INTRACELLULAR DYNAMICS OF ALKALINE PHOSPHATASE-CONTAINING GRANULES INELECTROPERMEABILIZED HUMAN NEUTROPHILS

Human neutrophils possess alkaline phosphatase-containing intracellula r granules which an upregulated to the cell surface upon stimulation. The mechanism that governs the intracellular dynamics of these granule s 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 g ranules using electropermeabilized cells. Biochemical assays using int act neutrophils showed that the alkaline phosphatase activity was upre gulated and exocytosed into the extracellular space upon stimulation w ith AIF(4)(-) 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 cel ls stimulated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), but not with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). Cytochem ically, alkaline phosphatase-containing granules were dispersed throug hout the cytoplasm in unstimulated electropermeabilized neutrophils. U pon stimulation with GTP gamma S, but not with GDP beta S, these granu les fused to form elongated tubular structures which eventually became associated with the plasma membrane. Nocodazole disturbed the reorgan ization of the alkaline phosphatase-containing granules in cells stimu lated with GTP gamma S. The results from this study indicate that GTP- binding proteins participate in the reorganization and exocytosis of a lkaline phosphatase-containing granules associated with the microtubul es in electropermeabilized human neutrophils.