Modulating effects of acepromazine on the reactive oxygen species production by stimulated equine neutrophils

Objective To investigate the effect of acepromazine (ACP) on reactive oxygen species (ROS) production by stimulated equine neutrophils. Study design Ex vivo biochemical experiments. Animals Isolated neutrophils from healthy untreated horses. Methods Neutrophils were incubated with ACP at concentrations of 10⁻⁴, 10⁻⁵ or 10⁻⁶ m and then stimulated with phorbol-myristate-acetate (PMA) before measurement of lucigenin-enhanced chemiluminescence (CL). In a second experiment neutrophils were incubated in the presence of α-keto-γ methylthiobutyric acid (KMB) and treated with ACP at concentrations of 10⁻⁴, 10⁻⁵ or 10⁻⁶ m. Subsequent PMA stimulation lead to neutrophilic ROS production and decomposition of KMB to ethylene, which is measured by gas chromatography. Electron paramagnetic resonance-spin trapping (EPR) analysis was performed with PMA-stimulated neutrophils in the presence of ACP (10⁻⁴, 10⁻⁵ or 10⁻⁶ m) directly added to the cell suspension. In the second experiment, the same concentrations of ACP were pre-incubated with neutrophils, then centrifuged to eliminate the excess of ACP and re-suspended in phosphate buffer before stimulation with PMA. In all experiments, the results of ACP-treated and ACP-untreated stimulated neutrophils were compared. Results Overall, results obtained with lucigenin-enhanced CL and KMB oxidation were in agreement with those seen in electron paramagnetic resonance spectroscopy. Acepromazine induced a dose-dependent inhibitory effect on neutrophilic ROS production. Electron paramagnetic resonance also showed, at high ACP concentration, the appearance of a cation radical derived from ACP. In contrast, electron paramagnetic resonance study performed with pre-incubated neutrophils showed an important dose-dependent inhibitory effect of ACP. Conclusion The results indicate that ACP can neutralize O˙⁻ ₂ or its by-products during the stimulation of neutrophils. Clinical relevance These findings may have a therapeutic relevance when phenothiazines are used in horses suffering from inflammatory diseases in which neutrophil activation and ROS production are implicated.