Rubidium transport in cultured monkey retinal pigment epithelium

Exp. Eye Res. (1992) 55, 289-296

Rubidium

Transport

in Cultured Epithelium BRIAN

Department

of Physiology

(Received

Monkey

Retinal

G. KENNEDY

and Biophysics, Northwest Center for Medical Education, School of Medicine, Gary, IN 46408, U.S.A.

Houston

2 July

Pigment

1991 and accepted

in revised

form 28 October

Indiana

University

1997)

Rb+ influx was used to assess Na-K-Cl cotransport and Na,K-ATPase activities in cultured monkey retinal pigment epithelium. Bumetanide-sensitive (Na-K-Cl cotransport-mediated) Rb+ influx exceeds ouabainsensitive (Na,K-ATPase-mediated) Rb+ influx, with these two transporters accounting for w 95 y0 of total Rb+ uptake. Half-maximal inhibition of Rb+ influx by bumetanide is attained at 75 nM bumetanide. The bumetanide-sensitive Rb+ influx depends on both extracellular Na+ and Cl-, and is activated by extracellular Rb’ with a relatively high afiinity. Na-K-Cl cotransport activity is stimulated (2.5fold) by increased extracellular osmolarity. Elevated CAMP content and glycolytic inhibition both depress cotransport activity. Cyanide application, however, had very little effect on Na-K-Cl cotransport activity. Monkey retinal pigment epithelial cells, maintained in culture, provide a system in which the activity and regulation of cation transport mechanisms can be examined. Key words : bumetanide; CAMP; Na-K-Cl cotransport : Na,K-ATPase : ouabain ; volume regulation.

1. Introduction The retinal pigment epithelium (RPE), a single layer of epithelial cells juxtaposed between the retina and choroid, serves a variety of metabolic functions. These cells actively phagocytize rod outer segments and store vitamin A. The epithelium also presents a diffusional barrier, which regulates ion, nutrient and water flow between photoreceptor cells and the choroidal blood supply (Steinberg and Miller, 1979). Many reports have examined the cation transport properties of the RPE (see Steinberg and Miller, 19 79, for a review). The RPE possesses both ouabain-sensitive Na,K-ATPase activity (Miller and Steinberg, 19 77 ; DiMattio, Degnan and Zadunaisky, 1983 ; Tsuboi, Manabe and Iizuka, 1986 ; Jaffe, Burke and Geroski, 1989) and Na-K-Cl cotransport activity (Frambach and Misfeldt, 198 3 ; Wiederholt and Zadunaisky, 1984; Tsuboi, Manabe and Iizuka, 1986; Miller and Edelman, 1990; Adorante and Miller, 1990; Kennedy, 1990; Joseph and Miller, 1991). These transporters perform crucial functions in maintenance of retinal homeostasis. The Na,K-ATPase of the RPE has been shown to regulate [K+] in the subretinal space (Linsenmeier and Steinberg, 1984 : Griff, Shirao and Steinberg, 1985 ; Miller and Edelman, 1990; Joseph and Miller, 1991), to sustain transepithelial fluid transport (Hughes, Miller and Machen, 1984), to catalyse transepithelial salt flux (Miller and Steinberg, 19 7 7 ; DiMattio, Degnan and Zadunaisky, 1983 ; Tsuboi, Manabe and Iizuka, 1986), and to maintain the Na+ electrochemical gradient which drives cotransport activities (Steinberg and Miller, 1979; Edwards, 1977; Ostwald and Steinberg, 1981). Though less well-characterized than the Na,K-ATPase, 0014-4835/92/080289+08 19

$08.00/O

Na-K-Cl cotransport has been implicated in fluid transport (Tsuboi and Pederson. 19 86), transepithelial Cl- flux (Frambach and Misfeldt, 1983: Tsuboi, Manabe and Iizuka, 198 6 ; Miller and Edelman, 1990), control of internal [Cl-] (Wiederholt and Zadunaisky, 1984; Joseph and Miller, 1991), and volume regulation (Adorante and Miller, 1990) in RPE. The present study examines Rb’ influx in cultured RPE isolated from monkey (MRPE). Rb’ is transported, with an affinity equivalent to that for K+, by both the Na,K-ATPase (Robinson and Flashner, 19 79) and the Na-K-Cl cotransport (O’Grady, Palfrey and Field, 198 7). Ouabain-sensitive Rb’ influx is routinely employed as an index of Na,K-ATPase activity (Robinson and Flashner, 19 79) while bumetanidesensitive Rb+ uptake serves as the index for Na-K-Cl cotransport activity (O’Grady, Palfrey and Field, 198 7 ; Haas, 1989). Rb’ influx has therefore ben used to assay Na,K-ATPase and Na-K-Cl cotransport activities in the MRPE cell culture system in order to characterize further the plasma membrane cation transport properties of the RPE.

2. Materials

and Methods

Cell Culture Primary cultures were established from monkey (A4ucaca fuscicularis) eyes obtained from animals employed in a non-related experimental investigation. Eyes were enucleated immediately after death, and then stored in saline at 4°C for up to 12 hr prior to dissection. Cultures were established as described by Yue and Fishman (1985). Briefly, the RPE-Bruch’s membrane-choroid complex was isolated and dis0 1992 Academic Press Limited EER55

290

sected into the - 0.2-cm” patches that were used to initiate cultures. RPE cells migrate out from these explants to form pure cultures (Yue and Fishman, 198 S), which are propagated as attachment-dependent monolayers on tissue culture plastic (25-cm” flasks, Falcon Primaria) at 3 7°C in a 9 5 % air/ 5 % CO, atmosphere. The culture medium was Eagle’s minimum essential medium supplemented with 10% fetal calf serum, 5% donor calf serum, essential and nonessential amino acids, 4 mM glutamine, amphotericin B and gentamycin sulfate. Upon attaining confluence (assessedby visual observation), cells were subcultured by trypsinization. All influx experiments employed confluent monolayers on 12-well culture plates (Corning). The work reported here was carried out with four separate cultures, each establishedfrom an individual animal. Cultures, unless noted, were used between the second and 13th passage. Solutions The standard solution used for uptake experiments is designated Na(Rb) and contained (in mM) NaCl (140) RbCl (S), CaCI, (2), MgCl, (1) glucose (5) Hepes (15) and Tris (8). A Na+-free solution used choline as an isosmotic replacement for Na’. The lowCl- solution (5 mM total Cl-) substituted sodium gluconate for NaCI. The Mg wash solution contained (mM) MgCI, (105). CaCl, (2), Hepes (15) and Tris (7). Solutions were adjusted to 290 mosmol kg-’ with a Wescor vapor pressure osmometer. The pH of all solutions was set to 7.4 at room temperature. Ouabain (Calbiochem) was diluted from a lo-mM stock solution in water to a final concentration of 100 ,UM. Bumetanide (a gift from Hoffman-LaRoche, Nutley, NJ) was diluted from a 20-mM stock solution in ethanol to give a final concentration of 10 ,uM. Stock solutions of isoproterenol (5 mM in water) and isobutylmethyl xanthine (IBMX, 50 mM in ethanol) were made on the day of the experiment. 86Rb as RbCl in water was purchased from Amersham. Other reagents were purchased from Sigma, Whittaker, Intergen, Hazelton and HyClone. Unidirectional @jRbInflux The protocol used in this investigation follows that of Kennedy and Lever (1984) as modified for use with cultured human RPE (Kennedy, 1990). Monolayers on 12-well plates were washed twice with Na(Rb) saline and then incubated for 10 min at 3 7°C in this solution. Uptake (10 min at 3 7°C) was initiated by addition of Na(Rb) saline containing 0.1-0.3 ,uCi mll’ 86Rb.Uptake was terminated by aspiration, followed by three rapid washes with ice-cold Mg wash buffer. As required, ouabain plus or minus bumetanide was present during both the incubation and uptake periods. Radioactivity was quantitated in a Packard Tri-carb liquid scintillation spectrometer, either by

6. G. KENNEDY 25 0 20 = zi $ Q z

15-

z

IO-

-

0/

:

0

/

5 ;/. 0

5

IO

15

20

Trne (mm)

FIG. 1. Time course for sGRbinflux into MRPE. Monolayers were preincubated at 37°C for 10 min in Na(Rb). “6Rb uptake was then determined, as described in Materials and Methods, for the time periods as noted. (Cells were from passage 16.)

Cherenkov radiation or with liquid scintillant (National Diagnostics, Ecoscint). ‘Zero time’ uptake, determined. by exposure of monolayers to 86Rb-labeledsaline followed immediately by the washing procedure described above, was subtracted from total uptake in each experiment. (‘Zero time’ uptake was < 5% of total uptake.) As shown in Fig. 1, Rb+ uptake was linear for 20 min at 37°C. Protein Determination Monolayers used for protein determination were treated in parallel with those used for uptake measurements. After the final Mg wash, monolayers were extracted for > 1 hr in a 2 % sodium carbonate/O.1 N NaOH solution. Protein was assayedin this extract by the method of Lowry et al. (19 5 l), with bovine serum albumin (Sigma, fraction V) as standard. Rb+ uptake was normalized to protein content. 3. Results Rb+ Uptake: Inhibition by Ouabainand Bumetanide Monolayer RPE cultures were establishedfrom four monkeys, and the results characterizing Rb’ influx into these cultures are summarized in Fig. 2. Ouabainsensitive (Na,K-ATPase-mediated) Rb’ influx, 0.469 + 0.033 (mean 4 s.E.)/Am01 Rb+ mg-’ protein hr-‘, comprises - 35 % of total Rb+ influx into these cultures. In comparison, bumetanide-inhibitable Rb+ influx is 0.730 f 0.030 (mean f S.E.) ,umol Rb+ mg-’ protein hr-I. Na-K-Cl cotransport activity thus comprises - 60% of Rb+ influx, a greater fraction than that mediated by Na,K-ATPase activity. Furthermore, the two transport mechanisms together account for approximately 9 5 % of Rb’ uptake in cultured MRPE. As noted in Materials and Methods, all experiments employed a HCO,--free, Hepes-buffered medium. In

RUBIDIUM

TRANSPORT

IN CULTURED

291

MRPE

I.50

T 2 T a

I 00

F B d 5

05c I o-00

I

o-10

,

I

I

I,

0 20 0.30

I

0.40

I

I

050

[Bumetonidel ooc Cotronsport

No, K-ATPose

FIG. 2. Ouabain-sensitive and bumetanide-sensitive Rb+ influxes. “6Rb uptake was determined in the Na(Rb) solution, as described in Materials and Methods. Where appropriate, transport inhibitors (ouabain 100 ym and bumetanide 10 ,uM) were present during both the lo-min incubation and the subsequent IO-min uptake periods. Each determination employed one 12-well plate, which was used to assay either ouabain-sensitive (total minus ouabain) or bumetanidesensitive (ouabain minus ouabain with bumetanide) activity. Values are meansfS.E. from 44 determinations for bumetanide-sensitive uptake, and from seven determinations for ouabain-sensitive uptake. Rb’ influx is almost completely eliminated in the presence of ouabain plus bumetanide. 0, Ouabain : q ( ouabain/bumetanide : q, bumetanide-sensitive : n , total ; q , ouabain-sensitive.

1.50( IF

-i 2 T a

r.ooc 3.

0.50( 3.

0

,

060

(I

,

0.70

,

/

0.80

IO

nw

FIG. 4 Concentration dependence for bumetanide inhibition of *‘jRb influx into MRPE. Uptake was determined as described in Fig. 2. Ouabain (100 pM) and bumetanide, at the concentration indicated, were present in both the incubation and uptake solutions. The curve is a non-linear least-squares fit (Entitter, Elsevier Biosoft) of the data to an equation of the form : uptake = ~V,W,,,)/(K,,, + bumetanide)) + R. K,,, is [bumetanide] for half-maximal inhibition. V, is total bumetanide-sensitive uptake and R is residual uptake. K,,, = 73 f 10 nM, V, = 0.76 f0.03, R =

0.05 + 0.02 pmol Rb+mg-’ protein hr-‘. but statistically significant, decrease in activity was noted as a function of time in culture. Though the effect is not large, it seemsthat Na-K-Cl cotransport activity may decrease as MRPE are maintained and passed in monolayer culture. (Further data, not included in this work, indicate that this downward trend continues in cells maintained up to passage20.) BumetanideSensitivity

.

E aD 2 zi

I

5

IO

I5

Passage

Fro. 3. Bumetanide-sensitive Rb’ influx as a function of time in culture. “Rb influx was determined as described in Fig. 2. Cultures were passed by trypsinization at 1-2-week intervals. All data in this Figure are from a single culture, which was passed 13 times. Values are means If: S.E. The line is the best least squares fit to the data. The negative slope ( - 2 % per passage) is significantly different from 0, P < 0.05 (Woolson, 1987).

cultured human RPE (Kennedy, 1990) 25 mM HCO,did not appreciably alter bumetanide-sensitive Rb’ influx. The data on Na-K-Cl cotransport activity was further analysed. as a function of tie in culture (number of cell passages).During the course of this study, bumetanide-sensitive Rb’ influx was measured in one culture line over 13 passages(Fig. 3). A slight,

To characterize the properties of Na-K-Cl cotransport activity in MRPE, Rb’ influx was assayed as a function of [bumetanide]. The data (Fig. 4) were fitted to a curve based on a single inhibitory site for bumetanide. Bumetanide acts with relatively high affinity: half-maximal inhibition is seenat 73 f 10 nM (mean f s.D.). In this work, 10 ,UM bumetanide is used to define Na-K-Cl cotransport activity. This concentration of bumetanide, while inhibiting > 99% of bumetanide-sensitive Rb+ uptake, is low enough only to minimally perturb other transporters (Haas, 1989). ionic Dependence of Bumetanide-sensitiveRb+ Influx Bumetanide was used, in the experiments summarized in Figs 24, to define the fraction of Rb+ influx that is mediated by Na-K-Cl cotransport activity. If this influx is, in fact, catalysed by the Na-K-Cl cotransporter, then it should require both extracellular Na+ and Cl-. As shown in Fig. 5, bumetanide-sensitive Rb’ influx clearly dependson both Na+ and Cl-. Na+ removal causesbumetanide-sensitive Rb’ influx to fall by + 90%. Moreover, bumetanide-sensitive Rb+ influx is virtually undetectable in a low-cl- solution (5 mM

292

B. G. KENNEDY 0.75

T

0.50 2

L F ir” 2

3.

0.25

0.00

0.10

0.20

0.30

0.40 External

0.50 [Rbl

OKI

0.70

080

J

IO’

m

6. Bumetanide-sensitive Rb’ influx as a function of extracelluar [Rb+]. 86Rb influx was determined as described in Fig. 2, except as noted. The standard Na(Rb) solution (see Materials and Methods) was modified so that [NaCl] was 130 mM. RbCl was varied from 1 to 10 mM, with choline chloride used as the isosmotic replacement (in all solutions pH was 7.4 and osmolarity was 290 mosmol kg-‘). These solutions are designated Na(x Rb) with x = 1,2, 5 or 10 mu. Monolayers were first incubated (37’C, 10 min) in the Na(5 Rb) solution and then washed twice with the desired Na(x Rb) solution. Uptake was then carried out (3 7°C. 5 min) in the same solution, supplemented with - 0.1 &i ml--‘, 86Rb. Ouabain was present in all incubation, wash and uptake solutions. Bumetanide, 1OpM when applied, was also present in the appropriate incubation, wash and uptake solutions. The line is a non-linear least-squares fit (Enzfitter, Elsevier Biosoft) of the data to the Michaelis-Menten equation, with Z$, = 1.3 mM and V, = 0.81 pmol Rb mg-l protein hr-‘. Cells were from passage 17. FIG.

Chol (Rb) Cl

No (Rb)

Cl

5. Ionic dependence

FIG.

No(Rb) Gluconatr

of Rb+ influx

in MRPE. 86Rb

influx was determinedas describedin Fig. 2. Monolayers were exposed to Na(Rb), chol(Rb) or Na(Rb, low Cl-) solution

at 37°C for 20 min (a IO-min

incubation

period

followed by a lo-min uptake period). All incubation and uptake solutions

contained

100

ouabain.

PM

Bumetanide

(10 pM when applied)was alsopresentin both incubation and

uptake

solutions.

All

solutions

were

adjusted

to

290 mosmolkg-‘, pH = 7.4. In the low-cl- solution,sodium gluconatewassubstitutedfor NaCl (final [Cl] was 5 mM). In the Na-free

solution

[chol(Na)],

choline

chloride

was

substitutedfor NaCl. Values are means+ S.E. q . ouabain; n , ouabain/bumetanide; q , bumetanide-sensitive. Cl-). This provides additional evidence that cultured MRPE possessNa-K-Cl cotransport activity. The Na-K-Cl cotransporter in a range of tissuesis stimulated by extracellular K+ or Rb’ (O’Grady, Palfrey and Field, 198 7). The effect of extracellular Rb+ (varied from 1 to 10 mM) on Na-K-Cl cotransport activity in MRPE is illustrated in Fig. 6. Extracellular Rb+ activates the transporter with relatively high affinity (K,,, = 1.3 mM). Uptake is saturated beyond 10 mM Rb’. Physiologically, extracellular [K+] can vary, in the range of 2-5 mM, during light/dark transitions in the intact mammalian retina (Steinberg, Oakley and Niemeyer, 1980; Linsenmeier and Steinberg, 1984). Osmolarity Na-K
CAMP The responseof the Na-K-Cl cotransporter to cyclic nucleotides (both CAMP and cGMP) is quite complex, varying from tissue to tissue and species to species (Haas, 1989). It has been shown that isoproterenol will elevate intracellular CAMP levels in cultured chick RPE (Koh and Chader, 1984) and in cultured human RPE (Hackett, Friedman and Campochiaro, 1986; Friedman, Hackett and Campochiaro, 1987). Furthermore, a phosphodiesteraseinhibitor, IBMX, also elevates CAMP content in the RPE (Miller and Farber, 1984). Basedon these observations, experiments were designedto assessthe effect of manipulations designed to elevate intracellular CAMP on Na-K-Cl cotransport activity in cultured MRPE. As shown in Table I, IBMX alone, or in combination with the /J-adrenergic agonist isoproterenol, significantly inhibits bumetanidesensitive Rb+ influx. Thus, manipulations designedto elevate CAMP levels seem to inhibit moderately ( N 3 5 %) Na-K-Cl cotransport activity in MRPE. Metabolic Dependence Na-K-Cl cotransport activity has been shown to depend on intracellular ATP (Russell, 1983 ; Ikehara et al., 1990). Figure 8 documents the effects of two distinct metabolic inhibitors on bumetanide-sensitive

RUBIDIUM

TRANSPORT

IN CULTURED

MRPE

293 1~001

\T \ o’--

T T l ----0

1

\

0

Bumetonide-sensitive

FIG. 7. Elevated external osmolarity and bumetanidesensitive Rb’ influx. Uptake was determined as described in Fig. 2. Monolayers were incubated with control (0, 290 mosmol kg-‘) or hypertonic ( n , 390 mosmol kg-l) media, containing ouabain (100 ,UM) with or without bumetanide (IO PM), for 5 min at 37°C. Uptake was then assayed for a further 10 min in an identical solution, plus afiRb. (Total exposure to the hypertonic media was thus 15 min, with uptake integrated over the final 10 min of exposure.} Control media was standard Na(Rb) media, while hypertonic mediawas Na(Rb)+ 100 mM mannitol. Values are meansf SE. TABLE I

Bumetanide-sensitive

___-Control

0.5 % ethanol

50 ,LLM isoproterenol 2 50 /LM IBMX 50 ILMisoproterenol/ 250 ,!4M IBMX

n

8 4 3 5 4

pmol

50 Time

(min

75

0

)

FIG. 8. Effect of metabolic inhibition on bumetanidesensitiveRb’ influx. s6Rbinflux was measuredasdescribed in Fig. 2. In each experiment, quadruplicate monolayers wereincubatedat 37°C for the timesasindicated,in Na(Rb) media(control) or in Na(Rb)mediapluseither 2 mMCN (0) or 5 mM 2-deoxy-n-glucose(DOG,0). (In the DOGsolution, 5 mM DOGwas substitutedfor the normal 5 mM glucose.) Ouabain (100 ,UM) plus or minus bumetanide( 10 pM) was addedto the incubation media10 min prior to the initiation of uptake. Uptakewas assayed,for 10 min. in an identical solutionsupplemented with ssRb.Time on the abscissa refers to the total incubation time, uptake beingassayedover the final 10 min of the incubation. Results(meansfS.E.) are expressed as a ratio : (uptake into the metabolically

Regulation of Na-K-Cl cotransport activity

Condition

25

inhibited

monolayer)/(uptake into its paired control monolayer). 0, CN; 0, DOG.

uptake

Rb mg Pr-’ hr^’ 0.598 kO.074 0.645 kO.124 0~510+0~105 *0.400 * 0.065 t0.383+_0.555

Values are presented as means f S.E. ‘“Rb uptake was determined as described in Materials and Methods. The control solution was Na(Rb). Additions. at the concentrations as indicated, were present during both the incubation (10 min) and uptake (10 min) periods. n refers to the number of experiments in which each condition was assayed. (Each experimental manipulation was not assayed in every experiment.) IBMX was added from a freshly made stock solution ( 50 mM) in ethanol. Final ethanol concentration in the presence of IBMX was thus 0.5%. A two-tailed Student’s t test for correlated groups was used to compare each of the experimental values to control. Experimental values which were found to differ from control beyond the P = 0.05 level are noted in the Table (*P < 0~01 : f P < 0.02).

Rb’ influx in MRPE. Na-K-Cl cotransport activity in MRPE does depend on ongoing glycolysis-the glycolytic inhibitor 2-deoxy-D-glucose (DOG) inhibits bumetanide-sensitive Rb+ influx. A decrease in influx is seen after only 20 min exposure to the inhibitor. In contrast, a respiratory inhibitor, cyanide (CN), has only a modest inhibitory effect on Na-K-Cl cotransport activity (solid symbols, Fig. 8).

4. Discussion The present work characterizes Rb+ (as an analog for K+) influx into RPE isolated from monkey, and maintained in cell culture. Rb’ influx into cultured MRPE is catalysed by both the Na,K-ATPase and the Na-K-Cl cotransporter, with these two transporters accounting for virtually all (95%) of the Rb’ uptake (Fig. 2). Bumetanide-sensitive Rb’ influx was dependent on the presence of both Na+ and Cl- in the external medium. Furthermore, several manipulations were shown to regulate Na-K-47 cotransport activity. Elevation of extracellular [Rb+], from 1 to 10 mM. approximately doubled transport activity. Transport was markedly (2.5-fold) stimulated by a 3 5 % increase in extracellular osmolarity. However, activity was decreasedby elevation of intracellular. CAMP, as well as by inhibition of glycolysis. On the other hand, Na-K-Cl cotransport was relatively insensitive to the mitochondrial inhibitor CN. Bumetanide inhibits Rb’ influx into cultured MRPE with a relatively high affinity (K,,, = 73 nM). In a variety of cell types, the bumetanide concentration producing half-maximal inhibition of Na-K-Cl cotransport ranges from 50 to 500 nM (Haas, 1989). The bumetanide sensitivity of Rb+ influ into MRPE is thus directly comparable to these values. Na,K-ATPase activity is also essential for RPE

294

function (Miller and Steinberg, 1977 ; DiMattio, Degnan and Zadunaisky, 198 3).-Of particular import is the RPE-dependent buffering of [K+] in the subretinal space (Miller, Steinberg and Oakley, 1978: Linsenmeier and Steinberg, 1984; Griff, Shirao and Steinberg, 198 5 ; LaCour, Lund-Andersen and Zeuthen, 1986 ; Miller and Edelman, 1990 ; Adorante and Miller, 1990; Joseph and Miller, 1991). Though not addressed in the present work, it should be noted that an apical localization of the Na,K-ATPase has been described in freshly isolated RPE-choroid preparations (Miller and Steinberg, 1977 ; Bok, 1982) but not in RPE cells maintained in culture (Rizzolo, 1990). Since control of synthesis and insertion of the Na,KATPase can be studied in cell culture (Karin and Cook, 198 3), cultured RPE could well provide a useful system in which factors determining epithelial polarity can be characterized. Several studies have described diuretic (furosemide or bumetanide)-sensitive ion transport in the RPE. This is consistent with the presence of Na-K-Cl cotransport activity in the RPE (Frambach and Misfeldt, 1983 ; Wiederholt and Zadunaisky, 1984; Tsuboi, Manabe and Iizuka, 1986 ; Miller and Edelman, 1990 ; Adorante and Miller, 1990 ; Kennedy, 1990; Joseph and Miller, 1991). The present study demonstrates a bumetanide-sensitive Rb’ influx which depends on extracellular Na+ and Cl--evidence for the presence of Na-K-Cl cotransport activity in cultured MRPE. The apparent affinity of the cotransporter for Rb+ (K,,? = 1.3 mM, Fig. 6) is in line with the affinities observed in a range of tissues (O’Grady, Palfrey and Field, 1987). Given its activation by extracellular Rb’, the Na-K-Cl cotransporter could function, with the Na,K-ATPase. in control of [K+] in the subretinal space. Cultured MRPE ,provide a useful system in which to study the Na-K-Cl cotransport due to the size, and excellent signal-to-background ratio of the bumetanide-sensitive influx (Fig. 2). The Na-K-Cl cotransporter plays a prominent role in the regulatory volume increase (RVI) displayed by a variety of cells (Haas, 1989). Figure 7 shows that the cotransport activity in MRPE is dramatically increased by elevated extracellular osmolarity. This response is consistent with the observations of Adorante and Miller (1990). Thus, increased external osmolarity would transiently decrease cell volume. Elevation of the Na-K-Cl cotransport activity, which would increase Cl-, Na’ and K+ influxes, would return cell volume to its resting level-the RVI. The effects of cyclic nucleotides (CAMP or cGMP) on Na-K-Cl cotransport activity. observed in a variety of cell types, are complex-both inhibition and activation have been described (Haas, 1989). The data in Table I would suggest, that in cultured MRPE, manipulations designed to elevate CAMP levels may serve to inhibit slightly Na-K-Cl cotransport activity. RPE possess adenylate cyclase activity which is stimulated by padrenergic activation (Koh and Chader, 1984;

5. G. KENNEDY

Friedman, Hackett and Campochiaro. 1987). Furthermore, CAMP has been shown to modulate RPE cell migration (Hackett, Friedman and Campochiaro, 1986) salt and water transport (Miller and Farber, 1984; Hughes, Miller and Machen, 1984) and membrane electrical parameters (Hughes et al., 19 8 8 : Nao-i, Gallemore and Steinberg, 1990). Thus, active absorption of Cl- across the frog RPE was inhibited by CAMP (Miller and Farber, 1984). The present work indicates that CAMP may also regulate the activity of a specific transport modality, the Na-KC1 cotransporter, in MRPE. However, it should be noted that the effect of isoproterenol plus IBMX is only slightly larger than the effect of IBMX alone (Table I). The relationship between regulation of this transport activity and other actions of CAMP (or possibly of cGMP) in the RPE requires further study. Na-K-Cl cotransport activity requires, though does not hydrolyse, ATP (Russell, 1983 ; Ikehara et al., 1990). The data presented in Fig. 8 are consistent with this ATP requirement : glycolytic inhibition depresses Na-KC1 cotransport activity. Of course, other perturbations which also result from glycolytic inhibition (changes in internal pH, Mg**, Ca2+ or Na’) could be important mediators of Na-K-Cl cotransport activity. It is notable that inhibition of respiration (with CN) has only minimal effects on Na-K-Cl cotransport activity. This would imply that, in cultured MRPE, at least one physiological function (Na-K-Cl cotransport) is demonstrably sensitive to glycolytic inhibition but little affected by interruption of respiration. Concerning 0, effects in the RPE. Arrindell. Horvath and Burke (1991) have shown that human RPE maintain a more epithelial morphology when cultured in a low (10%) 0, atmosphere. Similarly, Akeo. Ebenstein and Dorey (1989) have demonstrated O,dependent toxicity, in RPE cultured at moderate 0, tensions. Interestingly, work by Linsenmeier ( 19 8 6) indicated that 10 % 0, may more closely approximate the concentration present at the RPE in the intact eye, than the 20% 0, present in room air and routinely employed for cell culture. Furthermore, ongoing work (in cultured human RPE), has shown that Na,KATPase and Na-K-Cl cotransport activities, as well as ATP content, fall with glycolytic inhibition, but are relatively insensitive to CN treatment (Kennedy and Goldberg, 1991). However, it should be noted that in the RPE of the Gecko, Griff (1990) has documented a decrease in the TEP, and changes in apical and basolateral membrane potentials after CN exposure. Additional work to determine the effects of 0, tension and metabolic inhibition on ion transport in the RPE is required. Ion transport activity is critical to RPE function, and hence to retinal homeostasis. The present work demonstrates that one transporter which is central to this activity, the Na-K-Cl cotransporter, is regulated by extracellular osmolarity, cyclic nuclcotidc content and ceIlular metabolic activity. Future work must

RUBIDIUM

TRANSPORT

IN CULTURED

define how these parameters function ionic composition in the intact retina.

MRPE

to control

295

the

Acknowledgements I would like to thank Dr Stephen Echtenkamp for supplying the tissue from which the cultures used in this study were established. I would also like to thank Drs Carl Marfurt and Thomas Mueller for their helpful comments on the manuscript. This work was supported by a grant from the Northwest Center Medical Services Corporation.

References Adorante, J. S. and Miller, S. S. (1990). Potassium-dependent volume regulation in retinal pigment epithelium is mediated by Na,K,Cl cotransport. 1. Gem Physiol. 96, 1153-76. Akeo, K.. Ebenstein, D. B. and Dorey, C. K. (1989). Dopa and oxygen inhibit proliferation of retinal pigment epithelial cells, fibroblasts and endothelial cells in vitro. Exp. Eye Res. 49. 335-46. Arrindell. E. L., Horvath, K. and Burke, J. M. (1991). Effect of oxygen concentration on cytochrome oxidase activity in cultured human RPE. Invest. Ophthalmol. Vis. Sci. 32 (Suppl.), 1220 (Abstr.). Bok, D. (1982). Autoradiographic studies on the polarity of plasma membrane receptors in retinal pigment epithelial cells. In Structure of the Eye (Ed. Hollyfield, J. G.). Pp. 247-56. Elsevier: New York. DiMattio, J., Degnan, K. J. and Zadunaisky, J. A. (1983). A Model for transepithelial ion transport across the isolated retinal pigment epithelium of the frog. E;rp. Eye Res. 37, 409-20. Edwards, R. B. (19 77). Accumulation of taurine by cultured retinal pigment of the rat. Invest. Ophthalmol. Vis. Sci. 16. 201-8. Frambach, D. A. and Misfeldt, D. S. c.1983). Furosemidesensitive Cl transport in embryonic chicken retinal pigment epithelium. Am. 1. Physiol. 244, F679-85. Friedman, Z., Hackett, S. F. and Campochiaro, P. A. (198 7). Characterization of adenylate cyclase in human retinal pigment epithelial cells in vitro. Exp. Eye Res. 44, 471-9. Griff, E. R. (1990). Metabolic inhibitors reversibly alter the basal membrane potential of the gecko retinal pigment epithelium. Elcp. Eye Res. 50, 99-107. Griff. E. R.. Shirao. Y. and Steinberg. R. H. (1985). Ba2+ unmasks K+ modulation of the Na+-K+ pump in the frog retinal pigment epithelium. 1. Gen. Physiol. 86, 853-76. Haas, M. (1989). Properties and diversity of (Na-Kxl) cotransporters. Ann. Rev. Physiol. 51, 443-57. Hackett. S., Friedman, Z. and Campochiaro, P. A. (1986). Cyclic 3’, 5’-adenosine monophosphate modulates retinal pigment epithelial cell migration in vitro. Arch. Ophthalmol. 104, 1688-92. Hughes, B. A., Miller, S. S., Joseph, D. P. and Edelman, J. L. (1988). CAMP stimulates the Na-K pump in frog retina1 pigment epithelium. Am. 1. Physiol. 254, C84-98. Hughes, B. A., Miller, S. S. and Machen, T. E. (1984). Effects of cyclic AMP on fluid absorption and ion transport across frog retinal pigment epithelium. J. Gen. Physiol. 83. 875-Y9. Ikehara. T., Yamaguchi, H.. Hosokawa, K. and Miyamoto, H. (1990). Kinetic mechanisms of ATP action in Na-K-Cl cotransport of HeLa cells determined by Rb influx studies. Am. I. Physiol. 258, C599-609. Jaffe, G. J., Burke, J. M. and Geroski, D. H. (1989). Ouabainsensitive Na-K ATPase pumps in cultured human retinal pigment epithelium. Exp. Eye Res. 48, 61-8.

Joseph, D. P. and Miller, S. S. (1991). Apical and basal membrane ion transport mechanisms in bovine retinal pigment epithelium. 1. Physiol. 435, 439-63. Karin, N. J. and Cook, J. S. (1983). Regulation of Na,KATPase by its biosynthesis and turnover. Cut-r. Topics Membr. Trans. 19, 713-51. Kennedy, B. G. (1990). Na-K-C1 cotransport in cultured cells derived from human retinal pigment epithelium. Am. J. Physiol. 259, C29-34. Kennedy, B. G. and Goldberg, S. E. (1991). Transport activity and ATP content in cultured human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 32 (Suppl.), 83 7 (Abstr.). Kennedy, B. G. and Lever, J. E. (1984). Regulation of Na,KATPase activity in MDCK kidney epithelial cell cultures : role of growth state, cyclic AMP and chemical inducers of dome formation and differentiation. J. Cell Physiol. 121, 51-63. Koh. S.-W. and Chader. G. J. (1984). Retinal pigment epithelium in culture demonstrates a distinct /?adrenergic receptor. Exp. Eye Res. 38, 7-13. LaCour, M., Lund-Andersen, H. and Zeuthen, T. (1986). Potassium transport of the frog retinal pigment epithelium: autoregulation of potassium activity in the subretinal space. 1. Physiol. 375, 461-79. Linsenmeier. R. A. (1986). Effects of light and darkness on oxygen distribution and consumption in the cat retina. 1. Gen. Physiol. 88, 521-42. Linsenmeier, R. A. and Steinberg, R. H. (1984). Effect of hypoxia on potassium homeostasis and pigment epithelial cells in the cat retina. J. Gen. Physiol. 84, 945-70. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-75. Miller, S. S. and Edelman, J. L. (1990). Active ion transport pathways in the bovine retinal pigment epithelium. 1. Physiol. 424, 283-300. Miller, S. and Farber, D. (1984). Cyclic AMP modulation of ion transport across frog retinal pigment epithelium. J. Gen. Physiol. 83, 853-74. Miller, S. S. and Steinberg, R. H. (1977). Active transport of ions across frog retinal pigment epithelium. Exp. Eye Res. 25, 235-48. Miller, S. S., Steinberg, R. H. and Oakley, B. (1978). The electrogenic sodium pump of the frog retinal pigment epithelium. J. Membrane Biol. 44, 259-79. Nao-i, N., Gallemore, R. P. and Steinberg, R. H. (1990). Effects of CAMP and IBMX on the chick retinal pigment epithelium. Invest. Ophthulmol. Vis. Sci. 31, 54-66. O’Grady. S. M., Palfrey, H. C. and Field, M. ( 198 7). Characteristics and functions of Na-K-Cl cotransport in epithelial tissues. Am. J. Physiol. 253, Cl 77-92. Ostwald, T. J. and Steinberg, R. H. (1981). Transmembrane components of taurine flux across frog retinal pigment epithelium. Curr. Eye. Res. 1, 43 7-43. Rizzolo, L. J. (1990). The distribution of Na,K-ATPase in the retinal pigment epithelium from chicken embryo is polarized in vivo but not in primary cell culture. Exp. Eye Res. 51, 435-46. Robinson, J. D. and Flashner, M. S. (1979). The (Na+ K)ATPase. Enzymatic and transport properties. Biochim. Biophys. Actu 549, 145-76. Russell, J. M. (1983). Cation coupled chloride influx in squid axon. 1. Gen. Physiol. 81, 909-25. Steinberg, R. H. and Miller, S. S. (1979). Transport and membrane properties of the retinal pigment epithelium. In The Retinal Pigment Epithelium. (Eds Zinn. K. M. and Marmor. M. F.). Pp. 205-24. Harvard University Press: Cambridge, MA.

296

B. G. KENNEDY

Steinberg, R. H.. Oakley, B. and Niemeyer, G. (1980). Lightevoked changes in [K’], in retina of the intact cat eye. 1. Neurophys. 44, 897-921. Tsuboi, S., Manabe, R. and Iizuka, S. (1986). Aspects of electrolyte transport across isolated dog retinal pigment epithelium. Am. J. Physiol. 250, F781-4. Tsuboi, S. and Pederson,J. E. (1986). Experimentalretinal detachment.XI. Furosemide-inhibitable fluid absorption across retinal pigment epithelium in vivo. Arch. Ophthalmol.

104. 602-3.

Wiederholt. M. and Zadunaisky,J. A. (1984). Decreaseof intracellular chloride activity by furosemidein frog retinal pigmentepithelium. Curr. Eye Res. 3. 673-5. Woolson,R. F. (1987). Statistical Methods for the Analysis oj Biomedical Data. Pp. 280-313. John Wiley and Sons: New York. Yue, B. Y. J. T. and Fishman, G. A. (1985). Synthetic activities of cultured retinal pigment epithelial cells from a patient with retinitis pigmentosa. Arch. Ophthalmol.103, 1563-6.