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Biochemical Pharmacology (v.71, #8)

Editorial Advisory Board (pp. iii).

The challenge of drug discovery of a GPCR target: Analysis of preclinical pharmacology of histamine H₃ antagonists/inverse agonists by A.A. Hancock (pp. 1103-1113).

Although the histamine H₃ receptor was identified pharmacologically in 1983, and despite widespread pharmaceutical interest in the target, no compound interacting specifically with this site has undergone successful clinical examination to develop the necessary proof-of-concept data. Therefore, clinical knowledge of the therapeutic potential of H₃ receptor antagonists in neuropsychiatric diseases, in metabolic diseases or in sleep disorders has yet to determine if the preclinical data that show broad efficacy in animal models of the aforementioned states are relevant to current unmet medical needs. H₃ receptors are complex, with species-related sequence differences that impact pharmacological responses. The receptors have a complex gene organization that provides opportunity for multiple slice isoforms, most of which remain poorly characterized even within a species. H₃ receptors are constitutively active, although the extent of this could vary either between species and/or receptor splice isoforms, both of which may provide opportunity for preferential coupling to different G-proteins. Thus, it is not surprising that the pharmacological effects of known H₃ ligands are complex and diverse, since these agents may act both as agonists and antagonists in different systems. Moreover, other compounds show inverse agonism in some models but neutral antagonist activity in others. Some of this diversity may be related to different ligand-dependent receptor activation states or to the effects of key amino acids important for ligand recognition. This commentary provides an overview of these complexities as applied to the H₃ receptor and the challenges these intricacies create for drug discovery.

Keywords: H; ₃; receptor; Histamine; GPCR; Drug discovery; Cognition; Antagonist/inverse agonist

Hexadecylphosphocholine disrupts cholesterol homeostasis and induces the accumulation of free cholesterol in HepG2 tumour cells by Jos� M. Jim�nez-L�pez; Mar�a P. Carrasco; Carmen Marco; Josefa L. Segovia (pp. 1114-1121).

Hexadecylphosphocholine (HePC) is a synthetic lipid belonging to the alkylphosphocholines (APC), a new group of antiproliferative agents that are proving to be promising candidates in anticancer therapy. We reported in a previous study that HePC interferes with phosphatidylcholine (PC) synthesis in HepG2 cells via both CDP-choline and phosphatidylethanolamine (PE) methylation. We have subsequently extended our studies to show that HePC interferes with sphingolipid metabolism by hindering the formation of sphingomyelin (SM), an effect accompanied by a substantial increase in the incorporation of the exogenous lipogenic precursors into ceramides. Interestingly, we demonstrate for the first time that HePC strongly inhibits the esterification of free cholesterol (FC) by acting at the level of acyl CoA:cholesterol acyltransferase (ACAT) (EC 2.3.1.26) activity. This effect is accompanied by a considerable increase in the synthesis of cholesterol, which leads to a rise in the levels of FC in cells. We are left in no doubt that the imbalance in the metabolism of membrane-lipid components vital to cell survival may well be responsible for the observed DNA fragmentation and activation of caspase-3, an enzyme involved in the cell apoptosis found in this study.

Keywords: Abbreviations; HePC; hexadecylphosphocholine; APC; alkylphosphocholine; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PS; phosphatidylserine; SM; sphingomyelin; DAG; diacylglycerol; TAG; triacylglycerol; CE; cholesteryl ester; FC; free cholesterol; ACAT; acyl CoA:cholesterol acyltransferase; CEH; cholesteryl ester hydrolase; FBS; foetal bovine serum; MEM; minimum essential medium; DTT; dithiothreitol; TLC; thin-layer chromatographyAlkylphosphocholines; Apoptosis; Cholesterol metabolism; Cholesteryl esters; Phosphatidylcholine; Sphingolipids

Pyridine N-oxide derivatives inhibit viral transactivation by interfering with NF-κB binding by Miguel Stevens; Christophe Pannecouque; Erik De Clercq; Jan Balzarini (pp. 1122-1135).

Pyridine N-oxide derivatives represent a new class of anti-HIV compounds for which some members exclusively inhibit HIV-1 RT, whereas other members act, additionally or alternatively, at a post-integrational event in the replicative cycle of HIV. A prototype pyridine N-oxide derivative, JPL-32, inhibited tumor necrosis factor alpha (TNF-α)-induced HIV-1 expression in latently HIV-1-infected OM-10.1 and U1 cells, which could be reversed by the addition of N-acetyl-l-cysteine (NAC). The reversal of the antiviral activity of JPL-32 by NAC suggested the possible role of a redox-sensitive factor as target of inhibition. Indeed, when nuclear extracts of TNF-α-stimulated OM-10.1 and U1 cells cultured in the presence of JPL-32 were analyzed by an electrophoretic mobility shift assay (EMSA), a dose-dependent inhibition of DNA binding of nuclear NF-κB was observed, which could be reversed by the addition of NAC. JPL-32 did not inhibit the release and subsequent degradation of IκBα, nor did JPL-32 affect the nuclear translocation of NF-κB. EMSA revealed that the inhibition of the NF-κB DNA binding activity by JPL-32 could be reversed by the addition of reducing agents such as dithiothreitol or β-mercaptoethanol. Moreover, JPL-32 was able to directly oxidize the thiol groups on the purified p50 subunit of recombinant NF-κB. The oxidative modification of the thiol groups on NF-κB by JPL-32 could be ascribed to the intracellular pro-oxidant effect of JPL-32. Consequently, JPL-32 was able to increase the intracellular glutathione (GSH) levels and to induce apoptosis in a dose-dependent way.

Keywords: Abbreviations; BSO; buthionine sulfoximine; CMV; cytomegalovirus; DCFH-DA; 2′,7′-dichlorofluorescein diacetate; DTT; dithiothreitol; EMSA; electrophoretic mobility shift assay; GFP; green fluorescent protein; HIV; human immunodeficiency virus; IκBα; inhibitor of NF-κB; LTR; long terminal repeat; M/M; human primary monocytes/macrophages; NAC; N; -acetyl-; l; -cysteine; NF-κB; nuclear factor κB; NNRTI; non-nucleoside reverse transcriptase inhibitor; TNF-α; tumor necrosis factor α; TRX; thioredoxinPyridine; N; -oxide derivatives; Human immunodeficiency virus; Cytomegalovirus; Transactivation; NF-κB; Redox regulation

Cytotoxic efficacy of an anthraquinone linked platinum anticancer drug by R.A. Alderden; H.R. Mellor; S. Modok; T.W. Hambley; R. Callaghan (pp. 1136-1145).

Platinum complexes are widely used in cancer chemotherapy; however, they are associated with toxicity, high “non-specific�? reactivity and relatively poor pharmacokinetic profiles. In particular, their low cellular uptake and rapid metabolic inactivation means that the amount of “active�? drug reaching the nuclear compartment is low. Our strategy to facilitate nuclear accumulation was to introduce a hydrophobic anthraquinone (1C3) moiety to the Pt-complex. Anthraquinones are known to readily intercalate into DNA strands and hence, the Pt-1C3 complex may represent an effective system for the delivery of the platinum moiety to nuclear DNA. Efficacy of the complex was determined by measuring the extent and potency of cytotoxicity in comparison to cisplatin and an anthraquinone based anticancer drug, doxorubicin. The Pt-1C3 complex generated higher levels of cytotoxicity than cisplatin, with a potency of 19±4μM in the DLD-1 cancer cell line. However, this potency was not significantly different to that of the 1C3 moiety alone. To examine the reason for the apparent lack of platinum related cytotoxicity, the cellular distribution was characterised. Confocal fluorescence microscopy indicated that the Pt-1C3 complex was rapidly sequestered into lysosomes, in contrast to the nuclear localisation of doxorubicin. In addition, there was negligible DNA associated Pt following administration of the novel complex. Thus, the addition of a 1C3 moiety generated sequestration of the complex to lysosomes, thereby preventing localisation to the nucleus.

Keywords: Platinum; Chemotherapy; Cancer; Lysosomes; Anthraquinone; Cytotoxicity

The effects of cannabinoids on P-glycoprotein transport and expression in multidrug resistant cells by M.L. Holland; J.A. Panetta; J.M. Hoskins; M. Bebawy; B.D. Roufogalis; J.D. Allen; J.C. Arnold (pp. 1146-1154).

Cannabis is the most widely used illicit drug in the world. Cannabinoids are used therapeutically by some patients as they have analgesic, anti-emetic and appetite stimulant properties which palliate adverse symptoms. Use of these agents in an oncology setting raises the question of whether they act to modulate the effectiveness of concurrently administered anti-cancer drugs. The transporter, P-glycoprotein (P-gp) confers multiple drug resistance (MDR) by effluxing a diverse array of anti-cancer agents. This study was undertaken to examine the effect of cannabinoids on P-gp. Unlike the known P-gp inhibitor, PSC833, short 1h exposure to three plant-derived cannabinoids, cannabinol (CBN), cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (THC) and the synthetic cannabinoid receptor agonist, WIN55, 212-2 (WIN) did not inhibit the efflux of the P-gp substrate Rhodamine 123 (Rh123) in either a drug-selected human T lymphoblastoid leukaemia cell line (CEM/VLB₁₀₀) or in a mouse fibroblast MDR1 transfected cell line (77.1). However, in CEM/VLB₁₀₀ cells, prolonged 72h exposure to the cannabinoids, THC and CBD, decreased P-gp expression to a similar extent as the flavonoid, curcumin (turmeric). This correlated with an increase in intracellular accumulation of Rh123 and enhanced sensitivity of the cells to the cytotoxic actions of the P-gp substrate, vinblastine. Taken together, these results provide preliminary evidence that cannabinoids do not exacerbate P-gp mediated MDR. Further, plant-derived cannabinoids are moderately effective in reversing MDR in CEM/VLB₁₀₀ cells by decreasing P-gp expression.

Keywords: JEL classification; Antibiotics and chemotherapeuticsAbbreviations; MDR; multiple drug resistance; P-gp; P-glycoprotein; THC; Δ; ⁹; -tetrahydrocannabinol; CBD; cannabidiol; CBN; cannabinol; Rh123; Rhodamine 123; WIN; WIN 55, 212-2; Et-743; Ecteinascidin-743P-glycoprotein; Multiple drug resistance; Cannabinoids; Cannabidiol; Δ; ⁹; -Tetrahydrocannabinol; Palliative care

Circadian regulation of mouse topoisomerase I gene expression by glucocorticoid hormones by Yukako Kuramoto; Koujirou Hata; Satoru Koyanagi; Shigehiro Ohdo; Hiroshi Shimeno; Shinji Soeda (pp. 1155-1161).

Because glucocorticoid hormones modulate various biological processes, the endogenous rhythm of their secretion is thought to be an important factor affecting the efficacy and/or toxicity of many drugs. Topoisomerase I (Topo I) is a nuclear target of the anticancer drug camptothecin (CPT). In this study, we demonstrate that Topo I expression in tumor-bearing mice and the efficacy of CPT on the tumor are affected by the 24-h variation in circulating glucocorticoid levels. A single administration of corticosterone (CORT) to the tumor-bearing mice resulted in a significant increase in Topo I mRNA levels not only in the tumor masses but also in other healthy tissues such as liver and skeletal muscle. The CORT-induced increase in Topo I mRNA was suppressed by pretreating the mice with RU486, a glucocorticoid receptor antagonist. Significant 24-h oscillations in the Topo I mRNA levels were observed in the tumor and healthy liver without exogenous CORT, and were eliminated by adrenalectomy of the mice. This result suggests that endogenous glucocorticoid hormones are involved in the circadian regulation of Topo I gene expression. Furthermore, the anti-tumor efficacy of the Topo I inhibitor CPT-11 on the tumor-bearing mice was enhanced by administering the drug at the time when the Topo I activity was increased. Our present results demonstrate that glucocorticoid is involved in the 24-h oscillation mechanism of Topo I gene expression and suggest that monitoring the circadian rhythm in Topo I activity is useful for choosing the most appropriate time of day to administer of Topo I inhibitors.

Keywords: Abbreviations; Topo I; topoisomerase I; CPT; camptothecin; CORT; corticosterone; CPT-11; 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin; ICR; Institute of Cancer Research; RU486; 11β-(4-dimethyl-amino)phenyl-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-oneTopoisomerase I; CPT-11; Corticosterone; Adrenalectomy; Circadian rhythm; Chronopharmacotherapy

Mitochondrial localization and activity of P-glycoprotein in doxorubicin-resistant K562 cells by Eliza Munteanu; Mireille Verdier; Fabienne Grandjean-Forestier; Christophe Stenger; Chantal Jayat-Vignoles; Sylvie Huet; Jacques Robert; Marie-H�l�ne Ratinaud (pp. 1162-1174).

It is now well-established that P-glycoprotein 170 (P-gp), an efflux pump involved in multidrug resistance (MDR) is overexpressed at the plasma membrane of doxorubicin-resistant K562 leukemia cells. Nevertheless, several results suggested: (i) that P-gp-mediated drug efflux was not the only mechanism involved in resistance; (ii) that intracellular compartments could accumulate the drug, preventing it from reaching its nuclear targets; (iii) that agents able to reverse multidrug resistance may lead to intracellular drug redistribution. We have studied the localization of P-gp in mitochondria as well as its functional properties in this compartment. Using several monoclonal antibodies (MoAbs) directed against different P-gp epitopes, a protein was detected in the cytoplasm of two doxorubicin-resistant K562 sublines and, by confocal laser scanning microscopy, this protein was shown to co-localize in the Golgi apparatus and in mitochondria, in equivalent proportions. Purified mitochondria were isolated from K562 cell variants; the presence of a protein of about 170kDa and reacting with several anti-P-gp antibodies was assessed in MDR cells by Western blotting and flow cytometry. Functional assays have shown that mitochondrial P-gp was involved in doxorubicin accumulation inside the organelle but not in its efflux, suggesting an orientation of P-gp in the mitochondrial membrane inverse to that observed in the plasma membrane. A potential role for mitochondrial P-gp in MDR cells would be to protect the nucleus from doxorubicin. This is the first demonstration of the presence and functional activity of P-gp in mitochondria of MDR cells.

Keywords: Abbreviations; ABC; ATP-binding cassette protein; FITC; fluorescein isothiocyanate; LRP; lung resistance protein; MDR; multidrug resistance; PE; phycoerythrin; P-gp; P-glycoprotein 170Multidrug resistance; P-glycoprotein; Mitochondria; Doxorubicin; Intracellular P-gp

Stimulation of serum- and glucocorticoid-regulated kinase-1 gene expression by endothelin-1 by Sabine C. Wolf; Michael Schultze; Teut Risler; Timo Rieg; Florian Lang; Klaus Schulze-Osthoff; Bernhard R. Brehm (pp. 1175-1183).

The serum- and glucocorticoid-regulated kinase-1 (SGK1) participates in the regulation of sodium homeostasis and blood pressure by mineralocorticoids. Aldosterone rapidly induces SGK1 transcription, which contributes to the activation of renal epithelial sodium channels. Another important regulator of blood pressure is the vasoactive hormone endothelin-1 (ET-1) that is systemically upregulated in chronic renal failure. In the present study, we investigated whether ET-1 modulates SGK1 expression, and thereby might explain some of its hypertensive effects. As assessed by real-time PCR analysis, ET-1 triggered the rapid increase of SGK1 mRNA levels in A-10 smooth muscle cells and also in intact aortas of adult rats. In A-10 cells transcriptional activation was associated with a more than 6-fold upregulation of SGK1 protein expression and in similar range as found after treatment with aldosterone. A stimulatory effect of ET-1 was not only observed in isolated cells, but also in an animal model. Upon subtotal nephrectomy (SNX) of rats, myocardial ET-1 levels strongly increased, which was followed by a more than 2-fold induction of SGK1 expression in the left ventricle. The myocardial upregulation of SGK1 was completely abrogated by a specific ET_A receptor antagonist, thereby substantiating the in vivo role of ET-1 in SGK1 expression. Thus, these data demonstrate that ET-1 increases expression of SGK1 in vivo and in vitro, and therefore indicate that SGK1 upregulation might be involved in ET-1-dependent regulation of blood pressure and cardiac modelling during mild renal failure.

Keywords: Abbreviations; DIG; digoxigenin; ENaC; epithelial Na+ channel; ET-1; endothelin-1; PBST; phosphate-buffered saline/Tween-20; RT-PCR; reverse transcription polymerase chain reaction; SGK1; serum- and glucocorticoid-regulated kinase-1; SNX; subtotal nephrectomy; TBS; Tris-buffered salineSerum- and glucocorticoid-regulated kinase-1; Endothelin-1; Aldosterone; Smooth muscle cells; Myocardium; Blood pressure

Microarray profiling of gene expression in human adipocytes in response to anthocyanins by Takanori Tsuda; Yuki Ueno; Toshikazu Yoshikawa; Hitoshi Kojo; Toshihiko Osawa (pp. 1184-1197).

Adipocyte dysfunction is strongly associated with the development of obesity and insulin resistance. It is accepted that the regulation of adipocytokine secretion or the adipocyte specific gene expression is one of the most important targets for the prevention of obesity and amelioration of insulin sensitivity. Recently, we demonstrated that anthocyanins, which are pigments widespread in the plant kingdom, have the potency of anti-obesity in mice and the enhancement adipocytokine secretion and its gene expression in adipocytes. In this study, we have shown the gene expression profile in human adipocytes treated with anthocyanins (cyanidin 3-glucoside; C3G or cyanidin; Cy). The human adipocytes were treated with 100μM C3G, Cy or vehicle for 24h. The total RNA from the adipocytes was isolated and carried out GeneChip microarray analysis. Based on the gene expression profile, we demonstrated the significant changes of adipocytokine expression (up-regulation of adiponectin and down-regulation of plasminogen activator inhibitor-1 and interleukin-6). Some of lipid metabolism related genes (uncoupling protein2, acylCoA oxidase1 and perilipin) also significantly induced in both common the C3G or Cy treatment groups. These studies have provided an overview of the gene expression profiles in human adipocytes treated with anthocyanins and demonstrated that anthocyanins can regulate adipocytokine gene expression to ameliorate adipocyte function related with obesity and diabetes that merit further investigation.

Keywords: Abbreviations; ACOX1; acylCoA oxidase1; CCAAT/enhancer binding protein; C/EBP; C3G; cyanidin 3-; O; -β-; d; -glucoside; Cy; cyanidin; DMEM; Dulbecco's modified Eagle's medium; gp130; glycoprotein 130; IL; interleukin; OCM; oncostatin M; PAI-1; plasminogen activator inhibitor-1; PLN; perilipin; PPAR; peroxisome proliferator-activated receptor; TZD; thiazolidinediones; UCP2; uncoupling protein 2Anthocyanin; Cyanidin; Adipocyte; Gene expression profile; Adipocytokine; DNA microarray

Rengyolone inhibits inducible nitric oxide synthase expression and nitric oxide production by down-regulation of NF-κB and p38 MAP kinase activity in LPS-stimulated RAW 264.7 cells by Jin Hee Kim; Dong Hyun Kim; Seung Hwa Baek; Ho Jae Lee; Mee Ree Kim; Ho Jeong Kwon; Choong-Hwan Lee (pp. 1198-1205).

Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. Rengyolone, a cyclohexylethanoid isolated from the fruits of Forsythia koreana, exhibits anti-inflammatory activity with unknown mechanism. In this study, we found that rengyolone has a strong inhibitory effect on the production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α). Rengyolone also inhibited inducible nitric oxide synthase (iNOS) gene expression and cyclooxygenase 2 (COX-2) by lipopolysaccharide (LPS). In order to explore the mechanism responsible for the inhibition of iNOS gene expression by rengyolone, we investigated its effect on LPS-induced nuclear factor-κB (NF-κB) activation. The LPS-induced DNA binding activity of NF-κB was significantly inhibited by rengyolone, and this effect was mediated through inhibition of the degradation of inhibitory factor-κBα and phosphorylation of p38 MAP kinase. Furthermore, rengyolone suppressed the expression of ICE protein in IL-1β-treated D10S cells. Taken together, these results suggest that rengyolone attenuates the inflammation through inhibition of NO production and iNOS expression by blockade of NF-κB and p38 MAPK activation in LPS-stimulated RAW 264.7 cells.

Keywords: Rengyolone; Forsythia koreana; Nitric oxide; Inducible nitric oxide; Nuclear factor-κB; p38 MAPK

Inhibition of TNFα-induced activation of nuclear factor κB by kava ( Piper methysticum) derivatives by Florence Folmer; Romain Blasius; Franck Morceau; Jioji Tabudravu; Mario Dicato; Marcel Jaspars; Marc Diederich (pp. 1206-1218).

The inducible transcription factor nuclear factor κB (NF-κB) plays a central role in the regulation of immune, inflammatory and carcinogenic responses. While normal activation of NF-κB is required for cell survival and immunity, its deregulated expression is a characteristic of inflammatory and infectious diseases. In this study, we investigated the molecular mechanisms induced by lactones and chalcones isolated from Fijian kava ( Piper methysticum) used in traditional medicine against urinary tract infections and asthma. In order to understand underlying regulatory mechanisms, inhibition of both NF-κB-driven reporter gene expression and TNFα-induced binding of NF-κB to a consensus response element was achieved at concentrations of 320μM (flavokavain A), 175μM (flavokavain B) and 870μM (kavain and dihydrokavain). Moreover, kavain and flavokavains A and B treatment led to inhibition of both inhibitor of κB (IκB) degradation and subsequent translocation of p50 and p65 NF-κB subunits from the cytoplasm to the nucleus as shown by Western blot analysis. Additionally, kinase selectivity screening demonstrates that flavokavain A, but not kavain, nor flavokavain B, inhibits the IκB kinase (IKK) as well as PRAK (p38-regulated/activated kinase), MAPKAP-K3 (MAPK-activated protein kinase 3), DYRK1A (dual-specificity tyrosine-phosporylated and regulated kinase 1A) and Aurora B. Altogether, these results give a first insight into anti-inflammatory mechanisms triggered by traditionally used chemopreventive kava compounds.

Keywords: JEL classification; 6. Molecular PharmacologyAbbreviations; BSA; bovine serum albumin; DW; dry weight; DYRK1A; dual-specificity tyrosine-phosporylated and regulated kinase 1A; FCS; foetal calf serum; HPLC; high performance liquid chromatography; IκB; inhibitory protein of nuclear factor κB; IKK; IκB kinase; EMSA; electrophoretic mobility shift assay; Luc; luciferase; MAPK; mitogen-activated protein kinase; MAPKAP-K3; MAPK-activated protein kinase 3; NF-κB; nuclear factor κB; NMR; nuclear magnetic resonance; n.s.; non-specific binding; PBS; phosphate buffered saline; PRAK; p38-regulated/activated kinase; S.D.; standard deviation; TNFα; tumour necrosis factor αChalcones; Ethnopharmacology; Kava; Lactones; NF-κB; TNFα; K562

Histamine H2 receptor overexpression induces U937 cell differentiation despite triggered mechanisms to attenuate cAMP signalling by Federico Monczor; Natalia Fernandez; Eugenia Riveiro; Alejandro Mladovan; Alberto Baldi; Carina Shayo; Carlos Davio (pp. 1219-1228).

Knowing that cell-surface receptors that recognize and respond to extracellular stimuli are key components for the regular communication between individual cells required for the survival of any living organism, the aim of the present work was to investigate the effect of H2R overexpression on the U937 signal transduction pathway and its consequences on cell proliferation and differentiation. The overexpression of H2R led to an increase in cAMP basal levels, a leftward shift of agonist concentration–response curves, and similar maximal response to agonist treatment, suggesting that overexpressed H2Rs act as functional spare receptors. In this system cells triggered several mechanisms tending to restore cAMP basal levels to those of the naïve cells. H2R overexpression induced PDE activity stimulation and GRK2 overexpression. In spite of the onset of these regulatory mechanisms, H2 agonist and rolipram treatments induced the terminal differentiation of the H2R overexpressed clone, conversely to the naïve cells. Present findings show that stably H2R overexpression alters cAMP signalling as the result of not only the amounts of second messenger generated but also the activation or upregulation of various components of signalling cascade, leading to an adapted biologically unique system. This adaptation may represent an advantage or a disadvantage, depending on the biological system, but in any case, the existence of compensatory mechanisms should be considered when a clinical treatment is designed.

Keywords: Abbreviations; AC; adenylyl cyclase; ATRA; all trans retinoic acid; Ca; ²⁺; i; intracellular Ca; ²⁺; dbcAMP; dibutyryl cAMP; Fura 2-AM; Fura 2 acetoxymethyl ester; GPCR; G protein-coupled receptor; GRK; G protein-coupled receptor kinase; G-418; geneticin; H2R; histamine H2 receptor; HTMT dimaleate; 6[2-(4-imidazolyl)ethylamino]-; N; -(4-trifluoromethylphenyl)heptane-carboxamide; IBMX; isobutylmethylxanthine; PDE4; cyclic nucleotide phosphodiesterase 4; PGE2; prostaglandin E2; rhC5a; recombinant human C5aHistamine receptors; Receptor overexpression; cAMP; PDE; GRK; Cell differentiation

Inhibition of interleukin-4 production in activated T cells via the downregulation of AP-1/NF-AT activation by N-lauroyl-d-erythro-sphingosine and N-lauroyl-d-erythro-C₂₀-sphingosine by Jin Park; Seung Hyun Kim; Qian Li; Young-Tae Chang; Tae Sung Kim (pp. 1229-1239).

Allergic diseases are hypersensitivity disorders that are associated with the generation of specific immunoglobulin E (IgE) in response to environmental allergens. Interleukin (IL)-4, which is primarily produced by the CD4⁺ T cells, is an important stimulus for the switching of the antibody isotype to IgE in both mice and humans. In a previous study, we demonstrated that ceramide derivatives coupled with a lauroyl group exerted strong inhibitory effects on IL-4 production in T cells. In this study, we attempted to characterize the mechanisms underlying the inhibition of IL-4 production in T cells. Two ceramide derivatives, N-lauroyl-d-erythro-sphingosine and N-lauroyl-d-erythro-C₂₀-sphingosine (hereafter abbreviated as LES and LECS, respectively), were shown to significantly inhibit the production of IL-4 in both primary CD4⁺ T cells and EL4 T thymoma cells in a dose-dependent manner. LES and LECS also inhibited the activity of the IL-4 gene promoter in EL4 cells transiently transfected with IL-4 gene promoter constructs, but this effect was impaired in EL4 cells that had been transfected with an IL-4 promoter construct deleted of a P4 site harboring the AP-1 and NF-AT binding sites. Furthermore, LES and LECS inhibited the DNA binding activities of both AP-1 and NF-AT transcription factors. In addition, LES and LECS were demonstrated to suppress PMA-stimulated PKC activity, although they exerted no significant effects on the protein levels of the conventional PKCs. These results indicate that the ceramides, LES and LECS, may inhibit the production of IL-4 in the activated T cells, via the downregulation of AP-1/NF-AT activation and PKC activity.

Keywords: Abbreviations; AP-1; activating protein-1; BECS; N; -butyryl-; d; -erythro-C; ₂₀; -sphingosine; BES; N; -butyryl-; d; -erythro-sphingosine; ELISA; enzyme-linked immunosorbent assay; EMSA; electrophoretic mobility shift assay; IL; interleukin; KLH; keyhole limpet hemacyanin; LECS; N; -lauroyl-; d; -erythro-C; ₂₀; -sphingosine; LES; N; -lauroyl-; d; -erythro-sphingosine; NF-AT; nuclear factor of activated T cell; PKC; protein kinase C; PMA; phorbol 12-myristate 13-acetateCeramide; Interleukin-4; T cells; Allergy; Activating protein-1; Nuclear factor of activated T cell

Inhibition of neurite outgrowth in differentiating mouse N2a neuroblastoma cells by phenyl saligenin phosphate: Effects on MAP kinase (ERK 1/2) activation, neurofilament heavy chain phosphorylation and neuropathy target esterase activity by Alan J. Hargreaves; Maxine J. Fowler; Magdalini Sachana; John Flaskos; Mary Bountouri; Ian C. Coutts; Paul Glynn; Wayne Harris; W. Graham McLean (pp. 1240-1247).

Sub-lethal concentrations of the organophosphate phenyl saligenin phosphate (PSP) inhibited the outgrowth of axon-like processes in differentiating mouse N2a neuroblastoma cells (IC₅₀ 2.5μM). A transient rise in the phosphorylation state of neurofilament heavy chain (NFH) was detected on Western blots of cell extracts treated with 2.5μM PSP for 4h compared to untreated controls, as determined by a relative increase in reactivity with monoclonal antibody Ta51 (anti-phosphorylated NFH) compared to N52 (anti-total NFH). However, cross-reactivity of PSP-treated cell extracts was lower than that of untreated controls after 24h exposure, as indicated by decreased reactivity with both antibodies. Indirect immunofluorescence analysis with these antibodies revealed the appearance of neurofilament aggregates in the cell bodies of treated cells and reduced axonal staining compared to controls. By contrast, there was no significant change in reactivity with anti-α-tubulin antibody B512 at either time point. The activation state of the MAP kinase ERK 1/2 increased significantly after PSP treatment compared to controls, particularly at 4h, as indicated by increased reactivity with monoclonal antibody E-4 (anti-phosphorylated MAP kinase) but not with polyclonal antibody K-23 (anti-total MAP kinase). The observed early changes were concomitant with almost complete inhibition of the activity of neuropathy target esterase (NTE), one of the proposed early molecular targets in organophosphate-induced delayed neuropathy (OPIDN).

Keywords: Abbreviations; DFP; diisopropyl phosphorofluoridate; ERK; extracellular signal-regulated kinase; MAP kinase; mitogen-activated protein kinase; NFH; neurofilament heavy chain; NTE; neuropathy target esterase; OP; organophosphate; OPIDN; OP-induced delayed neuropathy; PSP; phenyl saligenin phosphate; T; O; CP; tri; ortho; cresyl phosphate; SC; O; TP; saligenin cyclic-; O; -tolyl phosphate; T; P; CP; tri; para; cresyl phosphatePhenyl saligenin phosphate; Mouse N2a neuroblastoma; Axon outgrowth; Neurofilament heavy chain; Neuropathy target esterase; MAP kinase (ERK1/2)

Pharmacological characterisation of the plant sesquiterpenes polygodial and drimanial as vanilloid receptor agonists by Eunice Andr�; Barbara Campi; Marcello Trevisani; Juliano Ferreira; �ngela Malheiros; Rosendo A. Yunes; Jo�o B. Calixto; Pierangelo Geppetti (pp. 1248-1254).

This study was designed to assess the participation of transient receptor potential vanilloid 1 (TRPV1) in the biological effects induced by the plant-derived sesquiterpenes polygodial and drimanial. In rat isolated urinary bladder, polygodial and drimanial produced a tachykinin-mediated contraction that was inhibited by combination of NK₁ and NK₂ tachykinin receptor antagonists, SR 140333 and SR 48968. Furthermore, two different TRPV1 antagonists, capsazepine and ruthenium red prevented the contraction induced by both compounds. In addition, capsaicin, polygodial and drimanial displaced in a concentration-dependent manner the specific binding sites of [³H]-resiniferatoxin to rat spinal cord membranes, with a IC₅₀ values of 0.48, 4.2 and 3.2μM, respectively. Likewise, capsaicin, polygodial and drimanial promoted an increase of [⁴⁵Ca²⁺] uptake in rat spinal cord synaptosomes. In cultured rat trigeminal neurons, polygodial, drimanial and capsaicin were also able to significantly increase the intracellular Ca²⁺ levels, effect that was significantly prevented by capsazepine. Together, the present results strongly suggest that the pharmacological actions of plant-derived sesquiterpenes polygodial and drimanial, seem to be partially mediated by activation of TRPV1. Additional investigations are needed to completely define the pharmacodynamic properties of these sesquiterpenes.

Keywords: Polygodial; Drimanial; TRPV1; Trigeminal ganglion neuron

The effect of methadone and buprenorphine on human placental aromatase by Olga L. Zharikova; Sujal V. Deshmukh; Tatiana N. Nanovskaya; Gary D.V. Hankins; Mahmoud S. Ahmed (pp. 1255-1264).

Methadone and buprenorphine (BUP) are used for treatment of the pregnant opiate addict. CYP19/aromatase is the major placental enzyme responsible for the metabolism of methadone to 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and BUP to norbuprenorphine (norBUP). The aim of this investigation was to determine the effects of methadone and BUP on the activity of placental microsomal aromatase in the conversion of its endogenous substrates testosterone to 17β-estradiol (E₂) and 16α-hydroxytestosterone (16-OHT) to estriol (E₃). The conversion of testosterone and 16-OHT by human placental microsomes exhibited saturation kinetics, and the apparent K_m values were 0.2±1 and 6±3μM, respectively. V_max values for E₂ and E₃ formation were 70±16 and 28±10pmol/mg proteinmin, respectively. Also, data obtained revealed that methadone and BUP are competitive inhibitors of testosterone conversion to E₂ and 16-OHT to E₃. The K_i for methadone inhibition of E₂ and E₃ formation were 393±144 and 53±28μM, respectively, and for BUP the K_i was 36±9 and 6±1μM. The higher potency of the two opiates and their metabolites in inhibiting E₃ formation is in agreement with the lower affinity of 16-OHT than testosterone to aromatase. Moreover, the metabolites EDDP and norBUP were weaker inhibitors of aromatase than their parent compounds. The determined inhibition constants of methadone and BUP for E₃ formation by a cDNA-expressed CYP19 preparation were similar to those for placental microsomes. Therefore, data reported here suggest that methadone, BUP, and their metabolites are inhibitors of androgen aromatization in the placental biosynthesis of estrogens.

Keywords: Abbreviations; BUP; buprenorphine; CYP; cytochrome P450; EDDP; 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine; EMDP; 2-ethyl-5-methyl-3,3-diphenylpyrroline; norBUP; norbuprenorphine; E; ₂; 17β-estradiol; 16-OHT; 16α-hydroxytestosterone; E; ₃; estriol; TCA; trichloroacetic acidHuman placenta; CYP19/aromatase; Methadone; Buprenorphine (BUP); Estrogen formation; Pregnant opiate addict

Potential role of short hairpin RNA targeting epidermal growth factor receptor in growth and sensitivity to drugs of human lung adenocarcinoma cells by Li Bai; Rong Zhu; Zhihong Chen; Lei Gao; Xin Zhang; Xiangdong Wang; Chunxue Bai (pp. 1265-1271).

Upregulation of expression and activation of epidermal growth factor receptor (EGFR) is involved in the development and progression of a wide range of human cancers. The present study aims at determining gene-silencing effects of vector-based short hairpin RNA (shRNA) targeting EGFR on receptor expression and cell growth and evaluating its modulation of responsiveness to drugs in human lung adenocarcinoma cells (HLAC). A vector-based polymerase 3-promotor system was used to express shRNA targeting EGFR in HLAC lines (A549 and SPC-A1). EGFR was detected by immunofluorescence staining and quantified by Western blot. The effect of shRNA targeting EGFR on tumor cell growth was assessed by colony formation assay, cell cycle and apoptosis by flow cytometry, and the responsiveness of HLAC lines to cytotoxic drugs by 3-[4,5-dimethylthiozol-2yl]-2,5-diphenyltetrazolium bromide [MTT] assay. Vectors expressing shRNA against EGFR significantly downregulated receptor expression by 74 and 85% and the colony number by 63 and 69% in A549 and SPC-A1, respectively. Vector-based shRNA against EGFR caused G1 arrest, induced apoptosis, and subsequently increased the sensitivity to cisplatin, doxorubicin and paclitaxel by about four- to seven-fold in both HLAC lines. Our data suggest that vector-based shRNA could be considered as an alternative to effectively inhibit EGFR expression in HLACs, probably with the higher efficacy in combination therapies with conventional chemotherapeutic drugs.

Keywords: EGFR; ShRNA; Lung carcinoma; Cisplatin; Doxorubicin; Paclitaxel

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Biochemical Pharmacology (v.71, #8)

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Biochemical Pharmacology (v.71, #8)