Modulating Ca2+ influx into adrenal chromaffin cells with short-duration nanosecond electric pulses.

Isolated bovine adrenal chromaffin cells exposed to single 2-, 4-, or 5-ns pulses undergo a rapid, transient rise in intracellular Ca2+ mediated by Ca2+ entry via voltage-gated Ca2+ channels (VGCCs), mimicking the activation of these cells in vivo by acetylcholine. However, pulse durations 150 ns or longer elicit larger amplitude and longer-lived Ca2+ responses due to Ca2+ influx via both VGCCs and a yet to be identified plasma membrane pathway(s). To further our understanding of the differential effects of ultrashort versus longer pulse durations on Ca2+ influx, chromaffin cells were loaded with calcium green-1 and exposed to single 3-, 5-, 11-, 25-, or 50-ns pulses applied at their respective Ca2+ activation threshold electric fields. Increasing pulse duration from 3 or 5 ns to only 11 ns was sufficient to elicit increased amplitude and longer-lived Ca2+ responses in the majority of cells, a trend that continued as pulse duration increased to 50 ns. The amplification of Ca2+ responses was not the result of Ca2+ release from intracellular stores and was accompanied by a decreased effectiveness of VGCC inhibitors to block the responses and a reduced reliance on extracellular Na+ and membrane depolarization to evoke the responses. Inhibitors of pannexin channels, P2X receptors, or non-selective cation channels failed to attenuate 50-ns-elicited Ca2+ responses, ruling out these Ca2+-permeable channels as secondary Ca2+ entry pathways. Analytical calculations and numerical modeling suggest that the parameter that best determines the response of chromaffin cells to increasing pulse durations is the time the membrane charges to its peak voltage. These results highlight the pronounced sensitivity of a neuroendocrine cell to pulse durations differing by only tens of nanoseconds, which has important implications for the future development of nanosecond pulse technologies enabling electrostimulation applications for spatially focused and graded in vivo neuromodulation.

High signal-to-noise imaging of spontaneous and 5 ns electric pulse-evoked Ca2+ signals in GCaMP6f-expressing adrenal chromaffin cells isolated from transgenic mice.

In studies exploring the potential for nanosecond duration electric pulses to serve as a novel modality for neuromodulation, we found that a 5 ns pulse triggers an immediate rise in [Ca2+]i in isolated bovine adrenal chromaffin cells. To facilitate ongoing efforts to understand underlying mechanisms and to work toward carrying out investigations in cells in situ, we describe the suitability and advantages of using isolated murine adrenal chromaffin cells expressing, in a Cre-dependent manner, the genetically-encoded Ca2+indicator GCaMP6f. Initial experiments confirmed that Ca2+ responses evoked by a 5 ns pulse were similar between fluorescent Ca2+ indicator-loaded murine and bovine chromaffin cells, thereby establishing that 5 ns-elicited excitation of chromaffin cells occurs reproducibly across species. In GCaMP6f-expressing murine chromaffin cells, spontaneous Ca2+ activity as well as nicotinic receptor agonist- and 5 ns evoked-Ca2+ responses consistently displayed similar kinetic characteristics as those in dye-loaded cells but with two-twentyfold greater amplitudes and without photobleaching. The high signal-to-noise ratio of evoked Ca2+ responses as well as spontaneous Ca2+ activity was observed in cells derived from Sox10-Cre, conditional GCaMP6f mice or TH-Cre, conditional GCaMP6f mice, although the number of cells expressing GCaMP6f at sufficiently high levels for achieving high signal-to-noise ratios was greater in Sox10-Cre mice. As in bovine cells, Ca2+ responses elicited in murine GCaMP6f-expressing cells by a 5 ns pulse were mediated by the activation of voltage-gated Ca2+ channels but not tetrodotoxin-sensitive voltage-gated Na+ channels. We conclude that genetically targeting GCaMP6f expression to murine chromaffin cells represents a sensitive and valuable approach to investigate spontaneous, receptor agonist- and nanosecond electric pulse-induced Ca2+ responses in vitro. This approach will also facilitate future studies investigating the effects of ultrashort electric pulses on cells in ex vivo slices of adrenal gland, which will lay the foundation for using nanosecond electric pulses to stimulate neurosecretion in vivo.

Ultrashort nanosecond electric pulses activate a conductance in bovine adrenal chromaffin cells that involves cation entry through TRPC and NALCN channels.

In whole-cell voltage clamped bovine adrenal chromaffin cells maintained at a holding potential of -70 mV, a single 5 ns, 5 MV/m pulse elicited an inward current carried mainly by Na+ that displayed inward rectification and a reversal potential near -3 mV, a voltage consistent with a non-selective cation current. The broad-spectrum inhibitors of transient receptor potential (TRP) channels, La3+ (10 µM), Gd3+ (10 µM), SKF-96365 (50 µM) and 2-aminoethoxydiphenyl borane (2-APB; 100 µM), inhibited the current similarly by ∼72%, ∼83%, ∼68% and ∼76%, respectively. Depleting membrane cholesterol with methyl-ß-cyclodextrin (MßCD; 1-6 mg/ml) or inhibiting phosphatidylinositol 4,5-bisphosphate (PIP2) synthesis with wortmannin (20 and 40 µM) produced a similar level of inhibition on the NEP-induced conductance as the broad spectrum TRP channel inhibitors. Moreover, no additive inhibitory effect was detected by combining MßCD (3 mg/ml), wortmannin (20 µM) and La3+ (10 µM), suggesting that each agent targeted different levels of the same pathway to exert a full effect. RT-PCR experiments revealed robust expression at the mRNA level of TRPC4, TRPC5 and TRPM7 channels for which specific blockers were available. Whereas the TRPM7 blocker FTY720 had no effect, the TRPC4/5 channel inhibitor M084 (20 µM) blocked the conductance by ∼50%, indicating that TRPC4 and/or TRPC5 channel(s) may be partially involved in mediating the NEP-induced current. CP-96345 (20 µM), a specific blocker of the sodium leak current channel (NALCN), also reduced the NEP-induced current. The inhibition was ∼30% and additive to that caused by the TRPC4/5 blocker M084. RT-PCR experiments confirmed the expression of this channel at the mRNA level. Taken as a whole, these data provide evidence that a large fraction of the current evoked by a 5 ns pulse in adrenal chromaffin cells may be carried by both TRPC4/5 channels and the NALCN channel. Understanding the biophysical properties of the NEP-elicited conductance in a neural-type cell will be extremely valuable for the future development of NEP stimulation approaches for neuromodulation.

5 ns electric pulses induce Ca2+-dependent exocytotic release of catecholamine from adrenal chromaffin cells.

Previously we reported that adrenal chromaffin cells exposed to a 5 ns, 5 MV/m pulse release the catecholamines norepinephrine (NE) and epinephrine (EPI) in a Ca2+-dependent manner. Here we determined that NE and EPI release increased with pulse number (one versus five and ten pulses at 1 Hz), established that release occurs by exocytosis, and characterized the exocytotic response in real-time. Evidence of an exocytotic mechanism was the appearance of dopamine-ß-hydroxylase on the plasma membrane, and the demonstration by total internal reflection fluorescence microscopy studies that a train of five or ten pulses at 1 Hz triggered the release of the fluorescent dye acridine orange from secretory granules. Release events were Ca2+-dependent, longer-lived relative to those evoked by nicotinic receptor stimulation, and occurred with a delay of several seconds despite an immediate rise in Ca2+. In complementary studies, cells labeled with the plasma membrane fluorescent dye FM 1-43 and exposed to a train of ten pulses at 1 Hz underwent Ca2+-dependent increases in FM 1-43 fluorescence indicative of granule fusion with the plasma membrane due to exocytosis. These results demonstrate the effectiveness of ultrashort electric pulses for stimulating catecholamine release, signifying their promise as a novel electrostimulation modality for neurosecretion.

2-ns Electrostimulation of Ca2+ Influx into Chromaffin Cells: Rapid Modulation by Field Reversal.

Cellular effects of nanosecond-pulsed electric field exposures can be attenuated by an electric field reversal, a phenomenon called bipolar pulse cancellation. Our investigations of this phenomenon in neuroendocrine adrenal chromaffin cells show that a single 2-ns, 16 MV/m unipolar pulse elicited a rapid, transient rise in intracellular Ca2+ levels due to Ca2+ influx through voltage-gated calcium channels. The response was eliminated by a 2-ns bipolar pulse with positive and negative phases of equal duration and amplitude and fully restored (unipolar-equivalent response) when the delay between each phase of the bipolar pulse was 30 ns. Longer interphase intervals evoked Ca2+ responses that were greater in magnitude than those evoked by a unipolar pulse (stimulation). Cancellation was also observed when the amplitude of the second (negative) phase of the bipolar pulse was half that of the first (positive) phase but progressively lost as the amplitude of the second phase was incrementally increased above that of the first phase. When the amplitude of the second phase was twice that of the first phase, there was stimulation. By comparing the experimental results for each manipulation of the bipolar pulse waveform with analytical calculations of capacitive membrane charging/discharging, also known as accelerated membrane discharge mechanism, we show that the transition from cancellation to unipolar-equivalent stimulation broadly agrees with this model. Taken as a whole, our results demonstrate that electrostimulation of adrenal chromaffin cells with ultrashort pulses can be modulated with interphase intervals of tens of nanoseconds, a prediction of the accelerated membrane discharge mechanism not previously observed in other bipolar pulse cancellation studies. Such modulation of Ca2+ responses in a neural-type cell is promising for the potential use of nanosecond bipolar pulse technologies for remote electrostimulation applications for neuromodulation.

Paradoxical effects on voltage-gated Na+ conductance in adrenal chromaffin cells by twin vs single high intensity nanosecond electric pulses.

We previously reported that a single 5 ns high intensity electric pulse (NEP) caused an E-field-dependent decrease in peak inward voltage-gated Na+ current (INa) in isolated bovine adrenal chromaffin cells. This study explored the effects of a pair of 5 ns pulses on INa recorded in the same cell type, and how varying the E-field amplitude and interval between the pulses altered its response. Regardless of the E-field strength (5 to 10 MV/m), twin NEPs having interpulse intervals ≥ than 5 s caused the inhibition of TTX-sensitive INa to approximately double relative to that produced by a single pulse. However, reducing the interval from 1 s to 10 ms between twin NEPs at E-fields of 5 and 8 MV/m but not 10 MV/m decreased the magnitude of the additive inhibitory effect by the second pulse in a pair on INa. The enhanced inhibitory effects of twin vs single NEPs on INa were not due to a shift in the voltage-dependence of steady-state activation and inactivation but were associated with a reduction in maximal Na+ conductance. Paradoxically, reducing the interval between twin NEPs at 5 or 8 MV/m but not 10 MV/m led to a progressive interval-dependent recovery of INa, which after 9 min exceeded the level of INa reached following the application of a single NEP. Disrupting lipid rafts by depleting membrane cholesterol with methyl-ß-cyclodextrin enhanced the inhibitory effects of twin NEPs on INa and ablated the progressive recovery of this current at short twin pulse intervals, suggesting a complete dissociation of the inhibitory effects of twin NEPs on this current from their ability to stimulate its recovery. Our results suggest that in contrast to a single NEP, twin NEPs may influence membrane lipid rafts in a manner that enhances the trafficking of newly synthesized and/or recycling of endocytosed voltage-gated Na+ channels, thereby pointing to novel means to regulate ion channels in excitable cells.

Stimulation or Cancellation of Ca2+ Influx by Bipolar Nanosecond Pulsed Electric Fields in Adrenal Chromaffin Cells Can Be Achieved by Tuning Pulse Waveform.

Exposing adrenal chromaffin cells to single 150 to 400 ns electric pulses triggers a rise in intracellular Ca2+ ([Ca2+]i) that is due to Ca2+ influx through voltage-gated Ca2+ channels (VGCC) and plasma membrane electropores. Immediate delivery of a second pulse of the opposite polarity in which the duration and amplitude were the same as the first pulse (a symmetrical bipolar pulse) or greater than the first pulse (an asymmetrical bipolar pulse) had a stimulatory effect, evoking larger Ca2+ responses than the corresponding unipolar pulse. Progressively decreasing the amplitude of the opposite polarity pulse while also increasing its duration converted stimulation to attenuation, which reached a maximum of 43% when the positive phase was 150 ns at 3.1 kV/cm, and the negative phase was 800 ns at 0.2 kV/cm. When VGCCs were blocked, Ca2+ responses evoked by asymmetrical and even symmetrical bipolar pulses were significantly reduced relative to those evoked by the corresponding unipolar pulse under the same conditions, indicating that attenuation involved mainly the portion of Ca2+ influx attributable to membrane electropermeabilization. Thus, by tuning the shape of the bipolar pulse, Ca2+ entry into chromaffin cells through electropores could be attenuated while preserving Ca2+ influx through VGCCs.

Different Membrane Pathways Mediate Ca2+ Influx in Adrenal Chromaffin Cells Exposed to 150-400 ns Electric Pulses.

Exposing adrenal chromaffin cells to 5 ns electric pulses (nsPEF) causes a rapid rise in intracellular Ca2+ ([Ca2+]i) that is solely the result of Ca2+ influx through voltage-gated Ca2+ channels (VGCCs). This study explored the effect of longer duration nsPEF on [Ca2+]i. Single 150, 200, or 400 ns pulses at 3.1 kV/cm evoked rapid increases in [Ca2+]i, the magnitude of which increased linearly with pulse width and electric field amplitude. Recovery of [Ca2+]i to prestimulus levels was faster for 150 ns exposures. Regardless of pulse width, no rise in [Ca2+]i occurred in the absence of extracellular Ca2+, indicating that the source of Ca2+ was from outside the cell. Ca2+ responses evoked by a 150 ns pulse were inhibited to varying degrees by ω-agatoxin IVA, ω-conotoxin GVIA, nitrendipine or nimodipine, antagonists of P/Q-, N-, and L-type VGCCs, respectively, and by 67% when all four types of VGCCs were blocked simultaneously. The remaining Ca2+ influx insensitive to VGCC inhibitors was attributed to plasma membrane nanoporation, which comprised the E-field sensitive component of the response. Both pathways of Ca2+ entry were inhibited by 200 µM Cd2+. These results demonstrate that, in excitable chromaffin cells, single 150-400 ns pulses increased the permeability of the plasma membrane to Ca2+ in addition to causing Ca2+ influx via VGCCs.

Adrenal Chromaffin Cells Exposed to 5-ns Pulses Require Higher Electric Fields to Porate Intracellular Membranes than the Plasma Membrane: An Experimental and Modeling Study.

Nanosecond-duration electric pulses (NEPs) can permeabilize the endoplasmic reticulum (ER), causing release of Ca2+ into the cytoplasm. This study used experimentation coupled with numerical modeling to understand the lack of Ca2+ mobilization from Ca2+-storing organelles in catecholamine-secreting adrenal chromaffin cells exposed to 5-ns pulses. Fluorescence imaging determined a threshold electric (E) field of 8 MV/m for mobilizing intracellular Ca2+ whereas whole-cell recordings of membrane conductance determined a threshold E-field of 3 MV/m for causing plasma membrane permeabilization. In contrast, a 2D numerical model of a chromaffin cell, which was constructed with internal structures representing a nucleus, mitochondrion, ER, and secretory granule, predicted that exposing the cell to the same 5-ns pulse electroporated the plasma and ER membranes at the same E-field amplitude, 3-4 MV/m. Agreement of the numerical simulations with the experimental results was obtained only when the ER interior conductivity was 30-fold lower than that of the cytoplasm and the ER membrane permittivity was twice that of the plasma membrane. A more realistic intracellular geometry for chromaffin cells in which structures representing multiple secretory granules and an ER showed slight differences in the thresholds necessary to porate the membranes of the secretory granules. We conclude that more sophisticated cell models together with knowledge of accurate dielectric properties are needed to understand the effects of NEPs on intracellular membranes in chromaffin cells, information that will be important for elucidating how NEPs porate organelle membranes in other cell types having a similarly complex cytoplasmic ultrastructure.

Nanosecond electric pulses differentially affect inward and outward currents in patch clamped adrenal chromaffin cells.

This study examined the effect of 5 ns electric pulses on macroscopic ionic currents in whole-cell voltage-clamped adrenal chromaffin cells. Current-voltage (I-V) relationships first established that the early peak inward current was primarily composed of a fast voltage-dependent Na+ current (INa), whereas the late outward current was composed of at least three ionic currents: a voltage-gated Ca2+ current (ICa), a Ca2+-activated K+ current (IK(Ca)), and a sustained voltage-dependent delayed rectifier K+ current (IKV). A constant-voltage step protocol was next used to monitor peak inward and late outward currents before and after cell exposure to a 5 ns pulse. A single pulse applied at an electric (E)-field amplitude of 5 MV/m resulted in an instantaneous decrease of ~4% in peak INa that then declined exponentially to a level that was ~85% of the initial level after 10 min. Increasing the E-field amplitude to 8 or 10 MV/m caused a twofold greater inhibitory effect on peak INa. The decrease in INa was not due to a change in either the steady-state inactivation or activation of the Na+ channel but instead was associated with a decrease in maximal Na+ conductance. Late outward current was not affected by a pulse applied at 5 MV/m. However, for a pulse applied at the higher E-field amplitudes of 8 and 10 MV/m, late outward current in some cells underwent a progressive ~22% decline over the course of the first 20 s following pulse exposure, with no further decline. The effect was most likely concentrated on ICa and IK(Ca) as IKV was not affected. The results of this study indicate that in whole-cell patch clamped adrenal chromaffin cells, a 5 ns pulse differentially inhibits specific voltage-gated ionic currents in a manner that can be manipulated by tuning E-field amplitude.

Enhanced Monitoring of Nanosecond Electric Pulse-Evoked Membrane Conductance Changes in Whole-Cell Patch Clamp Experiments.

Patch clamp electrophysiology serves as a powerful method for studying changes in plasma membrane ion conductance induced by externally applied high-intensity nanosecond electric pulses (NEPs). This paper describes an enhanced monitoring technique that minimizes the length of time between pulse exposure and data recording in a patch-clamped excitable cell. Whole-cell membrane currents were continuously recorded up to 11 ms before and resumed 8 ms after delivery of a 5-ns, 6 MV/m pulse by a pair of tungsten rod electrodes to a patched adrenal chromaffin cell maintained at a holding potential of -70 mV. This timing was achieved by two sets of relay switches. One set was used to disconnect the patch pipette electrode from the pre-amplifier and connect it to a battery to maintain membrane potential at -70 mV, and also to disconnect the reference electrode from the amplifier. The other set was used to disconnect the electrodes from the pulse generator until the time of NEP/sham exposure. The sequence and timing of both sets of relays were computer-controlled. Using this procedure, we observed that a 5-ns pulse induced an instantaneous inward current that decayed exponentially over the course of several minutes, that a second pulse induced a similar response, and that the current was carried, at least in part, by Na+. This approach for characterizing ion conductance changes in an excitable cell in response to NEPs will yield information essential for assessing the potential use of NEP stimulation for therapeutic applications.

Adrenal chromaffin cells do not swell when exposed to nanosecond electric pulses.

High intensity, nanosecond duration electric pulses (NEPs) permeabilize plasma membranes causing osmotic cell swelling that can elicit a wide variety of cellular effects. This study examined the possibility that cell swelling is the mechanism by which 5 ns NEPs trigger the release of catecholamines from neuroendocrine adrenal chromaffin cells. Swelling was assessed by comparing measurements of cell area obtained from bright field images of the cells before and at 10s intervals following exposure of the cells to 5 ns pulses at a field intensity of 5-6 MV/m. The results indicated that chromaffin cells do not swell in response to a single pulse or a train of ten pulses delivered at repetition frequencies of 10 Hz and 1 kHz. Swelling was also not observed in response to a train of 50 pulses whereas Jurkat T lymphoblast cell area increased 15% on average under the same NEP exposure conditions. These results demonstrating that chromaffin cells do not undergo swelling when exposed to 5 ns NEPs have important implications regarding the mechanism by which these pulses stimulate the release of catecholamines from these cells, namely that catecholamine secretion is most likely not caused by cell swelling.

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.

Nanosecond electric pulses: a novel stimulus for triggering Ca2+ influx into chromaffin cells via voltage-gated Ca2+ channels.

Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca(2+) entry into the cells via L-type voltage-gated Ca(2+) channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond pulse-induced effects on intracellular Ca(2+) level ([Ca(2+)](i)) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca(2+) entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude of the rise in [Ca(2+)](i) elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca(2+)](i) response of the cells, suggesting Ca(2+) influx occurs only via VGCCs. Lowering extracellular K(+) concentration from 5 to 2 mM or pulsing cells in Na(+)-free medium suppressed the pulse-induced rise in [Ca(2+)](i) in the majority of cells. Thus, both membrane potential and Na(+) entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca(2+) influx. However, activation of voltage-gated Na(+) channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca(2+)](i). These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple types of VGCCs in chromaffin cells in a manner involving Na(+) transport across the plasma membrane. Whether Na(+) transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined.

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.

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 Hank's 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.

Nicotinic stimulation modulates tyrosine hydroxylase mRNA half-life and protein binding to the 3'UTR in a manner that requires transcription.

Tyrosine hydroxylase (TH) expression increases in adrenal chromaffin cells treated with the nicotinic agonist, dimethylphenylpiperazinium (DMPP; 1 microM). We are using this response as a model of the changes in TH level that occur during increased cholinergic neural activity. Here we report a 4-fold increase in TH mRNA half-life in DMPP-treated cells chromaffin cells that is apparent when using a pulse-chase analysis to measure TH mRNA half-life. No increase is apparent using actinomycin D to measure half-life, indicating a requirement for ongoing transcription. Characterization of protein binding to the TH 3'UTR responsible for stabilization using labeled TH 3'UTR probes and electro-mobility shift assays shows the presence of two complexes both of which are increased by DMPP-treatment. The faster migrating complex (FMC) increases 2.5-fold and the slower migrating complex (SMC) increases 1.5-fold. Both changes are prevented by actinomycin D. Characterization of the protein binding to the TH UTR probes indicates SMC is disrupted by polyribonucleotides, poly (A) and poly (U), while binding to FMC is reduced by poly (CU). Separation of UV crosslinked RNA-protein complexes on SDS polyacrylamide gels shows FMC to contain a single protein whereas SMC contains three proteins. Northwesterns yielded similar results. Comparison of DMPP-induced protein binding with the poly C binding protein (PCBP) involved in hypoxia induced rat PC12 TH mRNA stability indicates none of the bovine UTR binding proteins are the PCBP. Thus, nicotinic stimulation produces a transcription-dependent increase in TH mRNA half-life that is mediated by previously unrecognized TH mRNA binding proteins.

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.

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.