Accumulation of γ-aminobutyric acid by horizontal cells isolated from the goldfish retina

Yirion Rcs. Vol. 27. No. 12, pp. 2027-2034,

1987 Copyright 0

Printed in Great Britain. All tights reserved

ACCUMULATION HORIZONTAL

0042-6989187 S3.00 + 0.00 1987 Pergamon Journals Ltd

OF y -AMINOBUTYRIC ACID BY CELLS ISOLATED FROM THE GOLDFISH RETINA

GEORGE S. AYOUB* and DOMINIC MAN-KIT La& The Center for Biotechnology, Cullen Eye Institute and Program in Neuroscience, Baylor College of Medicine, Houston, TX 77030, U.S.A. (Received 23 March 1987; in revised form

1 June 1987)

Abstract-In the goldfish retina, HI horizontal cells, which receive input predominantly from red sensitive cone photoreceptors, possess a single high-affinity uptake mechanism for y-aminobutyric acid (GABA). This GABA uptake is enhanced by light stimulation, which hyperpolarizes the Hl cells. The regulation of this uptake mechanism was examined in isolated horizontal cells by measuring the accumulation of exogenously supplied )H-GABA. Solutions containing elevated external K+ or glutamate were used to quantitatively depolarize the cells to reveal that the potential-sensitive GABA uptake is maximal under hyperpolarizing conditions and minimal with depolarization. The driving force for GABA uptake is derived from the Na+ electrochemical gradient, with approximately 2 Na+ ions being cotransported with each molecule of GABA. The results presented suggest that the uptake mechanism permits the synaptic concentration of GABA to be regulated by the membrane potential of the H 1 horizontal cells. This, then permits the presynaptic horizontal cell to modulate the synaptic concentration of transmitter in this tonically active synapse. GABA

Transmitter uptake

Potential-dependent

INTRODUCTION

Cone horizontal cells of the vertebrate retina are interneurons receiving input postsynaptically from cone photoreceptors and providing reciprocal input to cones (Baylor et al., 1971; Fuortes et al., 1973). Electrophysiological and morphological studies have shown that the Hl horizontal cells are postsynaptic to red-sensitive cones, and are depolarized in the dark and hyperpolarized by light stimulation (Kaneko, 1970; Hashimoto et al., 1976; Mitarai et al., 1974; Stell and Lightfoot, 1975; Stell et al., 1975). In the fish retina, there is considerable evidence that the type Hl cone horizontal cells may use GABA as their neurotransmitter (Lam, 1972; Murakami et al., 1978; Lam et al., 1980). Autoradiographic studies on the uptake of 3H-GABA in the fish retina reveal that steady or flickering light stimulation of all visible wavelengths increases the accumulation of the radio-

*Present address: Department of Physiology, University of California, San Francisco, CA 94143, U.S.A. tTo whom reprint requests should be addressed at: 4000 Research Forest Drive, The Woodlands, TX 77381, U.S.A.

Na+-driven

label by the Hl cell somas and axon terminals, while a dark incubation decreases the uptake of ‘H-GABA by these cells (Lam and Steinman, 1971; Marc et al., 1978; Lam et al., 1978). The accumulation of the labelled GABA by Hl horizontal cells is via a single, high-affinity which is Na+- and uptake mechanism temperature-dependent and has an apparent K,,, of 18.2pM (Lam et al., 1980). Because teleost retinas do not possess a low-affinity uptake mechanism for GABA (Lam and Steinman, 1971; Marc et al., 1978), the high-affinity uptake of GABA in goldfish horizontal cells may be examined in isolation of other factors. Since the amount of ‘H-GABA accumulated into Hl cells is determined by the combination of GABA uptake, metabolism, storage and release, it is difficult to determine the mechanism of the stimulation-dependent GABA accumulation by Hl cells in the intact retina. A direct approach to this problem is to dissociate the retina so as to obtain a population of cells enriched with horizontal cells (Lam, 1972, 1975; Lam and Ayoub, 1983). In this preparation, somas and axons of isolated horizontal cells in the goldfish retina retained their Na+- and temperature-dependent ‘H-GABA uptake, with depolarization diminishing the rate of this accu-

2027

2028

GEORGE

S.

AYOUB and DOMINICMAN-KIT LAM

mulation. The studies indicate that GABA uptake is increased by hyperpolarization and decreased by depolarization of the Hl horizontal cells. The active transport of amino acids and sugars across a cell membrane is driven by the electrochemical potential gradient of an electrolyte ion, usually Na+ in animal cells and H+ in microorganisms (Schultz and Curran, 1970; Heinz et al., 1975; Crane, 1977; West, 1980). The high-affinity uptake mechanism for several neurotransmitters is believed to fit this model of transport (Bennett et al., 1973; White; 1976). In particular, the high-affinity transport of GABA has been shown to be Na+- and potentialdependent (Martin and Smith, 1972; Martin, 1973; Kanner, 1978; Pastuszko et al.. 1982). These experiments confirm that GABA accumulation is enhanced by hyperpolarization and suggest that the driving force for uptake is dependent upon the Na+ gradient. In the study presented here, a population of isolated horizontal cells is used to examine the factors regulating GABA uptake into identified retinal neurons. This preparation has distinct advantages over the synaptosomal studies made previously in that identifiable horizontal cells are employed, rather that an unknown mixture of synaptic endings. The ionic regulation of uptake may also be controlled by manipulation of the bathing medium, providing a means by which the Na+- and potential-dependence of the accumulation may be quantitated. By altering the cellular membrane potential as well as the Na+ gradient, it is found that GABA uptake is driven by the Na+ electrochemical gradient, such that hyperpolarization enhances GABA accumulation. Additionally, the uptake requires approximately 2 Na+ ions for each GABA molecule translocated. The results suggest an importance of the uptake mechanism in regulation of the synaptic concentration of GABA, thereby providing a positive feedback system for this transmitter in the vertebrate retina. MATERIALS

AND METHODS

Preparation of isolated horizontal cells

Adult goldfish (Carassius auratus, 5-7 in long) were dark adapted, enucleated and the retinas dissected in saline solutions under dim red illumination. The saline composition was 120 mM NaCl, 3 mM KCl, 1.8 mM 1.1 mM MgC&, 5 mM dextrose, CaCl,, 1.OmM Na*PO,, 15 mM 4-(2-hydroxyethyl)- l-

piperazine ethanesulfonic acid (HEPES). and the pH was adjusted to 7.4 at 20°C with NaOH. Retinas were dissociated into single cells using a procedure described previously (Lam, 1972, 1975; Ayoub and Lam, 1984). Using the velocity sedimentation method (Lam, 1972, 1976) a cell suspension containing over 90% horizontal cells was routinely obtained. Earlier studies (Sarthy and Lam, 1979), using the trypan blue dyeexclusion test, showed that isolated retinal cells prepared by this procedure generally yield over 95% viable cells. ‘H-GABA

accumulation

GABA uptake into isolated horizontal cells was examined by incubating fractions enriched for these cells (>80%) at room temperature with isotonic saline solutions containing 1pCi/ml ‘H-GABA (New England Nuclear) for O-30 min. After the incubation, each sample was transferred to a filtration manifold where the cells were collected on Nuclepore filters (pore diameter 3 or 5 pm) and washed with 10 volumes of 4°C saline for 10 sec. Since incubation at 4°C decreased the uptake by 90%. addition of 10 volumes of the sold saline is a rapid and effective method of immediately curtailing further accumulation of the exogenous label. Each filter was then placed in a scintillation vial and dried at 100°C. Ten ml of scintillation fluid (ACS, Amersham) was added to each vial and the amount of radioactivity measured with liquid scintillation counting. The effects of alteration of the K+ and Na+ concentrations on GABA uptake were measured by appropriate changes of the incubating media. r_-Glutamic acid, which has been shown to depolarize Hl horizontal cells (Murakami et al., 1972; Lasater and Dowling, 1982), and to stimulate the release of ‘H-GABA (Ayoub and Lam, 1984), was added in concentrations of O.l-10OOpM to quantitate its effect on GABA accumulation. In these experiments, an incubation time of 10 min was utilized to ensure that a linear rate of uptake was measured. To compensate for the effects of ‘H-GABA release due to elevated external K+, the amount of release as determined by integrating the GABA release data (Ayoub and Lam, 1984), was added to each of the uptake values, to present simply the ‘H-GABA accumulation. Though this admittedly assumes a direct relationship between recently accumulated transmitter and its release, any error in this assumption will minify the response measured. In other words, if more

GABA uptake by isolated horizontal ceils

2029

label is added to the results than was actually released by these cells, it will appear as though there is less effect on the uptake than actually occurred. RESULTS

Accumulation of ‘H-GABA As shown in a previous study (Lam and Ayoub, 1983), the uptake of ‘H-GABA by isolated cells in normal saline solutions is approximately linear for 30min. For the uptake studies presented here, an incubation time of 10 min was chosen, to ensure that all observations were made within this linear range of the uptake rate. Additionally, preliminary kinetic analysis was accomplished (Lam and Ayoub, 1983) to show that these cells have retained the single, high-affinity site (apparent &, of 12 FM) for GABA uptake (Lam et al., 1980). To ensure that none of the accumulated ‘H-GABA was degraded during the course of the incubation, 100 PM aminooxyacetic acid (AOAA) was added to the cell suspension to inhibit the GABAtransaminase breakdown of applied 3H-GABA (Martin, 1976). No significant difference was observed in the level of jH-GABA accumulation under these conditions, hence the inclusion of AOAA in subsequent studies was deemed unnecessary. The accumulation of ‘H-GABA into isolated cells is strongly dependent upon the external

T

,;ii

/

60

90

Na*Conccnlrat~on

(mt.4)

120

Fig. I. H 1 horizontal cells isolated from the goldfish retina were incubated in ‘H-GABA for 10 min. and the amount of label accumulated as a function of the external Na+ concentration was examined. The K+ concentration for these experiments was held constant at 3 mM and isotonicity of the media was maintained with addition of choline chloride. The uptake of GABA was approximately linearly dependent upon the log of the Na+ concentration, implicating a Na+ driving force for GABA transport and/or a Na+-dependent loading of the GABA carrier as the most likely rationale for these data. The values represent the means f SE of at least 10 experiments.

-30

-25

-20

-15

-10

-5

0

5

10

t"mutes

Fig. 2. To assess the importance of the Na+ and K+ electrochemical gradients in controlling GABA transport, 10pM ouabain was added to the incubation media at various times before and after the initiation of uptake to break down the Na+ and K+ ionic gradients. The percent of uptake inhibition is charted as a function of the time difference between the addition of ouabain and the addition of ‘H-GABA. At time = 0, the labelled GABA is added to all samples. Ouabain is added up to 30min before GABA (time = -3O), where the greatest inhibition of uptake is seen. When ouabain and ‘H-GABA are added simultaneously (time = 0), there is approximately a 40% inhibition of uptake. Prolonged preincubation blocked 90% of the uptake, presumably through the breakdown of the Nat and K+ electrochemical gradients. The values represent means &SE of 12 experiments.

Na+ concentration (Fig. 1). In these experiments, choline chloride was substituted for NaCl to retain isotonicity. As the external Na+ was decreased to one-half its normal physiological value, the degree of accumulation of the label into the horizontal cells was decreased by 42%. When Na+ was fully removed from the incubating medium, little accumulation of the exogenous label was measured. To determine whether the uptake site is directly coupled to the Na+/K+ ATPase, or simply dependent upon the resultant ionic gradients, preincubations in 10 PM ouabain were employed. This drug immediately blocks the Na+/K+ ATPase and allows for a gradual degradation of the Na+and K+ electrochemical gradients (Skou, 1957). As seen in Fig. 2, ouabain indeed diminishes the accumulation of ‘H-GABA. Because the uptake is gradually diminished, this implies that the GABA accumulation is dependent upon the Na+ or K+ gradients, or both. L-Glutamic acid has previously been shown to be effective in depolarizing isolated Hl horizontal cells from teleost retinas (Lasater and Dowling, 1982; Ayoub and Lam, 1984; Ishida et al., 1984), and has been implicated as the possible transmitter from red cones to the Hl cells (Murakami et al., 1972). To examine the efficacy of L-glutamate in altering the uptake rate of the

2030

GEORGE S. AYOUB

r-Glutamic acid

and DOMINICMAN-KIT LAM

concentration f&t

Fig. 3. Addition of increasing concentrations of t-glutamic acid to the normal saline solution elicited a dose-dependent effect on the accumulation of ‘H-GABA. Glutamate, which elicits a depolarization of Hl horizontal cells in a dosedependent fashion, also decreases the uptake of the radioligand. The half-maximal concentration in this case is a few millimolar. The values represent means 2 SE of at least I2 experiments, and are plotted against the log of the glutamate concentration.

horizontal cells, this transmitter candidate was added to the incubation medium. Figure 3 shows that glutamate has a dose-dependent effect on 3H-GABA uptake, with significant reductions in accumulation measured when micromolar concentrations of glutamate were applied. Na + driving force

A simultaneous

assessment

6

of

the

con-

tributions of the membrane potential and the external Na+ concentration reveals that each are involved in regulating GABA uptake (Fig. 4). This suggests that the Na+ electrochemical gradient provides the driving forces for GABA uptake. Cells were incubated in media containing 3-60mM K’ and either 30. 60 or 90mM Na+, with isotonicity maintained by addition of choline chloride. This experiment was used to quantitate the dependence of the uptake rate upon each of these ions. As shown in Fig. 4(a), elevation of the external K+ concentration diminishes the rate of 3H-GABA accumulation, following approximately the same slope at each Na+ concentration. Concurrently, the external Na+ concentration is important in dictating the absolute displacement of these lines along the ordinate. In contrast, examination of the same data to observe uptake as a function of external Na+ is shown in Fig. 4(b). In this case, as the external Na+ concentration is increased, there is an increase in GABA uptake, with the extradetermining the cellular K + concentration vertical displacement of these lines along the ordinate. DISCUSSION

The large body of evidence in the teleost retina implicating GABA as the neuro-

50

a

n

I(+ concentration

tmM)

No+ concentration

(mM)

Fig. 4. The importance of the Na+ and K+ concentration gradients in controlling GABA uptake was examined in a series of experiments in which the concentrations of both of these salts were varied in the external media. In (A), the accumulation of ‘H-GABA is plotted as a function of the log of the external K* concentration. The Na+ concentrations in this series are: 90 mM Na+, top (dotted) line with the solid squares; 60 mM Na+, middle (soiid) line with open circles; 30 mM Na+, bottom (dashed) line with the open circles. In (B), the data are presented as a function of the log of the external Na+con~ntration. In this case, the respective K+ concentrations are: 3 mM K+, top {dotted) line with solid squares; 9 mM K+, middle (solid) line with solid circles; 15 mM K+, bottom (dashed) line with open circles. The isotonicity of the incubation media was maintained by the addition of choline chloride. The values represent means *SE of at least 12 experiments.

GABA uptake by isolated horizontal cells

transmitter used by the Hl horizontal cells (Lam et al., 1980) is supported by the uptake studies presented here. In addition to extending the results obtained from intact retina, uptake experiments on isolated horizontal cells permit examination of the intrinsic biophysical properties of these cells in the absence of complex neural interactions in the retina, as well as observation of the cellular mechanisms regulating the uptake and release of GABA in an identified CNS neuron. These cells retain the single high-affinity uptake mechanism for exogenous GABA present in intact retinas, as well as its Na+- and Accumulation of temperature-dependence. ‘H-GABA has been shown to be greatly diminished by preincubation in ouabain, which blocks the Na+/K+ ATPase and breaks down the Na+ and K+ ionic gradients. Because abolition of the uptake required prolonged incubation with this cardiac glycoside, the driving force for the GABA transport mechanism most likely depends upon the Na+ and K+ electrochemical gradients. Membrane potential regulation

Several means have been used to depolarize the horizontal cells and measure the change elicited in the rate of 3H-GABA accumulation. As determined by these experiments, the initial rate of GABA uptake into isolated GABA-ergic horizontal cells is influenced by the membrane potential, such that depolarizion diminishes the GABA uptake. In the intact, isolated goldfish retina, Lam and Steinman (1971) have shown that GABA accumulation into Hl horizontal cells is enhanced by light stimulation and decreased by darkness. Since Hl horizontal cells are hyperpolarized by light (Luminosity-type; Kaneko, 1970), the finding on GABA uptake in isolated cells is consistent with the hypothesis that the light-dependent stimulation of GABA accumulation in Hl horizontal cells can at least in part be explained by an increase in the rate of GABA uptake when these cells are hyperpolarized. Other factors which contribute to GABA accumulation by these cells are release (Ayoub and Lam, 1984) and metabolism. Since the ‘H-GABA taken up by the Hl cells is not significantly metabolized in 30 min (Lam, 1972), the accumulated ‘H-GABA predominantly represents the difference between the amount taken up and the amount released. Thus, the accumulation of 3H-GABA by Hl cells during hyper-

2031

polarization by light is due to the simultaneous enhancement of GABA uptake and depression of GABA release. The functional significance of a high-affinity mechanism for GABA uptake by GABA-ergic neurons has not been elucidated. Since the degradative enzyme for GABA (GABAtransaminase) has not been localized to any extracellular space or synaptic cleft examined (Varon et al., 1965; Salganicoff and DeRobertis, 1965), GABA uptake may play a role in facilitating clearance from the synaptic region. This role may be especially important in the teleost retina since (1) only the GABA-ergic neurons appear to possess this uptake system, (2) there is no low-affinity GABA uptake system, and (3) the Muller (glial) cells do not accumulate GABA in situ (Lam et al., 1980). In electrophysiological studies on goldfish isolated horizontal cells (Tachibana, 198 1, 1983a, b), it was found that the resting membrane potential is linearly dependent upon the logarithm of the external K+ concentration in a Nemstian fashion. Membrane potentials for each of the uptake data points reported here can be approximated from these studies. For example, resting potentials of - 30 and - 70 mV are expected in solutions containing about 40 and 6 mM external K+ , respectively (Tachibana, 1981). These values would be equal to the approximate range of membrane potential excursions of the horizontal cells in darkness and light (Kaneko, 1970; Kaneko and Shimazaki, 1975). Hence, these findings on isolated Hl cells are consistent with the observation that GABA accumulation in intact retinas is enhanced by light stimulation (Lam and Steinman, 1971; Marc et al., 1978). To confirm the membrane potential as a major factor in determining the rate of GABA accumulation, the isolated cells were depolarized by an additional method. Micromolar concentrations of L-glutamate, the putative transmitter to Hl cells (Murakami et al., 1972; Lasater and Dowling, 1982; Ishida et al., 1984), were effective in diminishing GABA uptake in a dose-dependent fashion. Since glutamate depolarizes the cells by opening a nonspecific cation channel (Tachibana, 1983a), addition of glutamate permits Na+ to enter the horizontal ceils and should also decrease the Na+ gradient to a small extent. This, too, will diminish the Na+ driving force and the GABA uptake, although its overall effect should be much less than that of the depolarization.

GEORGES. AYOUBand DOMINICMAN-KIT LAM

2032

Soditm drivingforce The data imply that transport may be dependent on the driving force for Na+ into the cehs (i.e. [V, - ENa]). The assumptions underlying such a mechanism are: (I) that GABA is transported in an uncharged form; (2) that the charge on the complex is derived from the Na ions, which are cot~nsported with the CABA molecules; and (3) since V, is approximately equal to Ex in this system, GABA transport is primarily dependent on [Ek - &$] and varies as a linear function of the logarithm of the Na’ or K* concentration. Although Na’ may function simply as a regular for loading of the uptake carrier, the data presented in Figs 1 and 4 provide support for the above assumptions and predictions that the driving force for GABA transport is derived from the Na+ electrochemical gradient. In fact, assuming EK approximates V,,,, the data in Fig, 4 may be examined as a transport system driven by Na+. Under these conditions the accumulation of GABA near equilibrium may be described as [GAB Ai] Kl P&l log [GABA,] = ’ log m + ’ log fNa,l where n is the number of Na+ ions transported with each GABA molecule. A value for n can be calculated from the above equation. The internal fK+] is approximately 118 mM (Tachibana, 1981) and internal [Na+] is assumed to be about 20 mM. The ratio of the intemal:extemal GABA concentrations is unknown in these experiments, so the rate of uptake is employed. Though the uptake data are clearly not equilibrium values, it is assumed that the use of a ratio of two rates will provide a close approximation for the estimation of n. In this case, simultaneous equations are solved to produce values for n between 1.4 and 2.7. This implies that the Na+ driving force may be used by the GABA uptake mechanism, with cotransport of about 2 Na+ ions providing the charge necessary to translocate each uncharged GABA molecule. These results compare favorably with synaptosomal studies, in which values for n have been between 1 and 3, also centering on 2 (Martin, 1973, 1976; Pastuszko et af., 1982). This agreement with synaptosomal studies is particularly si~ificant in that our preparation, which employs whole neurons, is inherently restricted in the range of treatments which may

be applied. This observation of a similar result for Hl cells and synaptosomes enhances the physiological implications of these earlier studies.

A possible function for the potentialdependence of the GABA uptake and release processes may be to enhance the positive feedback of this transmitter during neurotransmission. The suggestion of a membrane potential influence on the uptake of GABA or other amino acid transmitters has been made previously (Hammerstad and Cutler, 1972; Martin, 1976; Johnston, 1977), and appears to be a viable hypothesis for the HI cells as well. Based on the reduction in uptake observed upon depolarization of the cells, and the dependence on the Na’ concentration gradient, it appears that the driving force for GABA accumulation is the Na+ electrochemical gradient (e.g. Crane, 1977; West, 1980). This becomes of particular interest in light of the physiological condition of the Hi horizontal celfs. These neurons have resting potentials (in darkness) of about - 30 mV (Kaneko, 1970) and the stimulus received from illumination is the reduction of trans~tter from photore~ptors. In contrast to the well-studied neuromuscular junction, the horizontal cell synapse is tonicaliy active, and these same cells which release the transmitter also accumulate it. While GABA uptake is maximal under hy~~olarizing states (or bright illumination), the release of GABA is enhanced by deplaning conditions for darkness; Ayoub and Lam, 1984). The potential-d~~ndent changes in these two processes imply that the membrane potential of the Hl ceil may control the synaptic ~n~ntration of GABA. Hence, the GABA uptake system is capable of performing the function of a cellular positive feedback system: as the HI cell is depolarized, more GABA is released and less is a~umulated. This has the effect of enhancing the effective depolarizations and hyperpolarizations of this cell. In other words, since release and uptake are synergisticahy affected, even small excursions of the HI cell membrane potential should significantly affect the asynaptic GABA concentration. Because these cells mediate the lateral interactions in the outer retina, their dual effect on the synaptic transmitter concentration allows for a decrease of the time course of neurotransmission in this tonically active synapse. Since uptake is functioning in concert with

2033

GABA uptake by isolated horizontal cells

release, less time is required to attain each potential-dependent transmitter concentration than would be the case if uptake were a constant. In support of this hypothesis, several investigators have found that bloackade of GABA uptake in the hippocampus results in an increased time course of the inhibitory postsynaptic potential (Hablitz and Lebeda, 1985; Dingledine and Korn, 1985), implying that high-affinity uptake of this transmitter is essential in shaping the post-synaptic response in another synapse. Taken together, the studies presented on GABA transport in isolated horizontal cells provide a valuable tool for investigating questions of synaptic function. These studies show that the isolated cell preparation yields information consistent with what has been obtained in synaptosomal studies on GABA transport, as well as broadening the correlation of physiological responses in the intact retina with their underlying cellular mechanisms. wish to thank Professors Robert Marc, Sam Wu, Dan Johnstonand David Copenhagenfor ~Cknu~Iedge~enzs-We

their valuable suggestions and criticisms. This work was supported by grants from the National Institute of Health (EY2423) and the Retina Research Foundation (Houston).

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