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

Primary cultures of mammalian articular chondrocytes respond to treatm ent 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 t he induction of glucose-regulated proteins. The effects of low-dose TG (10 nM), measured by changes in [S-35]methionine labelling of newly s ynthesized proteins, can first be observed by 10 h and are maximal by 24 h. The pattern of changes induced by TG is shared with cyclopiazoni c acid, but effects of both perturbants differ significantly from chan ges induced by heat shock. Upon removal of TG, normal protein synthesi s 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 c onclude that inhibition of intracellular Ca2+-pump activity can elicit a stress response, which has important implications for the interpret ation of chronic use of Ca2+-pump inhibitors. In particular, the activ ation of the cellular shock response should be considered in interpret ing the regulation of protein synthesis and cell survival by Ca2+-pump inhibitors such as TG.