Generation of superoxide radicals by human peripheral neutrophils activated by chemotactic factor. Evidence for the role of calcium

Abstract

In response to activation by the synthetic chemotactic factor FMLP, human peripheral neutrophils generated superoxide radicals as assessed by ferricytochrome C reduction. A dose-dependent increase in the amount of superoxide induced by FMLP over the concentration range of 1 X 10(-8) M to 1.6 X 10(-7) M was observed. Examination of the kinetics of the response revealed large amounts of superoxide generated by 1 min of incubation at 37 degrees C at an optimal dose of FMLP and a plateau effect after 5 min of incubation. Divalent cations did not influence the binding of 3H-FMLP to the cell, but superoxide generation by FMLP-activated neutrophils was observed to be dependent on the presence of divalent cations in the medium. In the absence of Mg2+, increasing Ca2+ ion concentration in the medium led to progressive increases in superoxide generation up to 4 mM, after which the response declined slightly. Mg2+, 0.25 to 4 mM, increased FMLP-induced superoxide generation to a much lower extent than did Ca2+. Lanthanum ion, 0.1 to 1 mM, in the presence of 1 mM Ca2+ inhibited the production of superoxide by FMLP 4 X 10(-8 ) M. Over the concentration range 3.3 X 10(-5 M to 3 X 10(-4 M, verapamil, a drug which selectively blocks the calcium channel, caused a dose-dependent inhibition of superoxide production and calcium-45 uptake in response to FMLP. This effect of verapamil could be overcome by increasing the concentration of Ca2+ in the medium. These observations suggest that a calcium influx plays an important role in the superoxide-generating capacity of the neutrophil.