Incubation of endothelium with an increased epoxyeicosatrienoic acid (EET) concentration specifically augments the endothelium-dependent relaxation ascribed to endothelium-derived hyperpolarizing factor in porcine coronary arteries (Weintraub et al., Circ Res 1997;81:258-267). Experiments were designed to test whether such sustained increased levels of EETs in the environment of endothelial cells alters Ca2+ signaling. Changes in cytosolic Ca2+ were monitored in cultured porcine aortic endothelial cells (PAECs) and in the human endothelial EA.hy926 cell line after incubation (or not) with 5 microM 11,12-epoxyeicosatrienoic acid (EET). Although the mobilization of intracellular Ca2+ induced by 2 microM thapsigargin was not affected significantly, EET treatment augmented the capacitative Ca2+ entry evoked by the Ca(2+)-ATPase) inhibitor in both cell types. Similar observations were obtained by using histamine as a stimulant in EA.hy926 cells. As assessed in PAECs, 2 micrograms/ml triacsin C, a known inhibitor of the incorporation of EETs into phospholipids, did not significantly affect the potentiating action of EETs on Ca2+ signaling in response to thapsigargin. However, in solvent-control cells, triacsin C significantly reduced both the mobilization of Ca2+ from intracellular stores and the capacitative Ca2+ entry provoked by thapsigargin. Thus the EET-potentiating effect overcomes the inhibitory action of triacsin C on Ca2+ signaling in endothelial cells. Taken together, these results demonstrate that sustained increases in EETs may amplify Ca2+ signaling. However, contrary to the EET-induced augmentation of endothelium-dependent relaxation in the porcine coronary artery, resistance of this novel action of EETs to triacsin C suggests that the mechanism involved does not depend on incorporation into phospholipids.