Brain Research, 111 (1976) 79-93 © ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands

79

THE UPTAKE OF L-GLUTAMATE BY THE RETINA

RALPH D. WHITE and MICHAEL J. N E A L

Department of Pharmacology, The School of Pharmacy, London WC1N lAX (Great Britain) (Accepted December 7th, 1975)

SUMMARY

The accumulation of L-[14C]glutamate by the isolated rat retina has been studied. When retinae were incubated at 37 °C in a medium containing L-[14C]glutamate, tissue/medium ratios of about 40:1 were achieved after 60 min. The labelled Lglutamate was rapidly metabolised and after 10 min about 50 ~ of the radioactivity in the tissue amino acids was present as glutamine, aspartate, and 4-aminobutyrate (GABA). The process responsible for L-glutamate uptake showed many of the properties of an active uptake system: it was temperature sensitive, sodium dependent, inhibited by metabolic inhibitors and showed saturation kinetics. The saturable uptake process could be resolved into two components; a 'high' affinity process (apparent Km ----21 #M, Vmax -- 35 nmoles/min/g tissue) and a 'low' affinity process (Kin ----- 630 #M, Vmax ---- 881 nmoles/min/g tissue). The 'high' affinity and 'low' affinity uptake processes for L-glutamate appeared to have identical properties in the retina. The uptake of L-glutamate was not specific and was inhibited by other acidic amino acids including D-glutamate but not by neutral or basic amino acids. The retinal uptake of L-glutamate is not likely to be due to a homoexchange phenomenon because the retina was capable of achieving a large net uptake of glutamate and the efflux of L-[~4C]glutamate from the tissue was not increased by the addition of non-radioactive L-glutamate to the incubation medium. Autoradiographic studies indicated that the sites for glutamate uptake are largely in the neuroglial Miiller cells.

INTRODUCTION

In the retina it has been suggested that glutamate or aspartate may be an excitatory transmitter substance released from the photoreceptor terminals12,24. There is inferential evidence that the hyperpolarising response of horizontal cells to stimula-

80 tion by light might be due to a decrease in the release of an excitatory transmitter substanceS,Zg,4°: thus, the application of magnesium ions to the retina, which would be expected to reduce transmitter release, produces hyperpolarisation of the horizontal cells 6,12 and peroxidase, which is believed to be accumulated only by 'active' nerve endings, is taken up by rod terminals only in the dark adapted retina3L Furthermore, the continuous release of an excitatory transmitter from the photoreceptor in the dark would explain the low membrane potential of the second order neurones 38. The identity of this excitatory transmitter is unknown, but in the vertebrate retina it is unlikely to be acetylcholine, a catecholamine, or 5-hydroxytryptamine since these are not present in the outer synaptic layer15. 23 and acetylcholine has been shown to be without effect when applied to the horizontal cells of carp retina 24. If glutamate or aspartate is a photoreceptor transmitter substance, it may be inactivated after its release from receptor terminals by a reuptake process, as has been suggested previously for amino acid transmitters in other areas of the CNS 9,1°,zl. Although other studies have indicated that the retina can accumulate glutamate 1,13,z°,37,41,42, in this study the uptake of L-[14C]glutamate into the rat retina is examined in rather more detail. Some of this work has already been published in the form of preliminary communications 25-27. METHODS

Uptake of L-[14Cjglutamate by rat retina The uptake of L-[14C]glutamate by the retina was measured using methods described previously 14. Briefly, albino Wistar rats were light adapted and then killed by cervical dislocation. The eyes were enucleated and the retinae removed. Each retina was placed in 9.5 ml of ice-cold Krebs-Ringer bicarbonate medium contained in a 25 ml Erlenmeier flask. The air space was displaced with a mixture of oxygen (95 %) and carbon dioxide (5 %) and the flask was closed with a rubber stopper (Subaseal). The retinae were then given a preliminary incubation of 15 min at 37 °C. L[14C]Glutamate (25 nCi), in 0.5 ml medium previously warmed to the incubation temperature, was added to the flasks by injection through the stopper, to give a final concentration of 1.2 × 10-8M and the incubations were continued for various times. Each retina was recovered by rapid filtration and washed with 5 ml of icecold medium, during which time ( < 5 sec) there was negligible loss of radioactivity from the tissue. The tissue was dissolved in Soluene (Packard) and the radioactivity was estimated by liquid scintillation counting. Preliminary experiments indicated that residual radioactivity retained by the filter paper was very small ( < 20 disint./min) and could not be removed by further washing. The results were corrected for this filter blank.

Metabolism Groups of 4 retinae (total wet weight approximately 40 mg) were weighed and incubated with L-[14C]glutamate (1.2 × 10-TM) for either 10 or 60 min. The tissue and medium were separated by filtration and the medium was acidified by the addi-

81

tion of 1 M hydrochloric acid (1 ml). The tissue was rinsed with fresh, ice-cold medium, and then extracted by homogenization in 6 ~ perchloric acid (1 ml) and allowed to stand at 2 °C for 1 h. Unlabelled glutamate (25/~g) was added, and the acidified tissue and medium extracts were passed through columns (5 x 0.5 cm) of Amberlite CG 120 ion exchange resin (H + form) (200-400 # ) (BDH). After washing the columns with distilled water (15 ml) the amino acids were eluted with 2 M ammonium hydroxide (5 ml). Parallel control experiments were performed in which L-[14C]glutamate (50 nCi) was added to unlabelled homogenates or medium samples prior to placing on the columns. Aliquots were assayed at each stage for radioactivity. The ammonia eluates were evaporated to dryness by heating at 40 °C on a Biichi rotary evaporator. The residues were reconstituted in methanol:water (4:1 v/v) and spotted on Whatman no. 1 chromatography paper, together with a mixture of carrier amino acids (25/~g each of L-glutamate, L-glutamine, L-aspartate, glycine, taurine and GABA). The chromatograms were developed by descending chromatography, using (1) butanol:acetic acid:water (4:1:1), or (2) phenol:water:ammonia (phenol saturated with water, concentrated ammonia solution 0.5 ~ v/v, 8-hydroxyquinoline 0.1 ~ w/v) as the solvent system. After drying, the spots were visualised by spraying the standard runs with ninhydrin solution (0.25 ~ w/v in acetone) and heating to 60 °C for 15 min. The chromatograms were then cut into strips and the radioactivity measured with a strip scanner (Nuclear Chicago, Actigraph III). The pellet was dissolved in Soluene TM100 (Packard) and the radioactivity incorporated into the protein and lipoprotein was estimated by liquid scintillation counting.

Determination of free amino acids: net uptake of L-glutamate by rat retina Free amino acids were determined by ion-exchange chromatography using an automatic amino acid analyzer (Biocal BC100). Groups of 2 retinae (approximately 20 mg wet weight) were weighed and given a preliminary incubation at 37 °C for 15 rain in 4.5 ml medium. L-[14C]glutamate (50 nCi) with unlabelled L-glutamate in 0.5 ml medium was then added to some flasks to give a final concentration of 10-4M. To control flasks, 0.5 ml of medium was added. Both sets of flasks were incubated for a further 15 min. The retinae were quickly recovered from the medium and were rinsed briefly with ice-cold medium. Isoleucine (50 nmole for approximately 20 mg tissue) was added as an internal standard as preliminary determinations had shown it to exist in negligible quantities in the retina. Each group of retinae (20 mg tissue) was homogenized with 6 ~ perchloric acid (1.0 ml) in a hand-held Potter-Elvehjem ground glass homogenizer. If radioactivity was present, 50/A aliquots of both homogenate and medium were taken for estimation by liquid scintillation counting. The homogenate was allowed to stand at 2 °C for 1 h and was then centrifuged (1000 x g 10 min, 0 °C). The pellet was resuspended twice (2 x 0.5 ml 6 ~ perchloric acid followed by centrifugation) and the rinsings were pooled with the supernatant. The extract was adjusted to pH 4.0 with 0.5 N KOH. The potassium perchlorate precipitate was allowed to sediment at 2 °C overnight and the supernatant was recovered by centrifugation (I000 x g 10 min, 0 °C). Rinsings ( 2 x 0.5 ml) were also recovered and the pooled extract was dried down as described earlier. The residue was recon-

82 stituted in citrate buffer (pH 2.2, Biocal) to give a final concentration of approximately t0 mg original wet tissue in 1 ml buffer. Samples were run on an amino acid analyzer under conditions giving maximum resolution of the major glutamate metabolites. including GABA. The amino acid quantities so determined were corrected for recovery (normally > 96 ~ as judged by the recovery of the added isoleucine) and are expressed as #moles/g wet weight of retina.

A utoradiography Rat retinae were incubated at 37 °C for 30 min in oxygenated medium containing DL-[3H]glutamate (5 #M). At the end of the incubation the tissue was washed in ice-cold medium and fixed in 2.5 ~ glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 h at room temperature. Approximately 25 ~ of the radioactivity was lost during the fixation process. The fixed tissue was washed in 0.1 M cacodylate buffer (pH 7.4) and was processed for radioactivity as described by Iversen and Bloom 19.

Effiux of radioactivity from the retina Four retinae (total wet weight approximately 40 mg) were pooled and incubated with e-[14C]glutamate (2.4 × 10-7M) for 30 min at 37 °C. The retinae were recovered, briefly rinsed ( < 5 sec) in fresh medium, and transferred to 5 ml of fresh medium (37 °C) in a 25 ml Erlenmeyer flask. The air space was displaced with a mixture of oxygen (95 ~) and carbon dioxide (5 ~o) and the stoppered flask was placed in a shaking water bath. At various intervals a 200 #1 aliquot of medium was removed from the flask and assayed for radioactivity; a fresh 200 #1 sample of medium was added to the flask and the air space was again displaced with the gaseous phase before restoppering. The results were corrected for the small sequential diluting error.

Materials The incubation medium was a Krebs-Ringer bicarbonate solution of the following composition: NaCI, 118.4 mM: KCI, 4.74 mM; CaC12, 2.52 mM; MgSO4, 1.18 mM; KHzPOt, 1.19 mM; NaHCO3, 25 mM; equilibrated with a mixture of oxygen (95 ~) and carbon dioxide (5 ~), pH 7.4 and containing glucose 2 g/l. Universally labelled L-[14C]glutamic acid (270 mCi/mmole), DL-[3H]glutamic acid (1 Ci/mmole), [3H]inulin (300 mCi/mmole) and [14C]urea (62 mCi/mmole) were obtained from the Radiochemical Centre, Amersham. Laboratory reagents (AnalaR grade), and amino acids were purchased from either BDH Ltd. or Sigma (London) Chemical Co; L-serine-O-sulphate was prepared by the method of Dodgson et al. 11.

RESULTS

Time-course of L-[14C]glutamate uptake and the effect of temperature When individual retinae were incubated at 37 °C in a medium containing L-[14C]glutamate (1.2 × 10-aM) (Fig. 1) there was an initial phase of very rapid uptake (0 -5 rain) followed by a slower phase (5-50 rain). The maximum accumula-

83

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33

/.

~20 LIJ

2s~

/

/f

:

t/) DI5 II

o'c

8

0

o

10 20

30

40

TIME

50

IO 20 TIME (MIN)

60

70 80

30

90

(MIN)

Fig. 1. Time-course of L-[14C]glutamate uptake into retinae incubated in 10 ml medium at 37 °C with L-[14C]glutamate (1.2 × 10-aM). Figures by data points indicate the number of experiments at each time. Bottom line shows distribution of [all]inulin under identical conditions (4 independent determinations at each time). Inset shows the effect of temperature on the L-[14C]glutamate uptake (8 or more independent determinations at each point). Vertical bars indicate the S.E.M.'s

tion of radioactivity in the tissue occurred after 60 min incubation, when a tissue/ medium ratio (L-[14C]glutamate disint./min/gtissue: L-[14C]glutamate disint./min/ml medium) of 43 : 1 was achieved. Longer incubation times (90 min) produced a decline in the level of accumulated radioactivity. Because of the large volume of medium used (10 ml) compared with the small weight of tissue (10 mg), the concentration of L-[14C]glutamate in the medium fell by less than 5 ~ during a 60 min incubation. The extracellular space was determined by incubating retinae for various times with [3H]inulin. After 5 min a tissue/medium (T/M) ratio of 0.4 ± 0.05 °/o (mean ± S.E.M., n = 4) was obtained, and this was constant over the next 25 min (Fig. 1). This value corresponds to the T/M ratio obtained by extrapolating the uncorrected T/M ratios for L-[14C]glutamate uptake over short incubation times ( < 10 min) back to zero time. Unless otherwise stated the results were not corrected for the small amount of radioactivity accumulated in the extracellular space. The initial velocity of the uptake of L-[14C]glutamate (1.2 × 10-aM) was identical at 25 °C and 37 °C although after 30 min significantly more radioactivity was accumulated in retinae incubated at 25 °C (Fig. 1). When incubations were performed at 0 °C the rate of L-[14C]glutamate uptake was markedly reduced when compared with that of higher temperatures. However, some uptake of L-[14C]glutamate still occurred at this temperature giving a

84

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1.0

I | • 4, 0 +'+~'-'--" 0.6 1.0 1.2 1"4 1.6 1.8

V / S x10 3 Fig. 2. Kinetic analysis of effect of a range of L-glutamate concentrations on initial rate of L-[14C]glutamate uptake by retinae. Hofstee-Eadie linearising transformation, with v -- rate of L-[t4C] glutamate uptake (moles/min/g wet weight) and S = L-glutamate concentration (M). Each point is the mean of at least 9 experiments. The S.E.M. of the velocity determinations was less than 7 ~.

tissue m e d i u m ratio of 3:1 after a 30 m i n i n c u b a t i o n . I n c u b a t i o n s at 15 °C a n d 37 °C gave similar results. The values o b t a i n e d at 25 °C a n d 15 °C indicate a Qlo of approximately 0.6 for the initial u p t a k e process in this t e m p e r a t u r e range.

Effect of L-glutamate concentration The effect of the external glutamate c o n c e n t r a t i o n on the uptake of L-[14C] glutamate was studied over a range 1 0 - 6 M - 1 0 - 3 M at 37 °C. W h e n corrected for the 100

1'2x 10-8M

o iv.

~, 80

o u v "'

60

'"

4(1

3

2~

0

' 100

[Na]°/o Co,trot Fig. 3. Effect of sodium concentration on L-[14Clglutamate uptake by retinae incubated at 37 °C for 10 min with L-l14Clglutamate (l.2 × 10-8M, upper curve; or 10-SM, lower curve) in media in which various proportions of normal sodium content were replaced with Tris buffer (50 raM, pH "/.4) and choline chloride. Each point is the mean ::t: S.E.M. of at least 8 experiments.

85 TABLE I Amino acids and metabolic inhibitors affecting the uptake of L-{14C]glutamate into retinae

Retinae were preincubated at 37 °C for 15 min and then incubated for a further 10 min with L-[14C]glutamate (either 1.2 x 10-aM or 10-aM) in the presence of an amino acid, or a metabolic inhibitor (also present during preincubation). Values for per cent control uptake are means 4- S.E.M. of 4 or more independent determinations. Uptake of L-[14C]glutamate ( % control) 1.2 x IO-SM

IO-aM

(a) Amino acids (10-3M): L-aspartate L-Cysteate L-serine-O-sulphate D-glutamate GABA

35 4- 3 34 4- 2 51 4- 7 28 4- 4 123 4- 5

70 62 74 67 98

(b) Metabolic inhibitors: p-Chloromercuriphenylsulphonate (10-4M) Sodium iodoacetate (10-4M) Dinitrophenol (10-aM) Ouabain (10-SM)

42 4- 2 69 4- 7 119 4- 7 81 -4- 5

224- 2 39 + 10 61-4- 3 514- 7

44444-

4 2 5 5 7

label residing in the extracellular space, the accumulation of L-[14C]glutamate by the tissue was linear over at least the first 5 min of incubation at both extremes of the concentration range. Therefore, the values obtained after 5 min incubations were used to estimate the initial velocity of L-[14C]glutamate uptake at the various glutamate concentrations. These results were then plotted according to a linearising transformation of the Michaelis-Menten equation using least-squares regression analysis. [14C]Glutamate was accumulated in the retina by a saturable uptake process which could not, however, be interpreted as a single transport process ~s. A plot of V against v/S (Fig. 2) suggested that the uptake process consisted of two components and arbitrary division of the data at 10-SM was used to estimate kinetic parameters for a 'low' affinity (apparent Km = 630 #M, Vmax ---- 881 nmoles/min/g tissue) and a 'high' affinity (apparent Kra = 21 /~M, Vmax = 35 nmoles/min/g tissue) uptake process. In subsequent experiments, the characteristics of both the 'high' and 'low' affinity uptake processes were compared. Effect o f sodium ion concentration Retinae were incubated for 10 min in media in which Tris-hydrochloric acid buffer (pH 7.4, 50 m M ) was substituted for the usual bicarbonate buffer and bubbled with oxygen. Varying proportions of the normal sodium chloride content were replaced by choline chloride or sucrose. The uptake of L-[14C]glutamate (1.2 X 10-aM and 10-aM) was greatly dependent on the sodium concentration (Fig. 3) and in the absence of sodium ions the uptake was virtually abolished, being less than 4 % of the control values for both concentrations of L-[14C]glutamate. Subsequent incubation of

86 700

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Fig. 4. Double reciprocal analysis of the inhibition of L-[14C]glutamate uptake by L-aspartate. Uptake of two ranges of L-glutamate concentrations (10-6-10-4M, upper trace; and 5 × 10-5-10-SM, lower trace) was determined in the absence ( I ) and presence ( 0 ) of a single concentration of L-aspartate (25 F M and 650/~M, respectively). The data describe straight lines which intersect on the vertical axis indicating competitive inhibition. Velocity (v) is expresse~ as moles/g wet weight/min. Each point is the mean of at least 4 experiments and the S.E.M?s were less than 7 ~ .

retinae in a normal medium, after previously incubating in sodium-free medium, showed that the ability to accumulate glutamate was unimpaired, thus the transport process had not been irreversibly abolished by the procedure.

Effect of amino acids and analogues of L-glutamate Retinae were given a preliminary incubation for 15 min, then incubations were continued for 10 min after the addition of L-[14C]glutamate (final concentration: 2.1 × 10-SM or 10-aM) and various amino acids or analogues (1 mM). L-Aspartate, L-cysteate, L-serine-O-sulphate and D-glutamate reduced both the "high' and 'low' affinity uptake of L-[14C]glutamate; L-serine, L-glutamine, glycine, DL-homocysteate, L-histidine, 2,4-diaminobutyrate and the glutamate analogues dimethylglutamate, ?-methylglutamate, diethylglutamate and ?-ethylglutamate did not significantly affect the retinal accumulation of L-[14C]glutamate (P > 0,05 from Student's t-test). The presence of GABA (1 mM) had no effect on the 'tow' affinity uptake of L-[14C]glutamate, but significantly increased the 'high' affinity uptake (Table I). The effects of L-aspartate on the 'low' and 'high' affinity uptake processes for L-[14C]glutamate were examined in more detail by using two ranges of [14C]glutamate concentration: 10-SM-10-SM and 10-6M-10-4M, respectively. Because both Laspartate and L-glutamate exhibit non-linear Michaelis-Menten kinetics, extraction

87 .--

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10 20 30 40 50 60 70 80 90 100 TIME (MIN)

Fig. 5. Efltux of radioactivity from retinae previously incubated with L-[14C]glutamate (2.4 × ]0-TM) for 30 min at 37 °C. The release of radioactivity from the retinae is expressed as a percentage of the 14C taken up into the tissue during the 30 min incubation period. The upper trace (O) shows the spontaneous efflux of radioactivity from the tissue. In some experiments, the medium was replaced at 15 min with medium containing non-radioactive L-glutamate and the effect of this on the efflux of radioactivity was followed (©). Most of the points for these two curves were found to be identical ((D). The lower trace (A) shows the efflux of [14C]urea, a comparative demonstration of the loss of a freely diffusable material from the tissue. Each point is the mean of at least 8 independent determinations; S.E.M.'s were consistently less than 5 %. of meaningful kinetic parameters for inhibition is difficult. However, LineweaverBurk plots (Fig. 4) indicated that L-aspartate is a competitive inhibitor of both the 'high' and 'low' affinity uptake processes for L-glutamate.

Effect of metabolic inhibitors Retinae were preincubated for 15 min in media containing the metabolic inhibitor. L-[14C]glutamate and unlabelled L-glutamate were then added to give final concentrations of 1.2 × 10-aM or 10-3M and the incubations were continued for a further 10 min. The presence of sodium iodoacetate (0.1 mM), p-chloromercuriphenylsulphonate (0.1 m M ) and ouabain (0.01 m M ) significantly reduced the uptake of both high and low concentrations of L-[14C]glutamate. Dinitrophenol (1 m M ) increased the 'high' affinity uptake of L-[14C]glutamate (i.e., external concentration = 1.2 × 10-aM) but reduced the 'low' affinity uptake of L-[14C]glutamate (1 m M ) (Table I).

Metabolism and net uptake of L-[14C]glutamate by retina The metabolism of L-[z4C]glutamate was examined by incubating retinae for either 10 min or 60 rain at 37 °C in medium containing L-[14C]glutamate (1.2 × 10-TM). Chromatographic analysis of tissue extracts indicated that after both 10 min and 60 min a substantial proportion (48 % and 43 %, respectively) of the labelled glutamate accumulated by the retina was metabolised, principally to glutamine,

88 TABLE II Metabolism o f L-/laC]glutamate by rat retina

Retinae were incubated for either 10 or 60 min with L-[14C]glutamate(1.2 i 10 7M) in 10 ml medium at 37 °C. Tissue and medium were separately subjected to ion exchange and paper chromatography. Control blanks were run throughout. Results are the means ± S.E.M. of 6 experiments. N.D., not detected.

Tissue

14C re-

lncubation time (min)

Distribution o f 14C amongst amino acids (%) Glutamate

Glutamine

Aspartate

GABA

14C not covered retained by in insolu- column O~ ble residue (/o) (%)

10 60

52±5 57±2

28±4 12~2

11 =L1 18 ± 3

4tl 7± 1

2.6-5_-0.5 N.D. 7.3-l-0.5 N.D.

98 :~ 2 93 ± 3

N.D. N.D.

N.D. N.D.

N.D. N.D.

---

Medium 10 60

N.D. 10.2 ± 0.8

aspartate and a small amount of GABA. Because of the large amount of L - [ 1 4 C ] glutamate present in the medium, no amino acid metabolites could be detected in the medium after either l0 or 60 rain incubations, although after 60 rain small amounts of acidic and neutral 14C metabolites were detected and these amounted to approximately 10 ~ of the radioactivity remaining in the medium (Table II). The incorporation of L-[14C]glutamate or its metabotites into protein or lipoprotein was estimated by measuring the radioactivity associated with the insoluble protein precipitate of the tissue extracts. In 6 experiments, the average amounts of radioactivity incorporated into retinal protein following l0 min and 60 min incubations were 2.6 ± 0.6 ~o and 7.3 4- 0.5 ~ (mean 4- S.E.M.), respectively. To establish whether the L-glutamate uptake process could lead to a net increase in the total glutamate content of the tissue, retinae were incubated for 15 min at 37 °C in a medium containing L-[14C]glutamate (0.1 mM). At the end of the incubation, the retinae were extracted with acid and assayed for total radioactivity and total amino acids. The results (Table III) indicated that a considerable net uptake of L-glutamate occurred and also that the tissue levels of L-glutamate metabolites were increased. Thus, the total L-glutamate content of the tissue at the end of the incubation was 48 ~ higher than that of retinae incubated for the same time in glutamate free medium. Aspartate and glutamine levels were increased by 21 and 13 ~ , respectively, but there was no significant increase in the retinal G A B A concentration (Table IlI). The uptake of L-[14C]glutamate calculated from the uptake of 14C, was somewhat higher than the net increase in tissue glutamate and its major metabolites. A utoradiography

Light microscope autoradiography revealed that activity was present in all layers of the retina except the outer segments of the rods. The concentration of radio-

89 TABLE III

Net uptake of L-glutamate by rat retina Retinae were incubated for 15 min either with (test) or without (control) L-[14C]glutamate(10-4M) in 5 ml medium at 37 °C. Tissue extracts were analysed on an automatic amino acid analyser. 14C was determined by liquid scintillation counting. Results are expressed as/~moles/g wet weight tissue and are the means 4- S.E.M. of 8 (control) and 4 (test) experiments. P values from Student's t-test (two-tailed). N.S., not significant.

Amino acid

Control

Test

Taurine Aspartate Threonine Serine Glutamine Glutamate Glycine Alanine GABA

7.70 -4-0.500 1.08 4- 0.045 0.10 4- 0.013 0.32 4- 0.032 3.15 4- 0.109 1.20 4- 0.041 0.79 4- 0.041 0.25 ± 0.023 0.72 4- 0.055

7.07 4- 0.490 1.31 4- 0.056 0.16 4- 0.025 0.42 4- 0.055 3.55 4- 0.169 1.77 4- 0 . 0 8 7 0.95 4- 0.051 0.31 4- 0.052 0.76 -4- 0.069

Total net increase of endogenous amino acid Glutamate uptake calculated from accumulation of radioactivity

Difference

0.23 4- 0.085 0.06 4- 0.028 0.40 4- 0.210 0.57:3:0.094 0.16 4- 0.077

P N.S. < 0.025 < 0.010 N.S. < 0.10 < 0.001 < 0.10 N.S. N.S.

1.42

1.60 4- 0.030

1.60

activity was greatest in the ganglion cell layer, inner nuclear layer and at the outer limiting membrane. Much of the radioactivity appeared to be in striations running at right-angles to the retinal layers. The pattern of labelling suggests that a large proportion of the accumulated radioactivity was present in the neuroglial Miiller fibres. A predominantly glial uptake of [all]glutamate has also been reported in the rabbit 13 and frog 20 retina.

Efftux o f radioactivity f r o m retina The efflux of radioactivity from retinae which had previously been incubated with L-[14C]glutamate (2.4 X 10-TM) for 30 min and then transferred to fresh medium, is shown in Fig. 5. There was an initial fairly rapid loss of radioactivity from the tissue and after 20 min, 25 % of the radioactivity initially in the retinae had been lost. This was followed by a slower efflux and during the next 80 min only a further 17-18 % of radioactivity was released. The etBux of radioactivity was not increased by adding non-radioactive L-glutamate (final concentration = 1 mM) to the external medium (Fig. 5). Chromatographic analysis of the tissue and medium at the end of a 40 min etttux experiment indicated that only 31% of the radioactivity in the medium attributable to amino acids at the end of this period was glutamate, 56 % of the radioactivity was present as glutamine, the remainder being aspartate (7 ~ ) and G A B A (5 ~o) (Table IV). In contrast, the tissue retained only a small amount of glutamine (2 %) and over

90 T A B L E IV

Efflux ojL-~rl4C]ghttamate from rat retina: distribution 0 / r l c amongst major amino acid metabolite,~ in tissue and medium Retinae were incubated for 30 min with L-[14C]glutamate (1.2 × 10-VM) in 10 ml medium at 37 C

and were then transferred to 5 ml fresh medium. After a further 40 min incubation, tissue and medium were separately subjected to ion exchange and paper chromatography. Control blanks were run throughout. Results are the means ± S.E.M. of 4 experiments. N.D., not detected.

Tissue Medium

Distribution o f a4C amongst amino acids (%) . . . . . . . . . . . . . . . . . . . . . . . . . . . Glutamate Glutamine Aspartate GABA

HC not retained by column (%)

55 ± 3 31 ± 5

N.D. 41 ± 3

2 ± 1 56 ± 7

30 ± 2 7 ± 1

13 ~: 1 5 zL 1

half (55 ~o) the radioactivity remaining in the labelled tissue amino acids after the efflux period of 40 rain was glutamate. Aspartate and GABA represented 30 and 13 ~o of the radioactivity, respectively (Table IV). A considerable proportion of the total radioactivity present in the medium at the end of this time was attributable to nonamino acid metabolites such as carbon dioxide 7,42.

Uptake of glutamate by other regions of the eye The uptake of L-[r~C]glutamate by other ocular tissues was examined. Small slices (0.1 mm) of lens, cornea, or sclero-choroid, were subjected to identical incubation methods as the retinae, but even after 30 min incubation, negligible uptake of label could be demonstrated, and T/M ratios of less than 0.8 were obtained. DISCUSSION

The present results demonstrate that the retina possesses the ability to concentrate L-glutamate from an external medium by a transport process which is saturable, temperature sensitive, sodium dependent, inhibited by metabolic inhibitors, and is capable of producing a net uptake of the amino acid. The retinal transport process for L-glutamate is similar to those described previously for other areas of the central nervous system a,4,17,ao,86. Kinetic analysis of L-glutamate uptake by the retina indicated that the transport process does not follow simple Michaelis-Menten kinetics 2a. Two possible explanations, which are indistinguishable kinetically 16, are either that L-glutamate is taken up by a single transport system, which is activated by high concentrations of substrate or that two or more independent transport mechanisms exist for L-glutamate, which may correspond to the 'high' and 'low' affinity systems described by other investigators3,4,17, a6. Attempts to distinguish between the properties of the possible 'high' and 'low' affinity uptake systems did not reveal any differences and L-glutamate uptake at both high and low external concentrations was sodium depen

91 dent; reduced by metabolic inhibitors and exhibited the same specificity. The 'low' affinity process is not likely to be solely due to simple diffusion because, like the 'high' affinity component, it exhibited many of the features of an active uptake process. The uptake at both concentrations of L-glutamate was not specific and was inhibited by the same spectrum of acidic, but not basic or neutral amino acids. These results largely confirm similar experiments in other areas of the CNS 2-4,31 although there are some dissimilarities. Thus, the stereoisomer, D-glutamate, had a marked inhibitory effect on uptake not seen in the rat dorsal root ganglion (mainly glia) 81 or cerebral synaptosomes81 and only marginally apparent in rat brain slicesL Also in contrast to these 3 systems, DL-homocysteate had no effect on the retinal accumulation of L-glutamate. It is interesting that even a large molar excess of an effective amino acid was incapable of totally suppressing the accumulation of labelled Lglutamate: an observation explicable in terms of the complex transport kinetics involvedl6,2s. The extent to which homoexchange contributes to the 'high' affinity uptake of labelled GABA and glycine has been the subject of considerable speculationS,22,~5. We have shown that, in contrast to a previous report 7, a net uptake of L-glutamate can occur into retinae when the external concentration is relatively high41. Since the effiux of radioactivity from retinae loaded with labelled L-glutamate was not increased by exposure to unlabelled L-glutamate (1 mM) (but cf. van Harreveld and Fifkov~ 42,48), it would seem that homoexchange is not an important mechanism in ~he accumulation of L-glutamate by the retina. This contrasts with the finding that exogenous labelled L-glutamate is released from rat cerebral cortical slices exposed to a medium containing the unlabelled material ~9. The autoradiographic data indicate that in the rat retina, L-glutamate is extensively accumulated by the neuroglial MLiller fibres. This agrees with previous autoradiographic experiments in frog 20 and rabbit 13 retina, and other areas of the CNS where a glial uptake of glutamate has been reported ls,~3. However, it is possible that the large glial uptake seen in the present study could mask the uptake of glutamate by a small specific population of nerve endings. Under the conditions in which the metabolism of L-glutamate was studied (low concentration of L-[14C]glutamate, 37 °C, large volume of incubation medium compared with tissue) about half of the labelled amino acid was rapidly metabolised. After short incubations, glutamine was the major metabolite detected, together with smaller amounts of aspartate and GABA, but after long incubations, aspartate became the metabolite in the highest proportion. Thus qualitatively, the distribution of 14C amongst the labelled amino acid metabolites is similar to that previously described for the rat retinaT, 37, dorsal root ganglion80 and rat brain slices34. One quantitative difference was that the proportion of 14C incorporated into glutamine is consistently lower than that demonstrated elsewhere but this finding is probably explained by the effiux of labelled glutamine into the comparatively large volume of medium employed in our studies.

92 ACKNOWLEDGEMENTS We are grateful to the S.K.F. f o u n d a t i o n a n d to the Central Research F u n d of the University of L o n d o n for grants for a p p a r a t u s a n d radiochemicals. R . D . W . was in receipt of an M.R.C. scholarship.

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