Delta pH-dependent and -independent uptake of [3H]dopamine by synaptic vesicles isolated from rat brain

The dopamine (DA)-translocating mechanism of synaptic vesicles isolated from rat brain has been studied in the presence of an artificially imposed delta pH on the vesicle membranes (acidic inside with respect to the external medium) without the aid of ATP-Mg2+. Under the experimental conditions, [3H]DA uptake by the synaptic vesicles was driven by two different, i.e. delta pH-dependent and -independent processes. Both processes appeared to be carrier-mediated based on the inhibition by NEM (N-ethylmaleimide), an -SH reagent, and by nomifensine, a DA uptake blocker at nerve terminals. The DA carrier of the vesicles was similar to that of the nerve terminal plasma membrane with respect to their susceptibility to nomifensine. The delta pH-dependent uptake was transient and most of the incorporated DA was easily lost from the vesicles. On the other hand, the delta pH-independent uptake increased with time and the amine was retained in the vesicles. The initial rate of the delta pH-independent uptake was lower than that of the delta pH-dependent one but their extents were comparable with each other. These results indicate that rat brain synaptic vesicles have a DA uptake system requiring no ATP hydrolysis. A preparation of synaptic vesicles used here exhibited an inwardly directed proton translocation in the presence of ATP-Mg2+ when monitored by following changes in the fluorescence of ANS (8-anilino-1-naphthalene sulfonate). However, the time course of the delta pH-generation was not influenced by the addition of 1 mM DA.(ABSTRACT TRUNCATED AT 250 WORDS)     

5-Aminolevulinic acid inhibits [3H]muscimol binding to human and rat brain synaptic membranes

The interaction of 5-aminolevulinic acid (ALA) with GABA_A receptors has been proposed to underlie the neurological dysfunctions of ALA-accumulating disorders, such as acute intermittent porphyria. The effects of ALA on [³H]muscimol binding to human and rat cerebral cortical membranes were compared. ALA (0.1–10 mM) significantly inhibited the binding of [³H]muscimol (12 nM), with a similar potency in rat and human membranes (IC₅₀ = 199 vs. 228 M, respectively). Kinetical analysis revealed that ALA (1 mM) significantly increased the Kd and decreased the Bmax of [³H]muscimol to both rat (100 and 50%, respectively) and human (200 and 40%, respectively) membranes, indicating a mixed-type inhibition. The similarity in the potency and mechanism of the ALA-induced inhibition of muscimol binding in rat and human membranes indicate that rat studies are useful to evaluate the neurotoxic properties of ALA towards the human GABAergic system, and may help to understand the pathophysiology of porphyria.     

Characterization of a specific L-[3H]glutamic acid binding site on cilia isolated fromParamecium tetraurelia

Summary Cilia isolated fromParamecium tetraurelia possess a specific, high affinity L-[³H]glutamic acid binding site, defined by an ED₅₀ of 3.0×10^–8 M. The structural specificity of this site was probed by testing the competition between L-glutamate and various analogues for binding to cilia. The binding site is stereo-specific for L-glutamic acid, and requires the presence of all three ionizable groups on the glutamate molecule for optimal ligand: receptor interaction.Specific binding of L-[³H]glutamic acid to cilia is rapid in onset but transient, reaching peak values within 6 min, and then declining thereafter. This transience may represent a form of sensory adaptation during prolonged exposure to the ligand.     

Uptake of L-[propyl-2,3-3H]dihydroalprenolol by intact HeLa cells

This report describes the uptake of L-[propyl-2,3-3H]dihydroalprenolol, a beta-adrenergic antagonist, by HeLa (human adenocarcinoma) cells. [3H]Dihydroalprenolol binds to sites of high capacity and low affinity in intact HeLa cells. The binding achieves equilibrium rapidly and is rapidly reversible. Bound [3H]dihydroalprenolol is displaceable by beta-adrenergic antagonists in a nonstereoselective fashion, but is not displaceable by isoproterenol, an adrenergic agonist. Phentolamine, an alpha-adrenergic antagonist, and chloroquine, a lysosomotropic amine, also compete for [3H]dihydroalprenolol binding sites. [3H]Dihydroalprenolol binding is inhibited by metabolic inhibitors, but not by cytoskeletal blocking agents. The binding is sensitive to extracellular pH (less binding at lower pH) and is temperature-sensitive (less binding at lower temperatures). The bound radioligand is rapidly reversed following hypotonic lysis of the cells. These [3H]dihydroalprenolol binding sites in intact HeLa cells therefore do not have the characteristics expected for beta-adrenergic receptors. Further studies showed that beta-adrenergic receptors could be detected in a HeLa membrane preparation using [125I]iodohydroxybenzylpindolol, and that chloroquine had very low affinity for these receptors. We conclude that [3H]dihydroalprenolol diffuses across the plasma membrane of intact HeLa cells and accumulates in acidic intracellular compartments.     

Uptake of GABA by rat brain synaptic vesicles isolated by a new procedure.

Uptake of GABA was demonstrated in rat brain synaptic vesicles which were prepared by a new and efficient procedure. The uptake activity co-purified with the synaptic vesicles during the isolation procedure. The purity of the vesicle fraction was rigorously examined by analysis of marker enzymes and marker proteins and also by immunogold electron microscopy using antibodies against p38 (synaptophysin). Contamination by other cellular components was negligible, indicating that GABA uptake by the synaptic vesicle fraction is specific for synaptic vesicles and not due to the presence of other structure possessing GABA uptake or binding activities. GABA uptake was ATP dependent and similar to the uptake of glutamate, which was assayed for a comparison. Both uptake activities were independent of sodium. They were inhibited by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, indicating that the energy for the uptake is provided by an electrochemical proton gradient. This gradient is generated by a proton ATPase of the vacuolar type as suggested by the effects of various ATPase inhibitors on neurotransmitter uptake and proton pumping. Competition experiments revealed that the transporters for GABA and glutamate are selective for the respective neurotransmitters.     

Uptake of [3H]serotonin into plasma membrane vesicles from mouse cerebral cortex

Preparations of plasma membrane vesicles were used as a tool to study the properties of the serotonin transporter in the central nervous system. The vesicles were obtained after hypotonic shock of synaptosomes purified from mouse cerebral cortex. Uptake of [3H]serotonin had a Na+-dependent and Na+-independent component. The Na+-dependent uptake was inhibited by classical blockers of serotonin uptake and had a Km of 63-180 nM, and a Vmax of 0.1-0.3 pmol mg-1 s-1 at 77 mM Na+. The uptake required the presence of external Na+ and internal K+. It required a Na+ gradient ([Na+]out greater than [Na+]in) and was stimulated by a gradient of K+ ([K+]in greater than [K+]out). Replacement of Cl- by other anions (NO2-, S2O3-(2-)) reduced uptake appreciably. Gramicidin prevented uptake. Although valinomycin increased uptake somewhat, the membrane potential per se could not drive uptake because no uptake was observed when a membrane potential was generated by the SCN- ion in the absence of internal K+ and with equal [Na+] inside and outside. The increase of uptake as a function of [Na+] indicated a Km for Na+ of 118 mM and a Hill number of 2.0, suggesting a requirement of two sodium ions for serotonin transport. The present results are accommodated very well by the model developed for porcine platelet serotonin transport (Nelson, P. J., and Rudnick, G. (1979) J. Biol. Chem. 254, 10084-10089), except for the number of sodium ions that are required for transport.     

Endogenous inhibitor of [3H]kainate binding to synaptic membrane in rat brain

An understanding of the mechanism of kainic acid toxicity to neurons could provide important clues to pathogenesis of Huntington's chorea. The existence of high-affinity binding sites for kainate, a foreign compound, is suggestive of the existence of kainate-like substances in the brain. In addition to such neurotoxic kainate-like substances, and endogenous inhibitor of kainate binding may also exist in the brain to allow the synaptic function to operate normally. Based on this idea, the existence of molecules which inhibit [3H]kainate binding to synaptic membranes was examined in rat brain. An endogenous inhibitor of [3H]kainate binding to synaptic membranes was found in the supernatant obtained from synaptic membranes of rat brain. The inhibitor is a thermostable, basic protein with a relatively low molecular weight.     

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     

Uptake and binding of tritium from [chloroethyl 3H] cyclophosphamide by rat embryos in vitro

Day 10 rat embryos were exposed in vitro to [chloroethyl 3H] cyclophosphamide (3H-CP) at 4 micrograms/ml over a 24-hour period and the uptake and binding of labeled drug were monitored autoradiographically and biochemically. Autoradiographic analysis revealed that embryos exposed to 3H-CP and a complete activating system exhibited radioactivity distributed throughout the embryo. Subsequent analysis indicated that the distribution of autoradiographic grains on a per cell basis ranged from 7.7 in surface ectoderm to 13.4 in the neuroepithelium. No correlation was found between the sensitivity of various embryonic tissues to the cytotoxic effects of CP and the number of grains per cell. Direct radiochemical analysis of the amount of tritium taken up and bound by embryos under bioactivating conditions (3H-CP + S-9 + cofactors) confirmed the autoradiographic analysis. Autoradiographic and radiochemical analyses demonstrate that embryos exposed under bioactivating conditions take up and bind approximately three times more tritium than embryos exposed under nonactivating conditions (3H-CP + S-9 without cofactors). Additional studies have demonstrated that uptake and binding of tritium from bioactivated 3H-CP only are linear over the first 10 hours of incubation with no detectable increases thereafter.     

Uptake and metabolism ofl-[3H]glutamate andl-[3H]glutamine in adult rat cerebellar slices

Using very low concentrations (1 mumol range) of L-2-3-[3H]glutamate, (3H-Glu) or L-2-3-[3H]glutamine (3H-Gln), we have previously shown by autoradiography that these amino acids were preferentially taken up in the molecular layer of the cerebellar cortex. Furthermore, the accumulation of 3H-Glu was essentially glial in these conditions. We report here experiments in which uptake and metabolism of either (3H-Glu) or (3H-Gln) were studied in adult rat cerebellar slices. Both amino acids were rapidly converted into other metabolic compounds: after seven minutes of incubation in the presence of exogenous 3H-Glu, 70% of the tissue accumulated radioactivity was found to be in compounds other than glutamate. The main metabolites were Gln (42%), alpha-ketoglutarate (25%) and GABA (1,4%). In the presence of exogenous 3H-Gln the rate of metabolism was slightly slower (50% after seven minutes of incubation) and the metabolites were also Glu (29%), alpha-ketoglutarate (15%) and GABA (5%). Using depolarizing conditions (56 mM KCl) with either exogenous 3H-Glu or 3H-Gln, the radioactivity was preferentially accumulated in glutamate compared to control. From these results we conclude: i) there are two cellular compartments for the neurotransmission-glutamate-glutamine cycle; one is glial, the other neuronal; ii) these two cellular compartments contain both Gln and Glu; iii) transmitter glutamate is always in equilibrium with the so-called "metabolic" pool of glutamate; iv) the regulation of the glutamate-glutamine cycle occurs at least at two different levels: the uptake of glutamate and the enzymatic activity of the neuronal glutaminase.     

Effect of cholinomimetics on the release and uptake ofl-[3H] glutamic acid in rat neostriatum

The effects of cholinomimetics on the release and uptake of L-glutamic acid have been studied by means of local superfusion of rat neostriatum (in vivo) and using striatum slices (in vitro). Cholinomimetics regulate the release of L-glutamic acid through both muscarinic (m-) and nicotinic (n-) choline receptors according to the mechanism of negative feedback. Cholinomimetics regulate release through presynaptic receptors. The effect of n-cholinomimetics is mediated by neostriatum interneurons.     

Potassium and veratrine-stimulated l-[3H]cysteine sulfinate and l-[3H]glutamate release from rat brain slices

The release of l-[³H]cysteine sulfinic acid, l-[³H]glutamatic acid and [³H]GABA from preloaded slices of various rat brain regions in response to either 30 mM K⁺ or veratrin was investigated. All these aminoacids were released by both depolarizing agents, which did not produce any changes in the spontaneous efflux of [³H]lysine. The K⁺ stimulated cysteine sulfinate release from superfused slices was found partly Ca²⁺-dependent in the subiculum, and mainly Ca²⁺-independent in the hippocampus whereas the K⁺-elicited glutamate release was partly Ca²⁺-dependent in both regions. The veratrine-induced release of both cysteine sulfinate and glutamate was blocked by verapamil in a dose-dependent way, although a small verapamil concentration independent release remained. The release pattern of both amino acids was heterogeneous, but roughly correlated among brain regions, except in the subiculum and hypothalamus.These findings demonstrate the releasability of both substances from various brain regions and suggest that those releases occur from different pools, being probably mainly of neuronal origin. They give further evidence that cysteine sulfinate as well as glutamate may serve a neurotransmitter role in the CNS.     

Glutathione-induced inhibition of Na+-independent and -dependent bindings of L-[3H]glutamate in rat brain

Reduced glutathione (GSH, 10(-7)-10(-3) M) was found to exert a profound suppressive action on the Na+-independent and -dependent bindings of L-[3H]glutamic acid (Glu) in a temperature-independent manner. Similarly significant reduction of the bindings resulted from the addition of oxidized glutathione (GSSG). Scatchard analysis revealed that GSH as well as GSSG invariably decreased the affinity of the binding sites for [3H]Glu without significantly affecting the number of the binding sites. These results suggest that GSH (GSSG) may in part participate in the synaptic transmission at central Glu neurons through interaction with the receptors and/or the uptake sites for Glu.     

Studies on the uptake of [3H]dopamine by synaptic vesicle fraction isolated from bovine brain

1. A synaptic vesicle fraction isolated from bovine caudatolenticular nuclei showed enzymic activities of tyrosine hydroxylase [EC 1.14.16.2] and dopamine beta-hydroxylase [EC 1.14.17.1]. Tyrosine hydroxylase, whose subcellular localization is uncertain, appeared to be associated with the synaptic vesicles. 2. The vesicle fraction took up [3H]dopamine increasingly with time without the aid of ATP (6.3 pmol of [3H]dopamine per mg of vesicle proteins, or 3-4% of the added dopamine, at 30 min). In the presence of ATP, a transient accumulation of the amine was observed, reaching the highest level at 7-10 min (5.6 pmol dopamine/mg protein), and then a rapid release of the amine took place, obeying first-order kinetics with respect to the amine concentration. The amine uptake was strongly inhibited with NEM, regardless of the presence or absence of ATP. 3. The vesicle fraction also exhibited a weak ability to translocate protons inward and ATP-dependently, as monitored by an increase in the fluorescence intensity of ANS. The fluorescence enhancement persisted for at least 30 min and this time-dependent change was not consistent with that of the transient accumulation of [3H]dopamine mentioned above. Since the present vesicle preparation contained a small amount of mitochondrial ATPase (18-20% of the total activity), the proton translocating ability could be attributable to contaminating submitochondrial particles. 4. Therefore these results made it impossible to conclude that the transient uptake of [3H]dopamine by the synaptic vesicles was coupled to ATP hydrolysis.     

3H-dopamine accumulation by rat brain synaptic vesicles in a membrane-impermeable medium

3H-Dopamine (DA) accumulation by storage vesicles from whole rat brain was significantly stablized in a buffer system based upon the membrane-impermeant D-potassium tartrate. 3H-DA uptake saturated by twenty minutes (Km 2.1 X 10(-5)M) and remained stable for periods of 40-60 minutes. Accumulated DA was rapidly exchangeable with exogenous DA. Total levels of accumulation (pmol/mg protein) were 41.7 +/- 2.9 (37 degrees), 11.9 +/- 2.5 (4 degrees), 31.3 +/- 1.8 (absence of ATP), 26.3 +/- 2.7 (reserpine, 10(-6)M), 26.1 +/- 0.67 (no ATP + reserpine 10(-6), and 14.6 +/- 2.4 (carbonylcyanide-p-triflouromethoxyphenylhydrazone, FCCP, 10(-6)M). Depletion of endogenous DA levels by pretreatment of the animals with alpha-methyl-p-tyrosine greatly diminished the reserpine-insensitive DA accumulation. After depletion of endogenous DA, ATP-independent uptake was significantly retarded, but eventually reached near-control levels. This uptake was abolished in the presence of FCCP (10(-6)M). The results suggest that endogenous levels of DA and ATP contribute to the reserpine- and ATP-insensitive DA accumulation observed in vesicles from untreated animals. HPLC analysis demonstrated no conversion of DA to norepinephrine (NE) in the course of the experiments.     

Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture

The membrane complex lipids of human fibroblasts and differentiated rat cerebellar granule cells in culture were metabolically radiolabeled with [1-³H]sphingosine, L-[3-³H]serine and [9,10-³H]palmitic acid. A relevant efflux of radioactive sphingolipids and phosphatidylcholine was observed from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concentration and structure of the metabolic precursor administered to cells, and it was linearly time-dependent. The radioactive lipid patterns present in the medium were different from those present in the cells. Radioactive sphingomyelin and ganglioside GM3 containing short acyl chains were the main species present in the medium from human fibroblasts, while sphingomyelin and GD3 ganglioside in that from neuronal cells. In the absence of proteins in the culture medium, the efflux of complex lipids was much lower than in the presence of serum, and the patterns of released molecules were again different from those of cells. This work was supported by COFIN-PRIN, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy.