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

Abstract

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.

Significance

Nanosecond electric pulses (NEPs) have been gaining recognition as a potential electrostimulation approach for neuromodulation. Here, we report the ability to tune Ca²⁺ influx into neuroendocrine adrenal chromaffin cells by varying pulse duration over the course of only tens of nanoseconds, a sensitivity not found for other excitable cell types. We addressed the basis for the differential effects of ultrashort (<10 ns) versus longer-duration (up to 50 ns) pulses experimentally and discussed the results in the context of capacitive membrane charging wherein the time it takes the membrane to charge to the peak membrane voltage best explains the response of the cells to different pulse durations. Such information is crucial for developing novel neuromodulation approaches using NEPs.

Introduction

Electrostimulation continues to gain momentum as a safe and effective treatment modality. In addition to well-established clinical applications of electrostimulation for cardiac pacing (1,2), the medical use of electric fields has broadened to include, as examples, alleviation of symptoms of Parkinson’s disease (3,4,5) as well as treatment of psychiatric (6,7,8), neuromuscular (9), and visceral disorders (10). In the interest of expanding the range of non-invasive electrical stimulation approaches for neuromodulation beyond those that are currently being investigated, such as transcranial temporal interference stimulation (11,12), an approach that we and others have been pursuing involves the use of electric pulses that are in the nanosecond regime, namely, nanosecond electric pulses (NEPs). In addition to their capability to modulate neural cell excitability in distinctive ways by manipulating the pulse waveform (13,14,15,16) and the pulse delivery technique (17,18,19,20,21), such short-duration pulses also have the potential for remote and precise targeting of specific tissue sites within the body (22,23,24,25,26), thereby eliminating the need for implanting electrodes.

Studies conducted on isolated frog sciatic nerve serve as one of the best examples that such brief electric pulses hold promise of being a viable option to modulate neural activity. In this preparation, repeated tetanic NEP stimulation induces action potentials over the course of 1 min without causing nerve damage. Of note, damage-free nerve excitation was achieved regardless of the pulse duration that was used, which included pulses 12 ns in duration (27) and pulses that were much longer in duration, specifically 200, 300, and 700 ns (28). In another neural preparation, isolated rat nociceptor neurons, NEPs similarly caused non-deleterious excitation of action potentials. As found for the sciatic nerve, safe and effective NEP stimulation could be achieved regardless of whether pulses were 11 ns (29) or 350 ns in duration (30).

Studies conducted by our group on catecholamine-producing adrenal chromaffin cells, which are part of the sympathetic nervous system, further support the potential for NEPs to be used as a safe and effective electrostimulation modality. Using isolated bovine adrenal chromaffin cells for NEP exposure, in conjunction with Ca²⁺ fluorescence imaging to monitor intracellular Ca²⁺ ([Ca²⁺]_i), we found that a single 2-, 4-, or 5-ns pulse evoked a rise in [Ca²⁺]_i that was mediated by Ca²⁺ influx solely via voltage-gated Ca²⁺ channels (VGCCs) (31,32,33,34,35), the same mechanism underlying Ca²⁺ influx triggered in vivo by nicotinic acetylcholine receptor (nAChR) stimulation. Under the conditions of NEP exposure that were used, namely applying pulses at electric field (E-field) intensities at or just above the Ca²⁺ excitation threshold, i.e., the lowest E-field that elicits a rise of [Ca²⁺]_i in all exposed cells, catecholamines were released (32,35) with no evidence of undesirable cellular effects, such as cell swelling (31,34,36) or the electropermeabilization of membranes of internal Ca²⁺-storing organelles (37,38) that has been reported in other cell types (39,40,41,42). However, unlike NEP-evoked stimulation of sciatic nerve and nociceptor neurons, pulse duration had a marked effect on the nature of chromaffin cell Ca²⁺ responses elicited by NEP exposure. Specifically, a 150-ns pulse delivered to the cells at 0.31 MV/m, the Ca²⁺ activation threshold E-field for this pulse duration, elicited an increased amplitude and longer-duration Ca²⁺ response than that evoked by the shorter-duration 5-ns pulse delivered at the much higher E-field of 4 MV/m. These changes in Ca²⁺ response amplitude and duration did not involve release of Ca²⁺ from intracellular stores but instead were due to Ca²⁺ entry not only via VGCCs but also via another plasma membrane pathway (43).

Based on the well-documented finding that NEPs can cause a change in the integrity of the cell membrane, a biophysical process referred to as nanoelectroporation or nanoelectropermeabilization (44,45,46,47,48), in which nanometer-sized pores are formed in the lipid bilayer (44,46,47,48,49,50,51,52), we had proposed (43) that Ca²⁺ was crossing the chromaffin cell plasma membrane via nanopores and accumulating inside the cells (41,42,43,44,45,46,47,48,49,50,51,52,53,54,55). However, influx of the cell-impermeant fluorescent dye YO-PRO-1 that has typically been used to assess such membrane permeabilization following NEP exposure (44,48,55,56,57) was not observed in chromaffin cells exposed to a 150-ns pulse (43). While it is possible that the non-VGCC-mediated influx of Ca²⁺ could be a manifestation of a more subtle form of plasma membrane nanoelectropermeabilization that discriminates against YO-RPO-1 but not Ca²⁺ influx, in the present study we favored the idea instead that the second pathway of Ca²⁺ entry elicited by increasing pulse duration could be due to the activation of a membrane ion channel(s) that conducts Ca²⁺. This idea stems from our recent finding that a 5-ns pulse activates not only VGCCs but also transient receptor potential channels 4 and 5 (TRPC4/5) and the sodium leak current channel NALCN (58), suggesting that longer pulse durations could activate still other channels in the plasma membrane.

The present study was undertaken to understand the basis for the differential effects of ultrashort (i.e., less than 10 ns) versus longer-duration (up to 50 ns) NEPs on Ca²⁺ influx in these neural-type cells. We first determined the pulse duration at which the second pathway of Ca²⁺ entry is activated. For this determination, single pulses ranging in duration from 3 to 50 ns were delivered to the cells at their respective Ca²⁺ activation threshold E-fields, and the ability of VGCC inhibitors to block the evoked Ca²⁺ responses was assessed. We also determined the correlation between pulse duration and the reliance of the evoked Ca²⁺ responses on membrane depolarization and extracellular Na⁺, and the effect of blocking several non-voltage-gated Ca²⁺-conducting ion channels on the responses to NEPs. Finally, analytical calculations and numerical modeling were employed to explain, at least theoretically, the basis for the change in plasma membrane Ca²⁺ response characteristics by pulses that vary by only tens of nanoseconds in duration. Taken as a whole, these findings define a range of NEP parameters that can elicit precise and predictable cellular responses in a neural-type cell, which in general has implications for the future development of NEP stimulation approaches for modulating neural cell excitability. More specifically, given the critical role that chromaffin cells play in the control of circulating catecholamines under rest as well as during physiological stress, these findings contribute to efforts by us and others (59) to work toward modulating adrenal medullary function remotely in a way that could advance the treatment of stress-related disorders.

Materials and methods

Isolation, culturing, and preparation of bovine adrenal chromaffin cells

Adrenal chromaffin cells were isolated by collagenase digestion of the medulla of fresh bovine adrenal glands obtained from local slaughterhouses (Wolf Pack Meats, University of Nevada, Reno (UNR) and York Meats, Fallon, NV). The cells were maintained in suspension culture in Ham’s F-12 medium supplemented with 10% bovine calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL fungizone, and 6 μg/mL cytosine arabinoside at 37°C under a humidified atmosphere of 5% CO₂ as previously described (60). Cells were used up until 14 days in culture. For experiments, the large cell clusters that formed in culture were dissociated into single cells with the protease dispase (61) and plated onto fibronectin-coated 35-mm glass-bottom dishes. Once attached, cells were used for a period not exceeding 2 days.

Fluorescence imaging

For monitoring [Ca²⁺]_i, cells were incubated with the cell-permeant Ca²⁺-sensitive fluorescent indicator calcium green-1-AM (1 μM; 480/535 nm) for 45 min at 37°C in a balanced salt solution (BSS) with the following composition: 145 mM NaCl, 5 mM KCl, 1.2 mM NaH₂PO₄, 2 mM CaCl₂, 1.3 mM MgCl₂, 10 mM glucose, 15 mM HEPES, and 0.1% bovine serum albumin, pH 7.4. After incubation, the cells were washed twice with dye-free BSS and placed on the stage of an inverted microscope. As described in a previous study (35), imaging experiments were performed in two laboratories on the UNR campus, each using a different epifluorescence microscope system. One imaging system comprised a Nikon TE 2000 epifluorescence microscope equipped with a 100× air objective and an iXonEM⁺ DU-897 EMCCD camera (Andor Technology, Oxford Instruments, Belfast, UK) (34,37,38). Brightfield and fluorescence images of the cells were captured with open-source Micro-Manager software versions 1.4 and 2.0 gamma, with the exposure time of the camera set to 100 ms and fluorescence images captured at a rate of 10 frames/s. Sequences were analyzed using the public-domain image-processing program ImageJ. The other fluorescence imaging system was a Leica inverted DMi8 epifluorescence microscope equipped with a 63× air objective and an iXon Ultra 897 EMCCD camera (Andor Technology). Brightfield and fluorescence images were captured using Leica Application Suite X (LAS X) software, with the exposure time of the camera set to 150 ms and fluorescence images captured at a rate of ≈7 frames/s. Sequences were analyzed using LAS X software and a custom program written in MATLAB (35). In all experiments, continuous baseline fluorescence of the cells was monitored 10–15 s before stimulus application and continued for 50–75 s after the stimulus. Stimulus-evoked changes in cell fluorescence intensity were calculated by subtracting the cell-free background fluorescence from the fluorescence of the cell (F = F_cell − F_background). F was then normalized to the fluorescence intensity value (F₀) at the time when the stimulus was applied (F/F₀). Given that two microscope systems were being used in the present study, we verified that Ca²⁺ response amplitudes and durations compared quantitatively between the two imaging systems by first exposing cells to a short pulse, 5 ns, and also to a 10-fold longer pulse, 50 ns. For a 5-ns pulse, both imaging setups recorded an instantaneous average peak rise in [Ca²⁺]_i of about 1.6- to 1.8-fold, with [Ca²⁺]_i returning to baseline values typically by 25 s. For a 50-ns pulse, both imaging setups recorded an instantaneous average peak rise in [Ca²⁺]_i of about 2.1-fold, with [Ca²⁺]_i rarely returning to baseline values by 75 s post pulse.

For monitoring YO-PRO-1 uptake, cells were exposed to an NEP in BSS containing 2 μM of the dye. Fluorescence (491/506 nm) was monitored for 75 s after the exposure.

NEP exposure

Two different pulse generators were used to span the 3- to 50-ns pulse range examined in this study. One was a short-duration nanosecond bipolar pulse generator that provided pulses ranging from 3 to 11 ns (FPG 5-1NL10V2; FID, Germany; hereafter referred to as Pulser 1). The other was a longer-duration nanosecond bipolar pulse generator that provided pulses ranging from 12 to 50 ns (FPG 5-01NL100V2; FID; hereafter referred to as Pulser 2). Where noted, some experiments were also performed with the custom-fabricated, fixed-duration 5-ns pulse generator (Transient Plasma Systems, Torrance, CA; hereafter referred to as Pulser 3) used previously (37,38). Cells were exposed to NEPs that were 3, 5, 11, 25, or 50 ns in duration, and Fig. 1 shows the averaged pulse traces together with the full width at half maximum and rise times for each pulse. The waveform for a 3- and 5-ns pulse was bell-shaped (Fig. 1 A, left and middle panels), which was similar to the shape of the 5-ns pulse generated by Pulser 3 (Fig. 1 A, red trace in middle panel). The waveform for an 11-ns pulse was trapezoidal (Fig. 1 A, right panel), which was also the shape of the 25- and 50-ns pulses (Fig. 1 B, middle and right panels). To determine whether the difference in pulse shape resulted in different cell Ca²⁺ responses, cells were exposed to a trapezoidal 11-ns pulse or to a 12-ns pulse that was bell-shaped (Fig. 1 B, left panel), the latter generated by Pulser 2. Each pulse was delivered at its Ca²⁺ activation threshold E-field (see results). The determination revealed that the characteristics of the evoked Ca²⁺ responses were similar regardless of pulse shape (Fig. S1). Given this similarity, we opted to use the trapezoidal 11-ns pulse generated by Pulser 1 that resembled the trapezoidal 25- and 50-ns pulses generated by Pulser 2, thus providing overlap between the use of the two pulsers to cover the range of pulse durations used in this study.

Figure 1.

Pulse traces generated by each pulser. (A) Averaged pulse traces for 3-, 5-, and 11-ns pulses, respectively, generated by Pulser 1. The red trace in the middle is the averaged pulse trace for 5-ns pulses generated by Pulser 3. (B) Averaged pulse traces for 12-, 25-, and 50-ns pulses, respectively, generated by Pulser 2. All pulses, which were applied at threshold E-fields (see results), were captured using an oscilloscope. To see this figure in color, go online.

In all experiments, NEPs were applied by means of two cylindrical tungsten rod electrodes spaced 100 μm apart. The tips of the electrodes were immersed in the BSS and placed 40 μm above the bottom of the dish, with the cell being exposed to the pulse positioned in the center of the gap between the electrode tips. The pulsers were triggered externally by a custom program written in LabVIEW, and the camera was synchronized with the pulse generator to precisely determine the time at which the pulses were applied to the cells. Depending on pulse duration, pulses were delivered to the electrodes at amplitudes that produced E-fields ranging from 1.1 to 13.4 MV/m at the location of the cell (see results). The E-field distribution in the vicinity and at the location of the target cell was computed using the commercially available FDTD (finite-difference time-domain) software package Sim4Life (version 7.2; SPEAG, Zurich, Switzerland). Pulse traces were captured with an oscilloscope. Each cell was exposed only once to the applied E-field.

nAChR agonist stimulation

Cells were stimulated with the nAChR agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) applied with a pressure ejection system (Picospritzer III; Parker Hannifin, Cleveland, OH) that delivered brief (5 ms) applications of DMPP to individual cells via a glass micropipette (tip diameter: 1 μm). Pipette tips were positioned at a distance of one cell diameter away from a target cell using a motorized micromanipulator (MP-225; Sutter Instrument, Novato, CA).

Receptor and ion channel blockade

VGCCs were blocked with the inorganic non-selective blocker CdCl₂ by incubating the cells for 30 min at 37°C with 200 μM Cd²⁺. For experiments using selective VGCC inhibitors, cells were incubated for 60 min at 37°C in BSS containing a cocktail consisting of 100 nM ω-agatoxin IVA, 20 nM ω-conotoxin GVIA, and 20 μM nitrendipine to block P/Q-, N-, and L-type VGCCs, respectively. In some experiments, the cocktail also included 100 nM SNX-482 that blocks R-type VGCCs. Voltage-gated Na⁺ channels (VGSCs) were blocked by incubating cells for 20 min at room temperature in BSS containing 10 μM tetrodotoxin (TTX). Other blockers that were used included probenecid (200 μM) and ¹⁰Panx1 (200 μM) to assess the involvement of pannexin receptors (62), pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid tetrasodium salt (PPADS, 50 μM) to block P2X receptors (63), and La³⁺ (100 μM) to block the non-selective cation conductance identified by Cheek and Thorn (64). Cells were incubated with the blockers for 30 min at room temperature prior to experiments.

Ca²⁺- and Na⁺-free experiments

For experiments carried out under Ca²⁺-free conditions, the BSS lacked CaCl₂ and contained 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). For performing experiments under Na⁺-free conditions, Na⁺ in the extracellular solution was replaced with an equimolar concentration of tetramethylammonium hydroxide (TMA⁺) to maintain isotonicity. TMA⁺ was chosen because the osmolarity and conductivity of the TMA⁺ solution (58) were close to those of BSS (314.6 ± 0.3 mOsm and 298.3 ± 1.2 mOsm, for the osmolarities of BSS and TMA⁺, respectively; and 17.3 ± 0.2 mS/cm and 14.9 ± 0.5 mS/cm, for the conductivities of BSS and TMA⁺, respectively).

Patch-clamp electrophysiology

Perforated whole-cell patch-clamp was used in current clamp mode to measure the resting membrane potential. Recordings were obtained using an Axopatch 200B amplifier (Axon Instruments, San Jose, CA) and Digidata 1550B data acquisition system (Molecular Devices, San Jose, CA) with PClamp software (version 11, Molecular Devices) at a sampling rate of 20 kHz and low-pass filtering at 1 kHz. Cells were attached to coverslips and perfused first with BSS containing normal K⁺ (5 mM), followed by switching the BSS solution to one containing a reduced amount of K⁺ (2 mM). For the 2 mM K⁺ BSS, 3 mM NaCl was added to maintain isotonicity. Micropipettes with a tip size of 0.8–1.2 μm were fabricated from borosilicate glass (#BF150-110-7.5; Sutter Instruments) using a P-97 pipette puller (Sutter Instruments) and fire polished with a Narishige microforge (model MF-830; Narishige, Tokyo, Japan). The internal pipette solution consisted of 10 mM NaCl, 30 mM KCl, 110 mM K-gluconate, 1 mM MgCl₂, 10 mM EGTA, 3 mM MgATP, and 10 mM HEPES, pH 7.2. Nystatin was dissolved in dimethyl sulfoxide (5 mg/mL) and subsequently added to the internal solution at a final concentration of 200 μg/mL, using an ultrasonicator to facilitate solubilization.

Statistical analysis

Replicate experiments used cells from different cell preparations and cells from different days in culture. Unless otherwise stated, the normalized Ca²⁺ responses are presented as the mean ± standard error (SE). Statistical analysis was done with Origin 2022 (OriginLab Corporation, Northampton, MA) software using an unpaired Student's t-test when the means of two groups were compared, or a one-way ANOVA test followed by Tukey post hoc multiple-range tests when the means of multiple groups were compared. p < 0.05 was considered statistically significant unless otherwise specified.

Reagents

Ham’s F-12 medium, streptomycin, penicillin, and fungizone were obtained from Gibco Laboratories (Grand Island, NY). Dispase II and probenecid were obtained from Thermo Fisher Scientific (Waltham, MA). Bovine calf serum was obtained from Gemini Bio-Products (Sacramento, CA). Calcium green-1-AM and YO-PRO-1 were purchased from Invitrogen (Waltham, MA), tetrodotoxin from EMD Chemicals (San Diego, CA), and ω-conotoxin, ω-agatoxin, and SNX-482 from Alomone Labs (Jerusalem, Israel). ¹⁰Panx1 and Scrambled ¹⁰Panx1 were obtained from Sigma-Aldrich (Burlington, MA) and PPADS from Tocris Bioscience (Bristol, UK). Nystatin was obtained from Sigma-Aldrich (St. Louis, MO). All other chemicals were reagent grade and purchased from standard commercial sources.

Results

Increasing pulse duration from 3 to 50 ns decreased the Ca²⁺ activation threshold E-field

Based on our previous study reporting that the Ca²⁺ activation threshold E-field was significantly less for a 150-ns pulse than for a 5-ns pulse (43), we first determined the E-field threshold at which each pulse in the 3- to 50-ns range should be applied. As shown in Fig. 2 A, the threshold E-field intensity varied inversely with pulse duration, becoming progressively lower as pulse duration increased. The averaged E-field thresholds were 13.4 MV/m, 9.4 MV/m, 4.7 MV/m, 2.2 MV/m, and 1.1 MV/m for pulse durations of 3, 5, 11, 25, and 50 ns, respectively, and were obtained from the variation around the E-field threshold values for each pulse duration. Included in the plot is the threshold E-field also for a 12-ns pulse, which was 5.3 MV/m (Fig. 2 A, red circles). Thus, even though the 11-ns and 12-ns pulses were generated by different pulsers and had different shapes (trapezoidal shape versus bell-shaped, respectively; Fig. 1), the threshold E-field for each was similar. Fig. 2 B is a double-logarithmic plot of the same results showing that the relationship between pulse duration and E-field threshold was linear. Such linearity on a log-log plot is consistent with linear strength-duration curves reported for other types of excitable cells stimulated with NEPs (65,66). In all experiments, pulses were delivered at their respective E-field thresholds.

Figure 2.

Relationship between the Ca²⁺ activation threshold E-field and pulse duration. (A) Scatterplot where each circle represents an E-field value at its respective pulse duration that caused the cells to exhibit a response to the pulse. The equation represents a power function fit of the experimental data, where E is the E-field amplitude in MV/m and t is the pulse duration in nanoseconds. (B) Same plot as in (A) but on a log-log scale. The equation represents a linear fit of the experimental data, and R² represents the coefficient of determination of the linear regression model. On both plots, the red circles represent the 12-ns data recorded from Pulser 2. The red circles in (B) are obscured by the black circles. To see this figure in color, go online.

Ca²⁺ response characteristics started to deviate from those evoked by DMPP when pulse duration reached 11 ns

We next identified which pulse durations stimulated Ca²⁺ responses in chromaffin cells in a manner that most closely parallels that produced by the physiological stimulus, nAChR activation. As shown in Fig. 3 A, brief application of the nAChR agonist DMPP (5 μM) to a cell caused a rapid, transient rise in Ca²⁺ (mean Ca²⁺ peak amplitude F/F₀ = 1.82 ± 0.07; n = 13) with an average half-width value, which is the time interval for [Ca²⁺]_i to decline to 50% of the maximal value, of 3.5 s. The response was nearly abolished by the L-type VGCC blocker nitrendipine (Fig. 3 B).

Figure 3.

Ca²⁺ responses in cells exposed to DMPP. (A) DMPP (5 μM) was delivered to the cells (arrow) using a pressure ejection pipette. Traces show individual Ca²⁺ responses and the black line the averaged Ca²⁺ responses ±SE (n = 13). (B) Same as in (A) in the presence of 50 μM nitrendipine. The cells were incubated with nitrendipine for 60 min at 37°C prior to DMPP addition (arrow).

Fig. 4A, C, and E show that a 3-, 5-, or 11-ns pulse evoked similarly rapid Ca²⁺ responses, having mean Ca²⁺ peak amplitudes (F/F₀) that were 1.56 ± 0.03 (n = 55), 1.59 ± 0.04 (n = 28), and 1.71 ± 0.03 (n = 42), respectively. Ca²⁺ responses were, however, somewhat longer lived (half-widths 15.6, 16.0, and 28.4 for 3-, 5-, and 11-ns pulses, respectively) than DMPP-evoked Ca²⁺ transients, and in some cells multiple Ca²⁺ spikes were present after the initial Ca²⁺ transient had subsided, an observation previously reported in cells exposed to a 5-ns pulse (34). In some cells also, as shown in Fig. 4 B–D and F, Ca²⁺ responses exhibited half-widths that were longer than 30 s such that [Ca²⁺]_i never reached baseline during the 75-s post-pulse monitoring period. Of note is that the percentage of cells exhibiting short-lived Ca²⁺ responses was similar for a pulse that was 3 or 5 ns in duration (76% and 71%, respectively) but dramatically reduced (26%) for a pulse that was 11 ns in duration. Thus, increasing pulse duration from only 5 to 11 ns reversed the trend for NEP-elicited Ca²⁺ responses to resemble those evoked by DMPP, eliciting instead mainly longer-lived Ca²⁺ responses. Similar results were obtained for cells exposed to a 12-ns pulse where only 25% of cells exhibited transient Ca²⁺ responses (Fig. S1).

Figure 4.

Comparison of Ca²⁺ responses in cells exposed to pulses 3, 5, and 11 ns in duration. (A), (C), and (E) show representative transient Ca²⁺ responses together with the average ±SE (black line) of all cells exposed to a 3-, 5-, and 11-ns pulse, respectively. (B), (D), and (F) show representative longer-lived Ca²⁺ responses together with the average ±SE (black line) of all cells exposed to a 3-, 5-, and 11-ns pulse, respectively. The percentage of cells that displayed transient versus long-lived Ca²⁺ responses are summarized in Table 1. Arrows indicate the time when the pulse was applied.

When pulse duration was increased to 25 ns (Fig. 5 A), 94% of the cells exhibited long-lived Ca²⁺ responses (i.e., half-widths greater than 30 s). In addition, the mean Ca²⁺ peak amplitude of the responses (F/F₀) was 2.05 ± 0.06 (n = 17), which was higher than that observed in cells exposed to pulses 11 ns or less in duration. For a 50-ns pulse, long-lived Ca²⁺ responses were observed in all the cells (Fig. 5 B), where the mean Ca²⁺ peak amplitude of the responses (F/F₀) was 2.13 ± 0.05 (n = 31), a measurement similar to that observed for a 25-ns pulse.

Figure 5.

Comparison of Ca²⁺ responses in cells exposed to pulses 25 and 50 ns in duration. (A) and (B) show representative Ca²⁺ responses together with the averaged ±SE responses of all cells (black line) exposed to a 25- and 50-ns pulse, respectively. Arrows indicate the time when the pulse was applied.

To further highlight the effect of increasing pulse duration, both transient and long-lived Ca²⁺ responses for each pulse duration were combined into a single trace and plotted on the same graph (Fig. 6). While each pulse caused an immediate rise in [Ca²⁺]_i, both the amplitude and the duration of the responses increased for pulse durations beyond 5 ns, with 11 ns being the pivotal duration at which these changes came into play, as summarized in Table 1. A one-way ANOVA revealed a significant effect of increasing pulse duration on the magnitude of the Ca²⁺ response (Fig. 6; p < 0.001). Post hoc analysis showed no significant difference in the magnitude of the response for 3- versus 5-ns pulses (p = 0.98), for 5- versus 11-ns pulses (p = 0.20), and for 25- versus 50-ns pulses (p = 0.77). However, the Ca²⁺ responses evoked by a 25- or 50-ns pulse were significantly higher than those evoked by a 3-, 5-, and 11-ns pulse (p < 0.05).

Figure 6.

Comparison of the mean Ca²⁺ traces for the different pulse durations. Traces are plotted as the averaged ±SE Ca²⁺ responses of all cells exposed to pulses ranging from 3 to 50 ns in duration. The arrow indicates the time when the pulse was applied. To see this figure in color, go online.

Table 1.

Summary of Ca²⁺ responses elicited by pulses ranging from 3 to 50 ns in duration

Pulse duration (ns)	Total cells (n)	Mean Ca2+ peak ± SE (F/F0)	t-Test (p-value)a	Mean Ca2+ half-width (s)	Transient Ca2+ activity (%)b	Long-lived Ca2+ activity (%)b
3	55	1.56 ± 0.03	–	15.6	76	24
5	28	1.59 ± 0.04	0.55	16.0	71	29
11	42	1.71 ± 0.03	<0.001	28.4	26	74
25	17	2.05 ± 0.06	<0.001	48.4	6	94
50	31	2.13 ± 0.05	<0.001	>50.0	0	100

^aAn unpaired t-test was used to compare the peaks of each pulse duration to that of the 3-ns pulse.

^bDetermined from the total number of cells.

Release of Ca²⁺ from internal stores was not responsible for the increase in Ca²⁺ response amplitude and duration

To evaluate the possibility that the higher-amplitude and longer-duration Ca²⁺ responses could be due to Ca²⁺ release from internal stores, cells were exposed to pulses ranging from 5 ns to 50 ns in Ca²⁺-free BSS containing 1 mM EGTA. Under these experimental conditions, the cells are still capable of functional Ca²⁺ release, since either a brief stimulation of the cells with carbachol, which is an agonist of muscarinic receptors, or perfusion of the cells with 50 mM caffeine elicits an increase in intracellular Ca²⁺ (38). As shown in Fig. 7, the rise in [Ca²⁺]_i was abolished regardless of pulse duration, indicating that the source of Ca²⁺ was extracellular throughout the 5- to 50-ns pulse range. As in Fig. 6, post hoc analysis revealed a significant difference in the magnitude of the Ca²⁺ response under control conditions for a 50-ns pulse compared with that for an 11-ns (p < 0.05) and 5-ns (p < 0.05) pulse. No significant difference in the magnitude of the response was observed when comparing a 5-ns pulse with an 11-ns pulse (p = 0.09).

Figure 7.

Effect of extracellular Ca²⁺ on Ca²⁺ responses in cells exposed to a 5-, 11-, or 50-ns pulse. The bar graph represents the magnitude of the Ca²⁺ response amplitude as a function of pulse duration under control conditions (black bars) or under Ca²⁺-free conditions in which 1 mM EGTA was present (red bars). Data are expressed as the mean ± SE. n, number of cells. To see this figure in color, go online.

VGCC inhibitors were less effective at blocking Ca²⁺ responses as pulse duration increased

Bovine adrenal chromaffin cells express four main VGCC types, namely P/Q-, N-, and L-type Ca²⁺ channels (67), and in previous studies we had shown that a cocktail of VGCC inhibitors that included agents selective for each of these four Ca²⁺ channel types blocked the Ca²⁺response evoked by a 5-ns pulse (33). In contrast, the cocktail of inhibitors was only 60%–70% effective in blocking the rise in [Ca²⁺]_i evoked by a 150-ns pulse (43). Here we used the same cocktail, consisting of 100 nM ω-agatoxin IVA, 20 nM ω-conotoxin GVIA, and 20 μM nitrendipine, to block P/Q-, N-, and L-type Ca²⁺ channels, respectively (33,43), to determine its effectiveness to inhibit Ca²⁺ responses elicited by a 3-, 5-, 11-, 25-, or 50-ns pulse. As shown in Fig. 8, simultaneously blocking all four VGCC types abolished Ca²⁺ influx triggered by a 3-ns pulse (Fig. 8 A) and achieved near complete inhibition of Ca²⁺ influx evoked by a 5-ns pulse as reported previously (33). When pulse duration was increased to 11 ns, the inhibitory effect of the VGCC inhibitor cocktail was substantially reduced (Fig. 8 B), with Ca²⁺ influx detected in 65% of the cells (Table 2). Increasing pulse duration further to 25 and 50 ns resulted in even less blockade whereby 71% and 85% of the cells, respectively, underwent a rise in [Ca²⁺]_i in response to the pulse. An unpaired t-test was used to compare the peaks of control cells versus treated cells for each pulse duration. All Ca²⁺ responses were significantly different (p < 0.05) between the control cells and the cells treated with the cocktail of blockers. A one-way ANOVA revealed a significant effect of increasing pulse duration on the magnitude of the Ca²⁺ response in the presence of the cocktail of blockers (Fig. 8 B; p < 0.001). Post hoc analysis showed no significant difference in the magnitude of the response for 3- versus 5-ns pulses (p = 0.97), for 5- versus 11-ns pulses (p = 0.43), and for 11- versus 25-ns pulses (p = 0.99). However, the Ca²⁺ responses evoked by a 50-ns pulse were significantly higher than those evoked by a 3-, 5-, 11-, and 25-ns pulse (p < 0.05).

Figure 8.

Effect of VGCC inhibitors on Ca²⁺ responses in cells exposed to a 3-, 5-, 11-, 25-, or 50-ns pulse. Results are plotted as the averaged cell responses ±SE for all cells exposed to a 3-ns (n = 15C/8T), 5-ns (n = 27C/23T), 11-ns (n = 22C/31T), 25-ns (n = 14C/14T), and 50-ns pulse (n = 35C/26T) (C, control; T, treatment) in the absence (A) and presence (B) of a cocktail of VGCC inhibitors containing 100 nM ω-agatoxin IVA, 20 nM ω-conotoxin GVIA, and 20 μM nitrendipine. Arrows indicate the time when the pulse was applied. n, number of cells. (C) Bar graph showing the inhibition of Ca²⁺ responses by the cocktail of VGCC blockers, and for a 50-ns pulse, with the cocktail also containing 100 nM SNX-482. To see this figure in color, go online.

Table 2.

Comparison of the effect of blocking VGCCs and eliminating external Na⁺ on Ca²⁺ responses evoked by pulses increasing in duration from 3 to 50 ns

Pulse duration (ns)	VGCC inhibitor cocktail			TMA+
	Total cells (n)	Cells responding (%)	Inhibition relative to control (%)a	Total cells (n)	Cells responding (%)	Inhibition relative to control (%)a
3	8	0	100	15	7	94
5	23	26	89	22	22	74
11	31	65	81	16	56	68
25	14	71	74	13	62	60
50	26	85	46	32	91	47

^aCalculated from the average of all cells. A response was considered significant when Ca²⁺ fluorescence increased by more than 10%.

To investigate the possibility that the component of the Ca²⁺ response that remained in the presence of the VGCC inhibitor cocktail could be due to the selective activation of another type of VGCC, namely R-type channels that are resistant to P/Q-, N-, and L-type Ca²⁺ channel blockers (68), we carried out experiments in which the R-type channel inhibitor SNX-482 was also included in the cocktail. As shown in Fig. 8 C, there was no further inhibition of the Ca²⁺ response evoked by a 50-ns pulse when 100 nM SNX-482 was also present. These results indicate that for pulses less than 11 ns in duration (i.e., a 3- or 5-ns pulse), Ca²⁺ influx occurred primarily via P/Q-, N-, and L-type VGCC whereas for pulses 11 ns in duration and longer, Ca²⁺ influx occurred also via a non-VGCC-mediated pathway, leading to greater Ca²⁺ response amplitudes and durations as pulse duration increased. Because functional T-type Ca²⁺ channels have not been reported in adult bovine chromaffin cells (69,70,71,72,73), the potential involvement of these channels was not considered in this study.

In contrast to the VGCC inhibitor cocktail, Cd²⁺, an inorganic, non-selective inhibitor of VGCCs, eliminated NEP-evoked Ca²⁺ responses regardless of pulse duration (Fig. S2). We had observed a comparable inhibitory effect by Cd²⁺ on Ca²⁺ responses elicited by a 150-ns pulse (43). Given our thinking at the time, we had suggested that Cd²⁺ was blocking not only VGCCs but also Ca²⁺-permeable membrane nanopores. However, of note is that Cd²⁺ blockade is not restricted to VGCCs as it can block other membrane ion channels (74,75,76), the Na⁺/Ca²⁺ exchanger (77) as well as receptors for glutamate (78,79,80). Thus, the complete blockade of Ca²⁺ responses by Cd²⁺ could instead involve the interaction of this cation with an ion channel(s) activated by the longer-duration NEPs. The identity of such an ion channel is investigated in a later section.

Ca²⁺ responses relied less on membrane depolarization and extracellular Na⁺ as pulse duration increased

A likely mechanism underlying the VGCC-mediated influx of Ca²⁺ evoked by a 2-, 4-, and 5-ns pulse is a change in membrane potential that is sufficient in magnitude to reach VGCC activation thresholds. This possibility was investigated in a prior study (33) by exposing chromaffin cells to a 5-ns pulse under conditions in which the extracellular K⁺ concentration was decreased from 5 to 2 mM to drive the resting membrane potential to a more negative value (81,82), reasoning that the probability of VGCC activation thresholds being reached would decrease if the membrane potential was more hyperpolarized. Indeed, we found that lowering extracellular K⁺ to 2 mM either abolished Ca²⁺ responses completely or significantly reduced the amplitude of the rise in [Ca²⁺]_i in those cells that responded to the pulse. The same strategy was used here to determine the extent to which Ca²⁺ influx evoked by a 50-ns pulse, which included both a VGCC-mediated and non-VGCC-mediated pathway, was affected by driving membrane potential to a more hyperpolarized value. It was anticipated that the non-VGCC pathway Ca²⁺ influx, unlike that mediated by VGCCs, would not be affected by hyperpolarization and all cells exposed to a 50-ns pulse would show a Ca²⁺ response of some magnitude. Using the perforated whole-cell patch-clamp technique, we measured a statistically significant difference of 4 mV in membrane potential when the extracellular K⁺ concentration was decreased from 5 to 2 mM (V_m = −42.9 ± 1.4 vs. −47.1 ± 0.8 mV for 5 and 2 mM external K⁺, respectively; p ≤ 0.0005; Fig. 9 A). As shown in Fig. 9 B, this degree of hyperpolarization was sufficient to suppress spontaneous action potentials. When cells were exposed to a 5-ns pulse in 2 mM external K⁺, only 49% of the cells underwent an increase in [Ca²⁺]_i compared with 100% of the cells when the external concentration of K⁺ was 5 mM (Fig. 9 C). Moreover, in the responding cells, the amplitude of the rise in [Ca²⁺]_i was 64% less than that in cells exposed to the pulse in 5 mM external K⁺ (F/F₀ = 1.38 ± 0.15 vs. 2.05 ± 0.15 for 2 and 5 mM external K⁺, respectively), with the difference being statistically significant (p ≤ 0.0005). These findings are similar to those previously reported (33). When cells were exposed to a 50-ns pulse (Fig. 9 D), the NEP-evoked increase in [Ca²⁺]_i in the presence of 5 mM external K⁺ was greater in amplitude than that evoked by a 5-ns pulse under the same conditions, which reflected the contribution of the non-VGCC-mediated pathway to the Ca²⁺ response as shown in Fig. 8. As anticipated, reducing the external K⁺ concentration to 2 mM had no effect on the number of cells responding to the 50-ns pulse, since all the exposed cells underwent a rise in [Ca²⁺]_i. Thus, the non-VGCC-mediated pathway of Ca²⁺ influx activated by the longer-duration 50-ns pulse occurred in all cells regardless of membrane potential. Although reducing the external K⁺ concentration to 2 mM still significantly attenuated the rise in [Ca²⁺]_i mediated by a 50-ns pulse measured in 5 mM external K⁺, the reduction was approximately threefold smaller (22%; F/F₀ = 2.09 ± 0.14 vs. 2.39 ± 0.08 for 2 and 5 mM external K⁺, respectively) than that resulting from the same external K⁺ reduction for a 5-ns pulse. These results are consistent with the attenuation still being dictated by a reduced availability of VGCCs; however, this effect was proportionately smaller for a 50-ns pulse due to the presence of an additional sustained Ca²⁺ entry pathway through non-VGCCs (see discussion for further explanations).

Figure 9.

Effect of 2 and 5 mM external K⁺ on membrane potential, spontaneous action potentials, and Ca²⁺ responses evoked by a 5- or 50-ns pulse. (A) Averaged membrane potential ±SE for cells in 5 mM K⁺-BSS and then in 2 mM K⁺-BSS (n = 8). (B) Spontaneous action potential activity recorded from one of the cells in (A). At 90 s, the 5 mM K⁺-BSS was changed to 2 mM K⁺-BSS. The values for membrane potential presented in (A) were obtained at 0 s for 5 mM external K⁺ and at 350 s for 2 mM external K⁺. (C) Averaged cell responses ±SE for cells exposed to a 5-ns pulse in 5 mM K⁺-BSS (n = 26) and in 2 mM K⁺-BSS (n = 31). (D) Averaged cell responses ±SE for cells exposed to a 50-ns pulse (right) in 5 mM K⁺-BSS (n = 37) and in 2 mM K⁺-BSS (n = 32). ^∗∗∗p ≤ 0.0005. Pulser 3 was used to deliver the 5-ns pulses. To see this figure in color, go online.

Previously, we reported that for a 2- or a 5-ns pulse, the pulse-evoked rise in [Ca²⁺]_i in each case relied almost entirely on extracellular Na⁺ (33,35). Here, we investigated whether longer-duration pulses similarly relied on extracellular Na⁺ to elicit Ca²⁺ responses by exposing chromaffin cells to a 3-, 5-, 11-, 25-, or 50-ns pulse in Na⁺-free BSS in which Na⁺ was replaced with an equimolar concentration of TMA⁺. As shown in Fig. 10 and Table 2, Ca²⁺ responses were abolished in 93% of cells exposed to a 3-ns pulse and in 78% of cells exposed to a 5-ns pulse under Na⁺-free conditions, which is consistent with previous observations (33). Also consistent with previous observations is that in those few cells in which an increase in [Ca²⁺]_i was observed, Ca²⁺ response amplitudes were significantly reduced relative to the control. When cells were exposed to an 11-, 25-, or 50-ns pulse, the rise in [Ca²⁺]_i was abolished in fewer cells as pulse duration increased (44%, 38%, and 9% of the cells, respectively), with Ca²⁺ response amplitudes increasing concomitantly (F/F₀ = 1.48 ± 0.07, 1.56 ± 0.11, and 1.70 ± 0.09 for an 11-, 25-, and 50-ns pulse, respectively). An unpaired t-test was used to compare the peaks of control cells versus cells in no Na⁺ for each pulse duration. All Ca²⁺ responses were significantly different (p < 0.05) between the control cells and the cells bathed in TMA⁺. A one-way ANOVA revealed a significant effect of increasing pulse duration on the magnitude of the Ca²⁺ response in the presence of TMA⁺ (Fig. 10 B; p < 0.001). Post hoc analysis showed no significant difference in the magnitude of the response for 3- vs. 5-ns pulses (p = 0.99), for 5- vs. 11-ns pulses (p = 0.54), and for 11- vs. 25-ns pulses (p = 0.99). However, the Ca²⁺ responses evoked by a 50-ns pulse were significantly higher than those evoked by a 3-, 5-, 11-, and 25-ns pulse (p < 0.05). Thus, for pulses 3 ns and 5 ns in duration, NEP-induced Ca²⁺ influx was mainly dependent on external Na⁺. Increasing pulse duration to only 11 ns caused Ca²⁺ responses to become less dependent on external Na⁺, a trend that continued as pulse duration increased to 25 and 50 ns. Note the similarity of this pattern of behavior with that observed when VGCCs were blocked (Table 2).

Figure 10.

Effect of external Na⁺ on Ca²⁺ responses in cells exposed to a 5-, 11-, 25-, and 50-ns pulse. Results are plotted as the averaged cell responses ±SE for all cells exposed to a 3-ns (n = 15C/15T), 5-ns (n = 22C/22T), 11-ns (n = 14C/16T), 25-ns (n = 16C/13T), and 50-ns pulse (n = 28C/32T) (C, control; T, treatment) in the presence (A) and absence (B) of external Na⁺. Arrows indicate the time when the pulse was applied. n, number of cells. (C) Bar graph showing the inhibition of Ca²⁺ responses by TMA. To see this figure in color, go online.

Previously we had determined that for a 5-ns pulse, Na⁺ influx did not involve VGSCs, since blocking these channels with a high concentration (10 μM) of the VGSC inhibitor TTX failed to block the pulse-elicited Ca²⁺ responses (33). We repeated that experiment here and again found no effect of TTX on Ca²⁺ responses evoked by a 5-ns pulse (F/F₀ = 1.39 ± 0.06, n = 15 vs. 1.40 ± 0.06, n = 15 for control and TTX, respectively; p = 0.91, data not shown). A similar lack of effect of TTX was found when cells were exposed to a 50-ns pulse (F/F₀ = 1.89 ± 0.07, n = 16 vs. 1.84 ± 0.12, n = 15 for control and TTX, respectively; p = 0.72, data not shown), confirming that increasing pulse duration does not involve the activation of VGSCs in NEP-evoked Ca²⁺ responses.

Non-VGCC-mediated Ca²⁺ entry did not involve several known Ca²⁺-permeable ion channels

As stated earlier, the non-VGCC-mediated pathway of Ca²⁺ influx elicited by a 150-ns pulse had been suggested to be attributed to Cd²⁺-sensitive nanopores that formed as a result of membrane electropermeabilization (43). However, an observation inconsistent with this notion was that YO-PRO-1 uptake, which serves as an indicator of membrane nanoelectroporation to NEPs, was not observed in exposed cells (43). Even in the present study, YO-PRO-1 uptake was not detected in cells exposed to a single or train of ten 50-ns pulses (data not shown). Moreover, studies by others had failed to show that the conductance of ions flowing through plasma membrane nanopores following NEP exposure was blocked by Cd²⁺ (57,83). Given these considerations, an alternative possibility was that increasing pulse duration caused the activation of one or more voltage-independent ion channels that were permeable to Ca²⁺ and perhaps capable also of being inhibited by Cd²⁺. We investigated this possibility by focusing attention on Ca²⁺-permeable ion channels expressed in bovine chromaffin cells, using blockers of these channels to assess whether Ca²⁺ responses evoked by a 50-ns pulse as well as those evoked by DMPP (100 μM) were affected.

One candidate was the Na⁺- and Ca²⁺-permeable, constitutively active non-selective cation conductance described by Cheek and Thorn (64), which is voltage independent and is blocked by 100 μM La³⁺. In a previous study exploring the possibility that 5-ns-induced increases in [Ca²⁺]_i were mediated by Na⁺ entry via this channel, we found that La³⁺ at either 100 or 200 μM had no consistent effect on Ca²⁺ response amplitude (33), thereby eliminating the involvement of this channel in the response of the cells to the pulse. In the present study in which this channel was instead being considered as a potential pulse-evoked non-VGCC pathway of Ca²⁺ entry, we found that La³⁺ (100 μM) was ineffective as an inhibitor of Ca²⁺ responses evoked in cells exposed also to a 50-ns pulse (Table 3). The Ca²⁺ response of the cells to DMPP was also not affected by La³⁺.

Table 3.

Effect of blocking Ca²⁺-permeable ion channels on the Ca²⁺ responses evoked by a 50-ns pulse and DMPP (100 μM)

Channel targeted	Cells (n)	Mean Ca2+ peak ± SE(F/F0)	Reduction (%)	t-Test (p-value)a
50-ns pulse
Non-selective cation channel
No treatment	35	2.29 ± 0.08	3	0.53
La3+(100 μM)	27	2.25 ± 0.06
Pannexin channel
No treatment	31	2.26 ± 0.09	10	0.15
Probenecid (200 μM)	26	2.13 ± 0.13
No treatment	20	1.99 ± 0.09	0	0.75
10Panx1 (200 μM)	25	2.02 ± 0.1
P2X channel
No treatment	31	2.26 ± 0.09	2	0.79
PPADS (50 μM)	26	2.24 ± 0.1
DMPP
Non-selective cation channel
No treatment	12	1.90 ± 0.23	6	0.72
La3+(100 μM)	14	1.85 ± 0.15
Pannexin channel
No treatment	24	1.94 ± 0.07	39	0.0001b
Probenecid (200 μM)	24	1.57 ± 0.14
No treatment	20	1.85 ± 0.07	28	0.009b
10Panx1 (200 μM)	23	1.61 ± 0.05
P2X channel
No treatment	26	1.88 ± 0.10	19	0.02b
PPADS (50 μM)	26	1.71 ± 0.13

^aAn unpaired t-test was used to compare the peaks of control versus treatment.

^bSignificant difference.

Another candidate was the pannexin 1 channel that is permeable to Ca²⁺ and which has been shown to be activated by DMPP (62). To block this channel, we used probenecid (200 μM) and as well as the peptide ¹⁰Panx1 (200 μM), the results of which are shown in Table 3. Neither blocker reduced the Ca²⁺ response evoked by a 50-ns pulse (p > 0.05), indicating that pannexin 1 channels were not activated by the long-duration NEPs. However, in agreement with (62) in which pannexin channels were shown to play a role in amplifying Ca²⁺ responses evoked by DMPP, probenecid reduced the DMPP-mediated Ca²⁺ response by 39% (p < 0.0001), and ¹⁰Panx1 reduced it by 28% (p < 0.001); the ¹⁰Panx1 scrambled peptide that is inactive as an inhibitor was without effect (data not shown).

Lastly, we tested the potential involvement of ionotropic P2X purinoreceptors (84) in mediating Ca²⁺ influx elicited by a 50-ns pulse, using PPADS to block these channels. As shown in Table 3, PPADS (50 μM) had no significant effect on Ca²⁺ responses evoked by a 50-ns pulse (p > 0.05), suggesting that P2X receptors were also not responsible for the second pathway of Ca²⁺ entry. DMPP-evoked Ca²⁺ responses were, however, slightly reduced by blocking P2X receptors.

Discussion

As highlighted in this study, adrenal chromaffin cell Ca²⁺ responses evoked by a single NEP take on different characteristics over a very short range of pulse durations, on the order of tens of nanoseconds. Specifically, NEP-elicited increases in [Ca²⁺]_i more closely resemble those induced by nAChR stimulation, the physiological stimulus, when pulse duration is 3 or 5 ns, having a similarly transient kinetic profile and utilizing the same single pathway of Ca²⁺ entry into the cells, VGCCs. When pulse duration is increased from 5 ns to only 11 ns, the kinetic profile of the Ca²⁺ responses begins to shift to one in which the rise in [Ca²⁺]_i is longer lived, a trend that continues as pulse duration is increased further to 25 and 50 ns. Ca²⁺ responses also become greater in amplitude as pulse duration increases beyond 11 ns. The changes both in [Ca²⁺]_i amplitude and duration with increasing pulse duration are attributed to the activation of a non-VGCC-mediated Ca²⁺ entry pathway(s), the basis for which is discussed in the following sections.

Ca²⁺ release from internal stores does not contribute to the amplification of Ca²⁺ responses elicited by increasing pulse duration

As mentioned in results, Ca²⁺ release from intracellular stores is unlikely to contribute to the longer-duration and higher-amplitude Ca²⁺ responses elicited by pulses 11, 25, and 50 ns in duration (Fig. 7), particularly since each NEP was delivered to chromaffin cells at its respective Ca²⁺ activation threshold E-field. To emphasize the significance of this latter point for the present study, we had previously reported that when a 5-ns pulse was delivered to cells in the absence of extracellular Ca²⁺, intracellular Ca²⁺ mobilization from internal stores could be observed only when the magnitude of the E-field was increased well above the threshold eliciting Ca²⁺ influx (37,38). Here a similar determination was made for a 50-ns pulse applied to the cells at 8 MV/m, which is well above the Ca²⁺ activation threshold of 1.1 MV/m for this pulse duration. In the absence of extracellular Ca²⁺, we observed a robust Ca²⁺ response of the cells when a 50-ns pulse was applied at 8 MV/m (F/F₀ = 1.60 ± 0.12 (n = 7); data not shown) versus no Ca²⁺ response when the pulse was applied at 1.1 MV/m (Fig. 7). Thus, regardless of pulse duration, the NEP-elicited Ca²⁺ responses reported in this study are solely the result of plasma membrane-mediated Ca²⁺ influx, not Ca²⁺ mobilization from internal stores.

Analytical calculations and numerical modeling suggest that the length of time that the membrane charges to the peak voltage determines the nature of NEP-evoked Ca²⁺ responses

Our previous study in which adrenal chromaffin cells were stimulated with 2-ns unipolar and bipolar pulses was the first to demonstrate the sensitivity of these cells to electrostimulation by NEPs that differ in duration by only tens of nanoseconds (35). Using a 2-ns pulse as a stimulus, we found that the rise in [Ca²⁺]_i elicited by the pulse could be “canceled” if the pulse was immediately followed by the delivery of a second pulse of the same duration but with the opposite polarity. Notably, adding an interval of only 30 ns between the two opposite-polarity phases of the bipolar pulse caused full recovery of the Ca²⁺ response, a time frame considerably shorter than the microsecond range previously reported for full recovery of responses in other cell types (28,85). In fact, some recovery of the Ca²⁺ response in chromaffin cells was already evident when the interphase interval was only 10 ns (15), suggesting that the membrane events that lead to VGCC activation in this excitable cell type occur within 10 ns.

Understanding the sensitivity of chromaffin cell excitability to short-duration NEPs requires knowledge of the biophysical and molecular events occurring at the plasma membrane, the first cellular structure impacted by the E-field of an applied NEP. Essentially, the plasma membrane undergoes a rapid change of the transmembrane potential (TMP) during the pulse due to capacitive membrane charging, followed by TMP returning exponentially back to baseline by capacitive membrane discharging after the pulse has ended. Real-time measurements of the magnitude and dynamics of TMP changes of chromaffin cell membrane charging and discharging could yield valuable insight into how these changes culminate in downstream effects such as the opening of VGCCs and other Ca²⁺ entry pathways. However, membrane charging and discharging by NEPs occur on a nanosecond time scale, which for the very short pulse durations used in this study cannot be temporally resolved by current electrophysiological and imaging methods. In view of this limitation, we performed analytical calculations of capacitive membrane charging/discharging in chromaffin cells exposed to a 3-, 5-, 11-, 25-, and 50-ns pulse to explain, theoretically at least, why a pulse duration of 11 ns might be sufficient to begin evoking additional pathways of Ca²⁺ influx.

For the analytical calculations, we used a simplified spherical cell membrane model similar to that used in a previous study (15) and calculated the field-induced time-dependent membrane potential $V_{\mathrm{m}}\left(t\right)$ for each pulse duration as follows:

$$V_{\mathrm{m}}\left(t\right)=V_{\mathrm{m},0}+\left(V_{\mathrm{m},\text{induced}}-V_{\mathrm{m},0}\right)\left(1-\exp \left(-\frac{t-t_0}{{\tau }_{\mathrm{m}}}\right)\right)\text{,}$$ (1)

where $V_{\mathrm{m},0}$ is the initial TMP at time $t_0,V_{\mathrm{m},\text{induced}}$ is the induced TMP by the external E-field, and ${\tau }_{\mathrm{m}}$ is the membrane-charging time constant, which in the case of chromaffin cells is ≈100 ns (15,37). The induced TMP was calculated at the pole of the cell facing the anode where the TMP value is the highest. To investigate the effect of the different pulse durations on cell membrane charging, we used the integrated product of TMP and time (i.e., V.ns) during which the TMP is charging above some excitation threshold. For the latter, we used $V_{\mathrm{m},\text{critical}}=0.02\mathrm{V}$ as an arbitrary threshold voltage for which excitation would occur if this threshold was exceeded (86). The membrane charging and discharging parameters calculated for each pulse duration were the area up to the peak TMP, hereafter called the charging area (A_c), which represents the change in TMP when the pulse is “ON” (i.e., during pulse application), and the area from the peak TMP to 0.02 V, hereafter called the discharge area (A_d), which represents the change in TMP when the pulse is “OFF.” Actual pulse traces delivered to cells during experiments were used for the calculations. Fig. 11 shows a schematic representation of the time course of the calculated change in TMP highlighted by the shaded areas A_c and A_d, and the results obtained for the various pulse durations are summarized in Table 4.

Figure 11.

Schematic of the time course of the calculated change in the NEP-induced TMP. The red line indicates the threshold V_m,critical = 0.02 V used in the calculations of A_c and A_d. The highlighted areas are A_c and A_d for which the TMP charges up to the peak or discharges from the peak down to 0.02 V, respectively. To see this figure in color, go online.

Table 4.

Comparison of the effect of pulse duration on the NEP-induced peak TMP, A_c, and A_d

Pulse duration (ns)	Pulse amplitude (MV/m)	Calculations			Simulations
		Peak TMPa (V)	Ac	Ad	Peak TMPa (V)	Ac	Ad
3	13.4	4.2	11.6	407.9	3.7	8.5	617.6
5	9.4	4.0	12.0	387.4	3.2	9.5	570.8
11	4.7	4.4	27.9	424.0	3.5	21.8	691.5
25	2.2	3.9	47.1	385.1	3.4	46.3	727.5
50	1.1	3.5	130.1	343.7	3.2	85.8	700.3

^aAt the pole of the cell facing the anode where the TMP value is the highest.

Peak TMP

As shown in Table 4, the peak TMP was close to 4 V for all pulse durations, thereby ruling out an increase in peak TMP as a factor underlying the change in Ca²⁺ response characteristics with increasing pulse duration. Thus, by applying each pulse duration at its respective E-field threshold, a relatively consistent “dose” was delivered to the cells in each case, with VGCC activation occurring when the TMP reached a certain common level, which theoretically was 4 V but realistically is much lower and is currently unknown. Regarding the E-field threshold, of note is that applying a 5- or 50-ns pulse under conditions in which membrane potential was slightly more hyperpolarized resulted in a significant reduction in VGCC-mediated Ca²⁺ responses (Fig. 9), highlighting the sensitivity of the cells to a slight increase in the magnitude of membrane potential such that the probability of VGCC activation thresholds being reached was decreased (33).

Given the similarity of TMP values for the various pulse durations, one can speculate that the TMP threshold solely determines the point at which membrane potential-dependent components, such as VGCCs, are activated but not other non-voltage-dependent membrane events that lead to Ca²⁺ influx via other pathways.

A_c

In contrast to the consistency in peak TMP values for each pulse duration, the length of time for the membrane to reach the peak TMP increased markedly for pulse durations that exceeded 5 ns. As shown in Table 4, a 3- and 5-ns pulse had similar A_c values, which agrees with the experimental findings of similar Ca²⁺ response characteristics for each pulse duration. When pulse duration increased from 5 to 11 ns, A_c increased 2.8-fold relative to a 3-ns pulse. Thus, an 11-ns pulse exerted a larger effect than either a 3- or 5-ns pulse on the cells due to the longer period of time for the membrane to charge to a TMP of 4 V, leading to Ca²⁺ responses that started to become longer lived. For pulses that were 25 and 50 ns in duration, A_c, relative to a 3-ns pulse, increased even further (4.1- and 11.2-fold, respectively; Table 4), again consistent with the experimental findings showing that increasing pulse duration beyond 11 ns had even greater effects on Ca²⁺ response amplitude and duration. This point is highlighted in Fig. 12, where A_c values are normalized to the values calculated for a 3-ns pulse. Thus, the time it takes the membrane to charge to the peak TMP appears to be the critical parameter that determines whether only VGCCs are activated or whether non-VGCC pathways of Ca²⁺ influx are also triggered.

Figure 12.

Effect of increasing pulse duration on A_c. The bar graph represents the A_c values normalized to the values calculated for a 3-ns pulse, as a function of pulse duration, for both the calculations (black bars) and the simulations (red bars). To see this figure in color, go online.

A_d

Interestingly, A_d did not increase with pulse duration (Table 4). A possible explanation is that due to the lack of YO-PRO-1 uptake for any of the pulse durations, the membrane is assumed to remain “intact” (i.e., no nanoelectroporation) in each case. Hence, the TMP would discharge at a comparable rate for each pulse duration and A_d values would be similar.

Because neither TMP nor membrane discharging time appeared to be important for determining the nature of chromaffin cell membrane responses to NEP that increased in duration from 3 to 50 ns, we further investigated these findings by employing a more realistic representation of a chromaffin cell than the simple spherical cell model used for the analytical calculations, specifically the 2D numerical cell model previously developed by our group (37) that is based on the meshed transport network method developed by Smith and Weaver (52) and uses measured chromaffin cell properties (87). The calculated values for TMP, A_c, and A_d derived from the simulations are summarized in Table 4. Similar to the analytical calculation results, there was no effect of pulse duration on the peak TMP but a pronounced effect of pulse duration on A_c. The latter is further shown in Fig. 12, where A_c values are normalized to the values calculated for a 3-ns pulse. However, different from the analytical calculation results is that the cell membrane takes slightly longer to fully discharge for the longer-duration pulses (i.e., greater than 5 ns). That is, relative to a 3- or a 5-ns pulse, A_d is slightly increased for pulses 11 ns and longer in duration. Thus, although the time it takes the membrane to charge to the peak TMP appears to be the main parameter for determining the nature of chromaffin cell membrane responses as pulse duration increases from 3 to 50 ns, the time it takes the membrane to discharge may also play a role, albeit a minor one.

The nature of the non-VGCC-mediated pathway of Ca²⁺ entry remains to be determined

While analytical calculations and numerical modeling have suggested that membrane-charging time is the parameter that is responsible for triggering Ca²⁺ influx in a manner that is independent of VGCC activation (Fig. 8), leading to enhanced Ca²⁺ entry as pulse duration increases (i.e., as the time the membrane charges to peak voltage increases), a major challenge that remains is to elucidate the nature of this second Ca²⁺ influx pathway. In our studies describing the use of pulses 150–400 ns in duration as a stimulus for chromaffin cells (43), we had hypothesized that the second Ca²⁺ entry pathway activated by these longer-duration NEPs was the result of direct nanoelectropermeabilization of the plasma membrane to Ca²⁺ (i.e., nanopores that conduct Ca²⁺), which was in line with the thinking at the time (e.g., (46,48)). However, influx of YO-PRO-1 that serves as an indicator of membrane nanoelectroporation (44) was never observed unless cells were exposed to trains of 150-ns pulses delivered at very high E-fields (43).

Over the years it has become increasingly clear that what constitutes the exact nature of nanoelectroporation cannot be stated with certainty, as it can vary with cell type based on differences in lipid membrane composition and/or structural membrane differences and, perhaps, other unknown factors. Thus, in addition to a posit of simple lipid bilayer-spanning electropores (i.e., holes or lesions) that are around 1 nm in diameter (46), nanoelectroporation has been suggested to encompass a broad range of plasma membrane perturbations that include, as examples, complex electropore formation and structure (“electropermeome”) (88), lipid electropores with cytoskeletal constraints (89), impairment of the bilayer by lipid peroxidation (90,91,92) or scrambling (93), membrane disruptions based on permeabilization of membrane proteins rather than of the lipid bilayer (90,94), and electroconformationally altered membrane proteins (94,95).

Another possibility that we considered in the present study is that the NEP-induced conductance of Ca²⁺ across the plasma membrane of chromaffin cells may instead be due to the opening of some yet unidentified membrane ion channel that is selectively activated when pulse durations exceed 5 ns. Our data support the hypothesis that this additional Ca²⁺ entry pathway may be the product of one or more voltage-independent Ca²⁺ entry pathways. This suggestion is based first on the observation that for pulses longer than 5 ns, a large component of the Ca²⁺ response remains despite the blockade of P/Q-, N-, and L-type VGCCs, which are the main types of VGCC expressed in bovine chromaffin cells (67). Although R-type Ca²⁺ channels are expressed and functional in mouse (96,97,98,99) and rat chromaffin cells (73,100,101), their existence in the same cell type in the bovine species is controversial, with one group providing functional evidence (102) while other groups are failing to reveal their existence (103,104,105). To that end, we tested the effects of a 50-ns pulse on cells treated with inhibitors of the VGCCs described above as well as with SNX-482, a toxin known to inhibit R-type VGCCs with an IC₅₀ in the range of 15–30 nM (106). Regardless of the existence or not of R-type Ca²⁺ channels in bovine cells, we reasoned that the lack of an additional inhibitory effect of SNX-482 on the Ca²⁺ response evoked by a 50-ns pulse could be interpreted as indicating that the component of the Ca²⁺ response that remained would: 1) exclude a contribution from R-type Ca²⁺ channels; and 2) support the concept that this component is distinct from that produced by VGCCs. Consistent with the latter premises, adding 100 nM SNX-482 to the cocktail containing inhibitors for the other VGCCs did not lead to a greater attenuation of the Ca²⁺ response elicited by a 50-ns pulse.

A second argument in favor of a voltage-independent Ca²⁺ entry pathway triggered when pulse duration is increased beyond 5 ns was the observation that lowering external K⁺ concentration from 5 to 2 mM produced a differential effect on Ca²⁺ responses elicited by short (3 and 5 ns) versus long (11, 25, and 50 ns) NEPs. We first determined that lowering external K⁺ from 5 to 2 mM led to a 4 mV hyperpolarization of the membrane, as expected for K⁺ playing a major role in determining resting membrane potential (RMP) in this and many other excitable cells. In fact, we found that in chromaffin cells exhibiting spontaneous action potentials, which has been described by others (107,108,109), the 4 mV hyperpolarization of the membrane was sufficient in magnitude to attenuate or even suppress such activity. We next replicated our original finding that a 5-ns pulse was either unable to evoke a Ca²⁺ response in 2 mM external K⁺ or else triggered a Ca²⁺ response that was attenuated by more than 60% in 2 mM versus 5 mM external K⁺ (33). As illustrated in the working model presented in Fig. 13 A and B, we hypothesize that the hyperpolarization in 2 mM external K⁺ would push RMP further away from the threshold for activation of VGCCs (red solid line). Assuming that the magnitude of the depolarization mediated by both the 5- and 50-ns pulse is similar (NEP ΔV), the hyperpolarization would result in fewer Ca²⁺ channels being activated and a smaller Ca²⁺ response in each case (highlighted by the yellow area). However, the longer pulse would also stimulate an additional voltage-independent Ca²⁺ channel (VICC) pathway as demarked by the solid green line and area in Fig. 13 B. Although fewer VGCCs would be activated by the pulse when external K⁺ is lowered to 2 mM, this effect would be opposed by activation of VICC across which Ca²⁺ influx would be dictated by the driving force or electrochemical gradient for Ca²⁺ (Nernst potential for Ca²⁺ > +120 mV). If anything, the hyperpolarization mediated by lowering external K⁺ to 2 mM would have a small enhancing effect on Ca²⁺ influx due to a 4 mV increase in the driving force for Ca²⁺. Although this hypothesis will require further testing, regardless of the uncertainty about the relative magnitude of the conductances mediated by VGCCs and VICC, and the amount of depolarization mediated by NEPs, which could be different for the range of durations of NEPs tested, the working model presented in Fig. 13 A and B, can explain why the responses to longer NEPs were larger, graded, more sustained, and less affected by cell hyperpolarization than those evoked by ultrashort NEPs.

Figure 13.

Hypothetical working model explaining the differential effects of short- versus long-duration NEP on Ca²⁺ responses elicited in bovine chromaffin cells. (A) At 5 mM external K⁺ (left), a 5-ns pulse causes membrane depolarization (NEP ΔV) that triggers a Ca²⁺ conductance (I_Ca) due to activation of VGCCs (yellow area). At 2 mM external K⁺ (right) that hyperpolarizes the membrane, the activation threshold for VGCCs is shifted to the right (red arrowhead), causing fewer VGCCs to be activated. (B) At 5 mM external K⁺ (left), a 50-ns pulse similarly activates VGCCs by producing membrane depolarization but in addition stimulates a VICC conductance (green area) of unknown origin. At 2 mM external K⁺ (right) that hyperpolarizes the membrane, the activation threshold for VGCCs is shifted to the right (red arrowhead), causing fewer VGCCs to be activated, but there is little effect on VICC (green area) conductance, which is reliant on the driving force for Ca²⁺ (see text for a more detailed explanation). The resting membrane potential (RMP) values were taken from Fig. 9A. Hypothetical current-voltage relationships are shown for VGCCs (red traces) and VICCs (green traces). (C) Schematic diagram illustrating the activation of VGCC (red) and VICC (green) pathways of Ca²⁺ influx in relation to pulse duration. To see this figure in color, go online.

The schematic diagram in Fig. 13 C illustrating the activation of a VICC pathway of Ca²⁺ influx elicited by a 50-ns pulse raises the question of whether this Ca²⁺ entry pathway is produced by one or more ion channel proteins. While the involvement of several Ca²⁺-conducting ion channels known to be expressed in these cells (Table 3) such as the constitutively active non-selective cation conductance (64), the pannexin 1 channel (62), and P2X purinoreceptors (84) were eliminated in this study, there may be other Ca²⁺-conducting channels that we are unaware of and/or remain to be identified. To underscore this point, we previously reported that in cells exposed to a 2- or 5-ns pulse, the NEP-evoked rise in [Ca²⁺]_i relied almost entirely on extracellular Na⁺ (15,33), also shown here for a 3-ns pulse (Fig. 10), leading us to conclude that Na⁺ entry was responsible for membrane depolarization and subsequent VGCC activation. Given that Na⁺ influx occurred via a TTX-insensitive pathway (33), as also found in this study for a 3-ns pulse, we originally proposed that Na⁺ influx was occurring via nanopores (33), a finding that was supported by whole-cell patch-clamp experiments showing that a 5-ns pulse elicited an inward current carried primarily by Na⁺ (110). However, the suggestion that the formation of Na⁺-conducting nanopores was responsible for the inward Na⁺ current was inconsistent with the observation that Na⁺ entry occurred under conditions that did not also elicit YO-PRO-1 influx (34).

New insight into the nature of the NEP-elicited inward Na⁺ current has recently been reported by our group (58), where we showed that the inward current has characteristics consistent with a non-selective cation channel(s). More specifically, 70%–80% of the current was found to be due to the combined activation of TRPC4/5 channels and the NALCN. Except for the presence of TRPC4/5 channel mRNA transcripts that has been reported in bovine chromaffin cells (111), TRPC4/5 channels have as yet no established function in these cells. The NALCN, which is expressed in neurons where it regulates the RMP and neuronal excitability by affecting the resting Na⁺ permeability (112,113,114), was only first identified in mouse chromaffin cells in 2021 (115). In fact, we were the first group to report the presence of the NALCN in bovine chromaffin cells by finding that it is a target of a 5-ns pulse (58). Therefore, in the context of the present study, there may be other unidentified channel(s) that are activated by NEPs, promoting Ca²⁺ entry in a manner that is linked to increasing pulse duration. Whatever these channels may be, they are apparently blocked by Cd²⁺.

Conclusion

In this study, we again provide evidence of the remarkable sensitivity of isolated adrenal chromaffin cells to stimulation by short-duration NEPs, showing that increasing pulse duration by only tens of nanoseconds can modulate the amplitude and duration of increases in [Ca²⁺]_i. Thus, in addition to NEP-evoked Ca²⁺ influx via VGCCs that mimics the mechanism underlying Ca²⁺ influx triggered in vivo by nAChR stimulation, other voltage-independent Ca²⁺-permeable ion channels may well be activated when pulse durations exceed 5 ns. While analytical calculations and numerical modeling have provided a theoretical explanation for such activation, the identity of the ion channels that are involved, as well as the basis for their dependence on membrane-charging time for activation, remain unknown. Regardless, our data highlight how varying NEP duration over a very short range has the ability to enhance Ca²⁺ influx, the trigger for catecholamine release, a finding that has important implications for developing novel electrostimulation approaches using NEPs to modulate neurosecretion.

Author contributions

J.Z. and G.L.C. planned the research. J.Z. designed the experiments and plotted the experimental data. S.H.Y. and V.M. performed the Ca²⁺ imaging experiments and analyzed the experimental data. L.Y. performed the patch-clamp electrophysiology experiments and related Ca²⁺ imaging experiments and analyzed the experimental data. V.M. built custom scripts for experimental data plots. V.M and J.Z. performed the theoretical calculations and numerical modeling simulations, and J.Y. assisted with their interpretation. J.Z. and G.L.C. wrote the first draft of the manuscript. N.L. edited the manuscript and wrote additional sections pertaining to the electrophysiology experiments and their interpretation.

Editor: Manu Ben-Johny.