Secretion of catecholamines from single bovine chromaffin cells in culture was elicited by brief pressure ejections from a micropipette containing nicotine, carbamoylcholine, or potassium ions or by mechanical stimulation. Release was monitored electrochemically with a carbon-fiber microelectrode placed adjacent to the cell. Cyclic voltammetry was used to identify secreted species, whereas constant potential amperometry was used for improved temporal resolution (millisecond range) of catecholamine detection. During secretion, brief current spikes were observed, which were shown to be due to detection of catecholamines by electrooxidation. The spikes have the physical characteristics of multimolecular packets of catecholamines released at random times and locations from the surface of the single cell. The half-width of the spikes was found to increase with an increase in cell-electrode spacing. The properties of the catecholamine spikes correlate well with expectations based on secretion from individual storage vesicles. Spikes do not occur in the absence of Ca2+ in the buffer, and the majority of spikes are found to be distributed between 0.2 and 2 picocoulombs, corresponding to 1-10 attomoles of catecholamine detected. The frequency of the spikes increases with the intensity of the stimulus, but the average quantity of catecholamine in each spike is independent of the stimulus. Thus, these measurements represent timeresolved observation of quantal secretion of catecholamines and provide direct evidence for the exocytotic hypothesis.

Secretion of catecholamines is observed as a series of current spikes when measured at the surface of a bovine adrenal medullary cell in culture with a carbon-fiber microelectrode operated in the amperometric mode. Prior work has shown that these spikes are due to detection of concentrated packets of catecholamines which are released from individual vesicles after their fusion with the cell membrane, a process known as exocytosis. The shape of the individual current spikes, detected with a 5-microns spacing between the hemispherical cell and the electrode, has been compared to the shape generated by a theoretical model. The model consists of an instantaneous point source of material on a surface which subsequently diffuses to a disk that consumes the emitted material. The pertinent diffusion conditions have been evaluated with finite difference and random walk digital simulations. The two methods give identical results when the point source is located on a plane. The more realistic simulation geometry, emission from a hemispherical surface, was evaluated with the random walk method. The simulations allow a set of criteria to be established to evaluate diffusion-controlled broadening of spike shape. The broad range of spike widths observed experimentally and their individual shapes measured with 5-microns cell-electrode spacing are consistent with diffusion from point sources randomly distributed across a hemispherical surface. The data can be described with the diffusion coefficient for catecholamines in free solution. The model enables evaluation of signal-to-noise losses and correction for diffusional losses which are dependent on electrode radius.(ABSTRACT TRUNCATED AT 250 WORDS)

The stages of the complex events involved in exocytotic secretion after vesicle-cell membrane fusion have been examined at the level of individual vesicles. Catecholamine flux from single bovine adrenal medullary cells was measured with carbon-fiber microelectrodes firmly touching the cell surface. The data reveal that secretion during exocytotic events has three distinct stages: a small increase in catecholamine flux, a rapid, but not instantaneous, rise to a maximum, followed by an exponential decrease in the flux. These stages are interpreted in the following ways. The initial stage corresponds to catecholamine secretion through a fusion pore. The rate of pore expansion appears to control the rise time of the flux to its maximum value. The final exponential stage is consistent with chemical dissociation of the intravesicular matrix or gel.

The time course of extrusion of the vesicular contents during exocytosis has been examined at adrenal medullary cells with carbon-fiber microelectrodes. Two electrochemical techniques were used: cyclic voltammetry and amperometry. Spikes obtained by amperometry had a faster time course than those measured by cyclic voltammetry, consistent with the different concentration profiles established by each technique. However, the experimental data obtained with both techniques were temporally broadened with respect to dispersion of an instantaneous point source by diffusion. Measurements with the electrode firmly pressed against the cell surface established that the temporal broadening is a result of a rate-limiting kinetic step associated with extrusion of the vesicular contents at the cell surface. The data do not support a rate-limiting process due to restricted efflux from a small pore. When combined with previous results, the data suggest that the rate-limiting step for chemical secretion from adrenal medullary cells during exocytosis is the dissociation of catecholamines from the vesicular matrix at the surface of the cell.

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