Check out our New Publishers' Select for Free Articles

Biochemical Pharmacology (v.75, #5)

Editorial Advisory Board (pp. iii).

Mood disorders: Regulation by metabotropic glutamate receptors by Andrzej Pilc; Shigeyuki Chaki; Gabriel Nowak; Jeffrey M. Witkin (pp. 997-1006).

Medicinal therapies for mood disorders neither fully serve the efficacy needs of patients nor are they free of side-effect issues. Although monoamine-based therapies are the primary current treatment approaches, both preclinical and clinical findings have implicated the excitatory neurotransmitter glutamate in the pathogenesis of major depressive disorders. The present commentary focuses on the metabotropic glutamate receptors and their relationship to mood disorders. Metabotropic glutamate (mGlu) receptors regulate glutamate transmission by altering the release of neurotransmitter and/or modulating the post-synaptic responses to glutamate. Convergent biochemical, pharmacological, behavioral, and clinical data will be reviewed that establish glutamatergic neurotransmission via mGlu receptors as a biologically relevant process in the regulation of mood and that these receptors may serve as novel targets for the discovery of small molecule modulators with unique antidepressant properties. Specifically, compounds that antagonize mGlu2, mGlu3, and/or mGlu5 receptors (e.g. LY341495, MGS0039, MPEP, MTEP) exhibit biochemical effects indicative of antidepressant effects as well as in vivo activity in animal models predictive of antidepressant efficacy. Both preclinical and clinical data have previously been presented to define NMDA and AMPA receptors as important targets for the modulation of major depression. In the present review, we present a model suggesting how the interplay of glutamate at the mGlu and at the ionotropic AMPA and NMDA receptors might account for the antidepressant-like effects of glutamatergic- and monoaminergic-based drugs affecting mood in patients. The current data lead to the hypothesis that mGlu-based compounds and conventional antidepressants impact a network of interactive effects that converge upon a down regulation of NMDA receptor function and an enhancement in AMPA receptor signaling.

Keywords: Abbreviations; ACPD; 1-aminocyclopentane-; trans; -1,3; R; -dicarboxylic acid; ACPT-I; (1; S; ,3; R; ,4; S; )-1-aminocyclo-pentane-1,3,4-tricarboxylic acid; AMN082; N; ,; N; ′-dibenzyhydryl-ethane-1,2-diamine dihydrochloride; AMPA; α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; BDNF; brain-derived neurotrophic factor; l; -CCG-I; (2; S; ,1′; S; ,2′; S; )-2-(carboxycyclopropyl)glycine; CPPG; (; R; ,; S; )-alpha-cyclopropyl-4-phosphonophenyl glycine; CNS; central nervous system; DCG-IV; ((2; S; ,2′; R; ,3′; R; )-2-(2′,3′-dicarboxycyclopropyl)glycine); ECT; electroconvulsive treatment; EMQMCM; (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methanesulfonate; FST; forced swim test; iGlu; ionotropic glutamate; LY341495; 9; H; -xanthene-9-propanoic acid α-amino-α-[(1S,2; S; )-2-carboxycyclopropyl]-(α; S; )-(9CI); mGlu; metabotropic glutamate; MGS0039; 2-amino-3-[(3,4-dichlorophenyl)methoxy]-6-fluoro-, (1; R; ,2; R; ,3; R; ,5; R; ,6; R; )-(9CI); MPEP; 2-methyl-6-(phenylethynyl)pyridine; MTEP; 3-[(2-methyl-1,3-thiazol-4-ylethynyl]pyridine; NBQX; 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(; f; )quinoxaline; PHCCC; N; -phenyl-7-(hydroxyimino)cyclopropa[; b; ]chromen-1acarboxamide; (; R; ,; S; )-PPG; (; R; ,; S; )-4-phosphonophenylglycine; SSRI; selective serotonin reuptake inhibitor; TST; tail suspension testMetabotropic glutamate; mGlu receptors; Depression; LY341495; MGS0039; MPEP; MTEP

Pharmacological regulation of factor XII activation may be a new target to control pathological coagulation by Inger Schousboe (pp. 1007-1013).

FXII was identified 50 years ago as a coagulation protein in the intrinsic pathway of blood coagulation as FXII deficient patients had marked prolongation of the in vitro surface-activated coagulation time. However, series of investigations have convincingly shown that FXII has no role in normal hemostasis. Recently, experimentally induced thrombosis in factor XII-knockout mice has provided evidence that factor XII (FXII) deficient mice are protected against ischemic brain injury after obstructive clot formation. Based on these experiments it has, therefore, been suggested, that blocking of FXII could be a unique target to prevent obstructive clot formation in arterial thrombosis without side effect of increased bleeding. FXII deficiency has, however, not convincingly been shown to protect against arterial thrombosis in humans. The target mentioned above may either be an inhibition of FXII activation or an inhibition of its proteolytic activity. FXII is a zymogen of the proteolytic enzyme, FXIIa, the substrates of which are factor XI and prekallikrein. Thus, FXIIa is not only involved in the activation of the coagulation system, but is also associated with the kallikrein/kinin system. The activation of the latter is deeply involved in inflammation and pain sensation. Furthermore, FXIIa binds to endothelial cells and to the extracellular matrix, indicating a role in vascular repair. Therefore, a complete evaluation of all these properties of FXII and FXIIa has to be considered when formulating a strategy for blocking FXII activation.

Keywords: Factor XII; Pathological thrombosis; Contact activation; Deficiency

Synthesis and pharmacological evaluation of the novel pseudo-symmetrical tamoxifen derivatives as anti-tumor agents by Isamu Shiina; Yoshiyuki Sano; Kenya Nakata; Takaaki Kikuchi; Akane Sasaki; Masahiko Ikekita; Yukitoshi Nagahara; Yoshimune Hasome; Takao Yamori; Kanami Yamazaki (pp. 1014-1026).

Four pseudo-symmetrical tamoxifen derivatives, RID-B (13), RID-C (14), RID-D (15), and bis(dimethylaminophenetole) (16), were synthesized via the novel three-component coupling reaction, and the structure–activity relationships of these pseudo-symmetrical tamoxifen derivatives were examined. It was discovered that13 and16 strongly inhibit the viability of the HL-60 human acute promyelocytic leukemia cell line, whereas14 possesses a medium activity against the same cell line and15 has no effect on the cell viability. The global anti-tumor activity of13–16 against a variety of human cancer cells was assessed using a panel of 39 human cancer cell lines (JFCR 39), and it was shown that RID-B (13) strongly inhibited the growth of several cancer cell lines at concentrations of less than 1μM (at 0.38μM for SF-539 [central nervous system], at 0.58μM for HT-29 [colon], at 0.20μM for DMS114 [lung], at 0.21μM for LOX-IMVI [melanoma], and at 0.23μM for MKN74 [stomach]).

Keywords: Cancer; Tamoxifen; Symmetrical derivatives; HL-60; Panel of cancer cell lines; JFCR 39

Small-molecule inhibition of Aurora kinases triggers spindle checkpoint-independent apoptosis in cancer cells by Lei Sun; Dengwen Li; Xin Dong; Haiyang Yu; Jin-Tang Dong; Chuanmao Zhang; Xianyu Lu; Jun Zhou (pp. 1027-1034).

Aurora kinases are key regulators of mitotic progression and have also been implicated in tumorigenesis. Small molecules that inhibit Aurora kinases have shown impressive anticancer activity in preclinical studies and are currently under clinical evaluation. In this study, our data show that suppression of Aurora activity with a specific inhibitor prevents the proliferation of breast cancer cells. Molecular modeling studies indicate that the Aurora inhibitor suppresses Aurora activity by competitive displacement of ATP. Mechanistically, the Aurora inhibitor causes the accumulation of multinucleated cells, leading to profound apoptosis in the absence of caspase-3 activity. Further studies show that the sensitivity of cancer cells to the Aurora inhibitor is independent of the spindle checkpoint. In addition, the Aurora inhibitor acts synergistically with the vinca alkaloids but not with the taxanes in inhibiting cell proliferation and inducing apoptosis. These results suggest that Aurora inhibitors might be effective in spindle checkpoint-defective cancer cells and a combination of Aurora inhibitors with the vinca alkaloids is a promising approach for cancer chemotherapy.

Keywords: Aurora kinases; Breast cancer; Spindle checkpoint; Mitosis; Apoptosis

Synergic antiproliferative and antiangiogenic effects of EGFR and mTor inhibitors on pancreatic cancer cells by Amalia Azzariti; Letizia Porcelli; Giuliana Gatti; Angelo Nicolin; Angelo Paradiso (pp. 1035-1044).

The in vitro efficacy of both EGFR inhibitor gefitinib and mTor inhibitor rapamycin, either administrated alone or in different combination schedules, was analysed in four pancreas cancer cell lines. Both drugs were found to induce cell growth inhibition, apoptosis as well as a slight but stable accumulation of cells in the G0/G1 phase. In all cell lines, neither gefitinib nor rapamycin affected EGFR and the expression of its downstream effectors. By contrast, gefitinib inhibited in a fast and completely way p-EGFR and partially p-Akt while a 3 days-rapamycin exposure resulted in the inhibition of the expression of both mTor and p70S6K. Moreover, after early stimulation, the mTor inhibitor produced a progressive, and almost complete inhibition of p-Akt. The analysis of combined gefitinib and rapamycin administration showed a clear schedule-dependent activity which turned out to be synergic only when gefitinib was given before rapamycin. This synergism seemed to depend on increase of both p-Akt and p70S6K inhibition, the greater the induction of apoptosis, the higher the decrease in cell cycle rate. Moreover, the antiangiogenic activity of the two drugs given in combination was demonstrated by a strong reduction of VEGF release which turned out to be more pronounced in the synergic schedule, and HIF-1α inhibition-independent. Our results suggest that the schedule of gefitinib followed by rapamycin, acting at different levels of the EGFR cellular pathway, could induce antitumor and antiangiogenic effects of clinical interest in the pancreas cancer model.

Keywords: Pancreas cancer model; Gefitinib; Rapamycin; EGFR; mTor

Metabolic transformation has a profound effect on anti-inflammatory activity of flavonoids such as quercetin: Lack of association between antioxidant and lipoxygenase inhibitory activity by Wai Mun Loke; Julie M. Proudfoot; Scott Stewart; Allan J. McKinley; Paul W. Needs; Paul A. Kroon; Jonathan M. Hodgson; Kevin D. Croft (pp. 1045-1053).

Dietary flavonoids are thought to have health benefits possibly due to antioxidant and anti-inflammatory properties. Many previous in vitro studies examining the bioactivity of flavonoids have failed to consider the effects of metabolic transformation on flavonoid activity. In this study we examined the effect of quercetin and its major metabolites on the production of pro-inflammatory eicosanoids by human leukocytes. Studies comparing free radical scavenging, antioxidant activity and eicosanoid production demonstrate that there are different structural requirements for antioxidant and anti-inflammatory activity. We also investigated the effect of metabolic transformation on flavonoid bioactivity by comparing the activity of quercetin and its major metabolites to inhibit inflammatory eicosanoid production from human leukocytes. Quercetin was a potent inhibitor of leukotriene B₄ formation in leukocytes (IC₅₀∼2μM), and its activity was dependent on specific structural features, particularly the 2,3-double bond of the C-ring. Functionalisation of the 3′-OH group with either methyl or sulfate reduced inhibitory activity up to 50% while a glucuronide substituent at the 3-OH effectively removed the LTB₄ inhibitory activity. The major quercetin metabolite quercetin-3′- O-sulfate retained considerable lipoxygenase inhibitory activity (IC₅₀∼7μM) while quercetin-3- O-glucuronide maintained antioxidant activity but had no lipoxygenase inhibitory activity at physiological concentrations. In conclusion, we have found that structural modification of quercetin due to metabolic transformation had a profound effect on bioactivity, and that the structural features required for antioxidant activity of quercetin and related flavonoids were unrelated to those required for inhibition of inflammatory eicosanoids.

Keywords: Antioxidant; Anti-inflammatory; Metabolic transformation; Flavonoid structure; Leukotriene B; ₄; Prostaglandin E; ₂

SK-126, a synthetic compound, regulates the production of inflammatory cytokines induced by LPS in antigen-presenting cells by Kyeongah Kang; Hyeree Kim; Keun Il Kim; Young Yang; Do-Young Yoon; Joo-Hyon Kim; Je-Ho Ryu; Eun-Jung Noh; Sun-Duck Jeon; Jong-Seok Lim (pp. 1054-1064).

A variety of mediators released by immune cells triggers or enhances specific aspects of the inflammatory response. Dendritic cells (DCs) play an essential role in the innate immune system by shaping the adaptive immune responses and by controlling the production of cytokines in response to inflammatory stimuli. In the present study, we investigated whether SK-126, a pyridine derivative based on gentianine originated from a natural product, can affect the LPS-induced inflammatory cytokine production in DC. Interestingly, treatment of mouse bone marrow-derived dendritic cells (BMDCs) and the murine dendritic cell line, DC 2.4, with SK-126 completely suppressed LPS-induced TNF-α expression at both transcriptional and protein levels. In contrast to TNF-α, SK-126 enhanced IL-10 expression at both transcriptional and protein levels. To determine signaling pathways involved in the regulation of inflammatory cytokines, we examined the involvement of MAPK and the transcription factor, NF-κB. SK-126 enhanced ERK1/2 and p38 activation following LPS stimulation, but it did not induce phosphorylation of SAPK/JNK and NF-κB. Also, STAT3 phosphorylation after LPS stimulation was increased by SK-126 to a large extent. Using specific inhibitors, we confirmed that SK-126 has dual effects in which it suppresses TNF-α production and enhances IL-10 production via the up-regulation of ERK1/2 and p38. Finally, LPS-induced inflammatory responses such as TNF-α production in vivo were significantly reduced by treatment with SK-126. Therefore, our findings suggest that SK-126 may be a useful drug candidate to treat inflammatory diseases in which pro- or anti-inflammatory cytokines play a significant role in their pathogenesis.

Keywords: Abbreviations; DCs; dendritic cells; LPS; lipopolysaccharide; TNF; tumor necrosis factor; IL-10; interleukin-10; NF-κB; nuclear factor-κB; SAPK; stress-activated protein kinase; JNK; c-Jun N-terminal kinase; MAPK; mitogen-activated protein kinaseInflammation; LPS; TNF-α; IL-10; MAPK; Septic shock

Priming by lipopolysaccharide exaggerates acute lung injury and mortality in responses to peptidoglycan through up-regulation of Toll-like receptor-2 expression in mice by Naoyuki Matsuda; Hiromi Yamazaki; Ken-ichi Takano; Kazuhiro Matsui; Yasuo Takano; Osamu Kemmotsu; Yuichi Hattori (pp. 1065-1075).

Invasive infection mixed with Gram-positive and Gram-negative bacteria often results in severe sepsis and septic shock, the prognosis of which is extremely poor and the mortality is high. Here, we hypothesized that lipopolysaccharide (LPS) from Gram-negative bacteria may exert a priming effect on the innate immune response to peptidoglycan (PepG) from Gram-positive bacteria and if so, examined the molecular mechanism of this priming. We found that mice who underwent intratracheal instillation with PepG (5mg/kg) following prior administration of LPS (5mg/kg) had a marked decline in survival as compared with the animals given each bacterial cell wall component alone. Furthermore, blood gas exchange impairment and pulmonary vascular hyperpermeability were greatly enhanced in mice given PepG after LPS stimulation, indicating a severe development of acute lung injury. LPS significantly up-regulated the expression levels of Toll-like receptor (TLR)-2 mRNA and protein in mouse lungs. Translocation of TLR-2 to the membranes was also increased by LPS stimulation. This was supported by immunohistochemical examination showing that TLR-2 expression was changed from the cytoplasm to the luminal surface of bronchiolar epithelial cells following LPS stimulation. We also demonstrated an LPS-induced increase in TLR-2 mRNA expression in type-II pneumocytes by reverse transcription-polymerase chain reaction following laser-assisted microdissection. In vivo transfection of nuclear factor-κB (NF-κB) oligonucleotides strongly prevented the up-regulation of TLR-2 after LPS stimulation at pulmonary cellular and tissue levels. We conclude that the priming effect of LPS on PepG-induced lung injury and death is preceded by NF-κB-mediated up-regulation of TLR-2.

Keywords: Abbreviations; TLR; Toll-like receptor; LPS; lipopolysaccharide; PepG; peptidoglycan; NF-κB; nuclear factor-κB; ODN; oligodeoxynucleotide; GAPDH; glyceraldehydes-3-phosphate dihydrogenase; PBS; phosphate-buffered saline; RT-PCR; reverse transcription-polymerase chain reaction; IRAK; interleukin-1 receptor-associated kinase; TRAF; tumor necrosis factor-associated factor; IKK; inhibitor of nuclear factor-κB kinasePeptidoglycan; Lipopolysaccharide; Toll-like receptors; Acute lung injury; Nuclear factor-κB; Transcription factor decoy; Sepsis

A novel gender-related difference in the constitutive expression of hepatic cytochrome P4501A subfamily enzymes in Meishan pigs by Misaki Kojima; Masashi Sekimoto; Masakuni Degawa (pp. 1076-1082).

Constitutive expression levels of hepatic CYP1A subfamily enzymes, CYP1A1 and CYP1A2, in male and female Meishan pigs were examined at levels of the mRNA, protein, and enzyme activity. In mature (5-month-old) pigs, levels of hepatic CYP1A1 and CYP1A2 mRNAs, as determined by RT-PCR, were much higher in females than in males, but those of castrated male pigs were equivalent to female pigs. The gender-related differences in the levels of CYP1A mRNAs closely correlated with those of the corresponding apoproteins determined by Western blotting. Hepatic enzyme activities not only for the O-dealkylation of ethoxyresorufin and methoxyresorufin (typical substrates for CYP1A1 and CYP1A2, respectively) but also for the mutagenic activation of benzo[ a]pyrene and 2-amino-6-methyl-dipyrido[1,2- a; 3′,2′- d]imidazole (typical substrates for CYP1A1 and CYP1A2, respectively) were also much greater in female and castrated male pigs than in male pigs. In immature (1-month-old) pigs, no such gender-related differences were observed, and their gene expression levels of the CYP1A subfamily enzymes were almost the same as those of mature female pigs. Furthermore, treatment of immature pigs with testosterone resulted in a drastic decrease in the levels of the CYP1A1 and CYP1A2 mRNAs in both sexes. The present findings demonstrate a gender-related difference in the constitutive expression of hepatic CYP1A subfamily enzymes in Meishan pigs and further indicate that androgen down-regulates the constitutive gene expression of the enzymes.

Keywords: Cytochrome P450; CYP1A1; CYP1A2; Sex-difference; Liver; Meishan pigs

Inhibition of glioblastoma growth and angiogenesis by gambogic acid: An in vitro and in vivo study by Lei Qiang; Yong Yang; Qi-Dong You; Yan-Jun Ma; Lan Yang; Fei-Fei Nie; Hong-Yan Gu; Li Zhao; Na Lu; Qi Qi; Wei Liu; Xiao-Tang Wang; Qing-Long Guo (pp. 1083-1092).

Gambogic acid (GA) is the major active ingredient of gamboge, a brownish to orange resin exuded from Garcinia hanburryi tree in Southeast Asia. The present study aims to demonstrate that gambogic acid (GA) has potent anticancer activity for glioblastoma by in vitro and in vivo study. Rat brain microvascular endothelial cells (rBMEC) were used as an in vitro model of the blood–brain barrier (BBB). To reveal an involvement of the intrinsic mitochondrial pathway of apoptosis, the mitochondrial membrane potential and the western blot evaluation of Bax, Bcl-2, Caspase-3, caspase-9 and cytochrome c released from mitochondria were performed. Angiogenesis was detected by CD31 immunochemical study. The results showed that the uptake of GA by rBMEC was time-dependent, which indicated that it could pass BBB and represent a possible new target in glioma therapy. GA could cause apoptosis of rat C6 glioma cells in vitro in a concentration-dependent manner by triggering the intrinsic mitochondrial pathway of apoptosis. In vivo study also revealed that i.v. injection of GA once a day for two weeks could significantly reduce tumor volumes by antiangiogenesis and apoptotic induction of glioma cells. Collectively, the current data indicated that GA may be of potential use in treatment of glioblastoma by apoptotic induction and antiangiogenic effects.

Keywords: JEL classification; NeuropharmacologyGambogic acid; Apoptosis; Glioblastoma; Angiogenesis; Caspase; Mitochondrion

Peripheral elevation of IGF-1 fails to alter Aβ clearance in multiple in vivo models by Thomas A. Lanz; Christopher T. Salatto; Anthony R. Semproni; Michael Marconi; Tracy M. Brown; Karl E.G. Richter; Kari Schmidt; Frederick R. Nelson; Joel B. Schachter (pp. 1093-1103).

Increasing beta-amyloid (Aβ) clearance may alter the course of Alzheimer's disease progression and attenuate amyloid plaque pathology. Insulin-like growth factor I (IGF-1) augmentation has been suggested to increase Aβ clearance by facilitating transport of Aβ out of the brain. The availability of safe agents that increase IGF-1 levels therefore makes IGF-1 elevation an attractive target for disease modifying therapy in AD. The present series of studies sought to replicate published paradigms in which peripheral IGF-1 administration lowered brain Aβ acutely, with reduction in plaque pathology after chronic treatment. Thus Aβ levels were measured in several animal models following treatments that elevated IGF-1. Administration of IGF-1 to young or old rats for up to 3 days had no effect on Aβ levels in brain, CSF, or plasma. In adult beagles, 4 days of dosing with the growth hormone secretagogue, CP-424391, doubled baseline plasma IGF-1 levels, yet failed to alter CSF or plasma Aβ. 5-day treatment of young Tg2576 mice with IGF-1 produced robust elevations of IGF-1 levels in plasma, but no effects on Aβ were detected in brain, CSF, or plasma. Finally, 11-month-old Tg2576 mice were implanted with subcutaneous minipumps delivering IGF-1 for 1 month. No significant changes in Aβ (by ELISA or Western blot), plaque pathology, or phospho-tau epitopes were detected. These results do not demonstrate acute or chronic actions of peripherally administered IGF-1 on Aβ levels or the phosphorylation state of tau and therefore do not suggest any disease-modifying benefits of IGF-1 restorative therapy for AD through these mechanisms.

Keywords: Aβ; Alzheimer's disease; IGF-1; Clearance; Tg2576; Tau

Melittin exhibits necrotic cytotoxicity in gastrointestinal cells which is attenuated by cholesterol by Sam Maher; Siobhán McClean (pp. 1104-1114).

Melittin, a cationic antimicrobial peptide isolated from the venom of Apis mellifera, has shown potential as a permeability enhancer, transiently increasing intestinal permeability and enhancing the absorption of paracellular markers. Although it is cytotoxic to eukaryotic cells, its cytotoxicity is significantly lower in polarised epithelia compared to non-polarised cells. The aim of this study was to explore the mechanism of melittin cytotoxicity in gastrointestinal cells and to determine whether cytotoxicity was mediated by a necrotic or an apoptotic pathway. The role of cholesterol in melittin cytotoxicity was also examined. Using four distinct assays for apoptosis, phosphatidylserine translocation, caspase activation, DNA ladder formation and cell cycle analysis, no evidence of apoptotic pathway for cell death was observed with any of these approaches. It can therefore be concluded that cytotoxicity was likely to be mediated by necrosis in gastrointestinal epithelial cells. However, at low concentrations of melittin (<1μM), BRDU uptake was enhanced, demonstrating proliferative effects of melittin at sub-lethal concentrations. Furthermore, melittin cytotoxicity was further enhanced by depletion of cholesterol, using methyl-β-cyclodextrin, indicating that cholesterol depleting agents could be contradictory to its potential as an enhancer. Overall, although melittin appears to stimulate necrosis, with careful dosage selection the peptide could be considered for the oral delivery of poorly bioavailable drugs.

Keywords: Melittin; Necrosis; Paracellular permeability enhancer; Cholesterol; Cytotoxicity; Antimicrobial peptide; Intestinal epithelial cells

Differences in motilin receptor desensitization after stimulation with motilin or motilides are due to alternative receptor trafficking by Anna Mitselos; Pieter Vanden Berghe; Theo L. Peeters; Inge Depoortere (pp. 1115-1128).

The motilin receptor (MTLR) is an important therapeutic target for treatment of hypomotility disorders. The negative outcome in clinical trials with the motilin agonist, ABT-229, indicated that desensitization may limit the therapeutic usefulness of motilides. We therefore compared the mechanisms involved in the intracellular trafficking of the MTLR after stimulation with motilin, erythromycin-A (EM-A) or ABT-229.Desensitization was studied by measuring changes in Ca²⁺ rises and by receptor binding studies in CHO cells co-expressing the Ca²⁺ indicator apoaequorin and the MTLR, C-terminally tagged with EGFP. Receptor phosphorylation was studied by immunoprecipitation. MTLR-EGFP trafficking to organelles and translocation of β-arrestins were visualized by fluorescence microscopy.Agonist-induced desensitization of the MTLR was due to receptor internalization with potencies (p-int₅₀) in the order of: ABT-229 (8.3)>motilin (7.86)>EM-A (4.77) but with no differences in the internalization kinetics ( t_1/2: ∼25min). The percentage cell surface receptor loss was more profound after exposure to ABT-229 (88±1%) than to motilin (63±10%) or EM-A (34±2%). For motilin and EM-A MTLR phosphorylation probably occurs via G protein-coupled receptor kinases while for ABT-229 phosphorylation was also protein kinase C dependent. All agonists translocated cytosolic β-arrestin-2 with greater affinity to the plasma membrane than β-arrestin-1. After internalization the MTLR co-localized with transferrin but not with cathepsin D. After stimulation with motilin and EM-A the t_1/2 for MTLR resensitization was 3h and 1h, respectively but amounted 26h for ABT-229.Our results suggest that the resensitization kinetics determine the desensitization properties of the motilin agonists.

Keywords: Motilin; Motilides; Desensitization; Motilin receptor trafficking; Phosphorylation; β-Arrestins

Gene expression profiling elucidates a specific role for RARγ in the retinoic acid-induced differentiation of F9 teratocarcinoma stem cells by Dan Su; Lorraine J. Gudas (pp. 1129-1160).

The biological effects of all- trans-retinoic acid (RA), a major active metabolite of retinol, are mainly mediated through its interactions with retinoic acid receptor (RARs α, β, γ) and retinoid X receptor (RXRs α, β, γ) heterodimers. RAR/RXR heterodimers activate transcription by binding to RA-response elements (RAREs or RXREs) in the promoters of primary target genes. Murine F9 teratocarcinoma stem cells have been widely used as a model for cellular differentiation and RA signaling during embryonic development. We identified and characterized genes that are differentially expressed in F9 wild type (Wt) and F9 RARγ^−/− cells, with and without RA treatment, through the use of oligonucleotide-based microarrays. Our data indicate that RARγ, in the absence of exogenous RA, modulates gene expression. Genes such as Sfrp2, Tie1, Fbp2, Emp1, and Emp3 exhibited higher transcript levels in RA-treated Wt, RARα^−/− and RARβ₂^−/− lines than in RA-treated RARγ^−/− cells, and represent specific RARγ targets. Other genes, such as Runx1, were expressed at lower levels in both F9 RARβ₂^−/− and RARγ^−/− cell lines than in F9 Wt and RARα^−/−. Genes specifically induced by RA at 6h with the protein synthesis inhibitor cycloheximide in F9 Wt, but not in RARγ^−/− cells, included Hoxa3, Hoxa5, Gas1, Cyp26a1, Sfrp2, Fbp2, and Emp1. These genes represent specific primary RARγ targets in F9 cells. Several genes in the Wnt signaling pathway were regulated by RARγ. Delineation of the receptor-specific actions of RA with respect to cell proliferation and differentiation should result in more effective therapies with this drug.

Keywords: Retinoic acid receptor; Gene expression profiles; Differentiation; Retinol; sfrp5; Tie1

Molecular basis for the interaction of four different classes of substrates and inhibitors with human aromatase by Yanyan Hong; Michael Cho; Yate-Ching Yuan; Shiuan Chen (pp. 1161-1169).

Aromatase cytochrome P450 (CYP19) converts androgen to estrogen. In this study, the interactions of four classes of compounds, 17β-estradiol (the product of aromatase), 17-methyltestosterone (a synthetic androgen), dibenzylfluorescein (a synthetic substrate of aromatase), and coumestrol (a phytoestrogen), with aromatase were investigated through spectral analysis using purified human recombinant aromatase and site-directed mutagenesis studies using CHO cells expressing wild-type human aromatase or five aromatase mutants, E302D, D309A, T310S, S478T and H480Q. Spectral analysis showed that a type I binding spectrum was produced by the binding of 17-methyltestosterone to aromatase and a novel binding spectrum of aromatase was induced by dibenzylfluorescein. Mutagenesis experiments demonstrated that residues S478 and H480 in the β-4 sheet play an important role in the binding of all four compounds. Computer-assisted docking of these compounds into the three-dimensional model of aromatase revealed that: (1) weak interaction between 17β-estradiol and the β-4 sheet of aromatase facilitates the release of 17β-estradiol from the active site of aromatase; (2) 17-methyl group of 17-methyltestosterone affects its binding to aromatase; (3) dibenzylfluorescein binds to the active site of aromatase with its O-dealkylation site near the heme iron and residue T310; and (4) coumestrol binds to aromatase in a manner such that rings A and C of coumestrol mimic rings A and B of steroid. These structure–function studies help us to evaluate the structural model of aromatase, and to accelerate the structure-based design for new aromatase inhibitors.

Keywords: Abbreviations; E1; estrone; E2; 17β-estradiol; CYP; cytochrome P450; AIs; aromatase inhibitors; 3-D; three-dimensional; 17MT; 17-methyltestosterone; DBF; dibenzylfluorescein; WT; wild-type; CHO; Chinese hamster ovarian; 17ME2; 17-methylestradiol; ER; estrogen receptor; IC; ₅₀; 50% inhibitory concentrationAromatase inhibitor; Estrogen; Cytochrome P450; Mutagenesis; Structural model

Function of non-visual arrestins in signaling and endocytosis of the gastrin-releasing peptide receptor (GRP receptor) by Michael Schumann; Tomoo Nakagawa; Samuel A. Mantey; Brian Howell; Robert T. Jensen (pp. 1170-1185).

Little is known about the role of arrestins in gastrointestinal hormone/neurotransmitter receptor endocytosis. With other G protein-coupled receptors, arrestins induce G protein-uncoupling and receptor endocytosis. In this study, we used arrestin wild-type and dominant-negative mutant constructs to analyze the arrestin dependence of endocytosis and desensitization of the gastrin-releasing peptide receptor (GRP-R). Co-expression of the GRP-R with wild-type arrestin2 and arrestin3 increased not only GRP-R endocytosis but also GRP-R desensitization in arrestin-overexpressing cells. Co-expression of the dominant-negative mutants V53D-arrestin2 or V54D-arrestin3 reduced GRP-R endocytosis. Notably, different trafficking routes for agonist-activated GRP-R-arrestin2 and GRP-R-arrestin3 complexes were found. Arrestin3 internalizes with GRP-R to intracellular vesicles, arrestin2 splits from the GRP-R and localizes to the cell membrane. Also, the recycling pathway of the GRP-R was different if co-expressed with arrestin2 or arrestin3. Using different GRP-R mutants, the C-terminus and the 2nd intracellular loop of the GRP-R were found to be important for the GRP-R-arrestin interaction and for the difference in GRP receptor trafficking with the two arrestin subtypes. Our results show that both non-visual arrestins play an important role in GRP-R internalization and desensitization.

Keywords: Abbreviations; GPCR; G protein-coupled receptor; GRK; G protein-coupled receptor kinase; GRP receptor; gastrin-releasing peptide receptor; PLC; phospholipase C; DMEM; Dulbecco's modified Eagle's medium; FBS; fetal bovine serum; PBS; phosphate-buffered saline; Bn; bombesin; DTT; dithiothreitol; GFP; green fluorescent protein; IP; inositol phosphate; JF-1; GRP receptor mutant lacking carboxyl terminal serines and threoninesG protein-coupled receptor; Gastrin-releasing peptide; Arrestin; Receptor endocytosis; rab5

Correlation between PAMPA permeability and cellular activities of hepatitis C virus protease inhibitors by Cheng Li; Latha Nair; Tongtong Liu; Fangbiao Li; John Pichardo; Sony Agrawal; Robert Chase; Xiao Tong; Annette S. Uss; Stephane Bogen; F. George Njoroge; Richard A. Morrison; K.-C. Cheng (pp. 1186-1197).

Parallel artificial membrane permeability assay (PAMPA) and Caco-2 cells have been frequently used for the evaluation of in vitro permeability of new chemical entities. In this study we evaluated the correlation between permeability, assessed by both methods, and the cellular potency of 34 novel hepatitis C virus (HCV) protease inhibitors. Two types of assays were used to determine the potency of HCV protease inhibitors: a cell-free assay that evaluates the intrinsic affinity ( K_i) between the protease and the inhibitor and a cell-based replicon assay that determines the inhibitors’ IC₉₀. When the K_i/IC₉₀ ratios were compared with the PAMPA permeability and the Caco-2 permeability by linear regression analysis, a reasonable correlation was found between the K_i/IC₉₀ ratio and PAMPA permeability ( r²=0.76) but not with Caco-2 permeability ( r²=0.29). Correlations were also assessed between K_i/IC₉₀ ratios and the following physico-chemical properties: log P ( r²=0.41), log D ( r²=0.58), clog P ( r²=0.13), and mlog P ( r²=0.30). These results suggest that passive permeability may play a role in the uptake and cellular activity of these HCV protease inhibitors, and that PAMPA was more predictive of cellular activity than physico-chemical properties or Caco-2 permeability.

Keywords: HCV protease inhibitor; PAMPA assay; Caco-2 permeability; Cell-based activity; Physico-chemical property; Cell uptake

Binding thermodynamic characterization of human P2X₁ and P2X₃ purinergic receptors by Katia Varani; Annmarie Surprenant; Fabrizio Vincenzi; Alice Tosi; Stefania Gessi; Stefania Merighi; Pier Andrea Borea (pp. 1198-1208).

The present study was designed to perform binding and thermodynamic characterization of human P2X₁ and P2X₃ purinergic receptors expressed in HEK 293 cells. The thermodynamic parameters Δ G°, Δ H° and Δ S° (standard free energy, enthalpy and entropy) of the binding equilibrium of well-known purinergic agonists and antagonists at P2X₁ and P2X₃ receptors were determined. Saturation binding experiments, performed in the temperature range 4–30°C by using the high affinity purinergic agonist [³H]αβmeATP, revealed a single class of binding sites with an affinity value in the nanomolar range in both cell lines examined. The affinity changed with the temperature whereas receptor density was essentially independent of it. van’t Hoff plots of the purinergic receptors were linear in the range 4–30°C for agonists and antagonists. The thermodynamic parameters of the P2X₁ or P2X₃ purinergic receptors were in the ranges −31kJmol⁻¹≤Δ H°≤−19kJmol⁻¹ and 17JK⁻¹mol⁻¹≤Δ S°≤51JK⁻¹mol⁻¹ or −26kJmol⁻¹≤Δ H°≤36kJmol⁻¹ and 59≤Δ S°≤249JK⁻¹mol⁻¹, respectively. The results of these parameters showed that P2X₁ receptors are not thermodynamically discriminated and that the binding of agonists and antagonists was both enthalpy and entropy-driven. P2X₃ receptors were thermodynamically discriminated and purinergic agonist binding was enthalpy and entropy-driven while antagonist binding was totally entropy-driven. The analysis of such thermodynamic data makes it possible to obtain additional information on the nature of the forces driving the purinergic binding interaction. These data could be interesting in drug discovery programs aimed at development of novel and potent P2X₁ and P2X₃ purinergic ligands.

Keywords: Abbreviations; HEK; human embryonic kidney; [; ³; H]αβmeATP; α,βmethyleneATP; ATP; adenosine 5′ triphosphate; ADP; adenosine 5′ dihosphate; 2meSATP; 2methylSATP; BzATP′benzoylATP; TNP-ATP; 2′-3′-; O; -(2,4,6-trinitrophenyl)adenosine 5′-triphosphate; PPADS; pyridoxal 5-phosphate 6-azophenyl-2′,4′-disulphonic acid; NF023; 8,8′-[carbonylbis (imino-3,1-phenylene carbonylimino)]bis(1,3,5-naphthalene-trisulfonic acid); NF279; 8,8′-[carbonylbis(imino-4,1-phenylenecarbonyl-imino-4,1-phenylene carbonyl- imino)] bis-1,3,5-naphthalene-trisulphonic acid; A317491; 5-({[3-phenoxybenzyl][(1; S; )-1,2,3,4-tetrahydro-1-naphthalenyl]amino}carbonyl)-1,2,4-benzenetricarboxylic acidP2X; ₁; and P2X; ₃; purinergic receptors; HEK 293 cells; [; ³; H]αβmeATP binding; Binding thermodynamics; Enthalpy–entropy compensation; Agonist–antagonist discrimination

Cell shape, cell–cell contact, cell–extracellular matrix contact and cell polarity are all required for the maximum induction of CYP2B1 and CYP2B2 gene expression by phenobarbital in adult rat cultured hepatocytes by Hiroaki Oda; Yukiko Yoshida; Akino Kawamura; Atsushi Kakinuma (pp. 1209-1217).

The effect of cell shape, cell density, contact with extracellular matrix and cell polarity on the phenobarbital (PB)-induced gene expression of CYP2B1 and CYP2B2 (CYP2B1/2B2) in adult rat hepatocytes was investigated. High cell density enhanced the induction of CYP2B1/2B2 gene expression by PB. Hepatocytes cultured on EHS gel showed a spherical cell shape and highly enhanced the induction of CYP2B1/2B2 gene expression by PB. Although monolayer hepatocytes cultured on type I collagen (TIC) and type IV collagen exhibited poor induction of CYP2B1/2B2 gene expression by PB, monolayer cells on laminin showed substantial induction. The addition of soluble laminin to media did not show any effect on induction in monolayer hepatocytes cultured on TIC. Dishes coated with different concentrations of immovable laminin demonstrated complicated effects. Coating with higher concentrations of laminin resulted in greater induction of CYP2B1/2B2 gene expression by PB. On the other hand, when hepatocytes were cultured on dishes coated with lower concentrations of laminin, they became round and greater induction of CYP2B1/2B2 gene expression by PB was observed. Spherical hepatocytes cultured on low concentrations of TIC also showed highly enhanced induction of CYP2B1/2B2 gene expression by PB. EHS gel overlay to hepatocytes cultured on TIC and collagen sandwich configurations that are known to induce cell polarity enhanced the induction by PB. The induction of CYP2B1/2B2 gene expression needed cytoskeleton organization, such as actin filament, microtubule filament and intermediate filament. These results demonstrate that cell shape, cell density, contact with extracellular matrix and cell polarity all play critical roles in the induction of CYP2B1/2B2 gene expression by PB.

Keywords: Abbreviations; apo; apolipoprotein; CAR; constitutive activator receptor; CYP; cytochrome P-450; EHS; Engelbreth-Holm-Swan; IDPN; 3,3′-iminodipropionitrile; PB; phenobarbital; TIC; type I collagen; TIVC; type IV collagen; UGT; UDP-glucuronosyltransferaseHepatocyte; Phenobarbital; CYP2B; Cell shape; Cell–cell communications; Extracellular matrix; Cell polarity

Investigation of the metabolic fate of dihydrocaffeic acid by Laure Poquet; Michael N. Clifford; Gary Williamson (pp. 1218-1229).

The antioxidant dihydrocaffeic acid is a dietary constituent and a microbial metabolite of flavonoids. Orally administered to rats, dihydrocaffeic acid was very rapidly absorbed most probably by the gastric or duodenal epithelium and excreted in urine as free and conjugated forms. LC–MS² analysis of plasma and urine samples allowed confident identification of the dihydrocaffeic acid metabolites. The parent compound was glucuronidated, sulphated or methylated, on one of the hydroxyl groups present on its phenyl ring. All the dihydrocaffeic acid metabolites peaked in plasma within the first 30min following ingestion, suggesting a metabolism possibly by the gastric or duodenal cells and by the liver. Using in vitro and ex vivo models of the intestinal epithelium and the liver, the identity and source of the metabolites detected in vivo were examined. The data obtained suggest that, in rats, intestinal cells are more able to glucuronidate dihydrocaffeic acid, whereas liver favours sulphation. Moreover, glucuronidation, sulphation and methylation seem to be regio-selective, preferably on the 3-OH of dihydrocaffeic acid. The methyl conjugate, dihydroferulic acid, was shown to be oxidized into ferulic acid by intestinal and hepatic cells, which were also able to perform the reverse reaction, the reduction of ferulic acid into dihydroferulic acid. As a conclusion, the main form of dihydrocaffeic acid circulating in plasma after its ingestion is a mixture of different primary and secondary metabolites.

Keywords: Abbreviations; LC–MS; liquid chromatograph with mass spectrometry; MRM; multiple reaction monitoring; m; /; z; mass to charge ratio; TIC; total in countFerulic acid; Caco-2; Liver metabolism; Glucuronide; Sulfate

Expression of cytochrome P450 2A5 in a glucose-6-phosphate dehydrogenase-deficient mouse model of oxidative stress by Kathleen D. Nichols; Gordon M. Kirby (pp. 1230-1239).

Murine hepatic cytochrome P450 2A5 (CYP2A5), unlike most CYP enzymes, is upregulated during hepatitis and hepatotoxic conditions, but the common stimulus for its induction remains unknown. We investigated the involvement of oxidative stress in the regulation of CYP2A5 expression using an oxidative stress-sensitive glucose-6-phosphate dehydrogenase (G6PD)-deficient mouse model. Treatment of deficient and wild-type mice with the prototypical CYP2A5-inducer pyrazole for 72h led to a significantly greater degree of induction of CYP2A5 mRNA, protein and activity in deficient mice, with the greatest increase observed in animals homozygous for the deficiency. However, markers of oxidative stress including protein carbonyl, 8-hydroxydeoxyguanosine, malondiadehyde and 4-hydroxyalkenal levels were unaltered with pyrazole treatment. Furthermore, CYP2A5 expression was not altered in G6PD-deficient mice treated with the pro-oxidant menadione whereas DNA, lipid, and protein markers of oxidative stress were significantly increased. The antioxidant polyethylene glycol-conjugated catalase, while decreasing oxidative stress in menadione-treated mice, did not prevent the induction of CYP2A5 by pyrazole. Finally, the ER stress marker protein, GRP78, was increased following pyrazole treatment in G6PD-deficient compared to wild-type mice. These findings do not support a central role for generalized cellular oxidative stress in the regulation of CYP2A5 and suggest that additional factors related to G6PD-deficiency, such as ER stress, may be involved.

Keywords: Abbreviations; CYP; cytochrome P450; G6PD; glucose-6-phosphate dehydrogenase; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; Gclc; glutamate-cysteine ligase catalytic subunit; Gpx; glutathione-peroxidase; Gsr; glutathione reductase; GST; glutathione-; S; -transferase; 4-HAE; 4-hydroxyalkenal; MEN; menadione; MDA; malondialdehyde; Nqo1; NADPH:quinone oxidoreductase; PC; polyethylene glycol-conjugated catalase; PYR; pyrazole; Srxn1; sulfiredoxin; TCPOBOP; 1,4-bis-[2-(3,5,-dichloropyridyloxy)] benzeneCYP2A5; Oxidative stress; G6PD-deficiency; Gene regulation; Hepatotoxicity; ER stress

Identification of human UDP-glucuronosyltransferases catalyzing hepatic 1α,25-dihydroxyvitamin D₃ conjugation by Takanori Hashizume; Yang Xu; Michael A. Mohutsky; Jeffrey Alberts; Chad Hadden; Thomas F. Kalhorn; Nina Isoherranen; Margaret C. Shuhart; Kenneth E. Thummel (pp. 1240-1250).

The biological effects of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) are terminated primarily by P450-dependent hydroxylation reactions. However, the hormone is also conjugated in the liver and a metabolite, presumably a glucuronide, undergoes enterohepatic cycling. In this study, the identity of human enzymes capable of catalyzing the 1,25(OH)₂D₃ glucuronidation reaction was investigated in order to better understand environmental and endogenous factors affecting the disposition and biological effects of vitamin D₃. Among 12 different UGT isozymes tested, only UGT1A4≫2B4 and 2B7 supported the reaction. Two different 1,25(OH)₂D₃ monoglucuronide metabolites were generated by recombinant UGT1A4 and human liver microsomes. The most abundant product was identified by mass spectral and NMR analyses as the 25- O-glucuronide isomer. The formation of 25- O-glucuronide by UGT1A4 Supersomes and human liver microsomes followed simple hyperbolic kinetics, yielding respective K_m and V_max values of 7.3 and 11.2μM and 33.7±1.4 and 32.9±1.9pmol/min/mg protein. The calculated intrinsic 25- O-glucuronide M1 formation clearance for UGT1A4 was 14-fold higher than the next best isozyme, UGT2B7. There was only limited (four-fold) inter-liver variability in the 25- O-glucuronidation rate, but it was highly correlated with the relative rate of formation of the second, minor metabolite. In addition, formation of both metabolites was inhibited >80% by the selective UGT1A4 inhibitor, hecogenin. If enterohepatic recycling of 1,25(OH)₂D₃ represents a significant component of intestinal and systemic 1,25(OH)₂D₃ disposition, formation of monoglucuronides by hepatic UGT1A4 constitutes an important initial step.

Keywords: Abbreviations; 1,25(OH); ₂; D; ₃; 1α,25-dihydroxyvitamin D; ₃; UDPGA; uridine-5′-diphosphoglucuronic acid; PTAD; 4-phenyl-1,2,4-triazoline-3,5-dione; HLM; human liver microsomes; HPLC; high performance liquid chromatography; LC/MS/MS; liquid chromatography–tandem mass spectrometry; LC/MS; liquid chromatography–mass spectrometryHuman liver microsomes; High performance liquid chromatography; Liquid chromatographytandem mass spectrometry; Liquid chromatography–mass spectrometry

Sections

Personal tools

Biochemical Pharmacology (v.75, #5)

Sections

Personal tools

Local resources

Biochemical Pharmacology (v.75, #5)