THE INTRACELLULAR CA2+-PUMP INHIBITORS THAPSIGARGIN AND CYCLOPIAZONIC ACID INDUCE STRESS PROTEINS IN MAMMALIAN CHONDROCYTES

Primary cultures of mammalian articular chondrocytes respond to treatment with the intracellular Ca2+-pump inhibitors thapsigargin (TG) and cyclopiazonic acid by specific changes in protein synthesis consistent with a stress response. Two-dimensional gel electrophoresis of newly synthesized proteins confirmed that the response was consistent with the induction of glucose-regulated proteins. The effects of low-dose TG (10 nM), measured by changes in [S-35]methionine labelling of newly synthesized proteins, can first be observed by 10 h and are maximal by 24 h. The pattern of changes induced by TG is shared with cyclopiazonic acid, but effects of both perturbants differ significantly from changes induced by heat shock. Upon removal of TG, normal protein synthesis is restored by 48 h. Immunoblots showed increased concentrations of the stress proteins HSP90, HSP72/73 and HSP60 in chondrocytes treated with TG, but induction of newly synthesized heat-shock proteins by TG was not apparent on [S-35]methionine-labelled gels. The alterations in protein synthesis induced by Ca2+-pump inhibitors were unaffected by BAPTA-AM loading, which clamped cytosolic Ca2+ at resting levels. We conclude that inhibition of intracellular Ca2+-pump activity can elicit a stress response, which has important implications for the interpretation of chronic use of Ca2+-pump inhibitors. In particular, the activation of the cellular shock response should be considered in interpreting the regulation of protein synthesis and cell survival by Ca2+-pump inhibitors such as TG.