Inhibition of apoptosis in cultured porcine granulosa cells by inhibitors of caspase and serine protease activity

摘要

Protease inhibitors were used to test the hypothesis that caspases and other proteases were active during apoptosis in cultured porcine granulosa cells. Cells isolated from 3 to 6 mm follicles were cultured for 24 h in Dulbecco's modified Eagles medium: Hams Fl2 (1:1) containing 1% fetal bovine serum. Final inhibitor concentrations, added in 10 gL of dimethylsulfoxide, were 0, 1, 5, 25 and 125 #mu#M. Cells with compromised plasma membrane integrity, identified by uptake ethidium homodimer, increased during culture in the absence of inhibitors from 37% to 43%. Apoptotic (A_0) cells, identified by DNA fluorescence flow cytometry, increased (P < 0.05) from 1.7% to 29%. The serine protease inhibitor N-tosyl-L- phenylalanine chloromethyl ketone (TPCK) at 125 #mu#M was lethal increasing (P < 0.05) cells with compromised membranes to 92%. In response to TPCK, A_0 cells decreased from 55% to 1.2%; progesterone and estradiol production were decreased by 94% and 98%, respectively. The general caspase inhibitor, benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoro methylketone, decreased (P < 0.05) A_0 cells linearly from 33% to 3 % between 0 and 125 #mu#M without significant effect on steroidogenesis or on the percentage of cells with compromised plasma membranes. Other inhibitors only had a marginal effect on apoptosis; concentrations of >= 1 #mu#M decreased (P < 0.05) A_0 cells from 29% to 18% to 21% and had no significant effect on membrane integrity or steroid production. We conclude that caspases are associated with apoptosis in cultured porcine granulosa cells. Death induced by TPCK was through a nonapoptotic mechanism.