Stimulated release of [3H]β-alanine from the rabbit retina

Brain Research, 120 (1977) 447--457

447

© Elsevier/North-HollandBiomedicalPress, Amsterdam - Printed in The Netherlands

STIMULATED RELEASE OF [ZH]fl-ALANINE F R O M THE RABBIT RETINA

BIRGITTA BAUER and B. EHINGER Departments of Ophthalmology and Histology University of Lund, Lund (Sweden)

(Accepted May 18th, 1976)

SUMMARY The efflux of [aH]fl-alanine from rabbit retina after intravitreal injection has been studied. The site of uptake of [aH]fl-alanine into retina was checked by autoradiography and was found mainly in the inner plexiform layer and in cells with the position of amacrines and in some ganglion cells. When the preloaded retina was stimulated by light flashes the release of radioactivity increased significantly. Chromatography of the superfusate demonstrated a single radioactive spot which cochromatographed with authentic fl-alanine. The efflux of [aH]fl-alanine was affected by raising the K + concentration. The rate of efflux was also immediately increased when unlabelled fl-alanine or GABA was added to the superfusion medium. Glycine was much less effective. The present study shows that light stimulation releases [aH]fl-alanine from the retina and that fl-alanine may use the same transport system as GABA. ~lhis further supports the suggestion that fl-alanine may act as a 'false transmitter' replacing GABA in the r e t i n a .

INTRODUCTION The amino acid fl-alanine has recently been shown both in vivo and in vitro to be accumulated by certain amacrine cells and some ganglion cells in rabbit, rat, guinea-pig, cat, and human retina (in vitro)a,4, s. In this respect it behaves similarly to glycine (NH2CH2COOH) and GABA (NH2CH2CH2CH2COOH) to which fl-alanine (NH2CH2CH2COOH) is chemically related, Both glycine and GABA are now considered as neurotransmitters but fl-alanine is usually not regarded as a neurotransmitter, fl-Alanine is present in low amounts in the CNS 14,16,17 compared with glycine and GABA. However, the observation of a neuronal uptake of fl-alaninea,4,s, and neuronal receptors sensitive to fl-alanine6, indicates that it may act as a false trans-

448 mitter, which would have very interesting consequences. This investigation was undertaken to study this point by monitoring the efflux of/3-alanine from the retina under varying conditions, including light stimulation. METHODS AND MATERIALS

General experimental procedure Albino rabbits weighing about 1.5 kg were used. Ten #1 (== 10 #Ci) [3H]flalanine, or in some experiments 50 #1 (5 #Ci) [14C] t3-alanine was injected intravitreally into the eye. Two hours after the injection the rabbit was anaesthetized lightly with pentobarbitone and the eye was enucleated. The anterior segment and the vitreous were carefully removed.

Light stimulation The eye-cup was turned inside out and carefully placed in a specially designed water-jacketed superfusion chamber (about 0.75 ml). The eye-cup was superfused at 37 °C with 1 ml/min of the solution described by Ames 1 with 1 mM of unlabelled fl-alanine added. The retina was superfused in the dark for 20 min whereupon it was stimulated by light flashes during 8 min, 2 flashes/sec from a xenon flash tube about 15 cm from the retina. (The average illumination was 1.75 lux but the peak of the flash begun with an illumination of 2175 lux and decreased exponentially with a time constant of 0.4 msec.) The solution was gassed with a mixture of 95 ~o02 and 5 ~ COz. Up to the start of superfusion the experiment was run in ambient laboratory light and then in the dark.

Effiux studies The retinas were superfused for 30 min with Ames' salt solution without further additions whereupon various test substances were added to the superfusate. The substances and their concentrations in the superfusate were: fl-alanine (5 or 0.5 mM), GABA (5 or 0.5 raM), glycine (5 or 0.5 mM) and K + (40 raM, replacing Na+). The additives did not appreciably change the pH of the superfusate. The experiments were run in constant ambient artificial light (190 lux).

Analysis of the superfusate The radioactivity of the superfusate was determined in a liquid scintillation spectrometer. One minute samples were collected and 500 #1 of each was added to a commercial scintillation mixture and counted. Counting efficiency was monitored with the external standard method according to conventional principles. The efflux curves were fitted to each other with the aid of a computer, so that the average of the radioactivity measurements from the five minutes preceding the stimulation would attain a fixed position on the plot. Differences in the slopes of the curves were estimated by comparing with the Student's t-test the slopes of linear regression lines produced for appropriate segments of the curves. In the light-stimulation experiments with [14C]/~-alanine, 300/d of the samples were used for thin layer chromatography. The

449 samples were evaporated to dryness and the residue was reconstituted in 50 /~1 deionized distilled water, of which 5/zl was chromatographed on silica gel thin layer plates in n-propanol-water (70:30 v/v). The spots were detected by autoradiography and ninhydrin spraying. For tissue autoradiography pieces of retina were freeze-dried and further processed according to a previously described method 10. The radioactive amino acids were obtained from N E N Chemicals GmbH, Dreieichenhahn, Germany, the specific activities being 37.5 Ci/mmole for [3-3H(N)] fl-alanine and 4.29 mCi/mmole for [1-14C]fl-alanine. RESULTS

Autoradiography When fl-alanine was injected into the vitreous the radioactivity appeared after 2 h mainly in the inner plexiform layer and in cells with the position of amacrines and also in some ganglion cells (Fig. 1) in good agreement with previous results 4,8.

Fig. 1. Autoradiograph of rabbit retina, 3 h after injection of 20/~Ci [aH]fl-alanine. Radioactivity appears in certain cell bodies with the position of amacrines, and in the inner plexiformlayer (IPL). Two ganglion cells are also radioactive, but less than the amaerines. There is moderate radioactivity in the nerve fibre layer. Ph, photoreceptors; ONL, outer nuclear layer; OPL, outer plexiform layer; 1NL, inner nuclear layer; IPL, inner plexiform layer, G, ganglion cell layer. × 590.

450 There was also some r a d i o a c t i v i t y in the nerve fibre layer o f the retina, but little could be suspected to reside in the glia.

Thin layer chromatography The thin layer c h r o m a t o g r a m s o f the superfusate d e m o n s t r a t e d a single radioactive s p o t which c o c h r o m a t o g r a p h e d with a u t h e n t i c fl-alanine.

Light stimulation The s p o n t a n e o u s efflux o f r a d i o a c t i v i t y f r o m r a b b i t retinas k e p t in darkness is seen in Fig. 2. There is a r a p i d initial release followed by a slower phase which even-

rRae/C'~ivitcetyl 100 [

50 1

0

10

20

30

40

50

60 min

Fig. 2. Spontaneous el:flux in vitro of radioactivity in the dark from rabbit retinas preloaded in vivo with [aH]fl-alanine (2 h). Standard errors of the mean are indicated by vertical bars; 4 experiments. The time for the start of the superfusion is marked 0. The y-axis indicates relative radioactivity as the individual curves were fitted to each other as described in the text.

451 Relative radioactivity 100

50 \\ \

light

-10

flashes

J

i

0

10

20

30 min

Fig. 3. Effect of flashing light stimulation on the efflux of radioactivity in vitro from rabbit retinas preloaded in vivo with [aH]fl-alanine. The broken line is the spontaneous efflux of radioactivity in darkness (Fig. 2). The deviation caused by light stimulation is highly significant (P < 0.001). S.E.M.s are indicated by vertical bars; 8 experiments. The time for the start of stimulation is marked 0 (which was 20-25 rain from the start of the superfusion). tually approaches a steady state. Stimulation of the retina by light flashes increased the tritium release significantly (Fig. 3); P < 0.001 when the slope of curve during the period 0-5 min is compared with the same segment of the curve of efflux in darkness.

Effect of unlabelled fl-alanine The rate of efltux of [SH]fl-alanine was immediately increased when unlabelled fl-alanine was added to the standard superfusion medium. The effect was best seen when standard solution was replaced by one containing 5 m M fl-alanine ( 1 4 0 ~ increase) (Fig. 4 and Table I), but could also be demonstrated by one containing 0.5 m M fl-alanine (46 ~o increase) (Fig. 4 and Table I).

452

Relalive radioactivity 100

5o< -10

i

i

0

10

i

20

30 min

Fig. 4. Efflux of radioactivity in vitro from rabbit retinas preloaded in vivo with [ZH]~-alanine. At the time indicated by 0 (which was 30 min from the start of the superfusion) superfusion medium was changed from standard medium to one containing 5 mM/~-alanine (unbroken line); 3 experiments. and one 0.5 m M {kalanine (broken line); 3 experiments. S.E.M.s are indicated with vertical bars.

TABLE I

Effect of changes in the composition of the superfusion medium on the efflux of [aHj ~-alanine from rabbit retinas Experimental condition (n)

Increase %

5 m M fl-alanine 0.5 m M fl-alanine 5 mM GABA 0.5mMGABA 5 m M glycine 0.5 mMglycine 40 m M K +

140 ± 46 ± 110 419 422 ± NS* 132 4-

(3) (2) (4) (4) (4) (3) (3)

* NS: no significant change.

10.7 0.9 11.4 8.7 4.8 37.3

Relative radioactivity 100

50

-10

0

10

20

30 min

Fig. 5. Efflux of [aH]fl-alanine from rabbit retinas. At the time indicated 0 (30 min after the start of the superfusion), superfusion medium was changed from standard medium to one containing 5 m M GABA. S.E.M.s are indicated by vertical bars; 4 experiments.

Relative radioactivity 100

50

-10

0

10

~0

30 mln

,Fig. 6. Efflux of [aH]fl-alanine from rabbit retinas. At time indicated 0 (30 rain after the start of the superfusion), supcrfusion medium was changed from standard medium to one containing 5 r a m glycine. S.E.M.s are indicated by vertical bars; 4 experiments. The broken line is the spontaneous effiux in light.

454 Relative radioactivity IO0

50

-10

0

10

20

30 rain

Fig. 7. Efltux of [aH]/~-alaninefrom rabbit retinas. At time indicated 0 (30 min after the start of the superfusion), superfusion medium was changed from standard medium to standard medium containing 40 mM K +. S.E.M.s are indicated by vertical bars; 3 experiments.

Effect of glycine and GABA When the standard superfusion medium was exchanged for one containing GABA there was a prompt increase of the efflux of the radioactivity. The increased efflux was seen with 0.5 m M GABA (P < 0.001 for a change in slope) but was most pronounced with 5 m M GABA (P < 0.001 for a change in slope) (Fig. 5). The relative increases were 19 and 110~, respectively (Table I). The efflux of radioactivity was also highly significantly increased by 5 mM glycine in the superfusion medium (22 ~ increase) (Fig. 6, Table I) but the slope of the efflux curve was not significantly changed by 0.5 m M glycine.

Effect of high K + The effect of changes in the ionic composition of the superfusion medium is shown in Fig. 7. The superfusion medium was exchanged for one containing 40 m M K +. The etttux of radioactivity is immediately and pronouncedly increased (132~o increase) (Table I). DISCUSSION The uptake of fl-alanine into rabbit retina has previously been studied by Bruun et al 4. They found that fl-alanine was taken up into cells with position of amacrines

455 and some ganglion cells both in vitro and in vivo, and the present study corroborates the previous observation. The study of Bruun et al. was performed 4 h after the intravitreal injection of 50/zCi tritiated fl-alanine whereas we used 10/zCi and (for practical reasons) waited only 2 h, but with no significant change in results except that the most vitreal retinal layer, the nerve fibre layer, had a relatively higher radioactivity. The efflux of fl-alanine is affected by changes in the K ÷ increased the etflux of radioactivity (Table I). The same effect has been demonstrated on the release of [aH]GABA from rat retina 19, from brain slices 1~, of [3H]glycine from rabbit retina (Bauer and Ehinger unpublished), and from spinal cord 12, and the effect has often been interpreted as if it was a result of a K÷-induced neuronal activity mimicking normal neuronal activity. However, it now has also been shown that the treatment with high K ÷ will release amino acids from both glia and other tissues where no neurotransmitters are involved, and it is therefore possible that it is not directly related to neurotransmission 2. A significantly (P < 0.001) increased effiux of radioactivity could be demonstrated when stimulating the retina with light flashes (Fig. 3). Since the chromatographic experiments showed that it was mainly fl-alanine that was released and since it is also known that there is little or no metabolism of fl-alanine in the retina 4, we conclude that the increase in efflux of radioactivity means that fl-alanine can be released by normal, light-induced nerve activity in the retina. The autoradiography shows that the highest concentration of fl-alanine is in amacrine cells and it seems plausible that these neurons are the source of the fl-alanine released by light stimulation. However, we cannot exclude the possibility that glial cells may also be involved, even though they have a lower concentration of radioactive fl-alanine. The spontaneous efflux curve had initially a sharp drop and became progressively slower (Fig. 2). The initial drop is likely to represent a washout from extracellular and loosely bound radioactivity and the slower release is likely to be ofintracellular origin, as pointed out by several others 7,11As. The efflux of [3H]fl-alanine was increased by addition of unlabelled fl-alanine to the superfusion medium, presumably by displacing it or by competing for its re-uptake. Similar results have been reported from effiux studies of glycine from spinal cord 7, of GABA from frog spinal cord 5, from frog retina 13, and of glycine from rabbit retina (Bauer and Ehinger, unpublished). It is of considerable interest to study the influence of other amino acids on the efflux in order to find substances which can be transported by the same system. When GABA is added to the superfusion medium the rate of efflux is stimulated immediately. The effect is best demonstrated by 5 m M GABA but is highly significantly demonstrated also by 0.5 m M GABA. 5 m M GABA had nearly the same effect as 5 m M fl-alanine (Table I). Glycine, on the other hand, is very much less effective. This suggests that fl-alanine may use the same transport system as GABA but to a much lesser extent that of glycine. From the previous autoradiographic experiments it was not possible to judge whether/~-alanine entered those cells that would also accumulate glycine or the ones taking up GABA 4 but it was demonstrated that the uptake of fl-alanine is competitively inhibited by GABA but not by glycine in the retina 4. From

456 the previous a n d the p r e s e n t studies it thus seems likely t h a t /4-alanine a n d G A B A share a c o m m o n t r a n s p o r t system, but that fl-alanine to a m u c h smaller extent is t r a n s p o r t e d by the system o p e r a t i n g with glycine, In conclusion the e x p e r i m e n t s have shown t h a t flashing light s t i m u l a t i o n will release fl-alanine f r o m r a b b i t retinas in vitro, a n d t h a t the fl-alanine is p r e s u m a b l y c a r r i e d by the system also c a r r y i n g G A B A . This is further s u p p o r t for the suggestion t h a t in the r e t i n a fi-alanine m a y act as a 'false t r a n s m i t t e r ' r e p l a c i n g G A B A 4. There is evidence t h a t o t h e r f2-amino acids m a y behave similarly 9. ACKNOWLEDGEMENTS This s t u d y was s u p p o r t e d by grants f r o m Swedish M e d i c a l R e s e a r c h Council, Project N o 04X-032321, Swedish Society for M e d i c a l Research, R o y a l P h y s i o g r a p h i c Society a n d F a c u l t y o f Medicine, University o f L u n d .

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