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Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the
T H Steinberg1, A S Newman, J A Swanson
1Rover Laboratory of Physiology, Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, New York 10032.
Abstract:
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.
Insights
Macrophages release fluorescent dyes via probenecid-inhibitable transporters, suggesting a mechanism for exporting organic anions like prostaglandins. This process is energy-dependent and pH-sensitive.
Area of Science:
- Cell Biology
- Immunology
- Membrane Transport
Background:
- Macrophages play a crucial role in immune responses.
- Understanding how macrophages handle and transport molecules is vital.
- Previous research has not identified specific transporters for organic anions in macrophages.
Purpose of the Study:
- To investigate the fate of membrane-impermeant fluorescent dyes introduced into macrophages.
- To identify and characterize the transport mechanisms involved in dye efflux.
- To determine if macrophages possess organic anion transporters.
Main Methods:
- Loading J774 and primary mouse macrophages with fluorescent dyes (Lucifer Yellow, carboxyfluorescein, fura-2) via ATP permeabilization.
- Observing dye localization and release using microscopy.
- Investigating the effects of temperature, probenecid, extracellular pH, and ion co-transport on dye efflux.
Main Results:
- Fluorescent dyes were sequestered in cytoplasmic vacuoles and released extracellularly.
- Lucifer Yellow efflux was temperature-dependent, energy-requiring, and occurred against a concentration gradient.
- Probenecid inhibited Lucifer Yellow efflux in a dose-dependent and reversible manner.
- Efflux was not dependent on Na+ co-transport or Cl- antiport but was inhibited by low extracellular pH.
Conclusions:
- Macrophages possess probenecid-inhibitable transporters for organic anions.
- These transporters exhibit functional similarities to epithelial organic anion transporters.
- This discovery may explain the release of endogenous organic anions from macrophages.