guanosine-5--o-(3-thiotriphosphate) and Astrocytoma

SDF-1/CXCL12 binds to the chemokine receptors, CXCR4 and CXCR7, and controls cell proliferation and migration during development, tumorigenesis, and inflammatory processes. It is currently assumed that CXCR7 would represent an atypical or scavenger chemokine receptor which modulates the function of CXCR4. Contrasting this view, we demonstrated recently that CXCR7 actively mediates SDF-1 signaling in primary astrocytes. Here, we provide evidence that CXCR7 affects astrocytic cell signaling and function through pertussis toxin-sensitive G(i/o) proteins. SDF-1-dependent activation of G(i/o) proteins and subsequent increases in intracellular Ca(2+) concentration persisted in primary rodent astrocytes with depleted expression of CXCR4, but were abolished in astrocytes with depleted expression of CXCR7. Moreover, CXCR7-mediated effects of SDF-1 on Erk and Akt signaling as well as on astrocytic proliferation and migration were all sensitive to pertussis toxin. Likewise, pertussis toxin abolished SDF-1-induced activation of Erk and Akt in CXCR7-only expressing human glioma cell lines. Finally, consistent with a ligand-biased function of CXCR7 in astrocytes, the alternate CXCR7 ligand, I-TAC/CXCL11, activated Erk and Akt through β-arrestin. The demonstration that SDF-1-bound CXCR7 activates G(i/o) proteins in astrocytes could help to explain some discrepancies previously observed for the function of CXCR4 and CXCR7 in other cell types.

Topics: Animals; Animals, Newborn; Arrestins; Astrocytoma; beta-Arrestins; Calcium; Cell Proliferation; Cells, Cultured; Cerebral Cortex; Chemokine CXCL12; Chemotaxis; Embryo, Mammalian; Enzyme Inhibitors; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Mice; Mice, Knockout; Neuroglia; Rats; Rats, Sprague-Dawley; Receptors, CXCR; Receptors, CXCR4; RNA Interference; Signal Transduction; Sulfur Isotopes

Low-affinity A2B adenosine receptors (A2B ARs), which are expressed in astrocytes, are mainly activated during brain hypoxia and ischaemia, when large amounts of adenosine are released. Cytokines, which are also produced at high levels under these conditions, may regulate receptor responsiveness. In the present study, we detected A2B AR in human astrocytoma cells (ADF) by both immunoblotting and real-time PCR. Functional studies showed that the receptor stimulated adenylyl cyclase through Gs proteins. Moreover, A2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF-alpha) treatment (24- h) increased A2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF-alpha markedly reduced agonist-dependent receptor phosphorylation on threonine residues and attenuated agonist-mediated A2B ARs desensitization. In the presence of TNF-alpha, A2B AR stimulation in vitro induced the elongation of astrocytic processes, a typical morphological hallmark of in vivo reactive astrogliosis. This event was completely prevented by the selective A2B AR antagonist MRS 1706 and required the presence of TNF-alpha. These results suggest that, in ADF cells, TNF-alpha selectively modulates A2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A2B AR in response to brain damage.

Topics: Adenosine; Adenosine A2 Receptor Antagonists; Adenosine-5'-(N-ethylcarboxamide); Animals; Antineoplastic Agents; Astrocytes; Astrocytoma; Blotting, Western; Cell Growth Processes; Cell Line, Tumor; Cricetinae; Cricetulus; Cyclic AMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Immunoprecipitation; Neuroprotective Agents; Phenethylamines; Purines; Pyrimidines; Radioligand Assay; Receptor, Adenosine A2B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Threonine; Time Factors; Transfection; Triazoles; Tumor Necrosis Factor-alpha

The compartmentation of inositol phospholipids was examined by using a combination of radiolabelling approaches in intact and permeabilized 1321N1 astrocytoma cells. A 'chase' protocol was developed with whole cells in which phosphoinositide (PI) pools were labelled to steady state with [3H]inositol and the cellular [3H]inositol pool was then diluted selectively with non-radioactive inositol. In these cells muscarinic-receptor-stimulated phospholipase C (PLC) hydrolysed [3H]PI at approx. 1-2%/min. However, after the chase procedure the relative specific radioactivity of [3H]Ins(1,3,4)P3, a rapidly metabolized and sensitive marker of PLC activity, decreased only after more than 5 min and over a time course similar to that during which the labelling of each [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 declined by at least 50%. These results demonstrate a large receptor-responsive [3H]PI pool that is accessed by stimulated PLC without apparent metabolic compartmentation, despite its probable distribution between different membrane fractions. Support for this was obtained in intact cells by using an acute [3H]inositol labelling method in which increases in the specific radioactivity of [3H]inositol phosphates stimulated by carbachol occurred only in parallel with similar increases in the labelling of the bulk of cellular [3H]PI. In [3H]inositol-prelabelled cells permeabilized to deplete cytosolic proteins, carbachol and guanosine 5'-[gamma-thio]triphosphate stimulated the endogenous PLC to degrade only approx. 5% of [3H]PI. This was increased to approx. 30% in the presence of exogenous PtdIns transfer protein, which, at a concentration approx. 5-10% of that in 1321N1 cell cytosol, was sufficient to support PLC activity comparable with that observed in response to carbachol in whole cells. These and earlier results in 1321N1 cells suggest a model of integrated PI pools involving an obligatory role for lipid transport. Given the multifunctional capacity of PI in cellular signalling mechanisms, this model has important implications, particularly for the hypothesis that the ability of Li+ ions to influence these selectively might account for its therapeutic actions.

Topics: Astrocytoma; Carbachol; Cell Compartmentation; Cell Membrane Permeability; Cytosol; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Inositol; Kinetics; Models, Biological; Phosphatidylinositol Phosphates; Phosphatidylinositols; Receptors, Muscarinic; Tumor Cells, Cultured; Type C Phospholipases

Site- and phosphorylation state-specific antibodies are useful to analyze spatiotemporal distribution of site-specific phosphorylation of target proteins in vivo. Using several polyclonal and monoclonal antibodies that can specifically recognize four phosphorylated sites on glial fibrillary acidic protein (GFAP), we have previously reported that Thr-7, Ser-13, and Ser-34 on this intermediate filament protein are phosphorylated at the cleavage furrow during cytokinesis. This observation suggests that there exists a protein kinase named cleavage furrow kinase specifically activated at metaphase-anaphase transition (Matsuoka, Y., Nishizawa, K., Yano, T., Shibata, M., Ando, S., Takahashi, T., and Inagaki, M. (1992) EMBO J. 11, 2895-2902; Sekimata, M., Tsujimura, K., Tanaka, J., Takeuchi, Y., Inagaki, N., and Inagaki, M. (1996) J. Cell Biol. 132, 635-641). Here we report that GFAP is phosphorylated specifically at Thr-7, Ser-13, and Ser-34 by Rho-associated kinase (Rho-kinase), which binds to the small GTPase Rho in its GTP-bound active form. The kinase activity of Rho-kinase toward GFAP is dramatically stimulated by guanosine 5'-(3-O-thio)-triphosphate-bound RhoA. Furthermore, the phosphorylation of GFAP by Rho-kinase results in a nearly complete inhibition of its filament formation in vitro. The possibility that Rho-kinase is a candidate for cleavage furrow kinase is discussed.

Topics: Antibodies; Antibodies, Monoclonal; Astrocytoma; Glial Fibrillary Acidic Protein; Glutathione Transferase; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Intracellular Signaling Peptides and Proteins; Kinetics; Phosphopeptides; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Recombinant Fusion Proteins; Recombinant Proteins; rho-Associated Kinases; Substrate Specificity; Tumor Cells, Cultured

The cholinergic agonist carbachol induced the release of arachidonic acid in the 1321N1 astrocytoma cell line, and this was blocked by atropine, suggesting the involvement of muscarinic receptors. To assess the mechanisms of signalling involved in the response to carbachol, a set of compounds characterized by eliciting responses through different mechanisms was tested. A combination of 4beta-phorbol 12beta-myristate 13alpha-acetate and thapsigargin, an inhibitor of endomembrane Ca2+-ATPase that induces a prolonged elevation of cytosolic Ca2+ concentration, induced an optimal response, suggesting at first glance that both protein kinase C (PKC) and Ca2+ mobilization were involved in the response. This was consistent with the observation that carbachol elicited Ca2+ mobilization and PKC-dependent phosphorylation of cytosolic phospholipase A2 (cPLA2; phosphatide sn-2-acylhydrolase, EC 3.1.1.4) as measured by a decrease in electrophoretic mobility. Nevertheless, the release of arachidonate induced by carbachol was unaltered in media containing decreased concentrations of Ca2+ or in the presence of neomycin, a potent inhibitor of phospholipase C which blocks phosphoinositide turnover and Ca2+ mobilization. Guanosine 5'-[gamma-thio]triphosphate added to the cell-free homogenate induced both [3H]arachidonate release and cPLA2 translocation to the cell membrane fraction in the absence of Ca2+, thus suggesting the existence of an alternative mechanism of cPLA2 translocation dependent on G-proteins and independent of Ca2+ mobilization. From the combination of experiments utilizing biochemical and immunological tools the involvement of cPLA2 was ascertained. In summary, these data indicate the existence in the astrocytoma cell line 1321N1 of a pathway involving the cPLA2 which couples the release of arachidonate to the occupancy of receptors for a neurotransmitter, requires PKC activity and G-proteins and might operate in the absence of Ca2+ mobilization.

Topics: Arachidonic Acid; Astrocytoma; Brain Neoplasms; Calcimycin; Calcium; Carbachol; Carcinogens; Cytosol; Enzyme Activation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ionophores; Phospholipases A; Phospholipases A2; Protein Kinase C; Receptors, Muscarinic; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured

Thrombin stimulation of 1321N1 astrocytoma cells results in polyphosphoinositide hydrolysis, Ca2+ mobilization, AP-1-mediated transcriptional activation, and DNA replication. Thrombin stimulation also activates Ras as assessed by an increase in the proportion of Ras in a GTP bound state. We examined the functional requirement for endogenous Ras protein in mediating thrombin-induced responses. Microinjection of a dominant interfering mutant of H-Ras into 1321N1 cells inhibited DNA synthesis in response to thrombin as did microinjection of an inhibitory antibody to Ras. Stimulation of AP-1-mediated transcriptional activity was also reduced by the expression of interfering Ras mutants. However, neither the stimulation of polyphosphoinositide hydrolysis nor the mobilization of intracellular Ca2+ was dependent on endogenous Ras function. These observations indicate that thrombin stimulation of mitogenesis requires Ras protein function. Our data suggest that the G-protein-coupled thrombin receptor stimulates pathways, which in part are convergent with those stimulated by tyrosine kinase growth factor receptors.

Topics: Astrocytoma; Cell Division; DNA Replication; Genes, ras; Growth Substances; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Humans; Kinetics; Mutagenesis, Site-Directed; Phosphatidylinositols; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins p21(ras); Thrombin; Transfection; Tumor Cells, Cultured

The effect of adenosine on phosphoinositide hydrolysis was examined in 1321N1 human astrocytoma cells. Adenosine, L-N6-phenylisopropyladenosine (L-PIA), and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The potency order of adenosine analogues for inhibition of inositol phosphate accumulation was L-PIA greater than adenosine greater than NECA, a finding indicating that A1-class adenosine receptors are involved in the inhibition. The reduction in inositol phosphate accumulation by L-PIA was blocked by an adenosine receptor antagonist, 8-phenyltheophylline. Stimulation of A1-class adenosine receptors inhibited isoproterenol-stimulated cyclic AMP accumulation as well as histamine-induced inositol phosphate accumulation. Both inhibitory effects were blocked by pretreatment of the cells with pertussis toxin [islet-activating protein (IAP)]. L-PIA also inhibited guanosine 5'-(gamma-thio)triphosphate (GTP gamma S)-stimulated accumulation of inositol phosphates in membrane preparations, and 8-phenyl-theophylline antagonized the inhibition. L-PIA could not inhibit GTP gamma S-induced accumulation of inositol phosphates in IAP-treated membranes. Gi/Go, purified from rabbit brain, inhibited GTP gamma S-stimulated accumulation of inositol phosphates in a concentration-dependent manner in membrane preparations. These results suggest that stimulation of A1-class adenosine receptors interacts with the IAP-sensitive G protein(s), resulting in the inhibitions of phospholipase C as well as adenylate cyclase in human astrocytoma cells.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylate Cyclase Toxin; Adenylyl Cyclases; Astrocytoma; Cells, Cultured; Cyclic AMP; Dose-Response Relationship, Drug; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Histamine; Humans; Hydrolysis; Pertussis Toxin; Phosphatidylinositols; Receptors, Purinergic; Tumor Cells, Cultured; Type C Phospholipases; Vasodilator Agents; Virulence Factors, Bordetella

Carbachol, histamine and bradykinin activate phospholipase C in pertussis toxin-insensitive manner in human astrocytoma cells. Pretreatments of the cells with these agonists resulted in the reduction of GTP gamma S-induced accumulation of inositol phosphates in membrane preparations. Treatment of cells with carbachol mobilized GTP gamma S binding activities as well as muscarinic receptors from heavy membrane fraction to light fraction, reflecting from an agonist-induced desensitization. The treatment of the cells with agonists reduced a 32 kDa GTP binding protein in heavy membrane fraction, determined by a photoaffinity labeling with [35S]GTP gamma S. The data suggest that the 32 kDa GTP binding protein is involved in desensitization by agonists which activate phospholipase C in human astrocytoma cells.

Topics: Affinity Labels; Astrocytoma; Calcium Channel Agonists; Cells, Cultured; Centrifugation, Density Gradient; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Membranes; Neoplasms, Experimental; Protein Binding; Receptors, Muscarinic; Thionucleotides