Characterization of the carrier-mediated [3H]GABA release from isolated synaptic plasma membrane vesicles

Synaptic plasma membrane (SPM) vesicles were isolated under conditions which preserve most of their biochemical properties. Therefore, they appeared particularly useful to study the cytoplasmic GABA release mechanism through its neuronal transporter without interference of the exocytotic mechanism. In this work, we utilized SPM vesicles isolated from sheep brain cortex to investigate the process of [³H]GABA release induced by ouabain, veratridine and Na⁺ substitution by other monovalent cations (K⁺, Rb⁺, Li⁺, and choline). We observed that ouabain is unable to release [³H]GABA previously accumulated in the vesicles and, in our experimental conditions, it does not act as a depolarizing agent. In contrast, synaptic plasma membrane vesicles release [³H]GABA when veratridine is present in the external medium, and this process is sensitive to extravesicular Na⁺ and it is inhibited by extravesicular Ca²⁺ (1 mM) under conditions which appear to permit its entry. However, veratridine-induced [³H]GABA release does not require membrane depolarization, since this drug does not induce any significant alteration in the membrane potential, which is determined by the magnitude of the ionic gradients artificially imposed to the vesicles. The substitution of Na⁺ by other monovalent cations promotes [³H]GABA release by altering the Na⁺ concentration gradient and the membrane potential of SPM vesicles. In the case of choline and Li⁺, we observed that the fraction of [³H]GABA released relatively to the total amount of neurotransmitter released by K⁺ or Rb⁺ is about 28% and 68%, respectively. Since the replacement of Na⁺ by K⁺, Rb⁺, and Li⁺ causes different levels of membrane depolarization, and the replacement of Na⁺ by choline causes hyperpolarization of the vesicles, these results suggest that, in parallel to the [³H]GABA release, which is directly proportional to the level of membrane depolarization, this neurotransmitter can be released by decreasing the external Na⁺, which reflects an elevation of the Na⁺ concentration gradient (inout). Like veratridine-induced release, the depolarization-induced release of [³H]GABA by SPM vesicles is inhibited by Ca²⁺, which suggests that this divalent cation interfers with the cytoplasmic GABA release mechanism.Abbreviations used ATPase adenosine triphosphatase - GABA -aminobutyric acid - Mes 2 (N-morpholino)-ethanosulfonic acid - SPM synaptic plasma membranes - membrane potential     

Modulation of GABA-stimulated chloride influx into membrane vesicles from rat cerebral cortex by triazolobenzodiazepines

The effects of triazolobenzodiazepines on GABA-stimulated 36Cl- uptake by membrane vesicles from rat cerebral cortex were examined. Triazolam and alprazolam showed a significant enhancement of GABA-stimulated 36Cl- uptake at 0.01-10 microM. On the other hand, adinazolam showed a small enhancement at 0.1-1 microM followed by a significant inhibition of GABA-stimulated 36Cl- uptake at 100 microM. The enhancement of GABA-stimulated 36Cl- uptake by 1 microM alprazolam was antagonized by Ro15-1788, a benzodiazepine antagonist, but the inhibition of this response by 30 microM adinazolam was not antagonized by Ro15-1788. These results indicate that triazolobenzodiazepines enhanced GABA-stimulated 36Cl- uptake through benzodiazepine receptors. High concentrations of adinazolam inhibit GABA-stimulated 36Cl- uptake which may be due to the direct blockade of GABA-gated chloride channel.     

Release of [3H]serotonin from brain slices

1. Labelled serotonin ([³H]5-HT) accumulated by slices of rat brain either in vivo or in vitro is released by depolarizing procedures such as electrical stimulation or high external potassium concentrations. Electrical stimulation predominantly affects the liberation of the unchanged amine, rather than of its principal metabolite, 5-HIAA. 2. Release of [³H]5-HT does not appear to be calcium-dependent. 3. Amount of release parallels the density of serotonin-containing nerve terminals in each of several cerebral regions tested. Release from several extracerebral tissues was similar to that obtained from cerebral tissues having relatively little endogenous 5-HT. 4. Electrically induced release of [³H]5-HT is markedly inhibited by desipramine, chlorpromazine, LSD, lithium and ouabain.     

Uptake of L-[3H] glutamic acid by crude and purified synaptic vesicles from rat brain

Rat brain synaptic vesicles suspended in a medium comprised of potassium tartrate displayed saturable accumulation of L-[³H] glutamic acid at 37° (Km 2.0 × 10^?4M; 311±13 pmol/mg protein), which was stable for periods up to 60 min. The accumulation was temperature sensitive and partially ATP-dependent, uptake levels being reduced to 18.7±0.8 pmol/mg protein at 4°, and to 141±4 pmol/mg protein in the absence of ATP. Fractionation of a crude vesicle preparation on a discontinuous sucrose gradient demonstrated the accumulation to be specifically associated with the synaptic vesicle fraction.     

Regulatory sites and effectors ofd-[3H]aspartate release from rat cerebral cortex

To study the effect of agents interfering with the biosynthesis and/or the K⁺-evoked Ca²⁺-dependent release of neurotransmitter glutamate, rat cerebral slices were preincubated with Krebs-Ringer-HEPES-glucose-glutamine buffer (KRH buffer), loaded withd-[³H]aspartate and superfused with the preincubation medium in the presence or in the absence of Ca²⁺. The difference in radioactivity release divided by the basal release per min under the two conditions represented the K⁺-evoked Ca²⁺-dependent release. The agents used were: 1) Aminooxyacetic acid (AOAA), the inhibitor of transaminases, 2) Leucine (Leu), the inhibitor of phosphate activated glutaminase (PAG), 3) NH₄ ⁺, the inhibitor of PAG, 4) Phenylsuccinic acid (Phs), the inhibitor of the mitochondrial ketodicarboxylate carrier, 5) ketone bodies, the inhibitors of glycolysis, 6) the absence of glutamine, the substrate of PAG. The results show that Leu, NH₄ ⁺, Phs and the absence of Gln significantly increase the K⁺-evoked Ca²⁺-dependent release of radioactivity by 64%, 200%, 95% and 147% respectively, indicating that these agents are inhibitors of the K⁺-evoked Ca²⁺-dependent release of glutamate. Ketone bodies and AOAA had no effect. These results indicate that the major if not the exclusive biosynthetic pathway of neurotransmitter glutamate in rat cerebral cortex is through the PAG reaction and support a model for the pathway followed by neurotransmitter glutamate i.e. glutamate formed outside the inner mitochondrial membrane has to enter the mitochondrial matrix or is formed within it from where it can be extruded to supply the transmitter pool in exchange of GABA.     

Properties of [3H]taurine release from crude synaptosomal fractions of rat cerebral cortex

The release of previously accumulated [³H]taurine and [¹⁴C]GABA from crude synaptosomal (P₂) fractions isolated from rat cerebral cortex was studied using a superfusion system. The spontaneous efflux of [³H]taurine and [¹⁴C]GABA was stimulated by elevated concentrations of K⁺ (15–133 mM) in a concentration-dependent manner. This K⁺-stimulated release of [¹⁴C]GABA but not of [³H]taurine was enhanced in the presence of Ca²⁺. However, addition of 3 mM Ca²⁺ to the superfusion medium in the presence of the ionophore A 23187 resulted in a stimulation of the release of both [³H]taurine and [¹⁴C]GABA. These results are discussed in connection with the cellular localization of tourine in the central nervous system.     

Transport of 2-deoxy-d-[3H]glucose in microvessels isolated from bovine cerebral cortex

Microvessels were isolated from a bovine cortex and the transport of glucose was investigated by using 2-deoxy-d-[³H]glucose (2-DG). The apparentK _m for 2-DG transport was 118 M and therefore indicates a significant high affinity for the substrate. The inhibition of 2-DG uptake byd-glucose showed an apparentK _i of 222 M. Other sugars, e.g., 3-methyl-d-glucose andd-fructose, also inhibited the 2-DG uptake by 60.6 and 36.0%, respectively. Phloretin (1×10^–3 M) inhibited the 2-DG transport more than phlorizin (83.7 vs. 53.8%). Ouabain (1 and 5×10^–4 M) did not inhibit the uptake of 2-DG but 2,4-dinitrophenol (1×10^–4 M) did (78.0%). The uptake of 2-DG could not be demonstrated in homogenized microvessels. Adenine nucleotides (conc. 2 mM) had various effects on the 2-DG uptake by microvessels. ATP inhibited the uptake by 20.7%, ADP was virtually without effect, and AMP stimulated the uptake of 2-DG by 8.5%. It was also found that the decrease of adenylate energy charge favors the uptake of 2-DG. All these findings suggest that in cerebral microvessels of a bovine cortex, 2-DG is apparently transported by a specific, carrier-mediated transport system.Dedicated to Prof. Dr. R. Sammet on the occasion of his 60th birthday.     

Effect of pressure on [3H]GABA release by synaptosomes isolated from cerebral cortex

High hydrostatic pressure has been shown to produce neurological changes in humans which manifest, in part, as tremor, myoclonic jerks, electroencephalographic changes, and convulsions. This clinical pattern has been termed high-pressure nervous syndrome (HPNS). These symptoms may represent an alteration in synaptic transmission in the central nervous system with the inhibitory neural pathways being affected in particular. Since gamma-aminobutyric acid (GABA) transmission has been implicated in other seizure disorders, it was of interest to study GABAergic function at high pressure. Isolated synaptosomes were used to follow GABA release at 67.7 ATA of pressure. The major observation was a 33% depression in total [3H]GABA efflux from depolarized cerebrocortical synaptosomes at 67.7 ATA. The Ca2+-dependent component of release was found to be completely blocked during the 1st min of [3H]GABA efflux with a slow rise over the subsequent 3 min. These findings lead us to conclude that high pressure interferes with the intraterminal cascade for Ca2+-dependent release of GABA.     

Influence of pipecolic acid on the release and uptake of [3H]GABA from brain slices of mouse cerebral cortex

Recently, pipecolic acid (PA) has been involved in the functioning of the GABAergic system. In the present work we have studied the effect of PA on GABA uptake and release in cerebral cortex slices. PA (100 M) was able to increase the release of [³H]GABA (90%) stimulated by mild depolarization with 15 mM potassium. If during the labeling of the tissue with [³H]GABA, -alanine was present, PA also enhanced the release (42%). However, when nipecotic acid was present instead -alanine, no stimulation of [³H]GABA release by potassium was observed neither in the control nor in the presence of PA. Spontaneous release was not affected by PA in any of the experimental conditions tested. In uptake experiments, only when -alanine was present in the medium PA significantly diminished the uptake (36%) of [³H]GABA. These results suggest that the effect of PA is mostly at the presynaptic level, inhibiting the neuronal GABA uptake and/or enhancing its release.     

Distribution of [3H]noradrenaline and [3H]serotonin in photophores of Porichthys notatus

Summary Photophores of Porichthys notatus were examined by electron-microscopic radioautography following incubation in tritiated noradrenaline ([³H]NA) or serotonin ([³H]5-HT). Nerve varicosities surrounding the photocytes were found to accumulate [³H]NA but not [³H]5-HT, providing compelling evidence for the catecholaminergic nature of the monoaminergic innervation of photophores. The photocytes themselves appeared selectively labelled with both tracers, but the intensity of labelling after [³H]5-HT incubation was considerably greater than after [³H]NA. Stereological sampling of organelle content in photocytes showed ultrastructural differences between [³H]NA- and [³H]5-HT-labelled cells, probably related to light emission induced by NA. The main changes noted after incubation with [³H]NA were mitochondrial swelling and disorganization, increased coalescence of photocytic vesicles and extrusion of vesicular material into the extracellular matrix. With respect to the subcellular localization of [³H]NA and [³H]5-HT within the photocytes, statistical analysis of the distribution of silver grains disclosed a preferential affinity of both labels for appositional zones between mitochondria and coalescent vesicles. Moreover, in the case of 5-HT, selective affinity was also exhibited by sites comprising vesicular membrane and adjacent cytoplasm, suggesting binding of this biogenic amine to the entire membrane of photocytic vesicles.Supported by grants from the Natural Sciences and Engineering Research Council (M.A.), and Medical Research Council of Canada (L.D.). Dr. Pierre Legendre kindly provided advice on statistical methods     

Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes

Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1 × 10(-4)M concentration. On the other hand, the single addition of 1 × 10(-6)M, 1 × 10(-5)M and 1 × 10(-4)M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [(3)H]-ouabain binding (in %) to 87 ± 16; 74 ± 16 and 34 ± 17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [(3)H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased K(d) value whereas failed to modify B(max) value, indicating a competitive type interaction of the peptide at Na(+), K(+)-ATPase ouabain site. At variance, SR 48692 decreased B(max) value whereas it did not modify K(d) value. [(3)H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30 min earlier with 100 μg and 250 μg/kg SR 48692. It was observed that the 250 μg/kg SR 48692 dose led to 19% decrease in basal [(3)H]-ouabain binding. After SR 48692 treatments, addition of 1 × 10(-6)M led to additive or synergic effect. Results suggested that [(3)H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.     

Uptake of [3H]ouabain from the cell surface into the lysosomal compartment of HeLa cells

[3H]Ouabain specifically bound at sublethal concentrations to Na,K-ATPase on the surface of HeLa cells is taken up (internalized) by the cells at a rate of three membrane equivalents of labeled sites per generation. Immediately following a pulse label with the glycoside, codistribution of radioactivity with the surface marker 5'-nucleotidase is found in both conventional sucrose-gradient fractionation and in fractionation following a digitonin treatment. At appropriate concentrations digitonin increases the buoyant density of the HeLa surface membrane and solubilizes the lysosomal marker beta-hexosaminidase (Tulkens et al., 1974). After internalization, [3H]ouabain is also solubilized by digitonin. A shear analysis is described which shows internalized ouabain and beta-hexosaminidase to be codistributed in a particulate fraction that is homogeneous with respect to shear; extrapolation to zero-shear shows that little or none of either marker is found in the soluble fraction of the cytosol. Both markers are coreleased from the particulate fraction by osmotic shock. Although internalized ouabain is subsequently released from these cells with a half-time of about 70 hr, apparently by exocytosis, the shear sensitivity of the remaining cell-associated ouabain does not change for up to 72 hr. Thus ouabain (together with Na,K-ATPase?) appears to be taken up from the surface into a lysosomal compartment and, by at least one criterion, this compartment does not change its physical properties with time, i.e., does not "age."     

Incorporation of [3H]Leucine and [3H]Valine into Protein of Freshwater Bacteria: Uptake Kinetics and Intracellular Isotope Dilution

Incorporation of [³H]leucine and [³H]valine into proteins of freshwater bacteria was studied in two eutrophic lakes. Incorporation of both amino acids had a saturation level of about 50 nM external concentration. Only a fraction of the two amino acids taken up was used in protein synthesis. At 100 nM, the bacteria respired 91 and 78% of leucine and valine taken up, respectively. Respiration of ³H and ¹⁴C isotopes of leucine gave similar results. Most of the nonrespired leucine was recovered in bacterial proteins, while only up to one-half of the nonrespired valine occurred in proteins. In intracellular pools of the bacteria, [³H]leucine reached an isotope saturation of 88 to 100% at concentrations of >40 nM. For [³H]valine, an isotope equilibrium of about 90% was obtained at concentrations of >80 nM. Within an incubation period of typically 1 h, tritiated leucine and valine incorporated into proteins of the bacteria reached an isotope saturation of 2 to 6%. In a 99-h batch experiment, bacterial protein synthesis calculated from incorporation of leucine and valine corresponded to 31 and 51% (10 nM) and 89 and 97% (100 nM), respectively, of the chemically determined protein production. Measured conversion factors of 100 nM leucine and valine were 6.4 × 10¹⁶ and 6.6 × 10¹⁶ cells per mol, respectively, and fell within the expected theoretical values. The present study demonstrates that incorporation of both valine and leucine produces realistic measurements of protein synthesis in freshwater bacteria and that the incorporation can be used as a measure of bacterial production.     

Uptake and metabolism of [3H]norepinephrine in the cerebral hemispheres of chick embryos

Abstract— The uptake and metabolism of [³H]norepinephrine were studied in slices of cerebral hemispheres removed from chick embryos at 10, 15 and 20 days of embryonic age, as well as from 90-day-old hens. Brain tissue from all age groups concentrated [³H]norepinephrine to much greater levels at 37°C than at 0°C. There was a marked increase in the rate of accumulation of [³H]norepinephrine in tissues from 10 to 15 days of embryonic age, with no further increase in the rate observed from 15 to 20 days of embryonic age. Tissue slices were incubated for 20 min with [³H]norepinephrine, and the deaminated metabolites of norepinephrine were separated by paper chromatography. In tissues from all age groups, the neutral metabolites were produced in greater amounts than the acid metabolites. A significant increase in the amounts of deaminated metabolites formed was observed in the period from 10 to 15 days of embryonic age and a significant decrease in the amounts formed was observed in the period from 15 to 20 days of embryonic age. The deamination at 20 days was very similar to that observed in the adult. A significant decrease in the level of the deaminated metabolites was noted in all age groups in response to cocaine (an inhibitor of neuronal uptake mechanisms), an observation suggesting that mechanisms for neuronal uptake of NE are functional by 10 days of embryonic development in the chick. However, a significant increase in the level of deaminated metabolites in response to reserpine (an inhibitor of uptake of NE into storage granules) was observed only in slices taken from 20-day embryos and from the 90-day-old hen. The effect in the hen was more prominent than in the 20-day embryo. These results were interpreted to indicate that mechanisms for the uptake of NE develop at an earlier embryonic age than mechanisms for the storage of NE and that mechanisms for storage continue to develop after hatching.     

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.     

Photoaffinity labeling of [3H]flunitrazepam- and [3H]Ro15-4513-bound pellets in rat cerebral cortex and cerebellum

Irreversible incorporation of [3H]flunitrazepam and [3H]Ro15-4513 into GABA/benzodiazepine receptor subunits was studied by UV irradiation using ligand-bound membrane pellets from rat cerebral cortical and cerebellar synaptic membranes. Specific incorporation for [3H]flunitrazepam was greater in the pellet than in the suspension. The incorporation was identical for [3H]Ro15-4513 in both pellet and suspension. With the ligand-bound pellets, 50% of the available binding sites were photolabeled by both ligands in cortex and cerebellum. SDS polyacrylamide gel electrophoresis and fluorography of [3H]flunitrazepam photo-labeled receptor revealed the same number of major sites in both brain regions. In contrast, [3H]Ro15-4513 appears to label fewer sites in cortex and cerebellum. Photoaffinity labeling with [3H]flunitrazepam in ligand-bound membrane pellet provides a more selective and reliable method for studying the subunit structure of GABA/benzodiazepine receptor complex.     

Binding of β1-adrenoreceptor antagonist [3H]CGP 26505 in rat cerebral cortex and sinus atrial nodeantagonist [3H]CGP 26505 in rat cerebral cortex and sinus atrial node

Specific β₁-adrenoreceptors antagonist [³H]CGP 26505 binding was characterized in rat cerebral cortex and heart sinus atrial node. In both tissues [³H]CGP 26505 binding was maximal at 25°C, it was specific, saturable and protein concentration dependent. Scatchard analysis of saturation isotherms of specific [³H]CGP 26505 binding in cerebral cortex showed that [³H]CGP 26505 binds a single class of high affinity sites with a dissociation constant (K_D) of 1±0.3 nM and a maximal number of binding sites (B_max) of 40±2 fmol/mg of protein. In sinus atrial node, [³H]-CGP 26505 binds a single class of high affinity sites (K_D=1.9±0.4 nM, B_max=28±2 fmol/mg of protein).