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Biochemical Pharmacology (v.73, #5)

Editorial Advisory Board (pp. iii).

Focal adhesion kinase: A potential target in cancer therapy by Maroesja J. van Nimwegen; Bob van de Water (pp. 597-609).

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in signal transduction pathways that are initiated at sites of integrin-mediated cell adhesions and by growth factor receptors. FAK is a key regulator of survival, proliferation, migration and invasion: processes that are all involved in the development and progression of cancer. FAK is also linked to oncogenes at both a biochemical and functional level. Moreover, overexpression and/or increased activity of FAK is common in a wide variety of human cancers, implicating a role for FAK in carcinogenesis. Given the important role of FAK in a large number of processes involved in tumorigenesis, metastasis and survival signalling FAK should be regarded as a potential target in the development of anti-cancer drugs. Therefore, selective inhibitors of FAK need to be developed. Combination of these selective FAK inhibitors with cytotoxic agents could be a very promising anti-cancer therapy.

Keywords: Abbreviations; ASAP1; Arf-GAP containing SH3 domain, ankyrin repeats and pleckstrin homology domain; Bad; Bcl-2 associated death protein; CAS; Crk associated substrate; Cdk; cyclin dependent kinase; cPLA2; cytoplasmic phospholipase A2; ECM; extracellular matrix; EGF(R); epidermal growth factor (receptor); ERK; extracellular signal-regulated kinase; FAK; focal adhesion kinase; FERM; erythrocyte band four.1-ezrin-radixin-moesin; FKHR; forkhead transcription factor; FRNK; FAK related non-kinase; FAT; focal adhesion targeting; GRAF; GTPase regulator associated with FAK; Grb; growth factor binding protein; GSK3; glycogen synthase kinase 3; JNK; Jun N-terminal kinase; LD; paxillin leucine aspartate repeat; MAPK; mitogen-activated protein kinase; MMP; matrix metalloproteinase; NF-kappa B; nuclear factor kappa B; PDGF(R); platelet derived growth factor (receptor); PDK1; Phosphoinositide-dependent protein kinase-1; PI-3 kinase; phosphatidylinositol 3-kinase; PIP2; phosphatidylinositol 3,4 biphosphate; PIP3; phosphatidylinositol 3,4,5 triphosphate; PKB; protein kinase B; PKC; protein kinase C; PP1; Phospho-protein phosphatase 1; PTEN; phosphatase and tensin homolog deleted on chromosome 10; PYK2; proline rich tyrosine kinase 2; SH; Src homology; Shc; Src homology containing protein; Sos; Son of sevenless; uPA; urokinase plasminogen activatorFocal adhesion kinase; Cancer; Metastasis; Anti-cancer therapy

Mitochondria-mediated and p53-associated apoptosis induced in human cancer cells by a novel selenophene derivative, D-501036 by Her-Shyong Shiah; Wan-Shu Lee; Shin-Hun Juang; Pao-Chiung Hong; Chia-Chi Lung; Ching-Jer Chang; Kai-Ming Chou; Jang-Yang Chang (pp. 610-619).

D-501036 [2,5-bis(5-hydroxymethyl-2-selenienyl)-3-hydroxymethyl- N-methylpyrrol], a novel selenophene derivative, is a highly potent cytotoxic agent with broad spectrum antitumor activity. The present study was undertaken to explore the mechanism(s) through which D-501036 exerts its action mode on the cancer cell death. D-501036 was found to suppress the growth of KB and HepG₂ cells in an irreversible manner. The results of annexin-V assays and PARP cleavage studies were consistent with the D-501036-induced apoptosis. Findings provided a strong support for the induction of mitochondria-mediated apoptosis by this drug. The examination of two canonical pathways of initiation caspases, those for caspases -8 and -9, revealed that caspase-9 protein and the activities of caspases -9 and -3 were increased in a dose- and time-dependent manner. The concentrations of Fas/Fas-L and procaspase-8 and the activity of caspase-8 were not altered. Furthermore, the mitochondrial membrane potential permeability and the release of cytochrome c to the cytosol were both increased by D-501036. The concentrations of the pro-apoptotic protein Bax and translocation of Bax from the cytosol to the mitochondria were increased in response to D-501036, whereas the concentrations of the anti-apoptotic protein Bcl-2 were decreased. Two DNA damage-related pro-apoptotic proteins, Puma and Noxa, were upregulated in a dose- and time-dependent manner. These pro-apoptotic and anti-apoptotic proteins are downstream effectors of p53. Accordingly, the phosphorylated and total forms of p53 were induced and p53 was translocated from the cytosol to the mitochondria in response to D-501036 treatment. Collectively, we conclude that D-501036 induces cellular apoptosis through the p53-associated mitochondrial pathway.

Keywords: Selenophene derivative; Apoptosis; Mitochondria; Caspase; p53

Development of elastin-like polypeptide for thermally targeted delivery of doxorubicin by Gene L. Bidwell III; Izabela Fokt; Waldemar Priebe; Drazen Raucher (pp. 620-631).

The chemotherapeutic drug doxorubicin (Dox) is widely used as an antitumor agent in hematological malignancies and solid tumors. However, one of the limitations of its clinical use is that systemic administration of an effective dose of Dox results in nonselective cardiac toxicity and myelosuppression. In order to minimize this nonspecific toxicity, Elastin-like polypeptide (ELP) was examined for its ability to serve as a macromolecular carrier for thermally targeted delivery of Dox. The ELP-based doxorubicin delivery vehicle (Tat-ELP-GFLG-Dox) consists of: (1) a peptide derived from the HIV-1 Tat protein to facilitate its cellular uptake, (2) ELP to allow thermal targeting, and (3) the lysosomally degradable glycylphenylalanylleucylglycine (GFLG) spacer and a cysteine residue conjugated to a thiol reactive doxorubicin derivative. Cytotoxicity of Tat-ELP-GFLG-Dox in MES-SA uterine sarcoma cells was enhanced 20-fold when aggregation of ELP was induced with hyperthermia. The ELP delivered doxorubicin displayed a cytoplasmic distribution and induced temperature dependent caspase activation.

Keywords: Abbreviations; aa; amino acid; BSA; bovine serum albumin; DIC; differential interference contrast; Dox; doxorubicin; ELP; elastin-like polypeptide; FBS; fetal bovine serum; HPMA; N; -(2-hydroxypropyl)methacrylamide; M; _W; molecular weight; PBS; phosphate-buffered physiological saline; Tat; cell penetrating peptide derived from HIV-1 Tat protein; T; _t; transition temperatureElastin-like polypeptide; Doxorubicin; Thermal targeting; Macromolecule; Drug delivery

Sulfated cyclodextrins inhibit the entry of Plasmodium into red blood cells by Ian E. Crandall; Walter A. Szarek; Jason Z. Vlahakis; Yiming Xu; Rahul Vohra; Jie Sui; Robert Kisilevsky (pp. 632-642).

The effect of sulfated cyclodextrins on Plasmodium falciparum cultures was determined. α-, β-, and γ-Cyclodextrins having equal degrees of sulfation inhibited parasite viability to a similar degree, a result suggesting that the ring size of the cyclodextrin is not a critical factor for inhibitory activity. β-Cyclodextrins containing fewer than two sulfate groups had no inhibitory activity, however, compounds containing 7–17 sulfates were found to be active in the μM range. Examination of treated cultures indicated that intracellular forms of the parasite were unaffected; however, increased numbers of extracellular merozoites were present. Active compounds produced enhanced erythrocyte staining with cationic dyes that could be reduced by stilbene disulfonates, a result suggesting that sulfated cyclodextrins inhibit parasite growth by interacting with the anion transport protein, AE1. Compounds that were found to be active in P. falciparum cultures were also found to inhibit P. berghei merozoite entry and could reduce the parasitemia of P. berghei infection in a mouse model, results suggesting that these compounds inhibit a common step in the merozoite invasion process of at least two Plasmodium species.

Keywords: Malaria; Plasmodium falciparum; Merozoite; Sulfated cyclodextrin; AE1; Inhibitor

The novel atypical retinoid ST1926 is active in ATRA resistant neuroblastoma cells acting by a different mechanism by Angela Maria Di Francesco; Daniela Meco; Anna Rita Torella; Giuseppe Barone; Maurizio D’Incalci; Claudio Pisano; Paolo Carminati; Riccardo Riccardi (pp. 643-655).

E-3-(4′-Hydroxy-3′-adamantylbiphenyl-4-yl)acrylic acid (ST1926) is a novel orally available compound belonging to the class of synthetic atypical retinoids. These agents are attracting growing attention because of their unique mechanism of antitumor action that appears different from that of classical retinoic acid. This study aims at investigating the antitumor activity of ST1926 in neuroblastoma (NB) preclinical models. In vitro, ST1926 was more cytotoxic than both its prototype, CD437 and all- trans-retinoic acid (ATRA) and it was active in the SK-N-AS cell line, which is refractory to ATRA. We showed that unlike ATRA, ST1926 does not induce morphological differentiation in NB cells where it produces indirect DNA damage, cell cycle arrest in late S-G2 phases and p53-independent programmed cell death. DNA damage was not mediated by oxidative stress and was repaired by 24h after drug removal. The SK-N-DZ cell line appeared the most sensitive to the proapoptotic activity of ST1926, probably because both the extrinsic and intrinsic pathways appear involved in the process. Studies with Z-VAD-FMK, suggested that ST1926 might also mediate caspase-independent apoptosis in NB cells. In vivo, orally administered ST1926, appeared to inhibit tumor growth of NB xenografts with tolerable toxicity. Overall, our results support the view that ST1926 might represent a good drug candidate in this pediatric tumor.

Keywords: Neuroblastoma; Atypical retinoids; ST1926; Apoptosis; DNA-damage; Alkaline Comet assay

Preclinical efficacy of ST1976, a novel camptothecin analog of the 7-oxyiminomethyl series by Michelandrea De Cesare; Giovanni Luca Beretta; Stella Tinelli; Valentina Benedetti; Graziella Pratesi; Sergio Penco; Sabrina Dallavalle; Lucio Merlini; Claudio Pisano; Paolo Carminati; Franco Zunino (pp. 656-664).

In previous studies, we have documented the potential therapeutic advantages of camptothecin analogs modified at the 7-position, i.e., 7-oxyiminomethyl derivatives. The present study was performed to explore the therapeutic potential of novel hydrophilic derivatives of this series. With one exception (ST1976), the tested camptothecins exhibited a reduced antiproliferative activity and all compounds retained ability to stabilize the topoisomerase I-mediated cleavable complex. The two analogs (ST1976 and ST1968) characterized by the presence of a free amino group in the side chain also exhibited the formation of persistent cleavable complexes. The most potent compound, ST1976 (7-(4-aminobenzyl)oxyiminomethylcamptothecin), was selected for evaluation of its preclinical profile of antitumor activity in a large panel of human tumor xenografts. As expected on the basis of the introduction of a hydrophilic substituent, the novel camptothecin was a substrate for BCRP. However, in spite of an apparent recognition by BCRP, ST1976 was effective following oral administration. The antitumor activity was evaluated using various schedules and routes of administration (i.v. and p.o.). ST1976 exhibited a remarkable activity in all tested tumors and was effective in a number of tumors which are resistant to irinotecan. The biological and pharmacological profile of ST1976 supports the therapeutic potential of camptothecins containing hydrophilic substituents at the 7-position. On the basis of its excellent activity in preclinical models, ST1976 is a promising candidate for clinical development.

Keywords: Camptothecins; DNA topoisomerase I; Antitumor activity; Cyotoxicity; BCRP; DNA cleavage

Multidrug resistance related protein (ABCC1) and its role on nitrite production by the murine macrophage cell line RAW 264.7 by Daniela F.P. Leite; Juliana Echevarria-Lima; João B. Calixto; Vivian M. Rumjanek (pp. 665-674).

Multidrug resistance related protein 1 (MRP1/ABCC1) is an ABC transporter protein related to the extrusion of reduced glutathione (GSH), oxidized glutathione (GSSG) and GSH-conjugates, as well as leukotriene C₄ and cyclopentane prostaglandins. Inhibition of ABCC1 activity impairs lymphocyte activation. The present work studied ABCC1 expression and activity on a murine macrophage cell line, RAW 267.4 and the effects of ABCC1 classical inhibitors, as well as GSH metabolism modulators, on LPS induced activation. Approximately, 75% of resting cells were positive for ABCC1 and the classical ABCC1 reversors (indomethacin, 0.1–2mM; probenecid, 0.1–10mM and MK571, 0.01–1mM) were able to enhance intracellular CFDA accumulation in a concentration-dependent manner, suggesting ABCC1 inhibition. After LPS (100ng/ml) activation 50% of the population was positive for ABCC1, and this protein was still active. In LPS-activated cells, ABCC1 activity was also impaired by BSO (1mM), an inhibitor of GSH synthesis. Conversely, GSH (5mM) reversed the BSO effect. ABCC1 inhibition by indomethacin, probenecid or MK571 decreased LPS induced nitrite production in a concentration-dependent manner, the same result was observed with BSO and again GSH reversed its effect. The ABCC1 reversors were also able to inhibit iNOS expression. In conclusion, LPS modulated the expression and activity of ABCC1 transporters in RAW macrophages and inhibitors of these transporters were capable of inhibiting nitrite production suggesting a role for ABCC1 transporters in the inflammatory process.

Keywords: Multidrug resistance; ABCC1; Glutathione; Nitric oxide; Macrophage; RAW 264.7

Effect of lysophosphatidylglycerol on several signaling molecules in OVCAR-3 human ovarian cancer cells: Involvement of pertussis toxin-sensitive G-protein coupled receptor by Kyoung Sun Park; Mi-Kyoung Kim; Dong-Soon Im; Yoe-Sik Bae (pp. 675-681).

In this study, we observed that lysophosphatidylglycerol (LPG) stimulated intracellular calcium ([Ca²⁺]_i) increase in OVCAR-3 human ovarian cancer cells. LPG-stimulated [Ca²⁺]_i increase was inhibited by U-73122 but not by U-73343, suggesting that LPG stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca²⁺]_i increase by LPG, indicating the activation of PTX-sensitive G-proteins. LPG-induced [Ca²⁺]_i increase was only observed in OVCAR-3 ovarian cancer cells and SK-OV3 ovarian cancer cells among tested several cell types. LPG also induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation in OVCAR-3 ovarian cancer cells. Pertussis toxin did not affect the LPG-induced activation of ERK and Akt phosphorylation. We also found that LPG failed to stimulate NF-κB-driven luciferase activity in exogenously LPA₁, LPA₂, or LPA₃-transfected HepG2 cells. Taken together we suggest that LPG stimulates a membrane bound receptor which is different from well-known LPA receptors (LPA₁, LPA₂, and LPA₃), resulting in at least two different signaling cascades; one involves a pertussis toxin-sensitive and phospholipase C-dependent [Ca²⁺]_i increase, and the other involves a pertussis toxin-insensitive activation of ERK and Akt in ovarian cancer cells.

Keywords: Abbreviations; LPA; lysophosphatidic acid; GPCR; G-protein coupled receptor; LPG; lysophosphatidylglycerol; fura-2/AM; fura-2 pentaacetoxymethylester; PTX; pertussis toxin; ERK; extracellular signal regulated protein kinase; [Ca; ²⁺; ]; _i; intracellular calcium concentration; PLC; phospholipase C; NF-κB; nuclear factor kappa BLysophosphatidylglycerol; Ovarian cancer cell; G-protein coupled receptor; Intracellular calcium increase; Pertussis toxin-sensitive G-protein

Neuroprotective effects of PMC, a potent α-tocopherol derivative, in brain ischemia-reperfusion: Reduced neutrophil activation and anti-oxidant actions by George Hsiao; Jie-Jen Lee; Yi-Cheng Chen; Jiing-Harn Lin; Ming-Yi Shen; Kuang-Hung Lin; Duen-Suey Chou; Joen-Rong Sheu (pp. 682-693).

2,2,5,7,8-Pentamethyl-6-hydroxychromane (PMC) is the most potent analogue of α-tocopherol for anti-oxidation. It is more hydrophilic than other α-tocopherol derivatives and has potent free radical-scavenging activity. In the present study, PMC significantly attenuated middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. Administration of PMC at 20mg/kg, showed marked reductions in infarct size compared with that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in HIF-1α, active caspase-3, iNOS, and nitrotyrosine expressions in ischemic regions. These expressions were markedly inhibited by treatment with PMC (20mg/kg). In addition, PMC (4–12μM) inhibited respiratory bursts in human neutrophils stimulated by fMLP (800nM) and PMA (320nM). Furthermore, PMC (6, 12, and 60μM) also significantly inhibited neutrophil migration stimulated by leukotriene B₄ (160nM). An electron spin resonance (ESR) method was conducted on the scavenging activity of PMC on the free radicals formed. PMC (12μM) greatly reduced the ESR signal intensities of superoxide anion, hydroxyl radical, and methyl radical formation. In conclusion, we demonstrate a potent neuroprotective effect of PMC on MCAO-induced focal cerebral ischemia in vivo. This effect may be mediated, at least in part, by inhibition of free radical formation, followed by inhibition of HIF-1α activation, apoptosis formation (active caspase-3), neutrophil activation, and inflammatory responses (i.e., iNOS and nitrotyrosine expressions), resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, PMC treatment may represent a novel approach to lowering the risk or improving function in ischemia-reperfusion brain injury-related disorders.

Keywords: Abbreviations; ESR; electron spin resonance; fMLP; formyl-Met-Leu-Phe; HIF-1α; hypoxia-inducible factor-1α; iNOS; inducible nitric oxide synthase; LCL; lucigenin-enhanced chemiluminescence; MCAO; middle cerebral artery occlusion; ROS; reactive oxygen species; mAb; monoclonal antibodyPMC; MCAO; HIF-1α; Caspase-3; iNOS; Free radical-scavenger

Toxic effects of cobalt in primary cultures of mouse astrocytes by Olga Karovic; Ilaria Tonazzini; Nelson Rebola; Erik Edström; Cecilia Lövdahl; Bertil B. Fredholm; Elisabetta Daré (pp. 694-708).

Cobalt is suspected to cause memory deficit in humans and was reported to induce neurotoxicity in animal models. We have studied the effects of cobalt in primary cultures of mouse astrocytes. CoCl₂ (0.2–0.8mM) caused dose-dependent ATP depletion, apoptosis (cell shrinkage, phosphatidylserine externalization and chromatin rearrangements) and secondary necrosis. The mitochondria appeared to be a main target of cobalt toxicity, as shown by the loss of mitochondrial membrane potential (Δ Ψ_m) and release from the mitochondria of apoptogenic factors, e.g. apoptosis inducing factor (AIF). Pre-treatment with bongkrekic acid reduced ATP depletion, implicating the involvement of the mitochondrial permeability transition (MPT) pore. Cobalt increased the generation of oxygen radicals, but antioxidants did not prevent toxicity. There was also an impaired response to ATP stimulation, evaluated as a lower raise in intracellular calcium. Similarly to hypoxia and dymethyloxallyl glycine (DMOG), cobalt triggered stabilization of the α-subunit of hypoxia-inducible factor HIF-1 (HIF-1α). This early event was followed by an increased expression of HIF-1 regulated genes, e.g. stress protein HO-1, pro-apoptotic factor Nip3 and iNOS. Although all of the three stimuli activated the HIF-1α pathway and decreased ATP levels, the downstream effects were different. DMOG only inhibited cell proliferation, whereas the other two conditions caused cell death by apoptosis and necrosis. This points to cobalt and hypoxia not only inducing HIF-1α regulated genes but also affecting similarly other cellular functions, including metabolism.

Keywords: Hypoxia; Apoptosis; Mitochondrial damage; Reactive oxygen species; Cobalt; ATP

Carnosine protects against NMDA-induced neurotoxicity in differentiated rat PC12 cells through carnosine-histidine-histamine pathway and H₁/H₃ receptors by Yao Shen; Wei-wei Hu; Yan-yin Fan; Hai-bing Dai; Qiu-li Fu; Er-Qing Wei; Jian-hong Luo; Zhong Chen (pp. 709-717).

Since the histidine-containing dipeptide carnosine (β-alanyl-l-histidine) is believed to have many physiological functions in the brain, we investigated the neuroprotective effects of carnosine and its mechanisms of action in an in vitro model of neurotoxicity induced by N-methyl-d-aspartate (NMDA) in differentiated PC12 cells. Pretreatment with carnosine increased the viability and decreased the number of apoptotic and necrotic cells measured by MTT and Hoechst 33342 and propidium iodide (PI) double staining assays. Carnosine also can inhibite the glutamate release and increase HDC activity and the intracellular and extracellular contents of carnosine, histidine and histamine detected by high-performance liquid chromatography (HPLC). The protection by carnosine was reversed by α- fluoromethylhistidine, a selective and irreversible inhibitor of histidine decarboxylase (HDC). Pyrilamine and thioperamide, selective central histamine H₁ and H₃ antagonists also significantly reversed the protection of carnosine. Further, the inhibition of glutamate release by carnosine was reversed by thioperamide. Therefore, the protective mechanism of carnosine may not only involve the carnosine-histidine-histamine pathway, but also H₁/H₃ receptors and the effective inhibition of glutamate release. This study indicates that carnosine may be an endogenous protective factor and calls for its further study as a new antiexcitotoxic agent.

Keywords: Carnosine; Histamine; Neuroprotection; Neurotoxicity; N; -methyl-; d; -aspartate

Comparative effects of five bisphosphonates on apoptosis of macrophage cells in vitro by M.F. Moreau; C. Guillet; P. Massin; S. Chevalier; H. Gascan; M.F. Baslé; D. Chappard (pp. 718-723).

Bisphosphonates (BPs) inhibits bone resorption by reducing osteoclastic activity; they induce osteoclast apoptosis. Pathophysiology of prostheses loosening is complex and implies an inflammatory reaction secondary to the phagocytosis of wear debris by macrophages with a secondary increased bone resorption by osteoclasts. BPs inhibit proliferation and cause cell death in macrophages by induction of apoptosis. We have used mouse macrophage-like J774.1 cells to evaluate the effects of five BPs.J774A.1 cells were cultured in a standard culture medium for 2-days. BPs (alendronate, pamidronate, etidronate, risedronate, zoledronic acid) were added in the medium at concentration of 10⁻⁶ to 10⁻⁴M during 3 days. Cells were studied by fluorescence microscopy after staining with the fluorescent dye Hoescht H33342 and the percentage of apoptotic cells was determined on 300 nuclei. Cells were analyzed by flow cytofluorometry after staining with annexin V-FITC (for counting apoptotic cells) and propidium iodide (for necrotic/late-apoptotic cells) on 2000 cells.Etidronate did not cause significant apoptosis or necrosis, at any concentration. Alendronate and pamidronate caused apoptosis and death only at very high concentration [10⁻⁴M]. On the contrary, apoptotic and necrotic cells were evidenced with risedronate or zoledronic acid at lower concentrations. These effects were dose-dependant and occurred when concentration reached [10⁻⁵M]. The number of apoptotic cells was higher with zoledronic acid and then with risedronate. Cytofluorometry appeared superior to cytologic analysis in the investigation of macrophage apoptosis, since necrotic cells loose contact with the glass slides and are not identifiable in cytological counts. Some amino-BPs appear to induce apoptosis in macrophages.

Keywords: Bisphosphonates; Apoptosis; Necrosis; Macrophage; Flow cytometry

Sodium nitroprusside decreased leukotriene C4 generation by inhibiting leukotriene C4 synthase expression and activity in hepatic ischemia-reperfusion injured rats by Shu-Long Yang; Yi-Jia Lou (pp. 724-735).

The effects of NO on LTC4 generation during hepatic ischemia-reperfusion (I/R) are largely unclear. Sprague–Dawley rats were divided into control, I/R and sodium nitroprusside (SNP, 2.5, 5 and 10μg/kg/min)+I/R groups. Liver was subjected to I/R injury, saline or SNP administered intravenously. The protein expressions of LTC4 synthesis enzymes including LTC4 synthase (LTC4S), microsomal glutathione- S-transferase (mGST)2 and mGST3 were detected with immunoblotting, the LTC4 synthesis enzymes’ activities and LTC4 content were measured by RP-HPLC, the mRNA expressions of inducible nitric oxide synthase (iNOS) and endogenous nitric oxide synthase (eNOS) in liver were measured by RT-PCR. Tissue injuries were assessed by serum ALT and AST and histological changes. Serum NO₂⁻ and liver tissue GSH were also examined. Compared with I/R group, SNP markedly decreased LTC4 content, LTC4S protein and iNOS mRNA levels, and the LTC4 synthesis enzymes’ activities ( P<0.05), but significantly enhanced eNOS mRNA expression in liver ( P<0.05). The decline in serum ALT, AST and NO₂⁻ levels ( P<0.05) together with hepatic GSH elevation ( P<0.05) in SNP+I/R groups were also observed. LTC4S expression in hepatocytes and sinusoidal endothelial cells in SNP+I/R groups was lower than that in I/R group. But no significant differences in the protein expressions of mGST3 and mGST2 existed between control, I/R and SNP+I/R groups ( P>0.05). These results demonstrated that the decline in LTC4 production by SNP treatment during hepatic I/R could be partially resulted from SNP down-regulating the protein expression of LTC4S rather than mGST2 or mGST3 and its inhibiting the LTC4 synthesis enzymes’ activities.

Keywords: Abbreviations; SNP; sodium nitroprusside; NO; nitric oxide; eNOS; constitutively expressed endothelial NO synthase; iNOS; inducible NO synthase; I/R; ischemia and reperfusion; LT; leukotriene; 5-LO; 5-lipoxygenase; LTC4S; leukotriene C4 synthase; mGST; microsomal glutathione-; S; -transferase; LPS; lipopolysaccharides; RT-PCR; reverse-transcriptase polymerase chain reaction; RP-HPLC; reversed phase high-performance liquid chromatography; HTMP; 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-o; PAGE; SDS-polyacrylamide gel electrophoresis; ALT; alanine aminotransferase; AST; aspartate aminotransferase; GSH; reduced glutathioneNitric oxide; Sodium nitroprusside; Cysteinyl leukotrienes; Leukotriene C4 synthase; Microsomal glutathione-; S; -transferase; Ischemia-reperfusion injury; Liver

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Biochemical Pharmacology (v.73, #5)