Uptake Of Organic Solutes Research Articles

Aqueous pathways dominate permeation of solutes across Pisum sativum seed coats and mediate solute transport via diffusion and bulk flow of water.

The permeability of seed coats to solutes either of biological or anthropogenic origin plays a major role in germination, seedling growth and seed treatment by pesticides. An experimental set-up was designed for investigating the mechanisms of seed coat permeation, which allows steady-state experiments with isolated seed coats of Pisum sativum. Permeances were measured for a set of organic model compounds with different physicochemical properties and sizes. The results show that narrow aqueous pathways dominate the diffusion of solutes across pea seed coats, as indicated by a correlation of permeances with the molecular sizes of the compounds instead of their lipophilicity. Further indicators for an aqueous pathway are small size selectivity and a small effect of temperature on permeation. The application of an osmotic water potential gradient across isolated seed coats leads to an increase in solute transfer, indicating that the aqueous pathways form a water-filled continuum across the seed coat allowing the bulk flow of water. Thus, the uptake of organic solutes across pea testae has two components: (1) by diffusion and (2) by bulk water inflow, which, however, is relevant only during imbibition.

Changes in the Plasmodial Surface Anion Channel Reduce Leupeptin Uptake and Can Confer Drug Resistance in Plasmodium falciparum -Infected Erythrocytes

Cysteine protease inhibitors kill malaria parasites and are being pursued for development as antimalarial agents. Because they have multiple targets within bloodstream-stage parasites, workers have assumed that resistance to these inhibitors would not be acquired easily. In the present study, we used in vitro selection to generate a parasite resistant to growth inhibition by leupeptin, a broad-profile cysteine and serine protease inhibitor. Resistance was not associated with upregulation of cysteine protease activity, reduced leupeptin sensitivity of this activity, or expression level changes for putative cysteine or serine proteases in the parasite genome. Instead, it was associated with marked changes in the plasmodial surface anion channel (PSAC), an ion channel on infected erythrocytes that functions in nutrient and bulky organic solute uptake. Osmotic fragility measurements, electrophysiological recordings, and leupeptin uptake studies revealed selective reductions in organic solute permeability via PSAC, altered single-channel gating, and reduced inhibitor affinity. These changes yielded significantly reduced leupeptin uptake and could fully account for the acquired resistance. PSAC represents a novel route for the uptake of bulky hydrophilic compounds acting against intraerythrocytic parasite targets. Drug development based on such compounds should proceed cautiously in light of possible resistance development though the selection of PSAC mutants.

New perspectives for the treatment of cholestasis: Lessons from basic science applied clinically

Cholestasis is a clinical syndrome that results from the disturbances in the formation of bile, a unique and vital property of the liver. The causes of cholestasis are broad and range from rare genetic diseases and disruption of the normal development of the bile excretory anatomy, to progressive, ultimately fatal diseases such as primary biliary cirrhosis and sclerosing cholangitis(1). Cholestasis may also develop from mechanical obstructions in the extrahepatic bile ducts or during the course of viral hepatitis or the administration of certain drugs and hormones. Whatever the cause, the syndrome is manifested by the hepatic retention of products normally excreted into bile, in particular bile salts. As the process progresses with time, jaundice and hypercholesterolemia may follow. The syndrome is almost always characterized by an elevation in serum alkaline phosphatase and if progressive, fibrosis, cirrhosis and clinical signs of liver failure ultimately develop. During the past decade, as the molecular basis of bile formation has been clarified, the pathogenesis of many cholestatic disorders has also evolved resulting in the following paradigm. Determinants of bile secretion undergo an adaptive response during cholestasis which tend to minimize hepatic injury. This adaptation occurs by: (a) limiting hepatic uptake of bile acids and other organic solutes, (b) reducing bile acid synthesis, (c) accelerating bile acid detoxification and (d) up-regulating alternative pathways for excretion of bile salts and other solutes in liver, kidney and intestine Much of this information has resulted from the study of animal models of cholestasis, which with some exceptions have been confirmed in patients with cholestasis (1–3). The molecular basis of this process, which underlies current efforts to intervene clinically, has resulted from 3 fundamental developments: 1) The cloning of transporters that are the determinants of hepatobiliary secretion (greatly advanced by the completion of the human genome project) (1). 2) The discovery that mutations in some of these transporters result in cholestatic liver disease in patients (thereby providing “proof of principle” for their function (4) and 3) The evolving field of nuclear receptors which now enable a partial understanding of how these critical transporter and metabolism genes are transcriptionally regulated (5;6) (Fig 1). Fig 1 Drugs that regulate hepatic bile acid and other organic solute uptake, synthesis, detoxification and secretion. The figure illustrates their action as nuclear receptor ligands that inhibit or stimulate transcription of key transporters and enzymes in ... It is obvious that were these adaptive responses more robust, progressive cholestatic liver diseases might not occur or at least might become more protracted over time. Therefore the central question is whether therapy can be devised that might augment these potentially beneficial adaptive responses in expression of transporters and metabolic pathways? This commentary reviews the molecular basis of bile formation and the adaptive responses that are known to occur in cholestatic liver injury. It follows with a summary of current as well as possible future therapies for jaundice and cholestasis that are mediated by nuclear receptor ligands.

Utility of infertile male models for contraception and conservation

Epididymal defects in infertile domestic species and transgenic mice demonstrate the role of the epididymis in influencing sperm function. Spermatozoa from these males cannot negotiate the female tract as they fail to regulate their volume. The latter is necessary to counter the osmotic stresses encountered in the female tract. Reduced epididymal provision of osmolytes or their premature loss is discussed as probable causes of failed volume regulation. Measuring cell volume regulation for diagnosis of male infertility and blocking it as a means to male contraception are briefly considered. Unchecked human population growth is destroying habitats supporting vulnerable and endangered species. Genome resource banks have been established to preserve spermatozoa of genetically valuable individuals. As cryopreservation stresses spermatozoa osmotically, this process could jeopardise volume regulation with consequences for fertility. Knowledge of sperm volume regulation and the uptake of organic solutes may permit improvement in sperm storage and prevent osmolyte-related cryodamage.

Isolation and Functional Characterization of a Novel Organic Solute Carrier Protein, hOSCP1

We succeeded in isolating a novel organic solute carrier from a human placenta cDNA library. The isolated cDNA consisted of 1137 base pairs that encoded a 379-amino acid protein, hOSCP1. Northern blot and reverse transcription PCR analyses revealed that the hOSCP1 mRNA is expressed in the placenta and testis and weakly expressed in the thymus and small intestine. When expressed in Xenopus laevis oocytes, hOSCP1 mediated the high affinity transport of p-aminohippurate (PAH) (K(m) = 35.0 +/- 7.5 microm) and tetraethylammonium (K(m) = 62.3 +/- 12.2 microm) in a sodium-independent manner. However, the hOSCP1-expressing oocyte did not mediate the transport of L-carnitine. The transport of PAH by hOSCP1 was sensitive to pH, but the tetraethylammonium was not transported at the high pH examined. hOSCP1 transported prostaglandin E(2), prostaglandin F(2alpha), estrone sulfate, glutarate, L-leucine, L-ascorbic acid, and tetracycline. Thus, hOSCP1 also showed broad substrate specificity. A wide range of structurally unrelated organic compounds inhibited the hOSCP1-mediated PAH uptake. Immunohistochemical analysis revealed that the hOSCP1 protein is localized in the basal membrane of the syncytiotrophoblast in the human placenta. Our results suggest that hOSCP1 is a novel polyspecific organic solute carrier protein responsible for drug clearance from the human placenta.

An evolutionarily ancient Oatp: insights into conserved functional domains of these proteins.

Cellular uptake of organic solutes is mediated in large part by a gene family of membrane transporters called OATPs (SLC21A). To study the structural determinants and evolutionary development of the SLC21A family, we have cloned and functionally characterized a highly expressed evolutionarily primitive Oatp from the liver of the small skate, Raja erinacea. A full-length cDNA (2.3 kb) was obtained that encodes a protein of 689 amino acids. The characteristics of this novel skate Oatp, including tissue expression, subcellular localization, substrate selectivity, Na(+) dependence, and inhibitor selectivity were generally similar to liver-specific human OATP-C and rat Oatp4. However, sequence comparisons with other OATPs indicate that this skate Oatp shares only approximately 40-50% amino acid identity with the liver-specific OATPs/Oatps and with human OATP-F. Further computer analysis revealed that the highest amino acid identities reside in the first external (78%) and internal loops (75%) and transmembrane domains 2 (76%), 3 (62%), 4 (70%), and 11 (64%). We propose that the conserved regions of the SLC21A transporter family may be critical structural determinants of substrate specificity and function.

Expression cloning of two genes that together mediate organic solute and steroid transport in the liver of a marine vertebrate.

Uptake of organic solutes and xenobiotics by mammalian cells is mediated by ATP-independent transporters, and four families of transporters have now been identified. To search for novel organic solute transporters, a liver cDNA library from an evolutionarily primitive marine vertebrate, the little skate Raja erinacea, was screened for taurocholate transport activity by using Xenopus laevis oocytes. In contrast to the organic anion transporters identified to date, a transport activity was identified in this library that required the coexpression of two distinct gene products, termed organic solute transporter alpha and beta (Ostalpha, Ostbeta). Ostalpha cDNA encodes for a protein of 352 aa and seven putative transmembrane (TM) domains. Ostbeta contains 182 aa and has at least one and perhaps two TM domains. There is no significant sequence identity between Ostalpha and Ostbeta, and only low identity with sequences in the databases; however, Ostalpha bears a resemblance to some G protein-coupled receptors, and Ostbeta exhibits 22% amino acid identity with the C-terminal TM and intracellular domains of protocadherin-gamma, a cell surface glycoprotein. Xenopus oocytes injected with the cRNA for both Ostalpha and Ostbeta, but not each separately, were able to take up taurocholate, estrone sulfate, digoxin, and prostaglandin E(2), but not p-aminohippurate or S-dinitrophenyl glutathione. Transport was sodium-independent, saturable, and inhibited by organic anions and steroids, including the major skate bile salt, scymnol sulfate. These results identify an organic anion transporter composed of a putative seven-helix TM protein and an ancillary membrane polypeptide.

Organic Solute Uptake from Aqueous Solutions by Soil: A New Diffusion Model

A new diffusion model is proposed to describe the uptake of organic solutes from aqueous solutions by soil. The model considers that the uptake process is controlled by the pore diffusion of solutes within the soil pellets, the adsorption of solutes by the mineral fraction of soil, the surface diffusion of solutes along the mineral fraction surfaces, the partition of solutes into the soil organic matter, and the diffusion of solutes within the soil organic matter. An expression, in the Laplace domain, of the solute uptake by a soil pellet is obtained and with appropriate macroscopic conservation equations and standard numerical Laplace transform inversion techniques can be used to describe sorption and desorption as well as transport and distribution of organic solutes under a variety of conditions. On the basis of the uptake expression, a number of calculations are carried out with the objective to identify under which conditions the effect of diffusion into the soil organic matter, as well as the effects of dispersion and mineral adsorption, becomes important. Some comparisons of model predictions with experimental results are also provided to demonstrate the application of the model.

Uptake of organic compounds from aqueous solutions by soils—A comparison of two laplace transform inversion techniques

A general expression for the uptake of organic solutes from aqueous solutions by soil and sediment pellets in the Laplace domain is presented. This expression, together with the appropriate macroscopic conservation equation and initial and boundary conditions, provides a complete description of the movement and distribution of organic solutes through soil and subsurface systems. To obtain the numerical results for a specific system, the inversion from the Laplace domain into the time domain is required. In the present work, the use of two techniques, the fast Fourier transforms (FFT) and the Fourier series approximation (FSA), for inverting the Laplace transforms in connection with several problems concerning organic solute uptake by soils was explored. The FSA technique was found successful in all cases while the results by the FFT technique were strongly influenced by the selection of the algorithm parameters. In comparison with several numerical techniques for the direct solution of intraparticle diffusion equations, the FSA technique was found to have advantages in both accuracy and computational efficiency.

A model of organic solute uptake from aqueous solutions by soils

A model is proposed for the sorption of organic solutes from aqueous solutions by soils. This model is formulated by assuming that the uptake process is controlled by the pore diffusion of solutes within the soil pellets as well as the partition of solutes into the soil organic matter. A general expression of the solute uptake in the Laplace domain is obtained, which together with appropriate macroscopic conservation equations and standard Laplace transform inversion techniques, may be used to obtain the extent of sorption and the distribution of organics under a variety of conditions. Comparisons between model predictions and experiments indicate that the inclusion of the partition effect is indeed important in most of the cases.

Effect of Cutting on Solute Uptake by Plasma Membrane Vesicles from Sugar Beet (Beta vulgaris L.) Leaves

The uptake of sucrose, 3-O-methylglucose (3-O-MeG), and valine were studied in discs and in purified plasma membrane vesicles (PMV) prepared from sugar beet (Beta vulgaris L.) exporting leaves. The uptake capacities of freshly excised leaf discs were compared with the uptake in discs that had been floated for 12 h on a simple medium (aging) and with discs excised from leaves that had been cut from the plant 12 h before the experiments (cutting). After cutting, sucrose uptake amounted to twice the uptake measured in fresh discs, whereas the uptake of 3-O-MeG and valine remained unaffected. In aged leaf discs, there was a general stimulation of uptake, which represented 400, 300, and 400% of the uptake measured in fresh discs for sucrose, 3-O-MeG, and valine, respectively. Sucrose uptake in fresh discs was sensitive to N-ethylmaleimide (NEM), to p-chloromercuribenzenesulfonic acid (PCMBS), and to mersalyl acid (MA). Although cutting induced the appearance of a sucrose uptake system that is poorly sensitive to NEM but sensitive to PCMBS and MA, aging induced the development of an uptake system that is sensitive to NEM but poorly sensitive to PCMBS and MA. Autoradiographs of discs fed with [14C]sucrose show that cutting resulted in an increase of vein labeling with little effect in the mesophyll, whereas aging induced an increase of labeling located mainly in the mesophyll. The data show that cutting is sufficient to induce dramatic and selective changes in the uptake properties of leaf tissues and that the effects of cutting and aging on the uptake of organic solutes are clearly different. Parallel experiments were run with purified PMV prepared from fresh and cut leaves. The uptake of sugars and amino acids was studied after imposition of an artificial proton motive force (pmf). Comparison of the uptake properties of PMV and of leaf tissues indicate that the recovery of the sucrose uptake system in PMV is better than the recovery of the hexose and of the valine uptake systems. As observed with the leaf discs, cutting induced a 2-fold increase of the initial rate of sucrose uptake in PMV but did not affect the uptake of valine and 3-O-MeG. Cutting induced an increase of both Vmax and Km of the sucrose transport system in PMV. Measurements of the pmf imposed on the vesicles indicated that the increase of sucrose uptake induced by cutting was not due to a better integrity of the vesicles. Hexoses did not compete with sucrose for uptake in PMV from fresh and cut leaves, and maltose was a stronger inhibitor of sucrose uptake in PMV from cut leaves than in PMV from fresh leaves. The sensitivity of sucrose uptake to NEM, PCMBS, and MA in PMV from fresh and cut leaves paralleled that described above for the corresponding leaf discs. These data show that (a) the changes induced by cutting on sucrose uptake by leaf discs are due to membrane phenomena and not to the metabolism of sucrose; (b) the study of sucrose uptake with PMB gives a good account of the physiological situation; and (c) the specific effects induced by cutting on the sucrose uptake system are not lost during the preparation of the PMV.

Effect of chronic ethanol administration on the absorptive functions of the rat small intestine

Ethanol feeding to rats for 40 days significantly ( p < 0.01) depressed sodium-stimulated glucose and glycine uptakes at pH 5.5 and 7.2 without affecting sodium-independent solute transport in the rat intestine. Leucine uptake was essentially unaltered under these conditions. Absorption of bovine serum albumin and γ-globulin as detected by enzyme-linked immunosorbent blocking assay (ELISA) was markedly augmented ( p < 0.001) in ethanol-fed rats compared to the controls. These observations suggest that chronic ethanol intake differentially affects the uptake of organic solutes and macromolecules in the rat intestine.

Investigations on the hepatic uptake systems for organic cations with a photoaffinity probe of procainamide ethobromide

Azido procainamide methoiodide (APM), a photolabile derivative of the transport model compound procainamide ethobromide (PAEB), shows a close resemblance to PAEB from a physicochemical point of view. Like PAEB it is effectively taken up by the liver and excreted into bile. Kinetics of the uptake of APM in isolated hepatocytes revealed that in addition to a non-saturable process, two saturable uptake systems are involved ( K m1 = 3 μM, V mxl) = 80 pmol/min/10 6 cells K m2 =100 μM, V max2= 130 pmol/min × 10 6 cells). The uptake rate of APM was inhibited markedly in the presence of other organic cations. Organic anions and uncharged compounds generally had no inhibitory effect on the APM uptake. These results support the theory that there is a separate hepatic uptake system for organic cations like APM. Photoaffinity labeling of intact hepatocytes as well as plasma membrane sub-fractions enriched with sinusoidal domains disclosed two major binding polypeptides with apparent M r of 48,000 and 72,000. Such labeling patterns were not observed in membranes from hepatoma cells that are deficient in organic solute uptake. Differential photoaffinity labeling with other cationic compounds such as tributylmethyl ammonium and d-tubocurarine reduced the incorporation of APM in these polypeptides. The 48- and 72-kDa proteins might be involved in carrier-mediated transport of type I organic cations at the hepatic uptake level.

A nonselective cation channel in rat liver cells is activated by membrane stretch

A 16-pS channel was studied using patch-clamp electrophysiology in freshly dissociated rat liver cells and rat hepatoma cells. The channel was found to be cation selective and permeable to Na+, K+, and Ca2+. Its gating was unaffected by addition of the calcium ionophore A23187 (5 microM) in the presence of extracellular Ca2+ (2 mM). Ca2+ channel blockers, nifedipine, verapamil, and lanthanum, failed to inhibit the channel. The channel was activated by stretch, applied as suction to the interior of the patch pipette, and by cell swelling, induced by hypotonic shock or organic solute uptake (10 mM L-alanine). Channel activation by cell swelling was transient, lasting approximately 1 min. An elevation in cytosolic Ca2+ was evoked by hypotonic shock, as measured using the fluorescent indicator indo-1/AM. This change in intracellular Ca2+ concentration was dependent on extracellular Ca2+. Inasmuch as the time course for this response corresponded to that of channel activation, it is likely that hypotonic shock stimulated Ca2+ influx through the stretch-activated channel. To determine the role for Ca2+ influx in regulatory volume decrease (RVD), cell volume changes after hypotonic shock were studied using a Coulter counter. RVD was slightly but significantly inhibited by depletion of extracellular Ca2+. On the basis of these results it is proposed that stretch-activated channels in liver cells permit the transient influx of Ca2+, which in turn acts to trigger changes in ion conductance or cytoskeletal components involved in cell volume regulation.

Effect of nutrient enrichment on the heterotrophic uptake of organic solutes by microorganisms

Effect of nutrient enrichment on the heterotrophic uptake of organic solutes by microorganisms

Cellulase in Nereis virens.

IN their communication about direct uptake of organic solutes, Chapman and Taylor1 commented on the contradictory published accounts of the food and feeding of the polychaete Nereis virens Sars. Verrill2 stated that it is carnivorous, while Gross3 suggested that it is almost entirely herbivorous. Turnbull4 regarded the animal as omnivorous. The principal recognizable remains that we found in the guts of N. virens collected at Southend, Essex, were shells of the small gastropod Hydrobia, small fragments of small cockle (Cardium edule) shells, and the alga Enteromorpha intestinalis. When kept in aquaria, N. virens readily ate the flesh of cockles, mussels (Mytilus edulis), and shore crabs (Carcinus maenas), when these were placed near the entrances to the burrows. The shell-valves of cockles, approximately 5 mm in diameter, were broken by the jaws of the worms and ingested. Large cockles and mussels were eaten only when they died and gaped open. Moribund cockles and mussels occur in large numbers at Southend, resulting both from natural mortality and the activities of the “bait-diggers”, who accidentally bury these molluscs in their search for Nereis. Enteromorpha was also readily ingested, but it was observed during these experiments that the faecal pellets of the worms frequently contained strands of apparently undigested algae. This posed the question whether Nereis can digest the cell walls of algae which it has eaten or whether it can only utilize contents of cells broken mechanically.

Uptake of Organic Solutes by Nereis virens

WHILE investigating aspects of the trophic relationships of benthic animals, we have studied the uptake of amino-acids and other small organic molecules by the polychaete worm, N. virens, which lives in great numbers on the north side of the lower Thames Estuary1. Work on the isolation of possible organic solutes has already been reported2, and while no specific substances have yet been identified from surface water collected on Thames Estuary flats at Southend-on-Sea, other workers have reported the presence of quantities of organic material in sea water from various sources (for example, Duursma3).

Use of Glucose and Acetate by Bacteria and Algae in Aquatic Ecosystems

New methods employing C14—Labeled organic compounds measure the uptake of organic solutes by planktonic microorganisms. By testing uptake over a wide range of substrate concentrations, two separate mechanisms can be differentiated and their kinetics measured. Using filtration and evidence from experiments with laboratory cultures of planktonic bacteria and algae, specific transport systems effective at very low substrate concentrations were traced to the bacteria, and a diffusion mechanism, effective only at higher substrate concentrations, to the algae. Studies have shown that V, the maximum velocity of uptake by bacterial transport systems, gives information about the size and function of the bacterial populations. A diffusion constant, kd, gives information on the rate of uptake of solutes by the algal populations. Turnover times, derived from kinetic parameters, indicate that the algal uptake of glucose and acetate in Lake Erken, Sweden, is always less than 10% of the bacterial uptake, even though the algal biomass may be orders of magnitude greater than the bacterial. Two new types of bioassay employ the kinetics of bacterial uptake systems as the measuring reaction. Acetate and glucose were found in 1—10 mg/liter concentrations in the several natural waters tested. At these very low concentrations, algal uptake of glucose and acetate is so low that effective heterotrophy is impossible. In contrast, the bacteria effectively remove substrate from solution at these low levels and probably keep the substrate at these low concentrations. By doing this, the bacteria may prevent heterotrophic growth of algae in nature.

THE UPTAKE OF ORGANIC SOLUTES IN LAKE WATER1

Basic changes are made in an existing method using radioactive organic compounds for measuring the uptake of organic solutes by planktonic microorganisms. The changes give greater experimental flexibility and enable the maximum uptake velocity, V, to be found. Measurements over a range of substrate concentrations appear to show uptake by at least two mechanisms, by a transport system that becomes saturated at very low substrate concentrations and by simple diffusion, which shows a fairly constant increase in velocity of uptake with increasing substrate concentration. Because of this, the technique of measuring uptake at a single substrate concentration to give “relative heterotrophic potential” is questioned. The best estimate of heterotrophic potential may be V, the maximum uptake velocity attainable by planktonic populations with a transport system functioning according to the Michaelis laws. It is suggested that bacterial populations are responsible for uptake by means of transport systems and that algal populations show uptake by diffusion.