Fluoride Stimulation of Canine Neutrophils: The Role of Calcium Binding 1

Abstract

Abstract

Neutrophils are known to undergo a burst of metabolic activity in response to particulate and soluble stimuli. Most of these stimuli depend upon calcium in the extracellular medium to exert their maximal effects. We have shown that 24 mM sodium fluoride (NaF) stimulates the metabolic burst in canine neutrophils as measured by chemiluminescence and CO₂ formation from the carbon-1 position of glucose. Because these phenomena are calcium dependent, we determined the uptake of calcium by neutrophils activated with NaF. Neutrophils suspended in buffer were incubated with ⁴⁵CaCl₂ and exposed to NaF. This caused an increase in cell-associated calcium from 1.89 ± 0.43 × 10⁻¹⁰ mol/10⁷ cells to 1.06 ± 0.15 × 10⁻⁸mol/10⁷ cells in 2 min. In comparison, the calcium ionophore A23187 (10⁻⁶ M) caused only a modest increase in cell calcium, from 1.89 ± 0.43 × 10⁻¹⁰ mol/10⁷ cells to 4.89 ± 0.85 × 10⁻¹⁰ mol/10⁷ cells. When treated with EGTA (3 mM), NaF-stimulated neutrophils rapidly lost 90% of their cell-associated calcium. Verapamil (3 × 10⁻⁴ M), a calcium channel blocker, inhibited NaF stimulation of neutrophil metabolism but did not decrease the magnitude or the rate of the calcium association. These studies indicate that the mechanism of NaF stimulation of neutrophils involves massive calcium binding to the external membrane and a subsequent movement of some of this membrane calcium into the cell.