Glutamate and taurine uptake by retinal pigment epithelium during rat development

Camp. Biochem. Physiol. Vol. 104C, No. 2, pp. 31 l-316, 1993 Printed in Great Britain

0

0306~4492/93 $6.00 + 0.00 1993 Pergamon Press Ltd

GLUTAMATE AND TAURINE UPTAKE BY RETINAL PIGMENT EPIT~~L~UM DURING RAT DEVELOPMENT R.

SALCEDA*

and M. R.

SALDAI;IA

Departamento de Neurociencias, Instituto de Fisiologia Celular, Universidad National Autonoma de Mexico, Apartado Postai 7WXl0, 04510 Mexico D.F., Mexico (Tel. 525 550-5215; Fax 525 548-0387) (Received 20 August 1992; accepted for publication 25 September 1992) Ah&act-l. The rat retinal pigment epithelium accumulated glutamate and taurine by saturable, temperature and Na + -dependent mechanisms. 2. Glutamate and taurine showed high and low affinity transport systems, with a K,,,of 30pM and 80 hM, respectively. 3. The transport rates of both amino acids decreased during maturation of retinal pigment epithelial cells while their kinetic characteristics were not modified. 4. The results suggest an involvement of the retinal pigment epithelium in the regulation of glutamate and taurine levels in the neural retina and support its role as part of the blood retinal barrier.

INTRODUCTION

The blood retinal barrier consists of both an inner and outer barrier system (Cunha-Vaz, 1976). The outer barrier system, which results from the tight junctions between retinal pigment epithelial cells (RPE), restricts the flow of nutrients reaching the retina. Therefore the RPE plays an important, but little understood role in the transport of nutrients from the blood to the retina. Although different studies have focused on the transport properties for several nutrients (Miller and Steinberg, 1976; Zadunaisky and Degnan, 1976; Pascuzzo, Johnson and Pautler, 1980; Masterson and Chader, 1981; Khatami and Rockey, 1988; Pautler and Tengerdy, 1986), the transport of amino acids has not been extensively studied. We have chosen to study the transport of taurine and glutamate because of their importance in the retina. Taurine is present in high concentration in the retina of all vertebrates studied (Pasantes Morales et al., 1972; Orr et al., 1976) as well as in the RPE (Orr et al., 1976; Voaden et ai., 1977). In both tissues autoradiographic studies have shown a heavy uptake of taurine in different vertebrate retina (Lake et af., 1975, 1977; Voaden et al., 1977). In addition, tam-me is released from the retina as well as from isolated rod outer segments upon exposure to light (Pasantes-Morales et nl., 1973; Salceda et al., 1977). On the other hand, glutamic acid is thought to be the synaptic transmitter released from photoreceptor cells (Cervetto and MacNichol, 1972; Murakami et al., 1972; Sugawara and Negishi, 1973). In addition to a neurotransmitter role, glutamate can exert a neurotoxic action at high concent~tions (Olney et *To whom correspondence

should be addressed.

al., 1971; Olney, 1982). Therefore mechanisms which may contribute to the regulation of the extracellular amino acid concentrations in the retina are of considerable interest. The present studies were undertaken in order to characterize the transport of taurine and glutamate by the RPE during rat development.

MATERIALS

AND METROS

3H-taurine (25 Ci/mmol), ~-[3,4-~H] glutamic acid (54 Ci mmol-I), ~-[2,3-~H] aspartic acid (13 Ci mmol-i), i4C-inulin (0.05mCi 17mg-‘) were obtained from New England Nuclear (Boston, MA, U.S.A.). All other reagents were from Sigma Chemical Co. (St Louis, MO, U.S.A.). Retinal pigment epithelia (RPE) from Long Evans rats were used. The sclera was removed from the posterior position of the eye. The anterior part, including the retina, was eliminated and the RPE were gently peeled away using fine forceps and incubate at 37°C with 2ml of Krebs Ringerbicarbonate (118 mM NaCl, 1.2 mM RH,PO,, 4.7 mM KCl, 2.5 mM CaCl,, 1.17 mM MgSO,, 35mM NaHCO, plus 5.6mM glucose, pH 7.4) containing 0.25 PCi of either ‘H-taurine, ‘H-glutamate or 3H-D-aspartate. At the end of the incubation, RPE were washed with cold medium, weighted and dissolved in 0.5 ml of 1% sodium dodecyl sulfate. Radioactivity in the solubilized tissue was measured after the addition of 5 ml Tritosol and counts for radioactivity performed in a ligand scintillation spectrophotometer. The extracellular space was estimated as described by Ames et al., (1967) and Frank and ~hoffeniels (1972). RPE were incubated for 30 min with 1.0 @i ml-’ of i4C-inulin. After incubation, RPE were 311

R. SALCEDAand M. R. SAL.DARA

312

Table 1. Effect of different compounds taurine uptake by RPE Addition

nmoles g-’

&N (1 mM) Iodoacetate (I mM) 2, 4-dinitrophenol (1 mM) Ouabain (0.1 mM) Low tem~rature (4°C) Sodium free medium j?-&nine (1 mMf y-aminobutyric acid (I mM) Glycine (1 mM)

Time (min)

Fig. 1. Time course of taurine and glutamate uptake in rat RPE. The RPE of adult rats were incubated in a Krebs-bicarbonate medium, pH 7.4 in the presence of 3H-taurine (e), or )H-glutamate (0) at 2OpM final concentration. The values are the mean + SEM of 4-6 experiments.

weighted

and

their

radioactivity

measured

as de-

scribed above. The inulin space was calculated from the ratio of i4C-inulin content of RPE to r4C-inulin in the incubation medium. The obtained values which were around 30% through all the ages studied, were subtracted from taurine, glutamate and n-aspartate uptake values. Media depleted of sodium ions were prepared by replacement of NaCl and NaHCOS with isotonic quantities of choline chloride and KHCO,, respectively. RESULTS

Tuurine

The time course of taurine uptake by the adult rat RPE was linear for the first 5 min of incubation, reaching maximum levels of 15.6 nmol gg’ at 40 min (Fig. 1). The 3H-taurine saturation curve within a concentration range of S-500~M showed two transport systems with a I& of 80 p M and 400 FM and a V,,, of IOnmol g-r min-’ and 20nmol g-’ min-‘, respectively (Fig. 2). Uptake of 3H-taurine was remarkably reduced (90%) when the incubation was carried out at 4°C. Taurine accumulation was 30-40% inhibited by

250

500

on the

10.75 + 0.90 7.18+ 1.10 9.19-1 1.01 6.63 i I .01* 6.23 f 0.55* 1.21 * 0.09t 2.30 +_0.20t 3.99 * 0.43’ 6.80 * 0.70 15.11 f3.52

Incubation of RPE from adult rats was carried out for 20min in a Krebs bicarbonate medium. “H-taurine (20~M) was added at the same time as the drug. Each value is the average + SEM of at least four experiments. *P = 0.05 tf = 0.05

1.0 nM KCN, 1.0 nM dinitrophenol or 0.1 nM ouabain; while it was slightly decreased by 1.0 mM iodoacetate (Table 1). The uptake of taurine was not modified in the presence of 1.0 mM glycine while 1.0 mM /?-alanine or GABA reduced it by 60% and 30%, respectively (Table 1). An important effect on taurine accumulation was observed when the incubation was performed in the absence of sodium; under these conditions the uptake of taurine showed an 85% reduction through all ages studied (Table 1, Fig. 3). Taut-me accumulation was studied in RPE during rat development. Taurine uptake values in RPE were constant from 3-8 day old rats, then a remarkable increase (lOO”~) was observed at 12 days of age, which then decreased rapidly to reach the adult levels (Fig. 3a). L-Glutamate and D-aspartate

L-Glutamate accumulation by the adult rat RPE was found to be a saturable process, reaching equilibrium after IOmin at a maximum value of 7 nmol g-’ (Fig. I). As is shown in Fig. 4, the uptake of glutamate was ranging saturable at extracellular concentrations between 5 and 1OOOpM. Analysis of the double reciprocal plot revealed two transport systems

0.f

0.2

[ Taurine ] JIM

Fig. 2. rH-taurine a~umulation by adult rat RPE as a concentration function. incubation was carried out at 37°C for 2 min as described in “Methods”. Right: double reciprocal plot. The points are the mean of at least five experiments.

313

Amino acid transport

AGE (days)

AGEidays)

Fig. 3.(A) 3H-taurine and (B) 3H-glutamate uptake in RPE of developing rats. RPE were incubated for 20 min in a Krebs-bicarbonate medium in the presence of 20 yM of the amino acid. Normal medium (a). Na-free medium (0). Each point is the mean f SEM of at least five experiments.

40

1.0 -

P ‘1”

I 2 20 Lc c

0.5 [Glutamate]

0.5 /I/ -

1.0

0.1

mM

0.2

‘/Cs]yM

Fig. 4. ‘H-glutamate. uptake by adult rat RPE. The RPE were incubated for 2 min with different concentrations of glutamate. Right: Michaelis-Menten plot. The points are the mean of at least five experiments.

4):a high affinity system with an apparent K,,,for L-glutamate, with a K,,, of 80 PM and 500 PM, of 30 ~1M with a V,,,,, of 1.56 nmol gg ’ min-’ and a respectively (not shown). Uptake of L-glutamate low affinity system with a K,,, of 588 FM with a V,,,, and rr-aspartate was inhibited by 90% and 80%, of 34.09 nmol gg’ min-‘. Similar results were found respectively, by incubation at low temperature; for D-aspartate. (K, of 57 PM and 500 p M.) and was not significantly modified by 1 mM Kinetic studies on RPE from newborn rats (3-5 dinitrophenol, ouabain, iodoacetate or KCN days) showed high and low affinity transport systems (Table 2). (Fig.

Table 2. Effect of different compounds on the ‘H-glutamate and ‘H-o-aspartate bv RPE Addition Iodoacetate (I mM) 2, 4-Dinitrophenol (1 mM) KCN (1 mM) Ouabain (0. I mM) Low temperature (4°C) Sodium free medium L-Asp&ate (500 PM) L-Aspartate$-hydroxamate (500 PM) Glutamic acid-4-monohydroxamate (500 FM) a-Amino adipic acid (1 mM) Kainic acid (200 PM) y-Aminobutyric acid (1 mM) Taurine (I mM)

uptake

nmoles g - ’ L-Glutamate D-Asoartate 5.62 f 3.61 f 4.25 f 5.64 f 4.03 f 1.75f 2.42 + 2.71 f 3.45 f 6.34 f 4.48 + 5.90 f 6.13 k 4.80 f

0.52 0.09* 0.40 0.74 0.37 0.04t 0.12t 0.38’ 0.05* 0.25 0.30 0.70 0.17 0.52

6.48 + 4.97 f 6.60 + 7.45 + 4.76 f 2. I I f 3.69 + 3.95 i 5.05 f 6.36 f 8.48 f 7.32 + 7.71 f

0.60 0.50 0.60 1.10 0.38 0.20t O.lOt 0.57’ 0.85 0.50 0.80

I .40

0.72

Retinal pigment epithelium from adults rats was incubated for 20 min in a Krebs-bicarbonate medium in the presence of either ‘H-L-glutamate or ‘H-o-aspartate (20 PM). Each value is the mean f SEM of at least four experiments. ‘P = 0.05 tP = 0.005

R. SAL~E~Aand M. R. SALDA~~A

314

When the incubation was carried out in the absence of sodium, the accumulation of L-glutamate and D-aspartate showed a considerable reduction (65% and 85%, respectively) (Table 2). Glutamate uptake was absolutely Na + -dependent through all periods of age studied (Fig. 3B). Compounds known to act as competitive inhibitors of glutamate uptake in other systems (L-aspartate, dihydrokainic acid, glutamic acid4-monohydroxamate, L-aspartate-/?-hydroxamate, cc-amino adipic acid) as well as GABA and taurine were tested at concentrations of 200 p M to 1000 PM for their ability to inhibit n-aspartate and glutamate in RPE. Among these, only r.-aspartate and L-aspartate-~-hydroxamate showed a significant inhibition of L-glutamate and n-aspartate uptake (Table 2). The glutamate uptake was studied during various developmental stages in the rat RPE. During the first postnatal week, RPE accumulated around 13 nmol g-‘of glutamate, accumulation that showed a sharp decrease, reaching 10th postnatal day adult values at the 10th postnatal day (Fig. 3B). Similar results were found for D-aspartate (not shown). DISCUSSION

The tight junctions between RPE cells effectively block the diffusion of molecules such as amino acids through the intracellular space (Cunha-Vaz, 1976). Therefore a major portion of the nutrients required for the normal functioning of the retina must pass from the blood supply in the choriocapillaris, through the RPE and into the retina. The sulfur amino acid taurine, is the predominant free amino acid in the retina (Pasantes-Morales, 1972; Macaione et al., 1975; Orr et al., 1976). Its function is unknown but it appears essential for photoreceptor cell viability (Hayes et al., 197.5). In addition, taurine is released from the retina (Pasantes-Morales et al., 1973) and isolated rod outer segments (Salceda et al., 1977) upon light exposure. Autoradiographic studies have shown that in rats or frogs systemically injected with radioactive taurine the radioactivity is initially concentrated in the RPE, and subsequently it is detected in the retina (Young, 1969; Lake et al., 1977). High and low affinity sites for the uptake of taurine in RPE have been localized by light microscope autoradiography (Lake et al., 1977). The present findings demonstrate that rat RPE accumulates taurine by a saturable, temperature and sodium dependent system. Taurine uptake was inhibited by ouabain, in agreement with previous results (Miller and Steinberg, 1976) in which it was suggested that the sodium cotransport of taurine is modulated indirectly through the sodium pump, located in the apical membrane of the cells (Bok, 1982). In this context, a high affinity uptake system for taurine stimulated by sodium has been reported

in the isolated apical membranes from bovine RPE (Miyamoto et al., 1991; Sivakami et al., 1992). The data are consistent with a carrier mediated mechanism identified for the transport of taurine across cell membranes (Jacobsen and Smith, 1968), since it is of high affinity and inhibited by fi-alanine. The high affinity K,,, value found in rat RPE is in the same range as that reported for cultured rat (Edwards, 1977), baboon and bovine RPE (Hussain and Vaoden, 1985; Miyamoto et al., 1991). However, it is of higher affinity than that found in frog RPE (Miller and Steinberg, 1976). Taurine accumulation showed a remarkable increase in rat RPE at 12 days after birth and then decreased, remaining constant from day 30 until adult stage. Although we do not know the meaning of this taurine uptake increase, it coincides with the withdrawal of RPE proliferation (Stroeva and Mitashov, 1983). Similar results were observed in cultured chick RPE, in which a remarkable increase of taurine uptake was observed when cells achieved differentiation (Lopez-Colome et al., 1991). The characteristics of glutamate transport that we have observed in RPE are quite similar to those reported in other cell systems. First of all, the competition studies indicate that L-glutamate and D-aspartate are transported by a common carrier which has a low affinity for other amino acids (Table 2). This feature was corroborated by the uptake of ‘H-D-aspartate, a non-metabolized analog of glutamate, which uses the glutamate carrier in the central nervous system (Logan and Snyder, 1971). Secondly, we have shown a system which is sodium dependent and subject to inhibition by ouabain (Table 2). These results are in agreement with those of Paulter and Tengerdy (1986) in bovine RPE. They reported a glutamate flux from retina to choroid which is sodium dependent and ouabain sensitive. Of considerable interest is the high affinity of glutamate transport in RPE (30 p M). In most tissues the K, value for glutamate transport is in the mM range (Pardrige and Jefferson, 1975; Pardrige, 1977; Lerner, 1984). However, high affinity for glutamate transport has been reported in the central nervous system (Logan and Snyder, 1971) with Km values between 1 and 20 PM. Similarly, glutamate transport in the blood-brain barrier (BBB) is in the FM range (Pardrige, 1979). The apparent & value for glutamate does not show any significant change with age. In this context, Levi (1970) found in brain slices that the Km for glutamate and other amino acids is constant during chick development, but the capacity decreases. The diminished rate of accumulation of glutamate and taurine during development might be due to a selective adaptation in the barrier capacity to transport amino acids. In this way there is a selective decrease in the BBB capacity of adult animals to transport the acidic and small neutral amino acids

Amino acid transport (Pardrige, 1979; Sturman and Hayes, 1980). Interestingly, in a non-vascular&d retina, in which RPE has a major roIe in the blood-retinal barrier, glutamate a~umulation by RPE showed a six fold decrease from 7 to 20 day old chick embryos (Salceda and Saldaiia, (unpublished). The development of this retinal-blood barrier capacity of RPE might explain the retinal degeneration produced by monosodium glutamate in immature rats, which is not observed in adult animals (Olney er aZ., 1971; Olney, 1982). This inte~retation is in agreement with the fact that the low accumulation values of glutamate and triggering decrease of taurine uptake, are in parallel with the final differentiation of RPE (Stroeva and Mitashov, 1983). Our results support the essential involvement of RPE in the blood-retinal barrier. In addition, phagocytosis of RPE has been found to be stimulated by taurine (Ogino et al., 1973) and glutamate (Besharse et al., 1986). In order to subserve this function, a precise regulation of the extracellular concentration of these amino acids becomes necessary, and the high affinity uptake system might achieve such a function. However, to clarify this, further studies focused on both the basal and apicaf membranes of RPE are required. work was supported in part by grant P219CCOL880393tok. 8. The &hors would like to thank Mr Gustav0 Sanchez for his technical Acknowledgements-This

CONACY?‘,

assistance.

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