Heat Shock Inhibits the Hypoxia-Induced Effects on Iodide Uptake and Signal Transduction and Enhances Cell Survival in Rat Thyroid FRTL-5 Cells

Chronic hypoxia inhibits rat thyroid function in vivo. To determine possible mechanisms, we studied the effect of hypoxia on iodide uptake, the involvement of second messengers, and cell membrane permeability in rat thyroid FRTL-5 cells. Since sublethal heat stress protects tissues from ischemia, we also determined effects of heat stress. The initial rate of iodide uptake in untreated cells was between 12.98 and 15.28 pmol/μg DNA/min. Hypoxia (5% O₂) increased the rate of uptake in a time-dependent manner. Heating cells at 45°C for 15 min (heat shock) prior to exposure to hypoxia for 3 days inhibited the increase in the initial rate of I-uptake. Using fura-2, we found that the resting [Ca²⁺]_i in suspended FRTL-5 cells was 65 ± 7 nM (n = 16). [Ca²⁺]_i was not increased in cells exposed to hypoxia for 1 day, while a 3-day exposure increased [Ca²⁺]_i by 43 ± 4% (p < 0.05); no additional increase occurred after 7 days of exposure. When cells were heated prior to hypoxia exposure for 3 days, the hypoxia-induced increase in [Ca²⁺]_i did not occur. Similar observations were found with inositol trisphosphates (InsP₃). Exposure of cells to hypoxia for 3 days increased InsP₃ from 0.08 ± 0.02 (n = 5) to 0.32 ± 0.04% total cpm (n = 5, p < 0.05), but sublethal heating of cells prior to hypoxia exposure prevented the increase. Three-day hypoxia increased PKC activity in the membrane fraction (from 67 ± 7 to 86 ± 4% of total activity, p < 0.05), and heat shock inhibited these changes also. Immunoblots showed that hypoxia treatment alone and heat shock plus hypoxia resulted in the translocation of PKC-α, -δ, -ε, and -ζ isoforms, whereas heat shock alone translocated only PKC-βI, -βII, and -ζ Cell membrane integrity was assayed by trypan blue exclusion. Hypoxia alone for 3 days did not affect membrane permeability, but only 49 ± 3% of cells excluded trypan blue when a 3-day hypoxia exposure was followed by a 6 h reoxygenation. Heat shock prior to hypoxia and reoxygenation protected cell membrane function. Heat shock also induced heat shock protein 70 kDa (HSP-70) synthesis at the transcriptional level. Results suggest that heat shock protects FRTL-5 cells from hypoxic injury, perhaps by inhibiting the initial rate of iodide uptake and second messengers. It is likely that HSP-70 plays an essential role in the process of protection.