p-aminohippuric acid transport at renal apical membrane mediated by human inorganic phosphate transporter NPT1

Organic anions are secreted into urine via organic anion transporters across the renal basolateral and apical membranes. However, no apical membrane transporter for organic anions such as p-aminohippuric acid (PAH) has yet been identified. In the present study, we showed that human NPT1, which is present in renal apical membrane, mediates the transport of PAH. The K(m) value for PAH uptake was 2.66 mM and the uptake was chloride ion sensitive. These results are compatible with those reported for the classical organic anion transport system at the renal apical membrane. PAH transport was inhibited by various anionic compounds. Human NPT1 also accepted uric acid, benzylpenicillin, faropenem, and estradiol-17beta-glucuronide as substrates. Considering its chloride ion sensitivity, Npt1 is expected to function for secretion of PAH from renal proximal tubular cells. This is the first molecular demonstration of an organic anion transport function for PAH at the renal apical membrane.     

Luminal and peritubular steps in renal transport of p-aminohippurate

Why has the PAH transport system proven so difficult to characterize? The major problems appear to arise in three areas: species differences, differences in methodology, e.g., vesicle preparation techniques, and multiplicity of related transport systems. Species differences are certainly important in some areas, e.g., the luminal membrane where the same investigators have shown the presence of an anion exchanger capable of transporting PAH in rat and dog but not in rabbit. Since all of these species effectively secrete PAH, one must question whether or not the primary luminal component of the PAH secretory system has yet been identified. Other species differences have also been described. For example, the amphibian, Necturus, demonstrates bidirectional organic anion transport, including an uphill luminal step and the urinary bladders of certain species of crustaceans show net reabsorption, whereas the bladders of other species show net secretion of PAH. However, these differences may well prove to be important tools in assessing PAH transport, since amplification of specific pathways and the increased experimental control possible in intact tissue preparations from some of these species, e.g., flounder, snake and amphibian tubules or crustacean urinary bladder, may facilitate resolution of many of the remaining uncertainties. Species differences cannot explain the wide variety of results reported for the basolateral membrane transport step, since many of the conflicting studies were done in the same species. Difficulties inherent in vesicle techniques have been discussed above (Subsection III-A2), and emphasize the need to correlate such data with intact tissue preparations. However, the major source of confusion appears to be related to the ability of PAH to interact with several transport systems, directly or indirectly. Thus, despite the preponderance of evidence showing that the PAH transport system at the basolateral membrane is distinct from those for sulfate, mono- and dicarboxylic acids, acidic amino acids, and uric acid, there remains the real possibility that under physiological conditions: PAH may be a minor substrate for these other systems, substrates for other systems may inhibit PAH transport directly through competition for the PAH carrier or indirectly through competition for the same energy source, and entry of a substrate on one system may trans-stimulate PAH uptake on another. Furthermore, the existence of multiple systems may explain the inability of certain manipulations, e.g., Na gradient dissipation in vivo, to block PAH transport. PAH entry may simply increase via another pathway, e.g., anion exchange.(ABSTRACT TRUNCATED AT 400 WORDS)     

Riboflavin transport by isolated perfused rabbit renal proximal tubules

Rabbit renal proximal tubular transport of riboflavin(RF) was examined by using the in vitro isolated tubule perfusiontechnique. We found that proximal tubules actively reabsorbed(J_lb) and secreted (J_bl)RF. At 0.1 µM RF concentration, J_bl wassignificantly higher than J_lb, resulting in anet secretion. This net secretion of RF was decreased at 0.01 µM RFconcentration and increased at 1 µM RF concentration. BothJ_lb and J_bl wereinhibited by lowering temperature or by adding iodoacetate, a metabolicinhibitor, and lumichrome, an RF analog, suggesting the involvement ofcarrier-mediated transport mechanisms. J_bl wasinhibited by probenecid, an anion transport inhibitor, and bypara-aminohippuric acid, an organic anion, suggesting therelevance of RF secretion to renal organic anion transport.J_bl was also inhibited by alkaline pH (8.0) and by the calmodulin inhibitor trifluoperazine, indicating the influence of pH and Ca²⁺/calmodulin-dependent pathway on RFsecretion. Finally, we found that addition of chlorpromazine, aphenothiazine derivative, inhibited both J_lb andJ_bl, raising the concern about the nutritionalstatus in patients receiving such a type of medication.

     

Murine renal organic anion transporters mOAT1 and mOAT3 facilitate the transport of neuroactive tryptophan metabolites

Tryptophan metabolites such as kynurenate (KYNA), xanthurenate (XA), and quinolinate are considered to have an important impact on many physiological processes, especially brain function. Many of these metabolites are secreted with the urine. Because organic anion transporters (OATs) facilitate the renal secretion of weak organic acids, we investigated whether the secretion of bioactive tryptophan metabolites is mediated by OAT1 and OAT3, two prominent members of the OAT family. Immunohistochemical analyses of the mouse kidneys revealed the expression of OAT1 to be restricted to the proximal convoluted tubule (representing S1 and S2 segments), whereas OAT3 was detected in almost all parts of the nephron, including macula densa cells. In the mouse brain, OAT1 was found to be expressed in neurons of the cortex cerebri and hippocampus as well as in the ependymal cell layer of the choroid plexus. Six tryptophan metabolites, including the bioactive substances KYNA, XA, and the serotonin metabolite 5-hydroxyindol acetate inhibited [³H]p-aminohippurate (PAH) or 6-carboxyfluorescein (6-CF) uptake by 50–85%, demonstrating that these compounds interact with OAT1 as well as with OAT3. Half-maximal inhibition of mOAT1 occurred at 34 µM KYNA and 15 µM XA, and it occurred at 8 µM KYNA and 11.5 µM XA for mOAT3. Quinolinate showed a slight but significant inhibition of [³H]PAH uptake by mOAT1 and no alteration of 6-CF uptake by mOAT3. [¹⁴C]-Glutarate (GA) uptake was examined for both transporters and demonstrated differences in the transport rate for this substrate by a factor of 4. Trans-stimulation experiments with GA revealed that KYNA and XA are substrates for mOAT1. Our results support the idea that OAT1 and OAT3 are involved in the secretion of bioactive tryptophan metabolites from the body. Consequently, they are crucial for the regulation of central nervous system tryptophan metabolite concentration. kidneys; brain; macula densa; transforming growth factor; N-methyl-D-aspartate receptor     

Effects of anoxia and low pH on organic ion transport and electrolyte distribution in harbor seal (Phoca vitulina) kidney slices

Summary The present study was undertaken to examine the effects of anoxia and/or low pH on active organic ion transport and tissue water and electrolyte distribution in freshly prepared cortical slices from harbor seal and rat kidneys.p-Aminohippurate (PAH) and tetraethylammonium (TEA) were used as a representative organic anion and cation, respectively. The slice uptake of PAH was not influenced by the incubation medium pH over a range of 5.0–7.5 in the harbor seal while it was very sensitive to the medium pH in the rat, decreasing significantly with reductions in pH (Fig. 1). Although the TEA uptake decreased in anoxia in both species, it decreased considerably less in the harbor seal than in the rat (40% vs 74%) (Fig. 2). However, in contrast to the PAH transport system, the TEA uptake decreased significantly at low pH in both species. The magnitudes of increase in intracellular Na and of decrease in intracellular K in anoxia were consistently less in the harbor seal than in the rat, especially at low pH (Fig. 3). However, the pH dependence of Na–K-ATPase activity of the crude renal cortical homogenate was not different between the two species (Fig. 4). These results, together with the previous finding on the anoxic tolerance of the renal organic anion transport system, indicate that (1) the overall anoxic tolerance of active renal transport systems for both organic anions and cations is greater in the harbor seal than in the rat, and (2) the renal organic anion (but not cation) transport system is resistant to low pH in the harbor seal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetate - KRP Krebs-Ringer phosphate buffer - PAH para-aminohippurate - S/M slice to medium concentration ratio - TEA tetraethylammonium     

Effect of piperonyl butoxide on organic anion and cation transport in rabbit kidneys

Piperonyl butoxide has been shown to reduce accumulation of cephaloridine in rabbit renal cortex; however, the mechanism responsible for this effect remains unclear. Cephaloridine is a zwitterion and its accumulation in renal cortex has been suggested to be regulated by both organic anion and cation transport systems. Thus, it was of interest to determine the effect of piperonyl butoxide on renal transport of p-aminohippurate (PAH, an organic anion) and tetraethylammonium (TEA, an organic cation). Although pretreatment with piperonyl butoxide markedly inhibited renal cortical uptake of cephaloridine, the same treatment had less inhibitory effect on either PAH or TEA uptake. Efflux of PAH from preloaded renal cortical slices was enhanced by pretreatment with piperonyl butoxide; however, TEA efflux was unaffected. Thus, piperonyl butoxide appears to have effects on renal membrane functions which result in differential effects on PAH, TEA, and cephaloridine transport.     

Renal elimination of organic anions in rats with bilateral ureteral obstruction

Urinary tract obstruction is an important cause of acute renal failure. Several abnormalities in renal tubular function may occur in obstructive nephropathy. The tubular secretion of organic anions is an important function of the kidney that eliminates potentially toxic organic anions from the body, however, the mechanisms involved in organic anions renal elimination in rats with bilateral ureteral obstruction (BUO) have not been elucidated. In this study, it was evaluated the renal handling of p-aminohippurate (PAH) in adult male Wistar rats with BUO. A diminished renal clearance of PAH was observed in BUO rats as consequence of a diminution in the secreted load of this organic anion. The increase in the abundance of organic anions transporter 1 (OAT1) and the absence of modification in cortical renal blood flow, measured with fluorescence microspheres, do not explain the altered secretion of PAH. The diminished Na,K-ATPase activity in cortex from obstructed kidneys might condition OAT1 function. Additionally, it is also possible to conclude that in the presence of BUO, PAH clearance is not a good estimate of renal plasma flow.     

Facilitated diffusion of urate in avian brush-border membrane vesicles

Membrane transport pathways mediatingtranscellular secretion of urate across the proximal tubule wereinvestigated in brush-border membrane vesicles (BBMV) isolated fromavian kidney. An inside-positive K diffusion potential induced aconductive uptake of urate to levels exceeding equilibrium.Protonophore-induced dissipation of membrane potential significantlyreduced voltage-driven urate uptake. Conductive uptake of urate wasinhibitor sensitive, substrate specific, and a saturable function ofurate concentration. Urate uptake was trans-stimulated byurate and cis-inhibited by p-aminohippurate (PAH). Conductive uptake of PAH was cis-inhibited by urate.Urate uptake was unaffected by an outward -ketoglutarate gradient. In the absence of a membrane potential, urate uptake was similar in thepresence and absence of an imposed inside-alkaline pH gradient or anoutward Cl gradient. These observations suggest a uniporter-mediated facilitated diffusion of urate as a pathway for passive efflux acrossthe brush border membrane of urate-secreting proximal tubule cells.

     

Comparative mutant analysis of Arabidopsis ABCC-type ABC transporters: AtMRP2 contributes to detoxification, vacuolar organic anion transport and chlorophyll degradation

The enormous metabolic plasticity of plants allows detoxificationof many harmful compounds that are generated during biosyntheticprocesses or are present as biotic or abiotic toxins in theirenvironment. Derivatives of toxic compounds such as glutathioneconjugates are moved into the central vacuole via ATP-bindingcassette (ABC)-type transporters of the multidrug resistance-associatedprotein (MRP) subfamily. The Arabidopsis genome contains 15AtMRP isogenes, four of which (AtMRP1, 2, 11 and 12) clustertogether in one of two major phylogenetic clades. We isolatedT-DNA knockout alleles in all four highly homologous AtMRP genesof this clade and subjected them to physiological analysis toassess the function of each AtMRP of this group. None of thesingle atmrp mutants displayed visible phenotypes under controlconditions. In spite of the fact that AtMRP1 and AtMRP2 hadbeen described as efficient ATP-dependent organic anion transportersin heterologous expression experiments, the contribution ofthree of the AtMRP genes (1, 11 and 12) to detoxification ismarginal. Only knockouts in AtMRP2 exhibited a reduced sensitivitytowards 1-chloro-2,4-dinitrobenzene, but not towards other herbicides.AtMRP2 but not AtMRP1, 11 and 12 is involved in chlorophylldegradation since ethylene-treated rosettes of atmrp2 showedreduced senescence, and AtMRP2 expression is induced duringsenescence. This suggests that AtMRP2 is involved in vacuolartransport of chlorophyll catabolites. Vacuolar uptake studiesdemonstrated that transport of typical MRP substrates was reducedin atmrp2. We conclude that within clade I, only AtMRP2 contributessignificantly to overall organic anion pump activity in vivo.     

Lack of interaction between atrial natriuretic factor and renal organic anion transport system

The aim of the present investigation was to test the hypothesis that atrial natriuretic factor (ANF) is secreted into the proximal tubule lumen by the organic anion transport mechanism. The rationale for this hypothesis was the reported probenecid attenuation of the natriuretic effect of ANF. Probenecid is widely regarded as an inhibitor of organic acid transport in the proximal tubule. ANF was prepared in varying degrees of purity ranging from a relatively crude extract to a highly purified form. A commercially available form was also used. All forms were bioassayed using the anesthetized rat and a diuresis and natriuresis was observed in each case which was comparable to literature reports. Interaction of ANF with the organic acid transport system was evaluated using the renal cortical slice technique. Over a wide range of concentrations, there was no effect of ANF on cortical slice accumulation of either p-aminohippurate (PAH), the classical substrate of the organic anion transport system or tetraethylammonium (TEA), a typical organic cation. It is concluded that although ANF may indeed exert its effect at the luminal membranes of the nephron, access to the lumen is not mediated by the organic cation or anion transport system in the proximal tubule.     

Modulation of the basolateral and apical step of transepithelial organic anion secretion in proximal tubular opossum kidney cells. Acute effects of epidermal growth factor and mitogen-activated protein kinase

The organic anion transport system in the proximal tubule of the kidney is of major importance for the excretion of a variety of endogenous and potentially toxic exogenous substances. Furthermore, the clearance of model substrates (e.g. para-aminohippurate) of this system is used for the determination of renal blood flow. We investigated regulation of organic anion secretion in a way that allowed us to examine simultaneously regulation of overall transepithelial secretion and to estimate the separate contributions of regulation of the basolateral and apical transport steps to this overall regulation. The data were verified by measurement of initial basolateral uptake rate and initial apical efflux rate. Opossum kidney cells were used as a suitable model system for proximal tubule cells, and [14C]para-aminohippurate was utilized as an organic anion. Stimulation of protein kinase C inhibited transepithelial secretion because of inhibition of both apical efflux and basolateral uptake. Inhibition of the mitogen-activated protein kinase (MAPK) kinase MEK reduced transepithelial secretion via inhibition of basolateral uptake and apical efflux. Epidermal growth factor (EGF) enhanced transepithelial secretion via stimulation of basolateral uptake but did not affect apical efflux. EGF induced stimulation of basolateral uptake was abolished by inhibition of MEK. EGF led to phosphorylation of ERK1/2, which was also abolished by inhibition of MEK. Thus, EGF stimulated basolateral uptake of organic anions via MAPKs. Transepithelial organic anion secretion can be regulated at two sites, at least: basolateral uptake and apical efflux. Both steps are under control of protein kinase C and MAPK. The pathophysiologically relevant growth factor EGF enhances transepithelial secretion via stimulation of basolateral uptake. EGF stimulates basolateral uptake via MEK and ERK1/2. Thus, renal organic anion extraction may be modulated, especially under pathophysiological conditions.     

Decreased renal organic anion secretion and plasma accumulation of endogenous organic anions in OAT1 knock-out mice

The "classical" organic anion secretory pathway of the renal proximal tubule is critical for the renal excretion of the prototypic organic anion, para-aminohippurate, as well as of a large number of commonly prescribed drugs among other significant substrates. Organic anion transporter 1 (OAT1), originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J. G., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478), has physiological properties consistent with a role in this pathway. However, several other transporters (e.g. OAT2, OAT3, and MRP1) have also been proposed as important PAH transporters on the basis of in vitro studies; therefore, the relative contribution of OAT1 has remained unclear. We have now generated a colony of OAT1 knock-out mice, permitting elucidation of the role of OAT1 in the context of these other potentially functionally redundant transporters. We find that the knock-out mice manifest a profound loss of organic anion transport (e.g. para-aminohippurate) both ex vivo (in isolated renal slices) as well as in vivo (as indicated by loss of renal secretion). In the case of the organic anion, furosemide, loss of renal secretion in the knock-out results in impaired diuretic responsiveness to this drug. These results indicate a critical role for OAT1 in the functioning of the classical pathway. In addition, we have determined the levels of approximately 60 endogenous organic anions in the plasma and urine of wild-type and knock-out mice. This has led to identification of several compounds with significantly higher plasma concentrations and/or lower urinary concentrations in knock-out mice, suggesting the involvement of OAT1 in their renal secretion. We have also demonstrated in xenopus oocytes that some of these compounds interact with OAT1 in vitro. Thus, these latter compounds might represent physiological substrates of OAT1.     

Expression and function of TETRAN, a new type of membrane transporter

In contrast to transport across basolateral membranes, the mechanism governing transport of organic anions across the luminal membranes of proximal tubules has remained unclear. We recently found Tetracycline transporter-like protein (TETRAN), a human ortholog of yeast Tpo1p that can transport anionic Non-steroidal anti-inflammatory drugs (NSAIDs). In this study, we examine the expression and function of TETRAN. TETRAN mRNA is expressed in various human tissues, including kidney. When overexpressed in cultured cells, TETRAN was predominantly localized on cytoplasmic membranes. Immunohistochemical analysis of human and mouse kidney tissue showed that TETRAN was expressed at the luminal membranes of proximal tubules. Overexpression of TETRAN in cultured cells facilitated the uptake of organic anions such as indomethacin (a NSAID) and fluorescein. The results suggest that TETRAN is a novel human organic anion transporter, and that it serves as a transporter for some NSAIDs and various other organic anions at the final excretion step.     

Renal excretion of chlorphenol red and related organic acids in the intact flounder Pseudopleuronectes americanus

Renal clearance experiments were performed on unanesthetized winter flounder from which bladder urine was collected continuously and caudal vein blood was sampled periodically; renal tissue was also obtained terminally for comparison of test organic acid content in vivo and after incubation in vitro. Urine flow rates and inulin U/P (urine to plasma concentration) ratios were relatively constant for a given fish and averaged 1.0 ml/hr X kg fish and 2.6, respectively. In contrast, U/P ratios for all three test acids cycled from minima of near 100 to maxima of over 1000 roughly every 24 hr when plasma concentrations of unbound acid were below 1 micron; correction of plasma protein binding was required in the case of chlorphenol red, but not PAH or Diodrast. Both in vivo and in vitro the organic acid content of renal tissue was intermediate between plasma and urine concentrations. These results demonstrate that kidneys of intact flounder exhibit the remarkable concentrative capacity for exogenous organic acids previously observed with isolated tubules and suggest that the tubular urine concentration is established in two steps by cell transport first at the peritubular and second at the luminal membrane. The anterior kidney position and the magnitude of maximal PAH and Diodrast clearances, about 1000 ml/hr X kg flounder, are consistent with most of the cardiac output returning to the heart through the renal portal circulation; a regulatory shunt bypassing the peritubular capillaries is proposed to explain cycling of organic acid clearances to minimal values.     

Stereoselective renal secretion of carbenicillin in rabbits: Role of the organic anion transporter at the renal brush border membrane

Stereoselectivity in the renal secretion of carbenicillin (CBPC) was studied in rabbits. Significant renal secretion of CBPC was observed in vivo, with the secretion of the S-epimer being greater than that of the R-epimer. Stereoselective transport of CBPC was further studied in vitro using basolateral and brush border membrane vesicles prepared from rabbit kidneys. The transport of CBPC by the organic anion transporter into the basolateral membrane vesicles (BLMV) was not stereoselective. In contrast, a distinct stereoselectivity was observed in the transport of CBPC by the organic anion transporter into the brush border membrane vesicles (BBMV), with the transport of the S-epimer being more favorable. Significant epimer-epimer interactions were also observed in the transport into BBMV. The stereoselectivity of the transport of CBPC was calculated from the kinetic parameters with consideration of epimer-epimer interactions and was similar to that observed in vivo. It was concluded that the observed stereoselectivity in the renal secretion of CBPC in vivo reflected that of transport via the organic anion transporter located at the brush border membrane. Chirality 10:349–357, 1998. © 1998 Wiley-Liss, Inc.     

Nutrient Uptake and Organic Acid Anion Metabolism in Lupins and Peas Supplied with Nitrate

Unlike many plants reported in the literature, lupins do notexcrete OH^- in amounts equivalent to the net excess of inorganicanion uptake over inorganic cation uptake. To investigate themechanisms involved in the maintenance of charge balance, nutrientuptake and organic anion accumulation of lupins and peas suppliedwith a range of NO^-₃ concentrations, were compared. Lupins absorbed less NO^-₃ than peas on a dry weight basis, whichlargely accounted for the smaller excess of anion uptake overcation uptake in lupins than in peas at the same NO^-₃ supply.When anion uptake exceeded cation uptake, peas excreted an equivalentcharge of OH^-, whereas lupins excreted much smaller amountsof OH^- than the excess of anion over cation uptake. It was calculatedthat lupins excreted significant amounts of organic anions whenanion uptake exceeded cation uptake, whereas organic anion excretionfrom peas was negligible, regardless of their NO^-₃ supply andcation-anion balance. In this study, organic anion excretion was measured from lupinroots grown in near-sterile conditions while supplied with NO^-₃at 0, 500 and 2000 µM. Although complete sterility wasnot achieved, there was close agreement between the organicanion excreted and the excess anion over cation uptake.Copyright1994, 1999 Academic Press Lupinus angustifolius L., Pisum sativum L., organic acid, nutrient uptake     

Glutamine transport by the blood-brain barrier: a possible mechanism for nitrogen removal

Glutamine and glutamate transport activities were measuredin isolated luminal and abluminal plasma membrane vesiclesderived from bovine brain endothelial cells. Facilitativesystems for glutamine and glutamate were almost exclusivelylocated in luminal-enriched membranes. The facilitativeglutamine carrier was neither sensitive to2-aminobicyclo(2,2,1)heptane-2-carboxylic acid inhibition nor did itparticipate in accelerated amino acid exchange; it therefore appearedto be distinct from the neutral amino acid transport system L1. TwoNa-dependent glutamine transporters were found in abluminal-enrichedmembranes: systems A and N. System N accounted for ~80% ofNa-dependent glutamine transport at 100 µM. Abluminal-enriched membranes showed Na-dependent glutamate transport activity. The presence of 1) Na-dependent carrierscapable of pumping glutamine and glutamate from brain into endothelialcells, 2) glutaminase withinendothelial cells to hydrolyze glutamine to glutamate and ammonia, and3) facilitative carriers forglutamine and glutamate at the luminal membrane may provide a mechanismfor removing nitrogen and nitrogen-rich amino acids from brain.

     

Effect of S-(2-chloroethyl)-DL-cysteine on the transport of p-aminohippurate ion in renal plasma membrane vesicles

The effects of S-(2-chloroethyl)-DL-cysteine (CEC) (a potent nephrotoxin) on the transport of p-aminohippurate ion (PAH) in renal plasma membrane vesicles isolated from rat renal cortex were studied in vitro. The uptake of PAH was significantly reduced in a dose-dependent manner in both the brush border membrane (BBM) and basolateral membrane (BLM) vesicles. These results demonstrate that CEC is capable of interfering with the accumulation of PAH (a model organic anion for renal tubular transport system) by both energy-independent and energy-dependent carrier-mediated transport processes. Probenecid, a typical inhibitor of the organic anion transport system, showed the highest inhibition of PAH uptake in both the membranes vesicles. These data indirectly suggest that transport by renal tubular cells may result in the accumulation of CEC in renal cellular organelles eventually in toxic concentrations. Thus, CEC showed both dose- and time-dependent inhibition of the activities of gamma-glutamyl transferase (a BBM marker enzyme) and Na+, K(+)-ATPase (a BLM marker enzyme), while no such inhibition was noticed with probenecid. Pretreatment with probenecid prevented the inhibition of the gamma-glutamyl transferase activity due to CEC in BBM, but failed to do so for the Na+,K(+)-ATPase activity in BLM vesicles. Thus, the data suggest that the inhibition of the activities of these membrane-specific enzymes by CEC could lead to the initial development of its nephrotoxicity.     

p-Aminohippurate transport in canine tracheal epithelium

The unidirectional fluxes of 20, 100, 500, and 2,000 microM rho-aminohippurate (PAH) were measured under open- and short-circuit conditions in canine tracheal epithelium mounted as flat sheets in Ussing chambers. In tissues pretreated with mucosal indomethacin (10(-6) M) and amiloride (10(-4) M), unidirectional PAH fluxes under short-circuit conditions increased with increasing bath concentrations but there was no significant net PAH transport. After stimulation of chloride secretion by mucosal cyclic adenosine 3',5' -cyclic monophosphate (cAMP 10(-3) M), there was a significant increase in the secretory flux of PAH and a significant decrease in the absorptive flux of PAH. This resulted in net PAH secretion that demonstrated saturation kinetics with an apparent Michaelis-Menten constant of 754 microM by Lineweaver-Burk analysis. Intracellular concentrations of PAH were 0.4-1.2 times bath concentrations after pretreatment with indomethacin and amiloride and increased to 2.6-3.3 times bath concentrations after cAMP. Under open-circuit conditions, secretory PAH flux decreased and absorptive flux increased resulting in net PAH absorption. We conclude from these early studies that the canine tracheal epithelium possesses a specialized system for the transport of organic anions in the airways and that this transport system may share many similarities with organic anion transport in the kidney.     

Renal transport of gossypol in the rabbit

The present work was carried out to investigate the transport characteristics of gossypol, a toxic weak organic acid (pK = 7.2) contained in cottonseed, into the rabbit renal cortical slice. The uptake of gossypol increased linearly during a 2-hr incubation after which it leveled off with the average slice-to-medium concentration ratio (S/M) slightly above 20. In the presence of metabolic inhibitors, the S/M gossypol leveled off at about 9, suggesting an extensive binding of gossypol to tissue proteins. The uptake of gossypol was significantly inhibited by p-aminohippurate (PAH), probenecid, ouabain, and DIDS, all of which are known inhibitors of renal organic anion transport. However, the gossypol uptake was not affected by tetraethylammonium (TEA), a prototypical organic cation. Kinetic studies indicated that the apparent Km for gossypol transport is 0.28 mM, and also that probenecid inhibits gossypol transport in a competitive manner. It is concluded that gossypol is transported by the renal tubule through the classic organic anion system.