PGE1 abolishes the mitochondrial-independent cell death pathway induced by D-galactosamine in primary culture of rat hepatocytes.
OBJECTIVE
PGE1 reduces in vivo and in vitro D-galactosamine (D-GalN)-induced cell death in hepatocytes. The present study was undertaken to elucidate the intracellular pathway by which D-GalN induces cell death in cultured hepatocytes. In addition, we evaluated if PGE1 was able to modulate different parameters related to D-GalN-induced apoptosis in cultured rat hepatocytes.


METHODS
Hepatocytes were isolated from male Wistar rats (225-275 g) by the classical collagenase procedure. PGE1 (1 microM) was administered 2 h before D-GalN (5 mM) in primary culture of rat hepatocytes. Apoptosis was determined by DNA fragmentation and caspase-3, -6, -8 and -9 activation in hepatocytes. Caspase activation was evaluated by the detection of the related cleaved product and its associated activity. Cell necrosis was determined by the measurement of lactate dehydrogenase (LDH) activity in culture medium. To elucidate the role of mitochondria, we measured neutral (nSMase) and acid (aSMase) sphingomyelinase, as well as the expression of cytochrome c in mitochondria and cytoplasm fractions from D-GalN treated hepatocytes.


RESULTS
D-GalN induced caspase-3 activation and DNA fragmentation in hepatocytes. This apoptotic response was not associated with the activation of caspase-6, -8 or -9. The use of specific inhibitors confirmed that only caspase-3 was involved in D-GalN-induced apoptosis. D-GalN did not modify nSMase and aSMase activities, nor mitochondrial cytochrome c release in hepatocytes.


CONCLUSIONS
D-GalN induced apoptosis through caspase-3 activation but without modification of the activity of caspase-6, -8, -9, SMases or cytochrome c release. PGE1 appears to prevent D-GalN-induced apoptosis by a mitochondria-independent mechanism.
Siendones
Emilio
E
Clinical Unit of Digestive Apparatus, University Hospital Reina Sofía, Cordoba, Spain. emilio.siendones.exts@juntadeandalucia.es
Jiménez-Gómez
Yolanda
Y
Montero
José Luís
JL
Gómez-Díaz
Consuelo
C
Villalba
José Manuel
JM
Muntané
Jordi
J
D000527:Alprostadil / Q000502:physiology*; D000818:Animals; D017209:Apoptosis; D016923:Cell Death; D002478:Cells, Cultured; D005688:Galactosamine / Q000502:physiology*; D022781:Hepatocytes / Q000502:physiology*; D008297:Male; D008928:Mitochondria; D051381:Rats; D017208:Rats, Wistar
D005688:Galactosamine; D000527:Alprostadil