The Crucial Role of Chromogranins in Storage and Exocytosis Revealed Using Chromaffin Cells from Chromogranin A Null Mouse

Montesinos, Mónica S.; Machado, José David; Camacho, Marcial; Díaz, J. A.; Morales, Yezer G; Rosa, Diego Álvarez de la; Carmona, Emilia; Castañeyra, Agustín; Viveros, O. Humberto; O’Connor, Daniel T.; Mahata, Sushil K.; Borges, Ricardo

doi:10.1523/jneurosci.5292-07.2008

Cited by 121 publications

(155 citation statements)

References 34 publications

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“…1B). VNUT, ATP, and catecholamines exhibited a similar distribution (particularly in fractions [4][5][6][7][8][9][10][11], and VNUT coeluted with other chromaffin granule-specific and enriched proteins like CgB, CgA, and SgII. In contrast, the presence of the cytochrome c oxidase I (a mitochondrial protein) was negligible in all fractions recovered from the gradient (24).…”

Section: Resultsmentioning

confidence: 83%

“…For example, the catecholamine content of adrenal secretory granules (SGs), a type of large dense core secretory vesicles also known as chromaffin granules, was 0.8-1 M when measured directly in adrenal chromaffin cells using patch amperometry (5,6). In addition, SGs from chromaffin cells contain ATP at ∼150 mM (7), calcium ∼40 mM (8), about 2 mM of granins, ascorbate, peptides, and other nucleotides, all in an acidic pH ∼5.5 environment.…”

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confidence: 99%

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ATP: The crucial component of secretory vesicles

Estévez-Herrera

Domínguez

Pardo

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.exocytosis | purines | quantum size | secretory vesicles | VNUT V irtually most, and possibly all, types of secretory vesicles found in cells contain ATP, which often accumulates at high concentrations and, commonly, in conjunction with different types of neurotransmitters. However, the reason for this widespread distribution of ATP remains a mystery. Although ATP is present in all animal species, including primitive life forms like Giardia lamblia that lack Golgi complexes and mitochondria, the detection of ATP in the secretory vesicles of sympathetic neurons was considered to be the first example of cotransmission (1). However, given the ubiquitous accumulation of ATP in secretory vesicles, it might instead be considered that it is the other neurotransmitters that coincide with ATP, rather than the other way around (2). Indeed, perhaps ATP should be considered as the first molecule used as a transmitter in primitive forms of life.Astonishingly high concentrations of releasable species are stored inside secretory vesicles, far exceeding those in the cytosol (3, 4). For example, the catecholamine content of adrenal secretory granules (SGs), a type of large dense core secretory vesicles also known as chromaffin granules, was 0.8-1 M when measured directly in adrenal chromaffin cells using patch amperometry (5, 6). In addition, SGs from chromaffin cells contain ATP at ∼150 mM (7), calcium ∼40 mM (8), about 2 mM of granins, ascorbate, peptides, and other nucleotides, all in an acidic pH ∼5.5 environment.ATP possesses intrinsic chemical characteristics that make it relevant to the accumulation of soluble substances in secretory vesicles. The formation of weak complexes between monoamines and ATP, the two main soluble compounds in chromaffin granules, has been demonstrated in vitro by NMR (9), ultracentrifugation (10), infrared spectroscopy, and calorime...

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Section: Resultsmentioning

confidence: 83%

mentioning

confidence: 99%

ATP: The crucial component of secretory vesicles

Estévez-Herrera

Domínguez

Pardo

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Nonobese diabetic (NOD) mice were purchased from The Jackson Laboratory. ChgA −/− mice were originally obtained from Dr. Sushil Mahata (University of California, San Diego) (18). They were partially backcrossed to NOD mice by Dr. Katherine Haskins (National Jewish Health), who provided us with H-2 g7 +/+ /ChgA +/− mice from this cross.…”

Section: Methodsmentioning

confidence: 99%

N-terminal additions to the WE14 peptide of chromogranin A create strong autoantigen agonists in type 1 diabetes

Jin

Wang

Crawford

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Chromogranin A (ChgA) is an autoantigen for CD4 + T cells in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). The natural ChgA-processed peptide, WE14, is a weak agonist for the prototypical T cell, BDC-2.5, and other ChgA-specific T-cell clones. Mimotope peptides with much higher activity share a C-terminal motif, WXRM(D/E), that is predicted to lie in the p5 to p9 position in the mouse MHC class II, IA g7 binding groove. This motif is also present in WE14 (WSRMD), but at its N terminus. Therefore, to place the WE14 motif into the same position as seen in the mimotopes, we added the amino acids RLGL to its N terminus. Like the other mimotopes, RLGL-WE14, is much more potent than WE14 in T-cell stimulation and activates a diverse population of CD4 + T cells, which also respond to WE14 as well as islets from WT, but not ChgA −/− mice. The crystal structure of the IA g7 -RLGL-WE14 complex confirmed the predicted placement of the peptide within the IA g7 groove. Fluorescent IA g7 -RLGL-WE14 tetramers bind to ChgA-specific T-cell clones and easily detect ChgA-specific T cells in the pancreas and pancreatic lymph nodes of NOD mice. The prediction that many different N-terminal amino acid extensions to the WXRM(D/E) motif are sufficient to greatly improve T-cell stimulation leads us to propose that such a posttranslational modification may occur uniquely in the pancreas or pancreatic lymph nodes, perhaps via the mechanism of transpeptidation. This modification could account for the escape of these T cells from thymic negative selection. (T1D) is an autoimmune disease characterized by infiltration of T cells into the pancreatic islets of Langerhans, resulting in destruction of insulin-secreting beta cells (reviewed in ref. 1). Numerous autoantigens, including insulin/ proinsulin itself and chromogranin A (ChgA), have been reported to be T-cell targets in the disease in mice and/or humans (reviewed in refs. 2 and 3). In the nonobese diabetic (NOD) mouse model of T1D, epitopes in the insulin B chain have been shown to be essential for T1D development (4), and CD4 + T-cell clones specific for insulin or ChgA have been shown to be particularly potent in induction of T1D in immune-deficient NOD-scid mice (5). ChgA (gene name CHGA) is a member of the granin family of neuroendocrine secretory proteins. Full-length ChgA is proteolytically processed to yield several functional peptides including a small peptide, WE14 (6). Our group participated in the study that first identified ChgA as the autoantigen and the WE14 peptide as the key epitope for the prototypical BDC-2.5 and other NOD-derived CD4 + T cells (7). WE14 is a weak agonist for these ChgA-specific T cells, but we (7) and others (8, 9) have identified a series of mimotope peptides that are very strong agonists for these T cells. Comparison of the sequence of WE14 with these mimotope peptides reveals the presence of a common amino acid motif, WXRM(D/E), at the C terminus where it is predicted to occupy positions p5 to p9 in the IA g7 binding groove. WE14...

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“…However, a closer look reveals that the amount of material released from individual vesicles measured with patch amperometry is generally considerably larger than the amount measured using standard amperometry. A quick survey of the charges measured at chromaffin cells using amperometry gives values of ≈1 pC (Wightman et al 1991), 0·67 pC (Montesinos et al 2008) and 0·62 pC (Segovia et al 2010), and charges measured using patch amperometry are 2·2 pC (Albillos et al 1997), 1·8 pC (Montesinos et al 2008) and 3·2 pC (Segovia et al 2010). This suggests that each of these methods cater to a specific event, but it is difficult to make direct comparisons.…”

Section: Traditional Models Of Exocytosismentioning

confidence: 99%

“…This suggests that each of these methods cater to a specific event, but it is difficult to make direct comparisons. However, there are at least two experiments in the literature that carry both methods, one shows that the average charge measured using patch amperometry is nearly triple the values measured using amperometry (Montesinos et al 2008) and a second shows that the charge increases to more than five times when using patch amperometry (Segovia et al 2010). It appears that the patch influences the size of the quanta released.…”

Section: Traditional Models Of Exocytosismentioning

confidence: 99%

The evidence for open and closed exocytosis as the primary release mechanism

Ren

Mellander

Keighron

et al. 2016

Quart. Rev. Biophys.

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Abstract. Exocytosis is the fundamental process by which cells communicate with each other. The events that lead up to the fusion of a vesicle loaded with chemical messenger with the cell membrane were the subject of a Nobel Prize in 2013. However, the processes occurring after the initial formation of a fusion pore are very much still in debate. The release of chemical messenger has traditionally been thought to occur through full distention of the vesicle membrane, hence assuming exocytosis to be all or none. In contrast to the all or none hypothesis, here we discuss the evidence that during exocytosis the vesicle-membrane pore opens to release only a portion of the transmitter content during exocytosis and then close again. This open and closed exocytosis is distinct from kiss-and-run exocytosis, in that it appears to be the main content released during regular exocytosis. The evidence for this partial release via open and closed exocytosis is presented considering primarily the quantitative evidence obtained with amperometry.1. An overview of exocytosis and endocytosis 2 2. Techniques to monitor exocytosis 5 2.1. Patch clamp 6 2.2. Amperometry 6 2.3. Patch amperometry 7 2.4. Cyclic voltammetry 7 2.5. Fluorescence microscopy imaging 83. Traditional models of exocytosis 94. Evidence where partial release during exocytosis appears to be dominant 11 4.1. The total content of a secretory vesicle is greater than the amount released 11 4.2. The majority of exocytotic events are followed by immediate endocytosis 12 4.3. Full distention of the vesicle may not be necessary for quantal release 13 4.4. A flickering fusion pore might regulate quantal release and vesicle recycling 15 4.5. Nanometer-sized pores can be seen as 'feet' associated with amperometric spikes 17 3. Release appears to be only a fraction of vesicle content in mammalian brains 23 7.4. Impact cytometry of adrenal cell vesicles shows a significantly larger catecholamine content then that released 23 7.5. Impact cytometry has been used to measure vesicle content in live cells and this quantity is greater than the amount released 23 7.6. Full distention of the vesicle is not necessary for the measured amperometric current during exocytotic release 23 7.7. Fast changes in cell environment alter the amount of messenger released 23 7.8. Patch amperometry measurements reveal a greater amount of release 23 7.9. Postspike 'feet' are observed with an amperometric spike 23Acknowledgements 24

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The Crucial Role of Chromogranins in Storage and Exocytosis Revealed Using Chromaffin Cells from Chromogranin A Null Mouse

Cited by 121 publications

References 34 publications

ATP: The crucial component of secretory vesicles

ATP: The crucial component of secretory vesicles

N-terminal additions to the WE14 peptide of chromogranin A create strong autoantigen agonists in type 1 diabetes

The evidence for open and closed exocytosis as the primary release mechanism

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