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.
Adrenal chromaffin cells (ACCs) are the neuroendocrine arm of the sympathetic nervous system and key mediators of the physiological stress response. Acetylcholine (ACh) released from preganglionic splanchnic nerves activates nicotinic acetylcholine receptors (nAChRs) on chromaffin cells causing membrane depolarization, opening voltage-gated Ca2+ channels (VGCC), and exocytosis of catecholamines and neuropeptides. The serotonin transporter is expressed in ACCs and interacts with 5-HT1A receptors to control secretion. In addition to blocking the serotonin transporter, some selective serotonin reuptake inhibitors (SSRIs) are also agonists at sigma-1 receptors which function as intracellular chaperone proteins and can translocate to the plasma membrane to modulate ion channels. Therefore, we investigated whether SSRIs and other sigma-1 receptor ligands can modulate stimulus-secretion coupling in ACCs. Escitalopram and fluvoxamine (100 nM to 1 μM) reversibly inhibited nAChR currents. The sigma-1 receptor antagonists NE-100 and BD-1047 also blocked nAChR currents (≈ 50% block at 100 nM) as did PRE-084, a sigma-1 receptor agonist. Block of nAChR currents by fluvoxamine and NE-100 was not additive suggesting a common site of action. VGCC currents were unaffected by the drugs. Neither the increase in cytosolic [Ca2+ ] nor the resulting catecholamine secretion evoked by direct membrane depolarization to bypass nAChRs was altered by fluvoxamine or NE-100. However, both Ca2+ entry and catecholamine secretion evoked by the cholinergic agonist carbachol were significantly reduced by fluvoxamine or NE-100. Together, our data suggest that sigma-1 receptors do not acutely regulate catecholamine secretion. Rather, SSRIs and other sigma-1 receptor ligands inhibit secretion evoked by cholinergic stimulation because of direct block of Ca2+ entry via nAChRs.
Gi/o-coupled G protein-coupled receptors can inhibit neurotransmitter release at synapses via multiple mechanisms. In addition to Gβγ-mediated modulation of voltage-gated calcium channels (VGCC), inhibition can also be mediated through the direct interaction of Gβγ subunits with the soluble N-ethylmaleimide attachment protein receptor (SNARE) complex of the vesicle fusion apparatus. Binding studies with soluble SNARE complexes have shown that Gβγ binds to both ternary SNARE complexes, t-SNARE heterodimers, and monomeric SNAREs, competing with synaptotagmin 1(syt1) for binding sites on t-SNARE. However, in secretory cells, Gβγ, SNAREs, and synaptotagmin interact in the lipid environment of a vesicle at the plasma membrane. To approximate this environment, we show that fluorescently labeled Gβγ interacts specifically with lipid-embedded t-SNAREs consisting of full-length syntaxin 1 and SNAP-25B at the membrane, as measured by fluorescence polarization. Fluorescently labeled syt1 undergoes competition with Gβγ for SNARE-binding sites in lipid environments. Mutant Gβγ subunits that were previously shown to be more efficacious at inhibiting Ca2+-triggered exocytotic release than wild-type Gβγ were also shown to bind SNAREs at a higher affinity than wild type in a lipid environment. These mutant Gβγ subunits were unable to inhibit VGCC currents. Specific peptides corresponding to regions on Gβ and Gγ shown to be important for the interaction disrupt the interaction in a concentration-dependent manner. In in vitro fusion assays using full-length t- and v-SNAREs embedded in liposomes, Gβγ inhibited Ca2+/synaptotagmin-dependent fusion. Together, these studies demonstrate the importance of these regions for the Gβγ-SNARE interaction and show that the target of Gβγ, downstream of VGCC, is the membrane-embedded SNARE complex.
Release of neurotransmitters and hormones by calcium-regulated exocytosis is a fundamental cellular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. As such, there is significant interest in targeting neurosecretion for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistic insight coupled with increased experimental throughput. Here, we report a simple, inexpensive, reusable, microfluidic device designed to analyze catecholamine secretion from small populations of adrenal chromaffin cells in real time, an important neuroendocrine component of the sympathetic nervous system and versatile neurosecretory model. The device is fabricated by replica molding of polydimethylsiloxane (PDMS) using patterned photoresist on silicon wafer as the master. Microfluidic inlet channels lead to an array of U-shaped "cell traps", each capable of immobilizing single or small groups of chromaffin cells. The bottom of the device is a glass slide with patterned thin film platinum electrodes used for electrochemical detection of catecholamines in real time. We demonstrate reliable loading of the device with small populations of chromaffin cells, and perfusion/repetitive stimulation with physiologically relevant secretagogues (carbachol, PACAP, KCl) using the microfluidic network. Evoked catecholamine secretion was reproducible over multiple rounds of stimulation, and graded as expected to different concentrations of secretagogue or removal of extracellular calcium. Overall, we show this microfluidic device can be used to implement complex stimulation paradigms and analyze the amount and kinetics of catecholamine secretion from small populations of neuroendocrine cells in real time.
Abstract The dorsal root ganglia (DRG) house the primary afferent neurons responsible for somatosensation, including pain. We previously identified Jedi-1 (PEAR1/MEGF12) as a phagocytic receptor expressed by satellite glia in the DRG involved in clearing apoptotic neurons during development. Here, we further investigated the function of this receptor in vivo using Jedi-1 null mice. In addition to satellite glia, we found Jedi-1 expression in perineurial glia and endothelial cells, but not in sensory neurons. We did not detect any morphological or functional changes in the glial cells or vasculature of Jedi-1 knockout mice. Surprisingly, we did observe changes in DRG neuron activity. In neurons from Jedi-1 knockout (KO) mice, there was an increase in the fraction of capsaicin-sensitive cells relative to wild type (WT) controls. Patch-clamp electrophysiology revealed an increase in excitability, with a shift from phasic to tonic action potential firing patterns in KO neurons. We also found alterations in the properties of voltage-gated sodium channel currents in Jedi-1 null neurons. These results provide new insight into the expression pattern of Jedi-1 in the peripheral nervous system and indicate that loss of Jedi-1 alters DRG neuron activity indirectly through an intercellular interaction between non-neuronal cells and sensory neurons.
