Identification of functional PGH₂/TxA₂ receptors on human endothelial cells

Although functional receptors for thromboxane A₂ and prostaglandin H₂ (TxA₂/PGH₂) have been identified in platelets and vascular smooth muscle cells, receptor-mediated events in human endothelial cells stimulated by these endoperoxides have not been shown. Using cultured endothelial cells harvested from human umbilical or saphenous veins, we measured the effect of the TxA₂ mimetic U46619 on mobilization of cytoplasmic calcium ([Ca²⁺](i)), as well as release of prostacyclin and expression of the proto-oncogene c-fos, intracellular events that have been linked to [Ca²⁺](i) rise in stimulated endothelial cells. Addition of U46619 to confluent fura 2-loaded endothelial cells caused a concentration-dependent rise in intracellular [Ca²⁺](i), with agonist concentrations of 300 nM producing a maximal [Ca²⁺](i) rise. This [Ca²⁺](i) rise was a uniform response observed in all individual endothelial cells throughout the monolayer, as shown by microspectrofluorimetric visualization. Similar effects were seen with a structurally dissimilar endoperoxide analogue, I- BOP, and with the naturally occurring endoperoxide PGH₂. The initial [Ca²⁺](i) rise was not reduced when extracellular [Ca²⁺](i) was chelated with EGTA, but a later 'plateau' phase was eliminated. An antagonist of the receptor for TxA₂/PGH₂ (SQ29548) strongly inhibited [Ca²⁺](i) mobilization. Stimulation of endothelial cells with U46619 also transiently increased expression of the proto-oncogene c-fos, as determined by RNA hybridization, and induced a fivefold increase in prostacyclin release. Thus, endoperoxides can stimulate human venous endothelial cells by means of TxA₂/PGH₂ receptors, whose occupancy can activate intracellular events associated with functional changes.