Amino Acid Carrier System Research Articles

Vectorization of agrochemicals: amino acid carriers are more efficient than sugar carriers to translocate phenylpyrrole conjugates in the Ricinus system.

Producing quality food in sufficient quantity while using less agrochemical inputs will be one of the great challenges of the twenty-first century. One way of achieving this goal is to greatly reduce the doses of plant protection compounds by improving the targeting of pests to eradicate. Therefore, we developed a vectorization strategy to confer phloem mobility to fenpiclonil, a contact fungicide from the phenylpyrrole family used as a model molecule. It consists in coupling the antifungal compound to an amino acid or a sugar, so that the resulting conjugates are handled by active nutrient transport systems. The method of click chemistry was used to synthesize three conjugates combining fenpiclonil to glucose or glutamic acid with a spacer containing a triazole ring. Systemicity tests with the Ricinus model have shown that the amino acid promoiety was clearly more favorable to phloem mobility than that of glucose. In addition, the transport of the amino acid conjugate is carrier mediated since the derivative of the L series was about five times more concentrated in the phloem sap than its counterpart of the D series. The systemicity of the L-derivative is pH dependent and almost completely inhibited by the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP). These data suggest that the phloem transport of the L-derivative is governed by a stereospecific amino acid carrier system energized by the proton motive force.

Glycinergic-Fipronil Uptake Is Mediated by an Amino Acid Carrier System and Induces the Expression of Amino Acid Transporter Genes in Ricinus communis Seedlings.

Phloem-mobile insecticides are efficient for piercing and sucking insect control. Introduction of sugar or amino acid groups to the parent compound can improve the phloem mobility of insecticides, so a glycinergic-fipronil conjugate (GlyF), 2-(3-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-((trifluoromethyl)sulfinyl)-1H-pyrazole-5-yl)ureido) acetic acid, was designed and synthesized. Although the "Kleier model" predicted that this conjugate is not phloem mobile, GlyF can be continually detected during a 5 h collection of Ricinus communis phloem sap. Furthermore, an R. communis seedling cotyledon disk uptake experiment demonstrates that the uptake of GlyF is sensitive to pH, carbonyl cyanide m-chlorophenylhydrazone (CCCP), temperature, and p-chloromercuribenzenesulfonic acid (pCMBS) and is likely mediated by amino acid carrier system. To explore the roles of amino acid transporters (AATs) in GlyF uptake, a total of 62 AAT genes were identified from the R. communis genome in silico. Phylogenetic analysis revealed that AATs in R. communis were organized into the ATF (amino acid transporter) and APC (amino acid, polyaminem and choline transporter) superfamilies, with five subfamilies in ATF and two in APC. Furthermore, the expression profiles of 20 abundantly expressed AATs (cycle threshold (Ct) values <27) were analyzed at 1, 3, and 6 h after GlyF treatment by RT-qPCR. The results demonstrated that expression levels of four AAT genes, RcLHT6, RcANT15, RcProT2, and RcCAT2, were induced by the GlyF treatment in R. communis seedlings. On the basis of the observation that the expression profile of the four candidate genes is similar to the time course observation for GlyF foliar disk uptake, it is suggested that those four genes are possible candidates involved in the uptake of GlyF. These results contribute to a better understanding of the mechanism of GlyF uptake as well as phloem loading from a molecular biology perspective and facilitate functional characterization of candidate AAT genes in future studies.

The Effect of a Novel rBAT Mutation on the Expression and Function of System b0,+

To identify the causative mutations within a cohort of cystinuria patients, the two genes (SLC3A1 and SLC7A9) encoding the subunits (rBAT and b0,+AT) of the amino acid carrier System b0,+ were analysed. A novel homozygous missense mutation (M465K) in the heavy chain chaperone protein rBAT was identified in one patient and investigation of obligate heterozygotes showed an autosomal recessive penetrance of phenotype. Homology modelling was used to create a model of the extracellular domain of rBAT based on the known crystal structure of oligo‐1,6‐glucosidase from B. cereus (PDB code 1UOK). Using this model, M465K is predicted to be located on the 7th alpha helix of the triosephosphate isomerase (TIM)‐like structure. Protein expression and function were characterised following cRNA injection in Xenopus laevis oocytes by [3H]arginine uptake, western blotting, and immunocytochemistry. Reduced protein expression and arginine uptake were observed in M465K‐injected oocytes versus wild‐type rBAT suggesting a decrease in transporter trafficking to the oocyte membrane. M465K expression was dependent upon time (post‐injection) and quantity of cRNA injected and followed a pattern similar to that observed with the neighbouring mutation M467T, the most common cystinuria‐causing mutation in several European populations. Cystinuria in this patient likely results from reduced cystine reabsorption in the renal proximal tubule due to defective trafficking of M465K and decreased System b0,+ activity in vivo. Supported by the Medical Research Council and Northern Counties Kidney Research Fund.

Plasma free amino acid kinetics in rainbow trout (Oncorhynchus mykiss) using a bolus injection of 15N-labeled amino acids

To gain insight into the metabolic design of the amino acid carrier systems in fish, we injected a bolus of (15)N amino acids into the dorsal aorta in mature rainbow trout (Oncorhynchus mykiss). The plasma kinetic parameters including concentration, pool size, rate of disappearance (R(d)), half-life and turnover rate were determined for 15 amino acids. When corrected for metabolic rate, the R(d) values obtained for trout for most amino acids were largely comparable to human values, with the exception of glutamine (which was lower) and threonine (which was higher). R(d) values ranged from 0.9 μmol 100 g(-1) h(-1) (lysine) to 22.1 μmol 100 g(-1) h(-1) (threonine) with most values falling between 2 and 6 μmol 100 g(-1) h(-1). There was a significant correlation between R(d) and the molar proportion of amino acids in rainbow trout whole body protein hydrolysate. Other kinetic parameters did not correlate significantly with whole body amino acid composition. This indicates that an important design feature of the plasma-free amino acids system involves proportional delivery of amino acids to tissues for protein synthesis.

Synthesis and in vitro biological evaluation of valine-containing prodrugs derived from clinically used HIV-protease inhibitors

In an approach to improve the pharmacological properties and pharmacokinetic profiles of the current protease inhibitors (PIs) used in clinics, and consequently, their therapeutic potential, we performed the synthesis of PI–spacer–valine prodrugs (PI = saquinavir, nelfinavir and indinavir; spacer = –C(O)(CH 2) 5NH–), and evaluated their in vitro stability with respect to hydrolysis, anti-HIV activity, cytotoxicity, and permeation through a monolayer of Caco-2 cells (used as a model of the intestinal barrier), as compared with their parent PI and first generation of valine–PIs (wherein valine was directly connected through its carboxyl to the PIs). The PI–spacer–valine conjugates were prepared in two steps, in good yields, by condensing an acid derivative of the appropriate protected valine–spacer moiety with the PI, followed by deprotection of the valine protecting group. With respect to hydrolysis, we found that the PI–spacer–valine prodrugs were chemically more stable than the first generation of PI–Val prodrugs. Their stabilities correlated with the low to very low in vitro anti-HIV activity measured for those prodrugs wherein the coupling of valine–spacer residue to the PIs was performed onto the peptidomimetic PI's hydroxyl. Prodrugs wherein the coupling of the valine–spacer residue was performed onto the non-peptidomimetic PI hydroxyl displayed a higher antiviral activity, indicating that these prodrugs are also to some extent anti-HIV drugs by themselves. While the direct conjugation of l-valine to the PIs constituted a most appealing alternative, which improved their absorptive diffusion across Caco-2 cell monolayers and reduced their recognition by efflux carriers, its conjugation to the PIs through the –C(O)(CH 2) 5NH– spacer was found to inhibit their absorptive and secretory transepithelial transport. This was attributable to a drastic reduction of their passive permeation and/or active transport, indicating that the PI–spacer–valine conjugates are poor substrates of the aminoacid carrier system located at the brush border side of the Caco-2 cell monolayer.

Preparation and tumor affinity testing of the radioiodinated tetrahydroisoquinoline derivative [ 123I]TIC(OH) for targeting prostate cancer

This work describes the synthesis and tumor affinity testing of 8-[ 123I]iodo- l-1,2,3,4-tetrahydro-7-hydroxyisoquinoline-3-carboxylic acid [ITIC(OH)], a cyclic non-naturally occurring amino acid as an imaging probe for prostate cancer. Parameters for labelling were optimized with regard to the amount of precursor, the temperature and time. Thereafter, ITIC(OH) was evaluated in terms of its uptake in primary human PC-3 and DU-145 prostate cancer cells, followed by analysis of the underlying mechanisms of the radioactivity accumulation in tumor cells. No-carrier-added ITIC(OH) was obtained in 80±15% radiochemical yield and >98% radiochemical purity by a one-step radioiodination, using IODO-GEN as oxidant. The total synthesis time was less than 30 min, and compatible with a clinical routine production. ITIC(OH) accumulated intensively in primary human prostate cancer cells. The radioactivity incorporation in tumor following a 10-min incubation at 37 °C/pH 7.4 varied from 35% to 58% of the total loaded activity per 10 6 tumor cells (355–540 cpm/1000 cells). Inhibition experiments revealed that ITIC(OH) was taken up into tumor by an active transport different from the common amino acid carrier systems, including the sodium-dependent system A and B +,0, and the sodium-independent L- and ASC-type transporter. In contrast, the cellular incorporation was dependent on the membrane potential and correlated with the activity of the mitochondria. In conclusion, the specific and high-level accumulation of ITIC(OH) in human prostate carcinoma cells, indicates that the new radiopharmaceutical is a good candidate for further in vivo investigations to ascertain its potential as an imaging probe for prostate cancer by SPET.

Transport ofN-AcetylcysteineS-Conjugates of Methylmercury in Madin-Darby Canine Kidney Cells Stably Transfected with Human Isoform of Organic Anion Transporter 1

Recent studies have implicated the activity of the organic anion transporter 1 (OAT1) protein in the basolateral uptake of inorganic mercuric species in renal proximal tubular epithelial cells. However, very little is known about the potential role of OAT1 (and other OATs) in the renal epithelial transport of organic forms of mercury such as methylmercury (CH(3)Hg(+)). The present investigation was designed to study the transport of N-acetyl cysteine (NAC) S-conjugates of both methylmercury (CH(3)Hg-NAC) and inorganic mercury (NAC-Hg-NAC) in renal epithelial cells [Madin-Darby canine kidney (MDCK) cells] stably transfected with the human isoform of OAT1 (hOAT1). These mercuric species were studied because numerous mercapturates have been shown to be substrates of OATs. Data on saturation kinetics, time dependence, substrate specificity, and temperature dependence for the transport of CH(3)Hg-NAC and NAC-Hg-NAC indicate that both of these two mercuric species are indeed transportable substrates of hOAT1. Substrate specificity data also show that CH(3)Hg-NAC is a substrate of a transporter in MDCK cells that is not hOAT1. These data indicate that an amino acid carrier system is a likely candidate responsible for this transport. Furthermore, the rates of survival of the hOAT1-transfected MDCK cells were significantly lower than those of corresponding control MDCK cells when they were exposed to cytotoxic concentrations of CH(3)Hg-NAC or NAC-Hg-NAC. Collectively, the present data support the hypothesis that CH(3)Hg-NAC and NAC-Hg-NAC are transportable substrates of OAT1 and thus potentially transportable mercuric species taken up in vivo at the basolateral membrane of proximal tubular epithelial cells.

Preparation and investigation of tumor affinity, uptake kinetic and transport mechanism of iodine-123-labelled amino acid derivatives in human pancreatic carcinoma and glioblastoma cells

In developing radioiodinated agents for pancreatic and brain tumor imaging by single photon emission tomography (SPET), we prepared p-amino-3-[123I]iodo-l-phenylalanine (IAPA), p-[123I]iodo-l-phenylalanine (IPA), L-8-[123I]iodo-1,2,3,4-tetrahydro-7-hydroxyisoquinoline-3-carboxylic acid (ITIC) and L-3-[123I]iodo-α-methyl-tyrosine (IMT) in radiochemical yields up to 95%, and we investigated their uptake in human pancreatic carcinoma and glioblastoma cells as well as the mechanisms promoting the tumor uptake. The radiopharmaceutical uptake into tumor cells was rapid (t1/2 ≤ 5 min) and temperature- and pH-dependent. The radioactivity concentration in tumor cells varied from 10 to 33% of the total activity (105–310 cpm/1000 cells) following a 30-min incubation at 37°C (pH 7.4). In comparison, accumulation of the radiopharmaceuticals into normal brain and pancreatic tissue remained relatively low. Depolarizing the plasma membrane potential in high K+ buffer significantly altered the radioactivity concentration in the tumor cells, suggesting that membrane potential plays a certain role in the cellular uptake. Competitive inhibition experiments with specific amino acid transport inhibitors indicated that the uptake of IAPA, IPA and IMT into human pancreatic carcinoma and glioblastoma cells is predominantly mediated by the L and ASC transport systems, while no substantial involvement of the transport system A in their tumor uptake could be demonstrated. In contrast, results of the present investigation indicated that ITIC is not taken up into tumor cells via the common neutral amino acid carrier systems, including the A, L and ASC system. Furthermore, preloading with naturally occurring L-amino acids failed to stimulate the cellular uptake of the radiopharmaceuticals. These data indicate that the investigated radiopharmaceuticals exhibit interesting characteristics with promise for in vivo tumor investigations to ascertain their potential as radioligands for glioma and pancreatic carcinoma imaging by SPET.

Protein Synthesis Inhibition in Neocortical Grafts Evaluated by Systemic Amino Acid Uptake Autoradiography

The temporal pattern of protein synthesis inhibition was examined in grafted neocortical neurons using [3H]valine in vivo autoradiography. Neuronal uptake levels of systemically administered 3H-labeled amino acids which cross the blood–brain barrier (BBB) via endothelial cell neutral carriers have long been a hallmark in studies of experimental ischemic pathology; there is likely a strong correlation between persistent protein synthesis inhibition and the progression of cell damage. Because the grafting procedure involves the loss of blood flow and the subsequent reperfusion of the donor tissue there are, mechanistically, important similarities to reversible ischemia models. The effects of ischemic injury on grafted CNS neurons are not fully understood. Quantitative analysis of grain distribution in individual graft or control (adjacent host cortex) neurons indicated an initial breakdown of the amino acid barrier system, subsequent recovery, and progressive reduction of amino acid uptake by 1 year. Up to 3 weeks after surgery grafts were flooded with the [3H]valine tracer but individual neurons contained relatively few silver grains. After this time, the tracer was normally distributed within graft neurons but at significantly lower levels than in controls. Grain density gradually decreased over time such that 12-month grafted neurons had approximately half that compared to control and only 58% of that in 2-month grafts; the 12-month levels were comparable to those observed at early (10 days) postoperative times. Autoradiography of immunostained sections for MAP-2, SMI 311 (neurofilament marker), and neuron-specific enolase showed reduced expression of these proteins in neurons coupled with weak amino acid tracer uptake. The results further suggest that grafted neurons bear intriguing similarities to neurons placed at ischemic risk, particularly “penumbral” neurons, which are affected by reduced blood flow and are metabolically weakened. The loss of BBB properties in early grafts may also extend to the endothelial cell amino acid carrier system, and the delayed revascularization process could affect neuronal uptake mechanisms.

Pharmacokinetics of aniracetam and its metabolites in rat brain.

The pharmacokinetics of aniracetam (AP) and its main metabolites, 4-p-anisamidobutyric acid (ABA), 2-pyrrolidinone (PD) and p-anisic acid (AA), in 3 brain regions (cerebral cortex, hippocampus and thalamus) was investigated after single intravenous (i.v.) and oral administrations of AP to rats. AP, AA and PD were rapidly distributed into the 3 brain regions after i.v. administration of AP, but the amounts of AP were low. The concentrations of AP and AA in brain regions rapidly declined, whereas PD levels were higher and more sustained than those of AP and AA. ABA levels in the regions were below the detection limit. There were no significant differences in the distribution of these compounds in the 3 brain regions. The AUCbrain/AUCplasma ratio of PD was 53--55%, in contrast to the low ratio of AP (2.4--3.2%) and AA (3.9--4.2%). On oral administration of AP, the AUCbrain/AUCplasma ratio of PD was also higher than that of AA. When the transport of PD was tested using the in situ brain perfusion technique, it was clarified that PD was not transported across the blood-brain barrier (BBB) by a neutral amino acid carrier system. The high brain levels of PD and the low levels of AP suggest that the clinical efficacy of dosed AP may partly result from PD penetrating into the brain.

Synthesis and structure-activity relationships of some pesticides with an α-amino acid function

The synthesis of several derivatives of a xenobiotic (phenoxyalkanecarboxylic acid) and an α-amino acid ( l-lysine) is described. Various substituents were introduced in the aromatic ring of the phenoxyalkanecarboxylic acid and the side-chain was modified. They were tested for their effect on the transport of a neutral ( l-threonine), an acidic ( l-glutamic acid) and a basic ( l-lysine) amino acid, and a sugar (sucrose). All compounds markedly inhibited threonine uptake by leaf tissues of Vicia faba L. – and more specifically phloem loading – with two exceptions, when the aromatic ring was substituted in the 4-position by a primary amino group or when d-lysine was used instead of l-lysine. By contrast, the addition of a chlorine atom in the 4-position of the aromatic ring enhanced the inhibitory activity. Similar results were obtained for inhibition of glutamate uptake and, to a lesser extent, for lysine uptake. pH dependence of the inhibitory activity as well as electrophysiological data indicate that permeases mediating active transport of amino acids are the target of these conjugates. These, in addition to other data obtained with other xenobiotics, show that the amino acid carrier system is capable of recognizing a wide range of conjugates of various sizes, structures and octanol/water partition coefficients.

Nitric oxide generation from extracellularly applied NG-hydroxy- l-arginine in LPS-activated RAW 264 macrophages

Lipopolysaccharide (LPS)-activated but not control RAW 264 macrophages produced nitric oxide (NO) from extracellularly-applied N G-hydroxy- l-arginine ( l-NOHA) in a concentration-dependent manner, as measured by EPR spin trapping and assays for NO 2 − and NO 3 −. This production was inhibited by N G-nitro- l-arginine methyl ester and N G-monomethyl- l-arginine, NO-synthase inhibitors, as well as by l-lysine, a competitor for the y + amino acid carrier system. No significant differences were found between l-NOHA and l-arginine with respect to the rate of NO production and the effects of inhibitors. These results provide evidence that extracellular l-NOHA can enter LPS-activated RAW 264 macrophages via a cationic amino acid carrier system and be metabolized to NO by NO-synthase. The data also suggest that no alternative pathway exists for NO production from l-NOHA in non-activated RAW 264 macrophages.

L-[3H]nitroarginine and L-[3H]arginine uptake into rat cerebellar synaptosomes: kinetics and pharmacology.

Characteristics of the transport of the nitric oxide synthase substrate L-arginine and its inhibitor, NG-nitro-L-arginine (L-NOARG), into rat cerebellar synaptosomes were studied. Uptake of both L-arginine and L-NOARG was linear with increasing amount of protein (up to 40 micrograms) and time of incubation (up to 5 min) at 37 degrees C. Uptake of both compounds reached a steady state by 20 min. Maximal uptake of L-NOARG (650 pmol/mg of protein) was three to four times higher than that of L-arginine (170 pmol/mg of protein). L-NOARG uptake showed biphasic kinetics (Km1 = 0.72 mM, Vmax 1 = 0.98 nmol/min/mg of protein; Km2 = 2.57 mM, Vmax2 = 16.25 nmol/min/mg of protein). L-Arginine uptake was monophasic with a Km of 106 microM and a Vmax of 0.33 nmol/min/mg of protein. L-NOARG uptake was selectively inhibited by L-NOARG, NG-nitro-L-arginine methyl ester, and branched-chain and aromatic amino acids. L-Alanine and L-serine also inhibited L-NOARG uptake but with less potency. Uptake of L-arginine was selectively inhibited by NG-monomethyl-L-arginine acetate and basic amino acids. These studies suggest that in rat cerebellar synaptosomes, L-NOARG is transported by the neutral amino acid carrier systems T and L with high affinity, whereas L-arginine is transported by the basic amino acid carrier system y+ with high affinity. These data indicate that the concentration of competing amino acids is an important factor in determining the rates of uptake of L-NOARG and L-arginine into synaptosomes and, in this way, may control the activity of nitric oxide synthase.

Methylmercury transport across the placenta via neutral amino acid carrier

Methylmercury (MeHg) penetrates the placental barrier to affect developing fetuses in the uterus. However, the mechanism of placental MeHg transport is not well defined. To clarify the MeHg transport system that functions in the placenta, pregnant rats were intravenously administered MeHg on day 18 of gestation. The fetal blood was collected from the umbilical cord at 30 and 60 min after the administration, and its mercury concentration was measured. MeHg was found to be rapidly transported to the fetal blood in a time- and dose-dependent manner, and predominantly distributed in the blood cells there. MeHg transport was effectively suppressed by the co-injection of neutral amino acids, i.e., L-methionine and L-phenylalanine, suggesting that MeHg is actively transported as its cysteine conjugate via the neutral amino acid carrier system. The suppression by methionine was not so marked as by phenylalanine. Since methionine administration caused a rapid increase of the cysteine, which functioned as a predominant carrier in MeHg transport, in the maternal plasma, newly synthesized cysteine seemed to accelerate the mercury uptake. Accordingly, the acceleration by the extra cysteine would compensate partly the competitive effect of methionine as a neutral amino acid.

Renal Transport of Endogenous Amino Acids

Repeated adminisatration of triiodothyronine (T&lt;sub&gt;3&lt;/sub&gt;, 20 µg/l00 g b.w., once daily for 3 days) or dexamethasone (60 µg/l00 g b.w., once daily for 3 days) caused significant changes of amino acid plasma concentrations in young (10 days old) and adult rats (2 months old). After treatment with T&lt;sub&gt;3&lt;/sub&gt;, in young animals concentrations of β-alanine, aspartic acid, glutamic acid, ornithine, asparagine, leucine, taurine, serine, alanine, and glutamine were enhanced, whereas those of tyrosine, arginine, and threonine were significantly diminished. In adult rats only slight changes occurred after both T&lt;sub&gt;3&lt;/sub&gt; and dexamethasone. In the kidney, the reabsorbed fraction of amino acids was enhanced after both T&lt;sub&gt;3&lt;/sub&gt; and dexamethasone treatment in young rats, whereas in adult rats the two hormones were without influence on tubular reabsorption of amino acids. Taken together, both different plasma concentrations of amino acids and changes in amounts of amino acids reabsorbed in the tubuli, in adult rats the fractional excretion of amino acids was nearly unchanged following hormone treatment (exception: significant increase in taurine clearance). But in young animals the fractional excretion was reduced in 15 of 22 amino acids after dexamethasone and in 12 of 23 amino acids after T&lt;sub&gt;3&lt;/sub&gt; treatment, indicating stimulatory effects of both hormones on tubular amino acid carrier systems in immature animals.

Effects of amino acids on zinc transport in rat erythrocytes.

1. A significant proportion of plasma zinc exists complexed with amino acids. The effect of amino acids on the accumulation of radioactive zinc by rat erythrocytes was studied in vitro, to investigate the hypothesis that zinc might be transported into cells as an amino acid-zinc complex. 2. L-Histidine (500 microM-10 mM) stimulated 65Zn uptake; 50 mM-L-histidine gave a slight inhibition of uptake. D-Histidine (500 microM-10 mM) inhibited uptake in a dose-dependent manner. A non-zinc-binding amino acid, L-alanine, did not affect 65Zn uptake. 3. The effect of L-histidine was sodium dependent and temperature dependent, but was DIDS insensitive. These properties suggest that zinc is being transported as a zinc-histidine complex, utilizing an amino acid carrier system. Uptake of zinc in the presence of L-histidine differed from the previously described ionic mechanism, and may represent a physiological route of uptake. 4. L-Histidine stimulated efflux of 65Zn from pre-loaded cells. 5. The relevance of transport of a zinc-histidine complex is discussed with reference to histidinaemia, and as a significant zinc transport system in the presence of the very low ionic zinc concentrations found in plasma.

Acute cadmium uptake by rabbit kidneys: Mechanism and effects

Trapping of an arterial bolus in the kidney by 40 sec aortic occlusion permitted demonstration of diffusion-limited and Zn-sensitive renal uptake of nonfiltered but diffusible Cd from plasma: mercaptoethanol (ME) or 2,3-dimercaptopropanol (BAL) had been added to the bolus to prevent sequestration of the metals by plasma protein. Cadmium taken up by the kidney under these conditions, whether from blood or glomerular filtrate, unlike Zn, did not return to blood over a period of 2–3 min. Nor was Cd thus accumulated removed by EDTA; it had presumably been transferred into cells. The diffusion dependence of this internalization shows it to be a relatively slow process, as it is in jejunum. In contrast, uptake of Cd tightly bound in a lipid-soluble complex with diethyldithiocarbamate (DDTC) is rapid and flow dependent. Slow uptake in the presence of ME and its inhibition by Zn are therefore not likely to involve movement of undissociated ME complex across the cell membranes. Instead, it is suggested that (1) Cd-binding sites on the membrane possess a relatively high affinity for Cd and can compete for it with ME, and (2) it is the resulting Cd-membrane interaction which, as in jejunum, is depressed by Zn. During transient occlusion kidneys could be loaded with up to 25 μg Cd/g cortex without evidence of immediate malfunction. Inhibition of amino acid transport, as previously described, is seen only after an initiation period of 1–2 days following CdME injection, although cortical Cd levels at that time have decreased. The finding of a slowly developing inhibition of amino acid carrier systems suggests an indirect action of Cd; by implication, unique threshold concentrations should not be defined for Cd in renal cortex following subchronic exposures without reference to the duration of exposure.

Methyl Mercury Uptake Across Bovine Brain Capillary Endothelial Cells in Vitro: The Role of Amino Acids

Previous studies in the rat in vivo have demonstrated that co-injection of methyl mercury (MeHg) with L-cysteine into the common carotid artery enhances brain Hg levels following a single capillary pass through the CNS vasculature. In order to elucidate the relationship between MeHg transport and the neutral amino acid transport carrier system, regulatory aspects of MeHg transport across the bovine blood-brain barrier were investigated in isolated brain microvessel preparations. Following 1 hour co-incubations of 203Hg-MeHgCl with 0.1 mM L-cysteine at 37 degrees, 203Hg uptake by suspended microvessels was significantly increased (P less than 0.05) compared with controls. This enhanced capillary uptake of 203Hg was abolished by co-incubations of microvessels with 0.1 mM L-cysteine-L-methionine, or 0.1 mM L-cysteine plus AT-125 (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazolacetic acid), an irreversible inhibitor of gamma-glutamyl-transpeptidase. One hr co-incubations of bovine capillaries with 203Hg-MeHgCl and 0.1 mM D-cysteine at 37 degrees or 0.1 mM L-cysteine at 0 degrees did not increase rat of 203Hg uptake compare with controls. These results indicate that L-cysteine enhances the rate of capillary MeHg uptake. The accumulation of 203Hg in the bovine microvessels appears to be a carrier-mediated process. It is inhibited by L-methionine, a competitive substrate for neutral amino acid transport, and by AT-125. Capillary uptake of 203Hg is stereospecific to the L-enantiomorph of cysteine, suggesting selective uptake of MeHg across the blold-brain barrier. The data emphasize the relationship between the L-enantiomorph neutral amino acid carrier system and MeHg transport across the capillaries.

In vitro studies of the antitumor effect of melphalan and analogous aniline mustard derivatives

Melphalan and analogous aniline mustard derivatives were tested on antitumor activity in vitro. All compounds showed similar antiproliferative effects against melanoma B16, sarcoma 180 and leukemia P388D1. Thus, the singular clinical efficiency of melphalan may not simply be explained by a selective uptake employing amino acid carrier systems of tumor cells.

Kinetics and Characteristics of Absorption From an Equimolar Mixture of 12 Glycyl-Dipeptides in Human Jejunum

We have investigated rates of dipeptide disappearance during jejunal perfusion with an equimolar mixture of 12 glycyl-dipeptides and compared amino acid absorption rates from this mixture with those from the corresponding free amino acid mixture in healthy volunteers. All dipeptides contained glycine in the N-terminal position, which was designed to favor peptide absorption rather than hydrolysis. At 2 mM concentration there was little difference in disappearance among dipeptides, but at 6 mM there was some selectivity: Gly-Phe and Gly-Met exhibited the fastest, and Gly-Arg and Gly-His the slowest rates of disappearance. However, between these ends of the spectrum there were only modest differences in disappearance among dipeptides (Gly-Ala, Gly-Thr, Gly-Leu, Gly-Ile, Gly-Val, Gly-Lys, Gly-Pro, Gly-Trp). The amino acid absorption rates were generally smaller but more selective from the free amino acid than from the dipeptide mixture. We conclude that (a) the C-terminal amino acid residue influences intestinal assimilation of glycyl-dipeptides and (b) the considerably greater absorption rate of amino acids from the dipeptide than from the amino acid mixture appears to be the result of uptake by a system that has a greater transport capacity than amino acid carrier systems, thus minimizing competition among its substrates.