Thermotolerance Attenuates Heat-Induced Increases in [Ca 2+ ] i and HSP-72 Synthesis but not Heat-Induced Intracellular Acidification in Human A-431 Cells

Abstract

Background

Thermotolerance affects cell viability, retards translation of heat shock proteins, and protects RNA slicing mechanisms. We reported previously that heat shock-ing nonthermotolerant cells causes an intracellular acidifica-tion and an increase in cytosolic free Ca²⁺ ([Ca²⁺]_i) in addi-tion to an induction of heat shock protein 72 kDa (HSP-72) production. This study characterized heat-induced changes in cytosolic Ca²⁺, H⁺, and HSP-72 synthesis in thermotoler-ant A-431 cells.

Methods

We studied heat-induced changes in pH_i, [Ca²⁺]_i, and HSP-72 using thermotolerant A-431 cell mono-layers. 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 K_m 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 exter-nal Na⁺ concentration and was blocked by dichlorobenzamil, though thermotolerant cells were more sensitive to the inhibitor (IC₅₀ = 0.21 μmol/L for nonthermotolerant, 0.025 μmol/Lm for thermotolerant). Thermotolerant cells contained high resting levels of HSP-72. Heat shocking these cells atten-uated 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.