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To investigate the role of the intracellular C-terminal tail of the rat metabotropic glutamate receptor 1a (mGlu1a) in receptor regulation, we constructed three C-terminal tail deletion mutants (Arg847stop, DM-I; Arg868stop, DM-II; Val893stop, DM-III). Quantification of glutamate-induced internalization provided by ELISA indicated that DM-III, like the wild-type mGlu1a, underwent rapid internalization whilst internalization of DM-I and DM-II was impaired. The selective inhibitor of protein kinase C (PKC), GF109203X, which significantly reduced glutamate-induced mGlu1a internalization, had no effect on the internalization of DM-I, DM-II, or DM-III. In addition activation by carbachol of endogenously expressed (M)1 (muscarinic acetylcholine receptors, which induces PKC- and Ca[SUP2+]-calmodulin-dependent protein kinase II-dependent internalization of mGlu1a, produced negligible internalization of the deletion mutants. Co-expression of a dominant negative mutant form of G protein-coupled receptor kinase 2 (DNM-GRK2; Lys220Arg) significantly attenuated glutamate-induced internalization of mGlu1a and DM-III, whilst internalization of DM-I and DM-II was not significantly affected. The glutamate- induced internalization of mGlu1a and DM-III, but not of DM-I or DM-II, was inhibited by expression of DNM-arrestin [arrestin-2(319-418)]. In addition glutamate-induced rapid translocation of arrestin-2-Green Fluorescent Protein (arr-2-GFP) from cytosol to membrane was only observed in cells expressing mGlu1a or DM-III. Functionally, in cells expressing mGlu1a, glutamate-stimulated inositol phosphate accumulation was increased in the presence of PKC inhibition, but so too was that in cells expressing DM-II and DM-III. Together these results indicate that different PKC mechanisms regulate the desensitization and internalization of mGlu1a. Furthermore, PKC regulation of mGlu1a internalization requires the distal C terminus of the receptor (Ser894-Leu1199), whilst in contrast... [ABSTRACT FROM AUTHOR]

Background and Purpose NADPH oxidases ( NOXs) contribute to platelet activation by a largely unknown mechanism. Here, we studied the effect of the novel NOX inhibitor 2-acetylphenothiazine (2- APT) on human platelet functional responses and intracellular signaling pathways. Experimental Approach The generation of superoxide ions was assessed by single cell imaging on adhering platelets using dihydroethidium ( DHE), while other reactive oxygen species ( ROS) were detected with 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate ( CM- H2- DCFDA). Whole blood thrombus formation, washed platelet aggregation, integrin α IIbβ3 inside-out signalling, Syk phosphorylation and PKC activation were analysed to understand the functional consequences of NOX inhibition by 2- APT in platelets. Key Results Superoxide ion generation stimulated by platelet adhesion on collagen and fibrinogen was significantly inhibited by 2- APT in concentration-dependent manner ( IC50 = 306 n M and 227 n M, respectively), whereas cumulative ROS accumulation was not affected by this pharmacological agent. 2- APT also abolished collagen-dependent whole blood thrombus formation and washed platelet aggregation in response to collagen but not thrombin. The activation of integrin α IIbβ3 and PKC in response to the GPVI-specific agonist collagen-related peptide ( CRP) was significantly reduced, whereas the same responses to thrombin were not significantly affected by 2- APT. Finally, Syk activation in response to collagen but not thrombin was inhibited by 2- APT. Conclusions and Implications Taken together, our results suggest that 2- APT attenuates GPVI-specific signalling and is a novel inhibitor of collagen-induced platelet responses. Therefore, NOXs could represent a novel target for the discovery of anti-thrombotic drugs. [ABSTRACT FROM AUTHOR]

At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 µm; 30 min) the HA-mGluR1a underwent internalization to endosomes. Further quantification of receptor internalization was provided by ELISA experiments which showed rapid agonist-induced internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a internalization is an arrestin- and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319–418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 µm; 30 min) HA-mGluR1a internalization. In addition, wild-type arrestin-2-green fluorescent protein (arrestin-2-GFP) or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced internalization of mGluR1a is an arrestin- and dynamin-dependent process. [ABSTRACT FROM AUTHOR]

Vascular complications resulting from atherosclerosis development are a major cause of death. Reactive oxygen species (ROS) are produced by platelets during activation, and have been demonstrated to positively regulate platelet activatory responses. Zn2+ is also an important hemostatic cofactor in platelets, acting both as a platelet agonist and second messenger. Whilst the effect of Zn2+-dependent signaling mechanisms on ROS production in nucleated cells has been demonstrated, comparable roles in platelets have yet to be investigated. In this study we investigated the relationship between fluctuations in cytosolic Zn2 [Zn2+]i and platelet ROS production. Agonist-evoked ROS production, GSH levels and GPx activity are abrogated in platelets treated with the Zn2+-chelator, TPEN. Conversely, increasing platelet [Zn2+]i using Zn2+ ionophores potentiated ROS generation and decreased GSH levels and GPx activity. Zn2+-dependent ROS production was sensitive to pretreatment with DPI or mitoTEMPO, NADPH oxidase and mitochondria inhibitors respectively. Increasing [Zn2+]i resulted in increases of Erk1/2 and JNK phosphorylation. Our data are consistent with a functional association between [Zn2+]i and ROS production in platelets that could influence thrombus formation in a clinical context. [ABSTRACT FROM AUTHOR]

Redox regulation of angiogenesis is an important and emerging topic in vascular sciences; reactive oxygen species (ROS) modulate vascular endothelial cell (EC) proliferation and migration, but the molecular mechanism underlying this phenomenon remains largely unclear. We have shown micromolar concentrations of the pro-angiogenic metabolite deoxyribose-1-phosphate (dRP) detected by mass spectrometry (MS) and nuclear magnetic resonance (NMR) in the supernatants of collagen- and thrombin-stimulated platelets. The genetic silencing of dRP-generating enzymes uridine phosphorylase (UP) and thymidine phosphorylase (TP) in mouse platelets significantly reduced the levels of dRP in platelet supernatants and impaired the ability of protein-free platelet supernatants to enhance human umbilical vein endothelial cell (HUVEC) motility. Chick chorioallantoic membrane (CAM) vascularisation assays performed with protein-free supernatants suggested that the release of dRP by platelets stimulates angiogenesis in vivo.To understand the pro-angiogenic effect of dRP, we investigated further the molecular mechanism of action of dRP and whether this effect was observed in other nucleoside derivatives deoxyribose-5-phospate (dR5P), ribose-1-phosphate (R1P) and deoxyribose (dR). Using live cell real-time confocal imaging and fluorescence assays, HUVEC treatment with dRP significantly increased ROS generation. Similarly, dR5P and R1P also induced ROS accumulation in HUVECs, whereas dR did not. The protein expression of the redox stress marker heme oxygenase-1 (HO-1) and Integrin β3 were shown to be upregulated by dRP and its phosphorylated derivatives (except dR) by immunoblot experiments. Vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), interleukin 8 (IL-8), and stromal cell-derived factor 1α (SDF-1α) showed a similar effect. In parallel, only dRP and its phosphorylated derivatives increased HUVEC cell motility in scratch-healing assays and capillary tube formation using Matrigel™ assays. The causative link between integrin β3 upregulation and increased HUVEC motility/capillary tube formation was proved using integrin-specific inhibitory antibodies'. Furthermore the angiogenic potential of dRP was confirmed in vivo.In summary, dRP and its phosphorylated derivatives were shown to play a relevant role in the paracrine regulation of EC motility and pro-angiogenic activity by up-regulating integrin β3 in a ROS-dependent manner possibly signaling via VEGFR2. dRP is likely to play a role in the stimulation of postnatal angiogenesis and tissue regeneration. [ABSTRACT FROM PUBLISHER]

An abstract of the article "A succenturiate lobe with fused umbilical cord originated from vanishing twin syndrome," by N. Park, H. Bae, G. Cho, M. Oh, H. Kim, and S. Hong is presented.