Quantitative studies of norepinephrine uptake by synaptosomes
Abstract
Nerve-ending particles (synaptosomes) isolated from rat brain were studied as a model system for synaptic function with particular reference to the ionic conditions required for uptake of norepinephrine. For optimal uptake, Na+ and K+ were required at levels of 140 mM and 2–3 mM respectively, which are essentially physiological levels for these cations. The inhibition of norepinephrine uptake by reserpine and by higher concentrations of K+ was linear with the logarithm of the inhibitor concentration. The Vmax and Km for norepinephrine uptake were 0·10 μg/min/g and 0·56 × 10−6 M respectively.
References (18)
- S.H. Snyder et al.
Life Sci.
(1965) - E.W. Maynert et al.
Life Sci.
(1964) - D.F. Bogdanski et al.
Life Sci.
(1966) - C.O. Hebb et al.
J. Physiol., Lond.
(1958) - V.P. Whittaker et al.
Biochem. J.
(1964) - E. De Robertis et al.
J. Neurochem.
(1962) - R.W. Colburn et al.
Nature, Lond.
(1967) - A.F. Huxley et al.
J. Physiol. Lond.
(1951) - R.J. Baldessarini et al.
Science
(1966)
Cited by (83)
Characterization of norepinephrine accumulation by a crude synaptosomal-mitochondrial fraction isolated from rat heart
1991, Life SciencesNorepinephrine (NE) uptake into a heart synaptosomal-mitochondrial fraction was assessed under conditions where neuronal uptake (type 1) was linear with respect to both time and protein concentration. The NE accumulation process was sensitive to incubation temperature, sodium ion concentration and medium osmolality. Furthermore, NE uptake was attenuated by the neuronal uptake inhibitor desmethylimipramine (DMI) in a concentration dependent manner; the IC50 value was approximately 10 nM and maximum inhibition was obtained at 100 nM. In contrast, the extraneuronal uptake inhibitor, metanephrine did not significantly attenuate NE uptake. Kinetic analysis demonstrated that the DMI sensitive NE accumulation is saturable with a KM of approximately 400 nM and that NE uptake occurs via a single uptake process. This demonstration of neuronal type NE uptake by a synaptosomal-mitochondrial fraction constitutes a successful demonstration of the preparation of a rat heart subcellular fraction containing functional synaptosomes.
Abnormal norepinephrine uptake and release in brain synaptosomes in chronic renal failure
1989, Kidney InternationalAbnormal norepinephrine uptake and release in brain synaptosomes in chronic renal failure. Abnormalities in the function of the central nervous system exist in chronic renal failure (CRF) and some of these derangements may be related to excess parathyroid hormone (PTH) which causes a rise in brain calcium. The latter may affect metabolism of neurotransmitters such as norepinephrine (NE) in brain synaptosomes. We measured NE content, uptake and release in brain synaptosomes of CRF rats and studied whether excess PTH affects these parameters. Synaptosomes from rats with 21 days of CRF compared to those from normal animals have higher calcium content (11.4 ± 0.92 vs. 7.1 ± 0.50 nmol/mg protein, P < 0.01) and lower Na-K ATPase activity (6.5 ± 0.81 vs. 11.4 ± 0.76 µmol Pi/mg protein/hr, P < 0.01). NE content (11.0 ± 0.60 vs. 13.6 ± 0.55 pmol/mg protein/hr, P < 0.01), uptake (46 ± 4.5 vs. 110 ± 5.9 pmol/mg protein times 50 min, P < 0.01) and release (2.0 ± 0.2 vs. 5.1 ± 0.47 pmol/mg protein times 10 min, P < 0.01). Parathyroidectomy (PTX) in CRF rats kept normocalcemic reversed these abnormalities in brain synaptosomes; indeed calcium content, Na-K ATPase activity and NE content, uptake and release in synaptosomes from PTX-CRF rats were not different from those seen in normal rats. Administration of 1-84 PTH for 21 days produced abnormalities in brain synaptosomes similar to those seen in CRF rats; the synaptosomes from PTH-treated animals had significantly (P < 0.01) higher calcium content (10.0 ± 0.80 nmol/mg protein) and lower Na-K ATPase activity (8.3 ± 0.58 µmol Pi/mg protein/hr), and NE content (8.8 ± 0.51 pmol/mg protein), uptake (87 ± 4.0 pmol/mg protein times 50 min) and release (2.3 ± 0.27 pmol/mg protein times 10 min). However, in PTH-treated animals the initial rate of NE uptake was not different from normal but the steady state rate was reduced. In contrast, both the initial and steady state NE uptake were significantly reduced in CRF control rats. Despite this greater effect of CRF on NE uptake, the decrease in NE content in the synaptosomes of CRF control rats was significantly less than that in PTH-treated animals. The data show that abnormalities in the function of synaptosomes occur in CRF and they are most likely due in large part to excess PTH. The latter may act directly and/or mediate its effect through accumulation of calcium in the synaptosomes. Other factors associated with CRF or administration of PTH may also contribute to some of these abnormalities in synaptosomes function.
Vesiculated fragments of presynaptic plasma membranes have been isolated from the purely cholinergic electromotor nerve terminals of Torpedo marmorata. Synaptosomes, generated from the terminals by homogenization, were separated on a discontinuous Ficoll gradient and then lysed by osmotic shock at 2°C, pH 8.5 in the presence of 0.1 mM MgCl2. These conditions for lysis were optimal for choline transport. Electron micrographs of lysed synaptosomes showed vesiculated membranes with diameters smaller than those of synaptosomes; occasionally, synaptic vesicles were observed attached to them. Intact mitochondria or synaptosomes and basal laminae were not present. High-affinity (KT = 1.7 μM) uptake of choline into these vesiculated membrane fragments showed: (1) an absolute dependence on the Na+ gradient (outside > inside), (2) a transient Na+-gradient-dependent accumulation of choline over the equilibrium concentration (overshoot), (3) electrogenicity and rheogenicity, since the uptake was further stimulated in the presence of a Na+ gradient by valinomycin, (4) dependence on the presence of external Cl−, and partial dependence on a Cl− gradient (outside > inside), (5) high-affinity (Ki = 25 nM) inhibition by hemicholinium-3 and (6) temperature sensitivity. The plasma membranes were further purified by centrifugal density gradient fractionation on a 4–12% Ficoll gradient. Several enzymes and polypeptides copurified with the specific binding sites for choline present in the membranes. The fraction with the most binding sites was one denser than 12% Ficoll. This was also the fraction richest in acetylcholinesterase, 5′-nucleotidase and polypeptides of relative molecular mass, Mr (×10−3) of > 200, 140, 68 (doublet), 57, 54 and 28. Acetylcholinesterase was positively identified as a Mr 68 000 component by immune blot. By contrast the ouabain-sensitive ATPase showed a negative correlation with choline binding sites. When the solubilized proteins of the vesiculated membranes were transferred to liposomes, they conferred on the latter the capacity to take up choline in a manner closely resembling its transport in natural membranes but with an initial (one minute) rate of uptake approximately 10-times greater per mg of protein. Several proteins were selectively transferred to the liposomes including ones of Mr (×10−3) 34, 42, 47, 54, 60, 68, 92, 160 and > 200. The polypeptides of Mr (×10−3) 140, 57 and 28 were lost in the transfer. When a correlation was made between transport rates observed in the various liposome preparations tried and the presence of particular polypeptides, only three proteins correlated with the ability of liposomes to transport choline: the Mr (×10−3) > 200, 54 and 34 components.
Divalent cation, ATP-dependent [<sup>3</sup>H]Leu-enkephalin uptake by synaptic vesicle fraction isolated from bovine caudate nucleus
1982, Brain ResearchThe 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 apparentKm 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.
Glutamate, aspartate, and γ-aminobutyrate transport by membrane vesicles prepared from rat brain
1981, Archives of Biochemistry and BiophysicsTo prepare membrane vesicles, nerve terminal preparations (synaptosomes) isolated from rat cerebral cortex were first subjected to hypotonic lysis. After collecting the membranes contained in this fraction by centrifugation, membrane vesicles were then reconstituted during incubation in a potassium salt solution at 37 °C. The transport of glutamate, aspartate, or γ-aminobutyric acid (GABA) was measured by transferring vesicles to 10 vol of 0.1 m NaCl solution containing the radioactive substrate. Transport was temperature dependent and exhibited saturation kinetics with an apparent Km of 2.5 μm. The rates and extent of l-glutamate and l-aspartate uptake were equivalent and were greater than those for GABA. Valinomycin increased the rate of uptake of each of these substances suggesting a role for an electrogenic component in transport. Consonant with this notion, external K+ and Rb+ decreased uptake of all three compounds. External thiocyanate also increases the rate of glutamate, aspartate, and GABA transport. Uptake of these neuroactive amino acids was absolutely dependent on external Na+; no other monovalent cation tested substitutes for it. Gramicidin D and nigericin inhibit glutamate transport by abolishing both the Na+ and K+ gradients. Monensin inhibits uptake by selectively dissipating the Na+ gradient. For both glutamate and GABA transport, the Na+ and K+ gradients are synergistic and not additive.
Phenylacetate and brain dysfunction in experimental phenylketonuria: Synaptic development
1980, Life SciencesHigh affinity uptake of choline, GABA, norepinephrine and serotonin into synaptosomes and ganglioside content of cortices served as indices of synaptic development. Both parameters indicated that phenylacetate contributed to the retarded maturation of synapses in the cerebrum of the adult rat, previously treated with either phenylacetate or one of its precursors, phenylalanine (with p-CPA) and phenylethylamine, during the first 21 days of life. In these groups of rats, which also exhibited a pronounced deficit in learning capacity, the velocity of high affinity synaptosomal uptake of choline was reduced to a greater extent than that of GABA, and there was a profound decrease in the ganglioside content of cerebral cortex. In contrast, synaptic maturation and behavior of control and phenylpyruvate treated animals were similar. These findings lend strong support to our contention that phenylacetate, produced in excessive amounts in PKU, is most likely a primary cause of the mental retardation.