Indira Chatterjee

Nanosecond electric pulse-induced increase in intracellular calcium in adrenal chromaffin cells triggers calcium-dependent catecholamine release

Experimental results on the effect of a single 5-6 ns, 5-7 MV/m electric pulse on electrically excitable bovine chromaffin cells are presented. Effects on intracellular calcium level were assessed by loading the cells with the calcium-sensitive fluorescence indicator calcium green and imaging the cells during nanosecond field exposure in microelectrode chambers that were fabricated on a glass microscope slide with gold electrodes. Consistent with earlier findings that utilized different microelectrode chambers for pulse exposure, a single pulse elicited a rapid and transient rise in intracellular calcium by a mechanism that depends on extracellular calcium, which appears to enter the cells largely through voltage-gated calcium channels. In parallel experiments to assess catecholamine release, chromaffin cells were placed into electroporation cuvettes for nanosecond pulse exposure. As measured by high performance liquid chromatography coupled with electrochemical detection, a single pulse elicited an increase in both norepinephrine and epinephrine release that was also dependent on extracellular calcium and involved influx of calcium through voltage gated-calcium channels. Taken together these results indicate that a single nanosecond pulse can act as a stimulus to trigger calcium-dependent catecholamine release from chromaffin cells.

Likelihood of high rates of energy deposition in the human legs at the ANSI recommended 3-30-MHz RF safety levels

Based on physical measurements, the letter provides estimates of currents in human subjects exposed to ANSI C95.1-1982 recommended safety levels for RF EM fields. It is shown that such fields are likely to result in high SARs in the leg with values as high as 182 W/kg for the frequency band 3-30 MHz.

Modulation of intracellular Ca2+ levels in chromaffin cells by nanoelectropulses.

Exposing chromaffin cells to a single 5 ns, 5 MV/m pulse causes Ca(2+) influx and a rapid, transient rise in intracellular calcium concentration ([Ca(2+)](i)). A comparison of responses at room temperature versus 37°C revealed no effect of temperature on the magnitude of the increase in [Ca(2+)](i). The Ca(2+) transient, however, was shortened in duration almost twofold at 37°C, indicating that the rate of recovery was temperature-sensitive. Temperature also affected the interval required for a second pulse to elicit another maximal rise in [Ca(2+)](i), which was shorter at the higher temperature. In addition, a second pulse applied 5s after the first pulse was sufficient to cause cells at room temperature to become refractory to subsequent stimulation. At 37°C, cells became refractory after 5 pulses regardless of whether pulse delivery was at low (1 and 10 Hz) or high (1 kHz) rates. When refractory, cells showed no signs of swelling or uptake of the impermeant dye YO-PRO-1. These results demonstrate that temperature plays a role in determining how chromaffin cells respond to and become refractory to nanoelectropulses. They also indicate that despite the ultra-short duration of the pulses, pronounced effects on cell excitability result from the application of only very few pulses.

Mapping membrane-potential perturbations of chromaffin cells exposed to electric fields

The numerous and diverse responses of biological systems to electric fields continue to evoke interest in elucidating the fundamental mechanisms of cellular interactions with these fields. In order to address this issue for excitable cells, responses of bovine adrenal chromaffin cells exposed to DC electric fields in various orientations were investigated. Spatial variations in membrane potential were monitored in single cells as well as in complex clusters of cells using fluorescence video imaging and a voltage-sensitive dye, di-8-ANEPPS. Responses in isolated cells were in general agreement with that predicted by theory. Reversal of the electric field direction did not produce an exactly symmetrical reversal of fluorescent intensities, suggesting structural asymmetries within the cells. Clusters larger than a single cell exhibited more complex responses at the interfaces between cells that were dependent on the orientation of the regions of contact with the electric-field direction. Taken as a whole, the results of this study provide a foundation to understand the interaction of electric fields at the level of tissues and organs.

Measuring the growth of oxide films on low carbon steel at 500°C by impedance spectroscopy

Abstract Oxide films, grown on low carbon steel in air at 500°C, were evaluated by concurrent room-temperature measurements of weight gain and impedance spectroscopy (IS). Weight-gain measurements displayed the expected parabolic kinetics due to growth of a relatively thick inner layer of magnetite (Fe 3 O 4 ). The Nyquist impedance spectra exhibited depressed semicircles, which were curve-fitted to obtain values of low-frequency film resistance, R , and capacitance, C . R was linear with oxidation time due to growth of a thin, porous, high-resistivity outer layer of hematite (Fe 2 O 3 ) over the underlying low-resistivity magnetite. IS shows promise for in situ non-destructive measurement of oxide scales on large low carbon steel surfaces during service by periodic measurements of oxide impedance. Further experimental measures are discussed to adapt impedance measurements to in situ determinations of oxide thickness.

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.

A novel waveguide-based radio frequency/microwave exposure system for studying nonthermal effects on neurotransmitter Release-finite-difference time-domain modeling

A research effort is underway to identify specific radio frequency/microwave parameters in the frequency range 0.75-1.12 GHz that can produce nonthermal effects on the release of catecholamines from cultured bovine adrenal medullary chromaffin cells, an established in vitro model of neural-type cells. A well-characterized exposure system is crucial for interpreting the biological outcomes of experiments. This paper describes a novel waveguide-based exposure system that permits perfusion of the cells with a temperature-controlled balanced salt solution for online monitoring of catecholamine release from the cells during radio frequency/microwave exposure. The finite-difference time-domain method was used to optimize the exposure conditions, the goal being to achieve the maximum possible homogeneity in the distribution of the specific absorption rate at the location of the cells in the waveguide. At a frequency of 1 GHz, optimal coupling of the radio frequency/microwave field into the region containing the cells was obtained only when the cells were placed at the location of the electric field maximum of the standing wave pattern, with the electric field parallel to the plane containing the cells. In this case, the specific absorption rate distribution was found to be the highest as well as homogeneous to within 30%.

Numerical study of induced current perturbations in the vicinity of excitable cells exposed to extremely low frequency magnetic fields

Realistic three-dimensional cell morphologies were modelled to determine the current density induced in excitable cell culture preparations exposed to 60 Hz magnetic fields and to identify important factors that can influence the responses of cells to these fields. Cell morphologies representing single spherical adrenal chromaffin cells, single elongated smooth muscle cells and chromaffin cell aggregates in a Petri dish containing culture medium were modelled using the finite element method. The computations for a spherical cell revealed alterations in the magnitude and spatial distribution of the induced current density in the immediate vicinity of the cell. Maxima occurred at the equatorial sides and minima at the poles. Proximity of cells to each other as well as cell aggregate shape, size and orientation with respect to the induced current influenced the magnitude and spatial distribution of the induced current density. For an elongated cell, effects on the induced current density were highly dependent on cell orientation with respect to the direction of the induced current. These results provide novel insights into the perturbations in induced current that occur in excitable cell culture preparations and lay a foundation for understanding the mechanisms of interaction with extremely low frequency magnetic fields at the tissue level.

Design, Characterization, and Optimization of a Waveguide-Based RF/MW Exposure System for Studying Nonthermal Effects on Skeletal Muscle Contraction

A waveguide-based exposure system for studying in vitro nonthermal radio-frequency/microwave (RF/MW) effects on skeletal muscle contraction in the frequency range of 0.75-1.12 GHz was designed, characterized, and optimized. The design includes a vertical organ bath (OB) placed inside the waveguide for suspending an intact skeletal muscle from the hind limb of the mouse, i.e., flexor digitorum brevis, in an oxygenated Tyrode solution during the RF/MW exposures. Muscle contraction is stimulated by an electric field generated between two platinum electrodes and continuously measured before, during, and after RF/MW exposure by a force transducer. A temperature feedback system that controls the perfusion rate of the Tyrode solution works in conjunction with the outer water jacket of the OB to maintain the temperature of the solution to within plusmn0.2degC. Characterization and optimization of the RF/MW exposure setup were accomplished by a detailed numerical computation of the RF/MW fields and specific absorption rate (SAR) inside the inner chamber of the OB, where the muscle is suspended, using the finite-difference time-domain (FDTD) method. Analysis of the computed RF/MW fields within and immediately surrounding the skeletal muscle showed that the RF/MW fields and SAR exhibit the level of homogeneity required for performing well-controlled RF/MW exposure experiments

Design, Characterization, and Optimization of a Broadband Mini Exposure Chamber for Studying Catecholamine Release From Chromaffin Cells Exposed to Microwave Radiation: Finite-Difference Time-Domain Technique

A free-space in vitro exposure system for identifying specific microwave (MW) parameters in the frequency range of 1-6 GHz that can induce nonthermal effects on exocytosis, which is the process by which neurotransmitter release occurs, has been designed, constructed, characterized, and optimized. The exposure system is placed within an anechoic chamber and incorporates continuous online monitoring of basal and stimulated catecholamine release from cultured bovine adrenal-medullary chromaffin cells, which are well-established models of neural-type cells. The cells are immobilized inside a cell-perfusion apparatus (CPA) and are continuously superfused with temperature-controlled balanced salt solution, with the entire CPA placed within a mini exposure chamber (MEC) constructed out of a MW-absorbing material. All relevant equipment for carrying out the experiments is shielded from the MW field by being housed in an aluminum conductor box located behind the MEC. Detailed distributions of the electric field and the specific absorption rate (SAR) at the location of the cells within the MEC were computed using the finite-difference time-domain (FDTD) method. FDTD computations were also used for optimizing the exposure system, so that the highest intensity of electric field could be delivered under dynamic temperature control and with an acceptable degree of field homogeneity (to within 30%) over the entire frequency range of 1-6 GHz. A major finding is that maintaining an acceptable level of homogeneity of the electric field and SAR for exposing cells to 1-6 GHz MW fields requires a different distribution of the cells within the CPA for exposures carried out at the lower versus the higher end of the frequency range of interest