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Biochemical Pharmacology (v.80, #3)

Graphical Abstracts Contents (pp. e1-e4).

Editorial Advisory Board (pp. iii).

Urocortins in heart failure by Sowmya Venkatasubramanian; David E. Newby; Ninian N. Lang (pp. 289-296).

The effects of urocortins on multiple organ systems in heart failure: Urocortins, through different mechanisms exerts a multitude of beneficial effects on the various organ systems as demonstrated by animal and human models. ACTH: adrenocorticotrophic hormone; Ucn 1: urocortin 1; LAP: left atrial pressure; BP: blood pressure; PRA: plasma renin activity.Despite modern advances in the treatment of the causes and consequences of cardiovascular illness, heart disease and heart failure remain a leading cause of death in the western world. Many novel peptides are emerging as biomarkers and potential therapeutic tools for this debilitating condition. Urocortins represent one such group of peptides whose role in normal cardiovascular physiology and disease states is now increasingly being recognized. The cardiovascular effects of the urocortins are mediated via corticotrophin-releasing hormone (CRH) receptors through a variety of intra-cellular signaling pathways. Studies to date have demonstrated a favourable effect of urocortins on hemodynamic and neurohumoral regulation. They cause relaxation of the vasculature as well as having positive inotropic, chronotropic and lusitropic effects on the heart. This makes the urocortins a potentially attractive target in the treatment of heart failure. Indeed, a number of studies have demonstrated increased urocortin activity in experimental and clinical heart failure, with apparent augmented responses in these states. This article provides a review of the role of urocortins in normal cardiovascular physiology and in the pathophysiology of heart failure.

Keywords: Urocortins; Cardiovascular; Corticotrophin releasing hormone; Heart failure

Clinical utility of calcimimetics targeting the extracellular calcium-sensing receptor (CaSR) by Edward M. Brown (pp. 297-307).

Calcimimetics, which activate the extracellular calcium (Ca_o²⁺)-sensing receptor in the parathyroid and other tissues participating in Ca_o²⁺ homeostasis, were the first described allosteric activators of a G-protein-coupled receptor. Cinacalcet, the only calcimimetic currently approved for human use, is used clinically for treating secondary hyperparathyroidism (e.g., overactivity of parathyroid glands) in patients being dialyzed for chronic kidney disease. By sensitizing the parathyroids to Ca_o²⁺, cinacalcet lowers the circulating parathyroid hormone (PTH) level. It also reduces serum calcium and phosphate, changes increasing the percentage of patients achieving the guidelines recommended by the National Kidney Foundation (NKF) for these minerals. Studies are underway addressing whether better adherence to these guidelines in patients receiving cinacalcet reduces cardiovascular disease and related mortality, which are both common is the dialysis population. The second approved use of cinacalcet is for treating hypercalcemia in patients with inoperable parathyroid carcinoma. In this setting, it provides the first medical therapy chronically lowering serum calcium concentration in this condition, albeit not to normal in most patients. Its effect on the long-term prognosis of these patients, if any, is presently unclear. “Off-label” administration of cinacalcet [i.e., not yet approved by the US Food and Drug Administration (FDA)] effectively lowers serum calcium and/or PTH in various other forms of hyperparathyroidism and increases serum phosphate in renal phosphate-wasting syndromes by reducing PTH-induced phosphaturia. In the future, the drug could conceivably be utilized to modulate the activity of the CaSR in other tissues (i.e., kidney, colon) in therapeutically desirable ways.

Keywords: Abbreviations; CaSR; calcium-sensing receptor; Ca; _o; ²⁺; extracellular calcium concentration; PTH; parathyroid hormone; NKF; National Kidney Foundation; FDA; US Food and Drug Administration; CKD; chronic kidney disease; MGluR; metabotropic glutamate receptor; GABA; gamma aminobutyric acid; PLC; phospholipase C; MAPK; mitogen-activated protein kinase; ERK; extracellular signal-regulated kinase; JNK; c-jun N-terminal kinase; EGF; epidermal growth factor; 1,25(OH); ₂; D; ₃; 1,25-dihydroxyvitamin D; ₃; VDR; vitamin D receptor; EC; ₅₀; half-maximally effective concentration of a drug; KDOQI; Kidney Disease Outcomes Quality Initiative; ECD; extracellular domain; TMD; transmembrane domain; TIO; tumor-induced osteomalacia; XLH; X-linked hypophosphatemia; GFR; glomerular filtration rate; TP/GFR; renal threshold for phosphate corrected for GFR; ESRD; end stage renal disease; ADPKD; autosomal dominant polycystic kidney disease; PHEX; phosphate-regulating gene with homologies to endopeptidases on the X-chromosome; FGF-23; fibroblast growth factor-23; FHH; familial hypocalciuric hypercalcemia; NSHPT; neonatal severe hyperparathyroidism; PHPT; primary hyperparathyroidism; DCT; distal convoluted tubule; cTAL; cortical thick, ascending limb; BMD; bone mineral density; GPRC6A; G-protein-coupled receptor of family C, number 6ACalcimimetic; Calcium-sensing receptor; Parathyroid; Primary hyperparathyroidism; Secondary hyperparathyroidism; Parathyroid cancer; Stage 5 kidney disease; Renal transplantation; Lithium-induced hypercalcemia; Familial hypocalciuric hypercalcemia

Metabolism of short-chain ceramide by human cancer cells—Implications for therapeutic approaches by Jacqueline V. Chapman; Valérie Gouazé-Andersson; Maria C. Messner; Margaret Flowers; Ramin Karimi; Mark Kester; Brian M. Barth; Xin Liu; Yong-Yu Liu; Armando E. Giuliano; Myles C. Cabot (pp. 308-315).

Due to recent use of short-chain ceramides in preclinical studies, we characterized C6-ceramide metabolism in cancer cell lines and assessed metabolic junctures for enhancing efficacy. MDA-MB-231 breast cancer cells decreased the amount of C6-ceramide metabolized to C6-sphingomyelin (C6-SM) and increased the amount metabolized to C6-glucosylceramide (C6-GC) in response to increasing concentrations. A similar trend was seen in DU-145 (prostate cancer), PANC-1 (pancreatic cancer), and LoVo (colorectal cancer) cells. KG-1 leukemia cells favored C6-SM synthesis at low (0.6μM) and high-dose (12μM) C6-ceramide. Partnering C6-ceramide with tamoxifen, a P-glycoprotein antagonist that impedes ceramide glycosylation, was an effective regimen for enhancing cytotoxicity in cells. Experiments to assess the mechanism of cell death using KG-1 cells showed that tamoxifen inhibited synthesis of C6-GC and C6-SM from C6-ceramide by 80% and 50%, respectively, which was accompanied by enhanced apoptosis. Radiolabeling of KG-1 cells with [³H]palmitic acid produced a 2-fold increase in³H-long-chain ceramides when unlabeled C6-ceramide was added and a 9-fold increase when C6-ceramide and tamoxifen were added. The increase in³H-palmitate radiolabeling of long-chain ceramides was blocked by inclusion of a ceramide synthase inhibitor; however, inhibiting synthesis of long-chain ceramide did not rescue cells. These studies show that tamoxifen enhances the apoptotic effects of C6-ceramide. The proposed mechanism involves blocking short-chain ceramide anabolism to favor hydrolysis and generation of sphingosine. We propose that use of tamoxifen and other P-glycoprotein antagonists can be an effective means for enhancing cytotoxic potential of short-chain ceramides in the treatment of cancer.

Keywords: Ceramide; Glucosylceramide; Cancer cells; Tamoxifen

Ferritin H induction by histone deacetylase inhibitors by Wei Wang; Xiumin Di; Suzy V. Torti; Frank M. Torti (pp. 316-324).

Because both iron deficiency and iron excess are deleterious to normal cell function, the intracellular level of iron must be tightly controlled. Ferritin, an iron binding protein, regulates iron balance by storing iron in a bioavailable but nontoxic form. Ferritin protein comprises two subunits: ferritin H, which contains ferroxidase activity, and ferritin L. Here we demonstrate that ferritin H mRNA and protein are induced by histone deacetylase inhibitors (HDAC inhibitors), a promising class of anti-cancer drugs, in cultured human cancer cells. Deletion analysis and EMSA assays reveal that the induction of ferritin H occurs at a transcriptional level via Sp1 and NF-Y binding sites near the transcriptional start site of the human ferritin H promoter. Classically, HDAC inhibitors modulate gene expression by increasing histone acetylation. However, ChIP assays demonstrate that HDAC inhibitors induce ferritin H transcription by increasing NF-Y binding to the ferritin H promoter without changes in histone acetylation. These results identify ferritin H as a new target of HDAC inhibitors, and recruitment of NF-Y as a novel mechanism of action of HDAC inhibitors.

Keywords: Abbreviations; HDAC inhibitors; histone deacetylase inhibitors; TSA; trichostatin A; CHIP; chromatin immunoprecipitation; TRE; TSA responsive elementFerritin H; Histone acetylation; Chromatin immunoprecipitation; Cancer; HDAC inhibitors; Transcription

Transcriptional up-regulation of SOD1 by CEBPD: A potential target for cisplatin resistant human urothelial carcinoma cells by Tzyh-Chyuan Hour; Yan-Liang Lai; Ching-I Kuan; Chen-Kung Chou; Ju-Ming Wang; Huang-Yao Tu; Huei-Ting Hu; Chang-Shen Lin; Wen-Jeng Wu; Yeong-Shiau Pu; Esta Sterneck; A-Mei Huang (pp. 325-334).

Bladder cancer is the fourth most common type of cancer in men (ninth in women) in the United States. Cisplatin is an effective agent against the most common subtype, urothelial carcinoma. However, the development of chemotherapy resistance is a severe clinical problem for the successful treatment of this and other cancers. A better understanding of the cellular and molecular events in response to cisplatin treatment and the development of resistance are critical to improve the therapeutic options for patients. Here, we report that expression of the CCAAT/enhancer binding protein delta (CEBPD, C/EBPδ, NF-IL6β) is induced by cisplatin in the human bladder urothelial carcinoma NTUB1 cell line and is specifically elevated in a cisplatin resistant subline. Expression of CEBPD reduced cisplatin-induced reactive oxygen species (ROS) and apoptosis in NTUB1 cells by inducing the expression of Cu/Zn-superoxide dismutase (SOD1) via direct promoter transactivation. Several reports have implicated CEBPD as a tumor suppressor gene. This study reveals a novel role for CEBPD in conferring drug resistance, suggesting that it can also be pro-oncogenic. Furthermore, our data suggest that SOD inhibitors, which are already used as anti-angiogenic agents, may be suitable for combinatorial chemotherapy to prevent or treat cisplatin resistance in bladder and possibly other cancers.

Keywords: Abbreviations; CEBPD; CCAAT/enhancer binding protein delta; ROS; reactive oxygen species; SOD1; Cu/Zn-superoxide dismutase; TETA; triethylenetetramine dihydrochlorideC/EBP transcription factor; Cisplatin drug resistance; Superoxide dismutase; Urothelial carcinoma; SOD inhibitor

The NER proteins XPC and CSB, but not ERCC1, regulate the sensitivity to the novel DNA binder S23906: Implications for recognition and repair of antitumor alkylators by Céline J. Rocca; Virginie Poindessous; Daniele G. Soares; Karima El Ouadrani; Alain Sarasin; Eric Guérin; Aimery de Gramont; João A.P. Henriques; Alexandre E. Escargueil; Annette K. Larsen (pp. 335-343).

Cells resistant to S23906, a bulky monofunctional agent (left) show increased nucleotide excision repair activity (middle) which is accompanied by increased expression of CSB, XPC, but not ERCC1 (right).S23906 belongs to a novel class of alkylating anticancer agents forming bulky monofunctional DNA adducts. A unique feature of S23906 is its “helicase-like” activity leading to the destabilization of the surrounding duplex DNA. We here characterize the recognition and repair of S23906 adducts by the nucleotide excision repair (NER) machinery. All NER-deficient human cell lines tested showed increased sensitivity to S23906, which was particularly pronounced for cells deficient in XPC, CSB and XPA. In comparison, deficiencies in ERCC1 or XPF had lesser impact on the sensitivity to S23906. The sensitivity was, at least in part, linked to the conversion of unrepaired adducts into toxic DNA strand breaks as shown by single cell electrophoresis and gamma-H2AX formation. The pharmacological relevance of these findings was confirmed by the characterization of KB carcinoma cells with acquired S23906 resistance. These cells showed increased NER activity in vivo as well as toward damaged plasmid DNA in vitro. In particular, both global genome NER, as shown by unscheduled DNA synthesis, and transcription-coupled NER, as shown by transcriptional recovery, were up-regulated in the S23906-resistant cells. The increased NER activity was accompanied by up to 5-fold up-regulation of XPC, CSB and XPA proteins without detectable alterations of ERCC1 on the DNA, RNA or protein levels. Our results suggest that S23906 adducts are recognized and repaired by both NER sub-pathways in contrast to other members of this class, that are only recognized by transcription-coupled NER. We further show that NER activity can be up-regulated without changes in ERCC1 expression.

Keywords: Nucleotide excision repair; S23906; Monofunctional alkylators; XPC; ERCC1

Mechanistic insights into antitumor effects of new dinuclear cis Pt^II complexes containing aromatic linkers by Lenka Zerzankova; Hana Kostrhunova; Marie Vojtiskova; Olga Novakova; Tereza Suchankova; Miaoxin Lin; Zijian Guo; Jana Kasparkova; Viktor Brabec (pp. 344-351).

The primary objective was to understand more deeply the molecular mechanism underlying different antitumor effects of dinuclear Pt^II complexes containing aromatic linkers of different length, {[ cis-Pt(NH₃)₂Cl]₂(4,4′-methylenedianiline)}²⁺ (1) and {[ cis-Pt(NH₃)₂Cl]₂(α,α′-diamino-p-xylene)}²⁺ (2). These complexes belong to a new generation of promising polynuclear platinum drugs resistant to decomposition by sulfur nucleophiles which hampers clinical use of bifunctional polynuclear trans Pt^II complexes hitherto tested. Results obtained with the aid of methods of molecular biophysics and pharmacology reveal differences and new details of DNA modifications by1 and2 and recognition of these modifications by cellular components. The results indicate that the unique properties of DNA interstrand cross-links of this class of polynuclear platinum complexes and recognition of these cross-links may play a prevalent role in antitumor effects of these metallodrugs. Moreover, the results show for the first time a strong specific recognition and binding of high-mobility-group-domain proteins, which are known to modulate antitumor effects of clinically used platinum drugs, to DNA modified by a polynuclear platinum complex.

Keywords: Abbreviations; BBR3464; [{; trans; -PtCl(NH; ₃; ); ₂; }; ₂; -μ-; trans; -Pt(NH; ₃; ); ₂; (NH; ₂; (CH; ₂; ); ₆; NH; ₂; ); ₂; ]; ⁴⁺; BBR3610; [{; trans; -PtCl(NH; ₃; ); ₂; }; ₂; -μ-{; trans; -H; ₂; N(CH; ₂; ); ₆; NH; ₂; (CH; ₂; ); ₂; NH; ₂; (CH; ₂; ); ₆; NH; ₂; }]; ⁴⁺; bp; base pair; Cisplatin; cis; -diamminedichloridoplatinum(II); CFE; cell-free extract; CL; cross-link; Complex; 1; {[; cis; -Pt(NH; ₃; ); ₂; Cl]; ₂; (4,4′-methylenedianiline)}; ²⁺; Complex 2; {[; cis; -Pt(NH; ₃; ); ₂; Cl]; ₂; (α,α′-diamino-p-xylene)}; ²⁺; CT; calf-thymus; DPP; differential pulse polarography; EtBr; ethidium bromide; FAAS; flameless atomic absorption spectrophotometry; HMG; high-mobility-group; IC; ₅₀; concentration inhibiting cell growth by 50%; MTT; [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]; PAGE; polyacrylamide gel electrophoresis; r; _b; the number of molecules of the metal complex bound per nucleotide residue; r; _i; the molar ratio of free metal complex to nucleotide-phosphates at the onset of incubation with DNADinuclear platinum; Antitumor; DNA damage; Interstrand cross-link; Recognition

Alisol-B, a novel phyto-steroid, suppresses the RANKL-induced osteoclast formation and prevents bone loss in mice by Ji-Won Lee; Yasuhiro Kobayashi; Yuko Nakamichi; Nobuyuki Udagawa; Naoyuki Takahashi; Nam-Kyung Im; Hwa-Jeong Seo; Won Bae Jeon; Takayuki Yonezawa; Byung-Yoon Cha; Je-Tae Woo (pp. 352-361).

Osteoclasts, bone-resorbing multinucleated cells, are differentiated from hemopoietic progenitors of the monocyte/macrophage lineage. Bone resorption by osteoclasts is considered a potential therapeutic target to the treatment of erosive bone diseases, including osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we found that alisol-B, a phyto-steroid from Alisma orientale Juzepczuk, exhibited inhibitory effects on osteoclastogenesis both in vitro and in vivo. Although RT-PCR analysis showed that alisol-B did not affect the 1α,25(OH)₂D₃-induced expressions of RANKL, OPG and M-CSF mRNAs in osteoblasts, addition of alisol-B to co-cultures of mouse bone marrow cells and primary osteoblasts with 10⁻⁸M 1α,25(OH)₂D₃ caused significant inhibition of osteoclastogenesis. We further examined the direct effects of alisol-B on osteoclast precursors. Alisol-B strongly inhibited RANKL-induced osteoclast formation when added during the early stage of cultures, suggesting that alisol-B acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, alisol-B inhibited the phosphorylation of JNK, which are upregulated in response to RANKL in bone marrow macrophages, alisol-B also inhibited RANKL-induced expression of NFATc1 and c-Fos, which are key transcription factors for osteoclastogenesis. In addition, alisol-B suppressed the pit-forming activity and disrupted the actin ring formation of mature osteoclasts. In a hypercalcemic mouse model induced by 2-methylene-19-nor-(20 S)-1α,25(OH)₂D₃ (2MD), an analog of 1α,25(OH)₂D₃, administration of alisol-B significantly suppressed 2MD-induced hypercalcemia as resulting from the inhibition of osteoclastogenesis. Taken together, these findings suggest that alisol-B may be a potential novel therapeutic molecule for bone disorders by targeting the differentiation of osteoclasts as well as their functions.

Keywords: Alisol-B; Anti-resorptive agent; Osteoclast; RANKL; JNK

Altered phospholipid transfer protein gene expression and serum lipid profile by topotecan by Rudel A. Saunders; Kazuyuki Fujii; Leah Alabanza; Roald Ravatn; Tsunekazu Kita; Kazuya Kudoh; Masahiro Oka; Khew-Voon Chin (pp. 362-369).

Camptothecin (CPT) and its structural analogues including topotecan and irinotecan, are inhibitors of topoisomerase I. These drugs are clinically active against a broad spectrum of cancers. To understand the genesis of chemotherapeutic resistance to the CPT family of anticancer drugs, we examined by gene expression profiling the pharmacological response to topotecan in the human hepatoma HepG2 cells and found a striking induction of the phospholipid transfer protein (PLTP) gene expression by topotecan. We showed that activation of PLTP gene expression is specific to CPT and its analogues including specific enantiomers that inhibit topoisomerase I. PLTP-mediated lipid transfer to high-density lipoprotein (HDL) is thought to be important for shuttling and redistribution of lipids between lipoproteins, which are normally returned to the liver for metabolism via the reverse cholesterol transport pathway. Hence, we asked whether elevated PLTP levels might increase the transfer of drugs into HDL. We observed that CPT was not accumulated in HDL and other lipoproteins. In addition, topotecan treatment in mice caused a marked reduction in serum HDL that was accompanied by an increase in triglyceride and cholesterol levels. These results showed that PLTP does not mediate the transfer of topoisomerase I inhibitors to serum lipoproteins. However, elevated serum PLTP levels following treatment with topoisomerase I inhibitors in cancer patients may serve as a biomarker for monitoring the development of hypertriglyceridemia and acute pancreatitis.

Keywords: Topoisomerase I inhibitor; Camptothecin; Topotecan; Phospholipid transfer protein; High-density lipoprotein; Pancreatitis; Hyperlipidemia; Hypertriglyceridemia

Rimonabant-induced apoptosis in leukemia cell lines: Activation of caspase-dependent and -independent pathways by Dario Gallotta; Patrizia Nigro; Roberta Cotugno; Patrizia Gazzerro; Maurizio Bifulco; Maria Antonietta Belisario (pp. 370-380).

Rimonabant (SR141716), a cannabinoid CB1 receptor antagonist known for anti-obesity activity, has more recently been shown to inhibit tumor cell growth. Here we demonstrated the antitumor potential of SR141716 in leukemia-derived cell lines and its low toxicity in normal cells (PBMC). SR141716 (1–20μM range of doses) reduced Jurkat and U937 cell number by activating death signals as well as affecting cell cycle progression. The most prominent response in U937 to SR141716 was a G₀/G₁ block, while in Jurkat cells there was activation of cell death processes. SR141716-treated cells exhibited the morphological and biochemical features of apoptosis and to some extent necrosis. Apoptotic mode of cell death was confirmed in both cell lines by analysis of cell morphology, phosphatidylserine exposure and DNA fragmentation. Moreover, the drug was found to induce an early and robust mitochondrial membrane depolarization. In Jurkat cells the apoptotic process was typically caspase-dependent, while in U937 caspase-independent pathways were also activated. The contribution of PARP activation to SR141716-induced apoptosis in U937 was suggested by protein PARylation, AIF release and apoptosis reversal by PARP inhibitors. Moreover, SR141716 negatively modulated, especially in U937, the PI3K/AKT pathways. In conclusion, our data indicate that SR141716 elicits alternative response and/or cell death pathways depending on the cell type affected.

The structure of the C-terminal helical bundle in glutathione transferase M2-2 determines its ability to inhibit the cardiac ryanodine receptor by Ruwani Hewawasam; Dan Liu; Marco G. Casarotto; Angela F. Dulhunty; Philip G. Board (pp. 381-388).

Ca²⁺ release through cardiac ryanodine receptors is inhibited by the carboxy terminal domain of glutathione transferase M2-2. Therapeutics mimicking this structure may reduce excess Ca²⁺ release that occurs in arrhythmia.Ca²⁺ release from the sarcoplasmic reticulum through cardiac ryanodine receptors (RyR2) is essential for heart function and is inhibited by the carboxy terminal domain of glutathione transferase M2-2 (GSTM2-C) and derivative fragments containing helix 6. Since a peptide encoding helix 6 alone does not fold into a helix and does not inhibit RyR2 Ca²⁺ release, the importance of the structure of helix 6 and its role in stabilizing GSTM2-C was tested by inserting potentially destabilizing mutations into this helical segment. GSTM2-C preparations with D156A or L163A mutations were so insoluble that the protein could not be purified. Proteins with F157A and Y260A substitutions were soluble, but had lost their capacity to inhibit both RyR2 Ca²⁺ release from vesicles and RyR2 channels in bilayers. Circular dichroism studies indicated that these mutated proteins retained their helical secondary structure, although changes in their endogenous tryptophan fluorescence indicated that the F157A and Y160A mutations caused changes in their folding. The single channel studies were conducted with 2mM ATP and 10μM Ca²⁺ in the cytoplasmic solution, mimicking concentrations in the cytosol of cardiac myocytes. Wild type GSTM2-C inhibited RyR2 only at a potential of +40mV, which may develop during Ca²⁺ efflux, but not at −40mV. Together, the results indicate that the structure of helix 6 in the C-terminal fold is critical to the inhibitory action of GSTM2-2 and suggest that therapeutics mimicking this structure may reduce excess Ca²⁺ release during diastole, which can lead to fatal arrhythmia.

Keywords: Glutathione transferase GSTM2-2; Cardiac RyR2 channels; Skeletal RyR1 channels; Calcium release from cardiac sarcoplasmic reticulum; Calcium release from skeletal sarcoplasmic reticulum; Lipid bilayer single channel experiments

The anionic amphiphile SDS is an antagonist for the human neutrophil formyl peptide receptor 1 by Fredrik B. Thorén; Jennie Karlsson; Claes Dahlgren; Huamei Forsman (pp. 389-395).

The anionic amphiphil sodium dodecyl sulfate (SDS) is commonly used to activate the superoxide-generating NADPH-oxidase complex in cell-free systems, but very little is known about the effects of SDS on intact cells. It was, however, recently shown that SDS causes a translocation and an activation of Rac (a small G-protein) in intact cells, but this signal is not in its own sufficient to activate the oxidase (Nigorikawa et al. (2004) [1]). We found that SDS acted as an antagonist for FPR1, one of the neutrophil members of the formyl peptide receptor family. Accordingly, SDS reduced superoxide anion production induced by the chemoattractant formylmethionyl-leucyl-phenylalanine (fMLF). The receptor specificity of SDS was fairly high, but the concentration range in which it worked was narrow. The length of the carbohydrate chain as well as the charge of the molecule was of importance for the antagonistic effects. Signaling through FPR2, a closely related receptor also expressed in neutrophils, was not inhibited by SDS. On the contrary, the response induced by the FPR2-specific agonist WKYMVM was primed by SDS. The precise mechanism behind the primed state is not known, but might be related to the effects earlier described for SDS on the small G-protein Rac, that is of importance for a proper transduction of the down-stream signals from the occupied receptor.

Keywords: Pattern recognition; GPCR; FPR; Antagonist; NADPH-oxidase, Priming

Effects of γ-glutamyl linker on DPP-IV resistance, duration of action and biological efficacy of acylated glucagon-like peptide-1 by Barry D. Kerr; Peter R. Flatt; Victor A. Gault (pp. 396-401).

Liraglutide, a GLP-1 mimetic has recently been approved for clinical use in obesity-diabetes. The purpose of this study was to assess if acylation of Liraglutide via its γ-glutamyl linker contributes to DPP-IV inhibition and efficacy of the molecule, given that such an approach could be useful in prolonging bioactivity of related peptides. Liraglutide lacking the γ-glutamyl linker (Lira-γGlu) and Liraglutide exhibited enhanced DPP-IV resistance with extension of t_1/2 plus effective cAMP production (EC₅₀: 0.15±0.11 and 0.16±0.11nM, respectively) compared to GLP-1 (EC₅₀ 3.81±0.80nM). GLP-1, Lira-γGlu and Liraglutide increased insulin secretion compared to glucose (1.5–3.0-fold; p<0.05 to p<0.001). In vivo, Lira-γGlu and Liraglutide significantly lowered plasma glucose when administered 4 and 8h prior to a glucose load (1.3–1.9-fold; p<0.05 to p<0.001). Twice-daily administration of Lira-γGlu and Liraglutide for 14 days significantly decreased food intake (1.2-fold; p<0.05) and plasma glucose (1.1–1.6-fold; p<0.05 to p<0.01) whilst increasing plasma insulin (1.4–1.6-fold; p<0.05). At 14 days, Lira-γGlu and Liraglutide markedly improved glucose tolerance (1.4–3.4-fold; p<0.05 to p<0.001), insulin response to glucose (1.4–1.5-fold; p<0.05), insulin sensitivity (1.3–1.4-fold; p<0.05 to p<0.01), as well as increasing pancreatic insulin content (1.4-fold; p<0.05). Functional characteristics of Lira-γGlu and Liraglutide are almost indistinguishable, questioning necessity of γ-glutamyl linker in acylation for generation of long-acting incretin mimetics.

Keywords: Acylation; GLP-1 mimetic; Liraglutide; γ-Glutamyl spacer; Glucose homeostasis; Insulin secretion

Repeated nicotine administration robustly increases bPiDDB inhibitory potency at α6β2-containing nicotinic receptors mediating nicotine-evoked dopamine release by Andrew M. Smith; Marharyta Pivavarchyk; Thomas E. Wooters; Zhenfa Zhang; Guangrong Zheng; J. Michael McIntosh; Peter A. Crooks; Michael T. Bardo; Linda P. Dwoskin (pp. 402-409).

The novel nicotinic receptor (nAChR) antagonist, N, N′-dodecane-1,12-diyl- bis-3-picolinium dibromide (bPiDDB), and its chemically reduced analog, r-bPiDDB, potently inhibit nicotine-evoked dopamine (DA) release from rat striatal slices. Since tobacco smokers self-administer nicotine repeatedly, animal models incorporating repeated nicotine treatment allow for mechanistic evaluation of therapeutic candidates following neuroadaptive changes. The current study determined the ability of bPiDDB, r-bPiDDB and α-conotoxin MII (α-CtxMII), a peptide antagonist selective for α6β2-containing nAChRs, to inhibit nicotine-evoked [³H]DA release from striatal slices from rats repeatedly administered nicotine (0.4mg/kg for 10 days) or saline (control). Concomitant exposure to maximally effective concentrations of r-bPiDDB (1nM) and α-CtxMII (1nM) resulted in inhibition of nicotine-evoked [³H]DA release no greater than that produced by either antagonist alone, suggesting that r-bPiDDB inhibits α6β2-containing nAChRs. Repeated nicotine treatment increased locomotor activity, demonstrating behavioral sensitization. Concentration–response curves for nicotine-evoked [³H]DA release were not different between nicotine-treated and control groups. Maximal inhibition for α-CtxMII was greater following repeated nicotine compared to control ( I_max=90% vs. 62%), with no change in potency. bPiDDB was 3-orders of magnitude more potent in inhibiting nicotine-evoked [³H]DA release in nicotine-treated rats compared to control rats (IC₅₀=5pM vs. 6nM), with no change in maximal inhibition. Neither a shift to the left in the concentration response nor a change in maximal inhibition was observed for r-bPiDDB following repeated nicotine. Thus, repeated nicotine treatment may differentially regulate the stoichiometry, conformation and/or composition of α6β2-containing nAChRs mediating nicotine-evoked striatal DA release. Therefore, bPiDDB and r-bPiDDB appear to target different α6β2-containing nAChR subtypes.

Keywords: Abbreviations; nAChR; nicotinic acetylcholine receptor; DA; dopamine; NIC; nicotine; SAL; saline; MEC; mecamylamine; DHβE; dihydro-β-erythroidine; α-CtxMII; α-conotoxin MII; bPiDDB; N,N; ′-dodecane-1,12-diyl-; bis; -3-picolinium dibromide; r-bPiDDB; 1,12-; bis; (3-methyl-1,2,5,6-tetrahydropyridinyl)dodecane; ANOVA; analysis of varianceSmoking cessation; Nicotine; Nicotinic acetylcholine receptor; Dopamine release; Nicotinic receptor antagonist

Differential effect of covalent protein modification and glutathione depletion on the transcriptional response of Nrf2 and NF-κB by Alvin J.L. Chia; Christopher E. Goldring; Neil R. Kitteringham; Shi Quan Wong; Paul Morgan; B. Kevin Park (pp. 410-421).

CRMs activate Nrf2, but inhibit NF-κB, and GSH depletion without covalent modification activates both Nrf2 and NF-κB. This leads to cellular protection against the potentially harmful effects of redox perturbation.Liver injury associated with exposure to therapeutic agents that undergo hepatic metabolism can involve the formation of reactive metabolites. These may cause redox perturbation which can result in oxidative stress as well as protein modification leading to activation or inhibition of cellular transcriptional responses. Nevertheless, the effects of these challenges on more than one transcriptional pathway simultaneously remain unclear. We have investigated two transcription factors known to be sensitive to electrophilic stress and redox perturbation, Nrf2 and NF-κB, in mouse liver cells. Cellular stress was induced by the probes: N-acetyl-p-benzoquinineimine (NAPQI), the reactive metabolite of acetaminophen; dinitrochlorobenzene (DNCB), a model electrophile; and buthionine (S,R)-sulfoximine (BSO), an inhibitor of glutamate-cysteine ligase. NAPQI, DNCB and BSO can all cause glutathione (GSH) depletion; however only NAPQI and DNCB can covalently bind proteins. We also employed RNAi to manipulate Keap1 (the inhibitor of Nrf2), Nrf2 itself and NF-κB-p65, to understand their roles in the response to drug stress. All three chemicals induced Nrf2, but NF-κB binding activity was only increased after BSO treatment. In fact, NF-κB binding activity decreased after exposure to NAPQI and DNCB. While RNAi depletion of Keap1 led to reduced toxicity following exposure to DNCB, depletion of Nrf2 and NF-κB augmented toxicity. Interestingly, increased Nrf2 caused by Keap1 depletion was reversed by co-depletion of NF-κB. We demonstrate that Keap1/Nrf2 and NF-κB respond differently to electrophiles that bind proteins covalently and the redox perturbation associated with glutathione depletion, and that crosstalk may enable NF-κB to partly influence Nrf2 expression during cellular stress.

Keywords: Abbreviations; DILI; drug-induced liver injury; CRMs; chemically reactive metabolites; GSH; glutathione; Nrf2; nuclear factor-erythroid 2 (NF-E2)-related factor; NF-κB; NF-kappa B; ARE; anti-oxidant response element; NAPQI; N-acetyl-p-benzoquinineimine; DNCB; dinitrochlorobenzene; BSO; buthionine (S,R)-sulfoximine; RNAi; RNA interferenceNrf2; Keap1; NF-κB; RNAi; Cellular stress; Transcriptional response

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Biochemical Pharmacology (v.80, #3)

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Biochemical Pharmacology (v.80, #3)