Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells.

Wightman, R. Mark; Jankowski, Jeffrey A.; Kennedy, Robert T.; Kawagoe, Kirk T.; Schroeder, Timothy; Leszczyszyn, David J.; Near, Joseph A.; Diliberto, Emanuel J.; Viveros, O. Humberto

doi:10.1073/pnas.88.23.10754

Cited by 832 publications

(838 citation statements)

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“…Single cell amperometry, first introduced by Wightman et al, 23 employs a micrometer-sized electrode that, when placed on the top of a cell, can be used to quantify release from individual exocytosis release events (Figure 1a, top). Intracellular cytometry (Figure 1b, middle), introduced by us is a technique to probe the catecholamine content of single vesicles as they lyse on a nanotip microeloectrode inside of a living cell.…”

Section: Resultsmentioning

confidence: 99%

Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles

et al. 2016

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We report an approach to spatially resolve the content across nanometer neuroendocrine vesicles in nerve-like cells by correlating super high-resolution mass spectrometry imaging, NanoSIMS, with transmission electron microscopy (TEM). Furthermore, intracellular electrochemical cytometry at nanotip electrodes is used to count the number of molecules in individual vesicles to compare to imaged amounts in vesicles. Correlation between the NanoSIMS and TEM provides nanometer resolution of the inner structure of these organelles. Moreover, correlation with electrochemical methods provides a means to quantify and relate vesicle neurotransmitter content and release, which is used to explain the slow transfer of dopamine between vesicular compartments. These nanoanalytical tools reveal that dopamine loading/unloading between vesicular compartments, dense core and halo solution, is a kinetically limited process. The combination of NanoSIMS and TEM has been used to show the distribution profile of newly synthesized dopamine across individual vesicles. Our findings suggest that the vesicle inner morphology might regulate the neurotransmitter release event during open and closed exocytosis from dense core vesicles with hours of equilibrium needed to move significant amounts of catecholamine from the protein dense core despite its nanometer size. KEYWORDS: nanoimaging, NanoSIMS, vesicle content, electrochemistry, nanocompartments C hemical communication in eukaryotic cells relies heavily on loading and trafficking of secretory vesicles to the plasma membrane. Upon stimulation, the loaded, docked secretory vesicles fuse with the plasma membrane and discharge their neurotransmitter cargo into the extracellular space. This process, called exocytosis, is fundamental and highly regulated. 1,2 Regulation of exocytosis has been widely studied, and the understanding of its mechanism is still under debate. Several mechanisms have been reported such as all-or-nothing release during which vesicles completely fuse with the plasma membrane and release their total content. 3 Contrary to the all-or-nothing mode, partial release mechanisms have been proposed, suggesting the transient opening of the fusion pore between the cell membrane and secretory vesicle followed by closing again. 4 In addition to the release event, exocytosis is also regulated through vesicle loading. In general, this process involves a neurotransmitter transporter, an integral vesicle membrane protein that uses the pH gradient across the vesicle membrane, to drive uptake of neurotransmitters into the vesicle. 5−8 Defining the vesicle storage mechanisms would be an important piece of the exocytosis regulation puzzle. 5,9,10 Several cell types have been employed for loading studies among which neuroendocrine cells have had an important role as a model system. They possess two types of secretory vesicles, synaptic-like microvesicles (SLMVs) and so-called large densecore vesicles (LDCVs). The larger LDCVs typically have diameters around 150−300 nm and an inner ...

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Section: Resultsmentioning

confidence: 99%

Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles

et al. 2016

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“…IGF-II also The spikes elicited are of similar size and time course for the two stimulation protocols. This is expected for an exocytosis measurement because the amount released per vesicle and the amount detected in a spike will be independent of the method used to initiate exocytosis (30,31). In both cases, the spikes are superimposed over a broad envelope, although the envelope is more obvious with IGF-II stimulation.…”

Section: Resultsmentioning

confidence: 99%

“…3B). In previous studies, this method has been used to detect exocytosis from adrenal chromaffin cells, PC-12 cells, mast cells, and individual pancreatic ␤ cells (23,(30)(31)(32)(33)(34). The anodic current resulting from a secreted substance appears as a series of spikes that indicate concentration pulses occurring at the electrode surface.…”

Section: Resultsmentioning

confidence: 99%

Insulin-like growth factor II signaling through the insulin-like growth factor II/mannose-6-phosphate receptor promotes exocytosis in insulin-secreting cells

Zhang

Tally

Larsson

et al. 1997

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

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The insulin-like growth factor II (IGF-II)͞ mannose-6-phosphate (M-6-P) receptor is known to participate in endocytosis as well as sorting of lysosomal enzymes and is involved in membrane trafficking through rapid cycling between cytosolic membrane compartments and the plasma membrane. Here we demonstrate that IGF-II, acting through the IGF-II͞M-6-P receptor, promotes exocytosis of insulin in the pancreatic ␤ cell. The effect of IGF-II was evoked at nonstimulatory concentrations of glucose, was mediated by a pertussis toxin sensitive GTP-binding protein, was dependent on protein kinase C-induced phosphorylation, and was independent of changes in cytoplasmic free Ca 2؉ concentration. Since the applied concentration of IGF-II is within the range normally found free in circulation in humans, this novel signaling pathway for the IGF-II͞M-6-P receptor is likely to be involved in modulation of insulin exocytosis under physiological conditions.

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“…Release of catecholamines from a single vesicle can be detected electrochemically 7 . The initial flux of catecholamines through the early narrow fusion pore can be measured as an amperometric foot signal preceding the amperometric spike 3,5,8 .…”

mentioning

confidence: 99%

Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore

2007

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Release of charged neurotransmitter molecules through a narrow fusion pore requires charge compensation by other ions. It has been proposed that this may occur by ion flow from the cytosol through channels in the vesicle membrane, which would generate a net outward current. We tested this hypothesis in chromaffin cells using cell-attached patch amperometry measuring simultaneously catecholamine release from single vesicles and ionic current across the patch membrane. No detectable current was associated with catecholamine release indicating that <2% (if any) of cations enter the vesicle through its membrane. Instead we show that flux of catecholamines through the fusion pore, measured as an amperometric foot signal, decreases when the extracellular cation concentration is reduced. The results reveal that the rate of transmitter release through the fusion pore is coupled to net Na + influx through the fusion pore as predicted by electrodiffusion theory applied to fusion pore permeation and suggest a prefusion rather than postfusion role for vesicular cation channels.Neurotransmitter and hormone release occurs by exocytosis, which begins with the formation of a narrow fusion pore 1,2 . Fusion pores in mast cells and chromaffin cells have typically an initial conductance of ~330 pS 1,3 through which vesicular serotonin and catecholamines are released, respectively 4,5 . The initial fusion pore of a synaptic vesicle is typically >280 pS and release of neurotransmitter should occur with a time constant <500 μs due to the small vesicle volume 6 . Many neurotransmitters and hormones are organic ions that carry charge with them. Among these acetylcholine, serotonin and catecholamines are mostly monovalent cations at physiological or more acidic vesicular pH. Efflux of charged molecules through a narrow fusion pore would rapidly charge the small capacitance of the vesicle and a mechanism of charge compensation is required.Release of catecholamines from a single vesicle can be detected electrochemically 7 . The initial flux of catecholamines through the early narrow fusion pore can be measured as an amperometric foot signal preceding the amperometric spike 3,5,8 . The amplitude of amperometric foot currents is typically ~5 pA. Since most catecholamine molecules will be in monovalent cationic form but two electrons are transferred per molecule in the oxidation giving rise to the amperometric current 9 , the catecholamines released during the foot signal carry an ionic current of ~2.5 pA with them. This would charge a typical bovine chromaffin granule 4correspondence should be addressed to M.L. (ml95@cornell.edu).

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Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells.

Cited by 832 publications

References 24 publications

Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles

Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles

Insulin-like growth factor II signaling through the insulin-like growth factor II/mannose-6-phosphate receptor promotes exocytosis in insulin-secreting cells

Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore

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