| 1. |
- Taleat, Zahra, 1982, et al.
(author)
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Electrochemical Investigation of the Interaction between Catecholamines and ATP
- 2018
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 90:3, s. 1601-1607
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Journal article (peer-reviewed)abstract
- The study of the colligative properties of adenosine 5'-triphosphate (ATP) and catecholamines has received the attention of scientists for decades, as they could explain the capabilities of secretory vesicles (SVs) to accumulate neurotransmitters. In this Article, we have applied electrochemical methods to detect such interactions in vitro, at the acidic pH of SVs (pH 5.5) and examined the effect of compounds having structural similarities that correlate with functional groups of ATP (adenosine, phosphoric acid and sodium phosphate salts) and catecholamines (catechol). Chronoamperometry and fast scan cyclic voltammetry (FSCV) provide evidence compatible with an interaction of the catechol and adenine rings. This interaction is also reinforced by an electrostatic interaction between the phosphate group of ATP and the protonated ammonium group of catecholamines. Furthermore, chronoamperometry data suggest that the presence of ATP subtlety reduces the apparent diffusion coefficient of epinephrine in aqueous media that adds an additional factor leading to a slower rate of catecholamine exocytosis. This adds another plausible mechanism to regulate individual exocytosis events to alter communication.
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| 2. |
- Dunevall, Johan, 1984, et al.
(author)
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Characterizing the Catecholamine Content of Single Mammalian Vesicles by Collision-Adsorption Events at an Electrode
- 2015
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 137:13, s. 4344-4346
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Journal article (peer-reviewed)abstract
- We present the electrochemical response to single adrenal chromaffin vesicles filled with catecholamine hormones as they are adsorbed and rupture on a 33 mu m diameter disk-shaped carbon electrode. The vesicles adsorb onto the electrode surface and sequentially spread out over the electrode surface, trapping their contents against the electrode. These contents are then oxidized, and a current (or amperometric) peak results from each vesicle that bursts. A large number of current transients associated with rupture of single vesicles (86%) are observed under the experimental conditions used, allowing us to quantify the vesicular catecholamine content.
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| 3. |
- Dunevall, Johan, 1984
(author)
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New Approaches for Chemical Analysis of Single Cells and Vesicles
- 2018
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Doctoral thesis (other academic/artistic)abstract
- Exocytosis is the major cell-to-cell communication process in the nervous system, involving the conversion of an electrical signal (e.g. action potential) to a chemical one. Signaling molecules like neurotransmitters, hormones and/or peptides are stored in vesicles inside the cell. During exocytosis, calcium triggers the release of the vesicular cargo through SNARE-complex mediated fusion of the vesicles with the plasma (outer-membrane) of the cell. Consequently, a transient pore is formed through which the vesicular cargo is released into the extracellular space and is there able to interact with receptors of target cells. Most often, the pore closes again only allowing a fraction of the cargo to be released, so called partial release or kiss-and-run exocytosis. The extent of partial release is modulated by the intracellular calcium concentration, which can be regulated by the strength of the stimuli or with pharmaceuticals. Despite the importance of this process and the efforts that have been done to resolve the fundamental regulatory mechanisms of exocytosis, much remains unknown. In order to gain understanding of how the amount of vesicular cargo released is regulated, information about the total vesicular cargo (quantal content) has to be obtained. Until recently, no method aiming for quantification of the full quantal content existed. Our group has successfully developed an electrochemical method called vesicle impact electrochemical cytometry (VIEC) that allows direct quantification of the vesicular content in secretory granules as demonstrated with large dense-core granules from chromaffin and pheochromocytoma (PC12) cells. Chromaffin cells of the adrenal medulla are the body´s stress response output, and the best studied model system for exocytosis. The large-dense core vesicles of chromaffin cells contain a very important group of neurotransmitters and hormones, namely the catecholamines (e.g. dopamine, norepinephrine and epinephrine). The catecholamines are electroactive, and can readily be oxidized at the surface of a polarized electrode to give away two electrons per molecule. By counting the number of electrons passed through the system (charge) and knowing the charge of one mole of electrons (Faraday´s constant) the number of molecules can be quantified. In the intracellular or IVIEC method a conical nanotip carbon electrode is used to pierce into the cytosol of a living cell, allowing the vesicles to adsorb onto its surface. The vesicles burst open due to the electric field at the polarized electrode and the vesicular cargo is released towards the electrode surface and is oxidized, which allows the full content to be detected. VIEC is an electrochemical method that potentially can be applied to study the quantal content of the electroactive vesicular content of other cell types like mast cells and blood platelets, that contain both histamine and serotonin.
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| 4. |
- Dunevall, Johan, 1984, et al.
(author)
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Vesicle impact electrochemical cytometry compared to amperometric exocytosis measurements
- 2017
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In: Current Opinion in Electrochemistry. - : Elsevier BV. - 2451-9103 .- 2451-9111. ; 5:1, s. 85-91
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Journal article (peer-reviewed)abstract
- Three new tools are discussed for understanding chemical communication between cells and primarily to delve into the content and structure of nanometer transmitter vesicles. These are amperometric measurements of exocytosis, vesicle impact electrochemical cytometry, and intracellular vesicle impact electrochemical cytometry. These are combining in the end nanoscale mass spectrometry imaging to begin determination of vesicle structure. These methods have provided solid evidence for the concept of open and closed exocytosis leading to partial release of the vesicle content during normal exocytosis. They have also been used to discover cases where the fraction of transmitter released is not changed, and other cases where the vesicle transmitter fraction released is altered, as with zinc, thought to alter cognition. Overall, the combination of these methods is showing us details of vesicular processes that would not be measureable without these micro and nano electrochemical methods.
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| 5. |
- Kaya, Ibrahim, et al.
(author)
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On-Tissue Chemical Derivatization of Catecholamines Using 4-(N-Methyl)pyridinium Boronic Acid for ToF-SIMS and LDI-ToF Mass Spectrometry Imaging
- 2018
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 90:22, s. 13580-13590
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Journal article (peer-reviewed)abstract
- The analysis of small polar compounds with ToF-SIMS and MALDI-ToF-MS have been generally hindered by low detection sensitivity, poor ionization efficiency, ion suppression, analyte in-source fragmentation, and background spectral interferences from either a MALDI matrix and/or endogenous tissue components. Chemical derivatization has been a well-established strategy for improved mass spectrometric detection of many small molecular weight endogenous compounds in tissues. Here, we present a devised strategy to selectively derivatize and sensitively detect catecholamines with both secondary ion ejection and laser desorption ionization strategies, which are used in many imaging mass spectrometry (IMS) experiments. Chemical derivatization of catecholamines was performed by a reaction with a synthesized permanent pyridinium-cation-containing boronic acid molecule, 4-(N-methyl)pyridinium boronic acid, through boronate ester formation (boronic acid-diol reaction). The derivatization facilitates their sensitive detection with ToF-SIMS and LDI-ToF mass spectrometric techniques. 4-(N-Methyl)pyridinium boronic acid worked as a reactive matrix for catecholamines with LDI and improved the sensitivity of detection for both SIMS and LDI, while the isotopic abundances of the boron atom reflect a unique isotopic pattern for derivatized catecholamines in MS analysis. Finally, the devised strategy was applied, as a proof of concept, for on-tissue chemical derivatization and GCIB-ToF-SIMS (down to 3 μm per pixel spatial resolution) and LDI-ToF mass spectrometry imaging of dopamine, epinephrine, and norepinephrine in porcine adrenal gland tissue sections. MS/MS using collision-induced dissociation (CID)-ToF-ToF-SIMS was subsequently employed on the same tissue sections after SIMS and LDI mass spectrometry imaging experiments, which provided tandem MS information for the validation of the derivatized catecholamines in situ. This methodology can be a powerful approach for the selective and sensitive ionization/detection and spatial localization of diol-containing molecules such as aminols, vic-diols, saccharides, and glycans along with catecholamines in tissue sections with both SIMS and LDI/MALDI-MS techniques. © 2018 American Chemical Society.
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| 6. |
- Kaya, Ibrahim, et al.
(author)
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Spatial Lipidomics Reveals Region and Long Chain Base Specific Accumulations of Monosialogangliosides in Amyloid Plaques in Familial Alzheimer's Disease Mice (5xFAD) Brain
- 2020
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In: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 11:1, s. 14-24
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Journal article (peer-reviewed)abstract
- Ganglioside metabolism is significantly altered in Alzheimer's disease (AD), which is a progressive neuro-degenerative disease prominently characterized by one of its pathological hallmarks, amyloid deposits or "senile plaques". While the plaques mainly consist of aggregated variants of amyloid-beta protein (A beta), recent studies have revealed a number of lipid species including gangliosides in amyloid plaques along with A beta peptides. It has been widely suggested that long chain (sphingosine) base (LCBs), C18:1-LCB and C20:1-LCB, containing gangliosides might play different roles in neuronal function in vivo. In order to elucidate region-specific aspects of amyloid-plaque associated C18:1-LCB and C20:1-LCB ganglioside accumulations, high spatial resolution (10 mu m per pixel) matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) of gangliosides in amyloid plaques was performed in hippocampal and adjacent cortical regions of 12 month old 5xFAD mouse coronal brain sections from two different stereotaxic coordinates (bregma points, -2.2 and -2.7 mm). MALDI-IMS uncovered brain-region (2 and 3D) and/or LCB specific accumulations of monosialogangliosides (GMs): GM1, GM2, and GM3 in the hippocampal and cortical amyloid plaques. The results reveal monosialogangliosides to be an important component of amyloid plaques and the accumulation of different gangliosides is region and LCB specific in 12 month old 5xFAD mouse brain. This is discussed in relation to amyloid-associated AD pathogenesis such as lipid related immune changes in amyloid plaques, AD specific ganglioside metabolism, and, notably, AD-associated impaired neurogenesis in the subgranular zone.
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| 7. |
- Larsson, Anna, et al.
(author)
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Intracellular Electrochemical Nanomeasurements Reveal that Exocytosis of Molecules at Living Neurons is Subquantal and Complex
- 2020
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In: Angewandte Chemie-International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 59:17, s. 6711-6714
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Journal article (peer-reviewed)abstract
- Since the early work of Bernard Katz, the process of cellular chemical communication through exocytosis, quantal release, has been considered to be all or none. Recent evidence has shown exocytosis to be partial or "subquantal" at single-cell model systems, but there is a need to understand this at communicating nerve cells. Partial release allows nerve cells to control the signal at the site of release during individual events, for which the smaller the fraction released, the greater the range of regulation. Herein, we show that the fraction of the vesicular octopamine content released from a living Drosophila larval neuromuscular neuron is very small. The percentage of released molecules was found to be only 4.5 % for simple events and 10.7 % for complex (i.e., oscillating or flickering) events. This large content, combined with partial release controlled by fluctuations of the fusion pore, offers presynaptic plasticity that can be widely regulated.
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| 8. |
- Li, Xianchan, 1982, et al.
(author)
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Mechanistic Aspects of Vesicle Opening during Analysis with Vesicle Impact Electrochemical Cytometry
- 2017
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 89:17, s. 9416-9423
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Journal article (peer-reviewed)abstract
- Vesicle impact electrochemical cytometry (VIEC) has been used to quantify the vesicular transmitter content in mammalian vesicles. In the present study, we studied the mechanism of VIEC by quantifying the catecholamine content in single vesicles isolated from pheochromocytoma (PC12) cells. These vesicles contain about one tenth of the catecholamine compared with adrenal chromaffin vesicles. The existence of a prespike foot for many events suggests the formation of an initial transiently stable pore at the beginning of vesicle rupture. Increasing the detection temperature from 6 to 30 degrees C increases the possibility of vesicle rupture on the electrode, implying that there is a temperature-dependent process that facilitates electroporation. Natively larger vesicles are shown to rupture earlier and more frequently than smaller ones in VIEC. Likewise, manipulating vesicle content and size with drugs leads to similar trends. These data support the hypothesis that electroporation is the primary force for pore opening in VIEC. We further hypothesize that a critical step for initiating vesicle opening by electroporation is diffusion of membrane proteins away from the membrane region of contact with the electrode to allow closer contact, increasing the lateral potential field and thus facilitating electroporation.
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| 9. |
- Li, Xianchan, 1982, et al.
(author)
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Nanopore Opening at Flat and Nanotip Conical Electrodes during Vesicle Impact Electrochemical Cytometry
- 2018
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In: ACS Nano. - : American Chemical Society (ACS). - 1936-0851 .- 1936-086X. ; 12:3, s. 3010-3019
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Journal article (peer-reviewed)abstract
- The oxidation of catecholamine at a microelectrode, following its release from individual vesicles, allows interrogation of the content of single nanometer vesicles with vesicle impact electrochemical cytometry (VIEC). Previous to this development, there were no methods available to quantify the chemical load of single vesicles. However, accurate quantification of the content is hampered by uncertainty in the proportion of substituent molecules reaching the electrode surface (collection efficiency). In this work, we use quantitative modeling to calculate this collection efficiency. For all vesicles except those at the very edge of the electrode, modeling shows that ∼100% oxidation efficiency is achieved when employing a 33 μm diameter disk microelectrode for VIEC, independent of the location of the vesicle release pore. We use this to experimentally determine a precise distribution of catecholamine in individual vesicles extracted from PC12 cells. In contrast, we calculate that when a nanotip conical electrode (∼4 μm length, ∼1.5 μm diameter at the base) is employed, as in intracellular VIEC (IVIEC), the current-time response depends strongly on the position of the catecholamine-releasing pore in the vesicle membrane. When vesicle release occurs with the pore opening occurring far from the electrode, lower currents and partial oxidation (∼75%) of the catecholamine are predicted, as compared to higher currents and ∼100% oxidation, when the pore is close to/at the electrode surface. As close agreement is observed between the experimentally measured vesicular content in intracellular and extracted vesicles from the same cell line using nanotip and disk electrodes, respectively, we conclude that pores open at the electrode surface. Not only does this suggest that electroporation of the vesicle membrane is the primary driving force for catecholamine release from vesicles at polarized electrodes, but it also indicates that IVIEC with nanotip electrodes can directly assess vesicular content without correction. © 2018 American Chemical Society.
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| 10. |
- Li, Xianchan, 1982, et al.
(author)
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Quantitative Chemical Measurements of Vesicular Transmitters with Electrochemical Cytometry
- 2016
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In: Accounts of Chemical Research. - : American Chemical Society (ACS). - 0001-4842 .- 1520-4898. ; 49:10, s. 2347-2354
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Journal article (peer-reviewed)abstract
- Electrochemical cytometry adds a new dimension to our ability to study the chemistry and chemical storage of transmitter molecules stored in nanometer vesicles. The approach involves the adsorption and subsequent rupture of vesicles on an electrode surface during which the electroactive contents are quantitatively oxidized (or reduced). The measured current allows us to count the number of molecules in the vesicles using Faraday's law and to correlate this-to the amount of molecules released when single exocytosis events take place at communicating cells. The original format for this method involved a capillary electrophoresis separation step to singly address each vesicle, but we have more recently discovered that cellular vesicles tend to adsorb to carbon electrodes and spontaneously as well as stochastically rupture to give mostly single vesicle events. This approach, called impact electrochemical cytometry, even though the impact is perhaps not the important part of this process, has been studied and the vesicle rupture appears to be at the interface between the vesicle and the electrode and is probably driven by electroporation. The pore size and rate of content electrolysis are a function of the pore diameter and the presence of a protein core in the vesicles. In model liposomes with no protein, events appear extremely rapidly as the soft nanoparticles impact the electrode and the contents are oxidized. It appears that the proteins decorating the surface of the vesicle are important in maintaining a gap from the electrode and when this gap is closed electroporation takes place. Models of the event response times suggest the pores formed are small enough so we can carry out these measurements at nanotip electrodes and we have used this to quantify the vesicle content in living cells in a mode we call intracellular impact electrochemical cytometry. The development of electrochemical cytometry allows comparison between vesicle content and vesicular release and we have found that only part of the vesicle content is released in typical exocytotic cases measured by amperometry. This has led to the novel hypothesis that most exocytosis from dense core vesicles is via mechanism where vesicles fuse with the cell membrane, some content is released and then close again to be reloaded and reused. It leaves open the possibility that cells regulate release during individual events. This might be important in learning and memory and be a nonreceptor pharmaceutical target for brain related disorders. Indeed, the concept of the chemo-brain observed in cisplatin-treated cancer patients appears to be at least in part the result of changing the fraction of transmitter released and we have been able to show this by using the combined amperometric measurement of release and electrochemical cytometry at model cells.
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