Fluid phase endocytosis by isolated rabbit lacrimal gland acinar cells

It is well known that lacrimal gland acinar cells retrieve secretory vesicle membrane constituents from their apical plasma membranes after stimulated exocytosis of secretory proteins. There have also been indications of a recycling traffic involving the basal-lateral plasma membranes. In an effort to document this traffic, determine how it is regulated, and discern whether it involves more than one intracellular compartment, we studied internalization of the fluid phase marker, Lucifer Yellow, and its relationship to protein release in acinar cells isolated from rabbit lacrimal glands. Loading of intracellular vesicles was apparent with fluorescence microscopy. Stimulation with carbachol increased both the rate of internalization and the intracellular volume equilibrating with extracellular fluid, suggesting the loading of two compartments. A carbachol concentration of 10 μ m increased uptake by 80% during 20-min incubations at 37°C. Increasing the carbachol concentration to 1 m m reduced the response by 50%, and it appeared to do so by decreasing the intracellular volume accessible to extracellular fluid, rather than the rate of endocytosis. Carbachol affected protein release differently, increasing it by 50% at 10 μ m and 80% at 1 m m. Acceleration of endocytosis by 10 μ m carbachol was transient, becoming negligible after 60 min. Vasoactive intestinal peptide (VIP) and isoproterenol increased internalization 35% and 25% respectively; neither reduced uptake at the highest concentrations tested; and only isoproterenol significantly affected protein secretion. Combinations of VIP and carbachol exerted synergistic effects on both fluid phase internalization and protein release. Steady-state uptake at 18°C in the presence of 10 μ m carbachol was equal to uptake at 37°C in the absence of carbachol, suggesting a temperature block in the pathway to at least one endocytic compartment. Decreasing the temperature to 18°C eliminated the inhibitory action of excessive carbachol, suggesting that the compartment whose loading was impaired by excessive carbachol was positioned distal to the temperature block. Carbachol accelerated release of marker from preloaded cells, indicating that it stimulated recycling between the plasma membranes and endocytic compartments. This effect was maximal at a concentration of 10 μ m and unchanged with increasing concentrations. In accord with the hypothesis that traffic into and out of a certain compartment was particularly dependent