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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 microM) and CDP-Cho (300 microM) failed to significantly reduce incorporation of 28 microM [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 degrees C in permeabilized cells compared with controls while there was uniform leakage from permeabilized cells at 4 degrees 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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