TEMPORAL CHARACTERISTICS OF QUANTAL SECRETION OF CATECHOLAMINES FROM ADRENAL-MEDULLARY CELLS

Exocytotic release of vesicular catecholamine from individual bovine a drenal medullary cells was detected with carbon fiber microelectrodes. Release was elicited from cells permeabilized with 20 muM digitonin i n extracellular solutions of pH 5.5, 7.4, or 8.2, and with 100 muM nic otine at pH 7.4. Release detected amperometrically with a 6-mum radius electrode and 1-mum cell-electrode spacing was qualitatively similar for each pH and stimulus. However, amperometric detection with smaller electrodes (radius = 1 mum), cyclic voltammetry, or increased cell-el ectrode spacing with the larger electrode all resulted in a severe red uction in size and frequency of spikes detected at pH 5.5. Thus, the e xistence of a steep catecholamine concentration gradient at the cell s urface is necessary to cause dissociation of the vesicular matrix at l ow extracellular pH. At an extracellular pH of 7.4, the distribution o f amperometric spike widths measured with a 1-mum cell-electrode spaci ng was found to be inconsistent with that predicted for diffusional di spersion during transport from the cell surface to the electrode. Both of these results agree with the hypotheses that the chromaffin vesicl e matrix normally exists in an aggregated state that can be dissociate d by a chemical driving force. Some of the spikes exhibit a pre-spike feature. These were present more often following permeabilization in a cidic pH as opposed to more alkaline solutions, and were most prevalen t following exposure to nicotine at pH 7.4. The variability in the occ urrence of the pre-spike feature suggests it originates from free cate cholamine within the vesicle, since the molar fraction bound by the ve sicular matrix is regulated by the pH-dependent conformation and Ca2+- dependent binding affinity of chromogranin A, a major protein in the v esicle.