Selective Amperometric Recording of Endogenous Ascorbate Secretion from a Single Rat Adrenal Chromaffin Cell with Pretreated Carbon Fiber Microelectrodes

Abstract

Quantitative description of ascorbate secretion at a single-cell level is of great importance in physiological studies; however, most studies on the ascorbate secretion have so far been performed through analyzing cell extracts with high performance liquid chromatography, which lacks time resolution and analytical performance on a single-cell level. This study demonstrates a single-cell amperometry with carbon fiber microelectrodes (CFEs) to selectively monitor amperometric vesicular secretion of endogenous ascorbate from a single rat adrenal chromaffin cell. The CFEs are electrochemically pretreated in a weakly basic solution (pH 9.5), and such pretreatment essentially enables the oxidation of ascorbate to occur at a relatively low potential (i.e., 0.0 V vs Ag/AgCl), and further a high selectivity for ascorbate measurement over endogenously existing electroactive species such as epinephrine, norepinephrine, and dopamine. The selectivity is ensured by much larger amperometric response at the pretreated CFEs toward ascorbate over those toward other endogenously existing electroactive species added into the solution or ejected to the electrode with a micropuffer pipet, and by the totally suppressed current response by adding ascorbate oxidase into the cell lysate. With the pretreated CFE-based single-cell amperometry developed here, exocytosis of endogenous ascorbate of rat adrenal chromaffin cells is directly observed and ensured with the calcium ion-dependent high K-induced secretion of endogenous ascorbate from the cells. Moreover, the quantitative information on the exocytosis of endogenous ascorbate is provided.

Electrochemical Monitoring of Propagative Fluctuation of Ascorbate in the Live Rat Brain during Spreading Depolarization

“…For example, ascorbate can be directly transported into neurons through the sodium‐dependent vitamin C transporter‐2, or the oxidized form of ascorbate can be transported into astrocytes and subsequently reduced to ascorbate by glucose transporters . We and others have also demonstrated a vesicular‐mediated or volume‐sensitive organic anion channel (VSOAC) mediated release of ascorbate . Among those findings, the glutamate‐ascorbate hetero‐exchange mechanism is the most invoked, in which ascorbate is released to extracellular spaces to promote glutamate uptake into cells to alleviate excitotoxicity in some pathological processes, including SD.…”

Single-entity electrochemistry at confined sensing interfaces

“…For example, we found that VC can be released in the form of nanoscale vesicles, the main pathway that is commonly preserved in the regulated secretion of neurotransmitters by neurons (Figure 15(b)). [175] Moreover, we observed for the first time that the propagative nature of VC release following the spreading diffusion process, highlighting its importance as a neuromodulator in mediating neurotransmission (Figure 15(a)). [176] Furthermore, through our longterm collaboration with Peking University Third Hospital, which specializes in designing, establishing and evaluation of clinical relevant disease models, we demonstrated that VC plays more important roles other than just as free radical scavengers for neuro-protection in the events of tinnitus , olfactory dysfunction and Vertigo [177].…”

Nanopore Electrochemistry: A Nexus for Molecular Control of Electron Transfer Reactions

“… 69 , 70 Second, material released from vesicles or droplets provides a natural ex vivo mimic of extracellular release processes, e.g., neurotransmitters released from neurons. 71 , 72 Early work addressing single nanoparticle collisions was conducted on ultramicroeletrodes by Bard et al 73 and others. 74 − 76 Since these early reports, it has become possible to sequester a few nanoparticles, or even one.…”