Tyrosine phosphorylation of phosphatidylinositol 3-kinase and of the thromboxane A2 (TXA2) receptor by the TXA2 mimetic I-BOP in A7r5 cells

Thromboxane A2 (TXA2) interacts with its G-protein coupled receptor, the TP receptor, to produce contraction and proliferation of vascular smooth muscle cells. We have shown previously that proliferation of primary cultures of vascular smooth muscle cells initiated by [1S-(1alpha, 2beta(5Z), 3alpha(1E, 3R), 4alpha]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxab icyclo-[2.2.1]heptan-2yl]-5'-heptenoic acid (I-BOP), a stable TXA2 mimetic, is mediated by activation of mitogen-activated protein (MAP) kinase. In the present study, we examined further the intracellular mediators involved in TXA2 activation of vascular smooth muscle cells. Transient transfection of the cDNA for the TP receptor into A7r5 vascular smooth muscle cells resulted in expression of TP receptors with a receptor density, Bmax, of 0.7 +/- 0.2 pmol/mg protein and a receptor affinity, Kd, of 0.6 +/- 0.1 nM (N = 7). Mock transfected cells lacked significant receptor expression. In TP receptor transfected cells, I-BOP increased the activation of MAP kinase 2-fold, stimulated tyrosine phosphorylation of cellular proteins of relative molecular mass (Mr) of 140, 85, 60, 56, and 45 kDa, and increased the message for c-Jun, a nuclear transcription factor involved in mitogenesis, 2.6-fold. Immunoblot analysis indicated that the 85-kDa protein represented phosphoinositide 3-kinase (PI3-K), while the 60 kDa protein was the TP receptor. The activity of PI3-K was increased 3.5-fold by the addition of I-BOP (0.1 microM). In summary, the present study demonstrated that stimulation of the TP receptor results in tyrosine phosphorylation of the receptor and of PI3-K.