Temporal characteristics of quantal secretion of catecholamines from adrenal medullary cells

Abstract

Exocytotic release of vesicular catecholamine from individual bovine adrenal medullary cells was detected with carbon fiber microelectrodes. Release was elicited from cells permeabilized with 20 microM digitonin in extracellular solutions of pH 5.5, 7.4, or 8.2, and with 100 microM nicotine at pH 7.4. Release detected amperometrically with a 6-microns radius electrode and 1-micron cell-electrode spacing was qualitatively similar for each pH and stimulus. However, amperometric detection with smaller electrodes (radius = 1 micron), cyclic voltammetry, or increased cell-electrode spacing with the larger electrode all resulted in a severe reduction in size and frequency of spikes detected at pH 5.5. Thus, the existence of a steep catecholamine concentration gradient at the cell surface is necessary to cause dissociation of the vesicular matrix at low extracellular pH. At an extracellular pH of 7.4, the distribution of amperometric spike widths measured with a 1-micron cell-electrode spacing was found to be inconsistent with that predicted for diffusional dispersion during transport from the cell surface to the electrode. Both of these results agree with the hypotheses that the chromaffin vesicle matrix normally exists in an aggregated state that can be dissociated by a chemical driving force. Some of the spikes exhibit a pre-spike feature. These were present more often following permeabilization in acidic pH as opposed to more alkaline solutions, and were most prevalent following exposure to nicotine at pH 7.4. The variability in the occurrence of the pre-spike feature suggests it originates from free catecholamine within the vesicle, since the molar fraction bound by the vesicular matrix is regulated by the pH-dependent conformation and Ca(2+)-dependent binding affinity of chromogranin A, a major protein in the vesicle.