DEMONSTRATION OF ACETYLCHOLINE RELEASE BY MEASURING EFFLUX OF LABELLED CHOLINE FROM CEREBRAL CORTICAL SLICES

Abstract— To demonstrate release of ACh in the absence of inhibition of cholinesterase, slices of cerebral cortex were incubated with [³H]choline, after which they were placed in a tissue bath for superfusion. Hemicholinium (HC-3) increased the spontaneous efflux of [³H]choline. Electrical stimulation at 4/s increased the efflux of [³H]choline to the same extent whether the slices were stimulated early or late during superfusion. The effect of stimulation on efflux of [³H]choline was abolished by tetrodotoxin and by the absence of calcium. The extent of choline efflux resulting from stimulation, as calculated from the specific radioactivity of the incubation medium, was the same when the slices were incubated with 0.1 or 1.0mM choline, but was less with lower concentrations of choline. We conclude that the increased efflux of [³H]choline evoked by stimulation probably originates from stores of [³H]ACh synthetized during incubation.     

Potassium-Stimulated Release of Radiolabelled Taurine and Glycine from the Isolated Rat Retina

Abstract: The release of preloaded [³H]glycine and [³H]taurine in response to a depolarising stimulus (12.5-50 m M KCl) has been studied in the superfused rat retina. High external potassium concentration immediately increased the spontaneous efflux of [3H]glycine, the effect of 50 m M K⁺ apparently being abolished by omitting calcium from the superfusing medium. In contrast, although high potassium concentrations increased the spontaneous emux of [³H]taurine from the superfused rat retina, this release was not evident until the depolarising stimulus was removed from the superfusing medium. The magnitude of this "late" release of [³H]taurine was dependent on external K⁺ concentrations, and appeared immediately after cessation of the stimulus irrespective of whether it was applied for 4, 8, or 12 min. Potassium (50 m M )-induced release of taurine appeared partially calcium-dependent, being significantly reduced (p < 0.01) but not abolished by replacing calcium with 1 mM EDTA in the superfusate. High-affinity uptake systems for both [³H]glycine and [³H]taurine were demonstrated in the rat retina in vitro ( K _m values, 1.67 μ M and 2.97 μ M ; V_max values, 19.3 and 23.1 nmol/g wet weight tissue/h, respectively). The results are discussed with respect to the possible neuro-transmitter roles of both amino acids in the rat retina.     

THE EFFECT OF ELECTRICAL STIMULATION AND HIGH POTASSIUM CONCENTRATIONS ON THE EFFLUX OF [3H] γ-AMINOBUTYRIC ACID FROM BRAIN SLICES

Abstract— Brain slices were incubated with [³H]GABA in a medium containing aminooxyacetic acid to prevent metabolism of [³H]GABA by GABA-glutamate transaminase. The slices, which rapidly accumulated radioactivity, were then continuously perfused and the efflux of [³H]GABA from the tissue was measured. The spontaneous efflux of [³H]GABA consisted of an initial rapid phase followed by a much slower release of [³[H]GABA. After 40 min perfusion 90 per cent of the radioactivity remained in the tissue.
The slices were depolarized by electrical stimulation or by perfusion with a medium containing a high potassium concentration (40 mM). These procedures caused a striking increase in the efflux of [³H]GABA. The increased efflux produced by potassium, but not that produced by electrical stimulation, was dependent on calcium ions in the medium. The effect of electrical stimulation on [³H]GABA release was considerably reduced by a raised concentration (10 mM) of magnesium in the medium.
High potassium concentrations and electrical stimulation did not cause an increase in the efflux of [¹⁴C]urea, L-[³H]leucine or [¹⁴C]α-amino-isobutyric acid from brain slices. These results are consistent with the suggestion that GABA may be an inhibitory transmitter in the cerebral cortex.     

Identification of Strychnine Binding Sites in the Rat Retina

Abstract: [³H]Strychnine specifically binds to membrane fractions isolated from rat retinae. The binding is saturable, with an apparent dissociation constant, K _D, of 14.3 × 10⁻⁹ M and 205 fmol bound/mg protein. Specific binding is time-dependent and proportional to protein concentration. Glycine and taurine are equally potent inhibitors of [³H]strychnine binding ( K _i= 4 × 10⁻⁵ M); no other amino acids endogenously present in the retina inhibited [³H]strychnine binding.     

RELEASE OF [3H]GAMMA-AMINOBUTYRIC ACID FROM GLIAL CELLS IN RAT DORSAL ROOT GANGLIA.

Abstract— Desheathed rat dorsal root ganglia were incubated in a medium containing amino-oxyacetic acid and [³H]GABA. Under these conditions, [³H]GABA is taken up exclusively by the satellite glial cells in the ganglia. Efflux of [³H]GABA from the tissue was measured after passing the ganglia through a series of wash solutions. The spontaneous efflux of radioactivity, mostly [³H]GABA, was more rapid in the absence of amino-oxyacetic acid in the incubation and wash media.
Raising the potassium concentration in the wash media caused an increase in the efflux of [³H]GABA. This increase was sigmoidally related to the potassium concentration in the wash media, reaching a maximum at 64 m m -K⁺. The releasing effect of K⁺ was inhibited by removing calcium from the media. Reducing the calcium and raising the magnesium concentration in the wash solutions inhibited the increased efflux of [³H]GABA due to 64 m m -K⁺ by 48 per cent, while 5 mM-La³⁺ and diphenylhydantoin (0·005 and 0·5 m m ) had no effect on this increase.
Only a small increase in the efflux of [¹⁴C]glutamate was produced by 64 m m -K⁺ and it had no effect upon the effluxes of [³H]glycine, [³H]alanine or [³H]leucine. The efflux of lactate dehydrogenase was similarly unaffected by 64 mM-K⁺. The results suggest that glial cells in spinal ganglia can respond to depolarizing concentrations of potassium by releasing GABA in a calcium-dependent process.     

Accumulation of N-Arachidonoylethanolamine (Anandamide) into Cerebellar Granule Cells Occurs via Facilitated Diffusion

Abstract: N -Arachidonoylethanolamine (anandamide, AEA) is a putative endogenous ligand of the cannabinoid receptor. Intact cerebellar granule neurons in primary culture rapidly accumulate AEA. [³H]AEA accumulation by cerebellar granule cells is dependent on incubation time ( t _1/2 of 2.6 ± 0.8 min at 37°C) and temperature. The accumulation of AEA is saturable and has an apparent K _m of 41 ± 15 µ M and a V _max of 0.61 ± 0.04 nmol/min/10⁶ cells. [³H]AEA accumulation by cerebellar granule cells is significantly reduced by 200 µ M phloretin (57.4 ± 4% of control) in a noncompetitive manner. [³H]AEA accumulation is not inhibited by either ouabain or removal of extracellular sodium. [³H]AEA accumulation is fairly selective for AEA among other naturally occurring N -acylethanolamines; only N -oleoylethanolamine significantly inhibited [³H]AEA accumulation at a concentration of 10 µ M . The ethanolamides of palmitic acid and linolenic acid were inactive at 10 µ M . N -Arachidonoylbenzylamine and N -arachidonoylpropylamine, but not arachidonic acid, 15-hydroxy-AEA, or 12-hydroxy-AEA, compete for AEA accumulation. When cells are preloaded with [³H]AEA, temperature-dependent efflux occurs with a half-life of 1.9 ± 1.0 min. Phloretin does not inhibit [³H]AEA efflux from cells. These results suggest that AEA is accumulated by cerebellar granule cells by a protein-mediated transport process that has the characteristics of facilitated diffusion.     

Transport of G AB A, β-Alanine and Glutamate into Perikarya of Postnatal Rat Cerebellum

Abstract: Cells dissociated from the postnatally developing rat cerebellum retain their high-affinity carrier-mediated transport systems for [³H]GABA ( K _t=1.9 μM, V = 1.8 pmol/10⁶ cells/min) and [³H]glutamate ( K _t= 10 μM, V = 7.9 pmol/10⁶ cells/min). Using a unit gravity sedimentation technique it was demonstrated that [3H]GABA was taken principally into fractions that were enriched in inhibitory neurons (Purkinje, stellate and basket cells). [³H]β-alanine (which is taken up specifically by the glial GABA transport system) and [³H]glutamate were concentrated by glial-enriched fractions. However [³H]glutamate uptake was minimal in fractions enriched in precursors of granule cells, which may utilise this amino acid as their neurotransmitter. These results are discussed in relation to reports of high-affinity [³H]glutamate uptake by glia. The role of glutamate transport in glutamatergic cells is also considered. The data suggest that high-affinity glutamate transport is a property of glial cells but not granule neurons.     

THE RELEASE OF AMINO ACIDS FROM THE HEMISECTED SPINAL CORD DURING STIMULATION

Abstract— Isolated frog or toad hemicords were incubated for 40 min with either [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate. l -[¹⁴C]aspartate, l -[¹⁴C]serine, l [¹⁴C]threonine or l -[³H]leucine, and the release of these compounds from the cord was measured under resting conditions and during electrical stimulation. Stimulation of spinal roots produced no significant change in the efflux of any of the compounds tested. Direct stimulation of the rostral cord however, produced a large increase in the efflux of [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate and l -[¹⁴C]aspartate. These increased effluxes were calcium dependent, the effects of stimulation being reduced in a calcium-free, or magnesium-supplemented (10 mM) medium. Stimulation failed to produce an increase in the efflux of l -[¹⁴C]serine, l -[¹⁴C]threonine, l -[¹⁴H]leucine, [¹⁴C]mannitol or [¹⁴C]urea. These results are consistent with the suggestions that glycine, GABA, glutamate and aspartate may be synaptic transmitters in the spinal cord.     

Comparison of Solubilised Kainate and α-Amino-3-Hydroxy-5-Methylisoxazolepropionate Binding Sites in Chick Cerebellum

Abstract: [³H]Kainate bound to chick cerebellar membranes with a K _D of 0.6 μ M and with an exceptionally high B max of 165 pmol/mg of protein. In octylglucoside-solubilised extracts, the affinity of [³H]kainate was reduced ( K _D= 2.7 μ M ), but the B _max was relatively unchanged (130 pmol/mg of protein). The rank potency of competitive ligands was domoate > kainate > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > glutamate. Binding sites for α-[³H]amino-3-hydroxy-5-methylisoxazolepropionate ([³H]AMPA) were much less abundant, with K _D and B _max values in membranes of 86 n M and I pmol/mg of protein, respectively. The affinity of [³H]AMPA binding was also reduced on solubilisation ( K _D= 465 n M ), but there was an increase in the B _max (1.7 pmol/mg of protein). Quisqualate and CNQX were the most effective displacers of [³H]AMPA binding, but kainate was also a relatively potent inhibitor. However, in contrast to the displacement profile for [³H]kainate, domoate was markedly less potent than kainate at displacing [³H]AMPA. These results suggest that [³H]AMPA binds to a small subset of the kainate sites that, unlike the majority of the [³H]kainate binding protein, which has been reported to be located in the Bergmann glia, may represent neuronal unitary non- N -methyl-D-aspartate receptors.     

Characterization of 5,7-Dichlorokynurenate-Insensitive d-[3H]Serine Binding to Synaptosomal Fraction Isolated from Rat Brain Tissues

Abstract: To explore target sites for endogenous d -serine that are different from the glycine site of the N -methyl- d -aspartate (NMDA) type glutamate receptor, we have studied the binding of d -[³H]serine to the synaptosomal P2 fraction prepared from the rat brain and peripheral tissues in the presence of an excess concentration (100 µ M ) of the glycine site antagonist 5,7-dichlorokynurenate (DCK). Nonspecific binding was defined in the presence of 1 m M unlabeled d -serine. Association, dissociation, and saturation experiments indicated that d -[³H]serine bound rapidly and reversibly to a single population of recognition sites in the cerebellar P2 fraction in the presence of DCK, with a K _D of 614 n M and a B _max of 2.07 pmol/mg of protein. d -Serine, l -serine, and glycine produced a total inhibition of the specific DCK-insensitive d -[³H]serine binding to the cerebellum with similar K _i values. Strychnine and 7-chlorokynurenate failed to inhibit the binding at 10 µ M . The profiles of displacement of the DCK-insensitive d -[³H]serine binding by various amino acids and glutamate and glycine receptor-related compounds differ from those of any other defined recognition sites. DCK-insensitive d -[³H]serine binding was at high levels in the cerebral cortex and cerebellum but very low in the kidney and liver. The present findings indicate that the DCK-insensitive d -[³H]serine binding site could be a novel candidate for a target for endogenous d -serine in mammalian brains.     

GABA UPTAKE IN RAT CENTRAL NERVOUS SYSTEM: COMPARISON OF UPTAKE IN SLICES AND HOMOGENATES AND THE EFFECTS OF SOME INHIBITORS

Abstract— Fifty-two substances were tested as inhibitors of the uptake of [³H]GABA in slices of rat cerebral cortex. Among GABA analogues tested, only the 2-fluoro, 3-hydroxy and 2-amino compounds had affinities for the uptake mechanism comparable to that of GABA. [³H]GABA uptake was also potently inhibited by p -chloromercuriphenylsulphonate, N -ethylmaleimide, chlorpromazine and haloperidol. No inhibitors were found to act in a competitive manner with respect to GABA. [³H]GABA uptake was also examined in homogenates of cerebral cortex and other regions of CNS. There was a rapid uptake of [³H]GABA into particles when homogenate samples were incubated with the labelled amino acid; this uptake had similar kinetic properties and inhibitor sensitivity to that observed in slices of intact tissue. Density gradient centrifugation experiments indicated that the particles responsible for the uptake of [³H]GABA in homogenates were probably synaptosomes. Uptake of [³H]GABA also occurred in slices and homogenates of rat spinal cord, and evidence was obtained by the simultaneous labelling of homogenates with [¹⁴C]glycine and [³H]GABA that these two amino acids were taken up by different nerve terminals in this region.     

EVIDENCE FOR THE SYNTHESIS AND STORAGE OF 5-HYDROXYTRYPTAMINE IN TWO SEPARATE POOLS IN THE BRAIN

Abstract— Evidence is presented to support the hypothesis that 5-hydroxytryptamine (5-HT) in the rat brain is synthesized by two separate pathways and stored in two or more compartments. Lysergic acid diethylamide in doses down to 50 μg/kg was shown to reduce the formation of 5-[³H]HT from [³H]tryptophan in the presence of a monoamine oxidase inhibitor, although the total rate of accumulation of 5-HT was unchanged. Conversely, adrenalectomy was found to increase the total synthesis of 5-HT measured in the same way, although the amount of 5-[³H]HT formed suggested that there was no increase in the synthesis of the amine. In a third experiment it was found that electrical stimulation of 5-HT-containing nerves following labelling of 5-HT stores with [³H]tryptophan led to a biphasic disappearance of 5-[³H]HT. It is suggested that the method of measuring 5-HT synthesis by measuring 5-[³H]HT formed from[³H]tryptophan in the presence of a monoamine oxidase inhibitor may be a way of selectively measuring the turnover of the functional pool of 5-HT.     

Activating Mutations of the Serotonin 5-HT2C Receptor

Abstract: Site-directed mutagenesis was performed to create a mutant serotonin 5-HT_2C receptor that would mimic the active conformation of the native receptor. Structural alteration of receptor conformation was achieved by changing amino acid no. 312 from serine to phenylalanine (S312F) or lysine (S312K). After expression in COS-7 cells, the binding affinity of 5-HT for [³H]-mesulergine-labeled 5-HT_2C receptors increased from 203 n M (native) to 76 n M for S312F and 6.6 n M for S312K mutant receptors. 5-HT potency for stimulation of phosphatidylinositol (PI) hydrolysis increased from 70 n M (native) to 28 n M for S312F and 2.7 n M for S312K mutant receptors. The mutant receptors were constitutively active, stimulating PI hydrolysis in the absence of agonist. S312F and S312K mutations resulted in twofold and five-fold increases, respectively, in basal levels of PI hydrolysis. Mianserin and mesulergine displayed inverse agonist activity by decreasing basal levels of PI hydrolysis stimulated by S312K mutant receptors. [³H]5-HT and [³H]-mesulergine labeled the same number of S312K mutant receptors and 5'-guanylylimidodiphosphate had no effect on [³H]5-HT binding. These results indicate that serine → lysine mutation at amino acid no. 312 produces an agonist high-affinity state of the 5-HT_2C receptor that spontaneously couples to G proteins and stimulates PI hydrolysis in the absence of agonist.     

Increased auxin efflux in the IAA-overproducing sur1 mutant of Arabidopsis thaliana: A mechanism of reducing auxin levels?

With the aim of investigating the mechanisms that maintain auxin homeostasis in plants, we have monitored the net uptake and metabolism of exogenously supplied indole-3-acetic acid (IAA) and naphthalene-1-acetic acid (NAA) in seedlings of wild type and the IAA-overproducing mutant sur1 of Arabidopsis thaliana . Tritiated IAA and NAA entered the seedling tissues within minutes and were mostly accumulated as metabolites, probably amino acid and sugar conjugates. The mutant seedlings were marked by a strong increase of [³H]IAA metabolism and a reduction of the accumulation levels of both free [³H]IAA and [³H]NAA. The same characteristics were observed in wild-type seedlings grown on 5 μ M picloram. We measured [³H]NAA uptake in the presence of high concentrations of unlabeled NAA or the auxin efflux carrier inhibitor naphthylphthalamic acid (NPA). This abolished the difference in free [³H]NAA accumulation between the mutant or picloram-treated seedlings and wild-type seedlings. These data indicated that active auxin efflux carriers were present in Arabidopsis seedling tissues. Picloram-treated seedlings and seedlings of the IAA-overproducing mutant sur1 displayed increased auxin efflux carrier activity as well as elevated conjugation of IAA. There is previous evidence to suggest that conjugation is a means to remove excess IAA in plant cells. Here, we discuss the possibility of efflux constituting an additional mechanism for regulating free IAA levels in the face of an excess auxin supply.     

Inhibition of Neutral Amino Acid Transport Across the Human Blood-Brain Barrier by Phenylalanine

Abstract: The delivery of large neutral amino acids (LNAAs) to brain across the blood-brain barrier (BBB) is mediated by the L-type neutral amino acid transporter present in the membranes of the brain capillary endothelial cell. In experimental animals, the L-system transporter is saturated under normal conditions, and therefore an elevation in the plasma concentration of one LNAA will reduce brain uptake of others. In this study, we used positron emission tomography (PET) to determine the effect of elevated plasma phenylalanine concentrations on the uptake of an artificial neutral amino acid, [¹¹C]-aminocyclohexanecarboxylate ([¹¹C]ACHC), in human brain. PET scans were performed on six normal male subjects after an overnight fast and again 60 min after oral administration of 100 mg/kg of phenylalanine. The plasma phenylalanine concentration increased by an average of 11-fold between the first and second scans. This increase produced a reduction in [¹¹C]ACHC uptake in all brain regions but not in scalp. The mean ± SD influx rate constant for whole brain decreased after phenylalanine ingestion from 0.036 ± 0.002 to 0.019 ± 0.004 ml/g/min. Kinetic analysis of the effect of plasma phenylalanine concentration on the rate of [¹¹C]ACHC uptake is compatible with a model of competitive inhibition so that large increases in the concentration of one LNAA in plasma will reduce the brain uptake of other LNAAs across the human BBB.     

IN VIVO RELEASE OF ENDOGENOUSLY SYNTHESIZED CATECHOLAMINES FROM THE CAT BRAIN EVOKED BY ELECTRICAL STIMULATION AND BY d-AMPHETAMINE

Abstract— The cerebral ventricles of spinal-sectioned cats were perfused with artificial cerebrospinal fluid after the intraventricular administration of [³H]DOPA or [³H]tyrosine. Endogenously synthesized [³H]dopamine or [³H]norepinephrine were identified in the perfusate. Electrical stimulation of catecholaminergic nerve tracts in the hypothalamus increased the efflux of both catecholamines. The addition of d -amphetamine to the perfusing cerebrospinal fluid caused a large increase in [³H]dopamine and a small increase in [³H]norepinephrine appearing in the perfusate. Most of the endogenously synthesized [³H]catecholamines detected in the perfusate following stimuli originated from structures bordering the lateral cerebral ventricle. Thus, norepinephrine and dopamine can be synthesized in and released from catecholaminergic nerve terminals in structures bordering the cerebral ventricles.     

Neurochemical Studies of the Mesolimbic Dopaminergic Pathway: Somatodendritic Mechanisms and GABAergic Neurones in the Rat Ventral Tegmentum

Abstract: The rat ventral tegmentum (containing dendrites and somata of mesolimbic neurones) contained 1.3 μg/g of dopamine, which was reduced to 40% of the control level by reserpine. Slices of ventral tegmentum were able to accumulate and release (elevated potassium or protoveratrine A) exogenous [³H]dopamine. In parallel studies the uptake mechanism in ventral tegmentum was shown to be virtually identical to the nerve terminal uptake of [³H]dopamine by slices of nucleus accumbens. The release of [³H]dopamine was indistinguishable from that observed in substantia nigra, where there is substantial evidence for dendritic mechanisms. Basal adenylate cyclase activity was present, but dopamine-stimulated activity was not detected. A high GABA concentration (7.7 μmol/g) was present in ventral tegmentum, in conjunction with an uptake and a release mechanism for [³H]GABA. GABA and muscimol elicited a small, reproducible efflux of [³H]dopamine, but an interaction between dopamine and [³H]GABA efflux was not observed. The results are in accord with transmitter roles for dopamine and GABA in the somatoden-dritic area of mesolimbic dopaminergic neurons.     

CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [¹⁴C]tyrosine into the C-terminal position of α-tubulin of rat brain cytosol was 10-fold higher for non-assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non-assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.
[¹⁴C]Tyrosine was released from assembled and non-assembled [¹⁴C]tyrosinated tubulin by the action of an endogenous carboxypeptidase. Release from non-assembled tubulin was shown by incubating a tubulinyl-[¹⁴C]tyrosine preparation in the presence of CaCl₂ at a concentration that abolished microtubule formation. Release from microtubules was inferred from the observation that the percentages of [¹⁴C]tyrosine released and the decrease of the specific radioactivity of the recovered microtubules were practically identical and did not change after a 10-fold dilution of the incubated microtubules.
[³H]Phenylalanine was released from a preparation of tubulinyl-[³H]phenylalanine also by an enzymatic activity.
The capacity of a tubulin preparation to incorporate tyrosine was increased 43% by pre-treatment with endogenous carboxypeptidase.
Tubulin tyrosinated in vitro was assembled to the same extent as native tubulin. After a mixture of tubulinyl-[¹⁴C]tyrosine and tubulinyl-[³H]phenylalanine was partially assembled, the ratio of ¹⁴C/³H found in the microtubules was the same as in the non-assembled tubulin fraction.     

Comparison of [3H]WIN 35,428 Binding, a Marker for Dopamine Transporter, in Embryonic Mesencephalic Neuronal Cultures with Striatal Membranes of Adult Rats

Abstract: In contrast to striatal membranes of adult rats, where high- ( K _D1= 34 n M ) and low- ( K _D2= 48,400 n M ) affinity binding sites for [³H]WIN 35,428 are present, in primary cultures of ventral mesencephalon neurons (CVMNs) only low-affinity binding sites were found ( K _D= 336,000 n M ). The binding of [³H]WIN 35,428 in CVMNs prepared from rat embryos was reversible, saturable, and located in cytosol. Although dopamine (DA) uptake blockers inhibited [³H]DA uptake at nanomolar concentrations in CVMNs, the displacement of [³H]WIN 35,428 binding in CVMNs by DA uptake inhibitors required 100-8,000 times higher concentrations than were needed to displace [³H]WIN 35,428 binding in striatal membranes. Piperazine derivatives, e.g., GBR-12909, GBR-12935, and rimcazole, inhibited [³H]WIN 35,428 binding in CVMNs more effectively than did cocaine, WIN 35,428, mazindol, nomifensine, or benztropin. A positive correlation ( r = 0.779; p < 0.001) was found between drug affinities for the striatal membrane sites labeled by [³H]WIN 35,428 and their abilities to inhibit DA uptake in CVMNs, whereas no correlation existed between the IC₅₀ values of drugs that inhibited [³H]WIN 35,428 binding and [³H]DA uptake in CVMNs. The cytosolic [³H]WIN 35,428 binding sites may be a piperazine acceptor and may not be involved in the regulation of the DA transporter.     

Sodium and Chloride Ion-Dependent Transport of β-Alanine Across the Blood-Brain Barrier

Abstract: The characteristics of β-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β-[³H]alanine transport indicated that the transporter for β-alanine functions with K_t of 25.3 ± 2.5 µ M and J _max of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β-[³H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN₃) and low temperature reduced the uptake significantly. Furthermore, the uptake of β-[³H]alanine required Na⁺ and Cl⁻ in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β-alanine molecule. The Na⁺ and Cl⁻-dependent uptake of β-[³H]alanine was stimulated by a valinomycin-induced inside-negative K⁺-diffusion potential. β-Amino acids (β-alanine, taurine, and hypotaurine) inhibited strongly the uptake of β-[³H]alanine, whereas α- and γ-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of β-[³H]alanine was stimulated by preloading of β-alanine or taurine but not l -leucine. These results show that β-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β-amino acids.