Catecholamine exocytosis during low frequency stimulation in mouse adrenal chromaffin cells is primarily asynchronous and controlled by the novel mechanism of Ca²⁺ syntilla suppression

Abstract

Key pointsr Although the importance of asynchronous exocytosis is becoming clearer, not enough is known about its roles and mechanisms.r Here we describe the nature of exocytosis in mouse adrenal chromaffin cells during low frequency physiological stimulation, i.e. 0.5 Hz, providing new views.r We report that less than 10% of all catecholaminergic exocytosis during low frequency stimulation is synchronized to a simulated action potential (sAP), i.e. the dominant phase is asynchronous.r This asynchronous phase of exocytosis does not require Ca 2+ influx, requires the ryanodine receptor, RyR2, and comprises exocytic events with characteristics similar to those of spontaneous events.r We propose a novel mechanism of disinhibition wherein APs inhibit Ca 2+ syntillas, relieving their inhibition of spontaneous exocytosis, which leads to an increase in the asynchronous phase of elicited exocytosis.r The work has the specific physiological implication that basal sympathetic tone associated with the 'rest and digest' state is set in part by Ca 2+ syntillas. Furthermore, there is evidence that this regulation of exocytosis by Ca 2+ syntillas may be a general mechanism that extends to neurons.Abstract Adrenal chromaffin cells (ACCs), stimulated by the splanchnic nerve, generate action potentials (APs) at a frequency near 0.5 Hz in the resting physiological state, at times described as 'rest and digest' . How such low frequency stimulation in turn elicits sufficient catecholamine exocytosis to set basal sympathetic tone is not readily explained by the classical mechanism of stimulus-secretion coupling, where exocytosis is synchronized to AP-induced Ca 2+ influx. By using simulated action potentials (sAPs) at 0.5 Hz in isolated patch-clamped mouse ACCs, we show here that less than 10% of all catecholaminergic exocytosis, measured by carbon fibre amperometry, is synchronized to an AP. The asynchronous phase, the dominant phase, of exocytosis does not require Ca 2+ influx. Furthermore, increased asynchronous exocytosis is accompanied by an AP-dependent decrease in frequency of Ca 2+ syntillas (i.e. transient, focal Ca 2+ release from internal stores) and is ryanodine sensitive. We propose a mechanism of disinhibition, wherein APs suppress Ca 2+ syntillas, which themselves inhibit exocytosis as they do in the case of spontaneous catecholaminergic exocytosis.

Sustained Exocytosis after Action Potential-Like Stimulation at Low Frequencies in Mouse Chromaffin Cells Depends on a Dynamin-Dependent Fast Endocytotic Process

“…We did not observe evident signs of asynchronous exocytosis during several seconds after the application of AP ls (see Figure 8 ). The noise in our measurements would not allow the detection of single vesicle fusion, but important asynchronous exocytosis, as the one detected by Lefkowitz et al (2014) , should interfere in the monotonic decrease in capacitance, which is characteristic of our measurements. However, we cannot discard that asynchronous exocytosis may have a more significant contribution during prolonged trains of action potentials at low frequencies.…”

“…These results indicate that ETAP by itself can maintain exocytosis during low stimulation frequencies. In a recent paper, using amperometry, Lefkowitz et al (2014) proposed a mechanism by which spontaneous asynchronous exocytosis may contribute importantly to secretion during action potential waveforms applied at 0.5 Hz. We did not observe evident signs of asynchronous exocytosis during several seconds after the application of AP ls (see Figure 8 ).…”

Nanoelectrochemistry reveals how presynaptic neurons regulate vesicle release to sustain synaptic plasticity under repetitive stimuli

“…We succeeded in detecting a small number of synchronous exocytotic events (spikes occurring within 200 ms after EFS) and a mass of asynchronous exocytotic events (spikes occurring during a later period), which was in agreement with the exocytosis model of periglomerular neurons and adrenal chromaffin cells, 28,29 and all these events were considered in the following analysis. According to the shape and kinetic parameters of single exocytotic events, these spikes can be divided into two modes: simple spikes with a rising phase followed by a single falling phase (spike a, shown in Fig.…”

Cholinergic and peptidergic neurotransmission in the adrenal medulla: A dynamic control of stimulus‐secretion coupling

“…12 It is of note that a local gap junction-mediated communication between chromaffin also regulates hormone secretion. [13][14][15][16][17] Under basal ("unstressed") conditions, the sympathetic nervous system discharges at a moderate firing rate (<1 Hz 18 ), contributing to a normal blood pressure and to the homeostatic "rest and digest" status of energy storage. Accordingly, adrenal chromaffin cells release small amounts of catecholamines into the circulation.…”