Neuronal Uptake Sites Research Articles

Differences in uptake kinetics of cis-3-aminocyclohexane carboxylic acid into neurons and astrocytes in primary cultures

The uptake of [ 3H]ACHC and [ 3H]GABA into cultured neurons and astrocytes was studied. [ 3H]ACHC uptake was less efficient than that of GABA in both cell types and K m values for ACHC uptake into neurons and astrocytes were 40.3 μM and 210.8 μM, respectively. The corresponding V max values were 0.321 and 0.405 nmol·min −1·mg −1 cell protein, respectively. Kinetic studies of the effects of GABA on ACHC uptake and vice versa showed that GABA is a linear competitive inhibitor of ACHC uptake in both cell types with a K i value of 15 μM. On the other hand, ACHC turned out to be a complex inhibitor of astrocytic GABA uptake being competitive at lower concentrations and non-competitive at higher concentrations. ACHC inhibited GABA uptake into neurons competitively with a K i of 69 μM. It is concluded that ACHC acts primarily on neuronal GABA uptake sites but its uptake is much more complicated than hitherto anticipated.

Characterization of [3H]desipramine binding associated with neuronal norepinephrine uptake sites in rat brain membranes

A variety of evidence indicates that [3H]desipramine can label neuronal norepinephrine uptake sites in brain membranes. Pretreatment of rat cerebral cortical membranes with 0.3 M KCl increases the ratio of high affinity to low affinity saturable [3H]desipramine binding. With this improved tissue preparation, we have confirmed our earlier observation that the high affinity [3H]desipramine binding component (KD = 2 to 4 nM) is associated with norepinephrine neuronal uptake sites. The potencies of various antidepressant drugs in reducing [3H]desipramine binding correlate with their inhibition of neuronal [3H]norepinephrine accumulation. Like the norepinephrine uptake system, high affinity [3H]desipramine binding is dependent both on sodium and chloride, with half-maximal stimulation by 10 mM chloride. Although bromide can substitute for chloride to stimulate binding, other anions, including iodide, fluoride, acetate, citrate, and phosphate, are inactive. Comparable sodium and anion regulation of [3H]imipramine binding to serotonin uptake recognition sites also is observed. The association of [3H]desipramine binding sites with neuronal norepinephrine uptake sites is supported further by the selective abolition of high affinity [3H]desipramine binding following the destruction of central norepinephrine neurons by intraperitoneal administration of DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine). In vitro incubation of cerebral cortical membranes with DSP-4 also selectively abolishes the high affinity [3H]desipramine binding, an effect which cannot be reversed by repeated washing of the membranes, suggesting that DSP-4 alkylates neuronal norepinephrine uptake sites.

Depressed patients have decreased binding of tritiated imipramine to platelet serotonin "transporter".

The high-affinity tritiated (3H) imipramine binding sites are functionally (and perhaps structurally) associated with the presynaptic neuronal and platelet uptake sites for serotonin. Since there is an excellent correlation between the relative potencies of a series of antidepressants in displacing 3H-imipramine from binding sites in human brain and platelet, we have examined the binding of 3H-imipramine to platelets from 14 depressed patients and 28 age- and sex-matched controls. A highly significant decrease in the number of 3H-imipramine binding sites, with no significant change in the apparent affinity constants, was observed in platelets from the depressed patients compared with the controls. These results, coupled with previous studies showing a significant decrease in the maximal uptake of serotonin in platelets from depressed patients, suggest that an inherited or acquired deficiency of the serotonin transport protein or proteins may be involved in the pathogenesis of depression.

Inhibition of neuronal GABA uptake and glial β-alanine uptake by synthetic GABA analogues

Thirty three synthetic analogues of GABA were tested for their ability to act as inhibitors of neuronal or glial uptake sites for GABA and β-alanine, using [ 3H]GABA uptake by synaptosome preparations from rat cerebral cortex, and [ 3H]β-alanine uptake by cortical slices as test systems for neuronal and glial uptake sites, respectively. The results confirm that both uptake processes favour a substantially folded conformation. Neuronal uptake sites were significantly inhibited by analogues with long alkyl side chain substituents, such as dodecyl GABA-amide which was of comparable potency to GABA.

Changes in the uptake of GABA and taurine during neuronal and glial maturation.

Abstract: The influence of the time of culture on GABA and taurine uptake was investigated in spontaneously matured cultures of glial and neuronal origins and in cultures treated with cyclic nucleotides. In the spontaneously matured cultures the capacity of the high‐affinity neuronal GABA transport system increased with time in culture. Essentially opposite results were found for the uptake of GABA by glial cultures. In contrast with the neuronal uptake of GABA, the capacity of the taurine transport system was significantly decreased. Uptake of taurine into glia, however, exhibited a progressive increase with the period of culture. The values of Km, for the high‐affinity systems were always found to range around 10 μM. It is suggested that, in mature cells, neuronal uptake sites are of prime importance for GABA transport, while taurine uptake may be more specifically directed towards glial cells. When cultures were treated with cyclic nucleotide derivatives, a morphological differentiation was induced, which could not be linked to a stimulation of GABA or taurine uptake systems as compared with the non‐treated cultures.

GABA receptor binding with 3H (+) bicuculline–methiodide in rat CNS

NEUROTRANSMITTER receptors might be identified biochemically in binding studies with synaptic membrane preparations using labelled receptor agonists or antagonists as ligands1. In attempts to identify the GABA receptor 3H-GABA has been used as ligand. 3H-GABA, however, also binds to glial and neuronal GABA uptake sites and to enzymes metabolising GABA. Thus, Na+-dependent 3H-GABA-binding2,3, in contrast to Na+-independent 3H-GABA-binding is possibly not related to the GABA receptor4. In the present investigation the GABA antagonist 3H( + )bicuculline–methiodide (3H( + )BM) was used as ligand, which in contrast to bicuculline, is chemically sufficiently stable5. ( + )Bicuculline and its N-methyl-derivative antagonise reversibly and rather selectively the inhibitory synaptic action of GABA in the central nervous system (CNS) (refs 6, 7) without affecting the uptake or release of GABA or enzymes metabolising GABA (refs 8, 9). Thus 3H( + )BM might be more suitable for GABA receptor binding studies than GABA itself.

Control of hindlimb vascular resistance in rats chronically sympathectomized with 6-hydroxydopamine.

1. Vascular resistance was measured after acute surgical denervation in the blood-perfused hind limbs of adult normal rats and rats which had been treated with 6-hydroxydopamine in the first 2 weeks of postnatal life. Vascular resistance was significantly higher in the limbs of 6-hydroxydopamine treated rats. 2. No excess of vasoconstrictor materials was detected when blood from 6-hydroxydopamine treated rats was used to perfuse the hind limbs of untreated rats or other 6-hydroxydopamine treated rats. 3. Hind limb blood vessels of 6-hydroxydopamine treated rats showed increased sensitivity to normal amounts of circulating vasoconstrictors of adrenal origin and to intra-arterial infusions of noradrenaline and angiotensin. 4. Infusion of desipramine into the hindlimb blood vessels of 6-hydroxydopamine treated rats did not increase vasoconstrictor responses to infused noradrenaline. Infusion of desipramine into the limbs of untreated rats increased responses to noradrenaline but these never attained the magnitude and duration of responses seen in limbs of 6-hydroxydopamine treated rats. 5. The increased sensitivity to noradrenaline of hindlimb vessels from 6-hydroxydopamine treated rats could partly be accounted for by an absence of neuronal uptake sites and partly by the development of non-specific post-junctional supersensitivity.

γ-Aminobutyric Acid in the Rat Peripheral Nervous System, Pineal and Posterior Pituitary

activity accumulated was determined by scintillation spectrometry. To localize the cellular sites of uptake of y-[3H]aminobutyric acid individual organs were incubated with 5p~-y-[~H]aminobutyric acid, fixed in 5 % glutaraldehyde solution in the original incubation medium and prepared for light and electron-microscopic radioautography. In all four tissues accumulations of silver grains were observed exclusively over glial cell bodies and their processes, in contrast with the neuronal components which were completely devoid of label. The accumulation of y-[3H]-aminobutyric acid observed in biochemical experiments was greatly enhanced by the addition of amino-oxyacetic acid (10~~) to the incubation medium. Since at this concentration amino-oxyacetic acid potently blocks y-aminobutyrate-2-oxoglutarate aminotransferase without affecting y-aminobutyric acid uptake in brain slices (Snodgrass & Iversen, 1973), glial-rich tissues probably metabolize exogenously accumulated y-aminobutyric acid more rapidly than neuronal uptake sites in brain. The endogenous y-aminobutyric acid contents of the tissues were determined by using a radiochemical microassay similar to that described by Snodgrass & Iversen (1972) in which y-aminobutyric acid was measured as it radioactively labelled dansyl derivative. The activities of the enzymes E-glutamate 1-carboxy-lyase and y-aminobutyrate-2oxoglutarate aminotransferase were determined by sensitive radiochemical procedures, measuring the 14C02 evolved (Roberts & Simonsen, 1963) and the formation of labelled succinate from y-[14C]aminobutyric acid (Beart et al., 1972) respectively. All of the tissues examined contained significant amounts of endogenous y-aminobutyric acid, and L-glutamate 1-carboxy-lyase and y-aminobutyrate-2-oxoglutarate aminotransferase activities (Table 1). Further, the concentrations of endogenous y-aminobutyric acid were surprisingly high when compared with those in brain and spinal cord. In general the relative proportions of y-aminobutyric acid, L-glutamate 1-carboxy-lyase and

Inhibition of the uptake of 5-hydroxytryptamine, noradrenaline and dopamine into rat brain homogenates by various hydroxylated tryptamines.

AbstractRecent work has shown that intracerebral injections of 5,6‐dihydroxytryptamine (5,6‐DHT) lead to a fairly selective and long lasting depletion of 5‐HT in the rat CNS (BAUMGARTEN, BJORKLUND, LACHENMAYER, NOBIN and STENEVI, 1971; DALY, FUXE and JONSSON, 1973). This effect appears to result from a degeneration of the serotonin‐containing neurons (BAUMGARTEN and LACHENMAYER, 1972a). 5,6‐DHT does, however, to a lesser extent affect both NA and dopamine (DA) containing nerve terminals (BAUMGARTEN et al., 1971). In an attempt, therefore, to find compounds having a more specific toxic action we have investigated several other hydroxylated tryptamines. In order to obtain information about the differential affinities of these analogues for neuronal uptake sites we have examined their effects on the uptake of [3H]5‐HT and (±)‐[3H]NA into synaptosomes in homogenates of rat hypothalamus and of [3H]DA uptake into a similar preparation from the rat corpus striatum. It is known that the uptake of these putative transmitters in rat brain homogenates is predominantly into the synaptosome fraction (KANNENGIESSER, HUNT and RAYNAUD, 1973; COYLE and SNYDER, 1969).

Structure-activity relations for the inhibition of catecholamine uptake into synaptosomes from noradrenaline and dopaminergic neurones in rat brain homogenates.

1. The effects of various phenylethylamine analogues on the inhibition of (3)H-noradrenaline and (3)H-dopamine uptake into homogenates of rat hypothalamus and corpus striatum respectively, were examined.2. Phenolic hydroxyl groups and alpha-methylation of the side chain were both found to enhance the affinity for the neuronal uptake sites.3. Methoxylation, beta-hydroxylation and N-methylation were all found to reduce the ability of a compound to inhibit catecholamine transport.4. The noradrenaline and dopamine transport systems responded in a quantitatively different manner to the various phenylethylamine analogues. It was found that, in general, the noradrenaline uptake process was more sensitive to structural changes, both positive and negative, than the dopamine system.

Coupled transport of sodium and organic solutes.

Coupled transport of sodium and organic solutes.