A role for Na+/Ca2+ exchange in the generation of superoxide radicals by human neutrophils.
A Na+/Ca2+ exchange mechanism has been recently described in human neutrophils that constitutes the principal pathway for Ca2+ influx into resting cells. The potential role of this system in regulating the respiratory burst in response to activation by the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine was explored. In the presence of 1 mM Ca2+, a variety of di- and trivalent cations suppressed the generation of O(-2) radicals in a series of decreasing efficacy: La3+ approximately Zn2+ much greater than Sr2+ approximately Cd2+ greater than Ba2+ greater than Co2+ greater than Ni2+ approximately Mg2+. This sequence is similar to their rank order of activity in inhibiting 45Ca2+ influx via Na+/Ca2+ counter-transport. Benzamil, phenamil, and 2',4'-dichlorobenzamil, analogues of amiloride which selectively block Na+/Ca2+ exchange in neutrophils, likewise suppressed the release of O(-2) with apparent Ki values of approximately 30 microM. The effect of the cations was competitive with Ca2+, while the interaction between the benzamil derivatives and Ca2+ appeared to be noncompetitive in nature. Both the divalent cations and benzamil also inhibited the rise in cytoplasmic Ca2+ as monitored by fura-2 fluorescence: these agents reduced peak cytosolic Ca2+ levels after N-formyl-methionyl-leucyl-phenylalanine stimulation to values seen in the absence of extracellular Ca2+. These results are compatible with the hypothesis that the influx of Ca2+ via Na+/Ca2+ exchange contributes to the transient elevation in intracellular free Ca2+. The polyvalent cations block the entry of critical Ca2+ ions by competing with Ca2+ for binding to the translocation site on the exchange carrier, while benzamil acts by lowering the maximal transport rate. These studies emphasize that Na+/Ca2+ exchange through its effects on cytoplasmic Ca2+ plays a major regulatory role in activation of the respiratory burst in chemotactic factor-stimulated neutrophils.
Simchowitz
L
L
Department of Medicine, Veterans Administration Medical Center, St. Louis, Missouri.
Foy
M A
MA
Cragoe
E J
EJ
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000097:blood* / Q000494:pharmacology; D002412:Cations; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D012964:Sodium / Q000097:blood*; D013481:Superoxides / Q000097:blood*
D002412:Cations; C013407:benzamil; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000584:Amiloride; D012964:Sodium; D008274:Magnesium; D002118:Calcium