Endothelin-1 attenuates omega 3 fatty acid-induced apoptosis by inhibitionof caspase 3

Endothelin-1 (ET-1) may be involved in the induction of vascular hypertroph y in hypertension. ET-1 may also modulate vascular growth through the exert ion of antiapoptotic effects. The omega 3 fatty acids (omega 3 FAs), which have antiproliferative effects in various cell types, may have a beneficial role in hypertension. We tested the hypothesis that ET-1 could act as a su rvival factor against omega 3 FA-induced apoptosis and attempted to elucida te possible molecular mechanisms underlying the protective action of ET-1 o n docosahexaenoic acid (DHA)-induced apoptosis. Mesenteric vascular smooth muscle cells were stimulated with DHA, a representative omega 3 FA. Dose-re sponse curves of DHA at different apoptotic stages were assessed with the u se of flow cytometry: (1) very early: plasma membrane phosphatidylserine (P S) translocation; (2) early: change in mitochondrial transmembrane potentia l (Delta Psi m); and (3) late: cell cycle analysis. Expression of the proap optotic protein bar and the antiapoptotic protein bcl-2 was determined with Western blot assay. The activity and the expression of caspase 3, which as a critical proteolytic enzyme involved in the death-signaling pathway, wer e evaluated with a fluorometric immunosorbent enzyme assay and Western blot analysis, respectively. Apoptosis, which was detected with PS translocatio n, Delta Psi m disruption, and cell cycle analysis, was increased dose depe ndently by DHA. DHA-induced apoptosis was attenuated through exposure to ET -1 for 1 hour before DHA in cell cycle analysis. The interference of ET-1 w ith DHA-induced apoptosis, as detected with cell cycle analysis, was not ap parent at the membrane (PS translocation) or the mitochondrial (Delta Psi m ) level. The increase in bax/bcl-2 ratio in DHA-stimulated cells was not af fected by ET-1. However, DHA increased both caspase 3 activity and the acti ve forms of caspase 3 (20 and 17 kDa), resulting in enhanced DNA fragmentat ion as shown through Hoechst staining and fluorescence microscopy, which we re attenuated by ET-1 pretreatment. In conclusion, DHA, an omega 3 FA, indu ced apoptosis in vascular smooth muscle cells in a dose-dependent manner. E T-1 exerted important protective effects through the attenuation of DHA-ind uced caspase 3 activation and subsequent DNA fragmentation in the late stag es of apoptosis.