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Biochemical Pharmacology (v.70, #9)

Editorial Advisory Board (pp. iii).

Allosteric modulation of ligand-gated ion channels by Ron C. Hogg; Bruno Buisson; Daniel Bertrand (pp. 1267-1276).

Ligand-gated ion channels (LGICs) are cell surface proteins that play an important role in fast synaptic transmission and in the modulation of cellular activity. Due to their intrinsic properties, LGICs respond to neurotransmitters and other effectors (e.g. pH) and transduce the binding of a ligand into an electrical current on a microsecond timescale. Following activation, LGICs open allowing an ion flux across the cell membrane. Depending upon the charge and concentration of ions, the flux can cause a depolarization or hyperpolarization, thus modulating excitability of the cell. While our understanding of LGICs has significantly progressed during the past decade, many properties of these proteins are still poorly understood, in particular their modulation by allosteric effectors. LGICs are often thought as a simple on–off switches. However, a closer look at these receptors reveals a complex behavior and a wide repertoire of subtle modulation by intrinsic and extrinsic factors. From a physiological point of view, this modulation can be seen as an additional level of complexity in the cell signaling process.Here we review the allosteric modulation of LGICs in light of the latest findings and discuss the suitability of this approach to the design of new therapeutic molecules.

Keywords: Ligand-gated ion channels; Allosteric modulation; P2X receptors; Glutamate receptors; Nicotinic acetylcholine receptors; nAChRs; Divalent cations

Inhibition of Sp1 activity by a decoy PNA–DNA chimera prevents urokinase receptor expression and migration of breast cancer cells by Antonella Zannetti; Silvana Del Vecchio; Alessandra Romanelli; Stefania Scala; Michele Saviano; Gaetano Cali’; M. Patrizia Stoppelli; Carlo Pedone; Marco Salvatore (pp. 1277-1287).

Sp1 regulates the activation of many genes involved in tumor growth, apoptosis, and angiogenesis. We have previously shown the involvement of Sp1 in the up-regulation of urokinase receptor (uPAR) expression, a key molecule in tumor invasion and metastasis. Here, we investigated whether a marked down-regulation of Sp1 activity may inhibit uPAR expression and migration ability of MDA-MB-231 breast cancer cells. To this end, we tested the decoy ability of a novel peptide nucleic acid (PNA)–DNA chimera which carries a central DNA strand, containing Sp1-binding sequence, covalently linked to two PNA fragments at both ends (PNA–DNA–PNA, PDP). The chimera was synthesized, annealed with complementary DNA (PDP–DNA), and then tested for its ability to bind Sp1 both in vitro and in living MDA-MB-231 breast cancer cells in the presence of urokinase (uPA). This PDP–DNA decoy molecule efficiently competes for the binding to endogenous Sp1 in nuclear extracts, and upon transfection with liposomal vectors, causes a marked decrease of available Sp1 in both untreated and uPA-treated MDA-MB-231 cells. Accordingly, both uPA-dependent enhancement of uPAR expression and cell migration were strongly reduced in transfected cells. Interestingly, a detectable inhibitory effect is also observed in breast cancer cells exposed to PDP–DNA in the absence of transfection reagents. Finally, the inhibitory effect of PDP–DNA appeared to be stronger than that observed with oligonucleotides carrying Sp1 consensus sequence. Our findings show that this novel PNA–DNA chimera, containing Sp1 consensus sequence, effectively inhibits Sp1 activity, uPAR expression, and motility of breast cancer cells indicating its potential therapeutic use to prevent tumor dissemination.

Keywords: Abbreviations; ATF; amino-terminal fragment of urokinase; ECM; extracellular matrix; EMSA; electrophoretic mobility shift assay; ODN; oligodeoxynucleotide; PDP–DNA; hybrid of PDP chimera with complementary DNA; PDP; single strand PNA–DNA–PNA chimera; PNA; peptide nucleic acid; uPA; urokinase-type plasminogen activator; uPAR; urokinase-type plasminogen activator receptorSp1 transcription factor; Urokinase receptor; Peptide nucleic acid; PNA–DNA chimera; Breast cancer

The ribonucleotide reductase subunit M2B subcellular localization and functional importance for DNA replication in physiological growth of KB cells by Xiyong Liu; Bingsen Zhou; Lijun Xue; Jennifer Shih; Karen Tye; Christina Qi; Yun Yen (pp. 1288-1297).

Ribonucleoside diphosphate reductase (EC 1.17.4.1) (RR) is a potential target for antineoplastic agents due to its crucial role in DNA replication and repair. The expression and activity of RR subunits are highly regulated to maintain an optimal dNTP pool, which is required to maintain genetic fidelity. The human RR small subunit M2B (p53R2) is thought to contribute to DNA repair in response to DNA damage. However, it is not clear whether M2B is involved in providing dNTPs for DNA replication under physiological growth conditions. Serum starvation synchronized studies showed that a rapid increase of M2B was associated with cyclin E, which is responsible for regulation of G₁/S-phase transition. A living cell sorting study that used KB cells in normal growth, further confirmed that M2B increased to maximum levels at the G₁/S-phase transition, and decreased with DNA synthesis. Confocal studies revealed that M2B redistributed from the cytoplasm to the nucleus earlier than hRRM2 in response to DNA replication. Nuclear accumulation of M2B is associated with dynamic changes in dNTP at early periods of serum addition. By using M2B-shRNA expression vectors, inhibition of M2B may result in growth retardation in KB cells. We conclude that M2B may translocate from the cytoplasm into the nucleus and allow dNTPs to initiate DNA synthesis in KB cells under physiological conditions. Thus, our findings suggested that M2B might play an important role for initiating DNA replication of KB cells in normal growth.

Keywords: Abbreviations; hRR; human ribonucleotide reductase; hRRM1; human ribonucleotide reductase large subunit M1; hRRM2; human ribonucleotide reductase small subunit M2; M2B; a p53-dependent human ribonucleotide reductase small subunit M2B, it also called p53R2; RNR; yeast ribonucleotide reductase; shRNA; short hairpin interference RNA; dNTP; deoxyrinucleoside triphosphate; NDP; ribonucleoside diphosphate; CDP; cytidine 5′-diphosphate; dCDP; deoxycytidine 5′-diphosphate; NLS; nuclear localization sequence; DAPI; 4′,6′-diamidino-2-phenylindoleRibonucleotide reductase; DNA replication; Cell cycle; Cell proliferation; Small interference RNA; Subcellular localization

A novel LXR-α activator identified from the natural product Gynostemma pentaphyllum by Tom Hsun-Wei Huang; Valentina Razmovski-Naumovski; Noeris K. Salam; Rujee K. Duke; Van Hoan Tran; Colin C. Duke; Basil D. Roufogalis (pp. 1298-1308).

Liver X receptors (LXR) play an important role in cholesterol homeostasis by serving as regulatory sensors of cholesterol levels in tissues. The present study reports a novel LXR-α activator, (20 S)-2α, 3β, 12β, 24( S)-pentahydroxydammar-25-ene 20- O-β-d-glucopyranoside (TR1), a dammarane-type gynosaponin, isolated from the herbal medicine, Gynostemma pentaphyllum. Gynosaponin TR1 demonstrated greater selectivity toward activation of the LXR-α isoform than LXR-β in HEK293 cells. TR1 selectively enhanced LXR-mediated transcriptional activation and protein expression of ABCA1 and apoE gene expression and secretion in THP-1-derived macrophages. The selectivity of TR1 for LXR-α was consistent with ligand docking studies, which showed favourable interaction of TR1 in the LXR-α-binding domain, whereas the presence of the sugar substituent interfered with binding to the LXR-β site. Findings from the present study may provide insight into the development of pharmaceutical agents for treating atherosclerosis.

Keywords: Abbreviations; ABC; ATP-binding cassette; apoE; apolipoprotein E; CVD; cardiovascular disease; glu; glucose; HDL; high-density lipoprotein; LXR; liver X receptor; RCT; reverse cholesterol transport; RT; room temperature; VLDL; very-low-density lipoproteinLiver X receptor; ABCA1; Apolipoprotein E; Gynosaponin; Gypenoside; Gynostemma pentaphyllum

Contrasting effects of insulin and cellular differentiation on expression of the novel insulin receptor substrate APS in skeletal muscle by Rustam Rea; Samuel Gray; Richard Donnelly (pp. 1309-1311).

The novel insulin receptor substrate protein APS is highly expressed in insulin-sensitive tissues and plays an important role in insulin-mediated glucose uptake and GLUT4 translocation via the Cbl/CAP pathway. Tyrosine phosphorylation of APS leads to recruitment of c-Cbl and Crk, while overexpression of APS mutant inhibits GLUT4 translocation in response to insulin, but the regulation of APS expression in skeletal muscle has not been previously reported. L6 myoblasts were differentiated in 2% FBS and serum starved for 24h prior to stimulation for 24h with either insulin 1μM ( n=6), rosiglitazone 10μM ( n=6), resistin 500nM ( n=6) or the MAP kinase inhibitor PD098059 50μM ( n=6) for 30min, followed by insulin 1μM for 24h. Semi-quantitative real-time RT-PCR was used to determine the expression of APS mRNA relative to the control gene TF2D. APS expression was markedly upregulated by myoblast differentiation (0.55±0.08 versus 1.14±0.08, p=0.001), and this effect was augmented by addition of rosiglitazone 10μM for 24h to the differentiated myotubes (1.50±0.09, p=0.025). Insulin caused a 3.1-fold decrease in APS mRNA expression (0.37±0.01 versus 1.14±0.08, p=0.001), an effect that was attenuated by the MAP kinase inhibitor PD098059 (0.80±0.03, p=0.001). Exposure to resistin produced a modest decrease (1.4-fold) in myotube expression of APS (0.8±0.09, p=0.025). In conclusion, this is the first study to show that exposure to insulin markedly reduces the expression of APS in skeletal muscle via a MAP kinase dependent pathway, whereas myocyte differentiation and rosiglitazone increase APS expression. Changes in APS expression may be important in the aetiology and therapeutic reversal of insulin resistance in skeletal muscle.

Keywords: Abbreviations; APS; adapter protein with a pleckstrin homology and an Src homology 2 domain; CAP; cbl associated protein; IRS; insulin receptor substrate; PI3; phosphatidylinositol-3; PKC; protein kinase C; PPAR-γ; peroxisome proliferators activated receptor gammaAPS; Skeletal muscle; Insulin; Gene expression; Rosiglitazone; Resistin

Interferon-γ-induced STAT1-mediated membrane retention of NHE1 and associated proteins ezrin, radixin and moesin in HT-29 cells by Fernando Magro; S�nia Fraga; Patr�cio Soares-da-Silva (pp. 1312-1319).

This study evaluated the effect of interferon-γ (IFN-γ) upon the function and expression of type 1 Na⁺/H⁺ exchanger (NHE1) in human intestinal epithelial HT-29 cells, namely that concerning the abundance of surface NHE1 and NHE1 binding to the ezrin, radixin and moesin (ERM) family of proteins. HT-29 cells express endogenous NHE1 and the ERM family of proteins that retain the localization of NHE1 in the membrane. Long-term exposure (24h) of HT-29 cells to IFN-γ resulted in a concentration-dependent decrease in NHE1 activity. Inhibition of NHE1 activity by IFN-γ was absent after pretreatment with cariporide. The long-term exposure to IFN-γ was accompanied by increase in surface NHE1 and ERM abundance and no changes in total NHE1 and ERM abundance. Inhibition of signal transducer and activator transcription factor 1 (STAT1) with epigallocatechin-3-gallate (EGCG) prevented the inhibitory effect of IFN-γ. Treatment with IFN-γ activated phospho-STAT1 was markedly attenuated by EGCG. The IFN-γ-induced increase in surface NHE1 and ERM abundance was prevented by EGCG. In conclusion, long-term inhibition of NHE1 activity by IFN-γ involves STAT1 phosphorylation and is accompanied by increased abundance of surface NHE1 and the NHE1 membrane anchoring ERM proteins.

Keywords: Interferon-γ; NHE1; ERM proteins; HT-29 cells

Distinct effects of N-ethylmaleimide on formyl peptide- and cyclopiazonic acid-induced Ca²⁺ signals through thiol modification in neutrophils by Mei-Feng Hsu; Shu-Ping Sun; Yu-San Chen; Chi-Ren Tsai; Li-Jiau Huang; Lo-Ti Tsao; Sheng-Chu Kuo; Jih-Pyang Wang (pp. 1320-1329).

In this study, we demonstrate that N-ethylmaleimide (NEM), a cell permeable thiol-alkylating agent, enhanced the [Ca²⁺]_i rise caused by stimulation with cyclopiazonic acid (CPA), a sarcoplasmic-endoplasmic reticulum Ca²⁺-ATPase inhibitor, in rat neutrophils. In addition, NEM attenuated the formyl-Met-Leu-Phe (fMLP)-induced [Ca²⁺]_i rise whether NEM was added to cells prior to or after fMLP stimulation. Moreover, application of NEM after fMLP activation in the absence of external Ca²⁺ inhibited the Ca²⁺ signal upon addition of Ca²⁺ to the medium. Similar patterns were also obtained by using 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), a cell impermeable dithiol-oxidizing agent, which replaced NEM in the CPA- and fMLP-induced [Ca²⁺]_i rise experiments. Treatment with dithiothreitol (DTT), a cell permeable dithiol-reducing agent, N-acetyl-l-cysteine (NAC), a cell permeable monothiol-reducing agent, and tris-(2-carboxyethyl)phosphine (TCEP), a cell impermeable reductant without a thiol group, all rescued the fMLP-induced Ca²⁺ signal from NEM. Rat neutrophils express the mRNA encoding for transient receptor potential (TRP) C6, inositol trisphosphate receptor (IP₃R) 2 and IP₃R3. NEM had no effect on the mitochondrial membrane potential. NEM could restore the polarization and F-actin accumulation of fMLP-treated cells to those of the control. In the absence of external Ca²⁺, NEM rendered the CPA-induced [Ca²⁺]_i elevation persistently but inhibited the fMLP-induced Ca²⁺ spike, which was reversed by tris-(2-cyanoethyl)phosphine (TCP), a cell permeable reductant without a thiol group. DTNB did not affect the Ca²⁺ spike caused by fMLP. These results indicate that through protein thiol oxidation, NEM affects the receptor-activated and the store depletion-derived Ca²⁺ signals in an opposing manner.

Keywords: Abbreviations; CPA; cyclopiazonic acid; DTNB; 5,5′-dithiobis-2-nitrobenzoic acid; fMLP; formyl-Met-Leu-Phe; FPR; formyl peptide receptor; HBSS; Hanks’ balanced salt solution; IP; ₃; inositol trisphosphate; IP; ₃; R; inositol trisphosphate receptor; NAC; N; -acetyl-; l; -cysteine; NEM; N; -ethylmaleimide; PLC; phospholipase C; ROCE; receptor-operated Ca; ²⁺; entry; RT-PCR; reverse transcription-polymerase chain reaction; SERCA; sarcoplasmic-endoplasmic reticulum Ca; ²⁺; -ATPase; SOCE; store-operated Ca; ²⁺; entry; TCEP; tris-(2-carboxyethyl)phosphine; TCP; tris-(2-cyanoethyl)phosphine; TRP; transient receptor potential N; -Ethylmaleimide; Ca; ²⁺; entry; Ca; ²⁺; release; Cyclopiazonic acid; fMLP; Neutrophils

New class of linoleic acid metabolites biosynthesized by corn and rice lipoxygenases: Suppression of proinflammatory mediator expression via attenuation of MAPK- and Akt-, but not PPARγ-, dependent pathways in stimulated macrophages by Akira Murakami; Takashi Nishizawa; Kahori Egawa; Teruo Kawada; Yasushi Nishikawa; Kazuo Uenakai; Hajime Ohigashi (pp. 1330-1342).

In response to endogenous and exogenous stimuli, macrophages are activated to produce a cocktail of proinflammatory and anti-apoptotic mediators, thereby participating in the processes of inflammation-associated oncogenesis. Cereals, including corn and rice, have biological potentials to synthesize self-protective chemicals in order to repel the invasion of microorganisms and insects. We examined the suppressive effects of several fatty acids, including a new class of lipoxygenase metabolites of linoleic acid (LA) found in cereals, namely (±)-9-hydroxy- trans, cis-10,12-octadecadienoic acid (9-HOA from rice), (±)-13-hydroxy-10-oxo- trans-11-octadecenoic acid (13-HOA from corn), and (±)-10-oxo- trans-11-octadecen-13-olide (10-ODO from corn), on lipopolysaccharide (LPS)-induced mRNA expression of proinflammatory mediators in RAW264.7 murine macrophages. Each metabolite exhibited a suppressive activity toward nitrite production than LA, octadeca-9 Z,11 E-dienoic acid (a conjugated LA), and 13( S)-hydroxyoctadeca-9 Z,11 E-dienoic acid. LPS-up-regulated mRNA expression of inducible nitric oxide synthase, cyclooxygenase ( COX)-2, interleukin-6, and toll-like receptor- 2, - 4, and - 9 was also markedly attenuated without affecting the expression levels of several constitutive genes, including COX-1, as detected by reverse transcription-polymerase chain reactions. In addition, Western blot and luciferase reporter assay results showed that 13-HOA suppressed the phosphorylation of mitogen-activated protein kinases (extracellular signal-regulated kinasel/2, c-Jun N-terminal kinasel/2, p38 mitogen-activated protein kinase), and Akt (Ser⁴⁷³), and also attenuated degradation of inhibitor kappaB, nuclear translocation of nuclear factor kappaB (NFκB), and the transcriptional activities of NFκB and activator protein-1, both of which have essential roles in the transcription of numerous proinflammatory and oncogenic genes. In contrast, 13-HOA did not serve as a ligand for peroxisome proliferator-activated receptor-γ. Based on our findings, we propose that 13-HOA, a functionally novel LA-derivative, is a promising agent for anti-inflammatory and chemopreventive strategies with reasonable molecular mechanisms.

Keywords: Abbreviations; LA; linoleic acid; 9-HOA; (±)-9-hydroxy-; trans; ,; cis; -10,12-octadecadienoic acid; CLA; octadeca-9; Z; ,11; E; -dienoic acid; 13-HOA; (±)-13-hydroxy-10-oxo-; trans; -11-octadecenoic acid; 10-ODO; (±)-10-oxo-; trans; -11-octadecen-13-olide; LPS; lipopolysaccharide; CLA; conjugated linoleic acid; LOX; lipoxygenase; 13S-HODE; 13(; S; )-hydroxyoctadeca-9; Z; ,11; E; -dienoic; iNOS; inducible nitric oxide synthase; COX; cyclooxygenase; NO; nitric oxide; PG; prostaglandin; IL; interleukin; TLR; toll-like receptor; NFκB; nuclear factor-kappaB; AP-1; activator protein-1; NSAID; nonsteroidal anti-inflammatory drugs; IFN; interferon; DMEM; Dulbecco's modified eagle medium; FBS; fetal bovine serum; NO; ₂; ⁻; nitrite; PBS; phosphate-buffered saline; DMSO; dimethylsulfoxide; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; HPLC; high performance liquid chromatography; SD; standard deviation; RT-PCR; reverse transcription-polymerase chain reaction; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; TNF; tumor necrosis factor; TGF; transforming growth factor; GM-CSF; granulocyte-macrophage colony-stimulating factor; ERK; extracellular signal-regulated kinase; JNK; c-Jun; N; -terminal kinase; MAPK; mitogen-activated protein kinase; IκB; inhibitor kappaB; HRP; horseradish peroxidase; PPAR; peroxisome proliferator-activated receptor; IR; inhibitory rate; CV; cell viability; 15d-PGJ; ₂; 15-deoxy-deltal2,14-PGJ; ₂Linoleic acid; Macrophage; Anti-inflammation; NFκB; AP-1; IL-6; Lipoxygenase; Toll-like receptor; iNOS; COX-2

A comparison of the cyclooxygenase inhibitor-NO donors (CINOD), NMI-1182 and AZD3582, using in vitro biochemical and pharmacological methods by Delano V. Young; Edward D. Cochran; Vijay Dhawan; Richard A. Earl; James L. Ellis; David S. Garvey; David R. Janero; Subhash P. Khanapure; L. Gordon Letts; Terry L. Melim; Madhavi G. Murty; Matthew J. Shumway; Shiow-Jyi Wey; Irina S. Zemtseva; William M. Selig (pp. 1343-1351).

Cyclooxygenase (COX, EC 1.14.99.1) inhibitor-nitric oxide (NO) donor (CINOD) hybrid compounds represent an attractive alternative to NSAID and coxib therapy. This report compares two CINODs, NMI-1182 (naproxen–glyceryl dinitrate) and AZD3582 (naproxen– n-butyl nitrate), for their ability to inhibit COX-1 and -2, deliver bioavailable nitric oxide, and release naproxen, using in vitro biochemical and pharmacological methods. In human whole blood, both CINODs showed inhibition, comparable to naproxen, of both COX isozymes and slowly released naproxen. Both CINODs donated bioavailable NO, as detected by cGMP induction in the pig kidney transformed cell line, LLC-PK1, but NMI-1182 was more potent by 30–100 times than AZD3582, GTN, GDN, and ISDN and considerably faster in inducing cGMP synthesis than AZD3582. The nitrate groups of GTN, NMI-1182, and AZD3582 appeared to be bioactivated via a common pathway, since each compound desensitized LLC-PK1 cells to subsequent challenge with the other compounds. Similar cGMP induction also occurred in normal, untransformed cells (human renal proximal tubule epithelial cells and hepatocytes from man, rat, and monkey); again, NMI-1182 was superior to AZD3582. NMI-1182 was also the more metabolically labile compound, releasing more absolute nitrate and nitrite (total NO_x) in human stomach (in which NO is salutary) and liver S9 homogenates. Naproxen was also more rapidly freed from NMI-1182 than AZD3582 in human stomach, although liver S9 hydrolyzed both CINODs with similar rates. These in vitro tests revealed that NMI-1182 may be a better CINOD than AZD3582 because of its superior NO donating and naproxen liberating properties.

Keywords: Nitric oxide (NO); Nitric oxide donor; Cyclooxygenase (COX) inhibitor; CINOD; Coxib therapy; NaproxenAbbreviations; cGMP; cyclic guanosine 3′,5′-monophosphate; CINOD; COX inhibitor NO donor; COX; cyclooxygenase; GI; gastrointestinal; GDN; glyceryl dinitrate; GTN; glyceryl trinitrate; HPLC; high pressure liquid chromatography; ISDN; isosorbide dinitrate; LPS; lipopolysaccharide; NO; nitric oxide; NO; _x; nitrate and nitrite; NSAID; non-steroidal anti-inflammatory drug; RPTEC; renal proximal tubule epithelial cells; SOD; super oxide dismutase

Inhibition of LPS-induced NO production by taurine chloramine in macrophages is mediated though Ras-ERK-NF-κB by Jun Woo Kim; Chaekyun Kim (pp. 1352-1360).

Taurine is an abundant free amino acid in inflammatory cells that protects cells from inflammatory damages. Although the protection mechanism remains unclear, taurine chloramine (Tau-Cl) produced by the reaction between taurine and hypochlorous acid in neutrophils plays an important role. In this study, we investigated the mechanism(s) by which Tau-Cl inhibits LPS-induced NO production in macrophages. Tau-Cl inhibited LPS-induced iNOS expression and NO production in RAW 264.7 cells. LPS treatment elevated the level of active Ras-GTP, and Tau-Cl inhibited LPS-induced Ras activation. Tau-Cl also inhibited ERK1/2 activation in a dose-dependent manner in both RAW 264.7 cells and murine peritoneal macrophages, whereas it did not exert any effect on p38 MAPK activation. Furthermore, Tau-Cl inhibited NF-κB activation without affecting AP-1 activity. These results suggest that Tau-Cl suppresses LPS-induced NO production by inhibiting specific signaling pathways. Thus, Tau-Cl protects cells from inflammatory injury resulting from overproduction of NO in a signaling pathway-specific manner.

Keywords: Abbreviations; Tau-Cl; taurine chloramine; MPO; myeloperoxidase; NO; nitric oxide; iNOS; inducible nitric oxide synthase; TNF; tumor necrosis factor; IL; interleukin; NF-κB; nuclear factor κB; AP-1; activator protein 1; LPS; lipopolysaccharide; TLR; Toll-like receptor; MAPK; mitogen-activated protein kinase; ERK; extracellular signal-regulated kinase; MEK; MAPK/ERK kinase; GAPDH; glyceraldehydes-3-phosphate dehydrogenase; EMSA; electrophoretic gel mobility shift assay; AEBSF; 4-(2-aminoethyl)benzenesulfonyl fluoride; BCA; bicinchoninic acid; MTT; thiazolyl blue tetrazolium bromide; PI; propidium iodideTaurine chloramine; Nitric oxide; Ras; ERK1/2; NF-κB; AP-1

The protection of bioenergetic functions in mitochondria by new synthetic chromanols by Katrin Staniek; Thomas Rosenau; Wolfgang Gregor; Hans Nohl; Lars Gille (pp. 1361-1370).

α-Tocopherol is the most important lipophilic antioxidant of the chromanol type protecting biomembranes from lipid peroxidation (LPO). Therefore, α-tocopherol and its derivatives are frequently used in the therapy or prevention of oxygen radical-derived diseases. In the present study, novel chromanol-type antioxidants (twin-chromanol, cis- and trans-oxachromanol) as well as the well-known short-chain analogue of α-tocopherol, pentamethyl-chromanol, were tested for their antioxidative potency in rat heart mitochondria (RHM). Our experiments revealed that the bioenergetic parameters of mitochondria were not deteriorated in the presence of chromanols (up to 50nmol/mg protein). Exposure of RHM to cumene hydroperoxide and Fe²⁺ (final concentrations 50μM each), inducing LPO, significantly affected their bioenergetic parameters which were determined in the presence of glutamate and malate (substrates of mitochondrial complex I). Alterations of the bioenergetic parameters were partially prevented in a concentration-dependent manner by preincubating RHM with antioxidants before adding the radical-generating system. In the lower concentration range, twin-chromanol turned out to be more efficient than pentamethyl-chromanol, both being far more protective than cis- and trans-oxachromanol. Measurement of protein-bound SH groups and thiobarbituric acid-reactive substances revealed that this protective effect was due to their antioxidative action. Furthermore, HPLC measurements of α-tocopherol and α-tocopheryl quinone in rat liver mitochondria demonstrated an α-tocopherol-sparing effect of twin-chromanol. In conclusion, new chromanol-type antioxidants, especially twin-chromanol, were able to improve bioenergetic and biochemical parameters of mitochondria exposed to oxidative stress.

Keywords: Abbreviations; BHT; 3,5-di-; tert; -butyl-4-hydroxy-toluene; CumOOH; cumene hydroperoxide; DTNB; 5,5′-dithio-bis-(2-nitrobenzoic acid); LPO; lipid peroxidation; MPTP; mitochondrial permeability transition pore; OCA; oxachromanol; PMC; 2,2,5,7,8-pentamethyl-chroman-6-ol; RHM; rat heart mitochondria; RLM; rat liver mitochondria; ROS; reactive oxygen species; TBA; 2-thiobarbituric acid; TBARS; TBA-reactive substances; Toc; α-tocopherol; TQ; α-tocopheryl quinone; Twin; twin-chromanol (1,3,4,8,9,11-hexamethyl-6,12-methano-12H-dibenzo[d,g][1,3]dioxocin-2,10-diol)Mitochondria; Bioenergetics; Lipid peroxidation; Thiol oxidation; Chromanols; Tocopheryl quinone

Neuroprotective effects of TEMPOL in central and peripheral nervous system models of Parkinson's disease by Qinghua Liang; Amanda D. Smith; Stephen Pan; Vladimir A. Tyurin; Valerian E. Kagan; Teresa G. Hastings; Nina Felice Schor (pp. 1371-1381).

TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine- N-oxyl) is a stable nitroxyl antioxidant. Previous studies have suggested that TEMPOL is protective in acute disorders thought to involve reactive oxygen species (ROS), such as ischemic stroke and cardiac reperfusion injury. Oxidized TEMPOL can be recycled to its redox-active reducing form by co-administration with polynitroxylated albumin, making it a candidate as a pharmacological “reservoir�? for reducing potential of use in chronic disorders involving ROS. The present studies examine the efficacy of TEMPOL in cell culture and animal models of the central and peripheral dysfunction associated with Parkinson's disease, a disorder in the pathogenesis of which ROS generated from dopamine have been implicated. Antioxidants have been proposed as both preventive and symptomatic therapy for Parkinson's disease. TEMPOL protects MN9D dopaminergic mesencephalic cells in culture from 6-hydroxydopamine (6-OHDA)-induced apoptosis. Translocation of the p65 component of NF-κB to the nucleus accompanies protection by TEMPOL. In vivo, intraperitoneal TEMPOL protects mice from intrastriatal 6-OHDA-induced cell and dopamine metabolite loss in the striatum. TEMPOL also protects mice against the 6-OHDA-induced rotational behavior elicited by intrastriatal administration ofd-amphetamine. In addition, TEMPOL protects mice from the ptosis, activity level decrement, and mortality induced by intraperitoneal administration of 6-OHDA, a model of autonomic dysfunction in Parkinson's disease. Adjunctive use of polynitroxylated albumin enhances the in vitro and in vivo effects of TEMPOL.

Keywords: Abbreviations; TEMPOL; 4-hydroxy-2,2,6,6-tetramethylpiperidine-; N; -oxyl; PNA; polynitroxylated albumin; DOPAC; dihydroxyphenylacetic acid; HVA; homovanilic acid; TH; tyrosine hydroxylase; MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; i.p.; intraperitoneal; i.v.; intravenousParkinson's disease; Reactive oxygen species; Antioxidants; Neuroprotection; Dopamine; Autonomic nervous system

Radioligand binding studies of caloporoside and novel congeners with contrasting effects upon [³⁵S] TBPS binding to the mammalian GABA_A receptor by S. Abuhamdah; A. F�rstner; G. Lees; P.L. Chazot (pp. 1382-1388).

Caloporoside is a natural active fungal metabolite, which was isolated from Caloporous dichrous and was described to exhibit antibacterial, antifungal and phospholipase C inhibitory activity. We have previously reported evidence that related β-linked compounds, lactose and octyl-β-d-mannoside, bind and functionally modulate rodent GABA_A receptors, respectively. We have characterized the binding pharmacology of synthetic caloporoside and two further congeners, 2-hydroxy-6-{[(16R)-(β-d-mannopyranosyloxy)heptadecyl]} benzoic acid and octyl-β-d-glucoside on GABA_A receptors using a [³⁵S]- t-butylbicyclophosphoorothionate (TBPS) radioligand binding assay. Caloporoside and 2-hydroxy-6-{[(16R)-(β-d-mannopyranosyloxy)heptadecyl]} benzoic acid produced concentration-dependent complete inhibition of specific [³⁵S] TBPS binding with overall apparent IC₅₀ values of 14.7±0.1 and 14.2±0.1μM, respectively. In contrast, octyl-β-d-glucoside elicited a concentration-dependent stimulation of specific [³⁵S] TBPS binding ( E_max=144±4%; EC₅₀=39.2±22.7nM). The level of stimulation was similar to that elicited by diazepam ( E_max=147±6%; EC₅₀=0.8±0.1nM), and was occluded by GABA (0.3μM). However, the three test compounds failed to elicit any significant effect (positive or negative) upon [³H] flunitrazepam or [³H] muscimol binding, indicating that they did not bind directly, or allosterically couple, to the benzodiazepine or agonist binding site of the GABA_A receptor, respectively. The constituent monosaccharide, glucose, and both the closely related congeners octyl-β-d-glucoside or hexyl-β-d-glucoside have no significant effect upon [³⁵S] TBPS binding. These data, together, provide strong evidence that a β-glycosidic linkage and chain length are crucial for the positive modulation of [³⁵S] TBPS binding to the GABA_A receptor by this novel chemical class.

Keywords: Abbreviations; GABA; γ-aminobutyric acid; TBPS; t; -butylbicyclophosphoorothionate; SAR; structure activity relationship; TBOB; t; -butylbicycloorthobenzoate; DMSO; dimethylsulphoxideGABA; _A; receptor; Allosteric modulator; TBPS; SAR; Channel site; Antagonist; β-Linkage; Sugar

Effect of prolonged exposure to milnacipran on norepinephrine transporter in cultured bovine adrenal medullary cells by Koji Shinkai; Reiji Yoshimura; Yumiko Toyohira; Susumu Ueno; Masato Tsutsui; Jun Nakamura; Nobuyuki Yanagihara (pp. 1389-1397).

The antidepressants milnacipran and paroxetine are used clinically worldwide. In the present study, we report here the effects of treatment with milnacipran and paroxetine on the functional activity, binding sites, and mRNA of the norepinephrine (NE) transporter (NET) in cultured bovine adrenal medullary cells. In acute treatment with antidepressants for 20min, both milnacipran and paroxetine competitively inhibited NET function in cultured adrenal medullary cells. Prolonged treatment of adrenal medullary cells with milnacipran produced time (48–96h)- and concentration (35–355nM)-dependent increases in [³H]NE uptake and [³H]DMI binding without any increase in NET mRNA. At a high concentration (800nM, 72h), paroxetine suppressed [³H]NE uptake. To examine whether milnacipran-induced [³H]NE uptake is mediated by newly synthesized mRNAs or proteins, we used actinomycin D, an inhibitor of DNA-dependent RNA polymerase, and cycloheximide, an inhibitor of ribosomal protein synthesis. Cycloheximide (1μM, 72h) abolished the effect of milnacipran on [³H]NE uptake, while the stimulatory effect of milnacipran was observed in actinomycin D-treated cells. The present findings suggest that prolonged exposure to milnacipran up-regulates the NET function, probably through a post-transcriptional process of NET or other proteins.

Keywords: Abbreviations; KRH; Krebs-Ringer HEPES; DMI; desipramine; NET; norepinephrine transporter; SNRI; serotonin norepinephrine reuptake inhibitor; SSRI; selective serotonin reuptake inhibitor; RT-PCR; reverse transcriptase-polymerase chain reactionAdrenal medullary cells; Milnacipran; Norepinephrine transporter; Serotonin norepinephrine reuptake inhibitor; Up-regulation

Genipin-induced apoptosis in hepatoma cells is mediated by reactive oxygen species/c-Jun NH₂-terminal kinase-dependent activation of mitochondrial pathway by Byung-Chul Kim; Hong-Gyum Kim; Sin-Ae Lee; Seunghwan Lim; Eun-Hee Park; Seong-Jin Kim; Chang-Jin Lim (pp. 1398-1407).

Genipin, the aglycone of geniposide, exhibits anti-inflammatory and anti-angiogenic activities. Here we demonstrate that genipin induces apoptotic cell death in FaO rat hepatoma cells and human hepatocarcinoma Hep3B cells, detected by morphological cellular changes, caspase activation and release of cytochrome c. During genipin-induced apoptosis, reactive oxygen species (ROS) level was elevated, and N-acetyl-l-cysteine (NAC) and glutathione (GSH) suppressed activation of caspase-3, -7 and -9. Stress-activated protein kinase/c-Jun NH₂-terminal kinase 1/2(SAPK/JNK1/2) but neither MEK1/2 nor p38 MAPK was activated in genipin-treated hepatoma cells. SP600125, an SAPK/JNK1/2 inhibitor, markedly suppressed apoptotic cell death in the genipin-treated cells. The FaO cells stably transfected with a dominant-negative c-Jun, TAM67, was less susceptible to apoptotic cell death triggered by genipin. Diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, inhibited ROS generation, apoptotic cell death, caspase-3 activation and JNK activation. Consistently, the stable expression of Nox1-C, a C-terminal region of Nox1 unable to generate ROS, blocked the formation of TUNEL-positive apoptotic cells, and activation of caspase-3 and JNK in FaO cells treated with genipin. Our observations imply that genipin signaling to apoptosis of hepatoma cells is mediated via NADPH oxidase-dependent generation of ROS, which leads to downstream of JNK.

Keywords: Abbreviations; GSH; reduced glutathione; DPI; diphenyleneiodonium; HA; hemagglutinin; NAC; N; -acetyl-; l; -cysteine; ROS; reactive oxygen species; DCFH-DA; 2′,7′-dichlorohydrofluorescein diacetate; FACS; fluorescence-activated cell sorting; TUNEL; terminal deoxynucleotidyltransferase dUTP nick end labeling; SAPK; stress-activated protein kinase; JNK; c-Jun NH; ₂; -terminal kinase; MAPK; mitogen-activated protein kinase; ERK; extracellular signal-regulated kinase; PI3K; phosphoinositol 3-kinase; JNK; c-Jun NH; ₂; -terminal kinase; TGF-β1; transforming growth factor β1; PBS; phosphate-buffered salineGenipin; Apoptosis; Caspase; NADPH oxidase; Reactive oxygen species; Mitochondria

Serum thymic factor, FTS, attenuates cisplatin nephrotoxicity by suppressing cisplatin-induced ERK activation by Yuka Kohda; Yoshiko Kawai; Noriaki Iwamoto; Yoshiko Matsunaga; Hiromi Aiga; Akira Awaya; Munekazu Gemba (pp. 1408-1416).

Serum thymic factor (FTS), a thymic peptide hormone, has been reported to attenuate the bleomycin-induced pulmonary injury and also experimental pancreatitis and diabetes. In the present study, we investigated the effect of FTS on cis-diamminedichloroplatinum II (cisplatin)-induced nephrotoxicity. We have already demonstrated that cephaloridine, a nephrotoxic antibiotic, leads to extracellular signal-regulated protein kinase (ERK) activation in the rat kidney, which probably contributes to cephaloridine-induced renal dysfunction. The aim of this study was to examine the effect of cisplatin on ERK activation in the rat kidney and also the effect of FTS on cisplatin-induced nephrotoxicity in rats. In vitro treatment of LLC-PK₁ cells with FTS significantly ameliorated cisplatin-induced cell injury. Treatment of rats with intravenous cisplatin for 3 days markedly induced renal dysfunction and increased platinum contents in the kidney cortex. An increase in pERK was detected in the nuclear fraction prepared from the rat kidney cortex from days 1 to 3 after injection of cisplatin. FTS suppressed cisplatin-induced renal dysfunction and ERK activation in the kidney. FTS did not influence any Pt contents in the kidney after cisplatin administration. FTS has been shown to enhance the in vivo expression of heat shock protein (HSP) 70 in the kidney cortex. The beneficial role of FTS against cisplatin nephrotoxicity may be mediated in part by HSP70, as suggested by its up-regulation in the kidney cortex treated with FTS alone. Our results suggest that FTS participates in protection from cisplatin-induced nephrotoxicity by suppressing ERK activation caused by cisplatin.

Keywords: cis; -Diamminedichloroplatinum II (cisplatin); Nephrotoxicity; Serum thymic factor (FTS); ERK activation; HSP70

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Biochemical Pharmacology (v.70, #9)

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Biochemical Pharmacology (v.70, #9)