Miklos Gratzl

Release of dopamine and norepinephrine by hypoxia from PC-12 cells.

We examined the effects of hypoxia on the release of dopamine (DA) and norepinephrine (NE) from rat pheochromocytoma 12 (PC-12) cells and assessed the involvement of Ca2+ and protein kinases in stimulus-secretion coupling. Catecholamine release was monitored by microvoltammetry using a carbon fiber electrode as well as by HPLC coupled with electrochemical detection (ECD). Microvoltammetric analysis showed that hypoxia-induced catecholamine secretion (Po 2 of medium ∼40 mmHg) occurred within 1 min after the onset of the stimulus and reached a plateau between 10 and 15 min. HPLC-ECD analysis revealed that, at any level of Po 2, the release of NE was greater than the release of DA. In contrast, in response to K+ (80 mM), DA release was ∼11-fold greater than NE release. The magnitude of hypoxia-induced NE and DA releases depended on the passage, source, and culture conditions of the PC-12 cells. Omission of extracellular Ca2+ or addition of voltage-gated Ca2+ channel blockers attenuated hypoxia-induced release of both DA and NE to a similar extent. Protein kinase inhibitors, staurosporine (200 nM) and bisindolylmaleimide I (2 μM), on the other hand, attenuated hypoxia-induced NE release more than DA release. However, protein kinase inhibitors had no significant effect on K+-induced NE and DA releases. These results demonstrate that hypoxia releases catecholamines from PC-12 cells and that, for a given change in Po 2, NE release is greater than DA release. It is suggested that protein kinases are involved in the enhanced release of NE during hypoxia.

Continuous in Situ Electrochemical Monitoring of Doxorubicin Efflux from Sensitive and Drug-Resistant Cancer Cells

One of the least well understood problems in cancer chemotherapy is the cross-resistance of certain tumor cells to a series of chemically unrelated drugs. Multidrug resistance (MDR) can be attributed to several different biophysical processes, among them increased drug efflux. This has been found to correlate with overexpression of the cell surface 170-kDa P-glycoprotein that actively excludes cytotoxic drugs against their concentration gradient. To better understand MDR, experimental methods are needed to study drug efflux from cancer cells. Continuous measurement of efflux of nonfluorescent drugs on the same cell culture in situ, or assessing efflux from a few cells or even a single cell, is beyond the capabilities of existing technologies. In this work, a carbon fiber (CF) microelectrode is used to monitor efflux of doxorubicin from a monolayer of two cell lines: an auxotrophic mutant of Chinese hamster ovary cells, AUXB1, and its MDR subline, CHRC5. Because doxorubicin is both fluorescent and electroactive, the results could be validated against existing data obtained optically and with other techniques on the same cell lines, with good agreement found. The electrochemical detection, however, is capable of in situ monitoring with high temporal resolution and is suitable for single-cell studies.

Shape matters: the diffusion rates of TMV rods and CPMV icosahedrons in a spheroid model of extracellular matrix are distinct

Nanomaterial-based carrier systems hold great promise to deliver therapies with increased efficacy and reduced side effects. While the state-of-the-art carrier system is a sphere, recent data indicate that elongated rods and filaments have advantageous flow and margination properties, resulting in enhanced vascular targeting and tumor homing. Here, we report on the distinct diffusion rates of two bio-inspired carrier systems: 30 nm-sized spherical cowpea mosaic virus (CPMV) and 300×18 nm-sized tobacco mosaic virus (TMV) with a tubular structure, using a spheroid model of the tumor microenvironment and fluorescent imaging.

Impedance measurements for pressed-pellet electrode membranes based on silver iodide and silver iodide/silver sulfide with solution contacts

Abstract Impedance characteristics of pressed pelletm membranes based on silver iodide, mixed AgI Ag 2 S with molar ratios of 10:1, 1:1 and 1:10, and silver sulfide are investigated by using solution contacts and a computer-controlled automatic measuring system. As membrane bulk impedances were commensurable with those of contacting solutions, special regression methods were necessary for evaluation. Typical resistivities were, in the order indicated above, as follows: 14, 2, 0.3, 0.1 and 0.1 kΩ cm, respectively. Thus, mixing of silver sulfide into the silver iodide matrix decreases dramatically the membrane resistance. These measured bulk membrane resistances are not affected by changes in composition and concentration of bathing solutions, and even the low-frequencies parts of impedance plots were not influenced by different stirring conditions, even in presence of corroding solutions such as thiosulfate or cyanide. Sorption also had no manifest effect on the impedance characteristics, but increasing pressure during membrane preparation or heat treatment significantly increased membrane resistances.

Computational modeling and analysis of iron release from macrophages.

A major process of iron homeostasis in whole-body iron metabolism is the release of iron from the macrophages of the reticuloendothelial system. Macrophages recognize and phagocytose senescent or damaged erythrocytes. Then, they process the heme iron, which is returned to the circulation for reutilization by red blood cell precursors during erythropoiesis. The amount of iron released, compared to the amount shunted for storage as ferritin, is greater during iron deficiency. A currently accepted model of iron release assumes a passive-gradient with free diffusion of intracellular labile iron (Fe2+) through ferroportin (FPN), the transporter on the plasma membrane. Outside the cell, a multi-copper ferroxidase, ceruloplasmin (Cp), oxidizes ferrous to ferric ion. Apo-transferrin (Tf), the primary carrier of soluble iron in the plasma, binds ferric ion to form mono-ferric and di-ferric transferrin. According to the passive-gradient model, the removal of ferrous ion from the site of release sustains the gradient that maintains the iron release. Subcellular localization of FPN, however, indicates that the role of FPN may be more complex. By experiments and mathematical modeling, we have investigated the detailed mechanism of iron release from macrophages focusing on the roles of the Cp, FPN and apo-Tf. The passive-gradient model is quantitatively analyzed using a mathematical model for the first time. A comparison of experimental data with model simulations shows that the passive-gradient model cannot explain macrophage iron release. However, a facilitated-transport model associated with FPN can explain the iron release mechanism. According to the facilitated-transport model, intracellular FPN carries labile iron to the macrophage membrane. Extracellular Cp accelerates the oxidation of ferrous ion bound to FPN. Apo-Tf in the extracellular environment binds to the oxidized ferrous ion, completing the release process. Facilitated-transport model can correctly predict cellular iron efflux and is essential for physiologically relevant whole-body model of iron metabolism.

Serum cholinesterase assay using a reagent-free micro pH-stat

Enzyme activities in body fluids are often used as diagnostic markers for physiological conditions and diseases. Common enzyme assays use optical methods that often require the use of pseudosubstrates and associated dyes. Here we introduce a reagent-free micro pH-stat that can determine absolute enzyme activity without the need for exogenic reagents. This approach employs electrolysis for precise dosing of the requisite acid or base titrant to stat the pH of the sample. The micro pH-stat is based on the rotating sample system (RSS), a convection platform for microliter drops. Activities of serum cholinesterase in fetal bovine serum and human serum were analyzed with this approach. The performance of this system is comparable to that of standard techniques (r(2)=0.99), yet it offers a broader range of detection. The reagent-free micro pH-stat has potential to be developed as a miniaturized device for point-of-care testing.

Anomalies of deconvolution via discrete Fourier transform: a case study on assessing transport at live cell preparations

Deconvolution is one of the most frequently used mathematical operations in the evaluation of data in many branches of science and engineering. Analytical chemistry is no exception: deconvolution is part of routine methodologies in major instrumental techniques such as many modalities of spectroscopy and chromatography. The need for proper deconvolution, however, is often overlooked in the emerging field of bio-analytical chemistry as applied to live tissue in vivo, or cells in vitro. It is in this context that some of the major problems inherent to deconvolution, and to one of its most commonly used methodologies, deconvolution via discrete Fourier transform (DFT), are explored in this work. To illustrate the nature and severity of the difficulties involved and the resulting errors and potential for misinterpretation, the problem of assessing cellular processes with different microsensors is chosen as an example here. In particular, cancer multidrug resistance (MDR) modulated doxorubicin (DOX) efflux from drug sensitive cells derived from Chinese hamster ovary cancer, and their MDR subline, are discussed. DOX concentration monitored with a carbon fiber microelectrode at close proximity to a monolayer of these cells can be perceived as the result of convolution of cellular efflux with extracellular diffusion. Deconvolution via DFT to reconstruct the efflux process, however, leads to patterns that are very far from realistic. The observations and conclusions made using this example are relevant to other areas of analytical chemistry, and science and engineering in general that use deconvolution methods in processing experimental results of many types.

Simultaneous Visualization of Surface Topography and Concentration Field by Means of Scanning Electrochemical Microscopy Using a Single Electrochemical Probe and Impedance Spectroscopy

Scanning electrochemical microscopy visualizes concentration profiles. To determine the location of the probe relative to topographical features of the substrate, knowledge of the probe-to-sample distance at each probe position is required. The use of electrochemical impedance spectroscopy for obtaining information on the substrate-to-probe distance and on the concentration of interest using the electrochemical probe alone is suggested. By tuning the frequencies of interrogation, the probe-to-substrate distance can be derived followed by interrogation of processes that carry information on concentration at lower frequencies. These processes may include charge-transfer relaxation, diffusional relaxation at the electrode, and open-circuit potential at zero frequency. A potentiometric chloride sensing microprobe is used herein to reconstruct both topography and the concentration field at a microscopic diffusional source of chloride.

Modelling the response function of enzyme-based optical glucose-sensing capsules

A theoretical model for submillimetre-sized optical glucose sensors based on microscopic pH-sensitive optode beads and glucose oxidase (GOX) inside hydrophilic membrane capsules with ca. 12 μm thickness is presented. In this model, glucose influx and gluconic acid efflux across the capsule membrane are combined with enzymatic kinetics inside the capsule. Thereby, a simple model predicting the sensor responses with different permeabilities of the capsule membranes is obtained. The permeability of the capsule membranes for glucose and gluconic acid was successfully modified by changing the monomer ratio between 2-hydroxyethyl methacrylate (HEMA) and polyethylene glycol methacrylate (PEGMA) in the preparation of poly(HEMA-co-PEGMA)-based capsule membranes. Excellent agreements between the predicted sensor responses and the experimentally obtained ones were achieved in buffer solutions containing glucose at physiologically relevant concentrations. Consequently, this model can predict enhancement of the sensor response for glucose by reducing the gluconic acid efflux, and provides a general precept for the fabrication of enzyme-based optical sensors with enhanced responses.

Sample/reagent adsorption on alumina versus Pyrex substrates of microfabricated electrochemical sensors

Platinum redox sensors have been microfabricated for potentiometric detection in diffusional microtitration of microliter-size fluid samples. Two different materials, alumina ceramic and Pyrex glass, have been studied for their suitability as sensor substrates. Adsorptions of reagent and/or sample on an alumina ceramic substrate and a Pyrex glass substrate are compared. Using iodimetric redox titration, significant adsorption of both reagent and sample occurs on alumina ceramic substrates. This causes contamination and cross-contamination of samples, leading to highly irreproducible results. Reproducibility with a 1.6% coefficient of variation has been achieved using a platinum redox sensor deposited on a Pyrex substrate for the analysis of 20 μL sample microdroplets. Due to the large surface area relative to the small sample volume, surface processes can have a significant effect on the analysis of microsamples. Therefore, precautions must be taken to minimize problems arising from air interference and sensor/sample, sensor/reagent and reagent/sample interfaces.