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.     

Modeling Ca-Polyanion Crosslinking in Secretory Networks. Assessment of Charge Density and Bond Affinity in Polyanionic Secretory Networks

Materials released by secretory cells are stored inside intracellular membrane-bound vesicles. These moieties are not freely diffusible in the vesicle but remain immobilized in a Ca²⁺-crosslinked condensed-phase polyanionic polymer matrix. During exocytosis a Na⁺/Ca²⁺ ion exchange process triggers a volume phase transition resulting in massive swelling and release of the materials to the extracellular space. Here we formulate a simple model to assess Ca²⁺-ion binding from the swelling kinetics of polymer networks. We found the diffusivity of the networks (D) exhibits a power-law dependency on the Ca²⁺ concentration where D ∝ [Ca²⁺]^−2/3. The model yields an estimate of charge density and ionic affinity of the polymer chains. Studies of post-exocytic swelling kinetics in airway mucin granules, mast cell granules and granules from the microalga (Phaeocystis globosa) were used to validate predictions from our model. These results suggest that independent of the cell type, from animal to plant cells, a single polyelectrolyte interaction mechanism appear to be responsible for product release in exocytosis.     

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.     

Intracellular Electrochemical Nanomeasurements Reveal that Exocytosis of Molecules at Living Neurons is Subquantal and Complex

Since the early work of Bernard Katz, the process of cellular chemical communication through exocytosis, quantal release, has been considered to be all or none. Recent evidence has shown exocytosis to be partial or “subquantal” at single‐cell model systems, but there is a need to understand this at communicating nerve cells. Partial release allows nerve cells to control the signal at the site of release during individual events, for which the smaller the fraction released, the greater the range of regulation. Herein, we show that the fraction of the vesicular octopamine content released from a living Drosophila larval neuromuscular neuron is very small. The percentage of released molecules was found to be only 4.5 % for simple events and 10.7 % for complex (i.e., oscillating or flickering) events. This large content, combined with partial release controlled by fluctuations of the fusion pore, offers presynaptic plasticity that can be widely regulated.     

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.     

Vesicular release dynamics are altered by the interaction between the chemical cargo and vesicle membrane lipids

The release of the cargo from soft vesicles, an essential process for chemical delivery, is mediated by multiple factors. Among them, the regulation by the interaction between the chemical cargo species and the vesicular membrane, widely existing in all vesicles, has not been investigated to date. Yet, these interactions hold the potential to complicate the release process. We used liposomes loaded with different monoamines, dopamine (DA) and serotonin (5-HT), to simulate vesicular release and to monitor the dynamics of chemical release from isolated vesicles during vesicle impact electrochemical cytometry (VIEC). The release of DA from liposomes presents a longer release time compared to 5-HT. Modelling the release time showed that DA filled vesicles had a higher percentage of events where the time for the peak fall was better fit to a double exponential (DblExp) decay function, suggesting multiple kinetic steps in the release. By fitting to a desorption–release model, where the transmitters adsorbed to the vesicle membrane, the dissociation rates of DA and 5-HT from the liposome membrane were estimated. DA has a lower desorption rate constant, which leads to slower DA release than that observed for 5-HT, whereas there is little difference in pore size. The alteration of vesicular release dynamics due to the interaction between the chemical cargo and vesicle membrane lipids provides an important mechanism to regulate vesicular release in chemical and physiological processes. It is highly possible that this introduces a fundamental chemical regulation difference between transmitters during exocytosis.

The release of the cargo from soft vesicles, an essential process for chemical delivery, is mediated by multiple factors.     

NanoSIMS combined with fluorescence microscopy as a tool for subcellular imaging of isotopically labeled platinum-based anticancer drugs

Multi-elemental, isotope selective nano-scale secondary ion mass spectrometry (NanoSIMS) combined with confocal laser-scanning microscopy was used to characterize the subcellular distribution of ¹⁵N-labeled cisplatin in human colon cancer cells. These analyses indicated predominant cisplatin colocalisation with sulfur-rich structures in both the nucleus and cytoplasm. Furthermore, colocalisation of platinum with phosphorus-rich chromatin regions was observed, which is consistent with its binding affinity to DNA as the generally accepted crucial target of the drug. Application of ¹⁵N-labeled cisplatin and subsequent measurement of the nitrogen isotopic composition and determination of the relative intensities of platinum and nitrogen associated secondary ion signals in different cellular compartments with NanoSIMS suggested partial dissociation of Pt–N bonds during the accumulation process, in particular within nucleoli at elevated cisplatin concentrations. This finding raises the question as to whether the observed intracellular dissociation of the drug has implications for the mechanism of action of cisplatin. Within the cytoplasm, platinum mainly accumulated in acidic organelles, as demonstrated by a direct combination of specific fluorescent staining, confocal laser scanning microscopy and NanoSIMS. Different processing of platinum drugs in acidic organelles might be relevant for their detoxification, as well as for their mode of action.

NanoSIMS combined with fluorescence microscopy reveals subcellular structures in cancer cells where ¹⁵N-labeled cisplatin is accumulated, with implications for Pt–N bond integrity.     

Altered Lipid Composition of Secretory Cells Following Exposure to Zinc Can Be Correlated to Changes in Exocytosis

A micromolar concentration of zinc has been shown to significantly change the dynamics of exocytosis as well as the vesicle contents in a model cell line, providing direct evidence that zinc regulates neurotransmitter release. To provide insight into how zinc modulates these exocytotic processes, neurotransmitter release and vesicle content were compared with single cell amperometry and intracellular impact vesicle cytometry with a range of zinc concentrations. Additionally, time-of-flight secondary ion mass spectrometry (ToF-SIMS) images of lipid distributions in the cell membrane after zinc treatment correlate to changes in exocytosis. By combining electrochemical techniques and mass spectrometry imaging, we proposed a mechanism by which zinc changes the fusion pore and the rate of neurotransmitter release by changing lipid distributions and results in the modulation of synaptic strength and plasticity.     

Fabrication of positively charged catanionic vesicles from ion pair amphiphile with double-chained cationic surfactant

In this study, a pseudodouble-chained ion pair amphiphile, hexadecyltrimethylammonium-dodecylsulfate (HTMA-DS), was prepared from a mixture of cationic surfactant, hexadecyltrimethylammonium bromide, and anionic surfactant, sodium dodecylsulfate. Positively charged catanionic vesicles were then successfully fabricated from HTMA-DS with the addition of cationic surfactants, dialkyldimethylammonium bromide (DXDAB), including ditetradecyldimethylammonium bromide (DTDAB), dihexadecyldimethylammonium bromide, and dioctadecyldimethylammonium bromide (DODAB), with a mechanical disruption approach. The control of charge characteristic and physical stability of the catanionic vesicles through the variations of DXDAB molar fraction and alkyl chain length was then explored by size, zeta potential, and Fourier transform infrared analyses. It was found that the molecular packing and/or molecular interaction of HTMA-DS with DXDAB rather than the electrostatic repulsion between the charged vesicles dominated the physical stability of the mixed HTMA-DS/DXDAB vesicles. The presence of DTDAB, which possesses short alkyl chains, could adjust the packing of the unmatched chains of HTMA⁺ and DS^? and promote the vesicle formation. However, the weak molecular interaction due to the short chains of DTDA⁺ could not maintain the vesicle structures in long-term storage. With increasing the alkyl chain length of DXDAB, it was possible to improve the vesicle physical stability through the enhanced molecular interaction in the vesicular bilayer. However, the long alkyl chains of DODAB unmatched with those of HTMA-DS, resulting in the vesicle disintegration in long-term storage. For the formation of stable charged catanionic vesicles of HTMA-DS/DXDAB, a good match in hydrophobic chains and strong molecular interaction were preferred for the vesicle-forming molecules.     

Combined Amperometry and Electrochemical Cytometry Reveal Differential Effects of Cocaine and Methylphenidate on Exocytosis and the Fraction of Chemical Release

Amperometry with nanotip electrodes has been applied to show cocaine and methylphenidate not only trigger declines in vesicle content and exocytotic catecholamine release in a model cell line but also differentially change the fraction of transmitter released from each individual vesicle. In addition, cocaine accelerates exocytotic release dynamics while they remain unchanged after methylphenidate treatment. The parameters from pre‐spike feet for the two drugs are also in opposition, suggesting this aspect of release is affected differentially. As cocaine and methylphenidate are psychostimulants with similar pharmacologic action but have opposite effects on cognition, these results might provide a missing link between the regulation of exocytosis and vesicles and the effect of this regulation on cognition, learning, and memory. A speculative chemical mechanism of the effect of these drugs on vesicle content and exocytosis is presented.     

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

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.     

An Improved Semi-Preparative Liquid Chromatography Technique to Purify A Uremic Toxin Fraction Inhibiting Na+, K+-Atpase

Abstract

We reported previously inhibition of Na⁺,K⁺-ATPase by a fraction (fraction 2–3) of medium size uremic toxins. Due to considerable amount of sulfates in this fraction, a semi preparative high performance liquid chromatography turned out to be inadequate to isolate the active compound. Therefore we developed a novel chromatographic method using ion exchanger Sephadex QAE A25 in acidic medium. This method allows the elimination of sulfates and the partial purification of the active component.     

Zinc Regulates Chemical‐Transmitter Storage in Nanometer Vesicles and Exocytosis Dynamics as Measured by Amperometry

We applied electrochemical techniques with nano‐tip electrodes to show that micromolar concentrations of zinc not only trigger changes in the dynamics of exocytosis, but also vesicle content in a model cell line. The vesicle catecholamine content in PC12 cells is significantly decreased after 100 μm zinc treatment, but, catecholamine release during exocytosis remains nearly the same. This contrasts with the number of molecules stored in the exocytosis vesicles, which decreases, and we find that the amount of catecholamine released from zinc‐treated cells reaches nearly 100 % content expelled. Further investigation shows that zinc slows down exocytotic release. Our results provide the missing link between zinc and the regulation of neurotransmitter release processes, which might be important in memory formation and storage.     

Electrochemical Measurements of Optogenetically Stimulated Quantal Amine Release from Single Nerve Cell Varicosities in Drosophila Larvae

The nerve terminals found in the body wall of Drosophila melanogaster larvae are readily accessible to experimental manipulation. We used the light‐activated ion channel, channelrhodopsin‐2, which is expressed by genetic manipulation in Type II varicosities to study octopamine release in Drosophila. We report the development of a method to measure neurotransmitter release from exocytosis events at individual varicosities in the Drosophila larval system by amperometry. A microelectrode was placed in a region of the muscle containing a varicosity and held at a potential sufficient to oxidize octopamine and the terminal stimulated by blue light. Optical stimulation of Type II boutons evokes exocytosis of octopamine, which is detected through oxidization at the electrode surface. We observe 22700±4200 molecules of octopamine released per vesicle. This system provides a genetically accessible platform to study the regulation of amine release at an intact synapse.     

Concentration of stimulant regulates initial exocytotic molecular plasticity at single cells

Activity-induced synaptic plasticity has been intensively studied, but is not yet well understood. We examined the temporal and concentration effects of exocytotic molecular plasticity during and immediately after chemical stimulation (30 s K⁺ stimulation) via single cell amperometry. Here the first and the second 15 s event periods from individual event traces were compared. Remarkably, we found that the amount of catecholamine release and release dynamics depend on the stimulant concentration. No changes were observed at 10 mM K⁺ stimulation, but changes observed at 30 and 50 mM (i.e., potentiation, increased number of molecules) were opposite to those at 100 mM (i.e., depression, decreased number of events), revealing changes in exocytotic plasticity based on the concentration of the stimulant solution. These results show that molecular changes initiating exocytotic plasticity can be regulated by the concentration strength of the stimulant solution. These different effects on early plasticity offer a possible link between stimulation intensity and synaptic (or adrenal) plasticity.

Amperometric measurement of exocytosis (SCA) and vesicle content (IVIEC) over 15 s intervals reveals plasticity (none, potentiation, or depression), that is regulated by the concentration of stimulant solution (e.g., 30 s 10, 30, 50, and 100 mM K⁺).     

CO2-switchable vesicles-network structure transition and drug release property

A series of amphiphilic triblock polymers based on poly(ethylene glycol) (PEG) and two symmetrical poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) blocks was synthesized via the Atom Transfer Radical Polymerization (ATRP) method. Conductivity, pH, and viscosity tests demonstrated the CO₂-switchability jointly; Cryogenic transmission electron microscopy (Cryo-TEM), Dynamic light scattering (DLS) revealed the self-assembly morphology transformation from unilamellar vesicle to network structure when bubbling CO₂. These changes were all attributed to the protonation of tertiary amine groups in PDMAEMA blocks and the mechanism was proved by ^?H NMR. The vesicles have a relatively low release rate of drug; once stimulated by CO₂, the release rate will be accelerated. The polymeric vesicle has the possibility to find potential applications in drug delivery and release domains.     

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.     

Examining changes in cellular communication in neuroendocrine cells after noble metal nanoparticle exposure

As nanoparticles enjoy increasingly widespread use in commercial applications, the potential for unintentional exposure has become much more likely during any given day. Researchers in the field of nanotoxicity are working to determine the physicochemical nanoparticle properties that lead to toxicity in an effort to establish safe design rules. This work explores the effects of noble metal nanoparticle exposure in murine chromaffin cells, focusing on examining the effects of size and surface functionality (coating) in silver and gold, respectively. Carbon-fibre microelectrode amperometry was utilized to examine the effect of exposure on exocytosis function, at the single cell level, and provided new insights into the compromised functions of cells. Silver nanoparticles of varied size, between 15 and 60 nm diameter, were exposed to cells and found to alter the release kinetics of exocytosis for those cells exposed to the smallest examined size. Effects of gold were examined after modification with two commonly used 'bio-friendly' polymers, either heparin or poly (ethylene glycol), and gold nanoparticles were found to induce altered cellular adhesion or the number of chemical messenger molecules released, respectively. These results support the body of work suggesting that noble metal nanoparticles perturb exocytosis, typically altering the number of molecules and kinetics of release, and supports a direct disruption of the vesicle matrix by the nanoparticle. Overall, it is clear that various nanoparticle physicochemical properties, including size and surface coating, do modulate changes in cellular communication via exocytosis.     

The effect of resin particle size on the rate of ion release: interactions in mixed bed systems

The aim of the present study is to evaluate the influence of resin particle sizes on the rate of ions release from a mixture of ion-exchange resins (named NMTD) which supplies calcium, fluoride, and phosphate ions as the main mineral content, and to elucidate the different phenomena taking place through the related ion-exchange process. The final goal of the study, related to dental application (enamel restoration), is to limit the particle size range, since the rate of ion release is a key parameter in the successful achievement of such objective. Weak-type ion-exchange resins, loaded with the appropriate ions, were ground and sieved into granulometric fractions of bead diameters of 0.1–0.075, 0.075–0.063, and 0.063–0.05 mm. Particle size was controlled by a laser diffraction particle distribution analyzer. The experiments on the kinetics of ions release were carried out under batch conditions in artificial saliva desorption solution thermostatized at 37 °C. The release of Ca²⁺ and F^– was determined by corresponding ion-selective electrodes automatically controlled, whereas H₂PO₄^– was measured spectrophotometrically by the inductively coupled plasma–optical emission technique (ICP-OES). The results of this study show that the process of ion-exchange for the different particle size fractions of resins is critical for the study of the kinetics release of the ions immobilized in the corresponding mixed bed polymeric matrices. In fact, despite the apparent narrow range of particle sizes of the mixed bed systems studied, appreciable differences in the rate of ions release are obtained. Since the ion release rate is depending on the contact surface, an increase of factor of 2 in particle size represents an increase of an order of magnitude of the resin contact surface due to the resin porosity. In this concern, it has been observed that the rate of ions release increases when particle size decreases. The interactions occurring during the ion release from the mixed bed resins (containing calcium-, fluoride-, and phosphate-loaded resins) can be interpreted by the following phenomena: H₂PO₄^–, which hardly modifies its rate of release in the presence of Ca²⁺ and F^– in the mixture, promotes a considerable increase in the rate of Ca²⁺ release due to the formation of a calcium dihydrogen phosphate soluble complex. F^– also produces an acceleration in the rate of Ca²⁺ release due to the formation of solid CaF₂ on the surface of cationic resin particles, which in contrast leads to a decrease in the rate of F^– release.     

Visible Light and Microwave-Mediated Rapid Trapping and Release of Singlet Oxygen Using a Coordination Polymer

Due to its high reactivity and oxidative strength, singlet oxygen (¹O₂) is used in a variety of fields including organic synthesis, biomedicine, photodynamic therapy and materials science. Despite its importance, the controlled trapping and release of ¹O₂ is extremely challenging. Herein, we describe a one-dimensional coordination polymer, CP1 , which upon irradiation with visible light, transforms ³O₂ (triplet oxygen) to ¹O₂. CP1 consists of Cd^II centers bridged by 9,10-bis((E)-2-(pyridin-4-yl)vinyl)anthracene ligands which undergo a [4+2] cycloaddition reaction with ¹O₂, resulting in the generation of CP1−¹O₂ . Using microwave irradiation, CP1−¹O₂ displays efficient release of ¹O₂, over a period of 30 s. In addition, CP1 exhibits enhanced fluorescence and has an oxygen detection limit of 97.4 ppm. Theoretical calculations reveal that the fluorescence behaviour is dominated by unique through-space conjugation. In addition to describing a highly efficient approach for the trapping and controlled release of ¹O₂, using coordination polymers, this work also provides encouragement for the development of efficient fluorescent oxygen sensors.