European Journal of Pharmacology (v.698, #1-3)

Psychoactive “bath salts”: Not so soothing by Michael H. Baumann; John S. Partilla; Kurt R. Lehner (1-5).
Recently there has been a dramatic rise in the abuse of so-called “bath salts” products that are purchased as legal alternatives to illicit drugs like cocaine and 3,4-methylenedioxymethamphetamine (MDMA). Baths salts contain one or more synthetic derivatives of the naturally-occurring stimulant cathinone. Low doses of bath salts produce euphoria and increase alertness, but high doses or chronic use can cause serious adverse effects such as hallucinations, delirium, hyperthermia and tachycardia. Owing to the risks posed by bath salts, the governments of many countries have made certain cathinones illegal, namely: 4-methylmethcathinone (mephedrone), 3,4-methylenedioxymethcathinone (methylone) and 3,4-methylenedioxypyrovalerone (MDPV). Similar to other psychomotor stimulants, synthetic cathinones target plasma membrane transporters for dopamine (i.e., DAT), norepinephrine (i.e., NET) and serotonin (i.e, SERT). Mephedrone and methylone act as non-selective transporter substrates, thereby stimulating non-exocytotic release of dopamine, norepinephrine and serotonin. By contrast, MDPV acts as a potent blocker at DAT and NET, with little effect at SERT. Administration of mephedrone or methylone to rats increases extracellular concentrations of dopamine and serotonin in the brain, analogous to the effects of MDMA. Not surprisingly, synthetic cathinones elicit locomotor activation in rodents. Stimulation of dopamine transmission by synthetic cathinones predicts a high potential for addiction and may underlie clinical adverse effects. As popular synthetic cathinones are rendered illegal, new replacement cathinones are appearing in the marketplace. More research on the pharmacology and toxicology of abused cathinones is needed to inform public health policy and develop strategies for treating medical consequence of bath salts abuse.
Keywords: Cathinone; Designer drug; Dopamine; Serotonin; Monoamine transporter;

Excitotoxicity: Bridge to various triggers in neurodegenerative disorders by Ankita Mehta; Mayank Prabhakar; Puneet Kumar; Rahul Deshmukh; P.L. Sharma (6-18).
Glutamate is one of the most prominent neurotransmitter in the body, present in over 50% of nervous tissue and plays an important role in neuronal excitation. This neuronal excitation is short-lived and is followed by depression. Multiple abnormal triggers such as energy deficiency, oxidative stress, mitochondrial dysfunction, calcium overload, etc can lead to aberration in neuronal excitation process. Such an aberration, serves as a common pool or bridge between abnormal triggers and deleterious signaling processes with which central neurons cannot cope up, leading to death. Excitotoxicity is the pathological process by which nerve cells are damaged and killed by excessive stimulation by neurotransmitters such as glutamate and similar substances. Such excitotoxic neuronal death has been implicated in spinal cord injury, stroke, traumatic brain injury, hearing loss and in neurodegenerative diseases of the central nervous system such as stroke, epilepsy, multiple sclerosis, Alzheimer disease, Amyltropic lateral sclerosis, Parkinson’s disease, Huntington disease and alcohol withdrawal. This review mainly emphasizes the triggering events which sustain neuronal excitation, role of calcium, mitochondrial dysfunction, ROS, NO, chloride homeostasis and eicosanoids pathways. Further, a brief introduction about the recent research occurring in the treatment of various neurodegenerative diseases, including a summary of the presumed physiologic mechanisms behind the pharmacology of these disorders.
Keywords: Glutamate; Neurodegenerative diseases; Excitotoxicity; Oxidative stress; Mitochondrial dysfunction; Calcium overload; Therapeutics;

Regulation of the brain–gut axis by group III metabotropic glutamate receptors by Marcela Julio-Pieper; Richard M. O’Connor; Timothy G. Dinan; John F. Cryan (19-30).
l-glutamate is produced by a great variety of peripheral tissues in both health and disease. Like other components of the glutamatergic system, metabotropic glutamate (mGlu) receptors also have a widespread distribution outside the central nervous system (CNS). In particular, group III mGlu receptors have been recently found in human stomach and colon revealing an extraordinary potential for these receptors in the treatment of peripheral disorders, including gastrointestinal dysfunction. The significance of these findings is that pharmacological tools originally designed for mGlu receptors in the CNS may also be directed towards new disease targets in the periphery. Targeting mGlu receptors can also be beneficial in the treatment of disorders involving central components together with gastrointestinal dysfunction, such as irritable bowel syndrome, which can be co-morbid with anxiety and depression. Conversely, the development of more specific therapeutic approaches for mGlu ligands both centrally as in the gut will depend on the elucidation of tissue-specific elements in mGlu receptor signalling.Display Omitted
Keywords: Brain–gut axis; Metabotropic glutamate receptor; Gastrointestinal tract; Mood;

Obesity and type 2 diabetes are serious public health problems worldwide. Considerable efforts have highlighted the link between these two diseases. The high levels of pro-inflammatory cytokines and leptin, secreted by the adipose tissue, contribute actively to the insulin resistance induction; and the high levels of free fatty acids leads to an overproduction of reactive oxygen species that participate in pancreatic β cells failure and apoptosis. These two induced dysfunctions are the fundamental defects that precede type 2 diabetes. Genistein, an isoflavone present in a number of edible plants, has been reported as a potential therapeutic agent with anti-cancer, anti-oxidant, anti-inflammatory and anti-osteoporosis effects and proposed as a promising compound for the treatment of metabolic disorders. The pleiotropic effects of genistein are due to its multiple mechanisms of action and the multitude of cell signaling pathways involved. Here, we review the effects of genistein on obesity and type 2 diabetes and emphasize on its action on adipocyte life-cycle, obesity-related low-grade inflammation, oxidative stress and the protective effects on pancreatic β cells.
Keywords: Genistein; Adipocyte; β cell; Inflammation; Oxidative stress;

β-adrenoceptors are members of the G protein-coupled receptor superfamily which play a key role in the regulation of myocardial function. Their activation increases cardiac performance but can also induce deleterious effects such as cardiac arrhythmias or myocardial apoptosis. In fact, inhibition of β-adrenoceptors exerts a protective effect in patients with sympathetic over-stimulation during heart failure. Although β2-adrenoceptor is not the predominant subtype in the heart, it seems to importantly contribute to the cardiac effects of adrenergic stimulation; however, the mechanism by which this occurs is not fully understood. This review summarizes the current knowledge on the role of β2-adrenoceptors in the regulation of cardiac contractility, metabolism, cardiomyocyte survival and cardiac arrhythmias. In addition, therapeutic considerations relating to stimulation of the β2-adrenoceptor such as an increase in cardiac contractility with low arrythmogenic effect, protection of the myocardium again apoptosis or positive regulation of heart metabolism are discussed.
Keywords: Beta-adrenoceptors; Cardiac contractility; Cardiac arrhythmias; Cardiac metabolism; Cardiac apoptosis;

20-hydroxyecdysone-induced bone morphogenetic protein-2-dependent osteogenic differentiation through the ERK pathway in human periodontal ligament stem cells by Cong-Xiang Jian; Xiao-Fei Liu; Jun Hu; Chen-Jun Li; Gang Zhang; Yan Li; Ji-Wen Zhu; Ying-Hui Tan (48-56).
20-Hydroxyecdysone, an ecdysteroid hormone, can induce osteogenic differentiation in mesenchymal stem cells. Periodontal ligament stem cells (PDLS cells) have mesenchymal-stem-cell-like qualities and are considered as one of the candidates of future clinical application in periodontitis treatment. However, there are no studies describing the effect of 20-Hydroxyecdysone on PDLS cells. In this paper, we report a detailed study on the effect of 20-Hydroxyecdysone on PDLS cell proliferation in vitro. PDLS cells were developed from human PDL cells and were treated with 20-Hydroxyecdysone to understand different aspects of its effects. 20-Hydroxyecdysone promoted PDLS cell proliferation; significantly increased the gene expression levels of runt-related transcription factor 2, alkaline phosphatase (ALP), type I collagen, and osteocalcin. Moreover, 20-Hydroxyecdysone enhanced bone formation by PDLS cells and significantly increased bone morphogenetic protein-2 (BMP-2) mRNA and protein expression. However, 20-Hydroxyecdysonemediated increase in ALP activity was blocked with a BMP-2-specific neutralizing antibody or with the antagonist noggin; and20-Hydroxyecdysone mediated induction of BMP-2 expression and increase of ALP activity were abolished by the extracellular regulated protein kinase (ERK) MAPK pathway inhibitor PD98059. 20-Hydroxyecdysone also increased the phosphorylation of ERK. These findings provide evidence to state that 20-Hydroxyecdysone stimulates cell proliferation and induces osteogenic differentiation through the induction of BMP-2 expression in PDLS cells. It also shows that the ERK pathway is involved in 20-Hydroxyecdysone induced BMP-2 expression and osteogenic differentiation. These results are suggesting its potential as a drug for periodontal regenerative therapy.
Keywords: 20-Hydroxyecdysone; Periodontal ligament stem cells; Osteogenic differentiation; Bone morphogenetic protein-2; ERK;

We investigated the effect of galangin, a natural flavonoid, on osteoclastic bone destruction in collagen-induced arthritis and examined the molecular mechanisms by which galangin affects osteoclastogenesis in bone marrow derived macrophages. In mice with collagen-induced arthritis, administration of galangin significantly reduced the arthritis clinical score, edema and severity of disease without toxicity. Interestingly, galangin treatment during a later stage of collagen-induced arthritis, using mice with a higher clinical arthritis score, still significantly slowed the progression of the disease. Extensive cartilage and bone erosive changes as well as synovial inflammation, synovial hyperplasia and pannus formation were dramatically inhibited in arthritic mice treated with galangin. Furthermore, galangin-treated arthritic mice showed a significant reduction in the concentrations of IL-1β, TNF-α and IL-17 . We found that galangin inhibited osteoclastogenic factors and osteoclast formation in bone marrow-derived macrophages and osteoblast co-cultured cells, and increased osteoprotegerin (OPG) levels in osteoblasts. Galangin and NF-κB siRNA suppressed RANKL-induced phosphorylation of the c-jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), but not AKT and extracellular signal-regulated kinase 1/2 (ERK1/2). Also, the JNK inhibitor SP600125 and p38 inhibitor SB203580 reduced RANKL-induced expressions of phospho-c-Jun, c-fos and NFATc1 genes during osteoclast development. In addition, galangin suppressed RANKL-induced phosphorylation of NF-κB, phospho-IκBα, inflammatory cytokines and osteoclast formation in bone marrow-derived macrophages. Our data suggest that galangin prevented osteoclastic bone destruction and osteoclastogenesis in osteoclast precursors as well as in collagen-induced arthritis mice without toxicity via attenuation of RANKL-induced activation of JNK, p38 and NF-κB pathways.Display Omitted
Keywords: Galangin; Osteoclastogenesis; Bone destruction; RANKL; NF-κB;

The secreted Klotho protein restores phosphate retention and suppresses accelerated aging in Klotho mutant mice by Tso-Hsiao Chen; Makoto Kuro-o; Cheng-Hsien Chen; Yuh-Mou Sue; Yen-Cheng Chen; Ho-Han Wu; Chung-Yi Cheng (67-73).
Klotho was identified as the responsible gene in a mutant mouse line whose disruption results in a variety of premature aging-related phenotypes. Nonetheless, the related mechanisms were still unknown. Many studies report that dietary phosphate restriction and genetic ablation of vitamin D pathways indirectly reverse premature aging processes in these mice. Furthermore, transgenic overexpression of klotho in mice extends their life span through inhibition of insulin and IGF1 signaling. We found that intraperitoneal injection of recombinant soluble Klotho protein at dose of 0.02 mg/kg every other day effectively extends the life span of kl/kl mice by 17.4%. Soluble Klotho administration also ameliorated premature aging-related phenotype, such as growth retardation, premature thymus involution and vascular calcification, and effectively enhanced urinary phosphate excretion in kl/kl mice. Klotho treatment attenuated renal fibrosis through down-regulation of transforming growth factor-β signaling as well as reduced cellular senescence through down-regulation of p21-cip1 mRNA levels. In addition, soluble Klotho treatment significantly reduced both renal and aorta calcium deposits. In conclusion, our study shows the therapeutic potential of soluble Klotho protein to treat age-related disorders in mice.
Keywords: Klotho; Phosphate; Vascular calcification; Insulin; IGF1;

Dipeptidyl peptidase IV inhibition upregulates GLUT4 translocation and expression in heart and skeletal muscle of spontaneously hypertensive rats by Gisele Giannocco; Kelen C. Oliveira; Renato O. Crajoinas; Gabriela Venturini; Thiago A. Salles; Miriam H. Fonseca-Alaniz; Rui M.B. Maciel; Adriana C.C. Girardi (74-86).
The purpose of the current study was to test the hypothesis that the dipeptidyl peptidase IV (DPPIV) inhibitor sitagliptin, which exerts anti-hyperglycemic and anti-hypertensive effects, upregulates GLUT4 translocation, protein levels, and/or mRNA expression in heart and skeletal muscle of spontaneously hypertensive rats (SHRs). Ten days of treatment with sitagliptin (40 mg/kg twice daily) decreased plasma DPPIV activity in both young (Y, 5-week-old) and adult (A, 20-week-old) SHRs to similar extents (∼85%). However, DPPIV inhibition only lowered blood pressure in Y-SHRs (119±3 vs. 136±4 mmHg). GLUT4 translocation, total protein levels and mRNA expression were decreased in the heart, soleus and gastrocnemius muscle of SHRs compared to age-matched Wistar Kyoto (WKY) normotensive rats. These differences were much more pronounced between A-SHRs and A-WKY rats than between Y-SHRs and Y-WKY rats. In Y-SHRs, sitagliptin normalized GLUT4 expression in the heart, soleus and gastrocnemius. In A-SHRs, sitagliptin increased GLUT4 expression to levels that were even higher than those of A-WKY rats. Sitagliptin enhanced the circulating levels of the DPPIV substrate glucagon-like peptide-1 (GLP-1) in SHRs. In addition, stimulation of the GLP-1 receptor in cardiomyocytes isolated from SHRs increased the protein level of GLUT4 by 154±13%. Collectively, these results indicate that DPPIV inhibition upregulates GLUT4 in heart and skeletal muscle of SHRs. The underlying mechanism of sitagliptin-induced upregulation of GLUT4 in SHRs may be, at least partially, attributed to GLP-1.
Keywords: Dipeptidyl peptidase IV; Glucose transporter type 4; Hypertension; Heart; Skeletal muscle; Glucagon-like peptide-1;

Oligopeptides derived from autophosphorylation sites of EGF receptor suppress EGF-stimulated responses in human lung carcinoma A549 cells by Yoshihiro Kuroda; Nahoko Kato-Kogoe; Emi Tasaki; Eri Murata; Koyo Ueda; Mineo Abe; Kazuhide Miyamoto; Ikuhiko Nakase; Shiroh Futaki; Yumi Tohyama; Munetaka Hirose (87-94).
Epidermal growth factor (EGF) receptor plays a crucial role in the biology of human cancer, and is a highly appropriate target for anticancer agents. We have previously designed oligopeptides containing the amino acid sequences around autophosphorylation sites of EGF receptor to identify a specific inhibitor of this receptor. We found that Ac-ENAEYLR-NH2 and Ac-NYQQN-NH2 suppressed phosphorylation of purified EGF receptor in a non-ATP-competitive manner whereas Ac-QNAQYLR-NH2 and Ac-DYQQD-NH2 caused inhibition in an ATP-competitive manner. The aim of this study was to observe the effects of these peptides on the proliferation, cell death, and apoptosis of human lung carcinoma A549 cells. To facilitate transfer of these inhibitory peptides into A549 cells, the cell-penetrating peptide, human immunodeficiency virus type 1-transactivator of transcription (Tat), was linked to the peptides. When A549 cells were treated with each Tat-conjugated peptide, the peptides penetrated the cells and EGF-stimulated tyrosine phosphorylation of EGF receptor was significantly suppressed. These Tat-conjugated peptides played a suppressive role in EGF-stimulated A549 cell responses. In particular, Tat-epsilon-aminocaproic acid (acp)-ENAEYLR-NH2 significantly inhibited proliferation and showed cytotoxicity, while Tat-acp-NYQQN-NH2 and Tat-acp-DYQQD-NH2 suppressed the anti-apoptotic effect of EGF. In addition, we found that Tat-acp-ENAEYLR-NH2 also inhibited the phosphorylation of epidermal growth factor receptor 2 (ErbB2) as well as EGF receptor in A549 cells. In conclusion, membrane-permeable synthetic peptides derived from EGF receptor autophosphorylation sites have the potential to suppress EGF receptor function in A549 cells and to be developed into novel and useful agents for cancer therapy.
Keywords: A549; Cell-penetrating peptide; EGF receptor; ErbB2; Tyrosine kinase inhibitor;

Anti-tumor effect of germacrone on human hepatoma cell lines through inducing G2/M cell cycle arrest and promoting apoptosis by Yunyi Liu; Wei Wang; Bin Fang; Fengyun Ma; Qian Zheng; Pengyi Deng; Shasha Zhao; Mingjie Chen; Guangxiao Yang; Guangyuan He (95-102).
Germacrone is one of the main bioactive components in the traditional Chinese medicine Rhizoma curcuma. In this study, the anti-proliferative effect of germacrone on the human hepatoma cell lines and the molecular mechanism underlying the cytotoxicity of germacrone were investigated. Treatment of human hepatoma cell lines HepG2 and Bel7402 with germacrone resulted in cell cycle arrest and apoptosis in a dose-dependent manner as measured by MTT assay, flow cytometric and fluorescent microscopy analysis, while much lower effect on normal human liver cell L02 was observed. Flow cytometric analysis revealed that germacrone induced G2/M arrest in the cell cycle progression that was associated with an obvious decrease in the protein expression of cyclin B1 and its activating partner CDK1 with concomitant inductions of p21. Hoechst 33258 and Annexin V/PI staining results showed that the total cell number in apoptosis associated with a dose-dependent up-regulation of Bax and down-regulation of Bcl-2/Bcl-xl was increased. In the meantime, the up-regulation of p53 and reactive oxygen species increase were observed, which suggested that germacrone might be a new potent chemopreventive drug candidate for liver cancer via regulating the expression of proteins related to G2/M cell cycle and apoptosis, and p53 and oxidative damage may play important roles in the inhibition of human hepatoma cells growth by germacrone.
Keywords: Germacrone; Human hepatoma cell lines; Cell cycle arrest; Apoptosis; Reactive oxygen species;

Denbinobin induces human glioblastoma multiforme cell apoptosis through the IKKα–Akt–FKHR signaling cascade by Hsing-Yu Weng; Ming-Jen Hsu; Chien-Chih Chen; Bing-Chang Chen; Chuang-Ye Hong; Che-Ming Teng; Shiow-Lin Pan; Wen-Ta Chiu; Chien-Huang Lin (103-109).
Denbinobin, a phenanthraquinone derivative, was shown to exert antitumor activities in several types of cancer cell lines. However, the precise mechanism underlying denbinobin-induced cell death remains unclear. In this study, we investigated the apoptotic signaling cascade elicited by denbinobin in human glioblastoma multiforme (GBM) cells. Denbinobin concentration-dependently caused a decrease in the cell viability of GBM cells. A flow cytometric analysis of propidium iodide (PI)-stained cells demonstrated that denbinobin induced GBM cell apoptosis. Denbinobin evoked caspase-3 activation and degradation of poly (ADP-ribose) polymerase (PARP) and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor that prevented denbinobin-induced cell death. In addition, denbinobin-induced cell death was diminished by the transfection of wild-type (WT) Akt or IκB kinase (IKK) into GBM cells. Denbinobin reduced IKK phosphorylation in a time-dependent manner, and denbinobin-dephosphorylated IKK was accompanied by a decrease in Akt phosphorylation. The phosphorylation status of forkhead in rhabdomyosarcoma (FKHR), a downstream signal molecule of Akt, was also diminished by the presence of denbinobin. Furthermore, transfection of GBM cells with WT IKKα markedly suppressed the decreases in Akt and FKHR phosphorylation caused by denbinobin. In contrast, transfection with WT IKKβ only slightly affected denbinobin’s action against IKK, Akt, and FKHR. These results suggest that IKKα inactivation, followed by Akt and FKHR dephosphorylation and caspase-3 activation, contributes to denbinobin-induced GBM cell apoptosis.
Keywords: Denbinobin; Glioblastoma multiforme; Akt; IKK; FKHR;

When the prostate cancer cells become unresponsive to androgen therapy, resistance to chemotherapy becomes imminent, resulting in high mortality. To combat this situation, lycopodine, a pharmacologically important bioactive component derived from Lycopodium clavatum spores, was tested against hormone sensitive (LnCaP) and refractory (PC3) prostate cancer cells in vitro. This study aims to check if lycopodine has demonstrable anti-cancer effects and if it has, to find out the possible mechanism of its action. The MTT assay was performed to evaluate the cytotoxic effect. Depolarization of mitochondrial membrane potential, cell cycle, EGF receptor activity and apoptosis were recorded by FACS; profiles of different anti- and pro-apoptotic genes and their products were studied by semi-quantitative RT-PCR, indirect-ELISA, western blotting. Drug–DNA interaction was determined by CD spectroscopy. Administration of lycopodine down-regulated the expression of 5-lipoxygenase and the 5-oxo-ETE receptor (OXE receptor1) and EGF receptor, and caused up-regulation of cytochrome c with depolarization of mitochondrial inner membrane potential, without palpable change in p53 activity, resulting in apoptosis, cell arrest at G0/G1 stage and ultimately reduced proliferation of cancer cells; concomitantly, there was externalization of phosphotidyl serine residues. CD spectroscopic analysis revealed intercalating property of lycopodine with DNA molecule, implicating its ability to block cellular DNA synthesis. The overall results suggest that lycopodine is a promising candidate suitable for therapeutic use as an anti-cancer drug.Introduction of Lycopodine to the prostate cancer cells induces a massive apoptosis in a quick span of time by down regulating OXERI and 5 LOX gene, which in-turn depolarize mitochondrial inner membrane. Cyto-c release and Bcl-2, Bax ratio change favored caspase activation and initiation of apoptosis.Display Omitted
Keywords: Lycopodine; 5-Lipoxygenase; 5-Oxo-ETE receptor1; Mitochondrial membrane potential; Apoptosis; Drug–DNA interaction;

Effect of an all-trans-retinoic acid conjugate with spermine on viability of human prostate cancer and endothelial cells in vitro and angiogenesis in vivo by Dionissios Vourtsis; Margarita Lamprou; Eldem Sadikoglou; Anastassios Giannou; Olga Theodorakopoulou; Eliana Sarrou; George E. Magoulas; Stavros E. Bariamis; Constantinos M. Athanassopoulos; Dennis Drainas; Dionissios Papaioannou; Evangelia Papadimitriou (122-130).
Retinoids constitute a family of organic compounds that are being used for the treatment of various diseases, ranging from acne vulgaris to acute promyelocytic leukemia. Their use however is limited due to serious adverse effects and there is a great need for analogues with better safety profile. In the present work, the effect of N 1,N 12-bis(all-trans-retinoyl)spermine (RASP), a conjugate of all-trans-retinoic acid (atRA) with spermine, on angiogenesis in vivo and viability of human endothelial and prostate cancer cells in vitro were studied. Both atRA and RASP dose-dependently inhibited angiogenesis in the chicken embryo chorioallantoic membrane model. RASP was more effective and could be used in a wider dose range due to lower toxicity compared with atRA. Both retinoids decreased the number of human umbilical vein endothelial and prostate cancer LNCaP and PC3 cells in a concentration-dependent manner. RASP was more effective and potent compared with atRA, spermine, their combination, or conjugates of spermine with other acidic retinoids and/or psoralens in prostate cancer cells. The inhibitory effect of both atRA and RASP seems to be related to an increase of the tumour repressing gene retinoic acid receptor beta mRNA, was mediated by retinoic acid receptor alpha, and was proportional to endogenous retinoic acid receptor beta expression. These data suggest that RASP is more effective than atRA in decreasing angiogenesis and prostate cancer cell growth and identify retinoic acid receptor alpha as the receptor through which it causes retinoic acid receptor beta up-regulation and decrease of prostate cancer cell growth.
Keywords: Angiogenesis; All-trans-retinoic acid; Cancer; Prostate; Retinoids; Retinoid receptors;

Pharmacological characterization of receptor guanylyl cyclase reporter cell lines by Frank Wunder; Annette Woermann; Andreas Geerts; Markus Milde (131-136).
Receptor guanylyl cyclases are implicated in a growing number of pathophysiologies and, therefore, represent an important target class for drug development. We report here the generation and pharmacological characterization of three particulate guanylyl cyclase (pGC) reporter cell lines. Plasmid constructs encoding the natriuretic peptide receptors GC-A and GC-B, and the heat-stable enterotoxin receptor GC-C, were stably transfected in a parental reporter cell line expressing a cyclic nucleotide-gated (CNG) cation channel, acting as the biosensor for intracellular cGMP. In our reporter cell lines pGC activity can be monitored in living cells in real-time . By using different natural as well as synthetic receptor ligands of the natriuretic and guanylin peptide families, we show that our reporter assay monitors pGC activity with very high sensitivity. In contrast to previous findings, we could detect significant stimulation of GC-A and GC-B by each of the natriuretic peptides ANP, BNP and CNP. In addition, the clearance receptor ligand Cys-ANF(4–18) and the ANP receptor antagonist Arg-ANF(6–18) were characterized as partial GC-A agonists. The results imply that our novel pGC reporter cell lines are well suited for the characterization of receptor pharmacology and may be used for natural ligand characterization of guanylyl cyclase orphan receptors.
Keywords: Receptor guanylyl cyclase; cGMP; CNG channel; Reporter assay;

Omentin-1, a new adipokine released from adipose tissue, is associated with several key aspects of metabolic syndrome such as insulin sensitivity. However, it is not known whether omentin-1 affects cancer cell growth. In this study, we studied the influence of omentin-1 on two types of human hepatocellular carcinoma cells: HepG2 and HuH-7 cells. Cell viability assay showed that omentin-1 (1 and 2 μg/ml) significantly inhibited the proliferation of HepG2 and HuH-7 cells. Both annexin+PI staining and TUNEL assay showed that omentin-1 induced apoptosis in these cells. Moreover, omentin-1 treatment upregulated protein levels of p53 and p21, a main transcriptional target of p53. Interestingly, omentin-1 did not affect p53 mRNA level. Further mechanism study showed that omentin-1 upregulated p53 protein level through decreasing p53 deacetylation and thereby increasing the stability of p53 protein. Using small interfering RNA (siRNA)-mediated knockdown, we found that Sirt1 deacetylase, but not histone deacetylase 1 (HDAC1), was required for the effect of omentin-1 on p53 deacetylation and cancer cell proliferation. In omentin-1 treated HepG2 cells, the bax/bcl-2 protein ratio was increased, while the caspase-3 signaling pathway was also activated. Omentin-1 triggered JNK signaling but not p38 and ERK1/2 signaling pathways. Collectively, our data suggests that the novel adipokine omentin-1 may contribute to the therapeutic strategy for hepatocellular carcinoma.
Keywords: Adipokine; Omentin-1; HCC cells; p53; Acetylation; Sirt1;

Anti-inflammatory activity of anatabine via inhibition of STAT3 phosphorylation by Daniel Paris; David Beaulieu-Abdelahad; Laila Abdullah; Corbin Bachmeier; Ghania Ait-Ghezala; Jon Reed; Megha Verma; Fiona Crawford; Michael Mullan (145-153).
Previous investigations have demonstrated the anti-inflammatory effects of cholinergic agonists, such as nicotine. In the present study, we investigated the potential anti-inflammatory activity of anatabine, a minor tobacco alkaloid also present in plants of the Solanacea family which displays a chemical structural similarity with nicotine. Our data show that anatabine prevents STAT3 and NFκB phosphorylation induced by lipopolysaccharide (LPS) or TNF-α in SH-SY5Y, HEK293, human microglia and human blood mononuclear cells. Using human whole blood, we found that anatabine prevents IL-1β production induced by LPS. We assessed anatabine's anti-inflammatory activity in vivo using an acute model of inflammation by challenging wild-type mice with LPS. We observed that anatabine reduces pro-inflammatory cytokine production (IL-6, IL-1β and TNF-α) in the plasma, kidney and spleen of the animals following the injection of LPS and concomitantly opposes STAT3 phosphorylation induced by LPS in the spleen and kidney. We also investigated the impact of anatabine on neuroinflammation using a transgenic mouse model of Alzheimer’s disease (Tg APPsw) that displays elevated cytokine levels in the brain. Following a chronic oral treatment with anatabine, a reduction in brain TNF-α and IL-6 levels compared to untreated Tg APPsw mice was observed. Moreover, an increased STAT3 phosphorylation was detected in the brains of Tg APPsw mice compared to wild-type littermates and was inhibited by anatabine treatment. Overall our data show that the anti-inflammatory activity of anatabine in vitro and in vivo is mediated in part via an inhibition of STAT3 phosphorylation.
Keywords: Anatabine; Inflammation; Cytokine; STAT3; NFκB; LPS; Microglia; Alzheimer;

(−)-Epigallocatechin-3-gallate (EGCG), the principal constituent of green tea, protects neurons from toxic insults by suppressing the microglial secretion of neurotoxic inflammatory mediators. Voltage-gated proton channels are expressed in microglia, and are required for NADPH oxidase-dependent reactive oxygen species generation. Brain damage after ischemic stroke is dependent on proton channel activity. Accordingly, we examined whether EGCG could inhibit proton channel function in the murine microglial BV2 cells. EGCG potently inhibited proton currents with an IC50 of 3.7 μM. Other tea catechins, (−)-epigallocatechin, (−)-epicatechin and (−)-epicatechin-3-gallate, were far less potent than EGCG. EGCG did not change the kinetics of proton currents such as the activation and the deactivation time constants, the reversal potential and the activation voltage, suggesting that the gating process of proton channels were not altered by EGCG. EGCG is known to disturb lipid rafts by sequestering cholesterol. However, neither extraction of cholesterol with methyl-β-cyclodextrin or cholesterol supplementation could reverse the EGCG inhibition of proton currents. In addition, the EGCG effect was preserved in the presence of the cytoskeletal stabilizers paclitaxel and phalloidin, phosphatase inhibitors, the antioxidant Trolox, superoxide dismutase or catalase. The proton channel inhibition can be a substantial mechanism for EGCG to suppress microglial activation and subsequent neurotoxic events.
Keywords: Catechin; (−)-Epigallocatechin-3-gallate; Green tea; Microglia; Proton channel;

Scant information is available regarding the effects of cisplatin on the expression profile of tachykinin NK1 receptors and downstream signaling during cisplatin-induced emesis. Cisplatin causes peak early- and delayed-phase emesis in the least shrew at 1–2 and 33 h post-injection. To investigate the expression profile of NK1 receptor during both emetic phases, we cloned the cDNA corresponding to a ∼700 base pairs of mRNA flanked by two stretches of nucleotides conserved among different species and demonstrated that the shrew NK1 receptor nucleotide sequence shares ∼90% sequence identity with the human NK1 receptor. Of the 12 time-points tested, significant increases in expression levels of NK1 receptor mRNA in the shrew brainstem occurred at 2 and 28 h post-cisplatin injection, whereas intestinal NK1 receptor mRNA was increased at 28 h. Shrew brainstem and intestinal substance P mRNA levels also tended to increase during the two phases. Furthermore, expression levels of NK1 receptor protein were significantly increased in the brainstem at 2, 8, and 33 h post-cisplatin. No change in brainstem 5-HT3 receptor protein expression was observed. The temporal enhancements in NK1 receptor protein expression were mirrored by significant increases in the phosphorylation status of the brainstem ERK1/2 at 2, 8, and 33 h post-cisplatin. Phosphorylation of PKA significantly increased at 33rd and 40th hour. Our results indicate associations between cisplatin’s peak immediate- and delayed-phase vomiting frequency with increased: (1) expression levels of NK1 receptor mRNA and its protein level, and (2) downstream NK1 receptor-mediated phosphorylation of ERK1/2 and PKA signaling.Temporal associations in overexpression of NK1 receptor mRNA, protein levels and phosphorylation of ERK1/2 in the least shrew brainstem following cisplatin-induced peak immediate and delayed emesisemesis.Display Omitted
Keywords: Least shrew; Cisplatin; Emesis; Immediate; Delayed; Brainstem; Intestine; NK1 receptor; 5-HT3 receptor; Substance P; ERK1/2; PKA Intestine;

Opioid mediated activity and expression of mu and delta opioid receptors in isolated human term non-labouring myometrium by Rebecca A. Fanning; Jason P. McMorrow; Deirdre P. Campion; Michael F. Carey; John J. O'Connor (170-177).
The existence of opioid receptors in mammalian myometrial tissue is now widely accepted. Previously enkephalin degrading enzymes have been shown to be elevated in pregnant rat uterus and a met-enkephalin analogue has been shown to alter spontaneous contractility of rat myometrium. Here we have undertaken studies to determine the effects of met-enkephalin on in vitro human myometrial contractility and investigate the expression of opioid receptors in pregnant myometrium. Myometrial biopsies were taken from women undergoing elective caesarean delivery at term. Organ bath experiments were used to investigate the effect of the met-enkephalin analogue [d-Ala 2, d-met 5] enkephalin (DAMEA) on spontaneous contractility. A confocal immunofluorescent technique and real time PCR were used to determine the expression of protein and mRNA, respectively for two opioid receptor subtypes, mu and delta. DAMEA had a concentration dependent inhibitory effect on contractile activity (1×10−7  M–1×10−4  M; 54% reduction in contractile activity, P<0.001 at 1×10−4  M concentration). Mu and delta opioid receptor protein sub-types and their respective mRNA were identified in all tissues sampled. This is the first report of opioid receptor expression and of an opioid mediated uterorelaxant action in term human non-labouring myometrium in vitro.
Keywords: Human myometrium; Contractile activity; Pregnancy; Endogenous opioid peptide; Opioid receptor;

Participation of GABAA, GABAB receptors and neurosteroids in toluene-induced hypothermia: Evidence of concentration-dependent differences in the mechanism of action by Nayeli Paez-Martinez; Jorge Aldrete-Audiffred; Alfredo Gallardo-Tenorio; Mario Castro-Garcia; Erika Estrada-Camarena; Carolina Lopez-Rubalcava (178-185).
Toluene is a misused substance that modifies γ-aminobutyric acid (GABA) release and shares behavioral and molecular effects with GABAA and GABAB receptor agonists. GABAergic compounds are involved in thermoregulation processes and volatile substance users have reported that one of the reasons to inhale is to avoid feeling cold. At present, no studies have analyzed the effects of inhalants on body temperature and the mechanism of action involved. Thus, the main purpose of this study was to evaluate the effects of a (60 min) acute toluene inhalation (2000, 4000 and 6000 ppm) in core temperature. In addition, we tried to prevent the changes of temperature induced by toluene with the specific GABAA receptor blockers picrotoxin (0.01–0.1 mg/kg), bicuculline (0.1–0.3 mg/kg), and flumazenil (3–30 mg/kg); the GABAB receptor antagonist phaclofen (10–30 mg/kg) and the neurosteroid synthesis inhibitor finasteride (10–30 mg/kg). Results show that toluene reduced core temperature in mice in a concentration-dependent manner. The hypothermia produced by 4000 ppm toluene was prevented by picrotoxin, bicuculline, phaclofen and finasteride but not by flumazenil. In contrast none of these antagonists tested blocked the effects of 6000 ppm toluene. In conclusion, toluene decreases core temperature, GABA receptors and neurosteroids participate in toluene’s action at 4000 ppm; but other mechanisms of action are involved in the hypothermic effects of 6000 ppm toluene.
Keywords: Toluene; Hypothermia; GABAA receptors; GABAB receptors; Neurosteroids;

Hypothermia reduces calcium entry via the N-methyl-D-aspartate and ryanodine receptors in cultured hippocampal neurons by Kristin F. Phillips; Laxmikant S. Deshpande; Robert J. DeLorenzo (186-192).
Hypothermia is a powerful neuroprotective method when induced following cardiac arrest, stroke, and traumatic brain injury. The physiological effects of hypothermia are multifaceted and therefore a better knowledge of its therapeutic targets will be central to developing innovative combination therapies to augment the protective benefits of hypothermia. Altered neuronal calcium dynamics have been implicated following stroke, status epilepticus and traumatic brain injury. This study was therefore initiated to evaluate the effect of hypothermia on various modes of calcium entry into a neuron. Here, we utilized various pharmacological agents to stimulate major routes of calcium entry in primary cultured hippocampal neurons. Fluorescent calcium indicator Fura-2AM was used to compare calcium ratio under normothermic (37 °C) and hypothermic (31 °C) conditions. The results of this study indicate that hypothermia preferentially reduces calcium entry through N-methyl-D-aspartate receptors and ryanodine receptors. Hypothermia, on the other hand, did not have a significant effect on calcium entry through the voltage-dependent calcium channels or the inositol tri-phosphate receptors. The ability of hypothermia to selectively affect both N-methyl-D-aspartate receptors and ryanodine receptors-mediated calcium systems makes it an attractive intervention for alleviating calcium elevations that are present following many neurological injuries.
Keywords: Calcium; Hypothermia; Ryanodine receptor; NMDA receptor;

Alzheimer's disease pathomimetic toxicity could be induced in mice within one week after the intracerebroventricular (i.c.v.) injection of an aggregated preparation of the highly toxic and endogenous amyloid-β fragment Aβ25–35. It was recently reported that Aβ25–35 also provokes a modification of APP processing with accumulation of endogenous Aβ1–42. We here analyzed whether a γ-secretase inhibitor, BMS-299897, attenuated this Aβ25–35-induced Aβ1–42 seeding and toxicity. The compound was administered at 0.1–1 nmol/mouse, concomittantly with Aβ25–35 (9 nmol) in male Swiss mice. After one week, the contents in Aβ1–42 and Aβ1–40, and the levels in lipid peroxidation were analyzed in the mouse hippocampus. Mice were submitted to spontaneous alternation, passive avoidance and object recognition to analyze their short- and long-term memory abilities. Aβ25–35 increased Aβ1–42 content (+240%) but failed to affect Aβ1–40. BMS-299897 blocked the increase in Aβ1–42 content and decreased Aβ1–40 levels significantly. The compound did not affect Aβ25–35-induced increase in hippocampal lipid peroxidation. Behaviorally, BMS-299897 blocked the Aβ25–35-induced deficits in spontaneous alternation or novel object recognition, using a 1 h intertrial time interval. BMS-299896 failed to affect the passive avoidance impairments or novel object recognition, using a 24 h intertrial time interval. These results confirmed that Aβ25–35 injection provoked an accumulation in endogenous Aβ1–42, an effect blocked by γ-secretase inhibition. This Aβ1–42 accumulation marginally contributed to the toxicity or long-term memory deficits. However, since the seeded Aβ1–42 affected short-term memory, the rapid Aβ25–35 injection Alzheimer's disease model could be used to screen the activity of new secretase inhibitors.
Keywords: Alzheimer's disease; Amyloid-β peptide; γ-secretase inhibitor; Amyloid protein seeding; Drug discovery;

N-methyl-d-aspartate (NMDA) receptors can be inhibited by inhalational anesthetics in vitro at clinically relevant concentrations. Here, to clarify the role of NMDA receptors in anesthetic-induced unconsciousness, we examined the hypnotic properties of isoflurane, sevoflurane and nitrous oxide in NMDA receptor GluN2A subunit knockout mice. The hypnotic properties of inhalational anesthetics were evaluated in mice in the loss of righting reflex (LORR) assay by measuring the 50% concentration for LORR (LORR ED50). Knockout mice displayed isoflurane and sevoflurane LORR ED50 values similar to wild-type controls, indicating no significant contribution of these receptors to the hypnotic action of halogenated anesthetics. However, compared with wild-type controls, mutant mice displayed larger isoflurane LORR ED50 values in the presence of nitrous oxide, indicating a resistance to this gaseous anesthetic. Knockout mice have enhanced brain monoaminergic activity which occurs secondary to NMDA receptor dysfunction, and the observed resistance to the isoflurane LORR ED50-sparing effect of nitrous oxide could be abolished by pretreatment with the dopamine D2 receptor antagonist droperidol or with the serotonin 5-HT2A receptor antagonist ketanserin. Thus, resistance to nitrous oxide in knockout mice appears to be a secondary phenomenon of monoaminergic origin and not a direct result of impaired NMDA receptor function. Our results indicate that NMDA receptors are not critically involved in the hypnotic action of conventionally-used inhalational anesthetics. Also, they suggest that increased brain monoaminergic tone can diminish the effects of general anesthesia. Finally, they provide further evidence that changes secondary to genetic manipulation can explain the results obtained in global knockouts.
Keywords: N-methyl-d-aspartate receptor GluN2A subunit; Knockout mouse; Inhalational anesthetic; Nitrous oxide; Monoaminergic system; Secondary effect;

Neuropeptide Y is analgesic in rats after plantar incision by Suraj M. Yalamuri; Timothy J. Brennan; Christina M. Spofford (206-212).
Previous work has demonstrated that neuropeptide tyrosine (NPY), Y1 receptor and Y2 receptor are critical in modulation of pain after nerve injury. We hypothesized that NPY was important for nociception after surgical incision. As a model of postoperative pain, rats underwent a plantar incision in one hindpaw. Western blots were used to quantify changes in protein expression of NPY, Y1 receptor and Y2 receptor after incision in skin, muscle, and dorsal root ganglion (DRG). Pain-related behaviors were tested after incision in rats treated with intrathecal NPY, Y1 receptor antagonist (BIBO3304 – Chemical Name: N-[(1R)-1-[[[[4-[[(Aminocarbonyl)amino]methyl]phenyl]methyl]amino]carbonyl]-4-[(aminoiminomethyl)amino]butyl]-α-phenyl-benzeneacetamide ditrifluoroacetate), Y2 receptor antagonist (BIIE0246 – Chemical Name: N-[(1S)-4-[(Aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentaneacetamide), combined NPY+antagonists, morphine, or vehicle. Pain behaviors were tested after incision in rats treated with locally applied intraplantar injections of NPY, Y1 receptor and Y2 receptor antagonists or vehicle. NPY protein expression was significantly downregulated in muscle for two days after incision. In contrast, Y1 receptor and Y2 receptor protein expression was upregulated in both skin and muscle. A single intrathecal injection of NPY reduced cumulative guarding pain scores, as did morphine. The intrathecal administration of Y2 receptor antagonist also reduced pain scores; findings that were not observed when drugs were administered locally. Intrathecal Y2 receptor antagonists and NPY improved mechanical threshold and heat withdrawal latency 2 h after incision. Intrathecal administration of NPY and/or central blockade of Y2 receptor attenuated pain behaviors early after incision (postoperative day (POD) 1–2). Y1 receptor antagonist administration blocked the anti-hyperalgesic effect of NPY. Together these data suggest a role for spinal NPY in postoperative pain.
Keywords: NPY (Neuropeptide Y); Incision; Pain behavior; Gene; Protein expression;

The present study explored a link between spinal 5-HT7 and adenosine A1 receptors in antinociception by systemic amitriptyline in normal and adenosine A1 receptor knock-out mice using the 2% formalin test. In normal mice, antinociception by systemic amitriptyline 3 mg/kg was blocked by intrathecal administration of the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) 10 nmol. Blockade was also seen in adenosine A1 receptor +/+ mice, but not in −/− mice lacking these receptors. In both normal and adenosine A1 receptor +/+ mice, the selective 5-HT7 receptor antagonist (2R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine hydrochloride (SB269970) 3 μg blocked antinociception by systemic amitriptyline, but it did not prevent antinociception in adenosine A1 receptor −/− mice. In normal mice, flinching was unaltered when the selective 5-HT7 receptor agonist (2S)-(+)-5-(1,3,5-trimethylpyrazol-4-yl)-2-(dimethylamino)tetralin (AS-19) 20 μg was administered alone, but increased when co-administered intrathecally with DPCPX 10 nmol or SB269970 3 μg. Intrathecal AS-19 decreased flinching in adenosine A1 receptor +/+ mice compared to −/− mice. Systemic amitriptyline appears to reduce nociception by activating spinal adenosine A1 receptors secondarily to 5-HT7 receptors. Spinal actions constitute only one aspect of antinociception by amitriptyline, as intraplantar DPCPX 10 nmol blocked antinociception by systemic amitriptyline in normal and adenosine A1 receptor +/+, but not −/− mice. Adenosine A1 receptor interactions are worthy of attention, as chronic oral caffeine (0.1, 0.3 g/L, doses considered relevant to human intake levels) blocked antinociception by systemic amitriptyline in normal mice. In conclusion, adenosine A1 receptors contribute to antinociception by systemic amitriptyline in both spinal and peripheral compartments.
Keywords: Amitriptyline; Adenosine A1 receptor; Serotonin 5-HT7 receptor; Formalin test; Antinociception;

CNR1 gene deletion affects the density of endomorphin-2 binding sites in the mouse brain in a hemisphere-specific manner by Eszter Paldy; Erika Borcel; Alejandro Higuera-Matas; Gonzalo López-Montoya; Tibor Wenger; Geza Toth; Anna Borsodi; Emilio Ambrosio (220-227).
Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are two endogenous tetrapeptides with very high affinities for the μ-opioid receptor. Until recently, the precise neuroanatomical localization of the binding sites for these peptides was unknown. However, the recent synthesis of tritiated forms of these molecules has permitted these binding sites to be analysed with a very high degree of neuroanatomical specificity. Preliminary studies demonstrated a superior binding profile for EM-2, with less non-specific binding than EM-1. As the endogenous cannabinoid and opioid systems interact at several levels, we investigated how deletion of the CNR1 gene, which encodes the cannabinoid receptor 1 (CB1R) protein, affects the brain distribution of EM-2 binding sites. Our results revealed no differences in the average density of EM-2 binding sites in CB1 receptor knockout (CB1R KO) and WT mice. However, when both hemispheres were analysed separately, we detected specific alterations in the distribution of EM-2 binding sites in the right hemisphere of CB1R KO mice. While, the density of EM-2 binding sites in CB1R KO mice was higher in the CA3 hippocampal field and in the pontine tegmental nuclei, it was lower in the superior colliculus and ventral tegmental area than in WT controls. No differences were observed in the left hemisphere for any of the regions analysed. For the first time these findings demonstrate a lateralization effect on cerebral opioid binding sites that may be mediated by the central cannabinoid system.
Keywords: Endomorphins; CNR1; Opioid system; Cannabinoids; Lateralization;

Sub-anesthetic concentrations of (R,S)-ketamine metabolites inhibit acetylcholine-evoked currents in α7 nicotinic acetylcholine receptors by Ruin Moaddel; Galia Abdrakhmanova; Joanna Kozak; Krzysztof Jozwiak; Lawrence Toll; Lucita Jimenez; Avraham Rosenberg; Thao Tran; Yingxian Xiao; Carlos A. Zarate; Irving W. Wainer (228-234).
The effect of the (R,S)-ketamine metabolites (R,S)-norketamine, (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine on the activity of α7 and α3β4 neuronal nicotinic acetylcholine receptors was investigated using patch-clamp techniques. The data indicated that (R,S)-dehydronorketamine inhibited acetylcholine-evoked currents in α7-nicotinic acetylcholine receptor, IC50=55±6 nM, and that (2S,6S)-hydroxynorketamine, (2R,6R)-hydroxynorketamine and (R,S)-norketamine also inhibited α7-nicotinic acetylcholine receptor function at concentrations ≤1 μM, while ( R ,S)-ketamine was inactive at these concentrations. The inhibitory effect of (R,S)-dehydronorketamine was voltage-independent and the compound did not competitively displace selective α7-nicotinic acetylcholine receptor ligands [125I]-α-bungarotoxin and [3H]-epibatidine indicating that (R,S)-dehydronorketamine is a negative allosteric modulator of the α7-nicotinic acetylcholine receptor. (R,S)-Ketamine and (R,S)-norketamine inhibited (S)-nicotine-induced whole-cell currents in cells expressing α3β4-nicotinic acetylcholine receptor, IC50 3.1 and 9.1 μM, respectively, while (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine were weak inhibitors, IC50 >100 μM. The binding affinities of (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine at the NMDA receptor were also determined using rat brain membranes and the selective NMDA receptor antagonist [3H]-MK-801. The calculated K i values were 38.95 μM for (S)-dehydronorketamine, 21.19 μM for (2S,6S)-hydroxynorketamine and>100 μM for (2R,6R)-hydroxynorketamine. The results suggest that the inhibitory activity of ketamine metabolites at the α7-nicotinic acetylcholine receptor may contribute to the clinical effect of the drug.
Keywords: Dehydronorketamine; Hydroxynorketamine; Norketamine; Depression; Pain; Negative allosteric modifiers; Neuronal nicotinic acetylcholine receptors;

Chemical neuroanatomical and psychopharmacological evidence that κ receptor-mediated endogenous opioid peptide neurotransmission in the dorsal and ventral mesencephalon modulates panic-like behaviour by Juliana Almeida da Silva; Renato Leonardo de Freitas; Gustavo Cavalcanti Dutra Eichenberger; Cláudia Maria Padovan; Norberto Cysne Coimbra (235-245).
The chemical neuroanatomy and the effects of central administration of opioid antagonists on the innate fear-induced responses elicited by electrical (at escape behaviour threshold) stimulation of the midbrain tectum were determined. The aim of the present work was to investigate the interaction between the tecto-nigral endogenous opioid peptide-mediated disinhibitory pathways and nigro-tectal inhibitory links in the control of panic-like behaviour and their organisation in the continuum comprised by the deep layers of the superior colliculus (dlSC) and the dorsolateral columns of the periaqueductal grey matter (dlPAG). Beta-endorphin-labelled neurons and fibres were found in the dorsal midbrain and also in the substantia nigra. Opioid varicose fibres and terminal buttons were widely distributed in PAG columns and in all substantia nigra subdivisions. Microinjections of naltrexone (a non-selective opioid receptor antagonist; 5.0 μg/0.2 μl) or nor-binaltorphimine (a selective κ-opioid receptor antagonist; 5.0 μg/0.2 μl) in the dlSC/dlPAG continuum, in independent groups of animals, induced significant increases in the escape thresholds for midbrain tectum electrical stimulation. The microinjection of naltrexone or nor-binaltorphimine into the SNpr also increased the escape behaviour threshold for electrical stimulation of dlSC/dlPAG. These morphological and neuropharmacological findings support previous evidence from our team for the role played by the interaction between opioidergic and GABAergic mechanisms in the modulation of innate fear-induced responses. The present data offer a neuroanatomical basis for both intratectal axo-axonic/pre-synaptic and tecto-nigral axo-somatic opioid inhibition of GABAergic nigro-tectal neurons that modulate the dorsal midbrain neurons related to the organisation of fear-related emotional responses.
Keywords: Superior colliculus; Periaqueductal grey matter; Innate fear; Intra-tectal opioid pathway; Tecto-nigral opioid pathway; Panic-attack;

Effect of dopamine and serotonin receptor antagonists on fencamfamine-induced abolition of latent inhibition by Cilene Rejane Ramos Alves de Aguiar; Marlison José Lima de Aguiar; Roberto DeLucia; Maria Teresa Araujo Silva (246-251).
The purpose of this investigation was to verify the role of dopamine and serotonin receptors in the effect of fencamfamine (FCF) on latent inhibition. FCF is a psychomotor stimulant with an indirect dopaminergic action. Latent inhibition is a model of attention. Latent inhibition is blocked by dopaminergic agents and facilitated by dopamine receptor agonists. FCF has been shown to abolish latent inhibition. The serotonergic system may also participate in the neurochemical mediation of latent inhibition. The selective dopamine D1 receptor antagonist SCH 23390 (7-chloro-3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol), D2 receptor antagonists pimozide (PIM) and methoclopramide (METH), and serotonin 5-HT2A/C receptor antagonist ritanserin (RIT) were used in the present study. Latent inhibition was evaluated using a conditioned emotional response procedure. Male Wistar rats that were water-restricted were subjected to a three-phase procedure: preexposure to a tone, tone-shock conditioning, and a test of the effect of the tone on licking frequency. All of the drugs were administered before the preexposure and conditioning phases. The results showed that FCF abolished latent inhibition, and this effect was clearly antagonized by PIM and METH and moderately attenuated by SCH 23390. At the doses used in the present study, RIT pretreatment did not affect latent inhibition and did not eliminate the effect of FCF, suggesting that the FCF-induced abolition of latent inhibition is not mediated by serotonin 5-HT2A/C receptors. These results suggest that the effect of FCF on latent inhibition is predominantly related to dopamine D2 receptors and that dopamine D2 receptors participate in attention processes.
Keywords: Fencamfamine; Latent inhibition; Dopamine receptors; Serotonin receptors; Attention process;

Examination of methylphenidate-mediated behavior regulation by glycogen synthase kinase-3 in mice by Marjelo A. Mines; Eleonore Beurel; Richard S. Jope (252-258).
Abnormalities in dopaminergic activity have been implicated in psychiatric diseases, such as attention deficit hyperactivity disorder (ADHD), and are treated with therapeutic stimulants, commonly methylphenidate or amphetamine. Amphetamine administration increases glycogen synthase kinase-3 (GSK3) activation, which is necessary for certain acute behavioral responses to amphetamine, including increased locomotor activity and impaired sensorimotor gating. Here, we tested if modulating GSK3 by administration of the GSK3 inhibitor lithium or expression of constitutively active GSK3 altered behavioral responses to methylphenidate administered to mice acutely or daily for 8 days. Methylphenidate or amphetamine was administered to mice intraperitoneally for 1 or 8 days. Open-field activity and pre-pulse inhibition (PPI) were measured. In contrast to lithium’s blockade of acute amphetamine-induced locomotor hyperactivity, lithium treatment did not significantly reduce methylphenidate-induced locomotor hyperactivity in wild-type mice after acute or 8 days of repeated methylphenidate administration. Lithium treatment significantly increased the impairment in PPI caused by methylphenidate, but significantly reduced the amphetamine-induced PPI deficit. In GSK3 knockin mice, expression of constitutively active GSK3β, but not GSK3α, significantly increased locomotor hyperactivity after acute methylphenidate treatment, and significantly impaired PPI, preventing further methylphenidate-induced impairment of PPI that was evident in wild-type mice and GSK3α knockin mice. Lithium does not counteract locomotor activity and PPI responses to methylphenidate as it does these responses to amphetamine, indicating that different mechanisms mediate these behavioral responses to methylphenidate and amphetamine. Only active GSK3β, not GSK3α, modulates behavioral responses to MPH, indicating selectivity in the actions of GSK3 isoforms.
Keywords: Amphetamine; Attention deficit hyperactivity disorder; Glycogen synthase kinase-3; Methylphenidate; Pre-pulse inhibition;

Hippocampal synaptic plasticity restoration and anti-apoptotic effect underlie berberine improvement of learning and memory in streptozotocin-diabetic rats by Hamid Kalalian-Moghaddam; Tourandokht Baluchnejadmojarad; Mehrdad Roghani; Fatemeh Goshadrou; Abdolaziz Ronaghi (259-266).
Chronic diabetes mellitus initiates apoptosis and negatively affects synaptic plasticity in the hippocampus with ensuing impairments of learning and memory. Berberine, an isoquinoline alkaloid, exhibits anti-diabetic, antioxidant and nootropic effects. This study was conducted to evaluate the effect of berberine on hippocampal CA1 neuronal apoptosis, synaptic plasticity and learning and memory of streptozotocin (STZ)-diabetic rats. Long-term potentiation (LTP) in perforant path-dentate gyrus synapses was recorded for assessment of synaptic plasticity and field excitatory post-synaptic potential (fEPSP) slope and population spike (PS) amplitude. PS amplitude and fEPSP significantly decreased in diabetic group versus control, and chronic berberine treatment (100 mg/kg/day, p.o.) restored PS amplitude and fEPSP and ameliorated learning and memory impairment and attenuated apoptosis of pyramidal neurons in the CA1 area, as determined by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling method. In summary, chronic berberine treatment of STZ-diabetic rats significantly ameliorates learning and memory impairment and part of its beneficial effect could be attributed to improvement of synaptic dysfunction and anti-apoptotic property.
Keywords: Berberine; Diabetes mellitus; Streptozotocin; Learning and memory; Synaptic plasticity; Apoptosis;

It has been previously reported that the GABAB receptor agonist baclofen decreases food intake after oral administration and fat intake after intraperitoneal administration. The aim of the study was to investigate the effects of baclofen (1–4 mg/ kg) administered orally (Experiment 1) on food intake in non-deprived rats (n=6) and intraperitoneally (Experiment 2) on fat intake in non-deprived rats (n=8) that were naïve to baclofen (1st set of trials) and in the same group of rats after they were sub-chronically exposed to baclofen (2nd set of trials). The results from Experiment 1 show that baclofen had no effects on food intake during the 1st set of trials, but the 2 and 4 mg/kg doses significantly increased food consumption during the 2nd set of trials. Baclofen produced sedation during the 1st set of trials, but tolerance occurred to this effect and was not apparent during the 2nd set of trials. These observations suggest that the motor effects may have competed with the hyperphagic effects of baclofen during the 1st set of trials. The data from Experiment 2 show that baclofen had no effects on fat intake during either the 1st or 2nd set of trials. The results of the study thus indicate that orally administrated baclofen increases food intake and intraperitoneal administration has no effect on fat intake in non-deprived rats under the conditions used in this study. These findings may have important implications for research on the use of baclofen in studies concerned with ingestive behaviours.
Keywords: Baclofen; Food intake; Oral administration; Fat intake; GABAB; Feeding;

GPR88 is a novel orphan G protein-coupled receptor that is primarily located at the striatum. Genetic knockout studies reveal phenotypes of increased dopamine D2 receptor sensitivity in mice, suggesting that GPR88 receptors may be involved in the modulation of dopaminergic system. However, there is no study that examines the pharmacological effects of GPR88 receptor ligands in in vivo preparations. This study examined the effects of a GPR88 receptor agonist, (1R, 2R)-2-pyridin-2-yl-cyclopropane carboxylic acid ((2S, 3S)-2-amino-3-methyl-pentyl)-(4′-propylbiphenyl-4-yl)-amide (2-PCCA), on the motor activity in rats and on methamphetamine-induced hyperactivity and discriminative stimulus effects. 2-PCCA (0.1–3.2 mg/kg) dose-dependently decreased the locomotor activity in rats and, when studied in combination with 1.0 mg/kg methamphetamine, also dose-dependently decreased methamphetamine-induced hyperactivity. However, the dose of 2-PCCA that significantly attenuated methamphetamine-induced hyperactivity was also the dose that by itself markedly decreased the baseline locomotor activity. In rats discriminating 0.32 mg/kg methamphetamine, 2-PCCA (1–3.2 mg/kg) itself did not produce methamphetamine-like discriminative stimulus effects and, when studied in combination, did not alter the discriminative stimulus effects of methamphetamine. Together, these data have provided the first line of evidence that activation of GPR88 receptors does not alter the behavioral effects of methamphetamine. The potential implications of these findings are also discussed.
Keywords: GPR88; Methamphetamine; Locomotor activity; Discriminative stimulus; Rat;

The dopaminergic stabilizer pridopidine decreases expression of l-DOPA-induced locomotor sensitisation in the rat unilateral 6-OHDA model by Henrik Ponten; Johan Kullingsjö; Clas Sonesson; Susanna Waters; Nicholas Waters; Joakim Tedroff (278-285).
Treatment-limiting motor complications occur in patients with Parkinson's disease after chronic levodopa (l-DOPA) treatment, and represent an unmet medical need. We examined the motor and neurochemical effects of the dopaminergic stabilizer pridopidine (NeuroSearch A/S, Ballerup, Denmark) in the unilateral rodent 6-OHDA lesion model, which is often used to evaluate the potential of experimental compounds for such dopamine-related motor complications. In total, 72 rats were hemi-lesioned and allocated to receive twice-daily injections of either vehicle; 6.5 mg/kg l-DOPA; l-DOPA+25 μmol/kg pridopidine; or l-DOPA+25 μmol/kg (−)-OSU6162—a prototype dopaminergic stabilizer used previously in 6-OHDA hemi-lesion models. Animals were treated for 7, 14 or 21 days, and locomotor activity and ex vivo brain tissue neurochemistry analysed. In agreement with previous studies, l-DOPA sensitised the motor response, producing significantly more contralateral rotations than vehicle (P<0.05). Concomitant administration of pridopidine and l-DOPA significantly decreased the number of l-DOPA-induced contralateral rotations on day 7, 14 and 21 (P<0.05 versus l-DOPA alone), while still allowing a beneficial locomotor stimulant effect of l-DOPA. Concomitant pridopidine also reduced l-DOPA-induced rotation asymmetry (P<0.05 versus l-DOPA alone) and had no adverse effects on distance travelled. Brain neurochemistry was generally unaffected in all treatments groups. In conclusion, pridopidine shows potential for reducing motor complications of l-DOPA in Parkinson's disease and further testing is warranted.
Keywords: 6-OHDA; l-DOPA-induced dyskinesia; Locomotor sensitisation; Rotation; Pridopidine;

Kappa-opioid receptors mediate the antidepressant-like activity of hesperidin in the mouse forced swimming test by Carlos B. Filho; Lucian Del Fabbro; Marcelo G. de Gomes; André T.R. Goes; Leandro C. Souza; Silvana P. Boeira; Cristiano R. Jesse (286-291).
The opioid system has been implicated as a contributing factor for major depression and is thought to play a role in the mechanism of action of antidepressants. This study investigated the involvement of the opioid system in the antidepressant-like effect of hesperidin in the mouse forced swimming test. Our results demonstrate that hesperidin (0.1, 0.3 and 1 mg/kg; intraperitoneal) decreased the immobility time in the forced swimming test without affecting locomotor activity in the open field test. The antidepressant-like effect of hesperidin (0.3 mg/kg) in the forced swimming test was prevented by pretreating mice with naloxone (1 mg/kg, a nonselective opioid receptor antagonist) and 2-(3,4-dichlorophenyl)-Nmethyl-N-[(1S)-1-(3-isothiocyanatophenyl)-2-(1-pyrrolidinyl)ethyl] acetamide (DIPPA (1 mg/kg), a selective κ-opioid receptor antagonist), but not with naloxone methiodide (1 mg/kg, a peripherally acting opioid receptor antagonist), naltrindole (3 mg/kg, a selective δ-opioid receptor antagonist), clocinnamox (1 mg/kg, a selective μ-opioid receptor antagonist) or caffeine (3 mg/kg, a nonselective adenosine receptor antagonist). In addition, a sub-effective dose of hesperidin (0.01 mg/kg) produced a synergistic antidepressant-like effect in the forced swimming test when combined with a sub-effective dose of morphine (1 mg/kg). The antidepressant-like effect of hesperidin in the forced swimming test on mice was dependent on its interaction with the κ-opioid receptor, but not with the δ-opioid, μ-opioid or adenosinergic receptors. Taken together, these results suggest that hesperidin possesses antidepressant-like properties and may be of interest as a therapeutic agent for the treatment of depressive disorders.
Keywords: Hesperidin; Antidepressant; Forced swimming test; Opioid receptor; κ-opioid receptor;

Effects of amylin and bupropion/naltrexone on food intake and body weight are interactive in rodent models by Jason R. Clapper; Jennifer Athanacio; Carrie Wittmer; Pete S. Griffin; Lawrence D'Souza; David G. Parkes; Jonathan D. Roth (292-298).
Antagonism of opioid systems (e.g., with naltrexone) has been explored as an anti-obesity strategy, and is particularly effective when co-administered with dual inhibitors of dopamine and norepinephrine reuptake (e.g., bupropion). Previously, we demonstrated that amylin enhances the food intake lowering and weight loss effects of neurohormonal (e.g., leptin, cholecystokinin, melanocortins) and small molecule (e.g., phentermine, sibutramine) agents. Here, we sought to characterize the interaction of amylin with naltrexone/bupropion on energy balance. Wild-type and amylin knockout mice were similarly responsive to the food intake lowering effects of either naltrexone (1 mg/kg, subcutaneous) or bupropion (50 mg/kg, subcutaneous) suggesting that they act independently of amylinergic systems and could interact additively when given in combination with amylin. To test this, diet-induced obese rats were treated (for 11 days) with vehicle, rat amylin (50 μg/kg/d, infused subcutaneously), naltrexone/bupropion (1 and 20 mg/kg, respectively by twice daily subcutaneous injection) or their combination. We found that amylin+naltrexone/bupropion combination therapy exerted additive effects to reduce cumulative food intake, body weight and fat mass. In a separate study, the effects of amylin and naltrexone/bupropion administered at the same doses (for 14 days) were compared to a pair-fed group. Although the combination and pair-fed groups lost a similar amount of body weight, rats treated with the combination lost 68% more fat and better maintained their lean mass. These findings support the strategy of combined amylin agonism with opioid and catecholaminergic signaling systems for the treatment of obesity.
Keywords: Amylin; Contrave; Bupropion; Naltrexone; Obesity; Peptide;

Resveratrol mediates anti-atherogenic effects on cholesterol flux in human macrophages and endothelium via PPARγ and adenosine by Iryna Voloshyna; Ofek Hai; Michael J. Littlefield; Steven Carsons; Allison B. Reiss (299-309).
Resveratrol is a bioactive molecule used in dietary supplements and herbal medicines and consumed worldwide. Known cardioprotective and anti-inflammatory properties of resveratrol have spurred investigation of the mechanisms involved. The present study explored potential atheroprotective actions of resveratrol on cholesterol metabolism in cells of the arterial wall, including human macrophages and arterial endothelium. Using QRT-PCR and Western blotting techniques, we measured expression of the proteins involved in reverse cholesterol transport (ABCA1, ABCG1 and SR-B1) and the scavenger receptors responsible for uptake of modified cholesterol (CD36, SR-A1 and LOX-1). We analyzed the effect of resveratrol on apoA-1-and HDL-mediated cholesterol efflux in human THP-1 macrophages. The effect of resveratrol on oxLDL internalization and foam cell formation were evaluated using confocal and light microscopy. Our data indicate that resveratrol regulates expression of major proteins involved in cholesterol transport, promotes apoA-1 and HDL-mediated efflux, downregulates oxLDL uptake and diminishes foam cell formation. Mechanistically, resveratrol effects were dependent upon PPAR-γ and adenosine 2A receptor pathways. For the first time we demonstrate that resveratrol regulates expression of the cholesterol metabolizing enzyme cytochrome P450 27-hydroxylase, providing efficient cholesterol elimination via formation of oxysterols. This study establishes that resveratrol attenuates lipid accumulation in cultured human macrophages via effects on cholesterol transport. Further in vivo studies are needed to determine whether resveratrol may be an additional resource available to reduce lipid deposition and atherosclerosis in humans.
Keywords: Atherosclerosis; Anti-atherogenic function; Adenosin 2A receptor; PPAR-γ; Resveratrol; Reverse cholesterol transport; Scavenger receptor;

Reduced anti-contractile effect of perivascular adipose tissue on mesenteric small arteries from spontaneously hypertensive rats: Role of Kv7 channels by Rui Li; Ingrid Andersen; Josefin Aleke; Veronika Golubinskaya; Helena Gustafsson; Holger Nilsson (310-315).
Perivascular adipose tissue (PVAT) has been shown to produce vasoactive substances and regulate vascular tone. This function of PVAT has been reported to be altered in hypertension. However, the underlying mechanisms are not fully understood. In this study we used age-matched normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) as well as Sprague-Dawley rats and tested effects of PVAT on mesenteric small arteries. Vessels were mounted in a Mulvany–Halpern myograph and cumulative concentration–response relations to noradrenaline were determined in the presence or absence of PVAT. We found that PVAT has an anti-contractile effect on mesenteric small vessels, irrespective of strains. A reduced effect of PVAT was observed in SHR compared to WKY rats; the difference between strains was eliminated by 10 μM XE991, a blocker of Kv7 (KCNQ) voltage-dependent potassium channels. The anti-contractile effect of PVAT was not affected by depolarizing smooth muscle cells with high K+ solution. Sensitivities to exogenous vasodilators acetylcholine or sodium nitroprusside were not potentiated but reduced in vessels with PVAT. Our results suggest that the reduced anti-contractile effect of PVAT in SHR correlates with a deficiency in Kv7 channels. Diffusion hindrance of PVAT is also a factor that should be considered in investigations on rat mesenteric small arteries.
Keywords: Adipocyte-derived relaxing factor; Hypertension; Kv7 channel; Mesenteric artery; Perivascular adipose tissue; Vascular tone;

The sesame lignan sesamin attenuates vascular dysfunction in streptozotocin diabetic rats: Involvement of nitric oxide and oxidative stress by Tourandokht Baluchnejadmojarad; Mehrdad Roghani; Mohammad-Reza Jalali Nadoushan; Mohammad-Reza Vaez Mahdavi; Hamid Kalalian-Moghaddam; Farshad Roghani-Dehkordi; Sharareh Dariani; Safoura Raoufi (316-321).
The effect of chronic administration of sesamin was studied on aortic reactivity of streptozotocin diabetic rats. Male diabetic rats received sesamin for 7 weeks after diabetes induction. Contractile responses to KCl and phenylephrine and relaxation response to acetylcholine were obtained from aortic rings. Maximum contractile response of endothelium-intact rings to phenylephrine was significantly lower in sesamin-treated diabetic rats relative to untreated diabetics and endothelium removal abolished this difference. Meanwhile, endothelium-dependent relaxation to acetylcholine was significantly higher in sesamin-treated diabetic rats as compared to diabetic ones and pretreatment of rings with nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester significantly attenuated the observed response. Two-month diabetes also resulted in an elevation of malondialdehyde and decreased superoxide dismutase activity and sesamin treatment significantly improved these changes. Therefore, chronic treatment of diabetic rats with sesamin could prevent some abnormal changes in vascular reactivity in diabetic rats through nitric oxide and via attenuation of oxidative stress and tissue integrity of endothelium is necessary for its beneficial effect.
Keywords: Sesamin; Diabetes mellitus; Streptozotocin; Aorta;

Inhibition of pre-ischeamic conditioning in the mouse caudate brain slice by NMDA- or adenosine A1 receptor antagonists by Nikky K. Chauhan; Andrew M.J. Young; Claire L. Gibson; Colin Davidson (322-329).
Evidence suggests that pre-ischeamic conditioning (PIC) offers protection against a subsequent ischeamic event. Although some brain areas such as the hippocampus have received much attention, the receptor mechanisms of PIC in other brain regions are unknown. We have previously shown that 10 min oxygen and glucose deprivation (OGD) evokes tolerance to a second OGD event in the caudate. Here we further examine the effect of length of conditioning event on the second OGD event. Caudate mouse brain slices were superfused with artificial cerebro-spinal fluid (aCSF) bubbled with 95%O2/5%CO2. OGD was achieved by reducing the aCSF glucose concentration and by bubbling with 95%N2/5%CO2. After approximately 5 min OGD a large dopamine efflux was observed, presumably caused by anoxic depolarisation. On applying a second OGD event, 60 min later, dopamine efflux was delayed and reduced. We first examined the effect of varying the length of the conditioning event from 5 to 40 min and found tolerance to PIC increased with increasing duration of conditioning. We then examined the receptor mechanism(s) underlying PIC. We found that pre-incubation with either MK-801 or 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) reduced tolerance to the second OGD event. These data suggest that either N-methyl-d-aspartate (NMDA) or adenosine A1 receptor activation evokes PIC in the mouse caudate.
Keywords: Dopamine; Voltammetry; MK-801; DPCPX; TTC; HPLC;

Na+–H+ exchange inhibition attenuates ischemic injury in rat random pattern skin flap: The role of mitochondrial ATP-sensitive potassium channels by Hamed Emami; Saman Shafaat Talab; Behtash Ghazi Nezami; Azadeh Elmi; Solmaz Assa; Mohammad Reza Ostovaneh; Ahmad Reza Dehpour (330-334).
Necrosis of distal portion of skin flaps due to ischemia still remains a problem in plastic surgery. Following ischemia, a cascade of deleterious events including over-activity of Na+–H+ Exchanger (NHE) takes place. In present study we evaluated the effect of the potent NHE inhibitor, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) on ischemic tissue injury in a skin flap model, and investigated the role of mitochondrial ATP-sensitive K+ channels (KATP) in this phenomenon. Seventy-eight rats were randomly divided into thirteen treatment groups (6 rats each). Four groups received different doses of EIPA in the flap. EIPA/GLY group received an effective dose of a KATP channel blocker, glibenclamide (GLY, 0.3 mg/kg) intraperitoneally (i.p.) 30 min before raising the flap, and a local effective dose of EIPA (0.1 mM) immediately after raising the flap. EIPA/diazoxide group (EIPA/DIA) received a sub-effective dose of diazoxide (7.5 mg/kg i.p.) 30 min before raising the flap and a local sub-effective dose of EIPA (0.075 mM). EIPA 0.1 and 0.2 mM significantly increased flap survival area compared to control group (56.01±6.1%, P<0.001). The protective effect of EIPA (0.1 mM) was abolished by administration of glibenclamide (0.3 mg/kg i.p.). Co-administration of a sub-effective dose of EIPA (0.075 mM), with a sub-effective dose of diazoxide (7.5 mg/kg i.p.) significantly improved flap survival (P<0.05). We demonstrated that the NHE inhibitor, EIPA can increase random pattern skin flap survival. Administration of diazoxide potentiates this effect, while glibenclamide abolishes that, implicating that the protective effect of EIPA is mediated through mitochondrial-KATP channels.
Keywords: Skin flap; Amiloride; Ischemia injury;

In the search for new hypotesive agents a series of aroxyalkyl derivatives of 2-methoxyphenylpiperazine was obtained. The aim of the present study was to examine their hypotensive properties and to evaluate their mechanism of action. In the study their hypotensive activity after i.v. and p.o. administration, influence on the pressor responses to adrenaline, noradrenaline and methoxamine, direct spasmolytic and vasorelaxant effects were assessed. In the next step two compounds which were the most active and selective for α1-adrenoceptors were evaluated for their α1-adrenoreceptor subtypes selectivity in functional bioassays. The data from our experiments indicate that the hypotensive activity of tested aroxyalkyl derivatives of 2-methoxyphenylpiperazine is mainly a result of their α1- or α12-adrenoceptor blocking properties. The two most active compounds showed to be the competitive antagonists of α1-adrenoceptors with stronger activity at α1D-, α1A- and α1L- and weaker at α1B-subtype.
Keywords: Phenylpiperazine derivative; α1-adrenoceptor subtype; Hypotensive activity; Selectivity;

Late onset vascular dysfunction in the R6/1 model of Huntington’s disease by Awahan Rahman; Mari Ekman; Yulia Shakirova; Kristina E. Andersson; Matthias Mörgelin; Jonas S Erjefält; Patrik Brundin; Jia-Yi Li; Karl Swärd (345-353).
Huntington’s disease is a neurodegenerative disorder that also gives raise to widespread changes in peripheral organs and tissues. We tested the hypothesis that vascular dysfunction may occur in Huntington’s disease by studying R6/1 mice which express exon 1 of the mutant huntingtin gene. We assessed arterial function in R6/1 and wild type (WT) mice using myography. Arterial contractility was largely unaltered in R6/1 arteries at 15 and 32 weeks of age. By 40 weeks, contractility was impaired irrespective of which vasoconstrictor we tested. Endothelium-dependent relaxation was not affected, and we observed no changes in arterial geometry or expression of contractile proteins, such as myosin regulatory light chains or smooth muscle α-actin. The frequency of calcium oscillations in R6/1 arterial smooth muscle cells was higher than in WT control tissue, whereas myosin phosphorylation was unaltered. Impairment of force by the mitochondrial inhibitors cyanide and rotenone was less pronounced in R6/1 than in WT arteries and mitochondria were enlarged, in keeping with an effect related to altered mitochondrial function. Our results reveal that arteries in the R6/1 model of Huntington’s disease exhibit an age-dependent impairment of contractility and that they depend less on mitochondrial function when they contract.
Keywords: Huntington’s disease; R6/1; Vascular dysfunction; Mitochondria; Calcium waves.;

Effect of celecoxib on cyclooxygenase-1-mediated prostacyclin synthesis and endothelium-dependent contraction in mouse arteries by Bin Liu; Wenhong Luo; Yingzhan Zhang; Hui Li; Ningxia Zhu; Dongyang Huang; Yingbi Zhou (354-361).
This study aimed to determine whether a cyclooxygenase-2 (COX-2) inhibitor celecoxib influences endothelium-dependent contraction independent of its action on COX-2 and, if so, the underlying mechanism(s). Abdominal aortas and/or carotid arteries from C57BL/6 mice or those with genetic COX-2 deficiency (COX-2−/−) were isolated for functional and/or biochemical analyses. Result showed that following NO synthase inhibition celecoxib not only reduced the contraction evoked by acetylcholine in C57BL/6 abdominal aorta, but also that in COX-2 −/− mice showing a comparable magnitude. Notably, the IC50 of celecoxib obtained in COX-2 −/− abdominal aorta was only ∼0.364 μM. Also, celecoxib exhibited a similar effect on COX-2 −/− carotid arteries. Interestingly, celecoxib was not only found to inhibit the production of the prostacyclin (PGI2) metabolite 6-keto-PGF in COX-2 −/− aortas, but also caused a reduction in the contraction evoked by PGI2, by the α1-adrenergic agonist phenylephrine, or by 30 mM K+-induced depolarization in COX-2 −/− and/or C57BL/6 abdominal aorta. Moreover, N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS398), another COX-2 inhibitor, also reduced the contraction evoked by acetylcholine or by 30 mM K+-induced depolarization in COX-2 −/− mice. These results demonstrate explicitly that in mouse arteries celecoxib not only inhibits COX-1-mediated synthesis of PGI2 and probably some other prostanoids, but also causes a reduction in vessel contractility that is independent of either COX-2 or COX-1, leading to an inhibition of COX-1-mediated endothelium-dependent contraction with an IC50 value far below that of it considered for COX-1 . Also, our data suggest that such effects of celecoxib could be possibly shared by some other COX-2 inhibitors, such as NS398.
Keywords: COX-2; Inhibitor; Metabolite; PGI2; TP receptor; Contraction;

Reactive oxygen species contribute to regulating the excitability of vascular smooth muscle. This study investigated the actions of the relatively stable reactive oxygen species, H2O2, on nerve-evoked contractions of mouse distal tail artery. H2O2 (10–100 μM) increased nerve-evoked contractions of isometrically mounted segments of tail artery. Endothelium denudation increased nerve-evoked contractions and abolished the facilitatory effect of H2O2. Inhibition of nitric oxide synthase with l-nitroarginine methyl ester (0.1 mM) also increased nerve-evoked contractions and reduced the late phase of H2O2-induced facilitation. H2O2-induced facilitation of nerve-evoked contractions depended, in part, on synthesis of prostanoids and was reduced by the cyclooxygenase inhibitor indomethacin (1 μM) and the thromboxane A2 receptor antagonist SQ 29548 (1 μM). H2O2 increased sensitivity of nerve-evoked contractions to the α2-adrenoceptor antagonist idazoxan (0.1 μM) but not to the α1-adrenoceptor antagonist prazosin (10 nM). Idazoxan and the α2C-adrenoceptor antagonist JP 1302 (0.5–1 μM) reduced H2O2-induced facilitation. H2O2 induced facilitation of nerve-evoked contractions was abolished by the non-selective cation channel blocker SKF-96365 (10 μM), suggesting it depends on Ca2+ influx. In conclusion, H2O2-induced increases in nerve-evoked contractions depended on an intact endothelium and were mediated by activating thromboxane A2 receptors and by increasing the contribution of α2-adrenoceptors to these responses.
Keywords: Mouse tail artery; Sympathetic neurovascular transmission; Hydrogen peroxide; Endothelium;

2,3,4′,5-tetrahydroxystilbene-2-0-β-d glucoside (TSG) has been recognized to suppress the proliferation of vascular smooth muscle cells (VSMCs). The aim of the present study was to determine whether TSG inhibits neointimal hyperplasia in a rat carotid arterial balloon injury model. Balloon injury was induced in the left common carotid artery of rats. TSG (30, 60, 120 mg/kg/day) was treated from 3 days prior to, until 14 days after the induction of balloon injury. The ratio of intima-to-media was significantly reduced in the TSG-treated rats at 14 days after the induction of injury, which was associated with reduced expressions of proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA) and platelet-derived growth factor-BB (PDGF-BB), as markers of VSMCs proliferation and migration. Additionally, TSG significantly inhibited PDGF-BB induced cell migration in cultured VSMCs. Furthermore, we explored the underlying mechanisms for such effects of TSG. The result showed that TSG markedly reduced balloon injury-induced AKT, extracellular signal-regulated kinase (ERK1/2) and nuclear factor kappaB (NF-κB) activation as well as mRNA expressions of c-myc, c-fos and c-jun, which is important signal pathway for VSMCs proliferation. And in both vivo and vitro model, TSG markedly regulated matrix metalloproteinase-2, 9 expressions and collagen I, III expressions, which are key factors in extracellular matrix for VSMCs migration. These results suggest that the anti-proliferative and anti-migrative effects of TSG on VSMCs could help to explain the beneficial effects of TSG on neointima hyperplasia induced by balloon injury.
Keywords: 2,3,4′,5-tetrahydroxystilbene-2-0-β-d glucoside; Balloon injury; Proliferation; Migration; Vascular smooth muscle cell;

Comparative study of the quercetin, ascorbic acid, glutathione and superoxide dismutase for nitric oxide protecting effects in mouse gastric fundus by Peyman U. Ertuğ; Fatma Aydinoglu; Ozlem Goruroglu Ozturk; Ergin Singirik; Nuran Ögülener (379-387).
The aim of this work was to compare the preventing capacity of quercetin with Cu/Zn superoxide dismutase (Cu/Zn SOD), ascorbic acid and glutathione on nitric oxide (NO)-induced relaxation in mouse gastric fundus. Furthermore, the effects of the quercetin on the tissue level of total oxidant and antioxidant was investigated. Nitrergic stimulation (4 Hz, 25 V, 0.1 ms, 10 s-train) and exogenous NO (10 μM) induced relaxation. Pyrogallol (10 μM), hydroquinone (100 μM) and LY83583 (6-Anilino-quinolin-5,8-quinone, 5 μM) inhibited nitrergic relaxations. The inhibition observed with pyrogallol, hydroquinone and LY83583 was prevented by quercetin (0.1 μM). Also, ascorbic acid (500 μM), glutathione (100 μM) and Cu/Zn SOD (100 U/ml) prevented the inhibitory effect of superoxide anion generators on the relaxation to nitrergic stimulation and NO. Diethyldithiocarbamic acid (DETCA; 8 mM) inhibited nitrergic relaxations. DETCA-induced inhibition on nitrergic stimulation and NO-induced relaxation was prevented by quercetin, ascorbic acid, glutathione or Cu/Zn SOD. DETCA plus pyrogallol, hydroquinone or LY83583 strengthened the inhibition on the relaxations. Also, pre-treatment with quercetin, ascorbic acid and glutathione prevented the inhibitory effect of DETCA plus LY-83583 on the relaxation to nitrergic stimulation and NO but Cu/Zn SOD did not prevent this inhibition. Also, quercetin increased tissue total antioxidant capacity and decreased tissue oxidant level and oxidative stress index in DETCA-treatment group. These results indicate that quercetin has antioxidant effect and protects NO from endogenous superoxide anion-driven inactivation and enhances its biological activity, suggesting that quercetin may scavenge superoxide anion in a Cu/Zn SOD, glutathione or ascorbic acid-inhibitable manner.
Keywords: Antioxidants; Quercetin; Gastric fundus; Nitric oxide protecting effect; Superoxide generators; Tissue total antioxidant capacity;

We have previously shown that low-dose fenofibrate treatment has an ability to prevent diabetes-induced nephropathy in rats. We investigated here the comparative pre- and post-treatment effects of low-dose fenofibrate (30 mg/kg/day p.o.) in diabetes-induced onset of nephropathy. Rats were made diabetics by single administration of streptozotocin (STZ, 55 mg/kg i.p.). The development of diabetic nephropathy was assessed biochemically and histologically. Moreover, lipid profile and renal oxidative stress were assessed. Diabetic rats after 8 weeks of STZ-administration developed apparent nephropathy by elevating serum creatinine, blood urea nitrogen and microproteinuria, and inducing glomerular-capsular wall distortion, mesangial expansion and tubular damage and renal oxidative stress. Fenofibrate (30 mg/kg/day p.o., 4 weeks) pretreatment (4 weeks after STZ-administration) markedly prevented diabetes-induced onset of diabetic nephropathy, while the fenofibrate (30 mg/kg/day p.o., 4 weeks) post-treatment (8 weeks after STZ-administration) was less-effective. However, both pre-and post fenofibrate treatments were effective in preventing diabetes-induced renal oxidative stress and lipid alteration in diabetic rats though the pretreatment was slightly more effective. Conversely, both pre-and post fenofibrate treatments did not alter elevated glucose levels in diabetic rats. It may be concluded that diabetes-induced oxidative stress and lipid alteration, in addition to a marked glucose elevation, play a detrimental role in the onset of nephropathy in diabetic rats. The pretreatment with low-dose fenofibrate might be a potential therapeutic approach in preventing the onset of nephropathy in diabetic subjects under the risk of renal disease induction. However, low-dose fenofibrate treatment might not be effective in treating the established nephropathy in diabetic subjects.Display Omitted
Keywords: Diabetes mellitus; Hyperglycemia; Lipid alteration; Oxidative stress; Nephropathy; Fenofibrate; Low-dose strategy; Pre- and post-treatment strategies;

Oxidative stress is one of the major causative factors for development of benign prostatic hyperplasia (BPH). The aim of the present study is to evaluate the effect of 2-hydroxy-4-methoxy benzoic acid (HMBA), a potential antioxidant on testosterone induced BPH in rats. Male Wistar rats were divided into five groups (n=6), Group I—received saline, Group II—received testosterone (3 mg/kg/s.c.), Group III—received testosterone+finasteride (5 mg/kg/oral), Group IV and V received testosterone+HMBA (200 and 400 μg/kg/i.p.), respectively, for 21 days. Animals were weighed before and after the study period. On 22nd day, animals were humanly killed by cervical dislocation. Prostates were excised and weighed, and used for biochemical and histological studies. As a result, testosterone treated rats showed increased prostate weight; prostatic index accompanied with depleted antioxidant enzymes levels, elevated lipid peroxides and total nitrite and associated histology disruption. HMBA treatment at 200 μg/kg/i.p. and 400 μg/kg/i.p. significantly restored (P<0.05) antioxidant enzyme levels, lipid peroxide and total nitrite when compared to disease control animals. It also ameliorated the testosterone induced histological changes. The present study suggests the protective role of HMBA on testosterone induced BPH by virtue of its antioxidant potential.
Keywords: Oxidative stress; Testosterone; BPH; HMBA; Antioxidant;

Role of TRPV1 and TRPA1 in visceral hypersensitivity to colorectal distension during experimental colitis in rats by Wim Vermeulen; De Man Joris G.; De Schepper Heiko U.; Hidde Bult; Tom G. Moreels; Paul A. Pelckmans; De Winter Benedicte Y. (404-412).
The aim of the present study is to investigate the effects of TRPV1 and TRPA1 receptor antagonists and their synergism on the visceromotor responses during experimental colitis in rats. Colitis was induced in rats by a TNBS/ethanol enema at day 0 and was assessed at day 3 using endoscopy, histology and a myeloperoxidase assay. The visceromotor response to colorectal distension (10–80 mmHg) was evaluated in conscious rats before (control condition) and 3 days after 2,4,6-trinitrobenzene sulfonic acid (TNBS) administration (colitis condition). At day 3, visceromotor responses were assessed before and after treatment with a TRPV1 (BCTC) or TRPA1 (TCS-5861528) receptor antagonist either alone or in combination and either after intraperitoneal or intrathecal administration. Endoscopy, microscopy and myeloperoxidase activity indicated severe colonic tissue damage 3 days after TNBS administration. Colorectal distension-evoked visceromotor responses demonstrated a 2.9-fold increase during acute colitis (day 3) compared to control conditions. Intraperitoneal and intrathecal administration of BCTC or TCS-5861528 partially reversed the colitis-induced increase in visceromotor responses compared to control conditions (P<0.05). Intraperitoneal blockade of TRPA1 plus TRPV1 further decreased the enhanced visceromotor responses at high distension pressures (40–80 mmHg) compared to blockade of either TRPV1 or TRPA1 alone. This synergistic effect was not seen after combined intrathecal blockade of TRPA1 plus TRPV1. The present study demonstrates that in the rat, TRPV1 and TRPA1 play a pivotal role in visceral hypersensitivity at the peripheral and spinal cord level during acute TNBS colitis. Target interaction, however, is presumably mediated via a peripheral site of action.
Keywords: Colitis; Pain; Transient receptor potential ankyrin 1; Transient receptor potential vanilloid 1; Visceral hypersensitivity;

The role of PKC/ERK1/2 signaling in the anti-inflammatory effect of tetracyclic triterpene euphol on TPA-induced skin inflammation in mice by Giselle F. Passos; Rodrigo Medeiros; Rodrigo Marcon; Andrey F.Z. Nascimento; João B. Calixto; Luiz F. Pianowski (413-420).
Inflammation underlies the development and progression of a number of skin disorders including psoriasis, atopic dermatitis and cancer. Therefore, novel antiinflammatory agents are of great clinical interest for prevention and treatment of these conditions. Herein, we demonstrated the underlying molecular mechanisms of the antiinflammatory activity of euphol, a tetracyclic triterpene isolated from the sap of Euphorbia tirucalli, in skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mice. Topical application of euphol (100 μg/ear) significantly inhibited TPA-induced ear edema and leukocyte influx through the reduction of keratinocyte-derived chemokine (CXCL1/KC) and macrophage inflammatory protein (MIP)-2 levels. At the intracellular level, euphol reduced TPA-induced extracellular signal-regulated protein kinase (ERK) activation and cyclooxygenase-2 (COX-2) upregulation. These effects were associated with euphol's ability to prevent TPA-induced protein kinase C (PKC) activation, namely PKCα and PKCδ isozymes. Our data indicate that topical application of euphol markedly inhibits the inflammatory response induced by TPA. Thus, euphol represents a promising agent for the management of skin diseases with an inflammatory component.Display Omitted
Keywords: Euphol; TPA; Inflammation; PKC; MAPK; COX-2;

Evaluation of the effect of losartan and methotrexate combined therapy in adjuvant-induced arthritis in rats by Rowaida Refaat; Mona Salama; Elham Abdel Meguid; Ashgan El Sarha; Mennatallah Gowayed (421-428).
There is increasing body of evidence documenting the involvement of angiotensin II in inflammatory diseases. Moreover the up-regulation of angiotensin II AT1 receptors in the synovium of rheumatoid arthritis patients has been previously described.This study aimed at investigating the anti-inflammatory effect of losartan, the selective angiotensin II AT1 receptor blocker, and comparing the efficacy of methotrexate alone and in combination with losartan in adjuvant arthritis in rats. Twelve days post adjuvant injection, Sprague-Dawley rats were treated with methotrexate (1 mg/kg/week), losartan (20 mg/kg/day) and their combination for 15 days. Severity of arthritis was assessed by hind paw swelling, arthrogram scores. Serum was analyzed for measurement of albumin, C-reactive protein (CRP), nitrite/nitrate concentrations, interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), aspartate transaminase (AST) and alanine transaminase (ALT). Histopathological examination was done for hind paws and livers. Methotrexate and losartan monotherapies significantly reduced all parameters of inflammation and arthritis with better results in the methotrexate group except for the transaminases where losartan caused more significant reduction in their serum levels. The combined therapy showed better results than methotrexate and losartan alone. Hind paws showed better improvement of inflammatory cell infiltration and bone resorption in the combined therapy group. Disturbances in liver architecture and fibrosis caused by adjuvant arthritis were reverted to normal status in the combined therapy group in contrast to losartan and methotrexate monotherapies. In conclusion, methotrexate and losartan combined therapy provided more effective anti-inflammatory and hepatoprotective effects than either drug alone.
Keywords: Adjuvant-induced arthritis; Losartan; Methotrexate;

Infliximab reduces CD147, MMP-3, and MMP-9 expression in peripheral blood monocytes in patients with active rheumatoid arthritis by Jianlin Huang; Baozhao Xie; Qiuxia Li; Xujing Xie; Shangling Zhu; Mingxia Wang; Weixiang Peng; Jieruo Gu (429-434).
Recent studies have reported elevated expression levels in active rheumatoid arthritis patients of the cluster of differentiation (CD) 147 on CD14+ peripheral blood monocytes and as a result, CD147 may be a target for the development of a novel rheumatoid arthritis therapy. This report describes the inhibitory effects of infliximab on CD147 and metalloproteinases (MMP)-3 and MMP-9 overexpression in peripheral blood monocytes obtained from patients with active rheumatoid arthritis. Thirty patients with active rheumatoid arthritis that were refractory to methotrexate therapy were randomized at a 4:1 ratio into groups A and B, respectively. Group A received three to four infusions of infliximab (3 mg/kg) and group B participants received four infusions of placebo. Both groups were also treated with a stable background dose of methotrexate. The CD147 expression levels on CD14+ peripheral blood monocytes of rheumatoid arthritis patients was detected by flow cytometry. The expression of CD147, MMP-3, and, MMP-9 mRNA in peripheral blood mononuclear cells was assayed by real-time quantitative PCR, and the expression of MMP-3 and MMP-9 in serum was measured by a multiplexed microsphere-based flow assay. Results showed that the expression of CD147 and MMP-9 mRNA in group A decreased compared to group B. Expression of CD147 on CD14+ monocytes was reduced (P<0.05), and serum MMP-3 and -9 levels in group A were decreased by week 18. These data suggested that infliximab could inhibit CD147 expression on CD14+ monocytes as well as reduce the levels of MMP-3 and MMP-9 in peripheral blood monocytes.
Keywords: CD147; Matrix metalloproteinase; Rheumatoid arthritis; Infliximab; Therapy;

Berkeleyacetal C (BAC) isolated from Penicillium sp. which had isolated from a soil sample collected in Fukushima, inhibited NO production and induction of iNOS protein in RAW264.7 cells stimulated by the Toll-like receptor (TLR) 2 ligand, peptidoglycan (PGN) or TLR4 ligand, lipopolysaccharide (LPS). The other inflammatory mediator production by these stimulators was also suppressed by BAC in a concentration-dependent manner. BAC inhibited LPS- or PGN-activated nuclear translocation of nuclear factor (NF)-κB and MyD88-dependent signaling molecules. However, it showed no effect on LPS-induced nuclear translocation of interferon regulatory factor (IRF)-3, a MyD88-independent signaling molecule. To clarify the mechanistic basis for BAC ability to inhibit translocation of NF-κB and activated MyD88-dependent signaling molecules, we examined interleukin-1 receptor-associated kinase (IRAK)-4, existing to the most upstream on MyD88-dependent signaling molecules, in vitro kinase assay. BAC suppressed IRAK-4 kinase activity in a concentration-dependent manner. These findings suggest that BAC inhibits LPS- and PGN- induced NO production and iNOS expression by decreasing the level of the translocating of NF-κB in nuclear through inhibiting the kinase activity of IRAK-4 in inflammatory cells.
Keywords: Berkeleyacetal C; IRAK-4; Anti-inflammation; TLRs; NO;

The inclusion of antioxidant for the treatment of arthritis, especially under the therapy with immunosuppressant, is motivated because antioxidant plays an essential role in disease progression and moreover, immunosuppressive treatment suffers redox homeostasis balance of the organism. The aim of the present study was to evaluate the enhancement of anti-arthritic effect of dexamethasone in combination with epigallocatechin on the progression of adjuvant-induced arthritis in rats. Adjuvant arthritic rats were treated with dexamethasone (0.2 mg/kg), epigallocatechin (100 mg/kg) and combination of dexamethasone (0.1 mg/kg) with epigallocatechin (100 mg/kg) daily for a period of 28 days. Paw swelling changes, estimation of serum albumin level, alteration of bone mineral density, histopathological, and radiographical analysis were assessed to evaluate the anti-arthritic effect. Lipid peroxidation and antioxidant enzyme activities in joint tissue homogenate were performed along with the expression of different pro-inflammatory cartilage cytokines like TNF-α and IL-6. Dexamethasone and epigallocatechin combination potentiated both the antiarthritic (decrease of hind paw volume) and the antioxidant effect (lipid peroxidation, superoxide dismutase, glutathione reductase and catalase). In combination with dexamethasone, epigallocatechin markedly potentiated the beneficial effect of dexamethasone which resulted in more significant increment of serum albumin and bone mineral density. Improvement of anti-arthritic effect of combination therapy was supported by histopathological, radiographical alterations, and attenuation of over-expression of cartilage cytokines. Epigallocatechin act as potent antioxidant and combined administration of dexamethasone with epigallocatechin increased the anti-arthritic efficacy of basal dexamethasone therapy and suppressed the development phase of arthritic progression in rats.
Keywords: Dexamethasone; Epigallocatechin; Adjuvant arthritis; Antioxidant; Cytokines;

Anti-inflammatory effect and selectivity profile of AS1940477, a novel and potent p38 mitogen-activated protein kinase inhibitor by Masaomi Terajima; Tatsuo Inoue; Katsue Magari; Hitoshi Yamazaki; Yasuyuki Higashi; Hidekazu Mizuhara (455-462).
Given the key role p38 mitogen-activated protein kinase (MAPK) plays in inflammatory responses through the production of cytokines and inflammatory mediators, its inhibition is considered a promising therapeutic strategy for chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, and chronic obstructive pulmonary disease. Here, we evaluated the anti-inflammatory effect and selectivity profile of the novel p38 MAPK inhibitor AS1940477. AS1940477 inhibited the enzymatic activity of recombinant p38α and β isoforms but showed no effect against other 100 protein kinases including p38γ and δ isoforms. We also confirmed the selectivity of AS1940477 in the intracellular signaling pathway. In human peripheral blood mononuclear cells, AS1940477 inhibited lipopolysaccharide (LPS)- or phytohemagglutinin A (PHA)-induced production of proinflammatory cytokines, including TNFα, IL-1β, and IL-6 at low concentrations (LPS/TNFα, IC50=0.45 nM; PHA/TNFα, IC50=0.40 nM). In addition, equivalent concentrations of AS1940477 that inhibited cytokine production also inhibited TNFα- and IL-1β-induced production of IL-6, PGE2, and MMP-3 in human synovial stromal cells. AS1940477 was also found to potently inhibit TNF production in whole blood (IC50=12 nM) and effectively inhibited TNFα production induced by systemically administered LPS in rats at less than 0.1 mg/kg (ED50=0.053 mg/kg) with an anti-inflammatory effect lasting for 20 h after oral administration. Overall, this study demonstrated that AS1940477 is a novel and potent p38 MAPK inhibitor and may be useful as a promising anti-inflammatory agent for treating inflammatory disorders.
Keywords: p38; Mitogen-activated protein kinase; Inhibitor; Lipopolysaccharide; Tumor necrosis factor α; Interleukin-6;

Hydrogen sulfide inhibits oxidative stress in lungs from allergic mice in vivo by Leticia R. Benetti; Daiana Campos; Sonia A. Gurgueira; Anibal E. Vercesi; Cristiane E.V. Guedes; Kleber L. Santos; John L. Wallace; Simone A. Teixeira; Juliana Florenzano; Soraia K.P. Costa; Marcelo N. Muscará; Heloisa H.A. Ferreira (463-469).
Recent studies show that endogenous hydrogen sulfide (H2S) plays an anti-inflammatory role in the pathogenesis of airway inflammation. This study investigated whether exogenous H2S may counteract oxidative stress-mediated lung damage in allergic mice. Female BALB/c mice previously sensitized with ovalbumin (OVA) were treated with sodium hydrosulfide (NaHS) 30 min before OVA challenge. Forty eight hours after antigen-challenge, the mice were killed and leukocyte counting as well as nitrite plus nitrate concentrations were determined in the bronchoalveolar lavage fluid, and lung tissue was analysed for nitric oxide synthase (NOS) activity, iNOS expression, superoxide dismutase (SOD), catalase, glutathione reductase (GR) and glutathione peroxidase (GPx) activities, thiobarbituric acid reactive species and 3-nitrotyrosine containing proteins (3-NT). Pre-treatment of OVA-sensitized mice with NaHS resulted in significant reduction of both eosinophil and neutrophil migration to the lungs, and prevented the elevation of iNOS expression and activity observed in the lungs from the untreated allergic mice, although it did not affect 3-NT. NaHS treatment also abolished the increased lipid peroxidation present in the allergic mouse lungs and increased SOD, GPx and GR enzyme activities. These results show, for the first time, that the beneficial in vivo effects of the H2S-donor NaHS on allergic airway inflammation involve its inhibitory action on leukocyte recruitment and the prevention of lung damage by increasing endogenous antioxidant defenses. Thus, exogenous administration of H2S donors may be beneficial in reducing the deleterius impact of allergic pulmonary disease, and might represent an additional class of pharmacological agents for treatment of chronic pulmonary diseases.
Keywords: Hydrogen sulfide; Asthma; Mouse; iNOS; Glutathione peroxidase; Glutathione reductase;

Cycloart-23-ene-3β, 25-diol stimulates GLP-1 (7–36) amide secretion in streptozotocin–nicotinamide induced diabetic Sprague Dawley rats: A mechanistic approach by Sachin L. Badole; Sagar P. Mahamuni; Pranita P. Bagul; Rekha D. Khose; Anuja C. Joshi; Arvindkumar E. Ghule; Subhash L. Bodhankar; Chandrashekhar G. Raut; Vijay M. Khedkar; Evans C. Coutinho; Nilesh K. Wagh (470-479).
In previous study, we have reported cycloart-23-ene-3β, 25-diol is an active antidiabetic constituent isolated from stem bark of Pongamia pinnata (Linn.) Pierre. The objective of the present investigation was to evaluate cycloart-23-ene-3β, 25-diol stimulates glucagon like peptide-1 (GLP-1) (7–36) amide secretion in streptozotocin–nicotinamide induced diabetic Sprague Dawley rats. Molecular docking studies were performed to elucidate the molecular basis for GLP-1 receptor agonistic activity. Type 2 diabetes was induced in overnight fasted Sprague Dawley rats pre-treated with nicotinamide (100 mg/kg, i.p.) followed by administration of streptozotocin (55 mg/kg, i.p.) 20 min after. The rats were divided into following groups; I- non-diabetic, II- diabetic control, III- sitagliptin (5 mg/kg, p.o.), IV- cycloart-23-ene-3β, 25-diol (1 mg/kg, p.o.). The cycloart-23-ene-3β, 25-diol and sitagliptin treatment was 8 week. Plasma glucose was estimated every week (week 0 to week 8). Body weight, food and water intake were recorded daily. Glycosylated haemoglobin, lipid profile, plasma and colonic active (GLP-1) (7–36) amide, mRNA expression of proglucagnon GLP-1, plasma and pancreatic insulin, histology of pancreata as well as biomarkers of oxidative stress (superoxidase dismutase, reduced glutathione, malondialdehyde, glutathione peroxidase, glutathione S transferase) were measured after 8th week treatment. In acute study, active GLP-1 (7–36) amide release, plasma glucose and insulin were measured during oral glucose tolerance test. The docking data clearly indicated cycloart-23-ene-3β, 25-diol bind to the GLP-1 receptor. It decreased plasma glucose level, increased plasma and pancreatic insulin level as well as increased plasma and colonic active GLP-1 (7–36) amide secretion in streptozotocin–nicotinamide induced diabetic Sprague Dawley rats.Display Omitted
Keywords: Cycloart-23-ene-3β; 25-diol; Plasma glucose; GLP-1 (7–36) amide; mRNA proglucagon gene expression; Insulin; Pancreatic β cells; Oral glucose tolerance test; Antioxidant markers;

A novel indole derivative compound GY3 improves glucose and lipid metabolism via activation of AMP-activated protein kinase pathway by Meimei Si; Youyou Yan; Lei Tang; Honghai Wu; Bo Yang; Qiaojun He; Haoshu Wu (480-488).
The AMP-activated protein kinase (AMPK) is a ubiquitously expressed serine/threonine protein kinase that functions as an intracellular fuel sensor. It has been demonstrated to mediate the activities of a number of pharmacological and physiological factors that exert beneficial effects on type2 diabetes mellitus. GY3 is a novel synthesized indole compound derived from indomethacin, a non-steroid anti-inflammatory drug. In a previous study, we found that GY3 could improve insulin resistance and lower glucose levels in db/db mice, although its mechanism of action is not yet clear. In this study, we demonstrate that in vivo administration of GY3 improved serum triglyceride levels and decreased lipid accumulation in the livers of db/db mice. In vitro studies show that GY3 increased glucose consumption in HepG2 cells and 3T3-L1 adipocytes, decreased free fatty acid (FFA)-induced lipid accumulation in HepG2 cells and lipid accumulation in 3T3-L1 adipocytes. In vitro studies further show that GY3 improved glucose and lipid metabolism through an AMPK-dependent pathway but not the PI3K pathway. These findings suggest that GY3 is an effective agent for the improvement of glucose and lipid metabolism through AMPK pathway activation.GY3 increases glucose consumption and decreases lipid ccumulation in HepG2 hepatocytes and mature 3T3-L1 adipocytes through AMPK pathway. Control (CL), Metformine (Met), Rosiglitazone (Ros).Display Omitted
Keywords: Indole derivative compound; AMPK; Glucose metabolism; Lipid metabolism; Diabetes; Fatty liver;

Protective role of 20-OH ecdysone on lipid profile and tissue fatty acid changes in streptozotocin induced diabetic rats by Rajendran Naresh Kumar; Ramalingam Sundaram; Palanivelu Shanthi; Panchanatham Sachdanandam (489-498).
Hyperlipidemia is an associated complication of diabetes mellitus. The association of hyperglycemia with an alteration of lipid parameters presents a major risk for cardiovascular complications in diabetes. The present study was designed to examine the antihyperlipidemic effect of 20-OH ecdysone on lipid profile and tissue fatty acid changes in streptozotocin induced diabetic rats. The levels of blood glucose, cholesterol, triglycerides, free fatty acids, phospholipids, low density lipoprotein, very low density lipoprotein, high density lipoprotein, lipoprotein lipase, lecithin cholesterol acyl transferase, 3-hydroxy 3-methylglutaryl coenzyme A reductase and fatty acid composition were estimated in plasma, liver and kidneys of control and experimental groups of rats. Oral administration of 20-OH ecdysone at a dose of 5 mg/kg bodyweight per day to STZ-induced diabetic rats for a period of 30 days resulted in a significant reduction in fasting blood glucose, cholesterol, triglycerides, free fatty acids, phospholipids, low density lipoprotein, very low density lipoprotein, 3-hydroxy 3-methylglutaryl coenzyme A reductase and elevation of high density lipoprotein, lipoprotein lipase and lecithin cholesterol acyl transferasein comparison with diabetic untreated rats. Moreover, administration of 20-OH ecdysone to diabetic rats also decreased the concentrations of fatty acids, viz., palmitic, stearic (16:1) and oleic acid (18:1), whereas linolenic (18:3) and arachidonic acid (20:4) were elevated. The antihyperlipidemic effect of 20-OH ecdysone was compared with glibenclamide a well-known antihyperglycemic drug. The result of the present study indicates that 20-OH ecdysone showed an antihyperlipidemic effect in addition to its antidiabetic effect in experimental diabetes.
Keywords: Vitexnegundo; 20-OH ecdysone; Diabetes; Fatty acids;

In vivo characterization of intestinal effects of endomorphin-1 and endomorphin-2 in type 1 diabetic mice by Chang-lin Wang; Ying Zhou; Chao Guo; Ying Zhang; Rui Wang (499-504).
Previously, we have demonstrated that type 1 diabetes significantly attenuated the effects of endomorphins on mouse colonic contractions in vitro. In the present study, to further assess whether diabetes affects the in vivo effects of endomorphins on the mouse intestinal motility, we investigated the effects of endomorphins on colonic propulsion and large intestinal transit in diabetic mice. Both colonic bead expulsion and large intestinal transit were significantly delayed in 4 and 8 weeks diabetic mice compared to non-diabetic mice. Moreover, intracerebroventricular (i.c.v.) administration of EM-1 and EM-2 (0.5, 1.5 and 5 nmol/mouse) significantly increased bead expulsion latency in a dose-dependent manner both in non-diabetic and diabetic mice. Similar results were found in large intestinal transit. However, the inhibitory effects of colonic propulsion induced by endomorphins were significantly attenuated in diabetes compared to non-diabetes. It is noteworthy that the inhibition of distal colonic propulsion induced by EM-1 in 8-week diabetes was lower than that of in 4 weeks diabetes. Nevertheless, there was no significant influence on endomorphins-induced inhibition of large intestinal transit caused by diabetes. Co-administration of naloxone (10 nmol/mouse, i.c.v.) significantly attenuated the inhibitory effects of endomorphins (5 nmol/mouse, i.c.v.) on colonic bead expulsion and large intestinal transit in 4 weeks diabetes, indicating that opioid receptor involved in these effects. Our results indicated that type 1 diabetes attenuated the inhibition of distal colonic propulsion induced by endomorphins in mice, but not the large intestine. The central opioid mechanism was involved in the endomorphins-induced intestinal effects in diabetes.
Keywords: Endomorphins; Type 1 diabetes; Colonic propulsion; Large intestinal transit; μ-Opioid receptor;

Effects of telmisartan and valsartan on insulin sensitivity in obese diabetic mice by Kentarou Ushijima; Masashi Takuma; Hitoshi Ando; Eiko Ishikawa-Kobayashi; Masahiko Nozawa; Tomohiro Maekawa; Tsuyoshi Shiga; Akio Fujimura (505-510).
Telmisartan and valsartan have angiotensin II receptor blocking activity. Because telmisartan has also an agonistic action for peroxisome proliferators-activator receptor (PPAR)-γ, it is speculated that an effect of telmisartan on insulin sensitivity is different from that of valsartan, which lacks of PPAR-γ agonistic activity. To address the issue, effects of telmisartan and valsartan on insulin sensitivity, adipocytokines and PPAR-γ target genes were evaluated in obese diabetic mice. KK-A y mice were treated with telmisartan (5 mg/kg) and valsartan (15 mg/kg), once daily for 3 weeks. Insulin tolerance test was performed on day14, and plasma adiponectin concentration and mRNA expression levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in adipose tissues were measured on day 21. Time-course of plasma glucose level after the injection of insulin in mice with telmisartan was not significantly different from that of animals with valsartan. In addition, PPAR-γ antagonist did not diminished the improvement of insulin sensitivity by telmisartan. Telmisartan and valsartan elevated plasma adiponectin concentration and suppressed the mRNA expressions of TNF-α and IL-6 in adipose tissues. These variables of the telmisartan- and valsartan-treated groups did not significantly differ. Influence of telmisartan on the PPAR-γ target genes (ap2 and fatty acid synthase) mRNA expressions was not detected in adipose tissues under the present condition. These data suggest that the effect of telmisartan on insulin sensitivity is similar to that of valsartan, and a role of PPAR-γ-mediated stimuli is small in the telmisartan-induced improvement of insulin sensitivity.
Keywords: Adiponectin; Angiotensin II type I receptor blocker; Inflammatory cytokine; Peroxisome proliferators-activator receptor-γ;