Cystine/glutamate antiporter expression in retinal mu¨ller glial cells: Implications fordl-alpha-aminoadipate toxicity

Kato, Satoru; Ishita, S.; Sugawara, Kiyoshi; Mawatari, Kazuhiro

doi:10.1016/0306-4522(93)90080-y

Cited by 115 publications

(69 citation statements)

References 35 publications

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“…Earlier immunohistochemical studies from our laboratory [13] had detected system x c -in the ganglion cell layer of the intact mouse retina. In the present study, we repeated this experiment to confirm at a higher power magnification whether our affinity purified antibody against xCT detected the protein in ganglion cells.…”

Section: Discussionmentioning

confidence: 93%

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Expression of the cystine-glutamate exchanger (xc −) in retinal ganglion cells and regulation by nitric oxide and oxidative stress

et al. 2006

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Purpose-The cystine-glutamate transporter, system x c -, mediates the Na + -independent exchange of cystine into cells coupled to the efflux of intracellular glutamate. Within cells, cystine is reduced to cysteine, a component of glutathione, thus system x c -plays a critical role in glutathione homeostasis. Studies in brain had initially suggested that system x c -was present primarily in astrocytes and not neurons, but more recent work suggests that certain brain neurons have an active system x c -. In the retina, system x c -has been demonstrated in retinal Müller and RPE cells. Recent immunohistochemical work from our lab suggested that the two protein components of system x c -, xCT and 4F2hc, are present in ganglion cells of the intact retina. The purpose of this investigation was to determine whether system x c -was present in primary ganglion cells isolated from neonatal mouse retinas and, if so, to study its regulation by oxidative stress in a retinal ganglion cell line, RGC-5.Methods-The presence of xCT and 4F2hc in RGC-5 cells was established by RT-PCR, immunoblotting and immunohistochemistry and in primary mouse retinal ganglion cells by immunoblotting and immunohistochemistry; the function of the transporter in RGC-5 cells was established by measuring radiolabeled glutamate uptake in the absence of Na + . To assess regulation of system x c -by oxidative stress in ganglion cells, RGC-5 cells were incubated in the presence or absence of nitric oxide (NO) donors (3-nitroso-N-acetylpenicillamine (SNAP), Snitrosoglutathione (SNOG), or 3-morpholinosydnonimine hydrochloride (SIN-1) and reactive oxygen species (ROS) donors menadione sodium bisulfite, hydrogen peroxide and xanthine sodium salt/xanthine oxidase and system x c -was analyzed using functional assays, RT-PCR and immunoblotting.Results-RGC-5 cells and primary ganglion cells isolated from mouse retina express xCT and 4F2hc as demonstrated by RT-PCR, immunoblotting and immunohistochemistry. RGC-5 cells take up glutamate in the absence of Na + and this uptake was blocked by known inhibitors of system x c -, glutamate, cysteine, and cystine as well as quisqualic acid. Treatment of RGC-5 cells with NO donors and donors of ROS led to an increase in the functional activity of system x c -. Kinetic analysis of SNAP-treated RGC-5 cells compared to control cells showed that the increase was associated with an increase in the maximal velocity of the transporter with no significant change in the substrate affinity. Molecular analyses showed that the upregulation is associated with an increase in the expression of xCT with no detectable change in the expression of 4F2hc. Conclusions-RGC-5 cells and ganglion cells isolated from neonatal mice express the cystine/ glutamate transporter x c -(the light chain xCT and the heavy chain 4F2hc) as is evident from functional and molecular studies. Oxidative stress upregulates this transport system in RGC-5 cells and the process is associated with an increase in xCT mRNA and protein but no change in 4F2hc mRNA or...

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Section: Discussionmentioning

confidence: 93%

“…Work by Kato in the early 1990's [13] described system x cexpression in Müller cells, the predominant glial cell of the retina. These findings were confirmed by Pow in 2001 [14] using an antibody against aminoadipic acid, which is a selectively transported substrate for system x c -.…”

Section: Introductionmentioning

confidence: 99%

Expression of the cystine-glutamate exchanger (xc −) in retinal ganglion cells and regulation by nitric oxide and oxidative stress

et al. 2006

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“…In a subset of t-ACPD experiments, slices were perfused with the gliotoxin DL-2-aminoadipic acid (DL-AAA; 2 mmol/L) for 20 to 30 minutes before t-ACPD exposure to inactivate astrocytes. 19,20 Although our DL-AAA treatment protocol is well below the dose and incubation time (43 mmol/L and 2 hours) that has previously been shown to induce neuronal toxicity, 20 we cannot rule out the potential for neuronal toxicity in our experiments. However, we believe these data do not confound the conclusions of the study as (1) DL-AAA was used in t-ACPD experiments, which were designed to test the effect of hypertension on astrocytes and other downstream mGluR expressing cells (see schematic Supplementary Figure S1) and (2) although neurons also express mGluR, t-ACPD has been shown to suppress neuronal activity, 21,22 therefore, we would not expect neuronal toxicity to have an adverse effect on t-ACPD-induced dilations.…”

Section: Arteriole Cannulationmentioning

confidence: 90%

“…Because other components of the neurovascular unit, including the microvasculature, 25 also express mGluRs, we repeated t-ACPD experiments in the presence of the gliotoxin DL-aminoadipic acid (DL-AAA; 2 mmol/L) 19,20 to elucidate the contribution of astrocytes to mGluR-mediated vasodilations. After PA cannulation, DL-AAA was bath applied for 20 to 30 minutes, at which point the slice was exposed to t-ACPD in the continued presence of DL-AAA.…”

Section: Metabotropic Glutamate Receptor-induced Parenchymal Arteriolmentioning

confidence: 99%

Enhanced Parenchymal Arteriole Tone and Astrocyte Signaling Protect Neurovascular Coupling Mediated Parenchymal Arteriole Vasodilation in the Spontaneously Hypertensive Rat

Iddings

Kim

Zhou

et al. 2015

J Cereb Blood Flow Metab

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Functional hyperemia is the regional increase in cerebral blood flow upon increases in neuronal activity which ensures that the metabolic demands of the neurons are met. Hypertension is known to impair the hyperemic response; however, the neurovascular coupling mechanisms by which this cerebrovascular dysfunction occurs have yet to be fully elucidated. To determine whether altered cortical parenchymal arteriole function or astrocyte signaling contribute to blunted neurovascular coupling in hypertension, we measured parenchymal arteriole reactivity and vascular smooth muscle cell Ca 2+ dynamics in cortical brain slices from normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. We found that vasoconstriction in response to the thromboxane A 2 receptor agonist U46619 and basal vascular smooth muscle cell Ca 2+ oscillation frequency were significantly increased in parenchymal arterioles from SHR. In perfused and pressurized parenchymal arterioles, myogenic tone was significantly increased in SHR. Although K + -induced parenchymal arteriole dilations were similar in WKY and SHR, metabotropic glutamate receptor activation-induced parenchymal arteriole dilations were enhanced in SHR. Further, neuronal stimulation-evoked parenchymal arteriole dilations were similar in SHR and WKY. Our data indicate that neurovascular coupling is not impaired in SHR, at least at the level of the parenchymal arterioles. Keywords: astrocytes; brain slice; cerebral blood flow; hypertension; neurovascular coupling; parenchymal arterioles INTRODUCTION Functional hyperemia (FH), the process in which neuronal activation triggers local increases in cerebral blood flow (CBF), is of particular importance for cerebral homeostasis as it ensures that neuronal metabolic demands are met. 1 Despite recent controversy, 2,3 numerous in vitro and in vivo studies suggest that astrocytes act as intermediaries in the neurovascular coupling (NVC)-mediated vascular responses that govern the hyperemic response. Although NVC-mediated signaling can elicit both vasodilation and vasoconstriction, the focus of this study is to address NVC-mediated vasodilatory responses. During the orchestrated signaling among neurons, astrocytes, and the cerebral vasculature that occurs during NVC-mediated vasodilation, synaptically released glutamate binds to astrocytic metabotropic glutamate receptors (mGluR) increasing intracellular Ca 2+ , 1,4 which, in turn, results in the release of vasoactive signals such as arachidonic acid (AA) metabolites 4 and K + . 5 Although FH is impaired in hypertensive patients and animal models of hypertension, 6-9 the mechanisms by which hypertension disrupts NVC among cells in the neurovascular unit is not fully understood. A few elegantly designed studies, primarily evaluating pial arteriole function, have used regional CBF measurements to deliver valuable insight into the vascular mechanisms underlying NVC disruptions during acute, short-term Angiotensin II-induced hypertension 6,9 and chronic hypertension. 8 However,

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“…Because a large fraction (ϳ70%) of glutamate uptake in GLAST Ϫ/Ϫ mouse retina is Na ϩ -independent (Fig. 2) and Mü ller cells have been shown to express the glutamate-cystine exchanger (Kato et al, 1993;Pow, 2001b;Tomi et al, 2002Tomi et al, , 2003, we examined the involvement of the exchanger in glutamate uptake. For this study, [ 3 H]D-Asp uptake was carried out in Na ϩ -free medium in the presence of a known inhibitor of cystine-glutamate exchanger, CPG and the retinas were processed for autoradiography.…”

Section: Other Glutamate Transportersmentioning

confidence: 99%

Glutamate transport by retinal Müller cells in glutamate/aspartate transporter‐knockout mice

Sarthy

Pignataro

Pannicke

et al. 2004

Glia

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Glutamate transporters are involved in maintaining extracellular glutamate at a low level to ensure a high signal-to-noise ratio for glutamatergic neurotransmission and to protect neurons from excitotoxic damage. The mammalian retina is known to express the excitatory amino acid transporters, EAAT1-5; however, their specific role in glutamate homeostasis is poorly understood. To examine the role of the glial glutamate/ aspartate transporter (GLAST) in the retina, we have studied glutamate transport by Mü ller cells in GLAST Ϫ/Ϫ mice, using biochemical, electrophysiological, and immunocytochemical techniques. Glutamate uptake assays indicated that the K m value for glutamate uptake was similar in wild-type and GLAST Ϫ/Ϫ mouse retinas, but the V max was ϳ50% lower in the mutant. In Na ϩ -free medium, the V max was further reduced by 40%. In patch-clamp recordings of dissociated Mü ller cells from GLAST Ϫ/Ϫ mice, application of 0.1 mM glutamate evoked no current showing that the cells lacked functional electrogenic glutamate transporters. The result also indicated that there was no compensatory upregulation of EAATs in Mü ller cells. [ 3 H]D-Aspartate uptake autoradiography, however, showed that Na ϩ -dependent, high-affinity transporters account for most of the glutamate uptake by Mü ller cells, and that Na ϩ -independent glutamate transport is negligible. Additional experiments showed that the residual glutamate uptake in Mü ller cells in the GLAST Ϫ/Ϫ mouse retina is not due to known glutamate transporters-cystine-glutamate exchanger, ASCT-1, AGT-1, or other heteroexchangers. The present study shows that while several known glutamate transporters are expressed by mammalian Mü ller cells, new Na ϩ -dependent, high-affinity glutamate transporters remain to be identified.

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Cystine/glutamate antiporter expression in retinal mu¨ller glial cells: Implications fordl-alpha-aminoadipate toxicity

Cited by 115 publications

References 35 publications

Expression of the cystine-glutamate exchanger (xc −) in retinal ganglion cells and regulation by nitric oxide and oxidative stress

Expression of the cystine-glutamate exchanger (xc −) in retinal ganglion cells and regulation by nitric oxide and oxidative stress

Enhanced Parenchymal Arteriole Tone and Astrocyte Signaling Protect Neurovascular Coupling Mediated Parenchymal Arteriole Vasodilation in the Spontaneously Hypertensive Rat

Glutamate transport by retinal Müller cells in glutamate/aspartate transporter‐knockout mice

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