Extracellular ATP Regulates the Vesicular Pore Opening in Chromaffin Cells and Increases the Fraction Released During Individual Exocytosis Events

Abstract

Cellular communication is a prerequisite in multicellular organisms in order to survive. Many times, this communication occurs through the highly controlled and regulated release of chemical signals through a process known as exocytosis. This process consists of organelles known as vesicles fusing with the plasma membrane to release the content inside to the extracellular space. Although the basic underpinnings of this process are known, exactly how it is regulated is still largely unknown.This thesis covers studies performed on mammalian cell lines and invertebrate neurons with the aim to further understand regulation of exocytosis. Several IV

Recent Advancements in Bipolar Electrochemical Methods of Analysis

“…For example, nanoscale electrodes provide the resolution necessary to observe the millisecond-scale release of catecholamines from a single chromaffin cell. 104,105 Further, nanoconfinement leads to the formation of steep gradients in electrical potential and chemical species concentrations that can drive rapid or selective mass transport, which has been leveraged for kinetic measurements, 106 chemical separations, 107 and charge storage applications. 108 Finally, a key motivation for nanoscale electrochemistry is that measurements made on ensembles obscure the heterogeneity extant among single entities, [109][110][111] such as individual cells, enzymes, nanoparticle catalysts, 114,115 or nanobubbles.…”

Intracellular injection of phospholipids directly alters exocytosis and the fraction of chemical release in chromaffin cells as measured by nano-electrochemistry

“…Partial release has been shown to be the dominant mode of exocytosis where only a fraction of the vesicle load is released during an event. 2,11 Vesicular content and fraction released have been proposed to play an important role in regulation and plasticity in cellular communication. 2,12,[23][24][25] The fraction of catecholamine released was found to be 74%, 48%, 83%, and 41% for the control, and PC, PE, and LPC treated cells, respectively.…”

“…Single-cell amperometry (SCA) is the most used analytical technique to study quantitatively the individual exocytosis events at the single-cell level, allowing quantication of secreted electroactive neurotransmitters. [9][10][11] Moreover, the high temporal resolution of SCA provides important information about the dynamics of the fusion pore formed during the exocytosis process. Intracellular vesicle impact electrochemical cytometry (IVIEC), a method developed in the Ewing group, is another analytical technique, in this case offering quantication of vesicular neurotransmitter content inside the cytoplasm of individual living cells using a conical nanotip electrode.…”

“…12,13 The fraction of catecholamines secreted during exocytosis at the single-cell level can be calculated by combining the results of these two techniques. 10 Recently, combination of SCA and IVIEC at carbon nanotip electrodes has been successfully applied to study the effect of several pharmacological and chemical treatments on single cell exocytosis including investigation of the effects of extracellular ATP on exocytosis of bovine chromaffin cells, 11 and to study the inuence of the cognition altering drugs (cocaine and methylphenidate) on exocytotic release and vesicle content in pheochromocytoma (PC12) cells. 10 Also, a very recent study was done by combining these two techniques to reveal the possible link between the fraction of neurotransmitter release, synaptic strength and plasticity.…”

Electrochemical Quantification of Neurotransmitters in Single Live Cell Vesicles Shows Exocytosis is Predominantly Partial

“…Incubation of chromaffin cells with ATP increases exocytotic release apparently by resulting in a longer fusion pore expansion. The increase in rise time was dependent on the ATP concentration and a longer duration of pre‐spike feet was observed [34] . The effect of ATP on exocytotic release is regulated by purinergic receptors.…”