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Biochemical Pharmacology (v.71, #10)

Editorial Advisory Board (pp. iii).

Molecular targets of dietary agents for prevention and therapy of cancer by Bharat B. Aggarwal; Shishir Shishodia (pp. 1397-1421).

While fruits and vegetables are recommended for prevention of cancer and other diseases, their active ingredients (at the molecular level) and their mechanisms of action less well understood. Extensive research during the last half century has identified various molecular targets that can potentially be used not only for the prevention of cancer but also for treatment. However, lack of success with targeted monotherapy resulting from bypass mechanisms has forced researchers to employ either combination therapy or agents that interfere with multiple cell-signaling pathways. In this review, we present evidence that numerous agents identified from fruits and vegetables can interfere with several cell-signaling pathways. The agents include curcumin (turmeric), resveratrol (red grapes, peanuts and berries), genistein (soybean), diallyl sulfide (allium), S-allyl cysteine (allium), allicin (garlic), lycopene (tomato), capsaicin (red chilli), diosgenin (fenugreek), 6-gingerol (ginger), ellagic acid (pomegranate), ursolic acid (apple, pears, prunes), silymarin (milk thistle), anethol (anise, camphor, and fennel), catechins (green tea), eugenol (cloves), indole-3-carbinol (cruciferous vegetables), limonene (citrus fruits), beta carotene (carrots), and dietary fiber. For instance, the cell-signaling pathways inhibited by curcumin alone include NF-κB, AP-1, STAT3, Akt, Bcl-2, Bcl-X_L, caspases, PARP, IKK, EGFR, HER2, JNK, MAPK, COX2, and 5-LOX. The active principle identified in fruit and vegetables and the molecular targets modulated may be the basis for how these dietary agents not only prevent but also treat cancer and other diseases. This work reaffirms what Hippocrates said 25 centuries ago, let food be thy medicine and medicine be thy food.

Keywords: Abbreviations; AP-1; activator protein-1; CAPE; caffeic acid phenethyl ester; Cdk; cyclin-dependent kinase; COX-2; cyclooxygenase-2; cPLA; ₂; phospholipase A; CSF; colony-stimulating factors; DIM; 1,1-bis(3′-indolyl)-1-(; p; -substituted phenyl) methanes; DMBA; dimethyl-benz(; a; )anthracene; EGF; epidermal growth factor; EGCG; epigallocatechin-3-gallate; Epo; erythropoietin; ERK; extracellular signal-regulated kinase; FGF; fibroblast growth factor; HER2; human epidermal growth factor receptor 2; I-3-C; indole-3-carbinol; ICAM-1; intercellular adhesion molecule-1; IFN; interferon; IGF; insulin-like growth factor; IκBα; inhibitory kappa B alpha; IKK; IκBα kinase; IL-1; interleukin; iNOS; inducible nitric oxide synthase; JNK; c Jun N-terminal kinase; LPS; lipopolysaccharide; LOX; lipoxygenase; MMP; matrix metalloproteinase; MAPK; mitogen-activated protein kinases; NF-κB; nuclear factor-kappa B; PDK; pyruvate dehydrogenase kinase; PTK; protein tyrosine kinase; PKB; protein kinase B; p27KIP1; p27 kinase inhibitor protein 1; PDGF; platelet-derived growth factor; PARP; polyadenosine-5′-diphosphate-ribose polymerase; STAT; signal transducer and activator of transcription; TGF; transforming growth factor; THC; tetrahydrocurcumin; TNF; tumor necrosis factor; TPA; phorbol 12-; O; -tetradecanoate-13-acetate; TRE; TPA response elements; uPA; urokinase-type plasminogen activator; VEGF; vascular endothelial growth factorNF-κB; AP-1; MAP kinases; Apoptosis; Cell cycle; Cancer; Dietary agents

Preclinical analysis of the analinoquinazoline AG1478, a specific small molecule inhibitor of EGF receptor tyrosine kinase by A.G. Ellis; M.M. Doherty; F. Walker; J. Weinstock; M. Nerrie; A. Vitali; R. Murphy; T.G. Johns; A.M. Scott; A. Levitzki; G. McLachlan; L.K. Webster; A.W. Burgess; E.C. Nice (pp. 1422-1434).

The tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) is a potent and specific inhibitor of EGFR tyrosine kinase whose favourable preclinical profile supports progression towards clinical trials. Microphysiometric evaluation revealed a short (<24min) effective inhibition of cellular receptor response to EGF challenge in BaF/ERX cells indicating a need to maintain sustained levels of inhibitor. Initial pharmacokinetic evaluation in mice of novel AG1478 formulations in a beta-cyclodextrin (Captisol^®) showed monoexponential elimination from plasma (half-life 30min) following subcutaneous administration. A two-fold dose escalation gave a 2.4-fold increase in the total AUC. Bolus i.v. and 6h continuous infusion were investigated in rats to mimic a more clinically relevant administration regimen. Drug elimination following bolus i.v. administration was biphasic (terminal elimination half-life 30–48min). The linear relationship between dose and AUC_0→∞ ( r²=0.979) enabled the prediction of infusion rates and doses for sustained delivery using continuous 6h infusions, where steady state was reached in 120min. Plasma levels of AG1478>10μM were achieved over the duration of the infusion. At the lowest dose, plasma drug levels after the cessation of infusion declined with a half-life of approximately 43min. EGFR activity, measured both by autophosphorylation and downstream signalling, was inhibited in a dose-dependent manner by injection of AG1478 in mice bearing xenografts of the human glioblastoma cell line U87MG.Δ2-7, which expresses a constitutively active variant of the EGF receptor. Taken together, these experiments provide essential data to assess the anti-tumour efficacy of AG1478 and will assist in the rational design of dose regimens for clinical studies.

Keywords: EGFR receptor; AG1478; Pharmacokinetics; Tyrosine kinase inhibitor; Microphysiometer; Quinazoline

Blockade of IGF-1 receptor tyrosine kinase has antineoplastic effects in hepatocellular carcinoma cells by Michael Höpfner; Alexander Huether; Andreas P. Sutter; Viola Baradari; Detlef Schuppan; Hans Scherübl (pp. 1435-1448).

Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death worldwide. Due to very poor 5-year-survival new therapeutic approaches are mandatory. Most HCCs express insulin-like growth factors and their receptors (IGF-R). As IGF-1R-mediated signaling promotes survival, oncogenic transformation and tumor growth and spread, it represents a potential target for innovative treatment strategies of HCC. Here we studied the antineoplastic effects of inhibiting IGF-1R signaling in HCC cells by the novel IGF-1R tyrosine kinase inhibitor NVP-AEW541.NVP-AEW541 induced a time- and dose-dependent growth inhibition in the human hepatoblastoma and hepatocellular carcinoma cell lines SK-Hep-1, Hep-3B, Hep-G2 and Huh-7. Measurement of LDH-release showed that the antineoplastic effect of NVP-AEW541 was not due to cytotoxicity. Instead NVP-AEW541 induced apoptosis as evidenced by both caspase-3 and -8 activation as well as by apoptosis-specific morphological and mitochondrial changes. In addition, nuclear degradation was monitored by DNA-laddering. NVP-AEW541-treatment suppressed the expression of the antiapoptotic proteins Bcl-2 and survivin, while the expression of the proapoptotic protein BAX was stimulated in a dose-dependent manner. Moreover, NVP-AEW541 arrested the cell cycle at the G1/S checkpoint. When NVP-AEW541 was combined with cytotoxic chemotherapy or with a specific epidermal growth factor receptor antibody additive antiproliferative effects were observed.Inhibition of IGF-1R tyrosine kinase (IGF-1R-TK) by NVP-AEW541 induces growth inhibition, apoptosis and cell cycle arrest in human HCC cell lines without accompanying cytotoxicity. Thus, IGF-1R-TK inhibition may be a promising novel treatment approach in HCC.

Keywords: Abbreviations; TK; tyrosine kinase; MAPK; mitogen-activated protein kinase; PBS; phosphate-buffered NaCl solution; RT-PCR; reverse transcriptase-polymerase chain reaction; SDS; sodium dodecyl sulfate; PMSF; phenylmethylsulfonyl fluoride; ERK; extracellular signal-regulated kinase; BSA; bovine serum albumine; Tris; tris(hydroxymethyl)-aminomethane; MW; molecular weight; HCC; hepatocelluar carcinoma; IGFR; insulin-like growth factor receptor; FADD; Fas associated death domain; Ψ; mitochondrial membrane potential; EGFR epidermal growth factor receptor; IGFBP; insulin-like growth factor binding protein; HEPES; N; -(2-hydroxyethyl)piperazine-; N; ′-(2-ethane-sulfonic acid); DTT; dithiothreitolIGF-1 receptor; Tyrosine kinase; Apoptosis; Cell cycle; EGFR; Cytostatics

Pharmacological characterization of protease activated receptor-1 by a serum responsive element-dependent reporter gene assay: Major role of calmodulin by Luc De Vries; Christiane Palmier; Frederic Finana; Bruno Le Grand; Michel Perez; Didier Cussac (pp. 1449-1458).

We studied the protease activated receptor-1 coupling to a serum response element (SRE)-dependent luciferase activity readout in transfected COS-7 cells. Thrombin, with a pEC₅₀ of 10.5, was 3000-fold more potent than the peptide agonists SFLLR and its derived compound C721-40 in stimulating luciferase activity, although the three agonists exhibited similar efficacy at the maximal concentration tested. Interestingly, SFLLR- and C721-40-induced luciferase activity was biphasic, suggesting that at least two populations of G proteins couple to the receptor. Further pharmacological characterization of this system was performed using selective protease activated receptor-1 antagonists. SCH203099 and ER-112787 blocked SFLLR-induced luciferase activity with similar potencies (p K_B of 7), slightly higher than that exhibited by an arylisoxazole derivative compound from Merck (p K_B of 6.1). These values correlated with their affinities established by competition binding experiments using [³H]-C721-40 as radioligand for protease activated receptor-1. Transduction mechanisms of protease activated receptor-1 coupling to SRE-dependent luciferase activity were examined using specific inhibitors. The Ca²⁺ chelator BAPTA-AM, as well as the calmodulin inhibitors W-7 and ophiobolin A, robustly inhibited SFLLR-induced SRE activation. Overexpression of RGS2 and a dominant negative rhoA protein abolished the SFLLR signal in an additive manner, suggesting a major role of Gq and G12/13 proteins. Furthermore, inhibition of phospholipase C, MAP-kinases, phosphatidyl inositol-3 kinase, rho-kinase and Ca²⁺/calmodulin-dependent protein kinases, all downstream effectors of Gq and G12/13, partially blocked the SFLLR-induced luciferase signal. Taken together, this SRE-luciferase assay reveals a complex network of transduction pathways of protease activated receptor-1 in accordance with the pleiotrophic action of thrombin.

Keywords: Abbreviations; ALU; arbitrary luminescence units; CaM; calmodulin; CaMKII; calmodulin kinase II; CaMKK; calmodulin kinase kinase; DMEM; Dulbecco's modified Eagle's medium; ERK; extracellular regulated kinase; GPCR; G protein-coupled receptor; IP3; inositol trisphosphate; LUC; luciferase; MAPK; mitogen activated protein kinases; MEK; extracellular regulated kinase kinase; MLCK; myosin light chain kinase; PAR; protease activated receptor; PI3-kinase; phosphoinositol-3 kinase; PKC; protein kinase C; PLC; phospholipase C; PTX; pertussis toxin; RGS; regulator of G protein signaling; ROCK; rho-dependent kinase; SRE; serum response element; SRF; serum response factor; TRAP; thrombin receptor activating peptidePAR-1; G protein; Ca; ²⁺; Calmodulin; Serum response element; Kinase

Evaluation of ligand selectivity using reporter cell lines stably expressing estrogen receptor alpha or beta by Aurélie Escande; Arnaud Pillon; Nadège Servant; Jean-Pierre Cravedi; Fernando Larrea; Peter Muhn; Jean-Claude Nicolas; Vincent Cavaillès; Patrick Balaguer (pp. 1459-1469).

Estrogens control transcriptional responses through binding to two different nuclear receptors, estrogen receptor α (ERα) and β (ERβ). Since these two ER subtypes are thought to mediate different biological effects, there is intense interest in designing subtype-selective ER ligands. In this study, we evaluated the ERα and ERβ selectivity of 19 known estrogens and antiestrogens using reporter cell lines previously developed in our laboratory. The HELN-ERα and HELN-ERβ cells stably express full-length ERα and ERβ, respectively, and are derived from HELN cells (HeLa cells stably transfected with an ERE-driven luciferase plasmid). We report that 16α-LE2, PPT and 3β,5α-GSD have a high ERα-selective agonist potency while 8β-VE2, DPN, genistein and biochanin A show ERβ selectivity with 8β-VE2 being the most potent and selective ERβ agonist. We also tested ER antagonists and we showed that raloxifene and RU486 are ERα and ERβ-selective antiestrogens, respectively. In all cases, selectivity is due to differences in binding affinities as indicated by whole-cell ligand-binding assays. Very interestingly, we demonstrate that a combination of genistein and raloxifene produces a full-ERβ specific response. Together these results demonstrate the usefulness of our stably transfected cell lines to characterize ER ligands and indicate that treatments combining agonist/antagonist ligands produce full-ERβ selectivity.

Keywords: Abbreviations; AF; activation function; DCC; dextran-coated charcoal; DMEM; Dulbecco's modified Eagle's medium; DMSO; dimethyl sulfoxide; EC50 value; efficient concentration to obtain half maximum activity; ER; estrogen receptor; ERE; estrogen-responsive element; HELN; human cervix adenocarcinoma cell expressing luciferase under estrogen-response element control; IC50 value; concentration required to inhibit specific E2 binding by 50%; K; _d; dissociation constant; LBD; ligand-binding domain; Luc; firefly luciferase; RLU; relative luciferase units; RBA; relative binding affinity; RTC; relative transactivation capacity; SERM; selective estrogen receptor modulatorsEstrogen receptors; Ligand; Stable transfectants; Whole-cell ligand binding; Transactivation

Hormonal regulation of renal multidrug resistance-associated proteins 3 and 4 (Mrp3 and Mrp4) in mice by J.M. Maher; X. Cheng; Y. Tanaka; G.L. Scheffer; C.D. Klaassen (pp. 1470-1478).

Multidrug resistance-associated proteins 3 and 4 (Mrp3 and Mrp4) are expressed at much higher levels in female than male kidney. Sex steroids and sex-specific growth hormone (GH) secretion patterns often mediate gender-predominant gene expression. Thus, three models were used to investigate potential endocrine regulation of Mrp3 and Mrp4: (1) gonadectomized (GNX) mice with 17β-estradiol (E2) or 5α-dihydroxytestosterone (DHT) replacement; (2) hypophysectomized (HPX) mice receiving E2, DHT, or simulated male-pattern (MP) or female-pattern (FP) GH secretion; (3) lit/ lit mice, which have a spontaneous mutation in the growth-hormone releasing-hormone (GHRH) receptor, with simulated MP- or FP-GH secretion. GNX and HPX decreased Mrp3 mRNA levels compared with intact females. In both respective models E2 administration increased Mrp3 expression in GNX and HPX mice. DHT markedly repressed Mrp3 from GNX+placebo levels, however, this was not observed in the HPX model. In lit/ lit mice, Mrp3 expression was lower than in wild-type controls, and MP-GH and FP-GH simulation slightly increased Mrp3 expression. Whereas GNX increased Mrp4 in males to female levels, HPX actually increased Mrp4 expression in both genders +375% and +66%, respectively. In both models DHT markedly repressed Mrp4. Furthermore, Mrp4 was higher in lit/ lit than wild-type male mice, and simulation of MP-GH secretion suppressed female-predominant Mrp4 expression. In conclusion, these data indicate that E2 contributes to higher Mrp3 mRNA expression in females, yet a role for androgens in Mrp3 repression cannot be discounted. In contrast, Mrp4 mRNA is higher in females due to repression by both DHT and MP-GH secretion in males.

Keywords: Abbreviations; Mrp; multidrug resistance-associated protein; Cyp; cytochrome P450; ABCC; ATP-binding cassette transporter, subfamily C; DHT; 5α-dihydroxytestosterone; HPX; hypophysectomized; GNX; gonadectomized mice; E2; 17β-estradiol; GH; growth hormone; GHRH; growth hormone-releasing hormone; MP; male-pattern; FP; female-patternTransporter; Multidrug resistance; ABCC; Mrp; Hormonal regulation; Growth hormone

Relative effects of phenolic constituents from Yucca schidigera Roezl. bark on Kaposi's sarcoma cell proliferation, migration, and PAF synthesis by Ciro Balestrieri; Francesca Felice; Sonia Piacente; Cosimo Pizza; Paola Montoro; Wieslaw Oleszek; Vincenzo Visciano; Maria Luisa Balestrieri (pp. 1479-1487).

Yuccaols (A, B, C) are phenolic constituents isolated from Yucca schidigera bark characterized by unusual spirostructures made up of a C₁₅ unit and a stilbenic portion closely related to resveratrol. These novel compounds are of particular interest for their antioxidant and anti-inflammatory properties. However, their effects on cell proliferation, migration, and platelet-activating factor (PAF) biosynthesis remain unknown. PAF, a potent mediator of inflammation, is known to promote angiogenesis and in vitro migration of endothelial cells and Kaposi's sarcoma (KS) cells. The objective of our study was to determine the effect of Yuccaols and resveratrol on the vascular endothelial growth factor (VEGF)-induced proliferation, migration, and PAF biosynthesis in KS cells. The results indicated that Yuccaols (25μM) were more effective than resveratrol (25μM) in inhibiting the VEGF-induced KS cell proliferation. Western blot analysis revealed that Yuccaols reduced the VEGF-induced phosphorylation of p38 and p42/44, thus indicating a possible interference with the mechanism underlying the VEGF-stimulated cell proliferation. Furthermore, Yuccaols completely inhibited the VEGF-stimulated PAF biosynthesis catalyzed by the acetyl-CoA:lyso-PAF acetyltransferase and enhanced its degradation through the PAF-dependent CoA-independent transacetylase (250% of control). In addition, Yuccaol C abrogated the PAF-induced cell motility whereas Yuccaol A and Yuccaol B reduced the cell migration from 7.6μm/h to 6.1μm/h and 5.6μm/h, respectively. These results indicate that the anti-inflammatory properties attributed to Yucca schidigera can be ascribed to both resveratrol and Yuccaols and provide the first evidences of the anti-tumor and anti-invasive properties of these novel phenolic compounds.

Keywords: Kaposi's sarcoma; PAF; Stilbenic derivatives; Cell motility; Inflammation; VEGF

The peptide Trp-Lys-Tyr-Met-Val-D-Met activates neutrophils through the formyl peptide receptor only when signaling through the formylpeptde receptor like 1 is blocked by Jennie Karlsson; Huamei Fu; Francois Boulay; Johan Bylund; Claes Dahlgren (pp. 1488-1496).

Neutrophils express the G protein-coupled N-formyl peptide receptor (FPR) and its homologue FPRL1. The hexapeptide Trp-Lys-Tyr-Met-Val-D-Met-NH₂ (WKYMVm) activates HL-60 cells transfected either with FPRL1 or with FPR. The signaling through the stably expressed receptors was inhibited by specific receptor antagonists, cyclosporine H and WRWWWW (WRW₄) for FPR and FPRL1, respectively. The neutrophil release of superoxide was used to determine receptor preference, when these cells were triggered with WKYMVm. The response was not affected by the FPR specific antagonist suggesting that no signals are transduced through this receptor. The response was only partly inhibited by WRW₄, but this antagonist induced a receptor switch, perceptible as a change in sensitivity to the FPR antagonist. The activity remaining in the presence of WRW₄ was inhibited by cyclosporine H. A cell permeable peptide (PBP10) corresponding to the phosphatidyl-inositol-bisphosphate binding region of gelsolin, inhibited the FPRL1-, but not the FPR-induced cellular response and induced the same type of receptor switch. We show that an agonist that has the potential to bind and activate neutrophils through FPRL1 as well as through FPR, uses the latter receptor and its signaling route, only when the activating signal generated through FPRL1 is blocked. The receptor switch is achieved when signaling through FPRL1 is inhibited both by a receptor antagonist, and by an inhibitor operating from the inside of the plasma membrane. The phenomenon described is of general importance for proper interpretation of results generated through the use of different “silencing technologies�? in receptor operated signaling transduction research.

Keywords: Neutrophils; Formyl peptide receptors; Receptor antagonists; Signal transduction; NADPH-oxidase; Chemoattractant receptors

Extracellular ATP activates ERK1/ERK2 via a metabotropic P2Y₁ receptor in a Ca²⁺ independent manner in differentiated human skeletal muscle cells by Christopher May; Lukas Weigl; Anton Karel; Martin Hohenegger (pp. 1497-1509).

ATP is released at the neuromuscular junction to regulate development and proliferation. The sequential expression of P2X and P2Y receptors has been correlated to these effects in many species and cell lines. We have therefore investigated ATP mediated signalling in differentiated primary human skeletal muscle cells. ATP was capable to trigger Ca²⁺ transients in these cells via P2Y receptors which were not attributable to Ca²⁺ influx via P2X receptors. Instead, ATP propagated the formation of inositol phosphate (IP) with an EC₅₀ of 21.3μM. The Ca²⁺ transient provoked by ATP was abrogated roughly 75% by the phospholipase C (PLC) inhibitor, U73122. Interestingly, the ryanodine sensitive Ca²⁺ pool was not involved in ATP triggered Ca²⁺ release. On mRNA level and by a pharmacological approach we confirmed the presence of the P2Y₁, P2Y₂, P2Y₄ and P2Y₆ receptors. Substantially, ATP activated IP formation via a P2Y₁ receptor. In addition, ATP elicited extracellular signal regulated kinase (ERK)1/2 phosphorylation in a time and concentration dependent manner, again mainly via P2Y₁ receptors. The ATP mediated ERK1/2 phosphorylation was strictly dependent on phospholipase C and PI3 kinase activity. Importantly, ATP mediated ERK1/2 phosphorylation was Ca²⁺ independent. This observation was corroborated by the finding that conventional protein kinase C inhibitors did not suppress ATP triggered ERK1/2 phosphorylation. Taken together, these observations highlight the importance of ATP as a co-neurotransmitter at the neuromuscular junction via dual signalling, i.e. IP₃ receptor mediated Ca²⁺ transients and Ca²⁺ insensitive phosphorylation of ERK1/2.

Keywords: Abbreviations; PPADS; pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate; A3P5P; adenosine 3′,5′-diphosphate; XAC; xanthine amine congener; U73122; 1-(6-(17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl-1; H; -pyrrole-2,5-dione; U73343; 1-[6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione; 2-MeSATP; 2-methylthioadenosine 5′-triphosphate; 2-MeSADP; 2-methylthioadenosine 5′-diphosphate; CPK; creatine phosphokinase; CP; creatine phosphate; ERK1/2; extracellular signal-regulated kinase 1/2; PLC; the phospholipase C; IP; ₃; inositol 1,4,5-triphosphate; IP; inositol phosphate; RB; reactive blue 2; [Ca; ²⁺; ]; _i; intracellular calcium; MRS2179; 2′-deoxy-; N; ⁶; -methyl adenosine 3′,5′-diphosphate; LY294002; 2-(4-morpholinyl)-8-phenyl-4; H; -1-benzopyran-4-oneHuman skeletal muscle cells; P2Y receptor; ATP; MRS2179; Calcium signalling; ERK1/2

Inhibition of rabbit brain 4-aminobutyrate transaminase by some taurine analogues: A kinetic analysis by Lorenzo Ricci; Maria Frosini; Nicola Gaggelli; Gianni Valensin; Fabrizio Machetti; Giampietro Sgaragli; Massimo Valoti (pp. 1510-1519).

The use of the antiepileptic drug, 4-aminobutyrate transaminase (GABA-T) inhibitor vigabatrin (VIGA), has been recently cautioned because it is associated to irreversible field defects from damage of the retina. Since novel GABA-T inhibitors might prove useful in epilepsy or other CNS pathologies as VIGA substitutes, the aim of the present investigation was to characterize the biochemical properties of some taurine analogues (TA) previously shown to act as GABA-T inhibitors. These include (±)piperidine-3-sulfonic acid (PSA), 2-aminoethylphosphonic acid (AEP), (±)2-acetylaminocyclohexane sulfonic acid (ATAHS) and 2-aminobenzenesulfonate (ANSA). Kinetic analysis of the activity of partially purified rabbit brain GABA-T in the presence of VIGA and TA showed that PSA and AEP caused a linear, mixed-type inhibition ( K_i values 364 and 1010μM, respectively), whereas VIGA, ANSA and ATAHS behaved like competitive inhibitors ( K_i values 320, 434 and 598μM, respectively). Among the compounds studied, only VIGA exerted a time-dependent, irreversible inhibition of the enzyme, with K_i and k_inact values of 773μM and 0.14min⁻¹, respectively. Furthermore, the ability of VIGA and TA to enhance GABA-ergic transmission was assessed in rabbit brain cortical slices by NMR quantitative analysis. The results demonstrate that VIGA as well as all TA promoted a significant increase of GABA content. In conclusion, PSA, ANSA and ATAHS, reversible GABA-T inhibitors with K_i values close to that of VIGA, represent a new class of compounds, susceptible of therapeutic exploitation in many disorders associated with low levels of GABA in brain tissues.

Keywords: Abbreviations; AEP; 2-aminoethylphosphonic acid; ANSA; 2-aminobenzenesulfonate; ATAHS; (±); trans; -2; -; acetylaminocyclohexane sulfonic acid; GABA-T; 4-aminobutyrate transaminase (EC 2.6.1.19); PSA; (±)piperidine-3-sulfonic acid; TA; Taurine analogues; VIGA; 4-amino-5-hexenoic acid or γ-vinyl GABAGABA; 4-Aminobutyrate transaminase; Vigabatrin; Taurine analogues; 4-Aminobutyrate transaminase inhibitors

Effect of culture conditions on the expression and function of Bsep, Mrp2, and Mdr1a/b in sandwich-cultured rat hepatocytes by Ryan Z. Turncliff; Xianbin Tian; Kim L.R. Brouwer (pp. 1520-1529).

Rat hepatocytes cultured in a sandwich configuration form functional canalicular networks. The influence of extracellular matrix configuration, medium composition, and confluency on the expression and function of Bsep, Mrp2, and Mdr1a/b in sandwich-cultured (SC) rat hepatocytes was examined. Primary rat hepatocytes were: (1) maintained in various extracellular matrix sandwich configurations, (2) cultured in Dulbecco's modified Eagle's medium (DMEM), Modified Chee's medium (MCM) or Williams’ E medium (WME), and/or (3) plated at decreasing cell density. Bsep, Mrp2, and Mrdr1a/b expression in day 4 SC rat hepatocytes was assessed by Western blot; function was measured by accumulation of taurocholate, 5(and 6)-carboxy-2′,7′-dichlorofluorescein, and rhodamine 123, respectively, in canalicular networks. In general, the extracellular matrix conditions examined resulted in similar protein expression and function. Function of Bsep, Mrp2, and Mdr1a/b was higher in SC rat hepatocytes maintained in DMEM or WME. Mrp2 and Mdr1a/b expression, representative of total cellular content, did not always correlate directly with function, which should be reflective of canalicular membrane expression. Mrp2 expression decreased significantly as cell density decreased in SC hepatocytes. Low plating density in Biocoat™ plates resulted in poor canalicular network formation and reduced function of Mrp2 and Mdr1a/b. Expression and/or function of Mrp2 and Mdr1a/b in rat hepatocytes cultured in a sandwich configuration may be influenced by plating density and media type.

Keywords: Classification code; (9) Pulmonary; Renal and Hepatic PharmacologySandwich-cultured; Hepatocytes; Transport proteins; Biliary excretion; In vitro models

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Biochemical Pharmacology (v.71, #10)