title: Chemoattractant-induced activation of vacuolar 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 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.
authors.affiliation: Division of Cell Biology, Hospital for Sick Children, Toronto, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Nanda
authors.forename: S
authors.initials: S
authors.lastname: Grinstein
mesh_terms: D002118:Calcium / Q000502:physiology; D002630:Chemotactic Factors / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D009504:Neutrophils / Q000201:enzymology*; D010455:Peptides / Q000502:physiology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D017494:Proton Pumps / Q000502:physiology*; D006180:Proton-Translocating ATPases / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism; D014617:Vacuoles / Q000502:physiology
chemical_list: D002630:Chemotactic Factors; D010455:Peptides; D017494:Proton Pumps; D014443:Tyrosine; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D006180:Proton-Translocating ATPases; D002118:Calcium