LACK OF CORRELATION BETWEEN EARLY INTRACELLULAR CALCIUM-ION RISES ANDTHE ONSET OF APOPTOSIS IN THYMOCYTES

Apoptosis, a well-recognized process of cell death, is usually defined by chromatin condensation, plasma membrane blebbing, reduction in cel l volume, and in many cell types the cleavage of DNA into nucleosomal multiples, and finally the formation of apoptotic bodies. We have char acterized the time of onset and the range of concentrations at which t he toxins gliotoxin and thapsigargin induce apoptosis in thymocytes. W e also looked for early changes in cytosolic calcium ion concentration ([Ca2+](i)). Three methods were used to detect apoptosis: cellular mo rphology, DNA fragmentation and a flow cytometric method using ethidiu m bromide. Calcium fluxes were measured using both flow cytometry and bulk cell fluorimetry. Gliotoxin concentrations of 50 nmol/L to 10 mu mol/L induced significant numbers of cells to become apoptotic in a do se dependent manner. At these concentrations there was no observable i ncrease in [Ca2+](i) as determined by flow cytometry or in bulk cells. However, when thymocytes were treated with gliotoxin at concentration s greater than 500 mu mol/L, rises in [Ca2+](i) were apparent, but the se cells died by necrosis. Thapsigargin induced low levels of apoptosi s in thymocytes; the maximum effect observable after a 10 nmol/L treat ment. Thapsigargin is known to inhibit the Ca2+-ATPase in the endoplas mic reticulum thereby causing a sustained increase in [Ca2+](i) in thy mocytes. The rise in [Ca2+](i) observed was quantitatively similar whe n thymocytes were treated with thapsigargin concentrations ranging bet ween 10 and 100 nmol/L. These results led us to investigate the effect of dexamethasone on [Ca2+](i). In these experiments thymocytes showed no rises in [Ca2+](i) above the control over 85 min following treatme nt with 10 mu mol/L dexamethasone.