Syntillas Release Ca 2+ at a Site Different from the Microdomain Where Exocytosis Occurs in Mouse Chromaffin Cells

Spontaneous, short-lived, focal cytosolic Ca 2+ transients were found for the first time and characterized in freshly dissociated chromaffin cells from mouse. Produced by release of Ca 2+ from intracellular stores and mediated by type 2 and perhaps type 3 ryanodine receptors (RyRs), these transients are quantitatively similar in magnitude and duration to Ca 2+ syntillas in terminals of hypothalamic neurons, suggesting that Ca 2+ syntillas are found in a variety of excitable, exocytotic cells. However, unlike hypothalamic nerve terminals, chromaffin cells do not display syntilla activation by depolarization of the plasma membrane, nor do they have type 1 RyRs. It is widely thought that focal Ca 2+ transients cause “spontaneous” exocytosis, although there is no direct evidence for this view. Hence, we monitored catecholamine release amperometrically while simultaneously imaging Ca 2+ syntillas, the first such simultaneous measurements. Syntillas failed to produce exocytotic events; and, conversely, spontaneous exocytotic events were not preceded by syntillas. Therefore, we suggest that a spontaneous syntilla, at least in chromaffin cells, releases Ca 2+ into a cytosolic microdomain distinct from the microdomains containing docked, primed vesicles. Ryanodine (100 μM) reduced the frequency of Ca 2+ syntillas by an order of magnitude but did not alter the frequency of spontaneous amperometric events, suggesting that syntillas are not involved in steps preparatory to spontaneous exocytosis. Surprisingly, ryanodine also increased the total charge of individual amperometric events by 27%, indicating that intracellular Ca 2+ stores can regulate quantal size.