Chemoattractant‐induced activation of vaculoar H+ pumps and of an H+‐selective conductance in neutrophils

Abstract

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 Zn²⁺‐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 copuled to a heterotrimeric G protein. The signalling pathways downstream of the G proteins were also investigated. Stimulation of neither th V‐pump nor the conductace 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 (∞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. © 1995 Wiley‐Liss Inc.