Comparison of DABA and GABA Transport into Plasma Membrane Vesicles Derived from Synaptosomes

Abstract: Transport of GABA by a high-affinity transport system ( K _m≃ 10⁻⁵ M) is thought to terminate the action of this postulated neurotransmitter. 2,4-Diaminobutyric acid (DABA), a structural analogue, is taken up by neuronal elements and inhibits GABA uptake. Localization of [³H]DABA by auto-radiography has been used to identify neurons with the GABA high-affinity transport system. After reconstitution of lysed synaptosomal fractions in potassium salts, transfer of these membrane vesicles to sodium salts produces sodium and potassium ion gradients which drive [³H]GABA and [³H]DABA transport. For each, transport requires external sodium, is abolished by ionophores that dissipate the Na⁺ gradient, and is enhanced by conditions which make the intravesicular electromotive force more negative. Some characteristics of the transport of these substances, however, differ. For example, external chloride is required for GABA, but not DABA, transport. Internal potassium is required for DABA, but not GABA, transport. DABA is a competitive inhibitor ( K _i≃ 0.6 MM) of GABA transport into membrane vesicle and synaptosomes. GABA, however, is a feeble inhibitor of DABA uptake into the membrane vesicles. These differences suggest that the two substances are transported by different mechanisms and possibly by different carriers. In addition to these experiments, using enzymatic-fluorometric techniques, it was shown that the artificially imposed ion gradients drive net chemical transport of GABA into the vesicles.     

N-System Amino Acid Transport at the Blood-CSF Barrier

Abstract: Despite l -glutamine being the most abundant amino acid in CSF, the mechanisms of its transport at the choroid plexus have not been fully elucidated. This study examines the role of L-, A-, ASC-, and N-system amino acid transporters in l -[¹⁴C]glutamine uptake into isolated rat choroid plexus. In the absence of competing amino acids, approximately half the glutamine uptake was via a Na⁺-dependent mechanism. The Na⁺-independent uptake was inhibited by 2-amino-2-norbornane carboxylic acid, indicating that it is probably via an L-system transporter. Na⁺-dependent uptake was inhibited neither by the A-system substrate α-(methylamino)isobutyric acid nor by the ASC-system substrate cysteine. It was inhibited by histidine, asparagine, and l -glutamate γ-hydroxamate, three N-system substrates. Replacement of Na⁺ with Li⁺ had little effect on uptake, another feature of N-system amino acid transport. These data therefore indicate that N-system amino acid transport is present at the choroid plexus. The V _max and K _max for glutamine transport by this system were 8.1 ± 0.3 nmol/mg/min and 3.3 ± 0.4 m M , respectively. This system may play an important role in the control of CSF glutamine, particularly when the CSF glutamine level is elevated as in hepatic encephalopathy.     

Structural Specificity of Sugar Transport at the Blood-Nerve Barrier

Abstract: The major component of D-glucose transfer across the membranous sites of the blood-nerve barrier (BNB) occurs via a facilitative mechanism at a rate greater than twice the rate of D-glucose metabolism by nerve. To characterize further properties of monosaccharide transport at the BNB, unidirectional transfer constant (K) values were determined in vivo in tibial nerve of anesthetized rats for radiolabeled mannitol, L-glucose, and a series of D-glucose analogs. K values (× 10⁻⁴ ml s⁻¹ g^−l) equaled 4.8 for 2-deoxy-D-glucose, 3.7 for D-glucose, 2.3 for 3- O -methyl-D-glucose, 1.4 for D-man-nose, 0.6 for D-galactose, 0.2 for mannitol, and 0.19 for L-glucose. The rank order of ratios between K values of a D-hexose and D-glucose, which reflects the rank order of affinity of the system for individual sugars, was 2-deoxy-D-glucose > D-glucose > 3-O-methyl-D-glucose > D-mannose > D-galactose. The results demonstrate that the order of substrate affinity of the monosaccharide carrier at the BNB is similar to that at cerebral capillaries and at erythrocytes. At normal concentrations of plasma D-glucose, the contribution of simple passive diffusion to unidirectional D-glucose influx across the BNB equals 5%, which is greater than that at cerebral capillaries and reflects the greater permeability to hydrophilic nonelectrolytes of the endoneurial vasculature.     

Mechanisms of Sodium Transport at the Blood-Brain Barrier Studied with In Situ Perfusion of Rat Brain

Abstract: The mechanism of unidirectional transport of sodium from blood to brain in pentobarbital-anesthetized rats was examined using in situ perfusion. Sodium transport followed Michaelis-Menten saturation kinetics with a V _max of 50.1 nmol/g/min and a K _m of 17.7 m M in the left frontal cortex. The kinetic analysis indicated that, at a physiologic sodium concentration, ∼26% of sodium transport at the blood-brain barrier (BBB) was carrier mediated. Dimethylamiloride (25 µ M ), an inhibitor of Na⁺/H⁺ exchange, reduced sodium transport by 28%, whereas phenamil (25 µ M ), a sodium channel inhibitor, reduced the transfer constant for sodium by 22%. Bumetanide (250 µ M ) and hydrochlorothiazide (1.5 m M ), inhibitors of Na⁺-K⁺-2Cl⁻/NaCl symport, were ineffective in reducing blood to brain sodium transport. Acetazolamide (0.25 m M ), an inhibitor of carbonic anhydrase, did not change sodium transport at the BBB. Finally, a perfusate pH of 7.0 or 7.8 or a perfusate P co ₂ of 86 mm Hg failed to change sodium transport. These results indicate that 50% of transcellular transport of sodium from blood to brain occurs through Na⁺/H⁺ exchange and a sodium channel in the luminal membrane of the BBB. We propose that the sodium transport systems at the luminal membrane of the BBB, in conjunction with Cl⁻/HCO₃⁻ exchange, lead to net NaCl secretion and obligate water transport into the brain.     

Transmembrane Transport and Release of GABA in the Brain of Rats Subjected to Postnatal Hypoxia

We studied the effects of early postnatal hypoxia on the efficiency of active GABA transport through the plasma membrane of synaptic terminals (synaptosomes) isolated from the cerebral cortex, hippocampus, and thalamus of rats and on non-stimulated and Ca²⁺-stimulated GABA release. The state of hypoxia was induced by exposure of 10- to 12-day-old rats to a respiratory medium with low O₂ content (4% О₂ and 96% N₂) for 12 min (up to the initiation of clonico-tonic seizures). Animals were taken in the experiment 8 to 9 weeks after an episode of hypoxic stress. The intensity of transmembrane transport of GABA was estimated according to accumulation of [³Н]GABA in a coarse synaptosomal fraction. The process was characterized by calculation of the Michaelis constant K _m and also of the initial (within the 1st min) and maximum rates of accumulation of [³Н]GABA. The means of the initial rate of [³Н]GABA accumulation in preparations from the thalamus, cortex, and hippocampus were 205.5 ± 8.8, 266.2 ± 29.6, and 302.3 ± 31.2 pmol/min⋅mg protein, respectively. Hypoxic stress influenced the rates of accumulation of [³Н]GABA in synaptic terminals from the cortex and hippocampus but not in those from the thalamus. According to the characteristics of the response to hypoxic stress, all experimental animals could be classified into two groups. In some rats, accumulation of [³Н]GABA in both cortical and hippocampal synaptosomes decreased insignificantly (by about 15%), while in other animals this parameter increased significantly (by nearly 50%) for the cortex and decreased by 21.5%, on average, for the hippocampus. The affinity of the transporter with respect to [³Н]GABA in the cortex and hippocampus was nearly the same and showed no changes under the influence of hypoxia. The non-stimulated release of [³Н]GABA after the influence of hypoxia increased in all structures, while the depolarization-induced Ca²⁺-dependent release of [³Н]GABA was intensified only in synaptosomes from the cerebral cortex. The mechanisms of development of modifications of GABA-ergic processes under the influence of hypoxic stress in the course of the perinatal period are discussed. Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 293–302, July–August, 2008.     

Transport of Lead-203 at the Blood-Brain Barrier During Short Cerebrovascular Perfusion with Saline in the Rat

Lead transport at the blood-brain barrier has been studied by short (less than 1.5 min) vascular perfusion of one cerebral hemisphere of the rat with a buffered physiological salt solution at pH 7.4 without calcium, magnesium, or bicarbonate and containing 203 Pb-labelled lead chloride. In the absence of complexing agents, 203Pb uptake was rapid, giving a space of 9.7 ml/100 g of wet frontal cortex at 1 min. Lead-203 influx was linear with lead concentration up to 4 microM. Five percent albumin, 200 microM cysteine, or 1 mM EDTA almost abolished 203Pb uptake. Lead-203 entry into brain was uninfluenced by varying the calcium concentration or by magnesium or the calcium blocker methoxyverapamil. Similarly, 1 mM bicarbonate or 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid was without effect. Increasing the potassium concentration reduced 203Pb uptake. Vanadate at 2 mM, 2 microM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (a metabolic uncoupler), or 2 microM stannic chloride all markedly enhanced lead entry into brain, as did a more alkaline pH (7.80). In conclusion, there is a mechanism allowing rapid passive transport of 203Pb at the brain endothelium, perhaps as PbOH+. Lead uptake into brain via this system is probably made less important by active transport of lead back into the capillary lumen by the calcium-ATP-dependent pump.     

Transport of Zinc-65 at the Blood-Brain Barrier During Short Cerebrovascular Perfusion in the Rat: Its Enhancement by Histidine

Abstract: Zinc-65 transport into different regions of rat brain has been measured during short vascular perfusion of one cerebral hemisphere with an oxygenated HEPES-containing physiological saline at pH 7.40. The [Zn²⁺] was buffered with either bovine serum albumin or histidine. In each case uptake was linear with time up to 90 s. ⁶⁵Zn flux into brain in the presence of albumin followed Michaelis-Menten kinetics and for parietal cortex had a K _m of 16 n M and a V _max of 44 nmol/kg/min. Increasing concentrations of l -histidine enhanced ⁶⁵Zn flux into brain at [Zn²⁺] values between 1 and 1,000 n M . The combined effect of [histidine] and [Zn²⁺] was best accounted for by a function of [ZnHis⁺], i.e., flux = 64.4 · [ZnHis⁺]/(390 + [ZnHis⁺]) + 0.00378 · [ZnHis⁺], with concentrations being nanomolar. d -Histidine had an influence similar to that of l -histidine. ⁶⁵Zn flux in the presence of 100 µ M l -histidine was not affected by either 500 µ M l -arginine or 500 µ M l -phenylalanine. The results indicate specific transport of Zn²⁺ across the plasma membranes of brain endothelium. The enhancement due to histidine has been attributed to diffusion of ZnHis⁺ across unstirred layers "ferrying" zinc to and from transport sites.     

Kinetics of Neutral Amino Acid Transport Across the Blood-Brain Barrier

Neutral amino acid (NAA) transport across the blood-brain barrier was examined in pentobarbital-anesthetized rats with an in situ brain perfusion technique. Fourteen of 16 plasma NAAs showed measurable affinity for the cerebrovascular NAA transport system. Values of the transport constants (Vmax, Km, KD) were determined for seven large NAAs from saturation studies, whereas Km values for five small NAAs were estimated from inhibition studies. These data, together with our previous work, provide a complete set of constants for prediction of NAA influx from plasma. Among the NAAs, Vmax varied at least fivefold and Km varied approximately 700 fold. The apparent affinity (1/Km) of each NAA was related linearly (r = 0.910) to the octanol/water partition coefficient, a measure of NAA side-chain hydrophobicity. Predicted influx values from transport constants and average plasma concentrations agree well with values measured using plasma perfusate. These results provide accurate new estimates of the kinetic constants that determine NAA transport across the blood-brain barrier. Furthermore, they suggest that affinity of a L-alpha-amino acid for the transport system is determined primarily by side-chain hydrophobicity.     

Blood-Brain Barrier Transport of 1-Aminocyclohexanecarboxylic Acid, a Nonmetabolizable Amino Acid for In Vivo Studies of Brain Transport

Regional transport of 1-aminocyclohexanecarboxylic acid (ACHC), a nonmetabolizable amino acid, across the blood-brain barrier was studied in pentobarbital-anesthetized rats using an in situ brain perfusion technique. The concentration dependence of influx was best described by a model with a saturable and a nonsaturable component. Best-fit values for the kinetic constants of the frontal cortex equaled 9.7 X 10(-4) mumol/s/g for Vmax, 0.054 mumol/ml for Km, and 1.0 X 10(-4) ml/s/g for KD in the absence of competing amino acids. Saturable influx could be reduced by greater than 85% by either L-phenylalanine or 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with transport by the cerebrovascular neutral amino acid transport system. The transport Km for ACHC was one-fifth that for the more commonly used homologue, 1-aminocyclopentanecarboxylic acid, and was similar to values for several natural amino acids, such as L-methionine, L-isoleucine, and L-tyrosine. The results indicate that ACHC may be a useful probe for in vivo studies of amino acid transport into brain.     

Quantification of the permeability of the blood-CSF barrier to alpha-MSH in the rat

The ability of alpha-MSH to cross the blood-CSF barrier of the rat was assessed by measurement of the rate of appearance of immunoreactive alpha-MSH in a cerebrospinal fluid (CSF) perfusate following intravenous injection of peptide. Comparisons were made with the rate of appearance of a simultaneously administered dose of 14C-inulin which is poorly permeable at the blood-CSF barrier. Concentrations of drugs measured in plasma were fitted to two-compartment pharmacokinetic models, and those measured in the CSF perfusate to one-compartment open systems receiving an input from the plasma compartment. The rate constant for entry of alpha-MSH into CSF was 0.00087 min-1, which was not significantly different from that for inulin of 0.00055 min-1. As alpha-MSH penetrated into CSF at a rate comparable to inulin, it was concluded that the limited entry of peptide was by aqueous diffusion along with other water-soluble macromolecules.     

GABA对小鼠大脑皮质中GABA受体胚胎发育的调节

本文用ＧＡＢＡ及其受体激动剂和拮抗剂处理培养的胚胎小鼠大脑皮层神经细胞以及精确计时的妊娠小鼠,用放射配体结合法检测ＧＡＢＡＡ及ＧＡＢＡＢ的结合位点数目,研究了ＧＡＢＡ对小鼠大脑皮层ＧＡＢＡ受体胚胎发育的调节作用,结果表明：①ＧＡＢＡ可使培养１５—１７天妊龄的胚胎小鼠大脑皮层神经细胞及出生第１天的仔鼠大脑皮层中的ＧＡＢＡＡ及ＧＡＢＡＢ受体数目增加,这种作用可被蝇蕈醇（Ｍｕｓ）及巴氯芬（Ｂａｃ）分别模拟,对ＧＡＢＡＡ受体的作用可为荷包牡丹碱（Ｂｉｃ）所阻断;②用ＧＡＢＡ处理妊娠７—１３天的小鼠,仔鼠出生第１天其大脑皮层的ＧＡＢＡＡ及ＧＡＢＡＢ受体数目均无变化;③用ＧＡＢＡ处理妊娠１４—１９天的小鼠,仔鼠出生的第１天其大脑皮层中的ＧＡＢＡＡ受体数目增加而ＧＡＢＡＢ受体数目不变;④用ＧＡＢＡ处理妊娠７－１９天的小鼠,仔鼠出生第１天其大脑皮层中ＧＡＢＡＡ及ＧＡＢＡＢ受体数目增加。这说明在胚胎发育的特定时期内,ＧＡＢＡ可诱导其受体数目的增加,这个作用是由ＧＡＢＡ受体调节的。     

Phenylalanine Transport Across the Blood-Brain Barrier as Studied with the In Situ Brain Perfusion Technique

Unidirectional L-phenylalanine transport into six brain regions of pentobarbital-anesthetized rats was studied using the in situ brain perfusion technique. This technique allows both accurate measurements of cerebrovascular amino acid transport and complete control of perfusate amino acid composition. L-Phenylalanine influx into the brain was sodium independent and could be described by a model with a saturable and a nonsaturable component. Best-fit values for the kinetic constants in the parietal cortex equaled 6.9 X 10(-4) mumol/s/g for Vmax, 0.011 mumol/ml for Km, and 1.8 X 10(-4) ml/s/g for KD during perfusion with fluid that did not contain competing amino acids. D-Phenylalanine competitively inhibited L-phenylalanine transport with a Ki approximately 10-fold greater than the Km for L-phenylalanine. There were no significant regional differences in Km, KD, or Ki, whereas Vmax was significantly greater in the cortical lobes than in the other brain regions. L-Phenylalanine influx during plasma perfusion was only 30% of that predicted in the absence of competing amino acids. Competitive inhibition increased the apparent Km during plasma perfusion by approximately 20-fold, to 0.21 mumol/ml. These data provide accurate new estimates of the kinetic constants that describe L-phenylalanine transport across the blood-brain barrier. In addition, they indicate that the cerebrovascular transfer site affinity (1/Km) for L-phenylalanine is three- to 12-fold greater than previously estimated in either awake or anesthetized animals.     

Quantification of the GABA Shunt and the Importance of the GABA Shunt Versus the 2-Oxoglutarate Dehydrogenase Pathway in GABAergic Neurons

Abstract: We investigated the activity of the cerebral GABA shunt relative to the overall cerebral tricarboxylic acid (TCA) cycle and the importance of the GABA shunt versus 2-oxoglutarate dehydrogenase for the conversion of 2-oxoglutarate into succinate in GABAergic neurons. Awake mice were dosed with [1-¹³C]glucose, and brain extracts were analyzed by ¹³C NMR spectroscopy. The percent enrichments of GABA C-2 and glutamate C-4 were the same: 5.0 ± 1.6 and 5.1 ± 0.2%, respectively (mean ± SD). This, together with previous data, indicates that the flux through the GABA shunt relative to the overall cerebral TCA cycle flux equals the GABA/glutamate pool size ratio, which in the mouse is 17%. It has previously been shown that under the experimental conditions used in this study, the ¹³C labeling of aspartate from [1-¹³C]glucose specifically reflects the metabolic activity of GABAergic neurons. In the present study, the reduction in the formation of [¹³C]aspartate during inhibition of the GABA shunt by γ-vinyl-GABA indicated that not more than half the flux from 2-oxoglutarate to succinate in GABAergic neurons goes via the GABA shunt. Therefore, because fluxes through the GABA shunt and 2-oxoglutarate dehydrogenase in GABAergic neurons are approximately the same, the TCA cycle activity of GABAergic neurons could account for one-third of the overall cerebral TCA cycle activity in the mouse. Treatment with γ-vinyl-GABA, which increased GABA levels dramatically, caused changes in the ¹³C labeling of glutamate and glutamine, which indicated a reduction in the transfer of glutamate from neurons to glia, implying reduced glutamatergic neurotransmission. In the most severely affected animals these alterations were associated with convulsions.     

Inhibition, by 2-Oxo Acids That Accumulate in Maple-Syrup-Urine Disease, of Lactate, Pyruvate, and 3-Hydroxybutyrate Transport Across the Blood-Brain Barrier

Abstract: Data are presented in support of the transport of (-)- d -3-hydroxybutyrate across the blood-brain barrier (BBB) being a carrier-mediated process. The kinetic parameters in 21-day-old pentobarbital-anaesthetized rats were V_max 2.0 μmol.g⁻¹.min⁻¹, K _m 29 m M , and K _D 0.024 ml.g⁻¹.min⁻¹. The value for V_max was the same as that for l -lactate and pyruvate transport in animals of the same age. The transport of all three substrates was sensitive to inhibition by low concentrations of either 2-oxo-3-methylbutanoate or 2-0x0-4-methylpentanoate, the 2-oxo acids that can accumulate in patients with maple-syrup-urine disease. The K _m values for the 2-oxo acids were severalfold lower than the respective K _m values. 2-oxo-3-phenylpropionate was a poor inhibitor. The relative affinities of the various monocarboxylic acids for the transport system of the BBB distinguished it from similar systems described in brain, heart, and liver mitochondria; human erythrocytes; and Ehrlich ascites-tumour cells.     

Kinetic Analysis of Cerebrovascular Isoleucine Transport from Saline and Plasma

The concentration dependence of regional isoleucine transport across the blood-brain barrier was determined in anesthetized rats with the in situ brain perfusion technique of Takasato et al. [Am. J. Physiol. 247, H484-493 (1984)]. This technique allows, for the first time, accurate measurements of cerebrovascular amino acid transport in the absence of competing amino acids using saline perfusate, and in the presence of physiological concentrations of amino acids using plasma perfusate. Cerebrovascular isoleucine transport from saline perfusate followed Michaelis-Menten saturation kinetics where Vmax = 9 - 11 X 10(-4) mumol X s-1 X g-1 and Km = 0.054-0.068 mumol X ml-1 in six brain regions. A component of nonsaturable transport was not detected in any brain region even though perfusate isoleucine concentration was increased to greater than or equal to 150 times the normal plasma concentration. Isoleucine influx during plasma perfusion was only 8% of that predicted from the saline perfusion data due to transport inhibition by competing amino acids in plasma. Competitive inhibition increased the apparent Km for isoleucine transport from plasma by greater than or equal to 24-fold to 1.5-1.7 mumol X ml-1. These data provide accurate new estimates of the kinetic constants that describe amino acid transport across the blood-brain barrier. In addition, they indicate that the cerebrovascular transfer-site affinity (1/Km) for isoleucine is approximately fivefold greater than previously reported with the brain uptake index technique.     

Pretreatment of Rat Brain Synaptosomes with GABA Increases Subsequent GABA Uptake Via GABAB Receptor Activation

Pretreatment with 100 M GABA of synaptosomes purified from rat brain results in an increased uptake of the labelled neurotransmitter in subsequent incubations. The effect is blocked by a GABA_B receptor antagonist, 2-hydroxy-saclofen. The effect is mimicked by baclofen and the baclofen effect is blocked by saclofen too. Lower GABA concentrations (up to 50 M) do not result in an increase of subsequent GABA uptake. Treatment of synaptosomes with 8-Br-cAMP results in a decreased GABA uptake. Since the uptake incubations were run with saturating concentrations of labelled GABA, the data indicates that GABA_B receptor activation in brain synaptosomes up-regulates their GABA uptake capacity by an increase in V_max. This mechanism appears of physiological relevance under conditions of sustained GABA release and substantial increase of its extracellular concentration.     

Transport of valproate and its effects on GABA uptake in astroglial primary culture

The antiepileptic drug Na⁺-valproate (VPA) is a broadspectrum anticonvulsant. It has been proposed to be involved in the inhibitory mechanisms of GABA-ergic systems. In this study, transport of the drug and possible influence on the GABA uptake were investigated in primary astroglial cell cultures from newborn rat cerebral cortex. The results show a Na⁺ and K⁺ independent high affinity uptake for VPA, withK _m andV _max not significantly different from those observed for the GABA uptake. In the presence of the drug, the K_m-value of the GABA uptake increased. The GABA uptake inhibitors guvacine, (RS)-Cis-4-OH-nipecotic acid and 4,5,6,7-tetrahydroisoxazolo (4,5-c) pyridin-3-ol (THPO) did not influence upon the uptake of VPA, suggesting a transport mechanism for the drug, separated from the GABA uptake carrier.     

Effect of Inhibitors of GABA Aminotransferase on the Metabolism of GABA in Brain Tissue and Synaptosomal Fractions

Abstract: Five inhibitors of the GABA degrading enzyme GABA-aminotransferase (GABA-T), viz., gabaculine, γ-acetylenic GABA, γ-vinyl GABA, ethanolamine O -sulphate, and aminooxyacetic acid, as well as GABA itself and the antiepileptic sodium vdproate were administered to mice in doses equieffective to raise the electroconvulsive threshold by 30 V. The animals were killed at the time of maximal anticonvulsant effect of the respective drugs and GABA, GABA-T and glutamate decarboxylase (GAD) were determined in whole brain and synaptosomes, respectively. The synaptosomal fraction was prepared from brain by conventional ultracentrifugation procedures. All drugs studied brought about significant increases in both whole brain and synaptosomal GABA concentrations, and, except GABA itself, inhibited the activity of GABA-T. Furthermore, all drugs, except GABA and γ-acetylenic GABA, activated GAD in the synaptosomal fraction. This was most pronounced with ethanolamine O -sulphate, which induced a twofold activation of this enzyme but exerted only a weak inhibitory effect on GABA-T. The results suggest that activation of GAD is an important factor in the mechanism by which several inhibitors of GABA-T and also valproate increase GABA concentrations in nerve terminals, at least in the relatively non-toxic doses as used in this study.     

Regional Kinetic Constants for Blood–Brain Barrier Pyruvic Acid Transport in Conscious Rats by the Monocarboxylic Acid Carrier

The present investigation using labeled pyruvate describes the regional distribution and kinetics of the monocarboxylic acid carrier at the blood-brain barrier of conscious rats. The experimental procedure involved the arterial injection of a single bolus of 200 microliter containing [1-14C]pyruvate, [3H]water, and varying concentrations of unlabeled pyruvate into the common carotid via an indwelling externalized catheter. The hemisphere ipsi-lateral to the injection and rostral to the midbrain was removed and dissected into five regions. A kinetic analysis revealed no significant regional differences in Km values with an overall average of 1.37 mM. However, there was regional variation in the density of the monocarboxylic acid carrier as indicated by varied levels of the kinetic constant Vmax. The cortex showed the highest Vmax value of 0.42 +/- 0.08 mumol/min/g whereas values for the caudate/putamen, thalamus/hypothalamus, and remaining portion of hemisphere ranged significantly lower at 0.22-0.27 mumol/min/g. The Vmax for the hippocampus was intermediate at 0.37 +/- 0.12 mumol/min/g. The nonsaturable carrier described kinetically by KD had an overall average of 0.034 ml/min/g. The present study confirms quantitatively previous results suggesting a variable regional distribution of the monocarboxylic acid carrier.     

Comparative effects of the GABA uptake inhibitors,tiagabine and NNC-711, on extracellular GABA levels in the rat ventrolateral thalamus

The primary mechanism by which the action of synaptically released GABA is thought to be terminated is by re-uptake into neurones and glial cells, and the pharmacological inhibition of this uptake may be beneficial in conditions where decreased GABAergic transmission has been implicated, such as epilepsy. We have compared the effects of two of these uptake inhibitors, tiagabine and NNC-711, on extracellular GABA levels in the thalamus of the rat, after both systemic and local administration. Both compounds produced dose-dependent increases in GABA concentration irrespective of the route of administration, but the concentrations required to produce increased extracellular GABA levels were considerably higher than those known to be effective for anticonvulsant purposes. These data suggest that, initially at least, alternative GABA transporters, not susceptible to inhibition by the compounds used, may still be able to remove synaptically released GABA from the extracellular space. Special issue dedicated to Dr. Kinya Kuriyama.