Modulating Ca 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 Ca mediated by Ca entry via voltage-gated Ca 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 Ca responses due to Ca 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 Ca 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 Ca 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 Ca responses in the majority of cells, a trend that continued as pulse duration increased to 50 ns. The amplification of Ca responses was not the result of Ca 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 Ca responses, ruling out these Ca-permeable channels as secondary Ca 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.