scholarly journals Exocytotic exposure and recycling of membrane antigens of chromaffin granules: ultrastructural evaluation after immunolabeling.

1986 ◽  
Vol 102 (2) ◽  
pp. 510-515 ◽  
Author(s):  
A Patzak ◽  
H Winkler
Keyword(s):  
Plasma Membrane ◽  
Chromaffin Cells ◽  
Direct Evidence ◽  
Ultrastructural Level ◽  
Multivesicular Bodies ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Golgi Region ◽  
Membrane Antigens ◽  
Dense Vesicles

The exocytotic exposure and retrieval of an antigen of chromaffin granule membranes were studied with chromaffin cells isolated from bovine adrenal medulla. Cells were incubated with an antiserum against glycoprotein III followed by fluorescein- or gold-labeled anti-IgG. Immunofluorescence on the cell surface was present in a patchy distribution irrespective of whether bivalent antibodies or Fab fragments were used. During subsequent incubation these fluorescent membrane patches were internalized within 45 min. At the ultrastructural level immunogold-labeled patches were present on the surface of stimulated cells. During incubation (5 min to 6 h) these immunolabeled membrane patches became coated, giving rise to coated vesicles and finally to smooth vesicles. These latter vesicles were found spread throughout the cytoplasm including the Golgi region, but Golgi stacks did not become labeled. Part of the immunolabel was transferred to multivesicular bodies, which probably represent a lysosomal pathway. 30 min after incubation immunolabel was also found in electron-dense vesicles apparently representing newly formed chromaffin granules. After 6 h of incubation immunolabel was found in vesicles indistinguishable from mature chromaffin granules. These results provide direct evidence that after exocytosis membranes of chromaffin granules are selectively retrieved from the plasma membrane and are partly recycled to newly formed chromaffin granules, providing a shuttle service from the Golgi region to the plasma membrane.

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.

EM localization of chromaffin cell ATPase

1992 ◽  
Vol 50 (1) ◽  
pp. 816-817
Author(s):  
V. Kriho ◽  
G. D. Pappas
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Immunogold Labelling ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Cells In Culture ◽  
Membrane Bound ◽  
Bovine Chromaffin Cells

During exocytosis of the chromaffin granules, ATP is released. ATP can then be hydrolyzed by the ecto-ATPases of the plasma membrane to provide adenosine for reuptake or for activation of P1 purinoceptors. Chromaffin granule membranes also possess ATPase activity. This activity is linked to the uptake of catecholamines from the cytoplasm into the membrane-bound granule compartment.In this report we combine EM cytochemistry and immunogold labelling to provide further evidence for the presence of ATPase on both the plasma membrane and granule membranes of bovine chromaffin cells in culture.

scholarly journals Identification of rat hepatocyte plasma membrane proteins using monoclonal antibodies.

1985 ◽  
Vol 100 (4) ◽  
pp. 1115-1125 ◽  
Author(s):  
A L Hubbard ◽  
J R Bartles ◽  
L T Braiterman
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Ultrastructural Level ◽  
Isolated Hepatocytes ◽  
Plasma Membrane Proteins ◽  
Rat Hepatocyte ◽  
Molecular Weights ◽  
Domain Specific ◽  
Golgi Region ◽  
Surface Domains

We have localized and identified five rat hepatocyte plasma membrane proteins using hybridoma technology in combination with morphological and biochemical methods. Three different membrane preparations were used as immunogens: isolated hepatocytes, a preparation of plasma membrane sheets that contained all three recognizable surface domains of the intact hepatocyte (sinusoidal, lateral, and bile canalicular), and a glycoprotein subfraction of that plasma membrane preparation. We selected monoclonal IgGs that were hepatocyte specific and localized them using both immunofluorescence on 0.5-micron sections of frozen liver and immunoperoxidase at the ultrastructural level. One antigen (HA 4) was localized predominantly to the bile canalicular surface, whereas three (CE 9, HA 21, and HA 116) were localized predominantly to the lateral and sinusoidal surfaces. One antigen (HA 16) was present in all three domains. Only one antigen (HA 116) could be detected in intracellular structures both in the periphery of the cell and in the Golgi region. The antigens were all integral membrane proteins as judged by their stability to alkaline extraction and solubility in detergents. The apparent molecular weights of the antigens were established by immunoprecipitation and/or immunoblotting. In a related study (Bartles, J.R., L.T. Braiterman, and A.L. Hubbard, 1985, J. Cell. Biol., 100:1126-1138), we present biochemical confirmation of the domain-specific localizations for two of the antigens, HA 4 and CE 9, and demonstrate their suitability as endogenous domain markers for monitoring the separation of bile canalicular and sinusoidal lateral membrane on sucrose density gradients.

scholarly journals Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes.

1992 ◽  
Vol 116 (2) ◽  
pp. 359-365 ◽  
Author(s):  
D Scheuner ◽  
C D Logsdon ◽  
R W Holz
Keyword(s):  
Chromaffin Cells ◽  
Secretory Vesicle ◽  
Xenopus Laevis Oocytes ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Vesicle Membrane ◽  
Sequence Of Events ◽  
Dopamine Beta Hydroxylase ◽  
Granule Membrane ◽  
Adrenal Chromaffin

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.

Docking of chromaffin granules—A necessary step in exocytosis?

1987 ◽  
Vol 7 (4) ◽  
pp. 269-279 ◽  
Author(s):  
Theo Schäfer ◽  
Urs O. Karli ◽  
Felix E. Schweizer ◽  
Max M. Burger
Keyword(s):  
Plasma Membrane ◽  
Chromaffin Cells ◽  
Cell Types ◽  
Cell System ◽  
Secretory Vesicles ◽  
Chromaffin Granules ◽  
Permeabilized Cell ◽  
Permeabilized Cells ◽  
Pc 12 Cells

Putative docking of secretory vesicles comprising recognition of and attachment to future fusion sites in the plasma membrane has been investigated in chromaffin cells of the bovine adrenal medulla and in rat phaeochromocytoma (PC 12) cells. Upon permeabilization with digitonin, secretion can be stimulated in both cell types by indreasing the free Ca2+-concentration to μM levels. Secretory activity can be elicited up to 1 hr after starting permeabilization and despite the loss of soluble cytoplasmic components indicating a stable attachment of granules to the plasma membrane awaiting the trigger for fusion. Docked granules can be observed in the electron microscope in permeabilized PC 12 cells which contain a large proportion of their granules aligned underneath the plasma membrane. The population of putatively docked granules in chromaffin cells cannot be as readily discerned due to the dispersal of granules throughout the cytoplasm. Further experiments comparing PC 12 and chromaffin cells suggest that active docking but not transport of granules can still be performed by permeabilized cells in the presence of Ca2+: a short (2 min) pulse of Ca2+ in PC 12 cells leads to the secretion of almost all releasable hormone over a 15 min observation period whereas, in chromaffin cells, with only a small proportion of granules docked, withdrawal of Ca2+ leads to an immediate halt in secretion. Transport of chromaffin granules from the Golgi to the plasma membrane docking sites seems to depend on a mechanism sensitive to permeabilization. This is shown by the difference in the amount of hormone released from the two permeabilized cell types, reflecting the contrast in the proportion of granules docked to the plasma membrane in PC 12 or chromaffin cells. Neither docking nor the docked state are influenced by cytochalasine B or colchicine. The permeabilized cell system is a valuable technique for the in vitro study of interaction between secretory vesicles and their target membrane.

scholarly journals Exocytotic exposure and retrieval of membrane antigens of chromaffin granules: quantitative evaluation of immunofluorescence on the surface of chromaffin cells.

1984 ◽  
Vol 98 (5) ◽  
pp. 1817-1824 ◽  
Author(s):  
A Patzak ◽  
G Böck ◽  
R Fischer-Colbrie ◽  
K Schauenstein ◽  
W Schmidt ◽  
...  
Keyword(s):  
Cell Surface ◽  
Fluorescence Intensity ◽  
Chromaffin Cells ◽  
Quantitative Methods ◽  
Chromaffin Granules ◽  
Dopamine Beta Hydroxylase ◽  
Unstimulated Control ◽  
Membrane Antigens ◽  
Kinetics Of ◽  
Average Fluorescence

The exocytotic exposure of antigens of chromaffin granule membranes was studied with chromaffin cells isolated from bovine adrenal medulla. Antigens on the cell surface were visualized by indirect membrane immunofluorescence employing antisera against glycoprotein III and dopamine beta-hydroxylase. With unstimulated cells, only weak immunofluorescence on the cell surface was observed, whereas stimulated cells (with carbachol or Ba2+) exhibited much stronger reactions. In all cases the staining appeared as dots and patches. To quantitatively prove these observations, we analyzed the immunostained cells using a fluorescence-activated cell sorter. After stimulation, the average fluorescence intensity of the cell population was enhanced. This increase correlated with the degree of catecholamine secretion. The fluorescence intensity of stimulated cells varied over a broad range indicating that individual cells reacted variably to the secretagogues. When stimulated cells were incubated at 37 degrees C for up to 45 min after stimulation, a decrease of membrane immunofluorescence approaching that of unstimulated control cells was observed. Apparently, the membranes of chromaffin granules, which had been incorporated into the plasma membrane, were retrieved by a specific and relatively fast process. This retrieval of the antigen from the cell surface was blocked by sodium azide, but not influenced by colchicine, cytochalasin B, and trifluoperazine. The quantitative methods established in this paper should prove useful for further study of the kinetics of the exo-endocytotic cycle in secretory tissues.

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.

Immunogold labeling of the calcium binding protein synexin in isolated adrenal chromaffin granules and chromaffin cells

1990 ◽  
Vol 48 (3) ◽  
pp. 952-953
Author(s):  
Gemma A.J. Kuijpers ◽  
Harvey B. Pollard
Keyword(s):  
Adrenal Medulla ◽  
Calcium Binding ◽  
Chromaffin Cells ◽  
Cell Activation ◽  
Structural Information ◽  
Dependent Manner ◽  
Chromaffin Granules ◽  
Immuno Electron Microscopy ◽  
Ultrathin Sections ◽  
Adrenal Chromaffin

Exocytotic fusion of granules in the adrenal medulla chromaffin cell is triggered by a rise in the concentration of cytosolic Ca2+ upon cell activation. The protein synexin, annexin VII, was originally found in the adrenal medulla and has been shown to cause aggregation and to support fusion of chromaffin granules in a Ca2+-dependent manner. We have previously suggested that synexin may there fore play a role in the exocytotic fusion process. In order to obtain more structural information on synexin, we performed immuno-electron microscopy on frozen ultrathin sections of both isolated chromaffin granules and chromaffin cells.Chromaffin granules were isolated from bovine adrenal medulla, and synexin was isolated from bovine lung. Granules were incubated in the presence or absence of synexin (24 μg per mg granule protein) and Ca2+ (1 mM), which induces maximal granule aggregation, in 0.3M sucrose-40m MMES buffer(pH 6.0). Granules were pelleted, washed twice in buffer without synexin and fixed with 2% glutaraldehyde- 2% para formaldehyde in 0.1 M phosphate buffer (GA/PFA) for 30 min. Chromaffin cells were isolated and cultured for 3-5 days, and washed and incubated in Krebs solution with or without 20 uM nicotine. Cells were fixed 90 sec after on set of stimulation with GA/PFA for 30 min. Fixed granule or cell pellets were washed, infiltrated with 2.3 M sucrose in PBS, mounted and frozen in liquid N2.

Role of Exosomes in the Exchange of Spermatozoa after Leaving the Seminiferous Tubule: A Review

2020 ◽  
Vol 21 (5) ◽  
pp. 330-338
Author(s):  
Luming Wu ◽  
Yuan Ding ◽  
Shiqiang Han ◽  
Yiqing Wang
Keyword(s):  
Drug Delivery ◽  
Plasma Membrane ◽  
Seminiferous Tubule ◽  
Metabolic Diseases ◽  
Inflammatory Diseases ◽  
Multivesicular Bodies ◽  
Extensive Search ◽  
Neurological Research ◽  
The One

Background: Exosomes are extracellular vesicles (EVs) released from cells upon fusion of an intermediate endocytic compartment with the plasma membrane. They refer to the intraluminal vesicles released from the fusion of multivesicular bodies with the plasma membrane. The contents and number of exosomes are related to diseases such as metabolic diseases, cancer and inflammatory diseases. Exosomes have been used in neurological research as a drug delivery tool and also as biomarkers for diseases. Recently, exosomes were observed in the seminal plasma of the one who is asthenozoospermia, which can affect sperm motility and capacitation. Objective: The main objective of this review is to deeply discuss the role of exosomes in spermatozoa after leaving the seminiferous tubule. Methods: We conducted an extensive search of the literature available on relationships between exosomes and exosomes in spermatozoa on the bibliographic database. Conclusion: : This review thoroughly discussed the role that exosomes play in the exchange of spermatozoa after leaving the seminiferous tubule and its potential as a drug delivery tool and biomarkers for diseases as well.

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