The Serotonin Transporter Modulates the Kinetics and Quantal Size of Vesicular Fusion Events in Sympathoadrenal Chromaffin Cells

Abstract

The antidepressant‐sensitive serotonin transporter (SERT) is a key regulator of serotonin (5‐HT) signaling and availability in the CNS. The robust expression of SERT in adrenal chromaffin cells (ACCs), which comprise the neuroendocrine arm of the sympathetic nervous system, is less well understood. ACCs synthesize and secrete catecholamines (epinephrine, norepinephrine) which mediate the physiological response to stress. They do not synthesize 5‐HT, but do accumulate small amounts through SERT‐mediated uptake (5‐HT content is <0.15% of the epinephrine content). We hypothesized that the chromaffin cells utilize this 5‐HT for autocrine / paracrine control of the sympathoadrenal stress response. Consistent with this hypothesis, we previously reported that 5‐HT1A receptors inhibit catecholamine secretion by reducing the number of secretory vesicles that fuse with the plasma. The objective of the current study was to investigate an additional, receptor‐independent mechanism by which SERT controls adrenal catecholamine secretion.We used carbon fiber amperometry to analyze catecholamine secretion from ACCs that were isolated from either wild type mice, global SERT‐knockout mice (SERT‐/‐), or sympathoadrenal system‐specific SERT knockout mice (SERTΔTH). Transmitter release from individual vesicle fusion events can be resolved as amperometric spikes. There was no difference in the number or time‐course of amperometric spikes evoked by 30mM KCl. The charge of the spikes is directly proportional to the amount of transmitter released by a single vesicle (i.e. quantal size). Spike charge was significantly smaller (~35%) and spike duration (half‐width) was significantly shorter in SERT‐knockout cells compared to matched controls. This was surprising given that HPLC analysis revealed no change in the catecholamine content of adrenal glands isolated from knockout mice compared to wild‐type controls; the 5‐HT content was significantly reduced but this accounted for <0.15% of the total monoamine content. Changes in calcium entry can modulate the quantal size of catecholamine release in ACCs, but patch‐clamp recording revealed there was no significant difference in voltage‐gated calcium channel currents in SERT knockout cells and ratiometric calcium imaging found no difference in KCl‐evoked calcium entry. The decrease in quantal size was mimicked in wild‐type cells by treating for >24 hours with escitalopram (an SSRI antidepressant which blocks SERT), or by depleting extracellular 5‐HT from the culture medium for >24 hours (use of dialyzed serum in the culture media). In contrast, acute (minutes) or short‐term (<6‐8 hrs) treatment with escitalopram or 5‐HT depletion had no effect. Adrenal chromaffin cells lack the rate limiting enzyme for 5‐HT synthesis (tryptophan hydroxylase), but if the product of this enzyme (5‐hydroxytryptophan; 5‐HTP) is provided it can be converted into 5‐HT. Treating ACCs with 5‐HTP for 1‐3 hrs had no effect but 24 hr treatment rescued the reduced spike charge seen in SERT knockout cells. Together, our data suggest that, following SERT‐mediated uptake, intracellular 5‐HT modulates the kinetics and thus fraction of secretory vesicle content that is released during a fusion event.