Hackam D J, Rotstein O D, Zhang W J, Demaurex N, Woodside M, Tsai O, Grinstein S
Division of Cell Biology, The Hospital for Sick Children, Toronto Hospital, University of Toronto, Toronto M5G 1X8, Canada.
J Biol Chem. 1997 Nov 21;272(47):29810-20. doi: 10.1074/jbc.272.47.29810.
Vacuolar-type (V) ATPases are thought to be the main determinant of phagosomal acidification. In phagosomes containing mycobacteria, which ostensibly impair the delivery of V-ATPases to the phagosomal membrane, the pH would be expected to be near neutral. This prediction was tested by microfluorescence ratio imaging using macrophages from mice susceptible to mycobacterial infection. Although less acidic than their counterparts containing dead bacteria, phagosomes containing live Mycobacteria bovis were nearly 1 pH unit more acidic than the cytosol, suggesting the existence of alternate H+ transport mechanisms. We therefore investigated whether Na+/H+ exchange (NHE) contributes to phagosomal acidification. Immunoblotting, reverse transcriptase-polymerase chain reaction, and pharmacological studies indicated that NHE1 is the predominant isoform of the exchanger in macrophages. Fractionation revealed that NHE1 is incorporated into the phagosomal membrane, and measurements of pH indicated that it is functional in this location. Nevertheless, acidification of the lumen of phagosomes containing either latex beads or live M. bovis was insensitive to (3-methylsulfonyl-4-piperidinobenzoyl)-guanidine methanesulfonate, a potent inhibitor of NHE1. This may have been due to the absence of an appropriate lumen to cytosol Na+ gradient, because the phagosomal membrane was found to be devoid of Na+/K+ pumps. Unexpectedly, the acidification of M. bovis phagosomes was fully reversed by specific inhibitors of the vacuolar H+-ATPase, suggesting that ATPases are present only transiently or in reduced quantities in the phagosomal membrane. Alternatively, acid equivalents accumulated in endosomes by V-ATPases may be delivered to the mycobacterial phagosome by carrier vesicles devoid of ATPases.
液泡型（V）ATP酶被认为是吞噬体酸化的主要决定因素。在含有分枝杆菌的吞噬体中，表面上分枝杆菌会损害V-ATP酶向吞噬体膜的转运，因此预计其pH值接近中性。使用对分枝杆菌感染敏感的小鼠巨噬细胞进行微荧光比率成像，对这一预测进行了验证。尽管含有活牛分枝杆菌的吞噬体比含有死细菌的吞噬体酸性弱，但仍比细胞质酸性高近1个pH单位，这表明存在其他H⁺转运机制。因此，我们研究了Na⁺/H⁺交换（NHE）是否有助于吞噬体酸化。免疫印迹、逆转录聚合酶链反应和药理学研究表明，NHE1是巨噬细胞中该交换体的主要亚型。分级分离显示NHE1被整合到吞噬体膜中，pH测量表明它在该位置具有功能。然而，含有乳胶珠或活牛分枝杆菌的吞噬体腔的酸化对NHE1的强效抑制剂（3-甲基磺酰基-4-哌啶基苯甲酰基）-胍甲磺酸盐不敏感。这可能是由于缺乏合适的腔到细胞质的Na⁺梯度，因为发现吞噬体膜缺乏Na⁺/K⁺泵。出乎意料的是，液泡H⁺-ATP酶的特异性抑制剂能完全逆转牛分枝杆菌吞噬体的酸化，这表明ATP酶仅短暂存在于吞噬体膜中或数量减少。或者，V-ATP酶在内体中积累的酸当量可能通过不含ATP酶的载体囊泡传递到分枝杆菌吞噬体中。
