Ammo acid transport in the lens

Transcription

1 Ammo acid transport in the lens V. Everett Kinsey Most arnino acids usually found in other tissues are present in the lens. In the rabbit all of these compounds are in concentrations higher than those in the aqueous humors and much higher than concentrations in vitreous humor. Accumulation takes place through active transport by processes which require glucose, the anaerobic utilization of which can provide all the energy needed to maintain normal concentration gradients. The mechanisms responsible for transport of amino acids are probably confined to the epithelium, are highly temperature dependent, can be inhibited by various metabolic poisons and selectively, in varying degrees, by each other. At least three systems, one each for neutral, acidic, and basic amino acids, appear to be involved in their active transport. The steady state concentration of amino acids in the lens is determined by the balance between the rate of entrance through active transport and exit by diffusion. -L ifty years ago Van Slyke and Meyer 1 traced ammo acids from the intestine into the blood, then into the tissues where they were found to be concentrated five- to tenfold. Only in recent years has the concentration of amino acids in the lens been shown to exceed that in the ocular fluids which bathe it, 2 and have investigations of factors concerned with their accumulation led to some clarification of the kinetics and site of action of the systems responsible for active transport. 3 " 5 Accumulation and transport, when applied to exchange of substances between the lens and its environment, are not the same things. Accumulation is the difference between the quantity of a substance that enters and leaves it by any means, and is From the Kresge Eye Institute, Detroit, Mich. This study was supported in part by Research Grants B-1100 and B-2885 from the National Institute of Neurological Diseases and Blindness of the National Institutes of Health, United States Public Health Service; the United States Atomic Energy Commission, Contract No. COO , and Research to Prevent Blindness, Inc. 691 the variable usually determined experimentally. Transport refers to actual movement of a substance; it can be either active or passive. Christensen G described transport as "the process by which a solute is transferred from one phase to another." When movement is active, energy must be provided by cellular metabolism; when passive, it is dependent only on molecular mobility, although sometimes it is "facilitated" by mediation of cell structure or chemical groups. 7 Transport is considered to be active when it takes place against an electrochemical gradient. The electric gradient need not be produced by transfer of a charge on the substance in question, an amino acid, for example. It is essential, however, that the chemical gradient exists between the same species in free solution on either side of the boundary which separates two phases, in most instances a cell membrane. In the lens, Brindley s has shown the existence of a potential difference across the capsule, the inside being negative with respect to the outside. Neutral and acidic

2 692 Kinsey Investigative Ophthalmology August 1965 amino acids, which cany net negative charges, therefore must move across the capsule against an electric potential. Basic amino acids, which carry positive charges, move in the direction of the potential gradient and, therefore, cannot be considered to be actively transported solely on the basis of differences in chemical concentration between lens and environmental fluids. Amino acid composition The concentration of free amino acids in lenses of rabbits 2 and rats 9 has been determined by ion exchange chromatography on sulfonated polystyrene resins (Table I). The lens contains all of the amino. acids usually found in other tissues, with the exception of tryptophan, cysteine, and possibly hydroxyproline. The absence of cysteine seems unusual, because it is present in aqueous humor in small concentrations, and it is also a constituent of glutathione, a polypeptide known to be synthesized in relatively large quantities in situ. Possibly cysteine is bound to structural elements of the lens. Additional information concerning concentration of amino acids in lenses has been provided by Dardenne and Kirsten, 10 who analyzed the non-protein-bound ninhydrin-positive substances present in lenses of rats and cattle. The free amino acid composition of the lenses is not accurately reflected by their data because proteinfree extracts were analyzed only after being hydrolyzed for 24 hours in 6N HC1 and so include unknown quantities of amino acids from polypeptides, such as glutathione and ophthalmic acid. Distribution relative to ocular fluids The concentration of each amino acid is higher in the lens than in the anterior or posterior aqueous, and much higher than in the vitreous humor (Fig. I). 2 The concentration ratio of the two acidic amino acids, aspartic and glutamic, is highest; the ratios for the basic amino acids, arginine, lysine, and histidine, are among Table I. Concentration of free amino acids in lenses of rabbits 2 and rats'' Alanine /S-Alanine Y-Amino-n-butyric acid Arginine Asparagine and glutaminef Aspartic acid Cystathionine Cysteine-cystine Glutamic acid Glycine Histidine Hydroxyproline Lsoleucine Leucine Lysine Methionine Ornithine Phenylalanine Proline Serin e Taurine Threonine Tryptophan Tyrosine Valine Rabbits (mmoles/ Kg. lens icater) Trace.45* Rais (mmoles/ Kg. lens water) Trace In original text, 2 value was incorrectly given as fasparagine color equivalent used for analytical value. the lowest; and values for the neutral amino acids, except leucine and isoleucine, are intermediate between acidic and basic compounds. The ratio of the total quantity of amino acids and peptides in lenses of fresh calf and cattle eyes is likewise higher than that of aqueous humor, the values being 6.7 and 5.3, respectively.' 1 Rate of accumulation Evidence that the lens concentrates amino acids from the fluids which bathe it has been obtained from studies performed both in vivo and in vitro.' 1-5 The concentration of 14 C-labeled a-aminoisobutyric acid (a-aib) in lenses of live rabbits rises within 24 hours after its parenteral administration to 2 J /2 and 25 times that in aqueous and vitreous humor, respectively. 11 The rate of accumulation of a-aib in rabbit lenses, and hydroxyproline in calf lenses, cultured in media which contained

3 Amino acid transport in lens 693 Volume 4 Number 4 CONCENTRATION OF FREE (m mol«i ptr Kg. wotar) IN IN IN IN AMINO ACIDS VITREOUS POST. AO. LENS ANT. AO. PHENYLALANINE OCUTAMIC ACID O.YCINE Fig. 1. Relative concentration of free amino acids in lens, aqueous and vitreous humors of the rabbit eye. (From Reddy and Kinsey: INVEST. OPHTH. 1: 635, 1962.) no other amino acids, is shown in Fig. 2. Because of differences in weights of lenses and volume of culture fluid (5 ml. for rabbits and 10 ml. for calves), the data are not entirely comparable; however, it is apparent that the amino acids accumulate rapidly in lenses of both species. Factors affecting accumulation The effects of temperature, omission of glucose or calcium, and addition of various poisons to culture media, on accumula- tion of a-aib in rabbit lenses, are shown in Table II. Uptake of this amino acid is highly dependent upon temperature, the apparent Q10 being approximately "4. Absence of glucose from the medium caused a reduction in accumulation of a-aib to less than 20 per cent of normal. A similar observation was made by Kern4 on the uptake of hydroxyproline in calf lenses. The metabolic poisons, iodoacetate, ouabain, and cyanide, and omission of calcium also depress transport in varying de-

4 694 Kinsey In vestigative Ophthalmology August 1965 TIME IN HOURS Fig. 2. Accumulation of 1<l C-labeled a-aib in rabbit lenses and hydroxyproline in calf lenses cultured in media containing no other amino acids. (Data for calf lenses from Kern/ 1 recalculated.) grees. The inhibitory effect of ouabain on a-aib uptake occurred only after a delay of approximately one hour, an effect noted too with calf lenses. 4 Thoft and Kinoshita 12 found calcium was also required for normal accumulation of a-aib in rat lenses. These investigators reported that the leakage rate of a-aib increased only slightly when calcium was not present, and concluded that part of the reduction in uptake must be caused by inhibition of active transport. Kinsey and Reddy 5 reported accumulation of a-aib in rabbit lenses to be reduced approximately 30 per cent in the absence of oxygen, as it was also in the presence of 3 x 10~ 3 M dinitrophenol, suggesting that anaerobic processes are incapable of providing all of the energy required for its transport. These results differed from those of Kern, who showed that in calf lenses hydroxyproline concentrated in nitrogen as effectively as in air; and dinitrophenol, in concentrations as high as 0.67 mmole per liter, did not affect accumulation. They seemed inconsistent too, with the work of Kinoshita and coworkers, 13 who observed that, when glucose was included in the medium, oxygen was not required for maintaining the cations sodium and potassium at normal levels. Thus, in calf lenses sufficient energy can be obtained from anaerobic glycolysis to transport not only amino acids but cations as well. As a consequence, the question of whether uptake of a-aib in rabbit lenses can also be supported solely by energy derived anaerobically was reinvestigated. 9 Lenses were cultured in the usual manner under aerobic conditions, and uptake of tracer a-aib was measured and compared with that obtained when the gas phase was changed to 95 per cent nitrogen and 5 per cent carbon dioxide. Traces of oxygen were removed from the gas mixture in a manner similar to that employed by Kinoshita and co-workers, 13 in which the gas was passed serially through three washes of a solution containing vanadous sulfate. To minimize possible differences in temperature, the aerobic and anaerobic experiments were performed simultaneously in the same water bath. The effect of dinitrophenol was also reinvestigated. The dosage (10~ 4 M) was less than that employed in the earlier study. If it is assumed that the response of the lens to this poison Table II. Influence of various factors on accumulation of "C-labeled a-aib in rabbit lenses cultured for 24 hours in a medium containing no other amino acids 5 Condition or poison Control 37 C. Reduced temperature 23 C. Glucose absent Anaerobiosis Dinitrophenol Iodoacetate Ouabain Sodium cyanide Calcium absent 0 3 x l(hm 3 x i(hm 3 x 10-2M 5 x 10~ 3 M Number of lenses in parentheses. Concentration of a-aib, lens water medium, initially ±3.66 (67) 2.2 ±0.47 (12) 1.7 ±0.57 ( 7) 8.8 ±2.42 (12) 7.4 ±1.07 ( 6) 0.74 ± 0.19 ( 5) 3.4 ±0.88 ( 9) 1.55 ±0.40 (13) 5.1 ±1.2 ( 8) Concentration Control (%)

5 Volume 4 Number 4 Amino acid transport in lens 695 Table III. Influence of anaerobiosis and dinitrophenol (10~ 4 M) on accumulation of "C-labeled a-aib in rabbit lenses cultured for 24 hours in a medium containing no other amino acids Concentration of a-aib, Anaerobiosis Control, aerobic Dinitrophenol Control, no DNP lens water media, initially 13.1 ±2.7 (19) 13.2 ±2.0 (19) (15) 12.0 ±2.9 (15) is like that of other tissues, this concentration should be enough to uncouple oxidative phosphorylation but not to affect other metabolic processes. Contralateral lenses were used as controls in all instances. Table III shows that, contrary to results obtained previously, there was no difference in uptake of a-aib, either when oxygen was excluded from the gas phase or when dinitrophenol was present in the media. An explanation for the earlier observations, particularly those obtained under anaerobic conditions, is not apparent. Two sets of experiments had been performed more than a year apart, and accumulation consistently appeared to be decreased in the absence of oxygen. However, separate baths were used for the aerobic and anaerobic experiments, and possibly by chance the temperature of the baths differed. A variation of only two degrees would be enough to account for the observed difference in uptake. The inhibition in accumulation of a-aib with the higher concentration of dinitrophenol used in the earlier study could have been due to an effect on the lens, unrelated to energy metabolism. Whatever the explanation, it now seems that energy for amino acid transport in rabbit lenses also can be provided solely from, anaerobic metabolism, although, as KinoshitaV* studies suggest, it is very likely that under physiologic conditions ATP derived from respiration also contributes to the energy pool. The possibility of a close relationship between amino acid and potassium transport in the lens, as in tumor cells, was considered by Kern. 4 He noted that ouabain reduced the levels of both substances proportionately. However, he also observed that net movements of potassium and hydroxyproline were not always in the same direction. Kinsey and Reddy 0 measured the accumulation of tracer a-aib in rabbit lenses cultured in media containing no other amino acids, in which the concentration of potassium varied from 0 to 15 mmoles per liter, and also measured the rate of uptake of tracer potassium in media containing 5 mmoles per liter of potassium in which the concentration of a-aib varied from 0 to 15 mmoles per liter (Table IV). The accumulation of a-aib is not significantly affected by different concentrations of potassium, and uptake of the latter ion is just slightly greater (p < <.01) in the absence of amino acids. Thus there appears to be little, if any, interdependence of transport of amino acids and potassium in the lens. Uptake of tracer a-aib is affected by the presence of nonlabeled a-aib, glycine or methionine 5 in the culture media (Fig. 3). Accumulation of tracer a-aib decreases asymptotically with increased concentration of total amino acid, suggesting that the system responsible for transport of a-aib into the lens becomes saturated. 5 The existence of mutual competitive effects on transport was studied by determining the relative effect of 4.5 mmoles per liter of Table IV. Accumulation of ^'C-a-AIB as affected by potassium, and accumulation of 42 K as affected by nonlabeled a-aib, in cultured rabbit lenses (24 hours) Concentration of Kin media (mm.) Ratio of concentration of "'C-a-AlB, lens water media, initially ±3.2 (13) ± 1.7 ( 8) (12) Concentration of a-aib in media (mm.) Ratio of concentration of **K, lens water media, initially ±0.82 (8) ±0.92 (7) ±1.00 (7)

6 696 Kinsey Investigative Ophthalmology August CONCENTRATION AMINO ACID IN MEDIA (mmoles/l) Fig. 3. Effect of nonlabeled a-aib, glycine, and methionine in the media on the accumulation of ll C-labeled a-aib in lenses cultured for 24 hours. twenty-one L- and three D- amino acids on accumulation of tracer a-aib. The results (Fig. 4) are expressed on a scale of 100, where 100 units correspond to uptake when only tracer amino acid is present in the media. The gamma isomer of a-aib and both the basic and acidic amino acids have little or no effect in saturating the carrier system for a-aib (upper left-hand chart, Fig. 4). However, all neutral amino acids decrease accumulation in varying amounts, methionine being an even more effective inhibitor than a-aib. All D- forms of the neutral amino acids were less effective inhibitors than the L- isomers. This demonstration of the competitive effect of amino acids on accumulation led Kinsey and Reddy to conclude that at least three separate systems, perhaps involving carriers or carrier sites specific for neutral, acidic, and basic amino acids, are responsible for transporting them into the lens. Competition among amino acids for transport into the lens in species other than the rabbit has been demonstrated by several investigators. Thus, Kern 4 compared the rate of accumulation of hydroxyproline in calf lenses incubated in salt solution with that in TC 199 and in a salt solution containing 6 mmoles per liter of alanine, all as a function of the final concentration of hydroxyproline in the media (Fig. 5). He found that concentrations of 6 mmoles per liter of proline and methionine caused a reduction in uptake of hydroxyproline, the values being 36 and 53 per cent of normal, respectively; furthermore, the amino acids in TC 199 caused a decrease in accumulation of tyrosine and tryptophan. Kinoshita and colleagues 13 observed transport of a-aib in rat lenses to be effectively inhibited by methionine. Kinetics From the data in Fig. 3, the quantity of a-aib accumulated by rabbit lenses during a 24 hour period of culture was calculated on the assumption that lenses do not distinguish between labeled and nonlabeled amino acids. 5 The data (Fig. 6) show that accumulation increased with concentration in the media and reached a maximum at approximately 5.5 /^moles per lens. These values were then employed to calculate the apparent Michaelis-Menten constant, K m, and the maximum velocity of transport, V max. The reciprocal of the amount of a-aib in the lens was plotted against that of the concentration in the media using the method described by Lineweaver-Burk. This way of treating the data does not take into account the quantity of amino acid which has leaked out of the lens or changes in the concentration of labeled and nonlabeled amino acid in the culture media during the 24 hour period of culture. A detailed evaluation of the coefficients, employing methods similar to those used for calculating the same constants for potassium, 14 will be described more fully in a later paper. This evaluation gives a lower value for K m than previously reported, viz., 1.3 compared with 2.5 mmoles per liter, but approximately the same value for V max, viz., 0.23 yumole per lens per hour. The turnover rate of a-aib in rabbit lenses, also calculated by the newer method is 2.5 per cent per hour. Site of active transport Accumulation of amino acids is dependent upon the presence of the surface mem-

7 Volume 4 Number 4. Amino acid transport in lens 697 Fig. 4. lenses Effect of various amino acids (4.5 mm.) on the transport of 14 C-labeled a-aib into cultured for 24 hours. (From Kinsey and Reddy: INVEST. OPHTH. 2: 229, 1963.) HYDROXYPROLINE IN LENS (/im/ml ) 8- SALT MEDIUM J HYDROXYPROLINE IN MEDIUM Fig. 5. Effect of competing amino acids on the partition of hydroxyproline between calf lens and incubating medium. The period of incubation was 20 hours. (From Kern: INVEST. OPHTH. 1: 368, 1962.)

8 698 Kinsey Investigative Ophthalmology August 1965 TRACER ONLY 5mM 01 AIB 0.3 mm IODOACETATE Fig. 6. Accumulation of 14 C-labeled «-AIB by encapsulated lenses cultured in a medium containing no other amino acids. (From Kinsey and Reddy: INVEST. OPHTH. 2: 229, 1963.) TIME IN MINUTES Fig. 7. Effect of nonlabeled a-aib on the total amount of amino acid accumulated by lenses in 24 hours. (From Kinsey and Reddy: INVEST. OPHTH. 2: 229, 1963.) branes, the capsule, and epithelium. These membranes are also the site of the metabolically active processes concerned with active transport. Kern 4 found that the ratio of concentration of hydroxyproline in the lens plus its separated capsule, to that in the media, was 1.4 compared with 15 for an intact lens after 20 hours of incubation. Kinsey and Reddy 5 cultivated lenses without capsule and epithelia for various periods in media containing tracer a-aib only, tracer and 5 mmoles per liter of nonlabeled a-aib, and tracer with 0.3 mmole per liter of iodoacetate. They found that tracer a-aib rapidly entered decapsulated lenses and reached a maximum concentration within approximately one hour (Fig. 7). The presence of either nonlabeled a-aib or iodoacetate, both of which depress transport into intact lenses, had no appreciable effect on accumulation in lenses without capsule and epithelia. These results indicate that the mechanisms responsible for accumulation are associated with surface membranes, and, at least in decapsulated lenses, underlying structures are not involved to an appreciable extent. The question of whether the capsule or epithelium, or both, actively transport a-aib into the lens was investigated by studying separately 15 penetration across the anterior and posterior surfaces. Approximately two and one-half times as much 14 C-labeled a-aib was found in the lens within one hour following exposure of 60 per cent of the anterior surface to the isotope as when penetration occurred across the same area of the posterior surface (Table V). Removal of the epithelium and capsule caused a significant reduction in the rate of transport across the anterior surface, whereas there was only a slight increase in the quantity of a-aib that entered the lens through the posterior surface. Movement of a-aib through the anterior surface was reduced by nonlabeled a-aib, and by iodoacetate and ouabain, the latter to a lesser extent. Neither iodoacetate nor nonlabeled a-aib had any appreciable effect on penetration through the posterior surface. Thus all criteria suggestive of active transport of amino acids into the lens were shown to be associated with the anterior surface membranes. Since the anterior and posterior capsules appear to have similar physicochemical characteristics, the difference in behavior of the two sides of the lens led Kinsey and Reddy 13 to conclude that the single layer of epithelial cells, present only on the anterior surface, is the site of the mechanism responsible for active transport of amino acids in the lens. In conclusion, the lens behaves like a

9 Volume 4 Number 4 Amino acid transport in lens 699 Table V. Influx of "C-a-AIB in counts per minute per lens when a single surface is incubated for one hour in media containing 10 5 counts per minute per milliliter of radioactivity 15 Control Decapsulated Anterior Posterior 1,240 ± 370 (42) 540 ± 270 (37) (16) (10) Iodoacetate (14) 680 ± 230 (11) (3 x 10-3M) Ouabain ( 9) (10-5 M) Nonlabeled 605 ± 170 (13) (20) a-aib (HHM) Number of lenses shown in parentheses. pump-leak system whereby amino acids are actively transported into it by processes which depend on the presence of the epithelium, and leave by passive diffusion, probably chiefly across the posterior capsule. The balance between the rate of entrance through the action of the pump and exit by diffusion establishes their steady state concentration, which for all amino acids is higher in the lens than in the aqueous and vitreous humors. The amino acid pump requires energy which is derived primarily from the utilization of glucose; it can be saturated, and exhibits selectivity, suggesting that three or more systems are concerned in the active transport of amino acids. REFERENCES 1. Van Slyke, D. D., and Meyer, G. M.: The fate of protein digestion products in the body. III. The absorption of amino acids from the blood by the tissues, J. Biol. Chem. 16: 197, Reddy, D. V. N., and Kinsey, V. E.: Studies on the crystalline lens. IX. Quantitative analysis of free amino acids and related compounds, INVEST. OPHTH. 1: 635, Kinoshita, J. H., Kern, H. L., and Merola, L. O.: Factors affecting the cation transport of calf lenses, Biochim. et biophys. acta 47: 458, Kern, H. L.: Accumulation of amino acids by calf lens, INVEST. OPHTH. 1: 368, Kinsey, V. E., and Reddy, D. V. N.: Studies on the crystalline lens. X. Transport of amino acids, INVEST. OPHTH. 2: 229, Christensen, H. N.: Biological Transport, New York, 1962, W. A. Benjamin, Inc., p Danielli, J. F.: Morphological and molecular aspects of active transport, Symp. Soc. Exper. Biol. 8: 502, Brindley, G. S.: Resting potential of the lens, Brit. J. Ophth. 40: 385, Kinsey, V. E., and Reddy, D. V. N.: Unpublished. 10. Dardenne, U., and Kirsten, C: Presence and metabolism of amino acids in young and old lenses, Exper. Eye Res. 1: 415, Reddy, D. V. N., and Kinsey, V. E.: Transport of alpha aminoisobutyric acid into ocular fluids and lens, INVEST. OPHTH. 1: 41, Thoft, R. A., and Kinoshita, J. H.: The effect of calcium on rat lens permeability, INVEST. OPHTH. 4: 122, Kinoshita, J. H., Merola, L. O., and Hayman, S.: The effect of aldoses on accumulation of a-aminoisobutyric acid (a-aib) by the rabbit lens, J. Biol. Chem. 240: 310, Kinsey, V. E., and McLean, I.: Studies on the crystalline lens. XIII. Kinetics of potassium transport, INVEST. OPHTH. 3: 585, Kinsey, V. E., and Reddy, D. V. N.: Studies on the crystalline lens. XI. The relative role of the epithelium in transport, INVEST. OPHTH. 4: 104, 1965.