Translocation and turnover of phospholipid analogs in plasma membrane-derived vesicles from cell cultures

Yavin, Ephraïm; Zutra, Aliza

doi:10.1016/0005-2736(79)90298-0

Cited by 20 publications

(12 citation statements)

References 28 publications

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“…PtdCho is abundant in NG108-15 cells (99 nmol per mg of protein), turns over slowly (28), and moves rapidly by lateral diffusion in cell membranes above the phase transition temperature (29). If Ca 2 uptake by cells via Ca2+ action-potential channels were dependent on PtdCho, it seems unlikely that inhibition of PtdCho synthesis by DZ-SIBA or SIBA would rapidly and profoundly inhibit the activity of the channels.…”

Section: Discussionmentioning

confidence: 99%

Inhibitors of CDP-choline synthesis, action potential calcium channels, and stimulus-secretion coupling.

Blas¹,

Adler²,

Shih³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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The effects of putative transmethylation inhibitors were tested on stimulus-secretion coupling and neurotransmitter secretion at synapses between neuroblastoma X gjioma hybrid cells and myotubes. 5'-Deoxy-5"-isobutylthio-3-deazaadenosine or 5'-deoxy-$'-isobutylthioadenosine inhibited CDP-choline synthesis catalyzed by cholineplosphate cytidylyltransferase (CTP:cholinephosphate cytidylyjtransferase, EC 2.7.7.15) and thereby decreased the rate of phosphatidylcholine synthesis from CDP-choline, but did not affect -the transmethylation pathway for phosphatidylcholine synthesis. These compounds also inhibited 4Ca2+ uptake by hybrid cells mediated by voltage-sensitive Ca2+ channels, acetylcholine secretion at synapses, and signal transduction through cell membranes mediated by myotube nicotinic acetylcholine receptors.In contrast, 3-deazaadenosine or adenosine inhibited the transmethylation pathway for pgosphatidylcholine synthesis, but had no effect on Ca2+ action potentials, acetylcholine secretion, or signal transduction through cell membralles mediated by nicotinic acetylcholine receptors. These results show that the stimulus-secretion coupling and secretion reactions studied are not dependent on phospholipid methylation and suggest that the activity of action potential Ca2+ channels and the rate of neurotransmitter secretion are functionally coupled to the rate of phosphatidylcholine synthesis via the CDP-choline pathway.The synthesis of phosphatidylcholine (PtdCho) from phosphatidylethanolamine (PtdEtn) by transmethylation and concomitant translocation of the phospholipid from the inner to the outer leaflet of the plasma membrane has been proposed as a mechanism of signal transduction and secretion across cell membranes (1). Part of the evidence that the transmethylation pathway for PtdCho synthesis plays a role in signal transduction was obtained by the use of adenosine analogs such as 5'-deoxy-5'-isobutylthio-3-deazaadenosine (DZ-SIBA) on the assumption that these compounds are specific inhibitors of transmethylation reactions (2-5). However, with respect to DZ-SIBA the evidence for this assumption is inadequate (6, 7). In addition; questions have been raised about the hypothesized role of phpspholipid transmethylation (8, 9 RESULTSEffects of Adenosine Derivatives on Myotube Responses to Acetylcholine Secreted by NG108-15 Cells at Synapses. SAdenosylhomocysteinase (S-adenosyl-L-homocysteine hydrolase, EC 3.3.1.1) catalyzes the hydrolysis or synthesis of S-adenosylhomocysteine, a potent inhibitor of transmethylation reactions. Inhibition of S-adenosylhomocysteinase by various adenosine derivatives has been shown to increase cellular levels of S-adenosylhomocysteine and thereby inhibit transmethylation reactions (13).In Fig. 1A are shown the effects of adenosine derivatives on miniature end-plate potentials (MEPPs) of cultured rat myotubes innervated by NG108-15 cells. Myotube MEPPs were measured with intracellular microelectrodes; presumably, each myotube response is initiated by the secretion of acety...

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Section: Discussionmentioning

confidence: 99%

Inhibitors of CDP-choline synthesis, action potential calcium channels, and stimulus-secretion coupling.

Blas¹,

Adler²,

Shih³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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“…[11] However, another study claims its retention for up to three days. [20] To estimate the range of applicability for GPMVs, it is important to characterize them carefully. Further insight into the mechanisms leading to the formation of GPMVs is indispensable to infer how the membrane structure might be perturbed during this process.…”

Section: Introductionmentioning

confidence: 99%

PI(4,5)P₂ Degradation Promotes the Formation of Cytoskeleton‐Free Model Membrane Systems

Keller¹,

Lorizate²,

Schwille³

2009

ChemPhysChem

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Giant plasma membrane vesicles (GPMVs) are cell-derived model membrane systems that undergo large-scale lipid phase separation when cooled below room temperature. Because of their presumably more physiological lipid composition, they are increasingly used as alternatives to synthetic model membranes. However, the exact mechanism of GPMV formation, and thus, effects of this process on the physiological integrity of the membrane are still unclear. Herein, we identify the key steps of GPMV formation and characterize their differences with respect to the plasma membrane of intact cells. Addition of GPMV-inducing reagents triggers a steady Ca2+ influx that is accompanied by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] degradation and phosphatidyl serine (PS) externalization before detachment of the cytoskeleton and the onset of vesicle formation. When comparing GPMVs to other cell-derived model systems, PI(4,5)P2 is not detectable in phase-separating plasma membrane spheres (PMSs) either, but is present in non-phase-separating blebs. GPMVs differ from the physiological state of the plasma membrane in the presence of specific lipids, which limits their use as model systems. Furthermore, we propose that PI(4,5)P2 influences the phase-separation behavior.

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“…Mouse neuroblastoma clone NlE-115 and the glioma neuroblastoma hybrid NG-108-15 were originally obtained from M. Nirenberg. The cells were grown as described (21,22). Dishes were used 6-35 days after reseeding.…”

mentioning

confidence: 99%

Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons.

Grinvald¹,

Ross²,

Farber³

1981

Proc. Natl. Acad. Sci. U.S.A.

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Optical methods using changes in fluorescence and absorption ofvoltage-sensitive dyes were developed to record electrical activity from processes of nerve cells grown in monolayer culture. For transmission measurements, a merocyanine dye was discovered that was more sensitive than others previously tested on cultured neurons. Action potentials from the somata of these cells were detected without averaging, with a signal-to-noise ratio of 20:1. With this dye, electrical responses were simultaneously recorded from many points along the arborization of neuroblastoma cells by using a 10 x 10 array ofphotodiodes positioned in the microscope image plane. Frequently different processes had different shapes of electrical responses, suggesting regional specializations. Fluorescence measurements with an oxonol dye proved to be more sensitive than transmission measurements, particularly when recording from small processes. By changing the position of the cell relative to a laser microbeam while recording electrically from the cell body, it was possible to monitor the membrane potential in the cell body and in the process simultaneously. From the delay in reponse in the process, a lower limit for the mean conduction velocity of 0.2-0.6 m/sec was found for 2-to 7-,um processes. The mean space constants of processes were estimated by comparing the amplitudes of passive voltage responses in the cell body and growth cone. A lower limit of400-950 ,um was obtained for 4-to 7-,um processes. Many questions in neurobiology require an understanding of the regional properties ofneurons. For example, the integration of multiple synaptic inputs by single nerve cells requires a knowledge ofthe passive and active electrical membrane properties ofthe dendritic branches, soma, and axon (1-4); the space constants of each of the processes and the location and nature of specialized segments with localized excitability must be determined. Also, the role that given ions play in the regulation of neuronal development requires knowledge of the electrical properties of the developing neurites (5). Yet, the electrical events in the growth cone are not known.,Nerve cells maintained in tissue culture offer a simplified system for studying these electrical parameters, yet these questions are difficult to approach by current techniques because it is hard to record intracellularly from small processes. Extracellular recording from small processes ofcultured neurons has been reported (6-8). However, this technique is inadequate when studying graded potentials or regional variations in the form of the action potential (9, 10). Furthermore, simultaneous measurements of events in different regions within one neuron are very hard with classical recording methods. Some of the limitations inherent in these methods were recently discussed (10).Optical methods for measuring membrane potential by using voltage-sensitive dyes have been developed by Cohen and coworkers (11)(12)(13)(14) and by Waggoner (15) and colleagues. These dyes act as molecular transdu...

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Translocation and turnover of phospholipid analogs in plasma membrane-derived vesicles from cell cultures

Cited by 20 publications

References 28 publications

Inhibitors of CDP-choline synthesis, action potential calcium channels, and stimulus-secretion coupling.

Inhibitors of CDP-choline synthesis, action potential calcium channels, and stimulus-secretion coupling.

PI(4,5)P₂ Degradation Promotes the Formation of Cytoskeleton‐Free Model Membrane Systems

Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons.

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Translocation and turnover of phospholipid analogs in plasma membrane-derived vesicles from cell cultures

Cited by 20 publications

References 28 publications

Inhibitors of CDP-choline synthesis, action potential calcium channels, and stimulus-secretion coupling.

Inhibitors of CDP-choline synthesis, action potential calcium channels, and stimulus-secretion coupling.

PI(4,5)P2 Degradation Promotes the Formation of Cytoskeleton‐Free Model Membrane Systems

Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons.

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PI(4,5)P₂ Degradation Promotes the Formation of Cytoskeleton‐Free Model Membrane Systems