Gale L. Craviso

Nanosecond electric pulse-induced calcium entry into chromaffin cells.

Electrically excitable bovine adrenal chromaffin cells were exposed to nanosecond duration electric pulses at field intensities ranging from 2 MV/m to 8 MV/m and intracellular calcium levels ([Ca(2+)](i)) monitored in real time by fluorescence imaging of cells loaded with Calcium Green. A single 4 ns, 8 MV/m pulse produced a rapid, short-lived increase in [Ca(2+)](i), with the magnitude of the calcium response depending on the intensity of the electric field. Multiple pulses failed to produce a greater calcium response than a single pulse, and a short refractory period was required between pulses before another maximal increase in [Ca(2+)](i) could be triggered. The pulse-induced rise in [Ca(2+)](i) was not affected by depleting intracellular calcium stores with caffeine or thapsigargin but was completely prevented by the presence of EGTA, Co(2+), or the L-type calcium channel blocker nitrendipine in the extracellular medium. Thus, a single nanosecond pulse is sufficient to elicit a rise in [Ca(2+)](i) that involves entry of calcium via L-type calcium channels.

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.

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.

Protein Synthesis Blockade Differentially Affects the Degradation of Constitutive and Nicotinic Receptor‐Induced Tyrosine Hydroxylase Protein Level in Isolated Bovine Chromaffin Cells

Abstract: Continuous incubation of bovine adrenal chromaffin cells with the nicotinic receptor agonist 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP) causes a twofold increase in the steady‐state level of catalytically active tyrosine hydroxylase (TH) protein by 3‐4 days. The present study examined the processes that control the time course of enzyme induction. In cells exposed to DMPP for 36 or 54 h, incorporation of [3H]leucine into TH was increased 1.9‐ and 2.2‐fold, respectively, compared with control (non‐DMPP‐treated) cells. The increase correlated with a twofold rise in TH mRNA level, indicating the absence of translational control of TH synthesis by DMPP. Also absent was an effect by DMPP on the rate of degradation of TH protein because pulse‐chase analysis estimated a half‐life for TH of 26 ± 5 h in DMPP‐treated cells, a value that was (a) essentially the same as that estimated in control cells (29 ± 3 h), (b) within the same range as that estimated by approach to steady state (t1/2 = 19 ± 4 h), which measured the decline of TH protein content from the DMPP‐induced steady‐state level back to the basal value during deinduction with the nicotinic antagonist hexamethonium, and (c) consistent with the time course of accumulation of TH protein to a new steady‐state level in response to DMPP. However, different rates of degradation for TH protein were observed in control and DMPP‐treated cells under conditions in which protein synthesis was blocked. In control cells incubated with 100 μM puromycin or 20 μM cycloheximide for 3 days, the level of catalytically active TH protein failed to decline and exhibited a half‐life of ≥250 h. This finding indicated that TH protein was stabilized. TH protein level also failed to decline when cells were incubated for 3 days with a concentration of the transcription inhibitor α‐amanitin that caused a >90% loss of TH mRNA. Thus, degradation of constitutively expressed TH protein appears to be controlled by processes dependent on ongoing transcription and translation. In contrast, the increased amount of TH induced by DMPP was not stabilized but instead underwent a decline to the basal level following addition of puromycin or cycloheximide. It is important to note, however, that the decline occurred at a slower rate (t1/2≥ 45 h) than that measured during deinduction. Taken together, these data suggest that alterations in the rate of degradation of TH protein may play a role in controlling TH level when TH synthesis is blocked but not when TH synthesis is increased, such as during nicotinic receptor stimulation.

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.

Generation of functionally competent single bovine adrenal chromaffin cells from cell aggregates using the neutral protease dispase.

A simple and efficient procedure has been developed to enzymatically dissociate aggregates of bovine adrenal chromaffin cells in suspension culture into viable, responsive single cells. For dissociation, the neutral protease dispase is added directly to the culture medium for a minimum of 3 h, followed by incubation of the cells in Hanks calcium-magnesium-free balanced salt solution at 37 degrees C with intermittent trituration to facilitate dispersion. This procedure generates a population of phase-bright single cells that are round in morphology, take up the dye neutral red, exclude the dye trypan blue and readily attach to tissue culture dishes coated with collagen, fibronectin or polylysine, thereby permitting applications that require plated-down conditions. When transferred to culture medium, the cells begin to reaggregate. By altering the length of time the cells are incubated in culture medium prior to attachment, the degree of reaggregation can be controlled to obtain plate-down profiles that consist of both isolated cells and cells in aggregates of varying sizes. Returning dissociated cells to suspension culture results in the reformation of large cell aggregates. Several measures of chromaffin cell function were indistinguishable for dissociated cells placed either in monolayer culture or suspension culture versus non-dissociated cells, implying that the dissociation procedure does not alter cellular responses or cause cellular damage.

The transient nicotinic stimulation of tyrosine hydroxylase gene transcription in bovine adrenal chromaffin cells is independent of c-fos gene activation

The nicotinic agonist dimethylphenylpiperazinium (DMPP) transiently stimulates tyrosine hydroxylase (TH) gene transcription in cultured bovine adrenal chromaffin cells (Craviso et al., J. Neurochem., 59 (1992) 2285-2296). The present studies examined the mechanism of this stimulation, exploring the hypothesis that c-fos- and/or cyclic AMP-related mechanisms are involved. As determined by nuclear run-on assay, exposure of chromaffin cells to DMPP (1 microM) induced c-fos and TH gene transcription fivefold and twofold, respectively. Nitrendipine (20 microM) blocked both responses, indicating a similar dependency of each on extracellular calcium. Both c-fos and TH gene transcription rates were also elevated by entry of calcium due to the presence of the calcium ionophore A23187 (5 microM). Comparison of the time dependence of the DMPP stimulation of c-fos and TH gene transcription revealed similar time courses. Both were rapid and transient, peaking within 10-30 min of nicotinic receptor occupancy and returning to control values by 1 h. This simultaneous activation of the TH and c-fos genes indicates that Fos induction cannot be responsible for the stimulation of TH gene transcription. This conclusion was further supported by a failure of anisomycin (100 microM) pretreatment of chromaffin cells, which blocked protein synthesis 99%, to have any effect on either the rapid stimulation of TH gene transcription or the length of time that the TH gene was activated by DMPP. Thus, neither Fos nor other high turnover-rate transcription factors appear to be responsible for the stimulation, or return to control level, of TH gene activity following nicotinic stimulation of chromaffin cells. In other experiments, treating chromaffin cells with a combination of maximally effective concentrations of DMPP and forskolin was found to produce no greater stimulation of TH gene transcription than either agent alone, suggesting that DMPP acts through the same mechanism as forskolin. Taken together, these results support the conclusion that the mechanism of TH gene activation in chromaffin cells by DMPP involves a cyclic AMP-dependent process and not the induction of transcription factors such as Fos.

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.