scholarly journals Chromogranin A in the pancreatic islet: cellular and subcellular distribution.

1986 ◽  
Vol 34 (12) ◽  
pp. 1673-1682 ◽  
Author(s):  
M Ehrhart ◽  
D Grube ◽  
M F Bader ◽  
D Aunis ◽  
M Gratzl
Keyword(s):  
Pancreatic Islet ◽  
Subcellular Distribution ◽  
Chromogranin A ◽  
Chromaffin Cells ◽  
Protein A ◽  
Ultrastructural Level ◽  
Polyclonal Antiserum ◽  
Secretory Vesicles ◽  
The Matrix ◽  
Pancreatic Endocrine Cells

Chromogranin A (CGA) is the major soluble protein within secretory vesicles of chromaffin cells. A polyclonal antiserum was raised against bovine CGA and characterized in two-dimensional immunoblots. Cellular and subcellular distribution of CGA in bovine pancreatic islet was investigated by immunocytochemistry. At the light microscopic level, CGA-like immunoreactivity was found in the same cells that react with antibodies against insulin, glucagon, and somatostatin. A minority of cells containing pancreatic polypeptide also showed faint immunostaining. At the ultrastructural level (protein A-gold technique), CGA-like immunoreactivity was confined exclusively to the secretory vesicles. Whereas the hormones were localized mainly in the central part of the secretory vesicles, CGA was present predominantly in the periphery. These findings indicate that a CGA-like protein is a regular constituent of the matrix of secretory vesicles in pancreatic endocrine cells.

scholarly journals Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: suitability for immunogold ultrastructural localization of neural antigens.

1991 ◽  
Vol 39 (9) ◽  
pp. 1267-1279 ◽  
Author(s):  
M van Lookeren Campagne ◽  
A B Oestreicher ◽  
T P van der Krift ◽  
W H Gispen ◽  
A J Verkleij
Keyword(s):  
Monoclonal Antibodies ◽  
Rat Brain ◽  
Anhydrous Methanol ◽  
Protein A ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Polyclonal Antiserum ◽  
Double Labeling ◽  
Ultrastructural Studies

We examined the suitability of freeze-substitution and Lowicryl HM20 embedding of aldehyde-fixed rat brain to localize several neural antigens at the ultrastructural level. The following rabbit polyclonal and mouse monoclonal antibodies were used: affinity-purified polyclonal immunoglobulins G raised to B-50/GAP43 (a membrane-anchored, growth-associated protein); affinity-purified polyclonal immunoglobulins G to human glial fibrillary acidic protein (GFAP; a subunit of glial filaments); a polyclonal antiserum raised to adrenocorticotropic hormone[25-39] (a neuropeptide present in dense-core granules); a polyclonal antiserum raised to myelin basic protein (a protein present in compact myelin of the central nervous system); and mouse monoclonal antibodies to synaptophysin (an integral membrane protein of small synaptic vesicles). Rat mesencephalon was fixed by perfusion with buffered 2% glutaraldehyde and 4% paraformaldehyde, cryoprotected, and frozen in liquid nitrogen. Freeze-substitution of tissue was performed with anhydrous methanol and 0.5% uranyl acetate at -90 degrees C. Semi-thin Lowicryl sections were used for light microscopic visualization of B-50 in the ventromedial mesencephalic central gray substance. The procedure preserves well the ultrastructure of this region and the immunoreactivity of the selected antigens. This study shows that dehydration by freeze-substitution, combined with Lowicryl HM20 embedding at sub-zero temperature, provides a successful method of preparation of fixed brain tissue for ultrastructural studies, allowing immunogold localization of several neural antigens by double labeling in the same section and in serial sections.

Morphological Observations on the Granule Types in Rabbit Extra-Adrenal Chromaffin Cells.

1975 ◽  
Vol 33 ◽  
pp. 318-319
Author(s):  
Joe A. Mascorro ◽  
Robert D. Yates
Keyword(s):  
Chromaffin Cells ◽  
Sodium Phosphate ◽  
Ganglion Cells ◽  
Ultrastructural Level ◽  
Osmic Acid ◽  
Adrenal Chromaffin Cells ◽  
Potassium Iodate ◽  
Perfusion Fluid ◽  
New Zealand White Rabbits ◽  
Adrenal Chromaffin

Extra-adrenal chromaffin organs (abdominal paraganglia) constitute rich sources of catecholamines. It is believed that these bodies contain norepinephrine exclusively. However, the present workers recently observed epinephrine type granules in para- ganglion cells. This report investigates catecholamine containing granules in rabbit paraganglia at the ultrastructural level.New Zealand white rabbits (150-170 grams) were anesthetized with 50 mg/kg Nembutal (IP) and perfused with 3% glutaraldehyde buffered with 0.2M sodium phosphate, pH 7.3. The retroperitoneal tissue blocks were removed and placed in perfusion fluid for 4 hours. The abdominal paraganglia were dissected from the blocks, diced, washed in phosphate buffer and fixed in 1% osmic acid buffered with phosphate. In other animals, the glutaraldehyde perfused tissue blocks were immersed for 1 hour in 3% glutaraldehyde/2.5% potassium iodate buffered as before. The paraganglia were then diced, separated into two vials and washed in the buffer. A portion of this tissue received osmic acid fixation.

scholarly journals Evidence for Functional Localization of the Proenkephalin-Processing Enzyme, Prohormone Thiol Protease, to Secretory Vesicles of Chromaffin Cells*

Endocrinology ◽  
1999 ◽  
Vol 140 (8) ◽  
pp. 3744-3754 ◽  
Author(s):  
Vivian Y. H. Hook ◽  
Stephen Noctor ◽  
Catherine A. Sei ◽  
Thomas Toneff ◽  
Sukkid Yasothornsrikul ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Secretory Vesicles ◽  
Thiol Protease ◽  
Processing Enzyme ◽  
Functional Localization

Neurofilament proteins are co-expressed with desmin in heart conduction system myocytes

1990 ◽  
Vol 97 (1) ◽  
pp. 11-21
Author(s):  
M. Vitadello ◽  
M. Matteoli ◽  
L. Gorza
Keyword(s):  
Subcellular Distribution ◽  
Ultrastructural Level ◽  
Rabbit Heart ◽  
Cardiac Cells ◽  
Structural Components ◽  
Minor Population ◽  
Neuronal Proteins ◽  
Tissue Homogenates ◽  
A Minor

We have recently shown that specialized myocytes of the rabbit heart express a cytoskeletal protein similar to the M subunit of neurofilaments (NF). Since this result was obtained using a single anti-NF-M monoclonal antibody, we tested on conduction myocytes a panel of five anti-NF antibodies, specific for each of the three NF subunits and for phosphorylated and non-phosphorylated epitopes. Two antibodies, one specific for the L subunit and one for phosphorylated M subunit of NF, reacted with specialized myocytes in immunohistochemistry. In immunoblots on conduction tissue homogenates the two antibodies recognized two polypeptides with electrophoretic mobility and solubility properties identical to those of NF-L and NF-M in the sciatic nerve. The subcellular distribution of NF immunoreactivity in specialized myocytes was very similar to desmin localization; namely, it was distributed on large filamentous bundles and on fine filaments localized transversely at the level of the Z line. At the ultrastructural level, immunoreactive filaments were localized in the intermyofibrillar space and connected myofibrils with mitochondria. Co-expression of NF proteins and desmin was also observed in vitro in a minor population of cardiac myocytes cultured from embryonic rabbit heart. In most cases NF immunoreactivity co-localized with desmin, especially where filaments were well organized, but in some cells anti-NF and anti-desmin antibodies labelled different filamentous structures. These results indicate that NF proteins are structural components of the cytoskeleton of specialized myocytes and show a subcellular distribution very similar to desmin. Such a composition of intermediate filaments indicates that in these cardiac cells muscle differentiation is compatible with the expression of neuronal proteins.

scholarly journals Chromogranin A, the major lumenal protein in chromaffin granules, controls fusion pore expansion

2018 ◽  
Vol 151 (2) ◽  
pp. 118-130 ◽  
Author(s):  
Prabhodh S. Abbineni ◽  
Mary A. Bittner ◽  
Daniel Axelrod ◽  
Ronald W. Holz
Keyword(s):  
Plasma Membrane ◽  
Small Molecules ◽  
Chromogranin A ◽  
Chromaffin Cells ◽  
Fusion Pore ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Chromogranin B ◽  
Tissue Plasminogen ◽  
Pore Expansion

Upon fusion of the secretory granule with the plasma membrane, small molecules are discharged through the immediately formed narrow fusion pore, but protein discharge awaits pore expansion. Recently, fusion pore expansion was found to be regulated by tissue plasminogen activator (tPA), a protein present within the lumen of chromaffin granules in a subpopulation of chromaffin cells. Here, we further examined the influence of other lumenal proteins on fusion pore expansion, especially chromogranin A (CgA), the major and ubiquitous lumenal protein in chromaffin granules. Polarized TIRF microscopy demonstrated that the fusion pore curvature of granules containing CgA-EGFP was long lived, with curvature lifetimes comparable to those of tPA-EGFP–containing granules. This was surprising because fusion pore curvature durations of granules containing exogenous neuropeptide Y-EGFP (NPY-EGFP) are significantly shorter (80% lasting <1 s) than those containing CgA-EGFP, despite the anticipated expression of endogenous CgA. However, quantitative immunocytochemistry revealed that transiently expressed lumenal proteins, including NPY-EGFP, caused a down-regulation of endogenously expressed proteins, including CgA. Fusion pore curvature durations in nontransfected cells were significantly longer than those of granules containing overexpressed NPY but shorter than those associated with granules containing overexpressed tPA, CgA, or chromogranin B. Introduction of CgA to NPY-EGFP granules by coexpression converted the fusion pore from being transient to being longer lived, comparable to that found in nontransfected cells. These findings demonstrate that several endogenous chromaffin granule lumenal proteins are regulators of fusion pore expansion and that alteration of chromaffin granule contents affects fusion pore lifetimes. Importantly, the results indicate a new role for CgA. In addition to functioning as a prohormone, CgA plays an important role in controlling fusion pore expansion.

scholarly journals The post-translational processing of chromogranin A in the pancreatic islet: involvement of the eukaryote subtilisin PC2

1994 ◽  
Vol 298 (3) ◽  
pp. 521-528 ◽  
Author(s):  
S D Arden ◽  
N G Rutherford ◽  
P C Guest ◽  
W J Curry ◽  
E M Bailyes ◽  
...  
Keyword(s):  
Secretory Granule ◽  
Pancreatic Islet ◽  
Pancreatic Beta Cells ◽  
Chromogranin A ◽  
Time Course ◽  
Deae Cellulose ◽  
Neuroendocrine Cells ◽  
Time Period ◽  
Sds Page

The post-translational processing of chromogranin A (CGA) and the nature of the enzyme(s) involved were investigated in rat pancreatic islet and insulinoma tissue. Pulse-chase radiolabelling experiments using sequence-specific antisera showed that the 98 kDa (determined by SDS/PAGE) precursor was processed to an N-terminal 21 kDa peptide, a C-terminal 14 kDa peptide and a 45 kDa centrally located peptide with a rapid time course (t1/2 approx. 30 min) after an initial delay of 30-60 min. The 45 kDa peptide was, in turn, converted partially into a 5 kDa peptide with pancreastatin immunoreactivity and a 3 kDa peptide with WE-14 immunoreactivity over a longer time period. Incubation of bovine CGA with rat insulinoma secretory-granule lysate produced peptides of 18, 16 and 40 kDa via intermediates of 65 and 55 kDa. N-terminal sequence analysis indicated that cleavage occurred at the conserved paired basic sites Lys114-Arg115 and Lys330-Arg331, suggesting that cleavage of the equivalent sites (Lys129-Arg130 and Lys357-Arg358) in the rat molecule produced the initial post-translational products observed in intact pancreatic beta-cells. The enzyme activity responsible for the cleavage of bovine CGA co-chromatographed on DEAE-cellulose with the type-2 proinsulin endopeptidase and with PC2 immunoreactivity. The type-1 enzyme (PC1/3) appeared inactive towards CGA. The requirement for Ca2+ ions and an acidic pH for conversion was consistent with the involvement of a member of the eukaryote subtilisin family, and the composition of the released peptides in pulse-chase and secretion studies suggested that conversion occurred in the secretory-granule compartment. The overall catalytic rate as well as the relative susceptibilities of the Lys114-Arg115 and Lys330-Arg331 sites to cleavage were affected by pH, suggesting that the ionic environment of the processing compartment may play a role in the differential processing of CGA which is evident in various neuroendocrine cells.

scholarly journals Localization of bone sialoprotein (BSP) to Golgi and post-Golgi secretory structures in osteoblasts and to discrete sites in early bone matrix.

1993 ◽  
Vol 41 (2) ◽  
pp. 193-203 ◽  
Author(s):  
P Bianco ◽  
M Riminucci ◽  
G Silvestrini ◽  
E Bonucci ◽  
J D Termine ◽  
...  
Keyword(s):  
Secretory Granules ◽  
Bone Matrix ◽  
Ultrastructural Level ◽  
Bone Sialoprotein ◽  
Protein Glycosylation ◽  
Secretory Structures ◽  
Mineral Deposition ◽  
The Matrix ◽  
Random Manner ◽  
Intracellular Labelling

Bone sialoprotein (BSP), a bone matrix-enriched glycoprotein containing the Arg-Gly-Asp (RGD) motif and endowed with cell binding properties, was localized in osteoblasts and early bone matrix of developing rat bone at the ultrastructural level. Preliminary light microscopic observations indicated that intracellular labelling was restricted to a paranuclear dot corresponding to the "negative Golgi image" of classical histology. The same pattern was observed whether antisera against the fully glycosylated protein or a peptide antiserum to a stretch of amino acids in human BSP sequence were employed. At the EM level, we obtained labeling over the Golgi area of osteoblasts but not over the rER. The labeling was concentrated over distensions of the trans Golgi and over pro-secretory granules. In the matrix, BSP was distributed in a non-random manner. The label was concentrated over spherical aggregates of finely fibrillar material corresponding to the sites of early mineral deposition (so-called "mineralization nodules"). Such BSP-positive foci were seen both close to and away from the cell surface. The predominant association of BSP with Golgi and post-Golgi secretory structures and its absence from rER, as well as the reproducibility of the same pattern of localization with different antisera, might indicate a slow transit of the protein through the Golgi, not necessarily associated with protein glycosylation.

Chromogranin A in the Mammalian Merkel Cell: Cellular and Subcellular Distribution

1989 ◽  
Vol 93 (5) ◽  
pp. 641-648 ◽  
Author(s):  
Wolfgang Hartschuh ◽  
Eberhard Weihe ◽  
Ursula Egner
Keyword(s):  
Subcellular Distribution ◽  
Chromogranin A ◽  
Merkel Cell
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