scholarly journals Reversible effects of sphingomyelin degradation on cholesterol distribution and metabolism in fibroblasts and transformed neuroblastoma cells

1990 ◽  
Vol 271 (1) ◽  
pp. 121-126 ◽  
Author(s):  
M I Pörn ◽  
J P Slotte
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cell Line ◽  
Cholesterol Oxidase ◽  
Neuroblastoma Cell ◽  
Control Level ◽  
Neuroblastoma Cells ◽  
Long Term Effects ◽  
Human Lung Fibroblast ◽  
Acat Activity

Plasma-membrane sphingomyelin appears to be one of the major determinants of the preferential allocation of cell cholesterol into the plasma-membrane compartment, since removal of sphingomyelin leads to a dramatic redistribution of cholesterol within the cell [Slotte & Bierman (1988) Biochem. J. 250, 653-658]. In the present study we examined the long-term effects of sphingomyelin degradation on cholesterol redistribution in cells and determined the reversibility of the process. In a human lung fibroblast-cell line, removal of 80% of the sphingomyelin led to a rapid and transient up-regulation (3-fold) of acyl-CoA:cholesterol acyltransferase (ACAT) activity, and also, within 30 h, to the translocation of about 50% of the cell non-esterified cholesterol from a cholesterol oxidase-susceptible compartment (i.e. the cell surface) to oxidase-resistant compartments. At 49 h after the initial sphingomyelin degradation, the cell sphingomyelin level was back to 45% of the control level, and the direction of cell cholesterol flow was toward the cell surface, although the original distribution was not achieved. In a transformed neuroblastoma cell line (SH-SY5Y), the depletion of sphingomyelin led to a similarly rapid and transient up-regulation of ACAT activity, and to the translocation of about 25% of cell-surface cholesterol into internal membranes (within 3 h). The flow of cholesterol back to the cholesterol oxidase-susceptible pool was rapid, and a pretreatment cholesterol distribution was reached within 20-49 h. Also, the resynthesis of sphingomyelin was faster in SH-SY5Y neuroblastoma cells and reached control levels within 24 h. The findings of the present study show that the cellular redistribution of cholesterol, as induced by sphingomyelin degradation, is reversible and suggest that the normalization of cellular cholesterol distribution is linked to the re-synthesis of sphingomyelin.

scholarly journals Recombinant neutral endopeptidase-24.11 expressed in mouse neuroblastoma cells is associated with neurite membranes

1990 ◽  
Vol 267 (2) ◽  
pp. 447-452 ◽  
Author(s):  
G Lemay ◽  
M Zollinger ◽  
G Waksman ◽  
B P Roques ◽  
P Crine ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Neutral Endopeptidase ◽  
Rabbit Kidney ◽  
Synaptic Membranes ◽  
Membrane Domains ◽  
Mouse Neuroblastoma ◽  
Endopeptidase 24.11

Neutral endopeptidase-24.11 (EC 3.4.24.11) (NEP) is a transmembrane metallo-endopeptidase that has been shown to be involved in the degradation of several mammalian neuropeptides, including enkephalins. The enzyme has recently been found to be specifically associated with the axonal and synaptic membranes of neurons in the globus pallidus of the pig brain. This result suggests that neurons must possess mechanisms for targeting NEP to particular membrane domains. Study of these mechanisms would greatly benefit from the existence of an established neuron-like cell line capable of expressing and targeting NEP to specific membrane domains. For this reason we have used a retroviral vector containing the cDNA for rabbit kidney NEP to express this enzyme in a mouse neuroblastoma cell line (Neuro2A). Labelling of the cell surface with an antibody coupled to colloidal gold particles and examination of the cells by electron microscopy revealed a non-uniform distribution of NEP at the surface of the cells, the protein being preferentially associated with the membrane of neurites compared with the cell body. This observation suggests that Neuro2A cells possess a mechanism for targeting NEP to specific domains of the plasma membrane. This cell line could thus constitute a good model for studying the mechanisms responsible for targeting this enzyme to specialized regions of the plasma membrane.

TTX-Sensitive and -Resistant Na+ Currents, and mRNA for the TTX-Resistant rH1 Channel, Are Expressed in B104 Neuroblastoma Cells

1997 ◽  
Vol 77 (1) ◽  
pp. 236-246 ◽  
Author(s):  
Xiang Q. Gu ◽  
Sulayman Dib-Hajj ◽  
Marco A. Rizzo ◽  
Stephen G. Waxman
Keyword(s):  
Steady State ◽  
Cell Line ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Neuroblastoma Cell Line ◽  
Membrane Excitability ◽  
Rt Pcr ◽  
Α Subunit ◽  
Time To Peak ◽  
Steady State Inactivation

Gu, Xiang Q., Sulayman Dib-Hajj, Marco A. Rizzo, and Stephen G. Waxman. TTX-sensitive and -resistant Na+ currents, and mRNA for the TTX-resistant rH1 channel, are expressed in B104 neuroblastoma cells. J. Neurophysiol. 77: 236–246, 1997. To examine the molecular basis for membrane excitability in a neuroblastoma cell line, we used whole cell patch-clamp methods and reverse transcription-polymerase chain reaction (RT-PCR) to study Na+ currents and channels in B104 cells. We distinguished Tetrodotoxin (TTX)-sensitive and -resistant Na+ currents and detected the mRNA for the cardiac rH1 channel in B104 cells. Na+ currents could be recorded in 65% of cells. In the absence of TTX, mean peak Na+ current density was 126 ± 19 pA/pF, corresponding to a channel density of 2.7 ± 0.4/μ2 (mean ± SE). Time-to-peak (t-peak), activation (τm), and inactivation time constants (τh) for Na+ currents in B104 cells were 1.0 ± 0.04, 0.4 ± 0.06, and0.9 ± 0.04 ms at −10 mV. The peak conductance-voltage relationship had a V 1/2 of −39.8 ± 1.5 mV. V 1/2 for steady-state inactivation was −81.6 ± 1.5 mV. TTX-sensitive and -resistant components of the Na current had half-maximal inhibitions (IC50), respectively, of 1.2 nM and, minimally, 575.5 nM. The TTX-sensitive and-resistant Na+ currents were kinetically distinct; time-to-peak, τm, and τh for TTX-sensitive currents were shorter than for TTX-resistant currents. Steady-state voltage dependence of the two currents was indistinguishable. The presence of TTX-sensitive and-resistant Na+ currents, which are pharmacologically and kinetically distinct, led us to search for mRNAs known to be associated with TTX-resistant channels, in addition to the α subunit mRNAs, which have previously been shown to be expressed in these cells. Using RT-PCR and restriction enzyme mapping, we were unable to detect αSNS, but detected mRNA for rH1, which is known to encode a TTX-resistant channel, in B104 cells. B104 neuroblastoma cells thus express TTX-sensitive and -resistant Na+ currents. These appear to be encoded by neuronal-type and cardiac Na+ channel mRNAs including the RH1 transcript. This cell line may be useful for studies on the rH1 channel, which is known to be mutated in the long-QT syndrome.

scholarly journals Curcumin Analogue C1 Promotes Hex and Gal Recruitment to the Plasma Membrane via mTORC1-Independent TFEB Activation

2019 ◽  
Vol 20 (6) ◽  
pp. 1363 ◽  
Author(s):  
Alessandro Magini ◽  
Alice Polchi ◽  
Danila Di Meo ◽  
Sandra Buratta ◽  
Elisabetta Chiaradia ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Nuclear Translocation ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Jurkat Cells ◽  
Lipid Microdomains ◽  
Curcumin Analogue ◽  
Transcription Factor Eb ◽  
Increased Activity

The monocarbonyl analogue of curcumin (1E,4E)-1,5-Bis(2-methoxyphenyl)penta-1,4-dien-3-one (C1) has been used as a specific activator of the master gene transcription factor EB (TFEB) to correlate the activation of this nuclear factor with the increased activity of lysosomal glycohydrolases and their recruitment to the cell surface. The presence of active lysosomal glycohydrolases associated with the lipid microdomains has been extensively demonstrated, and their role in glycosphingolipid (GSL) remodeling in both physiological and pathological conditions, such as neurodegenerative disorders, has been suggested. Here, we demonstrate that Jurkat cell stimulation elicits TFEB nuclear translocation and an increase of both the expression of hexosaminidase subunit beta (HEXB), hexosaminidase subunit alpha (HEXA), and galactosidase beta 1 (GLB1) genes, and the recruitment of β-hexosaminidase (Hex, EC 3.2.1.52) and β-galactosidase (Gal, EC 3.2.1.23) on lipid microdomains. Treatment of Jurkat cells with the curcumin analogue C1 also resulted in an increase of both lysosomal glycohydrolase activity and their targeting to the cell surface. Similar effects of C1 on lysosomal glycohydrolase expression and their recruitment to lipid microdomains was observed by treating the SH-SY5Y neuroblastoma cell line; the effects of C1 treatment were abolished by TFEB silencing. Together, these results clearly demonstrate the existence of a direct link between TFEB nuclear translocation and the transport of Hex and Gal from lysosomes to the plasma membrane.

scholarly journals Localization of MOC-1 cell surface antigen in small-cell lung carcinoma cell lines: an immunohistochemical and immunoelectron microscopic study.

1993 ◽  
Vol 41 (9) ◽  
pp. 1303-1310 ◽  
Author(s):  
V Speirs ◽  
S Eich-Bender ◽  
C R Youngson ◽  
E Cutz
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Western Blot ◽  
Cell Line ◽  
Cell Lines ◽  
Carcinoma Cell ◽  
Small Cell ◽  
Cell Contact ◽  
Positive Staining ◽  
Carcinoma Cell Lines

Expression of cell surface antigens of the neural cell adhesion molecule (N-CAM) class was recently shown to be shared by both fetal and neoplastic neuroendocrine cells, including those of the lung. We investigated the expression and localization of MOC-1 antigen on small-cell (neuroendocrine) lung carcinoma cell lines with immunohistochemical methods at the light (LM) and electron microscopy (EM) level and by Western blot. At LM level, using monoclonal antibody (MAb) MOC-1 with the ABC method and immunofluorescence, positive staining was observed on surfaces of cells from all tumor lines examined. Strongest immunostaining was found on cell surfaces of pulmonary small-cell carcinoma-derived cell line NCI-H69 with the majority of cells showing positive staining. An adherent variant of NCI-H69 cell line, H69V, exhibited positive staining in about 60% of cells, whereas only occasional cells of NCI-H727 cell line derived from pulmonary carcinoid tumor were positive for MOC-1 antigen. Western blot analysis confirmed these findings, showing a strong MOC-1-specific band in cell extracts of NCI-H69, with weaker band densities for H69V and NCI-H727. Immunoelectron microscopy (IEM) revealed that MOC-1 was not uniformly distributed on the outer surface of plasma membrane; immunogold particles appeared concentrated in areas of thick cell surface "fuzz" coating, surface microvilli, and in areas of cell-cell contact. In some cells, areas of plasma membrane invaginations and a few intracytoplasmic vesicles were also labeled, suggesting endocytosis. Surface labeling for SEM confirmed the finding of more dense labeling over the microvilli, cell membrane folds, and in areas of cell-cell contact. The cell lines derived from pulmonary neuroendocrine cell tumors can provide a useful model to study the role and function of neural adhesion molecules in pulmonary neoplasia and during lung development.

Abstract P032: Dopamine Type 2 Receptor (D 2 R) Expression Modulates The Salt (NaCl) Induced Plasma Membrane Receptor Recruitment Of The Natriuretic Angiotensin Type 2 Receptor (AT 2 R) In Renal Proximal Tubule Cells Obtained From The Urine Of Salt Study Participants

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
John J Gildea ◽  
Peng Xu ◽  
Katherine Schiermeyer ◽  
Wei Yue ◽  
Robert M Carey ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Proximal Tubule ◽  
Cell Line ◽  
T Test ◽  
Proximal Tubule Cells ◽  
Membrane Recruitment ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

The effect of D2R expression on the natriuretic AT2R plasma membrane recruitment in high (190 mM NaCl) and normal (140 mM NaCl) salt conditions was measured in human renal proximal tubule cells (RPTC) obtained from urine provided by salt study diet volunteers that were either salt resistant (SR) or inverse salt sensitive (ISS) (increased blood pressure on a low salt diet). Basal D2R expression was 36.9% ± 2.6% lower in ISS vs SR control n=5, p<0.01). D2R expression in the SR was reduced by siRNA (37.1% ± 2.0% reduction in ISS vs SR control, n=5, p<0.01, t-test). The ISS D2R expression was returned to normal levels by D2R overexpression using BacMam technology (22.5% ± 0.9% increase for ISS vs SR control siRNA, n=5, p<0.01, t-test). Culturing SR and ISS cell lines in normal salt (NS, 140 mM NaCl) and high salt (HS, 190 mM NaCl) leads to the recruitment of AT2R to the cell surface in the ISS cell line (ISS Control: NS 47978 ± 2728 RFU vs HS 74056 ± 3002 RFU, n=5, p<0.001, t-test), but not in the SR cell line. Knockdown of D2R using siRNA in the SR cell line altered the HS response in the SR cell line to that measured in the ISS cell line (SR D2R siRNA: NS 48514 ± 2560 RFU vs HS 82599 ± 1492 RFU, n=5, p<0.001, t-test), and the difference between SR HS control siRNA and SR HS D2R siRNA is also highly significant (SR HS: control siRNA 60154 ± 3347 RFU vs SR HS D2R siRNA 82599 ± 1492 RFU, n=5, p<0.001, t-test). Further lowering of D2R in ISS with D2R siRNA still showed a significant translocation of AT2R to the cell surface under HS (ISS D2R siRNA: NS 47953 ± 3058 RFU vs HS 80284 ± 2173 RFU, n-5, p<0.001, t-test) but not a further enhancement over SR siRNA. Overexpression of D2R in the ISS cell line completely blocked the HS AT2R cell surface recruitment, thus converting the ISS HS phenotype of ISS to a cell line that resembled a SR cell line. In conclusion, we have shown that a clear ISS phenotype of HS AT2R plasma membrane recruitment can be replicated in an SR cell line by reducing D2R expression using siRNA to levels seen in an ISS cell line. Similarly, overexpressing D2R in an ISS cell line to a level seen in SR cells reverts the ISS cells back to a SR associated sodium induced AT2R cell surface recruitment.

Detection of dipeptidyl peptidase IV in glioma C6 and neuroblastoma SK-N-SH cell lines

1995 ◽  
Vol 73 (1-2) ◽  
pp. 113-115 ◽  
Author(s):  
Aleksi Sedo ◽  
Roberto P. Revoltella
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Human Neuroblastoma Cell ◽  
Fluorescent Substrate ◽  
Human Neuroblastoma ◽  
Dpp Iv

The dipeptidyl peptidase IV (DPP-IV) activity of the rat glioma cell line C6 and the human neuroblastoma cell line SK-N-SH was investigated. DPP-IV fluorescent substrate was cleaved by both cell lines. The pH reaction optimum determined was typical for DPP-IV described in other cell models. The reaction was inhibited by specific inhibitors diprotins A and B. Enzyme activity was localized, both on the cell surface and intracellularly. Most of the DPP-IV activity was membrane bound. However, soluble intra-cellular activity was found in both cell lines. Secreted activity was not detected in either cell line. In the C6 line, but not in the SK-N-SH line, we demonstrated depression of the ratio of cell surface to total cell DPP-IV activity at higher cell densities, indicating possible enzyme redistribution during cell growth in culture. Identification of DPP-IV activity is the first step in our study of the role of DPP-IV in the neural system.Key words: dipeptidyl peptidase IV, glioma, neuroblastoma.

Glycosphingolipid Domains on Cell Plasma Membrane

1999 ◽  
Vol 19 (3) ◽  
pp. 197-208 ◽  
Author(s):  
Maurizio Sorice ◽  
Tina Garofalo ◽  
Roberta Misasi ◽  
Vincenza Dolo ◽  
Giuseppe Lucania ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cell Activation ◽  
Human Peripheral Blood ◽  
Neuroblastoma Cells ◽  
Cell Types ◽  
Peripheral Blood Lymphocytes ◽  
Lymphoid Cells ◽  
Signaling Complex ◽  
3T3 Fibroblasts

In this study we analyzed by immunofluorescence, laser confocal microscopy, immunoelectron microscopy and label fracture technique the ganglioside distribution on the plasma membrane of several different cell types: human peripheral blood lymphocytes (PBL), Molt-4 lymphoid cells, and NIH 3T3 fibroblasts, which mainly express monosialoganglioside GM3, and murine NS20Y neuroblastoma cells, which have been shown to express a high amount of monosialoganglioside GM2. Our observations showed an uneven distribution of both GM3 and GM2 on the plasma membrane of all cells, confirming the existence of ganglioside-enriched microdomains on the cell surface. Interestingly, in lymphoid cells the clustered immunolabeling appeared localized over both the microvillous and the nonvillous portions of the membrane. Similarly, in cells growing in monolayer, the clusters were distributed on both central and peripheral regions of the cell surface. Therefore, glycosphingolipid clusters do not appear confined to specific areas of the plasma membrane, implying general functions of these domains, which, as structural components of a cell membrane multimolecular signaling complex, may be involved in cell activation and adhesion, signal transduction and, when associated to caveolae, in endocytosis of specific molecules.

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