Specific Glycine-Accumulating Synaptosomes in the Spinal Cord of Rats

Arregui, Alberto; Logan, William J.; Bennett, James P.; Snyder, Solomon H.

doi:10.1073/pnas.69.11.3485

“…This uneven distribution of the glycine uptake system is in accord with the high concentration of glycine (2), glycine receptors (35), and the Na+-dependent high-affinity glycine uptake system (37,38) in the spinal cord. Moreover, the differential distribution of these vesicular uptake systems is consistent with the observations that GABA, glycine, and glutamate are accumulated in different populations of nerve endings (9,10,39 …”

supporting

confidence: 73%

Active transport of gamma-aminobutyric acid and glycine into synaptic vesicles.

Kish

¹

,

Fischer-Bovenkerk

²

,

Ueda

³

1989

Proc. Natl. Acad. Sci. U.S.A.

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Although y-aminobutyric acid (GABA) and glycine are recognized as major amino acid inhibitory neurotransmitters in the central nervous system, their storage is poorly understood. In this study we have characterized vesicular GABA and glycine uptakes in the cerebrum and spinal cord, respectively. We present evidence that GABA and glycine are each taken up into isolated synaptic vesicles in an ATPdependent manner and that the uptake is driven by an electrochemical proton gradient. Uptake for both amino acids exhibited kinetics with low affinity (Km in the millimolar range) similar to vesicular glutamate uptake. The ATP-dependent GABA uptake was not inhibited by the putative amino acid neurotransmitters glycine, taurine, glutamate, or aspartate or by GABA analogs, agonists, and antagonists. Similarly, ATPdependent glycine uptake was hardly affected by GABA, taurine, glutamate, or aspartate or by glycine analogs or antagonists. The GABA uptake was not affected by chloride, which is in contrast to the uptake of the excitatory neurotransmitter glutamate, whereas the glycine uptake was slightly stimulated by low concentrations of chloride. Tissue distribution studies indicate that the vesicular uptake systems for GABA, glycine, and glutamate are distributed in different proportions in the cerebrum and spinal cord. These results suggest that the vesicular uptake systems for GABA, glycine, and glutamate are distinct from each other. y-Aminobutyric acid (GABA) and glycine are the major inhibitory neurotransmitters in the vertebrate central nervous system (1, 2). Recently, the primary structures of GABAA and glycine receptors have been deduced; the subunits of these receptors have been shown to have substantial sequence homology, particularly in the region thought to be involved in conducting chloride (3). GABA and glycine are released upon membrane depolarization, both in a calcium-dependent manner (4-6) and in a calciumindependent manner (4, 7). Recent evidence indicates that the calcium-dependent release of GABA originates from a noncytoplasmic compartment (8). There are also observations indicating that GABA and glycine are concentrated in distinct nerve terminals (9, 10). However, localization of endogenous amino acids in synaptic vesicles has not been clearly demonstrated, either with intact tissues or isolated vesicle preparations. In addition, there has been little study on the vesicular GABA and glycine uptake processes. We have previously provided evidence that glutamate is taken up into synaptic vesicles by a proton-motive force generated by a proton-pump ATPase in the vesicle (11-13). In this communication, we have studied vesicular GABA and glycine uptake, using a synaptic vesicle preparation different from that previously used for vesicular glutamate uptake. We provide evidence that suggests that GABA and glycine are also accumulated into synaptic vesicles in an ATP-dependent manner and that their uptake is driven by an electrochemical proton gradient. We have characterized these uptake systems with r...

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“…(1,6). Endogenous glycine probably suffices to explain the inhibition of strychnine binding in undialyzed whole homogenate and P2 fraction.…”

Section: Methodsmentioning

confidence: 99%

“…At 40 we examined the rate of dissociation using either 0.1 mM strychnine or 1.0 mM glycine to displace [3H]strychnine (Fig. 4) lates well with endogenous glycine, high-affinity synaptosomal glycine uptake into unique glycine-accumulating synaptosomes (4)(5)(6), and the ability of glycine to mimic natural inhibitory transmission (2).…”

Section: Subcellulkrmentioning

confidence: 99%

See 1 more Smart Citation

Strychnine Binding Associated with Glycine Receptors of the Central Nervous System

Young

¹

,

Snyder

²

1973

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACT['HIStrychnine binds to synaptic-membrane fractions of the spinal cord in a selective fashion, indicating an interaction with postsynaptic glycine receptors. Displacement of strychnine-by glycine and other amino acids parallels their glycine-like neurophysiologic activity. The regional localizition of strychnine binding in the central nervous system correlates closely with endogenous glycine concentrations. In subcellular fractionation experiments, strychnine binding is most enhanced in synaptic-membrane fractions. Strychnine binding is saturable, with affinity constants for glycine and strychnine of 10 and 0.03 MM, respectively.Biochemical (1) and neurophysiological (2) evidence suggests that glycine is a major inhibitory neurotransmitter in the mammalian central nervous system. Strychnine antagonizes the hyperpolarizing actions of glycine at spinal synapses, where it also antagonizes naturally occurring synaptic inhibition (3). Glycine best mimics a natural inhibitory transmitter in the spinal cord and brain stem, but not in the cerebral cortex (2). Similarly, endogenous glycine (4), highaffinity synaptosomal uptake of glycine (5), and unique glycine-accumulating synaptosomes (6) are most concentrated in the spinal cord and brain stem and not in the cerebral cortex.Here we report the binding of [3H]strychnine to membrane fractions of the spinal cord and brain stem, which appears to represent a specific interaction with the postsynaptic glycine receptor. METHODSStrychnine Was Labeled by catalytic tritium exchange at New England Nuclear Corp. 50 mg of strychnine sulfate was dissolved in 0.3 ml of trifluoroacetic acid, and to this was added 50 mg of 5% Rh/A1203 and 25 Ci of tritiated H20.The reaction mixture was stirred overnight at 800 and labile tritium was removed under reduced pressure with methanol as a solvent. After filtration from the solvent, the product was dissolved in 10 ml of methanol. In our laboratory, the product was purified by thin-layer chromatography on Silica Gel F-254 plates, 0.25 mm thickness (EM Laboratories, Inc., Elmsford, N.Y.), in three consecutive solvent systems (nbutanol-glacial acetic acid-H20 4:1:1, chloroform-methanol 1: 1, and methanol-H20 7:3). Purified Tissue Preparation. Male Sprague-Dawley rats (100-150 g) were decapitated and the brains and spinal cords were rapidly removed. For typical binding assays, the meninges were carefully removed and the medulla oblongata-pons and spinal cord were combined and homogenized in 20 volumes of ice-cold 0.32 M sucrose in a Potter-Elvehjem glass homogenizer fitted with a Teflon pestle. The whole homogenate was centrifuged for 10 min at 1000 X g. The pellet (crude nuclear fraction) was discarded, and the resultant supernatant fluid was centrifuged for 20 min at 17,000 X g. The pellet (crude mitochondrial fraction), after resuspension in 20 volumes of ice-cold distilled H20, was homogenized with a ground-glass homogenizer and centrifuged for 20 min at 9000 X g. The supernatant fluid was collected and the pellet, a bilayer with a so...

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“…

On the basis of neurophysiological and biochemical studies, it has been suggested that the amino acid, glycine, is a major inhibitory transmitter in the mammalian spinal cord (I, 23). The evidence in support of a role for glycine as a neurotransmitter includes the demonstration that glycine (a) mimics the action of naturally occurring inhibitory transmitter when it is iontophoretically applied to motor neurons (5, 6, 24), (b) is released after dorsal root stimulation (12), (c) is reduced in concentration when inhibitory interneurons are selectively destroyed (7), and (d) is taken up into brain slices and a distinct population of synaptosomes by a glycine-specific high-affinity transport system (2,16,17,22). This uptake into specific synaptosomes can be demonstrated only in those regions of the central nervous system in which glycine is iontophoretically active and present in high concentration.Electron microscope autoradiography has been used in attempts to identify synaptic terminals possessing high affinity uptake systems for glycine (10,11).

…”

mentioning

confidence: 99%

Glycine-specific synapses in rat spinal cord. Identification by electron microscope autoradiography.

Price¹,

Stocks

²

,

Griffin

³

et al. 1976

The Journal of Cell Biology

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On the basis of neurophysiological and biochemical studies, it has been suggested that the amino acid, glycine, is a major inhibitory transmitter in the mammalian spinal cord (I, 23). The evidence in support of a role for glycine as a neurotransmitter includes the demonstration that glycine (a) mimics the action of naturally occurring inhibitory transmitter when it is iontophoretically applied to motor neurons (5, 6, 24), (b) is released after dorsal root stimulation (12), (c) is reduced in concentration when inhibitory interneurons are selectively destroyed (7), and (d) is taken up into brain slices and a distinct population of synaptosomes by a glycine-specific high-affinity transport system (2,16,17,22). This uptake into specific synaptosomes can be demonstrated only in those regions of the central nervous system in which glycine is iontophoretically active and present in high concentration.Electron microscope autoradiography has been used in attempts to identify synaptic terminals possessing high affinity uptake systems for glycine (10,11). These autoradiographic studies of [SH] glycine uptake in vitro and in vivo have shown silver grains over nerve terminals with flattened vesicles (9, 14, 15). However, previous reports have not (a) provided quantitative evidence for selective labeling of synapses, (b) quantitatively analyzed the topographical distribution of labeled synapses, or (c) demonstrated that the label was in glycine and not in protein or metabolites.In the present investigation, synapses on rat ventral horn cells were labeled by [3H]glycine in vivo' and were identified by electron microscope autoradiography. Our findings indicate that (a) the grain density is greatest in synaptic terminals, (b) a high proportion of axosomatic and proximal axodendritic synapses take up [aH]glycine, (c) many of the labeled terminals have elliptical and pleomorphic vesicles, and (d) [SH]glycine is not metabolized significantly under the conditions of our experiment. MATERIALS AND METHODS Microinjection Technique and FixationSprague-Dawley rats weighing 150-300 g were anesthetized with chloral hydrate (40 mg/kg body weight), the lumbar spinal cord was exposed, and a small dural puncture 1.2 mm from midline was made over the L5 cord level with a fine shaq~ned needle. By means of a micromanipulator, a micropipette (tip diameter 15 ,m) was advanced perpendicularly 1.6 mm into the ventral horn. 0.4 ,l [aH]glycine (10 Ci/mmol, 4.5 • 10 5 dpm/,l, N~w England Nuclear, Boston, Mass.) in 0.1 M phosphate buffer (pH 7.3) was injected over 5 rain, using a screw-driven apparatus. The calculated concentration of [aH]glycine at the site of injection was 2.1 • 10 -2 raM. All animals were sacrificed 10 min after the beginning of the injection. Groups of animals were studied in three ways. Autoradiography was done on four female rats perfused with 2.5% glutaraldehyde in 0.1 M cacodylate buffer with 3% sucrose (Group I). By using a dissecting

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Specific Glycine-Accumulating Synaptosomes in the Spinal Cord of Rats

Cited by 33 publications

References 30 publications

Active transport of gamma-aminobutyric acid and glycine into synaptic vesicles.

Active transport of gamma-aminobutyric acid and glycine into synaptic vesicles.

Strychnine Binding Associated with Glycine Receptors of the Central Nervous System

Glycine-specific synapses in rat spinal cord. Identification by electron microscope autoradiography.

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