Abstract
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 (TPα) and the α 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 F2α with the TPα, in transfected HEK 293 cells and with the TPs expressed in platelets. Thus both the prostaglandin endoperoxide/TXA2 analog (U46619) and the 8-epi PGF2α were utilized as ligand probes of TPα activation. The results demonstrate that each ligand induced elevations of Ca++ i levels in HEK 293 cells, cotransfected with either the TPα and Gαq or the TPα and Gα11, and also in platelets. Initial stimulation of these cells with U46619 or 8-epi PGF2α desensitized a subsequent rise in [Ca++]i in response to U46619 or 8-epi PGF2α, respectively. Moreover, prestimulation with U46619 desensitized a subsequent rise in Ca++ iconcentration in response to 8-epi PGF2α, 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 PGF2α. After stimulation with either U46619 or 8-epi PGF2α, no significant rise in Ca++ i levels was observed in HEK 293 cells transfected with the TPα receptor only or in control cells transfected with the vector pCMV5. These results demonstrate that the TPα isoform functionally couples with either Gq or G11 in vivo, whether activated by a PG/TXA2 analog or by the F2 isoprostane 8-epi PGF2α.
Footnotes
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Send reprint requests to: D. J. O’Mahony, Elan Corporation Research Institute, Trinity College, Dublin 2, Ireland.
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↵1 This research was supported by grants from the Irish Heart Foundation, Forbairt, the Health Research Board of Ireland (BTK) and the Wellcome Trust (BTK & GAF).
- Abbreviations:
- AOSMC
- aortic smooth muscle cell
- Ca++i
- intracellular calcium
- COX
- cyclooxygenase
- DAG
- diacylglycerol
- 8-epi PGF2α
- 8-epi prostaglandin F2α
- HEK
- human embryonic kidney
- IP3
- inositol 1,4,5-triphosphate
- PAGE
- polyacrylamide gel electrophoresis
- PLC
- phospholipase C
- PPP
- platelet poor plasma
- PRP
- platelet rich plasma
- SDS
- sodium dodecyl sulphate
- TP
- thromboxane A2 receptor
- TXA2
- thromboxane A2
- Received August 26, 1996.
- Accepted January 17, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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