Lambert R W, Maves C A, Gierow J P, Wood R L, Mircheff A K
Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles 90033.
Invest Ophthalmol Vis Sci. 1993 Feb;34(2):305-16.
The purpose of this study was to examine internalization and recycling of plasma membrane constituents in lacrimal gland acinar cells.
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.
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.
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.
本研究旨在检测泪腺腺泡细胞中质膜成分的内化和再循环。
从兔泪腺中分离出腺泡细胞。在4℃下用磺基 - N - 羟基琥珀酰亚胺生物素对表面表达的反应基团进行生物素化。然后分别用抗生物素蛋白-辣根过氧化物酶复合物进行光学显微镜观察、用抗生物素蛋白 - 荧光素黄共轭物进行荧光显微镜观察和定量荧光测定、用抗生物素蛋白 - 铁蛋白共轭物进行电子显微镜观察，对掺入的生物素进行标记。
在4℃时，标记物保留在完整细胞的表面。当升温至37℃时，表面的抗生物素蛋白 - 荧光素黄显著减少，被点状的细胞质标记所取代。用抗生物素蛋白 - 铁蛋白标记后升温的细胞的电子显微镜观察显示，铁蛋白存在于质膜下方的光滑小泡、与高尔基体膜相邻的小泡以及多泡体中。在冷却并用抗生物素蛋白 - 荧光素黄标记之前于37℃孵育，使细胞结合抗生物素蛋白 - 荧光素黄的能力降低了95%，t0.5 < 0.5分钟。如果随后将细胞在37℃下与抗生物素蛋白 - 荧光素黄一起孵育，它们摄取标记物的时间进程表明，初始生物素的60%要么再循环回到质膜，要么保留在可被内吞的细胞外液到达的细胞内区室中。内化的生物素与细胞外抗生物素蛋白 - 荧光素黄的t0.5为2分钟，并且在10μmol/l和1mmol/l浓度的卡巴胆碱作用下这一过程加速。
腺泡细胞质膜成分参与了一个持续进行的、受促分泌素调节的再循环过程，该过程发生在小的表面表达池和大10至20倍的细胞内池之间。
