EFFECTS OF CALCIUM BUFFERING ON THE SYNTHESIS OF THE 26-KDA HEAT-SHOCK PROTEIN FAMILY

We have reported on the effect of heat in C127 cells having various basal levels of the Ca2+-binding proteins calmodulin (CaM) or parvalbumin [Evans, Simonette, Rasmussen, Means, and Tomasovic, J. Cell. Physiol. 142, 615-627 (1990)]. These studies suggested that induction of the synthesis of 26-kDa heat-shock protein (hsp-26) depended on increased intracellular free Ca2+ [Ca2+](i) and that induction was abrogated by increased Ca2+-binding capacity. To evaluate further the role of [Ca2+](i) in mediating the response to hyperthermia and the potential for Ca2+-buffering to affect these processes; we loaded C127 parental cells with the Ca2+ chelators BAPTA or quin-2 (5 μM for 60 min) and then immediately heated the cells (30 min at 43°C) and labeled them (3 h at 37°C) with [3H]leucine. Measurements of [Ca2+](i) with quin-2 and fura-2 showed that an increase in [Ca2+](i) occurred with this heat dose, but that the quin-2 buffered that increase. Two-dimensional gels showed that cells loaded with BAPTA and quin-2 had a reduced rate of synthesis of the most basic (nonphosphorylated) hsp-26a isoform. The apparent synthesis of the more acidic isoforms (hsp-26b, hsp- 26c) was less affected, but labeling studies with 32P showed this reflected continued accumulation of these phosphorylated isoforms, especially the most highly phosphorylated hsp-26c. Although it reduced hsp-26a synthesis, the temporary buffering of [Ca2+](i) did not alter the subsequent expression of heat killing or the extent of thermotolerance significantly, possibly because phosphorylated hsp-26 was still generated. These data support the hypothesis that perturbations of [Ca2+](i) directly modulate induction of hsp-26a synthesis.