European Journal of Pharmacology (v.757, #C)

Berberine reduces Toll-like receptor-mediated macrophage migration by suppression of Src enhancement by Wei-Erh Cheng; Miao Ying Chang; Jyun-Yan Wei; Yen-Jen Chen; Ming-Chei Maa; Tzeng-Horng Leu (1-10).
Berberine is an isoquinoline with anti-inflammatory activity. We previously demonstrated that there was a loop of signal amplification between nuclear factor kappa B and Src for macrophage mobility triggered by the engagement of Toll-like receptors (TLRs). The simultaneous suppression of lipopolysaccharide (LPS)-mediated upregulation of inducible nitric oxide synthase, cyclooxygenase 2, and cell mobility in berberine-treated macrophages suggested Src might be a target of berberine. Indeed, th reduced migration, greatly suppressed Src induction in both protein and RNA transcript by berberine were observed in macrophages exposed to LPS, peptidoglycan, polyinosinic-polycytidylic acid, and CpG-oligodeoxynucleotides. In addition to Src induction, berberine also inhibited LPS-mediated Src activation in Src overexpressing macrophages and S-nitroso-N-acetylpenicillamine (a nitric oxide donor) could partly restore it. Moreover, berberine suppressed Src activity in fibronectin-stimulated macrophages and in v-Src transformed cells. These results implied that by effectively reducing Src expression and activity, berberine inhibited TLR-mediated cell motility in macrophages.
Keywords: Berberine; Src; Toll-like receptor; Macrophage migration;

DSR-98776, a novel selective mGlu5 receptor negative allosteric modulator with potent antidepressant and antimanic activity by Taro Kato; Makoto Takata; Maiko Kitaichi; Momoe Kassai; Mitsuhiro Inoue; Chihiro Ishikawa; Wataru Hirose; Kozo Yoshida; Isao Shimizu (11-20).
Modulation of monoaminergic systems has been the main stream of treatment for patients with mood disorders. However, recent evidence suggests that the glutamatergic system plays an important role in the pathophysiology of these disorders. This study pharmacologically characterized a structurally novel metabotropic glutamate 5 (mGlu5) receptor negative allosteric modulator, DSR-98776, and evaluated its effect on rodent models of depression and mania. First, DSR-98776 in vitro profile was assessed using intracellular calcium and radioligand binding assays. This compound showed dose-dependent inhibitory activity for mGlu5 receptors by binding to the same allosteric site as 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a known mGlu5 inhibitor. The in vivo therapeutic benefits of DSR-98776 were evaluated in common rodent models of depression and mania. In the rat forced swimming test, DSR-98776 (1–3 mg/kg) significantly reduced rats immobility time after treatment for 7 consecutive days, while paroxetine (3 and 10 mg/kg) required administration for 2 consecutive weeks to reduce rats immobility time. In the mouse forced swimming test, acute administration of DSR-98776 (10–30 mg/kg) significantly reduced immobility time. This effect was not influenced by 4-chloro-DL-phenylalanine methyl ester hydrochloride-induced 5-HT depletion. Finally, DSR-98776 (30 mg/kg) significantly decreased methamphetamine/chlordiazepoxide-induced hyperactivity in mice, which reflects this compound antimanic-like effect. These results indicate that DSR-98776 acts as an orally potent antidepressant and antimanic in rodent models and can be a promising therapeutic option for the treatment of a broad range of mood disorders with depressive and manic states.
Keywords: mGlu5 receptor; Major depressive disorder; Mania; Bipolar disorder; Negative allosteric modulator;

Quinaldic acid inhibits proliferation of colon cancer HT-29 cells in vitro: Effects on signaling pathways by Ewa Langner; Katarzyna Walczak; Witold Jeleniewicz; Waldemar A. Turski; Grażyna Rajtar (21-27).
Quinaldic acid is presumed to be a derivative of kynurenic acid, a tryptophan metabolite with proven antiproliferative activity towards cancer cells in vitro. The aim of present study was to evaluate the activity of quinaldic acid in colon cancer cells. The antiproliferative potential of quinaldic acid was assessed in HT-29, LS180 and Caco-2 cells. Suppression of metabolic activity (IC50 of 0.5 mM for HT-29 and LS180 cells, 0.9 mM for Caco-2 cells) and DNA synthesis (IC50 of 2.7, 4.3, 2 mM for HT-29, LS180 and Caco-2 cells, respectively) were observed in all tested cell lines. It is noteworthy that quinaldic acid in antiproliferative concentrations was non-toxic to normal colon epithelium CCD 841 CoTr cells. Concomitantly, alterations in several signaling pathways in HT-29 cells were observed. Quinaldic acid led to changes in the phosphorylation level of extracellular-signal-regulated kinase (ERK) 1/2, p38, cAMP response element-binding protein (CREB) and Akt (protein kinase B) kinases. Moreover, changes in the CREB transcription factor were also found at the gene expression level. Antiproliferative activity and signaling pathways modulatory potential of quinaldic acid in colon cancer cells in vitro has been stated.
Keywords: Quinaldic acid; Kynurenic acid; Colon cancer; Cancer cells proliferation;

Rapamycin ameliorates brain metabolites alterations after transient focal ischemia in rats by Anjali Chauhan; Uma Sharma; Naranamangalam R. Jagannathan; Yogendra Kumar Gupta (28-33).
Rapamycin has been shown to protect against middle cerebral artery occlusion (MCAo) induced ischemic injury. In this study, the neuroprotective effect of rapamycin on the metabolic changes induced by MCAo was evaluated using nuclear magnetic resonance (NMR) spectroscopy of brain tissues. MCAo in rats was induced by insertion of nylon filament. One hour after ischemia, rapamycin (250 µg/kg, i.p.) in dimethyl sulfoxide was administered. Reperfusion was done 2 h after ischemia. Twenty-four hours after ischemia phospholipase A2 (PLA2) levels and metabolic changes were assessed. Perchloric acid extraction was performed on the brain of all animals (n=7; sham, vehicle; DMSO and rapamycin 250 µg/kg) and the various brain metabolites were assessed by NMR spectroscopy. In all 44 metabolites were assigned in the proton NMR spectrum of rat brain tissues. In the vehicle group, we observed increased lactate levels and decreased levels of glutamate/glutamine, choline containing compounds, creatine/phosphocreatine (Cr/PCr), taurine, myo-inositol, γ-amino butryic acid (GABA), N-aspartyl aspartate (NAA), purine and pyrimidine metabolites. In rapamycin treated rats, there was increase in the levels of choline containing compounds, NAA, myo-inositol, glutamate/glutamine, GABA, Cr/PCr and taurine as compared to those of vehicle control (P<0.05). Rapamycin treatment reduced PLA2 levels as compared to vehicle group (P<0.05). Our findings indicated that rapamycin reduced the increased PLA2 levels and altered brain metabolites after MCAo. These protective effects might be attributed to its effect on cell membrane metabolism; glutamate induced toxicity and calcium homeostasis in stroke.Display Omitted
Keywords: Focal cerebral ischemia; Metabolites; NMR spectroscopy; Rapamycin; Middle cerebral artery occlusion;

Production of interleukin (IL)-33 in the lungs during multiple antigen challenge-induced airway inflammation in mice, and its modulation by a glucocorticoid by Takeshi Nabe; Hiroki Wakamori; Chihiro Yano; Ayumi Nishiguchi; Rino Yuasa; Hitomi Kido; Yusaku Tomiyama; Ayumi Tomoda; Haruka Kida; Anna Takiguchi; Masaya Matsuda; Keiichi Ishihara; Satoshi Akiba; Susumu Ohya; Hiroyuki Fukui; Nobuaki Mizutani; Shin Yoshino (34-41).
Although interleukin (IL)-33 is a candidate aggravator of asthma, the cellular sources of IL-33 in the lungs during the progression of antigen-induced airway inflammation remain unclear. Furthermore, it has not been determined whether the antigen-induced production of IL-33 can be pharmacologically modulated in vivo. In this study, we examined the production of IL-33 in the lungs of sensitized mice during multiple intratracheal challenges with the antigen, ovalbumin. The 1st challenge clearly induced the IL-33 production in the lungs, and it was enhanced by the 2nd–4th challenges. IL-33 mRNA transcription was also induced after these challenges. An immunohistochemical analysis revealed that the cellular sources of IL-33 after the 1st challenge were mainly bronchial epithelial cells, while those after the 3rd challenge were not only the epithelial cells, but also inflammatory cells that infiltrated the lungs. Flow cytometric analyses indicated that approximately 20% and 10% of the IL-33-producing cells in the lungs were M2 macrophages and conventional dendritic cells, respectively. A systemic treatment with dexamethasone before the 1st challenge potently suppressed the IL-33 production. When dexamethasone was administered before the respective challenges, production of the IL-33 protein and the infiltration of IL-33-producing M2 macrophages and dendritic cells into the lungs in the 3rd challenge were also suppressed. In conclusion, the cellular sources of IL-33 in the lungs were dynamically altered during multiple challenges: not only bronchial epithelial cells, but also the M2 macrophages and dendritic cells that infiltrated the lungs produced IL-33. The production of IL-33 was susceptible to the glucocorticoid treatment.
Keywords: Interleukin-33; Asthma; M2 macrophages; Dendritic cells; Epithelial cells; Glucocorticoid;

Melatonin MT1 and MT2 receptors are Gi protein-coupled receptors and promising therapeutic targets for a number of diseases. A proportion of G protein-coupled receptor agonists and antagonists have been classified according to their duration of action, which influences their pharmacological efficacy. However, the duration of action of melatonin agonists remains unclear. In this study, we investigated the duration of action of melatonin agonists (melatonin, 2-iodomelatonin, ramelteon, and the ramelteon metabolite M-II) at the melatonin MT1 receptor, which is more resistant to agonist-induced desensitization than the melatonin MT2 receptor. In Chinese hamster ovary cells stably expressing the human melatonin MT1 receptor, significant differences in the duration of action were observed after 2-h pretreatment with agonists followed by washout. In contrast to melatonin and M-II, the agonist activities of ramelteon and 2-iodomelatonin were persistent (i.e. inhibition of forskolin-stimulated cAMP formation and increase in ERK 1/2 phosphorylation) even after repeated washouts. Similar activities were observed for INS-1 cells endogenously expressing the rat MT1 receptor. Further, we examined potential factors linked to the duration of action. Residual activities of melatonin agonists after washout strongly correlated with their dissociation rates from the human melatonin MT1 receptor, but not their lipophilicity or extent of desensitization. These data suggest that the in vitro duration of action significantly differs between melatonin agonists and might dictate dissociation kinetics. Characterization of these in vitro properties may facilitate further in vivo study of the duration of action.
Keywords: Dissociation rate; Duration of action; Melatonin; Melatonin agonist; Melatonin MT1 receptor; Ramelteon;

(+)-Borneol alleviates mechanical hyperalgesia in models of chronic inflammatory and neuropathic pain in mice by Jun Jiang; Ying Ying Shen; Jun Li; Yu Hui Lin; Chun Xia Luo; Dong Ya Zhu (53-58).
Chronic pain is a major public health problem categorized as inflammatory or neuropathic, each involving impaired GABAergic control in the spinal cord of mammals. (+)-Borneol, a bicyclic monoterpene present in the essential oil of plants, is used for analgesia and anesthesia in traditional Chinese medicine. It has been reported that (+)-borneol directly potentiates GABA activity at recombinant human GABAA receptors. Although borneol has antinociceptive effect on acute pain models, little is known about its effect on chronic pain and its mechanism. Here we report that (+)-borneol has remarkable anti-hyperalgesic effects on neuropathic and inflammatory pain in animal models. Neuropathic hypersensitivity was induced by segmental spinal nerve ligation (SNL), and inflammatory hypersensitivity was induced by intraplantar ( injection of complete Freund׳s adjuvant (CFA). Both oral administration (125, 250 or 500 mg/kg) and intrathecal injection (i.t.) (15, 30 and 60 μg) of (+)-borneol reduced mechanical hypersensitivity dose-dependently in SNL and CFA models. The anti-hyperalgesic effects of (+)-borneol were abolished by a selective GABAA receptor (GABAAR) antagonist bicuculline (i.t., at 30 min after (+)-borneol injection). Furthermore, (+)-borneol (500 mg/kg, p.o. or 60 μg, i.t.) did not influence motor function. These findings suggest that (+)-borneol may ameliorate mechanical hyperalgesia by enhancing GABAAR-mediated GABAergic transmission in the spinal cord, and could serve as a therapeutic for chronic pain.
Keywords: (+)-borneol; Neuropathic pain; Inflammatory pain; GABAA receptors;

SIRT1 inhibition in pancreatic cancer models: Contrasting effects in vitro and in vivo by Chern Ein Oon; Carina Strell; Keng Yoon Yeong; Arne Östman; Jai Prakash (59-67).
Gemcitabine remains the standard treatment for pancreatic cancer, although most patients acquire resistance to the therapy. Up-regulated in pancreatic cancer, SIRT1 is involved in tumorigenesis and drug resistance. However the mechanism through which SIRT1 regulates drug sensitivity in cancer cells is mainly unknown. We hypothesise that inhibiting SIRT1 activity may increase sensitivity of pancreatic cancer cells to gemcitabine treatment through the regulation of apototic cell death, cell cycle, epithelial-mesenschymal-transition (EMT) and senescence. We demonstrate that gemcitabine or 6-Chloro-2,3,4,9-tetrahydro-1 H-Carbazole-1-carboxamide (EX527) SIRT1 inhibitor reduces PANC-1 cell proliferation in vitro. EX527 enhanced sensitivity of PANC-1 cells to gemcitabine treatment through increased apoptosis. However, EX527 displayed no beneficial effect either as a monotreatment or in combination with gemcitabine in the modulation of cell cycle progression. Combination treatment did not reverse the two phenomena known to affect drug sensitivity, namely EMT and senescence, which are both induced by gemcitabine. Unexpectedly, EX527 promoted PANC-1 xenograft tumour growth in SCID mice compared to control group. Dual tX527 and gemcitabine displayed no synergistic effect compared to gemcitabine alone. The study reveals that SIRT1 is involved in chemoresistance and that inhibiting SIRT1 activity with EX527 sensitised PANC-1 cells to gemcitabine treatment in vitro. Sensitisation of cells is shown to be mainly through induction of micronuclei formation as a result of DNA damage and apoptosis in vitro. However, the absence of positive combinatorial effects in vivo indicates possible effects on cells of the tumor microenvironment and suggests caution regarding the clinical relevance of tissue culture findings with EX527.
Keywords: Pancreatic cancer; EX527; SIRT1; Sirtuin; Gemcitabine; Tumour resistance;

The interactive role of cannabinoid and vanilloid systems in hippocampal synaptic plasticity in rats by Lida Tahmasebi; Alireza Komaki; Ruhollah Karamian; Siamak Shahidi; Abdolrahman Sarihi; Iraj Salehi; Ali Nikkhah (68-73).
Long-term potentiation (LTP) has been most thoroughly studied in the hippocampus, which has a key role in learning and memory. Endocannabinoids are one of the endogenous systems that modulate this kind of synaptic plasticity. The activation of the vanillioid system has also been shown to mediate synaptic plasticity in the hippocampus. In addition, immunohistochemical studies have shown that cannabinoid receptor type 1 (CB1) and vanilloid receptor 1 (TRPV1) are closely located in the hippocampus. In this study, we examined the hippocampal effects of co-administrating WIN55-212-2 and capsaicin, which are CB1 and TRPV1 agonists, respectively, on the induction of LTP in the dentate gyrus (DG) of rats. LTP in the hippocampal area was induced by high-frequency stimulation (HFS). Our results indicated that the cannabinoid agonist reduced both field excitatory post-synaptic potential (fEPSP) slope and population spike (PS) amplitude after HFS with respect to the control group, whereas the vanilloid agonist increased these parameters along with the increased induction of LTP as compared to the control group. We also showed that the co-administration of cannabinoid and vanilloid agonists had different effects on fEPSP slope and PS amplitude. It seems that agonists of the vanilloid system modulate cannabinoid outputs that cause an increase in synaptic plastisity, while in contemporary consumption of two agonist, TRPV1 agonist can change production of endocannabinoid, which in turn result to enhancement of LTP induction. These findings suggest that the two systems may interact or share certain common signaling pathways in the hippocampus.
Keywords: Cannabinoid; Vanilloid; Capsaicin; WIN55-212-2; Long-term potentiation; Hippocampus; Rat;

Effects of dihydrotestosterone on rat dermal papilla cells in vitro by Jung-Il Kang; Sang-Cheol Kim; Min-Kyoung Kim; Hye-Jin Boo; Eun-Ji Kim; Guang-Jin Im; Young Ho Kim; Jin-Won Hyun; Ji-Hoon Kang; Young-Sang Koh; Deok-Bae Park; Eun-Sook Yoo; Hee-Kyoung Kang (74-83).
Androgenetic alopecia involves the action of dihydrotestosterone (DHT) on dermal papilla cells (DPCs) that line the base of the hair follicle. However, the mechanism of DHT action is not completely understood. The effects of DHT on DPCs, regulatory cells that function in follicle growth and the hair cycle, were examined in immortalized cells derived from rat vibrissa follicles. DHT did not affect the proliferation of immortalized DPCs. However, flow cytometry analysis revealed that DHT increased cell-cycle arrest in these cells, which was accompanied by an increase in the p27kip1 level and by decreases in cyclin E, cyclin D1, and cyclin-dependent kinase 2 levels. DHT treatment resulted in the phosphorylation and nuclear translocation of Smad2/3, a mediator of the transforming growth factor-β (TGF-β) signaling pathway, which leads to the catagen phase of the hair cycle. DHT also induced the phosphorylation and nuclear translocation of heat shock protein 27 (HSP27). Moreover, DHT decreased the levels of total and nuclear β-catenin, an important regulator of hair growth and proliferation, while lithium chloride, a glycogen synthase kinase-3β inhibitor, attenuated the DHT-induced downregulation of the β-catenin level. On the other hand, DHT increased the phosphorylation of mammalian target of rapamycin (mTOR), a regulator of proliferation, in immortalized DPCs. These results illustrate that DHT could shorten the duration of the hair growth cycle by initiating cell-cycle arrest, downregulating the β-catenin level, and upregulating the TGF-β/Smad and HSP27 level, whereas activation of mTOR by DHT could attenuate the inhibition of hair growth cycle in immortalized DPCs.Display Omitted
Keywords: Dihydrotestosterone; Immortalized dermal papilla cells; TGF-β/Smad; Heat shock protein 27; β-catenin; mTOR;

Desflurane increased the activity of excitatory amino-acid carrier 1 (EAAC1) expressed in Xenopus oocytes by Seong-Joo Park; Hyun-Jung Shin; Bon-Wook Gu; Kyoung-In Woo; Zhiyi Zuo; Sang-Hwan Do; Jung-Hee Ryu (84-89).
Desflurane is a volatile anaesthetic agent with neuroprotective properties. Excitatory amino-acid carrier 1 (EAAC1) may be neuroprotective by taking up glutamate and cysteine. Therefore, the effects of desflurane on EAAC1 activity were investigated in this study. EAAC1 was expressed in Xenopus laevis oocytes. Two-electrode voltage-clamping technique was used to record membrane currents upon exposure to l-glutamate (30 μM) in the presence or absence of desflurane (0.4, 1.0, 2.0, 2.6, or 3.2 mM). Currents were also measured in oocytes pre-exposed to a protein kinase C (PKC) activator (50 nM phorbol-12-myristate-13-acetate, PMA), PKC inhibitors (1 μM staurosporine or 50 μM chelerythrine), or phosphatidylinositol-3-kinase (PI3K) inhibitors (5 μM wortmannin or 10 μM LY294002).Desflurane significantly increased EAAC1 activity. The EC50 of desflurane for increasing the EAAC1 response was 0.75 mM. A kinetic study showed that desflurane significantly increased the V max but had no effect on the K m of the EAAC1 response for glutamate. Treatment of oocytes with desflurane plus PMA significantly increased the transporter currents compared to the control, but did not further increase the response compared to either agent alone. Staurosporine attenuated desflurane-enhanced transporter currents without decreasing the basal activity; chelerythrine did not decrease either. In addition, pretreatment of oocytes with two PI3K inhibitors (wortmannin or LY294002) significantly reduced desflurane-enhanced EAAC1 activity without decreasing basal activity. Our results suggest that desflurane increases EAAC1 activity via PKC or PI3K. This enhanced EAAC1 activity may be a mechanism for the neuroprotective effect of desflurane.
Keywords: Desflurane; Glutamate transporters; Protein kinase C; Xenopus oocyte; Phosphatidylinositol-3-kinase;