Neutrophil NADPH oxidase does not assemble on macropinocytic vacuole membranes

NADPH oxidase assembly is considered to occur mainly on the plasma membrane and phagolysosome membranes and to a lesser extent in intracellular granul es. Stimulation of neutrophils with phorbol 12-myristate 13-acetate (PMA) i n conjunction with chemiluminescent substrates has been widely used as a mo del to understand the sub-cellular locations of NADPH-oxidase activity. Int erpretation of data from such studies is complicated by observations that P MA does not trigger phagocytosis but results in formation of large macropin ocytic vacuoles. Here we show by laser-scanning confocal microscopy that PM A triggers uptake of lucigenin into macropinocytic vacuoles and that no che miluminescence (CL) can be detected at this location when NADPH oxidase ass embly on the plasma membrane still occurs. This shows that the macropinocyt ic vacuole membrane is distinct from the phagocytic vacuole membrane althou gh both compartments are derived from the plasma membrane. (C) 2000 Elsevie r Science B.V. All rights reserved.