Vol. 281, Issue 2, 957-964, 1997

The Human Thromboxane A₂ Receptor  Isoform (TP) Functionally Couples to the G Proteins G_q and G₁₁ In Vivo and is Activated by the Isoprostane 8-epi Prostaglandin F₂¹

B. Therese Kinsella, Daniel J. O'Mahony and Garret A. Fitzgerald

Department of Biochemistry (B.T.K.), University College Dublin, Dublin, Ireland; Elan Corporation Research Institute (D.J.OM.), Trinity College, Dublin, Ireland; Centre for Cardiovascular Science, Department of Medicine and Experimental Therapeutics (D.J.OM.), University College Dublin, Mater Hospital, Dublin, Ireland and Center for Experimental Therapeutics (G.A.F.), University of Pennsylvania, Philadelphia, Pennsylvania

To establish whether the thromboxane A₂ (TXA₂) receptor (TP) functionally couples to the G_q family of heterotrimeric G proteins in vivo, we have coexpressed the cDNAs coding for the human platelet/placental TP alpha isoform (TP) and the  subunits of G_q or G₁₁ in human embryonic kidney (HEK) 293 cells. TP activation in response to ligand stimulation was monitored by analyzing mobilization of intracellular calcium (Ca⁺⁺_i) in FURA2/AM-loaded transfected HEK 293 and in platelets. Second, we wished to examine the possible interaction of the isoprostane 8-epi prostaglandin F₂ with the TP, in transfected HEK 293 cells and with the TPs expressed in platelets. Thus both the prostaglandin endoperoxide/TXA₂ analog (U46619) and the 8-epi PGF₂ were utilized as ligand probes of TP activation. The results demonstrate that each ligand induced elevations of Ca⁺⁺_i levels in HEK 293 cells, cotransfected with either the TP and G_q or the TP and G₁₁, and also in platelets. Initial stimulation of these cells with U46619 or 8-epi PGF₂ desensitized a subsequent rise in [Ca⁺⁺]_i in response to U46619 or 8-epi PGF₂, respectively. Moreover, prestimulation with U46619 desensitized a subsequent rise in Ca⁺⁺_i concentration in response to 8-epi PGF₂, and vice versa. These responses were blocked by the TP antagonist SQ29,548 in both cell types. In contrast, prestimulation of the transfected HEK 293 cells or platelets with thrombin did not desensitize a subsequent rise in [Ca⁺⁺]_i in response to U46619 or 8-epi PGF₂. After stimulation with either U46619 or 8-epi PGF₂, no significant rise in Ca⁺⁺_i levels was observed in HEK 293 cells transfected with the TP receptor only or in control cells transfected with the vector pCMV5. These results demonstrate that the TP isoform functionally couples with either G_q or G₁₁ in vivo, whether activated by a PG/TXA₂ analog or by the F₂ isoprostane 8-epi PGF₂.