Inhibition of apoptosis in pulmonary endothelial cells by altered pH, mitochondrial function, and ATP supply.
We investigated the effect of altered extracellular pH, mitochondrial function, and ATP content on development of apoptosis in human pulmonary artery endothelial cells after treatment with staurosporine (STS). STS produced a concentration- and time-dependent increase in caspase-3 activity in pH 7.4 medium that reached a peak at 6 h. The increase in caspase activity was associated with significant DNA fragmentation. Fluorescent imaging of treated monolayers in pH 7.4 medium with Hoechst-33342-propidium iodide demonstrated a large percentage of apoptotic cells ( approximately 40%) with no evidence of necrosis. Caspase activity, DNA fragmentation, and percentage of apoptotic cells were reduced after STS treatment in acidic media (pH 7.0 and 6.6). The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM inhibited STS-induced apoptosis, whereas the rise in intracellular Ca2+concentration in STS-treated cells in pH 7.4 medium was reduced in pH 7.0 medium. These results suggest that one mechanism for inhibitory effects of acidosis may be a pH-induced alteration in Ca2+ signaling. Treatment with STS in the presence of oligomycin (10 microM), an inhibitor of the mitochondrial F(0)F(1)-ATPase, in glucose-free media abolished caspase activation and DNA fragmentation in association with severe ATP depletion ( approximately 2% of control cells). Imaging demonstrated a change in the mode of cell death from apoptosis to necrosis under these conditions. This change was linked to the level of ATP depletion, because STS treatment in the absence of glucose or the presence of oligomycin in media with glucose still leads to apoptosis in the presence of only moderate ATP depletion. These results demonstrate that pH, mitochondrial function, and ATP supply are important variables that regulate STS-induced apoptosis in human pulmonary artery endothelial cells.
Terminella
C
C
Pulmonary Disease Division, Department of Medicine, State University of New York/Downstate Health Sciences Center; and Department of Veterans Affairs Medical Center, Brooklyn, New York 11209, USA.
Tollefson
K
K
Kroczynski
J
J
Pelli
J
J
Cutaia
M
M
D000255:Adenosine Triphosphate / Q000172:deficiency / Q000502:physiology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D053938:DNA Fragmentation / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008928:Mitochondria / Q000502:physiology*; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D006859:Hydrogen; D000255:Adenosine Triphosphate; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine; D002118:Calcium