A ROLE FOR NA+/CA-2+ EXCHANGE IN THE GENERATION OF SUPEROXIDE RADICALS BY HUMAN NEUTROPHILS

  • 15 August 1990
    • journal article
    • research article
    • Vol. 265  (23) , 13449-13456
Abstract
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 O2- radicals in a series of decreasing efficacy: La3+ .apprx. Zn2+ .mchgt. Sr2+ .apprx. Cd2+ > Ba2+ > Co2+ > Ni2+ .apprx. Mg2+. This sequence is similar to their rank order of activity in inhibiting 45Ca2+ influx via Na2/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 O2- with apparent Ki values of .apprx. 30 .mu.M. 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-methionyleucyl-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.

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