Abstract

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_α) and the α 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 F_2α with the TP_α, in transfected HEK 293 cells and with the TPs expressed in platelets. Thus both the prostaglandin endoperoxide/TXA₂ analog (U46619) and the 8-epi PGF_2α were utilized as ligand probes of TP_α activation. The results demonstrate that each ligand induced elevations of Ca⁺⁺ _i levels in HEK 293 cells, cotransfected with either the TP_α and G_αq or the TP_α and G_α11, and also in platelets. Initial stimulation of these cells with U46619 or 8-epi PGF_2α desensitized a subsequent rise in [Ca⁺⁺]_i in response to U46619 or 8-epi PGF_2α, respectively. Moreover, prestimulation with U46619 desensitized a subsequent rise in Ca⁺⁺ _iconcentration in response to 8-epi PGF_2α, 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 PGF_2α. After stimulation with either U46619 or 8-epi PGF_2α, no significant rise in Ca⁺⁺ _i levels was observed in HEK 293 cells transfected with the TP_α receptor only or in control cells transfected with the vector pCMV5. These results demonstrate that the TP_α 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 PGF_2α.