Stutchfield J, Cockcroft S
Department of Experimental Pathology, School of Medicine, University College London.
Biochem J. 1988 Mar 1;250(2):375-82. doi: 10.1042/bj2500375.
The non-differentiated HL60 cell can be stimulated to secrete when Ca2+ and guanosine 5'-[gamma-thio]-triphosphate (GTP gamma S) are introduced into streptolysin-O-permeabilized cells. Secretion is accompanied by activation of polyphosphoinositide phosphodiesterase (PPI-pde). Both responses show a concentration-dependence on Ca2+ between pCa 8 and pCa 5. The half-maximal requirements for Ca2+ for PPI-pde activation and secretion are pCa 6.4 +/- 0.1 and pCa 6.2 +/- 0.2 respectively. The rank order of potency of the GTP analogues to stimulate PPI-pde activation and secretion is similar; GTP gamma S greater than guanosine 5'-[beta gamma-imido]-triphosphate greater than guanosine 5'-[beta gamma-methylene]triphosphate greater than XTP approximately equal to ITP, but the maximal response achieved by each compound compared with GTP gamma S is much greater for secretion than for PPI-pde activation. A dissociation of the two responses is obtained with 10 mM-XTP and -ITP; secretion is always observed but not inositol trisphosphate formation at this concentration. GTP, dGTP, UTP and CTP are inactive for both secretion and PPI-pde activation. Both GDP and dGDP are competitive inhibitors of both GTP gamma S-induced secretion and PPI-pde activation. Phorbol 12-myristate 13-acetate could not fully substitute for GTP gamma S in stimulating secretion, suggesting that the effect of GTP gamma S cannot result simply from the generation of diacylglycerol. In the absence of MgATP, secretion and PPI-pde activation is still evident, albeit at a reduced level. This also supports the hypothesis that protein kinase C-dependent phosphorylation is not essential for secretion. The effect of MgATP is to enhance secretion, and to reduce both the Ca2+ and GTP gamma S requirement for secretion. In conclusion, two roles for guanine nucleotides can be identified; one for activating PPI-pde (GP) and the other for activating exocytosis (GE), acting in series.
当将Ca2+和鸟苷5'-[γ-硫代]-三磷酸（GTPγS）引入经链球菌溶血素-O通透处理的细胞中时，未分化的HL60细胞可被刺激分泌。分泌伴随着多磷酸肌醇磷酸二酯酶（PPI-pde）的激活。两种反应均显示在pCa 8至pCa 5之间对Ca2+呈浓度依赖性。PPI-pde激活和分泌对Ca2+的半数最大需求量分别为pCa 6.4±0.1和pCa 6.2±0.2。刺激PPI-pde激活和分泌的GTP类似物的效力排序相似；GTPγS＞鸟苷5'-[βγ-亚氨基]-三磷酸＞鸟苷5'-[βγ-亚甲基]三磷酸＞XTP≈ITP，但与GTPγS相比，每种化合物实现的最大反应对于分泌而言比对PPI-pde激活要大得多。在10 mM-XTP和-ITP时可实现两种反应的解离；在此浓度下总是观察到分泌，但未观察到肌醇三磷酸的形成。GTP、dGTP、UTP和CTP对分泌和PPI-pde激活均无活性。GDP和dGDP均为GTPγS诱导的分泌和PPI-pde激活的竞争性抑制剂。佛波醇12-肉豆蔻酸酯13-乙酸酯在刺激分泌方面不能完全替代GTPγS，这表明GTPγS的作用不能简单地由二酰基甘油的生成导致。在不存在MgATP的情况下，分泌和PPI-pde激活仍然明显，尽管水平有所降低。这也支持了蛋白激酶C依赖性磷酸化对于分泌并非必不可少的假说。MgATP的作用是增强分泌，并降低分泌对Ca2+和GTPγS的需求。总之，可以确定鸟嘌呤核苷酸有两种作用；一种用于激活PPI-pde（GP），另一种用于激活胞吐作用（GE），二者串联起作用。
