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Journal Article 2008; 29(5): 790-793 PubMed PMID: 18987595 Citation  Keywords:  Adrenergic beta-Antagonists:pharmacology, Androstadienes:pharmacology, Calcimycin:pharmacology, Carbazoles:pharmacology, Humans, In Vitro Techniques, Male, N-Formylmethionine Leucyl-Phenylalanine:pharmacology, Neutrophils:drug effects, Oxidants:metabolism.   

OBJECTIVES: To compare three stimuli which activate human neutrophils with different signal transduction mechanisms, in order to better localize the effect of the beta-adrenoceptor antagonist carvedilol (CARV) on superoxide generation (O2*-) and myeloperoxidase release (MPO). The effect of CARV [0.1-100 micromol/l] on O2*- generation and MPO release from isolated human neutrophils was studied after specific receptor activator N-formyl-methionyl-leucyl-phenylalanine (fMLP) and nonreceptor phorbol-12-myristate-13-acetate (PMA) and calcium ionophor (A23187) stimuli.

METHODS: O2*- generation was measured as superoxide dismutase inhibitable reduction of cytochrome c and MPO release as the oxidation of o-dianisidine in the presence of hydrogen peroxide in a spectrophotometer Hewlet Packard 8452 A at respective 550 and 463 nm.

RESULTS: CARV had no effect on O2*- generation and MPO release in nonstimulated cells. In the concentration 10 and 100 micromol/l, it significantly decreased fMLP and PMA stimulated O2*- generation and MPO release. Incubation of neutrophils with CARV [100 micromol/l] caused significant inhibition of O2*- generation and MPO release induced by A23187. Wortmannin, a specific inhibitor of 1-phosphatidylinositol-3-kinase, inhibited significantly only fMLP stimulated O2*- generation. CARV [100 micromol/l] with wortmannin [50 nmol/l] further decreased O2*- generation after the same stimulus.

CONCLUSION: CARV decreased O2*- generation and MPO release from isolated human neutrophils both by membrane-operating stimulus - fMLP and membrane bypassing activators - PMA and A 23187. This fact, together with effect the of wortmannin, indicates that the inhibition may be attributed to the non-specific action of CARV and its interference with phospholipase D signaling pathway, which plays only a minor role in proteinkinase C stimulated O2*- generation....

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Journal Article 2008; 29(4): 529-535 PubMed PMID: 18766138 Citation  Keywords:  Androstadienes:pharmacology, Animals, Cells, Cultured, Chromones:pharmacology, Enzyme Activation, Extracellular Signal-Regulated MAP Kinases:metabolism, Gonadotrophs:cytology, Gonadotropin-Releasing Hormone:metabolism, Gonadotropins:genetics, Humans, Morp.   

OBJECTIVE: Phosphatidylinositol-3 kinase (PI3-kinase) has been known to play an important role in cell survival and proliferation by activation of its downstream target, Akt/protein kinase B (PKB). In this present study, we investigated the effects of PI3-kinase inhibitors on gonadotropin alpha-subunit gene expression in pituitary gonadotrophs.

METHODS: Alpha T3-1 cells, a pituitary gonadotroph cell line, were used in this study. alphaT3-1 cells were transfected with alpha-subunit promoter region-linked luciferase vector, and stimulated with GnRH in the presence or absence of two different PI3-kinase inhibitors, LY 294002 and wortmannin. Dose response effects of these inhibitors were also examined. Extracellular signal-regulated kinase (ERK) phosphorylation were determined by western blotting analysis.

RESULTS: Treatment of alphaT3-1 cells with PI3-kinase inhibitor, LY 294002, significantly increased alpha-subunit gene expression up to 6.89 +/- 0.26-fold, and showed additive effect with gonadotropin-releasing hormone (GnRH). The increasing effect of LY 294002 on alpha-subunit gene expression was observed at the concentration more than 1 microM. The experiment using another PI3-kinase inhibitor, wortmannin, showed similar effects, where wortmannin alone increased alpha-subunit gene expression by dose dependent manner and showed additive effect with GnRH. The inhibitor of PKB failed to modulate basal activity of alpha-subunit promoter as well as GnRH-induced promoter activities. Western blotting analysis using phosphorylated form specific antibody for ERK demonstrated that both LY 294002 and wortmannin increased ERK phosphorylation.

CONCLUSION: These results suggested that PI3-kinase inhibitor, LY 294002 and wortmannin increased gonadotropin alpha-subunit gene expression related with ERK activation....

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