ARTICLE |
Correspondence to: Ilan Hammel, Dept. of Pathology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel. E-mail: ilanh@patholog.tau.ac.il
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Summary |
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We 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 OsO4 and embedded in Araldite. After pilocarpine-stimulated degranulation, there was a marked diminution in the anionic charge density of immature and mature granules of the 4-hr group (43.0 gold particles/µm2) compared to the 8-hr mature granules group (
64.6 gold particles/µm2). 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.
(J Histochem Cytochem 49:11991204, 2001)
Key Words: pancreas, secretory granules, polyanions, cationic gold, quantitative cytochemistry
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Introduction |
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PANCREATIC ACINAR CELLS (
We have recently introduced a novel cytochemical ultrastructural approach for postembedding localization of acidic proteoglycans in mast cell secretory granules (
In the present study we used a similar histochemical approach to investigate the distribution of sulfated proteoglycans in pancreatic acinar cells and to demonstrate the possible correlation between granule profile size and anionic site density. For the localization of proteoglycans in pancreas, CCG was applied directly on Araldite sections of mouse pancreatic acinar cells fixed with aldehydes and OsO4.
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Materials and Methods |
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Isolation of Pancreas
Adult female ICR mice (910 weeks old, 2025 g body weight) were maintained on a diet of standard rodent chow (Mabaroth, Israel) and tapwater ad libitum. The animal experiments were conducted in accordance with the Tel Aviv University Institutional Animal Care and Use Committee and with guidelines prepared by the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (DHHS publication No. 86-23, revised 1985. Assurance number A5010-01, effective 1.10.1996 will expire on 30.9.2001). For these experiments, food was withheld overnight and then some of the mice were injected IP with pilocarpine (2.0 mg/mouse in 0.2 ml 0.9% NaCl; Sigma Chemical Company, St Louis, MO) to induce pancreatic acinar cell degranulation (
Electron Microscopic Histochemistry
A monodispersed suspension of colloidal gold with an average diameter of 15 nm was prepared by reduction of HAuCl4 with Na citrate (
Ultrathin sections (0.075 ± 0.015 µm) were prepared by a LKB III Ultratome, using a diamond knife, and the sections were mounted on Formvar-coated 200-mesh nickel grids. The sections were incubated with 1% bovine serum albumin solution for 10 min and labeled with the CCG at 1:100 dilution in PBS, for physiological pH. After staining for 1 hr at RT, the sections were rinsed with double-distilled water and poststained for 15 min with saturated uranyl acetate in 50% ethanol. Examination of the sections was carried out using a JEOL-100B transmission electron microscope at 80 kV.
Similar labeling procedures, carried out after preincubation of the sections in 100 µg/ml PLL and in the presence of a similar concentration of free PLL in the CCG solutions, served as specific controls.
Quantitative Microscopy
Morphometry of granules was performed on randomly obtained electron micrographs (x15,000) of secretory granules as previously described (Ni/
ai (
(ri2+1 - ri2). Statistical comparison analysis between the cumulative curves was performed by using the KolmogorovSmirnov test (
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Results |
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Postembedding labeling of ultrathin Araldite sections with CCG at pH 7.4 resulted in binding of the CCG particles to all mouse pancreatic acinar cells (Fig 1). The CCG binding at pH 7.4 was mainly restricted to the cytoplasmic granules and, to a considerably lesser extent, to nuclear heterochomatin (data not shown) and to RER (Fig 1). In the 4-hr group, the RER labeling was 8.3 particles/µm2, and in the 8-hr group it was 21.5 particles/µm2. Most of the labeling was along the RER membrane. In the Golgi zone it was significantly less for both groups (3.5 particles/µm2). Incubation with PLL followed by CCG solution resulted in no labeling.
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The frequency histogram of the CCG binding density in secretory granules is shown in Fig 2. The labeling density in the secretory granules exhibited considerable variations. The lowest density in the immature granules, 4 hr after pilocarpine injection, was calculated and found to be 35.9 ± 21.3 particles/µm2 (mean ± SD, 89 immature granule profiles), while in the mature granules, 8 hr after pilocarpine injection, it was increased by about 50% to a value of 64.6 ± 26.7 particles/µm2 (408 mature granule profiles). In the mature granules 4 hr after pilocarpine injection, the density was 43.0 ± 18.6 particles/µm2 (78 immature granule profiles). All three histograms differed significantly. This was done by the KolmogorovSmirnov test, which compares two size distributions and is independent of any size distribution assumptions. The KolmogorovSmirnov test compares the curves of two cumulative distributions by calculation of a variable (Dmax), which equals the largest observed distance between the curves. It is a sensitive test to examine differences in location, dispersion, and skewness (
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To check the labeling distribution within the granule profile, we have drawn a center-spaced grid of circles and projected it over circular granules from the 8-hr group. The granule profiles were divided by diameter size into four main groups, i.e., 196 granule profiles were selected from the 407 granule profiles because they were of a similar radius. The other granule profiles either deviated from the clear circular profile (maximal axial ratio 1:1.15) or were out of the rings' dimensions. The results (Fig 3) demonstrate that for each granule profile size group the gold particles were almost evenly distributed from the granule center. Small granule profiles (Fig 3 upper left panel; radius = 0.2 µm, 90 profiles) were evenly labeled with a mean density of 5560 gold particles/µm2. A similar distribution was observed in the largest granules (Fig 3 lower right panel; radius = 0.45 µm, 9 profiles). However, the mean density was lower, about 4550 gold particles/µm2. It should be noted that although the granule profiles are of equal size groups they result from granule sections of various diameters.
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Scattergram analysis of correlation between CCG particles mean density and the corresponding granule mean size is shown in Fig 4. The mean density of CCG particles in the immature granule profiles, derived 4 hr after pilocarpine injection, was 35 gold particles/µm2. On the mature granule profiles it was
40 gold particles/µm2, while 8 hr after pilocarpine injection the mean density of CCG particles in the mature granule profiles was increased up to
65 gold particles/µm2 (Fig 4; the broken line data were calculated from Fig 2). These results may suggest that in the hours after granule formation "new" anionic sites are expressed and might be either due to intragranule content redistribution or biogenesis of new anionic determinants, such as sulfated glycosaminoglycans. Our unpublished data in mast cells demonstrate similar behavior.
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Discussion |
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The classical model of secretory granule formation suggests that newly synthesized secretory proteins and sulfated proteoglycans are transported from the rough endoplasmic reticulum to the Golgi complex. Then they can undergo post-translational modification and are packaged for secretion by condensation within membrane-bound very small immature granules which fuse to form large immature granules (
The binding of CCG to mouse pancreatic acinar cell secretory granules indicates that the granule content is negatively charged. The charge properties of these constituents are preserved throughout the embedding procedure, which includes glutaraldehyde and OsO4 fixation, dehydration, and Araldite infiltration. The density is increased from the immature granules (= progranules) to the mature granules. Eight hours after activation, the granule charge density is higher compared to the 4-hr group. Binding of CCG along the secretory pathway, i.e., the RER or Golgi compartment, was significantly less than in the granule compartment and probably reflects sulfated anionic domains in various proteins. Careful observation of many micrographs suggests that most of the labeling in the pancreas is associated with the RER, mainly along the membrane.
It was previously demonstrated in other tissues that, at pH >7.0, CCG binds primarily to sulfated proteoglycans (
The density of attached CCGs in the different granules exhibited extreme variations that were generally in correlation with the size and granule "age." In general, the density of CCG exhibited a reverse correlation with the size of the granule profile, i.e., in each granule "age" group the CCG density was higher in the small profiles compared to the larger ones. These variations may be related to the differences in the relative density of sulfated proteoglycans and carboxyls in the granules.
The density of CCG varies among different cell types.
The increase in anionic site density on maturation of the granules (immature granules, 4-hr vs 8-hr groups) may be explained either by a progressive accumulation of sulfated proteoglycans or by a passive increase in their concentration due to condensation of the granule contents. The fact that the pancreatic acinar mature secretory granules are composed of quantal sizes (
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Acknowledgments |
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Support for this work was received from the Israel Ministry of Health Basic Science Foundation.
Received for publication September 20, 2000; accepted April 18, 2001.
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