Transport of acidic amino acids by the bovine pigment epithelium

Transport of acidic amino acids by the bovine pigment epithelium

Exp. Eye lies. (1986) 43, 207-214 Transport of Acidic Amino Acids by the Bovine Pigment Epithelium E. L. PAUTLER A N D C. T E N G E R D Y Departme...

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Exp. Eye lies. (1986) 43, 207-214

Transport of Acidic Amino Acids by the Bovine Pigment Epithelium E.

L. PAUTLER

A N D C. T E N G E R D Y

Department of Physioloyy and Biophysics: College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, U.S.A. (Received 30 September 1985 and in revised form 6 February 1986) The regulation of acidic amino-acid transport across the retinal pigment epithelium is of particular interest since glutamate and possibly aspartate have been identified as putative neurotransmitters in the retina, at the level of the photoreceptor ceil. The present study, designed to measure the rate of acidic amino-acid transport across the mammalian pigment epithelium (PE), shows that there is a net transi~rt of both glutamate and aspartate in the retina to choroid direction (R-C), with tile R-C unidirectional flux of glutamate being substantially larger than the corresponding aspartate flux. The ~ and C-R fluxes of glutamate were found to be inhibited by ouabain. Further investigations utilizing aspartate revealed t h a t the fluxes in both directions were inhibited when ouab,~in was present.on the retinal side of the tissue preparation. The R-C flux of glutamate was significantly reduced by lowered concentrations of l~a+, K + and Ca 2+, whereas the C~R flux was diminished only by the reduced concentration of Ca2+. The changes in K + concentration which markedly altered the ~ flux of glutamate were within the range of light-induced changes of K + which has been observed in the extracellular space of the photoreceptor cells. The transporting system appears to be. relatively specific for the acidic amino acids; tbr aspartate was an effective competitive inhibitor of glutamate transport whereas basic (lysine) and neutral (leucine) amino acids were not. The directionality, ouabain sensitivity, ionic dependence and sub.~trate specificity of the transmembrane fluxes tend to support the concept of active transport as a mechanism of acidic amino-acid removal from the neural retina. hey words: amino acids ; transport; a.upartate; glutamate; pigment epithelium.

1. I n t r o d u c t i o n

We have chosen to study the transport of the acidic amino acids (glutamate and aspartate) across the mammalian pigment epithelium (PE) since glutamate and p o s s i b l y a s p a r t a t e m a y b e i n v o l v e d in s y n a p t i c t r a n s m i s s i o n a t t h e l e v e l o f t h e p h o t o r e c e p t o r cell ( I s h i d a a n d F a i n , 1981 ; L a s a t e r a n d D o w l i n g , 1 9 8 2 ; B r a n d o n a n d L a i n , 1983), a n d m e c h a n i s m s w h i c h m a y c o n t r i b u t e t o t h e r e g u l a t i o n o f t h e 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 s in t h e r e t i n a a r e o f c o n s i d e r a b l e i n t e r e s t . The transport of amino acid5 across pigment epithelial tissues has not been e x t e n s i v e l y s t u d i e d . E d w a r d s (1977) has d e m o n s t r a t e d t h e a c t i v e a c c u m u l a t i o n o f t a u r i n e b y c u l t u r e d r e t i n a l p i g m e n t e p i t h e l i a l cells. M i l l e r a n d S t e i n b e r g ( 1 9 7 6 ) h a v e s t u d i e d t h e t r a n s p o r t o f t a u r i n c a n d L - m e t h i o n i n e in t h e f r o g P E a n d h a v e s h o w n t h a t t h e n e t f l u x o f t a u r i n e in t h e r e t i n a t o c h o r o i d d i r e c t i o n ( t L - C ) is g r e a t e r t h a n t h a t of L-methionine. The taurine flux was also modulated by the extracellular concentrat i o n o f p o t a s s i u m [K+]o, i n r a n g e s w h i c h c o ~ e s p o n d t o t h e l i g h t - i n d u c e d a l t e r a t i o n s o f K + l e v e l s s u r r o u n d i n g t h e p h o t o r e c e p t o r culls (Miller a n d S t e i n b e r g , 1 9 7 9 ) . I n a b s t r a c t f o r m , S e l l n a r a n d M a s l a n d (1.983) h a v e r e p o r t e d ' a n e t f l u x o f m s p a r t i c a c i d , l e u c i n e , l y s i n e , s e r i n e a n d t y r o s i n e in t h e r e t i n a - t o - c h o r o i d d i r e c t i o n o f t h e f r o g PE, but few details were provided on the ionic and metabolic dependence of the 0014-4835/86/080207 +08 $O3.OO/0 8

~)1986 Academic Press Inc. (London) Limited E~R 43

208

E.L. P A U T L E R

AND

C. T E N G ' E R D Y

transport systems. W e n o w report on the effect of ionic changes as well as competitive substrates and a metabolic inhibitor on the transport of acidic amino acids'across the" isolated bovine PE.

2. M a t e r i a l s a n d M e t h o d s B o v i n e eyes w e r e o b t a i n e d a t a local a b a t t o i r a n d t r a n s p o r t e d t o t h e l a b o r a t o r y in .~, ice-cold saline. T h e s c l e r a w a s r e m o v e d f r o m t h e p o s t e r i o r p o r t i o n o f t h e eye, a n d a 3- t o 4 c m 2 piece o f r e t i n a was excised. T h e n e u r a l r e t i n a w a s t h e n g e n t l y p e e l e d a w a y , a n d t h e r e m a i n i n g t i s s u e vonsisting o f R P E a n d c h o r o i d a l r e m n a n t s - - w a s m o u n t e d between two lucite plates. The mounting chip was coated with a thin covering of silicone g r e a s e a n d p l a c e d in a U s s i n g c h a m b e r . T h e c h a m b e r a n d c h i p d e s i g n w e r e s i m i l a r to t h o s e d e v e l o p e d b y Miller a n d S t e i n b e r g (1976), w~th t h e e x c e p t i o h o f a n i n c r e a s e in a p e r t u r e size f r o m 0"07- t o 2 c m ~. T h e c h a m b e r p e r f u s i o n s y s t e m c o n s i s t e d .of a p p r o x i m a t e l y 20 m l o f s a l i n e o n e a c h side o f t h e tissue. T h e t e m p e r a t u r e w a s m a i n t a i n e d a t 31°C b y m e a n s o f t h e r m o s t a t i c ally c o n t r o l l e d h e a t i n g e l e m e n t s in e a c h side o f t h e c h a m b e r . T h e t i s s u e is v i a b l e in r a n g e o f 3 0 - 3 4 ° C . T h e electrical p a r a m e t e r s d e t e r i o r a t e a t t e m p e r a t u r e s a b o v e 34°C. T h e ionic c o m p o s i t i o n o f t h e saline c o n s i s t e d o f 92 mM NaC1, 18 mM N a H C O a, 2"0 mM KCI, 1"8 mM CaCI2, I'0 mM MgSO4, 0.4 mM N a 2 H P O 4, 0"8 mM N a H 2 P O 4 , a n d 0'01 mM o f t h e c a r r i e r a m i n o acid. I n t h e ionic d e p e n d e n c y studies, K + . a n d Ca 2+ w e r e r e d u c e d b y 90 ~/o f r o m t h e s t a n d a r d c o m p o s i t i o n in b o t h sides o f t h e c h a m b e r . NaCI w a s r e p l a c e d t h r o u g h o u t t h e c h a m b e r w i t h LiCl r e s u l t i n g in a n 83 ~/o r e d u c t i o n . A f u r t h e r r e d u c t i o n w o u l d h a v e r e q u i r e d m a n i p u l a t i o n o f t h e b i c a r b o n a t e b u f f e r s y s t e m w h i c h we d i d n o t wish to p u r s u e a t t h e p r e s e n t t i m e . S o l u t i o n s on e a c h s i d e were a e r a t e d w i t h 95 ~ O~ a n d 5 ~/o C02, w h i c h m a i n t a i n e d t h e p H a t 7"4 a n d f a c i l i t a t e d fluid m i x i n g . T h e t r a n s e p i t h e l i a l electrical p o t e n t i a l ( T E P ) a n d s h o r t c i r c u i t c u r r e n t (SCC) were m o n i t o r e d b y a ~Vorld P r e c i s i o n I n s t r u m e n t s DVC-10O0 v o l t a g e - c u r r e n t c l a m p w h i c h also p e r m i t t e d c o m p e n s a t i o n f o r fluid resistance. T h e specific r e s i s t a n c e o f t h e tissue in w h i c h t h e P E cells h a d b e e n r e m o v e d b y b r u s h i n g w a s a p p r o x i m a t e l y 20 ~2 c m ~. All e x p e r i m e n t s w e r e p e r f o r m e d w i t h t h e T E P c l a m p e d t o zero in o r d e r t o e l i m i n a t e t h e T E P as a d r i v i n g force in t h e e v e n t ionic c o t r a n s p o r t w a s i n v o l v e d . T h e t r a n s e p i t h e l i a l flux e x p e r i m e n t s w e r e c o n d u c t e d b y a d d i n g 10/zCi o f 3[H]-labeled h i g h specific a c t i v i t y c a r r i e r a m i n o a c i d t o o n e side o f t h e c h a m b e r . T h e t o t a l c o n c e n t r a t i o n o f t h e c a r r i e r a m i n o a c i d o n b o t h sides o f t h e c h a m b e r w a s a d j u s t e d to 0-01 raM. T h e a m i n o acid carriers w e r e L - g l u t a m i c a c i d a n d L~-aspartic acid. T h e c o n c e n t r a t i o n o f t h e a m i n o a c i d s e m p l o y e d as c o m p e t i t i v e i n h i b i t o r s w a s 0"1 raM, w h i c h is I 0 t i m e s t h e carrier c o n c e n t r a t i o n , a n d w a s t h e s a m e o n b o t h sides o f t h e U s s i n g c h a m b e r . T h e a m i n o acids u s e d as c o m p e t i t i v e i n h i b i t o r s for g l u t a m a t e w e r e L - a s p a r t i c acid, L-lysine a n d L-leucine. T h e ~ o n c e n t r a t i o n o f o u a b a i n w a s 0"1 raM. O u a b a i n w a s a d d e d t o b o t h sides o f ~he U s s i n g c h a m b e r in t h e g l u t a m a t e e x p e r i m e n t s , w h e r e a s o u a b a i n w a s a d d e d t o e i t h e r t h e r e t i n a l side o r c h o r o i d a l side in t h e a s p a r t a t e studies. T h e effects o f t h e i n h i b i t o r s o n t h e u n i d i r e c t i o n a l fluxes w e r e m e a s u r e d in t h e r e t i n a ' t o - c h o r o i d ( R - C ) a n d c h o r o i d - t o - r e t i n a ( C - R ) d i r e c t i o n s . A m i n i m u m o f five o b s e r v a t i o n s o n d i f f e r e n t tissues w e r e m a d e u n d e r e a c h e x p e r i m e n t a l c o n d i t i o n . S a m p l e s o f 0-1 m l w e r e s i m u l t a n e o u s l y t a k e n f r o m e a c h side a t 5-, 10-, 15-, 20-, 30-, 40-, 50-, a n d 60 rain. T h e s a m p l e s were t h e n p r e p a r e d for q u a n t i f i c a t i o n in a B e c k m a n L S 9000 l i q u i d s c i n t i l l a t i o n c o u n t e r . U n i d i r e c t i o n a l fluxes w e r e d e t e r m i n e d f r o m t h v s l o p e

AMINO ACID TRANSPORT

209

of t he linear p o r t i o n of t he appearance curve, which occurred between 30 a n d 60 min, and were corrected for volum~ changes d u e to removal of samples d u r i n g the e xpe ri me nt . I n general, tissues were s t u d i e d i n pairs (two chambers) in which t h e TABLE I

Transport. of a~partate across the P E TEP

Condition Control Control Ouabain on retina side Ouabain on retina side Ouabain on choroid side Ouabain on choroid side

SCC

Specific resistance

Klux

(f/cm I}

(pmol cm -I hr)

Direction

N

(mV)

(pA cm-s)

R C

C R

5 5

10-6-b 1.9 8"8q-3"5

33.4-b 8"7 31"6-b'11-7

327=1=75"6 282q- 66~)

R

C

5

10"5 q- 2"0

37-4 -b 12"8

294 -{-48~)

*0.60 ± 0.26

C

R

5

8"82-b I-4

30"0-b 7-3

302-1-34"2

~ 2 7 ±0'05"

R

C

5

10"3-1-2"6

37"8~ 7-9

272-b25"9

1-04~0.18

C

R

5

8"6=[=2"9

31'2=[: 7"3

272-b44"4

0"31+0"09

1.12±0"22 0-39±0"06

R, r e t i n a ; C, ehoroid; T E P , transepithelial p o t e n t i a l ; scc, short circuit current. * P < 0 . 0 0 1 ; t P < : 0.02. T h e values are p r e s e n t e d as m e a n s ± s t a n d a r d deviations. T h e level of mgmficance was d e t e r m i n e d b y S t u d e n t ' s (two-tailed) t t e s t for d a t a with unequal v a r i a n c e s a n d t h e accepted level o f confidence was 0"02.

direction of t r a n s p o r t was opposite in each chamber. Occasionally, one tissue would be u n a c c e p t a b l e for s t u d y thereby d i s r u p t i n g the usual pairing of experiments. A n y tissue ha d to m a i n t a i n a specific resistance of a t least 200 ~ cm 2 t h r o u g h o u t t h e e x p e r i m e n t a l p r o c e d u r e in order to be utilized in d a t a analysis.

3. R e s u l t s

Electrical parameters As s h o w n in Tables I a n d I I , the initial transepithelial potentials (TEP), short circuit c u r r e n t s (SCC) a n d specific resistances (SR) of t~e control tissues were similar for t he e x p e r i / n e n t s in which t r a n s p o r t was m e a s u r e d in opposing directions. Thus, there is no c o n f o u n d i n g of t r a n s p o r t rates w i t h differences in electrical p a r a m e t e r s . The values of tl~e T E P and S R are higher than previously reported (Crosson and Pautler, 1982), resulting from a modification of the ionic composition of the incubating solution to concentrations similar to those employed by Miller and Steinberg (1976) on the frog PE. However, the S C C was not substantially changed by this alteration of the incubating solution. The T E P s and SCCs were significalitlyreduced w h e n incubated in low K + or Ca s+ solutions, whereas a reduction in N a + resulted in a decreased S C C and an increased S R without a detectable change in the T E P . However, without specificionic tracer studies, no definitive statements can be m a d e as to the actual ior~ietransport systems which account for the observed S C C in the m a m m a l i a n PE. The e m p l o y m e n t of ouabain after the initial measurements of T E P and SCC, 8-2

R C R 0 R C R C R C R C R C R C

C R C R C R C R C R C R C R C R

Direction 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

N '8.0+2'2 8"7±3"6 7'6± 1"8 8"5+4"3 *4"7+_1"7 *4.8+ 1'9 '1.8+0-6 '1.9+1.0 7'6+-1'6 8"7+-3"3 8.6±2.1 7-0±0.4 8.8+2.5 7"7± 1'9 8'7_+2.1 8"3±2'2

TEP (mV) 31.0± 8"3 31"8+10'5 27"4+ 7"2 26"8± 9'4 '16"5+ 6"5 '17'4± 6"7 *5.8± 1.1 *6.5+ 3"3' t19'2± 4"8 t24'7-+ 8'0 27.8_+ 6.1 24.8± 3'6 27.4_+ 5.5 24'8+ 6"4 26'6_+ 6-0 28"4+_. 7-2

SCCo (pA cm-~} 20-2± 5'6 23'2+13'6 3"3_+ 2'5 *2'8± 3"5 16"8+_18.0 15.3_+17.7 '1.2± 0.4 *7"7+_ 1.0 19-2+ 7'8 25.5+ 5"8 17.6_+ 5.8 20.6± 7'2 20.0± 4-6 15.0+_ 3'9 18"0_+ 8"4 22"8+ 8"3

SCCeo (pA cm-z)

266+37"4 274+52"6 289+48.0 306±55"8 294+80.8 280+80.5 297±46.0 307+95.0 '401+56'9 '345_+17.8 311_+33.2 278_+55-0 319_+4ff2 313±50-5 332+_29"7 292+22"5

Specific resistance (~ cmffi)

2"15+0"22 0"49+0-10 '0-72±0.14 *0.24±0-04 "1"10±0.30 ~28_+0.06 '1-00±0.25 0.39+0.14 t1"42+0"31 0.44_+0-10 ~99_+0-16 *0-24±0.04 1.86+-0.39 0.41_+0"13 1.76+_0.24 0.43+0.10

Flux (pmol em"ffihr)

TEP, transepithelial potential; SCCo, short-circuit current at beginningof experiment; SCCa0,short-circuit current at end of experiment. * P < 0.001; t P < 0.01. The values are presented ~ means+standard deviation. The levelof significancewas determined by Student's Itwo-tailed) t test for data with unequal variances and the accepted level of confidencewas 0.02. Data from the R-C and C-R directions were combined in analyzing the effects of reduced ion concentrations on the SCC and SR.

Control Control Ouabain on both sides Ouabain on both sides Ca2+reduction Ca2+reduction K+ reduction K + reduction Na+reduction Na+reduction Aspartate a8 inhibitor Aspartate as inhibitor Leucine as inhibitor Leucine as inhibitor Lysine as inhibitor Lysine as inhibitor

Condition

Transport of glutamate across the PE

TABL~II

Z

AMINO ACID T R A N S P O R T

21!

c o n s i s t e n t l y r e s u l t e d in a s i g n i f i c a n t r e d u c t i o n in t h e SCC ( o b s e r v e d a t t h e e n d o f t h e e x p e r i m e n t a l o b s e r v a t i o n p e r i o d ) as c o m p a r e d w i t h c o n t r o l values. T h i s c o n f i r m e d t h a t t h e c o n c e n t r a t i o n e m p l o y e d was w i t h i n an e f f e c t i v e range. T h e a d d i t i o n o f o t h e r a m i n o acids as c o m p e t i t i v e i n h i b i t o r s h a d n o s i g n i f i c a n t effect on a n y o f t h e electrical parameters. e 2400

2200

L~O0

~O00

1600

|400

a.

1200

I000 te

e0o

/ sI

4oo

/f -/ 0s

200 o/ o

,~

A

~)

,o,~2o

,,~

~)

TIME (rain)

s

B

TIME (rain)

Flo. 1. Typical appearance curves for glutamate are shown to demonstrate the lag time and linear phase of transport across the bovine pigment epithelium. In IA, the transport is in the R-C direction whereas I B is a paired experiment in the opposite direction. Note the lag time is consistently about 10 rain in both cases and the linear phase occurs between 30 and 60 rain. F l u x meazuremen~s

T y p i c a l a p p e a r a n c e c u r v e s a r e s h o w n in Fig. 1 for p a i r e d tissues in w h i c h t h e g l u t a m a t e t r a n s p o r t w a s d e t e r m i n e d in o p p o s i t e d i r e c t i o n s . T h e linear- o r s t e a d y - s t a t e p h a s e o c c u r r e d b e t w e e n 30 a n d 60 rain. N o t e t h e lag t i m e was c o n s i s t e n t l y a b o u t 10 m i n . This p a r a m e t e r c a n b e r e l a t e d t o t h e r a t e c o n s t a n t s o f t r a n s p o r t across t h e d i f f e r e n t m e m b r a n e s a n d p a r a c e l l u l a r r o u t e s in e p i t h e l i a . T h e precise m a t h e m a t i c a l form d e p e n d s upon the model of transport selecled for study. The model which i n c l u d e s p a r a c e l l u l a r t r a n s p o r t a n d u n s t i r r e d l a y e r s c a n n o t b e e m p l o y e d for t h e a n a l y s i s o f t h e usual e x p e r i m e n t a l d a t a ( B a r n e t t a n d L i v k o , 1977). T h e R - C flux w a s s i g n i f i c a n t l y g r e a t e r for b o t h a s p a r t a t e a n d g l u t a m a t e t h a n t h e C - R flux, r e s u l t i n g in a s u b s t a n t i a l n e t flux in t h e R - C d i r e c t i o n . H o w e v e r , t h e R - C g l u t a m a t e flux w a s a p p r o x i m a t e l y t w i c e as g r e a t as t h e a s p a r t a t e flux, s u g g e s t i n g t h a t

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g l u t a m a t e is preferred by this particular t r a n s p o r t system. There was no significant difference between the fluxes of aspartate a n d g l u t a m a t e in the C - R direction. An approximation of the a m o u n t of carrier being moved by simple diffusion can be m a d e by employing t h e diffusion coefficient we have established for L-glucose in over 75 separate tissue preparations. From this data, t h e simple diffusion of the amino acids should be in the range o f 0-08-0.10 n ~ hr -l cm ~. This would account for a b o u t 23 ~o o f the transport o f a s p a r t a t e in the R - C direction and 8 % in the F ~ 3 direction. The calculated percentages would be less for the glutamat~ transport. The addition of ouabain to both sides of t h e c h a m b e r significant|y reduce the Yi:-C flux a n d C - R flux for g l u t a m a t e . When ouabain was present on only the retinal side of the preparation, b o t h fluxes were similarly reduced for aspartate. Considering these observations, it appears t h a t both R - C and C-I~ retinal fluxes of t h e acidic amino acids are subject to regulation by an ATPase system, probably located in the apical m e m b r a n e of the PE. T h e transport of g l u t a m a t e in the R - C direction was significantly retarded by reducing the concentrations o f Na +, K + or Ca 2+, indicating a complex d e p e n d e n c e on specific ion species. The g l u t a m a t e flux in the C - R direction, however, did u o t reveal any dependence on Na + or K +, but was significantly reduced at t h e lower Ca ~+ concentration. A m i n o acids were tested as possible competitive inhibitors of g l u t a m a t e transport at concentrations I0 times thtJ.t of glutamate. Aspartic acid reduced both the R-C and C - R g l u t a m a t e fluxes b y approximately 50$/o, whereas lysine (basic) and leucine (neutral) amino acids did not significantly effect the g l u t a m a t e transport in either direction. This strongly suggests t h a t g l u t a m a t e and aspartate share c o m m o n transport systems in both t h e R-C and C - R directions which do not have a similar affinity for either lysine or leucine. 4. D i s c u s s i o n A t least six f u n d a m e n t a l amino acid transport systems have been identified in epithelial and non-epithelial cells (Christensen, 1975). One such system is primarily concerned with the transport of amino acids which are anionic at physiological p H such as g l u t a m a t e a n d aspartate. A s o d i u m - d e p e n d e n t system which handles Lg l u t a m a t e and L-aspartate at nearly equal affinities has been identified in hepatocytes (Christensen and Makowskie, 1983), lieurons (Drejer, Larrson and Schousboe, 1983) and in intestinal brush-border vesicles (Corcelli, Prezioso, Palmeri and Store||i, 1982; Corcelli and Storelli, 1983}. To our knowledge, the transpor~ properties o f the basolateral m e m b r a n e s o f the enterocytes have n o t as y e t been studied in regard to aspartate or glutamate. The characteristics ofacidlc amino-acid t r a n s p o r t t h a t we have observed across t h e retinal P]~ are quite similar to those reported in o t h e r cell systems. First of all, the competition studies indicate t h a t both asp~rtate a n d g l u t a m a t e are t r a n s p o r t e d by a c~)mmon carrier which has a low affinity for basic (lysine) and neutral (leucine) amino acids. This also indicates the preferences of t h e transport system for the acidic a m i n o acids. Secondly, we have shown a net t r a n s p o r t of both a s p a r t a t e and g l u t a m a t e in t h e R - C direction which is N a + - d e p e n d e n t and subject to inhibition by ouabain. These features are suggestive of an active transport system which is again similar to the acidic a m i n o acid transport m e c h a n i s m s of other celZs. F u r t h e r m o r e , the d e p e n d e n c e of t h e R - C transport o f g l u t a m a t e on K + in our

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213

experiments is consistent with the observation on intestinal brush-border m e m b r a n e vesicles t h a t the Na+-L-glutamate (L-aspartate) cotransport system is specifically activated by K + and CI- ions (Corcelli and Storelli, 1983). I t is of special interest t h a t potassium exerted a m o d u l a t i n g effect on the R - C flux of g l u t a m a t e which was similar to t h a t noted by Miller and Steinberg (1979) in their studies o f t a u r i n e transport across the frog PE. Specifically, the g l u t a m a t e R - C fiux was e x t r e m e l y sensitive to K + changes in the range of 0.2-2 m~, which corresponds to physiological changes in K + levels within ~he neural retina induced by photic stimulation (Oakley, 1977). I f g l u t a m a t e a n d / o r aspartate are indeed functioning as n e u r o t r a n s m i t t e r s for photoreeeptors (Brandon and Lain, 1983; I s h i d a and Fain, 19gl; Lasater a n d Dowling, 1982), and the photoreceptor cells are synapticaliy active in t h e d a r k (see Sillrnan, 1985, for discussion), then t h e level o f extracellular K + would function to facilitate the transport out o f the retinal spaces across the PE. Conversely, during light exposure when the cells are presumably less active, the transport function would be diminished. The sensitivity of the g l u t a m a t e transport system to K + in the R - C direction provides a regulatory mechanism which conceivably helps control the levels of g l u t a m a t e in t h e extracellular spaces. However, the sensitivity o£ the g l u t a m a t e transport in both directions t o Ca z+ m a y also implicate this ion as an i m p o r t a n t regulator of acidic amino-acid transport across the P E . The behavior o f g l u t a m a t e and a s p a r t a t e t r a n s p o r t in t h e C - R direction presents some difficulties in interpretation. The competition studies did reveal t h a t the transport system preferred t h e acidic amino acid as compared with the basic (lysine) and neutral (leueine) ones. However, no d e p e n d e n c e on Na + or K + was observed in the g l u t a m a t e flux in t h e C - R direction even though it w~s inhibited by ouabain. Instead, a lowered concentration of Ca ~+ significantly reduced this flux by a b o u t 40 %. Miller and Steinberg (1977) have reported t h a t t h e active transport o f Ca ~+ across the frog P E is sensitive to ouabain, b u t the effect is to increase the Ca ~+ flux in the C - R direction. W h e t h e r or n o t the active t r a n s p o r t of Ca z+ is a t all related to the transport of g l u t a m a t e in t h e R - C direction is an open question. I t should be recalled t h a t in these investigations we are measuring t h e flux across two functionally and structurally different m e m b r a n e s , apical and basal. The experim e n t a l d e t e r m i n a t i o n of valid kinetic p a r a m e t e r s for each individual m e m b r a n e will probably require the utilization o f vesicles m a d e up of separate apical a n d basal membranes, similar to those which have been successfully employed in t h e studies of intestinal epithelium (Stevens, K a u n i t z a n d Wright, I984). The m e t h o d of tissue preparation used in these studies does h a v e the a d v a n t a g e o f simulating the blood-retinal barrier p r o v i d e d by t h e P E in vlvo, a n d permits characterization of the transport properties of t h e system as well as being amenable to the s t u d y o f t h e metabolic a n d ionic r e q u i r e m e n t s necessary to sustain or regulate t h e transport. I n s u m m a r y , we have shown t h a t there is a n e t flux of aspartat~ and g l u t a m a t e in the R - ~ direction with t h e g l u t a m a t e flux being substantially greater t h a n t h a t of~spartate. The unidirectional flux of g l u t a m a t e is d e p e n d e n t on N a +, K ÷ a n d Ca ~+ and is ~ubject to inhibition by ouabain. The inhibition by ouabain was further localized to t h e retinal side. These findings suggest the existence of a t r a n s p o r t ATPase system located, probably, in the apical m e m b r a n e system. The exquisite sensitivity to K ÷ and Ca ~+, t a k e n along with the sensitivity to ouabain, the substrate specificity, a n d the direction of the n e t flux, implies a mechanism which m a y serve to help regulate t h e extracellular concentrations o f acidic amino-acid t r a n s m i t t e r s a n d other n e u r o t r a n s m i t t e r s within the neural retina.

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ACKNOWLEDGMENT This research was supported in part by NIH grant EY 04750. REFERENCES Barnett, G. and Livko, V. (1977). Transport across epithelia: a kinetic evaluation. Biochim. Biophy.~. Acts 464, 276-86. Brandon, C. and Lain, D. M. K. (1983). L-glutamic acid: a neurotransmitter candidate for cone photoreceptors in human and rat retinas. Proc. Nat. Acad, ~.WcL U.8.A. 80, 5117-2I. Christensen, N. H. (1975). BZOLOOlCAI,TaASSPORT (2nd edn). W. A. Benjamin: Reading, Massachusetts. Christensen, H. N. and Makowskie, M. (1983). Recognition chemistry of anionic amino acids for hepatocyte transport and for neurotransmittoff action compared. Life 8ci. 33, 2255-67. Corceili, A., Prezioso, G., Palmeri, F. and Storelli, C. (1982). Eteetroneutral Na + dicarb~xylie amino acid cotransport in rat intestinal brush border membrane vesicles. Biochim. Biophys. Acta 689, 97-105. Coreelli, A. and Storelli, C. (1983). The role of potassium and chloride ions on the Na+/acidic amino acid cotransport system in rat intestinal brush border membrane vesicles. Biochim. Biophys. Acts 732, 24-31. Crosson, C.E. and Pautler, E.L. (1982). Glucose transport across the isolated bovine pigment epithelium. Exp. Eye Res. 35, 371-7. Drejer, J., Larrson, O. M. and Schousboe, A. (1983}. Characterization of uptake and release processes for I)- and L-aspartate in primary cultures of astrocytes and eerebellar granule cells~ Neurochem. Res. 8, 231-44. Edwards, 1%,B. (i977). Accumulation of routine by cultured retinal pigment epithelium of the rat. lnvezt. Ophthalmol. 16, 201-8. Ishida, A. T. and Fain, G. L. (1981). D-aspartate potentiates the efli~et of L-glutamate on horizontal cells in goldfish retina. Proe. Nat. Acad. Sci. U.S.A. 781, 5890-4. Lasater, E. M. and Dowling, J. E. (1982). Carp horizontal cells in culture selectively respond to L-glutamate and its agonists. Proc. Nat. Acad. 8ci. U.8.A. 79, 936--40. Miller, S.S. and Steinberg, R.H. (1976). Transport of taurine, ~.-methionine and 3-0methyl-o-glucose across the frog retinal pigment epithelium. Exp. Eye Ree. 23, 177-89. 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. and Steinberg, R. H. (1979). Potassium modulation of taurine transport across the frog retinal pigment epithelium. J. Gen. Physiol. 74, 237-59. Oakley, B., II. (1977). Potassium and the photoreceptor-dependent pigment epithelial hyperpolarization. Ji Oen. Physiol. 70, 405--25. Sellner, P. A. and l~lasland, R. H. (1983). Movement of membrane precursors across the frog retinal pigment epithelium, lnvest. Ophthalmol. Vis. ,Sci. 24 (Suppl.), 69. Sillman, A.J. (I985). Current concepts in photoreceptor physiology. The Physioloyist 28, 122-8. Stevens, B. R., Kaunitz, J. D. and Wright, E. M. (1984). Intestinal transport of amino acids and sugars: advances umng membrane vesmles. Ann. Rev. Physiol. 46, 417-33. [

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