Abstract
Cells of the J774 mouse macrophage-like cell line possess organic anion transporter that transport fluorescent dyes such as Lucifer Yellow out of the cytoplasmic matrix of the cells; the dye is both sequestered in endosomes and secreted into the extracellular medium. Lucifer Yellow that is sequestered within endosomes is subsequently delivered to the lysosomal compartment. In the present studies we demonstrated that probenecid inhibited removal of Lucifer Yellow from the soluble cytoplasm and sequestration into membrane bound organelles by quantitating Lucifer Yellow fluorescence in both soluble and membrane-associated fractions of J774 cells. In addition, we examined the uptake of Lucifer Yellow into isolated subcellular organelles derived from J774 cells. Lucifer Yellow transport in the organellar fraction of J774 cell homogenates was temperature- and pH-dependent and did not require ATP. Subcellular organelles from J774 cells were fractionated into endosome- and lysosome-enriched fractions by Percoll density gradient centrifugation. Lucifer Yellow was preferentially taken up by vesicles of the endosome-enriched fraction, and this transport was inhibited by probenecid. These studies provide direct evidence that probenecid inhibits Lucifer Yellow transport out of the cytoplasmic matrix and into cytoplasmic vacuoles in J774 cells and that organic anion transport in isolated organelles derived from J774 cells occurs preferentially in endosome, rather than in lysosome-enriched fractions; they suggest that Lucifer Yellow is carried across membranes via a secondary active transport process that requires proton symptom or hydroxyl anion antiport.
Highlights
Of Medicine and the §Rover Physiology Laboratories, College of Physicians and Surgeons, New York, New Diseases, Washington University School of Medicine, Steinberg$lIli
Cellular Biophysics, of Medicine, Cells of the 5774 mouse macrophage-like cell line possess organic anion transporters that transport fluorescent dyes such as Lucifer Yellow out of the cytoplasmic matrix of the cells; the dye is both sequestered in endosomes and secreted into the extracellular medium
These studies provide direct evidence that probenecid inhibits Lucifer Yellow transport out of the cytoplasmic matrix and into cytoplasmic vacuoles in 5774 cells and that organic anion transport in isolated organelles derived from 5774 cells occurs preferentially in endosome, rather than in lysosome-enriched fractions; they suggest that Lucifer Yellow is carried across membranes via a secondary active transport process that requires proton symport or hydroxyl anion antiport
Summary
Of Medicine and the §Rover Physiology Laboratories, College of Physicians and Surgeons, New York, New Diseases, Washington University School of Medicine, Steinberg$lIli. Lucifer Yellow was preferentially taken up by vesicles of the endosome-enriched fraction, and this transport was inhibited by probenecid These studies provide direct evidence that probenecid inhibits Lucifer Yellow transport out of the cytoplasmic matrix and into cytoplasmic vacuoles in 5774 cells and that organic anion transport in isolated organelles derived from 5774 cells occurs preferentially in endosome, rather than in lysosome-enriched fractions; they suggest that Lucifer Yellow is carried across membranes via a secondary active transport process that requires proton symport or hydroxyl anion antiport. Macrophages secrete a number of inflammatory mediators and metabolites Some of these, such as leukotrienes, prostaglandins, glutathione, bilirubin, and lactate, are organic anions that must cross a cellular membrane in their exodus from the macrophage cytoplasmic matrix to the extracellular environment. We have shown that mouse peritoneal macrophages and cells of the 5774 mouse macrophage-like cell line transport water-soluble an-
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