Inhibition of apoptosis in cultured porcine granulosa cells by inhibitors of caspase and serine protease activity.
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 F12 (1:11 containing 1% fetal bovine serum. Final inhibitor concentrations, added in 10 microL of dimethylsulfoxide, were 0, 1, 5, 25 and 125 microM. Cells with compromised plasma membrane integrity, identified by uptake ethidium homodimer, increased during culture in the absence of inhibitors from 37% to 43%. Apoptotic (A0) 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 microM was lethal increasing (P < 0.05) cells with compromised membranes to 92%. In response to TPCK, A0 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) A0 cells linearly from 33% to 3 % between 0 and 125 microM 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 > or = 1 microM decreased (P < 0.05) A0 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 non-apoptotic mechanism.
Guthrie
H D
HD
Germplasm and Gamete Physiology Laboratory, Agricultural Research Service, Beltsville, MD 20705, USA. dave@ggpl.arsusda.gov
Garrett
W M
WM
Cooper
B S
BS
D000590:Amino Acid Chloromethyl Ketones / Q000737:chemistry; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000737:chemistry; D002462:Cell Membrane / Q000502:physiology; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry; D053938:DNA Fragmentation / Q000502:physiology; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D004958:Estradiol / Q000032:analysis; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary; D006107:Granulosa Cells / Q000502:physiology*; D007930:Leucine / Q000031:analogs & derivatives / Q000737:chemistry; D007976:Leupeptins / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000662:veterinary; D010664:Phenylmethylsulfonyl Fluoride / Q000737:chemistry; D011374:Progesterone / Q000032:analysis; D011863:Radioimmunoassay / Q000662:veterinary; D012044:Regression Analysis; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D013552:Swine / Q000502:physiology*; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000737:chemistry
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D015842:Serine Proteinase Inhibitors; C116501:acetyl-leucinal-leucinal-normethional; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D014108:Tosylphenylalanyl Chloromethyl Ketone; D011374:Progesterone; D004958:Estradiol; D010664:Phenylmethylsulfonyl Fluoride; D020169:Caspases; D007930:Leucine; C032854:leupeptin; C024974:E 64