Characterization of the amiloride-sensitive Na+-H+ antiport of human neutrophils.
In response to infection, neutrophils undergo a metabolic burst associated with a marked increase in acid generation. However, cytoplasmic pH (pHi) remains normal or even becomes slightly alkaline. Regulation of pHi is due, at least in part, to a Na+-H+ exchange mechanism. The basic properties of this antiport were investigated in human blood neutrophils using fluorescein derivatives as pHi indicators and by means of electronic cell sizing. Addition of external Na+ (Na+o) to acid-loaded cells resulted in intracellular alkalinization due to transmembrane H+ (equivalent) flux. The alkalinization was associated with Na+ uptake, and both processes were blocked by amiloride, suggesting Na+-H+ countertransport. The rate of amiloride-sensitive H+ efflux could be calculated from the rate of change of pHi, using a buffering power of 28 mmol X l-1 X pH unit-1, determined by titration with NH+4 or propionate-. The rate of Na+o-H+i exchange was a saturable function of Na+o (apparent Km = 73 mM). Forward (Na+o-H+i) exchange was inhibited by elevating external [H+] or internal [Na+] and competitively by amiloride (apparent Ki = 24 microM). The antiport was virtually inactive in unstimulated cells at pHi greater than or equal to 7.2 but was markedly stimulated by cytoplasmic acidification. This behavior is consistent with a role in pHi homeostasis. The possible mechanisms of stimulation of Na+-H+ countertransport in resting and activated neutrophils are discussed.
D000584:Amiloride / Q000494:pharmacology*; D002021:Buffers; D002469:Cell Separation; D003593:Cytoplasm / Q000378:metabolism; D004351:Drug Resistance; D006801:Humans; D006859:Hydrogen / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007474:Ion Exchange; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration; D012964:Sodium / Q000378:metabolism* / Q000502:physiology
D002021:Buffers; D000584:Amiloride; D006859:Hydrogen; D012964:Sodium