The mechanism of folate transport in rabbit reticulocytes

Abstract

A B S T R A C T Folate transport in phenylhydrazine-induced rabbit reticulocytes was studied with the nonmetabolized folate-analog, methotrexate. The time-course of methotrexate uptake into a mixed population of reticulocytes and mature erythrocytes is a two-component process consisting of a small, but rapid, initial uptake phase followed by a much slower uptake component which remains essentially constant over the period of observation. The velocity of the latter uptake component is directly proportional to the per cent reticulocytes and appears to represent a unidirectional influx of methotrexate into these cells. Uptake of methotrexate into reticulocytes was found to have the following characteristics: (a) temperature sensitivity, Qio of 4; (b) uptake velocity as a function of the extracellular methotrexate concentration approximated Michaelis-Menten kinetics with a maximum transport velocity of 48 pmoles/ min per g dry wt; the extracellular methotrexate level at which the uptake velocity was one-half maximum was 1.4 FM; (c) 5-formyltetrahydrofolate markedly inhibited methotrexate uptake but pteroylglutamic acid inhibition was weak; (d) uptake was stimulated in cells preincubated with 5-formyltetrahydrofolate, indicative of heteroexchange diffusion; (e) uptake was independent of extracellular sodium but was inhibited by anions including nitrate, phosphate, and glucose-6-phosphate; (f) uptake was enhanced by azide plus iodoacetate.These data indicate that folate transport in rabbit reticulocytes is mediated by a carrier mechanism which disappears with reticulocyte maturation. The mechanism of folate transport in rabbit reticulocytes is qualitatively similar to tumor cells previously studied; both appear to have an energy-dependent mechanism limiting folate uptake, and influx in both is .inhibited by structurally unrelated inorganic and organic anions. These studies suggest that circulating pteroylglutamic acid is of little importance in meeting the folate requirements of folate-

The Mechanism of 5-Methyltetrahydrofolate Transport by Human Erythrocytes

“…Uptake of 5-CH3-H4-folate was by erythrocytes, rather than other formed elements, because uptake did not change after addition or depletion of leukocytes and platelets, and was nearly identical in erythrocytes from normal individuals and in cells from patients with profound leukopenia, reticulocytopenia, and thrombocytopenia. Although the possibility must be considered that the observed uptake is the result of the few remaining reticulocytes (4,17), extrapolation of the linear relationship between uptake and reticulocyte count to zero reticulocytes predicts an uptake by mature erythrocytes comparable to our measured values. Thus, uptake in these studies is probably not significantly influenced by the small number of reticulocytes present.…”

“…Folate transport in mature human erythrocytes has not been extensively studied. We present evidence that mature erythrocytes transport-5-CH3-H4-folate by a carrier-mediated process similar to that found for folate and folate analogues in other normal and neoplastic cells (3)(4)(5)(6)(7)(8).…”

“…Uptake of 5-CH3-H4-folate by human erythrocytes increases after ATP depletion, thereby resembling Mtx uptake by rabbit reticulocytes (4) and L-1210 leukemia cells (19,27). However, in contrast to these experimental systems, wherein metabolic poisons apparently inhibit an energy-dependent efflux mechanism without altering influx (resulting in a net increase of uptake) (4, 19), we found efflux rate constants in control and F/CN-treated human erythrocytes were nearly identical.…”

Loss of Folic Acid Exporter Function with Markedly Augmented Folate Accumulation in Lipophilic Antifolate-resistant Mammalian Cells

“…After centrifugation (750 ϫ g for 2 min) at 4°C, cells were resuspended in ice-cold HBS, washed twice in the same buffer, and processed for determination of intracellular radiolabel with normalization to dry weight (22). Intracellular water of AA8 and Pyr R100 cells was determined using the membrane-impermeable [carboxyl- 14 C]inulin as the extracellular marker as described elsewhere (24). Efflux measurements were performed by first loading cells with [ Measurement of Plasma Membrane Potential-We have employed the method of equilibrium distribution of the lipid-soluble cation TPP ϩ across the plasma membrane to determine the membrane potential of AA8 and Pyr R100 cells (25).…”

Evidence for a Folic Acid Binding Protein in Human Cell Membrane

“…Its storage appears to be facilitated by folic acid binding proteins (FABP) inside the hé patocyte [5,21], In other cells such as leu kocytes and reticulocytes the presence of a 1 Supported by a grant from CNR 80.01524.96.…”