Intraocular transport of myoinositol

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1 Intraocular transport of myoinositol II. Accumulation in the rabbit lens in vitro S. D. Varma, B. Chakrapani, and V. N. Reddy The characteristics of myoinositol transport in the lens were studied by culturing them in a medium containing the tritiumlabeled compound. The accumulation in the lens takes place against a concentration gradient and is inhibited by ouabain, iodoacetate, phlorhizin, and at reduced temperature (Q, o = ). The energy for transport is derived from glycolysis. The transport system follows MichaelisMenten kinetics (V ma x = 0.02 xmoles per lens per hour, K m = 0.4 mm.) and requires the presence of Na + and K +. Although a relationship between Na+ concentration and inositol transport was observed, the lenses in Na + deficient media could not be maintained in a physiological state so that the decreased transport cannot be ascribed solely to the effect of this cation. Similar to the observations made in the ciliary body, the transport system in the lens is inhibited by inositol mono and hexaphosphates and shows the same specificity for the stereoisomers of inositol. Unlike the findings in the case of ciliary body, inositol accumulation in lens is inhibited by several amino acids. The reciprocal inhibition of aaminoisobutyric acid transport by 'inositol, however, could not be demonstrated. Key words: myoinositol transport, lens inositol, amino acids, metabolic inhibitors, rabbit, inositol isomers, active transport in lens, tissue culture medium, sodium, kinetics, aaib, glycine, serine, inositol efflux. 0 cular lens contains an unusually high concentration of myoinositol which is almost 70 times higher than that in the posterior chamber aqueous. 2 The role of high inositol concentration in the lens is not From the Institute of Biological Sciences, Oakland University, Rochester, Mich. This investigation was supported, in part, by Research Grant from the National Eye Institute of the National Institutes of Health and United States Atomic Energy Commission Contract No. AT( lll)20. Presented in part before the annual meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Fla., May to, 970. Manuscript submitted June 22, 970; revised manuscript accepted Aug. 2, known and the mechanism by which it is derived is not clearly understood. There is some evidence that inositol is accumulated in the lens against a concentration gradient and the process is inhibited by ouabain. 4 We have recently shown that myoinositol is actively transported into the ciliary body and across the bloodaqueous barrier by a highly specific carriermediated system. 2 The present study is designed to obtain further knowledge concerning the entrance of myoinositol into the lens and to characterize the transport system in greater detail. Methods Lenses from albino rabbits weighing between.8 and 2. kilograms were cultured under sterile conditions in ml. of KEIA medium (without Downloaded from iovs.arvojournals.org on 02209

IIINI Volume 9 Number 0 Myoinositol in rabbit lens 79 arnino acids) containing tritiumlabeled inositol. The culture medium contained 0 ic of [2 H] myoinositol per 00 milliliters.

2 IIINI Volume 9 Number 0 Myoinositol in rabbit lens 79 arnino acids) containing tritiumlabeled inositol. The culture medium contained 0 ic of [2 H] myoinositol per 00 milliliters. The specific activity of the compound was,470 mc per millimole. The radiochemical purity of inositol was established by chromatography. 2 Lens culture technique, composition of the media, and the method of determination of radioactivity in lenses have all been described previously. 0 The accumulation of myoinositol in the lens was expressed as a ratio of the concentration of radioactivity in lens water ( per cent) to that in the initial medium (ClCm). In experiments requiring addition of nonlabeled inositol or other compounds, equivalent amounts of sodium chloride were omitted from the media. The effect of various substances on the accumulation of myoinositol in the lens was studied by comparing the rates of accumulation in lenses from contralateral eyes which were cultured in the presence of labeled inositol only. The method used for determining the efflux of myoinositol from the lens was analogous to that used in an earlier study. 0 Lenses were preloaded by culturing in a complete medium (KEI4) containing 0 ic of [2^] myoinositol per 00 milliliters. The "runout" of the labeled compound was then measured in KEIA medium with or without 0 mm. nonlabeled inositol. Lipids from the lenses cultured in the presence of labeled inositol were extracted with chloroformmethanol mixture. 7 Analyses showed the absence of any radioactivity in these extracts. To investigate the possibility of metabolic breakdown of labeled myoinositol, samples of incubation medium and lens extracts, prepared by homogenization in barium hydroxide and zinc sulfate, were chromatographed. The chromatograms showed the presence of a single radioactive substance corresponding to the position of an authentic sample of myoinositol. Inorganic phosphorus was assayed by the method of Fiske and Subba Row. s Results Kinetics of transport. Fig. shows the time course of accumulation of myoinositol in the lens which occurs against a concentration gradient. The distribution ratio (ClCm) between the lens water and the initial medium after 24 hours of culture was approximately 2. Addition of increasing amounts of nonlabeled myoinositol to the incubation medium resulted in a progressive decline in the distribution ratio (Fig. 2), indicating that the system involved has a limited capacity for transporting the compound. s s s _ ( n J^t " (; I l l TIME (Hours) Fig.. Time course of accumulation of [2 H] myoinositol in lenses cultured in KEIA medium. The line through the data was generated with an analogue computer by solving the pumpleak equation (see text). Standard deviations and the number of lenses are indicated on the chart. CONC. NONIABEUED COMPOUND (mm) Fig. 2. Effect of nonlabeled myoinositol on the accumulation ratio of the labeled compound in lenses cultured in KEIA medium for 24 hours. Figures in parentheses indicate the number of lenses. The rates of efflux of labeled inositol from the lens in the presence or absence of nonlabeled inositol in the medium are shown in Fig.. When 0 mmoles per liter of nonlabeled inositol was added to the medium to prevent the reentry of the labeled compound into the lens, the rate of efflux was linear (lower curve) and the diffusion coefficient (Kd) was hr."\ Using this value of Kd, the coefficient of active transport (Kp) was estimated by solving the "pumpleak" equation of Kinsey and Mc Lean. 9 The value of Kp, which was found (.) Downloaded from iovs.arvojournals.org on 02209

3 79 Varma, Chakrapani, and Reddy Investigative Ophthalmology October 970 TIME (Hour Fig.. Rate of efflux of myoinositol from the lens. The lenses were preloaded with [2 H] myoinositol by culturing in KEI4 medium and the efflux determined in nonradioactive medium. Solid line indicates the rate of efflux in medium containing 0 mm. nonlabeled inositol; the dotted line is the rate of efflux in medium containing no inositol. Fig. 4. Relation between the velocity of active transport and the concentration of myoinositol in the culture medium. Fig.. LineweaverBurk plot of the data shown in Fig. 4. to give a good fit to the data (Fig, ) was 0.8 hrr ; the curve shown was generated by an analogue computer. The values of Kp at different substrate concentrations were also estimated by the same procedure, 9 assuming that Kd was unaffected by the concentration of the substrate in the medium. Appropriate corrections for the changing concentrations of both labeled and nonlabeled inositol in the medium during the 24 hour culture period were taken into account. The velocity of the pump at different substrate concentrations was then calculated as a product of the coefficient of active transport (Kp), concentration in the medium (C m ) and the volume of lens water (V t ). The data relating the velocity of the pump to the concentration of the medium are shown in Fig. 4. The LineweaverBurk plot of these data (Fig. ) gives a straight line, indicating that the transport process follows MichaelisMenten kinetics. The half saturation level (K m ) is 0.4 mm. and the maximum rate of transport (V ma x) is 0.02 jjnoles per lens per hour. Effect of temperature, metabolic inhibitors, and other factors. The accumulation of inositol in the lens was reduced in the presence of the metabolic inhibitors phlorhizin, iodoacetate, and ouabain (Table I). The results also show that the process of accumulation is reduced at lower temperature (Qio.0) and in the absence of glucose in the medium. That the energy for transport is derived from glycolysis follows from the observation that anaerobiosis and dinitrophenol have no effect on inositol accumulation. The distribution ratio (ClCm) attained in 24 hour culture in lenses from which capsule and epithelium were removed was less than, suggesting that the transport system involved resides in lens epithelium. 0 Effect of ions. In the absence of calcium or potassium ions, the accumulation of myoinositol in the lens was reduced (Table I). Lack of K + in the medium had a less pronounced effect. Partial replacement of Na + with tetra Downloaded from iovs.arvojournals.org on 02209

4 Volume 9 Number 0 Myoinositol in rabbit lens 797 Table I. Effect of various conditions of incubation and metabolic inhibitors on the accumulation of [2 H] myoinositol in the lens cultured for 24 hours in KEIA medium, expressed as percentage of contralateral lens (control) Condition of incubation Contralateral lens (control 7 C.) Reduced temperature (27 C.) Incubation without glucose Anaerobiosis Incubation without K + Incubation without Ca ++ 2,4Dinitrophenol Iodoacetate Ouabain Phlorhizin Phlorhizin No. of experiments 4 Inhibitor concentration (M) 0 4 x Accumulation (% of control ± S.D.) 00 4 ± 4 44 ± 4 0 ± 8 7 ± 27 ± 0 ± 4 8 ± 40 ± 40 ± 7 ± Fig.. Effect of partial replacement of Na + (0 meq. per liter) with various substituents on the accumulation of myoinositol in the lens cultured for four hours in KEIA medium expressed as percentage of contralateral lens (control). The values are given as the mean ± the standard deviation of the mean. ethyl ammonium (TEA + ), Tris + (hydroxymethyl amino methane), choline +, Li +, or sucrose also resulted in a significant reduction in the accumulation of inositol (Fig. ). The period of incubation in these experiments was four hours. The extent of inhibition, however, was different when the same amount of sodium chloride (0 mm.) was isoosmotically replaced with various substituents. It should be mentioned that the lenses cultured in these media became cloudy and the extent of accumulation of labeled inositol was inversely related to the degree of cloudiness. The minimum accumulation occurred in the medium containing TEA + in which lenses became most cloudy. The effect of changing Na + concentration on the accumulation of inositol was also investigated in Tyrode's medium where the concentration of the cation could be more easily varied over a wider range. Table II shows that accumulation of inositol in lenses incubated in this medium was directly related to the sodium ion concentration. However, the accumulation was affected differently when identical quantities of sodium ions were replaced by different substituents. As in the case of Na + deficient KEI medium, the lenses became cloudy and the accumulation of myoinositol was related to the extent of cloudiness. The effect of stereoisomers and inositol derivatives. In order to determine the specificity of the transport process, the effect of the stereoisomers of inositol and its phosphate esters on the accumulation of [2 H] myoinositol in the lens was investigated (Table III). Scylloinositol was found to significantly (P < 0.0) reduce the transport of myoinositol, whereas D and Lchiroinositols showed no effect. Both mono and hexaphosphate esters reduced the accumulation of labeled inositol. The inhibition by these esters was not due to nonlabeled inositol, liberated as a result of hydrolysis, as there was no change in the Downloaded from iovs.arvojournals.org on 02209

5 798 Varma, Chakrapani, and Reddy Investigative Ophthalmology October 970 Table II. Effect of changing concentration of sodium ions on the accumulation of [2 H] myoinositol in the lens cultured for 4 hours in Tyrode's solution* Concentration of No* (meq. per liter of the medium) Control, 49 (00%) 2t 80f 2 f Substances replacing Na+ {% of control) TEA Cl Choline Cl Trs HCl LiCl Sucrose 74 ±.2 49 ± 2. 2 ±.0 2. ± ±.0 9 ± ± ± ± 2. 8 ± ±.. ± ± 0 9 ± 2 7 ± 2.2. ±.0 80 ± 82 ± 4 ±.2 ± 'Results are expressed as percentage of contralateral lens cultured in the same medium containing 49 meq. per liter of sodium ions. The values are given as the mean ± S.D. fosmolarity was maintained by adding equivalent amounts of the substituents. Table III. Effect of nonlabeled isomers of inositol and its phosphate esters on the accumulation of [2 H] myoinositol in the lens cultured for 24 hours in KEI IA medium, expressed as percentage of contralateral lens (control) Compound Contralateral lens (control) Myoinositol Scylloinositol Dchiroinositol Lchiroinositol Inositol 2monophosphate Inositol hexaphosphate Inositol hexaphosphate No. of experiments Concentration (mm.) Accumulation (% of control ± S.D.) 00 ± 4 8 ± 94 ± 2 9 ± 2 8 ± 2 0 ± 9 ± Table IV. Effect of amino acids and other compounds on the accumulation of [2 H] myoinositol in the lens cultured for 24 hours in KEIIA medium, expressed as percentage of contralateral lens (control) Compound Contralateral lens (control) aaminoisobutyric acid Lalanine Glycine Lmethionine Proline Lserine Lthreonine Lvaline Ascorbic acid Methyl glucoside 0Methyl glucose No. of experiments 4 Concentration (mm.) Accumulation (% of control ± S.D.) ± 9 ± 44 ± 9 8 ± 7 ± 9 44 ± ± 0 ± 4 8 ± 9 4 ± 8 ± level of inorganic phosphorus in the incubation medium. To determine the nature of inhibition caused by phosphate esters, the accumulation of myoinositol at different substrate concentrations was studied in the presence and absence of either mono or hexaphosphate and the velocity of active transport (KpC m Vi) calculated in the usual manner. The double reciprocal plots of these data (Fig. 7), which have the same intercept on the ordinate, suggest that the inhibition of inositol transport by phosphate esters is of a competitive nature. Downloaded from iovs.arvojournals.org on 02209

Volume 9 Number 0 Myoinositol in rabbit lens 799 800 700 i 00 I 00 "I 400 E ^ 00 200 00 I I 2 4 i Ino.ilol Hexophospna B 7 8 X* 9 Monophosphate o Control Fig. 7. Competitive inhibition of myoinositol transport by inositol mono and hexaphosphates.

6 Volume 9 Number 0 Myoinositol in rabbit lens i 00 I 00 "I 400 E ^ I I 2 4 i Ino.ilol Hexophospna B 7 8 X* 9 Monophosphate o Control Fig. 7. Competitive inhibition of myoinositol transport by inositol mono and hexaphosphates. LineweaverBurk plots are derived from the rates of active transport of myoinositol in the lenses cultured in KEIA medium in the presence and absence of mm. monophosphate or.7 mm. hexaphosphate. The effect of amino acids and other compounds. Table IV shows the effect of the addition of mm. concentration of amino acids, ascorbic acid, and two derivatives of glucose on the amount of tracer myoinositol accumulated in the lens. In contrast to the observation made in the ciliary body, the transport system for inositol in the lens was inhibited by several amino acids. With the possible exception of methionine, all of the amino acids studied significantly (P < 0.0) decreased the accumulation of inositol. In view of this unexpected observation in the lens, experiments were designed to test if amino acid transport, in turn, was affected by myoinositol. However, in the presence of mm. concentration of nonlabeled inositol, the accumulation of 4 Clabeled aaib into the lens was unaffected. Approximately 40 per cent inhibition was observed with?methyl glucoside whereas 0methyl glucose and ascorbic acid had only negligible effect on the accumulation of labeled inositol. Discussion The findings that the accumulation of inositol is energydependent, occurs against 0 a concentration gradient, and shows saturation kinetics (K m = 0.4 mm.) are interpreted as favoring the existence of an active transport mechanism in the lens similar in nature to that demonstrated in other tissues. 2 X " 4 The high concentration of inositol found in the lens ' 2 must be derived chiefly by active transport from aqueous humor since it has been shown that there is no appreciable synthesis of this compound in the lens. The existence of two transport systems acting stepwise to move inositol from the blood into the lens is analogous to the situation with amino acids which are actively transported from the blood into the aqueous humor from which they are, in turn, concentrated by the lens. As observed previously, 4 the transport of inositol in the lens appears to require K + and is inhibited by ouabain. The sensitivity of the transport system to Na + has also been demonstrated in a variety of tissues. 2 " As in the case of the ciliary body, transport of inositol in the lens is affected differently depending on the substance used to replace the Na + in the medium. While there is a reduction in the accumulation of inositol with lowering of Na + concentration, the effect cannot be ascribed solely to the deficiency of this cation. The main difficulty in attempting to interpret the present findings, in which the accumulation of inositol is reduced by the partial replacement of Na +, is that the transparency of the lenses is not maintained. This may mean that the substituents have some effect on the transport mechanism itself or that they bring about changes in the normal physiology of the lens which result in lower uptake of inositol, possibly due to changes in the permeability characteristics of the lens membranes. Whatever the nature of such a mechanism, it is fortuitous that the change in lens physiology can be detected easily as a result of changes in its transparency. In view of these findings, one may raise the question about the physiological state of other tissues under similar experimental conditions. Downloaded from iovs.arvojournals.org on 02209

800 Varma, Chakrapani, and Reddy Investigative Ophthalmology October 970 The observations concerning the inhibition of myoinositol transport by the sterebisomers (See reference 2 for structures) and

7 800 Varma, Chakrapani, and Reddy Investigative Ophthalmology October 970 The observations concerning the inhibition of myoinositol transport by the sterebisomers (See reference 2 for structures) and the phosphate esters of inositol and by ^methyl glucoside are similar to those in the ciliary body. There is also a similarity in the lack of inhibition noted with ascorbic acid. These results suggest that the specificity of the transport process is essentially alike in the two tissues. However, the inhibitory effect of amino acids on inositol transport, which was observed in the lens, is in marked contrast to the results in the ciliary body where no such effect was found. The mechanism by which amino acids interfere with the transport of inositol in the lens is not entirely clear as reciprocal inhibition of the transport of aaib was not observed in the presence of mm. myoinositol. At the present time, one may only speculate regarding the possible mechanism involved. Transport of amino acids may involve more than a single carrier or carrier site. ' 7 While the bulk of a particular amino acid may be transported preferentially by a single carrier or site, smaller fractions may be translocated through the participation of additional carriers or carrier sites. Failure of inositol to inhibit the transport of aaib while its own uptake is reduced in the presence of the amino acid could be explained if it is assumed that the carrier site for myoinositol is close to the amino acid transporting site and is masked by the binding of the amino acid to its carrier. Alternatively, the carrier site, which is responsible for translocation of a small fraction of the amino acid, may be identical with the inositol transporting site, in which case the inhibition of the amino acid transport may go undetected. We wish to thank Mrs. Hermine Lowe and Mr. Martin Iverson for their technical assistance. REFERENCES. Van Heyningen, R.: mesolnositol in the lens of mammalian eyes, Biochem. J. : 24, Reddy, V. N., Varma, S. D., and Chakrapani, B.: Intraocular transport of myoinositol. I. Accumulation in rabbit ciliary body, INVEST. OPHTHAL. 9: 78, Cotlier, E.: Characteristics of an active transport system for inositol in the lens (Abst.), INVEST. OPHTHAL. 7: 8, Broekhuyse, R. M.: Changes in myoinositol permeability in the lens due to cataractous conditions, Biochim. Biophys. Acta : 29, 98.. Wachtl, C, and Kinsey, V. E.: Studies on the crystalline lens. VIII. A synthetic medium for lens culture and the effects of various constituents on cell division in the epithelium, Amer. J. Ophthal. 4: 288, 98.. Reddy, V. N.: Studies on intraocular transport of taurine. II. Accumulation in the rabbit lens, INVEST. OPHTHAL. 9: 20, Folch, J., Ascoli, I., Meath, J. A., and Le Barron, F. N.: Preparation of lipid extract from brain tissue, J. Biol. Chem. 9: 8, Fiske, C. H., and Subba Row, Y.: The colorimetric determination of phosphorus, J. Biol. Chem. : 7, Kinsey, V. E., and McLean, I. W.: Studies on the crystalline lens. XIII. Kinetics of potassium transport, INVEST. OPHTHAL. : 8, Kinsey, V. E., and Reddy, D. V. N.: Studies on the crystalline lens. XI. The relative role of the epithelium and capsule in transport, INVEST. OPHTHAL. 4: 04, 9.. Hauser, C: Energy and sodiumdependent uptake of inositol by kidney cortex slices, Biochem. Biophys. Res. Commun. 9: 9, Hauser, C: Myoinositol transport in slices of rat kidney cortex. I. Effect of incubation conditions and inhibitors, Biochim. Biophys. Acta 7: 27, 99.. Johnstone, R. M., and Sung, ChengPo: Transport of myoinositol in Ehrlich ascites cells, Biochim. Biophys. Acta : 02, Caspary, W. F., and Crane, R. K.: Active transport of myoinositol and its relation to the sugar transport system in hamster small intestine, Biochim. Biophys. Acta 20: 08, Kinoshita, J. H., Futterman, S., Satoh, K., and Merola, L. O.: Factors affecting the formation of sugar alcohols in ocular lens, Biochim. Biophys. Acta 74: 40, 9.. Reddy, D. V. N., and Kinsey, V. E.: Transport of alpha aminoisobutyric acid into ocular fluids and lens, INVEST. OPHTHAL. : 4, Kern, H. L., and Brassil, D.: Effect of sodium ions on the transport of neutral amino acids by calf lens, INVEST. OPHTHAL. 7: 42, 98. Downloaded from iovs.arvojournals.org on 02209

Intraocular transport of myoinositol. I. Accumulation in the rabbit ciliary body. V. JV. Reddy, S. D. Varma, and B. Chakrapani

Intraocular transport of myoinositol I. Accumulation in the rabbit ciliary body V. JV. Reddy, S. D. Varma, and B. Chakrapani [2- s H] Myoinositol accumidates in rabbit ciliary body-iris preparations in