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Biochemical Pharmacology (v.70, #2)

Editorial Advisory Board (pp. iii).

Receptor-independent actions of PPAR thiazolidinedione agonists: Is mitochondrial function the key? by D.L. Feinstein; A. Spagnolo; C. Akar; G. Weinberg; P. Murphy; V. Gavrilyuk; C. Dello Russo (pp. 177-188).

Agonists of the peroxisome proliferator activated receptor gamma (PPARγ) are currently used for treatment of type 2 diabetes due to their insulin sensitizing and glucose metabolism stabilizing effects. More recently some of these same agonists were shown to exert anti-inflammatory and anti-proliferative effects as well. Although PPARγ agonists can operate via receptor-mediated events occurring at the genomic level, thereby causing long lasting changes in gene expression patterns, recent studies demonstrate non-genomic as well as genomic actions, and receptor-dependent as well as receptor-independent effects of the thiazolidinedione (TZD) class of PPARγ agonists. In this review we will summarize data describing some of these novel, receptor independent actions of TZDs, review evidence that TZDs directly influence mitochondrial function, and attempt to reconcile how changes in mitochondrial function could contribute to other receptor-independent actions of these drugs.

Keywords: Abbreviations; AICAR; AMPK activator 5-aminoimidazole-4-carboxamide riboside; AMPK; adenosine 5′-monophosphate-activated protein kinase; AP1; activator protein 1; AUC; area under the curve; CBP; CREB-binding protein; CNS; central nervous system; COX; cyclooxygenase; CPT1; carnitine palmityl transferase 1; CREB; cyclic AMP-responsive element binding protein; DMSO; dimethyl sulfoxide; EGR-1; early growth response-1; ERK; extracellular signal regulated kinase; FA; fatty acid; FFA; free fatty acid; GADD45; growth arrest and DNA damage-inducible gene; GLUT; glucose transporter; HL; human leukemia; HSP; heat shock protein; HSR; heat shock response; IFN; interferon; IκB; Inhibitor of nuclear factor kappa B; IKK; IκB kinase; IL; interleukin; JNK; c-Jun NH2-terminal protein kinase; LPS; lipopolysaccharide; MalDC; malonyl-CoA decarboxylase; MAPK; mitogen-activated protein kinases; NAG-1; nonsteroidal anti-inflammatory drug-activated gene-1; NFκB; nuclear factor kappa B; NOS; nitric oxide synthase; PC; pyruvate carrier; NOS2; inducible form of NOS; PDH; pyruvate dehydrogenase; PG; prostaglandin; PGC1α; PPARγ coactivator 1α; PGJ2; prostaglandin J2; Pio; pioglitazone; PK; protein kinase; PPARγ; peroxisome proliferator activated receptor gamma; PPRE; PPAR DNA binding elements; ROS; reactive oxygen species; Rosi; rosiglitazone; siRNA; small interfering RNA; TNF; tumor necrosis factor; Trog; troglitazone; TZD; thiazolidinedione; VCAM-1; Vascular cell adhesion molecule-1; Δ; ψ; _m; delta psi; mitochondrial membrane potentialMitochondria; Glucose; Thiazolidinedione; Fatty acid; Insulin; Stress response; AMPK; Cytokines; Nitric oxide; Metabolism; Mitoneet

Stimulation of group I metabotropic glutamate receptors evokes calcium signals and c-jun and c-fos gene expression in human T cells by Gianluca Miglio; Federica Varsaldi; Chiara Dianzani; Roberto Fantozzi; Grazia Lombardi (pp. 189-199).

To study if the activation of group I mGlu receptors in human T cells modifies intracellular Ca²⁺ concentration ([Ca²⁺]_i) and cell function, we measured [Ca²⁺]_i on cell suspensions (spectrofluorimetric method) or single cell (digital Ca²⁺ imaging system) using fura-2 as indicator. Early-inducible gene ( c-jun and c-fos) expression was studied by reverse transcriptase-polymerase chain reaction assay as representative of Ca²⁺-sensitive gene expression. (1 S,3 R)-ACPD (100μM), the selective mGlu receptor agonist, evoked a significant increase (34.1±4.9%) of [Ca²⁺]_i, pharmacologically characterized as mediated by group I mGlu receptors, since both ( S)-3,5-DHPG (100μM), a selective group I mGlu receptor agonist and CHPG (1mM), the specific mGlu₅ receptor agonist, reproduced the effects, that were abolished by AIDA (1mM), a selective group I mGlu receptor antagonist. ( S)-3,5-DHPG-induced a rapid [Ca²⁺]_i rise (initial phase) followed by a slow decrease (second phase) to the baseline. Both extracellular Ca²⁺ and Ca²⁺ released from intracellular stores contribute to the [Ca²⁺]_i increase which depend on PLC activation. In a Ca²⁺-free buffer, the second phase rapidly return to the baseline; LaCl₃ (1–10μM), an inhibitor of extracellular Ca²⁺ influx, significantly reduced the second phase only; thapsigargin (1μM), by discharging intracellular Ca²⁺ stores, U 73122 (10μM) and D609 (300μM), by inhibiting PLC activity, prevented both phases. In our system, PTX pre-treatment increased ( S)-3,5-DHPG effects, demonstrating that PXT-sensitive G_i/o proteins are involved. Finally, specific stimulation of these receptors in Jurkat cells upregulates c-jun and c-fos gene expression, thus activating multiple downstream signalling regulating important T cell functions.

Keywords: Abbreviations; iGlu; ionotropic glutamate; mGlu; metabotropic glutamate; NMDA; N; -methyl-; d; -aspartate; AMPA; α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; PLC; phospholipase C; AC; adenylate cyclase; [Ca; ²⁺; ]; _i; intracellular Ca; ²⁺; concentration; mAb; monoclonal antibodies; PHA; phytohemagglutinin; ERK; extracellular signal-regulated kinase; PBMC; peripheral blood mononuclear cells; fura-2/AM; fura-2 acetoxymethyl ester; PTX; pertussis toxin; TG; thapsigargin; (1; S; ,3; R; )-ACPD; (1; S; ,3; R; )-1-aminocyclopentane-1,3-dicarboxylic acid; AIDA; (; R; ,; S; )-1-aminoindan-1,5-dicarboxylic acid; CHPG; (RS)-2-chloro-5-hydroxyphenylglycine; (; S; )-3,5-DHPG; (; S; )-3,5-dihydroxyphenylglycine; LY 367385; (S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid; MPEP; 2-methyl-6-(phenylethynyl)-pyridine; U 73122; 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1; H; -pyrrole-2,5-dione; U 73343; 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-pyrrolidine-2,5-dione; D609; tricyclodecan-9-yl xanthogenate; RT-PCR; reverse transcriptase-polymerase chain reaction; GAPDH; glyceraldehydes-3-phosphate dehydrogenase; VGCC; voltage-gated Ca; ²⁺; channels; TRPC; canonical transient receptor potential; HBSS; Hank's balanced salts solution; EAAT; excitatory amino acid transporters; APC; antigen-presenting cells

Retinoic acid blocks pro-inflammatory cytokine-induced matrix metalloproteinase production by down-regulating JNK-AP-1 signaling in human chondrocytes by Ling-Jun Ho; Leou-Chyr Lin; Li-Feng Hung; Shyu-Jye Wang; Chian-Her Lee; Deh-Ming Chang; Jenn-Haung Lai; Tong-Yuan Tai (pp. 200-208).

The development of osteoarthritis (OA) has recently been implicated as a result of immune-mediated damage of chondrocytes and their supporting matrixes. Pro-inflammatory cytokines like interleukin (IL)-1 and tumor necrosis factor alpha (TNF-α) play pivotal roles in immunopathogenesis of OA. Because vitamins preserving anti-oxidative effects are suggested to provide protection in OA patients from joint damage, in the present study, we examined the effects and mechanisms of all- trans retinoic acid ( t-RA) in suppressing pro-inflammatory cytokine-induced matrix metalloproteinases (MMPs) production in human chondrocytes. Chondrocytes were prepared from cartilage specimens of OA patients receiving total hip or total knee replacement. The protein concentration was measured by ELISA, the mRNA expression by reverse transcriptase-polymerase chain reaction, the protein expression by Western blotting, the transcription factor DNA-binding activity by electrophoretic mobility shift assay and the protein kinase activity by kinase assay. We showed that both MMP-1 and MMP-13 mRNA expression, protein production and enzyme activity induced by either IL-1 or TNF-α were suppressed by t-RA or different retinoid derivatives. The molecular investigation revealed that the t-RA-mediated suppression was likely through blocking p38 kinase and c-Jun N-terminal kinase-activator protein-1 signaling pathways. In contrast, t-RA had no effect on extracellular signal-regulated kinase activity, nuclear factor kappaB (NF-κB) DNA-binding activity and IκBα degradation. Furthermore, we showed that t-RA could reduce IL-1-induced TNF-α production in chondrocytes. Our results suggest that vitamin A may protect OA patients from pro-inflammatory cytokine-mediated damage of chondrocytes and their supporting matrixes.

Keywords: Abbreviations; AP-1; activator protein-1; EMSA; electrophoresis mobility shift assay; ERK; extracellular signal-regulated protein kinase; 4-HPR; N; -(4-hydroxyphenyl)retinamide; IκBα; I kappa B alpha; IL; interleukin; JNK; c-jun N-terminal kinase; mAb; monoclonal antibody; MMP; matrix metalloproteinase; NF-κB; nuclear factor kappa B; OA; osteoarthritis; RT-PCR; reverse transcriptase-polymerase chain reaction; t; -RA; all-; trans; retinoic acid; TIMP-1; tissue inhibitor of matrix metalloproteinase 1; TNF-α; tumor necrosis factor alphaOsteoarthritis; Cytokines; Chondrocytes; Matrix metalloproteinases; C-Jun N-terminal kinase; Activator protein-1

A synthesized cationic tetradecapeptide from hornet venom kills bacteria and neutralizes lipopolysaccharide in vivo and in vitro by Guo Yibin; Zheng Jiang; Zhou Hong; Lv Gengfa; Wang Liangxi; Wei Guo; Lu Yongling (pp. 209-219).

Sepsis is a complex clinical syndrome that results from a harmful host response to infection, in which foreign bacteria and lipopolysaccharide (LPS) are potent activators of different immune cells, including monocytes and macrophages. To date, there are currently few effective adjuvant therapies in clinical use except activated protein C focusing on the coagulation system. Mastoparans (MPs) are wasp venom cationic amphiphilic tetradecapeptides; these are capable of modulating various cellular activities, including stimulation of GTP-binding protein, phospholipase C and can bind to a phospholipid bilayer. Masroparan-1 (MP-1, INLKAIAALAKKLL-NH₂), a tetradecapeptide toxin isolated from hornet venom, was synthesized chemically. In this study, Escherichia coli 25922 ( E. coli 25922) and LPS were used to induce sepsis in an animal model. We found that MP-1 treatment at 3mg/kg protected mice from otherwise lethal bacteria and LPS challenges. MP-1 has antibacterial capabilities against Gram-negative and Gram-positive bacteria. Its antibacterial action against E. coli may result from the destruction of bacterial membrane structures. In addition, treatment of murine peritoneal macrophages with MP-1 potently inhibited the respiratory burst. This effect maybe related to an inhibition of NADPH oxidase in the membrane. Furthermore, MP-1, bound with high-affinity to LPS and lipid A with dissociation equilibrium constants of 484 and 456nM, respectively, and neutralized LPS in a dose-dependent manner. MP-1 also significantly reduced the expression of TLR4, TNF-α and IL-6 mRNA and the release of cytokines in LPS-stimulated murine peritoneal macrophages. Our results shows that the MP-1-mediated protection of mice from lethal challenge by live bacteria and LPS was associated with its bactericidal action and inhibition of inflammatory responses by macrophages to both bacteria and LPS (the release of cytokines and reactive oxygen species).

Keywords: Mastoparan-1; Lipopolysaccharide; Sepsis; Macrophage; Respiratory burst; Pro-inflammatory cytokines

Oral flavonoids delay recovery from experimental autoimmune encephalomyelitis in SJL mice by Richard Verbeek; Eric. A.F. van Tol; Johannes M. van Noort (pp. 220-228).

Flavonoids are food components that appear to have potential beneficial health effects. There is a range of in vitro studies supporting the anti-oxidant and anti-inflammatory properties of flavonoids. Previously, we demonstrated that in vitro flavonoids, including luteolin and apigenin, inhibit proliferation and IFN-γ production by murine and human autoimmune T cells. In the present study, we examined the effects of oral flavonoids as well as of curcumin on autoimmune T cell reactivity in mice and on the course of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis.Continuous oral administration of flavonoids significantly affected antigen-specific proliferation and IFN-γ production by lymph node-derived T cells following immunization with an EAE-inducing peptide. Both luteolin and apigenin suppress proliferative responses as they did in vitro, whereas IFN-γ production on the other hand was enhanced. Other flavonoids exerted differential effects on proliferation and IFN-γ production. The effects of flavonoids and curcumin on EAE were assessed using either passive transfer of autoimmune T cells or active disease induction. In passive EAE, flavonoids led to delayed recovery of clinical symptoms rather than to any reduction in disease. In active EAE, the effects were less pronounced but also, in this case, the flavonoid hesperitin delayed recovery. Oral curcumin had overall mild but beneficial effects. Our results indicate that oral flavonoids fail to beneficially influence the course of EAE in mice but, instead, suppress recovery from acute inflammatory damage.

Keywords: Abbreviations; PLP; proteolipid protein; IFN; interferon; MS; multiple sclerosis; EAE; experimental autoimmune encephalomyelitisFlavonoids; Apigenin; Luteolin; T cells; EAE

Emodin induces apoptosis in human lung adenocarcinoma cells through a reactive oxygen species-dependent mitochondrial signaling pathway by Yu-Ting Su; Huei-Ling Chang; Song-Kun Shyue; Shih-Lan Hsu (pp. 229-241).

Emodin, a natural anthraquinone derivative isolated from Rheum palmatum L., has been reported to exhibit anti-cancer effect on several human cancers such as liver cancers and lung cancers. However, the molecular mechanisms of emodin-mediated tumor regression have not been fully defined. In this study, we show that treatment with 50μM emodin resulted in a pronounced release of cytochrome c, activation of caspase-2, -3, and -9, and apoptosis in human lung adenocarcinoma A549 cells. These events were accompanied by the inactivation of ERK and AKT, generation of reactive oxygen species (ROS), disruption of mitochondrial membrane potential (Δ ψ_m), decrease of mitochondrial Bcl-2, and increase of mitochondrial Bax content. Ectopic expression of Bcl-2, or treatment with aurintricarboxylic acid, furosemide or caspase inhibitors markedly blocked emodin-induced apoptosis. Conversely, pharmacologic ERK and AKT inhibition promoted emodin-induced apoptosis. Furthermore, the free radical scavenger ascorbic acid and N-acetylcysteine attenuated emodin-mediated ROS production, ERK and AKT inactivation, mitochondrial dysfunction, Bcl-2/Bax modulation, and apoptosis. Take together, these findings suggest that in A549 cells, emodin-mediated oxidative injury acts as an early and upstream change in the cell death cascade to antagonize cytoprotective ERK and AKT signaling, triggers mitochondrial dysfunction, Bcl-2 and Bax modulation, mitochondrial cytochrome c release, caspase activation, and consequent leading to apoptosis.

Keywords: Abbreviations; Adv; adenovirus; ATA; aurintricarboxylic acid; DAPI; 4′,6-diamindino-2-phenylindole; DCF-DA; 2′,7′-dichlorofluorescensin diacetate; DEVD-AFC; Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin; DiOC; ₆; 3,3-dihexyloxacarbocyanine iodide; Emodin; 1,3,8-trihydroxy-6-methyl anthraquinone; ERK; extracellular signal-regulated protein kinase; HE; dihydroethidine; IETD-AFC; Ile-Glu-Thr-Asp-7-amino-4-trifluoromethyl coumarin; LEHD-AFC; Leu-Glu-His-Asp-7-amino-4-trifluoromethyl coumarin; MAPkinase; mitogen-activated protein kinase; NAC; N; -acetyl-cysteine; PI; ₃; kinase; phosphatidylinositol 3′-kinase; TUNEL; terminal transferase-mediated dUTP-fluorecensin nick end-labeling; U0126; 1,4-Diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene; YVAD-AFC; Tyr-Val-Ala-Asp-7-amino-4-trifluoromethyl coumarin; VDVAD-AFC; Val-Asp-Val-Ala-Asp-7-amino-4-trifluoromethyl coumarin; VEID-AFC; Val-Glu-Ile-Asp-7-amino-4-trifluoromethyl coumarin; z-VDVAD-fmk; z-Val-Asp-Val-Ala-Asp-fluoromethyl ketone; z-DEVD-fmk; z-Asp-Glu-Val-Asp-fluoromethyl ketone; z-IETD-fmk; z-Ile-Glu-Thr-Asp-fluoromethyl ketone; z-LEHD-fmk; z-Leu-Glu-His-Asp-fluoromethyl ketoneEmodin; ROS; Bcl-2 family; Cytochrome; c; Caspase; Apoptosis

Reactive oxygen species-mediated induction of apoptosis by a plant alkaloid 6-methoxydihydrosanguinarine in HepG2 cells by Hu-Quan Yin; Young-Ho Kim; Chang-Kiu Moon; Byung-Hoon Lee (pp. 242-248).

We have found in the previous study that 6-methoxydihydrosanguinarine (6ME), a benzophenanthridine alkaloid isolated from Hylomecon species, may have potential as a chemotherapeutic agent. However, the mechanisms of 6ME-induced cell death have not been investigated. The purpose of the present study was to determine the apoptosis-inducing potential of 6ME in human hepatocarcinoma HepG2 cells and the role of reactive oxygen species in 6ME-induced apoptosis. It can be concluded from the results that 6ME inhibits the growth of HepG2 cells in a concentration- and time-dependent manner (IC₅₀=3.8±0.2μM following 6h incubation). Treatment of HepG2 cells with 6ME resulted in the release of mitochondrial cytochrome c followed by the activation of caspase proteases, and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. 6ME increased the expression of p53 and bax and decreased the expression of bcl-2. The cytotoxic effect of 6ME is mediated by the time-dependent generation of reactive oxygen species. Our results also show that preincubation of HepG2 cells with vitamin C decreased the expression of p53 and bax and inhibited the release of cytochrome c, activation of downstream caspase and the cleavage of poly(ADP-ribose) polymerase, thus inhibiting the apoptosis inducing effect of 6ME.

Keywords: Abbreviations; 6ME; 6-methoxydihydrosanguinarine; ROS; reactive oxygen species; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Ac-DEVD-AMC; N; -acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin; Ac-IETD-AMC; N; -acetyl-Ile-Glu-Thr-Asp-7-amino-4-methylcoumarin; Ac-LEHD-AMC; N; -acetyl-Leu-Glu-His-Asp-7-amino-4-methylcoumarin; PI; propidium iodide; PARP; poly(ADP-ribose) polymerase; DCFH-DA; 2′,7′-dichlorofluorescin diacetate6-Methoxydihydrosanguinarine (6ME); Apoptosis; Reactive oxygen species (ROS); p53/bax; Cytochrome; c; Antioxidant; HepG2

Effect of DHEA on endocrine functions of adipose tissue, the involvement of PPARγ by Joanna Karbowska; Zdzislaw Kochan (pp. 249-257).

Dehydroepiandrosterone (DHEA), an adrenal steroid, is known to decrease body fat. Thus, it may also alter the endocrine functions of adipose tissue. The aim of this study was to determine if administration of DHEA might influence adiponectin gene expression and secretion from adipose tissue. We demonstrate here the inducing effect of exogenously administered DHEA on adiponectin gene expression in epididymal WAT and adiponectin levels in serum of rats fed a DHEA-containing diet (0.6%, w/w) for 2 weeks, accompanied by a reduction in epididymal adipose tissue mass. A corresponding increase in peroxisome proliferator-activated receptor γ (PPARγ) mRNA expression suggests that PPARγ may be involved in the up-regulation of adiponectin gene expression after DHEA treatment. The presented observations indicate that the positive effects of DHEA, which seems to play a protective role against insulin resistance and atherosclerosis, may be in fact indirect and due to up-regulation of adiponectin gene expression and stimulation of adiponectin secretion from adipose tissue.

Keywords: Adiponectin; Adiponectin receptor; Leptin; Dehydroepiandrosterone; PPARγ; Adipose tissue

Involvement of p38 MAP kinase-mediated cytochrome c release on sphingosine-1-phosphate (S1P)- and N-monomethyl-S1P-induced cell death of PC12 cells by Yuko Takashiro; Hiroyuki Nakamura; Yuuki Koide; Atsushi Nishida; Toshihiko Murayama (pp. 258-265).

d- erythro-Sphingosine-1-phosphate (S1P), a sphingolipid metabolite, affects various neuronal functions including cell fate. S1P appears to have contradictory effects in PC12 cells, a neuronal model cell line; neurite retraction and cell survival/differentiation. In the present study, we examined whether S1P induces cell death in undifferentiated PC12 cells. Culture with S1P at 20μM for 4h caused lactate dehydrogenase leakage 24h later. The response was reduced by an inhibitor of caspases and accompanied by the release of cytochrome c and DNA fragmentation. S1P caused the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) within 10min. An inhibitor of p38 MAPK (10μM SB203580) inhibited both the release of cytochrome c and DNA fragmentation induced by S1P. Treatment with nerve growth factor or pertussis toxin (PTX) decreased S1P-induced phosphorylation of p38 MAPK and cell death. These findings suggest that S1P-activated p38 MAPK acts as a death signal upstream of the release of cytochrome c. N-Monomethyl-S1P (MM-S1P), a weak agonist in cells expressing S1P₁ receptors, had marked effects (phosphorylation of p38 MAPK, release of cytochrome c and DNA fragmentation) at lower concentrations than S1P and in a PTX-sensitive manner. These findings show that the activation of S1P receptors by S1P and MM-S1P causes cell death accompanied by DNA fragmentation via the p38 MAPK pathway-mediated release of cytochrome c in PC12 cells. The potential of S1P and MM-S1P to act as agonists of S1P receptors and as intracellular messengers is discussed.

Keywords: Abbreviations; S1P; d; -; erythro; -Sphingosine-1-phosphate; MM-S1P; d; -; erythro; -; N; -Monomethyl-S1P; MAPK; mitogen-activated protein kinase; ERK1/2; extracellular regulated protein kinase 1/2; NGF; nerve growth factor; PTX; pertussis toxin; SB203580; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1; H; -imidazole; LDH; lactate dehydrogenase; ZVAD-fmk; z-Val-Ala-Asp(OMe)-fluoromethylketone; DMEM; Dulbecco's modified Eagle's medium; FBS; fetal bovine serum; TUNEL; TdT-mediated dUTP nick end labelingSphingosine-1-phosphate (S1P); N; -Monomethyl-S1P; p38 MAPK; Cytochrome; c; Apoptosis; PC12 cells

Synthesis of pyridoxal phosphate derivatives with antagonist activity at the P2Y₁₃ receptor by Yong-Chul Kim; Jung-Sun Lee; Katrin Sak; Frederic Marteau; Liaman Mamedova; Jean-Marie Boeynaems; Kenneth A. Jacobson (pp. 266-274).

We have synthesized a series of derivatives of the known P2 receptor antagonist PPADS (pyridoxal-5′-phosphate-6-azo-phenyl-2,4-disulfonate) and examined their ability to inhibit functional activity of the recombinant human P2Y₁₃ nucleotide receptor expressed in 1321N1 human astrocytoma cells co-expressing Gα₁₆ protein (AG32). Analogues of PPADS modified through substitution of the phenylazo ring, including halo and nitro substitution, and 5′-alkyl phosphonate analogues were synthesized and tested. A 6-benzyl-5′-methyl phosphonate analogue was prepared to examine the effect of stable replacement of the azo linkage. The highest antagonistic potency was observed for 6-(3-nitrophenylazo) derivatives of pyridoxal-5′-phosphate. The 2-chloro-5-nitro analogue (MRS 2211) and 4-chloro-3-nitro analogue (MRS 2603) inhibited ADP (100nM)-induced inositol trisphosphate (IP₃) formation with pIC₅₀ values of 5.97 and 6.18, respectively, being 45- and 74-fold more potent than PPADS. The antagonism of MRS 2211 was competitive with a pA₂ value of 6.3. MRS2211 and MRS2603 inhibited phospholipase C (PLC) responses to 30nM 2-methylthio-ADP in human P2Y₁ receptor-mediated 1321N1 astrocytoma cells with IC₅₀ values of >10 and 0.245μM, respectively. Both analogues were inactive (IC₅₀>10μM) as antagonists of human P2Y₁₂ receptor-mediated PLC responses in 1321N1 astrocytoma cells. Thus, MRS2211 displayed >20-fold selectivity as antagonist of the P2Y₁₃ receptor in comparison to P2Y₁ and P2Y₁₂ receptors, while MRS2603 antagonized both P2Y₁ and P2Y₁₃ receptors.

Keywords: Abbreviations; AG32; human astrocytoma 1321N1 cells co-expressing Gα; ₁₆; protein; AR-C67085MX; 2-(propylthio)-β,γ-dichloromethylene-ATP; CHO; Chinese Hamster ovary; DMEM; Dulbecco's Modified Eagle's Medium; FBS; fetal bovine serum; HRMS; high-resolution mass spectrometry; IP; ₃; inositol trisphosphate; KRH; Krebs–Ringer–HEPES; MRS 2211; pyridoxal-5′-phosphate-6-azo-(2-chloro-5-nitrophenyl)-2,4-disulfonate; MRS 2603; pyridoxal-5′-phosphate-6-azo-(4-chloro-3-nitrophenyl)-2,4-disulfonate; PLC; phospholipase C; PPADS; pyridoxal-5′-phosphate-6-azo-phenyl-2,4-disulfonatePPADS (pyridoxal-5′-phosphate-6-azo-phenyl-2,4-disulfonate); Pyridoxal phosphate derivatives; Adenine nucleotides; P2Y; ₁₃; receptor; Inositol trisphosphate; Purines

Reactive oxygen species generation and its role in the differential cytotoxicity of the arylhydroxylamine metabolites of sulfamethoxazole and dapsone in normal human epidermal keratinocytes by Piyush M. Vyas; Sanjoy Roychowdhury; Patrick M. Woster; Craig K. Svensson (pp. 275-286).

Cutaneous drug reactions (CDR) are responsible for numerous minor to life-threatening complications. Though the exact mechanism for CDR is not completely understood, evidence suggests that bioactivation of drugs to reactive oxygen or nitrogen species is an important factor in the initiation of these reactions. Several CDR-inducing drugs having an arylamine functional group, such as sulfamethoxazole (SMX) and dapsone (DDS), undergo bioactivation to reactive arylhydroxylamine metabolites. These metabolites can generate cellular oxidative stress by forming reactive oxygen species (ROS). Several studies have demonstrated a higher cytotoxicity with DDS hydroxylamine (DDS-NOH) compared to SMX hydroxylamine (SMX-NOH). To investigate the role of differential ROS generation in the higher cytotoxicity of DDS-NOH, hydroxylamine metabolites of SMX and DDS were synthesized and ROS formation by these metabolites determined. DDS-NOH and its analogues/metabolites consistently resulted in higher ROS formation as compared to SMX-NOH. However, comparison of the ROS generation and cytotoxicity of a series of arylhydroxylamine analogues of DDS did not support a simple correlation between ROS generation and cell death. Numerous ROS scavengers were found to reduce metabolite-induced ROS formation, with differences in the potency between the agents. The decrease in DDS-NOH-induced ROS generation in NHEK with ascorbic acid, N-acetylcysteine, Trolox, and melatonin was 87, 86, 44, and 16%, respectively. Similarly, the cytotoxicity and adduct formation of DDS-NOH in NHEK was reduced in the presence of ascorbic acid. In summary, these studies show that arylhydroxylamine metabolites of SMX/DDS induce ROS generation in NHEK, though such generation is not directly related to cytotoxicity.

Tangutorine induces p21 expression and abnormal mitosis in human colon cancer HT-29 cells by B.P.L. Liu; E.Y.Y. Chong; F.W.K. Cheung; Jin-Ao Duan; Chun-Tao Che; W.K. Liu (pp. 287-299).

A novel β-carboline alkaloid, tangutorine (benz[ f]indolo[2,3- a]quinolizidine) was isolated from the leaves of Nitraria tangutorum L. [Duan JA, Williams ID, Che CT, Zhou RH, Zhao RH, Tangutorine: a novel β-carboline alkaloid from Nitraria tangutorum. Tetrahedron Lett 1999;40:2593–6], and its unique structural characters led us to initiate a study of its potential anti-proliferation activity. The in vitro treatment with low doses of tangutorine slightly stimulated the proliferation of human colon cancer HT29 cells until at concentrations higher than 6.25μg/ml when the cell numbers, cellular MTT reduction, and cell proliferation by³H-thymidine incorporation decreased in a dose-dependent manner (IC₅₀=15μg/ml=48μM). Morphological studies of cells by fluorescence and electron microscopy did not show features for apoptosis but only large vacuoles, swollen mitochondria and dense cytoskeletal filaments bunching in the cytoplasm. Immunoblotting analysis revealed a dramatic induction of cyclin kinase inhibitor p21 as well as an inhibition of topoisomerase II expression at 25μg/ml tangutorine, thereby impeding cell progression from S to G2/M phase. Cells accumulated at G1 phase of the cell cycle at concentrations ≥50μg/ml tangutorine. Interestingly, some cells escaped from prolonged growth arrest without cell division and resulted in binucleated and polyploid G1 cells. Taken all results together, tangutorine induced a p21 suppression of all cyclins and their associated kinases, such as the topoisomerase II, and thus inhibited normal DNA replication and mitosis.

Keywords: Tangutorine; Cyclin kinase inhibitor p21; HT29 cells; Growth arrest; Abnormal mitosis

Inhibition of osteoclast differentiation by polycyclic aryl hydrocarbons is dependent on cell density and RANKL concentration by I. Voronov; J.N.M. Heersche; R.F. Casper; H.C. Tenenbaum; M.F. Manolson (pp. 300-307).

We investigated the effect of representative polycyclic aryl hydrocarbons (PAHs), benzo[ a]pyrene (BaP), and 7,12-dimethylbenz[ a]anthracene (DMBA) on osteoclast differentiation and function by using dispersed cancellous bone derived rabbit osteoclasts and the RAW264.7 cells. These cells differentiate into osteoclasts when exposed to receptor activator of NF-κB ligand (RANKL). The rabbit osteoclasts were exposed to 10⁻⁶ to 10⁻⁹M BaP or DMBA and the tartrate-resistant acid phosphatase (TRAP)-positive cells were counted. The effect of PAHs on osteoclast differentiation in dispersed rabbit osteoclast-containing stromal cell populations was cell density dependent, suggesting that the cell density of stromal cells, osteoclast precursors, and/or mature osteoclasts are factors regulating the effect of PAHs. To investigate the direct effect of BaP on osteoclast differentiation, RAW264.7 cells were exposed to 10⁻⁵ to 10⁻⁶M BaP. Treatment of RAW264.7 cells cultured with 25ng/ml soluble RANKL and 10⁻⁵M BaP for 5 days decreased osteoclast differentiation, TRAP activity levels, and resorption of bone-like substrata. The inhibition was prevented by 10⁻⁶ to 10⁻⁷M resveratrol, an aryl hydrocarbon receptor (AhR) antagonist, and by higher concentrations of RANKL. To investigate the ability of RANKL to reverse BaP-mediated inhibition, gene expression was determined by RT-PCR. Cytochrome P450 1B1 (CYP1B1) mRNA, one of the genes activated by BaP, was present only in the groups exposed to BaP; the levels of CYP1B1 mRNA decreased in the presence of increasing concentrations of RANKL. These results suggest that the inhibitory effects of PAHs on osteoclastogenesis are direct and likely involve interaction of the RANKL and PAH signaling pathways.

Keywords: Osteoclast; RANKL; Benzo[; a; ]pyrene; CYP1B1; TRAP

Functional analysis of the Ala67Thr polymorphism in agouti related protein associated with anorexia nervosa and leanness by Corine E. de Rijke; Pilgrim J. Jackson; Keith M. Garner; Rea J. van Rozen; Nick R. Douglas; Martien J.H. Kas; Glenn L. Millhauser; Roger A.H. Adan (pp. 308-316).

AgRP is a neuropeptide that stimulates food intake through inhibition of central melanocortin receptors (MCRs). In humans, the non-conservative amino acid substitution Alanine (Ala) 67 Threonine (Thr) has been associated with Anorexia Nervosa and with leanness. In the present study, the cellular distribution, processing and in vitro and in vivo activities of Ala67 and Thr67 AgRP were investigated. Western blots of media and lysates of BHK cells stably transfected with Ala67 or Thr67 expression constructs showed identical AgRP bands. Both Ala67 and Thr67 AgRP colocalised with the Golgi apparatus, but not with the ER or lysosomes when expressed in Att20 D16V cells. Also, no differences were observed between the potencies of bacterially expressed Ala67 and Thr67 AgRP to stimulate MC4R in a reporter gene assay or inhibit food intake in rats. Taken together, no evidence was found for a functional defect of Thr67 AgRP related to MC4R interactions.

Keywords: Agouti related protein; Melanocortin receptor; Polymorphism; Recombinant expression; Weight regulation; Anorexia

Synergistic effects of hydrogen peroxide and ethanol on cell viability loss in PC12 cells by increase in mitochondrial permeability transition by Chung Soo Lee; Yun Jeong Kim; Hyun Hee Ko; Eun Sook Han (pp. 317-325).

The promoting effect of ethanol against the cytotoxicity of hydrogen peroxide (H₂O₂) in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with H₂O₂ resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. In PC12 cells and dopaminergic neuroblastoma SH-SY5Y cells, the promoting effect of ethanol on the H₂O₂-induced cell death was increased with exposure time. Ethanol promoted the nuclear damage, change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents due to H₂O₂ in PC12 cells. Catalase, carboxy-PTIO, Mn-TBAP, N-acetylcysteine, cyclosporin A and trifluoperazine inhibited the H₂O₂ and ethanol-induced mitochondrial dysfunction and cell injury. The results show that the ethanol treatment promotes the cytotoxicity of H₂O₂ against PC12 cells. Ethanol may enhance the H₂O₂-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of ROS and depletion of GSH. The findings suggest that ethanol as a promoting agent for the formation of mitochondrial permeability transition may enhance the neuronal cell injury caused by oxidants.

Keywords: Abbreviations; carboxy-PTIO; 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; DCFH; ₂; -DA; 2′,7′-dichlorofluorescin diacetate; DiOC; ₆; (3); 3,3′-dihexyloxacarbocyanine iodide; DTNB; 5,5′-dithio-bis-(2-nitrobenzoic acid); Mn-TBAP; Mn(III) tetrakis(4-benzoic acid)porphyrin chloride; MPP; ⁺; 1-methyl-4-phenylpyridinium; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PMSF; phenylmethylsulfonylfluoride; ROS; reactive oxygen speciesHydrogen peroxide; Ethanol; PC12 cells; Mitochondrial membrane permeability; Cell injury

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Biochemical Pharmacology (v.70, #2)

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Biochemical Pharmacology (v.70, #2)