monensin and 5-methyltetrahydrofolate

Loss of folate receptor-α function is associated with cerebral folate transport deficiency and childhood-onset neurodegeneration. To clarify the mechanism of cerebral folate transport at the blood-cerebrospinal fluid barrier, we investigate the transport of 5-methyltetrahydrofolate in polarized cells. Here we identify folate receptor-α-positive intralumenal vesicles within multivesicular bodies and demonstrate the directional cotransport of human folate receptor-α, and labelled folate from the basolateral to the apical membrane in rat choroid plexus cells. Both the apical medium of folate receptor-α-transfected rat choroid plexus cells and human cerebrospinal fluid contain folate receptor-α-positive exosomes. Loss of folate receptor-α-expressing cerebrospinal fluid exosomes correlates with severely reduced 5-methyltetrahydrofolate concentration, corroborating the importance of the folate receptor-α-mediated folate transport in the cerebrospinal fluid. Intraventricular injections of folate receptor-α-positive and -negative exosomes into mouse brains demonstrate folate receptor-α-dependent delivery of exosomes into the brain parenchyma. Our results unravel a new pathway of folate receptor-α-dependent exosome-mediated folate delivery into the brain parenchyma and opens new avenues for cerebral drug targeting.

Topics: Adolescent; Adult; Animals; Cell Polarity; Child; Choroid Plexus; Cytoplasmic Vesicles; Dogs; Exosomes; Female; Folate Receptor 1; Folic Acid; Humans; Madin Darby Canine Kidney Cells; Male; Mice; Models, Biological; Monensin; Protein Transport; Proton-Coupled Folate Transporter; Rats; Tetrahydrofolates; Transcytosis; Transferrin; Young Adult

Previously we showed that the potocytosis of 5-methyltetrahydrofolate is dependent on the acidification of plasmalemmal vesicles created each time a caveolae, closes off from the cell surface. We now report that bafilomycin A1, which is a specific inhibitor of the V-type proton ATPase, inhibits 5-methyltetrahydrofolate uptake into MA104 cells (ED50 = 150 nM). The inhibitory effect was reversed within 30 min after removal of the drug from the cells. Bafilomycin A1 had no effect on the binding of folic acid to its receptor. A concentration of up to 200 nM bafilomycin A1 did not affect sequestration of folate receptors. Immunoblotting showed that the 70-kDa subunit of the V-type proton pump was localized to caveolae-rich fractions isolated from the plasma membrane of these cells. These results suggest that a V-type proton pump acidifies the lumen of plasmalemmal vesicles during potocytosis.

Topics: Acids; Ammonium Chloride; Animals; Anti-Bacterial Agents; Antimalarials; Biological Transport; Cell Compartmentation; Cell Line; Cell Membrane; Chloroquine; Enzyme Inhibitors; Epithelial Cells; Epithelium; Folic Acid; Haplorhini; Intracellular Membranes; Ionophores; Kidney; Macrolides; Monensin; Octoxynol; Oligomycins; Proton-Translocating ATPases; Protons; Tetrahydrofolates; Vacuoles

MA104 cells, as well as several other rapidly dividing tissue culture cells, have a folate-binding protein associated with their cell surface. The protein has the properties of a membrane receptor: (a) 5-methyl[3H]tetrahydrofolic acid binds with high affinity (Kd approximately equal to 3 nM); (b) the protein is an integral membrane protein; (c) it appears to deliver physiological concentrations of 5-methyl[3H]tetrahydrofolic acid to the inside of the cell; (d) binding activity is regulated by the concentration of folate within the cell. To better understand the mechanism of action of this receptor, we have studied the pathway of folate internalization. We present evidence that during internalization: (a) folate binds to the membrane receptor; (b) the ligand-receptor complex moves into the cell; (c) the ligand is released from the receptor in an acidic intracellular compartment and moves into the cytoplasm; and (d) the unoccupied receptor returns to the cell surface.

Topics: Ammonium Chloride; Animals; Binding, Competitive; Carrier Proteins; Cell Line; Cell Membrane; Chloroquine; Chromatography, Gel; Chromatography, High Pressure Liquid; Cytoplasm; Epithelium; Folate Receptors, GPI-Anchored; Folic Acid; Kinetics; Monensin; Receptors, Cell Surface; Tetrahydrofolates; Tritium