Regulation of dense core release from neuroendocrine cells revealed by imaging single exocytic events

Abstract

Using FM1-43 fluorescence, we have optically detected single exocytic and endocytic events in rat pituitary lactotrophs. About fifty discrete fluorescent spots abruptly appear around the entire surface of a cell bathed in FM1-43 and high-potassium saline. The spots, which also immunostain for prolactin, reflect the labeling of dense cores as well as membranes of exocytosed secretory granules. Stained cores are not released, but remain attached to the cell and are eventually endocytosed. However, in cells exposed to dopamine (or an analog, bromocriptine), the cores dissolve and are secreted after several seconds. Solubilization of dense cores is mediated through a reduction in cytoplasmic cyclic AMP. Thus, the composition of secretions from individual secretory granules is regulated.

Supramaximal cholecystokinin displaces Munc18c from the pancreatic acinar basal surface, redirecting apical exocytosis to the basal membrane

“…As shown in Figure 4, b-d, acquisition of fluorescent images at a frequency of one per second was possible, allowing the data to be analyzed in a similar fashion to that reported for the lactotroph (27,28). Since the epifluorescence images reflect all planes of the acinar cluster, these data are analogous to those obtained with the 3D-reconstructed confocal study (Figure 4a) and the projection of the confocal slices of a larger acinus in Figure 3.…”

“…The vast majority of the basal membrane hotspots exhibited these patterns. Like the lactotroph studies, these hotspots also appeared as solid spots, not rings, suggesting that the FM1-43 was staining both the ZG membranes and the granule cores, as was also reported in the lactotrophs (27,28). This increase in fluorescence eventually plateaued within 2 minutes with no further increase even with continued CCK stimulation of up to 10 minutes (see Figure 5b).…”

“…Epifluorescent images were analyzed using Merlin software. Fluorescence of individual spots was measured in the manner described by Angleson and colleagues (27,28). The mean pixel value inside a chosen "region of interest" circle centered around the "hotspot" (usual circle diameter, seven pixels) was normalized to the mean surface membrane fluorescence inside the circle at the same location measured for at least five frames immediately before the hotspot appearance.…”

“…This was performed as similarly described for the lactotroph study reported by Angleson et al (27,28). Pancreatic acini plated on glass coverslips were stimulated with 10 nM CCK for 15-20 minutes (37°C) and not washed nor permeabilized.…”

“…Fluorescence remained high for several minutes but began to weaken after 6 minutes and was significantly reduced within 15 minutes. The extended time of staining may reflect prolonged increases in membrane concentration on the apical surface or direct staining of granular contents, which may require time for dispersal (27,28).…”