An in vivo demonstration of the bicarbonate ion pump of rabbit corneal endothelium

Exp. Eye Res. (1979) 28, 699-707

An in vivo Demonstration of the Bicarbonate Ion Pump of Rabbit Cornea1Endothelium KENNETH R. MAYESAND

Twit

STUARTHODROE

ofElectron

Microscopy, Welsh Na,tio,d School of Me&&e, Heath Park, Carcliff CF4 &TN, U.K.

(Received 1.3 October 1978 a:& in revised form 12 December 1978, Lo&on,) The bicarbonate concentration in the stromal fluid of rabbit corneas in vivo is depressed by more than 50”, of its level in the aqueous humour. After allowing for ion-exchange effects, electrochemical potential effects, binding phenomena, compartmentalization and pH effects. it is concluded that most of the depression is caused by an active efflux of bicarbonate ions out of the stroma. Simple flux analysis suggests that the magnitude of the active bicarbonate efflux equals that previously measured to pass across the endothelium in t,he in vitro preparation. h’ej/ ~orrls: bicarbonate; ion pump; cornea1endothelilm.

1. Introduction Cornea1 stroma swells in aqueous solutions (Kinsey and Copan, 1942). In t,he living cornea, the hydration of the stroma is maintained constant by a transport process located in the endothelium which opposes the swelling tendency (Maurice, 19X3). The transport process has been claimed to be an electrogenic bicarbonate ion “pm11p” (Hodson and Miller, 1976). Hull, Green: Boyd and Wynn (1977) have confirmed the presence of a net bicarbonate flux across the endothelium into the lens-side and Mayes and Hodson (1978) have demonstrated that the active endothelial bicarbonate ion flux can be coupled to and, by inference, generate flows of water which are comparable in magnitude to those trans-endothelial fluid flows frequently observed in cornea,1 physiology (Davson, 1955; Mishima and Kudo, 1967; Dikstein and Maurice, 1972; Fischbarg and Lim, 1974). The present study concerns itself with measuring the concentration of bicarbonate in the stromal fluid. This measurement is considered to be a critical test of the Hodson and Miller (1976) hypothesis for the following reason. When a cornea is in its normal st#eatly state equilibrium, the flow of any (non-metabolized) molecule into the stroma must equa.1its flow out of the stroma. The hypothesis states that the endotheliuru is actively removing bicarbonate ions from the stroma.. If this actively elevated efflux is to be equalled in magnitude by a passive backflux into the stroma, t,hen the hackflux of bicarbonate must itself be elevated. This would occur if the concentration (or, more exactly, the electrochemical potential) of bicarbonate in the stromal fluid is lower t,han the concentration of bicarbonate in the aqueous humour. Such measurements of stromal bicarbonate concentration have the additional advantage that they may be made on freshly dissected corneas. Solutions of a simple flux equation will then indicate whether the endothelial bicarbonate “pump” operates in vivo, as well as in vitro where the activity may be measured more directly (Hodson and Miller. 1976; Hull et al., 1977; Mayes and Hodson. 1978). 0014~4%5/79/060699+09 $01.00/O F

0 1979 Academic Press Inc. (London) Limitetl

699

2. Materials and Methods Corneas were taken from 3%l-montli-olcl intravenous pentabarbitone.

Dutch

r:ll)bit,s which ha11 IWII

kill~.~l 11il h

Abbrehtions

Bicarbonate+glutathione Ringer consisted of XaCl, 111 nm; iYaHCO,, ST 111~1 : K(‘l. 5.15 mu: K,HPO,, 0.8 illM; JIgSO,, fW(i ml\I; CaCI,, 0.56 mn~; glucose, fG I~I\I ;Ltld reduced glutathione, 1 mu. Solutions were prepared on the day of use ar~l tnlhl)lecl to equilibrium pH 7.48 with a gas mixture of 88”;, X2, ‘is.0 0, ;rnd 5’);, CO,. I’PO is 2,5diphenyloxazole and POPOP is 1,4-di[2-(5-phen,vloxazolyl)]benzene. Ionic concentrations were determined by extracting the stromal fluid and then mea~uritrg the HCO, concentrations and, simultaneously, Cl- concentrations. In situ measuremel& were not attempted. The method of extracting the stromal fluid must be carefully chosen. Acid extraction? which gives accurate results for stromal Ir;a-I-, Kt and Cl- (Otori. 1967) is clearly unsuitable for HCO, evaluation, for most of the HCO, would be driven off as CO,. We chose aqueous extraction as our method and established that this method of estr;rction was efficient. Control experiments were conducted on cornea1 stroma denuded of epithelial and endothelial layers and clamped between pieces of steel mesh (thickness 0.7 trim arranged in a 1 mm square mesh) t,o control the hydration of the preparation.

The noll-cowpavtmentalization

of stromal

HCO, ad Cl-

Conventional efflux experiments (e.g. Ling, 1966) were carried out on de-epithelialized corneas equilibrated with radio-isotopes during maintenance under a specular microscope for 2-3 hr with perfusion of bicarbonate+glutathione Ringer solution including traces of HWO, and 36Clp. The dissected stroma was then carefully blotted to remove adherent fluid and then unloaded successively into 15 pots of 15 ml bicarbonate-glutathione Ringer over a period of 1 hr 10 min. A 1 ml sample from each pot was then pipetted into 9 ml-of scintillation fluid (5 g PPO plus (0.2 g POPOP in 1 1 toluene plus 500 ml Triton X-100). Activity in each pot was measured on a Packard tri-carb scintillation counter a~nl WITventional efflux curves were constructed. The resulting curves showed that 98:& of t*he HCO; and Cl- leaving the stroma is rron-corlipartnlerltalized (tl of efflux = 1.3 and 0.9 min respectively). There remained a small, slower efflux c,omponent? (t+ for HCO, and Cl- : z 14 and 6 min respectively) (Fig. 1). It is possible that this slow component represents the cellular compartment of the keratocytes of the stroma. These experiments show that all the stromal HCO, and Cl- is available for aqueous extraction.

The chemical measurement

of

HCO; ad C-

It is impractical to measure in vivo concentrations of stromal HCO; and Cl- using raciioisotope techniques, and so a chemical method of measurement was devised. The cornea1 preparation, denuded of epithelium and endothelium was blotted to remove adherent fluid, quickly weighed and then immersed in 150 ~1 of deionized water in a closed polypropylene, 1.6 ml capacity capsule. After 15 min of vigorous and regular shaking, when preliminary experiments showed that the concentrations of total CO, and Cl- in the aqueous extract had stabilized, 10 ~1 samples of the aqueous extract were analysetl on a Beckman Cl/CO, analyser. The (now swollen) stroma was removed from the extracting capsule and dried overnight at 105°C. Dry weight was determined the next day. The total water in the extracting capsule (= initial stromal water cont,ent, plus 150 ~1) was known. The initial total CO, and Cl- concentrations could then be calculated from the known concentrations in the 10 ~1 sample of aqueous extract, providing that the distribution of total CO, and Cl- between the stromal fluid and the aqueous extract was known. This

IN VIVO

ENDOTHELIAL

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PUMP

701

distribution was determined by experiments involving radio-isotopes. Denuded stroma was immersed in a loading solution of well-stirred Ringer (with either 37 mM-KaHCO, or 14 mM-NaHCO, buffered with Hepes) containing traces of NaF*CO, and Na3%1. After loading (40 min), blotting and weighing, the stroma was shaken in the usual manner in 1,50 ~1 of deionized water for 15 min. Triplicated samples (20 ~1) of the aqueous extract were each diluted to 1 ml with Ringer and counted in 9 ml scintillation fluid as previousl? described. This gave a measure of the 36C1- and H14COz; specific activities in the aqueous extract,. The stroma was blotted to remove adherent fluid (the loss of which was unimportant for the calculation of specific: activity) was weighed anI1 then immcrsetl for

2 E 2 z

2*5 7,$

‘& \I.\

-‘A

- y\,’ l \. \ B \ \,Ym .\. le5- \\lm 1. I \ \ ’ \, I.0- I ’ \ ’ I ’ \, o-5- I ’ ’ ‘1 ,\1 \\, 0 IO 20 30I L 40 50 2.0

Efflux time (mid

FIG. 1. Efflux curves for HYO; (0) and WY (m) from corneal stroma (loaded iu%inyer containing of traces NaHWO, and R’a36C1)into volumes of Ringer. Each set of points can be analysed to give the usual logarithmic-linear components. Here, each curve has two logarithmic-linear components. each of which is suggestive of one compartment. t+ of fast components: HCO, = 1.3 min; Cl- = O-9 min. tl of slon component: HCO, = 14 min; Cl- = 6 min. Logarithmic summation of the two component8 fits the dat,a points extremely well.

known periods in successive volumes (15 changes of 1.5 ml each, over a total period of 1 hl 10 min) of “cold” Ringer until, upon subsequent counting, the radio-activity in the Ringer volumes was not significantly above background levels. -4 1 1111sample was taken from each Ringer volume (1.5 ml), added to 9 ml scintillant and czounted as for the aqueous estrart. With 37 mM-NaHCO, in t#he loading Ringer solution, the HCO, level was 1.12 times (&2.4%, n = 2) greater in the stroma than in the water extract. With 14 mMKaHCO, in the loading Ringer solution, the HCO; levels were related by a factor of 1.16 (:*2.40,;, n = 2). With both Ringer solutions, the Cl- levels in the stroma and extract were related by a factor of 0.947 (f2”/& ra = 4). In the in vitro and in vivo experiments described below, the stromal total CO, levels were found to be low compared to the bathing level (cf. Table I) and the results were correrted by t,he factor found in the 14 mM-NaHCO,

‘ill”

K. IL NAYHS

-ASI)

s. 1IoT)SoS

Ringer experiments. In the experiments with t,he clamped tlenutlrtl stroma (c+. ‘1’;lblts I ) the total CO, level in the stronla wa,s fount1 to be similar to tlla,t in the b;rt,hillg ttletliutll and the correction fact#or from the 37 II~M-?;aHCO, Ringer tsperitnrtrt was asetl. ‘I’IIY errors on the values given in the results tables inclutle t,he errors ot, the wrrections. ‘1’111~ magnitude of the correction factors was sn~all compared to the tn;@t,u(le of t,he phvsiological phenomenon demonstrat,etl below.

Precuutions Other experiments confirmed that in the aqueous extract of t,he stromas there was tt(b “factor” which affected the accuracy of eit.her the Cl- or total CO2 measuretnents. Otle hundred and fifty ~1 of aqueous extract, taken in the usual manner from fresh tlenucle~l stroma, was analysed for total CO, and Cl- content. A solution of NaHCO, and XaCl W;LS made up having the same total CO, and Cl- content’ as the extract. The a&lition (Jf t tIv extract t,o an equal volume of the so&on did not change the measured total CO, at){1 Cl- content. Washing denuded st#roma for 1 hr in three changes of either deionized wat’er or irl (‘1 and total CO,-free Ringer (replaced by NO:;) with subsequent extracting in the usu;11 manner in 150 ~1 of deionized Jvater gave rise t)o a blank aqueous extract of tlw strotltii. Reroverv from sbromws equilibrated against high or low HCO, an(l Cl- Ringers estml~lisherl the lineiritv of the extraction t’echnique. We also ;nvest,igated the reliability of t.he Beckman U/CO, analyser. It proved to be linear over our range of interest.. For greatest, accuracy it was important to calihl*;lte the instrument immediately before a reading and then check its accuracy inmetli~~tely aft,et.. We found that the automatic acidification procedure, used to measure total CO,, mas not fully efficient in our buffered samples. Although a 37 m1\1 solution of NaHCO, whet1 sampled and analysed read 37 mM, the full Ringer, containing 37 nl~NaHC0,. ntensure(l on the Cl/CO, nnalyser gave a value of about 32 mnl. In each in vitro experiment nntl itt each clan~ped st,roma experiment (see below), a correction factor was found whilst makit+ up the Ringer. The mean of all the factors was 1.16 (il(?i). E x p eriments involving the me;~surement of total CO, in fresh aqueous humour before and after the addition of ktlo\v ti amounts of KaHCO,, both as solid and in solution, showetl that, the JRI~ correvtim f’xd or applied to itCpOUz: 1iutnOur. Measzrremerlt

of sfrowml

total CO, ad

Cb- rorbce7atratiom

i72 vim

Immediately after an animal was killed a hypodermic needle attached to a syringe v as inserted through the limbus into the anterior chamber. When the needle point was dire&l! below the eentre of the cornea, about 1UIspl of aqueous humour was collected. After removal from the eye, any air residue in the syringe barrel was expelled and the needle sealed with “plasticine”. The cornea was removed, cutting just inside the limbus of the eye, carefully blotted and immersed in silicone oil (Dow Corning silicone fluid DC 2WjlU cs) to prevent water loss. The epithelium was scraped off under oil with a scalpel blade and the endothelium was wiped off. The stroma was transferred to a balance after careful an(l rapid blotting (5 set) to remove the silicone oil and the wet weight (of the order of .iO mg) was measured to an accuracy of 0.1 mg. Total CO,, Cl- and hydration were then determined by the methods described above. The aqueous humour sample was also assayecl fur total CO and Cl- concentrations. Experiments established that the protein in the aqueous humour did not affect the determinations. Preliminary experiments showed that contact with the silicone oil did not affect the determinations of total CO, and Cl-. Oil was vigorously shaken with Ringer solution on a “whirlimixer” until small droplets had formed. After 5 min, when the layers had separated, the Ringer was aspirated into another vessel. Samples of this were analysed for Cl- and total CO, content. The results showed that no change had occurred as a consequence of intimate contact with the silicone oil.

IN Xeasurement

VIVO

of stromal

ENDOTHELIAL

BICARBOKATE

total CO, and Cl- concentrations

PUMP

TO3

in vitro

Corneas from eyes refrigerated in moist chambers for 1 to 24 hr were de-epithelialized by rotary brushing before excision from the intact globe. The de-epithelialized corneas plus sclerai rim were tied on to steel rings, mounted in a 35°C water jacket and examined with a specular microscope after the method of Dikstein and Maurice (1972). The endothelial surface was perfused with bicarbonate-glutathione Ringer at a ra,te of 1.8 ml. hrm1 and a hydrostatic pressure of 20 cm water applied to that surface. The bare stromal (deepithelialized) surface was covered with a layer of silicone oil in which t’he microscope obje&ve was immersed (Dikstein and Maurice, 1972). Aft’er the corneas had achieved and maintained a constant thickness up to SOY0above physiological levels (37(l-460 pm) after l--4 hr incubation, they were quickly removed from the specular microscope, blotted aIll immersed in silicone oil. The scleral rim was removed and t,he endothelium wiped off. Thf~ clenuderl stromas were transferred to a balance, after blotting to remove the oil: ancl weighed. Total COZ, Cl- and hvdration were determined as described above.

Corneas, de-epithelialized by rotary brushing, were excised from the intact globe, cutting ,just,inside the limbus. The endotheiium II-as wiped off and the denuded stroma clamped between two pieces of steel mesh (1 mm2 mesh aud ~7 mm thickness) to prevent swelliug beyond hydrations similar to those of Corneas in the in vitro experiments. The clamped denuded stroma was immersed in a vigorously-stirred 1OCJml volume of bicarbonnteglutathione Ringer in a closed vessel. Aft’er 1.5-2.5 hr, the denuded stroma was removed, blotted immediately and weighed. Total CO,, Cl- concentrations and hydration were cl~%erminedas described above. 3. Results The main results are given in Tables I and II. Aqueous humour values of total CO, and C- concentrations and stromal Cl- concentrations are in good agreement within a few percentage points of previously published estimations (Kinsey, 1951, 1953; Davson and Luck, 1956, 1957; Reddy and Kinsey, 1960; Otori, 1967). As far as we are aware there have been no previous measurements of stromal total CO, concentration. Proportions

of

HCO; and CO, in total CO, estimations

of stromal

jluid

It was possible to show by means of simple experiments that most of the stFoma1 total CO, was in the form of HCO, ions. Physically clamped denuded stromas were immersed in well-stirred Ringer in a closed vessel for 1.25 hr. Immediately upon removal from the Ringer, neutral red indicator (1% solution in saline) was dropped on to the strotnas. The st,romas took up a colour which corresponded to the upper end of the pH range 7-8 when compared to standard solutions of known pH. The colour of the stroma did not change, even upon subsequent exposure of the stromas to air for a period of hours. These experiments suggested, using the Henderson-Hasselbalch equation, that less than 4% of the total CO, was in the form of CO, and that greater than 96’$;, was in the form of HCO,. Loss of CO, through

the epithelial

surface

Lowered values of stromal total CO, concentrations could result from the loss of CO, to the atmosphere through the epithelial surface. Suppose there to be a mechanism which, by some unknown route, maintains the pH of t,he stromal fluid. Then.

itI4

Li. I?. hIAYES

AX0

S. HODSUS

if there were an ahundancc of carbonic anhydrase in the stroma. the cotltitluou< “blow off” of CO, could cause a rapid conversion of HCO; to (‘0,. If this w(‘tv tht case. low stromal total CO, would be clue to a through-flow of total (0, ilcrosd ttw preparation. This hypothesis was eliminated by experinlent. De-epithelializetl corneas were incubated under the specular microscope as described previously, with silicotlrt

Concentrutions of stromal total CO, in. passive (physically clamped derwded stroma) and iw “active” (in. viva and in ,vitro cor?beas)states Stromal

Experiment

Stromal fluid (mmol. 1-l)

Bathing solution (mmol. 1-l)

level

as Y,, bathing Ievt~l

Stromal hydral,ion (mg watt-r/ mg dry wt.)

--__ Clamped denuded stroma Equilibrated in vitro in viva

8444&0.9

:37.6&0.1*

92.6$2%

592.+0~24

17.2h1.2 18,2*1.2

38.0&0.1* 39.9’1.01

4x3&3.3 456&,:+7

518&0~“3 :1m$~o42

71= 4 for each experiment. Values are given *S.L * Bathing solution is bicarbonate-glutathionc Ringer. t Bathing solution is aqueous humour. TABLE

II

Concentrations of stromal chloride in passive (physically clamped denuded stroma) and in “active” (in viva and in vitro corneas) states

Experiment

Clamped denuded stroma Equilibratetl in vitro in viva

Stromal level as so bathing level

Strornal hydration (mg water/ m,o dry wt)

Stromal fluid (mEy .1-l)

Bathing solution (mEq. 1-l)

105*:3

118&l*

894*23

.592&0~24

11613 109&i

118&l* 108+2t

98.4+:+1 100~5*:Kt

518+0~2:3 3.23 &O.oy

1~= 4 for each experiment. Values are given *S.L * Bathing solut.ion is bicarbonate-glutathione Ringer. t Bathing solution is aqueous humour.

oil on the anterior (stromal) surface and Ringer solution perfusing the posterior (endothelial) surface. After 197 min, the oil covering one of the pair of corneas was bubbled vigorously for 19 min with a mixture of 88y,l N,, 70/, 0, and 5% CO,? identical to that with which the Ringer bathing the endothelium had been equilibrated. Any net, passive flows of gas across that preparation were thereby eliminated. The other cornea of the pair acted as a control. Anterior gassing had no effect on cornea1 hydration (Fig. 2). The corneas were removed from the specular microscope and

IN

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I 150 Time

705

PVMP

1 200

2 50

(min)

J?IG. 2. Thickness changes of a pair of de-epithelialized corneas mounted under the specular microscope with silicone oil on the anterior (stromal) surface and with Ringer (equilibrated with a gas mixture containing 5yb COz) bathing the posterior (endothelial) surface. After 197 min (arrow) the oil corering one of the corneas (0) is gassed with the same gas mixture containing 5 9;, CO,. Xo new trend it1 cornea1 thiehness was noted as a result.

stromnl levels of total CO, and Cl- were determined 1)~the methotl described above. The levels of total CO, found were not notably different in the gassed and non-gassed preparations (49.7 &l.99/, and 47*5&1.9O/, of the level in the bathing Ringer solut’ion respectively, ‘?! = 2). 4. Discussion When clamped stroma is bathed in an aqueous medium. then at equilibrium the anionic concentration in the stromal fluid is lower than that in the bathing medium (TaIAes I and II). This would be expected from the model of cornea1 swelling proposed by Hodson (1971) which predicts that at this cornea1 hydration (5*92), the Donnan potential would cause a non-selective depression of the anionic stromal concentration to 90yo of its value in the bathing medium. This compares favourably with the measured values of 92*6+2*6o/o for HCO; and 89.4&2*50/o for Cl- (Tables I and II). When the endothelial “pump” is used to maintain stromal hydration (instead of physical clamping methods) there is a considerable alteration in measured stromal anionic concentrations. HCO, is reduced by 50’3/, of levels in clamped denuded stroma. (Table I) whilst Cl- 1evels are in fact slightly (but significantly, P = 0.01) increased over levels in clamped denuded stroma (Table II? in vitro). We can think of only two explanations for this phenomenon. The first, that there is a continuous loss of CO, through the epithelial surface, we disproved by demonstrating that removing any possible CO, concentration difference across the cornea by anterior gassing had no effect on stromal level of total CO, or on cornea1 hydration. The explanation which we propose is that HCO, concentrations in the stroma of living rabbit, cornea are reduced mainly because of the presence of a mechanism located ill the endothelium which is continuously “pumping” HCO, ions out of the stroma. d straightforward analysis of the HCO; concentrations of the stroma and aqueous humour in vivo (bottom line, Table I) supports our interpretation. The depressed level of HCO, in the stroma in vitro and in vivo must represent a steady-state where ti back-flux of HCO, across the endothelium into the stroma “balances” HCO, remrr\,c,tl

706

Ii. H. AI AYES

XNU

S. HOUSOS

from the stroma hy the enclot~helial “~JUIU~J”. It, is pwsiMe t0 calculate t,ll(s sizcl 4jl’ 111~~ back-flux of HC’O, (and, since they are eclual. the magnitude of the “l~ut~~l~‘~)../. from the equation :

J =PAc

(11

where P = the permeability of the entlothelium to HCO:; (5.5 x 1Om2 cm. hr. I : Hodson and Miller, 1976). AC = the concentration difference of HC’O, across the endothelium (taken front Table I, in vivo, and adjusted for t,he Donnan effect; Hodson. 1971; = 15.5 x 10 6 Eq. en-3). The value calculated from this equation for passive back-flux is 1.16 x 10 Ii Eq en?. hrpl. We propose that this back-flux “balances” the active HC’O, flux out of the stroma across the endothelium. The magnitude of the total active HCO, e&s in vitro can be calculated from Hodson and Miller (1976) to be 1.01 x 1OP Eq. c111-~.hr.-l. The agreement between the values is very good. The evidence for the existence of the endothelial HCO, “pump” came mainly from the quantitative equality of the net trans-endothelial HCO, flux and the short circuit current (Hodson and Miller, 1976). The trans-endothelial HCO, and fluid fluxes have been shown to be coupled by a “local osmotic” mechanism in partially destromalized in vitro preparations (Mayes and Hodson, 1978). The present work demonstrates that the endothelial HCO, “pump” is operating both in vivo and in vitro to reduce the stromal HCO, level. We no longer hold the existence of the endothelial HCO, “pump” in doubt, although a detailed model of its action has still t’o be formulated. ACKNOWLEDGMENTS

This work was supported by the Wellcome Trust and by an equipment grant from the Royal Society. REFERENCES

Davson, H. (1955). The hydration of the cornea. Biochwm. J. 59,24-S. Davson, H. and Luck, C. P. (1956). A comparative study of the total carbon dioxide in the ocular fluids, cerebrospinal fluids and plasma of some mammalian species.J. Physiol. (London) 132, 454-64. Davson, H. and Luck, C. P. (1957). The effect of acetazoleamide on the chemical composition of the aqueous humour and cerebrospinal fluid of some mammalian species and on t,he rate of turnover of 24Na in these fluids. J. Physiol. (London) 137,279-93. Dikstein, S. and Maurice, D. M. (1972). The metabolic basis to the fluid pump in the cornea. J. Physiol. (London) 221, 2941. Fischbsrg, J. and Lim, J. J. (1974). Role of cations, anions and carbonic anhydrase in fluid transport across rabbit cornea1 endothelium. J. Physiol. (London) 241, 647-75. Hodson, S. (1971). Why the cornea swells. J. Theor. Biol. 33,419-27. Hodson, 8. and Miller, F. (1976). The bicarbonate ion pump in the endothelium which regulates the hydration of rabbit cornea. J. Physiol. (London) 263,563-77. bicarbonate Hull, D. S., Green, K., Boyd, M. and Wynn, H. R. (1977). Corneal endothelium transport and the effect of carbonic anhydrase inhibitors on endothelial permeability and fluxes and cornea1 thickness. Invest. Ophthalmol. 16, 883-92. Kinsey, V. E. (1951). The chemical composition and the osmotic pressure of the aqueous humour and plasma of the rabbit. J. Gen. Physiol. 34,389402. Kinsey, V. E. (1953). Comparative chemistry of aqueous humour in posterior and anterior chambers of rabbit eye. Arch. Ophthdnd. 50, 401-17. Kinsey, V. E. and Cogan, D. G. (1942). The cornea III. Hydration properties of excised cornea1 pieces. Arch. Ophthalmol. 28,272-84.

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Ling, G. N. (1966). Cell membrane and cell permeability. Ann. N. Y. Ad. Sci. 137, 837-59. Maurice, D. iPI- (1972). The location of the fluid pump in the cornea. J. Physid. (London) 221,43-54. iilayes, K. R. and Hodson, S. (1978). Local osmotic coupling to the active trans-endothelial bicarbonate flux in the rabbit cornea. Biochim. Biophys. Acta 514, 286-93. Mishima, S. and Kudo, T. (1967). In vitro incubation of rabbit cornea. Inmst. Ophthdmol. 6, 329-39. Otori. T. (1967). Electrolyte content of the rabbit cornea1 stroma. Exp. Eye Kes. 6, 356-67. Reddy, D. V. N. and Kinsey, V. E. (1960). Composition of the vitreous humour in relation to that of plasma and aqueous humour. Arch. Ophthulmol. 63, 715-20.