Real-time Monitoring of Chromaffin Cell Secretory Granules by Total Internal Reflection Fluorescence Microscopy Reveals a Novel Stimulatory Effect of Nanosecond Electric Pulses on Exocytosis

Abstract

Adrenal chromaffin cells are excitable neuroendocrine cells that have been widely used as a simple model of neurosecretion. In vivo, acetylcholine released from preganglionic neurons binds to nicotinic receptors, which are Na+ ionophores, causing Na+ influx that depolarizes the plasma membrane. Depolarization in turn causes voltage-gated calcium channels (VGCCs) to open, leading to an influx of Ca²⁺ that activates the fusion of secretory granules with the plasma membrane, resulting in catecholamine release that occurs within milliseconds. This Ca²⁺-dependent secretory process is referred to as exocytosis. Previous investigations exploring the potential for nanosecond electric pulses (NEPs) to serve as a novel bioelectric stimulus of neurosecretion in chromaffin cells have shown that in chromaffin cells exposed to 5 ns, 5 MV/m electric pulses, catecholamine release is stimulated in a manner that relies on Ca²⁺ influx via VGCCs. The goal of the present study was to further understand this novel neurosecretory stimulus by monitoring in real-time exocytosis evoked by NEPs and determining the extent to which exocytotic responses differed from those evoked by physiological stimulation, nicotinic receptor activation.

Chromaffin cells were exposed to single and multiple 5 ns and 150 ns pulses applied at different electric electric(E)-field amplitudes, and to single or multiple 5 ms applications of a nicotinic receptor agonist. The effects of each type of stimulus on intracellular Ca²⁺ levels were monitored by fluorescence imaging of cells loaded with the fluorescent Ca²⁺ indicator dye Calcium Green-1. Exocytosis was monitored by total internal reflection fluorescence microscopy (TIRFM) in cells in which secretory granules were labeled with the fluorescent dye acridine orange. TIRFM was also used to determine stimulus-evoked changes in the mobility of secretory granules that did not undergo exocytosis.

Both 5 ns and 150 ns pulses evoked longer-lived increases in intracellular Ca²⁺ compared with nicotinic receptor stimulation of the cells. As a result, NEP-stimulated exocytosis lasted for a longer period of time relative to that observed for the physiological stimulus. Of particular significance is that even though NEPs, like nicotinic receptor stimulation, caused an instantaneous rise in intracellular Ca²⁺, exocytosis occurred with a delay of several seconds. Measurement of granule motion revealed that the rise in intracellular Ca²⁺ that occurred in response to NEPs was responsible for increasing the mobility of granules not undergoing exocytosis, as was also found for nicotinic receptor stimulation.

These results indicate that NEPs are a novel stimulus for evoking exocytosis that can lead to new insights into basic processes that mediate neurosecretion. Additionally, the delay in exocytosis followed by an extended period during which exocytosis takes place raises the possibility that NEPs can potentially be used in therapeutic applications to produce a desired outcome not possible with existing electrostimulation approaches.