Superoxide generation and granule enzyme release induced by ionophore A23187: Studies on the early events of neutrophil activation

The responses of isolated human peripheral neutrophils to activation by the divalent cation ionophore A23187 (4 × 10⁻³M to 2 × 10⁻⁵M) were studied over the course of 15 min at 37 °C. Dose-dependent increases were noted in the following A23187-induced functions: (1) membrane depolarization, as assessed by a fluorescent cyanine dye, (2) altered membrane permeability to ²²Na⁺ and ⁴⁵Ca²⁺, (3) increased intracellular levels of cAMP, (4) O₂⁻ generation, and (5) granule enzyme release. Membrane depolarization and the cAMP response preceded the onset of O₂⁻ generation and exocytosis, which began after a latency period of approximately 30 sec. All five functions induced by 2 × 10⁻⁵M A23187 required ~1 mM Ca₀ for optimal expression and declined substantially as Ca₀ was reduced towards a nominal O mM. In contrast, the Na⁺ ionophore gramicidin D (0.1 to 10 μg/ml) caused cell depolarization and increased ²²Na⁺ uptake but did not stimulate ⁴⁵Ca²⁺ uptake, the cAMP response, O₂⁻ generation, or exocytosis of granule constituents. The observations dissociate membrane depolarization and altered Na⁺ permeability, on the one hand, from a Ca²⁺ influx, cAMP response, O₂⁻ release, and exocytosis on the other. These studies, then, are consistent with a hypothesis that a Ca²⁺ influx is required for the biologic expressions of cell activation.