Exptl Eye Res. (1967) 6, 79-92 .

S o l u t e M o v e m e n t a c r o s s the C o n s t i t u e n t M e m b r a n e s o f the C o r n e a *

KEITH GREEN

Ophthalmological Research Unit: The IV. K. Kellogg Foundation Laboratories, Wilmer Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland,

U.S.A.

(Received 14 March 1,966, a~td in revised form 1 October 1966)

A comparison has been made between the transcorneal, transepithelial, and transendo- thelial potentfals measured in normal Krebs-bicarbonate Ringer's solution. The trans- epithelial potential was approximately 67O//o of the total trarmcorneal potential, the differ. .ence being due to the greater anion shunt across the epithelium. The potential and short- circuit current of the isolated epithelium had the same characteristics as those of the whole cornea when bathed in normal Ringer's solution, sulfate Ringer's solution, and choline Ringer's solution. The transepithelial and transcorneal short.circuit currents were very similar, indicating tha t the transport system is located in the epithelium.

The unidirectional fluxes of sodium across the isolated epithelium and endothel ium were determined when the tissues were bathed in Krebs-bicarbonate Ringer's solution. The net flux of sodium across the epithelium and across the whole cornea were of the same order o f ms4~nitudo. The movement of sodium and chloride across the endothelium was equal in both direotions and was of a purely passive nature. The absence of any potential when the endo- thelium was bathed in media of different compositions emphasizes the absence of any transport process in the endothelium.

The results, therefore, show that all the transport characteristics of the cornea with regard to sodium and presumably to chloride are unquest ionably located in the epithelium, with no active ion transport characteristics in the endothelium.

1. I n t r o d u c t i o n

A c o r n e a l p o t e n t i a l n o r m a l l y ex i s t s , w i t h t h e e n d o t h e l i a l s u r f a c e p o s i t i v e w i t h r e s p e c t to t h e e p i t h e l i a l su r f ace . S e v e r a l w o r k e r s h a v e f o u n d , b o t h in r i v e a n d in v i t r o , t h a t d a m a g e t o t h e c o r n e a l e p i t h e l i u m c a u s e d a n a l m o s t c o m p l e t e d e p l e t i o n o f t h e t r a n s - c o r n e a l p o t e n t i a l ( F r i e d m a n a n d K u p f e r , 1960; P o t t s a n d Modre l l , 1957; D o n n , M a u r i c e a n d Mills, 1959). D a m a g e to t h e e n d o t h e l i u m , on t h e o t h e r h a n d , c a u s e d l i t t l e o r no c h a n g e in t h e t o t a l c o r n e a l p o t e n t i a l . T h i s a p p a r e n t l o c a l i z a t i o n o f t h e s o u r c e o f t h e t r a n s c o r n e a l p o t e n t i a l h a s n o t b e e n i n v e s t i g a t e d in a n y d e t a i l , p o s s i b l y b e c a u s e o f t h e a b s e n c e o f a n y o b v i o u s c o n n e c t i o n b e t w e e n t h i s p o t e n t i a l a n d t h e m a i n t e n a n c e o f a c o n s t a n t c o r n e a l t h i c k n e s s .

B a s e d o n t h e a s s u m p t i o n t h a t c o r n e a l t h i c k n e s s is c o n t r o l l e d b y a n a c t i v e s y s t e m - - a n d t h e r e a r e m a n y f i n d i n g s in t h e l i t e r a t u r e t h a t s u p p o r t t h i s c o n c e p t ( M a u r i c e , 1 9 6 2 ) - - p r o p o s i t i o n s w e r e m a d e t h a t t h i s a c t i v e s y s t e m w o u l d r e q u i r e a n a c t i v e m o v e m e n t o f f lu id o u t o f t h e s t r o m a i n t o e i t h e r t h e t e a r f i lm or t h e a q u e o u s h u m o r . T h e f ind ings s u m m a r i z e d a b o v e , h o w e v e r , s u g g e s t t h a t t h e r e is a n a c t i v e t r a n s f e r o f ions ac ross t h e comma. T h i s s y s t e m w a s s h o w n to be s o d i u m d e p e n d e n t (Modre l l a n d P o t t s , 1959), a n d t h e r e s u l t s o f e x p e r i m e n t s in w h i c h t h e e p i t h e l i u m w a s d a m a g e d i n d i c a t e d t h a t t h e p u m p w a s l o c a t e d in t h e e p i t h e l i l l m ( P o t t s a n d Modre l l , 1957;

* Reported in part at the Fall meeting of the American Physiological Society. Los Angeles, California, August 1965 (T~ PhysloloFist (1965) 8, 180). This work was aided by a PHS rematch grant (NB 04854) from the National Institute of NeurologieM Diseases and Blindness, U.S. P~blie Health S~rvice.

79

l ) l l l l i l I,t. i l l , , 1969). The Jl l i i l l l l wn~l n.bao ~t iowi l l ie lu l l . i ly Io lr i l l i ,~| lorl, ,~odillnl f ro l l i the t i,ili, I |hn ilitt.: I.li~, l l l i l ie l l l i ta l i l l l i lOi" (1) l lnn t,t. a l , , lt, il'Itt), l 'he eor i l l , i i l tDtiisilorl, ~y~tl~,ln htila liot, l l l ' tqi Ihl , t~illljl,cl, i l l ' liilil~o doi l i ih,I i .,il udle:~ i'l'lgiti~iiilt~, I lip aii o l l i ld l i l l l . i l i 'e o1" I ho triltll~|li~i'l ~yat.olll. l l i '0~l l l~ l i l l l ly l lei, i l l im, ~:1" the t l l lpnl 'o l l l t ' l l ihlr~ ot" ihe i'e~lllt.~ to l tr l lvidt~ l in ~×| l l i i i lu t io i l fll i, 1 lit, lt¢,f.ivo eol l l l ,o l li|" e.ol'ilenJ t hiekile~,~. The l l lek o f hl l0r l ,sl . hi t l i -m) i I~fi ' ;~, ' lr t~l l~l lorl, lvlatOlill~, llnl~ I~,aulll,d in il ~ i l l l l i lh ' l t l l l g i lp i l l tlil l" knowh, d~o o f lh~, t'li!il~.lit.lii|illll tit' t l l l,~l, ~)-l~l~llia i l i id l .heir ltO.~,~ibh • i~,Jnt io l ish ip t.o ihe l i l i ih l tonal l t ' t , lit" I?lll~lll'flJ | Jtiekni,.*m.-

T lu , ~tlih~l Jli~lilllll i lblo hns heon pnlp l isod tls the ai le of lhe Inelaht~l ic lnUnl l for an itel.ive eoilt~-itl i l l ' eoriil,i~| l.|iiekll~.~,a ( l l i l r i - i~ , ID157). Mi t l i r i ve ( ]967) , however , has l~ , vb , l~d t.h,, l~ll~, i~t" t lw Oll i lht, l i un l i l l l Jlo eontix~| o f i,(ll, i lel l l i.hi<,kl~ess and conehided t h l t t t Jii" I t lh , o f l h i ~ l l l i ) l l lh l ' l t l l~ ' ilt l i l l l l ! i . i~l lOl lm N o n e o f t h e ex l te l ' i l l l e l l t .~ report ed l i h o v e t h a i .~hfl'~'i,d l t l l nhl l , lwr ~ o f p~Hl, l l t i t l l in l ile ~,ndolhe l iu lu . h~w,, \ 'er, has de le rn l i ned tt-tl~,l ll~,l' l~r i lof l Itt,l'~, is :i eOul~Jt,d sod i l lm i~,lld ~-hlol i~h, pun lp in t he endo the l i nn l . Such

~v,~totll i~ ~ i m . i l i t he (i~h l ind l ~ l l h l i .t~lll bht ,hh, r ( l ) i an lo l l d , 1962, l~.~g.l ! and in l l le u lnr in l , t e | eo~t i'lal.e,~liile ( l lou,~e iliad (lri>t,.ll, i~,~qh), ll e~mph, d so. l iu lu qnd ehh~ride l l un l p ~1,~ i 'ostllt~ in th,, rlbs-eile~, of it molt.~ural)h- po l e l l l i l i l hi I tie tlreseuee o f a vo rv .~iitllifielil~ t, t . ln~l t l l~r t td' J~lth sodiu Il l l l l ld ehl~vdde, 'l'll~, a b~t,.l l¢t, TM ~1~ ~t. measl,.ra I~h, t~ll ~,n t ia i, t hel'ot~n,~ d~.'.~ u~l " l le~ms;t l ' i lv i n q d y the al!lenl~c, , i f : luv l rat lsl loYl p l l l l l ps i l l "1 ~,iven lltlt~lll lhyittllt),

T|l~" ~al l i d i~N! l~0t~?lrt a t h~' i~ ~x~t'h' 11 i ldol ' t4t k'ol l 1~I do | t i l l " i~./Ol~O pia,<-isely l.tle lllt~ix,(',lll~,li l of i~I118 n~-.l~x~a Ik~l.h t h~,' opi l .htqi t l t l t a uit lh~, e l ldof | l~ lh l l~ . Tt~ achie\-o th is a i l l l . I t ie a ul hor

A~.hdt nihin,~ rabbil~ xx-eitzhi~g 2-~ k,~ ~ r ~ k-i11~t x~:ith an intrax-enous injc~'tio:~ ,af s ~ i m n pentobsrl)~t~l (N~mh~tt~t} ,~imil~ist~.~t vi~ 1he roz ,x~rina] x-~in ,af an eat-. An eve w~s p~pt~)s~t ~.~ad ht~|d ~ i~d ly in th is p~sil.i~n. .h~ e~rt,~il~ ex~rir~enl,~. Oae epitht:li~n',~ x~n~.- ~ o x ~ w ~ at t.hi.~ si~x" hv ~',~.~hir~t 1.!he e,~,~nea q~tiekly wil.h the oxt~vi~n,ni-~al 2..,~er 's -.,~t~t~n a t .,~A~'X" a~d bx- ,~e~*a.~i~g - f f (,he :epit.heliura xvil)a a sl:raigl:t-~.-~wt ~-~:~ tpe.t h)ade. T,~ ~n.,aa~e t.h~t ~il the ¢.pi~a~.li~rn ~x~s n, rn,~x-~l.. ~\~t . ofl.he s t . r~ :~ was a l,-'~a eare611ty de t ached . ~ . x ~ i ~ t:h~. ~dc~hetiaa~a~ , ~ ] app~x~xi~at~ly l t ~ u of st.rx~na. The r~id~:gl s l a~na

..~dX~t~tm~o~s tie pex~. ~ l~a~ epit~l~elial ~ m o v a l I~.for~ < : o ~ a l e.we-i,*.~on, as l.h~. in~.raocmlar

• qOI,Uq'E 'I'RANSFEIt A(.'ROSS CORNEAl, ,ME~IBRANES 81

rmuoval of the cadet,helium, strolna] tissue was removed from the epithelium until the romainiug strom..l was deemed suflleient to provide mechanical support for the epithelium.

After separati(m of the cellular ]avers by either of the two proce~lure~s described, or a t i e r t h e r e m o v , d of t he who le c o r n e a , t h e t i ssue w a s m o u n t e d in a m o d i f i e d U . ~ i n g - Z e r a h n c h a m b e r (U,.~ing a n d Zer,fl~n, 1951) d e s i g n e d to ho ld t h e c o n v e x t i s sue ( G r e e n , 1965a; b'ig. 1). l , i lmra l a m o u n t s o f s i l icone ~,weas,.~ were a p p l i e d to t h e c h a m b e r f aces to o b t a i n , -omph , l e s ea l i ng at t h e ed.m, o f t h e p rcparu t . ion w i t h a m i n i m u m o f a p p l i e d pre.~sure. t,re-~s~ w,~s a p p l i e d to t he s u r f a c e s to p r e v e n t shor t -< , i rcu i t ing a r o u n d t h e e d g e o f t h e p r e p a r a t i o n whi le u s ing t h e m i n i m u m of a p p l i e d p r e s s u r e to t h e c h a m b e r s to c l a m p t h e c n r n e a . O n l y i n f r e q u e n t l y was v rease o b s e r v e d to ent~'r t h e c h a m b e r , a n d in t h e s e cases I he p r e p a r a t i , m w a s d i s c a r d e d . T h e v a l i d i t y o f t h e p o t e n t i a l m e a m t r e m e n t s m a d e in t h e c h : m t b e r was c h e c k e d us ing u n c h ~ m p e d c o r n e a s or p a r t s o f eonaeas . The.~e were p l a c e d on lhe Convoy c u r v a t u r e ,m ~:l~e o f t i le c h a m b e r s , w h i c h was f a c i n g u p w a r d s , t h e s u r f a c e o f w h i c h was well g reased : p m , , n t i a l s wer~, m e a s u r e d b y p l a c i n g t h e t ips o f t h e a g a r b r i d g e s in t h e s u r f a c e f i lms ,,f fluid a d h e r i n g to ~he t w o s ides of t h e *,.issue. No d i f f e r ence was ob- : o r v o d belx~-oo~ 1}le mea .~uren ;en r s m a d e u n d e r t i le t w o c o n d i t i o n s . V(heri an 'c pre=<~ttre was app l i ed t,, lh, , corne: l in th, , unK',re;-~sed c h a m b e r , s o m e d a r a a g e was d e t e c t e d irJ t h e e p i t h e l i u m by u s i n e ~.he d y e t luoresce in . Dama',.~e to t h e e n d o t h e l i u m was m o r e eas i ly

• two,.:gh~ a b o u t I,v t h e a p p l i c a t i o n ,.~f p ressure , a n d th i s d a m a g e b e c a m e vis ible in ~he f o r m t,f loe:~l ,,dem,,i of tlu. c o r n e a . I ) u r i n o _ ~he whMe p r o c e d u r e , we were carefxti n o t t~, aBow l h e c o r n e a Io bee,m~e w r i n k l e d , fo lded , or in a u v w a y p h y s i c a l J y da..umged, a l t h o u g h d u r i ~ c :'om:,x"at of l he s ~ m a frc,m I he i so l a t ed c o r n e a it was imposs ib l e t o a c h i e v e a , 'mnph , re a b s e n c e ,,f wr'~zk]ing: i~ was . h o w e v e r , k e p t a t a m i m m u m . A n y d a m a g e d come-as we.,x- discarded.

In c e r t a i n oxl~',ril~ael~l,s, t h e intztcl e¢~rP.ea wa~ moura red in t h e c h a m b e r a n d a lJowed t~ a c h i e v e e q u i h h r i u m for ~¢.'~ ~o 40na in . A f t e r ~.his t i m e . wh i l e t h e c~rne~ was s~.JJ] in t h e c h a m b e r , e i t h e r ';.he epi the . l ium or endo~,hel ium was expe_-dnaenladly d a m a g e d b y s c r a p i n g

: i t h :~ p:e,'," of si lver o r p l a ; i n u m u / r e . A h . h o u g h des-t,ruct.ion of t h e e~..]Ll~tar m e m b r a n e s , 'ouht no~ be assessed quan~a~at~vel.',', iI <'ou/d r e a d i ] v be dete~cxed, a s t h e c o r n e a b e c a m e , ' , ,mdv a~.d lbie-k u n d e r ~he d,,m.%e~d a rea . ] n *he e:xT.~riment~ u~iJ i r jng s u l f a t e or cho l i ne R i n g e r ' s s cdu~on as ]he ~,n~t~in~ "medium. ~.he eve was w a s h e d w i t h t,hese m e d i a for 3-.2 rnir~ b~fore ~he c o r n e a was r e m o v e d . Th i s ~echnJque gax'e m o r e r e p r o d u c i b l e r e s e t s ~ h a n we_re c,b~,nined w h e n lb.e eornea~ o.* corr, eal m e m b r a n e w a s g , ] ~ c l direct.Iv Jnxx~ ghe uppasa~us . c , m ~ i r S m g lb.e exI~rime:-~.a] mtwl ium.

h.#ler " ~he ~i.~sue was n-.ounred, r.he chambers were q-n_ickly filled wi%h t~e experimenvafl lq.i~'~w's scdm~on , l '~:e scQul~.o,.as w e r e .--'tfi.-,":r(~l a n d b u b b l e d w~Y3a a i r . ~--Vr~,-riag in e a c h ,-.h~mbe-r u-:~s pe.rform¢,.i bx a Tefion-e~a~ect m a g n e m e s-~,drrer. I rarned ax -$(~ roy!rob_) b y a borsesh,-~- m s n c e ~ , t.be ma~O~eT.¢ w e r e eorm.~T.ed to s h i e l d e d m o t o r s b y ~-in. , a l m . r a r o d s rha'~ be}d t j ' e m ch~e ~.o t h e l .uai~e e h a m b e x s .

T h e ec, rc, pos~.~.ic,n o f t h e ¢.xT~e.rimma*.~ .~<,}ntJon~. ba_,e~d on :K_vebs-bJ_e~a~-borm~e I ~ - ~ r ' s ~otu~.icm. h a v e been d~..~sx:r.q'~d T~rexqcms}y (Green . 1965): nil sa2d~e so lu l Jons we_~e a~. p H 7-4_ T h e R i n g e r ' s ~l~w:c, ns we.--e a~sed for ;ac,~ m<,re r2ama 5 dax-s a f -~r ~ret~artt~ion (e~oept. fo r c h o l i n e R incer : s .~',}urion. w h i c h w,~s -ased for. e m i r 2. d a y s aft~-~r p re~pa r~ ion ) : a tmffmient. v o l u m e ,~f sali~.e solm' ion for e a c h e .n~er imenl was b r o u g h t r,o "the re.qrm'e~ ~_mpea~xa~re tvnn~e.q.iar~.Iv / , r io t v-o. t h e ex-~er lmenra] pe r iod . _&If e ~ e n ~ _ a ~ w e r e -g~a'~o_rmed ~'t :F.~°C.

T h e pore,'~raM c~iffe.~e:t-e ~.nd sho~-cireujz. (--.v_r're.n~ "~zmea~.J;o.~'ats ~ere rngde via ~he • ~ma.u'~ xa~i~d, p,~ra.<mu-m ehlo=~ide-agar brid~es.. ~nd c~a]cmad e.lex-yarodes fft*r p o t ~ M differ- e~ee) a,r~5 paire~..'potz.~.;iurn e h l o r i d e - a g a r b.,"i.-~e~ a n d sa3~ex-~qver eb3emide elec~rcml~" f for sho~-c.ireu~:* cr~z-em.}. T h e "b.-dcl~es ~0ere ha.braced , ~ i o r r,o ~ e . trod bot3a t~be-ealome3 e)ee~,rode a n d l~ridge j n n c z i o n poT~.nwiats we..-~e me~- -m~d (azad e o r r e e X d J'o~, J:f ~ y ~.rke~d~) p r i o r ~.o rnem.-,mring ;.he "~,m:~nexnbrazae p ~ t ~ n ~ _ P o ' t ~ w~e~re ~m~a~_~a~r~ ~t l O - m i ~ mt~.7-x-als o v e r ~.t t e2m 2 hr . alt~c~ug_ h ~.bev ~ e r e e x m ~ y o h t ~ r r e d d u ~ g ~he ~ - r m e ~ a t $ ~ i o d so the : a n y x-aria~o~t~ i n ~he ~ ~ 1 ~ z t e ~ e ~ t t l d be_ not.,,ex]..

82 K. G R E E N

The potent ial difference was recorded on a high impedance null-point mii l ivol tmeter (pH Meter 4, Radiometer , Copenhagen, Denmark) . The potent ia l difference was measured to :J~0"2 mV and the short-circuit current to :~:0"5 f~A using a D.C. microammeter (Mode[ 741--60; Weston l tmtrumenta, Newark, N.J., U.S.A.), The resistance of the membrane. was determined f r o m the change in potent ia l resul t ing from a sudden exper imenta l change in the current runuing through the membrane; current changes of small magni tude were used because large sudden changes do not result in s teady potentials. By means of a potential divider circuit (see Ussing and Windhager , 1964), shown in Fig. 1, it. was possible

B I

t

0" 0

FJtcl. I . I)iggram of stpparatua used for the measurement of potential difference, short-circuit current, and reaist~nce of i s o l a t ~ rabbit corneal membranes.

a,~', War-saturated KC1 bridges; b,h', calomel electrodes; ~, cornea; d,d', si[ver-si.lver chloride elec- trod e~ ]~,B', batteries; L~L', Lucite chambers; M, high impedance millivoltmeter; O, osoitlosoope; S, t~epWitm resistance consisting of c a l i ~ ~ c e ~ for i u ~ i n g or d~t~asing the current through th~ ~ ttA, microal0ameimr; V,V', wariab|e resi~xtces.

t~ change the cur rent in five equal ly large steps sufficient to reduce the potent ia l to zero. Because of the small current, obta ined from the cornea, resistance calculat ions were often made only from the change in potent ia l resul t ing from the to ta l change in the applied ~ r r e n t , Dur /ng the resistance measure~ments, the potent ia l bridge~ of the chamber were connected to the i npu t of an oscilloscope (Type 502; T e ~ r o n i x Inc., Beaverton, Ore.) to record the signal. The sign convent ion in this paper for the definit ion of potent ia l differ~ enoes is t h a t the potent ia l of the serosal medium (endothel ial surface) has a lways been measured wi th the mucosal medium (epi thel ia l surface) as reference.

Unidirect /onal ~uxes of sodium were. de termined using Ringer ' s solution conta ining ~ N a (~pecifio ac t iv i ty , 1.2 t~o/ml) on ei ther the mucosal or sexosal side. 8s taples (25 ~xl) were t aken wi th a mieropipet te , in i t ia l ly from both ba th ing solutions bu t thereaf ter only .from the nonlabeled solution a t 1(~ or 20-rain intervals. Immedia te ly af ter removal from t'he-.~'aml)~r, t he sample~ were a ~ a y ~ l in a wel l , type scint i l la t ion counter (Baird Atomic Model ~810c ~ e d {n conjunct ion wi th Scaler T imer Model 135; Baird Atomic, Cambridge, Massachusetts , U.S.A.). A similar procedure was followed for tact flux de terminat ions ~ the endO~helin~, excep~ that the samples were as~yed using an end window tube.

S O L U T E T R A N S F E R A C R O S S C O R N E A L M E . M B R A N E S 83

Values of J , ~ (scrosal-to-mucosal flux) and J~n (mucosal-to-serosal flux) for sodium were calculated from the familiar two-compar tment analysis equations t h a t have been given by Ussing (1949). G iven a solution conta in ing radioact ive ions ba th ing the serosal side of a membrane in which the total concentrat ion (/~eq cm -s) of the ion species under s tudy is C. and the serosal concentrat ion of labeled ions is C* (counting rate, cm-a), then the rate of en t ry into the mucosal bathing solution of labeled ions is

A . c,* dt v . Cs

where A is the surface area (cm 2) across which ion exchange takes place, v is the volume of the mucos~ll solution (cm"), C* is the nmcosal concentrat ion of labeled ions, and J , ~ is the

• m

serosal-to-mucosal flux (#eq cm ~ uni t time) of the ion. In all the isotope measurements , identical solutions bathed both sides of the tissue, i.e. C~ -~ C,. The mucosal-to-serosal itux of the ion, J,,,.~. may be s imilar ly calculated by subst i tu t ing the appropriate, values in the equation. In all isotope flux determinat ions, the membrane was ma in t a ined in a constant ly short-circuited state to nullify the electrical gradient t h a t normally.would, be created by the potent ia l difference. As it.. was found tha t no potent ia l difference existed ~cross the endothel ium, no short-circuited current was necessary. Corrections were applied for the reduction in volume of the side from which samples were removed for counting.

Although biological membranes usually reach equil ibrium ~i~h any applied radio- isotope during the first 20-30 rain after application, the invest igat ion repor ted here was concerned not only with the position of the t ranspor t pump in the cornea bu t also with the changes t h a t t~ke place foUowing corneal excision. Becamse of thei r d~sign, therefore, the experiments necessitated the measuring of all the parameters of ac t iv i ty immedia te ly follo~-ing excision. The experimental procedure reported here results in more than the usual var ia t ion in the quant i ta t ive findings during the first 30 rain after isotope addit ion; the genera I quali tat i re in terpreta t ion of the results, however, is not altered by the technical difficulties (Green, 1965a, 1966).

3. R e s u l t s W h o l e cxn'~,ev,

The pot e n t i a l ~ shor t -c i rcu i t cu r ren t s , a n d ion m o v e m e n t s seen in t he ser ies of e x p e r i m e n t s repdvted here on the who | e cornea when b a t h e d in d i f ferent m e d i a w e r e s u b s t a n t i a l l y the same as those observed p rev ious ly (Green, 1965a, 1966). In t he e x p e r i m e n t s p resen ted here, t he m a j o r concern was dete,nmJning t h e locat ion w i t h i n t h e cornea of t he sod ium and chlor ide tram~port p u m p s t h a t a re responsibli~ for t h e or igin of t he p o t e n t i a l a n d the shor t -c i rcu i t cur ren t . F igu re 2 shows t h e effects of sc rap ing e i the r t he ep i t he l i nm or t he e n d o t h e l i u m whi le the whole cornea was m o u n t e d in t h e a p p a r a t u s a n d b a t h e d in no rma l R i n g e r ' s so lu t ion . D u r i n g t h e f i rs t 30 ~-9 rain, t he p o t e n t i a l difference, fol lowed the p a t t e r n shown b y nornxal corneas , r i~ing f rom 1-5 to 2-5 m V w i t h i n t h i s t ime . D a m a g e to t he e n d o t h e l i u m caused a n i m m e d i a t e decrease in t h e pot~Jat.ial difference to a b o u t 1-5 rnV, w h i c h s lowly r e t u r n e d to 1-8 m V a f t e r 10-15 rain; t h i s p o t e n t i a l d i f ference was m a i n t a i n e d for per iods of u p to 2 hr . Th i s va lue is approx- i raate ly two- th i rd s of t h e n o r m a l po t en t i a l . The short~J_reuit c u r r e n t of the whole cornea and of t h e cornea w i t h a d a m a g e d e n d o t h e l i - m were v e r y s imi lar , indice~ting t h a t t h e source of t h e ac t ive tratmqaort p ~ m p is loca ted ~ i t h i n t h e ep i the l ium. W h e n t h e e p i t h e l i u m was damaged , , however , t h e p o t e n t i a l d i f ference d ropped w i t h i n 10 ra in to 0-7 m V a n d t h e r e a f t e r d ropped s lowly t o a geeady level of 0-2-0-3 mY. T h e e x a c t d%gree of d a m a g e to each manfa~ of t h e oo rnea w a s diff icul t to assess, a l t h o u g h a n y d a m a g e t h a t d id occur w a s r e a d i l y v is ib le a f t e r

84 K . G R E E N

removal of the tissue from the appara tus , for the tissue became cloudy and swollen under the damaged area. The electrical resistance of the whole cornea ba thed in normal Ringer ' s solution was 565-]-25 ohms cm ~ (mean-~s tanda rd error of 8 experi- ments) , and wi th the epithelium d a m a g e d this value was only 60±5 ohms cm 2 (mean -~s tandard error of 8 experiments) . When only the endothel ium was damaged , however, the resistance was 450-1-20 ohms cm ~ ( m e a n ~ s t a n d a r d error of 8 experi- ments) , which indicates t h a t the main resistance is located in the epithelium.

E

o

C

0 D-

0

' " J " " ! . . . . i ~ " I . . . . I

't \

\ \

i , _ I I ! . I l IO 20 30 4 0 50 60

T i m e ( r a i n )

F I e . 2, E f fec t o f so rap ing d i f fe ren t p a r t s o f t h e co rnea on po t en t i a l difference. .0 • , P l o t o f p o t e n t i a l difference aga in s t t ime , o f corneas wi th e n d o t h e l i u m sc r ap e~ off, • . . . . • , p Io t o f po t en t i a l difference aga ins t t i m e o f co rneas wi th ep i the l ium .scraped off. E a c h po in t r e p r e s e n t s t h e m e a n , a n d t h e ve r t ioa l ba r s ind ica te t he s t a n d a r d e r rors o f t h e m e a n o f 6 ex I~ r i - . ~ o n ~ .

We have found t h a t when the excised cornea is ba thed in normal Krebs-bicarbon- a te Ringer ' s solution, l i t t l e or no swelling occurs following excision and mount ing in re~. t ion to the in r i v e measurements made before excision (0.00-0-02 m m swelling in 90 rain, resul t s o f 100 pa i red corneas; Green, unpubl ished da ta) . I n t h a t s tudy, the coxnea was mounted in a n appa ra tu s very s imilar to t h a t used in the exper iments repor ted here , b u t the appa ra tu s was modified so t h a t the corneal th ickness could be measured readily. In this way, d a m a g e to ei ther t he e p i t he l i um or endothel ium could b e detected readi ly as an increase in thlekness. Epi thel ia l damage resulted in in- c reases in thlelrness of abou t 25°/o of the total (0-38-0.46 rnrn), while damage to the e~dothel ium l e d to large increases of over 100~o in the corneal thickness. These inc reaseswere the m ~ i m a l ones, obta ined when the en t i re membrane was removed, but-smaLler increases in thickness were seen following sl ight damage to ei ther of the bOunding membranes . Since the main tenance of corneal thickness depends upon the in tegr i ty 0 f t h e bounding membranes , i t is obvious t h a t any damage to these membranes t l i a t : a l t e r s the i r p e r m e a b i l ~ e s causes an increase in corneal thickness. I t was a ppa ren t

S O L U T E T R A N S F E R A C R O S S C O R N E A L M E M B R A N E S 8 5

t ha t there was no a l tera t ion in the permeabi l i ty of the corneal membranes follcv~Ing excision of the cornea and ba th ing in an aerated, s t i rred Ringer ' s solution, for a permeabi l i ty change caused by damage to the membranes WOlLld have resulted in a change of thicl~mss. These observat ions indicate t h a t the solute t ransfer across the whole cornea, as found in the exper iments reported here, takes place in vi tro in the absence of any change in the corneal hydra t ion or thickness. This would tend to indicate t h a t the ion fluxes reflect those of the completely undamaged in vitro cornea.

Isolaze~l epithelium The experiments reported in this section are those in which the endothelium and

pa r t of the s t roma were removed from the cornea prior to mount ing the tissue in the appara tus .

5 1 J" I . . . . I " I . . . . I I 1 0

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T i m e ( m i n )

• - ~ ( } . 3. Compar ison o f potont /a l difference and shor t -c i rcui t cur ren t o f isolated epi thel ium and whole cornea.

O O , Po ten t ia l difference; • . . . . O , shor t -c i rcui t current ; C, control cornea; Ep, isolated epi thel ium. E a c h point represents the mean, and the vert ical bars indicate the s t a n d a r d errors o f the mean of 6 exper iments .

The results of the measurements of the potential difference and short~circuit current across the corneal epithelium, when bathed in normal ]Ringer's solution, are summarized in Fig. 3 and compared with the undamaged cornea from the other eye of the pair, which was used as a control. One interesting difference, however, is that in the epi thel ium the current s ta r ted a t a h igher value and fell to a s teady value, which was main ta ined for periods of 2 hr (compare Green, 1965a, 1966). There was a ne t inward t ransfer of sodium across the epithelium, the value of which was approxi- ma te ly equal to t h a t across the whole cornea (0.30/~eq Na/cm z p e r h r for the ep i the l i -m compared to 0.40/~eq Na/cm z per hr for the whole cornea). Both the etHux and influx across the epithelium were about the same as those across the whole cornea. The fluxes and short-circuit current are given in ]Fig. 4. The influx dropped m a r k e d l y dur ing the first 30 mln before reaching a s teady level, whereas the etHux was fair ly

86 K . G R F _ E N

constant throughout the experimental period. The short.circuit current, however, was lower than the net sodium transfer during this initial period. In view of the finding in the whole cornea tha t the difference was due to the initial presence of an active inward transport of chloride (Green, 1965a, 1966), i t can be reasonably assumed t lmt this explains the difference between the net sodium transfer and the short-circuit current. Four experiments were performed with the temperature of the bathing

I - Z I . . . . I " I - - " I ' 1

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T i m e ( m l n )

F I o . 4. Unid i rec t iona l sod ium fluxes a n d the shor t -e i rou / t c u r r e n t across t he i so la ted ep i the l ium. ~[, In f lux (epi the l ia l - f~-endothel ia l surface) ; E, offlux (endothol ia l - f~bepi thel ia l surfaoe) ; N, n e t flux;

S, shor t - e i r cu i t cu r ren t . E s ~ h p o i n t r ep re sen t s t he m e a n , a n d the vor t ica l ba r s ind ica te t h e s t an . d a r d e r rors o f t h e ~aean. N u m b e r o f e x p e r i m e n t s is g i v e n in pa ren theses .

solutions maintained at 30°C. The potential differences and short-circuit currents found under this condition were the same as those found at 25°C. In addition, four experi- ments were performed using medb,m TC199 (Grand Island Biological Company, Inc., Grand Island, N.Y., U.S.A.) as the bathing solution, and again the potential differ- ences and short-circuit currents were found to be the same as those seen in Ringer's solution.

The difference between the potential difference measured in the whole cornea and the potential difference of the isolated epithelium may be associated with the removal of the stroma. The removal of this diffusion barrier allow-~ the bathing medium to come into contact with the epithelial membrane facing the stroma, possibly allowing a n increased flux of a potential negating ion, for instance chloride.

S O L U T E T R A N S F E R A C R O S S C O R N E A L M E M B R A N E S ~ 87

When bathed in sul{ate Ringer's. solution, both the whole cornea and the epi- thelium had very similar short-circuit currents, whereas the potential difference, and hence the resistance, was greatly different in the two tissues (Figs. 5 and 6). The increase in potential difference ,~een in sulfate Ringerr's solution is probably due to the fact that the tissue is less permeable to sulfate than to chloride. V~rhen the isolated epi- thelium was bathed in choline Ringer's solution, the reeponse was similar to that of the normal cornea (compare Green, 1965a, 1966). The endothelial surface became

A

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I o IO

F I e . 5. P o t e n t i a l d i f f e r e n c e , s h o r t - c i r c u i t c u r r e n t , a n d r e s i s t a n c e o f w h o l e c o r n e a b a t h e d in s u l f ~ t ~ R i n g e r ' s s o l u t i o n .

• . . . . 0 , P o t e n t i a l d i f f erence ; • 0 , s h o r t - c i r c u i t current ; . . . . . . . . . , r e s i s t a n c e . T h e v a l u e s are t h e m e a n , a n d t h e v e r t i c a l bars i n d i o a t e t h e s t a n d a r d errors o f t h e m e a n o f 8 corneas .

negative, giving a potential difference of - 2 - 0 to - -3 .0 m V for 30 rain or so before returning to the zero level and remaining there for the duration of the experiment. The negative short-circuit current found in the isolated epithelium closely resembled that of the whole cornea, which supports the concept that the chloride transport sys tem i8 located in the epithelium.

88 K . G I t E E N

Isolated endothelium The results r e p o s e d in this section were obtJained from the tissue t ha t was separated

f rom the rest of the cornea as described under Materials and Methods before mount ing in the appara tus .

No potent ia l or short-circui t current was recorded from this tissue, and the electrical resistance Was low in the different ba th ing media used in the experiment~ reported here, a valkm of abou t 60 ohms cm a being found in all ba th ing media. Unidirectional sodium and chloride fluxes were measured in normal Ringer ' s solution, and the tissue

12 - - 6 . . . . . 8 . 0

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T i m e ( r a i n )

Fzo . 6. P o t e n t i a l difference, sho r t - c i r c u i t cu r r en t , a n d r e s i s t ance of i s o l a t e d e p i t h e l i u m b a t h e d in su l f a t e R i n g e r ' s so lu t ion .

~ - - - O , P o t e n t i a l difference; • • , shor t~c i rcu i t cu r r en t ; . . . . . . . . . , r e s i s t ance . T h e v a l u e s a r e t h e mean , a n d t h e v e r t i c a l b a r s i n d i c a t e t h e s t a n d a r d erT~rs o f t h e m e a n o f 8 ep i the l i a .

showed a passive behavior to bo th sodium and chlo=~de ions, the unidirectional fluxes being i~qual (within exper imenta l l i m ~ ) in each direction. The sodium influx was 1(~1 ~-t-0-32peq/em s per hr (meand- s t anda rd error of 8 ex]periments),and the efflux was 10-O=L(b25/zeq cmSpe r hr (mean-Fs t anda rd error of 8exper iments) ; the chloride fluxes were 5-OiOy 18/~cj]cm s per hr (mean d - s t anda rd error of 4 experiments)" for the influx and 4-9-4-0-12peq/cm ~ per hr (mean-bs ta~dard error of 4 experiments) for the el- flux. This would indicate t h a t the endothel ium is a nont ranspor t ing tissue, act ing s imply as a ba r r i e r coupled in series with the epi thel ium and the s t roma. F u r t h e r evidence t h a t the endothel i tun shows no ' t ransport ac t iv i ty was the domplete absentee of a potent ia l when the tissue was bgthed in ei ther choline or sulfate Ringer ' s solution.

I t was though t t h a t i f an endothelial p u m p existed, the temperature, m a y have bee~a too low for i t to function, a l though this was not the experience with the isolated e p i t h ~ . Some exper iments were performed, therefore: a t ,30°C; no potent ia l

S O L U T E T R A N S F E R A C R O S S C O R N E A L M E M B R A N E S 89

difference was de tec ted at th i s t empera tu re , even w i t h i n 1 ra in of the d e a t h of t he an imal . The sod ium flux ra tes in 4 expe r imen t s (2 for inf lux, 2 for cffiux) pe r ib rmed in no rma l R inge r ' s so lu t ion a t 30°C were the same as those found a t 25°C. A ~dmilar n u m b e r of expe r imen t s per formcd us ing m e d i u m TC199 a t 30°C on each side of the t i ssue gave t h e same flux ra tes .

The p e r m e a b i l i t y coefficients for 22Na ob ta ined for the whole cornea were calcu- l a t ed b y the m e t h o d of Maffiy, H a y s , L a m d i n and L e a f (1960) a n d were 6-3 x 10 -s cm[ hr for t he ilfflux and 3.5 x 10 -s e m / h r for the eflhxx. The p e r m e a b i l i t y coefficient for bo th inf lux and efttux across the e n d o t h e l i u m was 7 x 10 -z cm/hr , a n d t h a t ,~'or t h e ep i t he l i um was 4-8 x 10 -3 c m / h r for t he inf lux and 2.8 x 10 - s c m / h r for t he efflux.

I t is diff icult to e s t ima t e q u a n t i t a t i v e l y the a m o u n t of d a m a g e t h a t resu l t s f rom scrap ing the ep i the l ium, b u t some e s t ima te can be m a d e b y use of the d y e fluoreseein. Tiffs dye does no t p e n e t r a t e the i n t a c t cornea w h e n app l ied to the ep i the l ium. I n some of t he e x p e r i m e n t s t h a t inc luded sc rap ing of t he immersed c o r n e a whi le i t was in the a p p a r a t u s , app l i ca t ion of f luorescein p roduced a def in i te s ta in ing . In i t i a l l y , the en t i re ep i the l ium and s t roma were removed , and t h i s p rocedure was used d u r i n g the sodium flux d e t e r m i n a t i o n s on the endo the l imn . I t became a p p a r e n t , however , t h a t even the l i gh t e s t sc raping of t he corneal ep i the l ium caused a fall in p o t e n t i a l a n d shor t -c i rcu i t cur ren t , w i th a c o n c o m i t a n t decrease in res is tance . Smal l dJz turbances of the ep i the l ia l surface also lowered the p o t e n t i a l to v e r y low values, b u t on ly i f the ou te r surface of the ep i the l ium was punc tu red ; mere p h y s i c a l s t r e tch ing , however , does no t a l t e r t he measm'ed po ten t i a l . Several e x p e r i m e n t s were per formed in wh ich the whole u n d a m a g e d cornea was s t r e t ched b y the a p p l i c a t i o n of a b l u n t e d piece of p o l y e t h y l e n e t u b i n g to t he endo the l i a l surface. No difference f rom the no _rrnA1 was observed in e i t he r the p o t e n t i a l or t h e shor t -c i rcu i t c u r r e n t u n d e r these condi t ions .

4. Discussion

The cornea appea r s un ique in r e spec t to mos t ep i the l i a l t i s sues in h a v i n g t h e two epi the l ia l l ayers sepa ra t ed b y a la rge a m o u n t of col lagen and mucopolysacch_aride. Because of t he ua~ique a n a t o m y of t h e cornea, i t is poss ib le to detel-,~.ine va r ious cha rac te r i s t i c s of t h e s epa ra t e cel lular m e m b r a n e s and t h u s to charac te r i ze each m e m b r a n e i n d i v i d u a l l y and compare t he two, w h e n coupled in series w i t h each o ther , w i t h t he charac te r i s t i c s of t he whole cornea. I t shou ld be r emembered , however , t h a t w h e n " i so la ted ' : ep i the l ium or endot .hel inm is referred to, a m o u n t s o f s t rorn~ are a t t a c h e d to these membranes .

I t wa~ o r ig ina l ly t h o u g h t t h a t s epa ra t ion of the ep i t he l i um or t h e e n d o t h e l i n m f r o m the co rnea m i g h t de s t r oy t h e a b i l i t y of each to t r a n s p o r t ions. The series of expel-i- ment~ r epo r t ed here, however , showed t h a t t he i so la ted epithelil~rn survivors for a t least 2 h r in v i t ro a n d con t inues to t ra Impor t sod ium a t a c o n s t a n t r a t e d u r i n g t h i s t ime, a r a te t h a t is equa l to t h a t of t he whole cornea. Th i s f ind ing shows t h a t separa - t ioh of t he e p i t h e l i u m can be s u ~ y per formed w i t h o u t d e t r i m e n t t o t h e p h y s i .e*- logical cha rac te r i s t i c s of th i s m e m b r a n e . W e k-now t h a t t h e i so la ted cornea does n o t swell in v i t ro (see 3, Resu l t s ) and , therefore , t h a t m e m b r a n e s are n o t d a m a g e d b y the e x p e r i m e n t a l proe..edure d u r i n g m o u n t i n g of t h e cornea . The re w a s no di f ference be tween t h e two se ts of d a t a o b t a i n e d b y t h e d i f fe ren t t echn iques . T h e e p i t h e l i u m h a d t h e same cha rac te r i s t i c s e i t he r w h e n i t was c o m p l e t e l y i so la ted f r o m t h e co rnea or w h e n t h e e n d o t h e l i u m was d a m a g e d ; s imi la r ly , t h e c ~ o s o f t h e endo- t h e l i u m were t h e s a m e e i ther w h e n t h e e n d o t h e l i u m w a s c o m p l e t e l y i s o l a t e d f r o m t h e

90 K . G R E E N

cornea or when the epithelium was damaged. Since all the da ta concerning the two membranes , obtained by different techniques, agreed in each case, the au thor con- cludes t h a t separat ion of the epithelium or endothel ium from the remainder of the cornea does not a l ter the physiological character is t ics of ei ther cellular membrane. Since isolated endothel ium (plus a small a m oun t of s t roma) shows the same behavior as an endothel ium of a cornea with only the epithelium removed, it is considered unl ikely t h a t the membrane is damaged dur ing the isolation procedure. The same a rgumen t applies for the isolation of the epithelium.

Although the membranes have been shown to be unharmed by the procedures, two exper imental considerations remain to be discussed. Separat ion of the const i tuent membranes of the cornea, and bath ing each surface ~ i th the same medium, will not necessarily give the same quant i ta t ive da ta as t h o ~ obtained from the two different membranes while they remain a t t ached to the whole cornea. A certain difference is to be exl~cted, par t icu lar ly as the solutions ba th ing the membranes in the intact tissue do not have the same composition as normal Ringer ' s solution (Green, 1965b; T. Otori, i~ersonal communication). However, this difference will not al ter the basic quant i ta t ive findings regarding thn biophysical properties of the membranes . One fur ther difficulty in the analysis of the d a t a arises because any applie~l radioisotope normal ly reaches equilibrium with a biological membrane for the first 20-30 rain af ter addi t ion of the isotope. This problem has been discussed previously (Green, 1965a, 1966). I t was shown a t t h a t t ime t h a t there is quant i ta t ive ly more var ia t ion in the d a t a dur ing the 2 ~ 3 0 rain af ter addi t ion of the isotope t han is usual ly seen when a 30-rain equilibrium period wi th the isotope is allowed before samples are taken, and also t h a t the flux values m a y be lowered.

"The" d a t a in Fig . 7 show tha t when the epithelium is damaged or removed, there is no ne t solute t ransfer across the s t roma and endothelium. With the endothel ium damaged or removed, however, the ne t solute t ransfer (as judged by the maintenance of a s t eady short-circuit current and net sodium transfer) remains unchanged as com- pared with the whole cornea. The potent ia l decreases by about one-third of the original value, and tbla is due to the loss of the resistance t h a t is usually offered by the endo- thel ium and s t roma, for wi th the endothel ium damaged, ions are free to diffuse thr~dgh the s t roma. In the case of the isolated epithelium, the short-circuit current rema~nR unaffected by the removal of even large amounts of s troma. ~Vit.h the entire epithelium removed, the potent ial is zero, a value t h a t is independent of the e~mposi- tion of the b~thing medium.

A mlmmary of the findings of sodil~m mo~rement in normal Ringer ' s solution is given in Fig. 7. As can readi ly be seen, the unidirectional t ransfer ra tes of sodium in the isolated epithelium are similar to those of the whole cornea, and it is also evident t h a t there is a ne t t ransfer of sodium inward across the tissue in each case. The efltux of sodium in the epithelium is sl ightly higher t h a n t h a t in the whole cornea, owing to the removal of mos t of the s t romal diffusion barr ier and the endothelial diffusion barrier . The equal i ty of the flux values found in both the whole cornea and the isolated epi thel ium emphasizes the proposition t h a t all the t r anspor t ac t iv i ty of the cornea is, beyond doubt , located in the epithelb~m. The t r anspo r t pump, therefore, is seen to reside in the epithelium; no electrical ac t iv i ty or ne t ion t ransfer is found across the endothel inm, a result t h a t is in keeping wi th the findings obta ined by util~.ing micro- eleb%nmdes (Green and Green, unpubl ished da ta) .

I n a t t emp t s to localize the act ive t r anspor t pump in frog skin, using a technique s lmilar to t h a t used it, the exper iments repor ted here, l~ranz and van Bruggen (1946)

S O L U T E T R A N S F E R A C R O S S C O R N E A L M E M B R A N E S 91

dissected a w a y the basal cell layer of the serosal surface, the tela subcutanea. They found t h a t the potential difference decreased by 400/o, bu t t h a t the short-circuit current increased insignificantly. The influx of sodium across the remainder of the skin increased sl ightly following the removal of this cell layer, bu t the efllux increased greatly. The flux ratio (influx: efflux) decreased i'Tom 33.1 in the whole skin to 11-1 in the dissected skin, the change being due main ly to the increase in the passive

Sodium fluxes (/LeCl/CmZ/per hr)

Epitheliurn Endot heliurn

Influx 0"9

Influx 0-7

Efflux 0"5

Influx I I j Efflux ~ Efflux 0 "40 I0"0

F~o. 7. D i a g r a m ~howing t h e unid i rec t ional fluxes o f sod ium across t h e whole c o m e s , i so la ted epi- the l ium, a n d i so la ted endo the l ium.

efftux of sodium. The reduced potent ia l was due to the increased passive m o v e m e n t of chloride ions. This s i tuat ion in frog skin bears a close resemblance to the findings of the exper iments presented here in the r abb i t cornea, in which experiment6 the removal of the endothel ium (serosal bounding layer) caused a decrease in po ten t ia l , wi th litt le change in the act ive t r anspor t of sodium.

The fluxes of sodium and chloride ions across the endothel ium have been shown to be passive, despite repea ted a t t e m p t s to detect any ion t r anspor t ac t iv i ty . The resistance was low in all the ba th ing media employed in the exper iments repor ted here, and the tissue showed no electrical ac t iv i ty in a n y medillm. I f there existed a sodium p u m p and chloride pump, one of which could ac t independent ly of the other , then one or the other would create a potent ia l and a measurable short-circuit cur ren t when the endothel ium was ba thed in an appropr ia te medium; such an electrical ac t iv i ty was not found. The other explanat ion was t h a t there could have been a coupled sodium and ct~oride pump such as t h a t seen in t he gall b l adde r o f finh and r abb i t (Diamond, 1962, 1964) or in the mar ine teleost intest ine (House and Green, 1965). The sodium and chloride fluxes, as debermined in normal Ringer ' s solution, showed t h a t the movement of each ion was passive, and no evidence of an ac t ive t r anspor t p u m p could be found a t different t empera tu res or wi th r]i~erent b a t h i n g media. The previous emphasis placed on an act ive component in t he role of rnRinten- ance of corneal thickness (Harr is , 1957) must , therefore, be t r ea ted wi th cau t ion in

K. G R E E N

view of the present findings. The detai led exper iments reported here confirm the f inding t h a t there is no potent ia l across the endothel ium (Donn et al., 1959; Modrell and Ports, 1959; Green, 1966) and, in addi t ion, show tha t the ion movements are comple te ly passive. The endothel ium, therefore, appears to act s imply as a diffusion bar r ie r coupled in series wi th the epi thel ia l t ranspor t system and the stroma.

Al though the sc t ive ion h~nspo r t across the epi the l ium m a y p lay no par t in the direct ma in tenance of corneal th ickness other t han offering a t igh t diffusion barrier. the findings reported in the present paper show tha t the role of act ive t ransport in the main tenance of corneal hydra t ion , thickness, and t ransparency should not be neglected, because the active ion t ranspor t m a y serve to control th.e ionic environ- m e n t of the s t romal mucopolysaccharide. This would seem to be a logical explanat ion for the presence and funct ion of the inward t ranspor t p u m p in the epi thel ium. Since the s t roma consists of collagen fibrils, together wi th a mucopolysaccharide mat r ix , control of the ionic s t rength of the ba th ing f lu id in the s t roma by the pump systems migh t serve to exmtrol the water content of the stroma. Since the s t roma consti tutes 90~o of the total corneal thickness, i t is obvious tha t any d is turbance of the t ransport system, if i t is the controlling factor, would cause significant changes it/ corneal thickness in te rms of shr inkage or swelling. How this control is brought about is a t present ~mlrnown; i t m a y be t h a t the runs in some way interact with the muco- polysaccharides, thereby controll ing possible forces between the mucopolysaccharides (see Hedbys , 1961), or tha t the concentrat ion of the sodium in the bathh~g m e d i u m around the mucopolysaccharide in some ~vay controls thei r hydra t ion . Active ion t ranspor t m a y well be a factor in the control of the imbibi t ion of water of the corneal mucopolysaccharides, b y controll ing the ionic content of the intercel lular mat r ix .

A C K N O W L E D G M E N T S

I thank Mrs. M. A. Green for her valuable technical assistance during the course of this work. I also thank Dr. M. E. Langham and Mr. D. Andrews for their critical reading of the manuscript.

R E F E R E N C E S

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