Electrochemical detection of modulation of exocytosis from chromaffin cells monitored with amperometry.

摘要

Biological intercellular communication necessitates rapid and efficient processes. Cells must be able to relay and receive messages rapidly and this is accomplished readily through exocytosis. Chemical messengers are packaged into membrane-bound organelles known as vesicles or granules, which fuse with the outer plasma membrane upon an increase in intracellular [Ca2+] caused by influx from the extracellular space or from intracellular stores. Ca2+ increases can be effected through membrane depolarization or through receptor activation. The chemical messengers are then extruded into the extracellular space where they can bind to target cells and initiate a response. Because of the timescale of vesicular release and Ca2+ influx, it is necessary to utilize detection techniques that have sufficient temporal resolution. Additionally, the minute amounts that are secreted from these vesicles, whose dimensions are characteristically at the nanometer level, require a technique that possesses sensitivity sufficient to detect them. The electrochemical technique, constant potential amperometry, used in conjunction with carbon-fiber microelectrodes, offers excellent temporal and spatial resolution as well as sensitivity for electroactive chemical messengers. Moreover, the use of a fluorescent dye, fura-2 allows study of the Ca 2+ dynamics occurring during exocytosis. Both amperometry and fluorescence measurements via fura-2 allow for the characterization of exocytosis in bovine and murine chromaffin cells.; Amperometry has been advantageous in studies involving chromaffin cells. Using this technique, an autoreceptor effect that causes facilitation in bovine chromaffin cells was revealed. Since this autoreceptor effect appears to occur on very rapid timescales, the exquisite temporal resolution of amperometry has permitted the observation and manipulation of this effect with minimal perturbation of the cells under study. In experiments involving genetically altered mice, amperometry revealed aspects of the exocytotic process that may be affected by specific proteins. In studies conducted on R6/2 mice that serve as models for Huntington's Disease, it was noted that vesicular quanta was significantly diminished. Also, in other experiments conducted in mice that lack the intracellular phosphoprotein, synapsin, vesicular release frequency was significantly enhanced. The studies mentioned demonstrate that amperometry with carbon-fiber microelectrodes is a powerful tool in studying exocytotic processes.