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1.
In the mammalian central nervous system, the neurotransmitter, glycine, acts both on an inhibitory, strychnine-sensitive receptor (GlyR) and an excitatory, strychnine-insensitive site at the NMDA receptor. Here we present electrophysiological evidence that the strychnine-sensitive glycine agonists, glycine and taurine, and the antagonist, strychnine, affect the endodermal rhythmic potential (RP) system and that the ectodermal contraction burst (CB) pacemaker system is modulated by glycine and strychnine in hydra. The RP and CB pacemaker systems are responsible for the respective elongation and contraction of hydra's body column. Activity of the CB system, quantified by the rate of contraction bursts (CBs), the number of pulses per contraction burst (P/CB), and the duration of bursts, was decreased by glycine. Glycine, coadministered with the strychnine-insensitive glycine site blocker, indole-2-carboxylic acid (I2CA), decreased RPs but not CBs or P/CB. The effect was mimicked by taurine. Strychnine increased the duration of RP production, and decreased CB duration. The effect of glycine with I2CA was counteracted by strychnine. The results support the idea that a vertebrate-like GlyR may be involved in modulating activity of the endodermal RP system and suggest that a glycine site on an NMDA receptor may be involved in the CB system.  相似文献   

2.
Recent reports have provided evidence for the presence of amino acid neurotransmitter receptor/chloride channels in human and porcine spermatozoa and their involvement in the acrosome reaction (AR). In this work we investigated whether a glycine receptor (GlyR) was present in golden hamster sperm, and whether it had a role in the hamster AR. The neuronal GlyR agonist glycine, stimulated in a dose-dependent manner, the AR of hamster spermatozoa previously capacitated for at least 3 hr. This stimulation was completely inhibited by 50 microM (+)-bicuculline and by concentrations of strychnine as low as 10-50 nM; both agents are antagonists of neuronal GlyR when used at the concentrations reported in this study. beta-Alanine, another agonist of the neuronal GlyR, also stimulated the AR. The AR-stimulatory effect of this compound was completely abolished by 50 nM strychnine. The inhibitory effect of strychnine on the glycine-induced hamster sperm AR was completely overcome by subsequent treatment with the calcium ionophore ionomycin, demonstrating that the strychnine effect was specific for GlyR. Additional binding studies with (3)[H]-strychnine, the typical radioligand used to detect GlyR in several cells, demonstrated for the first time the presence of specific binding sites for strychnine in the hamster spermatozoa. Interestingly, binding increased during in vitro capacitation, particularly in those sperm suspensions showing high percentages of AR. Taken together these results strongly suggest the presence of a GlyR in the hamster spermatozoa, with a role in the AR when activated.  相似文献   

3.
Glycine and γ-aminobutyric acid (GABA) are depolarizing during early development but the purpose is unclear. We tested the effect of altering glycine signaling in zebrafish embryos by overexpressing the potassium-chloride co-transporter type 2 (KCC2) to reverse the chloride gradient or by blocking glycine receptors with strychnine or by selectively knocking down the embryonic glycine receptor (GlyR KD). Using a variety of markers we observed in all three cases a reduction of all types of spinal interneuron populations examined, indicating that glycine modulates their overall differentiation rather than choice of cell fate. Other cell populations (motor, sensory, and glial cells) were unaffected. As glycine appeared to act preceding neural and synaptic development, we examined the bandoneon (beo) mutant in which glycine receptors are functional but not clustered at synapses. Neural populations in beo embryos appeared normal, suggesting a paracrine action of circulating glycine in promoting interneuron differentiation.  相似文献   

4.
J Kuhse  V Schmieden  H Betz 《Neuron》1990,5(6):867-873
Agonist activation of the inhibitory glycine receptor (GlyR) in the adult vertebrate CNS is efficiently antagonized by the alkaloid strychnine. Here, we describe a novel rat GlyR alpha subunit cDNA (alpha 2*) that generates chloride channels of low strychnine sensitivity upon expression in Xenopus oocytes. Comparison with the highly homologous human alpha 2 polypeptide and site-directed mutagenesis identified a single amino acid exchange at position 167 that causes the altered pharmacology of alpha 2* receptors. Amplification by the polymerase chain reaction revealed a strong decrease in alpha 2* mRNA abundancy during postnatal spinal cord development. These data indicate that alpha 2* represents a ligand binding subunit of the previously identified neonatal GlyR isoform of low strychnine affinity.  相似文献   

5.
Phagocytes form engulfment pseudopodia at the contact area with their target particle by a process resembling cell volume (CV) regulatory mechanisms. We evaluated whether the osmoregulatory active neutral amino acid glycine, which contributes to CV regulation via activation of sodium-dependent neutral amino acid transporters (SNATs) improves phagocytosis in isotonic and hypertonic conditions in the murine microglial cell line BV-2 and primary microglial cells (pMG). In BV-2 cells and pMG, RT-PCR analysis revealed expression of SNATs (Slc38a1, Slc38a2), but not of GlyRs (Glra1–4). In BV-2 cells, glycine (5 mM) led to a rapid Na+-dependent depolarization of membrane potential (V mem). Furthermore, glycine increased CV by about 9 %. Visualizing of phagocytosis of polystyrene microspheres by scanning electron microscopy revealed that glycine (1 mM) increased the number of BV-2 cells containing at least one microsphere by about 13 %. Glycine-dependent increase in phagocytosis was suppressed by the SNAT inhibitor α-(methylamino)isobutyric acid (MeAIB), by replacing extracellular Na+ with choline, and under hypertonic conditions, but not by the GlyR antagonist strychnine or the GlyR agonist taurine. Interestingly, hypertonicity-induced suppression of phagocytosis was rescued by glycine. These findings demonstrate that glycine increases phagocytosis in iso- and hypertonic conditions by activation of SNATs.  相似文献   

6.
We have investigated the effect of chemical reagents that modify sulfhydryl groups on the ligand binding properties of the glycine receptor (GlyR). The Hill coefficient (nH) for the displacement of [3H]strychnine binding by glycine was increased from approximately 0.8 to values significantly above 1 (approximately 1.2-1.4) in membranes pretreated with the disulfide-reducing agent dithiothreitol or glutathione. However, the affinity of strychnine or glycine for the GlyR was not affected by these treatments. This indicates that several glycine binding sites interact cooperatively for displacing bound strychnine under such experimental circumstances. A similar increase in the nH for glycine has been observed when the temperature of the binding assay was increased to 37 degrees C. Combination of dithiothreitol pretreatment and increased binding temperature led to nH variations similar to those observed with either of these treatments alone, a finding suggesting that their mechanisms of action are not independent. Conversely, modification of rat spinal cord membranes or of purified and reconstituted GlyR preparations with the sulfhydryl-alkylating agent N-ethylmaleimide or fluorescein-maleimide decreased nH values to approximately 0.5, without affecting glycine or strychnine affinities. This effect may be caused by an increased heterogeneity of GlyR populations. It is interesting that occupancy of the receptor by glycine or beta-alanine (but not by antagonists) specifically protects from the effects of the different sulfhydryl reagents. Moreover, the presence of some of the Eccles' anions, i.e., anions that permeate through the channels associated with GlyRs and gamma-aminobutyric acidA receptors, seems to be required for the action of both dithiothreitol and N-ethylmaleimide.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

7.
Glycine is a major inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates and invertebrates. The postsynaptic receptor for this amino acid is an oligomeric glycoprotein which, upon binding of glycine, transiently forms an anion-selective transmembrane channel. Agonist-mediated receptor activation is antagonized by strychnine, a high-affinity ligand of the glycine receptor (GlyR). Biochemical and immunological data show that affinity-purified preparations of the mammalian GlyR contain three polypeptides of Mr 48,000, 58,000 and 93,000. These polypeptides have different functional properties and/or topologies in the postsynaptic membrane of the glycinergic synapse. The primary sequence of the Mr 48,000 subunit deduced by cDNA cloning exhibits structural and amino-acid homology to nicotinic acetylcholine and GABAa receptor proteins, indicating a common evolutionary relationship between the different neurotransmitter-gated ion channels of excitable membranes. Monoclonal antibodies against the GlyR allow its histochemical localization in different regions of the CNS. GlyR deficiencies have been implicated in the pathogenesis of spasticity and spinal cord degeneration in mouse and man.  相似文献   

8.
We studied 3H-glycine and 3H-strychnine specific binding to glycine receptor (GlyR) in intact isolated frog retinas. To avoid glycine binding to glycine uptake sites, experiments were performed at low ligand concentrations in a sodium-free medium. The binding of both radiolabeled ligands was saturated. Scatchard analysis of bound glycine and strychnine revealed a KD of 2.5 and 2.0 M, respectively. Specific binding of glycine was displaced by -alanine, sarcosine, and strychnine. Strychnine binding was displaced 50% by glycine, and sarcosine. Properties of the strychnine-binding site in the GlyR were modified by sarcosine. Binding of both radioligands was considerably reduced by compounds that inhibit or activate adenylate cyclase and increased cAMP levels. A phorbol ester activator of PKC remarkably decreased glycine and strychnine binding. These results suggest modulation of GlyR in response to endogenous activation of protein kinases A and C, as well as protein phosphorylation modulating GlyR function in retina.  相似文献   

9.
Two cDNAs encoding variants (alpha 1 and alpha 2) of the strychnine binding subunit of the inhibitory glycine receptor (GlyR) were isolated from a human fetal brain cDNA library. The predicted amino acid sequences exhibit approximately 99% and approximately 76% identity to the previously characterized rat 48 kd polypeptide. Heterologous expression of the human alpha 1 and alpha 2 subunits in Xenopus oocytes resulted in the formation of glycine-gated strychnine-sensitive chloride channels, indicating that both polypeptides can form functional GlyRs. Using a panel of rodent-human hybrid cell lines, the gene encoding alpha 2 was mapped to the short arm (Xp21.2-p22.1) of the human X chromosome. In contrast, the alpha 1 subunit gene is autosomally located. These data indicate molecular heterogeneity of the human GlyR at the level of alpha subunit genes.  相似文献   

10.
A novel truncated form (residues 1-214, with a randomized C-terminal tail) of the ligand-binding extracellular domain (ECD) of the human alpha1 glycine receptor (GlyR), with amino acids from the corresponding sequence of an acetylcholine binding protein (AChBP) substituted for two relatively hydrophobic membrane-proximal loops, was overexpressed using a baculovirus expression system. The mutant GlyR ECD, named GlyBP, was present in both soluble and membrane-associated fractions after cell lysis, though only the latter appeared to be in a native-like conformation capable of binding strychnine, a GlyR specific antagonist. The membrane-associated GlyBP was solubilized, and detergent/lipid/protein micelles were affinity purified. After detergent removal, GlyBP may be isolated in either aqueous or vesicular form. Binding assays and spectroscopic studies using circular dichroism and FRET are consistent with both forms adopting equivalent native-like conformations. Thus, GlyBP may be isolated as a soluble or membrane-associated assembly that serves as a structural and functional homologue of the ECD of GlyR.  相似文献   

11.
12.
The effects of the antihelmintic, ivermectin, were investigated in recombinantly expressed human alpha(1) homomeric and alpha(1)beta heteromeric glycine receptors (GlyRs). At low (0.03 microm) concentrations ivermectin potentiated the response to sub-saturating glycine concentrations, and at higher (> or =0.03 microm) concentrations it irreversibly activated both alpha(1) homomeric and alpha(1)beta heteromeric GlyRs. Relative to glycine-gated currents, ivermectin-gated currents exhibited a dramatically reduced sensitivity to inhibition by strychnine, picrotoxin, and zinc. The insensitivity to strychnine could not be explained by ivermectin preventing the access of strychnine to its binding site. Furthermore, the elimination of a known glycine- and strychnine-binding site by site-directed mutagenesis had little effect on ivermectin sensitivity, demonstrating that the ivermectin- and glycine-binding sites were not identical. Ivermectin strongly and irreversibly activated a fast-desensitizing mutant GlyR after it had been completely desensitized by a saturating concentration of glycine. Finally, a mutation known to impair dramatically the glycine signal transduction mechanism had little effect on the apparent affinity or efficacy of ivermectin. Together, these findings indicate that ivermectin activates the GlyR by a novel mechanism.  相似文献   

13.
The interaction of strychnine (1 mg/kg egg weight), glycine (100 mg/kg egg weight) and GABA (103 mg/kg egg weight) on spontaneous motor activity recorded by the method of Kovach (1970) in intact eggs was studied in chick embryos from the 11th to 21st day of incubation. In 11- and 13-day embryos, neither of the amino acids influenced strychnine activation of spontaneous motility. From the 15th incubation day, strychnine activation was distinctly affected by both amino acids, but the maximum effect was observed on the 19th day. Glycine had a stronger inhibitory effect, since it prevented strychnine convulsions from developing, whereas GABA only modified them. It can be concluded from the results that glycine-sensitive and GABA-sensitive mechanisms of embryonal spontaneous motility do not begin to take effect in chick embryos until the 15th day of incubation.  相似文献   

14.
15.
Y Lu  J Zhang  B Ma  K Li  X Li  H Bai  Q Yang  X Zhu  J Ben  Q Chen 《Neurochemistry international》2012,61(5):649-658
Glycine is a cytoprotector to protect cells against ischemic damage by counteracting neuronal depolarization. However, whether it can directly inhibit neuronal apoptosis is unknown. In this study, we demonstrated that glycine could attenuate ischemia/reperfusion (I/R) induced cerebral infarction and improved neurological outcomes in mice. The protective effect of glycine was associated with reduction of terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) positive cells, deactivation of phosphor-JNK, inhibition of caspase-3 cleavage, down-regulation of FasL/Fas, and up-regulation of bcl-2 and bcl-2/bax in the mouse I/R penumbra. The beneficial effect of glycine against oxygen and glucose deprivation (OGD) induced injury was also confirmed in SH-SY5Y cells as well as in primary cultured neurons, which was significantly dampened by knockdown of glycine receptor α1 (GlyR α1) with siRNA transfection or by preventing glycine binding with glycine receptor using a specific antibody against glycine receptor. These results suggest that glycine antagonize cerebral I/R induced injury by inhibiting apoptosis in mice. Glycine could block both extrinsic and intrinsic apoptotic pathways for which GlyR may be required.  相似文献   

16.
Glycine receptors (GlyRs) in the nucleus accumbens (nAc) have recently been suggested to be involved in the reinforcing and dopamine-elevating properties of ethanol via a neuronal circuitry involving the VTA. Apart from ethanol, both glycine and taurine have the ability to modulate dopamine output via GlyRs in the same brain region. In the present study, we wanted to explore whether yet another endogenous ligand for the GlyR, β-alanine, had similar effects. To this end, we monitored dopamine in the nAc by means of in vivo microdialysis and found that local perfusion of β-alanine increased dopamine output. In line with previous observations investigating ethanol, glycine and taurine, the competitive GlyR antagonist strychnine completely blocked the dopamine elevation. The present results suggest that β-alanine has the ability to modulate dopamine levels in the nAc via strychnine-sensitive GlyRs, and are consistent with previous studies suggesting the importance of this receptor for modulating dopamine output.  相似文献   

17.
Functional glycine receptors (GlyRs) are enriched in the hippocampus, but their roles in synaptic transmission are unclear. In this study, we examined the effect of GlyR activation on paired-pulse stimulation of the whole-cell postsynaptic currents (PSCs) in the Schaffer-CA1 synapses in rat hippocampal slices. Bath application of glycine reduced the amplitude of PSCs, accompanied by an increase in holding current and resting conductance. Moreover, glycine application increased the paired-pulse ratio (PPR) of PSCs significantly, an effect largely abolished by the GlyR specific antagonist strychnine. Interestingly, glycine application had no significant effect on either the amplitude or the PPR of excitatory postsynaptic currents (EPSCs). Our findings suggest that GlyR activation regulates hippocampal short-term plasticity by altering GABAergic neurotransmission.  相似文献   

18.
Cultured human cells were transfected with cloned rat glycine receptor (GlyR) 48 kd subunit cDNA. In these cells glycine elicited large chloride currents (up to 1.5 nA), which were blocked by nanomolar concentrations of strychnine. However, no corresponding high-affinity binding of [3H]strychnine was detected in membrane preparations of the transfected cells. Analysis by monoclonal antibodies specific for the 48 kd subunit revealed high expression levels of this membrane protein. After solubilization, the 48 kd subunit behaved as a macromolecular complex when analyzed by sucrose density centrifugation. Approximately 50% of the solubilized complex bound specifically to a 2-aminostrychnine affinity column, indicating the existence of low-affinity antagonist binding sites on most of the expressed GlyR protein. Thus, the 48 kd strychnine binding subunit efficiently assembles into high molecular weight complexes, resembling the native spinal cord GlyR. However, formation of functional receptor channels of high affinity for strychnine occurs with low efficiency.  相似文献   

19.
The cation‐chloride co‐transporters are important regulators of the cellular Cl homeostasis. Among them the Na+‐K+?2Cl? co‐transporter (NKCC1) is responsible for intracellular chloride accumulation in most immature brain structures, whereas the K+‐Cl? co‐transporter (KCC2) extrudes chloride from mature neurons, ensuring chloride‐mediated inhibitory effects of GABA/glycine. We have shown that both KCC2 and NKCC1 are expressed at early embryonic stages (E11.5) in the ventral spinal cord (SC). The mechanisms by which KCC2 is prematurely expressed are unknown. In this study, we found that chronically blocking glycine receptors (GlyR) by strychnine led to a loss of KCC2 expression, without affecting NKCC1 level. This effect was not dependent on the firing of Na+ action potentials but was mimicked by a Ca2+‐dependent PKC blocker. Blocking the vesicular release of neurotransmitters did not impinge on strychnine effect whereas blocking volume‐sensitive outwardly rectifying (VSOR) chloride channels reproduced the GlyR blockade, suggesting that KCC2 is controlled by a glycine release from progenitor radial cells in immature ventral spinal networks. Finally, we showed that the strychnine treatment prevented the maturation of rhythmic spontaneous activity. Thereby, the GlyR‐activation is a necessary developmental process for the expression of functional spinal motor networks. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 764–779, 2016  相似文献   

20.
Glycine and GABA are depolarizing during early development, but the purpose of this paradoxical chloride‐mediated depolarization remains unclear, especially at early stages. It was previously reported that suppressing glycine signaling from the beginning of development in zebrafish embryos caused an abnormal maintenance of the progenitor population and a specific reduction of spinal interneurons but not of other cell populations. Here, we show that cells including progenitors in the embryonic spinal cord had occasional spontaneous, glycine‐mediated calcium transients that were blocked by the glycine antagonist strychnine and the L‐type calcium channel blocker nifedipine. As shown previously for chronic block by strychnine, block of these transients by nifedipine reduced interneuron differentiation. Our results indicate that glycinergic depolarization of neural progenitors evokes spontaneous calcium transients that may enhance the interneuron neurogenic program. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013  相似文献   

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