Abstract
The intracellular pH (pHi) changes resulting from chemotactic factor-induced activation of Na+/H+ exchange in isolated human neutrophils were characterized. Intracellular pH was measured from the equilibrium distribution of [14C]-5,5-dimethyloxazolidine-2,4-dione and from the fluorescence of 6-carboxyfluorescein. Exposure of cells to 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (FMLP) in 140 mM Na+ medium at extracellular pH (pHo) 7.40 led to a rise in pHi along an exponential time course (rate coefficient approximately 0.55 min-1). By 10 min, a new steady-state pHi was reached (7.75-7.80) that was 0.55-0.60 units higher than the resting pHi of control cells (7.20-7.25). The initial rate of H+ efflux from the cells (approximately 15 meq/liter X min), calculated from the intrinsic intracellular buffering power of approximately 50 mM/pH, was comparable to the rate of net Na+ influx (approximately 17 meq/liter X min), an observation consistent with a 1:1 stoichiometry for Na+/H+ exchange. This counter-transport could be inhibited by amiloride (apparent Ki approximately 75 microM). When either the external ([Na+]o) or internal Na ([Na+]i) concentrations, pHo, or pHi were varied independently, the new steady-state [Na+]i and pHi values in FMLP-stimulated cells were those corresponding to a chemical equilibrium distribution of Na+ and H+ across the cell membrane. By analogy to other activated cells, these results indicate that an alkalinization of pHi in human neutrophils is mediated by a chemotactic factor-induced exchange of internal H+ for external Na+.
Highlights
2,4-dione and from the fluorescence of 6-carboxyflu- gain in internal Na+ measured chemically by flame photomorescein
PH, or pHi were variedindependently, the new steadystate [Na+Iiand pHi values in FMLP-stimulated cells. Neutrophils were those corresponding to a chemical equilibrium distribution of Na+ and H+ across the cell membrane
- - cate that an alkalinizatioonf pHiin human neutrophils cells
Summary
2,4-dione and from the fluorescence of 6-carboxyflu- gain in internal Na+ measured chemically by flame photomorescein. Thiscounter-transport could be inhibitedby The incubation media, cell isolation techniques, and reagents were amiloride (apparent K i 75 p ~ ) W. hen either the ex- the same as those described in detail inthe preceding report (1)with ternal ([Na+],) or internal Na ([Na+Ii)concentrations, the following additions Neutrophils were those corresponding to a chemical equilibrium distribution of Na+ and H+ across the cell membrane. Batches of Na+-loaded cells is mediated by a chemotactic factor-induced exchange ([Na+]; 50 or 70 meq/liter of cell water) were prepared We prepared a batch of Na+-depleted cells ([Na+]i5 2 meq/liter of cell water) by exposing cells for 1.5 h at 37°C to Na+-free medium in which N - In the preceding paper (l),I presented evidence that the chemotactic tripeptide FMLP’ activates an amiloride-sensitive Na+/H+ exchange in isolated human neutrophils. Batches of cells with differing pHi values
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