Mechanism of urate and p-aminohippurate transport in rat renal microvillus membrane vesicles

The transport of urate and p-aminohippurate (PAH) was evaluated in brush border membrane vesicles from the rat renal cortex. The binding of urate to the membranes was 6% of total uptake and no conversion of urate to allantoin was detected. The binding of PAH to the membranes was 24% of total uptake. In the presence of an outwardly directed hydroxyl ion gradient (pHi = 7.5, pHo = 6.0), the uptake of urate and PAH was stimulated relative to the absence of a hydroxyl ion gradient (pHi = pHo = 7.5) and the influx of urate resulted in a transient overshoot of the equilibrium value. The hydroxyl ion gradient-stimulated uptake of urate and PAH was not solely due to a change in membrane potential. Probenecid, DIDS, furosemide, and pyrazinoate inhibited the hydroxyl ion gradient-stimulated uptake of urate and PAH in a dose-dependent manner. The uptake of [14C]urate and [3H]PAH could be cis-inhibited and trans-stimulated by either unlabeled urate or PAH. In the presence of an outwardly directed bicarbonate gradient and 10% CO2 (outside HCO-3 = 5.4 mM, inside HCO-3 = 54 mM, pHo = 6.5, pHi = 7.5), the initial rate of urate uptake was faster and the initial rate of urate efflux was slower compared with vesicles that had the same pH gradient without bicarbonate or CO2. The effects of bicarbonate gradients on organic anion transport were not dependent on diffusion potentials. Finally, 100 mM extravesicular Na+, K+, Li+, or Cs+ did not affect urate or PAH uptake. These results indicate that brush border membrane vesicles from the rat kidney contain an anion exchange transport system with affinity for urate, PAH, hydroxyl ions, and bicarbonate. In addition there is no evidence for a sodium-urate or sodium-PAH cotransport mechanism in these membranes.     

Voltage-driven p-aminohippurate, chloride, and urate transport in porcine renal brush-border membrane vesicles

p-Aminohippurate (PAH) and urate are secreted into the proximal tubule lumen across the brush-border membrane. Here we used brush-border membrane vesicles from pig kidney to study PAH and urate transport. Efflux and influx of [3H]PAH were influenced by K+-diffusion potentials indicating electrogenic PAH transport. An outside>inside PAH concentration difference accelerated voltage-sensitive, Na+-coupled D-glucose uptake as efficiently as did an outside>inside Cl- concentration difference, suggesting comparable conductances for PAH and Cl- in brush-border membrane vesicles. Up to 1 mM of the uricosurics indacrinone, tienilic acid, losartan and probenecid, as well as of the stilbenes, DIDS and SITS, and of the loop diuretics furosemide and bumetanide inhibited voltage-driven PAH uptake, but not, or only slightly, voltage-driven Cl- uptake. Voltage-driven [14C]urate uptake, however, was inhibited by 0.1 mM DIDS, 0.2 mM losartan and 0.5 mM probenecid to a similar extent as [3H]PAH uptake. One millimolar pyrazinoic acid, oxonate, xanthine and adenosine inhibited neither [3H]PAH nor [14C]urate uptake. These results suggest that PAH and urate share an anion conductance which is distinct from the Cl- conductance and is probably not the same as a recently identified urate channel (Leal-Pinto E et a]. J Biol Chem 272:617-625, 1997).     

Sodium and chloride transport across rabbit ileal brush border. I. Evidence for Na-H exchange

A series of experiments were performed to demonstrate the presence of and characterize the Na-H exchanger on rabbit ileal brush border with a vesicle preparation. An outwardly directed proton gradient (pH 5.5 inside, pH 7.5 outside) stimulated Na uptake, and a fourfold "overshoot" was observed. Conversely, an inwardly directed proton gradient (pH 7.5 inside, pH 5.5 outside) inhibited Na uptake. This stimulation/inhibition of Na uptake could not be accounted for by a proton diffusion potential, because Na uptake was found to be potential insensitive. Amiloride and harmaline inhibited pH-stimulated Na uptake, but other transport inhibitors (acetazolamide, DIDS, SITS, furosemide, and bumetanide) had no effect. Amiloride also inhibited Na efflux in the presence and absence of a pH gradient. Proton gradient-stimulated Na uptake was saturable with a Km of 16.2 mM and a Vmax of 129 nmol X min-1 X mg protein-1. Tetramethylammonium did not affect pH-stimulated Na uptake, but other cations tested inhibited Na uptake, with NH4+ and Li+ causing greater inhibition than K+ or Cs+. Using the fluorescent probe acridine orange, an inwardly directed Na gradient was shown to stimulate proton efflux from the vesicles and an outwardly directed Na gradient stimulated proton influx.     

Transport of p-aminohippuric acid by plasma membrane vesicles isolated from rat kidney cortex

Summary Basal-lateral plasma membrane vesicles and brush border membrane vesicles were isolated from rat kidney cortex and the uptake of p-amino-hippuric acid (PAH) into these vesicles was studied by Millipore filtration techniques.Both membrane preparations take up PAH into an osmotically reactive intravesicular space. The transport across the brush border membrane seems to involve only simple diffusion whereas in the basal-lateral plasma membrane in addition a specific transport system exists which is inhibited competitively by probenecid. The apparent affinity of this transport system for PAH is 5.4×10^–4 M and for probenecid 5.4×10^–5 M.PAH uptake into basal-lateral plasma membrane vésicles is influenced by alteration of the membrane potential. Maneuvers which render the intravesicular space more positive-as for example replacement of chloride by sulfate in the presence of a sodium gradient directed into the vesicles and addition of valinomycin in the presence of a potassium gradient directed into the vesicles-stimulate the uptake of PAH. Replacement of a sodium chloride gradient by a sodium thiocyanate gradient reduces the uptake probably by reducing the inside positive membrane potential.In the absence of salt gradients anion replacement and replacement of sodium by potassium does not affect PAH transport by basal-lateral plasma membranes.These results suggest that in isolated basal-lateral membranes transfer of PAH across the membrane is accompanied by a transfer of negative charge. They furthermore provide no evidence for the existence of a sodium-PAH cotransport system in this membrane preparation.Part of these data have been presented on the Spring Meeting of the German Physiological Society in Bochum, 1975 [Pflügers Arch., Suppl.355, 99 (1975)].     

Changes in intestinal glucose transport over the lifespan of the rat

Age-related changes in intestinal glucose absorption were studied using everted intestinal sacs and brush border membrane vesicles prepared from male F344 rats. Glucose uptake by everted intestinal sacs was greatest in young (2-3-month-old) as compared with adult (12-14-month-old) and old (24-month-old) rats. The greatest decrease in glucose uptake occurred between 2 and 12 months. The addition of phloridzin reduced glucose uptake to similar levels in all age groups, suggesting that the age-related change was in the carrier-mediated component of glucose transport. In order to localize the site of decreased carrier-mediated glucose transport, experiments were performed using brush border membrane vesicles. Vesicular glucose uptake in the presence of Na was significantly greater in vesicles prepared from 2-month-old rats (133 +/- 18 pmol/mg/s), compared with those prepared from 12-month-old rats (82 +/- 13 pmol/mg/s). Kinetic studies performed under non-equilibrium conditions demonstrated that the major effect of age was on the Na-dependent component of the brush border transport system. There was a reduction in the Vmax from 335 +/- 37 pmol/mg/s in the young to 217 +/- 22 pmol/mg/s in the adult, but there was no change in the Km. Isotope exchange studies performed under equilibrium conditions confirmed a decrease in the activity of the glucose transporter with age. No age-related changes in Na uptake by brush border membrane vesicles were observed. These findings suggest that a decrease in the number and/or activity of Na-linked glucose carriers may account for the decrease in intestinal glucose transport with age.     

Inorganic anion transport in kidney and intestinal brush border and basolateral membranes

The efflux of inorganic anions from purified brush border and basolateral membrane vesicles from dog kidney cortex was measured under equilibrium exchange conditions. Marked differences in temperature sensitivity and effects of inhibitors were found between the Cl and SO4 transport pathways and between the two types of membranes. SO4 transport in both brush border and basolateral membranes was markedly reduced by cooling, but significant inhibition by 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) was only observed in basolateral vesicles. In contrast, Cl efflux from both types of vesicles was neither substantially inhibited by DIDS nor by lowering the temperature to 0 degrees C. Phosphate efflux from basolateral membrane vesicles was found to be only partially sensitive to DIDS. Attempts to label the stilbene-sensitive SO4 pathway in basolateral vesicles using [3H2]DIDS as a marker were unsuccessful due to the nonspecific labeling of many membrane components. The asymmetry in inorganic anion transport behavior exhibited by brush border and basolateral membrane vesicles from dog renal proximal tubule was also observed in equivalent vesicles prepared from rat small intestine.     

p-Aminohippurate/2-oxoglutarate exchange in bovine renal brush-border and basolateral membrane vesicles

The transport of the amphiphilic organic anion, P-aminohippurate (PAH), across the luminal (brush-border) and contraluminal (basolateral) membrane of renal proximal tubule cells was studied with membrane vesicles isolated from bovine kidney cortex. On the basis of the enrichment of specific activities of marker enzymes, leucine aminopeptidase and Na⁺/K⁺-ATPase, brush-border and basolateral membrane vesicles can be obtained from bovine kidneys in reasonably pure form. The uptake of [³H]PAH into both brush-border and basolateral membrane vesicles was trans-stimulated by intravesicular PAH and by 2-oxoglutarate. In the absence of Na⁺, [³H]PAH/2-oxoglutarate exchange was cis-inhibited by unlabelled 2-oxoglutarate in the medium. In the presence of an inward Na⁺ gradient, 10 M 2-oxoglutarate, but no other Krebs cycle derivative, cis-stimulated [³H]PAH uptake, indicating that a Na³-coupled dicarboxylate transporter and PAH/2-oxoglutarate exchanger cooperate in both membranes to enhance [³H]PAH uptake. [³H]PAH uptake showed a non-saturable and a saturable component with similar apparent K _m values in brush-border and basolateral membranes. Although one negatively charged PAH molecule exchanges with one doubly negatively charged 2-oxoglutarate molecule the exchange was electroneutral. Probenecid inhibited [³H]PAH/2-oxoglutarate exchange in brush-border and basolateral membrane vesicles with indistinguishable kinetics. We conclude that similar or identical PAH transporters are located in brush-border and basolateral membranes of bovine kidney proximal tubule cells. This arrangement seems species-specific since a Na⁺ gradient plus 2-oxoglutarate caused concentrative [³H]PAH uptake in brush-border membrane vesicles from bovine, but not from rat kidney.     

Intravesicular NAD has no effect on sodium-dependent phosphate transport in isolated renal brush border membrane vesicles

The effects of intravesicular NAD on Na⁺-dependent³²P_i uptake were investigated in isolated rat kidney brush border membrane vesicles (BBMV). NAD was introduced into the vesicles by osmotic shock, and extravesicular NAD was removed by passing the vesicles through a anion exchange column. The effectiveness of the osmotic shock procedure and the hydrolysis of extra- and intravesicular NAD were controlled by enzymatic analysis and thin layer chromatography. ADP-ribosylation of the membrane proteins was analyzed in vesicles osmotically shocked in the presence of either [adenylate-³²P]-NAD or [adenine-2,8-³H]-NAD by SDS-polyacrylamide gel electrophoresis.It was found that the Na⁺-dependent P_i uptake was inhibited when the BBMV were incubated with NAD at alkaline pH, which resulted in rapid NAD hydrolysis. When NAD was present in the intravesicular space only, the Na⁺-dependent P_i uptake was not inhibited.³²P from NAD was rapidly incorporated into a number of brush border membrane proteins, but no incorporation of³H-adenine could be detected.The results provide evidence that NAD does not inhibit P_i transport by a direct interaction with the cytoplasmic side of the brush border membrane. No evidence of ADP-ribosylation of the brush border membrane protein(s) was found.     

Uric acid transport in brush border membrane vesicles isolated from rabbit kidney.

The transport of uric acid was studied in brush border membrane vesicles isolated from rabbit kidney. The uptake of uric acid by the vesicles was osmotically sensitive and occurred in the absence of significant uric acid degradation. Under the conditions used to evaluate transport, urate binding to the membranes represented only 10--15% of the total uptake. The initial rate of uptake was linear over the concentration range 0.04--8 mM urate. Uptake of urage was Na+ gradient independent. It was dependent on external pH and temperature with Q10 near 3. The urate uptake was inhibited reversibly by p-chloromercuribenzoate. Probenecid, ouabain, cyclic adenosine 3',5'--monophosphate, and its dibutyryl derivative had no appreciable effects. Pyrazinoic acid and pyrazinamide stimulated urate uptake. Experiments performed with osmotically shocked vesicles demonstrated that this stimulatory effect resulted from increased binding of urate to the membranes. These results indicate that in several ways urate transport in vesicles resembles that observed with more physiologically intact preparations.     

Effect of SITS on organic anion transport in the rabbit kidney cortical slice

The effect of SITS (4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene) on the transport of organic ions in the rabbit kidney cortical slice was studied. SITS at a concentration of 10(-4) to 10(-3) M significantly decreased the slice-to-medium (S/M) concentration ratio of the organic anions p-aminohippurate (PAH), and 2,4,5-trichlorophenoxyacetate, but had no significant effect on that of the organic cation tetraethylammonium. The S/M ratio of PAH decreased to 0.52 +/- 0.03 (SE) in the presence of 10(-3) M SITS. The inhibition of PAH uptake caused by SITS was reversed in the presence of 0.5% bovine serum albumin in the medium. SITS at a concentration of 10(-4) M had no significant effect on the efflux of PAH. However, there was a small increase in PAH efflux at a concentration of 10(-3) M SITS. A Lineweaver-Burk analysis of the data indicates that SITS competitively inhibits PAH uptake and that SITS has a Ki value of 2.3 X 10(-4) M. SITS had no effect on the tissue water content, [14C]inulin space, or intracellular Na and K concentrations. It is suggested that the primary effect of SITS is to inhibit the entry of organic anions from the medium into the cell across the basolateral membrane.     

myo-Inositol transport in renal brush border vesicles and it inhibition by D-glucose

We examined the mechanism of myo-inositol uptake by rabbit renal proximal tubule brush border membrane vesicles and characterized the relationship between the transports of myo-inositol and D-glucose. A 100 mM Na+ electrochemical gradient (extravesicular medium > intravesicular medium) stimulated the initial rate of myo-inositol uptake 20- to 60-fold. Other cation gradients were ineffective. The Na+ myo-inositol co-transport system was shown to be electrogenic. The Na+ electrochemical gradient-dependent uptake of myo-inositol saturated at about 1 mM myo-inositol, with an apparent Km of 94 micro M at an initial 100 mM Na+ gradient. D-Glucose was an inhibitor of the Na+ gradient-dependent uptake of myo-inositol. D-Glucose, but not L-glucose, elicited accelerative exchange diffusion of myo-inositol. myo-Inositol did not significantly inhibit the Na+ gradient-dependent transport of D-glucose. We suggest that D-glucose inhibits myo-inositol uptake by dissipating the membrane potential and sharing the myo-inositol carrier. The inhibition of myo-inositol transport across the brush border membrane by D-glucose explains how glycosuria could produce inosituria in patients with diabetes mellitus.     

Transport of sulphate in rat jejunal and rat proximal tubular basolateral membrane vesicles

Basolateral membrane vesicles were isolated by a Percoll density gradient centrifugation method from small intestinal and renal proximal tubular epithelial cells. Transport of sulphate across the basolateral membrane was analyzed by measuring the uptake of tracer sulphate.In both membrane preparations, preloading the vesicles with sulphate-or hydroxyl-anions stimulated tracer sulphate uptake (trans-stimulation); an inwardly directed sodium gradient did not stimulate sulphate influx whether in the absence or in the presence of sulphate- or hydroxyl-iontrans-stimulation. Under sulphate trans-stimulation conditions, DIDS (10^–4 mol/l) inhibited sulphate influx.In intestinal membranes, trans-stimulation of sulphate influx was obtained by preloading the vesicles with chloride, in renal membranes by preloading with bicarbonate. Under sulphate trans-stimulation conditions, in intestinal membranes, sulphate influx was strongly inhibited by chloride, in renal membranes, chloride inhibition was absent. Under bicarbonate trans-stimulation conditions, in renal membranes, sulphate transport was inhibited by lactate.It is concluded that small intestinal and renal proximal tubular basolateral membrane vesicles contain a transport mechanism for sulphate that cannot be energized by a sodium gradient. The transport system in small intestinal basolateral membranes seems to be different from that in renal membranes. It is suggested that the observed interaction between inorganic and organic anion transport in renal basolateral membranes is indirect.Abbreviations DIDS 4,4-Diisothiocyano-2,2-disulfonic acid stilbene - DTT dithiothreitol - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - MES N-morpholinoethanesulfonic acid - PAH p-aminohippuric acid - PMSF phenylmethylsulfonyl fluoride - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate - TMA tetramethylammonium - Tris tris(hydroxymethyl)aminomethane     

Transport of organic cations in brush border membrane vesicles from rabbit kidney cortex

The transport of three organic cations, tetraethylammonium (TEA), morphine and N¹-methylnicotinamide (NMN) was studied in brush border membrane vesicles from rabbit kidney cortex under voltage clamp conditions. A proton gradient (pH_i=6.0, pH_o=7.4) produced a large stimulation of TEA and morphine uptake, yielding a transient overshoot of 190 and 220% respectively, as compared to equilibrium uptake values. No overshoot was observed under pH equilibrium conditions (pH_i=pH_o=7.4, control). These data suggest the presence of a proton-organic cation exchange mechanism in the rabbit renal cortical brush border membrane. Identical experimental conditions (proton gradient) failed however to stimulate significantly NMN transport above control values measured under pH equilibrium conditions. Proton gradient driven TEA transport showed an inhibition of 21% in the presence of NMN (1 mM) and of 63% in the presence of TEA (1 mM), and TEA transport was stimulated by preloading the vesicles with 1 mM TEA (305%) but not with 1 mM NMN (128%). NMN transport showed an inhibition of 39% in the presence of 1 mM TEA and of 27% in the presence of 1 mM NMN and its transport was stimulated by preloading the vesicles with 1 mM TEA (228%) and 1 mM NMN (178%). Our data suggest that TEA, NMN and morphine are transported by a common transport mechanism for which NMN has only a low affinity.     

Transport of adenosine by renal brush border membranes

The transport of adenosine into brush border membrane vesicles from rat kidney is demonstrated. A first, rapid stage of transport is completed by 20 s. It is stimulated by a gradient of sodium. The effect of the concentration of adenosine in the low micromolar range on the initial rate of uptake indicates a saturability. An apparent Km of 1.48 microM and a Vmax of 215 pmol/mg protein/min have been calculated. In the rapid stage of uptake, adenosine is transported into the intravesicular space with no significant binding to the membrane. The following slow uptake is not stimulated by sodium and reaches steady state after about one hour.     

Proton-coupled transport of organic solutes in animal cell membranes and its relation to Na+ transport

Recent studies on proton-coupled transport of organic solutes in animal cell membranes were reviewed. In the intestinal and renal brush border membranes, transport of intact small peptides (di- or tri-peptides) has been established to be cotransported with H+. The peptide transport is Na+-independent, dependent on a pH gradient, electrogenic as revealed by transport-associated membrane depolarization and conductance increase, and reveals a marked overshoot uptake when a sufficiently large proton gradient is imposed across the membrane. Similar properties are found for L-lysine transport by the brush border membrane vesicles from mullet kidney and for L-leucine transport in some cultured cells. Partial involvement of H+ in Na+-dependent transport has also been reported for some organic acids, L-glutamate, and citrate. The physiological meanings of these purely H+-dependent and partially H+-dependent transports have been discussed based on available data.     

Phosphorylation of rat kidney proximal tubular brush border membranes

A possible correlation between cyclic-AMP dependent protein phosphorylation and altered sodium dependent transport of inorganic phosphate was analyzed in isolated rat renal proximal tubular brush border membrane vesicles. In transiently opened vesicles (opened by an osmotic shock), the addition of gamma-32P-ATP leads to 32P-incorporation into several membrane proteins. The simultaneous addition of cyclic-AMP leads to increased phosphorylation of several proteins (e.g. apparent molecular weights: 40 kD, 46 kD, 55 kD). The addition of ATP, GTP and ITP to the osmotic shock medium leads to an (non-specific) inhibition of the sodium gradient dependent phosphate uptake. No further inhibition of the sodium dependent phosphate transport was observed when membrane vesicles were phosphorylated by ATP in the presence of cyclic-AMP. These data show a lack of correlation between cyclic-AMP dependent protein phosphorylation and altered sodium gradient dependent phosphate transport. Thus, there is no experimental support for the involvement of cyclic-AMP dependent protein phosphorylation as one of the final events in the regulation of phosphate transport across the rat renal proximal tubular brush border membrane.     

Effect of hydrogen ion-gradient on carrier-mediated transport of glycylglycine across brush border membrane vesicles from rabbit small intestine

Glycylglycine (Gly-Gly) transport across rabbit small intestinal brush border membrane vesicles was studied in the presence and absence of a Na+ gradient or a pH gradient. The transport was found to be entirely independent of Na+ but significantly stimulated by lowering extravesicular pH (pHo). The maximum stimulation was seen at pHo 5.5, where the uptake rate was about 2-times higher than the control value. FCCP, a protonophore, abolished the stimulating effect of low pHo, and low pHo conditions without a pH gradient did not stimulate the uptake rate. Overshoot uptake of Gly-Gly was observed when a pH gradient of 2 pH units was imposed across the vesicular membrane. Valinomycin-induced inside-negative K+ diffusion potential also had a stimulating effect on the uptake, and fluorescence measurements of vesicular suspensions containing diS-C3-(5) revealed the occurrence of depolarization of the vesicular membranes when Gly-Gly was added to the suspensions. Kinetic study showed that a pH gradient caused a decrease of Kt for Gly-Gly without affecting Vmax. All the data obtained indicate that Gly-Gly transport is independent of Na+, dependent on a H+ gradient, and electrogenic, suggesting the mechanism of cotransport with H+.     

Coupled sodium-chloride transport by rabbit ileal brush-border membrane vesicles

Uptake of Na and Cl into brush-border membrane vesicles isolated from rabbit ileal epithelial cells was investigated with a rapid filtration technique using 22Na and 36Cl as tracers. The rank order of anion dependence for Na uptake in the absence of anion gradients was SCN greater than NO3 greater than gluconate. The sequence of cation specificity for Cl uptake was Na congruent to Li greater than K greater than choline. The transport of Na and Cl were both inhibited by harmaline, furosemide, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid. Carrier mediation of Cl-stimulated Na transport was suggested by the competition for 22Na uptake with increasing concentrations of unlabeled Na in the presence of Cl but not when gluconate was the counterion. Chloride-dependent Na uptake had an apparent Km of 4.5 mM and a Vmax of 20 nmol . mg prot-1 . 15 s-1. Na-H exchange and Cl-OH (or HCO3) exchange were also demonstrated in these vesicles. These findings confirm the presence of an electrically neutral carrier-mediated, Na-Cl-coupled transport process in the apical cell membrane of rabbit ileal epithelial cells. The nature of the coupling of Na to Cl transport, i.e., NaCl symport or a process that combines Na-H antiport with Cl-OH (or HCO3) antiport, remains to be determined.     

Tetraethylammonium transport by snake renal brush-border membrane vesicles

Transport of tetraethylammonium (TEA) by snake (Thamnophis spp.) renal brush-border membrane vesicles (BBMV) was studied. An outwardly directed proton gradient (pH 6.0 in, pH 7.5 out) stimulated uptake of TEA into BBMV and supported concentrative accumulation. Uptake of radioactively labeled TEA was also stimulated by outwardly directed gradients of unlabeled TEA and choline. The initial rate of TEA uptake was a saturable process that was adequately described by Michaelis-Menten kinetics. TEA uptake was not influenced by changes in the electrical potential difference across the membranes. Although uptake of TEA was stimulated by an outwardly directed Na⁺ gradient and inhibited by an inwardly directed Na⁺ gradient, these effects were probably secondary to the generation of proton gradients via a Na⁺/H⁺ exchanger demonstrated in these same BBMV. In agreement with previous studies with intact snake renal tubules, the present results indicate that TEA transport across the brush-border membrane involves electroneutral countertransport for protons or organic cations.     

Modulation by anions of p-aminohippurate transport in bovine renal basolateral membrane vesicles

In the presence of 10 M 2-oxoglutarate (2-OG) and of an inward Na⁺ gradient, uphill [³H]p-aminohippurate (PAH) uptake occurs due to cooperation of the PAH/2-OG exchanger and the Na⁺-coupled 2-OG transporter in bovine renal basolateral membrane vesicles. Uphill PAH uptake is observed with Cl^–, but not with gluconate as the bulk anion. To determine specificity and nature of this anion effect [³H]PAH uptake was measured in the presence of several anions without and with ionophores to distinguish indirect from direct effects on the PAH transporter. Na⁺-gradient plus 2-OGstimulated [³H]PAH uptake is fast with Cl^–, intermediate with F^–, Br^–, I^–, NO₃ ^– and SCN^–, and slow in the presence of gluconate, SO₄ ^2– and HPO₄ ^2–. Stimulation by Cl^– (as compared to gluconate) is attenuated, but not abolished, by clamping electrical potential and pH differences to zero, suggesting a partial effect through charge compensation and a major effect of anions on the PAH transporter itself. Indeed, [³H]PAH/2-OG and [³H]PAH/ PAH exchange rates under voltage- and pH-clamped condition depend on bulk anions although the anion effects are less pronounced than with Na⁺-gradient plus 2-OG-stimulated [³H]PAH uptake. Since an inward Cl^– gradient does not drive [³H]PAH above or below equilibrium distribution, Cl^– ions are most probably not translocated by the PAH transporter. We propose that anions modulate the PAH transporter by interacting with a site not directly related to anion transport.