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5 ns Electric pulses evoke longer-lived calcium responses in adrenal chromaffin cells than the physiological stimulus: an experimental and computational study
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Nanosecond duration electric pulses of high electric field magnitudes (>1 MV/m) are being explored by our group as a novel type of electric stimulus for evoking catecholamine release from neuroendocrine adrenal chromaffin cells. The overall goal of this work is to understand why 5 ns, 5 MV/m pulses evoke longer-lived calcium responses than nicotinic cholinergic receptor activation, the physiological stimulus. In vivo, stimulation of chromaffin cells by acetylcholine causes calcium influx through voltage-gated calcium channels (VGCCs) that increases the concentration of calcium at sites where exocytosis of catecholamine-storing secretory granules occurs, evoking the release of catecholamines. This action is mimicked by the application of a 5 ns, 5 MV/m electric pulse. However, fluorescence imaging of chromaffin cells loaded with the fluorescent calcium indicator dye Calcium Green-1 has shown that the calcium responses evoked by a 5 ns pulse outlast those evoked by a nicotinic receptor agonist. Given the short duration and high electric field magnitude of the 5 ns pulse, it is possible that the externally applied electric field can affect internal organelles important for calcium clearance. Since in chromaffin cells mitochondria are the main organelle involved in the rapid clearing of intracellular calcium following calcium influx through VGCCs, and a disruption of mitochondrial membrane potential has been shown to delay calcium clearance, our first aim was to explore whether 5 ns pulses affect mitochondrial membrane potential using fluorescent mitochondrial membrane potential dyes. Control experiments established that treating the cells with a mitochondrial membrane potential disrupter altered the shape of the calcium responses evoked by nicotinic receptor stimulation. Experiments next established that in cells in which mitochondria were labeled with mitochondrial membrane potential dyes, cells treated with a mitochondrial membrane potential disrupter caused a decrease in fluorescence, which is indicative of a decrease in mitochondrial membrane potential. When cells were exposed to a single 5 ns, 5 MV/m pulse, no decrease in fluorescence was observed. However, a train of five or ten pulses applied at 10 Hz caused an average decrease of less than 2% in mitochondrial membrane potential fluorescence in approximately 31% and 60% of the cells, respectively. Moreover, a single pulse applied at a higher electric field of 10 MV/m caused an average decrease of less than 2% in mitochondrial membrane potential fluorescence in 44% of the cells, and less than 1.8% in 50% of the cells exposed to a pulse at 15 MV/m. These results suggest that a single 5 ns, 5 MV/m pulse does not affect mitochondrial membrane potential, and that increasing the number of pulses or the electric field magnitude has only minimal effects. A second aim of the research was to explore if there are differences in the electric field distribution between the two exposure systems used in our laboratory that could explain why calcium responses evoked by a 5 ns pulse varied in duration with each exposure system. Experiments by our group were initially conducted with a gold strip electrode exposure system where cells in suspension were placed between the electrodes on a glass slide. Our current delivery system is comprised of tungsten rod electrodes that are immersed at 45° to the vertical plane into a glass bottom culture dish in which the cells are attached. The tips of the electrodes are placed 40 µm above the bottom of the dish, with the target cell located at the center of the gap between the electrode tips. Although previous simulations performed in our laboratory using a coarse mesh (mesh size of 16 µm) had shown that the electric field was homogenous in the region containing the cell in both exposure systems, our goal here was to perform refined simulations with higher discretization in the region containing an exposed cell using the Huygens box (mesh size of 4 µm for the tungsten rod exposure system and 1 µm for the gold strip chamber exposure system). The results showed that at 60 MHz, the frequency that encompassed 99% of the pulse’s energy, there were only minor differences in the homogeneity of the electric field in the X and Y directions between the tungsten rod and gold strip chambers exposure systems, as well as in other electric field parameters, such as the direction of the electric field. These minor differences are likely not contributing to the different durations of the calcium responses evoked by each exposure system. In conclusion, a single 5 ns, 5 MV/m pulse produced no detectable effect on the mitochondrial membrane potential in adrenal chromaffin cells. Even multiple pulses and a single pulse applied at higher electric fields caused only an average decrease of less than 2% in fluorescence in a fraction of the cells studied, indicating that 5 ns pulses caused minimal mitochondrial toxicity. This finding indicates that other cellular mechanisms may be affected that will require further investigation, such as inhibitory effects on calcium extrusion, prolonged calcium-induced calcium release (CICR) from the endoplasmic reticulum (ER), and sustained membrane depolarization that may prolong the opening of VGCCs. In addition, our refined simulations showed that despite the very different geometries of the exposure systems, the electric field was homogeneous in the region containing the cell to the same extent, suggesting that other factors may be involved, such as differences in the current density between the electrodes. This and other possibilities will require further simulations.