Dissociation of human neutrophil activation events by prolonged treatment with amiloride

Human polymorphonuclear leukocytes (PMNs) can be stimulated to release granule contents and to produce superoxide anion. These functional responses are associated with cellular alkalinization and influx of Na⁺ in exchange for H⁺. Amiloride is a potassium-sparing diuretic that will inhibit stimulus-induced Na⁺-H⁺ exchange and prevent an increase in cell pH. Amiloride has been shown to inhibit a number of protein kinases including the calcium phospholipid-dependent protein kinase. Because PMA, which binds and activates C-kinase, is a potent stimulus of the PMN, this study was undertaken to investigate the effect of prolonged incubation of PMNs with amiloride on PMN stimulation by the chemotactic peptide N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP), the calcium ionophore A23187, opsonized zymosan particles, and the tumor promoter phorbol myristate acetate (PMA). Our results demonstrate that amiloride inhibits superoxide anion production by FMLP, A23187, and opsonized zymosan by causing a slower rate of release and lower maximal release without altering lag time. In contrast, amiloride, despite an inhibition of ²²Na⁺ influx, did not affect superoxide anion production stimulated by PMA. PMN degranulation, phagocytosis, arachidonic acid release, and early influx of calcium were unaffected by preincubation with amiloride. These data suggest that (1) PMN superoxide release induced by FMLP, A23187, and opsonized zymosan is likely modulated by amiloride-sensitive Na⁺-H⁺ exchange; and (2) phorbol ester-induced superoxide anion release and degranulation by any stimulant do not appear to be modulated by inhibition of an amiloride-sensitive Na⁺-H⁺ exchange.