title: Fibroblasts from methotrexate-sensitive mice accumulate methotrexate polyglutamates but those from methotrexate-resistant mice do not.
abstract: OBJECTIVE
  We and others have previously demonstrated that methotrexate (MTX) mediates its anti-inflammatory effects through an increase in cellular release of adenosine. Consistent with this observation, there is no increase in adenosine from exudates of mouse strains resistant to MTX. Because intracellular MTX polyglutamates inhibit AICAR transformylase (ATIC) activity and thereby promote adenosine release we determined whether there is any difference in the capacity of cells from MTX-resistant mice to accumulate MTX polyglutamates.
  
  
  METHODS
  Dermal fibroblasts (DF) from BALBc, MTX-sensitive, and DBA/1J, MTX-resistant, mice were cultured in the presence or absence of MTX. Adenosine concentration in the supernatant and intracellular MTX polyglutamate (MTXPG1-5) concentrations were measured by liquid chromatography. ATIC activity in DF was monitored spectrophotometrically by the formation of formytetrahydrofolate.
  
  
  RESULTS
  MTX (1 μM) increased adenosine production by DF from BALBc sensitive-mice from 269±40 nM to 446±4 nM. No adenosine production was found in supernates of cultured DF from DBA/1J mice regardless of MTX treatment. Intracellular MTX polyglutamates (MTXPG2-4) were detected only in BALBc DFs, not in DBA/1J DF. Further investigation demonstrated that ATIC activity was inhibited following MTX treatment in DF from BALBc mice.
  
  
  CONCLUSIONS
  These data suggest that resistance to the anti-inflammatory effects of MTX could be due to diminished MTX polyglutamate accumulation resulting in diminished ATIC inhibition and adenosine accumulation.
authors: Division of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital, Beijing, China. xn_you@yahoo.com
authors: Xin
authors: X
authors: You
authors: Adrienne
authors: A
authors: Williams
authors: Thierry
authors: T
authors: Dervieux
authors: Wenjie
authors: W
authors: He
authors: Bruce N
authors: BN
authors: Cronstein
mesh_terms: D000241:Adenosine / Q000276:immunology / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008811:Mice, Inbred DBA; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase / Q000037:antagonists & inhibitors*; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D011099:Polyglutamic Acid; D000249:Adenosine Monophosphate; C014085:methotrexate polyglutamate; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D000241:Adenosine; D008727:Methotrexate