Nanda A, Grinstein S
Division of Cell Biology, Hospital for Sick Children, Toronto, Canada.
J Cell Physiol. 1995 Dec;165(3):588-99. doi: 10.1002/jcp.1041650317.
Upon binding to their receptors on the surface of neutrophils, chemotactic peptides elicit a burst of metabolic activity. The excess acid generated by this burst must be rapidly extruded in order to maintain intracellular pH and preserve normal microbicidal responses. Recently, H(+)-pumping vacuolar-type ATPases (V-pumps) and a H(+)-selective conductance were described in the membrane of neutrophils. However, these systems are virtually quiescent in resting cells. In this report, we analyzed whether the V-pumps and the conductance become active and contribute to pH regulation following cell activation by chemoattractants. Formyl-Met-Leu-Phe (fMLP) was found to stimulate V-pumps, as assessed by the appearance of bafilomycin-sensitive H+ extrusion. Concomitantly, the chemoattractant also activated the H+ conductance, detected as a voltage-dependent and Zn(2+)-sensitive net H+ efflux. In both cases, activation was prevented by treatment with competing antagonistic peptides or with pertussis toxin, implying mediation by a receptor coupled to a heterotrimeric G protein. The signalling pathways downstream of the G proteins were also investigated. Stimulation of neither the V-pump nor the conductance required activation of protein kinase C. An elevation of cytosolic calcium ([Ca2+]i) comparable to that induced by fMLP did not suffice to trigger either transporter. Moreover activation of the conductance remained unaffected when the chemoattractant-induced increase in [Ca2+]i was precluded. In contrast, stimulation of the V-pump was substantially (approximately 50%) depressed when [Ca2+]i was prevented from rising. Tyrosine phosphorylation of several polypeptides accompanies stimulation by fMLP. Prevention of phosphotyrosine accumulation resulted in a pronounced inhibition of H(+)-pumping and of the H+ conductance. Together, these data indicate that engagement of surface receptors by chemotactic peptides can lead to stimulation of two voltage-sensitive pH regulatory pathways, a pump and a conductance, by a pathway that requires tyrosine phosphorylation. Both pathways are capable of sizable H+ extrusion, thereby contributing to pH regulation during the metabolic burst.
趋化肽与中性粒细胞表面的受体结合后，会引发一阵代谢活性。这种活性爆发产生的过量酸必须迅速排出，以维持细胞内pH值并保持正常的杀菌反应。最近，在中性粒细胞的膜中发现了H(+) - 泵浦空泡型ATP酶（V - 泵）和H(+) - 选择性电导。然而，这些系统在静息细胞中实际上是静止的。在本报告中，我们分析了在趋化因子激活细胞后，V - 泵和电导是否会变得活跃并有助于pH调节。通过巴弗洛霉素敏感的H+排出的出现评估发现，甲酰 - 甲硫氨酸 - 亮氨酸 - 苯丙氨酸（fMLP）可刺激V - 泵。同时，趋化因子还激活了H+电导，检测为电压依赖性和Zn(2+)敏感的净H+外流。在这两种情况下，用竞争性拮抗肽或百日咳毒素处理可阻止激活，这意味着由与异源三聚体G蛋白偶联的受体介导。还研究了G蛋白下游的信号通路。V - 泵和电导的刺激均不需要蛋白激酶C的激活。与fMLP诱导的细胞质钙（[Ca2+]i）升高相当的升高不足以触发任何一种转运体。此外，当趋化因子诱导的[Ca2+]i增加被阻止时，电导的激活仍然不受影响。相反，当[Ca2+]i的升高被阻止时，V - 泵的刺激显著（约50%）受到抑制。fMLP刺激会伴随几种多肽的酪氨酸磷酸化。磷酸酪氨酸积累的阻止导致H(+) - 泵浦和H+电导的明显抑制。总之，这些数据表明趋化肽与表面受体的结合可通过需要酪氨酸磷酸化的途径导致两种电压敏感的pH调节途径的刺激，一种是泵途径，另一种是电导途径。这两种途径都能够大量排出H+，从而有助于代谢爆发期间的pH调节。
