Active and passive properties of the rabbit corneal endothelium

Exp. Eye Res. (1973) 15, 615-638

Active and Passive Properties of the Rabbit Cornea1 Endothelium* J. FISCHBARG Department of Ophthalmology,

Columbia University, New York, N. Y. 10032, U.S.A.

Some of the physiological properties of rabbit cornea1 endothelium were investigated by studying: (a) fluid transport; (b) osmotic water flow; (c) electrical potential difference; and (d) electrical impedance, all of them across the endothelial layer; (e) biochemical parameters (ATP level and Na++K+-activated ATPase activity) of endothelial cells, and (f) morphological (light and electronmicroscopic) appearance of the endothelium. Several experimental manipulations which decreased or abolished fluid transport (total replacement of 43 m&r bicarbonate by Cl, of 5 mnr K by Na and of 150 mM Na by Li; treatment with 5 x 1O-6 M ouabain and with ZO/~/rnl cytochalesin B) also decreased or abolished the electrical potential difference. The electrical resistance of fresh preparations was 41&5 n = 14; range: 16-81 Sacma); scraping off theendothelium and treating it with Qcm2 (s.E.M., Ca-free media decreased the resistance to that of the supporting stromal layer (3-10 Qcm2) Cytochalasin B (20 pg/ml) markedly decreased the resistance, while ouabain did not affect it. Omission of all metabolites normally present in the perfusion medium (reduced glutathione, adenosine, and glucose) decreased the endothelial ATP level markedly, while addition of reduced glutathione spared ATP from depletion. Since the transport ceased even in the presence of near normal ATP levels and (Na++K+)-activated ATPase activity, other yet undefined factors are also probably involved in pump function. Osmotically induced water flows across the endothelium were not linear but followed a square root function of the driving force. Cytochalasin B induced profound changes in the shape of the endothelial cells, causing some of them to clump together. The values of electrical resistance, capacitance and water flows experimentally measured agreereasonably well with the numerical values calculated from a model. The results are consistent with the possibility that the fluid transport across the endothelium would be due to an electrogenic ionic pump, and that the intercellular spaces and their gap junctions would constitute the dominant “shunt” pathway for water and electrolytes.

1. Jntroduction The suggestion that an active process located in the cornea1 endothelium was instrumental in maintaining the cornea deturgesced, and therefore transparent, was advanced in 1949 (Davson, 1949). The experimental development of this original postulation went through several important stages (Davson, 1955; Harris and Nordquist, 1955 ; Langham and Taylor, 1956 ; Mishima and Kudo, 1967 ; Maurice, 1972 ; Dikstein and Maurice, 1972) until the present time. Nowadays there can be little doubt that the rabbit endothelium transports fluid from the stroma into the aqueous; a detailed description of the inner workings of this mechanism is, however, not available yet. The work to be reviewed here was undertaken largely in order to make possible an attack on this last problem. Several different orientations and collaborative efforts are being pursued for this purpose; hence, this system will be discussed on the basis of evidence drawn from: (a) fluid transport experiments; (b) experiments correlating physiology and biochemistry; (c) measurements of osmotically induced water flow; and (d) measurements of electrical potential difference and impedance across

tion

the preparation.

The evidence

of water and electrolytes

will be discussed in terms of a model for permea-

across this preparation.

Part of this work has

* This work was supported by Research Grant EY 00727 from the U.S.P.H.S. and, in part, by Fight for Sight, Inc., New York City. 615

J. FTSCHBARG

616

been published (Fischbarg, 1972); the rest is in preparation to be published in detail or in the press at the time of this writing.

2. Methods and Results Fluid transport across the endothelium was measured with a technique (Fig. 1; cf. also Fischbarg, 1972) which was basically the one developed by Dikstein and Maurice (ibid, 1972) after the pioneering work of Mishima (Mishima and Kudo, 1967).

Silicone

Perfuslc medium

To motor

FIG. 1. The cornea, with its epithelium scraped off, is shown clamped in place in a lucite chamber. A water immersion lens attached to a metallurgical microscope aliowa to me&sure stromal thickness (Dikstein and Maurice, 1972). The bottom of a lucite chamber previously described (Dikstein andMaurice 1972) has been modified to include a stirring device.

The most important modification, introduced after the initial experiments, consisted of a stirring device (Fig. 2) which ensured excellent mixing of the small volume of fluid enclosed between the endothelium and the lucite chamber (O-4 ml). Most of the experiments were done at 37°C with a pressure head of 20 cm of H,O on the aqueous side of the endothelium (inside) and without the epithelium present (it had been scraped off). In many instances, paired experiments were done at the same time with the two corneas of a rabbit, using one as the test preparation and the second one as a control (cf. Figs 5 and 8).

PROPERTIES

OF CORNEAL

617

EKDOTHELIUM

500 400 300 200

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600

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FIG. 2. (a) The denuded cornea1 stroma swelled by imbibition of the outside solution; after the outside solution was replaced by silicone oil, subsequent thickness readings measured the fluid transfer across the endothelium. The normal fluid transport in presence of 43 mx HCO; in the inside bathing solution is reversibly arrested when HCO; is totally replaced by Cl-; (b) the fluid pump is arrested by lowering the [Naf] in the inside perfusing medium from 150 to 100 rnM (replaced by Li+); (c) the fluid pump is reversibly arrested by totally replacing the K+ (5 m&f) in the inside perfusing medium by Na+ (from Fischbarg, 1972).

Pluid

transpovt

The fluid transport mechanism was known to be dependent on the ambient @CO;] (Dikstein and Maurice, 1972; Hodson, 1971); this line of evidence was pursued further with the results depicted in Figs 2 and 3. As can be seen there, the replacement of HCO; by Cl- done on the inside arrests transport; if the HCO;-free solution was not perfused for more than 30-45 min, then the transport mechanism could recover,

J. FISCHBARG

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[cl1 (mu/l) FIG. 3. The rates of deturgescence and swelling obtained at the different vs. the [HCO;]. Negative rates correspond to stromal swelling.

[HCO;]

tested are plotted

as seen in Fig. 2. In Fig. 3 all the rates of deturgescence and/or swelling that were measured in this series of experiments were collected into a dispersion diagram. The curve (drawn by the eye) across the set of points in this figure suggests a saturation effect by the HCO; ion on the rate of transport. Also in Fig. 3, the rate at which fluid leaks into the stroma from the aqueous (from inside to outside) at zero HCO; (c. 45 p./hr) is entirely comparable to the rate at which passive fluid movement occurs after ouabain poisoning (Trenberth and Mishima, 1968) and substrate deprivation (c. 40 p/hr, Anderson, Fischbarg and Spector, 1973); all of this supports the

600 -

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3

FIG. 4. Procedure as in Fig. 2; at the-arrow,

ouabain’10V

MIwas perfused on the inside.

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“pump-leak” theory for this preparation, as will be discussed later. The need for Naf in the solution perfusing the inside, noted before (Dikstein and Maurice, 1972) was confirmed; Naf was here replaced by Li+ (Fig. 2). The role of the ambient [K+] was also investigated; just like in other systems, here too the presence of this ion was seen to be needed for the integrity of the transport mechanism (Fig. 2). The effect of ouabain on endothelial transport, described earlier (Brown and Hedbys, 1965; Trenberth and Mishima, 1968) was confirmed here too (Fig. 4). A very interesting effect was observed using cytochalasin B, a cell-relaxing agent. Not only was transport apparently arrested (Fig. 5), but the image of the endothelial cells as observed under the specular microscope suffered pronounced changes (Fig. 6). Upon return to perfusion with fresh solution, the morphological effects could be reversed (Fig. 6) but the transport showed either a very small degree of recovery or no recovery at all (Fig. 5).

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FIG. 5. The stroma was swollen as explained in Fig. 2. Cytoohalasin B (20 pg/ml, solubilized indimethylsulfoxide at 26 mg/ml) was perfused at the arrow; after the microscopic image of the endothelial cells showed alterations (Fig. 6), the fluid transport was impaired. Upon return to regular solution, a long period of washing was required for the image of most endothelial cells to return to normal; this incomplete recovery was reflected by the subsequent low rate of fluid transport. At the arrow below the control curve, a solution with a concentration of dimethylsulfoxide equal to that used in the experiment was perfused without ill effects. After 5-6 hr of perfusion in vitro, both preparations deteriorated (from Fisohbarg, 1972).

In work which is presently in course (Kaye, Fenoglio, Fischbarg and Hoefle), the rate of fluid transport of some preparations is observed before and after addition of cytochalasin B (l-40 pg/ml) to the solution perfusing the endothelium; these preparations are subsequently fixed, and their morphology, as revealed by light microscopy, transmission and scanning electron microscopy, is compared to their physiological behavior. Preliminary results show that the drug produces profound deformation of some cells, and clumping of groups of cells which change their flat shape for a columnar

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appearance. There is excellent correspondence between morphology and physiological behavior. These morphological changes are sufficient to explain the effect of the drug on the transport rate as due t.o an increased leak (cf. Discussion); some other explanations previously advanced by the present author for the cytochalasin eifect (Fischbarg, 1972) appear unnecessarily elaborate.

FIG. 6. (a) Microphotograph from normal endothelial cells “in vitro”; (b) effect of cytochalasin B (5 pg/ml); (c) recovery after perfusing fresh solution. The three fields photographed during this experiment were not the same, but the appearance of the cells was homogeneous throughout the preparation at each condition (Kaye, Fenoglio, Fischbarg and Hoefle, in preparation).

Fluid transport and biochemistry In these experiments the ability of the endothelium to transport fluid was correlated with its ATP content and ATPase level in the presence of several combinations of nutrients (Anderson et al., 1973). Figure 7 depicts the experiments done to control and standardize the experimental procedures. The solutions employed were as follows: BS was a basal salt solution, containing only electrolytes and sucrose to compensate for osmolarity (300 m0sm). Addition of nutrients to this BS are

PROPERTIES

OF CORNEAL

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621

denoted, in short : ADN for adenosine, GSH for reduced glutathione (cf. Dikstein and Maurice, 1972), and GLU for glucose. Thus the complete medium (CM) was equivalent to BS +GLU +ADN +GSH. Figure 8 shows the behavior of the preparation under several conditions. Under extreme deprivation (absence of all nutrients), when the transport mechanism is apparently arrested or, more precisely, when the -

Fresh

Fresh

-

I Fresh

I Perfused

Fresh

Perfused I Perfused swelling

with

I

I Pefused with SWelllng then deturgesced to original thickness I

400

3 ‘0 300, k L I I I i ; 5001 E

400’

;

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T F o$--

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Time (hr) 0 m

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0 t-

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= Control = Test

!Li EXP

FIG. 7. Effect of standardized

no

experimental procedures on ATP levels and ATPase activities of rabbit endothelia. In each experiment the letters C and T refer, respectively, to control and a test cornea1 preparation obtained from a given pair of rabbit eyes. Each cornea was freed of epithelium, mounted in a chamber and, as indicated under Protocol, either taken immediately for analysis (fresh) or perfused. Legends above and below the experimental curves of thickness vs. time denote the fluids in contact with the outer (epithelial) and the inner (endothelial) surfaces, respectively, and the intervale during which they are employed. Individual thickness readings (solid circles) were accurate to 52 p (Anderson, Fischbarg and Spector, in preparation). H

J. FISCHRARG Test

600 BS

011

n l*

.

l.

“*ea&lP

CM BSfGSH

Time

600,

:CM

BS+ADN+GSH

(hr)

n 0= Control

m

= Test

n 3 Exp no

FIQ. 8. Effect of omissions of various nutrients on rates of fluid transport and endothelisl and ATPase activities. Details as in Fig. 7 (Anderson et al., in preparation).

ATP levels

rate of passive leak outwards overtakes the rate of fluid pumping inwards (in terms of the “pump-leak” theory, cf. Discussion), the ATP is depleted and the ATPases lowered. However, GSH plays some role in sparing this depletion of ATP (Fig. 8). It can also be seen from Fig. 8 that the glucose present in the CM lengthens the survival time of the preparation. It is to be noted that since the transport failure can occur while the biochemical parameters are still comparatively normal (cf. BS +AD + GSH, Fig. B), some factor other than the ATP level or the ATPase activity seems to be important in the preservation of the transport mechanism. On the other hand, from this and other evidence (as discussed later), an adequate ATP level appears to be a necessary prerequisite for the transport activity.

-6

-4

I

-2

I

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I 2

I 4 I 6

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FIG. 9. Perfusion with 30 mOsm hypertonic solution at a rate of 0.49iml/min, Zero time is the moment at which hypertonic solution rcaches:the chamber.

270

following

I IO

perfusion

I 12

I 16

I 18

I 20

2 with isotonic solution at the same rate.

I 14

624

J. E’ISCHBARC:

Osmotic jlow

For this type of work (Fischbarg and Warshavsky, in preparation), the chamber with rapid mixing was essential. At a given time, solutions made hypertonic with sucrose were perfused at a fast (0.5 ml/min) rate on the inside of the endothelium, and the ensuing changes in stromal thickness were recorded. The time course of the thickness change was plotted as shown in Fig. 9; from the time course, the initial rate of volume flow was found graphically. Interestingly, Fig. 10 shows that the volume flow is not linear with respect to its driving force. This had not been reported in previous similar experiments done with this preparation (Mishima and Hedbys, 1967). This nonlinear behavior is approximated reasonably well by a square-root function, as drawn in Pig. 10. The interesting implications of these findings will be discussed later. 0.3c

,-

0.25

I--

/o’ I IO Osmotic

I I I 20 30 40 pressure difference

I 50 (m OsmA)

I 60

FIG. 10. Plot of initial slopes of time transients (cf. Fig. 9) vs. osmotic pressure difference. The solid line is a square root function. The dotted lines represent laminar flows between parallel plates wit,h the following values for the width and depth of the slits: left: 300 A and 12 II; middle: 150 A and 12 11; right: 30 d and 0.15 p. The small square near the origin represents the passive leak into the stroma when the transport mechanism is arrested. The deviations are S.E.M.

Electrical potential difference and impedance Some attempts to measure a potential difference across the endothelium have been made in the past (Kikkawa, 1966a,b; Green, 1967; Hodson, 1971), but these results have been unclear. A small (0.5-1.0 mV, aqueous or inside negative) potential difference has been however recently found in this laboratory (Fischbarg, 1972). The experiments to be reported in this section were done in an attempt to correlate

PROPERTIES

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6%

ENDOTHELIUM

this potential difference to the fluid pumping activity of the endothelium. A diagram of the chamber used is shown in E”ig. 11. Practically all the conditions which affect fluid transport have also an effect on the potential difference (E’ischbarg, 1972). Figure 12 shows the effects of Na+, HCO, and Kt- replacements (by 21‘+, Cl- and Na+ respectively) on the potential difference, VAC t

4-O

i-

11. Schematic diagram of the chamber used to measure electrical potential differences. i, o: FIG. inlets and outlets for the perfusing solutions. P.D.,, PD.,: agar-solution bridges; th: thermistor; VAC: vacuum.

Regular soiutwn

Zero Na+

(b)

Zero K’

Regular solution

Fro. 12!(a) Efl’eot of replacing all the ambient Na+ by Li+ on the thelium. The curve was traced from the recorder’s chart: (b) effect potential ditference. Curve traced from recorded chart. The plot is of theibaseline; (0) effect of zero (K+) on transendothelial pokntial

(Cl

potential difference across the endoof zero [HCO;] on transendothelial interrupted by periodic calibrations difference.

626

J. FISCHBARG

and the degree to which these changes are reversible. Figure 13 depicts the effect of ouabain on the potential difference; when the exchange of solutions was done swiftly, this effect was so fast that it could not be resolved in time with the usual washing and recording methods employed (Fig. 13a) and the inhibition was estimated to have taken place in 10 set or less. In order to discard the possibility of side effects, a slower effect was obtained on purpose by placing a known volume of ouabain-containing solution on the upper part of the open chamber, thus allowing the drug to diffuse downwards and to reach the endothelium in a progressively increasing fashion (Fig. 13b). Cytochalasin B was also tested, and clearly decreased the potential difference in a reversible way exemplified in Fig. 13. The effect could be reproduced several times in the same ireparation, as shown in that figure.

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i FIG. 13. (a) Effect of 1O-6M ouabain placed on the inside on transendothelial potential difference (from Fischbarg, 1972); (b) effect of 5 x IO-& M ouabain placed slowly on the inside (cf. text) on transendothelial potential difference; (c) effect of cytochalasin B (20pg/ml) added to the inside bathing solution on the transendothelial potential difference (from Fischbarg, 1972).

In order to investigate further the properties of the endothelium, a four-electrode system was devised to measure its electrical impedance (Fischbarg and Lim, 1973, and in preparation). Figure 14 shows the impedance locus measured in one preparation at three different times. As can be seen, the semicircles that fit the sets of points best are centred below the R axis. The impedance was seen to decrease with time, which paralleled the decrease in potential difference. This is consistent with the possibility that an electromotive force which might be associated with an ionic pump would be increasingly shunted by a leak across the tissue. The resistance of freshly mounted preparations was about 4OQcma in the average (range: 16-81 Qcm2) and decreased to lo-40Qcm2 later in the experiments. Scraping off the endothelium immediately abolished the potential difference, decreased the resistance to some

PROPERTIES

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627

ENDOTHELIUM

(a) 40

Ohr n Ihr v 2,25hr l

t 30 20 IO

q

40 -

Control

(b)

(cl * Control 0 Ouabafn

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I 30

I 40

I 50 R (fix

I 60

I 70

I 80

I 90

I 100

I

cm21

FIG. 14. (a) Impedance locus of the endothelium at three different times during an experiment. The experimental points were fit by semicircles centered on a line below the R axis. The numbers adjacent to the points denote the frequency (in Hz) (Fisohbarg and Lim, in preparation); (b) impedance locus of the endothelium before and immediately after addition of cytochalasin B (20 pg/ml) to the inside bathing solution; (c) impedance loous of the endothelium before and immediately after addition of ouabain (5 x 10-5M) (Fischbarg and Lim, 1973).

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3-8QcnP, and produced an impedance pattern for the stroma which was indistinguishable from the pattern for free solution with the present methods. Ca-free solutions also decreased the resistance to the value of the stroma alone. Interestingly, the effects of cytochalasin B and ouabain on the electrical impedance were markedly different : cytochalasin B (20 pg/ml) clecreased the resistance by 50% or more almost immediately (Fig. 14), while ouabain left it clearly unchanged (Fig. 14). This evidence is entirely consistent with past and present morphological findings, as will be discussed. Lastly, the capacitance calculated for the enclothelium from the frequencies of maximum reactance (cf. Brown and Kastella, 1965) is about 0.4 pF/cm2. Assuming that this value arises from two capacitors in series which would represent the two cell membranes, (Fig. 15), the capacitance of the cell membrane wo&l be about 0.8 pF/ cm2.

Intercellular

300 A

“gap” junction

channel

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Aqueous

FIG. 15. (a) Schematic diagram of the intercellular spaces and the terminal bars of “gap junctions” in the endothelium. Dimensions obtained from electronmicrographs; (b) electrical model for the cornea1 endothelium. R8: resistance of the intercellular spaces; Rg: resistance of the gap junctions; R,: resistance of the cell cytoplasm; R,. . resistance of the cell membrane; R E: equivalent electrical resistance of the ionic pump; B: equivalent electromotive force of the ionic pump; C: capacitance of the cell membrane (Fischbarg and Lim, 1973).

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3. Discussion Since the process under study is ultimately that of a transport mechanism across a biological membrane, it seems pertinent to evaluate how well can such studies be conducted in the present preparation. The rabbit cornea1 endothelium has several obvious advantages when compared with other epithelia. The single layer of endothelial cells is in direct contact with the nutrient solution and is hence readily affected by the experimental manipulations imposed on that side, which is not the case with other preparations. This layer of cells can be easily detached from its supporting membrane and collected for analysis. The comparatively high rate of net fluid transfer across it (c. 604hr or the entire volume of the cells in some 5 min, cf. Maurice, 1972) and the possibility to actually see the cells under the microscope while they are active are other assets of the preparation. On the other hand, some of the disadvantages are : the relative difficulty in preserving it in good condition through dissection and in vitro mounting, the fact that the leak across the preparation is large and hence the potential difference across it is very small, and the presence of the stroma, which acts as an added barrier. In balance, the difficulties are not insurmountable, and the preparation is a useful tool for the investigation of transport processes in general. The nature of thejuk.3 pump (a) State of the problem. After Mishima’s work (Mishima and Kudo, 1967), most of the remaining doubts concerning the transport of fluid were dispelled by Maurice’s demonstration that the isolated endothelium pumps fluid against a head of pressure, and that the fluid movement can be followed directly from the displacement of a bubble in a capillary tube (Maurice, 1972). Using a different technique, this last results have been reproduced in a few experiments (Bourguet and Fischbarg, unpublished); in these, the isolated endothelium, with about half of the stromal layer dissected away, was also capable of pumping fluid for several hours against a pressure head of 20 cm H,O. (b) Electrogenic vs. neutral pump. From the evidence presented, the potential difference across the endothelium appears to be the result of an electrogenic pump. The extremely rapid abolition of the potential difference with ouabain (in 5 to 10 set) certainly points in that direction. Alternative explanations involving ionic concentration changes secondary to ouabain inhibition of an ionic pump, although possible in principle, appear more unlikely. From all the rest of the evidence coming from ionic substitutions and linking the potential difference with the fluid transport, the possibility that this fluid pump would be “electrically neutral” appears small indeed. As for the question of which ion or ions are being transported, from the direction of the potential difference (inside negative) and the fluid movement (towards the inside), the most logical candidates are the anions which are present, namely Cland HCO;. In view of this, the ouabain effect might appear puzzling, since this glycoside probably acts through its specific inhibition of the Na-K activated ATPase known to be present in the endothelium (Rogers, 1968), and there is no evidence as yet for hhe presence of anion-dependent ATPases in this preparation. In other preparations, however, ouabain inhibits Cl transport; such is the case for gastric mucosa (Cooperstein, 1959), South American frog skin (Zadunaisky, Candia and Chiarandini, 1963), bullfrog cornea (Candia, 1972), and the ascending limb of Henle’s loop in the kidney

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(Burg, 1972; Rocha and Kokko, 1972). If Cl- is indeed the ion being actively transported in the present preparation (just as it is in cornea1 epithelium, Zadunaisky, 1966), it is possible that the ouabain effect might take place directly on the transport, mechanism, as hypothesized by Cooperstein (1959) for the case of gastric mucosit. More indirect effects are also conceivable, but any such schemes would have to meet the stringent requirement of explaining an inhibition that takes place in a few seconds. It is also possible that HCO; would be transported or secreted into the inside. There is no known system so far where a HCO; pump would be present all by itself and would be responsible for the transfer of large amounts of fluid. There are, however, some systems where HCO, pumps have been described to be present together with other ionic pumps (cf. Brodsky and Schilb, 1967; Carlisky and Lew, 1970). An evaluation of which of these patterns, if any, applies to the present preparation (on which HCO; has the effects described above) will require further work. Biochemical aspects (a) Role of ATP and transport ATPase. Under conditions of extreme deprivation, in absence of all nutrients, the ATP stores of the cells are depleted in 0.5-1.5 hr, and the transport is arrested. No preparation was ever seen to transport fluid when the ATP content was drastically reduced; the converse, however, was not true, and since the endothelium can stop transporting fluid even with a normal amount of cellular ATP, factor or factors other than the basic energetics might also play a decisive role in the transport mechanism. This unknown factor or factors do not seem to correspond to the Na-K activated ATPase, the activity of which was near normal in presence of different combinations of nutrients and with the transport mechanism either operative or inoperative. The activity of this enzyme was decreased under extreme deprivation (absence of all nutrients), but, from what was said before, this decrease does not seem specifically linked to the transport failure but rather to general cellular deterioration. (b) Role of GSH. This compound, which has been described to have a beneficial effect for the transport of fluid in this preparation (Dikstein and Maurice, 19’72) was seen here to produce an actual increase in the initial rates of fluid transport. An effect by GSH in sparing ATP was also seen frequently, These two effects might be ascribed to two different sites at which GSH would act, or might be tied together in one single scheme involving oxido-reduction processes in the cell. (c) Energetics of the transport mechanism. The ATP contents of the cells has an interesting correspondence with the energy required for the fluid transport. In the “in vitro” preparation, the net fluid movement performs work against a 20 cm H,O pressure head. Assuming that the actual amount of fluid pumped would be the observed net (about 60 p/hr or 6 pl/hr cmz) plus the passive leak (about 4 pl/hr cm2, cf. discussion of “pump-leak” theory), the work done would be: W = P . d V = 4.7 x 10W6cal/hr cm2. As pointed out earlier (Maurice, 1969) the true expenditure of energy would probably be higher in account of the losses this system may have. On the other hand, the normal ATP content of these cells (Anderson et al., in preparation) is about 900 x lo-l2 M/cornea, or 3.9 x lo-lo M/cm2, which implies an available energy of: dp” = 7 x lo3

Cd/M

.

3.9

x

lo-lo

M/Cm”

=

2.7 x 1O-6 cal/cm2.

This energy would be enough for only half an hour of pumping. This is not far

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from what is experimentally observed when the cells are deprived of all nutrients. The fact that some preparations can pump fluid for somewhat longer times might be ascribed to the presence of endogenous reserves. Lastly, as pointed out earlier in this (Maurice, 1969) and other systems (cf. Martin and Diamond, 1966), the total energy available to the endothelium exceeds by far the amount needed for the fluid pump. Using for the 0, consumption of the endothelium a recent figure of 2-03 pl/crn” hr (Freeman, 1972) the available energy would be about 5 x lo9 cal/hr cm2 (cf. Maurice, 1969). The “‘pump-leak” theory A large amount of experimental evidence has accumulated over the years on passive and active properties of the endothelial layer. On one hand, it can act as a barrier and limit the diffusion of solutes and the flow of water across it; on the other hand, there is very strong evidence that the endothelium is the site of a metabolically dependent fluid pump. On the basis of this picture (Mishima, 1968; Maurice, 1969) the pertinent properties of the endothelium are best discussed in terms of the “pumpleak” theory (Maurice, 1972). Stated in very general terms, it means that the endothelial fluid pump would be working continuously while water would tend to flow passively back into the stroma across the same endothelium, driven by the swelling tendency of the stroma. Role of the intercellular spaces and the “gap junctions” In what follows, the evidence which will be discussed not only will be found to be consistent with the “pump-leak” theory, but will strongly point to the intercellular spaces and their terminal bars or “gap junctions” as the pathway for high electrical conductance and for passive water flow across the endothelium. This preparation appears to have many elements in common with the gall bladder, for which there has been strong evidence generated in recent years (Barry, Diamond and Wright, 1971; Diamond, Barry and Wright, 1971; Fromter and Diamond, 1972 ; Fromter, 1972 ; Machen and Diamond, 1972) that the high-conductance pathway for transepithelial ion permeation resides in the so-called “tight junctions”, and that these “tight junctions”, since they are permeable to La3+ (Machen, Erlij and Wooding, 1972), might present some hydraulic conductivity. There seems to be also a similarity with the proximal tubule of Necturus, in which the tight junction-lateral intercellular space has been hypothesized to constitute a pathway for a “shunt flow” (Bentzel, Parsa and Hare, 1969). Further, all three preparations display a high rate of fluid transport and very small electrical potential across them (cf. Orloff and Burg, 1971; Fischbarg, 1972). For the calculations reported below, the pertinent geometrical dimensions (cf. Fig. 15) were obtained from electronmicrographs (Kaye and Pappas, 1962; Kaye et al., in preparation; Smelser and Fischbarg, unpublished). The numerical values, symbols and formulas used were as follows: Average intercellular

space width : 300 A

(4

Average intercellular

space length : 12 p

(1)

Total cell perimeter length per unit area : 1200 cm cm-2

lb)

Approximate

gap junction width: 30 A

(4

Approximate

gap junction length: 0.24 p

(1)

Imbibition

pressure “in vitro”

at a cornea1 thickness of 450 p (cf. Hedbys,

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J. FISCHBARG

Mishima and Maurice, 1963) and with a pressure head of 20 cm H&J: 55 mmHg viscosity Equivalent

of water at 37°C: 6.9 x 10e3 poise conductance of NaCl at 37°C : 122 cm2/equiv.

Concentration of NaCl: 0.15 x 10d3 equiv./cm3

(P) (77) (4 Cc)

Laminar flow between parallel plates: a%P (a: width of channel; 1: depth; J, =b: length of plates) 1271 Electrical resistance : R=kA

c

(I: length; A : area ; intercellular spaces assumed filled with 0.15 M NaCl)

The evidence linking the passive leak pathway to the intercellular spaces and the “gap junctions” can then be listed as follows: (1) The intercellular channels fill up in a few minutes with horseradish peroxidase when the enzyme is added to the inside perfusing medium (Kaye, Hoefle and Sibley, 1972; Kaye, 1973). The marker is also seen inside the terminal bars, which in all likelihood means that these bars at the apical ends of the intercellular spaces are actually “gap junctions” (Revel and Karnovsky, 1967; Kaye, 1973), permeable to small molecules and with an overall diameter of 20-30 A*. The finding that these junctions were not always closed had been reported for the human endothelium some time ago (Iwamoto and Smelser, 1965) and the penetration of LaOH in the intercellular spaces has recently been documented in the cornea1 endothelium of the rat (Leuenberger, 1973). (2) The rate of passive fluid leak into the stroma resulting from several experimental manipulations is nearly the same in all cases (40-45 p/hr, as reported above). Strikingly, this same rate, when calculated from the figures listed above for laminar flow across the gap junctions comes out to be 41 p/h r, in complete agreement with the present experimental values. Barring a coincidence, this suggests that the gap junctions might indeed be the main restriction to water fiow under normal circumstances. Even further, if the rate of passive leak is calculated assuming that the gap junctions are fully open and the only restriction to fluid flow comes from the intercellular spaces, then the figure obtained is some 860 p/hr. This implies that those preparations which are unable to maintain their junctions intact (e.g. in Ca-free solutions) would swell very fast, and once again, the experimental findings are in complete agreement with this notion (Kaye, Mishima, Cole and Kaye, 1968). (3) The electrical potential difference and the impedance across the endothelium, as reported here, are closely related and decrease together as a function of time after the preparation is mounted. Yet, the effects of cytochalasin B and ouabain on these parameters (reported in the text) are dissimilar: cytochalasin B decreases both potential difference and impedance, while ouabain decreases only the potential difference. The present morphological evidence, in turn, shows that cytochalasin B produces deformations in the shape of the cells (Kaye et al., in preparation), while ouabain did not seem to affect these structural features (Kaye, Cole and Donn, 1965). As reported above, Ca-free solutions decreased the impedance to the value of the * Note added in proof: Some old results on endothelial permeability to dyes (quoted by Maurice, D. M., O$~thaZmoZ. Lit., 1953, 7, 3) are al&o consistent with these figures; from that evidence, the maximum size of a particle that could permeate the endothelium lies in the range lo-26 8.

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stroma, as could be expected. The simplest explanation consistent with the data presently reported is that the electrical potential difference would be generated by a ouabain-inhibitable electrogenic pump, and that this potential difference would be shunted to a varying degree depending on the resistance of the “leak” pathway. That this “leak” pathway seems to depend on cell geometry again points to the intercellular spaces as the site of the “leak”. (4) The electrical resistance calculated for a model such as the one in Fig. 15 agrees with the order of magnitude of the values experimentally measured. With the values listed above, the calculated resistance of the intercellular channels is 18Qcm”, and the resistance of the gap junctions is 4 Gems. The values experimentally measured range from 40 to 80SZcm2 for freshly mounted endothelia ; they then fall to some lo-dOQcn?- later in the experiment. If bhe terminal bars were fully closed, the resistance would be expected to approximate values more typical of cell membranes (c. 1000&n2) ; if they were instead fully open, the resistance would be expected to be approximately 25-50% of the value actually measured. Clearly the experimental values are close to the second alternative. It is of course possible that when the preparation is fresh, the resistance offered by the “gap junctions” might be larger than the value calcmated for them, but, aside from having to invoke such not too unreasonable an assumption, the present agreement in the order of magnitude constitutes per se good evidence for the proposed role of the “gap junctions” and the intercellular spaces as the site of the conductance pathway.

A modebfor tile en&he&cm The properties of the model shown in Fig. 15 require little further discussion. Although shown at the inside facing membrane, the actual location and value of the elect’romotive force representing the ionic pump (and the value of its equivalent internal electrical resistance) remain unclear. The non-linearity of the osmoticallyinduced water flow could be explained by variations in the hydraulic conductivity of the tissue (cf. Smulders, Tormey and Wright, 1972) but could also be due to the presence of solid fihrils inside the gap junction (depicted in Fig. 15), which would produce turbulent flow and therefore a square-root dependence of flow on the pressure applied at, relatively high rates of fluid flow (Reynolds, 1900). It seems worth pointing out t’hat’ the good aqreem.ent of both the electrical resistance and the water flow with the values expected from the model is highly suggestive of its validity, since otherwise of the t’wo simultaneous coincidences would have to be invoked. The feasibility model iu Fig. 15 is further highlighted by the value of 0.8 ,.~F/cma found for the membrane capacitance. That value is close to t#heusual 1 pF/cm2 found ubiquitously among cell membranes, and is curiously similar to the value reported for the erythrocyte membranc~ (0.81 @/cm2, Fricke, 1925). In the light of these consistent, patterns. this model. ahhough still incomplete, might constitute a useful tool for the understancling of actJive and passive properties of the endothelial layer and of other epithelia. ACKNOWLEDGMEh-TS The author is indebted to his colleagues at t,he Ophthalmology Research Institute for their material help in countless occasions. Useful discussiorls with Drs E. Anderson, L. Bit,o and G. Kaye are also acknowledged. This work was done with the able technical assistance of Miss Pegri Varjabedian; the technical excellence of the instrument maker, _Nr H. Rosskothen, and of the artist, Mr E. Bethke, also largely deserve a grateful mentIion.

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Anderson, E., Fischbarg, J. and Spector, A. (1973) Biochim. Biophys. Acta (in press). Barry, P. H., Diamond, J. M. and Wright, E. M. (1971). J. Membr. BioZ. 4, 358. Bentzel, C. J., Parsa, B. and Hare, D. K. (1969). Amer. J. Physiol. 217, 570. Brodsky, W. A. and Schilb, T. (1967). Fed. Proc. Fed. Amer. Sot. Exp. Biol. 26, 1314. Brown, A. C. and Kastella, K. G. (1965). Biophys. J. 5,591. Brown, S. I. and Hedbys, B. 0. (1965). Invest. OphthaZmoZ. 4, 216. Burg, M. (1972). V Int. Gong. Nephrol. Abst., p. 150. Candia, 0. A. (1972). Amer. J. Physiol. 223, 1053. Carlisky, N. J. and Lew, V. L. (1970). J. Physiol. (London) 206,529. Cooperstein, I. L. (1959). J. Gen. Physiol. 42, no. 6, 1233. Davson, H. (1955). Bioclwn. J. 59, 24. Davson, H. (1949). l&-it. J. OphthaZmoZ. 33, 175. Diamond, J. M., Barry, P. H. and Wright, E. M. (1971). In Symposium on Electrophysioology Epithelia. (Ed. Giebisch, G.). Schattauer, Stuttgart. D&stein, S. and Maurice, D. M. (1972). J. PhysioZ. (London) 221, 29. Fischbarg, J. (1972). B&him. Biophya. Acta 288,362. Fischbarg, J. and Lim, J. J. (1973). Biophys. J. (in press). Freeman, R. D. (1972). J. Physiol. (London) 225, 15. Fricke, H. (1925). J. Gen. Physiol. 9, 137. Fromter, E. (1972). J. Membr. Biol. 8, 259. Friimter, E. and Diamond, (1972). Nature New BioZ. 235, 9. Green, K. (1967). Ezp. Eye Res. 6, 79. Harris, J. E. and Nordquist, L. T. (1955). Amer. J. OphthaZmoZ. 40, 100. Hedbys, B. O., Mishima, S. and Maurice, D. M. (1963). Exp. Eye Res. 2, 99. Hodson, S. (1971). Exp. Eye Res. 11, 20. Iwamoto, T. and Smelser, G. (1965). Invest. OphtkZmoZ. 4, 270. Kaye, G. I. (1973) Exp. Eye Res. 15, 511. Kaye, G. I. and Pappas, G. D. (1962). J. CeZZBioZ. 12,457. Kaye, G. I., Cole, J. D. and Donn, A. (1965). Science 150, 1167. Kaye, G. I., Mishima, S., Cole, J. D. and Kaye, N. W. (1968). Invest. OphkZmoZ. 7, 53. Kaye, G. I., Hoefle, F. B. and Sibley, R. C. (1972). Arch. Mex. And. 39,23. Kikkawa, Y. (1966a). Ezp. Eye Res. 5, 21. Kikkawa, Y. (1966b). Exp. Eye Res. 5,31. Langham, M. E. and Taylor, I. S. (1956). Brit. J. Ophthalmol. 40,321. Leuenberger, P. M. (1973). Exp. Eye Res. 15, 85. Machen, T. E. and Diamond, J. (1972). J. Membr. Biol. 8,63. Machen, T. E., Edij, D. and Wooding, F. B. P. (1972). J. Cell BioZ. 54, 302. Martin, D. W. and Diamond, J. (1966). J. Gen. Physiol. 50, 295. Maurice, D. M. (1969). In The Eye, vol. 1. (Ed. Davson, H.). Academic Press, New York. Maurice, D. M. (1972). J. Physiol. (London) 221,43. Mishima, S. (1968). Survey Ophthul. 13, 57. Mishima, S. and Hedbys, B. 0. (1967). Exp. Eye Res. 6, 10. Mishima, S. and Kudo, T. (1967). Invest. OphthaZmoZ. 6, 329. Orloff, J. and Burg, M. (1971). Ann. Rev. Physiol. 33, 83. Revel, J. P. and Karnovsky, M. J. (1967) J. Cell. BioZ. 33, C7. Reynolds, 0. (1900). Scientifi Papera. Cambridge University Press, Cambridge. Rocha, A. S. and Kokko, J. P. (1972). I’. Int. Gong. Nephrol. A&., p. 79. Rogers, K. T. (1968). Biochim. Biophys. Aeta 163, 50. Smulders, A. P., Tormey, J. McD. and Wright, E. M. (1972). J. Membr. BioZ. 7, 164. Trenberth, S. M. and Mishima, S. (1968). Invest. OphthuZmoZ. 7,44. Zadunaisky, J. A., Candia, 0. A. and Chiarandini, D. J. (1963). J. Gen. Physiol. 47, 393. Zadunaisky, J. A. (1966). Amer. J. Physiol. 211, 506.

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Discussion Dr Maurice In this set-up we were able to measure the potential difference and fluid pump simultaneously and we find that at first there is a good correspondence between the potential difference and the fluid pump. Unfortunately, the potential seems to disappear about three-quarters of an hour before the pump stops, so I can’t altogether agree that the potential has anything to do with the pumps. I would like such to be the case since I calculated an expected value for the potential difference in the order now found as early as in 1951. Dr Fischbarg Well, your comment is absolutely right. There are gaps have to be filled in. The time course of the pump and them. But there are many explanations for this discrepancy. generated in some structural feature of the preparation before the actual pumping activity does.

in our knowledge which the potential is one of The potential might be which would disappear

Dr Rehm I’m ignorant about this field and I hesitate to ask these simple minded questions but did I understand you that you are postulating that this potential difference acting in between the cells in the intercellular spaces may be the force that is transferring the water? Dr Fischbarg There are two possibilities. Either there is an active transport or a substrate, in which case due to the polarity of the potential difference one can speculate that either chloride or bicarbonate would be pumped, generating the potential difference, and then having the counter-ion accompany it and moving subsequently a volume of fluid. In the second hypothesis, it could be that the peristaltic movements of the intercellular spaces would push the fluid out, perhaps through the terminal junctions, and would create a streaming potential. Dr Rehm Then you are not considering the possibility that there may be electro osmosis due to this half-to 1 mV causing fluid to flow through the intercellular spaces? Dr Fisch barg Yes, that is a possibility, generated?

but in that case where would the potential difference be

In the cells. I thought this was one of the hypotheses you were considering. Dr Fischbarg In other words, the cells would generate a potential difference which in turn would produce an electroosmotic movement!

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Dr Rehm The question that I want to ask is that if this is one of the hypotheses you are entertaining, then you ought to be able to test it by sending currents from an external source to reverse the flow through the intercellular spaces. Dr Fischbarg Yes, there is no question that several of these hypotheses can and will be tested. Dr Maurice Have you short circuited the endothelium’l Dr Fischbarg NC, I haven’t attempted to short circuit so far. Dr Rehm Well then one rather facetious question, what is wrong with old fashioned pinocytosis as the water pump of the endothelium? Dr Fischbarg Only the volume that it moves-there is nothing basically wrong with the idea, but the movement, the fantastic volume of solvent moved. I don’t think could be conveniently explained by the number of vesicles one actually sees in the electron microscopic pictures. That is my feeling about this. Yes, of course, it is a question of the turnover of the vesicles rather than their number. But even so . . . Dr Kaye One point of course is that even in one of the classic systems you cite of the neutral pump of the gall-bladder there was, when very careful measurements were made, a very small P.D. It was considered insignificant by most of the workers because it wasn’t the 40 to 60 mV that everybody expected, it was about 14 or 2 mV. Secondly, I hate to argue with a collaborator, but there are two problems with the whole pinocytotic system as well as with peristalsis of the intercellular space as a mechanism for moving fluid out. One is that pinocytosis as a rapid method of moving material is going in the wrong direction. Materials are going into the stroma that way. Secondly, when you follow the tracers that go with the pinocytotic vesicle they flow in, apparently under a pressure head into Descemet’s membrane. We have the old pictures showing the delta formation at the bottom of the intercellular space. In addition, if markers are put on the stromal side and are allowed to diffise as far as the margin of the endothelium and Descemet’s membrane they never enter the intercellular space. So if there is a flow in the intercellular space, whatever evidence we have at the moment indicates that it is in the wrong direction. Mr Geissler Would you care to speculate whether the one-half millivolt difference you found across the endothelium is actually due to the difference in the reflection coefficient and the sodium drag caused the volume flow!

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Dr Fischbarg Well, what you are saying in other words, is that there is a volume flow which is creating a potential difference. In this case it wouldn’t be the flow through channels with fixed charges that is causing the potential difference, but the charge separation would be achieved by other means. Yes, that is a possibility, of course, that intertwines with the possibilities I speculated on. Mr Geissh In which case we might be putting the cart before the horse in saying that the volume flow is due to the potential difference. Dr Handler A question about the effect of cytochalasin B which you said markedly changes the shape of the cells. In view of the importance which some people are attributing to the shape of the intercellular space, isn’t it possible that that change alone would be enough to inhibit the pump and thereby stop pumping without necessarily having peristaltic movements? Dr Fisclabarg That is one question we are investigating possibility.

at this point. Obviously there is such a

Dr Handler In other words the cell shape presumably is optimal as you have it and then if you distort it one way or another it conceivably could change the pump. And I might say as an addendum that someone has reported in abstract that colchicine B and other drugs which break up microtubules inhibit the water permeability in the toadbladder in response to cyclic-AMP. The same explanation, change in cell shape, may hold for the effect too, though I am not sure that either one is correct. Dr Maurice I don’t think these potential differences represent a streaming potential. We’ve done experiments raising the pressure, in which the 300~ due to the pump is stopped, and the potential doesn’t change, we can’t distinguish a change in the potential. I don’t think it is a diffusion potential either because the endothelium is like a piece of blotting paper; it has such a high permeability that it doesn’t distinguish between one ion species and another so I can’t think it is a diffusion potential. Dr Fisch barg Have you tried osmotic pressure difference on the potential difference! Is there an effect Z Dr Maurice No effect either. Dr Raye Relative to your comments on cytochalasins, one of the things that has gotten us

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into this is the possible relation between cytochalasin effects and calcium depletion effects which Bernfield and Wessels [(1970). Develop. Biol., 8uppl. 4,195] have discussed in the past. The calcium effect is so clearly on the junction and restoration of the junction and the reversal process appear to be related to the microfilaments that are present there. The classic work of Wessels [(1971). Science 171, 1351 shows that cytochalasin has a direct effect on microfilament structure and function, so that it may well be a change in shape of the cell itself or, at least, a change in relationship at the junction which is responsible for the effects shown by Dr Fischbarg. A propos of Dr Davson’s question before, this might be a very precisely regulated system at the junction, and cytochalasin effects might be another way of detecting it. Dr Dikstein I think that it is absolutely impossible to differentiate the actions of any poison or any metabolite. Is the effect important simply for normal cellular survival or is it a direct effect on the pump? Obviously if you influence the cells, their shape, their survival, their metabolism, deeply, so deeply that you destroy the viability of the cell, you also inlluence the pump, but so far as we have no idea of what is the pumping mechanism and what is the biochemistry of the pump we just cannot differentiate between these two alternatives. There is absolutely no way to differentiate whether cytochalasin has a morphological effect and as a consequence the pump is influenced by this or it also has a biochemical effect. Dr Handler A last comment, going back to the toad urinary bladder again which as you know, pumps sodium actively, colchicine does not affect sodium transport in the toad bladder at the same time that it blocks changes in water permeability elicited by cyclic-AMP. That is different than the example that we are talking about to day. However, in many tissues the effects of these agents are highly specific, and they are used at such low concentrations that I think we ought to pay more attention to them than we do when we throw in a drug at 10” M and then try to figure out what it is doing. Dr Fischbarg said that he did see changes in the shape of the cell, and so I think it is reasonable to attribute the changes to that rather than to a pump effect directly. Dr Zadunaisky Since mechanisms are not known for it, maybe we should forget about the term “neutral pump” for the moment and call it the “fluid pump” of the endothelium? Would that be acceptable! Dr Maurice Applause.