Inhibition of human neutrophil function by 6-aminonicotinamide: The role of the hexose monophosphate shunt in cell activation

Abstract

Listen

Translate article

Stimulation of polymorphonuclear leukocytes with phorbol myristate acetate (PMA) or chemotactic factors such as f-Met-Leu-Phe (fMLP) activates a membrane oxidase which results in the generation of the superoxide anion (O2−) and the oxidation of NADPH to NADP+. The subsequent reduction of NADP+ to NADPH is believed to be directly dependent upon activation of the hexose monophosphate shunt (HMPS). To further understand the role of the HMPS in the oxidative burst, we examined the kinetics of HMPS activation by fMLP and PMA. Both of these agents stimulate an increase in HMPS activity that parallels their production of O2−. To examine the role of the HMPS in cell activation, we treated polymorphonuclear leukocytes with the specific HMPS inhibitor, 6-aminonicotinamide. This pretreatment inhibited fMLP- and PMA-stimulated HMPS activity and O2− release by 80% and 60% respectively with a 50% inhibitory dose (ID50) of 5 × 10−7 M. Measurement of reduced NADPH using 350 nm ultraviolet light-stimulated fluorescence and flow cytometry indicated that 6-aminonicotinamide had no effect on resting levels of NADPH fluorescence but significantly inhibited the fluorescence recovery following stimulation with fMLP or PMA. In contrast, PMA- and fMLP-stimulated membrane depolarization measured with the carbocyanine dye 3,3′-dihexyloxacarbocyanine iodide and chemotaxis to fMLP were unaffected by 6-aminonicotinamide treatment. On the contrary, fMLP- or PMA-stimulated myeloperoxidase release by fMLP or PMA was enhanced by 30% and 150%, respectively, following treatment with 6-aminonicotinamide, suggesting a decreased oxidative inactivation of myeloperoxidase. These findings show that 6-aminonicotinamide can be used to preferentially inhibit the oxidative burst in neutrophils by inhibiting the HMPS and suggest that the HMPS functions primarily to reduce NADP+ for the production of O2− and has minimal involvement in neutrophil chemotaxis and enzyme release.