Changes in the Distribution of Anionic Constituents in Secretory Granules of Mouse Pancreatic Acinar Cells After Pilocarpine-induced Degranulation

Abstract

S U M M A R YWe used cationized colloidal gold (CCG) to investigate the distribution of anionic sites in different secretory granules of mouse pancreatic acinar cell regranulation. Localization of anionic sites with CCG was carried out on ultrathin sections of a mouse pancreas, fixed in Karnovsky's fixative and OsO 4 and embedded in Araldite. After pilocarpinestimulated degranulation, there was a marked diminution in the anionic charge density of immature and mature granules of the 4-hr group ( Ϸ 43.0 gold particles ր m 2 ) compared to the 8-hr mature granules group ( Ϸ 64.6 gold particles ր m 2 ). Scattergram analysis to investigate the correlation between section profile size and cationized gold labeling density revealed a reverse correlation, the small granule profiles demonstrated a higher density compared to the larger profiles of the same group. On the basis of these observations, it appears that a post-translational processing of secretory content influences the granule anionic charge and thus may affect the intragranular buffer capacity.

Localization of anionic constituents in mast cell granules of brachymorphic (bm/bm) mice by using avidin-conjugated colloidal gold

“…Morphometry of granules was performed on randomly obtained electron micrographs (×12,000) of secretory granules as previously described (Hammel et al 1989; Skutelsky et al 1995; Shoichetman et al 2001). Briefly, organelle cross-sectional areas were measured directly on the transmission electron micrographs.…”

“…A simple one-step incubation of sections with the cationic gold conjugate reveals subcellular sites having net negative charge (Skutelsky and Roth 1986) and the charge distribution can be examined with clear definition at a range of magnifications by employing cationic gold of various sizes (Skutelsky and Roth, 1986; Kashio et al 1992; Saga and Takahashi 1993; Shoichetman et al 2001). Accordingly, CCG has been used extensively for electron microscopic localization of extracellular and membrane-bound polyanionic constituents in a wide variety of cells and tissues (Vorbrodt 1989; Goode et al 1991; Skutelsky et al1992; Lawrenson et al 1994).…”

“…At physiological pH, CCG binds primarily to sulfated GAGs and can be used for the characterization of proteoglycans in various cellular compartments, cell membranes, and the extracellular matrix (ECM; Skutelsky et al 1995; Shoichetman et al 2001). Because of its high affinity for sulfated GAGs, we have used CCG for the post-embedding localization and quantification of sulfated GAGs in mouse and rat mast cell granules (Skutelsky et al 1995).…”

“…In using CCG for our prior studies of anionic charge distribution in the secretory granules of mast cells (Skutelsky et al 1995) and pancreatic acinar cells (Shoichetman et al 2001), we have noticed that this reagent gives relatively high levels of staining of structures other than granules, presumably reflecting the negative charge of these structures, e.g., ribosomes and cellular membranes. Electron-microscopic studies have demonstrated that avidin binds highly preferentially to individual mast cell granules rather than to other cellular structures (Bussolati and Gugliotta 1983; Jones et al 1987; Tharp et al 1985), and rodent or human mast cells are known to be readily stained with avidin conjugated to horseradish peroxidase (Bussolati and Gugliotta 1983; Kasper and Tharp 1987) or to certain fluorochrome dyes (Tharp et al 1985; Markey et al 1989; MacBride 1998).…”

Insulin receptor plays a central role in skin carcinogenesis by regulating cytoskeleton assembly

“…Skin samples or primary keratinocytes were collected, washed once in PBS, and then fixed with Karnovsky fixative (29) for 1 h at room temperature. Further sample processing was carried out following a standard procedure (30).…”