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PubMed1980

Isolation and Characterization of Glycine Hydroxamate-resistant Cell Lines of Nicotiana tabacum.

Seven lines of haploid Nicotiana tabacum tissue culture selected for resistance to normally toxic levels of the glycine analog glycine hydroxamate, a competitive inhibitor of the glycine decarboxylase reaction, were investigated. The presence of glycine hydroxamate greatly increased the intracellular concentration of both glycine and alanine in wild type and resistant cell lines, suggesting that the inhibitor blocks both glycine- and alanine-utilizing reactions. All the resistant cell lines, whether grown in the presence or absence of glycine hydroxamate, had high intracellular concentrations of the 12 free amino acids which were analyzed, including glycine and serine. (These lines averaged 3.6 times the total amino acid content of wild-type cells in the absence of the inhibitor). The resistant cell lines were indistinguishable from wild-type cell lines in their metabolism of radioactively labeled glycine hydroxamate and glycine. Comparison of the metabolism of radioactively labeled alanine, glycolate, and glyoxylate in wild-type and alpha resistant line also revealed no distinctive differences. Glycine decarboxylase activities were unaltered in the resistant cell lines. The cellular toxicity of glycine hydroxamate is considered in relation to (1) the competitive inhibition by glycine hydroxamate of the glycine- and alanine-utilizing enzymes and (2) the resultant imbalances caused by high intracellular concentrations of these amino acids. The significance of elevation of total free amino acid concentration in effecting resistance to the inhibitor is discussed.Plants were regenerated from 5 of these lines and callus cultures of explants were tested for glycine hydroxamate resistance. Plants from seedlings of two lines which retained the resistant characteristic in explanted callus did not have high amino acid levels in leaves.

Journal Article↗
PubMed2005

Dietary glycine inhibits bladder activity in normal rats and rats with spinal cord injury.

PURPOSE: The influence of dietary glycine on bladder activity, and on glutamate and glycine levels in the serum and lumbosacral cord was examined in rats with or without spinal cord injury (SCI). MATERIALS AND METHODS: A total of 84 female rats were divided into an intact and an SCI group. Each group of rats was divided into 7 subgroups. Two intact and 2 SCI subgroups were fed a standard diet and the remaining subgroups were fed diets containing 0.1% to 3% glycine. After 4 weeks isovolumetric cystometry was performed with rats under urethane anesthesia. Following cystometry glutamate and glycine levels in the serum and lumbosacral cord were measured as well as the glycine receptor alpha1 mRNA level in the lumbosacral cord. RESULTS: Dietary glycine (1% to 3%) prolonged the interval between bladder contractions in intact rats but did not change the amplitude of contractions. On the other hand, dietary glycine (1% to 3%) prolonged the interval and decreased the amplitude of bladder contractions in SCI rats. The glycine levels in the serum and lumbosacral cord of SCI rats on the standard diet were respectively 43% and 45% lower than those in intact rats on the standard diet. Dietary glycine (1% to 3%) increased the serum glycine level in intact and SCI rats but the glycine receptor alpha1 mRNA level in the lumbosacral cord was unchanged. CONCLUSIONS: Dietary glycine crosses the blood-brain barrier and inhibits the micturition reflex pathway in the lumbosacral cord but SCI and/or dietary glycine do not influence glycine receptor expression.

Animals↗
PubMed1999

Production of superoxide and TNF-alpha from alveolar macrophages is blunted by glycine.

Glycine blunts lipopolysaccharide (LPS)-induced increases in intracellular calcium concentration ([Ca(2+)](i)) and tumor necrosis factor-alpha (TNF-alpha) production by Kupffer cells through a glycine-gated chloride channel. Alveolar macrophages, which have a similar origin as Kupffer cells, play a significant role in the pathogenesis of several lung diseases including asthma, endotoxemia, and acute inflammation due to inhaled bacterial particles and dusts. Therefore, studies were designed here to test the hypothesis that alveolar macrophages could be inactivated by glycine via a glycine-gated chloride channel. The ability of glycine to prevent endotoxin [lipopolysaccharide (LPS)]-induced increases in [Ca(2+)](i) and subsequent production of superoxide and TNF-alpha in alveolar macrophages was examined. LPS caused a transient increase in intracellular calcium to nearly 200 nM, with EC(50) values slightly greater than 25 ng/ml. Glycine, in a dose-dependent manner, blunted the increase in [Ca(2+)](i), with an IC(50) less than 100 microM. Like the glycine-gated chloride channel in the central nervous system, the effects of glycine on [Ca(2+)](i) were both strychnine sensitive and chloride dependent. Glycine also caused a dose-dependent influx of radiolabeled chloride with EC(50) values near 10 microM, a phenomenon which was also inhibited by strychnine (1 microM). LPS-induced superoxide production was also blunted in a dose-dependent manner by glycine and was reduced approximately 50% with 10 microM glycine. Moreover, TNF-alpha production was also inhibited by glycine and also required nearly 10 microM glycine for half-inhibition. These data provide strong pharmacological evidence that alveolar macrophages contain glycine-gated chloride channels and that their activation is protective against the LPS-induced increase in [Ca(2+)](i) and subsequent production of toxic radicals and cytokines.

Animals↗
PubMed2001

Ethanol inhibition of glycine-activated responses in neurons of ventral tegmental area of neonatal rats.

The brain is particularly sensitive to alcohol during the period of its rapid growth. To better understand the mechanism(s) involved, we studied ethanol effects on glycine-activated responses of ventral tegmental area (VTA) neurons isolated from the newborn rat, using whole cell and gramicidin perforated patch-clamp techniques. Previously we reported that 0.1-40 mM ethanol enhances glycine-induced responses of 35% of VTA neurons. We now direct our attention to the inhibitory effects of ethanol observed in 45% (312 of 694) of neonatal VTA neurons. Under current-clamp conditions, 1 mM ethanol had no effect on the membrane potential of these cells, but it decreased glycine-induced membrane depolarization and the frequency of spontaneous action potentials. Under voltage-clamp conditions, 0.1-10 mM ethanol did not elicit a current but depressed the glycine-induced currents. The ethanol-induced inhibition of glycine current was independent of membrane potential (between -60 and +60 mV). Likewise, ethanol did not alter the reversal potential of the glycine-activated currents. Ethanol-mediated inhibition of glycine current depended on the glycine concentration. While ethanol strongly depressed currents activated by 30 microM glycine, it had no appreciable effect on maximal currents activated by 1 mM glycine. In the presence of ethanol (1 mM), the EC(50) for glycine increased from 32 +/- 5 to 60 +/- 3 microM. Thus ethanol may decrease the agonist affinity of glycine receptors. A kinetic analysis indicated that ethanol shortens the time constant of glycine current deactivation but has no effect on activation. In conclusion, by altering VTA neuronal function, ethanol-induced changes in glycine receptors may contribute to neurobehavioral manifestations of the fetal alcohol syndrome.

Aging↗
PubMed2001

Endothelial cells contain a glycine-gated chloride channel.

Glycine inhibited growth of B16 melanoma tumors in vivo most likely because of the inhibition of angiogenesis. Here, the hypothesis that the anticancer effect of glycine in vivo is due to expression of a glycine-gated Cl- channel in endothelial cells was tested. First, the effects of glycine on vascular endothelial growth factor-induced increases in intracellular Ca2+ concentration in a bovine endothelial (CPA) cell line were studied. Vascular endothelial growth factor (1 ng/ml) increased intracellular Ca2+ concentration, with peak values reaching 141 +/- 11 nM. Glycine blunted this increase dose dependently. Furthermore, the inhibitory effects of glycine were prevented by 1 microM strychnine, a glycine receptor antagonist, or when cells were incubated in Cl(-)-free buffer. Moreover, glycine increased influx of 36Cl into CPA cells approximately 10-fold; this reaction was also strychnine sensitive. Furthermore, mRNA similar to the beta-subunit of the glycine-gated Cl- channel from spinal cord was identified in endothelial cells by reverse transcription-polymerase chain reaction. In addition, Western analysis using antibody for the glycine receptor demonstrated expression of the beta-subunit of the glycine receptor. Importantly, glycine diminished serum-stimulated proliferation and migration of endothelial cells. Collectively, these data indicate that the inhibitory effect of glycine on growth and migration of endothelial cells is due to activation of a glycine-gated Cl- channel. This hyperpolarizes the cell membrane and blocks influx of Ca2+, thereby minimizing growth factor-mediated signaling.

Animals↗
PubMed1989

Interaction of 6-cyano-7-nitroquinoxaline-2,3-dione with the N-methyl-D-aspartate receptor-associated glycine binding site.

The interaction of newly described antagonist of the non-NMDA glutamate receptor 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) with the glycine site of the NMDA receptor complex has been investigated. In whole-cell patch recordings from hippocampal neurons maintained in culture, currents induced by N-methyl-D-aspartate (NMDA) were dependent on extracellular glycine. Responses to both NMDA (30 microM) and kainate (20 microM) were reduced by CNQX (10-30 microM). The antagonism by CNQX of NMDA, but not kainate, receptor-mediated responses could be reversed by increasing the concentration of glycine in the external medium. Glycine concentration-response curves constructed in the presence of 30 microM NMDA were shifted to the right by CNQX, suggesting that CNQX was competing with glycine for the glycine binding site. However, even at high concentrations of glycine (300 microM) the maximal NMDA current obtained in the presence of CNQX (10-30 microM) was not restored to control levels. Because CNQX had no effect on responses produced by supramaximal concentrations of NMDA (500 microM) and glycine (300 microM), it is suggested that CNQX also interacts with the NMDA recognition site. The antagonism of currents induced by NMDA was not dependent on the membrane potential, and the rapid onset and offset of the block suggested that there was little or no use dependence. Radioligand binding experiments were performed using [3H]glycine to label the strychnine-insensitive glycine regulatory site of the NMDA receptor complex in guinea pig brain frontal cortex membranes. CNQX displaced [3H]glycine binding in a concentration-dependent manner (IC50 = 5.7 microM). Scatchard analysis of the inhibition showed a decrease in the affinity (increase in Kd) of [3H]glycine binding, but no change in the number of binding sites (Bmax) in the presence of 5 microM CNQX, suggesting a competitive interaction. These data provide evidence that CNQX antagonizes NMDA receptor-mediated responses by competing with glycine for a modulatory site associated with the NMDA receptor complex. Furthermore, the results indicate that CNQX may not be as selective an antagonist for non-NMDA receptors as initially described, although its selectivity will depend on the concentration of the NMDA receptor ligand and may be enhanced by increasing the extracellular concentration of glycine.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2000

Cuneothalamic relay neurons are postsynaptic to glycine-immunoreactive terminals in the rat cuneate nucleus.

This study was aimed to clarify whether the cuneothalamic relay neurons (CTNs) in the rat cuneate nucleus contained glycine or whether the neurons were modulated directly by presynaptic glycine-IR terminals. For this purpose, retrograde transport of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) and immunoperoxidase labelling for glycine have been used to ascertain if the CTNs in the rat are glycine-immunoreactive (glycine-IR). Our results have shown that the WGA-HRP-labelled CTNs (mean area = 318 +/- 6.5 microm(2)) were not reactive for glycine. Glycine immunoreactivity, however, was localized in some small-sized neurons (mean area = 210 +/- 6.2 microm(2)) and axon terminals associated with the CTNs. The synaptic organization between the glycine-IR terminals and CTNs was further analyzed using anti-glycine postembedding immunogold labelling. By electron microscopy, the immunogold-labelled glycine-IR terminals containing pleomorphic synaptic vesicles formed symmetrical synaptic contacts with the dendrites, dendritic spines, and somata of CTNs. Quantitative estimation showed that the mean ratios of glycine-IR terminals to total terminals associated with the soma, proximal dendrites and distal dendrites of the CTN were 49.5, 45.2, and 45.8%, respectively. The higher incidence of glycine-IR terminals on the soma, however, was not significantly different from that of the proximal and distal dendrites. Notwithstanding the above, this study has shown a large number of glycine-IR terminals making direct synaptic contacts with CTNs, suggesting that glycine is one of the important neurotransmitters involved in postsynaptic inhibition on the cuneothalamic relay neurons to modulate incoming somatosensory information from forelimb areas in the rat.

Animals↗
PubMed2001

Afferent synaptic contacts on glycine-immunoreactive neurons in the rat cuneate nucleus.

This study was aimed to clarify whether the primary afferent terminals (PATs), GABAergic terminals, and glutamatergic terminals made direct synaptic contacts with glycine-IR neurons in the cuneate nucleus of rats. In this connection, injection of the anterograde tracer WGA-HRP into brachial plexus, antiglycine preembedding immunoperoxidase, and anti-GABA, along with antiglutamate postembedding immunogold labeling, were used to identify the PATs, glycine-IR neurons, GABA-IR terminals, and glutamate-IR terminals, respectively. The present results showed that HRP-labeled PATs, immunoperoxidase-labeled glycine-IR terminals, immunogold-labeled GABA-IR, and glutamate-IR terminals made axodendritic synaptic contacts with immunoperoxidase-labeled glycine-IR neurons. The latter three presynaptic elements also formed axosomatic synapses with glycine-IR neurons. Statistical analysis has shown that the minimum diameter of the glycine-IR dendrites postsynaptic to the above-mentioned four presynaptic elements did not differ significantly. In addition, the synaptic ratio of the glutamate-IR terminals on the glycine-IR dendrites was higher than that of GABA-IR terminals. The synaptic ratio of the GABA-IR terminals on glycine-IR dendrite was in turn higher than that of the PATs and glycine-IR terminals. It is suggested that the PATs and glutamate-IR terminals on the glycine-IR neurons may be involved in subsequent postsynaptic inhibition for spatial precision of lateral inhibition. On the other hand, the GABA-IR and glycine-IR terminals which make synaptic contacts with the dendrites of glycine-IR neurons may provide a putative means for disinhibition or facilitation to maintain the baseline neuronal activity in the rat cuneate nucleus. The results of quantitative analysis suggest that glutamate act as the primary excitatory neurotransmitter, while GABA, when compared with glycine, may serve as a more powerful inhibitory neurotransmitter on glycine-IR neurons in the rat cuneate nucleus.

Animals↗
PubMedDate not supplied

Localization of gephyrin and glycine receptor subunit immunoreactivity in the rabbit retina.

Being utilized by over 40% of the amacrine cells, glycine is considered to be a major inhibitory neurotransmitter in the retinas of all vertebrate species examined. Localization of gephyrin, which is a 93-kD peripheral membrane glycine receptor-associated anchoring protein, has been used in several studies to identify the sites of glycinergic interactions in the retina and other regions of the central nervous system. Recent studies have shown that gephyrin colocalizes with GABA(A) receptors which, like those for glycine, are also inhibitory amino acid receptors usually associated with a chloride channel. In the present study, we have used two antibodies which recognize either gephyrin (mAb7a), or the alpha and beta subunits of the glycine receptor (mAb4a) in order to determine to what extent gephyrin is associated with glycine receptors in the mammalian retina. Single-label studies showed extensive punctate staining throughout most of the inner plexiform layer with each antibody. Double labeling showed that nearly 90% of the glycine receptor sites were also immunoreactive for gephyrin. However, nearly 60% of the total punctae immunoreactive for gephyrin were not stained for glycine receptors. This distinction was most pronounced in the most proximal inner plexiform layer where only 24% of the gephyrin-immunoreactive sites were glycine receptor positive. This study suggests that although most glycine receptors in the rabbit retina colocalize with the anchoring protein gephyrin, a significant proportion of the gephyrin-labeled sites are not associated with glycine receptors. In light of studies showing gephyrin association with GABA(A) receptor subunits, the localization of gephyrin may be indicative of chloride-mediated inhibitory amino acid transmission in general and not solely that of glycinergic. Given several studies which show that bipolar cells express glycine receptors and respond to glycine but do not express gephyrin, the 10% of glycine receptors not colocalized with gephyrin shown in the present study may represent a subtype of glycine receptors found on bipolar cells which do not require gephyrin for the functional clustering of receptor subunits.

Animals↗
PubMed1994

Interactions in glycine and methionine uptake, conversion and incorporation into proteins in the preimplantation mouse embryo.

Glycine is the most concentrated amino acid in the female genital tract. In this study, we report its conversion and incorporation into proteins in the presence or absence of methionine, in both 1-cell and blastocyst mouse embryos. The uptake, incorporation and conversion of radiolabelled glycine were studied in the presence or absence of unlabelled methionine. For control purposes, the reciprocal experiment was performed with labelled methionine in the presence or absence of unlabelled glycine. At the 1-cell stage neither glycine uptake nor its incorporation into proteins is inhibited by methionine. Glycine is, however, highly used as an oxidisable energy substrate, via glycolate. At the blastocyst stage, glycine conversion into other amino acids is high and mainly utilised in the formation of glutamic acid. Glycine is highly incorporated into proteins, resulting in a poor exchange of glycine from the preloaded embryos. Methionine competes for glycine uptake and consequently reduces its overall incorporation into proteins. For methionine, neither its uptake nor its incorporation into proteins is reduced in the presence of glycine for the two embryonic stages tested here. The embryo has different mechanisms for incorporation and utilisation of methionine and glycine. Glycine, which has an important function in the embryo, has an inefficient transport system compared with methionine. We were unable to demonstrate the presence of methylglycine since SAM-glycine-methyltransferase (EC 2.1.1.20) was not detected. The same results were obtained when exogenous methionine was added. We therefore concluded that glycine does not compete in transmethylation within the embryo.

Amino Acids↗
PubMed1992

Rapid stimulation of the hepatic glycine-cleavage system in rats fed on a single high-protein meal.

Glycine catabolism was studied in isolated rat liver mitochondria by measuring the release of 14CO2 from [1-14C]glycine. Mitochondria isolated from rats fed on a high-protein (60% casein) diet for 5 days showed an enhanced ability to catabolize glycine compared with mitochondria from rats fed on a normal-protein (15% casein) diet. Glycine catabolism was also stimulated in normal protein-fed rats if they ingested a single high-protein meal for 2 h before being killed, thus illustrating the rapid response of the glycine-cleavage system to protein intake. The stimulation of glycine catabolism in rats given a high-protein diet or meal was not evident if the mitochondria were incubated in the absence of P(i) (omitting ADP had no effect on the rate). Mitochondria from high-protein- and normal-protein-fed rats did not differ in their ability to accumulate glycine, a process which occurred far too rapidly to impose a limit on the rate of flux through the glycine-cleavage system. The stimulation of glycine catabolism by high-protein feeding was not associated with a change in mitochondrial matrix volume. Furthermore, mitochondria from rats fed on a high-protein meal maintained an enhanced ability to catabolize glycine compared with those from rats fed on a normal-protein meal when incubated in hypo-osmotic solutions of very low osmolarity. When mitochondria from high-protein- or normal-protein-fed rats were maximally activated by incubation in the presence of 0.25 microM-Ca2+, the rates of glycine catabolism were high, but similar, showing that the stimulation of glycine catabolism by high-protein feeding does not involve an increase in the total capacity of the system. These findings show that hepatic glycine catabolism is stimulated rapidly by high-protein feeding, a response that we suggest is involved in the disposal of the excess glycine in the diet.

Amino Acid Oxidoreductases↗
PubMed2005

Glycine-ice nanolayers: morphology and surface energetics.

Ultrathin glycine-ice films (nanolayers) have been prepared in ultrahigh vacuum by condensation of H(2)O and glycine at 110 K and 150 K on single crystalline Al(2)O(3) surfaces and have been investigated by temperature programed thermal desorption, x-ray photoelectron spectroscopy, and work function measurements. Various layer architectures have been considered, including glycine-on-ice, ice-on-glycine, and mixed glycine-ice nanolayers. Low coverages of adsorbed glycine molecules on amorphous ice surfaces suppress the amorphous-to-crystalline phase transition in the temperature range 140-160 K in near-surface regions and consequently lead to a lower desorption temperature of H(2)O molecules than from pure ice layers. Thicker glycine overlayers on ice provide a kinetic restriction to H(2)O desorption from the underlying ice layers until the glycine molecules become mobile and develop pathways for water desorption at higher temperature (>170 K). Ice overlayers do not wet glycine film surfaces, but the glycine molecules on ice are sufficiently immobile at 110 K, so that continuous glycine overlayers form. In mixed glycine-ice nanolayers the glycine phase displays hydrophobic behavior and a phase separation takes place, with the accumulation of glycine near the surfaces of the films.

Computer Simulation↗
PubMed1989

Na- and Cl-dependent glycine transport in human red blood cells and ghosts. A study of the binding of substrates to the outward-facing carrier.

Na- and Cl-dependent glycine transport was investigated in human red blood cells. The effects of the carrier substrates (Na, Cl, and glycine) on the glycine transport kinetics were studied with the goal of learning more about the mechanism of transport. The K1/2-gly was 100 microM and the Vmax-gly was 109 mumol/kg Hb.h. When cis Na was lowered (50 mM) the K1/2-gly increased and the Vmax-gly decreased, which was consistent with a preferred order of rapid equilibrium loading of glycine before Na. Na-dependent glycine influx as a function of Na concentration was sigmoidal, and direct measurement of glycine and Na uptake indicated a stoichiometry of 2 Na:1 glycine transported. The sigmoidal response of glycine influx to Na concentration was best fit by a model with ordered binding of Na, the first Na with a high K1/2 (greater than 250 mM), and the second Na with a low K1/2 (less than 10.3 mM). In the presence of low Cl (cis and trans 5 mM), the K1/2-gly increased and the Vmax-gly increased. The Cl dependence displayed Michaelis-Menten kinetics with a K1/2-Cl of 9.5 mM. At low Cl (5 mM Cl balanced with NO3), the glycine influx as a function of Na showed the same stoichiometry and Vmax-Na but a decreased affinity of the carrier for Na. These data suggested that Cl binds to the carrier before Na. Experiments comparing influx and efflux rates of transport using red blood cell ghosts indicated a functional asymmetry of the transporter. Under the same gradient conditions, Na- and Cl-dependent glycine transport functioned in both directions across the membrane but rates of efflux were 50% greater than rates of influx. In addition, the presence of trans substrates modified influx and efflux differently. Trans glycine largely inhibited glycine efflux in the absence or presence of trans Na; trans Na largely inhibited glycine influx and this inhibition was partially reversed when trans glycine was also present. A model for the binding of these substrates to the outward-facing carrier is presented.

Biological Transport↗
PubMed1978

Glycine fluxes in squid giant axons.

1. The influx of a number of amino acids into squid giant axons has been studied. Particular emphasis has been placed on glycine and to a lesser extent glutamate. 2. To facilitate the study of the uptake of 14C-labelled amino acids a technique was devised in which the 14C taken up was measured directly in the intact axon with a glass scintillator fibre. This technique gave results similar to the usual technique in which the axoplasm was extruded for the assay of radioactivity. 3. The changes in glycine influx with extracellular glycine concentration suggests that two saturating components are present, one with high affinity and one with low affinity. 4. The glycine influx does not seem normally to be sensitive to the removal of extracellular sodium by replacement with choline. A Na-sensitive component appeared, however, after a period of immersion in artificial sea water. There was also some depression of glycine influx if Na were replaced by Li. 5. Glutamate uptake was greatly reduced by removal of extracellular Na in confirmation of work by Baker & Potashner (1973). Orthophosphate uptake was also greatly reduced by removal of extracellular Na. 6. CN reversibly inhibited glycine uptake after a delay, indicating that part of the uptake mechanism may require ATP. 7. 14C-labelled glycine injected into squid axons was found not to exchange to any serious extent with other compounds over periods of a few hours. The glycine efflux could therefore be studied. This was found to be markedly increased by extracellular glycine and by certain other neutral amino acids applied extracellularly in the artificial sea water. 8. The enhanced glycine efflux in extracellular glycine was not affected by ouabain and CN. 9. It is suggested that glycine uptake in squid axons involves two components. One is sensitive to CN and ouabain and probably derives energy from ATP break-down. The other is probably an ATP independent exchange diffusion system in which other amino acids as well as glycine can exchange for glycine. Both these systems are independent of extracellular Na concentration. A third Na-dependent system may appear under certain conditions.

Amino Acids↗
PubMed1990

Modulation of N-methyl-D-aspartic acid receptor desensitization by glycine in mouse cultured hippocampal neurones.

1. Responses to N-methyl-D-aspartic acid (NMDA) were recorded from mouse embryonic hippocampal neurones in dissociated culture, using the tight-seal, whole-cell, patch-clamp technique for voltage clamp. A rapid perfusion system, with an exchange time constant of less than 10 ms, was used to apply NMDA under conditions which minimized slow, calcium-sensitive desensitization. With no added glycine, responses to 100 microM-NMDA applied for 1.5 s declined by greater than 90%, due to an additional component of desensitization of time constant 250 ms. 2. Adding glycine to the extracellular solution, over the range 30 nM to 3 microM, both potentiated responses to NMDA and to L-glutamate, and reduced fast desensitization. In the presence of 3 microM-glycine responses to NMDA declined by only 10%. Similar potentiation and reduction of desensitization was obtained with 3 microM concentrations of the glycine analogues D-alanine and D-serine. 3. Analysis of dose-response curves for the action of glycine on responses to 100 microM-NMDA revealed a 3-fold higher potency of glycine for potentiation of peak versus steady-state responses, with concentrations for half-activation of 134 and 382 nM, respectively. The competitive glycine antagonist 7-chlorokynurenic acid produced a similar shift of both the peak and steady-state dose-response curves for glycine, consistent with an equilibrium dissociation constant of 280 nM for interaction of 7-chlorokynurenic acid with the glycine binding site on NMDA receptors. 4. In the presence of 100 nM-glycine, 10 microM-7-chlorokynurenic acid produced nearly complete block of the response to 3 nM-NMDA, suggesting that glycine is absolutely required for activation of the NMDA receptor channel complex. 5. In some neurones responses to NMDA showed essentially no desensitization in the presence of 3 microM-glycine. Under these conditions, 7-chlorokynurenic acid produced a concentration-dependent block of both the initial and equilibrium response to NMDA, with a 4-fold greater sensitivity for block of the steady-state current (IC50 = 2.25 microM) than for block of the peak current (IC50 = 8.96 microM). As a result, in the presence of 7-chlorokynurenic acid, responses to NMDA showed strong desensitization, even in the presence of 3 microM-glycine. 6. Our results show that glycine-evoked potentiation of NMDA receptor activity is accompanied by reduced desensitization.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

A kinetic analysis of the modulation of N-methyl-D-aspartic acid receptors by glycine in mouse cultured hippocampal neurones.

1. Responses to N-methyl-D-aspartic acid (NMDA) were recorded from mouse embryonic hippocampal neurones in dissociated culture, using whole-cell patch-clamp recording. A fast perfusion system, with an exchange time constant of less than 10 ms, was used to study modulation of NMDA receptor desensitization by glycine. 2. The onset of NMDA receptor desensitization was well fitted by a single-exponential function; with 30 nM-glycine the time constant was 250 ms, corresponding to a rate of 4 s-1. The rate of onset of desensitization became faster with increasing glycine concentration, with a slope of 0.87 x 10(7) M-1 s-1. Recovery from desensitization, studied with a twin-pulse technique, was also well fitted by a single-exponential function; with 30 nM-glycine the time constant of recovery was 1.95 s-1. The rate of recovery from desensitization became faster with increasing glycine concentration, with a slope of 0.76 x 10(7) M-1 s-1. These results are consistent with a model in which the effect of glycine occurs via an increase in the rate constant for recovery from desensitization, with little effect on the rate constant for onset of desensitization. Over the range 30-300 nM-glycine, the ratio of the rate constants calculated for recovery and onset of desensitization was a good predictor of the degree of desensitization recorded at equilibrium. 3. Concentration jump experiments with glycine were performed with 100 microM-NMDA present continuously, and for a single binding site model gave estimates of the association (1.1 x 10(7) M-1 s-1) and dissociation (3.1 s-1) rate constants for interaction of glycine with the NMDA receptor. In the presence of NMDA, concentration jumps from 3 microM-glycine to lower concentrations gave relaxations which became slower with decreasing glycine concentration over the range 1 microM-30 nM. A similar slowing of desensitization occurred when the glycine concentration was altered over the same range. 4. Glycine analogues of lower affinity produced desensitization with faster kinetics. D-Alanine, 150 nM, produced desensitization with a time constant of 175 ms, faster than recorded with an equipotent concentration of glycine (50 nM, time constant 259 ms). Responses of similar peak amplitude, recorded with 60 microM-L-alanine, and 500 microM-D,L-homoserine, did not produce strong desensitization, consistent with desensitization too rapid to resolve in our experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Alanine↗
PubMed1991

Inhibitory and potentiating influences of glycine on N-methyl-D-aspartate-evoked dopamine release from cultured rat mesencephalic cells.

In the presence of 1.2 mM Mg2+, glycine (30-100 microM) inhibited [3H]dopamine ([3H]DA) release stimulated by N-methyl-D-aspartate (NMDA), in fetal rat mesencephalic cell cultures. Strychnine (1 microM) blocked the inhibitory effect of 100 microM glycine, indicating an action via strychnine-sensitive inhibitory glycine receptors. A higher concentration of strychnine (100 microM), by itself, inhibited NMDA-evoked [3H]DA release in the presence or absence of Mg2+. Spontaneous [3H]DA release and [3H]DA release stimulated by kainate and quisqualate were unaffected by glycine (less than or equal to 100 microM) or strychnine (less than or equal to 100 microM), indicating that glycine and strychnine modulatory effects are only associated with the NMDA receptor subtype. [3H]DA release evoked by K+ (56 mM) was unaffected by glycine (less than or equal to 100 microM) but was attenuated by a high concentration of strychnine (100 microM). In the absence of exogenous Mg2+, glycine (30-100 microM) potentiated NMDA-evoked [3H]DA release by a strychnine-insensitive mechanism. A selective antagonist of the NMDA-associated glycine receptor, 7-chlorokynurenate (10 microM), attenuated NMDA-evoked [3H]DA release in the absence of Mg2+. The effect of 10 microM 7-chlorokynurenate was overcome by 1 microM glycine. Also, when tested in the presence of 1.2 nM Mg2+ and 1 microM strychnine, 100 microM 7-chlorokynurenate inhibited NMDA-evoked [3H]DA release, and this antagonism was overcome by 30 to 100 microM glycine. These results indicate that two distinct glycine receptors modulate NMDA-stimulated [3H]DA release from mesencephalic cells in culture. Manipulation of extracellular Mg2+ permits the differentiation of a strychnine-sensitive glycine response (inhibition of NMDA-evoked [3H]DA release) from a strychnine-insensitive glycine response (potentiation of NMDA-evoked [3H]DA release). It is suggested that voltage-dependent Mg2+ blockade of the NMDA response may allow for the expression of these opposing effects of glycine.

Amino Acids↗
PubMed1985

Effects of glycine on hemodynamic responses and visual evoked potentials in the dog.

To study the potential contribution of glycine toxicity in the transurethral resection syndrome, we evaluated hemodynamic and visual evoked potential responses to glycine infusion (1 g/kg) in 22 dogs anesthetized with halothane (1.0-1.2% end tidal. Three dogs received 5% glucose in normal saline without glycine; three received arginine (4 mg/kg) in normal saline without glycine; three received arginine (4 mg/kg) in normal saline without glycine; 10 received glycine (1 g/kg), then arginine (4 mg/kg) 120 min after the completion of glycine infusion; and six received arginine 30 min after the completion of glycine infusion. Arginine was infused to evaluate potential antagonistic effects of glycine toxicity. Blood levels of glycine, ammonia, arginine, urea, and formate were determined after infusions of glycine or arginine. All animals received about 5 ml X kg-1 X hr-1 of normal saline during the 2-4 hr of study. Immediately after glycine infusion, cardiac output increased 57%, whereas systemic vascular resistance and mean arterial pressure decreased 32% and 8%, respectively. Later cardiac output and mean arterial pressure were 41% and 18% less than control levels, whereas systemic vascular resistance returned to control levels. Both amplitude and latency of visual evoked potential waveforms were altered in the animals receiving glycine infusion but not in the control animals. These responses were associated with elevations of blood glycine (149 +/- 5 to 9591 +/- 809 microM/L, mean + SEM) and blood NH3 (10.5 +/- 2.8 to 100.0 +/- 13.6 microM/L), but not with formate levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia↗