Saturable Uptake Kinetics Research Articles

The putative proton-coupled organic cation antiporter is involved in uptake of triptans into human brain capillary endothelial cells

BackgroundTriptans are anti-migraine drugs with a potential central site of action. However, it is not known to what extent triptans cross the blood–brain barrier (BBB). The aim of this study was therefore to determine if triptans pass the brain capillary endothelium and investigate the possible underlying mechanisms with focus on the involvement of the putative proton-coupled organic cation (H+/OC) antiporter. Additionally, we evaluated whether triptans interacted with the efflux transporter, P-glycoprotein (P-gp).MethodsWe investigated the cellular uptake characteristics of the prototypical H+/OC antiporter substrates, pyrilamine and oxycodone, and seven different triptans in the human brain microvascular endothelial cell line, hCMEC/D3. Triptan interactions with P-gp were studied using the IPEC-J2 MDR1 cell line. Lastly, in vivo neuropharmacokinetic assessment of the unbound brain-to-plasma disposition of eletriptan was conducted in wild type and mdr1a/1b knockout mice.ResultsWe demonstrated that most triptans were able to inhibit uptake of the H+/OC antiporter substrate, pyrilamine, with eletriptan emerging as the strongest inhibitor. Eletriptan, almotriptan, and sumatriptan exhibited a pH-dependent uptake into hCMEC/D3 cells. Eletriptan demonstrated saturable uptake kinetics with an apparent Km of 89 ± 38 µM and a Jmax of 2.2 ± 0.7 nmol·min−1·mg protein−1 (n = 3). Bidirectional transport experiments across IPEC-J2 MDR1 monolayers showed that eletriptan is transported by P-gp, thus indicating that eletriptan is both a substrate of the H+/OC antiporter and P-gp. This was further confirmed in vivo, where the unbound brain-to-unbound plasma concentration ratio (Kp,uu) was 0.04 in wild type mice while the ratio rose to 1.32 in mdr1a/1b knockout mice.ConclusionsWe have demonstrated that the triptan family of compounds possesses affinity for the H+/OC antiporter proposing that the putative H+/OC antiporter plays a role in the BBB transport of triptans, particularly eletriptan. Our in vivo studies indicate that eletriptan is subjected to simultaneous brain uptake and efflux, possibly facilitated by the putative H+/OC antiporter and P-gp, respectively. Our findings offer novel insights into the potential central site of action involved in migraine treatment with triptans and highlight the significance of potential transporter related drug-drug interactions.Graphical

Competitive interactions among H, Cu, and Zn ions moderate aqueous uptake of Cu and Zn by an aquatic insect

The absorption of aqueous copper (Cu) and zinc (Zn) by aquatic insects, a group widely used to assess water quality, is unresolved. This study examined interactions among Cu, Zn, and protons that potentially moderate Cu and Zn uptake by the acid-tolerant stonefly Zapada sp. Saturation uptake kinetics were imposed to identify competitive mechanisms. Decreasing pH reduced the maximum transport capacity, Jmax, in both metals, had little effect on the Cu dissociation constant, KD, and increased the Zn KD. Partial noncompetitive (Cu) and partial mixed competitive (Zn) inhibitor models most closely tracked the observed Cu and Zn influx rates across pH treatments. The estimated values for acid dissociation constants for the binary (proton-receptor) and ternary (proton-metal-receptor) complexes indicated the strong inhibitory effect of protons on Cu and Zn. In neutral pH water, Cu inhibited Zn influx, but Zn had little effect on Cu influx. The mechanism of Cu-Zn interaction was not identified. Results from separate Zn experiments suggested that the insect's developmental stage may affect the apparent Jmax. The study underscores some of the challenges of modeling metal bioaccumulation and informs future research directions.

Species Differences in Human and Rodent PEPT2-Mediated Transport of Glycylsarcosine and Cefadroxil in Pichia Pastoris Transformants.

The proton-coupled oligopeptide transporter PEPT2 (SLC15A2) plays an important role in the disposition of di/tripeptides and peptide-like drugs in kidney and brain. However, unlike PEPT1 (SLC15A1), there is little information about species differences in the transport of PEPT2-mediated substrates. The purpose of this study was to determine whether PEPT2 exhibited a species-dependent uptake of glycylsarcosine (GlySar) and cefadroxil using yeast Pichia pastoris cells expressing cDNA from human, mouse, and rat. In such a system, the functional activity of PEPT2 was evaluated with [3H]GlySar as a function of time, pH, substrate concentration, and specificity, and with [3H]cefadroxil as a function of concentration. We observed that the uptake of GlySar was pH-dependent with an optimal uptake at pH 6.5 for all three species. Moreover, GlySar showed saturable uptake kinetics, with Km values in human (150.6 µM) > mouse (42.8 µM) ≈ rat (36.0 µM). The PEPT2-mediated uptake of GlySar in yeast transformants was specific, being inhibited by di/tripeptides and peptide-like drugs, but not by amino acids and nonsubstrate compounds. Cefadroxil also showed a saturable uptake profile in all three species, with Km values in human (150.8 μM) > mouse (15.6 μM) ≈ rat (11.9 μM). These findings demonstrated that the PEPT2-mediated uptake of GlySar and cefadroxil was specific, species dependent, and saturable. Furthermore, based on the Km values, mice appeared similar to rats but both were less than optimal as animal models in evaluating the renal reabsorption and pharmacokinetics of peptides and peptide-like drugs in humans.

Biogeochemical Controls of Uranium Bioavailability from the Dissolved Phase in Natural Freshwaters.

To gain insights into the risks associated with uranium (U) mining and processing, we investigated the biogeochemical controls of U bioavailability in the model freshwater species Lymnaea stagnalis (Gastropoda). Bioavailability of dissolved U(VI) was characterized in controlled laboratory experiments over a range of water hardness, pH, and in the presence of complexing ligands in the form of dissolved natural organic matter (DOM). Results show that dissolved U is bioavailable under all the geochemical conditions tested. Uranium uptake rates follow first order kinetics over a range encompassing most environmental concentrations. Uranium uptake rates in L. stagnalis ultimately demonstrate saturation uptake kinetics when exposure concentrations exceed 100 nM, suggesting uptake via a finite number of carriers or ion channels. The lack of a relationship between U uptake rate constants and Ca uptake rates suggest that U does not exclusively use Ca membrane transporters. In general, U bioavailability decreases with increasing pH, increasing Ca and Mg concentrations, and when DOM is present. Competing ions did not affect U uptake rates. Speciation modeling that includes formation constants for U ternary complexes reveals that the aqueous concentration of dicarbonato U species (UO2(CO3)2(-2)) best predicts U bioavailability to L. stagnalis, challenging the free-ion activity model postulate.

Confocal Imaging with a Fluorescent Bile Acid Analogue Closely Mimicking Hepatic Taurocholate Disposition

This study aimed to characterize the in vitro hepatic transport mechanisms in primary rat and human hepatocytes of the fluorescent bile acid derivative N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2'-aminoethanesulfonate (tauro-nor-THCA-24-DBD), previously synthesized to study the activity of the bile salt export pump (BSEP). The fluorescent bile acid derivative exhibited saturable uptake kinetics in suspended rat hepatocytes. Hepatic uptake was inhibited in the presence of substrates/inhibitors of the organic anion transporting polypeptide (Oatp) family and Na(+) -taurocholate cotransporting peptide (Ntcp). Concentration-dependent uptake of the fluorescent bile acid was also saturable in Chinese hamster ovary cells transfected with rNtcp, hNTCP, OATP1B1, or OATP1B3. The fluorescent bile acid derivative was actively excreted in the bile canaliculi of sandwich-cultured rat and human hepatocytes (SCRH and SCHH), with a biliary excretion index (BEI) of 26% and 32%, respectively. In SCRH, cyclosporin A significantly decreased the BEI to 5%. Quantification by real-time confocal imaging further confirmed canalicular transport of the fluorescent bile acid derivative (BEI = 75%). We conclude that tauro-nor-THCA-24-DBD is a useful probe to study interference of drugs with NTCP/Ntcp- and BSEP/Bsep-mediated transport in fluorescence-based in vitro assays.

Kinetics of bioaccumulation of heavy metals in Odontesthes bonariensis is explained by a single and common mechanism

Fish are often considered a very sensitive indicator of heavy metal contamination in aquatic ecosystems; however, the detailed mechanisms of bioaccumulation remain unclear. Here, we study the bioaccumulation processes of three relevant heavy metals (Cd2+, Cu2+ and Cr6+) in Odontesthes bonariensis using mathematical modelling. We developed a general compartmental kinetic model that describes the transport of heavy metals between the surrounding water and the gills and liver of fish. The general model was reduced to a simple one still capable of reproducing previous experimental data and suggesting a common mechanism for the three metals. The modelling results indicate that bioaccumulation of Cd2+, Cu2+, and Cr6+ is described by a combination of a concentration-independent and saturable uptake kinetics in both organs with a unidirectional path of elimination from gills to liver to waterborne. Finally, the good agreement between the parameter values predicted by the model and previously published data suggests that our modelling approach may shed light on the mechanisms of heavy metal bioaccumulation in other species.

Sodium fluorescein is a probe substrate for hepatic drug transport mediated by OATP1B1 and OATP1B3

The aim of this study was to characterize the in vitro hepatic uptake kinetics of sodium fluorescein (NaFluo) and identify the transporters involved. NaFluo exhibited saturable uptake kinetics in suspended rat and human hepatocytes as reflected by Km values of 22.5 and 14.1 µM, and Vmax values of 98.3 and 5.8 pmol/(million cells • min), respectively. Coincubation with known inhibitors (e.g. rifampicin) of organic anion transporting polypeptide (OATP/Oatp; SLCO gene family) significantly decreased NaFluo uptake in hepatocytes. In contrast, neither inhibitors/substrates of the organic cation transporter or organic anion transporter family nor depletion of extracellular sodium resulted in significant inhibition of NaFluo uptake. To explore the contribution of individual uptake transporters, NaFluo uptake was determined in Chinese hamster ovary cells transfected with OATP1B1, OATP1B3, and OATP2B1. Transporter‐mediated uptake of NaFluo was observed in OATP1B1‐ and OATP1B3‐transfected cells (Km = 4.2 and 10.9 µM; Vmax = 30.9 and 135 [pmol/(mg protein • min)], respectively). NaFluo can be used as a probe substrate to study Oatp/OATP1B‐mediated drug interactions in fluorescence‐based in vitro transport assays of rat and human liver. Labeling of drugs or bile salts with a fluorescein moiety can be expected to result in fluorescent conjugates with substantially altered hepatic uptake characteristics as compared with the unconjugated compounds. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:5018–5030, 2011

Comparison of the role of the foliar sheath in nutrient (ammonium and phosphate) acquisition by the seagrass Thalassia hemprichii (Ehrenb.) Aschers. at two different sites on tropical Hainan Island, China

Generally, the foliar sheaths of seagrass contribute a large biomass to the dry weight of plants, and are found to be above-sediment biomass or, sometimes, below-sediment biomass. However, the role of foliar sheaths of seagrass in nutrient uptake has not yet been established. Thus, this study was performed to test whether the growth form of foliar sheaths affects the nutrient uptake properties of the seagrass. Two separate sets of morphotypes of the seagrass Thalassia hemprichii were collected from two different tropical meadows in coastal Hainan Island, China in the South China Sea. Ammonium (NH4+) and phosphate (Pi) uptake by solely blades and roots (experiment I), and above and below-sediment tissues (experiment II) of the two sets of specimens were examined in partitioned chambers using laboratory incubations. Curve profiles of the blade and root saturation uptake kinetics were shown to be similar for the two morphotypes of T. hemprichii. However, the above and below-sediment tissues uptake kinetics had different characteristics between the two morphotypes. For plants with above-sediment foliar sheaths, uptake by the above-sediment tissues contributed an important part of the whole plants’ nutrient acquisition. In contrast, for plants with below-sediment foliar sheaths, the contribution of nutrient uptake by above-sediment foliar blade tissues seemed almost negligible. Therefore, the results demonstrated that foliar sheaths of the tropical seagrass T. hemprichii were able to absorb NH4+ and Pi. Especially interesting is that the capacity for uptake by robust foliar sheaths growing beneath the sediment was remarkable (we termed this the Zhang–Huang–Thorhaug effect). The role of sheaths in nutrient acquisition found in this study is critical in elucidating seagrass nutrient uptake strategies.

An Improved Nonlinear Model Describing the Hepatic Pharmacokinetics of Digoxin: Evidence for Two Functionally Different Uptake Systems and Saturable Binding

To develop a semi-distributed liver model for the evaluation of saturable sinusoidal uptake and binding kinetics of the Oatp1a4 substrate digoxin. In the perfused rat liver, two successive digoxin doses of 42 and 125 microg were administered, and the outflow concentration was determined by LC/MS/MS. [14C]-sucrose was used as vascular reference. The data were analyzed simultaneously by a population approach using sucrose to determine the sinusoidal mixing of digoxin. The results suggest the existence of a high-affinity, low-capacity system, and a low-affinity, high-capacity system for sinusoidal uptake with apparent Michaelis constants (K(M)) of 0.24 and 332 microg/ml, respectively. Incorporation of saturable sinusoidal binding of digoxin considerably improved the fit, and the parameter estimates were consistent with those of binding to hepatic Na,K-ATPase. Simpler models that neglect the concentration gradient in flow direction failed to describe the outflow data in the high dose range. The semi-distributed liver model with saturable uptake should be useful for a functional characterization of transporters in the in situ rat liver.

Cadmium bioaccumulation factors for terrestrial species: Application of the mechanistic bioaccumulation model OMEGA to explain field data

In environmental risk assessment of metals it is often assumed that the biota-to-soil accumulation factor (BSAF) is generic and constant. However, previous studies have shown that cadmium bioaccumulation factors of earthworms and small mammals are inversely related to total soil concentrations. Here, we provide an overview of cadmium accumulation in terrestrial species belonging to different trophic levels, including plants, snails and moles. Internal metal concentrations of these species are less than linearly related to total soil levels, which is in accordance with previously observed trends. The mechanistic bioaccumulation model OMEGA (Optimal Modeling for Ecotoxicological Applications) is used to provide a quantitative explanation of these trends in cadmium accumulation. Our results indicate that the model accurately predicts cadmium accumulation in earthworms, voles and shrews when accounting for geochemical availability of metals and saturable uptake kinetics.

Expression, purification and characterization of the human membrane transporter protein OATP2B1 from Sf9 insect cells

OATP2B1 is an important member of the organic anion transporting polypeptides (OATP) family and is implicated in the intestinal and hepatic disposition of endo- and xenobiotics. The purpose of this work was to produce a highly purified protein for use as a reference standard for quantification of OATP2B1 in human tissue and in vitro assay systems. Here, we report the successful expression, purification and characterization of OATP2B1 in a heterologous expression system. Protein expressed by the Sf9-baculovirus expression system is functionally active as demonstrated by saturable uptake kinetics with a Km of 5.9±0.76μM for estrone-3-sulfate. OATP2B1 was extracted from Sf9-membranes with ABS-14-4 detergent and purified using a one-step FLAG-tag purification method. Yield of OATP2B1 from Sf9 cells was 1.1mg per liter of culture, for a final recovery of 1.8%. SDS–PAGE resolution and Western blot of purified protein displayed multiple banding of OATP2B1-specific protein, which was thoroughly investigated to confirm homogeneity of the sample. C-terminal FLAG-tag purification and immunoblot detection, together with N-terminal sequencing, confirmed the presence of only full-length protein. Treatment with endoglycosidases had little effect on the migration pattern in SDS–PAGE, suggesting that multiple banding was not due to different glycosylation states of the protein. Amino acid analysis further confirmed the homogeneity of the protein with a calculated extinction coefficient of 80,387cm−1M−1. Physical, biochemical and functional characterization show that purified human OATP2B1 is pure, homogeneous and appropriate for use as a standard to quantitate expression of OATP2B1 in in vitro systems and tissue samples.

Uptake kinetics and assimilation of inorganic nitrogen by Catenella nipae and Ulva lactuca

The kinetics of NH 4 +, the assimilation of NH 4 + and nitrate uptake by Catenella nipae (Rhodophyta) were compared with Ulva lactuca (Chlorophyta). Both algal species demonstrated saturable NH 4 + and nitrate uptake kinetics. Uptake of NH 3 by simple diffusion across the plasmalemma could not account for the observed saturation uptake kinetics of ammonia-N (NH 3+NH 4 +), so NH 4 + was the chemical form being taken up by the transport systems of the cells. Although the V max of NH 4 + uptake by C. nipae and U. lactuca was high (≈550 and 450 μmol g −1 DW h −1, respectively), the K m for U. lactuca (≈85 μM) was much lower than that for C. nipae (≈692 μM). The K m and V max values for nitrate uptake were much lower than for NH 4 + for both C. nipae ( K m≈5 μM; V max≈8.3 μmol g −1 DW h −1) and U. lactuca ( K m≈34 μM; V max≈116 μmol g −1 DW h −1). Over the incubation times used (up to 28 min) there was no apparent induction of nitrate transport in either species. There was no evidence for induction of NH 4 + transport in C. nipae but incubation time did affect the kinetics of NH 4 + uptake in U. lactuca. At high concentrations of NH 4 +, U. lactuca rapidly assimilated it into organic N with limited build-up of intracellular NH 4 + whereas C. nipae accumulated large amounts of NH 4 + because uptake of NH 4 + overtook the rate of assimilation. The effects of species-specific differences and experimental design on uptake-kinetic estimates are discussed in the light of the results of this other comparable studies. C. nipae is promising as a bioindicator species of the N-status of estuaries but U. lactuca changes its N-status too quickly for it to be a useful bioindicator of environmental conditions.

Regulation of smooth muscle cell proliferation using paclitaxel-loaded poly(ethylene oxide)-poly(lactide/glycolide) nanospheres.

Available data suggest that drugs should be delivered to a vascular lesion at a high concentration over an extended period of time to control vascular smooth muscle cell (VSMC) proliferation. This study was undertaken to formulate a paclitaxel, an antimicrotubule agent, into a biodegradable poly (ethylene oxide)-poly(lactide/glycolide) (PEO-PLGA) nanosphere as a sustained drug delivery system and to study its effects on VSMC in culture. The paclitaxel-loaded nanospheres (PT/NS), prepared by an emulsion-solvent evaporation method, had an average diameter of approximately 150 nm and showed a sustained release profile over 4 weeks. The PT/NS exhibited antiproliferative effects comparable to those observed with free paclitaxel. The cellular internalization of nanospheres was visualized using confocal fluorescence microscopy, and from a flow cytometry study the progressive cellular uptake profile, uptake inhibition at low temperature, and saturation uptake kinetics (concentration dependency) were observed. These suggest that (adsorptive) pinocytosis is a major uptake mechanism of the nanospheres. The sustained drug release profile and cellular internalization results suggest that nanospheres loaded with paclitaxel may potentially be used as an endocytizable, local sustained drug delivery system for the prevention of restenosis.

Uptake of Benzoic Acid and Chloro-Substituted Benzoic Acids by Alcaligenes denitrificans BRI 3010 and BRI 6011

The mechanism of uptake of benzoic and 2,4-dichlorobenzoic acid (2,4-DCBA) by Alcaligenes denitrificans BRI 3010 and BRI 6011 and Pseudomonas sp. strain B13, three organisms capable of degrading various isomers of chlorinated benzoic acids, was investigated. In all three organisms, uptake of benzoic acid was inducible. For benzoic acid uptake into BRI 3010, monophasic saturation kinetics with apparent K(infm) and V(infmax) values of 1.4 (mu)M and 3.2 nmol/min/mg of cell dry weight, respectively, were obtained. For BRI 6011, biphasic saturation kinetics were observed, suggesting the presence of two uptake systems for benzoic acid with distinct K(infm) (0.72 and 5.3 (mu)M) and V(infmax) (3.3 and 4.6 nmol/min/mg of cell dry weight) values. BRI 3010 and BRI 6011 accumulated benzoic acid against a concentration gradient by a factor of 8 and 10, respectively. A wide range of structural analogs, at 50-fold excess concentrations, inhibited benzoic acid uptake by BRI 3010 and BRI 6011, whereas with B13, only 3-chlorobenzoic acid was an effective inhibitor. For BRI 3010 and BRI 6011, the inhibition by the structural analogs was not of a competitive nature. Uptake of benzoic acid by BRI 3010 and BRI 6011 was inhibited by KCN, by the protonophore 3,5,3(prm1), 4(prm1)-tetrachlorosalicylanilide (TCS), and, for BRI 6011, by anaerobiosis unless nitrate was present, thus indicating that energy was required for the uptake process. Uptake of 2,4-DCBA by BRI 6011 was constitutive and saturation uptake kinetics were not observed. Uptake of 2,4-DCBA by BRI 6011 was inhibited by KCN, TCS, and anaerobiosis even if nitrate was present, but the compound was not accumulated intracellularly against a concentration gradient. Uptake of 2,4-DCBA by BRI 6011 appears to occur by passive diffusion into the cell down its concentration gradient, which is maintained by the intracellular metabolism of the compound. This process could play an important role in the degradation of xenobiotic compounds by microorganisms.

Effect of total cadmium and organic complexing on the uptake of cadmium by the brine shrimp, Artemia franciscana

The effect of total cadmium and organic complexing on the rate of cadmium uptake by the brine shrimp, Artemia franciscana has been studied in chemically defined saltwater solutions. The uptake of cadmium from solution by the laboratory-reared brine shrimp displays saturable uptake kinetics. Uptake of cadmium is linear in time up to a total cadmium concentration of 200 μmoll-1 and saturates above 800 μmoll-1. Complexation of cadmium with organic ligands decreases the uptake of the metal by the brine shrimp. This is in agreement with the view that the availability of cadmium to aquatic organisms is related to the activity of the free cadmium ion in the solution. There is no evidence that the direct uptake of cadmium complexes is important in determining uptake of cadmium. Cadmium uptake is not, however, a mere function of the free cadmium ion activity in the solution, i.e., cadmium uptake rates may differ by an order of magnitude for the same free cadmium ion activity depending on the complexation conditions. In addition to controlling the free cadmium ion activity, the role of organic ligands in metal ion buffering and metal ion masking appear important factors in determining the availability of the metal to the organism.

Comparative analysis of sodium-dependent l-glutamate transport of synaptosomal and astroglial membrane vesicles from mouse cortex

Uptake of [ 3H] l-glutamate into membrane vesicles prepared from either mouse cortical astrocyte cultures or synaptosomes was found to be an electrogenic sodium- and potassium-dependent transport process with saturable uptake kinetics. Pharmacological differences were revealed by using a variety of substrate analogues. l- trans-PDC inhibited the synaptosomal glutamate transport 2–4-fold stronger than the astroglial uptake. The substrate analogues dl-threo-β-hydroxy-aspartate, dl-aspartate-β-hydroxamate, l-aspartate and d-aspartate inhibited glutamate transport of astroglial and neuronal membrane vesicles in a distinctive manner, whereas d-glutamate, quisqualate and dihydrokainate had no effect in either case. Immunoblotting and immunocytochemical labeling with antibodies against the rat brain glutamate transporter revealed the selective reaction of a band at about 75 kDa mol. wt. and a specific pattern of astrocyte immunostaining.

Uptake of selenium by freshwater phytoplankton

The uptake of three forms of Se, selenate, selenite and selenomethionine, was examined in three species of freshwater algae, Anabaena flos-aquae (Cyanophyceae), Chlamydomonas reinhardtii (Chlorophyceae), and Cyclotella meneghiania (Bacillariophyceae) in a defined medium using radiotracers at Se concentrations representative of contaminated systems. Based on the relative accumulation by live vs. heat-killed cells, and linear accumulation through time, selenate accumulation by all three species appears to be a physiological process. However, selenite accumulation at these concentrations appears to be due largely to sorption rather than active uptake, as shown by rapid initial accumulation and the fact that accumulation by heat-killed cells was nearly equal to that of dead cells. Both selenate and selenite uptake rates increased linearly with concentration over the range of 1 to 50 µg L−1. Selenomethionine uptake is a biological process with saturable uptake kinetics (Ks ranging from about 2 to 30 µg L−1 Se), with much greater uptake rates than the other two forms, and little inactive sorbtion to heat-killed cells.

Dissociation of a ferric maltol complex and its subsequent metabolism during absorption across the small intestine of the rat.

1. The fate and disposition of [59Fe]-ferric [3H]-maltol after intravenous administration were investigated in anaesthetized rats. Immediate dissociation of ferric iron from maltol took place in the circulation even with high doses of ferric maltol (containing 1 mg elemental iron). In plasma samples withdrawn within 1 min of injection and subjected to gel filtration, 59Fe eluted with the high molecular weight proteins whilst the tritium was associated with low molecular weight material. 2. The rates of elimination of 59Fe and of tritium from the plasma and their ultimate fate were very different. The half life for 59Fe in the plasma was around 70 min and 59Fe appeared mainly in the bone marrow and liver. There was an initial rapid exit of tritium from the plasma with a half life of around 12 min. This was followed either by a plateau or by a rise in tritium levels, involving entry of maltol metabolites into the circulation. These metabolites could be recovered in the urine. 3. Entry of 59Fe and of tritium into the blood plasma after intraduodenal administration of [59Fe]-ferric [3H]-maltol was also very different. At low doses of ferric maltol (containing 100 micrograms elemental iron), the tritium appeared in the plasma in highest amounts within seconds and then decreased whilst there was a slow rise in 59Fe levels. At higher doses of ferric maltol (containing 7 mg elemental iron), levels of 59Fe in the plasma were highest at 5 min and then fell whereas tritium levels rose steadily. Mucosal processing of 59Fe prevented further entry of iron at high dose into the circulation. 4. Initial rates of uptake of [3H]-maltol into isolatcd intestinal fragments were measured over a range of concentrations and revealed that maltol alone could diffuse freely into the tissues whereas maltol complexed to iron showed saturable uptake kinetics similar to those seen with the iron itself. 5. After intestinal uptake, 59Fe and tritium were associated with different subcellular fractions, maltol itself being metabolized to the glucuronide conjugate within the intestinal mucosa. 6. It is concluded that dissociation of metal and ligand takes place before entry into the intestinal mucosa. Iron is then taken up on the endogenous carrier and processed in the normal way whilst maltol enters by diffusion, its rate of entry being limited by the degree of dissociation. It is subsequently metabolized by conjugation and eliminated rapidly from the body in the urine.

Sodium and pH dependent carrier-mediated transport of antibiotic, fosfomycin, in the rat intestinal brush-border membrane.

The mechanism of intestinal absorption of an antimicrobial agent, fosfomycin (FOM), was investigated in rats using small intestinal brush-border membrane vesicles (BBMV). The uptake of [3H]FOM by BBMV was osmolarity- and temperature-sensitive and showed apparently saturable uptake kinetics consistent with the Michaelis-Menten equation, having Kt = 15.3 mM and Jmax = 7.78 nmol/30 s/mg protein at 37 degrees C. An overshoot uptake of FOM was observed in the presence of an inwardly direct Na+ gradient. The replacement of extravesicular Na+ with choline or mannitol significantly reduced the uptake. An addition of a protonophore, FCCP, significantly decreased the initial uptake of FOM in the absence of Na+ gradient but in the presence of a H+ gradient (pHin = 7.5, pHout = 6.0), whereas in the absence of a H+ gradient no significant difference was observed between the uptakes at an acidic pH (pHin = pHout = 6.0) and a neutral pH (pHin = pHout = 7.5). An inside negative potassium diffusion potential induced by valinomycin enhanced significantly the uptake of FOM. The uptake of FOM in the presence of both Na(+)- and H(+)-gradients was significantly inhibited by phosphate, arsenate and phosphonoformic acid (PFA), which are specific inhibitors of phosphate transport, but not by D-glucose. Based on these results, it is concluded that FOM transport in the small intestine is partially shared with the Na(+)-phosphate cotransport system and in part occurs via a H(+)-gradient dependent carrier-mediated system.

Methylamine uptake by zooxanthellae‐invertebrate symbioses: Insights into host ammonium environment and nutrition

Cnidarians with endosymbiotic algae (=zooxanthellae) take up dissolved inorganic nutrients from seawater, but neither the physiological mechanisms nor the effect of host nutrition on transport kinetics is known. We used the NH4+ analogue [14C]methylamine ([14C]MA) to examine these aspects of NH4+ uptake by a sea anemone (Aiptasia pallida) and a coral (Madracis decactis).Both intact symbioses and isolated zooxanthellae took up [14C]MA. In anemones, uptake rates per algal cell increased with time after feeding. Uptake rates for isolates from hosts unfed for 7– 10 d were linear for at least 200 min, slightly light‐dependent, and conformed to Michaelis‐Menten kinetics (Ks = 68 µM; V = 3.8 mol 10−18 cell−1 s−1). Isolates from well‐fed hosts took up [14C]MA much less rapidly at all concentrations tested and did not exhibit saturable uptake kinetics. NH4+ competitively inhibited [14C]MA uptake by isolated algae (inhibition constant = 4.0 µM) and reduced [14C]MA uptake by intact symbiotic anemones. We hypothesize that [14C]MA (and by analogy NH4+) uptake occurs by a “depletion‐diffusion” mechanism in intact symbiotic anemones with zooxanthellae maintaining very low intracellular [14C]MA and NH4+ concentrations in host tissue and that [14C]MA uptake kinetics will be useful in evaluating the nutritional status of corals and similar symbiotic associations under field conditions.