scholarly journals Insulin as a surface marker on isolated cells from rat pancreatic islets.

1983 ◽  
Vol 97 (2) ◽  
pp. 433-437 ◽  
Author(s):  
D R Kaplan ◽  
J R Colca ◽  
M L McDaniel
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Islet Cells ◽  
Secretory Granules ◽  
Immunoreactive Insulin ◽  
Isolated Cells ◽  
Intact Cells ◽  
Specific Receptors ◽  
Specific Membrane ◽  
Rat Pancreatic Islet

Immunoreactive insulin was shown to exist as a surface molecule in the plasma membrane of dispersed rat pancreatic islet cells. The intact cells were stained by immunofluorescence with a guinea pig antisera specific for insulin. The hormone on the cell surface could not be accounted for by insulin bound to specific receptors or nonspecifically absorbed to cells. Thus, surface insulin was demonstrated to be a specific membrane antigen for islet cells. Furthermore, the proportion of islet cells with insulin on the cell surface was directly correlated with insulin secretion in several different settings. This correspondence was demonstrated by varying the glucose concentration in the medium, by withholding Ca2+, which inhibits secretion, and by adding theophylline, which potentiates secretion. Consequently, these results suggested that insulin as a membrane protein was a marker for cells that actively secreted the hormone and may have been derived in the fusion process of secretory granules with the plasma membrane.

scholarly journals Reconstitution of constitutive secretion using semi-intact cells: regulation by GTP but not calcium.

1991 ◽  
Vol 112 (1) ◽  
pp. 39-54 ◽  
Author(s):  
S G Miller ◽  
H P Moore
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
G Protein ◽  
Secretory Granules ◽  
Intact Cells ◽  
Transport Reaction ◽  
Permeabilized Cells ◽  
Transmembrane Glycoprotein ◽  
Transport Vesicles ◽  
Constitutive Secretion

Regulated exocytosis in many permeabilized cells can be triggered by calcium and nonhydrolyzable GTP analogues. Here we examine the role of these effectors in exocytosis of constitutive vesicles using a system that reconstitutes transport between the trans-Golgi region and the plasma membrane. Transport is assayed by two independent methods: the movement of a transmembrane glycoprotein (vesicular stomatitis virus glycoprotein [VSV G protein]) to the cell surface; and the release of a soluble marker, sulfated glycosaminoglycan (GAG) chains, that have been synthesized and radiolabeled in the trans-Golgi. The plasma membrane of CHO cells was selectively perforated with the bacterial cytolysin streptolysin-O. These perforated cells allow exchange of ions and cytosolic proteins but retain intracellular organelles and transport vesicles. Incubation of the semi-intact cells with ATP and a cytosolic fraction results in transport of VSV G protein and GAG chains to the cell surface. The transport reaction is temperature dependent, requires hydrolyzable ATP, and is inhibited by N-ethylmaleimide. Nonhydrolyzable GTP analogs such as GTP gamma S, which stimulate the fusion of regulated secretory granules, completely abolish constitutive secretion. The rate and extent of constitutive transport between the trans-Golgi and the plasma membrane is independent of free Ca2+ concentrations. This is in marked contrast to fusion of regulated secretory granules with the plasma membrane, and transport between the ER and the cis-Golgi (Beckers, C. J. M., and W. E. Balch. 1989. J. Cell Biol. 108:1245-1256; Baker, D., L. Wuestehube, R. Schekman, and D. Botstein. 1990. Proc. Natl. Acad. Sci. USA. 87:355-359).

scholarly journals Clustering and Internalization of Toxic Amylin Oligomers in Pancreatic Cells Require Plasma Membrane Cholesterol

2011 ◽  
Vol 286 (41) ◽  
pp. 36086-36097 ◽  
Author(s):  
Saurabh Trikha ◽  
Aleksandar M. Jeremic
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Self Assembly ◽  
Surface Coverage ◽  
Islet Cells ◽  
Fold Increase ◽  
Water Soluble ◽  
Cholesterol Depletion ◽  
Β Cells ◽  
Pancreatic Hormone

Self-assembly of the human pancreatic hormone amylin into toxic oligomers and aggregates is linked to dysfunction of islet β-cells and pathogenesis of type 2 diabetes mellitus. Recent evidence suggests that cholesterol, an essential component of eukaryotic cells membranes, controls amylin aggregation on model membranes. However, the pathophysiological consequence of cholesterol-regulated amylin polymerization on membranes and biochemical mechanisms that protect β-cells from amylin toxicity are poorly understood. Here, we report that plasma membrane (PM) cholesterol plays a key role in molecular recognition, sorting, and internalization of toxic amylin oligomers but not monomers in pancreatic rat insulinoma and human islet cells. Depletion of PM cholesterol or the disruption of the cytoskeleton network inhibits internalization of amylin oligomers, which in turn enhances extracellular oligomer accumulation and potentiates amylin toxicity. Confocal microscopy reveals an increased nucleation of amylin oligomers across the plasma membrane in cholesterol-depleted cells, with a 2-fold increase in cell surface coverage and a 3-fold increase in their number on the PM. Biochemical studies confirm accumulation of amylin oligomers in the medium after depletion of PM cholesterol. Replenishment of PM cholesterol from intracellular cholesterol stores or by the addition of water-soluble cholesterol restores amylin oligomer clustering at the PM and internalization, which consequently diminishes cell surface coverage and toxicity of amylin oligomers. In contrast to oligomers, amylin monomers followed clathrin-dependent endocytosis, which is not sensitive to cholesterol depletion. Our studies identify an actin-mediated and cholesterol-dependent mechanism for selective uptake and clearance of amylin oligomers, impairment of which greatly potentiates amylin toxicity.

Expression of multiple plasma membrane Ca2+-ATPases in rat pancreatic islet cells

Cell Calcium ◽  
2000 ◽  
Vol 27 (4) ◽  
pp. 231-246 ◽  
Author(s):  
A. Kamagate ◽  
A. Herchuelz ◽  
A. Bollen ◽  
F.Van Eylen
Keyword(s):  
Plasma Membrane ◽  
Pancreatic Islet ◽  
Islet Cells ◽  
Pancreatic Islet Cells ◽  
Rat Pancreatic Islet

scholarly journals Delayed restoration of Mg2+ content and transport in liver cells following ethanol withdrawal

2009 ◽  
Vol 297 (4) ◽  
pp. G621-G631 ◽  
Author(s):  
Lisa M. Torres ◽  
Christie Cefaratti ◽  
Liliana Berti-Mattera ◽  
Andrea Romani
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Isolated Hepatocytes ◽  
Liver Cells ◽  
Ethanol Withdrawal ◽  
Adrenergic Stimulation ◽  
Plasma Membrane Vesicles ◽  
Isolated Cells ◽  
Intact Cells

Liver cells from rats chronically fed a Lieber-De Carli diet for 3 wk presented a marked decreased in tissue Mg2+ content and an inability to extrude Mg2+ into the extracellular compartment upon stimulation with catecholamine, isoproterenol, or cell-permeant cAMP analogs. This defect in Mg2+ extrusion was observed in both intact cells and purified liver plasma membrane vesicles. Inhibition of adrenergic or cAMP-mediated Mg2+ extrusion was also observed in freshly isolated hepatocytes from control rats incubated acutely in vitro with varying doses of ethanol (EtOH) for 8 min. In this model, however, the defect in Mg2+ extrusion was observed in intact cells but not in plasma membrane vesicles. In the chronic model, upon removal of EtOH from the diet hepatic Mg2+ content and extrusion required ∼10 days to return to normal level both in isolated cells and plasma membrane vesicles. In hepatocytes acutely treated with EtOH for 8 min, more than 60 min were necessary for Mg2+ content and extrusion to recover and return to the level observed in EtOH-untreated cells. Taken together, these data suggest that in the acute model the defect in Mg2+ extrusion is the result of a limited refilling of the cellular compartment(s) from which Mg2+ is mobilized upon adrenergic stimulation rather than a mere defect in adrenergic cellular signaling. The chronic EtOH model, instead, presents a transient but selective defect of the Mg2+ extrusion mechanisms in addition to the limited refilling of the cellular compartments.

scholarly journals SNAP-25 is expressed in islets of Langerhans and is involved in insulin release.

1995 ◽  
Vol 128 (6) ◽  
pp. 1019-1028 ◽  
Author(s):  
K Sadoul ◽  
J Lang ◽  
C Montecucco ◽  
U Weller ◽  
R Regazzi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Secretion ◽  
B Cell ◽  
Insulin Release ◽  
Islet Cells ◽  
Secretory Granules ◽  
Cell Types ◽  
Pancreatic B Cell ◽  
Pancreatic Endocrine Cells ◽  
Presynaptic Plasma Membrane

SNAP-25 is known as a neuron specific molecule involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane. By immunolocalization and Western blot analysis, it is now shown that SNAP-25 is also expressed in pancreatic endocrine cells. Botulinum neurotoxins (BoNT) A and E were used to study the role of SNAP-25 in insulin secretion. These neurotoxins inhibit transmitter release by cleaving SNAP-25 in neurons. Cells from a pancreatic B cell line (HIT) and primary rat islet cells were permeabilized with streptolysin-O to allow toxin entry. SNAP-25 was cleaved by BoNT/A and BoNT/E, resulting in a molecular mass shift of approximately 1 and 3 kD, respectively. Cleavage was accompanied by an inhibition of Ca(++)-stimulated insulin release in both cell types. In HIT cells, a concentration of 30-40 nM BoNT/E gave maximal inhibition of stimulated insulin secretion of approximately 60%, coinciding with essentially complete cleavage of SNAP-25. Half maximal effects in terms of cleavage and inhibition of insulin release were obtained at a concentration of 5-10 nM. The A type toxin showed maximal and half-maximal effects at concentrations of 4 and 2 nM, respectively. In conclusion, the results suggest a role for SNAP-25 in fusion of dense core secretory granules with the plasma membrane in an endocrine cell type- the pancreatic B cell.

scholarly journals SUBPLASMALEMMAL MICROFILAMENTS AND MICROTUBULES IN RESTING AND PHAGOCYTIZING CULTIVATED MACROPHAGES

1973 ◽  
Vol 59 (1) ◽  
pp. 12-27 ◽  
Author(s):  
Eve P. Reaven ◽  
Stanton G. Axline
Keyword(s):  
Plasma Membrane ◽  
Free Surface ◽  
Cell Surface ◽  
Cell Attachment ◽  
Close Association ◽  
Microtubule Organization ◽  
Thin Sections ◽  
Attached Cell ◽  
Specific Membrane

The subplasmalemmal organization of the free and glass-attached surfaces of resting and phagocytizing cultivated macrophages were examined in an attempt to define specific membrane-associated structures related to phagocytosis. From analysis of serial thin sections of oriented cells it was found that the subplasmalemmal region of the attached cell surface has a complex microfilament and microtubule organization relative to the subplasmalemmal area of the free surface. A filamentous network composed of 40–50-Å microfilaments extended for a depth of 400–600 Å from the attached plasma membrane. Immediately subjacent to the filamentous network was a zone of oriented bundles of 40–50-Å microfilaments and a zone of microtubules. Additional microtubules were found to extend from the plasma membrane to the interior of the cell in close association with electron-dense, channellike structures. In contrast, the free aspect of the cultivated macrophage contained only the subplasmalemmal filamentous network. However, after a phagocytic pulse with polystyrene particles (14 µm diam) microtubules and oriented filaments similar to those found on the attached surface were observed surrounding the ingested particles. The observations reported in this paper provide support for the hypothesis that microfilaments and/or microtubules play a role in the translocation of plasma membrane required for the functionally similar processes of phagocytosis and cell attachment to glass.

scholarly journals Inositol 1,4,5-trisphosphorothioate, a stable analogue of inositol trisphosphate which mobilizes intracellular calcium

1989 ◽  
Vol 259 (3) ◽  
pp. 645-650 ◽  
Author(s):  
C W Taylor ◽  
M J Berridge ◽  
A M Cooke ◽  
B V L Potter
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Intracellular Calcium ◽  
Racemic Mixture ◽  
Synthetic Analogue ◽  
Intact Cells ◽  
Surface Receptors ◽  
Prolonged Stimulation ◽  
Crude Preparation ◽  
Intracellular Messenger

D-Ins(1,4,5)P3 is now recognized as an intracellular messenger that mediates the actions of many cell-surface receptors on intracellular Ca2+ pools, but its complex and rapid metabolism in intact cells has confused interpretation of its possible roles in oscillatory changes in intracellular [Ca2+] and in controlling Ca2+ entry at the plasma membrane. We now report the actions and metabolic stability of a synthetic analogue of Ins(1,4,5)P3, DL-inositol 1,4,5-trisphosphorothioate [DL-Ins(1,4,5)P3[S]3]. In permeabilized hepatocytes, DL-Ins(1,4,5)P3[S]3 and synthetic DL-Ins(1,4,5)P3 stimulated Ca2+ release from the same intracellular stores, though the concentration required for half-maximal release was 3-fold higher for DL-Ins(1,4,5)P3[S]3. Since L-Ins(1,4,5)P3 neither antagonized the effects of D-Ins(1,4,5)P3 nor itself stimulated appreciable Ca2+ release, the activity of the racemic mixture of Ins(1,4,5)P3, and presumably also of Ins(1,4,5)P3[S]3, is attributable to the D-isomer. Under conditions where there was negligible metabolism of D-[3H]Ins(1,4,5)P3, both DL-Ins(1,4,5)P3 and DL-Ins(1,4,5)P3[S]3 elicited rapid Ca2+ release from intracellular stores, and the stores remained empty during prolonged stimulation. When cells were incubated at high density, both compounds stimulated rapid Ca2+ release, but while the stores soon refilled as Ins(1,4,5)P3 was degraded to Ins(1,4)P2, there was no refilling of the pools after stimulation with DL-Ins(1,4,5)P3[S]3. When DL-Ins(1,4,5)P3 or DL-Ins(1,4,5)P3[S]3 was treated with a crude preparation of Ins(1,4,5)P3 3-kinase and ATP, and the Ca2+-releasing activity of the products subsequently assayed, DL-Ins(1,4,5)P3 was completely inactivated by phosphorylation, but there was no loss of activity of the phosphorothioate analogue. In additional experiments, DL-Ins(1,4,5)P3[S]3 (10 microM) did not affect the rate of phosphorylation of D-[3H]Ins(1,4,5)P3 (1 microM). We conclude that Ins(1,4,5)P3[S]3 is a full agonist and only 3-fold less potent than Ins(1,4,5)P3 in mobilizing intracellular Ca2+ stores, but unlike the natural messenger it is resistant to both phosphorylation and dephosphorylation. We propose that this stable analogue will allow the direct actions of Ins(1,4,5)P3 to be resolved from those that require its metabolism.

scholarly journals Utilizing TAPBPR to promote exogenous peptide loading onto cell surface MHC I molecules

2018 ◽  
Vol 115 (40) ◽  
pp. E9353-E9361 ◽  
Author(s):  
F. Tudor Ilca ◽  
Andreas Neerincx ◽  
Mark R. Wills ◽  
Maike de la Roche ◽  
Louise H. Boyle
Keyword(s):  
Plasma Membrane ◽  
T Cell ◽  
Cell Surface ◽  
Cell Receptor ◽  
Target Cells ◽  
Intact Cells ◽  
Mhc I ◽  
Immunogenic Peptides ◽  
Presentation Pathway ◽  
Peptide Exchange

The repertoire of peptides displayed at the cell surface by MHC I molecules is shaped by two intracellular peptide editors, tapasin and TAPBPR. While cell-free assays have proven extremely useful in identifying the function of both of these proteins, here we explored whether a more physiological system could be developed to assess TAPBPR-mediated peptide editing on MHC I. We reveal that membrane-associated TAPBPR targeted to the plasma membrane retains its ability to function as a peptide editor and efficiently catalyzes peptide exchange on surface-expressed MHC I molecules. Additionally, we show that soluble TAPBPR, consisting of the luminal domain alone, added to intact cells, also functions as an effective peptide editor on surface MHC I molecules. Thus, we have established two systems in which TAPBPR-mediated peptide exchange on MHC class I can be interrogated. Furthermore, we could use both plasma membrane-targeted and exogenous soluble TAPBPR to display immunogenic peptides on surface MHC I molecules and consequently induce T cell receptor engagement, IFN-γ secretion, and T cell-mediated killing of target cells. Thus, we have developed an efficient way to by-pass the natural antigen presentation pathway of cells and load immunogenic peptides of choice onto cells. Our findings highlight a potential therapeutic use for TAPBPR in increasing the immunogenicity of tumors in the future.

scholarly journals Surface redistribution of 125I-insulin in cultured human lymphocytes.

1981 ◽  
Vol 91 (1) ◽  
pp. 17-25 ◽  
Author(s):  
J L Carpentier ◽  
E Van Obberghen ◽  
P Gorden ◽  
L Orci
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Human Lymphocytes ◽  
Freeze Fracture ◽  
Total Cell ◽  
Coated Pits ◽  
Villous Surface ◽  
Specific Receptors ◽  
Related Proteins ◽  
Preferential Association

The cultured human lymphocyte (IM-9) binds 125I-insulin by a receptor-mediated process; the receptor, in turn, is regulated by the ligand. In the present study we have examined quantitatively the morphologic events involved in 125I-insulin interaction with the surface of the lymphocyte. At 2 min of incubation of 15 degrees or 37 degrees C, the ligand localizes preferentially at the villous surface of the cell, whereas with longer periods of incubation, the ligand distributes indistinguishably between the villous and nonvillous surface. When rebinding is blocked, 125I-insulin localizes preferentially at the nonvillous surface of the cell. When the total cell surface is considered, there is little preferential association with coated pits; when only the nonvillous surface is considered, a preferential association with coated pits is found and is quantitatively increased in the absence of rebinding of the ligand. This cell has an abundant villous surface (approximately 55% of the total surface); and, as seen on freeze-fracture replicas, the plasma membrane of the villous surface contains a 60% greater density of intramembrane particles than the nonvillous surface. The data suggest an ordered pattern of insulin interaction with the cell surface (i.e., binding to villi followed by redistribution to the nonvillous portion of the cell containing coated pits). These events probably reflect the mechanism by which the cell segregates specific receptors and related proteins in the plane of the membrane so that they can be selectively removed.

scholarly journals Resynthesis of sphingomyelin from plasma-membrane phosphatidylcholine in BHK cells treated with Staphylococcus aureus sphingomyelinase

1988 ◽  
Vol 254 (3) ◽  
pp. 765-771 ◽  
Author(s):  
D Allan ◽  
P Quinn
Keyword(s):  
Staphylococcus Aureus ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Fluid Phase ◽  
Intact Cells ◽  
Prolonged Incubation ◽  
Bhk Cells ◽  
Streptolysin O ◽  
Incubation Experiments ◽  
Original Cell

About 60-65% of the total sphingomyelin in intact BHK cells is in a readily accessible pool which is rapidly degraded by Staphylococcus aureus sphingomyelinase. No more sphingomyelin is broken down in cells which have been fixed with glutaraldehyde or lysed with streptolysin O, suggesting that all the sphingomyelin which is available to the enzyme is on the cell surface. The inaccessible pool of sphingomyelin does not equilibrate with the plasma-membrane pool, even after prolonged incubation. Experiments using [3H]-choline show that much more phosphocholine is released from the intact cells treated with sphingomyelinase than can be accounted for by breakdown of the original cell-surface pool of sphingomyelin; the excess appears to be a consequence of the breakdown of sphingomyelin newly resynthesized at the expense of a pool of phosphatidylcholine which represents about 8% of total cell phosphatidylcholine and may reside in the plasma membrane. This would be consistent with resynthesis of cell-surface sphingomyelin by the phosphatidylcholine: ceramide phosphocholinetransferase pathway, which has previously been shown to be localized in the plasma membrane. However, in [3H]palmitate-labelled cells there appeared to be no accumulation of the diacylglycerol expected to be produced by this reaction, and no enhanced synthesis of phosphatidate or phosphatidylinositol; instead there was an increased synthesis of triacylglycerol. A similar increase in labelling of triacylglycerol was seen in enzyme-treated cells where the sphingomyelinase was subsequently removed, allowing resynthesis of sphingomyelin which occurred at a rate of about 25% of total sphingomyelin/h. Treatment of BHK cells with sphingomyelinase caused no change in the rates of fluid-phase endocytosis or exocytosis as measured with [3H]inulin.

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