Kinetic analysis of thapsigargin-induced thymocyte apoptosis

Thapsigargin addition to thymocytes increased cytosolic Ca2+ by a factor of 8.5 with a time for half-maximal effect (t(1/2)) of 2.5 min. Calcium signaling increased mitocondrial and endoplasmic reticulum nitric oxide synthase (NOS) activities by five and six times, with t(1/2) 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 t(1/2) of 13, 27, and 30 min, respectively. Mitochondrial dysfunction followed, and was characterized by decreased respiratory control, membrane depolarization, and cytochrome c release, processes with t(1/2) Of 101, 129, and 133 min, respectively. lncreased UDP-GT gene expression, observed by mRNA synthesis, and the enzymatic activity of this protein had t(1/2) Of 52 and 187 min, respectively. These events were followed by caspase-3 activation (t(1/2) - 210 min) and DNA laddering (t(1/2) = 260 min) at the completion of the cell death program. Preincubation of thymocytes with NOS inhibitors (N-G-methyl-L-arginine and L-Nomega-nitro-L-arginine methylester) halted the whole process through inhibition of mitochondrial and endoplasmic reticulum NOS activities and of DNA laddering. (C) 2004 Elsevier Inc. All rights reserved.