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Biochemical Pharmacology (v.79, #6)

Graphical Abstracts Contents (pp. e1-e4).

Editorial Advisory Board (pp. iii).

Good night and good luck: Norepinephrine in sleep pharmacology by Heather A. Mitchell; David Weinshenker (pp. 801-809).

Sleep is a crucial biological process that is regulated through complex interactions between multiple brain regions and neuromodulators. As sleep disorders can have deleterious impacts on health and quality of life, a wide variety of pharmacotherapies have been developed to treat conditions of excessive wakefulness and excessive sleepiness. The neurotransmitter norepinephrine (NE), through its involvement in the ascending arousal system, impacts the efficacy of many wake- and sleep-promoting medications. Wake-promoting drugs such as amphetamine and modafinil increase extracellular levels of NE, enhancing transmission along the wake-promoting pathway. GABAergic sleep-promoting medications like benzodiazepines and benzodiazepine-like drugs that act more specifically on benzodiazepine receptors increase the activity of GABA, which inhibits NE transmission and the wake-promoting pathway. Melatonin and related compounds increase sleep by suppressing the activity of the neurons in the brain's circadian clock, and NE influences the synthesis of melatonin. Antihistamines block the wake-promoting effects of histamine, which shares reciprocal signaling with NE. Many antidepressants that affect the signaling of NE are also used for treatment of insomnia. Finally, adrenergic receptor antagonists that are used to treat cardiovascular disorders have considerable sedative effects. Therefore, NE, long known for its role in maintaining general arousal, is also a crucial player in sleep pharmacology. The purpose of this review is to consider the role of NE in the actions of wake- and sleep-promoting drugs within the framework of the brain arousal systems.

Keywords: Abbreviations; 5-HT; serotonin; AMP; adenosine monophosphate; AR; adrenergic receptor; BDNF; brain-derived neurotrophic factor; DA; dopamine; DAT; dopamine transporter; Dbh; dopamine beta hydroxylase; DMH; dorsal medial hypothalamus; DRN; dorsal raphe nucleus; EEG; electroencephalogram; GABA; gamma-aminobutyric acid; GPCR; G protein-coupled receptor; H; histamine (receptor); KO; knockout; LC; locus coeruleus; MAOI; monoamine oxidase inhibitor; MPOA; medial preoptic area; MSA; medial septal area; MT; melatonin (receptor); NE; norepinephrine; NET; norepinephrine transporter; PFC; prefrontal cortex; PKA; protein kinase A; REM; rapid eye movement; SCN; suprachiasmatic nucleus; SERT; serotonin transporter; SSRI; selective serotonin reuptake inhibitor; TCA; tricyclic antidepressant; TMN; tuberomammillary nucleus; TrkB; tyrosine kinase receptor B; VLPO; ventrolateral preoptic area; vPAG; ventral periaqueductal grey; z-drug; drug that acts specifically on the α1 subunit of the benzodiazepine receptorNorepinephrine; Locus coeruleus; Sleep; Wake; Arousal

Effects of cisplatin on matrix metalloproteinase-2 in transformed thyroid cells by L. Urso; A. Muscella; N. Calabriso; C. Vetrugno; E. Jiménez; M. Montiel; S. Marsigliante (pp. 810-816).

We investigated the effects of cisplatin (cisPt) on matrix metalloproteinase-2 (MMP-2) gelatinolitic activity in transformed PC E1Araf rat thyroid cells. Cells incubated with increasing cisPt concentrations showed dose- and time-dependent decrease of the MMP-2 protein and activity. CisPt provoked the translocation from the cytosol to the plasma membrane of atypical protein kinase C-zeta (PKC-ζ) and the activation of PKB/AKT. The effect of cisPt on MMP-2 was dependent on PKC-ζ activation since it was potentiated by a myristoylated PKC-ζ pseudo substrate peptide or by PKC-ζ down-regulation by siRNA. Moreover, MMP-2 activity modulation by cisPt was also dependent on PKB/AKT activation since it was decreased by PKB/AKT down-regulation by siRNA or by pharmacological inhibition of PI3K, thus indicating the importance of the balance of PKB/AKT and PKC-ζ in regulating the cisPt effect on MMP-2 activity. The PC E1Araf cells displayed a migratory capacity that was blocked by MMP-2 down-regulation using siRNA or pharmacological inhibition . The inhibition of cell migration was also obtained with cisPt; in cisPt-treated cells the administration of MMP-2 active protein was able to restore cell migration capacity. In conclusion, the decrease of MMP-2 secretion after cisPt was allowed by PKB/AKT and counteracted by PKC-ζ; the cisPt-provoked inhibition of MMP-2 secretion ended in reduction of cell migration.

Keywords: MMP-2; Cisplatin; PKC-ζ; AKT/PKB; ERK1/2; PC E1Araf

Involvement of miR-326 in chemotherapy resistance of breast cancer through modulating expression of multidrug resistance-associated protein 1 by Zhongxing Liang; Hui Wu; James Xia; Yuhua Li; Yawei Zhang; Ke Huang; Nicholas Wagar; Younghyoun Yoon; Heidi T. Cho; Stefania Scala; Hyunsuk Shim (pp. 817-824).

Multidrug resistance-associated protein (MRP-1/ABCC1) transports a wide range of therapeutic agents and may play a critical role in the development of multidrug resistance (MDR) in tumor cells. However, the regulation of MRP-1 remains controversial. To explore whether miRNAs are involved in the regulation of MRP-1 expression and modulate the sensitivity of tumor cells to chemotherapeutic agents, we analyzed miRNA expression levels in VP-16-resistant MDR cell line, MCF-7/VP, in comparison with its parent cell line, MCF-7, using a miRNA microarray. MCF-7/VP overexpressed MRP-1 mRNA and protein not MDR-1 and BCRP. miR-326 was downregulated in MCF-7/VP compared to MCF-7. Additionally, miR-326 was downregulated in a panel of advanced breast cancer tissues and consistent reversely with expression levels of MRP-1. Furthermore, the elevated levels of miR-326 in the mimics-transfected VP-16-resistant cell line, MCF-7/VP, downregulated MRP-1 expression and sensitized these cells to VP-16 and doxorubicin. These findings demonstrate for the first time the involvement of miRNAs in multidrug resistance mediated by MRP-1 and suggest that miR-326 may be an efficient agent for preventing and reversing MDR in tumor cells.

Keywords: Abbreviations; miRNA; microRNA; MRP-1; multidrug resistance-associated protein 1; MDR; multidrug resistance; MDR-1; multidrug resistance 1; BCRP; breast cancer resistance protein; siRNA; small interfering RNA; RT-PCR; reverse transcription polymerase chain reactionmicroRNA; Multidrug resistance; MRP-1; Breast cancer

2-Methoxyestradiol induces endoreduplication through the induction of mitochondrial oxidative stress and the activation of MAPK signaling pathways by C.M. Ting; Y.M. Lee; C.K.C. Wong; A.S. Wong; H.L. Lung; M.L. Lung; K.W. Lo; R.N.S. Wong; N.K. Mak (pp. 825-841).

2-Methoxyestradiol (2ME2) is a normal physiological metabolite of 17β-estradiol with anti-proliferative and anti-angiogenic activities. The purpose of this study is to elucidate the mechanism whereby 2ME2 induces endoreduplication of the well-differentiated nasopharyngeal carcinoma (NPC) cells. We report here that 2ME2 induces G2/M phase cell cycle arrest followed by endoreduplication of the well-differentiated HK-1 cells. The increase in chromosome number was confirmed by cytogenetic study. Analysis of stress signaling pathways revealed the phosphorylation activation of ERK, JNK and p38 MAPKs at various times after 2ME2 treatment. Pre-treatment of 2ME2-treated HK-1 cells with JNK inhibitor (SP600125), ERK inhibitor (PD98059) and p38 MAPK inhibitor (SB203580) resulted in the reduction of endoreduplicating cells. Furthermore, the increase in the phosphorylation of JNK was accompanied by an increase in the reactive oxygen species. In addition, endoreduplication was observed in cells after treatment with superoxide donor, 2,3-dimethoxy-1,4-naphoquinone (DMNQ). Confocal microscopic analysis also revealed the increase in mitochondrial superoxide anion in 2ME2-treated HK-1 cells. Pre-treatment of HK-1 cells with superoxide dismutase mimetic 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) or overexpressing the mitochondrial enzyme MnSOD resulted in the reduction of phosphorylation of JNK and the formation of endoreduplicating cells. Furthermore, the tubulin filaments in cytoplasm remain intact in 2ME2-treated HK-1 cells after pre-treatment of TEMPO. Our results suggest that 2ME2 induces endoreduplication through the induction of oxidative stress and the activation of MAPK signal pathways. The biological significance of drug-induced endoreduplication will also be discussed.

Keywords: 2-Methoxyestradiol; Endoreduplication; Oxidative stress; Nasopharyngeal carcinoma; MAPK signaling pathways; Mitochondria

Reverse phase protein array identifies novel anti-invasion mechanisms of YC-1 by Bo Hong; Vivian W.Y. Lui; Edwin P. Hui; Yiling Lu; Horasis S.Y. Leung; Elaine Y.L. Wong; Suk-Hang Cheng; Margaret H.L. Ng; Gordon B. Mills; Anthony T.C. Chan (pp. 842-852).

YC-1 has recently been demonstrated to have potent anti-invasion and anti-metastatic activity in several cancer models, in addition to its anti-proliferation activity. However, the mechanism underlying its anti-invasion/anti-metastatic activity is largely unknown. Nasopharyngeal carcinoma (NPC) is a highly metastatic head and neck cancer in Southeast Asia. Here, we demonstrated that YC-1 inhibited invasiveness and proliferation of NPC cells, with the latter being accompanied by PARP cleavage, S-phase arrest and activation of Chk1/Chk2. We aimed at identifying novel anti-invasion mechanisms of YC-1 in NPC by a functional proteomic platform, the reverse phase protein array (RPPA). Our study revealed for the first time that multiple invasion-related signaling proteins (β-catenin, caveolin, Src and EGFR), as well as several growth-related proteins (AMPKα, phospho-acetyl-CoA carboxylase (p-ACC), HER-2 and mTOR), which were previously un-described signaling proteins altered by YC-1, were found to be down-modulated by YC-1 in NPC cells. We hypothesized that YC-1-mediated downregulation of these invasion proteins contributed to its anti-invasion activity in NPC cells. Overexpression of EGFR, activated Src or caveolin, but not β-catenin reversed the inhibitory effects of YC-1 on NPC cell invasion, with EGFR and activated Src having additional effects on rescuing NPC cells from YC-1-mediated growth inhibition. In summary, we have identified several novel anti-invasion mechanisms of YC-1 that could impact NPC, and possibly other cancers as well.

Keywords: Abbreviations; NPC; nasopharyngeal carcinoma; EBV; Epstein-Barr virus; RPPA; reverse phase protein array; EGF; epidermal growth factor; EGFR; epidermal growth factor receptor; MMP-2/-9; Matrix Metalloproteinases 2 and 9; ACC; acetyl-CoA carboxylase; AMPKα; AMP-activated protein kinase α; LMP1; latent membrane protein 1Anti-invasion; Anti-proliferation; Nasopharyngeal carcinoma (NPC); Reverse phase protein array; YC-1 mechanisms

AMPK-independent down-regulation of cFLIP and sensitization to TRAIL-induced apoptosis by AMPK activators by Celina García-García; Claudia Fumarola; Naveenan Navaratnam; David Carling; Abelardo López-Rivas (pp. 853-863).

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a TNF superfamily member that is being considered as a new strategy in anticancer therapy because of its ability to induce apoptosis, alone or in combination with other stimuli, in many cancer cells. AMP-activated protein kinase (AMPK) is an evolutionarily conserved key regulator of cellular energy homeostasis that protects the cell from energy depletion and stress by activating several biochemical pathways that lead to the conservation, as well as generation, of ATP. Here we report that a number of AMPK activators, including the small molecule activator A-769662, markedly sensitize TRAIL-resistant breast cancer cells to TRAIL-induced apoptosis. However, silencing AMPKα1 expression with siRNA or over-expression of DN-AMPKα1 does not inhibit AICAR, glucose deprivation, phenformin or A-769662-induced sensitization to TRAIL. Furthermore, the expression of constitutively active AMPK subunits does not sensitize resistant breast cancer cells to TRAIL-induced apoptosis. The cellular FLICE-inhibitory proteins (cFLIP_L and cFLIP_S) were significantly down-regulated following exposure to AMPK activators through an AMPK-independent mechanism. Furthermore, in cells over-expressing cFLIP_L, sensitization to TRAIL by AMPK activators was markedly reduced. In summary, our results indicate that AMPK activators facilitate the activation by TRAIL of an apoptotic cell death program through a mechanism independent of AMPK and dependent on the down-regulation of cFLIP levels.

Keywords: Abbreviations; cFLIP; cellular FLICE-inhibitory protein; TRAIL; tumor necrosis factor-related apoptosis-inducing ligand; TNF; tumor necrosis factor; DISC; death-inducing signalling complex; FADD; Fas-associated death domain; Cyt c; cytochrome; c; DD; death domain; DED; death effector domain; PARP; poly (ADP-ribose) polymerase; PFU; plaque-forming unitFLIP; Apoptosis; TRAIL; AMPK; Tumor

Mutations in the human kinesin Eg5 that confer resistance to monastrol and S-trityl-l-cysteine in tumor derived cell lines by Sergey Tcherniuk; Robert van Lis; Frank Kozielski; Dimitrios A. Skoufias (pp. 864-872).

Bipolar spindle formation in cells exposed to two chemical distinct mitotic inhibitors specific to Eg5 motor protein after expression of Eg5 point mutants in the drug binding pocket.The kinesin Eg5 plays an essential role in bipolar spindle formation. A variety of structurally diverse inhibitors of the human kinesin Eg5, including monastrol and STLC, share the same binding pocket on Eg5, composed by helix α2/loop L5, and helix α3 of the Eg5 motor domain. Previous biochemical analysis in the inhibitor binding pocket of Eg5 identified key residues in the inhibitor binding pocket of Eg5 that in the presence of either monastrol or STLC exhibited ATPase activities similar to the untreated wild type Eg5. Here we evaluated the ability of full-length human Eg5 carrying point mutations in the drug binding pocket to confer resistance in HeLa and U2OS cells to either monastrol or STLC, as measured by the formation of bipolar spindles. Both transfected cells expressing wild type Eg5 and untransfected cells were equally sensitive to both inhibitors. Expression of Eg5 single point mutants R119A, D130A, L132A, I136A, L214A and E215A conferred significant resistance to monastrol. Certain mutations inducing monastrol resistance such as R119A, D130A and L214A also conferred significant resistance to STLC. For the first time at a cellular level, the propensity of selected Eg5 point mutants to confer drug resistance confirms the target specificity of monastrol and STLC for Eg5. These data also suggest a possible mechanism by which drug resistance may occur in tumors treated with agents targeting Eg5.

Keywords: Eg5; Mitosis; Monastrol; STLC; Drug; Resistance

UDP-glucose acting at P2Y₁₄ receptors is a mediator of mast cell degranulation by Zhan-Guo Gao; Yi Ding; Kenneth A. Jacobson (pp. 873-879).

Inhibition of the P2Y₁₄ receptor may be a novel target for therapeutic intervention of asthma and allergic conditions.UDP-glucose (UDPG), a glycosyl donor in the biosynthesis of carbohydrates, is an endogenous agonist of the G protein-coupled P2Y₁₄ receptor. RBL-2H3 mast cells endogenously express a P2Y₁₄ receptor at which UDPG mediates degranulation as indicated by β-hexosaminidase (HEX) release. Both UDPG and a more potent, selective 2-thio-modified UDPG analog, MRS2690 (diphosphoric acid 1-α-d-glucopyranosyl ester 2-[(2-thio)uridin-5″-yl] ester), caused a substantial calcium transient in RBL-2H3 cells, which was blocked by pertussis toxin, indicating the presence of the G_i-coupled P2Y₁₄ receptor, supported also by quantitative detection of abundant mRNA. Expression of the closely related P2Y₆ receptor was over 100 times lower than the P2Y₁₄ receptor, and the P2Y₆ agonist 3-phenacyl-UDP was inactive in RBL-2H3 cells. P2Y₁₄ receptor agonists also induced [³⁵S]GTPγS binding to RBL-2H3 cell membranes, and phosphorylation of ERK1/2, P38 and JNK. UDPG and MRS2690 concentration-dependently enhanced HEX release with EC₅₀ values of 1150±320 and 103±18nM, respectively. The enhancement was completely blocked by pertussis toxin and significantly diminished by P2Y₁₄ receptor-specific siRNA. Thus, mast cells express an endogenous P2Y₁₄ receptor, which mediates G_i-dependent degranulation and is therefore a potential novel therapeutic target for allergic conditions.

Keywords: Abbreviations; BSA; bovine serum albumin; CHAPS; 3-[(3-cholamidopropyl) dimethylammonio]propanesulfonate; CHO; Chinese hamster ovary; DMEM; Dulbecco's modified Eagle's medium; DNP; 2,4-dinitrophenyl; EIA; enzyme immunometric assay; ERK1/2; extracellular signal-regulated protein kinases 1 and 2; FBS; fetal bovine serum; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; GPCR; G protein-coupled receptor; GTPγS; guanosine 5′-O-(γ-thio)triphosphate; HEX; β-hexosaminidase; LY294002; 2-(4-morpholino)-8-phenyl-4H-1-benzopyran-4-one; MAPK; mitogen-activated protein kinase; MRS2690; diphosphoric acid 1-α-; d; -glucopyranosyl ester 2-[(2-thio)uridin-5″-yl] ester disodium salt; PGE2; prostaglandin E2; PI3K; phosphoinositide 3-kinase; PIPES; piperazine-N,N′-bis(2-ethanesulfonic acid); PLC; phospholipase C; PTX; pertussis toxin; qRT-PCR; quantitative real-time polymerase chain reaction; RBL; rat basophilic leukemia; siRNA; small interfering RNA; TRIS; tris(hydroxymethyl)methylamine; U73122; 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione; UDP; uridine 5′-diphosphate; UDPG; uridin 5′-diphosphoglucose disodium saltNucleotide; P2Y receptor; Mast cell; Allergy; Inflammation; G protein-coupled receptor

Inhibition of soluble epoxide hydrolase enhances the anti-inflammatory effects of aspirin and 5-lipoxygenase activation protein inhibitor in a murine model by Jun-Yan Liu; Jun Yang; Bora Inceoglu; Hong Qiu; Arzu Ulu; Sung-Hee Hwang; Nipavan Chiamvimonvat; Bruce D. Hammock (pp. 880-887).

Dual inhibition of two branches of the AA acid cascade synergistically decreases inflammatory mediators.Inflammation is a multi-staged process whose expansive phase is thought to be driven by acutely released arachidonic acid (AA) and its metabolites. Inhibition of cyclooxygenase (COX), lipoxygenase (LOX), or soluble epoxide hydrolase (sEH) is known to be anti-inflammatory. Inhibition of sEH stabilizes the cytochrome P450 (CYP450) products epoxyeicosatrienoic acids (EETs). Here we used a non-selective COX inhibitor aspirin, a 5-lipoxygenase activation protein (FLAP) inhibitor MK886, and a sEH inhibitor t-AUCB to selectively modulate the branches of AA metabolism in a lipopolysaccharide (LPS)-challenged murine model. We used metabolomic profiling to simultaneously monitor representative AA metabolites of each branch. In addition to the significant crosstalk among branches of the AA cascade during selective modulation of COX, LOX, or sEH, we demonstrated that co-administration of t-AUCB enhanced the anti-inflammatory effects of aspirin or MK886, which was evidenced by the observations that co-administration resulted in favorable eicosanoid profiles and better control of LPS-mediated hypotension as well as hepatic protein expression of COX-2 and 5-LOX. Targeted disruption of the sEH gene displayed a parallel profile to that produced by t-AUCB. These observations demonstrate a significant level of crosstalk among the three major branches of the AA cascade and that they are not simply parallel pathways. These data illustrate that inhibition of sEH by both pharmacological intervention and gene knockout enhances the anti-inflammatory effects of aspirin and MK886, suggesting the possibility of modulating multiple branches to achieve better therapeutic effects.

Keywords: Abbreviations; AA; arachidonic acid; t; -AUCB; trans; -4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid; AUDA-BE; 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester; COX; cyclooxygenase(s); CYP450; cytochrome P450; DHET; dihydroxyeicosatrienoic acid; EETs; epoxyeicosatrienoic acids; FLAP; 5-lipoxygenase activation protein; 5-HETE; 5-hydroxyeicosatetrasanoic acid; 5-HpETE; 5-(S)-hydroperoxy-6,8,11,14-eicosatetraenoic acid; LOX; lipoxygenase; LPS; lipopolysaccharide; LT; leukotriene; MK 886; 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethyl propanoic acid; NSAID; nonsteroidal anti-inflammatory drug; PG; prostaglandin; sEH; soluble epoxide hydrolase; sEHI; sEH inhibitor; TX; thromboaxaneArachidonic acid; Eicosanoid; Interaction; Metabolomics; Synergism

Anti-allergic function and regulatory mechanisms of KR62980 in allergen-induced airway inflammation by Hee Yeon Won; Hyun Jung Min; Jin Hee Ahn; Sung-Eun Yoo; Myung Ae Bae; Jeong-Ho Hong; Eun Sook Hwang (pp. 888-896).

The ligand-activated transcription factor, peroxisome proliferator-activated receptor (PPAR)γ, and its ligands inhibit pro-inflammatory cytokine production by immune cells, thus exerting anti-inflammatory activity. As a non-thiazolidinedione PPARγ ligand, KR62980 has anti-diabetic and anti-adipogenic activities, but its anti-inflammatory function has yet to be characterized. In this study, we investigated the functions and mechanisms of KR62980 in the activation and differentiation of CD4+ T helper (Th) cells by comparing its effects with those of a thiazolidinedione PPARγ ligand, rosiglitazone. KR62980 dose-dependently and significantly suppressed TCR-triggered Th cell proliferation by suppressing IL-2/IL-2Rα-mediated signaling. Both KR62980 and rosiglitazone suppressed IFNγ production in a dose-dependent manner, whereas IL-4 gene expression was specifically suppressed by only KR62980. In addition, sustained KR62980 treatment diminished Th2 cytokine production by inhibiting c-Maf expression. In vivo administration of KR62980 in a model of allergic asthma significantly attenuated eotaxin-induced eosinophil infiltration, allergic cytokine production and collagen deposition in the lung. KR62980 also decreased goblet cell hyperplasia in the airway and mucous cell metaplasia in nasal epithelium, concurrent with decreases of allergic Th2 cytokines and IL-17 in the draining lymph node. In conclusion, a novel PPARγ ligand, KR62980, suppresses in vitro Th2 cell differentiation and attenuates in vivo OVA-induced airway inflammation, suggesting a beneficial role for KR62980 in the treatment of allergic asthma and allergic rhinitis.

Keywords: KR62980; PPARγ; Rosiglitazone; Th2 cell; Allergic airway inflammation

Development of tolerance in D3 dopamine receptor signaling is accompanied by distinct changes in receptor conformation by Ligia Westrich; Sara Gil-Mast; Sandhya Kortagere; Eldo V. Kuzhikandathil (pp. 897-907).

The D3 dopamine receptor tolerance property develops after the removal of the agonist and is accompanied by distinct changes in receptor conformation which uncouples the receptor from its signaling cascade.The D3 but not D2 dopamine receptors exhibit a tolerance property in which agonist-induced D3 receptor response progressively decreases upon repeated agonist stimulation. We have previously shown that the D3 receptor tolerance property is not mediated by receptor internalization, persistent agonist binding or a decrease in receptor binding affinity. In this paper, we test the hypothesis that alterations in D3 receptor conformation underlie the tolerance property. Structural models of wild type and mutant human D3 receptors were generated using the beta adrenergic receptor crystal structure as a template. These models suggested that the agonist-bound D3 receptor undergoes conformational changes that could underlie its tolerance property. To experimentally assess changes in receptor conformation, we measured the accessibility of native cysteine residues present in the extracellular and transmembrane regions of the human D3 receptor to two different thiol-modifying biotinylating reagents. The accessibilities of the native cysteine residues present in the D3 receptor were assessed under control conditions, in the presence of agonist and under conditions that induced receptor tolerance. By comparing the accessibility of D3 receptor cysteine residues to hydrophobic and hydrophilic thiol-modifying biotinylating reagents, we show that the alteration of D3 receptor conformation during tolerance involves the net movement of cysteine residues into a hydrophobic environment. Our results show that the conformation state of the D3 receptor during tolerance is distinct from the conformation under basal and agonist-bound conditions. The results suggest that the D3 receptor tolerance property is mediated by conformational changes that may uncouple the receptor from G-protein signaling.

Keywords: Desensitization; Drd3; Modeling; Biotinylation; Signaling; GPCR

Characterization of endogenous calcium responses in neuronal cell lines by Irina Vetter; Richard J. Lewis (pp. 908-920).

An increasing number of putative therapeutic targets have been identified in recent years for the treatment of neuronal pathophysiologies including pain, epilepsy, stroke and schizophrenia. Many of these targets signal through calcium (Ca²⁺), either by directly facilitating Ca²⁺ influx through an ion channel, or through activation of G proteins that couple to intracellular Ca²⁺ stores or voltage-gated Ca²⁺ channels. Immortalized neuronal cell lines are widely used models to study neuropharmacology. However, systematic pharmacological characterization of the receptors and ion channels expressed in these cell lines is lacking. In this study, we systematically assessed endogenous Ca²⁺ signaling in response to addition of agonists at potential therapeutic targets in a range of cell lines of neuronal origin (ND7/23, SH-SY5Y, 50B11, F11 and Neuro2A cells) as well as HEK293 cells, a cell line commonly used for over-expression of receptors and ion channels. This study revealed a remarkable diversity of endogenous Ca²⁺ responses in these cell lines, with one or more cell lines responding to addition of trypsin, bradykinin, ATP, nicotine, acetylcholine, histamine and neurotensin. Subtype specificity of these responses was inferred from agonist potency and the effect of receptor subtype specific antagonist. Surprisingly, HEK293 and SH-SY5Y cells responded to the largest number of agonists with potential roles in neuronal signaling. These findings have implications for the heterologous expression of neuronal receptors and ion channels in these cell lines, and highlight the potential of neuron-derived cell lines for the study of a range of endogenously expressed receptors and ion channels that signal through Ca²⁺.

Keywords: Abbreviations; Ca; ²⁺; calcium ion; GPCR; G-protein coupled receptor; ATP; adenosine triphosphate; TRPV1; transient receptor potential vanilloid 1; PAR; protease-activated receptor; GABA; γ-aminobutyric acid; DMEM; Dulbeccos’ Modified Eagle Medium; FBS; foetal bovine serum; PBS; phosphate buffered saline; PSS; physiological salt solution; EDTA; ethylenediaminetetraacetic acid; HAT; hypoxanthine, aminopterin and thymidine; db-cAMP; N6,2′-O-dibutyryladenosine 3′:5′ cyclic monophosphate; NGF; nerve growth factor; BAPTA; 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; AFU; arbitrary fluorescence unit; SEM; standard error of the mean; NMDA; N-methyl-; d; -aspartic acid; pFHHSiD; p; -fluorohexahydro-sila-difenidol hydrochloride; PLC; phopholipase C; Fluo-4-AM; Fluo-4 acetomethoxyester; HEK293; human embryonic kidney cells; RPMI; Roswell Park Memorial Institute; PDL; Poly-; d; -lysineEndogenous; Neuronal cell line; Characterization; Calcium; Receptor; Ion channel

Modulation of human placental P-glycoprotein expression and activity by MDR1 gene polymorphisms by Sarah J. Hemauer; Tatiana N. Nanovskaya; Sherif Z. Abdel-Rahman; Svetlana L. Patrikeeva; Gary D.V. Hankins; Mahmoud S. Ahmed (pp. 921-925).

Single nucleotide polymorphisms C1236T, C3435T, and G2677T/A in the MDR1 gene are associated with reduced placental P-gp protein expression yet increased transport of P-gp substrate, paclitaxel.The ABC transporter P-glycoprotein is a product of the MDR1 gene and its function in human placenta is to extrude xenobiotics from the tissue thus decreasing fetal exposure. The goal of this investigation was to examine the effect of three polymorphisms in the MDR1 gene on the expression and activity of placental P-gp. In 199 term placentas examined, the C1236T variant was associated with 11% lower P-gp protein expression than wild-type, while the C3435T and G2677T/A variants each were associated with a 16% reduction ( p<0.05). Homozygotes for the C1236T and C3435T variant allele (TT) were associated with 42% and 47% increase in placental P-gp transport activity, respectively ( p=0.04 and p=0.02) of the prototypic substrate, [³H]-paclitaxel. These findings indicate that the C3435T and G2677T/A SNPs in MDR1 are significantly associated with decreased placental P-gp protein expression, while the C1236T and C3245T homozygous variants are significantly associated with an increase in its efflux activity.

Keywords: Placenta; Membrane vesicle; p-Glycoprotein; MDR1; Polymorphism

The PI3K/Akt/FOXO3a/p27^Kip1 signaling contributes to anti-inflammatory drug-suppressed proliferation of human osteoblasts by Ching-Ju Li; Je-Ken Chang; Chia-Hsuan Chou; Gwo-Jaw Wang; Mei-Ling Ho (pp. 926-937).

Akt has been reported to suppress p27^Kip1 promoter activity through Forkhead box O (FOXO) in different kinds of cells. Previous studies indicated that anti-inflammatory drugs up-regulated p27^Kip1, and this effect might play an important role in anti-inflammatory drug-induced cell cycle arrest of human osteoblasts (hOBs). In this study, we hypothesized that these drugs might increase p27^Kip1 expression in hOBs by altering the Akt/FOXO signaling. We tested this hypothesis by examining the influences of three anti-inflammatory drugs on the levels and/or activities of Akt, FOXO and p27^Kip1 as well as the relationship between these factors and proliferation of hOBs. We tested the effects of indomethacin (10⁻⁵ and 10⁻⁴M), celecoxib (10⁻⁶ and 10⁻⁵M), and dexamethasone (10⁻⁷ and 10⁻⁶M) using PI3K inhibitor, LY294002 (10⁻⁵M) as the basis of comparison. The three drugs suppressed the canonical level of phosphorylated Akt in hOBs. This was accompanied by elevated FOXO3a level and increased promoter activity, mRNA expression and protein level of p27^Kip1. Furthermore, the anti-inflammatory drugs suppressed the EGF-induced increases in proliferation, phosphorylation, and nucleus translocation of Akt. Simultaneously, they suppressed EGF-induced decreases of FOXO3a nucleus accumulation and p27^Kip1 mRNA expression. On the other hand, FOXO silencing significantly attenuated the drug-induced up-regulation of p27^Kip1 and suppression of proliferation in hOBs. To the best of our knowledge, this study represents the first to demonstrate that Akt/FOXO3a/p27^Kip1 pathway contributes to suppression of hOB proliferation by anti-inflammatory drugs. We suggest that anti-inflammatory drugs suppress hOB proliferation, at least partly, through inactivating Akt, activating FOXO3a, and eventually up-regulating p27^Kip1 expression.

Keywords: Anti-inflammatory drugs; Human osteoblasts; PI3K/Akt signal transduction; Forkhead box O (FOXO); p27; ^Kip1; Proliferation

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Biochemical Pharmacology (v.79, #6)