Cromwell O, Bennett J P, Hide I, Kay A B, Gomperts B D
Department of Allergy and Clinical Immunology, National Heart & Lung Institute, London, UK.
J Immunol. 1991 Sep 15;147(6):1905-11.
The mechanisms of granule protein secretion have been studied in streptolysin-O-permeabilized guinea pig eosinophils. Secretion of the granule-associated enzyme N-acetyl-beta-D-glucosaminidase was dependent on both Ca2+ and a nonhydrolyzable GTP analogue, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), suggesting roles for both calcium and GTP binding proteins. Secretion was maximal by 7 min, and varied between 35 and 60% of the total enzyme activity. Other GTP analogues also elicited secretion, with rank order GTP-gamma-S greater than guanylyl-imidophosphate greater than guanylyl (beta-gamma-methylene-diphosphate). Unrelated nucleotide triphosphates showed little or no effect confirming the specificity of the G protein. Transmission electronmicroscopy confirmed that permeabilization alone did not result in loss of granules and that exocytosis was dependent on the addition of the effectors, Ca2+ and GTP-gamma-S. ATP enhanced the magnitude of the secretory response and also enhanced the effective affinities for both Ca2+ and GTP-gamma-S. In the presence of 10(-5) M GTP-gamma-S the ED50 (Ca2+) was pCa 5.57 +/- 0.04 (2.69 microM) in the absence of ATP and declined to pCa 6.16 +/- 0.03 (0.69 microM) in the presence of ATP (p less than 0.0001). Furthermore, ATP served to restore responsiveness in cells that had been rendered refractory by delaying stimulation after permeabilization. Pretreatment with PMA (an activator of PKC) inhibited the induction of a refractory state, whereas inhibition of PKC partially countered the ability of ATP to restore responsiveness, both observations pointing to a requirement for a specific component of the secretory mechanism to be in a phosphorylated state in order to condone the secretion process. These observations show that secretory mechanisms in eosinophils are similar to those in other myeloid cells, in particular neutrophils and mast cells, although the time course of secretion is more protracted.
已在经链球菌溶血素 - O通透处理的豚鼠嗜酸性粒细胞中研究了颗粒蛋白分泌的机制。颗粒相关酶N - 乙酰 - β - D - 氨基葡萄糖苷酶的分泌既依赖于Ca2 + ，也依赖于一种不可水解的GTP类似物，鸟苷 - 5'-O -（3 - 硫代三磷酸）（GTP - γ - S），这表明钙和GTP结合蛋白都发挥了作用。分泌在7分钟时达到最大值，占总酶活性的35%至60%。其他GTP类似物也能引发分泌，其活性顺序为GTP - γ - S大于鸟苷酰亚胺磷酸大于鸟苷酰（β - γ - 亚甲基二磷酸）。不相关的三磷酸核苷酸几乎没有影响或没有影响，这证实了G蛋白的特异性。透射电子显微镜证实，仅通透处理不会导致颗粒丢失，并且胞吐作用依赖于效应物Ca2 + 和GTP - γ - S的添加。ATP增强了分泌反应的幅度，也增强了对Ca2 + 和GTP - γ - S的有效亲和力。在存在10(-5) M GTP - γ - S的情况下，在不存在ATP时，ED50（Ca2 + ）为pCa 5.57 ± 0.04（2.69 microM），而在存在ATP时降至pCa 6.16 ± 0.03（0.69 microM）（p < 0.0001）。此外，ATP可恢复在通透处理后延迟刺激而变得不应答的细胞的反应性。用PMA（蛋白激酶C的激活剂）预处理可抑制不应答状态的诱导，而抑制蛋白激酶C部分抵消了ATP恢复反应性的能力，这两个观察结果都表明分泌机制的特定成分需要处于磷酸化状态才能支持分泌过程。这些观察结果表明，嗜酸性粒细胞中的分泌机制与其他髓样细胞，特别是中性粒细胞和肥大细胞中的分泌机制相似，尽管分泌的时间进程更长。
