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

Upon binding to their receptors on the surface of neutrophils, chemota ctic peptides elicit a burst of metabolic activity. The excess acid ge nerated by this burst must be rapidly extruded in order to maintain in tracellular pH and preserve normal microbicidal responses. Recently, H +-pumping vacuolar-type ATPases (V-pumps) and a H+-selective conductan ce were described in the membrane of neutrophils. However, these syste ms are virtually quiescent in resting cells. In this report, we analyz ed whether the V-pumps and the conductance become active and contribut e to pH regulation following cell activation by chemoattractants. Form yl-Met-Leu-Phe (FMLP) was found to stimulate V-pumps, as assessed by t he appearance of bafilomycin-sensitive H+ extrusion. Concomitantly, th e chemoattractant also activated the H+ conductance, detected as a vol tage-dependent and Zn2+-sensitive net H+ efflux. In both cases, activa tion was prevented by treatment with competing antagonistic peptides o r 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 d id not suffice to trigger either transporter. Moreover activation of t he conductance remained unaffected when the chemoattractant-induced in crease in [Ca2+](i) was precluded. In contrast, stimulation of the V-p ump was substantially (approximate to 50%) depressed when [Ca2+](i) wa s prevented from rising. Tyrosine phosphorylation of several polypepti des accompanies stimulation by fMLP. Prevention of phosphotyrosine acc umulation resulted in a pronounced inhibition of H+-pumping and of the H+ conductance. Together, these data indicate that engagement of surf ace receptors by chemotactic peptides can lead to stimulation of two v oltage-sensitive pH regulatory pathways, a pump and a conductance, by a pathway that requires tyrosine phosphorylation. Both pathways are ca pable of sizable H+ extrusion, thereby contributing to pH regulation d uring the metabolic burst. (C) 1995 Wiley-Liss, Inc.