Kiyoshi Sugawara

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

A cytotoxicity of glutamate or related amino acids (10 mM) mediated by a cystine/glutamate antiporter (system Xc) has recently been demonstrated in N18 neuroblastoma-rat retina hybrid (N18RE105) cells and C6 glioma cells. The antiporter usually transports glutamate outside and cystine inside, thereby maintaining cellular concentrations of glutathione. High concentrations of glutamate inhibit cystine uptake and lead to depletion of cellular levels of glutathione. Among related amino acids, DL-alpha-aminoadipic acid (DL-alpha-AAA), which is well known as a selective gliotoxin in the retina, is also toxic to these cells. However, this does not explain why DL-alpha-AAA acts gliospecifically on the retina. To answer this question we first examined the effects of DL-alpha-AAA on the [35S]cystine uptake with parental N18 neuroblastoma cells and rat retina of the hybrid cells. DL-alpha-AAA showed a competitive inhibition of [35S]cystine uptake in the rat retina but not in the N18 cells. Such a competitive inhibition of cystine uptake by DL-alpha-AAA could also be seen in the carp retina. The cystine uptake with carp retina was mainly Na(+)-independent and Cl(-)-dependent as already described as a characteristic ion dependency of the Xc antiporter. We next examined the effects of exogenous cystine on the glutamate release from the retina. Cystine (1 mM) actually induced a glutamate release approximately twice that of the control. Furthermore, the glutamate release induced by cystine was also Na(+)-independent and Cl(-)-dependent, and was blocked by DL-alpha-AAA. An autoradiogram of [35S]cystine uptake in the carp retina showed typical radial glial Müller cells. A large incorporation of [35S]cystine into retinal glutathione fraction was detected by a high pressure liquid chromatography method during a 1-4-h incubation. A significant or large decrease of retinal levels of glutathione was observed one day ater an intravitreal injection of 8 mumol DL-alpha-AAA or L-alpha-AAA, respectively. Buthionine sulfoximine (2.5 mumol), a specific inhibitor of glutathione synthesis, induced a large decrease of retinal levels of glutathione and a loss of electroretinographic b-wave 20-30 h after treatment. Taken together, our present data with rat and carp retinas strongly indicate that the expression of cystine/glutamate antiporter is enriched in the retina, particularly in the glial Müller cells which have a rapid turnover pool for glutathione. The gliotoxin DL-alpha-AAA inhibits cystine uptake through this antiporter on the glial cells and elicits reduction of cellular levels of glutathione.(ABSTRACT TRUNCATED AT 400 WORDS)

Gliotoxic effects of α-aminoadipic acid isomers on the carp retina: A long term observation

Abstract The glutamate analogue, α-aminoadipic acid was intravitreally administered in the d -, dl - and l -forms to carp ( Cyprinus carpio ) retina in vivo . To make a quantitative assessment of its gliotoxic action, the activity of glutamine synthetase, whose localization was confirmed in glial Muller cells by an immunohistochemical technique, was examined at various intervals over one month. Intravitreal injection of 8 μmol α-aminoadipic acids reduced the glutamine synthetase activity within 4 h and maximally by 24 h. The maximum reduction evoked by l -, dl - and d -forms was about 65, 45 and 28% in reduction, and their minimum effective dose was 0.8, 1.5 and 2.0 μmol, respectively. At three to four days after alpha-aminoadipic acids injection, sodium dodecyl sulphate gel electrophoresis suggested that some retinal proteins including glutamine synthetase were significantly reduced, whilst others were increased. These biochemical changes were fully reversed one to two weeks after administration of the d - or DL -forms, but not until one month with the l -form. The electroretinographic b-wave, reflecting glial activity, was completely blocked by 8 μmol α-aminoadipic acids within 4 h. The electroretinographic b-wave was recovered first in the case of d - and then of dl -form at two to three weeks after injection, but only 50% recovery was seen in the case of l -form even two months later. A high dose of dl -α-aminoadipic acid (16 μmol) induced as long lasting a suppression in the glutamine synthetase and electroretinographic b-wave activities as 8 μmol l -α-aminoadipic acid. Therefore, the gliotoxic efficacy of l -α-aminoadipic acid at micromol orders was two-fold higher than that of dl -α-aminoadipic acid. Differences in the time-course of recovery of the suppression of glutamate synthetase and electroretinographic b-wave activities induced by α-aminoadipic acids are discussed in terms of its gliotoxicity.

Neurotoxic effects ofl-α-aminoadipic acid on the carp retina: A long term observation

Abstract The hypothesis has been tested that the enantiomers of α-aminoadipic acid have different target effects; the l -isomer has both glio- and neurotoxic actions, while the dl -isomer has a gliospecific action in the CNS. Electrophysiological and morphological studies were carried out on the retina of the carp ( Cyprinus carpio ) for one to two months after intraocular injection with α-aminoadipic acids at various doses. Intracellular recording from horizontal cells and extracellular recording of spike discharges from ganglion cells in isolated retinal preparations were made from control and pretreated retinas at various intervals after intraocular injection with the enantiomers. In control retinas, application of 15 mM l -α-aminoadipic acid in the superfusate resulted in hyperpolarization of all horizontal cells and in a decrease in amplitude of their light responses (S-potentials). In the retinas pretreated with l -α-aminoadipic acid (8 μmol), low amplitude S-potentials were seen during an early phase 2–4 h after ocular injection, but the normal appearance of S-potentials was restored one day after injection. In control retinas, a brief period of iontophoretic application of l -α-aminoadipic acid resulted in a slight activation of the spontaneous spike firing of ganglion cells but a slight decrease in the rate of light-induced firing. In retinas pretreated with intraocular l -α-aminoadipic acid (4 μmol) 4 h prior to eye removal, however, light-induced spike discharges were abolished from nearly all spontaneously firing ganglion cells (90%). Their unresponsiveness to light stimuli lasted for more than two months after injection, and was accompanied by insensitivity to iontophoretically applied putative neurotransmitters. Use of the dl -α-aminoadipic acid, even at a higher dose (16 μmol), did not cause this semi-permanent loss of ganglion cell responses. Under light-microscopic examination, both dl -α-aminoadipic acid and l -α-aminoadipic acid were found to produce a marked swelling of glial Muller cells within one day after ocular injection, while the l -isomer additionally caused neuronal damage, particularly in the inner retina. The glial swelling completely disappeared in two to three weeks, and the neuronal damage disappeared gradually over one to two months. The present data show that the neurotoxicity caused by l -α-aminoadipic acid (4μmol) in the carp retina is toward neurons of the inner retina particularly to ganglion cells, and that the electrophysiologic effect is irreversible. Gliotoxicity of both l -and dl -α-aminoadipic acid, however, is reversible, the latter racemic mixture being gliospecific.

Reactive oxygen species uncouple external horizontal cells in the carp retina and glutathione couples them again

We have investigated the effect of free radicals on the electrical gap junctions between horizontal cells in the carp retina. In our previous study, L-buthionine sulfoximine, an inhibitor of glutathione synthesis, caused uncoupling of horizontal cells four days after injection. In the present study, we have used paraquat, a generator of exogenous reactive oxygen species, to investigate whether it was the depletion of glutathione or an increase in the level of reactive oxygen species which resulted in horizontal cell uncoupling after L-buthionine sulfoximine injection. Intracellular recordings were made from L-type horizontal cells at various time-points after intravitreal injection of paraquat. Injection of 25nmol paraquat caused an increase in response amplitude to central spot light stimuli by two days after injection, which continued for a further two to three days and had almost disappeared by seven days after injection. There was also a sharp increase in reactive oxygen species production, peaking at four days and disappearing by seven days after injection, and an accompanying depletion and a restoration of glutathione levels with a similar time-course. Marking cells with Lucifer Yellow clearly illustrated uncoupling of horizontal cells after paraquat injection. If paraquat and L-buthionine sulfoximine were injected simultaneously, the increase in response to central spots was observed as early as one day after injection. This response amplitude was not more enhanced than that observed after L-buthionine sulfoximine injection alone, although a dramatic increase in the level of reactive oxygen species was observed. From these results, we suggest that reactive oxygen species are involved in uncoupling, while recovery from uncoupling is dependent on glutathione. Furthermore, we conclude that a balance between glutathione and reactive oxygen species levels is the most important factor controlling gap junctional intercellular communication of L-type horizontal cells in the carp retina.

Altered emotional behaviors in the diabetes mellitus OLETF type 1 congenic rat.

GPR10 is a G-protein-coupled receptor expressed in thalamic and hypothalamic brain regions, including the reticular thalamic nucleus (RTN) and periventricular nucleus (Pev), and the endogenous ligand for this receptor, prolactin-releasing peptide (PrRP), has demonstrated regulatory effects on the stress response. We produced a congenic rat by introducing the Dmo1 allele from the OLETF rat which encodes the amino acid sequences of GPR10 with a truncated NH2-terminus, into the Brown-Norway background. Using receptor autoradiography, we determined a lack of specific [125I]PrRP binding in the RTN and Pev of these mutant rats compared to the control rats. Furthermore, intracerebroventricular injection of PrRP did not induce a significant increase of c-fos-like immunoreactivity in the paraventricular nucleus of the mutant rats compared to the control rats. The mutant rats also displayed a less anxious-like phenotype in three behavioral-based models of anxiety-like behavior (open field, elevated plus maze and defensive withdrawal test). These data show the mutant congenic rat, of which GPR10 neither binds nor responds to PrRP, expresses less anxious-like phenotypes. On the basis of these observations, the GPR10 might be a novel target for the developing new drugs against anxiety and/or other stress-related diseases.

Effects of locally applied chemicals on transretinal potential and horizontal cells in the isolated carp retina

Experiments were performed on isolated retinas of the carp (Cyprinus carpio) to investigate the effects of locally applied amino acids on the transretinal potential and the membrane potential of horizontal cells. A minute quantity of each solution of an amino acid was applied locally at different layers by means of pressure-microinjection. The responses were induced by alternating central and annular stimuli of white light. Amino acids applied locally at the distal layer (receptor surface or outer plexiform layer) in the centrally stimulated area, caused, at concentrations of 25–50 mM, rapid changes in the light-induced potential, whereas those microinjected into the inner plexiform layer did not. When applied locally at the distal layer, amino acids abolished the proximal PIII response and reduced the amplitude of S-potentials, particularly in response to a central stimulus. However, the resting potential of horizontal cells was depolarized only slightly by acidic amino acids. The results suggest that the passive membrane properties of horizontal cells are not altered significantly by the amino acids applied. Differential susceptibilities of the PII component to acidic and neutral amino acids were found; the former did not change or, in some cases, enlarged, whereas the latter abolished it. Possibly, neutral amino acids interfere with a neural mechanism generating the PII component.

A Multidisciplinary Approach to Investigating Optic Nerve Regeneration in the Goldfish

Unlike the mammalian central nervous system, the goldfish optic nerve can regenerate after transection. We have used this regenerative ability to examine possible factors involved in this process. By monitoring goldfish behavior, we found two phases of recovery. Simple behavioral patterns such as tilting and turning return one month after optic nerve transection (fast recovery phase), whereas more complex behaviors such as schooling behavior require up to 4 months or more to recover (slow recovery phase). We also demonstrated that severe hypertrophy occurs in retinal ganglion cells after optic nerve transection, taking about 4 months to recover. Our most recent investigations have looked at various aspects of the fast recovery phase using histochemical and molecular techniques. We have observed that there is an increase in the amount of the nitric oxide (NO) synthesizing enzyme (NOS) in retinal ganglion cells during the first month after optic nerve section. During optic nerve regeneration NO may play a role in strengthening regenerating connections between the retina and tectum. Finally, we have investigated changes in gene expression in the retina during optic nerve regeneration. Of the molecules that we have studied, transglutaminase (TG) and Na-K ATPase appear to be the most interesting. There is an increase in tissue TG gene expression in the retina at about one week after optic nerve section. Na-K ATPase alpha-3 subunit activity in the retina also peaked around this time. Using this multidisciplinary approach, we hope to gain a better understanding of many aspects of regeneration, from genetic control to behavioral modification.