ACTA O PHT HAL MO L OGIC A VOL. 55 1977

The Department of Ophthalmology (Head: E . Palm) and The Department of Histology (Head: B. Falck), University of Lund, Lund, Sweden

FACTORS AFFECTING THE SPONTANEOUS RELEASE OF [3H]GLYCINE FROM RABBIT RETINA BY

BlRGlTTA BAUER

The efflux of [sH]glycine was studied in superfused rabbit retina in the presence of various amino acids, ouabain, or high K+ or low Can+ concentrations in the superfusion medium. Unlabelled glycine evoked an accelerated efflux as did the structurally similar neutral a-amino acids. j3-alanine and GABA were ineffective. The results demonstrate a homoexchange of glycine, and a heteroexchange with the neutral a-amino acids. A low concentration of glutamic acid (10-5 M) will release glycine from the retina. This is an ATPase dependent process which is partially blocked by a high MgZ+/Caz+ ratio and which may be related to a retinal transmitter function of glutamic acid. A high concentration of K+ or the presence of ouabain in the superfusing medium greatly increases the rate at which glycine is lost from the retina.

Key words: retina - glycine - efflux - aminoacids - ouabain - potassium calcium.

GIycine is present in the retina as a free amino acid in concentrations ranging from 0.6 to 4.0 pmoleslg wet tissue, with some species differences (see Voaden 1976). In the rabbit, the concentration is 3.2 pmoleslg wet tissue. Exogenously applied glycine is taken up mainly into cells which correspond in location with amacrine cells (Bruun & Ehinger 1972, 1974; Marshall & Voaden 1974; Voaden et al. 1974), and an active high affinity uptake mechanism has also been deReceived June 25, 1977.

93 1

Birgitta Bauer

monstrated in rabbit and frog retina (Bruun & Ehinger 1972; Voaden et al. 1974). Further, glycine has been shown to inhibit the firing of retinal ganglion cells (Ames & Pollen 1969), whereas the glycine receptor blocker, strychnine (Curtis et al. 1968) has the opposite effect (Straschill 1968). Finally, light flashes release [3H]glycine from the retina, both in vivo and in vitro (Ehinger & Lindberg 1974; Ehinger & Lindberg-Bauer 1976). Thus, glycine is present in the retina, affects neuronal responses, can be released by stimulating the retina with’its proper stimulus, light, and there is a reuptake mechanism that may terminate the action of released glycine. This makes glycine a good candidate for being a neurotransmitter in the retina. Glycine is spontaneously released from the retina in the dark in a multiphasic pattern (Voaden 1974; Ehinger & Lindberg-Bauer 1976), but factors affecting this release have not been defined. In other parts of the CNS, homoexchange and heteroexchange of glycine have been demonstrated, for example in the spinal cord (Cutler et al. 1971), and evidence has also been given for carrier mediated transport systems (Levi et al. 1966). In order to better understand the release mechanisms for glycine from the retina we have studied the effect of different amino acids, ions and ouabain on the efflux of [SHIglycine from the rabbit retina.

Material and Methods General experimental procedure

Albino rabbits weighing about 1.5 kg were used. Ten p1 (10 pCi) [SHIglycine were injected intravitreally into one eye after topical anaesthesia. This in vivo labelling procedure was used in order to avoid prolonged experiments in vitro during which the retina might deteriorate before the experiment was finished. 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. The eye cup was then turned inside out and carefully placed in a specially designed water-jacketed superfusion chamber (Fig. 1). The everted retina was superfused at 37’C with 1 ml/min of the solution described by Ames (1965). The superfusion solution was aerated with 95 O/O 02 and 5 O/O CO, and the experiments were run in ambient laboratory light (about 190 lux). Efflux studies

The retina was initially superfused for 30 min with Ames’ salt solution and for the subsequent 5-15 min with a medium containing the test substances. The substances tested and their concentration in the solutions were: glycine (10-3 M 932

Retinal Glycine Release

Fig. 1. Schematic diagram of the water-jacketed superfusion chamber. The everted eye

is placed on the dome-shaped holder.

and 10-4 M), ,!I-alanine (10-3 M and 5 x 10-3 M), a-alanine (10-3 M), GABA (y-aminobutyric acid, 5 x 10-3 M), a-aminobutyric acid (10-5 M), L-lysine (5 x 10-3 M), L-leucine (10-6 M), valine (10-3 M), ouabain (10-4 M), K+ (40 mM) and a Ca2ffree medium with 2 mM EDTA. In a second series of experiments the retina was superfused as above with either the unmodified salt solution or with the latter containing 104 M ouabain or lo-’ M strychnine sulphate, or with low Ca2f (0.2 x 10-3 M) and high Mg2+ (20 x 104 M) concentrations. The stimulation test substance (glycine 10-5 M, or glutamic acid 10-5 M in the experiments with ouabain 104 M) was then applied for one min and then again for one min after 15 min. 2- [SHIglycine, 9.4 Ci/mmole was obtained from NEN Chemicals Gmbh, Dreieichenheim, G. F. R. Analysls of superfusate

The superfusate was collected in one min samples. The radioactivity of the effluent was monitored in a liquid scintillation spectrometer with quench corrections applied according to the external standard channels ratio method. The absolute levels of radioactivity in the effluent at any given time will vary from experiment to experiment because the amount of label in the retina will vary from eye to eye in these in vivo labelling experiments. The individual curves were therefore normalized so that they would always be in the same position 933 Acta ophthal. 55, 6

60

Birgitta Bauer

on the plot during the interval 25-30 min after the start of the experiment, immediately before the first stimulation. This normalization has applicability in all first order release processes where the release behaves as if it comes from a single compartment. Expressing the amount of [sHIglycine released per mg retina gives more variable results because different retinas are labelled to different degrees. Also, expressing release as a percentage of total radioactivity gives less precise results, because more measurements and calculations are involved, adding to the experimental errors. As applied, the normalization procedure gives a good representation of release rates. However, it gives no information about the absolute amounts released. Release rate constants were obtained by least squares fits of the observed efflux to the equation y = a s e - k t where y is the release rate at any given time t, a is the initial release rate when t = o and k is the rate constant. Over 10 min intervals the fit to the equation was good, with correlation coefficients of 0.95 or better. Over longer periods of time (30 min or more) it was clear that the efflux has several components (Voaden 1974; Ehinger & Lindberg-Bauer 1976) and cannot be represented by the simple equation above.

Ielative adioactivity

Fig. 2. Effect of 10-5 M unlabelled glycine on the efflux ([sH]glycine from rabbit retinas. S E M are indicated by vertical bars; 4 experiments. 934

Retinal Glycine Release

Since the efflux approximates a first order reaction over 10 min intervals, results a r e directly comparable when obtained i n normal retinas immediately after the normalization a t a fixed time i n the experimental procedure. W h e n the retinas have been pretreated for some time before normalization the efflux

Concentration 5x

10-3 M

10-3 M

lo4

10-5 M

M

amino acid

f 15 n = 4 P < 0.001

Glycine

79

P 7.2

f 1.1 n = 3 NS

5.7

n=4

P < 0.025 4 0 f 4.6 n = 4 2nd stim. P < 0.01

f 4.6 n = 3 NS 28

f 1.6 n = 3 P < 0.01

4.4 f 6.4 n = 4 NS 64 f 22 n = 5 1st stim. P 0.01 84 f 29 n = 5 2nd stim. P 0.01

Glutamic acid

<