Colocalization of prostacyclin synthase with prostaglandin H synthase-1 (PGHS-1) but not phorbol ester-induced PGHS-2 in cultured endothelial cells

The subcellular colocalization of prostacyclin synthase (PGIS) with prostag landin H synthase (PGHS) has not been delineated. To test the hypothesis th at its colocalization with PGHS is crucial for prostacyclin synthesis, we d etermined subcellular locations of PGIS, PGHS-1, and PGNS-2 in bovine aorti c endothelial cells by immunofluorescent confocal microscopy. PGIS and PGHS -1 were colocalized to nuclear envelope (NE) and endoplasmic reticulum (ER) in resting and adenovirus-infected bovine aortic endothelial cells. PGIS a nd PGHS-2 were also colocalized to ER in serum-treated or adenovirus-cycloo xygenase-2-infected cells. By contrast, PGIS was not colocalized with PGHS- 2 in cells induced with phorbol la-myristate 13-acetate where PGHS-2 was vi sualized primarily in vesicle-like structures. The lack of colocalization w as accompanied by failed prostacyclin production. Resting ECV304 cells did not produce prostacyclin and had no detectable PGHS-1 and PGIS proteins. Co nfocal analysis showed abnormal colocalization of PGIS and PGHS-1 to a fila mentous structure. Interestingly, the abundant PGIS and PGHS-1 expressed in adenovirus-infected ECV304 cells were colocalized to NE and ER, which synt hesized a large quantity of prostacyclin, These findings underscore the imp ortance of colocalization of PGHS and PGIS to ER and NE in prostacyclin syn thesis.