thromboxane-a2 and Astrocytoma

1321N1 human astrocytoma cells express thromboxane A2 (TXA2) receptors (TP). However, physiological consequences of TXA2 signaling in glial cells remain unclear. Herein, we show that TXA2 promotes interleukin-6 (IL-6) biosynthesis in glial cells. A TP agonist, 9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619), enhanced IL-6 production in both 1321N1 cells and cultured mouse astrocytes. It has been shown that IL-6 gene expression is regulated by various transcription factors. Among them, we found a significant increase in cyclic AMP-response element-binding protein (CREB) activity with its phosphorylation at Ser133 by U46619 in 1321N1 cells. Although U46619 increased IL-6 promoter activity, a mutation at cyclic AMP-response element (CRE) on the promoter clearly suppressed the effect, suggesting that CRE is involved in U46619-induced IL-6 expression. Furthermore, both CREB and IL-6 promoter activities were suppressed by SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole], a p38 mitogen-activated protein kinase (MAPK) inhibitor, and H89 [N-[2-(4-bromocinnamylamino)-ethyl]-5-isoquinoline], a protein kinase A (PKA) inhibitor, indicating involvements of p38 MAPK and PKA in CREB activation and IL-6 expression. To determine which G-proteins are implicated in the U46619-induced IL-6 synthesis, the interfering mutants of Galpha(q), Galpha12, or Galpha13 by were overexpressed in 1321N1 cells adenoviral approach. It is noteworthy that the Galpha(q) or Galpha13 mutant blocked the IL-6 production by U46619. The constitutively active mutant of Galpha(q), Galpha12, or Galpha13 enhanced IL-6 production, indicating that Galpha(q) and Galpha13 were involved in U46619-induced IL-6 production. In conclusion, TXA2 enhances the IL-6 biosynthesis via the PKA p38 MAPK/CREB pathway in 1321N1 cells. IL-6 induction depends on Galpha(q) and Galpha13 as well. This is the first report showing TP-mediated IL-6 production in glial cells.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Astrocytoma; Base Sequence; Blotting, Western; Cell Line, Tumor; CREB-Binding Protein; Cyclic AMP-Dependent Protein Kinases; DNA Primers; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Humans; Interleukin-6; Mitogen-Activated Protein Kinases; Nuclear Proteins; Reverse Transcriptase Polymerase Chain Reaction; Thromboxane A2; Trans-Activators

The desensitization mechanisms that regulate the response to thromboxane A2 (TXA2) were investigated in 1321N1 human astrocytoma cells. Exposure of the cells to 9,11-epithio-11, 12-methanothromboxane A2 (STA2), a stable TXA2 receptor agonist, at a concentration of 1 microM for 30 to 45 min resulted in about a 50% decrease in subsequent STA2-stimulated phosphoinositide hydrolysis and Ca2+ mobilization. However, exposure to STA2 for 0 to 5 hr did not change the binding of [3H]SQ29548, a TXA2 receptor antagonist. Because STA2-induced GTPase activation decreased and the GTP sensitivity in inhibition of [3H]SQ29548 binding by STA2 disappeared after the cells had been exposed to STA2 for 30 min, the TXA2 receptor desensitization during the short-term might result from G-protein-receptor uncoupling. STA2-induced desensitization was specific for the TXA2 receptor and homologous, because SQ29548 suppressed the desensitization and STA2 pretreatment did not affect the response to carbachol, a muscarinic cholinergic receptor agonist. Exposure to STA2 for 24 hr decreased [3H]SQ29548 binding sites to 20 to 30% of control and abolished STA2-stimulated phosphoinositide hydrolysis, indicating that long-term desensitization might induce down-regulation of the TXA2 receptor. However, exposure to STA2 for 1 to 24 hr did not change the level of TXA2 receptor mRNA. These results show that homologous desensitization of the TXA2 receptor in human astrocytoma cells can be divided into two stages; the early stage involves uncoupling of receptors from G-proteins and the late stage involves a loss of receptors in cells. The mRNA levels may not be controlled by stimulation of the TXA2 receptor.

Topics: Astrocytoma; Blotting, Northern; Calcium; Cells, Cultured; GTP-Binding Proteins; Humans; Inositol Phosphates; Receptors, Thromboxane; RNA, Messenger; Thromboxane A2

Western blot analysis was performed to clarify the presence of trimeric G protein subfamily in membranes derived from human astrocytoma cells (1321N1), cultured rabbit astrocytes and human platelets, using G protein antisera GS alpha, Gi alpha, Gq/11 alpha, and G beta were found to exist in membranes derived from human astrocytoma cells and rabbit astrocytes as well as human platelets. However, only small amount of G(o) alpha was detected in any membranes. Gq/11 alpha was expressed much more in human platelets than in human astrocytoma cells or rabbit astrocytes. 9,11-Epithio-11,12-methanothromboxane A2 (STA2), a stable analogue of thromboxane A2 (TXA2), activated GTPase in membranes derived from human astrocytoma cells, rabbit astrocytes and human platelets with different potencies. STA2-induced GTPase activation in human platelet membranes was partly inhibited by treatment with QL antibody at 0 degrees C for 90 min. Furthermore, STA2-induced GTPase activation in membranes derived from human astrocytoma cells and rabbit astrocytes were potently inhibited by treatment with QL antibody. The results obtained indicate that TXA2 receptors in human astrocytoma cells and rabbit astrocytes communicate with Gq/11 as well as in human platelets.

Topics: Animals; Astrocytes; Astrocytoma; Blood Platelets; Blotting, Western; Cells, Cultured; Enzyme Activation; GTP Phosphohydrolases; GTP-Binding Proteins; Humans; Rabbits; Receptors, Thromboxane; Thromboxane A2

Thromboxane A2 receptors of human astrocytoma cells and rabbit platelets were characterized by a binding of a new potent and selective thromboxane A2 antagonist, [3H]ONO NT-126. [3H]ONO NT-126 bound with a high affinity to membranes derived from astrocytoma cells and platelets. In astrocytoma cells, the nonspecific binding determined by 1 microM S-145 was observed in high percentage to total binding. Scatchard plots of both membranes were linear, expressing a single binding site with the Kd of 0.17 nM and Bmax of 46.4 fmol/mg protein in astrocytoma cell membranes and with the Kd of 0.28 nM and Bmax of 1585 fmol/mg protein in platelet membranes, respectively. [3H]ONO NT-126 binding was displaced most potently by ONO NT-126 among other thromboxane A2 analogues. [3H]ONO NT-126 binding was displaced by ONO NT-126, S-145, SQ29548, STA2 and U46619 with Ki values (nM) of 0.21, 1.14, 7.05, 23.5 and 235 in astrocytoma cell membranes and 0.23, 5.55, 259, 9.25 and 43.7 in platelet membranes, respectively. The characteristics of TXA2 receptors apparently differ in rabbit platelets and human astrocytoma cells.

Topics: Animals; Astrocytoma; Blood Platelets; Bridged Bicyclo Compounds; Cell Membrane; Fatty Acids, Monounsaturated; Fatty Acids, Unsaturated; Female; GTP-Binding Proteins; Humans; In Vitro Techniques; Male; Nervous System Neoplasms; Rabbits; Receptors, Prostaglandin; Receptors, Thromboxane; Thromboxane A2; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

Stimulation of thromboxane A2 (TXA2) receptors in human astrocytoma cells (1321N1) results in activation of phospholipase C via a pertussis toxin-insensitive G-protein. In the present study, the potency of a new TXA2 receptor antagonist, ONO NT-126, was examined with regard to receptor binding and phosphoinositide hydrolysis in human astrocytoma cells and was compared to that of the other known TXA2 antagonists. [3H]SQ29548 binding to membranes was inhibited by ONO NT-126 and the other TXA2 antagonists with Ki values (nM) of 0.09, 2.18, 8.35 and 25.9 for ONO NT-126, S-145, SQ29548 and ONO3708, respectively. STA2 and U46619, TXA2 receptor agonists, also inhibited [3H]SQ29548 binding with Ki (nM) of 25.1 and 233.5, respectively. STA2 and U46619 stimulated phosphoinositide hydrolysis in a concentration-dependent manner with EC50 values of 43.6 nM for STA2 and 1.2 microM for U46619, respectively. STA2 (1 microM)-induced phosphoinositide hydrolysis was also inhibited by TXA2 antagonists. The Ki values of TXA2 antagonists for the inhibition of phosphoinositide hydrolysis (nM) were 0.10 for ONO NT-126, 3.31 for S-145, 8.31 for SQ29548 and 19.49 for ONO3708 all of which were similar to those for receptor binding. The results indicate that ONO NT-126 is a potent and selective antagonist of TXA2 receptors in human astrocytoma cells.

Topics: Astrocytoma; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Fatty Acids, Monounsaturated; Fatty Acids, Unsaturated; Humans; Hydrazines; Hydrolysis; Inositol Phosphates; Phosphatidylinositols; Radioligand Assay; Receptors, Prostaglandin; Receptors, Thromboxane; Thromboxane A2; Tumor Cells, Cultured

The properties of thromboxane A2 (TXA2) receptors were examined in 1321N1 human astrocytoma cells. 9,11-Epithio-11,12-methanothromboxane A2 (STA2), a stable analogue of TXA2, stimulated the accumulation of inositol phosphates (IPs) with an EC50 of about 50 nM. The STA2-induced accumulation of IPs was inhibited concentration dependently by ONO3708, a TXA2 receptor antagonist, with an inhibition constant (Ki) of about 10 nM. Inositol trisphosphate (IP3) was accumulated more rapidly than inositol bisphosphate (IP2) in response to STA2. HPLC analysis indicated that inositol 1,4,5-trisphosphate accumulated in the presence of STA2. STA2 alone had no effect on the accumulation of IPs in membrane preparations but it potentiated the accumulation induced by GTP gamma S. [3H]SQ29548, a TXA2 receptor antagonist, bound specifically to TXA2 receptors, expressing a single binding site with a dissociation constant (Kd) of 10.9 nM. The competition curve for STA2 inhibition of [3H]SQ29548 binding was shifted to the right and was steeper in the presence of GTP gamma S. Pertussis toxin (IAP) elicited ADP-ribosylation of 41KD protein but had no effect on the sensitivity to GTP of the STA2 inhibition of SQ29548 binding or of STA2-induced accumulation of IPs. It is concluded from these results that the stimulation of TXA2 receptors results in activation of phospholipase C via a GTP binding protein and that the protein is not a substrate for IAP.

Topics: Adenosine Diphosphate; Astrocytoma; Bridged Bicyclo Compounds, Heterocyclic; Chromatography, High Pressure Liquid; Enzyme Activation; Fatty Acids, Unsaturated; GTP-Binding Proteins; Hydrazines; Inositol Phosphates; Pertussis Toxin; Phosphatidylinositols; Receptors, Prostaglandin; Receptors, Thromboxane; Ribose; Thromboxane A2; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella