Acidification is essential for maintaining the structure and function of lytic granules of CTL. Effect of concanamycin A, an inhibitor of vacuolar type H(+)-ATPase, on CTL-mediated cytotoxicity.
Acidification is essential for maintaining the structure and function of lytic granules of CTL. Effect of concanamycin A, an inhibitor of vacuolar type H(+)-ATPase, on CTL-mediated cytotoxicity.
复制标题
酸化对于维持 CTL 裂解颗粒的结构和功能至关重要。
作者：
T. Kataoka;K. Takaku;J. Magae;N. Shinohara;H. Takayama;S. Kondo;K. Nagai
空泡型H(+)- atp酶抑制剂concanamycin A （CMA）抑制CD8+ CTL克隆OE4的特异性细胞溶解活性。用CMA预处理效应细胞，而不是靶细胞，观察到抑制作用。CMA似乎没有抑制早期事件，因为效应物/靶偶联物的形成仍然不受影响。虽然CMA处理OE4会导致抗cd3刺激下颗粒胞吐效率的轻微下降，但最显著的效果是溶解颗粒中穿孔素活性和DNA降解活性的显著降低。Western blotting分析显示，cma处理的细胞中穿孔素的数量急剧减少。吖啶橙染色OE4的荧光显微镜观察表明，CMA提高了溶解颗粒的pH值。透射电镜下观察到CMA处理后OE4细胞质颗粒结构发生了显著的形态学变化。OE4溶解颗粒具有均匀染色的大核和充满周围区域的大量小泡。相比之下，cma处理的OE4溶解颗粒形状不规则，没有小泡，但核心变得粗糙和松散。偶见无结构的液泡。这些结果表明，通过液泡型H(+)- atp酶进行酸化对维持溶解颗粒的结构和功能至关重要。
An inhibitor of vacuolar type H(+)-ATPase, concanamycin A (CMA), inhibited the specific cytolytic activity of a CD8+ CTL clone, OE4. The inhibitory effect was observed when the effector cells, and not the target cells, were pretreated with CMA. CMA did not seem to inhibit early events, inasmuch as effector/target conjugate formation remained unaffected. Although CMA treatment of OE4 resulted in a slight decrease in the efficiency of granule exocytosis in response to anti-CD3 stimulation, the most prominent effect was a marked reduction of perforin activity and DNA degradation activity in lytic granules. Western blotting analysis indicated a drastic decrease in the amount of perforin in CMA-treated cells. Fluorescent microscopic observation of OE4 stained with acridine orange indicated that CMA raised the pH of the lytic granules. Under transmission electron microscopy, striking morphologic changes in cytoplasmic granular structures were observed after CMA treatment of OE4. The lytic granules of OE4 had homogeneously stained large cores and numerous small vesicles that filled peripheral areas. In contrast, the lytic granules of CMA-treated OE4 showed irregular shapes with no small vesicles, but with cores that became rough and loose. Vacuoles with no structure in them were seen occasionally. These results suggest that acidification through vacuolar type H(+)-ATPase is essential to maintain the structure and function of lytic granules.