Intracellular dynamics of alkaline phosphatase-containing granules in electropermeabilized human neutrophils

Human neutrophils possess alkaline phosphatase-containing intracellular granules which are upregulated to the cell surface upon stimulation. The mechanism that governs the intracellular dynamics of these granules is, however, poorly understood. The aim of the present study was to investigate the possible participation of GTP-binding proteins in the reorganization and exocytosis of the alkaline phosphatase-containing granules using electropermeabilized cells. Biochemical assays using intact neutrophils showed that the alkaline phosphatase activity was upregulated and exocytosed into the extracellular space upon stimulation with AIF4 and N-formyl peptide. This upregulation was inhibited by treatment of cells with pertussis toxin and botulinum toxin. Alkaline phosphatase activity was also upregulated in electropermeabilized cells stimulated with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), but not with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS). Cytochemically, alkaline phosphatase-containing granules were dispersed throughout the cytoplasm in unstimulated electropermeabilized neutrophils. Upon stimulation with GTPgammaS, but not with GDPbetaS, these granules fused to form elongated tubular structures which eventually became associated with the plasma membrane. Nocodazole disturbed the reorganization of the alkaline phosphatase-containing granules in cells stimulated with GTPgammaS. The results from this study indicate that GTP-binding proteins participate in the reorganization and exocytosis of alkaline phosphatase-containing granules associated with the microtubules in electropermeabilized human neutrophils.