Compartmental Analysis of Taurine Transport to the Outer Retina in the Bovine Eye

Abstract

purpose. To assess the relative resistance presented individually by Bruch’s membrane-choroid (BC) and the retinal pigment epithelium (RPE) to movement of taurine between the choroidal circulation and the outer retina. To quantify the effect of light-evoked changes in subretinal potassium concentration on the transepithelial transport of taurine across bovine RPE.

methods. Transport studies were performed in Ussing chambers with intact and RPE-denuded specimens of BC. RPE viability was monitored by recording transepithelial potential (TEP) and transepithelial resistance (TER). Taurine transport with substrate concentrations in the micro- and millimolar range, reflecting physiological taurine concentrations in plasma, retina, and subretinal space was quantified by high-performance liquid chromatography (HPLC) and radiotracer techniques. Taurine transport was also assessed after apical potassium concentration was lowered from 6.0 to 2.2 mM to mimic the effects of light.

results. Transport of taurine across RPE-BC at a 10-mM substrate concentration increased from 32.92 before to 111.72 nanomoles/4 mm per hour after removal of the RPE. Similarly, at 50 μM taurine, transport rates increased from 0.158 to 0.439 nanomoles/4 mm per hour after removal of the RPE. At both high (10 mM) and low (50 μM) substrate concentrations, lowering of apical potassium was associated with decreased transport of taurine across the RPE. For taurine concentrations greater than 42 μM, the rate-limiting compartment for transport of taurine to the outer retina was the RPE monolayer. Similar rates were observed across each compartment for concentrations <42 μM.

conclusions. The magnitude and directionality of taurine transport across the RPE is determined solely by the driving taurine concentration gradient and is modulated by subretinal levels of potassium. Such modulation may provide a mechanism for conserving retinal taurine. Processes that increase the resistance to diffusion across Bruch’s membrane such as human ageing and increased thickening and deposition of debris associated with age-related macular degeneration (AMD) are likely to affect transport across the RPE, culminating in a secondary retinal taurine deficiency.

The biological importance of taurine in the retina has been recognized ever since the original observation that taurine deficiency in cats results in retinal degeneration and blindness. ¹ 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. ² Taurine is the major constituent of the free amino acid pool in photoreceptor cells, reaching intracellular concentrations of 60 to 80 mM, ^{3 4} and it is essential for their maintenance. ⁵ Several roles of taurine in the retina have been suggested, including antioxidation, ^{5 6 7} membrane stabilization, ^{5 6 7} and osmoregulation. ^{5 8}  

In humans, pathologic conditions, such as bowel resection, compromise the intake of dietary taurine and lead to visual defects. ⁹ Human infants and children maintained on taurine-free total parenteral nutrition show abnormalities in the electroretinogram, and fundus changes first appear as mild granularity of the retinal pigment epithelium (RPE). ¹⁰ Although a precise physiological role of taurine in the retina has yet to be established, several studies have suggested the possible involvement of abnormal taurine homeostasis in the pathogenesis of certain retinal degenerations, including diabetic retinopathy and macular edema. ^{2 8 10 11} Because of its antioxidant properties, a possible involvement of taurine deficiency in the pathogenesis of age-related retinal degeneration has been suggested. ¹²  

In the macular region of the human fundus, maintenance of photoreceptor function and viability are inherently dependent on an adequate transport capacity for nutrients between the outer retina and the choroidal circulation. ^{13 14} The delivery pathway for taurine from the choroidal circulation to the photoreceptors encompasses the acellular Bruch’s membrane with the choroid and the cellular RPE as two serially connected compartments housing predominantly passive (free-diffusion) ¹⁵ and active (carrier-mediated) ^{3 4 16} transport pathways, respectively. An understanding of the relative contribution of Bruch’s membrane-choroid and the RPE to movement of solutes between the choroidal blood supply and the outer retina is therefore essential for understanding physiological and pathologic effects on the transport capacity of the intact system. 

There has been some controversy regarding the actual direction of taurine transport, as in vivo studies using intravascular injections of the radiolabeled amino acid demonstrated an initial rapid accumulation into the RPE followed by slower transfer to the retina. ¹⁷ In contrast, most in vitro attempts to establish the direction of taurine transport have shown net movement from the retina to the choroidal circulation. ¹⁸  

Our group has previously characterized both apical and basolateral high- and low-affinity taurine carriers in bovine RPE-Bruch’s choroid preparations and the kinetic parameters were used to construct a mathematical working model for the vectorial translocation of taurine across the RPE. ¹⁹ The model predicts that the magnitude and direction of taurine movement across the RPE is determined primarily by the apical and basolateral resting concentrations of taurine and the extracellular potassium concentration in the apical compartment. ¹⁹ The model did not, however, address the role of Bruch’s membrane in the transport process, a factor crucial to an understanding of abnormal transport homeostasis in the differential compartmental ageing scenario associated with human diseases, such as age-related macular degeneration. 

The present study was therefore designed to (1) assess the individual resistance of Bruch’s-choroid and the RPE to overall transport of taurine across the intact complex, (2) to quantify the effects of changes in the concentration of taurine in apical–basal compartments on the magnitude and directionality of transport, and (3) to evaluate the modulatory role of in vivo light-evoked changes in subretinal potassium concentration on the transport pathway. 

Materials and Methods

Preparation of Tissue for Transport Experiments

Fresh bovine eyes of Friesian cows aged 18 to 24 months were obtained from a local abattoir, and experiments were initiated within 6 hours 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-Aldrich, 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, discarded. The neural retina was then gently peeled away from the underlying RPE and cut at the optic nerve head. All samples for experimentation were obtained from the same midperipheral nontapetal fundus area close to the optic disc with an 8-mm trephine (Stiefel Laboratories, Buckinghamshire, UK). The RPE-Bruch’s membrane-choroid complex was then gently teased away from the sclera and floated onto hardened, ashless filter paper with a pore size of 20 to 25 μm (Grade 541; Whatman). The preparation was then mounted between the two halves of a Perspex insert cassette with a central 4-mm diameter hole (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, and 0.01% BSA (bovine serum albumin). In some experiments, the amount of potassium chloride was altered to give a Krebs’ medium of lowered potassium concentration. 

Each half compartment of the Ussing chamber contained 5 mL Krebs’ medium, gassed with humidified 10% O₂, 5% CO₂, and 85% N₂, because low oxygen increases the longevity of RPE function. ²⁰ 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 37°C 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). The Ussing chamber was then tilted by 25° with the apical side facing upward to minimize unstirred layers near the aperture of the internal cassette. RPE cell viability was monitored by measuring the short-circuit current and the transepithelial potential (TEP) at 10-minute intervals using a voltage clamp (EVC-4000; WPI) with voltage-sensing electrodes and current-passing bridges that 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 preparation was then allowed to equilibrate, and stabilization of bioelectrical parameters was usually achieved within 20 to 30 minutes of incubation. 

Transport Studies

Only tissue samples with a TER >100 Ω/cm² were considered for experimentation. Vectorial transport studies were conducted at two substrate concentrations of 10 mM and 50 μM, the former representing saturating conditions and the latter indicative of the physiological levels of plasma taurine. Taurine was added after equilibration to either the apical or the basal side of the preparation, resulting in a final concentration of 10 mM or 50 μM. The lower concentration incorporated a trace of tritiated taurine (49.9 μM cold taurine + 0.1 μM ³H-taurine). At timed intervals of 30, 60, 90, and 120 minutes, a 60-μL aliquot was withdrawn from each half chamber for quantification of taurine. 

The internal tissue insert cassette was then removed after the transport experiment and the RPE was gently brushed away from the underlying Bruch’s membrane using a fine sable-hair brush. After confirmation of the integrity of Bruch’s membrane, the same transport experiment as described earlier was repeated, measuring diffusion across the denuded Bruch’s membrane-choroid preparation. 

For further scrutiny of transport in the physiological micromolar concentration range, uptake of ³H-taurine across the basolateral surface of the RPE was determined at substrate concentrations of 1, 10, and 60 μM. Intact RPE-Bruch’s-choroid samples (three to six eyes per concentration) were mounted in Ussing chambers, and, after equilibration, taurine-containing and taurine-free solutions were added to basal and apical compartments, respectively. After a 5-minute incubation period, solutions were rapidly withdrawn and compartments filled with ice-cold taurine-free Krebs’ medium. After several washes, the chamber was dismantled and a 4-mm tissue disc trephined out and processed for scintillation counting. In some experiments, the RPE layer was brushed off and the residual counts in the remaining Bruch’s-choroid complex determined. This allowed a correction for nonspecific binding and uptake by choroidal cells. Incubations times were kept at a minimum to avoid complications from release of accumulated label by the RPE. These studies were supplemented by obtaining a diffusional transport profile across the isolated Bruch’s membrane-choroid complex in the concentration range of 1 to 100 μM taurine. 

In experiments with 10 mM taurine, the amount crossing the preparation was determined by high-performance liquid chromatography (HPLC) as described previously. ²¹ Briefly, samples (25 μL) were derivatized for 1 minute with o-phthalaldehyde (OPA; 50 μL) and then injected with an autosampler onto a column (25 cm × 4.6 mm; ODS2 Spherisorb; Waters, Milford, MA). Amino acids were eluted with a gradient generated with a pump (PU980; Jasco, Inc., Great Dunmow, UK) and gradient unit (PU980-02; Jasco, Inc.). The flow rate was 1 mL/min. The fluorescence detector was set at an excitation of 360 nm and emission of 430 nm. Results were analyzed with Borwin chromatography software (Jasco, Inc.). In experiments with substrate concentrations of taurine in the micromolar range, the more rapid HPLC detection method was not sufficiently sensitive and therefore radiolabel methods were used. With radiolabel experiments, the removed aliquot was added to scintillant (UltimaGold; Packard Instruments, Meriden, CT) and the amount of taurine determined by liquid scintillation spectrometry (Wallac 1409; LKB, Gaithersburg, MD). 

Assessment of Tissue Integrity

We carefully brushed the RPE away from the underlying Bruch’s membrane with a fine sable-hair brush. This method has been shown to preserve the integrity of Bruch’s membrane, as assessed by scanning electron microscopy. ¹⁵ To ascertain the integrity of Bruch’s membrane after this procedure, we devised an osmotic method to exclude destruction of the exclusion limit of Bruch’s membrane, previously determined to be 66 ± 10 kDa (Hussain AA, et al. IOVS 1999;40:ARVO Abstract 4852). Briefly, a 0.412-mM solution of a 162-kDa dextran (Sigma-Aldrich) constituted in PBS was placed in one half of the chamber, and an equal volume of PBS in the other, both solutions initially reaching the same height in the reservoir columns of both compartments. In an intact preparation, the compartment containing the impermeable dextran solution would exert an osmotic pressure, drawing fluid into it from the other compartment, leading to a discrepancy in the heights of the two solutions in the reservoir columns. Tissue damage, on the contrary, would lead to mixing of the dextran between the two compartments, thereby abolishing the osmotic response. Thus, integrity of preparations was assessed after an incubation of 3 hours and, if suitable, rinsed several times with PBS over the next 3 hours and then used in the experimental studies. 

Statistical Analyses

Statistical significance analyses were undertaken on computer (Fig.P Software Co., Durham, NC). Sampling theory of regression was used to assess the statistical significance between two regression lines. ²²  

Results

Transport of Taurine across the Intact and RPE-Denuded Bruch’s Membrane–Choroid Complex

Taurine Concentration Gradient of 10 mM.

In the presence of 10 mM taurine in the choroid-facing chamber of the Ussing assembly, the entry of this amino acid in the opposite chamber showed linearity of accumulation up to the 2 hours of incubation examined for both the intact and RPE-denuded complex. Across the intact RPE-Bruch’s-choroid complex, taurine was transported at a rate of 31.24 nanomoles/4-mm disc per hour in the choroid-to-retina direction. A repetition of the experiment after removal of the RPE layer resulted in transport rates across the isolated Bruch’s-choroid preparation of 112.7 nanomoles/4mm disc per hour, an increase by a factor of 3.61 (P < 0.005; n = 9; Fig. 1A ). 

A reversal in the driving concentration gradient resulted in taurine transport across the RPE-Bruch’s-choroid complex in the retina-to-choroid direction at a rate of 34.6 nanomoles/4mm disc per hour (Fig. 1B) . Thus at saturating concentrations of taurine, there was no discriminatory effect on the magnitude of transport in either direction across the RPE-Bruch’s-choroid complex. Again, removal of the RPE resulted in increased transport of taurine across the isolated Bruch’s-choroid complex in the retina-to-choroid direction of 110.74 nanomoles/4-mm disc per hour, a value 3.2 times higher than for the RPE-containing complex (P < 0.005; n = 7; Fig. 1B ). 

Taurine Concentration Gradient of 50 μM.

At a concentration gradient of 50 μM across the preparation, taurine was transported across the intact RPE-Bruch’s-choroid complex at an average rate of 0.158 nanomoles/4-mm disc per hour. Removal of the RPE layer resulted in an increased flux of 0.439 nanomoles/4-mm disc per hour across the isolated Bruch’s-choroid complex (Fig. 2) . 

Comparative Transport Rates at Physiological Concentrations of Taurine

The diffusional flux of taurine across the isolated RPE-denuded Bruch’s-choroid complex was determined over a substrate concentration range of 0 to 100 μM using the radiochemical techniques described earlier (Table 1 , Fig. 3 ). 

The RPE uptake of taurine across its basal aspects was determined at choroidal substrate concentrations of 1, 10, and 60 μM, also by using radiochemical techniques (Table 2 , Fig. 3 ). 

Superimposed on these data is the theoretical prediction of transport across the basal surface of the RPE according to the mathematical model of Kundaiker et al. ¹⁹ (Fig. 3 , dashed line). 

Results show that below a critical taurine concentration of approximately 42 μM, the relative rates of transport across Bruch’s-choroid and uptake into the RPE cell are similar. At higher concentrations, uptake by the RPE becomes limiting as the carriers become progressively saturated. 

K⁺-Modulation of Taurine Transport across Bovine RPE-Bruch’s-Choroid

Lowering apical K⁺ caused a highly significant reduction (P < 0.005) in the transport of taurine in both directions across the RPE complex at a 10 mM substrate concentration (Fig. 4) . Thus, in the choroid-to-retina pathway, basal transport rates (with 6.04 mM K⁺ in each half-compartment) were reduced from 33.74 to 26.18 nanomoles/4-mm disc per hour in samples incubated with lowered K⁺ on the apical side (Fig. 4A) . The retina-to-choroid pathway was much more severely affected, with a reduction from 37.26 to 19.0 nanomoles/4-mm disc per hour in a lowered apical K⁺ environment (Fig. 4B) . 

The effect of potassium modulation on taurine transport in the choroid-to-retina direction at physiological plasma concentrations was also investigated. Lowered apical K⁺ was again associated with a highly significant reduction in the transport of taurine across the RPE complex from 0.158 to 0.099 nanomoles/4-mm disc per hour (P < 0.005; Fig. 5 ). 

Discussion

Taurine transport, in either direction, between the choroidal blood supply and the outer retina is regulated by the intervening presence of the RPE-Bruch’s membrane compartment. ^{17 18 19 23} Taurine released from the fenestrated choriocapillaries encounters Bruch’s membrane as the first passive resistance barrier on its transit to the retina. ²³ In in vitro studies with Ussing chambers, the entire Bruch’s-choroid is a barrier, and, although evidence exists that the choroidal contribution is minimal, the artificial experimental setup must be considered in the interpretation of transport data. ²³  

Diffusional processes determine the rate of taurine transport across the compartment of Bruch’s-choroid and as a consequence of Fick’s first law of diffusion (F = D · dC/dx), where F = taurine flux, D = diffusional coefficient, dC/dx = taurine gradient across Bruch’s choroid. Flux is directly proportional to the driving concentration gradient. ^{23 24}  

Transport through the RPE has been shown to be carrier mediated, ^{18 19} and the rate of translocation is a nonlinear function of the substrate concentration and is described by the appropriate Michaelis-Menten equations for transporters on apical and basal surfaces of the RPE cell. ¹⁹ Thus, the difference in rate of transport between Bruch’s-choroid and the RPE in the intact complex is expected to be dependent on the driving taurine concentration gradient. 

In the present experimental study, transport across the complex was assessed at micromolar and at millimolar taurine concentration gradients. Micromolar levels reflect a plasma concentration in human of 44 to 62 μM ^{25 26 27} and in bovine of 18 to 89 μM, ^{28 29} on the basal aspects of the RPE and assuming apical levels to be similar to the 6.6 μM in cerebrospinal fluid. ³⁰ The need to examine transport at higher, millimolar concentrations of taurine arose from the consideration that the release of taurine by photoreceptor outer segments during a photoresponse into the small interphotoreceptor compartment could easily raise levels in the apical-facing RPE compartment to millimolar levels, ^{31 32 33} and the presence of low-affinity carriers on the apical surface of the RPE with Michaelis-Menten constants (K _m) in the millimolar range ¹⁹ could well serve to sequester rapidly the high levels of taurine released into the interphotoreceptor matrix. 

At a transcomplex taurine concentration gradient of 10 mM, the capacity for transport by bovine Bruch’s-choroid was nearly three times in excess of that across the RPE. Despite the presence and varied distribution of high- and low-affinity carriers on apical and basal surfaces of the RPE, ¹⁹ the direction and magnitude of net transport was only dependent on the driving concentration gradient of taurine across the complex (Figs. 1A 1B) . Similarly, at a mean plasma taurine concentration of 50 μM, the RPE was the rate-limiting step in the vectorial delivery of taurine (Fig. 2) . The linearity in transport over the 120 minutes of incubation shows that the initially established taurine concentration gradient was maintained throughout the experiment. Thus, the amount traversing the preparation was negligible compared with the amount in the initial taurine-containing half chamber. Similarly, the taurine transported out was diluted sufficiently in the chamber so that the concentration gradient was not perturbed significantly during the experiment. 

Furthermore, we found a temporary decline of TEP and TER after the addition of taurine, which was followed by a recovery to near baseline. There was a greater decline of TEP and TER when taurine was added to the apical side of the preparation compared with when taurine was added to the choroidal side of the preparation. This is principally in accordance with the findings of others ^{34 35} and our own previous results ¹⁹ describing a sodium-taurine cotransport uptake mechanism that brings positively charged sodium ions into the cell and thereby causes a decrease in TEP. However, Scharschmidt et al. ³⁵ reported a temporary decline of TEP and TER only when taurine was added to the apical side of their preparation of frog tissue, whereas we found a temporary decline of TEP and TER when taurine was added to either side of the preparation in most of our experiments; however, this effect showed a tendency to be more pronounced when taurine was added to the apical side (Figs. 1 4 ; Hillenkamp J, et al., unpublished data, 2002). 

Further scrutiny of the transport process was undertaken at micromolar levels of taurine by comparing the diffusional flux across the isolated Bruch’s-choroid complex with rate of accumulation of taurine across the basolateral surface of the RPE (Fig. 3) . For taurine concentrations <42 μM, transport rates across Bruch’s-choroid and the basal surface of the RPE were similar. With taurine levels higher than 42 μM, uptake by the RPE was considerably diminished (i.e., taurine was delivered at a faster rate across Bruch’s-choroid than it was taken up by the RPE). This level also has an important bearing on the mechanism of transport across the RPE. For example, the rate of uptake of taurine at 60 μM by the basolateral surface of the RPE was determined as 34.9 ± 2.3 picomoles/4 mm per 5 minutes. However, net transport across the RPE at 50 μM taurine (calculated from Fig. 2 ) was only 13.2 picomoles/4 mm per 5 minutes. Hence, most of the taurine taken across the basolateral surface is stored in the RPE and/or released back onto Bruch’s membrane. The efflux mechanism is supported by the presence of a low-affinity, sodium-independent carrier on the basal surface of the RPE cell. ¹⁹  

The second component of the present study assessed the effects of apical potassium on vectorial transport of taurine across the RPE. In vivo, light causes a reduction in the concentration of potassium in the interphotoreceptor matrix surrounding the apical membrane of the RPE cell. ^{36 37 38} The light-mediated alteration in K⁺ was simulated in vitro by lowering the concentration of apical K⁺ from 6.04 to 2.2 mM, and the effect of such a perturbation on taurine transport across intact RPE-Bruch’s-choroid complex was investigated. We first used a high taurine concentration (10 mM) in the transport analysis so that both high- and low-affinity carriers remained saturated, allowing maximal transport rates across the RPE. Irrespective of the direction of transport, by lowering apical potassium from 6.04 to 2.2 mM to mimic the effect of light, taurine transport across the RPE was reduced, with particularly marked effects in the retina-to-choroid direction (Fig. 4B) . Also at a physiological substrate concentration of 50 μM in the basolateral compartment, transport of taurine across the RPE was reduced after a reduction in apical potassium (Fig. 5) . Lowering apical potassium also resulted in a reduction of TEP and TER in most of our experiments (Figs. 4 5) . This is in accordance with Bialek and Miller, ³⁹ who found that a decrease in apical potassium induces a short initial increase followed by a longer decrease of TEP followed by a decline in intracellular potassium in the RPE of 19 mM, secondary to a net secretion of potassium into the subretinal space buffering the light-evoked decrease of subretinal potassium. This mechanism is possibly involved in the regulation of the hydration and chemical composition of the subretinal space after transitions between light and dark. ³⁹ Our results suggest that the flux of potassium and taurine between the outer retina, the subretinal space, and the RPE are linked and that both substances may play a role in the regulation of subretinal space volume. 

The action of potassium may be essential for conserving retinal levels of taurine. In the dark-adapted state, with plasma taurine in the range of 44 to 62 μM ^{25 26 27} in human and 18 to 89 μM ^{28 29} in bovine and interphotoreceptor matrix levels of 6.6 μM in bullfrog, ³⁰ taurine would be transported from the blood to photoreceptor cells. After light stimulation, the decrease in apical potassium and reduced taurine transport across the RPE would mean that the large efflux of retinal taurine into the apical compartment ^{31 32 33} remains in a position to protect outer segments, as taurine has been shown to protect photoreceptor cell membranes against oxidative damage. ^{6 7} This hypothesis is supported by recent work that showed that light deprivation slows retinal degeneration in taurine transporter knockout mice. ⁴⁰ Furthermore, reduced transport by the RPE would allow receptors to reuptake released taurine and limit the loss to the blood stream through the RPE. 

In summary, our data suggest that in the bovine eye, at taurine concentrations greater than 42 μM, the diffusional capacity of taurine across Bruch’s membrane well exceeds the active carrier-mediated transport capability of the RPE. The RPE is the rate-limiting step in the system of delivery of taurine to photoreceptor cells. At plasma taurine concentrations below 42 μM, transport rates across Bruch’s-choroid and RPE were similar. Furthermore, the decrease in taurine transport after a reduction in apical potassium may provide an important mechanism for conserving retinal stores of taurine. The information derived from this study is relevant to future evaluation of the effect of age on transport of taurine across Bruch’s-choroid and across the RPE and its effect on transport across the intact complex. Our findings may have a bearing on the delivery of taurine to the ageing human retina and on retinal diseases, such as age-related macular degeneration. Ageing is known to diminish the diffusional capacity of Bruch’s membrane, ¹⁵ and the exaggerated situation in age-related macular degeneration may predispose these patients to a secondary taurine deficiency. 

 Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, April 2004.

 Supported by Deutsche Forschungsgemeinschaft Grant Hi 758/1-1 (JH), the PPP Foundation and the Iris Fund (AAH), the Special Trustees of Guys and St. Thomas’ Hospital and The Allerton Trust, United Kingdom (TLJ), and the British Retinitis Pigmentosa Society (PAC).

 Submitted for publication May 31, 2004; revised July 18, 2004; accepted July 25, 2004.

 Disclosure: J. Hillenkamp, None; A.A. Hussain, None; T.L. Jackson, None; P.A. Constable, None; J.R. Cunningham, None; J. Marshall, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

(A) Transport of taurine across intact and RPE-denuded preparations of bovine RPE-Bruch’s-choroid at a 10-mM substrate concentration. Intact RPE-Bruch’s-choroid preparations were mounted in modified Ussing chambers and, after assessment of electrical stability, transport studies were initiated by addition of taurine to the choroid-facing compartment. Aliquots were removed at timed intervals, and the amount of taurine crossing the preparation was quantified by HPLC. The preparation was then denuded of RPE, and transport through the isolated Bruch’s-choroid was quantified. Thus, taurine transport at a concentration gradient of 10 mM for RPE and RPE-denuded preparations was determined as 31.24 and 112.7 nanomoles/4-mm disc per hour, respectively. TEP and TER were recorded at 10, 20, 30, 40, 50, 60, 90, and 120 minutes: mean TEP, 5.9 ± 0.31 (SEM); mean TER 283 ± 20.2 (SEM; n = 9). (B) Experiment conducted as in (A), except that taurine was added to the RPE-facing compartment. Taurine transport at a concentration gradient of 10 mM for RPE and RPE-denuded preparations was determined as 34.6 and 110.74 nanomoles/4-mm disc per hour, respectively. TEP and TER, recorded at the time points shown in (A): mean TEP 5.0 ± 0.15 (SEM); mean TER 220 ± 6.29 (SEM; n = 7).

Figure 2.

 

Transport of taurine across intact and RPE-denuded preparations of bovine RPE-Bruch’s-choroid at a 50-μM substrate concentration. Intact RPE-Bruch’s-choroid preparations were mounted in modified Ussing chambers and after assessment of electrical stability, transport studies were initiated by addition of taurine with a trace of tritiated taurine (49.9 μM cold taurine+0.1 μM ³H-taurine; final concentration, 50 μM) to the choroid-facing compartment. Aliquots were removed at timed intervals, and the amount of taurine crossing the preparation was quantified by liquid scintillation spectrometry. The preparation was then denuded of RPE, and transport through the isolated Bruch’s-choroid was quantified. Thus, taurine transport at a concentration gradient of 50 μM for RPE and RPE-denuded preparations was determined as 0.158 and 0.439 nanomoles/4-mm disc per hour, respectively. TEP and TER were recorded at 10, 20, 30, 40, 50, 60, 90, and 120 minutes: mean TEP, 7.4 ± 0.17 (SEM); mean TER, 196 ± 12.42 (SEM; n = 5).

Table 1.

 

Diffusion of Taurine across Bovine Bruch’s-Choroid

Taurine Substrate Concentration (μM)	Taurine Transport (picomoles/4 mm per 5 minutes)	SD
9.8	6.18	2.17
28.1	21.91	2.48
50	38.86	1.86
84.4	65.53	3.09
100	77.6	3.4

 The diffusional flux of taurine across the isolated RPE-denuded Bruch’s-choroid complex was determined over a substrate concentration range of 0–100 μM, using radiochemical techniques.

Figure 3.

 

Comparative analysis of taurine transport by bovine RPE and Bruch’s-choroid as a function of substrate concentration. The diffusional flux of taurine across the isolated RPE-denuded Bruch’s-choroid complex was determined over a substrate concentration range of 0 to 100 μM (Table 1) . RPE uptake of taurine across its basal aspects was determined at choroidal substrate concentrations of 1, 10, and 60 μM (Table 2) . Superimposed on these data is the theoretical prediction of transport across the basal surface of the RPE as calculated from the kinetic parameters of the high-affinity, Na⁺-dependent basal carrier of the RPE, according to the mathematical model of Kundaiker et al. ¹⁹ (dashed line): v = [V _max (S)]/[K _m + (S)], where v is picomoles per 4 millimeters per 5 minutes; (S) is taurine, (in micromolar); K _m is 29 μM; and V _max is 54.7 picomoles per 4 millimeters per 5 minutes.

Table 2.

 

Uptake of Taurine in Bovine RPE

Taurine Substrate Concentration (μM)	Taurine Uptake (picomoles/4 mm per 5 minutes)	SD
1	2.66	0.6
10	8.35	2.2
60	34.9	2.3

 RPE uptake of taurine across its basal aspects was determined at choroidal substrate concentrations of 1, 10, and 60 μM, using the radiochemical techniques.

Figure 4.

 

(A) K⁺ modulation of taurine transport across bovine RPE-Bruch’s-choroid in the choroid-to-retina direction at a 10-mM substrate concentration. Intact RPE-Bruch’s-choroid preparations were mounted in modified Ussing chambers and after assessment of electrical stability, transport studies were initiated by addition of taurine to the choroid-facing compartment. Aliquots were removed at timed intervals, and the amount of taurine crossing the preparation was quantified by HPLC. The light-mediated alteration in subretinal K⁺ was simulated in vitro by lowering the concentration of apical K⁺ from 6.04 to 2.2 mM. Basal transport rates (with 6.04 mM K⁺ in each compartment) were significantly reduced (from 33.74 to 26.18 nanomoles/4-mm disc per hour) in samples incubated with lowered K⁺ on the apical side (P < 0.005). TEP and TER were recorded at 10, 20, 30, 40, 50, 60, 90, and 120 minutes. Basal K⁺ of 6.04 mM: mean TEP, 6.8 ± 0.25 (SEM); mean TER, 332 ± 14.28 (SEM; n = 5). Low apical K⁺ of 2.2 mM: mean TEP, 4.4 ± 0.34 (SEM); mean TER, 215 ± 15.28 (SEM; n = 5). (B) K⁺-modulation of taurine transport across bovine RPE-Bruch’s-choroid in the retina-to-choroid direction at 10 mM substrate concentration. The experiments were conducted as in (A), except that the taurine was added to the RPE-facing compartment. Basal transport rates (with 6.04 mM K⁺ in each compartment) were significantly reduced (from 37.26 to 19.0 nanomoles/4-mm disc per hour) in samples incubated with lowered K⁺ on the apical side (P < 0.005). TEP and TER were recorded at 10, 20, 30, 40, 50, 60, 90, and 120 minutes. Basal K⁺ of 6.04 mM: mean TEP, 5.5 ± 0.15 (SEM); mean TER, 229 ± 5.4 (SEM; n = 4). Low apical K⁺ of 2.2 mM: mean TEP, 4.5 ± 0.17 (SEM); mean TER, 254 ± 14.7 (SEM; n = 4).

Figure 5.

 

K ⁺ modulation of taurine transport across bovine RPE-Bruch’s-choroid in the choroid-to-retina direction at a 50-μM substrate concentration. Intact RPE-Bruch’s-choroid preparations were mounted in modified Ussing chambers and after assessment of electrical stability, transport studies were initiated by addition of taurine with a trace of tritiated taurine (49.9 μM cold taurine+0.1 μM ³H-taurine, final concentration 50 μM) to the choroid-facing compartment. Aliquots were removed at timed intervals, and the amount of taurine crossing the preparation was quantified by liquid scintillation spectrometry. The light-mediated alteration in subretinal K⁺ was simulated in vitro by lowering the concentration of apical K⁺ from 6.04 to 2.2 mM. Basal transport rates (with 6.04 mM K⁺ in each compartment) were significantly reduced (from 0.158 to 0.099 nanomoles/4-mm disc per hour) in samples incubated with lowered K⁺ on the apical side (P < 0.005). TEP and TER were recorded at 10, 20, 30, 40, 50, 60, 90, and 120 minutes. Basal K⁺ 6.04 mM: mean TEP, 7.4 ± 0.17 (SEM); mean TER, 196 ± 12.42 (SEM; n = 5). Low apical K⁺ 2.2 mM: mean TEP, 3.4 ± 0.17 (SEM); mean TER, 122 ± 5.0 (SEM; n = 5).