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

Abstract

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-β-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.

Keywords: Acridine orange; Catecholamine release; Dopamine-β-hydroxylase; FM 1-43; Fluorescence imaging; Total internal reflection fluorescence microscopy.

Fig. 1

Plasma membrane localization of DβH following exposure of chromaffin cells to a 5 ns pulse. Cells were sham-exposed (left), stimulated with 20 μM DMPP (middle) or exposed to a single pulse (right), then probed for DβH immunofluorescence. Scale bar, 10 μm.

Fig. 2

Catecholamine release evoked by pulse pairs and trains of five and ten pulses. In (A) and (B) values for release of NE and EPI, respectively, were normalized to those obtained for a single pulse. Data represent the combined results of two independent experiments performed for each inter-pulse interval. All experiments included triplicate samples of sham-exposed cells (basal release), and cells exposed to either a single pulse (black bars) or to two pulses (gray bars). Error bars represent the standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001 vs. 1 pulse, by Student’s t-test. In (C), values for NE (closed circles) and EPI (open circles) release evoked by five and ten pulses delivered at a pulse repetition rate of 1 Hz were normalized to those obtained for a single pulse. Data represent the combined results of two independent experiments that were based on triplicate samples. Error bars represent the standard error of the mean. P < 0.01 vs. 1 pulse, by ANOVA.

Fig. 3

TIRFM images of AO labeled secretory granules and an exocytotic event. (A) A cell was exposed to a train of ten, 5 ns pulses at 1 Hz. The left panel shows a labeled secretory granule before exocytosis (arrow), and the right panel shows the resulting empty space (arrow) 2 s later. (B) Bright field image (left) of a cell (diameter ~16 μm) and its corresponding TIRFM footprint (right). Scale bar, 5 μm.

Fig. 4

Ca²⁺ responses and exocytotic events triggered by 100 μM DMPP versus 5 ns pulses. (A) and (B) show the mean rise in [Ca²⁺]_i ± SD triggered by ten applications of DMPP (n = 10) or ten 5 ns, 5 MV/m pulses (n = 10), respectively, at 1 Hz. (C) and (D) are histograms showing the number of exocytotic events triggered in cells exposed to ten applications of DMPP (n = 10) or to ten 5 ns pulses (n = 10), respectively, at 1 Hz. Each stimulus train was initiated at 10 s and ended at 20 s, as indicated by the arrows. All results were obtained using the Nikon imaging system.

Fig. 5

Ca²⁺ responses and exocytotic events triggered by a train of five 5 ns, 10 MV/m pulses at 1 Hz. (A) shows the mean rise in [Ca²⁺]_i ± SD (n = 10) and (B) is a histogram showing the number of exocytotic events (n = 10). The stimulus train was initiated at 10 s and ended at 15 s, as indicated by the arrows. All results were obtained using the Nikon imaging system.

Fig. 6

Monitoring plasma membrane labeling of chromaffin cells with FM 1-43. (A) and (B) are bright field images of a cell before and 5 min after labeling, respectively. (C) is a fluorescence image of the same cell captured at the end of the labeling protocol. Scale bar, 5 μm. (D) Cells were perfused with BSS containing 3 μM FM 1-43 as described in Methods. The trace represents the averaged whole-cell fluorescence ± SE (n = 7) during the perfusion protocol. The bars at the top of the trace indicate the time course over which each solution was perfused.

Fig. 7

Comparison of the effect of 56 mM K⁺ and a 5 ns pulse train on [Ca²⁺]_i and FM 1-43 fluorescence. (A) shows the mean rise in [Ca²⁺]_i ± SE (yellow trace; n = 17) and the averaged whole-cell fluorescence ± SE in FM 1-43-labeled cells (black trace; n = 12) triggered by perfusing high [K⁺]. (B) shows the mean rise in [Ca²⁺]_i ± SE (yellow trace; n = 5) and the averaged whole-cell fluorescence ± SE in FM 1-43-labeled cells (black trace; n = 9) triggered by ten 5 ns, 9 MV/m pulses applied at 1 Hz. In all plots, arrows indicate the time of stimulus application. All results were obtained using the Leica imaging system.

Fig. 8

Regions of interest (ROIs) used to investigate the effect of a train of 5 ns pulses on FM 1-43 labeled cells. (A) shows a fluorescence image of a cell with the ROI used for whole-cell fluorescence. ROI was drawn around the cell. Scale bar, 10 μm. (B) shows the ROI used for the line profile analysis. ROI dimensions are 5 μm width and 25 μm length. (C) shows the ROIs of the four different parts of the cell membrane based on their location relative to the electrodes. ROI dimensions are 2 μm width and 8 μm length.

Fig. 9

Effect of a train of ten 5 ns pulses applied at 1 Hz on FM 1-43 fluorescence on the anode versus cathode-facing sides of the cells. (A-C) show representative fluorescence traces in the presence of external Ca²⁺ before stimulus application (0 s; A), at the end of the pulse train (10 s; B) and 2 min post-stimulus (C). The corresponding fluorescence images are shown at the top of each trace. (D-F) show representative fluorescence traces in the absence of external Ca²⁺ before stimulus application (0 s; D), at the end of the pulse train (10 s; E) and 2 min post-stimulus (F). The corresponding fluorescence images are shown at the top of each trace.

Fig. 10

Effect of a train of ten 5 ns pulses delivered at 1 Hz on different parts of the FM 1-43-labeled cell membrane relative to the electrodes. (A) and (D) show the averaged FM 1-43 responses ± SE obtained using whole-cell fluorescence (black traces) in the presence (n = 9) and absence (n = 3) of external Ca²⁺, respectively. (B) and (E) show the averaged FM 1-43 responses ± SE at the anode-facing side (green traces) and cathode-facing (red traces) side of the cell in the presence and absence of external Ca²⁺, respectively. (C) and (F) show the averaged FM 1-43 responses ± SE at the left (blue traces) and right (orange traces) equators of the cell in the presence and absence of external Ca²⁺, respectively. The arrows indicate the start and the end of the pulse train.

Fig. 11

PS externalization assessed with lactadherin-FITC in cells exposed to a single or multiple trains of 5 ns pulses. (A) Averaged fluorescence traces ± SE (n = 4) for cells exposed to a single train of ten, 5 ns, 19 MV/m pulses delivered at 1 Hz. The arrows indicate the time the pulse train was applied. Error bars not visible are within the thickness of the lines. Green trace: anode-facing side, red trace: cathode-facing side. Bright field images show the appearance of a cell before (left) and after (right) stimulation with the pulse train. (B) shows a bright field image of a cell before stimulation with multiple trains of ten 5 ns pulses delivered at 10 Hz, followed by a series of fluorescence images taken after each of three successive pulse trains. Scale bar, 10 μm.