Quantifying Intracellular Single Vesicular Catecholamine Concentration with Open Carbon Nanopipettes to Unveil the Effect of L-DOPA on Vesicular Structure

Abstract

Understanding the regulatory mechanisms of exocytosis is essential for uncovering the pathologies of neuronal disorders and developing related pharmaceuticals. In this work intracellular vesicle impact electrochemical cytometry (IVIEC) measurements with different-sized (50-500 nm radius) open carbon nanopipettes (CNPs) were performed to quantify the vesicular content and release kinetics of specific vesicle populations grouped by orifice sizes. Intracellular vesicles with radius below 100 nm were captured and narrowed between 50 and 100 nm. On the basis of this, single vesicular catecholamine concentrations in the intracellular environment were quantified as 0.23-1.1 M. Our results with L-3,4-dihydroxyphenylalanine (L-DOPA)-exposure indicate that L-DOPA regulates exocytosis by increasing the dense core size and vesicular content while catecholamine concentrations did not show obvious alterations. These were all achieved simultaneously and relatively noninvasively with open CNPs.

Keywords: IVIEC; L-DOPA; exocytosis; open carbon nanopipettes; single vesicular catecholamine concentration.

Figure 1

A) IVIEC measurement with open CNPs and shape transformation of single spikes after cell exposure to L‐DOPA. B) Representative amperometric traces of 50 and 100 nm open CNPs obtained from L‐DOPA treated and non‐treated chromaffin cells at 700 mV vs. Ag/AgCl. (From top to bottom, amperometric traces obtained with 50, 100 nm CNPs for the control cells and 100 nm CNPs for L‐DOPA‐exposed cells; the inset shows an amplification of the spike labelled with the red asterisk; The red arrows indicate the moment CNPs were inserted into the cytoplasm, the recording was stopped when no more spikes were observed.)

Figure 2

A) Normalized frequency histograms of vesicular content obtained from L‐DOPA treated and non‐treated chromaffin cells with 100 nm radius open CNPs. B) The averages of vesicular content obtained from L‐DOPA‐treated and non‐treated chromaffin cells with 100 nm open CNPs. (Collected from 3 isolations of chromaffin cells; 9 cells were examined under each condition; 593 and 403 spikes for the control cells and L‐DOPA‐exposed cells, respectively. The value marked with **** are statistically different with p<0.0001 versus the control (unpaired t test).)

Figure 3

Histograms of A) vesicular content and B) release kinetics of the control cells and L‐DOPA‐exposed chromaffin cells derived from different‐sized open CNPs with linear trend lines (A) and second‐order polynomial trend lines (B), respectively. (Collected from 7 isolations of chromaffin cells, these results are based on the distributions presented in Figure S4. The values marked with **** and * are statistically different with p<0.0001 and P<0.05 versus the control, respectively (unpaired t test).)

Figure 4

Underlying mechanism of vesicular structure transformation with enlarged dense core (owing to the increase of electrostatic interactions between catecholamine cations and chromogranins carrying anions) and overall vesicular size after cell exposure to L‐DOPA.