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Effect of taurine and apical potassium concentration on electrophysiologic parameters of bovine retinal pigment epithelium
Experimental Eye Research 82 (2006) 258–264 www.elsevier.com/locate/yexer
Effect of taurine and apical potassium concentration on electrophysiologic parameters of bovine retinal pigment epithelium* Jost Hillenkampa,*,1, Ali A. Hussaina, Timothy L. Jacksona, Joanna R. Cunninghamb, John Marshalla a
Department of Ophthalmology, The Rayne Institute, St Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, UK b Department of Pharmacology, The Rayne Institute, St Thomas’ Hospital, Lambeth Palace Road, London, UK Received 11 January 2005; accepted in revised form 17 June 2005 Available online 18 August 2005
Abstract The purpose of this study was to assess the effect of taurine and apical potassium concentration modelling in vivo light evoked changes on the transepithelial potential (TEP) and the transepithelial resistance (TER) of isolated bovine retinal pigment epithelium (RPE). Isolated specimens of bovine non-tapetal RPE-Bruch’s-choroid (RPE-BC) were mounted in modified Ussing chambers. The apical and the basolateral side of the preparations were exposed to 10 mM and 10 mM concentrations of taurine in Krebs’ medium with either 6.04 or 2.2 mM potassium in the apical compartment. TEP and TER were recorded over 140 min. TEP and TER decreased with exposure to taurine over the course of 1 hr followed by a stabilisation. The degree of this response did not depend on the concentration of taurine but was more pronounced when taurine was added to the apical compartment. Lowering apical potassium from 6.04 to 2.2 mM further pronounced the decrease of TEP and TER. The data show that light-induced release of taurine from the outer retina and light-induced decrease of the potassium concentration in the subretinal space synergistically lead to a temporary decrease in TEP and TER. Thereby, taurine uptake into the RPE is reduced probably by a reduction of the activity of the electrogenic NaC/taurine co-transporter of the apical RPE cell membrane. The findings suggest a mechanism whereby the sustained presence of taurine in the interphotoreceptor matrix following exposure to light may protect photoreceptor outer segments from light-induced oxidative stress. q 2005 Elsevier Ltd. All rights reserved. Keywords: retinal pigment epithelium; taurine; electrophysiology; apical potassium
1. Introduction Taurine, an uncharged b-amino-sulfonic acid, is particularly important for normal vision. Its importance has been recognised ever since the original observation that taurine deficiency in cats results in retinal degeneration and blindness (Hayes et al., 1975). Taurine is essential for
*
Presented in part at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, USA, May 1–5th, 2005. * Corresponding author. E-mail address: [email protected] (J. Hillenkamp). 1 Present address: Universita¨ts-Augenklinik, Franz-Josef-StraussAllee 11, D-93042 Regensburg, Germany. Tel.: C49 941 944 9210; fax: C49 941 944 9202. 0014-4835/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2005.06.020
the maintenance of photoreceptor cells (Wright et al., 1986). Several roles for taurine in the retina have been suggested, including antioxidation (Wright et al., 1986; Bridges et al., 2001) membrane stabilisation (Wright et al., 1986), and osmoregulation (Wright et al., 1986; El-Sherbeny et al., 2004). In man, pathological conditions such as bowel resection compromise the intake of dietary taurine and lead to visual defects (Geggel et al., 1985). Human infants and children maintained on a taurine-free total parenteral nutrition show abnormalities in the electroretinogram and fundus changes are first seen as mild granularity of the retinal pigment epithelium (RPE) (Ameant et al., 1986). A mouse model with a disrupted gene coding for a taurine transporter exhibits severe retinal degeneration, suggesting that taurine is critical for normal retinal development and function (Heller-Stilb et al., 2002). Although a precise physiological role of taurine in the retina has yet to be established, its involvement in the pathogenesis of retinal
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degenerations, diabetic retinopathy and macular edema has been suggested (Geggel et al., 1985; Bridges et al., 2001; Heller-Stilb et al., 2002; El-Sherbeny et al., 2004). Taurine is highly concentrated (60–80 mM) in the outer nuclear layer of the vertebrate retina via a sodiumdependent uptake mechanism (Lake et al., 1975, 1977; Wright et al., 1986). In vivo, light stimulation causes a large efflux of retinal taurine into the subretinal space (Salceda et al., 1977; Schmidt, 1978; Neal et al., 1979) as well as a reduction of the concentration of potassium in the subretinal space (Oakley and Green, 1976; Oakley, 1977; Steinberg et al., 1980). It was the aim of the present study to further investigate the effect of potassium and taurine on the homeostasis of the subretinal space by analysing electrophysiologic parameters of the RPE in vitro.
2. Material and methods 2.1. Preparation of bovine tissue Fresh bovine eyes of Friesian cows aged 18–24 months were obtained from a local slaughterhouse and experiments initiated within 6 hr of death. Whole globes were dissected in a petri dish lined with filter paper (Grade 50; Whatman, Maidstone, UK), moistened with phosphate-buffered saline (PBS, Sigma Chemical Co., Poole, UK). The anterior portion of the eye was carefully removed by a circumferential incision at the pars plana, and the cornea together with the lens, iris and vitreous were discarded. The neural retina was then gently peeled away from the underlying RPE and cut at the optic nerve head. All samples were obtained from the same mid-peripheral non-tapetal fundus area close to the optic disc with an 8 mm trephine (Stiefel Laboratories, Buckinghamshire, UK). The RPE-Bruch’s membranechoroid complex was gently teased away from the sclera.
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Krebs’ medium on one side and 4 ml of Krebs’ medium on the other side (apical sideZRPE, basal sideZfilter paperchoroid). The solution was gassed on both sides with humidified 10% O2–5%CO2–85%N2, as low oxygen increases longevity of RPE function (Winkler and Giblin, 1983). The continuous gassing also stirred the solutions as demonstrated by adding one drop of fluorescein to the solution in a preliminary experiment. The temperature of the solutions was kept at 378C by constant circulation of heated water driven by a heating circulator (Julabo, Inc., Seelbach, Germany) through the jacketed circulation reservoirs of the Ussing chamber system (WPI, Aston, UK). The Ussing chamber was then tilted by 258 with the apical side facing upwards in order to minimise unstirred layers near the aperture of the internal cassette. We measured the shortcircuit current and the transepithelial potential (TEP) at regular intervals using a EVC-4000 voltage clamp (WPI, Aston, UK) with voltage-sensing electrodes and current passing bridges which consisted of 3 M KCl-agar. Transepithelial resistance (TER) was determined by clamping the transepithelial potential at 10 mV, recording the deflection of the short-circuit current, and applying Ohm’s law. The preparations were allowed to equilibrate. Stabilization of bioelectrical parameters was usually achieved within 20–30 min. Only tissue samples with a TERO100 Ucm2 were considered for experimentation. TEP and TER were recorded at K20, K10, 0, 10, 20, 30, 40, 50, 60, 90, and 120 min. The recordings taken during the first 20 min served as control. 1 ml of taurine in a 50 m M or 50 m M concentration was added at time point 0 min to either the apical or the basal side creating a 10 mM or 10 mM solution on one side of the preparation and 5 ml of volume on both sides. It has been shown that simple addition of taurine in a 10 mM concentration and adding 10 mM taurine while removing 5 mM NaCl lead to equivalent measurements of epithelial electrical properties (Scharschmidt et al., 1988). 2.3. Statistical analyses
2.2. Measurement of electrophysiologic parameters The isolated RPE-Bruch’s-choroid preparation was floated on hardened, ashless filter paper with a pore size of 20–25 mm (Grade 541; Whatman, Maidstone, UK). The preparation was then mounted between the two halves of a perspex insert cassette with a central 4 mm diameter aperture (Institute of Ophthalmology, London, UK). The cassette assembly was then clamped in a modified perspex Ussing chamber (WPI, Aston, UK) and both surfaces of the preparation were rinsed several times with Krebs’ medium of the following composition: (mM): NaCl 118, Glucose 6.6, NaHCO3 25, KCl 4.84, MgSO4 0.8, KH2PO4 1.2, CaCl2 1.8, 0.01% BSA (bovine serum albumin). In a part of the experiments the KCl-component of the medium was partially replaced by choline chloride to yield a solution with 2.2 mM KC on the apical side of the preparation. A 0.126 cm2 area of the tissue sample was exposed to 5 ml
Statistical analyses were undertaken using Graph Pad Prism 3.02 (Graph Pad Software, Inc., San Diego, CA, USA).
3. Results We measured TEP and TER with taurine concentrations of 10 mM and 10 mM added either to the apical side or to the basolateral side of the RPE-Bruch’s-choroid preparation. We conducted experiments with a potassium concentration of 6.04 mM on both sides and with a lower potassium concentration of 2.2 mM on the apical side (Tables 1 and 2). The addition of taurine to either the apical side or the basolateral side of the RPE-Bruch’s-choroid preparation resulted in a decrease of TEP and TER over the course of 1 hr followed by a stabilisation of TEP and TER during
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Table 1 Transepithelial potential (TEP) in experiments with 10 mM and 10 mM taurine on the apical or basolateral side of the RPE-Bruch’s-choroid preparation and 2.2 or 6 mM potassium on the apical side Transepithelial potential (TEP) (mV) (meanGSEM) Time (min)
10 mM taurine on basal side, K 6.04 mM both sides (nZ5)
10 mM taurine on apical side, K 6.04 mM both sides (nZ3)
10 mM taurine on basal side, K 6.04 mM both sides (nZ4)
10 mM taurine on apical side, K 6.04 mM both sides (nZ4)
10 mM taurine on basal side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ7)
10 mM taurine on apial side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ3)
10 mM taurine on basal side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ3)
10 mM taurine on apical side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ3)
K20 K10 0 10 20 30 40 50 60 90 120
8.9G0.43 8.7G0.29 8.9G0.33 7.68G0.64 7.4G0.62 7.14G0.76 7.2G0.9 6.7G1 6.42G1.18 5.92G1.41 5.74G1.55
7.63G1.15 7.53G1.11 7.47G1.12 5.27G1.22 4.83G1.07 4.33G1.19 4.13G1.33 3.93G1.33 3.8G1.3 3.93G1.44 4.17G1.48
7.33G0.67 7.15G0.7 7.3G0.78 6.83G0.79 5.83G0.69 5.1G0.51 4.5G0.43 4.3G0.58 3.98G0.66 3.1G0.12 3.43G0.19
7.45G0.56 7.4G0.56 7.3G0.55 5.88G0.41 5.63G0.45 5.4G0.62 5.22G0.78 5.05G0.87 4.85G0.99 5.55G0.92 6.18G0.93
6.59G0.68 6.59G0.7 6.51G0.7 5.51G0.78 4.8G0.58 4.61G0.48 4.57G0.41 4.49G0.38 4.47G0.34 4.64G0.22 5.17G0.47
4.47G0.33 4.6G0.3 4.63G0.29 4.3G0.17 5.17G0.34 6.13G0.57 6.53G0.67 6.17G0.65 5.9G0.75 6.17G0.92 5.7G0.1
7.65G0.35 7.55G0.25 7.6G0.4 5.45G0.85 4.45G0.55 4.1G0.4 3.95G0.35 3.75G0.45 3.65G0.55 3.65G0.75 3.75G0.55
6.3G1.02 6.33G1.01 6.43G1.07 5.03G0.83 4.76G1.1 4.5G0.19 4.33G1.23 4.1G1.23 3.93G1.07 4.1G1.02 5.23G1.42
the second hour of the experiments. This response was statistically significantly more pronounced for the first 10 min following the addition of taurine when taurine was added to the apical side as compared to when taurine was added to the basolateral side (Figs. 1 and 2). There was no statistically significant difference between TEP or TER when taurine was added in the 10 mM or the 10 mM concentration (Figs. 3 and 4). We pooled the data measured with the two different taurine concentrations (Figs. 1, 2, 5 and 6) and the data measured when taurine was added to the apical and the basolateral side (Figs. 3–6). TEP was statistically significantly lower before the addition of taurine and for the first 10–20 min following the addition of taurine when the potassium concentration on the apical side
was reduced from 6.04 to 2.2 mM. TER was statistically significantly lower at the time points K10 and 0 min with the lower potassium concentration on the apical side (Figs. 5 and 6).
4. Discussion In the present study, we used two different taurine concentrations in the millimolar and in the micromolar range to target both the high- an low-affinity taurine carriers previously identified in bovine RPE (Kundaiker et al., 1996) and to simulate the concentration of plasma taurine in the range of 44–62 mM in human (Hussain and Voaden, 1987;
Table 2 Transepithelial resistance (TER) in experiments with 10 mM and 10 mM taurine on the apical or basolateral side of the RPE-Bruch’s-choroid preparation and 2.2 or 6 mM potassium on the apical side Transepithelial resistance (TER) (Ucm2) (meanGSEM) Time (min)
10 mM taurine on basal side, K 6.04 mM both sides (nZ5)
10 mM taurine on apical side, K 6.04 mM both sides (nZ3)
10 mM taurine on basal side, K 6.04 mM both sides (nZ4)
10 mM taurine on apical side, K 6.04 mM both sides (nZ4)
10 mM taurine on basal side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ7)
10 mM taurine on apial side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ3)
10 mM taurine on basal side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ3)
10 mM taurine on apical side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ3)
K20 K10 0 10 20 30 40 50 60 90 120
462G42 504G51.4 462G42 399G61.2 352.8G78.1 352.8G78.1 346.8G81 332.4G86.4 315.2G92.4 281.4G93.8 277.2G92.6
399G116.9 378G126 378G126 246.7G87.4 239.3G90.5 193.7G60.8 190G62.9 181.7G66.7 181.7G66.7 216.7G101.7 216.7G101.7
420G74.3 367G30.3 420G74.3 404.3G82.8 288.8G50.3 231.8G32.5 190G25.4 190G25.4 174.5G28.3 131.7G8.3 133G12.9
362.3G90.5 351.8G95.2 362G90.6 236.3G26.3 236.3G26.3 231.8G32.6 220.8G38.4 216.3G41.1 208.3G45.4 227.5G50.5 258.3G66.7
378G79.7 332.5G68.7 339.5G66.5 273.9G62.5 226.9G34.6 211.9G21.3 198.6G15.3 196.9G12.8 190.9G9.4 204.3G11.1 234.3G23.5
190G10 180G0 172.6G7.3 190G10 238G14 350G70 350G70 280G70 280G70 294G64.2 252G0
420G0 336G84 336G84 262.5G52.5 210G0 180G0 158G0 158G0 158G0 160G20 160G20
394G236 394G236 394G236 260G87.4 246.7G87.4 234.7G93.1 228.7G95.8 228.7G95.8 228.7G95.8 228.7G95.8 315.3G157.3
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Fig. 1. Effect of taurine added to the apical side versus the basolateral side of RPE-Bruch’s-choroid on TEP. TEP was recorded for 140 min. The first 20 min served as control. Taurine was added at time 0 min to either the apical side (group A, triangles, nZ7) or the basolateral side (group B, squares, nZ9) of the RPE-Bruch’s-choroid preparation with a final concentration of 10 mM or 10 mM. The [KC]-concentration was 6 mM on both sides. There was a statistically significant difference between the measurements of group A and group B at 10 min (P!0.05). There was no statistically significant difference between all other measurements of group A and group B (PO0.05, Mann–Whitney-Test).
Trautwein and Hayes, 1990; Inoue et al., 2003) and 18–89 mM in bovine (Sakai and Nagasawa, 1992; Kelly et al., 2000), and interphotoreceptor matrix levels of 6.6 mM (Sellner, 1986) which increase to millimolar levels secondary to a large efflux of retinal taurine into the subretinal space following light stimulation (Salceda et al., 1977; Schmidt, 1978; Neal et al., 1979). Furthermore, we used two different apical potassium concentrations to simulate the light-induced reduction of the apical potassium concentration (Oakley and Green, 1976; Oakley, 1977; Steinberg et al., 1980). Exposure of either the apical or the basolateral side of the RPE to taurine lead to a decrease of TEP and TER
Fig. 2. Effect of taurine added to the apical side versus the basolateral side of RPE-Bruch’s-choroid on TER. TEP was recorded for 140 min. The first 20 min served as control. Taurine was added to either the apical side (group A, triangles, nZ7) or the basolateral side (group B, squares, nZ9) of the RPE-Bruch’s-choroid preparation with a final concentration of 10 mM or 10 mM. The [KC]-concentration was 6 mM on both sides. There was a statistically significant difference between the measurements of group A and group B at 10 min (P!0.05). There was no statistically significant difference between all other measurements of group A and group B (PO0.05, Mann–Whitney-Test).
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Fig. 3. Effect of taurine with a concentration of 10 mM versus 10 mM on TEP. TEP was recorded for 140 min. The first 20 min served as control. Taurine was added at time 0 min to the apical side or the basolateral side with a final concentration of 10 mM (group C, triangles, nZ8) or 10 mM (group D, squares, nZ8) of the RPE-Bruch’s-choroid preparation. The [KC]-concentration was 6 mM on both sides. There was no statistically significant difference between the measurements of group C and group D at any time point (PO0.05, Mann–Whitney-Test).
followed by a stabilisation. This effect was more pronounced when the the apical side was exposed to taurine (Figs. 1 and 2). This finding is principally in accordance with Kundaiker et al. who identified active high- and low-affinity taurine carriers on both the apical and the basolateral cell membranes of bovine RPE (Kundaiker et al., 1996). Our findings are partially in accordance with Scharschmidt et al. who found a depolarisation of the apical but not the basolateral membrane of frog RPE following exposure to a 10 mM taurine solution which only occurred in the presence of sodium in the medium (Scharschmidt et al., 1988). The effect of taurine on TEP and TER is probably mediated by a co-transport of positively charged sodium and taurine into the RPE (Scharschmidt et al., 1988). The mechanism of the later stabilisation of TEP and TER is not fully understood. With prolonged exposure to taurine, the
Fig. 4. Effect of taurine with a concentration of 10 mM versus 10 mM on TER. TER was recorded for 140 min. The first 20 min served as control. Taurine was added at time 0 min to the apical side or the basolateral side with a final concentration of 10 mM (group C, triangles, nZ8) or 10 mM (group D, squares, nZ8) of the RPE-Bruch’s-choroid preparation. The [KC]-concentration was 6 mM on both sides. There was no statistically significant difference between the measurements of group C and group D at any time point (PO0.05, Mann–Whitney-Test).
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Fig. 5. Effect of 2.2 versus 6.04 mM potassium on the apical side of RPEBruch’s-choroid on TEP. TEP was recorded for 140 min. The first 20 min served as control. [KC]-concentration on the apical side of the RPEBruch’s-choroid preparation was either 2.2 mM (group E, triangles, nZ15) or 6 mM (group F, squares, nZ16). [KC]-concentration on the basolateral side was 6 mM in all experiments. Experiments with taurine concentrations of 10 mM and 10 mM on the apical and the basolateral side (Tables 1 and 2) were pooled. There was a statistically significant difference between the measurements of group E and group F at K20, K10, 0, 10, and 20 min (P!0.05). There was no statistically significant difference between all other measurements of groups E and F (PO0.05) (Mann–Whitney test).
accompanying load of intracellular sodium may stimulate the electrogenic NaC/KC pump which then transports sodium and net positive charge out of the cell (Scharschmidt et al., 1988) and thereby counteracts the decrease of TEP caused by the sudden increase of the apical taurine concentration from micromolar levels to millimolar levels. By contrast, the basolateral side of the RPE is in vivo continuously exposed to a taurine concentration in the micromolar range (Hussain and Voaden, 1987; Trautwein and Hayes, 1990; Sakai and Nagasawa, 1992; Kelly et al., 2000; Inoue et al., 2003) without sudden drastic changes. The depolarising effect of
Fig. 6. Effect of 2.2 versus 6.04 mM potassium on the apical side of RPEBruch’s-choroid on TER. TER was recorded for 140 min. The first 20 min served as control. [KC]-concentration on the apical side of the RPEBruch’s-choroid preparation was either 2.2 mM (group E, triangles, nZ15) or 6 mM (group F, squares, nZ16). [KC]-concentration on the basolateral side was 6 mM in all experiments. Experiments with taurine concentrations of 10 mM and 10 mM on the apical and the basolateral side (Tables 1 and 2) were pooled. There was a statistically significant difference between the measurements of group E and group F at K10, and 0 min (P!0.05). There was no statistically significant difference between all other measurements of groups E and F (PO0.05) (Mann–Whitney test).
co-uptake of taurine and sodium at the basolateral side may be balanced by the NaC/KC pump. Furthermore, our findings suggest that the sodiummediated taurine-induced reduction of TEP and TER is a saturable mechanism as the two taurine concentrations used in the present study did not lead to a statistically significant difference in the magnitude of the reduction of TEP and TER (Figs. 3 and 4). Kundaiker et al. found that the highaffinity taurine carrier system of bovine RPE is characterised by a Michaelis–Menten constant (Km) of 23 mM and a Vmax of 86.7 pmol/5 min/4 mm disc and that it is sodiumdependent with a Hill coefficient of 2.0 indicating that two sodium ions are required for the uptake of one molecule of taurine. By contrast, the low-affinity system is characterised by a Km of 507 mM and a Vmax of 344 pmol/5 min/4 mm disc and it is heterogenous as it is only partially sensitive to sodium (Kundaiker et al., 1996). This suggests that the reduction of TEP is primarily mediated by the sodiumsensitive high-affinity system and it may explain why in the present study exposure to a millimolar taurine concentration did not lead to a greater reduction of TEP and TER as compared to a micromolar taurine concentration. We expected a significant change in the short circuit current following exposure to taurine. Although such a change was observed in some of the experiments the change was only slight. This may be due to the epithelium becoming leaky during the experiment. Further investigation is required to clarify this point. The observed changes of TEP and TER when the in vivo light-induced decrease of apical potassium was modelled (Figs. 5 and 6) are in accordance with Bialek and Miller (Bialek and Miller, 1994) who described a transient increase followed by a reduction of TEP when apical potassium was lowered from 5 to 2 mM. The reduction of TEP lasted until control solution with 5 mM potassium was returned to the apical side of the RPE. This effect was followed by a buffering net secretion of KC into the subretinal space which appeared to be electrically coupled to a basolateral increased ClK conductance (Bialek and Miller, 1994). Oakley and Steinberg described a duration of the reduced subretinal potassium concentration in response to maintained illumination of approximately 10 min after light onset in an in vitro preparation of bullfrog retina (Oakley and Steinberg, 1982). The exact duration and the magnitude of the decrease of subretinal [KC] following light onset in vivo in bovine or human is unknown. Oakley et al. have shown that in the toad retina the light-evoked decrease in [KC] in the outer plexiform layer is attenuated by the spatial buffering efflux of KC from Mu¨ller cells at that retinal depth (Oakley et al., 1992). Therefore, the results of the present in vitro study cannot be directly applied to the in vivo situation. However, they provide principal evidence that exposure to taurine and the light-induced reduction of the apical potassium concentration temporarily decrease TEP and TER. More specifically, the findings of the present study (Figs. 5 and 6) suggest that the light-induced decrease in
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subretinal [KC] further pronounces the temporary taurineinduced decrease of TEP and TER. The light-induced decrease of apical potassium together with taurine released from the retina could possibly have significant physiological functional importance by conserving retinal levels of taurine. The decrease of TEP leads to a decrease of the activity of the electrogenic taurine/sodium co-transporter of the RPE cell membrane (Scharschmidt et al., 1988) and thereby reduces the uptake of taurine from the subretinal space into the RPE. Indeed, it has been shown that a lowered apical [KC] is associated with reduced taurine uptake (Kundaiker et al., 1996) and reduced transport across the RPE (Miller and Steinberg, 1979; Hillenkamp et al., 2004). It can be hypothesised that taurine released into the subretinal space with the onset of light remains longer in position secondary to the decrease of TEP following the onset of light. Furthermore, reduced transport across the RPE would allow the outer retina to re-uptake released taurine and limit the loss to the blood stream via the RPE. As taurine has been shown to protect photoreceptor outer segment cell membranes against light-induced oxidative damage (Pasantes-Morales and Cruz, 1984; Wright et al., 1986; Keys and Zimmerman, 1999), this may be a physiological protective mechanism. This hypothesis is supported by recent work which showed that light deprivation slows retinal degeneration in taurine transporter knock-out mice (Rascher et al., 2004). In summary, our data suggest that taurine and apical potassium may synergistically act to keep taurine as a protection of photoreceptor outer segments against oxidative damage and osmotic imbalance longer in position in the subretinal space following the transition from dark to light.
Acknowledgements J.H. was supported by Deutsche Forschungsgemeinschaft (DFG) grant Hi 758/1-1. A.A.H. was supported by The PPP Foundation, UK. T.L.J. was supported by Special Trustees of Guys and St Thomas’ Hospital and The Allerton Trust.
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El-Sherbeny, A., Naggar, H., Miyauchi, S., Ola, M.S., Maddox, D.M., 2004. Osmoregulation of taurine transporter function and expression in retinal pigment epithelial, ganglion, and Mu¨ller cells. Invest. Ophthalmol. Vis. Sci. 45, 694–701. Geggel, H.S., Ameant, M.E., Heckenlively, J.R., Martin, D.A., Kopple, J.D., 1985. Hutritional requirement for taurine in patients receiving long term parenteral nutrition. N. Engl. J. Med. 312, 142–146. Hayes, K.C., Carey, R.E., Schmidt, S.Y., 1975. Retinal degeneration associated with taurine deficiency in the cat. Science 188, 949–951. Heller-Stilb, B., van Roeyen, C., Rascher, K., et al., 2002. Disruption of the taurine transporter gene (TauT) leads to retinal degeneration in mice. FASEB J. 16, 231–233. Hillenkamp, J., Hussain, A.A., Jackson, T.L., Constable, P.A., Cunningham, J.R., Marshall, J., 2004. Compartmental analysis of taurine transport to the outer retina in the bovine eye. Invest. Ophthalmol. Vis. Sci. 45, 4099–4105. Hussain, A.A., Voaden, M.J., 1987. Some observations on taurine homeostasis in patients with retinitis pigmentosa. Prog. Clin. Biol. Res. 247, 119–129. Inoue, H., Fukunaga, K., Tsuruta, Y., 2003. Determination of taurine in plasma by high-performance liquid chromatography using 4-(5,6dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent. Anal. Biochem. 319, 138–142. Kelly, M.T., Fabre, H., Perrett, D., 2000. Determination of taurine in plasma by capillary zone electrophoresis following derivatisation with fluorescamine. Electrophoresis 21, 699–705. Keys, S.A., Zimmerman, W.F., 1999. Antioxidant activity of retinol, glutathione, and taurine in bovine photoreceptor cell membranes. Exp. Eye Res. 68, 693–702. Kundaiker, S., Hussain, A.A., Marshall, J., 1996. Component characteristics of the vectorial transport system for taurine in isolated bovine retinal pigment epithelium. J. Physiol. 492, 505–516. Lake, N., Marshall, J., Voaden, M.J., 1975. Studies on the uptake of taurine by the isolated neuronal retina and pigment epithelium of the frog. Biochem. Soc. Trans. 3, 524–525. Lake, N., Marshall, J., Voaden, M.J., 1977. The entry of taurine into the neural retina and the pigment epithelium of the frog. Brain Res. 128, 497–503. Miller, S.S., Steinberg, R.H., 1979. Potassium modulation of taurine transport across the frog retinal pigment epithelium. J. Gen. Physiol. 74, 237–259. Neal, M.J., Collins, G.G., Massey, S.C., 1979. Inhibition of aspartate release from the retina of the anaesthetised rabbit by stimulation with light flashes. Neurosci. Lett. 14, 241–245. Oakley, B., 1977. Potassium and the photoreceptor-dependent pigment epithelial hyperpolarization. J. Gen. Physiol. 70, 405–425. Oakley, B., Green, D.G., 1976. Correlation of light-induced changes in retinal extracellular potassium concentration with c-wave of the electroretinogram. J. Neurophysiol. 39, 1117–1133. Oakley, B., Steinberg, R.H., 1982. Effects of maintained illumination upon [KC] in the subretinal space of the frog retina. Vision Res. 22, 767–773. Oakley, B., Katz, B.J., Xu, Z., Zheng, J., 1992. Spatial buffering of extracellular potassium by Mu¨ller (Glial) cells in the toad retina. Exp. Eye Res. 55, 539–550. Pasantes-Morales, H., Cruz, C., 1984. Protective effect of taurine and zinc on peroxidation-induced damage in photoreceptor outer segments. J. Neurosci. Res. 11, 303–311. Rascher, K., Servos, G., Berthold, G., Hartwig, H.G., Warskulat, U., HellerStilb, B., Haussinger, D., 2004. Light deprivation slows but does not prevent loss of photoreceptors in taurine transporter knock-out mice. Vis. Res. 44, 2091–2100. Sakai, T., Nagasawa, T., 1992. Simple, rapid and sensitive determination of plasma taurine by high performance liquid chromatography using precolumn derivative formation with fluorescamine. J. Chromatogr. 576, 155–157.
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Salceda, R., Lopez-Colome, A., Pasante-Morales, H., 1977. Lightstimulated release of (35S)-taurine from frog retinal rod outer segments. Brain Res. 135, 186–191. Scharschmidt, B.F., Griff, E.R., Steinberg, R.H., 1988. Effect of taurine on the isolated retinal pigment epithelium of the frog: electrophysiologic evidence for stimulation of an apical, electrogenic NaC–KC pump. J. Membr. Biol. 106, 71–81. Schmidt, S.Y., 1978. Taurine fluxes in isolated cat and rat retinas: effects of illumination. Exp. Eye Res. 26, 529–535. Sellner, P.A., 1986. The movement of solutes between the retina and pigment epithelium. Exp. Eye Res. 43, 631–639.
Steinberg, R.H., Oakley, B., Niemeyer, G., 1980. Light-evoked changes in KC in the retina of the intact cat eye. J. Neurophysiol. 44, 897– 921. Trautwein, E.A., Hayes, K.C., 1990. Taurine concentrations in plasma and whole blood in humans: estimation of error from intra- and interindividual variation and sampling technique. Am. J. Clin. Nutr. 52, 758–764. Winkler, B.S., Giblin, F.J., 1983. Glutathione oxidation in retina: effects on biochemical and electrical activities. Exp. Eye Res. 36, 287–297. Wright, C.E., Tallan, H.H., Lin, Y.Y., 1986. Taurine: biological update. Ann. Rev. Biochem. 55, 427–453.
Effect of taurine and apical potassium concentration on electrophysiologic parameters of bovine retinal pigment epithelium* Jost Hillenkampa,*,1, Ali A. Hussaina, Timothy L. Jacksona, Joanna R. Cunninghamb, John Marshalla a
Department of Ophthalmology, The Rayne Institute, St Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, UK b Department of Pharmacology, The Rayne Institute, St Thomas’ Hospital, Lambeth Palace Road, London, UK Received 11 January 2005; accepted in revised form 17 June 2005 Available online 18 August 2005
Abstract The purpose of this study was to assess the effect of taurine and apical potassium concentration modelling in vivo light evoked changes on the transepithelial potential (TEP) and the transepithelial resistance (TER) of isolated bovine retinal pigment epithelium (RPE). Isolated specimens of bovine non-tapetal RPE-Bruch’s-choroid (RPE-BC) were mounted in modified Ussing chambers. The apical and the basolateral side of the preparations were exposed to 10 mM and 10 mM concentrations of taurine in Krebs’ medium with either 6.04 or 2.2 mM potassium in the apical compartment. TEP and TER were recorded over 140 min. TEP and TER decreased with exposure to taurine over the course of 1 hr followed by a stabilisation. The degree of this response did not depend on the concentration of taurine but was more pronounced when taurine was added to the apical compartment. Lowering apical potassium from 6.04 to 2.2 mM further pronounced the decrease of TEP and TER. The data show that light-induced release of taurine from the outer retina and light-induced decrease of the potassium concentration in the subretinal space synergistically lead to a temporary decrease in TEP and TER. Thereby, taurine uptake into the RPE is reduced probably by a reduction of the activity of the electrogenic NaC/taurine co-transporter of the apical RPE cell membrane. The findings suggest a mechanism whereby the sustained presence of taurine in the interphotoreceptor matrix following exposure to light may protect photoreceptor outer segments from light-induced oxidative stress. q 2005 Elsevier Ltd. All rights reserved. Keywords: retinal pigment epithelium; taurine; electrophysiology; apical potassium
1. Introduction Taurine, an uncharged b-amino-sulfonic acid, is particularly important for normal vision. Its importance has been recognised ever since the original observation that taurine deficiency in cats results in retinal degeneration and blindness (Hayes et al., 1975). Taurine is essential for
*
Presented in part at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, USA, May 1–5th, 2005. * Corresponding author. E-mail address: [email protected] (J. Hillenkamp). 1 Present address: Universita¨ts-Augenklinik, Franz-Josef-StraussAllee 11, D-93042 Regensburg, Germany. Tel.: C49 941 944 9210; fax: C49 941 944 9202. 0014-4835/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2005.06.020
the maintenance of photoreceptor cells (Wright et al., 1986). Several roles for taurine in the retina have been suggested, including antioxidation (Wright et al., 1986; Bridges et al., 2001) membrane stabilisation (Wright et al., 1986), and osmoregulation (Wright et al., 1986; El-Sherbeny et al., 2004). In man, pathological conditions such as bowel resection compromise the intake of dietary taurine and lead to visual defects (Geggel et al., 1985). Human infants and children maintained on a taurine-free total parenteral nutrition show abnormalities in the electroretinogram and fundus changes are first seen as mild granularity of the retinal pigment epithelium (RPE) (Ameant et al., 1986). A mouse model with a disrupted gene coding for a taurine transporter exhibits severe retinal degeneration, suggesting that taurine is critical for normal retinal development and function (Heller-Stilb et al., 2002). Although a precise physiological role of taurine in the retina has yet to be established, its involvement in the pathogenesis of retinal
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degenerations, diabetic retinopathy and macular edema has been suggested (Geggel et al., 1985; Bridges et al., 2001; Heller-Stilb et al., 2002; El-Sherbeny et al., 2004). Taurine is highly concentrated (60–80 mM) in the outer nuclear layer of the vertebrate retina via a sodiumdependent uptake mechanism (Lake et al., 1975, 1977; Wright et al., 1986). In vivo, light stimulation causes a large efflux of retinal taurine into the subretinal space (Salceda et al., 1977; Schmidt, 1978; Neal et al., 1979) as well as a reduction of the concentration of potassium in the subretinal space (Oakley and Green, 1976; Oakley, 1977; Steinberg et al., 1980). It was the aim of the present study to further investigate the effect of potassium and taurine on the homeostasis of the subretinal space by analysing electrophysiologic parameters of the RPE in vitro.
2. Material and methods 2.1. Preparation of bovine tissue Fresh bovine eyes of Friesian cows aged 18–24 months were obtained from a local slaughterhouse and experiments initiated within 6 hr of death. Whole globes were dissected in a petri dish lined with filter paper (Grade 50; Whatman, Maidstone, UK), moistened with phosphate-buffered saline (PBS, Sigma Chemical Co., Poole, UK). The anterior portion of the eye was carefully removed by a circumferential incision at the pars plana, and the cornea together with the lens, iris and vitreous were discarded. The neural retina was then gently peeled away from the underlying RPE and cut at the optic nerve head. All samples were obtained from the same mid-peripheral non-tapetal fundus area close to the optic disc with an 8 mm trephine (Stiefel Laboratories, Buckinghamshire, UK). The RPE-Bruch’s membranechoroid complex was gently teased away from the sclera.
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Krebs’ medium on one side and 4 ml of Krebs’ medium on the other side (apical sideZRPE, basal sideZfilter paperchoroid). The solution was gassed on both sides with humidified 10% O2–5%CO2–85%N2, as low oxygen increases longevity of RPE function (Winkler and Giblin, 1983). The continuous gassing also stirred the solutions as demonstrated by adding one drop of fluorescein to the solution in a preliminary experiment. The temperature of the solutions was kept at 378C by constant circulation of heated water driven by a heating circulator (Julabo, Inc., Seelbach, Germany) through the jacketed circulation reservoirs of the Ussing chamber system (WPI, Aston, UK). The Ussing chamber was then tilted by 258 with the apical side facing upwards in order to minimise unstirred layers near the aperture of the internal cassette. We measured the shortcircuit current and the transepithelial potential (TEP) at regular intervals using a EVC-4000 voltage clamp (WPI, Aston, UK) with voltage-sensing electrodes and current passing bridges which consisted of 3 M KCl-agar. Transepithelial resistance (TER) was determined by clamping the transepithelial potential at 10 mV, recording the deflection of the short-circuit current, and applying Ohm’s law. The preparations were allowed to equilibrate. Stabilization of bioelectrical parameters was usually achieved within 20–30 min. Only tissue samples with a TERO100 Ucm2 were considered for experimentation. TEP and TER were recorded at K20, K10, 0, 10, 20, 30, 40, 50, 60, 90, and 120 min. The recordings taken during the first 20 min served as control. 1 ml of taurine in a 50 m M or 50 m M concentration was added at time point 0 min to either the apical or the basal side creating a 10 mM or 10 mM solution on one side of the preparation and 5 ml of volume on both sides. It has been shown that simple addition of taurine in a 10 mM concentration and adding 10 mM taurine while removing 5 mM NaCl lead to equivalent measurements of epithelial electrical properties (Scharschmidt et al., 1988). 2.3. Statistical analyses
2.2. Measurement of electrophysiologic parameters The isolated RPE-Bruch’s-choroid preparation was floated on hardened, ashless filter paper with a pore size of 20–25 mm (Grade 541; Whatman, Maidstone, UK). The preparation was then mounted between the two halves of a perspex insert cassette with a central 4 mm diameter aperture (Institute of Ophthalmology, London, UK). The cassette assembly was then clamped in a modified perspex Ussing chamber (WPI, Aston, UK) and both surfaces of the preparation were rinsed several times with Krebs’ medium of the following composition: (mM): NaCl 118, Glucose 6.6, NaHCO3 25, KCl 4.84, MgSO4 0.8, KH2PO4 1.2, CaCl2 1.8, 0.01% BSA (bovine serum albumin). In a part of the experiments the KCl-component of the medium was partially replaced by choline chloride to yield a solution with 2.2 mM KC on the apical side of the preparation. A 0.126 cm2 area of the tissue sample was exposed to 5 ml
Statistical analyses were undertaken using Graph Pad Prism 3.02 (Graph Pad Software, Inc., San Diego, CA, USA).
3. Results We measured TEP and TER with taurine concentrations of 10 mM and 10 mM added either to the apical side or to the basolateral side of the RPE-Bruch’s-choroid preparation. We conducted experiments with a potassium concentration of 6.04 mM on both sides and with a lower potassium concentration of 2.2 mM on the apical side (Tables 1 and 2). The addition of taurine to either the apical side or the basolateral side of the RPE-Bruch’s-choroid preparation resulted in a decrease of TEP and TER over the course of 1 hr followed by a stabilisation of TEP and TER during
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Table 1 Transepithelial potential (TEP) in experiments with 10 mM and 10 mM taurine on the apical or basolateral side of the RPE-Bruch’s-choroid preparation and 2.2 or 6 mM potassium on the apical side Transepithelial potential (TEP) (mV) (meanGSEM) Time (min)
10 mM taurine on basal side, K 6.04 mM both sides (nZ5)
10 mM taurine on apical side, K 6.04 mM both sides (nZ3)
10 mM taurine on basal side, K 6.04 mM both sides (nZ4)
10 mM taurine on apical side, K 6.04 mM both sides (nZ4)
10 mM taurine on basal side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ7)
10 mM taurine on apial side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ3)
10 mM taurine on basal side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ3)
10 mM taurine on apical side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ3)
K20 K10 0 10 20 30 40 50 60 90 120
8.9G0.43 8.7G0.29 8.9G0.33 7.68G0.64 7.4G0.62 7.14G0.76 7.2G0.9 6.7G1 6.42G1.18 5.92G1.41 5.74G1.55
7.63G1.15 7.53G1.11 7.47G1.12 5.27G1.22 4.83G1.07 4.33G1.19 4.13G1.33 3.93G1.33 3.8G1.3 3.93G1.44 4.17G1.48
7.33G0.67 7.15G0.7 7.3G0.78 6.83G0.79 5.83G0.69 5.1G0.51 4.5G0.43 4.3G0.58 3.98G0.66 3.1G0.12 3.43G0.19
7.45G0.56 7.4G0.56 7.3G0.55 5.88G0.41 5.63G0.45 5.4G0.62 5.22G0.78 5.05G0.87 4.85G0.99 5.55G0.92 6.18G0.93
6.59G0.68 6.59G0.7 6.51G0.7 5.51G0.78 4.8G0.58 4.61G0.48 4.57G0.41 4.49G0.38 4.47G0.34 4.64G0.22 5.17G0.47
4.47G0.33 4.6G0.3 4.63G0.29 4.3G0.17 5.17G0.34 6.13G0.57 6.53G0.67 6.17G0.65 5.9G0.75 6.17G0.92 5.7G0.1
7.65G0.35 7.55G0.25 7.6G0.4 5.45G0.85 4.45G0.55 4.1G0.4 3.95G0.35 3.75G0.45 3.65G0.55 3.65G0.75 3.75G0.55
6.3G1.02 6.33G1.01 6.43G1.07 5.03G0.83 4.76G1.1 4.5G0.19 4.33G1.23 4.1G1.23 3.93G1.07 4.1G1.02 5.23G1.42
the second hour of the experiments. This response was statistically significantly more pronounced for the first 10 min following the addition of taurine when taurine was added to the apical side as compared to when taurine was added to the basolateral side (Figs. 1 and 2). There was no statistically significant difference between TEP or TER when taurine was added in the 10 mM or the 10 mM concentration (Figs. 3 and 4). We pooled the data measured with the two different taurine concentrations (Figs. 1, 2, 5 and 6) and the data measured when taurine was added to the apical and the basolateral side (Figs. 3–6). TEP was statistically significantly lower before the addition of taurine and for the first 10–20 min following the addition of taurine when the potassium concentration on the apical side
was reduced from 6.04 to 2.2 mM. TER was statistically significantly lower at the time points K10 and 0 min with the lower potassium concentration on the apical side (Figs. 5 and 6).
4. Discussion In the present study, we used two different taurine concentrations in the millimolar and in the micromolar range to target both the high- an low-affinity taurine carriers previously identified in bovine RPE (Kundaiker et al., 1996) and to simulate the concentration of plasma taurine in the range of 44–62 mM in human (Hussain and Voaden, 1987;
Table 2 Transepithelial resistance (TER) in experiments with 10 mM and 10 mM taurine on the apical or basolateral side of the RPE-Bruch’s-choroid preparation and 2.2 or 6 mM potassium on the apical side Transepithelial resistance (TER) (Ucm2) (meanGSEM) Time (min)
10 mM taurine on basal side, K 6.04 mM both sides (nZ5)
10 mM taurine on apical side, K 6.04 mM both sides (nZ3)
10 mM taurine on basal side, K 6.04 mM both sides (nZ4)
10 mM taurine on apical side, K 6.04 mM both sides (nZ4)
10 mM taurine on basal side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ7)
10 mM taurine on apial side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ3)
10 mM taurine on basal side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ3)
10 mM taurine on apical side, K 2.2 mM on apical side, K 6.04 mM on basal side (nZ3)
K20 K10 0 10 20 30 40 50 60 90 120
462G42 504G51.4 462G42 399G61.2 352.8G78.1 352.8G78.1 346.8G81 332.4G86.4 315.2G92.4 281.4G93.8 277.2G92.6
399G116.9 378G126 378G126 246.7G87.4 239.3G90.5 193.7G60.8 190G62.9 181.7G66.7 181.7G66.7 216.7G101.7 216.7G101.7
420G74.3 367G30.3 420G74.3 404.3G82.8 288.8G50.3 231.8G32.5 190G25.4 190G25.4 174.5G28.3 131.7G8.3 133G12.9
362.3G90.5 351.8G95.2 362G90.6 236.3G26.3 236.3G26.3 231.8G32.6 220.8G38.4 216.3G41.1 208.3G45.4 227.5G50.5 258.3G66.7
378G79.7 332.5G68.7 339.5G66.5 273.9G62.5 226.9G34.6 211.9G21.3 198.6G15.3 196.9G12.8 190.9G9.4 204.3G11.1 234.3G23.5
190G10 180G0 172.6G7.3 190G10 238G14 350G70 350G70 280G70 280G70 294G64.2 252G0
420G0 336G84 336G84 262.5G52.5 210G0 180G0 158G0 158G0 158G0 160G20 160G20
394G236 394G236 394G236 260G87.4 246.7G87.4 234.7G93.1 228.7G95.8 228.7G95.8 228.7G95.8 228.7G95.8 315.3G157.3
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Fig. 1. Effect of taurine added to the apical side versus the basolateral side of RPE-Bruch’s-choroid on TEP. TEP was recorded for 140 min. The first 20 min served as control. Taurine was added at time 0 min to either the apical side (group A, triangles, nZ7) or the basolateral side (group B, squares, nZ9) of the RPE-Bruch’s-choroid preparation with a final concentration of 10 mM or 10 mM. The [KC]-concentration was 6 mM on both sides. There was a statistically significant difference between the measurements of group A and group B at 10 min (P!0.05). There was no statistically significant difference between all other measurements of group A and group B (PO0.05, Mann–Whitney-Test).
Trautwein and Hayes, 1990; Inoue et al., 2003) and 18–89 mM in bovine (Sakai and Nagasawa, 1992; Kelly et al., 2000), and interphotoreceptor matrix levels of 6.6 mM (Sellner, 1986) which increase to millimolar levels secondary to a large efflux of retinal taurine into the subretinal space following light stimulation (Salceda et al., 1977; Schmidt, 1978; Neal et al., 1979). Furthermore, we used two different apical potassium concentrations to simulate the light-induced reduction of the apical potassium concentration (Oakley and Green, 1976; Oakley, 1977; Steinberg et al., 1980). Exposure of either the apical or the basolateral side of the RPE to taurine lead to a decrease of TEP and TER
Fig. 2. Effect of taurine added to the apical side versus the basolateral side of RPE-Bruch’s-choroid on TER. TEP was recorded for 140 min. The first 20 min served as control. Taurine was added to either the apical side (group A, triangles, nZ7) or the basolateral side (group B, squares, nZ9) of the RPE-Bruch’s-choroid preparation with a final concentration of 10 mM or 10 mM. The [KC]-concentration was 6 mM on both sides. There was a statistically significant difference between the measurements of group A and group B at 10 min (P!0.05). There was no statistically significant difference between all other measurements of group A and group B (PO0.05, Mann–Whitney-Test).
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Fig. 3. Effect of taurine with a concentration of 10 mM versus 10 mM on TEP. TEP was recorded for 140 min. The first 20 min served as control. Taurine was added at time 0 min to the apical side or the basolateral side with a final concentration of 10 mM (group C, triangles, nZ8) or 10 mM (group D, squares, nZ8) of the RPE-Bruch’s-choroid preparation. The [KC]-concentration was 6 mM on both sides. There was no statistically significant difference between the measurements of group C and group D at any time point (PO0.05, Mann–Whitney-Test).
followed by a stabilisation. This effect was more pronounced when the the apical side was exposed to taurine (Figs. 1 and 2). This finding is principally in accordance with Kundaiker et al. who identified active high- and low-affinity taurine carriers on both the apical and the basolateral cell membranes of bovine RPE (Kundaiker et al., 1996). Our findings are partially in accordance with Scharschmidt et al. who found a depolarisation of the apical but not the basolateral membrane of frog RPE following exposure to a 10 mM taurine solution which only occurred in the presence of sodium in the medium (Scharschmidt et al., 1988). The effect of taurine on TEP and TER is probably mediated by a co-transport of positively charged sodium and taurine into the RPE (Scharschmidt et al., 1988). The mechanism of the later stabilisation of TEP and TER is not fully understood. With prolonged exposure to taurine, the
Fig. 4. Effect of taurine with a concentration of 10 mM versus 10 mM on TER. TER was recorded for 140 min. The first 20 min served as control. Taurine was added at time 0 min to the apical side or the basolateral side with a final concentration of 10 mM (group C, triangles, nZ8) or 10 mM (group D, squares, nZ8) of the RPE-Bruch’s-choroid preparation. The [KC]-concentration was 6 mM on both sides. There was no statistically significant difference between the measurements of group C and group D at any time point (PO0.05, Mann–Whitney-Test).
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Fig. 5. Effect of 2.2 versus 6.04 mM potassium on the apical side of RPEBruch’s-choroid on TEP. TEP was recorded for 140 min. The first 20 min served as control. [KC]-concentration on the apical side of the RPEBruch’s-choroid preparation was either 2.2 mM (group E, triangles, nZ15) or 6 mM (group F, squares, nZ16). [KC]-concentration on the basolateral side was 6 mM in all experiments. Experiments with taurine concentrations of 10 mM and 10 mM on the apical and the basolateral side (Tables 1 and 2) were pooled. There was a statistically significant difference between the measurements of group E and group F at K20, K10, 0, 10, and 20 min (P!0.05). There was no statistically significant difference between all other measurements of groups E and F (PO0.05) (Mann–Whitney test).
accompanying load of intracellular sodium may stimulate the electrogenic NaC/KC pump which then transports sodium and net positive charge out of the cell (Scharschmidt et al., 1988) and thereby counteracts the decrease of TEP caused by the sudden increase of the apical taurine concentration from micromolar levels to millimolar levels. By contrast, the basolateral side of the RPE is in vivo continuously exposed to a taurine concentration in the micromolar range (Hussain and Voaden, 1987; Trautwein and Hayes, 1990; Sakai and Nagasawa, 1992; Kelly et al., 2000; Inoue et al., 2003) without sudden drastic changes. The depolarising effect of
Fig. 6. Effect of 2.2 versus 6.04 mM potassium on the apical side of RPEBruch’s-choroid on TER. TER was recorded for 140 min. The first 20 min served as control. [KC]-concentration on the apical side of the RPEBruch’s-choroid preparation was either 2.2 mM (group E, triangles, nZ15) or 6 mM (group F, squares, nZ16). [KC]-concentration on the basolateral side was 6 mM in all experiments. Experiments with taurine concentrations of 10 mM and 10 mM on the apical and the basolateral side (Tables 1 and 2) were pooled. There was a statistically significant difference between the measurements of group E and group F at K10, and 0 min (P!0.05). There was no statistically significant difference between all other measurements of groups E and F (PO0.05) (Mann–Whitney test).
co-uptake of taurine and sodium at the basolateral side may be balanced by the NaC/KC pump. Furthermore, our findings suggest that the sodiummediated taurine-induced reduction of TEP and TER is a saturable mechanism as the two taurine concentrations used in the present study did not lead to a statistically significant difference in the magnitude of the reduction of TEP and TER (Figs. 3 and 4). Kundaiker et al. found that the highaffinity taurine carrier system of bovine RPE is characterised by a Michaelis–Menten constant (Km) of 23 mM and a Vmax of 86.7 pmol/5 min/4 mm disc and that it is sodiumdependent with a Hill coefficient of 2.0 indicating that two sodium ions are required for the uptake of one molecule of taurine. By contrast, the low-affinity system is characterised by a Km of 507 mM and a Vmax of 344 pmol/5 min/4 mm disc and it is heterogenous as it is only partially sensitive to sodium (Kundaiker et al., 1996). This suggests that the reduction of TEP is primarily mediated by the sodiumsensitive high-affinity system and it may explain why in the present study exposure to a millimolar taurine concentration did not lead to a greater reduction of TEP and TER as compared to a micromolar taurine concentration. We expected a significant change in the short circuit current following exposure to taurine. Although such a change was observed in some of the experiments the change was only slight. This may be due to the epithelium becoming leaky during the experiment. Further investigation is required to clarify this point. The observed changes of TEP and TER when the in vivo light-induced decrease of apical potassium was modelled (Figs. 5 and 6) are in accordance with Bialek and Miller (Bialek and Miller, 1994) who described a transient increase followed by a reduction of TEP when apical potassium was lowered from 5 to 2 mM. The reduction of TEP lasted until control solution with 5 mM potassium was returned to the apical side of the RPE. This effect was followed by a buffering net secretion of KC into the subretinal space which appeared to be electrically coupled to a basolateral increased ClK conductance (Bialek and Miller, 1994). Oakley and Steinberg described a duration of the reduced subretinal potassium concentration in response to maintained illumination of approximately 10 min after light onset in an in vitro preparation of bullfrog retina (Oakley and Steinberg, 1982). The exact duration and the magnitude of the decrease of subretinal [KC] following light onset in vivo in bovine or human is unknown. Oakley et al. have shown that in the toad retina the light-evoked decrease in [KC] in the outer plexiform layer is attenuated by the spatial buffering efflux of KC from Mu¨ller cells at that retinal depth (Oakley et al., 1992). Therefore, the results of the present in vitro study cannot be directly applied to the in vivo situation. However, they provide principal evidence that exposure to taurine and the light-induced reduction of the apical potassium concentration temporarily decrease TEP and TER. More specifically, the findings of the present study (Figs. 5 and 6) suggest that the light-induced decrease in
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subretinal [KC] further pronounces the temporary taurineinduced decrease of TEP and TER. The light-induced decrease of apical potassium together with taurine released from the retina could possibly have significant physiological functional importance by conserving retinal levels of taurine. The decrease of TEP leads to a decrease of the activity of the electrogenic taurine/sodium co-transporter of the RPE cell membrane (Scharschmidt et al., 1988) and thereby reduces the uptake of taurine from the subretinal space into the RPE. Indeed, it has been shown that a lowered apical [KC] is associated with reduced taurine uptake (Kundaiker et al., 1996) and reduced transport across the RPE (Miller and Steinberg, 1979; Hillenkamp et al., 2004). It can be hypothesised that taurine released into the subretinal space with the onset of light remains longer in position secondary to the decrease of TEP following the onset of light. Furthermore, reduced transport across the RPE would allow the outer retina to re-uptake released taurine and limit the loss to the blood stream via the RPE. As taurine has been shown to protect photoreceptor outer segment cell membranes against light-induced oxidative damage (Pasantes-Morales and Cruz, 1984; Wright et al., 1986; Keys and Zimmerman, 1999), this may be a physiological protective mechanism. This hypothesis is supported by recent work which showed that light deprivation slows retinal degeneration in taurine transporter knock-out mice (Rascher et al., 2004). In summary, our data suggest that taurine and apical potassium may synergistically act to keep taurine as a protection of photoreceptor outer segments against oxidative damage and osmotic imbalance longer in position in the subretinal space following the transition from dark to light.
Acknowledgements J.H. was supported by Deutsche Forschungsgemeinschaft (DFG) grant Hi 758/1-1. A.A.H. was supported by The PPP Foundation, UK. T.L.J. was supported by Special Trustees of Guys and St Thomas’ Hospital and The Allerton Trust.
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