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Biochemical Pharmacology (v.85, #6)

Editorial Board (pp. iii).

The Nrf2 cell defence pathway: Keap1-dependent and -independent mechanisms of regulation by Holly K. Bryan; Adedamola Olayanju; Christopher E. Goldring; B. Kevin Park (pp. 705-717).

The transcription factor Nrf2 (NF-E2-related factor 2) plays a vital role in maintaining cellular homeostasis, especially upon the exposure of cells to chemical or oxidative stress, through its ability to regulate the basal and inducible expression of a multitude of antioxidant proteins, detoxification enzymes and xenobiotic transporters. In addition, Nrf2 contributes to diverse cellular functions including differentiation, proliferation, inflammation and lipid synthesis and there is an increasing association of aberrant expression and/or function of Nrf2 with pathologies including cancer, neurodegeneration and cardiovascular disease. The activity of Nrf2 is primarily regulated via its interaction with Keap1 (Kelch-like ECH-associated protein 1), which directs the transcription factor for proteasomal degradation. Although it is generally accepted that modification (e.g. chemical adduction, oxidation, nitrosylation or glutathionylation) of one or more critical cysteine residues in Keap1 represents a likely chemico-biological trigger for the activation of Nrf2, unequivocal evidence for such a phenomenon remains elusive. An increasing body of literature has revealed alternative mechanisms of Nrf2 regulation, including phosphorylation of Nrf2 by various protein kinases (PKC, PI3K/Akt, GSK-3β, JNK), interaction with other protein partners (p21, caveolin-1) and epigenetic factors (micro-RNAs -144, -28 and -200a, and promoter methylation). These and other processes are potentially important determinants of Nrf2 activity, and therefore may contribute to the maintenance of cellular homeostasis. Here, we dissect evidence supporting these Keap1-dependent and -independent mechanisms of Nrf2 regulation. Furthermore, we highlight key knowledge gaps in this important field of biology, and suggest how these may be addressed experimentally.

Keywords: Abbreviations; DEA-NO/AM; acetoxymethylated diethylamine-NONO-ate; LC-ESI MS/MS; liquid chromatography electronspray ionisation tandem mass spectrometry; MRM; multiple reaction monitoring; tBHQ; tert-butylhydroquinone; CDDO-Me; methyl-2-cyano-3,12 dioxoolean-1,9 diene-28-oate; CDDO-Im; 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl] imidazoleNrf2; Keap1; Oxidative stress; Cell defence; Regulation

Role of DHEA in cardiovascular diseases by Jean-Pierre Savineau; Roger Marthan; Eric Dumas de la Roque (pp. 718-726).

Dehydroepiandrosterone (DHEA) is a steroid hormone derived from cholesterol synthesized by the adrenal glands. DHEA and its 3β-sulphate ester (DHEA-S) are the most abundant circulating steroid hormones. In human, there is a clear age-related decline in serum DHEA and DHEA-S and this has suggested that a relative deficiency in these steroids may be causally related to the development of a series of diseases associated with aging including cardiovascular diseases (CVD).This commentary aims to highlight the action of DHEA in CVD and its beneficial effect in therapy. We thus discuss the possible impact of serum DHEA decline and DHEA supplementation in diseases such as hypertension, coronary artery disease and atherosclerosis. More specifically, we provide evidence for a beneficial action of DHEA in the main disease of the pulmonary circulation: pulmonary hypertension. We also examine the potential cellular mechanism of action of DHEA in terms of receptors (membrane/nuclear) and associated signaling pathways (ion channels, calcium signaling, PI3K/AKT/eNos pathway, cGMP, RhoA/RhoK pathway). We show that DHEA acts as an anti-remodeling and vasorelaxant drug. Since it is a well-tolerated and inexpensive drug, DHEA may prove to be a valuable molecule in CVD but it deserves further studies both at the molecular level and in large clinical trials.

Keywords: Abbreviations; CH; chronic hypoxia; CVD; cardiovascular diseases; DHEA; dehydroepiandrosterone; DHEA-S; dehydroepiandrosterone sulphate; EC; endothelial cells; PAEC; pulmonary artery endothelial cell; PASMC; pulmonary artery smooth muscle cell; PH; pulmonary hypertension; SMC; smooth muscle cellsDehydroepiandrosterone; Steroid hormones; Cardiovascular diseases; Smooth muscle cell; Hypertension

Antivirals: Past, present and future by Erik De Clercq (pp. 727-744).

Vaccination is possible to prevent infections with some viruses: hepatitis B virus (HBV), varicella-zoster virus (VZV), influenza A and B viruses, Yellow fever virus and poliovirus; but not for others: human immunodeficiency virus (HIV), hepatitis C virus (HCV), herpes simplex virus (HSV), cytomegalovirus (CMV), and most hemorrhagic fever viruses (HFV) (except for Yellow fever virus). Antiviral therapy is obviously needed to control those infections that are not amenable to prophylaxis by vaccination, but is also highly desirable for those infections where vaccination has not been implemented or did not fulfill its premises for complete protection.

Keywords: Antivirals; Past; Present; Future; HIV; HCV; HBV; HSV; VZV; CMV; HFV

Live-cell imaging of p53 interactions using a novel Venus-based bimolecular fluorescence complementation system by Joana Dias Amaral; Federico Herrera; Pedro Miguel Rodrigues; Pedro Antunes Dionísio; Tiago Fleming Outeiro; Cecília Maria Pereira Rodrigues (pp. 745-752).

p53 plays an important role in regulating a wide variety of cellular processes, such as cell cycle arrest and/or apoptosis. Dysfunction of p53 is frequently associated with several pathologies, such as cancer and neurodegenerative diseases. In recent years substantial progress has been made in developing novel p53-activating molecules. Importantly, modulation of p53 interaction with its main inhibitor, Mdm2, has been highlighted as a promising therapeutic target. In this regard, bimolecular fluorescence complementation (BiFC) analysis, by providing direct visualization of protein interactions in living cells, offers a straightforward method to identify potential modulators of protein interactions. In this study, we developed a simple and robust Venus-based BiFC system to screen for modulators of p53–p53 and p53–Mdm2 interactions in live mammalian cells. We used nutlin-3, a well-known disruptor of p53–Mdm2 interaction, to validate the specificity of the assay. The reduction of BiFC signal mediated by nutlin-3 was correlated with an increase in Puma transactivation, PARP cleavage, and cell death. Finally, this novel BiFC approach was exploited to identify potential modulators of p53–Mdm2 complex formation among a commercially available chemical library of 33 protein phosphatase inhibitors. Our results constitute “proof-of-concept” that this model has strong potential as an alternative to traditional target-based drug discovery strategies. Identification of new modulators of p53–p53 and p53–Mdm2 interactions will be useful to achieve synergistic drug efficacy with currently used anti-tumor therapies.

Keywords: BiFC assay; p53–Mdm2 interaction; HCT116 cells; Drug discovery; Cancer

Inhibition of constitutive aryl hydrocarbon receptor (AhR) signaling attenuates androgen independent signaling and growth in (C4-2) prostate cancer cells by Cindy Tran; Oliver Richmond; LaTayia Aaron; Joann B. Powell (pp. 753-762).

The aryl hydrocarbon receptor is a member of the basic-helix-loop-helix family of transcription factors. AhR mediates the biochemical and toxic effects of a number of polyaromatic hydrocarbons such as 2,3,7,8,-tetrachloro-dibenzo-p-dioxin (TCDD). AhR is widely known for regulating the transcription of drug metabolizing enzymes involved in the xenobiotic metabolism of carcinogens and therapeutic agents, such as cytochrome P450-1B1 (CYP1B1). Additionally, AhR has also been reported to interact with multiple signaling pathways during prostate development. Here we investigate the effect of sustained AhR signaling on androgen receptor function in prostate cancer cells. Immunoblot analysis shows that AhR expression is increased in androgen independent (C4-2) prostate cancer cells when compared to androgen sensitive (LNCaP) cells. RT-PCR studies revealed constitutive AhR signaling in C4-2 cells without the ligand induced activation required in LNCaP cells. A reduction of AhR activity by short RNA mediated silencing in C4-2 cells reduced expression of both AhR and androgen responsive genes. The decrease in androgen responsive genes correlates to a decrease in phosphorylated androgen receptor and androgen receptor expression in the nucleus. Furthermore, the forced decrease in AhR expression resulted in a 50% decline in the growth rate of C4-2 cells. These data indicates that AhR is required to maintain hormone independent signaling and growth by the androgen receptor in C4-2 cells. Collectively, these data provide evidence of a direct role for AhR in androgen independent signaling and provides insight into the molecular mechanisms responsible for sustained androgen receptor signaling in hormone refractory prostate cancer.

Keywords: Dioxin; TCDD; AhR; Androgen receptor; Progression

Interference of a novel indolylmaleimide with microtubules induces mitotic arrest and apoptosis in human progenitor and cancer cells by Christian Eisenlöffel; Anne-Caroline Schmöle; Anahit Pews-Davtyan; Anne Brennführer; Sergei A. Kuznetsov; Rayk Hübner; Stefanie Frech; Catrin Schult; Christian Junghanss; Matthias Beller; Arndt Rolfs; Moritz J. Frech (pp. 763-771).

PDA-66 hinders proliferation of human progenitor and cancer cells by destabilising microtubules, leading to mitotic arrest and apoptosis. Thus indolylmaleimides provide a class of indoles to be used as anti-mitotic drugs.Indolylmaleimides display a broad spectrum of biological activity and offer great opportunity to influence several aspects of cell fate, as proliferation and differentiation. In this study we describe the effect of PDA-66, a newly synthesised indolylmaleimide, showing a strong dose dependent anti-proliferative effect on immortalised human progenitor and cancer cells. We demonstrated a highly depolymerizing effect on in vitro tubulin assembly and conclude that PDA-66 acts as microtubule destabilising agent. In addition we found that PDA-66 induces mitotic arrest of cells in the G₂/M phase of the cell cycle. Subsequently cells undergo apoptosis, indicating the major mechanism of the anti-proliferative effect. To prove a potential anti-cancer activity of PDA-66 we examined the effect of PDA-66 on human SH-SY5Y neuroblastoma and A-459 lung cancer cells, showing a significant reduction in cancer cell proliferation in a dose dependent manner. Thus PDA-66 is a new anti-mitotic compound with an indole-core with the potential to be used for cancer therapy.

Keywords: Cancer; Apoptosis; Microtubule; ReNcell VM; Indolylmaleimide

A novel recombinant fibrinogenase of Agkistrodon acutus venom protects against hyperacute rejection via degradation of complements by Xi Lin; Jie-zhen Qi; Ming-hui Chen; Bi-tao Qiu; Zhen-hua Huang; Peng-xin Qiu; Jia-shu Chen; Guang-mei Yan (pp. 772-779).

Hyperacute rejection (HAR) is a main barrier in xenotransplantation, which is mediated by the combination of natural antibody to the xenograft and complement activation. Current therapies have focus on the inhibition of complement by development of complement inhibitor and transgenic animal organ. Here, we investigated the effects of rFII, a recombinant fibrinogenase from Agkistrodon acutus venom, on complement and HAR. The degradation effect of rFII on complement was tested by SDS-PAGE, CH50 examination, ELISA Kit and cofocal immunofluorescence microscopy in vitro and in vivo. An ex-vivo rat-to-human perfusion model and a vivo guinea-pig-to-rat heat HAR model were used to determine the protection of rFII against HAR. Our investigation indicated that rFII could significantly degrade human C5, C6, and C9, decrease the activity of complement, and inhibit the MAC deposition on HUVECs membrane in vitro. In addition, serum levels of C1q, C3 and C4 in rat were gradually reduced after infusion of rFII. Importantly, in an ex vivo rat-to-human perfusion model, the survival of rat hearts perfused with human serum treated with rFII (83.36±16.63min) were significantly longer than that of hearts perfused with fresh human serum(15.94±4.75min). At the time of 15minutes after perfusion, functions of hearts added with 50ug/ml rFII sustained well with heart rates at 283±65.32 beats/minute and LVDP at 13.70±5.45Kpa, while that of hearts perfused with fresh human serum were severely damaged by HAR with heart rates at 107.77±40.31 beats/minute and LVDP at 1.01±0.83Kpa. We also found that rFII significantly decreased the levels of C1q, C3 and C4 in human fresh serum perfusate. In a vivo guinea-pig-to-rat heat HAR model, the survival of rat hearts treated with rFII were significantly longer than that of hearts perfused with normal saline; and relieved heart damage by complete activation. Our finding demonstrates the anti-complement property of rFII and its protection against HAR, indicating that rFII might be as a potential therapeutic agent for xenotransplantation.

Keywords: Abbreviations; ADP; adenosine diphosphate; ATII; antithrombin; CH50; complement hemolytic activity 50; CVF; cobra venom factor; HAR; hyperacute rejection; HUVEC; human umbilical vein endothelial cell; HR; heart rate; IgM; immunoglobulin M; LVDP; left ventricular developed pressure; LSCM; laser scanning confocal microscopy; MAC; membrane attack complex; rFII; recombinant fibrinogenase of Agkistrodon acutus venom; SCR1; soluble human complement receptor type1; XNAs; xenoreactive natural antibodiesXenotransplantation; Hyperacute rejection; Complement

The vasorelaxant effects of 1-nitro-2-phenylethane involve stimulation of the soluble guanylate cyclase-cGMP pathway by Teresinha S. Brito; Francisco J.B. Lima; Karoline S. Aragão; Rodrigo J.B. de Siqueira; Pergentino J.C. Sousa; José G.S. Maia; Jairo D. Filho; Saad Lahlou; Pedro J.C. Magalhães (pp. 780-788).

1-Nitro-2-phenylethane is the first organic NO₂-containing molecule isolated from plants. It possesses interesting hypotensive, bradycardic, and vasodilator properties, but the mode by which it induces vasorelaxation is still unknown. The underlying mechanism involved in the vasodilator effect of 1-nitro-2-phenylethane was investigated in rat aorta. The vasorelaxant effects of 1-nitro-2-phenylethane did not depend on endothelial layer integrity, and the effects were refractory to L-N^G-nitroarginine methyl ester (L-NAME)-induced nitric oxide synthase inhibition. Vasorelaxation was similarly resistant to treatment with indomethacin, cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine hydrochloride (MDL-12330A), and KT5720, indicating that neither prostaglandin release nor adenylyl cyclase activation is involved. Conversely, methylene blue- and ODQ-induced guanylate cyclase inhibition reduced the vasorelaxation induced by 1-nitro-2-phenylethane. The pharmacological blockade of K⁺ channels with tetraethylammonium, glybenclamide, and 4-aminopyridine also blunted vasorelaxation induced by 1-nitro-2-phenylethane. The effects of 1-nitro-2-phenylethane were reversed by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and comparable to the effects induced by sodium nitroprusside. In silico analysis using an Ns H-NOX subunit of guanylate cyclase revealed a pocket on the macromolecule surface where 1-nitro-2-phenylethane preferentially docked. In vitro, 1-nitro-2-phenylethane increased cyclic guanosine 3′,5′-monophosphate (cGMP) levels in rat aortic rings, an effect also reversed by ODQ. In conclusion, 1-nitro-2-phenylethane produces vasodilator effects by stimulating the soluble guanylate cyclase-cGMP pathway.

Keywords: Nitroderivative; Guanylate cyclase stimulation

Identification and characterization of MIP-1α/CCL3 isoform 2 from bovine serum as a potent monocyte/dendritic cell chemoattractant by Mieke De Buck; Mieke Gouwy; Paul Proost; Sofie Struyf; Jo Van Damme (pp. 789-797).

Bovine serum is a rich source of cytokines and growth factors supporting in vitro cell culture. Here, a novel bovine monocyte chemotactic factor (boMCF-1) was isolated from commercial bovine serum by a four step purification procedure including adsorption to silicic acid, heparin affinity and cation-exchange chromatography and reversed phase HPLC. Homogeneous boMCF-1 was characterized as a 7717Da protein by mass spectrometry and identified by Edman degradation as the predicted product of bovine macrophage inflammatory protein-1α gene (boMIP-1α/CCL3) isoform 2 (lacking three NH₂-terminal amino acids), belonging to the MIP subfamily of CC chemokines. Monocyte chemotactic activity of boCCL3 isoform 2 was completely desensitized by human CCL3 and CCL5, partially by CCL7 and not by CCL2. Its activity was better inhibited by CCR1 than by CCR5 blockade. BoCCL3 isoform 2, present in bovine serum at about 10ng/ml, functioned as a most potent chemoattractant for immature (but not mature) dendritic cells with a minimal effective concentration of 0.03ng/ml and a maximal chemotactic index of 30 at 0.3ng/ml. Its chemotactic activity on immature dendritic cells was significantly desensitized by human CCL3, CCL5 and CCL7. Blockade of CCR5 rather than CCR1 partially prevented chemotactic activity, whereas blockade of both further enhanced this inhibition. BoCCL3 isoform 2 was not chemokinetic but, like human CCL3, synergized with CXCL12 in monocytic cell chemotaxis. Since it cannot be deduced which is the exact human homolog of boCCL3 isoform 2, further research is required to study the biology of other boCCL3 family members.

Keywords: Abbreviations; Bo; bovine; CI; chemotactic index; CS; calf serum; EMEM; Eagle's minimum essential medium with Earle's salts; FPLC; fast protein liquid chromatography; MACS; magnetic cell sorting; MCF; monocyte chemotactic factor; MCP; monocyte chemotactic protein; MDDC; monocyte-derived dendritic cells; MIP; macrophage inflammatory protein; PBMC; peripheral blood mononuclear cells; RP-HPLC; reversed phase high performance liquid chromatographyChemokines; Macrophage inflammatory protein; Bos taurus; Chemotaxis; Immature monocyte-derived dendritic cells; Sequencing

Inhibition of Th1/Th17 responses via suppression of STAT1 and STAT3 activation contributes to the amelioration of murine experimental colitis by a natural flavonoid glucoside icariin by Feifei Tao; Cheng Qian; Wenjie Guo; Qiong Luo; Qiang Xu; Yang Sun (pp. 798-807).

Icariin, a natural flavonoid glucoside, attenuates dextran sulfate sodium (DSS)-induced experimental colitis in mice through inhibiting Th1/Th17 responses via suppression of STAT1 and STAT3 activation.Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the intestine which involves overproduction of pro-inflammatory cytokines and excessive functions of inflammatory cells. However, current treatments for IBD may have potential adverse effects including steroid dependence, infections and lymphoma. Therefore new therapies or drug candidates for the treatment of IBD are desperately needed. In the present study we found that icariin, a major bioactive compound from plants in Epimedium family, exerted protective effect on intestinal inflammation in mice induced by dextran sulfate sodium. Oral administration of icariin significantly attenuated the disease progression and alleviated the pathological changes of colitis. It also inhibited the production of pro-inflammatory cytokines and expression of p-p65, p-STAT1 and p-STAT3 in colon tissues. Further study showed that icariin dose-dependently inhibited the proliferation and activation of T lymphocytes, and suppressed pro-inflammatory cytokine levels of activated T cells. Moreover, icariin treatment inhibited the phosphorylations of STAT1 and STAT3 in CD4⁺ T cells, which were the crucial transcription factors for Th1 and Th17 respectively. Taken together, these results indicate that icariin is a potential therapeutic agent for IBD.

Keywords: Icariin; DSS-induced colitis; Th1/Th17; STAT1; STAT3

A p21-activated kinase (PAK1) signaling cascade coordinately regulates F-actin remodeling and insulin granule exocytosis in pancreatic β cells by Michael A. Kalwat; Stephanie M. Yoder; Zhanxiang Wang; Debbie C. Thurmond (pp. 808-816).

Human islet studies implicate an important signaling role for the Cdc42 effector protein p21-activated kinase (PAK1) in the sustained/second-phase of insulin secretion. Because human islets from type 2 diabetic donors lack ∼80% of normal PAK1 protein levels, the mechanistic requirement for PAK1 signaling in islet function was interrogated. Similar to MIN6 β cells, human islets elicited glucose-stimulated PAK1 activation that was sensitive to the PAK1 inhibitor, IPA3. Given that sustained insulin secretion has been correlated with glucose-induced filamentous actin (F-actin) remodeling, we tested the hypothesis that a Cdc42-activated PAK1 signaling cascade is required to elicit F-actin remodeling to mobilize granules to the cell surface. Live-cell imaging captured the glucose-induced cortical F-actin remodeling in MIN6 β cells; IPA3-mediated inhibition of PAK1 abolished this remodeling. IPA3 also ablated glucose-stimulated insulin granule accumulation at the plasma membrane, consistent with its role in sustained/second-phase insulin release. Both IPA3 and a selective inhibitor of the Cdc42 GTPase, ML-141, blunted the glucose-stimulated activation of Raf-1, suggesting Raf-1 to be downstream of Cdc42→PAK1. IPA3 also inhibited MEK1/2 activation, implicating the MEK1/2→ERK1/2 cascade to occur downstream of PAK1. Importantly, PD0325901, a new selective inhibitor of MEK1/2→ERK1/2 activation, impaired F-actin remodeling and the sustained/amplification pathway of insulin release. Taken together, these data suggest that glucose-mediated activation of Cdc42 leads to activation of PAK1 and prompts activation of its downstream targets Raf-1, MEK1/2 and ERK1/2 to elicit F-actin remodeling and recruitment of insulin granules to the plasma membrane to support the sustained phase of insulin release.

Keywords: Insulin granule; Exocytosis; Cdc42; F-actin remodeling; Islet β cell; PAK1

Point-to-point ligand–receptor interactions across the subunit interface modulate the induction and stabilization of conformational states of alpha7 nAChR by benzylidene anabaseines by Matthew D. Isaacson; Nicole A. Horenstein; Clare Stokes; William R. Kem; Roger L. Papke (pp. 817-828).

The homomeric α7 nicotinic acetylcholine receptor is a well-studied therapeutic target, though its characteristically rapid desensitization complicates the development of drugs with specific agonist effects. Moreover, some experimental compounds such as GTS-21 (2,4diMeOBA), a derivative of the α7-selective partial agonist benzylidene anabaseine (BA), produce a prolonged residual desensitization (RD) in which the receptor remains non-activatable long after the drug has been removed from extracellular solution. In contrast, the desensitization caused by GTS-21's dihydroxy metabolite (2,4diOHBA) is relatively short-lived. RD is hypothetically due to stable binding of the ligand to the receptor in its desensitized state. We can attribute the reduction in RD to a single BA hydroxyl group on the 4′ benzylidene position. Computational prediction derived from homology modeling showed the serine36 (S36) residue of α7 as a reasonable candidate for point-to-point interaction between BA compounds and the receptor. Through evaluating the activity of BA and simple derivatives on wild-type and mutant α7 receptors, it was observed that the drug–receptor pairs which were capable of hydrogen bonding at residue 36 exhibited significantly less stable desensitization. Further experiments involving the type II positive allosteric modulator (PAM) PNU-120596 showed that the various BA compounds’ preference to induce either a PAM-sensitive (D_s) or PAM-insensitive (D_i) desensitized state is concentration dependent and suggested that both states are destabilized by S36 H-bonding. These results indicate that the fine-tuning of agonists for specific interaction with S36 can facilitate the development of therapeutics with targeted effects on ion channel desensitization properties and conformational state stability.

Keywords: Abbreviations; ACh; acetylcholine; nAChR; nicotinic acetylcholine receptor; BA; benzylidene anabaseine; RD; residual desensitization; Ds; PAM-sensitive desensitization; Di; PAM-insensitive desensitization; PAM; positive allosteric modulator; LBD; ligand-binding domain; CRC; concentration–response curve; 2,4diMeOBA (GTS-21); 3-(2,4-dimethoxybenzylidene)anabaseine; 2,4diOHBA; 3-(2,4-dihydroxybenzylidene)anabaseine; PNU-120596; N-(5-Chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea; 4OHBA; (E)-3-(4-hydroxybenzylidene)anabaseine; 4MeBA; 3-(4-methylbenzylidene)anabaseineConformational states; Receptor desensitization; Site-directed mutation; Homology modeling

FXR-dependent and -independent interaction of glucocorticoids with the regulatory pathways involved in the control of bile acid handling by the liver by R. Rosales; M.R. Romero; J. Vaquero; M.J. Monte; P. Requena; O. Martinez-Augustin; F. Sanchez de Medina; J.J.G. Marin (pp. 829-838).

Treatment with glucocorticoids (GCs) may cause adverse effects, including cholestasis. The ability of dexamethasone, prednisolone and budesonide to affect the liver handling of bile acids (BAs) has been investigated. In rats treated with GCs for 4 days, altered serum and bile BA levels, changed conjugation pattern, and delayed and decreased ability to conjugate/secrete exogenously administered deoxycholate, were found using HPLC–MS/MS. RT-QPCR analyses revealed that GC treatment also induced a down-regulation of liver nuclear receptors (Fxr, Gr and Shp), transporters (Ntcp, Mrp4 and Bcrp) and enzymes (Cyp7a1 and Baat), whereas Bsep, Mrp2 and Cyp27a1 were up-regulated. Human HepG2 and Alexander cell lines were used as in vitro models of liver cells with and without constitutive FXR expression, respectively. In HepG2 cells, GCs induced a decreased expression of FXR and SHP, and inhibited the regulatory effect of GW4064 on FXR-target genes. In Alexander cells, only when they were transfected with FXR+RXR, GW4064 caused up-regulation of SHP and OSTβ, and a down-regulation of CYP27A1. GCs had the opposite effect on these genes, both in the absence and in the presence of FXR expression. Co-transfection of Alexander cells with IR-1-Luc and FXR+RXR revealed that GCs did not inhibit but moderately enhanced FXR activity. Moreover, GCs have a synergistic effect on GW4064-induced FXR activation, whereas chenodeoxycholate and GW4064 have an additive effect. In conclusion, GCs are able to directly or indirectly activate FXR but they also antagonize, through FXR-independent mechanisms, the expression of FXR and FXR target genes involved in the hepatic handling of BAs.

Keywords: Cholestasis; Liver; Metabolism; Nuclear receptors

Cloning, expression and analysis of the olfactory glutathione S-transferases in coho salmon by Herbert M. Espinoza; Laura M. Shireman; Valerie McClain; William Atkins; Evan P. Gallagher (pp. 839-848).

In this article, we characterized the GST profile in the peripheral olfactory system of salmon, including a novel rho-class isoform that protects against oxidative damage.The glutathione S-transferases (GSTs) provide cellular protection by detoxifying xenobiotics, maintaining redox status, and modulating secondary messengers, all of which are critical to maintaining olfaction in salmonids. Here, we characterized the major coho salmon olfactory GSTs (OlfGSTs), namely omega, pi, and rho subclasses. OlfGST omega contained an open reading frame of 720bp and encoded a protein of 239 amino acids. OlfGST pi and OlfGST rho contained open reading frames of 627 and 681nt, respectively, and encoded proteins of 208 and 226 amino acids. Whole-protein mass spectrometry yielded molecular weights of 29,950, 23,354, and 26,655Da, respectively, for the GST omega, pi, and rho subunits. Homology modeling using four protein-structure prediction algorithms suggest that the active sites in all three OlfGST isoforms resembled counterparts in other species. The olfactory GSTs conjugated prototypical GST substrates, but only OlfGST rho catalyzed the demethylation of the pesticide methyl parathion. OlfGST pi and rho exhibited thiol oxidoreductase activity toward 2-hydroxyethyl disulfide (2-HEDS) and conjugated 4-hydroxynonenal (HNE), a toxic aldehyde with neurodegenerative properties. The kinetic parameters for OlfGST pi conjugation of HNE were K_M=0.16±0.06mM and V_max=0.5±0.1μmolmin⁻¹mg⁻¹, whereas OlfGST rho was more efficient at catalyzing HNE conjugation ( K_M=0.022±0.008mM and V_max=0.47±0.05μmolmin⁻¹mg⁻¹). Our findings indicate that the peripheral olfactory system of coho expresses GST isoforms that detoxify certain electrophiles and pesticides and that help maintain redox status and signal transduction.

Keywords: Glutathione; S; -transferases; Coho salmon; Olfaction; 4-Hydroxynonenal; Oxidative stress; Methyl parathion

Acknowledgement (pp. 849-855).

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Biochemical Pharmacology (v.85, #6)