Simultaneous Quantification of Vesicle Size and Catecholamine Content by Resistive Pulses in Nanopores and Vesicle Impact Electrochemical Cytometry.

We have developed the means to simultaneously measure the physical size and count catecholamine molecules in individual nanometer transmitter vesicles. This is done by combining resistive pulse (RP) measurements in a nanopore pipet and vesicle impact electrochemical cytometry (VIEC) at an electrode as the vesicle exits the nanopore. Analysis of freshly isolated bovine adrenal vesicles shows that the size and internal catecholamine concentration of vesicles varies with the occurrence of a dense core inside the vesicles. These results might benefit the understanding about the vesicles maturation, especially involving the "sorting by retention" process and concentration increase of intravesicular catecholamine. The methodology is applicable to understanding soft nanoparticle collisions on electrodes, vesicles in exocytosis and phagocytosis, intracellular vesicle transport, and analysis of electroactive drugs in exosomes.

Pyroglutamate-amyloid-ß and glutaminyl cyclase are colocalized with amyloid-ß in secretory vesicles and undergo activity-dependent, regulated secretion.

BACKGROUND AND AIMS: N-truncated pyroglutamate (pGlu)-amyloid-ß [Aß(3-40/42)] peptides are key components that promote Aß peptide accumulation, leading to neurodegeneration and memory loss in Alzheimer's disease. Because Aß deposition in the brain occurs in an activity-dependent manner, it is important to define the subcellular organelle for pGlu-Aß(3-40/42) production by glutaminyl cyclase (QC) and their colocalization with full-length Aß(1-40/42) peptides for activity-dependent, regulated secretion. Therefore, the objective of this study was to investigate the hypothesis that pGlu-Aß and QC are colocalized with Aß in dense-core secretory vesicles (DCSV) for activity-dependent secretion with neurotransmitters. METHODS: Purified DCSV were assessed for pGlu-Aß(3-40/42), Aß(1-40/42), QC, and neurotransmitter secretion. Neuron-like chromaffin cells were analyzed for cosecretion of pGlu-Aß, QC, Aß, and neuropeptides. The cells were treated with a QC inhibitor, and pGlu-Aß production was measured. Human neuroblastoma cells were also examined for pGlu-Aß and QC secretion. RESULTS: Isolated DCSV contain pGlu-Aß(3-40/42), QC, and Aß(1-40/42) with neuropeptide and catecholamine neurotransmitters. Cellular pGlu-Aß and QC undergo activity-dependent cosecretion with Aß and enkephalin and galanin neurotransmitters. The QC inhibitor decreased the level of secreted pGlu-Aß. The human neuroblastoma cells displayed regulated secretion of pGlu-Aß that was colocalized with QC. CONCLUSIONS: pGlu-Aß and QC are present with Aß in DCSV and undergo activity-dependent, regulated cosecretion with neurotransmitters.

Chromogranin location in the adrenal glands of ISIAH rats.

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.

Controlling synaptotagmin activity by electrostatic screening.

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.

Relationship between amperometric pre-spike feet and secretion granule composition in chromaffin cells: an overview.

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.

pH modulation of large conductance potassium channel from adrenal chromaffin granules.

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.

Characterization and location of post-translational modifications on chromogranin B from bovine adrenal medullary chromaffin granules.

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.

A common mechanism for the regulation of vesicular SNAREs on phospholipid membranes.

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.

Secretory vesicles membrane area is regulated in tandem with quantal size in chromaffin cells.

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.

Chromaffin granule membranes contain at least three heme centers: direct evidence from EPR and absorption spectroscopy.

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).

Identification and characterization of novel chromogranin B-derived peptides from porcine chromaffin granules by liquid chromatography/electrospray tandem MS.

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.

Mechanisms underlying neuronal death induced by chromogranin A-activated microglia.

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.

Presence of dynamin--syntaxin complexes associated with secretory granules in adrenal chromaffin cells.

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.

Molecular cloning of endopin 1, a novel serpin localized to neurosecretory vesicles of chromaffin cells. Inhibition of basic residue-cleaving proteases by endopin 1.

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.

Chromogranin A from bovine adrenal medulla: molecular characterization of glycosylations, phosphorylations, and sequence heterogeneities by mass spectrometry.

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.

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".

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.

Neurotrophin activation of catecholamine storage vesicle protein gene expression: signaling to chromogranin a biosynthesis.

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.

Release of nontransmembrane full-length Alzheimer's amyloid precursor protein from the lumenar surface of chromaffin granule membranes.

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.

Highly sensitive and stable phosphatidylserine liposome aggregation assay for annexins.

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.

NSF and SNAP are present on adrenal chromaffin granules.

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.