Carbon Fiber Amperometry Research Articles

Insect haptoelectrical stimulation of Venus flytrap triggers exocytosis in gland cells

The Venus flytrap Dionaea muscipula captures insects and consumes their flesh. Prey contacting touch-sensitive hairs trigger traveling electrical waves. These action potentials (APs) cause rapid closure of the trap and activate secretory functions of glands, which cover its inner surface. Such prey-induced haptoelectric stimulation activates the touch hormone jasmonate (JA) signaling pathway, which initiates secretion of an acidic hydrolase mixture to decompose the victim and acquire the animal nutrients. Although postulated since Darwin's pioneering studies, these secretory events have not been recorded so far. Using advanced analytical and imaging techniques, such as vibrating ion-selective electrodes, carbon fiber amperometry, and magnetic resonance imaging, we monitored stimulus-coupled glandular secretion into the flytrap. Trigger-hair bending or direct application of JA caused a quantal release of oxidizable material from gland cells monitored as distinct amperometric spikes. Spikes reminiscent of exocytotic events in secretory animal cells progressively increased in frequency, reaching steady state 1 d after stimulation. Our data indicate that trigger-hair mechanical stimulation evokes APs. Gland cells translate APs into touch-inducible JA signaling that promotes the formation of secretory vesicles. Early vesicles loaded with H+ and Cl- fuse with the plasma membrane, hyperacidifying the "green stomach"-like digestive organ, whereas subsequent ones carry hydrolases and nutrient transporters, together with a glutathione redox moiety, which is likely to act as the major detected compound in amperometry. Hence, when glands perceive the haptoelectrical stimulation, secretory vesicles are tailored to be released in a sequence that optimizes digestion of the captured animal.

Adrenal chromaffin cell function in high‐altitude deer mice (Peromyscus maniculatus)

Hypoxia at high altitudes can activate the sympathetic nervous system, which could become maladaptive in high‐altitude natives if it is maintained over chronic periods. The deer mouse (Peromyscus maniculatus) has the largest altitudinal range of all North American mammals, with populations living above 4300m elevation. We hypothesized that high‐altitude deer mice might have altered the physiology of chromaffin cells in the adrenal gland to avoid chronic activation of the sympathetic response. We used captive breeding colonies derived from wild populations at high and low altitudes, and compared them in normoxia and after 6–8 weeks of acclimation to hypoxia (12kPa O2, simulating the PO2 at ~4300m). High‐altitude mice show lower rates of catecholamine secretion from adrenal slices (measured via carbon fibre amperometry) in response to stimulation by a nicotinic acetylcholine receptor agonist (nicotine). Here, we used ratiometric Ca2+ imaging of cultured chromaffin cells to assess whether the variation in catecholamine secretion resulted from variation in the Ca2+ signal for vesicular catecholamine release. The rise of intracellular Ca2+ in response to nicotine (1, 5, 25, 125μM) did not vary between populations and was not affected by hypoxia acclimation, suggesting that events downstream of Ca2+ influx likely contribute to population differences in catecholamine secretion. This appeared to include differences in catecholamine stores, which were lower in highlanders compared to lowlanders in cultured cells from normoxia‐acclimated mice. Our data therefore suggest that chronic sympathetic activation may be avoided in deer mice at high altitudes by a reduction in catecholamine storage and release from the adrenal gland.

An interplay between the serotonin transporter (SERT) and 5-HT receptors controls stimulus-secretion coupling in sympathoadrenal chromaffin cells

Adrenal chromaffin cells (ACCs), the neuroendocrine arm of the sympathetic nervous system, secrete catecholamines to mediate the physiological response to stress. Although ACCs do not synthesize 5-HT, they express the serotonin transporter (SERT). Genetic variations in SERT are linked to several CNS disorders but the role(s) of SERT/5-HT in ACCs has remained unclear. Adrenal glands from wild-type mice contained 5-HT at ≈ 750 fold lower abundance than adrenaline, and in SERT−/− mice this was reduced by ≈80% with no change in catecholamines. Carbon fibre amperometry showed that SERT modulated the ability of 5-HT1A receptors to inhibit exocytosis. 5-HT reduced the number of amperometric spikes (vesicular fusion events) evoked by KCl in SERT−/− cells and wild-type cells treated with escitalopram, a SERT antagonist. The 5-HT1A receptor antagonist WAY100635 blocked the inhibition by 5-HT which was mimicked by the 5-HT1A agonist 8-OH-DPAT but not the 5-HT1B agonist CP93129. There was no effect on voltage-gated Ca2+ channels, K+ channels, or intracellular [Ca2+] handling, showing the 5-HT receptors recruit an atypical inhibitory mechanism. Spike charge and kinetics were not altered by 5-HT receptors but were reduced in SERT−/− cells compared to wild-type cells. Our data reveal a novel role for SERT and suggest that adrenal chromaffin cells might be a previously unrecognized hub for serotonergic control of the sympathetic stress response.

Fusion Pore Size Limits 5-HT Release From Single Enterochromaffin Cell Vesicles.

Enterochromaffin cells are the major site of serotonin (5-HT) synthesis and secretion providing ∼95% of the body's total 5-HT. 5-HT can act as a neurotransmitter or hormone and has several important endocrine and paracrine roles. We have previously demonstrated that EC cells release small amounts of 5-HT per exocytosis event compared to other endocrine cells. We utilized a recently developed method to purify EC cells to demonstrate the mechanisms underlying 5-HT packaging and release. Using the fluorescent probe FFN511, we demonstrate that EC cells express VMAT and that VMAT plays a functional role in 5-HT loading into vesicles. Carbon fiber amperometry studies illustrate that the amount of 5-HT released per exocytosis event from EC cells is dependent on both VMAT and the H(+)-ATPase pump, as demonstrated with reserpine or bafilomycin, respectively. We also demonstrate that increasing the amount of 5-HT loaded into EC cell vesicles does not result in an increase in quantal release. As this indicates that fusion pore size may be a limiting factor involved, we compared pore diameter in EC and chromaffin cells by assessing the vesicle capture of different-sized fluorescent probes to measure the extent of fusion pore dilation. This identified that EC cells have a reduced fusion pore expansion that does not exceed 9 nm in diameter. These results demonstrate that the small amounts of 5-HT released per fusion event in EC cells can be explained by a smaller fusion pore that limits 5-HT release capacity from individual vesicles.

Oligophrenin-1 Connects Exocytotic Fusion to Compensatory Endocytosis in Neuroendocrine Cells.

Oligophrenin-1 (OPHN1) is a protein with multiple domains including a Rho family GTPase-activating (Rho-GAP) domain, and a Bin-Amphiphysin-Rvs (BAR) domain. Involved in X-linked intellectual disability, OPHN1 has been reported to control several synaptic functions, including synaptic plasticity, synaptic vesicle trafficking, and endocytosis. In neuroendocrine cells, hormones and neuropeptides stored in large dense core vesicles (secretory granules) are released through calcium-regulated exocytosis, a process that is tightly coupled to compensatory endocytosis, allowing secretory granule recycling. We show here that OPHN1 is expressed and mainly localized at the plasma membrane and in the cytosol in chromaffin cells from adrenal medulla. Using carbon fiber amperometry, we found that exocytosis is impaired at the late stage of membrane fusion in Ophn1 knock-out mice and OPHN1-silenced bovine chromaffin cells. Experiments performed with ectopically expressed OPHN1 mutants indicate that OPHN1 requires its Rho-GAP domain to control fusion pore dynamics. On the other hand, compensatory endocytosis assessed by measuring dopamine-β-hydroxylase (secretory granule membrane) internalization is severely inhibited in Ophn1 knock-out chromaffin cells. This inhibitory effect is mimicked by the expression of a truncated OPHN1 mutant lacking the BAR domain, demonstrating that the BAR domain implicates OPHN1 in granule membrane recapture after exocytosis. These findings reveal for the first time that OPHN1 is a bifunctional protein that is able, through distinct mechanisms, to regulate and most likely link exocytosis to compensatory endocytosis in chromaffin cells.

Positively charged amino acids at the SNAP-25 C terminus determine fusion rates, fusion pore properties, and energetics of tight SNARE complex zippering.

SNAP-25 is a Q-SNARE protein mediating exocytosis of neurosecretory vesicles including chromaffin granules. Previous results with a SNAP-25 construct lacking the nine C terminal residues (SNAP-25Δ9) showed changed fusion pore properties (Fang et al., 2008), suggesting a model for fusion pore mechanics that couple C terminal zipping of the SNARE complex to the opening of the fusion pore. The deleted fragment contains the positively charged residues R198 and K201, adjacent to layers 7 and 8 of the SNARE complex. To determine how fusion pore conductance and dynamics depend on these residues, single exocytotic events in bovine chromaffin cells expressing R198Q, R198E, K201Q, or K201E mutants were investigated by carbon fiber amperometry and cell-attached patch capacitance measurements. Coarse grain molecular dynamics simulations revealed spontaneous transitions between a loose and tightly zippered state at the SNARE complex C terminus. The SNAP-25 K201Q mutant showed no changes compared with SNAP-25 wild-type. However, K201E, R198Q, and R198E displayed reduced release frequencies, slower release kinetics, and prolonged fusion pore duration that were correlated with reduced probability to engage in the tightly zippered state. The results show that the positively charged amino acids at the SNAP-25 C terminus promote tight SNARE complex zippering and are required for high release frequency and rapid release in individual fusion events.

Pulmonary neuroepithelial bodies are polymodal airway sensors: evidence for CO2/H+ sensing.

Pulmonary neuroepithelial bodies (NEB) in mammalian lungs are thought to function as airway O2 sensors that release serotonin (5-HT) in response to hypoxia. Direct evidence that NEB cells also respond to airway hypercapnia/acidosis (CO2/H(+)) is presently lacking. We tested the effects of CO2/H(+) alone or in combination with hypoxia on 5-HT release from intact NEB cells in a neonatal hamster lung slice model. For the detection of 5-HT release we used carbon fiber amperometry. Fluorescence Ca(2+) imaging method was used to assess CO2/H(+)-evoked changes in intracellular Ca(2+). Exposure to 10 and 20% CO2 or pH 6.8-7.2 evoked significant release of 5-HT with a distinct rise in intracellular Ca(2+) in hamster NEBs. This secretory response was dependent on the voltage-gated entry of extracellular Ca(2+). Moreover, the combined effects of hypercapnia and hypoxia were additive. Critically, an inhibitor of carbonic anhydrase (CA), acetazolamide, suppressed CO2/H(+)-mediated 5-HT release. The expression of mRNAs for various CA isotypes, including CAII, was identified in NEB cells from human lung, and protein expression was confirmed by immunohistochemistry using a specific anti-CAII antibody on sections of human and hamster lung. Taken together our findings provide strong evidence for CO2/H(+) sensing by NEB cells and support their role as polymodal airway sensors with as yet to be defined functions under normal and disease conditions.

Controlling Stimulus-Secretion Coupling in Adrenal Chromaffin Cells: A Novel Role for the Serotonin Transporter?

Adrenal chromaffin cells, an important neuroendocrine component of the sympathetic nervous system, maintain homeostatic and acute stress responses through secretion of catecholamines, neuropeptides, and other hormones. Mounting evidence links depression and cardiovascular diseases, and some depressed patients exhibit elevated catecholamine levels, perhaps suggesting common molecular targets or mechanisms. The serotonin transporter (SERT) mediates reuptake of serotonin (5HT) and is an important target for anti-depressants. Notably, SERT is prominently expressed in adrenal chromaffin cells and 5HT co-localizes with epinephrine in chromaffin granules. However, the effects of 5HT/ SERT on chromaffin cell function remain unclear. Our data show that one role of SERT is to accumulate 5HT in chromaffin cells. The 5HT content of whole adrenal glands isolated from SERT knockout mice was reduced by ∼80% compared to wild-type littermates, but catecholamine content (∼1000 fold higher than 5HT) was unaltered. We used carbon fiber amperometry to investigate the regulation of stimulus-secretion coupling by 5HT/ SERT. Cells were isolated from wild-type or SERT knockout mice, secretion evoked by 30mM KCl, and the number of amperometric spikes (vesicular fusion events) over 60s was quantified. Under control conditions (no 5HT) there was no difference in the number of spikes in wild-type Vs SERT knockout cells. However, extracellular 5HT (25nM-1μM) significantly reduced secretion in SERT knockout compared to wild-type cells. Similarly, when SERT was acutely blocked in wild-type cells using escitalopram (1μM), 5HT inhibited secretion. Escitalopram alone (no 5HT) had no effect, while 5HT alone produced a slight increase in secretion, although this was not statistically significant. Patch clamp experiments demonstrated that neither 5HT nor escitalopram altered Ca2+ entry through voltage-gated calcium channels. Ongoing work will dissect the mechanisms underlying this complex regulation of chromaffin cells by 5HT/ SERT.

PC12 Cells that Lack Synaptotagmin I Exhibit Loss of a Subpool of Small Dense Core Vesicles

Neurons communicate by releasing neurotransmitters that are stored in intracellular vesicular compartments. PC12 cells are frequently used as a model secretory cell line that is described to have two subpools of vesicles: small clear vesicles and dense core vesicles. We measured transmitter molecules released from vesicles in NGF-differentiated PC12 cells using carbon-fiber amperometry, and relative diameters of individual vesicles using electron microscopy. Both amperometry and electron micrograph data were analyzed by statistical and machine learning methods for Gaussian mixture models. An electron microscopy size correction algorithm was used to predict and correct for observation bias of vesicle size due to tangential slices through some vesicles. Expectation maximization algorithms were used to perform maximum likelihood estimation for the Gaussian parameters of different populations of vesicles, and were shown to be better than histogram and cumulative distribution function methods for analyzing mixed populations. The Bayesian information criterion was used to determine the most likely number of vesicle subpools observed in the amperometric and electron microscopy data. From this analysis, we show that there are three major subpools, not two, of vesicles stored and released from PC12 cells. The three subpools of vesicles include small clear vesicles and two subpools of dense core vesicles, a small and a large dense core vesicle subpool. Using PC12 cells stably transfected with short-hairpin RNA targeted to synaptotagmin I, an exocytotic Ca2+ sensor, we show that the presence and release of the small dense core vesicle subpool is dependent on synaptotagmin I. Furthermore, synaptotagmin I also plays a role in the formation and/or maintenance of the small dense core vesicle subpool in PC12 cells.

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

Adrenal chromaffin cells (ACCs), stimulated by the splanchnic nerve, generate action potentials (APs) at a frequency near 0.5Hz 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 0.5Hz 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.

Pyrilamine inhibits nicotine-induced catecholamine secretion

Function of nicotine, which induces activation of all parts of the body including our brain, has been receiving much attention for a long period of time and also been actively studied by researchers for its pharmacological actions in the central nervous system. The modulation of nicotine concentration and the inhibition of nicotine binding on target receptors in the brain are the key factors for smoking addiction therapy. In previous studies showed that influx of nicotine at the blood–brain barrier was through the pyrilamine-sensitive organic cation transporters. But the direct interacting mechanism of pyrilamine on the nicotine binding target receptors has not yet been clarified. The aim of the present study is to investigate the direct binding mechanisms of a pyrilamine on the nicotinic acetylcholine receptors (nAChRs). We found that pyrilamine shares the same ligand binding pocket of nicotine (NCT) on nAChRs but interacts with more amino acid residues than NCT does. The extended part of pyrilamine interacts with additional residues in the ligand binding pocket of nAChRs which are located nearby the entrance of the binding pocket. The catecholamine (CA) secretion induced by nAChR agonist (NCT′) was significantly inhibited by the pyrilamine pretreatment. Real time carbon-fiber amperometry confirmed the inhibition of the NCT′-induced exocytosis by pyrilamine in a single cell level. We also found that pyrilamine inhibited the NCT′-induced [Ca2+]i. In contrast, pyrilamine did not affect the increase in calcium induced by high K+. Overall, these data suggest that pyrilamine directly docks into the ligand binding site of nAChRs and specifically inhibits the nAChR-mediated effects thereby causing inhibition of CA secretion. Therefore, pyrilamine may play an important role to explore new treatments to aid smoking cessation.

Huntingtin‐associated protein 1 regulates exocytosis, vesicle docking, readily releasable pool size and fusion pore stability in mouse chromaffin cells

Huntingtin-associated protein 1 (HAP1) was initially established as a neuronal binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking and cell signalling. In this study, we establish that HAP1 is important in several steps of exocytosis in adrenal chromaffin cells. Using carbon-fibre amperometry, we measured single vesicle exocytosis in chromaffin cells obtained from HAP1(-/-) and HAP1(+/+) littermate mice. Numbers of Ca(2+)-dependent and Ca(2+)-independent full fusion events in HAP1(-/-) cells are significantly decreased compared with those in HAP1(+/+) cells. We observed no change in the frequency of 'kiss-and-run' fusion events or in Ca(2+) entry. Whereas release per full fusion event is unchanged in HAP1(-/-) cells, early fusion pore duration is prolonged, as indicated by the increased duration of pre-spike foot signals. Kiss-and-run events have a shorter duration, indicating opposing roles for HAP1 in the stabilization of the fusion pore during full fusion and transient fusion, respectively. We use electron microscopy to demonstrate a reduction in the number of vesicles docked at the plasma membrane of HAP1(-/-) cells, where membrane capacitance measurements reveal the readily releasable pool of vesicles to be reduced in size. Our study therefore illustrates that HAP1 regulates exocytosis by influencing the morphological docking of vesicles at the plasma membrane, the ability of vesicles to be released rapidly upon stimulation, and the early stages of fusion pore formation.

Using a Cell Model to Study the Effect of Cholesterol on Exocytosis

Exocytosis is an essential cellular process in neuronal communication. This cellular function involves vesicle fusion and vesicle content release of neurotransmitter molecules. In the quest to determine the molecules affecting exocytosis, simplified model systems such as artificial cells are valuable tools. The models allow us to study the effect of different components such as various lipids on the process of exocytosis in a controlled manner. In this study the effect of cholesterol on membrane dynamics for exocytosis has been investigated. Membrane lipids including cholesterol provide a platform for carrying out and regulating exocytosis. Although cholesterol is a major component of membrane and certainly affects exocytosis, the overall biochemical and biophysical properties are not been fully understood.Two different artificial cell models have been applied in this work. The first cell model is based on pure lipid composition that provides total control of all the components in the system. The second cell model is constructed from cell plasma membrane from PC12 cells. This model gives the advantage of working with a system that is very much like a real cell and has almost all the components of the cell membrane including membrane proteins.Kinetic information of single vesicle release of dopamine is recorded using carbon fiber amperometry in both models. In the former model we have shown that adding cholesterol to the system slows the kinetics of the release in a concentration dependent manner. In the latter model, we show that depleting cholesterol from the membrane enhances the kinetics of the events. The result from the latter model not only validates the result from pure lipid-liposome system but also indicates the importance of lipids in the presence of all the membrane components including proteins.

Endotoxemia Enhances Catecholamine Secretion From Male Mouse Adrenal Chromaffin Cells Through an Increase In Ca2+ Release From the Endoplasmic Reticulum

Enhanced epinephrine secretion from adrenal chromaffin cells (ACCs) is an important homeostatic response to severe systemic inflammation during sepsis. Evidence suggests that increased activation of ACCs by preganglionic sympathetic neurons and direct alterations in ACC function contribute to this response. However, the direct effects of sepsis on ACC function have yet to be characterized. We hypothesized that sepsis enhances epinephrine secretion from ACCs by increasing intracellular Ca(2+) signaling. Plasma epinephrine concentration was increased 5-fold in the lipopolysaccharide-induced endotoxemia model of sepsis compared with saline-treated control mice. Endotoxemia significantly enhanced stimulus-evoked epinephrine secretion from isolated ACCs in vitro. Carbon fiber amperometry revealed an increase in the number of secretory events during endotoxemia, without significant changes in spike amplitude, half-width, or quantal content. ACCs isolated up to 12 hours after the induction of endotoxemia exhibited larger stimulus-evoked Ca(2+) transients compared with controls. Similarly, ACCs from cecal ligation and puncture mice also exhibited enhanced Ca(2+) signaling. Although sepsis did not significantly affect ACC excitability or voltage-gated Ca(2+) currents, a 2-fold increase in caffeine (10 mM)-stimulated Ca(2+) transients was observed during endotoxemia. Depletion of endoplasmic reticulum Ca(2+) stores using cyclopiazonic acid (10 μM) abolished the effects of endotoxemia on catecholamine secretion from ACCs. These findings suggest that sepsis directly enhances catecholamine secretion from ACCs through an increase in Ca(2+) release from the endoplasmic reticulum. These alterations in ACC function are likely to amplify the effects of increased preganglionic sympathetic neuron activity to further enhance epinephrine levels during sepsis.

Differences in the H2S-induced quantal release of catecholamine in adrenal chromaffin cells of neonatal and adult rats

Both catecholamine (CA) released from adrenal chromaffin cells and hydrogen sulfide (H2S) have been shown to play critical roles in the regulation of hypoxic stress response. Our previous study has demonstrated that exogenous H2S directly induced quantal CA released from adult rat adrenal chromaffin cells (ARACCs) by inhibiting Ca2+-activated K+ current [IK(Ca) current]. However, it is not clear now whether H2S can also directly induce quantal CA released from neonatal rat adrenal chromaffin cells (NRACCs). In the present study, we investigated whether exogenous H2S can stimulate quantal CA released from NRACCs, and whether there were differences in the kinetics of H2S-induced quantal CA released between ARACCs and NRACCs. Using carbon-fiber amperometry and whole-cell patch clamping techniques, our experimental results showed: (1) H2S can directly induce quantal CA released from NRACCs; (2) H2S induced the depolarization of membrane potential and inhibited IK(Ca) current; (3) compared with ARACCs, much smaller quantal size and faster quantal release were showed in NRACCs through the kinetic analysis of the single-vesicle secretion induced by H2S. Our results may not only help to further understand the H2S-induced CA released from adrenal chromaffin cells in the aspect of development, but also provide the insights for the clinical prevention and therapy for hypoxic stress-induced injury in neonates at birth.

Long‐lasting increase in basal catecholamine secretion from neonatal adrenal medullary chromaffin cells by chronic intermittent hypoxia

We previously reported that intermittent hypoxia (IH) facilitates hypoxia‐evoked catecholamine (CA) secretion from neonatal adrenal medullary chromaffin cells (AMC) in rat pups. Here, we examined the effects of repetitive hypoxia on CA secretion from AMC from IH exposed rat pups. Adrenal medullary slices were harvested from rat pups exposed to either IH or normoxia from ages P0‐P5, 8–9 hours/day. CA secretion was monitored by carbon fiber amperometry. In IH exposed pups, repetitive hypoxia (30s hypoxia followed by 90s normoxia × 5 episodes) facilitated CA secretion from AMC during each episode. Remarkably, basal CA secretion remained elevated for 30 min after terminating repetitive hypoxia. The long‐lasting increase in basal CA secretion was not seen in response to repetitive application of 40 mM K+. In striking contrast, in control pups, CA secretion during repetitive hypoxia was modest and after terminating the stimulus, promptly returned to basal levels. The long‐lasting increase in basal CA secretion in IH pups could be elicited in Ca2+ free medium and was prevented in presence of 10 μM ryanadine, which blocks ryanadine receptors (RyRs). These data suggest that neonatal IH results in long‐lasting activation of basal CA secretion by repetitive hypoxia, and this effect is mediated in part by RyRs and mobilization of intracellular Ca2+ (supported by HL‐90554, HL‐76537, HL‐86493 and HL‐089616).

Distinct Modes of Dopamine and GABA Release in a Dual Transmitter Neuron

We now know of a surprising number of cases where single neurons contain multiple neurotransmitters. Neurons that contain a fast-acting neurotransmitter, such as glutamate or GABA, and a modulatory transmitter, such as dopamine, are a particularly interesting case because they presumably serve dual signaling functions. The olfactory bulb contains a large population of GABA- and dopamine-containing neurons that have been implicated in normal olfaction as well as in Parkinson's disease. Yet, they have been classified as nonexocytotic catecholamine neurons because of the apparent lack of vesicular monoamine transporters. Thus, we examined how dopamine is stored and released from tyrosine hydroxylase-positive GFP (TH(+)-GFP) mouse periglomerular neurons in vitro. TH(+) cells expressed both VMAT2 (vesicular monoamine transporter 2) and VGAT (vesicular GABA transporter), consistent with vesicular storage of both dopamine and GABA. Carbon fiber amperometry revealed that release of dopamine was quantal and calcium-dependent, but quantal size was much less than expected for large dense core vesicles, suggesting that release originated from small clear vesicles identified by electron microscopy. A single action potential in a TH(+) neuron evoked a brief GABA-mediated synaptic current, whereas evoked dopamine release was asynchronous, lasting for tens of seconds. Our data suggest that dopamine and GABA serve temporally distinct roles in these dual transmitter neurons.

RCAN1 regulates vesicle recycling and quantal release kinetics via effects on calcineurin activity

We have previously shown that Regulator of Calcineurin 1 (RCAN1) regulates multiple stages of vesicle exocytosis. However, the mechanisms by which RCAN1 affects secretory vesicle exocytosis and quantal release kinetics remain unknown. Here, we use carbon fibre amperometry to detect exocytosis from chromaffin cells and identify these underlying mechanisms. We observe reduced exocytosis with repeated stimulations in chromaffin cells over-expressing RCAN1 (RCAN1(ox)), but not in wild-type (WT) cells, indicating a negative effect of RCAN1 on vesicle recycling and endocytosis. Acute exposure to calcineurin inhibitors, cyclosporine A and FK-506, replicates this effect in WT cells but has no additional effect in RCAN1(ox) cells. When we chronically expose WT cells to cyclosporine A and FK-506 we find that catecholamine release per vesicle and pre-spike foot (PSF) signal parameters are decreased, similar to that in RCAN1(ox) cells. Inhibiting calcineurin activity in RCAN1(ox) cells has no additional effect on the amount of catecholamine release per vesicle but further reduces PSF signal parameters. Although electron microscopy studies indicate these changes are not because of altered vesicle number or distribution in RCAN1(ox) cells, the smaller vesicle and dense core size we observe in RCAN1(ox) cells may underlie the reduced quantal release in these cells. Thus, our results indicate that RCAN1 most likely affects vesicle recycling and quantal release kinetics via the inhibition of calcineurin activity.

Exocytosis is impaired in mucopolysaccharidosis IIIA mouse chromaffin cells

Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disorder caused by a deficiency in the activity of the lysosomal hydrolase, sulphamidase, an enzyme involved in the degradation of heparan sulphate. MPS IIIA patients exhibit progressive mental retardation and behavioural disturbance. While neuropathology is the major clinical problem in MPS IIIA patients, there is little understanding of how lysosomal storage generates this phenotype. As reduced neuronal communication can underlie cognitive deficiencies, we investigated whether the secretion of neurotransmitters is altered in MPS IIIA mice; utilising adrenal chromaffin cells, a classical model for studying secretion via exocytosis. MPS IIIA chromaffin cells displayed heparan sulphate storage and electron microscopy revealed large electron-lucent storage compartments. There were also increased numbers of large/elongated chromaffin granules, with a morphology that was similar to immature secretory granules. Carbon fibre amperometry illustrated a significant decrease in the number of exocytotic events for MPS IIIA, when compared to control chromaffin cells. However, there were no changes in the kinetics of release, the amount of catecholamine released per exocytotic event, or the amount of Ca2+ entry upon stimulation. The increased number of large/elongated granules and reduced number of exocytotic events suggests that either the biogenesis and/or the cell surface docking and fusion potential of these vesicles is impaired in MPS IIIA. If this also occurs in central nervous system neurons, the reduction in neurotransmitter release could help to explain the development of neuropathology in MPS IIIA.

Endogenous H2S is required for hypoxic sensing by carotid body glomus cells

H(2)S generated by the enzyme cystathionine-γ-lyase (CSE) has been implicated in O(2) sensing by the carotid body. The objectives of the present study were to determine whether glomus cells, the primary site of hypoxic sensing in the carotid body, generate H(2)S in an O(2)-sensitive manner and whether endogenous H(2)S is required for O(2) sensing by glomus cells. Experiments were performed on glomus cells harvested from anesthetized adult rats as well as age and sex-matched CSE(+/+) and CSE(-/-) mice. Physiological levels of hypoxia (Po(2) ∼30 mmHg) increased H(2)S levels in glomus cells, and dl-propargylglycine (PAG), a CSE inhibitor, prevented this response in a dose-dependent manner. Catecholamine (CA) secretion from glomus cells was monitored by carbon-fiber amperometry. Hypoxia increased CA secretion from rat and mouse glomus cells, and this response was markedly attenuated by PAG and in cells from CSE(-/-) mice. CA secretion evoked by 40 mM KCl, however, was unaffected by PAG or CSE deletion. Exogenous application of a H(2)S donor (50 μM NaHS) increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in glomus cells, with a time course and magnitude that are similar to that produced by hypoxia. [Ca(2+)](i) responses to NaHS and hypoxia were markedly attenuated in the presence of Ca(2+)-free medium or cadmium chloride, a pan voltage-gated Ca(2+) channel blocker, or nifedipine, an L-type Ca(2+) channel inhibitor, suggesting that both hypoxia and H(2)S share common Ca(2+)-activating mechanisms. These results demonstrate that H(2)S generated by CSE is a physiologic mediator of the glomus cell's response to hypoxia.