FLUID-PHASE ENDOCYTOSIS BY ISOLATED RABBIT LACRIMAL GLAND ACINAR-CELLS

It is well known that lacrimal gland acinar cells retrieve secretory v esicle membrane constituents from their apical plasma membranes after stimulated exocytosis of secretory proteins. There have also been indi cations of a recycling traffic involving the basal-lateral plasma memb ranes. In an effort to document this traffic, determine how it is regu lated, and discern whether it involves more than one intracellular com partment, we studied internalization of the fluid phase marker, Lucife r Yellow, and its relationship to protein release in acinar cells isol ated from rabbit lacrimal glands. Loading of intracellular vesicles wa s apparent with fluoresence microscopy. Stimulation with carbachol inc reased both the rate of internalization and the intracellular volume e quilibrating with extracellular fluid, suggesting the loading of two c ompartments. A carbachol concentration of 10 mu M increased uptake by 80% during 20-min incubations at 37 degrees C. Increasing the carbacho l concentration to 1 mM reduced the response by 50%, and it appeared t o do so by decreasing the intracellular volume accessible to extracell ular fluid, rather than the rate of endocytosis. Carbachol affected pr otein release differently, increasing it by 50% at 10 mu M and 80% at 1 mM. Acceleration of endocytosis by 10 mu 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 o f VIP and carbachol exerted synergistic effects on both fluid phase in ternalization and protein release. Steady-state uptake at 18 degrees C in the presence of 10 mu M carbachol was equal to uptake at 37 degree s C in the absence of carbachol, suggesting a temperature block in the pathway to at least one endocytic compartment. Decreasing the tempera ture to 18 degrees C eliminated the inhibitory action of excessive car bachol, suggesting that the compartment whose loading was impaired by excessive carbachol was positioned distal to the temperature block. Ca rbachol accelerated release of marker from preloaded cells, indicating that it stimulated recycling between the plasma membranes and endocyt ic compartments. This effect was maximal at a concentration of 10 mu M and unchanged with increasing concentrations. In accord with the hypo thesis that traffic into and out of a certain compartment was particul arly dependent on stimulation, a fraction of the marker taken up by op timally stimulated cells at 37 degrees C was retained unless carbachol or VIP was present in the efflux medium. All of the marker taken up a t 18 degrees C in the presence of carbachol could be released in eithe r the presence or the absence of carbachol. These observations suggest an ongoing, secretagogue-accelerated recycling traffic which is disti nct from the traffic mediating retrieval of secretory vesicle membrane constituents; which involves basal-lateral membrane constituents; and which reaches distinct compartments located proximal and distal to an 18 degrees C temperature block.