December 2004
Volume 45, Issue 12
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Retina  |   December 2004
Taurine Uptake by Human Retinal Pigment Epithelium: Implications for the Transport of Small Solutes between the Choroid and the Outer Retina
Author Affiliations
  • Jost Hillenkamp
    From the Departments of Ophthalmology and
  • Ali A. Hussain
    From the Departments of Ophthalmology and
  • Timothy L. Jackson
    From the Departments of Ophthalmology and
  • Joanna R. Cunningham
    Pharmacology, The Rayne Institute, St. Thomas’ Hospital, London, United Kingdom.
  • John Marshall
    From the Departments of Ophthalmology and
Investigative Ophthalmology & Visual Science December 2004, Vol.45, 4529-4534. doi:10.1167/iovs.04-0919
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      Jost Hillenkamp, Ali A. Hussain, Timothy L. Jackson, Joanna R. Cunningham, John Marshall; Taurine Uptake by Human Retinal Pigment Epithelium: Implications for the Transport of Small Solutes between the Choroid and the Outer Retina. Invest. Ophthalmol. Vis. Sci. 2004;45(12):4529-4534. doi: 10.1167/iovs.04-0919.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To characterize the Michaelis-Menten kinetics of the taurine transporter (TT) in retinal pigment epithelium (RPE) freshly isolated from human donor eyes. To identify the rate limiting compartment in the pathway of taurine delivery from the choroidal blood supply to the outer retina composed by Bruch’s-choroid (BC) and the RPE in the human older age group.

methods. In human donor samples (4 melanoma-affected eyes, and 14 control eyes; age range, 62–93 years), radiochemical techniques were used to determine the RPE taurine accumulation at various exogenous concentrations. The transport capability of human RPE was obtained from a kinetic analysis of the high-affinity carrier over a substrate concentration of 1 to 60 μM taurine.

results. Uptake of taurine into human RPE at a taurine concentration of 1 μM was independent of donor age (P > 0.05) and averaged at 2.83 ± 0.27 (SEM) pmol/10 minutes per 6-mm trephine. Taurine transport by human RPE was mediated by a high-affinity carrier of K m 50 μM and V max of 267 pmol/10 minutes per 5-mm disc.

conclusions. In human donor RPE, uptake of taurine remained viable in the age range 62 to 93 years. Taurine transport rates in the RPE were lower than across the isolated BC complex, and thus the data suggest that the former compartment houses the rate-limiting step in the delivery of taurine to the outer retina.

Retinal photoreceptors sustain one of the highest rates of oxidative metabolism of any tissue in the body and, together with the continuous recycling of their outer segment proportions, require an uninterrupted and rapid supply of blood-borne metabolites. 1 2 Nutritional support is provided primarily by the choroidal circulation. The transport pathway between the choroidal capillaries and the outer retina comprises two serially connected compartments, the acellular Bruch’s membrane and the monolayer of retinal pigment epithelial (RPE) cells, the site of the outer blood–retinal barrier. Blood constituents released from the fenestrated capillaries of the choroid diffuse freely across Bruch’s membrane, provided that they are smaller than the exclusion limit of the membrane (Hussain AA, et al. IOVS 1999;40:ARVO Abstract 4852). 3 They may then diffuse across the RPE monolayer or are taken up at its basal surface by a battery of active and passive carriers for final delivery to photoreceptor cells. 4 5 6 7 8  
Our previous work with bovine eyes has identified the RPE as the rate-limiting step in taurine transport in the complex composed of Bruch’s-choroid and the RPE. These data were derived from young cows, a model devoid of gross ageing changes (Hillenkamp J, et al. IOVS 2004;45:ARVO E-Abstract 1091). 9  
The present investigation was designed to assess which of the two compartments in the transport pathway between the choroidal capillaries and photoreceptors is the rate-limiting step for delivery of small solutes in the older age group in humans. 
Taurine was chosen as the test substance for several reasons. First, it is taken up by the RPE cell by a sodium-dependent, high-affinity carrier, and hence this analysis would also provide an assessment of the functional status of the ageing cell. 8 Second, taurine is the major constituent of the free amino acid pool in photoreceptor cells and serves several functions including its role as an antioxidant, membrane stabilizer, and an osmoregulator. 10 11 12 Third, because a dietary deficiency in cats results in retinal degeneration, 13 and a similar deficiency in humans, because of pathologic conditions such as bowel resection or total taurine-free parenteral nutrition, can lead to visual defects. 14 15  
In our previous work, fresh bovine eyes maintained viability for several hours as assessed by measurement of transepithelial potential and resistance, and, as such, taurine flux measurements were obtained in a modified Ussing chamber initially with the RPE-Bruch’s-choroid complex followed by denudement of the RPE layer and a repeat estimation of flux in the isolated Bruch’s-choroid complex (Hillenkamp J, et al. IOVS 2004;45:ARVO E-Abstract 1091). 9 Similar experiments could not be undertaken in the present study with human donor eyes because a large enough intact sample for mounting in Ussing chambers could not be obtained. Even when such samples were obtained, they showed poor electrical viability. Despite a postmortem-related deterioration in cellular coupling, the biochemical parameters remained intact. Functional stability of the high-affinity carrier has been demonstrated for at least 48 hours after death in bovine, baboon, and human samples, 8 16 17 and therefore alternative procedures were followed. Taurine fluxes were estimated by radiolabel uptake studies, and the rates were compared with previously determined diffusional fluxes across isolated Bruch’s-choroid preparations. 18 19  
Material and Methods
Preparation of Tissue
Fourteen control human eyes from donors aged between 62 and 93 years (mean age, 75.4 years), with a postmortem time of up to 48 hours, were used (UK Transplant Support Service, Bristol Eye Bank, Bristol, UK). Informed consent for the use of the eyes for experimental studies was obtained, in accordance with the Declaration of Helsinki. Control donor eyes were obtained with the cornea removed for transplantation. The globes were dissected in a Petri dish lined with filter paper (Grade 50; Whatman, Maidstone, UK), moistened with Krebs’ bicarbonate buffer. The buffer composition was (mM): NaCl 118, glucose 6.6, NaHCO3 25, KCl 4.84, MgSO4 0.8, KH2PO4 1.2, CaCl2 1.8, 0.01% BSA. The posterior globe was inspected under a dissecting microscope for any evidence of subretinal or intraretinal blood, hard exudates, extensive drusen, irregular pigmentation of the RPE or any gross disease of the retina, and those exhibiting any abnormal appearance were discarded. The eyes were dissected at approximately the ora serrata to remove the remaining anterior portion, and then the globe was cut open to form a Maltese cross, the posterior segment being laid flat onto filter paper (Grade 50; Whatman). Vitreous was carefully removed, and the four quadrants (the arms of the cross) were removed and immersed in ice-cold Krebs’ buffer. Retina was then gently stripped from contact with the RPE and the RPE-choroid complex isolated from the sclera. Great care was taken when handling the aged tissue, as attachment of the RPE to Bruch’s membrane lessens with time after death. Samples were again carefully inspected under a dissecting microscope, and samples that had sustained damage to the RPE monolayer during dissection were discarded. The intact samples were used in the radiolabel uptake studies. In 6 of the 14 control donor eyes included in the study we could obtain only one intact trephine. In two eyes we obtained two intact trephines, in two eyes three intact trephines, and in four eyes four intact trephines. 
Four melanoma-affected human eyes were used. The donors were 61, 68, 69, and 74 years of age. These eyes were processed within a few hours after enucleation with the patient under general anesthesia. The anterior segment was first removed and the globe hemisected so that the melanoma-containing region could be processed for histopathology. The other half was cut into two or three segments and used for the kinetic characterization of taurine transport. 
Uptake of 3H-taurine into the RPE
RPE-choroid samples were incubated in pregassed Krebs’ buffer at 37°C, with a concentration of 10 nM 3H-taurine and 1 μM nonlabeled taurine. This concentration was chosen to target the high-affinity taurine carrier. 8 20 21 22 23 Three human samples from donors aged 62, 73, and 85 years were incubated for 10 and 20 minutes to confirm the linearity of the uptake mechanism. All other human samples were incubated for only 10 minutes. After incubation, the tissue samples were washed for several minutes in ice-cold Krebs’ medium to stop taurine uptake and to deplete label from the extracellular space. Samples were then floated onto filter paper and 6-mm discs were trephined (Stiefel Laboratories, Buckinghamshire, UK) from the tissue/filter paper preparation. Beta emissions were counted by scintillation spectrometry. Briefly, after addition of scintillant (UltimaGold; PerkinElmer, Boston, MA) the amount of taurine was determined by liquid scintillation spectrometry (Wallac 1409; Pharmacia-LKB Technology, Gaithersburg, MD). 
In five samples, we deliberately denuded Bruch’s choroid of RPE by brushing the surface with a fine sable hair brush before processing the tissue as described. These experiments were undertaken to measure uptake of radiolabeled taurine into cells of the choroid and unspecific binding, allowing the application of appropriate corrections to uptake by the RPE. 
Kinetic Characterization of Taurine Transport by Human RPE
Two to five 3- and 5-mm trephines of RPE-Bruch’s-choroid were obtained from each of a total of four donor eyes with melanoma. Incubations were performed at one or two taurine concentrations in the range 1, 10, 30, or 60 μM. 3H-taurine was added at a concentration of 1 nM. A parallel incubation was performed for each taurine concentration used, with samples that had been denuded of RPE to correct for unspecific binding, uptake by choroidal cells, and entrapment of label in the extracellular space. The procedure was identical with that described earlier. 
Statistical Analyses
The data in Figure 1 were analyzed by linear regression (Prism, ver. 3.02; Graph Pad Software, Inc., San Diego, CA). The kinetic data were analyzed by nonlinear regression to the Michaelis-Menten equation (Fig.P Software; Biosoft, Cambridge, UK). 
Results
Uptake of 3H-taurine into Human RPE
In Bruch’s-choroid preparations denuded of RPE, the uptake of taurine at an incubation concentration of 1 μM was determined to be 0.52 ± 0.04 (SEM) pmol/10 minutes per 6-mm disc (n = 5). These data relate to uptake by the choroidal mass and to unspecific binding and were subtracted from uptake measurements in intact RPE-Bruch’s-choroid preparations. 
Linearity of taurine uptake over 20 minutes of incubation was demonstrated in three donor human eyes at a substrate concentration of 1 μM. Thus, the amount of taurine accumulated over 10 and 20 minutes was determined to be 3.0 ± 0.38 and 5.9 ± 0.55 (SEM) pmol/6-mm disc (n = 3). All subsequent incubations were restricted to 10 minutes’ duration. The age of the donor was not associated with a statistically significant change in the transport of taurine by the RPE (P > 0.1; Fig. 1 ). The variation in uptake of taurine was considerable, with a range of 1.83 to 4.76 pmol/10 min per 6-mm disc over the studied age group. In the absence of statistical significance, the data were pooled to provide an average uptake rate at 1 μM substrate concentration of 2.89 ± 0.25 (SEM) pmol/10 min per 6-mm disc (n = 14). 
Kinetics of Taurine Transport by Human RPE
Kinetics were obtained over a substrate concentration of 1 to 60 μM taurine and used free-floating discs of RPE-Bruch’s-choroid. Thus, the uptakes were the sum of both apical and basal contributions of the RPE. An initial Lineweaver-Burk plot showed the presence of a single carrier. The data were therefore subjected to a non–linear-regression analysis using the single-carrier model of the Michaelis-Menten equation. The parameters of K m and V max for uptake of taurine by human RPE were determined to be 50 μM and 267 pmol/10 min per 5-mm disc, respectively (Fig. 2)
Discussion
Nutrient transport across the serially connected Bruch’s membrane and the RPE is crucial to survival of photoreceptors. An investigation of the relative functional contribution of the two compartments is essential for a fundamental understanding of the physiology of transport processes between the choroid and the outer retina. It may also be helpful in assessing the risk of nutritional deficiency in ageing and macular disease. 
It was the purpose of the present study to determine the uptake of taurine by human RPE and compare these results with our previous data 18 19 on transport through isolated human Bruch’s-choroid, so as to gauge the relative contribution of individual compartments to transport across the intact tissue complex. This analysis was undertaken at micromolar substrate concentrations, since only a single saturable high-affinity taurine transporter (K m range, 2–8.9 μM) has previously been localized in human RPE. 20 21 22 23  
There has been some controversy regarding the actual direction of taurine transport, as studies using intravascular injections of radiolabeled tracer demonstrated an initial rapid accumulation into the RPE followed by slower transfer to the retina. 24 In contrast, most in vitro attempts to establish the direction of taurine transport showed net flux from the retina to the choroidal circulation. 4 25 The working model for the vectorial transport of taurine across bovine RPE established by our group 8 and previous Ussing-chamber transport experiments 9 predict that under steady state conditions of extracellular potassium the net direction of taurine movement is determined by the relative apical and basolateral concentrations of taurine. 8 9  
Diffusional processes determine the rate of taurine transport across the extracellular 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, and dC/dx = taurine gradient across Bruch’s choroid), the rate is directly proportional to the driving concentration gradient. Transport through the RPE is carrier mediated, and the rate of translocation is a nonlinear function of the substrate concentration and is described by the appropriate Michaelis-Menten equation. Thus, the difference in rate of transport between Bruch’s-choroid and the RPE in the intact complex is dependent on the concentration of taurine. 
Studies comparable to our previous work with bovine samples 9 could not be undertaken with human donor tissue, because electrical stability of the RPE-monolayer could not be maintained in samples with postmortem times of 24 to 48 hours. Despite a postmortem-related deterioration of cellular coupling by tight junctions, the function of active taurine uptake remained unchanged for 48 hours after death. 16 17 An alternative approach was therefore followed to derive information on the relative contribution of the two transport compartments in the age group older than 60 years to delivery of taurine to the outer retina. This entailed measurements of (1) taurine uptake by the RPE in donors of various ages at a given substrate concentration and (2) determining the taurine transport kinetics of human RPE. 
In the present study, despite the gross morphologic alterations associated with ageing, the transport systems in the RPE for taurine uptake remained viable. The age of the donor had no effect on the accumulation of taurine by human RPE within the age group older than 60 years (linear regression, P > 0.1, Fig. 1 ). The changes in taurine transport after 60 years of age was not as dramatic as the decline with increasing age in hydraulic conductivity of Bruch’s membrane. 26 27  
This study represents the first kinetic characterization of the taurine transporter in human RPE freshly isolated from human donor eyes. All previous studies used human cell lines. 20 21 22 23 Our characterization of the high-affinity carrier in human RPE yielded a K m of 50 μM and V max of 267 pmol/10 minutes per 5-mm disc. This K m for freshly isolated human RPE was higher than the 2 to 8.9 μM quoted for human cell lines. 20 21 22 23 We have previously determined the effect of age on diffusional transport of taurine across the isolated human Bruch’s choroid and the calculated rates at birth and 90 years of age (at a concentration gradient of 10 mM) were 25.15 and 15.69 nmol/5 minutes per 4-mm disc, respectively. 19 These results were used to construct the differential transport profiles across the two transport compartments of human Bruch’s-choroid and the RPE shown in Figure 3
The V max used to construct the RPE taurine transport profile of Figure 3 was a summation of both apical and basal carrier capacity, because the kinetic analysis was undertaken with free floating tissue discs. Thus, the plot is an exaggeration of the capacity at apical or basal surfaces of the RPE. Nonetheless, as demonstrated in Figure 3 , at plasma taurine levels of 44 to 62 μM, 28 29 30 the transport capacity of ageing human Bruch’s-choroid remains in excess of the transport capacity of the RPE. A localization of the taurine transporter on both surfaces of the RPE cell can be assumed, because taurine has been shown to move in both directions. 4 8 9 17 25 Immunohistochemical methods localized the taurine transporter on the apical surface and throughout the cytoplasm of cultured human ARPE-19 cells. The authors did not seek to localize specifically the taurine transporter on the basal surface, which may technically have been impossible, because the investigated cells were cultured on plastic chamber supports. 23  
As stated earlier, several studies have determined the kinetic constants for transport of taurine in various human RPE cell lines. All showed the presence of a single NaCl-dependent high-affinity carrier for transport of taurine. 20 21 22 23 The maximum rate of transport (V max) in these studies was presented in milligrams of protein and are therefore not directly comparable with data in the present study obtained from discs of freshly isolated RPE. However, assuming that a 4-mm diameter disc of RPE is equivalent to 0.0157 mg protein (see legend to Table 1 ), it is possible to convert V max to units of picomoles per 5 minutes per 4-mm disc. Alternatively (because of the uncertainty of the protein conversion) V max can be calculated using the K m from each study and the uptake velocity determined at 1 μM for human RPE in the present study. Once, K m and V max are known, the Michaelis-Menten equation allows the calculation of transport rates for the RPE at an average plasma taurine concentration of 50 μM. 28 29 30 The corresponding transport of taurine at a concentration gradient of 50 μM across the isolated Bruch’s-choroid compartment can also be determined (Fig. 3)
An analysis combining the data of the present study with the kinetic constants of other research groups to compare our results is presented in Table 1 . It should be stressed that the calculated transport rates for human RPE are overestimated, because apical and basal contributions have been summed during the initial kinetic assessment. The description of carriers in cell lines and tissue preparations may not be directly comparable, and we have therefore characterized the kinetics of the taurine transporter in human tissue preparations (Fig. 2) . Despite the limited comparability of kinetic parameters obtained from cell line experiments and human tissue preparations and the gross variation in the various studies, Table 1 suggests that in human samples also, the rate-limiting compartment for transport of taurine is the RPE monolayer. Even in eyes 90 years of age, the transport rate of 80.6 pmol/5 min per 4-mm disc for Bruch’s-choroid was in excess of the handling capacity of the RPE. 
In summary, our data suggest that in the human eye, although the diffusional capacity of human Bruch’s membrane for small solutes such as taurine declines with age, it nevertheless remains in excess of the maximal calculated transport rate of human RPE under physiological conditions. Excessive impairment of the diffusional capacity of Bruch’s membrane may lead to an undersupply of the metabolic demand of the RPE. 
Further studies investigating taurine uptake in the younger age group and, ideally, examining transepithelial taurine transport across intact human RPE-Bruch’s-choroid preparations to further validate the calculations based on the data of the present study are needed. 
 
Figure 1.
 
High-affinity uptake of taurine by human RPE in the group aged >60 years (at a substrate concentration of 1 μM). One to four trephines were obtained from each donor eye. The decreasing trend in taurine uptake with increasing age was not statistically significant (P > 0.1). In the absence of statistical significance, the data were pooled to provide a mean ± SD of 2.89 ± 0.98 (n = 14) pmol/10 min per 6-mm disc for taurine uptake by human RPE, at a substrate concentration of 1 μM.
Figure 1.
 
High-affinity uptake of taurine by human RPE in the group aged >60 years (at a substrate concentration of 1 μM). One to four trephines were obtained from each donor eye. The decreasing trend in taurine uptake with increasing age was not statistically significant (P > 0.1). In the absence of statistical significance, the data were pooled to provide a mean ± SD of 2.89 ± 0.98 (n = 14) pmol/10 min per 6-mm disc for taurine uptake by human RPE, at a substrate concentration of 1 μM.
Figure 2.
 
Kinetics of taurine transport by human RPE cells. Tissue trephines (3- and 5-mm diameter) were obtained from four melanoma-affected human donor eyes. The donors were 61, 68, 69, and 74 years of age. Taurine uptake was determined in the concentration range of 1 to 60 μM, using three to five samples at each concentration. Nonlinear regression analysis in a single-carrier Michaelis-Menten model yielded a K m of 50 μM and V max of 267 pmol/10 min per 5-mm tissue disc.
Figure 2.
 
Kinetics of taurine transport by human RPE cells. Tissue trephines (3- and 5-mm diameter) were obtained from four melanoma-affected human donor eyes. The donors were 61, 68, 69, and 74 years of age. Taurine uptake was determined in the concentration range of 1 to 60 μM, using three to five samples at each concentration. Nonlinear regression analysis in a single-carrier Michaelis-Menten model yielded a K m of 50 μM and V max of 267 pmol/10 min per 5-mm tissue disc.
Figure 3.
 
Comparative analysis of taurine transport by human RPE and Bruch’s-choroid as a function of substrate concentration. Taurine transport (dotted lines) across isolated Bruch’s-choroid by diffusion was calculated as a function of age from our previous studies. 18 19 Transport across human RPE was shown not to be dependent on the age of the donor (Fig. 1) , and hence a single function (solid line) was plotted from the kinetics derived from Figure 2 . The transport rates across the RPE are likely to be an overestimate, because both apical and basal contributions were summed in the kinetic analysis. The single data point at 1 μM represents the average uptake by human RPE in the present study. At plasma taurine levels of 44 to 62 μM 28 29 30 the rate-limiting step, in even the elderly, for delivery of taurine to photoreceptors is the RPE compartment.
Figure 3.
 
Comparative analysis of taurine transport by human RPE and Bruch’s-choroid as a function of substrate concentration. Taurine transport (dotted lines) across isolated Bruch’s-choroid by diffusion was calculated as a function of age from our previous studies. 18 19 Transport across human RPE was shown not to be dependent on the age of the donor (Fig. 1) , and hence a single function (solid line) was plotted from the kinetics derived from Figure 2 . The transport rates across the RPE are likely to be an overestimate, because both apical and basal contributions were summed in the kinetic analysis. The single data point at 1 μM represents the average uptake by human RPE in the present study. At plasma taurine levels of 44 to 62 μM 28 29 30 the rate-limiting step, in even the elderly, for delivery of taurine to photoreceptors is the RPE compartment.
Table 1.
 
Taurine Transport Characteristics of Human RPE
Table 1.
 
Taurine Transport Characteristics of Human RPE
1 2 3 4 5 6 7
Study K m (μM) V max (original units of publication) Recalculated V max * (pmol/5min per 4-mm disc) Calculated Uptake Velocity at 50 μM Extracellular Taurine, † (pmol/5min per 4-mm disc) Calculated V max from Taurine Uptake at 1 μM, ‡ (pmol/5min per 4-mm disc) Calculated Uptake Velocity at 50 μM Extracellular Taurine, § (pmol/5min per 4-mm disc)
Present study Fresh donor RPE Sub. conc., 1–60 μM 50 267 pmol/10min per 5-mm disc 85.4 42.7 31.7 15.84
Leibach et al. 20 HRPE cell line sub. conc., 0.25–10 μM 2 93 pmol/mg protein per 15 minutes 0.49 0.47 1.86 1.79
Ganapathy et al. 21 HRPE cell line sub. conc., 0.5–10 μM 2.1 200 pmol/mg protein per 30 minutes 0.52 0.50 1.93 1.85
Stevens et al. 22 HRPE cell line sub. conc., 1–1000 μM 3.9 286 pmol/mg protein per minute 22.4 20.8 3.04 2.82
Bridges et al. 23 HRPE cell line, ARPE-19 sub. conc. 0.5–20 μM 8.9 194 pmol/mg protein per 15 minutes 1.02 0.86 6.15 5.22
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Figure 1.
 
High-affinity uptake of taurine by human RPE in the group aged >60 years (at a substrate concentration of 1 μM). One to four trephines were obtained from each donor eye. The decreasing trend in taurine uptake with increasing age was not statistically significant (P > 0.1). In the absence of statistical significance, the data were pooled to provide a mean ± SD of 2.89 ± 0.98 (n = 14) pmol/10 min per 6-mm disc for taurine uptake by human RPE, at a substrate concentration of 1 μM.
Figure 1.
 
High-affinity uptake of taurine by human RPE in the group aged >60 years (at a substrate concentration of 1 μM). One to four trephines were obtained from each donor eye. The decreasing trend in taurine uptake with increasing age was not statistically significant (P > 0.1). In the absence of statistical significance, the data were pooled to provide a mean ± SD of 2.89 ± 0.98 (n = 14) pmol/10 min per 6-mm disc for taurine uptake by human RPE, at a substrate concentration of 1 μM.
Figure 2.
 
Kinetics of taurine transport by human RPE cells. Tissue trephines (3- and 5-mm diameter) were obtained from four melanoma-affected human donor eyes. The donors were 61, 68, 69, and 74 years of age. Taurine uptake was determined in the concentration range of 1 to 60 μM, using three to five samples at each concentration. Nonlinear regression analysis in a single-carrier Michaelis-Menten model yielded a K m of 50 μM and V max of 267 pmol/10 min per 5-mm tissue disc.
Figure 2.
 
Kinetics of taurine transport by human RPE cells. Tissue trephines (3- and 5-mm diameter) were obtained from four melanoma-affected human donor eyes. The donors were 61, 68, 69, and 74 years of age. Taurine uptake was determined in the concentration range of 1 to 60 μM, using three to five samples at each concentration. Nonlinear regression analysis in a single-carrier Michaelis-Menten model yielded a K m of 50 μM and V max of 267 pmol/10 min per 5-mm tissue disc.
Figure 3.
 
Comparative analysis of taurine transport by human RPE and Bruch’s-choroid as a function of substrate concentration. Taurine transport (dotted lines) across isolated Bruch’s-choroid by diffusion was calculated as a function of age from our previous studies. 18 19 Transport across human RPE was shown not to be dependent on the age of the donor (Fig. 1) , and hence a single function (solid line) was plotted from the kinetics derived from Figure 2 . The transport rates across the RPE are likely to be an overestimate, because both apical and basal contributions were summed in the kinetic analysis. The single data point at 1 μM represents the average uptake by human RPE in the present study. At plasma taurine levels of 44 to 62 μM 28 29 30 the rate-limiting step, in even the elderly, for delivery of taurine to photoreceptors is the RPE compartment.
Figure 3.
 
Comparative analysis of taurine transport by human RPE and Bruch’s-choroid as a function of substrate concentration. Taurine transport (dotted lines) across isolated Bruch’s-choroid by diffusion was calculated as a function of age from our previous studies. 18 19 Transport across human RPE was shown not to be dependent on the age of the donor (Fig. 1) , and hence a single function (solid line) was plotted from the kinetics derived from Figure 2 . The transport rates across the RPE are likely to be an overestimate, because both apical and basal contributions were summed in the kinetic analysis. The single data point at 1 μM represents the average uptake by human RPE in the present study. At plasma taurine levels of 44 to 62 μM 28 29 30 the rate-limiting step, in even the elderly, for delivery of taurine to photoreceptors is the RPE compartment.
Table 1.
 
Taurine Transport Characteristics of Human RPE
Table 1.
 
Taurine Transport Characteristics of Human RPE
1 2 3 4 5 6 7
Study K m (μM) V max (original units of publication) Recalculated V max * (pmol/5min per 4-mm disc) Calculated Uptake Velocity at 50 μM Extracellular Taurine, † (pmol/5min per 4-mm disc) Calculated V max from Taurine Uptake at 1 μM, ‡ (pmol/5min per 4-mm disc) Calculated Uptake Velocity at 50 μM Extracellular Taurine, § (pmol/5min per 4-mm disc)
Present study Fresh donor RPE Sub. conc., 1–60 μM 50 267 pmol/10min per 5-mm disc 85.4 42.7 31.7 15.84
Leibach et al. 20 HRPE cell line sub. conc., 0.25–10 μM 2 93 pmol/mg protein per 15 minutes 0.49 0.47 1.86 1.79
Ganapathy et al. 21 HRPE cell line sub. conc., 0.5–10 μM 2.1 200 pmol/mg protein per 30 minutes 0.52 0.50 1.93 1.85
Stevens et al. 22 HRPE cell line sub. conc., 1–1000 μM 3.9 286 pmol/mg protein per minute 22.4 20.8 3.04 2.82
Bridges et al. 23 HRPE cell line, ARPE-19 sub. conc. 0.5–20 μM 8.9 194 pmol/mg protein per 15 minutes 1.02 0.86 6.15 5.22
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