Effects of ionizing irradiation on endothelial cell transglutaminase

Abstract

The activity of transglutaminase (TG) was measured in cultures of bovine aortic and capillary endothelial cells (EC) following exposure to γ-irradiation. Resting confluent EC express significant TG activity which fluctuates in growing cells. This activity was increased by 2-fold following non-lethal irradiation. The increase in TG activity was dose dependent up to 20 Gy and reached a plateau at 24–36 h after irradiation. Immunohistochemical studies showed a prominent increase in cytoplasmic TG following irradiation. Western blot analysis of whole cell extracts showed no increase in total cellular TG. Kinetic studies demonstrated that the affinity of the enzyme to its substrate was not altered, but the V max was increased. TG has previously been shown to be stored in an inactive form in EC membranes. This activity could be recovered in normal EC, but not in irradiated EC, by the addition of potassium thiocyanate and dithiothreitol or 0.8 M Nad. An inhibitor of TG was previously demonstrated in the 100,000 × g particulate fraction of EC. Following irradiation, a significant decrease in this inhibitory activity was demonstrated. These results imply that the post-irradiation enhancement of TG activity may be caused by activation of a latent cellular enzyme. This elevated TG activity may cross-link adjacent cytoplasmic and membrane proteins and may thus play an active role in the enhanced apoptosis observed following irradiation of EC.