Abstract
The relationship of N-formyl-methionyl-leucyl-phenylalanine-stimulated Na+/H+ exchange to the chemotactic responsiveness of human neutrophils was investigated. The pHi changes, measured from the equilibrium distribution of 5,5-dimethyloxazolidine-2,4-dione, were correlated with the migratory behavior of the cells as assessed by the leading front method. Exposure of cells to 10 nM FMLP caused activation of Na+/H+ exchange, leading to a rise in pHi from approximately 7.25 to approximately 7.75. This intracellular alkalinization was inhibited by amiloride and by three more potent analogues. All four compounds reduced the chemotactic response to FMLP with apparent Ki values similar to those for inhibition of the pHi transients, thereby suggesting that the blocking effect of the drugs on directed cell migration was related to inhibition of Na+/H+ exchange. The effect was specific for stimulated cell locomotion: FMLP-induced chemotaxis and chemokinesis were inhibited in parallel, whereas random motility was unimpaired. The relationship of pHi to function was also studied as the pHi of FMLP-activated cells was varied between 6.8 and 8.6 by altering the chemical gradients for Na+ and H+ across the cell membrane. There was a direct, positive correlation between the pHi value attained following FMLP-stimulation and the locomotor response to a chemotactic gradient. These results indicate that the motile functions of human neutrophils can be regulated by their pHi.
Highlights
This work complements a recent paper of ours [8]in which we demonstrated that an intracellular alkalinization facilitates thegeneration of superoxide radicals (OF)stimulated by inhibition of tphHe i transients, thereby suggesting thaFtMLP and thaptHi modulates this functional response
One of the early events following exposure of neutrophils to chemotactic factors is activation of an amiloride-sensitive Na+/H+ counter-transporstystem which mediates an alkalinization of pHi throughan exchange of internal H+for external Na+
If thisintracellular alkalinization wereinvolved in FMLP-induced functional responses, we predicted that (a) blockade of the activity of this Na+/H+ exchanger through specificdrug inhibitionshould prevent the functional expression, ( b ) the potency of amiloride andits analogues with respect to inhibition of Na+ fluxes, pHi transients, andfunctional capacity should be identical, and (c) varying the pHi in FMLP-stimulated cells by altering the Na+ andH' gradients across the cell membrane should regulate the functional response
Summary
Incubation Media-The standard medium used throughout this study had the followingcomposition: 140mM NaCl, 5 mM KCl, 1mM CaC12,0.5 mM MgC12, 5.6mM glucose, mM HEPES buffer, pH 7.40, and 1 mg/ml of crystalline bovine serum albumin. The intracellular Na+content of steady-state neutrophils batheidn standard medium (140 mM Na+,pH, 7.40) at 37 "C is -30 meq/liter of cell water [6, 10] These cells will be termed "normal Na+ cells." Neutrophils of different [Na+];were obtained by the following procedures: batches of Na+-depletedcells ([Na+Ii-2 or -15 meq/liter of cell water) were prepared by incubating cells for 1.5 or 0.5 h, respectively, a t 37 "C in Na+-free medium, where Na+was completely replaced by N-methyl-D-glucamine [10]; batches of Na+-loaded cells ([Na+Ii-45,-60, or -75 meq/liter cell water) were obtained by inulin was added as a marker for the extracellular space.The indicator content of the cells could be corrected for the medium trapped in the pellet. Net Migration without Drug x 100 derived pHi is a convenient measure of cytoplasmic pH: the contri- Assays of Granule Enzyme Release-Incubations were performed bution of the strongly acidic lysosomal subcompartment to average in duplicate in a volume of 1.0 ml in plastic microcentrifuge tubes pHi determinations using D M 0 can be discounted as negligibly small containing neutrophils (5 X 106/ml) and 100 nM FMLP. The pellets were isolated and counted in a liquid scintillation counter (Beckman LS 7000) after addition of 10
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