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.     

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.     

Vesicular exocytosis under hypotonic conditions shows two distinct populations of dense core vesicles in bovine chromaffin cells.

Several previous reports have discussed the effects of external osmolarity on vesicular exocytotic processes. However, few of these studies considered hypotonic conditions on chromaffin cells. Herein, the exocytosis of catecholamines by chromaffin cells was investigated in a medium of low osmolarity (200 mOsm) by amperometry at carbon fiber microelectrodes. It is observed that the frequency of the exocytotic events is significantly higher under hypotonic conditions than under physiological conditions (315 mOsm). This further confirms that the swelling of the polyelectrolytic matrix (which follows ionic exchanges) contained in dense core vesicles is the energetic driving force of the exocytotic phenomenon, being favored by a lower osmolarity. The mean amount of catecholamines released during secretory events also increases importantly under the hypotonic condition. This may be rationalized by the coexistence of two distinct populations of dense core vesicles with a relative content ratio of 4.7. The larger content population is favored under hypotonic conditions but plays only a side role under isotonic conditions.     

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.     

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.     

Synthesis,characterization and cytotoxicity of Pt(II), Pd(II), Cu(II) and Zn(II) complexes with 4’‐substituted terpyridine

Eight novel Pt(II), Pd(II), Cu(II) and Zn(II) complexes with 4’‐substituted terpyridine were synthesized and characterized by elemental analysis, UV, IR, NMR, electron paramagnetic resonance, high‐resolution mass spectrometry and molar conductivity measurements. The cytotoxicity of these complexes against HL‐60, BGC‐823, KB and Bel‐7402 cell lines was evaluated by MTT assay. All the complexes displayed cytotoxicity with low IC₅₀ values (<20 μm ) and showed selectivity. Complexes 3 , 5 , 7 and 8 exerted 9‐, 5‐, 12‐ and 7‐fold higher cytotoxicity than cisplatin against Bel‐7402 cell line. The cytotoxicity of complexes 3 , 5 , 6 , 7 and 8 was higher than that of cisplatin against BGC‐823 cell line. Complexes 3 , 7 and 8 showed similar cytotoxicity to cisplatin against KB cell line. Complex 7 exhibited higher cytotoxicity than cisplatin against HL‐60 cell line. Among these complexes, complex 7 demonstrated the highest in vitro cytotoxicity, with IC₅₀ values of 1.62, 3.59, 2.28 and 0.63 μm against HL‐60, BGC‐823, Bel‐7402 and KB cells lines, respectively. The results suggest that the cytotoxicity of these complexes is related to the nature of the terminal group of the ligand, the metal center and the leaving groups. Copyright © 2013 John Wiley & Sons, Ltd.     

Unveiling the Role of DJ‐1 Protein in Vesicular Storage and Release of Catecholamine with Nano/Micro‐Tip Electrodes

DJ‐1 protein deficiency caused by PARK7 gene mutation has been suggested to closely relate to Parkinson's disease (PD), mainly through the attenuation D2 dopamine receptor activity in mice; however, whether or how it affects the vesicular storage and exocytosis of neurochemicals remains unclear. By using electrochemical methods at a single vesicle/cell level with nano/micro‐tip electrodes, we for the first time find that DJ‐1 protein deficiency caused by PARK7 gene knockout (KO) in mice has little effect on vesicular catecholamine content but significantly prolongs the exocytotic events, especially the closing time of exocytotic fusion pores. Further studies suggest the inhibition of α‐synuclein aggregation by DJ‐1 protein might be one way that DJ‐1 protein acts on neurotransmission. This finding offers the first direct link between DJ‐1 protein deficiency and vesicular chemical storage and release of chemicals, providing a new chemical insight into the pathology of PD caused by PARK7 gene mutation.     

Imine‐N‐Heterocyclic Carbenes as Versatile Ligands in Ruthenium(II) p‐Cymene Anticancer Complexes: A Structure–Activity Relationship Study

A family of novel imine‐N‐heterocyclic carbene ruthenium(II) complexes of the general formula [(η⁶‐p‐cymene)Ru(C^N)Cl]PF₆⁻ (where C^N is an imine‐N‐heterocyclic carbene chelating ligand with varying substituents) have been prepared and characterized. In this imine‐N‐heterocyclic carbene chelating ligand framework, there are three potential sites that can be modified, which distinguishes this class of ligand and provides a body of flexibilities and opportunities to tune the cytotoxicity of these ruthenium(II) complexes. The influence of substituent effects of three tunable domains on the anticancer activity and catalytic ability in converting coenzyme NADH to NAD⁺ is investigated. This family of complexes displays an exceedingly distinct anticancer activity against A549 cancer cells, despite their close structural similarity. Complex 9 shows the highest anticancer activity in this series against A549 cancer cells (IC₅₀=14.36 μm ), with an approximately 1.5‐fold better activity than the clinical platinum drug cisplatin (IC₅₀=21.30 μm ) in A549 cancer cells. Mechanistic studies reveal that complex 9 mediates cell death mainly through cell stress, including cell cycle arrest, inducing apoptosis, increasing intracellular reactive oxygen species (ROS) levels, and depolarization of the mitochondrial membrane potential (MMP). Furthermore, lysosomal damage is also detected by confocal microscopy.     

Experimental verification of an equivalent circuit for the characterization of electrothermal micropumps: High pumping velocities induced by the external inductance at driving voltages below 5 V

Electrothermal micropumps (ETμPs) use local heating to create conductivity and permittivity gradients in the pump medium. In the presence of such gradients, an external AC electric field influences smeared spatial charges in the bulk of the medium. When there is also a symmetry break, the field‐charge interaction results in an effective volumetric force resulting in medium pumping. The advantages of the ETμP principle are the absence of moving parts, the opportunity to passivate all the pump structures, homogeneous pump‐channel cross‐sections, as well as force plateaus in broad frequency ranges. The ETμPs consisted of a DC‐heating element and AC field electrodes arranged in a 1000 μm × 250 μm × 60 μm (length × width × height) channel. They were processed as platinum structures on glass carriers. An equivalent‐circuit diagram allowed us to model the frequency‐dependent pumping velocities of passivated and nonpassivated ETμPs, which were measured at medium conductivities up to 1.0 S/m in the 300 kHz to 52 MHz frequency range. The temperature distributions within the pumps were controlled by thermochromic beads. Under resonance conditions, an additional inductance induced a tenfold pump‐velocity increase to more than 50 μm/s at driving voltages of 5 V_rms. A further miniaturization of the pumps is viewed as quite feasible.     

Direct Quantification of Nanoplastics Neurotoxicity by Single-Vesicle Electrochemistry

Nanoplastics are recently recognized as neurotoxic factors for the nervous systems. However, whether and how they affect vesicle chemistry (i.e., vesicular catecholamine content and exocytosis) remains unclear. This study offers the first direct evidence for the nanoplastics-induced neurotoxicity by single-vesicle electrochemistry. We observe the cellular uptake of polystyrene (PS) nanoplastics into model neuronal cells and mouse primary neurons, leading to cell viability loss depending on nanoplastics exposure time and concentration. By using single-vesicle electrochemistry, we find the reductions in the vesicular catecholamine content, the frequency of stimulated exocytotic spikes, the neurotransmitter release amount of single exocytotic event, and the membrane-vesicle fusion pore opening-closing speed. Mechanistic investigations suggest that PS nanoplastics can cause disruption of filamentous actin (F-actin) assemblies at cytomembrane zones and change the kinetic patterns of vesicle exocytosis. Our finding shapes the first quantitative picture of neurotoxicity induced by high-concentration nanoplastics exposure at a single-cell level.     

Glutathione modified CdTe quantum dots as a label for studying DNA interactions with platinum based cytostatics

Cisplatin, carboplatin, and oxaliplatin represent three generations of platinum based drugs applied successfully for cancer treatment. As a consequence of the employment of platinum based cytostatics in the cancer treatment, it became necessary to study the mechanism of their action. Current accepted opinion is the formation of Pt‐DNA adducts, but the mechanism of their formation is still unclear. Nanomaterials, as a progressively developing branch, can offer a tool for studying the interactions of these drugs with DNA. In this study, fluorescent CdTe quantum dots (QDs, λ_em = 525 nm) were employed to investigate the interactions of platinum cytostatics (cisplatin, carboplatin, and oxaliplatin) with DNA fragment (500 bp, c = 25 μg/mL). Primarily, the fluorescent behavior of QDs in the presence of platinum cytostatics was monitored and major differences in the interaction of QDs with tested drugs were observed. It was found that the presence of carboplatin (c = 0.25 mg/mL) had no significant influence on QDs fluorescence; however cisplatin and oxaliplatin quenched the fluorescence significantly (average decrease of 20%) at the same concentration. Subsequently, the amount of platinum incorporated in DNA was determined by QDs fluorescence quenching. Best results were reached using oxaliplatin (9.4% quenching). Linear trend (R² = 0.9811) was observed for DNA platinated by three different concentrations of oxaliplatin (0.250, 0.125, and 0.063 mg/mL). Correlation with differential pulse voltammetric measurements provided linear trend (R² = 0.9511). As a conclusion, especially in the case of oxaliplatin‐DNA adducts, the quenching was the most significant compared to cisplatin and nonquenching carboplatin.     

Mapping alternating current electroosmotic flow at the dielectrophoresis crossover frequency of a colloidal probe

AC electroosmotic (ACEO) flow above the gap between coplanar electrodes is mapped by the measurement of Stokes forces on an optically trapped polystyrene colloidal particle. E²‐dependent forces on the probe particle are selected by amplitude modulation (AM) of the ACEO electric field (E) and lock‐in detection at twice the AM frequency. E²‐dependent DEP of the probe is eliminated by driving the ACEO at the probe's DEP crossover frequency. The location‐independent DEP crossover frequency is determined, in a separate experiment, as the limiting frequency of zero horizontal force as the probe is moved toward the midpoint between the electrodes. The ACEO velocity field, uncoupled from probe DEP effects, was mapped in the region 1–9 μm above a 28 μm gap between the electrodes. By use of variously sized probes, each at its DEP crossover frequency, the frequency dependence of the ACEO flow was determined at a point 3 μm above the electrode gap and 4 μm from an electrode tip. At this location the ACEO flow was maximal at ～117 kHz for a low salt solution. This optical trapping method, by eliminating DEP forces on the probe, provides unambiguous mapping of the ACEO velocity field.     

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.     

Synergistic Photobactericidal Activity Based on Ultraviolet‐A Irradiation and Ferulic Acid Derivatives

Ultraviolet‐A (UV‐A)‐mediated bactericidal activity was enhanced by combined treatment with trans‐ferulic acid (trans‐FA, compound 1 ) or its derivatives. Derivative compounds 4 and 10 contain a phenyl group or an l ‐tyrosine HCl tert‐butyl ester, respectively, linked to the carboxyl group of trans‐FA. Of the three compounds, 10 exhibited the highest synergistic activity in a photobactericidal assay based on treating Escherichia coli with a derivative compound and UV‐A irradiation (wavelength 350–385 nm). Inactivation of viable cells at a 4.9 J cm^?2 UV‐A fluence increased from 1.90 to 5.19 logs in the presence of 10 (100 μm ); a 4.95‐log inactivation was achieved with 10 (5 μm ) and a 7.4 J cm^?2 UV‐A fluence. Addition of antioxidants significantly suppressed photosynergistic bactericidal activity, suggesting that reactive oxygen species (ROS) are involved in the combined bactericidal mechanism. Flow cytometry revealed that combined treatment with UV‐A and compound 10 , which showed the highest photobactericidal activity, generates an excess of oxidative radicals in bacterial cells. The bactericidal activity of compound 10 may be due to electrostatic interaction between the molecule's cationic moiety and the cell surface, followed by amplification of ROS generation in the cells.     

Electric‐field‐controlled synthesis of HPMA hydrogels containing self‐organized arrays of micro‐channels

We report the synthesis of poly N‐(2‐hydroxypropyl)methacrylamide ordered arrays of fluid filled channels. The polymerization and crosslinking reactions are carried out under the influence of a constant electric field (60 V/cm). A charged comonomer, immobiline (pK 3.6), and porogen, polyethylene glycol (PEG) are added to the pregel solutions. Scanning electron microscopy reveals that the channels have a typical diameter of 2–25 μm and are oriented parallel to the electric field employed during synthesis. The self‐organization of channels occurs around an optimal PEG concentration of 8.6 wt/vol %, whereas significantly higher or lower concentrations yield random, isotropic pore structures. Moreover, tensile strength measurements show that the mechanical stability increases with decreasing concentration of PEG. Rheology experiments reveal that the swelling degree of these superabsorbant hydrogels increases with increasing PEG. Possible applications of these microstructured hydrogels as bidirectional scaffolds for regenerating neurons in the injured spinal cord are discussed. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 2593–2600, 2007     

Monodispersed submicron porous silica particles functionalized with CD derivatives for chiral CEC

Rapid and efficient enantioseparation of halogen aryl alcohols and β‐blockers propranolol and pindolol in packed bed CEC (p‐CEC) using as‐prepared submicron porous silica chiral stationary phases (CSPs) has been achieved. Monodispersed 0.66 and 0.81 μm chiral submicron porous silica spheres were prepared using tetramethoxysilane and hexadecyltrimethylammonium bromide, followed by a hydrothermal treatment method with ammonia–ethanol to expand the pore of silica spheres without changing their spherical morphology. A proper specific surface of ca. 230 m²/g and pore sizes average of 6–8 nm were obtained by this method. The submicron porous silica spheres were modified with mono‐6‐phenylcarbamoylated β‐CD via thiol‐en radical addition. They were packed into 9 cm 50 μm id capillary columns with photopolymerized monolithic frits. These submicron CSPs showed greater column efficiency (about 476 000 plates/m for 4‐iodophenyl‐1‐ethanol) and higher resolution than the corresponding 3 μm CSP.     

Diazido Mixed‐Amine Platinum(IV) Anticancer Complexes Activatable by Visible‐Light Form Novel DNA Adducts

Platinum diam(m)ine complexes, such as cisplatin, are successful anticancer drugs, but suffer from problems of resistance and side‐effects. Photoactivatable Pt^IV prodrugs offer the potential of targeted drug release and new mechanisms of action. We report the synthesis, X‐ray crystallographic and spectroscopic properties of photoactivatable diazido complexes trans,trans,trans‐[Pt(N₃)₂(OH)₂(MA)(Py)] ( 1 ; MA=methylamine, Py=pyridine) and trans,trans,trans‐[Pt(N₃)₂(OH)₂(MA)(Tz)] ( 2 ; Tz=thiazole), and interpret their photophysical properties by TD‐DFT modelling. The orientation of the azido groups is highly dependent on H bonding and crystal packing, as shown by polymorphs 1 p and 1 q . Complexes 1 and 2 are stable in the dark towards hydrolysis and glutathione reduction, but undergo rapid photoreduction with UVA or blue light with minimal amine photodissociation. They are over an order of magnitude more potent towards HaCaT keratinocytes, A2780 ovarian, and OE19 oesophageal carcinoma cells than cisplatin and show particular potency towards cisplatin‐resistant human ovarian cancer cells (A2780cis). Analysis of binding to calf‐thymus (CT), plasmids, oligonucleotide DNA and individual nucleotides reveals that photoactivated 1 and 2 form both mono‐ and bifunctional DNA lesions, with preference for G and C, similar to transplatin, but with significantly larger unwinding angles and a higher percentage of interstrand cross‐links, with evidence for DNA strand cross‐linking further supported by a comet assay. DNA lesions of 1 and 2 on a 50 bp duplex were not recognised by HMGB1 protein, in contrast to cisplatin‐type lesions. The photo‐induced platination reactions of DNA by 1 and 2 show similarities with the products of the dark reactions of the Pt^II compounds trans‐[PtCl₂(MA)(Py)] ( 5 ) and trans‐[PtCl₂(MA)(Tz)] ( 6 ). Following photoactivation, complex 2 reacted most rapidly with CT DNA, followed by 1 , whereas the dark reactions of 5 and 6 with DNA were comparatively slow. Complexes 1 and 2 can therefore give rapid potent photocytotoxicity and novel DNA lesions in cancer cells, with no activity in the absence of irradiation.     

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.     

Comparison of A2E Cytotoxicity and Phototoxicity with all-trans-Retinal in Human Retinal Pigment Epithelial Cells†

All-trans-retinal is the precursor of A2E, a fluorophore within lipofuscin, which accumulates in human retinal pigment epithelial (hRPE) cells and contributes to age-related macular degeneration. Here we have compared the in vitro dark cytotoxicity and visible-light-mediated photoreactivity of all-trans-retinal and A2E in hRPE cells. All-trans-retinal caused distinct cytotoxicity in hRPE cells measured with cell metabolic activity (MTS) and lactate dehydrogenase release assays. Significant increases in intracellular oxidized glutathione (GSSG), extracellular GSH and GSSG levels and lipid hydroperoxide production were observed in cells incubated in the dark with 25 and 50 μm all-trans-retinal. Light modified all-trans-retinal’s harmful action and decreased extracellular glutathione and hydroperoxide levels. A2E (<25 μm ) did not affect cell metabolism or cytoplasmic membrane integrity in the dark or when irradiated. 25 μm A2E raised the intracellular GSSG level in hRPE cells to a much smaller extent than 25 μm all-trans-retinal. A2E did not induce glutathione efflux or hydroperoxide generation in the dark or after irradiation. These studies support our previous conclusions that although A2E may be harmful at high concentrations or when oxidized, its phototoxic properties are insignificant compared to those of all-trans-retinal. The endogenous production of A2E may serve as a protective mechanism to prevent damage to the retina by free all-trans-retinal.     

Impacts of Forced Convection Generated via High Precision Stirring on Scanning Electrochemical Microscopy Experiments in Feedback Mode

In this study, the effects of forced convection on scanning electrochemical microscopy (SECM) experiments in feedback mode using ferrocenemethanol as redox mediator are presented. Forced convection, which enhances the mass transfer inside the system, was generated via an electrical high precision stirrer integrated into the SECM setup. A thin‐film interdigitated array electrode serving as model substrate was investigated with probe scan curves in z‐direction and SECM imaging in constant height mode utilizing ultramicroelectrodes (UME) with diameters (d_probe) of 25 μm and 12.5 μm. It was found that forced convection increased the overall current during SECM imaging without distorting distinctive features of the imaged structure when working with a 25 μm UME at substrate‐to‐tip distances of 14 μm and 11 μm. Furthermore, the electrochemical contrast was improved under hydrodynamic conditions for a substrate‐to‐tip distance of 11 μm and scan rates of 5 μm s^?1, 10 μm s^?1, 20 μm s^?1 and 40 μm s^?1. When further decreasing the gap between the UME and the substrate to 9 μm almost no effects of the forced convection were observed. Consequently, for a 25 μm UME, forced convection led to higher currents and improved performance during SECM experiments in feedback mode at substrate‐to‐tip distances of 14 μm and 11 μm, whereas no effects were observed for a 12.5 μm UME at a distance of 8 μm.