Characterization of Intracellular Calcium Pools and Their Desensitization in Thermotolerant Human A-431 Cells

Abstract

Background

This study characterizes the intracellular Ca²⁺ pools in nonthermotolerant and thermotolerant human A-431 cells and the reduced cytotoxicity using the inhibitors of Ca²⁺ mobilizations.

Methods

Nonthermotolerant and thermotolerant cells were treated with different Ca²⁺ mobilizers in the absence of external Ca²⁺. The cytosolic Ca²⁺ concentration using fura-2 fluorescence probe was measured to identify the presence of intracellular Ca²⁺ pools. The cytotoxicity of the increase in [Ca²⁺]_i was studied using the colony forming efficiency assay.

Results

The resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the absence of extracellular Ca²⁺ was 42 ± 9 nm, determined by fura-2. Bradykinin (10 μmol/L), monensin (200 μmol/L), and ionomycin (1 μmol/L) sequentially treated to cells mobilized Ca²⁺ and increased [Ca²⁺]_i by 64 ± 23, 40 ± 6, and 59 ± 21 nm, respectively. The bradykinin effect was blocked by 5 μmol/L U-73122 (an inhibitor of inositol trisphosphate production); the ionomycin effect was inhibited by increasing intracellular pH (pH_i) or treatment with 100 μmol/L ryanodine while the monensin effect was enhanced by increasing pH_i, but was not inhibited by ryanodine. Cells that were made tolerant to lethal temperatures also responded to bradykinin, monensin, and ionomycin, but the magnitude of the response was diminished. Subsequent treatments with bradykinin, monensin, and ionomycin increased [Ca²⁺]; in thermotolerant cells to levels 68 ± 8, 44 ± 5, and 45±5%, respectively, of values found in nonthermotolerant cells. Higher concentrations of these agents did not further increase [Ca²⁺]_i. The bradykinin-induced increase in inositol trisphosphates in thermotolerant cells was also reduced, which perhaps accounts for the attenuation in Ca²⁺ mobilization. Unlike nonthermotolerant cells, the monensin effect was not enhanced when pH_i was increased. However, the ionomycin effect was still dependent on pH_i and was blocked by ryanodine at a higher concentration.

Conclusions

These results show that there are bradykinin-, monensin-, and ryanodine-sensitive pools and that thermotolerance attenuates Ca²⁺ mobilization stimulated by these three agents. Ionomycin at 10 μmol/L or NaCN at 10 mM for 1 hour demonstrated cytotoxicity. Pretreatment with 100 μmol/L ryanodine and/or 5 μmol/L U-73122 reduced cytotoxicity produced by either NaCN or ionomycin. These results suggest that an attenuation of [Ca²⁺]_i increases can diminish cytotoxicity