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The influence of ions, ouabain, propranolol and amiloride on the transepithelial potential and resistance of rabbit cornea
Exp. Eye Res. (1979) 28,413-426
The Influence of Ions, Ouabain, Propranolol and Amiloride on the Transepithelial Potential and Resistance of Rabbit Cornea C’. M.
A. W. FESTEN
Depnrtment of Ceil-Phllsioloyy,
ASD
J. F. G.
SLEGERS
Uwhersity of N[jnwcJe~~~S2j’megen. The Netherlards
(Received 27 June 1978. Sew I’ork) By means of in vitro ion substitution experiments the potential difference and resistance dependency of rabbit cornea1 epithelium on h’a +: I+ and Cl- activities have been established at the t,ear side as well as at the aqueous side. In normal conditions only a Ii+ selectivity at the aqueous side of the epithelium exists and a Na- and Cl- select,ivity at the tear side. The tear side was also K+ selective at Ii+ artivities greater than 38 maI. In Cl- Ringer ba,thing solutions a rectifying currenevoltage relationship is obtained. Ouahaingives rise to a two phase decrease in potential from 29.9i2.4 to 22.912.1 to 14.4h1.7 rn% (7~ -:= 9). In the first phase the resistance changes from 3.720.4 to 4.0&0.4 k&m” (n == 9) md in t,ht: second phase R decreased t,o 1.3hO.l kQcm2 (7~= 9). After ooabain the Sa+ selectivity has disappeared and the Cl- permeability has increased. The current-voltage relationship after ouabain has become linear. Amiloride induced a reversible change in resistance from 3~.SZ~O~fito 4,X-+,0+3 k&m” accompanied by a depolarization from 29.1 h2.7 to 22+iZ 1.5 mV (n = i), moreover the rectifying properties as exhibited in control cornea are not affected by amiloride. A voltage dependent Cl--conductivity of the tear side membranes of the cornea1 epithelium can be postulated as well as a voltage independent Cl--ronduc+irit> that can be induced by ouahain. The onabain effects are inhibited by propranolol. Key words: rabbit; cornea; epithelium ; ionic select,irity; ouabain; propranolol; amiloride: (~rrent-voltage relationship.
1. Introduction The degree of hydration of rabbit cornea and tht?relJ!iti; transparency is tlel~endent~ on the proper function of the endothelium (Mishima, and Kutlo: 1967 ; Maurice, 1972 : Fischbarg and Lim, 1974) as well as of the epithelium (Riley, 1971; Klyce, 1975). The epithelium is the place where most of the transcorneal pot,ential difference (p.(l.) and resistance (R) are located (Klyce, 1952; Ehlers. 1973; Clandia. Bentley and C!ook, 1974; Festen and Slegers, unpublished data). This layer is known to transport actively Na+ from the tear side (TS) towards the aqueous side (AS) (Donn. Maurice and Mills? 1959; Klyce, 1975; Van der Heyden, Weekers and Schoffeniels, 1975) and CY from AS to TS (Klyce, Neufeld and Zadunaisky, 1973; Klyce, 1975; Van tier Heyden et al., 1975). The contribution of the endothelium to p.d. and R is of minor importance. According to most authors the endothelium is characterized by bicarhonate transport (Fischbarg and Lim, 1974; Hodson and Miller, 1976). In order to obtain the ion-selectivities of the different constituting membranes of the cornea severa, authors have carried out microelectrode as well as ion substitution experiments in frog (Akaike, 1971) and rabbit (Akaike and Hori, 19iO; Fee and Edelhauser, 1970). These studies point towards a K+ and Cl- selectivity of the hasal cells. Frog transcornea1 p.d. and R both declined with increasing K-~ activity at the AS and basal cells of frog cornea1 epithelium showed a K+- and C!l--electrode behaviour (Graves. Sanders. Shoemaker and Rehm, 1975, 1976). In beef cornea1 epithelium lowering the TS Na+-activity decreased p.d. and increased R (Lindemann, 1968). Although at present most workers on cornea1 physiology use rabbit cornea no systematical 0014-4835/79/040413+14
:c] 1979 Academic Press Inc. (London) Limited
$01.00/0 413
114
C’. M. A. \v.
FESTEK
AN11 .l. F. t:. I;LEGERS
measurements of ion selectivities are known t,o exist for this tissue. In orclcr to characterize the rabbit cornea1 epitheliurn, pd. and R dependency on Na i-, K am1 Cl- activities are studied for both sides of the menlhrane.
2. Materials and Methods New Zealand white rabbits weighing 2-2.5 kg were killed by means of a blow ilI the neck and bleeding. The eyes were enucleated and kept in a moist atmosphere at 4’C. Storage of whole eyes at 4°C for several hours did not influence the studied electrophysiological parameters, i.e. pd. and R. The cornea including a scleral ritlg of 2 mm \GLS isolated without any wrinkling and transferred to a modified Vssing chamber (1Jssing and Zerahn, 1951) (cf. Fig. 1) adapted to fit the curvature of rabbit cornea. The edges were greased with silicone grease to improve sealing and minimize edge damage.
FIG. 1. A schematic diagram of the experimental set-up. For det,ails see text,.
The bathing solution contained the following concentration of substances in mu: NaCl, 110; NaHCO,, 39; KHCO,, 3%; KH,PO,. 1.0; CaCl,, 1.5; MgSO,. 7H,O, 1.0; glucose, 25; adenosine. 5.0; reduced glutathione, 1.0; I,-ascorbic acid, 1.5. Osmolalitv was adjusted to 315 mosmol with mannitol and the bathing solution was aerated witk 95% 0, and 5% CO, and adjusted to pH = 7.4. Temperature was kept at 35°C. Au intraocular pressure of 15cn1 H,O was obtained by raising the outflow opening 15 cm above the cornea (cf. results section). Changes in ion composition of the bathing solution were on equimolar basis, except for 110 rn?Y1Cl- which was replaced by 55 mM SO?-- with compensation for osmolality loss. Sctivity coefficients were obtained from Robinson and Stokes (1970). Flow rate of the bathing solution at TS and AS was 1 ml/mill ~(1 t,he outflowing solution was discarded. Changes in bathing solutions could be made without damage to p.d. or R. At the TS changes in ion composition had their maximal effect in less than 2 min, inclusive the time necessary for complete wash-out. Adenosine, glutathione, L-ascorbic, acid, ouabain (g-strophanthin, LUerck), inderal (propranolol, ICI) and amiloride (Merck, Sharp 8r. Dohme) were added to the bathing solution just before each experiment. With a four Ag/AgCl electrode system (cf. Fig. 1) trnnscorneal pd. and K could be measured. Electrical contact between the bathing solution and the Ag/AgCl electrode was made by means of 3~-KC1 filled microelectrodes with broken iips (diameter < 50 pm). Due to schematizing, the pd. measuring electrodes in Fig. 1 are situat,ed further from the cornea1 surface than they actually were. Via high
TKANSEPITHELIAL
P.D.
AND
R OF RABBIT
CORNEA
11.5
input impedance amplifiers (52 K, Analog Devices) the p.d. measuring electrodes were connected to a differential amplifier (52 K, Analog Devices) and the signal was recorded with a pen-writer. Liquid junction potentials between experimental bathing solutions connected via 3 ~-Kc1 agar bridges never exceeded 1 mV. The instantaneous R wi1.s determined measuring the transcorneal voltage response to bipolar current pulses of 100 msec duration and a magnitude < 5 PA/ems normally. The c,ontribution of polarization was neutralized by extrapolating the voltage response to t --f 0 (Augustus, 1978). Ripolar current pulses were obtained by int,roducing bipolar voltage pulses from it. Tektronix pulse generator (Tektronix inc.) to a voltage controlled current source using :I. 44 W amplifier (Analog Devices). Current pulse magnitude was measured by means of au amplifier (234 L, Analog Devices) in the ground return of the current loop. Current pulses and corresponding transcorneal voltage responses were monitored on a Tektronix storage oscilloscope. I/V-plots were obtained by varying the magnitude of the voltage pulses supplied to the voltage controlled current source. The measurement of a currelitvoltage relationship was completed within 6 min without affecting spontaneous trailscornea1 pd. xntl R. Temperature on both sides of the cornea was registered by means of thermocouples. The mean value of a parameher is always given with the standitr~l error of the inearl (S.E.M.). 3. Results Bfect of intrnoculur poressure Using the normal Ringer solution the potential differences and resistances are measured when p.d. and R had stabilized with a pressure gradient of 15 cmH,O (1.5 kPn), AS positive. Within 30 min pd. was 27.3k1.5 mV (TS negative) ant1 R 3.4~0.3 k&n” (mean &s.E.M., ~1, = 20). Incubating the cornea from the beginning without’ a pressure gradient gives statistically the same p.cl. and R values. The reason why a pressure drop is used in the presented experiments is twofold: it mimics the physiological situation and in planned experiments the pressure gradient will be needed for correct observation. Lowering the pressure gradient, in case t.he above mentioned stabilization was reached, resulted in a reversibly lower value for the 1j.d. as well as R. This effect cannot be attributed to a diminished streaming potential because lowering the temperature to zero. reduced the p.d. to zero in the absence or presence of a pressure gradient. In case of a streaming potential one would expect tJofind only a drop corresponding with the RTjF t,erm of the Nernst equation. Increasing the pressure gradient above 20 cmH,O at the AS irreversible pd. and K drops took place. In general these findings confirm the results of Ehlers and Ehlers (1968). The irreversible changes introduced at higher pressure probably may he due to edge damage effects. The reversible changes observed on lowering the pressure cannot~ be explained at present. A lack of sealing may be the reason, These findings indicate that once the cornea has stabilized the pressure gradient cannot be altcretl freelv without effect)s on pd. and R.
Figure 2 shows the results of equimolar substitution of Ii-+ for Na- at the AS of the cornea. Increase in K+ activity produces a depolarization of 25 mV per decade of the transcorneal p.d. The decrease in p.d. is accompanied by a decrease in R from 3-550.4 to 2*2*0*2 k&&n2 at a potassium activity of 89 rnM as is shown in Fig. 2 also. After changing the KT activity a steady state value of p.d. and R was reached within 8 min due to diffusive effects in the stromal layer as mentioned also t)v Graves
416
(1. .\I. A. \r.
VES’I’ES
ASI)
J. I’. c;. SLE(:EI(S
et al. (19’75) in frog cornea. Substituting Choline 1 for NX did not significallt,lv altot, p.d. or R. However, substituting Tetraet,hyl ammonium (TEAM+) t’or Na-; did protlu(:ta a reVersiiJle
depOhrizatiO1~
Of
7.5
lllV/deC
accOInpI~icd
hV
811 R
ilmws?.
Complete substitution of SC!:- for Cl on the AS produce(I an instwntanel~u~ depolarization of 7 mV, without any change in R for over a period of 15 min. Tht same result was obtained in case the epithelium and/or t,he enclnthclium had I~cll removed I )y scraping, with or without a pressure gradient. In symmetrical (‘lm or SOi- conditions with or without a pressure gradient when the epithelium had t)et,n scraped off a p.d. was obtainecl not significantly different from zero.
IOL
1
5
I
I
I
IO
50
100
UK+. AS
FIG. 2. The effect of changing the K+ activity (given in m&r) in the aqueous side bathing solution on the transcorneal p.d. (0-O in mV, tear side negative) and R (0-0, kQcm2), by means of Sa--/K+ substitution. The bar indicates S.E. of the mean. 11= number of observations.
Tear side ion substitution
Increase in K+ activity at the TM (Na+ substituted) gave a response in p.d. and R as shown in Fig. 3. Below 28 mM-K+- there is no significant change in p.d. and only a small decrease in R. Above 28 mM-K+ a hyperpolarization of transcorneal p.d. of 11 mV/dec existed with a simultaneous decrease in R from 26 ho.2 kQcm” to 1.6 ho.2 kOcm2 (,n = 6) at 89 maf K+ activity. When Na+ in the TS solution was replaced 1~~ choline+ or TEA+, a depolarization of 15 and 21 mV/dec was measured, respectively. At the same time R increased. However, the increase in R produced by Na+/TEA+substitution was greater than in tbe case of Nat/choline’- substitution, i.e. a decrease in Na+ activity to 32 mM gave an increase in R to 150 and 12094 of the original value respectively. Figure 4 shows the effect of Na+/TEA+ substitution at the TS on the p.d. and R. The combined effect of Na+ and K+ on the p.d. in the TS Na+/K+ substitution (vide supra) is shown in Fig. 5. Above 28 mM-K+ a pd. change of 29 mV/dec can be estimated. In an additional experiment with a potassium activity of 32 mni and Na+ substituted by TEA+, equimolar substitution of K+ for TEA+ gave a hyperpolarization of 21 mV/dec.
TRANSEPITHELIAL
P.D.
AND
R OF RABBIT
CORXEA
417
SOj- for Cl-- substitution gave rise to a transient hyperpolarization accompanied by an increase in R. The plateau of the hyperpolarization reached in 2 min was plotted against the Cl- activity (Fig. 6). At high C- activity a change of p.d. of 25 mV/dec was measured. The fall in pd. after the initial hyperpolarization had a t.+ of 2.7 min and started a few minutes after reaching the plateau. Decreasing the (‘I activity to 3 IllM, R increased to 6.5 +@7 kcme (II = 7) and p.tl. increased to $5.0&,34~ mV ‘I3 negative 02 = 7, cf. Fig. 6).
n=6
35 -
(Nat/K+)
30 1
25-
‘j E d ci
20I5 -
i-+--y
-t
---___ ---+
i ‘\ \
IO i
\. ‘.
50
i
I 5
I IO
I 50
\
I 100
’
UK+, TS
Fro. 3. The effect of changing the K+ activity (given in maa) in the tear side bathing solution on the transoorneal p.d. (O-O, in mV, tear side negative) and R (m-0, Mcm2) by means of Na+/K+ substitution. The bar indicates S.E. of the mean, 12= number of observations.
25
oNo +, TS FIG. 4. The effect of changing the Na+ activity (given in mM) in the tear side bathing solution on the transcorneal p.d. (O-O, in mV, tear side negative) and R (0-0, k&m?), by means of Na+/TEA+ substitution, )A = number of observations.
D
Addition of ouabain to the A8 resulted in a biphasic effect, on the p.tl. awl 1: ot cornea1 epithelium. Typical examples for 10e3 M and 1O-4 M ouabain are show in Fig. 7. The value of p.d. and R during 10m3RI and lo-* M ouabain iucul)ation in tJhck
50: 5
50
IO
100
OK+, TS FIG. 5. Figure 4 suggests the K+ effect, on p.d. and 11 to be greater than S~IIU n in Fix. :S. ‘l’hc~~~eli~~ the results of Fig. 3 and Fig. 4 are combined. The pd. part of Fig. 3 has bcru taken in this figu~.ts as a pwcrnt~age of the pd. for 4 mx K’ at the t’ear side (e-0). The pd. part of Fig. 4 has Iwu t,llwn a.c :L in the same way, only instead of expressing the p.d. as a function of the Saf acti\-ity it is es~wssc~tl function of the complement of the Ka+ activity, i.e. in this case the Ii7 activity (:3-c!). \\:h~t~ I IICSC plots are combined the real K+ effect is obtained for K+ actirit,,v changes at the tear side (?i: -*).
I 5
I IO
I 50
, 100
oCk+. TS (mkl)
FIG. 6. The effect of changing the Cl- activity (given in mM) in the tear side bathing solution, on transcorneal p.d. (O-0, in mV, tear side negative) and R (O-0, M2cm2), by means of Cl-/SO:-. substitution. The bar indicates S.E. of the mean, 1~= number of observations.
TRANBEPITHELIAL
P.D.
AND
R OF RABBIT
CORPL’EA
419
different phases did not differ significantly and were therefore taken together. At first, there was a decrease in p.d. from 29.912.4 to 22.9&2*1 mV and a small increase in R from 3.710~4 to 4.0-&0*3 kQcm2 (a = 9). Secondly, p.d. as well as R decreased. P.d. fell to 14*4&l-7 mV and R to 1*3&0.1 k.Qcm* (IZ = 9). The same effects were seen when ouabain was applied at the TS and the AS and when the endothelium was removed. The duration of the p.d. and R change due to ouabain was at 10-3 M 18 +l min (n = 5) for the first phase and 1352 min to a new steady-state in the second phase. At 1O-4 M-ouabain these values were 35&5 min and 32&7 min (1~ = 4). respe&ively.
1111
1
I
I
I
I
369
15
30
45
60
75
Time (mln after addltlon)
FIG. 7. ‘l’,ypiczl exunplrs of the effect of 10-4 JI ( ;:) and lo-” JI (C) ouabain on the aqueous sink. time nftcr addition in min; ordinate: transcorneal pd. as a pcrccntapr of the starting vale ( ~. --) ;tt~l R as a percentage of the starting ralur (- - -).
-kbwiss;i.:
In t,he second phase steady-state (10e3 Jr-ouabain) equimolar K-1 substitution for N;t+ gave a p.d. and R change as shown in Fig. 8. Increasing the Ii+ :totivit,v resulted in a depolarization of 16 mV/clec for a pota~ssimn activity ~.:.2%in11 and of ,“8 mV/dec for a potassium activity > 25 mu. At the Same time there was a IIIOP~ I)ronounce(l drop in R at higher potassium activities. TEA- nor ~‘holinc- for lYa 7 srllxtitut,ion at the AS did produce a change in 1j.d. Or R. Replacing Nar 1)~ TEA ’ Or (‘holine’ had no effect on p.d. or on R. So:- for (‘1 snhstitution did produce the s:ime effect as before ouabain, i.e. an instantaneous depolarization of 7 mV ill caqe of clrcrcnsc~ in n Cl -. ,4S from 89 to 3 11131.
Replacing Na+ by either K+ or TEA+ had no effect on the p.d. or R. Changing Clfor SCY-- gave a transient hyperpolarization and a simultaneous increase in R with essentially the same time course as before ouabain. The results are taken together in Fig. 9. At a Cl- activity of 3 mM p.d. increased to 51-O&3+0 mV and R to 5*2&O-3. kQcm2 (9~= 5). At high Cl- activity a hyperpolarization of 54 mV/dec existed.
The p.d. and R changes due to 20 min TS pre-incui,ation with IO- 5 propr’~1101~11 followed by incubation with 10~~M propranolol TX and lo-” M ouabain AS. compared to the effects of 10m3M ouabain AS alone are given in Table I as a percentage of t'hr original value. Propranolol did, not significantly, decrease p.d. and increase R. ( ‘OIIIbined further, propranolol and ouabain incubation as well as Oua~Jain alone decreascti the original p.d. to 489;;. However, due to propranolol and ouabain together R
1 4
15 IO -
-3 1
=
5 & d Ii
o-
f r i 2 Y k
-5-
-10I?=5 (No+MK+) after ouabom lncubctmn
-15 -20
-2
Id I
) 5
IO
xl
100
OK+. AS CrnM)
FIG. 8. The effect of changing the K+ activity (given in mM) in the aqueous side bathing solution kQcn,2) after ouabain addition, on transcorneal p.d. (O-O, mV, tear side negative) and IZ (m-6, by means of Na+/K+ substitution, 1~= number of observations.
30 -
20 -
n-5 (cl--so:-,
IO -
after cuabain incubation
t”l,
“\ ‘t
I 5
I
I 50
IO oCl-,TS
2
\ I _ loo’
(mu)
FIm. 9. The effect of changing the Cl- activity (given in mm) in the tear side bathing solution after vuabain addition, on transcorneal p.d. (O-O, mV, tear side negative) and R (e-e, kQcm*) by means of Cl-/SO:- substitution, n = number of observations.
TKANSEPITHELIAL
P.D.
AKD
H OF RABBIT
CORNEA
-121
increased by 13:/A while ouabain alone decreased R by 67.53;. Furthermore, the two phase decrease in p.d. after ouabain changed into a single exponential decrease with 1)ropranolol pre-incubation, t, being 15 min. TABLE I
‘I(, {If original valur of pd. and R after ouahain (1OF IN, AiS) proprauolol (IIJP M, ‘I%) : pro~wanolol in the stratlp state. 3lean *s.E.M. Number of ohscrrntions in pawnthescs.
and
ouahnin
Arniloride f$fects on pd. and R In CV as well as in SO?- Ringer bathing the cornea, there was no effect of 3 x 10m5M amiloride (TS) on R, but the p.d. decreased by a few mV. The results of addition of 5 x 10P4M amiloride at the TS are summarized in Table II. Within each experiment p.d. depolarized and R increased reversibly. The mean p.4. drop in four experiments was from 29.1k2.7 mV to 22.6kl.5 mV. R changed in t,hese experiments from 3.5 396 k1;2cm2to 48&0.8 kQcm2.
The effects of amiloride on trarwcorlzeal pd. wd K
The effect of amiloride (5;~ IO-* nr) at the tear sick on transcorueal in ltQcm2). Mean &s.E.>I. Kumbcr of observations in parentheses.
pd. (t(iwn in my) ;ud Ii (given
Cwrent-voltage relationships before and after owzbni~n In Fig. 10 the current-voltage relationships (Z/V plots) before and after ouabain are shown. In normal conditions, i.e. Cl- Ringer bathing the cornea rectifying properties can be assigned to the epithelium. Hyperpolarizing currents gave rise to smaller p.d. responses than depolarizing currents. As a result R, in hyperpolarizing direction (for Z > 20 pA/cm2) amounts to 2.2 k.Qcm2. In depolarizing direction R = 5.3 kQcm2. In the Z/V relationship three almost linear parts can be observed. i.e. for great de- and hyperpolarizing currents and the region between lOt~~/crn~
4”J?
(‘. Ill. A. b\.. FE:STEK
ANI)
.I. F. c;. Sl,E(:E’:Ks
depolarizing and 10 pA/cn’g hyperpolarizing (Breaking points are fountl at --YS tu\:, - 12 ,A/cn1” and +44 mV/ +14 ~A/cm2). In SOi Ringer the rectifying l)roptlrtif+ have disappeared and only for I > 30 ,&/cm’ symmetrical deflections take 11la.c~. R becoming smaller. In the presence of ouabain there are 110 rectifying lnq)e&s either. Only at high depolarizing current (1 > 40 ,&/cmz) an increase in R is fo~ntl. At the end of the first phase the same Z/IT relationship is obtained as before ouahain incubation.
Depol.
I (pA/cmz)
FIG. 10. Current voltage relationshipswhen Cl- Ringers cornea or in case ouabain is added to the aqueous side and taneous transoorneal p.d.‘s amounted to 25 (n. = 4), 50 negative = serosal potential negative. Abscissa: imposed potential change. dp.d. in mV (serosal potential positive).
Hyperpol.
(. . .) or SC)- Ringers (--) arc: bathing the Cl- Ringers bathing the cornea (- -. -). Span(I/ = 3) and 16 (1, = 3) mV resp. tear side current in ~A/cm”; ordinate: the resultin::
When propranolol (TS) or propranolol (TS) and ouahain (AS) together or amiloride (TS) are added to the bathing solution of the cornea the same rectifying properties exist as before addition of these substances. 4. Discussion The ion substitution experiments on rabbit cornea1 epitheiium clearly indicate different ionic selectivities of the limiting membranes at the AS and TS, i.e. the serosal and mucosal side of the epithelium respectively. The AS exhibits a passive K+selectivity only, as can be deduced from the fall in p.d. and R after increasing K+ activity at the AS. A passive Na+ selectivity at the AS could not be detected as is shown by the Cholinf for Na+ substitution. The small decrease in p.d. and increase in R after TEA+ for Naf substitution could be explained by the inhibiting effect of TEA+ on Na+-K+-ATPase as described in red blood cells (Sachs and Conrad, 1968).
TRAIYGEPITHELIAL
P.D.
AND
H OF RABBIT
(:ORKEA
4’3
In support, the change in p.d. and R is absent after ouabain. A Cl- selectivity at the AS could not be detected. During 15 min no R change was found. The p,d. change occurring after SO:- substitution was not due to the pernleability of one of the constituting membranes, because the same p.d. change took place even after the removal of the epithelium or endothelium. The pd. change could originat,e in the liquid junction between the AS and TS bathing solution separated by a stromal layer acting as a porous plug. In support of this is the finding of Van 0s and Slegers (1975) that a 1j.d. of 8.5 mV is developed when a 4 mu Cl- +4&S 111;~SO:- solution is connected via a 109 mM Cl- agar bridge to a 109 nlM Cl- solution. The pd. and R changes after SOi- for Cl- substitution are not in accordance with the finding of’ Klvce and Wang (1977) who found a small hyperpolarization (1 tnV) and an increase in R after 100 min, possibly due to cellular C- loss. Teleologically, any passive C’l- permeability of the AS which contains the Cl- pumping mechanism (Klyce et al.. 1977). would not be very efficient in transporbing Cl- from AS to TS. A TX S;L~. activity decrease induced p.d. and R changes, which suggest a Nazi selectivity of the TS membrane. In the same way a Kiselectivity can be attributed to the TS membrane. However, the selectivity does exist only for Ii-- activities greater than 35 1~1. Thih might be due to induction of selectivity or to a competitive effect at the Na-,-channels. The p.d. and R changes after SO:- for C- substitution indicate a Cl- selectivity of the TS meml)ranes of the epithelium. Other arguments for a (‘1~ selectivity of the TS are found in the 1/s’ relationships. As soon as Cl- was substit,uted by SO:- at the TS a, change in the 1/v plot occurred. An explanation can be found by accepting rect,ifying transport pathways as described by several authors for Dhe Na-t transljort, pathways in renal collecting tubules (Helman and Fischer. 1977) and for (‘lm- concluctance in toad skin (Bruus, Kristensen ancl Hviid Larsen, 1956; Hviicl Larsen ant1 Kristensen, 1978; Kristensen and Hviid Larsen. 1978). In toad skin hyperpolarizing currents increase the Cl- conductance. In SO:- conditions the rectifying (‘l- conductance of the membranes cannot interfere wit,h the current induced p.d. changes. As a consequence a linear 1/l’ relationship is obtained. Rectifying properties in thr cornea1 epithelium were also reported by Klyce (1972) in W Ringers. In corltrast to his findings we found for small de- and hyperpolarizing currents an almost linea’r part in the 1jv plot. In SOi- Ringers also a linear I/l’ plot was obtainecl. These tliscrepancies may be explained by the extrapolation procedures as mentioned in the met)hods section. Klyce (1972) also proposed a Cl ~ interference in the rect’ifying properties of the cornea. Tising the results of the ion substitution experiments an estimation can be made of the Nay-. K+, and Cl- resistances of the TS and AS membranes. In first instance the influence of a possible extracellular shunt-resistance (R,) is neglected. (El, = CO). Purthermore, no HCO, permeability is present in rabbit cornea1 epithelium as suggested by several authors (Ehlers, 1973; Klyce et al., 1973). In the epithelium a t !I-- resistance (R,J exists parallel to a Na+ resistance (R,,) at the TS and at the AS a K+ resistance (R,) is present in series with the former resistances. Figure 10 indicates a voltage independent R in SO:- conditions. Because of the Cl- free conditions R,., does not contribute to the total resistance which consequently consists of (R,, -t-R,). In Cl- conditions the total R is given by (l/RNa +1/R&l +R,. Extrapolation to zero Na+ activity in the TEA+ or Cholin+ for Na+ substitution experiments leads to a value of total R consisting of (R, +Rc,). The value of (RK + R,.,) includes the amiloride experiments also, because amiloride blocks the Na+ permeability. Moreover, (R, + R,:,) obtained by ion substitution experiments 4.5&O-6 kQcm2 (nz = 6), does not differ
494
(.‘. 11. A.
IT.
PES’I’~Es
AX I)
ti.
SLF:C:EI:S
significantly from the value obtained l),v the amiloritle espt4mcnt~+: i.e. 4.8 +O+ k!Z(:lll’ (7s = 4). ” R,:,+RIc = 6.5 l&‘cni” (‘1 (l/R,,+l/R,,)--l + R, = 3.4 kQcm” i”‘)
(RK +R,,) = 4.6 k.Qcm.”
(V
In open circuit the values of RSa, R,!, at the TS and R, at the AS can be calculatetl to be 5.1, 3.2 and 1.5 kficrn” respectively. The ratio of the TS resistance to total R(R,,/R,,,) amounts to 0.57 and is almost identical to the value of 0.60 publil;;M by Klyce (1973). The Na f, Cl- permeability ratio Ps;,/Pc!, = O-62 is in accordance with the P,,/P,., = 0.61 found for total epitheliurn as determined by nieans of tracer flux experiments (Klyce, 1975). In the calculation of PJP,., it, has been assumed that the translocation of Na i and Cl- across the AS membranes was no limiting factor for the tot,al Maimand (‘1 permeahility. When a finite shunt resistance consisting of a “tight junction” ant1 an “edge damage” part, is introduced Ijarallel to the comkinetl TS and AS resist,ances: the ratio of the above determined resistances does not change. However, the nt)solut,e value of the sepa,rate ion resistances increases. Another irilportant property of t#he shunt resistance and the tight junction part in particular, is its selectivity. The lack of pd. and R response after AS Cl- substitutions and the lack of p.d. and R chmges due to Na+ and K+ changes after ouahain point at a non-selective tight junction. In conclusion no interference of the tight junction with epithclial ion selectivity has t,cb he expected. Further information about the selectivity of the cpithelial membranes can IJ~ obtained by means of the ouabain incubation and subsequent ion substitution experiments. Ouabain gave rise to a two-phase step in p.d. decrease accompanied with a small R increase in the first and and a great decrease in the second phase. The still existing potential after ouahain incubation is probably the resilk of residual transport processes independent of Na -K+-ATPase (e.g. W-transport) but tlel)endent on active cell metabolism as corroborated by the reduction of t,he p.d. to zero after cooling. There are two possible explanations for the first’ phase increase in R. Firstly. the inhibition of the Nai-K+-ATPase by ouabain causes a rise in the cellular Na+ activity (Handler, Preston and Orloff. 1972) and thereby as a negative feetlhack mechanism, consisting of a decrease in permeable Nat-channels, a decrea,seof the X;n permeability and subsequently an increase in R (Hviitl Larsen, 1973; Lewis. and Diamond, 1976). The decrease in Na+ permeability is emphasized by t,he ahsenuc of changes in p.d. and R due to Na+ activity changes after ouabain. Moreover, t,he existen& of specific Na--sites in this tissue is corroborated by the amiloride induced increase in R (cf. Table II). On the other hand inhibition of ?Ja-:-K--ATPase could cause cell-swelling (Eggena, 1977) and the subsequent diminishing of the intercellular spaces could reduce the shunting effect of the paracellular pathway. The first phase drop in potential can be explained by the dissipation of the Na.7 and K+ gradients across the TS and AS membranes. Lewis. Eaton and Diamond (1976) suggested the second phase drop in R to be caused by cell lysis. Histologica, examination of cornea1 epithelium incubated in ouabain refuted this suggestion. The drop in R can be explained by means of the ion subsbitution experiments, in yart,icular SO:- for Cl- substitution before and after ouabain in the TS solution. The change in p.d. after ouabain, being about twice the change before ouabain, indicates a more pronounced Cl- permeability of the TS membrane. The increase in C!l-- permeability
TRANSEPlTHELIAL
P.D.
AND
B OF R,ARRIT
CORP;EA
4%
is compatible with the experiments on frog cornea (Candia, 1972; Candia, Bentley and Cook, 1974) where ouabain induced an increase in TS to AS Cl-flux and a transient increase in AS to TS Cl- flux followed by a decrease. possibly mediated by the inhihition of the Na+ transport. Additional arguments for an increase in Cl- permeability was found in experiments where t,he cornea was first treated with propranolol. In frog cornea this substance inhibits the stimulating effect of epinephrine on C’l- fluxes (Montoreano, Candia ant1 Cook, 1976). In rabbit cornea propranolol inhibited the Cl- permeability increase after onahain as can he concluded from the small increase in R instead of the large decrease. Furthermore, the I/V-relationships l)efore ouabain and after ouahain combined with propranolol do not differ markedlv. The I/ 1’ relationship aft’er ouabain is linear again, although the bathing medium contains (‘1 ~. A probable explanation i< t,he introduction of a non-rectifying C11-conductance neutralizing the tcctifying effect of the other or more simply a loss of the rectifving properties of the (‘1 cotIductance due t,o ouabain incubation. Any rect#ifying influence of the Na+ trallsport, pathways after inhibition of thrb Nat/K --ATPase may be neglected. The unchanged rectifying properties of thta cornea after amiloride point at a voltage independencv of the Ka+ channels also. Further experiments using micro-electrodes have to i,e performed in order to obtain the inbracellular p.d. and ion activities in different experimental conditiotis and t,herel)p to completje the model of cornea1 epithelium that describes t’hc ionic select,ivitic>s of t*hr different membranes and their transporting properties. ACKNO\T’LED(:,\IENl’S
This work was supported Pure Research (Z.W.O.).
by the Netherlands
Organization
for the Atlvatwetnrttt,
of
REFERENCES Aksikc. S. and Hori, &I. (1970). Effects of anions and cations on membmne potential of rabbit, cornea1 epit,helium. Am. J. Physiol. 219, 1811-18. Akaikr. i”\‘. (1971). The origin of the; basal cell potential in frog cornea1 epithelium. ./. P’hysiol.
(London) 219, 57-75. Augustus, J. H. M. G. (1978). Studies on a tight epithelium with a low specific resistance and A high potential difference. Ph.D. Thesis. University of Nijmegen. The Netherlands. Hruus, K., Kristensen, P. and Hviid Larsen, E. (1976). Pathways for chloride and sodium t,ransport across toad skin. Acta Physiol. Scar&. $V, 31-48. Cxndia, 0. A. (1972). Ouabain and sodium effects on chloride fluxes across the iso;a,trd bullfrog cornea. Am. J. Physiol. 223, 1053-7. Candia. 0. A., Bentley. P. J. and Cook, P. 1. (1974). Stimulation by amphothericin B of act,ive Na t,ransport across amphibian cornea. Sm. J. Physiol. 226, 143844. Davis. T. I,., Jackson, J. W., Day, B. E., Shoemaker, R. L. and Rehm, W. S. (1970). Potentials in frog cornea and micro-electrode artifact. Am. J. Physiol. 219, 178-83. Donn, A.. Maurice, M. D., Mills, N. L. (1959). Studies on the living cornea in vitro. II. Thr act’ive transport of sodium across the epithelium. Arch. Ophthdrnol. 62, 748-57. Eggena, P. (1977). Action of vasopressine, ouabain and cyanide on the volume of isolated toad bladder epithelial cells. J. Membrane Biol. 35, 29-37. Ehlers, N. and Ehlers, D. (1968). Effects of hydrostatic and colloid osmotic pressure on the electrical potential and short circuit current across the explanted rabbit cornea. Acta Ophthalmol. 46, 767-78. Ehlers, N. (1973). In vitro studies of trims- and intraepithelial potentials of t,he cornea. Bq. Eye RPX. 15, 553-65.
426
C’. 31. A. IV’. YES’L’ES
BSD
.I. F. (:. SLE(:ERS
Fee, J. I’. and Edelhauser, H. F. (1970). Intracellular electrical potentials in the rabbit coruc~il epithehum. Exp. f<‘ye Kes. 9, 234-40. Fischbarg, J. (1973). Artive and passive properties of the rabbit (*orneal cndot~heliunn. &.r/). /<‘;!I(’ Res. 15, 615638. Fischbarg, J.. Lim. J. J. (1974). Role of cations, anions and carbonic anhydrase in fluid transport across rabbit, cornea1 endothelium. J. Physiol. (London) 241, 647-75. Fromter, E. and Gebler, B. (1977). Electrical properties of amphibian urinary bladder epithclia. III. The cell membrane resistances and the effect of amiloride. Pfliigers Arch. 371,99--108. Fuchs, W., Larsen, E. H. and Lindemann, B. (1977). Current-voltage curve of sodium channels and concentration dependence of sodium permeability in frog skin. J. Ph?/sioZ. (Lo&on) 267, 137-66. Graves, C. N., Sanders, 8. S., Shoemaker, R. L. and Rehm, W. S. (1975). Diffusion resistance of endothelium and stroma of bullfrog cornea determined by potential response to K-’ . Biochinl. Biophys. Acta 389, 550-56. Graves, C. N., Sanders, 8. S., Shoemaker, R. L. and Rehm. W. S. (1976). An intact globe technique for elrotrophysiological studies on cornea. J. AppZ. Physiol. 40, 447-50. Handler, J. S., Preston, A. S. and Orloff, J. (1972). Effect of ADH, aldosterone, onabain and amiloride on toad bladder epithelial cells. An!. J. Physiol. 222, 10714. Helman. S. 1. and Fischer, R. S. (1977). Microelectrode studies of the act,ive Na t#ransport pathways of frog skin. J. Gen. Physiol. 69, 571-604. Hodson, S. and Miller, F. (1976). The bicarbonate ion pump in the endot’helium which regulates the hydration of rabbit cornea. J. Physiol. (London) 263, W-77. Hong, C. D. and Essig. A. (1976). Effects of 2-deoxy-n-glucose, amiloride, vasopressin and ouabain on active conductance and Exa in the toad bladder. J. Jlenrbrane Biol. 28, 121-42. Hviid Larsen, E. (1973). Effect of amiloride. cyanide and ouabain on the active transport pathway in toad skin. In Alfred Benzon Xymporlum V. Transport &lechanism.a in Epithelia (Eds ITssing, H. H., Thorn, N. A.). Copenhagen, Munksgaard. Hviid Larsen, E. and Kristensen, P. (1978). Properties of a conductive cellular chloride pathway in t’he skin of the toad (Hufo bufo). Actn Physiol. Scnnd. 102, l-21. Klyce, S. D. (1972). Elect,rical profiles in the eorneal epithelium. J. Physiol. (London) 226,407-29. Klyre, S. D.. Neufeld, A. H. and Zadunaisky, J. A. (1973). The activation of chloride transport by epinephrine and Db-cyclic AMP in t,he cornea of the rabbit. Iwcest. Ophthala~ol. 12,127-39. Klyce. S. D. (1975). Transport of Sa. Cl, and water by the rabbit, cornea1 epithelium at resting pot’mtial. Am. J. Phynid. 228, 1446-52. Klyce, S. D., ITong, K. S. (1977). Site and mode of adrenaline action on chloride t,ransport across the rabbit cornea1 epithelinm. J. Physiol. (London) 266,777-99. Kristensen, P. and Hviid Larsen, E. (1978). Relation between exchange diffusion and a conductive chloride pathway across t,he isolated skin of the toad (&fo bufo). Actn Physiol. ihwd. 102, 22234. Lewis, 8. A. and Diamond, J. M. (1976). Na t transport by rabbit urinary bladder: a tight. epithelium. J. &Zembra?ee Biol. 28, l-46. Lewis, S. A., Eaton, D. C. and Diamond, J. MI. (1976). The mechanism of Na transport by urinary bladder. J. Membrane Biol. 28, 41-71. Lindemann, B. (1968). Resting potential of isolated beef cornea. Exp. Eye Res. 7, 6269. Maurice, D. M. (1972). The location of the fluid pump in the cornea. J. Physiol. (Londor~) 221,43-54. Xshima, S. and Kudo, T. (1967). In vitro incubation of rabbit cornea. Incest. Ophthalmol. 6,329-39. Montoreano. R., Candia, 0. A. and Cook, P. (1976). Alpha- and beta-adrenergic receptors in regulation of ionic transport in frog cornea. Am. J. Physiol. 230, 1487-93. Riley, M. S. (1971). The role of the epithelium in control of cornea1 hydration. fiq. Eye Kes. 12, 128-37. Robinson, R. A. and Stokes, R. H. (1970). Electrolyte Solutions, 2nd ed. Butterworths, London. Sachs, J. R,. and Conrad, M. E. (1968). Effect of tetra-ethyl ammonium on the active cation transport system of the red blood cell. Am. J. Physiol. 215, 796-8. Ussing, H. H. and Zerahn, K. (1951). Active transport of sodium as the source of electric current in the short circuited isolated frog skin. Acta Physiol. Stand. 23, 110-27. Van der Heyden, Ch., Weekers, J. F. and Schoffeniels, E. (1975). Sodium and chloride transport across the isolated rabbit cornea. Exp. Eye Res. 20, 89-96. Van OS, C. H. and Slegers, J. F. G. (1975). The electrical potential profile of gallbladder epithelium. J. Membrane Biol. 24, 341-63.
The Influence of Ions, Ouabain, Propranolol and Amiloride on the Transepithelial Potential and Resistance of Rabbit Cornea C’. M.
A. W. FESTEN
Depnrtment of Ceil-Phllsioloyy,
ASD
J. F. G.
SLEGERS
Uwhersity of N[jnwcJe~~~S2j’megen. The Netherlards
(Received 27 June 1978. Sew I’ork) By means of in vitro ion substitution experiments the potential difference and resistance dependency of rabbit cornea1 epithelium on h’a +: I+ and Cl- activities have been established at the t,ear side as well as at the aqueous side. In normal conditions only a Ii+ selectivity at the aqueous side of the epithelium exists and a Na- and Cl- select,ivity at the tear side. The tear side was also K+ selective at Ii+ artivities greater than 38 maI. In Cl- Ringer ba,thing solutions a rectifying currenevoltage relationship is obtained. Ouahaingives rise to a two phase decrease in potential from 29.9i2.4 to 22.912.1 to 14.4h1.7 rn% (7~ -:= 9). In the first phase the resistance changes from 3.720.4 to 4.0&0.4 k&m” (n == 9) md in t,ht: second phase R decreased t,o 1.3hO.l kQcm2 (7~= 9). After ooabain the Sa+ selectivity has disappeared and the Cl- permeability has increased. The current-voltage relationship after ouabain has become linear. Amiloride induced a reversible change in resistance from 3~.SZ~O~fito 4,X-+,0+3 k&m” accompanied by a depolarization from 29.1 h2.7 to 22+iZ 1.5 mV (n = i), moreover the rectifying properties as exhibited in control cornea are not affected by amiloride. A voltage dependent Cl--conductivity of the tear side membranes of the cornea1 epithelium can be postulated as well as a voltage independent Cl--ronduc+irit> that can be induced by ouahain. The onabain effects are inhibited by propranolol. Key words: rabbit; cornea; epithelium ; ionic select,irity; ouabain; propranolol; amiloride: (~rrent-voltage relationship.
1. Introduction The degree of hydration of rabbit cornea and tht?relJ!iti; transparency is tlel~endent~ on the proper function of the endothelium (Mishima, and Kutlo: 1967 ; Maurice, 1972 : Fischbarg and Lim, 1974) as well as of the epithelium (Riley, 1971; Klyce, 1975). The epithelium is the place where most of the transcorneal pot,ential difference (p.(l.) and resistance (R) are located (Klyce, 1952; Ehlers. 1973; Clandia. Bentley and C!ook, 1974; Festen and Slegers, unpublished data). This layer is known to transport actively Na+ from the tear side (TS) towards the aqueous side (AS) (Donn. Maurice and Mills? 1959; Klyce, 1975; Van der Heyden, Weekers and Schoffeniels, 1975) and CY from AS to TS (Klyce, Neufeld and Zadunaisky, 1973; Klyce, 1975; Van tier Heyden et al., 1975). The contribution of the endothelium to p.d. and R is of minor importance. According to most authors the endothelium is characterized by bicarhonate transport (Fischbarg and Lim, 1974; Hodson and Miller, 1976). In order to obtain the ion-selectivities of the different constituting membranes of the cornea severa, authors have carried out microelectrode as well as ion substitution experiments in frog (Akaike, 1971) and rabbit (Akaike and Hori, 19iO; Fee and Edelhauser, 1970). These studies point towards a K+ and Cl- selectivity of the hasal cells. Frog transcornea1 p.d. and R both declined with increasing K-~ activity at the AS and basal cells of frog cornea1 epithelium showed a K+- and C!l--electrode behaviour (Graves. Sanders. Shoemaker and Rehm, 1975, 1976). In beef cornea1 epithelium lowering the TS Na+-activity decreased p.d. and increased R (Lindemann, 1968). Although at present most workers on cornea1 physiology use rabbit cornea no systematical 0014-4835/79/040413+14
:c] 1979 Academic Press Inc. (London) Limited
$01.00/0 413
114
C’. M. A. \v.
FESTEK
AN11 .l. F. t:. I;LEGERS
measurements of ion selectivities are known t,o exist for this tissue. In orclcr to characterize the rabbit cornea1 epitheliurn, pd. and R dependency on Na i-, K am1 Cl- activities are studied for both sides of the menlhrane.
2. Materials and Methods New Zealand white rabbits weighing 2-2.5 kg were killed by means of a blow ilI the neck and bleeding. The eyes were enucleated and kept in a moist atmosphere at 4’C. Storage of whole eyes at 4°C for several hours did not influence the studied electrophysiological parameters, i.e. pd. and R. The cornea including a scleral ritlg of 2 mm \GLS isolated without any wrinkling and transferred to a modified Vssing chamber (1Jssing and Zerahn, 1951) (cf. Fig. 1) adapted to fit the curvature of rabbit cornea. The edges were greased with silicone grease to improve sealing and minimize edge damage.
FIG. 1. A schematic diagram of the experimental set-up. For det,ails see text,.
The bathing solution contained the following concentration of substances in mu: NaCl, 110; NaHCO,, 39; KHCO,, 3%; KH,PO,. 1.0; CaCl,, 1.5; MgSO,. 7H,O, 1.0; glucose, 25; adenosine. 5.0; reduced glutathione, 1.0; I,-ascorbic acid, 1.5. Osmolalitv was adjusted to 315 mosmol with mannitol and the bathing solution was aerated witk 95% 0, and 5% CO, and adjusted to pH = 7.4. Temperature was kept at 35°C. Au intraocular pressure of 15cn1 H,O was obtained by raising the outflow opening 15 cm above the cornea (cf. results section). Changes in ion composition of the bathing solution were on equimolar basis, except for 110 rn?Y1Cl- which was replaced by 55 mM SO?-- with compensation for osmolality loss. Sctivity coefficients were obtained from Robinson and Stokes (1970). Flow rate of the bathing solution at TS and AS was 1 ml/mill ~(1 t,he outflowing solution was discarded. Changes in bathing solutions could be made without damage to p.d. or R. At the TS changes in ion composition had their maximal effect in less than 2 min, inclusive the time necessary for complete wash-out. Adenosine, glutathione, L-ascorbic, acid, ouabain (g-strophanthin, LUerck), inderal (propranolol, ICI) and amiloride (Merck, Sharp 8r. Dohme) were added to the bathing solution just before each experiment. With a four Ag/AgCl electrode system (cf. Fig. 1) trnnscorneal pd. and K could be measured. Electrical contact between the bathing solution and the Ag/AgCl electrode was made by means of 3~-KC1 filled microelectrodes with broken iips (diameter < 50 pm). Due to schematizing, the pd. measuring electrodes in Fig. 1 are situat,ed further from the cornea1 surface than they actually were. Via high
TKANSEPITHELIAL
P.D.
AND
R OF RABBIT
CORNEA
11.5
input impedance amplifiers (52 K, Analog Devices) the p.d. measuring electrodes were connected to a differential amplifier (52 K, Analog Devices) and the signal was recorded with a pen-writer. Liquid junction potentials between experimental bathing solutions connected via 3 ~-Kc1 agar bridges never exceeded 1 mV. The instantaneous R wi1.s determined measuring the transcorneal voltage response to bipolar current pulses of 100 msec duration and a magnitude < 5 PA/ems normally. The c,ontribution of polarization was neutralized by extrapolating the voltage response to t --f 0 (Augustus, 1978). Ripolar current pulses were obtained by int,roducing bipolar voltage pulses from it. Tektronix pulse generator (Tektronix inc.) to a voltage controlled current source using :I. 44 W amplifier (Analog Devices). Current pulse magnitude was measured by means of au amplifier (234 L, Analog Devices) in the ground return of the current loop. Current pulses and corresponding transcorneal voltage responses were monitored on a Tektronix storage oscilloscope. I/V-plots were obtained by varying the magnitude of the voltage pulses supplied to the voltage controlled current source. The measurement of a currelitvoltage relationship was completed within 6 min without affecting spontaneous trailscornea1 pd. xntl R. Temperature on both sides of the cornea was registered by means of thermocouples. The mean value of a parameher is always given with the standitr~l error of the inearl (S.E.M.). 3. Results Bfect of intrnoculur poressure Using the normal Ringer solution the potential differences and resistances are measured when p.d. and R had stabilized with a pressure gradient of 15 cmH,O (1.5 kPn), AS positive. Within 30 min pd. was 27.3k1.5 mV (TS negative) ant1 R 3.4~0.3 k&n” (mean &s.E.M., ~1, = 20). Incubating the cornea from the beginning without’ a pressure gradient gives statistically the same p.cl. and R values. The reason why a pressure drop is used in the presented experiments is twofold: it mimics the physiological situation and in planned experiments the pressure gradient will be needed for correct observation. Lowering the pressure gradient, in case t.he above mentioned stabilization was reached, resulted in a reversibly lower value for the 1j.d. as well as R. This effect cannot be attributed to a diminished streaming potential because lowering the temperature to zero. reduced the p.d. to zero in the absence or presence of a pressure gradient. In case of a streaming potential one would expect tJofind only a drop corresponding with the RTjF t,erm of the Nernst equation. Increasing the pressure gradient above 20 cmH,O at the AS irreversible pd. and K drops took place. In general these findings confirm the results of Ehlers and Ehlers (1968). The irreversible changes introduced at higher pressure probably may he due to edge damage effects. The reversible changes observed on lowering the pressure cannot~ be explained at present. A lack of sealing may be the reason, These findings indicate that once the cornea has stabilized the pressure gradient cannot be altcretl freelv without effect)s on pd. and R.
Figure 2 shows the results of equimolar substitution of Ii-+ for Na- at the AS of the cornea. Increase in K+ activity produces a depolarization of 25 mV per decade of the transcorneal p.d. The decrease in p.d. is accompanied by a decrease in R from 3-550.4 to 2*2*0*2 k&&n2 at a potassium activity of 89 rnM as is shown in Fig. 2 also. After changing the KT activity a steady state value of p.d. and R was reached within 8 min due to diffusive effects in the stromal layer as mentioned also t)v Graves
416
(1. .\I. A. \r.
VES’I’ES
ASI)
J. I’. c;. SLE(:EI(S
et al. (19’75) in frog cornea. Substituting Choline 1 for NX did not significallt,lv altot, p.d. or R. However, substituting Tetraet,hyl ammonium (TEAM+) t’or Na-; did protlu(:ta a reVersiiJle
depOhrizatiO1~
Of
7.5
lllV/deC
accOInpI~icd
hV
811 R
ilmws?.
Complete substitution of SC!:- for Cl on the AS produce(I an instwntanel~u~ depolarization of 7 mV, without any change in R for over a period of 15 min. Tht same result was obtained in case the epithelium and/or t,he enclnthclium had I~cll removed I )y scraping, with or without a pressure gradient. In symmetrical (‘lm or SOi- conditions with or without a pressure gradient when the epithelium had t)et,n scraped off a p.d. was obtainecl not significantly different from zero.
IOL
1
5
I
I
I
IO
50
100
UK+. AS
FIG. 2. The effect of changing the K+ activity (given in m&r) in the aqueous side bathing solution on the transcorneal p.d. (0-O in mV, tear side negative) and R (0-0, kQcm2), by means of Sa--/K+ substitution. The bar indicates S.E. of the mean. 11= number of observations.
Tear side ion substitution
Increase in K+ activity at the TM (Na+ substituted) gave a response in p.d. and R as shown in Fig. 3. Below 28 mM-K+- there is no significant change in p.d. and only a small decrease in R. Above 28 mM-K+ a hyperpolarization of transcorneal p.d. of 11 mV/dec existed with a simultaneous decrease in R from 26 ho.2 kQcm” to 1.6 ho.2 kOcm2 (,n = 6) at 89 maf K+ activity. When Na+ in the TS solution was replaced 1~~ choline+ or TEA+, a depolarization of 15 and 21 mV/dec was measured, respectively. At the same time R increased. However, the increase in R produced by Na+/TEA+substitution was greater than in tbe case of Nat/choline’- substitution, i.e. a decrease in Na+ activity to 32 mM gave an increase in R to 150 and 12094 of the original value respectively. Figure 4 shows the effect of Na+/TEA+ substitution at the TS on the p.d. and R. The combined effect of Na+ and K+ on the p.d. in the TS Na+/K+ substitution (vide supra) is shown in Fig. 5. Above 28 mM-K+ a pd. change of 29 mV/dec can be estimated. In an additional experiment with a potassium activity of 32 mni and Na+ substituted by TEA+, equimolar substitution of K+ for TEA+ gave a hyperpolarization of 21 mV/dec.
TRANSEPITHELIAL
P.D.
AND
R OF RABBIT
CORXEA
417
SOj- for Cl-- substitution gave rise to a transient hyperpolarization accompanied by an increase in R. The plateau of the hyperpolarization reached in 2 min was plotted against the Cl- activity (Fig. 6). At high C- activity a change of p.d. of 25 mV/dec was measured. The fall in pd. after the initial hyperpolarization had a t.+ of 2.7 min and started a few minutes after reaching the plateau. Decreasing the (‘I activity to 3 IllM, R increased to 6.5 +@7 kcme (II = 7) and p.tl. increased to $5.0&,34~ mV ‘I3 negative 02 = 7, cf. Fig. 6).
n=6
35 -
(Nat/K+)
30 1
25-
‘j E d ci
20I5 -
i-+--y
-t
---___ ---+
i ‘\ \
IO i
\. ‘.
50
i
I 5
I IO
I 50
\
I 100
’
UK+, TS
Fro. 3. The effect of changing the K+ activity (given in maa) in the tear side bathing solution on the transoorneal p.d. (O-O, in mV, tear side negative) and R (m-0, Mcm2) by means of Na+/K+ substitution. The bar indicates S.E. of the mean, 12= number of observations.
25
oNo +, TS FIG. 4. The effect of changing the Na+ activity (given in mM) in the tear side bathing solution on the transcorneal p.d. (O-O, in mV, tear side negative) and R (0-0, k&m?), by means of Na+/TEA+ substitution, )A = number of observations.
D
Addition of ouabain to the A8 resulted in a biphasic effect, on the p.tl. awl 1: ot cornea1 epithelium. Typical examples for 10e3 M and 1O-4 M ouabain are show in Fig. 7. The value of p.d. and R during 10m3RI and lo-* M ouabain iucul)ation in tJhck
50: 5
50
IO
100
OK+, TS FIG. 5. Figure 4 suggests the K+ effect, on p.d. and 11 to be greater than S~IIU n in Fix. :S. ‘l’hc~~~eli~~ the results of Fig. 3 and Fig. 4 are combined. The pd. part of Fig. 3 has bcru taken in this figu~.ts as a pwcrnt~age of the pd. for 4 mx K’ at the t’ear side (e-0). The pd. part of Fig. 4 has Iwu t,llwn a.c :L in the same way, only instead of expressing the p.d. as a function of the Saf acti\-ity it is es~wssc~tl function of the complement of the Ka+ activity, i.e. in this case the Ii7 activity (:3-c!). \\:h~t~ I IICSC plots are combined the real K+ effect is obtained for K+ actirit,,v changes at the tear side (?i: -*).
I 5
I IO
I 50
, 100
oCk+. TS (mkl)
FIG. 6. The effect of changing the Cl- activity (given in mM) in the tear side bathing solution, on transcorneal p.d. (O-0, in mV, tear side negative) and R (O-0, M2cm2), by means of Cl-/SO:-. substitution. The bar indicates S.E. of the mean, 1~= number of observations.
TRANBEPITHELIAL
P.D.
AND
R OF RABBIT
CORPL’EA
419
different phases did not differ significantly and were therefore taken together. At first, there was a decrease in p.d. from 29.912.4 to 22.9&2*1 mV and a small increase in R from 3.710~4 to 4.0-&0*3 kQcm2 (a = 9). Secondly, p.d. as well as R decreased. P.d. fell to 14*4&l-7 mV and R to 1*3&0.1 k.Qcm* (IZ = 9). The same effects were seen when ouabain was applied at the TS and the AS and when the endothelium was removed. The duration of the p.d. and R change due to ouabain was at 10-3 M 18 +l min (n = 5) for the first phase and 1352 min to a new steady-state in the second phase. At 1O-4 M-ouabain these values were 35&5 min and 32&7 min (1~ = 4). respe&ively.
1111
1
I
I
I
I
369
15
30
45
60
75
Time (mln after addltlon)
FIG. 7. ‘l’,ypiczl exunplrs of the effect of 10-4 JI ( ;:) and lo-” JI (C) ouabain on the aqueous sink. time nftcr addition in min; ordinate: transcorneal pd. as a pcrccntapr of the starting vale ( ~. --) ;tt~l R as a percentage of the starting ralur (- - -).
-kbwiss;i.:
In t,he second phase steady-state (10e3 Jr-ouabain) equimolar K-1 substitution for N;t+ gave a p.d. and R change as shown in Fig. 8. Increasing the Ii+ :totivit,v resulted in a depolarization of 16 mV/clec for a pota~ssimn activity ~.:.2%in11 and of ,“8 mV/dec for a potassium activity > 25 mu. At the Same time there was a IIIOP~ I)ronounce(l drop in R at higher potassium activities. TEA- nor ~‘holinc- for lYa 7 srllxtitut,ion at the AS did produce a change in 1j.d. Or R. Replacing Nar 1)~ TEA ’ Or (‘holine’ had no effect on p.d. or on R. So:- for (‘1 snhstitution did produce the s:ime effect as before ouabain, i.e. an instantaneous depolarization of 7 mV ill caqe of clrcrcnsc~ in n Cl -. ,4S from 89 to 3 11131.
Replacing Na+ by either K+ or TEA+ had no effect on the p.d. or R. Changing Clfor SCY-- gave a transient hyperpolarization and a simultaneous increase in R with essentially the same time course as before ouabain. The results are taken together in Fig. 9. At a Cl- activity of 3 mM p.d. increased to 51-O&3+0 mV and R to 5*2&O-3. kQcm2 (9~= 5). At high Cl- activity a hyperpolarization of 54 mV/dec existed.
The p.d. and R changes due to 20 min TS pre-incui,ation with IO- 5 propr’~1101~11 followed by incubation with 10~~M propranolol TX and lo-” M ouabain AS. compared to the effects of 10m3M ouabain AS alone are given in Table I as a percentage of t'hr original value. Propranolol did, not significantly, decrease p.d. and increase R. ( ‘OIIIbined further, propranolol and ouabain incubation as well as Oua~Jain alone decreascti the original p.d. to 489;;. However, due to propranolol and ouabain together R
1 4
15 IO -
-3 1
=
5 & d Ii
o-
f r i 2 Y k
-5-
-10I?=5 (No+MK+) after ouabom lncubctmn
-15 -20
-2
Id I
) 5
IO
xl
100
OK+. AS CrnM)
FIG. 8. The effect of changing the K+ activity (given in mM) in the aqueous side bathing solution kQcn,2) after ouabain addition, on transcorneal p.d. (O-O, mV, tear side negative) and IZ (m-6, by means of Na+/K+ substitution, 1~= number of observations.
30 -
20 -
n-5 (cl--so:-,
IO -
after cuabain incubation
t”l,
“\ ‘t
I 5
I
I 50
IO oCl-,TS
2
\ I _ loo’
(mu)
FIm. 9. The effect of changing the Cl- activity (given in mm) in the tear side bathing solution after vuabain addition, on transcorneal p.d. (O-O, mV, tear side negative) and R (e-e, kQcm*) by means of Cl-/SO:- substitution, n = number of observations.
TKANSEPITHELIAL
P.D.
AKD
H OF RABBIT
CORNEA
-121
increased by 13:/A while ouabain alone decreased R by 67.53;. Furthermore, the two phase decrease in p.d. after ouabain changed into a single exponential decrease with 1)ropranolol pre-incubation, t, being 15 min. TABLE I
‘I(, {If original valur of pd. and R after ouahain (1OF IN, AiS) proprauolol (IIJP M, ‘I%) : pro~wanolol in the stratlp state. 3lean *s.E.M. Number of ohscrrntions in pawnthescs.
and
ouahnin
Arniloride f$fects on pd. and R In CV as well as in SO?- Ringer bathing the cornea, there was no effect of 3 x 10m5M amiloride (TS) on R, but the p.d. decreased by a few mV. The results of addition of 5 x 10P4M amiloride at the TS are summarized in Table II. Within each experiment p.d. depolarized and R increased reversibly. The mean p.4. drop in four experiments was from 29.1k2.7 mV to 22.6kl.5 mV. R changed in t,hese experiments from 3.5 396 k1;2cm2to 48&0.8 kQcm2.
The effects of amiloride on trarwcorlzeal pd. wd K
The effect of amiloride (5;~ IO-* nr) at the tear sick on transcorueal in ltQcm2). Mean &s.E.>I. Kumbcr of observations in parentheses.
pd. (t(iwn in my) ;ud Ii (given
Cwrent-voltage relationships before and after owzbni~n In Fig. 10 the current-voltage relationships (Z/V plots) before and after ouabain are shown. In normal conditions, i.e. Cl- Ringer bathing the cornea rectifying properties can be assigned to the epithelium. Hyperpolarizing currents gave rise to smaller p.d. responses than depolarizing currents. As a result R, in hyperpolarizing direction (for Z > 20 pA/cm2) amounts to 2.2 k.Qcm2. In depolarizing direction R = 5.3 kQcm2. In the Z/V relationship three almost linear parts can be observed. i.e. for great de- and hyperpolarizing currents and the region between lOt~~/crn~
4”J?
(‘. Ill. A. b\.. FE:STEK
ANI)
.I. F. c;. Sl,E(:E’:Ks
depolarizing and 10 pA/cn’g hyperpolarizing (Breaking points are fountl at --YS tu\:, - 12 ,A/cn1” and +44 mV/ +14 ~A/cm2). In SOi Ringer the rectifying l)roptlrtif+ have disappeared and only for I > 30 ,&/cm’ symmetrical deflections take 11la.c~. R becoming smaller. In the presence of ouabain there are 110 rectifying lnq)e&s either. Only at high depolarizing current (1 > 40 ,&/cmz) an increase in R is fo~ntl. At the end of the first phase the same Z/IT relationship is obtained as before ouahain incubation.
Depol.
I (pA/cmz)
FIG. 10. Current voltage relationshipswhen Cl- Ringers cornea or in case ouabain is added to the aqueous side and taneous transoorneal p.d.‘s amounted to 25 (n. = 4), 50 negative = serosal potential negative. Abscissa: imposed potential change. dp.d. in mV (serosal potential positive).
Hyperpol.
(. . .) or SC)- Ringers (--) arc: bathing the Cl- Ringers bathing the cornea (- -. -). Span(I/ = 3) and 16 (1, = 3) mV resp. tear side current in ~A/cm”; ordinate: the resultin::
When propranolol (TS) or propranolol (TS) and ouahain (AS) together or amiloride (TS) are added to the bathing solution of the cornea the same rectifying properties exist as before addition of these substances. 4. Discussion The ion substitution experiments on rabbit cornea1 epitheiium clearly indicate different ionic selectivities of the limiting membranes at the AS and TS, i.e. the serosal and mucosal side of the epithelium respectively. The AS exhibits a passive K+selectivity only, as can be deduced from the fall in p.d. and R after increasing K+ activity at the AS. A passive Na+ selectivity at the AS could not be detected as is shown by the Cholinf for Na+ substitution. The small decrease in p.d. and increase in R after TEA+ for Naf substitution could be explained by the inhibiting effect of TEA+ on Na+-K+-ATPase as described in red blood cells (Sachs and Conrad, 1968).
TRAIYGEPITHELIAL
P.D.
AND
H OF RABBIT
(:ORKEA
4’3
In support, the change in p.d. and R is absent after ouabain. A Cl- selectivity at the AS could not be detected. During 15 min no R change was found. The p,d. change occurring after SO:- substitution was not due to the pernleability of one of the constituting membranes, because the same p.d. change took place even after the removal of the epithelium or endothelium. The pd. change could originat,e in the liquid junction between the AS and TS bathing solution separated by a stromal layer acting as a porous plug. In support of this is the finding of Van 0s and Slegers (1975) that a 1j.d. of 8.5 mV is developed when a 4 mu Cl- +4&S 111;~SO:- solution is connected via a 109 mM Cl- agar bridge to a 109 nlM Cl- solution. The pd. and R changes after SOi- for Cl- substitution are not in accordance with the finding of’ Klvce and Wang (1977) who found a small hyperpolarization (1 tnV) and an increase in R after 100 min, possibly due to cellular C- loss. Teleologically, any passive C’l- permeability of the AS which contains the Cl- pumping mechanism (Klyce et al.. 1977). would not be very efficient in transporbing Cl- from AS to TS. A TX S;L~. activity decrease induced p.d. and R changes, which suggest a Nazi selectivity of the TS membrane. In the same way a Kiselectivity can be attributed to the TS membrane. However, the selectivity does exist only for Ii-- activities greater than 35 1~1. Thih might be due to induction of selectivity or to a competitive effect at the Na-,-channels. The p.d. and R changes after SO:- for C- substitution indicate a Cl- selectivity of the TS meml)ranes of the epithelium. Other arguments for a (‘1~ selectivity of the TS are found in the 1/s’ relationships. As soon as Cl- was substit,uted by SO:- at the TS a, change in the 1/v plot occurred. An explanation can be found by accepting rect,ifying transport pathways as described by several authors for Dhe Na-t transljort, pathways in renal collecting tubules (Helman and Fischer. 1977) and for (‘lm- concluctance in toad skin (Bruus, Kristensen ancl Hviid Larsen, 1956; Hviicl Larsen ant1 Kristensen, 1978; Kristensen and Hviid Larsen. 1978). In toad skin hyperpolarizing currents increase the Cl- conductance. In SO:- conditions the rectifying (‘l- conductance of the membranes cannot interfere wit,h the current induced p.d. changes. As a consequence a linear 1/l’ relationship is obtained. Rectifying properties in thr cornea1 epithelium were also reported by Klyce (1972) in W Ringers. In corltrast to his findings we found for small de- and hyperpolarizing currents an almost linea’r part in the 1jv plot. In SOi- Ringers also a linear I/l’ plot was obtainecl. These tliscrepancies may be explained by the extrapolation procedures as mentioned in the met)hods section. Klyce (1972) also proposed a Cl ~ interference in the rect’ifying properties of the cornea. Tising the results of the ion substitution experiments an estimation can be made of the Nay-. K+, and Cl- resistances of the TS and AS membranes. In first instance the influence of a possible extracellular shunt-resistance (R,) is neglected. (El, = CO). Purthermore, no HCO, permeability is present in rabbit cornea1 epithelium as suggested by several authors (Ehlers, 1973; Klyce et al., 1973). In the epithelium a t !I-- resistance (R,J exists parallel to a Na+ resistance (R,,) at the TS and at the AS a K+ resistance (R,) is present in series with the former resistances. Figure 10 indicates a voltage independent R in SO:- conditions. Because of the Cl- free conditions R,., does not contribute to the total resistance which consequently consists of (R,, -t-R,). In Cl- conditions the total R is given by (l/RNa +1/R&l +R,. Extrapolation to zero Na+ activity in the TEA+ or Cholin+ for Na+ substitution experiments leads to a value of total R consisting of (R, +Rc,). The value of (RK + R,.,) includes the amiloride experiments also, because amiloride blocks the Na+ permeability. Moreover, (R, + R,:,) obtained by ion substitution experiments 4.5&O-6 kQcm2 (nz = 6), does not differ
494
(.‘. 11. A.
IT.
PES’I’~Es
AX I)
ti.
SLF:C:EI:S
significantly from the value obtained l),v the amiloritle espt4mcnt~+: i.e. 4.8 +O+ k!Z(:lll’ (7s = 4). ” R,:,+RIc = 6.5 l&‘cni” (‘1 (l/R,,+l/R,,)--l + R, = 3.4 kQcm” i”‘)
(RK +R,,) = 4.6 k.Qcm.”
(V
In open circuit the values of RSa, R,!, at the TS and R, at the AS can be calculatetl to be 5.1, 3.2 and 1.5 kficrn” respectively. The ratio of the TS resistance to total R(R,,/R,,,) amounts to 0.57 and is almost identical to the value of 0.60 publil;;M by Klyce (1973). The Na f, Cl- permeability ratio Ps;,/Pc!, = O-62 is in accordance with the P,,/P,., = 0.61 found for total epitheliurn as determined by nieans of tracer flux experiments (Klyce, 1975). In the calculation of PJP,., it, has been assumed that the translocation of Na i and Cl- across the AS membranes was no limiting factor for the tot,al Maimand (‘1 permeahility. When a finite shunt resistance consisting of a “tight junction” ant1 an “edge damage” part, is introduced Ijarallel to the comkinetl TS and AS resist,ances: the ratio of the above determined resistances does not change. However, the nt)solut,e value of the sepa,rate ion resistances increases. Another irilportant property of t#he shunt resistance and the tight junction part in particular, is its selectivity. The lack of pd. and R response after AS Cl- substitutions and the lack of p.d. and R chmges due to Na+ and K+ changes after ouahain point at a non-selective tight junction. In conclusion no interference of the tight junction with epithclial ion selectivity has t,cb he expected. Further information about the selectivity of the cpithelial membranes can IJ~ obtained by means of the ouabain incubation and subsequent ion substitution experiments. Ouabain gave rise to a two-phase step in p.d. decrease accompanied with a small R increase in the first and and a great decrease in the second phase. The still existing potential after ouahain incubation is probably the resilk of residual transport processes independent of Na -K+-ATPase (e.g. W-transport) but tlel)endent on active cell metabolism as corroborated by the reduction of t,he p.d. to zero after cooling. There are two possible explanations for the first’ phase increase in R. Firstly. the inhibition of the Nai-K+-ATPase by ouabain causes a rise in the cellular Na+ activity (Handler, Preston and Orloff. 1972) and thereby as a negative feetlhack mechanism, consisting of a decrease in permeable Nat-channels, a decrea,seof the X;n permeability and subsequently an increase in R (Hviitl Larsen, 1973; Lewis. and Diamond, 1976). The decrease in Na+ permeability is emphasized by t,he ahsenuc of changes in p.d. and R due to Na+ activity changes after ouabain. Moreover, t,he existen& of specific Na--sites in this tissue is corroborated by the amiloride induced increase in R (cf. Table II). On the other hand inhibition of ?Ja-:-K--ATPase could cause cell-swelling (Eggena, 1977) and the subsequent diminishing of the intercellular spaces could reduce the shunting effect of the paracellular pathway. The first phase drop in potential can be explained by the dissipation of the Na.7 and K+ gradients across the TS and AS membranes. Lewis. Eaton and Diamond (1976) suggested the second phase drop in R to be caused by cell lysis. Histologica, examination of cornea1 epithelium incubated in ouabain refuted this suggestion. The drop in R can be explained by means of the ion subsbitution experiments, in yart,icular SO:- for Cl- substitution before and after ouabain in the TS solution. The change in p.d. after ouabain, being about twice the change before ouabain, indicates a more pronounced Cl- permeability of the TS membrane. The increase in C!l-- permeability
TRANSEPlTHELIAL
P.D.
AND
B OF R,ARRIT
CORP;EA
4%
is compatible with the experiments on frog cornea (Candia, 1972; Candia, Bentley and Cook, 1974) where ouabain induced an increase in TS to AS Cl-flux and a transient increase in AS to TS Cl- flux followed by a decrease. possibly mediated by the inhihition of the Na+ transport. Additional arguments for an increase in Cl- permeability was found in experiments where t,he cornea was first treated with propranolol. In frog cornea this substance inhibits the stimulating effect of epinephrine on C’l- fluxes (Montoreano, Candia ant1 Cook, 1976). In rabbit cornea propranolol inhibited the Cl- permeability increase after onahain as can he concluded from the small increase in R instead of the large decrease. Furthermore, the I/V-relationships l)efore ouabain and after ouahain combined with propranolol do not differ markedlv. The I/ 1’ relationship aft’er ouabain is linear again, although the bathing medium contains (‘1 ~. A probable explanation i< t,he introduction of a non-rectifying C11-conductance neutralizing the tcctifying effect of the other or more simply a loss of the rectifving properties of the (‘1 cotIductance due t,o ouabain incubation. Any rect#ifying influence of the Na+ trallsport, pathways after inhibition of thrb Nat/K --ATPase may be neglected. The unchanged rectifying properties of thta cornea after amiloride point at a voltage independencv of the Ka+ channels also. Further experiments using micro-electrodes have to i,e performed in order to obtain the inbracellular p.d. and ion activities in different experimental conditiotis and t,herel)p to completje the model of cornea1 epithelium that describes t’hc ionic select,ivitic>s of t*hr different membranes and their transporting properties. ACKNO\T’LED(:,\IENl’S
This work was supported Pure Research (Z.W.O.).
by the Netherlands
Organization
for the Atlvatwetnrttt,
of
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(London) 219, 57-75. Augustus, J. H. M. G. (1978). Studies on a tight epithelium with a low specific resistance and A high potential difference. Ph.D. Thesis. University of Nijmegen. The Netherlands. Hruus, K., Kristensen, P. and Hviid Larsen, E. (1976). Pathways for chloride and sodium t,ransport across toad skin. Acta Physiol. Scar&. $V, 31-48. Cxndia, 0. A. (1972). Ouabain and sodium effects on chloride fluxes across the iso;a,trd bullfrog cornea. Am. J. Physiol. 223, 1053-7. Candia. 0. A., Bentley. P. J. and Cook, P. 1. (1974). Stimulation by amphothericin B of act,ive Na t,ransport across amphibian cornea. Sm. J. Physiol. 226, 143844. Davis. T. I,., Jackson, J. W., Day, B. E., Shoemaker, R. L. and Rehm, W. S. (1970). Potentials in frog cornea and micro-electrode artifact. Am. J. Physiol. 219, 178-83. Donn, A.. Maurice, M. D., Mills, N. L. (1959). Studies on the living cornea in vitro. II. Thr act’ive transport of sodium across the epithelium. Arch. Ophthdrnol. 62, 748-57. Eggena, P. (1977). Action of vasopressine, ouabain and cyanide on the volume of isolated toad bladder epithelial cells. J. Membrane Biol. 35, 29-37. Ehlers, N. and Ehlers, D. (1968). Effects of hydrostatic and colloid osmotic pressure on the electrical potential and short circuit current across the explanted rabbit cornea. Acta Ophthalmol. 46, 767-78. Ehlers, N. (1973). In vitro studies of trims- and intraepithelial potentials of t,he cornea. Bq. Eye RPX. 15, 553-65.
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.I. F. (:. SLE(:ERS
Fee, J. I’. and Edelhauser, H. F. (1970). Intracellular electrical potentials in the rabbit coruc~il epithehum. Exp. f<‘ye Kes. 9, 234-40. Fischbarg, J. (1973). Artive and passive properties of the rabbit (*orneal cndot~heliunn. &.r/). /<‘;!I(’ Res. 15, 615638. Fischbarg, J.. Lim. J. J. (1974). Role of cations, anions and carbonic anhydrase in fluid transport across rabbit, cornea1 endothelium. J. Physiol. (London) 241, 647-75. Fromter, E. and Gebler, B. (1977). Electrical properties of amphibian urinary bladder epithclia. III. The cell membrane resistances and the effect of amiloride. Pfliigers Arch. 371,99--108. Fuchs, W., Larsen, E. H. and Lindemann, B. (1977). Current-voltage curve of sodium channels and concentration dependence of sodium permeability in frog skin. J. Ph?/sioZ. (Lo&on) 267, 137-66. Graves, C. N., Sanders, 8. S., Shoemaker, R. L. and Rehm, W. S. (1975). Diffusion resistance of endothelium and stroma of bullfrog cornea determined by potential response to K-’ . Biochinl. Biophys. Acta 389, 550-56. Graves, C. N., Sanders, 8. S., Shoemaker, R. L. and Rehm. W. S. (1976). An intact globe technique for elrotrophysiological studies on cornea. J. AppZ. Physiol. 40, 447-50. Handler, J. S., Preston, A. S. and Orloff, J. (1972). Effect of ADH, aldosterone, onabain and amiloride on toad bladder epithelial cells. An!. J. Physiol. 222, 10714. Helman. S. 1. and Fischer, R. S. (1977). Microelectrode studies of the act,ive Na t#ransport pathways of frog skin. J. Gen. Physiol. 69, 571-604. Hodson, S. and Miller, F. (1976). The bicarbonate ion pump in the endot’helium which regulates the hydration of rabbit cornea. J. Physiol. (London) 263, W-77. Hong, C. D. and Essig. A. (1976). Effects of 2-deoxy-n-glucose, amiloride, vasopressin and ouabain on active conductance and Exa in the toad bladder. J. Jlenrbrane Biol. 28, 121-42. Hviid Larsen, E. (1973). Effect of amiloride. cyanide and ouabain on the active transport pathway in toad skin. In Alfred Benzon Xymporlum V. Transport &lechanism.a in Epithelia (Eds ITssing, H. H., Thorn, N. A.). Copenhagen, Munksgaard. Hviid Larsen, E. and Kristensen, P. (1978). Properties of a conductive cellular chloride pathway in t’he skin of the toad (Hufo bufo). Actn Physiol. Scnnd. 102, l-21. Klyce, S. D. (1972). Elect,rical profiles in the eorneal epithelium. J. Physiol. (London) 226,407-29. Klyre, S. D.. Neufeld, A. H. and Zadunaisky, J. A. (1973). The activation of chloride transport by epinephrine and Db-cyclic AMP in t,he cornea of the rabbit. Iwcest. Ophthala~ol. 12,127-39. Klyce. S. D. (1975). Transport of Sa. Cl, and water by the rabbit, cornea1 epithelium at resting pot’mtial. Am. J. Phynid. 228, 1446-52. Klyce, S. D., ITong, K. S. (1977). Site and mode of adrenaline action on chloride t,ransport across the rabbit cornea1 epithelinm. J. Physiol. (London) 266,777-99. Kristensen, P. and Hviid Larsen, E. (1978). Relation between exchange diffusion and a conductive chloride pathway across t,he isolated skin of the toad (&fo bufo). Actn Physiol. ihwd. 102, 22234. Lewis, 8. A. and Diamond, J. M. (1976). Na t transport by rabbit urinary bladder: a tight. epithelium. J. &Zembra?ee Biol. 28, l-46. Lewis, S. A., Eaton, D. C. and Diamond, J. MI. (1976). The mechanism of Na transport by urinary bladder. J. Membrane Biol. 28, 41-71. Lindemann, B. (1968). Resting potential of isolated beef cornea. Exp. Eye Res. 7, 6269. Maurice, D. M. (1972). The location of the fluid pump in the cornea. J. Physiol. (Londor~) 221,43-54. Xshima, S. and Kudo, T. (1967). In vitro incubation of rabbit cornea. Incest. Ophthalmol. 6,329-39. Montoreano. R., Candia, 0. A. and Cook, P. (1976). Alpha- and beta-adrenergic receptors in regulation of ionic transport in frog cornea. Am. J. Physiol. 230, 1487-93. Riley, M. S. (1971). The role of the epithelium in control of cornea1 hydration. fiq. Eye Kes. 12, 128-37. Robinson, R. A. and Stokes, R. H. (1970). Electrolyte Solutions, 2nd ed. Butterworths, London. Sachs, J. R,. and Conrad, M. E. (1968). Effect of tetra-ethyl ammonium on the active cation transport system of the red blood cell. Am. J. Physiol. 215, 796-8. Ussing, H. H. and Zerahn, K. (1951). Active transport of sodium as the source of electric current in the short circuited isolated frog skin. Acta Physiol. Stand. 23, 110-27. Van der Heyden, Ch., Weekers, J. F. and Schoffeniels, E. (1975). Sodium and chloride transport across the isolated rabbit cornea. Exp. Eye Res. 20, 89-96. Van OS, C. H. and Slegers, J. F. G. (1975). The electrical potential profile of gallbladder epithelium. J. Membrane Biol. 24, 341-63.