A High‐Affinity Fluorescent Sensor for Catecholamine: Application to Monitoring Norepinephrine Exocytosis

A fluorescent sensor for catecholamines, NS510, is presented. The sensor is based on a quinolone fluorophore incorporating a boronic acid recognition element that gives it high affinity for catecholamines and a turn‐on response to norepinephrine. The sensor results in punctate staining of norepinephrine‐enriched chromaffin cells visualized using confocal microscopy indicating that it stains the norepinephrine in secretory vesicles. Amperometry in conjunction with total internal reflection fluorescence (TIRF) microscopy demonstrates that the sensor can be used to observe destaining of individual chromaffin granules upon exocytosis. NS510 is the highest affinity fluorescent norepinephrine sensor currently available and can be used for measuring catecholamines in live‐cell assays.     

Exocytosis of a single bovine adrenal chromaffin cell: the electrical and morphological studies

Exocytosis of a single bovine adrenal chromaffin cell, triggered by histamine stimulation, was investigated via the electric responses detected with single-walled carbon-nanotube field-effect transistors (SWCNT-FET) and the morphological changes acquired by atomic force microscopy (AFM). Secretion of chromogranin A (CgA), stored in the vesicles of a single chromaffin cell, can be monitored in situ by the antibody against CgA (CgA-antibody) functionalized on the SWCNT-FET devices. The SWCNT-FET can further discriminate the amount of released CgA with different levels of histamine stimulations. The AFM morphological studies on a chromaffin cell indicate that the depression structures on the cell surface, caused by the histamine-evoked exocytotic fusion pores, appeared much more frequently than those without histamine stimulation or with the pretreatment of mepyramine before histamine stimulation. The vesicle diameters are about 50 nm calculated from the obtained three-dimensional AFM images. In comparison, the fusion pores of chromaffin cells stimulated by high-K (+) buffer solution were also investigated to have a wider-ranging distribution of vesicle diameters of 60-260 nm. This work demonstrates that the combination of novel techniques, SWCNT-FET and AFM, can provide further insights into the fundamental properties of exocytosis in neuroendocrine cells.     

Amperometric Measurements and Dynamic Models Reveal a Mechanism for How Zinc Alters Neurotransmitter Release

Zinc, a suspected potentiator of learning and memory, is shown to affect exocytotic release and storage in neurotransmitter‐containing vesicles. Structural and size analysis of the vesicular dense core and halo using transmission electron microscopy was combined with single‐cell amperometry to study the vesicle size changes induced after zinc treatment and to compare these changes to theoretical predictions based on the concept of partial release as opposed to full quantal release. This powerful combined analytical approach establishes the existence of an unsuspected strong link between vesicle structure and exocytotic dynamics, which can be used to explain the mechanism of regulation of synaptic plasticity by Zn²⁺ through modulation of neurotransmitter release.     

Simultaneous monitoring of monoamines,amino acids,nucleotides and neuropeptides by liquid chromatography‐tandem mass spectrometry and its application to neurosecretion in bovine chromaffin cells

The primary functions of adrenal medullary chromaffin cells are the synthesis and storage in their chromaffin vesicles of the catecholamines noradrenaline (NA) and adrenaline (AD), and their subsequent release into the bloodstream by Ca²⁺‐dependent exocytosis under conditions of fear or stress (fight or flight response). Several monoamines, nucleotides and opiates, such as leucine‐enkephalin (LENK) and methionine‐enkephalin (MENK), are also co‐stored and co‐released with the catecholamines. However, other neurotransmitters have not been studied in depth. Here, we present a novel high‐resolution liquid chromatography‐tandem mass spectrometry approach for the simultaneous monitoring of 14 compounds stored and released in bovine chromaffin cells (BCCs). We validated the analytical method according to the recommendations of the EMA and FDA by testing matrix effect, selectivity, sensitivity, precision, accuracy, stability and carry‐over. After testing on six batches of BCCs from different cultures, the method enabled simultaneous quantitative determination of monoamines (AD, NA, dopamine, serotonin, 5‐hydroxyindoleacetic acid, histamine and metanephrine), amino acids (L‐glutamic acid, γ‐aminobutyric acid), nucleotides (adenosine 5′‐diphosphate, adenosine 5′‐monophosphate, cyclic adenosine 5′‐monophosphate) and neuropeptides (LENK and MENK) in the intracellular content, basal secretion and acetylcholine induced secretion of BBCs. The high‐resolution approach used here enabled us to determine the levels of 14 compounds in the same BCC batch in only 16 min. This novel approach will make it possible to study the regulatory mechanisms of Ca²⁺ signaling, exocytosis and endocytosis using different neurotrophic factors and/or secretagogues as stimuli in primary BCC cultures. Our method is actually being applied to human plasma samples of different therapeutic areas where sympathoadrenal axis is involved in stress situations such as Alzheimer's disease, migraine or cirrhosis, to improve diagnosis and clinical practice. Copyright © 2016 John Wiley & Sons, Ltd.     

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

Using a nano-injection method, we introduced phospholipids having different intrinsic geometries into single secretory cells and used single cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC) with nanotip electrodes to monitor the effects of intracellular incubation on the exocytosis process and vesicular storage. Combining tools, this work provides new information to understand the impact of intracellular membrane lipid engineering on exocytotic release, vesicular content and fraction of chemical release. We also assessed the effect of membrane lipid alteration on catecholamine storage of isolated vesicles by implementing another amperometric technique, vesicle impact electrochemical cytometry (VIEC), outside the cell. Exocytosis analysis reveals that the intracellular nano-injection of phosphatidylcholine and lysophosphatidylcholine decreases the number of released catecholamines, whereas phosphatidylethanolamine shows the opposite effect. These observations support the emerging hypothesis that lipid curvature results in membrane remodeling through secretory pathways, and also provide new evidence for a critical role of the lipid localization in modulating the release process. Interestingly, the IVIEC data imply that total vesicular content is also affected by in situ supplementation of the cells with some lipids, while, the corresponding VIEC results show that the neurotransmitter content in isolated vesicles is not affected by altering the vesicle membrane lipids. This suggests that the intervention of phospholipids inside the cell has its effect on the cellular machinery for vesicle release rather than vesicle structure, and leads to the somewhat surprising conclusion that modulating release has a direct effect on vesicle structure, which is likely due to the vesicles opening and closing again during exocytosis. These findings could lead to a novel regulatory mechanism for the exocytotic or synaptic strength based on lipid heterogeneity across the cell membrane.

Amperometry and intracellular vesicle impact electrochemical cytometry with nanotip electrodes were used to monitor the effects on exocytosis and vesicular storage after nano-injection of phospholipids with different geometries into secretory cells.     

Reconstruction of Aperture Functions during Full Fusion in Vesicular Exocytosis of Neurotransmitters

Individual vesicular exocytosis of adrenaline by dense core vesicles in chromaffin cells is considered here as a paradigm of many situations encountered in biology, nanosciences and drug delivery in which a spherical container releases in the external environment through gradual uncovering of its surface. A procedure for extracting the aperture (opening) function of a biological vesicle fusing with a cell membrane from the released molecular flux of neurotransmitter as monitored by amperometry has been devised based on semi‐analytical expressions derived in a former work [C. Amatore, A. I. Oleinick, I. Svir, ChemPhysChem 2009 , 10, DOI: 10.1002/cphc.200900646]. This precise analysis shows that in the absence of direct information about the radius of the vesicle or about the concentration of the adrenaline cation stored by the vesicle matrix, current spikes do not contain enough information to determine the maximum aperture angle. Yet, a statistical analysis establishes that this maximum aperture angle is most probably less than a few tens of degrees, which suggests that full fusion is a very improbable event.     

Using Single‐Cell Amperometry To Reveal How Cisplatin Treatment Modulates the Release of Catecholamine Transmitters during Exocytosis

The pretreatment of cultured pheochromocytoma (PC12) cells with cis‐diamminedichloroplatinum (cisplatin), an anti‐cancer drug, influences the exocytotic ability of the cells in a dose‐dependent manner. Low concentrations of cisplatin stimulate catecholamine release whereas high concentrations inhibit it. Single‐cell amperometry reflects that 2 μm cisplatin treatment increases the frequency of exocytotic events and reduces their duration, whereas 100 μm cisplatin treatment decreases the frequency of exocytotic events and increases their duration. Furthermore, the stability of the initial fusion pore that is formed in the lipid membrane during exocytosis is also regulated differentially by different cisplatin concentrations. This study thus suggests that cisplatin influences exocytosis by multiple mechanisms.     

A Dual Functional Electroactive and Fluorescent Probe for Coupled Measurements of Vesicular Exocytosis with High Spatial and Temporal Resolution

In this work, Fluorescent False Neurotransmitter 102 (FFN102), a synthesized analogue of biogenic neurotransmitters, was demonstrated to show both pH‐dependent fluorescence and electroactivity. To study secretory behaviors at the single‐vesicle level, FFN102 was employed as a new fluorescent/electroactive dual probe in a coupled technique (amperometry and total internal reflection fluorescence microscopy (TIRFM)). We used N13 cells, a stable clone of BON cells, to specifically accumulate FFN102 into their secretory vesicles, and then optical and electrochemical measurements of vesicular exocytosis were experimentally achieved by using indium tin oxide (ITO) transparent electrodes. Upon stimulation, FFN102 started to diffuse out from the acidic intravesicular microenvironment to the neutral extracellular space, leading to fluorescent emissions and to the electrochemical oxidation signals that were simultaneously collected from the ITO electrode surface. The correlation of fluorescence and amperometric signals resulting from the FFN102 probe allows real‐time monitoring of single exocytotic events with both high spatial and temporal resolution. This work opens new possibilities in the investigation of exocytotic mechanisms.     

Cisplatin Inhibits Neurotransmitter Release during Exocytosis from Single Chromaffin Cells Monitored with Single Cell Amperometry

Chemotherapy with cisplatin induces side effects such as memory loss, confusion of thinking, and difficulties with multi-tasking. However, the mechanism of cisplatin inducing nervous dysfunction is still unknown. Herein, we examine whether and how cisplatin regulates the release of neurotransmitter during exocytosis in single chromaffin cells using single cell amperometry. The results show that cisplatin reduces the amount of transmitter released during exocytosis by reducing the duration of the exocytotic events, including the opening and closing time of the fusion pore. Furthermore, the stability of the initial fusion pore formed during exocytosis is also reduced by cisplatin. Our study holds the promise for understanding the side effects of cisplatin on the nervous system at single cell level.     

High Osmolarity and L‐DOPA Augment Release via the Fusion Pore in PC12 Cells

We have amperometrically measured dopamine release from rat pheochromocytoma cells (PC12 cells) in high osmolarity conditions with and without L ‐3,4‐dihydroxyphenylalanine (L ‐DOPA) treatment. We observe an increase in the number of release events displaying a prespike feature or “foot” when the cells are stimulated in high osmolarity saline. We also see an increase in foot area and duration when cells are stimulated in high osmolarity saline, or high osmolarity saline subsequent to incubation with the dopamine precursor L ‐DOPA in isotonic saline, which serves to increase the vesicle size. The data suggest that membrane biophysics are an important component in defining the rate, duration and amount of neurotransmitter release via the fusion pore.     

Catecholamine release from cultured bovine adrenal medullary chromaffin cells in the presence of 60-Hz magnetic fields

Effects of powerline frequency (50/60 Hz) electric and magnetic fields on the central nervous system may involve altered neurotransmitter release. This possibility was addressed by determining whether 60-Hz linearly polarized sinusoidal magnetic fields (MFs) alter the release of catecholamines from cultured bovine adrenal chromaffin cells, a well-characterized model of neural-type cells. Dishes of cells were placed in the center of each of two four-coil Merritt exposure systems that were enclosed within mu-metal chambers in matched incubators for simultaneous sham and MF exposure. Following 15-min MF exposure of the cells to flux densities of 0.01, 0.1, 1.0 or 2 mT, norepinephrine and epinephrine release were quantified by high-performance liquid chromatography (HPLC) coupled with electrochemical detection. No significant differences in the release of either norepinephrine or epinephrine were detected between sham-exposed cells and cells exposed to MFs in either the absence or presence of Bay K-8644 (2 microM) or dimethylphenylpiperazinium (DMPP, 10 microM). Consistent with these null findings is the lack of effect of MF exposure on calcium influx. We conclude that catecholamine release from chromaffin cells is not sensitive to 60-Hz MFs at magnetic flux densities in the 0.01-2 mT range.     

Screening populations of individual cells for secretory heterogeneity

Many common metabolic and neurological disorders are related to defective regulation of exocytosis at the level of single cells. In exocytosis, vesicles containing the secretory product of a given cell type fuse with the plasma membrane allowing release of the vesicular contents into the extracellular environment where the physiological action can be exerted. The typical secretory vesicle contains between 0.15 and 10 attomoles of material that is released on a millisecond timescale. Hence, detection of this process presents several chemical and analytical challenges. In this work, we utilize the native ATP, stored at high concentrations within the secretory vesicles of most neuroendocrine cells and co-released during exocytosis and during cell lysis, as a universal tracer of cellular secretion events. Organisms studied include pancreatic islets, mast cells, and Escherischia coli. Cellular processes investigated include exocytotic release, stimulated cell lysis, and programmed cell lysis.     

An ion conductor that recognizes osmotically-stressed phospholipid bilayers

A synthetic ion conductor (1), derived from cholic acid and spermine, has been found capable of recognizing osmotic stress in liposomes made from 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine [(C16:1)PC]. Thus, when large unilamellar vesicles of (C16:1)PC are placed under hypotonic conditions, the Na+/Li+ transport activity of 1 increases by as much as 1 order of magnitude, relative to isotonic conditions     

Budding and asymmetric protein microcompartmentation in giant vesicles containing two aqueous phases

We report the effect of external osmolarity on giant lipid vesicles containing an aqueous two-phase system (ATPS GVs). The ATPS, which is comprised of poly(ethyleneglycol) [PEG], dextran, and water, serves as a primitive model of the macromolecularly crowded environment of the cytoplasm. Coexisting PEG-rich and dextran-rich aqueous phases provide chemically dissimilar microenvironments, enabling local differences in protein concentration to be maintained within single ATPS GVs. The degree of biomolecule microcompartmentation can be increased by exposing the ATPS GVs to a hypertonic external solution, which draws water out of the vesicles, concentrating the polymers. Enrichment of a protein, soybean agglutinin, in the dextran-rich phase improves from 2.3-fold to 10-fold with an increase in external osmolarity from 100 to 200 mmol/kg. In some cases, budding occurs, with the bud(s) formed by partial expulsion of one of the two polymer-rich aqueous phases. Budding results in asymmetry in the internal polymer and biomolecule composition, giving rise to polarity in these primitive model cells. Budding is observed with increasing frequency as external ionic strength increases, when membrane elasticity permits, and can be reversed by decreasing external osmolarity. We note that the random symmetry-breaking induced by simple osmotic shrinkage resulted in polarity in both the structure and internal protein distribution in these primitive model cells. Budding in ATPS-containing GVs thus offers an experimental model system for investigating the effects of biochemical asymmetry on the length scale of single cells.     

ATP-stimulated transmitter release and cyclic AMP synthesis in isolated chromaffin granules.

ATP stimulates chromaffin granules from the bovine adrenal medulla to release epinephrine and specific soluble proteins. ATP analogs substituted in the beta-gamma position with either nitrogen or carbon were also found to be effective at inducing release from isolated chromaffin granules. However, an ATP analog substituted at the alpha-beta position with carbon was strongly inhibitory. Cyclic AMP was also found to be synthesized by isolated chromaffin granules under release conditions. ATP analogs were effective as substrates for adenylate cyclase in the same order as their efficiency for inducing release from vesicles. Hydrolysis at the beta-gamma linkage of ATP therefore is probably not necessary for release; however, hydrolysis at the alpha-beta position may be important in the release process. Cyclic AMP may be produced and play a regulatory role in this event.     

HPLC separation of catecholamines after derivatization with 9-fluorenylmethyl chloroformate

Summary Optimum conditions for the separation of 9-fluorenylmethyl chloroformate derivatized catecholamines by HPLC are described; three catecholamines (noradrenaline, adrenaline and dopamine) and an internal standard (epinine) were separated in less than 20 minutes under isocratic conditions. This method is 17 to 350 times more sensitive than electrochemical detection, depending on the test compounds. It has been applied to the analysis of catecholamines in urine. The sample was extracted by a metal-loaded silica prior to separation.     

ATP-induced lysis of rat parotid secretory granules: possible role of ATP in exocytotic release

Secretory vesicles isolated from a variety of mammalian tissues are known to lyse and thereby release their secretory products when exposed to ATP. This process, which will be termed ATP-induced lysis, has been studied most extensively using adrenal chromaffin-granule preparations. We report here that ATP causes the lysis of a highly purified preparation of rat parotid secretory granules. The rate of granule lysis was measured spectrophotometrically, and ATP-induced lysis was expressed as the increase in the rate of lysis (r = % lysis per min) when ATP was added. This lytic process was characterized with respect to pH, temperature, osmolarity, and the ionic composition of the media. ATP-induced lysis of parotid granules was found to have the following properties in common with the extensively characterized chromaffin-granule process: 1. It is a saturable function of ATP with half-maximal rates observed at 0.5 +/- 0.1 mM ATP. 2. It is temperature dependent, eg, r = 6.1 +/- 2.1%/min at 30 degrees C vs 12.2 +/- 2.5%/min at 37 degrees C. 3. It is inhibited in hyperosmotic media, eg, r = 5.3 +/- 0.3%/min at 0.3 OsM vs 0.8 +/- 0.2%/min at 0.4 OsM. 4. It shows a nucleotide preference of ATP = GTP greater than ADP greater than AMP greater than CTP = ITP. 5. It has an anion requirement. The above findings, combined with reports of ATP-induced lysis of cholinergic, insulin, and posterior-pituitary vesicles, imply that ATP-induced lysis may reflect an ATP-dependent property of all secretory vesicles, and as such, this vesicle property could play a similar role in each exocytotic release process. Using a model system, Miller and Racker [22] made a surprising finding that the extent of which liposomes fuse with a black lipid membrane depends on the osmotic gradient across the vesicle membrane. In view of the osmotic dependence of ATP-induced lysis in this and other secretory-vesicle preparations, we postulate that ATP may prime secretory vesicles for fusion with the plasma membrane by inducing and/or maintaining an osmotic gradient across the vesicle membrane.     

Simultaneous detection of vesicular content and exocytotic release with two electrodes in and at a single cell

We developed a technique employing two electrodes to simultaneously and dynamically monitor vesicular neurotransmitter storage and vesicular transmitter release in and at the same cell. To do this, two electrochemical techniques, single-cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC), were applied using two nanotip electrodes. With one electrode being placed on top of a cell measuring exocytotic release and the other electrode being inserted into the cytoplasm measuring vesicular transmitter storage, upon chemical stimulation, exocytosis is triggered and the amount of release and storage can be quantified simultaneously and compared. By using this technique, we made direct comparison between exocytotic release and vesicular storage, and investigated the dynamic changes of vesicular transmitter content before, during, and after chemical stimulation of PC12 cells, a neuroendocrine cell line. While confirming that exocytosis is partial, we suggest that chemical stimulation either induces a replenishment of the releasable pool with a subpool of vesicles having higher amount of transmitter storage, or triggers the vesicles within the same subpool to load more transiently at approximately 10–20 s. Thus, a time scale for vesicle reloading is determined. The effect of l-3,4-dihydroxyphenylalanine (l-DOPA), the precursor to dopamine, on the dynamic alteration of vesicular storage upon chemical stimulation for exocytosis was also studied. We found that l-DOPA incubation reduces the observed changes of vesicular storage in regular PC12 cells, which might be due to an increased capacity of vesicular transmitter loading caused by l-DOPA. Our data provide another mechanism for plasticity after stimulation via quantitative and dynamic changes in the exocytotic machinery.

Simultaneous measurements of IVIEC and SCA by two nanotip electrodes allows direct and dynamic comparison between vesicular transmitter content and vesicular transmitter release to shed light on stimulation-induced plasticity.     

Specific Features of Formation of Crystalline Silicoaluminophosphates in Grains Based on Kaolin and Phosphoric Acid

Transformations of granules based on kaolin and phosphoric acid under the conditions of vapor-phase crystallization at 170°C were studied. Specific features of the individual and cooperative action of a water vapor and a template dipropylamine on the granules were determined. It is shown that the regulation of competing processes of formation of dense non-porous phases (tridymite, cristobalite, berlinite) and microporous crystalline silicoaluminophosphates of structural types ATL and ATN can be carried out by creating transport pores in the volume of the initial granules. The formation of transport pores was provided by using of porogens (hydroxyethyl cellulose and carbon nanospheres) with the subsequent calcination of the granules. The presence of 40- and 60- nm macropores in the porous structure favored intensification of mass-exchange processes occurring within the granules under the vapor-phase crystallization conditions. This led to an increase in the selectivity of crystallization of the materials with crystal structure types AEL and ATN. The morphology of the crystals with the AEL and ATN structure and their chemical composition depend on the porogen used to prepare the starting granules.     

Cholesterol effects on vesicle pools in chromaffin cells revealed by carbon-fiber microelectrode amperometry

Cell–cell communication is often achieved via granular exocytosis, as in neurons during synaptic transmission or neuroendocrine cells during blood hormone control. Owing to its critical role in membrane properties and SNARE function, cholesterol is expected to play an important role in the highly conserved process of exocytosis. In this work, membrane cholesterol concentration is systematically varied in primary culture mouse chromaffin cells, and the change in secretion behavior of distinct vesicle pools as well as pool recovery following stimulation is measured using carbon-fiber microelectrode amperometry. Amperometric traces obtained from activation of the younger readily releasable and slowly releasable pool (RRP/SRP) vesicles at depleted cholesterol levels showed fewer sustained fusion pore features (6.1 ± 1.1% of spikes compared with 11.2 ± 1.0% for control), revealing that cholesterol content influences fusion pore formation and stability during exocytosis. Moreover, subsequent stimulation of RRP/SRP vesicles showed that cellular cholesterol level influences both the quantal recovery and kinetics of the later release events. Finally, diverging effects of cholesterol on RRP and the older reserve pool vesicle release suggest two different mechanisms for the release of these two vesicular pools.