Transport of ammo acids into the posterior chamber of the rabbit eye. V. Everett Kinsey and D.V. N. Recldy

Transcription

1 Transport of ammo acids into the posterior chamber of the rabbit eye V. Everett Kinsey and D.V. N. Recldy The rate of entrance of C-1-labeled alpha aminoisobutyric acid (a-aib) into the posterior chamber of rabbit eyes decreases asymptotically with increasing concentrations of nonlabeled compound in the blood to one fifth that found when tracer compound alone is given. The results indicate that approximately 0 per cent of the tracer a-aib enters by active transport and the remainder by passive diffusion. The agreement between experimental results and the hypothesis underlying Michaelis-Menten kinetics is in accord with the idea that a carrier is involved in the active transport mechanism. The following parameters of the transport mechanism have been determined or calculated: The coefficient of diffusion (k<t.,,h) is 0.63 per cent per minute. When the active transport system is saturated, i.e., when it is operating at maximum capacity, 2. x 10~ 6 mmoles per minute enter the posterior chamber, and, the concentration in the fluid believed to flow from the ciliary epithelium into the posterior chamber (C) is 0.72 mmoles per liter. The transport system for a-aib can be saturated to varying degrees by the administration of naturally occurring neutral amino acids but is not affected appreciably by either basic or acidic amino acids or by y-aminobutyric acid. The D and DL amino acids are less effective than the L form in saturating the transport system. The observations are compatible with the concept that at least three carrier systems must be involved in the active transport of naturally occurring amino acids across the ciliary epithelium. Llabeled (C-1) model amino acid, alpha aminoisobutyric acid (a-aib) entering the posterior chamber of the rabbit eye is reduced 70 per cent when nonlabeled a-aib (5 mmoles per kilogram) is administered 15 minutes before the tracer compound. 1 The amount of tracer a-aib entering the posterior chamber thus depends on the total concentration in the plasma; hence, it was concluded that this From the Kresge Eye Institute, Detroit, Mich. This study was supported in part by Research Crants B-1100 and B-25 from the National Institute of Neurological Diseases and Blindness, United States Public Health Service, by the United States Atomic Energy Commission Contract No. AT (11-1)-152, and by Research to Prevent Blindness, Inc. 355 amino acid is actively moved across the ciliary epithelial cells. That amino acids other than a-aib, which, incidentally, does not occur in nature and is not metabolized, are also actively transported into the posterior chamber was indicated by another study 2 in which the steady-state concentration of all but three of the amino acids in the aqueous humor of the posterior chamber was found to be higher than in plasma. The purpose of the present investigation is to determine: (1) the relative amounts of a-aib which enter the posterior chamber actively and passively; (2) the diffusion coefficient, k (1 )11, for a-aib from plasma to posterior chamber; (3) the concentration of a-aib in the "secreted fluid" when the active transport system is saturated; () the apparent Michaelis-Menten constant for the presumed carrier of a-aib; and (5)

2 356 Kinsey and Redcly Investigative Ophthalmology June 1962 the relative efficacy of naturally occurring amino acids and some D and DL compounds in saturating the mechanisms involved. Methods All the experiments were performed on conscious young albino rabbits which weighed between 1. and 2.3 kilograms. Aqueous humor was withdrawn from the posterior and anterior chambers under a local anesthetic, tetracaine hydrochloride (Pontocaine), with techniques described previously. 3 Blood was obtained by cardiac puncture, heparin was used as an anticoagulant. The animals were killed by air emboli, and immediately thereafter vitreous humor was withdrawn with an 1 gauge needle. Lenses were removed, homogenized in Nelson-Somogoyi reagent, and centrifuged to obtain a solution containing the free amino acids. No appreciable radioactivity was found in lens proteins. The amount of amino acids in the lens was calculated on the basis that 65 per cent of the total weight of the lens is water. Eight microcuries of C-1-labeled a-aib in 2 ml. of saline were given each rabbit. When radioactivity in lens and vitreous was determined, the dose was increased to 30 microcuries. One quarter of the initial dose was given intravenously and three quarters intraperitoneally. The samples were plated on copper planchets and radioactivity was measured with a thin window-flow gas counter, appropriate corrections being made for self-absorption. All of the nonlabeled amino acids including a-aib were given 15 minutes prior to administering the tracer a-aib. The proportion of the nonlabeled compound given intravenously and intraperitoneally was similar to that of tracer a-aib. Five millimoles per kilogram body weight of the naturally occurring amino acids was given as a 0.5 M solution when solubility limitations permitted; otherwise a less concentrated solution was employed and the volume of the solution injected increased accordingly. The concentration of nonlabeled a-aib in the plasma was estimated from the extent of isotopic dilution. The relationship between the estimated plasma concentration and the dose of nonlabeled a-aib given is shown in Fig. 1. The lack of linearity is probably due to saturation at increasing doses of those compartments in the body capable of taking up a-aib. In all calculations the volumes of the posterior chamber and vitreous were assumed to be 0.06 and 1.5 ml., respectively. We calculated the amount of radioactivity lost from the posterior chamber to the anterior chamber on the basis of a flow rate of 3.3 p\ per minute using the average concentration present during the 5 minute test period PLASMA CONCENTRATION (m moles/kg. H 2 O) Fig. 1. The relation between the amount of nonlabeled a-aib administered and the resulting concentration in the plasma as determined by isotopic dilution. The diffusion coefficient, k,i.,,i,, was calculated by subtracting the amount of a-aib that entered the posterior chamber in 5 minutes by active transport from the total quantity entering this chamber at the same time. It was assumed that the quantity entering by diffusion is directly proportional to the concentration in the plasma, which was shown to be essentially constant during this period. The method of calculation is as follows: k,i.,,u C,,, amount entering by diffusion k.i.,,1,, coefficient of diffusion C P, concentration in the plasma The concentration of a-aib in the "secreted fluid" in the ciliary epithelium under conditions in which the transport mechanism is saturated was calculated from a knowledge of the total amount of a-aib entering the posterior chamber by active transport: kfh C s amount entering by active transport. Flow rate km = 3.3 /(tl/min.; C s = concentration in the secreted fluid. Forty-five minutes was selected as a test period because it was believed that at this time any effect on the relation between concentration in the posterior chamber and time would be most sensitive to changes in rate of entrance (Fig. I 1 ). Results The effect of the concentration of nonlabeled a-aib in the plasma on the amount of C-1-labeled a-aib which enters the aqueous of the posterior chamber is shown in Fig. 2. The amount of the labeled compound is expressed as concentration rela-

3 Volume 1 Number 3 Amino acid transport into posterior chamber 357 tive to concentration in the plasma. The plasma levels were obtained by giving nonlabeled a-aib 15 minutes prior to injecting the tracer. The data show that the concentration of C-1-labeled amino acid in the posterior chamber decreases asymptotically with concentration in plasma. The lower limit approached is approximately 5 per cent of that in the plasma, or about one fifth that found when the concentration in plasma is essentially zero (i.e., tracer only given). Fig. 3 shows the effect of plasma concentration of a-aib on the amount of this amino acid present in the aqueous of the posterior chamber 5 minutes after injection. The solid line shows the total amount present; the dot-dashed and dashed lines show the amounts present which entered by diffusion and active transport, respectively. The values for the total amount present in the posterior chamber were calculated from a knowledge of the concentration of tracer a-aib in the posterior chamber for different plasma concentrations of a-aib as shown by the line in Fig. 2. For example, when the plasma concentration is 10 mmoles per kilogram water, the concentration of tracer compound in the posterior chamber was observed to be.2 per cent of this value. Thus, the total quantity present in the posterior chamber is 0.02 x 10 x 10~ M mmoles/ml. FLO x 0.06 ml. =.92 x 10" r> Fig. 2. The effect of concentration of nonlabeled a-aib in the plasma on the concentration of tracer a-aib in the posterior chamber after 5 minutes. i I I I! I I I I I I I PLASMA CONCENTRATION (m molet / kg. H,0) Fig. 3. The effect of plasma concentration of a-aib on the total amount of amino acid present. in the posterior chamber after 5 minutes and that entering by diffusion and active transport. mmoles. (The method of calculating the amount present in the posterior chamber which entered by diffusion and active transport is described under methods.) The linear increase in the total amount of a-aib present in the posterior chamber above plasma concentrations of approximately 10 mmoles per kilogram water is a necessary consequence of the relative independence of the amount of tracer entering the posterior chamber aqueous and the concentration of nonlabeled compound in plasma, as shown in Fig. 2. The slope of this portion of the line is a measure of the rate of accumulation resulting from diffusion alone. The quantity entering the posterior chamber by diffusion for any concentration in the plasma is represented by a line drawn parallel to the upper line through the point of origin, i.e., for zero concentration in plasma. The difference between the total amount of a-aib present in the posterior chamber and that which enters by diffusion is the result of active transport. It follows that the line showing entrance by diffusion must have a slope equal to about 20 per cent of that line showing active transport at substantially zero concentration in the plasma, i.e., where tracer compound only is involved. The circles in Fig. show the experimental results plotted according to the Lineweaver-Burk' 1 method of treating

4 35 Kinsey and Reddy Investigative Ophthalmology June 1962 I.I x icr M CONCENTRATION PLASMA ( m molei / kg. H 0) PLASMA CONCENTRATION ( kg. H20) Fig.. Lineweaver-Burk plot of the accumulation of a-aib in the posterior chamber at 5 minutes. The points represent the reciprocal of the amount present plotted against the reciprocal of the concentration of a-aib in the plasma. K,,, is the apparent Michaelis-Menten constant. Fig. 5. The effect of plasma concentration of a-aib on the total amount of amino acid which entered the posterior chamber during 5 minutes and that entering by diffusion and active transport io - o - Figs. 6 to 9. The concentration of C-1-labeled a-aib in the posterior chamber at 5 minutes when different amino acids (5 mmoles per kilogram) were given 15 minutes prior to the administration of the tracer compound.

5 Volume.1. Number 3 Amino acid transport into posterior chamber 359 It is assumed that all of the a-aib present in the vitreous at 5 minutes entered from the posterior chamber.,, _ 0,95 X 10- c mmoles/min. 1 n m M,. fktl.ph.= n ; ; : «- = / X 10-' mmoles/ml ml. Michaelis-Menten kinetics. These kinetics are expressly intended to describe a system which can be saturated (ordinarily involving an enzyme and substrate). The agreement between the experimental results and this type of hypothesis is compatible with the idea that a carrier may be involved in the transport mechanism. The value for K m which is the Michaelis-Menten value for half saturation is approximately 1.1 x 10~ M. The total quantity of a-aib actively transported across the ciliary epithelium is the sum of the amount present in the posterior chamber, vitreous,* and lens, plus that quantity which has flowed into the anterior chamber during the test period. The first three of these quantities were determined experimentally, and the last was calculated on the basis of a flow rate of 3.3 (A per minute. The results are shown in Fig. 5 by plots analogous to those in Fig. 3. The following values can either be taken directly from the graph or computed: When the active transport mechanism is saturated, 1.1 x 10" mmoles enter the posterior chamber by active transport in 5 minutes, or approximately 2. x 10~ G mmoles per minute. The concentration in the "secreted fluid," C s, is 0.72 mmoles per liter. When, for example, the concentration in the plasma is 25 mmoles per kilogram water, approximately 10~ 5 mmoles per minute cross the ciliary epithelium by diffusion or four times that which enters actively at this plasma concentration. The diffusion coefficient k (11,i, is 0.63 per cent per minute, f The effect of amino acids other than a-aib itself on accumulation of C-1- labeled a-aib was investigated to determine more about the specificity of the saturation phenomena, and, in turn, gain some insight into the mechanisms of transport of naturally occurring amino acids. Five mmoles per kilogram body weight of a number of amino acids were given separately 15 minutes prior to administration of the tracer compound, and the concentration of C-1-labeled a-aib in the posterior chamber was measured as a percentage of the plasma concentration 5 minutes after the injection of tracer a-aib. The data are presented in Table I. It is apparent that many of the naturally occurring amino acids, like a-aib itself, decrease the quantity of tracer a-aib transported into the posterior chamber. The block diagrams of Figs. 6 to 9 are concerned with the same data that appear in Table I, but show the effect of various amino acids on the amount of tracer a-aib entering the posterior chamber by active transport only. In constructing diagrams it was assumed, on the basis of data presented in Fig. 2, that one fifth of the total Table I. Shows the concentration of C-1-labeled a-aib 5 minutes after its parenteral administration in rabbits when various L-amino acids are similarly administered (5 mmoles per kilogram) 15 minutes prior to the tracer compound C-1 «-AIB only Alanine a-aib Arginine Ascorbic acid Aspartic acid Cysteine 7-AB Glutamine Clutamic acid Glycine Histidine Hydroxyproline Isoleucine Lysine Methionine Orni thine Phenylalanine Proline Serine Threonine Valine Range Per cent of plasma concentration in posterior chamber No. eyes Average

6 360 Kinsey and Reddy Investigative Ophthalmology June 1962 amount of tracer a-aib enters the posterior chamber by diffusion, independent of any saturation attributable to administration of nonlabeled amino acids. Thus, of the 2 per cent plasma level observed when tracer a-aib only was given,. per cent can be accounted for by that amount which enters the posterior chamber by diffusion and the remainder, 19.2 per cent, by active transport. Since this is the percentage concentration of tracer resulting from active transport in the absence of any measurable saturation (over and above that caused by the amino acids normally present in the plasma), this value has been used as a reference point and set equal to 100 in constructing the block diagrams. It appears as a background in the form of an open parallelepiped and is labeled "tracer only" in each of the figures. All other values are scaled accordingly. A sample calculation follows: The average absolute concentration of tracer a-aib present in the posterior chamber after administration of 5 mmoles per kilogram of nonlabeled a-aib as a result of both active and passive transport is 9.0. The transport of tracer a-aib in relative units is thus. ' x 100 = For ease of comparison, the first block diagram representing saturation by a-aib and having a height of 22 scale units is shown in Figs. 6 to. The second block diagram of Fig. 6 shows that T-aminobutyric acid, a structural analogue of a-aib, is without appreciable effect in saturating the transport mechanism of a-aib. The remaining diagrams show that the basic and acidic amino acids which occur in nature likewise do not affect a-aib transport. With the exception of isoleucine and phenylalanine, all of the neutral amino acids reduce the amount of tracer a-aib entering the posterior chamber (Figs. 7 and ). It is noteworthy that methionine is more effective in saturating the transport system than a-aib. The influence of isomerism on transport of tracer a-aib is shown in Fig. 9; the D and DL forms of the neutral amino acids are less effective than the L form in depressing transport. Discussion The fact that the amount of a-aib transported actively and passively could be determined separately made it possible to calculate the concentration, C s, of this amino acid in the secreted fluid without knowledge of its steady-state distribution. The ability to measure a parameter concerned with the system governing the formation of aqueous humor in the posterior chamber not heretofore assessed 3 was fortunate since it was not feasible to obtain such information for a-aib because of slowness of filling of the vitreous and the continued and prolonged uptake of this amino acid by the lens. 1 The latest method of calculation is applicable to any substance that has a transport mechanism capable of being saturated, e.g., ascorbic acid. 0 The present results are compatible with the concept that a carrier, having a limited capacity to transport a-aib, becomes progressively saturated as the concentration of nonlabeled compound in the plasma is increased, and that in addition to active transport there is a passive exchange between plasma and posterior aqueous which, when tracer a-aib alone is administered, contributes approximately 20 per cent to the total quantity of amino acid entering the posterior chamber. In previous papers*- ' the dual nature of entrance of other substances has been stressed and it was suggested that many, if not all, constituents of the aqueous enter the posterior chamber from the plasma both actively and passively. However, the mechanism of active transport of a-aib and active transport of other substances as discussed earlier is probably different. In the case of the movement of ions, and, presumably, other naturally occurring compounds, such as urea, we have suggested that fluid formed in the epithelium of the ciliary processes flows into the posterior chamber, or perhaps emerges from the epithelial cells by pinocytosis. No assumption

7 Volume 1 Number 3 Amino acid transport into posterior chamber 361 has been made concerning the exact location of this fluid before it enters the posterior chamber, but it may be contained in the vesicles shown by electron microscopy s> to be present in the ciliary epithelium. The transport mechanisms involved, and perhaps also the formation of this fluid, must require the expenditure of energy, so its unidirectional entrance may be defined as "active transport." It is in this sense that the expression active transport of various normal constituents of the aqueous humor was used. We have referred to this process as secretion, and the fluid involved as secreted fluid. If the above concept is correct, it seems likely that a-aib, and naturally occurring amino acids also are contained in the secreted fluid. We have, therefore, calculated the concentration of a-aib which would have to exist in the secreted fluid if the unidirectional entrance occurs in this manner, or in a closely similar one. However, the active transport mechanism for a-aib which is capable of saturation is not directly associated with the flow process, as we have shown experimentally by demonstrating that the flow rate is independent of the concentration of a-aib present in the plasma. Instead, it probably is associated with a carrier located on a cell or plasma membrane, perhaps bounding any vesicles concerned with unidirectional flow. Thus, the term active transport as used to describe the mechanism of entrance of a-aib does not have the same meaning as it did when discussing the entrance of ions or other constituents of the aqueous humor. Actually, the formation of the secreted fluid, so far as ions and other constituents of the aqueous is concerned, may likewise involve carriers, perhaps located on the same cellular structures as those for amino acids. These carrier systems, if indeed they exist, are probably already saturated at normal physiologic concentrations; therefore, it is not remarkable that they have yet to be demonstrated. When tracer a-aib only was given to the animals, 0 per cent of the total amount entered the posterior chamber actively and 20 per cent passively. Since many of the naturally occurring amino acids saturated the a-aib transport system, it follows that a still greater proportion of a-aib would have entered actively if no amino acids were present in the plasma normally. It follows, also, that the maximum amount of a-aib actively transported by the ciliary epithelial cells at saturation, as reported here, is not an accurate measure of the full capacity of the system involved, for the same carrier is simultaneously transporting unknown quantities of other amino acids. Thus, the amount of a-aib entering the posterior chamber under these conditions of saturation cannot be considered as a fundamental constant of the transport system, and so cannot be compared with a similarly measured rate of entrance of another substance. Moreover, the value for the Michaelis constant, K m, applies only to the half-saturation value of the system in the presence of other amino acids in the plasma. The high degree of selectivity of the blood-aqueous barrier in the ciliary processes is further illustrated by the present results. Not only is the active transport mechanism for amino acids highly specific with regard to chemical structure and isomeric form, but the rate of passage of substances through the barrier varies within wide limits, apparently independent of molecular size and solubility characteristics. For example, k<i.i.n for a-aib is 0.63 per cent per minute, whereas for sodium, chloride, and urea 10 it is 3.0, 15, and 3.6 per cent per minute, respectively. The values for the effect of administering different amino acids in depressing the amount of tracer a-aib transported, as shown in Table I and Figs. 6 to 9, must be considered only approximate. Although the same quantity of the naturally occurring amino acids was given, the resulting concentration in the plasma may not have been the same for each amino acid. However, these results, along with those reported elsewhere 2 which show that the concentra-

8 362 Kinsey and Recldy Investigative Ophthalmology June 1962 tion of most amino acids in the aqueous of the posterior chamber is greater than in plasma, suggest that amino acids in general are actively transported. The observations are compatible with the idea that, as for most cells, 11 the transport of amino acids across the ciliary epithelium involves three carrier systems, one each for basic, acidic, and neutral compounds. There may be others since phenylalanine and isoleucine, unlike other neutral amino acids, do not reduce the amount of a-aib entering the posterior chamber, and both of these compounds are present in higher concentrations in the aqueous humor of the posterior chamber than in plasma. We wish to thank Emilie Kay Hopkins, Beverley A. Skrentny, and Angell DeMeglio for their technical assistance. REFERENCES 1. Redcly, D. V. N., and Kinsey, V. E.: Transport of alpha aminoisobutyric acid into ocular fluids and lens, INVEST. OPHTH. 1: 1, Reddy, D. V. N., Rosenberg, C, and Kinsey, V. E.: Steady state distribution of free amino acids in the aqueous humors, vitreous body and plasma of the rabbit, Exper. Eye Res. 1: 175, Kinsey, V. E.: Comparative chemistry of aqueous humor in posterior and anterior chambers of rabbit eye: Its physiologic significance, A. M. A. Arch. Ophth. 50: Lineweaver, H., and Burk, D.: The determination of enzyme dissociation constants, J. Am. Chem. Soc. 56: 65, Kinsey, V. E., and Reddy, D. V. N.: An estimate of the ionic composition of the fluid secreted into the posterior chamber, inferred from a study of aqueous humor dynamics, Docum. Ophth. 13: 7, Kinsey, V. E.: Transfer of ascorbic acid and related compounds across the blood-aqueous barrier, Am. J. Ophth. 30: 1262, Kinsey, V. E.: Ion movement in the eye, Circulation 21: 96, Holmberg, A.: Ultrastructural change in the ciliary epithelium following inhibition of secretion of aqueous humor in the rabbit eye, Sodertalje, Sweden, 1957, Axlings Bok and Tidskriftryckeri. 9. Pappas, G. D., and Smelser, G. K.: Studies on the ciliary epithelium and the zonule. I. Electron microscope observations on changes induced by alteration of normal aqueous humor formation in the rabbit, Am. J. Ophth. 6: 299, Kinsey, V. E., Reddy, D. V. N., and Skrentny, B. A.: Intraocular transport of C-1-labeled urea and the influence of Diamox on its rate of accumulation in aqueous humors, Am. J. Ophth. 50: 1130, I960! 11. Christensen, H. N., Akedo, H., and Winter, C. G.: On the mechanism of amino acid transport into cells, Proceedings of the Conference on Free Amino Acids, City of Hope Medical Center, Duarte, California, May 19, 1961 (in press).