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Biochemical Pharmacology (v.74, #2)

Editorial Advisory Board (pp. iii).

Norepinephrine: The redheaded stepchild of Parkinson's disease by K.S. Rommelfanger; D. Weinshenker (pp. 177-190).

Parkinson's disease (PD) affects approximately 1% of the world's aging population. Despite its prevalence and rigorous research in both humans and animal models, the etiology remains unknown. PD is most often characterized by the degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNc), and models of PD generally attempt to mimic this deficit. However, PD is a true multisystem disorder marked by a profound but less appreciated loss of cells in the locus coeruleus (LC), which contains the major group of noradrenergic neurons in the brain. Historic and more recent experiments exploring the role of norepinephrine (NE) in PD will be analyzed in this review. First, we examine the evidence that NE is neuroprotective and that LC degeneration sensitizes DA neurons to damage. The second part of this review focuses on the potential contribution of NE loss to the behavioral symptoms associated with PD. We propose that LC loss represents a crucial turning point in PD progression and that pharmacotherapies aimed at restoring NE have important therapeutic potential.

Keywords: Dopamine β-hydroxylase; Neurodegeneration; Adrenoreceptor; Neuroprotection; Antioxidant; Alpha-synuclein

Cisplatin and doxorubicin repress Vascular Endothelial Growth Factor expression and differentially down-regulate Hypoxia-inducible Factor I activity in human ovarian cancer cells by Monique C.A. Duyndam; Maria P.A. van Berkel; Josephine C. Dorsman; Davy A.P. Rockx; Herbert M. Pinedo; Epie Boven (pp. 191-201).

Vascular Endothelial Growth Factor (VEGF) and its transcriptional regulator Hypoxia-inducible Factor 1 (HIF-1) play an important role in the process of angiogenesis in many types of cancer, including ovarian cancer. We have examined whether the DNA-damaging drugs cisplatin and doxorubicin and the microtubule inhibitors docetaxel and paclitaxel can affect VEGF expression and HIF-1 activity in three human ovarian cancer cell lines. We demonstrate that cisplatin and doxorubicin abolish hypoxia-induced VEGF mRNA expression in all cell lines, while basal VEGF mRNA expression was also downregulated. Transient transfection with a HIF-1-responsive luciferase construct indicated that cisplatin and doxorubicin inhibited hypoxic activation of HIF-1. Cisplatin repressed HIF-1α protein expression in all cell lines. Stimulation of HIF-1α protein degradation by cisplatin was observed in the only cell line expressing wild-type p53. Cisplatin also inhibited the synthesis of HIF-1α protein for which p53 was dispensable. Interestingly, cisplatin strongly reduced the protein levels of the HIF-1 coactivators p300 and CREB-binding protein (CBP) under hypoxia in all cell lines. Although doxorubicin inhibited hypoxic activation of HIF-1, this drug had no significant effect on the expression levels of HIF-1α and hypoxic expression of p300 and CBP was only weakly reduced. Docetaxel and paclitaxel did neither influence VEGF expression nor hypoxia-induced HIF-1 activity. In total, our findings indicate that cisplatin and doxorubicin can repress hypoxic induction of VEGF expression by inhibiting HIF-1 through different mechanisms. This knowledge may be useful for future treatment schedules including agents that target the HIF-1 signalling pathway.

Keywords: Abbreviations; VEGF; Vascular Endothelial Growth Factor l; HIF-1; Hypoxia-inducible Factor 1; HRE; hypoxia responsive element; VHL; Von Hippel-Lindau; wt; wildtype; mt; mutant; DFO; desferrioxamine; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CA; carbonic anhydrase; PI3K; phosphatidylinositol 3-kinase; mTOR; mammalian target of rapamycin; MAPK; Mitogen-Activated Protein KinaseHIF-1; VEGF; Taxanes; Cisplatin; Doxorubicin; P53

Effect of 15-lipoxygenase metabolites, 15-( S)-HPETE and 15-( S)-HETE on chronic myelogenous leukemia cell line K-562: Reactive oxygen species (ROS) mediate caspase-dependent apoptosis by Suraneni V.K. Mahipal; Jagu Subhashini; Madhava C. Reddy; Metukuri M. Reddy; Kotha Anilkumar; Karnati R. Roy; Gorla V. Reddy; Pallu Reddanna (pp. 202-214).

Growth inhibitory effects of 15-lipoxygenase-1 [13-( S)-HPODE and 13-( S)-HODE] and 15-lipoxygenase-2 [15-( S)-HPETE and 15-( S)-HETE] (15-LOX-1 and LOX-2) metabolites and the underlying mechanisms were studied on chronic myeloid leukemia cell line (K-562). The hydroperoxy metabolites, 15-( S)-HPETE and 13-( S)-HPODE rapidly inhibited the growth of K-562 cells by 3h with IC₅₀ values, 10 and 15μM, respectively. In contrast, the hydroxy metabolite of 15-LOX-2, 15-( S)-HETE, showed 50% inhibition only at 40μM by 6h and 13-( S)-HODE, hydroxy metabolite of 15-LOX-1, showed no significant effect up to 160μM. The cells exposed to 10μM of 15-( S)-HPETE and 40μM of 15-( S)-HETE showed typical apoptotic features like release of cytochrome c, caspase-3 activation and PARP-1 (poly(ADP) ribose polymerase-1) cleavage. A flow cytometry based DCFH-DA analysis and inhibitory studies with DPI, a pharmacological inhibitor of NADPH oxidase, NAC ( N-acetyl cysteine) and GSH revealed that NADPH oxidase-mediated generation of ROS is responsible for caspase-3 activation and subsequent induction of apoptosis in the K-562 cell line.

Keywords: Abbreviations; AA; arachidonic acid; DAPI; 4′,6-diamidino-2-phenylindole; DCFH-DA; 2′,7′-dichlorodihydrofluorescein diacetate; DPI; diphenylene iodonium; GSH; reduced glutathione; 13-(; S; )-HODE; 13-(; S; )-hydroxyoctadecadienoic acid; 13-(; S; )-HPODE; 13-(; S; )-hydroperoxyoctadecadienoic acid; 15-(; S; )-HETE; 15-(; S; )-hydroxyeicosatetraenoic acid; 15-(; S; )-HPETE; 15-(; S; )-hydroperoxyeicosatetraenoic acid; LA; linoleic acid; 15-LOX; 15-lipoxygenase; NAC; N; -acetyl cysteineApoptosis; 15-(; S; )-HPETE; 15-(; S; )-HETE; 15-Lipoxygenase; ROS

Chrysin inhibited stem cell factor (SCF)/c-Kit complex-induced cell proliferation in human myeloid leukemia cells by Seong Jin Lee; Joo-Heon Yoon; Kyoung Seob Song (pp. 215-225).

Stem cell factor (SCF) has important roles in the proliferation and differentiation of hematopoietic stem cells. The complex of c-Kit and its ligand SCF induce hematopoiesis, melanogenesis, and gametogenesis. However, the mechanism by which SCF induces cell proliferation in the human megakaryoblastic leukemia cell line, MO7e, and the signaling molecules involved, especially in downstream signaling of c-Kit, remain unclear. Here, we show that pharmacological inhibition of the PI3K pathway inhibits SCF/c-Kit signaling and cell proliferation. In addition, we find that the Shc/PDK1/PKC/Akt/c-raf signaling cascade is essential for SCF/c-Kit signal pathway. Our results also suggest that ERK5 is activated and translocated to the nucleus, activating CREB and STAT3. Interestingly, chrysin shuts down the SCF/c-Kit complex-induced signaling cascade. Taken together, these studies give additional insight into the molecular mechanism of SCF/c-Kit-induced cell proliferation and its inverse agonist, chrysin. Finally, these findings enhance our understanding of MO7e cell proliferation.

Keywords: SCF; c-Kit; PI3K; ERK5; Chrysin; MO7e cells; Cell proliferation

Y1068 phosphorylation is the most sensitive target of disulfonated tetraphenylporphyrin-based photodynamic therapy on epidermal growth factor receptor by Anette Weyergang; Pål Kristian Selbo; Kristian Berg (pp. 226-235).

Photodynamic therapy (PDT) is an anticancer therapy that utilizes the cytotoxic properties of a photosensitizer (PS) when combined with exposure to light. Photochemical internalization (PCI) is a drug delivery method for macromolecules based on PDT with endo-lysosomal localizing PSs, and synergistic effects can be obtained by PCI of EGFR targeting drugs. In this report the effects of PDT with two endo-lysosomal localizing PSs on EGFR are described. The experiments were performed in EGFR-positive cell-lines in vitro and also in a subcutaneous tumour-model in mice. In PCI, the PSs are transported from the plasma membrane to endocytic vesicles by endocytosis and some of the PS can therefore be retained at the plasma membrane. Two distinct treatment conditions with different amounts of the PS on the plasma membrane were therefore studied in vitro. The expression of total and phosphorylated EGFR was analyzed on Western blots and EGF-binding to EGFR was evaluated by fluorescence microscopy of Alexa488-labelled EGF. The results showed that PDT, as utilized in PCI, caused inhibition of EGF-stimulated EGFR phosphorylation on Y1068 in NuTu-19 cells, but not in WiDr cells. PDT performed with more PS on the plasma membrane of NuTu-19 cells caused in addition inhibition of EGF binding and also lack of recognition by antibodies towards sequences in the intracellular domain of EGFR. In vivo, total EGFR was reduced 24h after PDT in WiDr tumours. This report indicates EGF-stimulated phosphorylation on Y1068 as the most sensitive target on EGFR to PDT with amphiphilic PSs.

Keywords: Abbreviations; PS; photosensitizer; PDT; photodynamic therapy; PCI; photochemical internalization; ROS; reactive oxygen species; EGF; epidermal growth factor; EGFR; epidermal growth factor receptor; JNK; c-Jun NH2-terminal protein kinase; aa; amino acid; mAb; monoclonal antibody; TKI; tyrosine-kinase inhibitor; TPPS; _2a; meso-tetraphenylporphine with two sulfonate groups on adjacent phenyl rings; AlPcS; _2a; aluminium phthalocyanine with two sulfonate groups on adjacent phenyl rings; E-64; trans; -epoxysuccinyl-; l; -leucylamido(4-guanidino)butane; MTT; 3-[4,5-dimethylthiazol-2-y]-2,5 diphenyltetrazolium bromideEpidermal growth factor receptor; Photodynamic therapy; Combination therapy; Photochemical internalization

Cholesterol as a determinant of cooperativity in the M₂ muscarinic cholinergic receptor by Alejandro T. Colozo; Paul S.-H. Park; Chi Shing Sum; Luca F. Pisterzi; James W. Wells (pp. 236-255).

M₂ muscarinic receptor extracted from Sf9 cells in cholate–NaCl differs from that extracted from porcine sarcolemma. The latter has been shown to exhibit an anomalous pattern in which the capacity for N-[³H]methylscopolamine (NMS) is only 50% of that for [³H]quinuclidinylbenzilate (QNB), yet unlabeled NMS exhibits high affinity for all of the sites labeled by [³H]QNB. The effects can be explained in terms of cooperativity within a receptor that is at least tetravalent [Park PS, Sum CS, Pawagi AB, Wells JW. Cooperativity and oligomeric status of cardiac muscarinic cholinergic receptors. Biochemistry 2002;41:5588–604]. In contrast, M₂ receptor extracted from Sf9 membranes exhibited no shortfall in the capacity for [³H]NMS at either 30 or 4°C, although there was a time-dependent inactivation during incubation with [³H]NMS at 30°C; also, any discrepancies in the affinity of NMS were comparatively small. The level of cholesterol in Sf9 membranes was only 4% of that in sarcolemmal membranes, and it was increased to about 100% by means of cholesterol-methyl-β-cyclodextrin. M₂ receptors extracted from treated Sf9 membranes were stable at 30 and 4°C and resembled those from heart. Cholesterol induced a marked heterogeneity detected in the binding of both radioligands, including a shortfall in the apparent capacity for [³H]NMS, and there were significant discrepancies in the apparent affinity of NMS as estimated directly and via the inhibition of [³H]QNB. The data can be described quantitatively in terms of cooperative effects among six or more interacting sites. Cholesterol therefore appears to promote cooperativity in the binding of antagonists to the M₂ muscarinic receptor.

Keywords: Abbreviations; AIC; _C; second-order Akaike's information criterion; AFM; atomic force microscopy; CHL; cholesterol; DAMGO; [; d; -Ala; ²; ,; N; -MePhe; ⁴; ,Gly-ol; ⁵; ]enkephalin; BS; ³; bis[sulfosuccinimidyl]suberate; GMP-PNP; guanylylimidodiphosphate; GPCR; G protein-coupled receptor; GTPγS; guanosine 5′-; O; -[3-(γ-thio)triphosphate]; NMS; N; -methylscopolamine; p; -MPPF; (4-2′-methoxy)-phenyl-1-[2′-(; N; -2″-pyridinyl-; p; -fluorobenzamido]ethyl-piperazine; PMSF; phenylmethylsulfonylfluoride; QNB; (−)-quinuclidinylbenzilate; Sf; 9; Spodoptera frugiperdaM; ₂; muscarinic cholinergic receptor; G protein-coupled receptor; Cholesterol; Oligomers; Cooperativity; Mechanistic models

β-Cryptoxanthin, a novel natural RAR ligand, induces ATP-binding cassette transporters in macrophages by Akira Matsumoto; Hajime Mizukami; Satoshi Mizuno; Keizo Umegaki; Jun-ichi Nishikawa; Koichi Shudo; Hiroyuki Kagechika; Makoto Inoue (pp. 256-264).

Despite its serious adverse effects, recent accumulating evidence suggests that a physiological retinoic acid receptor (RAR) agonist, all- trans retinoic acid (atRA), exhibits preventive effects on atherogenesis. Therefore, the present study was designed to explore novel natural RAR ligands with anti-atherogenic effects in order to identify and develop a drug without severe side effects. Among xanthophylls and carotenoids studied, β-cryptoxanthin and lutein exhibited RAR ligand activity in yeast two-hybrid system that was found to be completely abolished by the RAR pan-antagonist LE540. Furthermore, these molecules can bind the RAR ligand-binding domain in the CoA-BAP system but not RXR ligand-binding domain. These results indicate that both β-cryptoxanthin and lutein serve as ligands for RAR, but not RXR, although their binding affinity was three orders of magnitude lower than that of atRA. Additionally, when applied to macrophages, β-cryptoxanthin indeed was found to induce the ATP-binding cassette transporter A1 (ABCA1) and ABCG1 mRNAs, which exert anti-atherosclerotic effects by preventing cholesteryl ester accumulation in macrophages. The induction of ABCA1 proteins by β-cryptoxanthin as well as atRA was abrogated by LE540. In summary, β-cryptoxanthin appears to be more an efficient provitamin A source than other carotenoids and xanthophylls including β-carotene, since β-cryptoxanthin can act not only as a RAR agonist but also a source of vitamin A. Taking into account that the pharmacodynamics difference between β-cryptoxanthin and atRA, β-cryptoxanthin appears to exert beneficial effects on atherogenesis through RAR activation in the manner different from atRA.

Keywords: β-Cryptoxanthin; Lutein; Xanthopyll; Retinoic acid receptor; Retinoid X receptor; ATP-binding cassette transporter

Amiodarone inhibits tocopherol-mediated human lipoprotein peroxidation by Domenico Lapenna; Giuliano Ciofani; Sante Donato Pierdomenico; Matteo Neri; Maria Adele Giamberardino; Franco Cuccurullo (pp. 265-272).

It is unknown whether lipoprotein tocopherol-mediated peroxidation (TMP) is influenced by peculiar drug physicochemical properties such as hydrophobicity. Thus, we studied the effect of the extremely hydrophobic agent amiodarone on human non-HDL TMP. The drug, albeit devoid of specific radical-scavenging effects, inhibited TMP at therapeutic concentrations and with an efficiency similar to that of the physiological co-antioxidant ascorbic acid, showing indeed an IC₅₀ of 5μM. A comparable efficiency was observed with human LDL, and with a pure LDL-VLDL mixture. TMP was also inhibited by other hydrophobic cationic amphiphiles without radical-scavenging activity, namely desethylamiodarone, chlorpromazine, clomipramine, promethazine, promazine, verapamil, bromhexine, propranolol, mepivacaine, metoprolol, tramadol and ranitidine, whose anti-TMP potency was far lower than that of amiodarone and related to drug hydrophobicity degree. Further, TMP was strongly inhibited by butylhydroxytoluene, a lipophilic radical scavenger. Hydrophobic acidic (diclofenac, indomethacin, ibuprofen and ketoprofen) or neutral ( n-hexane, anthracene, o-xylene and toluene) compounds could not instead inhibit TMP, indicating a stringent requirement for drug basicity in the pharmacological inhibition of TMP. Amiodarone effectiveness was lowered by lipoprotein α-tocopherol enrichment, suggesting some drug-α-tocopherol interaction and less lipid pharmacological protection at higher α-tocopheroxyl radical generation. Drug anti-TMP activity may so be related to electrostatic and hydrophobic interactions with lipoprotein α-tocopherol and lipid moiety, resulting in decreased radical phase transfer and lipid propensity to undergo radical-driven peroxidation. In conclusions, primarily drug basicity and then hydrophobicity are solely relevant to TMP inhibition. Amiodarone, at therapeutic concentrations, has anti-TMP properties, which could occur in the clinical setting.

Keywords: Abbreviations; AAPH; 2,2’-azobis(amidinopropane) dihydrochloride; α-TOH; α-tocopherol; α-TO; α-tocopheroxyl radical; BHT; butylhydroxytoluene; CDH; conjugated diene hydroperoxides; DPH; 1,1-diphenyl-2-pycrylhydrazyl; DTPA; diethylenetriaminepentaacetic acid; FOX; ferrous oxidation in xylenol orange assay; IC; _min; minimal drug concentration inhibiting significantly TMP; IC; ₅₀; drug concentration inhibiting by 50% TMP; log; P; octanol–water partition coefficient; LOOH; lipid hydroperoxides; PBS; phosphate buffered saline; TMP; tocopherol-mediated peroxidation; TBA; thiobarbituric acid; TBARS; thiobarbituric acid reactive substances; TNB; 5-thio-2-nitrobenzoic acidAmiodarone; α-Tocopherol; α-Tocopheroxyl radical; Tocopherol-mediated peroxidation; Lipoprotein oxidation; Atherosclerosis

The inhibitors of protein acylation, cerulenin and tunicamycin, increase voltage-dependent Ca²⁺ currents in the insulin-secreting INS 832/13 cell by Ying Zhao; Geoffrey W.G. Sharp; Susanne G. Straub (pp. 273-280).

As it has been suggested that protein acylation plays a role in nutrient stimulus–secretion coupling in the pancreatic β-cell, we examined the insulin-secreting INS 832/13 β-cell line for evidence that protein acylation was involved. The perforated whole-cell configuration was employed to voltage-clamp INS 832/13 cells. Voltage pulses were applied and Ca²⁺ currents measured in the presence and absence of the protein acylation inhibitors cerulenin and tunicamycin. Both inhibitors enhanced the peak amplitude of I_Ca,L. Both increased the peak inward current in the range between −40 and +30mV and shifted the apparent maximum current by 10mV in the hyperpolarizing direction without affecting the activation threshold of −40mV. The two drugs had qualitatively and quantitatively similar effects. Steady-state activation curves revealed that cerulenin and tunicamycin shifted the activation curves in the hyperpolarization direction. Activation time constants were significantly reduced in the presence of both drugs. The Ca²⁺ charge influx was increased by the drugs at all potentials tested. In contrast to these effects on the L-type Ca²⁺ channel, the two inhibitors of protein acylation had no effect on the ATP-sensitive K⁺ channel. The results suggest that protein acylation exerts a tonic inhibitory effect on L-type Ca²⁺ channel function in the insulin-secreting β-cell.

Keywords: Protein acylation; Cerulenin; Tunicamycin; Calcium channel; Pancreatic β-cell; Electrophysiology

De novo ceramide biosynthesis is associated with resveratrol-induced inhibition of ornithine decarboxylase activity by Sandra Ulrich; Andrea Huwiler; Stefan Loitsch; Helmut Schmidt; Jürgen M. Stein (pp. 281-289).

Previous studies could demonstrate, that the naturally occuring polyphenol resveratrol inhibits cell growth of colon carcinoma cells at least in part by inhibition of protooncogene ornithine decarboxylase (ODC). The objective of this study was to provide several lines of evidence suggesting that the induction of ceramide synthesis is involved in this regulatory mechanisms.Cell growth was determined by BrdU incorporation and crystal violet staining. Ceramide concentrations were detected by HPLC-coupled mass-spectrometry. Protein levels were examined by Western blot analysis. ODC activity was assayed radiometrically measuring [¹⁴CO₂]-liberation. A dominant-negative PPARγ mutant was transfected in Caco-2 cells to suppress PPARγ-mediated functions.Antiproliferative effects of resveratrol closely correlate with a dose-dependent increase of endogenous ceramides ( p<0.001). Compared to controls the cell-permeable ceramide analogues C2- and C6-ceramide significantly inhibit ODC-activity ( p<0.001) in colorectal cancer cells. C6-ceramide further diminished protein levels of protooncogenes c-myc ( p<0.05) and ODC ( p<0.01), which is strictly related to the ability of ceramides to inhibit cell growth in a time- and dose-dependent manner. These results were further confirmed using inhibitors of sphingolipid metabolism, where only co-incubation with a serine palmitoyltransferase (SPT) inhibitor could significantly counteract resveratrol-mediated actions. These data suggest that the induction of ceramide de novo biosynthesis but not hydrolysis of sphingomyelin is involved in resveratrol-mediated inhibition of ODC. In contrast to the regulation of catabolic spermidine/spermine acetyltransferase by resveratrol, inhibitory effects on ODC occur PPARγ-independently, indicating independent pathways of resveratrol-action. Due to our findings resveratrol could show great chemopreventive and therapeutic potential in the treatment of colorectal cancers.

Keywords: Abbreviations; BrdU; bromodeoxyuridine; DMEM; Dulbecco's modified Eagle's medium; DFMO; alpha-difluoromethylornithine; dnPPARγ; dominant negative PPARγ mutant; ELISA; enzyme-linked immunosorbent assay; MAPK; mitogen-activated protein kinase; NSAID; non-steroidal anti-inflammatory drugs; ODC; ornithine decarboxylase; PAO; polyamine oxidase; PPARγ; peroxisome-proliferator activated receptor γ; RXR; retinoid X receptor; SAMDC; S; -adenosylmethioninedecarboxylase; SMase; Sphingomyelinase; SPT; serine palmitoyltransferase; SSAT; spermine/spermidine acetyltransferase; TNF-α; tumor necrosis factor-αResveratrol; Ceramide; Ornithine decarboxylase; Colon Cancer; c-myc

Modulation of hepatic microsomal triglyceride transfer protein (MTP) induced by S-nitroso- N-acetylcysteine in ob/ob mice by Claudia P.M.S. Oliveira; Venâncio A.F. Alves; Vicência M.R. Lima; José Tadeu Stefano; Victor Debbas; Sandra Valéria Sá; Alda Wakamatsu; Maria Lúcia Corrêa-Giannella; Evandro Sobroza de Mello; Sofia Havaki; Dina G. Tiniakos; Evangelos Marinos; Marcelo G. de Oliveira; Daniel Giannella-Neto; Francisco R. Laurindo; Stephen Caldwell; Flair J. Carrilho (pp. 290-297).

We evaluated the effects of a potent NO donor, S-nitroso- N-acetylcysteine (SNAC), on microsomal triglyceride transfer protein (MTP) expression in ob/ob mice. NAFLD was induced in male ob/ob mice using a methionine–choline deficient diet (MCD) concomitantly with oral SNAC fed solution ( n=5) or vehicle (control; n=5) by gavage daily for 4 weeks. Livers were collected for histology and for assessing MTP by RT-qPCR, Western blot, immunohistochemistry and immunogold electron microscopy analyses. Histological analysis showed diffuse macro and microvesicular steatosis, moderate hepatocellular ballooning and moderate inflammatory infiltrate in ob/ob mice fed the MCD diet. With SNAC, mice showed a marked reduction in liver steatosis ( p<0.01), in parenchymal inflammation ( p=0.02) and in MTP protein immunoexpression in zone III ( p=0.05). Moreover, SNAC caused reduction of MTP protein in Western blot analysis ( p<0.05). In contrast, MTP mRNA content was significantly higher ( p<0.05) in mice receiving SNAC. Immuno-electron microscopy showed MTP localized in the rough endoplasmic reticulum of hepatocytes in both treated and untreated groups. However with SNAC treatment, MTP was also observed surrounding fat globules. Histological improvement mediated by a nitric oxide donor is associated with significantly altered expression and distribution of MTP in this animal model of fatty liver disease. Further studies are in progress to examine possible mechanisms and to develop SNAC as a possible therapy for human fatty liver disease.

Keywords: Abbreviations; NAFLD; nonalcoholic fatty liver disease; NASH; nonalcoholic steatohepatitis; MCD; methionine–choline deficient diet; SNAC; S; -Nitroso-; N; -acetylcysteine; MTP; microsomal triglyceride transfer proteinNAFLD; NASH; MCD diet; MTP; S; -Nitroso-; N-; acetylcysteine; SNAC

The anti-inflammatory actions of LCY-2-CHO, a carbazole analogue, in vascular smooth muscle cells by Feng-Ming Ho; Hao-Cheng Kang; Sho-Tone Lee; Yee Chao; You-Ci Chen; Li-Jiau Huang; Wan-Wan Lin (pp. 298-308).

LCY-2-CHO has anti-inflammatory actions on macrophages. To understand its therapeutic implication in atherosclerosis, we examined its effects on the expressions of anti-inflammatory and inflammatory proteins in cultured rat aortic vascular smooth muscle cells (VSMC). LCY-2-CHO is able to induce heme oxygenase-1 (HO-1) protein expression through a transcriptional action. The HO-1 inducting effect of LCY-2-CHO was inhibited by SB203580, N^G-nitro-l-arginine methylester (l-NAME), and wortmannin, but was not affected by U0126 or SP600125. In accordance LCY-2-CHO increased protein phosphorylation of p38, Akt, and eNOS. Nrf2 is a transcription factor essential for HO-1 gene induction and we showed that LCY-2-CHO is able to cause Nrf2 nuclear translocation and this action depends on p38, Akt and eNOS. In addition to induce anti-inflammatory HO-1, LCY-2-CHO reduced interleukin-1β (IL-1β)-induced inflammatory mediators, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), growth-related oncogene protein-alpha (GRO-α), and interleukin-8 (IL-8). Inhibitory effect on IL-1β-mediated NF-κB activation was evidenced by the diminishment of IκB kinase (IKK) phosphorylation and IκBα degradation. In contrast, IL-1β-mediated ERK and JNK activations were not changed by LCY-2-CHO, while p38 activation by IL-1β and LCY-2-CHO displayed the non-additivity. Taken together, given the overall anti-inflammatory properties of LCY-2-CHO in VSMC, in terms to induce HO-1 gene expression and inhibit inflammatory gene expression, these results highlight the therapeutic potential of LCY-2-CHO in atherosclerosis.

Keywords: LCY-2-CHO; HO-1; Anti-inflammation; Cell signaling; Vascular smooth muscleAbbreviations; ARE; anti-oxidant response element; COX-2; cyclooxygenase-2; GRO-α; growth-related oncogene protein-alpha; HO-1; heme oxygenase-1; IKK; IκB kinase; IL-1β; interleukin-1β; IL-8; interleukin-8; iNOS; inducible nitric oxide synthase; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; MBP; myelin basic protein; MTT; 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium; l; -NAME; N; ^G; -nitro-; l; -arginine methylester; NF-κB; nuclear factor κB; NO; nitric oxide; PGE; ₂; prostaglandin E; ₂; PGI; ₂; prostaglandin I; ₂; PI3K; phosphoinositide 3-kinase; RT-PCR; reverse-transcription polymerase chain reaction; TNF-α; tumor necrosis factor-α; VSMC; vascular smooth muscle cells

Probing the substrate specificity of the ergothioneine transporter with methimazole, hercynine, and organic cations by Silke Grigat; Stephanie Harlfinger; Sonia Pal; Ralph Striebinger; Stefan Golz; Andreas Geerts; Andreas Lazar; Edgar Schömig; Dirk Gründemann (pp. 309-316).

Recently, we have identified the ergothioneine (ET) transporter ETT (gene symbol SLC22A4). Much interest in human ETT has been generated by case-control studies that suggest an association of polymorphisms in the SLC22A4 gene with susceptibility to chronic inflammatory diseases. ETT was originally designated a multispecific novel organic cation transporter (OCTN1). Here we reinvestigated, based on stably transfected 293 cells and with ET as reference substrate, uptake of quinidine, verapamil, and pyrilamine. ETT from human robustly catalyzed transport of ET (68μl/(minmgprotein)), but no transport of organic cations was discernible. With ET as substrate, ETT was relatively resistant to inhibition by selected drugs; the most potent inhibitor was verapamil ( K_i=11μmol/l). The natural compound hercynine and antithyroid drug methimazole are related in structure to ET. However, efficiency of ETT-mediated transport of methimazole ( K_i=7.5mmol/l) was 130-fold lower, and transport of hercynine ( K_i=1.4mmol/l) was 25-fold lower than transport of ET. ETT from mouse, upon expression in 293 cells, catalyzed high affinity, sodium-driven uptake of ET very similar to ETT from human. Additional real-time PCR experiments based on 16 human tissues revealed ETT mRNA levels considerably lower than in bone marrow. Our experiments establish that ETT is highly specific for its physiological substrate ergothioneine. ETT is not a cationic drug transporter, and it does not have high affinity for organic cation inhibitors. Detection of ETT mRNA or protein can therefore be utilized as a specific molecular marker of intracellular ET activity.

Keywords: Abbreviations; ETT; ergothioneine transporter; LC; liquid chromatography; MPP; ⁺; 1-methyl-4-phenylpyridinium; MS; mass spectrometry; TEA; tetraethylammonium SLC22A4; Chronic inflammatory disease; Ergothioneine; Ergothioneine transporter; Methimazole; Organic cations

Discovery of Trp-Nle-Tyr-Met as a novel agonist for human formyl peptide receptor-like 1 by Hui-Xin Wan; Caihong Zhou; Yueyun Zhang; Meiling Sun; Xin Wang; Hong Yu; Xiaoke Yang; Richard D. Ye; Jing-Kang Shen; Ming-Wei Wang (pp. 317-326).

Formyl peptide receptor-like 1 (FPRL1) is a structural homologue of FPR, which binds chemotactic peptides as small as three amino acids (e.g., fMet-Leu-Phe, fMLF) and activates potent bactericidal functions in neutrophils. In comparison, FPRL1 ligands include peptides of 6-104 amino acids, such as Trp-Lys-Tyr-Met-Val-[d]Met (WKYMVm) and other synthetic peptides. To determine the core peptide sequence required for FPRL1 activation, we prepared various analogues based on WKYMVm and evaluated their bioactivities in an FPRL1-transfected cell line. Although substitution ofd-Met⁶ resulted in loss of activity, removal of Val⁵ together withd-Met⁶ produced a peptide that retained most of the bioactivities of the parent peptide. The resulting peptide, WKYM, represents a core structure for an FPRL1 ligand. Further substitution of Lys² with Nle slightly improved the potency of the tetrapeptide, which selectively activates FPRL1 over FPR. Based on these structure–activity relationship studies, we propose a model in which the modified tetrapeptide Trp-Nle-Tyr-Met (WNleYM) binds to FPRL1 through aromatic interactions involving the side chains of Trp¹ and Tyr³, hydrophobic interaction of Nle², and the thio-based hydrogen bonding of Met⁴, with the respective residues in FPRL1 which have not been identified. The identification of the core sequence of a potent peptide agonist provides a structural basis for future design of peptidomimetics as potential therapeutic agents for FPRL1-related disorders.

Keywords: Abbreviations; Nle; l; -norleucine; met; d; -methine; 1-Nap; l; -3-(1-naphthyl)-alanine; 3-Thi; l; -3-(3-thiophenyl)-alanine; Cha; l; -3-(cyclohexyl)-alanine; K (Ac) or Lys (Ac); l; -; ɛ; -; N; -acetyl-lysine; Y (4-Me) or Tyr (4-Me); l; -(4-methyl)-tyrosine; asn; d; -asparagine; Phe (4-F); l; -(4-fluoro)-phenylalanine; f; formyl; Ac; acetyl; FPR; formyl peptide receptor; FPRL1; formyl peptide receptor-like 1; WKYMVm; Trp-Lys-Tyr-Met-Val-[; d; ]Met; fMLF; N; -formyl-Met-Leu-Phe; Quin-C1; 4-butoxy-N-[2-(4-methoxy-phenyl)-4-; o; xo-1,4-dihydro-2H-quinazolin-3-yl]-benzamide; DMEM; Dulbecco's Modified Eagle's Medium; BSA; bovine serum albumin; Aβ; _(1–42); amyloid β-peptide (1–42); HIV; human immunodeficiency virus; LC–MS; liquid-phase chromatography mass spectrometry; NF-κB; nuclear factor κB; ACN; acetonitrileFormyl peptide receptor-like 1; Chemotactic peptides; Trp-Lys-Tyr-Met-Val-[; d; ]Met; Structure–activity relationship; Peptidomimetic

Irreversible cytoskeletal disarrangement is independent of caspase activation during in vitro azaspiracid toxicity in human neuroblastoma cells by Natalia Vilariño; K.C. Nicolaou; Michael O. Frederick; Mercedes R. Vieytes; Luis M. Botana (pp. 327-335).

Azaspiracid-1 (AZA-1) is a marine toxin discovered in 1995. Besides damage to several tissues in vivo, AZA-1 has been shown to cause cytotoxicity in a number of cell lines and alterations in actin cytoskeleton and cell morphology. We studied the reversibility of AZA-1-induced morphological changes in human neuroblastoma cells and their dependence on caspases and signaling pathways involved in cytoskeleton regulation. Morphological/cytoskeletal changes were clearly observed by confocal microscopy 24h after the addition of toxin, without recovery upon toxin removal. Interestingly, 2min of incubation with AZA-1 was enough for the cytoskeleton to be altered 24–48h later. The activation of caspases by AZA-1 was studied next using a fluorescent caspase inhibitor. A cell population with activated caspases was observed after 48h of exposure to the toxin, but not at 24h. Two fragments and a stereoisomer of AZA-1 were tested to analyze structure–activity relationship. Only ABCD- epi-AZA-1 was active with a similar effect to AZA-1. Additionally, regarding the involvement of apoptosis/cytoskeleton signaling in AZA-1-induced morphological effects, inhibition of caspases with Z-VAD-FMK did not affect AZA-1-induced cytoskeletal changes, suggesting, together with the activation kinetics, that caspases are not responsible for AZA-1-elicited morphological changes. Modulation of PKA, PKC, PI3K, Erk, p38MAPK, glutathione and microtubules with inhibitors/activators did not inhibit AZA-1-induced actin cytoskeleton rearrangement. The JNK inhibitor SP600125 seemed to slightly diminish AZA-1 effects, however due to the effects of the drug by itself the involvement of JNK in AZA-1 toxicity needs further investigation. The results suggest that AZA-1 binds irreversibly to its cellular target, needing moieties located in the ABCDE and FGHI rings of the molecule. Cytotoxicity of AZA-1 has been previously described without reference to the type of cell death, we report that AZA-1 induces the activation of caspases, commonly used as an early marker of apoptosis, and that these proteases are not responsible for AZA-1-induced cytoskeleton disarragement in human neuroblastoma cells.

Keywords: Abbreviations; AZA-1; Azaspiracid-1; EMEM; Eagle's Minimum Essential Medium; HBSS; Hank's balanced salt solution; FLICA; fluorescent inhibitor of caspases; DMSO; dimethylsulfoxide; BSA; bovine serum albumin; PBS; phosphate buffered saline; DSP; diarrhetic shellfish poisoning; i.p.; intraperitoneal; p.o.; per os or oral administration; PMA; phorbol 12-myristate 13-acetateAzaspiracid; Phycotoxin; F-Actin cytoskeleton; Caspase; Structure–activity; Human neuroblastoma

Irreversible binding of a novel phenylisothiocyanate tropane analog to monoamine transporters in rat brain by Vishakantha Murthy; Huw M.L. Davies; Simon J. Hedley; Steven R. Childers (pp. 336-344).

Irreversible tropane analogs have been useful in identifying binding sites of cocaine on biogenic amine transporters, including transporters for dopamine (DAT), serotonin (SERT) and norepinephrine (NET). The present study characterizes the properties of the novel phenylisothiocyanate tropane HD-205, synthesized from the highly potent 2-napthyl tropane analog WF-23. In radioligand binding studies in brain membranes, direct IC₅₀ values of HD-205 were 4.1, 14 and 280nM at DAT, SERT and NET, respectively. Wash-resistant binding was characterized by preincubation of HD-205 with brain membranes, followed by extensive washing before performing transporter radioligand binding. Results for HD-205 showed wash-resistant IC₅₀ values of 191, 230 and 840nM at DAT, SERT and NET, respectively. Saturation binding studies with [¹²⁵I]RTI-55 in membranes pretreated with 100nM HD-205 showed that HD-205 significantly decreased the B_max but not K_D of DAT and SERT binding. To further characterize its irreversible binding, an iodinated analog of HD-205, HD-244, was prepared from a trimethylsilyl precursor. The direct IC₅₀ of HD-244 at DAT was 20nM. [¹²⁵I]HD-244 was synthesized with chloramine-T, purified on HPLC, reacted with rat striatal membranes, and proteins were separated by SDS-PAGE. Results showed several non-specific labeled bands, but only a single specific band of radioactivity co-migrating with an immunoreactive DAT band at approx. 80 kilodaltons was detected, suggesting that [¹²⁵I]HD-244 covalently labeled DAT protein in striatal membranes. These results demonstrate that phenylisothiocyanate analogs of WF-23 can be used as potential ligands to map distinct binding sites of cocaine analogs at DAT.

Keywords: Abbreviations; DAT; dopamine transporter; SERT; 5-HT transporter; NET; norepinephrine transporter; WF-23; 2-β-propanoyl-3-β-(2-naphthyl)tropane; HD-205: 2-β-propanoyl-3-β-(2-naphthyl)-8-[(4-isothiocyanato)benzyl]nortropane; HD-206; 2-β-propanoyl-3-β-(2-naphthyl)-8-benzyl nortropane; HD-243; 2-β-propanoyl-3-β-(6-trimethylsilyl-2-naphthyl)-8-[(4-isothiocyanato)benzyl]nortropane; HD-244; 2-β-propanoyl-3-β-(6-iodo-2-naphthyl)-8-[(4-isothiocyanato)benzyl] nortropaneCocaine; Dopamine transporter; Serotonin transporter; Norepinephrine transporter; Tropane; Phenylisothiocyanate

The effect of quercetin phase II metabolism on its MRP1 and MRP2 inhibiting potential by Jelmer J. van Zanden; Hester van der Woude; Judith Vaessen; Mustafa Usta; Heleen M. Wortelboer; Nicole H.P. Cnubben; Ivonne M.C.M. Rietjens (pp. 345-351).

The present study characterises the effect of phase II metabolism, especially methylation and glucuronidation, of the model flavonoid quercetin on its capacity to inhibit human MRP1 and MRP2 activity in Sf9 inside-out vesicles. The results obtained reveal that 3′- O-methylation does not affect the MRP inhibitory potential of quercetin. However, 4′- O-methylation appeared to reduce the potential to inhibit both MRP1 and MRP2. In contrast, glucuronidation in general, and especially glucuronidation at the 7-hydroxylmoiety, resulting in 7- O-glucuronosyl quercetin, significantly increased the potential of quercetin to inhibit MRP1 and MRP2 mediated calcein transport with inhibition of MRP1 being generally more effective than that of MRP2. Overall, the results of this study reveal that the major phase II metabolites of quercetin are equally potent or even better inhibitors of human MRP1 and MRP2 than quercetin itself. This finding indicates that phase II metabolism of quercetin could enhance the potential use of quercetin- or flavonoids in general—as an inhibitor to overcome MRP-mediated multidrug resistance.

Keywords: ABC transporters; MRP1; MRP2; Quercetin; Flavonoids; Phase II metabolism

Comparison of 2-aminophenol and 4-nitrophenol as in vitro probe substrates for the major human hepatic sulfotransferase, SULT1A1, demonstrates improved selectivity with 2-aminophenol by Zoe Riches; Jackie C. Bloomer; Michael W.H. Coughtrie (pp. 352-358).

Sulfation, catalysed by members of the cytosolic sulfotransferase (SULT) enzyme family, is important in xenobiotic detoxification and in the biosynthesis and homeostasis of many hormones and neurotransmitters. The major human phenol sulfotransferase SULT1A1 plays a key role in chemical defence, is widely expressed in the body and is subject to a common polymorphism that results in reduced protein levels. Study of these enzymes in vitro requires robust probe substrates, and we have previously shown measurement of activity with the widely used SULT1A1 substrate, 4-nitrophenol, does not accurately reflect protein expression. Additionally, the high degree of substrate inhibition observed with this compound further reduces its value as a probe for SULT1A1. Here we show that 2-aminophenol is a more suitable probe substrate for quantifying SULT1A1 activity in human liver. This compound is sulfated at a high rate ( V_max with purified recombinant SULT1A1=121nmol/(minmg) and shows strong affinity for the enzyme ( K_m with purified recombinant SULT1A1=9μM) and, importantly, is a very poor substrate for the other major SULT1 enzyme expressed in liver, SULT1B1 (with V_max and K_m values of 17nmol/(minmg) and 114μM, respectively). Experiments with purified recombinant human SULTs and a panel of 28 human liver cytosols demonstrated that 2-aminophenol shows limited substrate inhibition with SULT1A1, and V_max values measured in liver cytosols correlated strongly with SULT1A1 enzyme protein levels measured by a quantitative immunoblot method. We therefore suggest that 2-aminophenol is a suitable substrate to use for quantifying SULT1A1 enzyme activity.

Keywords: Abbreviations; SULT; sulfotransferase; PAPS; 3′-phosphoadenosine 5′-phosphosulfateSulfotransferase; Sulfation; Drug metabolism; Detoxification; Human liver; Enzyme kinetics

Substrate specificity of MATE1 and MATE2-K, human multidrug and toxin extrusions/H⁺-organic cation antiporters by Yuko Tanihara; Satohiro Masuda; Tomoko Sato; Toshiya Katsura; Osamu Ogawa; Ken-ichi Inui (pp. 359-371).

The substrate specificities of human (h) multidrug and toxin extrusion (MATE) 1 and hMATE2-K were examined to find functional differences between these two transporters by the transfection of the cDNA of hMATE1 and hMATE2-K into HEK293 cells. Western blotting revealed specific signals for hMATE1 and hMATE2-K consistent with a size of 50 and 40kDa, respectively, in the transfectants as well as human renal brush-border membranes under reducing conditions. In the presence of oppositely directed H⁺-gradient, the transport activities of various compounds such as tetraethylammonium, 1-methyl-4-phenylpyridinium, cimetidine, metformin, creatinine, guanidine, procainamide, and topotecan were stimulated in hMATE1- and hMATE2-K-expressing cells. In addition to cationic compounds, anionic estrone sulfate, acyclovir, and ganciclovir were also recognized as substrates of these transporters. Kinetic analyses demonstrated the Michaelis–Menten constants for the hMATE1-mediated transport of tetraethylammonium, 1-methyl-4-phenylpyridinium, cimetidine, metformin, guanidine, procainamide, topotecan, estrone sulfate, acycrovir, and ganciclovir to be (in mM) 0.38, 0.10, 0.17, 0.78, 2.10, 1.23, 0.07, 0.47, 2.64, and 5.12, respectively. Those for hMATE2-K were 0.76, 0.11, 0.12, 1.98, 4.20, 1.58, 0.06, 0.85, 4.32, and 4.28, respectively. Although their affinity for hMATE1 and hMATE2-K was similar, the zwitterionic cephalexin and cephradine were revealed to be specific substrates of hMATE1, but not of hMATE2-K. Levofloxacin and ciprofloxacin were not transported, but were demonstrated to be potent inhibitors of these transporters. These results suggest that hMATE1 and hMATE2-K function together as a detoxication system, by mediating the tubular secretion of intracellular ionic compounds across the brush-border membranes of the kidney.

Keywords: Abbreviations; hOCT; human organic cation transporter; hMATE; human multidrug and toxin extrusion; MPP; 1-methyl-4-phenylpyridinium; TEA; tetraethylammoniumBrush-border membrane; Cephalexin; MATE1; MATE2-K; Organic cation

Interaction between the catalytic and modifier subunits of glutamate-cysteine ligase by Yi Yang; Ying Chen; Elisabet Johansson; Scott N. Schneider; Howard G. Shertzer; Daniel W. Nebert; Timothy P. Dalton (pp. 372-381).

Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione (GSH) biosynthesis pathway. This enzyme is a heterodimer, comprising a catalytic subunit (GCLC) and a regulatory subunit (GCLM). Although GCLC alone can catalyze the formation ofl-γ-glutamyl-l-cysteine, its binding with GCLM enhances the enzyme activity by lowering the K_m for glutamate and ATP, and increasing the K_i for GSH inhibition. To characterize the enzyme structure–function relationship, we investigated the heterodimer formation between GCLC and GCLM, in vivo using the yeast two-hybrid system, and in vitro using affinity chromatography. A strong and specific interaction between GCLC and GCLM was observed in both systems. Deletion analysis indicated that most regions, except a portion of the C-terminal region of GCLC and a portion of the N-terminal region of GCLM, are required for the interaction to occur. Point mutations of selected amino acids were also tested for the binding activity. The GCLC Cys248Ala/Cys249Ala and Pro158Leu mutations enzyme showed the same strength of binding to GCLM as did wild-type GCLC, yet the catalytic activity was dramatically decreased. The results suggest that the heterodimer formation may not be dependent on primary amino-acid sequence but, instead, involves a complex formation of the tertiary structure of both proteins.

Keywords: JEL classification; Protein interactionsAbbreviations; GCL; glutamate-cysteine ligase; Gclc; and GCLC; the mouse gene and cDNA/mRNA/enzyme, respectively, for the catalytic subunit of GCL; GCLC; and GCLC; the human gene and cDNA/mRNA/enzyme, respectively, for the catalytic subunit of GCL; Gclm; and GCLM; the mouse gene and cDNA/mRNA/enzyme, respectively, for the modifier subunit of GCL; GCLM; and GCLM; the human gene and cDNA/mRNA/enzyme, respectively, for the modifier subunit of GCL; GSH; reduced glutathione; GSSG; oxidized dimer of glutathione; SV40; simian virus-40; AD; activation domain; BD; DNA-binding domainGlutathione biosynthesis; Glutamate-cysteine ligase; Protein–protein interactions; Yeast two-hybrid system; Histidine-tagged protein; Nickel-NTA column; Oxidative stress

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Biochemical Pharmacology (v.74, #2)