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Biochemical Pharmacology (v.71, #1-2)

Editorial Advisory Board (pp. iii).

NRH:quinone reductase 2: An enzyme of surprises and mysteries by Fanny Vella; Gilles Ferry; Philippe Delagrange; Jean A. Boutin (pp. 1-12).

Quinone reductase 2 has been discovered in 1961 and rediscovered in 1997. Because of its sequence homology with quinone reductase 1, it has been suspected to detoxify quinones. Ten years later, evidences begin to point to a versatile role of this enzyme. Indeed, QR2 is strongly suspected to be the molecular target of anti-malarian drugs such as chloroquin or paraquine, and of red wine-derived resveratrol that might be responsible for the so-called French paradox. It also is identical to the melatonin binding site MT₃, and might therefore be a rationale explanation for the antioxidant role of melatonin. Finally QR2 might be implicated in the toxicity, in vivo, of quinones such as menadione. The present commentary attempts to summarize this information and discusses a series of hypotheses.

Keywords: Abbreviations; QR1; quinone reductase 1 (EC 1.6.99.2); QR2; NRH:quinone oxidoreductase 2 (EC 1.10.99.2); MT3; melatonin binding siteQuinone reductase 2; Quinone reductase 1; Melatonin; Resveratrol; Chloroquine; Molecular pharmacology

Sensitization of breast carcinoma cells to ionizing radiation by small molecule inhibitors of DNA-dependent protein kinase and ataxia telangiectsia mutated by Ian G. Cowell; Barbara W. Durkacz; Michael J. Tilby (pp. 13-20).

DNA-PK and ATM are members of the phosphatidylinositol 3′-kinase like kinase (PIKK) family of serine/threonine protein kinases and have critical roles in the cellular response to DNA double-strand breaks. Genetic loss of either activity leads to pronounced sensitivity to ionizing radiation (IR). Hence, these enzymes are potential targets to confer enhanced radiosensitivity on tumour cells. We show that novel inhibitors of either DNA-PK or ATM sensitize breast carcinoma cells to IR. Radiosensitization was accompanied by an apparent DNA repair deficit as measured by the persistence of IR-induced foci of phosphorylated histone H2AX (γH2AX foci). These specific inhibitors also allowed us to probe the biochemistry and kinetics of histone H2AX phosphorylation following γ-irradiation in breast cancer cells with the aim of validating H2AX as a biomarker for DNA-PK or ATM inhibition in vivo. ATM inhibition reduced the initial average intensity of γH2AX foci while inhibition of DNA-PK had only a small effect on the initial phosphorylation of H2AX. However, simultaneous treatment with both compounds dramatically reduced γH2AX focus intensity, consistent with the reported role of ATM and DNA-PK in IR induced phosphorylation of H2AX.

Keywords: Abbreviations; A-T; ataxia telangiectasia; DNA-PK; DNA-dependent protein kinase; DNA-PKcs; DNA-PK catalytic subunit; ATM; ataxia telangiectasia mutated; ATR; ataxia telangiectasia and Rad3 related; ATRIP; ATR-interacting protein; RPA; replication protein A; NHEJ; non-homologous end joining; DSB; DNA double-strand break; PIKK; phosphatidylinositol 3′-kinase like kinase; IR; ionising radiation; HRR; homologous recombination repairATM; DNA-PK; DNA repair; Histone H2AX; Radiosensitivity; Breast cancer

Inhibitory effect of DA-125, a new anthracyclin analog antitumor agent, on the invasion of human fibrosarcoma cells by down-regulating the matrix metalloproteinases by Hyen Joo Park; Hwa-Jin Chung; Hye-Young Min; Eun-Jung Park; Ji-Young Hong; Won Bae Kim; Soon Hoe Kim; Sang Kook Lee (pp. 21-31).

Matrix metalloproteinases (MMPs), zinc-dependent proteolytic enzymes, play a pivotal role in tumor metastasis by cleavage of extracellular matrix as well as non-matrix substrates. In this study, we examined the influence of DA-125, a new anthracyclin analog, on the gene expression of MMPs (MMP-2, MMP-9 and MT1-MMP), tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2) and in vitro invasiveness of human fibrosarcoma cells. Dose-dependent decreases of MMPs and TIMPs mRNA levels were observed in DA-125-treated HT1080 human fibrosarcoma cells detected by reverse transcriptase-polymerase chain reaction. Gelatin zymography analysis also showed a significant down-regulation of MMP-2 and MMP-9 expression in HT1080 cells treated with DA-125 compared to controls. In addition, DA-125 inhibited the invasion, motility and cell migration, and colony formation of tumor cells. These data, therefore, provide direct evidence for the role of DA-125 as a potential cancer chemotherapeutic agent, which can markedly inhibit the invasive capacity of malignant cells. Further, to clarify the transcriptional regulatory pathway, we primarily investigated the role of nuclear factor-κB (NF-κB) in the expression of MMPs by DA-125 in HT1080 cells. Electrophoretic mobility shift assay demonstrated that DA-125 modulates the binding activity of NF-κB. Using the luciferase reporter gene assay, a dose-dependent down-regulation of NF-κB-mediated luciferase expression was also observed. These results suggest that DA-125 down-regulates MMPs expression in HT1080 cells through the NF-κB-mediated pathway.

Keywords: Abbreviations; DMEM; Dulbecco's modified Eagle's medium; ECM; extracellular matrix; EMSA; electrophoretic mobility shift assay; MMPs; matrix metalloproteinases; MT1-MMP; membrane type metalloproteinase; NF-κB; nuclear factor κB; RT-PCR; reverse transcriptase-polymerase chain reaction; TIMP; tissue inhibitor of metalloproteinaseInvasion; Extracellular matrix; Matrix metalloproteinase; HT1080 cell; Anthracyclin analog; NF-κB

Cell cycle arrest and proapoptotic effects of the anticancer cyclodepsipeptide serratamolide (AT514) are independent of p53 status in breast cancer cells by Vanessa Soto-Cerrato; Beatriz Montaner; Marc Martinell; Marta Vilaseca; Ernest Giralt; Ricardo P�rez-Tom�s (pp. 32-41).

In a search for new anticancer agents, we have identified serratamolide (AT514), a cyclodepsipeptide from Serratia marcescens 2170 that induces cell cycle arrest and apoptosis in various cancer cell lines. A cell viability assay showed that the concentrations that cause 50% inhibition (IC₅₀) in human cancer cell lines range from 5.6 to 11.5μM depending on the cell line. Flow cytometry analysis revealed that AT514 caused cell cycle arrest in G₀/ G₁ or cell death, depending on the cell type and the length of time for which the cells were exposed to the drug. Subsequent studies revealed that AT514-induced cell death is caused by apoptosis, as indicated by caspases activation (8, 9, 2 and 3) and cleavage of poly (ADP-ribose) polymerase (PARP), release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria, and the appearance of apoptotic bodies and DNA laddering. Alterations in protein levels of Bcl-2 family members might be involved in the mitochondrial disruption observed. AT514 induced p53 accumulation in wild-type p53 cells but cell death was observed in both deficient and wild-type p53 cells. Our results indicate that AT514 induces cell cycle arrest and apoptosis in breast cancer cells irrespectively of p53 status, suggesting that it might represent a potential new chemotherapeutic agent.

Keywords: Abbreviations; AT514; serratamolide; HPLC; high performance liquid chromatography; KF; Kahalalide F; MALDI; matrix-assisted laser desorption/ionization; MPLC; medium pressure liquid chromatography; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; NMR; nuclear magnetic resonance; RT-PCR; real-time quantitative polymerase chain reaction; TOF; time of flightDepsipeptide; Cell cycle arrest; Apoptosis; Caspases; p53

Mechanisms involved in δ-aminolevulinic acid (ALA)-induced photosensitivity of tumor cells: Relation of ferrochelatase and uptake of ALA to the accumulation of protoporphyrin by Yoshiko Ohgari; Yuki Nakayasu; Sakihito Kitajima; Mari Sawamoto; Hajime Mori; Osamu Shimokawa; Hirofumi Matsui; Shigeru Taketani (pp. 42-49).

Photodynamic therapy (PDT) using δ-aminolevulinic acid (ALA)-induced accumulation of protoporphyrin IX is a useful approach to the early detection and treatment of cancers. To investigate the role of ferrochelatase in the accumulation of protoporphyrin, we first made mouse fibroblast Balb/3T3 cells highly expressing ferrochelatase and examined the ALA-induced photo-damage as well as the accumulation of porphyrin in the cells. When the ferrochelatase-transfected cells were treated with ALA and then exposed to visible light, they became resistant to the light without accumulating porphyrins, with a concomitant increase in the formation of heme. The accumulation of protoporphyrin was also abolished in human erythroleukemia K562 cells stably expressing mouse ferrochelatase. When mouse fibrosarcoma MethA cells, mouse fibroblast L929 cells and Balb/3T3 cells were treated with ALA, the greatest accumulation of protoporphyrin and the greatest level of cell death in response to the light were observed in MethA cells. The expression level of ferrochelatase was the lowest in MethA cells, while that of porphobilinogen deaminase was similar among all three cell lines. Moreover, an iron-chelator, desferrioxamine, which sequesters iron preventing the ferrochelatase reaction, enhanced the photo-damage as well as the accumulation of protoporphyrin in ALA-treated L929 cells. Thus, the light-induced cell death was tightly coupled with the accumulation of protoporphyrin caused by a decrease in ferrochelatase. Finally, we examined the uptake of ALA by MethA, L929 and Balb/3T3 cells. The extent of the uptake by MethA and L929 cells was greater, indicating a greater accumulation of protoporphyrin than in the Balb/3T3 cells. Taken together, not only the low level of ferrochelatase but also the augmented uptake of ALA contributes to the ALA-induced accumulation of protoporphyrin IX and subsequent photo-damage in cancer cells.

Keywords: Abbreviations; ALA; δ-aminolevlinic acid; DFO; desferrioxamine; DMEM; Dulbecco's modified Eagle's medium; FCS; fetal calf serum; HO-1; heme oxygenase-1; PBG; phorphobilinogen; PDT; Photodynamic therapy; PVDF; poly(vinylidene difluoride); SDS-PAGE; sodium dodecylsulfate-polyacrylamide gel electrophoresisFerrochelatase; δ-Aminolevlinic acid; Protoporphyrin; Photo-damage; PDT

Differential response of normal, dedifferentiated and transformed thyroid cell lines to cisplatin treatment by Antonella Muscella; Loredana Urso; Nadia Calabriso; Antonella Ciccarese; Danilo Migoni; Francesco Paolo Fanizzi; Bruno Di Jeso; Carlo Storelli; Santo Marsigliante (pp. 50-60).

The effects of cisplatin ( cisPt) on the extra cellular signal-regulated kinase (ERK) and the protein kinase B (PKB/Akt), known to play important roles in promoting cell survival and in down regulating apoptosis, were investigated in thyroid cell lines. The cytotoxic effect of cisPt was highest in normal PC-Cl3 cells, intermediate in dedifferentiated PC-E1A and PC-raf cells and lowest in fully transformed and tumorigenic PC-E1Araf cells. CisPt provoked ERK phosphorylation; such phosphorylation was unaltered by Gö6976, a conventional PKC inhibitor, whilst blocked by low doses (0.1μM) or high doses (10μM) of GF109203X, an inhibitor of all PKC isozymes, in PC-Cl3 and in PC-E1Araf cells, respectively. In PC-E1Araf, but not in PC-Cl3 cells, the cisPt-provoked ERK phosphorylation was also blocked by a myristoylated PKC-ζ pseudo substrate peptide (PS-ζ). The cytotoxic effects of cisPt increased when cells were pre-incubated with the mitogen-activated protein kinase (MEK) inhibitor PD98059. CisPt provoked the phosphorylation of PKB/Akt and this effect was blocked by LY294002, a PI3K inhibitor. In PC-Cl3 cells pre-incubated with LY294002 the effects of cisPt on ERK phosphorylation and cell mortality resulted unaffected; conversely, LY294002 reduced the ERK phosphorylation and increased cisPt cytotoxity of in PC-E1Araf cells. Furthermore, in PC-E1Araf cells pre-incubated with LY294002 and PS-ζ ERK phosphorylation was abolished and cisPt cytotoxicity was highest.Altogether results highlight a role for PKCs in the upstream regulation of ERK pathway facing the cell response to cisPt treatments. Understanding the mechanisms by which cells process cisPt provides important insights for designing more efficient platinum-based drugs.

Keywords: PC-Cl3; Cisplatin; ERK; PKB/Akt; PKC-ζ

Arylpiperazines displaying preferential potency against chloroquine-resistant strains of the malaria parasite Plasmodium falciparum by Carrie-Anne Molyneaux; Miriam Krugliak; Hagai Ginsburg; Kelly Chibale (pp. 61-68).

Arylpiperazines in which the terminal secondary amino group is unsubstituted were found to display a mefloquine-type antimalarial behavior in being significantly more potent against the chloroquine-resistant (W2 and FCR3) strains of Plasmodium falciparum than against the chloroquine-sensitive (D10 and NF54) strains. Substitution of the aforementioned amino group led to a dramatic drop in activity across all strains as well as abolition of the preferential potency against resistant strains that was observed for the unsubstituted counterparts. The data suggest that unsubstituted arylpiperazines are not well-recognized by the chloroquine resistance mechanism and may imply that they act mechanistically differently from chloroquine. On the other hand, 4-aminoquinoline-based heteroarylpiperazines in which the terminal secondary amino group is also unsubstituted, were found to be equally active against the chloroquine-resistant and chloroquine-sensitive strains, suggesting that chloroquine cross-resistance is not observed with these two 4-aminoquinolines. In contrast, two 4-aminoquinoline-based heteroarylpiperazines are positively recognized by the chloroquine resistance mechanism. These studies provide structural features that determine the antimalarial activity of arylpiperazines for further development, particularly against chloroquine-resistant strains.

Keywords: Malaria; Antimalarial agents; 4-Aminoquinolines; Drug resistance; Privileged structures; Arylpiperazines

6-Benzylthioinosine analogues: Promising anti-toxoplasmic agents as inhibitors of the mammalian nucleoside transporter ENT1 ( es) by Amol Gupte; John K. Buolamwini; Vikas Yadav; Chung K. Chu; Fardos N.M. Naguib; Mahmoud H. el Kouni (pp. 69-73).

Certain 6-benzylthioinosine analogues have been identified as potential chemotherapeutic agents against Toxoplasma gondii in cell culture and animal models. These compounds are selectively transported and metabolized by toxoplasma infected, but not uninfected, cells. In sharp contrast to mammalian nucleoside transporters, the toxoplasma adenosine/purine transporter (TgAT) allows the transport of these 6-benzylthioinosine analogues into infected cells. After entering the infected cell, these compounds act as subversive substrates for toxoplasma, but not the host, adenosine kinase (EC.2.7.1.20). Hence, 6-benzylthioinosine analogues become toxic to toxoplasma infected cells, but not uninfected host cells or animals. The basis for the lack of uptake of the anti-toxoplasmic 6-benzylthioinosines by uninfected host cells is currently unknown. These anti-toxoplasmic 6-benzylthioinosines may not be substrates for the mammalian nucleoside transporters or they may act as inhibitors of these transporters. Previous studies have shown that some 6-benzylthioinosines are inhibitors of the mammalian nucleoside transporter ENT1 ( es). Therefore, we examined the efficacy of promising anti-toxoplasmic 6-benzylthioinosines as inhibitors of ENT1 ( es) in an effort to elucidate the basis for the lack of uptake of anti-toxoplasmic 6-benzylthioinosines by uninfected host cells. The results showed that these compounds are inhibitors of ENT1 ( es). In general, electron-withdrawing substituents at the ortho, meta or para positions of the benzyl ring improved binding. The most potent inhibitors identified were m- and p-nitro-6-benzylthioinosine, which had K_i values in the subnanomolar range. Therefore, anti-toxoplasmic 6-benzylthioinosines are not only selectively toxic to parasites and parasite infected cells, they are also inhibitors of nucleoside transport in host cells. This inhibition of the host nucleoside transport is an added advantage for these 6-benzylthioinosine analogues as anti-toxoplasmic agents. Inhibitors of nucleoside transport in mammalian cells can selectively protect the host from the toxicity of toxic purine nucleosides that may be used in future combination therapy against toxoplasmosis or from metabolites of the 6-benzylthioinosine analogues that may be released by the destruction of infected cells. These findings further advance the rationale for developing 6-benzylthioinosine analogues as selective therapeutic agents for the treatment of toxoplasmosis.

Keywords: Abbreviations; 5-(SAENTA); 5′-; S; -(2-aminoethyl)-; N; ⁶; -(4-nitrobenzyl)-5′-thioadenosineToxoplasma; 6-Benzylthioinosine analogues; Nucleoside transport inhibitors; Chemotherapy

Characterization of the melatoninergic MT₃ binding site on the NRH:quinone oxidoreductase 2 enzyme by Fran�ois Mailliet; Gilles Ferry; Fanny Vella; Sylvie Berger; Francis Cog�; Pascale Chomarat; Catherine Mallet; Sophie-P�n�lope Gu�nin; G�rald Guillaumet; Marie-Claude Viaud-Massuard; Sa�d Yous; Philippe Delagrange; Jean A. Boutin (pp. 74-88).

Melatonin acts through a series of molecular targets: the G-protein coupled receptors, MT₁ and MT₂, and a third binding site, MT₃, recently identified as the enzyme NRH:quinone oxydoreductase 2 (QR2). The relationship between the multiple physiological functions of melatonin and this enzyme remains unclear. Because of the relationship of QR2 with the redox status of cells, these studies could bring the first tools for a molecular rationale of the antioxidant effects of melatonin. In the present paper, we used a QR2-stably expressing cell line and hamster kidneys to compare the 2-[¹²⁵I]-iodomelatonin and 2-[¹²⁵I]-iodo-5-methoxycarbonylamino- N-acetyltryptamine binding data, and to characterize the MT₃ binding site. We designed and tested compounds from two distinct chemicals series in a displacement assay of the two MT₃ ligands, 2-[¹²⁵I]-iodomelatonin and 2-[¹²⁵I]-iodo-5-methoxycarbonylamino- N-acetyltryptamine from their cloned target. We also tested their ability to inhibit QR2 catalytic activity. These compounds were separated into two classes: those that bind within the catalytic site (and being inhibitors) and those that bind outside it (and therefore not being inhibitors). Compounds range from potent ligands ( K_i=1nM) to potent inhibitors (14nM), and include one compound [NMDPEF: N-[2-(2-methoxy-6H-dipyrido[2,3-a:3,2-e]pyrrolizin-11-yl)ethyl]-2-furamide] active on both parameters in the low nanomolar range. To dissect the physio-pathological pathways in which QR2, MT₃ and melatonin meet, one needs more compounds binding to MT₃ and/or inhibitors of QR2 enzymatic activity. The compounds described in the present paper are new tools for such a task.

Keywords: Abbreviations; 2-I-MLT; 2-iodomelatonin; 2-[; ¹²⁵; I]-MLT; 2-[; ¹²⁵; I]-iodomelatonin; 2-[; ¹²⁵; I]-MCA-NAT; 2-[; ¹²⁵; I]-iodo-5-methoxycarbonylamino-; N; -acetyltryptamine; AFMK; N; ¹; -acetyl-; N; ²; -formyl-5-methoxy-kynurenamine; AMK; N; ¹; -acetyl-5-methoxykynurenamine; BNAH; dihydrobenzylnicotamide; CHO cells; Chinese Hamster Ovary; FAD; flavin adenine dinucleotide; hQR2-FAD; oxidative state of the FAD cofactor into hQR2 protein; hQR2-FADH; ₂; reductive state of the FAD cofactor into hQR2 protein; MCA-NAT; 5-methoxycarbonylamino-; N; -acetyltryptamine; MLT; melatonin; NAS; N; -acetylserotonin; hQR2; human quinone reductase 2; NRH; dihydronicotinamide riboside; QR1; quinone reductase 1 (NQO1); TCA; trichloroacetic acid; TFA; trifluoroacetic acidMelatonin; Quinone reductase 2; MT; ₃; Molecular pharmacology; Inhibitors; Binding

Differential interactions of G-proteins and adenylyl cyclase with nucleoside 5′-triphosphates, nucleoside 5′-[γ-thio]triphosphates and nucleoside 5′-[β,γ-imido]triphosphates by Andreas Gille; Jianxin Guo; Tung-Chung Mou; Michael B. Doughty; Gerald H. Lushington; Roland Seifert (pp. 89-97).

The regulatory G-proteins of adenylyl cyclase (AC), G_i and G_s, are not only activated by GTP and the stable GTP analogs, guanosine 5′-[γ-thio]triphosphate (GTPγS) and guanosine 5′-[β,γ-imido]triphosphate (GppNHp), but also by hypoxanthine, xanthine, uracil and cytidine nucleotides. The latter nucleotides were previously used to analyze distinct active G-protein states. Surprisingly, recent studies have shown that inosine 5′-[γ-thio]triphosphate and uridine 5′-[γ-thio]triphosphate can also inhibit AC directly. Therefore, we systematically compared the interactions of nucleoside 5′-triphosphates (NTPs), nucleoside 5′-[γ-thio]triphosphates (NTPγSs) and nucleoside 5′-[β,γ-imido]triphosphates (NppNHps) with G_i, G_s and AC. NTPγSs exhibited up to 26,000-fold higher affinity for G-proteins than NTPs and NppNHps. NTPγSs were up to 150-fold more potent direct AC inhibitors than NTPs and NppNHps. G-proteins exhibited striking preference for guanine nucleotides compared to other purine and pyrimidine nucleotides, whereas base-selectivity of various ACs, particularly the purified catalytic subunits C1·C2, was rather poor. GTP, GTPγS and GppNHp exhibited much higher selectivity for G-proteins relative to AC than all other purine and pyrimidine nucleotides. We have energetically characterized the interactions of purine and pyrimidine nucleotides with AC in silico, constructing pharmacophore models that correlate well with experimental affinities and have elucidated specific amino acid residues with greatest influence on nucleotide binding. Collectively, both G-proteins and ACs bind purine and pyrimidine nucleotides, with G-proteins showing much higher base-selectivity than AC. Thus, direct inhibitory effects of nucleotides on AC should be understood and considered when probing distinct active G-protein states with non-guanine nucleotides.

Keywords: Abbreviations; AC; adenylyl cyclase; AppNHp; adenosine 5′-[β,γ-imido]triphosphate; ATPαS; adenosine 5′-[α-thio]triphosphate; ATPγS; adenosine 5′-[γ-thio]triphosphate; β; ₂; AR-G; _sαL; fusion protein consisting of the β; ₂; -adrenoceptor and the long splice variant of the stimulatory G-protein of adenylyl cyclase, G; _sα; CTPγS; cytidine 5′-[γ-thio]triphosphate; FPR-G; _iα2; fusion protein consisting of the formyl peptide receptor and the inhibitory G-protein of adenylyl cyclase, G; _iα2; G; _iα; inhibitory G-protein of AC; G; _sα; stimulatory G-protein of AC; G; _sαL; long splice variant of G; _sα; GppNHp; guanosine 5′-[β,γ-imido]triphosphate; GTPγS; guanosine 5′-[γ-thio]triphosphate; IppNHp; inosine 5′-[β,γ-imido]triphosphate; ITPγS; inosine 5′-[γ-thio]triphosphate; NppNHp; nucleoside 5′-[β,γ-imido]triphosphate; NTPγS; nucleoside 5′-[γ-thio]triphosphate; NTP; nucleoside 5′-triphosphate; UppNHp; uridine 5′-[β,γ-imido]triphosphate; UTPγS; uridine 5′[γ-thio]triphosphate; XppNHp; xanthosine 5′-[β,γ-imido]triphosphate; XTPγS; xanthosine 5′-[γ-thio]triphosphateG-protein; Adenylyl cyclase; Nucleoside 5′-triphosphate; Nucleoside 5′-[γ-thio]triphosphate; Nucleoside 5′-[β,γ-imido]triphosphate; Molecular docking; Pharmacophore model

Selective allosteric ligand activation of the retinoid X receptor heterodimers of NGFI-B and Nurr1 by Kentaro Morita; Katsuyoshi Kawana; Mariko Sodeyama; Iichiro Shimomura; Hiroyuki Kagechika; Makoto Makishima (pp. 98-107).

NGFI-B, an orphan member of the NR4A subfamily of the nuclear receptors, recognizes specific sequences in the promoters of neuronal target genes as a monomer. Although NGFI-B also forms a heterodimer with the retinoid X receptor (RXR), a receptor for 9- cis retinoic acid (9CRA), endogenous targets of the heterodimer have not been identified. We investigated the role of RXR ligand binding in NGFI-B/RXR activation and found that dibenzodiazepine-derived ligands, such as the weak RXR agonist HX600, selectively activate NGFI-B/RXR heterodimers. HX600 also activated the heterodimer formed by RXR and Nurr1, another NR4A subfamily receptor. In an assembly assay that detects ligand-dependent reconstruction of the ligand-binding domain, HX600 and not 9CRA induced an allosteric ligand effect on NGFI-B through RXRα binding. The data indicate that the RXR heterodimers of NGFI-B and Nurr1 are selectively activated by the RXR ligand HX600, and that compounds such as HX600 will be valuable tools in investigating NGFI-B and Nurr1 function.

Keywords: Abbreviations; AF-2; activation function-2; 9CRA; 9-; cis; retinoic acid; DR; direct repeat; FXR; farnesoid X receptor; H; helix; HEK; human embryonic kidney; IR; inverted repeat; LBD; ligand-binding domain; LXR; liver X receptor; NBRE; NGFI-B response element; PPAR; peroxisome proliferator-activated receptor; RAR; retinoic acid receptor; RXR; retinoid X receptor; TR; thyroid hormone receptorNuclear receptor; NGFI-B; RXR; Dibenzodiazepine; Transcription; Ligand

Isoflavone genistein and daidzein up-regulate LPS-induced inducible nitric oxide synthase activity through estrogen receptor pathway in RAW264.7 cells by Mako Nakaya; Hirofumi Tachibana; Koji Yamada (pp. 108-114).

Isoflavones, such as genistein and daidzein, are found in abundance in soybeans. These plant-derived substances have estrogenic activities and can bind to the estrogen receptors (ERs). In this study, we investigated that the effects of 17β-estradiol (E2), genistein and daidzein on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) activity in RAW264.7 cells. We found that these isoflavones significantly increased lipopolysaccharide-induced NO production and iNOS expression as much as E2 at physiological concentrations. Moreover, E2 and isoflavone enhanced the production of tumor necrosis factor-α that is one of the important cytokines regarding NO production. The enhancing effects of E2 and isoflavones on NO production were markedly inhibited by not only N^G-nitro-l-arginine methyl ester (an inhibitor of NOS), but also ICI 182780 (ERs antagonist). Two types of ERs were identified as ERα and ERβ. An ERα agonist could increase iNOS expression in RAW264.7 cells, while an ERβ agonist could not. In conclusion, our results suggest E2, genistein and daidzein activate iNOS, and then up-regulate NO production. This enhancing effect is aroused through ERα pathway in RAW264.7 cells.

Keywords: Abbreviations; Dai; daidzein; E2; 17β-estradiol; ELISA; enzyme-linked immunosorbent assay; ER; estrogen receptor; FBS; fetal bovine serum; Gen; genistein; iNOS; inducible nitric oxide synthase; l; -NAME; N; ^G; -nitro-; l; -arginine methyl ester; LPS; lipopolysaccharide; PBS; phosphate buffered saline; PI3K; phosphatidylinositol 3-kinase; TNF-α; tumor necrosis factor αIsoflavone; iNOS; Macrophage; Estrogen receptor

CysLT₁ leukotriene receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors by Liaman Mamedova; Val�rie Capra; Maria Rosa Accomazzo; Zhan-Guo Gao; Silvia Ferrario; Marta Fumagalli; Maria P. Abbracchio; G. Enrico Rovati; Kenneth A. Jacobson (pp. 115-125).

Montelukast and pranlukast are orally active leukotriene receptor antagonists selective for the CysLT₁ receptor. Conversely, the hP2Y_{1,2,4,6,11,12,13,14} receptors represent a large family of GPCRs responding to either adenine or uracil nucleotides, or to sugar-nucleotides. Montelukast and pranlukast were found to inhibit nucleotide-induced calcium mobilization in a human monocyte-macrophage like cell line, DMSO-differentiated U937 (dU937). Montelukast and pranlukast inhibited the effects of UTP with IC₅₀ values of 7.7 and 4.3μM, respectively, and inhibited the effects of UDP with IC₅₀ values of 4.5 and 1.6μM, respectively, in an insurmountable manner. Furthermore, ligand binding studies using [³H]LTD₄ excluded the possibility of orthosteric nucleotide binding to the CysLT₁ receptor. dU937 cells were shown to express P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₃ and P2Y₁₄ receptors. Therefore, these antagonists were studied functionally in a heterologous expression system for the human P2Y receptors. In 1321N1 astrocytoma cells stably expressing human P2Y_1,2,4,6 receptors, CysLT₁ antagonists inhibited both the P2Y agonist-induced activation of phospholipase C and intracellular Ca²⁺ mobilization. IC₅₀ values at P2Y₁ and P2Y₆ receptors were <1μM. In control astrocytoma cells expressing an endogenous M3 muscarinic receptor, 10μM montelukast had no effect on the carbachol-induced rise in intracellular Ca²⁺. These data demonstrated that CysLT₁ receptor antagonists interact functionally with signaling pathways of P2Y receptors, and this should foster the study of possible implications for the clinical use of these compounds in asthma or in other inflammatory conditions.

Keywords: Abbreviations; DMEM; Dulbecco's modified Eagle's medium; DMSO; dimethylsulfoxide; FBS; fetal bovine serum; IP; ₃; inositol trisphosphate; CysLT; cysteinyl leukotriene; GPCR; G protein-coupled receptor; MRS2179; N; ⁶; -methyl-2′-deoxyadenosine-3′,5′-bisphosphate; MRS2279; (1′; R; ,2′; S; ,4′; S; ,5′; S; )-4-(2-chloro-6-methylamino-purin-9-yl)-1-[(phosphato)-methyl]-2-(phosphato)-bicyclo[3.1.0]hexane; PLC; phospholipase CMontelukast; Pranlukast; Purine receptors; Nucleotides; ATP; UDP

Inhibition of sphingolipid synthesis impairs cellular activation, cytokine production and proliferation in human lymphocytes by Norbert Blank; Martin Schiller; Christoph Gabler; Joachim R. Kalden; Hanns-Martin Lorenz (pp. 126-135).

The localisation of the T cell receptor and other signalling molecules in membrane microdomains (MM) is essential for the activation of T lymphocytes. These MM are stabilized by sphingolipids and cholesterol. It was recently shown that the activation of T lymphocytes leads to the confluence of small MM and the formation of an immunological synapse which is thought to be essential for a persistent activation and proliferation. We studied the effects of an inhibition of sphingolipid synthesis on T lymphocyte function. Both sphingolipid inhibitors, PDMP and myriocin, inhibited glucosphingolipids in whole cell lipid extracts and in MM. Both compounds inhibited the proliferation of superantigen-stimulated PBMC without inducing cell death. However, only the ceramide-like compound PDMP inhibited the expression of activation markers and the secretion of IFN-gamma which was not seen with myriocin treatment. The MM localisation of Lck and LAT was not significantly reduced in PDMP-treated cells.In conclusion, our results show that glucosphingolipids are necessary for cell growth of human T lymphocytes. However, inhibition of glucosphingolipid synthesis itself did not inhibit cellular activation. Our data show that glucosphingolipids – in contrast to cholesterol – are not essential for the stabilisation of MM.

Keywords: Abbreviations; CTB; cholera toxin B subunit; GM1; ganglioside GM1; LAT; linker of activation in T cells; MM; membrane microdomains; PDMP; 1-phenyl-2-decanoylamino-3-morpholino-1-propanol; SPT; serine palmitoyl transferaseMembrane microdomains; T lymphocytes; IFN-gamma

Estrogen receptor-independent inhibition of tumor necrosis factor-α gene expression by phytoestrogen equol is mediated by blocking nuclear factor-κB activation in mouse macrophages by Jong Soon Kang; Yeo Dae Yoon; Mi Hwa Han; Sang-Bae Han; Kiho Lee; Moo Rim Kang; Eun-Yi Moon; Young Jin Jeon; Song-Kyu Park; Hwan Mook Kim (pp. 136-143).

Equol has been suggested to possess protective effects on bone. However, the underlying mechanism of osteoprotective effect of equol has not been fully understood. In the present study, we examined the effect of equol on tumor necrosis factor-α (TNF-α) gene expression to elucidate a possible mechanism by which equol exerts osteoprotective effect. In vivo administration of equol inhibited TNF-α production by peritoneal macrophages isolated from LPS-treated mice. Equol also dose-dependently inhibited TNF-α production and TNF-α mRNA expression in LPS-stimulated mouse macrophages. Pretreatment of cells with ICI 182.780, an estrogen receptor antagonist, had no effect on the inhibitory efficacy of equol on LPS-induced TNF-α production. Further study demonstrated that equol inhibited NF-κB DNA binding and NF-κB-dependent reporter gene expression in activated RAW 264.7 cells. Moreover, equol blocked degradation of IκBα and IκBβ and nuclear translocation of p65 subunit of NF-κB in activated RAW 264.7 cells. These results suggest that the inhibitory effect of equol on TNF-α expression is mediated, at least in part, by blocking NF-κB activation and the inhibition of TNF-α expression by equol might be involved in its osteoprotective effect.

Keywords: Equol; TNF-α ;NF-κB; Osteoporosis

Polychlorinated biphenyls induce arachidonic acid release in human platelets in a tamoxifen sensitive manner via activation of group IVA cytosolic phospholipase A₂-α by Pontus K.A. Forsell; Anders O. Olsson; Erik Andersson; Laxman Nallan; Michael H. Gelb (pp. 144-155).

Polychlorinated biphenyls (PCBs) are stable compounds commonly found in nature as environmental pollutants. PCBs can affect the endocrine function of hormones such as steroid-hormones. Also, PCBs are known to be inducers of arachidonic acid release in various cells. We report, here, the effects of PCBs on eicosanoid formation, arachidonic acid release and cytosolic phospholipase A₂-α (cPLA₂-α) activation in human platelets. Ortho-substituted PCBs induced a time and dose-dependent release of arachidonic acid and the concomitant formation of 12( S)-hydroxy-5,8- cis-10- trans-14- cis-eicosatetraenoic acid (12-HETE) and 12( S)-hydroxy-5- cis-8,10- trans-heptadecatrienoic acid (12-HHT) in human platelets. The release of arachidonic acid and the formation of 12-HETE was completely blocked by the cPLA₂-α inhibitors AACOCF₃ or pyrrolidine-1. PCB-treatment of platelets demonstrated that the cPLA₂-α protein as well as PLA₂ activity translocated to the membrane fraction, independent of a rise in intracellular Ca²⁺. Furthermore, electrophoretic gel mobility shift analysis of cPLA₂-α on SDS-PAGE demonstrated a PCB-dependent phosphorylation of cPLA₂-α. The effects of 17β-estradiol and two structurally unrelated anti-estrogens, nafoxidin and tamoxifen on PCB-induced arachidonic acid release in platelets were also investigated. Both nafoxidin and tamoxifen inhibited PCB-induced arachidonic acid release as well as 12-HETE and 12-HHT formation. Interestingly, platelets incubated with PCBs did not aggregate despite the fact that robust release of arachidonic acid was observed. In summary, these results demonstrate that certain PCBs induce activation of cPLA₂-α independent of a rise in intracellular calcium and a robust release of arachidonic acid release with resulting eicosanoid formation in human platelets.

Keywords: Abbreviations; PCB; polychlorinated biphenyls; PLA; ₂; phospholipase A2; cPLA; ₂; cytosolic PLA; 12-HETE; 12(; S; )-hydroxy-5,8-; cis; -10-; trans; -14-; cis; -eicosatetraenoic acid; 12-HHT; 12(; S; )-hydroxy-5-; cis; -8,10-; trans; -heptadecatrienoic acid; AACOCF; ₃; arachidonyl trifluoromethyl ketone; 12-LO; 12-lipoxygenase; COX1; cyclooxygenase 1; TXA; ₂; thromboxane A2; PGH; ₂; prostaglandin H2; BAPTA-AM; 1,2-bis(; o; -aminophenoxy)ethane-; N; ,; N; ,; N; ′,; N; ′-tetraacetic acid tetra-(acetoxymethyl)ester; BEL; (E)-6-(bromomethylene)3-3(1-naphtalenyl)-2H-tetrahydropyran-2-onePolychlorinated biphenyls; Arachidonic acid; Phospholipase A; ₂; Platelets; Cyclooxygenase; Lipoxygenase

Identification of novel subtype selective RAR agonists by Fabrice Piu; Natalie K. Gauthier; Roger Olsson; Erika A. Currier; Birgitte W. Lund; Glenn E. Croston; Uli Hacksell; Mark R. Brann (pp. 156-162).

Drugs targeting retinoid receptors have been developed to treat a variety of therapeutic indications, but their success has been limited in part due to lack of selectivity. A novel functional cell-based assay R-SAT™ was employed to screen a small molecule chemical library and identify a variety of novel RAR agonists with various subtype selectivities, including RARβ/γ and RARγ selective agonists. A novel class of synthetic compounds that distinguishes between the different RARβ isoforms is described. This pharmacophore displays anti-proliferative activity and induces differentiation in a neuronal cell line, consistent with a classical retinoid mechanism of action while providing unique subtype selectivity. These novel subtype selective RAR agonists could serve as powerful tools to probe into subtype and isoform-specific retinoid function.

Molecular cloning and radioligand binding characterization of the chemokine receptor CCR5 from rhesus macaque and human by Carolyn Napier; Harriet Sale; Michael Mosley; Graham Rickett; Pat Dorr; Roy Mansfield; Mark Holbrook (pp. 163-172).

The aim of this study was to determine if macaque represents a suitable species for the pre-clinical evaluation of novel CCR5 antagonists, such as maraviroc (UK-427,857). To do this we cloned and expressed CCR5 from rhesus macaque and compared the binding properties of [¹²⁵I]-MIP-1β and [³H]-maraviroc with human recombinant CCR5. [¹²⁵I]-MIP-1β bound with similar high affinity to CCR5 from macaque ( K_d=0.24±0.05nM) and human ( K_d=0.23±0.05nM) and with similar kinetic properties. In competition binding studies the affinity of a range of human chemokines for macaque CCR5 was also similar to human CCR5. Maraviroc inhibited binding of [¹²⁵I]-MIP-1β to CCR5 from macaque and human with similar potency (IC₅₀=17.50±1.24nM and 7.18±0.93nM, respectively) and antagonised MIP-1β induced intracellular calcium release mediated through CCR5 from macaque and human with similar potency (IC₅₀=17.50±3.30nM and 12.07±1.89, respectively). [³H]-maraviroc bound with high affinity to CCR5 from macaque ( K_d=1.36±0.07nM) and human ( K_d=0.86±0.08nM), but was found to dissociate ∼10-fold more quickly from macaque CCR5. However, as with the human receptor, maraviroc was shown to be a high affinity, potent functional antagonist of macaque CCR5 thereby indicating that the macaque should be a suitable species in which to evaluate the pharmacology, safety and potential mechanism-related toxicology of novel CCR5 antagonists.

Keywords: Abbreviations; HIV; human immunodeficiency virus; MIP; macrophage inflammatory protein; HCC-1; hemofiltrate CC chemokine 1; MCP; monocyte chemotactic protein; RANTES; regulated on activation; normal T cell expressed and secreted; CTACK; cutaneous T cell-attracting chemokine; TARC; thymus and activation-regulated chemokine; LARC; liver and activation-regulated chemokine; ELC, EBI; Epstein-Barr virus induced molecule 1 ligand chemokine (MIP-3β); TECK; mature human thymus-expressed chemokineChemokine; CCR5; Receptor; Maraviroc; MIP-1β; Rhesus macaque; Radioligand binding

Molecular cloning, functional characterization of the porcine transient receptor potential V1 (pTRPV1) and pharmacological comparison with endogenous pTRPV1 by Toshio Ohta; Ryuichi Komatsu; Toshiaki Imagawa; Ken-ichi Otsuguro; Shigeo Ito (pp. 173-187).

In the present study, we cloned a porcine orthologue of transient receptor potential V1 (pTRPV1) and heterologously expressed it in human embryonic kidney (HEK) 293 cells to characterize its pharmacological properties. At the amino acid level, pTRPV1 was highly homologous (83–90%) to other orthologues of TRPV1. The expression of receptors was examined with current and [Ca²⁺]_i responses to capsaicin using whole-cell patch-clamp and fura-2 ratio imaging techniques, respectively, and by immunostaining with an anti-TRPV1 antibody. The receptors were characterized by changes in [Ca²⁺]_i in response to various vanilloid agonists, low pH and heat and by the effects of TRPV1 antagonists on them. The various TRPV1 agonists activated pTRPV1 in a dose-dependent manner in the order of potency of resiniferatoxin (RTX)>olvanil>capsaicin>phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), phorbol 12,13-dinonanoate 20-homovanillate (PDNHV). Isovelleral and scutigeral had no effect. Endogenous vanilloids (anandamide>15 (s)-HPETE≫NADA), low pH and noxious heat (>42°C) activated pTRPV1. Comparison of amino acid sequences with various mammalian TRPV1 homologues suggested some novel putative vanilloid recognition sites. TRPV1 antagonists, iodoRTX, ruthenium red and capsazepine suppressed capsaicin-induced responses. Similar to human TRPV1, but not rodent TRPV1, capsazepine was effective in blocking pH- and heat-induced responses. Similar pharmacological profiles were observed in cultured porcine dorsal root ganglion neurons. We discuss putative amino acid residues related to pharmacological differences among mammalian TRPV1 homologues.

Keywords: Calcium imaging; Capsaicin; Fura-2; Nociceptor; Noxious heat; Vanilloid

Synthesis and antinociceptive activity of cyclic endomorphin-2 and morphiceptin analogs by Anna Janecka; Jakub Fichna; Rafal Kruszynski; Yusuke Sasaki; Akihiro Ambo; Jean Costentin; Jean-Claude do-Rego (pp. 188-195).

Cyclic analogs of the opioid peptides endomorphin-2 and morphiceptin of the type Tyr-X-Phe-Phe-Y-NH₂ and Tyr-X-Phe-d-Pro-Y-NH₂ (X=Lys or Asp and Y=Lys or Asp), respectively, were synthesized in order to test their structure–activity relationships. Antinociceptive activity of the new analogs was assessed in the hot-plate test after intracerebroventricular administration in mice. The strong analgesic effect was observed for the analogs with Asp in position 2, while the analogs with Lys in the second position were inactive. Antinociception caused by Asp² analogs was dose-dependent and reversed by the concomitant administration of the universal opioid antagonist naloxone and by the selective κ antagonist, nor-BNI. However, receptor binding studies revealed poor affinity of all cyclic analogs at the μ-opioid receptor and no affinity at δ- and κ-opioid receptors. It is most likely that the new cyclic analogs produced their antinociception by the release of dynorphin A, which subsequently acted on the κ-opioid receptor.

Keywords: Binding studies; μ-, δ- and κ-opioid receptors; Hot-plate test; Locomotor activity; Solid phase peptide synthesis

Amitriptyline prevents thermal hyperalgesia and modifications in rat spinal cord GABA_B receptor expression and function in an animal model of neuropathic pain by Kenneth E. McCarson; Andrew Ralya; Scott A. Reisman; S.J. Enna (pp. 196-202).

Using an animal model of neuropathic pain, behavioral and biochemical experiments were performed to assess the effects of this condition on pain threshold and GABA_B receptor sensitivity and subunit gene expression in the rat lumbar spinal cord. The results indicate that partial sciatic nerve ligation decreases thermal and mechanical pain withdrawal latencies, and increases baclofen-stimulated [³⁵S]GTPγS binding and GABA_B receptor subunit gene expression in the rat lumbar spinal cord, suggesting that neuropathic pain may be due, in part, to a deficiency in GABAergic transmission. The experiments also demonstrate that daily administration (10mg/kg, i.p.) of amitriptyline, a tricyclic antidepressant used for the treatment of neuropathic pain, for 1 week after surgery prevents the decline in thermal pain threshold, the increase in GABA_B(2) gene expression, and development of increased GABA_B receptor function in spinal cord resulting from nerve damage. These findings indicate that the efficacy of amitriptyline as a treatment for neuropathic pain may be related to an ability to maintain spinal cord GABA_B receptor activity.

Keywords: Abbreviations; GABA; γ-aminobutyric acid; GABA; _B; γ-aminobutyric acid-B receptors; GABA; _B(1a); , GABA; _B(1b); , GABA; _B(2); γ-aminobutyric acid-B receptor subunits; GTP; guanosine triphosphateNociception; Neuropathic pain; Amitriptyline; GABA; _B; receptor; GABA; _B; receptor subunits; Spinal cord

Increased lysosomal uptake of methotrexate-polyglutamates in two methotrexate-resistant cell lines with distinct mechanisms of resistance by Lisa A. Marshall; Myung S. Rhee; Lars Hofmann; Alexey Khodjakov; Erasmus Schneider (pp. 203-213).

Methotrexate (MTX) resistance in mitoxantrone-selected MCF7/MX cells and in MTX-selected CEM/MTX cells is associated with reduced drug accumulation, albeit caused by different mechanisms. In addition, in both resistant cell lines the proportion of active long-chain MTX-polyglutamate (MTX-PG) metabolites is reduced relative to that in the respective parental cell line. Previous studies by others have implied that increased lysosomal uptake could affect the rate of MTX-PG hydrolysis, and hence the length distribution of the polyglutamate chains. However, in the two cell line pairs studied, the number of lysosomes per cell was not different between the corresponding parental and resistant cells. Instead, we observed a two- to three-fold increased facilitative uptake of MTX-Glu₄ by the lysosomes from these two independently derived MTX-resistant cell lines, compared to uptake by lysosomes from their corresponding parental cells. Enhanced lysosomal uptake of MTX-Glu₄ was reflected in an increased maximal uptake velocity, without a change in the apparent substrate affinity. In addition, the rate of MTX efflux from lysosomes from CEM/MTX cells was two-fold faster than from lysosomes from CEM cells. Consistent with this observation, the relative amount of short-chain MTX-Glu₁₊₂ species, as a fraction of the total amount of all MTX-Glu₁₋₄ species combined, was only half as large in lysosomes from CEM/MTX cells as in lysosomes from CEM cells. Together, these results suggest the possibility that increased lysosomal uptake, and hence enhanced sequestration of MTX-PGs in resistant cells, contributes to the development of high-level MTX resistance by decreasing the cytosolic levels of MTX-PGs.

Keywords: Abbreviations; FPGS; folylpoly-gamma-glutamate synthetase; gGH; gamma-glutamyl hydrolase; MTX; methotrexate; MTX-PG; methotrexate polyglutamateMethotrexate; Lysosomes; Drug transport; Drug resistance

Mobilization of iron from cells by hydroxyquinoline-based chelators by C. Mouralian; J.L. Buss; B. Stranix; J. Chin; P. Ponka (pp. 214-222).

With the aim of identifying an iron (Fe) chelator which is effective at mobilizing intracellular Fe, two novel ligands were synthesized and tested. Hydroxyquinoline is known to possess a high affinity for Fe and was thus chosen as the Fe binding motif for the hexadentate chelators, C1 (2,2′-[ethane-1,2-diylbis(iminomethylene)]diquinolin-8-ol) and C2 (2,2′-[cyclohexane-1,2-diylbis(iminomethylene)]diquinolin-8-ol). Both chelators are lipophilic, with Fe³⁺ complexes slightly more hydrophilic than the free ligands. C1 and C2 were equally toxic to K562 cells, and partial protection was afforded by supplementing the culture medium with human holotransferrin, suggesting that some of the toxicity of the ligands is due to cellular Fe depletion. Micromolar concentrations of both ligands effectively mobilized⁵⁹Fe from reticulocytes and K562 cells. In reticulocytes, 50μM C1 caused the release of 60% of the cells’ initial⁵⁹Fe uptake after a 4h incubation. Under the same conditions, C2 revealed a release of 50% of the⁵⁹Fe. Overall, both ligands merit in vivo study for oral activity. Their effectiveness at low concentrations makes them candidates for therapeutic use.

Keywords: Abbreviations; DFO; desferrioxamine; Fe; iron; HEPES; N; -2-hydroxyethylpiperazine-; N; ′-2-ethanesulfonic acid; 8-HQ; 8-hydroxyquinoline; L1; deferiprone; MEM; minimal essential medium; MO-8HQ; 7-morpholinomethyl-8-hydroxyquinoline; MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PIH; pyridoxal isonicotinoyl hydrazone; PMS; phenazine methosulfate; RPMI-1640; Rosewall Park Memorial Institute medium; Tf; transferrinCellular iron metabolism; Oral bioavailability; Intracellular iron chelatores; Iron chelator toxicity; Rational drug design

A protective role for proteinase activated receptor 2 in airways of lipopolysaccharide-treated rats by Silvana Morello; Valentina Vellecco; Fiorentina Roviezzo; Pasquale Maffia; Salvatore Cuzzocrea; Giuseppe Cirino; Carla Cicala (pp. 223-230).

Proteinase activated receptor-2 (PAR2), a seven transmembrane domain G protein coupled receptor, is expressed on airway epithelium and smooth muscle cells and over-expressed in human airways under pathological conditions, such as asthma and chronic obstructive pulmonary disease (COPD). However, the role of PAR2 in airways has not yet been defined. Aim of the present study, was to evaluate the in vitro rat bronchial response to a synthetic peptide activating PAR2 (PAR2-AP; SLIGRL), following an in vivo treatment with bacterial lipopolysaccharide (LPS). Bronchi from LPS-treated animals showed an increased relaxant response to PAR2-AP, compared to naïve animals, the effect was maximum after 20-h pre-treatment and reduced by epithelium removal. Western blot analysis showed an increased PAR2 protein expression on bronchi removed 20h after LPS treatment. PAR2-AP-induced bronchorelaxation was inhibited by ibuprofen, by the selective cyclooxygenase2 (COX-2) inhibitor, diisopropyl fluorophosphate (DFP) and partially by the calcitonin gene related peptide (CGRP) antagonist, rat-CGRP_[8–37]. Furthermore, there was a strong immunoreactivity for COX-2 on bronchial epithelium of LPS-treated rats. Prostaglandin E₂ (PGE₂) tissue release and CGRP tissue content were significantly increased following tissue incubation with PAR2-AP. The in vivo LPS treatment in rats strongly increases the bronchorelaxant effect of PAR2-AP, this effect correlates with an increased tissue protein receptor expression and the COX-2 localization on bronchial epithelium. Our work supports a role for PAR2 as a defence mechanism aimed to preserve bronchial functionality under systemic inflammatory conditions; both COX-2-derived PGE₂ and CGRP are involved in this effect.

Keywords: Airways; Calcitonin gene related peptide; Cyclooxygenase-2; Lipopolysaccharide; Proteinase activated receptor 2; Rat

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Biochemical Pharmacology (v.71, #1-2)