studies on intraocular transport of taurine · under the name of d. v. n. reddy. manuscript...

Studies on intraocular transport of taurineII. Accumulation in the rabbit lens

V. N. Reddy*

The accumulation of 1J<C-taurine in the lens was studied by culture technique under a varietyof conditions. This compound accumidates in the ocular lens against a concentration gradientby a process lohich can be readily saturated. The saturation mechanism does not conformto Michaelis-Menten kinetics. The transport process is highly dependent upon temperature(Qio = 5), and derives the energy required from anaerobic metabolism of glucose. The ac-cumulation of taurine in the lens is reduced by a number of metabolic inhibitors, and in theabsence of calcium, strontium ions may be substituted for calcium. Ouabain significantlydecreases the accumidation of taurine, and the effect is from the onset of lens culture sug-gesting that the amino acid transport is directly linked to the enzyme Na-K-ATPase. Ac-cumulation of taurine is unaffected in a potassium-free medium but is reduced by 50 percent if potassium ion in the lens is first partially depleted. Decrease in the concentration ofsodium ions in the medium also reduces the accumulation of taurine but the lenses couldnot be maintained in a physiological state so that decreased transport of taurine cannot beascribed to the specific effect of sodium ion. Transport of taurine is inhibited by a numberof structural analogues of this compound. Reciprocal inhibition of 0-alanine transport bytaurine was also observed suggesting a common carrier for the two amino acids. Of thevarious a-amino acids tested, only glycine, alanine, and arginine were found to compete forthe taurine transporting system. It is concluded that the high concentration of taurine normal-ly found in the lens is derived, at least in part, by active transport from intraoadar fluids.

Key words: Crystalline lens, active biological transport, taurine, carbon isotopes, tissue culture,culture media, concentration variations, temperature, metabolic inhibitor, ouabain, alanine.

.mong the free amino acids and re-lated compounds found in the lens, the

From the Institute of Biological Sciences, Oak-land University, Rochester, Mich.

This investigation was supported, in part, by Re-search Grant 08340 from the National EyeInstitute of the National Institutes of Health,the United States Atomic Energy CommissionContract No. AT(ll-l)-2012-2 and the BobHope Fight for Sight Award of Fight forSight, Inc.

""Previous publications of the author appearedunder the name of D. V. N. Reddy.

Manuscript submitted June 24, 1969; manuscriptaccepted Oct. 10, 1969.

concentration of taurine is one of the high-est.1 In certain tissues, taurine is presum-ably derived from the metabolism ofsulfur-containing amino acids by the seriesof reactions shown on the opposite page.

That taurine may be formed in the lensin a similar manner is suggested by astudy in which labeled taurine was de-tected following incubation of the lenswith 14C-labeled methionine.2 However, itis possible that taurine in the lens mayalso be derived from intraocular fluids byan active transport mechanism. Previousstudies3 in this laboratory have shown that

206

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Volume 9Number 3

Intraocular transport of taurine 207

Methionine r= — Homocysteine v=

Cystine =̂

Cystinedisulfoxide

Cystaminedisulfoxide

Hypotaurine

— Cystathionine

— Cysteine

Cysteinesulfinic acid

Cysteic acid

Taurine

this amino acid accumulates in the ciliarybody against a concentration gradient bya relatively specific carrier-mediated sys-tem.

In the present communication, evidenceis presented to show that the lens, too, iscapable of concentrating taurine from thebathing medium and that the character-istics of the transport system appear to besimilar, in many respects, to those found inthe ciliary body. These studies lead to theconclusion that the high concentration oftaurine found in the lens may be derived,at least in part, from intraocular fluids byan active transport mechanism.

MethodsLenses obtained from albino rabbits weighing

between 1.7 and 2.2 kilograms were culturedin 5 ml. of synthetic media in the presence of14C-labeled taurine.

Two types of media were employed; one whichcontained various amino acids at a concentrationnormally present in intraocular fluids (KEI-4),and another without the amino acids (KEI-1A).The composition of the media and the lens cul-ture techniques have been described previously.4"0

14C-taurine (specific activity 1.8 to 2.3 me.per millimole) was obtained from the New Eng-land Nuclear Corp. and its purity confirmed bychromatography.7 The concentration of the labeledamino acid present in the medium for all experi-ments other than those concerned with satura-tion by nonlabeled taurine itself was 2 x 10~5M,and the actual radioactivity in each tube was0.15 fie. When nonlabeled amino acids were usedfor saturation experiments or competitive inhi-bition, an equivalent amount of NaCl wasomitted from the medium.

Following the desired period of culture, lenseswere homogenized in 2 ml. of 10 per cent TCAand the radioactivity in supernatant fluid wasdetermined in a liquid scintillation counter andcompared with that in the initial medium (Cm).The concentration of radioactivity in the lens(Ci) was calculated on the basis of the lenswater (65 per cent of the lens weight). Theaccumulation of the amino acid in the lens isexpressed as a ratio of the concentration in

lens water to that in the initial medium (•— ).Cm

For comparison of the effect of various aminoacids on the accumulation of taurine, the ratesof accumulation of labeled taurine were mea-sured in the presence of 5 mM. concentrationof the amino acids in medium containing noother amino acids (KEI-1A). Contralateral lenses,


208 Reddy Investigative OphthalmologyMarch 1970

aUJ

<

OzoooX

COzUJ_)

dzoo

2

10

8

6

4

2

0

-

-

-

-

- A

/(I5)

A-H7> i

/

(i5)

i

1

(14)

1 1

^ *

* *

(15)

1 1 1

^ - " ^

(18)

1 1 1

(4

-

i

4)

-

-

-

1 1 -

8 18 20 22 2410 12 14 16

TIME IN HOURS

Fig. 1. Time course of accumulation of 11C-labeled taurine in lenses cultured in a mediumcontaining no other amino acids.

which were cultured in the presence of tracerquantities of labeled taurine only, served as con-trols.

Steady state levels of taurine were determinedby culturing lenses for approximately 24 hoursin KEI-4 medium containing 0.03 juc per milli-liter 14C-labeled taurine, then replacing it witha medium containing approximately 5 to 10 percent of the initial activity. The replacementmedium was allowed to mix for about two min-utes and a sample of 50 A was removed todetermine the exact level of the (initial) radio-activity in the medium. Culture was then con-tinued for an additional 24 hours. This pro-cedure reduced the time required to approxi-mate steady state. The radioactivity in the lensand the final "postculture medium" were deter-mined in the usual manner. Steady state condi-tions were considered to have been reachedwhen no significant increase or decrease of tracerlevels occurred in the medium during the secondculture period. Generally, such conditions wereattained when the concentration of 14C-taurinein the replacement medium was 8 per cent ofthat employed initially. In a series of experi-ments (13 lenses), the ratio of the concentrationin the lens water to that in the medium at theend of the second culture period was 70 ± 12.

For the measurement of the efflux rate, lenseswere "preloaded" in a medium (KEI-4) con-taining labeled taurine for approximately 24hours. The medium was then withdrawn andthe tubes and lenses were rinsed with 3 ml. of a

fresh medium containing no radioactivity byrocking the tubes for two to three minutes. Thefluid containing small amounts of radioactivitywas withdrawn and discarded. Five additionalmilliliters of nonradioactive medium were thenadded and 50 jil samples removed periodicallyto determine the amount of labeled amino acidthat had leaked out of the lenses. Radioactivityinitially in the sample withdrawn approximatelytwo minutes after the introduction of the mediumserved as the zero reading. At the end of 24hours, lenses were assayed for the remainingradioactivity in the usual maner and the totalradioactivity present initially in the lens wascalculated. Efflux of taurine into the mediumis expressed as a percentage of radioactivitypresent in the lens. Precautions were taken tomaintain the sterility throughout the cultureperiod in the various experiments.

Results

Time course of accumulation and ki-netics of transport. Taurine accumulatesin the lens against a concentration gra-dient (Fig. 1), the distribution ratio com-pared with the concentration in the initialmedium being approximately 11 at the endof 24 hours of culture in a medium con-taining no other amino acids. Fig. 2 showsthat the accumulation of the labeled com-


Volume 9Number 3

Intraocular transport of taurine

1

/ILD

HIT

IAL

l\'C

ON

C. I

N

o<\l

X

(O2LU_J

62Oo

10

8

6

4

2

0

f (25)

• 1 ( 3 )

1(4)

- V (4)

• t i i

(4)

i i i

-

m

•

m

•

(4)

10

NONLABELLED TAURINE (mM)Fig. 2. Effect of nonlabeled taurine on the accumulation ratio of the labeled compound inlenses cultured for 24 hours in a medium containing no other amino acids.

20

18

15

12

9

6

30

100

95 -

90 -

85 -

80 -

-

-

V(22) I*11*1-^*^^(16) ( S ^

1 1 1 1 1

(8)

0 2 ^ ^ "

(16)

1 1 1 1 1

(II) -T 1

" - 1 (20) _

-

(16) ~

1 1 1

10 12 14 16 20 22 24

TIME IN HOURS

Fig. 3. Rates of accumulation (A) and efflux (B) of 14C-labeled taurine by lenses culturedin KEI-4 medium at 37° C. Rate of accumulation is expressed as a ratio of the concentrationin the lens to that in the initial medium while the rate of efflux is a percentage of concentra-tion initially present in the lens. The lines drawn through the experimental data are generatedby the computer.


210 Recldy Investigative OphthalmologyMarch 1970

pound decreases with increasing concen-tration of the nonlabeled substance in themedium. The ratio decreases asymptoti-cally from a value of approximately 11 to0.4 over a range of concentration of 10mM. and the highest rate of decrease be-ing below 1 mM. concentration. These re-sults indicate that the transport system isreadily saturated by very low concentra-tions of the amino acid.

In order to learn about the transportprocess under conditions existing in theeye, the rates of accumulation (Fig. 3, A),efflux (Fig. 3, B), and steady state distri-bution ratio in the lens were measured ina more complete medium (KEI-4). It willbe seen that the accumulation ratio oftaurine in this medium, which contains aphysiological mixture of amino acids inconcentrations similar to those found in theaqueous humor, is even higher (18 at theend of 24 hours of culture) than in a medi-um free of amino acids. It seemed para-doxical that the ratio of accumulation inthe lens increased in the presence of aminoacids where competition would be ex-pected to reduce the uptake of labeledtaurine. Accordingly, a series of experi-

ments was performed in which contra-lateral lenses were employed to measurethe accumulation of taurine in both typesof media. For a period of approximately16 hours, the accumulation in the lenseswas found to be similar in both media.However, at culture periods beyond thistime the ratios were higher in the mediumcontaining other amino acids. These re-sults suggest that the medium lackingamino acids may be inadequate to main-tain the lens in a "physiological state" forextended periods of time.

The coefficients for active transport(kP) and diffusion (kd) for taurine in thelens were estimated from the "pump leakequation":

dC.—

V,C.

as described by Kinsey and McLean.8 Inthe above equation Cm and C{ denote theconcentration of taurine in the mediumand in the lens, respectively, while Vm andVt are equal to the respective volumes of

0 O.I 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

CONCENTRATION IN THE MEDIUM (mM)Fig. 4. Relation between the transfer coefficient by active transport (kp) and the concentra-tion of taurine in the medium. The values for kp are those which produce a fit to the experi-mental data of Fig. 2.


Volume 9Number 3


10

b

1.2

Fig. 5. Relation between the velocity of activetransport (pump) and the concentration of tau-rine in the culture medium.

the medium and lens water. The lines inFig. 3 are generated by solving the aboveequation with an analogue computer usingthe experimentally determined steadystate distribution ratio of 70 as a boundarycondition. Values of 0.188 hiv1 and 0.26hr.'1 for kp and kd, respectively, were foundto provide a good visual fit to the data. Insolving for the above equation, appro-priate corrections for changes in concen-trations of both labeled and nonlabeledtaurine in the medium during the 24 hourperiod of culture were taken into account.

To establish the relationship betweenthe velocity of the pump to the substrateconcentration, the values of kp at differentsubstrate concentrations were determinedby the procedure outlined by Kinsey andMcLean.8 It was assumed that kd is unaf-fected by the substrate concentration inthe medium. The relationship of kp to theconcentration of taurine in the medium isshown in Fig. 4.

The data relating the velocity of thepump (Kp ' Cm • Vi) to the concentrationof medium is shown in Fig. 5. The velocityincreases initially and then decreases be-yond a substrate concentration of 0.2 mM.indicating that the saturation process doesnot conform to Michaelis-Menten kinetics.

Table I. Effect of lack of potassium ion onthe accumulation of 14C-taurine in the lens(24 hours)*

Media | % of control

Control (5mM. K+) 100 (6)No K+ 100 ±14 (6)

> Preincubationf (22 hr.)> Control (5mM. K>) 100 (7)

No K> 48 ± 6 (7)

°Values are expressed as percentage of contralateralcontrols.fDuring the preincubation period, the media were replacedonce at the end of 7 hours and incubation continued for22 hours. Following preincubation the lenses were culturedfor a period of 24 hours in fresh media containing "C-taurine.

The effect of temperature, metabolicinhibitors, and other factors. The effectof reduced temperature, anaerobiosis, lackof glucose or calcium from the medium,and of various metabolic inhibitors on theaccumulation of taurine in the lens isshown in Figs. 6 and 7. Accumulation oftaurine is highly dependent upon tempera-ture and has an apparent Q10 of approx-imately 5. Absence of glucose and calciumfrom the medium reduced the accumula-tion of taurine. Anaerobiosis has little orno effect on uptake. The divalent strontiumion apparently can replace calcium in themedium which restores the ability of thelens to accumulate taurine and even ap-pears to have a stimulatory effect.

The metabolic inhibitors, cyanide, iodo-acetate, and ouabain, all reduce the ac-cumulation of taurine in the lens in vary-ing degrees, while dinitrophenol has littleor no effect (Fig. 7).

The effect of ouabain on taurine accum-ulation in the lens was also investigatedas a function of time. It will be seen fromFig. 8 that ouabain appears to inhibit ac-cumulation of taurine from the onset ofculture. These findings are in contrast tothe observations made on alpha-aminoiso-butyric acid (a -AIB) which was foundto be affected by ouabain only after a de-lay of two hours of culture.0

Effect of cations. In view of the imme-diate effect of ouabain on the accumula-tion of taurine, it was of interest to study



LLJ 1 0 0 -

crID<

O

u_ozo\-<

8 0 -

6 0 -

9 40-

OO

2 0 -

0 -

Fig. 6. Influence of various factors on the accumulation of 14C-labeled taurine in lenses cul-tured for 24 hours, expressed as percentage of contralateral controls. The value given by eachblock is an average for a minimum of 5 experiments.

a:

IO

100-

8 0 -

6 0 -

2 40-

2 0 -

O0—

0UABAIN

Fig. 7. Influence of various metabolic inhibitors on the accumulation of 14C-labeled taurinein lenses cultured for 24 hours, expressed as percentage of contralateral controls. The valuegiven by each block is an average for a minimum of 5 experiments.

the possible relationship between thetransport of this amino acid and potassiumand sodium. Absence of potassium ion inthe culture medium has no immediate ef-fect on the accumulation of taurine (TableI). However, when the potassium contentof the lens was first partially depleted byculturing the lens in a potassium-free

medium for 22 hours, taurine uptake wasreduced by approximately 50 per cent ina potassium-free medium.

The sensitivity of taurine transport tomodification of sodium ion in the mediais shown in Table II. These results indi-cate that the accumulation of taurine inthe lens is reduced by decreased sodium


Volume 9Number 3


n 6 -

5 -

4 -

3 -

2 -

TIME IN HOURS

Fig. 8. Effect of ouabain (filled circles) on the accumulation of 14C-taurine in lenses culturedin KEI-4 media. The data for experimental controls are given by the triangles. The numberof experiments and standard deviations are included in the chart.

Table. II. Effect of changing concentration of sodium ion on the accumulation of14C-taurine in the lens*

Media

KEI-1A (control)

Tyrodes (control)

Culture period(hr.)

A or 2424

4

44

Concentration(mEq. per liter)

Na+

14843

4373

1376834

Cholinechloride

105

10575

69103

Condition oflenses

ClearCloudy

CloudyPeripherally

cloudy

ClearCloudyCloudy

Taurineaccumulation

(% of control)

10019+ 3 (5)

22± 2 (5)65+16 (4)

10057± 7 (3)51+ 5 (3)

°Values are expressed as percentage of contralateral controls.

ion concentration in both KEI mediumand Tyrode's solution. However, the lenseswere quite cloudy in both media evenafter 4 hours of culture, suggesting thatthe lenses were highly abnormal and it isdoubtful if any conclusion is warrantedconcerning the specific effect of Na ionon the transport of taurine.

The effect of structural analogues oftaurine and other amino acids. The effectof a number of structural analogues and

other amino acids on the.transport of XIC-taurine was studied to determine the spe-cificity of the system responsible for itsaccumulation. The structural relationshipof the various analogues to taurine is

shown in Fig. 9. The ratio (^-) of radio-Cm

active taurine was determined in thepresence of a 5 mM, concentration ofeach substance and compared with thatin contralateral lenses cultured with tracer


214 Redcly Investigative OphthalmologyMarch 1970

S03HI

CH2

HC-NH2H

COOHICH2

HC-NH2H

SO2HICH2I

HC-NH2H

PO3H2ICH2I

HC-NH2H

TAURINE /3-ALANINE HYPOTAURINE 2-AMINOETHYLPHOSPHONIC

ACID

S03HICH2I

HC-N-CH3H H

N-METHYLTAURINE

COOHI

H3C-CHI

HC-NH2H

S-AMINOISOBUTYRIC

ACID

S03HICH2

HC-NH2ICOOH

CYSTEICACID

Fig. 9. Structural formulas of taurine and its analogues.

cr

LJ.O

<_l

IDOO

100—

8 0 -

6 0 -

40—

2 0 -

O—

TRACER ONLY

^ CD

SO3H

CH2I

HCHH

ETHANESULFONIC

ACID

Fig. 10. Effect of nonlabeled taurine and various amino acids (5 mM.) on the accumulationof 14C-labeled taurine in lenses cultured for 24 hours, expressed as percentage of contralateralcontrols which were cultured in the presence of 14C-labeled taurine only. The value givenby each block is an average for a minimum of 5 experiments.

taurine alone. It will be seen from Fig. 10that /?-alanine, which differs from taurinein having a carboxyl group instead of thesulfonic group, is almost as effective a com-petitor for the taurine transporting sys-tem as taurine itself. The higher homo-logues of /?-alanine show a decreasing com-petitive action for taurine transport as thelength of the carbon chain increases.

The effect of a number of other ana-logues of taurine was also investigatedand the results are shown in Fig. 11. In-troduction of a methyl group in the aminogroup of taurine (N-methyl taurine) re-duces taurine accumulation to a lesser de-gree than saturation of taurine by itself.Similarly, the inhibitory effect of jS-alanineon the accumulation of taurine is reduced


Volume 9Number 3


Fig. 11. Effect of various structural analogues of taurine (5 mM.) on the accumulation of14C-labeled taurine in lenses cultured for 24 hours, expressed as percentage of contralateralcontrols which were cultured in the presence of taurine only. The value given by each blockis an average for a minimum of 5 experiments.

100-

3

o

3OO<

1 i l i l i i

Fig. 12. Effect of various a-amino acids (5 mM.) on the accumulation of 1JC-labeled taurinein lenses cultured for 24 hours, expressed as percentage of contralateral controls which werecultured in the presence of 14C-labeled taurine only. The value given by each block is anaverage for a minimum of 5 experiments. Inhibition with alanine and glycine only was sig-nificant (P < < 0.01).

by the introduction of a methyl group inthe a = position of /?-alanine (/3-AIB). Theeffect of substituting the sulfonic acid oftaurine with other acid groups may alsobe noted on the accumulation of taurinein the lens (Fig. 11). Hypotaurine, whichresults from substitution of the SO3H groupof taurine with SO2H is a more effectivecompetitor for taurine than taurine itself.Introduction of a second acid group in

taurine molecule, as in cysteic acid, or re-moval of the ammo group (results notshown) renders the compound inactivesince these substances had no effect ontaurine accumulation.

Of the various a-amino acids studied,only alanine, glycine and arginine had anysignificant (P « 0.01) inhibitory effect onthe accumulation of taurine (Figs. 12 and13). While many of these observations on



UJ

?

3Oo

100-A

8 0 -

6 0 -

!=: 40—

2 0 -

0 -

Fig. 13. Effect of various a-amino acids (5 mM.) on the accumulation of 14C-labeled taurinein lenses cultured for 24 hours, expressed as percentage of contralateral controls which werecultured in the presence of 14C-labeled taurine only. The value given by each block is anaverage for a minimum of 5 experiments. The inhibition with arginine only was significant(P < < 0.01).

UJ

<r100-

Fig. 14. Effect of glycine and its N-methyl derivatives (5 mM.) on the accumulation of r e -labeled taurine in lenses cultured for 24 hours, expressed as percentage of contralateral con-trols cultured in the presence of 14C-labeled taurine only. The value given by each block isan average for a minimum of 5 experiments. The inhibition with glycine only was significant(P < < 0.01).

the lens are similar to those made on theciliary body, alanine and arginine werefound to be without competitive effecton the accumulation of taurine in the lat-ter tissue.

The competitive effect of glycine fortaurine is reduced by the introduction of

N-methyl groups as shown by the resultsin Fig. 14.

Discussion

The results of this study show that thelens can accumulate taurine against a con-centration gradient by a process which is


Volume 9Number 3 Intraocular transport of taurine 217

temperature and energy dependent, andis affected by a variety of metabolic poisons.These findings taken together with the ob-servation that the process responsible foraccumulation of taurine against a concen-tration gradient is readily saturated bytaurine suggests that the compound is ac-tively transported into the lens. Like theobservations made previously with regardto the transport of other amino acids intothe lens,9 the energy for active transportof taurine appears to be derived from theanaerobic metabolism of glucose.

The observation that lenses cultured ina medium containing no amino acids ac-cumulate less taurine than in the mediumcontaining amino acids deserves somecomment. The lower accumulation ratioof taurine in the former medium (KEI-IA) may be either due to decrease inactive transport or increase in the perme-ability of lens "membranes." It is possiblethat loss of some of the free amino acidsand other substances from the lens intothe medium in some manner affects theactive transport of taurine. Another pos-sibility is that the lens "membranes" maybecome more permeable to taurine in theincomplete medium. To explore the latterpossibility, lenses were preloaded with 14C-taurine in a complete medium (KEI-4)and the efflux determined using contra-lateral lenses in both types of media. Therates of efflux in the two media over aperiod of 24 hours were found to be in-distinguishable. Since the efflux rate is atleast as high in amino acid-free medium,despite a lower accumulation ratio, theseresults would be consistent with increasedpermeability of lens "membranes." How-ever, the possibility that the active trans-port itself may have decreased cannot beexcluded. In any case, when lenses are tobe cultured for extended periods of time,the medium of choice is the one whichcontains the amino acids (KEI-4).

The kinetics of taurine transporting sys-tem into the lens (Fig. 5) are more com-plex than those encountered in the case ofa-AIB9'10 and do not conform to the

Michaelis-Menten kinetics. Because of this,the values of Vmnx and Km could not bedetermined. However, taurine apparentlyhas a very low Km on the order of 0.1mM. While the exact nature of the satura-tion mechanism remains to be elucidated,it should be noted that the phenomenonobserved in Fig. 5 where the velocity in-creases initially and decreases beyond asubstrate concentration of 0.2 mM. maybe due to substrate inhibition. A numberof enzyme systems in which substrate in-hibition is involved are known to exhibitsimilar kinetics.11

The inhibitory effect of ouabain, whichaffects transport of taurine almost imme-diately, suggests a more direct involve-ment of the enzyme, Na-K-ATPase in thetransport of this amino acid. Previous stud-ies in this laboratory have shown that thetransport of a-AIB is also affected by oua-bain but only after a delay of a two hourculture period,7 suggesting the possibilitythat the effect of ouabain in this instancemay have been secondary.

Failure to detect any reduction in thetransport of taurine when cultured in apotassium-free medium probably meansthat taurine transport is not directly linkedto the transport of this cation. The obser-vation that accumulation of taurine is re-duced when potassium content of the lenswas partially depleted is interpreted tomean that a certain optimal concentrationof potassium ion is required for sodium-potassium activated ATPase and that theamino acid transport is reduced underthese conditions due possibly to a de-crease in the ATPase activity in the epi-thelium of the lens. It is interesting tonote that under similar experimental con-ditions, i.e., after partial depletion ofpotassium from the lens, the accumulationof a-AIB was unaffected, which wouldfurther suggest that Na-K-ATPase may notbe directly involved in the transport ofthis amino acid.

While the accumulation of taurine inthe lens decreased in media deficient insodium ion, all of the lenses were cata-



ractous indicating that the physiology ofthis organ is disrupted. It is, therefore,questionable if the reduced uptake ofamino acid can be ascribed to the specificeffect of sodium ion. There are a numberof studies3'12 in which a dependence ofamino acid transport on sodium ion hasbeen noted. In the ocular lens, too, it hasbeen reported that there is a stoichio-metric relation between the transport ofa-AIB and sodium ions.13 In all of thesestudies choline chloride was substitutedfor sodium ions in the medium, the im-plicit assumption being that choline chlo-ride is an inert substance. The results ofthe present investigation raise the ques-tion if such an assumption is justified. Theneed for caution in interpretation of theresults in which choline is used as a sub-stitute for sodium has also been stressedby Macey and Koblick.14

Because of the complex nature of thekinetics of taurine transport, it was notpossible to analyze the data with Line-weaver and Burk plots to ascertain if com-petitive inhibition was involved in theprocess by which /3-alanine and othercompounds reduce taurine accumulationin the lens. However, it was observed thatthe accumulation of jS-alanine, which alsooccurs against a concentration gradient inthe lens, was effectively reduced in thepresence of 5 mM. concentration oftaurine. In Ehrlich cells, it has been re-ported that the transport of y8-alanine wasinhibited by taurine only slightly but wasreduced by 1-alanine to a greater extentthan by /3-alanine itself.15'1G In the presentstudy, the competitive effect of 1-alaninefor jS-alanine was only small and of thesame order of magnitude as for taurine.Thus, in the ocular lens, a common carrieror carrier site appears to be involved inthe active transport of taurine and /?-alanine.

The results of the studies concernedwith the competition of various analoguesof taurine in the lens are similar to thoseobserved in the ciliary body. The replace-ment of sulfonic acid with other acid

groups results in quantitative differencesin the affinity of the molecules for thereactive site. For example, substitution ofsulfonic group with carboxyl (/3-alanine)or sulfinic (hypotaurine) does not appre-ciably change the affinity for the transportsite in question. Replacement with phos-phonic acid group, on the other hand,renders the molecule nearly inactive.

Introduction of a second acid group asin cysteic acid appears to abolish the re-activity of the molecule for the transportsite. Introduction of an a-methyl groupinto ^-alanine (/3-AIB) and an N-methylgroup into taurine makes these moleculessomewhat less reactive.

Although the reason for differences inthe reactivity of the various compounds tothe taurine transporting site is not known,the affinity bears no obvious relation totheir pK values.

Of the various a-amino acids studied,only glycine, alanine, and arginine hadany significant effect on the transport oftaurine. The results on the lens differ fromthose in the ciliary body in which tissueglycine alone showed any competitive ef-fect for taurine. They also differ fromthose of other investigators15"17 who couldfind no detectable interaction betweena-amino acids and the taurine transport-ing system in ascites cells. The observa-tion that arginine, a basic amino acid, andtwo neutral amino acids, glycine andalanine, all compete with taurine is unex-pected. While the competition of theseamino acids to taurine is small, there areno previous reports in which a neutraland basic amino acid have been shownto compete for the same carrier or carriersite.

Finally, the present study demonstratesthat taurine is actively transported intothe lens, and suggests that the high con-centration of this substance in the lensmay be derived, at least in part, by activetransport from aqueous humor. Whethersignificant quantities of taurine are formedin situ in the lens remains to be estab-lished.


Volume 9Number 3


I wish to acknowledge the technical assistanceof Mr. Larry Alder in conducting the experimentsreported, in this paper.

REFERENCES

1. Reddy, D. V. N.: Distribution of free aminoacids and related compounds in ocular fluids,lens, and plasma of various mammalian spe-cies, INVEST. OPHTHAL. 6: 478, 1967.

2. Dardenne, U., and Kirsten, G.: Presence andmetabolism of amino acids in young andold lenses, Exp. Eye Res. 1: 415, 1962.

3. Reddy, D. V. N.: Studies on intraoculartransport of taurine. I. Accumulation inrabbit ciliary body-iris preparation in vitro,Biochim. Biophys. Acta 158: 246, 1968.

4. Merriam, F. C, and Kinsey, V. E.: Studieson the crystalline lens. I. Technic for invitro culture of crystalline lenses and ob-servations on metabolism of the lens, Arch.Ophthal. 43: 979, 1950.

5. Wachtl, C, and Kinsey, V. E.: Studies onthe crystalline lens. VIII. A synthetic medi-um for lens culture and the effects of vari-ous constituents on cell division in the epi-thelium, Amer. J. Ophthal. 46: Part II, 288,1958.

6. Kinsey, V. E., and Reddy, D. V. N.: Studieson the crystalline lens. XI. The relative roleof the epithelium and capsule in transport,INVEST. OPHTHAL. 4: 104, 1965.

7. Reddy, D. V. N., Klethi, J., and Kinsey,V. E.: Studies on the crystalline lens. XII.Turnover of glycine and glutamic acid inglutathione and ophthalmic acid in the rab-bit, INVEST. OPHTHAL. 5: 594, 1966.

8. Kinsey, V. E., and McLean, I. W.: Studies

on the crystalline lens. XIII. Kinetics ofpotassium transport, INVEST. OPHTHAL. 3:585, 1964.

9. Kinsey, V. E., and Reddy, D. V. N.: Studieson the crystalline lens. X. Transport ofamino acids, INVEST. OPHTHAL. 2: 229, 1963.

10. Kinsey, V. E.: Studies on the crystallinelens. XIV. Kinetics of alpha amino isobutyricacid transport, Doc. Ophthal. 20: 30, 1966.

11. Webb, J. L.: Substrate and product inhibi-tion, in Webb, J. L., editor: Enzyme andmetabolic inhibitors, New York, 1963,Academic Press, Inc., Vol. I., pp. 111-139.

12. Wheeler, K. P., and Christensen, H. N.:Role of Na+ in the transport of amino acidsin rabbit red cells, J. Biol. Chem. 242: 1450,1967.

13. Cotlier, E., and Beaty, C : The role of Na+

ions in the transport of a-aminoisobutyricacid and other amino acids into the lens,INVEST. OPHTHAL. 6: 64, 1967.

14. Macey, R. I., and Koblick, D. C : Effects ofcholine and other quaternary ammoniumcompounds on Na movements in frog skin,Amer. J. Physiol. 205: 1063, 1963.

15. Paine, C. M., and Heinz, E.: The structuralspecificity of the glycine transport systemof Ehrlich carcinoma cells, J. Biol. Chem.235:1080, 1960.

16. Christensen, H. N.: Relations in the trans-port of /3-alanine and the a-amino acids inthe Ehrlich cell, J. Biol. Chem. 239:3584,1964.

17. Kromphardt, H.: Die Aufnahme von Taurinin Ehrlich-Ascites-Tumorzellen, Biochem. Z.339: 233, 1963.


studies on intraocular transport of taurine · under the name of d. v. n. reddy. manuscript...

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