Dietary goitrogen-induced changes in the transport of 2-deoxy-d-glucose and amino acids across the rat blood-brain barrier

The hypothesis that a defect in the rate-limiting blood-brain barrier (BBB) nutrient transport may be one of the factors responsible for the brain defects seen in some iodine deficiency disorders was tested in Wistar/NIN rats fed potassium thiocyanate (KSCN), a synthetic goitrogen. The BBB nutrient transport was measured by the brain uptake index (BUI) method. Feeding KSCN to female rats (from weaning) through their growth, pregnancy and lactation (G1) but not from conception (G2) or parturition (G3) resulted in a significant decrease (≈23%) in the BBB transport of 2-deoxy-d-glucose (2-DG) in their offspring at weaning, compared with controls (C). Post-weaning KSCN-feeding (G4) of control pups did not affect their BBB 2-DG transport (BUI: 36.2±4.98, vs 38.8±4.11). The effects of different KSCN regimens on BBB transport of leucine (leu), tyrosine (tyr) and sucrose (background marker) were inconsistent, of smaller magnitude (≈10%) and appeared to be of little significance. Withdrawing KSCN from the diet of chronically KSCN-fed (G1) mothers from conception (G5) or parturition (G6) prevented the impairment of BBB 2-DG transport in pups (BUI: 27.0±4.98, 20.8±3.27, 26.9±3.99 and 28.3±3.47 in C, G1, G5 and G6 pups, respectively); this was reversed by feeding the control diet to G1 pups from weaning. Withdrawal of dietary KSCN did not affect BBB transport of leu, tyr and sucrose. The decreased BBB transport of 2-DG in G1 pups appears to be due to a decrease in affinity (K_t app 5.46 vs 4.15 mM) rather than in the capacity (T_max app 0.94 vs 0.91 μmoles/g/min) of the transport system. Intracarotid injections of KSCN per se had no effect on BBB 2-DG transport, suggesting that the effects may be secondary to the altered thyroid status of the animal.     

Regional transport of phenylalanine across the blood-brain barrier

In adult rats, using the single-pass brain uptake technique with a tritiated water standard, L-phenylalanine was shown to enter brain across the blood-brain barrier (BBB) by both a saturable and diffusionary process. A kinetic analysis of the data revealed Michaelis constant (K_a) of 0.42 mM and V _max of 35 nmoles g^?1 min^?1 The saturable component of L-phenylalanine transport was adversely affected by dinitrophenol or low sodium concentration in the intracarotid injection solution. Intravascular ouabain did not affect transport and only partial inhibition was observed with 1.5 mM L-leucine. It appears that there is no regional difference in the capacity of the BBB to more L-phenylalanine from blood to brain. This process is directly or indirectly coupled to sodium movement and is partially dependent on cellular energy.     

Receptor types mediating the excitatory actions of exogenousl-aspartate andl-glutamate in rat olfactory cortex

Changes in potential between the pial and cut surfaces of rat olfactory cortex slices evoked by N-methyl-d-aspartate (NMDA), quisqualate, kainate,l-glutamate andl-aspartate and also by γ-aminobutyric acid (GABA) have been monitored using extracellular electrodes. All agonists produced a pial-negative potential response when superfused onto the pial surface, GABA,l-aspartate andl-glutamate being less potent than the others. Repeated applications of NMDA, but not of the other agonists, led to a progressive reduction in response to approximately 30% of the initial depolarization. The responses to NMDA (100 μM) were selectively abolished by(±)2-amino-5-phosphonopentanoic acid (APP; 100 μM) while depolarizations evoked byl-glutamate andl-aspartate (both at 10 mM) were only antagonized by21 ± 2 (n = 12) and36 ± 3 (n = 12) percent respectively (means ± S.E.M.). γ-d-Glutamylglycine (γ-DGG; 1 mM) and(±)cis-2,3-piperidine dicarboxylate (cis-PDA; 2 and 5 mM), in addition to antagonizing responses to NMDA, also partially blocked quisqualate- and kainate-evoked depolarizations. When a mixture of APP (100 μM), γ-DGG (1 mM) and cis-PDA (5 mM) was applied to preparations, although NMDA receptors were completely blocked and responses to both quisqualate and kainate antagonized by approximately 80%,l-glutamate andl-aspartate evoked depolarizations were only reduced by51 ± 7 (n = 4) and 49 ± 4 (n = 4) percent respectively (means ± S.E.M.). The results are discussed in terms of the contributions made by NMDA, quisqualate and kainate receptors to the composite responses evoked byl-aspartate andl-glutamate.     

Calcium accumulation precedes the degenerative effects ofl-glutamate on locust muscle fibres

Agonist-induced degeneration of locust muscle occurs only when desensitization of the excitatory glutamate receptors present on this tissue is inhibited. It has been suggested that an increase in intracellular Ca²⁺ is responsible for this degeneration. To test this proposal the accumulation of⁴⁵Ca by locusts muscle has been studied under various conditions, including those under which receptor desensitization was inhibited. Retractor unguis muscles from the metathoracic leg of locusts (Schistocerca gregaria) were used in these studies. All muscles exposed tol-glutamate exhibited an early increase in intracellular radioactivity but this was 2–3 times greater in muscle pretreated with concanavalin A (Con A) to block receptor desensitization. In the desensitizing system the increase in muscle radioactivity was not maintained, intracellular Ca²⁺-levels declining to control values after 30 min in⁴⁵Ca-saline-containing glutamate. In Con A-treated muscles intracellular Ca²⁺-levels plateaued well above control levels within 5 min of exposure to glutamate and were maintained at these levels throughout the period of glutamate treatment. These results support the contention that agonist-induced degeneration of locust muscle is triggered by entry of Ca²⁺ and a rise in intracellular concentration of this cation to a toxic level.     

Blood-brain barrier permeability to leucine-enkephalin,d-Alanine-d-leucine-enkephalin and their N-terminal amino acid (tyrosine)

The permeability of the blood-brain barrier to [ tyrosyl-3,5-³H]enkephalin-(5-l-leucine) (abbreviated to Leu-Enk) and of its synthetic analogued-alanine²-[ tyrosyl-3,5-³H]enkephalin-(5-d-leucine) (abbreviated tod-Ala²-d-Leu⁵-Enk) was studied, in the adult rat, by means of Oldendorf's²⁷ intracarotid injection technique. The brain uptake index (BUI) corrected for residual vascular radioactivity was about the same for both peptides, indicating a low extraction from the blood during a 5- or 15-s period of exposure to the peptides. Transport of Leu-Enk was not saturated by unlabelled Enk at a concentration as high as 5 mM but was completely abolished by 5 mM tyrosine and by the inhibitor of aminopeptidase activity, bacitracin (2 mM). Also the typicall-transport system substrate, 2-aminobicyclo(2,2,1)heptane-2 car☐ylic acid (BCH)⁹ at 10 mM concentration markedly reduced (by 80%) Leu-Enk uptake by the brain. In contrast, brain uptake ofd-Ala²-d-Leu⁵-Enk was reduced only to about one-half of its control value by bacitracin or by 25% by BCH. Brain uptake forl-tyrosine was typically large and markedly inhibited by BCH but not inhibited by 5 mM unlabelled Leu-Enk. These results show that the measurable but low first-pass extractions for enkephalins are not representative of the uptake of these peptides into the brain, but rather reflect their extreme sensitivity to enzymatic degradation with a release of the N-terminal tyrosine residue. The results also suggest that small amounts ofd-Ala²-d-Leu⁵-Enk might cross the blood-brain barrier in an intact form. It is concluded that the absence of a transport mechanism at the blood-brain barrier, together with a rapid enzymatic degradation, tends to prevent significant penetration of the barrier by Leu-Enk during the 5- or 15-s period of these studies.d-Ala²-d-Leu⁵-Enk however, shows a penetrance of the blood-brain barrier significantly greater than that of sucrose.     

Starvation-induced changes in transport of ketone bodies across the blood-brain barrier

The permeability of the blood brain barrier (BBB) to beta-hydroxybutyrate (beta-HB) was computed in fed and starved (five days) rats by the simultaneous measurement of cerebral blood flow (diffusible indicator method-123I-iodoantipyrine) and brain uptake of 14C-beta-HB (relative to a 3H2O reference). The results from the present study demonstrate that the movement of beta-HB across the BBB in rat is by a carrier-mediated process. During starvation, total movement (carrier-mediated and diffusionary) of this ketone body into brain was observed to be enhanced because of an increase in the diffusionary loss across the cerebral capillary. The calculated transport kinetics also suggest that the beta-HB molecule has a greater affinity for the transport (mediator) protein during starvation, although the maximal rate of uptake by brain due to a carrier processes mediated Vmax is decreased either because there is a smaller quantity of the mediating molecule or because there is trans inhibition by a high cellular concentration of beta-HB or some analog.     

Decarboxylation ofl-Dopa by cultured mouse astrocytes

Cultured astrocytes contain immunologically specific aromaticl-amino acid decarboxylase (AADC) protein and express the AADC gene. Following incubation withl-Dopa, glial cultures synthesize and metabolize dopamine. The addition of pyridoxal 5-phosphate did not change the rate of dopamine synthesis. The formation of dopamine was blocked by NSD-1015. These experiments show that mouse cultured astrocytes are capable to convertl-Dopa into dopamine in a dose-dependent fashion.     

Effect ofd- andl-α-aminoadipate on the efflux ofl-aspartate,l-glutamate and γ-aminobutyrate from superfused rat brain slices

d-α-Aminoadipate (d-AA) andl-α-aminoadipate (l-AA) were found to significantly reduce spontaneous efflux of [¹⁴C]l-aspartate from preloaded rat brain slices. Onlyd-AA significantly reduced spontaneous efflux of [¹⁴C]l-glutamate and [³H]γ-aminobutyric acid (GABA);l-AA reduced but not significantly the efflux of these 2 labeled amino acids.d-AA reduced K⁺-stimulated release of [¹⁴C]l-aspartate and [^14]C]l-glutamate significantly, andl-AA that of [³H]GABA significantly. Since bothd-AA andl-AA inhibit the uptake ofl-aspartate,l-glutamate and GABA, their effects on the efflux of these amino acids are more specific. These results also suggest that it is unlikely that the depressant effect ofd-AA, and the excitant effect ofl-AA on neurons when applied locally by iontophoresis are secondary to the accelerated or decelerated release of more specific transmitter amino acids from neighboring cells.     

Effects ofl-cysteine-sulphinate andl-aspartate, mixed excitatory amino acid agonists, on the membrane potential of cat caudate neurons

Responses evoked byl-cysteine-sulphinate (l-CSA) andl-aspartate (l-Asp) were recorded with intracellular electrodes from caudate neurons in halothane anesthetized cats.l-CSA andl-Asp were applied microiontophoretically to caudate cells and their effects on membrane and action potentials, as well as on cortically evoked synaptic potentials were evaluated.l-CSA andl-Asp induced depolarizations accompanied by regular firing resembling kainate (KA)- or quisqualate (QUIS)-induced excitation patterns (type 1) in 82% and 72% of the recorded neurons, respectively, and a mixed pattern consisting of aN-methyl-d-aspartate (NMDA)-like excitation (type 2) followed by a regular type 1 pattern in the remaining cells. In about a quarter of the cells the effects ofl-CSA andl-Asp, but not those of KA or QUIS, were partially antagonized by 2-amino-7-phosphonoheptanoate (AP-7), a specific NMDA receptor antagonist. Kynurenate, a broad spectrum excitatory amino acid antagonist, blocked responses elicited by eitherl-CSA or QUIS. The actions ofl-CSA andl-Asp on the firing pattern and membrane potential of cat caudate neurons in situ provide evidence in favor of their mixed agonist nature with respect to NMDA and non-NMDA excitatory amino acid receptors.     

Cations differentially affect subpopulations ofl-glutamate receptors in rat synaptic plasma membranes

Several cations were examined for their ability to specifically affect one of the 3l-glutamate (l-Glu) binding sites in rat forebrain synaptic plasma membranes (i.e. Na⁺-dependent, Cl⁻-dependent and Cl⁻-independent). Na⁺-dependent binding was potently inhibited by K⁺ and NH₄⁺ ions. Other monovalent cations testedd (Cs⁺, Li⁺, triethylammonium) had no effect on this binding site. Polyvalent cations (Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺ and Cr³⁺) also had little effect on the Na⁺-dependentl-Glu binding site. Cl⁻-dependentl-Glu binding was potently inhibited by Na⁺ ions but was not affected by other monovalent ions. All of the divalent cations were potent inhibitors of both Cl⁻-dependent and -independent binding. The results show that these binding sites ofl-Glu can be distinguished by their response to cations and suggest possible novel modes of regulation in vivo.     

Increased uptake ofl-cysteine andl-cystine by nerve growth factor in rat pheochromocytoma cells

Nerve growth factor is a neurotrophic factor which promotes cell survival and differentiation in the central and peripheral nervous system. The rat pheochromocytoma (PC12) cell has been frequently used to study the actions of nerve growth factor (NGF). Our previous studies demonstrate that pretreatment with NGF for 24 h protects PC12 cells from oxidative stress by increasing glutathione (GSH) concentrations and the activity of γ-glutamylcysteine synthetase, which is a rate-limiting enzyme in GSH synthesis. The synthesis of intracellular GSH is dependent on the availability of the precursor amino acid,l-cysteine. Cells take upl-cystine from the extracellular fluid and convert it tol-Cysteine intracellularly.l-Cysteine is then released from cells to maintain extracellularl-cysteine. Here we report that NGF increased the uptake ofl-cysteine orl-cystine. The increased concentrations ofl-Cysteine orl-cystine by NGF was responsible for the enhanced intracellular GSH concentrations. The increased GSH andl-cysteine concentrations by NGF also served as intracellular antioxidants. The protection of PC12 cells by NGF from oxidative stress was due to the stimulated increased levels of intracellular glutathione andl-cysteine orl-cystine.     

Dietary goitrogen-induced changes in the transport of 2-deoxy-d-glucose and amino acids across the rat blood-brain barrier

The hypothesis that a defect in the rate-limiting blood-brain barrier (BBB) nutrient transport may be one of the factors responsible for the brain defects seen in some iodine deficiency disorders was tested in Wistar/NIN rats fed potassium thiocyanate (KSCN), a synthetic goitrogen. The BBB nutrient transport was measured by the brain uptake index (BUI) method. Feeding KSCN to female rats (from weaning) through their growth, pregnancy and lactation (G1) but not from conception (G2) or parturition (G3) resulted in a significant decrease (≈23%) in the BBB transport of 2-deoxy-d-glucose (2-DG) in their offspring at weaning, compared with controls (C). Post-weaning KSCN-feeding (G4) of control pups did not affect their BBB 2-DG transport (BUI: 36.2±4.98, vs 38.8±4.11). The effects of different KSCN regimens on BBB transport of leucine (leu), tyrosine (tyr) and sucrose (background marker) were inconsistent, of smaller magnitude (≈10%) and appeared to be of little significance. Withdrawing KSCN from the diet of chronically KSCN-fed (G1) mothers from conception (G5) or parturition (G6) prevented the impairment of BBB 2-DG transport in pups (BUI: 27.0±4.98, 20.8±3.27, 26.9±3.99 and 28.3±3.47 in C, G1, G5 and G6 pups, respectively); this was reversed by feeding the control diet to G1 pups from weaning. Withdrawal of dietary KSCN did not affect BBB transport of leu, tyr and sucrose. The decreased BBB transport of 2-DG in G1 pups appears to be due to a decrease in affinity (K_t app 5.46 vs 4.15 mM) rather than in the capacity (T_max app 0.94 vs 0.91 μmoles/g/min) of the transport system. Intracarotid injections of KSCN per se had no effect on BBB 2-DG transport, suggesting that the effects may be secondary to the altered thyroid status of the animal.     

Uptake ofl-alanine, glycine andl-serine in the pigeon central nervous system

The uptake characteristics ofl-α-alanine, glycine andl-serine into crude mitochondrial fractions from pigeon telencephalon, tectum and spinal cord were determined. Sodium dependent high affinity uptake systems were found for all 3 amino acids in all 3 brain regions, except for glycine in the telencephalon. The mutual inhibition of the high affinity uptake by the 3 amino acids was measured in the tectum. Alanine uptake was inhibited competitively by serine and glycine. The inhibition of serine uptake by alanine and glycine was also competitive. In contrast, glycine uptake inhibition by alanine and serine was incomplete and neither competitive, non-competitive nor uncompetitive. The effect of various chemicals on the uptake of the 3 amino acids was measured in the tectum at a substrate concentration of 10⁻⁵M. Four groups could be distinguished: (1) Substances with no effect on the uptake of all 3 amino acids, (2) substances which inhibited the uptake of all 3 amino acids to a similar degree, (3) substances which inhibited the uptake of alanine and serine more than glycine and (4) substances which inhibited glycine uptake more than alanineand serine uptake.From these results we conclude: Alanine and serine are probably taken up by the same transport system. This system can possibly also use glycine as substrate. Most of the glycine high affinity uptake, however, is due to a specific glycine transport system. This system was found only in tectum and spinal cord and is probably the same uptake system as known in the spinal cord of other vertebrates.     

Novel recognition site forl-quisqualate sensitizes neurons to depolarization byl-2-amino-4-phosphonobutanoate (l-AP4)

Brief exposure of rat hippocampal slices tol-quisqualate sensitizes pyramidal neurons to depolarization byl-2-amino-4-phosphonobutanoate (l-AP4). We report here experiments designed to clarify the duration, pharmacology, mechanism, and pathway specificity of this ‘QUIS-effect’. The quisqualate-induced sensitization tol-AP4 decreases only 3-fold over a 4 h period. No compound besides quisqualate has been found to induce the QUIS-effect, including quisqualate analogues, potent excitatory amino acid agonists,l-glutamate,l-aspartate, and compounds known to stimulus second messenger systems in hippocampal slices. Of 43 compounds assayed here, only 5 are able to block the induction of the QUIS-effect. Although these blockers are also potent ligands at a chloride-dependent glutamate uptake site, the marked difference in rank ordering of compounds for QUIS-effect blockade and uptake site potency suggests that the QUIS-effect is not induced through this uptake site. The QUIS-effect can be induced in the CA1 region, the medial perforant path, and the lateral olfactory tract of the rat, and in the guinea pig CA1. It cannot be induced in thel-AP4-sensitive rat lateral perforant path (LPP), suggesting that the receptors forl-AP4 in the LPP may be distinct from those that are sensitized by quisqualate in the other pathways.     

Transfer of bromocriptine across the blood-brain barrier in man

The transfer of ¹⁴C-bromocriptine across the blood-brain barrier was studied in 10 patients using the double indicator single injection method. The extraction (E) of bromocriptine was 8 % (quartiles: 6 and 14 %). Based on the results the cerebral uptake velocity of bromocriptine was calculated assuming constant arterial drug concentration. Its T_1/2 was found to be 12–24 minutes, depending on the tissue-blood partition coefficient.     

Selective inhibition of glial versus neuronal uptake ofl-glutamic acid by SITS

SITS, an inhibitor of anion exchange, was found to be a potent and selective inhibitor ofl-glutamic acid uptake by cultured LRM55 glioma cells and rat brain astrocytes. Synaptosomal uptake of glutamate was relatively insensitive to inhibition by SITS. This differential effect indicates that the glutamate transport system in glia differs from that in neurons and that SITS may provide a tool for investigating the exclusive neuronal transport and metabolism ofl-glutamic acid.     

Chronic infusion ofl-glutamate causes neurotoxicity in rat striatum

Chronic infusion of a high dose of monosodium glutamate (approximately 8 nmol/min) into the rat left striatum, over a period of 1 week, caused degeneration of striatal neurones. This was accompanied by a significant loss of neurochemical markers for both GABAergic and cholinergic interneurones. These results indicate that the sustained presence of elevated concentrations of glutamate will, in time, give rise to changes similar to those seen in human neurodegenerative disorders, such as Huntington's disease.     

Manganese neurotoxicity: Effects ofl-DOPA and pargyline treatments

Single, monolateral injection into rat substantia nigra of manganese chloride produced within two weeks from its administration a loss of dopamine in the striatum ipsilateral to the injected side. The effect was dose-dependent and was not extended to serotoninergic terminals present in this brain area, whose content in serotonin and 5-hydroxyindoleacetic acid was not affected. Whenl-DOPA + carbidopa or pargyline were given to these animals the decrease of striatal dopamine was more marked. Moreover, rats treated two weeks before with a dose of manganese chloride that produced a 70–80% drop in striatal dopamine concentrations, rotated ipsilaterally to the dopamine-depleted striatum when injected with apomorphine, suggesting that in these animals the stimulatory effects of apomorphine were more relevant in striatum where presynaptic dopaminergic neurons were not affected by manganese chloride. These data indicate that the alterations of dopaminergic postsynaptic receptors may be different in parkinsonian and in manganese-intoxicated patients and that current therapy used for Parkinson's disease could be a hazard in treating manganese poisoning.     

Kinetics of blood-brain barrier dysfunction to albumin around small cerebral stab wounds

A small stab wound was made in the frontal lobe of the rat brain in order to study the amount of albumin extravasated at different times after the injury. Evans blue-labeled bovine albumin was injected at various times after the injury. The animal was perfused with buffered saline at predetermined times and the contents of exogenous bovine albumin and of endogeneous rat albumin in the brain were assayed by rocket immunoelectrophoresis.
There was an immense extravasation of bovine albumin at the time of injury and during the first 10 min post-operatively, but a very slight extravasation during the following three post-operative hours. At the 6th postoperative hour there was an increase in the extravasation of albumin, followed by a subsequent decrease during the 16th and 24th post-operative hours.
The amount of endogenous rat albumin present in the injured area of the brain did not change from 10 min to 3 h after injury. At the 6th post-operative hour there was an increase in the amount of extravasated rat albumin, reflecting the increased extravasation of macromolecules during the 6th hour after injury.
The results obtained indicate that most of the albumin present in the brain during the first post-operative day is extravasated through the immediate hemorrhage at the time of injury. Thus, only a small portion of the extravasation of albumin around cerebral stab wounds can be prevented by post-operative pharmacological treatment.     

Ultrastructural demonstration ofl-glutamate decarboxylase and cysteinesulfinic acid decarboxylase in rat retina by immunocytochemistry

The γ-aminobutyric acid (GABA) synthesizing enzyme,l-glutamate decarboxylase (GAD), and the taurine synthesizing enzyme, cysteinesulfinic acid decarboxylase (CSAD) have been localized in rat retina at the ultrastructural level by indirect immunoelectron microscopy. GAD immunoreactivity (GAD-IR) was seen only in some amacrine cells and their terminals. CSAD immunoreactivity (CSAD-IR) was found in most retinal neuronal types and their processes including photoreceptor cells (rod and cone cells), bipolar cells, amacrine cells and ganglion cells. The GAD-IR positive amacrine terminals have been found to make synaptic contact with other GAD-IR negative bipolar and amacrine terminals, and ganglion cell dendrites. Most of the GAD-IR positive terminals are presynaptic. Occasionally, GAD-IR positive amacrine terminals are postsynaptic to another amacrine terminal or ganglion cell body. In the inner plexiform layer, CSAD-IR positive amacrine terminals also make synaptic contacts with other nerve terminals, similar to that of GAD-IR positive amacrine terminals. In addition, CSAD-IR positive bipolar terminals make synaptic contact with some CSAD-IR positive as well as negative amacrine terminals. Both CSAD-IR positive amacrine and bipolar terminals are mostly presynaptic to other CSAD-IR negative terminals. In the outer plexiform layer, CSAD-IR was found to be associated with synaptic vesicles and the synaptic membrane in certain cone pedicles and rod spherules. It is concluded that only a fraction of amacrine cells in rat retina may use GABA as a neurotransmitter. The presence of CSAD-IR in some amacrine, bipolar, photoreceptor and ganglion cells in rat retina is compatible with the notion that taurine may play some important roles, such as those of neurotransmitter or neuromodulator in mammalian retina.