Macrophages secrete a host of molecules which are important in biological processes such as inflammation and immunity. A number of these molecules, notably prostaglandins and leukotrienes, are not prepackaged in exocytic granules; they are secreted across the plasma membrane, but this process has not been characterized. By following the disposition of fluorescent anionic dyes such as lucifer yellow after they are ATP-loaded into macrophages, I found that macrophages possess organic anion transport systems which may mediate the secretion of these substances. Lucifer yellow does not remain within the cells' cytoplasm, but is sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. These processes appear to be active transport processes, and are blocked by probenecid, which inhibits the transport of organic anions across many epithelia.
The aim of the proposed studies is to characterize organic anion transport in macrophages. Specific approaches include: 1) Determining whether probenecid-inhibitable organic anion transport mediates secretion of biologically relevant molecules such as leukotriene C4, prostaglandin E2, glutathione, and lactate in macrophages. 2) Reconstituting organic anion transport in cell homogenates and in isolated organellar fractions. 3) selecting J774 cells variant in organic anion transport by performing fluorescence activated cell sorting on cells loaded with lucifer yellow. 4) isolating and purifying the organic anion transporter after labelling it with radiolabelled, fluorescent, and photo- affinity substrates. Because this transporter can be specifically inhibited by a relatively non-toxic drug, probenecid, these studies may have clinical relevance for the control of inflammatory processes.
|Loike, J D; Kaback, E; Silverstein, S C et al. (1993) Lactate transport in macrophages. J Immunol 150:1951-8|
|Cao, C X; Silverstein, S C; Neu, H C et al. (1992) J774 macrophages secrete antibiotics via organic anion transporters. J Infect Dis 165:322-8|
|Beyer, E C; Steinberg, T H (1991) Evidence that the gap junction protein connexin-43 is the ATP-induced pore of mouse macrophages. J Biol Chem 266:7971-4|
|Di Virgilio, F; Steinberg, T H; Silverstein, S C (1989) Organic-anion transport inhibitors to facilitate measurement of cytosolic free Ca2+ with fura-2. Methods Cell Biol 31:453-62|
|Steinberg, T H; Silverstein, S C (1989) ATP permeabilization of the plasma membrane. Methods Cell Biol 31:45-61|
|Verma, J N; Goldfine, H (1985) Phosphatidylserine decarboxylase from Clostridium butyricum. J Lipid Res 26:610-6|