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

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