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Biochemical Pharmacology (v.79, #7)

Editorial Advisory Board (pp. iii).

Graphical Abstracts Contents (pp. e1-e5).

Endocrine disruptors and thyroid hormone physiology by Mary-Line Jugan; Yves Levi; Jean-Paul Blondeau (pp. 939-947).

Endocrine disruptors are man-made chemicals that can disrupt the synthesis, circulating levels, and peripheral action of hormones. The disruption of sex hormones was subject of intensive research, but thyroid hormone synthesis and signaling are now also recognized as important targets of endocrine disruptors. The neurological development of mammals is largely dependent on normal thyroid hormone homeostasis, and it is likely to be particularly sensitive to disruption of the thyroid axis. Here, we survey the main thyroid-disrupting chemicals, such as polychlorinated biphenyls, perchlorates, and brominated flame-retardants, that are characteristic disruptors of thyroid hormone homeostasis, and look at their suspected relationships to impaired development of the human central nervous system. The review then focuses on disrupting mechanisms known to be directly or indirectly related to the transcriptional activity of the thyroid hormone receptors.

Keywords: Abbreviations; T3; triiodothyronine; T4; thyroxine; TH; thyroid hormone; TSH; thyrotropin; CAR; constitutive androstane receptor; PPAR; peroxisome proliferator-activated receptors; PXR; pregnane X receptor; RXR; retinoid X receptor; TR; thyroid hormone receptor; N-CoR; nuclear corepressor; SRC; steroid receptor coactivator; SULT; sulfotransferase; UGT; glucuronosyltransferase; BBP; butylbenzyl phthalate; BPA; bisphenol-A; DBP; dibutylphthalate; DCHP; dicyclohexylphthalate; DEHP; di(2-ethylhexyl) phthalate; PBDE; polybrominated diphenyl ether; PCB; polychlorinated biphenyl; TBBPA; 3,3′,5,5′-tetrabromobisphenol-A; TCBPA; 3,3′,5,5′-tetrachlorobisphenol-A; TDC; thyroid-disrupting chemicalThyroid-disrupting compounds; Thyroid hormone receptors; Polychlorinated biphenyls; Flame-retardants; Neurodevelopment; Endocrine disruptors

Induction and intracellular localization of Nur77 dictate fenretinide-induced apoptosis of human liver cancer cells by Hui Yang; Nathan Bushue; Pengli Bu; Yu-Jui Yvonne Wan (pp. 948-954).

Fenretinide, a synthetic retinoid, is known to induce apoptosis in various cancer cells. However, the mechanism by which fenretinide induces apoptosis remains unclear. The current study examines the mechanisms of fenretinide-induced apoptosis in human hepatoma cells. The induction of Nur77 and the cytoplasmic distribution of Nur77 induced by fenretinide were positively correlated with the apoptotic effect of fenretinide in HCC cells. The sensitivity of Huh-7 cells was related to Nur77 translocation and targeting mitochondria, whereas the mechanism of resistance for HepG2 cells seemed due to Nur77 accumulating in the nucleus. The intracellular location of Nur77 was also associated with the differential capability of fenretinide-induced ROS generation in these two cell lines. In addition, the knockdown of Nur77 expression by siRNA greatly reduced fenretinide-induced apoptosis and cleaved caspase 3 in Huh-7 cells. Therefore, our findings demonstrate that fenretinide-induced apoptosis of HCC cells is Nur77 dependent and that the intracellular localization of Nur77 dictates the sensitivity of the HCC cells to fenretinide-induced apoptosis.

Keywords: Fenretinide; Nur77; Apoptosis; ROS; Nuclear receptor; Retinoids

Cannabidiol inhibits cancer cell invasion via upregulation of tissue inhibitor of matrix metalloproteinases-1 by Robert Ramer; Jutta Merkord; Helga Rohde; Burkhard Hinz (pp. 955-966).

Although cannabinoids exhibit a broad variety of anticarcinogenic effects, their potential use in cancer therapy is limited by their psychoactive effects. Here we evaluated the impact of cannabidiol, a plant-derived non-psychoactive cannabinoid, on cancer cell invasion. Using Matrigel invasion assays we found a cannabidiol-driven impaired invasion of human cervical cancer (HeLa, C33A) and human lung cancer cells (A549) that was reversed by antagonists to both CB₁ and CB₂ receptors as well as to transient receptor potential vanilloid 1 (TRPV1). The decrease of invasion by cannabidiol appeared concomitantly with upregulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Knockdown of cannabidiol-induced TIMP-1 expression by siRNA led to a reversal of the cannabidiol-elicited decrease in tumor cell invasiveness, implying a causal link between the TIMP-1-upregulating and anti-invasive action of cannabidiol. P38 and p42/44 mitogen-activated protein kinases were identified as upstream targets conferring TIMP-1 induction and subsequent decreased invasiveness. Additionally, in vivo studies in thymic-aplastic nude mice revealed a significant inhibition of A549 lung metastasis in cannabidiol-treated animals as compared to vehicle-treated controls. Altogether, these findings provide a novel mechanism underlying the anti-invasive action of cannabidiol and imply its use as a therapeutic option for the treatment of highly invasive cancers.

Keywords: Abbreviations; AM-251; N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; AM-630; (6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl) (4-methoxyphenyl)methanone; cannabidiol; 2-((1S,6S)-3-methyl-6-(prop-1-en-2-yl) cyclohex-2-enyl)-5-pentylbenzene-1,3-diol; CB; ₁; cannabinoid receptor 1; CB; ₂; cannabinoid receptor 2; DMSO; dimethyl sulfoxide; EDTA; ethylenediaminetetraacetic acid; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MAPK; mitogen-activated protein kinase; MMP; matrix metalloproteinase; PMSF; phenylmethylsulfonyl fluoride; RT-PCR; reverse transcriptase-polymerase chain reaction; siRNA; small-interfering RNA; THC; Δ; ⁹; -tetrahydrocannabinol; TIMP; tissue inhibitor of matrix metalloproteinase; TRPV1; transient receptor potential vanilloid 1; WST-1; 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1.6-benzene disulfonateCannabidiol; Matrigel cell invasion; Tissue inhibitor of matrix metalloproteinases-1; Human cancer cells; Experimental lung metastasis

Synthesis and pharmacological characterization of [¹²⁵I]MRS5127, a high affinity, selective agonist radioligand for the A₃ adenosine receptor by John A. Auchampach; Elizabeth T. Gizewski; Tina C. Wan; Sonia de Castro; Garth G. Brown Jr.; Kenneth A. Jacobson (pp. 967-973).

A recently reported selective agonist of the human A₃ adenosine receptor (hA₃AR), MRS5127 (1′R,2′R,3′S,4′R,5′S)-4′-[2-chloro-6-(3-iodobenzylamino)-purine]-2′,3′-O-dihydroxy-bicyclo-[3.1.0]hexane, was radioiodinated and characterized pharmacologically. It contains a rigid bicyclic ring system in place of a 5′-truncated ribose moiety, and was selected for radiolabeling due to its nanomolar binding affinity at both human and rat A₃ARs. The radioiodination of the N⁶-3-iodobenzyl substituent by iododestannylation of a 3-(trimethylstannyl)benzyl precursor was achieved in 73% yield, measured after purification by HPLC. [¹²⁵I]MRS5127 bound to the human A₃AR expressed in membranes of stably transfected HEK 293 cells. Specific binding was saturable, competitive, and followed a one-site binding model, with a K_d value of 5.74±0.97nM. At a concentration equivalent to its K_d, non-specific binding comprised 27±2% of total binding. In kinetic studies, [¹²⁵I]MRS5127 rapidly associated with the hA₃AR ( t_1/2=0.514±0.014min), and the affinity calculated from association and dissociation rate constants was 3.50±1.46nM. The pharmacological profile of ligands in competition experiments with [¹²⁵I]MRS5127 was consistent with the known structure-activity-relationship profile of the hA₃AR. [¹²⁵I]MRS5127 bound with similar high affinity ( K_d, nM) to recombinant A₃ARs from mouse (4.90±0.77), rabbit (2.53±0.11), and dog (3.35±0.54). For all of the species tested, MRS5127 exhibited A₃AR agonist activity based on negative coupling to cAMP production. Thus, [¹²⁵I]MRS5127 represents a new species-independent agonist radioligand for the A₃AR. The major advantage of [¹²⁵I]MRS5127 compared with previously used A₃AR radioligands is its high affinity, low degree of non-specific binding, and improved A₃AR selectivity.

Keywords: Abbreviations; AR; adenosine receptor; CHO; Chinese hamster ovary; DMEM; Dulbecco's modified Eagle's medium; IB-MECA; N; ⁶; -(3-iodobenzyl)-5′-; N; -methylcarboxamidoadenosine; I-AB-MECA; N; ⁶; -(4-amino-3-iodobenzyl)-5′-; N; -methylcarboxamidoadenosine; MRE 3008F20; 5-; N; -(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo [4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine; MRS1191; 1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylic acid, 3-ethyl-5-(phenylmethyl) ester; MRS1220; N; -[9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-yl]benzeneacetamide; MRS1523; 5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate; MRS5127; (1′R,2′R,3′S,4′R,5′S)-4′-[2-chloro-6-(3-iodobenzylamino)-purine]-2′,3′-O-dihydroxybicyclo-[3.1.0]hexane; MRS1754; 8-[4-[[(4-cyano)phenylcarbamoylmethyl]oxy]phenyl]-1,3-di-(; n; -propyl)xanthine; NECA; 5′-N-ethylcarboxamidoadenosine; PSB-11; 8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-oneNucleoside; G protein-coupled receptor; Adenosine receptor; Radioligand binding

Effects of AF3442 [ N-(9-ethyl-9 H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro by Annalisa Bruno; Luigia Di Francesco; Isabella Coletta; Giorgina Mangano; Maria Alessandra Alisi; Lorenzo Polenzani; Claudio Milanese; Paola Anzellotti; Emanuela Ricciotti; Melania Dovizio; Andrea Di Francesco; Stefania Tacconelli; Marta L. Capone; Paola Patrignani (pp. 974-981).

Inhibitors of microsomal prostaglandin (PG) E synthase-1 (mPGES-1) are being developed for the relief of pain. Redirection of the PGH₂ substrate to other PG synthases, found both in vitro and in vivo, in mPGES-1 knockout mice, may influence their efficacy and safety. We characterized the contribution of mPGES-1 to PGH₂ metabolism in lipopolysaccharide (LPS)-stimulated isolated human monocytes and whole blood by studying the synthesis of prostanoids [PGE₂, thromboxane (TX)B₂, PGF_2α and 6-keto-PGF_1α] and expression of cyclooxygenase (COX)-isozymes and down-stream synthases in the presence of pharmacological inhibition by the novel mPGES-1 inhibitor AF3442 [ N-(9-ethyl-9 H-carbazol-3-yl)-2-(trifluoromethyl)benzamide]. AF3442 caused a concentration-dependent inhibition of PGE₂ in human recombinant mPGES-1 with an IC₅₀ of 0.06μM. In LPS-stimulated monocytes, AF3442 caused a concentration-dependent reduction of PGE₂ biosynthesis with an IC₅₀ of 0.41μM. At 1μM, AF3442 caused maximal selective inhibitory effect of PGE₂ biosynthesis by 61±3.3% (mean±SEM, P<0.01 versus DMSO vehicle) without significantly affecting other prostanoids (i.e. TXB₂, PGF_2α and 6-keto-PGF_1α). In LPS-stimulated whole blood, AF3442 inhibited in a concentration-dependent fashion inducible PGE₂ biosynthesis with an IC₅₀ of 29μM. A statistically significant inhibition of mPGES-1 activity was detected at 10 and 100μM (38±14%, P<0.05, and 69±5%, P<0.01, respectively). Up to 100μM, the other prostanoids were not significantly affected. In conclusion, AF3442 is a selective mPGES-1 inhibitor which reduced monocyte PGE₂ generation also in the presence of plasma proteins. Pharmacological inhibition of mPGES-1 did not translate into redirection of PGH₂ metabolism towards other terminal PG synthases in monocytes. The functional relevance of this observation deserves to be investigated in vivo.

Keywords: Abbreviations; mPGES-1; microsomal prostaglandin (PG) E synthase-1; LPS; Escherichia coli; lipopolysaccharide; TX; thromboxane; COX; cyclooxygenase; AF3442; [; N; -(9-ethyl-9; H; -carbazol-3-yl)-2-(trifluoromethyl)benzamide]; AA; arachidonic acid; PGI; ₂; prostacyclin; cPGES; cytosolic PGE synthase; tNSAIDs; traditional non-steroidal anti-inflammatory drugs; GI; gastrointestinal; MAPEG; membrane-associated proteins involved in eicosanoid and glutathione metabolism; TXS; TX synthase; PMSF; phenylmethylsulfonyl fluoride; FCS; fetal calf serum; TBS; Tris-buffered saline; SDS-PAGE; sodium dodecyl sulphate-polyacrylamide gel electrophoresismPGE synthase-1; PGE; ₂; Monocytes; Inflammation; Human whole blood

Prostaglandin D₂ induces the production of human β-defensin-3 in human keratinocytes by Naoko Kanda; Takeko Ishikawa; Shinichi Watanabe (pp. 982-989).

Prostaglandin D₂ induces human β-defeinsin-3 production in human keratinocytes by activating AP-1 via the expression and phosphorylation of c-Fos through the CRTH2/G_i/ src/MEK/ERK pathway.The antimicrobial peptide human β-defensin-3 (hBD-3) is produced by epidermal keratinocytes and protects the skin from infections. This peptide induces the release of a lipid mediator, prostaglandin D₂ from dermal mast cells. Prostaglandin D₂ binds to cell-surface G protein-coupled receptors, D prostanoid receptor, and chemoattractant receptor-homologous molecule expressed on T helper cell type 2 (CRTH2). Both receptors are detected on epidermal keratinocytes. It is reported that prostaglandin D₂ is involved in cutaneous allergy, however, its role in antimicrobial defense is unknown. We examined the in vitro effects of prostaglandin D₂ on hBD-3 production in normal human keratinocytes. Prostaglandin D₂ enhanced hBD-3 secretion and mRNA expression in human keratinocytes. Prostaglandin D₂-induced hBD-3 production was suppressed by the CRTH2 antagonist ramatroban and by antisense oligonucleotides against c-Jun and c-Fos, components of a transcription factor, activator protein-1 (AP-1). Prostaglandin D₂ enhanced the transcriptional activity and DNA binding of AP-1, expression, phosphorylation, and DNA binding of c-Fos proteins in keratinocytes. Prostaglandin D₂-induced hBD-3 production, AP-1 activity, and c-Fos expression and phosphorylation were suppressed by U0126, PP2, and pertussis toxin, which are inhibitors of mitogen-activated protein kinase kinase (MEK), src, and G_i proteins, respectively. The phosphorylation of extracellular signal-regulated kinase (ERK), downstream kinase of MEK, was induced by prostaglandin D₂, and suppressed by ramatroban, pertussis toxin, PP2, and U0126. These results suggest that prostaglandin D₂ induces hBD-3 production in human keratinocytes by activating AP-1 through the expression and phosphorylation of c-Fos via the CRTH2/G_i/ src/MEK/ERK pathway. Prostaglandin D₂ may promote cutaneous antimicrobial activity via hBD-3.

Keywords: Prostaglandin D; ₂; CRTH2; Human β-defensin-3; Keratinocyte; c-Fos

Identification and characterization of small compound inhibitors of human FATP2 by Angel Sandoval; Aalap Chokshi; Elliot D. Jesch; Paul N. Black; Concetta C. DiRusso (pp. 990-999).

Fatty acid transport proteins (FATPs) are bifunctional proteins, which transport long chain fatty acids into cells and activate very long chain fatty acids by esterification with coenzyme A. In an effort to understand the linkage between cellular fatty acid transport and the pathology associated with excessive accumulation of exogenous fatty acids, we targeted FATP-mediated fatty acid transport in a high throughput screen of more than 100,000 small diverse chemical compounds in yeast expressing human FATP2 (hsFATP2). Compounds were selected for their ability to depress the transport of the fluorescent long chain fatty acid analogue, C₁-BODIPY-C₁₂. Among 234 hits identified in the primary screen, 5 compounds, each representative of a structural class, were further characterized in the human Caco-2 and HepG2 cell lines, each of which normally expresses FATP2, and in 3T3-L1 adipocytes, which do not. These compounds were effective in inhibiting uptake with IC₅₀s in the low micromolar range in both Caco-2 and HepG2 cells. Inhibition of transport was highly specific for fatty acids and there were no effects of these compounds on cell viability, trans-epithelial electrical resistance, glucose transport, or long chain acyl-CoA synthetase activity. The compounds were less effective when tested in 3T3-L1 adipocytes suggesting selectivity of inhibition. These results suggest fatty acid transport can be inhibited in a FATP-specific manner without causing cellular toxicity.

Keywords: Abbreviations; Acsl; acyl-CoA synthetase long chain; FAF BSA; fatty acid-free bovine serum albumin; FATP; fatty acid transport protein; C1-BODIPY-C12; 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid; HTS; high throughput screening; MEM; minimal essential media; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 2-NBDG; 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose; TEER; transepithelial electrical resistanceFatty acid uptake; High throughput screening; FATP; Humanized yeast; Caco-2; HepG2

Modulation of glucagon-like peptide-1 release by berberine: In vivo and in vitro studies by Yunli Yu; Li Liu; Xinting Wang; Xiang Liu; Xiaodong Liu; Lin Xie; Guangji Wang (pp. 1000-1006).

This study demonstrated berberine promoted GLP-1 level both in normal rats and NCI-H716 cells, which possibly based on the enhancement of GLP-1 secretion and biosynthesis.Glucagon-like peptide (GLP)-1 is a potent glucose-dependent insulinotropic gut hormone released from intestinal L cells. Our previous studies showed that berberine increased GLP-1 secretion in streptozotocin-induced diabetic rats. The aim of this study was to investigate whether berberine affected GLP-1 release in normal rats and in NCI-H716 cells. Proglucagon and prohormone convertase 3 genes regulating GLP-1 biosynthesis were analyzed by RT-PCR. Effects of pharmacological inhibitors on berberine-mediated GLP-1 release were studied. In vivo, 5-week treatment of berberine enhanced GLP-1 secretion induced by glucose load and promoted proglucagon mRNA expression as well as L cell proliferation in intestine. In vitro, berberine concentration-dependently stimulated GLP-1 release in NCI-H716 cells. Berberine also promoted both prohormone convertase 3 and proglucagon mRNA expression. Chelerythrine (inhibitor of PKC) concentration-dependently suppressed berberine-mediated GLP-1 secretion. Compound C (inhibitor of AMPK) also inhibited berberine-mediated GLP-1 secretion. But only low concentrations of H89 (inhibitor of PKA) showed inhibitory effects on berberine-mediated GLP-1 release. The present results demonstrated that berberine showed its modulation on GLP-1 via promoting GLP-1 secretion and GLP-1 biosynthesis. Some signal pathways including PKC-dependent pathway were involved in this process. Elucidation of mechanisms controlling berberine-mediated GLP-1 secretion may facilitate the understanding of berberine's antidiabetic effects.

Keywords: Abbreviations; GLP-1; glucagon-like peptide 1; PKC; protein kinase C; PKA; protein kinase A; AMPK; AMP-activated protein kinase; KRB; Krebs–Ringer bicarbonate buffer; FBG; fasted blood glucose; DPP-IV; dipeptidyl peptidase-IV; Compound C; 6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a] pyrimidine; H89; N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; ANOVA; analysis of varianceBerberine; Glucagon-like peptide-1; Prohormone convertase 3; Proglucagon; Protein kinase C-dependent pathway

Poly(ADP-ribose) polymerase (PARP) inhibition counteracts multiple manifestations of kidney disease in long-term streptozotocin-diabetic rat model by Hanna Shevalye; Roman Stavniichuk; Weizheng Xu; Jie Zhang; Sergey Lupachyk; Yury Maksimchyk; Viktor R. Drel; Elizabeth Z. Floyd; Barbara Slusher; Irina G. Obrosova (pp. 1007-1014).

Evidence for the important role for poly(ADP-ribose) polymerase (PARP) in the pathogenesis of diabetic nephropathy is emerging. We previously reported that PARP inhibitors counteract early Type 1 diabetic nephropathy. This study evaluated the role for PARP in kidney disease in long-term Type 1 diabetes. Control and streptozotocin-diabetic rats were maintained with or without treatment with the PARP inhibitor 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de] anthracen-3-one (GPI-15,427, Eisai Inc.), 30mgkg⁻¹d⁻¹, for 26 weeks after first 2 weeks without treatment. PARP activity in the renal cortex was assessed by Western blot analysis of poly(ADP-ribosyl)ated proteins. Urinary albumin, isoprostane, and 8-hydroxy-2′-deoxyguanosine excretion, and renal concentrations of transforming growth factor-β₁, vascular endothelial growth factor, soluble intercellular adhesion molecule-1, fibronectin, and nitrotyrosine were evaluated by ELISA, and urinary creatinine and renal lipid peroxidation products by colorimetric assays. PARP inhibition counteracted diabetes-associated increase in renal cortex poly(ADP-ribosyl)ated protein level. Urinary albumin, isoprostane, and 8-hydroxy-2′-deoxyguanosine excretions and urinary albumin/creatinine ratio were increased in diabetic rats, and all these changes were at least partially prevented by GPI-15,427 treatment. PARP inhibition counteracted diabetes-induced renal transforming growth factor-β₁, vascular endothelial growth factor, and fibronectin, but not soluble intercellular adhesion molecule-1 and nitrotyrosine, accumulations. Lipid peroxidation product concentrations were indistinguishable among control and diabetic rats maintained with or without GPI-15,427 treatment. In conclusion, PARP activation plays an important role in kidney disease in long-term diabetes. These findings provide rationale for development and further studies of PARP inhibitors and PARP inhibitor-containing combination therapies, for prevention and treatment of diabetic nephropathy.

Keywords: Poly(ADP-ribose) polymerase; Diabetic nephropathy; Streptozotocin-diabetic rat; Oxidative-nitrosative stress; Vascular endothelial growth factor; Transforming growth factor-β

Correlation of the time course of development and decay of tolerance to morphine with alterations in sodium pump protein isoform abundance by Peng Li; Hercules T. Maguma; Kathleen Thayne; Barbara Davis; David A. Taylor (pp. 1015-1024).

Opioid receptors acutely hyperpolarize myenteric neurons and activate signaling pathways that cause long-term alterations in production of selected proteins including the alpha₃ subunit of the sodium pump.Since the heterologous tolerance that develops after chronic morphine administration has been proposed to be an adaptive process, it follows that the time course of the change in the cellular components should coincide with the time course of the altered responsiveness. This study correlated the time course over which heterologous tolerance develops with changes in the abundance of selected proteins in the guinea-pig longitudinal muscle/myenteric plexus (LM/MP) preparation. Tissues were obtained at various times following a single surgical implantation procedure and heterologous tolerance confirmed by a significant reduction in the sensitivity of the LM/MP to inhibition of neurogenic twitches by morphine, DAMGO, and 2-CADO. Tolerance developed with a delayed onset (significant 2–5-fold reduction in sensitivity by day 4 after pellet implantation) that reached a maximum by 7 days (4–8-fold reduction in responsiveness) that was maintained through 14 days with normal sensitivity spontaneously returning by 21 days post-implantation. Dot blot analysis was used to examine the abundance of the alpha₁ and alpha₃ subunit isoforms of the Na⁺/K⁺ ATPase and beta-actin over the same time course. The results showed significant decreases in abundance of the alpha₃ subunit at 4, 7, and 10 days following pellet implantation but no change in beta-actin or the alpha₁ subunit at any time period. These data support the idea that heterologous tolerance following chronic morphine exposure results from a cellular adaptive change that may involve a change in the abundance of the alpha₃ subunit isoform of the Na⁺/K⁺ ATPase.

Keywords: Abbreviations; (Na; ⁺; -K; ⁺; ATPase); Na; ⁺; -K; ⁺; adenosine triphosphatase [EC 3.6.1.37]; (DAMGO); Tyr-; d; -Ala-Gly-N-methyl-Phe-Gly-ol; (2-CADO); 2-chloroadenosine; (LM/MP); longitudinal muscle/myenteric plexusMorphine tolerance; Longitudinal muscle/myenteric plexus; Sodium pump isoforms; Morphine treatment; Adaptation; Myenteric neurons; Opioids; Guinea-pig

Investigating the interaction of McN-A-343 with the M₂ muscarinic receptor using its nitrogen mustard derivative by Hinako Suga; Frederick J. Ehlert (pp. 1025-1035).

An allosteric interaction involving a site-directed electrophile can be studied over a wider range of ligand concentrations than that involving an orthosteric radioligand.We investigated whether the aziridinium ion formed from a nitrogen mustard derivative (4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophenyl)carbamate; BR384) structurally related to McN-A-343 (4-(trimethyl-amino)-2-butynyl N-(3-chlorophenyl)carbamate) interacts allosterically or orthosterically with the M₂ muscarinic receptor. Chinese hamster ovary cells expressing the human M₂ muscarinic receptor were incubated with the aziridinium ion of BR384 in combination with McN-A-343 or other known orthosteric and allosteric ligands for various incubation times. After removing unreacted ligands, we measured the binding of [³H]N-methylscopolamine to residual unalkylated receptors. Affinity constants, rate constants for alkylation, and cooperativity constants were estimated for the interacting ligands using a mathematical model. Receptor alkylation by BR384 was consistent with a two-step process. After rapidly equilibrating with the receptor (step one), the aziridinium ion–receptor complex became covalently linked with a first order rate constant of about 0.95min⁻¹ (step two). McN-A-343, acetylcholine and N-methylscopolamine competitively protected the M₂ receptor from irreversible alkylation by BR384. In contrast, the allosteric modulators, gallamine and WIN 51,708 (17-β-hydroxy-17-α-ethynyl-5-α-androstano[3,2-β]pyrimido[1,2-α]benzimidazole), allosterically inhibited or had no effect on, respectively, receptor alkylation by BR384. There was good agreement between affinity constants estimated from the kinetics of receptor alkylation and by displacement of [³H]N-methylscopolamine binding. Our results suggest that BR384 covalently binds to the orthosteric site of the M₂ receptor and that McN-A-343 binds reversibly at the same locus. Our method of analyzing allosteric interactions does not suffer from the limitations of more conventional approaches and can be adapted to detect allosteric interactions at receptors other than the muscarinic subtypes.

Keywords: M; ₂; muscarinic receptor; McN-A-343; Allosterism; Competitive inhibition; Irreversible binding

A microfluidic hepatic coculture platform for cell-based drug metabolism studies by Eric Novik; Timothy J. Maguire; Piyun Chao; K.C. Cheng; Martin L. Yarmush (pp. 1036-1044).

Within the global pharmaceutical and biotech industries, there is significant interest in identifying in vitro screening systems that are more human-relevant—i.e., that offer greater utility in predicting subcellular and cellular physiological responses in humans in vivo—and that thereby allow investigators to reduce the incidence of costly late-stage failures during pharmaceutical clinical trials, as well as to reduce the use of animals in drug testing. Currently incumbent in vitro screening methods, such as culturing human hepatocytes in suspension, while useful, are limited by a lack of long term cellular function. In order to address this limitation, we have established an integrated, microfluidic, in vitro platform that combines the patented HμREL^® microdevice with a hepatic coculture system. In the present report, we use this platform to study clearance and metabolite generation of a battery of molecular entities. The results show that the flow-based coculture system is capable of clearing, with improved resolution and predictive value, compounds with high, medium, and low clearance values. In addition, when coculture is coupled with flow, higher metabolite production rates are obtained than in static systems.

Keywords: Microfluidics; Human hepatocyte; Coculture; Hepatic clearance; Metabolism

Human CYP2E1 is regulated by miR-378 by Takuya Mohri; Miki Nakajima; Tatsuki Fukami; Masataka Takamiya; Yasuhiro Aoki; Tsuyoshi Yokoi (pp. 1045-1052).

Human CYP2E1 is one of the pharmacologically and toxicologically important cytochrome P450 isoforms. Earlier studies have reported that the CYP2E1 expression is extensively regulated by post-transcriptional and post-translational mechanisms, but the molecular basis remains unclear. In the present study, we examined the possibility that microRNA may be involved in the post-transcriptional regulation of human CYP2E1. In silico analysis identified a potential recognition element of miR-378 (MRE378) in the 3′-untranslated region (UTR) of human CYP2E1 mRNA. Luciferase assays using HEK293 cells revealed that the reporter activity of the plasmid containing the MRE378 was decreased by co-transfection of precursor miR-378, indicating that miR-378 functionally recognized the MRE378. We established two HEK293 cell lines stably expressing human CYP2E1 including or excluding 3′-UTR. When the precursor miR-378 was transfected into the cells expressing human CYP2E1 including 3′-UTR, the CYP2E1 protein level and chlorzoxazone 6-hydroxylase activity were significantly decreased, but were not in the cells expressing CYP2E1 excluding 3′-UTR. In both cell lines, the CYP2E1 mRNA levels were decreased by overexpression of miR-378, but miR-378 did not affect the stability of CYP2E1 mRNA. In a panel of 25 human livers, no positive correlation was observed between the CYP2E1 protein and CYP2E1 mRNA levels, supporting the post-transcriptional regulation. Interestingly, the miR-378 levels were inversely correlated with the CYP2E1 protein levels and the translational efficiency of CYP2E1. In conclusion, we found that human CYP2E1 expression is regulated by miR-378, mainly via translational repression. This study could provide new insight into the unsolved mechanism of the post-transcriptional regulation of CYP2E1.

Keywords: miRNA; Cytochrome P450; Post-transcriptional regulation; Liver; Interindividual variability

Thiopurine S-methyltransferase pharmacogenetics: Functional characterization of a novel rapidly degraded variant allozyme by Qiping Feng; Suda Vannaprasaht; Yi Peng; Susothorn Angsuthum; Yingyos Avihingsanon; Vivien C. Yee; Wichittra Tassaneeyakul; Richard M. Weinshilboum (pp. 1053-1061).

A novel human thiopurine S-methyltransferase (TPMT) variant allele, (319 T>G, 107Tyr>Asp, *27), was identified in a Thai renal transplantation recipient with reduced erythrocyte TPMT activity. The TPMT*27 variant allozyme showed a striking decrease in both immunoreactive protein level and enzyme activity after transient expression in a mammalian cell line. We set out to explore the mechanism(s) responsible for decreased expression of this novel variant of an important drug-metabolizing enzyme. We observed accelerated degradation of TPMT*27 protein in a rabbit reticulocyte lysate. TPMT*27 degradation was slowed by proteasome inhibition and involved chaperone proteins—similar to observations with regard to the degradation of the common TPMT*3A variant allozyme. TPMT*27 aggresome formation was also observed in transfected mammalian cells after proteasome inhibition. Inhibition of autophagy also decreased TPMT*27 degradation. Finally, structural analysis and molecular dynamics simulation indicated that TPMT*27 was less stable than was the wild type TPMT allozyme. In summary, TPMT*27 serves to illustrate the potential importance of protein degradation – both proteasome and autophagy-mediated degradation – for the pharmacogenetic effects of nonsynonymous SNPs.

Keywords: Thiopurine S-methyltransferase; TPMT*27; Pharmacogenetics; Protein degradation; Autophagy

Nematicidal activity of two monoterpenoids and SER-2 tyramine receptor of Caenorhabditis elegans by Jason Lei; Michael Leser; Essam Enan (pp. 1062-1071).

Two plant essential oil monoterpenoids (thymol and carvacrol) possess strong nematicidal activity, which might be mediated through a tyramine receptor.In vitro cultures of two nematodes ( Caenorhabditis elegans and Ascaris suum) were established to study the nematicidal activity of three monoterpenoids (thymol, carvacrol and p-cymene). Toxicity of thymol and carvacrol was found for the two nematodes tested. The study was then aimed to address whether nematode tyramine receptor (TyrR) could interact with the two compounds by using HEK293 mammalian cells transfected with a C. elegans TyrR (ser-2) sequence, in hope of developing a high-throughput cell-based platform for future screening of new antihelminthic compounds. SER-2 expression and functionality in the transfected cells was first confirmed by green fluorescent protein tagging, competitive receptor binding, intracellular cyclic AMP, and intracellular calcium [Ca²⁺]_i mobilization assays. Thymol and carvacrol were then tested and demonstrated to interact with TyrR in desensitizing SER-2 for tyramine activation in [Ca²⁺]_i mobilization assay, and in translocating SER-2 from membrane to cytoplasm in receptor internalization assay. Receptor internalization activity of thymol and carvacrol was significantly blocked in cells expressing mutant SER-2 with the S210A/S214A double mutations, thus confirming specificity of the interactions. In summary, the current study showed that the nematicidal activity of thymol and carvacrol might be mediated through TyrR as the two compounds could trigger the signaling cascade downstream from the receptor in cells expressing wild-type but not a mutant SER-2. The TyrR-expressing cell system may prove to be a good screening platform for developing new antihelmintic compounds that may overcome parasite drug resistance, especially when such chemicals are used in combination with commercial drugs.

Keywords: Thymol carvacrol; Plant essential oil monoterpenoids; SER-2 tyramine receptor; Caenorhabditis elegans; Nematode parasite; Ascaris suum

Cytosolic NADP⁺-dependent isocitrate dehydrogenase regulates cadmium-induced apoptosis by Seoung Woo Shin; In Sup Kil; Jeen-Woo Park (pp. 1072-1080).

Cadmium ions have a high affinity for thiol groups. Therefore, they may disturb many cellular functions. We recently reported that cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) functions as an antioxidant enzyme to supply NADPH, a major source of reducing equivalents to the cytosol. Cadmium decreased the activity of IDPc both as a purified enzyme and in cultured cells. In the present study, we demonstrate that the knockdown of IDPc expression in HEK293 cells greatly enhances apoptosis induced by cadmium. Transfection of HEK293 cells with an IDPc small interfering RNA significantly decreased the activity of IDPc and enhanced cellular susceptibility to cadmium-induced apoptosis as indicated by the morphological evidence of apoptosis, DNA fragmentation and condensation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. Taken together, our results suggest that suppressing the expression of IDPc enhances cadmium-induced apoptosis of HEK293 cells by increasing disruption of the cellular redox status.

Keywords: Cadmium; Antioxidant enzyme; siRNA; Apoptosis; Redox status

Corrigendum to “Involvement of sphingosine-1-phosphate and S1P₁ in angiogenesis: Analyses using a new S1P₁ antagonist of non-sphingosine-1-phosphate analog” [Biochem. Pharmacol. 77 (2009) 1011–1020] by Kiyoaki Yonesu; Yumi Kawase; Tatsuya Inoue; Nana Takagi; Jun Tsuchida; Yoh Takuwa; Seiichiro Kumakura; Futoshi Nara (pp. 1081-1082).

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Biochemical Pharmacology (v.79, #7)

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Biochemical Pharmacology (v.79, #7)