Camp. Biochem. Physiol. Vol. 81A, No. 2, pp. 427430, 1985 0300-9629/85 $3.00 + 0.00 Printed in Great Britain 0 1985 Pergamon Press Ltd

AMINO ACID TRANSPORT BY CORNEAL EPITHELIAL CELLS FROM THE TOAD, BUF’O kfARINUS*

DEBORAH F. COOPERSTEIN Department of Biology, Adelphi University, Garden City, NY 11530, USA

(Received 31 October 1984)

Abstract-l. cl-Aminoisobutyric acid is actively accumulated by the epithelium of the cornea of the toad, Bufo marinus, resulting in a tissue to medium ratio of 4 to 1 after 40min of incubation.

2. The accumulation of cl-aminoisobutyric acid is ouabain-sensitive and dependent upon the presence of extracellular sodium.

3. Transport is inhibited by carbon monoxide, 6-aminonicotinamide, arsenite and n-heptyl-3-hydroxy- quinoline-n-oxide and stimulated by diamide.

INTRODUCTION

The cornea is an avascular tissue bathed anteriorly by the tears and posteriorly by the aqueous humor. The epithelium of the cornea, which is exposed to the tear film, requires a significant supply of amino acids for protein synthesis because of its rapid proliferation (Friedenwald and Buschke, 1944). The amino acids necessary for this protein synthesis enter the cornea across the endothelial surface from the aqueous humor (Thoft and Friend, 1972; Scott and Fried- enthal, 1973; McGahan, 1981); the epithelial surface being relatively impermeable to water-soluble com- pounds. Approximately 90% of the AIB accumulated by the cornea is accumulated by the epithelial cells (McGahan, 1981). Although transport has been ex- amined in intact corneas of both rabbits (Riley et al., 1973; Riley, 1977) and toads (Friedenthal and Scott, 1973; McGahan, 1981) transport by isolated epi- thelial cells has not been studied. We employed the non-metabolizable amino acid, a-aminoisobutyric acid (AIB), to examine amino acid accumulation by the cornea1 epithelial cells of the toad, Bufo marinus.

MATERIALS AND METHODS

Toads, Bufo marinas, of Colombian origin were obtained through Tarpon Zoos, Inc. All radioactive materials were purchased from New England Nuclear Co. All inhibitors were supplied by Sigma Chemical Co.

Uptake in isolated epithelial cells

Epithelial cells were removed from 12 corneas by scraping the anterior surface with a sterile scalpel blade. Scraped cells were suspended in 10ml of modified Conway Ringer’s pH 7.6, composition: 83.5 mM NaCl, 17.7 mM NaHCO,, 4mM KCl, 0.8 mM MgSO,, 0.8 mM KH*PO,, 11 mM glucose, 1.5 mM EDTA. Clumps of cells were dispersed by passage through a syringe with a 21 gauge needle. The cell suspension was stirred constantly and aerated. For amino acid uptake studies, W-a-aminoisobutyric acid (W-AIB) (0.1 mCi/ml) and unlabeled AIB were added to the suspen- sions in concentrations ranging from 0.125 to 20 mM. At the end of the incubation period the cell suspension was filtered

*Supported by National Institute of Health (NIH) grant EY01485.

through Millipore Cellotate filters (0.5 pm). The cells caught on the filter were washed with 15.ml.of modified Conway Rinaer’s. dried, solubilized in Soluene 100 Packard) and counted in a Beckman Liquid Scintillation spectroph&om- eter. Media samples (0.1 ml) were treated identically. Samples from each experiment were taken for protein determination by the method of Lowry et al. (1951).

In experiments to determine cell volume 1.5 ml 3H,0 (0.25 mCi/ml) were added to the cell suspensions. The cells were treated as described above.

Uptake in the presence of metabolic inhibitors

In the experiments with carbon monoxide (CO), n-heptyl- 2-hydroxyquinoline-n-oxide (HOQNO), dinitrophenol (DNP) and diamide, AIB was added at the same time as the inhibitors. In some experiments with diamide, glucose in the media was replaced by sucrose. Cells were preincubated with arsenite for 20 min, then AIB was added and the incubations were continued for 10 or 30min. Cells were preincubated with cyanide for 10 or 20min, AIB was added and the incubations were continued for 10 min. 6-Aminonicotin- amide (6-AN) was dissolved in Ringer’s solution and 0.05 ml was injected into the anterior chamber of the eye. The final concentration of 6-AN in the anterior chamber was 1.5 mM. The contralateral eye received 0.05 ml of Ringer’s solution and served as the control. After 2 hr the epithelial cells were removed and suspended in EDTA- Ringer’s with ‘%-AIB. 6-AN (2mM) was added to the suspension of cells from eyes that had been pretreated with 6-AN and incubated for 10min.

Uptake in the whole cornea

Corneas were mounted as a membrane between two halves of a lucite chamber and bathed with modified Conway Ringer’s in which the EDTA was replaced with 1.5 mM CaCI,. In some experiments the corneas were denuded by removal of the epithelium as described pre- viously. Histological examination indicated that the epi- thelium was completely removed by this treatment. A cornea from each toad was exposed on one surface (endo- thelium or epithelium) to 0.1 PCi of 14C-AIB and 0.1 FCi of ‘H-mannitol. The contralateral control cornea was treated identically except that the epithelium was not removed. In some cases, a coat of silicone oil was placed on the denuded cornea and the contralateral cornea with its epithelium intact served as the control. At the end of the incubation period, 0.1 ml of Soluene-100 and lOm1 of a toluene base scintillation fluid was added and the samples were counted. The corneas -were removed from the chamber and a 6 mm

421

428 DEBORAH F. COOPER~TEIN

button was excised using a disposable trephine. The corneas were prepared for liquid scintillation counting in the same manner as the media.

RESULTS

The accumulation of AIB by isolated cornea1 epithelial cells was rapid during the first 10 min of incubation and was linear from 10 to 40 min of incubation (Fig. 1). Over this time period the intra- cellular water was constant and equaled 6.18 f 0.19 (SE) @/mg protein. The concentration of AIB within the cells was determined by comparing the cell water/ mg protein to the amount of AIB accumulated/mg protein. After 40min incubation the cells accumu- lated AIB to a concentration four times greater than that in the medium. This indicates that the cells are accumulating AIB by an active transport mechanism. Further delineation of this transport system as active was accomplished by studying the saturability and energy requirements of the system. Figure 2 shows that the accumulation of AIB is saturable. A linear least squares analysis of a Lineweaver-Burk plot of the data shows that the Km for AIB accumulation is 0.2 mM. An Eadie-Hofstee plot of the data also yields a K, of 0.2mM.

Christensen has classified the transport of AIB as occurring primarily via the A-system which depends upon the presence of extracellular Na+ (Oxender and Christensen, 1963) and an active Na+ + K+-ATPase (Shultz and Curran, 1970). Friedenthal and Scott (1973) had shown that AIB is transported primarily by the A-system in toad cornea. Therefore we studied the effect of removal of Na+ upon AIB accumulation. In these experiments, the NaCl in our Ringer’s solu- tion was replaced by choline chloride and KHCO, was substituted for NaHCO,. Removal of sodium completely inhibited active AIB accumulation so that intracellular levels were equimolar with extracellular levels (Table 1). When 1 mM ouabain was added to the incubation medium, AIB accumulation was in- hibited 51% but some active accumulation was still evident.

A series of metabolic inhibitors was employed to determine the energy requirements for AIB accumu- lation. Cyanide, rotenone, 2,4-dinitrophenol (DNP)

2 I 1 I I I I I

0 IO 20 30 40 50

Time (mid

Fig. 1. Time course of AIB accumulation by toad cornea1 epithelial cells. Each point represents the mean -h SE of at

least eight experiments.

SaturobiLity of AIB accumulation by cornea1 epithelial cells

IO r

CAIBI (mM)

Fig. 2. Saturability of AIB accumulation by cornea1 epi- thelial cells. Each point represents the mean ir SE of at least

eight experiments.

and iodoacetamide at concentrations of 2 mM and up to 40 min of incubation had no significant inhibitory effect upon AIB accumulation. The addition of 2 mM arsenite significantly reduced AIB accumulation after 40 min (Table 2). 0.2 mM n-Heptyl-3-hydroxy- quinoline-n-oxide (HOQNO) inhibited uptake 38% within 10 min of incubation. When carbon monoxide was used as an inhibitor, AIB accumulation was reduced 34%. 6-AN decreased AIB accumulation 61% and diamide, a glutathione oxidizing agent, increased amino acid transport 320%.

In order to determine the contribution of the epithelial cells to the active accumulation of amino acids observed in whole cornea, we compared the ability of intact and denuded corneas to accumulate AIB. In these experiments, corneas were mounted in lucite chambers so that each surface could be treated separately. When 14C-AIB and 3H-mannitol, which served as an extracellular space marker, were added to the endothelial bathing solution of the denuded cornea there was a decrease of approximately 96% in the amount taken up by the intact cornea (Table 3). When the denuded epithelial surface was coated with silicone oil, AIB accumulation was still reduced more than 90% as compared to intact control corneas indicating the importance of the epithelium in amino acid accumulation.

DISCUSSION

Our results confirm that the epithelial cells of the toad cornea are primarily responsible for the amino

Table 1. Sodium dependence and ouabain sensitivity of AIB accumulation by cornea1 epithelial cells

Uutake Inhibition Trial (nmol/n& protein) (%)

Conway Ringer’s Choline Ringer’s

Ouabain (mM) 0

10-d 10-2

7.0 f 0.8 2.6 i: 0.6 62*

9.5 * 1.7 7.8 * 0.8 17 6.2 + 1.1 34*

1 4.6 + 0.6 51*

*P IO.05

Accumulation was measured after 10min of incubation. Results represent the mean rt SE of four experiments.

Toad cornea1 amino acid transport 429

Table 2. Effects of metabolic inhibitors on AIB accumulation

Inhibitor Incubation time cmin1

AIB uptake control

(mnol/mg protein) + inhibitor

Change in uptake

(“/,)

6-AN A&n&e Arsenite co HOQNO Cyanide DNP Diamide

10 6.6 f 1.7 (8) 2.5 + 0.5 (8) -61* 10 5.6 k 0.2 (4) 3.6 + 0.4 (4) -35 30 6.7 + 1.2 (4) 3.2 + 0.8 (4) -52* 30 4.2 + 0.7 (8) 2.7 i. 0.3 (8) -34s 10 5.5 5 1.0 (8) 3.4 + 0.5 (8) -38* 10 4.6 i: 0.8 (14) 5.7 * 1.2 (7) +24 10 5.9 + 0.5 (4) 5.6 + 0.3 (4) -5

Wo’glu) 30 2.3 k 0.2 (4) 9.5 rt 0.3 (4) +320*

*P 50.05. All inhibitors were added to a final concentration of 2 mM except HOQNO and diamide

which are present at a concentration of 0.2mM. The incubation time is the time daring which the cells were exposed to AIB. The results are expressed as the mean + SE. The number of experiments are indicated in parentheses.

Table 3. AIB uptake by intact and denuded corneas

Uptake (nmo1/27 mm’) when radioactivity added to:

Treatment Endothelimn Epithelimn

Intact cornea 6.15 0 Denuded cornea 0.21 Denuded cornea with silicone

layer on Epi surface 0.56

Results are the means of at least four experiments.

acid accumulation demonstrated in intact corneas since removal of the epithelium virtually eliminates AIB accumulation. Riley (1977), using other meth- ods, concluded that amino acids are accumulated primarily by the epithelium of the rabbit cornea and to a much smaller extent by the keratocytes of the stroma. McGahan (198 l), by comparing efllux rates, estimated that the keratocytes and endothelial cells accounted for approximately 10% of the amino acid accumulated by intact toad corneas. Our results, using a more direct method, show that 1.0% of the amino acid accumulation is attributable to uptake by stromal keratocytes and endothelial cells. The de- creased accumulation observed when the epithelial surface was not coated with silicone oil is due to efflux of amino acid across the denuded surface.

Accumulation of AIB occurs against a concen- tration gradient reaching a tissue to medium ratio of 2 to 1 in 10 min and 4 to 1 after 40 min. This occurs without an increase in cell volume. The inhibition of transport by removal of extracellular Na+ or addition of ouabain is further indication of active transport occurring by a sodium-dependent system similar to that described in other tissues (Shultz and Curran, 1970) and previously described in this tissue (Friedenthal and Scott, 1973). McGahan (1981) found that ouabain stimulated AIB efflux from the cornea and proposed that this effect was due to increased intracellular Na+ concentrations. No effect on AIB efflux was found when Na+ was replaced by choline in the medium bathing the cornea. Because efflux is unaffected by .removal of extracellular Na+, the results presented here indicate that the uptake of AIB by toad cornea1 epithelial cells has a significant Na+-dependent component. This sodium dependency is less evident in intact corneas because of the time required to deplete interstitial Na+ (Friedenthal and Scott, 1973). The effects of ouabain are probably due

to a decreased uptake of AIB as well as an increase in AIB efflux. The relative’insensitivity to ouabain observed in this study and that of McGahan (1981) is probably due to the fact that toad Na+ + K+- ATPase is relatively insensitive to cardiac glycosides (Bonting and Canady, 1964).

AIB accumulation is not significantly affected by some inhibitors of aerobic metabolism, including rotenone, DNP and cyanide. However, HOQNO, which blocks the electron transport chain between cytochrome b and c (Slater, 1963), is an effective transport inhibitor, as is carbon monoxide.

6-AN markedly decreased AIB accumulation. This compound is incorporated into both NAD+ and NADP+ and because of the relatively higher concen- trations of intracellular NAD+ might be expected to cause a more significant decrease in NADP+ levels (Hasizume et al., 1975). It is possible that 6-AN is exerting its inhibitory effects through inhibition of both NAD+ and NADP+-dependent enzymes. Con- sidering the significant role played by the pentose phosphate pathway in cornea1 epithelial glucose metabolism (Kinoshita et al., 1955) this pathway may be involved in amino acid transport.

Diamide, a specific glutathione-oxidizing agent (Kosower et al., 1969), greatly enhanced AIB accumulation. This may be due to stimulation of the pentose phosphate pathway (Jacob and Jandel, 1966; Eggelston and Krebs, 1974) or to oxidation of intracellular and membrane sulfhydryl groups. The diamide-induced stimulation of AIB accumulation is diminished when glucose is present in the medium. This data is in agreement with that of Epstein and Kinoshita (1970) showing that the increase in lens GSSG caused by diamide is reduced when glucose is present.

The effects of arsenite are most likely due to its inhibitory effects on pyruvate dehydrogenase (Webb, 1966) but may also be attributed to inhibition of glutathione reductase (Mize and Langdon, 1962).

In summary, the epithelium is the major site of AIB accumulation in the toad cornea. This transport system depends upon the presence of extracellular sodium and the activity of the Na+ + K+-ATPase. Studies using metabolic inhibitors raise the possibility that metabolic pathways other than glycolysis and the Krebs cycle contribute to the process of active amino acid accumulation in cornea1 epithelial cells.

430 DEBORAH F. COOPERSTEIN

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