Differential Regulation of Granule-to-Granule and Granule-to-Plasma Membrane Fusion during Secretion from Rat Pituitary Lactotrophs

Abstract

We used fluorescence imaging of individual exocytic events together with electron microscopy to study the regulation of dense core granule-to-plasma membrane fusion and granule-to-granule fusion events that occur during secretion from rat pituitary lactotrophs. Stimulating secretion with elevated extracellular potassium, with the calcium ionophore ionomycin, or with thyrotropin releasing hormone or vasoactive intestinal polypeptide resulted in abundant exocytic structures. Approximately 67% of these structures consisted of multiple granules fused together sharing a single exocytic opening with the plasma membrane, i.e., compound exocytosis. For all of these stimulation conditions there appeared to be a finite number of plasma membrane fusion sites, ∼11 sites around each cellular equator. However, a granule could fuse directly with another granule that had already fused with the plasma membrane even before all plasma membrane sites were occupied. Granule-to-plasma membrane and granule-to-granule fusion events were subject to different regulations. Forskolin, which can elevate cAMP, increased the number of granule-to-granule fusion events without altering the number of granule-to-plasma membrane fusion events. In contrast, the phorbol ester PMA, which activates protein kinase C increased both granule-to-granule and granule-to-plasma membrane fusion events. These results provide a cellular mechanism that can account for the previously demonstrated potentiation of secretion from lactotrophs by cAMP- and PKC-dependent pathways.

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).…”