Glycine-gated Chloride Channels Research Articles

Glycine – an important neurotransmitter and cytoprotective agent

Glycine, the simplest of the amino acids, is an essential component of important biological molecules, a key substance in many metabolic reactions, the major inhibitory neurotransmitter in the spinal cord and brain stem, and an anti-inflammatory, cytoprotective, and immune modulating substance. Based on available literature, we discuss some of the important biological properties of glycine. In addition, we describe some clinical disorders where glycine plays a central role, either as an essential structural element, or through its metabolism or receptors. The past few years have witnessed a broadening of glycine research. The traditional prime interest in aspects related to its role as an inhibitory neurotransmitter in the central nervous system has been expanded to equally emphasize other organs and tissues. With the demonstration of glycine-gated chloride channels on neurons in the central nervous system, on most leukocytes, and subsequently on other cells as well, a unifying mechanism of action accounting for many of the widespread effects of glycine has been found. Glycine is a simple, easily available, and inexpensive substance with few and innocuous side-effects. The diversity of biological activities is well documented in the literature. Despite this, glycine has only gained a modest place in clinical medicine.

Benefit of Kupffer cell modulation with glycine versus Kupffer cell depletion after liver transplantation in the rat: effects on postischemic reperfusion injury, apoptotic cell death graft regeneration and survival*

Inhibition or destruction of Kupffer cells (KC) may protect against ischemia-reperfusion (IR) induced primary graft nonfunction (PNF) in liver transplantation. Besides KC activation, PNF is characterized by microvascular perfusion failure, intrahepatic leukocyte accumulation, cell death and hepatocellular dysfunction. KCs can be inactivated by different agents including gadolinium chloride (GdCl3), methyl palmitate (MP) and glycine. The effects of three KC inactivators on IR-injury after rat liver transplantation were compared in the present study. Lewis liver donors were treated with GdCl3, MP, glycine or saline (control). Liver grafts were transplanted following 24 h storage (UW solution). KC populations and IR damage were assessed by histologic analysis, quantitative real-time polymerase chain reaction (RT-PCR) and intravital microscopy. The number of hepatic ED-1 positive macrophages was diminished after GdCl3 (114.8+/-4.4/mm2 liver tissue) and MP treatment (176.0+/-5.0), versus the glycine (263.9+/-5.5) and control (272.1+/-5.6) groups. All three treatment modalities downregulated phagocytic activity for latex particles, paralleled by reduced microvascular injury (acinar perfusion index, GdCl3: 0.75+/-0.03; MP: 0.83+/-.03; glycine: 0.84+/-0.03; 0.63+/-0.03). Quantitative RT-PCR revealed elevated myeloperoxidase mRNA after glycine versus GdCl3 and MP pretreatment (3.2- and 3.4-fold, P=0.011, respectively), without difference to controls (2.9-fold of glycine). TNFalpha-mRNA was reduced after glycine- (5.2-fold), GdCl3- (19.7-fold), MP-treatment (39.5-fold) compared with controls. However, profound prevention of intrahepatic cell death and liver graft failure was solely achieved with glycine preconditioning. Different than GdCl3 and MP, glycine modulates rather than destroys KCs. Glycine appears to preserve cell viability and to TNFalpha/leukocyte dependent organ regeneration capacity, which is related to increase graft survival following liver transplantation.

Ginkgolide B preferentially blocks chloride channels formed by heteromeric glycine receptors in hippocampal pyramidal neurons of rat

It has been found recently that the platelet activating factor antagonist ginkgolide B is a selective use-dependent blocker of glycine-gated chloride channels. GABA A receptor antagonist picrotoxin is known to block α homomeric glycine (Gly) receptors, being less effective for heteromeric α1/β glycine receptors. Studying pyramidal hippocampal neurons of rat, we have confirmed that the effect of picrotoxin depends on the age of the animals. Its blocking ability was characterised by IC 50=140±12 μM and IC 50=354±43 μM for 7 and 14 days old rats, respectively, indicating at a possibly increased contribution of heteromeric receptors with animals age. We have revealed that the blocking action of ginkgolide B is subjected to a more drastic change in the same range of ages: the IC 50 value is decreased from 1.6±0.2 μM for 7days old rats to 0.27±0.01 μM for 14 days old rats. When measured on the background of ginkgolide B (1 μM), IC 50 for picrotoxin was 92±16 μM. Taken together, these findings indicate that ginkgolide B has higher affinity to heteromeric Gly receptor-gated channels than to the homomeric ones.

Ginkgolide B preferentially blocks chloride channels formed by heteromeric glycine receptors in hippocampal pyramidal neurons of rat

It has been found recently that the platelet activating factor antagonist ginkgolide B is a selective use-dependent blocker of glycine-gated chloride channels. GABAA receptor antagonist picrotoxin is known to block α homomeric glycine (Gly) receptors, being less effective for heteromeric α1/β glycine receptors. Studying pyramidal hippocampal neurons of rat, we have confirmed that the effect of picrotoxin depends on the age of the animals. Its blocking ability was characterised by IC50=140±12 μM and IC50=354±43 μM for 7 and 14 days old rats, respectively, indicating at a possibly increased contribution of heteromeric receptors with animals age. We have revealed that the blocking action of ginkgolide B is subjected to a more drastic change in the same range of ages: the IC50 value is decreased from 1.6±0.2 μM for 7days old rats to 0.27±0.01 μM for 14 days old rats. When measured on the background of ginkgolide B (1 μM), IC50 for picrotoxin was 92±16 μM. Taken together, these findings indicate that ginkgolide B has higher affinity to heteromeric Gly receptor-gated channels than to the homomeric ones.

Functional characterisation of the human α1 glycine receptor in a fluorescence-based membrane potential assay

In the present study, we have created a stable HEK293 cell line expressing the human homomeric α1 glycine receptor (GlyR) and characterised its functional pharmacology in a conventional patch-clamp assay and in the FLIPR ® Membrane Potential (FMP) assay, a fluorescence-based high throughput screening assay. In the patch-clamp assay, the α1 GlyR exhibited the properties expected from a strychnine-sensitive glycine-gated chloride channel. In the FMP assay exposure of the cell line to GlyR agonists elicited a concentration-dependent increase in fluorescent intensity, a signal that could be suppressed by pre-incubation with GlyR antagonists. Agonists and antagonists displayed EC 50 and K i values in good agreement with previously reported values from studies of recombinant α1 GlyRs and native α1β GlyRs. The rank orders of potencies was glycine > β-alanine > taurine for the agonists and RU 5135>strychnine>brucine>PMBA=picrotoxin>atropine for the antagonists. The actions of three allosteric modulators at the α1 GlyR cell line were also characterised in the FMP assay. Micromolar concentrations of Zn 2+ inhibited α1 GlyR signalling but in contrast to previous reports the metal ion did not appear to potentiate GlyR function at lower concentrations. Analogously, whereas pregnenolone sulphate inhibited α1 GlyR function, the potentiation of α1 GlyR by pregnenolone in electrophysiological studies could not be reproduced in the assay. In conclusion, the FMP assay may not be suited for sophisticated studies of GlyR pharmacology and kinetics. However, the assay offers several advantages in studies of ligand–receptor interactions. Furthermore, the assay could be highly useful in the search for structurally novel ligands acting at GlyRs.

Inhibition of hippocampal LTP by ginkgolide B is mediated by its blocking action on PAF rather than glycine receptors

Platelet-activating factor (PAF), a biologically active lipid (1- O-alkyl-2-acetyl-sn-glycero-3-phosphoholine), is identified in different regions of brain, including hippocampus. Specific PAF-activated receptors (PAFRs) are expressed in corresponding brain areas. PAF has been proposed to be a retrograde messenger of long-term potentiation (LTP): the antagonist of PAFRs, ginkgolide B (or BN52021) prevents induction of LTP. Recently it has been found that ginkgolide B is also an efficient blocker of the glycine receptor (GlyR) operated chloride channels (IC 50=270±10 nM in hippocampal pyramidal neurons). The question is as follows: is the alteration of LTP by BN52021 due to the PAF antagonism or to the inhibition of glycine-gated chloride channels? We have studied the effects of ginkgolides B and J on LTP induced in the CA1 area of rat hippocampus. Ginkgolide J which is the weakest blocker of PAFR (IC 50=54 μM, as compared to IC 50=2.5 μM for ginkgolide B) inhibits GlyR-operated channels with IC 50=2.0 μM. This assures a convenient concentration window which allows to inhibit GlyR-operated channels without affecting PAFRs. An amount of 5 μM of ginkgolide J did not prevent the induction of LTP, while ginkgolide B (5 μM) completely inhibited this phenomenon. The effect of ginkgolide B on LTP did not alter considerably if GlyRs were blocked by strychnine (2 μM). Strychnine itself had no significant effect on the induction of LTP. Both ginkgolides and strychnine significantly facilitated short-term potentiation (STP). Our data support a hypothesis according to which ginkgolides affect LTP by inhibiting PAFRs.

A wholly nutritional ‘multifocal angiostatic therapy’ for control of disseminated cancer

A great deal of effort is now being devoted to the development of new drugs that hopefully will control the spread of inoperable cancer by safely inhibiting tumor-evoked angiogenesis. However, there is growing evidence that certain practical nutritional measures have the potential to slow tumor angiogenesis, and it is reasonable to anticipate that, by combining several measures that work in distinct but complementary ways to impede the angiogenic process, a clinically useful ‘multifocal angiostatic therapy’ (MAT) might be devised. Several measures which might reasonably be included in such a protocol are discussed below, and include: a low-fat, low-glycemic index vegan diet, which may down-regulate the systemic IGF-I activity that supports angiogenesis; supplemental omega-3-rich fish oil, which has been shown to inhibit endothelial expression of Flk-1, a functionally crucial receptor for VEGF, and also can suppress tumor production of pro-angiogenic eicosanoids; high-dose selenium, which has recently been shown to inhibit tumor production of VEGF; green tea polyphenols, which can suppress endothelial responsiveness to both VEGF and fibroblast growth factor; and high-dose glycine, whose recently reported angiostatic activity may reflect inhibition of endothelial cell mitosis, possibly mediated by activation of glycine-gated chloride channels. In light of evidence that tumor-evoked angiogenesis has a high requirement for copper, copper depletion may have exceptional potential as an angiostatic measure, and is most efficiently achieved with the copper-chelating drug tetrathiomolybdate. If logistical difficulties make it difficult to acquire this experimental drug, high-dose zinc supplementation can achieve a slower depletion of the body’s copper pool, and in any case can be used as maintenance therapy to maintain an adequate level of copper depletion. A provisional protocol is offered for a nutritionally based MAT entailing a vegan diet and supplemental intakes of fish oil, selenium, green tea polyphenols, glycine, and zinc. Inasmuch as cox-2 is overexpressed in many cancers, and cAMP can boost tumor production of various angiogenic factors as well as autogenous growth factors, adjunctive use of cox-2-specific NSAIDS may be warranted in some cases.

The organochlorine pesticides γ-hexachlorocyclohexane (lindane), α-endosulfan and dieldrin differentially interact with GABA A and glycine-gated chloride channels in primary cultures of cerebellar granule cells

The neurotoxic organochlorine pesticides γ-hexachlorocyclohexane, α-endosulfan and dieldrin induce in mammals a hyperexcitability syndrome accompanied by convulsions. They reduce the GABA-induced Cl − flux. The strychnine-sensitive glycine receptor also regulates Cl −-flux inhibitory responses. We studied the effects of these compounds on Cl − channels associated with glycine receptors in cultured cerebellar granule cells in comparison to the GABA A receptor. Both GABA (EC 50: 5 μM) and glycine (EC 50: 68 μM) increased 36Cl − influx. This increase was antagonized by bicuculline and strychnine, respectively. Lindane inhibited with similar potency both GABA A (IC 50: 6.1 μM) and glycine (5.0 μM) receptors. α-Endosulfan and dieldrin inhibited the GABA A receptor (IC 50 values: 0.4 μM and 0.2 μM, respectively) more potently than the glycine receptor (IC 50 values: 3.5 μM and 3 μM, respectively). Picrotoxinin also inhibited the glycine receptor, although with low potency (IC 50>100 μM). A 3D pharmacophore model, consisting of five hydrophobic regions and one hydrogen bond acceptor site in a specific three-dimensional arrangement, was developed for these compounds by computational modelling. We propose that the hydrogen bond acceptor moiety and the hydrophobic region were responsible for the affinity of these compounds at the GABA A receptor whereas only the hydrophobic region of the molecules was responsible for their interaction with the glycine receptors. In summary, these compounds could produce neuronal hyperexcitability by blocking glycine receptors besides the GABA A receptor. We propose that two zones of the polychlorocycloalkane pesticide molecules (a lipophilic zone and a polar zone) differentially contribute to their binding to GABA A and glycine receptors.

Strychnine-sensitive glycine receptors depress hyperexcitability in rat dentate gyrus.

Previously we have shown that strychnine-sensitive glycine-gated chloride channels (GlyRs) are functionally expressed by CA1 pyramidal cells and GABAergic interneurons in mature rat hippocampal slices. We now report that glycine application to dentate granule cells and hilar interneurons recorded in acute slices from adolescent rats elicits a strychnine-sensitive current similar to glycine-mediated currents recorded in area CA1, indicating that GlyRs are also present on neurons in the dentate gyrus. This finding suggests that GlyRs have a widespread distribution in the hippocampal region. The physiological role of GlyRs in forebrain is unclear, but it is possible that these receptors mediate neuronal inhibition, similar to gamma-aminobutyric acid-A (GABA(A)) receptors and thus could be a novel target for antiepileptic therapy. Therefore we tested the hypothesis that activation of inhibitory GlyRs could suppress neuronal hyperexcitability in dentate, a brain region vulnerable to epileptic activity. In whole-cell current-clamp recordings of granule cells, we observed a membrane potential hyperpolarization followed by cessation of the action potential firing pattern in hyperexcitable slices induced by elevated extracellular K(+) or by blocking GABA(A) receptors with bicuculline. The GlyR antagonist, strychnine, prevented the antiepileptic effect of glycine. These results demonstrate that glycine, acting at GlyRs, elicits neuronal inhibition in dentate. Further, our findings suggest the possibility that these receptors could be a therapeutic target for the treatment of epilepsy.

Molecular evidence for a glycine-gated chloride channel in macrophages and leukocytes.

Recent studies have demonstrated that glycine blunts the response of Kupffer cells to endotoxin. Based on pharmacological evidence, it was hypothesized that Kupffer cells and other macrophages contain a glycine-gated chloride channel similar to the glycine receptor expressed in neuronal tissues. Moreover, glycine stimulates influx of radiolabeled chloride in Kupffer cells in a dose-dependent manner. RT-PCR was used to identify mRNA of both alpha- and beta-subunits of the glycine receptor in rat Kupffer cells, peritoneal neutrophils, and splenic and alveolar macrophages, similar to the sequence generated from rat spinal cord. Importantly, the sequence of the cloned Kupffer cell glycine receptor fragment for the beta-subunit was >95% homologous with the receptor from the spinal cord. Membranes of these cells also contain a protein that is immunoreactive with antibodies against the glycine-gated chloride channel. These data demonstrate that Kupffer cells, as well as other macrophages and leukocytes, express mRNA and protein for a glycine-gated chloride channel with both molecular and pharmacological properties similar to the channel expressed in the central nervous system.

Ethanol suppresses fast potentiation of glycine currents by glutamate.

Excitatory (glutamate) and inhibitory (GABA(A) and glycine) receptor/channels coexist in many neurons. To assess effects of ethanol on the interaction of glutamate and glycine receptors, glycine-induced current (I(Gly)) was recorded by a whole-cell patch-clamp technique from neurons freshly dissociated from the ventral tegmental area of rats. A conditioning prepulse of glutamate (1-3 s, 1 mM) significantly and reversibly potentiated responses to a pulse of glycine. This potentiation was increased when extracellular calcium was raised to 12 mM and reduced by including 10 mM 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the internal recording medium. It was not affected by 5 microM 1-N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a selective inhibitor of calcium/calmodulin-dependent protein kinase II. In a concentration-response analysis, a conditioning pulse of glutamate significantly lowered the EC(50) for glycine and increased the maximal I(Gly). Kinetic analysis of the currents indicated that glutamate slowed deactivation of glycine-gated chloride channels; therefore, glutamate may increase the affinity of glycine receptors for glycine. When coapplied with glycine, ethanol (10 mM) potentiated I(Gly) in 35% of neurons from the ventral tegmental area. In contrast, when coapplied with glutamate and glycine, ethanol suppressed the glutamate-induced potentiation of I(Gly) in these neurons. This suppression was also observed when ethanol and glycine were coapplied after a glutamate prepulse. A similar effect was observed when ethanol alone did not potentiate I(Gly). These findings suggest that glutamate-induced calcium influx modulates glycine receptors by a mechanism that can be blocked by ethanol.

Glycine blocks the increase in intracellular free Ca2+ due to vasoactive mediators in hepatic parenchymal cells.

Recently, glycine has been shown to prevent liver injury after endotoxin treatment in vivo. We demonstrated that ethanol and endotoxin stimulated Kupffer cells to release PGE(2), which elevated oxygen consumption in parenchymal cells. Because glycine has been reported to protect renal tubular cells, isolated hepatocytes, and perfused livers against hypoxic injury, the purpose of this study was to determine whether glycine prevents increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in hepatic parenchymal cells by agonists released during stress, such as with PGE(2) and adrenergic hormones. Liver parenchymal cells isolated from female Sprague-Dawley rats were cultured for 4 h in DMEM/F12 medium, and [Ca(2+)](i) in individual cells was assessed fluorometrically using the fluorescent calcium indicator fura 2. PGE(2) caused a dose-dependent increase in [Ca(2+)](i) from basal values of 130 +/- 10 to maximal levels of 434 +/- 55 nM. EGTA partially prevented this increase, indicating that either extracellular calcium or agonist binding is Ca(2+) dependent. 8-(Diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), an agent that prevents the release of Ca(2+) from intracellular stores, also partially blocked the increase in [Ca(2+)](i) caused by PGE(2), suggesting that intracellular Ca(2+) pools are involved. Together, these results are consistent with the hypothesis that both the intracellular and extracellular Ca(2+) pools are involved in the increase in [Ca(2+)](i) caused by PGE(2). Interestingly, glycine, which activates anion (i.e., chloride) channels, blocked the increase in [Ca(2+)](i) due to PGE(2) in a dose-dependent manner. Low-dose strychnine, an antagonist of glycine-gated chloride channel in the central nervous system, partially reversed the inhibition by glycine. When extracellular Cl(-) was omitted, glycine was much less effective in preventing the increase in [Ca(2+)](i) due to PGE(2). Phenylephrine, an alpha(1)-type adrenergic receptor agonist, also increased [Ca(2+)](i), as expected, from 159 +/- 20 to 432 +/- 43 nM. Glycine also blocked the increase in [Ca(2+)](i) due to phenylephrine, and the effect was also reversed by low-dose strychnine. Together, these data indicate that glycine rapidly blocks the increase in [Ca(2+)](i) in hepatic parenchymal cells due to agonists released during stress, most likely by actions on a glycine-sensitive anion channel and that this may be a major aspect of glycine-induced hepatoprotection.

Pharmacological characterization of glycine-gated chloride currents recorded in rat hippocampal slices.

An inhibitory role for strychnine-sensitive glycine-gated chloride channels (GlyRs) in mature hippocampus has been overlooked, largely due to the misconception that GlyR expression ceases early during development and to few functional studies demonstrating their presence. As a result, little is known regarding the physiological and pharmacological properties of native GlyRs expressed by hippocampal neurons. In this study, we used pharmacological tools and whole cell patch-clamp recordings of CA1 pyramidal cells and interneurons in acutely prepared hippocampal slices from 3- to 4-wk old rats to characterize these understudied receptors. We show that glycine application to recorded pyramidal cells and interneurons elicited strychnine-sensitive chloride-mediated currents (I(gly)) that did not completely desensitize in the continued presence of agonist but reached a steady state at 45-60% of the peak amplitude. Additionally, the inhibitory amino acid, taurine, which has been shown to activate GlyRs in other systems, activated GlyRs expressed by both pyramidal cells and interneurons, although with much less potency than glycine, having an EC(50) 10-fold higher. To examine the potential subunit composition of hippocampal GlyRs, we tested the effect of the GABA(A) receptor antagonist, picrotoxin, on I(gly) recorded from both cell types. At low micromolar concentrations of picrotoxin (< or =100 microM), which selectively block alpha homomeric GlyRs, I(gly) was partially attenuated in both cell types, indicating that alpha homomeric receptors are expressed by pyramidal cells and interneurons. At picrotoxin concentrations < or =1 mM, approximately 10-20% of the whole cell current remained, suggesting that alphabeta heteromeric GlyRs are also expressed because this subtype of GlyR is relatively resistant to picrotoxin antagonism. Finally, we examined whether hippocampal GlyRs are modulated by zinc. Consistent with previous reports in other preparations, zinc elicited a bidirectional modulation of GlyRs, with physiological zinc concentrations (1-100 microM) increasing whole cell currents and concentrations >100 microM depressing them. Furthermore, the same concentration of zinc that potentiates I(gly) suppressed currents mediated by the N-methyl-D-aspartate subtype of the glutamate receptor. Thus we provide a pharmacological characterization of native GlyRs expressed by both major neuron types in hippocampus and show that these receptors can be activated by taurine, an amino acid that is highly concentrated in hippocampus. Furthermore, our data suggest that at least two GlyR subtypes are present in hippocampus and that GlyR-mediated currents can be potentiated by zinc at concentrations that suppress glutamate-mediated excitability.

BN52021, a platelet activating factor antagonist, is a selective blocker of glycine-gated chloride channel

We have found that the platelet activating factor antagonist (BN52021) is an effective blocker of the glycine (Gly) receptor-mediated responses in the hippocampal pyramidal neurons of rat. Using the whole-cell voltage clamp and concentration clamp recording techniques, we investigated the mechanism underlying the inhibitory action of this terpenoid on the glycine-induced chloride current. BN52021 selectively and reversibly inhibits glycine current in a non-competitive and voltage-dependent fashion. The antagonistic effect of this substance is more pronounced at positive membrane potentials. At holding potential −70 mV and in the presence of 200 μM glycine IC 50 value for the blocking action of BN52021 was 270±10 nM. Repetitive applications of BN52021 reveal the use-dependence of its blocking action. When co-applied with strychnine (STR), a competitive glycine receptor antagonist, BN52021 does not alter the IC 50 value for strychnine. The inhibitory effect of BN52021 on gamma-aminobutyric acid (GABA) current is at least 25 times less potent than the effect on glycine current. This substance fails to affect AMPA and NMDA responses. It may be concluded that BN52021 inhibits glycine-gated Cl − channels by interacting with the pore region and does not compete for the strychnine-binding centre.

Dietary glycine prevents peptidoglycan polysaccharide-induced reactive arthritis in the rat: role for glycine-gated chloride channel.

Peptidoglycan polysaccharide (PG-PS) is a primary structural component of bacterial cell walls and causes rheumatoid-like arthritis in rats. Recently, glycine has been shown to be a potential immunomodulator; therefore, the purpose of this study was to determine if glycine would be protective in a PG-PS model of arthritis in vivo. In rats injected with PG-PS intra-articularly, ankle swelling increased 21% in 24 to 48 h and recovered in about 2 weeks. Three days prior to reactivation with PG-PS given intravenously (i.v.), rats were divided into two groups and fed a glycine-containing or nitrogen-balanced control diet. After i.v. PG-PS treatment joint swelling increased 2.1 +/- 0.3 mm in controls but only 1.0 +/- 0.2 mm in rats fed glycine. Infiltration of inflammatory cells, edema, and synovial hyperplasia in the joint were significantly attenuated by dietary glycine. Tumor necrosis factor alpha (TNF-alpha) mRNA was detected in ankle homogenates from rats fed the control diet but not in ankles from rats fed glycine. Moreover, intracellular calcium was increased significantly in splenic macrophages treated with PG-PS; however, glycine blunted the increase about 50%. The inhibitory effect of glycine was reversed by low concentrations of strychnine or chloride-free buffer, and it increased radiolabeled chloride influx nearly fourfold, an effect also inhibited by strychnine. In isolated splenic macrophages, glycine blunted translocation of the p65 subunit of NF-kappaB into the nucleus, superoxide generation, and TNF-alpha production caused by PG-PS. Further, mRNA for the beta subunit of the glycine receptor was detected in splenic macrophages. This work supports the hypothesis that glycine prevents reactive arthritis by blunting cytokine release from macrophages by increasing chloride influx via a glycine-gated chloride channel.

Dietary Glycine Prevents Peptidoglycan Polysaccharide-Induced Reactive Arthritis in the Rat: Role for Glycine-Gated Chloride Channel

Dietary Glycine Prevents Peptidoglycan Polysaccharide-Induced Reactive Arthritis in the Rat: Role for Glycine-Gated Chloride Channel

MOD-1 is a serotonin-gated chloride channel that modulates locomotory behaviour in C. elegans.

The neurotransmitter and neuromodulator serotonin (5-HT) functions by binding either to metabotropic G-protein-coupled receptors (for example, 5-HT1, 5-HT2, 5-HT4 to 5-HT7), which mediate 'slow' modulatory responses through numerous second messenger pathways, or to the ionotropic 5-HT3 receptor, a non-selective cation channel that mediates 'fast' membrane depolarizations. Here we report that the gene mod-1 (for modulation of locomotion defective) from the nematode Caenorhabditis elegans encodes a new type of ionotropic 5-HT receptor, a 5-HT-gated chloride channel. The predicted MOD-1 protein is similar to members of the nicotinic acetylcholine receptor family of ligand-gated ion channels, in particular to GABA (gamma-aminobutyric acid)- and glycine-gated chloride channels. The MOD-1 channel has distinctive ion selectivity and pharmacological properties. The reversal potential of the MOD-1 channel is dependent on the concentration of chloride ions but not of cations. The MOD-1 channel is not blocked by calcium ions or 5-HT3a-specific antagonists but is inhibited by the metabotropic 5-HT receptor antagonists mianserin and methiothepin. mod-1 mutant animals are defective in a 5-HT-mediated experience-dependent behaviour and are resistant to exogenous 5-HT, confirming that MOD-1 functions as a 5-HT receptor in vivo.

Dietary glycine blunts lung inflammatory cell influx following acute endotoxin.

Mortality associated with endotoxin shock is likely mediated by Kupffer cells, alveolar macrophages, and circulating neutrophils. Acute dietary glycine prevents mortality and blunts increases in serum tumor necrosis factor-alpha (TNF-alpha) following endotoxin in rats. Furthermore, acute glycine blunts activation of Kupffer cells, alveolar macrophages, and neutrophils by activating a glycine-gated chloride channel. However, in neuronal tissue, glycine rapidly downregulates chloride channel function. Therefore, the long-term effects of a glycine-containing diet on survival following endotoxin shock were investigated. Dietary glycine for 4 wk improved survival after endotoxin but did not improve liver pathology, decrease serum alanine transaminase, or effect TNF-alpha levels compared with animals fed control diet. Interestingly, dietary glycine largely prevented inflammation and injury in the lung following endotoxin. Surprisingly, Kupffer cells from animals fed glycine for 4 wk were no longer inactivated by glycine in vitro; however, isolated alveolar macrophages and neutrophils from the same animals were sensitive to glycine. These data are consistent with the hypothesis that glycine downregulates chloride channels on Kupffer cells but not on alveolar macrophages or neutrophils. Importantly, glycine diet for 4 wk protected against lung inflammation due to endotoxin. Chronic glycine improves survival by unknown mechanisms, but reduction of lung inflammation is likely involved.

A synthetic peptide based on a glycine-gated chloride channel induces a novel chloride conductance in isolated epithelial cells

CK 4-M2GlyR, an aqueous soluble peptide derived from the transmembrane M2 segment of the glycine-gated Cl − channel found in postsynaptic membranes of the central nervous system, has previously been shown to increase transepithelial Cl − and fluid secretion of epithelial monolayers. The goal of this study was to determine whether CK 4-M2GlyR exerts these effects via formation of a novel chloride conductance pathway, modulation of endogenous chloride channel activity, or a combination of these effects. Ionic currents were recorded from isolated epithelial cells before and after treatment with the peptide using the whole-cell configuration of the patch-clamp technique. CK 4-M2GlyR increased whole-cell Cl − currents in all epithelial cell lines that were studied, including: Madin–Darby canine kidney cells, a human colonic epithelial cell line (T84), and airway epithelial cells derived from a human cystic fibrosis patient (IB3-1). No evidence was found for modulation of endogenous Cl − channels by CK 4-M2GlyR based on both the electrophysiological properties of the observed currents and the pharmacological profile of the CK 4-M2GlyR-induced current. These results suggest that CK 4-M2GlyR increases Cl − permeability in epithelial cells directly, by forming a distinct conduction pathway in cell membranes.

Glycine-gated chloride channels in neutrophils attenuate calcium influx and superoxide production.

Recently, it was demonstrated that liver injury and TNF-alpha production as a result of endotoxin (lipopolysaccharide, LPS) were attenuated by feeding animals a diet enriched with glycine. This phenomenon was shown to be a result of, at least in part, activation of a chloride channel in Kupffer cells by glycine, which hyperpolarizes the cell membrane and blunts increases in intracellular calcium concentrations ([Ca(2+)](i)) similar to its action in the neuron. It is well known that hepatotoxicity due to LPS has a neutrophil-mediated component and that activation of neutrophils is dependent on increases in [Ca(2+)](i). Therefore, the purpose of this study was to determine if glycine affected agonist-induced increases in [Ca(2+)](i) in rat neutrophils. The effect of glycine on increases in [Ca(2+)](i) elicited either by the bacterial-derived peptide formyl-methionine-leucine-phenylalanine (FMLP) or LPS was studied in individual neutrophils using Fura-2 and fluorescence microscopy. Both FMLP and LPS caused dose-dependent increases in [Ca(2+)](i), which were maximal at 1 microM FMLP and 100 microgram/ml LPS, respectively. LPS increased intracellular calcium in the presence and absence of extracellular calcium. Glycine blunted increases in [Ca(2+)](i) in a dose-dependent manner with an IC(50) of approximately 0.3 mM, values only slightly higher than plasma levels. Glycine was unable to prevent agonist-induced increases in [Ca(2+)](i) in chloride-free buffer. Moreover, strychnine (1 microM), an antagonist of the glycine-gated chloride channel in the central nervous system, reversed the effects of glycine (1 mM) on FMLP- or LPS-stimulated increases in [Ca(2+)](i). To provide hard evidence for a glycine-gated chloride channel in the neutrophil, the effect of glycine on radioactive chloride uptake was determined. Glycine caused a dose-dependent increase in chloride uptake into neutrophils with an ED(50) of approximately 0.4 mM, an effect also prevented by 1 microM strychnine. Glycine also significantly reduced the production of superoxide anion from FMLP-stimulated neutrophils. Taken together, these data provide clear evidence that neutrophils contain a glycine-gated chloride channel that can attenuate increases in [Ca(2+)](i) and diminish oxidant production by this important leukocyte.