Exp. Eye Res. (1975) 20, 89-96
Sodium and Chloride Transport Across the Isolated Rabbit Cornea CHRISTINE
VAN DER HEYDEN,
J. 3’. WEEKERS
AND E. SCHOFFENIELS
Laboratoire de Biochimie gt+n&aleet compardee,Universite’ de Litge, 17 Place Delcour, B-4000 Likge, Be’gium (Received1 April 1974, and in revisedform 9 Octob.r 1974, London) Sodium and chloride unidirectional fluxes through rabbit corneas in vitro have been measured. The natural cornea curvature was simulated by applying a hydrostatic pressure on the endothelial side. Fluxes were correlated to saontaneous notential differences and shortcircuit currents in order to determine the components of passive and active transports. There is active transport of sodium toward the aqueous humour. Chloride ions are actively transported toward the tears. Both contributions do not add up to the short-circuit current. A tentative explanation in terms of non-steady-state conditions is proposed.
1. Introduction The rabbit cornea is the site of an electrical potential difference, the solution bathing the epithelial side being negative with respect to the endothelial side. Many experiments have been performed to measurethe electrical potential in vitro as well as in vivo: for a brief review seeWeekers and Van der Heyden (1973). As well as those mentioned in this previous paper, the results of Klyce and Zadunaisky (1970) obtained with microelectrodes should be referred to. According to Donn, Maurice and Mills (1959) the electrical potential difference is maintained by an active transport of Na from the tears to the aqueoushumor located within the epithelium and it is assumedto account for almost all the measuredshortcircuit current. Green (1966) hasalso suggestedthat a transport of both Na and Cl ion occurs from the tears to the stroma during the first hour after excision. On the other hand, the works of Hodson (1971) and Dikstein and Maurice (1972) seemto indicate that a fluid pump is located within the endothelium and the transport mechanismis calcium-, sodium- and bicarbonate-dependent. Since the importance of the hydrostatic pressure applied on the endothelial side upon the potential difference was not realized before the qualitative work of Ehlers and Ehlers (1968) and our subsequent quantitative analysis on rabbit corneas, it therefore seemedimportant to reconsider the ionic movements through the rabbit cornea subjected to the pressureconditions found in vivo. We report here results on ionic fluxes obtained under such an experimental condition. The problem of the steady-state conditions of the labelled ion fluxes is also examined. 2. Methods Adult rabbits were killed with an overdoseof Nembutal injected through the marginal ear veins. The cornea was detached by cutting the sclera 2 mm laterally to the limbus and the crystalline lens and iris were taken off. The cornea was then immediately clamped in the chamber as indicated in Fig. 1. The two faces of the corneawere irrigated by a TC Earle solution (NaCl 11’7.2mM; KCl: 5.4 mM; NaHCO, 26.2 mM; NaH,PO, 1.04 mm; CaCl, 1.8 mM; MgSO, 0.8 mm; glucose1 g/l; pH 7.4; t 30°C)and aeration with a mixture 95% CO, and 5% 0, provided oxygenation and a good circulation in the epithelial bath. The 89
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endothelial bath was stirred by a magnetic Teflon-coated mixer controlled by a magnet placed just under the chamber (En) and attached to an electric motor (not, shown in Fig. 1).
o a o,/co, :,i’;:-;‘.y I -.’.:,;,.,’ :‘...,-:..: .;a.:.’ i)’_,. SC -;.; Ep j; I,-
Pm. 1. Apparatus used to study the fluxes across the isolated cornea. C, cornea; SC, solera; En, endothelial chamber; Ep, epithelial chamber; a, injection of the gas mixture; M, manometer; R, rubber membrane; E,, E, and I’,, P,, agar-bridges and calomel electrodes to record potential; P,, P,, agarbridges used to inject electrical current from the battery B.
By injection of Ringer’s with the syringe S, we can apply a given pressure on the endothelial side. A manometer indicates the value of the internal pressure during the experiment. The endothelial chamber is entirely closed by a rubber membrane. The needle of a syringe can enter this membrane and penetrate the solution to remove samples. When taking samples, equal volumes of Ringer’s are added in the chamber through t,he external syringe S, so that the experimental pressure remains unchanged. Measurement of potential difference across the whole cornea was performed with a pair of calomel electrodes leading into a DC differential voltmeter (V) (John Fluke Model 881A). Using an external electromotive force, an electrical current of desired intensity was passed through the agar-bridge P,P, in order to abolish the spontaneous potential difference. The current was read on a DC microamperes Digitac. All our experiments were performed at 30°C. The area of the cornea exposed to the solution was O-923 cm2 and the volume of the epithelial chamber 10 ml, that of the endothelial one 5.2 ml. A hydrostatic pressure of some 18 cm H,O was applied on the internal side. Unidirectional sodium fluxes were determined separately with 24Na. One side of the chamber contained radioactive solution (50 $%/ml Ringer’s), the other was tiled as usual with the Ringer’s solution. Samples were removed from the nonlabelled solution with a micropipette (epithelial side) or by a syringe (endothelial side) every 30 min for 5 hr. One sample from the labelled solution was used as standard. Radioactive samples were then monitored in a scintillation counter.
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3Ieasurements of the unidirectional chloride fluxes were achieved with 36C1as described above. Samples were transferred to 10 ml of a scintillation counting fluid added to the solution (dioxane 1 M, naphthalene 100 g, PPO 10 g, POPOP 250 mg). The radioactivity was counted in a liquid Scintillation Spectrometer (Packard Tri Garb Model 3324). Values of P;a+ and Cl- unidirectional fluxes were then calculated after correction had been made to compensate for the decay of the 24Na during the experimental period. The uptake of 24Na
The cornea, mounted as usual, was in contact with a radioactive solution bathing the endothelial side, the epithelial side, or both of them. The corneawas withdrawn from the chamber at different times, dried on filter paper and rapidly weighed.The tissuewasthen digested with 200 ~1 of concentrated nitric acid (density 1.40) in a water-bath at 100%. This solution wasplaced in a volumetric flask and brought up to 10 ml with distilled water. An aliquot was taken off for radioactive measurementsand the total Na+ content of the solution was analysed after decay of the =Na. In somecases,the cornea1thickness has been measuredafter completion of an experiment by a method describedby Weekers (1974). The results show no difference with the thicknessof fresh corneas(360 pm). 3. Results Our experiments were performed under conditions of open- or short-circuit.
Table I indicates that, under open-circuit conditions, the flux J,, of Na from the lacrymal to the aqueoushumor side ranges between O-0462and O-0944pequiv.lhr-l cm-a and the opposite flux J,, from 0.0630 to O-0715pequiv./hr-r cm-2. The potential difference reaches an average value of 30.4 mV. In the last two columns, we report the mean value of the flux-ratio J,,/J,, found either experimentally or calculated according to Ussing and Zerahn (1951). As we can see,the experimental ratio exceeds unity and differs markedly from the calculated one. Such a comparison already shows that Na+ movements are not only driven by passive electrochemical forces, but also by an active transport mechanism directed towards the aqueous humor. Table I shows also the results obtained on 19 short-circuited corneas. The Na+influx increasesslowly from 0.0545 t,o O*lOlO pequiv./hr-l cm-2 and the Na+-outtlux from 0.0354 to 0.0902 pequiv./hr-l cm-2. The difference between the two fluxes (net flux) remains at a constant mean value of 0.0231pequiv./hr-l cmM2,and it represents an active transport of Na+ from the epithelial to the endothelial face. The comparison between net Na+-flux and short-circuit current indicates that the Na+-flux accounts only for 20% of the total short-circuit current. Chloridej6uxe.s Unidirectional fluxes of Cl- were also measured under open or short-circuit conditions. As we can see in Table II, when the membrane is not short-circuited, the intlux, i.e. lacrymal to aqueous humor, of chloride reaches 0.0859 and increasesto 0.1175 ~equiv./hr-l cm- 2; the Cl- outflux follows a very similar pattern, going from O-0505 to 0.0678 pequiv./hr--‘-l cm- 2. The average experimental flux-ratio is equal to 1.64 and also differs from the theoretical ratio, 2.70. When the potential difference is reduced to zero we observe an important increase in the Cl- outflux (mean value: O-1128pequiv./hr-l cme2) and a decreasein Cl- influx (mean value: 0.0569 pequiv./hr-1 cm-s). The net flux results in an active outward
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Sodium $uxes mross the rabbit cornea Open-circuit Time (min)
J 12 s=13 (pequiv/hr-l cm-s)
J 21 11=11 (*equiv/hr-1 cm-‘)
0.0462 0.0830 0.0944 0.0745
0.0674 0.0630 0.0715 0.0627
29.7 31.6 27.6 30.4
J n=lO (peq&/hr-l cmmz)
Flux ratio J,,/J,,
Net flux bewivl hr-1 cm-z)
0.0354 0.0687 0.0902 0.0626
1.54 1.36 1.12 1.39
0.0191 0.0247 0~0108 0.0231
45 166 285 Mean
Short-circuit Time (min)
55 175 295 Mean
J
n=9
(pequi:jhr-’
cm-2)
0.0545 0.0934 0.1010 0.0857
Flux ratlo Experimental
0.69 1.3” d 1.32 1.19
JIz/J,, Calculated
0.3” 0.30 0.35 I).?”. I
WC (pequiv/hr-l
cm-2)
0~1044 0.1126 0.1232 0.1145
J 1e and J,, represent the flux of Na from the tears to the aqueous humor and the opposite flux respectively; n is the number of experiments; AE is the average potential difference recorded every 30 min for 295 min on 24 oorneas. The radioisotope ie added to the solution when the potential difference has reached a steady value. Nineteen corneas were short-circuited before adding the isotope. TABLE
ChlorideJluxes Open-circuit Time (min)
50 170 290 Mean
Short-circuit Time (min)
80 200 290 Mean
II
across the rabbit corrw
J 12 n=ll (pequiv/hr-’ cm-2)
J n=9 (pequ:/hr-1 cm-z)
0~0859 0.0889 0.1175 0~0890
0.0505 0.0541 0.0678 0.0543
J 12 12=3 J (pequiv/hr-’ cme2) (pequi&’
0.0494 0.0679 0.0608 0.0569
71=4 cm-$)
0.0939 0.1161 0.1481 0.1128
Jle and Jzl represent the flux of Na from the spectively: n is the number of experiments; AE min for 290 min on 20 corneas. The radioisotope haa reached a steady value. Seven corneas were
Flux ratio Experimental
27.9 25.6 23.6 25.8
Flux ratio J,,/J,,
0.53 ov50 0.41 060
1.70 1.64 1.73 1.64
Net flux (fLequiv/ hr-’ cm-s)
0.0445 0.0582 0.0873 0.0566
J,,/JI1 Calculated
2.92 2.67 2.47 2.70
sc’c (pequiv/hrr’
cm-2)
0.1297 0.1191 0.1131 0.1208
tears to the aqueous humor and the opposite flux reis the average potential difference recorded every 30 is added to the solution when the potential difference short-circuited before adding the isotope.
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movement of Cl- from the aqueous to the tears (mean value: O-0566 pequiv./hr-l cn+). As in the case of Na ions, we observed a discrepancy between short-circuit current and net %ux: the Cl- net flux represents 35% of the short-circuit current after about 1 hr. As shown by our results, the unidirectional %ux as well as the net flux of Na+ and Cl- increase steadily with time. This could indicate that, within the time limits of our experiments, we do not reach a steady state as to the specific activity of the ion in the t)issue. In order to check this possibility we have loaded the cornea with tracers from the endothelial or the epithelial side as well as from both sides simultaneously. Figure 2 illustrates the evolution of the specific activity in the case of sodium ions. The ordinate gives the specific radioactivity expressed in per cent of the specific radioactivity of the bathing solution (1OOo/o). The specific radioactivity is expressed as activity (ctjmin) per pequiv. of Na in 1 g of cornea1 tissue, or in 1 ml of physiological solution. The full circles give the uptake of Naf after loading both sides of the cornea. The open circles and the squares correspond respectively to loading the preparation from the epithelium or the endothelium side.
Standard
Time
$
(hr)
Flo. L’. Isolated rabbit oornea. Specific radioactivity of Na ions versus time. Loading of the preparation from the endothelial side (full square), from both sides of the cornea simultaneously (full circles), or from the epithelial side (open circles).
It can be observed t,hat there is a great difference between the epithelial and the endothelial uptake. The loading from the endothelial or from the two cornea1 faces increases in the same way. The maximal load is obtained within 2 hr at 86.0 f 6*5%, thus never reaching the value of the incubating medium. In contrast, the speci%c radioactivity of the cornea loaded from the epithelial side reaches only 1.l4o/o of the standard specific radioactivity. The measurements of unidirectional %uxes as well as the loading of the cornea by labelled ions thus tend to prove that the cornea is not yet in a steady state after five hours and that the total replacement of cold ions by the radioisotope is a very slow phenomenon.
4. Discussion In the cornea1 epithelium, the magnitude of the normal resting potential, the short-circuit current and the resistance are still a point of controversy. According to several authors, the potential difference rises from 15 up to 40 mV during experiments in vitro (Donn et al., 1959; Ehlers and Ehlers, 1968) as well as in measurements in
94
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vivo (Friedmann, Kupfer and Boston, 1960). These authors report a short-circuit current value of 2.96 to 554 PAI cm2 and a resistance of 6-S kQ cm2. On the other hand, Green (1966) and Green and Otori (1970) report lower transcorneal potentjial values (5-6 mV) and much higher short-circuit current data (20 ~A/cm3). Some of the discrepancies observed between the data published by various amhors may be explained by the fact that the cornea is a very fragile structure. Any damage to the epithelial cells induces a fall in the potential difference as demonstrated by the results of Maurice (1967). Also it is clear that the electrical resistance to the sclera, being very low, any misfit between the two chambers clamping the isolated cornea would result in apparently low electrical resistance and potential with correspondingly higher short-circuit current values. As discussed elsewhere (Weekers and Van tlcr Heyden, 1973) a positive hydrostatic pressure has to be applied on the endot.helial side of the cornea in order to obtain reproducible values of potential differences ranging between 85 and 40 mV. In these circumstances, the electrical resistance of the preparation is always high (between 8 and 12 kQ cm2). Thus reproducible and consistent results can only be obtained if the cornea is correctly mounted in the experimental device and if a slight hydrostatic pressure, sufficient to give curvature of the cornea, is applied.
The flux ratio analysis in open- or short-circuit conditions clearly indicates that there is an active transport of sodium towards the aqueous humor. This confirms the results obtained by other workers in the field. An interesting observation is given by the fact that under short-circuit conditions, the influx of sodium goes up while the outflux remains practically constant. If the intlux was solely due to an active transport, this should be relatively insensitive to the electric field. Moreover, the outilux should decrease if it was essentially passive. One way of explaining our results is to assume that the specific activity of the intracornea1 pool of sodium controlling the influx is sensitive to the electric field. As shown above, the specific activity of the sodium in the cornea is only close to 1% of that of the solution bathing the epithelial side. If the abolition of the transcorneal potential difference results in an increase in the turnover of Na ions belonging to the active transport pool, the specific activity should accordingly increase with a concomitant increase in the influx of radioactivity. Since the specific activity of the sodium in the cornea is much higher (up to 80%) when the radioactivity is applied on the endothelial side, any increase in the turnover rate of sodium ions should have a less pronounced effect on the out&u of radioactive sodium. This interpretation is easily open to experimental verification if one measures the turnover rate of the sodium content of the cornea under short-circuit current conditions. Chloride
$uxes
The calculated flux ratio is 2.70 against an experimental value of 1.64 thus indicating that, though the net flux is in the direction tears-aqueous humor, an active transport of chloride ions directed outwards (i.e. to the tears) is taking place. That this is the caseis well demonstrated when the potential difference is clamped at 0 mV. In these circumstances the net flux of chloride is reversed. This is due to both a decreasein the influx and an increasein the e&x. The diminution in the inward movement is
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easily explained by the abolition of the electrical driving force since the aqueous humor is positive with respect to the tears. The increase in the e&x may also be explained by an increase in the passive component sensitive to the electric field. Our results dealing with the fluxes of chloride ions are in agreement with the data obtained by Zadunaisky (1966) on the frog cornea.
In a series of experiments Deuse (1973) has measured the fluxes of potassium ions in our experimental conditions. Her results obtained in our laboratory indicate that both influx and outflux of K ions are very small (close to 0*002 pequiv./hr-1 cm-s) and that these ions seem to move passively. Thus in open-circuit conditions there is a small net flux of K outwardly directed. If one adds up the various fluxes we have measured so far and compare the results with the value of the short-circuit current, it is apparent (Fig. 3) that the longer the experimental period the closer the ionic net fluxes of Na, K and Cl get to the shortcircuit current. Thus
&cc = I,,
+ I,, - I,
only after 4 hr. It is reasonable to assume that the delay we observe in establishing a steady state in the fluxes of the radioactive ions has to be explained by the fact that depending on the side of application of the tracer ion, the specific activity of the cornea for, let us say sodium ions, is very different.
Time i hr) FIG.
3. Comparison
between
short-circuit
current,
and ionic currents
(Na, Cl and Ii) as s function
of
time.
The epithelial s4Na uptake proceeds SO slowly that the excised cornea cannot reach its steady state during the experimental period. After 5 hr, only 1*14o/o of the total cornea1 Na+ has exchanged with labelled Na +. The recorded influx should then represent only a small fraction of the true inward movement except if the pool of cornea1 Na related to the active transport is small. This seems to be the case if one looks at the results of Fig. 3. The endothelial side shows greater permeability to the ions thus allowing better exchange with 24Na. The cornea1 specific radioactivity reaches rapidly a value close to the specific radioactivity of the labelled bathing solution, when the latter is placed on the endothelial side. Among many other possibilities, two hypotheses may be proposed to explain the slowly exchangeable or unexchangeable fraction of Na: (1) The sodium ions could be fixed on macromolecules in the extracellular or
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intracellular space. Such a hypothesis has already been suggested by Otori (196~). Sodium may also be sequestered within cellular organellcs. (2) Th e ep i tl le1’mm cells provide a very efficient barrier of low permeability to Na ions associated with a very low intracellular concentration in Na ions. Without excluding the first hypothesis, experiments now in progress seem to support the relevance of the second one. There is indeed a large increase in the permeahilit! of the cornea if the epithelial layers are removed (kcratectomy): the ion fluxes increase by a factor of 100. As discussed by Ussing (1972), it should however be emphasized that when ~011. sidering the flux ratio in non-steady-state conditions over short periods of time. it is still possible to decide whether or not one is dealing with an active transport. In our case, with Na and Cl ions it is clear that the isolated rabbit cornea is the site of an active transport for both ionic species. Cereijido and Rotunno (1967) as well as Aceves and Erlij (1971) have given a good account of tracer flux theory in the case of the amphibian skin where there is also a slowly exchangeable sodium compartment that does not contribute to the transport’ pool, a situation likely to occur in our experimental condit,ions. ACKXOWLEDGYEP;TS
This work has been aided by a grant from the Ophthalmologic Clinic of the University of Liege (Professor R. Weekers) and grant no. 790 from the ponds de la Rechew?be FondamentaleCollecti,ue to ProfessorE. Schoffeniels. REFERENCES
D. (1971). (London),212,195.
Aceves,
J. and Erlij,
Sodiumtransport
across the epithelium
of the frog skin.
J. Physio/.
Cereijido, M. and Rotmmo, C. A. (1967). Transport and distribut,ion of sodium across frcg skin. J. Physiol. (London) 190, 481. Deuse, J. (1973). Les caracteres de permeabilite de la corn&e de lapin. These cniversitb de Lihge. Dikstein, S. and ?rlaurice, D. M. (1972). The metabolic basis to the fluid pump in the cornea. J. Physiol. (London), 221, 29. Dorm, A., Maurice, D. M. and Mills, X. L. (1969). Studies on living cornea in vitro. Arch. Ophth&&. 62, 748. Ehlers, N. and Ehlers, D. (1968). Effect of hydrostatic pressure on electric potential and shortcircuit current across the explanted cornea. Acta Ophthulmol. 46, 767. Friedman. E., Kupfer, C. and Boston, M. D. (1960). Transcorneal potential in vivo. Arch. Ophthnlmol. 64, 892. Green, K. (1966). Ion transport across the isolated rabbit cornea. Exp. Eye Res. 5, 106. Green, K. and Otori, T. (1970). Studies on cornea1 physiology in vitro. Exp. Eye Res. 9, 268. Hodson, 6. (1971). Evidence for bicarbona,te dependent sodium pump in cornea1 endothelium. Exp. Eye Res. 11, 20. Klyce, S. D. and Zadunaisky, J. A. (1970). Microelectrode profile of rabbit cornea1epithelium. Biophys, J. 10, 199a. Maurice, D. M. (1967). Epithelial potential of the cornea. Exp. Eye I&s. 6, 138. Otori. T. (1967). Electrolyte content of the rabbit cornea1 stroma. Exp. Eye Res. 6, 356. Ussing, H, H. and Zerahn, K. (1951). Active transport of sodium as a source of electrical current in short-circuited isolated frog skin. Acta Physiol. Stand. 23, 110. Ussing, H. H. (1972). In Perspectives in Membrane Biophysics (Ed. Agin, D. P.). Gordon and Breach, Inc. New York. Weekers, J. F. and Van der Heyden, Ch. (1973). Recherches exp&imentales sur la viabilitb de la corn&e. Diffkrence de potentiel de la corn&e isolbe de lapin. Ann. Oculist. 206,495. Weekers, J. F. (1974). Reoherches expbimentales sur la genese des l&ions corneenes dues aux anesthbsiques. Arch. O~hthulmol. 34, 121. Zadunaisky, J. A. (1966). Active transport of chloride in frog cornea. Amer. J. Physiol. 211, 500.
Sodium and Chloride Transport Across the Isolated Rabbit Cornea CHRISTINE
VAN DER HEYDEN,
J. 3’. WEEKERS
AND E. SCHOFFENIELS
Laboratoire de Biochimie gt+n&aleet compardee,Universite’ de Litge, 17 Place Delcour, B-4000 Likge, Be’gium (Received1 April 1974, and in revisedform 9 Octob.r 1974, London) Sodium and chloride unidirectional fluxes through rabbit corneas in vitro have been measured. The natural cornea curvature was simulated by applying a hydrostatic pressure on the endothelial side. Fluxes were correlated to saontaneous notential differences and shortcircuit currents in order to determine the components of passive and active transports. There is active transport of sodium toward the aqueous humour. Chloride ions are actively transported toward the tears. Both contributions do not add up to the short-circuit current. A tentative explanation in terms of non-steady-state conditions is proposed.
1. Introduction The rabbit cornea is the site of an electrical potential difference, the solution bathing the epithelial side being negative with respect to the endothelial side. Many experiments have been performed to measurethe electrical potential in vitro as well as in vivo: for a brief review seeWeekers and Van der Heyden (1973). As well as those mentioned in this previous paper, the results of Klyce and Zadunaisky (1970) obtained with microelectrodes should be referred to. According to Donn, Maurice and Mills (1959) the electrical potential difference is maintained by an active transport of Na from the tears to the aqueoushumor located within the epithelium and it is assumedto account for almost all the measuredshortcircuit current. Green (1966) hasalso suggestedthat a transport of both Na and Cl ion occurs from the tears to the stroma during the first hour after excision. On the other hand, the works of Hodson (1971) and Dikstein and Maurice (1972) seemto indicate that a fluid pump is located within the endothelium and the transport mechanismis calcium-, sodium- and bicarbonate-dependent. Since the importance of the hydrostatic pressure applied on the endothelial side upon the potential difference was not realized before the qualitative work of Ehlers and Ehlers (1968) and our subsequent quantitative analysis on rabbit corneas, it therefore seemedimportant to reconsider the ionic movements through the rabbit cornea subjected to the pressureconditions found in vivo. We report here results on ionic fluxes obtained under such an experimental condition. The problem of the steady-state conditions of the labelled ion fluxes is also examined. 2. Methods Adult rabbits were killed with an overdoseof Nembutal injected through the marginal ear veins. The cornea was detached by cutting the sclera 2 mm laterally to the limbus and the crystalline lens and iris were taken off. The cornea was then immediately clamped in the chamber as indicated in Fig. 1. The two faces of the corneawere irrigated by a TC Earle solution (NaCl 11’7.2mM; KCl: 5.4 mM; NaHCO, 26.2 mM; NaH,PO, 1.04 mm; CaCl, 1.8 mM; MgSO, 0.8 mm; glucose1 g/l; pH 7.4; t 30°C)and aeration with a mixture 95% CO, and 5% 0, provided oxygenation and a good circulation in the epithelial bath. The 89
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endothelial bath was stirred by a magnetic Teflon-coated mixer controlled by a magnet placed just under the chamber (En) and attached to an electric motor (not, shown in Fig. 1).
o a o,/co, :,i’;:-;‘.y I -.’.:,;,.,’ :‘...,-:..: .;a.:.’ i)’_,. SC -;.; Ep j; I,-
Pm. 1. Apparatus used to study the fluxes across the isolated cornea. C, cornea; SC, solera; En, endothelial chamber; Ep, epithelial chamber; a, injection of the gas mixture; M, manometer; R, rubber membrane; E,, E, and I’,, P,, agar-bridges and calomel electrodes to record potential; P,, P,, agarbridges used to inject electrical current from the battery B.
By injection of Ringer’s with the syringe S, we can apply a given pressure on the endothelial side. A manometer indicates the value of the internal pressure during the experiment. The endothelial chamber is entirely closed by a rubber membrane. The needle of a syringe can enter this membrane and penetrate the solution to remove samples. When taking samples, equal volumes of Ringer’s are added in the chamber through t,he external syringe S, so that the experimental pressure remains unchanged. Measurement of potential difference across the whole cornea was performed with a pair of calomel electrodes leading into a DC differential voltmeter (V) (John Fluke Model 881A). Using an external electromotive force, an electrical current of desired intensity was passed through the agar-bridge P,P, in order to abolish the spontaneous potential difference. The current was read on a DC microamperes Digitac. All our experiments were performed at 30°C. The area of the cornea exposed to the solution was O-923 cm2 and the volume of the epithelial chamber 10 ml, that of the endothelial one 5.2 ml. A hydrostatic pressure of some 18 cm H,O was applied on the internal side. Unidirectional sodium fluxes were determined separately with 24Na. One side of the chamber contained radioactive solution (50 $%/ml Ringer’s), the other was tiled as usual with the Ringer’s solution. Samples were removed from the nonlabelled solution with a micropipette (epithelial side) or by a syringe (endothelial side) every 30 min for 5 hr. One sample from the labelled solution was used as standard. Radioactive samples were then monitored in a scintillation counter.
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3Ieasurements of the unidirectional chloride fluxes were achieved with 36C1as described above. Samples were transferred to 10 ml of a scintillation counting fluid added to the solution (dioxane 1 M, naphthalene 100 g, PPO 10 g, POPOP 250 mg). The radioactivity was counted in a liquid Scintillation Spectrometer (Packard Tri Garb Model 3324). Values of P;a+ and Cl- unidirectional fluxes were then calculated after correction had been made to compensate for the decay of the 24Na during the experimental period. The uptake of 24Na
The cornea, mounted as usual, was in contact with a radioactive solution bathing the endothelial side, the epithelial side, or both of them. The corneawas withdrawn from the chamber at different times, dried on filter paper and rapidly weighed.The tissuewasthen digested with 200 ~1 of concentrated nitric acid (density 1.40) in a water-bath at 100%. This solution wasplaced in a volumetric flask and brought up to 10 ml with distilled water. An aliquot was taken off for radioactive measurementsand the total Na+ content of the solution was analysed after decay of the =Na. In somecases,the cornea1thickness has been measuredafter completion of an experiment by a method describedby Weekers (1974). The results show no difference with the thicknessof fresh corneas(360 pm). 3. Results Our experiments were performed under conditions of open- or short-circuit.
Table I indicates that, under open-circuit conditions, the flux J,, of Na from the lacrymal to the aqueoushumor side ranges between O-0462and O-0944pequiv.lhr-l cm-a and the opposite flux J,, from 0.0630 to O-0715pequiv./hr-r cm-2. The potential difference reaches an average value of 30.4 mV. In the last two columns, we report the mean value of the flux-ratio J,,/J,, found either experimentally or calculated according to Ussing and Zerahn (1951). As we can see,the experimental ratio exceeds unity and differs markedly from the calculated one. Such a comparison already shows that Na+ movements are not only driven by passive electrochemical forces, but also by an active transport mechanism directed towards the aqueous humor. Table I shows also the results obtained on 19 short-circuited corneas. The Na+influx increasesslowly from 0.0545 t,o O*lOlO pequiv./hr-l cm-2 and the Na+-outtlux from 0.0354 to 0.0902 pequiv./hr-l cm-2. The difference between the two fluxes (net flux) remains at a constant mean value of 0.0231pequiv./hr-l cmM2,and it represents an active transport of Na+ from the epithelial to the endothelial face. The comparison between net Na+-flux and short-circuit current indicates that the Na+-flux accounts only for 20% of the total short-circuit current. Chloridej6uxe.s Unidirectional fluxes of Cl- were also measured under open or short-circuit conditions. As we can see in Table II, when the membrane is not short-circuited, the intlux, i.e. lacrymal to aqueous humor, of chloride reaches 0.0859 and increasesto 0.1175 ~equiv./hr-l cm- 2; the Cl- outflux follows a very similar pattern, going from O-0505 to 0.0678 pequiv./hr--‘-l cm- 2. The average experimental flux-ratio is equal to 1.64 and also differs from the theoretical ratio, 2.70. When the potential difference is reduced to zero we observe an important increase in the Cl- outflux (mean value: O-1128pequiv./hr-l cme2) and a decreasein Cl- influx (mean value: 0.0569 pequiv./hr-1 cm-s). The net flux results in an active outward
92
0. VAN
UER
HEYDEN,
J.
F.
W’EEKEKS TABLE
ASD
E.
YCHOFFENIELS
1
Sodium $uxes mross the rabbit cornea Open-circuit Time (min)
J 12 s=13 (pequiv/hr-l cm-s)
J 21 11=11 (*equiv/hr-1 cm-‘)
0.0462 0.0830 0.0944 0.0745
0.0674 0.0630 0.0715 0.0627
29.7 31.6 27.6 30.4
J n=lO (peq&/hr-l cmmz)
Flux ratio J,,/J,,
Net flux bewivl hr-1 cm-z)
0.0354 0.0687 0.0902 0.0626
1.54 1.36 1.12 1.39
0.0191 0.0247 0~0108 0.0231
45 166 285 Mean
Short-circuit Time (min)
55 175 295 Mean
J
n=9
(pequi:jhr-’
cm-2)
0.0545 0.0934 0.1010 0.0857
Flux ratlo Experimental
0.69 1.3” d 1.32 1.19
JIz/J,, Calculated
0.3” 0.30 0.35 I).?”. I
WC (pequiv/hr-l
cm-2)
0~1044 0.1126 0.1232 0.1145
J 1e and J,, represent the flux of Na from the tears to the aqueous humor and the opposite flux respectively; n is the number of experiments; AE is the average potential difference recorded every 30 min for 295 min on 24 oorneas. The radioisotope ie added to the solution when the potential difference has reached a steady value. Nineteen corneas were short-circuited before adding the isotope. TABLE
ChlorideJluxes Open-circuit Time (min)
50 170 290 Mean
Short-circuit Time (min)
80 200 290 Mean
II
across the rabbit corrw
J 12 n=ll (pequiv/hr-’ cm-2)
J n=9 (pequ:/hr-1 cm-z)
0~0859 0.0889 0.1175 0~0890
0.0505 0.0541 0.0678 0.0543
J 12 12=3 J (pequiv/hr-’ cme2) (pequi&’
0.0494 0.0679 0.0608 0.0569
71=4 cm-$)
0.0939 0.1161 0.1481 0.1128
Jle and Jzl represent the flux of Na from the spectively: n is the number of experiments; AE min for 290 min on 20 corneas. The radioisotope haa reached a steady value. Seven corneas were
Flux ratio Experimental
27.9 25.6 23.6 25.8
Flux ratio J,,/J,,
0.53 ov50 0.41 060
1.70 1.64 1.73 1.64
Net flux (fLequiv/ hr-’ cm-s)
0.0445 0.0582 0.0873 0.0566
J,,/JI1 Calculated
2.92 2.67 2.47 2.70
sc’c (pequiv/hrr’
cm-2)
0.1297 0.1191 0.1131 0.1208
tears to the aqueous humor and the opposite flux reis the average potential difference recorded every 30 is added to the solution when the potential difference short-circuited before adding the isotope.
IOK
TRANSPORT
ACROSS
RABBIT
CORKEA
93
movement of Cl- from the aqueous to the tears (mean value: O-0566 pequiv./hr-l cn+). As in the case of Na ions, we observed a discrepancy between short-circuit current and net %ux: the Cl- net flux represents 35% of the short-circuit current after about 1 hr. As shown by our results, the unidirectional %ux as well as the net flux of Na+ and Cl- increase steadily with time. This could indicate that, within the time limits of our experiments, we do not reach a steady state as to the specific activity of the ion in the t)issue. In order to check this possibility we have loaded the cornea with tracers from the endothelial or the epithelial side as well as from both sides simultaneously. Figure 2 illustrates the evolution of the specific activity in the case of sodium ions. The ordinate gives the specific radioactivity expressed in per cent of the specific radioactivity of the bathing solution (1OOo/o). The specific radioactivity is expressed as activity (ctjmin) per pequiv. of Na in 1 g of cornea1 tissue, or in 1 ml of physiological solution. The full circles give the uptake of Naf after loading both sides of the cornea. The open circles and the squares correspond respectively to loading the preparation from the epithelium or the endothelium side.
Standard
Time
$
(hr)
Flo. L’. Isolated rabbit oornea. Specific radioactivity of Na ions versus time. Loading of the preparation from the endothelial side (full square), from both sides of the cornea simultaneously (full circles), or from the epithelial side (open circles).
It can be observed t,hat there is a great difference between the epithelial and the endothelial uptake. The loading from the endothelial or from the two cornea1 faces increases in the same way. The maximal load is obtained within 2 hr at 86.0 f 6*5%, thus never reaching the value of the incubating medium. In contrast, the speci%c radioactivity of the cornea loaded from the epithelial side reaches only 1.l4o/o of the standard specific radioactivity. The measurements of unidirectional %uxes as well as the loading of the cornea by labelled ions thus tend to prove that the cornea is not yet in a steady state after five hours and that the total replacement of cold ions by the radioisotope is a very slow phenomenon.
4. Discussion In the cornea1 epithelium, the magnitude of the normal resting potential, the short-circuit current and the resistance are still a point of controversy. According to several authors, the potential difference rises from 15 up to 40 mV during experiments in vitro (Donn et al., 1959; Ehlers and Ehlers, 1968) as well as in measurements in
94
C. VAN
DER
HEYDEN,
J.
F.
WEEKNRS
AND
E.
SCHOPFESIELF
vivo (Friedmann, Kupfer and Boston, 1960). These authors report a short-circuit current value of 2.96 to 554 PAI cm2 and a resistance of 6-S kQ cm2. On the other hand, Green (1966) and Green and Otori (1970) report lower transcorneal potentjial values (5-6 mV) and much higher short-circuit current data (20 ~A/cm3). Some of the discrepancies observed between the data published by various amhors may be explained by the fact that the cornea is a very fragile structure. Any damage to the epithelial cells induces a fall in the potential difference as demonstrated by the results of Maurice (1967). Also it is clear that the electrical resistance to the sclera, being very low, any misfit between the two chambers clamping the isolated cornea would result in apparently low electrical resistance and potential with correspondingly higher short-circuit current values. As discussed elsewhere (Weekers and Van tlcr Heyden, 1973) a positive hydrostatic pressure has to be applied on the endot.helial side of the cornea in order to obtain reproducible values of potential differences ranging between 85 and 40 mV. In these circumstances, the electrical resistance of the preparation is always high (between 8 and 12 kQ cm2). Thus reproducible and consistent results can only be obtained if the cornea is correctly mounted in the experimental device and if a slight hydrostatic pressure, sufficient to give curvature of the cornea, is applied.
The flux ratio analysis in open- or short-circuit conditions clearly indicates that there is an active transport of sodium towards the aqueous humor. This confirms the results obtained by other workers in the field. An interesting observation is given by the fact that under short-circuit conditions, the influx of sodium goes up while the outflux remains practically constant. If the intlux was solely due to an active transport, this should be relatively insensitive to the electric field. Moreover, the outilux should decrease if it was essentially passive. One way of explaining our results is to assume that the specific activity of the intracornea1 pool of sodium controlling the influx is sensitive to the electric field. As shown above, the specific activity of the sodium in the cornea is only close to 1% of that of the solution bathing the epithelial side. If the abolition of the transcorneal potential difference results in an increase in the turnover of Na ions belonging to the active transport pool, the specific activity should accordingly increase with a concomitant increase in the influx of radioactivity. Since the specific activity of the sodium in the cornea is much higher (up to 80%) when the radioactivity is applied on the endothelial side, any increase in the turnover rate of sodium ions should have a less pronounced effect on the out&u of radioactive sodium. This interpretation is easily open to experimental verification if one measures the turnover rate of the sodium content of the cornea under short-circuit current conditions. Chloride
$uxes
The calculated flux ratio is 2.70 against an experimental value of 1.64 thus indicating that, though the net flux is in the direction tears-aqueous humor, an active transport of chloride ions directed outwards (i.e. to the tears) is taking place. That this is the caseis well demonstrated when the potential difference is clamped at 0 mV. In these circumstances the net flux of chloride is reversed. This is due to both a decreasein the influx and an increasein the e&x. The diminution in the inward movement is
ION
TRANSPORT
ACROSS
RABBlT
CORKEA
9.5
easily explained by the abolition of the electrical driving force since the aqueous humor is positive with respect to the tears. The increase in the e&x may also be explained by an increase in the passive component sensitive to the electric field. Our results dealing with the fluxes of chloride ions are in agreement with the data obtained by Zadunaisky (1966) on the frog cornea.
In a series of experiments Deuse (1973) has measured the fluxes of potassium ions in our experimental conditions. Her results obtained in our laboratory indicate that both influx and outflux of K ions are very small (close to 0*002 pequiv./hr-1 cm-s) and that these ions seem to move passively. Thus in open-circuit conditions there is a small net flux of K outwardly directed. If one adds up the various fluxes we have measured so far and compare the results with the value of the short-circuit current, it is apparent (Fig. 3) that the longer the experimental period the closer the ionic net fluxes of Na, K and Cl get to the shortcircuit current. Thus
&cc = I,,
+ I,, - I,
only after 4 hr. It is reasonable to assume that the delay we observe in establishing a steady state in the fluxes of the radioactive ions has to be explained by the fact that depending on the side of application of the tracer ion, the specific activity of the cornea for, let us say sodium ions, is very different.
Time i hr) FIG.
3. Comparison
between
short-circuit
current,
and ionic currents
(Na, Cl and Ii) as s function
of
time.
The epithelial s4Na uptake proceeds SO slowly that the excised cornea cannot reach its steady state during the experimental period. After 5 hr, only 1*14o/o of the total cornea1 Na+ has exchanged with labelled Na +. The recorded influx should then represent only a small fraction of the true inward movement except if the pool of cornea1 Na related to the active transport is small. This seems to be the case if one looks at the results of Fig. 3. The endothelial side shows greater permeability to the ions thus allowing better exchange with 24Na. The cornea1 specific radioactivity reaches rapidly a value close to the specific radioactivity of the labelled bathing solution, when the latter is placed on the endothelial side. Among many other possibilities, two hypotheses may be proposed to explain the slowly exchangeable or unexchangeable fraction of Na: (1) The sodium ions could be fixed on macromolecules in the extracellular or
C’. VAN
96
DER
HEYUEN,
J. F.
1vEEKEKS
AXI)
E. S(:HOPPEXlEI,S
intracellular space. Such a hypothesis has already been suggested by Otori (196~). Sodium may also be sequestered within cellular organellcs. (2) Th e ep i tl le1’mm cells provide a very efficient barrier of low permeability to Na ions associated with a very low intracellular concentration in Na ions. Without excluding the first hypothesis, experiments now in progress seem to support the relevance of the second one. There is indeed a large increase in the permeahilit! of the cornea if the epithelial layers are removed (kcratectomy): the ion fluxes increase by a factor of 100. As discussed by Ussing (1972), it should however be emphasized that when ~011. sidering the flux ratio in non-steady-state conditions over short periods of time. it is still possible to decide whether or not one is dealing with an active transport. In our case, with Na and Cl ions it is clear that the isolated rabbit cornea is the site of an active transport for both ionic species. Cereijido and Rotunno (1967) as well as Aceves and Erlij (1971) have given a good account of tracer flux theory in the case of the amphibian skin where there is also a slowly exchangeable sodium compartment that does not contribute to the transport’ pool, a situation likely to occur in our experimental condit,ions. ACKXOWLEDGYEP;TS
This work has been aided by a grant from the Ophthalmologic Clinic of the University of Liege (Professor R. Weekers) and grant no. 790 from the ponds de la Rechew?be FondamentaleCollecti,ue to ProfessorE. Schoffeniels. REFERENCES
D. (1971). (London),212,195.
Aceves,
J. and Erlij,
Sodiumtransport
across the epithelium
of the frog skin.
J. Physio/.
Cereijido, M. and Rotmmo, C. A. (1967). Transport and distribut,ion of sodium across frcg skin. J. Physiol. (London) 190, 481. Deuse, J. (1973). Les caracteres de permeabilite de la corn&e de lapin. These cniversitb de Lihge. Dikstein, S. and ?rlaurice, D. M. (1972). The metabolic basis to the fluid pump in the cornea. J. Physiol. (London), 221, 29. Dorm, A., Maurice, D. M. and Mills, X. L. (1969). Studies on living cornea in vitro. Arch. Ophth&&. 62, 748. Ehlers, N. and Ehlers, D. (1968). Effect of hydrostatic pressure on electric potential and shortcircuit current across the explanted cornea. Acta Ophthulmol. 46, 767. Friedman. E., Kupfer, C. and Boston, M. D. (1960). Transcorneal potential in vivo. Arch. Ophthnlmol. 64, 892. Green, K. (1966). Ion transport across the isolated rabbit cornea. Exp. Eye Res. 5, 106. Green, K. and Otori, T. (1970). Studies on cornea1 physiology in vitro. Exp. Eye Res. 9, 268. Hodson, 6. (1971). Evidence for bicarbona,te dependent sodium pump in cornea1 endothelium. Exp. Eye Res. 11, 20. Klyce, S. D. and Zadunaisky, J. A. (1970). Microelectrode profile of rabbit cornea1epithelium. Biophys, J. 10, 199a. Maurice, D. M. (1967). Epithelial potential of the cornea. Exp. Eye I&s. 6, 138. Otori. T. (1967). Electrolyte content of the rabbit cornea1 stroma. Exp. Eye Res. 6, 356. Ussing, H, H. and Zerahn, K. (1951). Active transport of sodium as a source of electrical current in short-circuited isolated frog skin. Acta Physiol. Stand. 23, 110. Ussing, H. H. (1972). In Perspectives in Membrane Biophysics (Ed. Agin, D. P.). Gordon and Breach, Inc. New York. Weekers, J. F. and Van der Heyden, Ch. (1973). Recherches exp&imentales sur la viabilitb de la corn&e. Diffkrence de potentiel de la corn&e isolbe de lapin. Ann. Oculist. 206,495. Weekers, J. F. (1974). Reoherches expbimentales sur la genese des l&ions corneenes dues aux anesthbsiques. Arch. O~hthulmol. 34, 121. Zadunaisky, J. A. (1966). Active transport of chloride in frog cornea. Amer. J. Physiol. 211, 500.
