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1.
Quantitative immunocytochemical studies were done by using the immunogold technique on sections of the intermediate lobe of rat pituitary. Antibodies raised (in rabbits) against the precursor proteins pro-opiomelanocortin (POMC) and ACTH were used. The results clearly indicate that the immature granules are the major site of POMC, as their antigenic density (gold beads/micron2) was almost 3 times as high as that of ACTH. In the mature granules, the antigenic density of ACTH was increased by 2.7-fold compared with the immature granules. Using a computer-assisted method, it was possible to categorize the granules' antigenic density according to their size. Using this approach it was found that the antigenic density of POMC remained constant in all mature granules of varied sizes, whereas the antigenic density of ACTH decreased with increasing granule size. The relationship between granule size, degree of maturation, and antigenic density is discussed. 相似文献
2.
When zymogen granules, the secretion granules of pancreatic acinar cells, fill, secretory product is accumulated in immature granules, condensing vacuoles. Mature granules are formed when this product (protein) condenses into an osmotically inactive aggregate and, bulk water is expelled. This hypothesis for granule morphogenesis has two elements. The first is that immature granules are precursors to mature granules. The second is that a particular maturational event, condensation, which involves the aggregation of protein, takes place. These hypotheses lead to two straightforward predictions. One, that condensing vacuoles on average, should contain less protein than filled or mature granules. And two, that, due to condensation, mature granules should contain protein at a common concentration. In the current work, both of these predictions were tested using measurements of the protein content of individual granules acquired by X-ray microscopy. Neither prediction was affirmed by the experimental results. First, there was no distinguishable difference in the distribution of protein between immature and mature granules. Second, the protein concentration of mature granules varied widely between preparations, although granules from the same preparation had similar concentrations. From the data we conclude that: 1) mature granules and condensing vacuoles are different, though not necessarily unrelated, types of secretory vesicle, and not two forms of the same object; 2) as such, condensing vacuoles are not precursors to mature granules; 3) all granules do not contain protein at one particular concentration when full, or mature; 4) granule maturation does not involve a condensation step; 5) concentration is not determined by such physical limits as the space available for protein packing or condensation; and 6) the amount of protein contained is physiologically regulated. 相似文献
3.
T Shoichetman E Skutelsky S Lew I Hammel 《The journal of histochemistry and cytochemistry》2001,49(10):1199-1204
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 (approximately 43.0 gold particles/microm2) compared to the 8-hr mature granules group (approximately 64.6 gold particles/microm2). 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. 相似文献
4.
D. V. Naik 《Cell and tissue research》1973,142(3):305-328
Summary Electron microscopic localization of adrenocorticotropin (ACTH) and melanocyte stimulating hormone (MSH) in light, dark and ACTH cells in the pars intermedia (PI) of rats and mice is attempted by using antisera to p 1–24, p 17–39 ACTH and b MSH with the immunoglobulin-peroxidase bridge technique. All of the PI parenchymatous cells (light, dark and ACTH cells), except the marginal cuboidal and the ependymal like cells, in rats and mice show very good localization of ACTH and MSH staining. In the light and dark cells, stain of varying intensity is seen on the secretory granules, vesicles and also in many places on the surface of the rough endoplasmic reticulum. There is no staining on the mitochondria, in the nuclei or in the granules inside and around the cisternae of the Golgi complex. Dark stained dense core granules become larger and larger as they appear farther and farther away from the Golgi complex. On the other hand, in the ACTH cells of the PI, ACTH antisera show stronger stained granules in the Golgi complex including the cisternae, similar to the pars distalis (PD) ACTH cells. From these observations it is concluded that the corticotropin in light and dark cells, is not packaged or condensed in the Golgi complex like that in the ACTH cells. MSH synthesis in light and dark cells also seems to be similar to that of ACTH synthesis. It is likely that the granules accumulate ACTH and MSH secretions after they are liberated from the Golgi cisternae, and thus become bigger and bigger in size. In case of ACTH cells of PI and PD, corticotropin may be packaged in Golgi cisternae and the size of the granule does not change much. This shows that there are distinct immunocytochemical differences between the light, dark and ACTH cells of the PI. At the moment, it is difficult to say whether ACTH and MSH are present in the same granule or not.The present and previous studies show that the ACTH and MSH secretion in the PI of rats and mice depends on the hypothalamic neural control.This study was supported by MRC of Canada Grant nos. MA-3759, and MA-5160.The author gratefully wishes to thank Drs. P. Desaulles and W. Rittel (CIBA, Basle, Switzerland) for the synthetic p 1–24 ACTH and b MSH, Dr. R. F. Phifer for p 17–39 ACTH, and Dr. S. S. Spicer for providing samples of rabbit anti-porcine 17–39 ACTH and anti-human ACTH sera, Drs. George Sétáló and Paul Nakane for their valuable advice. He also acknowledge the help of Mr. Shankar Nayak to prepare the antisera and the skilful technical assistance of Miss. Elise Poiré. He wishes to acknowledge Mr. Gatson Lambert for his photography. 相似文献
5.
Summary The size, number and volume per cell of secretion granules in rat exocrine pancreas have been measured using stereological techniques. The changes which occur as a result of feeding starved animals (90 min) or stimulating lobular fragments in vitro with carbachol are documented. In fasted animals mean acinar cell volume was estimated as 1670 m3 and the cells contained an average of around 450 secretion granules with a corrected mean diameter of 0.70 m. They occupied around 7% of cell volume. After feeding mean cell volume was about 1300 m3 and the cells contained an average of about 190 granules per cell with a mean diameter of 0.58 m. They occupied 3% of cell volume. A shift in the size frequency distribution of granule diameters occurred as a result of feeding. In vitro experiments in which lobules were induced to secrete with carbachol (10M, 3 h, 37° C) had a similar effect. Mean cell volume was reduced from around 1760 m3 to 1360 m3, mean granule number from around 420 per cell to 180 per cell and the volume density of granules was reduced from about 8% to 3% of cell volume. There was no significant change in mean granule diameter or shift in the size-frequency distribution of granule diameters. Incubation of tissues with cycloheximide (1 mM, 3 h, 37° C) did not prevent secretion by carbachol but it prevented replacement of granules. As a consequence, depletion by carbachol was greater in the presence of cycloheximide, the granules being reduced to around 110 per cell and to only 2.5% of cell volume. We conclude that feeding causes a preferential loss of larger granules and that during secretion replacement of granules occurs. Some of these granules are smaller than those evident in the glands of starved animals. 相似文献
6.
We used a computer-assisted morphometry approach to analyze quantitatively the process of cytoplasmic granule formation in mouse pancreatic acinar cells stimulated with pilocarpine to induce secretion. Our findings suggest that each condensing vacuole/immature granule of pancreatic acinar cells is formed by the progressive aggregation of 106 to 128 unit progranules of narrowly fixed volume, define a range of 7.7 to 9.2 for the factor of volume condensation between the largest immature granules and the mature unit granule, and predict that the formation of a single mature unit granule by the aggregation and fusion of unit progranules involves a net reduction of at least 95% in the amount of membrane surface area associated with these structures. 相似文献
7.
Placing the clawed toad Xenopus laevis on a black background stimulates the melanotrope cells in the pars intermedia of the pituitary gland to release proopiomelanocortin (POMC)-derived peptides, including -MSH and N-acetyl-endorphin. In this study three types of secretory granules, electron-dense(130 nm Ø), moderately electron-dense ( 160 nm Ø) and electronlucent ( 180 nm Ø), have been identified in these cells. Apparently, only dark granules are formed by the Golgi apparatus and lucent granules release their contents via exocytosis. Immuno-electron microscopy (immunogold double labelling) of glutaraldehyde-fixed and freeze-substituted material shows that desacetyl--MSH and N-acetyl--endorphin coexist in all three granule types. Quantification of immunostaining revealed that immunoreactivities to these peptides are lowest in the dark granules and highest in the light ones. It is proposed that intragranular processing of POMC to immunoreactive desacetyl--MSH and N-acetyl--endorphin involves an increase in granule size and a decrease in granule electron density. Black background-induced activation of the melanotrope cell is reflected by an increase in immunoreactivity of the secretory granules to each of the antisera. This suggests that cell activation stimulates the formation of peptides by intragranular processing of POMC and/or of intermediate POMC-processing products. In addition, cell activation evoked an increase in the percentage of the granule population that reacts with anti-N-acetyl--endorphin, probably by stimulating intragranular acetylation of -endorphin. Apparently, this acetylation is a regulated event that occurs in the cytoplasm, independently from the acetylation of desacetyl--MSH which takes place near the plasmalemma at the time of granule exocytosis. 相似文献
8.
The effect of fixation conditions on the ultrastructural appearance of gastrin cell granules in the rat gastric pyloric antrum 总被引:1,自引:0,他引:1
The ultrastructural appearance of gastrin cell (G cell) granules was studied after different fixation procedures. When the pH of prefixation was varied there was greater preservation of the electron density of granule cores after acidic (pH 5.0 and 6.0) than after neutral or alkaline (pH 7.0 and 8.0) prefixation. Increasing duration of prefixation at pH 7.3 resulted in progressive loss of electron density of the granule core with swelling and occasional rupture of the limiting membrane. In tissues where most granules had been rendered electron lucent by fixation, those granules remaining dense cored were preferentially located close to the Golgi zone. These findings indicate that the electron density of G cell granules is profoundly affected by conditions of fixation, and that immature granules are more resistant to loss of core density than mature granules. They also suggest that the gastrin granule in vivo, like other polypeptide granules, may have a "solid", osmotically inactive core. 相似文献
9.
Ilan Hammel David Lagunoff Stephen J. Galli 《Journal of cellular and molecular medicine》2010,14(7):1904-1916
Morphometric evidence derived from studies of mast cells, pancreatic acinar cells and other cell types supports a model in which the post-Golgi processes that generate mature secretory granules can be resolved into three steps: (1) fusion of small, Golgi-derived progranules to produce immature secretory granules which have a highly constrained volume; (2) transformation of such immature granules into mature secretory granules, a process often associated with a reduction in the maturing granule’s volume, as well as changes in the appearance of its content and (3) fusion of secretory granules of the smallest size, termed ‘unit granules’, forming granules whose volumes are multiples of the unit granule’s volume. Mutations which perturb this process can cause significant pathology. For example, Chediak–Higashi syndrome / lysosomal trafficking regulator (CHS)/(Lyst) mutations result in giant secretory granules in a number of cell types in human beings with the Chediak–Higashi syndrome and in ‘beige’ (Lystbg/Lystbg) mice. Analysis of the secretory granules of mast cells and pancreatic acinar cells in Lyst-deficient beige mice suggests that beige mouse secretory granules retain the ability to fuse randomly with other secretory granules no matter what the size of the fusion partners. By contrast, in normal mice, the pattern of granule–granule fusion occurs exclusively by the addition of unit granules, either to each other or to larger granules. The normal pattern of fusion is termed unit addition and the fusion evident in cells with CHS/Lyst mutations is called random addition. The proposed model of secretory granule formation has several implications. For example, in neurosecretory cells, the secretion of small amounts of cargo in granules constrained to a very narrow size increases the precision of the information conveyed by secretion. By contrast, in pancreatic acinar cells and mast cells, large granules composed of multiple unit granules permit the cells to store large amounts of material without requiring the amount of membrane necessary to package the same amount of cargo into small granules. In addition, the formation of mature secretory granules that are multimers of unit granules provides a mechanism for mixing in large granules the contents of unit granules which differ in their content of cargo. 相似文献
10.
Summary Paramylon is the -1,3 glucan storage carbohydrate in the euglenoid algae. Mature paramylon granules are highly crystalline, fibrillar, and have a complex substructure. X-ray diffraction was used to demonstrate that mature paramylon granules are much more crystalline than immature granules. Freeze-etch electron microscopy showed that in mature granules, the microfibrils are organized in highly ordered arrays while the microfibrils of immature granules are less organized. The data suggest that the high crystallinity of paramylon is due to higher-order aggregates of microfibrils and the interaction of water with the microfibrils. The dissolution of paramylon was recorded by darkfield videomicroscopy. In a 0.5 N NaOH solution, paramylon dissociates in a regular manner into its constituent 4 nm microfibrils, and the central region of the granule is the last remaining refractile area during the dissolution process.Abbreviation LN
liquid nitrogen 相似文献
11.
Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells 总被引:41,自引:0,他引:41
The pituitary cell line, AtT-20, synthesizes adrenocorticotropic hormone (ACTH) as a glycoprotein precursor that is cleaved into mature hormones during packaging into secretory granules. The cells also produce an endogenous leukemia virus (MuLV) that is glycosylated after translation similar to the glycosylation of the ACTH precursor. Our evidence suggests that the envelope glycoprotein and some precursor ACTH get to the cell surface in a vesicle different from the mature ACTH secretory granule. Viral glycoproteins and ACTH precursor are released from the cells much sooner after synthesis than mature ACTH. Isolated secretory granules do not contain significant amounts of the envelope glycoprotein or ACTH precursor. Exposing cells to 8Br-cAMP stimulates release of mature ACTH four to five fold, but has little effect on the release of the ACTH precursor or the viral glycoproteins. We propose that the viral glycoproteins and some of the ACTH precursor are transported by a constitutive pathway, while mature ACTH is stored in secretory granules where its release is enhanced by stimulation. 相似文献
12.
Summary The secretory granules of rat bronchiolar Clara cells were classified into different types by their ultrastructural appearances followed by immunocytochemistry using anti-rat 10 kDa Clara cell-specific protein (10 kDa CCSP) antibody. One predominant type was the oval to round granule (type A granule), of which the matrix was composed of a map-like mixture of electron-dense and less electron-dense material. Another predominant type was the rod-shaped granule (type B granule). The content of type B granules varied from a finely fibrillar (type B1 granule) to an electron-dense, rod-like (type B3 granule) structure. Various intermediate types (type B2 granule) between type B1 and B3 granules were also found. Small cytoplasmic vesicles were found occasionally in close proximity to type B2 or B3 granule. Another type of granule (type C granule) was large, up to 8 m in diameter, and contained a moderately electron-dense amorphous matrix. Both type A and C granules stained at a similar density with the antibody. The nascent form of type A granules, which was found in the vicinity to the trans face of the Golgi apparatus, was also labeled. On the other hand, the labeling density of type B granules varied: type B1 granules were almost devoid of immunolabeling, whereas type B3 granules were intensely labeled. Type B2 granules stained with the antibody; however, the labeling density was less than that of type B3 granules. The small cytoplasmic vesicles of type B2 granules were labeled. From these findings, it is suggested that the granules of rat Clara cells consist of two types of granules of distinct origin; one appears to derive from condensing vacuoles of Golgi origin, whereas the other may be formed by membranefusions with small cytoplasmic vesicles of unknown source. 相似文献
13.
An electron-microscope and freeze-fracture study of the egg cortex of Brachydanio rerio 总被引:2,自引:0,他引:2
Summary We have examined the cortex of the teleost (Brachydanio rerio) egg before and during exocytosis of cortical granules by scanning, transmission, and freeze-fracture electron microscopy. In the unactivated egg, the P-face of the plasma membrane exhibits a random distribution of intramembranous particles, showing a density of 959/m2 and an average diameter of 8 nm. Particles over P- and E-faces of the membranes of cortical granules are substantially larger and display a significantly lower density. An anastomosing cortical endoplasmic reticulum forms close associations with both the plasma membrane of the egg and the membranes of cortical granules. Exocytosis begins with cortical granules pushing up beneath the plasma membrane to form domeshaped swellings, coupled with an apparent clearing of particles from the site of contact between the apposed membranes. A depression in the particle-free plasma membrane appears to mark sites of fusion and pore formation between cortical granules and plasma membranes. Profiles of exocytotic vesicles undergo a predictable sequence of morphological change, but maintain their identity in the egg surface during this transformation. Coated vesicles form at sites of cortical granule breakdown. Differences in particle density between cortical granules and egg plasma membranes persist during transformation of the exocytotic profiles. This suggests that constituents of the 2 membrane domains remain segregated and do not intermix rapidly, lending support to the view that the process of membrane retrieval is selective (i.e., cortical granule membrane is removed). 相似文献
14.
15.
E. Bácsy C. Tougard A. Tixier-Vidal J. Marton E. Stark 《Histochemistry and cell biology》1976,50(2):161-174
Summary Monolayer cultures of trypsin-dispersed cells of the rat adenohypophysis were grown for 5 to 54 days. ACTH was localized by immunocytochemistry using an antiserum to synthetic ACTH1–28 prepared in rabbit and sheep anti-goat immunoglobulin coupled with peroxidase. ACTH content of the culture medium was measured by radioimmunoassay.Corticotrophs were found in the cultures and ACTH in the medium at each cultivation time. The corticotrophs retained their essential morphological characteristics. Immunological staining was found in the secretory granules, some tubular or saccular structures, parts of the rough endoplasmic reticulum, and the cytoplasmic matrix. Immature secretory granules in the Golgi apparatus as well as some Golgi elements showed different degrees of immunoreactivity. In agreement with the high ACTH content of the culture medium the number, size and shape of the secretory granules, the active Golgi apparatus, the high amount of extragranular ACTH as well as pictures suggesting granule extrusion claim for a high ACTH synthesis and transport (and low ACTH storage) in the cultured corticotrophs. 相似文献
16.
A. Balázs 《Cell and tissue research》1969,99(2):286-301
Summary Rat bone marrow was fixed in glutaraldehyde, postfixed in osmium tetroxide, and processed for electron microscopy. The myeloid cells were arranged in order of maturation according to their successive compartments.On the basis of their differences in form, substructure, volume, and density five morphologically distinct types of developing granules are to be observed in neutrophil, two in eosinophil, and four in basophil, cells. Primordial granules appear in the interphase of the myeloblast, respectively in the early promyelocytes. The first granules in the neutrophils are pale, of homogeneous structure. These granules grow gradually denser with increasing condensation. In the myelocyte stage polymorphism is more pronounced. In the granulocytes, vacuoles and dense-cored vacuoles indicate the sites of granules. In the eosinophil line, the basophilic bodies decrease in number during differentiation. The eosinophil granules show fewer variations in the course of maturation than the neutrophils. The immature forms of the basophil granules are relatively large, pale, and of globular structure; they undergo condensation and show gradually higher density.Sites of granulogenesis in the rat are first of all the Golgi apparatus and, possibly, the cisternae in the endoplasmic reticulum. On occasion, bodies in a transitional stage between a mitochondrium and a granule can be observed, but whether they may have a bearing on the problem of granulogenesis is an open question. 相似文献
17.
F. Dacheux 《Cell and tissue research》1984,235(3):623-633
Summary The fetal porcine pituitary was investigated by means of ultrastructural immunocytochemistry (1) to identify the first cells synthesizing the adenohypophyseal hormones, (2) to follow their differentiation during fetal development, and (3) to compare their ultrastructural characteristics with those of mature adult cells.The first ACTH-cells, which produced and stored ACTH, -LPH, -MSH, and - and -endorphin in the same granules, were very numerous at day 34 and displayed a uniform morphology. At day 50 and thereafter, until the end of gestation, the ACTH-cells differed in their appearance probably reflecting various stages of differentiation of one cell type. The GH-cells gained rapidly ultrastructural features comparable to those of mature GH-cells. In contrast, in the case of PRL-cells, which appeared only at the end of the gestation period as immature elements containing very small secretory granules, the morphological maturation seemed to take place only after birth. The first cells synthesizing the glycoprotein hormones (LH, LH, FSH and TSH) displayed ultrastructural features of immature cells. At day 50, their ultrastructural organization started to show a different pattern. At the end of gestation, the TSH-cells and the gonadotropic cells displayed the ultrastructural features of mature cells. 相似文献
18.
Distinct Molecular Events during Secretory Granule Biogenesis
Revealed by Sensitivities to Brefeldin A 总被引:3,自引:2,他引:1
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Carlos J. Fernandez Michael Haugwitz Benjamin Eaton Hsiao-Ping H. Moore 《Molecular biology of the cell》1997,8(11):2171-2185
The biogenesis of peptide hormone secretory granules involves a series of sorting, modification, and trafficking steps that initiate in the trans-Golgi and trans-Golgi network (TGN). To investigate their temporal order and interrelationships, we have developed a pulse–chase protocol that follows the synthesis and packaging of a sulfated hormone, pro-opiomelanocortin (POMC). In AtT-20 cells, sulfate is incorporated into POMC predominantly on N-linked endoglycosidase H-resistant oligosaccharides. Subcellular fractionation and pharmacological studies confirm that this sulfation occurs at the trans-Golgi/TGN. Subsequent to sulfation, POMC undergoes a number of molecular events before final storage in dense-core granules. The first step involves the transfer of POMC from the sulfation compartment to a processing compartment (immature secretory granules, ISGs): Inhibiting export of pulse-labeled POMC by brefeldin A (BFA) or a 20°C block prevents its proteolytic conversion to mature adrenocorticotropic hormone. Proteolytic cleavage products were found in vesicular fractions corresponding to ISGs, suggesting that the processing machinery is not appreciably activated until POMC exits the sulfation compartment. A large portion of the labeled hormone is secreted from ISGs as incompletely processed intermediates. This unregulated secretory process occurs only during a limited time window: Granules that have matured for 2 to 3 h exhibit very little unregulated release, as evidenced by the efficient storage of the 15-kDa N-terminal fragment that is generated by a relatively late cleavage event within the maturing granule. The second step of granule biogenesis thus involves two maturation events: proteolytic activation of POMC in ISGs and a transition of the organelle from a state of high unregulated release to one that favors intracellular storage. By using BFA, we show that the two processes occurring in ISGs may be uncoupled: although the unregulated secretion from ISGs is impaired by BFA, proteolytic processing of POMC within this organelle proceeds unaffected. The finding that BFA impairs constitutive secretion from both the TGN and ISGs also suggests that these secretory processes may be related in mechanism. Finally, our data indicate that the unusually high levels of unregulated secretion often associated with endocrine tumors may result, at least in part, from inefficient storage of secretory products at the level of ISGs. 相似文献
19.
Summary Synexin (annexin VII) is a Ca2+- and phospholid-binding protein which has been proposed to play a role in Ca2+-dependent membrane fusion processes. Using a monoclonal antibody against synexin, Mab 10E7, and immunogold, we carried out a semiquantitative localization study of synexin in bovine adrenal medullary chromaffin granules, and in resting and nicotine-stimulated adrenal chromaffin cells. Isolated chromaffin granules contained very little synexin, whereas chromaffin granules aggregated with synexin (24 g/mg) and Ca2+ (1 mM) clearly showed synexin-associated immunogold particles in the vicinity of the granule membrane (1.88 gold particles per granule profile). In isolated, cultured adrenal chromaffin cells, synexin was present in the nucleus (5.5 particles/m2) and in the cytosol (5.3 particles/m2), but mainly around the granule membrane in the granular cell area (11.7 particles/m2). During the active phase of cholinergically stimulated catecholamine secretion, the amount of synexin label was reduced by 33% in the nucleus, by 23% in the cytosol, and by 51% in the granule area. The plasma membrane contained a small amount of synexin, which did not significantly change upon stimulation of the cells. We conclude that synexin is involved in the secretory process in chromaffin cells. 相似文献
20.
Biogenesis of secretory granules. Implications arising from the immature secretory granule in the regulated pathway of secretion 总被引:9,自引:0,他引:9
S A Tooze 《FEBS letters》1991,285(2):220-224
In endocrine cells the regulated secretion of hormones, peptides, enzymes and neurotransmitters into the external medium occurs when mature secretory granules fuse with the plasma membrane. Secretory granules form at the trans-Golgi network (TGN) by envelopment of the dense-core aggregate of regulated secretory proteins by a specific membrane. The secretory granules initially formed at the TGN, referred to here as immature secretory granules, are morphologically and biochemically distinct from mature secretory granules. The functional similarities and differences between the immature secretory granule and the mature secretory granule, and the events involved in the maturation of the secretory granules are briefly discussed. 相似文献