Pryzwansky K B, Wyatt T A, Nichols H, Lincoln T M
Department of Pathology, University of North Carolina, Chapel Hill 27599-7525.
Blood. 1990 Aug 1;76(3):612-8.
The presence and physiologic role of cyclic GMP-dependent protein kinase (G-kinase) in human neutrophils was investigated by Western blot analysis and immunocytochemistry. Small quantities of G-kinase were found in the cytoskeletal-enriched fraction of neutrophil lysates as detected by Western blots using a polyclonal antibody raised against bovine aorta G-kinase. Immunofluorescence microscopy demonstrated in adherent neutrophils that G-kinase was localized diffusely within the cytoplasm, at the microtubule organizing center, and in the euchromatin of the nucleus. Because cyclic GMP is implicated as a modulator of neutrophil chemotaxis, G-kinase localization was investigated in neutrophils activated with N-formyl-methionyl-leucyl-phenylalanine (fMLP). fMLP stimulated transient focal changes in G-kinase localization that coincided with transient changes in cell shape. G-kinase translocated over a period of 5 minutes from diffuse staining of the cytosol to filaments within the uropod of polarized cells (1 minute), to bundles of filaments associated with loss of cell polarity (2.5 minutes), and finally to more intense staining of the nuclear euchromatin (5 minutes). Optical sectioning of neutrophils by confocal laser scanning microscopy confirmed that G-kinase was restricted to specific sub-cellular compartments during cell activation. This transient localization of G-kinase was disrupted by cytoskeletal inhibitors and was augmented by 8-Br-cyclic GMP. These data provide evidence for the first time that G-kinase plays a physiologic role in human neutrophils, and support the concept of compartmentalization of cyclic nucleotides during neutrophil activation.
通过蛋白质免疫印迹分析和免疫细胞化学方法，对环磷酸鸟苷依赖性蛋白激酶（G激酶）在人中性粒细胞中的存在情况及其生理作用进行了研究。使用针对牛主动脉G激酶产生的多克隆抗体进行蛋白质免疫印迹检测，结果发现在中性粒细胞裂解液富含细胞骨架的部分中存在少量G激酶。免疫荧光显微镜检查显示，在贴壁的中性粒细胞中，G激酶广泛分布于细胞质、微管组织中心以及细胞核的常染色质中。由于环磷酸鸟苷被认为是中性粒细胞趋化作用的调节因子，因此研究了用N-甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）激活的中性粒细胞中G激酶的定位情况。fMLP刺激后，G激酶的定位出现短暂的局部变化，这与细胞形状的短暂变化相吻合。G激酶在5分钟内发生移位，从胞质溶胶的弥漫性染色转移至极化细胞尾足内的细丝（1分钟），再转移至与细胞极性丧失相关的细丝束（2.5分钟），最后转移至核常染色质的更强染色区域（5分钟）。通过共聚焦激光扫描显微镜对中性粒细胞进行光学切片，证实了细胞激活过程中G激酶局限于特定的亚细胞区室。G激酶的这种短暂定位被细胞骨架抑制剂破坏，并被8-溴环磷酸鸟苷增强。这些数据首次证明G激酶在人中性粒细胞中发挥生理作用，并支持中性粒细胞激活过程中环核苷酸区室化的概念。
