Exocytosis In Chromaffin Cells Research Articles

Autocrine activation of P2X7 receptors mediates catecholamine secretion in chromaffin cells.

ATP is highly accumulated in secretory vesicles and secreted upon exocytosis from neurons and endocrine cells. In adrenal chromaffin granules, intraluminal ATP reaches concentrations over 100 mM. However, how these large amounts of ATP contribute to exocytosis has not been investigated. Exocytotic events in bovine and mouse adrenal chromaffin cells were measured with single cell amperometry. Cytosolic Ca2+ measurements were carried out in Fluo-4 loaded cells. Submembrane Ca2+ was examined in PC12 cells transfected with a membrane-tethered Ca2+ indicator Lck-GCaMP3. ATP release was measured using the luciferin/luciferase assay. Knockdown of P2X7 receptors was induced with short interfering RNA (siRNA). Direct Ca2+ influx through this receptor was measured using a P2X7 receptor-GCamp6 construct. ATP induced exocytosis in chromaffin cells, whereas the ectonucleotidase apyrase reduced the release events induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP), high KCl, or ionomycin. The purinergic agonist BzATP also promoted a secretory response that was dependent on extracellular Ca2+. A740003, a P2X7 receptor antagonist, abolished secretory responses of these secretagogues. Exocytosis was also diminished in chromaffin cells when P2X7 receptors were silenced using siRNAs and in cells of P2X7 receptor knockout mice. In PC12 cells, DMPP induced ATP release, triggering Ca2+ influx through P2X7 receptors. Furthermore, BzATP, DMPP, and KCl allowed the formation of submembrane Ca2+ microdomains inhibited by A740003. Autocrine activation of P2X7 receptors constitutes a crucial feedback system that amplifies the secretion of catecholamines in chromaffin cells by favouring submembrane Ca2+ microdomains.

Effects of reversible SERCA inhibition on catecholamine exocytosis and intracellular [Ca2+] signaling in chromaffin cells from normotensive Wistar Kyoto rats and spontaneously hypertensive rats

Intracellular Ca2+ ([Ca2+]i) signaling and catecholamine (CA) exocytosis from adrenal chromaffin cells (CCs) differ between mammalian species. These differences partly result from the different contributions of Ca2+-induced Ca2+-release (CICR) from internal stores, which boosts intracellular Ca2+ signals. Transient inhibition of the sarcoendoplasmic reticulum (SERCA) Ca2+ pump with cyclopiazonic acid (CPA) reduces CICR. Recently, Martínez-Ramírez et al. found that CPA had contrasting effects on catecholamine secretion and intracellular Ca2+ signals in mouse and bovine CCs, where it enhanced and inhibited exocytosis, respectively. After CPA withdrawal, exocytosis diminished in mouse CCs and increased in bovine CCs. These differences can be explained if mouse CCs have weak CICR and strong Ca2+ uptake, and the reverse is true for bovine CCs. Surprisingly, CPA slightly reduced the amplitude of Ca2+ signals in both mouse and bovine CCs. Here we examined the effects of CPA on stimulated CA exocytosis and Ca2+ signaling in rat CCs and investigated if it alters differently the responses of CCs from normotensive (WKY) or hypertensive (SHR) rats, which differ in the gain of CICR. Our results demonstrate that CPA application strongly inhibits voltage-gated exocytosis and Ca2+ transients in rat CCs, regardless of strain (SHR or WKY). Thus, despite the greater phylogenetic distance from the most recent common ancestors, suppression of endoplasmic reticulum (ER) Ca2+ uptake through CPA inhibits the CA secretion in rat CCs more similarly to bovine than mouse CCs, unveiling divergent evolutionary relationships in the mechanism of CA exocytosis of CCs between rodents. Agents that inhibit the SERCA pump, such as CPA, suppress catecholamine secretion equally well in WKY and SHR CCs and are not potential therapeutic agents for hypertension. Rat CCs display Ca2+ signals of varying widths. Some even show early and late Ca2+ components. Narrowing the Ca2+ transients by CPA and ryanodine suggests that the late component is mainly due to CICR. Simultaneous recordings of Ca2+ signaling and amperometry in CCs revealed the existence of a robust and predictable correlation between the kinetics of the whole-cell intracellular Ca2+ signal and the rate of exocytosis at the single-cell level.

Requirement of Cholesterol for Calcium-Dependent Vesicle Fusion by Strengthening Synaptotagmin-1-Induced Membrane Bending.

Cholesterol is essential for neuronal activity and function. Cholesterol depletion in the plasma membrane impairs synaptic transmission. However, the molecular mechanisms by which cholesterol deficiency leads to defects in vesicle fusion remain poorly understood. Here, it is shown that cholesterol is required for Ca2+ -dependent native vesicle fusion using the in vitro reconstitution of fusion and amperometry to monitor exocytosis in chromaffin cells. Purified native vesicles are crucial for the reconstitution of physiological Ca2+ -dependent fusion, because vesicle-mimicking liposomes fail to reproduce the cholesterol effect. Intriguingly, cholesterol has no effect on the membrane binding of synaptotagmin-1, a Ca2+ sensor for ultrafast fusion. Cholesterol strengthens local membrane deformation and bending induced by synaptotagmin-1, thereby lowering the energy barrier for Ca2+ -dependent fusion to occur. The data provide evidence that cholesterol depletion abolishes Ca2+ -dependent vesicle fusion by disrupting synaptotagmin-1-induced membrane bending, and suggests that cholesterol is an essential lipid regulator for Ca2+ -dependent fusion.

Two distinct pathways regulate chromaffin cell exocytosis.

JGP study reveals how the neurotransmitter PACAP induces a secretory response in chromaffin cells that differs from the one induced by acetylcholine.

Measurements of Calcium in Chromaffin Cell Organelles Using Targeted Aequorins.

The molecular mechanisms that mediate and regulate calcium (Ca2+) fluxes through the membranes of intracellular organelles play a key role in the generation and shaping of the local and global cytosolic Ca2+ signals triggering the process of regulated exocytosis in chromaffin cells. Beyond that role, intraorganellar Ca2+ homeostasis also regulates organelle-specific processes such as oxidative phosphorylation in mitochondria, maturation of secretory granules, or stress in the endoplasmic reticulum. In this chapter, we describe current methods to study mitochondrial, endoplasmic reticulum, and secretory vesicle calcium homeostasis in living chromaffin cells using engineered targeted aequorins.

CLC Anion/Proton Exchangers Regulate Secretory Vesicle Filling and Granule Exocytosis in Chromaffin Cells.

ClC-3, ClC-4, and ClC-5 are electrogenic chloride/proton exchangers that can be found in endosomal compartments of mammalian cells. Although the association with genetic diseases and the severe phenotype of knock-out animals illustrate their physiological importance, the cellular functions of these proteins have remained insufficiently understood. We here study the role of two Clcn3 splice variants, ClC-3b and ClC-3c, in granular exocytosis and catecholamine accumulation of adrenal chromaffin cells using a combination of high-resolution capacitance measurements, amperometry, protein expression/gene knock out/down, rescue experiments, and confocal microscopy. We demonstrate that ClC-3c resides in immature as well as in mature secretory granules, where it regulates catecholamine accumulation and contributes to the establishment of the readily releasable pool of secretory vesicles. The lysosomal splice variant ClC-3b contributes to vesicle priming only with low efficiency and leaves the vesicular catecholamine content unaltered. The related Cl-/H+ antiporter ClC-5 undergoes age-dependent downregulation in wild-type conditions. Its upregulation in Clcn3-/- cells partially rescues the exocytotic mutant defect. Our study demonstrates how different CLC transporters with similar transport functions, but distinct localizations can contribute to vesicle functions in the regulated secretory pathway of granule secretion in chromaffin cells.SIGNIFICANCE STATEMENT Cl-/H+ exchangers are expressed along the endosomal/lysosomal system of mammalian cells; however, their exact subcellular functions have remained insufficiently understood. We used chromaffin cells, a system extensively used to understand presynaptic mechanisms of synaptic transmission, to define the role of CLC exchangers in neurosecretion. Disruption of ClC-3 impairs catecholamine accumulation and secretory vesicle priming. There are multiple ClC-3 splice variants, and only expression of one, ClC-3c, in double Cl-/H+ exchanger-deficient cells fully rescues the WT phenotype. Another splice variant, ClC-3b, is present in lysosomes and is not necessary for catecholamine secretion. The distinct functions of ClC-3c and ClC-3b illustrate the impact of expressing multiple CLC transporters with similar transport functions and separate localizations in different endosomal compartments.

Glucagon-like peptide-1 receptor controls exocytosis in chromaffin cells by increasing full-fusion events.

Agonists for glucagon-like-peptide-1 receptor (GLP-1R) are currently used for the treatment of type 2 diabetes and obesity. Their benefits have been centered on pancreas and hypothalamus, but their roles in other organ systems are not well understood. We studied the action of GLP-1R on secretions of adrenal medulla. Exendin-4, a synthetic analog of GLP-1, increases the synthesis and the release of catecholamines (CAs) by increasing cyclic AMP (cAMP) production, without apparent participation of cAMP-regulated guanine nucleotide exchange factor (Epac). Exendin-4, when incubated for 24 h, increases CA synthesis by promoting the activation of tyrosine hydroxylase. Short incubation (20min) increases the quantum size of exocytotic events by switching exocytosis from partial to full fusion. Our results give a strong support to the role of GLP-1 in the fine control of exocytosis.

The interplay between α7 nicotinic acetylcholine receptors, pannexin‐1 channels and P2X7 receptors elicit exocytosis in chromaffin cells

Pannexin-1 (Panx1) forms plasma membrane channels that allow the exchange of small molecules between the intracellular and extracellular compartments, and are involved in diverse physiological and pathological responses in the nervous system. However, the signaling mechanisms that induce their opening still remain elusive. Here, we propose a new mechanism for Panx1 channel activation through a functional crosstalk with the highly Ca2+ permeable α7 nicotinic acetylcholine receptor (nAChR). Consistent with this hypothesis, we found that activation of α7 nAChRs induces Panx1-mediated dye uptake and ATP release in the neuroblastoma cell line SH-SY5Y-α7. Using membrane permeant Ca2+ chelators, total internal reflection fluorescence microscopy in SH-SY5Y-α7 cells expressing a membrane-tethered GCAMP3, and Src kinase inhibitors, we further demonstrated that Panx1 channel opening depends on Ca2+ signals localized in submembrane areas, as well as on Src kinases. In turn, Panx1 channels amplify cytosolic Ca2+ signals induced by the activation of α7 nAChRs, by a mechanism that seems to involve ATP release and P2X7 receptor activation, as hydrolysis of extracellular ATP with apyrase or blockage of P2X7 receptors with oxidized ATP significantly reduces the α7 nAChR-Ca2+ signal. The physiological relevance of this crosstalk was also demonstrated in neuroendocrine chromaffin cells, wherein Panx1 channels and P2X7 receptors contribute to the exocytotic release of catecholamines triggered by α7 nAChRs, as measured by amperometry. Together these findings point to a functional coupling between α7 nAChRs, Panx1 channels and P2X7 receptors with physiological relevance in neurosecretion.

Glucagon-Like Peptide-1 Receptor Controls Exocytosis in Chromaffin Cells by Increasing Full Fusion Events

Glucagon-like peptide-1 receptor (GLP-1R) agonists’ ability to increase insulin secretion and promote satiety have led to their development for the treatment of type 2 diabetes and obesity; while these beneficial effects have been attributed to receptors in the pancreas and hypothalamus, the function and importance of extra-pancreatic GLP-1Rs in other organ systems is not well understood despite the possibilities for targeting these receptors for other indications. We have analysed the contribution of GLP-1R to the secretory responses of adrenal medulla, a key player in stress responses. Exendin-4 (Ex-4), a synthetic analogue of GLP-1, increased the synthesis and the release of catecholamines (CA) by increasing cAMP production, without the participation of EPAC (Exchange Proteins Activated by cAMP). Ex-4 increased CA synthesis by promoting the activation of tyrosine hydroxylase (TH), and moreover increased the quantum size of exocytotic events by switching exocytosis from partial to full fusion. Our results give a strong support to the role of GLP-1 in the fine control of glycaemia and blood pressure by sympathetic tissues.

Extracellular and intracellular sphingosine‐1‐phosphate distinctly regulates exocytosis in chromaffin cells

Sphingosine-1-phosphate (S1P) is an essential bioactive sphingosine lipid involved in many neurological disorders. Sphingosine kinase 1 (SphK1), a key enzyme for S1P production, is concentrated in presynaptic terminals. However, the role of S1P/SphK1 signaling in exocytosis remains elusive. By detecting catecholamine release from single vesicles in chromaffin cells, we show that a dominant negative SphK1 (SphK1DN ) reduces the number of amperometric spikes and increases the duration of foot, which reflects release through a fusion pore, implying critical roles for S1P in regulating the rate of exocytosis and fusion pore expansion. Similar phenotypes were observed in chromaffin cells obtained from SphK1 knockout mice compared to those from wild-type mice. In addition, extracellular S1P treatment increased the number of amperometric spikes, and this increase, in turn, was inhibited by a selective S1P3 receptor blocker, suggesting extracellular S1P may regulate the rate of exocytosis via activation of S1P3. Furthermore, intracellular S1P application induced a decrease in foot duration of amperometric spikes in control cells, indicating intracellular S1P may regulate fusion pore expansion during exocytosis. Taken together, our study represents the first demonstration that S1P regulates exocytosis through distinct mechanisms: extracellular S1P may modulate the rate of exocytosis via activation of S1P receptors while intracellular S1P may directly control fusion pore expansion during exocytosis. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Synaptotagmin-7 Endows a Subpopulation of Chromaffin Granules with Distinct Calcium Sensing and Fusion Properties

Syt-7 is one of two major Ca2+-sensors for regulated release from adrenal chromaffin cells. Its unique biochemistry provides cells with a highly Ca2+ sensitive pool of dense core granules for exocytosis. Syt-7 also participates in limiting the rate at which fusion pores expand via mechanisms that are not fully clear. All previous imaging studies of Syt-7's actions during exocytosis in chromaffin cells have relied on overexpression of WT or mutant Syt protein. Here, we imaged exocytosis in mouse chromaffin cells without Syt-7 for the first time, so that properties which depend on the protein could be directly determined. Our data show that cells lacking Syt-7 exhibit at least a five-fold difference in fusion efficacy compared to WT cells in response to elevated K+ depolarization. Lumenal granule cargos are also released at faster rates from Syt-7 knockout (KO) cells, consistent with data from overexpression studies. Next, we measured the responses of WT and KO cells to endogenous secretogoguges of the sympatho-adrenal synapse, acetylcholine (ACh) and pituitary adenylate cyclase-activating peptide (PACAP). Compared to WT cells, ACh-evoked release in KO cells is significantly impaired with respect to granule fusion kinetics and fusion efficacy. PACAP-evoked release, on the hand, is only mildly suppressed. Our data provide evidence for Syt-7's role in two critical aspects of exocytosis – fusion and fusion pore expansion. Moreover, the separable responses of WT and KO cells to endogenous secretogogues suggest that Syt proteins occupy distinct roles in the physiology of the chromaffin cell system.

Issue Cover (November 2018)

Journal of NeurochemistryVolume 147, Issue 4 Issue CoverFree Access Issue Cover (November 2018) First published: 26 November 2018 https://doi.org/10.1111/jnc.14201 Read the full article: ‘Altered excitability and exocytosis in chromaffin cells from the R6/1 mouse model of Huntington's disease is linked to over-expression of mutated huntingtin’ by C. Martínez-Ramírez, A. M. Baraibar, C. Nanclares, I. Méndez-López, A. Gómez, Ma. P. Muñoz, A. M. G. de Diego, L. Gandía, M. J. Casarejos, A. G. García (J. Neurochem. 2018, vol. 147 (4), pp. 454–476) on doi: 10.1111/jnc.14585 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Front cover: Background: Neurodegenerative diseases are primarily linked to alterations of neurotransmitter release and synaptic plasticity in the brain. However, the peripheral sympatho-adrenal axis has been found to be also altered in some of those diseases. Here we present a study on morphological and functional alterations in adrenal chromaffin cells (CCs) of the R6/1 mouse model of Huntington's disease (HD). Main finding: We have found (i) huntingtin overexpression as nuclear aggregates in CCs; (ii) smaller CC size with decreased dopamine β-hydroxylase expression, indicating lesser number of chromaffin secretory vesicles; (iii) reduced adrenal tissue catecholamine content; (iv) reduced Na+ currents with (v) membrane hyperpolarisation and reduced ACh-evoked action potentials; (v) reduced [Ca2+]c transients with faster Ca2+ clearance; (vi) diminished quantal secretion with smaller vesicle quantal size; (vii) faster kinetics of the exocytotic fusion pore, pore expansion, and closure. Implications: As the sympatho-adrenal axis plays an essential role in adaptation to stress through the fight-or-flight response, this study suggests that such response could be sharply deteriorated in HD patients. Image Content: The image shows an immunohistochemical staining for mutated huntingtin (Htt) in adrenal medulla chromaffin cells of the R6/1 mice at predisease stages (2 months age, 2m) before phenotypic motor alterations appear. Read the full article ‘Altered excitability and exocytosis in chromaffin cells from the R6/1 mouse model of Huntington's disease is linked to over-expression of mutated huntingtin’ by C. Martínez-Ramírez, A. M. Baraibar, C. Nanclares, I. Méndez-López, A. Gómez, Ma. P. Muñoz, A. M. G. de Diego, L. Gandía, M. J. Casarejos, A. G. García (J. Neurochem. 2018, vol. 147 (4), pp. 454–476) on doi: 10.1111/jnc.14585 Volume147, Issue4November 2018 RelatedInformation

Altered excitability and exocytosis in chromaffin cells from the R6/1 mouse model of Huntington's disease is linked to over-expression of mutated huntingtin.

As the peripheral sympathoadrenal axis is tightly controlled by the cortex via hypothalamus and brain stem, the central pathological features of Hunting's disease, (HD) that is, deposition of mutated huntingtin and synaptic dysfunctions, could also be expressed in adrenal chromaffin cells. To test this hypothesis we here present a thorough investigation on the pathological and functional changes undergone by chromaffin cells (CCs) from 2-month (2m) to 7-month (7m) aged wild-type (WT) and R6/1 mouse model of Huntington's disease (HD), stimulated with acetylcholine (ACh) or high [K+ ] (K+ ). In order to do this, we used different techniques such as inmunohistochemistry, patch-clamp, and amperometric recording. With respect to WT cells, some of the changes next summarized were already observed in HD mice at a pre-disease stage (2m); however, they were more pronounced at 7m when motor deficits were clearly established, as follows: (i) huntingtin over-expression as nuclear aggregates in CCs; (ii) smaller CC size with decreased dopamine β-hydroxylase expression, indicating lesser number of chromaffin secretory vesicles; (iii) reduced adrenal tissue catecholamine content; (iv) reduced Na+ currents with (v) membrane hyperpolarization and reduced ACh-evoked action potentials; (v) reduced [Ca2+ ]c transients with faster Ca2+ clearance; (vi) diminished quantal secretion with smaller vesicle quantal size; (vii) faster kinetics of the exocytotic fusion pore, pore expansion, and closure. On the basis of these data, the hypothesis is here raised in the sense that nuclear deposition of mutated huntingtin in adrenal CCs of R6/1 mice could be primarily responsible for poorer Na+ channel expression and function, giving rise to profound depression of cell excitability, altered Ca2+ handling and exocytosis. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Cover Image for this issue: doi: 10.1111/jnc.14201.

Altered exocytosis in chromaffin cells from mouse models of neurodegenerative diseases.

Chromaffin cells from the adrenal gland (CCs) have extensively been used to explore the molecular structure and function of the exocytotic machinery, neurotransmitter release and synaptic transmission. The CC is integrated in the sympathoadrenal axis that helps the body maintain homoeostasis during both routine life and in acute stress conditions. This function is exquisitely controlled by the cerebral cortex and the hypothalamus. We propose the hypothesis that damage undergone by the brain during neurodegenerative diseases is also affecting the neurosecretory function of adrenal medullary CCs. In this context, we review here the following themes: (i) How the discharge of catecholamines is centrally and peripherally regulated at the sympathoadrenal axis; (ii) which are the intricacies of the amperometric techniques used to study the quantal release of single-vesicle exocytotic events; (iii) which are the alterations of the exocytotic fusion pore so far reported, in CCs of mouse models of neurodegenerative diseases; (iv) how some proteins linked to neurodegenerative pathologies affect the kinetics of exocytotic events; (v) finally, we try to integrate available data into a hypothesis to explain how the centrally originated neurodegenerative diseases may alter the kinetics of single-vesicle exocytotic events in peripheral adrenal medullary CCs.

Huntingtin overexpression is associated to altered excitability and exocytosis in chromaffin cells from the R6/1 mouse model of Huntington’s disease.

Huntingtin overexpression is associated to altered excitability and exocytosis in chromaffin cells from the R6/1 mouse model of Huntington’s disease.

Novel sulfoglycolipid IG20 causes neuroprotection by activating the phase II antioxidant response in rat hippocampal slices

Compound IG20 is a newly synthesised sulphated glycolipid that promotes neuritic outgrowth and myelinisation, at the time it causes the inhibition of glial proliferation and facilitates exocytosis in chromaffin cells. Here we have shown that IG20 at 0.3–10 μM afforded neuroprotection in rat hippocampal slices stressed with veratridine, glutamate or with oxygen plus glucose deprivation followed by reoxygenation (OGD/reox). Excess production of reactive oxygen species (ROS) elicited by glutamate or ODG/reox was prevented by IG20 that also restored the depressed tissue levels of GSH and ATP in hippocampal slices subjected to OGD/reox. Furthermore, the augmented iNOS expression produced upon OGD/reox exposure was also counteracted by IG20. Additionally, the IG20 elicited neuroprotection was prevented by the presence of inhibitors of the signalling pathways Jak2/STAT3, MEK/ERK1/2, and PI3K/Akt, consistent with the ability of the compound to increase the phosphorylation of Jak2, ERK1/2, and Akt. Thus, the activation of phase II response and the Nrf2/ARE pathway could explain the antioxidant and anti-inflammatory effects and the ensuing neuroprotective actions of IG20.

Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements.

Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles’ arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We also show that including the fusion-time statistics in our model does not produce any significant changes on the results. These findings indicate that the motion of the whole ensemble of vesicles towards the membrane is directed and reflected in the amperometric signals. Our results confirm the conclusions of previous imaging studies performed on single vesicles that vesicles’ motion underneath plasma membranes is not purely random, but biased towards the membrane.

Novel synthetic sulfoglycolipid IG20 facilitates exocytosis in chromaffin cells through the regulation of sodium channels.

In search of druggable synthetic lipids that function as potential modulators of synaptic transmission and plasticity, we synthesized sulfoglycolipid IG20, which stimulates neuritic outgrowth. Here, we have explored its effects on ion channels and exocytosis in bovine chromaffin cells. IG20 augmented the rate of basal catecholamine release. Such effect did not depend on Ca(2+) mobilization from intracellular stores; rather, IG20-elicited secretion entirely dependent on Ca(2+) entry through L-subtype voltage-activated Ca(2+) channels. Those channels were recruited by cell depolarization mediated by IG20 likely through its ability to enhance the recruitment of Na(+) channels at more hyperpolarizing potentials. Confocal imaging with fluorescent derivative IG20-NBD revealed its rapid incorporation and confinement into the plasmalemma, supporting the idea that IG20 effects are exerted through a plasmalemmal-delimited mechanism. Thus, synthetic IG20 seems to mimic several physiological effects of endogenous lipids such as regulation of ion channels, Ca(2+) signaling, and exocytosis. Therefore, sulfoglycolipid IG20 may become a pharmacological tool for investigating the role of the lipid environment on neuronal excitability, ion channels, neurotransmitter release, synaptic efficacy, and neuronal plasticity. It may also inspire the synthesis of druggable sulfoglycolipids aimed at increasing synaptic plasticity and efficacy in neurodegenerative diseases and traumatic brain-spinal cord injury. The novel synthetic sulfoglycolipid IG20 mimics several physiological effects of endogenous lipids such as regulation of ion channels, Ca(2+) signaling, and exocytosis. This profile may eventually drive enhanced synaptic plasticity and efficacy.

Calcium Channel Subtypes and Exocytosis in Chromaffin Cells at Early Life

Here we review the contribution of the various subtypes of voltage-activated calcium channels (VACCs) to the regulation of catecholamine release from chromaffin cells (CCs) at early life. Patch-clamp recording of inward currents through VACCs has revealed the expression of high-threshold VACCs (high-VACCs) of the L, N, and PQ subtypes in rat embryo CCs and ovine embryo CCs. Low-threshold VACC (low-VACC) currents (T-type) have also been recorded in rat embryo CCs and rat neonatal slices of adrenal medullae. Near full blockade by nifedipine and nimodipine of the K(+)-elicited secretion as well as the hypoxia induced secretion (HIS) supports the dominant role of L-VACC subtypes to the regulation of exocytosis at early life. Partial blockade by ω-conotoxin GVIA and ω-agatoxin IVA suggests a transient participation of N and PQ high-VACCs to the regulation of the HIS response at early stages of CC exposure to hypoxia. T-type low-VACC current did not elicit exocytosis triggered by electrical depolarising pulses applied to rat embryo CCs in one study, but largely contributed to the HIS response in neonatal rat adrenal slices in another. In spite of scarce available data, the sequence of events driving the HIS response in CCs at early life could be established as follows: (i) hypoxia blocks one or more K(+) channels; (ii) as a consequence, mild membrane depolarisation occurs; (iii) T-type low-VACCs open at membrane potentials more hyperpolarised than those required to recruit the high-VACCs; (iv) firing of action potentials then occurs; (v) fast-inactivating N and PQ high-VACCs transiently open and low-inactivating L high-VACCs remain open along the hypoxia stimulus; (vi) increase of cytosolic Ca(2+) takes place; and (vii) the exocytotic release of catecholamine occurs in two phases, an explosive initial phase, driven by Ca(2+) entry through L, N and PQ channels, followed by a more sustained catecholamine release at a slower rate driven by L-type channels.

Synaptotagmin-1 and Synaptotagmin-7 Differ in their Stimulus and Ca2+-Dependence of Activation

The secretory response of adrenal chromaffin cells varies in accordance with the stimulus applied. Mild stimulation favors limited release of granule cargo through a narrow fusion pore while strong stimulation leads to greater release through wide pore expansion. The trigger for release in these cells is a rise in intracellular Ca2+ through voltage-gated channels. The level of intracellular Ca2+ accumulation varies directly with the strength of depolarization. Ca2+ triggered exocytosis in chromaffin cells also requires the protein synaptotagmin (Syt) as a sensor. The two isoforms expressed in these cells - synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7) - have distinct Ca2+ affinities, are usually sorted to separate granules, and favor fundamentally different modes of exocytosis. Therefore, we wanted to determine whether changes in stimulation intensity drive release by selectively, or differentially, activating granules with Syt-1 or Syt-7. To address this question, we depolarized cells with varying levels of extracellular KCl. We found that while 56 mM KCl equally drove fusion of Syt-1 and Syt-7 granules, 10 and 25 mM KCl were far more effective at driving fusion of Syt-7 granules. To directly investigate Ca2+-dependence of Syt isoform fusion, we perfused permeabilized cells with Ca2+ buffered to different concentrations with EGTA. Both Syt granule populations fused effectively at highest concentrations of Ca2+ (30 µM and 100 μM), although there were small differences in their overall rate constants for activation. A greater fraction of docked Syt-7 granules fuse with significantly faster kinetics at a lower Ca2+ concentration (10 μM). Our results demonstrate chromaffin cells exploit Syt isoform diversity to modulate the secretory response. A significant consequence of synaptotagmin isoform segregation is that individual granules are endowed with unique Ca2+-sensitivities, allowing rapid and local control of the fusion mode based on local Ca2+ levels.