76 results on '"Chromaffin Granules chemistry"'
Search Results
2. Pyroglutamate-amyloid-β and glutaminyl cyclase are colocalized with amyloid-β in secretory vesicles and undergo activity-dependent, regulated secretion.
- Author
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Cynis H, Funkelstein L, Toneff T, Mosier C, Ziegler M, Koch B, Demuth HU, and Hook V
- Subjects
- Aminoacyltransferases analysis, Amyloid beta-Peptides analysis, Amyloid beta-Peptides chemistry, Cell Line, Tumor, Chromaffin Granules chemistry, Chromaffin Granules metabolism, Chromaffin Granules ultrastructure, Humans, Pyrrolidonecarboxylic Acid metabolism, Secretory Vesicles chemistry, Secretory Vesicles ultrastructure, Aminoacyltransferases metabolism, Amyloid beta-Peptides metabolism, Secretory Vesicles metabolism
- Abstract
Background and Aims: N-truncated pyroglutamate (pGlu)-amyloid-β [Aβ(3-40/42)] peptides are key components that promote Aβ peptide accumulation, leading to neurodegeneration and memory loss in Alzheimer's disease. Because Aβ deposition in the brain occurs in an activity-dependent manner, it is important to define the subcellular organelle for pGlu-Aβ(3-40/42) production by glutaminyl cyclase (QC) and their colocalization with full-length Aβ(1-40/42) peptides for activity-dependent, regulated secretion. Therefore, the objective of this study was to investigate the hypothesis that pGlu-Aβ and QC are colocalized with Aβ in dense-core secretory vesicles (DCSV) for activity-dependent secretion with neurotransmitters., Methods: Purified DCSV were assessed for pGlu-Aβ(3-40/42), Aβ(1-40/42), QC, and neurotransmitter secretion. Neuron-like chromaffin cells were analyzed for cosecretion of pGlu-Aβ, QC, Aβ, and neuropeptides. The cells were treated with a QC inhibitor, and pGlu-Aβ production was measured. Human neuroblastoma cells were also examined for pGlu-Aβ and QC secretion., Results: Isolated DCSV contain pGlu-Aβ(3-40/42), QC, and Aβ(1-40/42) with neuropeptide and catecholamine neurotransmitters. Cellular pGlu-Aβ and QC undergo activity-dependent cosecretion with Aβ and enkephalin and galanin neurotransmitters. The QC inhibitor decreased the level of secreted pGlu-Aβ. The human neuroblastoma cells displayed regulated secretion of pGlu-Aβ that was colocalized with QC., Conclusions: pGlu-Aβ and QC are present with Aβ in DCSV and undergo activity-dependent, regulated cosecretion with neurotransmitters., (© 2014 S. Karger AG, Basel.)
- Published
- 2014
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3. Chromogranin location in the adrenal glands of ISIAH rats.
- Author
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Buzueva II, Filyushina EE, Shmerling MD, Markel AL, and Yakobson GS
- Subjects
- Animals, Catecholamines analysis, Chromaffin Cells chemistry, Chromaffin Cells cytology, Immunohistochemistry, Male, Microscopy, Electron, Rats, Secretory Vesicles, Adrenal Glands chemistry, Chromaffin Granules chemistry, Chromogranins analysis, Hypertension metabolism
- Abstract
Comparative immunohistochemical and electron microscopic study of the adrenals from hypertensive ISIAH rats and normotensive WAG rats (control) showed a more intense reaction to chromogranin A in the ISIAH adrenal in comparison with the control. Electron microscopy and morphometric analysis showed high volume and numerical densities of the secretory granules in chromaffin cells of hypertensive rats. The results indicate stimulation of the adrenal medullary substance in ISIAH rats. Presumably, intensive accumulation of chromogranin A and secretory granules in chromaffin cells of hypertensive rats reflects a certain imbalance of chromogranin A and catecholamines biogenesis, this, in turn, leading to stable stimulation of the sympathoadrenal component and higher stress sensitivity of these animals.
- Published
- 2013
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4. Controlling synaptotagmin activity by electrostatic screening.
- Author
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Park Y, Hernandez JM, van den Bogaart G, Ahmed S, Holt M, Riedel D, and Jahn R
- Subjects
- Adenosine Triphosphate metabolism, Animals, Calcium metabolism, Cattle, Chromaffin Granules chemistry, Chromaffin Granules drug effects, Exocytosis physiology, Liposomes chemistry, Liposomes metabolism, Membrane Fusion, Phosphatidylinositol 4,5-Diphosphate metabolism, Phospholipids chemistry, Phospholipids metabolism, Polyphosphates chemistry, Polyphosphates metabolism, Rats, SNARE Proteins metabolism, Static Electricity, Synaptosomal-Associated Protein 25 metabolism, Syntaxin 1 metabolism, Chromaffin Granules metabolism, Synaptotagmin I metabolism
- Abstract
Exocytosis of neurosecretory vesicles is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxin-1, synaptobrevin and SNAP-25, with synaptotagmin functioning as the major Ca(2+) sensor for triggering membrane fusion. Here we show that bovine chromaffin granules readily fuse with large unilamellar liposomes in a SNARE-dependent manner. Fusion is enhanced by Ca(2+), but only when the target liposomes contain phosphatidylinositol-4,5-bisphosphate and when polyphosphate anions, such as nucleotides or pyrophosphate, are present. Ca(2+)-dependent enhancement is mediated by endogenous synaptotagmin-1. Polyphosphates operate by an electrostatic mechanism that reverses an inactivating cis association of synaptotagmin-1 with its own membrane without affecting trans binding. Hence, the balancing of trans- and cis-membrane interactions of synaptotagmin-1 could be a crucial element in the pathway of Ca(2+)-dependent exocytosis.
- Published
- 2012
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5. Relationship between amperometric pre-spike feet and secretion granule composition in chromaffin cells: an overview.
- Author
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Amatore C, Arbault S, Bonifas I, Guille M, Lemaître F, and Verchier Y
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- Animals, Cattle, Cells, Cultured, Chromaffin Cells chemistry, Chromaffin Cells ultrastructure, Chromaffin Granules chemistry, Secretory Vesicles chemistry, Chromaffin Cells physiology, Chromaffin Granules physiology, Conductometry methods, Exocytosis, Secretory Vesicles physiology
- Abstract
Amperometry is a simple and powerful technique to study exocytosis at the single cell level. By positioning and polarizing (at an appropriate potential at which the molecules released by the cell can be oxidized) a carbon fiber microelectrode at the top of the cell, each exocytotic event is detected as an amperometric spike. More particularly, a portion of these spikes has previously been shown to present a foot, i.e. a small pedestal of current that precedes the spike itself. Among the important number of works dealing with the monitoring of exocytosis by amperometry under different conditions, only a few studies focus on amperometric spikes with a foot. In this work, by coupling our previous and recent experiments on chromaffin cells (that release catecholamines after stimulation) with literature data, we bring more light on what an amperometric foot and particularly its features, represents.
- Published
- 2007
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6. pH modulation of large conductance potassium channel from adrenal chromaffin granules.
- Author
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Hordejuk R, Lobanov NA, Kicinska A, Szewczyk A, and Dolowy K
- Subjects
- Adrenal Medulla cytology, Animals, Cattle, Chromaffin Granules chemistry, Electric Conductivity, Hydrogen-Ion Concentration, Ion Transport physiology, Lipid Bilayers chemistry, Membrane Potentials physiology, Rubidium Radioisotopes analysis, Rubidium Radioisotopes metabolism, Adrenal Medulla physiology, Chromaffin Granules physiology, Potassium Channels physiology
- Abstract
We report here that large conductance K(+) selective channel in adrenal chromaffin granules is controlled by pH. We measured electrogenic influx of (86)Rb(+) into chromaffin granules prepared from bovine adrenal gland medulla. The (86)Rb(+) influx was inhibited by acidic pH. Purified chromaffin granule membranes were also fused with planar lipid bilayer. A potassium channel with conductance of 432+/-9 pS in symmetric 450 mM KCl was observed after reconstitution into lipid bilayer. The channel activity was unaffected by charybdotoxin, a blocker of the Ca(2+)-activated K(+) channel of large conductance. It was observed that acidification to pH 6.4 cis side of the membrane lowered the channel open probability and single channel conductance. Whereas only weak influence on the single channel current amplitude and open probability were observed upon lowering of the pH at the trans side. We conclude that a pH-sensitive large conductance potassium channel operates in the chromaffin granule membrane.
- Published
- 2004
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7. Characterization and location of post-translational modifications on chromogranin B from bovine adrenal medullary chromaffin granules.
- Author
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Gasnier C, Lugardon K, Ruh O, Strub JM, Aunis D, and Metz-Boutigue MH
- Subjects
- Amino Acid Sequence, Amino Acids, Acidic, Animals, Cattle, Chromogranins chemistry, Chromogranins genetics, Chromogranins isolation & purification, Molecular Sequence Data, Molecular Weight, Mutation, Protein Structure, Tertiary, Proteome, Proteomics methods, Sequence Analysis, Protein, Sequence Homology, Amino Acid, Adrenal Medulla chemistry, Chromaffin Granules chemistry, Chromogranins metabolism, Protein Processing, Post-Translational
- Abstract
Bovine chromoganin B (CGB)/secretogranin I, an acidic protein with a sequence of 626 residues and an isoelectric point of 5.2 is a major member of the chromogranin/secretogranin (CG/Sg) family. The difference between the theoretical molecular mass (76 kDa) and the value estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis results from post-translational modifications (glycosylation, phosphorylation and sulfation) and from the abundance of acidic residues (D 4.6%, and E 16.5%). Although the sequence of CGB is known, the structural analyses of the post-translational modifications have so far not been carried out. In the present study, using a combination of proteomic techniques including two-dimensional gel electrophoresis, Western blot, high-performance liquid chromatography purification, enzymatic digestion, sequencing, carbohydrate analysis, matrix-assisted laser desorption/ionization-time of flight and liquid chromatography mass spectrometry analysis, we have located 18 post-translational modifications on bovine CGB, isolated from adrenal medulla chromaffin granules. Furthermore, we have identified at the molecular level the presence of a mutation M/V on position 577 of natural CGB. All together these data reflect the complex structure of this protein marker of the neuroendocrine system.
- Published
- 2004
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8. A common mechanism for the regulation of vesicular SNAREs on phospholipid membranes.
- Author
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Hu K, Rickman C, Carroll J, and Davletov B
- Subjects
- Animals, Brain, Cattle, Chromaffin Granules chemistry, Liposomes chemistry, Membrane Proteins antagonists & inhibitors, Membrane Proteins metabolism, R-SNARE Proteins, Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins, Synaptophysin metabolism, Synaptophysin physiology, Carrier Proteins physiology, Lipid Bilayers chemistry, Membrane Proteins physiology, Phospholipids chemistry, Synaptic Vesicles chemistry, Vesicular Transport Proteins
- Abstract
The SNARE (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor) family of proteins is essential for membrane fusion in intracellular traffic in eukaryotic organisms. v-SNAREs (vesicular SNAREs) must engage target SNAREs in the opposing membrane to form the fusogenic SNARE complex. Temporal and spatial control of membrane fusion is important for many aspects of cell physiology and may involve the regulation of the SNAREs resident on intracellular membranes. Here we show that the v-SNARE synaptobrevin 2, also known as VAMP (vesicle-associated membrane protein) 2, is restricted from forming the SNARE complex in chromaffin granules from adrenal medullae to the same degree as in brain-purified synaptic vesicles. Our analysis indicates that the previously reported synaptophysin-synaptobrevin interaction is not likely to be involved in regulation of the v-SNARE. Indeed, the restriction can be reproduced for two distinct v-SNARE homologues, synaptobrevin 2 and cellubrevin/VAMP3, by reconstituting them in pure liposomal membranes. Overall, our data uncover a common mechanism for the control of SNARE engagement where intact phospholipid membranes rather than proteins down-regulate vesicular SNAREs in different cellular organelles.
- Published
- 2004
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9. Secretory vesicles membrane area is regulated in tandem with quantal size in chromaffin cells.
- Author
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Gong LW, Hafez I, Alvarez de Toledo G, and Lindau M
- Subjects
- Animals, Catecholamines analysis, Cattle, Cells, Cultured, Chromaffin Cells chemistry, Chromaffin Cells physiology, Chromaffin Granules drug effects, Exocytosis, Intracellular Membranes ultrastructure, Levodopa pharmacology, Neurotransmitter Agents analysis, Patch-Clamp Techniques, Reserpine pharmacology, Secretory Vesicles drug effects, Chromaffin Cells ultrastructure, Chromaffin Granules chemistry, Chromaffin Granules ultrastructure, Secretory Vesicles chemistry, Secretory Vesicles ultrastructure
- Abstract
The number of transmitter molecules released in a quantal event can be regulated, and recent studies suggest that the modulation of quantal size is associated with corresponding changes in vesicle volume (Colliver et al., 2000; Pothos et al., 2002). If so, this could occur either by distension of the vesicle membrane or by incorporation and removal of vesicle membrane. We performed simultaneous measurements of vesicle membrane area and catecholamine release in individual quantal events from chromaffin cells using cell-attached patch amperometry. Cells were treated with reserpine, a vesicular monoamine transport blocker that decreases quantal size, or l-dopa, a catecholamine precursor that increases quantal size. We show that decrease and increase in quantal size are associated with a respective decrease and increase in vesicle membrane area. These results point to a novel mechanism of vesicle membrane dynamics by which vesicles physically change their membrane area in response to changes in transmitter content such that the intravesicular concentration of transmitter is maintained.
- Published
- 2003
10. Mechanisms underlying neuronal death induced by chromogranin A-activated microglia.
- Author
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Ciesielski-Treska J, Ulrich G, Chasserot-Golaz S, Zwiller J, Revel MO, Aunis D, and Bader MF
- Subjects
- Animals, Apoptosis drug effects, Cattle, Cell Death drug effects, Cells, Cultured, Chromaffin Granules chemistry, Chromogranin A, Chromogranins isolation & purification, Coculture Techniques, Culture Media, Conditioned, Cytochrome c Group analysis, DNA Fragmentation, Enzyme Inhibitors pharmacology, Fas Ligand Protein, Imidazoles pharmacology, Kinetics, Membrane Glycoproteins immunology, Membrane Glycoproteins physiology, Microglia cytology, Microglia drug effects, Mitochondria physiology, Mitogen-Activated Protein Kinases metabolism, Neurons cytology, Neurons drug effects, Neurotoxins, Phosphatidylserines metabolism, Pyridines pharmacology, Rats, Time Factors, fas Receptor immunology, fas Receptor physiology, p38 Mitogen-Activated Protein Kinases, Apoptosis physiology, Chromogranins pharmacology, Microglia physiology, Neurons physiology
- Abstract
The neurotoxic effects of activated microglia in neurodegenerative diseases are well established. We recently provided evidence that chromogranin A (CGA), a multifunctional protein localized in dystrophic neurites and in senile plaques, induces an activated phenotype and secretion of neurotoxins by rat microglia in culture. In the present study, we focused on the mechanisms underlying neuronal degeneration triggered by CGA-activated microglia. We found that neuronal death exhibits apoptotic features, characterized by the externalization of phosphatidylserine and the fragmentation of DNA. Microglial neurotoxins markedly stimulate the phosphorylation and activity of neuronal p38 mitogen-activated protein kinase and provoke the release of mitochondrial cytochrome c, which precedes apoptosis. Inhibition of p38 kinase with SB 203580 partially protects neurons from death induced by CGA-activated microglia. Furthermore, neurons are also protected by Fas-Fc, which antagonizes the interactions between the death receptor Fas and its ligand FasL and by cell-permeable peptides that inhibit caspases 8 and 3. Thus, CGA triggers the release of microglial neurotoxins that mobilize several death-signaling pathways in neurons. Our results further support the idea that CGA, which is up-regulated in many neuropathologies, represents a potent endogeneous inflammatory factor possibly responsible for neuronal degeneration.
- Published
- 2001
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11. Chromaffin granule membranes contain at least three heme centers: direct evidence from EPR and absorption spectroscopy.
- Author
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Kamensky YA and Palmer G
- Subjects
- Adrenal Medulla chemistry, Adrenal Medulla ultrastructure, Animals, Ascorbic Acid chemistry, Cattle, Circular Dichroism, Electron Spin Resonance Spectroscopy, Intracellular Membranes chemistry, Intracellular Membranes ultrastructure, Oxidation-Reduction, Spectrum Analysis, Chromaffin Granules chemistry, Cytochrome b Group chemistry, Heme chemistry
- Abstract
Low-temperature electron paramagnetic resonance (EPR) spectroscopy, circular dichroism and two-component redox titration have previously provided evidence for two different ascorbate-reducible heme centers in cytochrome b(561) present in chromaffin granule membranes. These species have now been observed by room and liquid nitrogen temperature absorption spectroscopy. The visualization of these heme centers becomes possible as a consequence of utilizing chromaffin granule membranes prepared by a mild procedure. Additionally, a new redox center, not reducible by ascorbate, was discovered by both EPR and absorption spectroscopy. It constitutes about 15% of the heme absorbance of chromaffin membranes at 561 nm and has EPR characteristics of a well-organized highly axial low-spin heme center (thus making it unlikely that it is a denatured species). This species is either an alternative form of one of the hemes of cytochrome b(561) that has a very low redox potential or a b-type cytochrome distinct from b(561).
- Published
- 2001
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12. Identification and characterization of novel chromogranin B-derived peptides from porcine chromaffin granules by liquid chromatography/electrospray tandem MS.
- Author
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Wang Z, Vandenberghe I, Depreitere J, Devreese B, Clerens S, Nouwen EJ, Van Beeumen J, and De Potter W
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Chromatography, Affinity, Chromogranin B, DNA, Complementary genetics, Gas Chromatography-Mass Spectrometry methods, Molecular Sequence Data, Protein Processing, Post-Translational, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Swine, Chromaffin Granules chemistry, Chromogranins chemistry, Peptide Fragments isolation & purification
- Abstract
Chromogranin B (CgB) is a regulated secretory protein that is stored in endocrine and neuroendocrine cells. It can be processed proteolytically to small peptide fragments. In the present study three proteolytic products of porcine CgB were obtained after size-exclusion, immunoaffinity, and reversed-phase chromatography, and then identified by electrospray tandem MS. One novel peptide was identified as S586-R602 (SR-17) and is phosphorylated at one or two serine residues. Another novel peptide H603-Q636 (HQ-34), with molecular mass 3815.56 Da, was found to be oxidized at the methionine residue. In addition, a secretolytin-like peptide fragment (KR-11), which is two amino acids shorter than the bovine secretolytin, was found. This is the first report that the C-terminal region of CgB, the homologue of human CCB, is proteolytically processed further into three small peptide fragments.
- Published
- 2001
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13. Presence of dynamin--syntaxin complexes associated with secretory granules in adrenal chromaffin cells.
- Author
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Galas MC, Chasserot-Golaz S, Dirrig-Grosch S, and Bader MF
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- Adrenal Glands cytology, Animals, Cattle, Cells, Cultured, Chelating Agents pharmacology, Chromaffin Cells cytology, Chromaffin Granules chemistry, Detergents chemistry, Dimerization, Dynamin I, Dynamins, Fluorescent Antibody Technique, Guanosine 5'-O-(3-Thiotriphosphate) pharmacology, Guanosine Triphosphate metabolism, Guanosine Triphosphate pharmacology, Intracellular Membranes chemistry, Intracellular Membranes metabolism, Precipitin Tests, Protein Binding drug effects, Qa-SNARE Proteins, Adrenal Glands metabolism, Chromaffin Cells metabolism, Chromaffin Granules metabolism, GTP Phosphohydrolases metabolism, Membrane Proteins metabolism
- Abstract
Dynamin proteins have been implicated in many aspects of endocytosis, including clathrin-mediated endocytosis, internalization of caveolae, synaptic vesicle recycling, and, more recently, vesicular trafficking to and from the Golgi complex. To provide further insight into the function(s) of dynamin in neuroendocrine cells, we have examined its intracellular distribution in cultured chromaffin cells by subcellular fractionation, immunoreplica analysis, and confocal immunofluorescence. We found that dynamin, presumably the dynamin-2 isoform, is associated specifically with the membrane of purified secretory chromaffin granules. Oligomerization state analysis by sucrose density velocity gradients indicated that the granule-associated dynamin is in a monomeric form. Immunoprecipitation experiments coupled to double-labeling immunofluorescence cytochemistry revealed that the granular dynamin is associated with a syntaxin component that is not involved in the granule-bound SNARE complex. The possibility that dynamin participates in the coupling of the exocytotic and endocytotic reaction through the building of a granular membrane subset of proteins is discussed.
- Published
- 2000
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14. Molecular cloning of endopin 1, a novel serpin localized to neurosecretory vesicles of chromaffin cells. Inhibition of basic residue-cleaving proteases by endopin 1.
- Author
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Hwang SR, Steineckert B, Yasothornsrikul S, Sei CA, Toneff T, Rattan J, and Hook VY
- Subjects
- Adrenal Medulla chemistry, Amino Acid Sequence, Animals, Base Sequence, Cattle, Chromaffin Cells metabolism, Chromaffin Granules chemistry, Chromaffin Granules metabolism, Cloning, Molecular, Cysteine Endopeptidases metabolism, DNA, Complementary chemistry, DNA, Complementary genetics, Endopeptidases metabolism, Enkephalin, Methionine metabolism, Enkephalins metabolism, Fluorescent Antibody Technique, Gene Expression, Glycoproteins analysis, Hydrolysis, Molecular Sequence Data, Protease Inhibitors, Protein Precursors metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins physiology, Sequence Alignment, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Serpins analysis, Serpins physiology, Trypsin metabolism, Chromaffin Cells chemistry, Neurosecretory Systems chemistry, Serpins genetics
- Abstract
Serpins represent a diverse class of endogenous protease inhibitors that regulate important biological functions. In consideration of the importance of regulated proteolysis within secretory vesicles for the production of peptide hormones and neurotransmitters, this study revealed the molecular identity of a novel serpin, endopin 1, that is localized to neurosecretory vesicles of neuropeptide-containing chromaffin cells (chromaffin granules). Endopin 1 of 68-70 kDa was present within isolated chromaffin granules. Stimulated cosecretion of endopin 1 with chromaffin granule components, [Met]enkephalin and a cysteine protease known as "prohormone thiol protease," demonstrated localization of endopin 1 to functional secretory vesicles. Punctate, discrete immunofluorescence cellular localization of endopin 1 in chromaffin cells was consistent with its secretory vesicle localization. Endopin 1 contains a unique reactive site loop with Arg as the predicted P1 residue, suggesting inhibition of basic residue-cleaving proteases; indeed, trypsin was potently inhibited (K(i(app)) of 5 nM), and plasmin was moderately inhibited. Although endopin 1 possesses homology with alpha(1)-antichymotrypsin, chymotrypsin was not inhibited. Moreover, endopin 1 inhibited the chromaffin granule prohormone thiol protease (involved in proenkephalin processing). These results suggest a role for the novel serpin, endopin 1, in regulating basic residue-cleaving proteases within neurosecretory vesicles of chromaffin cells.
- Published
- 1999
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15. Chromogranin A from bovine adrenal medulla: molecular characterization of glycosylations, phosphorylations, and sequence heterogeneities by mass spectrometry.
- Author
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Bauer SH, Zhang XY, Van Dongen W, Claeys M, and Przybylski M
- Subjects
- Amino Acid Sequence, Animals, Cattle, Chromogranin A, Chromogranins genetics, Chromogranins isolation & purification, Disulfides analysis, Glycosylation, Molecular Sequence Data, Peptide Fragments chemistry, Peptide Fragments isolation & purification, Peptide Mapping, Phosphorylation, Protein Conformation, Protein Processing, Post-Translational, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods, Spectrometry, Mass, Secondary Ion methods, Trypsin, Adrenal Medulla chemistry, Chromaffin Granules chemistry, Chromogranins chemistry
- Abstract
Chromogranin A (CGA) is a member of a family of acidic glycoproteins present in endocrine and neuroendocrine tissues. One of its suggested physiological roles is being a precursor molecule for several peptide hormons. Further interest in this protein has recently originated from its potential role in pathophysiological processes of Alzheimer's disease. The concentration of CGA in the brain has been used for diagnosis of this disease, and CGA as an insoluble deposit has been found in the extracellular beta-amyloid plaques. By developing a new purification procedure we were able to isolate abundant CGA in high purity from bovine chromaffin cells. A MALDI-MS analysis of the intact protein revealed a heterogeneous molecular mass of ca. 50 kDa, indicating several structure modifications. By use of several subsequent proteolytic/chemical cleavage steps, HPLC isolation, a newly developed deglycosylation procedure, and several MS and MS-MS fragmentation approaches, the complete primary structure of CGA including four sequence heterogeneities, two O-glycosylations, five phosphorylations, and one disulfide bridge could be characterized. For both glycans six different forms could be identified. Ser167 was found to be mainly glycosylated by a trisaccharide, and Thr231 was found to be mainly glycosylated by a tetrasaccharide. Ser81, Ser124, and Ser297 residues were partially phosphorylated, whereas Ser372 and Ser377 were found completely phosphorylated. Sequence heterogeneities were identified in positions 293 (H/R), 301 (K/E), and 373 (Q/R) and at the partly missing C-terminal residue. Furthermore, a disulfide bridge between Cys17 and Cys38 was ascertained., (Copyright 1999 Academic Press.)
- Published
- 1999
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16. Alpha1-antichymotrypsin-like proteins I and II purified from bovine adrenal medulla are enriched in chromaffin granules and inhibit the proenkephalin processing enzyme "prohormone thiol protease".
- Author
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Hook VY, Tezapsidis N, Hwang SR, Sei C, Byrne M, and Yasothornsrikul S
- Subjects
- Adrenal Medulla ultrastructure, Animals, Cattle, Chymotrypsin metabolism, Enkephalin, Methionine analysis, Enkephalins metabolism, Fluorescent Antibody Technique, Isoelectric Point, Protein Precursors metabolism, Serine Proteinase Inhibitors pharmacology, alpha 1-Antichymotrypsin pharmacology, Adrenal Medulla chemistry, Chromaffin Granules chemistry, Cysteine Endopeptidases metabolism, Serine Proteinase Inhibitors isolation & purification, alpha 1-Antichymotrypsin isolation & purification
- Abstract
Proteolytic processing of inactive proenkephalin and proneuropeptides is essential for the production of biologically active enkephalins and many neuropeptides. The incomplete processing of proenkephalin in adrenal medulla suggests that endogenous protease inhibitors may inhibit proenkephalin processing enzymes. This study demonstrates the isolation and characterization of two isoforms of adrenal medullary alpha1-antichymotrypsin (ACT), referred to as ACT-like proteins I and II, which are colocalized with enkephalin in chromaffin granules and which inhibit the proenkephalin processing enzyme known as prohormone thiol protease (PTP). Subcellular fractionation demonstrated enrichment of 56- and 60-kDa ACT-like proteins I and II, respectively, to enkephalin-containing chromaffin granules (secretory vesicles). Immunofluorescence cytochemistry of chromaffin cells indicated a discrete, punctate pattern of ACT immunostaining that resembles that of [Met]enkephalin that is stored in secretory vesicles. Chromatography of adrenal medullary extracts through DEAE-Sepharose and chromatofocusing resulted in the separation of ACT-like proteins I and II that possess different isoelectric points of 5.5 and 4.0, respectively. The 56-kDa ACT-like protein I was purified to apparent homogeneity by Sephacryl S200 chromatography; the 60-kDa ACT-like protein II was isolated by butyl-Sepharose, Sephacryl S200, and concanavalin A-Sepharose columns. The proenkephalin processing enzyme PTP was potently inhibited by ACT-like protein I, with a K(i,app) of 35 nM, but ACT-like protein II was less effective. ACT-like proteins I and II had little effect on chymotrypsin. These results demonstrate the biochemical identification of two secretory vesicle ACT-like proteins that differentially inhibit PTP. The colocalization of the ACT-like proteins and PTP within chromaffin granules indicates that they could interact in vivo. Results from this study suggest that these ACT-like proteins may be considered as candidate inhibitors of PTP, which could provide a mechanism for limited proenkephalin processing in adrenal medulla.
- Published
- 1999
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17. Neurotrophin activation of catecholamine storage vesicle protein gene expression: signaling to chromogranin a biosynthesis.
- Author
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Mahata SK, Mahata M, Wu H, Parmer RJ, and O'Connor DT
- Subjects
- Alkaloids pharmacology, Animals, Base Sequence, Calcium-Calmodulin-Dependent Protein Kinases antagonists & inhibitors, Calcium-Calmodulin-Dependent Protein Kinases metabolism, Chamomile, Chromaffin Cells chemistry, Chromaffin Cells drug effects, Chromaffin Cells enzymology, Chromaffin Granules drug effects, Chromaffin Granules metabolism, Chromogranin A, Chromogranins biosynthesis, Enzyme Inhibitors pharmacology, Flavonoids pharmacology, Gene Deletion, Gene Expression drug effects, Humans, Hydroxymethylglutaryl-CoA Reductase Inhibitors pharmacology, Lovastatin pharmacology, Mice, Mitogen-Activated Protein Kinase Kinases, Molecular Sequence Data, Mutagenesis physiology, Oils, Volatile pharmacology, PC12 Cells, Peptidylprolyl Isomerase genetics, Phosphatidylinositol 3-Kinases metabolism, Phosphoinositide-3 Kinase Inhibitors, Plants, Medicinal, Promoter Regions, Genetic physiology, Protein Kinase Inhibitors, Protein Kinases metabolism, Proto-Oncogene Proteins genetics, RNA, Messenger metabolism, Rats, Receptor Protein-Tyrosine Kinases genetics, Receptor, trkA, Receptors, Nerve Growth Factor genetics, Transcription, Genetic physiology, Transfection, Type C Phospholipases antagonists & inhibitors, Type C Phospholipases metabolism, Carbazoles, Catecholamines metabolism, Chromaffin Granules chemistry, Chromogranins genetics, Indoles, Nerve Growth Factors pharmacology
- Abstract
Nerve growth factor differentiates precursor cells into sympathetic neurons. Does acquisition of a "neuronal" phenotype after nerve growth factor involve biosynthesis of chromogranin A, the major soluble protein in chromaffin granule cores? Nerve growth factor activated chromogranin A gene expression 7.6-fold in PC12 pheochromocytoma cells, and similarly activated PC12-transfected mouse, rat or human chromogranin A promoter/reporter constructs. Chromogranin A promoter 5'-deletions narrowed the nerve growth factor response element to a region from - 77 to - 61 bp upstream of the cap site, a region containing the chromogranin A cyclic AMP response element (TGACGTAA). Three different site-directed mutations of the cyclic AMP response element each reduced the nerve growth factor effect by >90%. Transfer of the cyclic AMP response element to a heterologous (thymidine kinase) promoter activated that promoter approximately 5-fold after nerve growth factor, while transfer of a cyclic AMP response element point-gap mutant (TGA-GTAA) to a heterologous promoter abolished the nerve growth factor effect. These findings indicate that the cyclic AMP response element in cis is, at least in part, both necessary and sufficient to activate the chromogranin A gene. Chemical blockade of the nerve growth factor receptor TrkA or the mitogen-activated protein kinase pathway component MEK substantially diminished nerve growth factor-induced expression of chromogranin A. By contrast, the response of chromogranin A to nerve growth factor was not impaired after blockade of phospholipase C-gamma or phosphoinositide-3 kinase. Chemical blockade of TrkA, Ras, MEK or mitogen-activated protein kinase similarly inhibited nerve growth factor activation of chromogranin A. Expression of constitutively activated Ras, Raf or MEK mutants increased chromogranin A promoter activity. Expression of dominant negative (inhibitory) mutants of Sos, Ha-Ras, Rafl, mitogen-activated protein kinase, ribosomal protein S6 serine kinase II (CREB kinase) or CREB (KCREB) each inhibited the nerve growth factor-induced increase in chromogranin A promoter activity. Thus, each component of the mitogen-activated protein kinase pathway is crucially involved in relaying the nerve growth factor signal in trans to the chromogranin A gene, in the following proposed sequence: nerve growth factor --> TrkA --> Shc/Grb2/Sos --> Ras --> Raf --> MEK --> mitogen-activated protein kinase --> ribosomal protein S6 serine kinase II --> CREB cyclic AMP response element.
- Published
- 1999
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18. Release of nontransmembrane full-length Alzheimer's amyloid precursor protein from the lumenar surface of chromaffin granule membranes.
- Author
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Tezapsidis N, Li HC, Ripellino JA, Efthimiopoulos S, Vassilacopoulou D, Sambamurti K, Toneff T, Yasothornsrikul S, Hook VY, and Robakis NK
- Subjects
- Adrenal Medulla, Amyloid beta-Protein Precursor chemistry, Amyloid beta-Protein Precursor drug effects, Animals, Azirines metabolism, Biotinylation, Cattle, Cell Membrane chemistry, Cell Membrane drug effects, Cell Membrane metabolism, Chromaffin Granules chemistry, Chromaffin Granules drug effects, Cross-Linking Reagents, Humans, Iodine Radioisotopes, Membrane Proteins chemistry, Photoaffinity Labels, Trypsin pharmacology, Amyloid beta-Protein Precursor metabolism, Chromaffin Granules metabolism, Membrane Proteins metabolism
- Abstract
We previously demonstrated the presence of a soluble form of full-length Alzheimer's amyloid precursor protein (APP) in the lumen of adrenal medullary chromaffin granules (CG). Furthermore, full-length APP is released from CG membranes in vitro at pH 9.0 by an enzymatic mechanism, sensitive to protease inhibitors [Vassilacopoulou et al. (1995) J. Neurochem. 64, 2140-2146]. In this study, we found that when intact CG were subjected to exogenous trypsin, a fraction of APP was not digested, consistent with an intragranular population of APP. To examine the substrate-product relationship between membrane and soluble full-length APP, we labeled CG transmembrane APP with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID), a lipophilic probe, specific for membrane-spanning domains of proteins. APP released from the membranes at pH 9.0 was not labeled with [125I]TID. In addition, this APP was not biotinylated in intact CG. Combined, the results indicate that APP released from CG membranes derives from a unique nontransmembrane population of membrane-associated APP, located in the lumenal side of CG membranes. Dithiobis(succinimidylpropionate) (DSP) cross-linking indicated that APP in CG is situated in close proximity with other proteins, possibly with APP itself. APP complexes were also detected under nonreducing conditions, without DSP cross-linking. These results, combined with our previous studies, indicate that full-length APP within CG exists as three different populations: (I) transmembrane, (II) membrane-associated/nontransmembrane, and (III) soluble. The existence of nontransmembrane populations suggests that putative gamma-secretase cleavage sites of APP, assumed to be buried within the lipid bilayer, could be accessible to proteolysis in a soluble intravesicular environment.
- Published
- 1998
- Full Text
- View/download PDF
19. Highly sensitive and stable phosphatidylserine liposome aggregation assay for annexins.
- Author
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Lee G and Pollard HB
- Subjects
- Animals, Annexin A1 metabolism, Annexin A5 metabolism, Biochemistry economics, Biochemistry methods, Calcium metabolism, Cattle, Chromaffin Granules chemistry, Liposomes metabolism, Nephelometry and Turbidimetry methods, Phosphatidylserines metabolism, Sensitivity and Specificity, Spectrophotometry economics, Spectrophotometry standards, Annexin A1 analysis, Annexin A5 analysis, Liposomes chemistry, Phosphatidylserines chemistry, Spectrophotometry methods
- Abstract
Annexins are a gene family of Ca(2+)-dependent membrane binding proteins which interact specifically with acidic phospholipids. We describe here details of highly sensitive and precise assays for annexins I and V, utilizing turbidometric analysis of phosphatidylserine liposome aggregation. In the case of annexin I, the new assay is 7-fold more sensitive than the conventional chromaffin granule aggregation assay in terms of threshold for detection and the rate of increase of initial absorbance is 15-fold greater. Annexin V, which binds but does not aggregate liposomes, can be assayed on the basis of inhibition of Ca(2+)-dependent liposome aggregation. Further comparative advantages of the assay include lower expense and increased shelf life of the liposome reagent.
- Published
- 1997
- Full Text
- View/download PDF
20. Existence of two heme B centers in cytochrome b561 from bovine adrenal chromaffin vesicles as revealed by a new purification procedure and EPR spectroscopy.
- Author
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Tsubaki M, Nakayama M, Okuyama E, Ichikawa Y, and Hori H
- Subjects
- Animals, Cattle, Electron Spin Resonance Spectroscopy, Models, Chemical, Oxidation-Reduction, Spectrophotometry, Adrenal Medulla chemistry, Chromaffin Granules chemistry, Cytochrome b Group chemistry, Heme chemistry
- Abstract
We have established a new purification procedure of cytochrome b561 from bovine adrenomedullary chromaffin vesicles. The heme content analysis of the purified sample indicated the presence of 1.7 molecules of heme B/cytochrome b561 molecule. EPR spectroscopy of the purified enzyme in oxidized state showed that there were three types of low spin heme species. Two of them showed usual EPR signals at gz = 3.14 and gz = 2.84 arising from the same heme and were interconvertible depending on pH. The other species showed a highly anisotropic low spin signal at gz = 3.70, with a lower redox potential than the others, and a temperature-sensitive character. These properties are very similar to low potential cytochrome b (bL or b566) of the mitochondrial complex III, indicating that the gz = 3.70 species is derived from a heme component different from the one that shows the usual low spin EPR signals. Based on our new structural model, these two heme B prosthetic groups are likely to be located on both sides of the membranes in close contact with the ascorbic acid- and semidehydroascorbic acid-binding sites, respectively, to facilitate the electron transfer across the membranes. This molecular architecture may provide a structural basis for the transmembrane electron transfer catalyzed by this hemoprotein.
- Published
- 1997
- Full Text
- View/download PDF
21. NSF and SNAP are present on adrenal chromaffin granules.
- Author
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Burgoyne RD and Williams G
- Subjects
- Animals, Cattle, Cell Fractionation, Centrifugation, Density Gradient, Chromaffin Granules ultrastructure, Dopamine beta-Hydroxylase analysis, Intracellular Membranes chemistry, Intracellular Membranes ultrastructure, N-Ethylmaleimide-Sensitive Proteins, Organelles ultrastructure, Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins, Adrenal Medulla chemistry, Carrier Proteins analysis, Chromaffin Granules chemistry, Membrane Proteins analysis, Vesicular Transport Proteins
- Abstract
N-ethylmaleimide sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs) are involved in many vesicular transport steps. It has been proposed that SNAPs and NSF associate with their membrane receptors only when vesicles dock on the target membrane. Analysis of NSF and alpha-SNAP distribution in fractionation of organelles from adrenal medulla indicated that a substantial amount of both proteins distributed with chromaffin granules. Further fractionation of intact granules and lysed granule membranes showed exact overlap of NSF and alpha-SNAP distribution with chromaffin granules. These results suggest that NSF and alpha-SNAP are associated with chromaffin granules and support the idea that they function prior to docking of the granules on the plasma membrane.
- Published
- 1997
- Full Text
- View/download PDF
22. Membrane composition of adrenergic large and small dense cored vesicles and of synaptic vesicles: consequences for their biogenesis.
- Author
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Winkler H
- Subjects
- Animals, Chromaffin Granules chemistry, Membrane Proteins chemistry, Sympathetic Nervous System chemistry, Synaptic Vesicles chemistry
- Abstract
The membrane proteins of adrenergic large dense cored vesicles, in particular those of chromaffin granules, have been characterized in detail. With the exception of the nucleotide carrier all major peptides have been cloned. There has been a controversy whether these vesicles contain antigens like synaptophysin, synaptotagmin and VAMP or synaptobrevin found in high concentration in synaptic vesicles. One can now conclude that large dense core vesicles also contain these peptides although in lower concentrations. The biosynthesis of large dense core vesicles is analogous to that of other peptide secreting vesicles of the regulated pathway. One cannot yet definitely define the biosynthesis of small dense core vesicles which apparently have a very similar membrane composition to that of large dense core vesicles. They may form directly from large dense core vesicles when their membranes have been retrieved after exocytosis. These membranes may become sorted in an endosomal compartment where peptides may be deleted or added. Such an addition could be derived from synaptophysin-rich vesicles present in adrenergic axons. However small dense core vesicle peptides may also be transported axonally independent of large dense core vesicles. For proving one of these possibilities some crucial experiments have been suggested.
- Published
- 1997
- Full Text
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23. Isolation and identification of intact chromogranin A and two N-terminal processing products, vasostatin I and II, from bovine adrenal medulla chromaffin granules by chromatographic and mass spectrometric methods.
- Author
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Bauer SH, Zhang XY, Liang F, De Potter WP, Claeys M, and Przybylski M
- Subjects
- Amino Acid Sequence, Animals, Cattle, Chromaffin Cells ultrastructure, Chromaffin Granules chemistry, Chromaffin Granules metabolism, Chromatography, High Pressure Liquid, Chromogranin A, Chromogranins genetics, Chromogranins metabolism, Electrophoresis, Polyacrylamide Gel, Mass Spectrometry, Molecular Sequence Data, Peptide Fragments genetics, Vasodilator Agents analysis, Vasodilator Agents metabolism, Chromaffin Cells chemistry, Chromogranins analysis, Peptide Fragments analysis
- Abstract
Chromogranin A (CGA) is the most abundant protein of the bovine adrenal medulla and plays an important role as precursor protein of several peptides that act as modulators for endocrine cell secretory activity. Furthermore, it is presumed to play a role in the targeting of peptide hormones and neurotransmitters to granules of the regulated pathway. However, its complete primary structure and proteolytic processing have not yet been identified. This study describes a rapid and efficient procedure for the high yield isolation of bovine CGA and its N-terminal processing products, vasostatin I and II. Using the lysate from bovine adrenal medulla chromaffin granules, the soluble proteins were purified by three consecutive HPLC steps, thereby avoiding the use of buffer solutions. The protein fractions were isolated and characterized by SDS-PAGE and Western blot analysis as well as by mass spectrometry. In the latter analysis, the efficiency of matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) was demonstrated, enabling the unequivocal and sensitive characterization of proteins from crude mixtures. Sufficient amounts of pure protein were obtained by the present procedure to form the basis for detailed structural studies by spectroscopic methods and X-ray crystallography.
- Published
- 1997
- Full Text
- View/download PDF
24. Protein from chromaffin granules promotes survival of mesencephalic dopaminergic neurons by an EGF-receptor ligand-mediated mechanism.
- Author
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Krieglstein K and Unsicker K
- Subjects
- 1-Methyl-4-phenylpyridinium pharmacology, Adrenal Glands chemistry, Adrenal Glands cytology, Animals, Astrocytes chemistry, Astrocytes cytology, Astrocytes drug effects, Carbachol pharmacology, Cattle, Cell Differentiation drug effects, Cell Division drug effects, Cell Survival drug effects, Cells, Cultured chemistry, Cells, Cultured drug effects, Cells, Cultured physiology, Chromaffin Cells chemistry, Chromaffin Cells metabolism, Chromaffin Cells ultrastructure, Dopamine physiology, Dopamine Agents pharmacology, Glial Cell Line-Derived Neurotrophic Factor, Glial Fibrillary Acidic Protein analysis, Immunohistochemistry, Ligands, Mesencephalon cytology, Nerve Growth Factors pharmacology, Nerve Tissue Proteins analysis, Neurons chemistry, Neurons drug effects, Neuropeptides pharmacology, Neuroprotective Agents analysis, Neurotoxins pharmacology, Parasympathomimetics pharmacology, Proteins pharmacology, Rats, Signal Transduction drug effects, Signal Transduction physiology, Solubility, Chromaffin Granules chemistry, ErbB Receptors physiology, Neurons cytology
- Abstract
Chromaffin cells grafted to the brain of animals with experimental parkinsonism and patients with Parkinson's disease can restore nigrostriatal functions. Mechanisms underlying these beneficial effects are unknown, but may include growth factors rather than the minute amounts of dopamine (DA) liberated from chromaffin cells. We now report that protein from chromaffin granules, which release their contents by exocytosis, promotes survival and uptake of 3H-DA of mesencephalic DAergic neurons in vitro and protect against N-methylpyridinium ion toxicity. This neurotrophic effect is accompanied by cell proliferation and mediated by astroglial cells induced in these cultures. Inhibition of cell proliferation and concomitant astrogliosis by 5-fluorodeoxyuridine and alpha-aminoadipic acid abolishes the trophic effect. Two highly specific inhibitors of the epidermal growth factor receptor (EGFR) signal transduction pathway, 4,5-dianilinophthalimide (10 microM) and tyrphostin B56 (10 microM), selectively block the neurotrophic capacity of chromaffin granule protein. As expected, they also block the mitogenic effects of EGF and TGF-alpha. However, these two mitogens do not mimic the pronounced mitogenic and trophic actions of chromaffin granule protein. Culture medium conditioned by mesencephalic cells pretreated with chromaffin granule protein promotes survival of DAergic neurons without increasing numbers of astroglial cells. The effective molecule is unlikely to be glial cell line-derived neurotrophic factor, whose mRNA is not detectable in cultures treated with chromaffin granule protein. We conclude that chromaffin granules contain a putatively novel growth factor, which signals through the EGFR and may be responsible for the known protective and restorative actions of chromaffin cell grafts to the lesioned nigrostriatal system.
- Published
- 1997
25. Vacuolar H(+)-ATPase: from mammals to yeast and back.
- Author
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Nelson N and Klionsky DJ
- Subjects
- Animals, Cattle, Chromaffin Granules chemistry, Chromaffin Granules metabolism, Cloning, Molecular, Eukaryotic Cells chemistry, Eukaryotic Cells metabolism, Fungal Proteins chemistry, Fungal Proteins metabolism, Gene Expression genetics, Models, Molecular, Proton-Translocating ATPases genetics, Proton-Translocating ATPases metabolism, Vacuoles metabolism, Proton-Translocating ATPases chemistry
- Abstract
Vacuolar H(+)-adenosine triphosphatase (V-ATPase) is composed of distinct catalytic (V1) and membrane (V0) sectors containing several subunits. The biochemistry of the enzyme was mainly studied in organelles from mammalian cells such as chromaffin granules and clathrin-coated vesicles. Subsequently, mammalian cDNAs and yeast genes encoding subunits of V-ATPase were cloned and sequenced. The sequence information revealed the relation between V- and F-ATPase that evolved from a common ancestor. The isolation of yeast genes encoding subunits of V-ATPase opened an avenue for molecular biology studies of the enzyme. Because V-ATPase is present in every known eukaryotic cell and provides energy for vital transport systems, it was anticipated that disruption of genes encoding V-ATPase subunits would be lethal. Fortunately, yeast cells can survive the absence of V-ATPase by 'drinking' the acidic medium. So far only yeast cells have been shown to be viable without an active V-ATPase. In contrast to yeast, mammalian cells may have more than one gene encoding each of the subunits of the enzyme. Some of these genes encode tissue- and/or organelle-specific subunits. Expression of these specific cDNAs in yeast cells may reveal their unique functions in mammalian cells. Following the route from mammals to yeast and back may prove useful in the study of many other complicated processes.
- Published
- 1996
- Full Text
- View/download PDF
26. Antibacterial activity of glycosylated and phosphorylated chromogranin A-derived peptide 173-194 from bovine adrenal medullary chromaffin granules.
- Author
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Strub JM, Goumon Y, Lugardon K, Capon C, Lopez M, Moniatte M, Van Dorsselaer A, Aunis D, and Metz-Boutigue MH
- Subjects
- Amino Acid Sequence, Animals, Anti-Bacterial Agents pharmacology, Cattle, Chromatography, High Pressure Liquid, Chromogranin A, Chromogranins pharmacology, Glycosylation, Molecular Sequence Data, Peptide Fragments pharmacology, Phosphorylation, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Adrenal Medulla chemistry, Anti-Bacterial Agents chemistry, Chromaffin Granules chemistry, Chromogranins metabolism, Peptide Fragments metabolism
- Abstract
Recently, we have isolated from bovine chromaffin granules and identified two natural peptides possessing antibacterial activity: secretolytin (chromogranin B 614-626) and enkelytin (proenkephalin-A 209-237). Here, we characterize a large natural fragment, corresponding to chromogranin A 79-431, that inhibits growth of both Gram-positive and Gram-negative bacteria. The aim of the present work was to determine the shortest active peptide located in the 79-431 chromogranin A region. Three peptides, which shared the same 173-194 chromogranin A sequence (YPGPQAKEDSEGPSQGPASREK) but differed in post-translational modifications, including O-glycosylation and tyrosine phosphorylation, were isolated. A detailed study using microsequencing and mass spectrometry allowed us to correlate their antibacterial activity with these post-translational modifications. The chromogranin A precursor fragment (79-431) and the active glycosylated and phosphorylated peptides were, respectively, named prochromacin and chromacin (P, G, and PG for phosphorylated, glycosylated, and phosphorylated-glycosylated form).
- Published
- 1996
- Full Text
- View/download PDF
27. Immunocytochemical localization of NCAM and catecholamine-synthesizing enzymes in rabbit intra- and extra-adrenal chromaffin tissue.
- Author
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Moftaquir A, Langley K, and Boutroy MJ
- Subjects
- Adrenal Medulla chemistry, Animals, Antibody Specificity, Chromogranin A, Chromogranins analysis, Immunohistochemistry, Rabbits, Adrenal Glands chemistry, Chromaffin Granules chemistry, Neural Cell Adhesion Molecules analysis
- Abstract
The expression of the neural cell adhesion molecule, chromogranin A, and catecholamine-synthesizing enzymes (tyrosine hydroxylase and phenylethanolamine N-methyl transferase) in adrenal medulla and para-aortic bodies (paraganglia) of the adult rabbit, was studied by immunofluorescence. The specificity of the neural cell adhesion molecule antibody employed was demonstrated on rabbit tissue by immunoblotting. Neural cell adhesion molecule was found to be expressed not only by adrenal medullary cells but also by extra-adrenal chromaffin cells present in para-aortic bodies. These paraganglionic cells were as intensely immunolabelled for chromogranin A as adrenal medullary chromaffin cells. They were also labelled for the catecholamine-synthesizing enzymes tested here. However, their levels of the adrenalin-synthesizing enzyme phenylethanolamine N-methyl transferase were lower than those of medullary chromaffin cells.
- Published
- 1996
- Full Text
- View/download PDF
28. Adrenal chromaffin cells contain functionally different SNAP-25 monomers and SNAP-25/syntaxin heterodimers.
- Author
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Höhne-Zell B and Gratzl M
- Subjects
- Adrenal Glands chemistry, Animals, Bacterial Proteins, Botulinum Toxins, Type A pharmacology, Cattle, Cell Membrane chemistry, Cells, Cultured, Centrifugation, Density Gradient, Chromaffin Cells ultrastructure, Chromaffin Granules ultrastructure, Dimerization, Exocytosis drug effects, Immunohistochemistry, Intracellular Membranes chemistry, Membrane Proteins chemistry, Membrane Proteins metabolism, Microscopy, Immunoelectron, Molecular Weight, Nerve Tissue Proteins chemistry, Nerve Tissue Proteins metabolism, Norepinephrine metabolism, Qa-SNARE Proteins, R-SNARE Proteins, Sodium Dodecyl Sulfate pharmacology, Streptolysins pharmacology, Synaptosomal-Associated Protein 25, Chromaffin Cells chemistry, Chromaffin Granules chemistry, Membrane Proteins analysis, Nerve Tissue Proteins analysis
- Abstract
Syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa), associated with the neuronal plasmalemma, and synaptobrevin, a membrane protein of synaptic vesicles, are essential components of the exocytotic apparatus of synaptic vesicles. All three can be proteolytically cleaved by tetanus and/or botulinum neurotoxins. As a consequence of their cleavage, exocytosis of neurotransmitters is blocked. In adrenal chromaffin cells botulinum neurotoxin A only incompletely inhibits exocytosis. This incomplete inhibition of exocytosis is associated with only partial cleavage of SNAP-25 by the toxin, indicating that distinct pools of SNAP-25 may exist in chromaffin cells which differ in their sensitivities to botulinum neurotoxin A. In line with this result we localized SNAP-25 by immunogold electron microscopy not only to the plasmalemma but also to the chromaffin vesicle membrane. Moreover, in addition to SNAP-25 monomers, stable SNAP-25/syntaxin heterodimers were found in chromaffin cells. Subfractionation studies revealed the presence of SNAP-25/syntaxin heterodimers in an enriched fraction of chromaffin vesicles. This complex proved to be stable in SDS, and SNAP-25 within heterodimers was resistant to proteolytic attack by botulinum neurotoxin A. We suggest that these preexisting heterodimers may serve as receptors of soluble NSF attachment proteins (SNAP receptors) during chromaffin vesicle exocytosis.
- Published
- 1996
- Full Text
- View/download PDF
29. SNAP-25 is present on chromaffin granules and acts as a SNAP receptor.
- Author
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Tagaya M, Genma T, Yamamoto A, Kozaki S, and Mizushima S
- Subjects
- Animals, Antibodies, Monoclonal immunology, Antibodies, Monoclonal metabolism, Antigens, Surface analysis, Antigens, Surface immunology, Cattle, Cell Fractionation, Chromaffin Granules metabolism, Exocytosis physiology, Immunoblotting, Immunohistochemistry, Membrane Proteins analysis, Membrane Proteins immunology, Microscopy, Electron, Nerve Tissue Proteins analysis, Nerve Tissue Proteins immunology, Precipitin Tests, R-SNARE Proteins, SNARE Proteins, Synaptosomal-Associated Protein 25, Syntaxin 1, Chromaffin Granules chemistry, Membrane Proteins metabolism, Nerve Tissue Proteins metabolism, Vesicular Transport Proteins
- Abstract
SNAP-25 is located on the plasma membrane and essential for exocytosis of neurotransmitters. It was suggested that SNAP-25 and syntaxin 1 via the interaction with VAMP-2 located on synaptic vesicles mediate the docking of the vesicles with the plasma membrane. In the present study, by means of biochemical and morphological analyses, we showed that SNAP-25 is present on chromaffin granules as well as on the plasma membrane. Reconstitution and immunoprecipitation analyses revealed that SNAP-25 on chromaffin granules has essentially the same properties as does SNAP-25 on the plasma membrane.
- Published
- 1996
- Full Text
- View/download PDF
30. Proteins from chromaffin granules promote survival of dorsal root ganglionic neurons: comparison with neurotrophins.
- Author
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Krieglstein K and Unsicker K
- Subjects
- Animals, Antibodies, Blocking pharmacology, Cattle, Cell Survival drug effects, Chick Embryo, Chromaffin Cells chemistry, Chromaffin Cells ultrastructure, Chromaffin Granules immunology, Cytokines pharmacology, Drug Synergism, ErbB Receptors metabolism, Ligands, Neurons, Afferent cytology, Neuroprotective Agents pharmacology, Neurotrophin 3, Neutralization Tests, Chromaffin Granules chemistry, Ganglia, Spinal cytology, Nerve Growth Factors pharmacology, Neurons, Afferent drug effects, Proteins pharmacology, Transforming Growth Factor beta immunology
- Abstract
Neurotrophins are established survival and differentiation factors for sensory dorsal root ganglionic (DRG) neurons. We have previously shown that proteins from the secretory granules of adrenal chromaffin cells have a capacity to promote the survival of cultured chick DRG neurons. Using DRG neurons from embryonic day (E) 8 chick embryos we show now that this material is (i) as effective as nerve growth factor (NGF), (ii) additive to NGF, neurotrophin-3, or -4, (iii) unlikely to be a neurotrophin, since the survival promoting effect can not be blocked by K252b, a specific inhibitor of the signal transduction pathways of neurotrophin high affinity receptors, (iv) partially blockable by antibodies to transforming growth factor-beta (TGF-beta) 1/2/3, and (v) more potent than any other out of 30 cytokines tested individually, including fibroblast growth factor (FGF)-5, epidermal growth factor (EGF), TGF-alpha, platelet-derived growth factor (PDGF)-AB, insulin-like growth factors (IGF)-I and -II, leukemia inhibitory factor (LIF), TGF-beta, glial cell line-derived neurotrophic factor (GDNF), stem cell factor, granulocyte-colony stimulating factor (G-CSF), oncostatin M, tumor necrosis factor (TNF)-alpha, and interleukins (IL)-1 through -12. We conclude that chromaffin cells, which are known to receive a sensory innervation, can provide (a) trophic factor(s), which, in addition to neurotrophins, may be relevant for the maintenance of DRG neurons.
- Published
- 1996
- Full Text
- View/download PDF
31. A chromaffin cell-derived protein induces the NADPH-diaphorase phenotype in cultured rat spinal cord neurons.
- Author
-
Huber KA, Krieglstein K, and Unsicker K
- Subjects
- Animals, Carbazoles pharmacology, Cattle, Cells, Cultured enzymology, Chromaffin Granules metabolism, Enzyme Inhibitors pharmacology, Fetus enzymology, Indole Alkaloids, NADPH Dehydrogenase genetics, Phenotype, Rats, Rats, Wistar, Time Factors, Chromaffin Granules chemistry, NADPH Dehydrogenase analysis, Neurons enzymology, Spinal Cord cytology
- Abstract
We have recently demonstrated that neurotrophins induce reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity in cultured spinal cord neurons. One prominent neuron population of the spinal cord expressing NADPH-diaphorase activity in vivo are preganglionic sympathetic neurons, including those innervating the adrenal medulla. These neurons receive trophic support from their target. We have shown previously that chromaffin cells contain as yet unidentified neurotrophic molecules, which may include releasable factors relevant for the survival and differentiation of developing preganglionic sympathetic neurons. We have studied the influence of proteins derived from bovine chromaffin cells and released by nicotine on NADPH-diaphorase expression in spinal cord cultures established from 16-day-old rat embryos. At this embryonic age, NADPH-diaphorase activity becomes apparent in the spinal cord and predominantly expressed in sympathetic nuclei. Similar to brain-derived neurotrophic factor and neurotrophin-4, a heat- and trypsin-sensitive component from chromaffin cells contained in granule preparations up-regulated the number of NADPH-diaphorase-positive neurons in spinal cord cultures. Combined application of this activity and neurotrophin-4 resulted in an additive effect, indicating that the effect of the chromaffin cell-derived active component is not mediated by one of the trk B ligands. This was confirmed by co-treatment studies with the trk-signalling pathway inhibitor K252b, which did not inhibit the effect of the chromaffin cell-derived protein(s). Further studies revealed that NADPH-diaphorase reactivity is inducible in spinal cord neurons at any time point throughout the entire culture period of six days, suggesting de novo induction of the enzyme rather than a survival-promoting effect of the activity from chromaffin cells. Culture supernatants from nicotine-stimulated bovine chromaffin cells induced NADPH-diaphorase-positive neurons at the same magnitude as the material obtained from chromaffin granule preparations. Our data suggest that chromaffin cell-derived proteins are capable of up-regulating NADPH-diaphorase activity or to induce de novo this transmitter phenotype in neuron populations of the spinal cord, which may include preganglionic sympathetic neurons.
- Published
- 1996
- Full Text
- View/download PDF
32. Syntaxin 1 (HPC-1) is associated with chromaffin granules.
- Author
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Tagaya M, Toyonaga S, Takahashi M, Yamamoto A, Fujiwara T, Akagawa K, Moriyama Y, and Mizushima S
- Subjects
- Amino Acid Sequence, Animals, Antigens, Surface immunology, Brain Chemistry, Cattle, Cell Fractionation, Cell Membrane chemistry, Intracellular Membranes chemistry, Microscopy, Immunoelectron, Molecular Sequence Data, Nerve Tissue Proteins immunology, Precipitin Tests, Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins, Syntaxin 1, Tissue Distribution, Adrenal Medulla chemistry, Antigens, Surface analysis, Carrier Proteins analysis, Chromaffin Granules chemistry, Membrane Proteins analysis, Nerve Tissue Proteins analysis, Vesicular Transport Proteins
- Abstract
Syntaxin 1 (HPC-1), a component of the receptor for SNAPs (soluble N-ethylmaleimide-sensitive factor attachment proteins), has been implicated in the docking and fusion of synaptic vesicles with the plasma membrane. It was reported that syntaxin 1 in rat brain and chromaffin cells (PC12) is exclusively located on the plasma membrane (Bennett, M. K., Calakos, N., and Scheller, R. H. (1992) Science 257, 255-259; Söllner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) Cell 75, 409-418). By means of biochemical and morphological analyses, we now show that syntaxin 1 is associated with chromaffin granules in the adrenal medulla. This finding raises the possibility that syntaxin 1 in chromaffin cells is a component of vesicle-SNAP receptor as well as one of target-SNAP receptor on the plasma membrane.
- Published
- 1995
- Full Text
- View/download PDF
33. Full-length and truncated Alzheimer amyloid precursors in chromaffin granules: solubilization of membrane amyloid precursor is mediated by an enzymatic mechanism.
- Author
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Vassilacopoulou D, Ripellino JA, Tezapsidis N, Hook VY, and Robakis NK
- Subjects
- Amyloid beta-Protein Precursor isolation & purification, Amyloid beta-Protein Precursor metabolism, Animals, Calcium pharmacology, Cations, Divalent, Cattle, Cysteine Endopeptidases metabolism, Electrophoresis, Polyacrylamide Gel, Endopeptidases metabolism, Hot Temperature, Hydrogen-Ion Concentration, Intracellular Membranes chemistry, Protease Inhibitors pharmacology, Serine Endopeptidases metabolism, Solubility, Zinc pharmacology, Adrenal Medulla ultrastructure, Amyloid beta-Protein Precursor analysis, Chromaffin Granules chemistry, Intracellular Membranes enzymology
- Abstract
The amyloid beta peptide (A beta) of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APPs), which are considered type I transmembrane proteins. Here we report that the soluble fraction of isolated adrenal medullary chromaffin granules (CG), a model neuronal secretory vesicle system, contains an antigen that immunochemically and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from full-length APP. A truncated APP fragment with intact A beta sequence was also detected in the soluble fraction of CG. In vitro experiments showed that full-length APP was solubilized from CG membranes at 37 degrees C as a function of pH, with a peak of activity between pH 8.5 and pH 9.0. Solubilization of full-length APP was inhibited by several protease inhibitors, including aprotinin, cystatin, and iodoacetamide, by the divalent cations Ca2+ and Zn2+, and by preheating of the membranes. These results are consistent with and suggest the involvement of an enzymatic mechanism in the solubilization of potentially amyloidogenic full-length APP. Production of A beta from a transmembrane APP predicts a proteolytic cleavage within the lipid bilayer, a site relatively inaccessible to proteases. Thus, the detected soluble, potentially amyloidogenic, full-length APP may be a substrate for the proteases producing A beta. The detection of soluble APP with intact A beta sequence in secretory vesicles is consistent with the extracellular topology of amyloid depositions.
- Published
- 1995
- Full Text
- View/download PDF
34. Coenzyme A glutathione disulfide. A potent vasoconstrictor derived from the adrenal gland.
- Author
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Schlüter H, Meissner M, van der Giet M, Tepel M, Bachmann J, Gross I, Nordhoff E, Karas M, Spieker C, and Witzel H
- Subjects
- Adrenal Medulla chemistry, Adrenal Medulla drug effects, Adrenal Medulla metabolism, Animals, Blood Pressure drug effects, Calcium analysis, Carbachol pharmacology, Cattle, Chromaffin Granules chemistry, Chromaffin Granules drug effects, Chromaffin Granules metabolism, Chromatography, High Pressure Liquid, Coenzyme A pharmacology, Hemodynamics, In Vitro Techniques, Kidney drug effects, Mass Spectrometry, Muscle, Smooth, Vascular chemistry, Muscle, Smooth, Vascular cytology, Perfusion, Rats, Spectrophotometry, Ultraviolet, Stimulation, Chemical, Vascular Resistance drug effects, Vasoconstrictor Agents pharmacology, Adrenal Glands chemistry, Coenzyme A isolation & purification, Vasoconstrictor Agents isolation & purification
- Abstract
The adrenal gland is involved in the regulation of vascular tone by secretion of vasoactive agents such as catecholamines, neuropeptide Y, or endogenous ouabain. A further potent vasoconstrictor is isolated from bovine adrenal glands and is identified by chromatography, mass spectrometry, UV spectroscopy, and enzymatic cleavage as coenzyme A glutathione disulfide (CoASSG). CoASSG is found in chromaffin granules of adrenal glands and is released from adrenal medulla slices by carbachol. At a concentration of 10(-12) mol/L CoASSG increases renal vascular resistance. Intra-aortic injection of 5 x 10(-10) mol CoASSG increases blood pressure in the intact animal. Besides its vasopressor properties, this substance potentiates the effects of angiotensin II on vascular tone. It is concluded that CoASSG could play a role in blood pressure regulation not only by direct effects but also by modulation of the action of angiotensin II.
- Published
- 1995
- Full Text
- View/download PDF
35. Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells.
- Author
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Liu Y, Schweitzer ES, Nirenberg MJ, Pickel VM, Evans CJ, and Edwards RH
- Subjects
- Adrenal Medulla chemistry, Adrenal Medulla ultrastructure, Amino Acid Sequence, Animals, CHO Cells, Cricetinae, Fluorescent Antibody Technique, Glycoproteins immunology, Male, Microscopy, Immunoelectron, Molecular Sequence Data, PC12 Cells, Rats, Rats, Sprague-Dawley, Synaptic Vesicles chemistry, Transfection, Vesicular Biogenic Amine Transport Proteins, Vesicular Monoamine Transport Proteins, Chromaffin Granules chemistry, Endocytosis, Endosomes chemistry, Glycoproteins analysis, Membrane Glycoproteins, Membrane Transport Proteins, Neuropeptides, Organelles chemistry
- Abstract
Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH-terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs.
- Published
- 1994
- Full Text
- View/download PDF
36. Ionic factors affecting the association of tyrosine hydroxylase with chromaffin granules in the adrenal medullary cell.
- Author
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Morita K, Hamano S, and Oka M
- Subjects
- Animals, Calcium pharmacology, Cattle, Chromaffin Granules chemistry, In Vitro Techniques, Osmolar Concentration, Potassium pharmacology, Sodium pharmacology, Adrenal Medulla metabolism, Cations pharmacology, Chromaffin Granules metabolism, Tyrosine 3-Monooxygenase metabolism
- Abstract
Chromaffin cells were treated with digitonin in medium containing various ions and the efflux of tyrosine hydroxylase from these permeabilized cells was then determined to elucidate a possible influence of cytoplasmic ionic environment on the association of this enzyme with the chromaffin granule. The enzyme efflux was observed with a distinct lag during exposure to low concentrations of digitonin in the medium containing isotonic sucrose. In contrast, a larger extent of the enzyme efflux was observed without any notable delay in the presence of isotonic NaCl. The results were thought to indicate the possibility that the dissociation of soluble enzyme from the granule surface within the permeabilized cells might occur in the presence of NaCl. Furthermore, the interaction between tyrosine hydroxylase and isolated chromaffin granule membranes was directly examined, and this interaction was shown to be inhibited by NaCl. However, the enzyme-granule membrane interaction was also inhibited by KCl and choline chloride. It therefore seems possible to consider that the inhibitory action of NaCl on the association of soluble enzyme with the granule may not be due to the specific action of Na+, but presumably due to the non-specific chaotropic effect of Cl-. On the other hand, the enzyme efflux was markedly reduced by the presence of Ca2+ in the permeabilizing medium, but the enzyme-granule membrane interaction was not affected by Ca2+ at the same concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1994
- Full Text
- View/download PDF
37. The post-translational processing of chromogranin A in the pancreatic islet: involvement of the eukaryote subtilisin PC2.
- Author
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Arden SD, Rutherford NG, Guest PC, Curry WJ, Bailyes EM, Johnston CF, and Hutton JC
- Subjects
- Adrenal Glands chemistry, Amino Acid Sequence, Animals, Cattle, Chromaffin Granules chemistry, Chromogranin A, Chromogranins chemistry, Cytoplasmic Granules metabolism, Immunosorbent Techniques, Insulinoma, Kinetics, Methionine metabolism, Molecular Sequence Data, Molecular Weight, Pancreatic Neoplasms, Peptide Fragments chemistry, Peptide Fragments metabolism, Proprotein Convertase 2, Rats, Sulfur Radioisotopes, Chromogranins metabolism, Islets of Langerhans metabolism, Protein Processing, Post-Translational, Subtilisins metabolism
- Abstract
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.
- Published
- 1994
- Full Text
- View/download PDF
38. Presence of laminin B chain-like protein in bovine and rat adrenal chromaffin granules.
- Author
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Fujino Y, Fujii T, and Daimon T
- Subjects
- Adrenal Medulla metabolism, Animals, Cattle, Cell Fractionation, Chromaffin Granules ultrastructure, Electrophoresis, Polyacrylamide Gel, Frozen Sections, Immunoblotting, Laminin chemistry, Male, Mice, Microscopy, Electron, Microscopy, Immunoelectron, Rats, Rats, Wistar, Sarcoma, Experimental chemistry, Chromaffin Granules chemistry, Laminin analysis
- Abstract
The presence of a glycoprotein laminin in bovine adrenal chromaffin granules was examined by SDS-PAGE followed by immunoblotting. The two chromaffin granule membrane fractions were obtained by linear sucrose gradient centrifugation followed by freezing and thawing and gel-filtration of the chromaffin granule-rich fraction, respectively. The purity of the granules in these fractions was examined by electron microscopy. These fractions contained laminin B chain-like immunoreactivity as a major immunoreactive component against anti-laminin. Laminin A chain-like immunoreactive protein was undetectable. The soluble fraction of the chromaffin granules contained no immunoreactive peptide. The presence of laminin-like immunoreactivity in the chromaffin granules was confirmed by immunocytochemical study. Laminin B chain-like immunoreactivity was also identified in the rat adrenal chromaffin granule fraction. Laminin A chain was hardly detected, as in the case of bovine adrenals. Structure of laminin in chromaffin granules in bovine and rat adrenals may be different from that of mouse Englebrethe-Holm-Swarm sarcoma laminin. The functional significance of laminin B chain-like protein in the granules is unknown at present.
- Published
- 1994
- Full Text
- View/download PDF
39. Intracellular and extracellular processing of chromogranin A. Determination of cleavage sites.
- Author
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Metz-Boutigue MH, Garcia-Sablone P, Hogue-Angeletti R, and Aunis D
- Subjects
- Adrenal Medulla chemistry, Amino Acid Sequence, Animals, Binding Sites, Cattle, Chromatography, High Pressure Liquid, Chromogranin A, Chromogranins chemistry, Electrophoresis, Gel, Two-Dimensional, Isoelectric Point, Molecular Sequence Data, Molecular Weight, Pancreatic Hormones metabolism, Peptide Fragments chemistry, Peptide Fragments metabolism, Potassium pharmacology, Chromaffin Granules chemistry, Chromogranins metabolism
- Abstract
Chromogranins are a family of acidic soluble proteins which exhibit widespread distribution in endocrine cells and neurons. Chromogranin A (CGA), the major soluble component of the secretory granules in chromaffin cells of the adrenal medulla, is a single polypeptide chain of 431 residues with an apparent molecular mass of 70-75 kDa and a pI of 4.5-5. In mature bovine chromaffin granules about 50% of the CGA has been processed. In the present paper, the structural features of the proteolytic degradation mechanism have been characterized with regard to the possible function of CGA as a prohormone, as suggested by recent studies. CGA-derived components present in chromaffin granules were subjected to either two-dimensional gel electrophoresis or HPLC and the N-terminal of each fragment was sequenced. Immunoblotting with antisera to specific sequences within the CGA molecule were used to characterize these fragments further at their C-terminal. In addition, a similar approach was performed to characterize CGA-derived fragments released into the extracellular space from directly depolarized bovine cultured chromaffin cells. Our results identified several proteolytic cleavage sites involved in CGA degradation. Intragranular processing occurs at 12 cleavage sites along the peptide chain located in both N- and C-terminal moieties of the protein; a preferential proteolytic attack in the C-terminal part was noted. We found that CGA processing also occurs in the extracellular space after release, generating new shorter fragments. The proteolytic cleavage sites identified in this study were compared with the cleavage points which are thought to be involved in generating CGA fragments with specific biological activity: pancreastatin, chromostatin and N-terminal vasostatin fragments. In addition, a new 12-amino-acid CGA-derived peptide corresponding to the sequence 65-76 was identified in the soluble core of purified chromaffin granules. This short peptide was released, together with catecholamines, after stimulation of cultured chromaffin cells suggesting its presence within the storage complex of chromaffin granules. The specific biological activity of this CGA-derived fragment remains to be determined.
- Published
- 1993
- Full Text
- View/download PDF
40. Novel peptides from adrenomedullary chromaffin vesicles.
- Author
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Sigafoos J, Chestnut WG, Merrill BM, Taylor LC, Diliberto EJ Jr, and Viveros OH
- Subjects
- Amino Acid Sequence, Animals, Cattle, Chromogranin A, Chromogranins chemistry, Decorin, Extracellular Matrix Proteins, Molecular Sequence Data, Proteins chemistry, Proteoglycans chemistry, Rats, Sequence Alignment, Swine, Adrenal Medulla chemistry, Chromaffin Granules chemistry, Peptides chemistry
- Abstract
The adrenal medulla chromaffin vesicle (CV) contains, on a weight basis, as much soluble protein and peptide as catecholamine. The bulk of the protein is accounted for by chromogranins (Cgr) A, B and C. Additionally, a large variety of neuropeptides and their precursor proteins have been found recently within these vesicles. Nevertheless, fractionation of CV lysates indicates the presence of many more peptides than previously reported. In the hope of finding novel bioactive peptides, we initiated a systematic isolation and characterisation of CV peptides. Bovine CV pellets were prepared by sucrose gradient centrifugation and immediately boiled in water to avoid degradation of native proteins and peptides. The water lysates were fractionated through a battery of reversed-phase and ion-exchange high-performance chromatographic steps. We fully or partially characterised a substantial number of novel peptides derived from CgrA and CgrB. A tetradecapeptide and a 13 kDa extended peptide were derived from the bovine homologue of rat secretogranin III. Peptides corresponding to C-terminal fragments of 7B2 and proteoglycan II were also found. Additionally, several sequences had no known precursors. Of the sequences derived from known precursors some corresponded to fragments bracketed by pairs of basic amino acids, but others were preceded or followed by single basic residues or by unusual putative cleavage sites. Some of these peptides were postranslationally modified (pyroglutamylation, glycosylation, phosphorylation, amidation). A significant degree of structural conservation of some of these peptides across species suggests that they may exert biological effects when cosecreted with catecholamines during splanchnic stimulation.
- Published
- 1993
41. The adrenal chromaffin granule: a model for large dense core vesicles of endocrine and nervous tissue.
- Author
-
Winkler H
- Subjects
- Animals, Antigens metabolism, Chromaffin Granules chemistry, Chromogranins metabolism, Exocytosis, Glycoproteins metabolism, Membrane Proteins metabolism, Neuropeptides metabolism, Adrenal Medulla metabolism, Chromaffin Granules metabolism, Endocrine Glands metabolism, Models, Biological, Neurons metabolism
- Abstract
More than 25 years have elapsed since R. E. Coupland made his classic observations on the ultrastructure of chromaffin granules, on the histochemical differentiation of noradrenaline and adrenaline storage granules and on their release by exocytosis. This essay attempts to demonstrate that subsequent studies on the biochemistry of chromaffin granules have yielded analytical and functional data relevant for all large dense core vesicles of endocrine and nervous tissue.
- Published
- 1993
42. Gamma-aminobutyric acid (GABA) immunoreactivity in the mouse adrenal gland.
- Author
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Oomori Y, Iuchi H, Nakaya K, Tanaka H, Ishikawa K, Satoh Y, and Ono K
- Subjects
- Adrenal Medulla cytology, Adrenal Medulla innervation, Animals, Immunohistochemistry, Male, Mice, Mice, Inbred BALB C, Microscopy, Fluorescence, Microscopy, Immunoelectron, Adrenal Medulla chemistry, Chromaffin Granules chemistry, Nerve Fibers chemistry, Norepinephrine analysis, gamma-Aminobutyric Acid analysis
- Abstract
Gamma-aminobutyric acid (GABA) immunoreactivity was revealed by immunocytochemistry in the mouse adrenal gland at the light and electron microscopic levels. Groups of weakly or faintly GABA immunoreactive chromaffin cells were often seen in the adrenal medulla. By means of immunohistochemistry combined with fluorescent microscopy, these GABA immunoreactive chromaffin cells showed noradrenaline fluorescence. The immunoreaction product was seen mainly in the granular cores of these noradrenaline cells. These results suggest the co-existence of GABA and noradrenaline within the chromaffin granules. Sometimes thick or thin bundles of GABA immunoreactive nerve fibers with or without varicosities were found running through the cortex directly into the medulla. In the medulla, GABA immunoreactive varicose nerve fibers were numerous and were often in close contact with small adrenaline cells and large ganglion cells; a few, however, surrounded clusters of the noradrenaline cells, where membrane specializations were formed. Single GABA immunoreactive nerve fibers, and thin or thick bundles of the immunoreactive varicose nerve fibers ran along the blood vessels in the medulla. The immunoreaction deposits were observed diffusely in the axoplasm and in small agranular vesicles of the GABA immunoreactive nerve fibers. Since no ganglion cells with GABA immunoreactivity were found in the adrenal gland, the GABA immunoreactive nerve fibers are regarded as extrinsic in origin.
- Published
- 1993
- Full Text
- View/download PDF
43. Chromogranin A: secretion of processed products from the stimulated retrogradely perfused bovine adrenal gland.
- Author
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Helle KB, Marley PD, Angeletti RH, Aunis D, Galindo E, Small DH, and Livett BG
- Subjects
- Acetylcholine pharmacology, Adrenal Glands drug effects, Adrenal Glands innervation, Adrenal Medulla chemistry, Animals, Aprotinin pharmacology, Blotting, Western, Cattle, Chromaffin Granules chemistry, Chromogranin A, Chromogranins isolation & purification, Electric Stimulation, Electrophoresis, Polyacrylamide Gel, Immunoblotting, In Vitro Techniques, Peptide Fragments metabolism, Perfusion, Adrenal Glands metabolism, Chromogranins metabolism
- Abstract
Chromogranin A (CGA) is a member of a family of highly acidic proteins co-stored and co-secreted with adrenaline and noradrenaline in the adrenal medulla. A number of biologically active fragments of CGA (CGAFs) have been characterized including a group of small N-terminal fragments collectively named vasostatins due to their vascular inhibitory activity. In the present study, the release of CGAFs, including CGA N-terminal fragments, from the isolated, retrogradely perfused bovine adrenal gland, has been studied under basal conditions and during nerve stimulation and perfusion with acetylcholine. The CGAFs were characterized by SDS-PAGE followed by immunoblotting with antisera to specific sequences within the CGA molecule. Many different CGAFs were released during stimulation of the glands. Antisera to CGA1-40 and CGA44-76 detected a 7 kD protein whose release was increased during stimulation. This component co-migrated with synthetic CGA1-76, was not immunoreactive to antisera to CGA79-113 or CGA124-143, and was seen whether or not the serine protease inhibitor aprotinin was present in the perfusion medium. The release of an approximately 18 kD component, which stained with antisera to CGA1-40, CGA44-76 and CGA79-113, but not to chromostatin (CGA124-143), was also increased during stimulation. Components of 22 kD and larger were detected with antisera to chromostatin, but not with antisera to CGA1-40, CGA44-76 and CGA79-113. Two of these components of 22 to 24 kD were enhanced during nerve stimulation in the presence of aprotinin. The results indicate that processed chromogranin A fragments are secreted from the bovine adrenal medulla during stimulation of chromaffin cells.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1993
- Full Text
- View/download PDF
44. The human platelet dense granule: serotonin uptake, tetrabenazine binding, phospholipid and ganglioside profiles.
- Author
-
Chatterjee D and Anderson GM
- Subjects
- Animals, Binding, Competitive, Centrifugation, Density Gradient, Chromaffin Granules chemistry, Cytoplasmic Granules chemistry, Gangliosides analysis, Humans, Kinetics, Phospholipids analysis, Species Specificity, Subcellular Fractions chemistry, Swine, Synaptic Vesicles, Blood Platelets metabolism, Cytoplasmic Granules metabolism, Ketanserin metabolism, Serotonin metabolism, Tetrabenazine metabolism
- Abstract
The functioning and composition of human platelet dense granules were studied using granules purified by Percoll fractionation. Reserpine-blockable serotonin (5-HT) uptake by the dense granule fraction was characterized and also demonstrated in the crude membrane fraction. Tetrabenazine (TBZ)-displaceable [3H]ketanserin binding was used to label the granular 5-HT transporter. Scatchard analyses, Hill plots, displacement curves, and binding kinetics indicated that TBZ-displaceable [3H]ketanserin binding labeled a site similar to that previously reported for chromaffin granules and synaptic vesicles. Analysis of phospholipid profiles in platelet fractions revealed that most platelet lysolecithin was associated with the dense granule fraction. Ganglioside analysis indicated that the predominant platelet ganglioside, GM3, was highly enriched in the dense granule fraction. The data lend further support to the idea that the 5-HT transporter complex is similar in platelet dense granules, chromaffin granules, and synaptic vesicles. However, the ganglioside and phospholipid findings clearly distinguish the types of storage sites and raise questions concerning the functional roles of dense granule GM3 and lysolecithin.
- Published
- 1993
- Full Text
- View/download PDF
45. Fusion of chromaffin granules with cardiolipin-containing phospholipid vesicles.
- Author
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Migallón MP, Ortiz A, Aranda FJ, and Gómez-Fernández JC
- Subjects
- Animals, Calcium pharmacology, Catecholamines metabolism, Cattle, Chromaffin Granules metabolism, Phospholipids chemistry, Cardiolipins chemistry, Chromaffin Granules chemistry, Liposomes chemistry, Membrane Fusion, Phospholipids metabolism
- Abstract
Fusion of chromaffin granule ghosts with model phospholipid vesicles is dependent on the composition of the vesicle membrane. Cardiolipin was found to make possible a process of fusion in the absence of calcium. This calcium-independent fusion appears to be partially protein-dependent. Upon interaction with pure cardiolipin vesicles calcium stimulates both fusion of chromaffin granule ghosts and release of catecholamines from intact chromaffin granules. We suggest that the release of catecholamines is not only a consequence of the fusion process. The relevance of protein-lipid interaction and the importance of the formation of HII phases or other non-lamellar phases, on the fusion of chromaffin granules are discussed.
- Published
- 1993
46. [Effects of NGF on chromaffin adrenaline-containing cells of adrenal medulla of rabbits transplanted into brains of mice].
- Author
-
Jousselin-Hosaja M and Derbin C
- Subjects
- Animals, Chromaffin Granules chemistry, Chromaffin Granules drug effects, Male, Mice, Rabbits, Adrenal Medulla cytology, Brain surgery, Chromaffin Granules transplantation, Chromaffin Granules ultrastructure, Epinephrine analysis, Nerve Growth Factors pharmacology
- Abstract
The graft of chromaffin adrenaline-containing (A) cells of rabbit adrenal medulla implanted to mouse brain and treated with NGF contains more survived cells 1 month after grafting than adrenal medulla alone. The cells developed either an intermediate (e.g. chromaffin cell and neuron) or a neuron-like phenotypes accompanied with a decrease in an immunoreactivity for PNMT (phenyletanolamine-N-methyltransferase). A gap junctions and attached plaques were found between grafted cells. The grafts received a synaptic input. The NGF influence on the fate of chromaffin A-containing cells is discussed.
- Published
- 1993
47. Small membrane-associated GTP-binding proteins of catecholamine-secreting cells.
- Author
-
Rhoads AR, Vu ND, and Carroll AG
- Subjects
- Adrenal Medulla chemistry, Adrenal Medulla cytology, Animals, Calcium pharmacology, Cattle, Cell Membrane chemistry, Chromaffin Granules chemistry, Electrophoresis, Gel, Two-Dimensional, Electrophoresis, Polyacrylamide Gel, PC12 Cells, Potassium pharmacology, Subcellular Fractions, Catecholamines metabolism, GTP-Binding Proteins isolation & purification
- Abstract
1. Four GTP-binding proteins (23-27 kDa) were identified in membranes from PC12 cells by [alpha 32P]GTP binding to nitrocellulose blots of SDS-polyacrylamide gels. 2. The GTP-binding proteins remained associated with membranes during stimulation of intact cells by K(+)-depolarization or even after addition of Ca2+ to digitonin-permeabilized cells. 3. By two-dimensional gel electrophoresis, six GTP-binding proteins were resolved and based on their mobility, their phosphorylation state appeared independent of Ca2+. 4. Fractionation of PC12 membranes showed that these GTP-binding proteins were broadly distributed in post-nuclear membranes with the plasma membranes containing the highest specific GTP-binding activity. 5. Membrane fractions from bovine adrenal medulla contain similar GTP-binding proteins with GTP-binding intensity also being highest in the plasma membrane. 6. The GTP-binding proteins could be concentrated in the detergent-rich fraction upon Triton X-114 phase separation.
- Published
- 1993
- Full Text
- View/download PDF
48. Regulation of the chromaffin granule catecholamine transporter in cultured bovine adrenal medullary cells: stimulus-biosynthesis coupling.
- Author
-
Desnos C, Laran MP, and Scherman D
- Subjects
- 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester pharmacology, Acetylcholinesterase analysis, Acetylcholinesterase metabolism, Adrenal Medulla chemistry, Animals, Biological Transport physiology, Carbachol pharmacology, Carrier Proteins analysis, Catecholamine Plasma Membrane Transport Proteins, Catecholamines pharmacokinetics, Cattle, Cells, Cultured, Chromaffin Granules metabolism, Chromaffin Granules ultrastructure, Chromogranin A, Chromogranins analysis, Chromogranins metabolism, Colforsin pharmacology, Cyclic AMP metabolism, Cycloheximide pharmacology, Dactinomycin pharmacology, Dopamine beta-Hydroxylase analysis, Dopamine beta-Hydroxylase metabolism, Membrane Proteins analysis, Membrane Proteins metabolism, Nifedipine pharmacology, Potassium pharmacology, Protein Kinase C metabolism, Tetrabenazine analogs & derivatives, Tetrabenazine metabolism, Time Factors, Tritium, Tyrosine 3-Monooxygenase analysis, Tyrosine 3-Monooxygenase metabolism, Adrenal Medulla cytology, Adrenal Medulla metabolism, Carrier Proteins metabolism, Chromaffin Granules chemistry, Membrane Transport Proteins
- Abstract
The transsynaptic induction of the monoamine transporter present on the membrane of chromaffin granules was studied in primary cultures of dissociated bovine adrenomedullary cells submitted to a chronic secretory stimulation. The amount of the vesicular monoamine transporter was assayed by binding of the specific ligand [3H]-dihydrotetrabenazine. After several days of incubation in the presence of high potassium, the concentration of [3H]-dihydrotetrabenazine binding sites was increased by a 1.5-2.5 factor. This increase was smaller in the presence of the cholinergic agonist carbachol. The long-term inductions of the vesicular monoamine transporter, of tyrosine hydroxylase, and of acetylcholinesterase were of similar magnitude. Under the same conditions, we found no variation in either the activities of other catecholamine biosynthetic enzymes (dopamine beta-hydroxylase and DOPA decarboxylase), or in metabolic enzymes such as lactate dehydrogenase and cytochrome c oxidase, and a decrease in the cellular content of chromogranin A and cytochrome b-561. The induction of the vesicular monoamine transporter was inhibited by the calcium channel antagonists, fluspirilene and nifedipine, and was increased by the agonist Bay K 8644. It was abolished by cycloheximide and actinomycin D. These results indicate that calcium entry into chromaffin cells increases the synthesis of the vesicular monoamine transporter, presumably by transcriptional activation. Elevation of intracellular cyclic AMP concentration or activation of protein kinase C also induced an increase in the expression of the vesicular monoamine transporter. Our results confirm that components of storage vesicle membranes are differentially regulated in response to secretory stimulation, as are several cytosolic or intravesicular soluble proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1992
- Full Text
- View/download PDF
49. Calcium-independent K(+)-selective channel from chromaffin granule membranes.
- Author
-
Arispe N, Pollard HB, and Rojas E
- Subjects
- Adrenal Medulla chemistry, Adrenal Medulla ultrastructure, Animals, Catecholamines metabolism, Cattle, Cell Fractionation, Charybdotoxin, Chromaffin Granules chemistry, Chromaffin Granules physiology, Exocytosis physiology, Intracellular Membranes physiology, Intracellular Membranes ultrastructure, Membrane Potentials physiology, Potassium Channels drug effects, Potassium Channels physiology, Scorpion Venoms pharmacology, Sodium pharmacology, Calcium physiology, Chromaffin Granules ultrastructure, Intracellular Membranes chemistry, Potassium Channels ultrastructure
- Abstract
Intact adrenal chromaffin granules and purified granule membrane ghosts were allowed to fuse with acidic phospholipid planar bilayer membranes in the presence of Ca2+ (1 mM). From both preparations, we were able to detect a large conductance potassium channel (ca. 160 pS in symmetrical 400 mM K+), which was highly selective for K+ over Na+ (PK/PNa = 11) as estimated from the reversal potential of the channel current. Channel activity was unaffected by charybdotoxin, a blocker of the [Ca2+]-activated K+ channel of large conductance. Furthermore, this channel proved quite different from the previously described channels from other types of secretory vesicle preparations, not only in its selectivity and conductance, but also in its insensitivity to both calcium and potential across the bilayer. We conclude that the chromaffin granule membrane contains a K(+)-selective channel with large conductance. We suggest that the role of this channel may include ion movement during granule assembly or recycling, and do not rule out events leading to exocytosis.
- Published
- 1992
- Full Text
- View/download PDF
50. Rapid, high-yield isolation of human chromogranin A from chromaffin granules of pheochromocytomas.
- Author
-
Syversen U, Waldum HL, and O'Connor DT
- Subjects
- Adrenal Gland Neoplasms ultrastructure, Amino Acid Sequence, Chromatography, Affinity, Chromatography, Gel, Chromogranin A, Chromogranins chemistry, Electrophoresis, Polyacrylamide Gel, Humans, Immunoblotting, Microscopy, Electron, Molecular Sequence Data, Pheochromocytoma ultrastructure, Adrenal Gland Neoplasms chemistry, Chromaffin Granules chemistry, Chromogranins isolation & purification, Pheochromocytoma chemistry
- Abstract
Chromogranin A (CgA) is a useful probe of human neuroendocrine neoplasia and exocytotic sympathoadrenal activity, but the application of CgA immunoassays has not been widespread because of limited availability of purified human CgA. Here we describe a rapid, high yield isolation of human CgA. After obtaining and lysing pheochromocytoma chromaffin granules, the soluble core proteins (chromogranins) were depleted of dopamine-beta-hydroxylase by passage over a concanavalin A-Sepharose affinity column, then lyophilized, resuspended in volatile buffer, and gel filtered on Sephacryl S-300. SDS-PAGE-analyzed column fractions contained homogeneous human CgA, which was verified structurally (N-terminal amino acid sequence) and immunologically (radioimmunoassay and immunoblot). The overall 22.6 mg yield of purified CgA represented 5.7% of the starting vesicle core protein. This preparation will be useful in evaluating the sympathoadrenal system and endocrine neoplasia in man.
- Published
- 1992
- Full Text
- View/download PDF
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