Plasma membrane internalization and recycling in rabbit lacrimal acinar cells.

PURPOSE

The purpose of this study was to examine internalization and recycling of plasma membrane constituents in lacrimal gland acinar cells.

METHODS

Acinar cells were isolated from rabbit lacrimal glands. Surface-expressed reactive groups were biotinylated at 4 degrees C with sulfo-N-hydroxysuccinimidyl-biotin. Incorporated biotin was then labeled with avidin-horseradish peroxidase complex for light microscopy, with avidin-lucifer yellow conjugate for fluorescence microscopy, and quantitative fluorometry, and with avidin-ferritin conjugate for electron microscopy.

RESULTS

At 4 degrees C labels remained at the surfaces of intact cells. Surface avidin-lucifer yellow decreased markedly, giving way to punctate cytoplasmic labeling, on warming to 37 degrees C. Electron microscopy of cells warmed after labeling with avidin-ferritin revealed ferritin in smooth vesicles underlying the plasma membranes, in vesicles adjacent to Golgi membranes, and in multivesicular bodies. Incubation at 37 degrees C before chilling and labeling with avidin-lucifer yellow decreased the cells' capacity to bind avidin-lucifer yellow by 95%, with t0.5 < 0.5 min. If cells were then incubated with avidin-lucifer yellow at 37 degrees C, they took up the marker with a time course that indicated that 60% of the initial biotin either recycled back to the plasma membrane or remained in intracellular compartments that could be reached by endocytosed extracellular fluid. Internalized biotin communicated with extracellular avidin-lucifer yellow with a t0.5 of 2 min, and this process was accelerated by carbachol at concentrations of 10 mumol/l and 1 mmol/l.

CONCLUSIONS

Acinar cell plasma membrane constituents participate in an ongoing, secretagogue-modulated recycling traffic between small surface-expressed pools and 10- to 20-fold larger intracellular pools.