Metabotropic glutamate receptor expression in olfactory receptor neurons from the channel catfish,Ictalurus punctatus

Metabotropic glutamate receptors (mGluRs) were identified in olfactory receptor neurons of the channel catfish, Ictalurus punctatus, by polymerase chain reaction. DNA sequence analysis confirmed the presence of two subtypes, mGluR1 and mGluR3, that were coexpressed with each other and with the putative odorant receptors within single olfactory receptor neurons. Immunocytochemical data showed that both mGluR subtypes were expressed in the apical dendrites and some cilia of olfactory neurons. Pharmacological analysis showed that antagonists to each mGluR subtype significantly decreased the electrophysiological response to odorant amino acids. α-Methyl-L -CCG1/(2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), a known antagonist to mGluR3, and (S)-4-carboxyphenylglycine (S-4CPG), a specific antagonist to mGluR1, each significantly reduced olfactory receptor responses to L -glutamate. S-4CPG and MCCG reduced the glutamate response to 54% and 56% of control, respectively, which was significantly greater than their effect on a neutral amino acid odorant, methionine. These significant reductions of odorant response by the antagonists, taken with the expression of these receptors throughout the dendritic and ciliated portions of some olfactory receptor neurons, suggest that these mGluRs may be involved in olfactory reception and signal transduction. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 94–104, 1998     

Induction of Bcl-2 by functional regulation of G-protein coupled receptors protects from oxidative glutamate toxicity by increasing glutathione

Glutamate treatment depletes hippocampal HT22 cells of glutathione, which renders the cells incapable to reduce reactive oxygen species and ultimately cumulates in cell death by oxidative stress. HT22 cells resistant to glutamate displayed increased phosphorylation of cAMP-response-element binding (CREB) and decreased ERK1/2 suggestive of differences in signal transmission. We investigated the amount of candidate G-protein-coupled receptors involved in this resistance and found an increase in mRNA for receptors activated by the vasoactive intestinal peptide VIP (VPAC₂, 12.6-fold) and glutamate like the metabotropic glutamate receptor mGlu₁ (5.3-fold). Treating cells with VIP and glutamate led to the same changes in protein phosphorylation observed in resistant cells and induced the proto-oncogene Bcl-2. Bcl-2 overexpression protected by increasing the amount of intracellular glutathione and Bcl-2 knockdown by small interfering RNAs (siRNA) increased glutamate susceptibility of resistant cells. Other receptors upregulated in this paradigm might represent useful targets in the treatment of neurological diseases associated with oxidative stress.     

Induction of Bcl-2 by functional regulation of G-protein coupled receptors protects from oxidative glutamate toxicity by increasing glutathione

Glutamate treatment depletes hippocampal HT22 cells of glutathione, which renders the cells incapable to reduce reactive oxygen species and ultimately cumulates in cell death by oxidative stress. HT22 cells resistant to glutamate displayed increased phosphorylation of cAMP-response-element binding (CREB) and decreased ERK1/2 suggestive of differences in signal transmission. We investigated the amount of candidate G-protein-coupled receptors involved in this resistance and found an increase in mRNA for receptors activated by the vasoactive intestinal peptide VIP (VPAC₂, 12.6-fold) and glutamate like the metabotropic glutamate receptor mGlu₁ (5.3-fold). Treating cells with VIP and glutamate led to the same changes in protein phosphorylation observed in resistant cells and induced the proto-oncogene Bcl-2. Bcl-2 overexpression protected by increasing the amount of intracellular glutathione and Bcl-2 knockdown by small interfering RNAs (siRNA) increased glutamate susceptibility of resistant cells. Other receptors upregulated in this paradigm might represent useful targets in the treatment of neurological diseases associated with oxidative stress.     

Cooperative action of glutamate transporters and cystine/glutamate antiporter system Xc- protects from oxidative glutamate toxicity

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for cell death by oxidative stress. In this paradigm, an excess of extracellular glutamate blocks the glutamate/cystine-antiporter system Xc-, depleting the cell of cysteine, a building block of the antioxidant glutathione. Loss of glutathione leads to the accumulation of reactive oxygen species and eventually cell death. We selected cells resistant to oxidative stress, which exhibit reduced glutamate-induced glutathione depletion mediated by an increase in the antiporter subunit xCT and system Xc- activity. Cystine uptake was less sensitive to inhibition by glutamate and we hypothesized that glutamate import via excitatory amino acid transporters and immediate re-export via system Xc- underlies this phenomenon. Inhibition of glutamate transporters by l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) and DL-threo-beta-benzyloxyaspartic acid (TBOA) exacerbated glutamate-induced cell death. PDC decreased intracellular glutamate accumulation and exacerbated glutathione depletion in the presence of glutamate. Transient overexpression of xCT and the glutamate transporter EAAT3 cooperatively protected against glutamate. We conclude that EAATs support system Xc- to prevent glutathione depletion caused by high extracellular glutamate. This knowledge could be of use for the development of novel therapeutics aimed at diseases associated with depletion of glutathione like Parkinson's disease.     

Up-regulation of Metabotropic glutamate receptor 3 (mGluR3) in rat fibrosis and cirrhosis model of persistent hypoxic condition

Glutamate is the major excitatory neurotransmitter in the central nervous system, and evidence for peripheral glutamatergic fibers in mammals is still lacking. However, glutamate receptors have been identified in peripheral organs, including taste buds, myenteric plexus, and pancreatic islet cell. Protection against anoxic damage could also be explained by mechanisms mediated by postsynaptic mGluR2 or mGluR3, such as the inhibition of membrane excitability resulting from a reduction of cAMP formation by a G-protein-dependent modulation of ion channels. In addition, activation of mGluR3 present in glial cells may contribute to neuroprotection by enhancing the production of death. Thus, mGluR2/3 behaves potentially as a major defensive mechanism anoxia-tolerant species. There are a few reports for the regional pattern of hypoxic damage, which was inversely related to the expression of mGluR2/3. The aim of this study was to characterize the expression of mGluR3 in hypoxic liver in experimental model of rat liver. Proteomic analysis of protein extracts from CCl₄–induced cirrhotic liver revealed the presence␣of the mGluR3. The presence of mGluR3 in the cirrhotic liver was confirmed by immunohistochemical analysis. There were a number of macrophages expressing mGluR3 mainly in the fibrous septa. After 2 weeks recovery, however, most of mGluR3 positive macrophages disappeared with collagen fibers. These results demonstrate that mGluR3 involved in the liver in response to persistent hypoxic status such as fibrotic/cirrhotic condition, and suggest that the expression of mGluR3 may be a key role functional metabolism and viability in the liver by interacting with the glutamate receptors in vivo.     

Selenoprotein P protects endothelial cells from oxidative damage by stimulation of glutathione peroxidase expression and activity

A major fraction of the essential trace element selenium circulating in human blood plasma is present as selenoprotein P (SeP). As SeP associates with endothelial membranes, the participation of SeP in selenium-mediated protection against oxidative damage was investigated, using the human endothelial cell line Ea.hy926 as a model system. Hepatocyte-derived SeP prevented tert-butylhydroperoxide (t-BHP)-induced oxidative cell death of Ea.hy926 cells in a similar manner as did sodium selenite, counteracting a t-BHP-induced loss of cellular membrane integrity. Protection was detected after at least 10 h of SeP supplementation and it peaked at 24 h. SeP time-dependently stimulated the expression of cytosolic glutathione peroxidase (cGPx) and increased the enzymatic activities of glutathione peroxidase (GPx) and thioredoxin reductase (TR). The cGPx inhibitor mercaptosuccinate as well as the γ-glutamylcysteine synthetase inhibitor buthionine sulfoximine counteracted the SeP-mediated protection, while the TR inhibitors cisplatin and auranofin had no effect. The presented data suggest that selenium supplementation by SeP prevents oxidative damage of human endothelial cells by restoring expression and enzymatic activity of GPx.     

Metabotropic glutamate receptor 5 and calcium signaling in retinal amacrine cells

To begin to understand the modulatory role of glutamate in the inner retina, we examined the mechanisms underlying metabotropic glutamate receptor 5 (mGluR5)-dependent Ca(2+) elevations in cultured GABAergic amacrine cells. A partial sequence of chicken retinal mGluR5 encompassing intracellular loops 2 and 3 suggests that it can couple to both G(q) and G(s). Selective activation of mGluR5 stimulated Ca(2+) elevations that varied in waveform from cell to cell. Experiments using high external K(+) revealed that the mGluR5-dependent Ca(2+) elevations are distinctive in amplitude and time course from those engendered by depolarization. Experiments with a Ca(2+) -free external solution demonstrated that the variability in the time course of mGluR5-dependent Ca(2+) elevations is largely due to the influx of extracellular Ca(2+). The sensitivity of the initial phase of the Ca(2+) elevation to thapsigargin indicates that this phase of the response is due to the release of Ca(2+) from the endoplasmic reticulum. Pharmacological evidence indicates that mGluR5-mediated Ca(2+) elevations are dependent upon the activation of phospholipase C. We rule out a role for L-type Ca(2+) channels and cAMP-gated channels as pathways for Ca(2+) entry, but provide evidence of transient receptor potential (TRP) channel-like immunoreactivity, suggesting that Ca(2+) influx may occur through TRP channels. These results indicate that GABAergic amacrine cells express an avian version of mGluR5 that is linked to phospholipase C-dependent Ca(2+) release and Ca(2+) influx, possibly through TRP channels.     

Differential negative coupling of type 3 metabotropic glutamate receptor to cyclic GMP levels in neurons and astrocytes

Metabotropic receptors may couple to different G proteins in different cells or perhaps even in different regions of the same cell. To date, direct studies of group II and group III metabotropic glutamate receptors' (mGluRs) relationships to second messenger cascades have reported negative coupling of these receptors to cyclic AMP (cAMP) levels in neurons, astrocytes and transfected cells. In the present study, we found that the peptide neurotransmitter N-acetylaspartylglutamate (NAAG), an mGluR3-selective agonist, decreased sodium nitroprusside (SNP)-stimulated cyclic GMP (cGMP) levels in cerebellar granule cells and cerebellar astrocytes. The mGluR3 and group II agonists FN6 and LY354740 had similar effects on cGMP levels. The mGluR3 and group II antagonists beta-NAAG and LY341495 blocked these actions. Treatment with pertussis toxin inhibited the effects of NAAG on SNP-stimulated cGMP levels in rat cerebellar astrocytes but not in cerebellar neurons. These data support the conclusion that mGluR3 is also coupled to cGMP levels and that this mGluR3-induced reduction of cGMP levels is mediated by different G proteins in cerebellar astrocytes and neurons. We previously reported that this receptor is coupled to a cAMP cascade via a pertussis toxin-sensitive G protein in cerebellar neurons, astrocytes and transfected cells. Taken together with the present data, we propose that mGluR3 is coupled to two different G proteins in granule cell neurons. These data greatly expand knowledge of the range of second messenger cascades induced by mGluR3, and have implications for clinical conditions affected by NAAG and other group II mGluR agonists.     

Activation of stimulatory heterotrimeric G proteins increases glutathione and protects neuronal cells against oxidative stress

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for cell death by oxidative stress, where an excess of extracellular glutamate inhibits import of cystine, a building block of the antioxidant glutathione. The subsequent decrease in glutathione then leads to the accumulation of reactive oxygen species (ROS) and programmed cell death. We used pharmacological compounds known to interact with heterotrimeric G-protein signalling and studied their effects on cell survival, morphology, and intracellular events that ultimately lead to cell death. Cholera toxin and phorbol esters were most effective and prevented cell death through independent pathways. Treating HT22 cells with cholera toxin attenuated the glutamate-induced accumulation of ROS and calcium influx. This was, at least in part, caused by an increase in glutathione due to improved uptake of cystine mediated by the induction of the glutamate/cystine-antiporter subunit xCT or, additionally, by the up-regulation of the antiapoptotic protein Bcl-2. Gs activation also protected HT22 cells from hydrogen peroxide or inhibition of glutathione synthesis by buthionine sulfoximine, and immature cortical neurones from oxidative glutamate toxicity. Thus, this pathway might be more generally implicated in protection from neuronal death by oxidative stress.     

Glutamate transport alteration triggers differentiation-state selective oxidative death of cultured astrocytes: a mechanism different from excitotoxicity depending on intracellular GSH contents

Recent evidence has been provided for astrocyte degeneration in experimental models of neurodegenerative insults associated with glutamate transport alteration. To determine whether astrocyte death can directly result from altered glutamate transport, we here investigated the effects of L-trans-pyrrolidine-2,4-dicarboxylate (PDC) on undifferentiated or differentiated cultured rat striatal astrocytes. PDC induced death of differentiated astrocytes without affecting undifferentiated astrocyte viability. Death of differentiated astrocytes was also triggered by another substrate inhibitor but not by blockers of glutamate transporters. The PDC-induced death was delayed and apoptotic, and death rate was dose and treatment duration-dependent. Although preceded by extracellular glutamate increase, this death was not mediated through glutamate receptor stimulation, as antagonists did not provide protection. It involves oxidative stress, as a decrease in glutathione contents and a dramatic raise in reactive oxygen species preceded cell loss, and as protection was provided by antioxidants. PDC induced a similar percentage of GSH depletion in the undifferentiated astrocytes, but only a slight increase in reactive oxygen species. Interestingly, undifferentiated astrocytes exhibited twofold higher basal GSH content compared with the differentiated ones, and depleting their GSH content was found to render them susceptible to PDC. Altogether, these data demonstrate that basal GSH content is a critical factor of astrocyte vulnerability to glutamate transport alteration with possible insights onto concurrent death of astrocytes and gliosis in neurodegenerative insults.     

The Nrf2 activator MIND4-17 protects retinal ganglion cells from high glucose-induced oxidative injury

Diabetic retinopathy (DR) is a leading cause of acquired blindness among adults. High glucose (HG) induces oxidative injury and apoptosis in retinal ganglion cells (RGCs), serving as a primary pathological mechanism of DR. MIND4-17 activates nuclear-factor-E2-related factor 2 (Nrf2) signaling via modifying one cysteine (C151) residue of Kelch-like ECH-associated protein 1 (Keap1). The current study tested its effect in HG-treated primary murine RGCs. We show that MIND4-17 disrupted Keap1–Nrf2 association, leading to Nrf2 protein stabilization and nuclear translocation, causing subsequent expression of key Nrf2 target genes, including heme oxygenase-1 and NAD(P)H quinone oxidoreductase 1. Functional studies showed that MIND4-17 pretreatment significantly inhibited HG-induced cytotoxicity and apoptosis in primary murine RGCs. Reactive oxygen species production and oxidative injury in HG-treated murine RGCs were attenuated by MIND4-17. Nrf2 silencing (by targeted small interfering RNA) or knockout (by CRISPR/Cas9 method) abolished MIND4-17-induced RGC cytoprotection against HG. Additionally, Keap1 knockout or silencing mimicked and abolished MIND4-17-induced activity in RGCs. In vivo, MIND4-17 intravitreal injection activated Nrf2 signaling and attenuated retinal dysfunction by light damage in mice. We conclude that MIND4-17 activates Nrf2 signaling to protect murine RGCs from HG-induced oxidative injury.     

Quiescence induced by iron challenge protects neuroblastoma cells from oxidative stress

The brain uses massive amounts of oxygen, generating large quantities of reactive oxygen species (ROS). Because of its lipid composition, rich in unsaturated fatty acids, the brain is especially vulnerable to ROS. Furthermore, oxidative damage in the brain is often associated with iron, which has pro-oxidative properties. Iron-mediated oxidative damage in the brain is compounded by the fact that brain iron distribution is non-uniform, being particularly high in areas sensitive to neurodegeneration. This work was aimed to further our understanding of the cellular mechanisms by which SHSY5Y neuroblastoma cells adapt to, and survive increasing iron loads. Using an iron accumulation protocol that kills about 50% of the cell population, we found by cell sorting analysis that the SHSY5Y sub-population that survived the iron loading arrested in the G(0) phase of the cell cycle. These cells expressed neuronal markers, while their electrical properties remained largely unaltered. These results suggest that upon iron challenge, neuroblastoma cells respond by entering the G(0) phase, somehow rendering them resistant to oxidative stress. A similar physiological condition might be involved in neuronal survival in tissues known to accumulate iron with age, such as the hippocampus and the substantia nigra pars compacta.     

Interleukin-6 protects PC12 cells from 4-hydroxynonenal-induced cytotoxicity by increasing intracellular glutathione levels

Oxidative stress plays an important role in neuronal cell death associated with many different neurodegenerative conditions, and it is reported that 4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, is a key mediator of neuronal cell death induced by oxidative stress. Previously, we have demonstrated that interleukin-6 (IL-6) protects PC12 cells from serum deprivation and 6-hydroxydopamine-induced toxicity. Therefore, in the present study, we examined the effects of interleukins on HNE toxicity in PC12 cells. Exposure of PC12 cells to HNE resulted in a decrease in levels of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, which was due to necrotic and apoptotic cell death. Addition of IL-6 24 h before HNE treatment provided a concentration-dependent protection against HNE toxicity, whereas neither IL-1β nor IL-2 had any effect. Addition of glutathione (GSH)-ethyl ester, but not superoxide dismutase or catalase, before HNE treatment to the culture medium protected PC12 cells from HNE toxicity. We found that IL-6 increases intracellular GSH levels and the activity of γ-glutamylcysteine synthetase (γ-GCS) in PC12 cells. Buthionine sulfoximine (BSO), an inhibitor of γ-GCS, reversed the protective effect of IL-6 against HNE toxicity. These results suggest that IL-6 protects PC12 cells from HNE-induced cytotoxicity by increasing intracellular levels of GSH.     

Inhibition of cPLA2 activation by Ginkgo biloba extract protects spinal cord neurons from glutamate excitotoxicity and oxidative stress-induced cell death

Ginkgo biloba extract (EGb761) has been shown to be neuroprotective; however, the mechanism by which EGb761 mediates neuroprotection remains unclear. We hypothesized that the neuroprotective effect of EGb761 is mediated by inhibition of cytosolic phospholipase A(2) (cPLA(2)), an enzyme that is known to play a key role in mediating secondary pathogenesis after acute spinal cord injury (SCI). To determine whether EGb761 neuroprotection involves the cPLA(2) pathway, we first investigated the effect of glutamate and hydrogen peroxide on cPLA(2) activation. Results showed that both insults induced an increase in the expression of phosphorylated cPLA(2) (p-cPLA(2)), a marker of cPLA(2) activation, and neuronal death in vitro. Such effects were significantly reversed by EGb761 administration. Additionally, EGb761 significantly decreased prostaglandin E(2) (PGE(2)) release, a downstream metabolite of cPLA(2). Moreover, inhibition of cPLA(2) activity with arachidonyl trifluromethyl ketone improved neuroprotection against glutamate and hydrogen peroxide-induced neuronal death, and reversed Bcl-2/Bax ratio; notably, EGb761 produced greater effects than arachidonyl trifluromethyl ketone. Finally, we showed that the extracellular signal-regulated kinase 1/2 signaling pathway is involved in EGb761's modulation of cPLA(2) phosphorylation. These results collectively suggest that the protective effect of EGb761 is mediated, at least in part, through inhibition of cPLA(2) activation, and that the extracellular signal-regulated kinase 1/2 signaling pathway may play an important role in mediating the EGb761's effect.     

CTRP3 protects against uric acid-induced endothelial injury by inhibiting inflammation and oxidase stress in rats

Hyperuricemia, which contributes to vascular endothelial damage, plays a key role in multiple cardiovascular diseases. This study was designed to investigate whether C1q/tumor necrosis factor (TNF)-related protein 3 (CTRP3) has a protective effect on endothelial damage induced by uric acid and its underlying mechanisms. Animal models of hyperuricemia were established in Sprague-Dawley (SD) rats through the consumption of 10% fructose water for 12 weeks. Then, the rats were given a single injection of Ad-CTRP3 or Ad-GFP. The animal experiments were ended two weeks later. In vitro, human umbilical vein endothelial cells (HUVECs) were first infected with Ad-CTRP3 or Ad-GFP. Then, the cells were stimulated with 10 mg/dL uric acid for 48 h after pretreatment with or without a Toll-like receptor 4 (TLR4)-specific inhibitor. Hyperuricemic rats showed disorganized intimal structures, increased endothelial apoptosis rates, increased inflammatory responses and oxidative stress, which were accompanied by reduced CTRP3 and elevated TLR4 protein levels in the thoracic aorta. In contrast, CTRP3 overexpression decreased TLR4 protein levels and ameliorated inflammatory responses and oxidative stress, thereby improving the morphology and apoptosis of the aortic endothelium in rats with hyperuricemia. Similarly, CTRP3 overexpression decreased TLR4-mediated inflammation, reduced oxidative stress, and rescued endothelial damage induced by uric acid in HUVECs. In conclusion, CTRP3 ameliorates uric acid-induced inflammation and oxidative stress, which in turn protects against endothelial injury, possibly by inhibiting TLR4-mediated inflammation and downregulating oxidative stress.     

PGC‐1α protects from myocardial ischaemia‐reperfusion injury by regulating mitonuclear communication

The recovery of blood supply after a period of myocardial ischaemia does not restore the heart function and instead results in a serious dysfunction called myocardial ischaemia‐reperfusion injury (IRI), which involves several complex pathophysiological processes. Mitochondria have a wide range of functions in maintaining the cellular energy supply, cell signalling and programmed cell death. When mitochondrial function is insufficient or disordered, it may have adverse effects on myocardial ischaemia‐reperfusion and therefore mitochondrial dysfunction caused by oxidative stress a core molecular mechanism of IRI. Peroxisome proliferator‐activated receptor gamma co‐activator 1α (PGC‐1α) is an important antioxidant molecule found in mitochondria. However, its role in IRI has not yet been systematically summarized. In this review, we speculate the role of PGC‐1α as a key regulator of mitonuclear communication, which may interacts with nuclear factor, erythroid 2 like ‐1 and ‐2 (NRF‐1/2) to inhibit mitochondrial oxidative stress, promote the clearance of damaged mitochondria, enhance mitochondrial biogenesis, and reduce the burden of IRI.