Identification of "Pits" and "Depressions" at the Apical Plasma Membrane of Live and Resting Acinar Cells.
The exposed apical region and the basolateral plasma membrane of isolated pancreatic acini and single cells were imaged by the BAFM. Whole cell images by the BAFM were difficult and almost impossible to obtain due to the large changes in height at different points on the cell, resulting in the loss of contact by the scanning cantilever (34). Fig. 2 is an image of the exposed apical cell plasma membrane in an acinus comprising 6 cells. At this resolution, the surface topology reveals microscopic projections as well as approximately 500- to 2000-nm pits that contain several depressions ranging from 100 nm to 200 nm in diameter. The pits and depressions were found to be present only at apical regions of the isolated acinar cell surface. At higher magnification, the depressions within pits are clearly identified (Fig. 2 Inset). Typically, the apical and basolateral regions of single acinar cells or acini composed of 2-4 cells were imaged by the BAFM. Scanning small surface areas (1-10 µm2) provided us with better-resolution images for accurate measurements of depressions. In Fig. 3a, a pit approximately 1.8 µm in diameter depicting 20 depressions is shown to illustrate depression size, range, and dynamics. Typically, pits have 2-6 depressions.
Pits or depressions were unidentified in isolated acinar preparations comprising 10-15 cells, where the apical secretory region faced inward toward the lumen, rendering it inaccessible to the BAFM cantilever. Only the basolateral parts of acinar cells in these large acini preparations were exposed to the scanning cantilever. The apical region of acinar cells could be accessed only in isolated cells or in partial acini comprising only 3-6 cells with an exposed lumen. This observation suggests that pits are located at the apical plasma membrane of pancreatic acinar cells, where regulated secretory vesicles have been implicated to dock and fuse (35, 36).
Dynamics at the Apical Plasma Membrane of Live Acinar Cells Following Stimulation of Secretion by Mas7.
In this study, dynamics of structures at the apical plasma membrane of acinar cells were examined following stimulation of secretion by the secretagogue Mas7. Changes were observed only in the depressions (Fig. 3). Diameters of pits and depressions, and the distance between depressions within a pit as well as the distance between pits, were calculated (Fig. 4). Trypan blue staining of isolated pancreatic acinar cells used in each experiment was performed to assess any physical damage. This staining confirmed that cells were intact after completion of scanning by the BAFM cantilever.
Following exposure of cells to 20 µM Mas7, a time-dependent increase in depression diameter was observed. Five minutes after stimulation of secretion, the diameter of the depressions increased significantly (P ± SEM, n = 28) (Fig. 3a, top and middle panels) to 211.23 ± 5.6 nm (Fig. 3b, top and middle panels). The approximate 35% increase in depression diameter (Fig. 4) was followed by a return toward control levels. Within 30 min, the depressions measured 171.5 ± 4.9 nm in diameter (Fig. 3c, top and middle panels), approximately a 20% decrease from the depression diameter observed 5 min after stimulation of secretion (Fig. 4). The enlargement of the depression diameter following exposure of cells to Mas7 correlated with a 129% increase in amylase secretion after 5 min over unstimulated control cells (the bottom panels of Fig. 3 a and b). No further increase in amylase secretion beyond 5 min after Mas7 exposure was seen. Examination of the diameter of pits and depressions, and the distance between depressions within a pit as well as the distance between pits, revealed no significant changes except in the depression diameter after stimulation of secretion (Fig. 4). No changes in membrane topology at the basolateral region of acinar cells were detected following stimulation of secretion by Mas7 (data not shown).
Plasma Membrane Depressions Regulated by Actin.
Exposure of acinar cells to cytochalasin B, a fungal toxin that inhibits actin polymerization, results in a 50-60% loss of stimulable amylase secretion. A significant decrease in depression diameter from 153.46 ± 3.07 nm to 125.88 ± 4.65 nm (P was observed following treatment of acinar cells with 20 µM cytochalasin B for 30 min at room temperature. Little increase in depression diameter (160.62 ± 3.6 nm), but a larger increase in relative depth of depression, was observed 5 min after Mas7 treatment of cytochalasin B pretreated acinar cells. Actin, therefore, may be an important component in maintenance and regulation of depression structure and dynamics involved in the exocytic process in pancreatic acinar cells.
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