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PI3K Inhibitor Wortmannin (pi3k + inhibitor_wortmannin)
Selected AbstractsThrombin potently enhances swelling-sensitive glutamate efflux from cultured astrocytesGLIA, Issue 9 2007Gerardo Ramos-Mandujano Abstract High concentrations of thrombin (Thr) have been linked to neuronal damage in cerebral ischemia and traumatic brain injury. In the present study we found that Thr markedly enhanced swelling-activated efflux of 3H -glutamate from cultured astrocytes exposed to hyposmotic medium. Thr (0.5,5 U/mL) elicited small 3H -glutamate efflux under isosmotic conditions and increased the hyposmotic glutamate efflux by 5- to 10-fold, the maximum effect being observed at 15% osmolarity reduction. These Thr effects involve its protease activity and are fully mimicked by SFFLRN, the synthetic peptide activating protease-activated receptor-1. Thr potentiation of 3H -glutamate efflux was largely dependent on a Thr-elicited increases in cytosolic Ca2+ (Ca2+i) concentration ([Ca2+]i). Preventing Ca2+i rise by treatment with EGTA-AM or with the phospholipase C blocker U73122 reduced the Thr-increased glutamate efflux by 68%. The protein kinase C blockers Go6976 or chelerythrine reduced the Thr effect by 19%,22%, while Ca/calmodulin blocker W7 caused a 63% inhibition. In addition to this Ca2+ -sensitive pathway, Thr effect on glutamate efflux also involved activation of phosphoinositide-3 kinase (PI3K), since it was reduced by the PI3K inhibitor wortmannin (51% inhibition). Treating cells with EGTA-AM plus wortmannin essentially abolished Thr-dependent glutamate efflux. Thr-activated glutamate release was potently inhibited by the blockers of the volume-sensitive anion permeability pathway, NPPB (IC50 15.8 ,M), DCPIB (IC50 4.2 ,M). These results suggest that Thr may contribute to the excitotoxic neuronal injury by elevating extracellular glutamate release from glial cells. Therefore, this work may aid in search of neuroprotective strategies for treating cerebral ischemia and brain trauma. © 2007 Wiley-Liss, Inc. [source] HER2 signaling enhances 5,UTR-mediated translation of c-Myc mRNAJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2004Enrico Galmozzi The increased levels of c-Myc protein observed previously in an ovarian carcinoma cell line stably transfected to express HER2 has suggested a role for the HER2 pathway in c-Myc expression. Analysis of HER2-transfected cells stimulated with heregulin ,1 (HRG) revealed increased c-Myc protein levels but not a corresponding increase in c-Myc mRNA expression or any change in c-Myc protein half-life. Transfection of HER2-overexpressing cells with a construct containing the 5, untranslated region (5,UTR) of c-Myc mRNA originated from the P2 promoter and placed upstream of the Renilla luciferase gene, enhanced reporter expression upon stimulation with HRG. The HRG-mediated increase in reporter activity correlated with the HRG-mediated induction observed for c-Myc protein, identifying the P2-derived leader (P2L) of c-Myc mRNA as the cis -element involved in c-Myc translational induction. Both the increase in c-Myc protein levels and P2L-enhanced translational activity were inhibited by the PI3K inhibitor wortmannin. Together, these results demonstrate that HRG stimulation of HER2 overexpressing cells leads to enhanced c-Myc protein synthesis through activation of the PI3K/Akt/mTOR pathway and that the P2L of c-Myc mRNA is the element responsible for induction of c-Myc translation. © 2004 Wiley-Liss, Inc. [source] Inhibition of nuclear factor ,B and phosphatidylinositol 3-kinase/Akt is essential for massive hepatocyte apoptosis induced by tumor necrosis factor , in miceLIVER INTERNATIONAL, Issue 5 2003Motoaki Imose Abstract: Background/aims: Tumor necrosis factor (TNF)-, itself does not induce liver injury in normal mice or hepatocytes. Rather, this event, especially in vitro, is explained by the fact that the TNF-,/TNF receptor system not only triggers downstream signals leading to apoptosis but also induces an antiapoptotic pathway through the activation of nuclear factor (NF)-,B. The aim of this study was to determine whether inhibition of antiapoptotic pathways influences the susceptibility of mice to TNF-,. Here, we focused on the roles of NF-,B and phosphatidylinositol 3-kinase (PI3K)-regulated serine/threonine kinase Akt. Methods: TNF-, was administered to BALB/c mice after treatment with an adenovirus expressing a mutant form I,B, (Ad5I,B), the PI3K inhibitor wortmannin, or both. Liver injury was assessed biochemically and histologically. The expression of Bcl-2 family members and caspase activity were examined. Results: In the mice livers, treatment with Ad5I,B or the wortmannin suppressed the activation of NF-,B or Akt, respectively. Suppression of either NF-,B or Akt showed a slight increase in transaminase levels and focal liver cell death after TNF-, administration. However, in mice treated with both Ad5I,B and wortmannin, TNF-, administration resulted in massive hepatocyte apoptosis and hemorrhagic liver destruction in mice. The combination of Ad5I,B, wortmannin, and TNF-, markedly increased the activation of caspase-3 and -9, and activated caspase-8 to a lesser degree, suggesting that TNF-,-induced hepatocyte apoptosis is dependent on type II cell death signaling pathway, probably through the mitochondria. Inhibition of the NF-,B and PI3K/Akt pathways had no effect on expression of Bcl-2 families. Conclusion: The inducible activation of NF-,B and constitutive activation of Akt regulate hepatocyte survival against TNF-,, which occurs independent of Bcl-2 families. [source] Inward relocation of exogenous phosphatidylserine triggered by IGF-1 in non-apoptotic C2C12 cells is concentration dependentCELL BIOCHEMISTRY AND FUNCTION, Issue 6 2005Cyril Rauch Abstract The plasma membrane is composed of two leaflets that are asymmetric with regard to their phospholipid composition with phosphatidylserine (PS) predominantly located within the inner leaflet whereas other phospholipids such as phosphatidylcholine (PC) are preferentially located in the outer leaflet. An intimate relationship between cellular physiology and the composition of the plasma membrane has been demonstrated, with for example apoptosis requiring PS exposure for macrophage recognition. In skeletal muscle development, differentiation also requires PS exposure in myoblasts to create cell,cell contact areas allowing the formation of multinucleate myotubes. Although it is clearly established that membrane composition/asymmetry plays an important role in cellular physiology, the role of cytokines in regulating this asymmetry is still unclear. When incubated with myoblasts, insulin-like growth factor I (IGF-1) has been shown to promote proliferation versus differentiation in a concentration dependent manner and therefore, may be a potential candidate regulating cell membrane asymmetry. We show, in non-apoptotic C2C12 cells, that relocation of an exogenous PS analogue, from the outer into the inner leaflet, is accelerated by IGF-1 in a concentration-dependent manner and that maintenance of membrane asymmetry triggered by IGF-1 is however independent of the PI3K inhibitor wortmannin. Copyright © 2005 John Wiley & Sons, Ltd. [source] Airway inflammation: chemokine-induced neutrophilia and the class,I phosphoinositide 3-kinasesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2005Matthew Abstract Class,I phosphoinositide 3-kinases (PI3K) are known to play a significant role in neutrophil chemotaxis. However, the relative contributions of different PI3K isoforms, and how these impact on lung inflammation, have not been addressed. In vitro studies using wild-type and PI3K, knockout neutrophils demonstrated the major role of the ,,isoform in chemotactic but not chemokinetic events. This was confirmed by a model of direct chemokine instillation into the airways in vivo. Within all studies, a low yet significant degree of neutrophil movement in the absence of PI3K, could be observed. No role for the ,,isoform was demonstrated both in vitro and in vivo using PI3K, kinase-dead knock-in mice. Moreover, further studies using the broad-spectrum PI3K inhibitors wortmannin or LY294002 showed no other class,I PI3K isoforms to be involved in these chemotactic processes. Here, we identify a contributory PI3K-independent mechanism of neutrophil movement, yet demonstrate PI3K, as the pivotal mediator through which the majority of neutrophils migrate into the lung in response to chemokines. These data resolve the complexities of chemokine-induced neutrophilia and PI3K signaling and define the ,,isoform as a promising target for new therapeutics to treat airway inflammatory diseases. [source] | |||||||||||||