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

ABSTRACT: 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/.

SUBMITTER: Jiang ZJ

PROVIDER: S-EPMC6591091 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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

Jiang Zhong-Jiao ZJ Delaney Taylor L TL Zanin Mark P MP Haberberger Rainer V RV Pitson Stuart M SM Huang Jian J Alford Simon S Cologna Stephanie M SM Keating Damien J DJ Gong Liang-Wei LW

Journal of neurochemistry 20190508 6

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 (SphK1<sup>DN</sup> ) reduces the number of amperometric spikes and increases the duration o ...[more]

PMID: 30963576

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Project description:Using a nano-injection method, we introduced phospholipids having different intrinsic geometries into single secretory cells and used single cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC) with nanotip electrodes to monitor the effects of intracellular incubation on the exocytosis process and vesicular storage. Combining tools, this work provides new information to understand the impact of intracellular membrane lipid engineering on exocytotic release, vesicular content and fraction of chemical release. We also assessed the effect of membrane lipid alteration on catecholamine storage of isolated vesicles by implementing another amperometric technique, vesicle impact electrochemical cytometry (VIEC), outside the cell. Exocytosis analysis reveals that the intracellular nano-injection of phosphatidylcholine and lysophosphatidylcholine decreases the number of released catecholamines, whereas phosphatidylethanolamine shows the opposite effect. These observations support the emerging hypothesis that lipid curvature results in membrane remodeling through secretory pathways, and also provide new evidence for a critical role of the lipid localization in modulating the release process. Interestingly, the IVIEC data imply that total vesicular content is also affected by in situ supplementation of the cells with some lipids, while, the corresponding VIEC results show that the neurotransmitter content in isolated vesicles is not affected by altering the vesicle membrane lipids. This suggests that the intervention of phospholipids inside the cell has its effect on the cellular machinery for vesicle release rather than vesicle structure, and leads to the somewhat surprising conclusion that modulating release has a direct effect on vesicle structure, which is likely due to the vesicles opening and closing again during exocytosis. These findings could lead to a novel regulatory mechanism for the exocytotic or synaptic strength based on lipid heterogeneity across the cell membrane.

Project description:Sphingosine-1-phosphate (SPP) is a bioactive lipid that has recently been identified as the ligand for the EDG family of G protein-coupled cell surface receptors. However, the mitogenic and survival effects of exogenous SPP may not correlate with binding to cell-surface receptors (Van Brocklyn, J.R., M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier, D.M. Thomas, P.J.P. Coopman, S. Thangada, T. Hla, and S. Spiegel. 1998. J. Cell Biol. 142:229-240). The recent cloning of sphingosine kinase, a unique lipid kinase responsible for the formation of SPP, has provided a new tool to investigate the role of intracellular SPP. Expression of sphingosine kinase markedly increased SPP levels in NIH 3T3 fibroblasts and HEK293 cells, but no detectable secretion of SPP into the medium was observed. The increased sphingosine kinase activity in NIH 3T3 fibroblasts was sufficient to promote growth in low- serum media, expedite the G(1)/S transition, and increase DNA synthesis and the proportion of cells in the S phase of the cell cycle with a concomitant increase in cell numbers. Transient or stable overexpression of sphingosine kinase in NIH 3T3 fibroblasts or HEK293 cells protected against apoptosis induced by serum deprivation or ceramide elevation. N,N-Dimethylsphingosine, a competitive inhibitor of sphingosine kinase, blocked the effects of sphingosine kinase overexpression on cell proliferation and suppression of apoptosis. In contrast, pertussis toxin did not abrogate these biological responses. In Jurkat T cells, overexpression of sphingosine kinase also suppressed serum deprivation- and ceramide-induced apoptosis and, to a lesser extent, Fas-induced apoptosis, which correlated with inhibition of DEVDase activity, as well as inhibition of the executionary caspase-3. Taken together with ample evidence showing that growth and survival factors activate sphingosine kinase, our results indicate that SPP functions as a second messenger important for growth and survival of cells. Hence, SPP belongs to a novel class of lipid mediators that can function inside and outside cells.

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

Publications

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

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