytes by Fernanda Cenci Vuaden; Luiz Eduardo Baggio Savio; Carolina Maria Alves Bastos; Maurício Reis Bogo; Carla Denise Bonan (212-217).
Adenosine 5′-triphosphate (ATP) released during inflammation presents proinflammatory properties. Adenosine, produced by catabolism of ATP, is an anti-inflammatory compound. Considering the role of ATP and adenosine in inflammation and the importance of ectonucleotidases in the maintenance of their extracellular levels, we investigated the effect of a selective agonist of the adenosine A2A receptor (CGS-21680) on ectonucleotidase activities and gene expression patterns in lymphocytes from mice submitted to an endotoxemia model. Animals were injected intraperitoneally with 12 mg/kg Lipopolyssacharide (LPS) and/or 0.5 mg/kg CGS-21680 or saline. Nucleotidase activities were determined in lymphocytes from mesenteric lymph nodes and analysis of ectonucleotidase expression was carried out by a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Exposure to endotoxemia promoted an increase in nucleotide hydrolysis. When CGS-21680 was administered concomitantly with LPS, this increase was prevented for ATP, adenosine 5′-monophosphate (AMP), and p-Nitrophenyl thymidine 5′-monophosphate (p-Nph-5′-TMP) hydrolysis. However, when CGS-21680 was administered 24 h after LPS injection, the increase was not reversed. The expression pattern of ectonucleotidases was not altered between LPS and LPS plus CGS-21680 groups, indicating that the transcriptional control was not involved on the effect exerted for CGS-21680. These results showed an enhancement of extracellular nucleotide catabolism in lymphocytes after induction of endotoxemia, which was prevented, but not reversed by CGS-21680 administration. These findings suggest that the control of nucleotide and nucleoside levels exerted by CGS-21680 could contribute to the modulation of the inflammatory process promoted by adenosine A2A agonists.
Keywords: Lipopolysaccharide; Lymphocytes; Nucleoside triphosphate diphosphohydrolase; Nucleotide pyrophosphatase/phosphodiesterase; 5′-Nucleotidase; Adenosine;

8-oxo-2′-deoxyguanosine suppresses allergy-induced lung tissue remodeling in mice by Jeong-Soon Kim; Dae-Yong Kim; Jin-Ku Lee; Jai-Youl Ro; Myung-Hee Chung (218-226).
We previously reported that 8-oxo-2′-deoxyguanosine (8-oxo-dG) suppressed airway hyperresponsiveness and allergy-associated immune responses in ovalbumin-induced allergic mice by inactivating Rac. In the present study, 8-oxo-dG was investigated for its suppression of inflammation and remodeling in lung tissues induced by allergic reaction in mice. Mice were sensitized and challenged with ovalbumin without or with oral administration of 8-oxo-dG. The mice without 8-oxo-dG administration showed the following inflammatory and airway remodeling signs: infiltration of inflammatory cells into peribronchial area, hyperplasia of mucus-secreting goblet cells in bronchial walls, increase of expressions of Muc5ac and vascular cell adhesion molecule (VCAM)-1, collagen deposition and protein expression, and matrix metalloproteinase (MMP)-2/-9 expressions. We also observed an increase of various inflammation-mediating proteins, namely IL-4, IL-5, IL-8, IL-13, TNF-α and IFN-γ, and activation of STAT1 and NF-κB. Production of reactive oxygen species and nitric oxide (NO.) was increased as indicated by a dramatic increase in formation of nitro-tyrosine. Importantly, Rac1 and 2 were also markedly activated. However, 8-oxo-dG suppressed all these inflammatory and tissue remodeling signs as well as activation of Rac1 and 2. These results indicate that 8-oxo-dG can inhibit allergy-induced inflammation and remodeling in airway and lung tissues through Rac inactivation.
Keywords: 8-oxo-2′-deoxyguanosine (8-oxo-dG); Lung tissue remodeling; Rac; Reactive oxygen species; NF-κB;

Pharmacologic profiling of corifollitropin alfa, the first developed sustained follicle stimulant by Pieter Verbost; Willem N. Sloot; Ursula M. Rose; Renato de Leeuw; Rob G.J.M. Hanssen; Gijs F.M. Verheijden (227-233).
Corifollitropin alfa (Elonva®, MSD, previously N.V. Organon or Schering-Plough Oss, The Netherlands) is a newly developed sustained follicle stimulant composed of the α subunit of human follicle-stimulating hormone (FSH) and a hybrid β subunit formed by fusion of the human chorionic gonadotropin β subunit carboxy terminal peptide with the β subunit of human FSH. Binding characteristics of corifollitropin alfa at the rat FSH receptor and transactivation properties at the rat FSH receptor, human luteinizing hormone (LH) receptor, and human thyroid-stimulating hormone receptor (TSH receptor) were assessed in vitro. Bioactivity of corifollitropin alfa in rats was also assessed. Serum corifollitropin alfa levels in rats and dogs were used to derive the main pharmacokinetic parameters of corifollitropin alfa. Binding and transactivation profile of corifollitropin alfa to rat FSH receptor was specific and comparable to that of recombinant human FSH, with no intrinsic TSH receptor or LH receptor activation. From pharmacokinetic studies, circulating half-life of corifollitropin alfa was calculated to be 17.3 h in rats and 46.9 h in dogs, 1.5- to 2-fold longer than recombinant FSH. Corifollitropin alfa demonstrated a 2- to 4-fold increase in bioactivity (ovarian weight, serum estradiol and progesterone, ovulated ova) over recombinant FSH across all in vivo parameters assessed. These data demonstrate that corifollitropin alfa is a specific ligand with high affinity for FSH receptor, lacking intrinsic activity for LH receptor and TSH receptor. By virtue of its increased in vivo half-life, corifollitropin alfa can be a valuable alternative to FSH by acting as a sustained follicle stimulant.
Keywords: Elonva®; Corifollitropin alfa; FSH (follicle stimulating hormone); CTP (carboxy terminal peptide); IVF (in vitro fertilization); Controlled ovarian stimulation;

The statins fluvastatin and pravastatin exert anti-flushing effects by improving vasomotor dysfunction through nitric oxide-mediated mechanisms in ovariectomized animals by Hideki Shuto; Koji Tominaga; Atsushi Yamauchi; Munehiko Ikeda; Kenji Kusaba; Daisuke Mitsunaga; Yasutoshi Hirabara; Takashi Egawa; Yukio Takano; Yasufumi Kataoka (234-239).
Statins have pleiotropic vascular protective effects that are independent of their cholesterol-lowering effects. The aim of the present study was to determine if statins have anti-flushing actions in an animal model of forced exercise-induced temperature dysregulation in menopausal hot flushes, and to clarify the critical role of statins in regulating vascular reactivity in the tail arteries of ovariectomized rats. Administration of fluvastatin or pravastatin (3 mg/kg/day for 7 days, p.o.) significantly ameliorated the flushing of tail skin in ovariectomized mice, and the effect of each statin was comparable with that of estrogen replacement (1 mg/kg/week for 3 weeks, i.m.). In phenylephrine-pre-contracted rat-tail arteries, ovariectomy inhibited acetylcholine-induced relaxation, but augmented sodium nitroprusside-induced relaxation. These ovariectomy-altered vasodilator responses were restored by fluvastatin treatment as well as by estrogen replacement. Nitrite/nitrate levels in the plasma of ovariectomized animals showed significantly lower values than those in sham-operated animals; this ovariectomy-reduced production of nitric oxide was improved by fluvastatin treatment. These data provide the first experimental evidence that statins such as fluvastatin and pravastatin exert anti-flushing effects by improving vasomotor dysfunction through nitric oxide-mediated mechanisms in ovariectomized animals. Thus, therapeutic methods that target improvement of vasomotor dysfunction could be novel strategies for reducing menopausal hot flushes.
Keywords: Estrogen; Fluvastatin; Hot flush; Nitric oxide; Pravastatin; Vascular reactivity;

Acarbose actions on insulin resistance and inflammatory parameters during an oral fat load by Giuseppe Derosa; Pamela Maffioli; Ilaria Ferrari; Elena Fogari; Angela D'Angelo; Ilaria Palumbo; Sabrina Randazzo; Lucio Bianchi; Arrigo FG Cicero (240-250).
The aim of this study was to evaluate the effects of acarbose on inflammatory biomarkers and insulin resistance in diabetic patients before and after a standardized oral fat load (OFL). Ninety six patients were assigned to take acarbose 50 mg three times a day and 92 to take placebo; after the first month acarbose was titrated to 100 mg three times a day. We evaluated the following parameters at the baseline, and after 1, 2 and 7 months: body mass index (BMI), glycemic control, fasting plasma insulin, post-prandial plasma insulin, homeostasis model assessment insulin resistance index (HOMA-IR), blood pressure, lipid profile, soluble intercellular adhesion molecule-1 (sICAM-1), interleukin-6 (IL-6), high-sensitivity C reactive protein (Hs-CRP), soluble vascular cell adhesion molecule-1 (sVCAM-1), and soluble E-selectin (sE-selectin). Furthermore, at the baseline and at the end of the study all patients underwent OFL, and an euglycemic hyperinsulinemic clamp to evaluate M value and total glucose requirement. Acarbose was better than placebo in improving glycemic and lipid profile, and HOMA-IR. Furthermore, acarbose gave a decrease of fasting plasma insulin, post-prandial insulin, s-ICAM-1, sVCAM-1, IL-6, and Hs-CRP, not observed with placebo, even if no significant differences between the two groups were observed. During the second OFL performed after the therapy with acarbose, we observed a significant decrease of all inflammatory parameters' peaks compared to the OFL administered at baseline. Acarbose was more effective than acarbose in reducing the post-OFL peaks of the various parameters included the inflammatory markers, after 7 months of therapy.
Keywords: Acarbose; Type 2 diabetes mellitus; Inflammation; Euglycemic hyperinsulinemic clamp; Oral fat load;