Thromboxane A 2 activates phospholipase C in astrocytoma cells via pertussis toxin-insensitive G-protein

Abstract

The properties of thromboxane A 2 (TXA 2) receptors were examined in 1321N1 human astrocytoma cells. 9,11-Epithio-11,12-methanothromboxane A 2 (STA 2), a stable analogue of TXA 2, stimulated the accumulation of inositol phosphates (IPs) with an EC 50 of about 50 nM. The STA 2-induced accumulation of IPs was inhibited concentration dependently by ONO3708, a TXA 2 receptor antagonist, with an inhibition constant (K i) of about 10 nM. Inositol triphosphate (IP 3) was accumulated more rapidly than inositol bisphosphate (IP 2) in response to STA 2. HPLC analysis indicated that inositol 1,4,5-triphosphate accumulated in the presence of STA 2. STA 2 alone had no effect on the accumulation of IPs in membrane preparations but it potentiated the accumulation induced by GTPγS. [ 3H]SQ29548, a TXA 2 receptor antagonist, bound specifically to TXA 2 receptors, expressing a single binding site with a dissociation constant (K d) of 10.9 nM. The competition curve for STA 2 inhibition of [ 3H]SQ29548 binding was shifted to the right and was steeper in the presence of GTPγS. Pertussis toxin (IAP) elicited ADP-ribosylation of 41KD protein but had no effect on the sensitivity to GTP of the STA 2 inhibition of SQ29548 binding or of STA 2-induced accumulation of IPs. It is concluded from these results that the stimulation of TXA 2 receptors results in activation of phospholipase C via a GTP binding protein and that the protein is not a substrate for IAP.