A novel physiological mechanism of glycine‐induced immunomodulation: Na<sup>+</sup>‐coupled amino acid transporter currents in cultured brain macrophages

Schilling, Tom; Eder, Claudia

doi:10.1113/jphysiol.2004.070763

Cited by 34 publications

(35 citation statements)

References 23 publications

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“…Reported values for intracellular Na + (Na + i ) in microglia range from 2-7 mM under resting conditions, to 5-11 mM after stimulation (e.g., with glycine). 42 To calculate E NCX , we first converted concentrations (c) to activities (a ion ) using the formula: a ion = f ion c ion where log f ion = (-0.51z 2 √I)_ (1+√I), and I is ionic strength. Setting Na + i at 5 mM, and Ca 2+ i at 70 nM (the mean resting value determined by calibrating the Fura-2 signal), and taking the bath concentrations of 135 mM Na + and 1 mM Ca 2+ , the calculated E NCX is +9 mV.…”

Section: Resultsmentioning

confidence: 99%

“…Extracellular glycine, which increases in ischemia, neurotrauma and epilepsy, elevates internal Na + in microglia through Na + -coupled neutral amino acid transporters. 42 In astrocytes, ischemia and reperfusion dramatically increase internal Na + through a Na + /K + /Cl -cotransporter, 57 59 Reversed Na + /Ca 2+ exchange is relevant to a broad range of CNS pathologies. It appears to mediate 'Ca 2+ -paradox injury', which is delayed cell death due to a persistent rise in internal Ca 2+ after cells are exposed to Ca 2+ -free solutions, then reperfused with normal Ca 2+ .…”

Section: Discussionmentioning

confidence: 99%

“…Especially under pathological conditions, several pathways can potentially cause a rise in cytoplasmic Na + . For instance, microglia have Na + -permeable ion channels, including non-selective cation channels, 42 and ionotropic purinergic receptors [50][51][52] (reviewed in refs. 15 and 53) that can be activated by ATP release from damaged cells.…”

Section: Discussionmentioning

confidence: 99%

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Reversed Na⁺/Ca²⁺Exchange Contributes to Ca²⁺Influx and Respiratory Burst in Microglia

Newell

Stanley²,

Schlichter

2007

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KEy WordSmicroglia activation, brain macrophage, reversed sodium-calcium exchange, Slc8a, NCX1-3, superoxide production, quantitative real-time RT-PCR, phagocytosis, Ca 2+ paradox, perforated patch, SBFI imaging, Fura-2 imaging AbSTrACTPhagocytosis and the ensuing NADPH-mediated respiratory burst are important aspects of microglial activation that require calcium ion (Ca 2+ ) influx. However, the specific Ca 2+ entry pathway(s) that regulates this mechanism remains unclear, with the best candidates being surface membrane Ca 2+ -permeable ion channels or Na + /Ca 2+ exchangers. In order to address this issue, we used quantitative real-time RT-PCR to assess mRNA expression of the Na + /Ca 2+ exchangers, Slc8a1-3/NCX1-3, before and after phagocytosis by rat microglia. All three Na + /Ca 2+ exchangers were expressed, with mRNA levels of NCX1 > NCX3 > NCX2, and were unaltered during the one hour phagocytosis period. We then carried out a biophysical characterization of Na + /Ca 2+ exchanger activity in these cells. To investigate conditions under which Na + /Ca 2+ exchange was functional, we used a combination of perforated patch-clamp analysis, fluorescence imaging of a Ca 2+ indicator (Fura-2) and a Na + indicator (SBFI), and manipulations of membrane potential and intracellular and extracellular ions. Then, we used a pharmacological toolbox to compare the contribution of Na + /Ca 2+ exchange with candidate Ca 2+ -permeable channels, to the NADPH-mediated respiratory burst that was triggered by phagocytosis. We find that inhibiting the reversed mode of the Na + /Ca 2+ exchanger with KB-R7943, dose dependently reduced the phagocytosis-stimulated respiratory burst; whereas, blockers of store-operated Ca 2+ channels or L-type voltage-gated Ca 2+ channels had no effect. These results provide evidence that Na + /Ca 2+ exchangers are potential therapeutic targets for reducing the bystander damage that often results from microglia activation in the damaged CNS. AbbrEviATioNSCR3, complement receptor 3; DPI, diphenylene iodonium; FBS, fetal bovine serum; HPRT1, hypoxanthine guanine phosphoribosyl transferase; IFNg, interferon-gamma; IL1b, interleukin 1 beta; NADPH, nicotinamide adenine dinucleotide phosphate; NCX, Na + / Ca 2+ exchanger; NMDG + , n-methyl d-glucamine; qRT-PCR, quantitative real-time reverse transcriptase polymerase chain reaction; SERCA, smooth endoplasmic reticulum calcium ATPase; SOC, store-operated Ca 2+ channels; TNFa, tumor necrosis factor alpha

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Reversed Na⁺/Ca²⁺Exchange Contributes to Ca²⁺Influx and Respiratory Burst in Microglia

Newell

Stanley²,

Schlichter

2007

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“…Effects of glycine on monocytes [12], Kupffer cells [13] or microglial cells [14,15] are mediated either by ionotropic glycine receptors (GlyRs) or by the Na + -dependent neutral amino acid transporter (SNAT; a member of the Slc38 System A amino acid transporter family). In Kupffer cells activation of GlyRs causes a hyperpolarization of the cell membrane potential (V mem ) [13,16], whereas a GlyRindependent, but SNAT-dependent effect of glycine appears in microglial cells [14,15]. We have shown in microglial cells that glycine uptake via SNAT depolarizes V mem due to Na + influx, which is unaffected by the GlyR antagonist strychnine, but is inhibited by the specific partial SNAT antagonist α-(methylamino)isobutyric acid (MeAIB).…”

Section: Introductionmentioning

confidence: 99%

Glycine Induces Migration of Microglial BV-2 Cells via SNAT-Mediated Cell Swelling

Kittl

Dobias

Beyreis

et al. 2018

Cell Physiol Biochem

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Background/Aims: The neutral, non-essential amino acid glycine has manifold functions and effects under physiological and pathophysiological conditions. Besides its function as a neurotransmitter in the central nervous system, glycine also exerts immunomodulatory effects and as an osmolyte it participates in cell volume regulation. During phagocytosis, glycine contributes to (local) cell volume-dependent processes like lamellipodium formation. Similar to the expansion of the lamellipodium we assume that glycine also affects the migration of microglial cells in a cell volume-dependent manner. Methods: Mean cell volume (MCV) and cell migration were determined using flow cytometry and trans-well migration assays, respectively. Electrophysiological recordings of the cell membrane potential (V_mem) and swelling-dependent chloride (Cl^-) currents (ICl_swell, VSOR, VRAC) were performed using the whole-cell patch clamp technique. Results: In the murine microglial cell line BV-2, flow cytometry analysis revealed that glycine (5 mM) increases the MCV by ∼9%. The glycine-dependent increase in MCV was suppressed by the partial sodium-dependent neutral amino acid transporter (SNAT) antagonist MeAIB and augmented by the Cl^- current blocker DCPIB. Electrophysiological recordings showed that addition of glycine activates a Cl^- current under isotonic conditions resembling features of the swelling-activated Cl^- current (ICl_swell). The cell membrane potential (V_mem) displayed a distinctive time course after glycine application; initially, glycine evoked a rapid depolarization mediated by Na⁺-coupled glycine uptake via SNAT, followed by a further gradual depolarization, which was fully suppressed by DCPIB. Interestingly, glycine significantly increased migration of BV-2 cells, which was suppressed by MeAIB, suggesting that SNAT is involved in the migration process of microglial cells. Conclusion: We conclude that glycine acts as a chemoattractant for microglial cells presumably by a cell volume-dependent mechanism involving SNAT-mediated cell swelling.

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“…GlyR 2-subunit transcripts predominate in the embryonic and neonatal brain and spinal cord, but are replaced Recently, several unexpected properties of glycine have been discovered. Like the structurally related nicotinic acetylcholine receptors [8], GlyR were shown to be expressed in peripheral, non-neuronal tissues and cells like macrophages and leukocytes suggesting immune-modulatory functions [9,10].Glycine is widely used in cosmetic products because of a suggested beneficial effect on skin collagen synthesis ("skin conditioning" according to INCI). Since glycine has no side chain, it can fit into many places where no other amino acid can.…”

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confidence: 99%

Glycine Receptors are Present in Human Epidermis

Booken¹,

Henrich-Kellner²,

Klein³

et al. 2008

TODJ

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Abstract:The inhibitory glycine receptor (GlyR) is a member of the nicotinoid receptor superfamily. This heteropentameric Cl -channel is composed of different (1-4) and a -subunit and mediates fast synaptic transmission in the central nervous system. Since glycine, the natural ligand of GlyR has been found to enhance epidermal barrier recovery; we aimed at characterizing GlyR distribution in human skin and their function in skin physiology. We detected differentsubunits and the -GlyR subunit on mRNA and protein level in human skin and cultured keratinocytes and fibroblasts. In cultured human keratinocytes but not in fibroblasts, glycine induced proliferation. Epidermis-equivalents were significantly thicker than control if cultured in the presence of glycine. In human skin, GlyR immunoreactivity (IR) was detected in the upper epidermal layers. In eczema and psoriasis, GlyR IR was reduced in areas with parakeratosis suggesting a role of GlyR in terminal differentiation and epidermal barrier control.Keywords: Epidermal barrier, chloride, strychnine, amino acid, skin. INTROCUCTIONStructurally, glycine (aminoethanoic acid, C 2 H 5 NO 2 ) is the simplest of the 20 standard proteinogenic amino acids, and yet it has a number of important and distinct functions in the body [1]. In addition to its function as part of peptides and proteins, glycine is a major inhibitory neurotransmitter in adult spinal cord and brain stem, where it controls both motor and sensory pathways. After its release from the presynaptic terminals of glycinergic interneurons, glycine exerts multiple functions in the central nervous system, as an inhibitory neurotransmitter through activation of specific, Cl --permeable, ligand-gated ionotropic receptors (GlyR) and as an obligatory co-agonist with glutamate on the activation of N-methyl-D-aspartate (NMDA) receptors [2,3]. The hyperpolarizing action of glycine is selectively antagonized by strychnine, the most potent GlyR antagonist known [4].The GlyR of different species are composed of two glycosylated integral membrane proteins, the 1-4-subunit of 48 kDa and the -subunit of 58 kDa. In addition, in neuronal tissues, there is an associated peripheral membrane protein of 93 kDa named gephyrin [5][6][7]. The primary structures of GlyR -and -subunits deduced by molecular cloning show significant sequence and structural similarity to nicotinic acetylcholine receptor (nAChR), -aminobutyric acid type A (GABA A) receptor, and serotonin type 3 (5HT3) receptor proteins. In situ hybridization showed that the different GlyR subunit genes exhibit unique spatial and temporal expression patterns in spinal cord, brain stem, and some higher brain regions. GlyR 2-subunit transcripts predominate in the embryonic and neonatal brain and spinal cord, but are replaced Recently, several unexpected properties of glycine have been discovered. Like the structurally related nicotinic acetylcholine receptors [8], GlyR were shown to be expressed in peripheral, non-neuronal tissues and cells like macrophages and leukocytes sugge...

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A novel physiological mechanism of glycine‐induced immunomodulation: Na⁺‐coupled amino acid transporter currents in cultured brain macrophages

Cited by 34 publications

References 23 publications

Reversed Na⁺/Ca²⁺Exchange Contributes to Ca²⁺Influx and Respiratory Burst in Microglia

Reversed Na⁺/Ca²⁺Exchange Contributes to Ca²⁺Influx and Respiratory Burst in Microglia

Glycine Induces Migration of Microglial BV-2 Cells via SNAT-Mediated Cell Swelling

Glycine Receptors are Present in Human Epidermis

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A novel physiological mechanism of glycine‐induced immunomodulation: Na+‐coupled amino acid transporter currents in cultured brain macrophages

Cited by 34 publications

References 23 publications

Reversed Na+/Ca2+Exchange Contributes to Ca2+Influx and Respiratory Burst in Microglia

Reversed Na+/Ca2+Exchange Contributes to Ca2+Influx and Respiratory Burst in Microglia

Glycine Induces Migration of Microglial BV-2 Cells via SNAT-Mediated Cell Swelling

Glycine Receptors are Present in Human Epidermis

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A novel physiological mechanism of glycine‐induced immunomodulation: Na⁺‐coupled amino acid transporter currents in cultured brain macrophages

Reversed Na⁺/Ca²⁺Exchange Contributes to Ca²⁺Influx and Respiratory Burst in Microglia

Reversed Na⁺/Ca²⁺Exchange Contributes to Ca²⁺Influx and Respiratory Burst in Microglia