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. NO did not modify the duration of feet. Effects were evident even on pre-fusioned granules, observed under hypertonic conditions, suggesting that the fusion pore is not the target for NO, which probably acts by modifying the affinity of catecholamines for the intragranular matrix. NO could modify the synaptic transmitter efficacy through a novel mechanism, which involves the regulation of the emptying of secretory vesicles.

Abstract-The effects of the antihypertensive agent hydralazine (1 to 100 nmol/L) on the exocytotic process of single adrenal chromaffin cells have been studied using amperometry. Hydralazine does not reduce the frequency of exocytotic spikes but rapidly slows the rate of catecholamine release from individual exocytotic events by reducing the quantal size of catecholamine exocytosis. Confocal and standard epifluorescence microscopy studies show that hydralazine rapidly accumulates within secretory vesicles. The blockade of the vesicular H ϩ pump with bafilomycin A 1 inhibits hydralazine uptake. Experiments with permeabilized cells show that hydralazine displaces catecholamines from secretory vesicles. The drug also displaces vesicular Ca 2ϩ , as shown by fura-2 microfluorimetry. These data suggest that hydralazine acts, at least partially, by interfering with the storage of catecholamines. These effects of hydralazine occurred within seconds, and at the tissue concentrations presumably reached in antihypertensive therapy; these concentrations are a thousand times lower than those described for relaxing vascular tissues in vitro. We proposed that these novel effects could explain many of the therapeutic and side effects of this drug that are likely exerted in sympathetic nerve terminals.

Amperometry is a powerful tool for studying the exocytotic process. Catecholamines released from a single secretory vesicle are oxidized by a carbon fiber microelectrode placed on a chromaffin cell membrane. This phenomenon can be recorded online as an amperometric signal. Each event of exocytosis is called a secretory spike. Several kinetic parameters can be extracted from spikes to get important information about catecholamine storage and to follow the time course of exocytosis. The large amount of data requires the use of computer programs. We describe software, written for Igor Pro (Wavemetrics, Lake Oswego, OR, USA), that allows the offline analysis of amperometric signals. It includes (1) the automatic analysis of a large collection of experiments without user intervention; (2) the visual check of located spikes; (3) data pooling from several experiments to create galleries with hundreds of thousands of spikes. In addition, we have designed a new filtering method for amperometric data. It provides an excellent tool to enhance the signal/noise ratio with minimal artifacts. This filter allows one to obtain more reliable spike parameters.

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