Exocytosis of Pinocytosed Fluid in Cultured Cells : Kinetic Evidence for Rapid Turnover and Compartmentation 716

The uptake and fate of pinocytosed fluid were investigated in monolayers of pulmonary alveolar macrophages and fetal lung fibroblasts using the fluid-phase marker, [14 C]sucrose. Initial experiments revealed that cellular accumulation of chromatographically repurified [14 C]sucrose was not linear with incubation time. Deviation from linearity was shown to be due to constant exocytosis of accumulating marker. Chromatographic analysis revealed that the cells were unable to metabolize sucrose and were releasing it intact by a process that was temperature-sensitive but not dependent on extracellular calcium and magnesium. A detailed analysis of the kinetics of exocytosis was undertaken by preloading cells with [ 1 'C] sucrose for various lengths of time and then monitoring the appearance of radioactivity into isotope-free medium. Results indicated that modeling the process of fluid-phase pinocytosis and subsequent exocytosis required at least two intracellular compartments in series, one compartment being of small size and turning over very rapidly (t 1/2 = 5 min in macrophages, 6-8 min in fibroblasts) and the other compartment being apparently larger in size and turning over very slowly (t 1/2 = 180 min in macrophages, 430-620 min in fibroblasts). Computer-simulation based on this model confirmed that the kinetics of efflux faithfully reflected the kinetics of influx and that the rate of efflux completely accounted for the deviation from linearity of accumulation kinetics. Moreover, the sizes of the compartments and magnitude of the intercom partment fluxes were such that the majority of fluid internalized in pinocytic vesicles was rapidly returned to the extracellular space via exocytosis. This result provides direct experimental evidence for a process previously thought necessary based solely on morphological and theoretical considerations. Furthermore, the turnover of pinocytosed fluid was so dynamic that accumulation deviated from linearity even within the first few minutes of incubation. We were able to show that the kinetics of exocytosis allowed calculation of the actual pinocytic rate, a rate that was nearly 50% greater than the apparent initial rate obtained from the slope of the uptake curve over the first 10 min. Pinocytosis may represent the afferent arm of a homeostatic mechanism for recycling the plasmalemma. The foundation for this hypothesis was laid by the work of Steinman et al. (37). Their stereological analysis of pinocytosis in macrophages and L-cells revealed that the volume and surface area of incoming pinocytic vesicles was ten times greater than that which the secondary lysosomal compartment was shown to accommodate and, …