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(Received for publication, February 20, 1996, and in revised form, September 5, 1996)
From the Department of Pathology, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107
Complete prevention of the killing of
L929 fibroblasts by tumor necrosis factor
Volume 271, Number 47,
Issue of November 22, 1996
pp. 29792-29798
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
(TNF) in the presence of
0.5 µg/ml actinomycin D (ActD) was obtained with cyclosporin A (CyA),
an inhibitor of the mitochondrial permeability transition (MPT), and
aristolochic acid (ArA), a phospholipase A2 inhibitor.
Peripheral benzodiazepine receptor (PBzR) agonists (PK11195, FGIN
1-27, or chlorodiazepam), agents known to potentiate induction of the
MPT, potentiated the cytotoxicity of TNF in the absence of ActD, an
effect prevented by CyA plus ArA. The MPT was demonstrated
independently of its effect on viability as the CyA-sensitive loss of
rhodamine 123 fluorescence from cells preloaded with the dye. Treatment
with TNF and ActD resulted in the loss of 80% of rhodamine
fluorescence within 6 h, a time prior to any loss of viability.
CyA plus ArA completely prevented this effect of TNF. Potentiation of
the cytotoxicity of TNF by PBzR agonists was associated with induction
of the MPT, as assessed by the loss of rhodamine fluorescence. CyA plus
ArA completely prevented the loss of rhodamine 123. Ceramide replaced TNF in killing L929 fibroblasts, an effect also prevented by CyA plus
ArA. Ceramide in the presence of ActD resulted in the loss of rhodamine
fluorescence, an effect that was again prevented by CyA plus ArA. In
addition, CyA plus ArA prevented the ability of PBzR agonists to
potentiate the cytotoxicity of ceramide. In the presence of each PBzR
agonist, ceramide caused the loss of rhodamine fluorescence, an effect
completely prevented by CyA plus ArA. D609, an inhibitor of
phosphatidylcholine-specific phospholipase C, completely prevented the
killing by TNF, but not by ceramide, in the presence of ActD. D609
prevented induction of the MPT occurring with TNF, but not with
ceramide. Inhibitors of endocytosis, as well as lysosomotropic amines,
prevented the cytotoxicity of TNF, but not that of ceramide. It is
concluded that the MPT is causally linked to the genesis of
irreversible cell injury with TNF. In the face of an inhibition of
protein synthesis, the MPT occurs as a consequence of the formation of
ceramide.
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