5 ns electric pulses induce Ca2+-dependent exocytotic release of catecholamine from adrenal chromaffin cells

Abstract

Previously we reported that adrenal chromaffin cells exposed to a 5 ns, 5 MV/m pulse release the catecholamines norepinephrine (NE) and epinephrine (EPI) in a Ca²⁺-dependent manner. Here we determined that NE and EPI release increased with pulse number (one versus five and ten pulses at 1 Hz), established that release occurs by exocytosis, and characterized the exocytotic response in real-time. Evidence of an exocytotic mechanism was the appearance of dopamine-β-hydroxylase on the plasma membrane, and the demonstration by total internal reflection fluorescence microscopy studies that a train of five or ten pulses at 1 Hz triggered the release of the fluorescent dye acridine orange from secretory granules. Release events were Ca²⁺-dependent, longer-lived relative to those evoked by nicotinic receptor stimulation, and occurred with a delay of several seconds despite an immediate rise in Ca²⁺. In complementary studies, cells labeled with the plasma membrane fluorescent dye FM 1-43 and exposed to a train of ten pulses at 1 Hz underwent Ca²⁺-dependent increases in FM 1-43 fluorescence indicative of granule fusion with the plasma membrane due to exocytosis. These results demonstrate the effectiveness of ultrashort electric pulses for stimulating catecholamine release, signifying their promise as a novel electrostimulation modality for neurosecretion.

Keywords: Acridine orange; Catecholamine release; Dopamine-β-hydroxylase; FM 1-43; Fluorescence imaging; Total internal reflection fluorescence microscopy.