U73122 and U73343 inhibit receptor-mediated phospholipase D activation downstream of phospholipase C in CHO cells

The aminosteroid 1-(6-{[17 β-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino}hexyl)-1 H-pyrrole-2,5-dione (U73122) and its inactive analogue 1-(6-{[17 β-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino}hexyl-2,5-pyrrolidine-dione (U73343) are widely used to study the involvement of G protein-coupled 1-phosphatidylinositol-phosphodiesterase, or phospholipase C, in receptor-mediated cell activation. The present work shows that both aminosteroids inhibit cholecystokinin-(26–33)-peptide amide (CCK-8)-induced phospholipase D activation equipotently in Chinese hamster ovary cells expressing the cholecystokinin-A receptor (CHO–CCK A cells). In addition, the two aminosteroids virtually completely inhibited thapsigargin- and 12- O-tetradecanoylphorbol 13-acetate (TPA)-induced phospholipase D activation. Since the latter two drugs mimic inositol 1,4,5-trisphosphate-mediated Ca 2+ mobilisation and 1,2-diacylglycerol-mediated protein kinase C activation, respectively, this suggests that both U73122 and U73343 act downstream of phospholipase C to inhibit receptor-mediated phospholipase D activation. U73122, but not U73343, effectively inhibited both TPA/Ca 2+-stimulated phospholipase D activation and TPA/phosphatidylserine-stimulated protein kinase C activation in a homogenate of CHO–CCK A cells. The data presented suggest that U73122 may act at the level of protein kinase C to inhibit activation of phospholipase D. The exact site of action of U73343 is presently unknown.