Molecular modeling of the weak glycine antagonist iso-THAO.

When compared to strychnine, a potent glycine antagonist, iso-THAO, a bicyclic 5-isoxazolol zwitterion, has been reported to be a weak glycine antagonist. Since there are so few glycine antagonists, and there is a striking lack of similarity between the structures of these two antagonists, iso-THAO was studied using current molecular modeling techniques and quantum mechanical calculations in order to compare the structural features and charge distributions of iso-THAO with glycine. The results of this study confirm our earlier hypothesis that an antagonist to inhibitory neurotransmitters like glycine and GABA has at least three binding sites to the natural receptor that are very similar to three such binding sites in the transmitter and its agonists, and each antagonist has an additional negative binding site. We speculate that the latter negative binding site can attach to the top of the chloride channel within the receptor complex. The diminished inhibitory activity of iso-THAO is attributed to its poor structural congruence with the three atom attachment sites used by glycine at its natural recognition site.     

Identification of a second glycine-like fragment on the strychnine molecule

Strychnine is a complex molecule that inhibits the physiological actions of glycine, an important inhibitory neurotransmitter in the spinal cord, brain stem, and other areas of many vertebrates. Since 1987, we have employed atomistic molecular modeling tools to find an explanation at the molecular level for how this antagonism works. We have located a second glycine-like fragment in the strychnine molecule that, when compared to glycine in a three pair atom analysis, provides an excellent topological and electronic charge congruence. The topological congruence in the second glycine-like fragment is much better than with the first fragment reported in 1987 when using a truncated strychnine molecule in the quantum mechanical analysis. A fourth negative atom, a characteristic of antagonists which we reported earlier (Aprison and Lipkowitz: J Neurosci Res 30:442–446, 1991; Aprison and Lipkowitz: J Neurosci Res 31:166–174, 1992) was found in strychnine. This result follows the pattern reported recently for the three weak glycine antagonists N, N-dimethylmuscimol, N-methyl-THIP, and iso-THAO, a bicyclic 5-isoxazolol zwitterion. © 1995 Wiley-Liss, Inc.     

Molecular dynamics of serotonin and ritanserin interacting with the 5-HT2 receptor.

A three-dimensional model of the serotonin (5-hydroxytrytamine; 5-HT) 5-HT2 receptor was constructed from the amino acid sequence by molecular graphics techniques, molecular mechanics energy calculations and molecular dynamics simulations. The receptor model has 7 alpha helical segments which form a membrane-spanning duct with a putative ligand binding site. Most of the synaptic domains and the ligand binding site were surrounded by negative electrostatic potentials, suggesting that positively charged ligands are attracted to the receptor by electrostatic forces. The cytoplasmic domains, except the C-terminal tail, had mainly positive electrostatic potentials. The molecular dynamics of the receptor-ligand complex was examined in 100 ps simulations with 5-HT or ritanserin at a postulated binding site. During the simulations the helices moved from an initial circular arrangement into a more oval arrangement, and became slightly tilted relative to each other. The protonated ligands neutralized the negative electrostatic potentials around Asp 120 and Asp 155 in the central core of the receptor. 5-HT had only weak interactions with Asp 155 but strong interactions with Asp 120 during the simulations, with the amino group of 5-HT tightly bound to the carboxylic side chain of Asp 120. Ritanserin showed similarly strong interactions with Asp 120 and Asp 155 during the simulations.     

Strychnine-insensitive [H] glycine binding to synaptosomal membranes from the chick retina

The pharmacology and kinetics of strychnine-insensitive [[3]H] glycine binding to synaptic membranes from the outer (P₁) and the inner (P₂) plexiform layers of chick retina was studied. Inhibition curves for glycine, d-serine, 1-amincyclopropanecarboxylic acid (ACPC) and strychnine were analyzed by non-linear regression. Hill slopes for glycine and d-serine were not different from unity, whereas those for ACPC were < 1 in both fractions, revealing heterogeneity of binding sites in these membranes. Non-linear regression analysis of time course and saturation experiments strengthen the idea that [[3]H] glycine binds to more than one class of sites, with similar affinities at equilibrium. Antagonists of strychnine-insensitive glycine receptors in the CNS did not inhibit [[3]H] glycine binding to these membranes, which demonstrates that NMDA receptors in the retina have different structural requirements for ligand interaction at these sites. pH affected the specific binding, in agreement with the participation of specific amino acid residues at glycine binding sites on NMDA receptors, and also with functional studies in which the modulation of affinity at this site by protons has been observed. These results support previous studies regarding CPP and MK-801 binding, and provide evidence which indicates that the pharmacophore for glycine and other NMDA-related ligands is distinct for the retina, compared to the CNS, mainly regarding the effects of glycine-site antagonists.     

Involvement of amino acids in periodic inhibitions of bulbar respiratory neurones

As previously demonstrated⁷, spontaneously firing bulbar inspiratory neurones are periodically inhibited either at the beginning of, or throughout expiration, while bulbar expiratory neurones are inhibited during inspiration. The aim of the present study was to test the hypothesis that amino acids act as transmitters of these periodic inhibitions.The study was performed using iontophoretic applications of drugs on bulbar respiratory neurones. On these neurones GABA and glycine-sensitive sites were identified and differentiated on the basis of the actions of agonist (muscimol) or antagonists (bicuculline, picrotoxin and strychnine). Using competitive antagonists (nipecotic acid, β-alanine) mechanisms responsible for GABA uptake were found in the close vicinity of respiratory-related neurones.Some but not all types of periodic inhibition were found to be reduced following application of GABA or glycine antagonists. Strychnine was found to reduce periodic inhibitions occurring at the beginning of expiration in inspiratory neurones. GABA antagonists had an effect on those periodic depressions which were prolonged throughout expiration. A different and complementary role of glycine-like and GABA-like systems in central respiratory mechanisms is proposed.     

Localization of high affinity [H]glycine transport sites in the cerebellar cortex

A study was made of [³H]glycine uptake sites in a preparation greatly enriched in large pieces of the cerebellar glomeruli (glomerulus particles) and in morphologically well preserved slices of rat cerebellum. Electron microscopic autoradiography revealed that of the neurones in the cerebellar cortex only Golgi cells transported [³H]glycine at the low concentration used. Glial cells also took up [³H]glycine but to a lesser extent than the Golgi neurons. It was also confirmed that under comparable conditions Golgi cells transport [³H]GABA. Kinetic studies utilizing the Golgi axon terminal-containing glomerulus particles showed that glycine is a weak non-competitive inhibitor of [³H]GABA uptake (K_i over 600 μM vs theK_t of about 20 μM) and that GABA is an even weaker inhibitor of [³H]glycine uptake. These observations indicated that glycine and GABA do not share the same carrier. Quantitative electron microscopic autoradiography showed that the uptake of the two amino acids, in terms of the unit area of labelled Golgi axon terminals, was not additive. In contrast, their uptake in terms of unit protein was strictly additive. These observations, the first relating to unit volume and the latter to the total volume of Golgi terminals, are consistent with the view that there are two biochemically separate populations of Golgi neurons, one transporting glycine the other GABA. Saturable [³H]strychnine binding was detected in the preparations of glomerulus particles, but in comparison with those from the spinal cord the affinity was lower and [³H]strychnine was not displaced by glycine. Available information on glycine receptors, however, suggest that this should not exclude the possibility of strychnine resistant glycine receptors in the rat cerebellum.     

Vocalization Responses After Spinal Administration of Bicuculline or Strychnine in Rats

Objective. Spinal administration of compounds decreasing inhibition of spinal nociceptive pathways, such as antagonists of GABA or glycine receptors, leads to vocalization. This can be quantified semiautomatically and could be used as a research model. Materials and Methods. Vocalization after intrathecal administration of bicuculline and strychnine was measured in Sprague-Dawley rats. Results. Both bicuculline and strychnine produced short, dose-related vocalization responses that were not significantly different between models of peripheral inflammation and neuropathic pain and normal controls, except for reduced strychnine-induced vocalization during inflammation. The strychnine-induced vocalization responses were also reduced in freely moving rats and increased by light tactile stimulation. Conclusions. Bicuculline-induced vocalization seems to be related to facilitation of nociceptive transmission, and could have limited use as a model of nociception at the spinal level. However, peripheral inflammation or neuropathic pain did not affect it. Strychnine-induced vocalization responses seem to be related to non-noxious somatosensory input.     

Identification of a glycine-like fragment on the strychnine molecule

Strychnine, a complex molecule, antagonizes in some unknown manner the action of glycine, an important inhibitory neurotransmitter in the spinal cord and brainstem of many vertebrates. To help understand the mechanism of this antagonism, we have employed modern computational methods to assess the similarities between these seemingly different molecules. An exhaustive comparison of topological and electronic features of both molecules was made. We have successfully located a glycine-like fragment in the strychnine molecule that, when compared to glycine, exhibits both topological and electronic charge congruence. The successful location of this glycine-like fragment allows us to speculate how the large strychnine molecule assumes its role as an antagonist against the inhibitory action of glycine, the simplest amino acid.     

Effect of penicillin, pentylenetetrazol and strychnine on the release of endogenous amino acids into the vitreous of the intact eye of the rat

The vitreous of the intact eye of the albino rat was perfused in vivo and the concentration of amino acids was measured in the vitreal effluent under various conditions.Elevated potassium concentration in the medium stimulated the release of glycine and taurine. Penicillin inhibited the K⁺-evoked release of both amino acids; pentylenetetrazol inhibited the release of only taurine; and strychnine did not inhibit the release of either amino acid. Penicillin did not inhibit the ouabain-evoked release of glycine.Part of the convulsant action of penicillin may reflect its ability to lower the concentration of inhibitory amino acids in the extracellular fluid of the nervous system under calcium-dependent depolarizing conditions.     

The central actions of glycine and strychnine on prolactin and LH secretion

Unanesthetized diestrous female rats were tested for the prolactin response following an intraventricular injection of several doses of the neuroinhibitory amino acid glycine, and the antagonist strychnine. Glycine in doses of 1.0 or 0.1 mumoles increased pituitary prolactin release, significantly elevating plasma hormone levels. Direct pituitary effects for glycine were not observed. Strychnine, a glycine antagonist, was effective in blocking the prolactin release caused by glycine in doses as low as 5 nmoles. Intraventricular glycine administration did not alter pituitary LH release significantly. These studies suggest a central stimulatory role for the neuroinhibitory amino acid glycine in provoking prolactin secretion, and that this effect is strychnine sensitive.     

The inhibitory neurotransmitter glycine modulates macrophage activity by activation of neutral amino acid transporters

Glycine, an important inhibitory neurotransmitter in the mammalian central nervous system (CNS), has been shown to modulate peripheral immune cell responses. In that respect, glycine levels are increased in several neuroinflammatory disorders, such as amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). In this study, we show that glycine modulates macrophage effector functions implicated in CNS inflammation and in other, related inflammatory conditions. We demonstrate that glycine does not affect the production of reactive oxygen species but stimulates myelin phagocytosis and the production of the proinflammatory mediators nitric oxide (NO) and tumor necrosis factor (TNF)‐α by rat macrophages. These effects of glycine are not mediated by the glycine receptor (GlyR) or by glycine transporters (GlyTs), as neither the GlyR antagonist strychnine nor the antagonist of GlyT1 (ALX5407) reverses the observed effects. In contrast, 2‐aminoisobutyric acid, a substrate of neutral amino acid transporters (NAATs), inhibits the glycine‐mediated enhancement of myelin phagocytosis as well as of NO and TNF‐α production. In conclusion, our findings demonstrate that glycine modulates macrophage function through activation of NAATs. Glycine may thereby influence immunological processes in inflammatory diseases involving macrophage activation and demyelination, including MS and related conditions associated with altered glycine levels. © 2010 Wiley‐Liss, Inc.     

The effect of glycine and gamma-aminobutyric acid on the excitatory postsynaptic potentials of the spinal motor neurons in the lamprey in the presence of antagonists

Effect of bath application of the inhibitory amino acids (glycine and GABA) on motoneurons of EPSPs was studied in the normal physiological solutions and after preliminary administration of antagonists: strychnine (10(-6) mol/l), bicuculline (10(-4) mol/l) or picrotoxin (10(-4) mol/l). All these antagonists diminished the depression of monosynaptic EPSPs which were elicited by both amino acids (glycine and GABA). Data obtained in this study and previously reported ones permit concluding that motoneuron membranes in the spinal cord of lamprey possess the unit receptor channel complex sensitive to both amino acids.     

Glycinergic input to carp retinal ganglion cells may be mediated by glycine receptors with homologous kinetics

Current responses of carp retinal ganglion cells (RGCs) retrogradely labeled and freshly dissociated to rapid application of glycine were recorded by whole-cell patch clamp techniques and effects of glycine antagonists on these responses were analyzed. The current response to maintained application of glycine at a concentration higher than 30 microM exhibited desensitization, which was well fitted to a monoexponential function. Strychnine (1 microM), a glycine receptor antagonist, completely blocked the response to 100 microM glycine. Strychnine at a concentration range between 10 and 200 nM suppressed the response to 100 microM glycine in a dose-dependent manner, and only a slow-activated and sustained current eventually remained in the presence of 200 nM strychnine. Power spectral density (PSD) analysis revealed no changes in the density-frequency dependence caused by strychnine. It was further shown that dissociation of strychnine from glycine receptors was rather slow. Moreover, Zn(2+) exerted similar dual action on this sustained response and the response in Ringer's: potentiating and reducing them at low and high concentrations of Zn(2+), respectively. 5,7-Dichlorokynurenic acid (DCKA, 500 microM), a selective blocker of the glycine recognition site at the NMDA receptor, partially reduced the glycine response, but without changing its kinetics. These results suggest that glycinergic input to carp ganglion cells may be mediated by strychnine-sensitive glycine receptors with homologous kinetics, and slow dissociation of strychnine from glycine receptors may partially account for the changes in glycine response kinetics occurring in the presence of strychnine.     

Development of sensitivity to GABA and glycine in cultured cerebellar neurons

The effects of iontophoretically applied gamma-aminobutyric acid (GABA) and glycine on developing cerebellar neurons cultured for 7-40 days were intracellularly investigated. All neurons tested dose-dependently responded to both GABA and glycine. In mature neurons (after 25 days in culture) these amino acids inhibited spontaneous spikes, decreased the membrane input resistance and induced either hyperpolarization or depolarization of membrane potential. The mean reversal potential was -47 mV for GABA and -43 mV for glycine. Immature neurons, 7-12 days in culture, which were not spontaneously firing, also behaved in a similar manner as the mature ones, though the membrane resistance was not so largely changed by GABA or glycine and the reversal potential was more positive (-39 mV for GABA, -37 mV for glycine). These reversal potentials were shifted toward 0 mV by lowering the external Cl- concentration in either mature or immature neurons. The effects of GABA and glycine on mature or immature neurons were more or less inhibited by all of picrotoxin, bicuculline and strychnine. The effective concentrations of these antagonists, however, were lower in general in immature neurons. In mature neurons, picrotoxin and bicuculline became more selective to GABA than glycine and strychnine became more selective to glycine than GABA. These results suggest that sensitivities to GABA and glycine differentiate into selective types in the course of maturing of cerebellar cultured neurons.     

Inhibitory postsynaptic actions of taurine, GABA and other amino acids on motoneurons of the isolated frog spinal cord.

The actions of glycine, GABA, alpha-alanine, beta-alanine and taurine were studied by intracellular recordings from lumbar motoneurons of the isolated spinal cord of the frog. All amino acids tested produced a reduction in the amplitude of postsynaptic potentials, a blockade of the antidromic action potential and an increase of membrane conductance. Furthermore, membrane polarizations occurred, which were always in the same direction as the IPSP. All these effects indicate a postsynaptic inhibitory action of these amino acids. When the relative strength of different amino acids was compared, taurine had the strongest inhibitory potency, followed by beta-alanine, alpha-alanine, GABA and glycine. Topically applied strychnine and picrotoxin induced different changes of post-synaptic potentials, indicating that distinct inhibitory systems might be influenced by these two convulsants. Interactions with amino acids showed that picrotoxin seletively diminished the postsymaptic actions of GABA, while strychnine reduced the effects of taurine, glycine, alpha- and beta-alanine. But differences in the susceptibility of these amino acid actions to strychnine could be detected: the action of taurine was more sensitively blocked by strychnine compared with glycine, alpha- and beta-alanine. With regard to these results the importance of taurine and GABA as transmitters of postsynaptic inhibition on motoneurons in the spinal cord of the frog is discussed.     

Glycine receptor internalization by protein kinases activation

Although glycine-induced currents in the central nervous system have been proven to be modulated by protein kinases A (PKA) and C (PKC), the mechanism is not well understood. In order to better comprehend the mechanism involved in this phenomenon, we tested the PKA and PKC activation effect on the specific [(3) H]glycine and [(3) H]strychnine binding to postsynaptic glycine receptor (GlyR) in intact rat retina. The specific binding constituted about 20% of the total radioligand binding. Kinetic analysis of the specific binding exhibited a sigmoidal behavior with three glycine and two strychnine binding sites and affinities of 212 nM for [(3) H]glycine and 50 nM for [(3) H]strychnine. Specific radioligand binding was decreased (60-85%) by PKA and PKC activation, an effect that was blocked by specific kinases inhibitors, as well as by cytochalasin D. GlyR expressed in the plasma membrane decreased about 50% in response to kinases activation, which was consistent with an increase of the receptor in the microsomal fraction when PKA was activated. Moreover, immunoprecipitation studies indicated that these kinases lead to a time-dependent receptor phosphorylation. Our results suggest that in retina, GlyR is cross-regulated by G protein-coupled receptors, activating PKA and PKC.     

Pharmacological blockade of the globus pallidus-induced inhibitory response of subthalamic cells in the rat

The aim of the present study was to investigate, with extracellular recording and microiontophoretic techniques, the possibility that gamma-aminobutyric acid (GABA) is the transmitter substance in the pallido—subthalamic (GP-STN) pathway. Experiments were carried out in 94 rats, anesthetized with ketamine. Among the 236 recorded STN neurons, 85 were inhibited by GP stimulation. This inhibition lasted 10–20 msec(mean duration±S.E.M.13.45 ± 0.5msec). Control stimulations located either in the internal GP or in the striatum never elicited the same type of response as they did in the STN. STN neurons were inhibited by microiontophoretically applied GABA or muscimol. Iontophoretically applied bicuculline or picrotoxin reversibly blocked the GP-evoked inhibition at doses which blocked the GABA inhibitory responses but not those produced by glycine. The results are consistent with the hypothesis that GABA is the transmitter released by the inhibitory pallido—subthalamic pathway, and are in agreement with recent biochemical data. Nevertheless, on a few cells, strychnine blocked the GP-induced inhibition. This result is discussed in relation to the specificity of GABA and glycine antagonists.     

Putative glycine receptors in Hydra: a biochemical and behavioural study

Glycine acts as an inhibitory transmitter in the lower brain stem and spinal cord of vertebrate species, while very few data are yet available to support a similar role in invertebrate nervous systems. Here we report the identification and characterization of glycine receptors in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa) by biochemical and behavioural studies. Saturation experiments revealed the occurrence of one population of binding sites of nanomolar affinity (KD = 33 nm) and low capacity (Bmax = 79 fmol/mg protein) for [(3)H]strychnine. The addition of glycine or taurine (0.1 microm-1 mm) produced a dose-dependent inhibition of [(3)H]strychnine binding. Beta-alanine (0.1-1 mm) did not significantly affect [(3)H]strychnine binding. The pharmacological properties of these receptors compare with those of vertebrate glycine receptors. Stimulation of Hydra polyps by reduced glutathione resulted in a significant increase in the duration of mouth opening in the presence of glycine, taurine or beta-alanine. The enhancement of the response was related both to amino acid (10-100 microm) and to glutathione concentration (1-10 microm). The effects of glycine or its agonists were suppressed by strychnine (1-10 microm). D-serine, a glycine agonist at the vertebrate NMDA receptor, produced opposite effects to those of glycine. The effects of d-serine were suppressed by 5,7-dichlorokynurenic acid but not by strychnine. In vitro, [(3)H]strychnine binding was not displaced by d-serine. These results indicate a dual action of glycine in Hydra tissues. The hypothesis that NMDA receptors may also be present in this elementary nervous system is proposed.     

Experimental hyperglycinaemia--an evaluation of the efficacy of strychnine therapy in nonketotic hyperglycinaemia

A hyperglycinaemic state resembling the human disease nonketotic hyperglycinaemia (NKH) was created in male adult albino rabbits by injecting subcutaneously a ten percent aqueous glycine solution. A single dose of 300 mg./kg. caused a transient rise of plasma glycine level, but CSF glycine concentration began to rise only after an injection of 1,000 mg./kg. glycine. Repetition of the latter dose every four hours for twenty-four hours caused a gradual rise of both plasma and CSF glycine. Progressive neurological symptoms, highly resembling the early stages of NKH, developed during this prolonged loading. Numerous therapeutic approaches have been made to NKH which, if not fatal during the neonatal period, leads to severe psychomotor retardation, but only strychnine treatment (100--200 micrograms./kg/. day) has resulted in some improvement of the clinical condition of these patients. However, even the highest tolerated dose of strychnine (1 mg./kg./day) did not significantly alter the progression of neurological symptoms during glycine loading in the present study. All but one of twenty-nine rabbits succumbed during glycine loading, and only three of twenty-one survived when the glucine-antagonist strychnine was administered during glycine loading. Therefore, it seems probable that the protective effect of strychnine in a hyperglycinaemic state is of limited value. Glycine loading influenced intermediate amino acid metabolism in several ways, plasma alpha-amino-N and ammonia level rose and moderate metabolic acidosis developed during the last phase of the experiment. Profound alterations of levels of amino acids involved in glycine metabolism occurred in both blood plasma and cerebropinal fluid.