Automatic analysis for amperometrical recordings of exocytosis.

Amperometry is a widely used technique for monitoring the secretion of catecholamines (CA) by exocytosis. The use of carbon fibre microelectrodes allows the on-line recording of CA released from a single secretory vesicle. Amperometric signals are generated by oxidation of the quantally released CA close to the electrode tip. Each event of exocytosis is called a secretory spike. Here we describe a program written for IGOR (Wavemetrics, Lake Oswego, OR, USA), which may be used to analyze amperometric signals off-line. The procedures allow, (i) digital filtering and analysis of the current noise, spike identification and calculation of spike kinetic parameters; (ii) spike review; (iii) pooling spikes and data to create galleries, tables and histograms of measured parameters which can be exported to a graphic format or files for further analysis.

Nitric oxide modulates a late step of exocytosis.

The effects of nitric oxide (NO) on the late phase of exocytosis have been studied, by amperometry, on Ba(2+)-stimulated chromaffin cells. Acute incubation with NO or NO donors (sodium nitroprusside, spermine-NO, S-nitrosoglutathione) produced a drastic slowdown of the granule emptying. Conversely, cell treatment with N(omega)-nitro-l-arginine methyl ester (a NO synthase inhibitor) or with NO scavengers (methylene blue, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium) accelerated the extrusion of catecholamines from chromaffin granules, suggesting the presence of a NO modulatory tone. The incubation with phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine or zaprinast) or with the cell-permeant cGMP analog 8-bromo-cGMP, mimicked the effects of NO, suggesting the involvement of the guanylate cyclase cascade. NO effects were not related to changes in intracellular Ba(2+). 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.

A new way for the analysis of the exocytosis.

Exocytosis is the most common way for cells to secrete substances. This is a wide distributed phenomenon that involves all cell types and all animal species from yeast to human. Catecholamine (CA) release from adrenal chromaffin cells occurs by the exocytosis of secretory vesicles also called chromaffin granules. Individual secretory events can be easily monitored by the use of carbon fibers encased in glass microelectrodes. Catecholamines oxidized at the electrode tip produce a transient electrical current wave called secretory spike. The typical secretory spike consists in a sharp elevation of current followed by a rapid exponential decrease to the basal level. A 35-50% of the spikes showed a pre-spike wave called "foot" which represents the CA release through the fusion pore. The time course of secretory spikes observed is altered by extracellular phenomena's like diffusion. It has been suggested that amperometric spikes can be described by the convolution of a Gaussian with a decreasing exponential. The exponential function is partially governed by the diffusion of the secreted substances towards the electrode. In this article a method to deconvolve both functions is proposed. This mathematical approach allows the observation of the original secretory profile--the Gaussian--without the distortion caused by the diffusional broadening of catecholamines.