Yonuschot G
Department of Biochemistry, University of New England, Biddeford, ME 04005.
Free Radic Biol Med. 1991;11(3):307-17. doi: 10.1016/0891-5849(91)90128-p.
The purpose of this study was to determine if there was an early increase in intracellular Ca++ which preceded generalized lysis of thymocytes during photodynamic permeabilization. A method was developed that facilitated the simultaneous measurement in real time of permeabilization of the thymocyte cell membrane to Ca++, Mn++, and ethidium bromide during photodynamic action. Quin-2 loaded cells were illuminated in the presence of erythrosin B and the change in the fluorescence emission of the calcium-quin-2 complex was used to determine how soon and to what extent intracellular Ca++ changed following illumination. In the presence of extracellular manganese, the same system was used to determine how soon the cells became permeable to Mn++ or quin-2. It was determined that the fluorescence emission of the ethidium bromide-DNA complex was strong enough to be measured in the presence of the calcium-quin-2 complex. This enabled the concomitant determination of the elapsed time following illumination before ethidium bromide entered the cell. It was established that increased intracellular Ca++ was an early event in the photodynamic permeabilization of thymocytes that preceded permeabilization of the cell membrane to ethidium bromide, Mn++ or quin-2, or lysis.
本研究的目的是确定在光动力通透化过程中，细胞内Ca++的早期增加是否先于胸腺细胞的全面裂解。开发了一种方法，该方法有助于在光动力作用期间实时同时测量胸腺细胞膜对Ca++、Mn++和溴化乙锭的通透性。在赤藓红B存在的情况下，对负载有喹啉-2的细胞进行光照，并利用钙-喹啉-2复合物荧光发射的变化来确定光照后细胞内Ca++多久以及在多大程度上发生变化。在存在细胞外锰的情况下，使用相同的系统来确定细胞对Mn++或喹啉-2的通透性何时开始。已确定溴化乙锭-DNA复合物的荧光发射强度足以在钙-喹啉-2复合物存在的情况下进行测量。这使得能够同时确定光照后溴化乙锭进入细胞之前所经过的时间。已确定细胞内Ca++增加是胸腺细胞光动力通透化过程中的早期事件，发生在细胞膜对溴化乙锭、Mn++或喹啉-2的通透化或裂解之前。
