Nucleoside triphosphate and cation requirement for dopamine uptake by plain synaptic vesicles isolated from rat cerebrums

doi:10.1016/0006-8993(76)90512-6

Brain Research

Volume 115, Issue 2, 15 October 1976, Pages 273-283

https://doi.org/10.1016/0006-8993(76)90512-6 Get rights and content

Summary

Plain synaptic vesicles were partially purified from rat cerebrums and their [³H]dopamine uptake was investigated. The addition of MgCl₂ plus ATP and of CaCl₂ plus ATP increased the uptake 16 times and 5.5 times, respectively, higher than the control level, whereas the separate addition of these agents augmented the level at most 2.5 times. GTP stimulated the uptake as well as ATP, whereas UTP was one-third as effective and CTP, ADP, and AMP were all ineffective. Adenylyl imidodiphosphate was not only ineffective, but strongly reduced the ATP-dependent uptake. The half-maximal level of the ATP-dependent uptake was reached within 1.5 min after the start of incubation at 25°C. The incorporation peaked at 5–10 min, then gradually declined to half-maximum at 45 min. This decline was prevented by the further addition of small amounts of CaCl₂. There was no uptake at 0°C, and the incorporation rate was at least 2 times faster at 37°C than it was at 25°C. The apparent Michaelis constant for [³H]dopamine was 1.6 μM. The half maximal inhibition of uptake was obtained at 0.1 μM reserpine; neither colchicine nor ouabain showed significant inhibition. The ATP-dependent uptake was not affected by K⁺, Na⁺, and Cl⁻, but was drastically decreased by isotonic phosphate buffer.

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Cited by (31)

Measurement of kinetically resolved vesicular dopamine uptake and efflux using rotating disk electrode voltammetry
2006, Journal of Neuroscience Methods
The vesicular monoamine transporter-2 (VMAT-2) sequesters cytoplasmic dopamine (DA) into vesicles for storage and subsequent release. VMAT-2 activity has traditionally been measured in small synaptic vesicles isolated from rat striatum by monitoring [³H] DA uptake and in cellular expression systems using fast scan cyclic voltammetry. This is the first report using rotating disk electrode (RDE) voltammetry to measure VMAT-2 DA uptake and efflux in small synaptic vesicles. DA uptake profiles followed mixed order kinetics with apparent zero order kinetics for the first 25 s and apparent first order kinetics thereafter. Vesicular DA uptake was temperature- and ATP-dependent and was blocked by the VMAT-2 inhibitor tetrabenazine. Initial velocities of DA uptake were kinetically resolved and displayed Michaelis–Menten kinetics with a K_m and V_max of 289 ± 59 nM and 1.9 ± 0.2 fmol/(s μg protein), respectively. Methamphetamine-induced DA efflux was blocked by tetrabenazine and kinetically resolved with an initial velocity of 0.54 ± 0.08 fmol/(s μg protein). These results suggest that RDE voltammetry can be used to make kinetically resolved measurements of vesicular DA uptake and efflux and will allow the design of experiments that could reveal important information about the kinetics of VMAT-2 activity and its inhibition.
Chloride ion increases [3H]dopamine accumulation by synaptic vesicles purified from rat striatum: inhibition by thiocyanate ion
1990, Brain Research
The effect of chloride ion on transport of [³H]dopamine into synaptic vesicles purified from rat striatum has been evaluated. The inclusion of 10 mM chloride ion in the incubation medium produced a 100% increase in temperature-sensitive [³H]dopamine uptake into synaptic vesicles from approximately 1800 pmol/mg to 3600 pmol/mg of protein. Half-maximal effects were observed with chloride ion at 4 mM concentration. The anion selectivity of stimulation supports the presence of anion channels within the membranes of dopaminergic storage organelles. Low concentrations of thiocyanate ion (<10 mM), 4-acetamido-4′-isothiocyano2-2′-disulfonic acid stilbene (100 μM), and duramycin (5 μg/ml) selectively blocked the chloride ion stimulated accumulation of [³H]dopamine. Higher concentrations of these agents are required to significantly reduce [³H]dopamine uptake in the absence of chloride ion. These results suggest that both components of the proton electrochemical gradient (ΔΨ and δpH) are important for dopamine uptake by brain vesicles. This article presents the first demonstration that chloride ion plays a role in the transport of dopamine into vesicles isolated from the CNS.
3H-Dopamine accumulation by rat brain synaptic vesicles in a membrane-impermeable medium
1983, Life Sciences
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 × 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°), 11.9 ± 2.5 (4°), 31.3 ± 1.8 (absence of ATP), 26.3 ± 2.7 (reserpine, 10⁻⁶M), 26.1 ± 0.67 (no ATP + reserpine 10⁻⁶), and 14.6 ± 2.4 (carbonylcyanide-p-triflouromethoxyphenylhydrazone, FCCP, 10⁻⁶M). Depletion of endogenous DA levels by pretreatment of the animals with α-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 (10⁻⁶M). 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.
Enzymatic activities of coated vesicle fractions from bovine and rat cerebrums
1983, Neurochemistry International
The changes in the enzymatic properties of coated vesicle fractions obtained from bovine cerebrums and rat forebrains were investigated as the preparation procedures were modified. Because Mg²⁺-dependent ATPase activity in the coated vesicle fractions that were prepared by conventional centrifugation methods appeared to derive from contaminating particulates, the activity was examined after further purification by column chromatography and confirmed by histochemical technique. Both the coat proteins and the vesicles enclosed in the coat networks failed to show the activity. Since plain synaptic vesicles are known to have ATPase activities, the results may indicate that the membrane structure of synaptic vesicles is modified between the coated vesicle stage and the plain vesicle stage of vesicle recycling as Heuser and Reese proposed (J. Cell Biol.57, 315–344, 1973). The comparison of the specific activity change in ATPase with those of acetylcholinesterase and NADPH-cytochrome c reductase suggested that there were two types of microsomes contaminating coated vesicle fractions that were prepared conventionally.
Divalent cation, ATP-dependent [3H]Leu-enkephalin uptake by synaptic vesicle fraction isolated from bovine caudate nucleus
1982, Brain Research
The mechanism of [³H]Leu-enkephalin uptake into synaptic vesicle fraction from bovine caudate nucleus was investigated. The simultaneous addition of 2 mM MgCl₂, 2 mM CaCl₂, and 2 mM ATP stimulated the uptake 22 times over the control, whereas the separate addition of these agents augmented the uptake 2.4 times at most. The addition of 2 mM MgCl₂ plus 2 mM ATP and of 2 mM CaCl₂ plus 2 mM ATP increased the uptake 6.6 times and 4.5 times, respectively. The cation, ATP-dependent uptake reached its half-maximal level within 10 min after the initiation of incubation at 25 °C, and little Leu-enkephalin was taken up at 0 °C. The apparentK_m for the uptake of [³H]Leu-enkephalin was1.8 × 10⁻⁷M. Guanosine triphosphate stimulated the uptake as well as ATP, whereas CTP and ITP were only one-fourth effective of ATP. The cation, ATP-dependent uptake was inhibited by 25% and 20% in the presence of 0.1 mM colchicine and 1 μM reserpine, respectively.
pH Dependence of [3H]norepinephrine uptake into catecholamine storage vesicles isolated from rat brain, heart and adrenal medulla
1981, Biochemical Pharmacology

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Summary

References (33)

Succinate dehydrogenase. I. Role of phospholipids

Biochim. biophys. Acta (Amst.)

Quantitative studies of norepinephrine uptake by synaptosomes

Biochem. Pharmacol.

Uptake of γ-amino butyric acid by mitochondrial and synaptosomal fractions from mouse brain

Brain Research

Protein measurement with the Folin phenol reagent

J. biol. Chem.

A low viscosity epoxy resin embedding medium for electron microscopy

J. Ultrastruct. Res.

Glucose-6-phosphatase from liver

Role of phospholipid in the activation of Na⁺, K⁺-activated adenosine triphosphatase of beef brain

Arch. Biochem. Biophys.

Monoamine oxidase (pig brain mitochondria)

Uptake and release of norepinephrine by rat brain tissue fractions prepared by ultrafiltration

J. Neurochem.

Catecholamine uptake by synaptosomes in homogenates of rat brain: stereospecificity of different area

J. Pharmacol. exp. Ther.

How do neurons secrete peptides? Exocytosis and its consequences, including “synaptic vesicle” formation, in hypothalamo-neurohypophyseal system

Chromaffin granules: effects of ions and ATP on catecholamine content, ATPase activity, and membrane phosphorylation

Experientia (Basel)

Evidences for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction

J. Cell Biol.

Regulation of uptake and release in isolated chromaffin granules

Trans. Amer. Soc. Neurochem.

GABA uptake in rat central nervous system: comparison of uptake in slices and homogenates and the effects of some inhibitors

J. Neurochem.

Isolation of coated vesicles, plain synaptic vesicles, and flocculent material from a crude synaptosome fraction of guinea pig whole brain

J. Cell Biol.

Cited by (31)

Measurement of kinetically resolved vesicular dopamine uptake and efflux using rotating disk electrode voltammetry

Chloride ion increases [<sup>3</sup>H]dopamine accumulation by synaptic vesicles purified from rat striatum: inhibition by thiocyanate ion

<sup>3</sup>H-Dopamine accumulation by rat brain synaptic vesicles in a membrane-impermeable medium

Enzymatic activities of coated vesicle fractions from bovine and rat cerebrums

Divalent cation, ATP-dependent [<sup>3</sup>H]Leu-enkephalin uptake by synaptic vesicle fraction isolated from bovine caudate nucleus

pH Dependence of [<sup>3</sup>H]norepinephrine uptake into catecholamine storage vesicles isolated from rat brain, heart and adrenal medulla