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Biochemical Pharmacology (v.73, #6)

Editorial Advisory Board (pp. iii).

“Phenotypic” pharmacology: The influence of cellular environment on G protein-coupled receptor antagonist and inverse agonist pharmacology by Carl P. Nelson; R.A. John Challiss (pp. 737-751).

A central dogma of G protein-coupled receptor (GPCR) pharmacology has been the concept that unlike agonists, antagonist ligands display equivalent affinities for a given receptor, regardless of the cellular environment in which the affinity is assayed. Indeed, the widespread use of antagonist pharmacology in the classification of receptor expression profiles in vivo has relied upon this ‘antagonist assumption’. However, emerging evidence suggests that the same gene-product may exhibit different antagonist pharmacological profiles, depending upon the cellular context in which it is expressed—so-called ‘phenotypic’ profiles. In this commentary, we review the evidence relating to some specific examples, focusing on adrenergic and muscarinic acetylcholine receptor systems, where GPCR antagonist/inverse agonist pharmacology has been demonstrated to be cell- or tissue-dependent, before going on to examine some of the ways in which the cellular environment might modulate receptor pharmacology. In the majority of cases, the cellular factors responsible for generating phenotypic profiles are unknown, but there is substantial evidence that factors, including post-transcriptional modifications, receptor oligomerization and constitutive receptor activity, can influence GPCR pharmacology and these concepts are discussed in relation to antagonist phenotypic profiles. A better molecular understanding of the impact of cell background on GPCR antagonist pharmacology is likely to provide previously unrealized opportunities to achieve greater specificity in new drug discovery candidates.

Keywords: Abbreviations; ADP(S; adenosine 5′-; O; -(2-thiotriphosphate); BPH; benign prostatic hyperplasia; CGP 12177A; (±)-4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazole-2-one; CGRP; calcitonin gene-related peptide; CHO; Chinese hamster ovary; CNS; central nervous system; CRLR; calcitonin receptor-like receptor; DPCPX; 8-cyclopentyl-1,3-dipropylxanthine; GPCR; G protein-coupled receptor; HEK; human embryonic kidney; mACh; muscarinic acetylcholine; MAP kinase; mitogen-activated protein kinase; mRNA; messenger RNA; NECA; 5′-; N; -ethylcarboxamidoadenosine; OAB; overactive bladder; p; FHHSiD; para; -fluorohexahydrosiladifenidol; PTx; pertussis toxin; RAMP; receptor activity-modifying proteins; SNP; single nucleotide polymorphismG protein-coupled receptor; Adrenoceptor; Muscarinic receptor; Cell background; Signal transduction; Antagonism; Inverse agonism; Receptor phosphorylation; Receptor dimerization

Functional studies on the activity of efflux transporters in an ex vivo model with chicken splenocytes and evaluation of selected fluoroquinolones in this model by Aneliya Milanova Haritova; Jan A. Schrickx; Johanna Fink-Gremmels (pp. 752-759).

The efflux proteins P-glycoprotein (P-gp), BCRP and members of the MRP-family (MRPs) are increasingly recognized as determinants of the absorption, tissue distribution and excretion of numerous drugs. A widely applied in vitro screening method, to assess the effect of these efflux transporters in transmembrane transport of drugs is based on the use of peripheral blood mononuclear cells (PBMC), in which the efflux of fluorescent dye Rhodamine 123 (Rh-123) can be easily measured. In avian species, the isolation of PBMCs is compromised by the presence of thrombocytes having approximately the same size. As an alternative, we validated the use of isolated splenocytes to assess Rhodamine 123 transport in the presence and absence of specific inhibitors for P-gp, MRPs and BCRP. Rh-123 efflux was concentration-dependent with the percentage of efflux that decreased with increasing concentrations. P-gp inhibitors, PSC833 and GF120918, significantly inhibit Rh-123 efflux, whereas inhibitors for MRPs and BCRP, MK571 and Ko-143, respectively, have a limited inhibitory effect. However, the effect of GF120918 was more pronounced as compared to PSC833, suggesting an additional role for BCRP next to P-gp in Rh-123 efflux. Moreover, fluoroquinolones were selected to test the applicability of the described model. None of these fluoroquinolones significantly inhibit P-gp function at concentrations up to 50μM, with exception of danofloxacin and danofloxacin mesylate that were found to reduce Rh-123 efflux by approximately 15%.

Keywords: Abbreviations; ABC; ATP-Binding Cassette; BCRP; breast cancer resistance protein; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; MDR1; multiple drug resistance 1; MRP2; multidrug resistance-associated protein 2; PBMC; peripheral blood mononuclear cells; P-gp; P-glycoprotein; Rh-123; Rhodamine 123ABC efflux transporters; Chicken lymphocytes; Rhodamine 123; Fluoroquinolones

Excess ribonucleotide reductase R2 subunits coordinate the S phase checkpoint to facilitate DNA damage repair and recovery from replication stress by Z. Ping Lin; Michael F. Belcourt; Rocco Carbone; Jana S. Eaton; Philip G. Penketh; Gerald S. Shadel; Joseph G. Cory; Alan C. Sartorelli (pp. 760-772).

Ribonucleotide reductase (RNR), which consists of R1 and R2 subunits, catalyzes a key step of deoxyribonucleoside triphosphate (dNTP) synthesis for DNA replication and repair. The R2 subunit is controlled in a cell cycle-specific manner for timely DNA synthesis and is negatively regulated by p53 in response to DNA damage. Herein we demonstrate that the presence of excess R2 subunits in p53(−/−) HCT-116 human colon cancer cells protects against DNA damage and replication stress. siRNA-mediated stable knockdown (>80%) of excess R2 subunits has no effect on proliferative growth but results in enhanced accumulation of γ-H2Ax and delayed recovery from DNA lesions inflicted by exposure to cisplatin and Triapine. This accentuated induction of γ-H2Ax in R2-knockdown cells is attributed to reduced ability to repair damaged DNA and overcome replication blockage. The lack of excess R2 subunits consequently augments chk1 activation and cdc25A degradation, causing impeded cell progression through the S phase and enhanced apoptosis in response to DNA damage and replication stress. In contrast, the level of R1 subunits appears to be limiting, since depletion of the R1 subunit directly activates the S phase checkpoint due to replication stress associated with impaired RNR activity. These findings suggest that excess R2 subunits facilitate DNA damage repair and recovery from replication stress through coordination with the S phase checkpoint in the absence of functional p53. Thus, the level of the R2 subunit constitutes an important determinant of the chemosensitivity of cancer cells and serves as a potential target for enhancement of DNA-damage based therapy.

Keywords: Abbreviations; RNR; ribonucleotide reductase; ATM; ataxia telangiectasia-mutated; ATR; ATM and Rad3-related; DSBs; DNA double-stranded breaks; IR; ionizing radiation; chk1; checkpoint kinase 1; γ-H2Ax; phosphorylated-histone 2Ax; sh/siRNA; short hairpin/short interference RNA; PARP; poly(ADP-ribose) polymerase; PCNA; proliferating cell nuclear antigen; MDC1; mediator of DNA damage checkpoint protein 1; 53BP; p53 binding protein 1; BRCA1; breast cancer 1 early onset; cdk2; cyclin dependent kinase 2; cdc25; cell division cycle 25; NBS1; Nijmegen breakage syndrome 1Ribonucleotide reductase; DNA damage checkpoint; γ-H2Ax; DNA repair; Cisplatin; siRNA

Mitochondria are primary targets in apoptosis induced by the mixed phosphine gold species chlorotriphenylphosphine-1,3-bis(diphenylphosphino)propanegold(I) in melanoma cell lines by Francesco Caruso; Raffaella Villa; Miriam Rossi; Claudio Pettinari; Francesco Paduano; Marzia Pennati; Maria Grazia Daidone; Nadia Zaffaroni (pp. 773-781).

Based on previous evidence indicating a selective cytotoxic activity of the mixed phosphine gold complex chlorotriphenylphosphine-1,3-bis(diphenylphosphino)propanegold(I) for melanoma cells, we investigated the cellular bases of its antiproliferative effect in a panel of human melanoma cell lines (JR8, SK-Mel-5, Mel-501, 2/60, 2/21 and GRIG). The drug consistently induced a dose-dependent inhibition of cell growth, with IC₅₀ values ranging from 0.8 to 2.3μM and, when tested under the same experimental conditions, its cytotoxic activity was higher than (from 2- to 5-fold) or comparable to that of cisplatin as a function of cell lines. The ability of the gold complex to activate programmed cell death was assessed in JR8 and 2/60 cells, and a dose-dependent increase in cells with an apoptotic nuclear morphology was observed in both cell lines (up to 40 and 66% of the overall cell population, for JR8 and 2/60 cell lines, respectively). Such an apoptotic response was mediated by a dose-dependent loss of mitochondrial membrane potential, cytochrome c and Smac/DIABLO release from mitochondria into cytosol and enhanced caspase-9 and caspase-3 catalytic activity. A reduced or completely abrogated expression of the anti-apoptotic proteins c-IAP1, XIAP and survivin in drug-treated cells was also observed. Overall, results from the study indicate that chlorotriphenylphosphine-1,3-bis(diphenylphosphino)propanegold(I) markedly inhibits melanoma cell growth by inducing mitochondria-mediated apoptosis and suggest it as a good candidate for additional evaluation as an anticancer agent against melanoma.

Keywords: Melanoma; Apoptosis; Mitochondria; Gold; Phosphine; CaspaseAbbreviations; SRB; sulforhodamine B; LEHD-AFC; Leu-Glu-His-Asp-7-amino-4-trifluoromethylcoumarin; Ac-DEVD-AMC; N; -acetyl-Asp-Glu-Val-Asp-aldehyde-7-amino-4-methylcoumarin; Δ; ψ; _mt; mitochondrial membrane potential

Genistein induces apoptosis in human hepatocellular carcinomas via interaction of endoplasmic reticulum stress and mitochondrial insult by Ting-Chun Yeh; Po-Cheng Chiang; Tsai-Kun Li; Jui-Ling Hsu; Chun-Jung Lin; Shih-Wei Wang; Chieh-Yu Peng; Jih-Hwa Guh (pp. 782-792).

Hepatocellular carcinoma is a very common malignancy and is chemoresistant to currently available chemotherapeutic agents. Endoplasmic reticulum (ER) stress-induced apoptotic pathway is suggested to be less affected by the resistance mechanisms, becoming a potential target of chemotherapeutic strategy. The anticancer effects and expression of GADD153, a transcription factor induced by ER stress, were examined in hepatocellular carcinoma Hep3B cells. The correlation between these two parameters was constructed under flavonoid stimulation with a correlation coefficient ( r) of 0.8. The data also showed that genistein (isoflavone) was the most effective one. Genistein induced the activation of several ER stress-relevant regulators, including m-calpain, GADD153, GRP78 and caspase-12. Furthermore, genistein-induced effect was inhibited in cells transfected with antisense GADD153 cDNA, indicating a functional role of GADD153. Notably, genistein induced the activation of caspase-2, whereas did not cause the DNA damage. It also triggered the production of ROS. The antioxidant trolox significantly reduced ROS accumulation, but did not modify genistein-induced apoptotic cell death. The long-term exposure (48h) of cells to genistein caused Mcl-1 down-regulation and Bad cleavage; furthermore, cyclosporin A (an inhibitor of mitochondrial permeability transition pore) almost completely abolished genistein-induced loss of mitochondrial membrane potential, and induced a 30% reverse of apoptosis caused by long-term treatment (48h) of genistein, suggesting the involvement of mitochondrial stress in the late phase of genistein-induced effect. Taken together, it is suggested that genistein induces the anticancer effect through a mechanism initiated by ER stress and facilitated by mitochondrial insult in Hep3B cells.

Keywords: Hepatocellular carcinoma; Flavonoid; Genistein; Endoplasmic reticulum; Mitochondria; GADD153

Protective effect of baicalein against endotoxic shock in rats in vivo and in vitro by Pao-Yun Cheng; Yen-Mei Lee; Yuan-Sheng Wu; Tz-Wei Chang; Jong-Shiaw Jin; Mao-Hsiung Yen (pp. 793-804).

Dried roots of Scutellaria baicalensis Georgi ( Huang qin) are widely used in traditional Chinese medicine. Baicalein is a major bioactive flavonoid component of H. qin that shows a wide range of biological activities, including antioxidant and anti-inflammatory actions. We evaluated therapeutic effects and possible mechanisms of action of baicalein on circulatory failure and vascular dysfunction during sepsis induced by lipopolysaccharide (LPS; 10mg/kg, i.v.) in anesthetized rats. Treatment of the rats with baicalein (20mg/kg, i.v.) significantly attenuated the deleterious hemodynamic changes of hypotension and tachycardia caused by LPS and significantly inhibited the elevation of plasma tumor necrosis factor α (TNF-α). Baicalein also decreased levels of inducible nitric oxide synthase (iNOS) and the overproduction of NO and superoxide anions caused by LPS. It also increased the survival rate of ICR mice (25–30g) challenged by LPS (60mg/kg). Moreover, infiltration of neutrophils into the liver and lungs of rats 6h after treatment with LPS was also reduced by baicalein. To investigate the mechanism of action of baicalein on sepsis, RAW 264.7 cells were used as a model. Baicalein inhibited iNOS protein production, and suppressed LPS-induced degradation of IκBα, the formation of a nuclear factor kappa B (NF-κB)-DNA complex and NF-κB-dependent reporter gene expression. Thus, the therapeutic effects of baicalein were associated with reductions in TNF-α and superoxide anion levels during sepsis. The inhibitory effects of baicalein on iNOS production may be mediated by inhibition of the activation of NF-κB. Baicalein may thus prove a potential agent against endotoxemia.

Keywords: Sepsis; Reactive oxygen species; Circulatory failure; Nitric oxide; NF-κB; Baicalein

Functional CRF receptors in BON cells stimulate serotonin release by Bengt von Mentzer; Yousuke Murata; Ingela Ahlstedt; Erik Lindström; Vicente Martínez (pp. 805-813).

BON cells are human, pancreatic carcinoid-derived, endocrine-like cells that share functional similarities with intestinal enterochromaffin (EC) cells. We investigated the presence of corticotropin-releasing factor (CRF) receptors, their signalling pathways and the functional effects of their stimulation in BON cells (clone #7). Expression analysis showed that BON cells contain mRNA for the CRF receptor types 1 and 2 (CRF_1/2), although CRF₂ mRNA levels were 23-fold higher than those of CRF₁ mRNA. The CRF_1/2 ligand, rat/human (r/h)CRF (EC₅₀=233nM), and the selective CRF₂ ligand, human urocortin 3 (Ucn 3) (EC₅₀=48nM), induced a dose-dependent increase in cAMP formation. Effects of r/hCRF were blocked by 44% with the selective CRF₁ antagonist DMP-696, while the selective CRF₂ antagonist antisauvagine-30 had only marginal effects. Both ligands (100nM) stimulated the release of serotonin with similar efficacy (3-fold increase over basal). Effects of r/hCRF, but not Ucn 3, were blocked by pre-incubation with antisauvagine-30. These observations demonstrate that the EC cell-related BON cells express functional CRF₂ receptors linked to the release of serotonin. This suggests that EC cells may be a target for CRF and/or Ucn 3 in the intestine during stress-related responses. Actions of CRF/Ucn 3 and EC cell-derived mediators, such as serotonin, might underlie several motor, secretory and/or sensory disorders of the gastrointestinal (GI) tract which may play a role in the pathophysiology of functional GI disorders, such as irritable bowel syndrome.

Keywords: Abbreviations; 5-HT; 5-hydroxytryptamine (serotonin); ASV-30; antisauvagine-30; cAMP; cyclic AMP; CGA; chromogranin A; CRF; corticotropin-releasing factor; CRF; ₁; CRF receptor type 1; CRF; ₂; CRF receptor type 2; EC; enterochromaffin; GI; gastrointestinal; IBS; irritable bowel syndrome; TPH; tryptophan hydroxylase; Ucn; urocortinBON cells; CRF; CRF receptors; 5-HT; Urocortin 3; Antisauvagine-30; DMP-696; Irritable bowel syndrome

Histamine augments β₂-adrenoceptor-induced cyclic AMP accumulation in human prostate cancer cells DU-145 independently of known histamine receptors by Judith Ramos-Jiménez; Luis-Enrique Soria-Jasso; Aurelio López-Colombo; Jorge-Alberto Reyes-Esparza; Javier Camacho; José-Antonio Arias-Montaño (pp. 814-823).

Androgen-independent prostate cancer cells DU-145 express a number of G protein-coupled receptors, including histamine H₁ receptors. There is evidence for the presence of β-adrenoceptors in the human prostate, and in this work we set out to characterise the expression of β-adrenoceptors by DU-145 cells, their linking to cyclic AMP (cAMP) formation and the possible modulation by histamine H₁ receptors of β-adrenoceptor function. Saturation [³H]-dihydroalprenolol binding indicated that DU-145 cells express moderate levels of β-adrenoceptors (22.7±2.5fmol/mg protein), which belong to the β₂-subtype as assessed by inhibition by the antagonists ICI-118,551 and CGP-20712A. Inhibition of [³H]-dihydroalprenolol binding by agonists (noradrenaline, adrenaline and isoproterenol) showed the presence of both high-(53–59%) and low-affinity binding sites. β-Adrenoceptor stimulation with isoproterenol resulted in robust [³H]-cAMP accumulation (10–30-fold of basal, EC₅₀ 142nM; pEC₅₀ 6.85±0.05). While not having effect of its own on basal [³H]-cAMP accumulation, histamine significantly augmented the β₂-adrenoceptor-induced response (overall effect 152±6% of isoproterenol alone) with EC₅₀ 1.35μM (pEC₅₀ 5.87±0.06). This effect was independent of extracellular Ca²⁺, insensitive to antagonists/agonists at H₁, H₂ or H₃/H₄ receptors and mimicked by drugs containing an imidazole ring in their chemical structure and by imidazole itself. Taken together, our results show that in DU-145 cells histamine augments β₂-adrenoceptor-induced cAMP independently of the activation of known histamine receptors. The effect may involve other mechanisms such as allosteric modulation of β₂-adrenoceptors by the imidazole moiety of histamine.

Keywords: DU-145 cells; Adrenoceptors; Histamine; cAMP; Adrenaline; Noradrenaline

Role of Tyr306 in the C-terminal fragment of Clostridium perfringens enterotoxin for modulation of tight junction by Chiaki Ebihara; Masuo Kondoh; Motoki Harada; Makiko Fujii; Hiroyuki Mizuguchi; Shin-ichi Tsunoda; Yasuhiko Horiguchi; Kiyohito Yagi; Yoshiteru Watanabe (pp. 824-830).

We previously reported that the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is a novel type of absorption enhancer that interacts with claudin-4 and that Tyr306 of C-CPE plays a role in ability of C-CPE to modulate barrier of tight junctions. In the current study, to investigate effects of Tyr306 on the C-CPE activity, we prepared some C-CPE mutants substituted Tyr306 with Trp (Y306W), Phe (Y306F) and Lys (Y306K). We found that Y306W and Y306F mutants of C-CPE had claudin-4 binding affinities and effects on the barrier function of tight junctions, whereas both of these properties were greatly reduced with the Y306K mutant. Finally, the Y306K but not the Y306F and Y306W mutants had reduced abilities to enhance absorption in rat jejunum. These results indicate that aromatic and hydrophobic properties, not hydrogen bonding potential, of Tyr306 are involved in the interaction of C-CPE with claudin-4 and in the modulation of the tight junction barrier function by C-CPE.

Keywords: Abbreviations; C-CPE; the C-terminal fragment of; Clostridium perfringens; enterotoxin; PSIF; protein synthesis inhibitory factor; TJ; tight junction; CPE; Clostridium perfringens; enterotoxin; TER; transepithelial electric resistance; C-CPE-PSIF; C-CPE fused to PSIF; PCR; polymerase chain reaction; LDH; lactate dehydrogenase; FD-4; fluorescein-isothiocyanate-dextran with a molecular weight of 4000Claudin-4; Absorption; Jejunum; Clostridium perfringens; enterotoxin; Tight junction

Nicotinamide inhibits B lymphocyte activation by disrupting MAPK signal transduction by Julien Daniel; Yoann Marechal; Frédéric Van Gool; Fabienne Andris; Oberdan Leo (pp. 831-842).

Nicotinamide (NAm) represents both a pharmacological agent known to express cell preserving and anti-inflammatory properties, and a useful investigational tool to elucidate cellular pathways regulating a wide range of cellular functions. We demonstrate in this study that exogenous NAm, when used at pharmacological doses, inhibits activation of primary murine B lymphocytes in response to multiple ligands. NAm appears to affect a membrane proximal event leading to MAPKs activation, a transduction pathway shared by multiple receptors including the antigen-specific B cell receptor, CD38, CD40 and TLR4 receptors. NAm inhibited phospho-ERK accumulation, and only marginally affected phospho-p38 and phospho-JNK induction upon BCR stimulation of naive B lymphocytes. Accordingly, NAm also affected the expression of known targets of the MAPK ERK pathway such as CD69 and cyclin D2. Based on a comparison with well-characterized pharmacological inhibitors, we suggest in this work that NAm may inhibit a post-translational modification mediated by a yet unidentified mono(ADP-ribose)transferase. Collectively, our observations indicate that in addition to its previously described effect on cells of the innate immune system, NAm is able to modulate the activity of B lymphocytes suggesting a potential role of this vitamin in regulating antibody-mediated autoimmune disorders.

Keywords: Abbreviations; NAD; nicotinamide adenine dinucleotide; NAm; nicotinamide; ART; mono(ADP-ribose)transferase; PARP; poly(ADP-ribose)polymeraseB lymphocytes; Vitamines; Nicotinamide; Immunosuppression; ADP-ribosyl transferases; Meta; -iodobenzylguanidine

Inhibition of PI3K and calcineurin suppresses chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2)-dependent responses of Th2 lymphocytes to prostaglandin D₂ by Luzheng Xue; Shân L. Gyles; Anna Barrow; Roy Pettipher (pp. 843-853).

Interaction of prostaglandin D₂ (PGD₂) with chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) triggers chemotaxis and pro-inflammatory cytokine production by Th2 lymphocytes. We have investigated the role of inhibitors of various cell-signalling pathways on the responses of human CRTH2⁺ CD4⁺ Th2 cells to PGD₂. Phosphatidylinositol 3-kinase (PI3K) and Ca²⁺/calcineurin/nuclear factor of activated T cells (NFAT) pathways were activated by PGD₂ in Th2 cells in a CRTH2-dependent manner. Inhibition of the PI3K pathway with LY294002 significantly reduced both PGD₂-induced cell migration and cytokine (interleukin-4, interleukin-5 and interleukin-13) production. The inhibitory effect of LY294002 on cell migration is likely to be related to cytoskeleton reorganization as it showed a similar potency on PGD₂-induced actin polymerization. The calcineurin inhibitors, tacrolimus (FK506) and cyclosporin A, had no effect on cell migration but completely blocked both cytokine production and the nuclear translocation of NFATc1 suggesting that Ca²⁺/calcineurin/NFAT is involved in CRTH2-dependent cytokine production but not chemotaxis. The promotion of NFAT nuclear location by PI3K activation may be mediated by negative regulation of glycogen synthase kinase-3β (GSK3β), since the PGD₂-stimulated increase in phospho-GSK3β was down-regulated by LY294002, and inhibition of GSK3β by SB216763 enhanced PGD₂-induced Th2 cytokine production and reversed the inhibitory effect of LY294002. These data suggest that PI3K and Ca²⁺/calcineurin/NFAT signalling pathways are critically involved in pro-inflammatory responses of Th2 cells to PGD₂.

Keywords: Abbreviations; PGD; ₂; prostaglandin D; ₂; CRTH2; chemoattractant receptor-homologous molecule expressed on Th2 cells (also named DP2); DP1; D prostanoid receptor 1; PI3K; phosphatidylinositol 3-kinase; NFAT; nuclear factor of activated T cells; GSK3β; glycogen synthase kinase-3β; FK506; tacrolimus; IL-4; interleukin-4; IL-5; interleukin-5; IL-13; interleukin-13; CaM; calmodulin; TP; thromboxane-like prostanoid receptor; DK-PGD; ₂; 13,14-dihydro-15-keto-PGD; ₂; CsA; cyclosporin A; PLCβ; phospholipase CβCRTH2; Th2 lymphocytes; Chemotaxis; Cytokines; PI3K; Calcineurin

Vanadate-induced activation of cytosolic phospholipase A₂α in L929 cells: Roles of tyrosine kinase, protein kinase C, and extracellular signal-regulated kinase by Tomoko Taniguchi; Masaya Shimizu; Hiroyuki Nakamura; Tetsuya Hirabayashi; Hiromichi Fujino; Takeshi Saito; Toshihiko Murayama (pp. 854-862).

Orthovanadate (Na₃VO₄), which acts as an inhibitor of protein tyrosine phosphatases, has a various pharmacological effects including the release of arachidonic acid (AA) from cells. We investigated roles of α-type cytosolic phospholipase A₂ (cPLA₂α), Src family kinases (Src) and protein kinase C (PKC) in the release of AA induced by Na₃VO₄ from a murine fibroblast cell line, L929. C12 cells, a variant of L929 that lacks expression of cPLA₂α, were used along with a clone of C12 cells that are stably expressing cPLA₂α (C12-cPLA₂α cells). In the presence of a Ca²⁺ ionophore (10μM A23187), 5 and 10mM Na₃VO₄ synergistically stimulated AA release from L929 and C12-cPLA₂α cells, and to a much lesser extent from control C12 cells. The release of AA by Na₃VO₄/A23187 was inhibited by a selective cPLA₂α inhibitor (3μM pyrrophenone). The release of AA by Na₃VO₄/A23187 was significantly inhibited by a PKC inhibitor (10μM GF109203X), in PKC-depleted cells, by a Src inhibitor (2μM PP2) and by an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) kinase (10μM U0126). The phosphorylation of ERK1/2 was stimulated by Na₃VO₄, and the response was significantly decreased by inhibitors of Src, PKC and ERK1/2 kinase. Our data show that Na₃VO₄ stimulates AA release largely via cPLA₂α activation in Ca²⁺-dependent manner, and the cross-talk between Src and PKC and the ERK-dependent pathways are involved in Na₃VO₄-induced AA release from L929 cells.

Keywords: Abbreviations; PTP; protein tyrosine phosphatases; Src; Src family kinases; ROS; reactive oxygen species; AA; arachidonic acid; PLA; ₂; phospholipase A; ₂; cPLA; ₂; α; α-type cytosolic PLA; ₂; PMA; 4β-phorbol 12-myristate 13-acetate; PKC; protein kinase C; U0126; 1,4-diamino-2,3-dicyano-1,4-bis-(; o; -aminophenylmercapto)butadiene; GF109203X; 2-[1-(3-dimethylamino-propyl)-1; H; -indol-3-yl]-3-(1; H; -indol-3-yl)-maleimide; PP2; 4-amino-5-(4-chlorophenyl)-7-(; t; -butyl)pyrazolo[3,4-; d; ]pyrimidine; PP3; 4-amino-7-phenylpyrazol[3,4-; d; ]pyrimidine; ERK1/2; extracellular signal-regulated kinases 1 and 2cPLA; ₂; α; Vanadate; Src; PKC; ERK1/2; Murine L929 cells

The changes of intracellular H₂O₂ are an important factor maintaining mitochondria membrane potential of antimycin A-treated As4.1 juxtaglomerular cells by Yong Whan Han; Sung Zoo Kim; Suhn Hee Kim; Woo Hyun Park (pp. 863-872).

We investigated an involvement of ROS, such as H₂O₂ and O₂⁻ and GSH in the As4.1 cell death by antimycin A and examined whether ROS scavengers rescue antimycin A-induced As4.1 cell death and its mechanism. Levels of intracellular H₂O₂ and O₂⁻ were markedly increased in antimycin A-treated cells. Antimycin A reduced the intracellular GSH content. A ROS scavenger, Tiron down-regulated the production of intracellular H₂O₂. However, the reduction of intracellular H₂O₂ level did not change the apoptosis parameters, such as sub-G1 DNA content and annexin V binding. Interestingly, treatment of Tiron could partially prevent the loss of mitochondrial transmembrane potential (Δ Ψ_m). Treatment of SOD and catalase also reduced the intracellular H₂O₂ and loss of mitochondrial transmembrane potential (Δ Ψ_m) without reducing O₂⁻ level and apoptosis in antimycin A-treated As4.1 cells. All the ROS scavengers, SOD and catalase did not inhibit GSH depletion induced by antimycin A, resulting in failure of preventing the apoptosis. In addition, all the reagents including antimycin A did not induce any specific phase arrest of cell cycle in As4.1 cells. In summary, these results demonstrate that antimycin A generates potently ROS, H₂O₂ and O₂⁻ and induces the depletion of GSH content in As4.1 JG cells, and that Tiron, SOD and catalase inhibited partially the loss of mitochondrial transmembrane potential (Δ Ψ_m) via the reduction of intracellular H₂O₂ level.

Keywords: Abbreviations; AMA; antimycin A; ROS; reactive oxygen species; NADPH; nicotine adenine diphosphate; XO; xanthine oxidase; SOD; superoxide dismutase; JGCT; juxtaglomerular cell tumors; FBS; fetal bovine serum; PBS; phosphate buffer saline; FITC; fluorescein isothiocyanate; H; ₂; DCFDA; 2′,7′-dichlorodihydrofluorescein diacetate; DHE; dihydroethidium; GSH; glutathione; NAC; N; -acetylcysteine; CMFDA; 5-chloromethylfluorescein diacetateAntimycin A; ROS; Cell cycle; Apoptosis; As4.1; ROS scavenger; SOD; Catalase

Hexachlorobenzene as hormonal disruptor—studies about glucocorticoids: Their hepatic receptors, adrenal synthesis and plasma levels in relation to impaired gluconeogenesis by Sandra M. Lelli; Nora R. Ceballos; Marta B. Mazzetti; Carmen A. Aldonatti; Leonor C. San Martín de Viale (pp. 873-879).

In Wistar rats, hexachlorobenzene (HCB) depresses the gluconeogenic enzyme phosphoenolpyruvate-carboxykinase (PEPCK). In the liver, glucocorticoids (GC) normally regulate the glucose synthesis by acting on PEPCK. Thus, the aim of this work was to investigate, in a time-course study, the effects of HCB on plasma GC, its adrenal synthesis and stimulation, and the kinetic parameters of its hepatic receptors (GR) in relation to the gluconeogenic blockage produced by HCB.Plasma corticosterone (CORT) concentration, urinary porphyrins and hepatic PEPCK were determined after 2, 4, 6 and 8 weeks of HCB-treatment. The effect of HCB on kinetic parameters of GR was studied in adrenalectomized porphyric rats after 2, 4 and 8 weeks of treatment. Additionally, adrenal CORT synthesis in the same weeks was measured with or without ACTH. Results show that plasma CORT in intoxicated animals dropped significantly after 2 and 4 weeks of treatment (23% and 58%, respectively), and then remained constant until the 8th week. HCB also promoted a reduction in the number of hepatic GR (50–55%) without modifying affinity. After 8 weeks, when porphyria was well established (40–50-fold increase in urinary porphyrins), a reduction (52%) in hepatic GR number, as well as a decrease in PEPCK activity (56%) were observed. Moreover, CORT biosynthesis in adrenals from intoxicated animals significantly decreased (60%) without changes in ACTH effect.Briefly, this paper shows that HCB causes a disruption in GC and GR. This disturbance could contribute to the negative effect on glucose synthesis through PEPCK regulation, thus modulating porphyria. These results enhance the knowledge about the hormonal disruption produced by chlorinated xenobiotics.

Keywords: Abbreviations; AA; arachidonic acid; ACTH; adrenocorticotropin; ALA-S; ALA-synthase; CORT; corticosterone; DEX; dexamethasone; GC; glucocorticoid; GR; glucocorticoid receptor; HCB; hexachlorobenzene; PCT; porphyria cutanea tarda; PEPCK; phosphoenolpyruvate carboxykinase; PL; phospholipid; PMSF; phenyl methyl sulfonylfluoride; SM; sphingomyelin; TCDD; 2,3,7,8-tetrachlorodibenzo-; p; -dioxinHexachlorobenzene; Corticosterone; Glucocorticoids receptor; Phosphoenol-pyruvate carboxykinase; Porphyria; Gluconeogenesis

Diclofenac hydroxylation in monkeys: Efficiency, regioselectivity, and response to inhibitors by Cuyue Tang; Yulin Fang; Catherine Booth-Genthe; Yuhsin Kuo; Scott D. Kuduk; Tom H. Rushmore; Brian A. Carr (pp. 880-890).

The catalytic efficiency, regioselectivity, and response to chemical inhibitors of diclofenac (DF) hydroxylation in three Old World monkey liver microsomes (rhesus, cynomolgus, and African green monkey) are different from those determined with human liver microsomes. In contrast to the high affinity–high capacity (low K_m–high V_max) characteristics of DF 4′-hydroxylation in humans, this reaction proceeded in all monkey species with catalytic efficiencies >20-fold lower. However, DF 5-hydroxylation, a negligible reaction in human liver microsomes, was kinetically favored in monkeys mainly due to the increased V_max values. Chemical inhibitors (reversible or mechanism-based) selective to human CYP3A4 and CYP2C9 failed to differentiate monkey orthologs involved in DF hydroxylation. Immunoinhibition studies with monoclonal antibodies against human CYPs revealed the major contribution of CYP2C and CYP3A to 4′- and to 5-hydroxylation, respectively, in rhesus and cynomolgus liver microsomes. However, in African green monkeys, in addition to CYP2C, CYP3A also appeared to be involved in 4′-hydroxylation. Further studies with recombinant rhesus and African green monkey CYP2C and CYP3A enzymes (rhesus CYP2C75, 2C74, and 3A64; African green monkey CYP2C9agm and CYP3A4agm) confirmed the major role of CYP enzymes of these two subfamilies in DF 4′- and 5-hydroxylation. Clearly, while monkey CYP2C and 3A enzymes retain the same substrate selectivity towards DF hydroxylation as their human orthologs, their altered catalytic efficiency and response to chemical inhibitors may indicate different structural features of active sites as opposed to human orthologs.

Keywords: Abbreviations; DF; diclofenac; 4′-OHD; 4′-hydroxy diclofenac; 5-OHD; 5-hydroxy diclofenac; CYPs; cytochrome P450s; K; _m; apparent Michaelis constant; K; _s; substrate inhibition constant; V; _max; maximal velocity; HLM; human liver microsomes; Rh-MLM; rhesus monkey liver microsomes; Cyno-MLM; cynomolgus monkey liver microsomes; AG-MLM; African green monkey liver microsomes; KTZ; ketoconazole; SLF; sulfaphenazole; TA; tienilic acidDiclofenac hydroxylation; Catalytic efficiency; Regioselectivity and response to chemical inhibitors; Monkey liver microsomes; Recombinant monkey CYP2C and CYP3A

Low-affinity uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)- N-methylpyridinium iodide (4-Di-1-ASP) in BeWo cells by Erik Rytting; Jordan Bryan; Marylee Southard; Kenneth L. Audus (pp. 891-900).

Understanding the mechanisms of transport processes in the placenta can improve the safety and efficacy of drug delivery during pregnancy. Functional studies of organic cation transporters (OCTs) are usually carried out using radioactivity, and a fluorescent marker would add flexibility to experimental methods. As a published substrate for OCT1 and OCT2, the fluorescent compound 4-(4-(dimethylamino)styryl)- N-methylpyridinium iodide (4-Di-1-ASP) was chosen as a candidate for studying placental OCT function in BeWo cells. The expression of OCT1 and OCT2 was also investigated in BeWo cells, an established human choriocarcinoma trophoblastic cell line frequently used as an in vitro model of the rate-limiting barrier for maternal–fetal exchange of drugs and nutrients within the placenta. 4-Di-1-ASP was taken up into BeWo cells by a low-affinity, carrier-mediated process exhibiting a K_m of 580±110μM and V_max of 97±9nmol/mg protein/30min, and asymmetric transport was observed, with greater permeability in the apical to basolateral (maternal-to-fetal) direction. However, RT-PCR revealed no expression of OCT1 or OCT2 in either BeWo cells or primary cultured human cytotrophoblast cells, and OCT substrates such as TEA and choline did not inhibit the uptake of 4-Di-1-ASP. Although the uptake of this fluorescent compound in BeWo cells is not mediated by an OCT, the colocalization experiments with fluorescence microscopy and inhibition studies confirmed significant mitochondrial uptake of 4-Di-1-ASP. Transport of 4-Di-1-ASP into the nuclear region of BeWo cells was also observed, which is likely mediated by a nucleoside transporter.

Keywords: Organic cation transporters; OCT1; OCT2; BeWo cells; Placenta; Drug transport

Interactions of human serum albumin with retinoic acid, retinal and retinyl acetate by Elena Karnaukhova (pp. 901-910).

Human serum albumin (HSA), a major plasma protein and plasma-derived therapeutic, interacts with a wide variety of drugs and native plasma metabolites. In this study the interactions between HSA and small lipophilic molecules all- trans retinoic acid (RA), all- trans retinaldehyde (retinal, RAL) and all- trans retinyl acetate (RAC) were investigated by UV–vis absorption spectroscopy, fluorescence spectroscopy and circular dichroism (CD). This paper focuses on investigation of the interactions between HSA and RA by the visible CD. RAL and RAC were used in this study due to their structural identity to RA to elucidate the importance of the end functional group for the complex formation. Our data demonstrate that RA specifically binds to HSA in a stable non-covalent complex at least at two internal binding sites with close but distinct affinities. Upon titration of HSA with RA, visible CD spectra clearly demonstrate the appearance of a well-defined induced positive Cotton Effect (CE) around 350nm. Beyond ligand-to-protein ratio of 0.8 and up to saturation (2.0), CD exhibits two major bands of opposite signs, suggesting exciton coupling between the chromophore molecules in the protein interior. The fluorescence quenching data suggest proximity of the primary RA binding site to tryptophan (W²¹⁴). RAC shows a weak association with HSA with stoichiometry close to that of RA, while interactions of RAL with HSA proceed non-specifically at multiple sites. Contrary to RA, the adducts of HSA with RAC and RAL do not show any induced chirality, thus indicating that despite their high structural similarity to RA, both compounds do not appear to occupy the internal binding sites, but associate with the protein exterior.

Keywords: Abbreviations; CD; circular dichroism; CE; Cotton Effect; HSA; human serum albumin; FA; fatty acid; L/P; ligand-to-protein (molar) ratio; PBS; phosphate buffer saline; RA; all; -; trans; retinoic acid; RAC; all; -; trans; retinyl acetate; RAL; all; -; trans; retinaldehyde; UV–vis; absorbanceHuman serum albumin; Retinoic acid; Retinal; Retinyl acetate; Circular dichroism; Protein-induced chirality

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Biochemical Pharmacology (v.73, #6)