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J B Pritchard

Publications and source records attributed to J B Pritchard.

At least 19 recordsLinked to original sources
PubMed1992

Functional expression of renal organic anion transport in Xenopus laevis oocytes.

Secretion of organic anions by the kidney plays a critical role in the elimination of toxic agents from the body. Recent findings in isolated membranes and intact tissue have demonstrated the participation of multiple transport proteins in this process. As a first step toward molecular characterization of these proteins through expression cloning, the studies reported below demonstrate functional expression of both fumarate- and lithium-sensitive glutarate and probenecid-sensitive p-aminohippurate transport in Xenopus oocytes injected with rat kidney poly(A)+ RNA. Maximal increase in substrate uptake over buffer-injected controls was reached by 5 days after mRNA injection. Expression of size-fractionated mRNA indicated that the active species with respect to both transport activities were in the range of 1.8 to 3.5 kb.

Animals↗
PubMed1991

Comparative insights into the mechanisms of renal organic anion and cation secretion.

Comparative models have played a major role in defining the mechanisms that enable vertebrate proximal tubules to transport organic anions and cations from the peritubular interstitium to the urine. The unique advantages of these models and their contributions to our understanding of organic anion and cation transport mechanisms are summarized here. Recent studies of the organic anion transport system suggest that transport is coupled to metabolic energy via indirect coupling to the sodium gradient. Organic anions enter the cell across the basolateral membrane in exchange for alpha-ketoglutarate (alpha-KG), and the alpha-KG is returned to the interior via Na-alpha-KG cotransport. Indirect coupling to Na has been demonstrated in both isolated membranes and intact renal epithelial cells of species ranging from marine crustaceans to mammals. This mechanism was shown to drive not only cellular accumulation but also secretory transepithelial fluxes of organic anions. Luminal exit of secreted organic anions appears to be carrier mediated but is, at present, poorly understood, with mediated potential-driven efflux and anion exchange-driven efflux implicated in some species. As for organic anions, the renal clearance of some organic cations approaches the renal plasma flow. Although there is considerable variation in the handling of specific substrates between species, the basic properties of organic cation transport include carrier-mediated potential-driven uptake at the basolateral membrane, intracellular sequestration that reduces the free concentration of the cation, and luminal exit by organic cation-proton exchange. Reabsorptive transport is also observed for some organic cations, but its mechanisms and driving forces are not well understood.

Animals↗
PubMed1991

Indirect coupling of organic anion secretion to sodium in teleost (Paralichthys lethostigma) renal tubules.

Recent findings in both rat and crab indicate that renal accumulation of p-aminohippurate (PAH) across the basolateral membrane can be coupled indirectly to the Na gradient through PAH-glutarate exchange and Na/glutarate cotransport. However, the role of this mechanism in net transepithelial PAH secretion was not examined. Therefore, proximal tubules from Southern flounder kidney were used to assess both the presence of indirect coupling in the fish and its relationship to net secretion. [14C]glutarate uptake by proximal tubular masses was concentrative, Na dependent, and Li inhibitable. Glutarate efflux from preloaded masses was stimulated by addition of PAH to the medium. Thus flounder tubules exhibited both Na/glutarate uptake and glutarate-PAH exchange. Furthermore, steady-state [3H]PAH accumulation was increased 50% by 10-50 microM glutarate, and this increase was abolished by Li, indicating indirect coupling of PAH entry to Na. To determine whether indirect coupling affected net secretion as well as tissue accumulation, the steady-state accumulation of an anionic dye, fluorescein (FL), was measured in individual renal tubules by use of epifluorescence microscopy and video-image analysis. FL accumulated in tubules to levels that were 20-40 times higher than the medium. In most fish, luminal fluorescence was measurably higher than cellular, and uptake in both compartments was markedly reduced by PAH and Li. Moreover, FL accumulation in cells and lumina was increased by 70-100% when 50 microM glutarate was added to the bathing medium. Thus glutarate not only stimulated uphill FL entry into the cells, but also stimulated active secretion into the tubular lumen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Relative roles of metabolism and renal excretory mechanisms in xenobiotic elimination by fish.

Renal clearance techniques were used to examine the relative contributions of metabolism and renal tubular transport in determining the rates of excretion of benzo(a)pyrene (BaP) and several of its phase I metabolites by southern flounder, Paralichthys lethostigma. Each compound (3H-labeled) was injected at a dose of 2.5 mumole/kg, producing plasma concentrations of 1 to 5 microM. Despite extensive plasma binding (greater than 95%), the uncorrected renal clearance of BaP-7,8-dihydrodiol exceeded the glomerular filtration rate (GFR) by more than 20-fold. Phenolic BaP metabolites also showed net secretion (1.5- to 3-fold). At times prior to 3 hr, BaP itself showed an average clearance of only 0.2 times the GFR. After 3 hr, BaP clearance increased to three times the GFR. Decreasing the dose of BaP injected also dramatically increased its clearance. Clearances of all four compounds studied were reduced by probenecid and other organic anion, including the herbicide 2,4-dichlorophenoxyacetic acid. HPLC analysis demonstrated that the bulk of the material excreted in the urine was not the parent compound, but sulfate or glucuronide conjugates of its phenolic or dihydrodiol metabolites. Excretion of sulfate conjugates predominated over the first 24 hr, whereas the glucuronide conjugates were the primary excretory products in succeeding days. In vitro, isolated renal tubules transported both glucuronide and sulfate conjugates, but sulfates were the preferred substrates. Isolated tubules were shown to be capable of catalyzing conjugation reactions, producing predominantly glucuronide conjugates. Liver slices produced both types of conjugates. Thus, the rapid excretion of BaP-7,8-dihydrodiol reflected a combination of two processes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Dehydroabietic acid, a major anionic contaminant of pulp mill effluent, reduces both active p-aminohippurate transport and passive membrane permeability in isolated renal membranes.

The renal organic anion transport system plays a pivotal role in elimination of potentially toxic anions. This system is driven by indirect coupling to the sodium gradient at the basolateral membrane, i.e., the organic anion enters the cell in exchange for internal alpha-ketoglutarate (alpha KG) and the in greater than out alpha KG gradient is regenerated by Na+/alpha KG cotransport. The resin acid, dehydroabietic acid (DHAA), is one of several anionic xenobiotics which enter the environment secondary to pulp and paper processing. Because it is largely ionized at neutral pH (pKa, 5.7), DHAA should share the organic anion system. Indeed, Na+/glutarate-coupled p-aminohippurate (PAH) uptake by renal basolateral membrane vesicles was inhibited competitively by DHAA (Ki congruent to 150 microM). Despite the reduced rate of PAH uptake, a substantial, but delayed, overshoot was observed, suggesting additional effects. Passive permeabilities to mannitol, PAH and sodium were all decreased by DHAA, consistent with a general tightening of the membrane. Decreased permeability extended the effective lifetime of imposed ion gradients. Thus, sodium driven glutarate uptake was stimulated by 200 microM DHAA, prolonging and more than doubling its overshoot. Because the immediate driving force for PAH uptake into basolateral membrane vesicles is the magnitude of the glutarate gradient, DHAA increased the driving force for PAH uptake and permitted a substantial overshoot despite the reduced rate of PAH uptake. These data indicate that DHAA has several distinctly different effects on the membrane.

Abietanes↗
PubMed1990

Sodium-coupled organic anion transport by Cancer borealis urinary bladder.

Recently, p-aminohippurate (PAH) transport by rat renal basolateral membrane (BLM) vesicles was shown to be coupled indirectly to the Na+ gradient through PAH-glutarate exchange and Na(+)-glutarate cotransport. We have examined the mechanism of PAH transport in Cancer borealis urinary bladder, a simple flat-sheet epithelium that is functionally analogous to vertebrate renal proximal tubule. These experiments indicate that crab bladder is capable of both Na(+)-coupled glutarate uptake and PAH-glutarate exchange and that PAH uptake may be coupled to Na+ in the intact tissue. First, glutarate uptake by intact bladder was lithium sensitive, Na(+) dependent, and inhibited by other dicarboxylates. In flux chambers, the bladder exhibited net glutarate secretion and tissue accumulation occurred primarily from the basolateral side. Furthermore, both BLM and brush-border membrane (BBM) vesicles also exhibited lithium-sensitive Na(+)-coupled glutarate uptake. Second, imposition of an in greater than out glutarate gradient markedly stimulated PAH uptake by bladder BLM vesicles, demonstrating PAH-glutarate exchange. In contrast, exchange was absent in BBM vesicles. Third, in intact bladder tissue, external glutarate increased the steady-state tissue-to-medium ratio for PAH from 14 +/- 1 to 19 +/- 1.5. This increase was both lithium inhibitable and Na+ dependent. Thus not only do bladder BLM show all the elements needed for indirect coupling of PAH transport to the Na+ gradient, but indirect coupling to Na+ can also drive uphill PAH transport in the intact epithelium.

Animals↗
PubMed1990

Rat renal cortical slices demonstrate p-aminohippurate/glutarate exchange and sodium/glutarate coupled p-aminohippurate transport.

In isolated basolateral membrane vesicles p-aminohippurate (PAH) transport may be coupled indirectly to the sodium gradient through PAH/glutarate [or alpha-ketoglutarate (alpha-KG)] exchange and Na/glutarate cotransport. In this study, rat renal cortical slices were used to examine indirect coupling of PAH transport to sodium in intact renal tissue. Like basolateral membrane vesicles, slices demonstrated avid uptake of 50 microM [14C]glutarate. Steady-state tissue/medium ratios of 30 were achieved by 90 to 120 min. Uptake was inhibited markedly by lithium and fumarate. PAH also inhibited glutarate accumulation, but through acceleration of glutarate efflux i.e., PAH/glutarate exchange, rather than direct inhibition of uptake. PAH-driven efflux of glutarate from slices was blocked by probenecid, which inhibits PAH/glutarate exchange in vesicles. Inasmuch as slices showed both Na/glutarate uptake and PAH/glutarate exchange, externally added glutarate should stimulate PAH uptake in slices. Indeed, in the presence of sodium, 50 microM external glutarate approximately doubled PAH accumulation by the slices. Stimulation by glutarate was abolished by either lithium or fumarate, or by elimination of sodium from the external buffer. The stimulatory effect was specific for glutarate or alpha-KG. Acetate, fumarate and succinate stimulated PAH uptake poorly, if at all. Neither fumarate nor lithium was able to eliminate concentrative PAH uptake completely, suggesting that a portion of PAH transport may occur without Na-dependent glutarate or alpha-KG recycling, i.e., that it may run directly off metabolic alpha-KG production or via a completely glutarate/alpha-KG independent mechanism.

Animals↗
PubMed1989

Organic anion and cation transport in crab urinary bladder.

Crab urinary bladder, a simple, flat-sheet epithelium, is structurally and functionally analogous to vertebrate renal proximal tubule. Like proximal tubule, crab bladder plays an important role in the excretion of potentially toxic, charged metabolites and xenobiotics. Bladders from Cancer borealis secrete monovalent, organic anions and cations in vivo and in vitro. For organic cations, secretion is a two-step process, with mediated and energetically downhill uptake into cells at the serosal membrane and uphill exit at the luminal membrane. The uptake step may be driven by the electrical potential difference across the serosal membrane, the luminal step by organic cation-proton exchange. Monovalent organic anions are also secreted by a separate two-step process. Recent experiments with intact bladder tissue and isolated membrane vesicles show that (as in mammalian proximal tubule) uphill serosal uptake can be coupled indirectly to the Na+ gradient. Organic anion (p-aminohippurate; PAH) uptake is driven by exchange for certain divalent organic anions, e.g., glutarate and alpha-ketoglutarate. The divalent anion gradient (in greater than out) is in turn maintained by Na+-coupled divalent uptake. The PAH exist step at the luminal membrane is mediated and downhill; it may involve anion exchange.

Animals↗
PubMed1988

2,4-Dichlorophenoxyacetic acid intoxication increases its accumulation within the brain.

Exposure to the phenoxyacetic acid herbicides has been shown to produce neurotoxicity. Therefore, adult mice (pregnant) and rabbits were used to examine the accumulation and regional distribution of 2,4-dichlorophenoxyacetic acid (2,4-D) within the brain following intraperitoneal injection of a low dose (0.2-0.4 mg/kg) of [14C]2,4-D. Controls, i.e. animals not previously exposed to 2,4-D, were compared to animals acutely pretreated with higher doses (40-160 mg/kg) of unlabeled 2,4-D. Both autoradiography and direct tissue analysis showed that in control animals brain levels were much lower than plasma in both adult (approximately 4%) and fetus (approximately 8%). In both species, small variations were seen between the brain regions, with brainstem and cerebellum somewhat higher than other regions. Pretreatment with unlabeled 2,4-D caused a 5- to 10-fold increase in accumulation of [14C]2,4-D in both mice and rabbits. On the other hand, 2-deoxyglucose entry into the brain was not altered by 2,4-D pretreatment. Thus, there was no generalized increase in blood-brain barrier permeability. Instead, increased 2,4-D accumulation appeared to be caused by its decreased elimination from the brain. Pretreatment with 40 mg/kg led to a CSF 2,4-D concentration of 10 microM, a concentration sufficient to inhibit choroid plexus transport of [14C]2,4-D by nearly 50% in vitro. These results suggest that exposure to organic anions like 2,4-D may lead to the retention of potentially toxic anions within the CNS via competitive inhibition of the organic anion transport system which normally reduces their brain and CSF concentrations to very low levels.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1988

Membrane potential drives organic cation transport into teleost renal proximal tubules.

The relationship between organic cation uptake and basolateral membrane potential was studied in renal tubules from two marine teleost fish, Southern flounder (Paralicthys lethostigma) and killifish (Fundulus heteroclitis). Carrier-mediated uptake of the model organic cation, tetraethylammonium (TEA), increased when K+ was changed from 2.5 to 0.2 mM and decreased when medium K+ was increased to 20 mM. Uptake was also reduced by the K+ channel blocker barium (1 mM). Furthermore, basolateral membrane potential hyperpolarized 15-25 mV in low-K+ medium and depolarized 30-40 mV in high-K+ medium. Barium also depolarized. Finally, basolateral membrane potential was depolarized in a concentration-dependent manner by addition of 100-500 microM TEA or Darstine. Thus treatments that hyperpolarize the basolateral membrane potential increase carrier-mediated TEA uptake, whereas those that depolarize basolateral membrane potential reduce uptake. Furthermore, organic cation transport into tubular cells involves the net influx of positive charge. Together, these findings support the argument that carrier-mediated organic cation uptake at the basolateral membrane is a potential-driven, electrogenic process.

Animals↗
PubMed1988

Coupled transport of p-aminohippurate by rat kidney basolateral membrane vesicles.

p-Aminohippuric acid (PAH) transport by basolateral membrane (BLM) vesicles isolated from rat renal cortex was stimulated very little by a Na+ gradient (out greater than in). However, when micromolar concentrations of glutaric acid or alpha-ketoglutaric acid were added in the presence of a out greater than in Na+ gradient, PAH uptake was accelerated greater than 20-fold and an overshoot of greater than fivefold was produced. Other anions, e.g., fumarate, stimulated PAH uptake very modestly under these conditions (approximately 2-fold), and that stimulation was totally prevented by short circuiting, i.e., with K+ (in = out) and valinomycin. Glutarate-stimulated uptake was inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and probenecid and was slightly stimulated by the imposition of an inside-negative membrane potential. Furthermore, even in the absence of a Na+ gradient, glutarate-loaded vesicles exhibited a marked acceleration of PAH uptake (5-fold) and a modest overshoot (2.5-fold). These results suggest an indirect coupling of BLM PAH uptake to the Na+ gradient by a cyclic accumulation (Na+-dependent) of glutarate followed by its efflux from the vesicle in exchange for PAH. This coupled system was absent in apical membranes. Thus net secretory transport of PAH may entail Na+-dependent, glutarate-driven PAH uptake at the BLM, followed by the exit of PAH into the lumen down its electrochemical gradient, probably in exchange for other anions, e.g., Cl-, HCO3-, or OH-.

Aminohippuric Acids↗
PubMed1987

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)

Aminohippuric Acids↗
PubMed1979

Toxic substances and cell membrane function.

The exposed location and functional importance of cell membranes make them particularly susceptible to the toxic effects of many chemicals. The likelihood of such effects has been appreciated for many years. However, the recent advent of new techniques has greatly increased our understanding of the complexities of membrane structure and function. These data make it quite clear that the interaction of toxic compounds with either the protein or the lipid component of cell membranes may substantially alter membrane function. This paper summarizes the current concepts of membrane structure and function and discusses the techniques currently in use to study cell membranes. Several examples are presented in which xenobiotics significantly alter membrane function. These include effects of heavy metals on passive ion permeability, impairment of osmoregulation and calcium transport by organochlorine pesticides, inhibition of the transport of neurotransmitter metabolites by phenoxyacetic acid herbicides in choroid plexus, and reduction in intestinal nutrient transport by heavy metals. Hence the study of the interactions of foreign compounds with membrane function may enhance our understanding of mechanisms both of toxicity and of basic membrane function.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1979

Determinants of the renal handling of 2, 4-dichlorophenoxyacetic acid by winter flounder.

The factors determining the renal handling of 2,4-dichlorophenoxyacetic acid (2,4-D) were examined in winter flounder, Pseudopleuronectes americanus, using isolated tubules and clearance techniques. In vitro, extensive energy-dependent uptake was seen with tissue/medium ratios of 30-fold at 1 micronM 2,4-D. The velocity of uptake was concentration-dependent with apparent Km and Vmax v,lues of 70 micronM and 3.6 micronmol/g of tubules per hr, respectively. Uptake was inhibited by other organic acids and 2,4-D competitively inhibited p-aminohippurate uptake. 2,4-D did not inhibit organic cation transport by the tubules. In vivo, 2,4-D was actively secreted with clearances of nearly 500 times the glomerular filtration rate at 1 micronM 2,4-D in plasma. At higher plasma concentrations (10-60 micronM) a transport maximum of 0.85 micronmol/g of kidney per hr was observed. Secretion was inhibited by other organic acids. 2,4-D also inhibited p-aminohippurate secretion in vivo. Little metabolism was noted; approximately 10% was excreted as the taurine conjugate. Plasma binding was 70%. Examination of the effects of added proteins on in vitro uptake showed that protein binding could limit 2,4-D transport but that flounder plasma (low in albumin) was far less effective than bovine serum albumin in binding and inhibition of transport. The roles of plasma binding, intracellular binding and metabolism in determining the rate of 2,4-D elimination by the kidney are discussed.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1978

Renal sugar transport in the winter flounder: V. secretion of 2-deoxy-D-galactose.

Isolated renal tubules and renal clearance techniques were used to characterize the renal handling of 2-deoxy-D-galactose (2-d-Gal) by the winter flounder (Pseudopleuronectes americanus). In vitro, energy-dependent, pH-sensitive uptake of 2-d-Gal (2-100 micron) was seen at the antiluminal face of the cell. Clearance measurements showed net secretion of 2-d-Gal in vivo. The mean clearance of 2-d-Gal in 18 fish was 0.98 +/- 0.16 ml/h while the glomerular filtration rate (GFR) was only 0.37 +/- 0.10 ml/h. Secretion was associated with marked renal accumulation of both 2-d-Gal and phosphorylated derivatives (2-d-Gal-1-phosphate). Tissue-to-plasma ratios (T/P) averaged 19 for free sugar and 59 for total sugar. Both clearance ratio and T/P were reduced to approximately 1 by injection of galactose (2.5 mmol/kg) simultaneously with 2-d-Gal (25 mumol/kg). Phlorizin (2.5 mumol/kg) increased net 2-d-Gal secretion, whereas glucose (2.5 mmol/kg) produced no change in secretion. Both compounds depressed 2-d-Gal T/P. This result suggests the presence of readsorptive transport at the brush border, sensitive to glucose and phlorizin.

Anaerobiosis↗
PubMed1978

Kinetic analysis of the renal handling of 2,2-bis(p-chlorophenyl) acetic acid by the rat.

The kinetics of uptake and efflux of organic acids in rat renal cortical slices were used to examine the affinity of 2,2-bis(p-chlorophenyl)acetic acid (DDA) for the organic acid transport system and to assess intracellular binding of this polar 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) metabolite. As judged by its ability to inhibit p-aminohippuric acid transport, DDA was a potent competitive inhibitor, almost as strong as probenecid, the classical inhibitor of this system. Efflux of DDA from slices demonstrated that the bulk (85%) of the DDA within the slice was reversibly bound to proteins or other macromolecules. Cortical slices incubated 60 minutes with 10 micron DDA contained a total concentration of 160 micron DDA within the tubular cells, but the actual free concentration in the cells was only 20 to 30 micron. Thus, although DDA was accumulated against a concentration gradient by the kidney, the gradient was much smaller than the measured tissue/medium ratio. Potential consequences of DDA exposure through its interaction with the organic acid system and roles of DDA binding sites in the toxicity and transport of DDA are discussed.

Animals↗
PubMed1977

Renal handling of the polar DDT metabolite DDA (2,2-bis[p-chlorophenyl] acetic acid) by marine fish.

The renal handling of 2,2-bis(p-chlorophenyl) acetic acid (DDA) was examined in the isolated tubules of the winter flounder (Pseudopleuronectes americanus) in vitro in conjunction with clearance studies in the flounder and in the aglomerular goosefish (Lophius americanus). In vitro, both uptake studies and autoradiography showed extensive energy-dependent accumulation within the cytoplasm of tubular cells and the tubular lumen. The uptake was strongly inhibited by p-aminohippurate and chlorophenol red. A second component of uptake was insensitive to metabolic inhibitors or organic acids and represented tissue binding. In vivo, both species showed net secretion which was inhibited by probenecid. Comparison of DDT and DDA distribution and excretion emphasized the importance of the greater water solubility of DDA and of its secretory transport, since DDA was excreted at over 200 times the rate of DDT. Liver, kidney, and bile also showed elevated DDA tissue-to-plasma ratios. Thus, the organic acid system mediates the accumulation and excretion of DDA in these fish.

Animals↗