The human thromboxane A2 receptor alpha isoform (TP alpha) functionally couples to the G proteins Gq and G11 in vivo and is activated by the isoprostane 8-epi prostaglandin F2 alpha.
To establish whether the thromboxane A2 (TXA2) receptor (TP) functionally couples to the Gq family of heterotrimeric G proteins in vivo, we have coexpressed the cDNAs coding for the human platelet/placental TP alpha isoform (TP alpha) and the alpha subunits of Gq 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 alpha with the TP alpha, 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 alpha were utilized as ligand probes of TP alpha activation. The results demonstrate that each ligand induced elevations of Ca++i levels in HEK 293 cells, cotransfected with either the TP alpha and G alpha q or the TP alpha and G alpha 11, and also in platelets. Initial stimulation of these cells with U46619 or 8-epi PGF 2 alpha desensitized a subsequent rise in [Ca++]i in response to U46619 or 8-epi PGF 2 alpha, respectively. Moreover, prestimulation with U46619 desensitized a subsequent rise in Ca++i concentration in response to 8-epi PGF 2 alpha, 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 2 alpha. After stimulation with either U46619 or 8-epi PGF 2 alpha, no significant rise in Ca++i levels was observed in HEK 293 cells transfected with the TP alpha receptor only or in control cells transfected with the vector pCMV5. These results demonstrate that the TP alpha isoform functionally couples with either Gq or G11 in vivo, whether activated by a PG/TXA2 analog or by the F2 isoprostane 8-epi PGF2 alpha.
Kinsella
B T
BT
Department of Biochemistry, University College Dublin, Ireland.
O'Mahony
D J
DJ
Fitzgerald
G A
GA
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary; D015237:Dinoprost / Q000031:analogs & derivatives* / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D017482:Receptors, Thromboxane / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013928:Thromboxane A2 / Q000031:analogs & derivatives / Q000494:pharmacology
D018076:DNA, Complementary; D011450:Prostaglandin Endoperoxides, Synthetic; D017482:Receptors, Thromboxane; D011994:Recombinant Proteins; C075750:8-epi-prostaglandin F2alpha; D013928:Thromboxane A2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D015237:Dinoprost; D019204:GTP-Binding Proteins; D002118:Calcium