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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Abstract: The antioestrogens, tamoxifen and its more recent homologue toremifene, are used in the therapy of breast cancer. Tamoxifen has been reported to cause retinal changes as side effects. Both compounds inhibited glutamate uptake in retinal pigment epithelial cells, and the present study was conducted to clarify the mechanism of this inhibition. Retinal pigment epithelial cells are part of the blood-retina barrier, and their glutamate transporters are essential for retinal glutamate homeostasis. Glutamate uptake was investigated in human retinal pigment epithelial cell line D407 and in cultured pig retinal pigment epithelial cells using L-[3H]glutamate as a tracer. The cells were exposed to 7.5 μM tamoxifen and toremifene. β-Hydroxyaspartate, a transportable inhibitor of glutamate transport, was used as a reference compound. In kinetic analyses, β-hydroxyaspartate increased the Km constant for glutamate transport. Tamoxifen and toremifene exhibited the same effect, which indicates that inhibition evoked by them is also competitive in nature. Both drugs were more effective in the human retinal pigment epithelial cell line than in the pig retinal pigment epithelial cells. The results show for the first time that the antioestrogens tamoxifen and toremifene could possibly hamper glutamate transport by replacing glutamate as the substrate.

The antioestrogenic drug tamoxifen has been successfully used in the hormonal therapy of breast cancer (Nayfield et al. 1991; Benshushan & Brzezinski 2002). Tamoxifen treatment has been reported to cause ocular side effects, e.g., retinal changes and impairment of vision (Pavlidis et al. 1992; Chern & Danis 1993; Alwitry & Gardner 2002).The other antioestrogen, toremifene, is pharmacologically and structurally similar to tamoxifen, differing only by a single chloride atom (Buzdar & Hortobagyi 1998). However, toremifene has been introduced later to clinical use, and thus there is less long-term experience compared to tamoxifen (Pukkala et al. 2002). One of the differences between the preclinical data of tamoxifen and toremifene is the non-genotoxicity and non-carcinogenity of toremifene in rats (Hard et al. 1993; Karlsson et al. 1996).

Glutamate is the major neurotransmitter in the retina. It is released from photoreceptors, bipolar cells and ganglion cells (Massey 1990). However, prolonged glutamate exposure can be harmful to retinal neurones (Olney 1982; Vorwerk et al. 1996). Glutamate transporters have the main role in preventing glutamate-induced retinal toxicity (Izumi et al. 2002). Their dysfunction promotes accumulation of glutamate in the extracellular space. Even low levels of extracellular glutamate may cause toxic effects when the transporter functions are compromised (Izumi et al. 2002).

Retinal pigment epithelium is a monolayer of cells between choroidal capillaries and photoreceptors. It is considered to be a component of the blood-retina barrier with tight junctions between epithelial cells (Törnquist et al. 1990) and it has a role in maintaining retinal glutamate homeostasis. In previous studies, retinal pigment epithelium has been found to be a sensitive target of antioestrogenic effects (Toimela et al. 1998; Pappas et al. 1999; Mannerström et al. 2001; Engelke et al. 2002; Mäenpääet al. 2002). We have recently shown that antioestrogenic drugs inhibit glutamate uptake in cultured retinal pigment epithelal cells (Mäenpääet al. 2002). Our aim was to analyse the mechanisms of inhibition of glutamate uptake by tamoxifen and toremifene exposure in cultured pig retinal pigment epithelial cells and in human retinal pigment cell line D407. β-Hydroxyaspartate, a transportable inhibitor of glutamate uptake (Danbolt 2001), was used as a reference compound. β-Hydroxyaspartate exhibited a marked inhibition in retinal pigment epithelial cells at the 100-μM concentration (Mäenpääet al. 2002).

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell cultures. The human transformed retinal pigment epithelial cell line D407 (Davis et al. 1995) was kindly donated by Dr. Hunt (University of South Carolina, U.S.A.). Passages 71–75 were used for the uptake experiments, in which 50,000 cells/cm2 were plated in 35 x 10 mm round culture dishes. The cell cultures in the dishes reached 80 % confluence in 2 days, and then the uptake was measured. The culture medium was Dulbecco's modified Eagle medium (DMEM) with 22 mM glucose supplied with 3 % foetal bovine serum (FBS) and 1 % antibiotic/antimycotic solution (all components from Gibco, Paisley, UK). The pig primary retinal pigment epithelial cell cultures were prepared from fresh pig eyes as previously described (Mäenpääet al. 2002). After 11–14 days, the primary cultures were further subcultured and passages 3–4 (60 000 cells/cm2 plated in round dishes) were used for the glutamate uptake assays when the subcultures reached the 80 % confluence. The growth medium for subcultures was DMEM with 5.6 mM glucose supplied with 10 % FBS and 1 % antibiotic/antimycotic solution.

Glutamate uptake assays. The glutamate uptake assays were performed as previously described (Mäenpääet al. 2002). L-[3H]Glutamate (1.55 PBq/mol, Amersham, Buckinghamshire, UK) was used as the label. The dishes were washed twice with oxygenated uptake buffer, Krebs-Ringer-Hepes-glucose medium (pH 7.4, 37 °) containing (mmol/l): NaCl 126.0, KCl 5.1, CaCl2 0.81, MgSO4 1.3, NaH2PO4 1.3, Hepes 15.0 and D-glucose 10.0. After 10 min. preincubation, from 5 to 1000 μM of glutamate (23 MBq/l) was added and the cell cultures were incubated at 37 ° for further 10 min. in the final volume of 2 ml in the dishes. The uptake was terminated by three washes with cold medium. After drying the dishes, the cells were dissolved in 0.4 M NaOH, and the radioactivity in the solubilised retinal pigment epithelium was measured with LKB Wallac 1219 Rackbeta liquid scintillation counter. The protein content of the samples was determined by a bicinchoninic acid -based modification of the Lowry method (BCA Protein Assay, Pierce, Rockford, USA). Bovine serum albumin was used as standard. The breakdown of L-[3H]glutamate was negligible during the experiments (Mäenpääet al. 2002).

Exposure to drugs. Tamoxifen and toremifene were kindly donated by Orion Pharma, Turku, Finland. The effects of tamoxifen and toremifene on the kinetics of glutamate transport were determined by incubating the cultures with a 7.5 μM concentration of both drugs. This concentration is based on our previous data, showing that the IC50 of the glutamate uptake inhibition is approximately 7.5 μM in D407 cells (Mäenpääet al. 2002). Dimethylsulfoxide (DMSO, 0.5 %, Merck, Darmstadt, Germany) was used as solvent. All controls were incubated with the same concentration of DMSO. The drugs were already added to the buffer at the beginning of pre-incubation in the glutamate uptake assays.

The kinetics of glutamate uptake were also studied in the presence of 100 μM β-hydroxyaspartate (Sigma, St. Louis, USA). β-Hydroxyaspartate was added at the beginning of pre-incubation in the glutamate uptake assays. Glutamate uptake was also investigated in cells exposed for 10 min. to the inhibitors, which were then washed off before adding labelled glutamate to the culture dishes. The concentrations of toremifene and β-hydroxyaspartate were the same as above. Tamoxifen concentrations were 7.5 and 30 μM.

Data analysis and statistics. In our study design, each experiment consisted of three totally independent assays done in duplicate on two parallel cell dishes. The data from these experiments was pooled together (n=6) and the S.E.M.s were calculated from this material. To determine the kinetic parameters of glutamate transport, the data were fitted (program Fig.P for Windows, version 2.2a) with the equation consisting of two components, saturable conforming Michaelis kinetics and non-saturable: v=V*s/(Km+s)+NSB*s, where v is the uptake velocity, V the maximal velocity of uptake, Km the Michaelis constant, s the glutamate concentration, and NSB the proportionality constant for non-saturable uptake. The computed constants were compared to the control constants without any inhibitors present (Mäenpääet al. 2002). In the case of linear competitive inhibition, Km (app)=Km (1+i/Ki), where Km (app) is the apparent Km constant in the presence of an inhibitor, i the inhibitor concentration and Ki the inhibition constant (the equilibrium constant of the reversible dissociation reaction of the transporter and inhibitor) (Kontro & Oja 1981). The statistical comparisons of the parameters were made with t-test (Graph Pad Prism, version 3.0).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Inhibition of glutamate uptake in human retinal pigment epithelial cell line D407.

The uptake of glutamate was saturable in human D407 retinal pigment epithelial cells within the concentration range of 5 to 1000 μM, although there was also a non-saturable uptake component with the proportionality constant of 0.92 min.−1 (tamoxifen), 1.29 min.−1 (toremifene) and 1.19 min.−1 (control). These constants did not significantly differ from each other. Saturable uptake was clearly decreased by 7.5 μM tamoxifen below the 300-μM concentration of glutamate, whereas 7.5 μM toremifene was inhibitory over the whole glutamate concentration range from 5 to 1000 μM (fig. 1). The kinetic parameters calculated for the saturable glutamate uptake are presented in table 1. Tamoxifen clearly increased the transport constant Km (more than 6 times), whereas the effect of toremifene was less pronounced (2.5 times). The maximal capacity of transport increased slightly in the presence of tamoxifen. Glutamate uptake was likewise decreased by 100 μM β-hydroxyaspartate at the low glutamate concentrations, but the uptake exceeded the control uptake at the high glutamate concentrations (fig. 1). Both Km (more than 10 times) and V (about 1.6 times) were increased by β-hydroxyaspartate (table 1). The inhibition constants Ki estimated from the data shown in table 1 were about 1.3, 4.9 and 8.3 μM for tamoxifen, toremifene and β-hydroxyaspartate, respectively.

image

Figure 1. Effects of 7.5 μM tamoxifen (circle), 7.5 μM toremifene (triangle down) and 100 μM β-hydroxyaspartate (triangle up) on saturable glutamate uptake in human retinal pigment epithelial cell line D407. Control glutamate uptake into untreated cells is shown with squares. The glutamate concentration varied from 5 to1000 μM and the incubation time was 10 min. In the inset the saturable uptake is shown within the glutamate concentrations of 5–100 μM. Mean values±S.E.M. (if it exceeds the size of symbols) are shown.

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Table 1.  Kinetic parameters of glutamate uptake in human retinal pigment epithelial cell line D407 and in pig retinal pigment epithelial cells
  Kinetic parameters
CellsInhibitorKm±S.E.M. (μM)V±S.E.M. (μmol kg−1min.−1)
  1. The concentrations of the inhibitors were as follows: 7.5 μM tamoxifen, 7.5 μM toremifene and 100 μM β-hydroxyaspartate. The results are calculated from the experiments shown in figs. 1–3. The Km constants shown are apparent Km constants in the presence of the inhibitor. β-Hydroxyaspartate caused so strong inhibition with pig retinal pigment epithelial cells that the Km constant was greater than 1500 μM and V could not be reliably estimated. 1 The control data are from Mäenpääet al. (2002). Statistical significance of the differences from the corresponding controls: *P<0.05; **P<0.01.

D407None (Control)19.0±4.01600.4±22.21
D407Tamoxifen125.0±47.6*787.8±76.6*
D407Toremifene48.1±9.9*548.0±19.6
D407β-Hydroxyaspartate247.6±29.7**975.2±42.5**
pig RPENone (Control) 58.3±17.51195.2±13.01
pig RPETamoxifen233.2±40.2**254.1±14.4*
pig RPEToremifene 97.3±43.5146.9±15.2
pig RPEβ-Hydroxyaspartate>1500

Inhibition of glutamate uptake in cultured pig retinal pigment epithelial cells.

Similar to human D407 cell line, the uptake of glutamate in pig retinal pigment epithelial cells exhibited saturable kinetics with a non-saturable component. The proportionality constants for non-saturable uptake were 0.97 min.−1 (tamoxifen), 1.06 min.−1 (toremifene) and 1.17 min.−1 (control). There were no statistically significant differences among them. The saturable uptake component is presented in fig. 2. Tamoxifen significantly increased both of the kinetic parameters Km (4 times) and V (1.3 times) (table 1). The corresponding constants in the presence of toremifene were markedly less affected. The inhibition constants Ki estimated from the data shown in table 1 were about 2.5 and 11.2 μM for tamoxifen and toremifene, respectively. The application of β-hydroxyaspartate to these cells abolished most of the saturable uptake of glutamate (fig. 3). The uptake was then almost linear within the concentration range of 5–1000 μM, and the kinetic parameters could not be estimated reliably (table 1).

image

Figure 2. Effects of 7.5 μM tamoxifen (circle) and 7.5 μM toremifene (triangle down) on saturable glutamate uptake in pig retinal pigment epithelial cells. The uptake into untreated cells is presented with squares. The glutamate concentration range was 5–1000 μM and the incubation time 10 min. In the inset the saturable uptake is depicted within the glutamate concentrations of 5–100 μM. Mean values±S.E.M. (if it exceeds the size of symbols) are shown.

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image

Figure 3. Effect of 100 μM β-hydroxyaspartate (stars) on glutamate uptake in pig retinal pigment epithelial cells. The uptake into untreated cells is presented with squares. The dashed line represents the share of non-saturable uptake in untreated cells. The glutamate concentration was 5–1000 μM and the incubation time 10 min. Mean values±S.E.M. (if it exceeds the size of symbols) are shown.

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Effect of the 10 min. drug preexposure on glutamate uptake in human retinal pigment epithelial cell line D407 and pig retinal pigment epithelial cells.

Preexposure to 7.5 μM tamoxifen or toremifene caused only minor changes in glutamate uptake by retinal pigment epithelial cells, except for a slight decrease in D407 cells due to tamoxifen (fig. 4). Preexposure to 100 μM β-hydroxyaspartate also affected glutamate uptake slightly in these experiments. The decrease was 28 % in pig retinal pigment epithelial cells. For comparison, in the experiments where the compounds were present throughout the uptake period (10 min.), 7.5 μM tamoxifen inhibited glutamate uptake by 36 % in D407 cells and by 48 % in pig cells and 7.5 μM toremifene 33 % and 46 %, respectively. With 100 μM β-hydroxyaspartate inhibition was 80 % in D407 cells and 69 % in pig cells. Preexposure to a very high concentration of tamoxifen was strongly inhibitory (fig. 4). A longer exposure (24 hr) to 7.5 μM tamoxifen and toremifene caused detachment of cells and trypan blue exclusion confirmed cell death (data not shown).

image

Figure 4. Effects of preexposure to tamoxifen, toremifene or β-hydroxyaspartate on total glutamate uptake in human retinal pigment epithelial cell line D407 and in pig retinal pigment epithelial cells. The glutamate concentration was 5 μM and the incubation time 10 min. The results±S.E.M. are presented as percentages of the control. Statistical significances compared to the controls: *P<0.05, **P<0.01, ***P<0.001.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In addition to human retinal pigment epithelial cell line D407 and pig retinal pigment epithelial cells, the presence of a glutamate transporter has also been demonstrated in rat retinal pigment epithelial cells (Salceda & Saldana 1993) and the human retinal pigment epithelial cell line 165 (Miyamoto & Del Monte 1994). Inhibitors of glutamate transport have been useful tools in studies of the characteristics of glutamate transporters. Most of them, e.g. β-hydroxyaspartate, are not subtype selective (Arriza et al. 1994; Danbolt 2001). Five glutamate transporter subtypes have been identified: GLAST, GLT-1, EAAC1, EAAT4 and EAAT5, the tissue distribution of these subtypes being different (Gegelashvili & Schousboe 1998; Danbolt 2001). The expression and function of glutamate transporters also differ in tumour and normal cells (McGivan 1998; Mäenpääet al. 2002).

To the best of our knowledge this is the first study made on the nature of inhibition of glutamate uptake by antioestrogens. Both tamoxifen and toremifene markedly increased the transport constant Km in the human D407 cell line. This clearly indicates that inhibition is competitive in nature (Kontro & Oja 1981). The results with 100 μM β-hydroxyaspartate, the well-recognised transportable inhibitor of glutamate uptake, which caused the same change in the calculated transport constant Km, corroborate this inference. The inhibition constants Ki for tamoxifen, toremifene and β-hydroxyaspartate were of the same order of magnitude. In cultured pig retinal pigment epithelial cells the inhibition of tamoxifen and toremifene was of the same type but less pronounced, whereas β-hydroxyaspartate was a stronger inhibitor in this preparation. We assume that these differences depend on transformation of the cell line rather than on species difference.

Since tamoxifen and toremifene are competitive inhibitors of glutamate uptake and thus interact with glutamate transporters, they may possibly act as substrates for the glutamate transporters in retinal pigment epithelial cells. The transportable inhibitors of glutamate uptake may also induce glutamate efflux by hetero-exchange (Velasco et al. 1996; Anderson et al. 2001). This antiport mechanism of glutamate may increase the extracellular concentration of glutamate in the retina and cause excitotoxic reactions in the neurons of the distal retina. Low intracellular concentration of glutamate could impede, for example, glutathione formation which is considered to be an important factor in the protection against oxidative stress (Sadzuka et al. 2001).

There was a minor tendency for the inhibition of glutamate uptake in the experiments with cells pre-exposed to tamoxifen. Washing of the cells before glutamate addition may not have completely removed the drugs owing to their tight binding to the cells. Furthermore, the very high concentration of tamoxifen (30 μM) may damage the cells, e.g. by inducing membrane changes (Engelke et al. 2002) that could affect glutamate uptake. Long exposures (24 hr) to 7.5 μM tamoxifen and toremifene have been found to hamper the viability of the cells and induce cytotoxicity (Mannerström et al. 2002). Only slight differences between the effects of these two amphiphilic, structurally very similar drugs have been discernible in retinal pigment epithelial cells (Toimela et al. 1998; Mannerström et al. 2001). A major different feature is the hepatocarcinogenity of tamoxifen in the rat (Hard et al. 1993). Tamoxifen has been shown to have many cellular targets, including protein kinase C (O'Brian et al. 1985), calmodulin (Hardcastle et al. 1996) and plasma membrane ion channels (Sahebgharani et al. 2001).

The mechanisms underlying tamoxifen retinopathy are poorly understood. Crystalline deposits in the retina and pigment epithelium are characteristic features (Wolfensberger 1998; Alwitry & Gardner 2002). Decreased lysosomal enzyme activity (Toimela et al. 1998) and phagocytosis (Mannerström et al. 2001) and changes in membrane lipids (Engelke et al. 2002) have been seen in the retinal pigment epithelial cells in in vitro studies. Tamoxifen is a partial agonist/antagonist of the oestrogen receptor. Oestradiol has been shown to potentiate the excitatory responses to glutamate (Smith 1989) but on the other hand, oestrogens and tamoxifen may reduce the excitatory amino acid –induced neuronal or glial injury (Goodman et al. 1996; Shy et al. 2000). Tamoxifen has been found to decrease the specific binding of glutamate to its N-methyl-D-aspartate (NMDA) class receptors in the brain region-dependently (Cyr et al. 2001). Tamoxifen also partially blocks the protective effect of oestrogens against kainate toxicity in cultured primary cortical neurons (Kajta & Lason 2000). It is not likely, however, that the oestrogen receptor is involved in the inhibition of glutamate uptake evoked by tamoxifen and toremifene. The present incubation times were too short to induce oestrogen receptor-mediated changes. Moreover, we have earlier shown that oestradiol does not change the glutamate uptake of pig retinal pigment epithelial cells (Mäenpääet al. 1997).

In conclusion, the present study shows that tamoxifen and toremifene are competitive inhibitors of glutamate transport in retinal pigment epithelial cells. The effects of these drugs were more pronounced in the human retinal pigment epithelial cell line than in the pig retinal pigment epithelial cells.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Professor Simo S. Oja for valuable discussions and Ms Paula Helpiölä and Ms Maiju Mallat for the expert technical assistance. We are grateful to Ms Virve Kajaste for checking the English language. This study was supported by the Medical Research Fund of Tampere University Hospital, Tampere Graduate School in Biomedicine and TEKES, the National Technology Agency.

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  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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