1. Large changes in intracellular pH and calcium observed during heat shock are not responsible for the induction of heat shock proteins in Drosophila melanogaster
I A Drummond, S A McClure, M Poenie, R Y Tsien, R A Steinhardt Mol Cell Biol. 1986 May;6(5):1767-75. doi: 10.1128/mcb.6.5.1767-1775.1986.
Heat shock caused significant changes in intracellular pH (pHi) and intracellular free calcium concentration [( Ca2+]i) which occurred rapidly after temperature elevation. pHi fell from a resting level value at 25 degrees C of 7.38 +/- 0.02 (mean +/- standard error of the mean, n = 15) to 6.91 +/- 0.11 (n = 7) at 35 degrees C. The resting level value of [Ca2+]i in single Drosophila melanogaster larval salivary gland cells was 198 +/- 31 nM (n = 4). It increased approximately 10-fold, to 1,870 +/- 770 nM (n = 4), during a heat shock. When salivary glands were incubated in calcium-free, ethylene glycol-bis(beta-aminoethyl ether)-N,N',N'-tetraacetic acid (EGTA)-buffered medium, the resting level value of [Ca2+]i was reduced to 80 +/- 7 nM (n = 3), and heat shock resulted in a fourfold increase in [Ca2+]i to 353 +/- 90 nM (n = 3). The intracellular free-ion concentrations of Na+, K+, Cl-, and Mg2+ were 9.6 +/- 0.8, 101.9 +/- 1.7, 36 +/- 1.5, and 2.4 +/- 0.2 mM, respectively, and remained essentially unchanged during a heat shock. Procedures were devised to mimic or block the effects of heat shock on pHi and [Ca2+]i and to assess their role in the induction of heat shock proteins. We report here that the changes in [Ca2+]i and pHi which occur during heat shock are not sufficient, nor are they required, for a complete induction of the heat shock response.
2. Thermotolerance attenuates heat-induced increases in [Ca2+]i and HSP-72 synthesis but not heat-induced intracellular acidification in human A-431 cells
J G Kiang, X Z Ding, D E McClain J Investig Med. 1996 Feb;44(2):53-63.
Background: Thermotolerance affects cell viability, retards translation of heat shock proteins, and protects RNA slicing mechanisms. We reported previously that heat shocking nonthermotolerant cells causes an intracellular acidification and an increase in cytosolic free Ca2+ ([Ca2+]i) in addition to an induction of heat shock protein 72kDa (HSP-72) production. This study characterized heat-induced changes in cytosolic Ca2+, H+, and HSP-72 synthesis in thermotolerant A-431 cells. Methods: We studied heat-induced changes in pH(i), [Ca2+]i, and HSP-72 using thermotolerant A-431 cell monolayers. pH(i) and [Ca2+]i were determined using fluorescence probes, and HSP-72 was measured by SDS-PAGE. The mRNA encoding HSP-72 was measured by Northern blots probed with a [32P]-labeled 2.3 kb fragment of an HSP-70 cDNA insert. Results: Heat shocking thermotolerant cells induced the same degree of intracellular acidification as that induced in nonthermotolerant cells, but the heat-induced increase in [Ca2+]i was less in thermotolerant cells. This diminished response was characterized by an increase in Km for external Ca2+ and was blocked by pretreatment with cycloheximide, indicating a newly synthesized protein is involved. Similar to what was seen in nonthermotolerant cells, the heat-induced increase in [Ca2+]i in thermotolerant cells depended on external Na+ concentration and was blocked by dichlorobenzamil, though thermotolerant cells were more sensitive to the inhibitor (IC50 = 0.21 mumol/L for nonthermotolerant, 0.025 mumol/Lm for thermotolerant). Thermotolerant cells contained high resting levels of HSP-72. Heat shocking these cells attenuated the HSF translocation from cytosol to nucleus and did not induce a further synthesis of HSP-72 mRNA and protein. Conclusions: The results suggest that thermotolerance desensitizes the machinery required for Ca2+ entry. Low [Ca2+]i levels probably result in diminished HSP-72 mRNA production and less HSP-72 synthesis.
3. Effects of calcium buffering on the synthesis of the 26-kDa heat-shock protein family
D P Evans, J R Corbin, S P Tomasovic Radiat Res. 1991 Sep;127(3):261-8.
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.
