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Biochemical Pharmacology (v.83, #11)

Editorial Board (pp. iii).

Targeting the Apo2L/TRAIL system for the therapy of autoimmune diseases and cancer by Luis Martinez-Lostao; Isabel Marzo; Alberto Anel; Javier Naval (pp. 1475-1483).
Apo 2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL), is a member of the TNF family of cytokines, which can induce apoptotic cell death in cells expressing at least one of their specific death receptors, DR4 (TRAIL-R1) or DR5 (TRAIL-R2). In the last decade, the Apo2L/TRAIL system of apoptosis has attracted significant interest as a potential drug-targeting pathway for human therapy, due to the ability of that cytokine to trigger apoptosis in various types of cancer cells while displaying low or no toxicity to normal cells. Recent results suggest that manipulating the Apo2L/TRAIL system may be also useful for the treatment of inflammatory disorders such as rheumatoid arthritis. For its possible therapeutic use, a number of receptor-specific Apo2L/TRAIL molecular variants and agonistic monoclonal antibodies have been developed, and some of them are in clinical trials. In addition, Apo2L/TRAIL-resistant tumors can be sensitized to Apo2L/TRAIL by selected novel or classical chemotherapeutic agents, opening new possibilities for combined therapies. We will briefly review the current status of Apo2L/TRAIL-based therapies for human disease, their promises and limitations.

Keywords: Apo2L; Apoptosis; Autoimmune; Cancer; TRAIL


The multifaceted exosome: Biogenesis, role in normal and aberrant cellular function, and frontiers for pharmacological and biomarker opportunities by Saumya Pant; Holly Hilton; Michael E. Burczynski (pp. 1484-1494).
Exosomes are nanometer sized cellular vesicles released into surrounding bio-fluids. Containing the parent cell's RNA, DNA, protein and lipid component, they reflect the cell's condition and hold promise as biomarkers of exquisite specificity.Exosomes are bioactive vesicles derived from the cell's endosomal membrane system and secreted into surrounding body fluids. Exosomes contain cell and cell-state specific cargos of protein, mRNA and miRNA. Exosome formation, cargo content, and delivery to surrounding cells is of immense biological interest considering the role that exosomes are believed to play in various pathological conditions. They aid antigen presentation by immune cells and can exhibit either anti-inflammatory or pro-inflammatory properties depending on the parent antigen-presenting cell's conditioning. Viruses can hijack a host cell's exosomal machinery to evade host defense systems aiding in the trans-infection of viruses.Tumor derived exosomes may help establish an oncogenic niche systemically via delivery of protein, mRNA, and miRNA that can aid angiogenesis, cell proliferation, and cell survival. Exosomes have also been implicated in the spread of neurodegenerative diseases.Studies have shown that exosomes are selectively taken up by cells distal from their release. They can reprogram the recipient cells due to their active molecular cargo. Cell-lineage and state-specific exosomes imply that they may therefore harbor body fluid-based biomarkers of unparalleled accuracy, particularly for tissues that are difficult or impossible to access.Exosome-specific membrane proteins provide markers enabling exosome identity and selection, while cell type and cell condition-specific protein, mRNA and miRNA cargo provide a rich potential source of biomarkers. This review serves to provide an overview of the current state of the science in the burgeoning field of exosome biology.

Keywords: Abbreviations; mRNA; messenger RNA; miRNA or miR; micro RNA; MVB; multivesicular bodies; ATPase; adenosine triphosphatase; DNA; deoxyribonucleic acid; TSG101; tumor susceptibility gene 101; ILV; intraluminal vesicles; Rab GTPases; “Ras-related in brain” GTPase; GTPase; guanosine triphosphatase; SNAREs; SNAP (soluble NSF attachment protein) receptor; AP2; clathrin adaptor protein; Hsc70; heat shock cognate 71; kDa protein; PI3K; phosphatidyl inositol-3-kinase; CD; cluster of differentiation; iDex; immature D cell derived exosomes; ICAM1; inter-cellular adhesion molecule 1; APCs; antigen presenting cells; TGF; tumor growth factor; TNF; tumor necrosis factor; IL; interleukin; MHC; major histocompatibility complex; DC; dendritic cell; LFA1; lymphocyte function-associated antigen 1; CXCL5; C-X-C motif chemokine 5; MIF; macrophage migration inhibitory factor; CCR1; chemokine (C–C motif) receptor 1; EMMPRIN; extracellular matrix metalloproteinase inducer; HIV; human immunodeficiency virus; EBV; Epstein Barr virus; VLP; virion-like particles; HLA-DR; MHC class II cell surface receptor; SARS; severe acute respiratory syndrome; VEGF; vascular endothelial growth factor; ESCRT; endosomal sorting complex required for transport; HLA-DR; MHC class II receptor encoded by human leukocyte antigen complex; TSPAN; tetraspanin; KRAS; protein involved in cell cycle regulation; EGFR; epidermal growth factor receptor; RT-PCR; reverse transcriptase PCR; PSA; prostate-specific antigen; PCA3; prostate CAncer gene 3; I/R; ischemia reperfusion; AQP1; aquaporin 1; NKCC2; sodium–potassium–chloride co-transporter; AKI; acute kidney injury; CKD; chronic kidney disease; ATF3; cyclic AMP-dependent transcription factor; WT1; Wilms tumor protein; ELISA; enzyme linked immunosorbent assay; FACS; fluorescence activated cell sortingExosomes; Biomarkers; Fluidome; Microenvironment; Secretome; Microvesicles


Kinetic stabilization of microtubule dynamics by indanocine perturbs EB1 localization, induces defects in cell polarity and inhibits migration of MDA-MB-231 cells by Sonia Kapoor; Dulal Panda (pp. 1495-1506).
Cell motility is an essential aspect of metastatic spread of cancer. Microtubule-targeted agents exhibit anti-metastatic properties, the underlying mechanism of which remains understudied. In this study, we have investigated the role of microtubule dynamics in migration of cancer cells using indanocine, a synthetic small molecule inhibitor of tubulin. We found that indanocine, at concentrations that did not visibly affect microtubule organization, suppressed dynamic instability of microtubules and reduced the rate of migration of highly metastatic MDA-MB-231 cells. Indanocine-treated cells were defective in lamellipodium formation and could not develop polarized morphology. The kinetic stabilization of microtubules was associated with a marked increase in their acetylation level and a perturbation in the localization of EB1, a microtubule plus end binding protein. Using standard scratch wound healing assay and immunofluorescence analysis; we found that microtubule acetylation occurred in the direction of migration in vehicle-treated cells, whereas indanocine treatment led to a global acetylation of microtubules. The results together suggested that selective stabilization of microtubules was perturbed in the presence of indanocine that possibly resulted in lack of cell polarization and a concurrent reduction in migration of cells. Moreover, microtubule stabilization by indanocine affected adhesion turnover and impaired the polarized pattern of adhesion sites in cells. Together the results indicated that the regulation of microtubule dynamics is required to coordinate cell polarization as well as adhesion asymmetry and support the hypothesis that the perturbation of microtubule dynamics by tubulin-targeted agents can be exploited to restrict the migration of tumor cells.

Keywords: Abbreviations; SRB; sulforhodamine B; BSA; bovine serum albumin; FBS; fetal bovine serum; ERK; extracellular signal-regulated kinase; FITC; fluorescein isothiocyanate; DMSO; dimethyl sulfoxide; IC; 50; half-maximal proliferation inhibitory concentration; EGFP; enhanced green fluorescent protein; GFP; green fluorescent protein; SD; standard deviation; PI; propidium iodide; DIC; differential interference contrastCell migration; Indanocine; Metastasis; Microtubule dynamics


Covalent binding of cisplatin impairs the function of Na+/K+-ATPase by binding to its cytoplasmic part by Miroslav Huličiak; Jan Vacek; Marek Šebela; Eva Orolinová; Joanna Znaleziona; Marika Havlíková; Martin Kubala (pp. 1507-1513).
We demonstrate that cisplatin, which exhibits nephrotoxicity, inhibits Na+/K+-ATPase, while the other less-nephrotoxic drugs do not. We propose that it is a consequence of cisplatin binding to the Cys367 near the phosphorylation site at Asp369.This study was aimed at verifying the hypothesis that acute kidney failure accompanying cisplatin administration in the cancer therapy could be due to cisplatin interaction with the cytoplasmic part of Na+/K+-ATPase. Our results demonstrated that cisplatin-binding caused inhibition of Na+/K+-ATPase, in contrast to other platinated chemotherapeutics such as carboplatin and oxaliplatin, which are known to be much less nephrotoxic. To acquire more detailed structural information, we performed a series of experiments with the isolated large cytoplasmic segment connecting transmembrane helices 4 and 5 (C45 loop) of Na+/K+-ATPase. Electrochemistry showed that cisplatin is bound to the cysteine residues of the C45 loop, mass spectrometry revealed a modification of the C45 peptide fragment GSHMASLEAVETLGSTSTICSDK, which contains the conserved phosphorylated residue Asp369. Hence, we hypothesize that binding of cisplatin to Cys367 can cause sterical obstruction during the phosphorylation or dephosphorylation step of the Na+/K+-ATPase catalytic cycle.

Keywords: Cisplatin; Nephrotoxicity; Na; +; ,K; +; -ATPase; Sodium pump; Cysteine


Minor structural modifications to alchemix influence mechanism of action and pharmacological activity by Qasem M.A. Abdallah; Roger M. Phillips; Fredrik Johansson; Thomas Helleday; Laura Cosentino; Hamdy Abdel-Rahman; Jasarat Etzad; Richard T. Wheelhouse; Konstantinos Kiakos; John P. Bingham; John A. Hartley; Laurence H. Patterson; Klaus Pors (pp. 1514-1522).
Alchemix is an exemplar of a class of anthraquinone with efficacy against multidrug resistant tumours. We have explored further the mechanism of action of alchemix and investigated the effect of extending its side arm bearing the alkylating functionality with regard to DNA binding and activity against multidrug resistant cancer cells. Increasing the distance between the intercalating chromophore and the alkylating functionality of ICT2901 (propyl), ICT2902 (butyl) and ICT2903 (pentyl), led to a higher number of DNA alkylation sites, more potent topoisomerase II inhibition and generated more apoptotic and necrotic cells when analysed in p53-proficient HCT116 cells. Intriguingly, alchemix, the compound with the shortest distance between its intercalative chromophore and alkylating functionality (ethyl), did not conform to this SAR. A different toxicity pattern against DNA repair defective CHO cell lines as well as arrest of cells in G1 supports a somewhat distinct mode of action by alchemix compared with its analogues. Importantly, both alchemix and ICT2901 demonstrated greater cytotoxic activity against anthraquinone-resistant MCF-7/adr cells than wild-type MCF-7 cells. Subtle synthetic modification in this anthraquinone series has led to significant changes to the stability of DNA-compound complexes and cellular activity. Given that the failure of chemotherapy in the clinic is often associated with MDR, the results of both alchemix and ICT2901 represent important advances towards improved therapies.

Keywords: Abbreviations; CHO; Chinese hamster ovary; CT DNA; calf thymus DNA; DMSO; dimethyl sulphoxide; DSB; double strand break; H2AX; histone-2AX; MDR; multidrug resistance; MTT; (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TdT; terminal deoxynucleotidyl transferase; Topo II; topoisomerase II; TUNEL; terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling; SAR; structure–activity relationshipAnthraquinone; H2AX phosphorylation; MDR; Topoisomerase II; NER; p53


The inhibitory glutathione transferase M2-2 binding site is located in divergent region 3 of the cardiac ryanodine receptor by Dan Liu; Ruwani Hewawasam; Yamuna Karunasekara; Marco G. Casarotto; Angela F. Dulhunty; Philip G. Board (pp. 1523-1529).
The muscle-specific glutathione transferase GSTM2-2 modulates the activity of ryanodine receptor (RyR) calcium release channels: it inhibits the activity of cardiac RyR (RyR2) channels with high affinity and activates skeletal RyR (RyR1) channels with low affinity. The C terminal domain of GSTM2-2 (GSTM2C) alone physically binds to RyR2 and inhibits its activity, but it does not bind to RyR1. We have now used yeast two-hybrid analysis, chemical cross-linking, intrinsic tryptophan fluorescence and Ca2+ release studies to determine that the binding site for GSTM2C is in divergent region 3 (D3) of RyR2. The D3 region encompasses residues 1855–1890 in RyR2. Specific mutagenesis shows the binding primarily involves electrostatic interactions with residues K1875, K1886, R1887 and K1889, all residues that are present in RyR2, but not in RyR1. The significant sequence differences between the D3 regions of RyR2 and RyR1 explain why GSTM2-2 specifically inhibits RyR2. This specific inhibition of RyR2 could modulate Ca cycling and be useful for the treatment of heart failure. RyR2 inhibition during diastole may improve filling of the SR with Ca2+ and improve contractility.

Keywords: Glutathione transferase GSTM2-2; Cardiac RyR2 channels; Divergent region 3 of RyRs; Skeletal RyR1 channels; Inhibition of RyR2 channels; Yeast two hybrid analysis


Dehydroepiandrosterone (DHEA) inhibits voltage-gated T-type calcium channels by M. Chevalier; G. Gilbert; P. Lory; R. Marthan; J.F. Quignard; J.P. Savineau (pp. 1530-1539).
Extracellular DHEA inhibits T-channels by a Gi protein dependent pathway. This effect accounts for DHEA inhibition of pulmonary artery contraction and thus accounts for its therapeutic action and/or physiological effects.Dehydroepiandrosterone (DHEA) and its sulfated form, DHEAS, are the most abundant steroid hormones in the mammalian blood flow. DHEA may have beneficial effects in various pathophysiological conditions such as cardiovascular diseases or deterioration of the sense of well-being. However to date, the cellular mechanism underlying DHEA action remains elusive and may involve ion channel modulation. In this study, we have characterized the effect of DHEA on T-type voltage-activated calcium channels (T-channels), which are involved in several cardiovascular and neuronal diseases.Using the whole-cell patch-clamp technique, we demonstrate that DHEA inhibits the three recombinant T-channels (CaV3.1, CaV3.2 and CaV3.3) expressed in NG108-15 cell line, as well as native T-channels in pulmonary artery smooth muscle cells. This effect of DHEA is both concentration (IC50 between 2 and 7μM) and voltage-dependent and results in a significant shift of the steady-state inactivation curves toward hyperpolarized potentials. Consequently, DHEA reduces window T-current and inhibits membrane potential oscillations induced by CaV3 channels. DHEA inhibition is not dependent on the activation of nuclear androgen or estrogen receptors and implicates a PTX-sensitive Gi protein pathway. Functionally, DHEA and the T-type inhibitor NNC 55-0396 inhibited KCl-induced contraction of pulmonary artery rings and their effect was not cumulative.Altogether, the present data demonstrate that DHEA inhibits T-channels by a Gi protein dependent pathway. DHEA-induced alteration in T-channel activity could thus account for its therapeutic action and/or physiological effects.

Keywords: Abbreviations; DHEA; dehydroepiandrosterone; DHEAS; sulfate of dehydroepiandrosterone; HP; holding potential; PASMC; pulmonary artery smooth muscle cell; T-channels; T-type voltage-activated calcium channelsDehydroepiandrosterone; T-type calcium channels; Gi protein; Pulmonary artery smooth muscle cell


3-(4-( tert-Octyl)phenoxy)propane-1,2-diol suppresses inflammatory responses via inhibition of multiple kinases by Tao Yu; Jaegal Shim; Yanyan Yang; Se Eun Byeon; Ji Hye Kim; Ho Sik Rho; Haeil Park; Gi-Ho Sung; Tae Woong Kim; Man Hee Rhee; Jae Youl Cho (pp. 1540-1551).
Novel anti-inflammatory compounds were synthesised by derivatization of militarin, a compound isolated from Cordyceps militaris that is an ethnopharmacologically well-known herbal medicine with multiple benefits such as anti-cancer, anti-inflammatory, anti-obesity, and anti-diabetic properties. In this study, we explored the in vitro and in vivo anti-inflammatory potencies of these compounds during inflammatory responses, their inhibitory mechanisms, and acute toxicity profiles. To do this, we studied inflammatory conditions using in vitro lipopolysaccharide-treated macrophages and several in vivo inflammatory models such as dextran sodium sulphate (DSS)-induced colitis, EtOH/HCl-induced gastritis, and arachidonic acid-induced ear oedema. Methods used included real-time PCR, immunoblotting analysis, immunoprecipitation, reporter gene assays, and direct kinase assays. Of the tested compounds, compoundIII showed the highest nitric oxide (NO) inhibitory activity. This compound also inhibited the production of prostaglandin (PG)E2 at the transcriptional level by suppression of Syk/NF-κB, IKKɛ/IRF-3, and p38/AP-1 pathways in lipopolysaccharide (LPS)-activated RAW264.7 cells and peritoneal macrophages. Consistent with these findings, compoundIII strongly ameliorated inflammatory symptoms in colitis, gastritis, and ear oedema models. In acute toxicity tests, there were no significant differences in body and organ weights, serum parameters, and stomach lesions between the untreated and compoundIII-treated mice. Therefore, this compound has the potential to be served as a lead chemical for developing a promising anti-inflammatory drug candidate with multiple kinase targets.

Keywords: Abbreviations; Na-CMC; sodium carboxyl methylcellulose; EIA; enzyme immunoassay; ELISA; enzyme linked immunosorbent assay; MTT; 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IRF-3; interferon regulatory factor-3; PI3K; phosphoinositide 3-kinase; STAT-1; signal transducer and activator of transcription; ALT; alanine aminotransferase; AST; aspartate aminotransferase; ALP; alkaline phosphatase; LDH; lactate dehydrogenase3-(4-(; tert; -Octyl)phenoxy)propane-1,2-diol; Anti-inflammatory effect; Inflammatory mediator; Syk; p38; IκB kinase ɛ


Novel fluorescent antagonist as a molecular probe in A3 adenosine receptor binding assays using flow cytometry by Eszter Kozma; T. Santhosh Kumar; Stephanie Federico; Khai Phan; Ramachandran Balasubramanian; Zhan-Guo Gao; Silvia Paoletta; Stefano Moro; Giampiero Spalluto; Kenneth A. Jacobson (pp. 1552-1561).
The physiological role of the A3 adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A3AR antagonist for application to cell-based assays in ligand discovery and for receptor imaging. Fluorescent pyrazolo[4,3- e][1,2,4]triazolo[1,5- c]pyrimidin-5-ylamine (pyrazolo-triazolo-pyrimidine, PTP) and triazolo[1,5- c]quinazolin-5-yl)amine (triazolo-quinazoline, TQ) AR antagonists were compared. A chain-extended and click-conjugated Alexa Fluor-488 TQ derivative (MRS5449) displayed a radioligand binding Ki value of 6.4±2.5nM in hA3AR-expressing CHO cell membranes. MRS5449 antagonized hA3AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner ( KB=4.8nM). Using flow cytometry (FCM), MRS5449 saturated hA3ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15nM, comparable to the Kd value of 6.65nM calculated from kinetic experiments. Ki values of known AR antagonists in inhibition of MRS5449 binding in whole cell FCM were consistent with radioligand binding in membranes, but agonist binding was 5-20 fold weaker than obtained with agonist radioligand [125I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A3AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA3AR characterization.

Keywords: Abbreviations; AF488; Alexa Fluor-488; CHO; Chinese hamster ovary; CGS15943; N; -[9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-; c; ]quinazolin-5-amine; CGS21680; 2-[; p; -(2-carboxyethyl)phenylethylamino]-5′-; N; -ethylcarboxamido-adenosine; Cl-IB-MECA; 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9; H; -purin-9-yl]-1-deoxy-; N; -methyl-β-; d; -ribofuranuronamide; CPA; (2R,3R,4S,5R)-2-[6-(cyclopentylamino)purin-9-yl]-5(hydroxymethyl)oxolane-3,4-diol; DMAP; 4-dimethylaminopyridine; DMEM; Dulbecco's Modified Eagle Medium; DMF; N; ,; N; -dimethylformamide; DMSO; dimethyl sulfoxide; PBS; phosphate buffered saline; DPCPX; 8-cyclopentyl-1,3-dipropylxanthine; EDC; 1-ethyl-[3-dimethylaminopropyl]carbodiimide; EDTA; ethylenediaminetetraacetic acid; EL; extracellular loop; FBS; fetal bovine serum; FCM; flow cytometry; FITC; fluorescein isothiocyanate; GPCR; G protein-coupled receptor; HEK; human embryonic kidney; [; 125; I]I-AB-MECA; [; 125; I]4-amino-3-iodobenzyl-5′-; N; -methylcarboxamidoadenosine; IB-MECA; 1-Deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9; H; -purin-9-yl]-; N; -methyl-β-; d; -ribofuranuronamide; MD; molecular dynamics; MESF; molecules of equivalent soluble fluorochrome; MOE; Molecular Operating Environment; MFI; mean fluorescence intensity; MRS1220; N; -[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-; c; ]quinazolin-5-yl]benzene acetamide; MRS5346; 5-((2-(2-(4-(3-(5-amino-2-(furan-2-yl)-7; H; -pyrazolo[4,3-; e; ][1,2,4]triazolo[1,5-; c; ]pyrimidin-7-yl)propyl)phenoxy)acetamido)-ethyl)carbamoyl)-2-(6-amino-3-iminio-4,5-disulfonato-3; H; -xanthen-9-yl)benzoate; MRS5449; 2-(6-amino-3-iminio-4,5-disulfonato-3; H; -xanthen-9-yl)-5-((6-(4-(4-((9-chloro-2-(furan-2-yl)-[1,2,4]triazolo[1,5-; c; ]quinazolin-5-yl)amino)-4-oxobutyl)-1; H; -1,2,3-triazol-1-yl)hexyl)carbamoyl)benzoate; NECA; 5′-; N; -ethylcarboxamidoadenosine; PSB10; 8-ethyl-1,4,7,8-tetrahydro-4-methyl-2-(2,3,5-trichlorophenyl)-5; H; -imidazo[2,1-; i; ]purin-5-one monohydrochloride; TM; transmembrane helix; XAC; xanthine amine congener,; N; -(2-aminoethyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1; H; -purin-8-yl)phenoxy]-acetamide hydrochloride; ZM241385; 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-; a; ][1,3,5]triazin-5-ylamino]ethyl)phenolPurines; Fluorescence; G protein-coupled receptor; A; 3; adenosine receptor; Flow cytometry


Characterisation of Nav types endogenously expressed in human SH-SY5Y neuroblastoma cells by Irina Vetter; Christine A. Mozar; Thomas Durek; Joshua S. Wingerd; Paul F. Alewood; Macdonald J. Christie; Richard J. Lewis (pp. 1562-1571).
The human neuroblastoma cell line SH-SY5Y is a potentially useful model for the identification and characterisation of Nav modulators, but little is known about the pharmacology of their endogenously expressed Navs. The aim of this study was to determine the expression of endogenous Nav α and β subunits in SH-SY5Y cells using PCR and immunohistochemical approaches, and pharmacologically characterise the Nav isoforms endogenously expressed in this cell line using electrophysiological and fluorescence approaches. SH-SY5Y human neuroblastoma cells were found to endogenously express several Nav isoforms including Nav1.2 and Nav1.7. Activation of endogenously expressed Navs with veratridine or the scorpion toxin OD1 caused membrane depolarisation and subsequent Ca2+ influx through voltage-gated L- and N-type calcium channels, allowing Nav activation to be detected with membrane potential and fluorescent Ca2 dyes. μ-Conotoxin TIIIA and ProTxII identified Nav1.2 and Nav1.7 as the major contributors of this response. The Nav1.7-selective scorpion toxin OD1 in combination with veratridine produced a Nav1.7-selective response, confirming that endogenously expressed human Nav1.7 in SH-SY5Y cells is functional and can be synergistically activated, providing a new assay format for ligand screening.

Keywords: Abbreviations; Na; v; voltage-gated sodium channel; TTX; tetrodotoxin; ProTxII; β/ω-theraphotoxin-Tp2a; Ca; 2+; calcium ion; DMEM; Dulbecco's Modified Eagle's Medium; FBS; foetal bovine serum; PBS; phosphate buffered saline; PSS; physiological salt solution; HBS; HEPES-buffered saline; AFU; arbitrary fluorescence unit; SEM; standard error of the mean; Fluo-4 AM; Fluo-4 acetoxymethylester; RPMI; Roswell Park Memorial Institute; BSA; bovine serum albumin; CCD; charge-coupled device; DAPI; 4′,6-diamidino-2-phenylindoleSH-SY5Y; Ca; 2+; Na; v; 1.7; FLIPR; ProTxII; OD1


Changes in morphine-induced activation of cerebral Na+,K+-ATPase during morphine tolerance: Biochemical and behavioral consequences by Luis G. Gonzalez; Willias Masocha; Cristina Sánchez-Fernández; Ahmad Agil; Maria Ocaña; Esperanza Del Pozo; José M. Baeyens (pp. 1572-1581).
There is ample evidence of the biological changes produced by the sustained activation of opioid receptors. We evaluated the adaptive changes of cerebral Na+,K+-ATPase in response to the sustained administration of morphine (minipumps, 45mg/kg/day, 6 days) in CD-1 mice and the functional role of these changes in opioid antinociception. The antinociceptive effect of morphine as determined with tail-flick tests was reduced in morphine-tolerant mice. There were no significant changes in the density of high-affinity Na+,K+-ATPase α subunits labeled with [3H]ouabain in forebrain membranes from morphine-tolerant compared to those of morphine-naive animals. Western blot analysis showed that there were no significant differences between groups in the changes in relative abundance of α1 and α3 subunits of Na+,K+-ATPase in the spinal cord or forebrain. However, the morphine-induced stimulation of Na+,K+-ATPase activity was significantly lower in brain synaptosomes from morphine-tolerant mice (EC50=1.79±0.10μM) than in synaptosomes from morphine-naive mice (EC50=0.69±0.12μM). Furthermore, adaptive alterations in the time-course of basal Na+,K+-ATPase activity were observed after sustained morphine treatment, with a change from a bi-exponential decay model (morphine-naive mice) to a mono-exponential model (morphine-tolerant mice). In behavioral studies the antinociceptive effects of morphine (s.c.) in the tail-flick test were dose-dependently antagonized by ouabain (1 and 10ng/mouse, i.c.v.) in morphine-naive mice, but not in morphine-tolerant mice. These findings suggest that during morphine tolerance, adaptive cellular changes take place in cerebral Na+,K+-ATPase activity which are of functional relevance for morphine-induced antinociception.

Keywords: Abbreviations; ANOVA; analysis of variance; AUC; area under the curve; B; max; maximum number of binding sites; EC; 50; concentration of drug that produces half-maximal stimulation; ED; 50; dose that produces 50% of maximum antinociception; E; max; maximum efficacy; K; D; dissociation constant; k; −1; rate constant reduction of enzymatic activity; t; 50; time required to reduce enzymatice activity by 50%; SEM; standard error of the mean; TCA; trichloracetic acidAnalgesia; Morphine; Opioid receptors; Ouabain; Sodium–potassium ATPase; Tolerance
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