Retentiveness of methotrexate polyglutamates in cultured L1210 cells: Evidence against a role for mediated plasma membrane transport outward Journal Article

  


Authors: Schlemmer, S. R.; Sirotnak, F. M.
Article Title: Retentiveness of methotrexate polyglutamates in cultured L1210 cells: Evidence against a role for mediated plasma membrane transport outward
Abstract: In the present report, studies are described examining the issue of methotrexate (MTX) polyglutamate retentiveness in cultured L1210 cells. Measurements made in intact L1210 cells showed that the rate of egress of [3H]MTX and [3H]MTX + G1 was different depending upon whether [3H]MTX and its polyglutamates were accumulated intracellularly following biosynthesis during growth (3 hr) in the presence of [3H]MTX or when cells were pulse loaded (5 min) with [3H]MTX or [3H]MTX + G1 just prior to measurement of egress. In the former case, [3H]MTX egressed with a T1 2 of 15 +- 2 min, while [3H]MTX + G1 egressed with a T1 2 of 50 ± 9 min. In pulse loaded cells, both [3H]MTX and [3H]MTX + G1 egressed with a T1 2 of 3.5 +- 0.5 min. The same rapid egress of [3H]MTX and [3H]MTX + G1 seen following pulse loading was also documented in cells grown for 3 hr in the presence of nonradioactive MTX to normalize conditions with respect to the intracellular accumulation of [3H]MTX polyglutamates seen during exposure of cells to [3H]MTX during growth. In light of these results, MTX, MTX + G1, MTX + G2 and MTX + G4 were examined directly as permeants for the outwardly-directed ATP-dependent pump (Schlemmer SR and Sirotnak FM, J Biol Chem 267: 14746-14752, 1992) mediating most of MTX efflux in intact L1210 cells using inside-out plasma membrane vesicles isolated from these cells. ATP-dependent efflux of [3H]MTX and [3H]MTX + G1 exhibited values for Km of 46-50 μM and values for Vmax of 102-106 pmol/min/mg protein. As competitive inhibitors of [3H]MTX and [3H]MTX + G1 efflux, MTX + G1 and MTX, respectively, exhibited Ki values of 43-47 μM, that is, Km ≈ Ki for both permeants. Also, values for Ki of 45-48 μM were obtained with MTX + G2 and MTX + G4 as competitive inhibitors of [3H]MTX efflux. From these results, we conclude that MTX and its polyglutamates are equivalent as copermeants for ATP-dependent efflux through the plasma membrane and that retentiveness of MTX polyglutamates is not determined at this level in these cells. © 1993.
Keywords: controlled study; nonhuman; comparative study; methotrexate; animal cell; mouse; animal; tumor cells, cultured; kinetics; drug accumulation; drug retention; cell culture; cell membrane; drug metabolism; radioisotope; adenosine triphosphate; drug transport; biological transport; concentration response; tritium; polyglutamic acid; leukemia l1210; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.; leukemia l 1210; methotrexate polyglutamate
Journal Title: Biochemical Pharmacology
Volume: 45
Issue: 6
ISSN: 0006-2952
Publisher: Elsevier Inc.  
Date Published: 1993-03-24
Start Page: 1261
End Page: 1266
Language: English
DOI: 10.1016/0006-2952(93)90278-5
PUBMED: 7682074
PROVIDER: scopus
DOI/URL:

https://www.scopus.com/inward/record.uri?eid=2-s2.0-0027447890&doi=10.1016%2f0006-2952%2893%2990278-5&partnerID=40&md5=594f892cd682aef156b8620753d75325
Notes: Source: Scopus
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MSK Authors
  1. Francis M Sirotnak
    184 Sirotnak
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