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Kinetic analysis of thapsigargin-induced thymocyte apoptosis

2004; Elsevier BV; Volume: 37; Issue: 9 Linguagem: Inglês

10.1016/j.freeradbiomed.2004.06.038

1873-4596

Juanita Bustamante, Eugenia Di Líbero, Mariana Fernandez-Cobo, Nicolás Monti, Enrique Cadenas, A Boveris,

Lipid Membrane Structure and Behavior

Thapsigargin addition to thymocytes increased cytosolic Ca2+ by a factor of 8.5 with a time for half maximal effect (t1/2) of 2.5 min. Calcium signaling increased mitocondrial and endoplasmic reticulum nitric oxide synthase (NOS) activities by five and six times, with t1/2 of 16 and 48 min, respectively, followed by increases of 140% in intracellular [H2O2], 73% in hydroperoxide content, and 250% in thiobarbituric reactive substance content, with t1/2 of 13, 27, and 30 min, respectively. Mitochondrial dysfunction followed, and was characterized by decreased respiratory control, membrane depolarization, and decrease cytochrome c content release, processes with t½ of 101, 129, and 133 min, respectively. Increased UDP-GT gene expression, observed by mRNA synthesis, and the enzymatic activity of this protein had t½ of 52 and 187 min, respectively. These events were followed by caspase-3 activation (t½=210 min) and DNA laddering (t½=260 min) at the completion of the cell death program. Preincubation of thymocytes with NOS inhibitors (NG-methyl-l-arginine and l-Nω-nitro-l-arginine methylester) halted the whole process through inhibition of mitochondrial and endoplasmic reticulum NOS activities and of DNA laddering.