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Biochemical Pharmacology (v.73, #10)

Editorial Advisory Board (pp. iii).

New paradigms in signal transduction by Kathleen M. Eyster (pp. 1511-1519).

Signal transduction is a dynamic field in which established pathways evolve and new pathways emerge. The purpose of this commentary is to highlight new paradigms of signal transduction that have developed over the past few years. This discussion proposes a third member of the generic models of membrane receptors in addition to the 7-transmembrane pass receptor and the enzyme-linked receptor: the non-enzymatic nucleating receptor. Also discussed are the new paradigms of signal transduction by proteolysis which includes signaling by Notch, signaling through the Hedgehog and Wnt pathways, signaling through histidine phosphorylation, and reactive oxygen species in signal transduction.

Keywords: Nucleating receptor; Signaling by proteolysis; Hedgehog; Wnt; Histidine kinase; Reactive oxygen species

Clotrimazole inhibits and modulates heterologous association of the key glycolytic enzyme 6-phosphofructo-1-kinase by Patricia Zancan; Alicia O. Rosas; Mariah C. Marcondes; Monica M. Marinho-Carvalho; Mauro Sola-Penna (pp. 1520-1527).

Clotrimazole is an antifungal azole derivative recently recognized as a calmodulin antagonist with promising anticancer effects. This property has been correlated with the ability of the drug to decrease the viability of tumor cells by inhibiting their glycolytic flux and consequently decreasing the intracellular concentration of ATP. The effects of clotrimazole on cell glycolysis and ATP production are considered to be due to the detachment of the glycolytic enzymes from the cytoskeleton. Here, we show that clotrimazole directly inhibits the key glycolytic enzyme 6-phosphofructo-1-kinase (PFK). This property is independent of the anti-calmodulin activity of the drug, since it is not mimicked by the classical calmodulin antagonist compound 48/80. However, the clotrimazole-inhibited enzyme can be activated by calmodulin, even though calmodulin has no effect on PFK activity in the absence of the drug. Clotrimazole alone induces the dimerization of PFK reducing the population of tetramers, which is not observed when calmodulin is also present. Since PFK dimers are less active than PFK tetramers, this can explain the inhibitory effect of clotrimazole on the enzyme. Additionally, clotrimazole positively modulates the association of PFK with erythrocyte membranes. Altogether, our data support a hitherto unrecognized action of clotrimazole as a negative modulator of glycolytic flux through direct inhibition of the key enzyme PFK.

Keywords: Abbreviations; CaM; calmodulin; CTZ; clotrimazole; PFK; 6-phosphofructo-1-kinase; phosphofructokinase; 48/80; compound 48/80Phosphofructokinase; Inhibition; Cytoskeleton; Erythrocyte; Membrane; Glycolysis; Calmodulin

Geldanamycin induces G2 arrest in U87MG glioblastoma cells through downregulation of Cdc2 and cyclin B1 by Naoko Nomura; Motohiro Nomura; Elizabeth W. Newcomb; David Zagzag (pp. 1528-1536).

Cell cycle progression requires precise expression and activation of several cyclins and cyclin-dependent kinases. Geldanamycin (GA) affects cell cycle progression in various kinds of cells. We analyzed GA-induced cell cycle regulation in glioblastoma cells. GA-induced G2 or M arrest in glioblastoma cells in a cell line-dependent manner. GA decreased the expression of Cdc2 and cyclin B1 in U87MG cells. And phosphorylated Cdc2 decreased along with Cdc2 in the GA-treated cells. This cell line showed G2 arrest after GA treatment. In contrast, GA failed to down-regulate these cell cycle regulators in U251MG cells. In U251MG cells, the cell cycle was arrested at M phase in addition to G2 by GA. Next, we analyzed the mechanism of the GA-induced regulation of Cdc2 and cyclin B1 in U87MG cells. Cdc2 and cyclin B1 were ubiquitinated by GA. MG132 abrogated the GA-induced decrease of Cdc2 and cyclin B1 indicating that these proteins were degraded by proteasomes. In conclusion, GA controls the stability of Cdc2 and cyclin B1 in glioblastomas cell species-dependently. Cdc2 and cyclin B1 might be responsible for the different responses of glioblastoma cell lines to GA.

Keywords: Abbreviations; Hsp90; heat shock protein 90; GA; geldanamycin; CHX; cycloheximide; DMSO; dimethyl sulfoxide; DMEM; Dulbecco's modified Eagle medium; PI; propidium iodide; FACS; fluorescence-activated cell sorter; Tyr 15; tyrosine 15; Thr 14; threonine 14Cell cycle arrest; G2/M; Cdc2; Cyclin B1; Glioblastoma; Geldanamycin

Trypanocidal nitroimidazole derivatives: Relationships among chemical structure and genotoxic activity by Annamaria Buschini; Federica Giordani; Cristina Northfleet de Albuquerque; Claudia Pellacani; Giorgio Pelosi; Carlo Rossi; Tânia Maria Araújo Domingues Zucchi; Paola Poli (pp. 1537-1547).

Human American trypanosomiasis is resurgent in Latin Americans, and new drugs are urgently required as current medications suffer from a number of drawbacks. Some nitroheterocycles have been demonstrated to exert a potent activity against trypanosomes. However, host toxicity issues halted their development as trypanocides. As part of the efforts to develop new compounds in order to treat parasitic infections, it is important to define their structure–activity relationship. In this study, 5-nitromegazol and two of its analogues, 4-nitromegazol, and 1-methyl-5-nitro-2-imidazolecarboxaldehyde 5-nitroimidazole-thiosemicarbazone, were tested and compared for in vitro induction of DNA damage in human leukocytes by the comet assay, performed at different pHs to better identify the types of damage. Specific oxidatively generated damage to DNA was also measured by using the comet assay with endonucleases. DNA damage was found in 5-nitromegazol-treated cells: oxidative stress appeared as the main source of DNA damage. 4-Nitromegazol did not produce any significant effect, thus confirming that 4-nitroimidazoles isomers have no important biological activity. The 5-nitroimidazole-thiosemicarbazone induced DNA damage with a higher efficiency than 5-nitromegazol. The central role in the reduction process played by the acidic hydrazine proton present in the thiosemicarbazone group but not in the cyclic (thiadiazole) form can contribute to rationalise our results. Given its versatility, thiosemicarbazone moiety could be involved in different reactions with nitrogenous bases (nucleophilic and/or electrophilic attacks).

Keywords: Abbreviations; FPG; formamidopyrimidine glycosylase; ENDOIII; endonuclease III; ALS; alkali-labile site; SB; strand break; TL; total lengthComet assay; Oxidatively damaged DNA; 5-Nitromegazol; Benznidazole; Thiosemicarbazone; Thiadiazole-5-nitrofuran

The synergistic interaction of gemcitabine and cytosine arabinoside with the ribonucleotide reductase inhibitor triapine is schedule dependent by J. Sigmond; J.A.E. Kamphuis; A.C. Laan; E.K. Hoebe; A.M. Bergman; G.J. Peters (pp. 1548-1557).

Gemcitabine and ara-C have multiple mechanisms of action: DNA incorporation and for gemcitabine also ribonucleotide reductase (RNR) inhibition. Since dCTP competes with their incorporation into DNA, dCTP depletion can potentiate their cytotoxicity. We investigated whether additional RNR inhibition by Triapine (3-AP), a new potent RNR inhibitor, enhanced cytotoxicity of gemcitabine and ara-C in four non-small-cell-lung-cancer (NSCLC) cell lines, using the multiple-drug-effect analysis.Simultaneous and sequential exposure (preexposure to 3-AP for 24h) in a constant molar ratio of 3-AP and gemcitabine was antagonistic/additive in all cell lines. Preexposure to 3-AP at an IC₂₅ concentration for 24h before variable concentrations of gemcitabine was synergistic. RNR inhibition by 3-AP resulted in a more synergistic interaction in combination with ara-C, which does not inhibit RNR.Two cell lines with pronounced synergism (SW1573) or antagonism (H460) for gemcitabine/3-AP, were evaluated for accumulation of the active metabolites, dFdCTP and ara-CTP. Simultaneous exposure induced no or a small increase, but ara-CTP increased after pretreatment with 3-AP, 4-fold in SW1573 cells, but not in H460 (<1.5 fold). Ara-C and gemcitabine incorporation into DNA were more pronounced (about 2-fold increased) for sequential treatment in SW1573 compared to H460 cells (<1.5 fold). This was not related to the activity and expression of deoxycytidine kinase and the M2 subunit of RNR.In conclusion, RNR inhibition by 3-AP prior to gemcitabine or ara-C exposure stimulates accumulation of the active metabolites and incorporation into DNA. The combination 3-AP/Ara-C is more synergistic than 3-AP/gemcitabine possibly because gemcitabine already inhibits RNR, but ara-C does not.

Keywords: Gemcitabine; Cytosine arabinoside; Triapine; Synergistic interaction; Ribonucleotide reductase; Deoxycytidine kinaseAbbreviations; dFdC; gemcitabine (2′,2′-difluorodeoxycytidine; Gemzar); Ara-C; cytosine arabinoside; dCK; deoxycytidine kinase; dFdCDP; gemcitabine diphosphate; dFdCTP; gemcitabine triphosphate; dCTP; deoxycytidine triphosphate; RNR; ribonucleotide reductase; dNTP; deoxynucleoside triphosphate; dCDA; deoxycytidine deaminase; dCMPD; deoxycytidylate deaminase; HU; hydroxyurea; HCT; a-(; N; )-heterocyclic carboxaldehyde thiosemicarbazone; Fe; iron; 5-HP; 5-hydroxypyridine-2-carboxaldehyde thiosemicarbazone; 3-AP; Triapine; 3-aminopyridine-2-carboxaldehyde thiosemicarbazone; HEPES; 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid; FCS; fetal calf serum; HBSS; Hank's balanced salt solution; TCA; trichloroacetic acid; SRB; sulforhodamine-B; CI; Combination Index; dATP; deoxyadenosine triphosphate; dGTP; deoxyguanosine triphosphate; dTTP; deoxythymidine triphosphate

Synthesis, biological evaluation and molecular modeling studies of N⁶-benzyladenosine analogues as potential anti-toxoplasma agents by Young Ah Kim; Ashoke Sharon; Chung K. Chu; Reem H. Rais; Omar N. Al Safarjalani; Fardos N.M. Naguib; Mahmoud H. el Kouni (pp. 1558-1572).

Toxoplasma gondii is an opportunistic pathogen responsible for toxoplasmosis. T. gondii is a purine auxotroph incapable of de novo purine biosynthesis and depends on salvage pathways for its purine requirements. Adenosine kinase (EC.2.7.1.20) is the major enzyme in the salvage of purines in these parasites. 6-Benzylthioinosine and analogues were established as “subversive substrates” for the T. gondii, but not for the human adenosine kinase. Therefore, these compounds act as selective anti-toxoplasma agents. In the present study, a series of N⁶-benzyladenosine analogues were synthesized from 6-chloropurine riboside with substituted benzylamines via solution phase parallel synthesis. These N⁶-benzyladenosine analogues were evaluated for their binding affinity to purified T. gondii adenosine kinase. Furthermore, the anti-toxoplasma efficacy and host toxicity of these compounds were tested in cell culture. Certain substituents on the aromatic ring improved binding affinity to T. gondii adenosine kinase when compared to the unsubstituted N⁶-benzyladenosine. Similarly, varying the type and position of the substituents on the aromatic ring led to different degrees of potency and selectivity as anti-toxoplasma agents. Among the synthesized analogues, N⁶-(2,4-dimethoxybenzyl)adenosine exhibited the most favorable anti-toxoplasma activity without host toxicity. The binding mode of the synthesized N⁶-benzyladenosine analogues were characterized to illustrate the role of additional hydrophobic effect and van der Waals interaction within an active site of T. gondii adenosine kinase by induced fit molecular modeling.

Keywords: Toxoplasma gondii; adenosine kinase; Toxoplasmosis; Chemotherapy; N; ⁶; -Benzyladenosine analogues; Molecular modeling

Comparison of the inhibitory activity of anti-HIV drugs on P-glycoprotein by Caroline Henrike Storch; Dirk Theile; Heike Lindenmaier; Walter Emil Haefeli; Johanna Weiss (pp. 1573-1581).

Human immunodeficiency virus 1 (HIV-1) infections are treated with HIV-protease inhibitors (PIs), nucleoside (NRTIs), non-nucleoside (NNRTIs), and nucleotide reverse transcriptase inhibitors (NtRTIs). The combined administration of antiretrovirals improves patient outcomes while increasing the likelihood of drug interactions. Indeed, as substrates, inhibitors, and occasionally also inducers of P-glycoprotein (P-gp) PIs may substantially alter the pharmacokinetics of co-administered drugs. However, the P-gp inhibitory potencies specified in the numerous publications are not comparable, because they were determined with different assays and cell lines. Moreover, data on the interaction of other anti-HIV drugs with P-gp are sparse and conflicting. We therefore aimed to clarify, which anti-HIV drugs inhibit P-gp and to compare the inhibitory potencies using two independent standard methods (calcein uptake assay, flow cytometric rhodamine123 efflux assay). In the calcein assay, all PIs, all NNRTIs, abacavir, and tenofovir disoproxil fumarate acted as P-gp inhibitors with largely differing potencies between compounds. In P388/dx cells the ranking order of inhibition was: nelfinavir>ritonavir>tipranavir>lopinavir>quinidine (positive control)>delavirdine>saquinavir>amprenavir>atazanavir>efavirenz>nevirapine>abacavir>tenofovir disoproxil fumarate. In conclusion this is the first study to provide comprehensive information on the P-gp interaction profile of anti-HIV drugs under identical assay conditions. Our study reveals that many compounds may indeed inhibit P-gp substantially and further indicates that of the various systems tested, the calcein assay in P388/dx/P388 cells is the most suitable and reliable in vitro model for the quantification of P-gp inhibition.

Keywords: Abbreviations; BCRP; breast cancer resistance protein; CYP; cytochrome P450 isozyme; f; 2; concentration needed to increase baseline fluorescence by factor 2; FCS; foetal calf serum; FTC; fumitremorgin C; HAART; highly active antiretroviral therapy; LY335979; zosuquidar; HBSS; Hanks’ balanced salt solution; HHBSS; Hanks’ balanced salt solution with 1% HEPES; HIV; human immunodeficiency virus; HIV-1; human immunodeficiency virus 1; PI; HIV protease inhibitor; MF; median fluorescence; MRP; multidrug resistance associated protein; NNRTI; non-nucleoside reverse transcriptase inhibitor; NRTI; nucleoside reverse transcriptase inhibitor; NtRTI; nucleotide reverse transcriptase inhibitor; pBCECs; porcine brain capillary endothelial cells; P-gp; P-glycoprotein; S.E.M.; standard error of the mean; tenofovir DF; tenofovir disoproxil fumarateP-glycoprotein; Protease inhibitors; NRTIs; NNRTIs; Calcein assay; Rhodamine123 assay

AT₁ receptor blocker-insensitive mutant AT_1A angiotensin receptors reveal the presence of G protein-independent signaling in C9 cells by László Szidonya; Katinka Süpeki; Eszter Karip; Gábor Turu; Péter Várnai; Adrian J.L. Clark; László Hunyady (pp. 1582-1592).

Although mutant receptors are highly useful to dissect the signal transduction pathways of receptors, they are difficult to study in physiological target tissues, due to the presence of endogenous receptors. To study AT₁ angiotensin receptors in their physiological environment, we constructed a mutant receptor, which differs only from the AT_1A receptor in its reduced affinity for candesartan, a biphenylimidazole antagonist. We have determined that the conserved S109Y substitution of the rat AT_1A receptor eliminates its candesartan binding, without exerting any major effect on its angiotensin II and peptide angiotensin receptor antagonist binding, internalization kinetics, β-arrestin binding, and potency or efficacy of the inositol phosphate response. To demonstrate the usefulness of this mutant receptor in signal transduction studies, we combined it with substitution of the highly conserved DRY sequence with AAY, which abolishes G protein activation. In rat C9 hepatocytes the S109Y receptor caused ERK activation with the same mechanism as the endogenous AT₁ receptor. After combination with the DRY/AAY mutation G protein-independent ERK activation was detected demonstrating that this approach can be used to study the angiotensin II-stimulated signaling pathways in cells endogenously expressing AT₁ receptors.

Keywords: Abbreviations; Ang II; angiotensin II; AT; ₁; -R; type I angiotensin receptor; AT; _1A; -R; type 1A angiotensin receptor; AT; _1B; -R; type 1B angiotensin receptor; MAP kinase; mitogen-activated protein kinase; ERK; extracellular signal-regulated kinase; TM; transmembrane domain; DMEM; Dulbecco's modified Eagle's medium; BRET; bioluminescence resonance energy transfer; eYFP; enhanced yellow fluorescent protein; eGFP; enhanced green fluorescent protein; SI-Ang II; [Sar; ¹; ,Ile; ⁸; ]Ang II; Rhod-Ang II; rhodamine-conjugated Ang II; Rluc; Renilla luciferase; EGF-R; epidermal growth factor receptor; PKC; protein kinase C; PI3K; phosphoinositide 3-kinaseAngiotensin receptor; BRET; Calcium signal; Candesartan; G protein; Receptor internalization

Inhibition of ADP-induced platelet adhesion to immobilised fibrinogen by nitric oxide: Evidence for cGMP-independent mechanisms by Nikolaus G. Oberprieler; Wayne Roberts; Anne M. Graham; Shervanthi Homer-Vanniasinkam; Khalid M. Naseem (pp. 1593-1601).

Nitric oxide (NO) is an established regulator of platelet function, although the processes by which NO modulates platelet adhesion are unclear. We studied the importance of Ca²⁺ and phosphoinositol-3-kinase (PI3kinase) as targets for NO signalling, in the physiological context of platelet adhesion using adenosine diphosphate (ADP)-stimulated adhesion to immobilised fibrinogen. DPTA-NONOate induced a time and concentration-dependent inhibition of adhesion, and reduced protein tyrosine phosphorylation. The action of NO was cGMP-independent despite activation of the cGMP-signalling cascade, as evidenced by VASP phosphorylation. Furthermore, the cGMP-independent mechanism did not involve PKA. Platelet activation by ADP requires Ca²⁺ and PI3kinase-dependent signalling pathways. We examined the effect of NO on these pathways using two approaches. Firstly, we dissected the signalling pathways using the P2Y₁-receptor antagonist A3P5P, and secondly, directly inhibited Ca²⁺ mobilisation and PI3kinase activity. ADP-induced adhesion was reduced but not abolished by A3P5P, suggesting signalling from P2Y₁₂ can induce adhesion. NO further reduced adhesion in the presence of A3P5P, indicating that NO inhibited adhesion independently of any effects on Ca²⁺ mobilisation. Dimethyl bis-( o-aminophenoxy) ethane-tetraacetic acid (BAPTA) and wortmannin both partially inhibited ADP-induced adhesion, but completely abolished adhesion when used in combination, demonstrating that ADP-induced adhesion requires Ca²⁺ and PI3kinase-regulated pathways. Combination of either dimethyl-BAPTA or wortmannin with DPTA-NONOate enhanced inhibition of both the Ca²⁺ and PI3kinase-dependent pathways when compared to the levels of inhibition with either agent alone. Thus, we demonstrate that NO inhibits α_IIbβ₃-mediated adhesion, by targeting both Ca²⁺ and PI3kinase pathways in a cGMP-independent manner.

Keywords: Nitric oxide; Platelets; ADP; cGMP; Integrin α; _IIb; β; ₃

Isoliquiritigenin inhibits IκB kinase activity and ROS generation to block TNF-α induced expression of cell adhesion molecules on human endothelial cells by Sarvesh Kumar; Amit Sharma; Babita Madan; Vandana Singhal; Balaram Ghosh (pp. 1602-1612).

Isoliquiritigenin (ILTG) is a flavonoid with chalcone structure (4,2′,4′-trihydroxychalcone), an active component present in plants like Glycyrrhiza and Dalbergia which showed various biological activities including anti-inflammatory, anti-carcinogenic and antihistamic. As very little is known in regard to the underlying mechanism involved in explaining the various activities of the compound, we carried out a detailed study on the effect of ILTG on the expression of cell adhesion molecules on human primary endothelial cells. We demonstrate here that ILTG inhibits TNF-α induced adhesion of neutrophils to endothelial monolayer by blocking the expression of ICAM-1, VCAM-1 and E-selectin. Since NF-κB is a major transcription factor involved in the transcriptional regulation of cell adhesion molecules, thus we studied the status of NF-κB activation in ILTG treated endothelial cells. We demonstrate that ILTG inhibits the translocation and activation of nuclear factor-κB (NF-κB) by blocking the phosphorylation and subsequent degradation of IκBα. As oxidative stress is also known to regulate the activation of NF-κB to modulate TNF-α signaling cascade, we tested the effect of ILTG on reactive oxygen species (ROS). We found that it inhibits TNF-α induced ROS production in endothelial cells. These results have important implications for using ILTG or its derivatives towards the development of effective anti-inflammatory molecules.

Keywords: Abbreviations; CAMs; cell adhesion molecules; ICAM-1; intercellular adhesion molecule-1; VCAM-1; vascular cell adhesion molecule-1; TNF-α; tumor necrosis factor-α; NF-κB; nuclear factor-κB; EMSA; electrophoretic mobility shift assay; HUVECs; human umbilical cord vein endothelial cells; NEMO; NF-κB essential modulatorCell adhesion molecules; Endothelial cells; IκBα; Isoliquiritigenin; NF-κB; ROS

Glutathione depletion and recovery after acute ethanol administration in the aging mouse by Barbara L. Vogt; John P. Richie Jr. (pp. 1613-1621).

Glutathione (GSH) plays an important role in the detoxification of ethanol (EtOH) and acute EtOH administration leads to GSH depletion in the liver and other tissues. Aging is also associated with a progressive decline in GSH levels and impairment in GSH biosynthesis in many tissues. Thus, the present study was designed to examine the effects of aging on EtOH-induced depletion and recovery of GSH in different tissues of the C57Bl/6NNIA mouse. EtOH (2–5g/kg) or saline was administered i.p. to mice of ages 6 months (young), 12 months (mature), and 24 months (old); and GSH and cyst(e)ine concentrations were measured 0–24h thereafter. EtOH administration (5g/kg) depleted hepatic GSH levels >50% by 6h in all animals. By 24h, levels remained low in both young and old mice, but recovered to baseline levels in mature mice. At 6h, the decrease in hepatic GSH was dose-dependent up to 3g/kg EtOH, but not at higher doses. The extent of depletion at the 3g/kg dose was dependent upon age, with old mice demonstrating significantly lower GSH levels than mature mice ( P<0.001). Altogether these results indicate that aging was associated with a greater degree of EtOH and fasting-induced GSH depletion and subsequent impaired recovery in liver. An impaired ability to recover was also observed in young animals. Further studies are required to determine if an inability to recover from GSH depletion by EtOH is associated with enhanced toxicity.

Keywords: Ethanol; Aging; Glutathione; Mouse

β-Naphthoflavone and 3′-methoxy-4′-nitroflavone exert ambiguous effects on Ah receptor-dependent cell proliferation and gene expression in rat liver ‘stem-like’ cells by Jiřina Zatloukalová; Lenka Švihálková-Šindlerová; Alois Kozubík; Pavel Krčmář; Miroslav Machala; Jan Vondráček (pp. 1622-1634).

Both natural and synthetic flavonoids are known to interact with the aryl hydrocarbon receptor (AhR); however, their agonist/antagonist properties in vitro have been so far studied mostly in the context of cytochrome P450 1A1 gene ( Cyp1a1) regulation. We investigated effects of two synthetic flavones known either as AhR agonist (β-naphthoflavone; BNF) or antagonist (3′-methoxy-4′-nitroflavone; 3M4NF), using an in vitro model of liver ‘stem-like’ cells, on expression of various AhR target genes and AhR-dependent cell proliferation. We found that the presumed antagonist 3M4NF induces a partial nuclear translocation and activation of AhR. Although inhibiting the 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced Cyp1a1 expression, 3M4NF alone induced a minor increase of CYP1A1 mRNA and protein. However, 3M4NF did not induce AhR binding to synthetic dioxin response elements (DRE). In contrast to Cyp1a1, 3M4NF induced a marked expression of other AhR-regulated genes, such as Cyp1b1 and Nqo1, as well as transcriptional repression of Cdh13 gene, confirming that its effects may be promoter-context specific. Like BNF, 3M4NF induced AhR-dependent cell proliferation of contact-inhibited rat liver ‘stem-like’ WB-F344 cells, associated with a marked upregulation of Cyclin A, as well as the downregulation of proteins involved in formation of cell–cell contacts. Based on these experimental findings, we conclude that partial agonists/antagonists of AhR can increase cell proliferation rate and AhR-dependent genes expression in both cell type- and gene-specific manner. The specificity of effects of flavones on diverse AhR targets should be taken into account, when studying AhR signaling using presumed AhR antagonists.

Keywords: Abbreviations; 3M4NF; 3′-methoxy-4′-nitroflavone; AhR; aryl hydrocarbon receptor; ARNT; AhR nuclear translocator; ANF; α-naphthoflavone; BNF; β-naphthoflavone; CYP; cytochrome P450; DRE; dioxin responsive elements; EMSA; electrophoretic mobility shift assay; FITC; fluorescein-isothiocyanate; NQO1; NAD(P)H-quinone dehydrogenase 1; PAGE; polyacrylamide gel electrophoresis; PARP; poly(ADP)-ribose polymerase; PBS; phosphate-buffered saline; RT-PCR; reverse transcription-polymerase chain reaction; siRNA; short interfering RNA; TCDD; 2,3,7,8-tetrachlorodibenzo-; p; -dioxinAryl hydrocarbon receptor; Flavones; Cell proliferation; Liver epithelial cells; Xenobiotic-metabolizing enzymes

Capsaicin causes protein synthesis inhibition and microtubule disassembly through TRPV1 activities both on the plasma membrane and intracellular membranes by Ping Han; Heath A. McDonald; Bruce R. Bianchi; Rachid El Kouhen; Melissa H. Vos; Michael F. Jarvis; Connie R. Faltynek; Robert B. Moreland (pp. 1635-1645).

TRPV1 is a non-selective cationic channel that is activated by capsaicin, acidic pH and thermal stimuli. Sustained TRPV1 channel activation causes severe cytotoxicity that leads to cell death. In this study, we investigated the mechanisms of capsaicin-induced cytotoxicity in HEK293 cells stably expressing TRPV1 with a focus on protein synthesis regulation and cytoskeleton reorganization. Capsaicin inhibited protein synthesis in TRPV1-expressing HEK cells with an IC₅₀ of 15.6nM and depolymerized microtubules within 10min after exposure. These effects were completely blocked by pretreatment of cells with the TRPV1 antagonist A-425619, both in the presence and absence of extracellular calcium. Protein synthesis inhibition induced by capsaicin was not a result of eIF2α hyperphosphorylation, but rather closely correlated with cytosolic calcium elevation caused by calcium flux through cell surface and intracellular TRPV1, and/or ER calcium depletion through intracellular TRPV1. Microtubule dependent cell process shrinkage may serve as a mechanism for rapid alteration of the neurotransmission network upon TRPV1 activation. Taken together, the present studies demonstrate that intracellular pool of TRPV1 plays an important role in regulating cell morphology and viability upon receptor activation.

Keywords: Abbreviations; TRPV; transient receptor potential channel type V; VR1; vanilloid receptor 1; CAP; capsaicin; DRG; dorsal root ganglion; FLIPR; Fluorometric Imaging Plate Reader; DOC; deoxycholic acid; NP-40; Nonidet P-40; ER; endoplasmic reticulum; PM; plasma membrane; [Ca; ²⁺; ]; _i; intracellular calcium levels; [Ca; ²⁺; ]; _o; extracellular calcium levels; RTX; resiniferatoxin; CPZ; capsazepine; A-425619; 1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea; RR; ruthenium redTRPV1; VR1; Calcium; Protein synthesis; Microtubule; Capsaicin

Novel agonistic action of mustard oil on recombinant and endogenous porcine transient receptor potential V1 (pTRPV1) channels by Toshio Ohta; Toshiaki Imagawa; Shigeo Ito (pp. 1646-1656).

Neurogenic components play a crucial role in inflammation and nociception. Mustard oil (MO) is a pungent plant extract from mustard seed, horseradish and wasabi, the main constituent of which is allylisothiocyanate. We have characterized the action of MO on transient receptor potential V1 (TRPV1), a key receptor of signal transduction pathways in the nociceptive system, using fura-2-based [Ca²⁺]_i imaging and the patch-clamp technique in a heterologous expression system and sensory neurons. In human embryonic kidney (HEK) 293 cells expressing porcine TRPV1 (pTRPV1), MO evoked increases of [Ca²⁺]_i in a concentration-dependent manner. A high concentration of MO elicited irreversible cell swelling. Capsazepine, ruthenium red and iodoresiniferatoxin dose-dependently suppressed the MO-induced [Ca²⁺]_i increase. MO elicited outward rectified currents in pTRPV1-expressing HEK 293 cells with a reversal potential similar to that of capsaicin. [Ca²⁺]_i responses to MO were completely abolished by the removal of external Ca²⁺. MO simultaneously elicited an inward current and increase of [Ca²⁺]_i in the same cells, indicating that MO promoted Ca²⁺ influx through TRPV1 channels. In cultured porcine dorsal root ganglion (DRG) neurons, MO elicited a [Ca²⁺]_i increase and inward current. Among DRG neurons responding to MO, 85% were also sensitive to capsaicin. The present data indicate that MO is a novel agonist of TRPV1 channels, and suggest that the action of MO in vivo may be partly mediated via TRPV1. These results provide an insight into the TRPV1-mediated effects of MO on inflammation and hyperalgesia.

Keywords: Allylisothiocyanate; Capsaicin; Dorsal root ganglion; Intracellular Ca; Vanilloid

The role of esterases in the metabolism of ciclesonide to desisobutyryl-ciclesonide in human tissue by Elaine Mutch; Ruediger Nave; Nigel McCracken; Karl Zech; Faith M. Williams (pp. 1657-1664).

Ciclesonide (CIC) is an inhaled glucocorticosteroid. This study aimed to identify esterases involved in the metabolism of CIC to the active metabolite desisobutyryl-ciclesonide (des-CIC), and to measure hydrolysis rates in human liver, lung and plasma and normal human bronchial epithelial (NHBE) cells in vitro. Ciclesonide (5μM and 500μM) was incubated with microsomal or cytosolic fractions from liver, lung and plasma ( n=4 for each) and des-CIC formation was determined by reverse-phase high-performance liquid chromatography with U.V. detection. The roles of carboxylesterase, cholinesterase and A-esterase in CIC hydrolysis were determined using a range of inhibitors. Inhibitor concentrations for liver and NHBE cells were 100μM and 5μM, respectively. Liver tissue had a higher activity for 500μM CIC hydrolysis (microsomes: 25.4; cytosol: 62.9nmol/gtissue/min) than peripheral lung (microsomes: 0.089; cytosol: 0.915nmol/gtissue/min) or plasma (0.001nmol/mLplasma/min), corresponding with high levels of carboxylesterase and cholinesterase in the liver compared with the lung. CIC (5μM) was rapidly hydrolyzed by NHBE cells (∼30% conversion at 4h), with almost complete conversion by 24h. In liver and NHBE cells, major involvement of cytosolic carboxylesterases, with some contribution by cholinesterases, was indicated. The highest level of conversion was found in the liver, the site of inactivation of des-CIC through rapid oxidation by cytochrome P450. Carboxylesterases in bronchial epithelial cells probably contribute significantly to the conversion to des-CIC in the target organ, whereas low systemic levels of des-CIC are a result of the high metabolic clearance by the liver following CIC inhalation.

Keywords: Ciclesonide; Carboxylesterase; Esterases; Liver; Lung; Metabolism

Intracellularly transported adenosine induces apoptosis in HuH-7 human hepatoma cells by downregulating c-FLIP expression causing caspase-3/-8 activation by Dongqin Yang; Takahiro Yaguchi; Hideyuki Yamamoto; Tomoyuki Nishizaki (pp. 1665-1675).

Extracellular adenosine induced apoptosis of HuH-7 cells, a Fas-deficient human hepatoma cell line. The adenosine action was inhibited by dipyridamole, an adenosine transporter inhibitor, or 5′-amino-5′-deoxyadenosine, an inhibitor of adenosine kinase to convert from adenosine to AMP, but it was not affected by inhibitors for adenosine A₁, A_2a, A_2b, and A₃ adenosine receptors. Adenosine activated caspase-3 and -8, but not caspase-9, in HuH-7 cells, and the activation was abolished by dipyridamole. In the real-time RT-PCR and Western blot analysis, extracellular adenosine downregulated mRNA and protein levels for c-FLIP, and the effect was suppressed by dipyridamole. Furthermore, overexpression of c-FLIP short in HuH-7 cells inhibited adenosine-induced caspase-8 activity. Taken together, these results suggest that intracellularly transported adenosine, perhaps converted AMP as the ensuing event, activates caspase-8 and the downstream effector caspase caspase-3 by neutralizing caspase-8 inhibition due to c-FLIP as a consequence of decreased c-FLIP expression, leading to apoptosis. This extends our understanding of adenosine-induced molecular apoptotic pathways.

Keywords: JEL classification; (9) Pulmonary; Renal and hepatic pharmacologyAbbreviations; FasL; Fas ligand; TNF-α; tumor necrosis factor-α; TNFR1; TNF receptor 1; FADD; Fas-associated death domain protein; TRADD; TNFR1-associated death domain protein; RIP1; receptor-interacting protein 1; FLIP; FADD-like interleukin-1β-converting enzyme inhibitory protein; c-FLIP; cellular FLIP; PKA; protein kinase A; AMPK; AMP-activated protein kinase; CHA; N; ⁶; -cyclohexyladenosine; DMPX; 3,7-dimethyl-1 propargylxanthine; CGS21680; 2-; p; -(2-carboxyethyl) phenetylamino-5′-; N; -ethylcarboxamidoadenosine hydrochloride; MRS1706; N; -(4-acethylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1; H; -purin-8-yl)phenoxy]acetamide; NECA; 5′-(; N; -ethylcarboxamido)adenosine; MRS1523; 3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate; AMDA; 5′-amino-5′-deoxyadenosine; PI; propidium iodide; DMEM; Dulbecco's modified Eagle's medium; 8-CPT; 8-cyclopentyltheophylline; EHNA; erythro-9 (2-hydroxy-3-nonyl)-adenosine; Cl-IB-MECA; 2-chloro-; N; ⁶; -(3-iodobenzyl)-adenosine-5′-; N; -methylurinamide; MTT; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2; H; -tetrazolium bromide; SDS; sodium dodecylsulfate; PBS; phosphate-buffered saline; EDTA; ethylenediamine-; N; ,; N; ,; N; ′,; N; ′-tetraacetic acid; RT-PCR; reverse transcription-polymerase chain reaction; SDS-PAGE; sodium dodecylsulfate polyacrylamide gel electrophoresis; c-FLIPS; c-FLIP short; ANOVA; analysis of varianceAdenosine; HuH-7 cell; Apoptosis; c-FLIP; Caspase-8

Mitochondria play an important role in adenosine-induced ATP release from Madin–Darby canine kidney cells by Keisuke Migita; Yumei Zhao; Takeshi Katsuragi (pp. 1676-1682).

We previously found that adenosine stimulates ATP release from Madin–Darby canine kidney (MDCK) cells, by activating an Ins(1,4,5)P₃ sensitive-calcium (Ca²⁺) pathway through the stimulation of A₁ receptors. Thus, we investigated the intracellular pathway of ATP efflux after the rise in intracellular Ca²⁺ in MDCK cells. Adenosine evoked an increase in mitochondrial Ca²⁺ using Rhod-2/AM, a mitochondrial Ca²⁺ indicator. Adenosine-induced ATP release was inhibited by mitochondrial modulators, such as oxidative phosphorylation modulators (carbonyl cyanide 3-chlorophenylhydrazone and oligomycin), mitochondrial ADP/ATP carrier inhibitors ( N-ethylmaleimide, carboxyatractyloside and bongkrekic acid), a mitochondrial Na⁺–Ca²⁺ exchange inhibitor (CGP-37157). In addition, mitochondrial modulators significantly reduced intracellular ATP content. On the other hand, 2-deoxy-glucose (2-DG) induced a greater decrease in intracellular ATP content than mitochondrial modulators. ATP release was still induced by adenosine in the presence of 5mM 2-DG. These results suggest that mitochondria play an important role in the signaling pathway of adenosine-triggered ATP release in MDCK cells.

Keywords: ATP release; Adenosine; Mitochondria; 2-Deoxy-glucose; Calcium; MDCK cells

Glucuronidation of fenamates: Kinetic studies using human kidney cortical microsomes and recombinant UDP-glucuronosyltransferase (UGT) 1A9 and 2B7 by Paraskevi Gaganis; John O. Miners; Kathleen M. Knights (pp. 1683-1691).

Mefenamic acid, a non-steroidal anti-inflammatory drug (NSAID), is used commonly to treat menorrhagia. This study investigated the glucuronidation kinetics of flufenamic, mefenamic and niflumic acid using human kidney cortical microsomes (HKCM) and recombinant UGT1A9 and UGT2B7. Using HKCM Michaelis–Menten (MM) kinetics were observed for mefenamic (Kmapp 23μM) and niflumic acid (Kmapp 123μM) glucuronidation, while flufenamic acid exhibited non-hyperbolic (atypical) glucuronidation kinetics. Notably, the intrinsic renal clearance of mefenamic acid (CL_int 17±5.5μL/minmg protein) was fifteen fold higher than that of niflumic acid (CL_int 1.1±0.8μL/minmg protein). These data suggest that renal glucuronidation of mefenamic acid may result in high intrarenal exposure to mefenamic acyl-glucuronide and subsequent binding to renal proteins. Diverse kinetics were observed for fenamate glucuronidation by UGT2B7 and UGT1A9. Using UGT2B7 MM kinetics were observed for flufenamic (Kmapp 48μM) and niflumic acid (Kmapp 135μM) glucuronidation and atypical kinetics with mefenamic acid. Similarity inKmapp between HKCM and UGT2B7 suggests that UGT2B7 may be the predominant renal UGT isoform catalysing niflumic acid glucuronidation. In contrast, UGT1A9 glucuronidation kinetics were characterised by negative cooperativity with mefenamic ( S₅₀ 449μM, h 0.4) and niflumic acid ( S₅₀ 7344μM, h 0.4) while atypical kinetics were observed with flufenamic acid. Additionally, potent inhibition of the renal glucuronidation of the UGT substrate ‘probe’ 4-methylumbelliferone by flufenamic, mefenamic and niflumic acid was observed. These data suggest that inhibitory metabolic interactions may occur between fenamates and other substrates metabolised by UGT2B7 and UGT1A9 in human kidney.

Keywords: Abbreviations; 4-MU; 4-methylumbelliferone; CL; _int; intrinsic clearance; h; Hill coefficient; HKCM; human kidney cortical microsomes; IC; ₅₀; concentration of inhibitor causing 50% inhibition; K; m; app; apparent Michaelis constant; MM; Michaelis–Menten; NSAIDs; non-steroidal anti-inflammatory drugs; OTC; over-the-counter; RPN; renal papillary necrosis; S; ₅₀; substrate concentration at half-maximal velocity; UDP; uridine diphosphate; UDPGA; UDP-glucuronic acid; UGT; UDP-glucuronosyltransferase; V; _max; maximal velocityUDP-glucuronosyltransferase; Glucuronidation kinetics; Human kidney; Flufenamic acid; Mefenamic acid; Niflumic acid

A thank you to our 2006 reviewers (pp. 1692-1696).

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Biochemical Pharmacology (v.73, #10)