NO-induced activation of cyclic GMP-dependent pathway down regulates ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) protein in rat C6 glioma.

In rat C6 glioma cells, the ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1), a modulator of purinergic receptor signaling, is down regulated after an increase in intracellular cAMP by addition of dibutyryl cAMP, a membrane-permeable cAMP-analog, or by activation of the β-adrenoceptor receptor with (-)-isoproterenol (Aerts et al., 2011, Eur. J.

The expression of ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (E-NPP1) is correlated with astrocytic tumor grade.

Objective: Astrocytic brain tumors are subdivided in four grades. The most aggressive and invasive one is grade IV or glioblastoma multiforme (GBM). Ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (E-NPP1), a membrane-bound enzyme, is involved in many cellular processes such as modulation of purinergic signalling, nucleotide recycling, regulation of extracellular pyrophosphate levels and stimulation of cell motility.

Cyclic AMP-dependent down regulation of ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) in rat C6 glioma.

In this communication, we demonstrate that an increase in intracellular cAMP by 1) addition of dibutyrylic cAMP (dbcAMP), a membrane-permeable cAMP-analogue, or 2) activation of the β-adrenoceptor with (-)-isoproterenol, down regulates the levels of ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) mRNA, NPP1 protein and ecto-NPPase activity in rat C6 glioma cells. DbcAMP and (-)-isoproterenol inhibit NPP1 expression in a time and dose-dependent manner. After 48h of stimulation, 1mM dbcAMP or 5μM (-)-isoproterenol decreases the amount of NPP1 protein by 75±3% and 81±1% respectively.

Integration of P2Y receptor-activated signal transduction pathways in G protein-dependent signalling networks.

The role of nucleotides in intracellular energy provision and nucleic acid synthesis has been known for a long time. In the past decade, evidence has been presented that, in addition to these functions, nucleotides are also autocrine and paracrine messenger molecules that initiate and regulate a large number of biological processes. The actions of extracellular nucleotides are mediated by ionotropic P2X and metabotropic P2Y receptors, while hydrolysis by ecto-enzymes modulates the initial signal.

Atypical PKCzeta is involved in RhoA-dependent mitogenic signaling by the P2Y(12) receptor in C6 cells.

When nucleotide hydrolysis is prevented, agonists of the P2Y(12) receptor enhance the proliferation of C6 glioma cells by RhoA-dependent, protein kinase C (PKC)-dependent activation of the extracellular signal-regulated kinase (ERK) pathway [Claes P, Grobben B, Van Kolen K, Roymans D & Slegers H (2001) Br J Pharmacol134, 402-408; Grobben B, Claes P, Van Kolen K, Roymans D, Fransen P, Sys SU & Slegers H (2001) J Neurochem78, 1325-1338]. In this study, we show that ERK1/2 phosphorylation was not affected by transfection of the cells with the Gbetagamma-subunit-scavenging adrenergic receptor kinase peptide [betaARK1-(495-689)] or with Rap1GAPII, indicating that P2Y(12) receptor stimulation enhances ERK1/2 phosphorylation by G(i)alpha subunit-mediated signaling independently of Rap1 activation. Inhibition of the RhoA downstream effector Rho-associated coiled-coil-containing kinase (ROCK) with Y-27632 did not affect the P2Y(12) receptor-induced increase in ERK1/2 phosphorylation but abrogated the mitogenic response.

P2Y12 receptor signalling towards PKB proceeds through IGF-I receptor cross-talk and requires activation of Src, Pyk2 and Rap1.

Previously it was shown that stimulation of the P2Y12 receptor activates PKB signalling in C6 glioma cells [K. Van Kolen and H. Slegers, J.

Investigation of the use of immobilised metal affinity chromatography for the on-line sample clean up and pre-concentration of nucleotides prior to their determination by ion pair liquid chromatography-electrospray mass spectrometry: a pilot study.

This study explored an alternative way to enrich and pre-purify biological samples containing nucleoside mono-, di- and triphosphates. These compounds were trapped by immobilised metal affinity chromatography (IMAC) on a Poros 20 MC IMAC-column, which was conditioned with Fe3+. The IMAC-column was implemented in a column switching set-up separating nucleoside mono-, di- and triphosphates on a Hypersil ODS 35 mm x 0.

P2Y12 receptor stimulation inhibits beta-adrenergic receptor-induced differentiation by reversing the cyclic AMP-dependent inhibition of protein kinase B.

Cyclic AMP-dependent induction of differentiation by activation of the beta-adrenergic receptor is correlated with inhibition of protein kinase B activity concomitant with growth arrest and increase in glial fibrillary acidic protein (GFAP) synthesis in rat C6 glioma cells. Costimulation of the beta-adrenergic receptor with purinergic receptors activated by 2-methylthio-adenosine-5'-diphosphate (2MeSADP) increased protein kinase B (PKB) phosphorylation above the level measured in non-stimulated cells and abolished cAMP-dependent differentiation. Transfection of cells with constitutively active PKB confirmed that reactivation of PKB is involved in the 2MeSADP-dependent inhibition of GFAP synthesis.

Reactive blue 2 inhibition of cyclic AMP-dependent differentiation of rat C6 glioma cells by purinergic receptor-independent inactivation of phosphatidylinositol 3-kinase.

Cyclic AMP-dependent differentiation of rat C6 glioma cells into an astrocyte type II is characterized by inhibition of cell growth and induction of glial fibrillary acidic protein (GFAP) synthesis. Activation of the P2Y(12) receptor with 2-methylthioadenosine-5'-diphosphate inhibited beta-adrenergic receptor-induced differentiation. The selective P2Y(12) receptor antagonist N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene ATP abolished the receptor-mediated effect on differentiation.

Influence of ionic and non-ionic radiographic contrast media on leukocyte adhesion molecules.

Background: Many papers have focused on the importance of granulocytes in the process of reperfusion and ischemia. Most of the clinical studies measured several parameters of this process during and after coronary angiography, without taking into account the effect of the radiographic contrast media (RCM) used during this procedure.

Materials And Methods: We performed a randomized patient study (n = 37) to evaluate the effect of ionic and non-ionic RCM on granulocyte adhesion during coronary angiography.

Physiological and pathophysiological functions of the ecto-nucleotide pyrophosphatase/phosphodiesterase family.

The ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) multigene family contains five members. NPP1-3 are type II transmembrane metalloenzymes characterized by a similar modular structure composed of a short intracellular domain, a single transmembrane domain and an extracellular domain containing a conserved catalytic site. The short intracellular domain of NPP1 has a basolateral membrane-targeting signal while NPP3 is targeted to the apical surface of polarized cells.

Rat C6 glioma as experimental model system for the study of glioblastoma growth and invasion.

Infiltration of the central nervous system by neoplastic cells in patients with glioblastoma multiforme (GBM) leads to neurological dysfunction and eventually to death. The elucidation of the mechanisms underlying the aggressive nature of GBM aims at improving radio-, chemo- and gene therapy. This review is focused on the use of rat C6 glioma as an experimental model system for GBM and provides an overview of the experimental data published in the literature using this cell line in elucidating the mechanism of tumor growth, angiogenesis and invasion, and in the design and evaluation of anticancer therapies.

Extracellular nucleoside diphosphate kinase NM23/NDPK modulates normal hematopoietic differentiation.

Objective: We previously demonstrated the presence of nucleoside diphosphate kinase NDPK/NM23 in normal human plasma. It also was reported that extracellular NM23 could inhibit differentiation of certain hematopoietic cell lines. We further investigated the extracellular effect of NM23 on hematopoiesis by adding recombinant NM23-H1, NM23-H2, and NM23-H3 proteins to in vitro differentiation assays of normal human hematopoietic progenitors.