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Biochemical Pharmacology (v.69, #11)

Editorial Advisory Board (pp. iii).

Role of PI3K/Akt and MEK/ERK signaling pathways in sulforaphane- and erucin-induced phase II enzymes and MRP2 transcription, G₂/M arrest and cell death in Caco-2 cells by Jana Jakub�kov�; J�n Sedl�k; Richard Mithen; Yongping Bao (pp. 1543-1552).

Isothiocyanate sulforaphane is an extensively studied cancer chemopreventive agent in human diet. In this study, the effects of sulforaphane (SFN) and its sulfide analog, erucin (ERN), have been examined on the induction of the phase II enzymes, quinine oxidoreductase (NQO1) and UDP-glucuronosyl transferase (UGT1A1), multidrug transporter (MRP2), cell cycle arrest and cell death in human colon adenocarcinoma Caco-2 cells. Additionally, the roles of PI3K/Akt and MEK/ERK signaling pathways have been assessed in these sulforaphane- and erucin-induced events. Although erucin and sulforaphane have similar IC₅₀ values (21 and 23μM after 72h treatment), erucin was more effective in the induction of G₂/M accumulation, depletion of mitochondrial potential, induction of cell death and mRNA induction of phase II enzymes and MRP2. Erucin (20μM) induced the mRNAs of NQO1, UGT1A1 and MRP2 by 11.1-, 11.6- and 6.7-fold, whereas sulforaphane (20μM) induced 3.3-, 5.3- and 2.2-fold, respectively. Both erucin and sulforaphane induced activation (phosphorylation) of ERK1/2 and Akt kinases but had no effect on JNK and p38 activation. Erucin-induced phase II enzyme transcriptions were decreased by PI3K and MEK1 inhibitors (LY294002 and PD98059), but the decreases in sulforaphane-induced transcription were less marked. Erucin induced a large increase in G₂/M cell number than sulforaphane. The ability of kinase inhibitors to overcome G₂/M block was low with the exception of PD98059 in sulforaphane-treated cells. Both, sulforaphane and erucin at high concentrations induced accumulation of sub-G₁ cells, cell death and dissipation of mitochondrial membrane potential. Taken together, these results demonstrate that PI3K/Akt and MEK/ERK signals are important intracellular mediators in erucin- and sulforaphane-mediated phase II enzyme transcription and cell cycle arrest in Caco-2 cells.

Keywords: Abbreviations; Akt; protein kinase B; ERKs; extracellular signal-regulated kinases; ITCs; isothiocyanates; JNK; c-Jun N-terminal kinase; MAPKs; mitogen-activated protein kinases; MRP2; multidrug resistance-associated protein 2; NF-κB; nuclear factor-kappa B; NQO1; NADP(H):quinine oxidoreductase; Nrf2; nuclear factor E2-related factor 2; PBS; phosphate-buffered saline; PI; propidium iodide; PI3K; phosphoinositide 3-kinase; UGTs; UDP-glucuronosyl transferases; TBS; Tris-buffered salineIsothiocyanate; Signaling pathway; Cell cycle; Apoptosis; Chemotherapeutics

The potential benefits of 1.5% hetastarch as a cardioplegia additive by James A. Coles Jr.; Daniel C. Sigg; Paul A. Iaizzo (pp. 1553-1558).

Myocardial edema is a clinically relevant problem found in post-ischemic reperfused hearts. The objective of this study was to understand the effects of hetastarch-supplemented cardioplegia on post-ischemic edema and cardiac function.Swine hearts were arrested with either St. Thomas Hospital cardioplegia with ( n=6) or without ( n=7) 1.5% hetastarch. Following hypothermic global ischemia, hearts were crystalloid reperfused in a four-chamber isolated working mode.Hetastarch decreased myocardial water content gains after three hours of reperfusion (control versus hetastarch, hour 0: 67�5% versus 67�3%, NS; hour 3: 82�2% versus 78�1%, p=0.1). Post-ischemic control group left ventricular end-diastolic pressures were elevated after 1h (in mm Hg, hour 0: 13�2, hour 1: 19�3, hour 2: 19�3, hour 3: 20�2) but remained stable (<16mm Hg) in the hetastarch group. Post-reperfusion creatine phosphokinase perfusate levels in the hetastarch treated hearts were decreased (control: 1.6IU/l/g versus hetastarch: 0.6IU/l/g, p=0.15).Hetastarch treatment delayed myocardial edema development and attenuated myocardial creatine kinase efflux, thereby preserving diastolic function.

Keywords: Cardiac; Ischemia; Reperfusion; Hetastarch; Edema; Swine

Antisense gene delivered by an adenoassociated viral vector inhibits iron uptake in human intestinal cells: Potential application in hemochromatosis by Fernando Ezquer; Marco Tulio N��ez; Yedy Israel (pp. 1559-1566).

Hereditary hemochromatosis (HH) is a condition in which intestinal iron absorption is greatly elevated. Present treatment is weekly phlebotomy, affecting quality of life and leading to recurrent infections. The iron transporter divalent metal transporter-1 (DMT-1) of enterocytes is responsible for iron uptake from the intestinal lumen; iron is further extruded into the blood by the basolateral transporter ferroportin-1. A therapeutic approach for HH could start with a long-term reduction of iron transport by reduction of DMT-1 levels. We designed an AAV vector coding for a short antisense RNA (AAV-DMT-1-AS) against DMT-1, which reduced iron uptake by 50–60% in human intestinal cells (Caco-2). At low infection levels, DMT-1 mRNA virtually disappeared, suggesting RNAi-like and/or RNase H antisense effects. DMT-1 mRNA levels returned to normal at higher infection levels, indicating that an additional mechanism of mRNA occupation, able to block DMT-1 translation and to avoid feedback regulation by iron responsive elements (IRE), also exists. Cell morphology was normal in all cases and no increases in the interferon-related responses, measured by (a) 2′-5′ A oligo synthetase (b) IFITM1 and (c) ISGF3γ mRNA levels, were observed. Studies presented herein indicate that enterocyte targeting with a gene coding for a short antisense against iron transport blocks enterocyte iron uptake, which may have therapeutic value.

Keywords: Hemochromatosis; Antisense; Adenoassociated vector; Gene therapy; Iron; Caco-2 cells

Inositol 1,4,5-triphosphate-mediated shuttling between intracellular stores and the cytosol contributes to the sustained elevation in cytosolic calcium in FMLP-activated human neutrophils by Ronald Anderson; Helen C. Steel; Gregory R. Tintinger (pp. 1567-1575).

The current study was designed to probe Ca²⁺ shuttling between intracellular stores and the cytosol as a potential mechanism contributing to the prolongation of elevated Ca²⁺ transients in N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-activated human neutrophils. Cytosolic Ca²⁺ concentrations and transmembrane fluxes of the cation were measured using spectrofluorimetric and radiometric procedures, respectively, while inositol 1,4,5-triphosphate (IP₃) was measured using a radioreceptor assay. The Ca²⁺-chelating agent, ethylene glycol-bis (β-aminoethyl ether) N, N, N′ N′-tetraacetic acid (EGTA; 10mM), was used to exclude store-operated influx of Ca²⁺ into neutrophils, while the IP₃ receptor antagonist, 2-aminoethoxydiphenyl borate (2-APB, 100μM), added to the cells 10s after FMLP (0.01 and 1μM), at which time the increases in IP₃ and cytosolic Ca²⁺ were maximal, was used to eliminate both sustained release from stores and influx of Ca²⁺. Addition of FMLP at 0.01 or 1μM resulted in equivalent peak increases in cytosolic Ca²⁺, while the increase in IP₃ was greater and the rate of clearance of Ca²⁺ from the cytosol slower, in cells activated with 1μM FMLP. Treatment of the cells with either EGTA or 2-APB following addition of 1μM FMLP, completely (EGTA) or almost completely (2-APB) abolished the influx of Ca²⁺ and accelerated the rate of clearance of the cation from the cytosol. Post-peak cytosolic Ca²⁺ concentrations were lower, and the Ca²⁺ content of the stores higher, in cells treated with 2-APB. The involvement of IP₃ was confirmed by similar findings in cells treated with U-73122 (1μM), a selective inhibitor of phospholipase C. Taken together, these observations are compatible with IP₃-mediated Ca²⁺ shuttling in neutrophils activated with FMLP.

Keywords: Abbreviations; 2-APB; 2-aminoethoxydiphenyl borate; DMSO; dimethylsulphoxide; EGTA; ethylene glycol-bis (β-aminoethyl ether); N; ,; N; ,; N; ′; N; ′-tetraacetic acid; FMLP; N; -formyl-; l; -methionyl-; l; -leucyl-; l; -phenylalanine; IP; ₃; inositol 1,4,5-triphosphate; U-73122; 1-[6[((17β)-3-methoxyestra-1,3,5(10)-trien-17-yl)amino]hexyl]-1-H-pyrrole-2,5-dione; U-73343; 1-[6[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-2,5-pyrrolidinedione2-Aminoethoxydiphenyl borate; Calcium; Calcium shuttling; Inositol 1,4,5-triphosphate; Neutrophils

Down-regulatory effect of quercitrin gallate on nuclear factor-κB-dependent inducible nitric oxide synthase expression in lipopolysaccharide-stimulated macrophages RAW 264.7 by Byung Hak Kim; Sung Min Cho; Alavala Matta Reddy; Yeong Shik Kim; Kyung Rak Min; Youngsoo Kim (pp. 1577-1583).

Quercetin 3- O-β-(2″-galloyl)-rhamnopyranoside (QGR) is a naturally occurring quercitrin gallate, a polyphenolic compound isolated from Persicaria lapathifolia (Polygonaceae). In the present study, QGR compound was discovered to have inhibitory effect on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophages RAW 264.7. QGR compound attenuated LPS-induced synthesis of both mRNA and protein of inducible nitric oxide synthase (iNOS), in parallel, and inhibited LPS-induced luciferase expression as a reporter of iNOS promoter activity in the macrophages. As a mechanism of the anti-inflammatory action shown by QGR compound, suppression of nuclear factor (NF)-κB activation has been documented. QGR compound exhibited inhibitory effect on LPS-mediated NF-κB transcriptional activity in macrophages RAW 264.7. Furthermore, the compound inhibited LPS-mediated nuclear translocation of NF-κB p65 and DNA binding activity of NF-κB complex, in parallel, but did not influence LPS-mediated IκBα degradation. Taken together, QGR compound suppressed LPS-mediated NF-κB activation, specifically to nuclear localization step of NF-κB p65, which was attributable to its down-regulatory action on LPS-induced NO production and iNOS expression.

Keywords: Abbreviations; IκB; inhibitory κB; IKK; inhibitory κB kinase; iNOS; inducible nitric oxide synthase; LPS; lipopolysaccharide; NF-κB; nuclear factor-κB; NO; nitric oxide; PDTC; pyrrolidine dithiocarbamate; QGR; quercetin 3-; O; -β-(2″-galloyl)-rhamnopyranoside; SEAP; secretory alkaline phosphatase; TLR4; Toll-like receptor 4Quercitrin gallate; Nitric oxide; Inducible nitric oxide synthase; Nuclear factor-κB; Anti-inflammation; Macrophages

Relative importance of apoptosis and cell cycle blockage in the synergistic effect of combined R115777 and imatinib treatment in BCR/ABL-positive cell lines by Takuji Miyoshi; Tadashi Nagai; Ken Ohmine; Makiko Nakamura; Yasuhiko Kano; Kazuo Muroi; Norio Komatsu; Keiya Ozawa (pp. 1585-1594).

The combination of imatinib and a farnesyltransferase inhibitor might be effective for reducing the number of BCR/ABL-positive leukemia cells. In this study, we examined the differences in the mechanisms of the growth inhibitory effect of the combination of imatinib and R115777 (Zarnestra™) among BCR/ABL-positive cell lines. Steel and Peckham isobologram analysis indicated that this combination had a strong synergistic inhibitory effect on growth in all imatinib-resistant cell lines and their parental cell lines. Levels of cleaved caspase 3 were increased by the combination treatment in all cell lines. However, both the level of cleaved PARP and the number of annexin-V-positive cells were much less increased in KCL22 and KCL22/SR cells than in K562, KU812, K562/SR and KU812/SR cells. The combination treatment promoted p27^KIP1 accumulation and induced a significant increase in the percentage of G0/G1 KCL22 and KCL22/SR cells. In other cell lines, the percentage of G0/G1 cells was not increased but rather decreased. The results indicate that induction of apoptosis and blockage of the cell cycle were major mechanisms of the synergistic inhibitory effect of the combination treatment, but the relative importance of these mechanisms differed among cell types. Additional treatment for overriding the G1 checkpoint may be required to eradicate leukemia cells, in which the combination induces cell cycle arrest.

Keywords: R115777; Farnesyltransferase inhibitor; Imatinib; BCR/ABL; Chronic myeloid leukemia; Drug resistance

Regulation of CYP26A1 expression by selective RAR and RXR agonists in human NB4 promyelocytic leukemia cells by Nadia Idres; Julie Marill; Guy G. Chabot (pp. 1595-1601).

All- trans retinoic acid (ATRA) can induce complete remission in acute promyelocytic leukemia (APL), but resistance to this treatment develops rapidly partly due to increased ATRA metabolism. Among the cytochrome P450s (CYPs) involved in ATRA metabolism, the ATRA-inducible cytochrome P450 26A1 (CYP26A1) is particularly active although the molecular mechanisms involved in its regulation are not well defined in the target leukemia cells. To study CYP26A1 expression and regulation in APL cells, we used the NB4 promyelocytic leukemia cell line. CYP26A1 constitutive expression was barely detectable in NB4 cells, but ATRA could induce high levels of CYP26A1 expression, which reached a maximum at 72h. To further define CYP26A1 induction mechanisms in the NB4 leukemia cells, we used RARs and RXR selective agonists. The RARα agonist BMS753 could elicit maturation, as expected, but not CYP26A1 expression. Treatment with the RARβ agonist BMS641, or the RARβ/γ agonist BMS961, could not elicit maturation, as expected, nor induce CYP26A1 expression. Because CYP26A1 expression could not be induced by RAR ligands alone, NB4 cells were then co-treated with the RXR agonist BMS649. The RXR agonist alone could not induce CYP26A1 expression, nor in combination with either the RARβ agonist or the RARβ/γ agonist. However, the combination of the RXR agonist and the RARα agonist could elicit a marked induction of CYP26A1 expression. In conclusion, we have shown that CYP26A1 induction is not essential for the granulocytic maturation of NB4 leukemia cells, and that CYP26A1 induction requires the activation of both RARα and RXR in these cells.

Keywords: Abbreviations; APL; acute promyelocytic leukemia; ATRA; all-; trans; -retinoic acid; 9-; cis; -RA; 9-; cis; -retinoic acid; 13-; cis; -RA; 13-; cis; -retinoic acid; CYP; cytochrome P450; CYP26A1; cytochrome P450 26A1; RAR; retinoic acid receptor; RXR; retinoid X receptor CYP26A1; regulation; NB4 leukemia cells; RAR; RXR; Maturation

Morin inhibits 12- O-tetradecanoylphorbol-13-acetate-induced hepatocellular transformation via activator protein 1 signaling pathway and cell cycle progression by Chien-Yun Hsiang; Shih-Lu Wu; Tin-Yun Ho (pp. 1603-1611).

Flavonoids are constituents of fruits, vegetables, and plant-derived beverages, as well as components in herbal containing dietary supplements. They exhibit a remarkable spectrum of biochemical and pharmacological activities. In this study, we examined morin (3,5,7,2′,4′-pentahydroxyflavone) for its effect on 12- O-tetradecanoylphorbol-13-acetate (TPA)-treated human hepatocytes. Morin inhibited TPA-induced cellular transformation in Chang liver cells in a dose-dependent manner. Luciferase assay and electrophoretic mobility shift assay revealed that morin suppressed TPA-induced AP-1 activity, and the inhibition of AP-1 activity by morin was mediated through the inhibition of p38 kinase. Moreover, morin induced the S-phase arrest and inhibited the DNA synthesis in TPA-treated hepatocytes, suggesting that a cell cycle checkpoint was activated by morin to block DNA synthesis in S phase. In conclusion, our results suggested that morin was a potent anti-hepatocellular transformation agent that inhibited cellular transformation by suppressing the AP-1 activity and inducing the S-phase arrest in human hepatocytes.

Keywords: Abbreviations; TPA; 12-; O; -tetradecanoylphorbol-13-acetate; AP-1; activator protein 1; MAP; mitogen-activated protein; ATCC; American Type Culture Collection; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMEM; Dulbecco modified Eagle medium; FBS; fetal bovine serum; INT; p; -iodonitrotetrazolium violet; RLU; relative luciferase unit; EMSA; electrophoretic mobility shift assay; JNKs; c-Jun N-terminal kinases; ERKs; extracellular signal-regulated kinases; MEK 1/2; MAP kinase kinase 1/2Morin; Hepatocyte; Transformation; Activator protein 1; Mitogen-activated protein kinase; Cell cycle

Dicoumarol relieves serum withdrawal-induced G_0/1 blockade in HL-60 cells through a superoxide-dependent mechanism by Rosario I. Bello; Consuelo G�mez-D�az; Guillermo L�pez-Lluch; Nathalie Forthoffer; Mar�a C. C�rdoba-Pedregosa; Pl�cido Navas; Jos� M. Villalba (pp. 1613-1625).

This work was set to study how dicoumarol affects the cell cycle in human myeloid leukemia HL-60 cells. Cells were accumulated in G_0/1 after serum deprivation. However, when cells were treated with 5μM dicoumarol in serum-free medium, a significant increment in the number of cells in S-phase was observed. Inhibition of G_0/1 blockade was confirmed by the increase of thymidine incorporation, the phosphorylation of retinoblastoma protein, and the promotion of cell growth in long-term treatments in the absence of serum. Dicoumarol treatment increased superoxide levels, but did not affect peroxide. Increase of cellular superoxide was essential for inhibition of G_0/1 blockade, since scavenging this reactive species with a cell-permeable form of SOD and the SOD mimetics 2-amino-3,5-dibromo- N-[ trans-4-hydroxycyclohexyl]benzylamine (ambroxol, 100μM) and copper[II]diisopropyl salicylate (CuDIPS, 10μM) completely abolished the effect of dicoumarol. However, N-acetyl-cysteine, overexpression of Bcl-2 or a cell-permeable form of catalase were not effective. 5-Methoxy-1,2-dimethyl-3-[(4-nitrophenol)methyl]-indole-4,7-dione (ES936), a mechanism-based irreversible inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), did not promote S phase entry, and dicoumarol still inhibited G_0/1 blockade in the presence of ES936. We demonstrate that dicoumarol inhibits the normal blockade in G_0/1 in HL-60 cells through a mechanism involving superoxide, but this effect is not dependent solely on the inhibition of the NQO1 catalytic activity. Our results send a precautionary message about use of dicoumarol to elucidate cellular processes involving oxidoreductases.

Keywords: Abbreviations; ES936; 5-methoxy-1,2-dimethyl-3-[(4-nitrophenol)methyl]-indole-4,7-dione; FCS; fetal calf serum; NAC; N; -acetyl-cysteine; NQO1; isoform 1 of the cytosolic NAD(P)H:(quinone acceptor) oxidoreductase; DT; diaphorase; ROS; reactive oxygen species; TNF-α; tumor necrosis factor-α; pRb; retinoblastoma tumor suppressor gene product (hypophosphorylated); pRb-P; retinoblastoma tumor suppressor gene product (hyperphosphorylated)Cell cycle; Dicoumarol; HL-60 cells; NAD(P)H:quinone oxidoreductase 1; Superoxide

Azaspiracid-4 inhibits Ca²⁺ entry by stored operated channels in human T lymphocytes by Amparo Alfonso; Yolanda Rom�n; Mercedes R. Vieytes; Katsuya Ofuji; Masayuki Satake; Takeshi Yasumoto; Luis M. Botana (pp. 1627-1636).

Azaspiracids (AZs) are a new group of phycotoxins discovered in the Ireland coast that includes the isolated analogues: AZ-1, AZ-2, AZ-3, AZ-4 and AZ-5 and the recently described AZ-6–11. Azaspiracid toxic episodes show gastrointestinal illness, but neurotoxic symptoms are also observed in mouse bioassay. Despite their great importance in human health, so far its mechanism of action is largely unknown. In this report, we present the first data about the effect of AZ-4 on cytosolic calcium concentration [Ca²⁺]_i in freshly human lymphocytes. Cytoslic Ca²⁺ variations were determined by fluorescence digital imaging microscopy using Fura2 acetoxymethyl ester (Fura2-AM). AZ-4 did not modify cytosolic Ca²⁺ in resting cells. However, the toxin dose-dependent inhibited the increase in cytosolic Ca²⁺ levels induced by thapsigargin (Tg). AZ-4 decreased Ca²⁺-influx induced by Tg but did not affect the Ca²⁺-release from internal stores induced by this drug. The effects of AZ-4 on Ca²⁺-influx induced by Tg were reversible and not regulated by adenosine 3′,5′-cyclic monophosphate (cAMP) pathway. When AZ-4 was added before, after or together with nickel, an unspecific blocker of Ca²⁺ channels, the effects were indistinguishable and additive. AZ-4 also inhibited maitotoxin (MTX)-stimulated Ca²⁺-influx by 5–10%. Thus, AZ-4 appeared to be a novel inhibitor of plasma membrane Ca²⁺ channels, affecting at least to store operated channels, showing an effect clearly different from other azaspiracid analogues.

Keywords: Abbreviations; AZ; Azaspiracid; [Ca; ²⁺; ]; _i; cytosolic calcium concentration; cAMP; adenosine 3′,5′-cyclic monophosphate; dbcAMP; N; ⁶; ,2′-; O; -dibutyryladenosine 3′,5′-cyclic monophosphate; DG; 1,2 diacylglycerol; FSK; forskolin; MTX; maitotoxin; PSS; physiological saline solution; SOC; channels store-operated Ca; ²⁺; channels; SQ22,536; 9-(tetrahydro-2-furanyl)-9; H; -purin-6-amine; Tg; thapsigarginLymphocytes; Azaspiracids; Cytosolic calcium; Calcium channel; Calcium pools

Muscarinic subtype affinity and functional activity profile of 1-methyl-2-(2-methyl-1,3-dioxolan-4-yl)pyrrolidine and 1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine derivatives by Silvia Dei; Piero Angeli; Cristina Bellucci; Michela Buccioni; Fulvio Gualtieri; Gabriella Marucci; Dina Manetti; Rosanna Matucci; Maria Novella Romanelli; Serena Scapecchi; Elisabetta Teodori (pp. 1637-1645).

Starting from two previously studied muscarinic full agonists, characterized by a 1,3-dioxolane ((+)−1) and a 1,3-oxathiolane ((+)−2) cycle, two new series of muscarinic ligands were designed, obtained by the steric complication of the parent compounds produced by freezing the aminoalkyl chain into a pyrrolidine ring. Both tertiary amines and the corresponding iodomethyl derivatives were synthesised and studied, and several compounds of the series which behaved as muscarinic agonists have been selected, on the basis of preliminary binding experiments on rat cortex homogenates, for the present work.Results are presented obtained from testing the affinity of the selected compounds against cloned human muscarinic receptors expressed in CHO cells, in order to evaluate subtype selectivity. Their functional activity on classical models of M₁–M₄ receptors, in guinea pig and rabbit tissues is also reported.With respect to parent compounds, the new molecules present some selectivity toward hm2 receptors; fair M₂ selectivity is also evident in functional studies, where these compounds behave as partial agonists. Among the other compounds of the series (2 S, 4′ R, 2′ S)-1,1-dimethyl-2-(2-methyl-1,3-dioxolan-4-yl)pyrrolidinium iodide(−)−3 and (2 R, 5′ S, 2′ S)-1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine(+)−5 present a promising pharmacological profile. Compound(−)−3 shows modest hm2 selectivity in binding experiments but a clearcut M₂ selectivity in functional tests, where it behaves as a weak antagonist on M₁ and M₄ subtypes, as a weak full agonist on the M₃ subtype and as a potent partial agonist on M₂ subtype. Tertiary amine(+)−5 presents a quite similar profile but appears more interesting since, lacking a permanent charge on the nitrogen atom, it may represent an interesting tool to study CNS muscarinic receptors.Our results confirm that sterical complication of parent compounds(+)−1 and(+)−2 produces more selective muscarinic agonists.

Keywords: Muscarinic agonist; Subtype selectivity; Dioxolane derivative; Oxathiolane derivative; Binding studies; Functional studiesAbbreviations; CHO; Chinese hamster ovary; hm1–hm5; human muscarinic subtypes; M; ₁; –M; ₅; muscarinic subtypes; K; _i; inhibitor equilibrium dissociation constant; p; K; _i; -log; K; _i; pD; ₂; -log; ED; ₅₀; K; _b; antagonist affinity from functional tests; p; K; _b; -log; K; _b; McN-A-343; 4-(; N; -[3-chlorophenyl]-carbamoyloxy-2-butynyltrimethylammonium chloride; pCl-McN-A-343; (4-(; N; -[4-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammonium chloride; NMS; N; -methylscopolamine; K; _D; affinity constant of the labeled ligand; B; _max; total receptor density; N; non specific binding; PSS; physiological salt solution

Hypoxia induces changes in expression of isoforms of the divalent metal transporter (DMT1) in rat pheochromocytoma (PC12) cells by Agnieszka Lis; Prasad N. Paradkar; Steve Singleton; Hung-Chieh Kuo; Michael D. Garrick; Jerome A. Roth (pp. 1647-1655).

Although hypoxia has been shown to increase the expression of a variety of proteins involved in iron homeostasis, including transferrin and its receptor, little is known about the effect of low oxygen on formation of isoforms of the major iron transport protein, divalent metal transporter 1, DMT1. Accordingly, we examined the effects of hypoxia on expression and subcellular distribution of the different isoforms of DMT1 in rat PC12 cells. Treatment with low oxygen modestly increased expression of protein and mRNA levels for both the +IRE and −IRE species of DMT1. In contrast, expression of the exon 1A containing species of DMT1 was greatly increased by hypoxia as indicated by Western blot and real-time RT-PCR analysis. Message levels for the 1A isoforms increased approximately 60-fold after exposure of PC12 cells to 1% oxygen for 5h. The subcellular distribution of exon 1A isoforms of DMT1 remained consistently in the cytoplasmic milieu of the cell after hypoxic exposure, as also did the distribution of +IRE species of DMT1. The −IRE species of DMT1, however, responded to hypoxia by becoming increasingly associated with the regions adjoining the outer cellular membranes, while a portion partially colocalized with an early endosomal marker (EEA). Hypoxia also caused a significant increase in the uptake of manganese in PC12 cells. In summary, these results demonstrate that hypoxia selectively increases expression of exon 1A containing species of DMT1 with lesser increases in either the +IRE or −IRE isoforms the transporter.

Keywords: Abbreviations; DMT1; divalent metal transporter 1; HIF; hypoxia inducing factor; HRE; hypoxic response elements; IRE; iron response element; IRP; iron response proteins; Tf; transferrin; TfR; transferrin receptorDivalent metal transporter 1; DMT1; PC12 cells; Hypoxia; HIF; Manganese; Iron response element

Reversal of in vitro cellular MRP1 and MRP2 mediated vincristine resistance by the flavonoid myricetin by Jelmer J. van Zanden; Anika de Mul; Heleen M. Wortelboer; Mustafa Usta; Peter J. van Bladeren; Ivonne M.C.M. Rietjens; Nicole H.P. Cnubben (pp. 1657-1665).

In the present study, the effects of myricetin on either MRP1 or MRP2 mediated vincristine resistance in transfected MDCKII cells were examined. The results obtained show that myricetin can inhibit both MRP1 and MRP2 mediated vincristine efflux in a concentration dependent manner. The IC₅₀ values for cellular vincristine transport inhibition by myricetin were 30.5±1.7μM for MRP1 and 24.6±1.3μM for MRP2 containing MDCKII cells. Cell proliferation analysis showed that the MDCKII control cells are very sensitive towards vincristine toxicity with an IC₅₀ value of 1.1±0.1μM. The MDCKII MRP1 and MRP2 cells are less sensitive towards vincristine toxicity with IC₅₀ values of 33.1±1.9 and 22.2±1.4μM, respectively. In both the MRP1 and MRP2 cells, exposure to 25μM myricetin enhances the sensitivity of the cells towards vincristine toxicity to IC₅₀ values of 7.6±0.5 and 5.8±0.5μM, respectively. The increase of sensitivity represents a reversal of the resistance towards vincristine as a result of MRP1 and MRP2 inhibition. Thus, the present study demonstrates the ability of the flavonoid myricetin to modulate MRP1 and MRP2 mediated resistance to the anticancer drug vincristine in transfected cells, indicating that flavonoids might be a valuable adjunct to chemotherapy to block MRP mediated resistance.

Keywords: Abbreviations; GSH; glutathione; GS-X; glutathione conjugate; IC; ₅₀; 50% inhibition concentration; MDR; multidrug resistance; MRP; multidrug resistance proteinMRP1; MRP2; Flavonoids; Vincristine; Multidrug resistance; Myricetin

Prevention of hemorrhagic shock-induced lung injury by heme arginate treatment in rats by Kyoichiro Maeshima; Toru Takahashi; Kenji Uehara; Hiroko Shimizu; Emiko Omori; Masataka Yokoyama; Toru Tani; Reiko Akagi; Kiyoshi Morita (pp. 1667-1680).

Hemorrhagic shock followed by resuscitation (HSR) induces oxidative stress, which leads to acute lung injury. Heme oxygenase (HO)-1 (EC 1.14.99.3), the rate-limiting enzyme in heme catabolism, is inducible by oxidative stress and is thought to play an important role in the protection from oxidative tissue injuries. In this study, we examined expression of HO-1 as well as tissue injuries in the lung, liver, and kidney after HSR in rats. We also pretreated animals with heme arginate (HA), a strong inducer of HO-1, and examined its effect on the HSR-induced lung injury. HO-1 expression significantly increased in the liver and kidney following HSR, while its expression in the lung was very low and unchanged after HSR. In contrast to HO-1 expression, tissue injury and tumor necrosis factor-α (TNF-α) gene expression was more prominent in the lung compared with those in the liver and kidney. HA pretreatment markedly induced HO-1 in pulmonary epithelial cells, and ameliorated the lung injury induced by HSR as judged by the improvement of histological changes, while it decreased TNF-α and inducible nitric oxide synthase gene expression, lung wet weight to dry weight ratio, and myeloperoxidase activity. In contrast, inhibition of HO-1 by tin-mesoporphyrin administration abolished the beneficial effect of HA pretreatment. These findings suggest that tissues with higher HO-1 may be better protected than those with lower HO-1 from oxidative tissue injury induced by HSR. Our findings also indicate that HA pretreatment can significantly suppress the HSR-induced lung injury by virtue of its ability to induce HO-1.

Keywords: Abbreviations; ALAS; δ-aminolevulinate synthase; ALI; acute lung injury; CO; carbon monoxide; HA; heme arginate; HO; heme oxygenase; HSR; hemorrhagic shock followed by resuscitation; iNOS; inducible nitric oxide synthase; IOD; integrated optical density; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; MPO; myeloperoxidase; SnMP; tin-mesoporphyrin; TNF-α; tumor necrosis factor-α; wet/dry; wet-weight to dry-weightAcute lung injury; Heme arginate; Heme oxygenase-1; Hemorrhagic shock; Inflammation; Oxidative injury

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Biochemical Pharmacology (v.69, #11)