Sequential NO production by mitochondria and endoplasmic reticulum during induced apoptosis.
Early stages of rat thymocyte apoptosis measured as annexin-V positive events and induced by methylprednisolone (MPS), etoposide, and thapsigargin, showed a sequential increase in nitric oxide (NO) production by mitochondrial and endoplasmic reticulum membranes. Thapsigargin induced the highest NO production, a sevenfold increase as compared with untreated thymocytes, in mitochondrial and microsomal membranes. MPS and etoposide were equally effective in increasing NO production by mitochondrial membranes by a factor of 4-5, with only a slight increase in NO production by endoplasmic reticulum membranes. Western blot analysis of both types of membrane indicated that a nitric oxide synthase (NOS) isoenzyme is present in mitochondrial membranes and reacts with antibodies to i-NOS (type II), while reactivity to antibodies to e-NOS (type III) was restricted to endoplasmic reticulum. The participation of endoplasmic reticulum during apoptosis was further determined by alterations in UDP-Glucosyltransferase (UDP-GT) and NADPH cytochrome P450 reductase. Increased UDP-GT activity was observed after thapsigargin treatment, and no changes were found after treatment with etoposide or MPS. NADPH cytochrome P450 reductase activity markedly decreased during apoptosis, being stronger after thapsigargin treatment. The latest stage of the apoptotic process was measured by caspase activities. Caspase 3 activity was markedly increased by the three apoptosis inducers; caspase 6 was only activated by MPS and etoposide, while caspase 8 was not activated by any of these inducers. It is clear that mitochondria and endoplasmic reticulum are involved in thapsigargin induced thymocyte apoptosis. Meanwhile, other thymocyte apoptotic pathways, such as those induced by MPS or etoposide, seem to centrally involve mitochondria but not endoplasmic reticulum.
Bustamante
Juanita
J
Laboratory of Free Radical Biology, Institute of Biochemistry Luis Federico Leloir, School of Natural and Exact Sciences, University of Buenos Aires, Junin 956, 1113 Buenos Aires, Argentina. juanitab@ffyb.uba.ar
Bersier
Geraldine
G
Badin
Romina Aron
RA
Cymeryng
Cora
C
Parodi
Armando
A
Boveris
Alberto
A
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D005434:Flow Cytometry; D005964:Glucosyltransferases / Q000378:metabolism; D008297:Male; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D009251:NADPH-Ferrihemoprotein Reductase; D005964:Glucosyltransferases