We introduced several membrane-impermeant fluorescent dyes, including Lucifer Yellow, carboxyfluorescein, and fura-2, into the cytoplasmic matrix of J774 cells and thioglycollate-elicited mouse peritoneal macrophages by ATP permeabilization of the plasma membrane and observed the subsequent fate of these dyes. The dyes did not remain within the cytoplasmic matrix; instead they were sequestered within phase-lucent cytoplasmic vacuoles and released into the extracellular medium. We used Lucifer Yellow to study these processes further. In cells incubated at 37 degrees C, 87% of Lucifer Yellow was released from the cells within 30 min after dye loading. The dye that remained within the cells at this time was predominantly within cytoplasmic vacuoles. Lucifer yellow transport was temperature dependent and occurred against a concentration gradient; therefore it appeared to be an energy-requiring process. The fluorescent dyes used in these studies are all organic anions. We therefore examined the ability of probenecid (p-[dipropylsulfamoyl]benzoic acid), which blocks organic anion transport across many epithelia, to inhibit efflux of Lucifer Yellow, and found that this drug inhibited this process in a dose-dependent and reversible manner. Efflux of Lucifer Yellow from the cells did not require Na+ co-transport or Cl- antiport; however, it was inhibited by lowering of the extracellular pH. These experiments indicate that macrophages possess probenecid-inhibitable transporters which are similar in their functional properties to organic anion transporters of epithelial cells. Such organic anion transporters have not been described previously in macrophages; they may mediate the release of naturally occurring organic anions such as prostaglandins, leukotrienes, glutathione, bilirubin, or lactate from macrophages.
Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix.
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T H Steinberg, A S Newman, J A Swanson, S C Silverstein; Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix.. J Cell Biol 1 December 1987; 105 (6): 2695–2702. doi: https://doi.org/10.1083/jcb.105.6.2695
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