CHEMOATTRACTANT-INDUCED ACTIVATION OF VACUOLAR H+ PUMPS AND OF AN H+-SELECTIVE CONDUCTANCE IN NEUTROPHILS

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 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 (approximate to 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. (C) 1995 Wiley-Liss, Inc.