Activation of proton pumping in human neutrophils occurs by exocytosis of vesicles bearing vacuolar-type H+-ATPases
Activation of proton pumping in human neutrophils occurs by exocytosis of vesicles bearing vacuolar-type H+-ATPases
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DOI：
10.1074/jbc.271.27.15963
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发表时间：
1996-07-05
影响因子：
4.8
通讯作者：
Grinstein, S
中科院分区：
文献类型：
--
作者：
Nanda, A;Brumell, JH;Grinstein, S
质子泵活性在未受刺激的中性粒细胞的质膜中是不可测量的，但在被可溶性激动剂激活后变得容易检测。本报告研究了泵激活的机制。在悬浮的中性粒细胞中，趋化肽和佛波酯可刺激V型H+泵的活性，这种活性被认为是巴弗洛霉素A（1）敏感的细胞质pH升高。细胞松弛素B（一种已知可增强中性粒细胞颗粒分泌的药物）可大大增强激动剂诱导的泵活性刺激。因此，我们使用流式细胞术和酶联免疫吸附试验，比较了泵激活的速率和程度与中性粒细胞中存在的四种类型的分泌细胞器的胞吐模式。分泌囊泡和次级和三级颗粒而不是初级颗粒的胞吐动力学表明泵活性的出现，使用V型ATP酶C亚基的抗体通过细胞分级分离和免疫印迹来定义泵的亚细胞定位。该泵丰富的三级颗粒，与显着的金额也存在于初级颗粒和分泌囊泡。该泵在次级颗粒中很少见，在细胞质中也检测不到。最后，激动剂不能刺激中性粒细胞胞质中的泵活性，中性粒细胞胞质是缺乏酸性颗粒的完整细胞碎片。总之，我们的结果表明，V型H+-ATP酶不是组成性地存在于中性粒细胞的质膜中，而是在细胞活化期间通过胞吐作用递送到表面膜。三级颗粒和分泌囊泡是V-ATP酶最可能的来源，在插入质膜后，泵准备有效地挤出在趋化性和细菌杀伤过程中产生的过量代谢酸。
Proton pump activity is not measurable in the plasma membrane of unstimulated neutrophils but becomes readily detectable upon activation by soluble agonists. The mechanism of pump activation was investigated in this report. V-type H+ pump activity, estimated as a bafilomycin A(1)-sensitive elevation of the cytosolic pH, was stimulated in suspended neutrophils by chemotactic peptides and by phorbol esters. Stimulation of pump activity induced by the agonists was greatly enhanced by cytochalasin B, an agent known to potentiate granular secretion in neutrophils. We therefore compared the rate and extent of pump activation with the pattern of exocytosis of the four types of secretory organelles present in neutrophils, using flow cytometry and enzyme-linked immunosorbent assay, The kinetics of exocytosis of secretory vesicles and secondary and tertiary granules but not primary granules paralleled the appearance of pump activity, The subcellular localization of the pump was defined by cellular fractionation and immunoblotting using an antibody to the C subunit of the V-type ATPase. The pump was abundant in tertiary granules, with significant amounts present also in primary granules and secretory vesicles. The pump was scarce in secondary granules and not detectable in the cytosol. Finally, the agonists failed to stimulate pump activity in neutrophil cytoplasts, which are intact cell fragments devoid of acidic granules, Together, our results suggest that the V-type H+-ATPase is not constitutively present in the plasma membrane of neutrophils but is delivered to the surface membrane by exocytosis during cellular activation, Tertiary granules and secretory vesicles are the most likely source of V-ATPases, Following insertion in the plasma membrane, the pump is poised to effectively extrude the excess metabolic acid that is generated during chemotaxis and bacterial killing.