Nano-analysis Reveals High Fraction of Serotonin Release during Exocytosis from a Gut Epithelium Model Cell.
TL;DR: In this article, the authors explore the exocytotic nature of serotonin (5-HT) release in human carcinoid BON cells, an in-vitro human enterochromaffin cell model, to understand the mechanisms operating the release of gut-derived 5-HT in the intestinal mucosal epithelium.
Abstract: Electrochemical methods were used to explore the exocytotic nature of serotonin (5-HT) release in human carcinoid BON cells, an in vitro human enterochromaffin cell model, to understand the mechanisms operating the release of gut-derived 5-HT in the intestinal mucosal epithelium. We show that the fractional vesicular 5-HT release in BON cells is 80 % compared to previous work in pancreatic beta cells (34 %). The fractional release increased from 80 % in control BON cells to 87 % with 5-HT preincubation and nearly 100 % with the combination of 5-HT and the 5-HT4 autoreceptor agonist, cisapride. Thus, partial release is the primary mechanism of exocytosis in BON cells, resulting in a variable amount of the vesicular content being released. Factors that control secretion of 5-HT from enterochromaffin cells or BON cells are important as partial release provides a mechanism for development of effective therapeutic strategies to treat gastrointestinal diseases.
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TL;DR: The discovery that in the presence of chaotropic anions (SCN−) the opening of nanometer biological vesicles at an electrified interface often becomes a two‐step process is speculated to be caused by an increase in the electrostatic attraction between protonated catecholamine and the negatively charged dense‐core following adsorption of SCN−.
Abstract: Abstract We report the discovery that in the presence of chaotropic anions (SCN−) the opening of nanometer biological vesicles at an electrified interface often becomes a two‐step process (around 30 % doublet peaks). We have then used this to independently count molecules in each subvesicular compartment, the halo and protein dense‐core, and the fraction of catecholamine binding to the dense‐core is 68 %. Moreover, we differentiated two distinct populations of large dense‐core vesicles (LDCVs) and quantified their content, which might correspond to immature (43 %) and mature (30 %) LDCVs, to reveal differences in their biogenesis. We speculate this is caused by an increase in the electrostatic attraction between protonated catecholamine and the negatively charged dense‐core following adsorption of SCN−.
8 citations
TL;DR: In this article , the authors show how changing the structure of carbon electrodes with methods such as chemical vapor deposition (CVD), wet-etching, direct laser writing (DLW), and 3D printing leads to different electrochemical properties.
6 citations
TL;DR: This study characterizes distinct physical and biological mechanisms by which released neurotransmitter escapes detection at the opposing microelectrode surface, while also revealing an important role for the NET in "presynaptic" modulation of neurotransmitter release.
Abstract: For decades, carbon-fiber microelectrodes have been used in amperometric measurements of neurotransmitter release at a wide variety of cell types, providing a tremendous amount of valuable information on the mechanisms involved in dense-core vesicle fusion. The electroactive molecules that are released can be detected at the opposing microelectrode surface, allowing for precise quantification as well as detailed kinetic information on the stages of neurotransmitter release. However, it remains unclear how much of the catecholamine that is released into the artificial synapse escapes detection. This work examines two separate mechanisms by which released neurotransmitter goes undetected in a typical amperometric measurement. First, diffusional loss is assessed by monitoring exocytosis at single bovine chromaffin cells using carbon-fiber microelectrodes fabricated in a recessed (cavity) geometry. This creates a microsampling vial that minimizes diffusional loss of analyte prior to detection. More molecules were detected per exocytotic release event when using a recessed cavity sensor as compared to the conventional configuration. In addition, pharmacological inhibition of the norepinephrine transporter (NET), which serves to remove catecholamine from the extracellular space, increased both the size and the time course of individual amperometric events. Overall, this study characterizes distinct physical and biological mechanisms by which released neurotransmitter escapes detection at the opposing microelectrode surface, while also revealing an important role for the NET in “presynaptic” modulation of neurotransmitter release.
6 citations
TL;DR: This work accurately describes the dynamic pore-opening process of individual vesicle which discloses the heterogeneity in electroporation of different sized vesicles which should allow us to examine the more complicated vesicular content transfer process between intravesicular compartments.
Abstract: Release from nanobiovesicles via a pore generated by membrane electroporation at an electrified interface can be monitored by vesicle impact electrochemical cytometry (VIEC) and provides rich information about the various vesicular content transfer processes, including content homeostasis, intraphase content transfer, or the transient fusion of vesicles. These processes are primarily influenced by the vesicular pore-opening dynamics at the electrified interface which has not been disclosed at the single nanobiovesicle level yet. In this work, after simultaneously measuring the size and release dynamics of individual vesicles, we employed a moving mesh-finite element simulation algorithm to reconstruct the accurate pore-opening dynamics of individual vesicles with different sizes during VIEC. We investigated the expansion times and maximal pore sizes as two characteristics of different vesicles. The pore expansion times between nanobiovesicles and pure lipid liposomes were compared, and that of the nanobiovesicles is much longer than that for the liposomes, 2.1 ms vs 0.18 ms, respectively, which reflects the membrane proteins limiting the electroporation process. For the vesicles with different sizes, a positive relationship of pore size (Rp,max) with the vesicle size (Rves) and also their ratio (Rp,max/Rves) versus the vesicle sizes is observed. The mechanism of the pore size determination is discussed and related to the membrane proteins and the vesicle size. This work accurately describes the dynamic pore-opening process of individual vesicles which discloses the heterogeneity in electroporation of different sized vesicles. This should allow us to examine the more complicated vesicular content transfer process between intravesicular compartments.
4 citations
TL;DR: In this paper , a review of single redox liposome electrochemistry is presented, based on the last published studies, and the limitations, applications, perspectives, and challenges are briefly discussed.
4 citations
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TL;DR: New work suggests that serotonin may regulate some processes, including platelet aggregation, by receptor-independent, transglutaminase-dependent covalent linkage to cellular proteins.
Abstract: Serotonin is perhaps best known as a neurotransmitter that modulates neural activity and a wide range of neuropsychological processes, and drugs that target serotonin receptors are used widely in psychiatry and neurology. However, most serotonin is found outside the central nervous system, and virtually all of the 15 serotonin receptors are expressed outside as well as within the brain. Serotonin regulates numerous biological processes including cardiovascular function, bowel motility, ejaculatory latency, and bladder control. Additionally, new work suggests that serotonin may regulate some processes, including platelet aggregation, by receptor-independent, transglutaminase-dependent covalent linkage to cellular proteins. We review this new “expanded serotonin biology” and discuss how drugs targeting specific serotonin receptors are beginning to help treat a wide range of diseases.
1,487 citations
TL;DR: Serotonin is an important gastrointestinal signaling molecule as mentioned in this paper, which is used by enterochromaffin (EC) cells to activate intrinsic and extrinsic primary afferent neurons to initiate peristaltic and secretory reflexes and transmit information to the central nervous system.
1,268 citations
TL;DR: Emerging evidence suggests that exploiting epithelial targets with nonabsorbable serotonergic agents could provide safe and effective therapies, and this work provides an overview of theseserotonergic actions and treatment strategies.
Abstract: Serotonin (5-HT) has been recognized for decades as an important signalling molecule in the gut, but it is still revealing its secrets. Novel gastrointestinal functions of 5-HT continue to be discovered, as well as distant actions of gut-derived 5-HT, and we are learning how 5-HT signalling is altered in gastrointestinal disorders. Conventional functions of 5-HT involving intrinsic reflexes include stimulation of propulsive and segmentation motility patterns, epithelial secretion and vasodilation. Activation of extrinsic vagal and spinal afferent fibres results in slowed gastric emptying, pancreatic secretion, satiation, pain and discomfort, as well as nausea and vomiting. Within the gut, 5-HT also exerts nonconventional actions such as promoting inflammation and serving as a trophic factor to promote the development and maintenance of neurons and interstitial cells of Cajal. Platelet 5-HT, originating in the gut, promotes haemostasis, influences bone development and serves many other functions. 5-HT3 receptor antagonists and 5-HT4 receptor agonists have been used to treat functional disorders with diarrhoea or constipation, respectively, and the synthetic enzyme tryptophan hydroxylase has also been targeted. Emerging evidence suggests that exploiting epithelial targets with nonabsorbable serotonergic agents could provide safe and effective therapies. We provide an overview of these serotonergic actions and treatment strategies.
728 citations
TL;DR: C cultured intestinal organoids are exploited together with single-cell measurements to elucidate intrinsic biophysical, pharmacological, and genetic properties of EC cells, showing that EC cells express specific chemosensory receptors, are electrically excitable, and modulate serotonin-sensitive primary afferent nerve fibers via synaptic connections, enabling them to detect and transduce environmental, metabolic, and homeostatic information from the gut directly to the nervous system.
509 citations
TL;DR: Nicotinic receptor-mediated secretion of catecholamines from individual cultured bovine adrenal medullary chromaffin cells was measured and characterized with a voltametric microelectrode placed adjacent to the cells, consistent with direct chemical measurement of single exocytotic events.
231 citations