Quantitative immunocytochemical analysis of the endocrine pancreas of the Nile crocodile

Four major pancreatic hormones were immunolocalized at the light and electron microscopic levels in the pancreas of the Nile crocodile, Crocodilus niloticus. Immunogold was used for electron microscopy, and peroxidase-antiperoxidase was used for light microscopy. Somatostatin-positive D-cells and pancreatic polypeptide-containing F-cells accounted for about 60% of the immunoreactive cells in the ventral pancreas. Glucagon-positive A-cells were the least frequent cell type in the ventral pancreas, about 15%, but were the predominant cell type, about 40%, in the pancreas that was dorsal in character. An expanded population of D-cells (relative to mammals and other higher vertebrates) in association with two very different numbers of A-cells can be expected to have important consequences for the homotropic control of secretory activity of the endocrine pancreas as well as for the function of the acinar pancreas. F-cells were absent from the dorsal part of the pancreas, whereas insulin-containing B-cells were slightly more abundant in this portion of the pancreas. The regional character of the endocrine pancreas was related to the complex looping of the proximal small intestine. Without immunolabeling, only B-granules were morphognomonic in electron micrographs. The insulin-reactive B-granules were the smallest (370 nm) of the secretory granules and were followed in size by somatostatin-positive D-granules (380 nm). The pancreatic polypeptide-containing secretory granules were the largest (580 nm). Glucagon-reactive A-granules (430 nm) sometimes exhibited a protuberance or extension of secretory granule matrix and limiting membrane. Such a morphological feature has previously been associated with secretion of glucagon and the initiation of insulin secretion. Taken together these studies indicate that protuberances have a significant, but as yet undefined, role in pancreatic endocrine cells.     

Formation and exocytosis of secretory granules in the endocrine cells of normal and transplanted pancreas: an electromicroscopic study

The mechanism of secretory granule formation and exocytosis in the endocrine cells of normal and transplanted rat pancreas was studied using electron microscopy. On the one hand, formation of secretory granules starts with the dilatation of the 2 ends or the vesicularization of the middle parts of rough endoplasmatic reticulum (RER). On the other hand, prohormone ribosomes condense into the vesicles of the GOLGI apparatus. This probably indicates that the GOLGI complex is not the only source of formation of secretory granules. Exocytosis occurs with the formation of an electron dense streak between the perigranular membrane and the apical cell membrane. This is followed by the rupture of the streak at this midpoint allowing the granule to extrude into the space between the cell membrane and the parenchymal basal membrane. This fusion-rupture-extrusion mechanism repeats itself at the parenchymal and capillary basal membranes and also at the endothelium until it gets into the capillary lumen, showing that hormones of pancreatic endocrine cells may be actively transported into circulation as intact secretory granules. There is no significant morphological difference between the mechanism of secretory granule formation in normal and transplanted pancreatic tissue.     

The ultrastructural identity of pancreatic polypeptide cells in chicks

A very similar ultrastructure has been attributed to pancreatic polypeptide and somatostatin cells in chickens. In order to characterize any possible differences between them, cells shown to be immunoreactive for these hormones in semi-thin sections of chick pancreas were identified in adjacent thin sections prepared for conventional electron microscopy. In this way the ultrastructural features of the immunoreactive cells could be determined. In general, in somatostatin-immunoreactive cells, granule profiles are almost exclusively round, whereas in pancreatic polypeptide cells there are elongate as well round profiles. Within cells of both types the electron density of the granule matrix varies from one granule to another, but the range of density is greater in pancreatic polypeptide granules. The latter are slightly smaller than somatostatin granules.     

The ultrastructural identity of pancreatic polypeptide cells in chicks

Summary A very similar ultrastructure has been attributed to pancreatic polypeptide and somatostatin cells in chickens. In order to characterize any possible differences between them, cells shown to be immunoreactive for these hormones in semi-thin sections of chick pancreas were identified in adjacent thin sections prepared for conventional electron microscopy. In this way the ultrastructural features of the immunoreactive cells could be determined. In general, in somatostatin-immunoreactive cells, granule profiles are almost exclusively round, whereas in pancreatic polypeptide cells there are elongate as well as round profiles. Within cells of both types the electron density of the granule matrix varies from one granule to another, but the range of density is greater in pancreatic polypeptide granules. The latter are slightly smaller than somatostatin granules.     

Mucous granule exocytosis and CFTR expression in gallbladder epithelium

A mechanistic model of mucous granule exocytosis by columnar epithelial cells must take into account the unique physical-chemical properties of mucin glycoproteins and the resultant mucus gel. In particular, any model must explain the intracellular packaging and the kinetics of release of these large, heavily charged species. We studied mucous granule exocytosis in gallbladder epithelium, a model system for mucus secretion by columnar epithelial cells. Mucous granules released mucus by merocrine exocytosis in mouse gallbladder epithelium when examined by transmission electron microscopy. Spherules of secreted mucus larger than intracellular granules were noted on scanning electron microscopy. Electron probe microanalysis demonstrated increased calcium concentrations within mucous granules. Immunofluorescence microscopic studies revealed intracellular colocalization of mucins and the cystic fibrosis transmembrane conductance regulator (CFTR). Confocal laser immunofluorescence microscopy confirmed colocalization. These observations suggest that calcium in mucous secretory granules provides cationic shielding to keep mucus tightly packed. The data also suggests CFTR chloride channels are present in granule membranes. These observations support a model in which influx of chloride ions into the granule disrupts cationic shielding, leading to rapid swelling, exocytosis and hydration of mucus. Such a model explains the physical-chemical mechanisms involved in mucous granule exocytosis.     

Immunocytochemical localization of secretory proteins in bovine pancreatic exocrine cells

The bovine exocrine pancreatic cell produces a variety of enzymes and proenzymes for export. Biochemical studies by Greene L.J., C.H. Hirs, and G.E. Palade (J. Biol. Chem. 1963. 238:2054) have shown that the mass proportions of several of these proteins in resting pancreatic juice and zymogen granule fractions are identical. In this study we have used immunocytochemical techniques at the electron microscope level to determine whether regional differences exist in the bovine gland with regard to production of individual secretory proteins and whether specialization of product handling occurs at the subcellular level. The technique used is a modification of one previously reported (McLean, J.D., and S.J. Singer. 1970. Proc. Natl. Acad. Sci U.S.A. 69:1771) in which immunocytochemical reagents are applied to thin sections of bovine serum albumin-imbedded tissue and zymogen granule fractions. A double antibody technique was used in which the first step consisted of rabbit F(ab')2 antibovine secretory protein and the detection step consisted of sheep (F(ab')2 antirabbit F(ab')2 conjugated to ferritin. The results showed that all exocrine cells in the gland, and all zymogen granules and Golgi cisternae in each cell, were qualitatively alike with regard to their content of secretory proteins examined (trypsinogen, chymotrypsinogen A, carboxypeptidase A, RNase, and DNase). The data suggest that these secretory proteins are transported through the cisternae of the Golgi complex where they are intermixed before copackaging in zymogen granules; passage through the Golgi complex is apparently obligatory for these (and likely all) secretory proteins, and is independent of extent of glycosylation, e.g., trypsinogen, a nonglycoprotein vs. DNase, a glycoprotein.     

Calsyntenins are secretory granule proteins in anterior pituitary gland and pancreatic islet alpha cells.

Calsyntenins are members of the cadherin superfamily of cell adhesion molecules. They are present in postsynaptic membranes of excitatory neurons and in vesicles in transit to neuronal growth cones. In the current study, calsyntenin-1 (CST-1) and calsyntenin-3 (CST-3) were identified by mass spectrometric analysis (LC-MS/MS) of integral membrane proteins from highly enriched secretory granule preparations from bovine anterior pituitary gland. Immunofluorescence microscopy on thin frozen sections of rat pituitary revealed that CST-1 was present only in gonadotropes where it colocalized with follicle-stimulating hormone in secretory granules. In contrast, CST-3 was present not only in gonadotrope secretory granules but also in those of somatotropes and thyrotropes. Neither protein was detected in mammatropes. In addition, CST-1 was also localized to the glucagon-containing secretory granules of alpha cells in the pancreatic islets of Langerhans. Results indicate that calsyntenins function outside the nervous system and potentially are modulators of endocrine function.     

Differential aggregation properties of secretory proteins that are stored in exocrine secretory granules of the pancreas and parotid glands

Low-pH- and calcium-induced aggregation of regulated secretory proteins has been proposed to play a role in their retention and storage in secretory granules. However, this has not been tested for secretory proteins that are stored in the exocrine parotid secretory granules. Parotid granule matrix proteins were analyzed for aggregation in the presence or absence of calcium and in the pH range of 5.5 to 7.5. Amylase did not aggregate under these conditions, although <10% of parotid secretory protein (PSP) aggregated below pH 6.0. To test aggregation directly in isolated granules, rat parotid secretory granules were permeabilized with 0.1% saponin in the presence or absence of calcium and in the pH range of 5.0 to 8.4. In contrast to the low-pH-dependent retention of amylase in exocrine pancreatic granules, amylase was quantitatively released and most PSP was released from parotid granules under all conditions. Both proteins were completely released upon granule membrane solubilization. Thus neither amylase nor PSP show low-pH- or calcium-induced aggregation under physiological conditions in the exocrine parotid secretory granules.     

Pituitary Secretory Granule Topography: Influence of Different Electron Microscopic Preparation Procedures on the Surface of Epon Sections

Human, rat and mouse pituitary tissues have been examined electron microscopically in transmission (TEM), scanning-transmission (STEM) and scanning (SEM) modes for the surface appearance of the secretory granules in tissue sections. Cryofixed and cryosectioned tissue showed only slightly protruding granule profiles which had a smooth surface. Cryofixed, freeze-dried and Epon embedded pituitaries, on the other hand, demonstrated swollen and furrowed surfaces over the granules after contact with water. This topography could also be seen after glutaraldehyde fixation but less after post-fixation in OsO₄. The surface alterations in the sections of pituitary secretory granules are thought to be due to differences in the homogeneity of the resin infiltration, leaving resin-free openings where water can enter. It also seems probable that the Epon resin is more influenced by water than has been previously assumed, based on the findings of efficient elimination of osmium from the granules after incubation of tissue sections in water for only 10 min.     

Pituitary Secretory Granule Topography: Influence of Different Electron Microscopic Preparation Procedures on the Surface of Epon Sections

Human, rat and mouse pituitary tissues have been examined electron microscopically in transmission (TEM), scanning-transmission (STEM) and scanning (SEM) modes for the surface appearance of the secretory granules in tissue sections. Cryofixed and cryosectioned tissue showed only slightly protruding granule profiles which had a smooth surface. Cryofixed, freeze-dried and Epon embedded pituitaries, on the other hand, demonstrated swollen and furrowed surfaces over the granules after contact with water. This topography could also be seen after glutaraldehyde fixation but less after post-fixation in OsO₄. The surface alterations in the sections of pituitary secretory granules are thought to be due to differences in the homogeneity of the resin infiltration, leaving resin-free openings where water can enter. It also seems probable that the Epon resin is more influenced by water than has been previously assumed, based on the findings of efficient elimination of osmium from the granules after incubation of tissue sections in water for only 10 min.     

Compartmentalization of secretory proteins in pancreatic zymogen granules as revealed by immunolabeling on cryo-fixed and molecular distillation processed tissue*

Summary— Immunogold labeling of amylase obtained over zymogen granules of rat pancreatic acinar cells processed through cryofixation, molecular distillation drying and embedding in resins was found to be of high intensity and displayed a particular pattern. Indeed, it was concentrated in certain areas of the granules leaving others devoid of gold particles. This pattern of labeling reflects a strong compartmentalization of the secretory proteins within each granule. In order to assess this phenomenon, we have compared the intensities and the pattern of distribution of the labelings in tissues processed through: chemical fixation with embedding in various resins, cryo-ultramicrotomy and cryo-fixation followed by molecular distillation drying. Serials sections and double labeling experiments were performed for further evaluation of the results and for assessing artefactual displacement of proteins during tissue preparation. The results obtained indicate that the secretory proteins are indeed segregated within the granule which appears thus as a well organized structure. Cryo-fixation combined with molecular distillation appears thus to be superior in terms of preservation of protein antigenicity and retention of cellular components close to their living state.     

Chromogranin B (secretogranin I), a neuroendocrine-regulated secretory protein, is sorted to exocrine secretory granules in transgenic mice.

Chromogranin B (CgB, secretogranin I) is a secretory granule matrix protein expressed in a wide variety of endocrine cells and neurons. Here we generated transgenic mice expressing CgB under the control of the human cytomegalovirus promoter. Northern and immunoblot analyses, in situ hybridization and immunocytochemistry revealed that the exocrine pancreas was the tissue with the highest level of ectopic CgB expression. Upon subcellular fractionation of the exocrine pancreas, the distribution of CgB in the various fractions was indistinguishable from that of amylase, an endogenous constituent of zymogen granules. Immunogold electron microscopy of pancreatic acinar cells showed co-localization of CgB with zymogens in Golgi cisternae, condensing vacuoles/immature granules and mature zymogen granules; the ratio of immunoreactivity of CgB to zymogens being highest in condensing vacuoles/immature granules. CgB isolated from zymogen granules of the pancreas of the transgenic mice aggregated in a mildly acidic (pH 5.5) milieu in vitro, suggesting that low pH-induced aggregation contributed to the observed concentration of CgB in condensing vacuoles. Our results show that a neuroendocrine-regulated secretory protein can be sorted to exocrine secretory granules in vivo, and imply that a key feature of CgB sorting in the trans-Golgi network of neuroendocrine cells, i.e. its aggregation-mediated concentration in the course of immature secretory granule formation, also occurs in exocrine cells although secretory protein sorting in these cells is thought to occur largely in the course of secretory granule maturation.     

Immunocytochemical characterization of glucagon-immunoreactive cells using monoclonal antibodies to pancreatic glucagon

Monoclonal antibodies raised to pancreatic glucagon were tested for their ability to detect glucagon-containing endocrine cells in material processed for light and electron microscopy. Samples from man, baboon and rat were used in this investigation. Two antibodies were specific for the pancreatic islet A cells, the remainder detected both pancreatic and enteric endocrine cells. In man and baboon the glucagon-containing cells were confined to the pancreas, lower small intestine and colon. In the rat the distribution was extended to include the corpus of the stomach and the jejunum. The cells identified in the ileum and colon were of three morphological types endocrine, paracrine (type 1) with a single basal process and paracrine (type 2) with multiple small cytoplasmic processes. These antibodies also detected cells in material fixed by conventional methods for electron microscopy. The ultrastructural appearance of the baboon pancreatic glucagon-containing ultracellular secretory granules were demonstrated to be clearly distinct from those described previously in man and rat. The secretory granules averaged 330 +/- 23 nm and lacked the characteristic clear outer halo seen in the other two species.     

Two-dimensional electrophoretic analysis of secretory-granule,granule-membrane,and plasma-membrane proteins of rat parotid cells

Secretory granules and plasma membranes were isolated from rat parotid cells and characterized enzymatically and by electron microscopy. The proteins of the secretory granule membranes, the secretory granules and the plasma membranes were characterized by two-dimensional polyacrylamide gel electrophoresis and visualized by silver staining. The granule membrane contains 166 polypeptides of which only 26 are also present in the granule contents. The membrane proteins have isoelectric points between 4.75 and 6.45 and apparent molecular weights of 17 000 to 190 000 Daltons. The granule content proteins are surprisingly complex and contain 122 polypeptides with molecular weights of 11 000 to 138 000 and isoelectric points of 4.8 to 6.55. Thirteen of these peptides are present as major species. The plasma membrane contains 172 polypeptide species with molecular weights from 17 000 to 200 000 Daltons and isoelectric points of 5.0 to 6.8. Thirty-five of the plasma membrane proteins are also present in the secretory granule membranes indicating that the two membranes have some enzymatic or structural properties in common. Thus, secretory granule membranes and plasma membranes from parotid cells have a more complex polypeptide composition than has previously been shown for membranes of this type. The systems developed are suitable for the analysis of regulatory events such as protein phosphorylation, proteolytic processing, and other types of post-translational modifications that may be important to the secretory mechanism.     

Immunocytochemical characterization of glucagon-immunoreactive cells using monoclonal antibodies to pancreatic glucagon

Summary Monoclonal antibodies raised to pancreatic glucagon were tested for their ability to detect glucagon-containing endocrine cells in material processed for light and electron microscopy. Samples from man, baboon and rat were used in this investigation. Two antibodies were specific for the pancreatic islet A cells, the remainder detected both pancreatic and enteric endocrine cells.In man and baboon the glucagon-containing cells were confined to the pancreas, lower small intestine and colon. In the rat the distribution was extended to include the corpus of the stomach and the jejunum. The cells identified in the ileum and colon were of three morphological types endocrine, paracrine (type 1) with a single basal process and paracrine (type 2) with multiple small cytoplasmic processes.These antibodies also detected cells in material fixed by conventional methods for electron microscopy. The ultrastructural appearance of the baboon pancreatic glucagon-containing ultracellular secretory granules were demonstrated to be clearly distinct from those described previously in man and rat. The secretory granules averaged 330±23 nm and lacked the characteristic clear outer halo seen in the other two species.     

Ultrastructure of reproductive accessory glands in the female sandfly Phlebotomus perniciosus Newstead (Diptera : Psychodidae)

The paired female accessory glands of Phlebotomus perniciosus (Diptera : Psychodidae) were investigated by light microscopy, and by scanning and transmission electron microscopy. These glands undergo morphological and functional changes during oocyte development. After the blood meal, the monostratified glandular epithelium differentiates and starts to secrete. Well-developed rough endoplasmic reticulum, Golgi complexes, and membrane-bounded exocytic vesicles suggest that these secretory cells are involved in protein synthesis. As the secretory cells differentiate, the glandular lumen increases in size and fills with secretory material, consisting of globular granules of different sizes in an amorphous electron-dense matrix. The granules have an electron-translucent core and an electron-dense cortex. The morphological characteristics of the glandular epithelium and the functional role of the glands are discussed in relation to their possible contribution to the reproductive process.     

The effects of colchicine on the ultrastructure of the dental epithelium and odontoblasts of teleost tooth buds

Secretory granule ultrastructure of teleost inner dental epithelial (IDE) cells has been reported to be similar to procollagen granules of other cells synthesizing collagen. This study describes the ultrastructure of secretory products in odontogenic cells during enameloid matrix formation in cichlids after inhibition of granule secretion with colchicine. Thirty-six fish were injected with 0.1 mg colchicine, then three were killed first at 2-hr intervals for 12 hr, then daily for 5 days. Tooth buds were processed for transmission electron microscopy, and ultrastructural alterations were assessed for each post-injection interval. Four hours post-injection, IDE cells contained increased numbers of secretory granules, lightly stained granules, dilated cisternae of the granular endoplasmic reticulum, and intercellular amorphous material. After 6 hr, the IDE intercellular amorphous material additionally contained electron dense deposits, and after 8 hr, the intercellular material had fibers similar in appearance to enameloid collagen. No ultrastructural changes were detected in odontoblasts that were in close proximity to the enameloid matrix. Only odontoblasts synthesizing predentin were affected by colchicine, and the observed alterations were similar to those seen in IDE cells. It is concluded that IDE cells synthesize and secrete ectodermal enameloid matrix collagen.     

Determinants for chromogranin A sorting into the regulated secretory pathway are also sufficient to generate granule-like structures in non-endocrine cells

In endocrine cells, prohormones and granins are segregated in the TGN (trans-Golgi network) from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner on specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures that resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of CgA (chromogranin A) is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP (green fluorescent protein) or a short epitope tag was expressed in COS-1 fibroblast cells and analysed by fluorescence and electron microscopy and pulse-chase labelling. Full-length CgA as well as deletion constructs containing the N-terminal 77 residues generated granule-like structures in the cell periphery that co-localized with co-expressed SgII (secretogranin II). These are essentially the same segments of the protein that were previously shown to be required for granule sorting in wild-type PC12 (pheochromocytoma cells) cells and for rescuing a regulated secretory pathway in A35C cells, a variant PC12 line deficient in granule formation. The results support the notion that self-aggregation is at the core of granule formation and sorting into the regulated pathway.     

A subset of p23 localized on secretory granules in pancreatic beta-cells.

Proteins on the membrane of secretory granules (SGs) involved in their biogenesis and exocytosis are poorly characterized compared with those of synaptic vesicle in neurons. Thus the secretory granule membrane was prepared from a mouse pancreatic beta-cell line MIN6 by subcellular fractionation, and protein constituents were analyzed by microscale two-dimensional liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Using this proteomics approach, one of the p24 family proteins, p23, was unexpectedly found in the granule fraction, although p24 proteins are generally regarded as functioning in the early secretory pathways between the endoplasmic reticulum and the Golgi apparatus. We further showed that p23 is expressed at high levels in endocrine cells. Furthermore, immunocytochemical analyses of pancreatic beta-cells at the light and electron microscopic levels demonstrated that a significant amount of p23 is localized on the insulin granule membrane, although it is most intensely concentrated at the cis-Golgi compartment as previously shown in non-endocrine cells. These findings suggest that a fraction of p23 enters post-Golgi compartments and may function in the biogenesis and/or quality control of SGs.     

Not all secretory granules are created equal: Partitioning of soluble content proteins

Secretory granules carrying fluorescent cargo proteins are widely used to study granule biogenesis, maturation, and regulated exocytosis. We fused the soluble secretory protein peptidylglycine alpha-hydroxylating monooxygenase (PHM) to green fluorescent protein (GFP) to study granule formation. When expressed in AtT-20 or GH3 cells, the PHM-GFP fusion protein partitioned from endogenous hormone (adrenocorticotropic hormone, growth hormone) into separate secretory granule pools. Both exogenous and endogenous granule proteins were stored and released in response to secretagogue. Importantly, we found that segregation of content proteins is not an artifact of overexpression nor peculiar to GFP-tagged proteins. Neither luminal acidification nor cholesterol-rich membrane microdomains play essential roles in soluble content protein segregation. Our data suggest that intrinsic biophysical properties of cargo proteins govern their differential sorting, with segregation occurring during the process of granule maturation. Proteins that can self-aggregate are likely to partition into separate granules, which can accommodate only a few thousand copies of any content protein; proteins that lack tertiary structure are more likely to distribute homogeneously into secretory granules. Therefore, a simple "self-aggregation default" theory may explain the little acknowledged, but commonly observed, tendency for both naturally occurring and exogenous content proteins to segregate from each other into distinct secretory granules.