热耐受性可减弱热诱导的人A-431细胞中[Ca2+]i升高和HSP-72合成，但不会减弱热诱导的细胞内酸化。

Thermotolerance attenuates heat-induced increases in [Ca2+]i and HSP-72 synthesis but not heat-induced intracellular acidification in human A-431 cells.

作者信息

Kiang J G, Ding X Z, McClain D E

机构信息

Department of Clinical Physiology, Walter Reed Army Institute of Research, Washington DC 20307-5100, USA.

出版信息

J Investig Med. 1996 Feb;44(2):53-63.

PMID:8689402

Abstract

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.

摘要

背景

热耐受性影响细胞活力，延缓热休克蛋白的翻译，并保护RNA剪接机制。我们之前报道过，热激非耐热细胞除了诱导72kDa热休克蛋白（HSP - 72）产生外，还会导致细胞内酸化和胞质游离Ca2+（[Ca2+]i）增加。本研究对耐热A - 431细胞中热诱导的胞质Ca2+、H+变化以及HSP - 72合成进行了表征。

方法

我们使用耐热A - 431细胞单层研究热诱导的pH(i)、[Ca2+]i和HSP - 72变化。使用荧光探针测定pH(i)和[Ca2+]i，通过SDS - PAGE测量HSP - 72。用[32P]标记的HSP - 70 cDNA插入片段的2.3 kb片段探测的Northern印迹法测量编码HSP - 72的mRNA。

结果

热激耐热细胞诱导的细胞内酸化程度与非耐热细胞相同，但耐热细胞中热诱导的[Ca2+]i增加较少。这种反应减弱的特征是细胞外Ca2+的Km增加，并被环己酰亚胺预处理所阻断，表明涉及一种新合成的蛋白质。与非耐热细胞中所见相似，耐热细胞中热诱导的[Ca2+]i增加取决于细胞外Na+浓度，并被二氯苯甲酰胺阻断，不过耐热细胞对该抑制剂更敏感（非耐热细胞的IC50 = 0.21 μmol/L，耐热细胞的IC50 = 0.025 μmol/L）。耐热细胞含有高水平的静息HSP - 72。热激这些细胞减弱了热休克因子从胞质向细胞核的转位，并且没有诱导HSP - 72 mRNA和蛋白质的进一步合成。