Nanda A, Grinstein S
Division of Cell Biology, Hospital for Sick Children, Toronto, Canada.
Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10816-20. doi: 10.1073/pnas.88.23.10816.
The rate of metabolic acid generation by neutrophils increases greatly when they are activated. Intracellular acidification is prevented in part by Na+/H+ exchange, but a sizable component of H+ extrusion persists in the nominal absence of Na+ and HCO3-. In this report we determined the contribution to H+ extrusion of a putative H+ conductive pathway and its mode of activation. In unstimulated cells, H+ conductance was found to be low and unaffected by depolarization. An experimental system was designed to minimize the metabolic acid generation and membrane potential changes associated with neutrophil activation. By using this system, beta-phorbol esters were shown to increase the H+ (equivalent) permeability of the plasma membrane. The direction of the phorbol ester-induced fluxes was dictated by the electrochemical H+ gradient. Moreover, the parallel migration of a counterion through a rheogenic pathway was necessary for the displacement of measurable amounts of H+ equivalents across the membrane. These findings suggest that the H+ flux is conductive. The effect of beta-phorbol esters was mimicked by diacylglycerol and mezerein and was blocked by staurosporine, whereas alpha-phorbol esters were ineffective. Together, these findings indicate that stimulation of protein kinase C induces the activation of an H+ conductance in the plasma membrane of human neutrophils. Preliminary evidence for activation of a separate, bafilomycin A1-sensitive H+ extrusion mechanism, likely a vacuolar type H(+)-ATPase, is also presented.
中性粒细胞被激活时，其代谢产酸速率会大幅增加。细胞内酸化部分通过Na⁺/H⁺交换得以防止，但在名义上不存在Na⁺和HCO₃⁻的情况下，仍有相当一部分H⁺排出持续存在。在本报告中，我们确定了一条假定的H⁺传导途径对H⁺排出的贡献及其激活方式。在未受刺激的细胞中，发现H⁺传导率较低且不受去极化影响。设计了一个实验系统，以尽量减少与中性粒细胞激活相关的代谢产酸和膜电位变化。通过使用该系统，发现β-佛波酯可增加质膜的H⁺（当量）通透性。佛波酯诱导的通量方向由电化学H⁺梯度决定。此外，抗衡离子通过生电途径的平行迁移对于跨膜置换可测量量的H⁺当量是必要的。这些发现表明H⁺通量是传导性的。二酰基甘油和大戟二萜醇可模拟β-佛波酯的作用，而星形孢菌素可阻断该作用，而α-佛波酯则无效。总之，这些发现表明蛋白激酶C的刺激可诱导人中性粒细胞质膜中H⁺传导的激活。还提供了激活一种单独的、对巴弗洛霉素A1敏感的H⁺排出机制（可能是液泡型H⁺-ATP酶）的初步证据。
