Influence of prostaglandins on cation movement in the lens

Exp. Eye Res. (1973) 15, 767-778

Influence of Prostaglandins on Cation Movement in the Lens* CHRISTOPHER

A. PATERSON

AND BEVERLY

A.

ECK

Division of Ophthulmology, University of ColoradoMedical Center, Denver, Coloroxio80220, U.S.A.

Rabbit lenses incubated at 36°C for 20 hr in a medium containing 1O-3 M prostaglandin a marked increase in sodium and decrease in potassium PG) E,, E, or %a exhibited content; 1O-3 M PGFlo was without effect. 1O-3 M PGE, caused a significant increase in cation permeability as evidenced by increased rubidium-86 efflux. Reduced rubidium-86 uptake was observed in lenses incubated in 10-a M PGE, and F I’ lo-* M prostaglandins (E,, E, and F,,) appeared to stimulate the lens cation transport mechanism. Recovery from a cold induced cation shift was enhanced, as was rubidium-86 uptake. Lenses subjected to reduced calcium level in the incubation medium survived better in the presence of lo-* M PG. It is suggested that the effects of 1O-4 M PG are mediated via the adenyl cyclase system. The effects of 1O-3 M PG might be due to a large non-specific increase in lens cation permeability.

1. Introduction Prostaglandins are widely distributed in biological tissues and exhibit a variety of effects (Horton, 1972; Ramwell and Shaw, 1970; Ramwell and Shaw, 1971). It has been demonstrated that irin is releasedinto the anterior chamber following irritation of the iris (Ambache, Kavanagh and Whiting, 1965). Irin is a mixture of E- and Ftype prostaglandins (Ambache, Brummer, Rose and Whiting, 1966; &ggard and Samuelsson, 1964; Waitzman, Bailey and Kirby, 1967). It is therefore possiblethat prostaglandins are released also during inflammatory conditions of the eye. Kaley and Weiner (1971) considerthat prostaglandins are an integral part of the inflammatory response. Lens changes subsequent to chronic ocular inflammatory diseaseare well documented (Duke-Elder, 1969). Since prostaglandins are known to influence the permeability of epithelial membranes (Ramwell and Shaw, 1970), the influence of these agents upon the lens was examined, A preliminary report (Paterson and Eck, 1971) described a deleterious effect of low3 M prostaglandin E, (PGE,) on lens cation balance. Also, Waitzman, Kuck and Woods (1972) have shown reduced galactose uptake by rat lensesincubated in a media containing prostaglandins E, and F,, at a concentration of 20-100 “9% (approximately 6 x low4 to 3 x 10e3M). This paper describesthe results of further experiments concerning the effect of prostaglandins on cation movement in the lens. 2. Materials and Methods Lenses were obtained from either freshly killed New Zealand strain albino rabbits or from the heads of rabbits supplied by A & B Rabbit Processors, Denver, Colorado. The heads were collected at the time of slaughtering and transported immediately to the laboratory. Comparison was made between the cation content of these lenses and those * This study Eye Institute.

was supported

by Public

Health

Research 767

Grant

EY

506-03,04

from

the National

768

(‘. A.

obtained from animals sacrificed lenses from both sources following

PATERSON

AND

R. A.

ECK

in the laboratory. In addition, the cation 20 hr incubation at 36°C was determined.

content

01

Iwubation Lenses were incubated at 36°C in 7 ml of a medium consisting of five parts TC 199 to two parts of a bicarbonate buffer with the following composition: 94 m&f NaHCO,; 20 mM NaCl; 4.9 mM CaCl,; 0.6’7 mM KH,PO,; 1.15 mM KCl; 5.5 mM dextrose. The pH was adjusted to 7.4 by bubbling with a 95% 0,/5% CO, gas mixture. St,reptomycin and penicillin were also added. 25 ml borosilicate screw top centrifuge tubes were used as incubation vessels. In a number of experiments the calcium concentration in the medium was altered. Since the TC 199 contains 5 mg% calcium, a final calcium concentration of 3.6 mg% in the medium was readily achieved by omitting CaCl, from the bicarbonate buffer. In the experiment where lenses were incubated in a medium containing 2 mg%, the regular incubation medium was substituted by a modified Tyrode’s solution with the following 23 rnnr NaHCO,; 0.42 rnM composition: 119 mM NaCl; 1 mM MgCl,, . 4.99 mM KHCO,; NaH,PO,. The calcium levels were adjusted to give 9.1 mg% in the control medium and 2.0 rng% in the experimental medium. Prostuglandins Prostaglandins were generously supplied by Dr Kalamazoo, Michigan. They were dissolved in 95% and stored at 0°C. In experiments where one lens containing prostaglandins, an equivalent amount of in which the control lenses were incubated.

John E. Pike of Upjohn Company, ethanol to give a solution of 20 mg% of a pair was incubated in a medium 95% alcohol was added to the medium

Rubidium-86 uptake Paired lenses were incubated at 36°C for 16 hr, one lens of the pair being exposed to varying concentrations of prostaglandin. After incubation for 16 hr, rubidium-86 (2 PC/ml) was added to each tube and the incubation continued for a further 4 hr. At the end of the total incubation period, the lenses were removed from the media, quickly blotted on filter paper and placed in pre-weighed tissue grinders. After re-weighing the tissue grinder, 2 ml of 10% trichloracetic acid were added and the lens homogenized. Radioactivity in the lens supernatant and in the incubation media was determined using a Picker Magnachanger 40 planchet counting system. Rubidium-86 uptake by control counts/min/ml of lens water and experimental lenses was expressed by the ratio counts/min/ml of medium ’ The sodium

and potassium

content

of the lens supernatant

was also determined.

Rubidium-86 e&x The efflux of rubidium from lenses was determined in a manner similar to that described at 36°C for 20 hr in a medium by Becker and Cotlier (1965). L enses were incubated containing 5 PC rubidium-86/ml. One lens of a pair was exposed to PGE, at 1O-3 M. At the end of the incubation with rubidium-86, the medium was replaced with 7 ml of an identical medium containing no rubidium-86, but containing lo-* M ouabain to prevent active reaccumulation of rubidium-86. 20 ~1 samples of the new medium were removed at 30 min, 1, 2 and 3 hr. At the end of the efflux period the lens was quickly blotted and uptake experiprocessed with trichloracetic acid, as described above for rubidium-86 ments. Per cent efflux of rubidium-86 from the lens was calculated as: total count/min total count/min

in medium at sampling times x 100. initially present in lens water

1

PROSTAGLANDINS

AND

THE

LEN,S

769

of sodiumand potassium Sodium and potassiumlevels in diluted lens supernatant and incubation media were determined by flame photometry using a Beckman Model B spectrophotometer with a flame attachment. Determination

Sodium-22exchange In one instance the exchange of sodium-22was determined in the presenceof 1O-4M PGE,. Paired lenseswere incubated for 20 hr at 36°Cin a medium containing sodium-22, one lens of the pair being exposedto lo-* M PGE,. The experimental details and determination of sodium exchangefrom the specificactivity ratio of lens to medium are given in a previous paper (Paterson, 1970). Tequerature reversal experiments During incubation at O”C, potassiumis lost and sodium is gained by the lens. On rewarming at 36°C the lens reconcentratespotassium and extrudes sodium. Essentially, the method describedand reviewed by Harris (1966)was employed in theseexperiments. However, considerableattention to detail was found necessaryin order to obtain reproducible results. The following method proved reliable and wasusedfor all experiments reported in this paper. Lenseswere placed in incubation tubes containing media at room temperature. One tube rack contained lensesto be removed after 20 hr incubation at 0°C; a secondrack contained those lenses which would undergo the cold incubation followed by 6 hr further incubation at 36°C. Both racks were placed in the bath at room temperature. The bath temperature wasthen loweredto 0°C over a period of about 20 min. After the required time interval, one rack wasremoved and the bath allowed to warm to 36°C over a period of again about 20 min. After a further 6 hr, the secondgroup of lenseswasremoved. Each lens was weighed and homogenizedin trichloracetic acid, and analyzed for sodium and potassiumas describedpreviously. Using the data on cation levels, the percent recovery wascalculated usingthe formula : per cent recovery = Gl---clY+3,~ x 100, where C is the ~,~--ccontrol sodium or potassiumcontent of the lens following the indicated procedures.The control cation content was taken as the mean of lensesincubated for 20 hr at 36°C.

3. Results Values for the cation content of lensesfrom freshly sacrificed laboratory animals and from heads obtained from a local rabbit processor are presented in Table I. There was no significant difference between the cation content of lensesobtained from either source. Also, there was no difference between lensesanalyzed immediately and those analyzed after incubation at 36°C for 20 hr. Incubation of rabbit lensesin a medium containing 10e3M PGE,, Es or F,, resulted in a marked increase in sodium and decreasein potassium content. Lensesincubated in 10m3M PGF,, did not exhibit a significant alteration in cation content (Table II). Lenses incubated in a medium containing 104~ PGE, and PGE, exhibited a reduction in sodium content and increasein potassium content. However, the change was not highly sign&ant; 0.05 < P < 0.1. lo-* M PGF,, and PGF,, did not cause any noticeable alteration in cation content. These findings are presented in Table II. 1O-3M PGE, significantly increased rubidium-86 efflux from the lens (Fig. 1) and 10”~ PGE, and PGF,, significantly reduced rubidium-86 uptake (Table III). PGF,, at 1O-3M did not significantly affect rubidium-86 uptake.

770

C!. A.

PATERSON

AND

B. A.

EC’K

Rubidium-86 uptake was significantly increased when lenses were incubated in a, medium containing 10d4 M PGE, or E, (Table III). Smaller increasesin rubidium-86 uptake seenwith 1O-4 M PGF,, and PGF,, were not statistically significant. In the presenceof 10e4M PGE,, sodium-22 exchange in the lens was reduced. For control lensesthe specific activity ratio (lens/medium) was 0*84*0.33 (5), and that for experimental lenseswas 0*77&0.13 (5) ; the difference was significant at the 950/x confidence level. Reduction of the calcium level in the incubation medium from 9.1 mg% to 36 or 2 rng% resulted in an increase in sodium and fall in potassium levels (Table IV). TABLE

Cation

Immediate

Following

removal

content”

I

of rabbit

lenses

Source

nt

Sodium

Potassium

A and BJ Laboratory$

20 10

17.7+04 17.9+1.2

131.9h2.2 131.7k2.9

A and B Laboratory

20 10

l&1*1.2 18.211.6

130.1&3.1 131.212.7

and analysis

20 hr incubation

at 36°C

* Mequiv./kg lens water; arithmetic mean&standard error of the mean. t Number of lenses. $ Heads from A and B Rabbit Processors. 8 Rabbits killed in the laboratory. There WBB no statistical difference between the cation content of lenses from was greater than 0.99 in every case.

either

source;

P value

When paired lenseswere incubated for 20 hr in a low calcium medium, the lens in the experimental medium containing 1O-4M PGE,, E, or F,, maintained better cation balance (Table V) ; the sodium content was lower and the potassium content higher. The results of the temperature reversal experiments suggestedthat low4 M PGE, and E, might stimulate the recovery from a cold induced cation shift. The results are shown in Table VI. In control lenses,the per cent recovery during the 6 hr incubation at 36°C was 88*1o/ofor sodium and 93*Oo/ofor potassium. In the presenoeof lo-* M PGE, the recovery was 91.4% and 96.4% for sodium and potassium respectively. In another experiment, paired lenses were preincubated at 0°C for 20 hr, one lens of each pair being exposed to 1O-4M PGE,. Control and experimental lenseswere rewarmed for 6 hr at 36°C. Comparison of cation levels in these lensesis shown in Table VI. The recovery was significantly better in the lensesincubated in the medium containing 1O-4M PGE,. 4. Discussion The use of lenses obtained from rabbit heads collected from a slaughter house presented no difficulty. The cation levels in those lensesanalyzed immediately and those incubated for 20 hr were statistically the same. The cation levels reported here agree with those published previously (Paterson, 1972). The economy of obtaining eyes from rabbit heads from a slaughter house should be obvious.

PROSTAGLANDINS

AND

THE

LENS

771

In confirmation of a preliminary report (Paterson and Eck, 1971), this study demonstrated a marked alteration in cation balance of lenses incubated in media containing 1OV M PGE,. In addition, it was shown that deranged cation balance was induced by 1O-3 M PGE, and F,, but not by PGF,,. The lack of effect of PGF,, on the lens in comparison to PGE,, E, and F,, seems to parallel its lack of effect in eliciting an ocular hypertensive effect following intracameral injection (Waitzman and King, 1967; Beitch and Eakins, 1969). However, Waitzman, Kuck and Woods (1972) reported reduced galactose uptake by rat lens incubated in a medium containing PGF,,. No explanation for the discrepancy can be offered at present. Control data for rubidium-86 uptake and efllux studies compare favorably with those reported by other investigators (e.g. Lambert, 1968; Kinoshita, Merola and TABLE

Injluence

II

of prostaglmdins on 20 hr incubated rabbit lens cation” balance nt

Sodium

Potassium

17.9,tl.l 45?5&9.0 < 0.01 lW,t6.6 2.1612.5 < 0.005 17.7+0.8 16+3&0.7 < 0.1

130.4*3.3 107.3*6.4 < 0.005 129.9*2.0 126.6Lir2.2 < 0.01 136.5k1.8 139.5&2.1 < 0.1

20.1 i 1.6 43.9+s.4 < 0.025 17.750.6 16.710.7 < 0.1 17.4*1.2 16.7hO.7 > 0.6

13O.Oh3.6 114.41t9.2 < 0.05 130.4&1.7 131.7&1+3 < 0.1 134.4h2.7 131.7+1++ > 0.6

19.3kO.9 22.2+2.3 > 0.1 18.7 18.6

123.311.1 119.9*0.7 < 0.1 134 131

20.6& 1.9 48.315.1 < 0905 16.QO.5 17.OhO.7 > 0.6

123%*2.5 99.4*7.0 < 0.005 127.4h3.8 126.9h3.6 > 0.7

PGE, Control IO-3 M PS Control

8 8

5X10-4M P

Control M

10

Control IO-3 M

5

lo-4

P

PGE,

P

Control 10-a M

9

P

Control 10-S M

4

P

PGF,, Control lo-’ M

7

P

Control 10-4 M

3

Control lo-3 M

6

P

Control lo-* M

5

P

* Mequiv./kg lens water; arithmetic t Number of paired lenses. $ P value obtained by paired t test.

mean&standard

error

of the mean.

C. A.

772

PATERSON

AND

B.

A.

ECK

Tung, 1968; Lambert and Kinoshita, 1967). The observed increase in rubidium-86 efflux from lenses exposed to 10e3 M PGE, may be interpreted as indicating an increase in the cation permeability of lens cell membranes. 1O-3 M PGE, and P,, reduced rubidium-86 uptake by the lenses to about 50% that of controls. Generally, rubidium86 uptake is considered an index of the activity of the active cation transport mechanism in the lens. However, as will be discussed later, a marked increase in passive permeability to rubidium-86 could reduce the lens/medium rubidium-86 uptake ratio, although the pump mechanism per se is not directly inhibited.

16 E 3 5 E

14-

.c

i2-

0

0.5

I

2 Time

3

4

(hr)

FIG. 1. Effect of 1O-3 M PGE, on rubidium-86 efflux from rabbit lenses. Each point represents the mean of *even lenses. Bars represent the standard error. Experimental lenses were exposed to PG for 20 hr at 36°C. The difference between control and experimental was statistically significant (paired t test; P < OfO5) at each time period.

Prostaglandins at 10h4M appeared to stimulate cation transport in the lens. Rubidium-86 uptake was significantly increased in the presence of lop4 M PGE, and Ea, and lenses incubated for 20 hr at 36°C in media containing lop4 M PGE, and E, exhibited a lower sodium and higher potassium content when compared to the controls. Also, sodium-22 exchange was reduced in the presence of lo--* M PGE,. More convincing demonstrations of the effects of lop4 M PG’s came from the results of experiments involving reduced calcium levels in the medium and temperature reversal experiments. Initially, the experiments with reduced calcium levels in the incubation media were designed to determine whether prostaglandins at lop4 M would have a toxic

PROSTAGLANDINS

AND

THE

773

LENS

effect upon lenses incubated in a medium containing a little less than an calcium level. Subsequently, it was found however, that in a low calcium 10w4~ prostaglandins (E,, E, and I?,,) tended to stimulate the lens to sodium and accumulate potassium, thereby retaining close to normal cation In control experiments, the increase in sodium and decrease in potassium lowered calcium was consistent with that reported previously in calf lens Kern and Kinoshita, 1960). TABLE

essential medium extrude balance. due to (Merola,

III

a6Rb uptake* by rabbit leases: in@uence of ~osta&m&ns

nt

Control

Experimental

Exp/Cont x 100

L-

1

P$value on difference

PGE, 10-a M 10-e M

7 3

3.71+0.27 3.66kO.09

57.1 110.3

< 0.01 < 0.025

10-4 M 10-s M IO-6 M

3 3 3

3.40*0.15 4.21 kO.20 3.71+0.16

3.7OhO.23 4.63 +0.07 395&0.20

108.8 109.9 103.7

< 0.025 < 0.1 > 0.2

IO-3 M IO-4 M

3 3

2.90*0.34 3.92hO.31

2.67hO.13 4~18,tO~ll

92.3 106.6

> 0.3 > 0.3

10-a M 10-4 M

3 3

3.60*052 3.35hO.03

1.64*0.04 3.55*0.13

45.5 105.9

< 0.01 > 0.1

PGE,

PGFla

PGF,z

* Figures given are the arithmetic t Number of paired lenses $ Obtained by paired t test.

mean*standard

TABLE

error

of the mean.

IV

Effect of calcium concentration in incubation medium on lens cation* balance

9.1 mgye 3.6 mgyo pi 9.1 mg% 2.0 mg%

nt

Sodium

Potassium

5

16.4kO.4 23.3f0.9 < 0.001

131.4&0+35 127.4*1+3 < 0.001

5

15.7&1.4 27.2h2.1 < 0.001

130.1+3.0 121.5f2.8 < 0.001

P

* Mequiv./kg lens water; arithmetic t Number of paired lenses. $ Obtained by paired t test.

mean-&standard

error

of the mean.

774

C. A. PATERSON

AND

B. A.

ECK

The temperature reversal experiments also were designed initially to look for a toxic effect of 10-4 M prostaglandins. Harris (1966) has demonstrated and remarked upon the fact that the temperature reversal technique is a more sensitive method for study% the influence of various factors on lens cation balance. For example, prednisolone phosphate at 5 x 1O-4 M partially inhibits the recovery from a cold TABLE

V

E$ect of 1O-4 M prostaghdins on cation* content of lensesincubated in low calcium medium

3.6 mg Ca2+% Control 1O-4 M PGE, PI.

lo-&

Control M PGF*,

nt

Sodium

Potassium

6

225hO.8 20.1*0.3 < 0.025

131.2kl.2 133.1f1.2 < 0.001

5

21.7hO.9 19?5*0.5 < 0.025

124.1&l+ 126.4+1.8 < 0.05

26.4& 1.2 22.1 kO.6 0.05

136.1*3.2 141.4*3.1 < 0.025

P

2 mg Caa+% Control lo-” M PGE,

6

P

* Mequiv./kg lens water; arithmetic t Number of paired lenses. $ Calculated by paired t test. TABLE

mean&standard

error

of the mean.

VI

Temperature reversal experiments

Control 0” for 20 hr 0” for 20 hr and 36” for 6 hr Mean y0 recovery Experimental I: 1OW 0” for 20 hr 0” for 20 hr and 36” for 6 hr Mean y0 recovery Experimental 0”+36”; 0”+36”;

n*

Sodium?

Potassium

11

35.2*3.8 20.4*0.9

111.9*3.0 128+x*1.0

88.1 M

93.0

PGE,

II Control lo-* M PGE, PS

5

35.9+2.1 19+*0.5

107.3&3.9 129.1*2.2

91.4

10

96.4

22.1 h1.3 19.6&0.6 < 0.01

* Number of paired lenses. t Mequiv./kg lens water; arithmetic mean*standard $ P value obtained by paired t test.

1266f2.4 129.6+2.5 < 0.05

error

of the mean.

PROSTAGLANDINS

AND

THE

LENS

775

induced cation shift, but an effect of this steroid after 24 hr incubation at 37°C is seen only at a concentration of 2 x 10m3M. It was therefore quite surprising to find that 10m4 M PGE, and Es appeared to significantly enhance recovery from the cold induced cation shift. Our results in summary, demonstrate that, at low3 M, prostaglandins are toxic to the lens in that they cause increased sodium and reduced potassium content. However, at 1O-4M, prostaglandins appear to stimulate, in someway, the mechanism responsible for accumulating potassium and extruding sodium. What mechanisms can be involved in these effects? Let us consider first the “beneficial” effect of lop4 M prostaglandins. Several studies on frog skin and toad bladder have demonstrated that PGE, can stimulate sodium transport. Maximal stimulation in toad bladder was obtained with 10e4M PGE, (Lipson, Hynie and Sharp, 1971). An excellent review on this subject is provided by Ramwell and Shaw (1970). The postulated mechanism for the stimulation of sodium transport across epithelial membranes is that PGE, displaces membrane bound calcium ions, favoring an influx of sodium thereby activating the adenyl cyclase system, which in turn stimulates production of cyclic-AMP (adenosine3’, 5’ monophosphate). Cyclic-AMP appears to be the mediator of many hormonal effects on membrane transport and permeability (see Robison, Butcher and Sutherland, 1971). Adenyl cyclase has been demonstrated in lens tissue, predominantly in the epithelium (Makman and Kern, 1971). It is possibletherefore, that, in the lens, prostaglandin at 10e4M stimulates the adenyl cyclase system, and that cyclic-AMP stimulates the cation transport mechanism. However, it is necessary to demonstrate whether (a) exogenous cyclic-AMP stimulates lens cation transport; and whether (h) lo-* M prostaglandins influences lens epithelial adenyl cyclase activity. It has been frequently demonstrated that increasing the external calcium level can inhibit adenyl cyclase activity (seeRamwell and Shaw, 1970). The smaller effect of 1O-4M PG on the cation balance of lensesincubated for 20 hr in a medium containing 9.1 mg Ca2+% as opposed to 36 or 2 mg Caz+% might therefore be due to partial inhibition of adenyl cyclase. However, stimulation of cation transport following a cold induced cation shift occurred in 9.1 mg Ca2+%media. Perhaps it is necessary that the lens cation balance be altered before an effect of 10e4M PG on cation balance becomesobvious. The mechanismof the deleterious effect of 10e3M prostaglandins on the lenspresents another question for which few answersare available. The experimental data presented in this paper suggestincreased membrane permeability and reduced pump activity. No inhibition by PGE, of ATPases prepared from erythrocytes (Fassinaand Contessa, 1967) or calf heart (Fassina, Carpendo and Santi, 1969) could be demonstrated. Therefore, the reduced rubidium-86 uptake might be due solely to increasedrubidium86 leakage from the lens. The effect of high concentrations of prostaglandins in reducing galactose uptake in rat lenses (Waitzman, Kuck and Woods, 1972) may possibly be explained in terms of increasedlens cell membrane permeability. Whether high concentrations ( 10e3M) of prostaglandins are capable of inhibiting lens Na-KATPase remains to be established. The possibility that prostaglandins at low3M are exerting a detergent effect should also be considered.However, in view of their similarity in structure, one would expect that if prostaglandins were exertiug a detergent action, 1O-3M PGF,, would be equally as effective as the other prostaglandins. This was not found to be the case.

776

C. A.

PATERSON

AND

B. A.

ECK

Perhaps the most logical explanation, at this time, for the action of 10~~ M prostaglandins on the lens is that like 10e4 M, they displace membrane calcium to such an extent that passive permeability is greatly increased and the balance between the cation transport mechanism and passive permeability is upset. We have demonstrated that prostaglandins have apparently quite opposite effects on the lens, depending on the concentration employed. At present, however, we have insufficient data to more than simply guess at the mechanisms involved. Whether the release of prostaglandins into the aqueous humor plays any role in the development of lens changes during chronic ocular inflammation is an open question. The level of prostaglandins attained in the aqueous humor during uveitis is in the order of 50 rig/ml (E a k ins, pers. comm.). The result of long term exposure of the lens to such levels of prostaglandins is not known. ACKNOWLEDGMENT

We wish to thank Dr John E. Pike of Upjohn Company, Kalamazoo, Michigan, for the continued supply of prostaglandins. REFERENCES Ambache, N., Brummer, H. C., Rose, J. G. and Whiting, J. M. C. (1966). J. Physiol. (London) 185, 77. Ambache, N., Kavanagh, L. and Whiting, J. M. C. (1965). J. Physiol. (London) 176,378. linggard, E. and Samuelsson, B. (1964). B&hem. Pharmacol. 13,281. Becker, B. and Cotlier, E. (1965). Invest. Ophthalmol. 4, 117. Beitch, B. R. and Eakins, K. E. (1969). Brit. J. Pharmacol. 37, 158. Duke-Elder, W. S. (1969). System of Ophthalmology, vol. XI. C. V. Mosby, St. Louis. Fassina, G. and Contessa, A. R. (1967). B&hem. Pharmad. 16, 1447. Fassina, G., Carpendo, F. and Santi, R. (1969). Life Sci. 8, 181. Harris, J. E. (1966). Trans. Amer. Ophthul. Sot. 64, 675. Horton, E. W. (1972). Prostaglandins, vol. 7. (Ed. Gross, F., Labharl, A., Mann, T., Samuels, L. T. and Zander, J.). Springer-Verlag, New York. Kaley, G. and Weiner, R. (1971). Ann. N.Y. Acad. Sci. 180, 338. Kinoshita, J. H., Merola, L. 0. and Tung, B. (1968). Exp. Eye Rea. 7, 80. Lambert, B. W. and Kinoshita, J. H. (1967). Invest. OphthuZwwZ. 6, 624. Lambert, B. W. (1968). Arch. OphthaZmoZ. 80, 230. Lipson, L., Hynie, S. and Sharp, G. (1971). Ann. N. Y. Acud. Sci., vol. 180, p. 261. (Ed. Ramwell, P. W. and Shaw, J. E.). N.Y. Acad. Sci., New York. Makman, M. H. and Kern, H. L. (1971). Fed. Proc. 30, 895. Merola, L. O., Kern, H. L. and Kinoshita, J. H. (1960). Arch. Ophthdmol. 63, 830. Paterson, C. A. (1970). Exp. Eye Res. 10, 151. Paterson, C. A. and Eck, B. (1971). Ophthalmic Re.s. 2, 246. Paterson, C. A. (1972). Dot. Ophthdnwl. 31, 1. Ramwell, P. and Shaw, J. E. (Eds.) (1971). Ann. N.Y. Ad. Sci., vol. 180. Ramwell, P. and Shaw, J. E. (1970). Rec. Progr. Horm. Res. 26, 139. Robison, G. A., Butcher, R. W. and Sutherland, E. W. (Eds.) (1971). Cyclic-AMP. Academic Press, New York. Waitzman, M. B., Bailey, W. It., Jr. and Kirby, C. G. (1967). Exp. Eye Res. 6, 130. Waitzman, M. B. and King, C. D. (1967). Amer. J. Physiol. 212, 329. Waitzman, M. B., Kuck, J. F. R., Jr. and Woods, W. D. (1972). Fed. Proc. 31,936.

Discussion

Dr

Iiinsey

I suspect that prostaglandins normally exert a significant physiological effect at concentrations somewherebetween one hundred thousandth to one millionth the dose

PROSTAGLANDINS

ANDTHE

LENS

777

used by Dr Paterson, from which I would draw the tentative conclusion that the lens is unusually insensitive to prostaglandins. I’d like to ask Dr Neufeld to comment on this since I know he has done extensive work in this field. Dr Neufeld Actually anything above 1O-6or 10e5M in prostaglandins is very unusual, Can you explain having used 1O-4? Dr Paterson. I certainly wish I had a precise explanation, but I don’t. Ramwell and Shaw [(1970). Rec. Progr. Horm. Res. 26, 1391reviewed experiments on sodium transport in frog skin using prostaglandins at concentrations in the region of 10e4and 10mKM. I think that part of the problem is that in lens one is looking for small changesin a large bulk of tissue. In the experiments on frog skin, stimulation of sodium transport by prostaglandins was detected by changes in short circuit current and this is certainly a more sensitive measure of alterations in transport. Dr Neufeld I think that you are just not working with that sensitive a change. Dr Paterson I think if one could work with an isolated lens capsule-epithelium preparation, one may be able to seesome of the effects at much lower concentrations of prostaglandins. Dr Neufeld O.K., let me just make one quick suggestion, possibly these are rabbit lenses?You might try pretreating your rabbits with aspirin and then using the lenses.That way you could increase your sensitivity. Dr Paterson, I’ve been using aspirin in conjunction with prostaglandins for someother purposes so I will follow up your suggestion. Dr Zadtcnaisky On that point of the sensitivity, the short-circuit current and the epithelial potential of isolated frog and rabbit corneas increaseswith prostaglandins at 10M6 and 10m5M. The lens is obviously lesssensitive. Dr Paterson This is certainly something that I would like to do, to look at changes in the electrical activity of the lens with prostaglandins. Dr Dikstein First of all I totally agree with Dr Kinsey that the pharmacological range of the prostaglandins is within 1OW and lo-'M, but as to the relative insensitivity of the lens we tried the prostaglandins on the endothelium of the cornea in the range of

778

C.A.PATERSOK

AND B.A.ECK

10e6and 1O-5M and couldn’t find any effect, so probably the cornea1endothelium is also insensitive to the prostaglandins. Dr Paterson I would just like to say that one talks about the high concentrations one useswith prostaglandins and perhaps we could draw somekind of corollary between the high concentrations one usesto induce changesin the lens with steroids and more recently with antibiotics. Dr Kaye Do you find any reversibility or attenuation in these effects with time or with removal of the prostaglandin or do you find any effects of the prostaglandin inhibitors or antagonists? Dr Paterson Well, the prostaglandin antagonists have been something of a headache; we started using polyphloretin phosphate, and we found that if one incubated the lens in a concentration of polyphloretin phosphate anything more than O*Olo/oit appeared to be toxic to the lens. We then tried SC 19220, but this is such an insoluble drug that we were just unable to get any useful concentrations at all. More recently I’ve been working with ‘i-oxa 13 prostynoic acid and this again appears to be somewhat toxic to the lens. So, you see, I have been having a few problems with the prostaglandin antagonists; they are antagonizing me.