Physiology, structure, and regulation of the cloned organic anion transporters.

1. The transport of negatively charged drugs, xenobiotics, and metabolites by epithelial tissues, particularly the kidney, plays critical roles in controlling their distribution, concentration, and retention in the body. Thus, organic anion transporters (OATs) impact both their therapeutic efficacy and potential toxicity. 2. This review summarizes current knowledge of the properties and functional roles of the cloned OATs, the relationships between transporter structure and function, and those factors that determine the efficacy of transport. Such factors include plasma protein binding of substrates, genetic polymorphisms among the transporters, and regulation of transporter expression. 3. Clearly, much progress has been made in the decade since the first OAT was cloned. However, unresolved questions remain. Several of these issues--drug-drug interactions, functional characterization of newly cloned OATs, tissue differences in expression and function, and details of the nature and consequences of transporter regulation at genomic and intracellular sites--are discussed in the concluding Perspectives section.

Development and characterization of immobilized human organic anion transporter-based liquid chromatographic stationary phase: hOAT1 and hOAT2.

This paper reports the development of liquid chromatographic columns containing immobilized organic anion transporters (hOAT1 and hOAT2). Cellular membrane fragments from MDCK cells expressing hOAT1 and S2 cells expressing hOAT2 were immobilized on the surface of the immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The resulting stationary phases were characterized by frontal affinity chromatography, using the marker ligand [3H]-adefovir for the hOAT1 and [14C]-p-aminohippurate for the hOAT2 in the presence of multiple displacers. The determined binding affinities (Kd) for eight OAT1 ligands and eight OAT2 ligands were correlated with literature values and a statistically significant correlation was obtained for both the hOAT1 and hOAT2 columns: r2=0.688 (p<0.05) and r2=0.9967 (p<0.0001), respectively. The results indicate that the OAT1 and OAT2 have been successfully immobilized with retention of their binding activity. The use of these columns to identify ligands to the respective transporters will be presented.

Mercapturic acids (N-acetylcysteine S-conjugates) as endogenous substrates for the renal organic anion transporter-1.

Mercapturic acids are N-acetyl-L-cysteine S-conjugates that are formed from a range of endogenous and exogenous chemicals. Although the kidney is a major site for elimination of mercapturic acids, the transport mechanisms involved have not been identified. The present study examined whether mercapturic acids are substrates for the renal basolateral organic anion transporter-1 (Oat1) from rat kidney. This carrier mediates uptake of organic anions from the bloodstream in exchange for intracellular alpha-ketoglutarate. Uptake of [(3)H]p-aminohippuric acid (PAH) in Oat1-expressing Xenopus laevis oocytes was strongly inhibited by S-(2,4-dinitrophenyl)-N-acetyl-L-cysteine (DNP-NAC) and by all other mercapturic acids tested, including the endogenous mercapturic acid N-acetyl-leukotriene E(4). Inhibition by the mercapturic acids was competitive, which is consistent with the hypothesis that these compounds are substrates for Oat1. This conclusion was supported by the direct demonstration of saturable [(35)S]DNP-NAC uptake in Oat1-expressing oocytes. [(35)S]DNP-NAC uptake was inhibited by PAH and other mercapturic acids and was stimulated in oocytes preloaded with glutarate. The apparent K(m) value for DNP-NAC uptake was only 2 microM, indicating that this mercapturic acid is a high affinity substrate for Oat1. Together, these data indicate that clearance of endogenous mercapturic acids is an important function of the renal organic anion transporter.

Ventricular choline transport: a role for organic cation transporter 2 expressed in choroid plexus.

To determine whether organic cation transporter (OCT) family members might mediate choline transport in choroid plexus (CP), the handling of choline by cloned transporters and by intact CP isolated from the adult rat was investigated. Expression of OCT1 and OCT2 in Xenopus oocytes increased hemicholinium-3-sensitive choline uptake. In contrast, OCT3 did not mediate choline transport. Estimated K(m) values for choline in rOCT1-, rOCT2-, and hOCT2-expressing oocytes were 346 +/- 50, 441 +/- 67, and 102 +/- 80 microm, respectively. Membrane potential was the major driving force for choline uptake in rat and human OCT2-expressing oocytes and in intact CP in vitro. Lowering of medium pH (6 versus 7.4) was equally effective at inhibiting choline uptake in CP, suggesting that there might be a non-OCT component of choline uptake that is responsive to an H(+) gradient. However, choline efflux from CP was not stimulated by a trans-applied H(+) gradient. Choline uptake by CP was Na(+)-independent with an estimated K(m) of 183 microm. Reverse transcriptase-polymerase chain reaction detected OCT2 and OCT3, but not OCT1, mRNA expression in CP. Transfection of intact CP with a rOCT2/green fluorescent protein fusion construct resulted in strong apical membrane fluorescence with no detectable signal in the basal and lateral plasma membranes. These data indicate that OCT2 mediates choline transport across the ventricular membrane of CP.

Characterization of a novel cationic drug transporter in human retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells transport a variety of solutes, but the capacity of human RPE cells to transport drugs and xenobiotics is not well understood. As an initial step to address this issue, we have examined human RPE transport of verapamil. Transport of [3H]verapamil was measured in two human RPE cell lines (RPE/Hu and ARPE-19) grown to confluence on 12-well culture plates. Verapamil uptake by RPE/Hu cells was highly concentrative, reaching cell-to-medium ratios as high as 42 by 1 h. Uptake was saturable, with an apparent K(m) of 7.2 microM. Verapamil uptake decreased in the presence of metabolic inhibitors, low temperature, and organic cations, including quinidine, pyrilamine, quinacrine, and diphenhydramine. However, other organic cations, including tetraethylammonium and cimetidine failed to inhibit. Verapamil uptake was also inhibited by the cationic antiglaucoma drugs diltiazem, timolol, and propranolol. Verapamil uptake was insensitive to changes in membrane potential. However, transport was markedly altered by changes in pH. Decreasing external pH inhibited uptake, whereas efflux was stimulated. Intracellular acidification via NH4Cl prepulse also stimulated uptake. Identical findings were obtained using the commercially available cell line ARPE-19. In view of its unique specificity, the RPE cell verapamil transporter described above is a novel, heretofore undescribed, organic cation transporter, distinct from the known members of the OCT family of organic cation transporters.

Basolateral localization of organic cation transporter 2 in intact renal proximal tubules.

The localization of organic cation transporter 2 (OCT2) within renal cells is the subject of considerable controversy, resulting in marked uncertainty as to its function. To resolve this issue, we made an OCT2/green fluorescent protein (GFP) fusion construct (rOCT2-GFP) and determined its localization within Xenopus laevis oocytes and renal cells using confocal microscopy. Oocytes expressing rOCT2-GFP exhibited plasma membrane fluorescence as well as greatly increased specific, potential-driven uptake of [(14)C]tetraethylammonium (TEA). Polarized monolayers of renal epithelial cell lines [LLC-PK(1) and Madin-Darby canine kidney (MDCK)] transiently transfected with pEGFP-C3, which codes for a cytoplasmic GFP, showed a diffuse, evenly distributed cytoplasmic signal with no plasma membrane fluorescence. In contrast, cells transiently transfected with pEGFP-C3/rOCT2 (the vector coding for rOCT2-GFP) showed predominantly plasma membrane fluorescence, which was most prominent in the lateral membrane. MDCK cells stably expressing rOCT2-GFP (MDCK/rOCT2-GFP) maintained in long-term culture showed a greatly increased basal and lateral membrane fluorescence. When grown on porous supports, MDCK/rOCT2-GFP monolayers showed specific, potential-driven TEA uptake from the basal side. Finally, expression and distribution of rOCT2-GFP were investigated in isolated killifish (Fundulus heteroclitus) renal proximal tubules. On expression of rOCT2-GFP, transfected tubules showed marked basal and lateral membrane fluorescence, with no detectable signal at the apical membrane. In contrast, tubules expressing a luminal sodium-dicarboxylate cotransporter (rbNaDC-1)-GFP construct showed apical membrane fluorescence, and tubules expressing cytoplasmic GFP had a diffuse cytoplasmic fluorescence. These results indicate that rOCT2 is basolateral in renal proximal tubule cells.

rOCT2 is a basolateral potential-driven carrier, not an organic cation/proton exchanger.

The driving forces mediating tetraethylammonium (TEA) transport were systematically assessed in Xenopus oocytes and MDCK cells expressing organic cation transporter (OCT) 2 cloned from rat kidney (rOCT2). In rOCT2 cRNA-injected oocytes, uptake of [14C]TEA was saturable, with an estimated Michaelis constant (Km) of 393 microM, and was specifically inhibited by organic cations. Furthermore, TEA uptake demonstrated two distinct components, one that was potential sensitive and one that was pH sensitive. When membrane potential was intact, TEA uptake was largely unaffected by changes in medium pH; when the oocyte membrane was depolarized (K+ in = out = 102.5 mM, plus valinomycin), decreasing external medium pH significantly reduced TEA uptake. Consistent with the potential sensitivity of uptake, electrophysiological analysis of rOCT2-injected oocytes demonstrated movement of positive charge into the oocyte upon TEA addition. To further evaluate the nature of the pH effect and assess the properties of rOCT2 in a renal epithelium, rOCT2 was introduced into MDCK cells. A stably transfected single cell clone (MDCK-rOCT2) showed mediated, potential-sensitive, pH-sensitive TEA uptake (Km = 48 microM). TEA efflux from preloaded MDCK-rOCT2 cells was stimulated by externally applied (trans) tetramethylammonium but was trans-inhibited by H+ (external pH 5.4). The effect of external H+ was to modulate rOCT2-mediated transport. Thus rOCT2 is a potential-driven transporter, not an organic cation/H+ exchanger, consistent with a physiological role in the basolateral entry step in renal organic cation secretion.

Mechanism of organic anion transport across the apical membrane of choroid plexus.

The mechanism and membrane localization of choroid plexus (CP) organic anion transport were determined in apical (or brush border) membrane vesicles isolated from bovine choroid plexus and in intact CP tissue from cow and rat. Brush border membrane vesicles were enriched in Na(+),K(+)-ATPase (20-fold; an apical marker in CP) and demonstrated specific, sodium-coupled transport of proline, glucose, and glutarate. Vesicular uptake of the anionic herbicide 2, 4-dichlorophenoxyacetic acid (2,4-D) was markedly stimulated by an inward sodium gradient but only in the presence of glutarate, indicating the presence of apical dicarboxylate/organic anion exchange. Consistent with this interpretation, an imposed outward glutarate gradient stimulated 2,4-D uptake in the absence of sodium. Under both conditions, uptake was dramatically slowed and overshoot was abolished by probenecid. Likewise, apical accumulation of 2,4-D by intact bovine choroid plexus tissue in vitro was stimulated by external glutarate in the presence of sodium. Glutarate stimulation was abolished by 5 mM LiCl. Identical findings were obtained using rat CP tissue, which showed both sodium/glutarate-stimulated 2,4-D (tissue/medium (T/M) approximately 8) and p-aminohippurate (T/M = 2) transport. Finally, since the renal exchanger (rROAT1) has been cloned in rat kidney, a rROAT1-green fluorescent protein construct was used to analyze exchanger distribution directly in transiently transfected rat CP. As predicted by the functional studies, the fluorescently tagged transporter was seen in apical but not basolateral membranes of the CP.

The antiviral nucleotide analogs cidofovir and adefovir are novel substrates for human and rat renal organic anion transporter 1.

Nephrotoxicity is the dose-limiting clinical adverse effect of cidofovir and adefovir, two potent antiviral therapeutics. Because renal uptake likely plays a role in the etiology of cidofovir- and adefovir-associated nephrotoxicity, we attempted to identify a renal transporter capable of interacting with these therapeutics. A cDNA clone was isolated from a human renal library and designated human organic anion transporter 1 (hOAT1). Northern analysis detected a specific 2.5-kilobase pair hOAT1 transcript only in human kidney. However, reverse transcription-polymerase chain reaction revealed hOAT1 expression in human brain and skeletal muscle, as well. Immunoblot analysis of human kidney cortex demonstrated that hOAT1 is an 80- to 90-kilodalton heterogeneous protein modified by abundant N-glycosylation. Xenopus laevis oocytes expressing hOAT1 supported probenecid-sensitive uptake of [(3)H]p-aminohippurate (K(m) = 4 microM), which was trans-stimulated in oocytes preloaded with glutarate. Importantly, both hOAT1 and rat renal organic anion transporter 1 (rROAT1) mediated saturable, probenecid-sensitive uptake of cidofovir, adefovir, and other nucleoside phosphonate antivirals. The affinity of hOAT1 toward cidofovir and adefovir (K(m) = 46 and 30 microM, respectively) was 5- to 9-fold higher compared with rROAT1 (K(m) = 238 and 270 microM, respectively). These data indicate that hOAT1 may significantly contribute to the accumulation of cidofovir and adefovir in renal proximal tubules and, thus, play an active role in the mechanism of nephrotoxicity associated with these antiviral therapeutics.

Localization of an organic anion transporter-GFP fusion construct (rROAT1-GFP) in intact proximal tubules.

The organic anion transporter, rROAT1, is a dicarboxylate/organic anion exchanger, a function associated with the basolateral membrane in rat proximal tubule. To directly establish the subcellular localization of rROAT1 in renal epithelia, we made a rROAT1-green fluorescent protein (GFP) fusion construct (rROAT1-GFP). Plasma membrane-associated fluorescence was observed in rROAT1-GFP-expressing Xenopus oocytes examined by confocal microscopy. Uptake of 3H-labeled p-aminohippurate (PAH) increased 2. 5-fold in rROAT1-GFP-expressing Xenopus oocytes, and this increase was abolished by 1 mM probenecid. Thus the construct was capable of specific organic anion transport. Cultured renal epithelial cell lines (MDCK and LLC-PK1) transfected with the vector pEGFP-C3 showed a diffuse, evenly distributed cytoplasmic signal. However, when transfected with pEGFP-C3/rROAT1 (vector coding for rROAT1-GFP), both cell lines showed predominantly plasma membrane fluorescence. The expression and distribution of rROAT1-GFP in intact renal proximal tubules was also investigated. Isolated killifish (Fundulus heteroclitus) renal tubules transfected with pEGFP-C3/rROAT1 showed marked basal and lateral membrane-associated fluorescence, but no detectable signal in the nucleus or the apical pole of tubule cells. Tubules transfected with pEGFP-C3 showed diffuse cytoplasmic fluorescence. Function of the rROAT1-GFP construct was demonstrated in transfected killifish tubules by fluorescein transport assay. These results demonstrate the basolateral subcellular localization of rROAT1 in polarized renal epithelia and validate a new technique for localizing cloned transporters within intact renal tubules.

Organic cation transport in rat choroid plexus cells studied by fluorescence microscopy.

Quinacrine uptake and distribution were studied in a primary culture of rat choroid plexus epithelial cells using conventional and confocal fluorescence microscopy and image analysis. Quinacrine rapidly accumulated in cells, with steady-state levels being achieved after 10-20 min. Uptake was reduced by other organic cations, e.g., tetraethylammonium (TEA), and by KCN. Quinacrine fluorescence was distributed in two cytoplasmic compartments, one diffuse and the other punctate. TEA efflux experiments indicated that more than one-half of intracellular organic cation was in a slowly emptying compartment. The protonophore monensin both emptied that TEA compartment and abolished punctate quinacrine fluorescence, suggesting that a large fraction of total intracellular organic cation was sequestered in acidic vesicles, e.g., endosomes. Finally, quinacrine-loaded vesicles were seen to move within the cytoplasm and to abruptly release their contents at the blood side of the cell; the rate of release was greatly reduced by the microtubule disrupter nocodazole.

A transfected cell model for the renal toxin transporter, rOCT2.

A cDNA for the organic cation transporter (rOCT2) of the rat kidney was inserted into the retroviral plasmid pLXSN. This plasmid was used to stably transfect NIH3T3 cells. The transfected cell line exhibited an enhanced rate of tetraethylammonium (TEA) uptake and efflux compared to wild-type NIH3T3 cells. Uptake of TEA by the transfected cells was markedly reduced upon incubation at 4 degrees C. When the extracellular pH was lowered from 8.1 to 5.9, uptake was also reduced, suggesting inhibition of rOCT2 by extracellular protons. The apparent K(m) for TEA in the transfected cells was 141 microM. The classical organic cation transport inhibitors, cyanine 863 and cimetidine, produced noncompetitive inhibition with apparent Ki values of 0.81 and 198 microM, respectively. Daunomycin, vinblastine, and the deoxyadenosine analogs, 2'-deoxytubercidin and 2-chlorodeoxyadenosine, did not appear to be substrates for rOCT2. However, the anticancer drug, cisplatin, competitively inhibited TEA uptake by rOCT2 with an apparent Ki value of 925 microM, suggesting that rOCT2 may play a role in its renal secretion. In summary, transfected NIH3T3 cells provide a facile system by which this and other organic ion transporters can be studied.

Dual pathways for organic anion secretion in renal proximal tubule.

Transport on the "classical" organic anion system in renal proximal tubule is specific, active, Na-dependent, and ouabain sensitive. Here we review recent studies using intact teleost proximal tubules and laser scanning confocal microscopy which show that the secretion of large organic anions, such as, fluorescein-methotrexate (FL-MTX, Mw 923 Da) is handled by a separate and distinct organic anion transport system. In contrast to the classical system, FL-MTX uptake into cells and secretion into the tubular lumen was ouabain insensitive and largely Na-independent. KCN did not affect cellular uptake but abolished secretion into the lumen. PAH and probenecid, potent inhibitors of transport on the classical system, were weak inhibitors of FL-MTX transport. Uptake and secretion of FL-MTX were inhibited by micromolar concentrations of other organic anions (MTX, folate, bromocresol green, bromosulfonphthalein). FL-MTX secretion into the lumen was inhibited by leukotriene C4 and cyclosporine A, neither of which affected transport of the model substrate for the classical system, fluorescein. Thus, FL-MTX secretion is specific, but largely Na-independent and ouabain-insensitive. Both the basolateral and luminal steps in FL-MTX transport differ from those associated with fluorescein and P-aminohippurate secretion.

Expression cloning and characterization of ROAT1. The basolateral organic anion transporter in rat kidney.

Expression cloning in Xenopus laevis oocytes was used to isolate an organic anion transport protein from rat kidney. A cDNA library was constructed from size-fractionated poly(A)+ RNA and screened for probenecid-sensitive transport of p-aminohippurate (PAH). A 2, 227-base pair cDNA clone containing a 1,656-base pair open reading frame coding for a peptide 551 amino acids long was isolated and named ROAT1. ROAT1-mediated transport of 50 mu M [3H]PAH was independent of imposed changes in membrane potential. Transport was significantly inhibited at 4 degrees C, or upon incubation with other organic anions, but not by the organic cation tetraethylammonium, by the multidrug resistance ATPase inhibitor cyclosporin A, or by urate. External glutarate and alpha-ketoglutarate (1 mM), both counterions for basolateral PAH exchange, also inhibited transport, suggesting that ROAT1 is functionally similar to the basolateral PAH carrier. Consistent with this conclusion, PAH uptake was trans-stimulated in oocytes preloaded with glutarate, whereas the dicarboxylate methylsuccinate, which is not accepted by the basolateral exchanger, did not trans-stimulate. Finally, ROAT1-mediated PAH transport was saturable, with an estimated Km of 70 mu M. Each of these properties is identical to those previously described for the basolateral alpha-ketoglutarate/PAH exchanger in isolated membrane vesicles or intact renal tubules.

Functional characterization of choroid plexus epithelial cells in primary culture.

The objective of this study was to develop and evaluate a primary culture system for choroid plexus epithelial cells as an in vitro model for studying organic cation transport. Cells were dispersed from choroid plexus of neonatal rats by enzymatic digestion and grew as differentiated monolayers when plated on solid or permeable support. Electron microscopy showed that cultured cells were morphologically similar to intact choroid plexus epithelium, having apical tight junctions between cells, numerous mitochondria, basal nuclei and apical microvilli and cilia. As previously demonstrated for intact choroid plexus, immunocytochemistry showed that Na+,K+-ATPase was localized to the apical membrane, and GLUT-1, the facilitative glucose transporter, was localized to the basolateral membrane of cultured cells. Apical transport of L-proline by cultured cells was mediated by a sodium-dependent, electrogenic process, as in whole tissue. 14C-Tetraethylammonium (TEA), a prototypic organic cation, was accumulated by isolated choroid plexus in a time-dependent manner; uptake was inhibited by tetrapentylammonium (TePA). In cultured cells, apical TEA transport was mediated by a saturable process coupled to cellular metabolism. Unlabeled TEA and other organic cations (TePA, N1-methylnicotinamide and mepiperphenidol) inhibited TEA transport; the organic anion, p-aminohippurate, had no effect. Finally, TePA-sensitive transport of 14C-TEA was stimulated after preloading the cells with unlabeled TEA. Based on the morphological, biochemical and functional properties of these cultured cells, we conclude that this primary culture system should be an excellent in vitro model for experimental characterization of choroid plexus function.

Mechanism mediating basolateral transport of 2,4-dichlorophenoxyacetic acid in rat kidney.

The organic anions, p-aminohippurate (PAH) and fluorescein, are transported across the basolateral membrane of the renal proximal tubule in exchange for intracellular alpha-ketoglutarate (alpha KG), a mechanism indirectly coupled to sodium via Na+/alpha KG cotransport. To determine whether this mechanism mediates the basolateral transport of other organic anions, transport of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), was examined in rat renal cortical slices and basolateral membrane vesicles. In slices, uptake of 2,4-D increased steadily over time, approaching steady-state tissue/medium ratios of approximately 8 after 60 min. Probenecid, PAH and chlorophenol red inhibited steady-state uptake of 2,4-D. Accumulation of 10 microM 2,4-D was stimulated 2-fold by 60 microM glutarate; other dicarboxylic acids failed to stimulate uptake. In the presence of sodium, the addition of 5 mM LiCl or 2 mM ouabain to the bathing medium abolished glutarate stimulation. Removal of sodium from the bathing medium reversibly inhibited uptake as much as 75%. Furthermore, PAH inhibited 2,4-D uptake by slices in a dose-dependent manner, and increasing the external 2,4-D concentration decreased the inhibitory potency of PAH. In basolateral membrane vesicles, unlabeled 2,4-D inhibited sodium glutarate-coupled uptake of 3H-labeled PAH and 2,4-D in a concentration-dependent manner. Moreover, concentrative uptake of 2,4-D into vesicles could be driven by an outwardly directed gradient of glutarate or alpha KG that was generated by lithium-sensitive Na+/dicarboxylate cotransport or imposed experimentally. An outwardly directed gradient of unlabeled 2,4-D or PAH also stimulated uptake of 2,4-D. Based on these data, basolateral accumulation of 2,4-D by the renal proximal tubule is mediated by 2,4-D/alpha KG exchange, a mechanism energetically coupled to Na+/alpha KG cotransport and shared with PAH.

Renal secretion of organic anions and cations.

The renal proximal tubule actively transports charged, potentially toxic xenobiotics from blood to lumen. Basolateral uptake of organic anions is indirectly coupled to the sodium gradient through Na-dicarboxylate cotransport and dicarboxylate-organic anion exchange. Upon entry, a significant fraction of intracellular organic anion is sequestered within vesicles. Disruption of the cellular microtubular network can lead to both diminished vesicular movement and reduced transepithelial secretion. Luminal efflux of organic anions is energetically downhill, but carrier mediated. Both anion exchange and potential driven transport are present, but neither completely accounts for transport from cell to lumen. For organic cations, basolateral entry is downhill via potential driven facilitated diffusion. Intracellular sequestration of organic cations in vesicles is substantial, but its role in secretion is uncertain. Multiple carriers are available to drive organic cations uphill into the tubular lumen. The classical system indirectly taps the energy of the luminal Na gradient to drive organic cation efflux via Na(+)-H+ and proton-organic cation exchange. In addition, the multidrug resistance ATPase can pump organic cations into the tubular lumen. Thus, although much detailed information has been added over the last 50 years, it is not yet possible to provide a detailed, quantitative understanding of these important excretory systems.

Intracellular alpha-ketoglutarate controls the efficacy of renal organic anion transport.

Renal organic anion secretion is driven by indirect coupling to the Na+ gradient at the basolateral membrane through Na(+)-dicarboxylate cotransport and dicarboxylate-organic anion exchange. The impact of changing intracellular alpha-ketoglutarate (alpha KG) concentrations and gradient on p-aminohippurate (PAH) transport was assessed in rat renal cortical slices. Fluorimetric analysis of alpha KG indicated that freshly isolated slices averaged 137 +/- 4 nmol/g wet weight (approximately 265 microM in cellular water). This value was sustained over several hours at 4 degrees C. On incubation at 22 degrees C, intracellular alpha KG concentrations rose steadily, reaching levels of 2 (air) to 4 (100% O2) times that of fresh tissue. When internal alpha KG was increased by preincubation and PAH uptake was determined at a fixed gradient, PAH transport increased with increasing internal alpha KG. Conversely, at a fixed internal alpha KG concentration, PAH uptake was a linear function of the driving force provided by the alpha KG gradient. Thus, intracellular alpha KG is a major determinant of the efficacy of renal organic anion transport, and events that alter internal alpha KG concentration, gradient, or both are poised to exert significant control over organic anion secretion. Kinetic analysis of alpha KG-PAH exchange indicated that the Km for alpha KG was 151 microM in basolateral membrane vesicles and 131 microM in slices. Because PAH transport increased at intracellular alpha KG concentrations that should have been saturating, this finding indicates that cytoplasmic alpha KG levels must be substantially lower than total tissue concentration, i.e., that much intracellular alpha KG must be sequestered.

Nocodazole inhibition of organic anion secretion in teleost renal proximal tubules.

The impact of the microtubule-disrupting drug nocodazole on renal tubular secretion of organic anions was examined in vitro using proximal tubular masses from teleost fish. Nocodazole reversibly inhibited 20-30% of the tubular accumulation of two model organic anions, p-aminohippurate and fluorescein (FL), by winter flounder tubular masses. However, the drug had no effect on the initial rate of organic anion uptake. Thus it did not reduce transport into the cells at the basolateral membrane, either directly by affecting basolateral organic anion transport proteins or indirectly by altering metabolism or ion gradients. Instead, epifluorescence video microscopy and digital image analysis of killifish tubules showed that nocodazole greatly reduced luminal accumulation of FL and had a smaller effect on cellular dye accumulation. Luminal FL accumulation returned to control levels when tubules were incubated in drug-free medium. Confocal fluorescence microscopy confirmed the marked reduction in luminal FL concentration and demonstrated that intracellular punctate FL accumulation was also markedly reduced. Finally, immunohistochemistry with an anti-tubulin antibody showed that the concentrations of nocodazole used in the above experiments reversibly disrupted microtubules within renal epithelial cells. These data indicate that a component of organic anion secretion in teleost proximal tubule is dependent on an intact microtubular network.