Resumen de In Vitro Induction of Apoptosis in Rat Hepatocytes by Cyclosporine A
Armin Wolf, Sibylle Grub, Elke Persohn, Wolfgang E. Tommer
In rat hepatocytes and isolated liver mitochondrial fractions, CsA is often used as a specific inhibitor of mitochondrial Ca2+ release and as a specific blocker of mitochondrial membrane potential and permeability transition (MPT), which are all processes involved in the inhibition of apoptosis. However, neither inhibition nor induction of apoptosis by CsA has yet been described in the rat hepatocyte primary culture during incubation for 4 and 20 hours. It was the purpose of the present study to examine by means of morphological and biochemical criteria the effects of CsA on apoptosis, and to characterize the underlying mechanisms. Rat hepatocytes were cultured for 4 or 20 hours with CsA at concentrations of 0, 10, 25 and 50 ?M. Chromatin condensation and fragmentation, DNA fragmentation (TUNEL), membrane phosphatidylserine distribution (Annexin V), caspase-1, -3 and -6 activity, mitochondrial membrane potential (Rhodamine 123), and cytochrome c release into the cytosol were investigated. Four hours after CsA treatment, chromatin condensation and fragmentation, and the number of TUNEL- and Annexin V-positive cells increased dose dependentlywithout any observable enzyme leakage, which indicated the integrity of the outer cell membrane. After 20 hours of CsA incubation apoptosis parameters were further increased and were accompanied by the increased activity of the cysteine protease, caspase-3 (CPP 32), and slightly increased caspase-6 (Mch 2), but not caspase-1 (ICE). The specific caspase-3 inhibitor, Ac-DEVD-CHO, inhibited caspase-3 activation and attenuated CsA-induced apoptosis and LDH leakage. The caspase-6 inhibitor, Ac-VEIDCHO, only marginally inhibited CsA-induced apoptosis. Decreased mitochondrial membrane potential and cytochrome c release went in parallel with ultrastructural mitochondrial changes and might be regarded as early events which trigger the apoptosis cascade. Transmission electron microscopy (TEM) confirmed an increase in the number of necrotic cells after 20 hours, but not after 4 hours, compared with controls.
