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

GLIOTOXIC EFFECTS OF r -AMINOADIPIC ACID ISOMERS ON THE CARP RETINA: A LONG TERM OBSERVATION S.

KATO,*

K. SUGAWARA,

T. MATSUKAWA

and K.

NEGISHI

Molecular Neurobiology Group, Department of Neurophysiology, Neuroinformation (NIRI), University of Kanazawa School of Medicine. 13-l Takara-machi. Ka!Iazawa.

Research Institute Ishikawa 920. Japan

Abstract-The glutamate analogue, ~-aminoadipic acid was intravitre;~liy administered in the W, UL- and L-forms to carp (f~pri~tus curpio) retina in I+~. To make a quantitative assessment of its gliotoxic action, the activity of gl&amine synthetase, whose localization was confirmed in glial Miiller cells by an immunohistochemical technique, was examined at various intervals over one month. lntravitreal injection of 8 pmol r-aminoadipic acids reduced the glutamine synthetase activity within 4 h and maximally by 24 h. The maximum reduction evoked by L-. IIL- and r>-forms was about 65, 45 and 28% in reduction. and their minimum effective dose was 0.8, 1.5 and 2.0~~mol, respectively. At three to four days after alpha-alninoadip~c 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 o- or uL-forms, but not until one month with the L-form. The elcctroretinographic b-wave, reflecting glial activity, was completely blocked by 8 ~~rnmolr-aminoadipic acids within 4 h. The electroretinographic b-wave was recovered tirst in the case of D- and then of i,L-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-z-aminoadipic acid (16 pmoi) induced as long lasting a suppression in the glutamine synthetase and eiectroretinog~phic h-wave activities as 8 /Imol L-a-amilloadipic acid. Therefore. the gliotoxic efficacy of L-a-aminoadipic acid at micromol orders was two-fold higher than that of DL-a-aminoadipic acid. Differences in the time-course of recovery of the suppression of glutamate synthetase and electroretinographic b-wave activities induced by n-aminoadipic acids are discussed in terms of its gliotoxicity.

The gliotoxic effects of ~-aminoadipic acid (r-AAA), a six-carbon homologue of the excitatory amino acid, L-glutamate, have attracted considerable attention. Studies, both in riro and in vitro, have evaluated 2 -AAA as a gliotoxin in parts of the CNPL,” and on a glioma cell line.’ In the retina, r-AAA has been reported to cause loss of the b-wave of the electroretinogram (ERG) and structural damage to glial Miiller cells of many vertebrates.‘,‘8.24.“.‘8 With regard to its isomers, recent reports have indicated that the racemic DL-X-AAA has specific gliotoxic effects in the retina and that D- and L-isomers individually also exhibit gliotoxic actions. while the L-form is also found to cause neuronal injury.2,!‘,‘x Almost all previous studies have focused upon short term morphologic and electroretinographic effects of intravitreal a-AAA treatment. In a few studies, biochemical analyses of the gliotoxic and electrophysiolo~ical analyses of the neurotoxic effects of r-AAA on the retina have been conducted.

A long term observation of glio-. and neurotoxicity induced by I*-AAA has never been reported. Long term observation affords an opportunity to examine the reversibility or recovery of the retina from its toxicity. Therefore, we investigated in the present study with carp retinas, the long term biochemical and electroretinographic effects of cr-AAA isomers on glutamine synthetase (GS) and ERG b-wave activities, respectively, and further, in the following paper.” the electrophysiological effects of I -AAA isomers on individual retinal neuronal cell activities. In the present study, we explored the effects of a-AAAs on the GS activity and ERG b-wave in the carp retina for a long term (one to two months). Since the retina is a nervous tissue with a high activity of GS,‘.* which is immunohistochemically localized exclusively in glial Miiller cell~,‘“~‘~~*”and since the pioneering work by Svaetichin,21 the fish has been one of the best studied vertebrates for retinal electrophysiology. we have used the carp retina to evaluate the gliotoxicity and neurotoxicity induced by r-AAAs.

*To whom correspondence

should be addressed, alpha-aminoadipic acid: ABC, avidin-biotin peroxidase complex; BSA. bovine serum albumin; CBB. Coomassie Brilliant Blue; EDTA, ~thyienediamilletetra-acetate; ERG, ei~ctroretinogram~ FITC. fluorescein isothiocyanate~ GS. glutamine synthetase: PAGE. poiyacrytamide gel electrophoresis; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulphate.

.4hhreriurions

: r -AAA,

EXPERIMENTAL

PROCEDURES

L-[‘~C]Glutamic acid (55.3 mCi~mmo1) was obtained from New England Nuclear. DL-. D- and L-a-AAAs were purchased from Sigma. L-Glutamine synthetase (sheep brain, type III), used as an immunogen. was also from Sigma. 145

Carp (C:lprinus curpio. body weight 600 8OOg) were purchased from a local dealer. Their left eyes were injected intravitreally with SO ~1 solution containing various concentrations of a-AAAs dissolved in phosphate-buffered saline (PBS), pH 7.4 (treated). The drug concentration introduced into the vitreous were when using e.g. 8 pmol of the a-AAAs is. in fact. 160mM. Their right eyes were injected with an equivalent volume of PBS and served as control. This did not influence the ERG, GS activity or morphological analyses as compared with the untreated eye. Eyes were enucleated under MS 222 (Sankyo) anacsthesia and the retinas were thus isolated for biochemical and electroretinographic examinations.

Retina was homogenized in 0.5 ml of 10 mM imidazole HCI (pH 7.2), containing 0.5 mM EDTA, 5 mM 2-mercaptoethanol and 0.5 mM phenylmethylsulphonyl fluoride. The homogenate was then centrifuged at 27,000g for 60 min at 4 C and the supernatant (crude retina1 extract) was saved. GS was assayed by the method of Prusiner and Milner.” The assay system contained the following components in a final volume of 100~1 (pH 7.2): imidazole HCI (pH 7.2). 40mM; MgCIZ, l5mM; KCI, 15mM; NH,CI. 5mM; 2-mercaptoethanol, 2.2 mM; ATP, IO mM; r.-[‘4C]glutamic acid. 4 mM (0.2 pCi): and crude extract (50-60 pg protein). The mixture was placed into a 25.C water-bath, and after 20 min the reaction was stopped by adding 1 ml of ice-cold 20 mM imidazole ( pH 7.2) containing 30 mM t_-glutamic acid and 30 mM r-glutamine. The mixture was then applied to a Dowex I x 4 (Cl form, 200-400mesh) column (3.0 cm x 0.8 cm). Glutamine was recovered by washing the column with 3 ml of elution buffer which contained IO mM imidazole HCI (pH 7.2) and 30 mM L-glutamine. The eluate (2 ml) was collected in a scintillation vial and I4 ml of liquid scintillation cocktail was added. Radioactivity was quantified using a Beckman LS-9000 scintillation counter. The protein content of crude retinal extract was measured according to the method of Lowry c/ nl.” with bovine serum albumin (BSA) as a standard. Prepurution

und

churac/eri-_urion

o/

anti-glutnmine

s,vn -

literuse .~erunr Preparation: anti-GS serum was produced in a New Zealand white rabbit as follows. GS (sheep brain, type III) was mixed with an equal volume of Freund’s complete adjuvant and injected into the foot pads and backs in a rabbit (60pgjinjection) every week for four weeks. One week after the last injection, an anti-serum was obtained. The specificity of anti-GS serum was analysed by an Ouchterlony double-immunodiffusion test” and immunoprecipitation test. Ouchterlony double-immunodiffusion test was carried out in 1% agar containing PBS. Immunoprecipitation was performed as follows: 2Ogl of the antiserum at various dilutions were added to 80~1 of crude retinal extracts containing carp retinal GS. After I6 h at 4 C. the samples were centrifuged for 30 min at 15,000 g and the GS activities were determined in the supernatants. The pellets (immunoprecipitates) were washed three times with 20 mM TrissHCl (pH 7.4) and were analysed in sodium dodecyl sulphateepolyacrylamide gel electrophoresis (SDS PAGE) (see Fig. I). Immunohlotting. Retinal crude extracts containing GS were first subjected to SDS-PAGE. Proteins separated on the gels were then transferred to a nitrocellulose paper according to the method of Towbin PI a/,26 For detection of the transferred proteins, strips of nitrocellulose paper after blotting were soaked in 3% BSA in PBS for I h at 37 C, and rinsed three times for IOmin with 0.02% TweenPBS. The blotted paper was then incubated at 37 ‘C for 1 h with anti-GS serum or preimmune serum diluted (I : 250) and washed four times for IO min with TweenPBS,

and incubated at 37 C for I h with biotin-conjugatrJ goat anti-rabbit IgG diluted (I: 100) into 3”/u BSA in PBS. Aftet four additional washes. the sheet was incubated for- 30 min with avidin conjugated-peroxidasc diluted (I: 10.000) with 34/, BSA in 0.1 M TrisHCl (pH 9.0). After washing, the sheet was visualized at room temperature for IOmin in 0.5 mg/ml of 3,3’-diaminobenzidine-tetrahydrochloride. 0.1% H,O, in 100 mM TrisHCl ( nH 7.5). Iadire;,t ~mmunohi.~tochemi.~tr~,. Carp retinas were ltxed m ice-cold Zamboni’s solution overnight and washed in 30L!il sucrose containing 0. I M phosphate buffer (pH 7.4) at 4 C. Retinas were cryosectioned at I5 pm thickness in a radial direction, mounted on chrome alum gelatin coated cover slips and treated with anti-GS serum (at dilutions from I :250 to 1:2000) overnight. The sections were rinsed with ice-cold PBS for IO min and then with 1.0% Triton X-100 containing PBS for IO min. After the washing procedure was repeated twice, the sections were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (I : 1000) overnight at room temperature, washed in PBS and mounted in glycerin-PBS mixture. Sections were examined under a fluorescence microscope (Nikon EF) using a B (495 nm) excitation filter and a 5 I5 W ( > 5 I5 nm) absorption filter. Photomicrographs were taken on Tri-X pan films (Kodak). Sodium

dodeq4

.sulphatr-po!,ucr~,lumide

gel elecrrophoresic

Samples were mixed with a half volume of the sotubilizing buffer: I75 mM Tris-HCI ( pH 6.5). 5% SDS, 0.25 M dithiothreitol, 12.5% glycerin and 0.1 “IOBromophenol Blue and subjected to SDS-PAGE in 12% polyacrylamide gel according to Laemmli.g After electrophoresis. the slab gels were stained with Coomassie Brilliant Blue G-250 (CBB). The following proteins were used as molecular weight standards: phosphorylase B (92,500mol. wt). bovine serum albumin (66,200 mol. wt). ovalbumin (45,000 mol. wt), carbonic anhydrase (3 1,000 mol. wt), trypsin inhibitor (2 I.500 mol wt) and lysozyme (14.400 mol. at) Electroretinogrum

recording

procedure

Carp were dark-adapted for at least 1h and eyes were then enucleated under anaesthesia. The retina was isolated under dim red light and placed photoreceptor-side up in a recording chamber, superfused with a physiological solution containing 119.5 mM NaCI, 3.6 mM KCI, 1.15 mM CaCIZ, 1.04 mM MgSO,, 22.6 mM NaHCO,. 0. I mM NaH2P0,. 0.4mM NazHPO, and 10mM glucose. The solution was adjusted to pH 7.8 by bubbling with 97% O2 and 3% CO,. The ERG was recorded by means of Ag--AgCl electrodes placed on both sides of the retinal preparation. The electrodes were connected with a conventional d.c. amplifier. The light stimulus was delivered from a Xenon arc lamp which produced an unattenuated intensity of 0.7 PWicm’ in the plane of the retina. A large spot (8.0 mm diameter) was used for evoking the ERG. The duration of the light stimulus was 2.0 sand its intensity, controlled by interposing neutral density filters, was 2.0 log units below the unattenuated intensity (0 log unit). Details of the methods used for light stimulation and recording have been described elsewhere.’ Five retinas were used for ERG at various time intervals after intravitreal injection of %-AAAs. RESULTS

Glutamine cells

synthetase

localization

in retinal

Miiller

Initially, we prepared an antiserum against GS to determine its localization and its protein content in the fish retina. The specificity of anti-GS serum was examined by an Ouchterlony double immunodiffusion and an immunoprecipitation method, in

Gliotoxic

effect of r-AAAs

147

in the retina

D

-45K

44K-

I

3

I

3

Fig. I. Characterization of the anti-serum against GS. A. Ouchterlony’s double-immunodiffusion test. Well I, 100 pg of carp retinal crude extracts; well 2, rabbit anti-serum at dilution x I; well 3, dilution x 2: well 4. dilution x 4; well 5. dilution x 8; well 6, dilution x 16: well 7, preimmune serum. Twenty microlitres of antigen solution (well I) and 20~1 of variously diluted anti-serum (wells 2 6) or preimmune serum (well 7) were allowed to diffuse for 24 h. The plate was then washed for 72 h in PBS (pH 7.5). stained with 0.5% CBB for I h and destained. A single precipitation line was clearly observed as the reaction with the anti-sera. but not with the preimmune serum. B. SDSgel electrophoresis (12% polyacrylamide) showing protein profiles with I pg of sheep brain GS (lane l), 40 lg of the pellets after immunoprecipitation (lane 2) and 40 llg of crude retinal extracts (lane 3). The number at the left indicates a molecular weight of 44.000. C and D. Immunoblotting with the anti-serum. Proteins of sheep brain GS (lane I) and retinal crude extracts (lane 3) separated on SDS-PAGE (as shown in B) were transferred onto a nitrocellulose paper by a Western blotting method, reacted with the anti- (C) or preimmune(D) serum. and then visualized by an ABC method (see Experimental Procedures). A protein having a molecular weight of 45,000 in a monomeric form was identified as retinal GS (C. lane 3).

crude retinal extracts containing GS were reacted with the anti-serum or preimmune serum. The anti-serum showed a single precipitation line against the starting extracts (Fig. IA) but not with preimmune serum. Sheep brain GS and carp retinal extracts were subjected to 12% polyacrylamide SDS gel electrophoresis. and the proteins were stained with CBB. Sheep brain GS showed a single major protein band. whose molecular weight was 44,000 (Fig. lB, lane I), while the carp retinal extracts showed many protein bands (Fig. IB, lane 3). However, when the carp retinal proteins subjected to SDS-PAGE were transferred onto a nitrocellulose paper by the Western blotting method and reacted with anti-GS serum, a single protein band could be visualized by the ABC (avidin-biotin peroxidase complex) method (Fig. IC. lane 3). The mobility of this carp retinal protein was slightly slower than that of sheep brain GS. whose molecular weight as a monomeric form was 45,000 in SDS-PAGE. No protein bands could which

be seen when the blotted proteins were reacted with preimmune serum (Fig. ID). Direct immunoprecipitation was performed by mixing the extracts and the anti-serum. The mixture was centrifuged and the supernatants and pellets were obtained. The GS activity in the supernatant was clearly inhibited by an addition of the anti-serum diluted I:10 (data not shown). When the pellets (immunoprecipitates) were subjected to 12% SDS-_ PAGE, a single major protein band, having a molecular weight of 45,000, was observed, with minor contaminants (Fig. IB, lane 2). The single band protein (45,000 mol. wt) revealed by reacting with anti-GS serum was thus the GS molecule of carp retina. In confirmation, immunohistochemistry revealed GS localization in glial Miiller cells (Fig. 2). Polygonal-shaped fluorescing cell perikarya were clearly outlined in the inner nuclear layer (Fig. 2A). Positively stained Miiller cell processes were traced from

Fig. 2. Indirect immunohistochemlstr~ oI‘GS III the fsh retina. Frozen sections of carp retina arc rcactcti with anti-GS serum (A) and prei~isnulle serum (8) diluted at I: 500, and further reacted uith FiT(conjugated anti-rabbit IgG diluted at I : 1000. After washing with PBS, the sections were cover-slipped with glycerin PBS and photographed hy fluorescence microscopy. Scale bar = 30 ,(n>

the inner nuclear layer through the inner plexiforn? layer as narrow parallel columns ending in funneishaped deposits in the ganglion cell and nerve fibre layers, Positive Miiller cell processes also extended through the outer plexiform and outer nuclear layer to terminate along the outer limiting mcmbranc. .4 yellow non-specific fuorescence also seen in retinas treated with preinlmune serum (Fig. 2B), was observed in the photoreceptor cell layer.

Eight micromole aliquots of cc-AAA isomers were injected intravitreally into the carp eyes and the GS activity in the retina was measured 4 h to one month

later (Fig. 3). The maximal reducti~~n in activity (65%) was seen one to two days after injection of I.-sc-AAA and remained for three to four days; thereafter the activity was gradually restored reaching original levels about one month later. On the other hand, the maximal reduction induced by the same dosage of D- and DL-I-AAA was about one third (28%) and tao thirds (45%) of that of L-form, and the recovery from D- and IX-r-AAA treatment occurred in four to seven days and seven to 10 days, respectively. The minimum effective dose of I.-, IX.and D-form, determined from dose--response studies (Fig. 4), about 0.8, 1.5 and 2.Ojlmol. respectively. A high dose (> 8 pmol) of I>-a-AAA and L-X-AAA induced a 30 and 75% reduction of GS activity. respectively. Thus L-x-AAA at i~rnol doses was equivalent in its reduction of GS activity to that 01 two-fold dose of DL-a-AAA. The racemic mixture. IX-r-AAA showed an intermediate potency.

Retinal protein chungrs in&w’

h!. x -urninoud~ic* wid

We further examined the etfects of jntravitrcal x-AAA isomers on retinal soluble proteins in the

2

I

-m- L-a-AAA

8,umol

2 041;;t-;--;-;i72Tr%iDays after inlrawutar

injection

of U-AAAs

Fig. 3. GS activities at various time intervals after injection of a-AAA. Each isomer of cc-AAAs (8-16+mol) was injected into the carp eye at 0 hand the GS activity (~moijmg protein per 20 min) was followed from 4 h to one month after injection. The GS activity was rapidly reduced in all cases. The recovery with 8 pmol u- (A) and DL-N-AAA(0) was rapid (five to 10 days), while it was rather gradual with L-u-AAA (m). However. the delay tn recovery from GS reduction with 16pmol DL-a-AAA (0) was comparable to that with 8 pmol L-a-AAA (about one month). Each point is the mean + S.D. of three to five determinations. The range of values of GS activity in the control retinas pretreated with saline is shown by the broken horizontal lines.

.-

2

s

-

-m- DL-a-AAA

t

-I-

04r

L -IX-AAA ”

2.0 0.810 d(-Aminoadipic

LO 8.0 10.0 16.0 acid (~~mol )

Fig. 4. Dose-response curves of GS activities after injection of r-AAAs at various doses (0.8-16 pmol). The GS activities were measured at one day after injection of t- (I), DL- (of and a-a-AAA (A), respectively. Each point is the mean value (with SD.) of three determinations.

Gliotoxic

effect of r-AAAs

149

in the retina

DL

x IO3

I

2

3

-

92.5

-

66.2

-

21.5

-

14.4

4

Fig. 5. Retinal protein profiles after injection of r-AAAs. Retinal crude extract (50 pg protein) obtained four days after intravitreal injection of 8 pmol cc-AAAs was applied to 12”/0 SDS-PAGE. Some retinal proteins were clearly reduced (90,000, 58,000, 45,000 mol. wt) whereas others were increased (marked with dots) in the retinas treated with L- and UL-c-AAA (lanes I and 3). as compared with control (lane 4). The protein changes were not as conspicuous in the retina treated with u-r-AAA (lane 2). Molecular weight is indicated by arrows.

supernatant

(105,000~).

injection, induced

a large

Three

change

by administration

AAA.

Some

10.000

mol.

retinal wt,

to

of 8 pmol proteins

marked

four

in the protein

by

of L- and

(68,000, dots)

days profile

were

27,000 clearly

after was DL-S(-

and in-

mol. wt) were decreased as compared with controls (Fig. 5. lane C). The protein changes induced by the L-form (lane L) were more drastic than that due to the Dt.-form (lane DL), while changes were less evident with n-r*-AAA (lane D). To test whether or not the protein changes were reversible, the protein profile revealed by SDS-- PAGE was followed at various time intervals after L-r-AAA injection. The changes in retinal proteins first appeared at three to four days

creased

and

(90,000.

60.000,

45,000

and

38.000

after injection, were maintained for one week, and finally disappeared three to four weeks later (Fig. 6). The protein changes induced by the rx-form recovered by seven to IO days (data not shown). The GS protein (45,000 mol. wt) itself was clearly reduced to less than half of that in controls. This was further confirmed by the immunoblot data with anti-GS serum. in which there was a less dense single 45,000 mol. wt protein band in treated retinas as compared to control retinas (Fig. 7). Loss of’electroretinogram acid injection

h-ware after x -unzinoadipic~

Four hours after intravitreal administration of any one of 8 /Irnol a-AAA isomers. the ERG b-wave was

150

Mr

c

3d

Iw

92.5 66.2

,GS

45

31

21.5 14.4

123456789 Fig. 6. Retinal proteins at extract (50 pg protein) of week (lane 4), three weeks from untreated (control)

various time intervals after injection of L-a-AAA. SDS-PAGE profiles of crude retinas, treated with intravitreal L-a-AAA (8 pmol) for three days (lane 3), one (lanes 5 and 6) and four weeks (lanes 7-9) prior to eye removal. Protein profile retina is shown in lane 2. At three to seven days after injection, increase or

decrease of retinal proteins was conspicuous. Three to four weeks after injection (lanes S-9) the protein changes were essentially recovered. Molecular weight markers (M,) were indicated at lane 1.

markedly reduced or absent and the a-wave increased compared with control eye (Fig. 8, 4 h). The ERG b-wave recovered with a variable time course depending on the a-AAA isomer used. The loss of ERG b-wave caused by D-U-AAA began to recover about one week later, and was fully restored to a normal level by three weeks after injection (Fig. 8, column D-a-AAA). In contrast, the recovery after L-c(-AAA injection was severely delayed and only a small positive deflection was evident even one month later (column L-a-AAA). The negative-going a-wave was grossly abnormal one month after administration of the L-isomer. Even at two months after injection of L-u-AAA, only 50% of recovery of ERG b-wave could be seen. The extent and rate of recovery from DL-a-AAA treatment was intermediate (column DLa-AAA) as compared with those of D- and L-forms. However, an injection of 16 pmol DL-a-AAA caused as long-lasting a suppression of ERG b-wave as with 8 pmol of L-GL-AAA. The relationship between the amplitude of ERG b-wave and intervals (days) after injection of a-AAA isomers (8 and 16pmol) are summarized in Fig. 9. The dose of 16 pmol DL-CYAAA was almost equivalent to 8 pmol L-a-AAA with

respect to both suppression b-wave.

and recovery

of the ERG

DISCUSSION GIatamine synthetase in the fish retina GS obtained from sheep brain, rat liver or chicken retina has a molecular weight of 350,000-450,000 and is compased of eight identical subunits, each with a molecular weight of 42,000-50,000 in the monomeric form.*‘.*’ Carp retinal GS (45,000mol. wt) appeared to be slightly different from that of sheep brain (44,000 mol. wt) in molecular structure, but with comparable antigenicity. Here we were able to confirm that GS was exclusively localized to glial Miiller cells in the carp retina by immunohistochemistry as seen previously in the bony fish retinas.‘” Retinal GS is a useful and specific marker for the study of Miiller cell biochemistry and for quantitative evaluation of the effect of such gliotoxins as a-AAAs. Gliotoxic action of a-aminoadipic acid in the,fish retina Although a-AAAs may be general gliotoxins, their actions would appear to be limited to Miiller cells in

Gliotoxic

effect of r-AAAs

L

I

2

Fig. 7. Immunoblotting of GS protein in the retina pretreated with injection of L-c(-AAA. The 45,000 mol. wt GS protein was clearly reduced one week after injection of X Ilrnol L-a-AAA (lane 2) in comparison with control (lane I) retina. The GS protein was visualized by an immunoblotting method with anti-GS serum.

the carp retina. Unlike most mammalian retinas which contain two distinct neuroglial cell types, Miiller cells and astrocytes. the avascular retina of

151

in the retina

fish does not contain astrocytes. Many investigators have shown a selective swelling of retinal Miiller cells with pycnosis in response to ~~-AAAs.‘~,“.‘~ Four hours after intraocular injection of DL-X-AAA into the adult rat eye, Miiller cells could no longer be identified.“.‘” Biochemically, the lesion was accompanied by a 55% reduction in the high affinity uptake of p-alanine which was thought to be selectively taken up by glial cells. On the other hand, the high affinity uptake of choline as well as the activities of choline acctyltransferase and glutamic acid decarboxylase. which were associated with neuronal cells. were unaffected by r-AAAs. Electrophysiologic changes inducing the loss of the ERG b-wave and a disturbance of the light response of Miiller cells have been reported in many vertebrates 2-4 h after treatment with z-AAAs.‘~‘~.‘~ Morphological changes in glial cells after intraocular injection of CX-AAAs (8-16 LImoI), presented in the following paper.” further confirm the biochemical and electroretinographical changes shown in the present study. Since the dose of a-AAAs used was of the order of micromoles, the intraocular concentration of r-AAA would be of the order of mM assuming a vitreous volume of I ml. This concentration was comparable to those used in previous studies of retina? and glioma cell lines.h Our present long term observation in the carp retina showed that the reduction of GS activity. the decrease or increase of retinal protein components and the loss of the ERG b-wave were induced by intravitreal injection of z-AAA isomers in micromolar amounts. Most changes were transient, but the recovery of ERG b-wave was so delayed after injection of 8 [lrnol L-c(-AAA or I6 LLrnol IIL-Z-AAA.

Control

L-a-AAA

1

2

3

DL- a -AAA

4hr

4 hr

D-a-AAA

4hr -i-f+

lm

2w +-mw--

2m-

-L-l--

-‘w*rusIIcI

-J 100Jlv 1 set

‘“h

Fig. 8. Electroretinograms from the carp retinas at various intervals after intravitreal injection of Z-AAAs. The ERGS were recorded from the retinas before and after treatment with L-, DL or D-r-AAA. 8 pmol. Four hours after injection of one of the three isomers, no ERG b-wave was recordable. The loss of the b-wave induced by D-. DL- and L-a-AAA continued for four to five days, seven to IO days and two to three weeks, respectively. Thereafter the amplitude of the b-wave gradually and partially recovered over the periods indicated.

Days

after

rntraocular

injection

of &-AAAs

Fig. 9. Relative amplitudes of ERG b-wave at various intervals after injection of cc-AAAs. Each isomer of r-AAAs (8-16 pmol) was injected jnto the carp eye at 0 h and the b-wave of ERG was followed from 4 h to two months after injection. The ERG b-wave was rapidly abolished by all isomers. The full recovery from I>-x-AAA (A) treatment was seen at three to four weeks after injection, while the gradual and partial recovery from VL-n-AAA (0, 8 pmol) treatment took place over two months. The recovery from L-x-AAA (u, 8k~mol) treatment was only 50% even at two months after injection, and similarly with 16ymoI of DL-X-AAA (0). PI al.” reported that in the albino rabbit retina a low voltage ERG was observed at a later stage (two weeks after administration of us-a-AAA). In our present study with carp retina, about 25% recovery of ERG b-wave at the corresponding time periods after pretreatment with I6 pmol us-a-AAA. They suggested that this probably reflected a general retinotoxic action of the drug. The drug might have a significant effect on the functional integrity of the visual cells and pigment epithelium cells,27 even if the gliotoxic effects had dissipated within these time periods, abnormal receptoral activity would have exerted a profound effect on the b-wave of the ERG. As they did not study longer (> two weeks) effects of z-AAA on the ERG b-wave of the rabbit eye, we could not compare our results, in which the ERG b-wave was gradually recovered and 50% recovery was obtained two months after administration of high doses of DL-X-AAA. The carp retina has germinal cells, which are potential cells to mature and differentiate any types of retinal cells {see also the following paperZ2). This may account for the discrepancy between the both species.

Welinder

gliotoxic potency estimated from the rcductron oi GS activity was in the order L-X-AAA (h5’1,tI reduction) > ILL-z-AAA (45% j > o-3 -AA/\ c.?X”‘~~~ 1 The same order of potency was obtained ti>r the suppressive effects on the ERG b-wave (anuthcr glial parameter), and for the r-AAAs-induced dccreasc 01 increase in retinal proteins. Reversal of the effects on retinal proteins and on GS activity occurred in the opposite order; that induced by 8 pmmolof IX-x-AAA required about seven to IO days while IL-x-AAA changes did not recover until three to four weeks. The data suggest that changes to retinal proteins resulted from general damage to glial cells. The increased amounts of 68,000+27.000 and 10,000 mol. wt proteins seen after DL- or I.-u-AAA injection are not likely to bc associated with the GS protein (45,000 mol. wt) since the latter was reduced in content as seen in the immunoblots. The increases 01 decreases of retinal proteins, including GS. may refect a substantial destruction of glial Miiller cells caused by L- or ILL-r-AAA as a gliotoxic agent. As noted previously, structural damage to Miiller cells have been reported. Further studies of retinal protein changes produced by a-AAAs must be conducted in detail in order to identify the spccihc proteins affected by LX-AAAs. A secondary aim of this study, was to examine the possibility that the o-isomer antagonizes the neurotoxic effects of the L-isomer:‘4 if so the ut-isomer would be expected to act as a pure gliotoxin. We compared the effects of g pm01 L-a-AAA with those of 16pmol us-a-AAA. If the above possibility was correct, both L-isomer and the racemic mixture would deliver a comparable degree of gliotoxicity at 8 Aimol, while at that dose only the L-isomer would produce an additional degree of neurotoxicity. Indeed. the equivalent efficacy of 8 ktmol L-isomer and of I6 pm01 oL-isomer was seen in the suppressing effect vn the GS and ERG b-wave activities. It can be concluded that the gliotoxic effect (causing a long term suppression of ERG b-wave) of 8 Airno L-XAAA is more potent than that of 8 LimoI DL-r-AAA, since it contains only 4 /~rnol of the gliotoxic ~-form.

.4t.lmuM,ie~g~~e~6~-We thank Dr Y. Hayashi and Dr S. Hatakenaka for their valuable suggestions on preparing GS Quantitative aspects qf gliotoxicity induced by CL- anti-serum and Dr A. W. Spira for his correction of our ffrnino~d~~~c acid isomers English. We also thank Mrs Tami Urano for her secretarial assistance. This work was supported in part by research The present study has aimed to quantify the grants (Nos. 59570055, 60770056, 61480105 and 01659506 gliotoxic action of a-AAA enantiomers on the retina to SK.) from the Ministry of Education, Sdence and by using a biochemical glial marker, GS. The Culture. Japan.

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