title: Similar characteristics of folate analogue transport in vitro in contrast to varying dihydrofolate reductase levels in epithelial cells at different stages of maturation in mouse small intestine.
abstract: We describe studies of folate analogue transport in purified epithelial cell fractions isolated from mouse small intestine. Fractionation of these cells into immature proliferative and mature absorptive components and two components representative of intermediate stages of maturation was carried out by stepwise, nonenzymatic stripping of the everted organ. In contrast to the proliferative-specific enzyme markers, thymidine kinase and dihydrofolate reductase, folate analogue transport did not vary with the alteration in proliferative potential of these cells during maturation. The brush-border enzyme, alkaline phosphatase, was used as a positive marker for maturation. Initial influx of [3H]-aminopterin into both mature and immature cell fractions showed the same kinetics and did not exhibit pH dependence within the range of 6.0 to 7.8. A single saturable component (Km = 16 +/- 3 microM; V37 = 57 +/- 8 pmol/min/10(7) cells) was delineated, with the same temperature dependence (Q10 27-37 degrees = 3.2 +/- 0.4; Arrenhius constant = 11.1 +/- 3 kcal/mol) and same specificity for various folate compounds. Initial efflux of [3H]aminopterin was also similar in both cell types. Efflux was first-order (t1/2 37 degrees = 1.1 to 1.3 min; K37 = 0.53 +/- 0.04 min-1) and equal to the decay-time constant for approach to steady-state during accumulation of [3H]aminopterin, but showed higher-temperature dependence (Q10 27-37 degrees = 6.7 +/- 0.8; Arrenhius constant = 25.3 +/- 4 kcal/mol). Under the conditions used which do not allow polyglutamylation of [3H]aminopterin, steady-state levels of accumulation of exchangeable drug at 37 degrees in each cell fraction were accounted for by the various kinetic parameters for each flux. At all concentrations of [3H]aminopterin examined, both types of epithelial cells appeared to maintain a negative electrochemical gradient under physiological conditions. Overall, the data conform to a two-carrier model for folate analogue transport in which each flux is mediated by a separate system. However, specificity and saturability of influx for folate compounds, and inhibition of this flux by various agents was markedly different from that reported for various tumor cells.
mesh_terms: D000630:Aminopterin / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002455:Cell Division; D004261:DNA Replication; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007421:Intestine, Small / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008727:Methotrexate / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D005493:Folic Acid Antagonists; D013762:Tetrahydrofolate Dehydrogenase; D000630:Aminopterin; C017178:10-deazaaminopterin; D008727:Methotrexate