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 Ca(2+)-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 Ca(2+)-binding capacity. To evaluate further the role of [Ca2+]i in mediating the response to hyperthermia and the potential for Ca(2+)-buffering to affect these processes, we loaded C127 parental cells with the Ca2+ chelators BAPTA or quin-2 (5 microM for 60 min) and then immediately heated the cells (30 min at 43 degrees C) and labeled them (3 h at 37 degrees 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.
Evans
D P
DP
Department of Tumor Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Corbin
J R
JR
Tomasovic
S P
SP
D000064:Acclimatization / Q000187:drug effects / Q000502:physiology; D000634:Aminoquinolines; D000818:Animals; D002021:Buffers; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002614:Chelating Agents; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D016257:Fura-2; D006360:Heat-Shock Proteins / Q000096:biosynthesis*; D006358:Hot Temperature
D000634:Aminoquinolines; D002021:Buffers; D002614:Chelating Agents; D006360:Heat-Shock Proteins; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C035739:Quin2; D002118:Calcium; D016257:Fura-2; D004121:Dimethyl Sulfoxide