Phorbol esters stimulate macropinocytosis and solute flow through macrophages

Abstract

The morphology and kinetics of pinocytosis by bone marrow-derived macrophages were studied to determine how stimulation by phorbol esters increases net solute accumulation. Application of phorbol myristate acetate (PMA) increased both the abundance of macropinosomes and the rate of solute flow through the endocytic compartment. The large pinosomes originated as ruffles at the cell margins that folded back on themselves, internalizing extracellular medium and solutes. I examined how stimulation affects the kinetics of pinocytic influx, accumulation, and subsequent efflux of the fluorescent dye Lucifer Yellow (LY) in macrophages. Both the accumulation of LY and its subsequent efflux were temperature-dependent and directly proportional to the concentration of LY in the extracellular medium. Macrophages incubated in PMA and LY for 2 h accumulated four to six times more LY than did macrophages in LY alone. If after pinocytosis the macrophages were washed and reincubated in unlabeled medium for a 1h chase period, some of the internalized LY was regurgitated from the cells. Inclusion of PMA in the chase medium increased efflux of LY. In contrast, a smaller percentage of LY was regurgitated from macrophages which were both loaded and chased in the presence of PMA. This indicates that although efflux is increased by PMA, influx increases more, and therefore more of the LY entering by pinocytosis is retained within the cell. I suggest that macropinocytosis increases the size difference between pinosomes and efflux vesicles, and that that difference increases greatly both solute accumulation and membrane flow through the endocytic compartment.