Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix.

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.

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Downregulation of organic anion transporters OAT1 and OAT3 correlates with impaired secretion of para-aminohippurate after ischemic acute renal failure in rats

AJP Renal Physiology ◽

10.1152/ajprenal.00473.2006 ◽

2007 ◽

Vol 292 (5) ◽

pp. F1599-F1605 ◽

Cited By ~ 64

Author(s):

R. Schneider ◽

C. Sauvant ◽

B. Betz ◽

M. Otremba ◽

D. Fischer ◽

...

Keyword(s):

Renal Failure ◽

Acute Renal Failure ◽

Organic Anion ◽

Organic Anions ◽

Organic Anion Transporters ◽

Protein Levels ◽

Anion Transporters ◽

Pah Clearance ◽

Ischemic Acute Renal Failure

Ischemic acute renal failure (iARF) was described to reduce renal extraction of the organic anion para-aminohippurate (PAH) in humans. The rate-limiting step of renal organic anion secretion is its basolateral uptake into proximal tubular cells. This process is mediated by the organic anion transporters OAT1 and OAT3, which both have a broad spectrum of substrates including a variety of pharmaceutics and toxins. Using a rat model of iARF, we investigated whether impairing the secretion of the organic anion PAH might be associated with downregulation of OAT1 or OAT3. Inulin and PAH clearance was determined starting from 6 up to 336 h after ischemia-reperfusion (I/R) injury. Net secretion of PAH was calculated and OAT1 as well as OAT3 expression was analyzed by RT-PCR and Western blotting. Inulin and PAH clearance along with PAH net secretion were initially diminished after I/R injury with a gradual recovery during follow-up. This initial impairment after iARF was accompanied by decreased mRNA and protein levels of OAT1 and OAT3 in clamped animals compared with sham-operated controls. In correlation to the improvement of kidney function, both mRNA and protein levels of OAT1 and OAT3 were upregulated during the follow-up. Thus decreased expression of OAT1 and OAT3 is sufficient to explain the decline of PAH secretion after iARF. As a result, this may have substantial impact on excretion kinetics and half-life of organic anions. As a consequence, the biological effects of a variety of organic anions may be affected after iARF.

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A prelysosomal compartment sequesters membrane-impermeant fluorescent dyes from the cytoplasmic matrix of J774 macrophages.

The Journal of Cell Biology ◽

10.1083/jcb.107.3.887 ◽

1988 ◽

Vol 107 (3) ◽

pp. 887-896 ◽

Cited By ~ 45

Author(s):

T H Steinberg ◽

J A Swanson ◽

S C Silverstein

Keyword(s):

Plasma Membrane ◽

Lucifer Yellow ◽

Organic Anion ◽

Buoyant Density ◽

Endocytic Pathway ◽

Cytoplasmic Matrix ◽

Cytoplasmic Vacuoles ◽

J774 Cells ◽

Membrane Bound ◽

Texas Red

After the membrane impermeant dye Lucifer Yellow is introduced into the cytoplasmic matrix of J774 cells, the dye is sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. In the present work we studied the intracellular transport of Lucifer Yellow in J774 macrophages and the nature of the cytoplasmic vacuoles into which this dye is sequestered. When the lysosomal system of J774 cells was prelabeled with a Texas red ovalbumin conjugate and Lucifer Yellow was then loaded into the cytoplasm of the cells by ATP-mediated permeabilization of the plasma membrane, the vacuoles that sequestered Lucifer Yellow 30 min later were distinct from the Texas red-stained lysosomes. After an additional 30 min Lucifer Yellow and Texas red colocalized in the same membrane bound compartments, indicating that the Lucifer Yellow had been delivered to lysosomes. We next prelabeled the plasma membrane of J774 cells with anti-macrophage antibody and Texas red protein A before Lucifer Yellow was loaded into the cells. The phase-lucent vacuoles that subsequently sequestered Lucifer Yellow also stained with Texas red, showing that they were part of the endocytic pathway. J774 cells were fractionated on percoll density gradients either 15 or 60 min after Lucifer Yellow was introduced into the cytoplasmic matrix of the cells. In cells fractionated after 15 min, Lucifer Yellow was contained within the fractions of light buoyant density that contain plasma membrane and endosomes; the dye later appeared in vesicles of higher density which contained lysosomes. Secretion of Lucifer Yellow from the cytoplasmic matrix of J774 cells is inhibited by the organic anion transport blocker probenecid. We found that probenecid also reversibly inhibited sequestration of dye, indicating that sequestration of dye within cytoplasmic vacuoles was also mediated by organic anion transporters. These studies show that the vacuoles that sequester Lucifer Yellow from the cytoplasmic matrix of J774 cells possess the attributes of endosomes. Thus, in addition to their role in sorting of membrane bound and soluble substances, macrophage endosomes may play a role in the accumulation and transport of molecules resident in the soluble cytoplasm.

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Mg2+-Malate Co-Transport, a Mechanism for Na+-Independent Mg2+ Transport in Neurons of the Leech Hirudo medicinalis

Journal of Neurophysiology ◽

10.1152/jn.01221.2004 ◽

2005 ◽

Vol 94 (1) ◽

pp. 441-453 ◽

Cited By ~ 2

Author(s):

Dorothee Günzel ◽

Karin Hintz ◽

Simone Durry ◽

Wolf-Rüdiger Schlue

Keyword(s):

Organic Anion ◽

Inhibitory Effect ◽

Organic Anions ◽

Hirudo Medicinalis ◽

Disulfonic Acid ◽

Organic Anion Transporters ◽

Experimental Conditions ◽

Physiological Conditions ◽

Anion Transporters ◽

Proposed Model

Mg2+-extrusion from Mg2+-loaded neurons of the leech, Hirudo medicinalis, is mediated mainly by Na+/Mg2+ antiport. However, in a number of leech neurons, Mg2+ is extruded in the nominal absence of extracellular Na+, indicating the existence of an additional, Na+-independent Mg2+ transport mechanism. This mechanism was investigated using electrophysiological and microfluorimetrical techniques. The rate of Na+-independent Mg2+ extrusion from Mg2+-loaded leech neurons was found to be independent of extracellular Ca2+, K+, NO3−, HCO3−, SO42−, HPO42−, and of intra- and extracellular pH. Na+-independent Mg2+ extrusion was not inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), furosemide, ouabain, vanadate, iodoacetate, 4-amino-hippurate, or α-cyano-4-hydroxycinnamate and was not influenced by changes in the membrane potential in voltage-clamp experiments. Na+-independent Mg2+ extrusion was, however, inhibited by the application of 2 mM probenecid, a blocker of organic anion transporters, suggesting that Mg2+ might be co-transported with organic anions. Extracellularly, of all organic anions tested (malate, citrate, lactate, α-ketoglutarate, and 4-amino-hippurate) only high, but physiological, concentrations of malate (30 mM) had a significant inhibitory effect on Na+-independent Mg2+ extrusion. Intracellularly, iontophoretically injected malate, citrate, or fura-2, but not Cl−, α-ketoglutarate, glutamate, succinate, or urate, were stimulating Na+-independent Mg2+ extrusion from those neurons that initially did not extrude Mg2+ in Na+-free solutions. Our data indicate that Mg2+ is co-transported with organic anions, preferably with malate, the predominant extracellular anion in the leech. The proposed model implies that, under experimental conditions, malate drives Mg2+ extrusion, whereas under physiological conditions, malate is actively taken up, driven by Mg2+, so that malate can be metabolized.

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Molecular physiology of renal organic anion transporters

AJP Renal Physiology ◽

10.1152/ajprenal.00439.2004 ◽

2006 ◽

Vol 290 (2) ◽

pp. F251-F261 ◽

Cited By ~ 156

Author(s):

Takashi Sekine ◽

Hiroki Miyazaki ◽

Hitoshi Endou

Keyword(s):

Organic Anion ◽

Organic Anions ◽

Organic Anion Transporter ◽

Transepithelial Transport ◽

Gene Localization ◽

Organic Anion Transporters ◽

Molecular Physiology ◽

Anion Transporters ◽

Anion Transporter ◽

Molecular Information

Recent advances in molecular biology have identified three organic anion transporter families: the organic anion transporter (OAT) family encoded by SLC22A, the organic anion transporting peptide (OATP) family encoded by SLC21A ( SLCO), and the multidrug resistance-associated protein (MRP) family encoded by ABCC. These families play critical roles in the transepithelial transport of organic anions in the kidneys as well as in other tissues such as the liver and brain. Among these families, the OAT family plays the central role in renal organic anion transport. Knowledge of these three families at the molecular level, such as substrate selectivity, tissue distribution, and gene localization, is rapidly increasing. In this review, we will give an overview of molecular information on renal organic anion transporters and describe recent topics such as the regulatory mechanisms and molecular physiology of urate transport. We will also discuss the physiological roles of each organic anion transporter in the light of the transepithelial transport of organic anions in the kidneys.

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Immunolocalization of Multispecific Organic Anion Transporters, OAT1, OAT2, and OAT3, in Rat Kidney

Journal of the American Society of Nephrology ◽

10.1681/asn.v134848 ◽

2002 ◽

Vol 13 (4) ◽

pp. 848-857 ◽

Cited By ~ 1

Author(s):

Ryoji Kojima ◽

Takashi Sekine ◽

Masanao Kawachi ◽

Seok Ho Cha ◽

Yoshio Suzuki ◽

...

Keyword(s):

Rat Kidney ◽

Organic Anion ◽

Basolateral Membrane ◽

Organic Anions ◽

Proximal Tubules ◽

Transepithelial Transport ◽

Thick Ascending Limb ◽

Apical Surface ◽

Organic Anion Transporters ◽

Anion Transporters

ABSTRACT. Recently, a family of multispecific organic anion transporters has been identified, and several isoforms have been reported. However, the physiologic and pharmacologic roles of each isoform, except OAT1, in the transepithelial transport of organic anions in the kidney remain to be elucidated. To address this issue, it is essential to determine the intrarenal distribution and membrane localization of each OAT isoform along the nephron. In this study, the intrarenal distributions of rOAT1, rOAT2, and rOAT3 were investigated by an immunofluorescence method that used frozen rat serial kidney sections. Confocal microscopic analysis showed that immunoreactivity for rOAT1 was detected exclusively in the proximal tubules (S1, S2, S3) in the cortex with basolateral membrane staining. rOAT2 was detected in the apical surface of the tubules in the medullary thick ascending limb of Henle’s loop (MTAL) and cortical and medullary collecting ducts (CD). rOAT3 was localized in the basolateral digitation of the cell membrane in all the segments (S1, S2, and S3) of the proximal tubules, MTAL, cortical TAL, connecting tubules, and cortical and medullary CD. These results on the distribution of each OAT isoform will facilitate the understanding of the role of OATs in the renal processing of organic anions.

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