sta-2 and Astrocytoma

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