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Biochemical Pharmacology (v.70, #4)

Editorial Advisory Board (pp. iii).

Use-dependent blockade of Ca_v2.2 voltage-gated calcium channels for neuropathic pain by Raymond J. Winquist; Jennifer Qian Pan; Valentin K. Gribkoff (pp. 489-499).

The translocation of extracellular calcium (Ca²⁺) via voltage-gated Ca²⁺ channels (VGCCs) in neurons is involved in triggering multiple physiological cell functions but also the abnormal, pathophysiological responses that develop as a consequence of injury. In conditions of neuropathic pain, VGCCs are involved in supplying the signal Ca²⁺ important for the sustained neuronal firing and neurotransmitter release characteristic of these syndromes. Preclinical data have identified N-type VGCCs (Ca_v2.2) as key participants in contributing to these Ca²⁺ signaling events and clinical data with the peptide blocker Prialt™ have now validated Ca_v2.2 as a bona fide target for future drug discovery efforts to identify new and novel therapeutics for neuropathic pain. Imperative for the success of such an endeavor will be the ability to identify compounds selective for Ca_v2.2, versus other VGCCs, but also compounds which demonstrate effective blockade during the pathophysiological states of neuropathic pain without compromising channel activity associated with sustaining normal housekeeping cellular functions. An approach to obtain this research target profile is to identify compounds, which are more potent in blocking Ca_v2.2 during higher frequencies of firing as compared to the slower more physiologically-relevant frequencies. This may be achieved by identifying compounds with enhanced potency for the inactivated state of Ca_v2.2. This commentary explores the rationale and options for engineering a use-dependent blocker of Ca_v2.2. It is anticipated that this use-dependent profile of channel blockade will result in new chemical entities with an improved therapeutic ratio for neuropathic pain.

Keywords: Abbreviations; VGCC; voltage-gated calcium channel; Ct; conotoxin; V; _Rest; resting membrane potential; V; _Threshold; threshold membrane potential for activationNeuropathic pain; Voltage-gated calcium channels; Use-dependent block

Interactions of mefloquine with ABC proteins, MRP1 (ABCC1) and MRP4 (ABCC4) that are present in human red cell membranes by Chung-Pu Wu; Antonios Klokouzas; Stephen B. Hladky; Suresh V. Ambudkar; Margery A. Barrand (pp. 500-510).

Human erythrocyte membranes express the multidrug resistance-associated proteins, MRP1, MRP4 and 5, that collectively can efflux oxidised glutathione, glutathione conjugates and cyclic nucleotides. It is already known that the quinoline derivative, MK-571, is a potent inhibitor of MRP-mediated transport. We here examine whether the quinoline-based antimalarial drugs, amodiaquine, chloroquine, mefloquine, primaquine, quinidine and quinine, also interact with erythrocyte MRPs with consequences for their access to the intracellular parasites or for efflux of oxidised glutathione from infected cells. Using inside-out vesicles prepared from human erythrocytes we have shown that mefloquine and MK-571 inhibit transport of 3μM [³H]DNP-SG known to be mediated by MRP1 (IC₅₀ 127 and 1.1μM, respectively) and of 3.3μM [³H]cGMP thought but not proven to be mediated primarily by MRP4 (IC₅₀ 21 and 0.41μM). They also inhibited transport in membrane vesicles prepared from tumour cells expressing MRP1 or MRP4 and blocked calcein efflux from MRP1-overexpressing cells and BCECF efflux from MRP4-overexpressing cells. Both stimulated ATPase activity in membranes prepared from MRP1 and MRP4-overexpressing cells and inhibited activity stimulated by quercetin or PGE₁, respectively. Neither inhibited [α-³²P]8-azidoATP binding confirming that the interactions are not at the ATP binding site. These results demonstrate that mefloquine and MK-571 both inhibit transport of other substrates and stimulate ATPase activity and thus may themselves be substrates for transport. But at concentrations achieved clinically mefloquine is unlikely to affect the MRP1-mediated transport of GSSG across the erythrocyte membrane.

Keywords: Abbreviations; BCECF; 2′,7′-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein; BCECF-AM; BCECF acetoxy-methyl ester; BeFx; Beryllium fluoride; calcein-AM; calcein acetoxy-methyl ester; DNP-SG; dinitrophenyl S-glutathione conjugate; GSH; reduced glutathione; GSSG; oxidised glutathione; MK-571; (3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-(dimethyl amino-3-oxo propyl)thio)methyl)thio)propanoic acid; MRP; multidrug resistance-associated proteinATP hydrolysis; Mefloquine; Multidrug resistance-associated proteins 1 and 4; MK-571; Erythrocyte membranes

Mechanism of action of a novel “combi-triazene�? engineered to possess a polar functional group on the alkylating moiety: Evidence for enhancement of potency by Fouad Brahimi; Zakaria Rachid; James P. McNamee; Moulay A. Alaoui-Jamali; Ana M. Tari; Bertrand J. Jean-Claude (pp. 511-519).

Previous studies showed that SMA41, a 3-methyltriazene termed “combi-molecules�? possessing a dual epidermal growth factor receptor (EGFR)/DNA targeting properties induced potent antiproliferative activity against alkylating-agent-resistant cells expressing EGFR in vitro. However, despite its marked potency, its antitumour activity in vivo was significantly hampered by its poor hydrosolubility and the moderate reactivity of its alkylating moiety. To circumvent this problem, we designed the quinazolinotriazene ZRBA1 to contain a N, N-dimethylaminoethyl group grafted to the 3-position of the triazene chain where it could serve both as a water soluble and a more potent alkylating moiety. ZRBA1 exhibited five-fold stronger EGFR tyrosine kinase (TK) inhibitory activity (IC₅₀=37nM) than SMA41, decomposed into a 6-amino-quinazoline FD105 (IC₅₀=200nM) and preferentially blocked EGF- over platelet-derived growth factor (PDGF)-or serum-induced cell growth. ZRBA1 induced DNA damage, concomitantly blocked EGF-stimulated EGFR phosphorylation by a partially irreversible mechanism in MDA-MB-468 breast cancer cells, and induced partially irreversible antiproliferative activity. It also prevented EGFR-mediated MAP kinase activation and, in contrast to FD105 and SMA41, induced high levels of apoptosis. Furthermore, ZRBA1 showed significantly greater antitumor activity ( p<0.05) than SMA41 in the human MDA-MB-468 breast cancer xenograft model. The results in toto indicate that the appendage of N, N-dimethylaminoethyl to combi-triazenes may be an alternative to the reduced hydrosolubility and also to the lack of potency of monofunctional combi-triazenes against resistant tumours.

Keywords: EGFR signaling; Quinazolines; DNA damage; Apoptosis; O; ⁶; -alkylguanine transferase; In vivo efficacy

Effect of statins on the proteasomal activity in mammalian endothelial and vascular smooth muscle cells by Antje Ludwig; Britt Friedel; Susanne Metzkow; Silke Meiners; Verena Stangl; Gert Baumann; Karl Stangl (pp. 520-526).

Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, known as statins, effectively prevent cardiovascular events. In addition to their lipid lowering properties, a variety of pleiotropic effects on cardiovascular cells were demonstrated in vitro and in vivo. It has been hypothized that statins deploy a part of their effects by targeting the proteasome. Statin-induced effects remarkably overlap with effects obtained by inhibition of the proteasome in endothelial and vascular smooth muscle cells (e.g., endothelial nitric oxide synthase (eNOS)-upregulation, attenuation of nuclear factor kappa B (NF-κB) activation, inhibition of proliferation). We therefore examined, whether statins modulate the proteasomal activity of vascular cells. We studied the effect of simvastatin, atorvastatin, and pravastatin as well as of the proteasome inhibitor clasto-lactacystin on morphology, proliferation, viability, and proteasomal activity in two mammalian endothelial cell lines (CPAE and Ea.hy962), and in primary vascular smooth muscle cells (VSMCs). Both statins and lactacystin induced comparable morphological changes and attenuated proliferation of calf pulmonary artery cell line (CPAE). Whereas the statin-induced effects were reversed by mevalonic acid, however, the lactacystin-induced alterations were not influenced by mevalonic acid. As expected, lactacystin caused a significant loss of proteasomal activity measured in the extract of treated CPAE cells, whereas the extracts of statin-treated CPAEs exhibited unchanged activities. This result was also confirmed in Ea.hy926 cells and in primary rat VSMCs. We show here, that even high doses of statins do not modulate the activities of purified human 20S proteasomes. We conclude that the similar biological effects of statins and proteasome inhibitors in vascular cells are not due to a common inhibitory mechanism of action on the proteasome.

Keywords: Abbreviations; CaspL; caspase-like activity; ChTL; chymotrypsin-like activity; DMSO; dimethylsulfoxide; eNOS; endothelial nitric oxide synthase; HMGR; 3-hydroxy-3-methylglutaryl-coenzyme A reductase; MVA; mevalonic acid; NF-κB; nuclear factor kappa B; TL; trypsin-like activity; VSMC; vascular smooth muscle cellStatins; Proteasome; Endothelial cells; Smooth muscle cells

S-15261, a new anti-hyperglycemic agent, reduces hepatic glucose production through direct and insulin-sensitizing effects by M. Ca�zac; C. Kohl; J. Girard; J.P. P�gorier (pp. 527-534).

S-15261 is a new oral anti-hyperglycemic agent that increases insulin sensitivity in various insulin-resistant animal models. The aim of this study was to determine the short- and long-term effects of S-15261 and its metabolites (S–15511 and Y-415) on fatty acid and glucose metabolism in hepatocytes isolated from 24-h starved rats. During short-term exposure (1h) neither S-15261 nor its metabolites affected fatty acid oxidation whatever the concentration used. By contrast, S-15261 and its two metabolites reduced the rates of glucose production from lactate/pyruvate and dihydroxyacetone. Using crossover plot analysis, it was shown that Y-415 reduced hepatic gluconeogenesis upstream the formation of dihydroxyacetone phosphate. After 48h in culture, S-15261 and its two metabolites reduced the rates of glucose production from lactate/pyruvate secondarily to a decrease in PEPCK and Glc-6-Pase mRNA levels. A part of these effects on gene expression could be due to a drug-induced reduction in PGC-1 gene expression. When hepatocytes were cultured in the presence of a submaximal concentration of insulin (10⁻⁹M), S-15261, through its metabolite S-15511, enhanced insulin sensitivity both on gene expression (PEPCK, Glc-6-Pase, PGC-1) and on gluconeogenesis. Furthermore, S-15261 and S-15511 induced the expression of GK and FAS genes as the result of an increased in SREBP-1c mRNA levels. Finally, S-15511 enhanced the stimulatory effect of insulin on GK mRNA level through an additional increase in SREBP-1c gene expression.In conclusion, this work reveals that S-15261 via its metabolites reduces hepatic glucose production through direct and insulin-sensitizing effects on genes encoding regulatory proteins of hepatic glucose metabolism.

Keywords: Anti-diabetic agent; Gluconeogenesis; Fatty acid oxidation; Gluconeogenic; Glycolytic and lipogenic gene expression

Targeting neutrophil collagenase/matrix metalloproteinase-8 and gelatinase B/matrix metalloproteinase-9 with a peptidomimetic inhibitor protects against endotoxin shock by Jialiang Hu; Philippe E. Van den Steen; Chris Dillen; Ghislain Opdenakker (pp. 535-544).

Gram-negative sepsis, bacterial meningitis and endotoxin shock are life-threatening disorders, associated with the rapid release of neutrophil enzymes. Neutrophil collagenase/matrix metalloproteinase-8 (MMP-8) and gelatinase B/matrix metalloproteinase-9 (MMP-9) are contained in granules, are quickly exocytosed upon granulocyte activation and efficiently cleave intact and denatured collagens, respectively. Genetic ablation of gelatinase B protects against endotoxin-induced mortality. Therefore, we designed and synthesized a peptidomimetic gelatinase B inhibitor Regasepin1, and compared the selectivity for the collagenases MMP-1, MMP-8 and MMP-13. Regasepin1 was found to inhibit, almost to the same degree, the neutrophil enzymes MMP-8 and MMP-9 and the monocytic tumor necrosis factor-α (TNF-α) converting enzyme (TACE/ADAM-17) in vitro. With the use of mass spectrometry analysis, the plasma half-life of inhibitor levels was determined after an intraperitoneal bolus injection in mice. Plasma peak levels of the inhibitor were reached at 50min after intraperitoneal injection and the subsequent half-life in the circulation exceeded 40min. Regasepin1 protected mice against lethal endotoxinemia by intraperitoneal and intravenous injection routes. This proofs the principle that early neutrophil MMP inhibition followed by TACE blockade may become a treatment strategy of gram-negative sepsis, endotoxinemia and other life-threatening inflammatory reactions.

Keywords: Abbreviations; MMP; matrix metalloproteinase; TIMP; tissue inhibitor of metalloproteinase; LPS; lipopolysaccharide; PGN; peptidoglycan; TLR; Toll-like receptor; TNF-α; tumor necrosis factor-α; TACE; TNF converting enzyme; DMSO; dimethylsulphoxide; ip; intraperitoneal; iv; intravenousMatrix metalloproteinase; Lipopolysaccharide; Sepsis; Inflammation; Neutrophil; Endotoxin

Effects of ethanol on cytokine generation and NFκB activity in human lung epithelial cell by Anne-Sofie M. Johansson; Johan Lid�n; Sam Okret; Jan E.W. Palmblad (pp. 545-551).

Alcohol abuse is associated with enhanced risk for pulmonary infections, but the mechanisms remain obscure. We assessed whether ethanol reduced generation of cytokines from a human lung epithelial cell line (A549) in vitro and if effects on the NFκB transcription factor were involved. Exposure of A549 to ethanol (0.1–1%) dose-dependently inhibited (by 15–49%) the release of G-CSF and IL-8, but not of M-CSF, triggered by IL1β or TNFα. Ethanol also inhibited by 49% the IL-1β stimulated translocation of the p65 subunit of NFκB from the cytoplasm into the nucleus. Using a κB binding and luciferase coupled construct, transfected into A549 cells, we found that 1% ethanol specifically reduced IL-1β and TNFα induced luciferase activity with 34 and 40%, respectively. Thus, in vitro exposure of lung epithelial cells to ethanol reduced the generation of cytokines, as well as translocation and gene activation by NFκB.

The relationship between the anti-inflammatory effects of curcumin and cellular glutathione content in myelomonocytic cells by Eva-Maria Strasser; Barbara Wessner; Nicole Manhart; Erich Roth (pp. 552-559).

Oxidative stress plays an important role during inflammatory diseases and recent therapies have focused on antioxidant administration to diminish oxidative stress and to arrest inflammatory processes. In this study, we investigated the impact of the GSH modulating effects of curcumin, a naturally derived polyphenol, on inflammatory processes in myelomonocytic U937 cells.One hour after administration of 10μmol/l curcumin reactive oxygen species (ROS) production was significantly increased in undifferentiated U937 cells (+43%). Twenty-four hour after addition of curcumin, a significantly decreased ROS concentration was found (−32%), whereas GSH (+110%) and GSSG (+88%) content increased. A higher concentration of curcumin (25μmol/l) caused an even stronger increase of GSH (+145%) and GSSG (+101%), but significantly decreased percentage of living cells to 84%.The increased GSH content of differentiated U937 cells after pre-incubation with curcumin was associated with lowered ROS production, nuclear factor kappa B (NFκB) activation (−34%) and tumor necrosis factor alpha (TNF-α) secretion (−51%) after LPS exposure. Curcumin inhibited TNF-α formation was also seen after GSH depletion by buthionine sulfoximine (BSO).This study shows that the antioxidative effects of curcumin are preceded by an oxidative stimulus, which is time and dose-dependent. Excessive concentrations of curcumin may even harm cells, as cell viability was decreased, in spite of elevated GSH contents. There was no clear relationship between intracellular GSH concentrations and the anti-inflammatory effects of curcumin.

Keywords: Abbreviations; BSO; buthionine sulfoximine; DMSO; dimethyl sulfoxide; EMSA; electrophoretic mobility shift assay; GSH; glutathione; GSHt; total glutathione; GSSG; glutathione disulfide; LPS; lipopolysaccharide; MCF; mean channel fluorescence; NFκB; nuclear factor kappa B; PMA; phorbol 12-myristate 13-acetate; ROS; reactive oxygen species; TNF-α; tumor necrosis factor-alphaCurcumin; Antioxidative; Prooxidative; Anti-inflammatory; Glutathione; Reactive oxygen species

The effect of AZT and chloroquine on the activities of ricin and a saporin–transferrin chimeric toxin by A.R. Lizzi; A.M. D’Alessandro; N. Zeolla; F. Brisdelli; G. D’Andrea; G. Pitari; A. Oratore; A. Bozzi; R. Ippoliti (pp. 560-569).

This study deals with the combination of chloroquine (CQ, an anti-malaric drug) and 3′-azido-3′-deoxythymidine (AZT, anti-human immuno-deficiency virus (HIV) drug) with a chimeric toxin (TS) obtained by chemical linking of saporin (a ribosome inactivating protein from the plant Saponaria officinalis) and human transferrin, in the intoxication of the human chronic myeloid leukaemia cells (K562). Our data demonstrate that AZT, at concentrations comparable to those reached in the blood of HIV-infected patients under pharmacological treatment with this drug, can increase the toxicity of TS in cooperation with CQ inducing an increased effect on protein synthesis in K562 cells (∼50% inhibition of protein synthesis for TS alone, and TS with AZT and ∼70% with both AZT and CQ). Furthermore, pre-treatment of cells with AZT alone can induce an increase of apoptosis in K562 cells intoxicated with TS. By comparing data obtained with the model toxin ricin, we get indications that the two toxins partially differ in their intracellular routes, also suggesting that chimeric constructs containing ricin-like toxins (i.e. immunotoxins) could be coupled with the use of common and cheap drugs for the treatment of cancer in HIV-infected patients.

Keywords: AZT; Chloroquine; Toxins; Transferrin; Ricin; K562 cells

TCR pathway involves ICBP90 gene down-regulation via E2F binding sites by Abdul-Qader Abbady; Christian Bronner; Kawtar Bathami; Christian D. Muller; Micha�l Jeanblanc; Eric Mathieu; Jean Paul Klein; Ermanno Candolfi; Marc Mousli (pp. 570-579).

Antigen-induced cell death is essential for function, growth and differentiation of T-lymphocytes through legation of the T cell receptor. Since TCR-induced cell death occurs at late G1 checkpoint of the cell cycle and considering that ICBP90 is critical for G1/S transition, we studied the ICBP90 regulation through the TCR pathway in Jurkat cells. ICBP90 expression was strongly decreased after TCR triggering concomitantly to cyclin D3 and topoisomerase IIα expression decreases. Cell stimulation with PMA and/or calcium ionophore A23187 down-regulated ICBP90 expression. The decrease of ICBP90 protein and mRNA expressions was accompanied with cell growth arrest. A luciferase reporter assay demonstrated that activation of TCR pathways inhibit ICBP90 gene promoter activity. Three consensus E2F binding sites (called from E2F-a to E2F-c) were identified in the ICBP90 gene promoter and were subjected to mutations. The E2F-a, located in a highly active promoter fragment, shows a strong positive functional activity in proliferating cells. E2F-a and E2F-c binding sites are involved in the TCR-induced down-regulation of ICBP90 gene transcription. Altogether, our data demonstrate that TCR signaling pathways regulate ICBP90 gene expression through pRb/E2F complex. We propose that ICBP90 down-regulation is a key event in G1 arrest preceding T cell death.

Keywords: Abbreviations; AID; antigen-induced cell death; ICBP90; inverted CCAAT box binding protein of 90; kDa; TopoIIα; topoisomerase IIα; NIRF; Np95/ICBP90 ring fingerApoptosis; Cell cycle; Gene regulation; T cells; Signal transduction; Transcription factors

17-Allylamino-17-demethoxygeldanamycin overcomes TRAIL resistance in colon cancer cell lines by Irina A. Vasilevskaya; Peter J. O’Dwyer (pp. 580-589).

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a promising candidate for treatment of cancer, but displays variable cytotoxicity in cell lines. The mechanisms of sensitivity and resistance have not been fully elucidated; both AKT and NF-κB pathways may modulate cytotoxic responses. We have shown that the Hsp90 inhibitor 17-AAG enhances the cytotoxicity of oxaliplatin in colon cancer cell lines through inhibition of NF-κB. We analyzed the effects of TRAIL and 17-AAG in combination in a series of nine colon cancer cell lines and characterized activation of the pathways to apoptosis. IC₅₀ values for a 72h exposure to TRAIL ranged from 30 to 4000ng/ml. Cytotoxicity assays demonstrated additivity or synergism of the TRAIL/17-AAG combination in all cell lines, with combination indices at IC₅₀ ranging from 0.53 to 1. The sensitizing effect of 17-AAG was greater in the TRAIL-resistant cell lines. In TRAIL-resistant cell lines, the combination of 17-AAG and TRAIL resulted in activation of both extrinsic and intrinsic apoptotic pathways, though with quantitative differences between HT29 and RKO cells: differential effects of 17-AAG on AKT and NF-κB characterized these cell lines. In both cell lines, the combination also led to down-regulation of X-linked inhibitor of apoptosis protein (XIAP) and enhanced activation of caspase-3. We conclude that either AKT or NF-κB may promote resistance to TRAIL in colon cancer cells, and that the ability of 17-AAG to target multiple putative determinants of TRAIL sensitivity warrants their further investigation in combination.

Keywords: Abbreviations; hsp90; 90; kDa heat-shock protein; 17-AAG; 17-allylamino-17-demethoxygeldanamycin; TRAIL; tumor necrosis factor-related apoptosis-inducing ligand; DR4 and DR5; death receptors 4 and 5, respectively; CI; combination index; DISC; death-inducing signaling complex; FLIP; FLICE inhibitory protein; c-IAP1/2; cellular inhibitors of apoptosis 1/2; XIAP; X-linked IAP; AIF; apoptosis-inducing factor; AP-1; activator-protein 1; NF-κB; nuclear factor kappa B; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideTRAIL; 17-AAG; AKT; NF-κB; Apoptosis; Colon cancer

Sequential induction of heme oxygenase-1 and manganese superoxide dismutase protects cultured astrocytes against nitric oxide by Eunyung Son; Jaeyoon Jeong; Jayoung Lee; Dae Young Jung; Gyeong Jae Cho; Wan Sung Choi; Myung-Shik Lee; Sang-Hyun Kim; In Kyeom Kim; Kyoungho Suk (pp. 590-597).

Nitric oxide (NO) is a widely recognized mediator of physiological and pathophysiological signal transmission. In an attempt to better understand the molecular actions of NO in astrocytes, stress protein expression in response to NO donor sodium nitroprusside was investigated. Heme oxygenase-1 (HO-1) has been identified as an inducer of manganese superoxide dismutase (MnSOD), playing a cytoprotective role under the condition of nitrosative stress. We present evidence that the sequential induction of HO-1 and MnSOD protects astrocytes from NO toxicity: (1) both HO-1 and MnSOD expression were induced by NO; (2) NO-mediated increase in MnSOD activity was partly abolished by HO-1 inhibitor Zn(II) protoporphyrin IX (ZnPP); (3) pretreatment of astrocytes with a nontoxic dose of NO protected the cells against the later treatment with a toxic dose of NO; (4) inhibition of HO-1 by ZnPP sensitized astrocytes to the nontoxic dose of NO resulting in a marked cytotoxicity; and (5) adenovirus-mediated overexpression of MnSOD protected astrocytes from the NO toxicity. The molecular action of NO in astrocytes appears to be dose-dependent. While a high dose of NO exerts cytotoxicity leading to the tissue damage in the central nervous system, a low dose of NO may act as an important signaling molecule in astrocytes with concurrent induction of cytoprotective proteins such as HO-1 and MnSOD.

Keywords: Nitric oxide; Cell death; Inflammation; Signal transduction; Cytoprotection; Astrocyte

Evidence against adenosine analogues being agonists at the growth hormone secretagogue receptor by Stina Johansson; Bertil B. Fredholm; Charlotta Hjort; Torbj�rn Morein; Bj�rn Kull; Ping-Sheng Hu (pp. 598-605).

Adenosine and adenosine analogues have been reported to act as agonists or partial agonists at the growth hormone secretagogue receptor 1a (GHSR1a). We have re-examined this question. A concentration-dependent increase in intracellular calcium concentration ([Ca²⁺]_i) was observed in GHSR1a transfected HEK 293-EBNA cells stimulated with adenosine (EC50: 0.2μM) or 2-chloroadenosine (EC50: 1.1μM) but also in untransfected HEK 293-EBNA cells stimulated with 2-chloroadenosine (EC50: 0.67μM) or 5′- N-ethylcarboxamidoadenosine (NECA) (EC50: 0.045μM). These findings support endogenous expression of adenosine receptors, presumably A_2B receptors in HEK 293-EBNA cells. In GHSR1a transfected CHO cells, lacking adenosine receptors, the GHSR1a agonist hGhrelin (EC50: 2.4nM) increased [Ca²⁺]_i, but no effects of adenosine, 2-chloroadenosine or NECA were detected. An inverse agonist of GHSR1a, [d-Arg-1,d-Phe-5,d-Trp-7, 9, Leu-11] substance P, reduced hGhrelin effects but adenosine, 2-chloroadenosine or 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) did not. NECA increased the [Ca²⁺]_i in co-transfected (GHSR1a and A_2B receptor) CHO cells (EC50: 0.053μM), but no additive or synergistic effects on [Ca²⁺]_i or cAMP formation were observed after stimulation with NECA in the absence or in the presence of hGhrelin. In binding studies on GHSR1a transfected CHO cell membranes, [¹²⁵I]-hGhrelin binding could be displaced by the GHSR1a agonist MK-0677 (IC50: 0.34nM), hGhrelin (IC50: 1.5nM), and the substance P analogue (IC50: 0.64μM) but not by adenosine or 2-chloroadenosine. We conclude that adenosine and analogues do not act as agonists or partial agonists at the GHSR1a and that cross-talk between the GHSR1a and A_2B receptors is limited.

Keywords: GHSR1a; Adenosine A; _2B; receptor; Ghrelin; CHO cells; HEK 293 cells

Cloning and functional characterization of the rat α2B-adrenergic receptor gene promoter region: Evidence for binding sites for erythropoiesis-related transcription factors GATA1 and NF-E2 by St�phane Schaak; Daniel Cussac; St�phane Labialle; Vincent Mignotte; Herv� Paris (pp. 606-617).

In the rat, the α2B-adrenergic receptor (α2B-AR) is encoded by the rat non-glycosylated (RNG) gene and is primarily expressed in the kidney, brain and liver of adult animals. High levels of α2B-AR are also found during fetal life in the placenta, liver and blood, where it is borne by cells of the erythropoietic lineage. As a first step to define the mechanisms responsible for the spatio-temporal pattern of α2B-AR expression, a genomic fragment containing 2.8kb of the 5′-flanking region, the ORF and approximately 20kb of the 3′-flanking region of the RNG gene was isolated. RNase protection assays performed on RNA from placenta or kidney using a series of riboprobes permitted to locate the transcription start site 372 bases upstream from the start codon. Transient transfection of various cells, including rat proximal tubule in primary culture, with constructs containing luciferase as a reporter gene demonstrated that: (i) the 5′-flanking region exhibited a strong and sense-dependent transcriptional activity and (ii) the 332bp fragment (−732/−401 relative to the start codon), which lacks a TATA box but contains Sp1 sites, is sufficient to drive expression. Analysis of chromatin susceptibility to DNaseI digestion identified two hypersensitive sites (HS1 and HS2) located 1.7 and 1.0kb, respectively, upstream from ATG and containing recognition sequences for erythroid transcription factors. EMSA showed specific binding of GATA1 and NF-E2 to these elements. Taken together, the results suggest that the chromatin environment in the vicinity of these boxes plays a critical role for α2B-AR expression during fetal life.

Keywords: α2b-adrenergic receptor; RNG; Promoter; Transcription factors; GATA1; NF-E2

Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by phenolic (3 E)-4-(2-hydroxyphenyl)but-3-en-2-one in RAW 264.7 macrophages by Lo-Ti Tsao; Pei-Shan Tsai; Ruey-Hseng Lin; Li-Jiau Huang; Sheng-Chu Kuo; Jih-Pyang Wang (pp. 618-626).

The large amount of nitric oxide (NO) produced by inducible NO synthase (iNOS) contributes to cellular injury in inflammatory disease. In the present study, a novel synthetic compound (3 E)-4-(2-hydroxyphenyl)but-3-en-2-one (HPB) was found to inhibit lipopolysaccharide (LPS)-induced NO generation, but not through the inhibition of iNOS activity, in RAW 264.7 macrophages. Administration of HPB into mice also inhibited the LPS-induced increase in serum nitrite/nitrate levels. To evaluate the underlying mechanisms of HPB inhibition of NO generation, the expression of the iNOS gene in RAW 264.7 macrophages was examined. HPB abolished the LPS-induced expression of iNOS protein, iNOS mRNA and iNOS promoter activity in a similar concentration-dependent manner. LPS-induced nuclear factor-κB (NF-κB) DNA binding and NF-κB-dependent reporter gene activity were both significantly inhibited by HPB. This effect was mediated through the inhibition of inhibitory factor-κBα (IκBα) phosphorylation and degradation, and of p65 nuclear translocation. HPB had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinases (MAPK), and c-Jun NH₂-terminal kinase (JNK). However, HPB suppressed the LPS-induced intracellular reactive oxygen species (ROS) production. These results indicate that HPB down-regulates iNOS gene expression probably through the inhibition of LPS-induced intracellular ROS production, which has been implicated in the activation of NF-κB.

Keywords: Abbreviations; DCFH-DA; 2′,7′-dichlorofluorescein diacetate; EMSA; electrophoretic mobility shift assay; ERK; extracellular signal-regulated kinase; HPB; (3; E; )-4-(2-hydroxyphenyl)but-3-en-2-one; IκB; inhibitory factor-κB; IKK; IκB kinase; iNOS; inducible NO synthase; JNK; c-Jun NH; ₂; -terminal kinase; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinases; NAC; N; -acetyl-; l; -cysteine; NF-κB; nuclear factor-κB; NO; nitric oxide; ROS; reactive oxygen species; and RT-PCR; reverse transcriptase-polymerase chain reactionHPB; Nitric oxide; Nitric oxide synthase; Nuclear factor-κB; Antioxidant; RAW 264.7 macrophages

Structure activity relationships and quantitative structure activity relationships for the flavonoid-mediated inhibition of breast cancer resistance protein by Shuzhong Zhang; Xinning Yang; Robert A. Coburn; Marilyn E. Morris (pp. 627-639).

Breast cancer resistance protein (BCRP) is a newly identified ABC transporter, which plays an important role in drug disposition and represents an additional mechanism for the development of MDR. Flavonoids, a major class of natural compounds widely present in foods and herbal products, have been shown to be BCRP inhibitors. The objective of the present study was to elucidate the SAR and derive a QSAR model for flavonoid–BCRP interaction. The EC₅₀ values for increasing mitoxantrone accumulation in MCF-7 MX100 cells for 25 flavonoids, from five flavonoid subclasses, were determined in this study or obtained from our previous publication [Zhang S, Yang X, Morris ME. Combined effects of multiple flavonoids on breast cancer resistance protein (ABCG2)-mediated transport. Pharm Res 2004;21(7):1263–73], and ranged from 0.07±0.02μM to 183±21.7μM. We found that the presence of a 2,3-double bond in ring C, ring B attached at position 2, hydroxylation at position 5, lack of hydroxylation at position 3 and hydrophobic substitution at positions 6, 7, 8 or 4′, are important structural properties important for potent flavonoid–BCRP interaction. These structural requirements are similar but not identical to those for potent flavonoid–NBD2 (P-glycoprotein) interaction, indicating that inhibition of BCRP by flavonoids may involve, in part, the binding of flavonoids with the NBD of BCRP. In addition, a QSAR model consisting three structural descriptors was constructed, and both internally and externally validated, suggesting the model could be used to quantitatively predict BCRP inhibition activity of flavonoids. These findings should be useful for predicting BCRP inhibition activity of other untested flavonoids and for guiding the synthesis of potent BCRP inhibitors for potential clinical application.

Keywords: Abbreviations; ABC; ATP binding cassette; BCRP; breast cancer resistance protein; FTC; fumitremorgin C; MDR; multidrug resistance; MRP1; multidrug resistance-associated protein 1; MRP; multidrug resistance-associated protein 2; MX; mitoxantrone; QSAR; quantitative structure activity relationship; SAR; structure activity relationship; NBD; nucleotide binding domainBreast cancer resistance protein; Flavonoids; Structure activity relationships; Quantitative structure activity relationships; Mitoxantrone transport; Membrane transport

Transport of dietary phenethyl isothiocyanate is mediated by multidrug resistance protein 2 but not P-glycoprotein by Yan Ji; Marilyn E. Morris (pp. 640-647).

We demonstrated recently that phenethyl isothiocyanate (PEITC), a potent anticarcinogen present in cruciferous vegetables, inhibited P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) and that MRP1 can transport PEITC and/or its metabolites. In this study, we have examined whether PEITC is transported by P-gp and MRP2, two transporters with high expression in human intestine, liver and kidney. Using¹⁴C-PEITC, no significant difference was observed for the intracellular accumulation of PEITC in human breast cancer MCF-7/sensitive (control) and MCF-7/ADR (P-gp overexpressing) cells at PEITC concentrations of 1, 10 and 50μM. Moreover, the presence of verapamil or PSC833, two P-gp inhibitors, had no significant effect on the intracellular accumulation of PEITC in P-gp overexpressing MCF-7/ADR and MDA435/LCC6MDR1 cells, indicating that PEITC may not be a substrate for P-gp. In contrast,¹⁴C-PEITC intracellular accumulation in the kidney epithelial MDCK II/MRP2 cells (transfected with human MRP2) was significantly lower than in the wild-type MDCK II/wt cells at PEITC concentrations of 1, 5, 10 and 50μM. The presence of MK571, an MRP inhibitor, significantly enhanced¹⁴C-PEITC accumulation in MDCK II/MRP2 but not MDCK II/wt cells. Furthermore, depletion of intracellular glutathione (GSH) following treatment with buthionine sulphoximine, an inhibitor of GSH biosynthesis, significantly increased¹⁴C-PEITC intracellular accumulation in a concentration-dependent manner. Transcellular transport studies also demonstrated that depletion of intracellular GSH reduced the mean ratio of basal-to-apical transport to apical-to-basal transport of PEITC in MDCK II/MRP2, but not MDCK II/wt cell monolayers. These results indicate that GSH plays an important role in the MRP2-mediated transport of PEITC. The findings provide new information concerning the interactions between PEITC and membrane transporters and suggest the possibility of PEITC interactions with xenobiotics that are MRP2 substrates.

Keywords: Abbreviations; ABC; ATP-binding cassette; BCRP; breast cancer resistance protein; BSO; buthionine sulphoximine; GSH; glutathione; GST; glutathione-; S; -transferase; ITC; isothiocyanate; MRP; multidrug resistance protein; PEITC; phenethyl isothiocyanate; P; _app; apparent permeability; P-gp; P-glycoproteinPhenethyl isothiocyanate; P-glycoprotein; MRP2; Membrane transport

Corrigendum to “Differences in binding properties of two proton pump inhibitors on the gastric H⁺, K⁺-ATPase in vivo�? [Biochemical Pharmacology 68 (11) (2004) 2117–2127] by Jai Moo Shin; George Sachs (pp. 648-648).

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Biochemical Pharmacology (v.70, #4)