283

Journal of Physiology (1990), 424, pp. 283-300 With 6figure8 Printed in Great Britain

ACTIVE ION TRANSPORT PATHWAYS IN THE BOVINE RETINAL PIGMENT EPITHELIUM

BY SHELDON S. MILLER AND JEFFREY L. EDELMAN From the University of California, School of Optometry and Department of Molecular and Cell Biology, Division of Biophysics and Cell Physiology, Berkeley, CA 94720, USA

(Received 31 May 1989) SUMMARY

1. Radioactive tracer flux measurements demonstrate that active ion transport across the isolated bovine retinal pigment epithelium (RPE)-choroid preparation can be maintained for hours after the eye is enucleated and the tissue removed from the eye. 2. It has been shown that 86Rb tracer fluxes can be used to monitor potassium (K+) transport across bull-frog RPE. In bovine RPE, net 86Rb (K+) absorption is zero. Apical barium (Ba2+) elevated active K+ absorption from zero to t 03 gequiv cm-2 h-1. This Ba2+-induced increase in active K+ absorption was inhibited either by ouabain or bumetanide in the apical bath. 3. In control Ringer solution, buffered with bicarbonate and CO2' the RPEchoroid actively absorbs chloride (Cl-) at a rate of 05 /sequiv cm-2 h-1. In contrast, sodium (Na+) is secreted at a rate of 0O5 juequiv Cm-2 h-. Chloride absorption was inhibited by apical bumetanide, and Na+ secretion was inhibited by apical ouabain. These drugs were only effective when placed in the solution bathing the apical or retinal side of the tissue. 4. Net Cl- absorption requires an exit mechanism at the basolateral membrane. DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid) in the basal bath completely inhibited net Cl- absorption in bicarbonate-free Ringer solution. 5. These experiments show that the chloride transport pathway contains at least two components: (1) a bumetanide-sensitive uptake mechanism at the apical membrane; and (2) an efflux mechanism at the basolateral membrane that is blocked by DIDS. 6. Three apical membrane mechanisms were identified that could help modulate [K+]0 in the subretinal or extracellular space that separates the distal retina and the RPE apical membrane. They are: (1) an ouabain-sensitive Na+-K+ pump; (2) a bumetanide-sensitive mechanism, the putative Na+-K+-Cl- co-transporter; (3) a barium-sensitive K+ channel that recycles, to the apical bath, most or all of the potassium that is actively taken up by the Na+-K+ pump and the co-transporter. 7. These data suggest that light-induced alterations in subretinal potassium that occur in vivo can activate the chloride transport pathway and help modulate RPE intracellular Cl- during transitions between the light and dark. -

MS 7720

284

S. S. MILLER AND J. L. EDELMAN INTRODUCTION

The retinal pigment epithelium (RPE) is made up of a single layer of cells that lie in the back of the vertebrate eye and separate the distal retina and the choroidal blood supply. The apical membrane faces the photoreceptor outer segments and their surrounding extracellular, or subretinal, space. The proximal boundary of this subretinal space is formed by the Muller (glial) cells that tightly surround the basal portion of the photoreceptor inner segments and extend to the vitreal surface of the retina. All three of these cells help regulate the chemical composition of the subretinal space. For example, it has been shown that the photoreceptors play a fundamental role in determining the time course of the potassium (K+) concentration changes that occur in the subretinal space during light-dark transitions (Oakley & Green, 1976; Oakley, 1977; Oakley, Steinberg, Miller & Nilsson, 1977; Shimazaki & Oakley, 1984). The RPE apical membrane and the Muller cells also contain a variety of K+dependent transport mechanisms that can modulate, or be modulated by, subretinal potassium concentration ([K+]O) changes during the light-dark transitions (Miller & Steinberg, 1977a, 1979, 1982; Miller, Steinberg & Oakley, 1978; Newman, 1985; Immel & Steinberg, 1986; la Cour, Lund-Andersen & Zeuthen, 1986; Edelman, Miller & Hughes, 1988; Adorante & Miller, 1989; Lin & Miller, 1989). Information about retina-RPE interactions in the light and dark have come from a variety of mammalian and non-mammalian preparations (Griff & Steinberg, 1982; Linsenmeier & Steinberg, 1982; Steinberg, Linsenmeier & Griff, 1985). In contrast, in vitro studies of RPE ion and fluid transport have mainly, but not exclusively, utilized amphibian preparations (Lasansky & De Fisch, 1966; Miller & Steinberg, 1977 a, b; Steinberg, Miller & Stern, 1978; Hughes, Miller & Machen, 1984; Miller & Farber, 1984; Hughes, Miller & Farber, 1987; Segawa, 1987). Recently, more information, using transepithelial potential measurements has become available in several mammalian preparations (Paulter & Tengerdy, 1986; Tsuboi, Manabe & lisuka, 1986; Tsuboi, 1987; Frambach, Valentine & Weiter, 1988). The experiments presented here provide a first step in the analysis of the membrane mechanisms that control ion transport at the apical and basolateral membranes of the bovine RPE. We investigated three apical membrane mechanisms that help regulate K+ transport across the RPE; one of them is a bumetanidesensitive mechanism that actively absorbs potassium (K+) and chloride (Cl-) across the apical membrane. The other two are a Na+-K+ pump and a Ba2+-sensitive K+ channel. These three mechanisms, along with the basolateral Cl- channel, have been analysed at the single-membrane level in a separate series of electrophysiological experiments (D. Joseph and S. S. Miller, unpublished). These transport mechanisms are exquisitely sensitive to small changes in apical [K+]. (3 mM), similar to those produced in the subretinal space of the intact eye following transitions between light and dark (Miller, Joseph & Edelman, 1988). A preliminary account of some of this work has been presented in abstract form (Edelman & Miller, 1987).

ACTIVE TRANSPORT IN BOVINE PIGMENT EPITHELIUM

285

METHODS

The bovine eyes used in these experiments were obtained from a local slaughterhouse 15-40 min after death, placed in ice-cold Ringer solution and transported to the laboratory. The Ringer solution used for transporting and dissecting the eyes was the same as the control Ringer solution used during the experiments. Each eye was sectioned behind the lens into posterior and anterior portions. The posterior portion, consisting of retina, pigment epithelium, choroid and sclera. was then cut into sections of 4 cm2. Each piece was pinned retina-side up in a wax-lined Petri dish filled with saline. A stainless-steel circular punch was used to obtain a piece of retinal pigment epithelium-choroid-sclera of area 1-13 cm2. The neural retina was discarded and the choroidalseleral connections gently cut and teased away. The pigment epithelium-choroid preparation was then placed on a supportive mesh and mounted in a chamber adapted from the design of Miller & Steinberg (1977 a, b) with the following changes. A larger exposed tissue area was used in this study (03 cm2) and the chamber had a water-jacketed flow system controlled by a Lauda K-2/R temperature circulator so that the bathing solutions were kept at 38 + 0-1 'C. The composition of the bathing solutions was 120 mM-NaCl, 23 mM-NaHCO3, 5 mM-KCl, 1-0 mMMgCl2, 1-8 mM-CaCl2, 10 mM-glucose, 1-0 mM-glutathione and gassed with 85 % N2 10% 02 and 5% CO2. Nominally HCO3 /C02-free Ringer solution was made by substituting 23 mM-NaHCO3 with 13 mM-sodium cyclamate and 10 mM-N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES), titrated to pH 7-5 with NaOH. This solution was equilibrated with room air. Gas was injected into apical and basal bathing solutions via stainless-steel 30 gauge needles which facilitated mixing and kept pH at approximately 7-4. The combination of low 02 (10%) and glutathione was used since it dramatically increased the longevity of the preparation presumably by protecting the bovine pigment epithelium against oxidation (Winkler & Giblin, 1983). In the intact cat eye it has been shown that the 02 tension is 80-100 mmHg at the RPE (Linsenmeier, 1986). In the absence of glutathione and at high 02 levels the bovine RPE preparation could not hold stable flux measurements and tissue resistance for more than 2 h. In the presence of 1 mmglutathione and at lowered 02 levels the preparation held stable tissue resistance and steady-state flux values for 5 h or longer (not shown). Neither addition of glutathione or lower 02 levels alone aided in preparation longevity. Ringer-agar bridges were placed on each side of the tissue to measure transepithelial potential (TEP, apical side positive) and pass short-circuit current (SCC). A voltage clamp (Physiologic Instruments VCC600, San Diego, CA, USA) was used to measure SCC, periodically pass 5 /tA current pulses across the tissue, and measure the current-induced changes in TEP. Transepithelial resistance (RJ) was calculated from the current-induced TEP changes. In the short-circuited state compensation for the fluid resistance (100 Q) was provided by the voltage clamp. The choroidal resistance, 9 Q cm2, was small enough to ignore. Tissue pairs were electrically 'matched' and taken from the same eye in more than 90 % of the experiments. In these tissue pairs the initial SCC never differed by more than 25 % and the initial values of Rt did not differ by more than 15 %. Flux measurements were only carried out using tissues with Rt > 100 Q cm2. This criterion was chosen on the basis of a series of observations using ouabain. In RPE, unlike almost all other epithelia, the Na'-K' pumps are located on the apical membrane (Miller et al. 1978; Ostwald & Steinberg, 1980; Bok, 1982; Miller & Steinberg, 1982; Hughes, Miller, Joseph & Edelman, 1988). This conclusion was tested during the course of an electrophysiological study of transport mechanisms in the apical and basolateral membranes of the bovine RPE. In these experiments (D. Joseph & S. S. Miller, unpublished) ouabain was added to the basal bathing solution and, in most cases, there was no change in apical or basolateral membrane potential. However, for tissues with Rt less than 90 Q cm2 (TEP < 2 mV), basal ouabain depolarized the apical membrane at a greater rate than the basolateral membrane, as if the inhibitor had diffused to the apical bath. This suggests the presence of a significant amount of edge damage, which probably occurred during the process of mechanically sealing the tissue in its holder. The method for determining transepithelial unidirectional fluxes of 22Na, 36Cl and 86Rb (42K substitute) was similar to that of Miller & Steinberg (1982) except that in most experiments 'cold' samples were taken every 10 min. In all cases in which ouabain was added to the apical bathing solution tissue integrity was lost within 40 min; experiments with apical ouabain were therefore sampled every 5 min. Samples were assayed using a Beckman LS 7500 scintillation counter. 22Na -

286

S. S. MILLER AND J. L. EDELMAN

and 86Rb were obtained from Amersham Corp. and 36CL- from ICN. Ouabain, BaCl2 and DIDS (4,4'diisothiocyanostilbene-2,2'-disulphonic acid) were obtained from Sigma Chemical Co. and bumetanide was a generous gift from Hoffman-La Roche. RESULTS

The active transport of sodium, chloride and potassium across the RPE was assessed by measuring the steady-state unidirectional fluxes of 22Na, 36Cland 86Rb from the retinal-to-choroidal (apical-to-basal, A -- B) and choroidal-to-retinal (basalto-apical, B --HA) side of the tissue. Table 1 summarizes the results from a series of experiments using paired tissues from the same eye. For each isotope at least nine pairs of tissues were used (numbers in parentheses, columns 2-4). In a few cases one of the paired tissues lost its seal in the middle of an experiment and had to be discarded but in most experiments both tissues remained in the steady state for an hour or more. Table 1 also shows that the electrical properties (TEP, apical side positive; Rt and SCC) of these paired tissues are not significantly different. In the case of 22Na, Table 1 shows that the B -- A flux is significantly greater than the A-R B flux (P < 005) and that there is a net secretion of sodium, 0 48 tequiv cm-2 h-1, in the choroid-to-retina direction. In contrast, the RPE actively absorbs chloride and the mean retina-to-choroid flux is 0-51 ,tequiv cm-2 h-1. Finally Table 1 shows that the net potassium flux, as measured by 86Rb, is not significantly different from zero. This result differs from that of the bull-frog RPE where it has been shown that the tissue absorbs potassium at a small but significant rate, mainly through the apical membrane Na+-K+ pump (Miller & Steinberg, 1979, 1982).

Chloride transport Figure 1 shows the data from a typical 36C1 flux experiment. The bottom half of the figure summarizes the steady-state unidirectional fluxes (O and 0) and in this case the net rate of chloride absorption was t 0-5 gequivncm-2 h-1. The data in this figure give a clear picture of the variability in the steady-state unidirectional flux measurements and allow one to compare this variability with the magnitude of the net flux and the magnitude of subsequent drug-induced changes in net flux (Figs 2 and 3). The upper half of the figure shows the short-circuit current as a function of time for each tissue (O and 0). The values of TEP and Rt are given at the top of the figure and the values in parentheses are for the experiment summarized with closed circles. During the course of this experiment the transepithelial resistance was practically unchanged in both tissues while the SCC and TEP decreased by ; 30 %, a common finding in these experiments. In a variety of epithelia it has been shown that chloride absorption (or secretion) is coupled to that of sodium and potassium through a bumetanide-inhibitable electroneutral Na+-K+-CP- co-transporter (O'Grady, Palfrey & Field, 1987). In bovine (and bull-frog) RPE we have observed that bumetanide significantly inhibits the net absorption of both chloride and potassium (see below), suggesting that K+ and Cl- may also be coupled at the RPE apical membrane. Figure 2 shows that apical bumetanide (10-4 M) completely inhibited net chloride

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absorption across the bovine RPE. Prior to the addition of bumetanide to the apical was 0-53 tequiv cm-2 h-1 and this net flux was brought to zero, mainly by a reduction in the retina-to-choroid unidirectional flux. Bumetanide also caused a small decrease in the choroid-to-retina unidirectional flux.

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The decrease in SCC, ; 0 5 /equiv cn-2 h-1 (Table 2), was somewhat smaller than the decrease in net 36CI flux suggesting that bumetanide may have also inhibited the active transport of some other ion(s). Table 2 summarizes a series of these experiments (eight tissues, four eyes) in which the net flux was brought to approximately zero in each case. It is worth noting that bumetanide at 10-6 M also inhibited chloride absorption by ~ 30 % (not shown). Net chloride absorption requires an exit mechanism at the basolateral membrane. There is strong electrophysiological evidence for a basolateral membrane Clconductance that is blocked by DIDS (Miller et al. 1988). If this conductive mechanism provides the pathway for 36CI exit, then the retina-to-choroid flux should also be blocked by basal DIDS. However, it has been shown, in frog RPE and in other systems, that DIDS also blocks HC03-{C1P or Na'-dependent HCO3-Cl-

ACTIVE TRANSPORT IN BOVINE PIGMENT EPITHELIUM TEP (mV) 3-2 (4-7) Rt(W cm2) 138 (156)

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60 80 100 120 Time (min) Fig. 2. Effect of apical bumetanide (10-4 M) on unidirectional 36CI fluxes, retina-to-choroid (0) and choroid-to-retina (@). Bumetanide (10-4 M) was added at t = 60 min to the apical bathing solution of two tissues, from the same eye. Net active 36CI absorption decreased from 0 53 sequiv cm-2 h-' to approximately zero, mainly due to a decrease in the retinato-choroid unidirectional flux (0). SCC was decreased by practically the same amount (see also Table 2). Otherwise as in Fig. 1.

TABLE 2. Apical bumetanide-induced changes in 36CI transport Retina to choroid (A -* B) Control Bumetanide Unidirectional flux (Izequiv cm-2 h-') 3-98 + 0-36 (n = 4) 3-13 + 043 SCC (,uequiv cm-2 h-') 1P28+0-27 0-84+0'21 TEP (mV) 5.1+1 1 2'8+0'5 R (Q cm2) 152+12 130+ 7 Choroid to retina (B -O A) Control Bumetanide Unidirectional flux (,uequiv cm-2 h-') 3-29 + 0 09 (n = 4) 3'02 + 0'31 SCC (tequiv cm-2 h-1) 1P12+0-19 0-62+0-08 TEP (mV) 4*5+0-6 2'3+06 152+4 128+13 R, (Q cm2) Net flux (,uequiv cm-2 h-') 0'69 (A B)* 0'11 (A+B)**

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Values are the mean+s.E.M. for six steady-state flux measurements per tissue over 1 h. The number of paired tissues (n) and eyes is listed in the control column. The levels of significance (unpaired t test) are given in the far right-hand column. Apical bumetanide (10-4 M) reduced the net 36C1 flux (last row) from 0'69 Sequiv cm-2 h-' (*P = 0'03) to a value not significantly different from zero (**P = 0-61; unpaired t test). The bumetanide-induced change in net 36Cl flux was 0-58 sequiv cm-2 h-'. 10

PHY 424

S. S. MILLER AND J. L. EDELMAN

290

exchangers (Fong, Bialek, Hughes & Miller, 1988; Hughes, Adorante, Miller & Lin, 1989). To minimize the possible effects of HC03--dependent transport mechanisms, the experiment summarized in Fig. 3 was carried out in HEPES buffer. In the absence of substrate these putative HC03--dependent chloride mechanisms should not contribute to the movement of 36Cl across either membrane. TEP (mV) 7.6 (6-6) Rt(Q cm2) 216 (204)

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Figure 3 shows that basal DIDS (2 mM) completely inhibited net chloride absorption across the bovine RPE. Prior to the addition of DIDS to the basal bathing solution the net flux was 05 jequiv Cm-2 h-1 and this net flux was brought to zero, mainly by a reduction in the retina-to-choroid unidirectional flux. In contrast, DIDS had relatively little effect on the choroid-to-retina unidirectional flux. The DIDS-induced changes in SCC, TEP and Rt were small and difficult to quantify. This is not unexpected because the apical and basolateral membranes are tightly coupled electrically and the conductance of the paracellular pathway is eight times that of the cellular pathway (D. Joseph and S. S. Miller, unpublished). In a total of six tissues basal DIDS significantly reduced the A -÷ B unidirectional flux by 0 73 + 0 14 jequiv cm-2 h-1 (mean + s.E.M.; P = 0 02). The B -*A flux was also reduced, but this decrease was not statistically significant (0-20 + 0 09 tequiv cm-2 h-1, P = 0-12). In summary, these experiments show that the chloride transport pathway across the RPE contains at least two components: (1) a bumetanide-sensitive uptake mechanism at the apical membrane; and (2) an efflux mechanism at the basolateral membrane that is blocked by DIDS.

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TABLE 3. Apical ouabain-induced changes in 22 Na transport Retina to choroid (A -* B) Control Ouabain = Unidirectional flux (,zequiv cm-2 h-1) + 2-52 0-24 (n 4) 3-32+0 35 SCC (,sequiv cm-2 h-') 1-62 + 0-32 0-34+0-09 TEP (mV) 5*0+1-0 0.5+0.1 R. (1 cm2) 119+7 115+3 Control Choroid to retina (B -- A) Ouabain Unidirectional flux (,uequiv cm-2 h-') 3-04+0-27 (n = 3) 3-17+0-24 SCC (sequiv cm-2 h-') 1-65+0-36 0-40+0.10 TEP (mV) 6-0+1-2 0-6+0-3 R, (Q cm2) 141+17 138+ 16 0-52 (B--A)* 0.15 (A- B)** Net flux (/tequiv cm-2 h-1) 1

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Values are the mean+ S.E.M. for six to eight steady-state flux measurements per tissue over 40-60 min. Otherwise as in Table 2. Apical ouabain (10-5 M) reduced net 22Na flux (last row) from 0-52 (sequiv cm-2 h-) (*P < 0-05) to a value not significantly different from zero (**P = 0-71; unpaired t test). The ouabain-induced change in net 22Na flux was 0-67,uequiv cm-2 h-'. 10-2

292

S. S. MILLER AND J. L. EDELMAN

Sodium transport In bull-frog RPE it has been demonstrated that the apical membrane contains a Na+-K+ pump that is, in large part, responsible for the active movement of sodium and potassium across the epithelium (Miller et al. 1978; Ostwald & Steinberg, 1980; Bok, 1982; Miller & Steinberg, 1982). Some evidence for an apical membrane TABLE 4. Apical ouabain inhibition of 86Rb (K+) transport P Control Ouabain Retina to choroid (A -* B) + flux 014 0-15 + 0-046 0031 0 39 Unidirectional (,uequiv cm-2 h-') (n =4) 0.11+0-02 002 SCC (/zequiv cm2 h-) 108+0-18 TEP (mV) 3-4+1 2 005 0.1+0.1 R (1 cm2) 125+15 108+5 0-20 Control Ouabain P Choroid to retina (B -O A) 0'15 Unidirectional flux (#equiv cm-2 h-1) 0-17+0-019 (n = 3) 0-19+0-017 0 30+ 011 0 05 SCC (,#equiv cm 2 h-') 1-56+040 TEP (mV) 4-6+2-1 05+0-14 0-02 R ( cm2) 109+12 103+7 0-34 0-028 (B--A)* 0-036 (B-.A)** Net flux (,uequiv cm-2 h-') Values are the mean+S.E.M. for eight steady-state flux measurements per tissue over 40 min (sample period, 5 min). Format otherwise as in Table 2. Apical ouabain (10-5 M) had no significant effect on either of the unidirectional fluxes (column 5) or on net 86Rb (K+) flux (*P = 061, **P = 0 35; unpaired t test). t

ouabain-sensitive mechanism has also been obtained in cat and dog RPE (Steinberg et al. 1978; Tsuboi et al. 1986). In this paper we present a more detailed analysis of the bovine Na+-K+ pump and leak system. Figure 4 shows the steady-state unidirectional fluxes of 22Na across paired tissues from the same eye (bottom panel, 0 and 0). During the first hour the net secretion was approximately 0 5 sequiv cm2 h1 and at t = 60 mi10 M-ouabain was added to the solution bathing the apical membrane. The secretary flux was largely unaffected but the absorptive flux increased such that the net active sodium transport was brought to zero. This pattern of change induced by ouabain has also been observed in bull-frog and dog RPE (Miller & Steinberg, 1977a; Tsuboi et al. 1986). Figure 4 also shows that ouabain rapidly decreased the TEP towards zero but had relatively little effect on the transepithelial resistance. The data summarized in Table 3 corroborate the results shown in Fig. 4. These data were obtained from seven tissues and four different eyes and from three of these eyes we were able to obtain a complete set of flux measurements (A -+ B and B -> A) from electrically well-matched tissues. Column 2 in Table 3 (last row) shows that under short-circuit conditions sodium is actively secreted across the RPE at a rate of t 0-52 /equiv cm-2 h-1 and column 3 (last row) shows that this net flux is not significantly different from zero in the presence of apical ouabain (10-5 M). This result is consistent with the location of an ouabain-inhibitable Na+-K+ pump at the apical membrane (Discussion).

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Fig. 5. Effect of apical barium (1 mM) on unidirectional 8f6Rb (K+) fluxes in retina-tochoroid (0) and choroid-to-retina (@) directions. In paired tissues from the same eye apical barium, added at t = 60 min, increased net 86 Rb (K+) absorption from zero to t 0 4 /equiv cm-2 h-1. TEP also decreased. Otherwise as in Fig. 1.

TABLE 5. Ba2+ induced changes in 86Rb (K+) transport Retina to choroid (A -, B) Control Ba2+ p Unidirectional flux (,uequiv cm-2 h-1) 0-15+0-016 (n = 4) 049+00064 0 005 SCC (,uequiv cm-2 h-1) 1-42 + 030 0-58+0-14 0.01 TEP (mV) 4*3+0*9 1-8+0-5 0*01 Rt (Q cm2) 117+2 113+ 1 0-20 Choroid to retina (B -*A) Control Ba2+ p Unidirectional flux (#equiv cm-2 h-1) 0-16+0-020 (n =4) 0-16+ 0-019 0-38 SCC (,uequiv cm-2 h-1) 1.91+0-28 0-83 + 0-02 0003 TEP (mV) 5-7+1.0 2-2+0-4 0-02 Rt (Qj cm2) 116+ 5 116+8 0-97 Net flux (,uequiv cm-2 h-') 0-01 (B-÷ A)* 033 (A -.B)** Values are the mean+ S.E.M. for six steady-state flux measurements per tissue over 1 h. Format otherwise as in Table 2. In control Ringer solution the net 86Rb flux (last row) was not significantly different from zero (*P 088). Apical Ba2+ (1 mM) elevated the net 86Rb (K+) flux from zero to 0 33 #uequiv cm2 h' (**P < 0e001). =

= | I ~ ~ ~ OAK- Bflux S. S. MILLER AND J. L. EDELMAN

294

Potassium transport If the apical membrane does contain a Na+-K+ pump that mediates transepithelial sodium and potassium transport one would also expect apical ouabain to inhibit active 86Rb (K+) absorption. Table 1 (column 4, last row) and Table 4 (column 2) TEP

Rt ( cm2) 2.0 00

>

Ci)

0~

1.0

L +

3-9 161

4.1 154

4.8 147

13 Ba2' (apical)

0@6~

l 0

-0-5 105

0

|

O.

X,

-0.3 126

Ouabain (apical)

0

X

0.1 147

1-4 154

~~I 20

40 Time (min)

0

I

60

o

T

___

80

Fig. 6. Effect of apical ouabain (10-5 M) on the unidirectional retina-to-choroid 86Rb (K+) flux. The tissue was pre-treated (t = 0) with apical barium (1 mM), which elevated the steady-state 86Rb flux to 0 55 lsequiv cm-2 h-1. At t = 40 min, 10-5 M-apical ouabain reduced the Ba2+-stimulated flux to control levels, 0-15 sequiv cm-2 h-. Otherwise as in Fig. 1. -

TABLE 6. Apical ouabain inhibition of Ba2+-stimulated 86Rb (K+) transport P Ba2+ Ouabain Retina to choroid (A-H B) 0 009 0-19 + 0-017 0'53 + 0-066 Unidirectional flux (Izequiv cm-2 h-1) 0-004 0-32+0-05 1-01+0-12 SCC (,uequiv cm-2 h-1) 0.01 0-8+0.1 3-6+04 TEP (mV) 0.10 131+11 140+8 Rt(Qcm2) tissue over 40 min flux measurements per Values are the mean+ S.E.M. for eight steady-state (n = 8 tissues). Format otherwise as in Table 2. A comparison of columns 2 and 3 shows that ouabain (10-5 M) significantly inhibited the Ba2+-induced increase in 86Rb (K+) flux.

show that in control Ringer solution the net flux of 86Rb (K+) is not measurably different from zero. Furthermore, ouabain had no effect on either of the unidirectional fluxes (Table 4; Discussion). These results can be understood if one assumes that potassium is actively taken up

ACTIVE TRANSPORT IN BOVINE PIGMENT EPITHELIUM

295

at the apical membrane by the Na+-K+ pump and then recycled to the apical bath via an apical membrane K+ channel that is barium inhabitable. It has been shown that the apical membranes of bull-frog and bovine RPE both contain an ouabainsensitive Na+-K+ pump and that the relative K+ conductance is very large and almost completely blocked by Ba2+ (Miller & Steinberg, 1982; Hughes et al. 1988; D. Joseph and S. S. Miller, unpublished). The data shown in Figs 5 and 6 strongly support this interpretation. In Fig. 5 we used electrically paired tissues from the same eye and measured the unidirectional 86Rb fluxes in both directions across the tissue (0 and 0, bottom panel). Consistent with the data summarized in Tables 1 and 4 the net 86Rb (K+) flux was zero. At t = 60 min, I mM-barium was added to the solution bathing the apical membrane. This caused an immediate decrease in SCC and TEP with no apparent change in Rt. As noted above, most of the transepithelial current pulse ( 84 %) for measuring Rt moves through the paracellular pathway. Most importantly, Fig. 5 clearly shows that blockade of the apical membrane K+ channels significantly increased the active absorption of 86Rb (K+), from zero to 0-51 jtequiv cm-2 h-1. This result was corroborated in a series of four experiments using electrically matched tissues from four eyes (Table 5). Column 1 shows that in control Ringer solution the net active absorption of rubidium is practically zero and that the addition of 1 mM-barium to the apical solution (column 3) increased the mean absorption rate from zero to 0-32 jequiv cm-2 h-1. In order to test this hypothesis further we measured the Ba2+_stimulated, 'active', retina-to-choroid flux of 86Rb, first in the absence and then in the presence of apical ouabain. The choroid-to-retina or 'passive' fluxes were not measured in this set of experiments because they are unaffected by barium or ouabain (Tables 4 and 5). Figure 6 shows that in the presence of 1 mM-Ba2+ the retina-to-choroid flux is elevated to 0-5 /tequiv cm-2 h-1, consistent with the results summarized in Table 5 and Fig. 5. At t = 40 min 10-5 M-ouabain was added to the solution bathing the apical membrane. This caused an immediate reduction in the retina-to-choroid unidirectional flux and brought the steady-state flux almost back to the level observed in control Ringer solution, 0415 Itequiv cm-2 h-1 (Tables 1 and 5). This result was corroborated in the set of experiments summarized in Table 6. In five experiments using electrically matched tissues from five eyes the mean steadystate flux in Ba2+ Ringer solution was 0 53 tequiv cm-2 h-1 and this flux was reduced by apical ouabain (10-5 M) to 0 19 /aequiv cm-2 h-1. Since barium and ouabain had practically no effect on the choroid-to-retina fluxes (Tables 4 and 5) the flux changes observed in Fig. 6 and Table 6, 0 34 jtequiv cm-2 h-1, are equivalent to changes in net active transport. Although Table 6 shows that a large fraction of the Ba2+-induced increase in active 86Rb absorption is blocked by apical ouabain some potassium could have entered the cell via the putative Na+-K+Cl- co-transporter. If such a co-transporter is operating then apical bumetanide should, at least partially, inhibit net 86Rb flux across the RPE. Consequently, we compared the barium-stimulated retina-to-choroid 86Rb flux in the absence and presence of apical bumetanide (10-4 M). In five experiments the barium-stimulated unidirectional 86Rb (K+) flux was 060+0005 ,tequiv cm-2 h-1 (mean+S.E.M.) and this was reduced to 0-39+0-02 tequiv cm-2 h-1 by apical -

296

S. S. MILLER AND J. L. EDELMAN bumetanide. Again, this difference, 021 /sequiv cm-2 h-1, represents a change in net active transport because barium (Table 5) and bumetanide (data not shown) had no appreciable effect on the unidirectional choroid-to-retina 86Rb fluxes. In summary, both ouabain and bumetanide inhibited the Ba2+-stimulated active 86Rb absorption across the bovine RPE, by 034 and 0-21 /sequiv cm-2 h-1, respectively. These results suggest, but do not prove, that the bumetanide-sensitive mechanism helps mediate net 86Rb (K+) absorption across the RPE apical membrane (see below). DISCUSSION

Active ion transport across the isolated bovine RPE-choroid can be maintained for hours after the eye is enucleated and the tissue removed from the eye. The present experiments show that the apical membrane contains at least three mechanisms that can be modulated by (or modulate) the light-induced changes in subretinal [K+]o: (1) a ouabain-sensitive Na+-K+ pump; (2) a bumetanide-sensitive mechanism, the putative Na+-K+{Cl- co-transporter; and (3) a Ba2+-sensitive K+ channel. Evidence for a basolateral membrane DIDS-sensitive Cl- exit mechanism is presented here, and more directly by showing that basal membrane chloride diffusion potentials are inhibited by basal DIDS (Miller et al. 1988). Given the directions of active transport, the basolateral membrane must also contain transport proteins that allow K+ exit and Na+ entry. Indirect evidence is also provided for a HCO3- absorption mechanism (see below). Ouabain-senrsitive transport Evidence for the apical membrane location of the Na+-K+ pump is twofold. First, sodium is actively secreted across the RPE at a rate of ; 050 ,uequiv cm-2 h- and this active transport is completely blocked by apical ouabain (Tables 1 and 3, and Fig. 4). Secondly, in the presence of apical Ba2+, there is a large active absorption of 86Rb (K+), which is also inhibited by apical ouabain (Fig. 6, Tables 5 and 6). Although ouabain blocked net transport its effect on the unidirectional fluxes was unexpected. Net flux was brought to zero by an increase in the A -* B unidirectional flux with practically no change in the B -+ A or pump direction (Table 3). One might have expected ouabain to decrease the unidirectional flux in the pump direction, as observed in other epithelia. One possibility is that ouabain inhibited the pump and also decreased the shunt resistance. This could have reduced the cellular pump flux and increased, in approximately equal amounts, both unidirectional fluxes through the paracellular pathway. This notion, which would explain the data in Table 3, remains to be tested. A very similar pattern of changes has been observed in bull-frog RPE (Miller & Steinberg, 1977 b). In control Ringer solution, the net 86Rb (K+) flux across this epithelium is zero and it was striking to observe that apical Ba2+ increased the active K+ flux from zero to 032 ,uequiv cm-2 h-1 (Table 4). This indicates that most of the 86Rb (K+) that enters the apical membrane via the K+ limb of the pump then recycles back to the apical bath through the apical membrane K+ channel. Additional support for this notion comes from experiments in frog RPE where it was shown, using double-barrelled K+-

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specific microelectrodes, that apical Ba2+ significantly increased intracellular K+ (Hughes et al. 1988). The data summarized in Fig. 6 and Table 6 show that most of the Ba2+-stimulated 86Rb flux is ouabain inhabitable, as expected if the Na+-K+ pump is involved in the recycling process at the apical membrane. Since net K+ flux is zero in control Ringer solution one might have expected apical ouabain to alter net K+ flux, at least transiently. However, in control Ringer solution, ouabain had no effect on either of the unidirectional 86Rb fluxes (Table 4).

Bicarbonate absorption Table 3 shows that the decrease in sodium secretion only accounts for approximately 50% of the ouabain-induced decrease in SCC. Since ouabain had no effect on active Cl- (n = 2, not shown) or 86Rb transport (Table 4) the only other ions that are available to carry current across this tissue are H+ in the secretary direction or HCO3- in the absorptive direction. There is strong, indirect evidence for HCO3absorption in toad and bull-frog RPE (Lasansky & De Fisch, 1966; Miller & Steinberg, 1977 a; Hughes et al. 1984; Miller & Farber, 1984) and recently it has been shown that the apical membrane of the bull-frog RPE contains an electrogenic, Na+-HCO3- co-transporter that regulates intracellular pH and helps transport HC03- across the RPE in the absorptive direction (Hughes et al. 1989; Lin & Miller, 1989). In the frog, this co-transporter, along with a chloride-dependent mechanism, provides the main driving force for fluid absorption across the RPE (Hughes et al. 1987). A similar set of mechanisms exist in the bovine RPE (D. Joseph & S. S. Miller, unpublished).

Bumetanide-sensitive transport Tables 1 and 2 and Figs 1-3 show that the bovine RPE actively absorbs chloride and that this chloride transport system is completely inhibited by apical bumetanide (10-4 M). Much lower concentrations of this drug (10-6 M) also inhibited chloride absorption by t 30% (not shown). Table 2 also shows that the decrease in net 36C1 flux and the decrease in short-circuit current are practically identical suggesting that the bumetanide-sensitive mechanism is entirely responsible for active chloride absorption across the RPE apical membrane. On the basis of unidirectional chloride flux data and the inhibitory action of furosemide or bumetanide it has been suggested that the RPE apical membrane contains a NaCl or Na+-K+-Cl- co-transporter (Frambach & Misfeldt, 1983; Tsuboi et al. 1986). However, other interpretations are possible because these inhibitors can also block Cl--HCO3- antiporters and anion channels (Widdicombe, Nathanson & Highland, 1983; Knickelbein, Aronson, Schron, Seifter & Dobbins, 1985; Evans, Marty, Tan & Trautmann, 1986; O'Grady et al. 1987; Pirani, Evans, Cook & Young, 1987). More importantly, this hypothesis can only be verified by a variety of additional experiments that establish the obligatory coupling of all three ions, at a fixed stoichiometry (Geck & Heinz, 1986; O'Grady et al. 1987). In other systems it has been shown that the Na+-K+-Cl- co-transporter is electroneutral and moves two or three chloride ions per potassium ion (Russell, 1983;

S. S. MILLER AND J. L. EDELMAN 298 O'Grady et al. 1987). From the present data one can estimate the K+: Clstoichiometry of the co-transporter if it is assumed that bumetanide has no direct or secondary effects on the Na+-K+ pump. Support for this assumption comes from the observation that bumetanide had no effect on the unidirectional Na+ fluxes, either in bovine or bull-frog RPE (not shown). This is contrary to what one would observe if bumetanide significantly inhibited the pump. The K+: Cl- stoichiometry was estimated by comparing the inhibitory effects of bumetanide on the active absorption of K+ and Cl- across the RPE. During the 86Rb+ experiments K+ recycling was blocked with apical barium which, by itself, has no effect on Cl- transport in the bovine RPE (n = 1; not shown). Apical bumetanide reduced net Cl- absorption by 010-58 #sequiv cm-2 h-' (Table 2) and it also reduced the Ba2+-stimulated K+ flux by 021 ,tequiv cm-2 h-1 (Results). If K+ and Cl- are coupled then these results suggest a K+: Cl- co-transport stoichiometry of 1: 2 or 1: 3. Clearly, additional kinds of experiments will be required to confirm this conclusion. Co-transporter role in retinal pigment epithelium physiology The present paper shows that the putative Na+-K+-Cl- co-transporter is responsible for the active uptake of Cl- and perhaps K+ at the apical membrane. It has been shown (Miller et al. 1988; D. Joseph and S. S. Miller, unpublished) that intracellular Cl- is significantly altered in response to apical [K+]o changes, similar to those that occur in the subretinal space of the intact eye following transitions between light and dark (Steinberg et al. 1985). These light-dark transitions produce a component of the DC-recorded electroretinogram (ERG) or electro-oculogram (EOG) known as the 'fast oscillation' (FO) (Steinberg et al. 1985), which is clinically useful for detecting RPE-retinal pathology (Weleber, 1989). This component of the ERG (or EOG) is probably generated by the chloride transport pathway (Miller et al. 1988). In a variety of systems, epithelial and non-epithelial, it has been shown that the Na+-K+Cl- co-transporter is under cyclic nucleotide regulation and provides the driving force for fluid transport and volume regulation (O'Grady et al. 1987). In the RPE, it has been shown that fluid transport is driven, in part, by active Cl- transport and inhibited following a rise in cell cyclic AMP (Miller & Farber, 1984; Hughes et al. 1984, 1987; Tsuboi, 1987). Recently, it has been shown that the Na+-K+-Cl- co-transporter provides the driving force for volume-regulatory changes in frog RPE (Adorante & Miller, 1989). These regulatory changes in cell volume are dependent on apical [K+]. over the same range (2-5 mm) produced by light-dark transitions in the intact eye; and they are blocked by apical bumetanide or the removal of extracellular Na+ or Cl- from the apical bathing solution. The functions of these volume-regulatory changes remain to be determined. They may, for example, play a role in retinal-RPE adhesivity or protect the cell during the phagocytic ingestion of photoreceptor outer segments. The present in vitro data make it seem likely that the putative Na+-K+-Cl- cotransporter plays a crucial role in the intact eye during light-dark transitions. Given the strategic location of this co-transporter at the retina-RPE interface, it could be an important source or target of retinal-RPE pathophysiology. This work was supported by NIH grants EY02205 (SM), RCDA EY00242 (SM) and Core Grant EY03176.

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