Summary. The accumulation of 5‐methyl[3H]tetrahydrofolic acid (5CH3[3H]FH4) by phytohaemagglutinin stimulated lymphocytes (PHA‐L) cultured in folate free media was investigated to determine the mechanism of uptake of 5CH3FH4 and the requirement of the cells for this vitamin as assessed by monitoring de novo thymidine synthesis. When grown in 20 nm 5CH3[3H]FH4 PHA‐L accumulate radiolabel at a rate of 0·04 pmol/h/106 cells. This doubles the endogenous folate pool of unstimulated cells (0·6±0·16 pmol/106 cells) in about 15 h. Uptake proceeded via a saturable process, independent of a high affinity folate receptor as assessed by ligand binding and by Northern and Western blot analysis. However, transport was blocked by probenecid, which is consistent with an anion carrier mechanism. Unstimulated cells lacked folylpolyglutamate synthetase (FPGS) activity and did not express significant amounts of FPGS mRNA. After 48 h of mitogen stimulation there was a 4–10‐fold increase in FPGS mRNA and folylpolyglutamate formation (Glu≥5) was essentially simultaneous with 5CH3[3H]FH4 transport. Increasing extracellular folate to 2μm only increased intracellular folate 8‐fold, but the length of the folylpolyglutamates decreased. The increased folate did not increase de novo thymidine synthesis compared to cells grown in physiological folate. We conclude that mitogen stimulation activates the process(es) for folate accumulation, especially FPGS, and that physiological uptake (0·04 pmol/h/106 cells) is adequate for meeting the cells' need for the vitamin.
|Original language||English (US)|
|Number of pages||7|
|Journal||British Journal of Haematology|
|State||Published - Aug 1993|
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