Nanosecond Electric Pulses: A Novel Stimulus for Triggering Ca²⁺ Influx into Chromaffin Cells Via Voltage-Gated Ca²⁺ Channels

Abstract

Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca²⁺ entry into the cells via L-type voltage-gated Ca²⁺ channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond pulse-induced effects on intracellular Ca²⁺ level ([Ca²⁺]_i) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca²⁺ entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude of the rise in [Ca²⁺]_i elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca²⁺]_i response of the cells, suggesting Ca²⁺ influx occurs only via VGCCs. Lowering extracellular K⁺ concentration from 5 to 2 mM or pulsing cells in Na⁺-free medium suppressed the pulse-induced rise in [Ca²⁺]_i in the majority of cells. Thus, both membrane potential and Na⁺ entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca²⁺ influx. However, activation of voltage-gated Na⁺ channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca²⁺]_i. These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple types of VGCCs in chromaffin cells in a manner involving Na⁺ transport across the plasma membrane. Whether Na⁺ transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined.