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Biochemical Pharmacology (v.71, #5)

Editorial Advisory Board (pp. iii).

Redox modifications of protein–thiols: Emerging roles in cell signaling by Saibal Biswas; Asiya Seema Chida; Irfan Rahman (pp. 551-564).

Glutathione represents the major low molecular weight antioxidant redox recycling thiol in mammalian cells and plays a central role in the cellular defence against oxidative damage. Classically glutathione has been known to provide the cell with a reducing environment in addition to maintaining the proteins in a reduced state. Emerging evidences suggest that the glutathione redox status may entail dynamic regulation of protein function by reversible disulfide bond formation. The formation of inter- and intramolecular disulfides as well as mixed disulfides between protein cysteines and glutathione, i.e., S-glutathiolation, has now been associated with the stabilization of extracellular proteins, protection of proteins against irreversible oxidation of critical cysteine residues, and regulation of enzyme functions and transcription. Regulation of DNA binding of redox-dependent transcription factors such as nuclear factor-κB, p53, and activator protein-1, has been suggested as one of the mechanisms by which cells may transduce oxidative stress redox signaling into an inducible expression of a wide variety of genes implicated in cellular changes such as proliferation, differentiation, and apoptosis. However, the molecular mechanisms linking the glutathione cellular redox state to a reversible oxidation of various signaling proteins are still poorly understood. This commentary discusses the emerging concept of protein- S-thiolation, protein- S-nitrosation and protein–SH (formation of sulfenic, sulfinic and sulfonic acids) in redox signaling during normal physiology and under oxidative stress in controlling the cellular processes.

Keywords: Abbreviations; AP-1; activator protein-1; ASK-1; apoptosis signaling kinase-1; GSNO; S; -nitrosoglutathione; GAPDH; glyceraldehyde-3-P-dehydrogenase; H; ₂; O; ₂; hydrogen peroxide; OH; hydroxyl radical; NO; nitric oxide radical; NF-κB; nuclear factor-kappa B; ONOO; ⁻; peroxynitrite; PKC; protein kinase C; PrSH; protein–Cys–SH; PTP-1B; protein-tyrosine phosphatases 1B; O; ₂; ⁻; superoxide radicalRedox; Glutathione; Thioredoxin; S; -thiolation; NF-κB; S; -nitrosylation

Contribution of an unidentified sodium-dependent nucleoside transport system to the uptake and cytotoxicity of anthracycline in mouse M5076 ovarian sarcoma cells by Katsuhito Nagai; Kazuki Nagasawa; Mineto Koma; Yukiko Kihara; Sadaki Fujimoto (pp. 565-573).

In the present study, we investigated whether an unidentified system for Na⁺-dependent nucleoside transport is expressed by mouse M5076 ovarian sarcoma cells, besides concentrative nucleoside transporter 2 (CNT2_M), and is involved in the uptake and cytotoxicity of anthracyclines. In a transport assay involving CNT2_M-transfectants, CNT2_M was found to transport [³H]cytidine in a Na⁺-dependent manner, and 500μM cytidine completely inhibited the Na⁺-dependent uptake of [³H]uridine via the transporter. In contrast, the Na⁺-dependent [³H]uridine uptake by M5076 cells decreased with 500μM cytidine only to 70% of the control level. Furthermore, transfection of CNT2_M-specific siRNAs into M5076 cells resulted in a reduction in the Na⁺-dependent uptake of [³H]uridine by only 23%, although the expression of CNT2_M mRNA and Na⁺-dependent uptake of [³H]cytidine disappeared in the cells. The uptake of pirarubicin (THP), an anthracycline, by M5076 cells requiring extracellular Na⁺ was significantly inhibited by 500μM uridine, but not 500μM cytidine. The Na⁺-dependent and cytidine-insensitive uptake of [³H]uridine and the that of THP by M5076 cells significantly increased on cotreatment with both cholate and taurocholate, and the enhancement of THP uptake by the bile acids was reversed by cotreatment with 500μM uridine. Furthermore, the cytotoxicity of THP and doxorubicin, which were previously reported to be taken up via the same transporter, toward M5076 cells was enhanced by cotreatment with both the bile acids. Therefore, it was indicated that an unidentified Na⁺-dependent transport system for nucleosides is expressed by M5076 cells, and contributes to the uptake and cytotoxicity of the anthracyclines.

Keywords: Anthracycline; M5076 cell; Nucleoside transport system; de novo resistance; Chemotherapy

Fibroblast heterogeneity in collagenolytic response to colchicine by Berit Mathisen; Thrina Loennechen; Tobias Gedde-Dahl; Jan-Olof Winberg (pp. 574-583).

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in various physiological and pathological conditions, including those that involve homeostasis of collagen. Drug induced regulation of MMP-1, other MMPs and TIMPs is critical in treatment of various diseases, e.g. the use of the plant alkaloid, colchicine. One possible factor that might explain the failure in colchicine-treatment of some patients is interindividual variability on the cellular level. To investigate the possible individual heterogeneity in response to colchicine, we studied the effect of colchicine-induced synthesis of collagenase from 32 different human skin fibroblast strains derived from both healthy individuals as well as individuals with different skin diseases. We showed that colchicine induced an increased synthesis of collagenase in 22 of 32 cases. This heterogeneity occurred in fibroblasts from healthy as well as diseased individuals. To determine if colchicine also affected the fibroblast synthesis of gelatinase, stromelysin and tissue inhibitors of MMPs, we investigated several individuals from a single family. The results showed that both colchicine responsive and non-responsive fibroblasts with respect to collagenase synthesis responded to colchicine by an increased stromelysin synthesis, while the synthesis of gelatinase and TIMP-1 were unaffected. As a whole, our results indicate that individual heterogeneity in collagenase response to colchicine treatment may partly explain some of the controversial results obtained with colchicine as a drug.

Keywords: Abbreviations; MMPs; matrix metalloproteinases; TIMPs; tissue inhibitors of MMPs; SBTI; soybean trypsin inhibitor; CLCS; collagenase colchicine sensitivityMatrix metalloproteinases; Collagenase; Tissue inhibitors of matrix metalloproteinases; Colchicine; Fibroblast heterogeneity; Drug response

Schisandrin B enhances doxorubicin-induced apoptosis of cancer cells but not normal cells by Ling Li; Qinghua Lu; Yanwei Shen; Xun Hu (pp. 584-595).

The dose-dependent cardiotoxicities of doxorubicin (DOX) significantly limits its anti-cancer efficacies. One of the ways to augment the efficacies of DOX at a relatively low cumulative dose is to use a chemical sensitizer. Here, we demonstrated that schisandrin B (Sch B) significantly enhanced DOX-induced apoptosis of SMMC7721, a human hepatic carcinoma cell line, and of MCF-7, a human breast cancer cell line. This enhancement was irrelevant to the action of Sch B on P-glycoprotein or other drug-transporters, but associated with the activation of caspase-9 rather than caspase-8. The loss of mitochondria membrane potential was observed when cells were treated with DOX and Sch B combined. On the other hand, at the same experimental conditions, Sch B did not enhance the DOX-induced apoptosis of primary rat cardiomyocytes and primary human fibroblasts. Therefore, it is speculative that Sch B may bring benefit to clinical chemotherapy by reducing significantly the cumulative doses of DOX and its associated cardiotoxicities.

Keywords: Abbreviations; Sch B; Schisandrin B; DOX; doxorubicin; MDR; multidrug resistance; P-gp; P-glycoprotein; MRP1; multidrug resistance related protein 1; FACS; fluorescence-activated cell sorting; MTT; 3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide; RT-PCR; reverse transcription-PCR; PARP; poly(ADP-ribose) polymerase; MMP; mitochondria membrane potentialApoptosis; Doxorubicin; Schisandrin B; Caspase; Cardiomyocytes

Anti-Tat and anti-HIV activities of trimers of n-alkylglycines by Nieves M�rquez; Roc�o Sancho; Antonio Macho; Alejandra Moure; Isabel Masip; Angel Messeguer; Eduardo Mu�oz (pp. 596-604).

Transcription of human immunodeficiency virus (HIV-1) is activated by viral Tat protein which regulates HIV–LTR transcription and elongation. In the present report, the evaluation of the anti-Tat activity of a combinatorial library composed of 5120 N-trialkylglycines is reported. The antiviral activity was studied through luciferase-based assays targeting the HIV-1 promoter activation induced by the HIV-1 Tat protein. We identified five peptoids with specific anti-HIV-1 Tat activity; none of these peptoids affected the binding of HIV-1 Tat protein to the viral TAR RNA. Using a recombinant-virus assay in which luciferase activity correlates with the rate of HIV-1 transcription we have detected that one of the five selected peptoids, NC37-37-15C, is a potent inhibitor of HIV-1–LTR transcription in both primary T lymphocytes and transformed cell lines. The inhibitory effect of NC37-37-15C, which is additive with azidothymidine (AZT), correlates with its ability to inhibit CTD phosphorylation and shows a suitable profile for development of novel anti-HIV-1 drugs. Likewise, the structural simplicity of N-alkylglycine oligomers makes these peptidomimetics amenable to structural manipulation, thus facilitating the optimisation of lead molecules for drug-like properties.

Keywords: Abbreviations; AIDS; acquired immunodeficiency syndrome; AZT; azidothymidine; CDK; cyclin-dependent kinase; CTD; C-terminal domain of the RNA polymerase II; HIV-1; human immunodeficiency virus; LTR; long terminal repeat promoter; Tat; Trans-activator of transcription; TAR; transactivating response element; VSV; vesicular stomatitis virusCombinatorial libraries; Peptoids; CDK; HIV-1; LTR; Tat

Effects of rosiglitazone and atorvastatin on the expression of genes that control cholesterol homeostasis in differentiating monocytes by Gemma Llaverias; Alba Rebollo; Jordi Pou; Manuel V�zquez-Carrera; Rosa M. S�nchez; Juan C. Laguna; Marta Alegret (pp. 605-614).

We studied the effects of 5μM atorvastatin, 2μM rosiglitazone and their combination on intracellular cholesterol levels and on the expression of genes controlling cholesterol trafficking in human monocytes during their differentiation into macrophages. Our results show that treatment with rosiglitazone caused an increase in CD36 mRNA and protein levels (2.7- and 2.9-fold, P<0.001), but significantly induced the expression of most genes related to cholesterol efflux: ABCA1 mRNA (23%, P<0.05) and protein (2.4-fold, P<0.05), apo E protein (2.4-fold, P<0.05), caveolin-1 mRNA (2.6-fold, P<0.001) and SR-BI mRNA (1.9-fold, P<0.001) and protein (3-fold, P<0.01). As a consequence, rosiglitazone treatment reduced intracellular free cholesterol levels by 22% ( P<0.01). Treatment with 5μM atorvastatin caused the opposite effect on the expression of cholesterol efflux-related genes, which was generally reduced: ABCA1 mRNA (71%, P<0.05), apo E mRNA (46%, P<0.001) and protein (5.6-fold, P<0.001), and CYP27 mRNA (15%, P<0.05). Despite these reductions, intracellular total and free cholesterol levels were also reduced by 30% ( P<0.01), an effect that can be attributed to the inhibition of de novo cholesterol synthesis by the statins. The combination of rosiglitazone with atorvastatin attenuated CD36 induction, and caused reductions similar to those caused by the statin alone on the expression of genes involved in cholesterol efflux and on intracellular cholesterol levels.

Keywords: Abbreviations; ABC; ATP-binding cassette transporter; apo E; apolipoprotein E; ATV; atorvastatin; LXR; liver X receptor; oxLDL; oxidized LDL; PPAR; peroxisome proliferator-activated receptor; ROSI; rosiglitazone; SR-A; scavenger receptor class A; SR-BI; scavenger receptor class B type I; TC; total cholesterolMonocytes; Rosiglitazone; Atorvastatin; Cholesterol; CD36; ABCA1; SR-BI

Diethyl pyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH₃ cells by Sheng-Nan Wu; Han-Dong Chang (pp. 615-623).

The ATP-sensitive K⁺ (K_ATP) channels are composed of sulfonylurea receptor and inwardly rectifying K⁺ channel (Kir6.2) subunit. These channels are regulated by intracellular ADP/ATP ratio and play a role in cellular metabolism. Diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, is known to modify the histidine residues of the structure of proteins. The objective of this study was to determine whether DEPC modifies K_ATP-channel activity in pituitary GH₃ cells. Steady-state fluctuation analyses of macroscopic K⁺ current at −120mV produced power spectra that could be fitted with a single Lorentzian curve in these cells. The time constants in the presence of DEPC were increased. Consistent with fluctuation analyses, the mean open time of K_ATP-channels was significantly increased during exposure to DEPC. However, DEPC produced no change in single-channel conductance, despite the ability of this compound to enhance K_ATP-channel activity in a concentration-dependent manner with an EC₅₀ value of 16μM. DEPC-stimulated K_ATP-channel activity was attenuated by pretreatment with glibenclamide. In current–clamp configuration, DEPC decreased the firing of action potentials in GH₃ cells. A further application of glibenclamide reversed DEPC-induced inhibition of spontaneous action potentials. Intracellullar Ca²⁺ measurements revealed the ability of DEPC to decrease Ca²⁺ oscillations in GH₃ cells. Simulation studies also demonstrated that the increased conductance of K_ATP-channels used to mimic DEPC actions reduced the frequency of spontaneous action potentials and fluctuation of intracellular Ca²⁺. The results indicate that chemical modification with DEPC enhances K_ATP-channel activity and influences functional activities of pituitary GH₃ cells.

Keywords: Abbreviations; DEPC; diethyl pyrocarbonate; K; _ATP; -channel; ATP-sensitive K; ⁺; channel; [Ca; ²⁺; ]; _i; intracellular Ca; ²⁺; [Ca; ²⁺; ]; _ER; Ca; ²⁺; in the endoplasmic reticulum; erg; ether-à-go-go-related gene; I–V; current–voltageDiethyl pyrocarbonate (DEPC); K; _ATP; -channel; Action potentials; GH; ₃; cells

14-3-3ζ interacts with human thromboxane receptors and is involved in the agonist-induced activation of the extracellular-signal-regulated kinase by Weili Yan; Yunfei Ding; Hsin-Hsiung Tai (pp. 624-633).

Thromboxane receptor (TP) signaling results in a broad range of cellular responses including kinase activation and subsequent nuclear signaling events involved in cell transformation, proliferation, and cell survival. Proteins that may participate in the early signaling following receptor activation remain to be identified. We found that 14-3-3ζ is a novel protein interacting with TP intracellular loop 3 (i3) by yeast two-hybrid system. This interaction was further confirmed by GST pull-down and co-immunoprecipitation methods. Site-directed mutagenesis studies indicated that Pro-236 of the TP-i3 was involved in the binding to the 14-3-3ζ. Co-immunoprecipitation studies in the same cell lysate by TP antibody showed that TP binds not only with the 14-3-3ζ but also with the Raf-1. Our data also demonstrated that TP receptor activation induced by agonist rapidly recruited 14-3-3ζ and Raf-1 to form a complex with the TP on the plasma membrane. The significance of assembling this protein complex was examined by TP agonist-induced extracellular-signal-regulated kinase (ERK) phosphorylation in intact cells. TP agonist, I-BOP, induced ERK phosphorylation in HEK 293 cells expressing wild type TPα but significantly lower in those expressing TPα-P236V mutant. Attenuation of the expression of 14-3-3ζ by 14-3-3ζ siRNA decreased I-BOP-induced ERK phosphorylation indicating the involvement of the 14-3-3ζ in the signal transduction process. These results suggest that 14-3-3ζ may serve as a scaffold protein to form a protein complex consisting of TP, 14-3-3ζ, and Raf-1, and that this protein complex may be involved in the activation of ERK pathway following TP receptor activation.

Keywords: Abbreviations; BLAST; basic local alignment search tool; ct; carboxyl terminal tail; DMEM; Dulbecco modified Eagle's medium; EGFR; epidermal growth factor receptor; ERK; extracellular-signal-regulated kinase; FBS; fetal bovine serum; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; GPCR; G-protein-coupled receptor; GSH; glutathione; GST; glutathione; S; -transferase; HRP; horseradish perioxidase; i3; intracellular loop 3; I-BOP; [1S-α,2α(Z),3β(1E,3S),4α]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptenoic acid; IP; immunoprecipitation; MAPK; mitogen-activated protein kinases; 15-PGDH; 15-hydroxyprostaglandin dehydrogenase; PMSF; phenylmethylsulfonyl fluoride; PVDF; polyvinylidene fluoride; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TXA; ₂; thromboxane A2; TPα/β; thromboxane A2 receptor α or β form14-3-3ζ; Thromboxane receptor; Raf-1; Signal transduction; ERK

Inhibition of transcription factor NF-κB signaling proteins IKKβ and p65 through specific cysteine residues by epoxyquinone A monomer: Correlation with its anti-cancer cell growth activity by Mei-Chih Liang; Sujata Bardhan; Emily A. Pace; Diana Rosman; John A. Beutler; John A. Porco Jr.; Thomas D. Gilmore (pp. 634-645).

Transcription factor NF-κB is constitutively active in many human chronic inflammatory diseases and cancers. Epoxyquinone A monomer (EqM), a synthetic derivative of the natural product epoxyquinol A, has previously been shown to be a potent inhibitor of tumor necrosis factor-α (TNF-α)-induced activation of NF-κB, but the mechanism by which EqM inhibits NF-κB activation was not known. In this report, we show that EqM blocks activation of NF-κB by inhibiting two molecular targets: IκB kinase IKKβ and NF-κB subunit p65. EqM inhibits TNF-α-induced IκBα phosphorylation and degradation by targeting IKKβ, and an alanine substitution for Cys179 in the activation loop of IKKβ makes it resistant to EqM-mediated inhibition. EqM also directly inhibits DNA binding by p65, but not p50; moreover, replacement of Cys38 in p65 with Ser abolishes EqM-mediated inhibition of DNA binding. Pretreatment of cells with reducing agent dithiothreitol dose-dependently reduces EqM-mediated inhibition of NF-κB, further suggesting that EqM directly modifies the thiol group of Cys residues in protein targets. Modifications of the exocyclic alkene of EqM substantially reduce EqM's ability to inhibit NF-κB activation. In the human SUDHL-4 lymphoma cell line, EqM inhibits both proliferation and NF-κB DNA binding, and activates caspase-3 activity. EqM also effectively inhibits the growth of human leukemia, kidney, and colon cancer cell lines in the NCI's tumor cell panel. Among six colon cancer cell lines, those with low amounts of constitutive NF-κB DNA-binding activity are generally more sensitive to growth inhibition by EqM. Taken together, these results suggest that EqM inhibits growth and induces cell death in tumor cells through a mechanism that involves inhibition of NF-κB activity at multiple steps in the signaling pathway.

Keywords: Abbreviations; Ac-DEVD-AMC; N; -acetyl-Asp-Glu-Val-Asp-AMC (7-amino-4-methylcoumarin); DHMEQ; dehydroxymethylepoxyquinomicin; DISC; death-inducing signaling complex; DMEM; Dulbecco's modified Eagle's medium; DTT; dithiothreitol; EMSA; electrophoretic mobility shift assay; EqM; epoxyquinone A monomer; FBS; fetal bovine serum; FLAG; flu antigen; GI; ₅₀; dose for 50% cell growth inhibition; GST; glutathione; S; -transferase; ID; ₅₀; dose for 50% inhibition; IKKβ; IκB kinase β; JD; jesterone dimer; NCI; National Cancer Institute; NF-κB; nuclear factor-κB; PARP; poly(ADP-ribose) polymerase; PMSF; phenylmethylsulfonyl fluoride; TNF-α; tumor necrosis factor-α; IκBα; inhibitor of κBαNF-kappaB; IkappaB; IkappaB kinase; Epoxyquinone A monomer; Fungal metabolite; Epoxyquinoid

Niacin induces PPARγ expression and transcriptional activation in macrophages via HM74 and HM74a-mediated induction of prostaglandin synthesis pathways by Helen J. Knowles; Robert Te Poole; Paul Workman; Adrian L. Harris (pp. 646-656).

HM74 and HM74a have been identified as receptors for niacin. HM74a mediates the pharmacological anti-lipolytic effects of niacin in adipocytes by reducing intracellular cyclic AMP (cAMP) and inhibiting release of free fatty acids into the circulation. In macrophages, niacin induces peroxisome proliferator-activated receptor γ (PPARγ)-dependent and cAMP-dependent expression of genes mediating reverse cholesterol transport, although via an unidentified receptor. We describe constitutive expression of HM74a mRNA and hypoxia- and IFNγ-inducible expression of HM74 and HM74a in human monocytic cell lines and primary cells in culture. In U937 cells niacin-induced expression of 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂), the most potent endogenous ligand of PPARγ. Both niacin and the structurally distinct HM74/HM74a ligand acifran-induced nuclear expression of PPARγ protein and enhanced PPARγ transcriptional activity. Niacin-induced PPARγ transcriptional activity was pertussis toxin sensitive and required activity of phospholipase A₂ (EC 3.1.1.4), cyclo-oxygenase (EC 1.14.99.1) and prostaglandin D₂ synthase (EC 5.3.99.2). Niacin also induced PPARγ transcriptional activity in HM74 and HM74a CHO cell transfectants, although not in vector-only control cells. This was sensitive to pertussis toxin and to inhibition of phoshoplipase A₂ and cyclo-oxygenase activity. Additionally, niacin increased intracellular cAMP in U937 via a pertussis toxin and cyclo-oxygenase-sensitive mechanism. These results indicate that HM74 and HM74a can mediate macrophage responses to niacin via activation of the prostaglandin synthesis pathway and induction and activation of PPARγ. This suggests a novel mechanism(s) mediating the clinical effects of pharmacological doses of niacin.

Keywords: HM74a; Niacin; Macrophage; PPARγ; Prostaglandin; Cyclic AMP

Hydrolytic metabolism of pyrethroids by human and other mammalian carboxylesterases by Matthew K. Ross; Abdolsamad Borazjani; Carol C. Edwards; Philip M. Potter (pp. 657-669).

Pyrethroid chemicals are attractive alternatives to the organophosphates (OPs) because of their selective toxicity against pests rather than mammals. The carboxylesterases (CEs) are hepatic enzymes that metabolize ester-containing xenobiotics such as pyrethroids. The primary aim of this study was to gain insight into the catalytic properties of the CE enzymes in humans that metabolize pyrethroids, while a secondary aim was to investigate pyrethroid metabolism using CEs from other mammalian species. Pure human CEs (hCE-1 and hCE-2), a rabbit CE (rCE), and two rat CEs (Hydrolases A and B) were used to study the hydrolytic metabolism of the following pyrethroids: 1 R trans-resmethrin (bioresmethrin), 1 RS trans-permethrin, and 1 RS cis-permethrin. hCE-1 and hCE-2 hydrolyzed trans-permethrin 8- and 28-fold more efficiently than cis-permethrin (when k_cat/ K_m values were compared), respectively. In contrast, hydrolysis of bioresmethrin was catalyzed efficiently by hCE-1, but not by hCE-2. The kinetic parameters for the pure rat and rabbit CEs were qualitatively similar to the human CEs when hydrolysis rates of the investigated pyrethroids were evaluated. Further, a comparison of pyrethroid hydrolysis by hepatic microsomes from rats, mice, and humans indicated that the rates for each compound were similar between species, which further supports the use of rodent models for pyrethroid metabolism studies. An eight-fold range in hydrolytic rates for 11 individual human liver samples toward trans-permethrin was also found, although this variability was not related to the levels of hCE-1 protein in each sample. We also determined that the CE inhibitor 2-chloro-3,4-dimethoxybenzil blocked hCE-2-catalyzed trans-permethrin hydrolysis 36 times more potently than hCE-1. Thus, this inhibitor will be useful in future studies that examine CE-mediated metabolism of pyrethroids. While there are likely other esterases in human liver that hydrolyze pyrethroids, the results of this study clearly demonstrate that hCE-1 and hCE-2 are human pyrethroid-hydrolyzing CEs.

Keywords: Abbreviations; CDMB; 2-chloro-3,4-dimethoxybenzil; hCE-1; human carboxylesterase 1; hCE-2; human carboxylesterase 2; rCE; rabbit carboxylesterase; alpha-Cypermethrin; α; RS; -cyano-(3-phenoxyphenyl)methyl (1; RS; )-; cis; -3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropanecarboxylate; deltamethrin; α; S; -cyano-(3-phenoxyphenyl)methyl (1; R; )-; cis; -3-(2,2-dibromovinyl)-2,2-dimethyl-cyclopropanecarboxylate; trans; -permethrin; 3-phenoxbenzyl (1; RS; )-; trans; -3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate; cis; -permethrin; 3-phenoxbenzyl (1; RS; )-; cis; -3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropanecarboxylate; bioresmethrin; (5-benzyl-3-furyl)methyl (1; R; )-; trans; -2,2-dimethyl-3-(2-methylprop-1-enyl)-cyclopropanecarboxylate; 4-MUBA; 4-methyl umbelliferyl acetate; p; -NPA; para; -nitrophenyl acetate; p; -NPV; para; -nitrophenyl valerate; p-; NPB; para; -nitrophenyl valerate; p; -NPB; para; -nitrophenyl butyrate; 3PBAlc; 3-phenoxybenzyl alcohol; Cl; ₂; CA; cis; /; trans; -3-(2’,2’-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid; 3PBAld; 3-phenoxybenzaldehyde; hAChE; human acetylcholinesterase; hBChE; human butyrylcholinesteraseCarboxylesterases; Pyrethroids; Human metabolism

N-(4-Trifluoromethylphenyl)amide group of the synthetic histamine receptor agonist inhibits nicotinic acetylcholine receptor-mediated catecholamine secretion by Dong-Chan Kim; Yong-Soo Park; Dong-Jae Jun; Eun-Mi Hur; Sun-Hee Kim; Bo-Hwa Choi; Kyong-Tai Kim (pp. 670-682).

The therapeutic targeting of nicotinic receptors requires the identification of drugs that selectively activate or inhibit a limited range of nicotine acetylcholine receptors (nAChRs). In this study, we identified N-(4-trifluoromethylphenyl)amide group of the synthetic histamine receptor ligands, histamine-trifluoromethyltoluide, that act as potent inhibitors of nAChRs in bovine adrenal chromaffin cells. Catecholamine secretion induced by the nAChRs agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), was significantly inhibited by histamine-trifluoromethyltoluide. Real time carbon-fiber amperometry confirmed the ability of histamine-trifluoromethyltoluide to inhibit DMPP-induced exocytosis in single chromaffin cells. We also found that histamine-trifluoromethyltoluide inhibited DMPP-induced [Ca²⁺]_i and [Na⁺]_i increases, as well as DMPP-induced inward currents in the absence of extracellular calcium. Histamine-trifluoromethyltoluide had no effect on [³H]nicotine binding or on calcium increases induced by high K⁺, bradykinin, veratridine, histamine, and benzoylbenzoyl ATP. Among the synthetic histamine receptor ligands, clobenpropit exhibited similarity. In addition, 4′-nitroacetanilide also significantly attenuated nAChR-mediated catecholamine secretion. In conclusion, the N-(4-trifluoromethylphenyl)amide group of the histamine-trifluoromethyltoluide might be the critical moiety in the inhibition of nAChR-mediated CA secretion.

Keywords: Abbreviations; CA; catecholamine; DMPP; 1,1-dimethyl-4-phenylpiperazinium iodide; EP; epinephrine; nAChR; nicotinic acetylcholine receptor; NA; norepinephrine; VSCC; voltage-sensitive calcium channel; VSSC; voltage-sensitive sodium channelCatecholamine; DMPP; Ca; ²⁺; influx; Histamine-trifluoromethyltoluide; Nicotinic acetylcholine receptor; Chromaffin cells

Binding kinetics and duration of in vivo action of novel prolyl oligopeptidase inhibitors by Jarkko I. Ven�l�inen; J. Arturo Garcia-Horsman; Markus M. Forsberg; Aaro Jalkanen; Erik A.A. Wall�n; Elina M. Jarho; Johannes A.M. Christiaans; Jukka Gynther; Pekka T. M�nnist� (pp. 683-692).

Prolyl oligopeptidase (POP) is a serine protease that specifically hydrolyses small peptides at the carboxyl end of the proline residue. POP has gained pharmaceutical interest, since its inhibitors have been shown to have antiamnesic properties in rat. We examined the effect of the 2( S)-substituents CN and COCH₂OH at the P1 site of the parent inhibitors isophthalic acid 2( S)-(cyclopentanecarbonyl)pyrrolidine-l-prolyl-pyrrolidine amide and 4-phenylbutanoyl-l-prolyl-pyrrolidine and bulky 5- t-butyl group at the P2 sitel-prolyl residue of the parent inhibitor 4-phenylbutanoyl-l-prolyl-pyrrolidine on the binding kinetics to the enzyme. In addition, we studied the duration of POP inhibition in the rat tissues in vivo after i.p. administration. CN and COCH₂OH substituents at the P1 site pyrrolidine group were found to greatly increase the affinity of the inhibitor and the enzyme–inhibitor complex half-life. In addition, 5- t-butyl group at the P2 sitel-prolyl residue increased the dissociation half-life of the enzyme–inhibitor complex, without much affecting the inhibitory potency. The duration of the inhibition in the rat tissues followed the inhibition kinetic properties in that the compounds with fast dissociation produced shorter inhibition in the rat tissues than the compounds with slow dissociation. The duration of POP inhibition of compounds was evidently not governed by their serum clearance. The fact that the in vivo pharmacodynamic behaviour of POP inhibitors can be predicted by their in vitro-properties may be of importance when designing therapeutically useful POP inhibitors.

Keywords: Prolyl oligopeptidase; Inhibitor; Tight binding inhibition; Slow binding inhibition; Pharmacokinetics

Proximal HNF1 element is essential for the induction of human UDP-glucuronosyltransferase 1A1 by glucocorticoid receptor by Toru Usui; Takuya Kuno; Hisao Ueyama; Iwao Ohkubo; Takaharu Mizutani (pp. 693-701).

Previous study showed noinduction of the reporter gene (−3174/+14) of UGT1A1 in HepG2 by bilirubin, but induction by dexamethasone (DEX). This induction was enhanced seven-fold by the co-expression of human glucocorticoid receptor (GR) and was inhibited by a GR antagonist, RU486, indicating stimulation by DEX-GR. Meanwhile, we could not detect stimulation by β-estradiol, phenobarbital or rifampicin (RIF) in the presence of GR. We investigated the position playing a role in this induction by GR in the promoter region of UGT1A1 using deletion mutants, and clarified the essential sequence (−75/−63) for the binding site of hepatocyte nuclear factor 1 (HNF1). However, GR did not bind directly to this sequence, because UGT-PE2 did not compete for binding to a glucocorticoid responsive element (GRE) probe in an electrophoretic mobility shift assay (EMSA) method. Labeled [³²P]DNA probe of HNF1 binds with nuclear extracts as shown by the EMSA. This shift of the complex of probe–protein was not inhibited by unlabeled GRE but was inhibited by unlabeled HNF1 element. This shift was not influenced by the addition of anti-GR, but was super-shifted by the addition of anti-HNF1. GR did not stimulate the induction of HNF1, because we detected no-elevation of the mRNA level of HNF1 by reverse transcription-polymerase chain reaction (RT-PCR). Therefore, the induction of UGT1A1 by DEX-GR did not depend on the elevation of HNF1 but on the interaction of GR with HNF1 or the activation of HNF1 through the transcription of other proteins. Also given the lack of evidence of binding of DEX-GR to HNF1 in the EMSA, the data suggest that the mechanism of DEX-GRE effect on HNF1 is indirect by whatever mechanisms.

Keywords: Abbreviations; UGT; UDP-glucuronosyltransferase; PB; phenobarbital; PBREM; PB response enhancer module; DE; distal element; DEX; dexamethasone; GR; human glucocorticoid receptor; GRE; glucocorticoid responsive element; CAR; constitutive androstane receptor; PXR; human pregnenolone xenobiotic receptor; HNF; hepatocyte nuclear factor; BIL; bilirubin; RIF; rifampicin; NE; nuclear extracts; PE; proximal element; PCN; pregnenolone 16α-carbonitrile; RT-PCR; reverse transcription-polymerase chain reactionDexamethasone; UGT1A1; Bilirubin; Glucocorticoid receptor; Hepatocyte nuclear factor; Pregnenolone xenobiotic receptor

Inhibition of RNAse A family enzymes prevents degradation and loss of silencing activity of siRNAs in serum by J�rg Haupenthal; Christina Baehr; Simone Kiermayer; Stefan Zeuzem; Albrecht Piiper (pp. 702-710).

Small interfering RNAs (siRNA), RNA duplexes of ∼21 nucleotides, offer a promising approach to specifically degrade RNAs in target cells by a process termed RNA interference. Insufficient in vivo-stability is a major problem of a systemic application of siRNAs in humans. The present study demonstrated that RNAse A-like RNAses degraded siRNAs in serum. The susceptibility of siRNAs towards degradation in serum was strongly enhanced by local clustering of A/Us within the siRNA sequence, i.e. regions showing low thermal stability, most notably at the ends of the molecule, and by 3′-overhanging bases. Importantly, inhibition of RNAse A family enzymes prevented the degradation and loss of silencing activity of siRNAs in serum. Furthermore, the degradation of siRNAs was considerably faster in human than in mouse serum, suggesting that the degradation of siRNAs by RNAse A family enzymes might be a more challenging problem in a future therapeutic application of siRNAs in humans than in mouse models. Together, the present study indicates that siRNAs are degraded by RNAse A family enzymes in serum and that the kinetics of their degradation in serum depends on their sequence. These findings might be of great importance for a possible future human therapeutic application of siRNAs.

Keywords: siRNA; Double-stranded RNA; Serum; RNAi; RNAse A

Corrigendum to “A comparison of the cyclooxygenase inhibitor-NO donors (CINOD), NMI-1182 and AZD3582, using in vitro biochemical and pharmacological methods�? [Biochem. Pharmacol. 70/9 (2005) 1343–1351] 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. 711-711).

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Biochemical Pharmacology (v.71, #5)