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

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1 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 vitro against a concentration gradient. The transport system is energy and temperature dependent (Q l0 = 2.) and demonstrates saturation kinetics (K m = 0.9 mm.). It is inhibited by ouabain, iodoacetate, phlorhizin, and shows requirements for Na +, K+, and Ca ++. The transport system appears highly specific; various amino acids, ascorbic acid, glucoascorbic acid, and sugar alcohols were without effect on the accumulation of inositol. The stereoisomer scylloinositol was found to inhibit the transport of myoinositol. D- and x.-chiroinositols had no effect. The phosphate esters of inositol competitively inhibited transport of myoinositol. The steady state concentrations of inositol in the lens, ciliary body-iris, aqueous humors, and plasma were determined. The concentration in posterior aqueous was higher than in the anterior aqueous and still higher than in plasma. Evidence is presented to show that the transport of inositol across the blood-aqueous barrier in vivo is a carrier-mediated process and it is suggested that the high concentration of inositol in the aqueous humors is the result of active transport from plasma into posterior aqueous. Key words: myoinositol, inositol, scylloinositol, chiroinositol, amino acids, active transport, ciliary body-iris, steady state distribution, competitive inhibition, blood-aqueous barrier, aqueous humor, posterior aqueous, rabbit. M. yoinositol,* a carbohydrate analogue, is known to be present at a much higher concentration in the aqueous humor of the From the Institute of Biological Sciences, Oakland University, Rochester, Mich. This investigation was supported, in part, by Research Grant 008 from the National Eye Institute of the National Institutes of Health and the National Society for the Prevention of Blindness. Presented before the Annual Meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Fla., May -, 970. Manuscript submitted June 22, 970; revised manuscript accepted Aug. 2, 970. "The term is used interchangeably with inositol. 78 anterior chamber and in the lens than in blood plasma. ' 2 The high concentration of inositol in ocular tissues raises the question whether it is the result of active transport or local synthesis. Surprisingly little attention appears to have been paid to the mechanism by which myoinositol is derived in intraocular fluids, although recent studies in kidney cortex ' and ascites cells have shown this compound to be transported against a concentration gradient. Ciliary body-iris preparation, incubated in vitro, has been employed previously G " s to study the mechanisms of transport of a number of substances and to relate this information to the transport of the same substances in vivo across the blood-aqueous barrier. This report deals principally with the

2 786 Recldy, Warma, and Chakrapani Investigative Ophthalmology October 970 accumulation of labeled myoinositol in the ciliary body-iris preparation and the characteristics of the transport system involved. Evidence is also presented to show that the transport of inositol across the bloodaqueous barrier in vivo can be saturated; it is suggested that it occurs against a concentration gradient from plasma into the posterior chamber. Methods Ciliary body-iris preparations were obtained from albino rabbits weighing between.8 and 2. kilograms. The details of tissue preparation, incubation procedure, and the tissue culture medium (KEI) employed were identical to those given in a previous paper. s The incubation tubes were gassed throughout the experiment with a mixture of per cent carbon dioxide, 7 per cent oxygen, and 88 per cent nitrogen in aerobic experiments. A gas mixture containing per cent carbon dioxide and 9 per cent nitrogen, which was first passed through vanadyl sulfate to remove all traces of oxygen, was employed for anaerobic experiments. The final ph of the medium in all experiments was 7.. [2- H] Myoinositol (specific activity 70 mc per millimole) was obtained from New England Nuclear Corporation. Radiochemical purity was established by paper chromatography in three different solvent systems (phenol: water (:6; w/v); n-butanol:ethanol:water (0:2:8); n-butanol: acetic acid:water (::)). The amount of radioactivity present in the 2 ml. medium for each tube was 0.2 fic. After incubation the ciliary body-iris was removed, blotted gently on a filter paper, weighed, homogenized in ml. of 0 per cent trichloroacetic acid, and subsequently centrifuged. The radioactivity in the tissue extract and the initial medium was determined in a liquid scintillation counter. The concentration of radioactivity in the tissue was calculated on the assumption that the tissue contains 8 per cent water. 0 The results are expressed as a ratio of the radioactivity in tissue water to that in the medium (Ct/Cm). In experiments where the effects of various conditions or substances were investigated, the rates of accumulation of [2- H] myoinositol in tissues obtained from contralateral eyes were compared since agreement between the eyes of the same animal was much closer than the results obtained from different animals. When nonlabeled compounds were used for saturation experiments or competitive inhibition, an equivalent amount of sodium chloride was omitted from the medium. Thhe steady-state concentrations of nonlabeled myoinositol existing in vivo in plasma, intraocular fluids, ciliary body-iris, and the lens were determined by gas liquid chromatography. Samples of aqueous humor were obtained under local anesthesia from posterior and anterior chambers by techniques described previously. After obtaining a blood sample by cardiac puncture, the animals were put to death by air embolism and ciliary body-iris preparations and lenses removed following enucleation of the eyes. All samples from each animal were obtained within approximately 0 minutes and kept at zero degrees until they were pooled. Equal quantities of aqueous humors and plasma obtained from each of the four rabbits were pooled separately. Lenses and ciliary bodyiris preparations from the same rabbits were also pooled from each set of animals and the proteins from the tissues and the fluids were precipitated with barium hydroxide and zinc sulfate. The total time elapsed for the entire operation was approximately 60 minutes. After centrifugation, aliquots of the supernatants were evaporated in vacuo and the myoinositol content was determined. 0 * The concentrations of myoinositol in various fluids and tissues are expressed as millimoles per kilogram of water. The water content of the lens and plasma was assumed to be 6 and 9.7 per cent, respectively; no corrections were found to be necessary for the aqueous humors. The method for the determination of the rate of accumulation of [2- H] myoinositol in the posterior chamber aqueous following parenteral administration of labeled compound to rabbits was essentially similar to that reported previously. One group of animals was injected with 0 pc of labeled inositol per kilogram of body weight (0 per cent intravenously and 60 per cent intraperitoneally). The second group was administered mmoles of nonlabeled compound per kilogram of body weight in addition to the labeled substance. The proportionate amounts of nonlabeled inositol given intravenously and intraperitoneally were the same as those used when the labeled substance was given alone. An additional 0 per cent of the total initial dose was injected intraperitoneally at the end of 0 minutes to both groups of animals. This method of administration kept the plasma level approximately constant. Ninety minutes after initial injections, samples of plasma and of aqueous humor from the posterior chamber were obtained and their radioactivity was determined in a liquid scintillation counter after solubilization in 0. ml. of hydroxide of hyamine (Packard Instrument Co.). Results In vitro studies. The distribution ratio of radioactivity in the tissue water to that in "The analyses by gas liquid chromatography were performed in the laboratory of Dr. Jin H. Kinoshita, Howe Laboratory of Ophthalmology, Boston, Mass.

3 Volume 9 Number 0 Myoinositol in rabbit ciliary body 787 the medium (Ct/Cm) increased linearly with time for a period of 90 minutes when the ciliary body-iris preparation was incubated in the presence of [2- H] myoinositol (Fig. ). At the end of 90 minutes the ratio attained was approximately eight. For all other experiments, an incubation period of 60 minutes was chosen as a convenient time interval. The addition of nonlabeled myoinositol to the medium decreased the distribution ratio of the labeled compound. The velocity of accumulation was calculated from the distribution ratio and the substrate concentration in the medium (Ct/Cm x S). Since the weight of the ciliary body varied in different experiments, the values for accumulation of inositol at different substrate concentrations were adjusted for the average weight of the ciliary body (0 mg.). The velocity of accumulation increased asymptotically with increase in inositol concentration in the medium (Fig. 2). The Lineweaver-Burk plot of these data (shown in the inset) gives a straight-line relationship and suggests a carrier-mediated accumulation process. The V max for the system was found to be 0.08 ^mole per ciliary body per hour and the Km was 0.90 mmole per liter. In these calculations, the diffusion component was not taken into account; failure to consider diffusion out of the ciliary body as a factor has the effect of reducing V mnx. The possibility that part of the inositol transported into the tissue may be incorporated into phospholipids was considered. In one such experiment, four ciliary bodyiris preparations, after incubation, were homogenized with barium hydroxide and zinc sulfate. The residue obtained after centrifugation of the homogenate was washed repeatedly with water and recentrifuged. Finally, lipids were extracted with a chloroform-methanol mixture (2:). No radioactivity could be detected in these extracts, suggesting that, under the experimental conditions employed, no inositol was incorporated into the tissue lipids. In order to study the possibility of ex- J TIME (MINUTES) Fig.. Time course of accumulation of [2- H] myoinositol in ciliary body-iris in vitro in synthetic medium without amino acids (KEI-A) at 7 C. t o 2 y bod o < 60 - "o 0 20 V 0 20 " Vmox ^ ole»/c.b/hr..90 mm ^ ^ 80 - ^ ^ 0 ^ ^ /l I 08.0 S(mM) Fig. 2. Effect of increasing concentration of myoinositol in the medium on its rate of accumulation in the ciliary body-iris in vitro incubated at 7 C. for 60 minutes. The inset is the Lineweaver- Burk (double reciprocal) plot of same data. I 2 ^ T HI change of tritium with water or formation of "metabolic water" from catabolism of inositol, samples of incubation media and tissue extracts were evaporated to dryness. Radioactivity in the residue was compared with that in the unevaporated samples. No loss of radioactivity was observed in the residue, indicating that no tritium-labeled water was formed in the experiment. Moreover, paper chromatography of tissue extracts gave rise to a single spot of radioactivity corresponding to the position of an authentic sample of radioactive inositol. Thus, no catabolism of inositol took place in these experiments and the radioactivity

4 788 Reddy, Varma, and Chakrapani Investigative Ophthalmology October 970 determined in tissue extracts represents the true concentration of free inositol. The influence of reduced temperature, the absence of glucose from the incubation medium, anaerobiosis, and the addition of metabolic inhibitors on the accumulation of myoinositol in the ciliary body are shown Table I. Effect of various factors on the accumulation of [2- H] myoinositol in the ciliary body-iris incubated for 60 minutes in vitro, expressed as percentage of contralateral eye (control) Condition of incubation Contralateral eye (control, 7 ) Reduced temperature (27 ) No glucose Anaerobic DNP Iodoacetate Ouabain Phlorhizin Phlorhizin Phlorhizin No. of cases 6 Inhibitor concentration (M) x0- x0- X 0- X 0- X 0- lxlo- Control ± S.D. (%) ± ± ±. 2 ±.8 20 ±.2 7 ±.9 ±.8 0 ± 7. in Table I. The accumulation was found to decrease at reduced temperature and in the absence of glucose. The observation that anaerobiosis has little or no effect suggests that the metabolic energy for transport is derived from glycolysis. This conclusion is supported by the further observation that dinitrophenol was without effect on the process of accumulation of myoinositol. The metabolic inhibitors, iodoacetate, ouabain, and phlorhizin, significantly reduced the accumulation of labeled inositol in the ciliary body. All of the aforementioned observations suggest that the accumulation of myoinositol is dependent on metabolic energy. They also indirectly suggest that the accumulation of this compound in the tissue against a concentration gradient is probably not due to binding since it is most unlikely that such a process would be affected under the variety of conditions studied. Accumulation of myoinositol in the ciliary body was also found to be sensitive to changes in the concentration of various cations in the incubation medium. In the absence of calcium ion, the accumulation Fig.. Effect of partial replacement of Na+ in the medium with various substances on the accumulation of [2- H] myoinositol in ciliary body-iris in vitro incubated at 7 C. for 60 minutes. 0 meq. per liter of Na + were iso-osmotically replaced by the cations or sucrose and the results are expressed as percentage of contralateral eyes incubated in media containing 8 meq. per liter Na +. The values are given as the mean - the standard deviation of the mean.

5 Volume 9 Number 0 Myoinositol in rabbit ciliary body 789 was lowered to per cent, while omission of potassium from the medium reduced the accumulation to 22 per cent of that in the controls. Partial substitution of sodium ions (0 mmoles) by different substances resulted in a significant but unequal reduction in the accumulation of myoinositol in the tissues (Fig. ). These results suggest that, in addition to the effect of sodium ions, the transport system may be affected by the replacement ions. The specificity of the transport system was investigated by studying the effect of a number of polyhydric compounds, amino acids, and stereoisomers and structural analogues of myoinositol. Sugar alcohols, /?-methyl glucoside, ascorbic acid, glucoascorbic acid, and several amino acids were found to be without effect on the accumulation ratio of labeled inositol when mmoles of these substances were added to the incubation medium (Table II). Of the stereoisomers of inositol* (Fig. ) studied, "Samples of scylloinositol and of D- and L-chiroinositol were kindly supplied by Dr. Laurens Anderson, Department of Biochemistry, University of Wisconsin, Madison, Wis. Table II. Effect of various compounds on the accumulation of [2- H] myoinositol in the ciliary body-iris incubated at 7 for 60 minutes in vitro, expressed as percentage of contralateral (eye) control Compound Contralateral eye (control) Xylitol Sorbitol Dulcitol /-methyl glucoside -0-Methyl glucose Ascorbic acid Glucoascorbic acid a-aib Glycine Serine Myoinositol Scylloinositol D-chiroinositol L-chiroinositol Inositol monophosphate Inositol monophosphate Inositol monophosphate Inositol hexaphosphate Inositol hexaphosphate No. of cases Concentration (mm.) Control ± S.D. (%) ± 98 ±8 97 ±2 80 ±2 87 ± 06 ±8 06 ± 2 99 ± 99 ± 9 ± 8 22 ± 9 ± 97 ±6 0 ± 7 ± 7 8 ± 22 ± 7 8± 7 8 ±0 D-CHIROINOSITOL HO' \ \.OH OH OH HO OH L-CHIROINOSirOl Fig.. Structures of the stereoisomers of inositol. OH

6 790 Reddy, Varma, and Chakrapani Investigative Ophthalmology October x_x Hexaphosphate O O Monophosphate Control o 20 " 80 E -l> 0 S(mM) Fig.. Competitive inhibition of myoinositol transport by inositol mono- and hexaphosphate. The rates of accumulation of myoinositol by the ciliary body-iris in vitro were determined in the presence or absence of either mm. concentration of monophosphate or.7 mm. hexaphosphate. Time of incubation was 60 minutes' at 7 C. and each point in the data represents an average of four experiments. Velocities are calculated tor the average weight of the ciliary body (0 mg.). 0 Fig. 6. Concentration (millimoles per kilogram of water) of free myoinositol in plasma, aqueous humors, ciliary body, and lens of rabbits. Results shown are averages of duplicate determinations on pooled samples from four rabbits. only scylloinositol significantly (P < 0.0) reduced the accumulation of [2- H] myoinositol. D- and L-chiroinositols did not affect the transport system. Both mono- and hexaphosphates of inositol decreased the uptake of myoinositol in the ciliary body-iris (Table II). The possibility that this effect may have been due to nonlabeled inositol liberated as a result of hydrolysis was ruled out since there was no increase in inorganic phosphorus in the medium in these experiments following incubation. In order to determine the nature of this inhibition, accumulation of myoinositol in the ciliary body at various concentrations was determined in the presence and absence of either mm. monophosphate or.7 mm. hexaphosphate (phytic acid). The double reciprocal plots for the data which have the same intercept on the ordinate (Fig. ) indicate that competitive inhibition is involved. It is of some interest to note that when hexaphosphate was used below.7 mm. concentration a precipitate appeared in the medium which was presumably due to insoluble calcium phytate. Above this concentration, there was no precipitate, suggesting the formation of a soluble complex salt. In vivo studies. The results of the steady-state distribution of myoinositol in intraocular fluids and tissues (Fig. 6) clearly demonstrate that, despite the extremely low levels of inositol in the plasma (trace quantities), the intraocular chambers and the tissues are able to maintain large concentration gradients. The possibility that myoinositol is actively transported across the blood-aqueous barrier by a carrier-mediated system was investigated by administering mmoles of nonlabeled inositol per kilogram of body weight just prior to giving the labeled sub-

7 Volume 9 Number 0 Myoinositol in rabbit ciliary body 79 stance to determine whether the transport of myoinositol was dependent on plasma concentration. It was observed that the accumulation of labeled myoinositol in the posterior chamber (six eyes) following administration of nonlabeled substance was about 2 per cent of plasma compared with 8 per cent of plasma in animals given only trace amounts of inositol. This suggests that the transport process in vivo across the blood-aqueous barrier can be saturated. Discussion The evidence presented suggests the existence of an active transport mechanism for myoinositol in the ciliary body. The transport process in vitro is carrier-mediated with a Km of 0.9 mm. and a V raax of 0.0 /xmole per ciliary body per hour. The mechanism is energy and temperature dependent (Q l0 = 2.) and is inhibited by metabolic poisons. The energy required for transport is largely derived from the anaerobic metabolism of glucose. As demonstrated in several other tissues, the transport mechanism is sensitive to ouabain ' F>> 2> and shows dependence on the concentration of sodium and potassium ions in the medium, suggesting the involvement of membrane adenosine triphosphase. (ATPase). Although inositol transport shows a dependence on sodium ion concentration, the observation that accumulation of inositol was affected differently when the same amount of sodium was replaced by other cations or sucrose would suggest that the substituents themselves affect the transport mechanism to some extent. The transport system for inositol in the ciliary body appears to be highly specific. Various amino acids, ascorbic acid, glucoascorbic acid, sugar alcohols, and some derivatives of glucose were studied for possible competition with the inositol transport system; none of these compounds had a significant (P > 0.0) effect on inositol transport. After this work was completed, Caspary and Crane reported that the transport of myoinositol in the intestine was inhibited 8 per cent in the presence of /?-methyl glucoside. In view of this finding, we repeated our experiments with (- methyl glucoside using the same concentration as employed in the aforementioned study (2 mm.). Under these conditions, inositol transport in the ciliary body was inhibited 0 per cent. Failure of amino acids to influence myoinositol transport in the ciliary body would suggest that different carrier systems may be involved in the transport of these compounds. However, in studies to be described in the following paper, 0 it was observed that the transport of myoinositol into the ocular lens is affected by some of the neutral amino acids. In previous studies 7 a relationship between amino acid transport and inositol has been suggested. In cells grown in medium deficient in inositol, it was observed that the transport of glycine and serine into these cells was reduced. The decrease in the capacity of the cells grown in inositol-deficient medium to accumulate amino acids was explained on the basis of the impairment of membrane structure rather than a direct involvement of the carrier system responsible for the transport of both amino acids and inositol. The high degree of selectivity of the myoinositol transport system in the ciliary body is further illustrated by the fact that only scylloinositol competes for the transport of myoinositol whereas the other two stereoisomers, namely D- and L-chiroinositols, are without effect (Table II). An examination of the structural characteristics of these compounds (Fig. ) indicates that the configurations around the various carbon atoms in scyllo- and myoinositol are similar except at position, where the hydroxyl group is "equatorial" in scyllo- and "axial" in myoinositol. Since both isomers compete for the carrier, the configuration around carbon atom does not appear to be a stereo-specific requirement. The molecular configurations in D- and L-chiroinositol differ from myoinositol chiefly with respect to the hydroxyl group at position

8 792 Reddy, Varma, and Chakrapani Investigative Ophthalmology October 970. The ineffectiveness of D- and L-chiroinositol on the transport of myoinositol suggests that,- diequatorial configurations of hydroxyl groups is present in myoand scylloinositol may be essential for the molecule to be bound to the carrier and thus be transported. The capacity of the ciliary body to accumulate a substance does not necessarily mean that it is actively transported from plasma into the posterior chamber. It has been observed that substances which are actively transported across the bloodaqueous barrier in either direction, i.e., from plasma into the posterior aqueous or vice versa, are concentrated by ciliary epithelial cells. G " s The observation that the steady-state concentration of inositol in the posterior aqueous far exceeds that in the plasma suggests that the transport may take place against a concentration gradient. However, it does not rule out the possibility that the high concentration in the posterior aqueous may be a result of leakage from the ocular lens where myoinositol is found in high concentration (Fig. 6). The observation that the presence of nonlabeled inositol in the blood plasma produces reduction in the rate of accumulation of labeled compound in the aqueous humor of the posterior chamber suggests that the mechanism of transport becomes saturated as the absolute concentration of myoinositol in the plasma is raised. From similar evidence, it has been demonstrated that the mechanism concerned with the secretion of ascorbic acid 7 ' 9 and aminb acids ' 20 could be saturated. A value of less than (0.8) obtained for the ratio aqueous/plasma when labeled inositol was injected is due to the non-steady-state condition (90 minutes after injection) and does not exclude the possibility of transport against a concentration gradient. It is suggested that the high concentration of inositol found in the aqueous humors is the result of an active transport system across the blood-aqueous barrier involving a highly specific carriermediated system. We wish to thank Mrs. Hermine Lowe and Mr. Martin Iverson for their technical assistance. REFERENCES. Krause, A. C, and Weekers, R.: Inositol in the ocular tissues, Arch. Ophthal. 20: 00, Van Heyningen, R.: meso-inositol in the lens of mammalian eyes, Biochem. J. 6: 2, 97.. Hauser, G.: Energy- and sodium-dependent uptake of inositol by kidney cortex slices, Biochim. Biophys. Res. Comm. 9: 696, 96.. Hauser, G.: Myo-inositol transport in slices of rat kidney cortex. I. Effect of incubation conditions and inhibitors, Biochim. Biophys. Acta 7: 27, Johnstone, R. M., and Sung, Cheng-Po: Transport of myo-inositol in Ehrlich ascites cells, Biochim. Biophys. Acta : 02, Becker, B., and Forbes, M.: Iodopyracet (Diodrast) transport by the rabbit eye, Amer. J. Physiol. 200: 6, Becker, B.: Ascorbate transport in guinea pig eyes, INVEST. OPHTHAL. 6: 0, Reddy, D. V. N.: Studies on intraocular transport of taurine. I. Accumulation in rabbit ciliary body-iris preparation in vitro, Biochim. Biophys. Acta 8: 26, Kinsey, V. E.: Comparative chemistry of aqueous humor in posterior and anterior chambers of rabbit eye, Arch. Ophthal. 0: 0, Hayman, S., Lou, M. F., Merola, L. O., and Kinoshita, J. H.: Aldose reductase activity in the lens and other tissues, Biochim. Biophys. Acta 28: 7, Reddy, D. V. N., and Kinsey, V. E.: Transport of alpha aminoisobutyric acid into ocular fluids and lens, INVEST. OPHTHAL. :, Broekhuyse, R. M.: Changes in myoinositol permeability in the lens due to cataractous condition, Biochim. Biophys. Acta 6: 269, Cotlier, E.: Characteristics of an active transport system for inositol in the lens (Abst.), INVEST. OPHTHAL. 7: 8, Hauser, G.: Myoinositol transport in slices of rat kidney cortex. II. Effect of the ionic composition of the medium, Biochim. Biophys. Acta 7: 267, 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, Varma, S. D., Chakrapani, B., and Reddy, V. N.: Intraocular transport of myoinositol. II.

9 Volume 9 Number 0 Myoinositol in rabbit ciliaiy body 79 Accumulation in the rabbit lens in vitro, INVEST. OPHTHAL. 9: 79, Lembach, K., and Charalampous, F. C.: Metabolic functions of myo-inositol. VI. Impairment of amino acid transport in KB cells caused by inositol deficiency, J. Biol. Chem. 22: 2606, Angyal, S. J., and Anderson, L.: The cyclitols, Advances Carbohyd. Chem. :, Kinsey, V. E.: Transfer of ascorbic acid and related compounds across blood-aqueous barrier, Amer. J. Ophthal. 0: 262, Reddy, D. V. N.: Distribution of free amino acids and related compounds in ocular fluids, lens, and plasma of various mammalian species, INVEST. OPHTHAL. 6: 78, 967.