Organic Anion Transport in Macrophage Membrane Vesicles*

Cells of the 5774 mouse macrophage-like cell line possess organic anion transporters that transport fluorescent dyes such as Lucifer Yellow out of the cyto- plasmic matrix of the cells; the dye is both sequestered in endosomes and secreted into the extracellular me- dium. Lucifer Yellow that is sequestered within endo- somes 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 5774 cells. In addition, we examined the uptake of Lucifer Yellow into isolated subcellular or- ganelles derived from cells. Lucifer Yellow transport in the organellar fraction of

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.
The mechanisms by which these organic anions cross macrophage membranes are unknown. We have shown that mouse peritoneal macrophages and cells of the 5774 mouse macrophage-like cell line transport water-soluble an- ionic fluorescent dyes such as Lucifer Yellow from the cells' cytoplasmic matrix. Although Lucifer Yellow is membraneimpermeant, it can be introduced into the cytoplasmic matrix of mouse macrophages or 5774 cells by reversible permeabilization of their plasma membrane with ATP4-(1). Dye introduced into the cytoplasmic matrix by this method does not remain in this location: it is sequestered within endosomal vacuoles (2) and secreted into the extracellular medium (3). Some of the Lucifer Yellow sequestered within endosomal vacuoles is subsequently delivered to lysosomes. Lucifer Yellow sequestration and secretion appear to be active transport processes and are blocked by probenecid and sulfinpyrazone, compounds which inhibit organic anion transport in polarized epithelia (3

Probenecid Inhibits Secretion and Vacuolar Sequestration of Lucifer Yellow in J774 Cells-We
showed previously that 5774 cells both sequester Lucifer Yellow within endosomes and secrete the dye into the extracellular medium and that probenecid inhibits both sequestration and secretion of the dye (2,3). In the present studies, we have directly quantitated dye sequestration by cellular organelles. To accomplish this, 5774 cells were loaded with Lucifer Yellow by ATP permeabilization, allowed to reseal, and incubated in Lucifer Yellowfree medium at 37 "C for varying intervals in the presence or absence of 5 mM probenecid. The cells were homogenized and the large membrane-bound organelles ("organellar fraction") were separated from the soluble portion ("cytosolic fraction") of the postnuclear supernatant by centrifugation at 20,000 x g for 10 min. We then measured Lucifer Yellow fluorescence associated with the total cell homogenate and with various subcellular fractions. Cells incubated in the absence of probenecid rapidly secreted Lucifer Yellow into the surrounding medium as demonstrated by a rapid decrease in Lucifer Yellow content of the cell homogenate, whereas cells incubated in the presence of prnbenecid did not demonstrate a decrease in Lucifer Yellow content (Fig. 1). The cytosolic fractions of cells incubated in the absence of probenecid also demonstrated a rapid decline in Lucifer Yellow content at the same time that the organellar fraction from these cells accumulated dye. In contrast, both the cell homogenate and the cytosolic fraction of cells incubated in medium containing probenecid showed no change in Lucifer Yellow content during this period; moreover, their organellar fractions did not demonstrate an increase in Lucifer Yellow content, even though at the times studied the cytoplasm of the probenecid-treated cells contained a higher concentration of dye than did the cytoplasm of cells incubated in the absence of probenecid.
This experiment provided quantitative evidence that 5774 cells simultaneously sequester Lucifer Yellow into membranebound vacuoles and clear the dye from the cytoplasmic matrix and that probenecid inhibits these processes. In addition, it confirmed that Lucifer Yellow sequestering vacuoles could be isolated from 5774 cells without being disrupted. That pro- benecid blocked Lucifer Yellow sequestration into the organellar fraction shows that dye uptake is not an artefact of the homogenization procedure. Lucifer Yellow Sequestration in Cell Homogenates is Temperature-dependent--Lucifer YellOw secretion from intact cells is markedly reduced at 4 "C (3). To determine whether isolated cellular organelles accumulate Lucifer Yellow and whether this uptake is temperature-dependent, 5774 cells were homogenized, the nuclei were removed by centrifugation, and the postnuclear supernatant was centrifuged at 20,000 X g for 10 min at 4 "C to sediment large membrane bound vesicles. These vesicles were incubated in KC1 buffer containing 0.5 mg/ml Lucifer Yellow at 4 or 37 "C. The samples then were centrifuged, and the Lucifer Yellow content of the sedimented organelles was measured. The Lucifer Yellow content of samples incubated at 37 "C increased with time (Fig. 2). The most rapid increased in the uptake of Lucifer Yellow into vacuoles occurred during the first 5-10 min, with a much slower increase occurring thereafter. There was little Lucifer Yellow uptake into the organellar fraction at 4 "C. The small amount of Lucifer Yellow uptake that did occur at 4 "C exhibited linear kinetics and did not reach the same level as that observed at 37 "C, even after 120 min of incubation (Fig. 2). This suggests that Lucifer Yellow uptake at 4 "C was due to diffusion. The difference between the uptake that occurred at 37 and at 4 "C yielded an estimate of the amount of Lucifer Yellow carried by specific membrane transporters (Fig. 2, broken line).
We performed two kinds of experiments to confirm that the Lucifer Yellow associated with sedimentable vesicles in the studies described above was due to transport of Lucifer Yellow into a membrane bound compartment and not merely The postnuclear supernatant of 5774 cells was incubated with 0.5 mg/ml-Lucifer Yellow (LY) at either 37 or 4 "C for the indicated times. Organelles contained in this fraction were washed in cold KC1 homogenization buffer (pH 7.4) three times by sedimentation, solubilized in 0.05% Triton X-100, and analyzed spectrofluorometrically.
The Lucifer Yellow content was expressed as nanograms of Lucifer Yellow/mg of protein. l ,37 "C; H, 4 "C; broken line, 37 "C -4 "C.
to membrane binding of the dye. First, we examined the effects of repeatedly freezing and thawing the samples on their retention of Lucifer Yellow. Vesicles derived from the postnuclear supernatant were incubated with Lucifer Yellow as described above and washed twice. One aliquot of the sedimented vesicles was solubilized in 2 ml of 0.05% Triton X-100, whereas a second aliquot was resuspended in KC1 buffer, frozen in liquid nitrogen three times, and centrifuged at 20,000 X g for 10 min at 4 "C. The sedimented material was solubilized in 2 ml of 0.05% Triton X-100 and its Lucifer Yellow content compared with that of the vesicles pelleted without being subjected to freezing and thawing. The Lucifer Yellow content of the vesicles subjected to repeated freezethaw cycles was reduced by 80% compared to the Lucifer Yellow content of the other samples. Second, we exposed membrane vesicles to hypotonic medium and quantitated the release of Lucifer Yellow from these vesicles. Vesicles derived from the postnuclear supernatant were incubated with Lucifer Yellow as above, washed at 4 "C, and resuspended either in KC1 buffer or in dilute (1.5 mM) KC1 buffer. We then centrifuged the vesicles and quantitated their Lucifer Yellow content. The Lucifer Yellow content of vesicles that were exposed to hypotonic medium was reduced by 76% compared to vesicles that had been resuspended in isotonic medium. These two experiments suggest that the bulk of the sequestered Lucifer Yellow was contained in membrane bound vesicles and was not merely bound to their membranes.
We also examined by fluorescence microscopy samples of the organellar fraction that had been incubated at 37 "C with Lucifer Yellow before and after freezing and thawing. Examination of the homogenate prior to freeze-thawing revealed numerous vesicular structures of heterogenous size containing fluorescent dye (not shown). These fluorescent vesicles were not seen after freezing and thawing. This provided morphological confirmation that the Lucifer Yellow incorporated into the organellar fraction in cell homogenates was contained within membrane delimited compartments.
Lucifer Yellow Sequestration in Cell Homogenates Is pHdependent-Lucifer Yellow efflux from intact 5774 cells is inhibited by acidification of the extracellular medium, and is maximal when the cells are incubated in medium with pH 2 7.5 (3). To determine whether Lucifer Yellow transport into cytoplasmic vesicles is also pH-sensitive, we measured the effect of pH on Lucifer Yellow uptake into these organelles. The postnuclear supernatant of 5774 cells was incubated in medium containing 0.5 mg/ml Lucifer Yellow at pH 6.2, 7.4, or 8.6 (Fig. 3). There was a marked increase in the quantity of Lucifer Yellow recovered in the organellar fraction as the pH was reduced. Vesicles incubated at pH 6.2 took up four times more Lucifer Yellow than did vesicles incubated at pH 7.4 and six times more dye than vesicles incubated at pH 8.6.
This result is consistent with the inhibitory effect of acidifying the extracellular medium on Lucifer Yellow efflux from intact cells. In both intact cells and isolated organelles Lucifer Yellow transport occurred in the same direction as the proton gradient. In intact cells incubated in alkaline medium this gradient is directed outward; lowering the external pH decreases this gradient and results in less Lucifer Yellow efflux from the cells. In vesicles isolated in neutral buffer and incubated in acidic medium, the pH gradient was directed inward, raising the pH in the medium surrounding the vesicles diminished the inward-directed proton gradient and also diminished Lucifer Yellow transport. This suggests that Lucifer Yellow transport in both the intact cell homogenate is dependent on H+ symport or OH-antiport.
To determine whether ATP stimulates Lucifer Yellow uptake into cytoplasmic vacuoles, the postnuclear supernatant from 2 X 10' 5774 cells was centrifuged at 20,000 X g for 10 min at 4 "C, and the pelleted organelles were resuspended in KC1 buffer. Aliquots of this suspension were incubated in KC1 buffer containing 0.5 mg/ml Lucifer Yellow in the presence or absence of 1 mM MgATP for 10 min at 37 "C, centrifuged at 10,000 X g for 10 min at 4 "C, and the amount of Lucifer Yellow taken up into the sedimented organelles was assayed fluorometrically.
An equal amount of Lucifer Yellow was incorporated into organelles incubated in the presence and absence of ATP, indicating that Lucifer Yellow transport into these vesicles did not require ATP.
Lucifer Yellow Transport in Endosome-and Lysosome-enriched Fractions-In intact 5774 cells, Lucifer Yellow is first transported from the cytoplasmic matrix into a prelysosomal vacuolar compartment and is subsequently transferred from this compartment to lysosomes (2). This prelysosomal compartment possesses many of the characteristics of endosomes. Lysosomes and endosomes can be separated from each other on Percoll density gradients because the buoyant density of lysosomes is greater than that of endosomes (8). To determine whether there are differences in Lucifer Yellow transport into endosomes and lysosomes, we separated the post nuclear supernatant fraction of 5774 cells into endosome-and lysosome-enriched fractions and studied dye transport into the membrane bound organelles contained in each of them. The postnuclear supernatant of 5774 cells was incubated in KC1 buffer containing 0.5 mg/ml Lucifer Yellow (LY) at pH 6.2, 7.4, or 8.6 for 15 min at 37 "C. Organelles contained in this fraction were washed in cold KC1 buffer of the same pH three times, solubilized with Triton X-100, and their Lucifer Yellow content was measured. on a 27% Percoll density gradient and centrifuged for 60 min at 15,000 x g. The resulting endosome-and lysosome-enriched bands were collected and centrifuged for 60 min at 100,000 x g to sediment the Percoll. The organelles formed a layer on top of the Percoll pellet. They were resuspended in KC1 buffer for further studies.
The morphological characteristics of the two fractions were examined by electron microscopy (Fig. 4). The less dense fraction (r = 1.04 g/ml) contained membrane-bound vacuoles with electron-lucent interiors that were heterogenous in size. A few strips of rough endoplasmic reticulum were occasionally seen in this fraction. The fractions of greater density (r = 1.08 g/ml) consisted of a homogenous population of membrane-delimited structures possessing electron dense interiors characteristic of lysosomes.
In a separate experiment, the postnuclear supernatant from 1.5 x 10' 5774 cells was layered on a 27% Percoll gradient and centrifuged for 60 min at 15,000 x g. One ml fractions were collected and each fraction was assayed for the presence of enzymatic markers of plasma membrane (alkaline phosphodiesterase I), mitochondria (cytochrome c oxidase), and lysosomes (@-glucuronidase) (Fig. 5). The peak of alkaline phosphodiesterase activity was located in the less dense fractions. Although a small proportion of the total @-glucuronidase activity was in these fractions as well, the major peak of &glucuronidase activity was located in the fractions of greatest buoyant density and accounted for >90% of the total @glucuronidase activity. Cytochrome c oxidase activity was distributed in a broad band throughout the fractions of intermediate buoyant density.
From the distribution of the enzyme markers and the morphological characteristics of the fractions observed by electron microscopy, we concluded that the lysosomes were contained in the fractions of greatest buoyant density ("lysosomal fractions") and that the plasma membrane derived and FM;. 4 early endocytic vesicles were located in the fractions of least buoyant density ("endosome-enriched fractions"). These conclusions are consistent with the work of Galloway et al. (8).
Lucifer Yellow transport was studied in the endosome-and lysosome-enriched fractions of 5774 cells. Samples were incubated in medium containing 0.5 mg/ml Lucifer Yellow at pH 7.5 and 37 'C for various times and the membrane associated fluorescence determined. At 5 and 15 min there was significantly less uptake of Lucifer Yellow into the lysosomal fraction compared with the endosome-enriched fraction (Fig.   6).
The buoyant density of vacuoles sequestering Lucifer Yellow also was assessed in another manner. 5774 cells were homogenized and the postnuclear supernatant was incubated with 0.5 mg/ml Lucifer Yellow at 37 "C for 5 min. The membrane-bound organelles contained in the postnuclear supernatant were washed twice in KC1 homogenization buffer by sedimentation, layered on a 27% Percoll density gradient, and centrifuged at I5,OOO x g for 60 min, all at 4 'C. One-ml fractions were collected from the bottom of the tube, and the Percoll was removed from each sample by ultracentrifugation (100,000 X g X 60 min). The pellet from each sample was solubilized in 2 ml of 0.05% Triton X-100, and its Lucifer Yellow content was measured (Fig. 5). Almost all of the Lucifer Yellow was contained within the fractions of lowest density, coincident with the alkaline phosphodiesterase activity.
Probenecid Inhibits Lucifer Yellow Transport in Endosomeenriched Subcellular Fractions-5 mM probenecid markedly inhibits Lucifer Yellow sequestration and secretion in intact cells (3). Studies described above using the postnuclear supernatant fraction of 5774 cells demonstrated that Lucifer Yellow uptake was inhibited by 5 mM probenecid.
We also assessed the effect of probenecid on the sequestration of Lucifer Yellow into the endosome-enriched fraction of 5774 cell homogenates. The endosome-enriched fraction was collected and incubated with 0.5 mg/ml Lucifer Yellow at 37 "C in the presence or absence of 5 mM probenecid (pH 7.5) (Fig. 7). In the absence of probenecid, Lucifer Yellow uptake averaged 311 f 82 ng of Lucifer Yellow/mg of protein at 5 min and 517 f 77 ng of Lucifer Yellow/mg of protein at 15 min. In the presence of 5 mM probenecid, uptake at 5 min averaged 162 f 44 ng of Lucifer Yellow/mg of protein and at 15 min, 293 + 83 ng of Lucifer Yellow/mg of protein. This represents a 40% reduction in Lucifer Yellow transport in the presence of probenecid.

DISCUSSION
Organic anion transport has been studied primarily in polarized epithelia, including renal proximal tubular cells (9) and hepatocytes (10). In renal proximal tubular cells, organic anions such as urate and p-aminohippurate are transported across both the basolateral and apical plasma membranes of the cells, resulting in net secretion of these molecules (11). Several different organic anion transporters mediate these processes, and polarized cells may have different transport systems in their apical and basolateral membranes (9).
We have shown previously that Lucifer Yellow secretion from intact 5774 cells is most efficient when cells are incubated in an alkaline medium. In the present experiments, dye sequestration into isolated cytoplasmic vesicles was most efficient when the medium in which they were suspended was acidic. These results are consistent with one another, since in both instances Lucifer Yellow is transported in the direction of the proton gradient. These findings suggest that Lucifer Yellow transport in 5774 cells is a secondary active transport process involving either H+ symport or OH-antiport. Secondary active transport involving H+ symport or OH-antiport has been described in polarized cells. Blomstedt and Aronson (12) found that transport of urate and p-aminohippurate into dog renal microvillus membrane vesicles was stimulated by an inwardly directed proton gradient. Similarly, Blitzer et al. (13) demonstrated similar pH dependence for cholate uptake in vesicles derived from rat basolateral liver plasma mem-branes. Finally, Olsnes et al. (14) described a pH-regulated anion antiporter in Vero cells. Lucifer Yellow is first sequestered into an early endocytic compartment and not into lysosomes in intact 5774 cells. Similarly, isolated vesicles in the endosome-enriched fraction accumulated more dye than did isolated lysosomes. The reason for preferential uptake into endosomes is not clear. If the transmembrane proton gradient is a primary determinant of organic anion transport, then it is possible that Lucifer Yellow transport does not occur directly into lysosomes because of their low internal pH. This line of reasoning suggests that Lucifer Yellow sequestration into endosomes of intact cells must occur before the endosomes have had an opportunity to acidify (i.e. early endosomes). Schmid et al. (15) have shown that early endosomes acidify more slowly and to a lesser extent than do late endosomes.
It is possible also that the organic anion transporters are present in endosomes but not in lysosomes. The transporters may cycle between plasma membrane and endosomes or reside permanently in the endosomal compartment, but may never enter the lysosomal compartment.
(The transferrin receptor exemplifies a plasma membrane protein that eschews lysosomes.) This is consistent with our suggestion that organic anion transporters are located in early endosomes. The physiologic significance of the 5774 organic anion transporter is not known. As noted previously, macrophages secrete or are involved in the metabolism of a wide variety of substances, many of which are organic anions. These include prostaglandins (16), leukotrienes (17), glutathione (18), lactate (19), and bilirubin (20). That prostaglandins and leukotrienes are major secretory products of macrophages is underscored by the fact that arachidonate comprises 25% of macrophage membrane fatty acids (16).
Probenecid-sensitive transporters mediate prostaglandin transport in other tissues. Prostaglandin release by renal proximal tubule cells is inhibited by probenecid as is leukotriene Cq transport across the choroid plexus (21). It is conceivable, therefore, that the organic anion transporter we have described in 5774 macrophages is involved in the secretion of arachidonate metabolites by these cells.