Phorbol esters stimulate macropinocytosis and solute flow through macrophages JOEL A. SWANSON Department of Anatomy and Cellular Biology, Harvard Medical School, 220 Longioood Avenue, Boston, MA 02115, USA Summary The morphology and kinetics of pinocytosis by bone marrow-derived macrophages were studied to determine how stimulation by phorbol esters in- creases 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, internaliz- ing 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 macro- phages. Both the accumulation of LY and its sub- sequent 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 lh chase period, some of the internalized LY was regurgi- tated 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 in- creases more, and therefore more of the LY enter- ing 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. Key words: pinocytosis, macrophage, phorbol ester, Lucifer Yellow. Introduction Macrophages are capable of continuous internalization of plasma membrane by pinocytosis. Stereological measure- ments indicate that they internalize the equivalent of their cell surface area once every 33 min (Steinman et al. 1976), yet they maintain a flattened morphology with a high cell surface to volume ratio. After endocytosis, membrane receptors and some fluid solute probes of pinocytosis return to the cell surface, indicating that internalized membrane is recycled. Despite such partial recycling, however, solute probes accumulate inside macrophages linearly for many hours, indicating that much of the fluid volume entering the cell does not recycle. How does the macrophage maintain steady state rates of solute accumulation without internalizing more membrane than it recycles? Treatment of macrophages with the tumor-promoting phorbol ester phorbol myristate acetate (PMA) stimu- lates pinocytosis. Within several minutes of addition of PMA, macrophage accumulation of the fluid phase solute probes horseradish peroxidase or Lucifer Yellow (LY) Journal of Cell Science 94, 135-142 (1989) Printed in Great Britain © The Company of Biologists Limited 1989 increases several fold (Phaire-Washington et al. 1980; Swanson e? al. 1985). Increased accumulation is continu- ous for many hours, and occurs without any noticeable loss,of cell surface area. Indeed, surface area appears to increase during the first hour of stimulation (Phaire- Washington et al. 1980). Examination of the kinetics of stimulation in thioglycollate-elicited murine peritoneal macrophages revealed that PMA stimulated both the rate of influx and the net intracellular retention of LY (Swanson et al. 1985). It was proposed that PMA increased the efficiency of pinosome-lysosome fusion, possibly via the extension of lysosomes into more periph- eral regions of cytoplasm (Swanson et al. 1987). The present work considers constitutive and stimu- lated pinocytosis in light of their effect, or lack of effect, on cell shape. It was prompted first by the observation that in bone marrow-derived macrophages PMA in- creases LY accumulation without noticeable redistri- bution of lysosomes. Using time-lapse video microscopy and measurement of the kinetics of LY pinocytosis, I report that PMA stimulates macropinocytosis and in- creases the net rate of flow through the endocytic 135