Blood-Brain Transfer of l-Phenylalanine Declines After Peripheral but Not Central Nervous Administration of Vasopressin

To determine whether a previously reported effect of vasopressin on blood-brain transfer of leucine extends to other large neutral amino acids, we measured the regional blood-brain transfer of L-phenylalanine with the integral technique. Intravenous co-injection of L-phenylalanine and arginine vasopressin (30 nmol to 10 pmol) resulted in a decrease of the permeability-surface area (PaS) product of phenylalanine of between 11 and 39%. In addition, the peptide elicited a decrease of the cerebral blood flow of between 11 and 56% combined with a drastic decrease of the cardiac output (32-64%) and an elevation of the blood pressure to approximately 150% of control values. However, we found no changes of the cardiac output, the blood pressure, or the PaS product of phenylalanine after microdialysis (30 min, 5 microliters min-1) of arginine vasopressin (15 mumol L-1) into the dorsal hippocampus, but cerebral blood flow was decreased. The results support the hypothesis that arginine vasopressin receptors at the blood-brain barrier are involved in the regulation of large neutral amino acid transfer from blood to brain and indicate that these receptors are located at the luminal membrane of the endothelial cells.     

Effects of Microdialysis-Perfusion with Anisoosmotic Media on Extracellular Amino Acids in the Rat Hippocampus and Skeletal Muscle

Changes in the levels of amino acids have been implicated as being important in osmoregulation both within and outside the CNS. The present study addressed the question of whether changes in osmolarity affect the extracellular concentration of amino acids in the rat hippocampus and femoral biceps muscle (FBM). Microdialysis probes were implanted in these tissues and perfused with standard physiological saline. Amino acid concentrations in the dialysate were determined with HPLC separation of o-phthaldialdehyde derivatives and fluorescence detection. The osmolarity of the perfusion buffer was gradually decreased by reduction of the concentration of NaCl from 122 to 61 to 0 mM. In other experiments, the osmolarity was increased by elevation of the NaCl level from 122 to 183 to 244 mM or by addition of mannitol. Glutamate, aspartate, gamma-aminobutyrate, and alanine levels in dialysate from the hippocampus increased when the concentration of NaCl was decreased by 61 mM, and they were further elevated when NaCl was omitted. Taurine and phosphoethanolamine (PEA) levels were maximally elevated at the intermediary decrease of NaCl concentration, and glutamine in particular but also methionine and leucine were suppressed by perfusion with hypoosmolar medium. The amino acid response of the FBM differed substantially from that of the hippocampus. The aspartate content increased slightly, and there was a marginal transient increase in PEA level. Perfusion with media containing high concentrations of NaCl induced diminished dialysate levels of taurine, PEA, and glutamate, whereas levels of other amino acids were either unaffected or increased. Mannitol administration via the perfusion fluid led to reduced levels of taurine, PEA, glutamate, and aspartate. In contrast to the effects of high NaCl levels, hyperosmotic mannitol did not induce increases in level of any of the amino acids detected. The results suggest that taurine and PEA are involved in osmoregulation in the mammalian brain. From a quantitative viewpoint, taurine seems to be most important. Transmitter amino acids may also be involved in the maintenance of the volume of neural cells subjected to severe disturbances in osmotic equilibrium.     

In Vivo Substitution of Choline for Sodium Evokes a Selective Osmoinsensitive Increase of Extracellular Taurine in the Rat Hippocampus

Recent investigations have demonstrated that taurine and phosphoethanolamine (PEA) are the amino acids most sensitive to microdialysis-perfusion with reduced concentrations of NaCl. The aim of the present work was to assess the importance of Na+ deficiency in evoking this response. Further, the previously described selectivity of replacement of Cl- with acetate with respect to amino acid release was reinvestigated. The hippocampus of urethane-anesthetized rats was dialyzed with Krebs-Ringer bicarbonate buffer, and amino acid concentrations of the perfusate were determined. Choline chloride was then stepwise substituted for NaCl, and, in some cases, mannitol (122 mM) was included in low sodium-containing media. In other experiments, NaCl was replaced with sodium acetate. The dialysate levels of taurine increased selectively in response to Na+ substitution. The elevation of taurine was linearly related to the increase in choline chloride, and maximal levels amounted to 335% of basal levels. The increase in extracellular taurine was not inhibited by perfusion with medium made hyperosmotic with mannitol. Replacement of Cl- with acetate stimulated the release of taurine to 652% of resting levels. In addition, PEA levels increased to 250% of control concentration. Other amino acids were unaffected by Cl- substitution. The results show that taurine transport is considerably more sensitive to Na+ depletion than glutamate transport, which also is known to be Na+ dependent. The taurine increase evoked by low Na+ is not caused by cellular swelling as it was unaffected by hyperosmolar medium. Finally, substitution of acetate for Cl- causes a specific elevation of extracellular taurine and PEA, possibly as a result of cytotoxic edema.     

Elevation of the Extracellular Concentrations of Glutamate and Aspartate in Rat Hippocampus During Transient Cerebral Ischemia Monitored by Intracerebral Microdialysis

Rats were implanted with 0.3-mm-diameter dialysis tubing through the hippocampus and subsequently perfused with Ringer's solution at a flow rate of 2 microliter/min. Samples of the perfusate representing the extracellular fluid were collected over 5-min periods and subsequently analyzed for contents of the amino acids glutamate, aspartate, glutamine, taurine, alanine, and serine. Samples were collected before, during, and after a 10-min period of transient complete cerebral ischemia. The extracellular contents of glutamate and aspartate were increased, respectively, eight- and threefold during the ischemic period; the taurine concentration also was increased 2.6-fold. During the same period the extracellular content of glutamine was significantly decreased (to 68% of the control value), whereas the concentrations of alanine and serine did not change significantly during the ischemic period. The concentrations of gamma-aminobutyric acid (GABA) were too low to be measured reliably. It is suggested that the large increase in the content of extracellular glutamate and aspartate in the hippocampus induced by the ischemia may be one of the causal factors in the damage to certain neurons observed after ischemia.     

Changes in Extracellular Amino Acids and Spontaneous Neuronal Activity During Ischemia and Extended Reflow in the CA1 of the Rat Hippocampus

This study addresses the possible involvement of an agonist-induced postischemic hyperactivity in the delayed neuronal death of the CA1 hippocampus in the rat. In two sets of experiments, dialytrodes were implanted into the CA1 either acutely or chronically (24 h of recovery). During 20 min of cerebral ischemia (four-vessel occlusion model) and 8 h of reflow, we followed extracellular amino acids and multiple-unit activity. Multiple-unit activity ceased within 20 sec of ischemia and remained zero during the ischemic insult and for the following 1 h of reflow. During ischemia, extracellular aspartate, glutamate, taurine, and gamma-aminobutyric acid increased in both acute and chronic experiments (seven- to 26-fold). Multiple-unit activity recovered to preischemic levels following 4-6 h of reflow. In the group with dialytrodes implanted acutely, the continuous increase in multiple-unit activity reached 110% of basal at 8 h of reflow. In the group with dialytrodes implanted chronically, multiple-unit activity recovered faster and reached 140% of control at 8 h, paralleled by an increase in extracellular aspartate (5.5-fold) and glutamate (twofold). In conclusion, the postischemic increase of excitatory amino acids and the recovery of the neuronal activity may stress the CA1 pyramidal cells, which could be detrimental in combination with, e.g., postsynaptic impairments.     

Changes in Extracellular Amino Acids During Soman-and Kainic Acid-Induced Seizures

Extracellular amino acid levels in the rat piriform cortex, an area highly susceptible to seizure-induced neuropathology, were determined by means of intracranial microdialysis. Seizures were induced by systemic administration of either soman (O-1,2,2-trimethylpropyl methylphosphonofluoridate), a potent inhibitor of acetylcholinesterase, or the excitotoxin kainic acid. Extracellular glutamate levels increased in animals with seizures shortly after administration of either convulsant, but this change was statistically significant only in the case of soman-treated animals. Extracellular taurine levels increased markedly, reaching two- and fourfold baseline levels during the second hour of soman- and kainic acid-induced seizures, respectively. Taurine levels did not increase in the subpopulation of soman-treated animals without seizures, a finding indicating that elevation of extracellular taurine level is seizure related. Thus, we propose that taurine efflux may be a physiological cellular response to neuronal changes produced by excitotoxic chemicals, either directly or as a consequence of seizures.     

A Possible Interrelationship Between Extracellular Taurine and Phosphoethanolamine in the Hippocampus

The effect of guanidinoethane sulfonic acid (GES), an inhibitor of taurine uptake, was examined with respect to endogenous amino acids in the hippocampus of the freely moving rabbit. GES increased the extracellular levels of both taurine and phosphoethanolamine (PEA), other amino acids being unaffected. However, long-term oral administration of GES selectively reduced endogenous taurine levels. The effect of GES on PEA appeared to be a consequence of the elevated extracellular taurine as exogenously administered taurine per se increased PEA levels in the extracellular space. The findings are discussed in conjunction with the proposed membrane-stabilizing effects of taurine.     

Saturable Retention of Vasopressin by Hippocampus Vessels In Vivo, Associated with Inhibition of Blood-Brain Transfer of Large Neutral Amino Acids

Vasopressin receptors have been reported in the endothelium of brain capillaries. The function of these receptors is not known. To test the prediction that vasopressin receptors in brain capillary endothelium affect amino acid transport across the blood-brain barrier and to assess the role of vasopressin transport across the cerebral vascular endothelium, we measured (a) the endothelial permeability to the large neutral amino acid leucine in the absence and presence of arginine vasopressin (AVP) and (b) the permeability of the blood-brain barrier to AVP relative to manitol. In brain regions protected by the blood-brain barrier, after circulation for 20 s, coinjection of leucine and AVP intravenously led to a decrease of leucine transport unrelated to changes of blood flow. The decrease was most pronounced in hippocampus (42%) and least pronounced in olfactory bulb and colliculi (17 and 19%, respectively). In the latter regions, the endothelial permeability to AVP did not significantly exceed that of mannitol. In hippocampus and in regions with no blood-brain barrier (pituitary and pineal glands), AVP retention in excess of mannitol retention was blocked by unlabeled AVP. The findings do not contradict the hypothesis of a role for AVP in the regulation of large neutral amino acid transfer into brain tissue.     

Concentrations of Amino Acids in Extracellular Fluid After Opening of the Blood-Brain Barrier by Intracarotid Infusion of Protamine Sulfate

Abstract: This article evaluates the influence of an opening of the blood-brain barrier (BBB) on compounds in brain extracellular fluid. The concentrations of amino acids and some other primary amines were determined in dialysates sampled from the right parietal cortex of rats before and after an intracarotid infusion of protamine sulfate. Extravasated plasma proteins were visualized by Evans blue/albumin and immunohistochemistry. CSF albumin— an indicator of blood-CSF barrier opening—was quantified with immunoelectrophoresis. The brains were macroscopically edematous after 10 mg but not after 5 mg of protamine sulfate. The higher dose led to a 50% death rate. The concentrations of amino acids did not change 10 min after the BBB opening. No significant alterations in the amino acid concentrations were observed after the lower dose. The concentrations of glutamate, aspartate, GABA, glycine, taurine, and phosphoethanolamine increased significantly within 50–80 min after the infusion of 10 mg of protamine sulfate. CSF albumin levels were significantly increased 1 h after infusion. We conclude that a dysfunction of the BBB, of a degree known to induce brain edema (10 mg of protamine sulfate), significantly increases the extracellular concentration of excitatory amino acids, GABA, taurine, and phosphoethanolamine in the extracellular space.     

Extracellular Amino Acid Concentrations in the Dorsal Spinal Cord of Freely Moving Rats Following Veratridine and Nociceptive Stimulation

In vivo microdialysis was used to sample extracellular concentrations of amino acids in the dorsal lumbar spinal cord of freely moving rats. Changes in the extracellular concentrations of amino acids were measured in response to infusion of veratridine (180 microM), a sodium channel activator, as well as during acute noxious stimulation by an injection of 5% formalin into the metatarsal region of the hindleg. Veratridine produced a tetrodotoxin (TTX)-sensitive increase in the extracellular concentration of Glu. Concentrations of Asp, taurine, Ala, Asn, and Gly were not significantly elevated following veratridine stimulation. Intradermal injection of formalin produced a TTX-sensitive increase in Asp concentration and a non-TTX-sensitive increase in Glu concentration. These data support the hypothesis that Glu and Asp are dorsal horn neurotransmitters involved in nociception.     

Changes in the Extracellular Concentrations of Amino Acids in the Rat Striatum During Transient Focal Cerebral Ischemia

Abstract: Although considerable evidence supports a role for amino acids in transient global cerebral ischemia and permanent focal cerebral ischemia, effects of transient focal cerebral ischemia on the extracellular concentrations of amino acids have not been reported. Accordingly, our study was undertaken to examine the patterns of changes of extracellular glutamate, aspartate, GABA, taurine, glutamine, alanine, and phosphoethanolamine in the striatum of transient focal cerebral ischemia, as evidence to support their pathogenic roles. Focal ischemia was induced using the middle cerebral artery occlusion model, with no need for craniotomy. Microdialysis was used to sample the brain's extracellular space before, during, and after the ischemic period. One hour of middle cerebral artery occlusion followed by recirculation caused neuronal damage that was common in the frontoparietal cortex and the lateral segment of the caudate nucleus. During 1 h of ischemia, the largest increase occurred for GABA and moderate increases were observed for taurine, glutamate, and aspartate. Alanine, which is a nonneuroactive amino acid, increased little. After recirculation, the levels of glutamate and aspartate reverted to normal baseline values right after reperfusion. Despite these rapid normalizations, neuronal damage occurred. Therefore, uptake of excitatory amino acids can still be restored after 1 h of middle cerebral artery occlusion, and tissue damage occurs even though high extracellular levels of glutamate are not maintained.     

Extracellular Glucose Concentrations in the Rat Hippocampus Measured by Zero-Net-Flux

Abstract : The concentration of glucose in the brain's extracellular fluid remains controversial, with recent estimates and measurements ranging from 0.35 to 3.3 m M . In the present experiments, we used the method of zero-net-flux microdialysis to determine glucose concentration in the hippocampal extracellular fluid of awake, freely moving rats. In addition, the point of zero-net-flux was measured across variations in flow rate to confirm that the results for glucose measurement were robust to such variations. In 3-month-old male Sprague-Dawley rats, the concentration of glucose in the hippocampal extracellular fluid was found to be 1.00 ± 0.05 m M , which did not vary with changes in flow rate. Three-month-old and 24-month-old Fischer-344 rats both showed a significantly higher hippocampal extracellular fluid glucose concentration, at 1.24 ± 0.07 and 1.21 ± 0.04 m M , respectively ; there was no significant difference between the two age groups. The present data demonstrate variation in extracellular brain glucose concentration between rat strains. When taken together with previous data showing a striatal extracellular glucose concentration on the order of 0.5 m M , the data also demonstrate variation in extracellular glucose between brain regions. Traditional models of brain glucose transport and distribution, in which extracellular concentration is assumed to be constant, may require revision.     

Effects of Methylprednisolone on Extracellular Lactic Acidosis and Amino Acids After Severe Compression Injury of Rat Spinal Cord

Abstract: We evaluated in rats with severe spinal cord compression at T8–9 the influence of methylprednisolone (MP) on lactic acidosis and extracellular amino acids, which may cause secondary, perifocal injuries of the cord. MP (30 mg/kg) was given intravenously 30 min before compression and hourly thereafter (15 mg/kg). Other rats with compression, given saline, served as controls. Samples from the extracellular fluid of one dorsal horn were collected by microdialysis and analyzed by HPLC. Microdialysis was performed for 1.5 h to establish basal levels. Samples were collected for 3 h after compression. MP-treated rats showed a reduction of dialysate lactic acid and arginine levels during the first 1–2 h after trauma. The mean dialysate levels of glutamate in MP-treated rats were lower than those of the controls, but the difference was not statistically significant. MP treatment did not influence dialysate levels of aspartate, glutamine, histidine, glycine, threonine, taurine, alanine, GABA, and tyrosine. Our study shows that MP has several effects, including reduced lactic acid formation, reduced levels of arginine (the substrate for nitric oxide production), and a trend toward decreased extracellular accumulation of the excitotoxic amino acid glutamate. We conclude that MP has the capacity to change the composition of the extracellular edema fluid after trauma to the spinal cord. These changes may counteract free radical formation and may be important mechanisms by which MP exerts its beneficial actions.     

Regional Cerebral Glucose Phosphorylation and Blood Flow After Insertion of a Microdialysis Fiber Through the Dorsal Hippocampus in the Rat

Local cerebral glucose metabolism (LCMRglc) and local cerebral blood flow (LCBF) were studied following implantation of a microdialysis fiber in rat dorsal hippocampus. Recovery time after implantation varied from 0 to 24 h. All rats showed pronounced disturbances in LCMRglc and LCBF during the first 2 h of implantation. The changes consisted of (a) a general decrease in blood flow and glucose phosphorylation and (b) small areas (spots) around the fiber with increased glucose phosphorylation and decreased blood flow. Animals allowed to recover for 24 h demonstrated a near normalization of LCMRglu and LCBF, and the focal disturbances (spots) of glucose phosphorylation and blood flow disappeared. The slight reduction in blood flow and glucose metabolism at this time must be accepted, because extension of the recovery period beyond 24 h may give interpretation problems due to the developing gliosis.     

Taurine transport systems in the ciliate protozoanTetrahymena pyriformis

Summary Tetrahymena pyriformis cultivated in the presence of 1 mM taurine prior to transfer of the cells to non-nutrient medium express an enhanced capacity for concentrative taurine uptake and for taurine diffusion compared to cells grown without added taurine. The unidirectional taurine influx in taurine-grown cells comprises a saturable component with K_m -257M, V_max = 21 n-moles · g dry wt^–1 sd min^–1, and a diffusion component with a diffusion constant of 0.20 ml · g dry wt^–1 · min^–1. At extracellular taurine concentrations <30M, 20% of the influx is via the saturable system and 80% is via the diffusion system. 19% of the influx in Na⁺-dependent, Cl^–-independent, and not inhibitable with structural analogues to taurine, suggesting that the transport system responsible for the saturable component in Tetrahymena is different from the Na⁺- and Cl^–-dependent taurine translocating system (the-system) described in vertebrate cells. The unidirectional taurine influx is reduced by 80% by 1mM DIDS (inhibitor of anion exchange and anion channels) and by 1 mM MK196 (indachrinone, inhibitor of anion channels) indicating that taurine diffusion inTetrahymena is via a channel, which is permanently active and which resembles the swelling-induced taurine channel seen in mammalian cells. Taurine influx is stimulated by the forskolin analogue 1,9-dideoxyforskolin and by arachidonic acid, and this stimulation is in both cases sensitive to DIDS and MK196.Abbreviations DDF dideoxyforskolin - DIDS 4,4-diisothiocyano-2,2-stilbene disulfonic acid - GABA gamma amino butyric acid - HEPES N(2-hydroxyethyl)piperazine-N-(2-ethane sulfonic acid) - MK196 indachrinone - MOPS 3-(N-morpholino)propane sulfonic acid - NMDG n-methyl-dglucamonium - OPA ortho-phtalaldehyde - PCA perchloric acid - TES N-tris(hydroxy methyl)-methyl-2-amino ethane sulfonic acid - TRIS tris(hydroxy methyl)amino methane     

Role of Endogenous Taurine on the Glutamate Analogue-Induced Neurotoxicity in the Rat Hippocampus In Vivo

The glutamate analogues N-methyl-D-aspartate (NMDA), kainic acid (KA), and quisqualic acid (QA), prepared in different hypertonic media, were perfused in vivo in the hippocampal CA1 field of rats using a microdialysis technique. Extracellular taurine levels, estimated after analysis of the taurine content of dialysates, increased during perfusion of all three agonists but varied according to the osmolarity of the medium. The NMDA-induced increase in extracellular taurine content was only slightly inhibited by perfusion of 150 and 300 mM sucrose. The KA-evoked increase was partially dependent on extracellular osmolarity, because addition of 50 and 150 mM sucrose caused a dose-dependent inhibition that was not augmented using higher sucrose concentrations. QA caused a taurine increase that was totally abolished by addition of 50 mM sucrose. These results indicate that the rise in extracellular taurine level elicited by QA and part of the increase elicited by KA are probably due to a release caused by the cellular swelling that these substances evoke, a finding substantiating the previously proposed osmoregulatory role of taurine. However, almost all the increase in extracellular taurine content caused by NMDA and all the osmotically insensitive part of the KA-evoked rise cannot be explained as release triggered by cell swelling and may reflect a function of taurine other than osmoregulation.     

The Role of Taurine in the Central Nervous System and the Modulation of Intracellular Calcium Homeostasis

The effects of taurine in the mammalian nervous system are numerous and varied. There has been great difficulty in determining the specific targets of taurine action. The authors present a review of accepted taurine action and highlight recent discoveries regarding taurine and calcium homeostasis in neurons. In general there is a consensus that taurine is a powerful agent in regulating and reducing the intracellular calcium levels in neurons. After prolonged L-glutamate stimulation, neurons lose the ability to effectively regulate intracellular calcium. This condition can lead to acute swelling and lysis of the cell, or culminate in apoptosis. Under these conditions, significant amounts of taurine (mM range) are released from the excited neuron. This extracellular taurine acts to slow the influx of calcium into the cytosol through both transmembrane ion transporters and intracellular storage pools. Two specific targets of taurine action are discussed: Na⁺-Ca²⁺ exchangers, and metabotropic receptors mediating phospholipase-C.     

Endogenous Glutamate Increases Extracellular Concentrations of Dopamine, GABA, and Taurine Through NMDA and AMPA/Kainate Receptors in Striatum of the Freely Moving Rat: A Microdialysis Study

Abstract: Interactions between glutamate (Glu), dopamine (DA), GABA, and taurine (Tau) were investigated in striatum of the freely moving rat by using microdialysis. Intrastriatal infusions of the selective Glu uptake inhibitor l - trans -pyrrolidine-3,4-dicarboxylic acid (PDC) were used to increase the endogenous extracellular [Glu]. Correlations between extracellular [Glu] and extracellular [DA], [GABA], and [Tau], and the effects of a selective blockade of ionotropic Glu receptors, were studied. PDC (1, 2, and 4 m M ) produced a dose-related increase in extracellular [Glu]. At the highest dose of PDC, [Glu] increased from 1.55 ± 0.35 to 6.11 ± 0.88 µ M . PDC also increased extracellular [DA], [GABA], and [Tau]. The increasing [Glu] was correlated significantly with increasing [DA], [GABA], and [Tau]. PDC also decreased extracellular concentrations of DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxyphenylacetic acid (HVA). Perfusion with the NMDA-receptor antagonist 3-[( R )-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (1 m M ) or the AMPA/kainate-receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) (1 m M ) attenuated the increases produced by PDC (4 m M ) on [DA], [GABA], and [Tau], and decreases in [DOPAC] and [HVA]. DNQX also attenuated the increases in [Glu] induced by PDC. These data show that endogenous Glu plays a role in modulating the extracellular concentrations of DA, GABA, and Tau in striatum of the freely moving rat.     

Threonine Entry into Rat Brain After Diet-Induced Changes in Plasma Amino Acids

Abstract: Passage of amino acids across the blood-brain barrier is modified by the amino acid composition of the blood. Because blood amino acid concentrations respond to changes in protein intake, we have examined associations among diet, plasma amino acid patterns, and the rate of entry of threonine into the brain. Rats were adapted for 8 h/ day for 7–10 days to diets containing 6, 18 , or 50% casein before receiving a single, independently varied, final meal of a diet containing 0, 6, 18 , or 50% casein. After 4–7 h, they were anesthetized and infused intravenously with [¹⁴C]threonine for 5 min before plasma and brain samples were taken for determination of radioactivity and amino acid content. Plasma and brain threonine concentrations decreased as protein content increased in the diets to which the rats had been adapted. Plasma threonine concentrations increased twofold, from 1.6 to 3.0 m M , when rats adapted to 6% casein meals received a single 50% casein meal rather than a nonprotein meal; a fivefold increase, from 0.13 to 0.69 m M , occurred when rats had been previously adapted to 50% casein meals. Increasing the protein content of the final meal did not increase brain threonine concentrations. Highest and lowest rates of threonine entry into the brain occurred, respectively, in rats adapted to 6 and 50% casein meals. Changes in plasma threonine concentrations and threonine flux into brain reflected protein content of both pretreatment and final meals.