Phospholipid effects on the rat lens transport systems

Exp. Eye Res. (1978) 26, 657465

Phospholipid

Effects on the Rat Lens Transport PETER

I?. KADOR

AND

(Received 21 September

JIN H.

Systems

KINOSHITA

1977, New York)

The in vitro effects of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl seriae, sphingomyelin, lysophosphatidyl choline, lysophosphatidyl ethanolamine, lysophosphatidyl serine, glycerophosphoryl choline, and phosphatidic acid on rubidium uptake aad amino acid transport of rat lens are presented. None of these lipids affected the rubidium uptake mechanism; however, a large increase in the apparent leak-out of rubidium was observed with lysophosphatidyl choline. Lysophosphatidyl choline also inhibited all of the amino acid transport systems (A, ASC, L, Gly, Ly and j3) as well as the uptake of inositol. Key words: phospholipids; lysophosphatidyl choline; amino a,cid tra.nsport inhibition; rubidium transport; cataract.

1. Introduction The phospholipid composition of the lens has been found to change with age (Broekhuyse, 1969, 1971, 1974). Increasesin certain phospholipids have been reported in senile cataracts (Obara, Cotlier, Kim, Lueck and Tao, 1976 and Broekhuyse, Roelfzema, Breimer and Karlsson, 1974) and alterations in the membrane structure and lipid metabolism of the cataractous lens have been detected (Broekhuyse, 1974). The biochemical effects of the alteration of the phospholipid composition, however, are still not well understood. Various studies suggest that the lipid environment determines or controls several biochemical functions of membrane bound components that are involved in catalytic and/or transport functions (Fourcaas an.d Jain, 1974). Recently, correlations between serum phospholipids and cataract formation have Ijeen proposed (Goswa.my, Mathur and Agarwal, 1971 and Kahn et al., 1977). Xoreover, changesin the phospholipid concentrations of aqueoushumor in alloxan diabetic rabbits (Varma and Reddy, 1972) well as in rabbits with experimentally induced uveitis (Cotlier, Baskin, Kim and Lueck, 1976) have been reported. Although Broekhuyse (1968) has shown phospholipid synthesis to occur in the lens, the possibility of phospholipid incorporation into the lens has not been ruled out. Here we report the in vitro elects of phospholipids on amino acid trai?sport and rubidium upt,ake of rat lens. 2. Methods Lenses were obtahed from Sprague-Dawley rats weighing 125-150 g suffocated with carbon dioxide. Paired lenses were cultured according to a modified proced.ure of Obazawa, MeroIa and Kinoshita (1974) in 4 ml of synthetic media containing amino acids (TC-199) or without amino acids (balanced salt) in the presence oflabeled compounds or radionuclide. All phospholipids obtained from either Applied Science Laboratories Inc. or Supelco, Inc. were used without further purification. Generally the solvents were removed in vacua and the phospholipids were sonicated under argon in a small aliquot of media at lo-20°C 657

658

P. F. KADOR

AND

J. H.

KINOSHITA

until the solution became transparent to only slight,ly translucent. This was then diluted to the desired volume with media warmed to 37°C and saturated with 10% carbon dioxide-air. Some difficulty in the sonication of phosphatidyl ethanolamine, phosphatidyl serine, sphingomyelin, lysophosphatidyl ethanolamine and lysophosphatidyl serine was encountered and some precipitation of lipid upon incubation was observed. [14C]cl-Anlinoisobutyric acid (AIB, specific activity 90-500 PCi), [r4C]aminocyclopentane1 -carboxylic acid (cycloleucine, specific activity 230 $?i/mg), [r4C]arginine (specific activity 1320 $Zi/mg), [14C]ta,urine (specific activity 450 pCi/mg), [l%]sarcosine (specific activity 40 $X/mg), [3H]myoinositol (specific activity 100 @i/mg), and s6Rb mere obtained from New England Nuclear. The amount of radioactivity in the medium of each tube was 0.11-0.25 ,JZi. Following the desired period of culture the lens were homogenized in 1.00 ml of 10% TCA and the radioactivity in the supernatant was determined in a liquid scintillation counter using hydromix (Yorktown Research) as the fluor. The total concentration of radioactivity in the lens was calculated on the basis of lens water (62% by weight). The accumulation of tracer (L/M) was expressed as the ratio of the amount accumulated in the lens water (L) to that in the media (M).

3. Results The phospholipid effects on the in vitro incubations of rat lenses were observed by several methods. The cumulative effect of phospholipid insult on the active transport systems was observed by adding tracer at the beginning of long-term incubation (%4 hr or more). The net effect of the phospholipid insult after a specified period of incubation was observed by adding tracer to the media and incubating an additional 4 hr (short-term incubation). TABLE

1

The eflect of yhospho- madsphingolipids 01186Rbaccumulation in rat lenses iwubatecl in TC-199 median

Lipid

Concentration

(mx)

Length

of incubation

Accumulation Control

(L/M) Experimental

PC

064

24 hr tracer 20 hr+4 hr tracer 44 hr + 4 hr tracer

34.7+2.6 3.9hO.5 4.2&0.4

30.9h2.3” 4.461.3 54+1.0

PE

0.65

24 hr tracer

31.7k2.8

30.2h2.1

24 hr tracer

36.313.9

:21.7+4.3

PC-PE mixture

0-32-0~34

PS

0.64

20 hr+4 44 hr+4

hr tracer hr tracer

3.QO.3 4.0*0,5

:+4*0.2 4.4&O-5

PA

0.14

20 hr -; 4 hr tracer 44 hr 7-4 hr tracer

3+l*O.6 4.3f0.8

3+3&0.4 2.9fO.9

SM

0.3::

24 hr tracer

PC, phosphatidyl choline (bovine); PA, phosphatidic

(bovine); PE, phosphatidyl ethanolaminc acid (plant); SM, sphingomyelin (bovine).

28.1*1G3

“5.2&1.9

(bovine); PS, phosphatidyl * P < 0.01, ?L = 4-S.

serine

PHOSPHOLIPID

EFFECTS

ON

RST

659

LENS

The effect.s of phospho- and sphingolipids on ssRb accumulation in rat lenses incubated in TC-199 media are shown in Table I. Similarly, the effect of lysophospholipids and L-a-glycerophosphoryl choline (GPC) is shown in Table II. Incubating with 640 +t-phosphatidyl choline (PC) produced a slight but significant decrease in the 24 hr accumulation of S6Rb. No difference, however, was found in the 4 hr incorporation of sGRb after 20 or 44 hr exposure to PC indicating that the difference II

TABLE

The

effect

of dysophospholipids

and

glycerophosphoryl

esters

on

a6Rb accumdation

in

ra.t

lenses incubated in TC-199 media.

Lipid

Concentration

LPC

(mix)

Length

Accumulation Control

of incubation

24 hr tracer

I.0

34.514.7 10~2~1~0

1,6*0.2” 10~9&2~1

20 hr+4 hr tracer 48 hr tracer 44 hr+4 hr tracer

31.614.4 4.751.2 38.7+7~0 4.31-0.5

X.5&4.6* 4.3k1.5 11.3&5.6* 3.8&1.2

hr tracer

17 hr+7

24 hr tracer

0.1

(L/M) Experimental

LPE

0.22

24 hr tracer

32.3h2.5

33.7*1.2

LIT

0.20

24 hr tracer

38,6&l?

41.913.6

GPC

0.36

24 hr tracer

29.0+1.s

30.2k3.5

LIT, Lysophosphatidyl choline (egg) ; LPE. lysophosphatidyl phosphatidyl swine (bovine); QPC L-a-glycerophosphoryl choline

ethanolamine (synthetic).

in the accumulation observed is probably not due to increase in the leak-out of tracer from the lens. A similar was observed by incubating with a mixture containing Iamine (PE) and 320 PM-PC although incubation with

the uptake process but to an decrease ins6Rb accumulation 340 PM-phosphatidyl ethano670 PM-PE alone produced no

TABLE

The

efleect

Lipid

PC PE L’s PA SM

of phospho-

and

Concentration

0.64 0.67 0.64 0.14 0.33

(miv)

LPS,

lyso-

n = 4--s.

III

on [14C]AIB incubated in TG-199 media

sphingoli@ls

Length

(bovine); * P < 0.01,

of incubation

accuwulation

Accumulation Control

24 hr tracer 24 hr tracer 24 hr tracer

13.6kl.9 13,4+2.2

24 hr tracer 24 hr tracer

l-3.8&2.1

12.7k2.2 13.1h1.3

in

rat

lenses

(L/M) Experimental

13.2*1.7 12.2k2.8 11.911.8 14.332.1 X2.7&2.5

PC, phosphatidyl choline (bovine); PE, phosphatidyl ethanolamine (bovine); PS, phosphatidyl serine (bovine); PA, phosphatidic acid (plant); SM, sphingomyelin (bovine). * P < 0.01, n, = s-12.

660

P. F. KADOR

AND

J. H.

KINOSHITA

difference. A significant difference in the 24 hr accumulation of s6Rb along with lens opacification was also observed upon incubation with 100~” of lysophosphatidyl choline (LPC). This difference was increased when the concentration of LPC was increased to 1000 PM. Furthermore, this difference in the accumulation appears to be a dose-dependent response directly linked to the in vitro rate of formation of lens opacities. However, even in those lenses which developed opacities there was no difference in the short-term accumulation of @jRb. The difference in the accumulation of tracer is probably the result of an increase in the leak-out of ssRb caused by an alteration of cation permeability since the osmolarity of the media was not altered by LPC. No difference in the rate of accumulation of tracer was observed with either 640 PM-phosphatidyl serine (PS), 140 pv-phosphatidic acid (PA), 330 PulM-sphingomyelin (SM), 220 PM-lysophosphatidyl et,hanolamine (LPE), 200 PM-lysophosphatidyl serine (LPS), or 360 ,UM-GPC. TABLE

IV

The effect of lysophospholipids and glycerophosphoryl esters on [14C]AIB in rat lenses incubated in TC-199 media

Lipid LPC

Concentration 0.10

(mix)

Length

of incubation

Accumulation Control

24 hr tracer

(L/M) Experimentnl 3.1*0.4* 0.9*0.3* 3.051.1" 0~9*0~2*

20 hr+4 hr tnwer 48 hr tracer 44 hr+4 hr tracer LPE

nccunwdation

0.22

24 hr tracer

0.11

24 hr tracer

0.22-1.27

24 hr tracer

LPS

0.20

24 hr tracer

11.9&2+3

12~Q3.3

GPC

0.36

24 lx tracer

15.9k2.8

l-1.1&1.9

LPE-PC mixture

LPC, Lysophosphatidyl choline (egg); LPE, lysophosphatidyl phosphatidyl serinc (bovine); GPC L-a-glycerophosphoryl choline

11.213.7

ethanolamine (bovine); LPS, lyso(synthetic). * P < 0.01. n = 3-8.

The effects of phospho- and sphingolipids on [14C]BIB accumulation in rat lenses incubated in TC-199 media are shown in Table III while the similar effects of lysophospholipids and GPC are shown in Table IV. A striking effect on the accumulation of [W2]AIB was observed upon incubation with LPC. At a concentration of 100 PMLPC the L/M ratio of the 24 hr accumulation of [r4C]AIB decreasedby 700/,. A doseresponsecurve on the effect of LPC on the 24 hr accumulation of [14C]AIB in TC-199 media (Fig. 1) indicated a rapid decreasein the effect of LPC at a concentration of 50 PM with no significant difference at 12.5 PM. No significant difference in the rate of accumulation of [14C]AIB was observed with either 64Opm-PC, 670 PM-PE, 640 PM-PS, 140 PM-PA, 330 ,UM-SM, 200~~-LPS or 360~~-GPC. A very small difference in the accumulation of [14C]AIB was observed with 220+-LPE. No difference, however, could be observed with a reduced concentration of 110PM-LPE or a mixture of lipid containing 220 PM-LPE and 1270,UM-PC.

PHOSPHOLIPID

EFFECTS

100

80

ON

60

FIG.

FIG.

1. Dose-response curve of the effect of LPC on the 24 hr accumulation in TC-199 media (71 = 3-5).

2. Effect of 100~~~LPC (TL = 3-7). 0-O Control;

of [W]AIB

in rat lenses

(hr)

on the accumulation of [W]AIB @- - -A, experimental.

in rat

lenses incubated

in TC-199

q controt q Experimental

5% 3.0 2 s .z a 2.0 z a s I-0 o-o

661

QUA)

Incubation

media

LENS

40

LPC

incubated

RAT

0

I Preincubotion

FIG. 3. Effect of preincubation with 100 Pnr-LPC ncubated in TC-199 media (n = S-20).

2

3 fhr)

on the 4 hr accumulation

of [W]AIB

in rat lenses

662

P. F. KADOR

AP\‘D

J.

H. KINOSHITA

A rate study of the effect of 100 ,UM-LPC on the accumulation of [14C)J141B(Fig. 2) indicated that the amount of [14C]AIB accumulated in the rat lens remained constant after 4 hr of incubation. Preincubation with 100 PM-LPC in TC-199 media’ followed by 4 hr accumulation of [i4C]AIB (Fig. 3) indicated a significant decrease in the accumulation of tracer after 1 hr of preincubation. Furthermore. active transport

-I 5

IO

20

I5

Incubation

25

(hr)

FIG. 4. Effect of 100 PM-LPC on the leak-out of [W]AIB from rat lenses incubated The [W]AIB had previously been accumulated over a 24 hr period in TC-199 [14C]AIB (~1 = 4). a-0, Control; A- - -A, experimental.

in W-199 media. media containing

essentially stopped after 2 hr of preincubation. TO insure that this [14C]AIB decrease was not due to an increased permeability in the LPC incubated lens a leak-out experiment was conducted. As seen in Fig. 4 no significant difference in the leak-out of [14C]AIB was noted after 8 hr incubation with 100 PM-LPC. However, increased leak-out was seen after 24 hr of incubation. TABLE

V

Attempts at reversing the effect of 0.1 ELM LPC on [l4C]AIB incubated in TC-199 media

a~ccumnulation i,r~rat lenses

Accumulat,ion C’ontrol

(L/M) Experimental

___I.

II.

of LPC

during incubation 1 hr LPC media+4 hr LPC free media 2 hr LPC media+4 hr LPC free media 3 hr LPC media+4 hr LPC free media Washout

Increasing the concentration of -4IB 24 hr tracer only 24 hr tracer+O.l mnr cold AIB 24 hr tracer+ 1.0 rniv cold BIB

* P < 0.01, 1~ = 4-6.

and tracer and tracer and tracer

1.9*0.2 1.5kO.3 1.6&0.X

1.6&0.2* 1.210,1* 0.9+0,1*

8.6&3.1 7.0+1.0

3.1*0+s*

9.2+0.5

3.3kO.5” 4.0&1.5”

PHOSPHOLIPID

EFFECTS

OX

RAT

LENS

663

As seen in Table V this effect of 100 PM-LPC on the accumulation of [i%]BIl3 appears difficult to reverse by either removal of the exposed lens from the LPC ~ontailljng media, or by increasing the concentration of AIB. Incubating rat lens in LPC-free media conta.ining [14C]AIB for 4 hr after a 1 hr incubation with LPC Ta BLB I,‘1

Balanced Control

3 hr iilcubatio11tl [I”C]AIB trace1

salt media Expcrimentai

hr 54&l+!

1?5*0~5*

3 hr incubation+4 hr [**Clcyclulrucine t,racer

8.3&1.0

4.2&l@

3 Ii r iwxbation f4 sarcosine tracer

6.9&l.?:

1.3*0.6*

hr [‘“C!-

jndicated a significant decrease in the accumulation of tracer. Sfter 3 hr of incubation the a,ctive transport of [14C]AIB was destroyed. Increasing the concentration of BIB by the addition of up to 1.0 mwAIB could not reverse the effect of 100 q-LPC on the 34 hr accumulation of [14C]AIB.

Tlze pJect of 0.1 1~1~1LPC on, the accumula~tion of [14C]aryinine, [14G]tnurine myoinositol ZYLmt lenses incubated in TC-199 media

Length

Arginine

of incubution

24 hr tracer

4 hr t.rscer I hr+4 hr tracer 3 hrf4 hr tracer Taurine

24 hr tracer 4 hr trizcer

1 hr+4 3 hrG-4 Myoinositol

* P < 0.01,

71, =

3-9.

hr tracer hr tracer

24 hr tracer 20 hr+4 hr tracer 4 hr tracer I hr+4 hr tracer

Accumdstion Control

7.31-1.2 64&O,Y

7.3kO.i 6.9+0,9 62.5* 10.9 12.3h1.6 11~2+1~1 9.3+o+i 30.213.7 5.2&1.4 4.1 &OS 445 I.2

(L/M) Experimental

wed [aH]-

664

P. F. KADOR

AND

J. H.

KIlNOSHITA

The transport of amino acids in the lens has been classified into six systems (Kern, Ho and Ostrove, 1977). Since AIB appears t,o exhibit overlapping affinity for several of these systems (A, L, ASC and /3), incubation . wit’h more specific amino acids was investigated in order to determine the specificity of LPC for these va,rious systems. As seen in Table VI lOO+f-LPC appeared to decrease t,he accumulation of cycloleucine (L) and sarcosine (Gly, ASC). After 3 hr of preincubation with LPC in TC-199 media the active transport of [r4C]sarcosine lvas essentially stopped and the transport of [i4C]cycloleucine was significantly decreased. A 24 hr incubation of [14C]cycloleucine, however, showed an accumulation value of 2.650.6 vs. 7.3h1.6 for t,he control indicating that the active transport a,lso appears to be eventually stopped. Similar results for the effect of 100 FM-LPC were obtained upon incubat’ion in the amino acid-free balanced salt media, although the rate of incorporation of tracers was much higher. As seen in Table VII the accumulation of arginine (Ly) and taurine (8) in TC-199 media were also significantly affected by 100 ,ULM-LPC with the active transport of taurine being essentially stopped after 3 hr of incubation. The effect of 100 ~xLPC on the inositol transport system was also invest,igated since this system has been reported to interact w&h certain amino acids (Varma et al.; 1970). As seen in Table VII the accumulation of [3H]nlyoinositol was rapidly decreased by 100 PM-LPC with active transport being stopped after 1 hr of incubation. 4. Discussion The ability of the lens to accumulate *6Rb has been reported to be a good index of the effectiveness of the eationic pump mechanism (Becker, 1962). Short-term incubation of lenses previously exposed to LPC indicated no significant change in the accumulation of a6Rb. Therefore, LPC does not appear to affect the rubidium upt,ake process. However, when the s6Rb accumulation was followed for 24 hr in the presence of LPC a decrease in the L/M ratio was observed. Since the uptake process appears to be unaffected, this result is probably due t’o an increase in cation permeability. This increase in the apparent permeability is accompanied by opacification of the lens. Cotlier, Baskin and Kresca, 1975 and Cotlier et al. (1976) in studyicg the effects of LPC on rabbit lenses have reported that an increased level of LPC in uveitis leads to cataract formation. Furthermore, by measuring the sodium and potassium ion concentrations, they have reported permeability changes in rabbit lenses incubated in vitro with LPC. D’Ermo and Secchi (1977) h ave also reported an efflux of proteins from rabbit lens incubated with LPC. The findings here confirm and extend these previous studies. None of the other lipids appeared to inhibit the cation uptake mechanism however, a, slight increase in the leak-out of 86Rb was also observed in lenses incubated with PC. In this study LPC has been shown to affect the amino acid transport systems in the lens (A, AX, L, Gly, Ly and ,@. Th’is inhibition cannot be decreased by increasing the concentration of amino acid, indicating that LPC does not compete with the amino acid for the amino acid transport site. Furthermore; the inhibitory effect of LPC could not be reversed by the “washing out” of LPC from the lens indicating either that LPC is strongIy bound to the lens or that its effect is irreversible. The inositol transport system of the lens was also inhibited in the presence of LPC. A dose-response curve of the inhibitory effect of LPC on the accumulation of [14C]AIB in TC-199 media indicated a rapid decrease in inhibitory activity at 50 /*x with no significant inhibition at 12.5 p&t. Cotlier (1976) has reported that a con-

PHOSPHOLIPID

EFFECTS

ON

RAT

LENS

665

centration of 16~31-LPC was necessary to produce permeability changes in the rabbit lens. The inhibitory effect on the amino transport systems is rather specific for LPC and it appears that both the fatty acyl and phosphorylcholine groups are essent,ial for the observed inhibition since no inhibition was observed with either PC, PE, PX, LPS or GPC. A similar specificity by membrane-bound galactosyl-transferase in rat liver has been reported (Xookerjea and Yung, 1974). LPC activates this enzyme probably through solubilization of the membrane and consequent enhanced interaction of t’he enzyme with substrate. A similar mode of action for LPC in the lens appears possible with LIT selectively altering alipid-protein complex in t’he transport syst.ems necessary for activity. REFERENCES Becker, 47. (1962). =Iccumulation of “@Rb by the rabbit lens. fnoest. O$~i~aZn~oi. I, 602-6. Broekhuyse, R. M. (1974). Membrane lipids and proteins in ageing lens and cataract. Ciba Foundafion S~ln~posiwx Series 19 ASP. Pp. 135-49. Elsevier, Xorth-Holland, Amsterdam. Broekhuyse. R,. 81.. Roelfzema, H., Breimer, M. E. and Karlsson, K.--4. (1974). Lipids in tissues of the eye S. Molecular species of sphingomyelins from different parts of calf lens in relation to differentiation and ageing. Exip. Eye Res. 19, 47%84. Broekhuyse, 12. IVI. (1971). Lipids in tissue of the eye IV. Influence of age and species difference on t,he phospholipid composition of the lens. Biochinl. Bioph,ys. .&a 218, 546-S. Broekhuyse, It. M. (1969). Phospholipids in tissues of the eye III. Composition and metabolism of phospholipids in human lens in relation to age and cataract formation. Biochim. Biophys. Acta, 187, 354-65. Broekhuyse, R. M. and Veerkamp, J. H. (1968). Phosphoiipids in t,he tissues of the eye II. Composition and incorporation of ‘“Pi of phospholipid of normal rat and calf lens. BiocAim. Biophys. Actn 152, 316-24. Cotlier, E., Baskin, X, Kim; J. 0. and Lueck, K. (1976). Lysophosphatidyl choline and cataracts in uveitis. Arc71. OphthalnzoZ. 94, 1159-62. Cotlier, E., Baskin, M. and Kresca, L. (1975). Effects of lysophosphatidyl choline and phospholipase A on the lens. Invest. Ophthalmol. 14, 697-TOI. D’Ermo, F. and Secchi, A. G. (1977). Phospholipase A und Lysophosphatidylcholin in der Pathogenese der “permeabilitaetskatarakt”. liZi?b. XbE. Atcgexheik 170, 43340. Fourcans, B. and Jain, M. K. (1974). Role of phospholipids in transport and enzymatic reactions. In B&xwzres in Lz&‘d Resewch, Vol. 12 (Eds Paoletti, P,. and Kritchevsky, D.). Academic Press. New York. Goswamy, S., Xathur> R. L. and Agarmal, L. P. (1971). Lipoproteins of the crystalline lens and serum factors in senile cataracts. Indian J. Ned. Res. 59, 1460~. Khan, H. A., Leibowitz, H. &‘I., Manley, J. I?.. Kit& M. X., Colton, T., Nickerson, R. S. and Dawber, T. R. (1977). The Framingham Eye Study II. Association of ophthalmic pathology with single variables previously measured in the Framingham Heart Study. Am. J. JSpidenaiol. 106, 33-41. Kern, H. L.. Ho. C.-K. and Ostrove, S. A. (197i). Comparison of transport at the anterior a,nd posterior surfaces of the calf lens. Ezp. Eye Res. 24, 559-70. Mookerjea, 8. and Yung, J. 11’. M. (1954). A study 011 the effect of lysolecithin and phospholipa,se A on the membrane bound gaIxctosyl-transferase. Can. J. Biochenr. 52, 1083-66. Obara, Y., Cotlier, E., Kim, J. O., Lueck, K. and Tao, R. (1976). Sphingomyelin species stored in human senile cataract. Invest. Ophthdmol. 15, 966-9. Obazawa, H., l!terola, L. 0. and Kinoshita, J. H. (1974). The effects of xylose on the isolated lens. Invest. Ophthalmol. 13, 204-Q. Varma, S. D. and Reddy, V. N. (1972). Phospholipid composition of aqueous humor, plasma, and lens in normal a,nd alloxan diabetic rabbits. Exp. Eye Res. 13, 120-5. Varma. S. D., Chakrapani, B. and Reddy, V. N. (1970). Intraocular transport of myoinositol IIAccumulation in the rabbit lens in vitro. Invest. Ophthalmol. 9, ‘i94-800.