Potassium Modulation of Taurine Transport across the Frog Retinal Pigment Epithelium S H E L D O N S. M I L L E R and ROY H. S T E I N B E R G From the School of Optometry, University of California, Berkeley, California 94720 and the Departments of Physiologyand Ophthalmology,Universityof California, San Francisco,California 94143

A BS T R AC T Net taurine transport across the frog retinal pigment epitheliu mchoroid was measured as a function of extracellular potassium concentration, [K+]o. The net rate of retina-to-choroid transport increased monotonically as [K+]o increased from 0.2 m M to 2 m M on the apical (neural retinal) side of the tissue. No further increase was observed when [K+]o was elevated to 5 raM. The [K+]o changes that modulate taurine transport approximate the light-induced [K+]o changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium. The taurine-potassium interaction was studied by using rubidium as a substitute for potassium and measuring active rubidium transport as a function of extracellular taurine concentration. An increase in apical taurine concentration, from 0.2 m i to 2 m i , produced a threefold increase in active rubidium transport, retina to choroid. Net taurine transport can also be altered by relatively large, 55 raM, changes in [Na+]o. Apical ouabain, 10~4 M, inhibited active taurine, rubidium, and potassium transport; in the case of taurine, this inhibition is most likely due to a decrease in the sodium electrochemical gradient. In sum, these results suggest that the apical membrane contains a taurine, sodium co-transport mechanism whose rate is modulated, indirectly, through the sodium pump. This pump has previously been shown to be electrogenic and located on the apical membrane, and its rate is modulated, indirectly, by the taurine co,transport mechanism. INTRODUCTION In the v e r t e b r a t e r e t i n a the p h o t o r e c e p t o r o u t e r segments a n d the apical surfaces o f the p i g m e n t epithelial cells share the same ionic e n v i r o n m e n t . T h e y can, therefore, alter the activity o f one a n o t h e r b y causing ion or m e t a b o l i t e c o n c e n t r a t i o n changes in the space t h a t separates t h e m . In frog, for e x a m p l e , it is n o w k n o w n t h a t light-evoked changes in p h o t o r e c e p t o r activity can alter the e x t r a c e l l u l a r c o n c e n t r a t i o n o f potassium ([K+]o), which t h e n changes the m e m b r a n e p o t e n t i a l o f the p i g m e n t epithelial cells. In particular, light causes a decrease in [K+]o that h y p e r p o l a r i z e s the apical j. GEN. PHYSIOL.9 The Rockefeller University Press 9 0022-1295/79/08/0237/23 $1.00 Volume 74 August 1979 237-259

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m e m b r a n e of the epithelial cells and provides the principal voltage source of the c-wave of the electroretinogram (Oakley and Green, 1976; Oakley, 1977; Oakley et al., 1977). Potassium m a y also affect pigment epithelial function in other ways. It is well known, for example, that sugar and amino acid transport across cell membranes can be significantly altered by changes in the extracellular concentration of potassium or sodium (Riggs et al., 1958; Crane, 1962, 1964; Schultz and Curran, 1970; Heinz, 1972). For potassium, extracellular concentration changes of 10-40 m M are usually required to cause sizable changes in the rate of nonelectrolyte transport. There exist some systems, however, for which changes of a few millimolar in [K+]o can significantly alter the rate of metabolite transport (Reddy, 1968; Dantzler, 1974). We were interested in determining, therefore, whether amino acid transport across the epithelium would be affected by changes in potassium concentration that were similar to those produced by light (Oakley and Green, 1976; Oakley, 1977). T h e amino acid taurine was chosen for study because of its importance in the retina (Starr and Voaden, 1972; Pasantes-Morales et al., 1974; Hayes et al., 1975; Cunningham and Miller, 1976; Orr et al., 1976; Schmidt, 1978), and because it undergoes a large net transport across the retinal pigment epithelium in the apical ~ basal (retina ~ choroid) direction (Miller and Steinberg, 1976). Recently, it has been shown that there is a light-stimulated release of taurine from frog rod outer segments (Salceda et al., 1977). METHODS These studies were performed on the isolated retinal pigment epithelium-choroid of the bullfrog, Rana catesbeiana. The retinal pigment epithelium (RPE) consists of a single layer of cuboidal epithelial cells. The basal surface of the RPE faces the choroid, which consists mainly of blood vessels and melanocytes dispersed in a fibrous stroma. Individual RPE cells measure approximately 15/~m in width and depth (Porter and Yamada, 1960; Nilsson, 1964; Steinberg, 1973). The junctional complexes connecting these cells in frogs differ from those of other species by being absent at the cellular apices; they begin about halfway down the lateral surfaces of each cell (Porter and Yamada, 1960; Hudspeth and Yee, 1973). The apical surface of the RPE faces the sensory retina, and is covered with villous-like processes that are 60-90/zm long. These processes are closely apposed to the photoreceptors, and extend all the way down to their inner segments (Nilsson, 1964). In these in vitro studies, the tissue was mounted as a sheet separating two separate compartments, each of which contained a modified Ringer's solution. The fluid bathing the apical surface was referred to as the apical solution; that bathing the basal surface was referred to as the basal solution. The composition of these two solutions could be separately controlled. The bullfrogs were obtained from Californian and Midwestern suppliers, and kept from several days to several weeks in running tap water at 17.5 ~ on an alternating 12-h cycle of light and darkness. Pieces of pigment epithelium-choroid, 6.5 mm square, were isolated from dark-adapted eyes and mounted in a lucite chamber, so that the surface of the choroid and the apical surface of the pigment epithelium were immersed in separate 1.8-ml baths. The chamber design and the techniques used for dissecting the tissue and mounting it between two lucite plates were identical to those used previously (Miller and Steinberg, 1977 a, b).

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The composition of the control bathing solution was 82.5 mM NaCl, 27.5 mM NaHCOs, 2.0 m M KCI, 1.0 mM MgCI2.6H~O, 1.8 mM CaCI2, 10.0 mM glucose; the solution was gassed with 95% 02/5% COs to a pH of 7.4 :t: 0.1. The osmolarity of this solution was 227 mosM. In some experiments, the potassium concentration was varied by equimolar exchange of KCI and NaCI. In other experiments the concentration of potassium was increased by adding small amounts of KC1 (3 mM or less) to the solutions bathing the apical or basal membranes. In all experiments "cold" taurine (0.1-5 mM) was added to one or both solutions. The sodium concentration was varied by equimolar replacement of NaCI and NaHCOs with tris chloride (tris [hydroxymethyl] aminomethane hydrochloride) and choline bicarbonate, respectively. Two pairs of Agar-Ringer bridges on each side of the tissue were used to monitor the transepithelial potential (TEP) and to pass current both for short-circuiting (shortcircuit current, SCC) and to measure the transepithelial resistance (Rt). Short-circuit current was monitored continuously on a pen recorder. Readings of TEP were obtained hourly by briefly interrupting the short circuit, and Rt also was obtained at the same time by passing 1/~A pulses transepithelially and recording the changes in TEP. For the determination of the transepithelial unidirectional fluxes of taurine, a tracer amount of the isotope [aSS]taurine or [3H]taurine, was added either to the solution facing the choroid--the basal solution--or to the apical solution. The fluxes, apical --* basal (retina --~ choroid) or basal --* apical (choroid --* retina) were then measured by sampling the "cold" solution on the opposite side of the tissue every 40 min. The sample size was 100 #l, and it was replaced with an equal amount of"cold" solution. The fluid in each chamber was stirred and oxygenated by a stream of watersaturated gas bubbles. Samples were assayed by conventional counting techniques, using a Packard TriCarb liquid scintillation spectrometer (Packard Instrument Co., Inc., Downers Grove, Ill.). The unidirectional fluxes Were calculated in nmol/cm2h from the rate of tracer appearance on the "cold" side and the specific activity of the "hot" side; the area refers to the area of the window between the two lucite chambers. RESULTS Potassium and Taurine Fluxes W e previously showed t h a t the retinal p i g m e n t e p i t h e l i u m actively transports t a u r i n e in the retina-choroid direction a n d t h a t this net transport takes place over a wide range o f t a u r i n e concentrations, 1.7 • 10 -4 to 3 • 10-2M (Miller a n d Steinberg, 1976). These experiments were p e r f o r m e d with an external potassium c o n c e n t r a t i o n ([K+]o) o f 2.0 m M in b o t h the apical a n d basal solutions. T o d e t e r m i n e w h e t h e r unidirectional taurine fluxes were affected by a decrease in [K+]o, we first p e r f o r m e d experiments in which [K+]o was changed, in one or b o t h solutions, to 0.2 m M . These d a t a have been plotted in Figs. 1 a n d 2 (symbols) along with the d a t a previously o b t a i n e d at 2 m M [K+]o (lines). Fig. 1 shows t h a t a 10-fold reduction in [K+]o did not appreciably c h a n g e the choroid-retina t a u r i n e flux regardless of w h e t h e r the [K+]o was altered in both solutions or in only the apical or basal solution. By contrast, Fig. 2 shows t h a t there was a d r a m a t i c decrease in the retina-choroid flux w h e n [K+]o was reduced on the apical side alone (F1) or on both sides (C)) of the tissue. This decrease, to a p p r o x i m a t e l y 20% of the level in 2.0 m M [K+]o (line), occurred across the whole range o f taurine concentrations. T h e points

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at 1.85 m M (marked by an arrow on the abscissa) represent a decrease in taurine flux of 70 nM/cmZh. Fig. 2 also shows that the reduction of [K+]o in the basal solution alone (A) did not affect the retina-choroid flux, across the entire range of taurine concentrations. It is only apical [K+]o, therefore, that affects the transepithelial movement of taurine. Because the experiments in Fig. 2 each required a period of hours to complete, it was important to determine if the observed effects were reversible. T h a t the tissues can regain their previous rate of taurine transport after e,J

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FIGURE 1. Unidirectional, choroid --~ retina, taurine fluxes, as a function of taurine concentration, in 0.2 mM [K+]o (log-log plot). The solid line is the flux curve at 2.0 mM [K+]o (Miller and Steinberg, 1976). Each point is the mean steady-state unidirectional flux from one tissue. There were at least four steadystate measurements per point, and the SEMs were never more than + 0.1 log units. The potassium concentration was maintained at 0.2 mM on both sides of the tissue in one group of experiments (0). In other experiments it was maintained at 0.2 mM on only one side of the tissue: ( I ) retina side, (&) choroid side; with 2.0 mM on the opposite side. The arrow on the abscissa is at 1.85 mM. readmission of potassium is demonstrated in Fig. 3. The retina-choroid flux in 0.3 m M [K+]o was only 10 n M / c m Z h after 3 h, but a sudden increase in [K+]o to 2.0 m M in both solutions immediately increased the flux, as well as the short-circuit current, the flux reaching a steady state of 80 n M / c m 2 h after about 2 h. This is the rate expected for a tissue in 2.0 m M [K+]o that had not been exposed previously to low potassium (Figs. 2 and 4). We next examined the effect of much smaller increases and decreases in [K+]o around the 2.0 m M point and at a fixed taurine concentration of 1.85 mM. T h e latter was chosen because it represented a point on the curves (Figs. 1 and 2), where the unidirectional flux was high but not maximal. Fig. 4 presents the data for a total of 66 experiments; 57 in the retina-to-choroid direction and 9 in the opposite direction. In most cases [K+]0 was kept constant

MILLER AND STEINBERG Potassium-ModulatedTaurine Transport

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on both sides of the tissue during the course of the experiment. These experiments show that the retina-choroid taurine flux (O, I-'1) is significantly altered by small changes in [K+]o, while the choroid-to-retina flux ( I , A, O) was approximately constant over the entire range of [K+]o changes. T h e average retina-choroid flux was sensitive to [K+]o changes as small as 0.5 mM, the smallest increment tested, and there was a sharp fall in flux when [K+]o was decreased below 1.0 m M either in both solutions (C)) or in the apical solution alone (r-I). We concluded, therefore, that from 0.2 to 5 mM, apical [K+]0 is an important determinant of net taurine transport across the R P E J

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Log "routine Goncn (Id) FiouaE 2. Unidirectional, retina --* choroid, taurine fluxes, as a function o f taurine concentration, in 0.2 m M [K+]o (log-log plot). Potassium concentration at 0.2 m M on both sides o f the tissue (C)); or on only the retina side ([]), or on the choroid side (A). T h e open circle and square at 1.85 m M (marked by an arrow on the abscissa) represent the average o f three experiments (mean and SEM). Otherwise as Fig. 1. Fig. 4 s h o w s t h a t t h e a v e r a g e flux w a s m a x i m u m at a p p r o x i m a t e l y 2 m M [K+]o a n d t h a t this w a s n o t s i g n i f i c a n t l y a l t e r e d w h e n [K+]o w a s i n c r e a s e d t o 5 m M . I n o r d e r t o d e t e r m i n e t h a t t h e r e w a s a s i g n i f i c a n t i n c r e a s e in flux b e t w e e n 1 a n d 2 m M [K+]o, a s e p a r a t e series o f e x p e r i m e n t s w a s p e r f o r m e d in t i n Figs. 3 and 5, a decrease in transepithelial resistance (Rt) accompanies the increase in [K+]o (see legends). This inverse relationship holds over the whole range of [K+]o changes and must result from a decrease in cell membrane (apical or basal) resistance, shunt resistance or a combination of the two (Miller and Steinberg, 1977 a). The decrease in Rt could also signify an altered tissue taurine permeability, which might explain the rise in retina to choroid flux as a function of [K+]o (Fig. 4). This seems very unlikely, however, since the choroid to retina fluxes were unaffected by apical or basal changes in [K+]o.

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w h i c h the average fluxes at these c o n c e n t r a t i o n s were b o t h m e a s u r e d on a single tissue. T h e t a u r i n e flux in 1.0 m M [K+]o was m e a s u r e d first a n d t h e n after r e a c h i n g the steady state [K+]o was increased to 2.0 m M (Fig. 5). T h i s was r e p e a t e d on six tissues, a n d the results are s u m m a r i z e d in T a b l e I. In five out o f six tissues, t h e r e was a significant increase in the steady-state retinac h o r o i d flux at 2.0 m M [K§ the difference b e t w e e n the m e a n s was 30 n M / cm2h. A similar set o f m e a s u r e m e n t s was m a d e b e t w e e n 2 a n d 3 or 5 m M

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FIGURE 3. Taurine flux in the retina ---) choroid direction at 0.3 and 2.0 m M [K+]o. The taurine concentration was 1.85 m M in both apical and basal solutions. Tracer [a~S]taurine, was added to the apical solution at t -- 0 and the retina ~ choroid (apical to basal) flux was sampled at 40-min intervals (0). The flux was first measured with 0.3 m M [K§ on both sides of the tissue and at 3 h 20-rain the potassium concentration, on both sides, was elevated to 2.0 raM, and monitored until it reached a steady state (..... ). Throughout the experiment the transepithelial potential (TEP) was short-circuited to zero, and the short-circuit current, SCC in nM/cm2h, was monitored continuously (t~---'--Q). The T E P and transepithelial resistance (Rt) were monitored at 40- or 80-min intervals by briefly interrupting the short-circuit current and passing a 1-~tA current pulse. During the first part of the experiment the average T E P was 7.4 mV and the average Rt was 5.5 k~. After [K+]o was elevated to 2 m M the T E P rose transiently to 10.6 mV and Rt fell to 4.7 k~. Subsequently, the T E P fell to 8.5 mV and Rt remained constant. [K+]o, a n d the results, p l o t t e d in Fig. 4, show t h a t the r e t i n a - t o - c h o r o i d t a u r i n e flux in 2 m M [K+]o was not a p p r e c i a b l y different t h a n in 3 or 5 m M [K§

Sodium and Taurine Fluxes Since, in m a n y systems, the active a c c u m u l a t i o n o f nonelectrolytes is c o u p l e d to the flow o f s o d i u m d o w n its e l e c t r o c h e m i c a l p o t e n t i a l gradient ( W h e e l e r a n d Christensen, 1967; S c h u l t z a n d C u r r a n , 1970; H e i n z , 1972; K i m m i c h et al., 1977; Schultz, 1977), we next e x a m i n e d u n i d i r e c t i o n a l t a u r i n e fluxes as a f u n c t i o n o f e x t e r n a l s o d i u m c o n c e n t r a t i o n . T h e solid lines in Figs. 6 a n d 7

MII~LE~AND STEINBERG Potassium-Modulated Taurine Transport

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specify u n i d i r e c t i o n a l t a u r i n e flux as a f u n c t i o n o f t a u r i n e c o n c e n t r a t i o n ; in these e x p e r i m e n t s the e x t e r n a l s o d i u m c o n c e n t r a t i o n , [Na+]o, was set at 110 m M in b o t h the apical a n d basal solutions. W e first d e t e r m i n e d w h e t h e r a large c h a n g e in s o d i u m c o n c e n t r a t i o n , from 110 to 11 m M , c o u l d affect the t a u r i n e u n i d i r e c t i o n a l fluxes. Fig. 6 shows t h a t t h e r e was n o c h a n g e in the c h o r o i d ~ r e t i n a flux w h e n [Na+]o was r e d u c e d to 11 m M o n b o t h sides or 2"

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[K +]0 mM FIGURE 4. Taurine flux as a function of external potassium concentration. The open symbols represent retina --~ choroid fluxes and the closed symbols choroid ---* retina fluxes. The taurine concentration was 1.85 m M in both solutions for all the experiments shown. The circles are experiments in which [K+]o was the same on both sides of the tissue; the squares are experiments in which [K+]o was altered in the apical solution only and maintained at 2.0 m M in the basal solution. The triangles represent experiments in which [K+]o was 2 m M in the apical solution and 0.2 or 5 m M in the basal solution. Each point with error bars is the mean __. SEM of steady-state measurements on 3-10 tissues. Points without error bars are from single experiments. In most experiments [K+]o was held fixed on both sides of the tissue throughout the measurement period. In four experiments, however, the taurine fluxes were measured at two different [K+]o concentrations on the same tissue. In these experiments (open circles at 2 mM), both sides of the tissue were first bathed in 2 m M [K+]o Ringer's for approximately 489 h and then [K§ was increased on one or both sides to 3 o r 5 m M (O, A, 1"7); the flux measurements were then continued for another 3 h.

only on one side o f the tissue. In contrast, Fig. 7 shows t h a t the r e t i n a --~ c h o r o i d fluxes were m u c h r e d u c e d in low s o d i u m across the entire r a n g e o f t a u r i n e c o n c e n t r a t i o n s . T h e r e d u c t i o n o f [Na+]o to 10% o f its n o r m a l level r e d u c e d the t a u r i n e fluxes to a p p r o x i m a t e l y 3% o f their value in 110 m M [Na+]o. T h e d a t a at 1.85 m M t a u r i n e ( m a r k e d b y the a r r o w on the abscissa) i n d i c a t e d t h a t most or all o f this c h a n g e was caused b y the r e d u c t i o n o f [Na+]o at the apical side (['7) o f the tissue. Fig. 8 shows the v a r i a t i o n in retina-

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FIGURE 5. Taurine flux in the retina - * choroid direction with either 1.0 m M [K+]o or 2.0 m M [K+]o in the apical and basal solutions. T h e steady-state flux (first interrupted horizontal line) was first measured with 1.0 m M [K+]o in both solutions. [K+]o was then elevated to 2.0 m M in both solutions, which increased the mean unidirectional flux by 52 nM/cm2h (second interrupted horizontal line). Otherwise as in Fig. 3. During the first part of the experiment the average T E P was 12.0 m V and the average Rt was 4.5 k~. After [K+]o was elevated to 2 m M , Rt decreased to 3.8 k~ and the average T E P fell slightly to 11.8 mV.

TABLE ELEVATION

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1.0 m M [K+]o Expt.

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56 97 51 86 63 65

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70 3= 7 1013=12

Elevation of taurine flux by A[Ig.+]o in individual tissues. For each experiment columns 4 and 5 present the average taurine flux, in the retina ~ choroid direction, as a function of external potassium concentration. Potassium concentration was changed simultaneously on both sides of the tissue. C o l u m n s 2 and 7 and 3 and 6 are the average transepithelial potentials and resistances, respectively. T h e change in taurine flux produced by elevating [K*]o is highly significant (P < 0.001, paired t test).

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Potassium-ModulatedTaurine Transport

to-choroid taurine flux as a function of [Na+]o, and at a fixed taurine concentration of 1.85 mM. These data demonstrate that relatively large changes in [Na+]o (55 mM) are needed to reduce appreciably the taurine flux. In m a n y of the experiments (Fig. 4), [K+]o was altered by exchanging NaCI for KCI, and it was important to determine whether the small increase in [Na+]o, for example, 1.8 m M for a [K+]o decrease from 2.0 to 0.2 raM, caused the change in taurine flux. This was shown not to be the case because almost identical changes in taurine flux could be produced by holding [Na+]o constant

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FIGURE 6. Unidirectional, choroid --* retina, taurine fluxes as a function of taurine concentration in 11 mM [Na+]o (log-log plot). The solid line is the flux curve at 110 mM [Na+]o in both solutions, apical and basal (Miller and Steinberg, 1976). Each point is the mean steady-state unidirectional flux from one experiment. The SEMs did not exceed 4- 0.3 log units. The sodium concentration was maintained at 11 mM on both sides of the tissue in one group of experiments (O). In other experiments, it was maintained at 11 mM on only one side of the tissue: (11), retina side, (A) choroid side; with 110 mM on the opposite side. The sodium concentration was varied by equimolar replacement of NaCI and NaHCO3 with tris chloride (tris [hydroxymethyl] aminomethane hydrochloride) and choline bicarbonate, respectively. The arrow on the abscissa denotes a taurine concentration of 1.85 mM. and increasing [KCI]o, from 0.3 to 2.0 m M (Fig. 3). (This effect of increasing [KCI]o was not an osmotic one, since taurine fluxes were not altered by adding 3 m M choline chloride or tris chloride to the apical or basal solution.) In other experiments, small changes in [Na+]o (10 m M or less) produced no measurable change in taurine transport. Taurine and Rubidium Fluxes It has been shown in other epithelia (Schuhz and Curran, 1970; Ahearn, 1976; Schuhz, 1977) that Na-nonelectrolyte (sugars and amino acids) interactions are reciprocal; that is, a change in the concentration of one of these

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Log Tourine Concn (M) FIGURE 7. U n i d i r e c t i o n a l , r e t i n a ---* choroid, t a u r i n e fluxes as a function of t a u r i n e c o n c e n t r a t i o n , in 1 1 m M [Na+]o. T h e s o d i u m c o n c e n t r a t i o n was m a i n t a i n e d at 11 m M on b o t h sides o f the tissue in one group o f e x p e r i m e n t s (O). In o t h e r experiments, it was m a i n t a i n e d at 11 m M on o n l y one side o f the tissue: (I-1) r e t i n a side, (LX) choroid side; with 110 m M on the opposite side. O t h e r w i s e as in Fig. 6.

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FIOURE 8. T a u r i n e flux in t h e r e t i n a --* c h o r o i d direction as a function o f external s o d i u m concentration. E a c h p o i n t w i t h o u t an error b a r is the m e a n steady-state u n i d i r e c t i o n a l flux from one tissue. T h e r e were at least four steadystate m e a s u r e m e n t s p e r point, a n d the S E M s were never m o r e t h a n + 0.1 log units. T h e s y m b o l at 110 m M represents the average --4-S E M for 10 tissues. T h e symbols are defined as in Fig. 7. In each e x p e r i m e n t the t a u r i n e c o n c e n t r a t i o n was 1.85 m M .

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substances c a n alter the active t r a n s p o r t o f the o t h e r substance. It was, therefore, o f general interest to e x a m i n e the possibility o f a reciprocal interaction b e t w e e n [Na+]o or [K+]o a n d taurine. O f m o r e specific interest, with r e g a r d to R P E function, is the possibility t h a t changes in the c o n c e n t r a t i o n o f t a u r i n e in the e x t r a c e l l u l a r space b e t w e e n the rod p h o t o r e c e p t o r s a n d the apical m e m b r a n e o f the R P E c o u l d affect the transepithelial m o v e m e n t s o f s o d i u m a n d / o r potassium. W e studied, therefore, the changes in u n i d i r e c t i o n a l c a t i o n flux as a f u n c t i o n o f external t a u r i n e c o n c e n t r a t i o n . W e f o u n d t h a t the u n i d i r e c t i o n a l s o d i u m fluxes were not significantly affected, 2 b u t t h e r e was a large effect o n active r u b i d i u m t r a n s p o r t . T h e i n t e r a c t i o n b e t w e e n t a u r i n e a n d p o t a s s i u m t r a n s p o r t was e x a m i n e d b y m e a s u r i n g S6Rb u n i d i r e c t i o n a l fluxes across the R P E as a f u n c t i o n o f extracellular t a u r i n e c o n c e n t r a t i o n ) In these e x p e r i m e n t s b o t h sides o f the tissue were initially b a t h e d in n o r m a l Ringer's (Methods) c o n t a i n i n g no taurine. Flux m e a s u r e m e n t s were m a d e over a 2 - 3 - h period, a n d t h e n t a u r i n e was a d d e d to the apical or basal solutions, a n d the e x p e r i m e n t c o n t i n u e d for a n o t h e r 2 - 3 h. In Fig. 9 the SSRb flux, r e t i n a to choroid, is p l o t t e d as a f u n c t i o n o f time. T h e initial steady-state flux in zero t a u r i n e Ringer's was 81 n M / c m 2 h , a n d after 3 h 5 m M t a u r i n e was a d d e d to the solutions b a t h i n g b o t h sides o f the tissue. T h e flux quickly rose to 145 n M / c m 2 h . T h e transepithelial p o t e n t i a l a n d resistance also decreased slightly. T h e decrease in T E P , n o t e d in the legend to Fig. 9, was d u e to a d e p o l a r i z a t i o n o f the apical membrane.* T h e r u b i d i u m fluxes were also m e a s u r e d at o t h e r t a u r i n e c o n c e n t r a t i o n s , a n d Fig. l0 is a s u m m a r y o f these data. E a c h d a t a point represents the m e a n s t e a d y - s t a t e flux in a s e p a r a t e e x p e r i m e n t . T h e r e were a total o f 25 experiments, 17 in the r e t i n a - t o - c h o r o i d direction (open symbols) a n d 8 in the c h o r o i d to r e t i n a d i r e c t i o n (closed symbols). Fig. 10 shows t h a t the choroid-tor e t i n a flux o f SSRb was u n a f f e c t e d w h e t h e r t a u r i n e was e l e v a t e d o n o n e or b o t h sides o f the tissue. In contrast, t h e r e t i n a - t o - c h o r o i d flux o f r u b i d i u m was 2 We assume, by analogy with many other systems, that a rise in cell sodium concentration will stimulate this pump (Schwartz et al., 1975; Joiner and Lauf, 1978). In nine experiments apical taurine (2 mM) increased the net choroid-to-retina sodium flux by an average of 110 nM/cm2h but this increase was not statistically significant. The presumed increase in Na-K pump rate, however, could have been masked by an alteration in apical and/or basal membrane sodium conductance. a Rubidium is a convenient substitute for potassium because it is cheaper and has a much longer half-life. This substitution is justified since rubidium and potassium compete for the same conductance channels in other systems (Solomon, 1952; Sjodin, 1959; Cavaggioni et al., 1973), and this seems to be also true at the apical and basal membrane of the RPE (unpublished observations). In addition, the RPE actively transports aeRb and 42K in the retina-to-choroid direction; in both cases the active transport was inhibited by ouabain, acting at the apical membrane (Miller, S. S., and Steinberg, R. H., manuscript in preparation). This is also true in a variety of other systems (Bonting, 1970). We also bathed the tissue in gbCl Ringer's and verified that the net taurine transport could be inhibited by reducing the concentration of rubidium from 2 to 0.2 mM. The net taurine flux was reduced by a factor of 2.5 as compared to 3.5 in potassium Ringer's (Fig. 4). 4 Miller, S. S., and R. H. Steinberg. Unpublished observations.

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e l e v a t e d at e v e r y c o n c e n t r a t i o n tested, f r o m 0.2 to 5 m M . C o m p a r i n g the a~Rb u n i d i r e c t i o n a l fluxes at zero t a u r i n e c o n c e n t r a t i o n shows t h a t r u b i d i u m is actively t r a n s p o r t e d across the retinal p i g m e n t e p i t h e l i u m at a p p r o x i m a t e l y 125 n M / c m 2 h . T h i s active t r a n s p o r t r a t e was s t i m u l a t e d b y increasing the t a u r i n e c o n c e n t r a t i o n in o n e (apical) or b o t h o f the b a t h i n g solutions a n d the m a x i m u m s t i m u l a t i o n o c c u r r e d at a c o n c e n t r a t i o n o f 1-3 m M . T h e s e d a t a i n d i c a t e t h a t the a6Rb, t a u r i n e i n t e r a c t i o n o c c u r r e d at the R P E apical membrane. 5

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TIME (h) FIGURE 9. Rubidium flux in the retina - * choroid direction with either 0 m M taurine or 5 m M taurine in the apical and basal solutions. These solutions contained 2 m M potassium and no "cold" rubidium. T h e steady-state flux was first measured with no taurine in the bathing solutions (first interrupted horizontal line) and at 3 h the taurine concentration was elevated to 5 m M in both solutions. The mean steady-state flux was increased by 64 nM/cm2h (second interrupted horizontal line). Otherwise as in Fig. 3. During the first part of the experiment the average T E P was 9.5 m V and the average Rt was 4.7 k~. After the taurine concentration was elevated to 5 m M , these values decreased to 6.5 m V and 4.25 kfl, respectively. I n f o r m a t i o n a b o u t the specificity o f the t a u r i n e t r a n s p o r t site was previously o b t a i n e d b y c o m p a r i n g the net t r a n s p o r t o f t a u r i n e a n d L - m e t h i o n i n e across the R P E (Miller a n d S t e i n b e r g , 1976). T h e net t r a n s p o r t o f L - m e t h i o n i n e was significantly s m a l l e r t h a n t a u r i n e , did not s a t u r a t e at h i g h c o n c e n t r a t i o n s (1.5 5 In Fig. 9 the tissue was bathed in Ringer's that contained 2 mM potassium and no "cold" rubidium. In the experiments summarized in Fig. 10, the Ringers contained 2 mM rubidium and no potassium. This difference in the composition of the Ringer's accounts for the difference in steady-state rubidium flux at 5 mM taurine (Miller, S. S., and R. H. Steinberg, manuscript in preparation).

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X 10-2M) and was not, in contrast to taurine, inhibited by apical ouabain. These results and others strongly suggested that taurine and L-methionine were transported across the R P E by different mechanisms. T h e specificity of the taurine transport site has been elaborated on in the course of the present study by two kinds of experiments. As in other systems (Christensen et al., 1954; Christensen, 1964; Reddy, 1968), it was shown (seven experiments) that ~-alanine, a ~-amino acid, competitively inhibited net taurine transport. This transport was 60% inhibited by 0.2 m M fl-alanine 600 ~E

500

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[Tourine ] o mM FIGURE 10. Rubidium flux as a function of external taurine concentration. The open symbols represent retina --+ choroid fluxes and the closed symbols choroid ~ retina fluxes. The rubidium concentration was 2 mM in both solutions for all the experiments shown. The circles represent experiments in which the taurine concentration was the same on both sides of the tissue; the squares and triangles represent experiments in which the taurine concentration was varied in one solution only (apical or basal) and maintained at 0 mM in the opposite solution. There were at least five steady-state measurements per experiment; the standard error in each was typically 8-15 nM/cm2h and never exceeded 30 nM/cm2h. The open and closed circles with standard error bars represent an average of 17 and 8 experiments, respectively.

and was 90% inhibited when fl-alanine was greater than or equal to 5 mM. T h e taurine concentration was 1.85 m M in these experiments. In the second class of experiments, the specificity of the interaction between rubidium and taurine was examined by measuring the retina to choroid flux of SeRb before and after the addition of 2 m M L-methionine tO the solutions bathing both sides of the tissue. In four experiments L-methionine caused no significant increase in the retina-to-choroid rubidium flux. This was in striking contrast to the effect of 2 m M taurine which increased the SeRb flux by 250 n M / c m 2 h (Fig. 10).

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DISCUSSION

The retinal pigment epithelium actively transports taurine, potassium, rubidium, and sodium (Miller and Steinberg, 1976; Miller and Steinberg, 1977 b; Fig. 10). The present study shows that: (a) small (1 raM) changes in extracellular potassium concentration modulate the active transepithelial movement of taurine; (b) there is a reciprocal interaction between taurine and potassium, that is, 1 m M changes in extracellular taurine concentration modulate the active transepithelial movement of rubidium (potassium); (c) relatively large (55 mM) changes in extracellular sodium concentration are required to alter active taurine transport, while the addition of taurine (2 raM) does not significantly alter active sodium transport; (d) these changes in ion or amino acid concentration are effective only from the apical (or neural retinal) side of the tissue; (e) the range of potassium ion concentration change, which modulates net taurine transport, approximates the narrow range known to be produced by light (Oakley and Green, 1976; Oakley, 1977).

Cation-Linked Transport of Organic Solutes A mechanism for the coupling of amino acids and other metabolites to cation transport was first suggested by Riggs and his co-workers (1958). They showed that Ehrlich mouse acites tumor cells concentrated amino acids to many times that of the environment. On the basis of this data it was suggested that potassium efflux from the cell drove amino acid influx by exchange diffusion (counter-transport). They realized that the exchange diffusion could also have been driven by sodium influx (co-transport), and in recent years the sodium gradient hypothesis has received support in most, but not all, systems (Schultz and Curran, 1970; Heinz, 1972; Schultz, 1977). In general, the electrochemical gradients of both sodium and potassium, along with a direct link to metabolic processes (ATP hydrolysis), could be used to accumulate amino acids, sugars, and other metabolites. The active accumulation of these solutes has been measured as a function of extracellular potassium concentration in a variety of preparations (Riklis and Quastel, 1958; Kleinzeller and Kotyk, 1961; Abadom and Scholefield, 1962; Bihler and Crane, 1962; Fox et al., 1964; Dantzler, 1974; Dantzler and Bentley, 1975). For example, in isolated guinea pig intestine potassium changes from 15 to 5 m M or 15 to 30 m M significantly inhibited the active absorption of glucose (Riklis and Quastel, 1958). These effects were apparently not due to permeability changes. As in the pigment epithelium, it was found that rubidium could effectively substitute for potassium in inhibiting transport. As another example, Sellstr6m and Hamberger (1975) showed that uptake of yaminobutryic acid (GABA) by glia-enriched fractions was maximal at 5.0 raM, the normal level of [K+]o; but the two comparison levels were 0 and 30 raM. It is characteristic of almost all of the above quoted studies that the potassium concentration changes were relatively large, compared to the present experiments, and therefore the cells were more vulnerable to nonspecific osmotic effects. Reddy (1968) studied the effects of small changes in extracellular potassium

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concentration on taurine accumulation in the rabbit ciliary body-iris preparation. He found that this accumulation could be increased monotonically, from approximately 3 to 100% of control, by increasing extracellular potassium in small steps, from 0 to 5 mM. In comparison, relatively large changes in extracellular sodium (148 --~ 73 mM) had a much smaller effect on taurine accumulation (28% of control). Similar relationships exist in the R P E between changes in [K+]o, [Na+]o, and taurine transport (Figs. 4 and 9). Reddy also showed that the active accumulation of taurine was inhibited by ouabain and fl-alanine. This is also true for the RPE (Miller and Steinberg, 1976; Fig. 11) and further suggests a basic similarity in the underlying transport mechanism. This similarity would not be surprising since the retinal pigment epithelium and ciliary body are anatomical extensions of one another and are embryologically of similar origin.

Na +- and~or K+-Linked Taurine Transport The dependence of taurine transport on small changes in [K+]o could implicate a direct link between taurine movement and the potassium electrochemical gradient. If taurine is carried across the R P E apical membrane by a K § counter-transport mechanism, which is driven by the apical K § electrochemical gradient, then increasing that gradient should increase the net apical to basal transport of taurine. From electrophysiological experiments on this tissue, the potassium electrochemical gradient (2 m M K § on both sides of the tissue) can be estimated as V,n - EK ----- + 12 mV, driving K § out of the cell. Vm is the apical membrane resting potential, and EK ---- --100 m V is the potassium equilibrium potential (Miller and Steinberg, 1977 a; Oakley et al., 1978). Decreasing [K+]o from 2 --* 0.2 m M would increase this driving force by 40-50 inV. Therefore, a carrier mechanism which accumulates taurine by counter-transport of potassium would cause an increase in the net apical-tobasal transport of taurine, not the observed decrease. We next considered the possibility of a Na, taurine co-transport mechanism. If sodium and taurine movements are coupled by co-transport on a shared carrier, then the net taurine transport should be reduced by a decrease in the sodium electrochemical potential gradient (Alvaredo and Mahmood, 1974; Christensen et al., 1974; Heinz, 1972; Schultz and Curran, 1970; Ahearn, 1977). It is known that the apical membrane of the RPE contains a Na-K p u m p whose electrogenicity can be removed by a reduction in [K+]o a n d / o r a rise in [K+]i (Miller et al., 1978; Oakley et al., 1978). It is possible, therefore, that lowering [K+]o (Fig. 4) reduced Na-pump efflux, increased internal sodium, and thereby caused a reduction in the sodium electrochemical potential gradient across the apical membrane. This mechanism does not require that the change in cell sodium be very large. The size of this change can be estimated by assuming a reasonable range of values for the cell sodium concentration (10-30 mM; Kernan and MacDermott, 1976; Zeuthen, 1978). For these concentrations the sodium equilibrium potential, ENa, would be 60 and 33 mV, respectively. These values can be used in Fig. 8 to calculate the reduction in [Na+]o, needed to produce

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a given reduction in taurine flux. For example, a reduction of approximately 60 n M / c m 2 h was produced by a decrease in apical [K+]o from 2.0 to 0.2 mM. According to Fig 8, this decrease could also be achieved by reducing [Na§ from 110 to 70 mM. In the case that cell sodium is 10 raM, this would decrease ENa from 60 to 49.5 mV. O n e can now calculate the increase in cell sodium that would be required to achieve this same reduction in ENa. From the Nernst equation, cell sodium would only have to increase from 10 to 15.7 mM. (In the case that cell sodium is 30 mM, the increase would be to 47 mM.) Fig. l0 also can be understood in terms of the sodium gradient hypothesis. This understanding follows from Figs. 1 and 2 (solid lines), which show that net taurine transport is stimulated as the taurine concentration increases from 0 to 2 mM. Sodium co-transport into the cell, across the apical membrane, therefore, would also be stimulated. If this caused a rise in internal sodium, stimulating the Na-K pump, then one could expect an increase in the active retina-to-choroid rubidium (potassium) flux. 2 We have assumed here that the sodium p u m p and the Na, taurine cotransport mechanism are spatially separate entities. This would not be true if there were a direct link between taurine transport and the apical m e m b r a n e bound Na-K ATPase (or the Na-K pump). In order to distinguish between these possibilities, the following experiment was performed. A microelectrode was placed in a RPE cell and the concentration of taurine in the apical bath was increased from 0 to 2 or 10 mM. The apical membrane potential began to depolarize as soon as the first taurine molecules arrived at the apical surface (not shown). Since taurine is a neutral amino acid, this suggests the stimulation of a Na +, taurine carrier, but it is also consistent with a taurine co-transport mechanism involving Na-K ATPase. If the observed voltage response depended directly on the Na-K ATPase (or the Na-K pump), then the prior addition of ouabain, 10-4M, to the apical bath should have eliminated this response. In fact, it was not eliminated and its size was unchanged 10-15 min after the application of ouabain. This experiment suggests therefore that the carrier and the p u m p are located at separate sites on the apical membrane. The Driving Force, Chemical or Electrical? Since some co-transport systems seem to be mainly driven by chemical or electrical gradients while others have contributions from both (Geck and Heinz, 1976; Crane, 1977; Kimmich and Carter-Su, 1978), it was of interest to determine whether the R P E falls into the former or latter category. It has been shown that the net retina--choroid flux of taurine is ouabain inhibitable (Miller and Steinberg, 1976). In these experiments, however, the first flux measurements were obtained 20 rnin after the application of ouabain, so that the inhibition may have been due to a decrease in the apical m e m b r a n e potential, the sodium chemical potential, or both. More recently, these measurements were repeated using a much larger (by a factor of 5) n u m b e r of microcuries on the "hot" side. This allowed the first flux measurements to be m a d e at a time when the apical m e m b r a n e electrogenic p u m p was still

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depolarizing. Previous electrophysiological experiments showed that the onset of this ouabain-induced depolarization coincided with the arrival of ouabain molecules at the apical membrane and that the time-course of this "fast" depolarization, 2V2-3 rain, was determined by the diffusion of ouabain molecules through the unstirred layer to the apical membrane (Miller et al., 1978). Since these experiments also indicated that the Na, K gradients did not start running down until 15 min after the application of ouabain, the flux measurements can provide evidence for the membrane potential contribution to taurine transport. One of the experiments is shown in Fig. 11, where the average steady-state flux before ouabain was 87 nM/cmZh. After approximately 4 h, 10-4M ouabain was added to the apical bath (see figure legend for details), and a flux sample was taken 5 min after the SCC began to change. The flux had fallen to 38 nM/em2h, which was significantly smaller than the pre-ouabain value. In five experiments the average pre-ouabain flux ( • SEM) in the retina to choroid direction was 94 + 12 nM/cm2h, and in the first flux period, 2V25 min after ouabain began to alter the apical membrane potential, the average flux was 18 • 12 nM/cm2h. The average basal to apical flux (five experiments) was 23.6 • 2 n M / c m 2 h and was not significantly altered by ouabain. Therefore, the net decrease in taurine flux coincided in time with the arrival of ouabain molecules at the apical membrane. (In control experiments, without ouabain, the steady-state flux obtained from a 5-rain sample was indistinguishable from the more usual 20- or 40-rain samples.) This means that the apical membrane potential forms part of the driving force responsible for active taurine transport. But if the potential change were the only factor controlling taurine transport, one could not explain, in Fig. 4, the observed increase in taurine flux with the apical membrane depolarization that accompanies an increase in [K+]o (Mi!ler and Steinberg, 1977 a). These data indicate, therefore, an electrical and chemical contribution to transepithelial taurine transport. At present, however, we cannot say to what extent each component is involved in the movement of taurine across the RPE.

Taurine and [K+]o in the Dark and Light It is now well established that taurine is the most abundant amino acid in the vertebrate retina, that a very substantial portion of this amino acid is located in the photoreceptors, and that it is actively accumulated by the pigment epithelium (Pasantes-Morales et al., 1972; Cohen et al., 1973; Kennedy and Voaden, 1974; Orr et al., 1976; Lake et al., 1977; Pourcho, 1977; Voaden et al., 1977). In cat it has been shown that a dietary deficiency of taurine disrupts outer segment structure and eventually leads to photoreceptor cell death (Schmidt et al., 1976; Berson et al., 1976). Retinal activity also can be altered by small changes in extracellular taurine, 0.1-5 mM. For example, extracellular concentration changes in this range can reversibly alter b-wave and ganglion cell activity in the retina (Pasantes-Morales et al., 1974; C u n n i n g h a m and Miller, 1976; Mandel et al., 1976). In the dark-adapted frog retina, the concentration of potassium in the

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subretinal space is a p p r o x i m a t e l y 3-4 m M (Oakley a n d Green, 1976; Oakley, 1977). A bright flash of light produces several effects in a dark a d a p t e d eye: (a) on a fairly rapid time scale (milliseconds), it severely reduces the entry of Na + into the outer segments causing the photoreceptors to hyperpolarize (Bortoff, 1964; Hagins et al., 1970; T o y o d a et al., 1970; K o r e n b r o t a n d Cone, 1972; Brown a n d Pinto, 1974); (b) on a longer time scale (seconds), it causes a decrease in potassium c o n c e n t r a t i o n outside the photoreceptors (Cavaggioni I

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TIME (h) FIGURE 11. Taurine flux in the retina --* choroid direction. The taurine concentration was 2 mM in both solutions. Otherwise as in Fig. 3. Tracer, a[H] taurine (50-75/~Ci) was added to the apical solution at t -- 0, and the apical-tobasal flux was sampled at 40-min intervals; the taurine flux reached a steadystate at o~2 h (interrupted horizontal line). The last pre-ouabain sample was taken at t = 4 h and is plotted at the midpoint of that sample period (t = 32/3 h). Approximately 2 min prior to t --- 4 h, 10-4M ouabain was added to the apical solution. This 2-min interval allowed the ouabain to reach the apical membrane and begin its effect on the membrane potential. The first postouabain sample period began with the onset of the change in TEP and was 5 min long. After this period the flux was sampled at 20-min intervals. Prior to the addition of ouabain, the average TEP was 11.3 mV and the average Rt was 3.5 k~. 1 h after the addition of ouabain, the TEP decreased to 2 mV and Rt rose to 4.1 k~. et al., 1973; Oakley a n d Green, 1976; Oakley, 1977). According to the d a t a s u m m a r i z e d in Fig 4, the potassium decrease should alter the net transport of t a u r i n e out of the retina, across the p i g m e n t epithelium. T h e situation in viva would be complex, however, since the past history of light exposure a n d presence or absence of other substances (taurine, for example) should also affect taurine transport. T h e past history of light exposure will set [K+]o at a particular level, a n d it will be the m a g n i t u d e a n d d u r a t i o n of the change from this level that will d e t e r m i n e the effect. An e x a m i n a t i o n of Fig. 4, for

MILLER AND STEINBERG Potassium-Modulated Taurine Transport

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example, shows that a 1.5 m M decrease in [K*]o from 3.0 to 1.5 m M should have a considerably smaller effect on taurine transport than the decrease from 2.0 to 0.5 raM. Although the in vivo relationship between taurine and potassium is undoubtedly complex, it seems worthwhile to incorporate what is known about the accumulation and transport of these substances into some suggestions about the possible functional significance of taurine inside the photoreceptors and in the extracellular space surrounding them.

Possible Functions of Taurine It has been shown in a variety of cells that osmotic swelling is counteracted by a substantial release of intracellular taurine (Fugelli and Zachariassen, 1976; Hoffman and Hendil, 1976). It is also known that light causes isolated outer segments to swell by approximately 6% (Enoch et al., 1973) and to release taurine (Salceda et al., 1977). This release m a y indicate the presence of an isoosmotic regulating mechanism that helps preserve osmotic balance during light stimulation. T h e increase in extracellular taurine due to a light-evoked photoreceptor release might be magnified by an effect on pigment epithelial transport. A bright flash of light, for example, can lower [K§ below 1 m M (Oakley, 1977), which would decrease the net taurine transport across the RPE (Table I and Fig. 4), and tend to increase the concentration of taurine in the extracellular space between the photoreceptors and the RPE. According to the data summarized in Fig. 10, any increase of taurine concentration between 0 and 2 m M should lead to an increase in the active transport of K § out of the retinal space and therefore help keep [K+]o at a relatively low level. After the light flash goes off, the taurine concentration outside the photo~ receptors should fall since the net efflux of taurine from the outer segments decreased to its dark level (Salceda et al., 1977). This fall would be enhanced by the rise in [K+]o (Oakley and Green, 1976; Oakley, 1977), which stimulates the transport of taurine out of the retinal space (Fig. 4). In order to assess critically the osmoregulatory function of taurine, one would need to compare, in the light and dark, the changes in extracellular ionic activity with the changes in intracellular and extracellular taurine activity. It would also be important to determine the mechanism that underlies the light-dependent movement of taurine across the outer segment membrane. We wish to thank Herman Chibnik and Paul Blum for technical assistance, and also Terry Machen for his helpful comments on the manuscript. This work was supported by research grants EY-02205 to Dr. Miller and EY-01429 to Dr. Steinberg from the National Eye Institute, National Institutes of Health.

Receivedfor publication 19 October 1978. REFERENCES ABADOM, P. N., and P. G. SC:HOLEFIELD.1962. Amino acid transport in brain cortex slices. III.

The utilization of energy for transport. Can.J. Biochem. Physiol. 40:1603-1618. AHEARN, G. A. 1976. Co-transport of glycine and sodium across the mucosal border of the

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