A role for Na+/Ca2+ exchange in the generation of superoxide radicals by human neutrophils

• Published in: The Journal of biological chemistry 1990-08, Vol.265 (23), p.13449-13456
• Main Authors: L Simchowitz, M A Foy, E J Cragoe, Jr
• Format: Article
• Language: English
• Subjects: Amiloride - analogs & derivatives; Amiloride - pharmacology; Biological and medical sciences; Calcium - blood; Calcium - pharmacology; Cations; Cytosol - metabolism; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Hydrogen-Ion Concentration; Immunobiology; In Vitro Techniques; Kinetics; Magnesium - pharmacology; Myeloid cells: ontogeny, maturation, markers, receptors; N-Formylmethionine Leucyl-Phenylalanine - pharmacology; Neutrophils - drug effects; Neutrophils - metabolism; Polynuclears; Sodium - blood; Superoxides - blood
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)77368-7

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.