Differences in the release ofl-glutamate andd-aspartate from primary neuronal chick cultures

Primary neuronal cultures were made from eight-day-old embryonic chick telencephalon. Ten-day-old cultures were used to study the release ofd-[³H]aspartate andl-[³H]glutamate. Thed-[³H]aspartate release was stimulated by increasing potassium concentrations, but it was not calcium dependent. In contrast, the potassium dependentl-[³H]glutamate release was calcium dependent, and furthermorel-[³H]glutamate release was optimal at potassium concentrations<30 mM. The inhibitors of glutamate uptake, dihydrokainate and 1-aminocyclobutane-trans-1,3-dicarboxylic acid (CACB), also referred to as cis-1-aminocyclobutane-1,3-dicarboxylate, were used in the release experiments. Dihydrokainate had no effect on aspartate release, whereas CACB increased both the basal efflux ofd-[³H]aspartate and the potassium evoked release. CACB had no effect on the potassium stimulatedl-glutamate release. We believe thatl-glutamate is released mainly by a vesicular mechanism from the presumably glutamatergic neurons present in our culture.d-aspartate release observed by us, could be mediated by a transporter protein. The cellular origin of this release remains to be assessed.     

Substrate regulation of ascorbate transport activity in astrocytes

Astrocytes possess a concentrativel-ascorbate (vitamin C) uptake mechanism involving a Na⁺-dependentl-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellularl-ascorbate on the activity of this transport system. Initial rates ofl-ascorbate uptake were measured by incubating primary cultures of rat astrocytes withl-[¹⁴C]ascorbate for 1 min at 37°C. We observed that the apparent maximal rate of uptake (V _max) increased rapidly (<1 h) when cultured cells were deprived ofl-ascorbate. In contrast, there was no change in the apparent affinity of the transport system forl-[¹⁴C]ascorbate. The increase inV _max was reversed by addition ofl-ascorbate, but notD-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures withl-ascorbate did not affect uptake of 2-deoxy-D-[³H(G)]glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.     

Neurochemical,pharmacological, and developmental studies on cerebellar receptors for dicarboxylic amino acids

Specific binding ofl-[³H]glutamate ([³H]Glu) andl-[³H]asparate ([³H]Asp) to cerebellar membranes represented a time-, temperature- pH- and protein-dependent interaction which was both saturable and reversible. Binding sites for both radioligands appeared maximally enriched in synaptosomal fractions isolated by gradient centrifugation. Kinetically derived dissociation constant (K _off/K _on=K _d) for [³H]Glu binding to this fraction indicated high-affinity (443 nM). Competition experiments employing analogs of excitatory amino acids, including new antagonists, helped identify binding sites for [³H]Glu and [³H]Asp as receptors with differential pharmacological, specificities. Membrane freezing reduced numbers of both receptor types, but binding activity could be recovered partially by incubation at 37°C. Glu receptors exhibited a pronounced deleterious sensitivity to thiol modifying reagents andl-Glu (50–1000 M) provided protection, against these compounds during co-incubation with cerebellar membranes. It is suggested that cold storage may induce partially reversible receptor inactivation by promoting sulfhydryl group/bond modification. Rat cerebellar glutamatergic function (endogenous Glu content, Glu uptake and receptor sites) exhibited an apparent ontogenetic peak between days 8–12 postpartum with a plateauing profile from day 30 to adulthood. The accelerated development (days 8–12) coincides with the first demonstrable Glu release and kainic acid neurotoxicity, as described previously.     

Sodium gradient- and sodium plus potassium gradient-dependentl-glutamate uptake in renal basolateral membrane vesicles

Summary A membrane preparation enriched in the basolateral segment of the plasma membrane was isolated from the rat renal cortex by a procedure that included separation of particulates on a self-generating Percoll gradient. The uptake ofl-glutamate by the basolateral membrane vesicles was studied. A Na⁺ gradient ([Na⁺] _o >[Na⁺] _i ) stimulated the uptake ofl-glutamate and provided the driving force for the uphill transport of the acidic amino acid, suggesting a Na⁺-l-glutamate cotransport system in the basolateral membrane. A K⁺ gradient ([K⁺] _i >[K⁺] _o ) increased the uptake additionally. This effect was specific for K⁺ (Rb⁺). The action of the K⁺ gradient in enhancing the uptake ofl-glutamate had an absolute requirement for Na⁺. In the presence of Na⁺, but in the absence of a Na⁺ gradient. i.e., [Na⁺] _o =[Na⁺] _i , the K⁺ gradient also energized the concentrative uptake ofl-glutamate. This effect of the K⁺ gradient was not attributable to an alteration in membrane potential. The finding of a concentrative uptake system forl-glutamate energized by both Na⁺ ([Na⁺] _o >[Na⁺] _i and K⁺ ([K⁺] _i >[K⁺] _o ) gradients in the basolateral membrane, combined with previous reports of an ion gradient-dependent uphill transport system for this amino acid in the brush border membrane, suggests a mechanism by whichl-glutamate is accumulated intracellularly in the renal proximal tubule to extraordinarily high concentrations.     

Release of [3H]l-glutamate and [3H]l-glutamine in rat cerebellum slices: A comparison of the effect of veratridine and electrical stimulation

Depolarization-elicited release of neurotransmitter glutamate was studied in rat cerebellar slices previously loaded with either [³H]l-glutamate or [³H]l-glutamine. Both depolarization conditions used (e.g. long-lasting tonic depolarization elicited by veratridine, or short repetive electrical pulses) increased 6 to 8 folds the release of labelled glutamate and of another compound, presumably alpha-ketoglutarate, without modifying the release of labeled glutamine. Because of the position of the label in the precursor radioactive molecules, GABA was weakly labeled and aspartate was unlabeled. The properties of the evoked glutamate release from cerebellar slices were those of a neurotransmitter since it was inhibited by tetrodotoxin and was Ca²⁺-dependent. Alpha-ketoglutarate is either coreleased from nerve terminals or is released from astrocytes and could participate in glutamate recycling. The data confirm the generally accepted model implying the presence of two neurotransmitter glutamate pools, a neuronal pool of newly synthesized glutamate and an astrocytic storage pool, but in addition indicate that the former is in rapid isotopic equilibrium with the extracellular compartment. Our present results also indicate that the glutamate/glutamine cycle is not activated in depolarizing conditions.With the technical assistance of O. LEVY¹ and K. WINDISCH²     

Studies on isolated subcellular components of cat pancreas

Summary Transport of alanine was studied in isolated plasma membrane vesicles from cat pancreas using a rapid filtration technique. The uptake is osmotically sensitive and the kinetics ofl-alanine transport are biphasic showing a saturable and a nonsaturable component. The saturable component is seen only when a sodium gradient directed from the medium to the vesicular space is present. Under this condition an overshooting uptake ofl-but not ofd-alanine occurs. The Na⁺ gradient stimulated uptake ofl-alanine is inhibited byl-serine andl-leucine and stimulated when the membrane vesicles had been preloaded withl-alanine,l-serine orl-leucine.The ionophore monensin inhibits stimulation of uptake caused by a sodium gradient. In the presence of valinomycin or carbonyl cyanidep-trifluoromethoxyphenylhydrazone (CFCCP), the sodium-dependent transport is augmented in vesicles preloaded with K₂SO₄ or H⁺ ions (intravesicular pH 5.5), respectively. In the presence of different anions, the Na⁺-dependent transport is stimulated according to increasing anionic penetration through membranes (lipid solubility). We conclude that a sodium dependent electrogenic amino acid transport system is present in pancreatic plasma membranes.     

Glutamate-mediated synaptic transmission between neuron B4 and salivary cells ofHelisoma trivolvis

In this study, we have further characterized the morphology and physiology of the neuroglandular synapse between the identified buccal neuron, B4, and the salivary gland ofHelisoma. We demonstrate that the coupling coefficient between salivary cells within an individual acinus is approximately 1.0. We also demonstrate that synapses within the salivary gland are located near a superficial muscle layer. We examine the effects of glutamate on the salivary gland and on the B4-salivary gland EPSP.l-glutamate produces a transient, rapid onset depolarization of salivary gland cells. The response is mimicked by high concentrations ofl-homocysteic acid, but not by NMDA,l-aspartate,d-glutamate or kainate. The response is blocked by the presence ofl- ord-glutamate in the bath, but not by CNQX, DNQX, DGG,d-AP5, orl-AP3. The depolarization is primarily dependent on the presence of calcium in the bathing solution. When eitherl- ord-glutamate is present in the bathing solution, the amplitude of the B4-salivary gland EPSP is reversibly reduced. The similar pharmacological properties of the response of the salivary gland to glutamate and the B4 epsp indicate thatl-glutamate is a strong candidate for the fast excitatory neurotransmitter at theHelisoma neuroglandular synapse.     

Analysis of a Vesicular Glutamate Transporter (VGLUT2) Supports a Cell-leakage Mode in Addition to Vesicular Packaging

VGLUT2 is one of three vesicular glutamate transporters that play crucial roles in glutamatergic excitatory neurotransmission. We explored the functional properties of the rat VGLUT2 by heterologous expression of VGLUT2 in Xenopus oocytes. Immunocytochemical analysis indicated that most VGLUT2 protein was expressed in intracellular compartments but that some expression occurred also on the plasma membrane. Functional analysis revealed VGLUT2 to be active in two independent modes, namely, uptake into intracellular organelles and efflux at the plasma membrane. VGLUT-specific transport was identified based on the strong preference for glutamate over aspartate—in contrast to plasma-membrane or mitochondrial glutamate transporters—and sensitivity to known VGLUT blockers. VGLUT2 expression in oocytes (1) stimulated the influx of l-[³H]glutamate, but not d-[³H]aspartate, into digitonin-permeabilized oocytes and (2) stimulated efflux of l-glutamate, but not l-aspartate, from intact oocytes preinjected with ³H-labeled amino acids. In the latter assay, cellular efflux of glutamate (which was blocked by rose bengal and trypan blue) may be analogous to vesicular packaging of glutamate. Our data are consistent with VGLUT2-mediated H⁺/l-glutamate antiport, but not antiport with chloride. Expression of mammalian VGLUT1 and VGLUT3 also stimulated l-[³H]glutamate efflux from Xenopus oocytes, suggesting that this phenomenon is a general feature of vesicular glutamate transporters. Our findings support the idea that vesicular glutamate transporters, when transiently expressed on the neuronal plasma membrane, may mediate Ca²⁺-independent glutamate leakage in addition to their traditional role of packaging glutamate into synaptic vesicles for Ca²⁺-dependent exocytosis. Special issue article in honor of Dr. Frode Fonnum.     

Acute neurotoxicity ofl-glutamate induced by impairment of the glutamate uptake system

We examined the effect of the glutamate uptake inhibitorl-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) on the neurotoxicity ofl-glutamate in organotypic cultures of rat spinal cord. Eighteen-day-old cultures were incubated with 500 μMl-glutamate, 1 mM PDC, or both. After 72 hours, the tissues were stained for acetylcholinesterase (AChE), and the ventral horn AChE-positive neurons (VHANs) analyzed using morphometry. Neitherl-glutamate nor PDC affected AChE staining, but in combination they produced markedly reduced AChE staining in the dorsal horn and a significant decrease in the number of VHANs (especially the smaller VHANs) as compared with the control. Moreover, treatment with 200 μM PDC for 2 weeks preferentially affected the smaller VHANs. The neurotoxicity ofl-glutamate plus PDC was blocked by the N-methyl-d-aspartate (NMDA) antagonist 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). Results suggest that glutamate uptake system has an important protective function in the aggravation of acute neuronal damage.     

Amino acid and monoamine transport in primary astroglial cultures from defined brain regions

The uptake ofl-[³H]glutamate,l-[³H]aspartate, -[³H]aminobutric acid (GABA), [³H]dopamine,dl-[³H]norepinephrine and [³H]5-hydroxytryptamine (5-HT) was studied in astrocytes cultured from the cerebral cortex, striatum and brain stem of newborn rat and grown for 2 weeks in primary cultures. The astrocytes exhibited a high-affinityl-glutamate uptake withK _m values ranging from 11 to 110 M.V _max values were 4.5 in cerebral cortex, 39.1 in striatum, and 0.4 in brain stem, nmol per mg cell protein per min. There was a less prominent high-affinity uptake ofl-aspartate withK _m values from 88 to 187 M.V _max values were 7.4 in cerebral cortex, 37.1 in striatum, and 3.1 in brain stem, nmol per mg cell protein per min. The high-affinity GABA uptake exhibitedK _m values ranging from 5 to 17 M andV _max values were 0.01 for cerebral cortex, 0.04 for striatum, and 0.1 for brain stem, nmol per mg cell protein per min. No high-affinity, high-capacity uptake was found for the monoamines. The results demonstrate a heterogeneity among the astroglial cells cultivated from the different brain regions concerning the uptake capacity of amino acid neurotransmitters. Furthermore, amino acid transmitters and monoamines are taken up by the cells in different ways.     

Diverse Presynaptic Mechanisms Underlying Methyl-β-Cyclodextrin-Mediated Changes in Glutamate Transport

The effect of the cholesterol-depleting agent methyl-β-cyclodextrin (MβCD) on exocytotic, transporter-mediated, tonic release, the ambient level and uptake of l-[¹⁴C]glutamate was assessed in rat brain synaptosomes using different methodological approaches of MβCD application. The addition of 15 mM MβCD to synaptosomes (the acute treatment, AT) immediately resulted in the extraction of cholesterol and in a two times increase in the extracellular l-[¹⁴C]glutamate level. When 15 mM MβCD was applied to synaptosomes for 35 min followed by washing of the acceptor (the long-term pretreatment, LP), this level was only one-third higher than in the control. The opposite effects of MβCD on tonic l-[¹⁴C]glutamate release and glutamate transporter reversal were found in AT and LP. Tonic release was dramatically enlarged in AT, but decreased after LP. Transporter-mediated release was increased several times in AT, but attenuated in LP. Depolarization-evoked exocytotic release of l-[¹⁴C]glutamate was completely lost in AT, whereas after LP, it was decreased by half in comparison with the control. Na⁺-dependent l-[¹⁴C]glutamate uptake was decreased by ~60% in AT, whereas in LP, it was lowered by ~40% only. The presence of MβCD in the incubation media during AT caused dramatic dissipation of the proton gradient of synaptic vesicles that was shown with the pH-sensitive dye acridine orange, whereas after LP, no statistically significant changes were registered in synaptic vesicle acidification. It was concluded that the diverse changes in glutamate transport in AT and LP were associated with the difference in the functional state of synaptic vesicles.     

Involvement of the glutamine synthetase/glutamate synthase pathway in ammonia assimilation by the wood-rotting fungusPleurotus ostreatus

The mycelium of the wood-rotting fungus,P. ostreatus, contains NAD-dependent glutamate synthase inhibited by azaserine.l-Glutamine andl-glutamate are the most important free amino acids in the mycelium. Feeding of the mycelium with nitrogenous substrates showed thatl-glutamate,l-aspartate andl-alanine are interconnected by way of transaminases. After the inhibition of glutamine synthetase by methionine-S-sulfoximine the synthesis ofl-glutamate was inhibited and the level of all free amino acids decreased. The¹⁵N-NMR spectra of mycelia after the addition of¹⁵NH₄Cl confirmed that the GS/GOGAT is the only pathway of ammonia assimilation inP. ostreatus and NAD-glutamate dehydrogenase should be the deaminating enzyme.     

Na+-independentl-aspartate binding sites in chick brain

1. One binding component with aK _d value of 200×10^–9 M and half-life of the ligand binding component of 30 min was found. 2. Chloride ions produced a significant increase ofl-[³H]aspartate andl-[³H]glutamate binding. 3.l-Glutamate,l-ibotenate,l-quisqualate, anddl-homocysteic acid were potent inhibitors ofl-[³H]aspartate binding. 4. In all brain regions major increases of binding were observed during the third week of the in ovo period of life.     

<Emphasis Type="Italic">In vivo</Emphasis> Quinolinic Acid Increases Synaptosomal Glutamate Release in Rats: Reversal by Guanosine

Glutamate, the main excitatory neurotransmitter in the mammalian central nervous system (CNS), plays important role in brain physiological and pathological events. Quinolinic acid (QA) is a glutamatergic agent that induces seizures and is involved in the etiology of epilepsy. Guanine-based purines (GBPs) (guanosine and GMP) have been shown to exert neuroprotective effects against glutamatergic excitotoxic events. In this study, the influence of QA and GBPs on synaptosomal glutamate release and uptake in rats was investigated. We had previously demonstrated that QA “in vitro” stimulates synaptosomal L-[³H]glutamate release. In this work, we show that i.c.v. QA administration induced seizures in rats and was able to stimulate synaptosomal L-[³H]glutamate release. This in vivo neurochemical effect was prevented by i.p. guanosine only when this nucleoside prevented QA-induced seizures. I.c.v. QA did not affect synaptosomal L-[³H]glutamate uptake. These data provided new evidence on the role of QA and GBPs on glutamatergic system in rat brain.     

Properties of the uptake and release of neurotransmitter glutamate in cerebral cortical tissue of guinea pigs

In order to investigate the dynamics of glutamate as a neurotransmitter and to avoid a complication by its metabolism, we studied the uptake and release of labeled non-metabolizabled-isomers of aspartate and glutamate in cerebral cortical slices and synaptosome preparation from guinea-pigs. The rate of uptake ofd-aspartate and glutamate was mutually inhibited in a non-competitive fashion, indicating that their uptake mechanisms are not exactly the same. By ouabain (0.05 mM), the uptake ofd-aspartate and glutamate into synaptosome preparation was less inhibited than that into cerebral slices. In synaptosome preparation most of the preloadedd-aspartate and glutamate was released by high-potassium (50 mM) stimulation, whereas in cerebral slices only a slight release was observed. However, when the slices were superfused with a medium free of sodium ions, which are absolutely necessary for the uptake, after preloaded with the labeled amino acids in the standard medium, a distinct release of radioactivity was induced by high-potassium stimulation. This potassium-induced release corresponded to only about 20% of the radioactivity accumulated in the slices. The accumulation ofd-aspartate and glutamate into cerebral slices was much larger on the basis of their protein content than that into synaptosome preparation, when a high concentration (1 mM) of the amino acids was added to the medium. These observations suggest that the uptake system ofd-aspartate and glutamate in cerebral slices is quite different from that in synaptosome preparation, and that the accumulation into cerebral slices is mainly localized in glial cells. In vivo the glial cell uptake is probably more important in removing the released neurotransmitter glutamate.Dedicated to Professor Yasuzo Tsukada.     

Metabolism ofl-proline toN-acetyl-d-glucosamine during germ tube formation ofCandida albicans

The metabolism ofl-proline toN-acetyl-d-glucosamine (GlcNAc) during germ tube formation ofCandida albicans (C. albicans) ATCC 1002 was studied. In uptake experiments, 6.9 nmol ofl-[¹⁴C]proline were taken up by 1×10⁶ cells during 3 h of incubation at 37°C. The percentage of germ tube formation was 94 under the same condition. The presence of GlcNAc reduced the uptake ofl-proline to 3.0 nmol. The percentage of germ tube formation was 95 in the presence and absence of GlcNAc. The [³H]GlcNAc uptake was 3.0 nmol and was constant whetherl-proline was present or not. After the preparation of a chitin fraction from germ tubes that were labeled withl-[¹⁴C]proline, the radioactivity froml-proline was detected in the glucosamine (GlcN) fraction by thin-layer chromatography (TLC). The metabolism ofl-proline to GlcNAc in chitin during germ tube formation was confirmed in this experiment.     

Single Taste Stimuli Elicit Either Increases or Decreases in Intracellular Calcium in Isolated Catfish Taste Cells

Taste cells are specialized epithelial cells that respond to stimulation with release of neurotransmitters onto afferent nerves that innervate taste buds. In analogy to neurotransmitter release in other cells, it is expected that neurotransmitter release in taste cells is dependent on an increase in intracellular Ca²⁺ ([Ca²⁺] _i ). We have studied changes in [Ca²⁺] _i elicited by the taste stimuli l- and d-arginine in isolated taste cells from the channel catfish (Ictalurus punctatus). In a sample of 119 cells, we found 15 cells responding to l-arginine, and 12 cells responding to d-arginine with an increase in [Ca²⁺] _i . The response to l-arginine was inhibited by equimolar d-arginine in cells where d-arginine alone did not cause a change in [Ca²⁺] _i , which is consistent with mediation of this response by a previously characterized l-arginine-gated nonspecific cation channel antagonized by d-arginine [31]. However, we also found that these taste stimuli elicited decreases in [Ca²⁺] _i in substantial number of cells (6 for l-Arg, and 2 for d-Arg, n= 119). These observations suggest that stimulation of taste cells with sapid stimuli may result in simultaneous excitation and inhibition of different taste cells within the taste bud, which could be involved in local processing of the taste signal. Received: 25 May 1995/Revised: 29 September 1995     

Modulation of [<Superscript>3</Superscript>H]Dopamine Release by Glutathione in Mouse Striatal Slices

Glutathione (γ-glutamylcysteinylglycine, GSH and oxidized glutathione, GSSG), may function as a neuromodulator at the glutamate receptors and as a neurotransmitter at its own receptors. We studied now the effects of GSH, GSSG, glutathione derivatives and thiol redox agents on the spontaneous, K⁺- and glutamate-agonist-evoked releases of [³H]dopamine from mouse striatal slices. The release evoked by 25 mM K⁺ was inhibited by GSH, S-ethyl-, -propyl-, -butyl- and pentylglutathione and glutathione sulfonate. 5,5′-Dithio-bis-2-nitrobenzoate (DTNB) and l-cystine were also inhibitory, while dithiothreitol (DTT) and l-cysteine enhanced the K⁺-evoked release. Ten min preperfusion with 50 μM ZnCl₂ enhanced the basal unstimulated release but prevented the activation of K⁺-evoked release by DTT. Kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) evoked dopamine release but the other glutamate receptor agonists N-methyl-d-aspartate (NMDA), glycine (1 mM) and trans-1-aminocyclopentane-1,3-dicarboxylate (t-ACPD, 0.5 mM), and the modulators GSH, GSSG, glutathione sulfonate, S-alkyl-derivatives of glutathione, DTNB, cystine, cysteine and DTT (all 1 mM) were without effect. The release evoked by 1 mM glutamate was enhanced by 1 mM GSH, while GSSG, glutathionesulfonate and S-alkyl derivatives of glutathione were generally without effect or inhibitory. NMDA (1 mM) evoked release only in the presence of 1 mM GSH but not with GSSG, other peptides or thiol modulators. l-Cysteine (1 mM) enhanced the glutamate-evoked release similarly to GSH. The activation by 1 mM kainate was inhibited by S-ethyl-, -propyl-, and -butylglutathione and the activation by 0.5 mM AMPA was inhibited by S-ethylglutathione but enhanced by GSSG. Glutathione alone does not directly evoke dopamine release but may inhibit the depolarization-evoked release by preventing the toxic effects of high glutamate, and by modulating the cysteine–cystine redox state in Ca²⁺ channels. GSH also seems to enhance the glutamate-agonist-evoked release via both non-NMDA and NMDA receptors. In this action, the γ-glutamyl and cysteinyl moieties of glutathione are involved.     

Inhibition by excitatory sulphur amino acids of the high-affinityl-glutamate transporter in synaptosomes and in primary cultures of cortical astrocytes and cerebellar neurons

A detailed kinetic study of the inhibitory effects ofl- andd-enantiomers of cysteate, cysteine sulphinate, homocysteine sulphinate, homocysteate, and S-sulpho-cysteine on the neuronal, astroglial and synaptosomal high-affinity glutamate transport system was undertaken.d-[³H] Aspartate was used as the transport substrate. Kinetic characterisation of uptake in the absence of sulphur compounds confirmed the high-affinity nature of the transport systems, the Michaelis constant (K _m) ford-aspartate uptake being 6 M, 21 M and 84 M, respectively, in rat brain cortical synaptosomes and primary cultures of mouse cerebellar granule cells and cortical astrocytes. In those cases where significant effects could be demonstrated, the nature of the inhibition was competitive irrespective of the neuronal versus glial systems. The rank order of inhibition was essentially similar in synaptosomes, neurons and astrocytes. Potent inhibition (K _iK _m) of transport in each system was exhibited byl-cysteate, andl- andd-cysteine sulphinate whereas substantially weaker inhibitory effects (K _i>10–1000 times the appropriateK _m value) were exhibited by the remaining sulphur amino acids. In general, inhibition: (i) was markedly stereospecific in favor of thel-enantiomers (except for cysteine sulphinate) and (ii) was found to decrease with increasing chain length. Computer-assisted molecular modelling studies, in which volume contour maps of the sulphur compounds were superimposed on those ofd-aspartate andl-glutamate, demonstrated an order of inhibitory potency which was, qualitatively, in agreement with that obtained quantitatively by in vitro kinetic studies.Special issue dedicated to Dr. Elling Kvamme     

Stimulation of mucosal uptake of selenium from selenite by some thiols at various sites of rat intestine

The influence of several thiols (conc. 1 mmol/L) on mucosal uptake of⁷⁵Se from⁷⁵Se-labeled selenite (conc. 10 μmol/L) across the brush border of rat jejunum and cecum was investigated in vitro using a short-term uptake technique.l-Cysteine (l-Cys) stimulated⁷⁵Se uptake in the mid- and distal jejunum and cecum, but not in the proximal jejunum. The effect was maximal in the distal jejunum.d-Cys was less effective in the jejunum and similarly effective in the cecum.l-Leucine (l-Leu) andl-glutamic acid significantly reduced the stimulatory effect ofl-Cys on Se uptake in the distal jejunum, whereas the respective effect ofd-Cys was not diminished byl-Leu. Cysteamine stimulated mucosal⁷⁵Se uptake at all intestinal sites tested, whereas the effect of mercaptopyruvate was restricted to the distal jejunum. Thioglycolate also enhanced⁷⁵Se uptake in the distal jejunum. The stimulatory effects ofl-Cys, mercaptopyruvate, and thiologlycolate were Na⁺-dependent, whereas the effect of cysteamine also occurred in the absence of Na⁺. Mercaptosuccinate,d-penicillamine, ergothioneine, and thiosulfate did not enhance mucosa⁷⁵Se uptake. It is concluded from these findings that the reaction of some thiols with selenite results in Se compounds that are rapidly absorbed by the intestinal epithelium through various Na⁺-dependent and Na⁺-independent, mechanisms. The high bioavailability of Se from selenite found by others might thus be the result of the presence of thiols in the gastrointestinal tract.