Phosphatidylcholine biosynthesis in cultured glioma cells: evidence for channeling of intermediates

T. P. George; S. C. Morash; H. W. Cook; D. M. Byers; F. B.St C. Palmer; M. W. Spence

doi:10.1016/0005-2760(89)90075-1

Phosphatidylcholine biosynthesis in cultured glioma cells: evidence for channeling of intermediates

T. P. George, S. C. Morash, H. W. Cook, D. M. Byers, F. B.St C. Palmer, M. W. Spence

Medicine

Medicine

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Abstract

The major pathway of choline (Cho) incorporation into phosphatidylcholine (PtdCho) in mammalian cells is sequential conversion of Cho to phosphocholine (PCho), cytidinediphosphate choline (CDP-Cho) and PtdCho. In intact cells, this sequence is usually demonstrated using radiolabeled Cho since PCho and CDP-Cho do not enter the cell intact. We have studied the incorporation of radiolabeled Cho, PCho and CDP-Cho into rat glioma (C6) cells following electropermeabilization. C6 cells were permeable as judged by [U-¹⁴C]sucrose and Erythrosin B uptake and more rapid incorporation of [1,2,3-³H]glycerol into cell lipids, and viable as assessed by uptake and incorporation of [methyl-³H]Cho, [1-¹⁴C]oleate and [1,2,3-³H]glycerol into complex lipids. Despite rapid incorporation of [methyl-³H]Cho into PtdCho in permeabilized cells, there was no incorporation of [methyl-¹⁴C]PCho or CDP-[methyl-¹⁴C]Cho into PtdCho. PCho (300 μM) and CDP-Cho (300 μM) failed to significantly reduce incorporation of 28 μM [methyl-³H]Cho into PtdCho. Radioactivity in PtdCho of cells prelabeled with [methyl-³H]Cho prior to permeabilization could be chased with 4 mM Cho but not with 4 mM PCho or 4 mM CDP-Cho. The water-soluble products of Cho metabolism - PCho, CDP-Cho and glycerophosphocholine - were retained at 37°C in permeabilized cells compared with controls while there was uniform leakage from permeabilized cells at 4°C. Hemicholinium-3, an inhibitor of high-affinity Cho transport, decreased [methyl-³H]Cho incorporation into PtdCho in permeabilized cells, as in controls, suggesting that even in permeabilized cells, Cho incorporation into PtdCho is linked to the transport system. We propose that individual steps of the cytidine pathway of PtdCho biosynthesis are functionally linked and that reaction intermediates are not freely diffusible within the cell but are channeled to PtdCho biosynthesis.

Original language	English
Pages (from-to)	283-291
Number of pages	9
Journal	Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume	1004
Issue number	3
DOIs	https://doi.org/10.1016/0005-2760(89)90075-1
Publication status	Published - Aug 22 1989

Bibliographical note

Funding Information:
This work was supportedb y a ProgramG rant (PG-16), a Pre-doctoralF ellowship( S.C.M.) and a Career Investigator Award (M.W.S.) from the Medical Re-searchC ouncil of Canada and a Natural Sciencesa nd EngineeringR esearchC ouncil (Canada) SummerU n-dergraduateS cholarship( T.P.G.). The expertt echnical assistanceo f Mr Robert Zwicker in culturingc ells and Mr. Jim Baker in constructiono f the electropermeabili-zationa pparatusis gratefullya cknowledged.

ASJC Scopus Subject Areas

Biophysics
Biochemistry
Endocrinology

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10.1016/0005-2760(89)90075-1

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@article{b5a68b1f4a6e4df5841533f02d4b2d2c,

title = "Phosphatidylcholine biosynthesis in cultured glioma cells: evidence for channeling of intermediates",

abstract = "The major pathway of choline (Cho) incorporation into phosphatidylcholine (PtdCho) in mammalian cells is sequential conversion of Cho to phosphocholine (PCho), cytidinediphosphate choline (CDP-Cho) and PtdCho. In intact cells, this sequence is usually demonstrated using radiolabeled Cho since PCho and CDP-Cho do not enter the cell intact. We have studied the incorporation of radiolabeled Cho, PCho and CDP-Cho into rat glioma (C6) cells following electropermeabilization. C6 cells were permeable as judged by [U-14C]sucrose and Erythrosin B uptake and more rapid incorporation of [1,2,3-3H]glycerol into cell lipids, and viable as assessed by uptake and incorporation of [methyl-3H]Cho, [1-14C]oleate and [1,2,3-3H]glycerol into complex lipids. Despite rapid incorporation of [methyl-3H]Cho into PtdCho in permeabilized cells, there was no incorporation of [methyl-14C]PCho or CDP-[methyl-14C]Cho into PtdCho. PCho (300 μM) and CDP-Cho (300 μM) failed to significantly reduce incorporation of 28 μM [methyl-3H]Cho into PtdCho. Radioactivity in PtdCho of cells prelabeled with [methyl-3H]Cho prior to permeabilization could be chased with 4 mM Cho but not with 4 mM PCho or 4 mM CDP-Cho. The water-soluble products of Cho metabolism - PCho, CDP-Cho and glycerophosphocholine - were retained at 37°C in permeabilized cells compared with controls while there was uniform leakage from permeabilized cells at 4°C. Hemicholinium-3, an inhibitor of high-affinity Cho transport, decreased [methyl-3H]Cho incorporation into PtdCho in permeabilized cells, as in controls, suggesting that even in permeabilized cells, Cho incorporation into PtdCho is linked to the transport system. We propose that individual steps of the cytidine pathway of PtdCho biosynthesis are functionally linked and that reaction intermediates are not freely diffusible within the cell but are channeled to PtdCho biosynthesis.",

author = "George, {T. P.} and Morash, {S. C.} and Cook, {H. W.} and Byers, {D. M.} and Palmer, {F. B.St C.} and Spence, {M. W.}",

note = "Funding Information: This work was supportedb y a ProgramG rant (PG-16), a Pre-doctoralF ellowship( S.C.M.) and a Career Investigator Award (M.W.S.) from the Medical Re-searchC ouncil of Canada and a Natural Sciencesa nd EngineeringR esearchC ouncil (Canada) SummerU n-dergraduateS cholarship( T.P.G.). The expertt echnical assistanceo f Mr Robert Zwicker in culturingc ells and Mr. Jim Baker in constructiono f the electropermeabili-zationa pparatusis gratefullya cknowledged.",

year = "1989",

month = aug,

day = "22",

doi = "10.1016/0005-2760(89)90075-1",

language = "English",

volume = "1004",

pages = "283--291",

journal = "Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids",

issn = "0005-2760",

publisher = "Elsevier",

number = "3",

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TY - JOUR

T1 - Phosphatidylcholine biosynthesis in cultured glioma cells

T2 - evidence for channeling of intermediates

AU - George, T. P.

AU - Morash, S. C.

AU - Cook, H. W.

AU - Byers, D. M.

AU - Palmer, F. B.St C.

AU - Spence, M. W.

N1 - Funding Information: This work was supportedb y a ProgramG rant (PG-16), a Pre-doctoralF ellowship( S.C.M.) and a Career Investigator Award (M.W.S.) from the Medical Re-searchC ouncil of Canada and a Natural Sciencesa nd EngineeringR esearchC ouncil (Canada) SummerU n-dergraduateS cholarship( T.P.G.). The expertt echnical assistanceo f Mr Robert Zwicker in culturingc ells and Mr. Jim Baker in constructiono f the electropermeabili-zationa pparatusis gratefullya cknowledged.

PY - 1989/8/22

Y1 - 1989/8/22

N2 - The major pathway of choline (Cho) incorporation into phosphatidylcholine (PtdCho) in mammalian cells is sequential conversion of Cho to phosphocholine (PCho), cytidinediphosphate choline (CDP-Cho) and PtdCho. In intact cells, this sequence is usually demonstrated using radiolabeled Cho since PCho and CDP-Cho do not enter the cell intact. We have studied the incorporation of radiolabeled Cho, PCho and CDP-Cho into rat glioma (C6) cells following electropermeabilization. C6 cells were permeable as judged by [U-14C]sucrose and Erythrosin B uptake and more rapid incorporation of [1,2,3-3H]glycerol into cell lipids, and viable as assessed by uptake and incorporation of [methyl-3H]Cho, [1-14C]oleate and [1,2,3-3H]glycerol into complex lipids. Despite rapid incorporation of [methyl-3H]Cho into PtdCho in permeabilized cells, there was no incorporation of [methyl-14C]PCho or CDP-[methyl-14C]Cho into PtdCho. PCho (300 μM) and CDP-Cho (300 μM) failed to significantly reduce incorporation of 28 μM [methyl-3H]Cho into PtdCho. Radioactivity in PtdCho of cells prelabeled with [methyl-3H]Cho prior to permeabilization could be chased with 4 mM Cho but not with 4 mM PCho or 4 mM CDP-Cho. The water-soluble products of Cho metabolism - PCho, CDP-Cho and glycerophosphocholine - were retained at 37°C in permeabilized cells compared with controls while there was uniform leakage from permeabilized cells at 4°C. Hemicholinium-3, an inhibitor of high-affinity Cho transport, decreased [methyl-3H]Cho incorporation into PtdCho in permeabilized cells, as in controls, suggesting that even in permeabilized cells, Cho incorporation into PtdCho is linked to the transport system. We propose that individual steps of the cytidine pathway of PtdCho biosynthesis are functionally linked and that reaction intermediates are not freely diffusible within the cell but are channeled to PtdCho biosynthesis.

AB - The major pathway of choline (Cho) incorporation into phosphatidylcholine (PtdCho) in mammalian cells is sequential conversion of Cho to phosphocholine (PCho), cytidinediphosphate choline (CDP-Cho) and PtdCho. In intact cells, this sequence is usually demonstrated using radiolabeled Cho since PCho and CDP-Cho do not enter the cell intact. We have studied the incorporation of radiolabeled Cho, PCho and CDP-Cho into rat glioma (C6) cells following electropermeabilization. C6 cells were permeable as judged by [U-14C]sucrose and Erythrosin B uptake and more rapid incorporation of [1,2,3-3H]glycerol into cell lipids, and viable as assessed by uptake and incorporation of [methyl-3H]Cho, [1-14C]oleate and [1,2,3-3H]glycerol into complex lipids. Despite rapid incorporation of [methyl-3H]Cho into PtdCho in permeabilized cells, there was no incorporation of [methyl-14C]PCho or CDP-[methyl-14C]Cho into PtdCho. PCho (300 μM) and CDP-Cho (300 μM) failed to significantly reduce incorporation of 28 μM [methyl-3H]Cho into PtdCho. Radioactivity in PtdCho of cells prelabeled with [methyl-3H]Cho prior to permeabilization could be chased with 4 mM Cho but not with 4 mM PCho or 4 mM CDP-Cho. The water-soluble products of Cho metabolism - PCho, CDP-Cho and glycerophosphocholine - were retained at 37°C in permeabilized cells compared with controls while there was uniform leakage from permeabilized cells at 4°C. Hemicholinium-3, an inhibitor of high-affinity Cho transport, decreased [methyl-3H]Cho incorporation into PtdCho in permeabilized cells, as in controls, suggesting that even in permeabilized cells, Cho incorporation into PtdCho is linked to the transport system. We propose that individual steps of the cytidine pathway of PtdCho biosynthesis are functionally linked and that reaction intermediates are not freely diffusible within the cell but are channeled to PtdCho biosynthesis.

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DO - 10.1016/0005-2760(89)90075-1

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JO - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids

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IS - 3

ER -

Phosphatidylcholine biosynthesis in cultured glioma cells: evidence for channeling of intermediates

Abstract

Bibliographical note

ASJC Scopus Subject Areas

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