Kiang J G, Koenig M L
Department of Clinical Physiology, Walter Reed Army Institute of Research, Washington DC 20307-5100, USA.
J Investig Med. 1996 Aug;44(6):352-61.
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.
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.
The resting intracellular Ca2+ concentration ([Ca2+]i) in the absence of extracellular Ca2+ was 42 +/- 9 nm, determined by fura-2. Bradykinin (10 mumol/L), monensin (200 mumol/L), and ionomycin (1 mumol/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 mumol/L U-73122 (an inhibitor of inositol trisphosphate production); the ionomycin effect was inhibited by increasing intracellular pH (pHi) or treatment with 100 mumol/L ryanodine while the monensin effect was enhanced by increasing pHi, 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+]i 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 pHi was increased. However, the ionomycin effect was still dependent on pHi and was blocked by ryanodine at a higher concentration.
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 mumol/L or NaCN at 10 mM for 1 hour demonstrated cytotoxicity. Pretreatment with 100 mumol/L ryanodine and/or 5 mumol/L U-73122 reduced cytotoxicity produced by either NaCN or ionomycin. These results suggest that an attenuation of [Ca2+]i increases can diminish cytotoxicity.
本研究对非耐热型和耐热型人A - 431细胞中的细胞内钙池进行了表征，并使用钙动员抑制剂研究了细胞毒性的降低情况。
在无细胞外钙的情况下，用不同的钙动员剂处理非耐热型和耐热型细胞。使用fura - 2荧光探针测量胞质钙浓度，以确定细胞内钙池的存在。使用集落形成效率试验研究细胞内钙浓度升高的细胞毒性。
通过fura - 2测定，在无细胞外钙时，静息细胞内钙浓度（[Ca²⁺]i）为42±9 nM。依次用缓激肽（10 μmol/L）、莫能菌素（200 μmol/L）和离子霉素（1 μmol/L）处理细胞，可动员钙并使[Ca²⁺]i分别增加64±23、40±6和59±21 nM。缓激肽的作用被5 μmol/L U - 73122（肌醇三磷酸生成抑制剂）阻断；离子霉素的作用通过提高细胞内pH（pHi）或用100 μmol/L 兰尼碱处理而受到抑制，而莫能菌素的作用在提高pHi时增强，但不受兰尼碱抑制。对致死温度产生耐受的细胞对缓激肽、莫能菌素和离子霉素也有反应，但反应幅度减小。随后用缓激肽、莫能菌素和离子霉素处理使耐热型细胞中的[Ca²⁺]i分别升高至非耐热型细胞中所测值的68±8%、44±5%和45±5%。这些试剂的更高浓度并未进一步增加[Ca²⁺]i。耐热型细胞中缓激肽诱导的肌醇三磷酸增加也减少，这可能解释了钙动员的减弱。与非耐热型细胞不同，当pHi升高时，莫能菌素的作用并未增强。然而，离子霉素的作用仍依赖于pHi，且在更高浓度下被兰尼碱阻断。
这些结果表明存在对缓激肽、莫能菌素和兰尼碱敏感的钙池，并且热耐受性减弱了这三种试剂刺激的钙动员。10 μmol/L的离子霉素或10 mM的NaCN处理1小时具有细胞毒性。用100 μmol/L兰尼碱和/或5 μmol/L U - 73122预处理可降低由NaCN或离子霉素产生的细胞毒性。这些结果表明细胞内钙浓度升高的减弱可降低细胞毒性。
