Kiang J G, Wang X D, Ding X Z, Gist I D, Smallridge R C
Department of Clinical Physiology, Walter Reed Army Institute of Research, Washington DC 20307-5100, USA.
Thyroid. 1996 Oct;6(5):475-83. doi: 10.1089/thy.1996.6.475.
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/micrograms DNA/min. Hypoxia (5% O2) increased the rate of uptake in a time-dependent manner. Heating cells at 45 degrees 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-alpha, -delta, -epsilon, and -zeta isoforms, whereas heat shock alone translocated only PKC-beta I, -beta II, and -zeta. 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.
慢性低氧在体内抑制大鼠甲状腺功能。为确定可能的机制，我们研究了低氧对大鼠甲状腺FRTL-5细胞碘摄取、第二信使的参与情况及细胞膜通透性的影响。由于亚致死性热应激可保护组织免受缺血损伤，我们还确定了热应激的作用。未处理细胞中碘摄取的初始速率在12.98至15.28 pmol/μg DNA/分钟之间。低氧（5% O₂）以时间依赖性方式增加摄取速率。在暴露于低氧3天之前，将细胞在45℃加热15分钟（热休克）可抑制碘摄取初始速率的增加。使用fura-2，我们发现悬浮的FRTL-5细胞中静息[Ca²⁺]i为65±7 nM（n = 16）。暴露于低氧1天的细胞中[Ca²⁺]i未增加，而暴露3天使[Ca²⁺]i增加了43±4%（p < 0.05）；暴露7天后未出现进一步增加。当细胞在低氧暴露3天之前进行加热时，低氧诱导的[Ca²⁺]i增加未发生。对于肌醇三磷酸（InsP3）也有类似观察结果。细胞暴露于低氧3天使InsP3从0.08±0.02（n = 5）增加至总cpm的0.32±0.04%（n = 5，p < 0.05），但在低氧暴露之前对细胞进行亚致死性加热可防止这种增加。三天低氧增加了膜组分中的PKC活性（从总活性的67±7%增加至86±4%，p < 0.05），热休克也抑制了这些变化。免疫印迹显示，单独低氧处理以及热休克加低氧导致PKC-α、-δ、-ε和-ζ同工型易位，而单独热休克仅使PKC-βI、-βII和-ζ易位。通过台盼蓝排斥法测定细胞膜完整性。单独低氧3天不影响膜通透性，但在3天低氧暴露后再进行6小时复氧时，只有49±3%的细胞排斥台盼蓝。低氧和复氧之前的热休克保护细胞膜功能。热休克还在转录水平诱导70 kDa热休克蛋白（HSP-70）合成。结果表明，热休克可能通过抑制碘摄取初始速率和第二信使来保护FRTL-5细胞免受低氧损伤。HSP-70很可能在保护过程中起重要作用。
